Journal of Ethnopharmacology 76 (2001) 201– 214 www.elsevier.com/locate/jethpharm Review Lippia: traditional uses, chemistry and pharmacology: a review M.E. Pascual a, K. Slowing a,*, E. Carretero a, D. Sánchez Mata b, A. Villar a b a Departamento de Farmacologı́a, Facultad de Farmacia, Uni6ersidad Complutense de Madrid, Ciudad Uni6ersitaria, 28040 Madrid Spain Departamento de Biologı́a Vegetal II, Facultad de Farmacia, Uni6ersidad Complutense de Madrid, Ciudad Uni6ersitaria, 28040 Madrid Spain Received 13 September 1999; accepted 20 October 2000 Abstract The chemical composition, pharmacological activity and traditional uses of 52 species attributed to the genus Lippia (Verbenaceae) as used in the South and Central America, and Tropical Africa, were revised and compared. A survey of the available literature shows that these species are used mostly for the treatment of gastrointestinal and respiratory disorders and as seasoning. Additionally, some of these Lippia species showed antimalarial, spasmolitic, sedative, hypotensive and, anti-inflammatory activities. Generally, the essential oil or the phenolic compounds (flavonoids) from these plant extracts are assumed to be the active principles. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chemical composition; Lippia species; Traditional uses; Verbenaceae 1. Introduction The genus Lippia (Verbenaceae) includes approximately 200 species of herbs, shrubs and small trees. The species are mainly distributed throughout the South and Central America countries, and Tropical Africa territories (Terblanché and Kornelius, 1996). Most of them are traditionally utilized as gastrointestinal and respiratory remedies (Morton, 1981). Some Lippia species have shown antimalarial (Gasquet et al., 1993), antiviral (Abad et al., 1995) and cytostatic activities (López et al., 1979; Slowing Barillas, 1992; Klueger et al., 1997),... Besides, the leaves from the majority of these species are utilized as seasoning for food preparations (Morton, 1981). With regard to these culinary purposes, it is necessary highlight the importance of the specie Lippia dulcis Trevir., a sweet plant, which principal and non toxic component was isolated by Kinghorn * Corresponding author. E-mail addresses: m.pascual.010@recol.es (M.E. Pascual), karlas@eucmax.sim.ucm.es (K. Slowing). and coworkers: (+ )-hernandulcin, showed it to be more than 1000 times as sweet as sucrose. The purpose of this paper is to list and to compare the chemical composition, pharmacological aspects and traditional uses which have been identified in the literature concerning to Lippia species, in order to uncovering records of other compounds, new flavouring substances and their relationships. There are few studies published about the chemical composition and pharmacological aspects. The composition of essential oils of this genus has been most throughly investigated. However, there are many references about the traditional uses of these species, often these are contradictory activities. But only few pharmacological studies has been made. L. multiflora Moldenke and L. alba (Mill.) N.E.Brown are the most widely studied species. In general, the genus appears to present a consistent profile of chemical composition, pharmacological activities and folk uses. In most cases, the parts used are leaves or aerial parts, and flowers. They are commonly prepared as an infusion or decoction, and administered orally. 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 3 4 - 3 202 Table 1 Species of Lippia reviewed, by Moldenke (1971, 1980) Synonymya L. abyssinica (Otto & A. Dietr.) Cuf. L. alba (Mill.) N.E. Brown L. adoënsis Hochst.; L. adoensis Hochst. ex Schau.; H Lantana abyssinica Otto. & A. Dietr. L. alba (Mill.) N.E. Brown; ex Britton & Wilson, L. S asperifolia A. Rich.; L. crenata Sessé & Moc.; L. geminata microphylla Griseb.; L. germinata H.B.K.; L. glabriflora Kuntze; L. ha6anensis Turcz; L. lantanoides Coult.; L. trifolia Sessé & Moc.; Lantana alba Mill.; Lantana canescens Hort.; Lantana geminata (H.B.K.) Spreng.; Lantana geminata Spreng.; Lantana lippioides Hook. & Arn.; Phyla geminata H.B.K.; Verbena lantanoides Willd. L. balsamea Mart.; L. citrata Cham.; L. citrata H Willd.; L. geminata Kunth; L. geminata H.B.K.; L. globiflora Kuntze L. brasiliensis A.S. Müller; Lantana odora Mart. L. capitulis pyramidatis L.; L. floribunda H.B.K.; L. floribunda Kunth; L. hemisphaerica Cham.; L. pauciserrata Turcz..; L. pyramidata Crantz L. alba 6ar. globiflora (L%Hér.) Moldenke L. alnifolia Schau. L. americana L. L. car6iodora Meikle L. car6iodora var. minor Meikle L. che6alieri Moldenke L. dauensis (Chiov.) Chiov. L. formosa T. S. Brandegee L. grandifolia Hochst. [ex A. Rich] L. grata Schau. L. gra6eolens H.B.K. L. grisebachiana Moldenke L. integrifolia (Griseb.) Hieron. L. ja6anica ( N.L. Burm.) Spreng. L. junelliana (Moldenke) Tronc. Lantana microphyla Hutch. & Bruce; Lantana petitiana Glover L. che6alieri Moldenke L. dauensis Chiov.; L. ellenbeckii Loes.; L. ellenbeckii var. pinnatifida Loes.; Lantana dauensis Chiov. Plant L. asperifolia Marthe; L. asperifolia H.H.W. Pearson; L. asperifolia var. anomala Moldenke; L. capensis Spreng.; L. la6andulaefolia Schwaegr.; L. scabra Hochst. L. crenata (Griseb.) Kuntze; L. crenata Kuntze; L. lantanifolia var. crenata Griseb.; Lantana junelliana Moldenke References Africa Elakovich et al. (1987); Terblanché and Kornelius (1996) Craveiro et al., (1981); Morton (1981); Cáceres et al., (1991); Heinrich et al., (1992); Zamora-Martı́nez and Nieto de Pascaul (1992); Munir (1993); Aguilar and Morales (1995); De Abreu Matos et al., (1996); Pino et al. (1996); Terblanché and Kornelius (1996) Florida, Texas, Central and South America Central and South America Zamora-Martı́nez and Nietode Pascaul (1992); Forestieri et al., (1996) Brazil Central and South America Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Kenya Republic of Somali, Kenya Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) H Occidental Africa Ethiopia, Republic of Somali, Kenya Mexico Central and Oriental Africa Brazil S Texas and Central America Pousset (1989); Gasquet et al., (1993) Mwangi et al., (1992); Samuelsson et al., (1993); Terblanché and Kornelius (1996) Dimayuga and Keer Garcı́a (1991) Mwangi et al., (1992) Souza Brito and Souza Brito (1993); Terblanché and Kornelius (1996) Morton (1981); Compadre et al., (1987); Domı́nguez et al., (1989); Germosen-Robineau (1995); Terblanché and Kornelius (1996) L. adoënsis Auct.; L. adoënsis R. E. Fries; L. amentacea M.E. Jones; L. berlandieri Schau.; L. berlandieri Millsp.; L. bolandieri Schau.; L. gra6eolens Schau.; L. gra6eolens Kunth; L. tomentosa Sessé & Moc.; Lantana gra6eolens Crutchfield & Johnston; Lantana origanoides Mart. & Gal. L. asperifolia argentiniensis Gill.&Schau.; L. lantanaefolia Griseb. L. turbinata var. integrifolia Griseb. Distribution S Argentina Terblanché and Kornelius (1996) Argentina Central and South Africa Catalán et al., (1994); Terblanché and Kornelius (1996); Fricke et al., (1999) Rimpler and Sauerbier (1986); Mwangi et al., (1992); Hutchings and van Staden (1994); Terblanché and Kornelius (1996) Argentina Terblanché and Kornelius (1996) M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 Specie Table 1 Species of Lippia reviewed, by Moldenke (1971, 1980) Synonymya Plant Distribution References L. micromera Schau. L. origanifolia Kunth S South America L. micromera var. helleri (Britton) Moldenke L. microphylla Cham. L. cuneifolia Sessé & Moc.; L. helleri Britton Central America Morton (1981); Germosen-Robineau (1995); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) L. microphylla Cham. & Schlecht.; L. microphylla Mart.; Lantana microphylla Mart. ex Uphof Guyana, Brazil Lemos et al., (1992) H Central Africa S Colombia, Venezuela, Brazil Brazil Pham Huu Chanh et al., (1988a, 1988b); Pousset (1989); Valentin et al., (1995); Taoubi et al., (1997); Abena et al., (1998) Morton (1981); Terblanché and Kornelius (1996) Grieve (1995) Zaire, Namibia, South Africa Paris and Moyse (1971) Brazil Macambira et al., (1986); Lemos (1990, 1992); Souza Brito and Souza Brito (1993); Terblanché and Kornelius (1996) Mwangi et al., (1992); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) L. multiflora Moldenke L. origanoides H.B.K. L. pseudo-thea (St. Hil.) Schau. L. rehmanni H.H.W. Pearson L. sidoides Cham. L. berterii Spreng.; L. organoides H.B.K. Lantana pseudo-thea St. Hil.; Lantana pseudothea St. Hil. L. baurii H. H. W. Pearson; L. bazeiana H.H.W. Pearson; Lantana bazeiana H.H.W. Pearson; Lantana rehmanni H.H.W. Pearson L. multicapitata Mart. L. somalensis Vatke L. thymoides Mart. & Schau. L. turbinata Griseb. L. turbinata f. magnifolia Moldenke L. ukambensis Vatke L. wilmsii H.H.W. Pearson a S Republic of Somali, Kenya L. micromera var. paludicola Moldenke; L. ocymoides Mart. & Schau. L. aprica Phil.; L. disepala Phil.; L. microphylla f. glabriuscula Kuntze; L. poleo Lillo Lippia fissicalyx Tronc. Brazil L. africana Moldenke; Lantana ochroleuca Dinter; Lantana wilmsii H.H.W. Pearson Central and South Africa Peru, Chile, Argentina Argentina Tanzania, Kenya Rimpler and Sauerbier (1986); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Mwangi et al.,(1992); Terblanché and Kornelius (1996) Mwangi et al., (1992); Terblanché and Kornelius (1996) M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 Specie Usual synonomous names cited in pharmacognosy works. Plant: H = herb; S = shrub. 203 204 M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 1.1. Species included in the re6iew As mentioned earlier, there are about 200 species of Lippia and 60 infraspecific described taxa in the world (Terblanché and Kornelius, 1996). The much higher number of species attributed to this genus by some authors is probably due to taxonomic inclusion of several taxa with some characters common to Lippia species. For instance, R. A. Howard considered it a genus of about 400 species. Moldenke compared the generic descriptions given to this genus by varios authors over the years (1849– 1969) and concluded that ‘‘pressumably all these authors included Acantholippia, Aloysia and Phyla in their concept of Lippia’’. Besides, some species of genus Lantana has been considered as Lippia, because both these genera are closely related (Munir, 1993). Due to taxonomic problems between Lippia and other genera, we follow the taxonomic approach of Moldenke (1971, 1980). However, it is essential to recall that several authors, include some species belonging to the genus Aloysia, Lantana, Acantholippia, Phylla or Junellia in the genus Lippia; from what we have listed the species as they have reported. Also, we followed for authors abbreviations involved in taxonomic nomenclature the Stafleu and Cowan compilation (1988) . The list of species reviewed and their geographical distribution and synonymy is incorporated in Table 1, and the species found in the literature reviewed including the Moldenke rejected names are incorporated in Table 2. The following species are not cited by Moldenke (1971, 1980): 1. Lippia affinis aristata Schau. (Craveiro et al., 1981; Terblanché and Kornelius, 1996) from Brazil. Moldenke recognizes only Lippia affinis Schau. from Brazil and Bolivia, and includes in the rejected names Lippia affinis Briq. [ = Aloysia sellowii (Briq.) Moldenke], excluding any intraspecific variability in Lippia affinis Schau. 2. Lippia affinis sidoides Cham. (Craveiro et al., 1981; Terblanché and Kornelius, 1996) from Brazil. 3. Lippia palmeri S. Wats. var. palmeri (Dimayuga and Keer Garcı́a, 1991) from Mexico: Moldenke only cites L. palmeri S. Wats. excluding any intraspecific variability in this specie. 4. Lippia gracilis H.B.K. (Lemos et al., 1992): Moldenke cites L. gracilis Schau. from Brazil, and in the rejected names includes: L. gracilis Phil. [= Acantholippia trifida (Gay) Moldenke]. 5. Lippia grandis Scham. (Di Stasi et al., 1994) from Brazilian Amazon. Besides, the relationships of Lippia alba (Mill.) N.E. Brown and Lippia geminata H.B.K. are in dispute. Some authors think that they are synonymous (Morton, 1981) and others considere both as differents species (Zamora-Martı́nez and Nieto de Pascual, 1992). Moldenke (1971, 1980) recognizes Lippia geminata H.B.K. as a synonym of Lippia alba var. globiflora (L%Hér.) Moldenke. Also, the species L. multiflora Moldenke, L. adoënsis Hochst. and L. grandifolia Hochst. were accepted as synonymous by some authors (Mwangi et al., 1992; Terblanché and Kornelius, 1996) whereas Moldenke (1971, 1980) recognized them as different species. 1.2. Traditional uses of Lippia The most common use of Lippia species is for the treatment of respiratory disorders, as Table 3 shows. For this use the plant is commonly prepared as decoction. In Central and South America (Guatemala, Venezuela, Brazil) it is also employed as a remedy for colds, grippe, bronchitis, coughs and asthma: L. alba N.E. Brown, L. dulcis Trevir., L. che6alieri Moldenke, L. gra6eolens H.B.K., L. micromera, Schauer., L. microphylla Cham., L. multiflora Moldenke, L. nodiflora (L.) Michx. and L. origanoides H.B.K. (Morton, 1981; Pousset, 1989; Lemos et al., 1992; Forestieri et al., 1996). Out of these seven medicinal plants, two are commonly used as pectoral remedy: L. alba (Mill.) N.E.Brown and L. dulcis Trevir., in the Mesoamerican region (Morton, 1981; Compadre et al., 1986; CEMATFARMAYA, 1996). Besides, in Africa, milk or water infusions of L. ja6anica (N.L. Burm.) Spreng. are drunk for colds and their leaves and roots are used for shortness of breath and chest (Hutchings and van Staden, 1994). The leaves from L. alba (Mill.) N.E.Brown, L. citriodora (Ort.) H.B.K., L. gra6eolens H.B.K., L. micromera Schau and L. origanoides H.B.K. (Morton, 1981; De Vincenzi et al., 1995) are utilized as seasoning for food preparations. Additionally, the leaves of L. citriodora (Ort.) H.B.K. are used for flavouring beverages (De Vincenzi et al., 1995); the licorice-flavored root from L. dulcis Trevir. is chewed in Central America, and cigarette paper was dyed with the juice of this plant in Cuba (Morton, 1981). In Brazil, L. pseudo-thea Schau is used as a substitute for tea and its fruit is eaten (Grieve, 1995). In Venezuela the decoction of L. origanoides H.B.K. is taken to stimulate the appetite (Morton, 1981). Also, the decoction and infusion of these plants is taken as gastrointestinal remedy throughout South and Central America, Tropical Africa and some European countries. Some of them are considered very useful in the treatment of stomach ache and indigestion, as L. alba ( Mill.) N.E. Brown, L. citriodora H.B.K. or L. triphylla (L%Hér.) Kuntze, L. dulcis Trevir., L. micromera Schau, L. origanoides H.B.K., L. reptans H.B.K., L. stoechadifolia H.B.K. and L. turbinata Griseb. (Paris and Moyse, 1971; Bandoni et al., 1972; Morton, 1981; Girón and Cáceres, 1994; Nakamura et al., 1997; Ballero et al., 1998). Several of them, M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 205 reports about the use of the L. alba (Mill.) N.E. Brown leaves infusion to soothe vesicle ache in Mexico (Zamora-Martı́nez and Nieto de Pascual, 1992) and the choleretic activity of L. multiflora Moldenke (Pham Huu Chanh et al., 1988a). In Guatemala and Brazil, several Lippia species are applied externally to treat cutaneous diseases, burns, wounds, ulcers: L. alba N.E. Brown and L. gracilis H.B.K. (Girón et al., 1991; Lemos et al., 1992; Girón acts as carminatives: L. alba (Mill.) N.E. Brown, L. triphylla (L%Hér.) Kuntze and L. reptans H.B.K. (Heinrich et al., 1992; Girón and Cáceres, 1994; Nakamura et al., 1997). Most frequently these plant contain essential oils that might produce the mentioned effects. In Middle America and Brazilian Amazon an infusion or decoction of L. alba (Mill.) N.E. Brown or L. grandis Scham. (Morton, 1981; Di Stasi et al., 1994) is taken to treat hepatic diseases. Furthermore, there are Table 2 Other species found, included as rejected names by Moldenke (1971, 1980) Planta Scientific Name Found Scientific Name by Moldenke Lippia aristata Schau. Lantana aristata (Schau.) Briq. Lippia canescens Kunth Phyla nodiflora var. canescens (H.B.K.) Moldenke Lippia chamaedrifolia Steud. Aloysia chamaedryfolia Cham. Lippia chamissonis D. Dietr. Lantana chamissonis (D. Dietr.) Benth. Aloysia triphylla (L%Hér.) Britton S South America Lippia dulcis Trevir. Phyla scaberrima (Juss.) Moldenke Central and South America Lippia hastulata (Griseb.) Hieron. Lippia ligustrina (Lag.) Britton Acantholippia hastulata Griseb. Lippia citriodora (ort.) H.B.K. or L. citriodora Kunth or L. citriodora H.B.K. or L. triphylla ( L%Hér.) Kuntze Junellia ligustrina (Lag.) Moldenke Lippia lycoides (Cham.) Steud. Aloysia gratissima (Gill. & or L. lycioides (Cham.) Steud. Hook.) Tronc. Lippia nodiflora (L.) Greene or Phyla nodiflora (L.) Greene L. nodiflora (L.) Michx. Lippia polystachia Griseb. Lippia reptans H.B.K. Lippia sellowii Briq. Lippia seriphioides A. Gray Lippia stoechadifolia H.B.K. Lippia trifida Gay a Plant: H = herb; S= shrub. Aloysia polystachya (Griseb.) Moldenke Phyla nodiflora var. reptans (H.B.K.) Moldenke Aloysia sellowii (Briq.) Moldenke Acantholippia seriphioides (A. Gray) Moldenke Phyla stoechadifolia (L.) Small Acantholippia trifida (Gay) Moldenke H H Distribution References Brazil, Bolivia, Paraguay, Argentina EEUU, Central and South America, France, Asia, Central and North Africa Brazil, Uruguay, Argentina. Terblanché and Kornelius (1996) Tomás-Barberán et al., (1987); De Bolós (1990) North Carolina, California, Central and South America, Spain, India, China Bolivia, Argentina Argentina S H Texas, New Mexico, Mexico, South America Around the World Argentina Around the World South America Argentina S Florida, Venezuela, Central America Chile Terblanché and Kornelius (1996) Souza Brito and Souza Brito (1993) Paris and Moyse (1971); Rimpler and Sauerbio (1986); Skaltsa and Shammas (1988); Hutchings and van Staden (1994); De Vincenzi et al., (1995); Terblanché and Kornelius (1996); Nakamura et al., (1997) Morton (1981); Compadre et al., (1985, 1986); Mori and Kato (1986); Kaneda et al., (1992); Cáceres et al., (1993); Guevara et al., (1994); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996); USDA (1998) Rimpler and Sauerbier (1986); Tomás-Barberán et al., (1987); Mukherjee (1991); Forestieri et al., (1996); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Girón and Casceres(1994) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Morton (1981); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) 206 M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 and Cáceres, 1994); and also in cases of fever: L. alba N.E. Brown (Morton, 1981). The use of L. nodiflora (L.) Michx. and L. geminata H.B.K. to treat gonorrhea and L. alba (Mill.) N.E. Brown (and/or L. geminata H.B.K.) to treat syphilis are also reported (Morton, 1981; Zamora-Martı́nez and Nieto de Pascual, 1992; Forestieri et al., 1996). Some of these species are valuated to treat diarrhea and dysentery: L. alba (Mill.) N.E. Brown, L. dulcis Trevir., L. gra6eolens H.B.K. and L. reptans H.B.K. (Morton, 1981; Compadre et al., 1986; Cáceres et al., 1993; Girón and Cáceres, 1994). Besides, from Yucatan, the leaf decoction of L. gra6eolens H.B.K. is taken as an intestinal antiseptic (Morton, 1981). Most of them are used as antispasmodic tea: L. alba (Mill.) N.E.Brown, L. citriodora (Ort.) H.B.K. or L.triphylla L%Hèr., L. dulcis Trevir., L. geminata H.B.K., L. gra6eolens H.B.K. and L. nodiflora (L.) Michx. (Schauenberg and Paris, 1977; Morton, 1981; Hutchings and van Staden, 1994; De Abreu Matos et al., 1996; Forestieri et al., 1996; Klueger et al., 1997). In Zimbabwe the leaves and roots of L. ja6anica (N.L. Burm.) Spreng. are used for abdominal pains (Hutchings and van Staden, 1994). L. alba (Mill.) N.E. Brown, L. dulcis Trevir., L. geminata H.B.K., L. gra6eolens H.B.K., L. ja6anica (N.L. Burm.) Spreng., L. nodiflora (L.) Michx. and L. triphylla L%Hèr. are used as analgesic, antiinflammatory and/or antipyretic remedies (Morton, 1981; Cáceres et al., 1993; Di Stasi et al., 1994; Hutchings and van Staden, 1994; Germosén-Robineau, 1995; CEMATFARMAYA, 1996; Forestieri et al., 1996). In Africa, several Lippia species: L. che6alieri Moldenke, L. multiflora Moldenke and L. nodiflora (L.) Michx. are used in folk medicine as antimalarials (Mukherjee, 1991; Gasquet et al., 1993; Valentı́n et al., 1995). The decoction of L. alba (Mill.) N.E. Brown and milk is taken in Costa Rica to treat worms (Aguilar and Morales, 1995). In Central America L. stoechadifolia H.B.K. is planted around houses to repel insects (Morton, 1981). The African specie, L. multiflora Moldenke is used for the treatment of arterial hypertension (Pham Huu Chanh et al., 1988a; Abena et al., 1998). Whereas, in Guatemala: L. dulcis Trevir., in Trinidad L. micromera Schau and the African L. nodiflora (L.) Michx. are used as diuretic (Morton, 1981; Forestieri et al., 1996). L. alba Mill. N.E. Brown and/or L. geminata are used in Brazil as sedatives (De Abreu Matos et al., 1996; Klueger et al., 1997). In view of the above reports, it is unusual that L. che6alieri Moldenke is taken as stimulant tea in Senegal, while in Nigeria the same infusion is drunk as a sedative and relaxing remedy (Pousset, 1989). Several Mexican species displayed a abortifacient and antifertility activity. This is especially true of L. dulcis Trevir. and L. gra6eolens H.B.K. Most authors associ- ated these activities with the relatively high amount of the toxic monoterpenes: camphor, p-cymene and bornyl acetate (Compadre et al., 1986, 1987; Domı́nguez et al., 1989). L. alba (Mill.) N.E. Brown, L. dulcis Trevir., L. geminata H.B.K., L. gra6eolens H.B.K. L. grandis Scham. and L. nodiflora (L.) Michx. are used as a menstrual disorders remedy in Brazil and Middle America (Morton, 1981; Compadre et al., 1986; Di Stasi et al., 1994; Forestieri et al., 1996). In Mexico the decoction of L. gra6eolens H.B.K. is also taken as an effective treatment for diabetes (Morton, 1981). 1.3. Chemical composition The chemical composition of the essential oils from the many of Lippia species (see Table 4) has been investigated by means of gas chromatographic techniques. From these data, we can see the components which were found in the highest frequency in the Lippia essential oils are: limonene, i-caryophyllene, p-cymene, camphor, linalool, h-pinene and thymol. Some species showed variable oil composition, these differences often separate them into several chemotypes. As noted above, this is the case of L. alba (Mill.) N.E. Brown, where distinct citral and carvone types are identified (De Abreu Matos et al., 1996). An additional chemotype has recently been reported from Cuba (Pino et al., 1996). L. ukambensis Vatke is another specie where a camphor and a cineole chemotypes have been identified (Mwangi et al., 1992). Furthermore, a remarkably sweet compound was isolated from L. dulcis Trevir.: (+ )-hernandulcin (Fig. 1) (Compadre et al., 1985), a sesquiterpene which its absolute configuration was established by Mori and Kato (1986). By means of latter studies were isolated other related sesquiterpenes as (+)-4i-hernandulcin and (-) — epihernandulcin (Kaneda et al., 1992). The essential oil from L. integrifolia (Griseb.) Hieron. and L. polystachya Griseb. contains several characteristic sesquiterpenes as lippifoli-1(6)-en-5-one (Fig. 2) (Catalán et al., 1994; Terblanché and Kornelius, 1996). In regard to the phenolic acids, a wide variety of caffeic acid derivatives have been identified, including verbascoside or acteoside from L. dulcis Trevir., L. multiflora Moldenke and L. triphylla (L%Hér.) Kuntze (Pham Huu Chanh et al., 1988a,b; Kaneda et al., 1992; Nakamura et al., 1997; Taoubi et al., 1997), isoverbascoside, derhamnosylverbascoside, and a cafeic acid ester linked to 3,4–dihydroxyphenylethanol whose chemical structure has not been elucidated yet (Li 1, from L. multiflora Moldenke), with a marked and long-lasting hypotensive effect (Taoubi et al., 1997). Additional phenolic compounds found in Lippia species are derived from p-coumaric acid, ferulic acid, or synapic acid (Slowing Barillas, 1992). There are fewer references about these less common phenolic compounds. (Table 5). M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 207 Table 3 Traditional uses and pharmacological activities of Lippia speciesa Species L. affinis sidoides Cham. L. alba (Mill.) N.E. Brown L. geminata H.B.K. L. aristata Schau. L. chamissonis D. Dietr. L. che6alieri Moldenke L. citriodora (Ort.) H.B.K. L. dauensis (Chiov.) Chiov. L. dulcis Trevir. Traditional uses Pharmacology References O Craveiro et al. (1981) A, B, C, D, E, G, H, A, B, D, M, N, K, Q, R, S T, V A, B, D, G, K, Q, S A O Q, J López et al., (1979); Morton (1981); Cáceres et al., (1991); Heinrich et al., (1992); Slowing Barillas (1992); Zamora-Martı́nez and Nieto de Pascaul (1992); Di Stasi et al., (1994); Girón and Cáceres (1994); Abad et al., (1995); Germosén-Robineau (1995); CEMAT-FARMAYA (1996); De Abreu et al., (1996); Klueger et al., (1997) Zamora-Martı́nez and Nieto Pascaul (1992); Forestieri et al., (1996) Craveiro et al., (1981) Souza Brito and Souza Brito(1993) B, P, R, S, P Pousset (1989); Gasquet et al., (1993) A, B, C, G, I, Q A, B, Y Paris and Moyse (1971); Schauenberg and Paris (1977); Hutchings and van Staden (1994); De Vincenzi et al. (1995); Nakamura et al., (1997); Ballero et al., (1998) Mwangi et al., (1992) L A, C, D, G, I, K, R, W C, M L. formosa T. S. Brandegee L. gracilis H.B.K. E L. grandifolia Hochst. L. grata Schau. L. gra6eolens H.B.K. A, C, D, K, Q, R, U, W L. ja6anica (N.L. A, Q, R Burm.) Spreng. L. micromera Schau C, G, I, R, L. microphylla R Cham. L. multiflora H, J, P, R Moldenke L. nodiflora (L.) A, I, K, M, P, R, Q, Michx. S L. origanoides C, G, R H.B.K. L. palmeri S. Wats. var. palmeri L. reptans H.B.K. D, G L. sidoides Cham. M Morton (1981); Compadre et al., (1985, 1986, 1987); Cáceres et al., (1991, 1993); Kaneda et al., (1992) Dimayuga and Keer Garcı́a (1991) M L, M Lemos et al., (1992) Mwangi et al., (1992) Q Souza Brito and Souza Brito (1993) Morton (1981); Compadre et al., (1987); Domı́nguez et al., (1989) L, M Mwangi et al., (1992); Hutchings and van Staden (1994) M Morton (1981) Lemos et al., (1992) L. somalensis Vatke L. stoechadifolia H.B.K. L. turbinata Griseb. L. ukambensis Vatke L. wilmsii H. H. W. Pearson L F, J, N, P M Pham Huu Chanh et al., (1988a, 1988b); Pousset (1989); Valentin et al., (1995); Taoubi et al., (1997); Abena et al., (1998) Mukherjee (1991); Forestieri et al., (1996) Morton (1981) F, M B, F, J, M, Q, Z G, L G L, M L Dimayuga and Keer Garcı́a (1991) Girón and Cáceres (1994) Macambira et al., (1986); Lemos et al., (1990); Souza Brito and Souza Brito (1993) Mwangi et al., (1992) Morton (1981) Bandoni et al., (1972) Mwangi et al., (1992) Mwangi et al., (1992) a A: analgesic/antiinflammatory/antipyretic; B: sedative; C: culinary seasoning; D: remedy for diarrhoea and dysentery; E: cutaneous diseases treatment; F: antifungal; G: remedy for gastrointestinal disorders; H: treatment of hepatic diseases/choleretic/vesicle ache remedy; I: diuretic; J: antihypertensive; K: menstrual disorders remedy; L: larvicidal/repellant; M: antimicrobial; N: antiviral; O: molluscicidal; P: antimalarial; Q: antispasmodic; R: respiratory diseases treatment; S: treatment of syphilis and gonorrhoea; T: cytostatic; U: diabetes remedy; V: anticonvulsant; W: abortifacient; X: stimulant, Y: pro-convulsant; Z: local anaesthetic. There are very few studies detailing the Lippia flavonoid substituents. The majority of flavonoids identified are flavones, frequently 6-hydroxylated flavones and methoxyflavones, but some flavone sulphates have been reported from a few Lippia species (mono- and disulphates). There are references about the flavones 208 M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 Table 4 Main essential oil constituents of the Lippia speciesa Plant Monoterpenes Sesquiterpenes References L. affinis aristata Schau. Sabinene, limonene, p-cymene, l-3-carene, h-pinene, h-thujene, k-terpinene Thymol, h-phellandrene, p-cymene, myrcene, carvacrol, k-terpinene (a) h-terpineol, i-pinene, k-terpinene, carvone, 1,8-cineole, p-cymene, limonene, linalool, piperitone, thymol (a) Borneol, camphor, 1,8-cineole, citronellol, geranial, linalool, myrcene, neral, piperitone, sabinene, 2-undecanone i-caryophyllene, k-cadinene, k-elemene Craveiro et al., (1981); Terblanché and Kornelius (1996) i-cariophyllene Craveiro et al., (1981); Terblanché and Kornelius (1996) Elakovich and Oguntimein (1987); Terblanché and Kornelius (1996) L. affinis sidoides Cham. L. adoënsis Hochst. L. alba (Mill.) N.E. Brown L. alnifolia Schau. L. americana L. L. aristata Schau. L. car6iodora Meikle L. car6iodora Meikle var. Minor Carvacrol, p-cymene, methyl thymol, thymol, k-terpinene Sabinene, limonene, l-3-carene, h-pinene, linalool (a) Carvone,carvyl acetate, p-cymene, limonene, linalool Limonene L. chamaedrifolia Steud. 1,8-cineole, linalool, sabinyl acetate, sabinene, limonene L. citriodora Kunth Citral-A, citral-B, 1,8-cineole, geraniol, linalool, limonene i-ocimene, ipsenone, myrcene, (Z)-tagetone Camphor, camphene, limonene, terpinolene, h-pinene, (B) lippiol L. dauensis (Chiov.) Chiov. L. dulcis Trevir. Piperitone oxide, limonene, pulegone, piperitone, carvone L. gracilis H.B.K. Thymol, carvacrol, p-cymene,4-terpenil acetate L. grandifolia Hochst. ex. A. Rich. Linalool, thymol, p-cymene L.grandis Mart. & Schau. (b) p-cymene, carvacrol, thymol L. grata Schau. p-cymene, carvacrol, thymol, k-terpinene L. gra6eolens H.B.K. (a) i-phellandrene, carvacrol, 1,8.cineole, p-cymene, methyl thymol, thymol L. grisebachiana Moldenke (b) h-terpineol, dihydrocarvone, linalool, myrcene, piperitone, pulegone, linalyl acetate L. hastulata (Griseb.) Hieron. (b) h-thujone, i-thujone, thujyl alcohol L. integrifolia (Griseb.) Hieron. Camphor, limonene, camphene, methyl isoeugenol (a) l-cadinene, i-cariophyllene, Copaene, germacrene-D, nerolidol, (a) h-muurolene, i-caryophyllene, i-cubebene, i-elemene, k-cadinene, allo-aromadendrene, caryophyllene oxide. i-caryophyllene, h-humulene, allo-aromadendrene Cadinenol i-caryophylene, k-elemene, k-cadinene, h-humulene h-copaene Craveiro et al., (1981); Morton (1981); Terblanché and Kornelius (1996) i-cubebene, i-elemene, kmuurolene, h-humulene Globulol, spathulenol, k-elemene, i-caryophyllene, i-cubebene Caryophyllene oxide Terblanché and Kornelius (1996) L. junelliana (Moldenke) Tronc. L. ligustrina (Lag.) Britton Myrcene, myrcenone, ocimene, (E)-tagetenone, (Z)-tagetenone, cis-tagetone Cis-dihydrocarvone, myrcenone, camphor, limonene, myrcene, (E)-tagetenone (b) 1,8-cineole, h-pinene, vanillin (1996) (1996) (1996) (1996) Terblanché and Kornelius (1996) De Vincenzi et al., (1994); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) (a) h-copaene, i-cariophyllene, l-cadinene, (+)–hernandulcin, (+)-4i-hernandulcin, (−)–epihernandulcin Compadre et al., (1985, 1986); Mori and Kato (1986); Kaneda et al., (1992); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) h-copaene, i-cubebene Lemos et al., (1992) i-cubebene i-caryophyllene, h-copaene, h-muurolene h-humulene, i-caryophyllene, i-bisabolene, aromadendrene Mwangi et al., (1992) Terblanché and Kornelius (1996) Craveiro et al., (1981); Terblanché and Kornelius (1996) Compadre et al., (1987); Terblanché and Kornelius (1996) i-caryophyllene Terblanché and Kornelius (1996) L. fissicalyx Tronc. L. ja6anica (N.L. Burm.) Spreng. Craveiro et al., (1981); Terblanché and Kornelius Terblanché and Kornelius Craveiro et al., (1981); Terblanché and Kornelius Terblanché and Kornelius Terblanché and Kornelius (1996) Lippifoli-1(6)-ene-5-one, D8-africanene, bicyclo germacrene, germacrene-D, h-humulene, asteriscane derivatives i-cariophyllene Bicyclogermacrene, i-caryophyllene, spathulenol Catalán et al. (1993, 1994); Catalán and Lampasona 1995; Terblanché and Kornelius (1996); Fricke et al. (1999) Mwangi et la., (1992); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 209 Table 4 (Continued) Plant Monoterpenes L. lycoides (Cham.) Steud. (b) h-pinene, camphor, carvone, 1,8-cineole, citral, p-cymene,geraniol,limonene, menthone (b) Geranial, neral Carvacrol L. micromera Schau. L. micromera var. helleri (Britton) Moldenke L. microphylla Cham. L. multiflora Moldenke L. nodiflora (L.) Greene L. origanoides H.B.K. L. polystachia Griseb. L. sellowii Briq. L. seriphioides A. Gray L. sidoides Cham. L. somalensis Vatke L. stoechadifolia H.B.K. L. thymoides Mart. & Schau. L. trifida Gay L. turbinata Griseb. L. ukambensis Vatke L. wilmsii H.H.W. Pearson 1,8-cineole, h-terpineol, terpinen-4-ol, methyl thymol, sabinene, k-terpinene, thymol Linalool, 1,8-cineole Sesquiterpenes References Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) i-caryophyllene, h-humulene Nerolidol, i-farnesene, germacrene-D, i-cariophyllene 2-phenethyl alcohol, 1-octen-3-ol, Calamenene, i-caryophyllene, linalool, 2,6-dimethyloctane, methyl h-copaene, h bergamotene, salicylate, p-cymen-8-ol l-cadinene, i-bisabolene (a) h-terpinene, k-terpinene, i-caryophyllene, umbellulone 1,8-cineole, p-cymene, thymol, thymyl acetate h-thujone, carvone, limonene, i-caryophyllene, sabinene lippifoli-1(6)-ene-5-one 1,6-germacradien-5-ol (b) carvacrol, citral, p-cymene, geranial, piperitone, thymol (a) Carvacrol, p-cymene, i-caryophyllene, h-copaene, h-terpinene, thymol h-humulene (a) 1,8-cineole,l-3-carene, i-cubebene myrcene,limonene Pulegone oxide Methyl thymol (b) h-terpinene, h-terpineol, citronellal, citronellol, thymol h-thujone, carvone, limonene, (a) i-caryophyllene oxide, bornyl acetate, camphor spathulenol, i-caryophyllene, germacrene-D, bicyclogermacrene (a) h-terpineol, l-3-carene, (a) i-cubebene camphene, camphor, 1,8-cineole, p-cymene, trans-sabinene hydrate, terpinen-4-ol (a) k-terpinene, 1,8-cineole, (a) i-caryophyllene, i-elemene p-cymene, limonene, linalool, piperitone, piperitenone Lemos et al., (1992) Valentı́n et al. (1995) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Lemos et al., (1990); Terblanché and Kornelius (1996) Mwangi et al., (1992); Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Terblanché and Kornelius (1996) Mwangi et al., (1992); Terblanché and Kornelius (1996) Mwangi et al., (1992); Terblanché and Kornelius (1996) a In order of decreasing %: (a) =the % of these compounds are variable reported; (b) =positive identification. We do not have the facts about their %. trisulphates content in populations of L. nodiflora (L.) Michx. from Malaysia and Saudi Arabia (Tomás-Barberán et al., 1987). Moreover, some flavanones (pinocembrin and naringenin) were identified from L. gra6eolens H.B.K. (Domı́nguez et al., 1989). The most common flavonoids reported include (Table 5): salvigenin, eupatorin, eupafolin, luteolin, hispidulin, diosmetin, cismaritin, cirsiliol, pectolin-arigenin, 6-hydroxyluteolin from L. citriodora (Ort.) H.B.K. (Skaltsa and Shammas, 1988; De Vincenzi et al., 1995); jaceosidin, nepetin, hispidulin, hydroxyluteolin and nodifloretin mono- and disulphates, and flavone aglycones (nepetin, jaceosidin and hispidulin) from L. nodiflora (L.) Michx. L. canescens Kunth showed a flavonoid pattern very similar to that of L. nodiflora (L.) Michx. (Tomás-Barberán et al., 1987). Furthermore, there are references about some flavonoid 4-sulphates from a butanolic fraction of L. alba (Mill.) N.E. Brown, as possible active principles with neuropharmacological activity (Klueger et al., 1997). Other components reported from the literature are: the Fig. 1. Hernandulcin (Compadre et al., 1985). M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 210 Fig. 2. Lippifoli-1 (6)-en-5-one (Catalán et al., 1994). carcinogenic naphthoquinoid ‘lapachenol’ (found in methanolic extracts of aerial parts of L. gra6eolens H.B.K. and L. sidoides Cham. (Macambira et al., 1986; Domı́nguez et al., 1989), and other naphthoquinoids as isocatalponol and 6-oxo-3,4,4a,5-tetrahydro-3-hydroxy2,2-dimethyl naphtho-1,2-pirane, from L. sidoides Cham., (Macambira et al., 1986). In 1986 Rimpler and Sauerbier (Rimpler and Sauerbier, 1986) identified several iridoids from nine species of Lantana, Lippia, Aloysia and Phyla, by means of Fig. 3. (Rimpler et al., 1986). Iridoid, R1, R2; Geniposide, CH3, H; Theveside-Na, Na, OH; Theviridoside, CH3, OH. chromatography methods (CC, TLC, HPLC and GC) folllowed by GC/MS, IR, 1H NMR and/or 13C NMR; and they proposed the use of this iridoid glucosides as taxonomic determinants. Lantana and Lippia seem to be very similar: the main constituents of them are geniposide-type and lamiide-type. L. ja6anica (N.L. Table 5 Other components from Lippia species Species Flavonoids Other compounds References L. alba (Mill.) N.E. Brown L. geminata H.B.K. L. canescens Kunth Flavonoid 4-sulphates Tannins (low%), geniposide (iridoid), triterpenic saponins, resin, mucilages Alkaloids, tannins, saponins, sterols Heinrich et al., (1992); Slowing Barillas (1992); Klueger et al., (1997) Forestieri et al., (1996) Tomás-Barberán et al., (1987) Verbascoside (or acteoside) Skaltsa and Shammas (1988); De Vincenzi et al. (1995); Nakamura et al., (1997) Verbascoside (acteoside), alkaloids Kaneda et al., (1992); Cáceres et al., (1993) Domı́nguez et al., (1989) Hutchings and van Staden (1994) L. citriodora (Ort.) H.B.K. Flavone aglycones, flavone monoand di-sulphates Salvigenin, eupatorin, eupafolin, hispidulin, 6-hydroxyluteolin, luteolin 7-O-i- glucoside, luteolin, cismaritin, diosmetin, cirsiliol, chrysoeriol apygenin, pectolin-arigenin L. dulcis Trevir. L. gra6eolens H.B.K. L. ja6anica (N.L. Burm.) Spreng. L. multiflora Moldenke Naringenin and pinocembrin Lapachenol (naphthoquinoid) Icterogenin Flavonoids L. nodiflora (L.) Michx. Nepetin, jaceosidin and hispidulin aglycones. Hispidulin, jaceosidin, nepetin, hydroxyluteolin and nodifloretin mono and disulphates. Lippiflorin A and B glycosides; nodifloretin A and B, nodiflorin A and B, nodifloridin A and B glucosides Verbascoside, derhamnosylverbascoside, isoverbascoside, Li 1 ( a cafeic acid ester linked to 3,4-dihydroxyphenyl-ethanol), sterols, carotenoids Alkaloids, resin, sugars, stigmasterol, i-sitosterol L. rehmanni H.H.W. Pearson L. sidoides Cham. Triterpenic compounds: icterogenin, Paris and Moyse (1971) rehmannic acid and desoxyicterogenin 6,7-dimethoxy-5,4%-dihydroxyflavone Naphthoquinoids: lapachenol, Macambira et al., (1986) isocatalponol, and 6-oxo-3,4,4a,5-tetra-hydro-3-hydroxy-2,2 -dimethylnaphtho-1,2-pirane Leucoanthocyanidins, steroidic and Bandoni et al., (1972) triterpenic compounds, alkaloids and cardenolides (traces) L. turbinata Griseb. Pham Huu Chanh et al., (1988a, 1988b); Taoubi et al., (1997) Tomás-Barberán et al., (1987); Forestieri et al., (1996) M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 Burm.) Spreng. and L. turbinata Griseb. contains theveside-Na and theviridoside (Fig. 3), Aloysia triphylla (L%Hér.) Britton contains geniposidic acid-Na, geniposide and mussaenoside, and Phyla nodiflora (L.) Greene accumulate the quinol glucoside cornoside (Fig. 4). The iridoid geniposide has also been reported from L. alba (Mill.) N.E. Brown (Heinrich et al., 1992). Alkaloids have been reported from L. dulcis Trevir., L. geminata H.B.K., L. nodiflora (L.) Michx. and L. turbinata Griseb. (Bandoni et al., 1972; Cáceres et al., 1993; Forestieri et al., 1996). However, there are a study from L. dulcis Trevir. in which alkaloids were not present (Compadre et al., 1986). Finally, triterpene and steroidic derivatives, usually identified as saponins, have been reported from L. alba (Mill.) N.E. Brown and/or L. geminata H.B.K., L. ja6anica (N.L. Burm.) Spreng., L. rehmanni H. H. W. Pearson and L. turbinata Griseb. (Paris and Moyse, 1971; Bandoni et al., 1972; Slowing Barillas, 1992; Hutchings and van Staden, 1994; Forestieri et al., 1996). The tannins and resin from L. alba (Mill.) N. E. Brown and/or L. geminata H.B.K. has been examined (Slowing Barillas, 1992; Forestieri et al., 1996). L. dulcis Trevir. contains tannin and a camphor-like substance which was name ‘‘Lippiol’’ (Compadre et al., 1986). There is a report of traces of cardenolides from L. turbinata Griseb. (Bandoni et al., 1972). 1.4. Pharmacology In vitro screening has demonstrated an reproducible inhibitory activity against many of the Gram-positive bacteria most frequently responsible for respiratory infections in humans (Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes), and, thus, may provide a scientific basis for the ethnomedical use of L. alba (Mill.) N.E. Brown and L. dulcis Trevir. against bacterial respiratory infections (Cáceres et al., 1991). The essential oils from Lippia species have also been extensively shown to exhibit antimicrobial activity against other microorganisms: L. sidoides Cham. shows a inhibitory activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium smegmatis, a low activity against Pseudomonas aeruginosa (Lemos et al., 1990), and against some microorganisms living on the skin of feet and armpits (Lacoste et al., Fig. 4. Cornoside (Rimpler et al., 1986). 211 1996). The essential oils of L. gracilis H.B.K. and L. microphylla Cham. is not as broadly active (Lemos et al., 1992). The ethanolic extracts of L. palmeri S.Wats. var. palmeri and L. formosa T. S. Brandegee exhibit activity against Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis (Dimayuga and Keer Garcı́a, 1991). Some extracts of L. dulcis Trevir. shows antimicrobial activity against enteropathogenic Salmonella typhi and Shigella flexneri (Cáceres et al., 1993), but in a study made in Peru this specie showed no in vitro effect against Vibrio cholerae 01, and this suggests that its use for the treatment of cholera is not efficient (Guevara et al., 1994). There are reports of the bactericidal activity of L. grandifolia Hochst. ex A. Rich., L. ja6anica (N.L. Burm.) Spreng., L. nodiflora (L.) Michx. and L. ukambensis Vatke (Mwangi et al., 1992; Forestieri et al., 1996). Species as L. multiflora Moldenke, L. palmeri S. Wats. 6ar. palmeri and L. sidoides Cham. exhibited a in vitro antifungal activity (Lemos et al., 1990; Dimayuga and Keer Garcı́a, 1991; Valentı́n et al., 1995; Lacoste et al., 1996). Moreover, L. alba (Mill.) N.E. Brown and L. multiflora Moldenke showed a certain antiviral activity (Abad et al., 1995; Valentı́n et al., 1995). The cytostatic activity of the volatile oil of L. alba (Mill.) N.E. Brown has been reported (López et al., 1979; Slowing Barillas, 1992; Klueger et al., 1997). It is possible to find studies of in vitro antimalarial activity of decoction and infusion obtained from L. multiflora Moldenke (Valentı́n et al., 1995) and in vitro and in vivo activity from L. che6alieri Moldenke (Gasquet et al., 1993), on Plasmodium falciparum and P. berghei that support their ethnomedical uses. Besides, the essential oils of Brazilian Lippia species and the residual waters from the steam distillation of L. aristata Schau. and L. aff. sidoides Cham., have also shown molluscicidal activity against Biomphalaria glabrata, the most important host of Schistosoma mansoni in Brazil (Craveiro et al., 1981; Mwangi et al., 1992). In a study made in Kenya (Mwangi et al., 1992), the essential oils of a variety of Lippia species demonstrated a larvicidal activity against Aedes aegypti larvae and a maize weevil (Sitophilus zeamais Motsch) repellancy. The active species included: L. dauensis (Chiov.) Chiov., L. grandifolia Hochst., L. ja6anica (N.L. Burm.) Spreng., L. somalensis Vatke, L. ukambensis Vatke (the most active as repellant and with a low larvicidal activity) and L. wilmsii H. H. W. Pearson (the most active larvicide and with a low repellancy). There are reports about the local anaesthetic activity of the essential oil compounds (thymol) from L. sidoides Cham. (Souza Brito and Souza Brito, 1993). Other essential oil compounds identified in Lippia species showed a significant antispasmodic activity: Lippia chamissonis D. Dietr., L. grata Schau., L. ja6anica (N.L. Burm.) Spreng. and L. sidoides Cham. (Souza Brito and Souza Brito, 1993; Hutchings and van Staden, 1994). L. alba (Mill.) N.E. Brown reportedly 212 M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 shows a reduction in intestinal tonus (Klueger et al., 1997). L. alba (Mill.) N.E. Brown, L. geminata H.B.K. and L. nodiflora (L.) Michx. showed a significant analgesic, antiinflammatory and antipyretic activities (Costa et al., 1989; Slowing Barillas, 1992; Forestieri et al., 1996; Klueger et al., 1997; Vale, 1997). And all extracts proved (0.5 mg/kg) from the african L. geminata H.B.K. and L. nodiflora (L.) Michx. did not cause gastric lesion or hyperaemia (Forestieri et al., 1996). Furthermore, a lyophilisated powder obtained from an infusion of dried leaves of L. multiflora Moldenke caused a muscle relaxant effect (in the traction test), an analgesic activity (by using acetic acid and hot plate methods), but did not cause modification of rectal temperature (Abena et al., 1998). A compound from L. triphylla (L%Hér.) Kuntze: acteoside, exhibit analgesia (writhing and tail pressure methods) and a weak sedation (prolongation of pentobarbital-induced anesthesia and methamphetamine-enhanced locomotor activity) by the oral administration (Nakamura et al., 1997). However, in a study about the anxiolytic and sedative properties of infusions of Aloysia triphylla (L’Hér.) Britton in normal volunteers, no difference was observed between plant or placebo (Wannmacher et al., 1990). Additionally, the essential oil of L. sidoides Cham., the infusion of L. multiflora Moldenke and different L. alba (Mill.) N.E. Brown extracts, shows a sedative effect (Souza Brito and Souza Brito, 1993; Klueger et al., 1997; Abena et al., 1998). The anticonvulsant activity of Brazilian L. geminata H.B.K., stands in seeming contrast to proconvulsant effect of L. citriodora (Klueger et al., 1997). Some active principles of Brazilian and African species, essential oil of L. chamissonis D. Dietr., phenolic compounds of L. multiflora Moldenke and residual water resulting from the steam distillation from L. sidoides Cham. induced a hypotensive effect (Macambira et al., 1986; Pham Huu Chanh et al., 1988a,b; Souza Brito and Souza Brito, 1993; Taoubi et al., 1997). L. rehmanni H. H. W. Pearson and L. ja6anica (N.L. Burm.) Spreng. are the only reported toxic Lippia species (Paris and Moyse, 1971; Hutchings and van Staden, 1994). The toxic principle is assumed to be icterogenin. The first of them is reported to produce hepatic disorders, jaundice and photosensibilization in the sheeps (Paris and Moyse, 1971). 2. Discussion and conclusion As mentioned earlier, there are a higher number of species attributed to the genus Lippia by some authors. Moldenke compared the generic descriptions given to this genus by varios authors and concludes that ‘‘pres- sumably all these authors included Acantholippia, Aloysia and Phyla in their concept of Lippia’’. Also, some species of genus Lantana has been considered as Lippia, because both these genera are closely related (Munir, 1993). Besides, the synonymy of these species, included in the works here reviewed, they change according to the authors. For instance, the species L. multiflora Moldenke, L. adoënsis Hochst. and L. grandifolia Hochst. were accepted as synonymous by some authors (Mwangi et al., 1992; Terblanché and Kornelius, 1996) whereas Moldenke (1971, 1980) recognised them as three distincts species. Throughout our literature review we can see that, generally, the species of the genus Lippia show a variable content of essential oils, the components wich were found in these oils in the highest frequency were the monoterpenes: limonene, p-cymene, linalool, camphor and the sesquiterpene i-cariophyllene. The phenolic compounds has been less studied. The flavonoids found in these species are 6-hydroxylated and methoxy flavones for the most part, and also has been identified some flavone sulphates; but these has been studied and identified only in a few of them. The remaining chemical composition of these species has been investigated a little, excepting that relating to the essential oils. The traditional uses of the American species has been reviewed and published extensively. They show little tendency to toxicity. Whereas, there are few and confused references about the folk uses of African species (L. dauensis (Chiov.) Chiov.) is used in Somalia ‘‘to treat a patient who is affected by demons and cannot speak or move’’ (Samuelsson et al., 1993). Other seeming contradictions include the following traditional uses: L. che6alieri is taken as both a stimulant and a sedative (Pousset, 1989). L. alba (Mill.) Brown is taken both to treat gastritis (Zamora-Martı́nez and Nieto de Pascual, 1992) and is a reported to produce a gastric irritant effect (CEMAT-FARMAYA, 1996). It is also difficult to reconcile the traditional use of L. gra6eolens H.B.K. as seasoning for food preparations (Morton, 1981) and its alleged antifertility effect (Compadre et al., 1987; Domı́nguez et al., 1989). In conclusion, there are few studies about the pharmacological activity of these species. Most of studies to date have focussed their attention on the antimicrobial, antifungal, repellant or larvicidal effects of essential oils or different extracts. There is a deplorable scarcity of detailed isolation studies works published or detailed examinations of their pharmacological activities. Thus, we believe that the isolation of new active principles from these species would be of great scientific merit. In addition, the scientific validation for the popular use of them deserves to be further investigated. M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 Acknowledgements The authors wish to thank Drs. Michael Day, A. A. Munir and C. Beecher for their helpful assistance with the taxonomic and others aspects of this work. References Abad, M.J., Sánchez, S., Bermejo, P., Villar, A., Carrasco, L., 1995. Antiviral activity of some medicinal plants. Methods and Findings 17(Suppl. A), 108. Abena, A.A., Ngondzo-Kombeti, G.R., Bioka, D., 1998. Psychopharmacologic properties of Lippia multiflora. Encephale 24, 449 – 454. Aguilar, R. Morales, F., 1995. Manual de Flora de Costa Rica. 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Chemistry, Biological and Pharmacological Properties of Medicinal Plants from the Americas. Poster Session, 2: B23. Lacoste, E., Chaumont, J.P., Mandin, D., Plumel, M.M., Matos, F.J., 1996. Antiseptic properties of essential oil of Lippia sidoides Cham. Application to the cutaneous microflora. Annales Pharmaceutiques Françaises 54, 228 –230. Lemos, T.L.G., Matos, F.J.A., Alencar, J.W., Craveiro, A.A., Clark, A.M., McChesney, J.D., 1990. Antimicrobial activity of essential oils of Brazilian plants. Phytotherapy Research 4, 82 – 84. Lemos, T.L.G., Monte, F.J.Q., Matos, F.J.A., Alencar, J.W., Craveiro, A.A., Barbosa, R.C.S.B., Lima, E.O., 1992. Chemical composition and antimicrobial activity of essential oils from Brazilian plants. Fitoterapia 63, 266 – 268. 214 M.E. Pascual et al. / Journal of Ethnopharmacology 76 (2001) 201–214 López, A.A.M., Rojas, H.N.M., Jiménez, M.C.A., 1979. Plants extracts with cytostatic properties growing in Cuba. Revista Cubana de Medicina Tropical 31, 105 – 111. Macambira, L.M.A., Andrade, C.H.S., Matos, F.J.A., Craveiro, A.A., 1986. Naphthoquinoids from Lippia sidoides. Journal of Natural Products Lloydia 49, 310 – 312. Moldenke, H.N. (1971) A Fifth Summary of the Verbenaceae, Avicenniaceae, Stilbaceae, Dicrastylidaceae, Symphoremaceae, Nyctanthaceae and Eriocaulaceae of the World as to Valid Taxa, Geographic Distribution and Synonymy. U.S.A. Moldenke, H.N. (1980) Phytologia Memoirs II. A Sixth Summary of the Verbenaceae, Avicenniaceae, Stilbaceae, Chloanthaceae, Symphoremaceae, Nyctanthaceae and Eriocaulaceae of the World as to Valid Taxa, Geographic Distribution and Synonymy. U.S.A. Mori, K., Kato, M., 1986. Synthesis and absolute configuration of ( + )-hernandulcin, a new sesquiterpene with intensely sweet taste. Tetrahedron Letters 27, 981 – 982. Morton, 1981. Atlas of Medicinal Plants of Middle America, vol. I. Springfield, Illinois, USA, pp. 745 – 750. Mukherjee, T., 1991. Antimalarial herbal drugs. A review. Fitoterapia 62, 197 – 204. Munir, A.A., 1993. A taxonomic revision of the genus Lippia (Houst. Ex) Linn. (Verbenaceae) in Australia. Journal of Adelaide Botanic Garden 15, 129 – 145. Mwangi, J.W., Addae-Mensah, I., Muriuki, G., Munavu, R., Lwande, W., Hassanali, A., 1992. Essential oils of Lippia species in Kenya. IV. Maize weevil (Sitophilus zeamais) repellancy and larvicidal activity. International Journal of Pharmacognosy 30, 9– 16. Nakamura, T., Okuyama, E., Tsukada, A., Yamazaki, M., Satake, M., Nishibe, S., Deyama, T., Moriya, A., Maruno, M., Nishimura, H., 1997. Acteoside as the analgesic principle of Cedron (Lippia triphylla), a Peruvian medicinal plant. Chemical and Pharmaceutical Bulletin 45, 499 – 504. Paris, R.R., Moyse, H. 1971 Matière Médicale. Saint-Germain, Paris, vol III, pp. 253 – 254. Pham Huu Chanh, Koffi, Y., Pham Huu Chanh, A., 1988a. Comparative hypotensive effects of compounds extacted from Lippia multiflora leaves. 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Plants of the families Passifloraceae-Zygophyllaceae. Journal of Ethnopharmacology 38, 1 – 29. Schauenberg, P., Paris, F., 1977. Guı́a de las Plantas Medicinales. Ediciones OMEGA, Barcelona, p. 266. Skaltsa, H., Shammas, G., 1988. Flavonoids from Lippia citriodora. Planta Medica 54, 465. Slowing Barillas, K.V. 1992. Estudio de la Actividad Antiinflamatoria de Diversas Especies de la Flora de Guatemala. Facultad de Farmacia. Memoria Doctoral. Universidad Complutense de Madrid. Souza Brito, A.R.M., Souza Brito, A.A., 1993. Forty years of Brazilian medicinal plant research. Journal of Ethnopharmacology 39, 53 – 67. Stafleu, F.A. and Cowan, R.S., 1988. Taxonomic Literature. Regnon Vegetabile. 2nd ed. V Utreach vols II. Taoubi, K., Fauvel, M.T., Gleye, J., Moulis, C., Fourasté, I., 1997. Phenylpropanoid glycosides from Lantana camara and Lippia multiflora. Planta Medica 63, 192 – 193. Terblanché, F.C., Kornelius, G., 1996. Essential oil constituents of the genus Lippia (Verbenaceae)-A literature review. Journal of Essential Oil Research 8, 471 – 485. Tomás-Barberán, F.A., Harborne, J.B., Self, R., 1987. Twelve 6-oxygenated flavone sulphates from Lippia nodiflora and Lippia canescens. Phytochemistry 26, 2281 – 2284. USDA (United States Department of Agriculture), 1998. Query the Plants Database. Vale, T.G. 1997. Antinociceptive and antiedematogenic effects of three chemotypes of Lippia alba (Mill.) N.E. Brown from Northeast Brazil. Abstracts. International Joint Symposium. Chemistry, Biological and Pharmacological Properties of Medicinal Plants from the Americas. Poster Session 2: B24 Valentı́n, A., Pélissier, Y., Benoit, F., Marion, C., Kone, D., Mallie, M., Bastide, J.M., Bessières, J.M., 1995. Composition and antimalarial activity in vitro of volatile components of Lippia multiflora. Phytochemistry 40, 1439 – 1442. Wannmacher, L., Fuchs, F.D., Paoli, C.L., Fillman, H.S., Gianlupi, A., Lubianca, J.F., Hassegawa, C.Y., Guimaraes, F.S., 1990. Plants employed in the treatment of anxiety and insomnia: II. Effect of infusions of Aloysia triphylla, on experimental anxiety in normal volunteers. Fitoterapia 61, 449 – 453. Zamora-Martı́nez, M.C., Nieto de Pascual, C., 1992. Medicinal plants used in some rural populations of Oaxaca, Puebla and Veracruz, Mexico. Journal of Ethnopharmacology 35, 229 – 257. Journal of Ethnopharmacology 76 (2001) 215– 221 www.elsevier.com/locate/jethpharm Effects of the aqueous and methylene chloride extracts of Bidens pilosa leaf on fructose-hypertensive rats Théophile Dimo a,*, Jacqueline Azay b, Paul V. Tan a, Jacques Pellecuer c, Gérard Cros b, Marc Bopelet a, Jean Jacques Serrano b a Department of Animal Biology and Physiology, Faculty of Sciences, Uni6ersity of Yaounde I, PO Box 812, Yaounde, Cameroon Laboratoire de Pharmacologie et Physiopathologie Expérimentales, Faculté de Pharmacie, 34 060 Montpellier-Cedex 2, France c Laboratoire de Matière Médicale, Faculté de Pharmacie, 34 060 Montpellier-Cedex 2, France b Received 3 July 2000; received in revised form 3 March 2001; accepted 14 March 2001 Abstract We investigated the effects of the aqueous (150– 350 mg/kg) and methylene chloride (150– 300 mg/kg) extracts of Bidens pilosa on fructose-induced hypertension in rats. Food and liquid intake were measured as well as systolic blood pressure and plasma levels of glucose, insulin, cholesterol, triglycerides and creatinine. Fructose feeding for 6 weeks induced hypertension, hyperinsulinemia and increased plasma triglyceride levels in male Wistar rats. The aqueous and methylene chloride extracts of B. pilosa reversed the high blood pressure and hypertriglyceridemia developed due to fructose feeding but did not have any effects on plasma levels of insulin and glucose. High doses of the extracts reduced plasma creatinine levels and tended to increase plasma cholesterol. These results suggest that the extracts of B. pilosa possess hypotensive effects whose mechanism of action is not related to insulin sensitivity. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bidens pilosa; Fructose-induced hypertension; Hypotensive effects; Rats 1. Introduction Bidens pilosa L. (Asteraceae) is one of the plant species that is extensively used in traditional medicine in Africa (Bouquet and Debray, 1974). Chemical analyses of the leaves have revealed the presence of flavonoids, alkaloids, saponins, phenyl acetylenes and terpenes (Bouquet and Debray, 1974; Hoffmann and Hölzi, 1988; Brandao et al., 1997). Amvam Zollo et al. (1995) indicated the presence of 1-phenyl hepta 1,3,5triyne in the leaves as well as essential oils with anti-microbial and anti-inflammatory properties. The Zulu of South Africa also chew the leaves for their anti-inflammatory properties (Jäger et al., 1996). All parts of the plant are medicinally used. According to Adjanohoun et al. (1996), the leaves are used in the treatment of jaundice, threatened abortion, conjunctivitis, toothache, cough, intestinal helminthiasis and fever. The leaves with the flowers are used to treat * Corresponding author. flank pains, while the whole plant is used to treat fractures and febrile convulsion. The flower is used in the treatment of diarrhoea, dysentery and upset stomach in food poisoning (Bouquet, 1969; Kerharo and Gadam, 1973; Adjanohoun et al., 1982). In the Peruvian Amazon, B. pilosa is used for aftosa, angina, diabetes, dysentery, dysmennorrhea, edema, hepatitis, water retention and dropsy (Duke and Vasquez, 1994). B. pilosa also forms part of a compound mixture of herbs used for the treatment of hypertension (Girault, 1984). The plant is well known in the western region of Cameroon for the management of problems related to high blood pressure and we (Dimo et al., 1998) have shown that the aqueous extract of the leaf possesses aortic smooth muscle relaxant activity. High fructose or high sucrose diets have been documented to increase blood pressure in experimental rats (Bunnag et al., 1997; Cosenzi et al., 1999). Hypertension develops in Wistar rats fed a high fructose diet as early as 2 weeks after initiation of the diet (Vasdev et al., 1994). The hypertension is accompanied by the metabolic abnormalities of hyperinsulinemia, insulin 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 2 9 - X 216 T. Dimo et al. / Journal of Ethnopharmacology 76 (2001) 215–221 resistance, and hypertriglyceridemia (Lee et al., 1994; Iyer and Katovich, 1996; Navarro-Cid et al., 1996). Our previous studies showed that the methanol leaf extract of B. pilosa is effective in preventing the increase in blood pressure induced by salt-loading in rats (Dimo et al., 1999). The aim of the present study was to evaluate whether the aqueous and methylene chloride extracts of B. pilosa are effective in preventing and reversing the high blood pressure and metabolic abnormalities induced by a diet rich in fructose. 2. Materials and methods 2.1. Animals Male albino Wistar rats (160–180 g) were used. The rats were kept in a laboratory animal unit with a 12 h light/dark cycle. Throughout the experiment, room temperature was maintained at 25°C. The rats were maintained on a standard chow diet and fresh water ad libitum prior to dietary manipulation. They were trained for the first week to become acclimated to the procedure of indirect blood pressure measurement. 2.2. Preparation of plant extracts Fresh leaves of B. pilosa were collected around the campus of the University of Yaounde I. A voucher specimen (no. HNC/58742) is deposited at the national herbarium, Yaounde. The leaves were sun-dried and ground into a powder. The aqueous extract was obtained by adding 1 l of distilled water to 100 g of the fine powder. The mixture was allowed to stand and infuse. The resulting decoction was filtered after 24 h and freeze-dried to obtain 15 g of a black powder. The methylene chloride extract was prepared by macerating 2 kg of the air-dried powdered leaves in a mixture of methanol/methylene chloride (1:1). Removal of the solvent under reduced pressure yielded 80 g of a dark extract, which was fractionated with methylene chloride giving 32 g of the methylene chloride extract. 2.3. Pharmacological analysis 2.3.1. Fructose-induced hypertension and effect of Bidens pilosa Forty-two male Wistar rats were divided into seven groups of six animals each. Control groups were given ordinary drinking water ad libitum throughout the whole treatment course and the remaining groups were given 10% fructose solution to drink ad libitum. Three weeks later, the fructose-treated animals were assigned the following treatment regimens: fructose-fed, fructose plus nifedipine (10 mg/kg), fructose plus aqueous extract at the dose of 150 mg/kg or 350 mg/kg, and fructose plus methylene chloride extract at the dose of 150 mg/kg or 300 mg/kg. The animals received these treatment regimens for the next 3 weeks. The plant extracts were administered daily by gastric intubation. Body weight, pulse rate and systolic blood pressure (SBP) were measured every 3 days. In addition, food and fluid intake were recorded each day. 2.3.2. Blood pressure measurement procedure Systolic blood pressure and the heart rate were measured using the tail-cuff method with an electrosphygmograph (Le 5002 Storage Pressure Meter). The animals were pre-warmed to 36°C using an Le 5650/6 Heater & Scanner for 10– 15 min before the readings were taken. Seven SBP measurements were carried out on each animal during each measurement with the maximum and the minimum values being rejected. 2.3.3. Biochemical measurements Concentrations of glucose, cholesterol, triglycerides, and creatinine were measured in plasma samples at the end of the experiment using a Hitachi 704 automatic plasma analyzer. Plasma insulin levels were measured by a radioimmunoassay method (Herbert et al., 1965) using rat insulin as standard. 2.4. Statistical analysis Data were analyzed using a one-way analysis of variance followed by the Student’s t-test for comparison between groups. Values for P 50.05 were considered significant. Values in tables are expressed as mean9 SEM. 3. Results 3.1. Fluid and food intake Fluid and food intake of the various groups of rats are shown in Tables 1 and 2. When compared with the controls, fructose in drinking water had no significant effect on fluid and food intake throughout the 6-week period. The plant extracts did not affect food intake in animals treated with fructose. The fluid intakes of fructose-fed rats given the leaf aqueous extract of B. pilosa (150 –350 mg/kg) were significantly higher compared with the control and alone fructose treatment groups. On the other hand, the fluid intake of the fructose-fed rats given the methylene chloride extract was significantly higher (P B0.05) compared with the T. Dimo et al. / Journal of Ethnopharmacology 76 (2001) 215–221 control and alone fructose groups only at the dose of 150 mg of extract. 3.2. Systolic blood pressure and pulse rate The effects of fructose and the aqueous and methylene chloride extracts of B. pilosa on SBP are shown in Figs. 1 and 2. Fructose treatment was associated with a significant increase (P B0.01) in SBP. Chronic treatment with the aqueous or methylene chloride extract of B. pilosa completely blocked the elevation of blood pressure in fructose-treated rats and provoked a decline toward control values. At the end of the study, SBP was lower in groups treated with both doses of the two plant extracts compared with the fructose-fed group. Body weight was similar in all groups at the beginning and at the end of the study. No significant difference in the heart rate was observed in animals treated with fructose alone or fructose plus the plant extracts compared with the control diet (Table 3). 217 3.3. Biochemical parameters Fructose in drinking water had no influence on the plasma glucose, cholesterol and creatinine levels. After a 6-week feeding period, none of the treatment groups had significantly different plasma glucose levels. Plasma insulin levels were increased by the high fructose diet, and this effect persisted in rats treated with the plant extracts. Fructose feeding led to a small, but not significant increase in plasma triglycerides which was seen neither in the rats treated with the aqueous leaf extract (350 mg/kg) nor in those given both doses of the methylene chloride extract. Cholesterol levels were not affected by fructose feeding, but were significantly increased in animals treated with the methylene chloride extract at 150 mg/kg and with nifedipine (10 mg/kg). Plasma creatinine levels were slightly lower in rats treated with the high doses of the plant extracts (Table 3), and the differences were statistically significant compared with the control and the high fructose diets. Table 1 Fluid intake (ml) of rats given a fructose-rich diet with or without aqueous or methylene chloride extracts of B. pilosa leafa Groups 1st week 2nd week 3rd week 4th week 5th week 6th week Control HF HF+A150 HF+A350 HF+M150 HF+M300 HF+Ni10 29.98 91.98 26.46 90.69 29.99 93.17 29.91 92.61 30.07 9 1.91 28.62 9 4.24 28.99 9 2.06 26.60 9 1.84 25.52 9 1.11 32.71 9 3.71 29.60 9 3.63 28.73 9 1.24 29.50 9 3.82 24.74 9 1.09 27.46 9 1.83 26.20 9 0.37 32.71 9 2.74 31.46 9 2.84 32.909 1.36 32.50 9 1.32 26.07 9 1.93 28.329 1.24 29.179 2.25 52.64 9 5.24b,c 39.79 9 5.90b,c 47.08 9 6.19b,c 36.88 9 3.82 25.06 9 0.85 29.50 9 1.82 30.35 9 3.73 60.239 3.82b,c 50.079 6.02b,c 44.06 9 7.56b,c 32.61 9 2.17 28.71 9 1.24 32.50 9 0.93 31.46 9 1.39 56.66 9 6.04b,c 56.85 9 7.72b,c 47.58 9 5.39b,c 32.80 9 1.54 27.02 91.27 a Data are given as mean 9 SEM of fluid intake recordings (one daily recording per rat) corresponding to the period and animal group indicated. HF – high fructose; HF+A150 – high fructose plus aqueous extract, 150 mg/kg; HF+A350 – high fructose plus aqueous extract, 350 mg/kg; HF+M150 – high fructose plus methylene chloride extract, 150 mg/kg; HF+M300 – high fructose plus methylene chloride extract, 300 mg/kg. Ni10 – nifedipine (10 mg/kg). b Statistically significant difference (PB0.05) vs control group. c Statistically significant difference (PB0.05) vs fructose only group. Table 2 Food intake (g) of rats given a fructose-rich diet with or without aqueous or methylene chloride extracts of B. pilosa leafa Groups 1st week 2nd week 3rd week 4th week 5th week 6th week Control HF HF+A150 HF+A350 HF+M150 HF+M300 HF+Ni10 22.39 9 0.98 21.48 9 1.06 22.12 9 1.22 21.70 9 0.99 22.39 9 1.24 23.01 9 2.44 20.749 1.78 23.29 9 1.84 20.58 9 1.27 21.27 9 0.83 21.78 9 0.87 23.29 9 1.84 20.65 9 1.18 19.16 91.63 23.22 9 1.83 19.93 9 0.52 20.76 9 0.80 22.32 9 1.37 23.22 9 2.10 19.05 9 1.79 21.28 9 1.35 22.67 9 1.78 21.22 9 0.70 21.04 9 1.34 19.04 9 2.78 20.31 90.93 20.79 9 1.17 22.24 9 1.83 23.10 9 1.44 20.57 9 0.76 20.02 9 1.37 18.74 9 0.90b 20.169 2.22 19.61 9 1.35 22.80 9 1.64 21.64 9 1.48 20.67 9 0.64 20.90 9 1.97 19.19 9 2.81 20.15 9 2.05 18.41 91.93 21.719 1.18 a Data are given as mean 9 SEM of food intake recordings (one daily recording per rat) corresponding to the period and animal group indicated. HF – high fructose; HF+A150 – high fructose plus aqueous extract, 150 mg/kg; HF+A350 – high fructose plus aqueous extract, 350 mg/kg; HF+M150 – high fructose plus methylene chloride extract, 150 mg/kg; HF+M300 – high fructose plus methylene chloride extract, 300 mg/kg; Ni10 – nifedipine (10 mg/kg). b Significant difference (PB0.05) vs control group. 218 T. Dimo et al. / Journal of Ethnopharmacology 76 (2001) 215–221 Fig. 1. The effects of fructose and the aqueous extract of B. pilosa leaf on SBP in rats. HF – high fructose; HF + A150 – high fructose plus aqueous extract, 150 mg/kg; HF+ A350 – high fructose plus aqueous extract, 350 mg/kg; HF + M150 – high fructose plus methylene chloride extract, 150 mg/kg; HF+ M300 – high fructose plus methylene chloride extract, 300 mg/kg; Ni10 – nifedipine (10 mg/kg). (*) Statistically significant difference (PB 0.05) vs control group. (§) Statistically significant difference (P B 0.05) vs fructose group. Fig. 2. The effects of fructose and the methylene chloride extract of B. pilosa leaf on systolic blood pressure (SBP) in rats. HF – high fructose; HF+ A150 – high fructose plus aqueous extract, 150 mg/kg; HF+ A350 – high fructose plus aqueous extract, 350 mg/kg; HF +M150 – high fructose plus methylene chloride extract, 150 mg/kg; HF+ M300 – high fructose plus methylene chloride extract, 300 mg/kg; Ni10 – nifedipine (10 mg/kg). (*) Statistically significant difference (P B 0.05) vs control group. (§) Statistically significant difference (P B 0.05) vs fructose group. T. Dimo et al. / Journal of Ethnopharmacology 76 (2001) 215–221 4. Discussion Several anti-hypertensive drugs effectively prevent and reverse the increase in blood pressure induced by high fructose diets (Bhanot et al., 1994; Iimura et al., 1995; Rösen et al., 1997). The results of this study demonstrate that 3 weeks of high fructose feeding in rats resulted in a hypertensive state that was normalized by chronic treatment with the leaf aqueous or methylene chloride extract of B. pilosa. The extracts reversed the increase in SBP in a dose-dependent manner. The results of our study confirm that fructose feeding can induce hypertension in normal Wistar rats. Plasma insulin levels were higher in these rats while plasma glucose levels were not significantly different from the controls, suggesting that the rats were insulin-resistant. A number of studies favour the hypothesis that hypertriglyceridemia, insulin resistance or both, contribute to the development of hypertension in these rats (Erlich and Rosenthal, 1996; Bunnag et al., 1997; Fang and Huang, 1998). Hyperinsulinemia and insulin resistance could induce elevation of blood pressure levels via a variety of mechanisms including sodium– water retention, sympathetic nerve stimulation, changes in transmembrane ion traffic, and direct stimulation of smooth muscle cell growth (Ferrannini and Natali, 1991; Suzuki et al., 1997). Moreover, other reports have shown that reducing insulin levels in these rats leads to a reduction in blood pressure and correction of other metabolic abnormalities (Verma et al., 1994; Bhanot and McNeill, 1996; Rösen et al., 1997). Thus, vanadate, an agent that reduces insulin levels, lessens the severity of hypertension in fructose hypertensive and spontaneously hypertensive rats (Bunnag et al., 1997). In our study, the plant extract reduces SBP in fructose-fed animals without any effect on plasma insulin levels, suggesting that the mechanism(s) by which B. pilosa contribute to lowering blood pressure is not related to insulin sensitivity. 219 In our previous study, we (Dimo et al., 1998) have demonstrated that the leaf aqueous extract of B. pilosa possesses aortic smooth muscle relaxant properties. It is well known that drugs that possess vasodilatory effects, also lower blood pressure in hypertensive animals (Xu et al., 1982; Ho et al., 1989; Ninomiya et al., 1989; Dimo et al., 1999). The effect of nifedipine in the present study further supports this concept. Calcium channel blockers are arterial vasodilators that lower cytosolic calcium concentrations mainly through a reduction of transmembraneous calcium influx in vascular smooth muscle cells. This action could lower elevated blood pressure. The anti-hypertensive effect of the plant extracts may be due to their ability to reduce vascular resistance. The methylene chloride extract prevented the elevation of plasma triglyceride levels in fructose-treated rats whereas plasma cholesterol concentrations tended to be higher in the extract-treated animals. The increase in plasma cholesterol levels may be due to the presence of essential oils in the extracts (Amvam Zollo et al., 1995). There was also a decrease in plasma triglycerides in animals treated with the higher dose of the leaf aqueous extract of B. pilosa compared with the animals given fructose alone. It is known that high-fructose feeding leads to hypertriglyceridemia, probably through decreased very-low-density lipoprotein-triglyceride catabolism and/or increased hepatic very-low-density lipoprotein triglyceride secretion (Lee et al., 1994). The extracts of B. pilosa may be capable of interrupting this sequence thus preventing the development of hypertriglyceridemia in fructose-hypertensive rats. We observed that fructose treatment did not affect plasma creatinine levels but that there was a significant decrease in plasma creatinine levels in animals treated with the higher doses of the plant extracts compared with the controls. This result suggests a possible protective effect of the extracts against muscle destruction. Table 3 Effect of B. pilosa leaf extract on body weight, heart rate, and plasma levels of glucose, insulin, cholesterol, triglycerides, and creatinine in fructose-fed ratsa Parameters Control HF HF+Ni10 HF+A150 HF+A350 HF+M150 HF+M300 Body weight (g) Heart rate (b/min) Glucose (mM) Insulin (pM) Cholesterol (mM) Triglycerides (mM) Creatinine (pM) 3789 15 3359 8 6.569 0.35 105.83 915 1.369 0.05 1.039 0.15 49 93 362 9 9 3349 9 7.19 9 0.58 166.11 9 18b 1.35 9 0.15 1.80 9 0.55 48 9 2 353 97 347 911 6.06 90.43 198.60 9 15b 1.77 90.13b,c 2.00 90.29b 50 9 2 356 9 17 357 9 8 6.19 9 0.59 283.60 928b,c 1.33 9 0.07 2.05 9 0.43b 47 9 2 369 9 12 349 9 12 6.21 9 0.34 270.27 9 16b 1.60 90.05 1.17 9 0.11 44 9 0b,c 373 9 19 370 9 4 7.15 9 0.22 321.66 950b,c 1.62 9 0.07b,c 0.94 9 0.18c 47 9 2 368 9 13 378 98 7.019 0.21 196.94 9 12 1.50 9 0.04 1.05 9 0.89 41 9 3b,c a Data are given as mean 9 SEM for six animals per group. HF – high fructose; HF+A150 – high fructose plus aqueous extract, 150 mg/kg; HF+A350 – high fructose plus aqueous extract, 350 mg/kg; HF+M150 – high fructose plus methylene chloride extract, 150 mg/kg; HF+M300 – high fructose plus methylene chloride extract, 300 mg/kg; Ni10 – nifedipine (10 mg/kg). b Statistically significant difference (PB0.05) vs control group. c Statistically significant difference (PB0.05) vs fructose group. 220 T. Dimo et al. / Journal of Ethnopharmacology 76 (2001) 215–221 In conclusion, the present study shows that after drinking a high fructose solution for 6 weeks, normal rats exhibited significant increases in blood pressure and plasma insulin levels. Treatment with the methylene chloride and aqueous extracts of B. pilosa blocked the continued elevation of blood pressure and provoked a return to normal values. The reversal of the development of hypertension was not accompanied by a reduction of plasma insulin levels, suggesting that the vascular effects observed during our earlier study may be responsible for the hypotensive effects. The extracts also prevented the establishment of hypertriglyceridemia, which accompanied chronic feeding with the high fructose diet. Acknowledgements The authors are grateful to the Fondation Simone et Cino Del Duca, Paris, for the research grant awarded to Théophile Dimo. References Adjanohoun, E.J., Ake Assi, L., Ali Ahmed, Eyme, J., Guinko, S., Kayonga, A., Keita, A., Lebras, M., 1982. Médecine Traditionnelle et Pharmacopée. Contribution aux Etudes Botaniques et Floristiques aux Comores. Rapport Agence de Coopération Culturelle et Technique, Paris, pp. 243. 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Metabolic effects of troglitazone on fructose-induced insulin resistance in the rat. Diabetes 43, 1435 – 1439. Navarro-Cid, J., Maeso, R., Perez-Vizcaino, F., Casal, M.C., Cachofeiro, V., Ruilope, L.M., Tamargo, J., Lahera, V., 1996. Effects of antihypertensive drugs on blood pressure and metabolic alterations in the fructose-induced hypertensive rat. Journal of Hypertension 9, 669 – 674. Ninomiya, M., Tani, T., Nakajima, S., Ueda, M., 1989. Effects of S-312, a new calcium antagonist, on the mechanical and electrophysiological responses of isolated cardiovascular preparations. Japanese Journal of Pharmacology 51, 227 – 238. T. Dimo et al. / Journal of Ethnopharmacology 76 (2001) 215–221 Rösen, P., Ohly, P., Gleichmann, H., 1997. Experimental benefit of moxonidine on glucose metabolism and insulin secretion in fructose-fed rat. Journal of Hypertension 15 (Suppl. 1), S31 – S38. Suzuki, M., Nomura, C., Odaka, H., Ikeda, H., 1997. Effect of an insulin sensitizer, pioglitazone, on hypertension in fructosedrinking rats. Japanese Journal of Pharmacology 74, 297 – 302. Vasdev, S., Prabhakaran, V.M., Whelan, M., Ford, C.A., Longerich, L., Parai, S., 1994. Fructose-induced hypertension, hypertriglyceridemia and elevated cytosolic calcium in rats: . 221 prevention by Deuterium oxide. Artery 21 (3), 124 – 147. Verma, S., Bhanot, S., McNeill, J.H., 1994. Antihypertensive effects of metformin in fructose-fed hyperinsulinemic, hypertensive rats. Journal of Pharmacology and Experimental Therapeuties 271 (3), 1334 – 1337. Xu, B.S., Huang, M.Y., Lau, N.B.C., Wat, K.H.C., Kong, C.Y., 1982. Hypotensive effect of Dehydroevodiamine from Evodiae fructus. American Journal of Chinese Medicine X (1 –4), 75 – 85. Journal of Ethnopharmacology 76 (2001) 223– 228 www.elsevier.com/locate/jethpharm Neuropharmacological profile of ethnomedicinal plants of Guatemala C. Morales Cifuentes, M.P. Gómez-Serranillos *, I. Iglesias, A.M. Villar del Fresno Departamento de Farmacologı́a, Facultad de Farmacia, Uni6ersidad Complutense de Madrid, 28040 Madrid, Spain Received 14 August 2000; received in revised form 30 March 2001; accepted 03 April 2001 Abstract We carried out the Irwin’s test with some different extracts of the aerial parts of Thidax procumbens L., the leaves of Neurolaena lobata (L.) R. Br., bark and leaves of Byrsonima crassifolia (L.) Kunth. and Gliricidia sepium Jacq. Walp., and root and leaves of Peti6eria alliacea L. At dosage of 1.25 g dried plant/kg weight aqueous extracts of bark and leaves of Byrsonima crassifolia (L.) Kunth. and Gliricidia sepium Jacq. Walp. demonstrated the most activity: decrease in motor activity, back tonus, reversible parpebral ptosis, catalepsy and strong hypothermia. These extracts of both plants were assayed for effects on CNS and they caused very significant reductions in spontaneous locomotor activity, exploratory behavior and rectal temperature and they increased the sodium pentobarbital-induced sleeping time. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethnomedicinal plants; Guatemala; CNS activity 1. Introduction Tridax procumbens L. (Asteraceae), Neurolaena lobata (L.) R. Br.(Asteraceae), Byrsonima crassifolia (L.) Kunth. (Malphighiaceae), Gliricidia sepium Jacq. Walp. (Fabaceae), and Peti6eria alliacea L are species widely distributed in Central and South America; pharmacology books and other publications contain little information about these plants. Pharmacological studies had reported the hepatoprotective activity of Tridax procumbens (Pathak et al., 1991), diuretic (Gupta, 1995) and fungicide and the antimalarial activity of Neurolaena lobata (Rai and Upadhyay, 1988). Extracts obtained of Byrsonima crassifolia has been described as bactericide (Martı́nez Vázquez et al., 1999) and fungicide (Cáceres et al., 1991, 1993) and extracts from Gliricidia sepium as antiaterogenic (Mendieta and Amo, 1981), antimalarial (Castro et al., 1996) and fungicide (Cáceres et al., 1991, 1993). Peti6eria alliacea has antiinflammatory and analgesic activity (Distasi et al., 1988), antitumor and antimicotic (Von Szczepanski et al., 1975). * Corresponding author. Fax: + 34-913-941764. E-mail address: pserra@eucmos.sim.ucm.es (M.P. Gómez-Serranillos). In our efforts to find new plants with biological activity, we present this study, which attempts to study the neuropharmacologic effects of some extracts of these five plants in laboratory animals. After the realization a preliminary screening using the Irwin’s test, a series of pharmacological test was used to explore their activity on CNS. 2. Materials and methods 2.1. Plant material and extraction procedure T. procumbens, B. crassifolia, G. sepium and P. alliacea were collected in Samayac (Suchitepéquez) and N. lobata in Livingston (Izabal), and herbarium samples are authenticated in the University of Valley Herbarium (Dra. Elfriede de Pöll), and deposited in Farmaya Laboratory Herbarium (Guatemala) (voucher number: 372, 369, 233 and 456, respectively). Plants were dried in the dark and powered finely. The power was extracted with hexane for 1 or 2 weeks and the defatted material was percolated with chloroform for 1 or 2 weeks. The marc was percolated again with MetOH or EtOH. Hexane, chloroform and MetOH extracts were 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 3 5 - 5 C.M. Cifuentes et al. / Journal of Ethnopharmacology 76 (2001) 223–228 224 evaporated using a rotary evaporator. The defatted material was extracted with boiling distilled water and lyophilized, obtaining the aqueous extract. Residues were weighed and kept at 4°C. Table 1 show the result (%) of the extraction procedure. 2.2. Test animals Male and female Swiss albino mice (20– 25 g) (Charles Rives, Paris) were used. Animals were housed in community cages (approximately 22°C, 12-h light:12h dark cycle) with approved bedding materials, and kept under standard laboratory conditions. Animals had free access to standard rodent diet and clean drinking water. They were acclimatized for at least 2 days before the start of experiments, in a sound-less room. 2.3. Drugs and dosage Table 1 Yield of extracts Extracts (%) Hexane Tridax procumbens (aerial part) Byrsonima crassifolia (bark) Byrsonima crassifolia (leaf) Gliricidia sepium (bark) Gliricidia sepium (leaf) Peti6eria alliacea (leaf) 2.4. Preliminary neuropsychopharmacologic screening Doses of the test material were administered to groups of mice (n=5) and grossly observable behavioral effects were observed and quantified as described by Irwin (1962). The Irwin’s test is a comprehensive procedure that makes it possible to quantify and collate, in each animal, a wide variety of grossly observable changes produced by drugs (e.g. behavioral, neurological, autonomic and toxic). After the treatment with drugs, mice were observed every 30 min up to 6 h for studying behavioral changes. Before the treatment with extracts, we conducted the test with some standard reference drugs that has very known effects on CNS. 3. Activity on the central nervous system The following drugs and dosage were used: carbachol HCl (Sigma) 2 mg/kg; atropine sulfate (Sigma) 2.5 mg/kg; scopolamine HBr (Sigma) 2 mg/kg; epinephrine bitartrate (Sigma) 1 mg/kg; amphetamine bitartrate 5 mg/kg; lidocaine HCl (Sigma) 40 mg/kg; morphine HCl 80 mg/kg; haloperidol (Syntex latino, SA) 1 and 6 mg/kg; pentobarbital sodium 40 and 60 mg/kg; T. procumbens chloroform and aqueous extracts, N. lobata hexane, MeOH and aqueous extracts, B. crassifolia hexane, MeOH and aqueous extracts, G. sepium hexane, MeOH and aqueous extracts and P. alliacea hexane, MeOH and aqueous extracts. Extract dosage was 1.25 g dried plant/kg weight. Each drug was dissolved in isotonic saline solution (0.9% NaCl) just before use, and the resulting solution Plant was administered by intraperitoneal (i.p.) injection of 0.2 ml. Cloroform Methanol 3.26 Aqueous 11.70 2.61 29.56 9.56 1.24 12.10 11.22 0.67 4.23 7.54 1.84 11.83 14.74 1.37 9.4 13.21 The activity of the aqueous bark and leaf extract of B. crassifolia and G. sepium on the CNS was evaluated by performing assays of its effects on spontaneous motor activity, exploratory conduct (hole-board test), body temperature (as rectal temperature) and sodium pentobarbital-induced hypnosis. 3.1. Spontaneous motor acti6ity The spontaneous motor activity of the animals was measured using an actimetre photoelectric of J. Boissier and J.C. Montagné (APELAB). This activity cage contains photoelectric cells that are sensitive to any motion within it. Mice were divided into six groups of five. Four groups were given i.p. leaf and bark aqueous extract of B. crassifolia and G. sepium. Haloperidol (1 mg/kg i.p.) was used as a standard reference drug. Animals in the remaining group received the vehicle as control. Then 30 min after receiving the extracts as i.p. injection, saline or haloperidol, each group of five animals was placed into an activity cage and their spontaneous motor activity was recorded for 10 min sessions along 90 min. 3.2. Exploratory conduct (hole-board test) The method described by Boissier and Simon (1967) was used. Mouse was placed on an automated holeboard (16 holes, APELAB) with automatic counting of the animals’ movements across the board (photoelectric cell). The assays were performed 30 min after administration of the extracts (five mice per dose). Haloperidol (1 mg/kg) was used as a standard reference drug. Exploratory behavior was recorded every 5 min for an hour. C.M. Cifuentes et al. / Journal of Ethnopharmacology 76 (2001) 223–228 225 3.5. Statistical analysis Statistical (P B0.05) differences between values of these assays: exploratory conduct, rectal temperature and sodium pentobarbital-induced hypnosis, were calculated using the analysis of variance (ANOVA) method, into the program Statgrafics v.4.1. 4. Results Fig. 1. Effects of bark aqueous extract of Byrsonima crassifolia on spontaneous motor activity. Activity was monitored every 10 min for 90 min. The extract was administered at t= 0. Each bar represents the mean response of four groups of five (treated or control) animals. * P B0.05, statistically significant difference between the animals receiving extracts. Fig. 2. Effects of the aqueous extract of leaves of Byrsonima crassifolia on spontaneous motor activity. Activity was monitored every 10 min for 90 min. The extract was administered at t= 0. Each bar represents the mean response of four groups of five (treated or control) animals. * PB 0.05, statistically significant difference between the animals receiving extracts. 3.3. Rectal temperature Rectal temperature was measured with a digital thermometer connected with a thermoelectric probe, which was carefully inserted into the rectum of the animals about 2 cm depth. Temperature was recorded just before (T0) the administration of the extracts and at 30, 60, 90 and 120 (T30, T60, T90, T120, respectively) after administration. 4.1. Preliminary neuropsychopharmacologic screening The i.p. administration of hexane and chloroform extracts of T. procumbens had no effects on behavioral, neurological and autonomic signs. Aqueous leaf extract of N. lobata decreased lightly spontaneous motor activity and muscle tone but these effects were not over 2 h. Mice exhibited diuresis. Aqueous extract of bark of B. crassifolia produced pronounced effects on gross behavior. Decreased motor activity and general flaccidity, positive piloerection and parpebral ptosis was observed. Extract also induced reduced sound and touch responses. Aqueous extract of leaves retained all of the effects observed with the bark extract and also produced awareness, catalepsy (reversible by stimuli), and decreased righting reflex, ipsilateral flexor reflex and respiratory rate. These effects were not over 24 h. The administration of aqueous bark and leaf extracts of G. sepium decreased locomotor activity, general muscle tone and righting reflex and also induced reduced sound and touch responses. These extracts produced awareness, positive piloerection and parpebral ptosis. These effects persisted for at least 6 h after administration. One to three out of five animals receiving these extracts died along 48 h after administration. Necropsy revealed multiple hemorrhages. P. alliacea’s root extracts only lightly decreased spontaneous motor activity and leaf extracts showed hiperexcitability. 4.2. Effects on central ner6ous system The present results provide strong indications that the aqueous bark and leaf extracts of B. crassifolia and G. sepium are CNS depressants since they showed activity in the entire predictive test applied. 3.4. Sodium pentobarbital-induced hypnosis 4.3. Spontaneous motor acti6ity Sodium pentobarbital (60 mg/kg) was injected i.p., 30 min after administration of the extracts. Following this barbiturate injection the disappearance and reappearance of the righting reflex were considered to indicate onset and duration of hypnosis, respectively. Sleeping time was considered to be the time interval between disappearance and reappearance of the righting reflex. Both aqueous extracts of B. crassifolia and G. sepium reduced spontaneous motor activity immediately after i.p. administration. The mean activity count in each extract-treated group was significantly reduced with respect to the control group. Results are shown in Figs. 1 – 3. 226 C.M. Cifuentes et al. / Journal of Ethnopharmacology 76 (2001) 223–228 4.4. Exploratory conduct Exploratory conduct was significantly reduced by the four extracts. This effect was noted at 30 min after injection and was increased during the rest of the experiment as shown in Figs. 4 and 5. 4.5. Rectal temperature Fig. 3. Effects of leave and bark aqueous extracts of Gliricidia sepium on spontaneous motor activity. Activity was monitored every 10 min for 90 min. The extracts was administered at t = 0. Each bar represents the mean response of four groups of five (treated or control) animals. * PB 0.05, statistically significant difference between the animals receiving extracts. All of the extracts tested produced a significant hypothermia. Also 30 min after the treatment, the temperature was significantly reduced when compared with, that of the control group. The temperature remained low for more than 2 h after treatment. Figs. 6 and 7 show the effect of the two extracts of both plants on rectal temperature. Fig. 4. Effect of bark and leave aqueous extracts of Byrsonima crassifolia on exploratory conduct; measurements were taken every 5 min for 1 h. The extracts was administered at t= 0. Each bar represents the mean response of five animals. * PB 0.05, statistically significant difference between the animals receiving extracts. Fig. 5. Effect of bark and leave aqueous extracts of Gliricidia sepium on exploratory conduct; measurements were taken every 5 min for 1 h. The extracts was administered at t= 0. Each bar represents the mean response of five animals. * PB 0.05, statistically significant difference between the animals receiving extracts. C.M. Cifuentes et al. / Journal of Ethnopharmacology 76 (2001) 223–228 227 Fig. 6. Variations in rectal temperature over time in response to the bark and leaves aqueous extracts of Byrsonima crassifolia. The effect is expressed with respect to temperature at t= 0. Each point represents the mean response of five animals. Standard: haloperidol 1 mg/kg. * PB 0.05, statistically significant difference between the animals receiving extracts. Fig. 7. Variations in rectal temperature over time in response to the bark and leaves aqueous extracts of Gliricidia sepium. The effect is expressed with respect to temperature at t= 0. Each point represents the mean response of five animals. Standard: haloperidol 1 mg/kg. * PB 0.05, statistically significant difference between the animals receiving extracts. Table 2 Effects of bark and leave aqueous extracts of Byrsonima crassifolia and Gliricidia sepium on pentobarbital-induced hypnosis Treatment Dosage (mg/kg) (g dried plant) Loss time of righting of reflex (min) (mean 9 S.E.M.) Sleeping time (min) (mean 9 S.E.M.) Controla B. crassifolia (Leave) G. sepium (Bark) G. sepium (Leave) 60 1.25 4 90.7 2.7 9 0.41 63.7 9 15.6 113.7 9 33.8 1.25 2.4 9 0.4 142.6 9 64.1 1.25 2.7 9 0.5 80.6 9 45.1 The values show mean time elapsed before disappearance of the righting reflex9 S.E.M. of five mice. a Control: pentobarbital sodium. 4.6. Sodium pentobarbital-induced hypnosis 5. Discussion Aqueous extracts of bark and leaves of B. crassifolia and G. sepium appeared to decrease the onset of disappearance time of the righting reflex, and increased pentobarbital-induced sleeping time. Results are shown in Table 2. Irwin’s test was realized on some different extracts of five South American plants for knowing its effects on CNS. T. procumbens did not show neurological effects. N. lobata, and P. alliacea caused little effects on behavioral, neurological and autonomic signs. On the other 228 C.M. Cifuentes et al. / Journal of Ethnopharmacology 76 (2001) 223–228 hand, bark and leaf aqueous extracts of B. crassifolia produced significant variations on gross behavior. They decreased motor activity and sound and touch response, with back tonus, parpebral ptosis, catalepsy (awake) and decreased righting reflex. G. sepium bark and leaves aqueous extracts decreased motor activity and muscle tone and reactivity, and produced piloerection and parpebral ptosis. They showed toxicity signs. These results indicate that these aqueous extracts of B. crassifolia and G. sepium have important effects on CNS. This activity was well confirmed. Bark and leaf aqueous extracts of both plants reduced spontaneous motor activity, exploratory conduct in mice and they caused hypothermia significantly. Both plants increased the pentobarbital-induced sleeping time. In conclusion, it can be seen that the assayed extracts of B. crassifolia and G. sepium possess significant depressant effects on CNS. Acknowledgements The authors wish to thank CYTED and the Chemical Sciences and Pharmacy Faculty of San Carlos University of Guatemala for supplying all of the extracts. References Boissier, J.R., Simon, P., 1967. Le test de la curiosité pour l’etude . du psicolépticos. Therapie 22, 467 – 471. Cáceres, A., Lopez, B., Girón, M.A., 1991. Plants used in Guatemala for the treatment of dermatophytic infections. 1. Screening for antimycotic activity of 44 plant extracts. Journal of Ethnopharmacology 31, 263 – 276. Cáceres, A., Lopez, B., Juárez, X., 1993. Plants used in Guatemala for the treatment of dermatophytic infections. 2. Evaluation of antifungal activity of seven American plants. Journal of Ethnopharmacology 40, 207 – 213. Castro, O., Barrios, M., Chinchilla, M., 1996. Chemical and biological evaluation of the effect of plant extracts against Plasmodium berghei. Revista de Biologia Tropical 44, 361 – 367. Distasi, L.C., Costa, M., Medacolli, L.J., Kirizana, M., Gómes, C., Trolin, G., 1988. Screening in mice of some medicinal plants used for analgesic purposes in the state of Sao Paulo. Journal of Ethnopharmacology 24 (2/3), 205 – 211. Gupta, M.P., 1995. 270 Plantas Medicinales Iberoamericanas, Ed. Presencia. Ltda. Santa Fe’ de Bogota, D.C. Irwin, S., 1962. Drug screening and evaluative procedures. Science 136, 123 – 126. Martı́nez Vázquez, M., González Esquinca, A.R., Cazares Luna, L., 1999. Antimicrobial activity of Byrsonima crassifolia (L.) H.B.K. Journal of Ethnopharmacology 66, 79 – 82. Mendieta, R.M., Amo, S. del., 1981 Plantas medicinales del estado de Yucatán, Xálapa. INIREB, p. 137. Pathak, A.K., Sarae, S., Dixit, V.K., 1991. Hepatoprotection activity of Tridax procumbens. Part I. Fitoterapia 62, 307 – 313. Rai, M.K., Upadhyay, S., 1988. Screening of medicinal plants of Chhindwara district against Trichophyton mentagrophytes: a causal organism of Tenia pedis. Hindustan Antibiotics Bulletin 30 (1/2), 33 – 34. Von Szczepanski, C., Zgorzelak, P., Hoyer, G.A., 1975. Isolation, structure determination and synthesis of an antimicrobial substance from Peti6eria alliacea. Arzneimittel Forschung 22, 147 – 148. Journal of Ethnopharmacology 76 (2001) 229– 232 www.elsevier.com/locate/jethpharm Effect of garlic (Allium sati6um L.) extract on tissue lead level in rats S.K. Senapati, S. Dey, S.K. Dwivedi *, D. Swarup Laboratory of Comparati6e System of Medicine, Di6ision of Medicine, Indian Veterinary Research Institute, Izatnagar, 243 122 U.P. India Received 25 September 2000; received in revised form 20 March 2001; accepted 3 April 2001 Abstract The prophylactic efficacy of garlic (Allium sati6um L.) extract to reduce tissue lead (Pb) concentration was evaluated experimentally in rats. Thirty female rats were divided into five groups, keeping group A as a healthy control. Rats of groups B, C, D and E received lead acetate orally at the rate of 5 mg per kg body weight daily for 6 weeks. The garlic extract was tried in three doses, viz. 100 (low), 200 (medium) and 400 mg (high) per kg body weight orally and given simultaneously with lead salt to the rats of group C, D and E, respectively. Mean blood lead concentrations in lead-exposed rats ranged between 0.13 9 0.02 and 0.96 9 0.06 mg/ml, whereas in garlic-treated rats, the range was between 0.16 90.01 and 0.80 9 0.05; 0.13 90.01 and 0.71 90.06 and 0.14 9 0.01 and 0.60 90.05 mg per ml in low, medium and high dose groups, respectively. The mean lead concentration in liver, kidneys, brain and bone of lead exposed rats was 2.943 90.206, 4.780 9 0.609, 1.019 9 0.100 and 44.0759 2.60 mg per ml, respectively. Concomitant use of garlic extract at the three different doses was found to reduce lead concentration considerably indicating the potential therapeutic activity of garlic against lead. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Allium sati6um (garlic) juice; Ameliorative potential; Blood lead; Rats; Tissue lead accumulation 1. Introduction Lead (Pb), a non-biodegradable heavy metal, continues to pose health hazards in man and animal in India and elsewhere in the world. It affects each and every organ and system in the body (Goyer, 1990). Being a cumulative poison, Pb is accumulated in various organs, viz. liver, kidneys, brain, bone and haemopoietic system (Klaassan, 1991). Several metal chelators have been used to manage Pb toxicity in the event of exposure, but none is suitable in reducing Pb burden in chronic Pb exposure (Osweiler, 1999). Moreover, these chelators have a toxic potential in themselves (Gilman et al., 1991) and often fail to remove Pb from all body tissues (Bratton et al., 1981; Cory-Slechta et al., 1987). In the Ancient Indian system of medicine (Ayurveda), a number of plants and herbs have been indicated for amelioration of metal * Corresponding author. Present address: National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana. Tel.: + 91-166232955 fax: + 91-1662-76217. E-mail address: kdwivedi@nde.vsnl.net.in (S.K. Dwivedi). poisoning (Dwivedi, 1995). However, these have not been evaluated scientifically to date, and their use is mainly restricted to observable recovery. Garlic (Allium sati6um L.) is cultivated all over India and is used as a vegetable. The traditional ethnoveterinary practitioners consider garlic as an excellent natural product that has immense therapeutic potential in many pathological conditions. The bulb of garlic is used as an antirheumatic and stimulant besides its use in conditions like paralysis, forgetfulness, tremor colicky pain and chronic fever (Nadkarni, 1994). Internally, it is indicated in affections of the nervous system. In this study, garlic extract was tested for its potential to reduce body lead residues in experimented Pb-exposed rats. 2. Material and method 2.1. Source of garlic Fresh garlic bulbs were collected from a natural habitat around Bareilly during February – March 1997. 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 3 7 - 9 S.K. Senapati et al. / Journal of Ethnopharmacology 76 (2001) 229–232 230 They were identified at The Department of Botany, Bareilly college (Rohilkhand University), Bareilly, where voucher reference specimens were deposited. through the outer canthus of the eye. The sample was pooled into nitric acid-washed heparinized vials for estimation of lead. 2.2. Extract preparation 2.5.2. Tissue samples Portions of liver, kidneys, brain and long bone were collected in polyethylene bags without any preservative after sacrifice on the day after the last dose of Pb. The samples were subsequently subjected to acid digestion for Pb estimation. Garlic bulbs were separated, peeled and washed with distilled water. After drying in a shed, about 500 g of clean garlic bulb were crushed with the help of an electric grinder. The extract was strained through muslin cloth after squeezing the crushed material. The concentration of the juice was calculated as: Gram equivalent= weight of the fresh garlic . weight of the juice 2.3. Animals and treatment The experiment was conducted on 30 female rats IVRI 2CQ, bred in the Laboratory Animal Research Division of this institute. They were housed in plastic cages with proper bedding and maintained on a standard ration and ad libitum water. They were acclimatized for 30 days before starting of the experiment. 2.4. Experimental protocol The rats were divided into five groups of six animals each. The details of the experimental protocol are shown in Table 1. The lead was administered orally as a 1% aqueous solution once daily at the morning hours before feeding. Garlic extract was administered at two divided doses: half of the required doses in the morning and the other half in the evening. All treatments were given daily for 6 weeks. 2.6. Estimation of lead All samples were wet-digested (AOAC, 1984) using a 5:1 mixture of concentrated nitric acid and 70% perchloric acid in a heating block under a low heat. The concentration of Pb in the acid digest was estimated by atomic absorption spectrophotometry (AAS-4129, Electronic Corporation of India Limited) at 217 nm wavelength following the instrument’s instruction manual. An air– acetylene mixture was used as the oxidant gas. The analytical quality was maintained by repeated analysis of the reference standards (Sigma Chemicals, St. Louis, MO). The results were expressed as mg/g of sample. The ameliorative potential of the test extract was assessed on the basis of its effect on Pb concentration in blood and in different tissues of the body. 2.7. Statistical analysis The data were analysed statistically to determine whether there was any significant difference among different treatment groups using Fisher’s t-test and two-way analysis of variance (ANOVA) following standard methods (Snedecor and Cochran, 1989). 2.5. Sampling 3. Results 2.5.1. Blood Samples of blood were collected from individual rats on day 0 of the experiment and thereafter at weekly intervals for 6 weeks from the orbital plexus using heparinized microhaematocrit capillaries piercing The blood Pb concentrations in rats of different treatment groups are presented in Table 2. The mean blood Pb concentration in healthy rats (Group A) ranged between 0.1490.01 and 0.3190.02 mg/ml. Oral Pb exposure increased blood Pb concentration from Table 1 Details of the experimental protocol Treatments Groups A B Pb acetate (mg/kg b. wt.) Nil 5.0 Garlic extracts (mg/kg b. wt.) Placebo Nil Distilled water Nil Nil C D 5.0 100 Nil E 5.0 200 Nil 5.0 400 Nil S.K. Senapati et al. / Journal of Ethnopharmacology 76 (2001) 229–232 231 Table 2 Blood lead concentration (mg/ml) in garlic treated rats Groups A B C D E Weeks of exposure 0 1 2 3 4 5 6 0.14 9 0.01 0.13 90.02 0.169 0.01 0.139 0.01 0.149 0.01 0.23 9 0.09 0.539 0.01** 0.279 0.02*B 0.21 9 0.01*B 0.18 9 0.01B 0.27 9 0.01 0.80 9 0.04** 0.539 0.03**A 0.309 0.03**B 0.279 0.02**B 0.28 9 0.02 0.80 9 0.02** 0.63 9 0.03**A 0.45 9 0.03**B 0.39 9 0.04**B 0.28 90.02 0.91 90.03** 0.70 9 0.04**A 0.56 9 0.04**A 0.45 9 0.05**B 0.29 9 0.01 0.94 9 0.04** 0.759 0.04**A 0.629 0.06**A 0.539 0.07**B 0.31 9 0.02 0.96 9 0.06** 0.809 0.05**A 0.719 0.06**A 0.609 0.05**B *Differ significantly (P50.05) compare to the day ‘0’ value of the same group. **Differ significantly (P50.01) compared to the day ‘0’ value of the same group. ADiffer significantly (P50.05) compared to group B. BDiffer significantly (P50.01) compared to group B. 0.13 90.02 to 0.9690.06 mg/ml in rats of group B. The mean blood Pb concentration in garlic-treated rats was estimated in the range of 0.1690.01 to 0.80 9 0.05, 0.1390.01 to 0.719 0.06 and 0.149 0.01 to 0.60 9 0.05 mg/ml in groups C, D and E respectively. Concomitant use of garlic juice significantly reduced blood Pb concentration in rats in a dose-dependent manner. Table 3 shows the tissue Pb accumulation pattern in the experimental rats. In healthy rats (Group A), the mean Pb concentration was estimated as 0.9159 0.038, 1.2449 0.180, 0.5789 0.068, and 8.54290.834 mg/g in liver, kidneys, brain and femur, respectively. Exposure of Pb acetate for 6 weeks led to an increased tissue Pb concentration to 2.94390.214, 4.7809 0.609, 1.01990.100 and 44.0759 2.600 mg/g in liver, kidneys, brain and femur, respectively, in rats of Group B. Administration of garlic juice significantly (P B0.01) reduced tissue Pb accumulation. The liver Pb concentration was 1.71990.300, 0.9209 0.121 and 0.9659 0.081mg/g in rats of groups C, D and E respectively. The mean Pb concentration in kidneys was recorded as 2.4499 0.352, 1.29890.152 and 1.420 90.372 mg/g in groups C, D and E, respectively. The Pb concentration in brain decreased significantly (P B0.05) in group D (0.90090.130 mg/g), and the reduction was highly significant (PB 0.01) in group E (0.7439 0.091). The Pb concentration of the femur was non-significantly reduced in group C (41.4729 10.250 mg/g), whereas it was significantly (P B0.01) reduced in rats of group D (30.86597.450 mg/g) and group E (29.6609 5.650 mg/g). receiving garlic along with Pb. The highest dose (400 mg per kg body weight) decreased blood Pb concentration most efficiently, almost to the values recorded in healthy rats. Garlic juice contains sulfur-containing amino acids like S-allyl cystine, S-allyl mercaptocystein and alliin (Horie et al., 1992). Sulfur-containing amino acids like cystein have already been reported for their chemoproplylactic use in Pb toxicosis (Quarterman et al., 1980; Latta and Donaldson, 1986; Tandon et al., 1988; Rai and Raizada, 1988). The efficiency of garlic was perhaps due to the presence of these sulfur-containing amino acids and compounds having free carboxyl (C0) and amino (NH2) groups in their structures. These biologically active compounds might have chelated Pb and enhanced its excretion from the body resulting in reduced Pb accumulation in tissues and blood. Further published results also showed that garlic extracts increased the Pb concentration in the urine as well as the faeces of these rats (Senapati, 1997) lending credence to this hypothesis. Besides chelation, other components of garlic extracts (S-allyl cystein and S-allyl mercaptocystein and some micronutrients) also prevent absorption of Pb from the gastro-intestinal tract (Crowe and Morgan, 1996). It can be suggested, therefore, that the ameliorative potential of garlic juice was perhaps due to combined effects both on metal absorption and on excretion from the body. Table 3 Lead concentration (mg/g) in different organs of lead- and garlictreated rats Group Organ 4. Discussion The Pb level was increased significantly in liver, kidneys, brain, femur and blood of rat receiving Pb acetate alone. However, the concomitant use of garlic juice prevented the accumulation of Pb in these organs. The weekly blood Pb profiles revealed a significant and constant dose-dependent decreasing trend in the rats A B C D E Liver Kidney Brain Long bone 0.915 90.038 2.943 90.214 1.17990.300** 0.92090.121** 0.965 90.081** 1.244 90.18 4.780 90.609 2.449 90.353** 1.29890.152** 1.42090.372** 0.578 90.068 1.019 90.100 1.00090.030 0.900 90.130** 0.743 9 0.091** 8.542 90.834 44.075 92.600 41.472910.25 30.86597.45** 29.660 95.650** *Differ significantly (P50.05) compare to group B. **Differ significantly (P50.01) compared to group B. 232 S.K. Senapati et al. / Journal of Ethnopharmacology 76 (2001) 229–232 Our findings have also revealed that garlic juice had the ability to reduce residues of Pb in soft tissues (liver, kidneys, brain) as well as in the bone sink in the body. Hanafy et al. (1994) have also reported on the efficacy of garlic in reducing Pb content in chicken tissue. Therefore, it is concluded that garlic can be used for amelioration of chronic Pb toxicity in man and animal. Further studies are required, however, to establish the dose and the molecular basis of the anti-toxic mechanism and the components of garlic involved in it. Acknowledgements The financial assistance given to the senior author in the form of ICAR Jr. Fellowship by The Indian Council of Agricultural Research is thankfully acknowledged. We acknowledge the technical assistance of Mr. Brijesh Tyagi in estimation of Pb in biosamples. References Association of Official Analytical Chemists, 1984. Official Methods of Analysis, 14th ed. AOAC International, Artington, pp. 444 – 476. Bratton, G.R., Zmudzki, J., Bell, M.C., Warnoch, L.G., 1981. Thiamine effects on lead intoxication and deposition of lead in tissues — therapeutic potential. Toxicology and Applied Pharmacology 59, 164 – 172. Cory-Slechta, D.A., Weiss, B., Cox, C., 1987. Mobilization and redistribution of lead over the course of calcium disodium ethylene diamine tetraacetate chelation therapy. Journal of Pharmacology and Experimental Therapeutics 243, 804 – 813. Crowe, A., Morgan, E.H., 1996. Interaction between tissue uptake of lead and iron in normal and iron deficient rats during development. Biological Transition Element Research 52, 249 – 261. Dwivedi, S.K., 1995. Scope of Indian System of Medicine in Amelio- . ration of Heavy Metal Toxicity. Symposium on Man, Animal and Environment. December 21 –23, New Delhi, Proceedings, pp. 195 – 198. Gilman, A.G., Rall, T.W., Nies, A.S., Taylor, P., 1991. Goodman & Gilman’s the Pharmacological Basis of Therapeutics. Pergamon, New York, pp. 1592 –1614. Goyer, R.A., 1990. Lead toxicity: from overt to subclinical to subtle health effects. Environmental Health Perspectives 86, 177 – 181. Hanafy, M.S.M., Shalaby, S.M., Elfonly, M.A.A., Abdel Aziz, M.I., Soliman, F.O., 1994. Effect of garlic on lead contents in chicken tissue. Deutsche — Tierarztlide-wochenschrift 101, 157 – 158. Horie, T., Awazu, S., Itakura, Y., Fuwa, T., 1992. Identified diallyl polysulfides from an aged garlic extract protects the membrane from lipid peroxidation. Planta Medica 58, 468 – 469. Klaassan, C.D., 1991. Heavy metal and heavy metal antagonist. In: Gilman, A.G., Rall, T.W., Nilb, A.S., Taylor, P. (Eds.), Pharmacological basis of Therapeutics. Pergamon, New York, pp. 1592 – 1614. Latta, D.M., Donaldson, W.E., 1986. Lead toxicity in chicks: Interaction with dietary methionine and choline. Journal of Nutrition 116, 1561 – 1568. Nadkarni, A.K., 1994. Indian Materia Medica, vol. I and II. Popular Prakashan, Bombay, pp. 66 – 71. Osweiler, G.D., 1999. Toxicology. Williams and Wilkins, Philadelphia, PA, pp. 195 –196. Quarterman, J., Humphries, W.R., Morrison, J.N., Morridon, E., 1980. The influence of dietary amino acids on lead absorptions. Environmental Research 23, 54 – 67. Rai, L.E., Raizada, M., 1988. Impact on chromium and lead on Nostoc Muscorum regulation of toxicity by ascorbic acid, glutathione, and sulfur containing amino acid. Ecotoxicology and Environmental Safety 15, 195 – 205. Senapati, S.K., 1997. Evaluation of Medicinal Herbs in Amelioration of Plumbism in Rats. Indian Veterinary Research Institute, Izatnagar, p. 107. Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods, eighth ed. Iowa State University Press, Ames, IA, pp. 217 – 272. Tandon, S.K., Sharma, B.C., Singh, S., 1988. Chelation in metal intoxication XXVII: chelating agents containing vicinal thioether groups as antidotes of lead toxicity. Drug and Chemical Toxicology 11, 71 – 84. Journal of Ethnopharmacology 76 (2001) 233– 238 www.elsevier.com/locate/jethpharm Traditional Indian anti-diabetic plants attenuate progression of renal damage in streptozotocin induced diabetic mice J.K. Grover a,*, V. Vats a, S.S. Rathi b, R. Dawar c a Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110049, India b St. Boniface Institute of Cardio6ascular Sciences, Winnipeg, Canada c Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110049, India Received 20 June 2000; received in revised form 10 March 2001; accepted 11 April 2001 Abstract The purpose of the study was to investigate the effects of daily oral feeding Momordica charantia (MC) (200 mg/kg), Eugenia jambolana (EJ) (200 mg/kg), Mucuna pruriens (MP) (200 mg/kg) and Tinospora cordifolia (TC) extracts for 40 days on blood glucose concentrations and kidney functions in streptozotocin (STZ)-diabetic rats. Plasma glucose levels, body weight, urine volume and urinary albumin levels were monitored on every 10th day over a 40-day period while plasma creatinine levels were assessed at the beginning and end of experiment. Renal hypertrophy was assessed as the ratio between the kidney weight and total body weight. Plasma glucose concentrations in STZ-diabetic mice were reduced by the administration of extracts of MC, EJ, TC and MP by 24.4, 20.84, 7.45 and 9.07%, respectively (P B 0.005 for MC, EJ, MP and P B0.05 for TC). Urine volume was significantly higher (P B 0.005) in diabetic controls and MC, EJ, MP and TC treatment prevented polyuria (P B 0.001, 0.0001, 0.01 and 0.001, respectively). After 10 days of STZ administration urinary albumin levels (UAE) were over 6 fold higher in diabetic controls as compared to normal controls. Treatment with MC, EJ, MP and TC significantly prevented the rise in UAE levels from day 0 to 40 in comparison to diabetic controls (P B0.0001, 0.0001, 0.05, 0.05, respectively). Renal hypertrophy was significantly higher in diabetic controls as compared to non-diabetic controls. MC and EJ partially but significantly (P B0.05) prevented renal hypertrophy as compared to diabetic controls. TC and MP failed to modify renal hypertrophy. Results indicate that these plant drugs should be studied further. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Momordica charantia; Eugenia jambolana; Tinospora cordifolia; Mucuna pruriens; Experimental diabetes; Diabetic nephropathy; Streptozotocin 1. Introduction For various reasons in the recent years, the popularity of complimentary medicine has increased. Dietary measures and traditional plant therapies as prescribed by Ayurvedic and other indigenous systems of medicine have been used commonly in India. Surveys conducted in Australia and US indicate that almost 48.5 and 34% of respondents had used at least one form of unconventional therapy including herbal medicine (Eisenberg et al., 1993; Maclennan et al., 1996). Indian figures are not * Corresponding author. Tel.: + 91-11-6594897; fax: + 91-116862663. E-mail address: jkgrover@hotmail.com (J.K. Grover). available. The World Health Organization (1980) has also recommended the evaluation of the effectiveness of plants in conditions where we lack safe modern drugs (Upadhayay and Pandey, 1984). The primary objective of this study was to assess the efficacy of selected traditional anti-diabetic plants in diabetic nephropathy. Momordica charantia (MC), commonly known as bitter gourd belongs to Cucurbitaceae family. It is a widely used vegetable in India and its fruit, leaves and roots are recommended for treating diabetes mellitus (Warier, 1995). The hypoglycemic activity of extract of MC fruit has been previously confirmed in experimental animals and reviewed elsewhere (Gupta and Seth, 1962; Leatherdale et al., 1981; Raman and Lau, 1996). Seeds of MC have been 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 4 6 - X 234 J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238 shown to exert hypoglycemic activity comparable to glibenclamide (Kedar and Chakrabarti, 1982). Eugenia jambolana (EJ) belongs to the Myrtacae family and is commonly called Black Berry. Jamun seeds have been used by the Indian natives as an anti-diabetic remedy (Chopra et al., 1958). Various medicinal properties of EJ including its astringent, stomachic, astringent, diuretic and anti-diabetic properties have been described in traditional medicine (Nadkarni, 1992). Mucuna pruriens (MP), commonly known as Cowitch in English belongs to the Papillonaceae family and has been reported to be useful in diabetes (Dhawan et al., 1980). Hypoglycemic effects of seeds of MP have been demonstrated in normal rats (Pant and Joshi, 1970) but not in alloxan treated rats (Joshi and Pant, 1970). Tinospora cordifolia (TC) belongs to Menispermaceae and has been indicated in Ayurvedic treatment as a tonic, vitalizer and anti-diabetic (Nadkarni, 1954; Chopra et al., 1958). Scientific reports describing anti-diabetic (Gupta et al., 1967), immunomodulatory (Atal et al., 1986), anti-hepatotoxic (Peer and Sharma, 1989), anti-pyretic (Vedavathy and Rao, 1991) and anti-stress activity (Sarma et al., 1996) are available. 2. Materials and methods 2.1. Preparation of extracts 2.1.1. Aqueous extracts Fruit of MC and kernels of EJ were purchased from the local market in June 1997 and were authenticated by Head, Department of Botany, Miranda House, University of Delhi (India). The green outer skin (i.e. cuticle) of the MC fruit was peeled off and the rest was macerated in an electric mixer (Electro Com, New Delhi). The kernels of EJ were ground in an electric grinder and macerated as described above. This pulp (1 kg each) was then soaked separately in equal amount of water (1 l) and stirred intermittently and then left overnight. This pulp was then filtered through a coarse sieve and the filtrate was dried at reduced temperature. Respective yields MC and EJ after partial drying was 80.4 g/kg of fruit and 73 g/kg of kernel. To increase the shelf life and uniformity, the extracts were completely lyophilised by continuous freeze drying operation of 54 h (Christ freeze dryer, alpha 1-4, Germany), yielding 64 and 51 g/100 g; of MC and EJ extract. 2.1.2. Extracts of Tinospora cordifolia and Mucuna pruriens Alcoholic and aqueous extracts of TC and MP were received as gift from Brawn Pharmaceuticals Ltd., Faridabad (India). All the extracts were suspended in 1% carboxymethylcellulose (Central Drug House, New Delhi) and given orally. 2.2. Experimental design Albino mice (30–50 g) of both sexes were obtained from the experimental animal facility of the All India Institute of Medical Sciences. Before the start and during the experiment, mice were fed standard chow diet. The animals were randomised in the following groups: Mice in group I received saline plus CMC daily and served as normal control. Mice in group II–VI received a single intraperitoneal injection of 150 mg/kg STZ. Group II received saline daily and served as a diabetic control. Group III received 200 mg of lyophilised powder of MC, group IV received 200 mg of lyophilised powder of EJ, and group V received 400 mg of aqueous extract of TC and group VI received 200 mg/day of alcoholic extract of MP. All the extracts were dissolved in CMC and given PO everyday for 40 days by force-feeding using a 5-ml syringe. After randomisation into various groups, the rats were acclimatised for a period of 2– 3 days before initiation of experiment. Animals described as fasting had been deprived of food for at least 16 h but had been allowed free access to drinking water. 2.3. Preparation of diabetic nephropathic animals The method of Yotsumoto et al., 1997 was used for experimental induction of diabetic nephropathy. After acclimatisation, overnight fasted animals were injected a bolus of STZ (150 mg/kg of dissolved in 3 mM citrate buffer pH 4.5) intraperitoneally. Ten days after the single dose of STZ injection, only those mice exhibiting plasma glucose levels \ 300 mg/dl were included in the study. 2.4. Sample collection Fasting blood sample was collected in fresh vials containing sodium fluoride and sodium oxalate as anticoagulant/anti-glycolytic agents, retro-orbitally every 10th day till the end of experiment (i.e. 50th day) from the inner canthus of the eye under light ether anaesthesia using capillary tubes (Micro Hematocrit Capillaries, Mucaps). Plasma was separated in a T8 electric centrifuger (Remi Udyog, New Delhi) at 2000 rpm for 2 min. 2.5. Biochemical analysis 2.5.1. Plasma glucose Glucose levels were estimated by commercially available glucose kits based on glucose oxidase method (Trinder, 1969) (Autopak®, Bayer Diagnostics, Baroda). J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238 Table 1 The effect of 40 days treatment with selected doses of four different plant extracts on glucose levels (mg/%) and serum creatinine (mmol/l) in STZ (150 per mg/kg) diabetic mice Serum glucose 10th day Serum creatinine 50th day 10th day NC 94.98 9 4.67 92.03 9 4.32 DC 441.16 9 19.90b 439.5 9 14.25b MC 449.33 9 22.33 339.66 9 14.22b (24.4) EJ 456.5 9 22.90 361.83 9 31.192b (20.84) TC 464.66 9 15.57 430 9 14.97b (7.45) MP 462.83 9 14.48 420.83 9 13.36a (9.07) 50th day 41.5 9 3.08 46.6 9 3.05 46.8 94.49 50.0 9 1.67 45.3 91.63 47.5 9 3.08 46.3 94.63 42.3 9 3.92 235 2.5.4. Renal hypertrophy On the 40th day, animals were sacrificed and the kidney weight was determined gravimetrically and the degree of renal hypertrophy was expressed as the ratio of the weight of the two kidneys to total body weight. 2.6. Statistical analysis All results are expressed as the mean9 SD. The results were analysed for statistical significance by one way ANOVA test using computerised software, Microcal Origin version 2.9, Northampton, USA. 46.0 9 6.32 47.1 9 7.54 42.8 9 3.90 51.0 9 5.36 3. Results Values are given as mean 9 SD for groups of six animals each. Diabetic control was compared with the corresponding value of the non-diabetic controls. Experimental groups were compared with their corresponding values on the 0th day. NC: non-diabetic control; DC: diabetic control; MC: Momordica charantia (lyophilised powder 200 mg/kg); EJ: Eugenia jambolana (lyophilised powder 200 mg/kg); MP: Mucuna pruriens (alcoholic extract 200 mg/kg) and TC: Tinospora cordifolia (aqueous extract 400 mg/day). a Values are statistically significant at PB0.05. b Values are statistically significant at PB0.005. 3.1. Plasma glucose 2.5.2. Urinary albumin and creatinine le6els Albumin and creatinine were measured by commercially available kits (based on quantitative colorimetric assay) (Spectrum Medical India Pvt. Ltd, New Delhi). 3.2. Body weight 2.5.3. Body weight and urine 6olume Weight of individual animals was measured gravimetrically on every 10th day. For urine collection, animals were placed individually in metabolic cages for 24-h urine collection and urine volume was gravimetrically measured. The effect of STZ and the plant extracts on plasma glucose levels is shown in Table 1. The plasma glucose levels were markedly raised (up to 4.5 times) in the diabetic controls as compared with non-diabetic controls on the 10th and 40th day of the experiment (PB 0.005). Treatment with MC, EJ, TC and MP for 40 days significantly reduced the plasma glucose levels (PB 0.005 for MC, EJ, MP and P B0.05 for TC) with percentage reduction of 24.4, 20.84, 7.45 and 9.07, respectively. The effect of STZ and different plant extracts on the body weight of mice is summarised in Table 2. The basal values in the controls and treated groups were not significantly different from each other. The percentage increase in the body weight in the non-diabetic controls was significantly higher as compared to diabetic controls (27.5192.55 versus 14.969 11.76; P B0.05). Treatment with plant extracts did not affect the body weight through out the experimental period. Table 2 The effect of 50 days treatment with selected doses of four different plant extracts on body weight (g) in STZ (150 per mg/kg) diabetic mice NC DC MC EJ MP TC 0th day 10th day 20th day 30th day 40th day % Rise 26.66 9 1.96 28 9 2.44 26.66 9 3.77 28 9 2.44 26.66 9 2.58 25 9 3.16 28.16 9 2.22 29.83 9 2.56 28.83 9 2.04 29.83 9 2.56 27.5 9 2.42 26.16 9 3.25 30.16 9 2.40 30.33 9 3.26 29.66 9 2.58 30.33 9 3.26 29.5 9 2.34 279 3.93 32.33 9 2.87 32.33 9 4.36 30.5 9 3.01 32.339 4.36 31.33 9 2.50 29.83 9 3.43 34.0 9 2.52 32.5 93.50 32.16 9 4.02 33.5 9 3.50 32.83 9 2.48 30.2 9 3.30 27.51 9 2.55 14.96 9 11.76a 20.07 9 13.87 21.49 12.5 19.61 9 5.98 23.55 9 9.06 Values are given as mean 9 SD for groups of six animals each. Diabetic control was compared with the normal. Abbreviations as for Table 1. a Values are statistically significant at PB0.05. J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238 236 Table 3 The effect of 40 days treatment with selected doses of four different plant extracts on mean urine volume (ml) in six STZ (150 per mg/kg) diabetic mice NC DC MC EJ MP TC 0th day 10th day 20th day 30th day 40th day Mean rise from days 0–40 1.56 9 0.28 21.95 9 0.28b 17.43 9 3.07 20.83 9 1.83 21.16 9 1.47 22.13 9 0.75 1.56 9 0.13 23.96 9 4.10b 19.41 9 1.09 20.16 9 1.72 22.01 9 2.72 24.12 9 1.35 1.36 9 0.20 31.01 9 1.93b 19.58 9 1.78 19.83 9 0.75 22.1 9 3.72b 28.5 9 2.12 1.47 9 0.07 31.96 9 0.39b 20.96 9 4.08 21.16 9 2.04 25.16 9 2.99b 33.4 9 1.1 1.53 9 0.19 32.00 9 0.30b 20.45 9 3.08 20.33 9 1.5 26.05 9 3.59b 30.23 9 0.65 Negligible 10.59 0.432 1.93 9 2.24b 1.5 9 1.56c 4.889 3.32a 8.08 9 0.94b Values are given as mean 9 SD for groups of six animals each. Diabetic control was compared with the normal on 0, 10th, 20th, 30th, 40th day and the mean rise in urine volume from day 0–40. Abbreviations as for Table 1. a Values are statistically significant at PB0.01. b Values are statistically significant at PB0.001. c Values are statistically significant at PB0.0001. Table 4 The effect of 40 days treatment with selected doses of four different plant extracts on urinary albumin (mg/24 h) in STZ (150 per mg/kg) diabetic mice NC DC MC EJ MP TC 0th day 10th day 20th day 30th day 40th day Mean rise in UAE levels from days 0–40 170.83 9 22.01 1055.33 9 18.6 1043.59 29.69 1097.16 9 0.87 1064.66 9 20.39 1079.16 9 52.65 205.33 9 11.48 1552.66 9 48.68 1396.33 9 64.83 1465.59 59.19 1527 9 26.55 1449.839 72.41 369.66 917.85 1504.8 9150.78 1159.3 9 151.85 1151.5 9 74.54 1510 9 59.294 1410.5 9 47.60 474.83 9 21.67 1488.6 9 102.27 1078.83 945.66 1083.83 9 54.13 1420.33 9 82.62 1406.33 9 15.76 582.83 9 23.7 1411.33 9 72.69 1072.839 73.49 1041.5 991.492 1310 9 90.54a 1377.5 918.9 352.33 9 153.15 387 9 70.9 46.66 9 45.79b 59.33 9 57.34b 246.16 9 102.64a 298.33 9 63.74a Values are given as mean 9 SD for groups of six animals each. Diabetic control was compared with the normal on 0, 10th, 20th, 30th and 40th day. Abbreviations as for Table 1. a Values are statistically significant at PB0.05. b Values are statistically significant at PB0.0001. 3.3. Urine 6olume The effect of STZ on urine volume of mice is shown in Table 3. Ten days after STZ injection, urine volume was significantly higher (PB 0.005) in diabetic controls as compared to non-diabetic controls (21.959 0.28 versus 1.5690.28 ml/day, respectively). The polyuria in diabetic animals continued till the end of experiment (31.9890.29 versus 1.5391.96 ml/day, respectively, PB 0.005). The mean rise in urine volume from day 0 to 40 in diabetic control was 1090.43 ml and it was significantly more than compared to MC, EJ, MP and TC treated groups (1.9392.24, 1.591.56, 4.889 3.32 and 8.08 9 0.94, respectively; respective P values were B0.001, B 0.0001, B 0.001 and B0.001). 3.4. Urinary albumin le6els Urinary albumin levels in the STZ treated and nondiabetic control is shown in Table 4. The mean rise in diabetic controls over the period of 40 days was : 3 folds over the baseline values (170.839 22.01 versus 582.83 923.7 on 0th and 40th day, respectively; PB 0.005). The basal levels in the diabetic controls were over 6 fold high as compared to normal controls on 0 day (1055.339 18.6 versus 170.83922.01, respectively). The mean rise in UAE levels from day 0 to 40 in diabetic control was 3879 70.9 mg/24 h and it was significantly more in comparison to MC, EJ, MP and TC treated groups (46.669 45.79, 59.33957.34, 246.169 102.64 and 298.33963.74; respective P values were B 0.0001, B 0.0001, B0.05, B0.05). 3.5. Renal hypertrophy Table 5 summarises the effect of STZ and treatment with different plant extracts on renal hypertrophy. The net two weight of kidneys was significantly higher in diabetic controls as compared to controls (0.50119 0.0286 versus 0.5969 0.0508; P B0.005). Treatment with MC and EJ partially but significantly (PB 0.05) prevented renal hypertrophy as compared to diabetic controls along with reduction in kidney weight. TC and MP failed to modify renal hypertrophy. Renal hypertrophy measured as the ratio of kidney weight to total body weight in control, diabetic controls, MC, EJ, MP and TC treated groups was 0. 0147, 0.0183, 0.0161, 0.0153, 0.0167 and 0.0191, respectively. J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238 3.6. Serum creatinine The effect of STZ and treatment with plant extracts on the creatinine levels is shown in Table 1. No significant difference between normal controls and diabetic control animals or the treated groups was noted throughout the duration of the experiment. 4. Discussion STZ induced diabetes in rodents results in development of nephropathy similar to early stage clinical diabetic nephropathy (Rasch and Mogensen, 1980; Sassy-Prigent et al., 1995). Yotsumoto et al. (1997) showed that the use of mice was beneficial as they developed nephropathy earlier than rats (Mauer et al., 1978) and required a lower less amount of the diabetogenic agent. Therefore, mice were used in the present study. The effects of STZ induced diabetes in mice in the present case were similar to those reported earlier (Yotsumoto et al., 1997). Therefore, the present study reinforces the findings of Yotsumoto et al. (1997) and recommends the use of mice to produce experimental diabetic nephropathy. Furthermore, STZ has no longterm effects on the kidney other than those mediated by diabetes mellitus (Rasch, 1979). Urinary albumin levels are a selective marker of glomerular injury and elevated rates of albumin excretion are a harbinger of progressive nephropathy (Viberti et al., 1982), Rodents such as rats and mice exhibit albuminuria normally (Mauer et al., 1978; Yotsumoto et al., 1997) and during a period of 40 days, normal controls showed a rise in UAE by over 3 folds. UAE levels in the diabetic group, however, were many folds higher than normal controls. Treatment with MC, EJ, MP and TC prevented the rise in UAE levels with varying degree as seen in diabetic controls. The effect was more with MC and EJ and less with MP and TC. The observations of the present study indicate that the extracts of MC and EJ protect glomerulous from the injurious effects of diabetes. The histopathological examination of the kidneys from different groups sup- 237 ports this point as MC and EJ treated kidneys were within normal limits. Diabetes induction by STZ has been known to produce increase in kidney weight relative to body weight (Seyer-Hansen, 1976; Kang et al., 1982). In the present study, the average net weight of both kidneys of diabetic controls was significantly higher than non-diabetic controls. This is consistent with the previous finding of Yotsumoto et al., 1997. Treatment with MC and EJ prevented renal enlargement while MP and TC failed to bring any effect on this parameter. Previous studies on this respect have correlated the degree of renal enlargement with the degree of glycemic control (Rasch, 1979) while others have contradicted the glycemic theory (Wiseman et al., 1985), However, in the present study although MC and EJ exerted anti-hyperglycemic effect and prevented renal enlargement, the serum glucose levels remained well above 300 mg/dl. Thus, it is likely that mechanism or mechanisms independent of anti-hyperglycemic properties played a role in preventing renal enlargement. Rasch (1980), reported that the rise in body weight was far less in the poorly controlled diabetic rats as compared to well-controlled diabetic rats. Similar observations were made in this study. However, body weight was not significantly affected by any of the plant extracts, possibly due to poor glycemic control offered by the treatment. No statistical significant intra-group variation was seen in serum creatinine levels. Polyuria is a characteristic symptom of diabetes. Twenty-four hour urine volume was significantly increased in diabetic controls versus normal controls. MC, EJ, MP and TC treatment prevented polyuria in comparison with diabetic controls. Administration of plant extracts (notably MC and EJ) to diabetic mice prevented the increase in urine volume, UAE excretion, renal hypertrophy as well as caused a marginal reduction in plasma glucose levels. The results of the present study cannot be explained entirely on the basis of glycemic theory as treatment with MC and EJ was unable to achieve an euglycemic state and yet halted the progression of diabetic nephropathy (prevented renal enlargement in animals Table 5 The effect of 50 days treatment with selected doses of four different plant extracts on renal hypertrophy i.e. kidney weight (g) in STZ (150 per mg/kg) diabetic mice Two kidney weight (g) Ratio (kidney weight/total body weight) NDC DC MC EJ MP TC 0.5011 9 0.0286 0.0147 0.596 9 0.0508b 0.0183 0.5193 9 0.0264a 0.0161 0.515 9 0.0219a 0.0153 0.549 9 0.0379 0.0167 0.577 9 0.307 0.0191 Values are given as mean 9 SD for groups of six animals each. Diabetic control was compared with the normal. Experimental groups were compared with diabetic control. Abbreviations as for Table 1. a Values are statistically significant at PB0.05. b Values are statistically significant at PB0.005. 238 J.K. Gro6er et al. / Journal of Ethnopharmacology 76 (2001) 233–238 and attenuated the rate of increase in microalbuminuria along with reduction in renal hypertrophy). Regardless of the mechanism of action, the present study indicates that these plant drugs (MC and EJ) should be studied further. References Atal, C.K., Sharma, M.L., Kaul, A., Khajuria, A., 1986. Immunomodulating agents of plant origin. 1. Preliminary screening. J. Ethnopharmacol. 18, 133 –141. Chopra, R.N., Chopra, I.C., Handa, K.L., Kapur, L.D., 1958. Indigenous Drugs of India, second ed. Dhar and Sons, Calcutta, pp. 686 – 689. Dhawan, B.N., Dubey, M.P., Mehrotra, B.N., Rastogi, R.P., Tandon, J.S., 1980. Screening of Indian plants for biological activity. Part IX. Indian J. Exp. Biol. 18, 594 – 606. Eisenberg, D.M., Kessler, R.C., Foster, C., Norlock, F.E., Calkins, D.R., Delbanco, T.L., 1993. Unconventional al medicine in the United States. Prevalence, costs, and patterns of use. New Engl. J. Med. 328, 246 – 252. Gupta, S.S., Seth, C.B., 1962. Effect of Momordica charantia Linn. (Karela) on glucose tolerance in albino rats. J. Indian Med. Assoc. 39, 581 – 584. Gupta, S.S., Verma, S.C.L., Garg, V.P., Rai, M., 1967. Anti-diabetic effect of Tinospora cordifolia part I. Effect on fasting blood sugar level, glucose tolerance and adrenaline induced hyperglycemia. Indian J. Med. Res. 55, 733 – 745. Joshi, L.D., Pant, M.C., 1970. Hypoglycemic effect of Glycine soja, Dolichos biflorus and Mucuna pruriens seed in albino rats. Annual Conference of Indian Pharmacological Society. Lucknow, India. Indian J. Pharmacol. 2, 29. Kang, S.S., Fears, R., Noirot, S., Mbanya, J.N., Yudkin, J., 1982. Changes in metabolism of rat kidney and liver caused by experimental diabetes by dietary sucrose. J. Diabetol. 22, 285 – 288. Kedar, P., Chakrabarti, C.H., 1982. Effects of bittergourd (Momordica charantia) seed and glibenclamide in streptozotocin induced diabetes mellitus. Indian J. Exp. Biol. 20, 232 – 235. Leatherdale, B.A., Panesar, R.K., Singh, G., Atkins, T.W., Bailery, C.J., Bignell, A.H.C., 1981. Improvement in glucose tolerance due to Momordica charantia (Karela). BMJ 282, 1823 – 1824. Maclennan, A.H., Wilson, D.H., Taylor, A.W., 1996. Prevalence and cost of alternative medicine in Australia. Lancet 347, 569 – 573. Mauer, S.M., Brown, D.M., Matas, A.J., Steeffes, M.W., 1978. Effects of pancreatic islets transplantation on the increased urinary albumin excretion rates in intact and unephrectomized rats with diabetes mellitus. Diabetes 27, 959 – 964. Nadkarni, A.K., 1954. Indian Materia Medica. Popular Prakashan, Bombay, p. 1221. Nadkarni, A.K., 1992. Indian Materia Medica, vol. I. Popular Prakashan, Bombay, p. 157. . Pant, M.C., Joshi, L.D., 1970. Identification of pharmacologically active substances in the seeds of Mucuna pruriens (Abs). Proceedings of Annual Conference of Indian Pharmacological Society. Lucknow, India. Indian J. Pharmacol. 2, 24. Peer, F., Sharma, M.C., 1989. Therapeutic evaluation of Tinospora cordifolia on blood glucose and total lipid levels of normal and alloxan diabetic rats. Plant. Med. 58, 131 – 136. Raman, A., Lau, C., 1996. Antidiabetic properties and phytochemistry of Momordica charantia L. (Cucurbitaceae). Phytomedicine 2, 349 – 362. Rasch, R., 1979. Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment. Kidney size and glomerular volume. Diabetologia 16, 124 – 128. Rasch, R., 1980. Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment. Diabetologia 18, 413 – 416. Rasch, R., Mogensen, C.I., 1980. Urinary excretion of albumin and total protein in normal and streptozotocin diabetic rats. Acta Endocrinol. 95, 376 –381. Sassy-Prigent, C., Heudes, D., Jouquey, S., Auberval, D., Belair, M.F., Michel, O., Hamon, G., Bariety, J., Bruneval, P., 1995. Morphometric detection of incipient glomerular lesions in diabetic nephropathy in rats. Protective effects of ACE inhibition. Lab. Invest. 73, 64 – 71. Sarma, D.N.K., Khosa, R.L., Chansouria, J.P.N., Sahai, M., 1996. Anti-stress activity of Tinospora cordifolia Miers and Centella asiatica (L.) extracts. Phytotherapy Res. 10, 181 –183. Seyer-Hansen, K., 1976. Renal nephropathy in streptozotocin diabetic rats. Clin. Sci. Mol. Med. 51, 551 –555. Trinder, P., 1969. Determination of glucose using glucose oxidase with an alternative oxygen acceptor. Annu. Clin. Biochem. 6, 24 – 27. Upadhayay, V.P., Pandey, K., 1984. Ayurvedic approach to diabetes mellitus and its management by indigenous resources. In: Bajaj, J.S. (Ed.), Diabetes Mellitus in Developing Countries. Interprint, New Delhi, pp. 375 – 377. Vedavathy, S., Rao, K.N., 1991. Antipyretic activity of six indigenous medicinal plants of Tirumala Hills, Andhra Pradesh, India. J. Ethnopharmacol. 33, 193 –196. Viberti, G.C., Hill, R.D., Jarrett, R.J., Argyropoulous, A., Mahmud, U., Keen, H., 1982. Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus. Lancet 1, 1430 – 1432. Warier, P.K., 1995. Momordica charantia Linn. In: Warrier, P.K., Nambiar, V.P.K., Ramankutty, C. (Eds.), Indian Medicinal Plants. Orient Longman, Chennai, pp. 48 – 51. Wiseman, M.J., Saunders, A.J., Keen, H., Viberti, G., 1985. Effect of blood glucose control on increased glomerular filtration rate and kidney size in insulin dependent diabetes. New Engl. J. Med. 312, 617 – 621. World Health Organization, 1980. Second Report of the WHO Expert Committee on Diabetes Mellitus. Technical report series. 646,66. Yotsumoto, T., Naitoh, T., Shikada, K., Tanaka, S., 1997. Effects of specific antagonists of angiotensin II receptors and captopril on diabetic nephropathy in mice. Jpn. J. Pharmacol. 75, 59 – 64. Journal of Ethnopharmacology 76 (2001) 239– 245 www.elsevier.com/locate/jethpharm In vitro antiplasmodial activity and cytotoxicity of ethnobotanically selected South African plants E.A. Prozesky a, J.J.M. Meyer a,*, A.I. Louw b b a Department of Botany, Uni6ersity of Pretoria, Pretoria 0002, South Africa Department of Biochemistry, Uni6ersity of Pretoria, Pretoria 0002, South Africa Received 14 August 2000; received in revised form 15 March 2001; accepted 11 April 2001 Abstract The resistance of Plasmodium spp. to currently used drugs has become a serious problem and efforts are being directed in obtaining new drugs with different structural features. One option favoured is the search for new plant derived antimalarial drugs. Bark and leaves of 20 extracts from 14 South African plant species were tested for in vitro antiplasmodial activity by means of the flow cytometric test. The most active extract of each species giving more than 70% inhibition at 50 mg/ml was selected for determination of IC50 values. Two extracts had IC50 values below 2 mg/ml, another seven had IC50 values between 2 and 5 mg/ml while one had an IC50 of 10.1 mg/ml. Chloroquine had an IC50 of 0.043 mg/ml. Cytotoxicities of the five most active extracts at 50 mg/ml were determined with the monkey kidney cell toxicity test and the ID50 values ranged between 35 and 100 mg/ml. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: South African; Medicinal plants; Antiplasmodial activity; Cytotoxicity; Ethnobotanical 1. Introduction The use of medicinal plants as a source for relief from illness can be tracked back over five millennia to written documents of the early civilisations in China, India and the Near East, but it is doubtless an art as old as mankind (Hamburger and Hostettmann, 1991). It has been estimated that 80% of people living in developing countries are almost completely dependent on traditional medical practices for their primary health care needs, and higher plants are known to be the main source of drug therapy in traditional medicine. Malaria still remains one of the world’s biggest killers with more than two million people dying from the disease each year. Present drugs have become ineffective because parasites are developing resistance to most of them (Peters, 1998). The success of artemisinin isolated from Chinese traditional medicine with a different chemical structure than quinine, has stimulated the search for new antimalarial drugs from traditional remedies (Qinghaosu Antimalarial Coordinating Re* Corresponding author. E-mail address: marion@scientia.up.ac.za (J.J.M. Meyer). search Group, 1979). Efforts are now being directed in obtaining drugs with different structural features. Since many modern drugs originated from plants, the investigation of the chemical components of traditional medicinal plants could lead to the development of new antimalarial drugs. South Africa with its rich floral resources and ethnobotanical history is an ideal place to screen plants for antiplasmodial activity. The recognition and validation of traditional medical practices and the search for plant-derived drugs could lead to new strategies in malaria control. It is necessary to obtain more scientific information concerning the efficacy and safety of the remedies in use, because many people in third world countries already use and depend on herbal medicines for the treatment of malaria. Health planners in developing countries also require such information for evaluating the position and effectiveness of traditional medicine (Gessler et al., 1994). At present very little is known about the antiplasmodial activity of extracts from South African plant species. Various medicinal plants tested for antiplasmodial activity in other parts of Africa have shown potential as Western medicine and many related members of those plants also occur in South Africa. Very few studies on 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 4 5 - 8 240 E.A. Prozesky et al. / Journal of Ethnopharmacology 76 (2001) 239–245 the antiplasmodial activity of these plants have been done in South Africa. 2. Methodology 2.1. Selection and collection of plant material Ethnobotanically described antimalarial South African plants were selected, as well as South African representatives of plants used elsewhere against malaria. The plants analysed in this study were collected mainly from two areas in the malaria distribution region of South Africa, namely Venda and Northern Kwazulu-Natal. Voucher specimens are preserved in the H.G.W.J. Schweickerdt herbarium at the University of Pretoria (Table 1). 2.2. Preparation of extracts Samples of stem bark and leaves were collected and left at room temperature for two weeks to dry. Samples were chopped into smaller pieces and then ground into powder with an IKA dry mill (N.T. Laboratory Supplies, Cape Town). For each extraction, 5– 20 g of powdered leaves or stem bark were put in a round-bottomed flask and 150 ml of solvent (acetone, ethanol, dichloromethane, chloroform or petroleum ether) added. The extracts were then stirred with a magnetic stirrer for 24 h, filtered and dried by rotary vacuum evaporation. The mass of the extracts was determined, they were then re-dissolved in ethanol or dimethyl sulfoxide to concentrations of 100 or 200 mg/ml and stored at 4°C. In the flow cytometric analysis (Schulze et al., 1997), extracts were tested in duplicate (average of two experiments) at 50 mg/ml and serial dilutions made from this concentration for the determination of the IC50 values. The most active extract of each species with an inhibition of more than 70% at 50 mg/ml was selected for determination of the IC50 values. Previous experience in our laboratory has shown that extraction solvents influence biotests and solvent controls were therefore also tested for antiplasmodial activity. Extraction solvents were concentrated to the same degree as the extracts and dissolved in the same solvents as was the case with the extracts. These concentrated solvents were then diluted equivalently to the highest extract dilutions (500 and 1000 times) giving 0.2 and 0.1% solvent percentages and included in the flow cytometric test. 2.3. Antiplasmodial screening The South African isolate (PfUP1, a chloroquine resistant strain) of the malaria parasite Plasmodium falciparum was used in the bioassays. For continuous in vitro culturing a slightly modified version of the Trager and Jensen method was employed (Trager and Jensen, 1976; Hoppe, 1993). The wash medium consisted of 10.4 g RPMI 1640 L-glutamine, 5.94 g HEPES buffer, 4.0 g D-glucose, 44 mg hypoxanthine, 5% sodium hydrogen carbonate and 4 mg of gentamycin dissolved in 900 ml deionised sterile water. For use as culture medium, this wash medium was supplemented with 10% human serum of a rhesus positive blood group after heat inactivation at 56°C for 20 min. The parasite culture was then suspended in 10 ml of this culture medium, in a 75 ml culture flask (Sterilin). The culture, consisting of parasites and culture medium was then further supplemented with fresh, uninfected O+ blood group human erythrocytes. To determine the activity of extracts against P. falciparum in an accurate in vitro assay, the flow cytometric method of (Schulze et al., 1997) was used. Parasites (0.5% parasitemia, 5% haematocrit) in 96-well plates were pre-fixed after 48 h incubation, by adding fixing solution (1:1) after which they were incubated at 4°C for at least 18 h. The fixing solution consisted of 10% formaldehyde and 4% D-glucose formulated in a Tris– saline buffer (10 mM Tris, 150 mM sodium chloride and 10 mM sodium azide). After incubation at 4°C for 18 h or longer, 50 ml fixed parasite culture was added to 1 ml phosphate buffered saline (PBS) containing 0.25 mg thiazole orange (Molecular Probes, OR), in plastic tubes (Corning). The samples were then placed on ice to inhibit further staining of the parasite DNA prior to flow cytometric analysis. A volume of 200 ml of prepared parasite sample was analysed by the flow cytometer (Coulter Epics II, Coulter electronics, Hialeah, FL) and a total of 100 000 erythrocytes were counted in each sample. 2.4. Cytotoxicity screening Microtitre plates with vervet monkey kidney cells (Pretoria Biomedical Research Center) were used for testing the plant extracts for cytotoxicity. Multilayer cells present in the tissue culture plates were first rinsed three times with 10% PBS, before adding 3 ml of 0.1% Trypsin EDTA (National institute of Virology, Johannesburg) to loosen the cells adhering to the bottom of the plates. The plates were then incubated for 5 min at 37°C. Fresh maintenance medium (7–8 ml) was poured in the tissue culture plates and the contents were transferred to a test tube. This medium consisted of 97 g of Eagle’s MEM (National institute of Virology, Johannesburg) with Earle’s salts (Highveld Biological), 47.06 g nitrogen-2-hydroxyethylpiperasinenitrogen-2-ethanesulfonic acid (Hepes BSS), 8.5 g sodium hydroxide made up in 10 l sterile distilled water, supplemented with 1–2% heat inactivated (56°C for 30 min) fetal calf serum (FCS) (Delta Bioproducts, Kempton Park, Table 1 Plant species analysed for antimalarial activity in this study and their medicinal uses reported in the literature Family Plant species and herbarium voucher number Medicinal uses reported in the literature Other medicinal uses Related species, O. insignis in Tanzania (Weenen et al., 1990) Related species, B. aegyptica in Tanzania (Weenen et al., 1990) Used to dress hair (Pooley, 1993), for venereal diseases and as a general cleanser for men Used as an emetic (Palmer and Pitman, 1972), an arrow poison (Mabogo, 1990) and as a molluscicide (Pretorius et al., 1988) Taken as emetics for chronic coughs (Bryant, 1966) and as emetics for respiratory ailments (Watt and Breyer-Brandwijk, 1962) Used for various stomach complaints, fevers and wounds (Pooley, 1993), as abortifacients and anthelmintics and also for stomachache, colic and fevers (Irvine, 1961). Also used for infertility, intestinal worms and as a laxative for wounds, snakebite and difficult confinements (Mabogo, 1990) Used as antiviral and antitussive medicines (Irvine, 1961) and for asthma and syphilitic sores (Watt and Breyer-Brandwijk, 1962). Used for swellings and abscesses (Pujol, 1990), to clear wounds of maggots and for toothache (Mabogo, 1990) None found Anacardiaceae O. engleri R.A. Fernandes (E.A. Prozesky 95 PRUa) Balanitaceae B. maughamii Sprague (E.A. Prozesky 89 PRU) Celastraceae M. senegalensis (Lam.) Excell (E.A. Prozesky 79 PRU) Stem bark used in Tanzania (Gessler et al., 1994). Included as a positive plant control Combretaceae Combretum molle R.Br. ex G.Don (E.A. Prozesky 81 PRU) Related species, C. aff. Psidioides subsp. Psilophyllum in Tanzania (Gessler et al., 1994) Euphorbiaceae C. pseudopulchellus Pax (E.A. Prozesky 91 PRU) Related species, C. megalobotrys used by Vhavenda (Mabogo, 1990) Fabaceae E. lysistemon Hutch (E.A. Prozesky 103 PRU) Related species, E. sacleuxii in Tanzania (Weenen et al., 1990) Meliaceae E. caudatum (Sprague) Sprague (E.A. Prozesky 100 PRU) Meliaceae T. emetica Vahl (E.A. Prozesky 76 PRU) Related species E. bussei in Tanzania (Weenen et al., 1990) Trichilia glabra tested by MacKinnon et al. (1997). Mimosaceae Acacia xanthoploea Benth. (E.A. Prozesky 96 PRU) Rhamnaceae R. prinoides L’Hérit. (E.A. Prozesky 104 PRU) Rhamnaceae Z. mucronata Willd. (E.A. Prozesky 70A PRU) Powdered bark and roots are used by Zulus in emetics for malaria and are also taken prophylactically (Watt and Breyer-Brandwijk, 1962) Used In Kenya (Prof. A.E. van Wyk personal communication) Selected as closely related genus to Rhamnus Used as enemas for stomach and intestinal complaints (Bryant, 1966) and for lumbago, rectal ulceration and dysentery (Watt and Breyer-Brandwijk, 1962). Administered for kidney ailments, as blood cleansers and for intestinal worms (Mabogo, 1990) and used for fever and as purgatives (Gelfand et al., 1985). Bark decoctions are taken for sickle cell anemia in Tanzania (Chhabra et al., 1984) E.A. Prozesky et al. / Journal of Ethnopharmacology 76 (2001) 239–245 Against malaria Used for sprains (Bryant, 1966) and as blood purifiers (Watt and Breyer-Brandwijk, 1962) Used for chronic coughs (Bryant, 1966) and for respiratory aliments (Watt and Breyer-Brandwijk, 1962). Used for pain and infertility (Mabogo, 1990). For measles, scarlet fever, boils, carbuncles and dysentery. Used for gonorrhea (Hutchings et al., 1996), treating snakebite, rheumatism and stomach ailments (Kokwaro, 1976) 241 242 Table 1 (Continued) Plant species and herbarium voucher number Medicinal uses reported in the literature Against malaria Rubiaceae Catunaregam spinosa (Thunb.) Tirvengadum subsp. Spinosa (E.A. Prozesky 82 PRU) Solanaceae S. panduriforme E. Mey. (E.A. Prozesky 94 PRU) Verbenaceae L. ja6anica (Burm. f.) Spreng. (E.A. Prozesky 78 PRU) a b University of Pretoria Herbarium, Pretoria. National Herbarium, Pretoria. Used by Zulus for fever (Gerstner, 1941) Other medicinal uses Used as emetics for fevers (Gerstner, 1941), as an aphrodisiac, for gonorrhea (Verdcourt and Trump, 1969), headaches (Watt and Breyer-Brandwijk, 1962), snakebite, nausea, various respiratory, febrile and gynecological ailments, epilepsy and dizziness (Gelfand et al., 1985) Leaf infusions of a related species S. nigrum are used Used for skin infections (A.R. Bain 4145 PREb), by the Vhavenda (Mabogo, 1990) and is also used in wounds and ulcers (Mabogo, 1990), toothache and Zimbabwe (Watt and Breyer-Brandwijk, 1962) are taken for hemorrhoids (Watt and Breyer-Brandwijk, 1962) Used by Vhavenda (Mabogo, 1990) Used for coughs and colds (Hutchings et al., 1996) and to treat febrile rashes (Doke and Vilakazi, 1972). Used in washes and poultices for chest ailments (Roberts, 1990). Used for a variety of ailments including asthma, headaches, febrile and respiratory complaints, convulsions, weak joints, cataracts and sore eyes (Gelfand et al., 1985). Used for scabies and backache, and as anthelmintics, for febrile ailments and as prophylactics against dysentery, diarrhea and skin diseases (Dalziel, 1937; Irvine, 1961) E.A. Prozesky et al. / Journal of Ethnopharmacology 76 (2001) 239–245 Family E.A. Prozesky et al. / Journal of Ethnopharmacology 76 (2001) 239–245 South Africa) and containing 100 U/ml penicillin and 100 mg/ml streptomycin. The test tube was then centrifuged for 5 min at 3000 rpm (Girst et al., 1979). After discarding the supernatant fresh minimal essential medium (MEM) was added and mixed thoroughly with the cells. 100 ml from these freshly mixed cells in MEM was transferred to microtitre plates and incubated at 37°C. The MEM was essentially the same as the maintenance medium except that it contained 10% FCS. The MEM was poured off after 24 h and replaced with 200 ml of plant extract diluted to the desired concentration with MEM. Stock solutions of plant extracts were made up to a concentration of 100 mg/ml. Serial dilutions of a 1000fold diluted stock solution were added to monkey kidney cells. Cells were incubated for 6 days and a visual estimate of the inhibition compared to the control was taken on day 2, 4 and 6. Three controls; a negative control (just the media), dimethyl sulfoxide 0.1% and ethanol 0.1% were used. 243 mg/ml. The most active extract of each species with an inhibition of more than 70% at 50 mg/ml was selected for determination of the IC50 values. Results from this experiment are shown in Table 3. Among the plant species tested, the strongest antiplasmodial activity was found in the dichloromethane extract of Ozoroa engleri with an IC50 of 1.7 mg/ml. This plant, together with Balanites maughamii belonged to the category of plants with the highest antiplasmodial activity (IC50 values B2 mg/ml). The extracts of Croton pseudopulchellus, Entandrophragma caudatum, Erythrina lysistemon, Lippia ja6anica, Solanum panduriforme, Trichilia emetica and Ziziphus mucronata had IC50 values between 2 and 5 mg/ml, while Acacia zanthoploea was the only one of the selected plants with an IC50 above 10 mg/ml. Results from the cytotoxicity tests are shown in Table 4. The highest cytotoxicity in the monkey kidney cell test was found with the dichloromethane extract of O. engleri with an ID50 of 35 mg/ml. The lowest cytotoxicity was found with the dichloromethane extract of E. caudatum with an ID50 of 100 mg/ml. 3. Results 4. Discussion and conclusions Results obtained from the flow cytometric analysis are shown in Table 2. Most extracts had more than 50% inhibition of malaria parasite proliferation at 50 Little is known about the chemistry of B. maughamii but a related species B. aegyptiaca did show antiplas- Table 2 In vitro antimalarial activity of plant extracts as determined with the flow cytometric test. Concentrated extraction solvent and solvent controls had no inhibition at 0.1% except concentrated acetone which had a 5% inhibition at 0.1% Plant species Plant part Solvent Yield% (w/w) Minimum inhibition% at 50 mg/ml A. xanthoploea Stem bark B. maughamii C. spinosa C. molle Stem bark Stem bark Stem bark C. pseudopulchellus Stem bark Entandophragma caudatum Stem bark E. lysistemon L. ja6anica M. senegalensis Stem bark Leaves Stem bark O. engleri Stem bark R. prinoides S. panduriforme T. emetica Z. mucronata Leaves Leaves Stem bark Stem bark Aa Eb DMc A A E A CFd PEe DM A A A E PE DM A A DM A 20.23 17.18 1.71 4.55 8.55 10.06 8.25 7.91 1.57 3.47 8.27 9.85 10.4 8.94 6.19 7.90 10.84 3.97 6.73 8.51 75 60 70 40 53 62 82 82 80 82 82 78 60 50 75 80 48 75 82 73 a Acetone. Ethanol. c Dichloromethane. d Chloroform. e Petroleum ether. f Standard error. b ( 9 4.6)f ( 9 9.1) ( 9 5.6) (9 9.4) (9 6.3) ( 9 7.5) ( 9 4.9) ( 9 6.1) (9 12.4) ( 9 11.7) ( 9 13.5) (9 12.1) ( 9 8.7) ( 9 6.8) ( 9 8.3) ( 9 6.7) ( 9 9.2) (9 5.7) ( 9 8.3) ( 9 8.9) E.A. Prozesky et al. / Journal of Ethnopharmacology 76 (2001) 239–245 244 Table 3 IC50 values of extracts with more than 70% inhibition at 50 mg/ml Species Plant part Solvent IC50 mg/ml A. xanthoploea Stem bark Stem bark Stem bark Stem bark Stem bark Leaves Stem bark Leaves Stem bark Stem bark Aa 10.10 7.51–12.67 DMb 1.94 1.20–2.68 CFc 3.45 2.91–3.99 DM 2.90 1.62–4.18 A 4.80 2.73–6.87 A DM 4.26 1.70 2.73–5.79 1.32–2.08 A DM 3.62 3.29 2.96–4.28 2.94–3.64 A 4.13 2.95–5.31 0.043 0.028–0.058 B. maughamii C. pseudopulchellus E. caudatum E. lysistemon L. ja6anica O. engleri S. panduriforme T. emetica Z. mucronata Chloroquine 95% confidence intervald a Acetone. Dichloromethane. c Chloroform. d 95% confidence intervals were determined with Student’s t-test (Snedecor and Cochran, 1980). b modial activity between 10 and 50 mg/ml (Weenen et al., 1990). Bark from B. aegyptiaca is reported to be toxic to fish, but not to man (Lewis and Elvin-Lewis, 1977), while seeds have shown significant anthelmintic activity (Ibrahim, 1992). The antiplasmodial activity of B. maughamii in this study was five times higher than the antiplasmodial activity from B. aegyptiaca. This could be due to different Plasmodium strains used or variation in the amount of active principle(s) present in the different plant species. Several species of Croton are known to be toxic (Lewis and Elvin-Lewis, 1977), but conflicting reports on the toxicity of South African Croton species were reported (Hutchings et al., 1996). Various Croton species are used as antimalarials throughout the world and the results obtained with C. Table 4 In vitro cytotoxicity of plant extracts tested on monkey kidney cells Extract ID50 mg/ml monkey kidney cells C. pseudopulchellus 64 (chloroform) E. caudatum (dichloromethane) 100 E. lysistemon (acetone) 69 O. engleri (dichloromethane) 35 T. emetica (dichloromethane) 50 pseudopulchellus warrants further investigation into the antimalarial activity of C. megalobotrys used traditionally against malaria by the Vhavenda (Mabogo, 1990). Various species of Meliaceae are used traditionally against malaria and the highly active antiplasmodial compound gedunin has been isolated from some species (MacKinnon et al., 1997). Very little information is available about the traditional uses of E. caudatum and this coupled with its good antiplasmodial activity, might warrant further investigation. Little is known about O. engleri’s medicinal activity, but O. insignis tested in Tanzania did show some antiplasmodial activity between 10 and 49 mg/ml (Gessler et al., 1994). This is lower than the result obtained with O. engleri in this study and might be due to a different parasite strain used or active compounds present in different concentrations in these two plant species. The genus, Solanum contains some very poisonous species (Hutchings et al., 1996). Despite this, S. nigrum is used as a traditional antimalarial (Mabogo, 1990). Our results obtained with S. panduriforme confirm the antiplasmodial activity in some species of the genus and further investigation into the use of S. nigrum might be worthwhile. Various Trichilia species have shown antiplasmodial activity (MacKinnon et al., 1997) and results from T. emetica confirm the general antiplasmodial activity of this genus. The genus Rhamnus and Ziziphus are very closely related morphologically. Although Rhamnus prinoides is used as a traditional antimalarial in Kenya, no reports on the antimalarial use of Z. mucronata could be found. Investigation of the genus Ziziphus might lead to interesting findings. Erythrina species are widely used as antimalarials in Madagascar and East Africa (Gessler et al., 1994). Results from E. lysistemon in this study confirm the antiplasmodial activity of this genus. Lippia species are widely used as traditional medicine including against malaria (Valentin et al., 1995). The antiplasmodial activity found in L. ja6anica supports its use as a traditional medicine in South Africa. The antiplasmodial activity found in the acetone extract of A. xanthoploea supports the use of this plant as a traditional antimalarial remedy. Although the antiplasmodial activity of B. maughamii and A. xanthoploea are similar at 50 mg/ml, the IC50 values differ 5-fold. This might be due to a toxic effect influencing the antiplasmodial activity at higher concentrations. The relatively similar IC50 values of the other extracts might be due to selection based on ethnobotanical knowledge as well as selection of only the most active extracts. The highest cytotoxicity in the monkey kidney cell test was found with the dichloromethane extract of O. engleri with an ID50 of 35 mg/ml. The lowest cytotoxicity was found with the dichloromethane extract of E. caudatum with an ID50 100 mg/ml. The therapeutic index is expressed as the antiplasmodial activity (flow E.A. Prozesky et al. / Journal of Ethnopharmacology 76 (2001) 239–245 cytometric test) to cytotoxicity (monkey kidney cell test) ratio. The index values of the extracts were in the range 35–100 and therefore they have poor selectivity indices. The best ratios were obtained with the dichloromethane extract of E. caudatum (100) and the acetone extract of E. lysistemon (69). It has been proposed that the ratio for a good therapeutic remedy should be \ 1000, as found for example with quinine (Likhitwitayawuid et al., 1993). In vitro cytotoxicity as determined in this study, is not always a clear indication of toxicity in vivo. Kirby et al., (1993) describe the extreme cytotoxicity in vitro of highly active antiplasmodial quassinoids isolated from Brucea species, although human clinical studies using crude preparations of Brucea fruits have shown antimalarial efficiency without toxicity (Kirby et al., 1993). It is therefore possible that the extracts investigated in this study will have less cytotoxicity in vivo, as well. This warrants further investigations of their potential as antimalarials. Acknowledgements The National Research Foundation and the University of Pretoria are thanked for financial support. Department of Virology of the University of Pretoria for monkey kidney cell toxicity tests. References Bryant, A.T., 1966. Zulu Medicine and Medicine-Men. C. Struik, Cape Town. Chhabra, S.C., uiso, F.C., Mshiu, E.N., 1984. Phytochemical screening of Tanzanian medicinal plants. Journal of Ethnopharmacology 11, 157 – 179. Dalziel, J.M., 1937. The Useful Plants of West Tropical Africa. Crown Agents, London. Doke, C.M., Vilakazi, B.W., 1972. Zulu – English Dictionary, second ed. Witwatersrand University Press, Johannesburg. Gelfand, M., Mavi, S., Drummond, R.B., Ndemera, B., 1985. The Traditional Medical Practitioner in Zimbabwe. Mambo Press, Gweru, Zimbabwe. Gerstner, J. 1941. A preliminary checklist of Zulu names of plants with short notes. Bantu Stud. 12 (3): 215 –236. Gessler, M.C., Nkunya, M.H.H., Mwasumbi, L.B., Heinrich, M., Tanner, M., 1994. Screening Tanzanian medicinal-plants for antimalarial activity. Acta Tropica 56, 65 – 77. Girst, N.R., Bell, E.J., Follette, E.A.C., Urquhart, G.E.D., 1979. Diagnostic Methods in Clinical Virology, third ed. Blackwell Scientific Publications, Oxford, pp. 60 –79. Hamburger, M., Hostettmann, K., 1991. Bioactivity in plants: the link between phytochemistry and medicine. Phytochemistry 30, 3864 – 3874. . 245 Hoppe, H.C., 1993. Identification and characterisation of selected meroziote-stage antigens in southern African isolates of Plasmodium falciparum. PhD Thesis, University of Pretoria, Pretoria. Hutchings, A., Scott, A.H., Lewis, G, Cunningham, A.B., 1996. Zulu Medicinal Plants. University of Natal Press, Pietermaritzburg. Ibrahim, A.M., 1992. Anthelmintic activity of some Sudanese medicinal plants. Phytotherapy Research 7, 348 –351. Irvine, F.R., 1961. Woody Plants of Ghana. Oxford University Press, London. Kirby, G.C., Warhurst, D.C., Phillipson, J.D., 1993. Plants as a source of novel antimalarial drugs. Transactions of the Royal Society of Tropical Medicine and Hygiene 87 (4), 370. Kokwaro, J.O. 1976. Medicinal plants of East Africa. East African Literature Bureau. Lewis, W.W.H., Elvin-Lewis, M.P.F., 1977. Medical Botany. Wiley, New York. Likhitwitayawuid, K., Angerhofer, C.K., Cordell, G.A., Pezzuto, J.M., 1993. Cytotoxic and antimalarial bizbenzyl-isoquinoline alkaloids from Stephania erecta. Journal of Natural Products 56 (1), 30 – 38. Mabogo, D.E.N., 1990. The Ethnobotany of the Vhavenda. Unpublished MSc. Thesis, University of Pretoria, Pretoria. MacKinnon, S., Durst, T., Arnason, J.T., 1997. Antimalarial activity of tropical meliaceae extracts and gedunin derivatives. Journal of Natural Products 60, 336 –341. Palmer, E., Pitman, N., 1972. Trees of Southern Africa, vol. 2. Balkema, Cape Town. Peters, W., 1998. Drug resistance in malaria parasites of animals and man. Advances in Parasitology 41, 1 – 4. Pooley, E., 1993. The Complete Field Guide to Trees of Natal, Zululand and Transkei. Natal Flora Publications Trust, Natal Herbarium, Durban. Pretorius, S.J., Joubert, P.H., Evans, A.C., 1988. A re-evaluation of the molluscicidal properties of the torchwood tree, Balanites maughamii Sprague. South African Journal of Science 84, 201 – 202. Pujol, J., 1990. Natur Africa: The Herbalist Handbook. Jean Pujol Natural Healers Foundation, Durban. Qinghaosu Antimalarial Coordinating Research Group, 1979. Antimalaria studies on qinghaosu. Chinese Medical Journal 92, 811 – 816. Roberts, M., 1990. Indigenous Healing Plants. Southern Book Publishers, Halfway House. Schulze, D.L.C., Makgatho, E.M., Coetzer, T.L., Louw, A.I., Van Rensburg, C.E.J., Visser, L., 1997. Development and application of a modified flow cytometric procedure for rapid in vitro quantitation of malaria parasitaemia. South African Journal of Science 93, 156 – 158. Snedecor, G.W., Cochran, W.G., 1980. Statistical Methods, seventh ed. Iowa State University Press, Hemisphere. Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 674. Valentin, A., Pelissier, Y., Benoit, F., Marion, C., Kone, D., Mallie, M., Bastide, J.-M., Bessiere, J.-M., 1995. Composition and antimalarial activity in vitro of volatile components of Lippia multiflora. Phytochemistry 40 (5), 1439. Verdcourt, B., Trump, E.C., 1969. Common Poisonous Plants of East Africa. Collins, London. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern and Eastern Africa, second ed. Livingstone, London. Weenen, H., Nkunya, M.H.H., Bray, D.H., Mwasumbi, L.B., Kinabo, L.S., Kilimali, V.A.E.B., 1990. Antimalarial activity of Tanzanian medicinal plants. Planta Medica 56, 369. Journal of Ethnopharmacology 76 (2001) 247– 252 www.elsevier.com/locate/jethpharm Effects of Sclerocarya birrea (A. rich) hochst (anacardiaceae) leaf extracts on calcium signalling in cultured rat skeletal muscle cells R.G. Belemtougri a, B. Constantin b, C. Cognard b, G. Raymond b,*, L. Sawadogo a a Faculté des Sciences et Techniques, Laboratoire de Physiologie Animale, Uni6ersity of Ouagadougou, Ouagadougou BP 7021, Burkina Faso b L.B.S.C., CNRS UMR 6558, Uni6ersity of Poitiers, Bâtiment P, 40 a6enue du Recteur Pineau, F-86022, Poitiers Cedex, France Received 30 August 2000; received in revised form 14 March 2001; accepted 11 April 2001 Abstract Sclerocarya birrea is a plant used widely to treat many diseases in Burkina Faso, although no scientific data has been reported about its mechanism of action. In the present study the effects of its leaf extracts were investigated on calcium signalling in rat cultured skeletal muscle cells. The results show that the different extracts (crude decoction, aqueous, ethanolic and chloroformic extracts) have significant antagonistic effect on caffeine-induced calcium release from sarcoplasmic reticulum. Crude decoction is the most active followed by ethanolic, aqueous and chloroformic extracts in dose-dependent manner and can partly justify the use of the plant in traditional medicine. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sclerocarya birrea; Anacardiaceae; Caffeine; Skeletal muscle cells; Intracellular calcium; Sarcoplasmic reticulum 1. Introduction Many medicinal plants were described in African pharmacopoeia (Adjanohoun, 1990). According to Nacoulma-Ouédraogo (1996), more than 445 medicinal plants are used to treat many diseases in the central plateau of Burkina-Faso. Sclerocarya birrea is one of plant species used widely in traditional medicine in Africa against many diseases and affections such as hypertension, dysentery, stomach-ache or gastro-enteritis. According to Guinko (1984), the bark decoction is used as anticough; the leaves, the pulp of fruit and mistletoe are used for hypertension and other affections. The bark and leaves are also used as antihyperglycemic (Nacoulma-Ouédraogo, 1996). Chemical analyses of the plant resulted in the isolation of (−)-Epicatechin-3-galloyl ester which exhibited secretagogue activity (Galvez et al., 1992), crude oil, carbohydrate, crude protein, fibre and saponins (Ogbobe, 1992), minerals (Smith et al., 1996) and ascorbic acid (Eromosele et al., 1991). Its gum has local techno* Corresponding author. Tel.: + 33-5-49453666; fax: + 33-549454014. E-mail address: guy.raymond@univ-poitiers.fr (G. Raymond). logical applications. The methanolic, evaporated crude water and unevaporated crude water extracts exhibited molluscicidal activity (Kela et al., 1989). Our recent chemical screening showed alkaloids in chloroformic extract, anthocyans, flavonoids and tannins in ethanolic extract, tannins and saponosides in aqueous extract. According to the unifying ‘ionic hypothesis’ (see Resnick, 1999) the large variety of clinical responses reported in human hypertensive disease, can be explained by a steady-state elevation of cytosolic free Ca2 + and decrease in Mg2 + level that alter the function of many tissues. Among them blood vessels exhibit vasoconstriction, cardiac tissue hypertrophy and skeletal muscle insulin resistance. One of the possible ways of action of Sclerocarya leave extract could be a decrease in the internal Ca2 + concentration in accordance to the previous observation (unpublished results) that crude decoction from the leaves induced relaxation on vascular smooth muscles. In addition, the presence of a large amount of ascorbic acid, a powerful antioxidant, could be of relevance in the counteraction of some of the clinical features associated with hypertension (May, 2000). Calcium transients are mainly responsible for the intracellular cascades leading to contraction of muscle cells therefore we aimed to explore the effects of crude 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 4 8 - 3 248 R.G. Belemtougri et al. / Journal of Ethnopharmacology 76 (2001) 247–252 decoction and different extracts on calcium signalling systems in order to dissect the mechanism(s) of action of the plant compounds with regard to its relaxing effect. This was done in mammalian skeletal muscle cell in culture for several reasons: (i) the different mechanisms involved in the control of Ca2 + homeostasis are well-defined and abundantly documented, (ii) skeletal muscle cells in culture share with smooth and cardiac muscle some specific Ca2 + -signalling pathways (Reyes & Jaimovich, 1996; Capiati et al., 2000) which are absent in adult skeletal muscle, (iii) antioxidants such ascorbic acid are susceptible to directly act on the muscle cell which produces oxygen radicals and vasoactive NO (Reid, 1996; Thomas et al., 1998), (iv) the skeletal muscle cell is the most important source of voltage-dependent Ca2 + channels, one possible target of the plant compounds. The present results describe for the first time the inhibition of calcium release from sarcoplasmic reticulum by S. birrea leaf extracts on rat skeletal muscle cells. This could contribute to the identification of the mechanisms underlying its action. 2. Materials and methods 2.1. Plant collection Fresh leaves of S. birrea were collected from Gampéla (Burkina-Faso, West Africa) in July 1997. The plant was identified by Dr Millogo-Rasolodimby, Department of Botany, University of Ouagadougou. A voucher specimen has been deposited in this Department. 2.2. Preparation of plant extracts Crude decoction was prepared from the dried leaves. Six grams of leaves powder were first macerated in deionized water with agitation for 24 h at room temperature and then boiled for 10 min. After cooling, the resulting extract was filtered through whatman c2 and evaporated. The chloroformic, ethanolic and aqueous extracts were prepared as follows: 40 g of leaves powder were macerated with agitation in chloroform (200 ml) for 24 h. After filtration and evaporation, 0.7134 g of final extract yielded was obtained. The leaves powder submitted to chloroformic extract was dried at room temperature and macerated in ethanol (200 ml) with agitation for 24 h. The resulting extract was filtered through whatman c2 and evaporated; 1.5611 g of final extract yielded was obtained. The leaves powder submitted to ethanol extract was dried and macerated in deionized water (200 ml) at room temperature for 24 h. After filtration, the resulting extract was lyophilized. 2.3. Cell culture Primary cultures of rat skeletal muscle cells were initiated from satellite cells. As previously detailed (Cognard et al., 1993a; Mouzou et al., 1999) these satellite cells were obtained by trypsinisation of small muscle pieces of 1–2 day-old rats hind-limbs. The pieces were washed in a calcium- and magnesium-free medium (spinner) medium containing (mM): NaCl 116; KCl 5.3; Na2HPO4 8; NaHCO3 22.6. D glucose 1 g and phenol red 10 mg were added. The pieces were incubated in spinner with 7.5% trypsin for 10 min at 37°C and the supernatant was removed. Two or three successive incubations for 20 min were carried out and the successive cell suspensions were cooled and centrifuged (500×g, 15 min). The cell pellets were resuspended in growth medium (Ham F12, Gibco) supplemented with 10% fetal calf serum (Boehringer, Mannhein, Germany), 10% heat-inactivated horse serum (Gibco) and 1% antibiotics: (Penicillin G 100 U/ml, Sigma and Streptomycin 50 U/ml, Sigma). After filtration, the cells were numbered, diluted to an appropriate concentration and preplated in large plastic dishes (90 mm) for 90 min (37°C, 5% CO2, water saturated air atmosphere) to remove most of the fibroblasts which rapidly adhere. The cells were plated in 35 mm plastic dishes on glass coverslips and placed in the same condition as for preplating. After 96 h, the growth medium was exchanged for a medium constituted by Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% heat-inactivated horse serum and 1% antibiotics which elicited the fusion of mononucleated myoblasts into multinucleated myotubes. Experiments were carried out on 3– 5 day-old myotubes (never more than 2 myotubes per Petri dish) selected from 15 different primary cultures. 2.4. Intracellular free calcium measurements Intracellular free calcium was measured with the fluorescent dye Indo-1. Cells were loaded with 3 mM of the lipophilic form of Indo-1 (AM form, dissolved in dimethylsulfoxide) diluted in control solution. The cells were incubated for 45 min in the dark at room temperature, washed twice with control solution and incubated 10 min at 37°C to obtain a complete de-esterification of the probe. Fluorescence was recorded at room temperature using an OSP100CA microscopic photometry device (Olympus). Excitation of Indo-1 was set in UV range (band-pass filter centered to 360 nm) by means of a xenon lamp. Fluorescence emission was acquired through a dichroic filter (455 nm) by means of two photomultipliers tubes with two band-pass filters centered to 405 nm for the emission of the calcium bound form and to 485 nm for the calcium free form of the probe. R.G. Belemtougri et al. / Journal of Ethnopharmacology 76 (2001) 247–252 2.5. Experimental solutions The experiments were performed in a control solution containing (mM): NaCl 130; KCl 5.4; CaCl2 2.5; MgCl2 0.8; Glucose 5.6; HEPES 10 and adjusted at pH 7.4 with Tris. The test solutions were made by adding caffeine (final concentration 10 mM) (caffeine solution) and then 1– 10 mg/ml of the plant extracts to the caffeine solution (caffeine+Sclerocarya solution). Rapid changes of the solutions in the surroundings of interrogated cells were achieved by means of a homemade microperfusion system. 249 induced by caffeine, but with different efficiencies. For example at 3 mg/ml, the ethanolic extract which contains anthocyans, flavonoids and tannins decreased the calcium release by 40% against 20% for the aqueous extract in which there are tannins and saponosides and only 5% for the chloroformic extract which contains alkaloids. 2.6. Statistical analysis Results of the experiments were expressed as mean9 SEM, and paired or unpaired Student’s t-test was used to test for difference significance between the means. P B0.05 was considered as significant. 3. Results The different extracts of S. birrea (SB), when applied alone, failed to display any activity on the resting calcium level, regardless of the duration of incubation (not shown). That indicates that they have no effect on the mechanisms controlling the resting internal Ca2 + homeostasis. Then application of SB has been performed during caffeine stimulation in order to search for antagonistic effect of SB on calcium release. As already reported (Cognard et al., 1993b) the application of caffeine (10 mM) in 3–5 day-old myotubes induced intracellular calcium transients which reflected the emptying of the sarcoplasmic reticulum Ca2 + stores. For each interrogated cell, 10 mM caffeine was applied two times (Fig. 1A) with a 2 min interval in order to test for the stability of the response and this response was determined as the control one. Then after another recovery period of 2 min, the caffeine+ SB extract solution was superfused. As illustrated in Fig. 1B, for a concentration of 10 mg/ml, crude extract of SB decreased the amplitude of calcium transients by comparison with control responses. This indicates that the extracts have a significant antagonistic effect on caffeine-induced calcium release from the sarcoplasmic reticulum. The crude decoction at 1, 3 and 10 mg/ml produced a dose dependent reduction in caffeine-induced calcium-release. This extract decreased intracellular calcium release from 29% at 1 mg/ml to 62% at 10 mg/ml (Fig. 2). Similar experiments have been performed with the different extracts (see methods) of which the main chemical compounds had been determined (Table 1). The results, summarized in Fig. 2, show that all the extracts exerted an inhibitory effect on the Ca2 + signal Fig. 1. Examples of recordings showing effects of caffeine (A) and caffeine plus crude decoction (B) on intacellular calcium release from sarcoplasmic reticulum different time of recording on the same myotube (caf., caffeine and CD, crude decoction). R.G. Belemtougri et al. / Journal of Ethnopharmacology 76 (2001) 247–252 250 Fig. 2. Percentage inhibition of intacellular calcium release by different leaf extracts of Sclerocarya birrea at three different doses. This led to the following observations: 1. The crude decoction showed the largest inhibition of intracellular calcium release with an IC50 value around 3.5 mg/ml whereas for other extracts it was above than 10 mg/ml. 2. Among the semi-purified extracts the ethanolic extract was the most active with the following order of efficiency: crude decoction\ethanolic extract\ aqueous extract\ chloroformic extract. 4. Discussion This work was performed to investigate for possible interferences of different extracts of S. birrea with Ca2 + mobilizing systems in muscle cells. As any of them failed to display any activity on the resting calcium level it seems they have no effect on the mechanisms controlling the resting internal Ca2 + homeostasis. As in skeletal muscle cell the main source of internal Ca2 + ions is the sarcoplasmic reticulum, we used caffeine which remains the best known activator of sarcoplasmic reticulum calcium release channels (HerrmannFrank et al., 1999) to explore for a possible action on the mechanisms responsible for Ca2 + release and activation of muscular contraction. The main result of the present work was the observation of a decrease in the net caffeine-induced calciumrelease flux from sarcoplasmic reticulum and that the four extracts tested here were dose-dependently effective. On a dose-to-dose basis, the crude decoction was the most effective, followed by the ethanolic extract, the aqueous extract and then the chloroformic extract. This difference in the action could be due to the variation of the polarity and chemical nature of the compounds present in the extract used. The difference between the actions of the different extracts may be due to differential extraction or phase partition of active compounds during the extraction process. Preparation methods of medicinal remedies determine their activity and effectiveness. Most of the plants used in traditional medicine are administered as decoctions or infusions, thus the biologically active components must be water-soluble. This is consistent with the positive correlation between the crude decoction and the inhibition of intracellular calcium release demonstrated in the present study. The decrease in the calcium release from sarcoplasmic reticulum to myoplasm could result from some competition between SB extracts and the Ca2 + -releasing channel leading to a dose-dependent block of calcium release from sarcoplasmic reticulum. Interaction of the extracts with some proteins involved in the regulation of internal Ca2 + cycling or handling could not be ruled out. For example a stimulatory effect on the sarcoplasmic reticulum Ca2 + -pump would have a similar decreasing effect on the net myoplasmic Ca2 + increase measured during the caffeine response. With regard to this, tannins which are present in the two most active fractions (ethanolic and aqueous) could play some role since tannins have been several times reported to stimulate the sarcoplasmic reticulum Ca2 + ATPase of heart (Chiesi & Schwaller, 1994). However Coll et al. (1999) have shown that, contrary to that observed in cardiac SR, tannin failed to activate SR Ca2 + -ATPase from adult rabbit skeletal muscle. On the other hand, the presence of flavonoids in the extracts could also explain the observed results. The pharmacological effects of flavonoids in mammals resulted mainly from inhibition of certain enzymes and their antioxidant activity and they have been reported to inhibit phospholipase C (PLC) and protein kinase C Table 1 Summarizes chemical screening of Sclerocarya birrea leaf extracts Chemical compounds Anthocyans Alkaloids Flavonoids Coumarins Quinones Tannins Steroids/Terpenoids Saponosides Extracts Aqueous extract Ethanolic extract Chloroformic extract − + − − − + − + − − − − − − − + + − − − − + − nd +, presence; −, absence; nd, not determined. R.G. Belemtougri et al. / Journal of Ethnopharmacology 76 (2001) 247–252 (de Groot & Rauen, 1998). In cultured skeletal myotubes, functional expression of muscarinic M1 receptors coupled to PLC and to internal Ca2 + stores has been reported (Reyes & Jaimovich, 1996) and a PKCinduced increase in internal calcium (Capiati et al., 2000) as well. In addition, quercetin, a plant flavonoid has been reported to have a depressing effect on skeletal muscle contraction (Apisariyakul et al., 1999). All these known actions of flavonoids, are thus in favor of a decrease in internal Ca2 + release form the SR. 5. Conclusion The results reported here show that different leaf extracts of S. birrea possess active constituents capable of inhibiting calcium release from sarcoplasmic reticulum. Such antagonistic effect on the net calcium release could be at least partly at the origin of the vasorelaxant effect observed on rat arterial vessels since calcium ions are responsible for the contraction of smooth muscle and since an internal calcium increase sensitive to caffeine has been demonstrated to be involved in contraction of arterial tissue (Itoh et al., 1982) like in other muscular systems. So far, two studies on S. birrea have been reported: one of them (Galvez et al., 1993) concerned the ethanol extract from leaves which inhibited angiotensin converting enzyme (ACE). Clearly this might explain the antihypertensive effect in traditional medicine (Duncan et al., 1999) but an additionnal decrease in the internal Ca2 + mobilization could also be relevant. Further investigations will be done leading to isolation of active components and also acute toxicity experiments to clarify the degree of toxicity of the plant. Acknowledgements We thank Dr Samate, D. A., Pr Nacoulma-ouedraogo, O. G. & Mr Lamien, E. for the screening of the plant leaf extracts. Thanks are also due to Dr MillogoRasolodimby for the identification of the plant. References Adjanohoun E. 1990, Etat d’évolution de l’ethnopharmacopée africaine, Bulletin Médicine Tradational Pharmacology Vol. 1. Apisariyakul, A., Chaichana, N., Takemura, H., 1999. Dual effect of quercetin on contraction in cardiac and skeletal muscle preparations. Research Communication Molecular Pathology Pharmacology 105, 129 – 138. Capiati, D.A., Vasquez, G., Tellez Inon, M.T., Boland, R.L., 2000. Role of protein kinase C in 1,255OH)(2)-vitamin D(3) modulation of intracellular calcium during development of skeletal muscle cells in culture. Journal of Cellular Biochemistry 77, 200 – 212. 251 Chiesi, M., Schwaller, R., 1994. Reversal of phospholamban-induced inhibition of cariac sarcoplasmic reticulum Ca2 + -ATPase by tannin. Biochemistry Biophysics Research Communication 202, 1668 – 1673. Cognard, C., Constantin, B., Rivet-Bastide, M., Imbert, N., Besse, C., Raymond, G., 1993a. Appearance and evolution of Ca2 + currents and contraction during the early post-fusional stages of rat skeletal muscle cells developing in primary culture. Development 117, 1153 – 1161. Cognard, C., Constantin, B., Rivet-Bastide, M., Raymond, G., 1993b. Intracellular Ca2 + transients induced by different kinds of stimulus during myogenesis of rat skeletal muscle cells studied by laser cytofluorimetry with Indo-1. Cell Calcium 14, 333 – 348. Coll, K.E., Johnson, R.G., McKenna, E., 1999. Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum adenosinetriphosphatase. Biochemistry 38, 2444 –2451. de Groot, H., Rauen, U., 1998. Tissue injury by reactive oxygen species and the protective effects of flavonoids. Fundamental Clinical Pharmacology 12, 249 –255. Duncan, A.C., Jäger, A.K., Van staden, J., 1999. Screening of Zulu medicinal plants for angiotensin converting enzyme (ACE) inhibitors. Journal of Ethnopharmacology 68, 63 – 70. Eromosele, I.C., Eromosele, C.O., Kuzhkuzha, D.M., 1991. Evaluation of mineral elements and ascorbic acid contents in fruits of some wild plants. Plant Foods Human Nutral April 41 (2), 151 – 154. Galvez, P.J., Zarzuelo, A., Busson, R., Cobbaert, C., de Witte, P., 1992. ( − ) — Epicatechin — 3 — galloyl ester: a secretagogue compound from the bark of Sclerocarya birrea. Planta Medica April 58 (2), 174 – 175. Galvez, J., Crespo, M.E., Zarzuelo, A., de Witte, P., Spiessens, C., 1993. Pharmacological activity of a procyanidin isolated from Sclerocarya birrea bark: antidiarrhoeal activity and effects on isolated guinea-pig ileum. Phytotherapy Research 7, 25 – 28. Guinko S., 1984. La végétation de Haute-Volta. Tome 1. Thèse de Doctorat: Université de Bordeaux III, p. 316 Herrmann-Frank, A., Lüttgau, H.C., Stephenson, D.G., 1999. Caffeine and excitation-contraction coupling in skeletal muscle: a stimulating story. Journal Muscle Research Cell Motility 20, 223 – 237. Itoh, T., Kajiwara, M., Kitamura, K., Kuriyama, H., 1982. Roles of stored calcium on the mechanical response evoked in smooth muscle cells of the porcine coronary artery. Journal Physiology 322, 107 – 123. Kela, S.L., Ogunsusi, R.A., Ogbogu, V.C., Nwude, N., 1989. Screening of some Nigerian plants for molluscicidal activity. Review Elevation Medical Vetinary Pays Tropical 42 (2), 195 – 202. May, J.M., 2000. How does ascorbic acid prevent endothelial dysfunction? Free Radical Biology Medicine 28, 1421 – 1429. Mouzou, A.P., Bulteau, L., Raymond, G., 1999. The effects of Securidaca longepedunculata root extract on ionic current and contraction of cultured rat skeletal muscle cells. Journal Ethnopharmacology 65, 157 –164. Nacoulma-Ouédraogo O.G., 1996. Plantes médicinales et pratiques médicales traditionnelles au Burkina Faso: cas du plateau central. Thèse de Doctorat Es Sciences Naturelles: Université de Ouagadougou, tome II, p. 285 Ogbobe, O., 1992. Physico-chemical composition and characterisation of the seed and seed oil of Sclerocarya birrea. Plant Food Human Nutral July 42 (3), 201 – 206. Reid, M.B., 1996. Reactive oxygen and nitric oxide in skeletal muscle. NIPS 11, 114 – 119. Resnick, L., 1999. The cellular basis of hypertension and allied clinical conditions. Progress Cardiovascular Display 42, 1 – 22. Reyes, R., Jaimovich, E., 1996. Functional muscarinic receptors in cultured skeletal muscle. Archives Biochemistry Biophysics 331, 41 – 47. 252 R.G. Belemtougri et al. / Journal of Ethnopharmacology 76 (2001) 247–252 Smith, G.C., Clegg, M.S., Keen, C.L., Grivetti, L.E., 1996. Mineral values of selected plant foods common to southern Burkina Faso and Niamey, Niger West Africa. Institute Journal Food Science Nutral, January 47 (1), 41 –53. . Thomas, G.D., Sander, M., Lau, K.S., Huang, P.L., Stull, J.T., Victor, R.G., 1998. Impaired matabolic modulation of h-adrebnergic vasoconstriction in dystrophin-deficient skeletal muscle. Proceedings Natural Academic Science USA 95, 15090 –15095. Journal of Ethnopharmacology 76 (2001) 253– 262 www.elsevier.com/locate/jethpharm Influence of Podophyllum hexandrum on endogenous antioxidant defence system in mice: possible role in radioprotection A. Mittal, V. Pathania, P.K. Agrawala, J. Prasad, S. Singh, H.C. Goel * Radiation Biology Laboratory, Institute of Nuclear Medicine and Allied Sciences, Lucknow Road, Timarpur, Delhi 110054, India Received 18 December 2000; received in revised form 3 April 2001; accepted 11 April 2001 Abstract Podophyllum hexandrum, a Himalayan herb with known radioprotective and anti-tumour properties, was investigated for its mechanism of action. Glutathione S-transferase (GST), catalase, superoxide dismutase (SOD) activities and lipid peroxidation (LPx) were determined in the liver, jejunum and ileum at various time intervals, with and without the aqueous extract of P. hexandrum rhizome (200 mg/kg b.w. i.p.) in unirradiated and whole body irradiated (10 Gy,-2 h) male Swiss albino mice. Pre-irradiation treatment with P. hexandrum enhanced liver GST (P B 0.01) and SOD (P B 0.05) at 12 h post irradiation, the intestinal SOD (P B 0.00005) at 84 h post irradiation was significantly elevated. However, no significant change was manifested in the catalase activity in the liver, at any of the post irradiation intervals (0, 12 and 84 h). The antioxidant defence with Podophyllum sp. treatment in mice can explain to some extent its protective action manifested in terms of survival against whole body lethal irradiation. However, some other possible mechanisms that may strengthen radioprotective action of the Podophyllum sp. extract need to be investigated further. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Radioprotection; Antioxidants; Podophyllum hexandrum; Superoxide dismutase; Catalase; Glutathione S-transferase 1. Introduction Radiation injuries are manifested as a result of increased production of reactive oxygen species (ROS) − like O− 2 , OH , H2O2, etc. due to oxidative stress (Sies, 1983). ROS are highly mutagenic and damage cellular macromolecules like DNA, proteins and lipids. To maintain the redox balance in order to protect themselves, the living cells have evolved an endogenous antioxidant defence mechanism which include non enzymatic entities like glutathione, ascorbic acid, uric acid, etc. and also enzymes like catalase, superoxide dismutase (SOD), glutathione peroxidase, etc. Ayurveda, the Indian system of medicine, has exploited different herbs for the treatment of various diseases including free radical generated pathologies like atherosclerosis, arthritis, geriatric problems etc. Many herbal preparations in the recent past have been reported to act as good radioprotectors by scavenging the free radicals. These agents can also modulate antioxi* Corresponding author. Fax: + 91-11-3919509. E-mail address: radbiol@nda.vsnl.net.in (H.C. Goel). dant defence system of the body tissues by upregulation/ downregulation of the antioxidant gene expression (Suzuki et al., 1997). Podophyllum hexandrum (Family, Berberidaceae) has been widely used in the treatment of genital warts, malignant and non-malignant tumours and infections of bacterial and viral origin including AIDS (Blasko and Cordell, 1988). Recently, it has also been reported to elicit significant protection against whole body lethal irradiation in mice. It was found to scavenge the free radicals and decrease free radical mediated reactions like lipid peroxidation (LPx) under in vitro conditions (Kumar and Goel, 2000) and in vivo micronuclei formation (Goel et al., 1999) in our laboratory. Therefore, to understand the mode of action of P. hexandrum under in vivo conditions it was necessary to investigate its effect on the antioxidant defence system with special reference to enzymes like glutathione Stransferase (GST) (neutralises electrophiles by conjugation with glutathione and making them readily excretable from the body), catalase (inactivates H2O2 to form water), and SOD (scavenges superoxide anion to form H2O2) and also LPx during g-irradiation and its consequences in post-irradiation scenario. 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 4 3 - 4 254 A. Mittal et al. / Journal of Ethnopharmacology 76 (2001) 253–262 Liver is the primary organ of drug metabolism and is mainly responsible for the detoxification of damaging electrophiles generated during oxidative stress, hence it is rich in endogenous antioxidants and related enzymes (Sipes and Gandolfi, 1983). Similarly, gastro-intestinal tissue has highly proliferating stem cells which is radiosensitive (Potten, 1990) and is also having an antioxidant defence mechanism to combat oxidations occurring. It comprises of the jejunum and ileum which perform slightly different physiological functions of digestion and assimilation, respectively. Therefore, the present study was aimed to unravel the mechanism of radiation protection by P. hexandrum by investigating the antioxidant defence system in liver and intestinal tissues of mice. 2. Methodology 2.1. Chemicals 1-Chloro-2,4-dinitrobenzene (CDNB), 5% dithiobis-2nitrobezoic acid (DTNB), bovine serum albumin (BSA), reduced nicotinamide adenine dinucleotidesodium salt (NADH), 2-thiobarbituric acid (TBA), reduced glutathione (GSH), phenazine methosulphate (PMS), nitroblue tetrazolium (NBT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The rest of the chemicals used were procured locally and were of analytical grade. 2.2. Preparation of plant extract Dried rhizome of P. hexandrum supplied by Field Research Laboratory, Leh (Jammu and Kashmir, India) was powdered mechanically and 1 g of powder mixed in 100 ml of distilled water was kept at 3791°C in an incubator for 24 h and filtered thereafter using whatman filter paper no. 1. The filtrate was passed through millipore filter (0.12 mm), lyophilised and dried and then was resuspended in triple distilled water (vehicle) in desired quantities before i.p. administration. The dose administered as mg/kg b.w. of experimental animal refers to the dried lyophilised extract of rhizome of P. hexandrum. 2.3. Animals About 6 –8 weeks old, male Swiss albino mice, weighing approximately 25 g, were maintained in our experimental facility under controlled temperature 2592°C and were provided standard animal feed for laboratory rodents (Amrut Laboratory Animal Feed, India) and regular tap water were provided ad libitum. Four to five animals were housed together in polypropylene cages. All procedures involving animals were carried out in strict compliance with the Animal Ethics Committee rules and regulations followed in this institute. 2.4. Irradiation of animals Irradiation of mice was carried out at room temperature in a plastic container in a Co60 g-chamber (Model 220, Atomic Energy of Canada Ltd.) having a dose rate of 2 cGy s − 1, to deliver the desired whole body dose. To avoid any decrease in O2 concentration in the irradiated chamber, air was circulated through a rubber tube connected to an air pump. Dosimetry was carried out with Baldwin Farmer secondary dosimeter and Fricke’s chemical dosimetry. 2.5. Experimental design 2.5.1. Whole body sur6i6al studies Different groups of mice (n= 5– 16) were administered desired doses of P. hexandrum extract (i.p.) 2 h prior to whole body 10 Gy g-irradiation and the survival was observed daily for upto 30 days post irradiation. Data was presented as percentage survival as compared with untreated control. 2.5.2. Antioxidant parameters Animals were divided into four groups as under and sacrificed by cervical dislocation at 0, 12 and 84 h post-irradiation in all the groups. The experiments were repeated thrice. Since these experiments were conducted over a span of time, therefore, for comparison of different experimental groups the control value was considered as one and the experimental value was considered as a proportion of that. Group I (n =4 ×3) — 0.2 ml vehicle (i.p.) and sham irradiated. Group II (n =4 ×3) — 0.2 ml (200 mg/kg b.w.) P. hexandrum in vehicle 2 h before sham irradiation. Group III (n =4 ×3) — 10 Gy whole body irradiation. Group IV (n= 4× 3) — 0.2 ml (200 mg/kg b.w.) P. hexandrum in vehicle 2 h before 10 Gy whole body irradiation. 2.6. Preparation of homogenates 2.6.1. Li6er The liver was perfused in situ immediately with cold 0.9% NaCl and thereafter removed, and was rinsed in chilled 0.15 M Tris–KCl (pH 7.4). The liver was then blotted dry, weighed and homogenised in ice cold 0.15 M Tris – KCl buffer (pH 7.4) to yield a 10% w/v homogenate. Aliquots (1 ml) of this homogenate was used for assaying LPx, while the remainder was centrifuged at 10 000×g for 30 min at 4°C and the supernatant was used for analysing the specific activities of SOD, catalase and GST. A. Mittal et al. / Journal of Ethnopharmacology 76 (2001) 253–262 2.6.2. Intestine Jejunum (first 1/3 part of the small intestine) and ileum (remaining 2/3 part of the small intestine) were removed, cut into 2–3 pieces each and flushed thoroughly with chilled 0.9% NaCl. The intestines were now split open longitudinally and gently scraped on prechilled slides dipped in buffer, to separate the mucosal layer to estimate the enzymes. The jejunum and ileum mucosal layers were then suspended in 4 ml of 0.15 M Tris –KCl (pH 7.4) and homogenised. The aliquots for the estimation of LPx were taken and the remaining homogenate was centrifuged at 10 000×g for 30 min at 4°C. The supernatant was used for determining the specific activities of SOD, catalase and GST. 2.7. Lipid peroxidation (LPx) assay The LPx level in the microsomal suspension was estimated as the number of nmoles of malondialdehyde (MDA) formed by the method of Pryor (1976). The reaction mixture comprised 0.2 ml homogenate, 1.8 ml Tris buffer (pH 7.4), 0.5 ml 30% trichloroacetic acid (TCA) and 0.5 ml 52 mM thiobarbituric acid (TBA), heated for 30 min at 95°C and cooled in ice immediately. It was then centrifuged for 10 min at 5000 rpm and the absorbance was read at 531 nm. The level of LPx was estimated by using an extinction coefficient of 0.152 mM − 1 cm − 1 and data was expressed as mmoles MDA formed per mg of protein. 2.8. Superoxide dismutase (SOD) assay The SOD activity was measured according to McCord and Fridovich (1969)) based on the SOD mediated inhibition of NBT formazan formation from NBT in the presence of O− generator, phenazine methosulphate 2 (PMS). The reaction was initiated by the addition of PMS to a 50 mM Tris 0.1 M NaCl buffer (pH 8.3) containing 0.20 ml of 500 mM NBT, 0.20 ml of 780 mM NADH and suitable aliquots of supernatant to make a final volume of reaction mixture of 2 ml. The increase in absorbance was followed for 5 min at 560 nm. The activity of SOD in samples was determined from a calibration curve drawn by taking the amount of inhibition of NBT formazan formation in presence of aliquots of sample versus absence of sample (100% NBT reduction). One unit of enzyme activity was defined as the amount of SOD required to cause 50% decrease in NBT reduction. Data was expressed as units of SOD activity per mg of protein. 255 contained 1.95 ml 10 mM H2O2 in 60 mM phosphate buffer (pH 7.0). The reaction was started by adding 0.05 ml supernatant and the absorbance was followed for 3 min at 240 nm. 60 mM phosphate buffer (pH 7.0) was used as a reference. The extinction coefficient of 0.04 mM − 1 cm − 1 was used to determine the specific activity of catalase. The data was expressed as mmoles H2O2 consumed per min per mg of protein. 2.10. Glutathione S-transferase (GST) assay The GST activity in the supernatant was determined spectrophotometrically at 37°C according to the procedure of Habig et al., 1974). The reaction mixture (3 ml) contained 1.7 ml of 100 mM phosphate buffer (pH 6.5), 0.1 ml of 30 mM GSH and 0.1 ml of 30 mM CDNB. After preincubating the reaction mixture at 37°C for 2 min, the reaction was started by the addition of 0.1 ml diluted supernatant and the absorbance was followed for 3 min at 340 nm. Reaction mixture without the enzyme was used as the blank. The specific activity of GST was expressed as mmole GSH– CDNB conjugate formed per min per mg of protein using an extinction coefficient of 9.6 mM − 1 cm − 1. The protein content was measured according to Schacterle and Pollack (1973)), using BSA as a standard. 2.11. Statistical analysis Statistical analysis was performed using analysis of variance (ANOVA) to test the significant difference between the groups. Figures have been drawn using data expressed as mean9standard deviation and comparing it with respect to the control, taken as one, for observations at a particular time interval. 3. Results 3.1. Whole body sur6i6al studies All irradiated animals without Podophyllum species treatment showed 100% mortality within 15 days (Fig. 1). Different doses of P. hexandrum were administered to mice 2 h before irradiation (10 Gy).Maximum survival of 93% (in 30 days) was achieved by administration of 200 mg/kg b.w. of P. hexandrum. Treated mice showed an improvement in their gain of body weights and malaise as compared with the irradiated animals. 2.9. Catalase assay 3.2. Antioxidant parameters The supernatant activity of catalase activity was determined spectrophotometrically according to the protocol of Claiborne (1985). The reaction mixture (2 ml) 3.2.1. Catalase acti6ity The catalase activity in the liver was unaffected by 256 A. Mittal et al. / Journal of Ethnopharmacology 76 (2001) 253–262 Fig. 1. The effect of different doses of extract (aq.) of Podophyllum hexandrum rhizome administered i.p. 2 h prior to 10 Gy whole body irradiation on whole body survival (30 days) in Swiss albino mice. A. Mittal et al. / Journal of Ethnopharmacology 76 (2001) 253–262 Podophyllum species treatment alone (group II), radiation (group III) or by treatment with P. hexandrum before irradiation (group IV)as compared with the con- 257 trol (group I) or by inter group comparison, at all time intervals (Fig. 2). In the jejunum, by P. hexandrum treatment alone Fig. 2. Effect of extract (aq.) of Podophyllum hexandrum rhizome administration on catalase activity in Swiss albino mice. — Group I; control administered 0.2 ml vehicle. - - Group II; Podophyllum hexandrum administered 200 mg/kg b.w. i.p. in 0.2 ml vehicle. – - Group III; 10 Gy whole body irradiation. Group IV; Podophyllum hexandrum 200 mg/kg b.w. administered i.p. 2 h prior to 10 Gy whole body irradiation. Catalase activity of the groups has been represented as a proportion of the control, taken as 1. Data expressed as mean 9 standard deviation. 258 A. Mittal et al. / Journal of Ethnopharmacology 76 (2001) 253–262 (group II) at 0 h time interval, the mice expressed significantly depressed catalase activity (0.4890.006; P B0.005) as compared with the control (taken as 1). At 12 and 84 h the catalase activity was back within the range of control. After irradiation (group III), the catalase activity in the jejunum of mice in comparison to the control remained significantly reduced at 0, 12 and 84 h post irradiation (0.6890.01; PB 0.01, 0.65 9 0.016; PB 0.0001and 0.6890.005; PB 0.0005, respectively). After preirradiation treatment with Podophyllum sp. (group IV), at 0 and 84 h time intervals, the mice expressed significantly depressed catalase activity (0.3890.0002; PB 0.001 and 0.569 0.004; PB 0.0005, respectively) as compared with the control. At 12 h post irradiation, the significant depression had been alleviated and the value was comparable with the control. In the ileum, though the Podophyllum species treatment alone (group II) expressed enhanced catalase activity than the control (taken as 1), there was a significant elevation only at 0 h time interval (1.359 0.03; P B0.05).The irradiated group (group III) and the preirradiated P. hexandrum treated group (group IV) showed significantly depressed catalase activity (0.60 90.013; P B0.005 and 0.5390.014; P B0.05, respectively) as compared with the control and to Podophyllum species alone treated animals (group II, 1.18 90.10; P B0.05) at 84 h time interval. 3.2.2. Glutathione S-transferase acti6ity In the liver of P. hexandrum treatment alone (group II), though the GST activity was more enhanced than the control, the difference was not significant at any time interval (Fig. 3). A similar trend was followed by the irradiation group (group III) though initially at 0 h it was lesser than the control. The GST activity in the preirradiation Podophyllum sp. treated group (group IV) was significantly more enhanced than the control (1.499 0.008; P B0.01) at 12 h post irradiation, and remained insignificantly different at 0 and 84 h post irradiation from the control. In the jejunum, the GST activity in the Podophyllum sp. alone treated animals (group II) remained unaffected as compared with the control (considered as 1) at all time intervals. Irradiation (group III) and preirradiation Podophyllum sp. treatment (group IV) remained unaffected at 0 and 12 h post irradiation but at 84 h both the groups had significantly more depressed GST activity than the control (0.6690.01; P B0.01 and 0.6590.003; PB 0.005, respectively). In the ileum, the GST activity in the P. hexandrum alone treated mice (group II) was significantly lesser than the control (taken as 1) at 0 and 84 h (0.749 0.001 and 0.9259 0.0003, respectively; PB 0.0005) but was insignificantly different at 12 h. The other two groups (group III and IV) were unaffected either by radiation alone or by Podophyllum sp. preirradiation treatment at all time intervals as compared with the control. 3.2.3. Superoxide dismutase acti6ity In the liver, the SOD activity was unaffected in the Podophyllum sp. alone treated mice (group II) as compared with the control (taken as 1) at all time intervals (Fig. 4). By irradiation (group III) at only 12 h post irradiation the mice expressed reduced SOD activity as compared with the control (0.6890.003; P B 0.001) and to the preirradiated treatment group with Podophyllum species (group IV, 0.9090.004; P B 0.005), while at other time intervals it was unaffected. The preirradiated treatment with Podophyllum sp. (group IV) expressed insignificantly different catalase activity as compared with the control at all time intervals, though at 84 h it was enhanced than the control. In the jejunum, the P. hexandrum alone treated animals (group II) at 0 h time interval expressed significantly depressed SOD activity (0.769 0.005; P B0.005) as compared with the control (taken as 1), became insignificantly different from the control at 12 h and then was significantly enhanced at 84 h (1.529 0.09; P B0.05) as compared with the control. Irradiation (group III) after 12 h post irradiation only had significantly depressed SOD activity (0.6690.05; P B 0.05) as compared with the control. Determination of SOD activity showed no significant difference in the Podophyllum sp. pretreated irradiated animals (group IV) as compared with the control after 0 and 12 h but at 84 h, there was a significant enhancement (1.269 0.00009; P B0.00005). In the ileum, at 0 h, Podophyllum species treatment only (group II) had significantly enhanced the SOD activity (1.3490.05; P B0.05), but was significantly reduced at 12 h (0.869 0.003; P B0.05) as compared with the control and at 84 h was comparable to the control value. With irradiation (group III) though the SOD showed a steady decline, it remained insignificantly different as compared with the control at all time intervals. With Podophyllum hexandrum preirradiation treatment (group IV) the SOD was significantly enhanced at 0 h post irradiation (1.249 0.01; P B0.05) and then remained unchanged as compared with the control at 12 and 84 h post irradiation. 3.2.4. Lipid peroxidation In the liver, there was no significant change in the LPx level in groups II and III at all time intervals as compared with the control (taken as 1) though the LPx in the irradiated group was more than the control (Fig. 5). Determination of the LPx in the liver showed a A. Mittal et al. / Journal of Ethnopharmacology 76 (2001) 253–262 259 Fig. 3. Effect of extract (aq.) of Podophyllum hexandrum rhizome administration on GST activity in Swiss albino mice. — Group I; Control administered 0.2 ml vehicle. - - Group II; Podophyllum hexandrum administered 200 mg/kg b.w. i.p. in 0.2 ml vehicle. – - Group III; 10 Gy whole body irradiation. Group IV; Podophyllum hexandrum 200 mg/kg b.w. administered i.p. 2 h prior to 10 Gy whole body irradiation. GST activity of the groups has been represented as a proportion of the control, taken as 1. Data expressed as mean 9standard deviation. significant enhancement at 12 h post irradiation in the preirradiated group treated P. hexandrum group (group IV; 1.449 0.009; PB 0.01) as compared with the control. The LPx in the jejunum and ileum remained significantly unchanged among all the groups at all time intervals (0, 12 and 84 h post irradiation). 4. Discussion There have been several reports on the radioprotective action of plant extracts (Narimanov et al., 1992; Devi and Ganasoundari, 1995) and maximum survival for 30 days in mice against lethal radiation was around 260 A. Mittal et al. / Journal of Ethnopharmacology 76 (2001) 253–262 70%. In our laboratory, P. hexandrum (aqueous) at the dose of 200 mg/kg b.w. (i.p.) was found to render more than 90% survival against whole body lethal irradiation (10 Gy). Therefore, it was necessary to elucidate its mechanism of action by investigating the antioxidant profile which may help expediting its clinical application. P. hexandrum treatment enhanced GST activity in liver at 12 h post irradiation period and it reverted to the normal level after sometime and, therefore, no effect was observed at 84 h period. Probably it might have upregulated the enzymatic gene expression during oxidative stress (Suzuki et al., 1997). Initial detoxification of electrophiles produced by radiation would warrant enhanced antioxidant levels. P. hexandrum was unable to maintain the enhanced activity for an extended period of time. The short life of free radicals would have made the Fig. 4. Effect of extract (aq.) of Podophyllum hexandrum rhizome administration on SOD activity in Swiss albino mice. — Group I; Control administered 0.2 ml vehicle. - - Group II; Podophyllum hexandrum administered 200 mg/kg b.w. i.p. in 0.2 ml vehicle. – - Group III; 10 Gy whole body irradiation. Group IV; Podophyllum hexandrum 200 mg/kg b.w. administered i.p. 2 h prior to 10 Gy whole body irradiation. SOD activity of the groups has been represented as a proportion of the control, taken as 1. Data expressed as mean 9 standard deviation. A. Mittal et al. / Journal of Ethnopharmacology 76 (2001) 253–262 261 Fig. 5. Effect of extract (aq.) of Podophyllum hexandrum rhizome administration on LPx level in Swiss albino mice. — Group I; Control administered 0.2 ml vehicle. - - Group II; Podophyllum hexandrum administered 200 mg/kg b.w. i.p. in 0.2 ml vehicle. – - Group III; 10 Gy whole body irradiation. Group IV; Podophyllum hexandrum 200 mg/kg b.w. administered i.p. 2 h prior to 10 Gy whole body irradiation. LPx level of the groups has been represented as a proportion of the control, taken as 1. Data expressed as mean 9 standard deviation. GST activity redundant at 84 h post irradiation period and, therefore, through the feedback mechanism the GST level could have reverted to normal. At 12 h, the radiation induced decrease in catalase activity in the intestine was enhanced by P. hexandrum treatment. Catalase is known to quench H2O2 (Claiborne, 1985). The enhanced activity of catalase in P. hexandrum treated irradiated animals may explain its radioprotective action to some extent. At 84 h after the irradiation the catalase activity returned to normal as probably no further quenching of H2O2 remained necessary. The effect of preirradiation treatment with P. hexandrum resulted in the increase of the SOD activity in 262 A. Mittal et al. / Journal of Ethnopharmacology 76 (2001) 253–262 liver and intestinal tissues at 12 h post irradiation period. Enhanced SOD activity could have provided reasonable radioprotection to the organism against the highly damaging O− 2 electrophile produced as a consequence of irradiation. The present data on SOD activity explains to some extent the radioprotective effect of P. hexandrum. Different responses of jejunum and ileum to P. hexandrum treatment may be explained due to different physiological roles played during digestion. LPx which also reflects the oxidative damage, was not observed to be enhanced at 12 h post irradiation period. Possibly, the lipid hydroperoxide radicals formed due to irradiation were quenched by 12 h post irradiation since the LPx have been reported to revert back to control values within a shorter time span (Devi and Ganasoundari, 1998). It is also possible that Podophyllum species treatment does not act via LPx quenching and, therefore, we could not find any decrease in LPx level at 12 h post irradiation period. Observations at durations shorter than 12 h after irradiation, may unravel the mode of action of P. hexandrum in this direction. Thus, the present results indicate that Podophyllum species may be manifesting its radioprotective effect on whole body survival by antioxidant mechanism associated with other possible mechanisms both of which need to be investigated further. References Blasko, G., Cordell, G.A., 1988. Recent developments in the chemistry of plant derived anticancer agents. In: Wagner, H., Hiroshi, H., Fransworth, N.R. (Eds.), Economic and Medicinal Plant Research. Academic Press, London. . Claiborne, A.L., 1985. Catalase activity. In: Greenwals, R.A. (Ed.), Handbook of Methods for Oxygen Radical Research. CRC Press, London. Devi, P.U., Ganasoundari, A., 1995. Radioprotective effect of leaf extract of Indian medicinal plant Ocimum sanctum. Indian Journal of Experimental Biology 33, 205. Devi, P.U., Ganasoundari, A., 1998. Modulation of glutathione and antioxidant enzymes by Ocimum sanctum and its role in protection against radiation injury. Indian Journal of Experimental Biology 37, 262 – 268. Goel, H.C., Prasad, J., Sharma, A.K., 1999. Protective effect of Podophyllum against radiation damage. Advances in Radiation Biology and Peace (Suppl.) 2, 27 – 33. Habig, W.H., Pabst, N.V., Jakoby, W.B., 1974. GST, first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130 – 7139. Kumar, I.P., Goel, H.C., 2000. Iron chelation and related properties of Podophyllum hexandrum, a possible role in radioprotection. Indian Journal of Experimental Biology, 38. McCord, J.M., Fridovich, I., 1969. The utility of superoxide dismutase in studying free radical reactions. I. Radicals generated by the interaction of sulfite, dimethyl sulfoxide, and oxygen. Journal of Biological Chemistry 244, 6056 – 6063. Narimanov, A.A., Popova, O.I., Murav’eva, D.A., 1992. Changes in the sensitivity of mice to the action of gamma irradiation by Viscum album L. polysaccharide. Radiobiologiia 32, 868. Potten, C.S., 1990. A comprehensive study of the radiobiological response of the murine (BDF1) small intestine. International Journal of Radiation Biology 58, 925 – 973. Pryor, W.A., 1976. The role of free radical reactions in biological systems. In: Pryor, W.A. (Ed.), Free Radicals in Biology. Academic Press, New York. Schacterle, G.R., Pollack, R.L., 1973. A simplified method for the quantitative assay of small amounts of protein in biologic material. Analytical Biochemistry 51, 654 –655. Sies, H., 1983. In: Sies, H. (Ed.), Oxidative Stress. Academic Press, New York. Sipes, G.I., Gandolfi, A.J., 1983. Biotransformation of toxicants. In: Klaassen, C.D., Amdur, M.O., Doull, J. (Eds.), Toxicology: The Science of Poisons. Macmillan, New York. Suzuki, V.J., Forman, H.J., Sevanian, A., 1997. Oxidants as stimulators of signal transduction. Free Radicals in Biology and Medicine 22, 269 – 285. Journal of Ethnopharmacology 76 (2001) 263– 268 www.elsevier.com/locate/jethpharm Plants used as abortifacients in the Sangmelima region of Southern Cameroon E. Noumi *, N.Y.C. Tchakonang Laboratoire de Biologie Végétale, Ecole Normale Supérieure, Uni6ersité de Yaoundé I, B.P.47 Yaoundé, Cameroon Received in revised form 10 April 2001; accepted 16 April 2001 Abstract Twenty species from 16 plant families used to induce abortion in the Sangmelima region (South Province of Cameroon) are reported with local names, modes of uses and their side effects, if known, with a discussion on pharmacological effect based on published experimental literature. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethnobotany; Abortive plants; Cameroon; Sangmelima 1. Introduction The South Province of Cameroon covers an area of 47 190 km2. It includes the Dja-et-Lobo, Vallée du Ntem, Ocean and Mvila Divisions. Dja-et-Lobo Division is the homeland of the Maka, Fang and Beti. The indigenous people of Sangmelima Region are the Bulu, a subtribe of the Beti tribe. The primary means of livelihood of these people are hunting, gathering and traditional agriculture. Unemployment, low income and poor living conditions lead to sex prostitution work among females. The local sex market has promoted the use of plants as aphrodisiacs (Noumi et al., 1998) for men, abortifacients for women. Abortions, which take place outside of medical facilities, often result in maternal mortality, septicemia, sterility. The objective of the present study was to survey the plants used as abortifacients in the Sangmelima region and to evaluate the pertinence of their stated effect based on a literature review. 2. Study area Sangmelima is the main town of Dja-et-Lobo Division, which consists of six subdivisions, namely, Beng* Corresponding author. E-mail address: jtames@uycdc.uninet.cm (E. Noumi). bis, Djoum, Oven, Mintom, Sangmelima and Zoetele (Fig. 1). Its total population was estimated at 12 105 in 1987, representing 32.3% of the population of South Province, and 11% of the total population of Cameroon (Cameroun/FNUAP, 1987). The Dja, a tributary of Congo River, crosses the region. The study area includes Sangmelima, Zoetele, Djoum, Oven and Bengbis subdivisions. It extends within 11°38% – 13°43% E longitude and 2°11% – 3°27% N latitude and includes a part of the Dja biosphere reserve. The climate is equatorial (Guinean type) with 4 seasons. The average annual rainfall, temperature and humidity are 1654 mm, 24°C and 79.8%, respectively. The forests are evergreen, semi-evergreen and deciduous. 3. Methodology The study was carried out for the period of 1996 – 1997 in 12 localities: Bengbis, Djoum, Oven, Sangmelima, Zoetele, Mvog-Meka, Meyome-Messala and Mekom. The interview data on the name and part of the plants used, the mode of preparation, administration, and side effects were collected during field trips. Young men, older men and women, local herbalists and traditional healers were interviewed, using a structured interview form, after consent was given. Data on the side effects of the abortifacients were obtained from 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 5 2 - 5 264 E. Noumi, N.Y.C. Tchakonang / Journal of Ethnopharmacology 76 (2001) 263–268 users or individuals who had some experience with the use of the plant. Only the plants on which information was in agreement in at least three villages were retained in the present study. Collected plants were identified using published literature (Koechling, 1965; Keraudren, 1969; Fouilloy, 1974; Vivien and Faure, 1985; Biholong, 1986) and authenticated by Dr Achoundong of the National Herbarium of Cameroon (HNC), Ministry of Agriculture. Information was compared with data found in Cameroonian and African medicinal plant Fig. 1. The study area in Dja-et-Lobo Division. 1, Study area; 2, part of the reserve forest of Dja. E. Noumi, N.Y.C. Tchakonang / Journal of Ethnopharmacology 76 (2001) 263–268 books and ethnopharmacological literature (Cousteix, 1961; Kerharo and Adam, 1974; Oliver-Bever, 1982). Voucher herbarium specimens were deposited in the Department of Biological Sciences, Higher Teachers’ Training College, University of Yaoundé I, Cameroon. 4. Results The plants collected and the results of the interviews are listed below in alphabetical order by the plant scientific name. Each entry gives the following data: species (family), voucher specimen number, local name in quotes (locality), purpose of its use and plant part used. Side effects, if stated, are given. The mode of preparation and route of administration are listed, and the relevant phytochemical and pharmacological literature data of some plants, in support of their effectiveness, are also given. Concerning dosages, a spoonful means approximately 10 g, a handful 40– 50 g, a cup 250 ml. Adenia cissampeloides Harms (Passifloraceae); Noumi 983. ‘Nom angakomo’ (Sangmelima, Ebolowa); abortion, fish poison; root, stem. A concentrated macerate of the roots is regularly administered in the vagina until the start of bleeding (3 days maximum). To use, leafy stems can be roasted under hot ashes and transformed into a small ball which is introduced into the vagina. A report said that the foetus died in the uterus and was ejected. Bleeding from uterus was said to be very abundant and the patient became very weak and was shaken for about 2 weeks. Albizia ferruginea (Guill. et Perr.) Benth. (Mimosaceae); Noumi 985. ‘Evuvus-esaka’ (Sangmelima, Yaoundé, Ebolowa); abortion; leaves. Crushed fresh leaves are rolled into small balls, then one ball is introduced into the vagina, preferably before going to bed. The vagina is not cleaned until the onset of bleeding. The treatment is often repeated. It was reported that this treatment provoked irritations, burns, redness and even wounds and infections in the vagina. Basella alba L. (Basellaceae); Noumi 1003. ‘Ele lengue’ (Sangmelima, Yaoundé), ‘lakguegwik’ (Batoufam); oxytocic; fresh leaves. Two spoonfuls of the juice of crushed leaves are drunk. The treatment is repeated as needed. It was indicated that the administration of this drug often led to multiple tears (lacerations) of the vulva. Bidens pilosa L. (Asteraceae); Noumi 977. ‘Mfeg-zoa’ (Sangmelima), ‘Okpadi’ (Yaoundé); oxytocic; leaves. To use, three handfuls of leaves are boiled in 1.5 l of water until the water is reduced to 1 l. Three cups are taken orally to induce oxytocic effect within 3 h. The indicated side effects are the same as those of B. alba. Ceiba pentandra (L.) Gaertn. (Bombacaceae); Noumi 995. ‘Douma’ (Sangmelima, Ebolowa), ‘Dum’ 265 (Yaoundé), ‘Bouma’ (Douala), ‘Djam’ (Bangangte); abortion; leaf, stembark. A petiole is soaked in water for 6 h, until a resinous gum appears at the enlarged extremity, or a fragment of stembark is soaked in water, until a resinous gum is produced at the edges. Then, the petiole is introduced into the vagina such that the gum is in contact with the cervix or the gum is mechanically placed into contact with the cervix. A report said that bleeding of the uterus started after 3 h accompanied by the opening of the cervix. Hospital nurses and illicit abortionists use speculum to introduce the side of the gummy petiole into the cervix. Another report stated that an abundant bleeding occurred for many days, before and after the expulsion of the foetus. Citrus aurantifolia (Christm.) Swingle (Rutaceae); Noumi 998. ‘Ofumbi beti’ (Sangmelima, Yaoundé); abortion; fruit (juice). There are four recipes of drug preparation: (1) 200 ml each of whisky and C. aurantifolia juice are mixed, the mixture is then saturated with powdered rock salt and filtered; the solution is drunk on an empty stomach, preferably before going to bed; one report said that a violent dizziness and headache were experienced for at least 3 days; (2) kitchen salt is dissolved in a cup of water to saturation, then one cup of C. aurantifolia juice is added and the mixture boiled; the warm decoction is then introduced into the vagina before going to bed; irritation and burning sensation are the reported side effects; (3) half a cupful of bush honey and half a cupful of C. aurantifolia juice are mixed; the mixture is then drunk on an empty stomach; light abdominal pains were reported as side effects; (4) a cupful of C. aurantifolia juice is saturated with powdered rock salt, homogenised and filtered; the solution is then drunk on an empty stomach; light abdominal pains were reported as side effects. Preparations of C. aurantifolia juice are also taken when menstruation is 3 or 4 weeks late. In such a case, bleeding is said to begin about 10 h after drinking such preparations. Another report stated that the use of C. aurantifolia leads to menstrual cycle disorders. Desmodium ramosissimum G. Don (Fabaceae); Noumi 978. ‘Owondo-bekon’ (Sangmelima, Ebolowa, Yaoundé); abortion; leaves. Two handfuls of leaves are macerated in 1 l of water, then, a cupful is drunk once a day for 2 or 3 days. This recipe was claimed to be effective when added with B. alba or B. pilosa. Drymaria cordata Will. ex Roem. et Schult. (Caryophyllaceae); Noumi 1002. ‘Oyaya’ (Sangmelima, Ebolowa), ‘Lomtokia’ (Bayangam); abortion. Fresh leaves are heated under hot ashes and rounded into a small ball which is introduced into the vagina, preferably before going to bed. A report said that bleeding of the uterus began 12 h after insertion, and burning sensation and irritations were experienced. Erythrophleum guineensis G. Don (Caesalpiniaceae); Noumi 990. ‘Elon’ (Sangmelima, Ebolowa, Yaoundé), 266 E. Noumi, N.Y.C. Tchakonang / Journal of Ethnopharmacology 76 (2001) 263–268 ‘Tom’ (Bangangte); abortion, stembark. A piece of the stembark is placed at the bottom of the panties as a pad. It was reported that the contact with the vulva provokes the expulsion of the foetus, even if it is 6 months old. Other reported side effects included irritation and the reddening of the vulva, and that the expulsion of the foetus was accompanied by violent and persistent contractions. Maesopsis eminii Engl. (Rhamnaceae); Noumi 1005. ‘Nkala’ (Sangmelima), ‘Nkangela’ (Yaoundé), ‘Lando’ (Abong-Mbang); abortion; stembark. A crushed piece of stembark is macerated for 2 h in a cup of palm wine and the solution taken orally. The authors entered the abortionist’s house just when a young pregnant girl drank the maceration, and bleeding of the uterus began 2 h later. Manihot esculenta Crantz (Euphorbiaceae); Noumi 988. ‘Mbom’ (Sangmelima, Yaoundé), ‘Nkwamba’ (Douala), ‘Kasinga’ (Bangangte); abortion; leaves. A fresh leaf of the bitter variety is harvested and the enlarged end of the petiole is introduced into the vagina such that the white latex comes in contact with the cervix. Bleeding was claimed to start 6 h after. The preparation should be maintained until the foetus has been expulsed. Momordia charantia L. (Cucurbitaceae); Noumi 980. ‘Eyel zom’ (Sangmelima, Yaoundé); abortion; seeds, leaves. To produce abortion, ten seeds are ground and the paste divided into three small balls using saliva; a ball is then introduced into the vagina, once a day, preferably before going to bed. It was reported that the application of M. charantia preparation resulted in a high fever; during this time no drug was allowed to be administered; a putrid vaginal discharge resulted for 2– 4 days. Musa sp. (Musaceae); Noumi 986. ‘Ekoan’ (Sangmelima, Akonolinga, Yaoundé); oxytocic effect; young plant. A young plant is cut and the juice drunk by a woman in labour to accelerate delivery. Its claimed effect is to empower the drug effects of other plants. Musanga cecropioides R. Br. (Cecropiaceae); Noumi 979. ‘Assam’ (Sangmelima, Ebolowa), ‘Asseng’ (Yaoundé), ‘Bossengue’ (Douala), ‘Lisseng’ (Bangangte); oxytocic effect; stipule protecting young leaves. About four stipules are macerated in 1 l of water, then, the maceration is drunk as needed. This recipe is claimed to empower the drug effects of other plants. Nicotiana tabacum L. (Solanaceae); Noumi 981. ‘Tah’ (Sangmelima, Ebolowa, Yaoundé), ‘Depah’ (Bafoussam, Foumban); abortion; leaves. 3/4 cup of hot ashes and 1/4 cup of rock salt are dissolved in four cupfuls of water, then five to ten crushed leaves of N. tabacum are added and left to stand for 4 h. The mixture is homogenized and filtered and the filtrate is injected into the vagina before going to bed. It was reported that the intoxication due to N. tabacum is terrifying in two ways: exciting and paralyzing. The patients would go into a coma for many days and some die in the process. Ocimum gratissimum L. (Lamiaceae); Noumi 1001. ‘Mesep’ (Yaoundé, Sangmelima, Abong-Mbang), ‘Masepo’ (Douala); oxytocic effect; leaves. Three handfuls of leaves are boiled in 1.5 l of water and the solution is concentrated to 1 l. This is drunk, three cups thrice a day. Abortionists also add O. gratissimum leaves to other abortifacient plants to reinforce their effects. Pentaclethra macrophylla Benth. (Mimosaceae); Noumi 997. ‘Ebae’ (Sangmelima, Ebolowa, Yaoundé); abortive effect; fruits, stembark. A handful of the stembark is boiled in 2 l of water and the solution is concentrated to 1 l. This is drunk, three cups thrice a day. It was reported that the effect is very painful accompanied by agonies. Persea americana Mill. (Lauraceae); Noumi 996. ‘Fia’ (Sangmelima, Yaoundé); leaves. Three handfuls of leaves are boiled in 1 l of water, the liquid drunk, one cup thrice a day. It was reported that the effect involves abundant and painful bleeding. Piptadeniastrum africanum (Hook. f.) Brenan (Mimosaceae); Noumi 993. ‘Atui’ (Sangmelima, Yaoundé); oxytocic effect; stembark. A decoction is prepared from a handful of stembark in 2 l of water and 250 ml is applied as an enema at the onset of labour, which may be repeated, if necessary. The remainder of the decoction is used for external massage of the pregnant woman’s belly, from top to bottom. The effect of P. africanum is claimed to reinforce the abortive effects of other plants. Saccharum officinarum L. (Poaceae); Noumi 989. ‘Nkog’ (Sangmelima, Yaoundé); abortion; stem. The juice is sucked, which in large amounts increases the body’s content of sucrose which is claimed to have a dilating action on the cervix. A report said that bleeding of the uterus started with pains and provoked disorders in the pregnancy condition, but the expulsion of the foetus is not a certainty. The patient is said to suffer intensely for many months with anemia and the risk of dying. 5. Discussion As a result of our field studies among the Bulu, Fang and Maka, three ethnic groups of Sangmelima, 20 plant species with abortive properties were collected and documented. Much of the information reported in this communication, particularly on A. cissampeloides, A. ferruginea, C. pentandra, D. ramosissimum, D. cordata, M. eminii, M. esculenta, P. macrophylla and S. officinarum was found to be new to the literature of Cameroon medicinal plants (Cousteix, 1961; Chenu and Ake Assi, 1992; Chenu et al., 1992; Noumi et al., 1998, E. Noumi, N.Y.C. Tchakonang / Journal of Ethnopharmacology 76 (2001) 263–268 1999). Likewise, the use of A. cissampeloides, P. macrophylla, M. charantia, and M. cecropioides is similar to its use by other African people (Raponda-Walker and Sillans, 1961; Bouquet, 1969; Bouquet and Debray, 1974; Ake Assi et al., 1978, 1985), thus lending support to their use in the abortion practices. The experimental literature gives scientific basis for the use of plant species as abortifacients. Some act by their toxicity. For example, cyanogenetic principles lead to emetic chronic intoxications, abdominal troubles and collapse. Thus, A. cissampeloides contains cyanogenetic glucoside (Watt and Breyer-Brandwijk, 1962), C. pentandra, a cyanogenetic acid (Watt and Breyer-Brandwijk, 1962), M. esculenta, hydrogen cyanide (Wood, 1965) and a cyanogenetic glucoside (linamarine; Freize, 1938), while E. guineensis erythrophleine, a frog’s cardiac toxin (Kerharo and Adam, 1974). N. tabacum leaves contain nicotine which can kill cold-blooded animals (insects, frogs) and toxic to some warmblooded animals (dog, cat, pig and human) (Planchon and Bretin, 1946). Henry (1949) reported that the seed husk of P. macrophylla contains paucine, a toxic substance. A 10% stembark decoction provokes, on isolated guinea-pig uterus, durable and intensive contractions (Correia Da Silva et al., 1960). Other plant species act by their pharmacodynamic properties and confirm their effect in inducing an abortion. According to Jamwall and Anand (1962), root preparations of M. charantia have an arbotive action, while Chopra et al. (1938) reported a case of abortion during 7th month of pregnancy with a root decoction. Pousset (1992) mentioned the presence of an essential oil with thymol and engerol in O. gratissimum, as well as substances which also provoke contraction of guinea-pig’s ileum and rat’s intestine, raising the blood pressure. Pharmacodynamic tests conducted by Feng et al. (1962), on aqueous and alcoholic extracts of P. americana gave the following results: toxicity in the mouse by the intraperitoneum route at doses between 0.5 and 1 g of dry leafy stems; a spasm effect on isolated intestine of a guinea pig at between 0.01 and 0.1 mg, and on the rat’s uterus at 0.01 mg. As to plant sources, they are generally obtained from wild populations. However some species, such as, B. alba, C. aurantifolia, M. esculenta, Musa sp., N. tabacum, P. americana and S. officinarum are grown by many people in their homestead (Erdelen et al., 1999). During fieldwork it was surprising to notice that men, women and young people could equally identify abortive plants, and that they all praised the abortive power of M. charantia, nicknamed ‘Miss efficient’. The practice of sexual relation by adolescent belongs to a syndrome of behavioral risk. During that period, adolescents wish to be recognized for their independence and their opinion. Because sexual maturity is reached before psychological maturity and they do not under- 267 stand the risk due to their sexuality, they have no appreciation for contraceptive practices (Kamtchouing et al., 1997); as a result, girls are more likely than boys to drop out of school prematurely (Lloyd et al., 1998). The first sex acts occur without contraceptive protection (Mensch et al., 1999). Those first relations can determine the future reproductive health of adolescents through undesired pregnancies and sexually transmitted diseases (including AIDS) (Leke, 1989). In Sangmelima, girls turn to illicit abortion. The sepsis, the hemorrhages and the traumatism that affect vital organs are their frequent complications. They also use a variety of other materials to provoke abortion, including kerosene, fork, hook, tablets of nivaquin, and fermented urine saturated with kitchen salt applied to the vagina. They swallow salted water in saturation, mixture of water, and rock salt and egg. Many cases of illicit abortion end in a hospital. In spite of the availability of intensive care, the power of antibiotics, the help of blood transfusion, some women die from elements used to abort. Damages to reproductive organs have been reported in the literature (Dixon Mueller and Wasserheit, 1996; Leke, 1998). Drugs destroy many parts of the genital system and lead to infertility. Deadly accidents are frequent in spite of the prohibitions of the law and religious action of the prelates in discouraging the practice. Consequently, when the population of Cameroon passed from 5 836 000 in 1970 to 10 493 655 in 1987 (a 79.8% population growth), for the same period, the population of Sangmelima region only increased from 108 000 to 121 059 inhabitants (a 12% population growth; MINPAT, 1985; Cameroun/FNUAP, 1987). Practices in which plants are used to produce sterility, death, and low population growth must be prohibited. The present study has raised a patch of veil on a disturbing situation: the issues of sex in the Sangmelima region and its disastrous consequences. Its inhabitants need a special sex education. Acknowledgements The authors express their thanks to all informants who participated in this survey, who generously provided information on abortive plants. They also thank Mr Oyono Jean Marc and Mr and Mrs Kwayep Jacop, for their hospitality at Sangmelima, and Mrs Sitze Esther for her cooperation during fieldwork and for her translation services. References Ake Assi, L., Abeye, J., Guinko, S., Giguet, R., Bangavou, X.J., 1978. Contribution à l’Identification et au Recencement des Plan- 268 E. Noumi, N.Y.C. Tchakonang / Journal of Ethnopharmacology 76 (2001) 263–268 tes Utilisées dans la Médécine Traditionnelle et la Pharmacopée en Empire Centrafricaine. Edition ACCT, Paris, p. 139. Ake Assi, L., Abeye, J., Guinko, S., Giguet, R., Bangovou, X., 1985. Contribution aux Etudes Ethnobataniques et Floristiques en République Centrafricaine. Edition ACCT, Paris, p. 139. Biholong, M., 1986. Contribution à l’Etude de la Flore du Cameroun: les Astéracées. Thèse Doctorat d’Université. Université de Bordeaux III, p. 365. Bouquet, A., 1969. Féticheurs et Médécines Traditionnelles du Congo (Brazzaville). Mémoire ORSTOM, 36, Paris, p. 282. Bouquet, A., Debray, M., 1974. Plantes Médécinales de la Côte d’Ivoire. Edition ORSTOM, Paris, p. 231. Cameroun/FNUAP, 1987. Demo 87. Edition SOPECAM Yaoundé, p. 30. Chenu, J., Ake Assi, L, 1992. Plantes Médicinales Tropicales et Camerounaises. Tome 1. Edition Ren-Ronche Berrebi Véronique, Barcelone, p. 222. Chenu, J., Oury, R., Lavegne, R., Seguele, J., Mumbe, J., 1992. Plantes Médicinales Tropicales et Camerounaises. Tome 2. Edition Haricot, Moubain, p. 134. Chopra, R.N., Chopra, I.C., Handra, K.L., Kapur, L.D., 1938. Chopras Indigenous Drugs of India, vol. 1, 2nd edition. Dhur and Sons Edition, Calcutta. Correia Da Silva, A.C., Correia Alves, A., Nogueira Prista, L., 1960. Nota previa acerca da acçao da casca de Pentaclethra macrophylla Benth. sobre do utero de cobaia isolado. Anais da Faculdade de Farmacia do Porto 21, 3 – 7. Cousteix, P.J., 1961. L’Art et la Pharmacopée des Guérisseurs Ewondo (Région de Yaoundé). Recherches et Etudes Camerounaises, Yaoundé, p. 87. Dixon Mueller, R., Wasserheit, J., 1996. La culture du Silence: Les Infections du Tractus Génital chez les Femmes des Pays du Sud. International Women’s Health Coalition, New York. Erdelen, W.R., Adimihardja, K., Moesdarsono, H., Sidik, 1999. Biodiversity, traditional medicine and the sustainable use of indigenous medicinal plants in Indonesia. Indigenous Knowledge and Development Monitor 7(3), 3 – 6. Feng, P.C., Haynes, L.J., Magnus, K.E., Plimmer, J.R., Sherratt, H.S.A., 1962. Pharmacological screening of some west Indian medicinal plants. Journal of Pharmacy and Pharmacology 14, 556 – 561. Fouilloy, R., 1974. Lauracées, Myristicacées, Monimiacées, vol. 18. Flore du Cameroun, Yaoundé, p. 120. Freize, F.W., 1938. Therapeutically utilizable constituents of Manihot utilissima and Mercurialis annua or perennis. Chemical Abstract 32, 2288. Henry, T.A., 1949. The Plant Alkaloids, vol. 1, 4th edition. Churchill, London. Jamwall, K.S., Anand, H.K., 1962. Preliminary screening of some reported abortifacient indigenous plants. Indian Journal of Medical Research 24, 218 –220. . Kamtchouing, P., Takougang, I., Ngoh, N., Yakam, I., 1997. Sexualité des adolescents en milieu scolaire à Yaoundé. Contraception, Fertility and Sex 25 (10), 11 –20. Keraudren, M., 1969. Cucurbitacées, vol. 6. Flore du Cameroun, Yaoundé, p. 192. Kerharo, J., Adam, J.G., 1974. La Pharmacopée Sénégalaise Traditionnelle, Plantes Médicinales et Toxiques. Edition Vigot-Frères, Paris, p. 1011. Koechling, J., 1965. Scitaminales: Musacées, Strélitziacées, Zingibéracées, Cannacées, Marantacées, vol. 4. Flore du Cameroun, Yaoundé, p. 162. Leke, R.J.L., 1989. Commentary on unwanted pregnancy and abortion complications in Cameroon. International Journal of Gynecology and Obstetrics (Supplement) 3, 33 – 35. Leke, R.J.L., 1998. Sexualité et Santé Reproductrice durant l’Adolescence en Afrique avec une Attention Particulière au Cameroun. Les Adolescentes et l’Avortement. Ediconseil INC Boucherville, Quebec, p. 394. Lloyd, C.B., Mensch, B.S., Clark, Wesley, H., 1998. The Effects of Primary School Quality on the Educational Participation and Attainment of Kenyan Girls and Boys. Working Papers, Population Council No. 116, p. 49. Mensch, B.S., Clark, W.H., Lloyd, C.B., Erulkar, A.S., 1999. Premarital Sex and School Dropout in Kenya: Can Schools Make a Difference? Working Papers, Population Council, No. 124, p. 51. MINPAT (Ministère du Plan et de l’Aménagement du Térritoire), 1985. Annuaire Statistique du Cameroun, édit. SOPECAM, Yaoundé, p. 44. Noumi, E, Amvan Zollo, P.H., Lontsi, D., 1998. Aphrodisiac plants used in Cameroon. Fitoterapia 69 (2), 125 – 134. Noumi, E., Houngue, F., Lontsi, D., 1999. Traditional medicines in primary health care: plants used for the treatment of hypertension in Bafia, Cameroon. Fitoterapia 70, 134 – 139. Oliver-Bever, B., 1982. Medicinal plants in tropical West Africa. 1. Plants acting on the cardiovascular system. Journal of Ethnopharmacology 5, 1 – 71. Planchon, L., Bretin, P., 1946. Précis de Matières Médicales, vol. 2. Maloire, Paris. Pousset, J.L., 1992. Plantes Médicinales Africaines; Possiblités de Développement. Edition ACCT, Paris, p. 159. Raponda-Walker, A., Sillans, R., 1961. Plantes Utiles du Gabon. Edition Paul Lechevalier, Paris, p. 614. Vivien, J., Faure, J.J., 1985. Arbres des Forêts denses d’Afrique Centrale. Edition ACCT, Paris, p. 565. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern and Eastern Africa, vol. 1, 2nd edition. Churchill, London, p. 1457. Wood, T., 1965. The toxic and nutritional qualities of Cassava. African Pharmacology 7, 2 – 4. Journal of Ethnopharmacology 76 (2001) 269– 277 www.elsevier.com/locate/jethpharm Effect of an antidiabetic extract of Catharanthus roseus on enzymic activities in streptozotocin induced diabetic rats Som Nath Singh *, Praveen Vats, Shoba Suri, Radhey Shyam, M.M.L. Kumria, S. Ranganathan, K. Sridharan Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi 110054, India Received 9 October 2000; received in revised form 1 April 2001; accepted 24 April 2001 Abstract Hypoglycemic activity was detected in dichloromethane:methanol extract (1:1) of leaves and twigs of Catharanthus roseus (family Apocynaceae), a traditionally used medicinal plant, using streptozotocin (STZ) induced diabetic rat model. Extract at dose 500 mg/kg given orally for 7 and 15 days showed 48.6 and 57.6% hypoglycemic activity, respectively. Prior treatment at the same dose for 30 days provided complete protection against STZ challenge (75 mg/kg/i.p.× 1). Enzymic activities of glycogen synthase, glucose 6-phosphate-dehydrogenase, succinate dehydrogenase and malate dehydrogenase were decreased in liver of diabetic animals in comparison to normal and were significantly improved after treatment with extract at dose 500 mg/kg p.o. for 7 days. Results indicate increased metabolization of glucose in treated rats. Increased levels of lipid peroxidation measured as 2-thiobarbituric acid reactive substances (TBARS) indicative of oxidative stress in diabetic rats were also normalized by treatment with the extract. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antidiabetic activity; Catharanthus roseus; Streptozotocin; Glucose metabolism; Glutathione; Lipid peroxidation 1. Introduction Diabetes mellitus is a metabolic disease as old as mankind and its incidence is considered to be high (4– 5%) all over the world (Pickup and Williams, 1997). In spite of the introduction of hypoglycemic agents, diabetes and related complications continue to be a major medical problem. Since time immemorial, patients with non-insulin requiring diabetes have been treated orally in folk medicine with a variety of plant extracts. In India a number of plants are mentioned in ancient literature (Ayurveda) for the cure of diabetic conditions known as ‘madhumeha’ and some of them have been experimentally evaluated and the active principles isolated (Chopra et al., 1956; Rajashekharan and Tuli, 1976; Chattopadhyay et al., 1993; Pugazhenthi and Murthy, 1996; Chattopadhyay, 1999; Joy and Kuttan, 1999) * Corresponding author. Fax: + 91-11-3932869. E-mail address: shoba72@yahoo.com (S.N. Singh). Cajtharanthus roseus belonging to family Apocynaceae is known with various names in India and all over the world. Hot water decoction of the leaves and/or the whole plant is used for treatment of diabetes in several countries i.e. Brazil, Cook Islands, Dominica, England, Jamaica, Mozambique, Pakistan, Taiwan, Thailand and West Indies (Don, 1999). In India seven flowers/leaves are used at a time whereas in The Cook Islands 18 leaves boiled in a kettle of water and in The West Indies roots of plants infused in whiskey are used traditionally. Preliminary reports indicate blood glucose lowering activity in alcoholic extract of leaves (Ghosh and Gupta, 1980; Chattopadhyay et al., 1991, 1992). In the present study we have evaluated antidiabetic activity of a dichloromethane – methanol (DCMM) extract of leaves and twigs (L&T) and its effect on enzymes of carbohydrate metabolism to find out possible mechanism of hypoglycemic action. In addition to this the effect of extract was evaluated on glutathione levels, related enzymes and lipid peroxidation as oxidative stress is known to occur in diabetes. Effect of extract on other enzymes of pharmacological importance i.e. as- 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 5 4 - 9 270 S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269–277 partate aminotransferase (AST), alanine aminotransferase (ALT), acid and alkaline phosphatases were also evaluated. 2. Materials and methods in blood sugar level in comparison to that of untreated controls was taken as antidiabetic activity. Five animals at a time were used and experiments were repeated twice in identical conditions with same number of control animals making a total of 8– 10 animals for each study parameter. Changes in body weight of untreated controls and experimental animals were recorded at the same time, i.e. in fasting state. 2.1. Plant material Flowering twigs of C. roseus were collected after authentication by Dr. J. Yadava, Defence Agricultural Research Laboratory, Pithoragarh. Voucher specimen of plant sample is available in herbarium file of the institute (HERB/DIP 99/Vinca 13). Collected plant material was washed thoroughly with water and dried in the shade. Dried leaves, twigs and flowers were made into powder in a grinder and were extracted with 10 volumes of dichloromethane:methanol (1:1) by continuous stirring for 48 h. After 48 h the solvent was filtered and remaining parts were re-extracted with the same volume of solvent. Extracts were pooled and evaporated to dryness at 60 °C. The yield of this extract was 17% on dry weight basis. Extracts thus prepared were stored at 4 °C. For preparation of aqueous crude extract, known quantities of wet leaves and twigs were homogenized with distilled water in a mixer grinder and centrifuged at 1000×g for 15 min, supernatants were freeze dried and used as crude extract (yield 20– 24% of wet wt). 2.2. Experimental animals and induction of diabetes Male Sprague–Dawley rats weighing 250– 300 g were used in the present study. Animals were maintained at 2292 °C with 12 h light and dark cycle, fed on standard pellet diet supplied by Lipton India Ltd. Animals had free access to diet and water. After initial determination of 12 h fasting blood glucose levels (blood drawn from retro orbital plexus) animals were given single i.p. injection of streptozotocin at dose 75 mg/kg (freshly dissolved in physiological saline) and blood glucose was monitored after 24 h and thereafter at weekly intervals after 12 h fasting in case of all the experiments reported here. 2.3. Determination of antidiabetic acti6ity and change in body mass Animals showing blood glucose levels \200 mg/dl 48 h after STZ treatment were selected for study. Animals were treated orally for different duration at different doses of test extracts suspended in distilled water. Fasting blood glucose levels were monitored weekly along with untreated controls. Any reduction 2.4. Prophylactic acti6ity against STZ challenge For determination of prophylactic activity, normal animals were treated with test extracts (aqueous extract and DCMM) at a dose of 500 mg/kg (p.o.) for 30 days. After monitoring blood glucose levels on day 30, animals were challenged to streptozotocin at a dose of 75 mg/kg ×1 (i.p.). Blood glucose levels after 48 h were monitored for induction of hyperglycemia along with untreated control animals (fed with distilled water) challenged with STZ. 2.5. E6aluation of effect on biochemical 6ariables For in vivo study of effect of DCMM extract on biochemical variables, diabetic animals were treated (n= 8-10) with the extract at a dose of 500 mg/kg for 7 consecutive days which showed around 50% hypoglycemic activity. Diabetic animals with similar elevated blood glucose levels and normal animals were kept as diabetic and normal controls respectively (n = 8– 10). All the three groups were sacrificed by cervical dislocation on day 8 post treatment after fasting overnight. Blood was drawn from the heart. The liver was removed, washed with chilled saline, small weighed portion of the liver were processed for determination of glycogen and glutathione immediately after their removal. Ten percent homogenate (w/v) of liver was prepared in 150 mM KCl using Potter– Elvehjem homogenizer at 4 °C. Two milliliter aliquots of crude liver homogenates were used for assay of lipid peroxidation and rest of the homogenates were centrifuged at 3000×g for 15 min at 4 °C and supernatants were divided into aliquots and frozen at −20 °C until assayed for different enzymes. Blood plasma was recovered by centrifugation at 1000 g for 10 min at 4 °C. Effect of extract on activities of different enzyme of glucose metabolism were evaluated in 6itro using liver homogenate/plasma of normal animals as enzyme source. Extract was dissolved in methanol (10 mg/ml). An aliquot of 10–30 ml as required was added to the assay mixture to give a final concentration of 100 mg/ml. Samples were incubated for 10 min at 37 °C before assay. All assays were performed in triplicate with suitable amount of enzyme and assay mixtures S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269–277 containing solvent (methanol) in place of extract served as control. Blood glucose was estimated by method of Nelson as described by Ashwell (1957). Effect on oral glucose tolerance was evaluated by feeding 10 g/kg glucose after 3.5 h of treatment with 500 mg/kg DCMM extract (Chattopadhyay et al., 1991). Plasma fructosamine was determined by the method of Johnson et al. (1982) with slight modification. In brief, reduction of nitroblue tetrazolium (NBT) was monitored at 530 nm using an assay mixture containing 50 ml plasma, 1.75 ml Tris buffer pH 10.6 (pH adjusted with 0.1 N NaOH) and 0.2 ml of NBT (1 mg/ml). Liver glycogen was measured by method of Montgomery (1957). The specific activities of enzymes viz. succinate dehydrogenase (EC 1.3.99.1) (Slater and Bonner, 1952), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), malate dehydrogenase (EC 1.1.1.37) (Shonk and Boxer, 1964), glucokinase (EC 2.7.1.2) (Parry and Walker, 1966), glycogen synthase (EC 2.4.1.11) (Leloir and Goldenberg, 1962), lactate dehydrogenase (EC 1.1.1.27), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) (King, 1965), acid phosphatase (EC 3.1.3.2), alkaline phosphatase (EC 3.1.3.1) (Lowry et al., 1954), g-glutamyl transpeptidase (EC 2.3.2.2) (Orlowsky and Meister, 1963), glutathione S-transferase (EC 2.5.1.18) (Habig and Jakoby, 1981) were estimated with standard colorimetric and photometric techniques. Glutathione in blood and liver was estimated colorimetrically by using DTNB (Ellman, 1959) and lipid peroxidation in crude homogenates as 2-thiobarbituric acid reactive substances (TBARS) by method of Utley et al., (1967). Protein content of enzyme samples were determined colorimetrically (Lowry et al., 1951). 2.6. Statistical analysis Values are expressed as mean9 SEM. Unpaired Student t-test was used for statistical comparison. In case of in vivo studies comparison were made between normal and diabetic, diabetic versus diabetic treated animals. Changes were considered significant if the P-value was less than 0.05. Table 1 Effect of DCMM extract of C. roseus (leaves and twigs) on blood glucose levels (mg/dl) of STZ induced diabetic rats Dose (mg/kg p.o.×days) 500×7 500×15 Blood glucose Control diabetic Treated diabetic 291.36 9 14.6 237.98 9 19.17 149.879 25.99a 100.80 9 16.9a Values are expressed as mean 9 SEM. a PB0.001 in comparison with control diabetics. 271 3. Results 3.1. Antidiabetic and prophylactic acti6ity Diabetic rats treated with crude aqueous extract at oral dose of 1 g/kg for 21 days showed 20.2% reduction in blood glucose in comparison to untreated diabetic rats. Blood glucose levels of control diabetic and treated animals were 315915.7, 215.3925.0 mg/dl respectively (P B0.05). DCMM extract showed 57.6 and 48.6% antidiabetic activity (P B0.001) at dose 500 mg/kg given orally for 15 and 7 days respectively (Table 1). Changes in body weight and nitroblue tetrazolium (NBT) reduction assay a measure of fructosamine in plasma are depicted in Fig. 1. Normal animals treated with extract at dose 500 mg/kg (p.o.) and given oral glucose 10 g/kg showed delay in peak blood glucose levels by 30 min in comparison to untreated rats (Fig. 2). Prior to treatment for 30 days with aqueous and DCMM extract at dose of 500 mg/kg (p.o.) before STZ challenge provided 27.7 and 100% protection, respectively, in comparison with control animals which received saline daily (Table 2). 3.2. Effect on biochemical 6ariables 3.2.1. Effect on glucose metabolism In vivo effect of treatment with DCMM extract at dose 500 mg/kg for 5 days on glycogen levels and glucose metabolizing enzymes are given in Tables 3 and 4. In vitro effect of DCMM extract at conc. 100 mg/ml in assay mixture on enzymic activities are given in Table 5. There was significant stimulation of glucokinase activity whereas other enzymic activities remain almost unaffected. 3.2.2. Effect on glutathione and other biochemical 6ariables No significant change in glutathione (GSH) levels of diabetic, diabetic DCMM treated animals were observed in comparison to normal animals. Lipid peroxidation was 22.7% more in diabetic animals in comparison with normal animals but was reduced in animals treated with extract at 500 mg/kg (p.o.)×7 days. Enzymic activities of g-glutamyl transpeptidase, glutathione S-transferase (GST) and enzymes of toxicological importance, i.e. AST, ALT, acid and alkaline phosphatase activities are given in Table 6. 4. Discussion For the study of antidiabetic agents, STZ induced hyperglycemia in rodents is considered to be a good 272 S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269–277 Fig. 1. Effect of DCMM extract on change in body weights and plasma NBT reduction. Values are mean (n =10), * PB 0.05, in comparison with initial and normal in case of body weight and NBT reduction respectively. † P B0.05 in comparison with diabetic. preliminary screening model (Ivorra et al., 1989) and is widely used. STZ, N-[methylnitrocarbamoyl]-D-glucosamine is a potent methylating agent for DNA and acts as nitric oxide donor in pancreatic cells. b cells are particularly sensitive to damage by nitric oxide and free radicals because of their low levels of free radical scavenging enzymes (Lukic et al., 1998; Spinas, 1999). Leaves and flowers of C. roseus are used traditionally by diabetic patients in India and are taken as water decoction. Due to this reason crude aqueous extract was given orally at dose of 1 g/kg (generally used oral dose for primary testing of crude products at our laboratory) for a period of 21 days and 20.2% glucose lowering activity was observed. Although the activity is apparently low, it has significance for further studies as STZ treated animals represent chronic model of IDDM and marked destruction of pancreatic structures was observed (slide not shown). Significant hypoglycemic activity was detected in DCMM extract of leaves and twigs (Table 1) along with 100% prophylactic action against STZ challenge during primary screening and need special attention (Table 2). DCMM extract was better tolerated in primary toxicity study and oral LD50 was found more than 5000 mg/kg body weight in rats. Other studies on hydroalcoholic extracts of leaves and twigs also indicate fairly higher margin of safety (Chattopadhyay et al., 1992; Chattopadhyay, 1999) Nitroblue tetrazolium (NBT) reduction assay as a marker of fructosamine levels (protein– ketoamine product) also showed decrease in animals treated with DCMM extract (Fig. 1). This assay is a measure of long term glycemic control (Baker et al., 1985). The extract was evaluated in vitro as well as in vivo on several biochemical variables. Glycogen levels in liver which were low in diabetic animals, increased several folds in DCMM treated diabetic animals (Table 3). Although glycogen synthetase activity decreased in diabetic animals significantly, the treatment however could not normalize activity. Glycogen content of normal animals in fasting stage was only slightly higher than diabetic animals and this may be due to degradation of glycogen to maintain normal blood glucose levels, whereas glycogen levels in diabetics were found to be very low despite high blood glucose levels possibly due to lower levels of glycogen synthase activity. Accumulation of glycogen in liver of treated animals is somewhat similar to that reported during insulin therapy. When insulin therapy is instituted, hepatic glycogen accumulation begins rapidly and glycogen content rises to 300% of normal levels within 24 h and this inordinate accumulation of glycogen may account for upto 60% of dry liver weight in diabetic animals (Osborn et al., 1953; Spiro et al., 1958; Steiner and King, 1964; Anderson, 1974). Activity of glucose-6-phosphate dehydrogenase, the first regulatory enzyme of pentose phosphate pathway was found to be decreased in diabetic animals and increased in DCMM extract treated animals, the activity was higher in comparison to untreated diabetic animals indicating improvement in glucose utilization by this pathway (Table 4). S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269–277 273 Fig. 2. Effect of DCMM extract on blood glucose levels of normal rats after feeding glucose (10 g/kg). Values are mean (n = 8), P B 0.05 in comparison with control. Table 2 Prophylactic activity of C. roseus extracts against STZ induced diabetes in rats (n = 10) Blood glucose (mg/dl) Untreated control Aqueous extract Dichloromethane: methanol extract Initial 48 h Post STZ challenge 74.14 9 4.05 85.00 9 3.11 70.50 9 4.81 350.09 10.44 336.56 9 7.94 91.769 1.77a No. of animals diabetic/total % Animals showing hyper-glycemia % Protection in comparison to untreated control 10/12 6/10 0/12 83 60 0 – 27.7 100 Animals were treated with extract at dose of 500 mg/kg, p.o. for 30 consecutive days prior to STZ challenge. Values expressed as mean 9SEM (n = 10). a PB0.001 in comparison with untreated control group. S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269–277 274 Table 3 Effect of DCMM extract of C. roseus on liver glycogen levels and glycogen synthase activity of STZ induced diabetic rats Group Liver glycogen (mg/g wet tissue) Normal 4.84 90.74 4.37 9 0.93 Diabetic Diabetic treated (500 22.19 9 6.69b mg/kg P.O. x 7 days) unit B and isozymes 2,3 and 5 in STZ induced diabetes in Chinese hamsters is reported. Increase in lactate dehydrogenase activity is also reported in hypoglycemic rats (Chang et al., 1977; Lemieux et al., 1984). Malate dehydrogenase plays an important role in the citric acid cycle by providing oxaloacetate for the formation of citrate with acetyl-CoA for generating malate which can feed the cytosolic gluconeogenic pathway (Murray et al., 1998). In the present study, decrease in malate dehydrogenase in liver and plasma of diabetic animals was observed. Levels increased significantly in liver of treated animals whereas in plasma it reached almost normal level. Succinate dehydrogenase activity which was decreased to almost half in diabetic animals was found to be increased in treated animals and levels were even more than normal (Table 5). Increase in succinate and malate dehydrogenase activities in treated animals indicates better utilization of energy yielding intermediates by TCA cycle. These enzymes are reported to be inhibited in tissues of diabetic animals in several studies (Chen and Ianuzzo, 1981; Lemieux et al., 1984; Ianuzzo and Armstrong, 1976). Our results indicate that treat- Glycogen synthase (mmol UDP formed/min/mg protein) 3.01 90.19 0.77 90.18a 1.23 9 0.19a, NS Values expressed as mean 9 SEM (n = 8). a PB0.001 in comparison with normal. b PB0.05 and NS-not significant when compared with diabetic. Activity of glucokinase, the first regulatory enzyme of glycolytic pathway was also increased by extracts in vitro (Table 5). Decreased activity of glucokinase is reported in diabetes (Storey and Bailey, 1978; Chang et al., 1977). We found no change in lactate dehydrogenase activity in liver of diabetic animals in the present study (Table 5). Increase in lactate dehydrogenase subTable 4 Effect of DCMM extract of C. roseus on enzymes of glucose metabolism Enzyme Specific activity Glucose-6-phosphate dehydrogenase Liver (nmol NADP+ reduced/min/mg protein) Lactate dehydrogenase Liver (nmol pyruvate formed/min/mg protein) Succinate dehyrogenase Liver (nmol Pot. Ferricyanide reduced/min/mg protein) Malate dehydrogenase Liver (mmol NADH oxidized/min/mg protein) Plasma (mmol NADH oxidized/min/ml) Normal Diabetic Diabetic treated 19.33 9 1.31 14.329 1.09** 15.46 90.76*a,NS 63.16 9 2.58 65.49 9 2.84 62.809 5.93 4.16 9 0.40 2.92 9 0.39* 5.84 9 0.68††b 1.56 9 0.13 1.16 90.07* 3.85 9 0.21***, ††† 0.88 9 0.13 0.52 9 0.10* 0.80 90.18 Values expressed as mean 9 SEM (n = 10). a * PB0.05, ** PB0.01, *** PB0.001 in comparison with normal. b †† PB0.01, ††† PB0.001, NS — not significant in comparison with diabetic. Table 5 In vitro effect of DCMM extract of C. roseus on enzymatic activities of liver Addition Glycogen synthase1b Glucokinase2 Lactate dehydrogenase3 Succinate dehydrogenase4 Malate dehydrogenase5 Control MeOH Extract (100 mg) 4.62 9 0.37 5.01 9 0.29 3.029 0.03 4.02 9 0.03a 65.74 92.47 62.24 9 2.77 4.16 9 0.49 4.73 9 0.74 1.77 9 0.04 1.49 9 0.17 Values expressed as mean 9 SEM (n = 10) a PB0.001 in comparison with MeOH control. b (1) mmol UDP formed/min/mg protein, (2) nmol NADP converted to NADPH/min/mg protein, (3) nmol pyruvate formed/min/mg protein, (4) nmol potassium ferricyanide reduced/min/mg protein, (5) mmol NADH oxidized/min/mg protein. S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269–277 275 Table 6 Effect of DCMM extract of C. roseus on glutathione, MDA levels and activities of g-GT, GST, AST, ALT, acid and alkaline phosphatase Variable Glutathione (GSH) Blood (mg/dl) Liver (mg/g wet tissue) Lipid peroxidation Liver (mmol MDA/g wet tissue) g-Glutamyl transpeptidase Liver (nmol p-nitroaniline released/min/mg protein) Plasma (nmol p-nitroaniline released/min/ml) Glutathione S-transferase Liver (mmol thioester formed/min/mg protein) Aspartate aminotransferase Liver (nmol pyruvate /min/mg protein) Plasma (nmol pyruvate formed/min/ml) Alanine aminotransferase Liver (nmol pyruvate /min/mg protein) Alkaline phosphatase Liver (nmol p-nitrophenol released/min/mg protein) Acid phosphatase Liver (nmol p-nitrophenol released/min/mg protein) Normal Diabetic Diabetic treated (500 mg/kg (p.o.)×7 days) 108.8 9 19.7 105.6 9 9.5 121.5 9 24.2 2822.4 9 186.0 3262.5 9 292.0 3381.5 9 544.0 13.70 90.40 16.84 9 0.20** 4.61 90.27 2.76 9 0.68*a 14.199 1.35 22.349 2.05** 17.58 9 3.00 0.599 0.05 0.45 90.06 0.44 9 0.07 88.889 7.79 73.47 9 8.37 78.14 9 7.03 79.189 7.03 84.559 2.22 163.73 924.25** 79.26 98.25 33.88 9 1.40 5.759 0.95† 107.09 9 27.21 80.33 9 8.94 0.54 90.06 1.06 9 0.10*** 1.17 9 0.15*** 9.26 9 0.16 9.46 9 0.83 9.98 9 0.67 Values expressed as mean 9 SEM (n = 10) a * PB0.05, ** PB0.01, *** PB0.001 in comparison with normal, † PB0.05 in comparison with diabetic. ment with DCMM extract of C. roseus increases utilization of circulating glucose by liver. Oxidative stress appears to be a key element of the production of secondary complications in diabetes (Wohaieb and Godin, 1987; Godin et al., 1988; Wolff et al., 1991; Thomson and McNeil, 1993; Thornalley et al., 1996). Glutathione, a tripeptide present in millimolar concentrations in all the cells is an important antioxidant (Meister and Anderson, 1983; DeLeve and Kaplowitz, 1991; Lu, 1999). Decreased glutathione levels in diabetes have been considered to be an indicator of increased oxidative stress (Wolff, 1987; McLennan et al., 1991). In the present study not much change was observed in GSH levels either in blood or liver of diabetic animals, however in treated animals GSH levels were marginally high in both blood as well as liver. Lipid peroxidation was found to be increased in liver of diabetic animals which became normal in DCMM treated animals. This indicates that the extract may be helpful in the prevention of damage caused by oxygen free radicals. We have not studied oxidized glutathione levels and this is a limitation of this study. There was not much change in glutathione S-trans- ferase activity in diabetic and diabetic treated animals. g-Glutamyl transpeptidase activity was decreased significantly in liver of diabetic animals and with DCMM extract treatment activity increased (Table 6). g-Glutamyl transpeptidase has a key role in amino acid transport across membranes and catalyzes the initial step in breakdown of glutathione, i.e. transfer of g-glutamyl moiety of glutathione to a variety of amino acids and peptides (Meister, 1983). Increase in g-glutamyl transpeptidase activity in plasma is an indicator of impairment in liver function. In the present study there was an increase in g-glutamyl transpeptidase activity in plasma of diabetic animals. In C. roseus treated animals activity of this enzyme shows a decrease in plasma and was close to normal activity. Measurement of enzymic activities of aminotransferases (AST and ALT) and phosphatases (acid and alkaline) is of clinical and toxocological importance as changes in their activities are indicative of tissue damage by toxicants or in disease conditions. AST and ALT activities in liver of diabetic animals remain unchanged in liver though AST activity was little less than normal. Plasma levels of AST were increased around 276 S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269–277 twice that of normal in diabetic animals and diabetic animals treated with extract show improvement. Recovery of plasma AST levels of diabetic rats towards normal shows that the DCMM extract has no adverse effect on liver functions. Liver alkaline phosphatase activity was found to be significantly increased in diabetic animals. Treatment with extract further caused increase in activity (Table 6). Increase in alkaline phosphatase activity in testes and prostate at 300 mg/kg for 24 days by ethanolic extract of C. roseus leaves is reported in normal animals (Chauhan et al., 1979). Acid phosphatase activity of liver of diabetic rats was also found to be increased. At low dose 75 mg/kg for 24 days ethanolic extract is reported to inhibit acid phosphatase activity and at higher doses i.e. 300 mg/kg stimulation is reported (Chauhan et al., 1979). Detection of hypoglycemic activity in DCMM extract along with protective effect against STZ challenge and preventive action on lipid peroxidation provides scientific rationale of use of C. roseus as antidiabetic plant. Antidiabetic activity seems to be a result of increase in glucose utilization. Further chromatographic fractionation of the extract may be useful in improving activity and reduction of dose. In preliminary toxicity study it is safe, however chronic toxicity evaluation will be required for human use. Prophylactic activity observed in present study is of much importance and there is a need for further studies on this aspect. Acknowledgements The investigators express their sincere thanks to Dr. P.K.Banerjee, Sc’F’ and Dr. W. Selvamurthy, Director DIPAS, for their constant interest and encouragement throughout the present study. References Anderson, J.W., 1974. Alterations in metabolic fate of glucose in the liver of diabetic animals. American Journal of Clinical Nutrition 27, 746 – 755. Ashwell, G., 1957. Colorimetric analysis of sugars. Methods in Enzymology 3, 378 – 386. Baker, J.R., Metcalf, P.A., Holdway, I.M., Johnson, R.N., 1985. Serum fructosamine as a measure of blood glucose control in type I (insulin dependent) diabetes mellitus. British Medical Journal 290, 352 – 355. Chang, A.Y., Noble, R.E., Wyse, B.M., 1977. Streptozotocin induced diabetes in the Chinese hamster. Diabetologia 13, 595 – 602. Chattopadhyay, R.R., 1999. 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Effect of vanadyl sulfate feeding on susceptibility to peroxidative changes in diabetic rats. Research Communication in Chemical Patholology and Pharmacolology 80, 180 – 200. Thornalley, P.J., McLellan, A.C., Lo, T.W., Benn, J., Sonksen, P.H., 1996. Negative association between reduced glutathione concentration and diabetic complications. Medical Science 91, 575 – 582. Utley, H.G., Berheim, F., Hochstein, P., 1967. Effect of sulphydryl reagents on peroxidation of microsomes. Archives of Biochemistry and Biophysics 118, 29 –32. Wohaieb, S.A., Godin, D.V., 1987. Alterations in free radical tissue defence mechanisms in streptozotocin induced diabetes in rat. Effect of insulin treatment. Diabetes 36, 1014 – 1018. Wolff, S.P., 1987. The potential role of oxidative stress in diabetes and its complications: novel implication for theory and therapy. In: Crabbe, M.J.C. (Ed.), Diabetic complications. Scientific and Clinical aspects. Churchill Livingstone, Edinburgh, pp. 167 – 220. Wolff, S.P., Jiang, Z.Y., Hunt, J.V., 1991. Protein glycation and oxidative stress in diabetes mellitus and ageing. Free Radical Biology and Medicine 10, 339 – 352. Journal of Ethnopharmacology 76 (2001) 279– 284 www.elsevier.com/locate/jethpharm A pharmacological study of Cecropia obtusifolia Bertol aqueous extract Concepción Pérez-Guerrero *, Marı́a Dolores Herrera, Rafael Ortiz, Marı́a Alvarez de Sotomayor, Marı́a Angeles Fernández Department of Pharmacology, Faculty of Pharmacy, Uni6ersity of Se6ille, Se6ille, Spain Received 10 December 2000; received in revised form 11 April 2001; accepted 24 April 2001 Abstract Cecropia obtusifolia (Cecropiaceae) is a species from tropical America and its leaves are used in traditional medicine for the treatment of diabetes and as an anti-inflammatory agent. In the present study, the analgesic, anti-inflammatory and central nervous system depressant effects of the aqueous extract from the leaves of C. obtusifolia were investigated in different experimental models, with the purpose of validating its ethnomedical uses. The results obtained with the extract from the leaves of C. obtusifolia reflect a low toxicity, a substantial central depressor effect and analgesic activity and significant motor incoordination and muscle relaxant activity. Concerning the analgesic activity, using the hot plate test, the extract did not produce any effect, however it showed a significant effect on the pain induced by chemical stimuli (acetic and formalin test); this suggests the peripheral analgesic effect of the extract. The extract also showed a topical and systemic anti-inflammatory effect. Thus this work could justify the popular use of C. obtusifolia in rheumatic and kidney inflammation pathologies and reveals that this plant is an interesting species © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Analgesic activity; Anti-inflammatory activity; Cecropia obtusifolia Bertol 1. Introduction Cecropia obtusifolia (Cecropiaceae) is a species from tropical America known by the popular names of ‘grayumbo’, ‘trompeto’, ‘guarumo’, ‘yagrumo’, ‘guarumbo’, ‘hormiguillo’ and ‘chancarro’ in El Salvador (Berg, 1978). There is not much information reported about the phytochemistry of C. obtusifolia. It is reported that the leaves and bark contain alkaloids, cardiotonic glycosides, flavonoids, tannins, triterpenoids and saponin glycosides (Morton, 1981). The leaves of this plant are used in an infusion for the treatment of diabetes and as an anti-inflammatory agent. Previous pharmacological research carried out with the aqueous extract from C. obtusifolia pointed out that this plant might have anti-hypertensive, hypoglycaemic and diuretic effects (Cáceres et al., 1987; Salas et al., 1987a,b; Vidrio et al., 1982). In El Sal* Corresponding author. Tel.: + 34-5-4556720; fax: 4233765. E-mail address: cperez@fafar.us.es (C. Pérez-Guerrero). + 34-5- vador, decotions of its leaves are used as sedatives and for the treatment of arthritis and rheumatism (Morton, 1981). However, there are no previous studies of its anti-inflammatory, analgesic and depressant effects. In the present study, the analgesic, anti-inflammatory and central nervous system depressant effects of the aqueous extract from the leaves of C. obtusifolia were investigated in different experimental models, with the purpose of validating its ethnomedical uses. 2. Material and methods 2.1. Plant material Leaves of C. obtusifolia were collected and identified at the University of Costa Rica in August 1998 (USJ 52907). The leaves were dried in an oven at 40°C. Dry leaves were powdered in a Wiley grinder and then sieved. 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 5 3 - 7 280 C. Pérez-Guerrero et al. / Journal of Ethnopharmacology 76 (2001) 279–284 2.2. Extract preparation Extraction was carried out in the Research Center of Natural Products (CIPRONA). Dry powered leaves of C. obtusifolia (600 g) were extracted with 30 l of distilled water at 2% and at 7092°C for 30 min. The aqueous extract was filtered using a cotton funnel and filtered again under reduced pressure on a Whatman 4 filter paper. Water was removed using a rotatory evaporator and the remainder was later lyophilized yielding 77.6 g of extract. The dry residue obtained was diluted in isotonic NaCl solution for the pharmacological study. The doses employed are expressed in terms of dried plant material (mg/kg wt.) 2.3. Animals Male Swiss albino mice weighing 20– 25 g and male Wistar rats weighing 200– 250 g (n =5 –6 per group) were used. Animals were kept under standard conditions and fasted 18 h prior to their use. 2.4. Acute toxicity Toxicity was assessed by determination of the median lethal dose, LD50. (Miller and Tainter, 1944). Increasing doses of the extract (1, 1.25, 1.375, 1.5 and 1.75 mg/kg) were administered by i.p. injection. The animals were kept under observation for a period of 48 h. 2.5. Depressant effect on the central ner6ous system The activity of C. obtusifolia on the CNS was evaluated by performing assays of its effects on exploratory capacity (hole-board test), muscular relaxation (traction-test), motor coordination (rotarod test) and escape instinct (evasion test). 2.5.1. Hole-board test (Boissier and Simon, 1960) The animals were placed on a board (40×40 cm) with 16 holes (symmetrically distributed in four rows) 30, 60 and 90 min after i.p. administration of C. obtusifolia aqueous extract (125 and 250 mg/kg), vehicle (saline solution) or standard drug, diazepam (2 mg/kg), and the number of times that the head dipped into the hole during 5 min was registered. 2.5.2. Traction test (Cour6oisier, 1956) Mice were suspended by their hind legs from a taut metal wire and the time taken to bring their front legs up the wire was recorded for 120 s. The test was performed 30, 60 and 90 min after i.p. injection of the C. obtusifolia extract (125 and 250 mg/kg), vehicle or diazepam. 2.5.3. E6asion test (Simon et al., 1982) The animals were kept in a rectangular box having an inclined plane. The time taken to escape was noted for 120 s. The assessment was performed 30 min after i.p. injection of C. obtusifolia aqueous extract (125 and 250 mg/kg), vehicle or diazepam. 2.5.4. Rota-rod test (Dunham and Miya, 1957) Mice were placed on a rota-rod (Letica), and those staying on the rod at 10 rpm for more than 2 min were selected for the experiment. The selected mice were injected (i.p.) with C. obtusifolia extract (125 and 250 mg/kg), vehicle or diazepam and 30, 60 and 90 min later were placed on the rod (initial speed 5 rpm, with a gradual increase to 8 rpm). The length of time they remained on the rod was recorded. 2.6. Analgesic study 2.6.1. Acetic acid-induced writhing test (Santos et al., 1994) The total number of writhings following i.p. administration of a solution of 1% acetic acid, was recorded over a period of 5 min, starting 7 min after acetic acid injection. The mice were treated i.p. with the aqueous extract from C. obtusifolia (500, 250, 125 and 50 mg/kg) or saline solution (0.9% w/v NaCl) or the standard drug methamizol (200 mg/kg) 30 min before administration of the acetic acid. 2.6.2. Formalin test (Hunskaar et al., 1985) The animals were injected with formalin (1%, 20 ml) into the suplantar area of the right hind paw. The duration of the paw licking, an index of nocicepcion, was measured at 1–5 min (early phase) and 20– 25 min (late phase) after formalin administration. Animals were i.p. treated with the aqueous extract (50, 125 and 250 mg/kg), saline solution or methamizol. 2.6.3. Hot plate test (Eddy and Leimbach, 1953) The respective times (s) for licking and jumping on a hot plate at 55°C were registered. Animals were i.p. treated with the aqueous extract from C. obtusifolia (250 and 500 mg/kg), vehicle or standard drug (morphine 10 mg/kg) 30 min before being placed on the hot plate. 2.7. Anti-inflammatory acti6ity 2.7.1. Rat paw carrageenan-induced edema The method used was similar to that described by Winter (Winter et al., 1962). The extract was administered orally (125, 250 and 500 mg/kg). The paw vol- C. Pérez-Guerrero et al. / Journal of Ethnopharmacology 76 (2001) 279–284 ume of the rats was measured plethismographically (LETICA-7500) before administering carrageenan (V0) and 1, 3 and 5 h after (VT). The amount of paw swelling was determined for each rat and the difference at VT (1, 3 and 5 h) from V0 was taken as the edema value. Indomethacin (25 mg/kg) was used as standard drug. The percentages of inhibition were calculated according to the following formula: %Inhibition= (VT − V0)control − (VT − V0)treated group ×100 (VT − V0) 2.7.2. 12 -O-tetradecanoylphorbol acetate (TPA) induced mouse ear edema (De Young et al., 1989) Each mouse received 2.5 mg of TPA dissolved in 20 ml of EtOH 70%. This was applied by an automatic pipette in 10 ml volumes to both anterior and posterior surfaces of the right ear. The left ear (control) received the same volume of solvent (EtOH 70%). Extract was applied topically at doses of 1 and 2 mg/ear in EtOH 70%, simultaneously with TPA. Diclofenac (0.5 mg/ear) was used as standard drug. Inflammation was allowed to develop for 4 h, after which the animals were killed by cervical dislocation and a section (6 mm diameter) of the central portion of both ears was obtained and weighed. The swelling induced by TPA was assessed in terms of the increase in the weight of the right ear punch biopsy over that of the left ear. 2.8. Statistical analysis The data are expressed as mean9S.E.M. Significance of differences with respect to controls was evaluated using the Student t-test. 2.9. Drugs 12-O-Tetradecanoylphorbol (TPA) and carrageenan type IV, were purchased from Sigma- Aldrich Quı́mica (Madrid. Spain), methamizol (Nolotil®, Europharma, Barcelona. Spain), diclofenac (IBSA, Lugano SwitzerProdes, land), diazepam (Diazepam-Prodes®, Barcelona. Spain), morphine (Sevredol®, Asta Medica, Madrid. Spain). 3. Results 3.1. Acute toxicity The median lethal dose (LD50) of aqueous extract from C. obtusifolia after i.p. administration was measured as 14509 70 mg/kg animal (11.2190.52 g of plant/kg of weight). We observed in the animals a general relaxed state, sleepiness and piloerection. 281 3.2. Depressant acti6ity on the central ner6ous system 3.2.1. Hole-board test The results at 30 min are shown in Fig. 1A. The head dip responses were significantly reduced in the groups treated with the extract of C. obtusifolia compared with control group. 3.2.2. Traction test In the traction test, the aqueous extract from C. obtusifolia exerted a important effect with all the doses assayed, compared with the control group and had a similar effect to the reference drug, diazepam (Fig. 1B). 3.2.3. E6asion test The extract showed a significant dose-dependent effect on the escape capacity of the mice with the two doses assayed (Fig. 1C). 3.2.4. Rota-rod test On the rota-rod test the fraction significantly reduced the time that mice were able to stay on the rod at all dosages and 125 mg/kg in the similar magnitude than diazepam (Fig. 1D). 3.3. Analgesic acti6ity 3.3.1. Acetic acid-induced writhing test Table 1 shows that the aqueous extract from C. obtusifolia significantly (PB 0.001) [MP1]reduced the number of writhing movements induced by the i.p. administration of the acetic acid solution in a dose-dependent way. Doses of 500, 250 and 125 mg/kg produced similar effects as methamizol (100 mg/kg). 3.3.2. Formalin test The aqueous extract from C. obtusifolia inhibited both phases of the response to formalin. This inhibition was higher for the second phase of the response, reaching values of 99.32% of inhibition (Table 1). 3.3.3. Hot plate test Table 1 shows the results of the hot plate test. The C. obtusifolia extract did not affect both licking and jumping times. 3.4. Anti-inflammatory acti6ity 3.4.1. Rat paw carrageenan-induced edema As shown in Fig. 2A, the administration of aqueous extracts of C. obtusifolia significantly decreased the edema induced by carrageenan in rat paw at all the doses assayed (125, 250 and 500 mg/kg) compared to control group. The indomethacin effect was significantly higher than that of the aqueous extract in decreasing the edema. Percentage of inhibition on C. Pérez-Guerrero et al. / Journal of Ethnopharmacology 76 (2001) 279–284 282 carrageenan-induced rat paw edema is presented in Fig. 2B. 3.4.2. Mouse ear TPA-induced edema The topical administration of aqueous extracts of C. obtusifolia significantly decreased the TPA-induced mouse ear edema at both doses used, compared to the control group and diclofenac, standard drug shown higher effect than extract (Fig. 2C). 4. Discussion and conclusions The results obtained with the extract from the leaves of C. obtusifolia reflect a low toxicity, a substantial central depressor effect and anti-inflammatory and analgesic activity. The depressor effect was reflected by a positive response to the C. obtusifolia extract on the hole board, rota-rod, evasion and traction tests. In the hole board test, the extract significantly reduced the Fig. 1. Depressant activity of aqueous extract of C. obtusifolia on the central nervous system. (A) Effect on responses in the hole-board test. (B) Effect on responses in the traction test. (C) Effect on responses in the evasion test. (D) Effect on responses in the rota-rod test. Each point represent the mean of five– six animals. Significance of differences with respect to controls was evaluated using the Student t-test. *PB 0.05, **PB 0.01, ***PB 0.001. Table 1 Analgesic activity of C. obtusifolia on responses to the acetic acid-induced writhing test, formalin test and hot plate test, showing the mean times9 S.E.M. of six mice Acetic acid Formalin test Hot plate test Treatment No writhings Early phase Late phase Licking time Jumping time Control 50 mg/kg 125 mg/kg 250 mg/kg 500 mg/kg Methamizol Morphine 17.809 1.39 5.809 1.59*** 1.009 0.44*** 0.209 0.20*** 0.409 0.24*** 1.609 0.67*** – 76.609 8.22 36.00 9 5.39** 18.20 9 4.20*** 12.60 9 2.06*** – 21.20 9 9.28*** – 29.509 6.11 14.009 5.80n.s 4.40 9 1.56** 0.209 0.20*** – 0.609 0.60*** – 14.20 9 1.40 – – 15.50 9 1.10 n.s 15.5 9 1.60 n.s – 12.50 9 1.71 n.s 66.70 9 4.10 – – 66.8 9 7.72 n.s 51.8 9 5.30 n.s – 122.30 9 12.6** Significance of differences with respect to controls was evaluated using the Student t-test. *PB0.05, **PB0.01, **PB0.001. C. Pérez-Guerrero et al. / Journal of Ethnopharmacology 76 (2001) 279–284 Fig. 2. Anti-inflammatory activity of aqueous extract of C. obtusifolia. (A) Effect on carrageenan-induced edema in rat paw (volume increased in ml). (B) Percentage of inhibition on carrageenan-induced edema. (C) Effect on TPA-induced mouse ear edema. Each point represents the mean of five–six animals. Significance of differences with respect to controls was evaluated using the Student t-test. *PB 0.05, **PB 0.01, ***PB 0.001. exploratory capacity. This effect appeared 30 min after administration and was sustained throughout the experiments. This response corresponded to the effect on the escape capacity, that also was significantly supressed by the extract at the two doses assayed. Concerning the muscle relaxant activity (rota-rod and traction test) C. obtusifolia was found to produce 283 significant motor incoordination and muscle relaxant activity. Analgesic activity was evaluated using thermal (hot plate test) and chemical stimuli (formalin and acetic acid) in order to determine the type of analgesic activity. In the hot plate test C. obtusifolia hardly produced any effect. It did not significantly increase the latency time of licking or jumping. However, C. obtusifolia showed a significant effect on the pain induced by chemical stimuli (acetic and formalin test), suggesting the induction of a peripheral analgesic effect. The extract of C. obtusifolia also showed a significant anti-inflammatory action, evaluated by the edema reduction with carrageenan (oral route) and the edema induction with TPA (topical administration). In the carrageenan test, the extract significantly reduced the inflammation (1, 3 and 5 h after the administration), showing activity from the first measurement. Carrageenan produces an inflamatory response of a twophase type (Di Rosa et al., 1971). In the beginnig of its administration, there is sudden elevation of paw volume in relation with histamine mediators (Geen, 1964). After 1 h the inflammation increases gradually and was elevated during the later 3 h. This second phase could be due to the kinins and prostaglandins (PGs) liberated. According to these results, it can be suggested that the mechanism of anti-inflammatory action of the extract occurs by interfering with the synthesis or liberation of mediators: histamine and PGs. In the TPA-test the extract also produced a significant edema inhibition with all the doses assayed and again this effect was not dose-dependent. In summary, the results of this preliminary study have showed that the aqueous extract from leaves of C. obtusifolia possesses an important central depressor effect, although apparently it is not related to its analgesic properties, as unlike morphine (hypnotic reference drug), the extract was not capable of modifying the latent jumping time in the hot plate test. The extract also showed a topical and systemic antiinflammatory effect. Thus, this work could justify its popular use in rheumatic and kidney inflammation pathologies, which reveals that C. obtusifolia is an interesting species. Chemical and pharmacological studies are now in progress to isolate the active compounds responsible for these actions, and to determine their mechanism of action. References Berg, C.C., 1978. Cecropiaceae, a new family of Urticales. Taxon. 27, 39 – 44. Boissier, J.R., Simon, P., 1960. Planche à trous automatisée. Thérapie 15, 1170 –1174. Cáceres, A., Girón, A., Martinez, A.M., 1987. Diuretic activity of plants used for the treatment of urinary ailments in Guatemala. Journal of Ethnopharmacology 19, 233 – 245. 284 C. Pérez-Guerrero et al. / Journal of Ethnopharmacology 76 (2001) 279–284 Courvoisier, S., 1956. Pharmacodynamic properties of chlorpromazine. Journal of Clinical Experimental Psychopathology 17, 25 – 37. De Young, L., Kheifets, J.B., Ballaron, S.J., Young, J.M., 1989. Oedema and cell infiltration in the phorbol ester-treated mouse ear are temorally separated and can be differentially modulated by pharmacologic agents. Agents Actions 26, 335 – 341. Di Rosa, M., Giroud, J.P., Willoughby, D.A., 1971. Studies of the mediators of acute inflammatory sites by carrageenan and turpentine. Journal of Pathology 104, 15. Dunham, M.W., Miya, T.S., 1957. A note of a simple apparatus detecting neurological deficit in rats and mice. Journal of American Pharmaceutical Science 46, 208 –209. Eddy, N.B., Leimbach, D., 1953. Synthetic analgesics, II. Dithienylbutenyl and dithienylbutenylamines. Journal of Pharmacology and Experimental Therapeutics 107, 385 – 393. Geen, K.L., 1964. Role of endogenous catecholamines in the antiinflamatory activity of alpha-adrenoceptor blocking agents. British Journal of Pharmacology 51, 45 – 53. Hunskaar, S., Famer, O.B., Hole, K., 1985. Formalin test in mice: a useful technique for evaluating wild analgesics. Journal of Neuroscience Methods 14, 69 – 76. Miller, L.L.C., Tainter, T.L., 1944. Stimation of the DL50 and its error by means of logarithmin-probits graphpaper. Proceding of Society of Experimental Biology and Medicine 57, 261 – 264. . Morton, J., 1981. Atlas of Medicinal Plants of Middle America. Charles C. Thomas Publishers, USA. Salas, I., Brenes, J.R., Orlando, M., Morales, M., 1987a. Antihypertensive effect of Cecropia obtusifolia (Moraceae) leaf extract on rats. Revista de Biologia Tropical 35 (1), 127 – 130. Salas, I., Orlando, M., Morales, M., Brenes, J.R., 1987b. Effect of chronic administration of Cecropia obtusifolia (Moraceae) on mean arterial pressure in rats. Revista de Biologia Tropical 35, 359 – 362. Santos, A.R.S., Cechinel Filho, V., Niero, R., Viana, A.M., Moreno, F.N., Campos, M.M., Yuunes, R.A., Calixto, J.B., 1994. Analgesic effects of callus culture extracts from selected of Phyllantus in mice. Journal of Pharmacie and Pharmacology 46, 755 – 759. Simon, P., Chermat, R., Doare, L., Bourin, M., Farinotti, R., 1982. Unexpected interactions of some psychotropic drugs with barbital and pentobarbital effects in mice. Journal of Pharmacology 13, 241 – 242. Vidrio, H., Garcı́a-Márquez, F., Reyes, J., Soto, R.M., 1982. Hypotensive activity of Cecropia obtusifolia. Journal of Pharmaceutical Science 71, 475 – 476. Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenan-induced edema in hind paw of the rat as an assay for anti-inflammatory drugs. Procedings of Society of Experimental Biology and Medicine 3, 544 – 547. Journal of Ethnopharmacology 76 (2001) 285– 291 www.elsevier.com/locate/jethpharm Short communication Antioxidant potential of two polyherbal. preparations used in Ayurveda for the treatment of rheumatoid arthritis M. Ira Thabrew a,*, Lakshmi Senaratna b, Nirma Samarawickrema b, C. Munasinghe c a Department of Biochemistry & Clinical Chemistry, Faculty of Medicine, Uni6ersity of Kelaniya, 6 Talagolla Road, Ragama, Sri Lanka b Bandaranayake Memorial Ayur6edic Research Institute, Nawinna, Sri Lanka c Department of Biochemistry, Faculty of Medicine, Uni6ersity of Sri-Jayawardenapura, Sri Jayawardenapura, Nugegoda, Sri Lanka Received 4 December 2000; received in revised form 17 January 2001; accepted 6 February 2001 Abstract Reactive oxygen intermediates (ROI) are together with prostanoids, leukotrienes and proteases, believed to be the mediators of inflammation and responsible for the pathogenesis of tissue destruction in RA. Antioxidant (AO) activity is one of the mechanisms by which many conventional drugs used in day to day treatment of RA alleviate the painful symptoms associated with this disease. An investigation has been carried out to compare the antioxidant potentials of two polyherbal formulations, Maharasnadhi quathar (MRQ) and Weldehi choornaya (WC), used by Ayurvedic medical practitioners in Sri-Lanka for the treatment of RA patients. AO potentials of these preparations were assessed by their effects in RA patients on: (a) activities of the AO enzymes superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase; (b) lipid peroxidation (as estimated by thiobarbituric acid reacting substances (TBARS) generation); and (c) concentrations of serum iron and haemoglobin (Hb), and the total iron binding capacity (TIBC). The overall results of the study demonstrate that MRQ has much greater AO potential than WC. Thus, on treatment with MRQ for 3 months, the initial activities of plasma SOD, GPX and catalase, were enhanced by 44.6, 39.8 and 25.2%, respectively. There was no significant improvement in any of these enzyme activities in patients treated with WC for the same time period as MRQ. Although the extent of lipid peroxidation in plasma of RA patients could be decreased by both drug preparations, the reduction mediated in 3 months by MRQ (34%) was markedly greater than that due to WC (21.8%). The total serum iron and Hb concentrations and TIBC in the RA patients included in the study could be significantly improved by treatment with MRQ but not by WC. Thus, at the end of 3 months treatment with MRQ, concentrations of the total serum iron and Hb, and the TIBC of patients improved by 26.8, 24.8 and 16.1%, respectively. Possible reasons for differences in the AO potentials of MRQ and WC are discussed. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Maharasnadi quathar; Weldehi choornaya; Rheumatoid arthritis; Antioxidants 1. Introduction Rheumatoid arthritis (RA) is a multisystem, chronic, relapsing, inflammatory disease affecting 1% of the world’s population (Cotran et al., 1989). ROI produced by activated phagocytes in the inflamed joints have been implicated along with prostanoids, leukotrienes and proteases, as mediators of inflammation and the * Corresponding author. E-mail address: mrthab@dynaweb.lk (M. Ira Thabrew). pathogenesis of tissue destruction (Blake et al., 1981, 1989; Halliwell and Gutteridge, 1989; Krane et al., 1990). At the onset of inflammation in RA, there is a rapid drop in the serum iron level followed by an increased iron deposition in the synovial fluid. Such an increase in metal ion availability in the synovial fluid is thought to accelerate ROI mediated reactions and thus contribute to increased disease severity (Gutteridge, 1986). Many drugs commonly used in the day to day treatment of RA are believed to mediate their therapeutic 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 1 3 - 6 286 M. Ira Thabrew et al. / Journal of Ethnopharmacology 76 (2001) 285–291 actions by multiple mechanisms, one of them being suggested is a reduction of oxidant damage at sites of inflammation by drugs either acting as ROI scavengers or inhibitors of ROI production by phagocytes (Wasil, 1987; Aruoma, 1996). Weldehi choornaya (WC) and Maharasnadi quathar (MRQ) are two polyherbal preparations (Ayurveda Pharmacopoeia, 1975) used by Ayurvedic and other traditional medical practitioners in Sri-Lanka for the treatment of RA patients. During the past decade, research conducted in many different laboratories have shown that plants are very important sources of antioxidant and radical scavenging components (Scott et al., 1993; Joyeaux et al., 1995; Mitra et al., 1999). In view of: (a) the involvement of ROI in the pathogenesis of tissue destruction associated with RA; and (b) the role of antioxidant drugs in the prevention of ROI mediated tissue destruction, an investigation has been carried out to determine whether the beneficial effects of WC and MRQ in RA patients are mediated at least in part, through alterations in the activities of their antioxidant defences. As a preliminary step towards achieving this objective, the effects of WC and MRQ on the activities of the antioxidant (AO) enzymes catalase, glutathione peroxidase (GPX) and superoxide dismutase (SOD) in blood of RA patients, as well as their effect on the iron status of these patients have been investigated. 2. Materials and methods 2.1. Chemicals All reaction kits for assessment of SOD and GPX activities and iron status were purchased from Tanyo Medical (Pvt), Colombo, Sri-Lanka. All other chemicals were purchased from Sigma, Poole, Dorset, UK. 2.2. Patient selection Patients for the study were selected from individuals attending the routine RA clinics at the BMARI, based on criteria recommended by the American Rheumatism Association. A total of 100 patients (50 for each treatment regime) with uncomplicated RA were selected. All patients who participated in the study were females whose ages ranged from 35 to 65 years. No male patients could be selected, because the few who attended the RA clinics during the trial period had other complications such as diabetes or cardiac problems. When choosing patients for the study, preference was given to those who were not on any medication at the time of selection and were free of any other systemic disease which required specific treatment. A 2-week wash out period was advised for patients who had been on other forms of treatment for RA prior to selection for the present study. 2.3. The composition of MRQ and WC The composition of these formulations is shown below: Maharasnadi quathar Alpinia calcarata rhizome Sida cordifolia root Ricinus communis root Cedrus deodara bark Kaempteria galanga rhizome Acorus calamus rhizome Weldehi choornaya Smilax glabra tuber Myristica fragrans seed Piper nigrum seeds Zingiber officinale rhizome Michelia chammpaca flower buds Eugenia caryophyllus dried flower buds Adhatoda 6asica root Zingiber officinale Mace rhizome Terminalia chebula dried Triticum sati6um seeds fruit Piper chaba hunter roots Hyoseyamus reticulatus seeds Cyperus rotundus tuber Nigella sati6a seeds Boerhaa6ia diffusa roots Azima tetracantha roots Tinospora cordifolia Terminalia catappa seeds stem Argyra speciosa stem Tinospora cordifolia stem Finiculum 6ulgare seeds Gycyrrhiza glabra stem Tribulus terrestrin whole Melochia corchorifolia root plant Withania somnifera Gymnema lactiferum root rhizome Aconitum heterophyllum rhizome Cassia fistula bark Asparagus racemosus rhizome Nigella sati6a seeds Barleria prionitis root Coriandrum sati6um seeds Solanum melongena roots Solanum xanthocarpum whole plant Tragia in6olucrata whole plant 2.4. Preparation and dosage of plant drugs The parts of plants used in the preparation of the MRQ and WC were collected, sundried and mixed in the required proportions by the Pharmacist at the BMARI according to instructions in the Ayurveda M. Ira Thabrew et al. / Journal of Ethnopharmacology 76 (2001) 285–291 Pharmacopoeia (1975). The preparation of the drug formulations for patient treatment and the daily dosage of each drug formulation administered to patients were also in accordance with information in the Ayurveda Pharmacopoeia (1975). 2.4.1. MRQ The plant mixture containing 80 g of Alpinia calcarata plus 40 g of all other plant material (1.6 g of each) was boiled in 3.84 l water until the volume was reduced to 480 ml. The extract was divided into three equal portions of 160 ml each, and administered orally to patients in three doses (two doses at 06:30 and 18:30 h of each day and the third, at 06:30 h of the following day). The decoction was prepared fresh each day. 2.4.2. WC The sun dried plant material mixture (containing 480 g Smilax glabra, 40 g Myristica fragrans, 30 g Piper nigrum, 25 g each of Zingiber officinale and Michelia chammpaca, 20 g of a mixture of Eugenia caryophyllus and mace, plus 1.6 g each of all other plants in this mixture) was ground to a very fine powder. Half a teaspoon of the powder mixed with a little bees honey was administered orally to patients twice a day, as recommended in the Ayurveda Pharmacopoeia (1975). 2.5. Blood collection Blood samples (5 ml each) were collected from each patient before treatment, and at 1 and 3 months after starting the treatment. Although 50 patients for each treatment regime were selected initially, due to poor patient attendance at the clinics, the final results could be computed by analysing blood from only 45 MRQ patients and 20 WC patients. Blood samples (5 ml each) were also collected from a group (n =35) of healthy volunteers (ages between 25 and 58 years) for comparison of the activities of SOD, GPX and catalase and levels of serum iron, Hb and TIBC in normal individuals with initial values of these parameters in the patients selected for the study. Informed, written consent was obtained from each patient included in the trial before collection of blood. 2.6. Assessment of antioxidant acti6ities 2.6.1. Glutathione peroxidase (GPX) assay GPX activity, was estimated by the method of Paglia and Valentine (1967), using commercially available reagent kits purchased from Randox Laboratories. Enzyme activity was expressed as U/g haemoglobin. 287 2.6.2. Superoxide dismutase (SOD) assay Erythrocyte SOD activity was assayed according to the method described in the assay kit purchased from Randox, UK. In this method, xanthine and xanthine oxidase are used to generate SOD radicals which react with 2-(4-iodophenyl)-3-(4nitrophenol)-5-phenyl tetrazolium chloride to form a red formazan dye. SOD activity is then measured by the degree of inhibition of this reaction. Enzyme activity was expressed as U/g haemoglobin. 2.6.3. Catalase Catalase activity was determined according to the colorimetric method recommended by Goth (1991). 2.6.4. Serum iron and total iron binding capacity (TIBC) Serum iron and TIBC were estimated according to Ceriotti and Ceriotti (1980), by use of reagent kits purchased from Randox. 2.6.5. Lipid peroxidation as assessed by production of thiobarbituric acid reacti6e substances (TBARS) Plasma TBARS concentration was estimated by reaction with thiobarbituric acid in the presence of butylated hydroxytoluene. and measuring the absorbance at 535 rim of the pink coloured complex formed, against a reagent blank (Aust, 1985) 2.6.6. Estimation of blood haemoglobin concentration The haemoglobin concentration was estimated by the cyanomethaemoglobin photometric method recommended by the WHO (1980). 2.7. Statistical analysis Results are presented as the Means9 S.D. Significance of differences between groups was assessed by use of one way ANOVA. 3. Results 3.1. The antioxidant enzyme concentrations and iron status of patients before and after commencement of treatment A comparison of: (a) activities of the antioxidant enzymes SOD, GPX and catalase; and (b) the levels of serum iron and TIBC, in the patients selected for the study before any drug treatment, with corresponding values in a normal group of individuals is shown in Table 1. It is apparent that in the RA patients included in the study, blood SOD, GPX and catalase activities as well as the mean serum iron concentration and TIBC were significantly lower than normal. The mean 288 M. Ira Thabrew et al. / Journal of Ethnopharmacology 76 (2001) 285–291 Table 1 A comparison of: (a) activities of blood SOD, GPX and catalase; and (b) serum iron and TIBC levels of the RA patients before commencement of treatment with corresponding values in normal controlsa Parameter Normal controls RA patients SOD (U/g Hb) 915.40 9 212.2 (742.1–1240.5) 58.40 9 10.1 (22.4–85.6) 1.15 9 0.05 (0.5–1.8) 77.50 9 10.4 (41.0–90.5) 0.321 9 0.140 (0.262–0.322) 272.60 9 45.7b (174.8–411.1) 33.50 912.6b (18.4–58.3) 0.95 9 0.08c (0.4–1.6) 64.70 9 8.1c (35.0–78.2) 0.280 9 0.052c (0.239–0.329) GPX (U/g Hb) CATALASE (KIA units) Serum iron (mg/dl) with MRQ or WC for a period of 3 months are presented in Table 2. In patients treated with MRQ, there was a gradual, statistically significant rise in the activities of all three enzymes. Although there appeared to be a slight rise in these enzyme activities mediated by WC, these changes were not statistically significant. 3.3. Effects of MRQ and WC on lipid peroxidation and iron status of patients haemoglobin level (11.3 g/dl 91.2) in the patients under investigation was also significantly (P B0.001) less than that of the healthy controls (12.990.8 g/dl). These results indicate that RA patients included in the study had lower than normal plasma antioxidant enzyme levels, as well as a lower serum iron concentration and TIBC. Table 3 summarizes the effects of MRQ or WC treatment on the extent of lipid peroxidation (as assessed by TBARS generation), serum iron concentration and the total iron binding capacity in the patients. While both drug preparations were able to cause a significant decrease in TBARS generation, a greater effect was evident in the MRQ treated group than in the WC treated group. With respect to the total iron concentration and TIBC, MRQ treatment resulted in a significant improvement in the serum iron and TIBC levels. The mean Hb concentration also increased from 11.391.2 to 14.1 g/dl. No improvement in the above parameters were observed in the WC treated group. 3.2. Changes in antioxidant enzyme concentrations with treatment. 4. Discussion A summary of changes in activities of the antioxidant enzymes GPX, SOD and catalase on treatment Reactive oxygen intermediates (ROI) such as hydro’ gen peroxide (H2O2), superoxide radicals (O2− ) and TIBC (mg/dl) a Results are expressed as the Means 9 S.D. of 65 RA patients and 20 normal controls. b Significantly different from controls at PB0.001. c Significantly different from controls at PB0.05. Table 2 Activities of the antioxidant enzymes GPX, SOD and catalase in blood of RA patients after treatment with either Maha Rasnadi Quathar (MRQ) or Weldehi choornaya (WC)a Group Enzyme SOD GPX (Activities expressed as a% of the values in patients blood starting treatment with MRQ or WC) MRQ treated (n = 45) Before commencement of treatment After 1 month of MRQ After 3 months of MRQ WC treated (n = 20) Before commencement of treatment After 1 month of WC After 3 months of WC a Catalase 100% 120.3 9 17.4%c (112.5–136%) 144.6 9 16.5%d (115.5–168.5%) 100% 126.1 9 19.1%d (104.5–149.1%) 139.8 9 14.2%d (110.7–161.2%) 100% 112.6 9 7.8%d (100.6–134%) 125.2 9 23.8%d (117.5–150.1%) 100% 112.5 9 12.4%NSb (101.8–122.5%) 114.2 9 10.4%NSb 100% 114.5 9 12.9%NSb (100.5–129.2%) 120.4 9 18.2% NSb 100% 106.3 9 6.8%NSb (101.2–110.2%) 111.2 (102.1–130%) (106.4–148.4%) 9 12.4%NSb (102.4–132.1) Results are expressed as the Mean 9S.D. (determined by the Student’s t-test). Significance between groups was analysed by one way ANOVA. NS, not significantly different from before commencement of treatment with the respective drug. c Significantly different from corresponding values before commencement of treatment at PB0.001. d Significantly different from corresponding values before commencement of treatment at PB0.05. b M. Ira Thabrew et al. / Journal of Ethnopharmacology 76 (2001) 285–291 289 Table 3 Effects of Maharasnadi Quathar (MRQ) and Weldehi Choornaya (WC) on the concentrations of plasma thiobarbituric acid reactive substances (TBARS), serum iron and total iron binding capacity (TIBC) in RA patientsa Group MRQ treated (n = 45) Before commencement of treatment After 1 month of MRQ After 3 months of MRQ TBARS Serum Iron TIBC 100% 100% 100% 75.5 9 11.0%** (50.2 – 85.3%) 104.8 9 4.4% (100 – 114.6%) 103.9 9 3.2% (100 – 114.6%) 66.0 9 14.5%* (38.5 – 76%) 126.8 9 10.2%* (110.5 – 138.2%) 116.1 9 5.1%* (112.5 – 122.3%) WC treated (n = 20) Before commencement of treatment After 1 month of WC After 3 months of WC 100% 100% 83.6 9 12.2%** (72.0 – 94.4%) 100.5 9 2.1% ( 98.2 – 104.8%) 78.2 9 10.8%* (62.4 – 86.2%) 93.4 9 5.2%* (89.1 – 100.2%) 100% 95.3 9 7.4% (88.1 – 108.2%) 88.4 9 9.6%* (76.3 – 102.4%) a Results are expressed as the Means 9 S.D. as determined by the Student’s t-test. Significant differences between groups was determined by one way ANOVA. and * represent significant differences from values before treatment at pB0.001 and pB0.05 respectively. hydroxyl radicals (OH’) are unstable, highly reactive chemical species that are produced in biological systems by various Redox reactions that occur during metabolism. In humans, there are intrinsic enzymatic and non-enzymatic AO detoxifying mechanisms that help to maintain a low ROI concentration in the body (Halliwell and Gutteridge, 1989; Seiss, 1991). Enzymatic mechanisms include a superoxide dismutase (SOD) which dismutates superoxide radicals, and catalase and glutathione peroxidase (GPX) that reduce H2O2 to water and molecular oxygen. Vitamins A, C, E and glutathione are some of the major non-enzymatic AO’s in the body. Considerable evidence accumulated in the past two decades indicates that an imbalance in the AO: pro-oxidant concentration in the body may be responsible for the pathogenesis of many different types of disease conditions including rheumatoid arthritis (Arthur, 1988; Halliwell, 1994). Results of the present investigation demonstrates that, of the two drug formulations under investigation, Maharasnadhl Quathar (MRQ) can mediate a greater rise in the antioxidant enzyme activities of RA patients than Weldehi choornaya (WC). Thus, MRQ could markedly enhance activities of the enzymes SOD, GPX and catalase and mediate a decrease in the plasma TBARS concentration. WC could only mediate a decrease in the TBARS concentration, without any significant influence on the activities of SOD, GPX or catalase. Reasons for the MRQ mediated increase in plasma antioxidant enzyme activities is not clear. It may reflect an increase in antioxidant defences by MRQ at rheumatoid joints to protect against continuing free-radical mediated tissue destruction. SOD, GPX and catalase constitute a co-ordinated system of defence against reactive oxygen species (Halliwell and Gutteridge, 1989; Seiss, 1991). A wide variety of chemical and environmental factors are known to induce these AO enzymes (Halliwell and Gutteridge, 1989; Aruoma, 1993). Whether the improvement in the activities of the above enzymes by MRQ is an actual transcriptional activation, as has been reported for Panax ginseng (Chang et al., 1999) or not, remains to be investigated. Data from clinical assessment of patients (based on criteria stipulated by the American Rheumatism Association) also demonstrates that MRQ was of greater therapeutic benefit to RA patients than WC (data obtained from patient records kept at the RA clinics at the BMARI). At the end of the 3 month drug treatment period, 93% of the patients treated with MRQ had experienced: (a) a marked reduction in pain, stiffness and inflammation; and (b) improvement in the mobility of the affected joints. Of the patients who consumed WC for a period of 3 months, a slight reduction of inflammation was apparent in 12%, but no improvement of the pain or mobility of the affected joints was seen in any. The lack of clinical improvement may have been the main reason why the majority of patients in the WC treated group stopped attending the clinics after the first month of treatment while those on MRQ continued to attend even after the end of the 3 month trial period. Anti-inflammatory drugs including those used in RA (e.g. penicillamine, phenylbutazone, and non-steroidal anti-inflammatory drugs such as piroxicam) could decrease oxidant damage at sites of inflammation by acting as scavengers of ROI and thus decrease the concentrations of tissue destroying oxidants produced by neutrophils, monocytes and macrophages (Wasil, 1987; Aruoma, 1996). Effects of the drug formulations under investigation on activities of antioxidant enzymes indicates that MRQ might also increase resistance to oxidants.It is an accepted fact that RA is accompanied by abnormalities in body iron metabolism. At the onset of inflammation, there is a rapid fall in the ‘total 290 M. Ira Thabrew et al. / Journal of Ethnopharmacology 76 (2001) 285–291 iron’ content of blood plasma followed by an increased deposition of iron proteins in the synovial fluid. The drop in plasma iron correlates closely with the activity of the inflammatory process (Gutteridge, 1986). In the synovial fluid of inflamed joints, the iron released during necrosis, might catalyze the formation of OH’ (hydroxyl) radicals from H2O2, thus contributing to an increase in inflammation. The total serum iron content, TIBC and haemoglobin levels in RA patients selected for this study were also less than the corresponding values in the normal control group of individuals. Of the two drugs under investigation, only MRQ was able to mediate significant improvements in the above parameters in RA patients. In patients on WC, the total serum iron and TIBC continued to decrease even during treatment. Whether this effect of MRQ can be attributed to iron in the herbs used in its preparation or some other mechanism remains to be investigated. Since the activities of SOD, GPX and catalase were enhanced despite an increase in the serum iron content, improvement of the antioxidant status of RA patients by MRQ probably occurs by mechanisms independent of effects on circulating iron levels. The overall results of this investigation demonstrate that MRQ is a more effective drug than WC for providing relief to RA patients. Changes in the AO reserve of these patients is probably one of the mechanisms by which MRQ mediates its beneficial effects. MRQ is constituted from 26 herbs, with Alpinia calcarata as the major component (comprises 70% of the total mixture). Although WC is a mixture of 16 herbs, no Alpinia is used in its formulation. Scientifically controlled studies have shown that Alpinia calcarata extract exerts significant anti-inflammatory activity in both rodents and human arthritic patients (Ranasingha, 1979). Although no information regarding the AO potential of this plant is available in published literature, components from a close relative, A1pinia galanga has been shown to possess significant AO activity. Thus, Cheah and Gan (2000) have reported that by mixing raw minced beef with an extract of Alpinia galanga (0– 0.10% wt./wt.), lipid oxidation that occurs during refrigerated storage of the meat can be reduced significantly, and thus help to extend its shelf life. The inclusion of Alpinia calcarata as the major component in the MRQ mixture may be one of the principal reasons for the greater benefit of this formulation to RA patients, than WC. Acknowledgements This research was supported by a University of Kelaniya research grant. Our appreciation to the Director of the Bandaranayake Memorial Ayurvedic Institute (BMARI), Nawinna, for granting permission to use patients attending RA clinics at the institute, for our study. Assistance given by Ayur. Dr Sewwandi of the BMARI, in the collection of patient data, and the technical support given by C.S. Senarath and C.P.K.S. Keerthisena, department of Biochemistry and Clinical Chemistry, is also gratefully acknowledged. References Arthur, M.P.J., 1988. Reactive oxygen intermediates and liver injury. Journal of Hepatology 6, 125 – 131. Aruoma, O.I., 1993. Use of DNA damage as a measure of pro-oxidant actions of antioxidant food additives and nutrient components. In: Halliwell, B., Aruoma, O.I. (Eds.), DNA and Free Radicals, Ellis Horwood, London, pp. 315 – 327. Aruoma, O.I., 1996. Characterization of drugs as antioxidant prophylactics. Free Radical Biology and Medicine 20, 675 – 705. Aust, S.D., 1985. Lipid peroxidation. In: Greenwald, R.A. (Ed.), Handbook of Methods for Oxygen Radical Research, CRC Press, Boca Raton, FL, pp. 203 – 207. The Ayurveda Pharmacopoeia, 1975. Department of Ayurveda Publication, Colombo, Sri-Lanka. Blake, D.R., Hall, N.D., Bacon, P.A., Dieppe, P.A., 1981. The importance of iron in rheumatoid disease. Lancet 2, 1142 – 1144. Blake, D.R., Merry, P., Unsworth, J., Kidd, B.L., Outhwaite, J.M., Ballard, R., Morris, C.J., Gray, L., Lunec, J., 1989. Hypoxicreperfusion injury in the inflamed human joint. Lancet 1, 289 – 293. Ceriotti, F., Ceriotti, G., 1980. Improved direct specific determination of serum iron and total iron binding capacity. Clinical Chemistry 26, 327 – 331. Chang, M.S., Lee, S.G., Rho, H.M., 1999. Transcriptional activation of Cu/Zn dismutase and catalase genes by panaxadiol ginsenoides extracted from Panax ginseng. Phytotherapy Research 13, 641 –644. Cheah, P.B., Gan, S.P., 2000. Antioxidant and antimicrobial effects of galangal and alpha-tocopherol in minced beef. Journal of Food Protein 63, 404 – 407. Cotran, L.S., Kumar, V., Robbins, S.L., 1989. Robbins Pathologic Basis of Disease, fourth ed., p. 1349 Goth, L., 1991. A simple method for determination of serum catalase activity and revision of reference range. Clinica Chemica Acta 196, 143 – 152. Gutteridge, J.M.C., 1986. Antioxidant properties of the proteins ceruloplasmin, albumin and transferrin A. A study of their activity in serum and synovial fluid from patients with rheumatoid arthritis. Biochemistry and Biophysics Acta 869, 119 – 127. Halliwell. B., Gutteridge, J.M.C., 1989. In: Free Radicals in Biology and Medicine, second ed. Clarendon Press, Oxford. Halliwell, B., 1994. Free radical antioxidants in human disease. Curiosity, cause or consequence. Lancet 344, 721 – 724. Joyeaux, M., Lobstein, A., Anton, R., Mortier, F., 1995. Comparative antillpoperoxldant, anti-necrolic and scavenging properties of terpenes and biflavones from Gingko and some flavanoids. Planta Medica 61, 126 –129. Krane, S.M., Conca, W., Stephenson, M.I., Amento, E.P., Goldring, M.B., 1990. Mechanisms of matrix degradation in rheumatoid arthritis. Annals of the New York Academy of Sciences 580, 340 – 354. Mitra, S.K., Venkataranganna, M.V., Sundaram, R., Gopumadha- M. Ira Thabrew et al. / Journal of Ethnopharmacology 76 (2001) 285–291 van, S., 1999. Antioxidant activity of AO-8, a herbal formulation in vitro and in vivo experimental models. Phytotherapy Research 13, 300 – 303. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative characteristics of erythrocyte glutathione peroxidase. Journal of Laboratory Clinical Medicine 70, 158 –169. Ranasingha, S.G., 1979. Clinical and experimental studies on antiarthritic property of Alpinia calcarata Rosc (Sri Lankan Rasna). Thesis submitted for the degree of MD. (Ay.), Dept. Kayachikitsa, Institute of Medical Sciences, Baranas Hindu University, India, I – 244. . 291 Scott, B.C., Butler, J., Halliwell, B., Auroma, O., 1993. Evaluation of the anti-oxidant actions of ferulic acid and catechins. Free Radical Research Communications 19, 241 – 253. Seiss, H., 1991. Oxidative stress: From basic research to clinical applications. American Journal of Medicine 91 (3C), 375 – 385. Wasil, M., 1987. Biologically significant scavenging of the myeloperoxidase derived oxidant hypochlorous acid by some anti-inflammatory drugs. Biochemical Pharmacology 36, 3847. World Health Organization, 1980. Haemoglobin: cyanomethaemoglobin, photometric method. Manual of Basic Techniques for a Health Laboratory, Geneva, pp. 371 – 374. Journal of Ethnopharmacology 76 (2001) 293– 297 www.elsevier.com/locate/jethpharm Short communication Ethnomedicinal and obnoxious grasses of Rajasthan, India S.S. Katewa *, B.D. Guria, Anita Jain Laboratory of Ethnobotany and Agrostology, Department of Botany, College of Science, M.L. Sukhadia Uni6ersity, Udaipur-313 001, Rajasthan, India Received 14 August 2000; received in revised form 21 February 2001; accepted 14 March 2001 Abstract The present communication is an account of 24 ethnomedicinal and 10 obnoxious grasses of Rajasthan. The ethnomedicinal grasses are used by forest dwellers for various ailments in their daily life. In spite of the fact that certain grasses are of high medicinal value, some grasses are also alleged to be obnoxious. These obnoxious grasses hamper the economic balance of tribal as well as rural communities. The necessity to popularise the identity and utility of grasses and the need to build up a database on local knowledge/information held by tribes is stressed. Local name, Botanical name, plant parts used, causing injuries and methods of application or administration are given. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Grasses; Ethnomedicinal; Obnoxious; Rajasthan 1. Introduction Rajasthan is the largest state of India located in the north-western part of India (Fig. 1). Geographically it lies between 23°3% to 30°12%N longitude and 69°30% and 78°17%E latitude. The vegetation of Rajasthan has considerable diversity on account of a variety of ecosystems found in different parts of the state. Apart from the predominant desert ecosystem, there are grasslands, scrub jungles, wetlands and deciduous forests each supporting a characteristic assemblage of plant species. Many tribes are scattered in different parts of Rajasthan, but the southern part of the state is the homeland of several tribes (Fig. 1) which have their own separate identities. Considerable published work on grasses are that of Kirtikar and Basu (1935), Caius (1937), Chopra et al. (1956, 1965), Bor (1960), and Chaudhari and Pal (1975). Also in Rajasthan, a lot of work has been done on ethnomedicinal plants used for various ailments by tribal communities (Sebastian and Bhandari, 1984, 1988; Joshi, 1995; Katewa and Arora, 1997; Katewa and Guria, 1997; Katewa and Sharma, 1998; Singh and Pandey, 1980). Perusal of the literature indicated that no comprehensive ethnobotanical work has been pub* Corresponding author. lished yet on grasses as compared to other wild dicotyledonous plants. Therefore, in the present paper an attempt has been made to record ethnomedicinal and obnoxious properties of certain grasses of Rajasthan hitherto unreported. 2. Methodology An ethnobotanical survey was conducted repeatedly during 1994 – 1999 in different seasons and areas. Before actually launching into the fieldwork, a rapport was established with one or two people, preferably the chief, guidance sought and contact was then established with other tribes in the locality. The local informants were medicinemen, men and women working in the field, village headmen, priests and other prominent leaders. Field sites were visited with the local medicineman. People mostly above the age of 60 have accurate information regarding their old traditions. Generally, two types of interviews were conducted, firstly of individuals and secondly of groups. Of individuals, people were selected at random on the way to or entering a hut, thus finding knowledgeable individuals from the village or also the bhopa (village priest) or the headman. In group interviews more than one individual was approached, our purpose explained and interviews con- 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 3 3 - 1 294 S.S. Katewa et al. / Journal of Ethnopharmacology 76 (2001) 293–297 ducted. In forests surrounded by ambient vegetation the tribes were prompted to remark on the usefulness of species, especially when accompanied by a group. In smaller groups, women were less in number, but both sexes were represented in larger groups and this resulted in heterogeneity of information. To determine the authenticity of information collected during field work, repeated verification of data from different informants and in different areas at different times was done. Thus, only specific and reliable information that was crosschecked with at least 14 informants has been incorporated in the present study. Participating in their feasts, festivals, other social events, etc., was of great use in collecting information on plants and observing how they are used. During the survey some interesting folk uses of grasses have come to light which are not mentioned in important medicinal literature. The collected medicinal and obnoxious grasses were identified up to species level at the Herbaria of Forest Research Institute, Dehradun. All the collected specimens were deposited in the Herbarium of Laboratory of Ethnobotany and Agrostology, Department of Botany, College of Science, M.L. Sukhadia University, Udaipur for authentication of information and further references. Numbers of voucher specimens is also provided. The information about the medicinal and obnoxious properties of grasses are given by mentioning their botanical name, local name, mode of administration of medicinal grasses and harmful effect of obnoxious grasses. 3. Results and discussion Ethnomedicinal and obnoxious studies on the grasses of Rajasthan are of considerable interest as the economy of the state mostly depends on the grass. Although much ethnomedicinal studies on plants other than the grasses has been carried out, such properties of grasses is restricted to a limited number of people of the study Fig. 1. Map of Rajasthan showing the localities of tribals. S.S. Katewa et al. / Journal of Ethnopharmacology 76 (2001) 293–297 Table 1 Ethnomedicinal grasses of Rajasthan Botanical name Local name Herbarium number Ethnomedicinal uses Alloteropsis cimicina (Linn.) Stapf Apluda mutica Linn. Basanti ghass Tamta bheda EA 13 Root paste is used in toothache EA 12 Capillipedium heugelii (Hack.) Blatt. et McCann. Cynodon dactylon (Linn.) Pers Rohis EA 56 Doob EA 50 Coix lacryma-jobi Linn. Garelo EA 41 Cymbopogon martinii (Roxb.) Wats. Rhoido EA 58 Dactyloctenium aegyptium (Linn.) P. Beauv. Dactyloctenium sindicum (Boiss.) Cenchrus ciliaris Linn. Malicha EA 65 Poultice of whole plant is used to cure mouth sores of cattle. It is also given to cattle with small fishes to cure flatulence. Essential oil is extracted from this grass and used for massage in rheumatism. Aqueous extract of plant with sugar is given to persons suffering from nostril haemorrhage. Paste of young leaves with sugar is used to stop bleeding from cuts and wounds. Leaf juice with a pinch of common salt is taken orally in stomach-ache. Decoction of whole plant is given orally to cure menstrual problem. The seeds are boiled in water and then eaten to cure dysentery. The leaf juice is given orally in urinary complaints. Paste of leaves is warmed with mustard oil (Brassica compestris) and then used in massage to relieve rheumatic pain. Oil extracted from leaves is used to cure skin diseases. Decoction of leaves is given in cold and fever. Its smoke is used as rodent and mosquito repellent. Crushed leaves and stem is given to children in stomach-ache. Decoction of leaves is used as diuretic. Leaf extract is used to remove insect from the ear. Grain powder mixed in water is used in stomach-ache. Gathia/ Mokraghas Anjan ghas/ Dhaman EA 72 Desmostachya bipinnata (Linn.) Stapf. Daab/ Dabra EA 59 Echinochloa crusgalli (Linn.) P. Beauv. Eleusine indica (Linn.) Gaertn. Hemarthria compressa (Linn.) R.Br. Heteropogon contortus (Linn.) P. Beauv. Imperata cylindrica (Linn.) P. Beauv. Panicum antidotale Retz. Paspalum scrobiculatum Linn. Batti EA 88 Chitki EA 83 Kataru/ Ghora Doob EA 104 Lapia/ Lapida EA 93 Siru EA 119 Mall/Muret EA 340 Kodra EA 360 Perotis indica (Linn.) O. Ktze. – EA 373 Phragmites karka (Retz.) Trin. Saccharum bengalense Retz. – EA 398 Munj EA 412 EA 54 Plant extract is taken orally in rheumatism. Decoction of roots is given to children suffering from intestinal worms. Paste of whole plant is taken orally to cure dysentery. Leaf paste is used to cure cuts and wounds. The root paste is useful in asthma and jaundice. Juice of whole plant is taken orally to cure nostril haemorrhage Paste of whole plant along with water is taken orally in fever. Root paste is taken orally with a pinch of common salt during indigestion. Root paste is taken orally in snake and dog bite. Poultice of root paste is also tied on the bite portion for early cure. Root paste is externally applied in scorpion bite. Poultice of paste of the whole plant is tied over wounds for early cure. Paste of whole plant is used for skin diseases. It is also effective on boils and sores. Decoction of dried inflorescence is taken orally to stop bleeding after child birth. The leaf juice along with water is used to cool the body. Approx. 100 g fresh leaves are taken and cut into small pieces. Leaves are then boiled in approx. two cups of water till it is reduced to half cup and then filtered. This filtrate is taken orally for 3 days to cure menstrual complaint. It is also a good remedy for abortion. The juice of leaves is taken orally in fever. 295 S.S. Katewa et al. / Journal of Ethnopharmacology 76 (2001) 293–297 296 Table 1 (Continued
Botanical name Local name Herbarium number Ethnomedicinal uses Sorghum halepense (Linn.) Pers. Themeda quadri6al6is (Linn.) O. Ktze Baru EA 425 Godli ghas EA 433 Veti6eria zizanioides (Linn.) Nash Zea mays Linn. Khas/Baliya ghass Makki EA 440 Juice of whole plant is taken orally with a pinch of common salt in fever. Paste of whole plant is warmed and used to cure the septic wounds. Small fishes moulded in this grass is fed to cattle with flatulence. Root paste along with water is taken orally in anthelmintic problems Juice of leaves is taken orally to cure renal disorders. The root decoction is also useful in ulcers. EA 445 area. As a result of this study, the ethnomedicinal uses of approximately 24 grasses have been reported for the first time in the study area (Table 1). Similarly, 10 obnoxious grasses have also been reported from the study area (Table 2). The present study shows that grasses like Cynodon dactylon, Cymbo- pogon martinii, Saccharum bengalense, Coix lacrymajobi, Desmostachya bipinnata and Echinochloa crusgalli have remarkable medicinal properties. Hence the role of ethnobotanical surveys and field work is of crucial importance as some miraculous medicines are known to the tribes and aboriginals and much acquired Table 2 Obnoxious grasses of Rajasthan Botanical name Local name Herbarium number Obnoxious properties Aristida adscensionis Linn. Lampro EA 6 Aristida funiculata Trin. et. Rupr. Lamp EA 9 A6ena fatua Linn. Jei EA 23 Cenchrus biflorus Roxb. Bhurat EA 53 Dendrocalamus strictus Nees. Bans EA 92 Heteropogon controtus (Linn.) P. Beauv. Lapia/ Laplo EA 93 Paspalum scrobiculatum Linn. Kodra EA 360 Pennisetum purpureum Schumach. Rottboellia exaltata Linn. f. Sorghum halepense Linn. Pers. Elephant grass EA 332 – EA 404 Baru EA 425 Stiff, fine awns causes injuries by lodging between the teeth of cattle and thus pus formation takes place. It is quite painful due to its typically contorted and very troublesome awns since their pointed callus spears through the clothes. Development of hair balls takes place occasionally in the stomach of horses which are fed upon the straw of this grass. The stiff bristles causes injury by lodging between the teeth, lips and even mouth and throats of grazing animals which results in pus formation. It also poses a great difficulty to the farmers while working in their field. The very fine stiff fragile hairs on the young sheaths are harmful to skin, as its hairs causes irritation. The mature grass causes mechanical injury to the cattle. The stiff powerful awns and basal calli when adhered to the soft parts of grazing animals, abscesses may occur. The grains are used as famine food by the tribals of Rajasthan but it contains a poisonous narcotic constituent which causes vomiting and vertigo. This grass causes mechanical injury to cattle, because of sharp edges on the leaf and thus cutting of the flesh. The stiff hairs of this grass adhere to the skin and cause irritation. This grass is very poisonous in the early stages of growth. HCN is present in the young stage of growth which is fatal to cattle. S.S. Katewa et al. / Journal of Ethnopharmacology 76 (2001) 293–297 knowledge through the ages is usually passed on from generation-to-generation as a guarded secret of certain families. Therefore, it is necessary to popularise the identity and utility of the grasses. More efforts should be done for identification of such medicinal grasses through ethnobotany. As population and demand for agricultural land by modern man increase, the forest areas are gradually cleared and converted into townships, agriculture land and plantation. This process of encroachment on the rich forest of Rajasthan has been steadily increasing, with the result that today we are faced with the possibility of total extermination of the forest and the floristic wealth they contain. Also, as the domain of civilised man extends into the forest lands, the tribal culture is gradually lost. This process has already led to our loosing forever a wealth of empirical knowledge the forest tribes have had, particularly with respect to medicinal plants. It is therefore pertinent that ethnobotanical studies be undertaken so that we may preserve and record the knowledge about useful plants and conserve the plants themselves before both are lost in society’s quest for land to raise cash and plantation crops. The knowledge of poisonous grasses is very old and yet it is surprising that no attempt has hitherto been made in India to study poisonous grasses, particularly in Rajasthan where frequent incidents of sheep death due to grass poisoning occurs. Many of these grasses are known to be nourishing, palatable forest grasses, but under certain conditions, such as in the process of wilting or under conditions of drought, become deadly poisonous because of large quantities of prussic acid (HCN) released in their tissues. The grains of certain grasses are eaten by tribes of south Rajasthan. It is possible that those who use grains as food may often become seriously ill. Many grasses cause mechanical injury to cattle, causing severe wounds, thus less production of meat and even death in some cases. So the importance of the study of poisonous grasses to a state like Rajasthan where the economy depends on livestock . 297 cannot be overrated. It is therefore of fundamental importance to any country to gain a complete knowledge of their distribution, chemical constituents and physiological action. Acknowledgements The authors are thankful to the Indian Council of Agricultural Research, New Delhi for providing financial support for the present study. References Bor, N.L., 1960. Grasses of Burma, Ceylon, India and Pakistan. Pergamon Press, London. Caius, J.F., 1937. The medicinal and poisonous grasses of India. J. Bombay Nat. Hist. Soc. 38, 540 – 584. Chaudhari, R.H.N., Pal, D.C., 1975. Less known uses of some grasses of India. Bull. Bot. Soc. Bengal 32, 48 – 53. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Medicinal Plants of India. Coun. Sci. and Ind. Res, New Delhi. Chopra, R.N., Badhwar, R.L., Singh, S., 1965. Poisonous Plants of India, vol. II. ICAR, New Delhi. Joshi, P., 1995. Ethnobotany of the Primitive Tribes in Rajasthan. Printwell, Jaipur. Katewa, S.S., Arora, A., 1997. Some plants in folk medicine of Udaipur district (Rajasthan). Ethnobotany 9, 48 – 51. Katewa, S.S., Guria, B.D., 1997. Ethnomedicinal observations on certain wild plants from southern Aravalli hills of Rajasthan. Vasundhara 2, 85 – 88. Katewa, S.S., Sharma, R., 1998. Ethnobotanical observations from certain watershed areas of Rajasthan. Ethnobotany 10, 46 – 49. Kirtikar, K.R., Basu, B.D., 1935. Indian Medicinal Plants, 2nd ed. Lalit Mohan Basu, Allahabad 4 Vols. Sebastian, M.K., Bhandari, M.M., 1984. Medicinal plant lore of Udaipur district, Rajasthan. Bull. Med.-ethnobot. Res. 5, 122 – 134. Sebastian, M.K., Bhandari, M.M., 1988. Medicinal plant lore in Udaipur district, Rajasthan. Bull. Med. Ethno. Bot. Res. 5 (3-4), 133 – 134. Singh, V., Pandey, R.P., 1980. Medicinal plant lore of the tribals of eastern Rajasthan. J. Econ. Tax. Bot. 1, 137 – 147. Journal of Ethnopharmacology 76 (2001) 299– 304 www.elsevier.com/locate/jethpharm Short communication The effect of fennel essential oil on uterine contraction as a model for dysmenorrhea, pharmacology and toxicology study S.N. Ostad a,*, M. Soodi a, M. Shariffzadeh a, N. Khorshidi b, H. Marzban c a Department of Toxicology and Pharmacology, Faculty of Pharmacy, Uni6ersity of Tehran Medical Sciences, PO Box 14155 /6451, Tehran, Iran b Department of Embryology and Anatomy, Faculty of Medicine, Uni6ersity of Tehran Medical Sciences, PO Box 14155 /6451, Tehran, Iran c Department of obstetrics, Faculty of Medicine, Uni6ersity of Iran Medical Sciences, PO Box 14155 /6451, Tehran, Iran Received 14 August 2000; received in revised form 3 March 2001; accepted 11 April 2001 Abstract Increasing the ectopic uterine motility is the major reason for primary dysmenorrhea. This motility is the basis for several symptoms including for pain is the main complaints of patients with primary dismenorrhea. There are several mechanisms, which initiate dysmenorrhea. Therefore, different compounds can be employed to control its symptoms. In long-term therapy, combination of oestrogens and progestins may be useful. In short-term therapy, dysmenorrhea sometimes non-steroidal anti-inflammatory drugs (NSAIDs) are used. Most of NSAIDs in long-term therapy show severe adverse effects. In an attempt to find agents with less adverse effect the fennel essential oil (FEO) was chosen for this investigation. In this article, effects of FEO on the uterine contraction and estimation of LD50 in rat were described. For assessment of pharmacological effects on the isolated rat uterus, oxytocin (0.1, 1 and 10 mu/ml) and prostaglandin E2 (PGE2) (5× 10 − 5 M) were employed to induce muscle contraction. Administration of different doses of FEO reduced the intensity of oxytocin and PGE2 induced contractions significantly (25 and 50 mg/ml for oxytocin and 10 and 20 mg/ml PGE2, respectively). FEO also reduced the frequency of contractions induced by PGE2 but not with oxytocin. LD50 of FEO was obtained in the female rats by using moving average method. The estimated LD50 was 1326 mg/kg. No obvious damage was observed in the vital organs of the dead animals. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dysmenorrhea; Fennel essential oil; Pain 1. Introduction Menstrual pain that occur in the absence of visible organic pelvic origin is nominated primary dysmenorrhea. More than half of all postmenarcheal women complain of dysmenorrhea at least once during their life. About 10% of these women cannot have normal life for 1 – 3 days in each month (Dawood, 1990). It is shown that the pain associated with this disorder is caused by hypercontractility of uterine muscle, subse* Corresponding author. Tel.: + 98-21-6112316; fax: + 98-216461178. E-mail address: snostad@yahoo.com (S.N. Ostad). quent reduction in blood flow and concomitant uterine ischemia (Akerlund, 1979). Several factors may involve in the uterine hypercontractility including: ovarian steroids, cervical obstruction, pituitary hormones and most notably prostaglandins. It has been demonstrated that prostaglandin production in the uterine lining is up to seven times greater in women with clinically diagnosed dysmenorrhea as compared with controls. For this reason, successful treatment of this disorder has been achieved with drugs that inhibit prostaglandin synthesis and reduce uterine hypercontractility (Dawood, 1990). Tocolytic agents such as b2 stimulator or calcium channel blockers are employed to inhibit muscular contraction and increase uterine blood flow. In 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 4 9 - 5 300 S.N. Ostad et al. / Journal of Ethnopharmacology 76 (2001) 299–304 spite of their effectiveness, side effects of these drugs in long-term therapy limit its clinical uses (Hansen and Secher, 1975; Anderson et al., 1978). Fennel (foeniculum vulgare mill) is a well-known umbelliferous plant. The seeds of this plant have been known to be able to promoter of menstruation, alleviate the symptoms of female climacteric, and increase libido (Albert-Puleo, 1980). FEO also possesses emnenagague and galactagogue properties (Hare et al., 1916). It has been previously reported that FEO used in the pediatric colic and some respiratory disorders due to its anti-spasmodic effects (Reynolds, 1982). Seeds of fennel are used in folk remedies for treatment of dysmenorrhea. This traditional usage might be related to anti-spasmodic effects of FEO. The present study was designed to examine the scientific basis of traditional use of FEO by routine pharmacological and toxicological studies. 2. Methods and materials 2.1. E6aluation of pharmacological acti6ity Uterine strips were obtained from virgin Wistar rats (180 –250 g) kept in a controlled temperature (259 1 °C) and illumination (12 h on, 12 h off) room. The animals were treated with oestradiol valerate (5 mg/kg, s.c.) 24 h before the experiments. Animals were then be scarified by cervical dislocation and the uterine horns were removed and placed in dejalon solution (NaCl, 154 mmol/l; KCl, 5.6 mmol/l; CaCl2, 0.3 mmol/l; MgCl2, 1.4 mmol/l; NaHCO3, 1.7 mmol/l and glucose, 5.5 mmol/l). Uterine strips (15-mm long) free from adhering tissues were mounted in 50 ml jacketed organ bath containing dejalon solution, maintained at 31 °C and continuously aerated. One end of tissue was tied to perspex holder and the other end of the tissue was tied under 1 g resting tension to grass f-50 isometric transducer. Tension was recorded on a physiograph recorder as described by Calixto and Yunes (1991). Following an equilibration period of 30– 40 min, cumulative curves (Von Rossum, 1963) for oxytocin (obtained from Pars Minoo Co., Ltd., Iran) were constructed. After the concentration– response curves became constant, different concentration of FEO (25, 50 and 100 mg/ml final concentration in the organ bath, obtained from Barij Essence Co. Ltd., Iran) were added to the bath and left in contact with the tissue for 10– 20 min. Different concentrations of FEO (10, 20 and 40 mg/ml) were employed to inhibit response of uterine when was treated by 5× 10 − 5 M of prostaglandin E2 (PGE2) (Prostin ER 2 , Upjohn, USA) final concentration in organ bath. Oxytocin and PGE2 were added to separate strips and control experiments were performed with the vehicle (90% ethyl alcohol added to the organ bath). 2.2. Acute toxicity assay The routine acute toxicity assay was performed using female Sprague–Drawly rats, which were bred on house and acclimated 2 weeks before experiment (Ecobichon, 1997). Due to possible oral use of FEO for dysmenorrhea, the oral LD50 protocol was performed for it. FEO was dissolved in minimum amount of 90% ethyl alcohol and used throughout experiments. Due to small amount of FEO, it was used with normal saline. Vehicle group was treated in the same manner without using FEO. To obtain toxic dose level and reducing the number of animals, which should be used, five rats were chosen randomly with average weight of 183934 g. Four doses of 50, 500, 1000 and 2000 mg/kg of FEO and vehicle were administered by gavages to each rat. Animals were starved for 16 h before each experiment. Furthermore, the volume of gavages for each experiment was constant by using different concentration of FEO in 90% ethyl alcohol. Maximum amount of alcohol, which was used in the test group, was chosen for vehicle control. Animals were evaluated for any possible toxic effects. Dosages for final experiment was chosen according to the results obtained from observed our experiment. 2.2.1. LD50 assay LD50 assay was studied in eight groups of rats. Each group contained five rats with average weight of 1809 25 g. These groups were divided into control, vehicle, 1000, 1125, 1250, 1375 and 1500 mg/kg of FEO, respectively. After administration of FEO animals were observed closely during first 48 h and then every 12 h. Lung, liver, kidney, stomach and intestine of dead animals were examined for any pathological sign by using histological methods. Alive animals were observed for any behavioural, respiratory and cutaneous changes. 2.3. Statistical analysis Statistical analysis of the pharmacological data was performed with an analysis of variance followed by the Newman – Keuls test. Differences with P B0.05 and 0.01 were considered statistically significant. The moving average method was employed for determination of LD50. 3. Results 3.1. Pharmacological study Figs. 1 and 2 show the inhibitory effect of fennel essential oil (FEO) on the response of uterine when treated by oxytocin and PGE2, respectively. As it is S.N. Ostad et al. / Journal of Ethnopharmacology 76 (2001) 299–304 301 shown in Fig. 1, 25 mg/ml of FEO caused no remarkable effect in uterine contraction. FEO at 50 mg/ml caused a parallel and concentration dependent rightward displacement of oxytocin (0.1– 10 mu/ml) dose – response curve (P B0.01). Fig. 2 shows the significant inhibitory effect of FEO on the response caused by 5× 10 − 5 M PGE2 (PB 0.01). In the case of PGE2, both the frequency and intensity of uterine contraction decreased during experiment but in the case of oxytocin Table 1 Results of animal death in the LD50 final measurement Fig. 1. Effects of different concentrations of FEO on oxytocin-stimulated rat uterine muscle contraction. The muscle strip was pretreated either with vehicle () or FEO 25 ( ) and 50 mg/ml ( ) immediately before different doses of oxytocin (0.1, 1 and 10 mU/ml). (**) Data are presented as mean 9 SEM of three separate experiments, PB 0.01 different from control group. Fig. 3. Transverse section of liver capsule with port area in control group. H, hepatocytes; CV, central veins; PS, portal sinusoid; S, sinusoid (H&E, 100 × ). Group number Dose (mg/kg) Number exposed Number of death Total (%) 1 2 3 4 5 6 7 8 Control Vehicle 750 1000 1125 1250 1375 1500 5 5 5 5 5 5 5 5 0 0 0 0 1 0 3 5 0 0 0 0 20 0 60 100 only the intensity decreased by increasing the dose of FEO. 3.2. Toxicity study Fig. 2. Effects of different concentrations of FEO on PG E2-stimulated rat uterine muscle contraction. The muscle strop was pretreated either with vehicle (A) or FEO 10 (B), 20 (C) and 40 mg/ml (D) immediately before PG E2 (5× 10 − 5 M). (**) Data are presented as mean9 SEM of three separate experiments, PB 0.01 different from control group. In present study, during the first 24 h animals in groups of 1500 and 2000 mg/kg FEO died. Based on these results, the final test was performed in the range of 750– 1500 mg/kg. Table 1 shows the result of final test. The value of LD50 was 1326 mg/kg that was calculated by probit analysis method. Animals in group 8 showed prostration, sedation, respiratory distress, movement disorder, and unresponsiveness to external stimulation, hind limb weakness, tremor and fasciculation in dorsal muscles during first 24 h after FEO ingestion. The severity of toxicity in groups 7, 6 and 5 decreased respectively and other groups showed no significant toxicity. In groups 4, 5 and 6, the most dominant adverse effect was sedation. In all groups, the amount of 24 h urine increased parallel to the amount of given FEO. Histological observations in different tissues of dead animals did not show any significant tissue damage (Figs. 3–12). 302 S.N. Ostad et al. / Journal of Ethnopharmacology 76 (2001) 299–304 4. Discussion The present results demonstrate that FEO can inhibit contraction of isolated uterus that was induced by Fig. 7. Transverse section of the Bronchi in control group. Al, aleolar terminal; BR, bronchiole respiratory; TB, terminal bronchiole (H&E, 100 × ). Fig. 4. Transverse section of liver capsule with port area, which is treated with 2000 mg/kg fennel oil, notes no remarkable histological changes as compared with control group (H&E, 100 × ). Fig. 8. Transverse section of the bronchi in the group, which is treated with 2000 mg/kg fennel oil, notes no remarkable histological changes as compared with control group (H&E, 100 × ). Fig. 5. Transverse section of the kidney cortex in control group. P, proximal convoluted tubule; D, distal convoluted tubule; G, glumerule; C, capsule of Boumen (H&E, 100 × ). Fig. 9. Transverse section of the cardiac muscle in control group noted normal endocardium. CM, cardiac muscle; Endocardium (H&E, 100× ). Fig. 6. Transverse section of the kidney cortex group, which is treated with 2000 mg/kg fennel oil, notes no remarkable histological changes as compared with control group (H&E, 100 ×). oxytocin and PGE2. The pattern of response during the administration of oxytocin and PGE2 showed that the mechanism of FEO inhibition was probably different in these two cases. These effects also confirm the previous S.N. Ostad et al. / Journal of Ethnopharmacology 76 (2001) 299–304 303 report about uterine relaxation after FEO administration (Saleh et al., 1996). Although all NSAIDs can reduce this contractility by inhibition of arachidonic acid pathway but some NSAIDs such as diclofenac, piroxicam and indomethacin, which have direct muscle relaxation effect, seems to be more efficient. In the case of diclofenac, the contraction of uterine muscle, which is induced by 4 mg/ml oxytocin, was inhibited by 81 mg/ml in vitro (Perez et al., 1990). The optimum dose of FEO in our experiment for inhibition of similar contraction is 100 mg/ml suggesting that the mechaFig. 12. Transverse section of the stomach in the group, which is treated with 2000 mg/kg fennel oil, notes no remarkable histological changes as compared with control group (H&E, 100 × ). Fig. 10. Transverse section of the cardiac muscle in the group, which is treated with 2000 mg/kg fennel oil, notes no remarkable histological changes as compared with control group (H&E, 100 × ). Fig. 11. Transverse section of the stomach in control group note normal epithelial tissue cell coat mucosa. ET, epithelial tissue; M, cell coat mucosa (H&E, 100 × ). nism of action is possibly similar to diclofenac. Although the mechanism of action of fennel oil is unknown but it seems the uterine smooth muscles are directly influenced by FEO as well as its central action in intact animal. Furthermore, the effect can be contributed to oestrogenic activity of FEO. It has been shown that the isometric developed tension of spontaneous contractions in isolated rat uterine strips is clearly influenced by sex hormones (Gimeno et al., 1979; Sterin-Speziale et al., 1980; Chaud et al., 1984). Moreover, uterine preparations isolated from ovariectomised rats treated with 17-beta-oestradiol (17-b-E2), exhibited a more diminished spontaneous motility than the same time from 17-b-E2 untreated animals (SterinSpeziale et al., 1980). The main constituent of fennel oil is anol or dimethylated anethole that may have mild oestrogen-like activity (Albert-Puleo, 1980) and induces oestrus in mice (Dodds and Lawson, 1937) inhibit spasms in smooth muscles (Forster et al., 1980). The results of acute toxicity test showed that the FEO could be classified in the group of slightly toxic substance on the basis for classification of chemical substances. No pathological toxicity was seen in organs of dead animals, which indicate that death may be due to metabolic effects of metabolite imbalance or some kind of nervous toxicity. The emotional and behavioural toxicity in intoxicated animals may confirm this conclusion. Anethole is the major fragrant in the FEO (Duke, 1965). It has been shown that anethole induce ataxia and hypnotic activity in rat (Boissier et al., 1967). Several pathophysiological phenomena can cause dysmenorrhea. Although the reason of dysmenorrhea is not fully understood but it seems irregularity in the peristaltic movement in the uterine muscle should be the major cause of dysmenorrhea. PGs may be one of the groups that cause these pathophysiological disorders and so for several years prostaglandin inhibitors were employed to control symptoms of dysmenorrhea. NSAIDs with muscle relaxant property are more useful 304 S.N. Ostad et al. / Journal of Ethnopharmacology 76 (2001) 299–304 and therefore the major factor for control of symptoms can be muscle relaxation. Increasing muscle relaxation may lead to increase in volume of total bleeding but this increase is not too much problem for normal women (Khorshidi et al., unpublished data). If this negative point outweigh with effect of FEO on reducing dysmenorrhea pain, FEO may be of use for alleviation of dysmenorrhea sequalee. Acknowledgements Authors would like to thank the undersecretary for medical research of Ministry of Health and Medical Education of Iran for their financial support of this project, Dr Ebrahim Azizi for his advice and Barij essence Co. Ltd. for giving standard FEO generously. References Albert-Puleo, M., 1980. Fennel and anise as estrogenic agent. Journal of Ethnopharmacology 2, 337 –344. Akerlund, M., 1979. Pathophysiology of dysmenorrhea. Acta Obstetrics and Gynaecology of Scandinavia 87, 27 – 35. Anderson, A.B.M., Haynes, P.J., Fraser, I.S., Turnbull, A.C., 1978. Trial of prostaglandin synthetase inhibitors in primary dysmenorrhea. Lancet 1, 345 –348. Boissier, J.R., Simon, P., Bourhis, B.L., 1967. Experimental psychotropic effect of isomeric cis and trans anetholes. Therapeutics 22 (2), 309 – 323. Calixto, J.B., Yunes, R.A, 1991. Antagonism of kinin induced contraction of isolated rat uterus by the crude hydroalcoholic extract from manderilla illustris. General Pharmacology 22 (1), 99 – 101. Chaud, M., Gonzalez, E.T., Franchi, A.M., Gimeco, M.F., Gimeno, A.L., 1984. Sex hormones and the motility of and prostaglandin . output from uterine horns of immature rats. Prostaglandins Leukemia and Medicine 15, 35 – 44. Dawood, M.Y., 1990. Dysmenorrhea. Clincical Obstetrics and Gynaecology 33 (1), 168 – 187. Dodds, E.C., Lawson, W., 1937. A simple oestrogenic agent with an activity of the same order as that controls. Nature (London) 139, 267. Duke, J.A., 1965. Fennel. Handbook of Medicinal Herbs. CRC Press, Boca Raton, FL, pp. 198 –199. Ecobichon, D.J., 1997. The Basis of Toxicity Testing, second ed. CRC Press, Boca Raton, FL. Forster, C., Whalley, E.T., Mohan, J., Dutton, J., 1980. Vascular smooth muscle response to fibrinogen degradation products and 5-hydroxytryptamine: possible role in cerebral vasospasm in man. Br J Clin Pharmacol 10 (3), 231 – 236. Gimeno, M.F., Sterin-Speziale, N., Landa, A., Bonacossa, A., Gimeno, A.L., 1979. Is the spontaneous motility of isolated rat uterus controlled by prostaglandin E? Prostaglandins 17, 673 – 682. Hansen, M.K., Secher, N.J., 1975. B-receptor stimulation in essential dysmenorrhea. American Journal of Obstetrics and Gynaecology 121, 566. Hare, H.A., Caspari, C.E., Rusky, H.H., 1916. The National Standard Dispensatory. Lee and Febiger, Philadelphia, p. 1129. Perez, V.J.R., Cantabrana, B., Hidalgo, A., 1990. Mechanisms involved in the effects of phenidone, diclofenac and ethacrynic acid in rat uterus in vitro. General Pharmacology 22 (3), 435 – 441. Reynolds, E.F.J., 1982. Essential Oils and Aromatic Carminatives, Martindale, The Extra Pharmacopeia, 28th ed. Royal Pharmaceutical Society, London, pp. 670 – 676. Saleh, M.M., Hashem, F.A., Garace, M.M., 1996. Volatile oil of Egyption sweet fennel and its effect on isolated smooth muscles. Pharmacy and Pharmacology Letters 6 (1), 6 – 7. Sterin-Speziale, N., Gimeno, M.F., Bonacossa, A., Gimeno, A.L., 1980. The effect of oestradiol in isolated rat uterine motility and on prostaglandin generation. Prostaglandins 20, 233 – 300. Von Rossum, J.M., 1963. Cumulative dose-response curves: the technique for the making of dose-response curves isolated organs and the evaluation of drug parameters. Archives of International Pharmacology and Therapy 143, 299 – 330. Journal of Ethnopharmacology 76 (2001) 305– 308 www.elsevier.com/locate/jethpharm Short Communication Antibacterial activity of Marula (Sclerocarya birrea (A. rich.) Hochst. subsp. caffra (Sond.) Kokwaro) (Anacardiaceae) bark and leaves J.N. Eloff * Department of Pharmacology, Uni6ersity of Pretoria, Pretoria, South Africa Received 14 March 2001; received in revised form 10 April 2001; accepted 12 May 2001 Abstract Marula bark is widely used for bacteria-related diseases by indigenous cultures in Africa. This study was undertaken to investigate whether the ethnobotanical use can be validated by laboratory studies. Bark and leaves were extracted with acetone and MIC values were determined using a microplate serial dilution technique with Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis as test organisms. All extracts were active with MIC values from 0.15 to 3 mg/ml. Based on minimum inhibitory concentration values, inner bark extracts tended to be the most potent followed by outer bark and leaf extracts, but the differences were not statistically significant. There were two major bioactive components visible after bioautography of leaf extracts: one strongly polar and the other highly non-polar. The bioactive components could be separated from 92% of the non-active dry matter by solvent– solvent fractionation into the carbon tetrachloride, chloroform and n-butanol fractions; these fractions, however, still contained many different compounds. Using bark may be detrimental to the plant, but leaf material can also be used for antibacterial application. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sclerocarya birrea subsp. Caffra; Bark; Leaf; Antibacterial; Bioactivity 1. Introduction The Marula (Sclerocarya birrea (A. Rich.) Hochst. subsp. caffra (Sond.) Kokwaro) is one of the most highly valued indigenous trees in southern Africa. The Tonga people celebrate the Feast of the First Fruits by pouring a drink offering of the fresh juice of the fruit over the tombs of dead chiefs (Palgrave, 1983). The pulp of the fruit is delicious and the large nut is also edible. Some tribes such as the Pedi make a relish from the leaves (Fox and Young, 1982). Some attempts have been made to improve the plant by selection and breeding and it has become a commercial fruit crop recently. Watt and Breyer-Brandwijk (1962), Oliver-Bever (1986) * Tel.: + 27-12-319-2139; fax: + 27-12-319-2411. E-mail address: jneloff@medic.up.ac.za (J.N. Eloff). and Hutchings et al. (1996) identified a number of medicinal uses in southern, eastern and tropical West Africa. The presence of antimicrobial constituents may be inferred from the following ethnomedical uses: the Zulu people use bark decoctions administered as enemas for diarrhoea. Traditional Zulu healers wash in bark decoctions before treating patients with gangrenous rectitis and also administer the decoction to the patient (Bryant, 1966). Bark decoctions are taken in 300 ml doses for dysentery and diarrhoea in unspecified parts of southern Africa (Watt and Breyer-Brandwijk, 1962). Bark has also been used in treating proctitis. The Vhavenda use bark for treating fevers, stomach ailments and ulcers (Mobogo, 1990). Roots are used for many purposes including sore eyes in Zimbabwe (Gelfand et al., 1985). In East Africa, roots are an ingredient in an alcoholic medicine taken to treat an 0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 6 0 - 4 306 J.N. Eloff / Journal of Ethnopharmacology 76 (2001) 305–308 internal ailment known as kati while bark is used for stomach disorders (Kokwaro, 1976). The Hausas in West Africa use a cold infusion of the bark along with native natron as a remedy for dysentery (Oliver-Bever, 1986). Galves et al. (1993) could show that extracts inhibit diarrhoea in mice. Bark yields 3.5– 20.5% tannin, 10.7% tanning matter and traces of alkaloids (Watt and Breyer-Brandwijk, 1962). The fruit is rich in ascorbic acid and juice extracts yield 33 sesquiterpene hydrocarbons (Pretorius et al., 1985). Kernels yield 54–60% of a non-drying oil and contain as much as 28% protein and some iodine (Watt and Breyer-Brandwijk, 1962). The oil-rich seeds contain 64% oleic acid, myristic, stearic and amino acids with a predominance of glutamic acid and arginine (Busson, 1985). The gum is rich in tannin. Tannins and flavonoids are present in leaves but no alkaloids, steroids or triterpenoids have been detected (Gueye, 1973). Only one paper published on the effect of S. birrea plant extracts on in vitro antimicrobial activity (Hussein and Deeni, 1991) was found. In a preliminary study these authors examined 34 plant species for antimicrobial activity. Methanolic extracts from dried powdered stembark had strong antibacterial activity (zone of inhibition\ 8 mm) against Corynebacterium diphtheriae, Pseudomonas aeruginosa and a Streptococcus spp. and less activity against unidentified Neisseria, Streptobacillus and Salmonella species. The extracts were inactive against Staphylococcus aureus and Escherichia coli. The authors indicated that their results differed from results obtained by earlier authors without specifying the differences or authors. Unfortunately no voucher specimens of the plants were retained. Subsequently, subspecies of Sclerocarya were recognised. It is therefore not clear which taxon Hussein and Deeni (1991) investigated. With a widely distributed plant that is valued highly for edible purposes and for treating many other ailments, Marula could be considered a ‘power plant’ (Balick, 1990). Power plants have been defined as widely used plants without any apparent or demonstrable pharmacological basis for their use. Some workers have eliminated ‘power plants’ from their list of plants to investigate. A disadvantage of using bark as source for the medicinal component is that injudicial removal of bark can lead to the death of the plant. If bark can be sold for a high price, valuable plant populations may be collected to extinction in nature. The aim of this paper is to determine if the use of extracts of Marula for ailments that may be related to bacterial pathogens can be substantiated in in vitro experiments and to compare the antibacterial activity of leaf and bark extracts. 2. Materials and methods Plant material was collected after fruiting in late May from a single tree growing in Pretoria. A voucher specimen was deposited at the National Herbarium (J N Eloff 503, PRE). Stem bark was collected by making two longitudinal cuts ca. 1 cm apart and ca. 25 cm long and removing the bark from the stem. The inner bark had a light buff colour and was separated from the dark brown flaky outer bark. Bark and leaves were dried indoors at ambient temperature and then ground to a fine powder in a Jankel and Künkel Model A10 mill. The finely ground plant material (0.5 g) was extracted (5 ml) in a centrifuge tube and the marc collected by centrifuging at 300×g for 5 min. The extraction was repeated two times on the marc. The acetone was removed by a stream of cold air and redissolved in acetone to yield a concentration of 50 mg/ml. The plant extracts were analysed by TLC (5 ml of 100 mg extract/ml solution) on Merck TLC F254 plates with chloroform/ethyl acetate/formic acid (5:4:1) as eluent. Plates were sprayed with 0.5 g vanillin dissolved in 100 ml sulphuric acid/ethanol (40:10). Minimum inhibitory concentrations (MIC) were determined by two-fold serial dilution of extracts beyond the concentration where no inhibition of growth of Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 25922 was observed. The wells in the dilution series were inoculated with the relevant cultures, incubated overnight at 37 °C and 0.2 mg/ml p-iodonitrotetrazolium violet [INT] (Sigma) was added. After further incubation bacterial growth was indicated by the red colour of the INT formazan produced (Eloff, 1998). For bioautography, developed TLC plates were dried overnight and sprayed with a concentrated suspension of actively growing S. aureus cells, before incubating at 38 °C in a chamber at 100% relative humidity. Plates were sprayed with a 2 mg/ml solution of p-iodonitrotetrazolium violet (Sigma). Clear zones on the chromatogram indicated inhibition of growth (Begue and Kline, 1972). The acetone extract was taken to dryness in a rotary evaporator under reduced pressure and this extract was dissolved in a 1:1 mixture of chloroform/water. The water fraction was extracted with an equal volume of n-butanol to yield the water (W) and butanol (B) fractions. The chloroform fraction was taken to dryness in a rotary evaporator under reduced pressure and extracted with a 1:1 mixture of hexane and 10% water in methanol. The 10% water in methanol extract was diluted to 20% water in methanol and extracted with carbon tetrachloride to yield the carbon tetrachloride (CT) fraction. The 20% water in methanol extract was J.N. Eloff / Journal of Ethnopharmacology 76 (2001) 305–308 diluted to 35% methanol in water and extracted with chloroform to yield the chloroform (CHL) fraction and the 35% water in methanol (MW) fractions (Suffness and Douros, 1979). In all cases equal volumes of the solvents were used and the extraction was repeated with a small volume ca. three more times or until all the colour was extracted. In some instances centrifugation was used to separate the fractions and a small quantity of an insoluble pellicle was formed that was discarded. 3. Results and discussion Acetone extracted 6.4% of the leaf, 9.4% of the outer bark and 9.8% of the inner bark. Extracts from leaves, inner bark and outer bark inhibited the growth of all the test organisms (Table 1). The average values for leaf, inner bark and outer bark for all the test organisms were 1.4 (SD=1.33), 0.49 (SD=0.51) and 0.81 (SD = 1.23) mg/ml, respectively. The extremely high standard deviations are caused by combining MIC values for the different organisms. The standard deviation of MIC values with the same test organisms were much lower (Table 1). The MIC values for the positive control, gentamycin, ranged from 0.02 to 0.6 mg/ml. One microwell where the bacterial growth inhibition was not noted would lead to a doubling of the MIC value. Although the differences were not statistically significant, the inner bark extract tended to be more active (i.e. have a lower MIC) than the outer bark and the leaf extracts. Based only on MIC values, one could speculate that leaves would also be a useful source for treating ailments caused by bacteria. It should be kept in mind that extracts were dried and made up to a known concentration before bioassay and the MIC values found does not relate to the total quantity present in the different plant parts. The total antibacterial activity present in different parts of the plant can be calculated 307 by dividing the quantity extracted from one gram of plant material with the MIC value in mg/ml (Eloff, 2000) (Table 1). The average total activity for the four test organisms, with leaf extracts is 56, with outer bark is 189 and with inner bark is 385 ml/g. Staphylococcus aureus was the most sensitive with a total activity of 653 ml/g. This means that the antibacterial compounds present in 1 g of inner bark material diluted to 653 ml would still inhibit the growth of the test organisms. When the total activity is compared it explains why Marula leaves are not traditionally used for ailments related to bacteria. The total activity values obtained here are in the same order of magnitude as that found for leaves of members of the Combretaceae and Celastraceae (Eloff, 1999, 2000). To obtain some information on the active component(s) a larger quantity of dried leaves was extracted in acetone and fractionated by solvent–solvent extraction. After solvent– solvent extraction, most of the extract (85%) was in hexane, the least polar fraction. In increasing order of polarity the carbon tetrachloride fraction contained 2%, chloroform 1%, n-butanol 5%, 35% water in methanol 3% and water 3% of the dry weight of the original extract. Most of the antibacterial activity was in highly non-polar compounds in the chloroform and carbon tetrachloride fractions (Rf =0.93). There was also some activity in the n-butanol fraction (Rf = 0.04) with chloroform/ethylacetate/formic acid (5:4:1) as eluent. According to the bioautograms there are only two major inhibiting compounds present in the leaf extracts. The solvent– solvent fractionation was effective in concentrating more than 95% of the activity of the non-polar compound in 3% of the original dry mass extracted (2% in carbon tetrachloride and 1% in the chloroform fraction). More than 95% of the activity of the polar compound was concentrated in 5% of the original dry weight extracted in the n-butanol fraction. According to the TLC separation, the components present in the bark and leaves differed (results not Table 1 The MIC values in mg/ml of acetone extracts of the inner bark, outer bark and leaves of Marula using Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis as test organisms and total activity present in different plant parts Organism and extract tested S. S. S. P. P. P. E. E. E. E. E. E. aureus leaves aureus inner bark aureus outer bark aeruginosa leaves aeruginosa inner bark aeruginosa outer bark coli leaves coli inner bark coli outer bark faecalis leaves faecalis inner bark faecalis outer bark Average SD Quantity (mg extracted from 1 g) Total activity (mg/ml) 1.15 0.15 0.50 1.27 0.37 0.47 3.00 1.33 2.43 1.58 0.60 0.67 0.38 0.05 0.24 0.53 0.08 0.16 1.64 0.41 1.89 1.55 0.22 0.21 64 98 94 64 98 94 64 98 94 64 98 94 56 653 188 50 265 200 21 74 39 41 163 140 308 J.N. Eloff / Journal of Ethnopharmacology 76 (2001) 305–308 Fig. 1. Left bioautogram of Marula acetone leaf extract separated into different fractions by solvent – solvent extraction. TLC plate developed in chloroform/ethylacetate/formic acid (5:4:1) and sprayed with E. faecalis culture, incubated overnight then sprayed with INT. Growth inhibition indicated by colourless areas on TLC. Right TLC of fractions sprayed with vanillin – sulphuric acid. Lanes from left to right 35% MeOH in water, hexane, water, butanol, chloroform and carbon tetrachloride fractions. shown), but in both cases there were compounds present with Rf values similar to the antibacterial compounds visible on bioautograms of leaf extracts (Fig. 1). This may mean that the same compounds are responsible for the antibacterial activity in leaves and stem bark. Attempts are under way to isolate and characterise these compounds. The results substantiate the ethnobotanical use of Marula bark for bacteria-related diseases. The results also show that leaf material is also useful for antibacterial uses. Because leaves are a more sustainable resource than bark it could be used without any detrimental effect on the plant. Bark is probably preferred because it contains a larger total quantity of antibacterial activity and because it is easier to transport and store for trading purposes. Acknowledgements Maryna Steinmann and Nataly Martini gave valuable technical assistance. The Research Committee of the Faculty of Medicine, University of Pretoria, provided financial support. References Balick, M.J., 1990. Ethnobotany and the identification of therapeutic agents from the rainforest. In: Chadwick, D.J., Marsh, J. (Eds.), Bioactive Compounds from Plants. Wiley, Chichester, pp. 22 – 39. Begue, W.J., Kline, R.M., 1972. The use of tetrazolium salts in bioautographic procedures. Journal of Chromatography 64, 182 – 184. Bryant, A.T., 1966. Zulu medicine and medicine men. C. Struik, Cape Town. Busson, F., 1985. 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