Natural Toxins Nat. Toxins 6: 1±14 (1998) REVIEW ARTICLE A Review of the Toxicosis and Biological Properties of the Genus Eupatorium Om P. Sharma,1* Rajinder K. Dawra,1 Nitin P. Kurade1 and Pritam D. Sharma2 1 Indian Veterinary Research Institute, Regional Station, Palampur, Kangra Valley, Himachal Pradesh, India 2 University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India ABSTRACT Eupatorium genus grows wild in many parts of the world. A number of species of Eupatorium are toxic to grazing animals. Milk sickness in humans is caused by ingestion of milk of the animals reared on the pastures infested with Eupatorium rugosum (white snakeroot). While some information is available on the toxins in various species of Eupatorium, ambiguities still persist in extrapolation of the data to ®eld incidence of toxicosis. Eupatorium genus has been used for its medicinal properties for many decades. A number of bioactive natural products have been reported in the extracts of Eupatorium spp. and the genus is a promising bioresource for preparation of drugs and value-added products.  1998 John Wiley & Sons, Ltd. Key words: Eupatorium; Ageratina; Chromolaena; toxicosis; hepatotoxicity; milk sickness; trembles; sesquiterpenes; sesquiterpene lactones; bioactive compounds INTRODUCTION The genus Eupatorium belongs to the Eupatorieae, one of the thirteen tribes of the Asteraceae (Woerdenbag, 1993; Frohne and Jensen, 1979). Over 1000 Eupatorium species are listed in the Index Kewensis but now the genus has been taxonomically revised to contain 44 species (Hooker and Jackson, 1960; Robinson and King, 1985). Comparison of American, Asian and African samples of Eupatorium odoratum using descriptive taxonomy showed little differentiation. However, chemotaxonomy studies using enzymatic markers showed that plants from south America (French Guiana) were different from those of Asian origin (Lanaud et al., 1991). Eupatorium species have very luxuriant growth, exert allelopathic action and have come up as major intractable weeds in many parts of the world (Table 1). The fast growth of plants of the Eupatorium genus is disturbing the overall ecology by encroaching upon pastures and replacing the forest biomass; it is a major concern for farmers, agriculture, and animal and forestry scientists (Sharma and Dawra, 1994). The overt impact on farmers’ economy is in terms of loss of pastures. In addition, the huge Eupatorium biomass is not useful in agriculture and animal husbandry operations. The various species of the genus Eupatorium which have been investigated in different parts of the world are listed in Table 1. Eupatorium spp. are known to have a number of adverse interactions with livestock and humans. Much work has been done on the toxicosis, CCC 1056±9014/98/010001±14 $17.50  1998 John Wiley & Sons, Ltd. medicinal properties of plant extracts and bioactive compounds of plants belonging to the Eupatorium genus (Beier and Norman, 1990; Woerdenbag, 1993; Sharma and Dawra, 1994). Here we review the present state of the art on this genus in order to open up vistas for future research and development. TOXICOSIS Eupatorium spp. have vigorously invaded pastures and forests in many parts of the world (Woerdenbag, 1993; Sharma and Dawra, 1994). There is a lot of variation in the chemistry of the natural products of the different species of the Eupatorium genus. So, different toxicosis syndromes have been observed in different parts of the world. The clinical signs on ingestion of various Eupatorium spp. by grazing animals and experimental animals, and the present knowledge about the nature of the toxins, are summarized in Table 2. The following species of Eupatorium have been recognized as causing serious problems to livestock and humans when in contact with infested pastures. *Correspondence to: Dr Om P. Sharma, Indian Veterinary Research Institute, Regional Station Palampur, Kangra Valley, Himachal Pradesh, India 176 061. Received 18 June 1997; Accepted 27 April 1998. SHARMA ET AL. 2 Table 1. Distribution of plants belonging to the genus Eupatorium Plant Place/country E. adenophorum syn. E. trapezoideum, Ageratina adenophora, E. glandulosum common name: Crofton weed E. odoratum syn. Chromolaena odorata common name: Siam weed India, Nepal, China, Australia E. riparium common name: mist flower India, Burma, Indonesia, China, Africa, Ivory Coast, South Africa, South America, West Indies, Malaysia Northeastern India, Australia, Sri Lanka E. cannabinum E. stoechadosmum Europe, USA Vietnam, Japan E. compositifolium common name: Yankeeweed E. inulifolium E. japonicum E. lancifolium E. semiserratum E. pilosum E. buniifolium E. chinense syn. E. reeversii E. altissimum USA E. formosanum Hay E. perfoliatum E. capillifolium E. triplinerve syn. E. ayapana E. rugosum syn. E. urticaefolium, Ageratina altissima common name: white snakeroot E. coelestinum E. salvia E. tinifolium E. cyrili-nelsonii E. tashiroi Hayata E. maculatum Indonesia Japan Arkansas Florida Florida Northeastern and central Argentina India, China Ansari et al. (1983), Jha and Yadav (1985), Chen and Zhao (1984) Parsons and Cuthbertson (1992) Muniappan et al. (1990), Zmarlicki (1984), Sipayung et al. (1991), Wu et al. (1984), M’Boob (1991), Quencez and Vernou (1983), Liggit (1983), Ooi et al. (1988) Rai and Tripathi (1984) Gibson and O’Sullivan (1984) Bandara et al. (1992) Woerdenbag (1993) Trang et al. (1993a), Furuya and Hikichi (1973) Hanselka et al. (1984) West Virginia, Mississippi Da-Tung-Shan (Taiwan) Germany USA India, Vietnam Eastern and central Bahri et al. (1988) Nagao et al. (1981) Herz et al. (1981) Herz et al. (1981) Herz et al. (1981) Muschietti et al. (1994) Gopalakrishnan et al. (1990), Rastogi and Mehrotra (1991) Jakupovic et al. (1987), Herz et al. (1978) Lee et al. (1972) Vollmar et al. (1986) Rao and Alvarez (1981) Yadav and Saini (1990), Trang et al. (1993b) Beier and Norman (1990) Louisiana Chile Colombia Honduras Tawain Canada Van and Pham (1979) Gonzalez et al. (1990) D’Agostino et al. (1990) Molina (1978) Wu et al. (1985) Biovin and Benoit (1987) E. adenophorum E. adenophorum is native to Mexico but has now spread to Hawaii, the Philippines, China, Thailand, Australia, New Zealand, India, Nepal, and California (Chen and Zhao, 1984; Oelrichs et al., 1995; Fuller, 1981). In the 1920s, the plant was suspected of causing chronic respiratory disease in horses in Hawaii. By 1940, outbreaks of the same syndrome were recorded in New South Wales and southern Queensland in Australia where the disease was called Numinbah Horse Sickness or Tallebudyera Horse Disease (Jones, 1954; Oelrichs et al., 1995). Horses of both sexes between 18 months and 12  1998 John Wiley & Sons, Ltd. Reference(s) years of age were affected. Coughing exacerbated by exercise was the first clinical sign followed by decrease in exercise tolerance, heaving respiration and loss of condition. The lungs of the chronically affected field cases of horses were firm and did not collapse when the thoracic cavity was opened. The visceral pleura was white and thickened over a large area of the lung surface. There were some focal adhesions to the parietal pleura (O’Sullivan, 1979). Histopathological examination revealed that alveolar lining cells had proliferated in all animals forming sheets of epithelial-like cells lining the alveoli. In more chronic cases, clumps of inspissated protein were present in alveoli. Many bronchioles and Nat. Toxins 6: 1–14 (1998) TOXICOSIS AND BIOLOGICAL PROPERTIES OF EUPATORIUM 3 Table 2. Toxicosis of different spp. of Eupatorium Plant Animal affected E. adenophorum Horses Clinical signs/lesions Toxin(s) n.i. Reference Cattle Mice Coughing, heaving respiration, exercise intolerance Anorexia, photosensitization Jaundice, hepatotoxicity Rats Jaundice, cholestasis, hepatotoxicity n.i. Verma et al. (1987) 9-oxo-10,11, Oelrichs et al. (1995) dehydroagerohporone n.i. Katoch (1995), E. riparium Horses Same as for E. adenophorum n.i. Gibson and O’Sullivan (1984) E. rugosum Cattle – Beier and Norman (1990) – Smetzer et al. (1983), Olson et al. (1984), White et al. (1985) Tremetola Couch (1927), Christensen (1965), Beier and Norman (1990) Beier and Norman (1990) Trembles, acetone breath, constipation, difficult respiration, profuse nasal discharge Horses Unsteady gait, difficulty in swallowing, acetone breath, cardiac arrhythmias, constipation, trembling of muscles Sheep Trembles, anorexia, laboured respiration stiffness of limbs, ataxia Goats Depression, anorexia, photosensitization, no trembles Chickens/ Trembles, cyanosis, acetone breath, chicks severe acidosis E. inulifolium Cattle Rats Liver cirrhosis Hepatotoxicity E. odoratum Mammals – – O’Sullivan (1979,1985) Tremetola Butler (1946), Beier and Norman (1990) n.i. n.i. Bahri et al. (1988) Bahri et al. (1988) High levels of nitrate, Liggitt (1983), Sajise et al. (1974) Antinutritional factors Nwokolo (1987) n.i., not identified. Mixture of at least two steroids and three ketones. a surrounding alveoli were filled with neutrophils and eosinophils (O’Sullivan, 1979). The respiratory problem caused by ingestion of E. adenophorum has also been reported from New Zealand (Connor, 1977). In Australia, there are a large number of farms infested with E. adenophorum with a history of toxicosis in horses (Everist, 1981). Moreover, respiratory disease in horses in Australia was found only in areas which were infested with E. adenophorum, more commonly called Crofton weed (O’Sullivan, 1979). Similar epidemiological correlation is not available for other countries where E. adenophorum is a serious weed. O’Sullivan (1979) did experimental feeding studies on horses (two), rabbits, sheep and rats. The plant was collected from Numinbah Valley, Queensland, from March to October and was fed daily with lucerne chaff. One horse exhibited coughing after eating flowering plants for one month. The pathological profile of lungs was found to be similar to the field cases. No gross abnormality was found in the lungs of rabbits fed a mixture of lucerne chaff, E. adenophorum leaves and flowers and proprietary pellets in the ratio 2:1:2 for about 8 months. Histopathological examination showed con 1998 John Wiley & Sons, Ltd. solidated areas with marked proliferation and desquamation of alveolar lining cells, infiltration of eosinophils and mononuclear cells (O’Sullivan, 1979). Sheep fed E. adenophorum did not show any abnormality or lesions. Rats fed E. adenophorum mixed in feed similar to rabbits during October–November for 52 days did not show any abnormality or lesions (O’Sullivan, 1979). These feeding experiments confirmed the association of E. adenophorum with the respiratory disease in horses but raised many questions regarding species variation in susceptibility to E. adenophorum toxicosis, route of entry of toxins, and reversibility of toxicosis. Subsequently, more detailed studies confirmed that E. adenophorum is toxic to horses and is responsible for the field problem of respiratory disease in E. adenophorum infested areas. The plant in the flowering stage is more toxic to horses than in the non-flowering stage. It was also concluded that putative toxins acted via the bloodstream and not by inhalation (O’Sullivan et al., 1985) and the disease condition is irreversible. Toxins responsible for the E. adenophorum toxicosis in horses have not been identified so far. Freeze-dried powdered leaves of E. adenophorum collected in the Numimbah Valley region of Queensland Nat. Toxins 6: 1–14 (1998) SHARMA ET AL. 4 Figure 1. Chemical structure of toxins in Eupatorium genus in Australia when fed to male mice induced lesions in the liver but not in the lungs (Sani et al., 1992). The plant sample was given as a water suspension orally at a dose of 50, 100 and 150 mg kgÿ1 or mixed in the diet at 10 and 16 %. A single dose of freeze-dried E. adenophorum leaf powder elicited lesions in bile ducts. Hepatic injury involved multiple areas of focal necrosis of the parenchyma associated with degeneration and loss of the epithelial cells lining the small bile ducts (Sani et al., 1992). Oelrichs et al. (1995) did systematic purification studies for characterization of the E. adenophorum toxins which elicit liver injury in mice. The plant samples were collected from the Brookfield area near Brisbane, Queensland, Australia. The leaves were freeze-dried, powdered and stored at ÿ30 °C; 100 g of the plant sample gave 737 mg of the pure toxin which was chemically characterized as 9-oxo-10,11-dehydroagerophorone (Figure 1). The highest non-fatal dose of the toxin was 350 mg kgÿ1 body weight. The intoxicated mice became icteric within 2 days of dosing at 350 mg kgÿ1 body weight and the jaundice persisted for at least 3 weeks. On necropsy, pale areas could be seen on the visceral surface of the liver. Histopathological examination showed that there was degeneration and loss of the epithelium of most of the intrahepatic bile ducts. The duct walls were thickened due to oedema and fibrosis (Oelrichs et al., 1995). The lesions elicited by the isolated toxin were identical to those caused by the freeze-dried E. adenophorum leaves observed earlier by Sani et al. (1992). E. adenophorum has encroached upon a vast expanse of pastures and forest areas in the northeastern and northwestern sub-Himalayan region in India (Verma et al., 1987; Sharma and Dawra, 1994). In northeastern India it colonises rapidly after jhum (shifting) cultivation. The species is unpalatable to grazing cattle (Verma et al., 1987). In experimental feeding studies, when E. adenophorum was offered to calves, they did not accept it well in the beginning but after 5–6 days consumed up to 5 kg on a fresh weight basis. The first symptoms of toxicity, which appeared after 14 days, were anorexia, suspension of rumination and photosensitization. On continuing  1998 John Wiley & Sons, Ltd. feeding, the initial symptoms disappeared but the growth rate did not improve (Verma et al., 1987). In the Kangra Valley of Himachal Pradesh in India, grazing sheep, goats and cattle browse E. adenophorum. No epidemiological data are available on the toxicosis in these species of ruminants but farmers consider the plant as undesirable and noxious. No toxic effects were seen in goats when E. adenophorum comprised up to 67 % of their food ration but at higher concentrations voluntary intake was reduced due to low palatability (Neopane et al., 1992). E. adenophorum, sometimes grows very near human habitations and some people have reported allergic problems during the flowering months, i.e. March to April, in the Kangra Valley of Himachal Pradesh. Systematic investigations are under way on the nature of toxins in E. adenophorum in this valley (Sharma and Dawra, 1994). In the first study phase plant leaf samples were collected during the flowering stage, freeze-dried and fed to male rats in the diet at the 25 % level for two consecutive days. The animals became sedated and icteric after 24 h; the ictericity became more distinct at 48 h of observation and the animals were sacrificed. Postmortem examination revealed that the liver had yellow coloration and the epidermis was yellow, presumably due to binding of bilirubin to the proteins. The stomach was filled with gas, the intestines contained only small amounts of solid, the large intestines had hard faecal pellets and the urinary bladder was invariably full of urine (Katoch, 1995). There was a marked increase in the level of conjugated and total bilirubin in the blood plasma of the intoxicated animals (Katoch, 1995). The increase was more in the conjugated form of bilirubin which is characteristic of cholestasis (Cornelius, 1980). Histopathological examination of the liver of intoxicated animals revealed bile duct proliferation. The increase in bilirubin level and subcellular lesions implies that liver is the target organ of E. adenophorum toxins (Katoch, 1995). During the second phase of studies, E. adenophorum leaf samples collected from the vicinity of Indian Veterinary Research Institute laboratories were ovendried at 60 °C and fed to male rats at 25 % in their diet. The results were identical to those obtained with freezedried leaf samples. The animals developed jaundice within 24–30 h, accompanied by appearance of bilirubin in urine; they were sacrificed at 48 h after dosing (Kaushal, 1996). Postmortem observations were similar to those recorded in the animals offered freeze-dried leaves of E. adenophorum (Katoch, 1995). There was an increase in the total and conjugated form of bilirubin and the activities of alkaline phosphatase, 5'-nucleotidase, aminotransferases and lactate dehydrogenase in blood plasma of intoxicated animals. Histopathological studies revealed that liver had proliferation and dilatation of bile ducts with infiltration of mononuclear cells. Gastric mucosa had superficial necrosis. Similar biochemical and Nat. Toxins 6: 1–14 (1998) TOXICOSIS AND BIOLOGICAL PROPERTIES OF EUPATORIUM histopathological changes were observed in rats offered methanolic extract or sesquiterpene fractions extracted by the protocol of Oelrichs et al. (1995). These studies showed that E. adenophorum leaves elicit hepatotoxicity and cholestasis in rats and the putative toxins are stable to heating at 60 °C (Kaushal, 1996). E. riparium E. riparium is a perennial weed in southeastern Queensland and northeastern New South Wales in Australia (Everist, 1981). It flourishes in high rainfall areas, on damp hillsides and in moist places (Gibson and O’Sullivan, 1984). Field cases of E. riparium toxicity have not been reported. Gibson and O’Sullivan (1984) fed air-dried plant samples from flowering and nonflowering stages to two horses at the rate of 6 g kgÿ1 liveweight. The lesions in the animals were comparable to those observed in the field cases and experimental feeding experiments using E. adenophorum, but were less severe. The authors opined that the lower toxicity of E. riparium might be due to the fact that the dose of plant sample in these experiments was smaller compared to the experiments using E. adenophorum plant samples (O’Sullivan, 1979, 1985; Gibson and O’Sullivan, 1984). It was also suggested that both these plants share a common toxin capable of causing respiratory disease in horses. No further investigations have been done on the toxicosis of E. riparium. E. inulifolium E. inulifolium grows in low and high areas in Sumatra and Java in Indonesia (Bahri et al., 1988). The plant has been suspected of causing hepatotoxicity in grazing animals. Normally, animals do not browse this plant but during the dry season, when other vegetation dries up, E. inulifolium remains green and grazing livestock consume it. Bahri et al. (1988) studied the toxicity of oven-dried (50 °C) leaves of E. inulifolium in male rats at 12.5 % and 2.5 % in diets for a period of 3 weeks. One week after the start, one rat fed 25 % E. inulifolium leaf powder in the feed had anorexia, weakness and weight loss. On postmortem, the liver was found to be pale with a nodular surface. Histopathological examination of animals in both the groups showed megalocytosis and necrosis of hepatocytes and proliferation of bile ducts (Bahri et al., 1988). The toxins in E. inulifolium have not been identified. Hepatotoxicity in ruminants has not been reproduced by experimental feeding of E. inulifolium. E. odoratum E. odoratum is native to Mexico, the West Indies and South America from where it has spread to other parts of  1998 John Wiley & Sons, Ltd. 5 the world (Liggitt, 1983). It is a serious weed in West and South Africa, southern parts of India, Sri Lanka, Australia, Indonesia, South America, Ivory Coast, Ghana and China (Liggitt, 1983; Moni and George, 1959; Hall et al., 1972; Holm et al., 1977; Sipayung et al., 1991; Fadiga and Akpagni, 1983; Quencez and Vernou, 1983; M’Boob, 1991; Liu and Fu, 1990). E. odoratum is relatively unpalatable possibly due to its aromatic smell, but may sometimes be heavily browsed particularly when other green vegetation is scarce. Mammals known to browse E. odorartum include cattle, goats, nyala, eland and bushbuck (Liggitt, 1983). Young plants contain five or six times the toxic levels of nitrate (Sajise et al., 1974). Nitrate is not itself toxic but is converted to nitrite in the rumen. Nitrite ions oxidize ferrous iron in haemoglobin to produce methaemoglobin. Methaemoglobin cannot react with oxygen and this leads to anoxia and death (Liggitt, 1983; Cheeke and Shull, 1985). Leaf meal of E. odoratum was evaluated as a nutrient source in poultry diets. Mineral as well as amino acid availability was low which implied the presence of antinutritional factors (Nwokolo, 1987). E. rugosum E. rugosum (commonly called white snakeroot) is distributed in central and eastern USA (Scotts, 1984; Beier and Norman; 1990). Trembles in animals and milk sickness in humans were encountered by the early settlers in the United States (Couch, 1927). White snakeroot has been identified as the etiological agent responsible for both these syndromes, viz. trembles in grazing animals and milk sickness in humans who consume milk only from animals which graze in white snakeroot infested areas (Snively and Furbee, 1966). Milk sickness was the principal cause of death and morbidity in many communities in the midwest and upper south of the USA during the 18th and 19th centuries. The illness sometimes wiped out entire villages in the affected areas (Beier and Norman, 1990). Abraham Lincoln’s mother died of milk sickness in 1818. Other names for milk sickness are ‘sick stomach’ and ‘slows’. Historical aspects of the disease in humans and grazing animals have been traced by Beier and Norman (1990). The early evidence for involvement of white snakeroot with trembles and milk sickness was based on the epidemiology of these two syndromes and the infestation of plant. In 1962, two children having acidosis were diagnosed as having milk sickness. They had been taking fresh milk from cows which had access to white snakeroot (Hartmann et al., 1963). White snakeroot is still a problem in horses and goats in the USA (Beier and Norman, 1990). In the spring of 1985, 53 angora goats died of white snakeroot poisoning (Beier and Norman, 1990). Systematic animal feeding Nat. Toxins 6: 1–14 (1998) 6 SHARMA ET AL. experiments were started at the beginning of the 20th century (Moseley 1906, 1909, 1917; Wolf et al., 1918; Couch, 1928). Wolf et al., (1918) reported the use of white snakeroot extracts in animal feeding experiments and adduced evidence that these extracts caused trembles. Couch (1927) reported the isolation of the toxin from extracts of white snakeroot and called it tremetol. This was erroneously thought to be a pure compound for many years until Bonner and colleagues (Bonner et al., 1961; Bonner and DeGraw, 1962) isolated three main ketones, tremetone, hydroxytremetone and dehydrotremetone from the ketone fraction of tremetol (Figure 1). Tremetone constituted the major part of the crude fraction tremetol and was presumed to be the principal toxin responsible for causing trembles in cattle (Beier and Norman, 1990). However, using synthetic tremetone, Bowen et al., (1963) observed that tremetone was not toxic in the bioassays. Thus, the question regarding the identity of toxins in white snakeroot persisted till the early 1990s. Beier et al. (1987, 1993) observed that metabolic (microsomal) activation of tremetone might be responsible for the toxicosis of the plant. In these studies microsomal preparation for biotransformation of tremetone was prepared from rat liver. There is marked difference in the microsomal transformation system of rat and many ruminants (Watkins and Klaassen, 1986). It is still not certain if the ruminants bring about comparable biotransformation and activation of tremetone in white snakeroot. Thus, the identity of white snakeroot toxins still remains uncertain. It has been suggested that whatever the ultimate nature of the toxin, it is elaborated in the milk, and interferes with the fatty acid metabolism of the consumer (Plummer, 1992). The clinical profile of white snakeroot toxicosis in different species of animals and milk sickness in humans has been described by Beier and Norman (1990). The victims of white snakeroot poisoning have been cattle, horses, sheep and goats. One consistent clinical feature in cattle, sheep and horses is trembles and ‘acetone breath’ (Table 2). In horses, gross postmortem findings are limited to myocardial haemorrhages and greyish streaks on myocardium. Detailed blood clinical chemistry has been investigated in white snakeroot toxicosis in horses. There is increase in the activity of creatinine phosphokinase, lactate dehydrogenase, serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase and alkaline phosphatase (Smetzer et al., 1983; Olson et al., 1984; White et al., 1985; Thompson, 1989). Goats are highly susceptible to white snakeroot poisoning but they do not show the clinical sign of trembles. Goats fed white snakeroot had depression, anorexia, head pressing and death in 12–36 h. Goats browsing white snakeroot in pastures for 3 or more days had photosensitization. In experimental feeding studies,  1998 John Wiley & Sons, Ltd. it has been observed that there was marked increase in the activity of lactate dehydrogenase and glutamate pyruvate transaminase, and histological studies revealed lesions in liver. In an attempt to get a suitable laboratory animal model, studies have been done using chicks, mice, rabbits and guinea pigs (Beier and Norman, 1990). Chickens simulated all the symptoms of white snakeroot poisoning as observed in field animals and experimental studies using crude toxin tremetol (Butler, 1946). The results using other experimental animals have been inconsistent. Studies have been done to understand the biochemical basis of ketoacidosis in white snakeroot poisoning. It appears that the toxin inhibits the activity of citrate synthase which in turn limits the utilization of acetyl CoA in the citric acid cycle. Acetyl CoA is then converted into ketone bodies like acetone, acetoacetic acid and bhydroxybutyrate which are responsible for ketoacidosis and ‘acetone breath’ (Hartmann et al., 1963; Beier and Norman, 1990; Plummer, 1992). E. chinense Green parts of E. chinense caused chronic poisoning in rabbits and guinea pigs associated with necrotic degeneration of liver, tubular nephritis and glycosuria (Pak and Read, 1937). Many spp. of Eupatorium are rich in pyrrolidizine alkaloids (PAs) (Woerdenbag, 1993). PAs are known to cause a number of adverse effects like hepatotoxicity, carcinogenesis and genotoxicity (Mattocks, 1968, 1971; Smith and Culvenor, 1981). The hepatotoxicity of PAs is related to the formation of pyrrole metabolities in the liver (Mattocks, 1971, 1972; Westendorf, 1992; Rizk and Kamel; 1991). There is a lot of variation in the susceptibility of different animal species to PA toxicosis (Cheeke and Shull, 1985). This has been ascribed to the variation in the rate of pyrrole production and the ruminal biotransformation of PAs (Cheeke and Shull, 1985; Craig et al., 1986; Wachenheim et al., 1992). However, there is incomplete correspondence between the rate of pyrrole formation from PAs and susceptibility to PA toxicosis (Cheeke and Shull, 1985). The rate of biotransformation of PAs expressed as mg mlÿ1 rumen fluid hÿ1 has been found to be 2.9 for bovine, 25.6 for caprine, and 19.2 for ovine species. The number of PA biotransforming bacteria in the rumen fluid of different species was 1.1  107 for bovine, 2.4  107 for caprine and 3.0  107 for the ovine species (Wachenheim et al., 1992). The presence of PAs in a number of Eupatorium species and the species variation in susceptibility to PAs toxicosis (Woerdenbag, 1993; Cheeke and Shull, 1985) appears to be the reason for the variation of the response of various animal species to Eupatorium-induced toxicosis (Beier and Norman, 1990; O’Sullivan, 1979; Verma et al., 1987). Many spp. of Eupatorium are rich in sesquiterpene Nat. Toxins 6: 1–14 (1998) TOXICOSIS AND BIOLOGICAL PROPERTIES OF EUPATORIUM lactones containing an a-methylene g-lactone (Woerdenbag, 1993). Such compounds cause degranulation of mast cells and liberate histamine (Elissalde et al., 1983). Thus, toxicosis in ruminants grazing Eupatorium spp. containing sesquiterpene lactones in addition to PAs, cadinenes, tremetol and other noxious constituents can be of additive type. Manifestation of the clinical picture would depend upon the relative abundance of the different toxins in the species prevalent in a particular geographical region. ALLELOPATHY Weeds usually produce phytotoxins which give them competitive advantage by inhibiting the growth of neighbouring vegetation (Rice, 1979). This phenomenon, known as allelopathy, is involved in the dominance and rapid spread of weeds in agroecosystems (Putnam and Duke, 1978). E. adenophorum is the most noxious weed in central Nepal. It is so prolific that indigenous Himalayan species are being crowded out by this weed (Jha and Yadav, 1985). Aqueous extracts and leachates of fresh leaves and litter of E. riparium suppressed the germination and radicle and plumule growth of Galinsoga ciliata and G. parviflora (Rai and Tripathi, 1984). Aqueous extracts of E. chinense considerably reduced radical and hypocotyl growth of lettuce seedlings (Takahashi et al., 1995). Cadinenes and b-sitosterol isolated from E. adenophorum Spreng. inhibited the germination and seedling growth of Allium cepa cv. red globe, Raphanus sativus cv. jap white and Cucumis sativus cv. long green (Baruah et al., 1994). Aqueous extracts of E. odoratum have been shown to suppress the growth of wheat (Triticum aestivum L.) and fenugreek (Trigonella foenumgraecum L.) seedlings (Ambika and Jayachandra, 1980). Leaf and stem exudates of E. odoratum inhibited the growth of seedlings of the weeds Crassocephalum crepidiodes, Ageratum conyzoides, Cynodon dactylon and Cynodon odorata (Nakamura and Nemoto, 1993). E. odoratum extracts reduced the germination of the seeds of spinach, Chinese cabbage, rape and Capsicum frutescens (Sahid and Sugau, 1993). Petroleum spirit, diethyl ether and ethyl acetate fractions of acidic and basic methanolic extracts of the aerial parts of E. odoratum were inhibitory to tomato seed germination (Tijani-Eniola and Fawusi, 1989). Tissue extracts (1%) of E. capillifolium reduced germination and seedlings growth of Lollium multiflorum and Medicago sativa and the plant has been rated as potentially allelopathic (Smith, 1991). Systematic investigations on the purification and characterization of the allelochemicals in the various species of Eupatorium have not been done so far.  1998 John Wiley & Sons, Ltd. 7 MEDICINAL PROPERTIES Various spp. of Eupatorium plant have been used in the traditional system of medicine in different parts of the world (Woerdenbag, 1993). E. cannabinum Extracts of E. cannabinum have been used against spleen, liver and biliary diseases, diarrhoea, snakebites, ulcers, wound healing, fever, as a diuretic, anthelmintic and as a repellent against poisonous animals (Woerdenbag, 1993; Madaus, 1938). Extracts of leaves and roots have choleretic, laxative and appetising actions (Woerdenbag, 1993; Hoppe, 1975; Woerdenbag et al., 1991). Aqueous extracts of E. cannabinum had choleretic and hepatoprotective activity in mice against carbon tetrachlorideinduced hepatotoxicity (Lexa et al., 1989, 1990). The aerial parts of E. cannabinum are used as immunostimulating agents in cases of influenza infection, as a remedy against obstipation, for decreasing the level of cholesterol and as a diuretic (Roeder, 1995). The plant is currently used as an ingredient in immunostimulatory drugs (Siebertz et al., 1989). E. perfoliatum Extracts of E. perfoliatum have been used against fever, bronchial infections, migraine, intestinal worms, colds, catarrh, influenza, rheumatism, malaria and constipation (Madaus, 1938; Locock, 1990; Tyler, 1987; Hall, 1974; Woerdenbag, 1993). Other properties of the plant are diaphoretic, laxative, emetic and cathartic (Baker, 1983). Extracts of E. cannibinum and E. perfoliatum are known to stimulate defence mechanisms against viral infections (Weiss, 1991). Both species are used in homeopathic medicine systems against fevers, hepatobiliary diseases and rheumatism (Woerdenbag, 1993). Heteroglycans isolated from E. cannabinum and E. perfoliatum showed significant immunostimulant effect (Vollmar et al., 1986; Wagner, 1991; Wagner et al., 1985). Other Species E. adenophorum is used in India as an antiseptic and as a blood coagulant. A decoction of the plant has been recommended to treat jaundice and ulcers (Ansari et al., 1993). Rootstock of E. aromaticum is known to have diuretic and antispasmodic properties (Uphof, 1968). E. chinese L is used in southern China to treat syndromes caused by summer heat and humidity including headache, fatigue, anorexia, nausea, vomiting and diarrhoea. The plant serves as a diuretic and anthelmintic and is used for the treatment of diphtheria (Woerdenbag, 1993; Zhao et al., 1987). E. buniifolium, a medicinal plant found in Nat. Toxins 6: 1–14 (1998) 8 SHARMA ET AL. Figure 2. Chemical structure of sesquiterpene lactones northeastern and central Argentina, is used as a tincture and for its hepatoprotective and disinfectant properties; and the ethanolic extracts of the plant also show good antiherpetic activity against herpes simplex virus. (Garcia et al., 1990; Muschietti et al., 1994). E. cuneifolium, E. rotundifolium and E. semiserratum have been traditionally used for the treatment of cancer (Herz et al., 1981, Kupchan et al., 1967, 1968, 1969; Woerdenbag, 1993). E. formosanum is used in Formosan folk medicine for its antileukemic, antipyretic, anti-inflammatory and anticancer properties (Lee et al., 1972, 1977; Woerdenbag, 1993). E. ayapana is used in Vietnam to treat diarrhoea and ulcers and as an insect repellent (Trang et al., 1993b).  1998 John Wiley & Sons, Ltd. An aqueous extract of E. triplinerve is known to serve as a cardiac stimulant. A leaf decoction has hemostatic action which is ascribed to coumarins. Hot infusion of E. triplinerve is a tonic, expectorant, diaphoretic, laxative and emetic (Kapoor, 1990). E. fortunei Turcz is used in Chinese medicine as a diuretic, antipyretic and emmenagogue (Haruna et al., 1986). Ageratina viscosa (Eupatorium sp.), a shrub which grows in Colombia, is used in folk medicine. The root extract is known to exhibit medicinal properties against syphilis while the leaf extract is used to treat fevers and swelling (Torrenegra et al., 1990). E. odoratum, introduced into Nigeria nearly 30 years Nat. Toxins 6: 1–14 (1998) TOXICOSIS AND BIOLOGICAL PROPERTIES OF EUPATORIUM Figure 3. Chemical structure of bioactive sesquiterpenes and ¯avonoids from Eupatorium genus ago, is used locally for the treatment of skin diseases, to arrest bleeding from cuts, for wound dressing and as a cure for malaria and cough (Irobi, 1992; Akah, 1990; Metwally and Ekejiuba, 1981). The plant oils have antibacterial and antifungal activities (Irobi, 1992). The leaf extract significantly reduced the bleeding time in guinea pigs and rabbits. The effect was attributed to the vasoconstrictor activity of the leaf extract which was similar to that of adrenaline (Akah, 1990). The studies suggested that the hemostatic action of E. odoratum may be partially due to the a-receptor mediated vasoconstriction (Akah, 1990). BIOACTIVE CONSTITUENTS Bioactive constituents belonging to the groups sesquiterpene lactones, sesquiterpenes and flavonoids have been investigated in various species of Eupatorium (Table 3, Figure 2(a), 2(b), Figure 3). Sesquiterpene lactones are known to have antitumor, cytotoxic, antimicrobial, phytotoxic and antifeedant activities (Rotriguez et al., 1976; Woerdenbag, 1986). These natural products are known to cause livestock poisoning and contact dermatitis (Rotriguez et al., 1976). Cytotoxic agents eupatolide and eupaformin and antileukemic principle eupaformosanin have been reported from E.  1998 John Wiley & Sons, Ltd. 9 formasonum collected from Wootai-Pingtong, Taiwan (Lee et al., 1972, 1977; McPhail et al., 1974; McPhail and Onan, 1975, 1976). Analysis of the above-ground parts of E. lancifolium collected from west of Chidchester, Arkansas gave the cytotoxic and antileukemic bioactive compounds eupacunolin, eupacunin and desacetyleupacunin (Herz et al., 1981). Likewise, the analysis of above-ground parts of E. semiserratum DC collected from south of Bloutstown, Calhoun Co., Florida gave antileukemic germacrdienolide desacetyleupaserrin and flavones eupatorin, pectolinarigenin, hispidulin and salvigenin (Herz et al., 1981; Kupchan et al., 1973a). Tumor inhibiting sesquiterpene lactones including eupacunin have also been reported from E. cuneifolium (Kupchan et al., 1971; 1973b). E. hyssopifolium contains the sesquiterpene lactone eupahyssopin which has immunostimulating, anti-inflammatory and cytotoxic activities (Wagner, 1984; Hall et al., 1979; 1980, Lee et al., 1976). Eupahyssopin lowered serum cholesterol and triglycerides, inhibited synthesis of nucleic acids, proteins and cholesterol and inhibited the activities of DNA polymerase and thymidylate synthetase (Hall et al., 1978a,b). Extracts of the above-ground parts of E. altissimum L. collected from Osborne Oktibbeba, Co. Mississippi, which showed antileukemic effect, contained two flavones eupatorin and 3',4',6,7-tetramethoxyflavone (Herz et al., 1978). Eupatoriopicrin, a major sesquiterpene lactone in E. cannabinum, has been investigated in detail for the cytotoxic and antitumor action (Woerdenbag, 1989). It was concluded that the oncolytic action of eupatoriopicrin is of short duration and rather nonspecific. It does not discriminate much between the tumor tissue and the normal tissue. Thus, eupatoriopicrin itself may not find applications for cancer treatment but there is a vast scope for new drug development by derivatization. The flavone nobiletin isolated from E. coelestinum exhibited strong fungistatic activity towards Deuterophoma tracheiphila which causes ‘mal secco’ disease of citrus varieties (Van and Pham, 1979). Chloroform extracts of E. odorartum showed in vitro antimicrobial activity against Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Aspergillus niger (Iwu and Chiori, 1984). Essential oil from E. odoratum exhibited activity against Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The main constituents of the oil were a-pinene, b-pinene and pregeijerene (Bamba et al., 1993). Similarly, the essential oil from E. triplinerve showed antimicrobial activity against a number of bacterial and fungal species. It exhibited high activity against Escherichia coli and Proteus vulgaris, and moderate activity against Bacillus anthracis, Salmonella stanley, S. pullorum, S. richmond, Staphylococcus aureus and B. subtilis. The essential oil was less active against Pseudomonas aeruginosa, Klebsiella pneumoNat. Toxins 6: 1–14 (1998) SHARMA ET AL. 10 Table 3. Bioactive constituents isolated from various species of Eupatorium plant Compound Plant species Sesquiterpene lactones Eupatoriopicrin Eupahyssopin Eupaserrin Desacetyleupaserrin Eupaformin Eupaformosanin Eupatolide Eupacunolin Eupacunin Eucannabinolide Desacetyleupacunin Euponin Sesquiterpenes Cadinenes Costic acid Flavonoids Eupatorin Nobiletin E. cannabinum E. hyssopifolium E. semiserratum E. semiserratum E. formosanum E. formosanum E. formosanum E. lancifolium E. lancifolium E. cuneifolium E. lancifolium E. lancifolium E. japonicum E. adenophorum E. capillifolium E. altissimum E. semiserratum E. coelestinum Biological activity Cytotoxic, antitumor Immunostimmulating, antiinflammatory, cytotoxic Antileukemic Antileukemic Cytotoxic Antileukemic Cytotoxic Cytotoxic Cytotoxic Antileukemic Antileukemic Cytotoxic Inhibits insect development Woerdenbag et al., 1987 a,b, 1989 a,b) Wagner (1984), Hall et al. (1979, 1980) Lee et al. (1976) Kupchan et al. (1973a) Kupchan et al. (1973b) Lee et al. (1977) Lee et al. (1977) Lee et al. (1972, 1977) Herz et al. (1981) Herz et al. (1981) Kupchan et al. (1971, 1973b) Herz et al. (1981) Herz et al. (1981) Nakajima and Kawazu (1978) Antifeedant Seed germination and growth inhibition Antibacterial Bordoloi et al. (1985) Baruah et al. (1994) Cytotoxic Herz et al. (1978) Herz et al., (1981) Van and Pham (1979) Fungistatic niae, Streptococcus agalactiae, Salnonella newport, Aspergillus niger, Rhizopus stolonifer and Fusarium oxysporum (Yadava and Saini, 1990). 7-Methoxycoumarin isolated from aerial parts of E. ayapana showed insect antifeedant activity (Dutta et al., 1989). Root extracts of E. riparium exhibited antifungal activity against Cladosporium cladosporiodes. The active constituent appeared to be methylripariochromene A (6acetyl-7,8-dimethoxy-2,2-dimethylchromene; Bandara et al., 1992). FUTURE RESEARCH . A number of species of Eupatorium cause toxicosis in different parts of the world. E. rugosum toxicosis in livestock and the associated problem of milk sickness in humans has been of utmost significance in the USA for the last 100 years or more (Beier and Norman, 1990). Using rat liver microsomes and the cytotoxicity as bioassay, Beier et al. (1993) showed that E. rugosum toxins undergo microsomal activation. These studies remain to be extrapolated to ruminants and horses which are the victim of natural toxicosis. The nature of E. rugosum toxins remains uncertain and warrants future research. E. adenophorum causes hepatotoxicity in mice, cholestatis in rats and respiratory disease in horses. The toxin which causes hepatotoxicity in mice  1998 John Wiley & Sons, Ltd. Reference(s) . . . . Rao and Alvarez (1981) has been characterized while the toxin(s) responsible for the toxicosis in rats and horses remain to be elucidated. Similarly, toxins present in other plants like E. inulifolium have not been characterized. It is germane to do investigations on the chemotaxonomy of Eupatorium genus for obviating ambiguities in nomenclature based on descriptive taxonomy ((Lanaud et al., 1991). A number of the species of Eupatorium genus are known to exhibit allelopathic action. Purification and characterization of allelochemicals would help find leads for development of novel biocides. Extracts of various spp. of Eupatorium have been used in traditional medicine for decades. The plant has spread and become very wild in many parts of the world and provides an attractive bioresource for drug research using the natural products. A number of isolated constituents from Eupatorium spp. have promising biological activities and hold potential for further research by chemical derivatization. ACKNOWLEDGEMENTS We thank Dr O.S. Tomer, the Director of the Indian Veterinary Research Institute, for the facilities and Dr B. Nat. 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