Antibacterial Activity of a Stearic Acid Derivative from Stemodia foliosa Lourinalda Luiza Dantas da Silva2, Mµrcia Nascimento1, Dulce Helena Siqueira Silva2, Maysa Furlan2, Vanderlan da Silva Bolzani2 Abstract Affiliation: 1 Departamento de Antibióticos ± Centro de Ci†ncias Biológicas, Universidade Federal de Pernambuco, PE, Brazil ´ 2 NuBBE (N‚cleo de Bioensaio, Biossíntese e Ecofisiologia de Produtos Naturais), Instituto de Química, Universidade Estadual Paulista ± UNESP, Araraquara, SP, Brazil Correspondence: Vanderlan da Silva Bolzani ´ NuBBE (N‚cleo de Bioensaio, Biossíntese e Ecofisiologia de Produtos Naturais) ´ Instituto de Química ´ Universidade Estadual Paulista ´ CP 355, CEP 14801-970 ´ Araraquara ´ SP, Brazil ´ E-mail: bolzaniv@iq.unesp.br Received: April 8, 2002 ´ Accepted: July 28, 2002 Bibliography: Planta Med 2002; 68: 1137±1139 ´  Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943 Letter ¼ Planta Med 2002; 68: 1137 ± 1139 1137 Heruntergeladen von: University of Illinois at Chicago. Urheberrechtlich geschützt. In the course of our systematic work on Brazilian medicinal plants, we have investigated Stemodia foliosa (Scrophulariaceae), a shrub popularly named ªmeladinhaº, which is a folk medicine known in Northeastern Brazil to treat asthma and respiratory tract diseases and also used as domestic bioinsecticide [1]. There are about 40 Stemodia species growing naturally in Asia, Africa, Australia and America and until now, only three species have been chemically investigated: S. maritima, S. durantifolia and S. chilensis, which afforded diterpenes as main secondary metabolites [2], [3], [4]. In Brazil, Stemodia species are widely distributed and some of them (S. humilis, S. stricta, S. trifoliata, S. lanceolata, S. viscosa and S. foliosa) are used as domestic insecticide to kill chicken flea [5], although no phytochemical or biological work has been undertaken on such species yet. On the basis of its folk uses, S. foliosa was selected in our search for antibacterial compounds. This paper describes the isolation and structure elucidation of the new stearic acid derivative 1, which inhibited the growth of the Gram-positive bacteria Bacillus cereus and B. subtilis, and the fast-acid bacterium Mycobacterium fortuitum. Letter From the hexane-soluble fraction of an ethanol extract from leaves and stems of Stemodia foliosa (Scrophulariaceae), the new stearic acid 4-[(n-pentoxy)phenethyl] ester (1) was isolated. This compound exhibited antibacterial properties at 10 mg/mL concentration by using disc diffusion method against Gram-positive bacteria Bacillus cereus and Bacillus subtilis and fast-acid bacterium Mycobacterium fortuitum. The structure of the new compound was elucidated by spectroscopic methods and by chemical conversion. 1138 Table 1 NMR data for compound 1 (CDCl3; 500 MHz for 1H- and 125 MHz for 13C-NMR)a Position dH (J in Hz) b dCc 1 ± 173.9 s 2 2.27 t (7.6) 34.4 t 1.58 C-1, C-3, C-4 3 1.58 m 24.9 t 2.27, 1.20 C-1 4 ± 15 1.25 m 29.3 t 29.1 ± 29.7 t 1.58, 1.25 16 1.25 m 31.9 t 1.58 17 1.32 m 22.7 t 0.86 C-18 18 0.86 t (6.5) 14.1 q 1.32, 1.25 C-17, C-16 COSY 1H-1H HMBC ± 1¢ ± 130.0 s ± ± 2¢ , 6¢ 7.07 d (8.0) 130.3 d 6.76 C-1¢, C-4¢, C-7¢ 3¢ , 5¢ 6.76 d (8.0) 115.1 d 7.07 C-1¢, C-4¢ 4¢ ± 154.3 s ± 7¢ 2.85 t (7.1) 34.3 t 4.23 C-1¢,C-2¢, C-8¢ 8¢ 4.23 t (7.1) 64.9 t 2.85 C-1¢, C-1 1¢¢ 3.64 t (6.4) 63.1 t 1.55 C-4¢, C3¢¢ 2¢¢ 1.55 m 32.8 t 3.64, 1.25 C-1¢¢, C-4¢¢ 3¢¢ 1.25 m 29.7 t 1.55 C-5¢¢, C-1¢¢ 4¢¢ 1.28 m 22.7 t 1.28, 0.88 C-5¢¢, C-3¢¢ 5¢¢ 0.88 t (6.0) 14.0 q 1.28 C-3¢¢, C-4¢¢ a Chemical shifts refer to TMS (d = 0.00). Assignments were based mostly on HOMOCOSY experiments. c Multiplicities were determined from DEPT 1358 analysis. Assignments were based mostly on gHMQC and gHMBC experiments. b Letter ¼ Planta Med 2002; 68: 1137 ± 1139 lations between H-1¢¢ and C-4¢ as well as between H-7¢ and C-1¢ were evidenced in the gHMBC spectrum and corroborated the 4(n-pentoxy)-phenethyl moiety of compound 1. Additionally, correlation of H-8¢ with C-1¢ and C-1 evidenced the stearic acid bonding to the phenethyl moiety through an ester linkage. Subsequent attempts to confirm the structure of compound 1 through ES-MS analysis disclosed the base peak with m/z 267 and minor peaks in good agreement with the expected fragmentation pattern for compound 1. Additional evidence was obtained from mild basic hydrolysis of 1 followed by methylation, achieved in good yield, to produce the expected stearic methyl ester, which was identified from GC-MS analysis. Thus, 1 was established as stearic acid 4-[(n-pentoxy)-phenethyl] ester. This compound was tested against Gram-positive bacteria Staphylococcus aureus, Bacillus cereus, B. anthracis and B. subtilis; Gram-negative bacteria Klebisiella pneumoniae and Pseudonocardia termophyla and fast-acid bacterium Mycobacterium fortuitum due to the inhibition displayed by the crude ethanol extract against these strains. However, the new isolate 1 only showed significant activity against Gram-positive bacteria Bacillus cereus and B. subtilis and fast-acid bacterium Mycobacterium fortuitum (Table 2) and gave no inhibition against the other strains at the concentration of 10 mg/mL, which were compared with the positive control claritomycin at the concentration of 1.28 mg/mL [7]. Materials and Methods Silica gel 60 F254 sheets (Merck and Sigma) were used for TLC and open Column Chromatography (CC) was performed on silica gel (60 ± 230 mm, Merck). Solvents: EtOH, hexane, MeOH, EtOAc, n-BuOH and DMSO (Merck and Sigma-Aldrich) were used for chromatographic procedures, bioassays and UV/IR analysis; CDCl3 (Merck) was used for NMR purposes. IR and UV spectra were recorded on a FT-1750 Perkin Elmer and UV/Vis Hitachi U-3000 spectrometers. ES-MS spectra were obtained at 70 eV on a VG platform II spectrometer; 1H- and 13C-NMR spectra were recorded on a Varian-Inova Unit 500 spectrometer using TMS as internal standard. Stemodia foliosa Benth was collected in July, 1998 at the Campus of Federal University of Pernambuco (UFPE), in Recife, Northeastern Brazil. The botanical material was identified by Dr. Alda Chiappeta, from Departamento de Antibióticos-UFPE, and a voucher specimen was deposited in the Herbarium of Departamento de Botânica-UFPE, under the number UFPE-19 810. Whole aerial parts of this shrub (1.0 kg) were dried at room temperature, powdered, extracted with ethanol and concentrated to give a crude extract (52.0 g). Aliquots from the ethanol extract were separated for acute toxicity and antibacterial bioassays. The antibiotic assay was performed with Gram-positive bacteria: Staphylococcus aureus (DAUFPE 01), Bacillus cereus (DAUFPE 11), B. anthracis (DAUFPE 09) and B. subtilis (DAUFPE 016); Gramnegative bacteria: Klebisiella pneumoniae (DAUFPE 396*) and Pseudonocardia thermophyla (DAUFPE 3517) and fast-acid bacterium Mycobacterium fortuitum (DAUFPE 405). Bacteria were incubated on a nutrient agar-slant (stationary culture) for 48 h Heruntergeladen von: University of Illinois at Chicago. Urheberrechtlich geschützt. Letter A concentrated ethanol extract of the leaves and stems of S. foliosa was dissolved in n-BuOH and successively extracted with hexane. The hexane-soluble phase was concentrated and submitted to chromatographic procedures to afford the new compound 1. Stearic acid 4-pentoyloxyphenethyl ester (1) was obtained as an amorphous powder and its molecular formula, C32H54O3 was established from ES-MS, 1H- and 13C-NMR data. The ES-MS exhibited a molecular ion peak at m/z 475 [M + H]+ and representative ion peaks at m/z 207, 191, 177 and 163, which are consistent with the 4-(n-pentoxy)-phenethyl moiety proposed for 1. An IR band at 1710 cm±1 and one signal at d 173.9 in the 13C-NMR spectrum suggested an aliphatic ester. The 1H-NMR spectrum (Table 1) showed two ortho-coupled doublets due to a 4-O-substituted phenethyl unit and three triplets for six hydrogens at d 4.23, d 3.64 and d 2.85 due to the oxygen-bearing methylenes (H-8¢ and H-1¢¢) and to the benzylic methylene (H-7¢), respectively. Moreover, signals at d 2.27, d 1.58, d 1.55 and d 1.25 corresponding to several methylenes, and two triplets at d 0.86 and d 0.88, attributed to two terminal methyl groups suggested, as part of 1, two aliphatic methylene chains. The 13C-NMR spectrum (Table 1) showed signals which confirmed the phenethyl moiety and two aliphatic methylene chains in compound 1. The HOMOCOSY experiment evidenced the spins system associated with aromatic ortho-coupled hydrogens and with methylene hydrogens H-8¢ and H-7¢. Additional couplings between methylene hydrogens at d 3.64 and d 1.55 as well as at d 2.27, d 1.58 and d 1.25 were also evidenced by the COSY spectrum and gave clear indication of signals belonging to each methylene chain. Precise assignments of all hydrogens and carbons followed from detailed analysis of gHMQC and gHMBC spectra (Table 1) and allowed the location of the two aliphatic moieties in compound 1. Corre- References Antibacterial activity of stearic acid 4-[(n-pentoxy)phenethyl] ester (1)a Microorganism Ethanolic Extractb 1c Claritomycin Staphylococcus aureus (DAUFPE 01) 12 ± Bacillus cereus (DAUFPE 11) 10 10 8 Bacillus subtilis (DAUFPE 16) 13 10 7 Bacillus anthracis (DAUFPE 09) NT NT 17 Klesbisiella pneumoniae (DAUFPE 396)d 13 ± ± Pseudonocardia thermophylla (DAUFPE 3 517) 10 ± 11 ± 10 ± 20 Mycobacterium phlei (DAUFPE 70) ± ± 35 Mycobacterium fortuitum (DAUFPE 405) 10 8 20 a size of inhibition zones, mm. Claritomycin at 1.28 mg/mL used as positive control. inhibition detected at 200 mg/disk; (-) no inhibition at 1000 mg/disk for the ethanolic extract. c inhibition detected at 10 mg/disk. (-) no inhibition at 20 mg/disk. d DAUFPE 396 = ATCC 29 665 b Barroso G. Sistemµtica de Angiospermas do Brasil.; Vol. 3 VicËosa: Universidade Federal de VicËosa, 1991: 124 ± 30 2 Chamy MC, Piovano M, Garbarino JA, Gambaro V. Stemodane diterpenoids from Stemodia chilensis. Phytochemistry 1991; 30: 1719 ± 21 3 Hufford CD. H-1-NMR and C-13-NMR Assignments for the Stemodia diterpenes stemodin, stemodinone and maritimol. J Nat Prod 1988; 51: 367 ± 9 4 Weniger B, Haagberrurier M, Anton R. Plants of Haiti used as antifertility agents. J Ethnopharmacol 1982; 6: 67 ± 84 5 Correa MP. Dicionµrio das Plantas Úteis do Brasil e das Exóticas Cultivadas. Rio de Janeiro: MinistØrio da Agricultura, IBDF, 1978: 182 ± 97 6 Watt B, Collee JG. Bacterial challenges and evolving antibacterial drug strategy. Postgrad Med J 1992; 68: 6 ± 21 7 National Committee for Clinical Laboratory Standards, 3rd. ed., Approved Standard M7-A3, NCCLS, Villanova, PA. 1993 8 Holland HL, Diakow PRP, Taylor GJ. C-13 Nuclear magnetic-resonance spectra of some C-19-hydroxy, C-5,6 epoxy, C-24 ethyl, and C-19-norsteroids. Can. J. Chem. 1978; 56: 3121 ± 7 9 Mahato SB, Kundu AP. C-13 NMR spectra of pentacyclic triterpenoids ± A compilation and some salient features. Phytochemistry 1994; 37: 1517 ± 75 at 37 8C followed by inoculation in Mueller Hinton Agar (MHA) medium. Bacteria strains were supplied by Departamento de Antibióticos-UFPE. The antibacterial activity was demonstrated using the disc-diffusion method originally described by Watt and Collee [6]. The stock solution of each test compound/extract in DMSO was diluted at 10 mg/mL in nutrient broth. All the tests were done by placing the disc (9 mm diameter) impregnated with extracts, fractions or pure compound 1 on the MHA surface previously inoculated with 10 mL of (MHA) liquid medium with Gram-positive, Gram-negative and fast acid bacteria. DMSO was used as negative control and standard antibiotic claritomycin (1.28 mg/disc) was used as reference or positive control. Plates were incubated at 37 8C for 48 h and the inhibition zones around each disc were measured. Letter Micrococcus luteus (DAUFPE 06) 1 1139 The ethanol extract (52.0 g) was partitioned between H2O and nBuOH to give the aqueous and n-BuOH phases. After evaporation of the solvents, the n-BuOH residue (41.0 g) was dissolved in 80 % aqueous MeOH and sequentially partitioned with n-hexane, CH2Cl2 and EtOAc. The n-hexane soluble phase was concentrated to give a bioactive n-hexane extract (9.4 g), which was subjected to silica gel (300.0 g) column chromatography, eluted with nhexane/EtOAc gradient: 9 : 1, 8 : 2 and 75 : 25, (1000 mL of each eluent). Fractions were tested and those obtained upon elution with n-hexane/EtOAc (8 : 2) proved to contain bioactive compounds. They were analyzed by TLC (n-hexane/EtOAc 8 : 2, UV detection at 254 nm), pooled (2.1g) and further submitted to silica gel (200 g) column chromatography using a n-hexane/EtOAc gradient: 95 : 5, 9 : 1, 8 : 2 and 7: 3, (300 mL of each eluent). Fractions eluted with n-hexane/EtOAc 95 : 5 were pooled and further purified by preparative TLC (n-hexane/EtOAc 9 : 1), yielding the new bioactive compound 1 (35.0 mg, Rf 0.26), in addition to a mixture of sitosterol and stigmasterol (100 mg), lupeol (90 mg) and lupeol acetate (12 mg), which have been identified from analyzes of their NMR spectra and comparison with literature data [8], [9]. Copies of the original spectra are obtainable from the author of correspondence. Letter ¼ Planta Med 2002; 68: 1137 ± 1139 Heruntergeladen von: University of Illinois at Chicago. Urheberrechtlich geschützt. Table 2