6650009-3130/13/4904-0665 �2013 Springer Science+Business Media New York

Chemistry of Natural Compounds, Vol. 49, No. 4, September, 2013 [Russian original No. 4, July–August, 2013]

FUNGAL TRANSFORMATION OF METHYLTESTOSTERONEBY THE SOIL ASCOMYCETE Acremonium strictum TO SOMEHYDROXY DERIVATIVES OF 17-METHYLSTEROID

N. Nassiri-Koopaei,1 M. Mogharabi,1 M. Amini,2 UDC 547.918A. Shafiee,2 and M. A. Faramarzi1,2*

The ascomycete Acremonium strictum was used for the biotransformation of methyltestosterone (1), apharmaceutical steroid substance, into some steroid derivatives (6�-hydroxy-17�-methyltestosterone (2),6�,12�-dihydroxy-17�-methyltestosterone (3), 7�-hydroxy-17�-methyltestosterone (4), 6�,17�-dihydroxy-17�-methylandrosta-1,4-dien-3-one (5), and 3,17�-dihydroxy-17�-methylestra-1,3,5(10)-triene (6). Thefermentation was carried out in Sabouraud-dextrose broth (SDB) supplemented with 1 mM of the substrate,and the temperature and aeration rate were adjusted to 30�C and 150 rpm, respectively. The biotransformationcharacteristics observed were hydoxylations at C-6�, C-7�, and C-12�, 1,2-dehydrogenation, and ring Aaromatization. The best fermentation conditions, such as temperature, substrate concentration, pH, incubationperiod, and aeration, were found to be 25�C, 1 mM, pH 6.5, 6 days, and 150 rpm, respectively, for maximumbiotransformation of 1.

Keywords: Acremonium strictum, biotransformation, methyltestosterone, steroid, fungus, ascomycete.

Steroid compounds rank among the most widely marketed products in the pharmaceutical industry. Ongoing effortsare being made to increase the efficiency of the existing biotransformations as well as to identify new potentially usefulbiomodifications [1], as highly specific reactions are required to produce functionalized steroid compounds having therapeuticuse and commercial value [2].

Acremonium Link et Fries (formerly Cephalosporium) 1821 belongs to a very large group of white or pink moldswith wet heads of conidia produced from the tips of straight hyphae or lateral nipples. In some species, the conidia buds�secondary spores and the colony become yeast-like. They are the cosmopolitan fungi in the environment and are found in soil,plant debris, water, and foodstuffs. The taxon of A. strictum is genetically diverse [3]. One of the keratinophilic fungi and adermatophyte [4], it is not commonly associated with human diseases, but it has been identified as a pathogen in cases ofmycetoma, keratomycosis, postoperative endophthalmitis, onychomycosis, and meningitis [5]. It is also a pathogen of maize[6] and button mushrooms [7]. The biotransformation of steroid compounds such as hydrocortisone [8], progesterone [9],androst-1,4-dien-3,17-dione [10], nandrolone decanoate [11], and prednisolone [12] by A. strictum has been reported.

Methyltestosterone (1, MT), one of the most important androgenic compounds, is representative of this class oftherapeutic agents used for hypogonadism and other diseases. Ongoing attempts are being made to introduce new derivativesof methyltestosterone possessing therapeutic activity.

In the present work, methyltestosterone (1) was biotransformed to metabolites by A. strictum during a 7-day period ofincubation, which was the optimum bioconversion interval. The fermentation process resulted in five major metabolites 2–6.TLC profile revealed that the metabolites were more polar and hydrophilic than the precursor substance, methyltestosterone.

1) Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, TehranUniversity of Medical Sciences, P. O. Box 14155-6451, 14176, Tehran, Iran, fax: +98 21 66954712, e-mail: faramarz@tums.ac.ir;2) Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, 14176, Tehran, Iran. Published in KhimiyaPrirodnykh Soedinenii, No. 4, July–August, 2013, pp. 571–575. Original article submitted March 29, 2012.

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The fermentation medium was extracted by chloroform; then the solvent was evaporated. Products were purifiedchromatographically and characterized by applying the following melting point, retention time, and spectroscopic methods:1H NMR, 13C NMR, MS, and FTIR. The HPLC profile of the fermentation extract provided well-resolved and sharp peaks(chromatogram not shown), and some small peaks were also detected, indicating the existence of minor metabolites. 13C NMRsignal assignments of substrate 1 and its metabolites are presented in Table 1.

The EI-MS of metabolite 2 exhibited an M+ at m/z 318, corresponding to the formula C20H30O3, 16 a.m.u. higherthan 1, revealing the incorporation of an oxygen atom. The IR spectrum showed no new functional groups. In the 1H NMRspectra of the metabolites with a hydroxyl group at the 6�-position, a narrow triplet of the 6�-proton, and the downfield shiftof the 19-CH3 signal (about 0.2 ppm with respect to the substrate) confirmed that a 6�-hydroxyl group was introduced. The6�-proton chemical shift of compound 2 was 4.35 ppm, which was in accordance with those reported by Kirk and colleagues[13] and Hanson and colleagues [14] for 6�-hydroxymethyltestosterone. Moreover, the 13C NMR spectrum showed an additionalOH-bearing carbon at � 72.3 and disappearance of the 6-methylene signal at 32.8 in comparison with compound 1 [14].

The MS of metabolite 3 showed an M+ at m/z 334, in agreement with the formula C20H30O4, 32 a.m.u. higher than1, confirming two new oxygen atoms. The position and shape of the CHOH signal at � 3.81 resembled those found in the12�-hydroxy-17�-methyltestosterone spectrum. The triplet peak at � 4.42 in downfield for H-4 suggested the presence of a6�-hydroxyl group. The downfield shift of signal for 18-CH3 (0.10 ppm) corresponded to that calculated for6�,12�-dihydroxy-17�-methyltestosterone (3). In addition, the 13C NMR spectrum showed two extra OH-bearing carbon atomsat � 73.1 and 65.4, while 6- and 12-methylene signals at 32.8 and 31.3 were lacking, respectively, compared to compound 1 [15].

TABLE 1. 13C NMR Signal Assignments of the Substrate (1) and the Metabolites (2–6)

C atom 1 2 3 4 5 6 DEPT of 6

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20

35.6 33.9 199.6 123.7 171.4 32.8 31.5 36.4 53.7 38.6 20.6 31.3 45.3 50.0 23.1 38.7 81.3 13.9 17.3 25.7

36.1 35.7 199.6 124.5 171.1 72.3 38.6 33.9 50.6 32.8 20.5 31.5 45.1 53.8 22.4 28.7 79.1 15.4 17.5 26.2

37.1 34.3

200.3 126.4 168.2 73.1 38.9 31.4 53.6 38.2 30.5 65.4 50.1 53.6 23.2 38.8 81.6 13.9 19.5 25.8

34.9 33.4 200.3 127.2 166.9 43.8 65.9 40.9 47.1 39.8 21.5 30.7 46.3 48.8 23.7 38.4 81.3 14.8 18.5 24.8

155.4 128.8 185.3 122.9 170.1 71.5 32.7 36.3 52.7 42.9 23.4 31.2 45.9 51.5 23.5 38.4 81.2 15.9 16.7 26.4

127.2 113.2 155.8 116.1 131.5 28.3 26.4 39.5 45.4

138.8 27.3 28.7 50.3 47.3 23.5 38.9 81.4 14.5 30.8

–

CH CH C

CH C

CH2 CH2 CH

CHC CH2 CH2

C CH CH CH2 CH2

C CH3 CH3

–

1: R1 = R2 = R3 = H; 2: R1 = R3 = H, R2 = OH3: R1 = R2 = OH, R3 = H; 4: R1 = R2 = H, R3 = OH

5 6

18

36

18 12

19

17

OR2

R3

R1 OH

1 - 4

OOH

OH OH

HO

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The MS of 4 showed the molecular-ion peak at m/z 318 (C20H30O3), which suggested that one oxygen atom (16 unitsincreased) was introduced into the starting compound 1. The 1H NMR spectrum signals for 18-CH3, 19-CH3, 20-CH3, and H-4of compound 4 confirmed that the primary substrate backbone was unaltered. The presence of a carbon resonance at � 65.9in the 13C NMR of 4 (see Table 1) and a downfield shift for the 7�-proton at � 3.91 in the 1H NMR spectrum, in addition to thelack of the signal related to the 7-methylene group at � 31.5 in compound 1 in the 13C NMR spectrum, made the structuralelucidation decisive, based on the reported data [16].

The MS of metabolite 5 revealed an ion peak at m/z 316, corresponding to the formula C20H28O3. It was 14 unitsmore than that of methyltestosterone (1), not exactly confirming the insertion of a hydroxyl group. The IR spectrum showed3475 cm–1 (OH group).The peaks in 1668, 1617, and 1605 cm–1 showed the presence of the 1,4-dien-3-one system in the ringA. The 1H NMR spectrum signals for H-4, H-2, and H-1 of compound 5 confirmed that the primary substrate backbone waschanged in agreement with the IR spectrum, suggesting C1-C2 bond unsaturation. C18, C19, and C20 were not modified. The13C NMR of 5 (see Table 1), the downfield shift of the C3 carbonyl group at � 185.3, the peaks for C1 and C2 at 155.4 and128.8, respectively, and the absence of 13C NMR spectrum signals related to the mentioned carbon atoms in compound 1better conveyed the structure. The 71.4 peak indicated insertion at the 6�-position, as with compounds 2 and 3 [17].

The mass spectroscopy data of compound 6 showed an ion peak at m/z 286 in accordance with the formula C19H26O2.It was 14 units less than that of methyltestosterone (1), suggesting the possible omission of a methyl group. The IR spectrumshowed 3287 cm–1 for the hydroxyl group. The peaks in 1612 and 1574 cm–1 showed that an unsaturated and reduced systemwas present in the ring A, while the indicative peak at 1665 cm–1 for 3-ketone group was absent. The 1H NMR spectrumsignals for H-4, H-2, and H-1 of compound 6 confirmed that the primary substrate backbone was changed to an aromatic(phenolic) ring, in agreement with the IR spectrum indicating 3-ketone reduction. The lack of the peak at � 199.6 of the parentcompound 1 for the C3 carbonyl group in 13C NMR of 6 (see Table 1), in addition to the absence of the 13C NMR spectrumsignals of 1 related to the C1, C2, and C10 carbons at � 35.6, 33.9, and 38.6, respectively, confirmed the structure of ring A forcompound 6. The data from 13C NMR, 1H NMR, and IR were in agreement with that reported by Poirier and co-workers [18].The DEPT spectrum also confirmed the structure.

Time Course Experiment and the Influences of Temperature, pH, and the Substrate Concentration. Thin-layerchromatography was applied to detect the production of 2 to 6 as a function of incubation time. A. strictum transformedmethyltestosterone with concentration 1 mM into different metabolites within 6 days. The TLC profile, determined quantitativelyby HPLC (Fig. 1), showed that compounds 2 and 3 appeared in the broth from the first day while compounds 4 to 6 wereproduced from the second day. After 10 days of incubation, the substrate concentration was no longer detectable, and theconcentration of metabolites reached a plateau. The effect of substrate concentration in the range of 0.15 to 5 mM onmethyltestosterone biotransformation by A. strictum was also studied. The optimum substrate concentration was 1 mM, andconcentrations above 5 mM inhibited methyltestosterone biotransformation to metabolites. Shaking speed at 150 rpm providedthe highest bioconversion rate of methyltestosterone. The rate and yield of compounds 5 and 6 did not increase with a higheraeration rate. For compounds 2 to 4, although higher shaking speeds resulted in higher production rates, such speeds concurrentlydiminished the overall yield.

0 2 4 6 8 100.0

0.2

0.4

0.8

1.0

0.6

1

23456

Time, daySt

eroi

d co

ncen

tratio

n, m

M

Fig. 1. Time course profile for the biotransformation of17�-methyltestosterone (1) by A. strictum.

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The highest bioconversion rate was obtained within the pH range 6–7. The optimal pH for production of almost allmetabolites was 6.5. Higher pH (beginning at pH 3.5) potentiated the biomass production rate, and after biotransformationreached its maximum value at pH 6–7, thereafter decreasing gradually, and ultimately stopped at pH 10. The bioconversionreaction proceeded smoothly at 25�C for the production of all the metabolites, and the substrate was thoroughly wielded at thementioned temperature for 6 days. At 40�C, methyltestosterone stayed unchanged in the medium.

A. strictum transformed methyltestosterone into some steroid metabolites (2 to 6) within 6 days. Results showed thatcompounds 2 and 3 emerged sooner in the medium in comparison to compounds 4 to 6. Large amounts of the substrateinhibited the biotransformation process. Shaking speed did not influence the rate and yield of compounds 5 and 6, but itbrought about a potentiating impact on compounds 2 to 4 and diminished the overall yield. The optimal pH for production ofalmost all metabolites was 6.5.

Previous studies have subjected methyltestosterone to biotransformation by different fungi and bacteria. Hydroxylationhas emerged as the most prominent path for the bioconversion reactions in the form of both mono- and dihydroxylation [16].The results of the present study showed that A. strictum has hydroxylated MT by means of both mono- and dihydroxylation, inthe positions of C-6�, C-7�, and C-12� (2–4). The capacity for dehydrogenation of C1-C2 in ring A was also observed (5).Such a reaction has been observed for methyltestosterone to produce methandienone [19]. In the metabolite under investigation,a 6�-hydroxyl group was also introduced; this metabolite was obtained from human metabolism of methandienone [20].Another interesting metabolite was the aromatized metabolite of MT (6). Aromatization of androgenic compounds is a naturalprocess in the human metabolic pathway; testosterone is converted to estradiol by the aromatase enzyme in physiologic andpathologic conditions [21]. Aromatization of similar compounds like 2-methyleneandrostenedione, androstenedione, and16�-hydroxyandrostenedione in human placental microsomes has been investigated [22]. Conversion of 17�-methyltestosteroneto 17�-methylestradiol has been reported in fish [23]. Arthrobacter sp., Corynebacterium sp., Mycobacterium sp., Nocardia sp.,and Pseudomonas sp. have been reported to aromatize various steroids. In all the metabolites characterized, ring D was stillidentical to that of 1.

In conclusion, A. strictum generated hydroxylated metabolites of MT as the dominant products. It also yielded reducedand aromatized metabolites as minor products. The aromatized structure 6 may serve as the starting material for further steroidsulfatase inhibitor synthesis [24]. Compound 2 can exert antagonistic effects on androgenic receptors, with the effect ofaugmentation by 7-OH substitution by low hindrance groups [16], and the hydroxylation metabolism can be applied for theproduction of MT metabolites for pharmacokinetic studies [25, 26]. In short, A. strictum yields interesting compounds, givingthe strain potential biocatalyst activity. Characteristics such as hydoxylations on C-6�, C-7�, and C-12�, 1-dehydrogenation,and ring A aromatization prompt us to conduct more in-depth studies on the role of microorganisms in biotransformations ofsteroids.

EXPERIMENTAL

Chemicals and Instrumental Analyses. Methyltestosterone was donated by Abu-Reihan Pharmaceutical Company(Tehran, Iran). Sabouraud-2% dextrose broth (SDB) and Sabouraud-4% dextrose agar (SDA) were purchased from Merck(Darmstadt, Germany). Analytical grade chemicals and solvents were purchased from Merck (Darmstadt, Germany) andSigma-Aldrich (St. Louis, MO, USA). Melting points (mp) were determined on a (Gallenkamp, UK) hot stage melting pointapparatus and are uncorrected. Both 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded using a BrukerDRX (Avance 500) spectrometer (Rheinstetten, Germany), using CDCl3 as solvent and tetramethylsilane (TMS) as the internalstandard. Chemical shifts are given in parts per million (ppm) relative to TMS at 0 ppm. The coupling constant (J) is expressedin hertz (Hz). Infrared (IR) spectra were recorded in KBr discs with a Magna-IR 550 Nicolet FTIR spectrometer (Madison,WI, USA). Mass spectra (MS) were obtained with a Finnigan MAT TSQ-70 instrument (Bremer, Germany) operating in theelectron impact (EI) mode at 70 eV. Thin-layer chromatography (TLC) and preparative TLC were performed, respectively, on0.25 and 0.5 mm thick layers of silica gel G (Kieselgel 60 HF254 + 366, Merck). Layers were prepared on glass plates andactivated at 105�C, 1 h before use. Chromatography was performed with ethyl acetate–chloroform–methanol (80:20:5, v/v/v)and visualized by spraying the plates with a mixture of methanol–sulfuric acid (6:1, v/v) and heating them in an oven at 100�Cfor 3 min until the colors appeared. The compounds were also visualized under a UV lamp (Strstedt-Gruppe HP-UVIS) at 254nm. The high-performance liquid chromatography (HPLC) apparatus consisted of a Smartline HPLC pump 1000, a PDAdetector 2800, and a degasser 5000, all from Knauer (Berlin, Germany). Samples were withdrawn from vials prepared for an

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autosampler 3950 with a 100 �L sample loop. The data were acquired and processed by means of ChromGate software(version 3.3.1) from Knauer (Berlin, Germany). Chromatographic separation was achieved on a Lichrospher 100 RP & EC C8reverse-phase column (C8, 25 0.46 cm i.d., 5 �m particle size) from Teknokroma (Barcelona, Spain). PTFE MembraneFilters (0.45 �m), purchased from Schleicher & Schull (Germany), were used.

The Fungus and Incubation Conditions. The examined fungus was a strain of Acremonium strictum PTCC 5282[8, 18], a locally isolated fungus that was grown and maintained in sterile Sabouraud-dextrose agar (SDA) medium plates andfreshly subcultured before use in a biotransformation experiment. The organism was subcultured every 2 months. Spores ofA. strictum, preserved at low temperature, were inoculated into 50 mL of the sterilized culture medium of SDB in a conicalflask. The flask was then incubated to grow for a period of 24 h in an incubator shaker (150 rpm) at 30�C. Methyltestosteronewas dissolved in chloroform (1 mL chloroform for each flask) and evenly distributed between 20500 mL conical flasks, eachcontaining 100 mL of the SDB medium. The final concentration of the substrate was 0.03% in each flask. The pH was thenadjusted at 7.0. The flasks were further incubated in an orbital incubator shaker at 30�C and 150 rpm for 10 days.

Product Isolation and Analyses. The fermentation mixture was harvested and extracted exhaustively by three roundsof equal volumes of chloroform. The collected extracts were dried over anhydrous sodium sulfate and evaporated to drynessunder reduced pressure. The crude extract was then submitted to preparative TLC and elution by an ethyl acetate–chloroform–methanol (80:20:5, v/v/v) solvent system, and the metabolites were extracted. Purified metabolites were identified by meltingpoints and spectral data (13C NMR, 1H NMR, FTIR, and MS) after crystallization in order to obtain highly purified metabolites.

Time Course Experiment and the Influences of Temperature, pH, and the Substrate Concentration.Pre-cultured A. strictum was transferred into a 500 mL Erlenmeyer flask containing 100 mL of the SDB medium supplementedwith 50 mg of the substrate dissolved in 1 mL of chloroform. The fermentation process was allowed to proceed for 10 dayswith the conditions kept constant. Sampling was carried out on subsequent days to follow the course of biotransformation.Parallel control samples were similarly conducted without inoculation of the microorganism. Experimental studies wereperformed to determine the optimum pH, temperature, and concentration of substrate to be transformed to the products. Thetemperature investigated ranged from 25� to 40�C. The influence of pH on the biotransformation procedure was studied innon-buffered media by adjusting the pH from 3 to 11 with NaOH and HCl. The concentration of the substrate varied from 0.15to 5 mM. In all experiments the parameter being studied varied while the others were kept constant.

HPLC Analysis. HPLC was applied to the quantitative studies of time course experiments, the influence of temperature,pH, and substrate concentration, and also to obtain the biotransformation yield of each metabolite. A total of 10 �L of theconcentrated extract was injected into a C8 reverse-phase column. Elution was conducted by an isocratic method using methanol–water (25:75, v/v) with a flow rate of 1.0 mL/min at 30�C. Detection was performed by a Knauer PDA detector 2800 at 240 nm.Stock solutions were prepared individually by dissolving 10 mg of the substrate 1 as well as purified metabolites 2–6 in 10 mLmethanol.

6�-Hydroxy-17�-methyltestosterone (2). Crystallized from chloroform; yield 28.4%; mp 250–252�C. IR (max,cm–1): 3436, 2929, 1685, 1454. EI-MS (m/z, Irel, %): 318 (M+, C20H30O3; 14), 307 (40), 289 (26), 265 (57), 246 (100), 234(93), 174 (61), 162 (29), 145 (20), 133 (22), 119 (27), 105 (31), 93 (24), 71 (32), 55 (13). 1H NMR (CDCl3, �, ppm): 0.93(3H, s, 18-CH3), 1.32 (3H, s, 17-CH3), 1.40 (3H, s, 19-CH3), 4.35 (1H, t, H-6�), 5.73 (1H, s, H-4). UV (MeOH, �max, nm):240. Rf in ethyl acetate–chloroform–methanol (80:20:5, v/v/v): 0.8; retention time in methanol–water (25:75, v/v): 4.64 min.

6�,12�-Dihydroxy-17�-methyltestosterone (3). Crystallized from chloroform; yield 24.3%; mp 260–262°C.IR (max, cm–1): 3446, 2964, 1675, 1451. EI-MS (m/z, Irel, %): 334 (M+, C20H30O4; 14), 307 (40), 289 (26), 265 (57), 246(100), 234 (93), 174 (61), 162 (29), 145 (20), 133 (22), 119 (27), 105 (31), 93 (24), 71 (32), 55 (13). 1H NMR (CDCl3, �,ppm): 1.08 (3H, s, 18-CH3), 1.34 (3H, s, 17-CH3), 1.46 (3H, s, 19-CH3), 3.81 (1H, m, H-12�), 4.42 (1H, t, H-6�), 5.81 (1H,s, H-4). UV (MeOH, �max, nm): 240. Rf in ethyl acetate–chloroform–methanol (80:20:5, v/v/v): 0.5; retention time inmethanol–water (25:75, v/v): 4.18 min.

7�-Hydroxy-17�-methyltestosterone (4). Crystallized from chloroform; yield 16.7%; mp 253–255�C. IR (max,cm–1): 3446, 2964, 1671, 1624. EI-MS (m/z, Irel, %): 318 (M+, C20H30O3; 18), 300 (100), 282 (18), 243 (48), 124 (55), 91(52), 67 (35), 55 (30). 1H NMR (CDCl3, �, ppm): 0.98 (3H, s, 18-CH3), 1.30 (3H, s, 19-CH3), 1.40 (3H, s, 17-CH3), 3.91 (1H,m, H-7), 5.81 (1H, s, H-4). UV (MeOH, �max, nm): 240. Rf in ethyl acetate–chloroform–methanol (80:20:5, v/v/v): 0.7;retention time in methanol–water (25:75, v/v): 4.48 min.

6�,17�-Dihydroxy-17�-methylandrosta-1,4-dien-3-one (5). Crystallized from chloroform; yield 9.5%; mp 227–229�C.IR (max, cm–1): 3475, 2965, 1668, 1451. EI-MS (m/z, Irel, %): 316 (M+, C20H28O3; 14), 299 (17), 282 (17), 242 (21), 147(95), 122 (25), 91 (31), 43 (48). 1H NMR (CDCl3, �, ppm, J/Hz): 0.95 (3H, s, 18-CH3), 1.26 (3H, s, 17-CH3), 1.33 (3H, s,

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19-CH3), 4.38 (1H, t, H-6�), 5.98 (1H, br.s, H-4), 6.18 (1H, d, J = 10, H-2), 7.08 (1H, d, J = 10, H-1). UV (MeOH, �max, nm):240. Rf in ethyl acetate–chloroform–methanol (80:20:5, v/v/v): 0.31; retention time in methanol–water (25:75, v/v): 5.23 min.

3,17�-Dihydroxy-17�-methylestra-1,3,5(10)-triene (6). Crystallized from chloroform; yield 7.6%; mp 186–188�C.IR (max, cm–1): 3247, 2926, 1612, 1574, 1441. EI-MS (m/z, Irel, %): 286 (M+, C19H26O2; 14), 269 (17), 228 (41), 201 (27),187 (14), 173 (14). 1H NMR (CDCl3, �, ppm): 0.86 (3H, s, 18-CH3), 1.35 (3H, s, 17-CH3), 2.75 (2H, m, H-6), 4.76 (1H, br,phenolic OH), 6.53 (1H, d, H-4), 6.65 (1H, dd, H-2), 7.15 (1H, d, H-1). UV (MeOH, �max, nm): 280. Rf in ethyl acetate–chloroform–methanol (80:20:5, v/v/v): 0.39; retention time in methanol–water (25:75, v/v): 6.9 min.

ACKNOWLEDGMENT

This work was supported by Grant No. 91-02-90-18474 awarded to M.A.F. by the Biotechnology Research Center,Tehran University of Medical Sciences, Tehran, Iran.

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