The biotransformation of ß-estradiol (1,3,5-estratriene-3,17,ß-diols) by Bacillus subtillus resulted in four metabolites which were (1) 3,5-estratriene-3-ol–17–one, (2) 1,3,5-estratriene-3,16,17-triol, (3) 1,3,5,7- estratetra-ene-3-ol-17-one and (4) 1,3,5,6, estratetra-ene-3-ol-17-one. The structures were elucidated on the basis of spectroscopic techniques. Compound 3 showed good inhibitory activity against lipoxygenase enzyme.Key words: Microbial transformation, ß-estradiol (1,3,5-estratriene-3,17,ß-diols), Bacillus subtillus, Aspergillus niger, lipoxygenase

Ahmad, S

Ayaz, S

Haider, S

Hussain, J

Jamila, N

Khan, N

Muhammad, Z

Ullah, R

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 10(28), pp. 5529-5533, 20 June, 2011     Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.2716 ISSN 1684–5315 © 2011 Academic Journals     Full Length Research Paper  Biotransformation of ß-estradiol isolated from Sonchus eruca  Javid Hussain1,5*, Zia Muhammad1, Riaz Ullah1,4 , Nargis Jamila3, Shabir Ahmad1, Naeem Khan1, Sultan Ayaz2 and Sajjad Haider4  1Department of Chemistry, Kohat University of Science and Technology, Kohat-26000, Pakistan. 2Department of Zoology, Kohat University of Science and Technology, Kohat-26000, Pakistan. 3School of Chemical Sciences USM University of Sains Malaysia Penang, Malaysia. 4Department of Chemical Engineering, College of Engineering, King Saud University, P.O BOX 800, Riyadh 11421, KSA. 5Department of Biological Sciences and Chemistry, College of Arts and Sciences University of Nizwa, Oman.  Accepted 25 March, 2011  The biotransformation of ß-estradiol (1,3,5-estratriene-3,17,ß-diols) by Bacillus subtillus resulted in four metabolites which were (1) 3,5-estratriene-3-ol–17–one, (2) 1,3,5-estratriene-3,16,17-triol, (3) 1,3,5,7-estratetra-ene-3-ol-17-one and (4) 1,3,5,6, estratetra-ene-3-ol-17-one. The structures were elucidated on the basis of spectroscopic techniques. Compound 3 showed good inhibitory activity against lipoxygenase enzyme.  Key words: Microbial transformation, ß-estradiol (1,3,5-estratriene-3,17,ß-diols), Bacillus subtillus, Aspergillus niger, lipoxygenase.   INTRODUCTION  Biotransformation is increasingly exploited as a useful and often unique reaction step in the semi synthesis of pharmaceuticals or for structural modification of complex natural compounds (Jovetic et al., 1998). In a chemical synthesis, a variety of catalysts and protecting group are needed for well defined chemical transformation. An alternative way of structural modification of substrate is the use of some bio-cataylst (isolated enzymes, cell extract, plants and animals, whole cell of bacteria, yeast, micro algae and fungi) under mild conditions collectively called biotransformation. The transformation of substrate that occurred by this pathway are regio-and stereo-selective or sometimes novel (Javid et al., 2010). ß-Estradiol was isolated from chloroform soluble fraction of Sonchus eruca belonging to the family of asteraceae. The asteraceae or sunflower family (formerly composite, also known as the Aster family) is a family of dicoty-ledonous flowering plants. It is the largest family of angiosperms  and  comprises  of  over  1535  genera and     *Corresponding author. E-mail: javidhej@yahoo.com, afridiriaz@yahoo.com. Tel: +923339623430. 23000 species, distributed in three sub-families and 17 tribes. It is also the largest plant family in Pakistan, repre- sented by over 650 species distributed in 15 tribes (Carvalho et al., 2006). ß-estradiol is the most potent and naturally occurring estrogen in human beings followed by estrone and estriol (Bulent and Inci, 2002). Estradiol is one of the most abundant and important estrogens occurring in animals and man (Jan et al., 2002). Estrogen plays a central role in the development and maintenance of the female reproductive organs, mammary glands and other sexual characteristics. The decreased production of estrogen has been linked to a series of postmenopausal pathologies, such as mood alterations, sleeplessness, hot flushes, sweats and sexual dysfunction (Muhammad et al., 2005).  In continuation of the work with microbial transfor-mation of steroid, incubation of compound 1 with Bacillus subtilus and Aspergillus niger resulted in the formation of four metabolites (Scheme 1) which were reported for the first time via B. substillus. Metabolite 2 underwent reduction at C-19, metabolite 3 underwent hydroxylation at C-18, metabolite 4 underwent reduction at C-19 and also exhibited dehy-drogenation at C-7 and C-8, and metabolite 5 underwent reduction at C-19 and also exhibited  5530         Afr. J. Biotechnol.    ehydrogenation  at C-6 and C-7.   MATERIALS AND METHODS  Plant materials  The plant Sonchus eruca was collected at Parachinar Kurram agency, N.W.F.P Pakistan, in July 2008 and was identified by plants taxonomist. The voucher specimen has been deposited in the herbarium of the Botany Department of Kohat University of Science and Technology Kohat (KUST). The whole plant was air-dried for 10 days and milled into powder with an electrical grinder and finally, was stored in airtight bottles before analysis.   General experimental procedure  The electron ionization mass spectra (EI-MS) were measured on a Varian MAT 311 A mass spectrometer. The HR-EI-MS spectra were recorded on Joel JMS-600H mass spectrometer. The 1H NMR spectra ( in ppm, J in Hz) were obtained in deuterated solvent (CDCl3) at 400 MHz on Bruker Avance-400 NMR spectrometer, while 13C NMR spectra were recorded in CDCl3 on the same instru-ment at 100 MHz. The FTIR were recorded on Shimadzu FTIR-8900 spectrophotometer. UV spectra were recorded on advance UV spectrophotometer. Optical rotation measurement was perfor-med on Jasco DIP-360 digital polarimeter. Melting point was taken on Buchi 535 apparatus. The metabolites were purified on column chromatography on silica gel. The purity of sample was checked on precoated TLC plates (Si 60, PF 254, 20 × 20, 0.25 mm, E. Merck).    Preparation of media  The medium for B. subtillus (NRRL-68751) was prepared by mixing the following reagents and distilled water (2 L): glucose (10 g), peptone (2 g), yeast extract (10 g) and sucrose (4 g). The media for A. niger (NRRL-68751) was prepared by mixing glucose (20 g), peptone (10 g), KH2PO4 (10 g), yeast extract (10 g), glycerol (10 ml) and NaCl (10 g) in distilled water (2 L) and the pH was maintained at 5.6.   Culture and fermentation procedure  The following procedure was used for both B. subtllus and A. niger. Two days old spores of the bacteria were aseptically transferred into the broth medium flask containing 100 ml of freshly prepared and auto claved medium. The seed flask thus obtained was incubated on a shaker table at 30°C for two days. The two days old broth culture from the 100 ml seed flask was inoculated into 20 media flask (250 ml) containing 100 ml of the medium and fermen-tation was continued for further 24 h. Compound 1 (700 mg) was diluted with acetone (10 ml) and the resulting solution was evenly distributed among 20 conical flask having the shake culture and the fermentation was continued for 10 days for B. subtillus and for 12 days for A. niger. The mycelia were filtered, washed with ethyl acetate and the broth thus obtained was extracted with ethyl acetate. The extract was dried over anhydrous sodium sulphate and was concentrated in vacuum to obtain gums. These were adsorbed on equal quantity of Silicon (Si) gel and were eluted with various solvents, gradients of petroleum ether, EtoAc and MeOH. The obtained compounds 2, 3, 4 and 5 had 14.7, 6.2, 20.2 and 10.2% yield, respectively. The remaining compound (compound 1) was recovered unchanged. The yield of compounds 2 and 3 from A. niger was 3.2 and 5.4%, respectively.      Assay of lipoxygenase inhibition  Lipoxygenase inhibiting activity was conveniently measured by slightly modifying the spectrometric method developed by Tappel (Tappel, 1962). Lipoxygenase (1.13.11.12) type I-band linoleic acid was purchased from Sigma (St. Louis, MO, USA). 160 µl of 0.1 mM sodium phosphate buffer (pH 7.0), 10 µl of the test-compound solution and 20 µl of lipoxygenase solution were mixed and incubated at 25°C for 5 min. The reaction was then initiated with the addition of 10 µl of linoleic acid (substrate) solution, and resulted in the formation of (9Z, 11E)-(13S)-13-hydroperoxyoctadeca-9, 11-die-noate, the change in absorbance was read after 10 min at a wavelength of 234 nm. The test compound and control were dissolved in 50% EtOH. All reactions were performed in triplicate. The IC50 values were then calculated using the EZ-fit enzyme kinetics program (Perrella Scientific Inc., Amherst, USA).   RESULTS AND DISCUSSION  The fermentation of compound 1 with B. subtillus for 10 days yielded the reductive metabolite 2. The molecular formula of compound 2 determined from HR-EI-MS was found to be C18H22O2 and showed M+ at m/z 270.3706. The IR spectrum of compound 2 showed kenotic and hydroxyl absorption at 1720 and 3335 cm-1, respectively. The 13C NMR data of compound 2 was the same to the authentic sample except for the absorption at C-17. The unusual 13C NMR spectrum (100MHz, C5D5N) has absor-ption at δ 219.6 for C-17 instead of absorption at δ 81.22 for the authentic sample (Table 2), due to the presence of five member ring that caused angle strain. 1H NMR further confirmed this by the disappearance of absorption at  3.92 (J = 8 Hz, H-17) in compound 2 as absent in the authentic sample (Table1). The molecular formula of compound 3 determined from HR-EI-MS was found to be C18H22O3, and showed M+ at m/z 288.3858. The NMR data of compound 3 was also the same to the authentic sample but only was different in 1H NMR absorption at H-17 and H-16 in 1H NMR spectrum and C-17 and C-16 in the 13C NMR spectrum. The 1H NMR spectrum of compound 3 had an overlapped peak at 4.68 (H-17) instead of a peak at 3.92 (J = 8 Hz, H-17) in the authentic sample (Table 1). This assumption was further confirmed with 13C NMR spectrum having absorption at δ 82.30 for C-16 of methyne, instead of absorption at δ 30.90 for C-16 methylene (Table 2). Fur-thermore, no interaction was observed in NOE between H-16 and H-17, which confirmed that H-16 and H-17 had different stereochemistry; H-16 was ß while H-17 was . In NOE spectrum, the position of 16-OH was further confirmed through COSY interaction of H-16 with H-17 (Horeau and Kagan, 1964), and was used to assign the stereochemistry of the newly formed hydroxyl group at position-17 of the compound 3. The molecular formula of compound 4 determined from HR-EI-MS was found to be C18H20O2, and showed M+ at m/z 268.3548. The IR spectrum of compound 4 showed olefinic, ketonic and hydroxyl absorption at 1604, 1665 and 3335 cm-1,  respectively.  The  1H  NMR  spectrum  of  Hussain et al.         5531    Table 1. 1H NMR assignments of compound (1) and its metabolites 2 to 5.  Proton 1 2 3 4 5 H-1 7.09 d (12.6) 7.06 d (12.6) 7.27 d (12.6) 7.11 d (12.6) 7.04 d (12.6) H-2 7.00 dd (10.4, 6.6) 6.99 dd (10.4, 6.6) 6.58 dd (10.4, 6.6) 7.00 dd (10.4, 6.6) 6.78 dd (10.4, 6.6) 3-OH 8.70s 8.71s 8.70 s 8.69 s 8.69 s H-4 6.99 dd (16.2, 4.3) 7.00 dd (16.2, 4. 3) 6.98 dd (16.2, 4. 3) 7.01 dd (16.2, 4. 3) 6.72 dd (16.2, 4. 3) H-5 - - - - - H-6 2.81t (2.5, 6.2) 2.82 t (2.5, 6.2) 2.85 t (2.5, 6.2) 3.42 t (2.5, 6.2) 5.56 d H-7 1.26, 1.78 1.27, 1.79m 1.40, 1.66 m 5.41 m 5.48 dd H-8 1.29 m 1.30 m 1.43 m - 3.38 m H-9 2.09 m 2.08 m 2.08 m 3.43 t 2.10 m H-10 - - - -  H-11 2.17, 1.29 m 2.29, 1.37m 2.24, 1.30 m 2.21, 1.63 m 2.23, 1.28 m H-12 1.12, 1.73d (11.7) 1.29, 1.80 d (11.7) 1.46, 1.66 d (11.7) 1.10, 1.64 d (11.7) 1.16, 1.80 d (11.7) H-13 - - - - - H-14 1.79 m 1.44 m 1.50 m 2.43 m 1.52 m H-15 1.79 m 1.79 m 1.66, 1.08 m 1.64, 1.08 m 1.58, 1.28 m H-16 1.78, 1.32 m 1.93, 1.43t (2.2, 25) 4.03 m 1.94,173 t 1.84, 1.30 t (2.2, 25) 16-OH - - 4.66 s - - H-17 3.89 t (17.1, 8.5) - 4.05 d (20.08) - - 17-OH 3.92 s - 4.68 s - - H-18 0.98 0.80 1.06 0.70 0.86  The signal appeared in 1H-NMR spectrum recorded in C5H5N.    Table 2. 13C NMR chemical shifts and multiplicities of metabolites 1 to 5.  Carban 1 2 3 4 5 C-1 126.8 (CH) 126.9 (CH) 126.8 (CH) 128.6 (CH) 128.4 (CH) C-2 113.8 (CH) 113.9 (CH) 113.9 (CH) 115.0 (CH) 115.0 (CH) C-3 156.6 (Q) 156.8(Q) 156.7 (Q) 157.0 (Q) 157.3 (Q) C-4 116.2 (CH) 116.3 (CH) 116.3 (CH) 116.0 (CH) 116.0 (CH) C-5 138.1 (Q) 138.0 (Q) 138.1 (Q) 136.5 (Q) 135.9 (Q) C-6 29.9 (CH2) 29.8 (CH2) 29.7 (CH2) 30.1 (CH2) 126.9 (CH) C-7 26.8 (CH2) 26.3 (CH2) 26.3 (CH2) 122.9 (CH) 116.3 (CH) C-8 39.4 (CH) 38.7(CH) 38.8 (CH) 123.2 (Q) 40.4 (CH) C-9 43.7 (CH) 44.3 (CH) 45.0 (CH) 49.5 (CH) 49.5 (CH) C-10 131.4 (Q) 130.8 (Q) 130.9 (Q) 129.3 (Q) 129.2 (Q) C-11 27.6 ( CH2) 26.8 (CH2) 26.9 (CH2) 30.1 (CH2) 30.1 (CH2) C-12 37.4 (CH2) 35.9 (CH2) 36.9 (CH2) 35.5 (CH2) 36.7 (CH2) C-13 43.7 (Q) 44.3 (Q) 44.5 (Q) 49.5 (Q) 49.6 (Q) C-14 50.2 (CH) 50.3 (CH) 50.4 (CH) 50.7(CH) 50.7 (CH) C-15 23.4 (CH2) 21.6 (CH2) 22.6 (CH2) 19.9 (CH2) 20.9 (CH2) C-16 30.9 (CH2) 32.1 (CH2) 82.3 (CH) 32.7 (CH2) 32.7 (CH2) C-17 81.2 (CH) 219.0 (C=O) 82.2 (CH) 218.1 (C=O) 220.2 (C=O) C-18 11.6 (CH3) 13.8 (CH3) 12.2 (CH3) 13.7 (CH3) 13.6 (CH3)  Multiplicities were determined by DEPT experiment.    compound 4 was similar to that of compound 2; the only difference was the olefinic group at C-7 and C-8 position. The 1H NMR data of compound 4 had only difference in absorption at δ 5.41(m) H-7 for H-7 methine instead of at δ1.27 for H-7 methyne (Table 1). This assumption was further   confirmed   through   13C NMR  spectrum  with  a 5532         Afr. J. Biotechnol.    HOHHOHHOHHO(1)(2)HOHHOH(3)HO(4)1- Bicillus subtilusBicillus subtilusBicillus subtilus2- Aspergillus nigerHHHOHOHOOBicillus subtilus(5)125 78911121013141516171834 6 Scheme 1: Microbial transformation of compound 1.    signal for C-7 at δ 122.95 (CH) instead of a signal at  δ 26.88(CH2), while C-8 was at  δ 123.25(q) instead of at δ 39.40(CH) (Table 2). Further, the position of the double bond was confirmed by the HMBC interaction of the signal of H-6 at  5.41 (overlap), with C-8, C-9 and C-14, respectively. The molecular formula of compound 5 determined from HR-EI-MS was found to be C18H20O2, and showed M+ at m/z 268.3467. The IR spectrum of compound 5 showed olefinic, ketonic and hydroxyl absorption at 1607, 1667 and 3338 cm-1, respectively. The 1H NMR spectrum of compound 5 was similar to that of compound 4. The only difference was for the olefinic group which shifted from C-7 and C-8 in compound 4 to C-6 and C-7 positions in compound 5. The 1H NMR spectrum of compound 5 showed absorption peak for H-6 at δ 5.56(CH) instead  of at δ 3.42(CH2) (Table 1). The assumption was further confirmed through 13C-NMR spectrum with a signal for C-6 at δ 126.93 (CH), instead of at δ 30.17(CH2) in compound 4 (Table 2). Further, the position of the double bond between C-6 and C-7 was confirmed by the HMBC interaction experiment. Compounds 1 to 5 were screened for the inhibition of lipoxygenase enzyme. Lipoxygenase constitute a family of non-haem iron containing dioxygenases that are widely distributed in animals and plants. In mammalian cells, these are key enzymes in the biosynthesis of variety of bioregulatory compounds such as hydroxyeicosa- tetraenoic     Table 3. Inhibitory activity (IC50 (µM)) of compounds 1 to 5 against lipoxygenase enzyme.  Compound Lipoxygenase IC50 1 374.4±5.2 2 Inactive 3 168.4±3.2 4 197.4±4.1 5 195.4±1.3 Baicalcin (control) 22.4±1.3    acids (HETEs), leukotrienes, lipoxins and hepaxylines. Lipoxygenases are therefore, regarded as an attractive target for the rational drug design and discovery of mechanism-based inhibitors of treatment of variety of disorders such as bronchial asthma, inflammation, cancer and autoimmune diseases ( Lili  et al., 2004). The IC50 values of metabolites 1 to 5 are shown in Table 3, along with the positive control of lipoxygenase (Baicalcin). The biological screenings revealed that, metabolite 3 had more inhibitory potential against the lipoxygenase enzyme than -estradiol, while metabolites 4 and 5 also had moderate inhibitory activity against the lipoxygenase enzyme (Table 3). Similarly, compound 1 showed non- significant potential followed by compound 2 which was inactive.     ACKNOWLEDGEMENT  The authors wish to thank the Higher Education Commission, Government of Pakistan for providing financial support for the current study under the National Research Program for Universities (NRPU). Hussain et al.         5533    REFERENCES  Bulent S, Inci B (2002). Voltammetric investigation of -estradiol. J. Pharmaceut. Biomed. Anal., 28: 753-759.  Carvalho DCCCR, Fonseca DMMR  (2006). Biotechnology Advances Horeau A, Kagan HB (1964). Tetrahedron,  20: 2431-2433. Javid H, Zia M, Riaz U, Faman U (2010). Evaluation and chemical composition of  Sonchus eruca and Sonchus asper. J. Am. Sci. 6(9): 231-235. Jan Cz, Dobrowolski, Nina Sadlej S and Marek T (2002) Family of the estradiol chiral stereoisomers can they all exist? J. Mol. Structure, (Theochem), 618: 109-116. Jovetic S, Tramper J, Marinelli F (1998). Biotransformation of the lipoglycopeptide antibiotic A40926 with immobilized cells of Actinoplanes teichomyceticus-in situ supply of nutrients. Enzyme Microbial Technol. 22: 117-121. Lili S, Yuan Z, Li G, Shengshi Z (2004). Li Molecular electronegativity-distance vector (MEDV-4): a two-dimensional QSAR method for the estimation and prediction of biological activities of estradiol derivatives. J. Mol. Structure, 679: 107-113. Muhammad IC, Zafar AS, Ghulam S, Sarfaraz AN, Atta UR (2005). Microbial transformation of prednisone. Natural Prod. Res. 19(4): 311-317. Tappel AL (1962 b). In Methods in Enzymology, 5:,p 539. Ed. by Colowick SP, Kaplan N0. New York and London: Academic Press.                   

Biotransformation of ß-estradiol isolated from Sonchus eruca

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Biotransformation of ß-estradiol isolated from Sonchus eruca

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