Int J Pharm Pharm Sci, Vol 9, Issue 10, 171-175Original Article


MICROBIAL OXIDATION OF FINASTERIDE WITH MACROPHOMINA PHASEOLINA(KUCC 730)

SAIRA ERUM1, SADIA SULTAN2,3,4*, SYED ADNAN ALI SHAH2,3,4, MUHAMMAD ASHRAF5, MUHAMMAD IQBAL CHOUDHARY1

1H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan, 2Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia, 3Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia, 4Pharmaceutical and Life Sciences Core, Universiti Teknologi MARA, Shah Alam, 40450 Shah Alam, Selangor Darul Ehsan, Malaysia, 5Merck Pharmaceutical Private Limited, F-126, S. I. T. E. 75350, Karachi Pakistan
Email: drsadia@puncakalam.uitm.edu.my

Received: 20 Jun 2016 Revised and Accepted: 21 Sep 2017

ABSTRACT

Objective: New microbial oxidative derivatives of finasteride [17β-(N-tert-butylcarbamoyl)-4-aza-5α-androst-1-en-3-one] (1) has been investigated with Macrophomina phaseolina (ATCC730).

Methods: Fermented media of Macrophomina phaseolina (ATCC730) was prepared to cultivate the fungal cultures. Substrate 1 was incubated in liquid media for 16 d. After sixteen days, filtration and extraction of the fermented media were carried out with 9 L DCM in three portions. The resulting organic extract was dried using anhydrous (Na2SO4), and evaporated to afford a brown gum (950 mg). This on chromatographic purification with MeOH in CH2Cl2 afforded the metabolites 2-4.

Results: Three oxidised metabolites of finasteride (1) which were identified as 15-oxo-finasteride (2), 11α-hydroxyfinasteride (3), and 15β-hydroxyfinasteride (4). Metabolite 2 was found to be new. The structure of the oxidised metabolites was elucidated by 1-D (1H, 13C) and 2-D NMR (COSY, HMBC, HMQC, NOESY) techniques and MS analyses.

Conclusion: As a result of these study, oxidation at C-7, C-11 and C-15 positions were found. Metabolite 2 was identified as a new metabolite.

Keywords: Microbial transformation, Finateride, Oxidised metabolites, Macrophomina phaseolina

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2017v9i11.13576

INTRODUCTION

Finasteride [17β-(N-tert-butylcarbamoyl)-4-aza-5α-androst-1-en-3-one] (1) is a potent inhibitor of 5α-reductase, an enzyme which catalyzes the conversion of testosterone into dihydrotestosterone in many organs [1]. Finasteride (1) is used in the treatment of hair loss [2], and benign prostate hyperplasia, both these disorders are associated with in-situ high levels of increased dihydrotestosterone. It is also employed for the prevention of prostate cancer [3]. Previously, biotransformation of finasteride (1) has been reported by various fungi, including Selenastrum capricornutum [4], Mortierella isabellina, Cunninghamella elegans, and Bacillus megaterium [5].

Conversion of steroidal compounds by microbial transformation has turned out to be an important milestone in the research and development of steroidal drug industry. Steroids are also extensively used as anti-inflammatory drugs [6] and antiviral activity has also been reported from a steroidal glycoside Torvoside H, was isolated from the Solanum turvum [7]. Since then, steroidal drugs have emerged as the second largest category of drugs in the pharmaceutical industry with about 300 steroidal drugs in the market [8]. Many reports on microbial conversion of steroids have been published during the last half-century [9, 6, 10-21] focusing mainly on steroidal dehydrogenation, hydroxylations, esterification, halogenations, isomerization, methoxylation, and side-chain cleavage [22, 23]. In continuation of our work on micro-biotransformation [24-28] for the first time, we report here the fermentation of finasteride (1) with Macrophomina phaseolina (ATCC730) in order to obtain further microbial derivatives of finasteride which can be future candidates of drugs.

Scheme 1: Oxidation of finasteride (1) with Macrophomina phaseolina

MATERIALS AND METHODS

General

Finasteride (1) was isolated from the drug Proscar, manufactured by Merck Sharp and Dhome Limited of Pakistan. Melting points were determined on a Yanaco MP-S3 apparatus. UV spectra were measured on a Shimadzu UV 240 spectrophotometer. JASCO DIP–360 Digital polarimeters was used to measure the optical rotations in chloroform by using 10 cm cell tube. FTIR-8900 Spectrophotometer was used to record IR spectra in CHCl3. The 1H-NMR and 2D NMR spectra were recorded on a Bruker Avance III 500 spectrometer, while 13C-NMR spectra were recorded on Bruker Avance III 500 spectrometer operating at 125 MHz using CDCl3 as a solvent. chemical shifts were reported in δ (ppm), relative to SiMe4 as an internal standard, and coupling constants (J) were measured in Hz. The HREI MS were measured on Jeol HX 110 mass spectrometer. TLC was performed on Si gel precoated plates (PF254, 20 × 20, 0.25 mm, Merck, Germany). Ceric sulphate in 10% H2SO4 spraying reagent was used for the staining of compounds on TLC. All reagents used were of analytical grades.

Chemicals and reagents

Silica gel GF-254, preparative TLC (0.5 mm) solvents, Ingredients for media, reagents and substrates were purchased from E. Merck, Aldrich, and Fluka.

General fermentation and extraction procedure

The substrate was dissolved in acetone and the substrate solution was evenly distributed among all the flasks (20-30 mg/0.5 ml). Fermentation was continued for further 12-16 d on a rotary shaker (128 rpm) at 25-28 °C. Time course studies were carried out at regular intervals for analyzing the extent of fermentation on TLC. Fermentation media were harvested by filtration to separate the broth from the mycelium and extracted thrice with organic solvents such as dichloromethane or ethyl acetate. Sodium sulfate (Na2SO4) was added in the organic extracts for drying, filtered and then it is concentrated in vaccum to afford a brown gummy crude extract which was analyzed by thin layer chromatographic techniques.

Fungi and culture conditions

Microbial culture Macrophomina phaseolina (ATCC730) purchased from American Type Culture Collection (ATCC) from the USA was grown on Potato dextrose agar (PDA) at 25° C and stored at 4° C. M. phaseolina (ATCC730) medium was prepared by adding Glucose (10.0 g), peptone (5.0 g), KH2PO4 (5.0 g), yeast extract (5.0 g), NaCl (5.0 g) and glycerol (10 ml) into distilled water (1 L) and maintained pH at 5.6.

General stage II fermentation and extraction procedure

Cultures grown on the agar slants were transferred into a broth medium flask containing freshly prepared sterilized medium (100 ml in 250 ml flask). The seed flask was then incubated in a shaker for two days at 25 °C. The remaining flasks were inoculated from seed flasks and incubated for further 2-3 d. After 2 d substrate 1 (450 mg/13 ml acetone) was equally transferred among 25 flasks containing stage II fermentation medium of Macrophomina phaseolina. Fermentation was allowed and a time course study was carried out after each 24 h to analyze the degree of transformation on TLC. After four days, filtration and extraction of the fermented media was carried out with 9 L DCM in three portions. Resulting organic extract was dried using anhydrous (Na2SO4), and evaporated to afford a brown gum (950 mg). This on chromatographic purification with MeOH in CH2Cl2 afforded the metabolites 2-4. 2 (5.4 mg); MeOH/CH2Cl2 (3:97), 3 (8.6 mg); MeOH/CH2Cl2 (5:95) and 4 (23.4 mg); MeOH/CH2Cl2 (7:93).

15-Oxofinasteride (2): colourless solid (5.4 mg) (MeOH: CH2Cl2, 3:97), M. p.: 132-133 οC

UV (MeOH) λmax nm (log ∈ε): 243 (3.9), [α][25]D: +73.3° (c = 0.12, CHCl3); Rf: 0.55 (DCM/MeOH = 85:15); IR (CHCl3) λmax cm-1: 1672, 2934, and 1726 EI-MS m/z (rel. int., %): m/z 387 [M+H+] (100), 386 (10), 368 (18), 353 (28), 266 (12), 244 (23), 212 (16), 199 (15), 180 (55), 168 (14), 159 (31), 105 (42), 81 (61), 67 (12), 53 (22). HREI-MS m/z (mol. formula, calcd value): 386.2545 (C23H34N2O3, 386.2543). 1H-NMR: table 1 13C-NMR: : δ 150.5 (C-1), 166.3 (C-3), 59.6 (C-5), 25.6 (C-6), 27.5 (C-7), 47.5 (C-9), 39.5 (C-10), 38.2 (C-12), 42.3 (C-13), 64.2 (C-14), 212.8 (C-15), 37.4 (C-16), 52.9 (C-17), 14.3 (C-18), 12.1 (C-19), 168.8 C-20), 51.7 (C-22), 29.0 (C-23).

11α-Hydroxyfinasteride (3): colourless solid (8.6 mg), M. p.: 141-143 °C UV (MeOH) λmax nm (log ∈ε): 207 (3.6), [α][25]D: +69.3° (c = 0.14, CHCl3); Rf: 0.63 (DCM/MeOH = 85:15); IR (CHCl3) λmax cm-1: 3336, 3311, 1730, 1678, 1600; EI-MS: m/z (rel. int., %): 389 [M+H+] (100), 388 (16), 370 (8), 355 (7), 270 (31), 256 (32), 240 (13), 222 (46), 180 (35), 168 (54), 159 (21), 109 (39), 105 (22), 81 (21), 67 (2), 53 (29). HREI-MS m/z (mol. formula, calcd value): 388.2744 (C23H36N2O3, 388.2726). 1H-NMR: table 1; 13C-NMR: δ 167.0 (C-3), 53.5 (C-9), 44.0 (C-10), 69.2 (C-11), 49.9 (C-12), 40.5 (C-13).

15β-Hydroxyfinasteride (4): colourless solid (23.4 mg), M. P.: 142-145 °C UV (MeOH) λmax nm (log ∈ε): 202 (3.8), [α][25]D: +65.3° (c = 0.14, CHCl3); Rf: 0.42 (DCM/MeOH = 80:20); IR (CHCl3) λmax cm-1: 3342, 2935, 1666, 1595; EI-MS: m/z (rel. int., EI-MS: m/z (rel. int., %): 389 [M+H+] (18), 388 (55), 370 (58), 355 (7), 288 (12), 270 (37), 260 (12), 256 (10), 232 (100), 157 (53), 128 (85), 115 (24), 110 (72), 72 (39), 58 (46). HREI-MS m/z (mol. formula, calcd value): 388.4848 (C20H36N2O3, 388.4823). 1H-NMR: table 1;13C-NMR: δ 166.9 (C-3), 31.6 (C-8), 43.5 (C-13), 59.7 (C-14), 69.6 (C-15), 40.9 (C-16), 57.7 (C-17).

RESULTS AND DISCUSSION

Metabolism of finasteride (1) with Macrophomina phaseolina KUCC730 for 16 d resulted in three oxidative products, 2-4 (Scheme 1). Structures of the metabolites deduced through the comparative spectroscopic studies with the substrate(1). The HREI MS of metabolite 2 exhibited the molecular ion (M+) at m/z 316.1536, corresponding to the formula C20H28O3, The EI-MS of the compound 2 exhibited the M+at m/z 386, 14 amu. higher than the substrate 1, indicating the introduction of an oxygen atom, as carbonyl functionality. The HREI-MS of metabolite 2 exhibited a M+at m/z 386.2545, corresponding to the formula C23H34N2O3 (Calcd 386.2543). The IR spectrum of 2 showed the presence of an amide, an olefinic, and carbonyl functionalities by showing absorptions at 1672, 2934, and 1726 cm-1, respectively. The UV spectrum exhibited an strong absorption at 243 nm, indicating an α,β-unsaturated amidic moiety. The 1H-NMR spectrum of the metabolite 2 was almost similar to the parent compound 2, except a downfield shift of the H-14 signal from δ 1.08 to 1.64, indicating an oxidation at its vicinal carbon. The 13C-NMR spectrum of 2 was also quite similar to the substrate 1, and showed resonances for all 23 carbons, including seven methine, five methylene, five methyl, and six quaternary carbons. Additionally, it also exhibited a new quaternary ketonic carbon signal, resonated at δ 212.7 (C-15). The position of the newly introduced ketonic moiety was deduced to be at C-15 through the HMBC correlations of H-14 (δ 1.64) and H-16 (δ 2.91, 2.71) with δ 212.7 (C-15). Based on the above mentioned spectral details the metabolite 2 was deduced to be a new compound and characterized as 15-oxo-finasteride (2).

Metabolite 3 was obtained as a colorless solid (Scheme 1). The HREI-MS analysis of metabolite 3 displayed an M+at m/z 388.2744, which is corresponded to the formula C23H36N2O3 (Calcd 388.2726), indicating the introduction of an oxygen atom in the metabolite. The 1H-NMR spectrum of 3 was quite similar to the substrate 1, except for a new downfield hydroxyl-bearing methine proton signal at δ 4.04 (m, W1/2 = 23.7 Hz). A downfield shift of H-1 from δ 6.80 to 7.94 was also observed, which indicated the appearance of a newly introduced OH functionality at C-11. The 13C-NMR spectrum of 3 also exhibited a new methine signal resonating at δ 69.2 (C-11), whereas a β-downfield shift of C-9 (δ 53.5), and C-12 (δ 49.9) suggested the introduction of the hydroxyl group in their vicinity. Two-dimensional NMR techniques (HMBC and COSY 45 °) were further employed to assign the location of the new hydroxyl group. In the HMBC spectrum, 2J correlations of H-11 (δ 4.04) with C-9 (δ 53.5), and C-12 (δ 49.9), and 3J correlation of H-11 with C-13 (δ 40.5) was observed. The COSY 45° showed the cross peaks between H-11 (δ 4.04) and H-9 (δ 1.17), and H-12 (δ 1.31, 2.20). The stereochemistry of this newly introduced hydroxyl group was assigned through NOESY correlations between H-11 (δ 4.04), and Me-18 (δ 0.68), and Me-19 (δ 1.04). Based on the above mentioned spectral data, the structure of the known metabolite 3 was identified as 11α-hydroxy finasteride. Metabolite 3 was earlier obtained by biotransformation of finasteride with Selenastrum capricornutum [4].

Metabolite 4 was obtained as a crystalline solid (Scheme 1). The formula of the metabolite 4 was deduced as C23H36N2O3 (Calcd 388.4848), through HREI-MS (m/z 388.4823). This indicated a mono hydroxylation in the substrate 1.

The 1H-NMR spectrum of compound 4 was quite similar to the substrate 1, except an additional downfield methine proton signal, resonated at δ 4.14 (dt, J 15e,16a = 7.3 Hz, J 15e,14a = 2.0 Hz), indicating the introduction of a hydroxyl group at a secondary carbon. The 13C-NMR spectrum showed an additional downfield methine signal resonated at δ 69.6 (C-15), and downfield shifts of C-14 (δ 59.7), and C-16 (δ 40.9), suggested the hydroxylation at C-15. The presence of the hydroxyl functionality was further deduced by HMBC and COSY 45 ° spectrum. In HMBC spectrum, 2J-heteronuclear couplings of H-15 (δ 4.14) with C-14 (δ 59.7) and C-16 (δ 40.9), and 3J-heteronuclear couplings C-13 (δ 43.5) were observed indicating the hydroxylation at C-15.

In COSY 45° homonuclear interactions between H-15 (δ 4.14) and H2-16 (δ 2.01, 1.95), and H-14 (δ 0.83), further encouraged the assigned position of hydroxylation at C-15. The stereochemistry of C-15 hydroxyl group was assigned on the basis of NOESY correlations of H-15 (δ 4.14) and H-14 (δ 0.83), and on the comparison with the reported data [5] as β (axial) hydroxyl group.

Metabolite 4 (15β-hydroxyfinasteride) was previously obtained by the microbial transformation of finasteride with Mortierella isabellina [5].

Table 1: 1 H-NMR (600 MHz, CDCl3) chemical shifts assignments of Compounds 2-4

Carbon No 2 δH (J = Hz) 3 δH (J = Hz) 4 δH (J = Hz)
1 6.79, d (9.9) 7.94, d (10.2) 6.74, d (9.9)
2 5.81, d (9.8) 5.68, d (10.2) 5.69, d (9.9)
3 - - -
4 5.53, br. s 5.26, br. s 5.36, br. s
5 3.33, t (8.2) 3.33, dd (11.4, 4.5) 3.26, m
6 1.60, m; 1.61, d (6.2) 1.20, m; 2.05, d (6.20) 1.55, m; 1.53, d (6.2)
7 2.81, m; 1.71, m 1.71, m; 1.01,m 2.04, m; 1.20, m
8 1.78, m 1.37, m 1.77, m
9 1.01, m 1.17, m 0.98, m
10 - - -
11 2.03, m; 1.80, m 4.04, m (W ½ = 23.7) 1.66, m; 1.38, m
12 3.10, d (6.2); 1.99, m 2.20 dd (11.4, 4.5); 1.31 1.80, m; 1.79 m
13 - - -
14 1.64, m 1.35, m 0.83, m
15 - 2.01, m; 1.22, m 4.14, dt (7.3, 2.0)
16 2.91, m; 2.71, m 1.63, m; 1.52, m 2.01, m; 1.95, m
17 2.41,m 2.06, m 1.92, m
18 0.76, s 0.68, s 0.84, s
19 0.96, s 1.04, s 0.89
20 - - -
21 5.16, br. s 5.13, br. s 6.13, br. s
22 - - -
23 1.36, s 1.32 1.24, s

CONCLUSION

In conclusion, the biotransformation of finasteride (1) by fungal culture Macrophomina phaseolina yielded metabolites 2-4. The main oxidation and hydroxylations occurred in rings B C and D especially at C-7, C-11 and C-15 positions. The metabolite 2 was identified as the new metabolites of the fermentation. As a result of these studies, the regio-and stereoselective hydroxylation at C-11α and C-15β positions were found. These transformed products provide accesses to regions of the steroidal skeletons, which are difficult to be functionalized by conventional chemical methods.

ACKNOWLEDGMENT

Sadia Sultan would like to acknowledge Universiti Teknologi MARA for the financial support under the reference number UiTM 600-IRMI/FRGS 5/3 (0119/2016) and LESTARI grant 600-RMI/ DANA5/ 3 LESTARI (92/2015).

CONTRIBUTION

The first author has carried out the research. Second third fourth and fifth authors have provided study conception, the design of work, drafting of the manuscript and critical revision.

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. Rasmusson GH, Reynolds GF, Steinberg NG, Walton EGF, Patel T. Azasteroids: structure-activity relationships for inhibition of 5 alpha-reductase and of androgen receptor binding. J Med Chem 1986;29:2298-315.

  2. Heizl S. Androgenetic alopecia: finasteride-treated hair loss. Med Monatsschr Pharm 1999;22;124-7.

  3. Murtola TJ, Tammela TLJ, Maattanen L, Hakama M, Auvinen A. Prostate cancer risk among users of finasteride and alpha-blockers-a population based case-control study. Eur J Cancer 2007;43:775-81.

  4. Venkataramani ES, Carlinn JR, Dolling U, Christofalo P, Magliette RJ. Biotransformation of finasteride (MK-0906) by selenastrum capricornutum green algae. Analys New York Acad Sci 1994;30:51-60.

  5. Pamidi C, Jia Q. 4-aza-steroids as inhibitors of testosterone-5-alpha-reductase. WO Patent; 1998;9850419.

  6. Mahato SB, Majumder I. Current trends in microbial steroid biotransformation. Phytochemistry 1993;34:883-98.

  7. Arthan D, Svasti J, Kittakoop P, Pittayakhachonwut D, Tanticharoen M. Thebtaranonth Y. Antiviral isoflavonoid sulfate and steroidal glycosides from the fruits of Solanum torvum. Phytochemistry 2002;59:459-63.

  8. Zhang YQ, Wang DQ. Advances in the microbial transform of phytosterol into steroid medicine intermediates. Microbiology 2006;33:142-6.

  9. Murray HC, Peterson DH. Oxygenation of steroids by Mucorales fungi. U. S. Patent; 1952;2602769.

  10. Fernandes P, Cruz A, Angelova B, Pinheiro HM, Cabral JMS. Microbial conversion of steroid compounds: recent developments. Enzyme Microb Technol 2003;32:688-705.

  11. Atif M, Sultan S, Shah SAA, Choudhary MI. Solid phase microbial reactions of sex hormone, trans-androsterone with filamentous fungi. Int J Pharm Pharm Sci 2015;7:385-8.

  12. Atif M, Shah SAA, Sultan S, Choudhary MI. Solid phase microbial fermentation of anabolic steroid dihydrotestosterone with ascomycete fungus fusarium oxyporum. Int J Pharm Pharm Sci 2015;7:104-7.

  13. Sultan S, Zaimi M, Anouar EH, Shah SAA, Salim F. Structure and absolute configuration of 20β-hydroxy prednisolone a biotransformed product of a prednisolone by a marine endophytic fungus Peniciliumlapidosum: experimental and DFT approaches. Molecules 2014;19:13775-87.

  14. Choudhary MI, Atif M, Shah SAA, Sultan S, Erum S, Khan SN, et al. Biotransformation of dehydroabietic acid with microbial cell cultures and α-glucosidase inhibitory activity of resulting metabolites. Int J Pharm Pharm Sci 2014;6:375-8.

  15. Choudhary MI, Atif M, Sultan S, Shah SAA, Atta-ur-Rahman. Microbial metabolism of an anti-HIV and anti-malarial natural product andrographolide. Int J Pharm Pharm Sci 2014;6:195-8.

  16. Shah SAA, Sultan S, Hassan NB, Muhammad FKB, Faridz MABM. Biotransformation of 17α-ethynyl substituted steroidal drugs with microbial and plant cell cultures. Steroids 2013;78:1312–24.

  17. Shah SAA, Sultan S, Adnan HS. Solid phase microbial transformation of cortexolone and prolyl endopeptidase inhibitory activity of the transformed products. Int J Pharm Pharm Sci 2011;3:1-6.

  18. Choudhary MI, Sultan S, Jalil S, Anjum S, Atta-ur-Rahman, Fun HK. Microbial transformation of mesterolone. Chem Biodiversity 2005;2:392-400.

  19. Choudhary MI, Sultan S, Khan MTH, Atta-ur-Rahman. Microbial transformation of 17alpha-ethynyl-and 17alpha-ethylsteroids, and tyrosinase inhibitory activity of transformed products. Steroids 2005;70:798-802.

  20. Choudhary MI, Sultan S, Yaqoob M, Musharraf SG, Yasin A, Shaheen F, et al. Transformation of cortisol and prolyl endopeptidase activity of transformed products. Nat Prod Res 2003;17:389-95.

  21. Choudhary MI, Sultan S, Hassan MTH, Yasin A, Shaheen F, Atta-ur-Rahman. Biotransformation of (+)-Androst-4-ene-3, 17-dione. Nat Prod Res 2004;18:529-35.

  22. Chakravarthy VA, Bbv S, Kumar P. Stability indicating reverse phase high-performance liquid chromatographic method for simultaneous estimation of labetalol and its degradation products in tablet dosage forms. Asian J Pharm Clin Res 2016;9:202-5.

  23. Yu Han L, Min S, Taijun H. Development of an LC-MS/MS method for determination of 2-oxo clopidogrel in human plasma. J Pharm Anal 2015;5:12-7.

  24. Shah SAA, Sultan S, Adnan HS. A whole-cell biocatalysis application of steroidal drugs. Orient J Chem 2013;29:389-403.

  25. Shah SAA, Tan HL, Sultan S, Faridz MABM, Shah MABM, Nurfazilah, et al. Microbial-catalyzed biotransformation of multifunctional triterpenoids derived from phytonutrients. Int J Mol Sci 2014;15:12027-60.

  26. Shah SAA, Sultan S, Noor ZM. Biotransformation of tissue-specific hormone tibolone with fungal culture trichothecium roseum. J Mol Struct 2013;1042:118–22.

  27. Sultan S, Choudhary MI, Khan SN, Fatima U, Atif M, Ali RA, et al. Fungal transformation of cedryl acetate and α-glucosidase inhibition assay, quantum mechanical calculations and molecular docking studies of its metabolites. Eur J Med 2013;62:764–70.

  28. Sultan S, Ghani NA, Shah SAA, Ismail NH, Noor MZ, Naz H. Microbial transformation of bioactive anthraquinones–a review. Biosci Biotechnol Res Asia 2013;10:577-82.