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Inhibitory Effects of Isoflavonoids on Rat Prostate Testosterone 5α-Reductase
J Acupunct Meridian Stud 2012;5(6):319e322
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Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com
- BRIEF
REPORT
-
Inhibitory Effects of Isoflavonoids on Rat Prostate Testosterone 5a-Reductase Mijeong Bae 1, Mijin Woo 1, Irawan Wijaya Kusuma 2, Enos Tangke Arung 2, Chae Ha Yang 3, Yong-ung Kim 1,* 1
Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-industry, Daegu Haany University, Daegu, Republic of Korea 2 Department of Forest Products, Faculty of Forestry, Mulawarman University, Samarinda, Indonesia 3 Department of Preparatory Oriental Medicine, College of Oriental Medicine, Daegu Haany University, Daegu, Republic of Korea Available online Aug 9, 2012 Received: Jan 18, 2012 Revised: Apr 5, 2012 Accepted: Apr 11, 2012 KEYWORDS bacteria; isoflavone; O-methylated isoflavone; soybean; testosterone 5areductase inhibitor; 30 ,40 ,7trihydroxyisoflavone
Abstract Testosterone 5a-reductase inhibitors represent important therapeutic drugs for use against androgen-dependent diseases such as benign prostatic hyperplasia, male pattern baldness, and acne. We have searched for inhibitors of rat prostate testosterone 5areductase in the cultured broths of many kinds of soil bacteria, and have found that cultured soybean-casein digest broths of certain bacterial strains have a potent inhibitory effect on the enzyme. We tested 10 selected isoflavonoids, including isoflavones and O-methylated isoflavones, for inhibitory effects on rat prostate testosterone 5a-reductase to determine the important structural elements for inhibition of the enzyme. Genistein, biochanin A, equol, and 30 ,40 ,7-trihydroxyisoflavone showed considerably higher inhibitory effects whereas daidzein, formononetin, glycitein, prunetin, ipriflavone, and 40 ,7-dimethoxyisoflavone showed lower inhibitory effects. The IC50 values of genistein, biochanin A, equol, 30 ,40 ,7-trihydroxyisoflavone, and riboflavin, a positive control, for rat prostate testosterone 5a-reductase were 710 mM, 140 mM, 370 mM, 690 mM, and 17 mM, respectively. Daidzein, genistein, biochanin A, formononetin, and equol are already known to be testosterone 5a-reductase inhibitors, but this is the first characterization of 30 ,40 ,7-trihydroxyisoflavone as an inhibitor of the enzyme.
* Corresponding author. Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-industry, Daegu Haany University, 290 Yugok-dong, Gyeongsan-si, Gyeongsangbuk-do 712-715, Republic of Korea. E-mail: [email protected] Copyright ª 2012, International Pharmacopuncture Institute pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2012.07.022
320
M. Bae et al.
1. Introduction
2.3. Testosterone 5a-reductase assay
Testosterone 5a-reductase (reduced nicotinamide adenine dinucleotide phosphate [NADPH]: D4-3-ketosteroid 5aoxidoreductase: EC 1.3.99.5) catalyzes the NADPHdependent reduction of D4-3-ketosteroid to 3-ketosteroid, including the conversion of testosterone, the major circulating androgen, to 5a-dihydrotestosterone in target tissues. The type 1 and the type 2 isozymes of testosterone 5a-reductase are present in humans and rats, but with different tissue distributions [1]. Androgen-dependent diseases, such as benign prostatic hyperplasia, male pattern baldness, and acne, may be associated with testosterone 5a-reductase activity in many tissues [1]. Therefore, testosterone 5a-reductase inhibitors represent important therapeutic drugs for use against such androgendependent diseases. Certain natural products, especially dietary and medicinal plants, are known to produce many kinds of bioactive compounds that have the ability to inhibit testosterone 5a-reductase. In addition, microbial transformations of the compounds by microorganisms are known to provide new bioactive compounds. A variety of polyphenols are known to have the ability to inhibit testosterone 5a-reductase activity, and specific isoflavones from soybean have been identified as efficient inhibitors [2e4]. Specific fermentation broths of microorganisms have remarkable inhibitory effects on testosterone 5a-reductase, and potent inhibitors have been identified from those microbial species [5e10]. We have searched for inhibitors of rat prostate testosterone 5a-reductase in the cultured broths of many kinds of soil bacteria, and have found that cultured soybean-casein digest broths of certain bacterial strains have a potent inhibitory effect on the enzyme. In the current study, we investigated 10 selected isoflavonoids, including isoflavones and O-methylated isoflavones, for their inhibitory effects on testosterone 5a-reductase prepared from rat prostates to determine the important structural elements for the inhibitory effects of those isoflavonoids.
The enzyme suspension of testosterone 5a-reductase was prepared from the homogenate of the ventral prostates of five male Sprague-Dawley rats by using a previously reported method [11]. The testosterone 5a-reductase inhibitory activity was measured using a previously reported testosterone 5a-reductase assay method [11]. Each enzyme reaction was carried out in duplicate, and the half maximal inhibitory concentration (IC50) was calculated from the values of the inhibitory activities at several concentrations by using a linear regression analysis.
2. Materials and methods 2.1. General experimental procedures Thin layer chromatography was performed using precoated silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany), and spots were visualized under ultraviolet light at 254 nm.
2.2. Chemicals Riboflavin, daidzein, genistein, and glycitein were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Formononetin, prunetin, and ipriflavone were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Biochanin A, 40 ,7-dimethoxyisoflavone, and 30 ,40 ,7-trihydroxyisoflavone were purchased from Alfa Aesar (Ward Hill, MA, USA). Equol was purchased from LC Laboratories (Woburn, MA, USA).
3. Results and discussion Inhibition of testosterone 5a-reductase represents an important pharmacologic approach against androgendependent diseases such as benign prostate hyperplasia, male pattern baldness, and acne, which are mediated by 5a-dihydrotestosterone in target tissues. Most testosterone 5a-reductase inhibitors that have been developed are steroid derivatives that have various undesirable hormonal effects, and the purpose of our research is to find new nonsteroidal testosterone 5a-reductase inhibitors from dietary and medicinal natural products such as microorganisms and plants. We tested 10 selected isoflavonoids including isoflavones and O-methylated isoflavones for their inhibitory effect on testosterone 5a-reductase prepared from rat prostates to determine the important structural elements for the inhibitory effects of those isoflavonoids. In the ventral prostate of the rat, both type 1 and type 2 isozymes were detected in comparable amounts [1]. Because the rat prostate extracts for measuring the testosterone 5areductase inhibition were used in a neutral pH buffer, in this study, both isozymes were assayed due to differences in their biochemical properties. The structures of the isoflavonoids tested are shown in Fig. 1. The inhibitory effects of those isoflavonoids are shown in Table 1. Genistein, biochanin A, equol, and 30 ,40 ,7trihydroxyisoflavone showed considerably higher inhibitory effects, whereas daidzein, formononetin, glycitein, prunetin, ipriflavone, and 4´,7-dimethoxyisoflavone showed lower inhibitory effects. The IC50 values of genistein, biochanin A, equol, and 30 ,40 ,7-trihydroxyisoflavone for rat prostate testosterone 5a-reductase were 710 mM, 140 mM, 370 mM, and 690 mM, respectively. Riboflavin, a potent nonsteroidal inhibitor of testosterone 5a-reductase, was used as a positive control and showed greater activity, with an IC50 value of 17 mM, than these isoflavonoids. From a comparison of the structures and the inhibitory activities of daidzein and genistein or of biochanin A and formononetin, the presence of a 5-hydroxyl group in the Aring appears to enhance the inhibitory activity. Similarly, from a comparison of the structures and the inhibitory activities of daidzein and 30 ,40 ,7-trihydroxyisoflavone, the presence of a 30 -hydroxyl group in the B-ring appears to enhance the inhibitory activity. From a comparison of the structures and the inhibitory activities of isoflavones, such as daidzein and genistein, and their O-methylated isoflavones, such as biochanin A, formononetin, prunetin, and
5a-Reductase inhibitory effects of isoflavonoids 1
8
R3
O
2
7 R2
321
2' 6 5 R1
3'
4 O
6'
4' 5'
Daidzein Genistein Biochanin A Formononetin Glycitein Prunetin Ipriflavone 4',7-Dimethoxyisoflavone 3',4',7-Trihydroxyisoflavone
HO
R4
1'
3
R5
R1
R2
R3
R4
R5
H OH OH H H OH H H H
H H H H OCH3 H H H H
OH OH OH OH OH OCH3 OCH(CH3)2 OCH3 OH
H H H H H H H H OH
OH OH OCH3 OCH3 OH OH H OCH3 OH
O
OH
Equol
Figure 1 Chemical structures of isoflavonoids, including isoflavones and O-methylated isoflavones, tested for their inhibitory effects on rat prostate testosterone 5a-reductase.
40 ,7-dimethoxyisoflavone, O-methylation of the 7-hydroxyl group in the A-ring or of the 40 -hydroxyl group in the Bring does not seem to potentiate the inhibitory activity. From a comparison of the structures and the inhibitory activities of daidzein and glycitein, the presence of an
additional 6-methoxy group in the A-ring seems to substantially inactivate the inhibitory activity. The inhibitory effects of daidzein, genistein, biochanin A, formononetin, and equol on testosterone 5a-reductase have already been reported [3,4]. According to our
Table 1 Inhibitory effects of the tested isoflavonoids, including isoflavones and O-methylated isoflavones, on rat prostate testosterone 5a-reductase*. Compound
Inhibition (%) at a concentration of compound (mM) 100 mM
Daidzein Genistein Biochanin A Formononetin Glycitein Prunetin Ipriflavone Equol 40 ,7-Dimethoxyisoflavone 30 ,40 ,7-Trihydroxyisoflavone Riboflavin
28.1 34.3 45.1 23.8 14.9 33.8 24.8 17.9 37.5 31.1 68.7
IC50 (mM)
1000 mM 11.3 4.4 3.6 16.4 2.0 7.5 20.2 16.7 5.6 1.8 28.9
* Values are the means of the results from two different experiments.
36.5 52.8 81.7 27.5 1.4 40.1 23.0 74.1 27.8 53.6 93.3
1.3 12.5 2.2 0.8 24.1 15.8 13.0 4.4 17.3 8.8 0.9
>1000 710 140 >1000 >1000 >1000 >1000 370 >1000 690 17
322 testosterone 5a-reductase assay, the inhibitory effects of daidzein and formononetin were found to be considerably less than those of genistein, biochanin A, and equol. The inhibitory effect of equol, which is the reduced isoflavone metabolite of daidzein due to bacterial flora in the intestines [12], was considerably higher than that of daidzein itself, which has an isoflavone skeleton. Equol has been reported to show a blocking effect on the growth of the ventral prostates induced by 5a-dihydrotestosterone in castrated male rats [13]. As far as we know, the current report is the first characterization of 30 ,40 ,7-trihydroxyisoflavone as a testosterone 5a-reductase inhibitor. 30 ,40 ,7-Trihydroxyisoflavone was previously isolated from the heartwood of Machaerium villosum (Leguminosae) [14] and as a metabolite of Streptomyces sp. OH-1049 grown in media containing soybean meal, which shows antioxidant activity [15,16]. Also, this compound and 30 ,40 ,5,7-tetrahydroxyisoflavone were isolated from culture filtrates of a marine Streptomyces sp. 060524 in soybean meal glucose medium, and were produced through 30 hydroxylation from daidzein and genistein, respectively, by Streptomyces avermitilis MA-4680 [17,18]. Further studies are in progress to investigate both the structure-activity relationships of metabolites of soybean isoflavones produced by specific bacterial strains and of active components from certain bacterial strains grown in soybean-casein digest broths.
Acknowledgments The authors thank Dahye Jeon, Soohyun Lee, and Hyeonsuk Im for providing comments on the work. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20100008259).
References 1. Russell DW, Wilson JD. Steroid 5a-reductase: two genes/two enzymes. Annu Rev Biochem. 1994;63:25e61. 2. Komoda Y. Isolation of flavonoids from Populus nigra as D4-3ketosteroid (5a) reductase inhibitors. Chem Pharm Bull. 1989;37:3128e3130. 3. Evans BAJ, Griffiths K, Morton MS. Inhibition of 5a-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids. J Endocrinol. 1995;147:295e302. 4. Hiipakka RA, Zhang HZ, Dai W, Dai Q, Liao S. Structure-activity relationships for inhibition of human 5a-reductases by polyphenols. Biochem Pharmacol. 2002;63:1165e1176.
M. Bae et al. 5. Nakayama O, Yagi M, Tanaka M, Kiyoto S, Okuhara M, Kohsaka M. WS-9659 A and B, novel testosterone 5a-reductase inhibitors isolated from a Streptomyces. I. Taxonomy, fermentation, isolation, physico-chemical characteristics. J Antibiot. 1989;42:1221e1229. 6. Nakayama O, Shigematsu N, Katayama A, Takase S, Kiyoto S, Hashimoto M, et al. WS-9659 A and B, novel testosterone 5areductase inhibitors isolated from a Streptomyces. II. Structural elucidation of WS-9659 A and B. J Antibiot. 1989;42: 1230e1234. 7. Nakayama O, Arakawa H, Yagi M, Tanaka M, Kiyoto S, Okuhara M, et al. WS-9659 A and B, novel testosterone 5areductase inhibitors isolated from a Streptomyces. III. Biological characteristics and pharmacological characteristics. J Antibiot. 1989;42:1235e1240. 8. Nakayama O, Yagi M, Kiyoto S, Okuhara M, Kohsaka M. Riboflavin, a testosterone 5a-reductase inhibitor. J Antibiot. 1990; 43:1615e1616. 9. Takamatsu S, Rho M-C, Masuma R, Hayashi M, Komiyama K, Tanaka H, et al. A novel testosterone 5a-reductase inhibitor, 80 ,90 -dehydroascochlorin produced by Verticillium sp. FO-2787. Chem Pharm Bull. 1994;42:953e956. 10. Kim Y, Kim CY, Son HK, Song HK, Han J, Lee SS, et al. Testosterone 5a-reductase inhibitors, menaquinone 7 produced by a Bacillus and phenazine methosulfate. Biol Pharm Bull. 1999; 22:1396e1399. 11. Kim S, Kim Y, Ma E. Synthesis and 5a-reductase inhibitory activity of C21 steroids having 1,4-diene or 4,6-diene 20-ones and 4-azasteroid 20-oximes. Molecules. 2012;17:355e368. 12. Kim M, Marsh ENG, Kim S-U, Han J. Conversion of (3S,4R)-tetrahydrodaidzein to (3S)-equol by THD reductase: proposed mechanism involving a radical intermediate. Biochemistry. 2010;49:5582e5587. 13. Lund TD, Munson DJ, Haldy ME, Setchell KDR, Lephart ED, Handa RJ. Equol is a novel anti-androgen that inhibits prostate growth and hormone feedback. Biol Reprod. 2004;70: 1188e1195. 14. De Oliveira AB, Gottlieb OR, Ollis WD. A Quimica de Leguminosas Brasileiras. XVII. Constituintes do Machaerium villosum. An Acad Bras Cienc. 1968;40:147e150. 15. Komiyama K, Funayama S, Anraku Y, Mita A, Takahashi Y, Omura S. Isolation of isoflavonoids possessing antioxidant activity from the fermentation broth of Streptomyces sp. J Antibiot. 1989;42:1344e1349. 16. Funayama S, Anraku Y, Mita A, Komiyama K, Omura S. Structural study of isoflavonoids possessing antioxidant activity isolated from the fermentation broth of Streptomyces sp. J Antibiot. 1989;42:1350e1355. 17. Hu SC, Hong K, Song YC, Liu JY, Tan RX. Biotransformation of soybean isoflavones by a marine Streptomyces sp. 060524 and cytotoxicity of the products. World J Microbiol Biotechnol. 2009;25:115e121. 18. Roh C, Seo SH, Choi KY, Cha M, Pandey BP, Kim JH, et al. Regioselective hydroxylation of isoflavones by Streptomyces avermitilis MA-4680. J Biosci Bioeng. 2009;108:41e46.
Available online at www.sciencedirect.com
Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com
- BRIEF
REPORT
-
Inhibitory Effects of Isoflavonoids on Rat Prostate Testosterone 5a-Reductase Mijeong Bae 1, Mijin Woo 1, Irawan Wijaya Kusuma 2, Enos Tangke Arung 2, Chae Ha Yang 3, Yong-ung Kim 1,* 1
Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-industry, Daegu Haany University, Daegu, Republic of Korea 2 Department of Forest Products, Faculty of Forestry, Mulawarman University, Samarinda, Indonesia 3 Department of Preparatory Oriental Medicine, College of Oriental Medicine, Daegu Haany University, Daegu, Republic of Korea Available online Aug 9, 2012 Received: Jan 18, 2012 Revised: Apr 5, 2012 Accepted: Apr 11, 2012 KEYWORDS bacteria; isoflavone; O-methylated isoflavone; soybean; testosterone 5areductase inhibitor; 30 ,40 ,7trihydroxyisoflavone
Abstract Testosterone 5a-reductase inhibitors represent important therapeutic drugs for use against androgen-dependent diseases such as benign prostatic hyperplasia, male pattern baldness, and acne. We have searched for inhibitors of rat prostate testosterone 5areductase in the cultured broths of many kinds of soil bacteria, and have found that cultured soybean-casein digest broths of certain bacterial strains have a potent inhibitory effect on the enzyme. We tested 10 selected isoflavonoids, including isoflavones and O-methylated isoflavones, for inhibitory effects on rat prostate testosterone 5a-reductase to determine the important structural elements for inhibition of the enzyme. Genistein, biochanin A, equol, and 30 ,40 ,7-trihydroxyisoflavone showed considerably higher inhibitory effects whereas daidzein, formononetin, glycitein, prunetin, ipriflavone, and 40 ,7-dimethoxyisoflavone showed lower inhibitory effects. The IC50 values of genistein, biochanin A, equol, 30 ,40 ,7-trihydroxyisoflavone, and riboflavin, a positive control, for rat prostate testosterone 5a-reductase were 710 mM, 140 mM, 370 mM, 690 mM, and 17 mM, respectively. Daidzein, genistein, biochanin A, formononetin, and equol are already known to be testosterone 5a-reductase inhibitors, but this is the first characterization of 30 ,40 ,7-trihydroxyisoflavone as an inhibitor of the enzyme.
* Corresponding author. Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-industry, Daegu Haany University, 290 Yugok-dong, Gyeongsan-si, Gyeongsangbuk-do 712-715, Republic of Korea. E-mail: [email protected] Copyright ª 2012, International Pharmacopuncture Institute pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2012.07.022
320
M. Bae et al.
1. Introduction
2.3. Testosterone 5a-reductase assay
Testosterone 5a-reductase (reduced nicotinamide adenine dinucleotide phosphate [NADPH]: D4-3-ketosteroid 5aoxidoreductase: EC 1.3.99.5) catalyzes the NADPHdependent reduction of D4-3-ketosteroid to 3-ketosteroid, including the conversion of testosterone, the major circulating androgen, to 5a-dihydrotestosterone in target tissues. The type 1 and the type 2 isozymes of testosterone 5a-reductase are present in humans and rats, but with different tissue distributions [1]. Androgen-dependent diseases, such as benign prostatic hyperplasia, male pattern baldness, and acne, may be associated with testosterone 5a-reductase activity in many tissues [1]. Therefore, testosterone 5a-reductase inhibitors represent important therapeutic drugs for use against such androgendependent diseases. Certain natural products, especially dietary and medicinal plants, are known to produce many kinds of bioactive compounds that have the ability to inhibit testosterone 5a-reductase. In addition, microbial transformations of the compounds by microorganisms are known to provide new bioactive compounds. A variety of polyphenols are known to have the ability to inhibit testosterone 5a-reductase activity, and specific isoflavones from soybean have been identified as efficient inhibitors [2e4]. Specific fermentation broths of microorganisms have remarkable inhibitory effects on testosterone 5a-reductase, and potent inhibitors have been identified from those microbial species [5e10]. We have searched for inhibitors of rat prostate testosterone 5a-reductase in the cultured broths of many kinds of soil bacteria, and have found that cultured soybean-casein digest broths of certain bacterial strains have a potent inhibitory effect on the enzyme. In the current study, we investigated 10 selected isoflavonoids, including isoflavones and O-methylated isoflavones, for their inhibitory effects on testosterone 5a-reductase prepared from rat prostates to determine the important structural elements for the inhibitory effects of those isoflavonoids.
The enzyme suspension of testosterone 5a-reductase was prepared from the homogenate of the ventral prostates of five male Sprague-Dawley rats by using a previously reported method [11]. The testosterone 5a-reductase inhibitory activity was measured using a previously reported testosterone 5a-reductase assay method [11]. Each enzyme reaction was carried out in duplicate, and the half maximal inhibitory concentration (IC50) was calculated from the values of the inhibitory activities at several concentrations by using a linear regression analysis.
2. Materials and methods 2.1. General experimental procedures Thin layer chromatography was performed using precoated silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany), and spots were visualized under ultraviolet light at 254 nm.
2.2. Chemicals Riboflavin, daidzein, genistein, and glycitein were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Formononetin, prunetin, and ipriflavone were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Biochanin A, 40 ,7-dimethoxyisoflavone, and 30 ,40 ,7-trihydroxyisoflavone were purchased from Alfa Aesar (Ward Hill, MA, USA). Equol was purchased from LC Laboratories (Woburn, MA, USA).
3. Results and discussion Inhibition of testosterone 5a-reductase represents an important pharmacologic approach against androgendependent diseases such as benign prostate hyperplasia, male pattern baldness, and acne, which are mediated by 5a-dihydrotestosterone in target tissues. Most testosterone 5a-reductase inhibitors that have been developed are steroid derivatives that have various undesirable hormonal effects, and the purpose of our research is to find new nonsteroidal testosterone 5a-reductase inhibitors from dietary and medicinal natural products such as microorganisms and plants. We tested 10 selected isoflavonoids including isoflavones and O-methylated isoflavones for their inhibitory effect on testosterone 5a-reductase prepared from rat prostates to determine the important structural elements for the inhibitory effects of those isoflavonoids. In the ventral prostate of the rat, both type 1 and type 2 isozymes were detected in comparable amounts [1]. Because the rat prostate extracts for measuring the testosterone 5areductase inhibition were used in a neutral pH buffer, in this study, both isozymes were assayed due to differences in their biochemical properties. The structures of the isoflavonoids tested are shown in Fig. 1. The inhibitory effects of those isoflavonoids are shown in Table 1. Genistein, biochanin A, equol, and 30 ,40 ,7trihydroxyisoflavone showed considerably higher inhibitory effects, whereas daidzein, formononetin, glycitein, prunetin, ipriflavone, and 4´,7-dimethoxyisoflavone showed lower inhibitory effects. The IC50 values of genistein, biochanin A, equol, and 30 ,40 ,7-trihydroxyisoflavone for rat prostate testosterone 5a-reductase were 710 mM, 140 mM, 370 mM, and 690 mM, respectively. Riboflavin, a potent nonsteroidal inhibitor of testosterone 5a-reductase, was used as a positive control and showed greater activity, with an IC50 value of 17 mM, than these isoflavonoids. From a comparison of the structures and the inhibitory activities of daidzein and genistein or of biochanin A and formononetin, the presence of a 5-hydroxyl group in the Aring appears to enhance the inhibitory activity. Similarly, from a comparison of the structures and the inhibitory activities of daidzein and 30 ,40 ,7-trihydroxyisoflavone, the presence of a 30 -hydroxyl group in the B-ring appears to enhance the inhibitory activity. From a comparison of the structures and the inhibitory activities of isoflavones, such as daidzein and genistein, and their O-methylated isoflavones, such as biochanin A, formononetin, prunetin, and
5a-Reductase inhibitory effects of isoflavonoids 1
8
R3
O
2
7 R2
321
2' 6 5 R1
3'
4 O
6'
4' 5'
Daidzein Genistein Biochanin A Formononetin Glycitein Prunetin Ipriflavone 4',7-Dimethoxyisoflavone 3',4',7-Trihydroxyisoflavone
HO
R4
1'
3
R5
R1
R2
R3
R4
R5
H OH OH H H OH H H H
H H H H OCH3 H H H H
OH OH OH OH OH OCH3 OCH(CH3)2 OCH3 OH
H H H H H H H H OH
OH OH OCH3 OCH3 OH OH H OCH3 OH
O
OH
Equol
Figure 1 Chemical structures of isoflavonoids, including isoflavones and O-methylated isoflavones, tested for their inhibitory effects on rat prostate testosterone 5a-reductase.
40 ,7-dimethoxyisoflavone, O-methylation of the 7-hydroxyl group in the A-ring or of the 40 -hydroxyl group in the Bring does not seem to potentiate the inhibitory activity. From a comparison of the structures and the inhibitory activities of daidzein and glycitein, the presence of an
additional 6-methoxy group in the A-ring seems to substantially inactivate the inhibitory activity. The inhibitory effects of daidzein, genistein, biochanin A, formononetin, and equol on testosterone 5a-reductase have already been reported [3,4]. According to our
Table 1 Inhibitory effects of the tested isoflavonoids, including isoflavones and O-methylated isoflavones, on rat prostate testosterone 5a-reductase*. Compound
Inhibition (%) at a concentration of compound (mM) 100 mM
Daidzein Genistein Biochanin A Formononetin Glycitein Prunetin Ipriflavone Equol 40 ,7-Dimethoxyisoflavone 30 ,40 ,7-Trihydroxyisoflavone Riboflavin
28.1 34.3 45.1 23.8 14.9 33.8 24.8 17.9 37.5 31.1 68.7
IC50 (mM)
1000 mM 11.3 4.4 3.6 16.4 2.0 7.5 20.2 16.7 5.6 1.8 28.9
* Values are the means of the results from two different experiments.
36.5 52.8 81.7 27.5 1.4 40.1 23.0 74.1 27.8 53.6 93.3
1.3 12.5 2.2 0.8 24.1 15.8 13.0 4.4 17.3 8.8 0.9
>1000 710 140 >1000 >1000 >1000 >1000 370 >1000 690 17
322 testosterone 5a-reductase assay, the inhibitory effects of daidzein and formononetin were found to be considerably less than those of genistein, biochanin A, and equol. The inhibitory effect of equol, which is the reduced isoflavone metabolite of daidzein due to bacterial flora in the intestines [12], was considerably higher than that of daidzein itself, which has an isoflavone skeleton. Equol has been reported to show a blocking effect on the growth of the ventral prostates induced by 5a-dihydrotestosterone in castrated male rats [13]. As far as we know, the current report is the first characterization of 30 ,40 ,7-trihydroxyisoflavone as a testosterone 5a-reductase inhibitor. 30 ,40 ,7-Trihydroxyisoflavone was previously isolated from the heartwood of Machaerium villosum (Leguminosae) [14] and as a metabolite of Streptomyces sp. OH-1049 grown in media containing soybean meal, which shows antioxidant activity [15,16]. Also, this compound and 30 ,40 ,5,7-tetrahydroxyisoflavone were isolated from culture filtrates of a marine Streptomyces sp. 060524 in soybean meal glucose medium, and were produced through 30 hydroxylation from daidzein and genistein, respectively, by Streptomyces avermitilis MA-4680 [17,18]. Further studies are in progress to investigate both the structure-activity relationships of metabolites of soybean isoflavones produced by specific bacterial strains and of active components from certain bacterial strains grown in soybean-casein digest broths.
Acknowledgments The authors thank Dahye Jeon, Soohyun Lee, and Hyeonsuk Im for providing comments on the work. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20100008259).
References 1. Russell DW, Wilson JD. Steroid 5a-reductase: two genes/two enzymes. Annu Rev Biochem. 1994;63:25e61. 2. Komoda Y. Isolation of flavonoids from Populus nigra as D4-3ketosteroid (5a) reductase inhibitors. Chem Pharm Bull. 1989;37:3128e3130. 3. Evans BAJ, Griffiths K, Morton MS. Inhibition of 5a-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids. J Endocrinol. 1995;147:295e302. 4. Hiipakka RA, Zhang HZ, Dai W, Dai Q, Liao S. Structure-activity relationships for inhibition of human 5a-reductases by polyphenols. Biochem Pharmacol. 2002;63:1165e1176.
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