J Acupunct Meridian Stud 2012;5(6):319e322

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 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 Abstract Revised: Apr 5, 2012 Testosterone 5a-reductase inhibitors represent important therapeutic drugs for use Accepted: Apr 11, 2012 against androgen-dependent diseases such as benign prostatic hyperplasia, male pattern baldness, and acne. We have searched for inhibitors of rat prostate testosterone 5a- KEYWORDS reductase in the cultured broths of many kinds of soil bacteria, and have found that bacteria; cultured -casein digest broths of certain bacterial strains have a potent inhibitory isoflavone; effect on the enzyme. We tested 10 selected isoflavonoids, including isoflavones and O-methylated O-methylated isoflavones, for inhibitory effects on rat prostate testosterone 5a-reduc- isoflavone; tase to determine the important structural elements for inhibition of the enzyme. Gen- soybean; istein, , , and 30,40,7-trihydroxyisoflavone showed considerably higher a testosterone 5 - inhibitory effects whereas , , , , ipriflavone, and reductase inhibitor; 0 0 0 4 ,7-dimethoxyisoflavone showed lower inhibitory effects. The IC50 values of , 3 ,4 ,7- 0 0 trihydroxyisoflavone biochanin A, equol, 3 ,4 ,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 adenine The enzyme suspension of testosterone 5a-reductase was dinucleotide phosphate [NADPH]: D4-3-ketosteroid 5a- prepared from the homogenate of the ventral prostates of oxidoreductase: EC 1.3.99.5) catalyzes the NADPH- five male Sprague-Dawley rats by using a previously re- dependent reduction of D4-3-ketosteroid to 3-ketosteroid, ported method [11]. The testosterone 5a-reductase inhib- including the conversion of testosterone, the major circu- itory activity was measured using a previously reported lating androgen, to 5a- in target testosterone 5a-reductase assay method [11]. Each enzyme tissues. The type 1 and the type 2 isozymes of testosterone reaction was carried out in duplicate, and the half maximal 5a-reductase are present in humans and rats, but with inhibitory concentration (IC50) was calculated from the different tissue distributions [1]. Androgen-dependent values of the inhibitory activities at several concentrations diseases, such as benign prostatic hyperplasia, male by using a linear regression analysis. pattern baldness, and acne, may be associated with testosterone 5a-reductase activity in many tissues [1]. Therefore, testosterone 5a-reductase inhibitors represent 3. Results and discussion important therapeutic drugs for use against such androgen- a dependent diseases. Inhibition of testosterone 5 -reductase represents an Certain natural products, especially dietary and important pharmacologic approach against androgen- medicinal plants, are known to produce many kinds of dependent diseases such as benign prostate hyperplasia, bioactive compounds that have the ability to inhibit male pattern baldness, and acne, which are mediated by a testosterone 5a-reductase. In addition, microbial trans- 5 -dihydrotestosterone in target tissues. Most testosterone a formations of the compounds by microorganisms are known 5 -reductase inhibitors that have been developed are to provide new bioactive compounds. A variety of poly- derivatives that have various undesirable hormonal phenols are known to have the ability to inhibit testos- effects, and the purpose of our research is to find new a terone 5a-reductase activity, and specific isoflavones from testosterone 5 -reductase inhibitors from soybean have been identified as efficient inhibitors [2e4]. dietary and medicinal natural products such as microor- Specific fermentation broths of microorganisms have ganisms and plants. remarkable inhibitory effects on testosterone 5a-reduc- We tested 10 selected isoflavonoids including isoflavones tase, and potent inhibitors have been identified from those and O-methylated isoflavones for their inhibitory effect on a microbial species [5e10]. testosterone 5 -reductase prepared from rat prostates to We have searched for inhibitors of rat prostate determine the important structural elements for the testosterone 5a-reductase in the cultured broths of many inhibitory effects of those isoflavonoids. In the ventral kinds of soil bacteria, and have found that cultured prostate of the rat, both type 1 and type 2 isozymes were soybean-casein digest broths of certain bacterial strains detected in comparable amounts [1]. Because the rat a have a potent inhibitory effect on the enzyme. In the prostate extracts for measuring the testosterone 5 - current study, we investigated 10 selected isoflavonoids, reductase inhibition were used in a neutral pH buffer, in including isoflavones and O-methylated isoflavones, for this study, both isozymes were assayed due to differences their inhibitory effects on testosterone 5a-reductase in their biochemical properties. The structures of the iso- prepared from rat prostates to determine the important flavonoids tested are shown in Fig. 1. The inhibitory effects of those isoflavonoids are shown in Table 1. structural elements for the inhibitory effects of those 0 0 isoflavonoids. Genistein, biochanin A, equol, and 3 ,4 ,7- trihydroxyisoflavone showed considerably higher inhibitory effects, whereas daidzein, formononetin, glycitein, pru- 2. Materials and methods netin, ipriflavone, and 4´,7-dimethoxyisoflavone showed lower inhibitory effects. The IC50 values of genistein, bio- chanin A, equol, and 30,40,7-trihydroxyisoflavone for rat 2.1. General experimental procedures prostate testosterone 5a-reductase were 710 mM, 140 mM, 370 mM, and 690 mM, respectively. Riboflavin, a potent Thin layer chromatography was performed using precoated nonsteroidal inhibitor of testosterone 5a-reductase, was silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany), used as a positive control and showed greater activity, with m and spots were visualized under ultraviolet light at 254 nm. an IC50 value of 17 M, than these isoflavonoids. From a comparison of the structures and the inhibitory activities of daidzein and genistein or of biochanin A and 2.2. Chemicals formononetin, the presence of a 5-hydroxyl group in the A- ring appears to enhance the inhibitory activity. Similarly, Riboflavin, daidzein, genistein, and glycitein were purchased from a comparison of the structures and the inhibitory from Wako Pure Chemical Industries, Ltd (Osaka, Japan). activities of daidzein and 30,40,7-trihydroxyisoflavone, the Formononetin, prunetin, and ipriflavone were purchased presence of a 30-hydroxyl group in the B-ring appears to from Sigma-Aldrich Company (St. Louis, MO, USA). Biochanin enhance the inhibitory activity. From a comparison of the A, 40,7-dimethoxyisoflavone, and 30,40,7-trihydroxyisoflavone structures and the inhibitory activities of isoflavones, such were purchased from Alfa Aesar (Ward Hill, MA, USA). Equol as daidzein and genistein, and their O-methylated iso- was purchased from LC Laboratories (Woburn, MA, USA). flavones, such as biochanin A, formononetin, prunetin, and 5a-Reductase inhibitory effects of isoflavonoids 321

8 1 R3 O 2 7 2' 1' R4 6 3 R2 45 3'

R1 O 6' 4' R5 5'

R1 R2 R3 R4 R5

HHOHHOH Daidzein Genistein OH H OH H OH Biochanin A OH H OH H OCH3 Formononetin HHOHHOCH3 Glycitein HOCH3 OH H OH Prunetin OH H OCH3 HOH HHOCH(CH) HH 4',7-Dimethoxyisoflavone 3 2 3',4',7-Trihydroxyisoflavone HHOCH3 HOCH3 HHOHOHOH

HO 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 additional 6-methoxy group in the A-ring seems to group in the A-ring or of the 40-hydroxyl group in the B- substantially inactivate the inhibitory activity. ring does not seem to potentiate the inhibitory activity. The inhibitory effects of daidzein, genistein, biochanin From a comparison of the structures and the inhibitory A, formononetin, and equol on testosterone 5a-reductase activities of daidzein and glycitein, the presence of an 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)IC50 (mM)

100 mM 1000 mM Daidzein 28.1 11.3 36.5 1.3 >1000 Genistein 34.3 4.4 52.8 12.5 710 Biochanin A 45.1 3.6 81.7 2.2 140 Formononetin 23.8 16.4 27.5 0.8 >1000 Glycitein 14.9 2.0 1.4 24.1 >1000 Prunetin 33.8 7.5 40.1 15.8 >1000 Ipriflavone 24.8 20.2 23.0 13.0 >1000 Equol 17.9 16.7 74.1 4.4 370 40,7-Dimethoxyisoflavone 37.5 5.6 27.8 17.3 >1000 30,40,7-Trihydroxyisoflavone 31.1 1.8 53.6 8.8 690 Riboflavin 68.7 28.9 93.3 0.9 17 * Values are the means of the results from two different experiments. 322 M. Bae et al. testosterone 5a-reductase assay, the inhibitory effects of 5. Nakayama O, Yagi M, Tanaka M, Kiyoto S, Okuhara M, daidzein and formononetin were found to be considerably Kohsaka M. WS-9659 A and B, novel testosterone 5a-reductase less than those of genistein, biochanin A, and equol. inhibitors isolated from a Streptomyces. I. Taxonomy, The inhibitory effect of equol, which is the reduced fermentation, isolation, physico-chemical characteristics. J e isoflavone metabolite of daidzein due to bacterial flora in Antibiot. 1989;42:1221 1229. 6. Nakayama O, Shigematsu N, Katayama A, Takase S, Kiyoto S, the intestines [12], was considerably higher than that of Hashimoto M, et al. WS-9659 A and B, novel testosterone 5a- daidzein itself, which has an isoflavone skeleton. Equol has reductase inhibitors isolated from a Streptomyces. II. Struc- been reported to show a blocking effect on the growth of tural elucidation of WS-9659 A and B. J Antibiot. 1989;42: the ventral prostates induced by 5a-dihydrotestosterone in 1230e1234. castrated male rats [13]. 7. Nakayama O, Arakawa H, Yagi M, Tanaka M, Kiyoto S, As far as we know, the current report is the first charac- Okuhara M, et al. WS-9659 A and B, novel testosterone 5a- terization of 30,40,7-trihydroxyisoflavone as a testosterone reductase inhibitors isolated from a Streptomyces. III. Biolog- 5a-reductase inhibitor. 30,40,7-Trihydroxyisoflavone was ical characteristics and pharmacological characteristics. J e previously isolated from the heartwood of Machaerium vil- Antibiot. 1989;42:1235 1240. losum (Leguminosae) [14] and as a metabolite of Strepto- 8. Nakayama O, Yagi M, Kiyoto S, Okuhara M, Kohsaka M. Ribo- flavin, a testosterone 5a-reductase inhibitor. J Antibiot. 1990; myces sp. OH-1049 grown in media containing , 43:1615e1616. which shows antioxidant activity [15,16]. Also, this 0 0 9. Takamatsu S, Rho M-C, Masuma R, Hayashi M, Komiyama K, compound and 3 ,4 ,5,7-tetrahydroxyisoflavone were iso- Tanaka H, et al. A novel testosterone 5a-reductase inhibitor, lated from culture filtrates of a marine Streptomyces sp. 80,90-dehydroascochlorin produced by Verticillium sp. FO-2787. 060524 in soybean meal glucose medium, and were produced Chem Pharm Bull. 1994;42:953e956. through 30 hydroxylation from daidzein and genistein, 10. 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