Volume 3, Issue 2, Pages 116-118 (June 2010)

8 of 13

ABSTRACT

FULL TEXT

FULL-TEXT PDF (140 KB)

5α-Reductase Inhibitory Components as Antiandrogens From Herbal Medicine

Received 10 February 2010; accepted 12 April 2010.

Abstract

We investigated medicinal plant sources with 5α-reductase inhibitory activity. These compounds have been used in several remedies against androgen-dependent diseases including benign prostatic hyperplasia. The 50% ethanol extract of Polygonum multiflorum Thunb (Polygoni Multiflori Radix; Polygonaceae) showed potent 5α-reductase inhibitory activity. The fraction responsible for this activity was purified, and the active constituent was isolated and identified as emodin, an anthraquinone compound. Although emodin showed considerably less potent inhibitory activity than riboflavin, the inhibitory activity of the compound was more potent than that of alizarin (1,2-dihydroxyanthraquinone), an anthraquinone-type positive control. Also, anthraquinone itself was substantially inactive against 5α-reductase, in dicating that the hydroxyl group on the structure of emodin is an important structural moiety for displaying inhibitory activity.

Key Words: androgen-dependent diseases , anthraquinone , emodin , Polygoni Multiflori Radix , 5α-reductase inhibitor , Polygonum multiflorum Thunb

Article Outline

• Abstract

• 1.. Introduction

• 2.. Materials and Methods

• 2.1.. General experimental procedures

• 2.2.. Plant material

• 2.3.. Preparation of extracts and isolation of active compound

• 2.4.. Enzymatic assay

• 3.. Results

• 4.. Discussion

• Acknowledgment

• References

• Copyright

1. Introduction

Dihydrotestosterone, also known as 5α-dihydrotestosterone acts as a more active androgen than testosterone in many tissues, including the prostate. Therefore, inhibitors of 5α-reductase, which catalyzes the reductive conversion of testosterone to 5α-dihydrotestosterone, may be useful in the selective treatment of androgen-dependent diseases, such as benign prostatic hyperplasia, male pattern baldness and acne [1]. Most developed 5α-reductase inhibitors are steroidal compounds, which bind to steroid receptors, act as agonists or antagonists, and may produce various undesirable hormonal effects. Therefore, several 5α-reductase inhibitory active constituents from various plant sources have been isolated [2, 3, 4, 5, 6, 7, 8, 9, 10, 11]. In this paper, we describe the inhibitory activity of the active component in a 50% ethanol extract of Polygoni Multiflori Radix (PMR; Polygonum multiflorum Thunb., Polygonaceae) on the activity of 5α-reductase prepared from rat prostate.

2. Materials and Methods

2.1. General experimental procedures

Mass spectrometry spectra from the electron impact of 70 eV were obtained with JMS AX505WA (JEOL Ltd., Tokyo, Japan) mass spectrometer. 1H- and 13C-NMR spectra at 400 MHz and 100 MHz were obtained on a JEOL JNM-AL400 spectrometer (JEOL Ltd.) with internal TMS as standard. Column chromatography was performed with silica gel (Merck & Co., Inc., Whitehouse Station, NJ, USA). TLC was performed on precoated Silica gel 60 F254 plates (Merck & Co., Inc.), and spots were visualized using UV light at 254 nm. Riboflavin and alizarin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Anthraquinone was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

2.2. Plant material

Plant samples of PMR were purchased from the Herbal Cosmeceutical Material Bank, Korea National Research Resource Center. Voucher specimens have been deposited at the herbarium of the Natural Products Chemistry Laboratory, Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-industry, Daegu Haany University, Korea.

2.3. Preparation of extracts and isolation of active compound

The dried and chopped radix of PMR (500 g) was extracted with 50% ethanol (1.0 L×3) at room temperature for 3 days. After filtration, the extract was evaporated under reduced pressure and lyophilized to give a 50% ethanol extract (135.5 g). The 50% ethanol extract was partitioned between ethyl acetate and water to give an ethyl acetate-soluble fraction (11.4 g) and an aqueous fraction, respectively. The ethyl acetate-soluble fraction from PMR was mixed with silica gel and then fractionated by silica gel column chromatography with n-hexane-ethyl acetate to give active Compound 1 (66.1mg).

2.4. Enzymatic assay

Homogenate of the ventral prostate of male Sprague-Dawley rats was prepared and 5α-reductase inhibition was measured using the methods previously reported [12].

3. Results

We investigated medicinal plant sources having 5α-reductase inhibitory activity used in several remedies against androgen-dependent diseases, including benign prostatic hyperplasia. The 50% ethanol extract of PMR showed substantial 5α-reductase inhi bitory activity. A 500 μg/mL solution of the dried 50% ethanol extract of PMR showed an 80.7% inhibition of the enzyme. The inhibitory activity of the 50% ethanol extract of this crude drug was superior to that of Davallia mariesii Moore (Davalliaceae) and Panax ginseng C.A. Meyer (Araliaceae), both of which have been used in several phytotherapeutic preparations in the treatment of androgen-dependent diseases. When the 50% ethanol extract was partitioned between ethyl acetate and water, the ethyl acetate-soluble portion exhibited inhibition of the enzyme. The portion was separated by repeated silica gel chromatography with the guidance of rat prostate 5α-reductase inhibitory activity to give the active constituent, Compound 1, as an orange powder. Electron ionization mass spectrometry of the compound showed characteristic fragmentations at m/z 270 (M+, 100%), which can be assigned to peaks corresponding to an emodin moiety. Compound 1 was finally identified by inspection of 1H- and 13C-NMR spectra as emodin (Figure 1).

View Large Image
Download to PowerPoint

Figure 1. Chemical structures of emodin (1), from the radix of Polygonum multiflorum, anthraquinone and alizarin.

4. Discussion

In traditional Chinese medicine, PMR is prescribed to treat weak bones, premature graying of hair, hair loss, and also to tonify the kidneys, and balance a fragile yin. PMR is also described to have a laxative effect when taken internally. It has been shown to contain anthraquinones, such as emodin and stilbene glycosides, which are similar to resveratrol but have superior antioxidant activity [13]. However, the 5α-reductase inhibitory effect of PMR is reported here for the first time. The isolated emodin was evaluated for inhibitory potency on rat prostate 5α-reductase. Emodin inhibited the enzyme activity in a dose-dependent manner (Table 1). As shown in our data, although emodin showed considerably less potent inhibitory activity than riboflavin, the inhibitory activity of the compound was more potent than that of alizarin, a naturally occurring 5α-reductase inhibitor with an anthraquinone backbone [14]. Anthraquinone itself was substantially inactive against 5α-reductase indicating that the anthraquinone part lacks inhi bitory activity. This observation implies that the hydroxyl group in the structure of emodin is an important structural moiety for displaying inhibitory activity. Thus it may be necessary to examine the activity of a series of anthraquinone analogs to study the structure-activity relationship for enhancing inhibitory activity.

Table 1.

IC50 values of emodin (1) from the radix of Polygonum multiflorum and anthraquinone on rat prostate 5α-reductase activity*


	Compound	IC50 (μM)
	Emodin	40
	Anthraquinone	>1000
	Alizarin	330
	Riboflavin	1.6

Value represents the mean of two experiments.

Acknowledgments

This research was supported by a grant from Daegu Haany University Kylin Foundation in 2009.

References

1. 1 Russell DW , Wilson JD . Steroid 5a-reductase: two genes/two enzymes . Annu Rev Biochem . 1994;63:25–61 . MEDLINE

2. 2 Komoda Y . Isolation of flavonoids from Populus nigra as D4-3-ketosteroid (5a) reductase inhibitors . Chem Pharm Bull . 1989;37:3128–3130 .

3. 3 Lesuisse D , Berjonneau J , Ciot C , Devaux P , Doucet B , Gourvest JF , et al. Determination of oenothein B as the active 5-a-reductase-inhibiting principle of the folk medicine Epilobium parviflorum . J Nat Prod . 1996;59:490–492 . MEDLINE | CrossRef

4. 4 Shimizu K , Fukuda M , Kondo R , Sakai K . The 5a-reductase inhibitory components from heartwood of Artocarpus incisus: structure-activity investigations . Planta Med . 2000;66:16–19 . MEDLINE | CrossRef

5. 5 Shimizu K , Kondo R , Sakai K , Shoyama Y , Sato H , Ueno T . Steroid 5a-reductase inhibitory activity and hair regrowth effects of an extract from Boehmeria nipononivea . Biosci Biotechnol Biochem . 2000;64:875–877 . MEDLINE | CrossRef

6. 6 Ishiguro K , Oku H , Kato T . Testosterone 5a-reductase inhibitor bisnaphthoquinone derivative from Impatiens balsamina . Phytother Res . 2000;14:54–56 . CrossRef

7. 7 Matsuda H , Yamazaki M , Matsuo K , Asanuma Y , Kubo M . Anti-androgenic activity of Myricae Cortex-Isolation of active constituents from bark of Myrica rubra . Biol Pharm Bull . 2001;24:259–263 . MEDLINE | CrossRef

8. 8 Seo EK , Kim KH , Kim MK , Cho MH , Choi E , et al. Inhibitors of 5a-reductase type I in LNCaP cells from the roots of Angelica koreana . Planta Med . 2002;68:162–163 . MEDLINE | CrossRef

9. 9 Kim Y , Son HK , Song HK , Ahn MJ , Lee SS , Lee SK . Inhibition of 5a-reductase activity by diarylheptanoids from Alpinia officinarum . Planta Med . 2003;69:72–74 . MEDLINE | CrossRef

10. 10 Park WS , Son ED , Nam GW , Kim SH , Noh MS , Lee BG , et al. Torilin from Torilis japonica, as a new inhibitor of testosterone 5a-reductase . Planta Med . 2003;69:459–461 . MEDLINE | CrossRef

11. 11 Liu J , Kurashiki K , Shimizu K , Kondo R . 5alpha-reductase inhibitory effect of triterpenoids isolated from Ganoderma lucidum . Biol Pharm Bull . 2006;29:392–395 . MEDLINE

12. 12 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;22:1396–1399 . MEDLINE

13. 13 Li-Shuang LV , Gu X , Ho CT . Stilbene glycosides from the roots of Polygonum multiflorum Thunb and their antioxidant activities . J Food Lipids . 2006;13:131–144 .

14. 14 Hiipakka RA , Zhang HZ , Dai W , Dai Q , Liao S . Structure-activity relationships for inhibition of human 5a-reductases by polyphenols . Biochem Pharmacol . 2002;63:1165–1176 . MEDLINE | CrossRef

a Department of Medical Management, College of Health and Therapy, Daegu Haany University, Gyeongsangbuk-do, Korea

b Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-industry, Daegu Haany University, Gyeongsangbuk-do, Korea

Corresponding author. Department of Herbal Pharmaceutical Engineering, College of Herbal Bio-industry, Daegu Haany University, 290, Yugok-dong, Gyeongsan-si, Gyeongsangbuk-do 712-715, Korea

PII: S2005-2901(10)60021-0

doi:10.1016/S2005-2901(10)60021-0

8 of 13