Inhibitors of 5α -Reductase Type I in LNCaP Cells from the Roots of Angelica koreana

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

A prenylated coumarin, osthenol (1) and a sesquiterpene, bisabolangelone (2) have been isolated as active principles with 5α-reductase type I inhibitory effects in LNCaP cells from the roots of Angelica koreana Max. by bioassay-guided chromatographic fractionation. Osthenol exhibited a highly potent inhibitory activity on 5α-reductase type I in LNCaP cells with an IC₅₀ value of 0.1 μg/ml, which is about 200 times more potent than the positive control, finasteride (IC₅₀ = 19.8 μg/ml). Bisabolangelone also inhibited the activity of 5α-reductase type I in LNCaP cells (IC₅₀ = 11.6 μg/ml), indicating that these compounds are possible candidates for the development of new drugs to treat human endocrine disorders associated with overproduction of DHT by 5 α-reductase type I. In addition, four compounds isooxypeucedanin, oxypeucedanin hydrate, oxypeucedanin and isoimperatorin were also isolated and found to be inactive in the 5α-reductase assay systems used in the present study.

The plant Angelica koreana Max. is found in Korea and the northeast region of China, and has been used in traditional medicines for headaches, paralysis, fever, arthritis, and rheumatism. It was reported that other Angelica species such as A. pubescens, A. dahurica, A. furcijuga and A. gigas possess anti-platelet aggregation [1], antimicrobial [2], hepatoprotective [3], inhibition of nitric oxide synthesis [4], and antiatherogenesis properties [5]. However, to the best of our knowledge, a 5α-reductase inhibitory activity of these species has never been reported before. Two isozymes (type I and II) of steroid 5α-reductase have been cloned, expressed and characterized [6]. It has been reported that male pattern baldness is associated with an increase in dihydrotestosterone (DHT) production in the scalp and primary cultured benign prostatic hyperplasia (BPH) cells have the capacity to express both 5α-reductase isozymes [7]. LNCaP cells are from an androgen-sensitive human prostatic cancer cell line widely available for use in preclinical investigations and it has been reported that 5α-reductase type I is expressed in LNCaP cells but not 5α-reductase type II [8]. The existence of androgen sensitivity along with 5α-reductase activity in LNCaP cells offered us the opportunity to study the effects of inhibitors in the enzymatic and in vitro functional LNCaP responses to androgens in a single human cell line.

During the course of our research to find potential agents to treat human endocrine disorders associated with the overproduction of DHT using 5α-reductase inhibitory assay systems, hexane and ethyl acetate extracts from the roots of A. koreana exhibited significant activity. Therefore, each extract of the plant was subjected to a bioassay-guided chromatographic fractionation procedure. A coumarin, osthenol (1) was obtained and identified [9], [10] as an active constituent from the hexane extract along with the inactive isooxypeucedanin [11], and a sesquiterpene, bisabolangelone (2) was also isolated and identified [12] as the active principle along with inactive oxypeucedanin hydrate [13], oxypeucedanin [11], [14] and isoimperatorin [11], [14] from the ethyl acetate extract of A. koreana. The structures of these compounds were compared with the literature values for structure identifications.[]

The purified compounds were tested for the activity of 5α-reductase type I in LNCaP cells. Among isolates, osthenol (1) showed a strong inhibitory effect on 5α-reductase type I with an IC₅₀ value of 0.1 μg/ml, which is about 200 times more potent than the positive control, finasteride (IC₅₀ = 19.8 μg/ml) as shown in the Table [1]. To the best of our knowledge, this seems to be the first report about a coumarin with 5α-reductase inhibitory activity. A sesquiterpene, bisabolangelone (2) also exhibited significant inhibitory activity against the 5α-reductase type I in LNCaP cells with an IC₅₀ value of 11.6 μg/ml. The cytotoxicity test showed that these inhibitory effects of osthenol and bisabolangelone were not due to a direct cytotoxic action of the compounds in 5α-reductase assay (Table [1]). It has been reported that benign prostate hyperplastic tissue expresses 5α-reductase type I as well as type II [8]. Therefore, our results indicate that osthenol and bisabolangelone from A. koreana may be potential candidates to treat possibly prostate diseases as well as male pattern hair loss.

Materials and Methods

The roots of Angelica koreana were cultivated in Kangwon-do, Korea and a voucher specimen (No. NPRI-A88) is maintained at the Natural Products Resource Depository of our institute. The dried roots of A. koreana (500 g) were extracted three times with MeOH (3 × 2 L) overnight at room temperature. The MeOH extracts (58 g) were concentrated in vacuo and suspended in water. It was partitioned with equal volume of n-hexane (3 × 300 mL) and ethyl acetate (3 × 300 mL), subsequently. The n-hexane extract (5.2 g) was subjected to a silica gel (200 g) column chromatography, eluting with a stepwise gradient of n-hexane-ethyl acetate mixtures (100 : 0 → 50 : 50) to give 5 fractions (500 mL, each fraction). Fraction 4 (650 mg) was further separated by high performance liquid chromatography (Phenomenex Econo-prep, C-18, 1.0 × 25 cm, 5 μm) with MeOH-water gradient (40 : 60 → 100 : 0, total 75 min, flow rate: 4 ml/min, UV detection: 254 nm) to yield, in the elution order, osthenol (1) (87 mg, t_R 42 min) [9], [10] and isooxypeucedanin (144 mg, t_R 66 min) [11]. One gram of ethyl acetate extract was separated by high performance liquid chromatography (Phenomenex Econo-prep, C-18, 1.0 × 25 cm, 5 cm, flow rate: 4 ml/min, UV detection: 254 nm) with 30 % acetonitrile in water to afford, in the elution order, oxypeucedanin hydrate (S form) {14 mg, t_R 19 min, [α]²⁵ _D: + 69.9° (c 0.014, MeOH)} [12], oxypeucedanin (S form) {22 mg, t_R 29 min [α]²⁵ _D: -17.1° (c 0.022, MeOH)} [11], [14], isoimperatorin (40 mg, t _R 45 min) [11], [14], and bisabolangelone (2) {10 mg, t_R 32 min, [α]²⁵ _D: + 195.3° (c 0.044, MeOH)} [12]. The characterization of the isolates from A. koreana was performed by analysis of 1D and 2D NMR data as well as by comparisons of their physical and spectral data such as m. p., UV, IR, ¹H- and ¹³C-NMR with those of literature values.

LNCaP (ATCC CRL-1740) cells were used for 5α-reductase type I enzyme assay. Enzyme assays were based on the report of Smith et al. [15] and modified. In brief, cells were seeded at a density of 2 × 10⁵ cells/ml/well in 24-well plates and cultured for 24 h in RPMI 1640 media containing 5 % charcol-stripped, heat-inactivated fetal bovine serum. Then cells were treated with [1,2,6,7 - ³H] testosterone (Dupont - New England Nuclear, Boston, MA) and test samples (to yield 0.5 % DMSO solution). After an additional 18 hrs of incubation, [³H]-T and [³H]-DHT in medium was subjected to TLC with silica gel (Silica 60W, Merck) using chloroform/methanol (96 : 4, v/v) as the solvent system. The measurement of [³H]-T and [³H]-DHT was performed by using an image plate reader (BAS-1500, Fuji). The conversion from T to DHT was calculated from the ratio of the radioactivity of DHT to the sum of the radioactivities of T and DHT.

For the determination of cytotoxic effects, LNCaP cells were plated at a density of 1 × 10⁶ cells/ml in 96 well-plates (200 μl), cultured for 24 hrs, and measured by the mitochondrial-dependent reduction of MTT to formazan [16] using the same treatment procedures as the determination of enzyme activity.

Acknowledgements

This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. (HMP-97-D-4 - 0023)

References

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Prof. Ph. D. Woongchon Mar

Natural Products Research Institute

Seoul National University

Seoul 110-460

Korea

Phone: +82-2-740-8911

Fax: +82-2-3672-5488

Email: mars@snu.ac.kr

References

• 1 Liu J H, Xu S X, Yao X S, Kobayashi H. Angelol-type coumarins from Angelica pubescens F. biserrata and their inhibitory effect on platelet aggregation.  Phytochemistry;. 1995;  39 1099-101
  CrossrefPubMedSearch in Google Scholar
• 2 Kwon Y S, Kobayashi A, Kajiyama S I, Kawazu K, Kanzaki H, Kim C M. Antimicrobial constituents of Angelica dahurica roots.  Phytochemistry. 1997;  44 887-9
  CrossrefPubMedSearch in Google Scholar
• 3 Matsuda H, Murakami T, Kageura T, Ninomiya K, Toguchida I, Nishida N, Yoshikawa M. Hepatoprotective and nitric oxide production inhibitory activities of coumarin and polyacetylene constituents from the roots of Angelica furcijuga .  Bioorg. Med. Chem. Lett.. 1998;  18 2191-6
  PubMedSearch in Google Scholar
• 4 Choi Y E, Ahn H, Ryu J H. Polyacetylenes from Angelica gigas and their inhibitory activity on nitric oxide synthesis in activated macrophages.  Biol. Pharm. Bull.. 2000;  23 884-6
  PubMedSearch in Google Scholar
• 5 Zhui Y, Jing-Ping O Y, Yongming L, Lei W, Shuzheng T, Hailu Y, Hanqiao Z, Xiaoho Y. Experimental study of the antiatherogenesis effect of Chinese medicine angelica and its mechanisms.  Clin Hemorheol Microcirc.. 2000;  22 305-10
  PubMedSearch in Google Scholar
• 6 Russell D W, Wilson J D. Steroid 5 alpha-reductase: two genes/two enzymes.  Annu. Rev. Biochem.. 1994;  63 5-61
  PubMedSearch in Google Scholar
• 7 Hirsch K S, Jones C D, Audia J E, Andersson S, McQuaid L, Stamm N B, Meubauer B L, Pennington P, Toomey R E, Russel D W. LY191704: A selective, nonsteroidal of human steroid 5α-reductase type I. Proc. Natl. Acad. Sci.  USA. 1993;  90 5277-81
  PubMedSearch in Google Scholar
• 8 Negri-Cesi P, Poletti A, Colciago A, Magni P, Motta M. Presence of 5α-reductase isozymes and aromatase in human prostate cancer cells and in benign prostate hyperplastic tissue.  The Prostate. 1998;  34 283-91
  CrossrefPubMedSearch in Google Scholar
• 9 Macias F A, Hernandez-Galan R, Massanet G M, Rodriquez-Luis F, Vasquez M, Salva J. Carbon-13 NMR of coumarins. V. 3-Prenylated coumarins.  Magn. Reson. Chem.. 1990;  28 732-5
  CrossrefPubMedSearch in Google Scholar
• 10 Seo E -K, Chai H B, Chagwedera T E, Farnsworth N R, Cordell G A, Pezzuto J M, Kinghorn A D. 5-(4-Hydroxyphenethenyl)-4,7-dimethoxycoumarin, a new constituent of Monotes engleri .  Planta Med.. 2000;  66 182-4
  PubMedSearch in Google Scholar
• 11 Kim T J, Lee S I, Yoon Y S, Ko J S. A study on structure and quantitation of furanocoumarins from Angelica koreana Max.  J. Kor. Soc. Anal. Sci.. 1989;  2 337-45
  PubMedSearch in Google Scholar
• 12 Bae K H, Ji J M, Kang J S, Ahn B Z. A cytotoxic component from Angelicae koreanae radix against L1210 and HL-60 cells.  Arch. Pharm. Res.. 1994;  17 45-7
  PubMedSearch in Google Scholar
• 13 Steck M, Mazurek M. Clinical and biochemical parameters of androgen action in normal healthy Caucasian versus Chinese subjects.  Lloydia. 1972;  35 418-39
  PubMedSearch in Google Scholar
• 14 Bergendorff O, Dekermendjian K, Nielsen M, Shan R, Witt R, Ai J, Sterner O. Furanocoumarins with affinity to brain benzodiazepine receptors in vitro .  Phytochemistry. 1997;  44 1121-4
  CrossrefPubMedSearch in Google Scholar
• 15 Smith C M, Ballard S A, Worman N, Buettner R, Masters J R. 5 Alpha-reductase expression by prostate cancer cell lines and benign prostatic hyperplasia in vitro .  J. Clinical Endo. Metabol.. 1996;  81 1361-6
  PubMedSearch in Google Scholar
• 16 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.  J. Immunol. Methods. 1983;  65 55-63
  CrossrefPubMedSearch in Google Scholar

Prof. Ph. D. Woongchon Mar

Natural Products Research Institute

Seoul National University

Seoul 110-460

Korea

Phone: +82-2-740-8911

Fax: +82-2-3672-5488

Email: mars@snu.ac.kr