Geranylated Coumarins From Thai Medicinal Plant Mammea siamensis With Testosterone 5α-Reductase Inhibitory Activity

Geranylated coumarin constituents, kayeassamin I (1) and mammeasins E (2) and F (3) were newly isolated from the methanol extract of the flowers of Mammea siamensis (Calophyllaceae) originating in Thailand, along with five known isolates, such as mammea E/BC (23), deacetylmammea E/AA cyclo D (31), deacetylmammea E/BB cyclo D (32), mammea A/AA cyclo F (34), and mammea A/AC cyclo F (35). These compounds (1–3) were obtained as an inseparable mixture (ca. 1:1 ratio) of the 3″R and 3″S forms, respectively. Among the isolated coumarins from the extract, mammeasins E (2, 22.6 μM), A (4, 19.0 μM), and B (5, 24.0 μM), kayeassamins E (9, 33.8 μM), F (10, 15.9 μM), and G (11, 17.7 μM), surangin C (13, 5.9 μM), and mammeas A/AA (17, 19.5 μM), E/BB (22, 16.8 μM), and A/AA cyclo F (34, 23.6 μM), were found to inhibit testosterone 5α-reductase.


Plant Material
The flowers of Mammea siamensis were collected from the Nakhonsithammarat Province, Thailand, in September 2006, as described previously (Morikawa et al., 2012;Ninomiya et al., 2016). The plant material was identified by one of the authors (Y. P.). A voucher specimen (2006.09. Raj-04) for this plant has been deposited in our laboratory.

Assay for Testosterone 5α-Reductase Inhibitory Activity
The experiment was performed in accordance with previously reported methods (Matsuda et al., 2001;Lee et al., 2012;Koseki et al., 2015) with slight modifications. In brief, the assay was performed in 48-well microplates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan). The reaction solution was pre-incubated with or without a test sample (5 µL/well, dissolved in DMSO), in a potassium phosphate buffer (40 mM, pH 6.5, 490 µL/well) containing substrate (0.35 nmol of testosterone, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and NADPH (10 nmol, Oriental Yeast Co., Ltd., Tokyo, Japan) at room temperature (25 • C) for 20 min. The enzymatic reaction was initiated by the addition of rat liver S9 fractions (10 µL/well, dissolved in the phosphate buffer, 20.6 µg/well, Oriental Yeast Co., Ltd., Tokyo, Japan, lot no. 109031513) at 37 • C for 30 min. After incubation, the reaction mixture was immediately heated in boiling water for 2 min to stop the reaction. Then the reaction solution of each well was transferred to a microtube and extracted with 500 µL of EtOAc. After the microtube was centrifuged (10,000 rpm, 5 min), an aliquot of each EtOAc phase (300 µL) was transferred into another tube. The solvent in the tube was evaporated and the residue was dissolved in 30 µL of acetonitrile containing an internal standard (I.S.) fludrocortisone acetate (20 µg/mL, Sigma-Aldrich, Co., LLC, St. Louis, USA). An aliquot of 2 µL was injected into the HPLC under the following conditions [Instrument: a series LC-20A Prominence HPLC system (Shimadzu Co., Kyoto, Japan); Detection: UV (254 nm); Column: Cosmosil 5C 18 -MS-II (Nakalai Tesque Inc., Kyoto, Japan, 5 µm particle size, 2.0 mm i.d. × 150 mm); Column temperature: 40 • C; Mobile phase: MeOH-H 2 O (60:40, v/v); Flow rate: 0.2 mL/min; retention time: 13.5 min for testosterone and 8.0 min for I.S. A similar procedure that described above was carried out for the control tubes. The 5α-reductase inhibitory activity was determined from the following equation using the peak area ratios (r = testosterone/I.S.). Experiments were performed in triplicate or quadruple, and IC 50 values were determined graphically. The 5α-reductase inhibitor finasteride (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was used as a reference compound.

Statistics
Values are expressed as mean ± S.E.M. One-way analysis of variance (ANOVA), followed by Dunnett's test, was used for statistical analysis. Probability (p) values <0.05 were considered significant.

Effects of the Methanol Extract From the Flowers of M. siamensis on Testosterone 5α-Reductase
The male sex hormones, androgens, play a crucial role in the development, growth and function of the prostate, and other androgen-sensitive peripheral tissues. In the prostate gland, androgens are involved in benign prostatic hyperplasia and prostate cancer, as well as in skin disorders, such as acne, seborrhea, androgenic alopecia, and hirsutism. Among the androgens, testosterone is the most abundant in serum and secreted primarily by the testicles and ovaries. The enzyme steroid 5α-reductase catalyzes the conversion of testosterone to the most potent natural androgen, 5α-dihydrotestosterone (Yamana et al., 2010;Yao et al., 2011;Azzouni et al., 2012). Therefore, inhibition of testosterone 5α-reductase could be useful for the treatment of the above diseases. To date, three types of 5α-reductases, chronologically named types 1, 2, and 3 5αreductases, have been described (Yamana et al., 2010;Azzouni et al., 2012;Titus et al., 2014). A type 2 and 3 5α-reductase inhibitor, finasteride, is currently marketed worldwide as a drug for benign prostatic hyperplasia and is also used in the treatment of hair loss (Heinzl, 1999;Tosti and Piraccini, 2000) and in the prevention of prostate cancer (Coltman et al., 1999). Therefore, 5α-reductase is considered a useful therapeutic target in the treatment and prevention of the above deceases. In particular, many heterocyclic compounds based on oxygen and nitrogen atoms often have good antiproliferative activity against a variety of solid tumor cell lines and are expected to be seeds of new anticancer agents (Sharma et al., 2018;Petel et al., 2019). During our characterization studies on bioactive constituents from Thai natural medicines (Manse et al., 2017;Morikawa et al., 2018;Tanabe et al., 2018;Kobayashi et al., 2019), a methanol extract of the flowers of M. siamensis was found to inhibit 5αreductase activity (IC 50 = 2.4 µg/mL). In order to investigate new 5α-reductase inhibitors, we conducted a search for the bioactive constituents from the flowers of M. siamensis.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.