DOI: 10.14457/tu.the.2016.1470
title: Cytotoxic and anti-estrogenic activities of five Thai medicinal plants called ‘Hua-Khao-Yen’ and their isolated compounds

description: The rhizomes of Hua-Khoa-Yen have been used in Thai traditional medicine as an ingredient formula for cancer treatment especially liver and breast cancer which is the most cancer found in Thailand. Based on in vitro cytotoxicity of the ethanolic and water extract of Hua-Khoa-Yen, which are Dioscorea birmanica, Dioscorea membranacea, Smilax corbularia, Smilax glabra and Pygmaeopremna herbacea against hepatocellular carcinoma (HepG2), cholangiocarcinoma (KKU-M156) and breast cancer (MCF-7 and T47D) cells, both extracts of D. membranacea showed high potency specific. Aim of the study is to investigate the cytotoxic and estrogenic/anti-estrogenic activities of ethanolic and water extracts and constituents of D. membranacea to support its traditional use. The SRB assay was used to determine the cytotoxic activity against hepatocellular carcinoma (HepG2), cholangiocarcinoma (KKU-M156), breast cancer (MCF-7 and T47D) cells and one normal human keratinocyte immortal cells (HaCaT) with its extract and isolated compounds. The bioassay-guided fractionation of the rhizome of ethanolic extract of D. membranacea led to the isolation of two napthofuranoxepins [dioscorealide A (DME1) and dioscorealide B (DME2)], phenanthraquinone [dioscoreanone (DME3)], two phenanthrenes [5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (DME4) and 2,5,6-trihydroxy-3,4-dimethoxy, 9, 10-dihydrophenanthrene (DME5)], a known steroid [panthogenin B (DME6)] and a novel steroid [epipanthogenin B (DME7)]. The crude water extract of D. membranacea was purified by HPLC technique to give thirteen compounds namely six known steroids saponins; three spirostane steroids [25 (R)-spirost-5-en-3β,14α-diol-3-O-β-ᴅ-glucopyranoside or polygodoside E  (DMW1), 25 (R)-spirost-5-en-3, 14-diol -3-O-α-L-rhamnopyranosyl-(1→2)-O-β-ᴅ-glucopyranoside or 25 (R,S)-dracaenoside F (DMW2) and diosgenin-3-O-α-ʟ-rhamnosyl (1→2)-β-ᴅ-glucopyranoside or prosapogenin A of dioscin  (DMW3)] and three furostane steroids [26-O--ᴅ-glucopyranosyl- 25(R)-furost-5-en-3,14,22, 26-tetrol -3-O--L-rhamnopyranosyl-(12)--ᴅ-glucopyranoside  or 25(R,S)-dracaenoside N (DMW4), 26-O-β-ᴅ-glucopyranosyl-22-hydroxyfurost-5-ene-3β,26-diol-3-O-α-ʟ-rhamnopyranosyl (1 → 2)-β-ᴅ-glucopyranoside (DMW5) and 3β, 26-dihydroxy-25 (R)-furosta-∆5,20(22)-diene-3-O-α-ʟ-rhamnopyranosyl-(1→2)-O-β-ᴅ-glucopyranoside (DMW6)], in addition to seven compounds which were isolated from ethanolic extract (DME1-DME7). Their structures were elucidated by spectroscopic methods. The cytotoxicity of all isolated compounds against cholangiocarcinoma (KKU-M156), hepatocellular carcinoma (HepG2) and breast cancer (MCF-7 and T47D) were tested. Dioscorealide B (DME2) showed the highest potency against KKU-M156 (IC50 = 1.67 ± 0.21 µM), HepG2 (IC50 = 2.87 ± 0.10 µM), MCF-7 (IC50 = 2.17 ± 0.01 µM) and T47D (IC50 = 1.70 ± 0.17 µM) cells and had no toxicity against normal cell (HaCaT) with IC50 &gt;100 µM but less toxic on normal cell with the SI value of &gt;14.9 and &gt;58.82, respectively. DME3 also showed high cytotoxicity against these four cell lines but toxic on immortal keratinocyte cell (HaCat). For the estrogenic and anti-estrogenic activities against E2-enhance T47D cell proliferation, no extracts showed estrogenic activitity through the concentration of 0.01-10 µg/mL. The crude ethanolic extract (DME) showed anti-estrogenic activity at 0.1 µg/mL, and crude water extract (DMW) was recognized the anti-estrogenic activity at 100 times less concentration (&lt; 0.01 µg/mL). The dihydrophenanthrene (DME4) and steroid spirostane saponin (DMW1) showed strong inhibition against T47D cell at low concentrations with their iEqE1 values of &lt;0.1 µM and the highest activity than tamoxifen (iEqE1 = 9 µM). Thus, this study supports Thai traditional use of this plant for treatment cancer patients. DME2 should be devoped as anti-cancer drug for liver and bile duct cancer patients. In addition, this compound should be as anti-cancer drug for breast cancer because it displayed anti-estrogen.