Studies on the Anticancer Effects of Solanum Lyratum Thunb. and Its Activity Constituents

Abstract: The medicinal plant Solatium lyratum Thunb. belonging to the family of Solanaceae distributed widely in south areas of Changjiang River in China. The whole plant of Solatium lyratum has long been used as traditional Chinese folk medicine to treat malarias, jaundices, edemas, rheumatisms and furuncles. It was also used as an anti-tumor medicine in some folk remedy.This dissertation was divided into 5 aspects as following: (1) the anti-tumor activity in vitro and vivo of aqueous extract and ethanol extract of Solanum lyratum, (2) the screening of anti-tumor activity fractions and its anti-tumor effect in vivo, (3) the isolation of anti-tumor activity compounds by bioassay-directed fractionation and measurements of anti-tumor of isolated compounds, (4) the molecular mechanisms of anti-tumor effect of 16-dehydropregnenolone, (5) the establishment of quantitative methods of two active constituents, diosgenin and 16-dehydropregnenolone.The antiproliferative activities of aqueous extract and ethanol extract against human cervix epithelial cell line HeLa, human malignant melanoma cell line A375-S2, murine fibrosarcoma cell line L929, human breast cancer cell line MCF7, human hepatocarcinoma cell line BEL-7402, human gastric carcinoma cell line SGC-7901 and the myeloid leukemia cell line U937 were evaluated by MTT colorimetric assay in vitro. The results showed the aqueous extract and ethanol extract possessed the antiproliferative activity against different tumor lines in a dose-dependent manner. Compared with the cytotoxic activity of aqueous extract, the ethanol extract showed stronger cytotoxic activity. The inhibitory effects of aqueous extract and ethanol extract on tumor growth were observed by the models of implanted sarcoma 180 (S180) and hepatoma 22 (H22) in mice. Aqueous extract and ethanol extract restrained obviously the growth of tumor in mice. The inhibitory rates of aqueous extract on the growth of S180 and H22 at dose of 62.4 g·kg~(-1) were 41.4% and 45.4%, respectively. The inhibitory rates of ethanol extract on the growth of S180 and H22 at doses of 62.4 g·kg~(-1) were 39.0 % and 37.2 %, respectively.The anticancer activities of petroleum ether, EtOAc, n-BuOH and aqueous fraction were screened by MTT using human cervix epithelial cell line HeLa, human malignant melanoma cell line A375-S2, murine fibrosarcoma cell line L929, human breast cancer cell line MCF7, human hepatocarcinoma cell line BEL-7402, and human gastric carcinoma cell line SGC-7901. The results showed that EtOAc fraction exhibited strong antitumor activity; n-BuOH fraction possessed medium antitumor activity; petroleum ether fraction and aqueous fraction had no activity. In the meanwhile, the inhibitory effects of EtOAc fraction and n-BuOH fraction on tumor growth were observed by the models of implanted sarcoma 180 (S180) and hepatoma 22 (H22) in mice. EtOAc fraction was proved to be effective on the tumor-bearing mice of S180 and H22, and the inhibitory rate of the dose of 62.4g·kg~(-1) were 42.7%and 46.0%, respectively, n-BuOH fraction exhibited weak effect on the tumor-bearing mice of S180, and the inhibitory rate of the dose of 62.4 g·kg~(-1) were 33.3%. n-BuOH fraction exhibited no effect on the tumor-bearing mice of H22.Bioassay-directed fractionation of the EtOAC fraction and n-BuOH fraction led to isolation of 20 compounds from Solanum Lyratum with silica gel, sephadex LH20, polyamide, HPLC etc. Among them, 15 compounds were elucidated on the basis of physi-chemical and spectral methods. They identified as 16-dehydropregnenolone (1), 16-dehydropregnenolone3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucpyranosid-uroni c acid (2), 3-hydroxy-5-pregn-16-en-20-one (3), protocatechuic acid (4), vanillic acid (5), caffeic acid (6), diosgenin (7), diosgenin 3-O-β-D-glucopyranosiduronic acid methyl ester (8), diosgenin 3-O-β-D-glucopyranosiduronic acid (9), diosgenin 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosiduronic acid (10), diosgenin 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuroniduronic acid methyl ester (11), quercetin (12), rutin(13),β-sitosterol(14), scopoletin (15). Among them, compound 2 and 8 are new compounds, 9 is a new nature compound, 1 and 3 are isolated from the genus for the first time, and 4, 7, 12, 14 are isolated from the plant for the first time. The antiproliferative activities of 13 isolated compounds against human cervix epithelial cell line HeLa, human malignant melanoma cell line A375-S2, human hepatocarcinoma cell line BEL-7402 and human gastric carcinoma cell line SGC-7901 were evaluated by MTT. The result suggested compound 1, 7, 10, 11, 12 exhibited antiproliferative activity, and the anti-tumor activity of compound 1, 10, 11 are reported for the first time. In the present studies, We examined the molecular mechanism by which 16-dehydropregnenolone could induce apoptosis in HeLa cells. The characteristic changes of apoptosis, i.e., shrinkage of cells, blebbing nuclei and granular apoptotic bodies, were observed by phase contrast microscopy and Hoechst 33258 staining. When the DNA from HeLa cells incubated with 16-dehydropregnenolone was analyzed by agarose gel electrophoresis, it generated a characteristic “ladder pattern” of disconstinuous DNA fragments.Cell cycle analysis of HeLa cells was investigated by using FACS after PI staining. The results showed that 16-DHP-treated HeLa cells led to a marked accumulation in G_0/G_1 phase after 24h treatment and sub-G_1 apoptotic fraction was increased compared with control. It suggested that the growth inhibitory effect of 16-dehydropregnenolone was the result of arrest in G_0/G_1 phase and apoptosis.To determine whether Caspase-3 participates in 16-dehydropregnenolone induced apoptosis, HeLa cells were treated with 30μM 16-dehydropregnenolone for 24h in the absence or presence of caspase-3 inhibitor, z-DEVD-fmk. Z-DEVD-fmk effectively inhibited 16-dehydropregnenolone-induced HeLa cell death. In the following investigation, we assessed the expressions of caspase-3 substrates PARP and ICAD. The results showed that PARP (116 kDa) expression was down-regulated in a time dependent manner and minor 85 kDa fragment was increased, and ICAD was also down-regulated in time dependent manner, suggesting that caspases participated in 16-DHP induced HeLa cell death.Mitochondrial Bcl-2 family is a series of proteins that regulate apoptosis. Some of these proteins such as Bax and Bak, promote apoptosis, whereas others like Bcl-2 and Bcl-X_L inhibit apoptosis. After incubation with 16-dehydropregnenolone, expression of Bcl-2 protein was down-regulated, on the contrary, the level of Bax protein was increased. These results suggested that the mitochondrial pathway of cell death might be involved in HeLa cells death induced by 16-dehydropregnenolone.Diosgenin and 16-dehydropregnenolone, two anti-tumor activity constituents, were also determined by RP-HPLC. The linear range of them were 0.04-0.2 mg-mL~(-1) (r=0.9999) and 5.06-45.54μg·mL~(-1) (r=1.0000), respectively. The average recoveries of of them were 104.5%(RSD=1.4%)and 98.8%(RSD=1.7%), respectively. These methods were simple, rapid, accurate and provided a quantitative for the quality assessment of Solanum lyratum…
Key words: Solatium lyratum Thunb; anti-tumor activity; cell apoptosis; cell cycle; RP-HPLC; 16-dehydropregnenolone; diosgenin

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