Studies On Screening Antitumor Active Fractions In Vitro And Chemical Constituents In Active Fractions From Solanum Nigrum L.

The medicinal plant Solanum nigrum L. belonging to the family of Solanaceae had the property of heat-clearing and detoxifying, promoting blood circulation, inducing diuresis to alleviate edem, relieving cough and asthma. The whole plant of Solanum nigrum L.was widely used to treat inflammation, diarrhea, flu fever, toothache, functional uterine bleeding, snakebite, furunculosis and herpes zoster.This dissertation conducted to reveal the antitumor material base of Solanum nigrum L. was divided into three aspects as following:(1) The screening of anti-tumor activity fractions from Solanum nigrum L. in vitro,(2) chemical constituents analysis of activity fractions,(3) studies on the antitumor effect and its mechanism of active monomer components belonging to alkaloid.The first part:The screening of anti-gastric carcinoma and anti-hepatoma activity fractions from Solanum nigrum L. in vitro.1. The antiproliferative activities of aqueous extract and ethanol extract on human hepatocellular carcinoma cell line SMMC-7721, HepG2and human gastric carcinoma cell line MGC-803were evaluated by MTT colorimetric assay in vitro with IC50as index. The results showed that aqueous extract and ethanol extract of Solanum nigrum L. possessed the antiproliferative activity on the three kinds of tumor cell lines in a dose-dependent manner. The significant changes of cellular morphology of above-mentioned cell lines after treating with aqueous extract and ethanol extract were observed by inverted phase contrast microscope.2. The ethanol extract of Solanum nigrum L. was separated into five different fractions as following:petroleum ether fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction and water-soluble fraction. Their anticancer effect on human hepatocellular carcinoma cell line SMMC-7721and HepG2and human gastric carcinoma cell line MGC-803were studied by MTT. The results showed that petroleum ether fraction and n-butanol fraction exhibited significant antiproliferative activities in a dose-dependent manner. The cellular morphology was also changed markedly after being exposed to petroleum ether fraction and n-butanol fraction respectively3. Two types of alkaloids and saponins were isolated from n-butanol fraction by acid dissovlving and base precipitation, macroporous adsorption resin and acetone precipitation reseparately. Water-soluble polysaccharide and alkali-soluble polysaccharide were extrated by traditional decocting method, and purified by sevag and H2O2-decoloring method. The anti-gastric carcinoma and anti-hepatoma activities in vitro of these components were also evaluated by MTT colorimetric assay and observing cell morphology under inverted phase contrast microscope. The experimental results indicate that alkaloids and saponins showed significantly antiproliferative effects on SMMC-7721cells, HepG2cells and MGC-803cells and lead to cell morphology changes. Water-soluble polysaccharide and alkali-soluble polysaccharide exhibited no effete on the selected cell lines.The second part:The chemical components analysis of activity fractions from Solanum nigrum L..1. The essential oil of petroleum ether fraction was obtained by steam distillation.The chemical components of essential oils were determined by GC-MS,49compounds in the essential oil were identified, which account for97.32%of total peak area. And the major constituents included esters, alkanes, fatty acid and ketones.2. Chemical identify methods were used to analyze the property of alkaloids and saponins. The contents of alkaloids, saponins and polysaccharide of Solanum nigrum L. were determined by the acid dyestuff method, vanillin-perchloric acid colorimetry and phenol-sulfuric acid colorimetry reseparately.The third part:Studies on the antitumor effect and its mechanism of monomer component belonging to alkaloid in Solanum nigrum L..1. The main monomer components of alkaloid in Solanum nigrum L.include solasonine, solamargine and ε-solanine. The antiproliferative activities of these monomer components on SMMC-7721. HepG2and MGC-803cells were studied by MTT colorimetric assay. The results showed that solamargine had the significant cytotoxic effect on SMMC-7721and MGC-803cells. The diversity changes of cell morphology and granular apoptosis bodies were observed by inverted phase contrast microscope.2. Cell cycle analysis of SMMC-7721cells was investigated by using FACS after PI staining. The results showed that solamargine-treated SMMC-7721cells led to a marked accumulation in G2/M phase in dose-dependent manner. Solamargine can induce apoptosis at the concentration of15μg/mL. It indicated that antitumor effect of solamargine may be related to arrest in G2/M phase and apoptosis.