| The study of Chinese traditional medicine is a hot topic for Anti-tumor medicine. Because the for western medicine cancer treatment bear side effects to the body, So Chinese traditional medicine has become new area of the field of cancer therapy. Chinese traditional medicine, Many have anti-tumor effect, but its chemical structure was complex, and displayed distinct anti-tumor mechanisms. Chinese traditional medicine displayed a huge potential and bright prospects for tumor treatment.The fruit of the Hydrocaryaceae plants, known as water chestnut, or water caltop. Its planting has more than 3000-year histories from the Zhou Dynasty. Water caltrop was growed in the shallow lake or pond , quite widely distributed in China and has rich resources. Water caltrop is of very wide range of food and medicinal value. In civil remedies, Water caltop has a very good effect to treat liver ascites. In recent years, with the accelerated pace of modernization of Chinese traditional medicine, the anti-tumor effect of water caltrop has been gradually attracted people's attention.Based on the literature report and the previous studies, this experiment have separated and extracted the chemical substances of water caltrop, preliminarily studied on the anti-tumor effect and mechanism of the water caltrop. Our aim is to develop water caltop as an efficient and low toxicity antineoplastic agents.Objective: To study the anti-tumor activity elements of water caltrop, and study the effect in vitro anti-tumor and apoptosis mechanism.Methods:(1) Ethanol fractionation method was used to separate crude polysaccharides from Water Extract; Steam distillation method was used to separate the volatile oil from water caltrop; Thin-layer chromatography, column chromatography, liquid chromatography were used to isolated the anti-tumor compounds.(2) MTT method was used to Evaluate the crude polysaccharide and volatile oil of water caltrop in vitro anti-tumor activity; analyzed using the flow cytometry method was used to detecet that Polysaccharide F3 derivn the apoptosis of U251 cells; agarose gel electrophoresis method was used to analysis that the volatile oil derivn apoptosis of Hela cell.Results:1 Ethanol precipitation was used to extract three kinds of polysaccharide compounds from water extract of water caltrop, their sugar content: 19.07%, 25.20%, 39.25% for sugar content.2 High performance liquid chromatography, silica gel column chromatography were used , and a single compounds were isolated from water caltrop. Based on physicochemical properties and spectral analysis to analysis and identify its chemical structure.3 GC-MS was used and 16 components was detected and separated from volatile oil of water caltrop. From MS peaks of each scan, the standard MS library was searched and eight compounds were identified. 4 MTT assay showed that the crude Polysaccharide of water caltrop can significantly inhibit Hela and U251 cell proliferation, decrease the cell survival, and the dose-effect relationship was obtained. The maximum inhibitory concentration of crude Polysaccharide for 24 hours all above 70%. IC50 for U251 cells: 24.06μg/mL,71.33μg/mL and 41.68; IC50 for Hela cells: 52.3μg/mL,66.39μg/mL and 51.87μg/mL. Volatile Oil inhibited cell proliferation on Hela cells ,IC50 :244.7μg/mL.5 The results of flow cytometry showed that Polysaccharide F3 500μg/mL concentration caused 50.18% of U251 cells apoptosis. There is a significant difference between the control group and experiment group.6 The results of agarose gel electrophoresis showed that volatile oil of water caltrop can induce apoptosis to Hela cells. Conclusion:The results showed:1 The results proved that the crude polysaccharide inhibit the U251 cell proliferation, and induce apoptosis U251 cells.2 The results proved that the essential oil can induce apoptosis to Hela cells.3 An anti-tumor compound was obtained -- dibutyl phthalate. The chemical composition and pharmacological activity of preliminary study will provid the theoretical basis for the further development of water caltrop as an antineoplastic agent.
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