Pharmaceutical And Anti-tumor Mechanism Research Of Arachniodes Exilis Ching

Arachniodes exilis Ching is mainly distributed in Yangze River surrounding areas of China, which belongs to the Arachniodes Dryopteridaceae. So far we can see little of reports about the chemical constituents and bioactivity of Arachniodes exilis Ching. In the interest of exploreing the folk medicinal resource and investigating its bioactivity, the exploration on the identification of crude drug, the extraction and purification technology, anti-tumor activity in vitro and in vivo of the crude flavonoids from Arachniodes exilis Ching had been studied. Furtherly, its molecular mechanism of anti-tumor activity was also evaluated. Meanwhile, the quality standard of Arachniodes exilis Ching was established.In the identification studies, the source, character, microscopic, chemical composition systems pretest and UV absorption characteristics and other aspects of Arachniodes exilis Ching had been identified. According to the results of microscopic identification, the organizational structure of the petiole cross-section was special and had better scientific significance for the identification of the Arachniodes exilis Ching. The results of system pre-test Chemical composition showed that there were flavonoids, phenolic compounds, terpenoids, sugar and other ingredients in Arachniodes exilis Ching. On the contral, there were no saponins and alkaloids. And UV scanning lines of the different parts of the polar solvent extraction had been used to express their characteristics combined by using Single UV Spectral Method.The preparation method by using Ultrasonic-assisted extraction method and Orthogonal experiment had been established. So was its purification method of total flavonoids by Polyamide resin purification method. The best extraction technology:60%ethanol as the extraction solvent,1:20solid-liquid ratio, ultrasonic extraction30minutes with three times. The best purification technology:the sample solution:pH value is5, the concentration of sample solution is2.0mg/ml, using70%ethanol as eluent; the elution ratr is2.0BV/h.In the comparative study of the anti-tumor activity of total extract and crude flavonoids from Arachniodes exilis Ching via observing under inverted phase contrast microscope and CCK-8colorimetric method in vitro, the results showed that crude flavonoids had better anti-tumor activity than total extract from Arachniodes exilis Ching in HepG2and A-549cell. Moreover, the potential mechanism of cytotoxic effect had been evaluated on HepG2cell induction by the total flavonoids from Arachniodes exilis Ching in HepG2cells. On the basis of flow cytometry, fluorescence microscope and Western blot methods, the results showed that crude flavonoids from Arachniodes exilis Ching induced apoptosis in HepG2cells resulting in inhibition, and induced the increased level of intracellular ROS and the loss of Δψm. Western blot assay showed that the rude flavonoids increased cytochrome c release, activated caspase-3,-9and significantly down-regulated the ratio of Bcl-2/Bax. Further analysis showed that the rude flavonoids stimulated the mitogen activated protein kinase (MAPK). However, treatment with NAC (reactive oxygen species scavenger) and MAPK-specific inhibitors (SP600125and SB203580) could reverse the changes of these apoptotic-related proteins. All results suggested that crude flavonoids from Arachniodes exilis Ching remarkably inhibited the growth of HepG2cells through ROS-mediated mitochondrial dysfunction involving MAPK pathway.In the study of the anti-tumor activities of the crude flavonoids from Arachniodes exilis Ching in vivo, the human hepatocellular tumor xenograft model had been used to establish with HepG2cell line. On the basis of analysis of the inhibiting ratio of tumor growth, biochemical indicators detection, HE staining method, immunohistochemistry and western blot analysis, it was showed that the treatment of the crude flavonoids from Arachniodes exilis Ching could considerably inhibited the growth of HepG2tumor in tumor-bearing model nude mice without significant toxicity. The mechanism of antitumor activity may be related to induction of apoptosis of tumor cells and inhibition of tumor tissue angiogenesis.Besides, the quality standard of Arachniodes exilis Ching had been established by analyzing the characteristics, identification method, content determination, the extract, total ash and moisture content of the herb on the basis of2010China Pharmacopoeia.