Study On The Chemical Constituents And Anti-tumor Activity Of Macrothelypteris Viridifrons (Tagawa)Ching

Macrothelypteris viridifrons (Tagawa) Ching is widely distributed in south of China, which belongs to the genus Macrothelypteris and has been frequently used as a folk medicine for the effects of heat-cleaning and detoxification, the treatment of diseases such as hydropsy and traumatic bleeding. Up to now, there was little report about the chemical constituents and bioactivity of M. viridifrons. In order to explore this folk medicinal resource and research its bioactivity, we did the investigation on the extraction and purification technology, chemical constituents, anti-tumor activity in vitro and in vivo of the crude flavonoids from M. viridifrons. Moreover, the effect on inducing apoptosis of a novel non-aromatic B-ring flavonoid (DHEC) and its putative molecular mechanism of action were evaluated. Meanwhile, the quality standard of M. viridifrons was established.We investigated the extraction and purification technology of the crude flavonoids from M. viridifrons and established its preparation method via the merge alkali-soluble and acid-isolation with macroporous resin, so that the final flavonoids yield was not less than56.57%, the content of flavonoids was not less than53.37%. The best extraction process:using70%ethanol,1:20solid-liquid ratio, reflux extraction60min with twice at80℃; The best purification process:alkali-soluble and acid-isolation method (the best alkali-soluble PH=7, the best acid-isolation PH=1-2), macroporous resin method (the best resin: HPD500, the best PH of sample solution:5, the best concentration of sample solution:4.0mg/mL, the best adsorption velocity:2.0BV/h, the best eluent:70%ethanol; the best elution volume:3.0BV, the best elution velocity:2.0BV/h)We comparatively studied the anti-tumor activities of total extract and crude flavonoids from M. viridifron by MTT colorimetric method in vitro and S180sarcoma mouse model in vivo. Research results showed the better anti-tumor activity of crude flavonoids from M. viridifrons. Furthermore, we deeply analyzed the in vitro anti-tumor activity of crude flavonoids from M. viridifrons with MTT method for detecting cell proliferation, PI method for detecting cell cycle, Annexin V-FITC/PI method and Hoechst33258method for detecting cell apoptosis in human hepatoma HepG2cell, and its in vivo anti-tumor effect with the inhibiting ratio of tumor growth, the detection of biochemical indicators, HE dyeing method, immunohistochemical method in H22hepatoma mouse model. Research results showed that crude flavonoids from M. viridifrons could induce apoptosis and G2/M phase arrest in HepG2cell, and could block the growth of tumor and inhibit the formation of blood vessels in H22hepatoma mouse model. All together, crude flavonoids from M. viridifrons exhibits potential anti-tumor activity in vitro and in vivo, which may highly be associated with the effect on induction of apoptosis and its anti-angiogenic activity.On the basis of normal and reverse phase silica gel and Sephadex LH-20gel column chromatography, nineteen flavonoids (containing nine non-aromatic B-ring flavonoids and one new compound) were isolated from the rhizomes of M. viridifrons and identified as5,7-dihydroxychromone(1);5,7-dihydroxy-2-(1-hydroxy-2,6-dimethoxy-4-oxo-cyclohex)-chromen-4-one(2);5,7-dihydroxy-2-(1,2-isopropyldioxy-4-oxocyclohex-5-enyl)-chromen-4-one(3); protoapigenone(4); apigenin(5); narigenin (6); kaempferol(7);2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5-hydroxy-7-ethoxy-chromone(8);2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5,7-dihydroxy-chromone (9); quercetin(10);2-(trans-1,4-dihydroxy-cyclohexyl)-5,7-dihydroxy-chromone(11); protoapigenin(12); apigenin-4'-O-glucoside(13); narigenin-4'-O-glucoside(14); kaempferol-3-O-glucoside(15);5,7-dihydroxy-2-(1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-chromone-4'-O-glucoside(16); protoapigenin-4'-O-glucoside(17); kaempferol-3-O-rutinose(18); quercetin-3-O-rutinose(19). All compounds were firstly obtained from this plant.Moreover, on the basis of MTT assay, flow cytometry, microscope and Western blot methods, the effect of2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5-hydroxy-7-ethoxy-chromone (DHEC) on induction of apoptosis and its putative molecular mechanism of action in human colon HT-29cancer cell were evaluated. After treatment of HT-29cell with DHEC, we observed the inhibition of proliferation, the happen of apoptosis, the loss of MMP, the accumulation of intracellular ROS, the releasing of cytochrome c, the cleavage of PARP, the activation of caspase-3,-8and-9, the increase of expression of Bax and Bad, the decrease of expression of Bcl-2, Bcl-xl, and the phosphorylation of MAPK members (ERK, JNK, P38MAPK). All results suggest that DHEC exhibits potential anti-tumor activity in HT-29cell through induction of apoptosis, which may highly be associated with ROS-mitochondrial dysfunction and involving of caspases family, as well as activation of MAPK signaling pathway.Besides, on the basis of2010China Pharmacopoeia, we analyzed the traits and microscopic characteristics, the identification method, the content determination, the extract, total ash and moisture content. Thus, the quality standard of M. viridifrons was established.