Chemical Composition,Activities Of Antioxidation And Anticancer Of Tetrastigma Hemsleyanum Diels Et Gilg

Tetrastigma hemsleyanum Diels et Gilg(also called ―Sanyeqing‖ in Chinese),a family member of Vitaceae and Tetrastigma obtectum Genus,is a well-known rare medicinal plant,which is distributed mainly in the southern China.As an popular Chinese herbal medicine,Tetrastigma hemsleyanum are widely used for the treatment of some diseases due to its anti-inflammatory,analgesic and antipyretic properties.Modern pharmacological research shows that Tetrastigma hemsleyanum possess various bioactivities such as anti-tumor,anti-inflammatory activities and hepatoprotective effect.However,the studies on phytochemicals,antioxidant and anti-tumor effects as well as the underlying mechanism of the Tetrastigma hemsleyanum are not comprehensive,and most of them are focused on its root(RT)rather than its leaves(THL).Up to now,there is no study reported the metabolites of the Tetrastigma hemsleyanum during the in vitro digestion,colonic fermentation and in rat after oral administration as well as the structure-function relationship between the metabolites and bioactivities.Thus,develop rapid,accurate and sensitive methods to analysis the major bioactives and metabolites,select the suitable chemical,celluar and animal model to study the bioactivies of Tetrastigma hemsleyanum and the corresponding mechanism,are very necessary.The objectives of the present project are to: investigate the antioxidant(DPPH,FRAP,ABTS)and antiproliferative activities(MTT assay)of Tetrastigma hemsleyanum extracts by different solvents(80% methanol,ethyl acetate and hexane);qualitative and quantitative analysis of the phytochemicals and major bioactives of Tetrastigma hemsleyanum by using a combination of chemical assays,LC-Qq Q-MS(liquid chromatography triple quadrupole tandem mass spectrometry)and LC-QTOF-MS(liquid chromatography orthogonal acceleration quadrupole time-of-flight mass spectrometry)techniques;explore the process of digestion,absorption and metabolism of Tetrastigma hemsleyanum based on in vitro digestion,colonic fermentation and animal model;investgate the in vivo antioxidant effects of Tetrastigma hemsleyanum in rat model;study on the in vitro anticancer activities of Tetrastigma hemsleyanum and the related molecular mechanisms of anti-proliferation and apoptosis by using human hepatoma cell Hep G2;investigate the effects and the underlying mechanism of Tetrastigma hemsleyanum in H22 tumor-bearing mice;optimize the extraction technology of total flavonoids from Tetrastigma hemsleyanum leaves by response surface methodology;find a kind of selenium-riched Tetrastigma hemsleyanum formula by the compatibility of other Chinese medicine.The major results obtained in this study are concluded as follows:1.The antioxidant(DPPH,FRAP,ABTS)and antiproliferative activities(MTT assay)of Tetrastigma hemsleyanum extracts by different solvents(80% methanol,ethyl acetate and hexane)were investigated by DPPH,FRAP,ABTS and MTT assays.Resluts showed that 80% methanol extract exhibited the highest antioxidant activities(DPPH: 2.76 mmol trolox/g DW,ABTS: 1.34 mmol trolox/g DW,FRAP: 1.15 mmol Fe SO4/g DW for the roots;DPPH: 3.32 mmol trolox/g DW,ABTS: 1.38 mmol trolox/g DW,FRAP: 1.85 mmol Fe SO4/g DW for the leaves).Thus,the 80% methanol extract may contain the bioactives and need further anaylsis.2.The phytochemicals of Tetrastigma hemsleyanum were identified and quantified by using a combination of chemical assays,LC-Qq Q-MS and LC-QTOF-MS techniques.A total of 58 compounds were identified or tentatively characterized in RT and 75 compounds were found in THL.Moreover,the positive correlation between the total phenolic/flavonoid contents and the antioxidant/ antiproliferative activities were found by regression analysis.Thus,nine phenolic compounds of RT and six phenolics of THL were quantified.Results showed that kaempferol-rutinoside(21.63 mg/g DW),rutin(11.17 mg/g DW),isoquercitrin(6.38 mg/g DW),catechin(5.78 mg/g DW)were the major bioactives in RT.Meanwhile,5-caffeoylquinic acid(38.47 mg/g DW),orientin(11.30 mg/g DW),isovitexin(10.94 mg/g DW),vitexin-2″-rhamnoside(10.21 mg/g DW),isoorientin(10.17 mg/g DW),vitexin(8.28 mg/g DW)were the main bioactives in THL.3.The changes of Tetrastigma hemsleyanum phenolics during the in vitro gastrointestinal digestion and colonic fermentation were investigated by a combination of chemical assays,LC-Qq Q-MS and LC-QTOF-MS techniques.Results showed that the Tetrastigma hemsleyanum phenolics were degraded and metabolized during the gastrointestinal digestion and colonic fermentation.4.The metabolites of Tetrastigma hemsleyanum in rat plasma and urine after oral administration were identified by UPLC-ESI-QTOF-MS/MS.A total of 20 absorbed compounds and 18 metabolites were found in RT-treated group,while 30 absorbed compounds and 26 metabolites were detected in THL-treated group.Results revealed that the bioactives of Tetrastigma hemsleyanum extracts could be absorbed and metabolized by rats.The main metabolic reactions of Tetrastigma hemsleyanum phenolics in rats were deglycosylation,hydroxylation,reduction,hydrolysis reaction,demethylation and loss of oxygen in phase I and sulfation,methylation,glucuronidation in phase II metabolism.5.The in vivo antioxidant activities of Tetrastigma hemsleyanum extracts were assessed by measuring MDA and GSH contents and activities of antioxidant enzymes SOD,GSH-Px and T-AOC in a aging rat model induced by D-galactose.It is found that Tetrastigma hemsleyanum extracts could significantly enhance the antioxidant effects in rat,reduce the MDA content,increase the T-AOC,SOD,GSH-Px level and rise the GSH content.6.The phenolics of THL could inhibite the proliferation of Hep G2 cell and induce apoptosis.The underlying mechanism of the anticancer effects of THL phenolics were mainly induced apoptosis by both of extrinsic death receptor pathway and intrinsic mitochondria pathway.Meanwhile,the MAPK pathway also involved the anticancer effects of THL phenolics.7.The phenolic-rich extracts of RT and THL(250 and 500 mg/kg BW)could significantly inhibit the growth of tumor transplanted in mice,the inhibitory rates were 23.53-45.52% for RT and 22.25-51.92% for THL,respectively.Moreover,the Tetrastigma hemsleyanum phenolics showed their anti-tumor effects by inducing apoptosis,improving the immune function,increasing the antioxidant effects and inhibiting tumor angiogenesis.Additionally,the related mechanism of Tetrastigma hemsleyanum phenolics was induced tumor cell apoptosis by regulating the expression of Bcl-2 family proteins and the activation of caspase family proteins,improved the immunity system by increasing T lymphocyte counts,CD4+/CD8+ ratio,NK cell levels,the serum TNF-α production and IL-2 levels,enhanced the antioxidant effects by improving the antioxidant enzyme activity and inhibiting lipid peroxidation.Furthermore,Tetrastigma hemsleyanum phenolics could suppresse tumor angiogenesis by inhibiting VEGF production.