| Objective:Mainly use modern scientific research methods of traditional Chinese medicine,in vitro and flow cytometry,fluorescent staining,q RT-PCR,Western Blot,etc.and biological experiment techniques to explore the extraction process and in vitro antioxidant capacity of Tetrastigma hemsleyanum polysaccharides(THP).And preliminary study of its effect on human ovarian adenocarcinoma cells SKOV3(hereinafter referred to as SKOV3 cells),to provide a rationale for further effects of new and anti-human related diseases.Methods:(1)THP was extracted by hot water method,and L9(3~4)orthogonal experiment was conducted on the basis of single factor experiment to optimize the extraction process parameters and determine the optimal extraction conditions.The polysaccharide was purified by AB-8 macroporous resin.(2)The antioxidant capacity of THP in vitro was studied by investigating the total antioxidant capacity,reducing power,inhibition rate of superoxide anion radical,scavenging rate of DPPH radical and inhibition rate of hydroxyl radical.(3)The effect of THP on the proliferation of SKOV3 cell was detected by CCK-8 technique;the effect of THP on the morphology of SKOV3 cell was observed by inverted microscope;the effect of THP on the apoptosis rate of SKOV3 cell was detected by flow cytometry PI single staining method;The effect of THP on apoptosis of SKOV3 cell was observed by Hoechst 33342 and AO-EB staining;The expression of apoptosis-related genes and apoptosis-related proteins in SKOV3 cell was detected by q RT-PCR and Western blot method.Result:(1)The optimal extraction technology of THP:solid-liquid ratio1:35(g/m L),extraction times 1,extraction time 1.5 h,extraction temperature90℃.Under the optimal extraction conditions,the extraction rate of polysaccharide was 7.34%.The purity of polysaccharide was 61.47%after purification with AB-8 macroporous resin.(2)The results of anti-oxidation test in vitro showed that the total anti-oxidation activity of the polysaccharide was VC>THP>LBP≥APS in the concentration range of 0.1～1 mg/m L;the results of anti-oxidation were BHT>THP>APS≥LBP;the inhibition rate of superoxide anion radical was VC>THP>LBP>APS;the inhibition rate of DPPH radical was LBP≥APS>THP>BHT.In the range of 0.05～0.4 mg/m L,the inhibition rate of hydroxyl radical was VC>THP>APS≥LBP.(3)The CCK-8 method test results showed that after 24,48,and 72 hours of the action on SKOV3 cells,the inhibition rate of each concentration group reached(0.00±0.00)%、(7.90±0.30)%、(13.8±2.81)%、(22.1±0.26)%,、(34.1±0.40)%、(47.4±0.87)%,(0.00±0.00)%、(13.0±0.82)%、(21.2±1.30)%、(31.7±0.40)%、(45.1±2.66)%、(67.4±0.21)%,(0.00±0.00)%、(15.6±0.25)%、(25.1±0.25)%、(35.6±0.59)%、(50.8±0.26)%、(71.1±0.36)%;the results of inverted microscope morphologically prove that the THP has an inhibitory effect on the proliferation of SKOV3 cells;the results of PI testing,G0/G1 in the blank control group and the low,medium,and high concentration groups The cell proportions of S,G2/M phases are(72.07±2.21)%、(21.54±1.04)%、(6.39±2.67)%;(66.41±1.14)%、(28.55±3.16)%、(5.04±3.04)%;(63.25±1.06)%、(31.05±1.60)%、(5.70±2.09)%,(58.61±1.09)%、(35.47±0.85)%、(5.89±0.85)%;Annexin V-FITC/PI In the double staining method,the early,late and total apoptosis rates of the blank control group and the low,medium and high concentration groups were(1.28±0.11)%、(2.09±0.10)%、(3.37±0.19)%,(2.46±0.64)%、(3.63±0.58)%、(6.09±1.17)%,(3.99±0.32)%、(4.61±0.56)%、(8.60±0.46)%,(5.12±0.39)%、(6.80±0.15)%、(11.92±0.26)%;Hoechst 33342and AO/EB showed that SKOV3 cells appeared granular bright blue and pyknotic orange-red deep staining;The results of q RT-PCR showed that THP could up-regulate the expression of Bax and Caspase-3 m RNA in SKOV3 cells and down-regulate the expression of Bcl-2 and Survivin m RNA(P<0.05);Western blot The experiment showed that THP could up-regulate the expression of Bax and Caspase-3 protein and down-regulate the expression of Bcl-2 and Survivin protein(P<0.05).Conclusion:Under the optimum conditions of 1:35(g/m L)ratio of solid to liquid,extraction time 1.5h,extraction temperature 90°C,the extraction rate of polysaccharide was 7.34%,the purity of polysaccharide was 61.47%after purification by AB-8 macroporous resin.THP antioxidant capacity in vitro,which could inhibit the proliferation of human ovarian adenocarcinoma cell SKOV3,block it in S phase,and promote its apoptosis.The mechanism of apoptosis might be related to up-regulation of Bax,Caspase-3 expression and down-regulation of Bcl-2 and Survivin expression. |
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