The Extraction And Separation Of Flavone In Dongzao Jujube As Well As The Study Of Their Anti-oxidation And Anti-tumor Activities

Phytochemicals,especially flavonoids,in fruits and vegetables are thought to be the major bioactive compounds beneficial for human health.Flavonoids,originated in fruits,vegetables and tea,were shown to have antioxidant activity,antianaphylaxis, bacteriostasis and inhibiting cancer cell proliferation,which may therefore play a key role in reducing chronic diseases.In recent years,accumulating evidence show that treatment with a low concentration of flavanone or 2-OH flavanone can improve the efficacy of doxorubicin.Flavanone and 2-OH flavanone may have an in vivo inhibitory effect on the lung tumor development without any apparent sign of toxicity primarily based on the data of body weight.There are abundant flavonoids in Dongzao Jujube,but few anti-tumor researches have been done using Dongzao jujube before.This study was designed to optimize the extraction and purification conditions of flavonoids in Dongzao jujube(DJF),to study it's antioxidant effect and possible mechanisms,as well as to investigate the inhibition of DJF on S180 sarcoma,to explore therapeutic and commercial potentials of Dongzao jujube.The main research content and results are as follows:1.Using ethanol as the extractant,through the single test of oscillation by heating extraction,ultrasonic-assisted extraction and microwave-assisted extraction methods, main impact factors of the three methods were determined,respectively.And then the extraction condition of each method was optimized by using orthogonal test.The results showed that 75%of the ethanol solutions under the microwave power at 468W each extract 20s and extracted 9 times was the optimized condition for obtaining DJF with the extraction ratio of DJF about 1.46%.2.By comparing the five kinds of macroporous resin adsorption and desorption ability for DJF,the optimal separation and purification condition were determined. Results show that X-5 macroporous resin was the most appropriate resin in all for the preliminary purification of DJF.The best technology of preliminary purification using X-5 macroporous resin is as follows:first 2-times the column volume of 50%ethanol solution to elute and then 1.5-times the column volume of 70%ethanol solution for gradient elution.Under the optimized process conditions,the elution rate is 82.42%.3.The capacity of DJF scavenging DPPH·,superoxide radical(O·₂^-) and·OH of DJF were evaluated with DPPH·system,O·₂^- system and·OH system.The inhibition ability of DJF to lipid oxidation and serum protein oxidative modificated ability were evaluated with the ferric chloride-ascorbic acid system and the Cu2+ catalyst system. Results indicate that different solvent extracts of DJF have effect of scavenging DPPH·,O·₂^- and·OH,showing good dose-effect relationship at a certain concentration range.The ethanol extract(ECF),ethyl acetate extract(ETF) and extracted more than water-soluble material(WF) can effectively inhibit the Fe²⁺-induced membrane analog soybean phospholipid liposome peroxidation, reducing the generation of malondialdehyde,showing good dose-response relationship.ECF,ETF,WF can effectively postpone the launch of oxidized lipoprotein time,inhibit oxidation process.Results showed that DJF could scavenge reactive oxygen species and inhibit lipid peroxidation in some modified chemical systems.The capabilities of quenching free radicals and reduction or chelate iron ion and copper ion linked with oxidation were revealed to be the mechanism of inhibiting oxidation of DJF.4.Dongzao jujube flavonoids' antioxidant activity in vitro was evaluated with HaOa-induced oxidation of model.MDA content and GSH-Px,ATP-ase of vitality were determined with malondialdehyde(MDA) Kit,GSH-Px Kit and ATP-ase Kit. RBC hemolysis and liver mitochondrial swelling of the mice was determined with Spectrophotometry.Effects of DJF on blood lymphocyte DNA,and VECs of oxidative damage were measured with Comet assay,cell culture method separately. The results showed that DJF could inhibit H₂O₂-induced blood lymphocyte DNA, VECs oxidative damage,inhibit the swelling of mice liver mitochondria and the hemolysis of mice RBC induced by H₂O₂ as well.DJF could also heighten the GSH-Px,Na⁺—K⁺—ATPase,Ca²⁺—Mg⁺—ATPase of vitality,and inhibit the formation of MDA in mice liver homogenate induced by Fe²⁺+ H₂O₂.These results showed that DJF has the antioxidant effect on mice/rat,cell and tissue levels in vitro.5.S₁₈₀ tumor-bearing mice models were established to explore the inhibitory effects of the DJF and its possible mechanism.Sarcoma weight showed that DJF could inhibit S₁₈₀ sarcoma growth in vivo and the inhibiting rate of 100-400 mg/Kg·d DJF were between 20.23%and 30.01%.DJF may promote tumor-bearing mice to promote apoptosis of tumor tissue gene expression of Bax.At the same time,DIF can reduce the inhibitor of apoptosis gene Bcl-2 expression,increased Bax and Bcl-2 ratio, thereby promoting tumor cell apoptosis.DJF can enhance the spleen index,thymus index,serum IL-2 content,and increase splenocyte proliferation of tumor-bearing mice.6.Horn method was used to measure the DJF half lethal dose(LD50).Results show that DJF does not have evident toxicity with its LD50 more than 5000mg·Kg^-1.DJF is safe.It can inhibit the growth of S₁₈₀ sarcom.The anti-tumor effect of DJF to S₁₈₀ tumor-bearing mice related to promoting tumor cell apoptosis,significantly strengthening the immune system and antioxidant activities.