Purification And Function Activity Of Flavonoids From Seed Shell Of Juglans Mandshurica

Juglans mandshurica is not only precious wood species but also medicinal herbs resource plants and possesses many kinds of bioactivities,especially antineoplastic activity.So far,some compounds including quinines,flavonoids,diaryl group hepta-hydrocarbons,polyphenols,erpenoid and volatile oils and so on have been separated from the bark,leave,root and green peel of Juglans mandshurica.The research systematically focused on the separation,purification,safety and bioactivities of flavonoids from seed shell of Juglans mandshurica(FSSJM),which were produced as garbage during processing kernel in the food industry.The results were as follows.The Box-Behnken Design(BBD)combined with response surface methodology(RSM)was used as flavonoids yield as response value.The optimal conditions of ultrasonic-assisted extraction for total flavonoids from seed shell of Juglans mandshurica were obtained as follows:ultrasonic power was 250 W,ultrasonic time was 31 min and solid-liquid ratio was 1:31(g/mL).The flavonoids yield was 6.62 mg/g under optimal conditions.HPD-600 macroporous resin was selected for purifying FSSJM through static adsorption and desorption performance test of 6 kinds of macroporous resins.By kinetic absorption and desorption performance analysis of HPD-600 macroporous resin,the optimal conditions of purification for one time were as follows:absorption time 2 h,sample solution pH6,volume of sample 1/3BV,sample solution concentration of flavonoids 1.2 mg/mL,bed of macroporous resin were eluted using 4BV distilled water and 2BV 90%ethanol,recovery yield was 81.6%.And FSSJM-1 were obtained,the purity was increased from 18.6%to 37.7%.And 40%ethanol was selected for second time purification of flavonoids by HPD-600 macroporous resin,recovery yield of flavonoids was 85.9%.And FSSJM-2 were obtained,the purity were further reached 54.39%.Rutin and quercetin were determined in FSSJM-2 through High Performance Liquid Chromatography analysis.The antioxidant activity of FSSJM-1 and FSSJM-2 were investigated using spectrophotometry in different oxidizing reaction systems.Besides metal ion chelate,FSSJM-1 could increase the scavenging capability of ABTS,DPPH and hydroxyl radicals compared with the crude extraction and BHT.Compared with Trolox and VC,FSSJM-2 showed the better scavenging capability of ABTS,DPPH,hydroxyl radicals and lipid peroxidation inhibition effect,there was a dose-effect relationship.It was excited that the lipid peroxidation inhibition of FSSJM-2 was better than VC.The safety of FSSJM-2 was assessed in vivo and in vitro through mouse acute toxicity,Ames and micronucleus of mouse bone marrow cell tests.The acute toxicity test showed that FSSJM-2 had not toxicity(LD50>16 g/kg).The mutation of Salmonella typhimurium TA98 and TA100 were not induced by FSSJM-2,and the micronucleus of polychromatic erythrocytes(MPCE)was not significantly increased by FSSJM-2 in mice.They demonstrated that FSSJM-2 had no mutagenicity and genetics toxicity.Through Ames test,MTT test and cell morphology observation,antimutagenic and antitumor activities of FSSJM-2 were investigated.FSSJM-2 could significantly inhibit the back mutation of TA98 and TA100 induced by MNNG,4NQO directly and B(a)P,Trp-P-1 indirectly and displayed a dose-effect relationship.In addition,FSSJM-2 could also significantly inhibit the proliferation of HepG-2B,N87,MDA-MB-231and HepG-2 cells,and promote degenerative morphology changes in these four kinds of cells,and there was a dose-effect relationship.These results showed that FSSJM-2 possessed antimutagenic and antitumor activities.The mechanism of induced-apoptosis of FSSJM-2 was studied by MTT test,apoptotic morphology,flow cytometry and Westen Blot in HepG-2 cells.The results showed that FSSJM-2 could inhibit the proliferation of HepG-2 cell in a certain time-eff-ect manner,but not significantly inhibit the growth of normal liver cell(Chang Liver cell).These results demonstrated that FSSJM-2 could selectively inhibit proliferation in malignant tumor cells and not in normal cells.FSSJM-2 could change HepG-2 cell nuclear morphology,remarkably increased early apoptosis,late apoptotic cells ratio and ratio of Sub-Gl phase cells,and induce apoptosis of HepG-2 cells.FSSJM-2 could activate caspase-3 through decreasing Bcl-2 and increasing Bax proteins expression and then induce apoptosis of HepG-2 cells via mitochondria-dependent pathway in HepG-2 cells.FSSJM-2 could arrest HepG-2 cells at G0/G1 and G2 phases during inducing apoptosis.In conclusion,the results demonstrated that FSSJM-2 could significantly inhibit proliferation of HepG-2 cells through induced-apoptosis and arrested-cell cycle.