Separation,In-vitro Antioxidant And K562 Cells Inhibition With Ingredients From Solanum Nigrum L.Berries

Solanum nigrum L.,which belong to Angiophyta,Dicotyledoneae,Solanaceae,Solanum.Solanum nigrum L.contains a variety of active ingredients,with antioxidant,anti-cancer,anti-inflammatory,liver protection,improving immunity,other physiological activity and possessing high development value.The extraction technology of active ingredients from Solanum nigrum L.berries were optimized;The antioxidant capacity of different active ingredients and their inhibitory effect on K562 cells were studied;The secondary purification process of Solanum nigrum L.saponins were optimized;The inhibition mechanism of Solanum nigrum L.saponins on K562 cells was explored.（1）The extraction process of active ingredients of Solanum nigrum L.were optimized by the single-factor test,the full factorial test design,steepest ascent and the central composite design with response surface method.The yield of alkaloids reached（0.824±0.001）mg/g by using 80%ethanol at 57.5℃and the ratio of material to liquid 1:20.5 g/mL to extract 4 h.The yield of saponins reached（8.253±0.270）mg/g by using 20%ethanol at 90.6℃and the ratio of material to liquid 1:33.5 g/mL to extract 2 h.The yield of polysaccharide reached（24.27±0.21）%by using distilled water at 59.14℃and the ratio of material to liquid 1:8 g/mL to extract 2.15 h.（2）The purification process of active ingredient of Solanum nigrum L.were optimized by screening resin and the single-factor test.AB-8 resins were used,loading concentration 0.03mg/mL,loading pH 9,diameter to length ratio 1∶10,maximum loading volume of 0.6 BV,elution with 3.0 BV pH 3 70%ethanol and elution flow rate of 2.0 B/h,and the purity of alkaloids could increase 9.44 times.AB-8 resins were used,loading concentration 0.4 mg/mL,loading pH 7,diameter to length ratio 1∶25,maximum loading volume of 0.625 BV,elution with 3.0 BV pH 5 100%ethanol and elution flow rate of 2.0 BV/h,which increased the purity of saponins 2.93 times.NKA-Ⅱresins were used,loading concentration 20 mg/mL,diameter to length ratio 1∶30,the maximum loading volume of 0.54 BV,elution with 3.5 BV pH 7 60%ethanol,the elution flow rate of 2.0 BV/h,and the flavonoids purity increased 7.60 times.2%papain was used and the proteins were removed from the crude polysaccharides of Solanum nigrum L.After 2 h enzymolysis at pH 7 and 60℃,the purity of polysaccharides were further enhanced by precipitation with 90%ethanol at a concentration of 1∶3.（3）To compare the antioxidative capacity of the active ingredients of Solanum nigrum L,scavenging DPPH·and HO·activity,lipid peroxidation inhibitory activity and total reducing power as the indexes,the antioxidant capacity of each active ingredients from strong to weak were as follows:anthocyanins>alkaloids>flavonoids>saponins>polysaccharides,and all showed concentration-dose dependence.（4）The correlation between the antioxidative capacity of Solanum nigrum L.saponins and the inhibitory ability on chronic leukemia K562 cells were studied.There was a significant positive correlation between the inhibition rate on K562 cells and the antioxidative capacity,especially,the correlation between the inhibitory ability on K562 cells and scavenging HO·was significantly positive（P<0.01）.（5）The secondary purification process of Solanum nigrum L.saponins was optimized by single factor test.30⁶0 mesh polyamide resins were used,loading concentration 0.2 mg/mL,loading pH 7,diameter to length ratio 1∶30,the maximum loading volume of 0.9375 BV,3.0BV pH 6 distilled water,20%,40%,60%ethanol gradient elution successively,and the elution flow rate of 2.0 BV/h,under these conditions,the purity of saponins reached 75.99%.（6）The inhibitory mechanism on K562 cells were investigated.Solanum nigrum L.saponins had strong inhibitory effect on K562 cells and showed dose-response dependence at low dose.saponins could induce K562 cell apoptosis by blocking cell cycle in G₂/M phase.