| In this paper,natural plant-derived Lycium ruthenicum Murr.was selected as the main research object.Currently,reported studies on the active componen ts of LR.focus on protein,LR polysaccharide,polyphenols,anthocyanins,flavo noids,trace elements and other natural components and various nutrients.Studies on its functions mostly focus on hypoglycemic,anti-oxidation,hypidemia,eye protection,anti-cancer,intestinal protection,anti-aging,fatigue,depression and other aspects.There are no relevant reports on antibacterial activity and mecha nism of action.As for anthocyanins with excellent antioxidant effect,its antiox idant mechanism is not detailed.In addition,there are still many problems in a nthocyanin extraction,such as environmental protection,low extraction rate and high cost.Response surface optimization（RSM）was used to extract anthocya nins from LR.and explore its antibacterial and antioxidant mechanism to broad en its application pathway,providing theoretical basis for the further developme nt of medicinal products,food and cosmetics.The main research contents and results are as follows:（1）Extraction of LRA.by ultrasonic microwave;In this study,the optimal extraction conditions were determined by single factor and response surface de sign:ultrasonic power 216 W,microwave power 89 W,time 26 min,liquid-so lid ratio 17 m L/g.Under these conditions,the predicted content of the model was 10.157mg/g,and the actual value was basically consistent with the predict ed value.AB-8 was used to separate and purify crude anthocyanins with macroporo us resin.The optimal purification conditions were determined as follows:loadin g concentration 5 mg/m L,loading flow rate 0.5 b V/h,eluent 1%acidic distill ed water and 1%acidic 70%ethanol,elution flow rate 2 b V/h.Under these c onditions,the purified sample is purple-black powder,and the color value of t he purified sample is 214,which is about 4 times higher than that of the puri fied sample.（2）Antibacterial action and mechanism of anthocyanins of Lycium barbaru m l.:In this study,staphylococcus aureus,Escherichia coli,Aspergillus Niger and Penicillium were used as experimental strains,and MIC of purified and c rude extracts of LRA to bacteria and fungi were determined by micro-broth dil ution method and constant broth dilution method.Overall,the antibacterial effect was S.aureus>E.coli>Penicillium>Aspergillus Niger,and the anthocyanin purified product had the best antibacterial effect on S.ureus,with MIC of 3.125mg/m L.Anthocyanins had a dose-dependent effect on the growth of S.areus bacteria.When anthocyanins were treated with thalli,the hydrophobicity of thal li surface increased,indicating that anthocyanins affected thalli aggregation and thus affected thalli growth.The conductivity,leakage of small molecule（K+）a nd large molecule（protein）in the bacterial suspension were measured by cond uctivity method and BCA kit detection method.The results showed that anthoc yanins caused a large amount of potassium ion leakage in the bacterial cell an d increased the conductivity.After treated with MIC and 2MIC LRA for 9h,th e total soluble protein in the bacteria decreased by 5.5%and 25.7%,respective ly.SDS-PAGE showed that LRA could inhibit protein synthesis.The effect of LRA on cell membrane permeability was observed by inverted fluorescence mi croscope,and it was found that after MIC and 2MIC treatment,the fluorescenc e intensity increased significantly,indicating that LRA can destroy the cell me mbrane.The same results were obtained by flow cytometry for cell mortality.Wi th the increase of concentration,fluorescence intensity increased,the number of bacteria stained by PI increased,and the P3 region of dead bacteria increased to 77.21%.In conclusion,the antibacterial mechanism of anthocyanins is relate d to the change of membrane permeability and integrity.（3）S.aureus ATCC29213 was used to establish biofilm model of the expe rimental strain,which had good film-producing ability.MTT and crystal violet methods were used to study the scavenging ability of LRA on S.aureus biofil m increased with increasing concentration.The silver staining method observed that with the increase of concentration,the biofilm supported on the glass sur face was continuously removed and the black masses became fewer and looser.Scanning electron microscopy（SEM）observed that the complete biofilm was a mature and compact Three dimensional structure structure with smooth surfac e.After MIC treatment,the three-dimensional structure of the biofilm was dest royed and the structure became loose.After 2MIC treatment,the aggregation d egree became higher and higher,and the bacteria broke and a large number of contents leaked on the surface.（4）The scavenging ability and total reducing ability of LRA on DPPH·,·OH,O2ˉ·and ABTS·free radicals were investigated,and VC was used as co ntrol test.The results showed that the higher the anthocyanin concentration wa s,the stronger the antioxidant capacity was.The antioxidant level of anthocyan in in the range of 4 mg/m L was almost the same as that of VC in the same concentration.When the LRA concentration was less than 250μg/m L,the cell survival r ate was higher than 90%,and there was no toxicity to cells.Anthocyanin conc entration of 50,100 and 250μg/m L was selected as low,medium and high d ose groups.When HepG2 cells were treated with 850μM for 3 h,the surviva l rate of HepG2 cells was 50.96%.The survival rate of injured model cells in creased to 64.82%and 82.51%after treatment with medium and high doses of anthocyanin,indicating that anthocyanin can reverse the damage of H2O2to H ep G2 cells,and the higher the concentration,the greater the protection degree.In addition,anthocyanins could significantly reduce the activity of lactate dehy drogenase（LDH）and the content of malondialdehyde（MDA）,and increase the level of superoxide dismutase（SOD）in cells（P<0.05）.These results indicat ed that the anthocyanins of Lycium ruthenicum Murr.could improve the antiox idant activity of liver cells and protect the cells from oxidative damage. |
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