| Duchesnea indica(Andr.)Focke are used as Chinese herbal medicine.The whole plant is used for medicinal purposes.It has the functions of dispersing blood stasis and detumescence,astringent and hemostasis,clearing away heat and detoxification.Some studies have shown that Duchesnea indica has activities of antitumor,anti-inflammatory,antibacterial,antioxidation and others.Duchesnea indica has numerous effective ingredients including terpenoids,flavonoids,phenolic acid and phenolic acid lipids,sterols,polysaccharides,tocopherols,etc.,It was reported that this herbal shows some prospective active for prevention and treatment on some infectious diseases induced by some bacteria,parasites or virus in livestock and birds.However,there are few of reports on comparative researches of active function between the different extracts,and it is still unclear that the pharmacological role of its polysaccharide in RAW264.7 cells induced by LPS.In this study,the main chemical composition,antibacterial activity,antioxidant activity of Duchesnea indica different extracts,and anti-inflammatory activity of Duchesnea indica polysaccharide in LPS-induced mouse peritoneal macrophages(RW264.7)and its potential molecular mechanism were investigated.Experiment 1:Four alcohol extracts were obtained by reflux extraction with 60%,70%,80%and100%ethanol at 85℃respectively and the yield differences were not significant.The contents of total polysaccharide,reducing sugar,total flavonoids and total polyphenols of 100%ethanol extract were significantly lower than those of other alcohol extracts(P<0.05).The three aqueous extracts were obtained at 70℃,85℃and boiling water and the yield differences were not significant.The contents of total polysaccharides,reducing sugars and total polyphenols of aqueous extract extracted under boiling water condition was higher than those of water extracts at other temperature conditions.Duchesnea indica(Andr.)Focke polysaccharide was prepared by water extraction and alcohol precipitation.The results showed that the total polysaccharide content was 32.62±0.91%,the reducing sugar content was13.41±0.18%,the total flavonoids content was 1.07±0.07%and the total polyphenols content was12.16±0.27%.Experiment 2:The antibacterial activities of alcohol extract,aqueous extract and polysaccharide from Duchesnea indica(Andr.)Focke were determined by Oxford cup method and MIC method.Antioxidant activities of alcohol extract,aqueous extract and polysaccharide were determined by FRAP,ABTS,DPPH and superoxide anion radical scavenging method.The results showed that the polysaccharide of Duchesnea indica had no antibacterial activity,the alcohol extract of Duchesnea indica had antibacterial activity against 4 strains of Gram-positive bacteria and 3 strains of Gram-negative bacteria,and the aqueous extract of Duchesnea indica had antibacterial activity against 3strains of Gram-positive bacteria and 3 strains of Gram-negative bacteria.The ABTS and DPPH free radical scavenging rates of polysaccharide were significantly higher than those of alcohol extract and aqueous extract(P<0.05),and this active effect was similar to vitamin C.The total antioxidant capacity(FRAP)of alcohol extract,aqueous extract and polysaccharide were not significant difference,and there was significant lower than vitamin C or BHA(P<0.05).The superoxide anion radical scavenging rate of alcohol extract,aqueous extract and polysaccharide were no significant difference.Experiment 3:CCK-8 method was used to determine the working concentration of the polysaccharide on RAW264.7 cells.The NO content,MDA content and total SOD activity in the supernatant of cells treated with Duchesnea indica polysaccharide and LPS for 24 hours were determined.The m RNA levels of inflammatory mediators i NOS,COX-2,TNF-α,IL-6 and IL-1βwere determined by q PCR,and the protein levels of NF-κB-p65,ERK,p-ERK,p38 and p-p38 were detected by Western blot.The results showed that Duchesnea indica polysaccharide of 400μg/m L and dexamethasone of 100 ng/mL can significantly inhibit the increase of NO and MDA contents induced by LPS(P<0.05),and significantly improved the change of SOD activity.Duchesnea indica polysaccharides significantly inhibit the m RNA expression of i NOS,COX-2,TNF-α,IL-6 and IL-1βin cells induced by LPS,and the expression of NF-κB-p65,p-ERK,p-p38 proteins in cells induced by LPS.In conclusion,there was no significant difference in the yield and the contents of main chemical components of the alcohol extracts from Duchesnea indica prepared by 60-80%ethanol.The parameters including the yield and main chemical components of aqueous extract prepared under boiling water condition were better than those prepared at 70℃and 85℃.The polysaccharide of Duchesnea indica had no antibacterial activity in vitro,and the alcohol and aqueous extracts of Duchesnea indica had antibacterial effect on Cryptobacterium pyogenes and other G~+bacteria and salmonella and other G~-bacteria.The antioxidant activity of Duchesnea indica polysaccharide in vitro was better than that of Duchesnea indica alcohol extract and aqueous extracts,which could improve the LPS-induced decrease in the antioxidant capacity of mouse peritoneal macrophages.Duchesnea indica polysaccharide may play an anti-inflammatory role by regulating the overexpression of pro-inflammatory factors such as IL-6,IL-1βand TNF-αthrough NF-κB/ERK/p38 signaling pathway. |
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