Research On The Protective Effects Of Stipuleanoside R₁?R₂ On Carbon Tetrachloride-induced Acute Liver Injury And The Change In Intestinal Flora

Panax stipuleanatus is root and rhizome of Panax stipuleanatus H.T.Tsai et K.M.Feng,a kind of ginseng plant of the acanthaceae,which has the effect of"dispersing blood stasis,relieving swelling and pain",and contains oleanane pentacyclic triterpenoid saponins.Panax stipuleanatus is a medicinal plant resource with high economic value,but its main pharmacological activity is not clear.Therefore,this study preliminarily investigated the biological activities of anticoagulant,analgesic and anti-inflammatory of stipuleanoside R₁ and stipuleanoside R₂,and further studied the protective effect of stipuleanoside R₁ and stipuleanoside R₂ on the carbon tetrachloride-induced acute liver injury in mice and the effect on the changes of intestinal flora in mouse feces.The main methods and results of this research are as follows:1.Preliminary study on the biological activities of stipuleanoside R₁?SP-R₁?and stipuleanoside R₂?SP-R₂?1.1 Anticoagulant effect of stipuleanoside R₁?R₂ in vitro.SP-R₁?SP-R₂significantly increased prothrombin time?PT?and activated partial thrombin time?APTT??p<0.01,p<0.001?,and had a dose-effect relationship in the range of 0.5²0mg/mL concentration.1.2 Analgesic effect of stipuleanoside R₁?R₂.SP-R₁?SP-R₂ can increase the threshold of pain,which indicating that SP-R₁?SP-R₂ have significant analgesic activity.1.3 Inhibitory effect of stipuleanoside R₁?R₂ on ear edema induced by xylene in mice.The inhibition ratios of SP-R₁ on ear edema at 1,5 and 10 mg/kg were 29.73%,18.18%and 21.38%,respectively.The inhibition ratios of SP-R₂ were 38.57%,30.63%and 35.63%,respectively,at doses of 1,5 and 10 mg/kg.The inhibitory effects of s SP-R₂ on ear edema in mice were more significant than SP-R_1.1.4 Inhibitory effect of stipuleanoside R₁?R₂ on paw edema induced by carrageenan in mice.SP-R₁?SP-R₂ can significantly inhibit the increase of TNF-??IL-6?MDA?NO in paw tissues?p<0.05,p<0.01,p<0.001?,and reduce the damage of inflammation to paw tissues.Histological analysis showed that SP-R₁?SP-R₂ reduced the paw edema caused by carrageenan,tissue damage,and significantly reduced the infiltration of inflammatory cells in connective tissue.1.5 Protective effect of stipuleanoside R₁?R₂ on acute liver injury induced by carbon tetrachloride?CCl₄?in mice.SP-R₁?SP-R₂?1,10 mg/kg?could significantly reduce the activity of alanine aminotransferase?ALT??p<0.05,p<0.01?;SP-R₁?SP-R₂ significantly reduced the activity of aspartate aminotransferase?AST?at 1,5 and10 mg/kg?p<0.05,p<0.01?.2.Protective effect of stipuleanoside R₁?R₂ against CCl₄-induced acute liver injury in mice2.1 Stipuleanoside R₁?R₂ decreased the levels of tumor necrosis factor?TNF-??,interleukin-1??IL-1??and interleukin-6?IL-6?.SP-R₁?SP-R₂ significantly inhibited the levels of TNF-?and IL-1?at doses of 1,5,and 10 mg/kg?p<0.05,p<0.01,p<0.001?.SP-R₁?SP-R₂ both inhibited the level of IL-6,and SP-R₂ significantly inhibited IL-6at dose of 10 mg/kg?p<0.01?.2.2 Inhibited oxidative stress and improved the antioxidant capacity.SP-R₁?SP-R₂ significantly inhibited the release of MDA at doses of 1,5 and 10 mg/kg?p<0.001?.Meanwhile,SP-R₁?SP-R₂ can promote the release of glutathione?GSH?and superoxide dismutase?SOD?,increase the resistance to oxidation and the ability of scavenging free radicals,SP-R₁ at doses of 1,10 mg/kg,and SP-R₂ at dose of 1 mg/kg could significantly promote the release of GSH?p<0.05?,while SP-R₁ at dose of 1mg/kg,SP-R₂ at dose of 5 mg/kg could significantly increase SOD activity?p<0.05,p<0.05?.2.3 Western Blot analysis showed that SP-R₁?SP-R₂ could up-regulate the expression of I?B?and down-regulate the expression of NF-?B.2.4 Histological analysis.SP-R₁?SP-R₂ can restore the normal function and morphology of liver cells and reduce the degree of cell necrosis,among which SP-R₂has a best protective effect on liver cells.3.Effect of stipuleanoside R₂ on the changes of intestinal flora in mice with CCl₄-induced acute liver injury.SP-R₂ could improve the diversity and richness of fecal intestinal flora in mice with acute liver injury induced by CCl₄.The species composition of faecal intestinal flora of mice with acute liver injury induced by CCl ₄was changed at the level of phylum,class and order to make it similar to the control group.According to the PCoA analysis based on weighted Unifrac,the colony structure of SP-R₂ group was closer to the control group than the model group.LEfse analysis revealed the groups with the largest difference in efficacy among groups.