Study On The Biological Activity Of Ethanol Extract Of Physalis Calyx Seu Fructus

Objective:We have assessed the effect of the general ethanol extract from Physalis Calyx Seu Fructus(P)as well as its five major individual components:fraction-1(P1),fraction-2(P2),fraction-3(P3),fraction-4(P4)and fraction-5(P5)on anti-inflammatory activity in lipopolysaccharide(LPS)-stimulated RAW264.7 macrophages.In addition,we have also assessed their anti-Mycoplasma gallisepticum(MG)activity in vitro.Methods:The extract P was isolated from the dried calyces of Physalis Calyx Seu Fructus with 80%EtOH under reflux.The five individual fractions(P1,P2,P3,P4 and P5)were obtained using macroporous resin separation from the entired extract(P).HPLC were used to detect the luteoloside from the original Physalis Calyx Seu Fructus and extracted P,P1,P2,P3,P4 and P5.LPS-stimulated RAW264.7 cells were treated with P,P1,P2,P3,P4 and P5 respectively and then ELISA was used to detect the inhibitory effects of each extract compound on IL-6 and TNF-a.GRIESS was used to detect the secretion of NO.RT-PCR was used to detect the influence on gene expression of each compound on IL-6,TNF-a and iNOS.The minimum inhibitory concentration(MIC)and minimum mycoplasmacidal concentration(MMC)of P,P1,P2,P3,P4 and P5 against MG-F were determined in vitro by a broth microdilution method.Finally,using the determined MIC,we assessed the inhibitory effects of P,P2,P3,P4 and P5 on the growth of MG at MIC.Results:Luteoloside in Physalis Calyx Seu Fructus was detected as 0.1735%,which is h:igher than the minimal requirement of 0.10%according to the Chinese Veterinary Phannacopoeia.The detected luteoloside in P(1.39%)is 8 times of that in Physalis Calyx Seu Fructus.Luteoloside in P2 is the highest with 2.548%.2.IL-6,TNF-a and NO were affected following the treatment on LPS-stimulated RAW264.7 cells with P,P1,P2,P3,P4 and P5 respectively.P3 effectively inhibited the LPS-induced IL-6,TNF-a and NO secretion(P<0.05).The gene copy number of IL-6,TNF-a and iNOS decreased at maximum safe concentration(P<0.05).3.The MICs of P,P2 and P3 against MG-F were 0.625 mg/mL,1.25 mg/mL for P4,2.5 mg/mL for P5,0.3125 ?g/mL for Tylosin.P2 and P3 had the lowest MMC(5 mg/mL).The MMC of P and P4 was 10 mg/mL,0.625 ?g/mL for Tylosin.The MMC of P5 was 20 mg/mL.The growth of MG was inhibited by P4 in the MIC when the concentration of MG is less than 106 CCU/mL.The growth of MG was inhibited by P,P2 and P3 in the MICs when the concentration of MG is less than 105 CCU/mL.Conclusion:1.P3 is the most effective anti-inflammatory component of P.It effectively inhibited the expression of LPS-induced IL-6,TNF-a and NO as well as the gene copy number of IL-6,TNF-a and iNOS.2.P2,P3 and P4 inhibited the growth of MG-F.The inhibitory effects of P2 and P3 were stronger than that of P4.