Anti-Escherichia Coli And Anti-inflammatory Activity Of Ethyl Acetate Extract From The Root Of Rosa Roxburghii Tratt

Rosa roxburghii Tratt(Ci Li)was one of the members of rosaceous plant(Rosa roxburghii Tratt.f.roxburghii),and it was a unique wild plant resource in Yunnan-Guizhou Plateau and Western Sichuan Plateau in China.The fruits of R.roxburghii Tratt(Ci Li)contained abundant compounds and nutrients,and had many pharmacological effects including anti-tumor activity,anti-inflammatory activity,anti-apoptosis activity,anti-radiation activity,and anti-oxidation activity.The decoction of the the root of R.roxburghii Tratt(Ci Li Gen)recorded in "Zhonghua Bencao" was reported to be used to treat chronic gastritis,stomachache,acute enteritis,diarrhea,and white diarrhea in human and livestock.However,until now,the components in the Ci Li Gen and their pharmacological effects have never been reported.Therefore,in this study,the antibacterial and anti-inflammatory components of Ci Li Gen were isolated and identified,and the relevant mechanism was studied.1.Isolation and purification of effective antibacterial components from the ethyl acetate extract of Ci Li GenThe ethyl acetate extract of Ci Li Gen was prepared by the dipping method.After concentrated under reduced pressure,it was dried at 40℃ and stored at-20℃.High-performance liquid chromatography(high performance liquid chromatography,HPLC),high-resolution mass spectrometry(high resolution mass spectrometry,HRMS)and nuclear magnetic resonance(nuclear magnetic resonance,NMR)were used to isolate and identify the effective antibacterial components in the ethyl acetate extract of Ci Li Gen,respectively.The results showed that the compound was strictinin isomers.Its molecular formula was C26H20O19.2.Antibacterial activity and antibacterial mechanism of strictinin isomers against Escherichia coli ATCC 25922 in vitroThe antibacterial activity of strictinin isomers was evaluated by the determination of the minimum inhibitory concentration(MIC)and the time-kill curve.The antibacterial mechanisms were mainly explored by the following methods:the differences in protein expression of Escherichia coli ATCC 25922 were analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE)combined with NanoLC-ESI-MS/MS technology.The intracellular esterase activity was detected by Flow cytometry,and the ultrastructural changes of the E.coli were observed by scanning electron microscope(SEM).Compared with the negative control group,the results of SDS-PAGE and NanoLC-ESI-MS/MS showed that the up-regulated proteins of the strictinin isomers treated group were Metal-binding protein ZinT,30S ribosomal protein S4 and 50S ribosomal protein L4,while the down-regulated protein was hydroperoxide reductase subunit C.Moreover,in the strictinin isomers treated group,the esterase activity in the E.coli cells was reduced and the bacteria E.coli became atrophied,pitted and contorted,and the surface of E.coli was rough and blurred.According to above results,the antimicrobial mechanism of strictinin isomers against E.coli ATCC25922 were oxidative stress and protein synthesis disorder,which inhibited the activity of the enzymes required for bacterial growth and metabolism,achieveing bacteriostatic effects through multiple ways.3.The anti-inflammatory activity of strictinin isomers in vivoThe anti-inflammatory effect of strictinin isomers was evaluated by acute inflammatory model of carrageene-induced paw swelling in mice.Thirty-six ICR mice were randomly divided into six groups.Three strictinin isomers groups(dose:12.50,25,50 mg/kg bw),blank control group and carrageene control group(the same volume of ultra-pure water),DMSO control group(the same volume of 0.1%DMSO),positive control group(dexamethasone 5 mg/kg bw)were administrated continuously for 7 days.One hour after the last oral administration,30 μL of normal saline was injected subcutaneously into the right plantar of the healthy control group,and 30 μL 1%carrageenan was injected into the other groups.The thickness changes of the oedema of the hind paw of rats were measured by vernier caliper at 1h,2h,3h,4h after the injection of carrageenan.After 4 hours,normal and inflammatory feet were excised from the hind limbs of the mice along the ankle joint,and the mass was weighed to calculate the degree of swelling of the paw and its inhibition rate.The results showed that,compared with the carrageene control group,the paw swelling degree of the dexamethasone group,the medium-dose group and the high-dose group was significantly reduced,and the inhibition rates of the paw swelling of each group were 50.82%,62.44%,and 49.25%(P<0.001),respectively.The low-dose group did not reduce the degree of paw swelling.4.The anti-inflammatory activity of strictinin isomers in vitroThe inflammatory model of RAW264.7 cells induced by LPS was used to further explore the anti-inflammatory activity of strictinin isomers in MTT method was used to determine the effect of strictinin isomers on cell viability,and RT-PCR method was used to determine its effects on the mRNA expression of IL-1β、IL-6、TNF-α、iNOS and COX-2.The results showed that the safe concentration range of strictinin isomers on RAW264.7 cells was 0-100 p,g/mL.Compared with the LPS group,strictinin isomers significantly inhibited the mRNA expression level of IL-1β and IL-6 at 6.25μg/mLand 12.5 μg/mL(P<0.001).