Study On The Effect Of Hippophae Rhamnoides Linn. Extract On Gastrointestinal Microorganism

Rich in antibacterial active ingredients,Hippophae rhamnoides Linn.,conventionally used for improving gastrointestinal inflammations,is a characteristic plant resource in Qinghai-Tibet Plateau.In this paper,we evaluated the anti-Helicobacter pylori activity by Hippophae rhamnoides Linn extracts,and explored the potential mechanism in vitro,and analyzed the influence by Hippophae rhamnoides Linn.on inflammation,signaling pathway,gastrointestinal microbiota of H.pylori-infected animals of BALB/c mice.Results were following.1.According to the inhibition zone test in vitro,extracts by distilled water,80%ethanol,methanol or ethyl acetate,showed strong antibacterial effect and noticeable inhibition zone.Results indicated that minimum inhibitory concentration(MIC)of three extracts by distilled water,80% ethanol,methanol were from 5 to 10 mg/m L which valued for by the double dilution method,and from 2.5 to 5 mg/m L for ethyl acetate extract.Minimal bactericidal concentration(MBC)ranged from 5 to 10mg/m L for all extracts.Hippophae rhamnoides Linn.juice was observed to barely inhibit H.pylori in inhibition zone test,rather,it has opposite result in the double dilution test,both MIC and MBC ranged from 3.125% to 6.25%.2.The Hippophae rhamnoides Linn.water extract(HLW)can turn the morphology of H.pylori from screw into ball,which was observed by scanning electron microscope technology.In addition,HLW was observed to inhibit the activity of H.pylori urease(HPU)of both culture fluid and HPU solution,and its inhibitory rate was positively time-dependent and dose-dependent in a certain range.Kinetic analyses showed that type of urease inhibition by HLW was noncompetitive for HPU.At the same time,neither thiol protectors nor inorganic compounds played a role in recovering the activity of suppressed urease.Furthermore,molecular biology experiments indicated that m RNA expression levels of urease accessory protein E(Ure-E),urease accessory protein H(Ure-H),urease subunit α(Ure-α),urease subunit beta(Ure-β),cytotoxin associated gene A(Cag A),the signal peptide and middle region of vacuolating(Vac)were markedly decreased,as presence of HLW of different concentrations in culture medium of H.pylori,thus,the growth of H.pylori was inhibited by HLW.3.The antibacterial effect of HLW on H.pylori in vivo was evaluated by model mice of H.pylori,it was found that oral administration of different concentration of HLW,80% ethanol extract and Hippophae seed oil could decrease the serum IL-1βlevel and the activity of urease in gastric mucosa.The result of histopathological analysis revealed that Hippophae rhamnoides Linn.extract(HLE)could improve gastric mucosal inflammation in BALB/c mice.Molecular biology experiment showed that HLE decreased the expression levels of two inflammatory factors,NF-κB p65 and IκB-α in NF-κB inflammatory signaling pathway.According to immunohistochemical experiment,HLE reduced the expression level of NF-κB p65 protein and up-regulated the expression of IκB-α protein,and then improving gastritis induced by H.pylori in mice.Taken together,HLW,with concentration of 80 mg/m L,had the effect on treating gastritis of model mice,which was similar to the triple therapy group by omeprazole,amoxicillin and metronidazole inhibitors.4.In Animal experiment,based on isolation and culture of intestinal characteristic bacteria and high-throughput sequencing analysis techniques,H.pylori infection led to a decline in the number of Lactobacillus,however,it hadn’t apparent effect on the diversity and stability of intestinal flora.Triple therapy,evidently reducing the diversity of flora,increasing the relative abundance of Escherichia and Enterococcus,had a negative impact on the intestinal flora in BALB/c mice,in addition to those results,the treatment noticeably decreased strings of functional abundance alike,for example,transport and metabolism of nucleotides,coenzymes and lipids,signal transduction and defense mechanisms.Choosing HLE instead of triple therapy,the intestinal flora structure remained relatively balanced,B/E value increased,species diversity increased significantly and the variation of functional abundance kept stable.Among several samples tested,80 mg/m L ethanol extract worked best,followed by 80 mg/m L water extract,which clearly decreased the relative abundance of Helicobacter.Moreover,Hippophae seed oil barely had obvious effect on intestinal floras of H.pylori-inoculated mice,but specifically increased the relative abundance of several bacteria genera,among which Akkermansia behaved most markedly.Results of normal mice experiment showed that the B/E value increased,species diversity rose noticeably and functional abundance reached its peak in the presence of 80 mg/m L HLW.