Research Of Some Natural Substances For The Therapy Of Helicobacter Pylori Infection

Backgrounds:Helicobacter pylori(Hp) are isolated from the gastric of chronic gastritis patients.It is closely associated with gastritis and peptic ulcers and is even a bacterial risk factor for gastric cancer and MALT lymphoma.Antibiotics are used presently for Hp infections.But some problems occurred,such as the bad obedience of patients,high cost of the drugs,high ratio of toxicity and drug resistance.Natural substances can overcome these difficulties and that's why we have this study.Mastic gum,garlic oil and coptis extraction were selected in this study.They have broad-spectrum antimicrobial effects,which were certified in the in vitro antimicrobial tests, and the anti-Hp activity was proved through the eradicative test in the infected mice.Drug resistances were tested by resistant inducing reaction,drug safeties were tested by toxicology research,and pharmacokinetics parameters were tested by pharmacokinetics research.Methods:1.Research on the in vitro antimicrobial activity of natural substances.MICs were determined by two-fold dilution of agar plate,MBCs were determined by two-fold dilution of test tube,killing curves were determined by colony count.MICs were also determined on condition of varied inoculation and varied pH of the culture.The morphous change of the bacteria when the substances were added were determined by scan electron microscope and transmission electron microscope.2.The eradicative test of Hp infected mice.(1) Research on the dose selection of mastic gum used in the treatment of Hp infection.Mongolian gerbil were used,15mg mastic gum group,7.5mg mastic gum group,3.75mg mastic gum group,dissolvent group no disposal group or 3mg Omeprazole and 2mg Clarithromyein group were setted,the infected mice were treated by these substances for 2 weeks.Mice were sacrificed on the 8th day of treatment,1st day after treatment and 20 days after treatment,and the stomachs were obtained for the close behind tests such as urease test,PCR test,culture test and histopathology test The eradication rate were obtained through these tests.(2)Research on the therapy effect selection of natural substances.Mongolian gerbil were used,15mg mastic gum group,15mg mastic gum and Hp whole bacteria vaccine group,15mg garlic oil group,15mg Coptis extraction group,15mg berberine group,dissolvent group no disposal group or 3mg Omeprazole and 2mg Clarithromycin group were setted,the infected mice were treated by these substances for 2 weeks.The mice in the 15mg mastic gum and Hp whole bacteria vaccine group were immunized through intramuscular injection in the 1st week,2ed week,and 3rd week of infection,and were treated like other groups in the following test.Mice were sacrificed on the 8th day of treatment,1st day after treatment and20 day after treatment,and the stomachs were obtained for the close behind tests such as urease test,PCR test,culture test and histopathology test The eradication rate were obtained through these tests.3.Research on the in vitro drug resistance test of Hp by multi-step inducing method. Metronidazole that is high resistant in use and Clarithromycin that has good effect in use were used as control.Mastic gum,garlic oil,berberine,Coptis extraction,Metronidazole and Clarithromycin were tested.Hp were cultured in the culture with gradually increased concentration of these substances,MIC changes were recorded.The MICs of the inducing drugs were determined after the induced bacteria with decreased sensitivity were cultured in culture with no drugs,and the MICs of mastic gum,garlic oil,berberine and Coptis extraction were also determined at that time.4.Research on the pharmacokinetics parameter of mastic gum by bacteriostasis effect method.5.Research on the toxicology of mastic gum by acute toxicity test and chronic toxicity test.Mice were drenched with maximum tolerance dose,the death condition were observed for 7 days.Rats were drenched with different doses everyday for 30 days,blood indexes, biochemical indexes,weight of rats,weight of organs,and pathology changes were detected.Results:1.The four natural substances all have in vitro antimicrobial activity.For mastic gum, garlic oil,Coptis extraction and berberine,the MICs for Hp SS1 were 125ug/ml,82.5ug/ml,1000ug/ml and 500ug/ml respectively,the MICs for Staphylococcus aureus ATCC25923 were 1000ug/ml,82.5ug/ml,4000ug/ml and 2000ug/ml respectively, the MICs for Escherichia coli ATCC25922 were 2000ug/ml,330ug/ml,8000ug/ml and 2000ug/ml respectively,the MBCs for Hp SS1 were 500ug/ml,165ug/ml,1000ug/ml and 500ug/ml respectively,the MBCs for Staphylococcus aureus ATCC25923 were 2000ug/ml, 330ug/ml,8000ug/ml and 4000ug/ml respectively,and the MBCs for Escherichia coli ATCC25922 were 8000ug/ml,1320ug/ml,32000ug/ml and 8000ug/ml respectively.From the killing curve,we saw that the three strains growed slower than control strain under condition of 1/2MIC and 1/4MIC.When the concentration of drugs raised to 2MIC and 4MIC,they all have comparatively strong antimicrobial effect against the three strains,thus they have obviously dose-effect relationship.The antibacterial effect of mastic gum can be affected by inoculated dose of bacteria and pH.This effect decreased when the inoculated dose increased to 10⁹CFU/ml or the pH decreased to 4.0 or the pH increased to 9.0.Under electron microscope,we saw that the Hp shrinked and had sparse periplas,we also saw that the cell walls were damaged,and the periplas went outside.2.The eradicative test of Hp infected mice.(1) For the 15mg mastic gum group,7.5mg mastic gum group and 3.75mg mastic gum group,the eradicated rate of Hp were all 0%on the 8th day of treatment,which were all 100%on the 1st day after treatment,while on the 20th day after treatment,the eradicated rate were 100%,50%and 25%respectively.The eradicated rate of 3mg Omeprazole and 2mg Clarithromycin group were all 100%at the three time points.(2) For the four medicine,the eradicated rates of Hp were 90%,70%,70% and 60%respectively on the 1st day after treatment.For them,the eradicated rates of Hp were 100%,80%,70%,70%,70%and 100%respectively on the 20th day after treatment. The eradicated rate for 3mg Omeprazole and 2mg Clarithromycin were all 100%at the two time points.3.In the in vitro drug resistance test,the MICs of mastic gum,garlic oil,Coptis extraction and berberine stayed the same,which means that they didn't induce resistant strain,and they can still grow in the culture having 64MIC concentration of Metronidazole. The sensitivity of strain for Metronidazole decreased gradually on the process,MIC raised up to 64μg·mL^-1 at last.Metronidazole resistance strain(MR) was cultured for 10 generations in culture without drugs,and this strain was named MR',MIC for this strain stayed the same.Mastic gum,garlic oil,Coptis extraction and berberine has no effect on the MIC.4.The pharmacokinetics parameter of mastic gum were as follows:Ka=0.666 h^-1, Ke=0.143 h^-1,t_1/2(Ka)=1.041 h,_1/2(Ke)=4.856h,Lagtime=0.574h,T(peak)=3.0,C(max)=0.9503 ug/ml,AUC=3.2605ug/ml·h,CL/F=38.03g/kg·h/(ug/ml),V/F=266.8(g/kg)/(ug/ml).5.Mice survived in seven days.Rats' general condition,hemogram,biochemical indexes,weight of organs and weight indexes were normal,and there was no abnormal change in pathological section.Conclusions:1.Mastic gum,garlic oil,Coptis extraction and berberine all have antimicrobial effects against Staphylococcus aureus,Escherichia coli and Helicobacter pylori.2.Mastic gum is effective in the therapy of Hp infection in vivo,15mg everyday can eradicate 100%,and there is no recurrence,but it recurs in low dose group.15mg mastic gum,garlic oil,Coptis extraction,berberine and Hp whole vaccine are all effective in vivo, but mastic gum is the most effective one.The together use of mastic gum and Hp whole vaccine don't induce antagonistic effect.3.Mastic gum,garlic oil,Coptis extraction and berberine will not induce resistance to Hp,while Metronidazole induces high level of resistance.The Metronidazole resistant strain is still susceptible to mastic gum,garlic oil,Coptis extraction and berberine.Mastic gum,garlic oil,Coptis extraction and berberine have the potential for the treatment of Hp in future.4.The pharmacokinetics of mastic gum is one compartment open model.It absorbs quickly and eradicate slowly.5.Mastic gum has no obviously toxically effect in acute toxicity test and long-term toxicity test.The intended dose is safe.