Antimicrobial Susceptibility Of Helicobacter Pylori Strains To Chinese Herbal Medicines In Vitro

Helicobacter pylori(H.pylori)is an important pathogen responsible for gastroduodenal diseases in humans.Although the eradication of H.pylori using antibiotics often improves gastroduodenal diseases,resistance to the antibiotics is emerging.Finding healing powers in plants is an ancient idea.Many hundreds of plant extracts have been tested for in vitro antibacterial activity.Nine Chinese herbal medicines were used in the study,including Coptis Chinensis, Scutellaria baicalensis,Phellodendron.chinese Schneid.,Rheum palmatum L., Forsythia suspensa Vahl.,Evodia rutaecarpa Benth.,Citrus reticulata Blanco,Folium Artemisiae Argyi,Perilla Frutescens L..Initial screenings of the nine herbal medicines for possible antimicrobial activities began by using crude alcohol extractions,which have been obtained by successive extractions with ethanol.Crude products were used in broth dilution assays to test for antibacterial properties.Ten H.pylori strains were used in this study.StrainⅠ～Ⅸwere isolated from human gastric biopsy specimens at Anhui University of Science and Technology Hospital.NCTC 11637 was a kind gift from Dr.Hu(Department of Gastroentreology,Beijing University First Hospital). Culture was carried out on gastric biopsy samples obtained from 11 of 13 patients with H.pylori infection.The main inclusion criteria were H.pylori positive by at least two of ¹⁴C urea breath test,urease test and histology.Primary H.pylori culture and antimicrobial susceptibility testing were performed using GENbag incubated at 37℃.Susceptibility testing was performed using the broth dilution assays to determine the minimum inhibitory concentrations(MICs).Twelve sterile tubes were placed in a rack and labeled,the eleventh tube was growth control, and the twelfth tube was sterility control.5ml of the Brucella broth with 5%BSA (bovine serum albumin)was added to the each tube,5ml of crude alcohol extraction was added to the first tube.The concentration of the crude product in this tube was 250mg/ml.Mixed the contents thoroughly,and transferred 5ml from this tube into the second tube(125 mg/ml).Continued the dilution process through tube number 10.The eleventh and twelfth received no crude product.Added 0.1ml of the H.pylori suspension(1×10⁷CFU/ml～1×10⁸CFU/ml)into to the crude product containing broth tubes 1-10 and to the growth control tube.The rack was shaked gently to mix the tube contents and incubated at 37℃for 72h with GENbag.The activities of nine crude products against H.pylori strains were tested and seven crude products were identified that affected growth.The effects of seven crude products were further analyzed with different activities on H.pylori strains viability. Coptis Chinensis and Scutellaria baicalensis showed strong bactericidal activities.The MIC₅₀and MIC₉₀of Coptis Chinensis after 72h against 10 strains of H.pylori are 7.81 mg/ml and 10.42 mg/ml respectively,The MIC₅₀and MIC₉₀of Scutellaria baicalensis after 72h against ten strains of H.pylori are 2.60 mg/ml and 3.26 mg/ml respectively Coptis Chinensis and Scutellaria baicalensis had been reported to contain a wide range of compounds.The major constituent of Coptis Chinensis and Scutellaria baicalensis were Berberine and Baicalin.Berberine and Baicalin were extracted by using organic extraction methods and the purity was analyzed by high-performance liquid chromatography(HPLC).The contents of Berberine and Baicalin were 8.87g and 5.01g from 100g Coptis Chinensis and Scutellaria baicalensis.The purities of Berberine and Baicalin were 97.2%and 96.8%.Berberine and Baicalin were tested for their ability to inhibit H.pylori in vitro by using the broth dilution assays.Baicalin exhibited the strongest anti-H.pylori activities.The MIC₅₀and MIC₉₀of Baicalin against ten strains of H.pylori are 1.04 mg/ml and 1.30 mg/ml respectively.The MIC₅₀ and MIC₉₀of berberine against ten strains of H.pylori are 14.58 mg/ml and 16.67 mg/ml respectively.Compared with Baicalin,the MICs of berberine increases by 13 to 14 times.The antimicrobial activities of Baicalin was greater than that observed with berberine.The antimicrobial activities of Baicalin was greater than that observed with Scutellaria baicalensis.The effect of Coptis Chinensis and berberine against 10 strains of H.pylori is equivalent.These results demonstrate that the Baicalin,Coptis Chinensis,Scutellaria baicalensis and Berberine are bactericidal against H.pylori,suggesting that Chinese herbal medicines may represent a new source of agents designed for inhibition H.pylori in eradicated therapy.