Study On The Antibacterial Activity,Isolation, Purification Of Active Component And The Inhibiting Mechanism Of Prinsepia Utilis Royle

Prinsepia Utilis Royle is a plant fruit which belongs to the family of rosebush,it possesses versatile medicinal properties including dropping blood sugar,dropping blood fat,enhancing immunity,staying consenescence,antitumour activity and antibacterial activity.The most investigations relate to the edible field and health care in recently. However,there is no information about the antibacterial activity,isolation,purification of active component and the inhibiting mechanism of Prinsepia Utilis Royle.In order to develop a new botan-bactericide and utilize the Prinsepia Utilis Royle resource,this thesis focus on the antibacterial activity of crude extracts of Prinsepia Utilis Royle against bacteria in vitro,chemical composition and stability of the aqueous extract were discussed by the numbers.The screening for active part,isolation and purification for active component were studied by a bioassay-directed fractionation and its inhibiting mechanism against S.aureus.The main results are as follows:1.Study on the antibacterial activity of crude extracts of Prinsepia Utilis Royle against bacteria in vitro:Prinsepia Utilis Royle was extracted in different polarity solvents(petroleum ether,ethanol and water),and study the best active extract by the method of antibacterial test in vitro.The result showed that the rates of aqueous extract, ethanol extract and petroleum ether extract were 30.5%,18.2%and 8.7%,respectively. The antibacterial activities of three extracts of 800 mg/mL were obviously difference, strong-polar aqueous extract had the strongest inhibitory capacity,medium-polar ethanol extract had a certain degree of inhibitory effect,low-polar petroleum ether extract had the weakest inhibition.The aqueous extract of Prinsepia Utilis Royle was a good part of the antibacterial activity. The results showed that inhibitory rate of different concentrations of Prinsepia Utilis Royle aqueous extract against three standard bacteria.Among them,the extract had the highest inhibitory effect to S.aureusATCC25923,the second one was E.coli44102,the third one was Salmonella50041.For the three drug-resistant bacteria,inhibitory rate of S.aureusGL17 was the highest,followed by E.coliEYAC08-56 and SalmonellaSTQD2G when the concentration of aqueous extract was 253 mg/mL,whereas at the concentration of 338 to 800 mg/mL,inhibitory rate of E.coliEYAC08-56 was the highest,followed by S.aureusGL17 and SalmonellaSTQD2G..The Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of aqueous extract was measured by test-tube double dilution method.The results showed that the MIC and MBC values of the aqueous extract against three standard bacteria (S.aureusATCC25923,E.coli 44102,Salmonella50041) were 25,25,50 mg/mL and 25,50, 100 mg/mL,the MIC and MBC values of three drug-resistant bacteria(S.aureusGL17, E.coliEYAC08-56,SalmonellaSTQD2G.) were 50 mg/mL and 100 mg/mL,respectively.2.Study on the systematic examination on chemical composition and stability of Prinsepia Utilis Royle aqueous extract:Chemical composition of Prinsepia Utilis Royle aqueous extract was tested by the method of test tube and thin-layer chromatography.The result showed that aqueous extract contained organic acid,alkaloids,flavonoids,lactones and coumarins,cardiac glycosides,terpenoid and steroid,it may contain saponins,have no quinones.Stability study made done with aqueous extract had shown that it possessed thermal stability and UV radiation stability,but its inhibitory effect was impacted by pH value,it showed good inhibitory effect under the acidic condition,of which the pH value of 4 to 6 for the best.3.Study on the antibacterial active site of Prinsepia Utilis Royle aqueous extract: Aqueous extract was precipitated by ethanol and extracted by the method of system solvent (petroleum ether,acetic ether,n-butanol),and study its active site by the mothod of antibacterial test in vitro.The result showed that antibacterial active components were mainly in the upper solution of ethanol,polysaccharides,proteins and other macromolecules almost had no antibacterial activity after precipitating.Antibacterial activity of different extracts were as follows:Acetic ether extract had antibacterial activity against all tested bacteria,and it was observed that the antibacterial activity of acetic ether extract was significantly higher(P＜0.05) than other extracts; n-Butanol and Water extracts had antibacterial activity against standard and drug-resistant S.aureus and E.coli,while it had no distinct antibacterial activity against standard and drug-resistant Salmonella at the concentration of 450 mg/mL;Petroleum ether extract had non-inhibitory effect on standard and drug-resistant Salmonella,it had shown inhibition for standard and drug-resistant S.aureus and E.coli,but inhibitory effect was relatively weak, which was significantly lower(P＜0.05) than other extracts.These results showed that the acetic ether extract,n-butanol and water extracts were all effect on bacteria in vitro.The acetic ether extract displayed relatively better antibacterial activity than other extracts.4.Study on the isolation and purification of active component from Prinsepia Utilis Royle aqueous extract:Bioassay-directed fractionation of acetic ether extract of Prinsepia Utilis Royle was conducted by the separation with silica gel column chromatography and macroporous adsorption resin technology.Acetic ether extract was separated into seven fractions through silica gel column chromatography,which some of the major activity fractions focus on the former four fractions.The inhibition zones of the first four parts against three standard strains were greater than 10 mm at the concentration of 200 mg/mL.The recrystallization with acetone resulted in the isolation of a white snowflake compound A from the first fraction,the TLC indicated that Compound A was only one spot by petroleum ether,chloroform and ethanol.The inhibition zones of compound A against three standard bacteria were 14.03 mm,11.54 mm and 12 mm, respectively,respectively.The MIC and MBC values were 1.25mg/mL,2.5mg/mL, 20mg/mL and 1.25mg/mL,5mg/mL,bigger than 20mg/mL,respectively.The results of screening for macroporous resin indicated that AB-8 resin was to be the best adsorbent for separation and purification of acetic ether extract of Prinsepia Utilis Royle.The antibacterial active component was enriched in 30%ethanol elution after AB-8 macroporous resin elution,the inhibition zone of 30%ethanol elution against three standard bacteria was located between 19 mm and 21 mm,three drug-resistant bacteria was located between 17 mm and 20 mm.Chemical composition of 30%ethanol elution was mostly alkaloids,may contain organic acids and flavonoids,which was further to be purified by the method of acid-alkaline-lipophilic solvent extraction.The results showed that chloroform layer had strong inhibitory effect against bacteria,especially the standard S.aureus ATCC25923.The MIC and MBC values of chloroform layer(6.25,6.25mg/mL) were lower than that of the 30%ethanol elution(12.5,12.5mg/mL).The result revealed that active component of Prinsepia Utilis Royle not only may be related to alkaloids,but also was associated with flavonoids,organic acid and so on.5.Study on the inhibitory mechanism of the active site of Prinsepia Utilis Royle: Inhibitory mechanism of the active site of Prinsepia Utilis Royle was discussed by taking standard S.aureusATCC25923 as instruction bacteria.The Transmission Electron Microscope results showed that the thalli of S.aureus effected by Prinsepia Utilis Royle was metamorphosed,cytoplasm was not uniformity,agglomerated and some were degenerative solution to appear cavum,membrane was ruptured,and some cell wall were blurred or even disappeared.Consequently,this suggested that inhibitory mechanism of the active site of Prinsepia Utilis Royle could undermine the integrity of the cell wall and membrane.