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ActaAgriculturaeZhejiangensis17255～5920051213100292310031MTT3PMNNewbouldS-305410.05.02.51.0mg/mlPMN1mg/ml3P0.05P0.01MTT1mg/ml3P0.05P0.01PMNPMNtP0.053MTTS858.23A1004-1524200502-0055-05EffectofwaterextractofLeontopodiumleontopodioidesonthebactericidalactivityofbovinepolymorphonuclearleukocytesinvitroHUANGLi-quan1WUYi-hang21CollegeofAnimalScienceZhejiangUniversityHangzhou310029China2CollegeofPharmaceuticalScienceZhe-jiangUniversityHangzhou310031ChinaAbstractTheEffectsofthreewater-solublefractionssolutionofwater-extractionalcoholprecipitationandthewater-sol-ublefractionofthealcoholextractionofLeontopodiumleontopodioidesonthebactericidalactivityofbovinepolymorphonu-clearleukocytesfrommilkinvitroweredeterminedbycolony-countingmethodandMTTmethylthiazolyltetrazoliumas-say.Therewere4concentrations1.02.55.0and10.0mg/mlineachfraction.Theresultsdeterminedbythetwomethodsshowedthatthethreewater-solublefractionssignificantlyincreasedthebactericidalactivityofbovinepolymor-phonuclearleukocytesfrommilkinvitroandtheincreaseimprovedwithincreasingthedrugconcentrations.Theresultsde-terminedbythetwomethodswereconsistentwhichsuggestingthatthethreewater-solublefractionshadagoodadjustingef-fectonthebactericidalactivityofbovinepolymorphonuclearleukocytesfrommilkinvitro.KeywordsLeontopodiumleontopodioideswaterextractbovinepolymorphonuclearleukocytescolony-countingmethodMTTassaybactericidalactivityinvitroLeontopodiumleontopodioides12004-02-151957-2～4570%95%6～91011caffeicacidvanillicacidprotocatechuicthans-cinnamicacidferulicacidβ-β-sitosterolphenformin712～1511.11.1.11.1.2NewbouldS-3052×108cfu/ml1.1.3PMN10PMN90%95%RPMI-16401.0×107cfu/mlⅠ2.0×107cfu/mlⅡ4℃16171.1.4101.2PMN18PMN19MTT20PMN1.2.1ODODOD1.2.23MBC15.63mg/ml125.00mg/ml21.62mg/mlPMN3PBS201052mg/ml50µl50µlPBS537℃2h21221.2.3PMN⑴PMN550µl50µlⅠ1:1050µlRPMI-164037℃1h2100µlPBS1×1041×1031×102cfu/ml320µl237℃18hPMN⑵PMN25µl25µlⅡ50µl25µlⅡ75µlRPMI-164025µl75µlRPMI-164025µl25µlⅡ50µlRPMI-1640537℃1h2100µlPMN65172005PMNPMN1.2.4MTTPMN1PMN250µlⅠ50µl1:1037℃1h50µl2mg/ml5minPMN50µlMTT2mg/ml37℃1h3000r/min10min150µlDMSO37℃30min578nmOD50µlRPMI-1640ODPMN2PMN25µlⅡ25µl50µl37℃1hPMN50µlRPMI-164050µlRPMI-164025µlⅡ25µl50µlRPMI-164037℃1hPMN22.1ODOD11ODr=0.9968y=3.6956x+0.0325PMNPMNODPMN181ODFig.1RelationcurvebetweenbacterialcountsandtheODvalues2.2332h1P0.05313n1=n2=5Table1EffectsofthreeextractingreagentsonthesurvivalofStaphlococcusaureus305strain/mgml-1/1.0×108cfuml-10CK0.95±0.0510.95±0.0510.95±0.0511.00.94±0.1130.92±0.0370.88±0.0972.50.89±0.0910.87±0.0850.93±0.1025.00.92±0.0890.90±0.0710.87±0.09910.00.79±0.1590.81±0.1480.84±0.135CKP0.05752.3PMNMTTPMN2PMNMTTP0.052PMN1mg/mlPMNP0.051.02.5mg/ml5.0mg/ml2.55.010.0mg/mlPMNP0.055.010.0mg/ml1.02.510.0mg/mlPMNP0.011.0mg/mlPMNP0.05P0.012MTTPMNTable2ComparisonofbactericidalactivityofPMNpolymor-phonuclearleukocytesfrommilkbythecolonycountingmethodwithMTTassaymethod/mgml-1PMN/%MTT0100000041.8±3.2440.5±4.251.051.2±7.17*47.9±4.10*2.549.6±4.14*48.1±2.85*5.052.3±6.19**51.4±3.71**10.052.9±5.71**52.7±5.12**1.048.2±2.75*46.5±4.322.548.7±3.10*47.1±3.41*5.050.3±4.93*48.4±3.74*10.051.5±5.21**51.3±5.81**1.045.9±3.1252.0±6.73**2.547.3±3.20*50.9±5.40**5.049.7±4.55*51.3±4.35**10.050.8±5.38*53.4±5.86***P0.05**P0.01MTTPMN1.0mg/mlPMNP0.051.02.5mg/ml2.55.0mg/ml1.0mg/mlPMNP0.055.010.0mg/ml10.0mg/ml2.55.010.0mg/mlPMNP0.01MTT1mg/mlP0.05P0.01PMN2.4PMN2PMN3MTT2PMNFig.2EffectsofthedifferentreagentconcentrationsonthebactericidalactivityofPMNdetectedbythecolonycountingmethod3MTTPMNFig.3EffectsofthedifferentreagentconcentrationsonthebactericidalactivityofPMNdetectedbyMTTassay85172005PMNMTTPMNP0.05PMNPMN31.75M.1979.2.M.1996.3.M.1993.4.M.1997.5.M.1977.6.J.19972811673-675.7.J.199717210-11.8.J.200012324-27.9.J.1994193174-176.10.45J.1987711669-670.11.J.1994296280.12.ⅠJ.19841537-8.13.J.19849631-32.14.J.198813234-35.15.J.200017225.16.M.2000.17.M.1997.18.D.2000.19StevensMGKehrliMECanningPC.AcolorimetricassayforquantitatingbovineneutrophilbactericidalactivityJ.VeterinaryImmunologyandImmunopathology19912845-56.20.MTTJ.1996165306-307.21.PMNJ.1994141277-279.22.CLMPOPMNJ.1994142121-123.95