Studies On Extraction,Mechanism And Purincation Of Antibacterial Substances From Amomum Tsao-ko

This article studied the extraction procedure and mechanism of antibacterial substance from Amomum tsao-ko for. And the purification of active compound was studied, which sieved high performance antibacterial compound, accelerated the development of Amomum tsao-ko nature food preservative. Adopt single-factor method and response surface method for optimizing the extraction procedure of antibacterial substance from Amomum tsao-ko, and liquid-liquid separating method for gradient abstraction of crude extract from Amomum tsao-ko. Determin the minimum inhibitory concentration (MIC) of testing bacterial, the effect on auxodrome, culture solution conductivity, protein and reducing sugar, and the effect on cell breathing rate and cell shape for the mechanism of Amomum tsao-ko extract. Adopt silica gel column chromatography and thin-layer chromatography for purification of antibacterial substance, antibacterial activity testing for sieving high performance antibacterial compound, analysis of qualitation and ration by gas chromatography-mass spectrum (GC-MS). The results as below:1. Regarding soak time, ethanol concentrate, dosage liquor ratio, micro-ultrasound extract temperature, micro-ultrasound extract time as factors, and inhibition zone as index, adopt single-factor method and response surface method for optimizing Amomum tsao-ko antibacterial substance extract procedure, the optimal extraction conditions:soap time20h, ethanol concentration65%, dosage liquor ratio25:1(ml/g), micro-ultrasound extract temperature57.5℃, micro-ultrasound extraction time52.5min. The inhibition zone diameter of Amomum tsao-ko antibacterial substance was14.2mm.2. Adopt liquid-liquid separating method for gradient abstraction of crude extract from Amomum tsao-ko, getting ligroin extractant, chloroform extractant, acetidin extractant, n-butanol extractant. The MIC:ligroin extractant on Staphylococcus aureus2.5mg/ml, on escherichia coli5mg/ml; chloroform extractant on Staphylococcus aureus1.25mg/ml, on escherichia coli2.5mg/ml; acetidin extractant on Staphylococcus aureus1.25mg/ml, on escherichia coli1.25mg/ml; n-butanol extractant on Staphylococcus aureus5mg/ml, on escherichia coli5mg/ml.3. The results of effect ofAmomum tsao-ko extractant on testing bacterial auxodrome showed that, Amomum tsao-ko extractant could weaken the exponential growth phase, make thalli grow slowly. Regarding Staphylococcus aureus as testing bacterial, the antibacterial ability of Amomum tsao-ko extractant was:acetidin extractant>chloroform extractant>ligroin extractant>n-butanol extractant; regarding escherichia coli as testing bacterial, the antibacterial ability of Amomum tsao-ko extractant was:acetidin extractant>chloroform extractant> n-butanol extractant> ligroin extractant.4. The results of effect of Amomum tsao-ko extractant on testing bacterial culture conductivity showed that, Amomum tsao-ko extractant could raise cell membrane permeability, improve the value of testing bacterial culture conductivity. Regarding Staphylococcus aureus and Escherichia coli as testing bacterial, the antibacterial ability of Amomum tsao-ko extractant was:acetidin extractant>chloroform extractant>ligroin extractant>n-butanol extractant.5. The results of effect ofAmomum tsao-ko extractant on testing bacterial culture protein content showed that, the antibacterial ability of acetidin extractant for Staphylococcus aureus and Escherichia coli was best, protein content significantly increased, the final concentration was respectively863ug/ml,811ug/ml. The chloroform extractant was following, protein final concentration was respectively801ug/ml,751ug/ml.6. The results of effect of Amomum tsao-ko extractant on testing bacterial culture revertose content showed that, the antibacterial ability of acetidin extractant for Staphylococcus aureus and Escherichia coli was best, revertose content significantly increased, the final concentration was respectively16.5mg/ml,16mg/ml. The chloroform extractant was following, revertose final concentration was respectively14.5mg/ml,13.4mg/ml.7. The results of effect of Amomum tsao-ko extractant on cell respiratory rate showed that, the Amomum tsao-ko extractant was the same with three model inhibitors, could inhibit cell respiration, regarding Staphylococcus aureus and Escherichia coli as testing bacterial. The superposition rate of Amomum tsao-ko extractant and model inhibitor indicated that, the Amomum tsao-ko extractant inhibited cell respiration by krebs cycle path, because of the minimum superposition rate.8. The results of effect of Amomum tsao-ko extractant on cell shape by scanning electron microscope showed that, Amomum tsao-ko extractant affected cell membrane of testing bacterial. Acetidin extractant acted8h, the Staphylococcus aureus seriously adhesion, leading ambiguity of cell membrane. Acetidin extractant and chloroform extractant acted4h, Escherichia coli was fractured.9. Adopt kiese gel pillar for purification of acetidin extractant, sieve EA-1-6, EA-3-1, EA-3-2, EA-3-3fractions for third purification by antibacterial experiment, for GC-MS analysis. EA-1-6fraction mainly included:o-Cymene (1.34%)、Catechol (62.86%); EA-3-1fraction mainly included:Maltol (8.7%)、2-Cyclopenten-l-one,2-(2-butenyl)-4-hydroxy-3-methyl-,(Z)-(1.54%)、9-Octadecenoamide,(Z)-(4.13%); EA-3-2fraction mainly included:2,3,4,5-Tetrahydropyridazine (5.02%)、Maltol (8.62%)、9-Octadecenoamide,(Z)-(4.28%); EA-3-3fraction mainly included: Grandisol(9.34%)、2-acetyl-Cyclopentanone(2.32%)、(-)-Spathulenol(4.46%)、9-Octadecenoamide,(Z)-(3.69%)...