| In traditional chinese medicine bitter melon can clear heat, improve eyesight, detoxify. Modern studies also had shown that bitter melon has a high medicinal value. This paper studied the extraction technology of bitter melon antimicrobial active substances, used silica gel column and HPLC separation bitter melon’s antimicrobial active substances, and, then used the nuclear magnetic resonance (NMR) spectrum technology identified some material’s structure.This study Can provided theoretical basis for making better use of the bitter melon resources, developing bitter melon natural bacteriostatic agent and founding the bacteriostatic activity guide materia. The main results were as follows:1、Staphylococcus aureus is the most sensitive among all the test strains to the bitter melon,has a minimal inhibitory concentrations (MIC) of12.5mg/ml, bitter melon also has a good inhibition to Bacillus subtilis, which has a MIC of25mg/ml,However, the inhibitory effect on Escherichia coli, Yeast is very weak. Thers is almost no inhibition to the Penicillium and Aspergillus niger. Bitter melon has a strong inhibition to gram-positive bacterium, but the inhibition to gram-negative bacteria and yeast is weak, there is almost no inhibitory effect on mold. There was a good dose-effect relationship between the extract concentration and antimicrobial effect in the range of0.2-1.5g/mL. The higher extract concentration, the better inhibitory effect. when the treatment temperature of bitter melon slices was set to60℃, no adverse effect on antibacterial activity was observed, the drying pretreatment time can be cut down, and the color of bitter melon slices is fine than it was treated by50℃and55℃,moreover,the bacteriostatic activity is also the bset. Evaluation antimicrobial activity by oxford cup method was better than the filter method, the inhibition zone diameter was bigger and more stable. Bitter melon’s inhibitory effect was far better than common preservative sodium benzoate and potassium sorbate to bacterial, but weaker than preservative to yeast and mold.2、Different solvents extracted results showed:to Staphylococcus aureus,water extract has the best antibacterial effect,70%ethanol extract was slightly weak. To Bacillus subtilis,70%ethanol extract bacteriostatic effect was best. Ethyl acetate extract also had strong inhibitory effect to Staphylococcus aureus and Bacillus subtilis, which also showed a weak inhibition to E. coli and Yeast. To mold, there were no inhibition had been observed of all the solvent extracts. Different extract methods results showed:to all the strain, ultrasound was superior to the circumfluence, circumfluence was better than leaching. Among the three varieties (Guinongke-3, Bizhu-6and Bite-111) Bite-111had the strongest bacteriostasis ability.3、 Basis on the single factor experiment, used the response surface method for the further optimize of the ultrasonic extraction technology, the results showed:under the conditions of ultrasonic power348.74W, extracted time61.21min, extracted temperature55.89℃, the antibacterial activity reached the maximum value,which was15.764mm, and experimental value coincide well with the predicted values.4、Phytochemistry qualitative appraisal indicated that bitter melon extract contained saponins, flavonoids, polysaccharides, alkaloids and polyphenols. Used spectrophotometric method for quantitative determination, the results showed that the content of polyphenols in balsam pear extract was1910mg/100g, flavonoids was1120mg/100g, polysaccharide was17500mg/100g.5、70%ethanol extract of bitter melon were successively extracted by petroleum ether, ethyl acetate, n-butyl alcohol and water, then the antibacterial activity were tested. The results showed:the antibacterial activity of the ethyl acetate phase was the strongest, When the concentration is100mg/ml, the inhibition zone diameter was come to14.44mm to Staphylococcus aureus,12.68mm to Bacillus subtilis,9.14mm to Escherichia coli. Chose the ethyl acetate phase, used the silica gel column for the further separation, parts Ⅱ, Ⅳ, Ⅷ had the strongest antibacterial activity after silica gel column separation, to Staphylococcus aureus, the inhibition zone diameter of Ⅷ was16.36mm, to Bacillus subtilis the inhibition zone diameter of Ⅱ was16.22mm, to E. coli the inhibition zone diameter of Ⅳ was10.80mm, to Yeast Ⅳ was8.46mm. This was far bigger than the crude extract. Which indicated that silica gel column can separate and enrichment the bacteriostatic active substances of balsam pear.6、Further separation used HPLC technology, compound A was obtained from IV. compounds B, C, D, E were isolated from Ⅷ. According to H-NMR、 C-NMR spectrum and refer to some related literatures, identified A, B, C, D,E were:4-ethoxybenznoic acid、4-propoxybenznoic acid、4-butoxybenznoic acid、 p-hydroxy phenylethanol and4-(3-hydroxypropoxy) phenol。... |
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