Isolation And Structure Analysis Of Monoamine Oxidase Inhibitors Synthesized By Lactobacillus Casei JH23

Some neurodegenerative diseases, such as Parkinson’s disease which due to thehyperactivity of monoamine oxidase, were gradually become serious threat to both humanhealth and social prosperity. Therefore, monoamine oxidase inhibitors with therapeutic effectto some nervous diseases were under extensive study. However, a few undesirable side-effectsof MAO Inhibitors already in clinical therapy were gradually out of the market. So MAOinhibitors from natural resources and with no undesirable side-effects was of great interests.Most of the natural MAO inhibitors reported had been resourced from plants as well asmetobolites from fungi. But none has been reported regarding the metobolites from Lacticacid bacteria on MAO inhibition.In this paper, the purification and chemical analysis of active components of aLactobacillus casei strain JH23were studied which was selected in this lab in previous study.(1) The crude enzyme of MAO was prepared from livers of aged male rats viahomogenization and differential centrifugation The protein content and enzyme activity wasassayed to calculate specific enzymic activity. MAO-A and MAO-B was obtained by additionof individual selective inhibitors to the crude enzyme of MAO.(2) Single-factor tests were carried out to analyze different culture time, fermentationtemperature and inocula rates on the biosynthesis of MAO inhibitor by Lactobacillus caseiJH23. An optimal fermentation conditions composed of32.5℃, for21hours with3.5%inocula was obtained by the response surface method.(3)The optimal organic solvents for extraction of active components from the culturedsupernatant of Lactobacillus casei strain JH23were studied by comparison the extractionefficiency among different solvents, and ethyl acetate was chosen. The pooled ethyl acetatephase was washed with distilled water to remove hydrophobic substances. The aqueous phasewas freeze-dried and its IC50for MAO-A and MAO-B were1.69mg/ml and4.93mg/mlrespectively, which showed a3.98times stronger in MAO inhibition than thoses of the initialfermentation broth.(4) The molecular of the active components against MAO were determined to be lessthan1KD via dialyze(molecular cut off was1KD), Then the aqueous phase containing theactive components was applied on a Sephadex G-15column and eluted with distilled water,the fraction with MAO was collected and pooled. The IC50of the eluted active components toMAO-A and MAO-B were0.327mg/ml and1.13mg/ml respectively, which was about4times lower than those of the aqueous phase.(5) The eluted active components from Sephadex G-15column was furtherchromatographed by HPLC and2active peaks remarked with Peak1and Peak2wereobtained.The Peak1was further analyzed by IR, NMR, and MS-MS. All the dataobtainedindicated that the purified component was succinic acid, and was finally justified byits HPLC-MS profiles the same as that of AR succinic acid (Sigma Co.).