Screening Of Antibacterial Components With Alanine Racemase As Target In Traditional Chinese Medicine

Alanine racemase (EC5.1.1.1) is a pyridoxal5’-phosphate (PLP) dependent enzyme that catalyzes the interconversion of L-and D-alanine isomers. D-alanine is essential for peptidoglycan biosynthesis in the cell wall of gram-positive and gram-negative bacteria. In recent years, alanine racemase was regarded as an target for the development of antibacterial medicines. Traditional Chinese medicines are paid more attention for wild source, little toxic side effect and non-resistance. Screening inhibitors of alanine racemase from traditional Chinese medicines (TCM), can provide basis for searching non-toxic, low cost of antimicrobial agents and easy to understand the antibacterial mechanism for traditional Chinese medicines.In this paper,89anti-inflammatory traditional Chinese medicines were screened by His-Alr as target which from Pseudomonas purida YZ-26. Firstly, determination the activity of Alr with or without the extraction of TCM was used by polarimeter. It indicated that25of89traditional Chinese medicines could inhibit the alanine racemase activities in different degrees. Secondly, the antimicrobial activitys of TCM against Pseudomonas purida YZ-26were performed by multiple proportion dilute method under cultivation. Among them23TCM had inhibitory effect on the growth of Pseudomonas purida YZ-26, except lalang grass rhizome and tangerine peel. Tree peony bark showed strongest inhibing rates, with68.3%, followed by Rhizoma Polygoni Cuspidati, with54.55%. In the present of D-alanine, the inhibition was relieved except tree peony bark, pubescent holly root and radix aucklandiae. Rhizoma Polygoni Cuspidati has the obvious remission action, about30%.Furtherly, due to the different dissolution character, the effective ingredients of Rhizoma Polygoni Cuspidati were isolated by chemical regagents. The activity of Alr was assayed by the polarimeter with or without extracts of95%alcohol, or petroleum ether, or acetic ether, or N-butanol and alcohol soluble substance, alcohol heavy component, respectively. The results displayed that the extracts of95%alcohol, petroleum ether, acetic ether and alcohol heavy component could inhibit the activity of Alr. The strongest inhibition effect among them was alcohol heavy component, which inhibit rate was49.51%. The alcohol heavy component was mainly composed of polysaccharide and protein. With the increase of polysaccharide concentration, its inhibtion on the activity of Alr has been also enhanced when the protein was removed by the Sevag method. So polysaccharide was the effective ingredients in Rhizoma Polygoni Cuspidati. When the polysaccharide was oxidized and decomposed with H2O2in microwave, its inhibtion on the activity of Alrr was disappeared. The polysaccharide was the uncompetitive inhibition determined by fluorospectro photometer, and the IC50was2.425mg·ml-1. The polysaccharide could obviously inhibit the growth of YZ-26, and the half of the minimal inhibitory concentrations(MIC50) was0.4925mg·ml-1Alr was coupled to Spharose4B activated by CNBr, then incubated with extracts of Rhizoma Polygoni Cuspidati. The activity of Alr coupling with Spharose4B was measured by polarimeter. It was indicated that some component of Rhizoma Polygoni Cuspidati binding to Sepharose-4B-Alr could inhibited the activity of Alr, the inhibition rate was40%. The component was identified as polysaccharide by fehling’s reagent, Wagner’s reagent, KOH solution and FeCl3solution.There were uronic acid in the polysaccharide measured by sulphuric acid-carbazole method, and-NH2measured by ninhydrin reaction. The result of ultraviolet spectrum exhibited that the polysaccharide had specific absorbing peak at300nm. The polysaccharide contained a-glycosidic bond, C-O-C,-OH, C=C,-CH2, and-NH2by infrared radiation.