Separation And Identification Of Anti-MRSA From Rhizoma Polygoni Cuspidati And Investigation Of Their Biological Activities

Background:The discovery and useage of antibiotics provides human beings the powerful weapon to treat infectious diseases. However, the over use even abuse of antibiotics lead to more and more bacterial resistance strains. Among these bacterial resistance strains, especially methicillin-resistant Staphylococcus aureus occurs(MRSA) presents the characteristics of high pathogenic rate and high mortality, which is very difficult to treat. Therefore, it is very urgent to research of new drugs to treat MRSA infection.At present, there are two strategies to develop anti-MRSA drugs: Firstly, the direct antibacterial strategies, including research for new antibiotics and modifing the structures of existing antibiotics such as linezolid, daptomycin and so on. Secondly, the the indirect antibacterial strategies, to look for the agents to enhance the effect of the existing antibiotics. These agents have no antibacterial activity, but they can increase the antibiotic’ zntibacterial activities.Our group engages on the work of separation and extraction of antibacterial active substance from traditional Chinese medicine herbs for a long time. In the present experiments, firstly, 37 traditional Chinese herbs were screened targeting PBP2 a and then the herbs with high affinity with PBP2 a were selected. Secondly, on the directional of anti-MRSA activity the root of the Polygonum cuspidatum with the best anti-MRSA activity was selected. Thirdly, focusing on the root of the polygonum cuspidatum, the components and monomers with anti-MRSA activity were isolated on the directional of anti-MRSA activity, and then their activities were investigated.Objectives:To screen the herbs with the high anti-MRSA activities from traditional Chinese herbs, and identify the material basis related to anti-MRSA activities, witch will shed a light on the further study of new antibacterial drugs.Methods:1. Traditional Chinese herbs with high affinities for PBP2 a were screened from 37 herbs based on targeting PBP2 a using biosensor technology;2. The anti-MRSA activities of water extracts of polygonum cuspidatum, ET-RPC(ethereal extract from alcohol extract of rhizoma polygoni cuspidati), and monomer compounds activities of anti-MRSA were investigated based on MIC, IZD, and growth curves experiments;3. Separation and identification of the monomer compounds from the ET-RPC by HPLC-MS technology;4. The affinity of the monomer compound binding to PBP2 a was measured using biosensor technology.5. Transmission electron microscopy was used to observe morphological changes in the cell wall of MRSA252.Results:1. Eight traditional Chinese medicine herbs with high affinities with PBP2 a were selected from total 37 herbs, they were Chinese pulsatilla root, polygonum cuspidatum(root and leaves), sargentg loryvine stem, cortex lycii, cinnamon, rheum officinale, radix paeoniae rubra, and gardenia;2. The anti-MRSA experiments showed the water extracts of root of polygonum cuspidatum and cortex lycii had strong anti-MRSA activities, the anti-MRSA activity of the former possessed stronger activity than the latter;3. ET-RPC had a direct anti-MRSA activity against MRSA in vitro;4. There were 7 monomer compounds in ET-RPC: polydatin, resveratrol-4-O-D-(6′-galloyl)- glucopyranoside, resveratrol, torachryson-8-O-glucoside, emodin-8-Oglucoside, 6- hydroxyl-emodin and emodin;5. Emodin had strongly direct activity against MRSA, which demonstrated that emodin was the main anti-MRSA substances in ET-RPC.6. Emodin had strong anti-MRSA activity against MRSA standard and clinical strains; the KD value for PBP2 a was 6.78 × 10-5 M.7. Emodin could damage the integrity of cell wall of MRSA252.Conclusion:ET-RPC had significant anti-MRSA activities of ET-RPC against MRSA252 and 17 clinical MRSA strains. Seven major compounds of ET-RPC obtained by HPLC-MS were polydatin, resveratrol-4-O-D-(6′-galloyl)-glucopyranoside, resveratrol, torachryson-8-O-glucoside, emodin-8-O-glucoside, 6-hydroxyl-emodin and emodin. However, only emodin had strong anti-MRSA activity, demonstrating emodin was the main anti-MRSA substance of ET-RPC. Emodin possessed strong anti-MRSA activity not only against standard strain but also against clinical strains. Emodin could damage the integrity of cell wall.The KD value of emodin for PBP2 a was 6.78 × 10-5 M.