Inhibitory Effect Of Epigallocatechin-3-gallate On Mycobacterium Smegmatis Mc~2155

Mycobacterium tuberculosis (M.TB)，a species of the genus Mycobacteria is thepathogen of tuberculosis (TB). The cell wall of M.TB is thick, hard and hydrophobic.It serves to protect the organism from the environment and makes it highly impermeableto conventional antimicrobial agents, so it is difficult to develop new antimicrobialagents. The mycobacterial cell wall is a unique structure for mycobacterial survival andgrowth in the host. The core of mycobacterial cell wall consists of inner layerpeptidoglycan, middle layer arabinogalactan and outer layer mycplic acid.Arabinogalactan is an important structure to connect PG and mycolic acid and to keepthe integrity of the cell wall. Therefore, people choose arabinogalactan as an adealtarget to develop new effective anti-TB drugs.Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, andis also a potent antioxidant that may have therapeutic properties for many diaordersincluding infection, cancer and aging. It is one of the hot spot in the research in recentyears. How it play the role to the mycobacterium tuberculosis, with special structure tocell wall, common drug very difficult to reach the target function have not been reported.Mycobacterium smegmatis was used in this study. The strain has similar geneticcharacteristics as rapid growth, but without infectivity as mycobacterium bacteria.Therefore M. smegmatis is a model on research of EGCG bacteriostasis.Objectives:1. Extracting green tea extraction from green tea.2. Studing the bacteriostasis of green tea extraction to mycobacterium smegmatismc2155.3. Studing the bacteriostasis of EGCG to mycobacterium smegmatis mc2155.4. Studing the metabolic influence of AG after EGCG treatment. Further discussing the bacteriostasis of EGCG to mycobacterium smegmatis mc2155.Methods:1. Preparing crude green tea extract containing epigallocatechin gallate (EGCG)by ethyl acetate extraction under neutral condition. Testing the content of EGCG byHPLC.2. Inhibitory effect from crude extract against Mycobacterium smegmatis mc2155was determined by disk agar diffusion method. Using of enzyme standard analyzer tomeasure MIC of green tea extraction.3. Determining the inhibitory effect from EGCG against Mycobacteriumsmegmatis mc2155by disk agar diffusion method. The changes of structure of cell wallwere observed by transmission electron microscopy (TEM).4. Extracting AG from Mycobacterium smegmatis mc2155. Testing the metabolicinfluence of AG after EGCG treatment.Result:1. Successfully extracting green tea extraction from green tea, what was rich inEGCG.2. Crude extra showed significant growth inhibition of Mycobacterium smegmatismc2155from both approaches, and the inhibitions were dose-dependent. MIC of greentea extraction was measured by enzyme standard analyzer.3. Antibacterial circle experiment testified EGCG showing significant growthinhibition of Mycobacterium smegmatis mc2155. The changes cell wall structure wereconfirmed by TEM.4. TLC testified the significantly metabolic influence of AG after green teaextraction treatment.Conclusion:We found that EGCG showed significant growth inhibition of Mycobacteriumsmegmatis mc2155. It could change the cell wall structure of Mycobacterium smegmatismc2155. It could influence the metabolism of AG. We infer that EGCG may be throughinfluencing the metabolism of AG and damaging the cell wall structure ofMycobacterium smegmatis mc2155to play its bactriostasis.Future research:(1) Deeply studing the metabolic influence of AG after EGCG treatment. QuantifyAnalysing AG by HPLC. Observing the ratio change between Ara and Gal.(2) I plan to culture THP-1in vitro and add EGCG to THP-1after the infection of Mycobacterium smegmatis mc2155. The cell morphology will be observed bymicroscope and the viability will be detected using MTT method. To determine theinhibition of macrophage apoptosis, M.tuberculosis-infected cells exposed toantimycobacterial drugs will be stained with propidium iodide (PI), then detected usingflow cytometry. Finally, I will judge if EGCG can increasing the apoptosis ofmacrophages of Mycobacterium smegmatis mc2155or not.