West Effective Constituents In Total For The Treatment Of Infective Wounds And Mechanism Research

ObjectiveIn this study, based on traditional Chinese and modern medicine theory,effective compositionof chebulae fructus immaturus was extracted、seperated and purified in order to select theoptimal processe for the first time by using modern separation&analysis technology andscreening of effective composition in vitro method; With the combination of vivo and vitropharmacology, the antibacterial test、 anti-oxidation test and acute toxicity test wereundertaken to the resulting effective composition whose mechanism was assessed for the fisrttime, which can be provided as theoretical and experimental basis for the future research ofChebula fructus immaturus.Methods1. Using modern pharmacognostical identification method, we check the plantmorphology、 macroscopic and microscopic characteristics of the chebulae fructusimmaturus used in the study to confirm the same source of the chebulae fructusimmaturus.2. We collected extracts from chebulae fructus immaturus and selected staphylococcusaureus and klebsiella pneumonia, to observe the effects of different compositions onbacteria growth by plating-digging-perfusion method; the inhibition effect of serumcontainning effective composition (total tannin)on two bacteria growth was tested basedon serum pharmacology.3. For the first time, we used improved Casein method to determine the total tannins ofantibacterial effective composition; we sort out the optimal extract process and conditionswith the application of single factor exploration and L9(34)orthogonal test.4. For the fisrt time, we determined the total tannin and gallic acid content by UV andHPLC method according to Chinese new drug guideline and Pharmacopoeia, and usedstatic test to sort out the macro porous adsorption resin type, and dynamic test todetermine the optimal separation and purification conditions of the macro porousadsorption resin for total tannin.5. We used column chromatography and half-prepared chromatography to separatemonocase compounds from effective composition which had been extracted by optimalprocess, and determine the chemical structure by physical and chemical method andspectral analysis etc.6. We tested the gallic acid and ethyl gallate content of effective composition by HPLCmethod for the first time.7．For the first time,we evaluated effective composition’s treatment for infectious wound and its working mechanism from4aspects: antibacterial, anti-oxidation, acute toxicity andwound healing. As for antibacterial, effective composition’s MIC, MBC and killing curveof two bacterias were surveyed. And also effective composition’s effects on bacteriagrowth at different times were explored by scan and transmission electron microscope toevaluate the antibacterial effect from mechanism point of view; As for anti-oxidationactivities, ABT, FRAP and DPPH were used to evaluate effective composition’santi-oxidation activities in vitro; As for acute toxicity, acute toxicity test and pathologicalsection were conducted to mice; during wound healing experiment, a systematic studywas conducted which included effective composition’s treatment for the mice woundhealing, mice pathological section, VEGF and β-FGFmRNA expression andimmunohistochemistry.Results1. Through the check of the plant morphology, macroscopic and microscopic characteristics,the chebulae fructus immaturus used in the study were identified to be dry immature fruitof Terminalia chebula Retz.2. By plating-digging-perfusion method and serum pharmacology method, it was found thattatol tannin and serum both had antibacterial effect on two bacterias, and also proved thattotal tannin was the effective composition.3. Before extraction, total tannin content of main antibacterial effective composition inchebulae fructus immaturus was only9.57%; however, produced from selected optimalextraction processes, the tannin content can reach up to30%, and the optimal extractionconditions are:15times amount aqueous solvent, soaking for20min,and extract in3times, each for1h.4. For UV method which was used for the separation and purification of total tannin, thebest conditions were as followings: AB-8resin, resin balde diameter: length: ratio is1:1.5,eluent is50%ethonal, elute speed is1ml/min.For HPLC method, the best conditions areas followings: eluent is50%ethonal, use HPD resin, sample volume is40ml.5.13compounds were extracted from effective composition by optimal separation andpurification process. And11of them were identified and chemical structures determined:9phenolic acids,1triterpenes compound. And one of them has never been reported before,and the rest were firstly obtained in the chebulae fructus immaturus separation.6. The gallic acid and ethyl gallate content of effective composition produced from optimalprocess were determined by HPLC to be0.2%and0.392%.7. Effective composition’s and ethyl gallate’s treatment for wound healing and relativemechanism were evaluated from4aspects: antibacterial, anti-oxidation, acute toxicityand wound healing. It was found that effective composition and ethyl gallate both hadinhibition effects on bacteria growth, and the anti-oxidation effects were obvious, and proved to be minor toxic and highly safe by acute toxicity test in vitro; And the effectivecomposition were proved to have wound healing effect as a result of the evaluationmentioned above.Conclusions1．The optimal extraction process of effective composition (total tannin) from Chebulaefructus immaturus is stable, feasible and suitable for pilot and scale-up experiment.2．The chemical composition of the effective composition was fisrtly separated and itschemical structure determined. Single active composition was tested, which provides amaterial basis for the further research.3．Pharmacology in vivo and vitro proved that the effective composition of chebulae fructusimmaturus had the characteristic of antibacterial, anti-oxidation, wound healing, minortoxic and high-safety.4．For the first time, electron microscope was used to evaluate the antibaterial mechanism.With the application of immunohistochemistry, working mechanism of the effectivecomposition’s treatment for wound healing was insightly assessed.