| Objective: To investigate pharmacognosy of jiejia tincture composing prescriptionsChinese medicine; establish a method of identification of the effective components injiejia tincture; determine the optimum extraction and purification process; investigateantibacterial and antioxidant activity in vitro; observe the treatment effect of scald inrats and explore the possible mechanism.Methods:1. Confirmation of catechu, scutellaria and phellodendron in Jiejia tincture throughcharacters and microscopic identification.2. To determined effective components in Jiejia tincture by HPLC. Orthogonal designmethod was used to optimize the extraction process. Macroporous resin adsorptionmethod was used to determine the best purification process.3. Tube double dilution method was used to examine the antibacterial activity ofeffective components of jiejia tincture. Electrical conductivity, scanning electronmicroscopy and transmission electron microscopywere used to study the possiblemechanism of the antibacterial activity.4. Folin method was used to evaluate the content of total phenol of effectivecomponents of jiejia tincture. To estimate the anti-oxidation activity of effectivecomponents of jiejia through FRAP, potassium ferricyanide reduction, DPPH andABTS, hydroxyl radical and superoxide anion free radical.5. Rats scald model was Established to observe the effect of effective component ofjiejia tincture. SOD, MDA, NO and TNF-α, IL-1β, IL-6in serum wasdeterminationed to study the possible mechanism. Results:1. Catechu, scutellaria and phellodendron were identified by Dalian Medical Universityassociate professor DiaoYunPeng.2. The best extraction process was30℃,70%ethanol extracting3times, each time toextract30min according to Orthogonal design; RH-HPLC established in this studywas simple, accurate and reproducible. Catechin showed a Good linearity at therange of100~800μg/mL, baicalin showed a Good linearity at the range of15~120μg/mL, and berberine at the range of7~56μg/mL. HP20macroporous resin wasused for gradient elution, and catechins, baicalin, berberine were eluted by10%ethanol,30%ethanol,50%ethanol, respectively.3. In vitro antibacterial experiment,10%ethanol extraction of jiejia tincture had higherantimicrobial activity. MBC was0.5mg/mL.The inhibition rate of10%ethanolextraction of jiejia tincture (0.5mg/mL for18h) was up to100%. By conductivityand transmission electron microscopy and scanning electron microscopy, we foundthat the antibacterial activity was related to the destruction of bacterial cell wall andthe enhancement of membrane permeability.4. Content of the total phenol from Jiejia tincture purification:10%ethanolpurification>30%ethanol purification>50%ethanol purification; Among them,10%ethanol purification had the strongest antioxidant capacity; Antioxidant capacitygradually increased with the increase of the total phenolic content.5. In vivo scald experiment, jiejia tincture effective component had notable treatmenteffect. Wound area was significantly reduced and healing time was significantlyshortened. Meanwhile, jiejia tincture effective component could inhibit the growth ofinflammatory factors, promote the regeneration of fibroblast, granulation tissueandhair follicle, etc.；Jiejia tincture effective components could increase the activity ofSOD and decrease the levels of MDA、NO, reduce lipid peroxidation injury; Jiejiatincture effective components could reduce the level of inflammatory mediators,such as TNF-α, IL-1β, IL-6.Conclusion: Chinese Herbal Medicine jiejia tincture recipe were identified in thissubject; the optimum extraction process of effective components of jiejia tincturewas Established; the most effective way of purification was Selected; HPLC wasused to determine the content of effective component; effective components of jiejiatincture had good antibacterialand antioxidant activity in vitro; moreover, effective components of jiejia tincture had significant therapeutic effect on scalded rats. |
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