Study On The Antifungal Effect Of Viridin And Its Tincture Preparation

In modern life,due to the increase in organ transplant,broad-spectrum antibiotics,hormone drugs,wide range of application and diabetes,AIDS patients,the body's immune function appeared some damage,the fungal infection more easily,the incidence rate increased year by year.In response to fungal infection,existing antifungal drugs are being hepatotoxicity,nephrotoxicity,prone to drug resistance problems,while Chinese medicine has little side effect,wide source,low price,less resistance and other natural advantages.Therefore,the research and development of antifungal drugs of traditional Chinese medicine has become a research hotspot in recent years,with the in-depth study found some problems exist in the traditional Chinese Medicine Anti fungal drug development,including:the research content is scattered,extensive,the overall research lacks main guide,the results of many experiments to compare and repeated studies of more,antifungal traditional Chinese medicine antibacterial mechanism of depth is not enough,evaluation index and evaluation system is not perfect,most research results not as drug listed and not effectively translated into productivity.Beriberi,tinea corporis is clinically more common superficial fungal disease,infected with superficial fungal disease on the patient's daily life caused great influence,and it has difficult to prevent,easy infection,the incidence rate is high,easy to relapse and other characteristics.In ethnic medicine resources investigation we found that Guizhou in Qiandongnan Dong use Viola yedoensis Makino for treating beriberi,tinea corporis curative effect is distinct,and applied to the clinic has a time of 10 years.However,a main component of V.yedoensis antifungal,extraction process and form selection mechanism without the system research and preliminary research by DNA barcoding technique we found that in the Guizhou Qiandongnan area collected V.yedoensis often mixed with long calyx violet,two the appearance of morphological identification can make the basic distinction,however,may be due to the impact of local growth environment,two of the appearance variation phenomenon,occasionally mixed production occurs,leading to the distinction between plants can not be accurate,the urgent need to find than DNA barcoding technique more rapid and simple identification methods,distinguish and mixed in V.yedoensis long calyx Viola,to ensure accurate provide the basis for medicinal sources.This article in view of the problems mentioned above,the fluorescent identification,TLC and IR identification of V.yedoensis and long calyx violet identification;on the basis of previous research,literature data and experimental verification,aesculetine selection element as herba violae antifungal extraction process optimization index;by consulting literature and experimental verification,tincture as herba violae antifungal drugs target dosage forms and of V.yedoensis tincture were quality inspection and safety evaluation;Dong medicine traditional medicine and tincture medicine clinical data collection,clinical efficacy compared with.The main results are as follows:(1)V.yedoensis and long calyx violet fluorescence identification results:the violet water extract and 50%alcohol extract in 365 nm were observed under UV light was significantly different,liquid extraction are bright blue spots of V.yedoensis,Viola long calyx extract of spot color is slightly darker violet spot color;TLC results the optimal conditions:Violae and TLC:Viola long calyx mobilephase conditions:toluene ethyl acetate formic acid(8:3:1)of the upper solution,in view of 365 nm UV light,V.yedoensis shift value minimum red spots,the Rf value is 0.23,long calyx rfvalue Viola minimal red spot.The Rf value is 0.26,which can identify V.yedoensis and long calyx violet;infrared identification results:50%alcohol and viola viola verecunda long calyx extract and petroleum ether extraction part,chloroform extract,ethyl acetate extraction part Have peak shape,peak position and peak intensity of differences.Among them,V yedoensis ethyl acetate extract of the second derivative infrared spectrum peak of the strongest peak of the peak position in 1467 cm-1,while the long calyx violet samples the peak position in 1384 cm-1,the most obvious difference between,which can be identification of V.yedoensis and long calyx violet.(2)By single factor screening and orthogonal test and aesculetine elements rate as screening index,V.yedoensis extraction conditions for ethanol concentration is 50%,the ratio of material to liquid 1:15,extraction time is 180 min and extracted two times.(3)Early by scanning electron microscopy of V yedoensis extract effect on Trichophyton rubrum and Trichophyton rubrum morphology changes were studied on the basis of this,by transmission electron microscopy,at the cellular level,to explore the mechanism of V.yedoensis anti Trichophyton rubrum,observe the small purple extracts from Trichophyton rubrum after the change of cell structure of Trichophyton rubrum Trichophyton rubrum,found increase in cell wall thickness,cell wall contours appear transparent,then the cell wall becomes rough,the cell matrix fades,intracellular matrix vacuolization,finally,cell wall,cell membrane rupture,cell death,the pharmacological effects of to V.yedoensis antifungal may be through the destruction of cell wall and cell membrane to achieve;different concentrations of V.yedoensis extract on Trichophyton rubrum,ergosterol content was:low dose group:0.0038 ?g/?L middle dose group 0.0032 ?g/?L The high dose group,0.0023 ?g/?L,ergosterol content growth in control group was 0.0082 ?g/?L,relative to the growth of low dose group,the ergosterol content in high dose group,were significantly reduced(P<0.01),indicating the ergosterol biosynthetic pathway by V.yedoensis e extract can influence of Trichophyton rubrum and play the role of anti fungal;V.yedoensis extract solution of different concentration gradient,can make the Trichophyton rubrum squalene epoxidase??-(1,3)-Dglucan synthase and succinate dehydrogenase decreased the activity of succinate dehydrogenase,and with the increase of the concentration of V.yedoensis extract solution,three kinds of enzyme activity decreased significantly;resistance the role of fungi in V.yedoensis may be the mechanism of multiple targets including:by reducing the squalene epoxidase activity in Trichophyton rubrum not normal ergosterol;Trichophyton rubrum strains ?-(1,3)-D-glucan synthase activity reduce will directly lead to cell wall glucan synthesis is blocked,fungal cell osmotic pressure instability,reach the effect of bacteriostasis and sterilization;by inhibiting the activity of succinate dehydrogenase,affecting the respiratory chain electron transfer system,interference of Trichophyton rubrum strains of respiration,hamper the energy metabolism,inhibit the growth of Trichophyton rubrum strains,lead to the death.(4)Through the literature consult and preliminary experimental screening and selection of dosage forms of traditional Chinese medicine tincture as target formulation,the selective dissolution to prepare tinctures,with the optimal extraction conditions of V.yedoensis were extracted,the extract after being dried and crushed,dissolved with 50%ethanol into concentration 12.5 mg/mL concentration(MIC)solution,ultrasonic 30 min,six layers of gauze filtration,bottling.The preparation of light brown V.yedoensis tincture,clarification,smell and the smell of alcohol.Ethanol content conforms to the Pharmacopoeia of the people's Republic of China(2015)tincture under the relevant provisions,V.yedoensis tincture 1mL quite to crude drugs 52 mg(V.yedoensis extract extract rate is 24%),the measured content of Aesculetin as 1.405 mg/g;V.yedoensis tincture of normal experimental group and experimental group in the skin of guinea pigs and the average irritation scores damaged were 0,indicating that the V.yedoensis tincture of guinea pig skin without irritation,allergy test results show Tincture on rat skin did not cause allergic,experimental process,guinea pigs and rats weight,spirit,diet and skin and mucous membrane and other.No abnormal changes were found.That is a topical application of tincture of V.yedoensis safe.Clinical results show that the V.yedoensis tincture of the cure rate is 48.39%,of the first group(Dong medicine traditional medicine)cure rate 42.86%and the curative effect of V.yedoensis tincture optimized to better than the first group(Dong medicine traditional medicine)the effect of.