Study On Anti-HSV-â…  Active Fractions Of Styela Plicata

Objective:Styela plicata is an ascidian which belongs to class Ascidiacea, subphylum urochordata,that has fifteen hundred kinds distributed in the maritime space all over the world,from the coastal water to the ocean bed.Because its special status in the evolution, Styela plicata is highly regarded by animal scholars,evolution scholars and endocrinology scholars,becoming one of the hotspots that the scientist compete to research. Since the eighties of the twentieth century, many chemical active compositions such as peptide, alkaloids, macrolide, terpenoid, and so on, have been found in ascidian. Most of them have strong biological activity, such as anti-tumor, anti-virus, antibiosis, immunity modulation and biocatalysis, etc.There are abundant ascidian resources that have been found sixty-six species in the South China Sea.Styela plicata is one of them. It is one of the edibile ascidians,the researches about its nutritional ingredient have been reported at home and abroad,such as amine acid and polysaccharides.For the past few years, we made a foundational empirical study on the ethanol extract of the Styela plicata.The experimental results indicates that it has good inhibition on HBV in vivo and vitro which is DNA virus. In order to study the antiviral activity of Styela plicata farther, we made a study on the ethanol extract of the Styela plicata,in order to eximane the antiviral effect of it against herpes simplex virus type-Ⅰwhich also is DNA virus. And its antiviral activity and mechanism experiments of the ethanol extract of the Styela plicata were carried out in this study.Method:The whole task has three parts: 1 Anti-HSV-Ⅰpharmacodynamics study of Styela plicata in vitroTo stuty the anti-HSV-Ⅰeffect of the ethanol extract of the Styela plicata in vitro,we take advantage of model of vero cell in vitro.First,MTT methods was carried out to determine the 50% toxic concentration of the ethanol extract of the Styela plicata.The vero cells in the exponential growth phase were digested and counted,then cultivated in the 96 holes of the cultivate plate.When the cells grow in the monolayer form,the culture solution coule be thrown away. Six different concentration of the ethanol extract of the Styela plicata were put in the holes of the cultivate plate,four holes for each concentration,four nomal compared holes were set at the same time.The information of cytopathic effect was observed by the inverted microscope everyday.After three days of cultivation,20μL of the MTT solution was put into each hole,and continuted to cultivate for four hours.Then 150μL of DMSO solution was put into each hole antigrading fully.The OD values of each group at 570nm was detenmined by ELISA analyzer,reflecting the ethanol extract of the Styela plicata's impact on active of the vero cells. Second, the mixture of the different concentrations of the ethanol extract of the Styela plicata and virus has been directly put to infect the cells,four holes for each concentration.The anti-virus effects of the the ethanol extract of the Styela plicata on HSV-Ⅰin vero cells were evaluated by MTT test after 72 hours'cultivation,the positive control group,nomal cell group and the virus cell group were set. The TC50 value of the ethanol extract of the Styela plicata and ACV to the vero cell and the IC50 value and TI value of them were calculated by Reed-Muench method.2 Anti-HSV-Ⅰpharmacodynamics study of Styela plicata in vivoTo stuty the anti-HSV-Ⅰeffect of the ethanol extract of the Styela plicata in vivo,we discuss the treatment of the ethanol extract of the Styela plicata against the local and systemic infection of the virus.The local infection was Herps Simplex keratitis(HSK).Twenty guinea pigs were selected.By the cornea's scarification method, the animals were anesthetized by intraperitoneal injection of pentobarbital sodium for 7.5ml per kg,then draw by the syringe needle.One drop of the virus solution was put into the eyes.Then close the animal's eyes and massage for 15-30 seconds.After two days,the conea were coloretured by the 1% fluorescein sodium and the keratopathy imformation could be observed by the slit lamp microscope. According to the severity of disease,the animals were divided into three groups,each group for twelve eyes,then administrated by physiological saline,ACV and the ethanol extract of the Styela plicata respectively for fifteen days,five times each day. Every three day,the conea were coloretured by the 1% fluorescein sodium and the keratopathy imformation could be observed by the slit lamp microscope,and the treatment indicator is pathological changes of the cornea.The systemic infection model of the HSV-Ⅰwas through abdominal cavity of mice to observe the inflences of the ethanol extract of the Styela plicata on the mortality rate, body weight. And the influences of the ethanol extract of the Styela plicata on pathological changes in the liver were observed by histological method in infected mice,setting up the nomal control group and viral control group at the same time.3 Anti- HSV-Ⅰmechanism of action study of Styela plicataWith vero cells as host cell,CPE method and MTT method were applied to study the antiviral mechanism of the ethanol extract of the Styela plicata,with the different concentration of the ethanol extract of Styela plicata affecting the different links of the replicative cycle of the virus replication,in order to explore the mechanism of action of the influence on the virus replication in the host cells.Results:The experimental results by MTT test indicates that the TC50 value of the ethanol extract of the Styela plicata is 6.868mg/mL and 0.253 mg/mL for ACV.The maximum concentration of the ethanol extract of the Styela plicata and ACV in the pharmacodynamics experiment is 2mg/ml and 0.04 mg/ml, respectively. The survival rate of the cells in these concentration is greater than 80%. The experimental result by MTT test indicates that the ethanol extract of the Styela plicata has good inhibition on HSV-Ⅰin vero cells. The IC50 value is 0.230 mg/mL for Styelec Plicata and its therapeutic indices was 29.86. The IC50 value is 0.008mg/mL for ACV and its therapeutic indices was 31.75. The effectiveness of them are nearly.The experimental results indicates that from the third day of the treatment of the ethanol extract of the Styela plicata,pathological changes of the cornea of the animal improved quickly,the healing time was shorten and recover soon. Compared the virus control group,the mean rank of the experimental group is lower significantly,has the statistics differences among the groups by Kruskal-Wallis H method (P<0.05).Then test the Styela plicata group and the control group by the two independent samples tests on the third,sixth,ninth,twelfth,fifteenth day after administration.The results indicates that the difference has statistical significance from the third to ninth day (P<0.0167).But the difference has not statistical significance from the twelfth to fifteenth day (P>0.0167).Because some of the infected cornea of the experiental animal in the control group recover by themselves naturally,the gap of the mean rank between the control group and the experiemental group is narrow.The preliminary results show that the pathological changes of the animal's cornea reduce significantly after the treatment of the ethanol extract of the Styela plicata.The ethanol extract of the Styela plicata is sensitive to HSK, possessing antiviral activity and it has apparente curative effect on the HSK.In the experiment of systemic infection model,the result indicates that compared with the control group, the ethanol extract of the Styela plicata significantly decreased mortality rate(P<0.05) and lighten the pathological damage of liver organs in the infected mice,the body weigh of the mice in the Styelec Plicata group is higher than the mice in the control group at the sixth day afer being infected,the difference has statistical significance.The experimental result by MTT test indicates that the different concentration of the ethanol extract of the Styela plicata has direct killing role on HSV-Ⅰ. When the concentration was for 2mg/ml,the cumulative survival rate was 96.44%. The inhibition on virus strengthen in the wake of the increasing concentration;The difference of the OD value between the virus group and each concentration's group has statistical significance(P<0.01).The high concentration of the ethanol extract of the Styela plicata can inhibit the replication of the virus,but the low concentration of it did not, the difference of the OD value between the virus group and each concentration's group has statistical significance(P<0.05). The cumulative survival Rate is 74.52% at the maximum concentration for 2mg/mL. The influence on the viral adsorption is little. The cumulative survival rate is 40.01% at the maximum concentration for 2mg/mL. The difference of the OD value between the virus group and group which concentration is higher than 0.5mg/ml has statistical significance(P<0.05).Conclusion:This experiment confirms that the ethanol extract of the Styela plicata has good pharmacodynamic action on HSV-Ⅰin vero cells. The drug action strengthen along with the increasing concentration, possessing favourable dose-effect relationship. Compared to positive control drug, the toxicity of the ethanol extract of the Styela plicata on the cells was low and its therapeutic indices was high. The ethanol extract of the Styela plicata has direct killing role on HSV-Ⅰ,and has certain extent on the absorption, biosynthesis of the virus.In the model of the local and systemic infection of the HSV-Ⅰin vivo, the ethanol extract of the Styela plicata has the antiviral activity.The results and characteristic of the experiment are consistent with request of the potenc's evaluation of the drugs.It shows that the ethanol extract of the Styela plicata can be studied in the rearch of experiment of the anti-HSV further.The results of this task remined us again that Styelec Plicata is a natural product,and it has the low-toxic, efficient characteristics, is different from the frequently used antiviral agents.It has good quality while the synthetic drug has not,and has the vital significance in the development of new drug to resist herpes simplex virus.Therefore,we suppose that the Styelec Plicata,as the antiviral drugs with low-toxic, and high performance, deserves to be researched and developped, and possess broad prospect.