| The virus and tumor diseases are serious harmful to human and animals'health. Most of tumors were associated with virus. The present antiviral and anti-tumor drugs have many side-effects, so how to find a new drug with better curative effect and less side-effects is the main problem medicine worker concerning. Chinese traditional medicine has affirmative curative effect and almost least toxic reaction. The resource of Chinese traditional medicine is abundant. So Chinese traditional medicine has particular advantages in treating diseases. Research of the past few decades showed that the comparative advantages of Chinese medicines in preventing and treating viral infection are as follows. (1)They have a wide spectrum effect of killing or inhibiting the viruses. (2)They can initiate the preventative ability of organism and regulate the immune function.(3)They are more safe and do not have obvious poisonous or side effect. The disadvantage of dysbacteriosis and double infection is very small.Ginseng is one of the most important Chinese medicines used in the world, which has function in tonics to combat stress and cancer, disturbances of the central nervous system, and so on. It contains many dammarane and oleanane saponins,polyacetylene derivatives and polysaccharides of which the biological activity has been studied widely. Ginsenoside (GS) is the major effective ingredient of Ginseng. Ginsenoside has extensive biological activities. Ginsenoside not only has stimulatory effects on the components of specific the cellular and humeral immune responses, but also presents some non-specific immune reactions such as monocyte proliferation and improving the ability of the macrophages;Long-term clinical practice studies proved that Ginsenoside had surely anticancer and antiviral effect。Marek's Disease (MD), a highly infectious lymphoproliferative disease in chickens, is caused by an alphaherpesvirus-Marek's disease virus (MDV), resulting in T cell lymphomas in visceral organs and peripheral nerves. Now, MD still is a major threat to the poultry production in the world. Vaccines against MDV are available, but immune defeat sometimes happens, resulting in MD breaking in some regions. And moreover, MD, served as an important animal model of viral oncogenesis,has offered important contributions to veterinary medicine basic science and comparative oncology.In order to examine the antiviral effect of ginsenoside and its derivatives against Marek's disease virus and find the medicine which has excellent antiviral effects, in vitro and in vivo., Antiviral experiments of Ginsenoside and its derivatives against Marek's disease virus were carried out in this study.The first, the model of chick embryo fibroblast cell infected by MDV was established in vitro. The viral titer was determined by tissue culture infectious dose 50 (TCID50) assay; The plaque forming method of CEF infected by MDV was established.The results showed that MDV could infect CEF cell and emerge typical cell pathological changes. The titer TCID50 of MDV in CEF cell is 10-5.23/0.1mL. The plaque forming method of MDV in CEF cell was successfully established.The determination of safety concentration of ginsenoside and its derivatives on chick embryo fibroblast cells. The safety concentrations of ginsenoside and its derivatives on CEF were studied by cytopathic effect (CPE) and Neutral red assay. Ginsenoside, its derivatives and morpholinebiguanide(ABOB) were diluted with DMEM medium into series concentrations and used for cultivating chick embryo fibroblasts (CEF) in two ways, at the beginning of culture and after CEF monolayer formation. Their maximal safety concentrations to CEF were judged according to OD value and morphological variety of the cell.The results showed that the safety concentration of ginsenoside, derivative one, derivative two, derivative three, derivative four, derivative five, derivative six, derivative seven, derivative eight and ABOB were 375μg/mL,93.75μg/mL,46.88μg/mL,93.75μg/mL,23.44μg/mL,46.88μg/mL,23.44μg/mL,187.5μg/mL,93.75μg/mL和750μg/mL, respectively;The cytotoxic concentrations 50%(CC50) of ginsenoside, derivative one, derivative two, derivative three, derivative four, derivative five, derivative six, derivative seven, derivative eight and ABOB were 568μg/mL,170μg/mL,59μg/mL,104μg/mL,27μg/mL,71μg/mL,29μg/mL,286μg/mL,112μg/mL and 900μg/mL respectively. And the toxicity of derivatives were larger than that of Ginsenoside. Under safe concentration, the ginsenoside and its derivatives could stimulate cell proliferation at certain concentrations and time points, which had relations to dose and time effect.Assay for antiviral activity in vitro: Cytopathic effect (CPE) reduction, Neutral red staining and plaque forming reduction method were employed for antiviral assay. ABOB was used as positive controls in the antiviral assay. The ginsenoside and its derivatives in various concentrations were applied to CEF cell infected by MDV. Then the in vitro anti-MDV activity of drugs was observed from 3 aspects: (1) inhibiting the virus directly; (2) obstructing absorption; (3) inhibiting replication. The CPE inhibition data were expressed as the 50% effective (viral CPE-inhibitory) concentration (IC50), and selectivity index (SI), determined as the CC50/IC50.The results indicated that Ginsenoside and its derivatives could inhibit multiplication on MDV in vitro. When adding medicine and virus together to the single cell layer, ginsenoside, derivative two and derivative six could significantly suppress MDV's multiplication. The 50% effective (viral CPE-inhibitory) concentrations (IC50) of ginsenoside, derivative two and derivative six were 170.55μg/mL, 13.49μg/mL and 8.66μg/mL, and selectivity indexes (SI) were 3.33, 4.37, and 3.35 respectively. When adding the virus after the interaction of the medicine and cell, ginsenoside, derivative one, derivative six, and derivative seven have significant antiviral effects.The 50% effective (viral CPE-inhibitory) concentration (IC50) of ginsenoside, derivative one, derivative six, and derivative seven were 64.96μg/mL, 34.55μg/mL, 6.16μg/mL and 20.83μg/mL, and selectivity indexes (SI) were 8.74, 4.92, 4.71 and 13.73 respectively. For adding the medicine after attachment of the virus, ginsenoside, derivative seven and ABOB can significant inhibit MDV. The 50% effective (viral CPE-inhibitory) concentrations (IC50) of ginsenoside, derivative seven and ABOB were 100.89μg/mL, 61.98μg/mL and 117.25μg/mL, and selectivity indexes (SI) were 5.63, 4.61 and 7.68 respectively. It suggested that they possibly behaved by the mechanism on inhibiting virus directly or promoting cell antiviral activities or both.To study the interaction of between medicine and virus directly in vitro: Using the transmission electron microscope, we observed the morphologic change of the viral particles with negative staining after the interaction of medicine and MDV, and in order to study medicinal antiviral mechanisms, ultrastructural myocardial changes of CEF cell infected by MDV were observed in different times after medicine treatment.The negative staining results showed that drugs have significant damaging effect on the morphosis, superficial components and dispersion degree of the viral particles. The viral particles deformed, and the sizes the particles were not uniform, the envelopes were denuded or damaged. The viral particles were fused into masses, and the structure was blurred. The observation results of ultrastructure of CEF infected by MDV demonstrated that ginsenoside and its derivative seven could apparently inhibit MDV. The microscopic ultrastructural myocardial damage of medicinal treatment groups were lesser, the range of focus was smaller than that in the viral control group. To observe the results of antiviral effects of ginsenoside and derivative seven against MDV in vivo: The MD animal model was established with chicken in 1day.ABOB was employed as a positive control drug. Anti-MDV efficacy was assessed by morbidity rate, survival rate; mean day to death of chicken dying prior to 65 day.The animal experiments confirmed that ginsenoside and derivatives seven indeed showed antiviral principle activity in vivo. Drugs were effective on preventing death, the survival rate of ginsenoside and derivatives seven were 75% and 80% respectively. The survival rate of treatment groups was higher than viral control group, which is 45%(p<0.05). In tumor happening rate, the ginsenoside and derivatives seven were 205% and 185% respectively. The tumor happening rate of ginsenoside and derivatives seven was lower than those viral control group (304%) or positive drug control group (245%), and the difference rate is less than 0.05. Hence, the anti-tumor effects form high to low is derivatives seven, ginsenoside and ABOB one by one. The weight changes showed that the increasing weight of treatment groups was bigger than that of viral control group, furthermore, the treatment groups of ginsenoside and derivatives seven is more remarkableness in body weight increasing than that the treatment group of ABOB. This showed that the Chinese medicine on ginsenoside and derivatives seven could promote body growth and raise immunity.To further study the mechanisms of ginsenoside and derivative seven against Marek's disease Virus. The focus of the experiments was on the remedial action of ginsenoside and derivative seven to MDV infection about variety of viscera pathology by HE staining and ultrasreucture observation; The antigen's amount and distribution between treatments groups and viral control group was observed by immunofluorescence. The distribution and appearing time of viral nucleic acid was examined by polymerase chain reaction; the defense effect was assessed with thymus index and bursal index.Treated the tissue by HE staining and ultrasreucture observed the morphological changes. The results demonstrated that ginsenoside and derivative seven could decrease the pathological changes in viscera, eliminate the virus amount notably; The immunofluorescence assay indicated that the antigen's amount and distribution in treatments groups is less than those in viral control group; The PCR experiment found that the appearing time and distribution of viral DNA is the same as those of viral control group. In treatment group, the thymus index and bursal index is bigger than those of viral control group, but the difference is not significance (p>0.05).As set forth, the ginsenoside and its derivatives have antiviral function against Marek's disease virus.The derivatives have higher antiviral effects than those of ginsenoside. In vitro, the medicines could decrease viral infecting to CEF cell, and in vivo, the drugs can bring down the death rate and reduce tumor happening rate of chicken infected by experimenter. The antiviral mechanism of ginsenoside and its derivatives should raise the defense ability, or directly damage to virus. This experiment has laid a strong foundation in order to explore definite antiviral component of ginsenoside, increase antiviral effect, and develop a highly effect and lowly toxic medicine used as antiviral and anti-tumor drugs.
|
PDF Full Text Request