Antivirus And Immune-Enhancing Activity Of Several Chmis And Their Prescriptions

For a long time, viral diseases have greatly threatened breeding industry and it is short of available medicines to cure. At present, vaccination is the important measure to prevent the diseases and the cooperation of vaccine with immunopotentiator can further improve immunization effect. However, some chemical immunopotentiators such as oil emulsion, aluminum hydroxide commonly have disadvantages, like side effects, strong local stimulation and carcinogenesis, complicated preparation, or not improving the antigenity of infirm antigen. Therefore, it becomes an urgent problem to research and develop antiviral drugs and new immune adjuvants with high efficiency and low toxicity. Many researches proved that Chinese herbal medicine had the effects of antivirus and immunoenhancement, moreover, it is safe to utilize. Based on literature reports, seven Chinese herbal medicinal ingredients (CHMIs) with antiviral activity and immunoenhancement, oxymatrine(OM), astragalus polysaccharide (APS) and so on, and their prescriptions were selected to compare the effect of them on resisting Newcastle disease virus (NDV) and immune response of NDV vaccine in chickens. This study contained the following 8 parts.Test 1 Effect of seven CHMIs on safety concentration and growth of CEF Seven CHMIs, oxymatrine (OM), glycyrrhizic acid (GA), chlorogenic acid (CA), geniposide (GP), polydatin (PD), luteolin (LT) and astragalus polysaccharide (APS), were diluted into 10 concentrations with MM, OM, APS, GP and PD,2000-3.91μgty·mL-1, GA and CA,1000-1.95μg·mL-1, LT,50-0.09μg·mL-1 and added into monolayer chick embryo fibroblasts (CEF). The effects of CHMIs on growth of CEF were tested by MTT method and morphological variety of cells. The results showed that the maximal safety concentrations (μg·mL-1) of PD, APS and GA, GP and CA, OM, LT were 500,250,125, 31.25 and 3.13 respectively. All of seven CHMIs could promote the cellular proliferation within the suitable concentartions, in which GA was the strongest, the next were CA, PD, OM, GP, APS and LT, successively.Test 2 Effect of seven CHMIs on cellular infectivity of NDV Seven CHMIs were diluted into high, middle and low concentrations (μg·mL-1) within safety concentrations, PD:500,250,125; GA:250,125,62.5; APS and CA:125,62.5,31.25; OM and GP:32,16, 8; LT:1.6,0.8,0.4. These CHMIs and Newcastle disease virus (NDV) were added into CEF in three different ways (pre-, post- and synchronously). The effects of seven CHMIs on cellular infectivity of NDV were assayed by MTT method. The results showed that OM, GA and APS at high and middle doses and GP at high dose could inhibit NDV to infect CEF significantly in pre-adding CHMIs. OM at high and middle doses and APS at middle and low doses could obviously inhibit the infectivity of NDV in post-adding CHMIs. APS at high, middle and low doses, OM at high and middle doses, and GA at high dose could inhibit significantly the infectivity of NDV in simultaneous adding CHMIs and NDV after mixed. In general comparison, the order of inhibiting cellular infectivity of NDV was OM>APS>GA>GP.Test 3 Effect of four compound CHMIs on safety concentration and growth of CEF Compound CHMIs (cCHMIs), OM-APS, OM-APS-GP,GA-APS and GA-APS-GP, were combined with OM, APS, GP and GA according to certain proportion. Four cCHMIs were diluted into 10 concentrations,from 1000 to 1.95μg·mL-1, with MEM and added into monolayer CEF. The effects of four cCHMIs on the growth of CEF were assayed by MTT method. The results showed that safety concentration (μg·mL-1) of OM-APS, OM-APS-GP and GA-APS-GP were 1000μg·mL-1, GA-APS,500μg·mL-1.OM-APS, OM-APS-GP, GA-APS at 500-1.96μg·mL-1 and GA-APS-GP at 500-3.92μg·mL-1 could promote the growth of CEF significantly, in which the effect of OM-APS-GP was the strongest, the next was OM-APS.Test 4 Effect of four cCHMIs on cellular infectivity of NDV Four cCHMIs, OM-APS, OM-APS-GP, GA-APS and GA-APS-GP, were diluted into high, middle and low concentrations (500,250 and 125μg·mL-1) within safety concentrations. Four cCHMIs and NDV were added into CEF in three different ways (pre-, post- and synchronous), respectively. The effects of four cCHMIs on cellular infectivity of NDV were assayed by MTT method. The results showed that in pre-adding mode, all cCHMIs could significantly inhibit NDV to infect CEF at three doses. In post-adding mode, OM-APS at three doses, OM-APS-GP at middle and low doses, GA-APS at high and middle doses and GA-APS-GP at high and low doses could inhibit NDV infection obviously. In synchronous mode, OM-APS at high and middle doses, OM-APS-GP at middle and low doses, GA-APS at high and low doses and GA-APS-GP at high dose could inhibit NDV infection significantly. Comprehensively, the order of NDV infection was OM-APS> GA-APS> OM-APS-GP> GA-APS-GP.Test 5 Effects of two cCHMIs and their principal drug in controlling Chicken ND Five hundred and seventy 26-day-old non-immunized Roman cocks were randomly divided into 19 groups and blank control group were isolated housed. Vaccine control group were vaccinated with NDV-IV vaccine. At 30 old days, three preventive administration groups were given OM-APS, OM-APS-GP and OM with 1 mg by oral administration for 7 d, three times per day. At 33 old days, all the groups were injected with virulent NDV by intramuscular injection, three groups were given OM-APS, OM-APS-GP and OM with lmg by oral administration for 4 d, three times per day. Twelve hours after challenge, nine treatment groups were given OM-APS, OM-APS-GP and OM at high, middle and low doses (2 mg, lmg and 0.5mg) and in ribavirin control group were given 10 mg ribavirin by oral administration for 4 d, three times per day. Virus and vaccination control groups were not treated. Seven days after challenge, effects of compounds and their principal components on preventing and treating chicken ND were evaluated by mortality, serum antibody titers, organ hemorrhage rate of dead chickens, total weight gains and average body weight gains of chickens. The results showed that mortality of OM-APS at three doses, OM-APS-GP at high, low doses and preventive administration group were significantly lower than that of virus control group, OM-APS at high dose and preventive administration group, OM-APS-GP at low dose and preventive administration group being significantly lower than that of ribavirin control group. Serum antibody titers of fiveteen groups were higher than that of virus control group, OM-APS at high and low doses and preventive administration group, OM-APS-GP at high and middle doses being higher than that of ribavirin control group. Cecal tonsil hemorrhage rate in OM-APS at high, middle and low doses, preventive administration group of OM-APS-GP were significantly lower than those of virus control and ribavirin control groups. Gastric hemorrhage rate of two compounds at three doses and preventive administration group and successive administration group were significantly lower than that of virus control group. Intestine mucosas of OM-APS group at middle and low doses and OM-APS-GP at high dose were significantly lower than that of virus control group. Percentage of total weight gains of OM-APS at high dose and preventive administration groups of OM-APS-GP, average weight gains of OM-APS at high and middle doses and preventive administration group of OM-APS-GP were significantly higher than those of ribavirin control and virus control groups. This was manifested that two compounds had better prevention and treatment effect against ND and the effect of OM-APS was the best.Test 6 Effects of four CHMIs and their compounds on proliferation of chicken splenic lymphocyte Four CHMIs, OM, APS, GA and GP and their four compounds, OM-APS, OM-APS-GP, GA-APS and GA-APS-GP, were diluted with RPMI1640 into 5 concentrations within safety concentrations from 15 to 0.9μg·mL-1. They and concanavalin A (ConA) were added into the 96-well culture plate with splenic lymphocyte. The changes of splenic lymphocyte proliferation were tested by MTT method. The results showed that A570 values of APS, OM, GA, OM-APS, OM-APS-GP and GA-APS at five concentrations, GA-APS-GP at 0.9μg·mL-1 were significantly higher than those of cell control and ConA control groups (P< 0.05). This manifested that four CHMIs and their compounds could promote splenic lymphocyte proliferation, in which the effects of APS and OM-APS were the best, moreover the effects of compound CHMI were better than that of single CHMI.Test 7 Effects of cCHMIs and their components on immune response of ND vaccine Five hundred and ninety-four 14-day-old non-immunized Roman cocks were randomly divided into 11 groups. Except for blank control group, other groups were vaccinated with NDV-Ⅳvaccine by intraocular-nasal route, repeated vaccination at 28 days old. At the same time of the first immunization, the chickens in experimental groups were given OM-APS, OM and APS at high, middle and low doses (4,2 and 1 mg·mL-1) respectively by oral administration,1 mL each chichen, in vaccine and blank control groups with physiological saline, once a day for three days. On 14,21,28,35 and 42 day old, peripheral T lymphocyte proliferations and serum antibody titers were determined by MTT method andβ-microscopic method respectively. On 14,21,28 and 35 day old, the organ index of spleen and bursa were determined. At 69 days old, all chickens were injected with virulent NDV and the protective rate was observed. The results showed that at all time point T lymphocyte proliferation of OM-APS, OM and APS at high and middle doses groups and serum antibody titer of OM-APS at high and middle doses groups were obviously higher than those of vaccine control group. At all time point, spleen indexs of OM-APS group at three doses and bursal indexs of OM-APS group at high and middle doses were significantly higher than those of vaccine control group. Protective rates of OM-APS and APS groups at three doses, group OM at middle and low doses were higher than that of vaccine control group.Test 8 Effects of cCHMIs and their components on IL-2 and IFN-y level in chicken vaccinated with ND vaccine Five hundred and ninety-four 14-day-old non-immunized Roman cocks were randomly divided into 11 groups. Except for blank control group, other groups were vaccinated with NDV-Ⅳvaccine by intraocular-nasal route, repeated vaccination at 28 days old. At the same time of the first vaccination, groups 1 to 9 were given OM-APS, OM and APS at high, middle and low doses (4,2 and 1 mg·mL-1) respectively with oral administration, in vaccination and blank control groups with physiological saline, once a day for three days. At 14,28 and 42 day old, concentrations of serum IL-2 and IFN-y were determined by double-antibodies-sandwich-ELISA method. The results showed that concentrations of serum IL-2 in OM-APS group at three dose groups, OM at high, low dose groups and APS at high, middle dose groups were significantly higher than that of vaccine control group at all time point (p<0.05). Concentrations of serum IFN-y in OM-APS at high and middle dose groups at all time point, low dose group at 42 day-old were obviously higher than that of vaccine control group (p<0.05). Concentrations of IFN-y in serum APS at high and middle dose at 28 and 42 day-old and OM at middle dose at 42 day-old were obviously higher than that of vaccine control group (p<0.05). This manifested that OM-APS, OM and APS could promote the secretion of IL-2 and IFN-y in chicken and the effects of prescriptions were better than that of single ingredients.