| ObjectiveInfluenza is a kind of acute respiratory infectious diseases caused by influenza virus that characterized by fast spread and powerful infectivity. It has caused worldwide pandemic several times, which seriously threatened human daily life and health. At present, there is not a good method that can safely and effectively prevent and treat influenza. The antiviral research of garlic oil has made initial progress, such as cytomegalovirus, enterovirus, mouse hepatitis virus and herpes progenitalis, but not influenza virus. Experiments in vitro and in vivo will be conducted to explore the anti-influenza virus effect of garlic oil and mechanism, which will provide reference for the research on garlic oil anti-influenza.Methods1. Multiplication of influenza virus:0.2mL human seasonal H1N1influenza virus (influenza virus) was injected into chick embryo allantoic cavity and incubated for44-48h at34℃, and then incubated for6h at4℃before harvest. Sterile allantoic fluid was inoculated in the next chick embryo as described above, until blood clots titer of allantoic fluid was higher than1:128. Then make allantoic fluid mixed and saved at-80℃.2. Experiment in vitro on garlic oil anti-influenza virus:both the cytopathic effect (CPE) and3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were used to determine0%toxicity concentration (TCo) of garlic oil against MDCK cells, and50%cell culture infectious dose (CCID50) of influenza virus against MDCK cells. Then observe the antivirus effect of garlic oil in vitro experiments against H1N1in four dosing ways:precaution group, direct effect group, anti-adsorption group and anti-biosynthesis group. Every dosing way included blank control, virus control, solvent control (dimetylsulfoxide, DMSO), positive drug control (ribavirin) and a series of garlic oil diluted. Respectively calculated effective rate (ER) and therapeutic index (TI) on garlic oil anti-influenza virus.3. Constructing the animal model of influenza virus:10KM male mice were inoculated influenza virus by nasal cavity. After4days, the mice whose infectious signs were serious were killed. Pneumonic homogenate that were confirmed to have infected influenza virus by real time fluorescence quantitative polymerase chain reaction (FQ-PCR) were injected in chick embryo allantoic cavity. Allantoic fluid that had the highest blood titer was inoculated in the next generation mice, until infected mice appeared obvious infectious signs and calculated50%lethal dose (LD50). The dose of IOLD50was inoculated into the mice to establish infection model. Then, observed general state, weight, lung index, pathological sections and detected viral RNA by FQ-PCR.4. Precaution effect and death effect of garlic oil on mice infected influenza virus: The mice were randomly divided7groups which respectively were blank control, virus control, solvent control (corn oil), positive drug control (amantadinehydr-ochloride), low dose of garlic oil, middle dose of garlic oil and high dose of garlic oil, and constructed respectively experimental models. The mice infected by H1N1on the body weigh、lung index、average survival time and death protection rate were deserved to estimate the anti-influenza activities of garlic oil.5. Immunoregulation effect of garlic oil against mice infected influenza virus: The mice were randomly divided7groups as above, and constructed respectively experimental models. Observed lymphocyte transformation function stimulated by ConA and NK cytoactive. Assayed the content of TNF-α, IFN-γ and IL-10.6. The methods of statistics:Assay excel data base and SPSS16.0statistics software to process data, all the test level was a=0.05.Results1. Influenza virus was handed down for4generations, and the blood clots titer reached1:128.2. CCID50of influenza virus against MDCK cells was10-3/0.1mL48h later. TC0of garlic oil against MDCK cells was0.13mg/L, and50%toxicity concentration (TC50) of garlic oil was5.87mg/L. In precaution group, the most effective antiviral concentration was0.1mg/L, ER was72.5%which was higher than positive drug group. In direct effect group, the most effective antiviral concentration was0.13mg/L, ER was51.4%which was lower than ribavirin(.P<0.05). In anti-adsorption group, the value of ER heightened as concentration increased, but still lower than ribavirin group (P<0.05). The most effective antiviral concentration was O.OOlmg/L. In anti-biosynthesis group, TI of garlic oil was higher than positive control ribavirin in all groups (P<0.05). The value of TI was7159which was the highest in all groups.3. Influenza virus was handed to the fourth generation whose LD50was10-2.41/0.05mL. Mice infection model of influenza virus was established, with serious infectious signs, such as tachypnea, crouch, anorexia and activities slow, and70%of death rate. Moreover, hyperemia obviously, inflammatory cell infiltration, lung index increase. Results of FQ-PCR were all positive.4. Precaution effect and death effect of garlic oil on mice infected influenza virus: Compared with the virus control group, garlic oil could inhibit the mice infected by H1N1from losing weight, reducing lung index, relieving the inflammation of lung. In the experiment of death protection, garlic oil could reduce the death rate and extend survival time (P<0.05).5. In group of middle dose garlic oil, the function of lymphocyte transformation stimulated by ConA was higher than blank control, virus control and solvent control (P<0.05). The group of high dose garlic oil had the highest cytoactive (P<0.05). The content of TNF-α was no difference among of groups (P>0.05). Three garlic oil group had lower content of IFN-γ and IL-10than the other groups (P<0.05).Conclusions1. Garlic oil has the strong antivirus effect on influenza virus in vitro, and has the high safety. This is related to its blocking influenza virus to invade cells and the effect of killing virus and preventing viral absorption.2. Garlic oil has the strong antivirus effect on influenza virus in vivo, which is considered that garlic oil can improve NK cytoactive and cellular immunity. |
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