Antiviral, Antioxidant And Hepatoprotective Activities Of The Protein Extraction From The Larvae Of Housefly

Housefly(Musca domestica vicina M.) distributed all over the world.The propagation coefficient of housefly is very high and their life history is short.The technology to breed is very simple and they are listed as the top new type resource insect enriched of protein.Housefly lives in the condition of pathogen propagating and completes its life history in decaying organic material.There is a large number of bacterium in the larvae of housefly both in vitro and in vivo.They can spread various diseases between humans and other animals,while they can thrive without disease.Their strong disease-resisting abilities become a new topic of general interest.In this study, bioactive proteins were extracted from the larvae of housefly and their chemical composition was analyzed.Moreovr,their antioxidant,hepatoprotective and antiviral activities were also detected.The major results were as following:1.Antiviral activity of tissue homogenate of the housefly larvaeThe method of chicken embryo and the diagnostic reagent kit for surface antigen of Hepatitis B virus(HBV) were adopted to detect the antiviral activity of tissue homogenate of the housefly larvae in vitro.The results showed that different concentrations of tissue homogenate had significant effects on inhibiting the proliferation of influenza virus in chicken embryo.The results of the diagnostic reagent kit also indicated that the tissue homogenate had remarkable destructive effect on HBV and the destructive efficiency reaches 87.5%.2.Isolation and characteristics of active protein from the larvae of houseflyFive sorts of proteins were isolated from the body of the housefly larvae according to their solubility based on the defatted powder and their antioxidant activities and antiviral activities were measured.Further isolation was processed on the component with high content and well activity of antiviral and antioxidant.An optimal technology for preparation was determined.Further isolation was processed on the protein extraction by cation exchange chromatography and the antiviral activity of each fraction was detected. The results showed that albumin was the main component of the housefly defatted powder and it had better antioxidant and antiviral activities than others.Therefore,we further isolated and obtained the protein extraction.The optimal extracted condition for NaC1 0.18 mol/1 and pH value 7.Four fractions(A1-4) were isolated from the protein extraction.The antiviral results showed that all of the fractions inhibited the proliferation of influenza virus in some degree,but the effects had not been improved compared with the protein extraction.3.Antiviral activity against Newcastle disease virus(NDV) The method of chicken embryo was adopted to detect the depressive effect of the protein extraction on the proliferation of Newcastle disease virus in chicken embryo.The result of toxicity test showed that the survival rates of all group were 100%after injection of protein extraction with different concentrations.This indicated that protein extraction had no effect on the growth and development of chicken embryo and it had no toxicity.The antiviral results showed that the protein extraction inhibitted the proliferation of Newcastle disease virus in chicken embryo at different concentrations.4.Antiviral activity against Poliomyelitis virus(PV)The method of cell culture was adopted to detect the antiviral activity of protein extraction in vitro.End-point dilution method was adopted to detect the effect of protein extraction on Poliomyelitis virus after neutralization of protein extraction and virus in vitro.We also detected the minimum depressive concentration of protein extraction on 100个TCID₅₀ virus and the selectivity index(SI) was calculated.The MTT method indicated that protein extraction had minimal cytotoxicity against Hela cells and the CC₅₀ was 331.13μg/mL.The preliminary studies on the inactivation of PV-1 indicated that the loss of infectious capacity was more than two log(10) units when the virus suspensions were pre-incubated for 2 h with protein extraction prior to the cell infection.Protein extraction strongly elicit its antiviral activity against PV-1 in Hela cells with an EC₅₀ of 10μg/mL and the SI was calculated to be more than 33.All of the results indicated that protein extraction had antiviral activity against PV-1 virus.5.Antiviral activity against avian influenza virus(AIV)The interaction between virus and cell made it possible to detect anti-influenza virus agents on the basis of the modification of the HA test.The method of HA test was adopted to detect the antiviral activity of protein extraction on avian influenza virus H₉N₂ in vitro.The results showed that protein extraction had little toxic effect on red blood cells and no cytopathic effects were observed below the concentration of 5 mg/mL. Different doses of protein extraction exhibited,to some degree,inhibition of viral adsorption of RBC and the end point of haemagglutinin dilution was greatly decreased compared with the control.6.Antiviral activity against herpes simplex virus(HSV)The method of cell culture was adopted to detect the antiviral activity of protein extraction in vitro.Three actions were detected as follows:neutralization of protein extraction and virus in vitro,simultancous effect of protein extraction and viruses and protective effects of protein extraction on Vero cells.Protein extraction exhibit remarkable antiviral activities against herpes simplex virus on the first two effect patterns. While the result of protective effects on cells showed that protein extraction had no effect on the infectivity of virus.This suggested that the effect of protein extraction could not due to the protective effects on cells.7.Antiviral activity against influenza A virusThe method of cell culture was adopted to detect the antiviral activity of protein extraction on influenza A virus in vitro.HA test was used to detect the virus titer in the supernatant of the cells culture infected by influenza A virus.The MTT method indicated that protein extraction had minimal cytotoxicity against Vero cells and the CC₅₀ was 284.45μg/mL.Protein extraction remarkably depressed the infectivity of virus after the neutralization action with virus,which was better than the positive drug Ribavirin.Both protein extraction and Ribavirin significantly inhibited the proliferation of influenza A virus in MDCK cells when drugs and viruses acted simultaneously.Both protein extraction and Ribavirin had no protective effect on the MDCK cells and could not depress the proliferation of virus effectively.Ribavirin had strong therapeutic effect on the virus infection,while protein extraction had virucidal activity on influenza A virus. This might be related to the mechanism of their action.8.Antioxidant effects of protein extractionThe Fe³⁺-ascorbate-EDTA-H₂O₂ system and the auto-oxidation system of pyrogallol were adopted to detect the antioxidant activity of protein extraction.The antioxidant results showed that the protein extraction had good scavenging activities on both hydroxyl radical and superoxide anion radical.The 50%hydroxyl radical scavenging concentration of the protein extraction was 1.23 mg/mL.The 50%superoxide anion radical scavenging concentration of the protein extraction was 34.63μg/mL calculated from the dose-activity curves.At the concentration of 1 mg/mL,superoxide anion radicals were completely scavenged.9.Hepatoprotective effect of protein extractionThe protective effects of protein extraction on CCl₄-induced hepatotoxicity were studied from both biochemical measurements and morphological observations.The results showed that the model of hepatic injury was successful.The AST and ALT activities of serum were significantly decreased and the ability of hepatocytes to synthesis protein was greatly improved after treated with the protein extraction.Photomicrographs of haematoxylin-eosin stained liver tissue showed that the single administration of CCl₄ caused serious liver damage in all animals of this group.The hepatocytes around the central vein were complete necrosis and the cellular boundary was lost.No histological abnormalities were observed in rats of the control group and also the groups of administration of various doses of protein extraction pretreated with CCl₄.The histological changes associated with the hepatoprotective activity in three prescriptions of protein extraction supported the estimation of the biochemical results.In conclusion,the hepatoprotective effects of protein extraction from the larvae of M.domestica were demonstrated by the biochemical measurements and the morphological observations using the acute hepatic damage model.In conclusion,protein extraction isolated from the larva of housefly,had remarkable antioxidant,hepatoprotective and antiviral activity in vitro.It had no adverse reaction on our body and it had preliminary clinical and applicable value in medicine.