Safety Evaluation Of Musca Domestica Larvae Powder

Introduction: Musca domestica larvae is a kind of traditional Chinese medicine, which name is Oriental Latrine Fly Larvina, Common name is Maggot. In the preliminary work of this research group, the shells of third instar Musca domestica larvae is processed into chitosan, the other solution is vacuum dried into maggot protein powder. Maggot protein powder and chitosan are mixed into Musca domestica Larvae Powder (MDLP), which can anti-hyperlipidemia and anti-atherosclerosis, especially in 10:1 ratio of combination. In this study, Maggot protein powder and chitosan are mixed into 10:1 ratio of combination.Kunming mice, Sprague Dawley (SD) rats are used as study subjects.Acute toxicity test,Bone marrow cell micronucleus test,Mice sperm abnormality test,Teratogenicity study,Chronic toxicity test are used to preclinical safety accessement of MDLP; measurement of angle of repose, content uniformity test, Moisture test, disintegration test, Microbial limit test, are used in Initial Quality Evaluation of MDLP.Objective:To study the toxicity of Musca domestica Larvae Powder (MDLP) in rodents and have A preliminary quality evaluation of MDLP capsules,evaluate the preclinical safty of MDLP,speculate the possible Toxic effects or adverse reactions of MDLP in clinical trials , Provide experimental evidence for clinical trials of MDLP.Methods:1.Preparation of MDLP. Feed the musca domestica larvae with milk powder,wheat bran and yeast powder.when they grow into 3 instar,stimulate them by cold ,then collect them . they are smashed into solution and shell by ultrasound. Solution is vacuum dried into maggot protein powder and shell is processed into Chitosan. 10 dividers 0f maggot protein powder and 1 dividers of chitosan are mixed into MDLP.2.Acute toxicity test of MDLP. MDLP are dissoved into 0.5% acetic acid solution,which is saturated into 0.275g/ml.The mice are divided into 2 groups to intragastric administration: MDLP maximun tolerated dose group; 0.5% acetic acid solvent control group. General state, appearance , body weight growth and death are study. The mice are sacrificed and dissected , the grass condition and histopathology specimen of liver and spleen of them are study.3.Bone marrow cell micronucleus test of MDLP. MDLP are dissoved into 0.5% acetic acid solution. The mice are divided into 5 groups to intragastric administration:High dose group of MDLP,middle dose group of MDLP,low dose group of MDLP, solvent control group, cyclophosphamide positive control group. The mice are sacrificed and their sternum marrow cells were collected , fixed with methanol and stained with Giemsa staining solution. The frequency of micronuclei peripheral reticulocytes (MNRETs) was counted.4. Mouse sperm abnormality test of MDLP. MDLP are dissoved into 0.5% acetic acid solution.The male mice are divided into 5 groups as the Bone marrow cell micronucleus test to intragastric administration. The mice are sacrifice and the filtrate of epididymises are prepared into sperm slides, fixed with methanol and stained with 1% eosin solution, the abnormal sperm rates of mice are counted under microscope.5. Teratogenicity test of MDLP.MDLP are mixed to animal feeds to be prepared high dose of MDLP animal feeds, middle dose of MDLP animal feeds,low dose of MDLP animal feeds. In each cage,2 female eugamic rats and 1 male eugamic rat are put together to mate. After conception, the pregnant rats are divided into the following group: high dose of MDLP animal feeds group, middle dose of MDLP animal feeds group,low dose of MDLP animal feeds group,common animal feeds group.growth information of body weight of the above 4 group pregnant rats are weighed.At the endpoint, pregnant rats are sacrificed, uteruses of them are weighed,absorpted foetus ,earlier died foetus before delivery, later died foetus before delivery and living foetus are counted ,taken notes. Appearance, Sex ,body weight and stem length of Each fetus are taken notes.Bone and internal organs specimen of rats'fetus are prepared and being taken photoes.6.Chronic toxicity test of MDLP. MDLP animal feeds are prepared as above test. The rats are randomly divided into 4 groups: high dose of MDLP animal feeds group, middle dose of MDLP animal feeds group, low dose of MDLP animal feeds group,common animal feeds group. The general state of health,body weight growth and food consumption are taken notes.The blood cells,blood biochemical indicator,urine biochemical indicator and blood coagulation are tested.The organ weight rate(in proportion to body weight) and histopathology examination are tested in 2/3 rats.The other rats are tested as above rats after stop the MDLP animal feeds. Expression of superoxide dismutase gene and endogenous controlβ-actin gene of liver specimen is examined by real-time quantitative Polymerase chain reaction at the endpoint.7. Quality analysis of MDLP. MDLP's angle of repose is measured with fixed-funnel method to assess its flowability; The moisture of MDLP is measured by weighing its weight before baking and after baking; Microbe content of MDLP is measured by microbial limit test;The quality of MDLP capsule is accessed by disintegration test;The homogeneity of MDLP is evaluated by content uniformity test.Results:1. Under the conditions of this acute oral test, Maximun tolerated dose for MDLP acetic acid solution is greater than 33.0g/kg bw. In comparison to the control group,no clinical abnormalities or deaths and no significant differences in body weight gain for either male or female mice. In addition, compared to the solvent control group,the findings of gross necropsy performed at study termination did not reveal any signs of toxicity;no significant differences in liver or spleen weight and no organic changes are found in histopathology evaluation.2. No significant differences in the frequencies of MNRETs were found between treated mice and the controls in the micronucleus test. Therefore, MDLP have no significant mutagenicity in mice.3. The frequencies of sperm abnormalities for three different dose groups were not significantly different compared with that of the negative group. The present test show that MDLP have no significantly Genetic damage to male reproductive cells of mice.4. In teratogenicity test, growth information of body weight and uteruses weight of pregnant rats, fetus'appearance,fetus'sex , fetus'body weight, fetus'stem length, fetus'bone and fetus'internal organs specimen for three different dose groups were not significantly different compared with that of the negative group.No absorpted foetus ,earlier died foetus before delivery, later died foetus before delivery for three different dose groups.5. Throughout the chronic oral toxicity study, there were no clinical signs or effects on survival that could be attributed to administration of MDLP in rats. no significant differences in animal weights and feed consumption were observed between any of the treatment groups and the control groups. There were no differences in mean organ weight, organ-to-body weight between controls and treated animals. histopathological analysis revealed no significant changes between treated and control animals. Blood cell count ,blood biochemistry levels , urine biochemistry levels and blood coagulation factor levels are all within normal ranges of SD rats. no significant differences in superoxide dismutase gene Expression levels between control group and high dose group in male rats; superoxide dismutase gene Expression levels for high dose group in female rats are up-regulation compared to control group.6. In quality analysis of MDLP, MDLP's angle of repose is lower than 45 degree,so its flowability is good;The moisture of MDLP is lower than 9%;No microorganism growth in MDLP; Disintegrated time of MDLP capsules is lower than 30min;Content uniformity of MDLP capsules is lower than 10%,so it is homogeneity.conclusion:No Acute organic toxicity, mutagenicity, teratogenicity or non-reversible chronic toxicity for MDLP; There are no Pathogenic micro-organisms in MDLP; MDLP is stable and relialbe, so it may be produced into anti-hyperlipidemia and anti-atherosclerosis drug capsules.