Screening And Evaluation Of Traditional Chinese Medicine Compound For Promoting Animalgrowth And Enhancing Immunity

Feed additives refer to substances added to animal diets that can enrich the nutritional components of animal diets,improve the nutrition and health level and performance of the body,improve the quality of animal products,and reduce the breeding cost.Chinese herbal medicine has the advantages of natural safety and abundant resources.As a feed additive in animal production,Chinese herbal medicine has the functions of resisting bacteria and viruses,enhancing animal disease resistance and improving organism growth and development.Since the country entered the period of no resistance and no resistance,the research and development of Chinese herbal medicine feed additives and related products have attracted more and more attention from the industry.In this paper,the cell experiments were conducted to determine the promoting effects of several traditional Chinese medicines on cell growth,and then different compounds were formed.In vitro experiments and in vivo experiments were conducted to verify the effects of different traditional Chinese medicines on organism growth and immune enhancement.Finally,an effective,economical and green traditional Chinese medicine compound which can promote animal weight gain and improve animal immunity was determined.The acute oral toxicity test in mice proved that the traditional Chinese medicine compound was actually a non-toxic product.The research contents and results of this topic are as follows.1.Screening and formulation of traditional Chinese medicine compound prescriptionIn this study,through CCK-8 cell viability experiment,it was found that the best action time of each traditional Chinese medicine on C₂C₁₂cells was 24 h,and the best action concentrations at this time were:Atractylodes macrocephala 1.95 mg/m L,Codonopsis pilosula 7.80 mg/m L,Angelica sinensis 3.91 mg/m L,Glycyrrhiza uralensis0.98 mg/m L,Radix Isatidis 7.80 mg/m L,Astragalus membranaceus 7.80 mg/m L,Hawthorn 7.80 mg/m L and Ligustrum lucidum 7.80 mg/m L.The last few traditional Chinese medicines were combined into five different compounds at the best action time and concentration,and the effects of traditional Chinese medicines on promoting weight gain and improving immunity were explored from cell experiment and animal experiment respectively.The five kinds of compound prescription are:Compound 1:Astragalus membranaceus,Angelica sinensis,Codonopsis pilosula,Radix Isatidis,Atractylodes macrocephala and Glycyrrhiza uralensis.Compound 2:Astragalus membranaceus,Hawthorn,Atractylodes macrocephala and Ligustrum lucidum.Compound:Astragalus membranaceus,Hawthorn,Angelica sinensis and Ligustrum lucidum.Compound:Astragalus membranaceus,Hawthorn,Angelica sinensisCompound 5:Astragalus membranaceus and Hawthorn2.Chinese herbal compound can promote the proliferation of C₂C₁₂cells.In this study,the effects of five different Chinese herbal formulas on the proliferation of C₂C₁₂cells were investigated by Edu cell proliferation experiment.It was found that the number of C₂C₁₂cells in the five Chinese herbal formulas increased,and the number of cells in S phase increased,which indicated that Chinese herbal formulas could promote cell division and proliferation.Compound 2,compound 3 and compound 4 are more obvious.3.Traditional Chinese medicine compound can enhance the phagocytosis of RAW264.7 cells.The neutral red staining method was used to explore the effects of five different traditional Chinese medicines on the phagocytosis of RAW264.7 cells.It was found that the number of cells in each compound group increased,the red area expanded and the red degree deepened,which indicated that traditional Chinese medicines could promote the phagocytosis of neutral red dye by RAW264.7 cells.After the effects of traditional Chinese medicines,compound 1,compound 3 and compound 5 could significantly improve the macrophage activity and enhance the phagocytosis.4.Effect of traditional Chinese medicine compound on growth and development of mice.Compound extracts of five kinds of traditional Chinese medicines were prepared,and the mice were gavaged at the concentration of 3 g/kg,with the dosage of 10 m L/kg b.w.and the feeding period was 14 days.During the experiment,mice were weighed at the same time every day,and the weight of feed and drinking water were weighed every other day.Through the analysis of the weight of mice,it was found that compared with the blank control group,five kinds of traditional Chinese medicine compounds could promote the weight growth of mice,among which compound 1 to compound 4 increased by 7.4%,9.1%,7.8%and 8.2%respectively,and compound 5 group increased by 12.5%（p>0.05）.Through the analysis of feed weight and drinking water weight,it was found that the feed intake and drinking water consumption of compound five groups were higher than those of other groups（p>0.05）.By calculating the ratio of feed intake to body weight of mice,it was found that compared with the blank control group,each compound group could reduce the feed-weight ratio,among which compound 2,compound 4 and compound 5 decreased by 38.7%,40.0%and 41.1%respectively.After feeding,the mice were dissected.By extracting RNA from muscle tissue and liver tissue,the effects of traditional Chinese medicine compound on m RNA expression of related genes in mice were detected.It was found that the expression levels of Myod and Myog in muscle of compound 1 group increased significantly,the expression level of Myf5 increased（p>0.05）,and the expression level of Mstn decreased（p>0.05）.The expression levels of Myod,Myog and Myf5 in the muscles of the two groups increased（p>0.05）,while the expression level of Mstn decreased（p>0.05）.The expression levels of Myod,Myog and Myf5 in muscle were up-regulated in the three groups（p>0.05）,and the expression level of Mstn was down-regulated（p>0.05）.In the four groups,the expression level of Myf5 in muscle and GH in liver were significantly increased,the expression level of Mstn in muscle was decreased（p>0.05）,and the expression level of IGF-1 in liver was increased（p>0.05）.The expression levels of Myog and Myf5 in muscle,Mstn and Myod in liver,IGF-1 and GH in the five groups increased significantly（p>0.05）.The total protein of muscle tissue was extracted,and the effect of traditional Chinese medicine compound on the expression level of MSTN in mouse muscle tissue was detected by Western Blot.It was found that all compound groups down-regulated the expression level of MSTN in mouse muscle tissue,and compound groups 1,2 and 5 could significantly inhibit the expression of MSTN.5.Effect of traditional Chinese medicine compound on immune function of mice.After feeding,the mice were dissected,the spleen and thymus were harvested,the blood and water on the organ surface were dried with clean gauze,the organ weight was weighed,and the immune organ index was calculated.It was found that Compound 5could significantly increase the spleen weight,and the spleen index increased by 49.5%,while Compound 1,Compound 2 and Compound 4 could increase the spleen weight,and the spleen index increased by 38.1%,29.1%and 39.6%respectively,but there was no significant difference.The four groups significantly increased thymus weight,thymus index increased by 63.4%,thymus weight of mice of Compound 1,Compound 2 and Compound 5 also increased,thymus organ index increased by 61.3%,25.7%and 46.3%respectively.RNA was extracted from spleen tissue,and the effects of various Chinese herbal formulas on the expression levels of IL-2 and IL-1βin mouse spleen were detected.It was found that the expression levels of IL-1βin spleen were significantly up-regulated in four groups,and those of IL-1βand IL-2 in other groups were also increased（p>0.05）.6.Acute toxicity test of traditional Chinese medicine compound to miceThe above experimental results show that compound Chinese medicine 5 has a good comprehensive effect.The acute oral toxicity test of compound Chinese medicine 5 was carried out in mice,and the 50%lethal dose(LD₅₀)and the maximum tolerance dose（MTD）of compound Chinese medicine extract were determined to evaluate the toxic and side effects of compound Chinese medicine on mice and judge the safety of the medicine.Traditional Chinese medicine compound 5 was orally administered to mice at a dose of 20g/kg b.w.for three consecutive times,with an interval of 4-6 hours.It was found that the mental state,appetite and behavior of mice were normal,and there was no poisoning or death.Finally,it was determined that the maximum tolerance of compound Chinese medicine 5 in mice was 60 g/kg,which was an actual nontoxic drug.7.Study on the application of traditional Chinese medicine compound to broilers.The above experimental results show that Shanqi compound（Chinese medicine compound 5）composed of hawthorn and astragalus has the best effect and is safe and nontoxic to mice.A listed positive drug,Ligustrum lucidum extract powder,was purchased as a positive control group,and a blank control group,0.5%Shanqi powder group,1%Shanqi powder group and 1%Shanqi extract group were set up to explore the effect of Shanqi compound on target animals and the difference of effects of different administration forms.The results showed that the feed-weight ratio of broilers in each drug group decreased,and 0.5%Shanqi powder and 1%Shanqi extract could significantly increase the weight of broilers,increasing by 22.6%and 19.8%respectively,and the food intake and water consumption of the two groups also increased,suggesting that compound prescription might promote the weight gain of animals by promoting the food intake and water consumption of broilers.Ligustrum lucidum extract powder group can significantly increase the m RNA expression of Myog,0.5%Shanqi powder can significantly increase the m RNA expression of Myf5 and IGF-1,1%Shanqi powder can significantly increase the m RNA expression of GH,and 1%Shanqi extract can significantly increase the m RNA expression of IGF-1 and GH.Each drug group can promote the weight of broiler spleen,thymus and bursa of fabricius,and increase the immune organ index.0.5%Shanqi powder can significantly increase the weight of broiler spleen,and the spleen index is increased by 29.3%.1%Shanqi extract can significantly increase the m RNA expression level of IL-2,while Ligustrum lucidum extract powder and 1%Shanqi powder can significantly increase the m RNA expression level of IFN-γ.These results show that each drug group can promote the growth and development of broilers by increasing the expression of myogenic regulatory factors and growth-related factors and inhibiting the expression of myostatin,improve the storage and utilization efficiency of nutrients,improve the growth performance of broilers,and improve the immune performance of broilers by promoting the development and maturity of immune organs,increasing the level of immune cytokines.