Study On Antioxidant And Bacteriostasis Effects Of Water Extracts From Artemisia Argyi Leaves

As people attach importance to health and advocate green,natural and pollution-free animal products,antibiotics have been restricted to be used as antibacterial drugs and growth regulators in livestock breeding,which undoubtedly brings tests to livestock breeding and management.At present,only plant-derived additives are allowed,because they are natural and low toxicity.Also restricted are feed additives,with only plant-derived additives still allowed.Therefore,it is of far-reaching significance to develop substances that can replace antibiotics or regulate animal growth in plants.Artemisia argyi(A.argyi)is a genus of Artemisia in the Compositae family.A.argyi leaves are considered as the main source of active substances of A.argyi,and rich volatile oil is considered as the main active ingredient.As the extraction technology,separation technology and identification technology are improved,the non-volatile components of the A.argyi leaves were also studied in various fields.However,the study lacks a systematic,mostly in the phase of the total extract,and less research on the specific components.Based on the above problems,this paper took A.argyi leaf as the test material and its extract as the research object,and set up five experiments.Experiment 1:Extraction of non-volatile components from A.argyi leaves with different polar solvents and preliminary identification of the main components.Experiment 2:Extracts of A.argyi leaves with different polarity were screened by antioxidant activity and antibacterial activity.Experiment 3:The effects of active components were evaluated by the growth and intestinal microflora of mice.Experiment 4:The effects of active components were evaluated by constructing in vitro model of bovine mammary epithelial cells inflammation.Experiment 5:The role of active components was evaluated by establishing an in vivo model of mammary gland inflammation in mice.In order to provide some guidance for the determination of active components and efficient development of A.argyi extracts,and provide the oretical basis for the development of natural drugs.The main research contents are as follows:1.According to similarity-intermiscibility theory,in order of polarity,Ethyl acetate(EAC),n-butanol(NBA),Ethanol(EA)and Water were used to extract A.argyi leaves powder without volatile components.The contents of total phenol,flavonoids and polysaccharides in different extracts were determined by Folin-Ciocalteu(FC)reagent method,NaNO2-AlCl3 complexometric spectrophotometry and phenol-sulfuric acid method,respectively.The results showed that the contents of total phenol,total flavonoids and polysaccharides were different in different polar parts.Total phenol,total flavonoids and polysaccharides were detected in total extract(TE)of A.argyi leaves extracted by 70%ethanol.The contents of total phenols and flavonoids in water extract(WE)were significantly higher than those in other polar components(P<0.05).The polysaccharide content of WE and EA extract(EAE)were similar,and were significantly higher than that of ethyl acetate extract(EACE)and N-butanol extract(NBAE)(P<0.05).The contents of total phenols and flavonoids in EACE and NBAE were also at low levels.2.According to previous studies,it can be observed that there were differences in the composition of compounds in different polar parts.Therefore,in order to further screen the active components of different polar fractions,the antioxidant capacity of different polar fractions was evaluated by Fe3+-reducing antioxidant power(FRAP)method and DPPH radical scavenging activity assay(DPPH).Then,Escherichia coli(E.coli)was used as the test bacteria to evaluate the bacteriostatic ability of different polar parts.Finally,the main components of the active components were separated and identified by high performance liquid chromatography tandem mass spectrometry(HPLC-MS)according to the evaluation results.TE had a certain antioxidant capacity,and the reduction ability of Fe3+and DPPH radical scavenging ability of WE were significantly higher than those of other polar components(P<0.05).The antioxidant capacity of EACE and NBAE was at a low level.It was found that TE,EAE and WE were all sensitive to E.coli by bacteriostatic evaluation of drug sensitive tablets,and the results showed concentration-dependent,in which WE was the most sensitive to E.coli.Therefore,WE showed the most outstanding performance in combination with antioxidant capacity and bacteriostatic capacity,and WE was selected for subsequent experiments.The antibacterial capacity of different concentrations of WE was further compared and the results showed that the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of WE were 2.5 mg/mL and 5 mg/mL respectively.Also,E.coli proliferation was completely inhibited when WE concentration was higher than 5 mg/mL.Further study found that E.coli biofilm formation had a significant inhibitory effect when WE concentration was not less than 1.25 mg/mL(P<0.05).Finally,11 main components in WE were identified by HPLC-MS,including 6 phenolic components and 5 flavonoids.3.Combined with the previous two experiments,it was found that WE(After called ALE)had high antioxidant capacity and bacteriostatic capacity,and most studies showed that substances with such functions could regulate the growth of experimental animals.Therefore,in order to further explore the function of ALE,40 BALB/c SPF female mice aged 6-8 weeks were fed with different concentrations of ALE(High dose:300 mg/kg,medium dose:150 mg/kg,low dose:75 mg/kg)and equal volume of normal saline as control for 60 days.The effects of ALE were analyzed by growth performance,blood related indexes and intestinal morphology of mice.The mechanism of ALE action was further explored from the microecology by analyzing the intestinal flora of mice.The results showed that there was no significant difference in average weight gain from mice(P>0.05),the medium-dose group finally showed a trend better than the control group.By measuring the average growth rate,the change of body weight of mice could be observed more intuitively.It was found that the growth rate of medium dose group was significantly higher than that of control group(P<0.05),and the growth rate of high dose at 10,30 and 40 days decreased significantly compared with the control group(P<0.05).It is worth mentioning that there was no significant change in the growth rate in the low-dose group compared with the control group(P>0.05).By dissecting the mice,it was found that ALE had little effect on the viscera of mice and the results of blood indexes showed that high dose ALE significantly up-regulated the levels of WBC,GRAN,ALT and AST(P<0.05),suggesting that high doses of ALE may have caused an inflammatory response in mice.Further sections of different intestinal tracts were made.The crypt depth and villus length of the intestine were measured in different intestinal sections of mice.It was found that high dose ALE significantly increased duodenal crypt depth(P<0.05),but had no significant effect on jejunum and ileum(P>0.05),and significantly decreased villus length in duodenum and jejunum(P<0.05),but had no significant effect on ileum(P>0.05).Medium doses of ALE showed the opposite results.It significantly reduced crypt depth of duodenum(P<0.05)and significantly increased the villus length of duodenum,jejunum and ileum(P<0.05).The above experimental results showed that medium dose ALE had a positive effect on the growth of mice.Through the analysis of intestinal flora of medium dose ALE,it was found that ALE regulated the proportion of intestinal flora Firmicutes and Bacteroidetes.It also increased the abundance of Akkermansia and Bifidobacterium,and then had a positive effect on intestinal function of mice.4.The positive effects of ALE on oxidation,bacteriostasis and growth of mice were demonstrated in previous experiments.E.coli is one of the main pathogenic bacteria of bovine mastitis,and inflammation is usually accompanied by oxidative disorder.Based on the above results,lipopolysaccharide(LPS)from E.coli was used in this study to construct the inflammation of bovine mammary epithelial cells(BMECs).The expression of related genes and the expression level of inflammation-related pathway proteins were measured by adding ALE with appropriate concentration,and the mechanism of ALE was explored.In the experiment,the rough concentration of ALE treatment was determined to be lower than 0.25 mg/mL by CCK-8 method,and the final concentration of ALE treatment was further determined to be 0.062 mg/mL by expression of related genes.LPS treatment time was also confirmed according to the expression of related genes,and the results showed that it should be controlled within 9h-12h.Combined with the above conditions,the final test was divided into 4 groups.The results showed that ALE significantly down-regulated LPS-induced BMECs inflammatory cytokines IL6,IL8,IL1β and TNF-α(P<0.05),and significantly downregulated TLR2 in Toll-like receptors(P<0.05),and there was no significant difference on TLR4(P>0.05),NO synthase iNOS was also significantly regulated(P<0.05).Similarly,ALE significantly down-regulated the levels of p-IκBα and p-p65 in NF-κB signaling pathway and p-p38,p-JNK and p-ERK in MAPK signaling pathway(P<0.05).5.The above experiments constructed an inflammatory model of BMECs through LPS,explored the action mechanism of ALE,and discovered the effects of ALE on inflammatory factors and oxidation-related enzymes in BMECs.Therefore,in order to further determine the role of ALE in vivo,LPS was injected into the fourth mammary gland of mice to establish mice mastitis model,and the regulatory role of ALE in vivo was explored by evaluating the expression levels of serum oxidation indexes,mammary histopathological indexes and mammary tissue related genes.First,the successful establishment of LPS model was confirmed by observing the morphology of mammary gland in LPS-induced mice,and it was found that ALE could alleviate LPS-induced mammary gland lesions in mice.Next,pathological section results showed that ALE alleviated LPS-induced inflammatory factor infiltration.MPO activity was consistent with the above results,ALE significantly downregulated LPS-induced neutrophil increase(P<0.05).Oxidative stress is closely related to inflammation.LPS caused oxidative stress imbalance and significantly down-regulated the activities of SOD and GSH-PX(P<0.05),while ALE could significantly recover the decreased activities of SOD and GSH-PX caused by LPS(P<0.05).Then,changes of T-NO synthase and NO content were consistent.ALE significantly down-regulated LPS-induced upregulation of T-NOS activity and NO content(P<0.05).Finally,ALE significantly downregulated the expression of LPS-induced inflammatory cytokines IL6,IL1β and TNFα by determining the expression levels of related genes in mammary tissues(P<0.05),and significantly down-regulated toll-like receptor connector molecules MyD88,TLR2 and TLR4 related to LPS-induced inflammatory transmission(P<0.05).ALE also positively regulated NO synthase in mammary tissue and significantly down-regulated LPS-induced excessive elevation(P<0.05).As a key gene of NF-κB signaling pathway,IκB was significantly elevated under LPS induction(P<0.05),however,ALE significantly reduced the effects of LPS(P<0.05).In conclusion,the non-volatile components of A.argyi leaves also have a variety of biological activities.ALE in this study has antioxidant and antibacterial ability,and has a positive effect on the growth of mice by regulating intestinal flora.Also,ALE has a positive effect on LPS-induced inflammation,specifically by alleviating oxidative stress and the expression of related proteins and genes regulating NF-κB and MAPK signaling pathways.