Reaserch On The Stems And Leaves Of Scutellaria Baicalensis With Anti-bacterial And Anti-inflammation Properties And Its Effects On Mice Mastitis

Following the development of the traditional Chinese medicine(TCM)industry,a huge amount of solid waste has been generated in the TCM production and deep processing,leading to serious social,environmental,and ecological problems,as well as great waste of natural resources.As a presentative case,the stem and leave of Scutellaria baicalensis generated during the production of Scutellariae Radix(Huang Qin in Chinese)is still not utilized rationally to date.In the present study,the stem and leave of S.baicalensis was investigated focusing on its anti-bacterial,anti-inflammatory properties,as well as its potential effects on mouse mastitis.And a practically scientific foundation promoting the rational utilization of S.baicalensis was highly expected.This dissertation is comprised of four chapters:Chapter 1 Documentary researchDuring this part,the chemical constituents of the stems and leaves of S.baicalensis including the detailed information for reported flavonoids were summarized.The potential utilization strategies for the stems and leaves of S.baicalensis were discussed as well.Meanwhile,the present situation of bovine mastitis was briefly summarized to provide a reliable bacround.Chapter 2 Optimization and analysis of extraction process of resource chemical substances from the stems and leaves of S.baicalensis.1.The extraction technology of the stems and leaves of S.baicalensis was studied by orthogonal method and taking the total content of total flavonoids in the stems and leaves of S.baicalensis as the index.The results showed that the optimum extraction process of the stems and leaves of S.baicalensis was 70%ethanol refluxing,the ratio of material to liquid was 1≤15,and the extraction time was 2 h each time.2.The components in the stems and leaves of S.baicalensis were quantitatively analyzed by UPLC-MS spectrometry.The flavonoids in the stems and leaves of S.baicalensis were higher than those in phenolic acids.Among them,the content of scutellarin was the highest,19.71 mg/g..Chapter 3 Evaluation of Bacteriostatic and Anti-inflammatory activity Resources in the stems and leaves of S.baicalensis.1.The inhibitory effect of the stems and leaves of S.baicalensis on Staphylococcus aureus,Shigella dysenteriae,Escherichia coli,Streptococcus agalactiae was evaluated by the method of drug sensitive paper and microbroth dilution.The results showed that the inhibitory effect of the stems and leaves of S.baicalenssi on the Staphylococcus aureus and Shigella dysenteriae was superior to that of Escherichia coli and Streptococcus agalactiae.2.The inhibitory effect of the stems and leaves of S.baicalensis combined with Ampelopsis grossedentata on Staphylococcus aureus and Shigella dysenteriae was evaluated by checkerboard method and time-kill curve method.FIC index was used as the evaluation standard.The results showed that the FIC index was 0.625 for Staphylococcus aureus and 0.75 for Shigella dysenteriae,which had additive effect on Staphylococcus aureus and Shigella dysentery.It is proved that the combination of the two can effectively improve the bacteriostatic effect and promote the effective application of the stems and leaves of S.baicalensis.3.Based on the three kinds of inflammatory models,the effects of antibacterial and anti-inflammation of the stems and leaves of S.baicalensis were evaluated by using the indexes of the organ index,the pathological changes of the tissues and the inflammatory factors.1)Establish the model of S.aureus-infected mice,evaluate the regulation effect of the stems and leaves of S.baicalensis on inflammation caused by S.aureus infection.The results showed that the hepatocytes in the high dose group were clearly visible,the size was uniform,the arrangement level was more regular,the hepatocyte edema,the fat cells were basically disappeared,and a small amount of inflammatory cell infiltration was occasionally seen in some parts.Spleen morphology,good structure recovery,clear boundary of white pulp and red pulp,small amount of infiltrating inflammatory cells.The alveolar wall is slightly thickened,there are infiltrating inflammatory cells in some parts,and a few parts have hyperemia.The results showed that the expression of TNF-α and IL-6 in plasma(P<0.05)could be significantly reduced by the stems and leaves of S.baicalensis,and the expression of TNF-α and IL-6 in the liver,lung and kidney could be reduced(P<0.05).The results showed that the stems and leaves of S.baicalensis could regulate the expression of inflammatory factors and regulate the infection of bacteria by improving the injury of multiple organs infected by S.aureus,regulating the gene level of inflammatory factors in organs and regulating the expression of inflammatory factors.2)the systemic inflammation model of mice infected with LPS was established.The effects of the stems and leaves of S.baicalensis on systemic inflammation were evaluated by taking the changes of mortality,organ index and cytokines as indexes.The stems and leaves of S.baicalensis could reduce the mortality of mice,and the index of heart and spleen in SBSL group was significantly higher than that in model group(P<0.05).Compared with the model group,the H-SBSL could significantly decrease the levels of cytokines TNF-α and IL-6 in serum of mice(P<0.05).The results showed that the stems and leaves of S.baicalensis could protect mice infected with LPS by improving organ index and reducing the expression of cytokines.3)The inflammatory bowel disease model of mice infected with DSS was established.The regulatory effect of the stems and leaves of S.baicalensis on inflammatory bowel disease was evaluated by using DAI score,pathological changes,cytokines and intestinal flora as indexes.The results showed that the stems and leaves of S.baicalensis significantly alleviated the DSS-induced inflammatory reaction,as well as intestinal inflammatory symptoms such as weight loss,intestinal damage and colon shortening.The results of HE section staining showed that the colonic mucosa congestion,edema and the area of inflammatory cell infiltration of SBSL group was reduced.The stems and leaves of S.baicalensis can regulate the levels of peroxidation and colonic inflammatory factors.The stems and leaves of S.baicalensis can restored intestinal homeostasis by up-regulating the level of beneficial intestinal bacteria,enhancing the immune response of the body,promoting the growth of probiotics in the intestinal tract,and inhibiting the colonization of pathogenic bacteria.The results showed that the stems and leaves of S.baicalensis can alleviate the symptoms of diarrhea and bloody stool in mice with DSS-induced colitis.The stems and leaves of S.baicalensis can improve the inflammatory bowel disease induced by DSS by improving the damage of colon tissue and regulating the expression of inflammatory factors in colon.Chapter 4 Evaluation of the effect of the stems and leaves of S.baicalensis and its compatibility on mastitis in mice.1.The bacteriostatic activity of the stems and leaves of S.baicalensis,seeds of Sophora flavescens,leaves of Lonicera japonica and Lonicera japonica Thunb.was evaluated by paper method and the diameter of antibacterial circle was taken as the evaluation index.The results showed that all the tested drugs had inhibitory effect on S.aureus,and the extract of the stems and leaves of S.baicalensis and Lonicera japonica Thunb.had a strong inhibitory effect on S.aureus at the working concentration of 100 mg/mL.Among the inhibitory effects of each tested drug on Shigella dysenterace,only the stems and leaves of S.baicalensis and seeds of Sophora flavescens had certain inhibitory effect.2.The model of the infection of S.aureus in mice was successfully established by using the method of breast injetion.The model was established with 107 CFU/mL according to the analysis of the results.3.Based on the model of S.aureus infection in the mouse mastitis,the effects of the changes of the pathological sections,the organ index and the change of the cytokines on the mastitis of the mice were evaluated.The results showed that after the intervention of the stems and leaves of S.baicalensis and Lonicera japonica Thunb.separately,the infiltration of the inflammatory cells could be partially reduced,but some of the acinar necrosis and the shedding of the epithelial cells were observed.The protective effect of the combination is superior to each drug alone.The results showed that the level of IL-6 in the mammary gland of the mice was lower than that of each drug alone group,and the effect of the combination was better than that of each drug alone group.The effect on TNF-a expression level was significantly different between the combination group and the model group,and there was a significant difference between the combination group and the single groupThe experimental results showed that the stems and leaves of S.baicalensis and Lonicera japonica Thunb could decrease the level of IL-6 in the breast tissue of the mice induced by S.aureus.The combination of the stems and leaves of S.baicalensis with Lonicera japonica Thunb can significantly improve the breast tissue damage in the mice induced by S.aureus.