Optimal Extraction Process And Therapeutic Effect Studies On Mice Model Mastitis In Vivo Of Effective Components From Nymphaea Candida

Objective and significance:This experiment was conducted with Nymphaea candida as material,and response surface methodology（RSM）combined with ultrasonic-assisted extraction was used to study and analyze the ethanol extraction process of total flavonoids from Nymphaea candida and the water extraction and alcohol precipitation process of crude polysaccharides from Nymphaea candida.In addition,the antibacterial and anti-inflammatory effects of total flavonoids and crude polysaccharides from Nymphaea candida in vivo were explored through the establishment of a mouse model of Staphylococcus aureus mastitis.The results will provide data support for the further research of the effective components of Xinjiang local medicinal material Nymphaea candida and the development of new drugs for dairy cow mastitis.Methods:The experiment using the extraction rate of total flavonoids/crude polysaccharides of Nymphaea Candida as the study standard,ultrasonic-assisted extraction was applied to study the impact of the five single factors（ethanol concentration,ultrasonic power,extraction time,solid-liquid ratio,extraction temperature）/（solid-liquid ratio,extraction temperature,ultrasonic power,extraction time,extraction times）on the extraction rate of total flavonoids/crude polysaccharides of Nymphaea Candida.The optimal extraction conditions on the extraction rate of total flavonoids/crude polysaccharides of Nymphaea Candida was studied with response surface methodology on the most effective factor.After delivering 3～10 days,75 Kunming mice were randomly divided into blank control group,model group,positive control group,total flavones of Nymphaea candida group and crude polysaccharides of Nymphaea Candida group,There were 5 groups,15 in each group.Except the blank control group without any treaement,the remaining four groups were injected with Staphylococcus aureus to establish mouse mastitis model.After successful modeling,the groups were given gastric saline,0.13 g/kg ciprofloxacin,0.5 g/kg total flavones and 0.5 g/kg crude polysaccharides of Nymphaea Candida,twice a day,for consecutive 7days.The content of colony for units（CFU）of Staphylococcus aureus,tumor necrosis factor-α（TNF-α）and Interleukin-6（IL-6）concentration in the mammary glands tissue of each test group and the histopathological changes of mouse mammary glands were observed at the 3^rd,5^th,and 7^th day.Results:（1）By single factor experiment,the three most significant factors for the extraction rate of total flavonoids of Nymphaea Candida were ethanol concentration,ultrasonic power and extraction time.The optimal extraction process of total flavonoids by response surface methodology was as follows:temperature of extraction was 60℃,a ratio of solid liquid was 1:30 g/m L,optimal ethanol concentration was 81%,extraction time was 20 min and ultrasound power was 60%.Under these conditions,the extraction rate of total flavonoids from Nymphaea candida was 12%,which was close to the predicted value.（2）By single factor experiment,the three most significant factors for the extraction rate of crude polysaccharides of Nymphaea Candida were solid liquid ratio,extraction temperature and ultrasonic power.The optimal extraction process of crude polysaccharides by response surface methodology was as follows:ultrasound treatment time was 30 min,times of extraction was twice,a ratio of solid liquid was 1:14 g/m L,extraction temperature was 57℃,and ultrasound power was 70%.Under these conditions,the extraction rate of crude polysaccharides from Nymphaea candida was 51.78%,which was close to the predicted value.（3）Compared with the blank control group,mice in model group were more depressed,the consumption of feed and water was reduced,there are many erythrocytes and inflammatory cells in the mammary glands tissue,and the content of CFU of Staphylococcus aureus,TNF-αand IL-6 in the mammary glands tissue were significantly increased（P<0.01）.Compared with model group,the mental state,feed and water consumption of mice in positive control group,total flavonoids and crude polysaccharides groups of Nymphaea Candida gradually recovers,their inflammatory cells and erythrocytes were significantly reduced in the mammary glands tissue,and the content of CFU of Staphylococcus aureus,TNF-αand IL-6in the mammary glands tissue were significantly decreased（P<0.01）.Conclusion:The optimum conditions of the total flavonoids of Nymphaea candida were as follows:extraction temperature of 60℃,solid-liquid ratio of 1:30 g/m L,ethanol concentration of 81%,extraction time of 20 min and ultrasonic power of 60%.And the optimum conditions of crude polysaccharides of Nymphaea candida were as follows:extraction time of 30 min,extraction times of 2 times,solid-liquid ratio of 1:14 g/m L,extraction temperature of 57℃and ultrasonic power of 70%.The total flavonoids/crude polysaccharides of Nymphaea candida can improve the damage of mammary tissue in the mice model of Staphylococcus aureus mastitis,and have significant bacteriostatic antibacterial and anti-inflammatory effects in vivo.