Study Of Extraction And Purification Of Schisandrin B In Schisandra Chinensis(Turcz.) Baill Leaves And Combined With Paclitaxel To Inhibit Cancerstudy And Compound Antioxidant With Total Flavonoids Of Citrus Peel

Schisandra chinensis?Turcz.?Baill is a characteristic plant in the north of China,with a large planting area.It contains schisandra b has a variety of biological activities,such as antioxidant,anti-inflammatory anti-cancer and other important activities.In the food direction,Schisandra chinensis?Turcz.?Baill can be used as quality food for us to eat.In addition to being a daily edible food,Schisandra chinensis?Turcz.?Baill also has medicinal value in the medical field.The effectiveness of these two aspects has become a research hotspot in recent years.In the process of producing and processing Schisandra chinensis?Turcz.?Baill products,a large number of its leaves are often wasted as wastes.If used reasonably,it can not only reduce the production cost and realize the effective utilization of resources,but also reduce pollution and save energy consumption.Our previous research results showed that there was schisandrin b in the cotyledon of northern wuweizi,and its inhibitory effect on breast cancer proliferation was preliminarily explored.And then continue to study the synergistic and detoxifying mechanism of paclitaxel against breast cancer.The total flavonoids in citrus peel can also increase the sensitivity of cancer cells and enhance the anti-cancer effect of taxol.Therefore,this paper further developed the compounds of acanthopanax cotyledon and citrus peel total flavonoids with antioxidant activity as the index.But in the experiment,we found that the stability of total flavonoids in citrus peel was not good,so,the stability of total flavonoids extracted from citrus peel was optimized.This paper intends to extract,separate and purify schisandrin b from the cotyledon and qualitative and quantitative analysis.The mechanism of its combination with paclitaxel against breast cancer was studied to develop new resources for anti-cancer research.Before the combination of b.schisandrae and total flavonoids of citrus peel,the extraction process optimization and stability of total flavonoids from citrus peel were studied.To ensure the accuracy and stability of the composition of the compound,and to provide a scientific basis and experimental basis for the development of new compound products.The content and results of the study are as follows:this paper,with schisandra leaves as the raw material and the crude extract of schisandrin b from schisandra leaves was extracted by ultrasonic wave.Silica gel column chromatography purification,HPLC identification of its content and purity.Single factor experiment and response surface optimization were carried out to study the extraction effect.The bioactivity of schisandrin against cancer was further investigated.The mechanism of reducing toxicity and enhancing efficacy of paclitaxel in the treatment of breast cancer was explored.To establish a mouse model of breast cancer in vivo and in vitro.They were randomly divided into the blank group,negative group,PTX group,SchB group,PTX and SchB combined group.The contents of interleukin 10?il-10?and tumor necrosis factor-??TNF-??were detected by ELISA.Superoxide dismutase?SOD?,glutathione?GSH?and malondialdehyde?MDA?were detected with the kit.The morphological state of breast cancer cells and surrounding muscle tissues was detected by HE staining.The expression of protein kinase?p38?was detected by immunohistochemistry.The expression of vascular endothelial growth factor?VEGF?and matrix metalloproteinase-2?mmp-2?was detected by Western blot.To explore the extraction rate and stability of total flavonoids from citrus peel and compare the effects of ultrasonic and microwave on extraction rate of total flavonoids from citrus peel.Compound design was carried out to combine schisandrin b of the schisandra leaves with total flavonoids of citrus peel to explore its antioxidant biological activity.The optimal technological conditions for extracting schisandrin b from schisandra leaves are as follows:The ratio of liquid to material V:m?mL/g?is 20:1,75%ethanol and ultrasonic power is 400W,60?for 30 min.Schisandrin b in schisandra leaves was obtained by Silica gel chromatography and the content was 3.056mg/g by HPLC.Purity of schisandrin b:98.26%.A breast cancer mouse model was established in vivo and in vitro.Compared with the blank group,the survival time of mice in the PTX group,the SchB group and the combined group was significantly prolonged and the number of lung metastases was significantly reduced?P<0.05?.The change in the combination group was significantly greater than that in the PTX group?P<0.05?,and there was no significant difference between negative group and blank group?P>0.05?.Compared with the blank group,the solid tumor weight of the PTX group was significantly reduced?P<0.05?,while that of the negative group and the SchB group was not significant?P>0.05?.Compared with the PTX group,the solid tumor weight of the combined group was significantly reduced?P<0.05?.Compared with the blank group,the level of TNF-?and MDA in serum of breast cancer mice was significantly increased after the administration of PTX.The contents of il-10,SOD and GSH and the expressions of p38,VEGF and mmp-2 in tumor tissues were significantly decreased?P<0.05?.The number of spindle-shaped cancer cells decreased and the degree of invasion of surrounding muscle tissue decreased.Compared with the blank group,after the action of SchB and it can significantly reduce the content of TNF-?,il-10,MDA and the expression of p38,VEGF and mmp-2 protein in the serum of breast cancer mice.The number of spindle-shaped cancer cells also decreased,and the degree of invasion of muscle tissue also decreased.The contents of SOD and GSH were significantly increased?P<0.05?.After the combined action of SchB and PTX group and the levels of TNF-?,il-10 and MDA in serum and the expressions of p38,VEGF and mmp-2 in tumor tissues were significantly decreased and the contents of SOD and GSH were significantly increased.The number of spindle cancer cells decreased significantly,and the degree of muscle tissue invasion decreased significantly.Compared with PTX group,the difference was significant?P<0.05?.There was no significant difference between the negative group and the blank group?P>0.05?.The optimum conditions for extraction of total flavonoids from citrus peel by ultrasonic and microwave were determined by single factor and response surface optimization.Optimum process conditions of ultrasonic assisted extraction as follows:80%ethanol concentration,liquid than V:m?mL/g?40:1,35 min ultrasonic time,extraction temperature 50?,the extraction rate of total flavonoids from citrus peel was 13.785%±0.03%;The optimum conditions for microwave extraction were:ethanol concentration 70%,liquid to solid ratio V:m?mL/g?40:1,irradiation time 8min,microwave power 400W,the extraction rate of total flavonoids from citrus peel was 13.154%±0.36%.The effects of temperature,pH and other indicators on the stability of total flavonoids in citrus peel were discussed.Results showed that the flavonoids in shows the good heat stability under 60?,PH=5.0-8.0 was stable.The oxidant H₂O₂,reducing agent Na₂SO₃,sugars and natural light affect its stability.Citric acid and NaCl had little effect and Vitamin C promoted the preservation of flavonoids.Single factor test and response surface optimization optimization of the compound compound of the optimal process conditions:The mixture ratio(m _{schisandrin b}:m _flavonoids)was3:1,40%ethanol,compound with pH8.0.The average clearance rate was 92.063%,close to the predicted value?92.276%?.The scavenging effect of pair·OH was as follows:complex solution>schisandrin b solution>ascorbic acid solution>total flavonoid solution,the scavenger action on O₂^-.Was complex solution>ascorbic acid solution>schisandrin solution>total flavonoids solution.