Extraction,Purification And Physiological Activity Of Total Flavonoids In Leaves Of Rosa Davurica Pall. In Different Picking Periods

Rosa davurica Pall.is a pure natural plant of the genus Rosaceae.It is widely grown in the mountainous and hilly areas of Northeast China.Its active ingredients mainly include flavonoids,triterpenoids and some essential amino acids.Modern pharmacological research shows that Rosa davurica Pall.has the effects of anti-oxidation,anti-aging and enhancing immunity.And it has extremely high medicinal value.In recent years,most of the research reports focus on the fruit of Rosa davurica Pall.,but there are few reports on the research of the leaves of Rosa davurica Pall..Therefore,this study takes the total flavonoids from the leaves of Rosa davurica Pall.as the research object.On this basis,the physiological effects of total flavonoids in leaves of Rosa davurica Pall.was studied from the aspects of antioxidant,immune enhancement and anti-aging mechanisms.The extraction conditions of total flavonoids in leaves of Rosa davurica Pall.were optimized by alcohol-extracted method and orthogonal test method.The total flavonoids were purified by using D101 macroporous resin.On this basis,the changes of total flavonoids in the leaves of Rosa davurica Pall.were measured in different harvesting seasons.The anti-aging effect of total flavonoids in leaves of Rosa davurica Pall.was mainly detected in the anti-oxidation of·DPPH free radicals,·OH free radicals,O₂^-·free radicals and ABTS⁺·free radicals.The aging model was established in the anti-oxidation test in vivo.60 SPF kunming mice,male and female half and were randomly divided into blank group,aging model group,vitamin E group（50 mg/（kg·d））and total flavonoids in leaves of Rosa davurica Pall.low,medium and high dose group（1.0、2.0、4.0 g/（kg·d））,a total of 6 group（n=10）,the blank group mice were gavaged and subcutaneoused injection with an equal volume of normal saline.The other groups of mice were injected with the back of the neck subcutaneous of D-galactose 125 mg/kg.After 6weeks,the enzyme activities of SOD,GSH-Px and MDA content in liver,serum and brain tissues of each group were determined.Secondly,the immune organ index of each group was tested from the aspect of immune enhancement;The concentrations of cytokines IL-2,IL-4,IL-6 and TNF-αin the serum of each group mice were detected by ELISA;Morphological changes of brain tissue in mice of each group were observed by HE staining;And to investigate the proliferation of mice spleen lymphocytes induced by ConA,and then explore the immune enhancement mechanism of total flavonoids from the leaves of Rosa davurica Pall..The contents and results of this study are as follows:1.The optimum extraction technology of total flavonoids in leaves of Rosa davurica Pall.was determined by single factor analysis combined with orthogonal experiment:80%ethanol,ratio of material to liquid 1:30 g/mL,water bath temperature 80°C,water bath time 2 h,extraction rate 4.976%;through the determination of total flavonoids between May and September,the total flavonoids content of the thorns in the middle of August is the highest,reaching 33.84 mg/g;The purification condition of D101 macroporous resin was determined as follows:The concentration of the upper column was 4 times diluted with the original extract,the adsorption rate was 2 BV/h,the pH value of the upper solution was 4,the desorption solution was 70%ethanol,the elution rate was 2 BV/h,the amount of eluent was 7 BV.Under this condition,the purity of the obtained total flavonoids was 57.3%.2.The results of in vitro antioxidant activity determination of total flavonoids in leaves of Rosa davurica Pall.showed that:The total flavonoids in leaves of Rosa davurica Pall.had good scavenging effects on four kinds of free radicals,including·DPPH,·OH,O₂^-·and ABTS⁺·.The scavenging rate increased with the increase of sample concentration,and its antioxidant capacity was close to V_C;In vivo antioxidant studies of total flavonoids in leaves of Rosa davurica Pall.showed that:Compared with the aging group mice,the medium and high doses of total flavonoids in leaves of Rosa davurica Pall.could significantly or extremely significantly increase the activity of SOD in the liver of aging model mice（P<0.05,P<0.01）,and the medium and high dose groups could also be significant or extremely significant reduce the MDA content in the liver of mice（P<0.05,P<0.01）;The medium and high doses of total flavonoids in leaves of Rosa davurica Pall.could significantly or extremely significantly increase the activity of SOD and GSH-Px in the serum of aging model mice（P<0.05,P<0.01）,and the middle and high dose groups could also significantly or extremely significantly reduced MDA content in serum（P<0.05,P<0.01）;The high dose group of total flavonoids in leaves of Rosa davurica Pall.could significantly increase the activity of GSH-Px in brain tissue of aging model mice（P<0.05）,and the high dose group could also significantly reduce the MDA content in brain tissue（P<0.05）;There were significant differences in SOD,GSH-Px enzyme activity and MDA content in liver,serum and brain tissue of vitamin E group（P<0.01）.3.In terms of immune enhancement,compared with the aging model group,the brain index of rats in the aging model group was significantly increased in the medium and high dose groups of total flavonoids in leaves of Rosa davurica Pall.（P<0.01）,and the spleen and thymus index of mice in the aging model group were significantly increased in the low,medium and high dose groups of total flavonoids in leaves of Rosa davurica Pall.（P<0.05）.The concentrations of cytokines IL-2 and IL-4 in serum of the medium and high dose groups of total flavonoids in leaves of Rosa davurica Pall.were significantly or extremely significantly higher than those of the aging model group（P<0.05,P<0.01）,while the concentrations of cytokines IL-6 and TNF-αin the serum of the low,medium and high dose groups of total flavonoids in leaves of Rosa davurica Pall.were significantly or extremely significantly lower than those of the aging model group（P<0.05,P<0.01）;The serum concentrations of cytokines IL-2,IL-4,IL-6 and TNF-αin the vitamin E group were significantly different（P<0.01）.Compared with the aging group mice,the low,medium and high dose groups total flavonoids in leaves of Rosa davurica Pall.dose group were dose-dependent on the number of neurons in brain tissue and the reduction of cell necrosis and degeneration.The total flavonoids in leaves of Rosa davurica Pall.had proliferative effects on mice spleen cells and the proliferation of T-lymphocytes in ConA-induced spleen cells in mice.According to the above results,the total flavonoids in leaves of Rosa davurica Pall.have a good anti-aging effect.Its mechanism of action is mainly to delay the aging effect on the body from the aspects of antioxidant and immune enhancement.This study provides a theoretical basis and scientific basis for the rational development and utilization of leaves of Rosa davurica Pall.and their bioactive substances.It has positive significance for the application of leaves of Rosa davurica Pall.resources in the field of food processing and clinical application.