Epimedium Flavonoid Compounds Active Substances In Vitro Model Of Screening And Antioxidant Research

This paper analyses bioactive components in flavonoids extracts from Herba Epimedii at the level of chemical composition and cell biology, and builds the models of oxidative stress and blood-brain barrier in vitro. At the same time, this paper processes screening, analysis, identification activity and antioxidant activities in vitro of bioactive compounds in flavonoids extracts from Herba Epimedii, in order to offer new ideas for modernization and screening cardiovascular and central nervous system (CNS) related diseases drugs.The research works are as follows:1. The foundation of oxidative stress screening model, and screening of active compounds from flavonoids extracts from Herba Epimedii.In order to screen active compounds, oxidative stress model is used to screen active compounds related cardiovascular diseases from Chinese herbal medicine. The principle of the oxidative stress model is the oxidative-damaged ECV304cells induced by H2O2combine active compounds which are screened from Chinese herbal medicines, and the unbound components and superfluous components are washed and discarded from cell surface of ECV304cells. Then the special binding components release from cell membrane receptors by changing pH. The oxidative stress model combines with LC-MS technique is applied in screening bioactive compounds from flavonoids from Herba Epimedii extracts. The results show that there are6active components which are bound and captured by ECV304membrane receptors, and the3active compounds are identified as epimedin B, icariin and icariside II.2. The foundation of blood-brain barrier screening model, and screening of active compounds from flavonoids extracts from Herba Epimedii.The CNS has formed the unique structure in the human evolution, and the CNS avoids the influence of many kinds of molecular drugs, simultaneously this kind of isolation influences the function of medicine to cure CNS diseases. The active compounds which can permeate into blood-brain barrier model in vitro are screened from Chinese herbal medicine. The ECV304/C6co-culture model is applied to simulate the blood-brain barrier in vivo, and the trans-epithelial electrical resistance (TEER) and permeability coefficient of ECV304/C6co-culture are analyzed. The results show that ECV304/C6co-culture has the characters of blood-brain barrier in vivo. The blood-brain barrier model combines with LC-MS technique is applied to screen activity compounds which can go through the blood-brain barrier from flavonoids extracts from Herba Epimedii. The screened candidate drugs are analyzed and identified with LC-MS technique. The result shows that2candidate drugs can go through blood-brain barrier and confirmed by LC-MS, the2screened bioactive compounds are icariin and icariside II. Further more, the results prove that ECV304/C6co-culture model can screen the active compounds which can go through blood-brain barrier from Chinese herbal medicine extracts, and the method is helpful in guiding screening and developing new drugs of CNS targets.3. Antioxidant properties of icariin and icariside II in vitro.Icariin, a major active flavonoid isolated from Herba Epimedii, possesses wide pharmacological actions, especially in protecting cardiovascular system, immunological function modulation, anti-tumor, anti-osteoporosis and improvement of sexual dysfunction. Icariside II is metabolites of icariin, and its function is hardly understood. We analysed the antioxidant activity of the icariin and icariside II using different assays including:total antioxidant activity and reducing power, hydroxyl radical scavenging assay, superoxide radical scavenging assay,1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay and lipid peroxidation assay which uses egg yolk as the lipid-rich source. Total antioxidant capacity was determined by the assay based on the decrease in absorbance of β-carotene by the sample. These various antioxidant activities were compared to standard antioxidants such as BHT and Vc.The experiment results show icariin exhibites strong DPPH radical, superoxide radical scavenging activity, reducing power and total antioxidant activity, icariside II has stronger DPPH radical scavenging capacity than icariin, and effective inhibition of lipid peroxidation. The radical-scavenging and inhibition effects of icariin and icariside II are in a dose-dependent manner. A positive correlation is found between dose of the drugs and antioxidant effects, and the effects change unobviousely once the scavenging or inhibition ratio reaches some level. The effects of radical scavenging of icariin and icariside II are different, and the reason probably is the difference of number and position of two compounds'phenolic hydroxyl and glycosyl. The experiment provided the reference for the study of the pharmacological activity mechanism of icariin and icariside II, and experiment foundation for potential antioxidants'exploitation and use.