Study On Flavonoids Of Inula Britannica L.

The Traditional Chinese Medicines (TCM) plays an important role in the human disease prevention and treatment. The science and technology in 21st century develops more rapidly. The development of the life science, the information science and the material science is promoting the economical prosperity and the transform of the humanity culture. As theirs research achievement are applied to the further developments of TCM, innovational research of TCM will be strengthened. That will enormously promote the process of modernization of TCM. The practices have been proved all over the world that TCM are invariably the important source of innovational research of drugs. In China, it will be proved to be a shortcut that we develop and research new drugs from wild plants by using thousands of valuable experiences of TCM. Bonding innovational research of drugs and modernization of TCM will certainly form the Chinese characteristic.Inula Britannica L. has been used in Chinese folk medicine for the treatment of bronchitis, nausea, hiccup, excessive sputum and inflammation, it belongs to the genus inula of family Compositae and distributes in China, Korea, Japan and other region of world. At present, It was reported that more than 40 chemical compositions had been identified from Inula Britannica L., mainly included flavonoids, sterols, coumarin and terpenoids, etc. Its extracts were reported to have anti-inflammatory, antibacterial, antihepatitic, antitumor and antioxidant activities. This prompted us to search for active compounds from this plant.This paper established a method for determining of the total flavonoids from Inula Britannica L. by UV-spectrophotometry for the first time. Taking the content of total flavonoids as index, we chose the optimum extraction conditions by L9(3)~4 orthogonal test and selected best macroporous resins and purification parameters by static and dynamic experiments; At the same time the biological activities of total flavonoids were studied. Repeated chromatography of the n-butanol fraction and ethyl acetate fraction obtained from the alcohol extract of Inula Britannica L. led to the isolation and the structural determination of twelve compounds. In this study, a reversed phase high performance liquid chromatography (HPLC) method for simultaneous determination of five flavonoids from Inula Britannica L. was developed.Part one: Studies on the extraction, separation and determining of total flavonoids from Inula Britannica L.Section one: Studies on the extraction, seperation and purification of total flavonoids from Inula Britannica L.Objective: To study the process of extracting total flavonoids from Inula Britannica L., and the adsorption technology in seperation and purification of extract with macroporous resins.Methods: (1) To study the extraction process of total flavonoids from Inula Britannica L. According to the properties of flavonoids, the appropriate extraction solvent was chosen from 80%,60%,30% alcohol and water, the best extraction method was chosen from three extraction methods (ultrasonic extraction, refluxing extraction and soxhlet extraction). The extraction process was optimized by orthogonal test and the optimum extracting conditions were verified.(2) To study the adsorption technology of total flavonoids from Inula Britannica L. The solution of Inula Britannica L. was prepared according to the optimum extracting conditions. We first pretreated seven kinds of macroporous resins (X-5, S-8, NKA, NKA-9, AB-8, D4020, SP700), then tested on their static equilibrium adsorption quantity and static adsorptive dynamic kinetics. A resin was chosen as the suitable medium to enrich total flavonoids from Inula Britannica L.. And the best technological parameters for enrichment of total flavonoids from Inula Britannica L. were ascertained.(3) To compare the purified method by AB-8 macroporous resin and the polyamide. The same quantity of samples were adsorbed on polyamide column (eluting with 95% alcohol) and AB-8 macroporous resin column (eluting with 70% alcohol), respectively, then determine the content of total flavonoids and the recovery rate.Results: The results indicated that the optimum process was to extract 3 times (each for one hour) at reflux with 10:1 (water:materials). The extract times has the significance influence on the technology. The results of verification test was consistent with the orthogonal test.Seven types of macroporous resins were studied and compared. The results indicated that SP700 was eminent among all these resins, but it was import production, and the AB-8 macroporous resin also had good effect for absorption and enrichment, and AB-8 macroporous resin was applicable to industrial production. So we chose AB-8 macroporous resin to separate total flavonoids from Inula Britannica L.The optimum technological parameters for separation and purification of total flavonoids from Inula Britannica L. were as follow: the concentration of sample (0.2g/mL), absorbing rate (2mL/min), eluent (70% alcohol) and the eluting rate (3mL/min).According to the optimum extracting conditions, the content of total flavonoids from Inula Britannica L. was 10.9%. After adsorption on polyamide and desorption by 95% alcohol, the content of total flavonoids was 78.2% and recovery rate was 49.9%; After adsorption on AB-8 macroporous resin and desorption by 70% alcohol, the content of total flavonoids was 89.52% and recovery rate was 92.8%.Conclusion: The total flavonoids from Inula Britannica L. was extracted by refluxing method and the extraction process was optimized by orthogonal test. AB-8 macroporous resin was chosen and the optimum technological parameters were studied. After adsorption by AB-8 macroporous resin, the content of total flavonoids from Inula Britannica L. was 89.5%.Section two: Quantitative determination of the total flavonoids from Inula Britannica L. by UV spectrophotometry .Objective: To establish a method for quantitative determination of the total flavonoids from Inula Britannica L Methods: Using detection wavelength at 510nm and rutin as standard, a simple and reproducible UV spectrophotometry was developed to determine total flavonoids in Inula Britannica L. from different sources.Results: The standard curve was linear in the concentration range of 10.3 to 51.5μg/mL. The regression equation was Y=12.2X-0.3×10-3, r=0.9999. The average recovery and relative standard deviation were 99.1% and 1.5% respectively. The contents of total flavonoids in Inula Britannica L. from seven different habitat were determined.Conclution: This paper established a method for determining of the total flavonoids from Inula Britannica L. by UV spectrophotometry for the first time, and had carried on the determination to seven different habitat. This method was simple, the result was accurate, the repeatability was good. It can be used for the quality control of Inula Britannica L.Part two: Studies on separating flavonoids from Inula Britannica L. and determining contentSection one: Studies on separating and purification of flavonoids from Inula Britannica L.Objective: Individual components of ethyl acetate and n-butanol fraction were isolated and purified by various chromatographic techniques. Their structures were identified based on the physico-chemical properties and spectral data.Methods: Air-dried Inula Britannica L. were extracted with 95% alcohol, and extract was distributed with petroleum, chloroform, ethyl acetate and n-butanol successively. The ethyl acetate and the n-butanol fraction were isolated and purified via column chromatography on silica gel, polyamide, Sephadex LH-20, macroporous resin. Their structures were identified based on the physico-chemical properties and spectral data (EI-MS, IR, H-NMR, C-NMR, H-H COSY, DEPT, HMQC, HMBC).Results: A chemical study on flavonoids from Inula Britannica L was carried out, and twelve compounds have been isolated and identified as spinacetin, quercetin, luteolin, 6-methoxyluteolin, isorhamnetin, isoquercitrin, quercitrin, kaempferol, rutin, patulitrin, chlorogenic acid and berberine.Conclution: 12 compounds were isolated and their structures were identified. 5 compounds such as berberine,rutin,isoquercitrin,quercitrin and isorhamnetin were isolated from Inula britannica L. for the first time.Section two: Simultaneous determination of five flavonoids from Inula Britannic L. by HPLCObjective: To establish a method for the assay of spinacetin, quercetin, luteolin, 6-methoxyluteolin and isorhamnetin from Inula Britannica L. in order to provide a guide line for their quality control.Methods: Chromatographic conditions: An Inertsil ODS-3 column (250mm×4.6mm, 5μm) at a temperature of 40℃was applied for all analyses. Detection wavelength was set at 360nm. The mobile phase consisted of methanol(A) and acetonitrile(B) and 0.49% aqueous citric acid(C) (v/v/v) using a gradient elution. The flow rate was 1.0 mL.min-1 and aliquots of 20μL were injected. The developed method was successfully applied to the analysis of 7 samples of Inula Britannica L. collected from different parts of China.Results: The analytical method was utilized for the determination of the five flavonoids (spinacetin, quercetin, luteolin, 6-methoxyluteolin and isorhamnetin) in 7 commercial samples from different parts of China. The results showed that there was a significant variability in the contents of flavonoids among 7 samples which might be derived from the genetic variations and plant origins, and some other external factors, such as seasonal and environmental factors, drying process and storage conditions.Conclution: It is the first time to establish the reversed-phase HPLC method to determine simultaneously the contents of the five flavonoids from Inula Britannica L., and further, to quantify these 5 compounds from 7 different samples of Inula Britannica L.. Based upon our validation results, this analytical technique was a convenient method to simultaneous quantify numerous active compounds derived from Inula Britannica L, featuring good quantification parameters, accuracy and precision.Part three: Studies on biological activities of total flavonoids from Inula Britannica L.Section one: Effects of total flavonoids from Inula Britannica L. on the proliferation and migration of cultured rat vascular smooth muscle cellsObjective: To study the effects of total flavonoids from Inula britannica L. on the proliferation and migration of cultured rat vascular smooth muscle cells of thoracic aorta.Methods: Vascular smooth muscle cells of rat were cultured in vitro and the cells of 4-7 passages of cultured VSMC were used in the experiment and were divided into control group and total flavonoids group according to different concentrations of total flavonoids from Inula britannica L.,MTT assay, and microscope were used respectively to observe the ratio of cell proliferation and migration.Result: Compared with the control group, the ratio of cell proliferation were inhibited in groups of TFIB in a dose-dependent manner. The distance of VSMC migration were decreased compared with the control group.Conclution: The total flavonoids from Inula britannica L. can significantly inhibit the vascular smooth muscle cell proliferation and migration in vitro.Section two: Protective effects of TFIB against permanent focal cerebral ischemia-reperfusion injury in ratsObjective: To investigate the effects of TFIB on MDA content and SOD activity on focal cerebral ischemia-reperfusion injury in rats.Methods: The treatment groups were given three different doses by ranine vein. The animal model of the right middle cerebral artery occlusion ischemia-reperfusion was established using suture. MDA content and SOD activity were observed after cerebral ischemia 2h followed by reperfusion 4h.Result: TFIB significantly decreased the level of MDA and improved the activity of SOD in brain.Conclution: TFIB has protective effect on focal cerebral ischemia reperfusion in rats, and its mechanism may relate with its effective scavenging of free radicals.