Isolation Of Galuteolin From Elsholtiza Bodinieri Vaniot And Its Biological Activities

Elsholtiza Bodinieri Vaniot（EBV） is a Chinese specie of the genus Elsholtzia（Labiatae） that abundantly distributed in Yunnan, Sichuan and Guizhou in China,known as “Fengwei Tea”, ”Yeshan Tea”, “brush grass”. Reportedly, the genus Elsholtzia has been widely used as food and folk medicine. When considered as a folk medicine, EBV has been used to cure common cold, headache, toothache, dyspepsia,diarrhea, sore throat, acute conjunctivitis, anuria and hepatitis. EBV is believed to contain bioactive phytochemicals such as essential oils, flavonoids, triterpenoids,phenolic constituents, steride, β-carotene, vitamin C and inorganic elements and so on.Previous reports showed that flavonoids were rich in EBV. It is well-known that flavonoids are a large group of plant polyphenol secondary metabolites and found widespread occurrence in plants with profuse bioactivities, such as anti-bacterial,anti-oxidants, anti-inflammation, anti-tumo. The consumption of flavonoids lowers the risk of cardiovascular disease, diabetes, arthritis and cancer due.The study for EBV mostly focused on the essential oil, but the flavonoids have not been fully researched at present. Therefore, this paper mainly studied the extraction, isolation the flavonoids from EBV and their biological activities to provide effective basis for the further development and utilization of EBV.Chapter one mainly introduced the research status of EBV including chemical components and biological activities. The extraction and separation methods of flavonoids were also presented.Chapter two researched the extraction and quantitative determination of the flaconoids from EBV. The content of flaconoids was measured with NaNO₂-AL（NO₃）₃-NaOH method. The extraction process of flaconoids from EBV was optimized by orthogonal design using extraction amount as target.First of all, the total flavonoids were extracted effectively by ethanol reflux and liquid-liquid extraction. The extraction process was optimized by orthogonal experiments L9（3⁴） using extraction amount as the target index. The temperature,reflux time and liquid-material ratio with three levels were chosen in the orthogonal experiments. Orthogonal test was carried out according to the table of factor levels,the yield of the extraction was calculated, then variance analysis was done. Through the above steps, the influence of these three factors on the extraction amount was concluded and the best extraction technology was determined, listed as followed:extraction temperature 60 ℃, reflux time 3 h, liquid-material ratio 1:20（g/mL）.Chapter three studied the chemical composition of flavonoids from EBV.Galuteolin was isolated and identified.The composition of flavonoids from EBV was analyzed by HPLC. Galuteolin was separated as the purity of 94 % by Prep-HPLC. The compound was identified according to its retention time of HPLC, mass spectra, 13C-NMR spectra, 1H-NMR spectra. It was referred as galuteolin.Chapter four evaluated the biological activities such as antioxidant,anti-inflammatory and immune activities of the galuteolin.Antioxidant activity was appraisaled by 1,1-diphenyl-2-picrylhydrazyl radical（DPPH） and ferric reducing antioxidant power（FRAP） assays. Anti-inflammatory and immune activities were investigated by in vitro and in vivo experiments, respectively.First, the antioxidant activity of galuteolin was determined using the DPPH radical scavenging activity method and Prussian Blue method. The results suggested that galuteolin had potential antioxidant activity. Galuteolin exhibited good DPPH radical scavenging activity, and there was a strong correlation y=11.9095x-6.4155,（R=0.9978） between the radical scavenging rate and concentration of galuteolin. The IC50 of DPPH radical scavenging of galuteolin was 5.14 μg/mL. Besides, galuteolin exhibited certain reducing power activity, and there was a strong correlation y=0.06768x+0.0855,（R=0.9954） between the reducing power and concentration of galuteolin. Also, the reducing power increased with the concentration of galuteolin,and there was a concentration-dependent increase.Secondly, the anti-inflammatory activity in vitro of galuteolin was detected through the effect of galuteolin on NO releases of RAW264.7 stimulated by LPS. The result showed that the clearance ratio of NO was 31.1-79.7 % at the range of concentrations of 5-500 μg/mL. The clearance rate increased with the concentration of galuteolin.Galuteolin significantly reduced the release of NO in cells, indicating that it had good capability of in vitro anti-inflammatory. The anti-inflammatory activity in vivo of galuteolin was evaluated by comparing the ear swelling and the content of PGE2 between experimental groups and model group. The result showed that ear swelling was inhibited by galuteolin within a concentration range of 40-80 mg/kg. The inhibition rate of ear swelling was ranged from 43.2% to 81.4 %. Compared with control group, the ear swelling of mice in the middle-dose group and high-dose group decreased high significantly（P < 0.01）. Besides, the content of PGE2 was reduced significantly in experimental groups, compared with model control group. High-dose group（80 mg/kg） has the extremely significant difference（P < 0.01）. So we concluded that galuteolin possessed anti-inflammatory activity.Besides, immune activity of galuteolin was investigated by in vitro and in vivo experiments. The murine macrophage cell line RAW264.7 was chose as cellular model to observe the effect of galuteolin on the phagocytic activity. The result showed that galuteolin could improve phagocytosis of RAW264.7 at the range of concentrations of 100-350 μg/mL. Its immune activity in vivo was researched by carbon clearance test. The carbon clearance index K and phagocytic ratio α increased significantly in the high-dose group（8 mg/kg） and middle-dose group（6 mg/kg）.Thus, galuteolin possessed good immune activity.