Extraction Of Vitex Negundo L.var.cannabifolia Flavonoids By Ionic Liquid And It’s Antioxidant Activity

Vitex negundo L.cannabifolia（sib.et Zucc.）Hand.-Mazz.Vitex negundo volatile oil is the main effective component of Vitex negundo pharmacopoeia.At present,studies on the pharmacological activity of Vitex negundo mainly focus on its volatile oil,while there are few reports on its non-volatile components,leading to incomplete utilization of Vitex negundo resources,resulting in resource waste.In order to make full use of Vitex negundo resources,the flavonoids in Vitex negundo were studied in this paper,and the contents were as follows:1.A green p H-responsive Functionalized ionic liquid surfactant（Functionalized surface active ionic liquid,FSAILs）was prepared,and the extraction process of Vitex negundo ultrasound-assisted functional ionic liquid surfactant（FSAILs S-UAE）was established.The extraction process of total flavonoids from Vitex negundo was optimized by single factor and BBD experiments.At a liquid-solid ratio of 10:Under the optimized conditions of 1 m L/g,[N-14,N’-CO2H-Im][Br]concentration of 42 mmol/L,extraction temperature of 40℃,ultrasonic time of 39 min,and ultrasonic power of 400 W,and sonication time of 39 min,the extraction yield of flavonoids from Vitex negundo reached 9.96 mg/g.FSAILs could be used for up to 6cycles,and the extraction yield was still above 6.41 mg/g.2.Eight batches of Vitex negundo flavonoids were tested for their antioxidant activity in vitro.The results showed that Vitex negundo flavonoids had better antioxidant activity than ascorbic acid by scavenging hydroxyl radicals,ABTS radicals,DPPH radicals and thiobarbital acid method.In order to further clarify the main antioxidant active ingredients,vitexin,isovitexin,hyperoside,luteolin,quercetin and apigenin were screened by HPLC-DPPH combined with UPLC-MS.3.A HPLC method for the determination of six flavonoids in vitexin,isovitexin,hyperoside,luteolin,quercetin and apigenin in vitexin leaves was established.Aglient 1260 high performance liquid chromatography-Ecosil C₁₈（4.6 mm×250 mm,5μm）column,liquid detection conditions:The mobile phase consisted of 0.2%aqueous phosphoric acid（A）-hexonitrile（B）,gradient elution（0-15 min,8%-8%B;15-20 min,8%-15%B;20-30 min,15%-15%B;30-50 min,15%-40%B;50-60 min,40%-8%B）at a flow rate of 1.0 m L/min;The column temperature was 35℃.The detection wavelength was 320 nm.The injection volume was 10μL.4.Six macroporous adsorption resins（D100,D101,HPD-100,HPD-300,X-5,AB-8）were screened and the enrichment process was optimized.The optimization results were as follows:The loading concentration was 0.996 mg/m L,loading volume was 1 BV,loading flow rate was1 BV/h,and the desorption solution concentration was 1 BV with 60%ethanol solution and 2BV with 90%ethanol solution.The results showed that HPD-100 macroporous adsorption resin had a good enrichment effect on flavonoids in Vitex leaves.After enrichment,the purity of total flavonoids in Vitex leaves increased from 19.54%to 71.06%,which was 3.64 times higher than that before purification.5.The antioxidant effects of eight batches of Vitex leaf flavonoids on aging mice induced by D-galactose were studied.The organ index,T-AOC and MDA contents in serum and liver tissues,SOD and CAT contents in heart and kidney tissues were measured.The pathological changes of heart and kidney tissues were observed by HE staining.The results showed that vitex leaf flavonoids promoted ROS scavenging in mice,and the activities of T-AOC,SOD and CAT were increased,while the level of MAD was decreased.The changes of these indexes indicated that the activities of antioxidant enzymes in mice were affected by the flavonoids of Vitex negundo,which could prevent cell damage and effectively remove ROS generated in the metabolic process in vivo,thus effectively alleviate and improve the aging of mice.The results of antioxidant activity in and outside the complex showed that Vitex negundo samples from Baoding City of Hebei Province were selected as the best batch.The T-AOC activity in serum of mice was 31.03 U/m L,and the MDA content was 9.51 U/m L.The T-AOC activity in liver was 38.78 U/m L,and the MDA content was 21.17 U/m L.The activity of SOD and CAT in mouse heart was 11.56 U/m L and 44.23 U/m L,respectively.The activity of SOD and CAT in mouse kidney was 24.23 U/m L and 62.19 U/m L.This study realized the efficient utilization of Vitex negundo resources.The established FSAILs-UAE extraction process is not only convenient,efficient,but also green and sustainable,which meets the requirements of green chemistry.The purified Vitex negundo flavonoids have good antioxidant activity,which provides a theoretical reference for the development of natural antioxidants from Vitex negundo and the improvement of the added value of Vitex negundo resources.