Identification And Evaluation On Antioxidant Activities Of Flavonoids From Choerospondias Axillaris

Choerospondias axillaris(Roxb.) Burtt et Hillbelonging to thefamily Anacardiaceaeb and mainly distributes in Tibet, Guangxi, Yunnan and Guizhou Provinces of China and South East Asia. The dry fruit of C. axillarishas variety of physiological activities, and the components responsible for the effect are always thought to be the total flavonoids. But so far the study of flavonoids in C. axillarismostly focus on its pharmacological activities, and the reports are rare at its chemical compositions and biological activities.This study is made up of four parts:(1)The optimal conditions for microwave-assisted extraction of flavonoids from the flesh fruits of C. axillariswere established by using single factor experiments(consist of five factors including ethanol concentration,ratio of solvent to material, ratio of solvent to material, extraction time and microwave power) and response surface analysis as follows: extraction temperature,75 ℃; ratio of solvent to material, 62:1(m L/g); ethanol concentration, 62%; microwave power 500 W; and extraction time,7 min. Under the optimum conditions, the yield of flavonoids was16.41%.(2) The flow of the sample solution, p H value of adsorption solution, flow of the elution agent(ethanol)and the elution agent concentration were studied by using dynamic adsorption/desorption. The purification of crudeflavonoids from C. axillaris was also investigated using preparative chromatography. The results show that the optimal adsorption/desorption conditions for purifying the crude flavonoids with AB-8macroporous adsorbent resin were determined as follows: the extract solution at p H 2 was passed through the column at aflow rate of 2 bed volumes/h(BV/h), and then the column was eluted with 60% alcohol at 3 BV/h. On this condition, the content of flavonoids in the purified extract increased from 28.5% to 67.3%. The purification of crudeflavonoids from C. axillaris was separated into two fractions(F1 and F2) using preparative chromatography and the feasibility was confirmed bythe results of HPLC analysis.(3) Qualitative analysis of the flavonoids in C. axillariswas carried out by using liquid chromatography-series time of flight mass spectrometry(LC-Q-TOF-MS) and liquid chromatography-triple level 4 pole mass spectrometry(LC-QQQ-MS/MS). Combined with comparison of the retention times and mass spectral fragment with standard substances or related literature, 10 phenolic acids and five flavonoids were identified. Quantitative analysis of the flavonoids was performed by LC-QQQ-MS/MS in multiple reactions monitoring(MRM) mode. Sixcomponentswere found in F1, includingquinic acid(399.76±16.69 μg/g), citric acid(874.26±24.13 μg/g),gallic acid(1313.32±46.85 μg/g),chlorogenic acid(14.39±0.34 μg/g),catechins(27.31±0.27 μg/g)and protocatechuic acid(1282.53±52.63 μg/g). Elevencomponentswere found in F2, including catechins(117.18 ± 3.44 μg/g), chlorogenic acid(18407.81 ± 260.39 μg/g),vitexin(12.52 ± 0.29 μg/g), 1-caffeoylquinicacid(148.12 ± 5.90 μg/g), isoquercitrin(45.16 ± 1.16 μg/g), ellagic acid(806.50±28.12 μg/g), quercetin(484.83±20.39 μg/g), astragalin(60.00±0.80 μg/g), 3-O-methylellagicacid(583.84 ± 14.87 μg/g), 3,3’-di-O-methylellagic acid(662.41±16.62 μg/g) and isorhamnetin(46.31±0.53 μg/g).(4)The total antioxidant activity, DPPH radical scavenging activities, hydroxyl radical scavenging activities and superoxide anion radical scavenging activities of the total flavonoids, F1 and F2 were determined, respectively, and the results werecompared to those of ascorbic acid(Vc). In addition,the antioxidant activities of the flavonoids wereevaluated by using the cell model. The results clearly demonstrated that the flavonoids from C. axillaris possess significantly antioxidant activities, which werebetween F1 and F2. The DPPH radical scavenging activities of F1 and the hydroxyl radical scavenging activities of F1 and F2 were higher than those of Vc,but the activities of total antioxidant and superoxide anion radical scavenging of F1 and F2 were lower than Vc. According to the results of the cell model, the flavonoids from C. axillaris showed non-toxic in the concentration of 1~500 μg/m L and significant antioxidant activity in mice macrophage(RAW264.7).