Extraction And Purification And Biological Activities Of Flavonoids From Fruit Of Actinidia Auguta

The northeast wild Actinidia arguta is an important wild fruit resource. Actinidia arguta Sieb.et Zucc belongs to Actinidiaceae, Actinidia is perennial and Deciduous liana. This wild plant distributes in the Northeast China, North China, Shandong, Northwest China and Yangtze valley, and chiefly concentrated in the southern part of the mountainous area in the Northeast. Its root, stem and fruit are all useful for us in their medical value and healthy function. Flavonoid is an important natural polyphenol product, which has extensive biological activities, existing in largely in various plants. Making deep research on the fruit of Actnidia arguta is good for resource protection and comprehensive exploitation.At present, preliminary purification and component has been studied on Actnidia arguta of different varieties, but there has no research on the preparation and bioactivities of the northeast wild Actinidia arguta. Therefore, in this paper, some systematic studies on flavonoids are made and the main results are as follows:1. Two different extraction methods, including ultrasonic-assisted extraction and microwave-assisted extraction are compared on the extraction efficiency of flavonoids from Actinidia arguta by response surface methodology, and ultrasonic-assisted extraction is selected as the extraction method in the end. The results showed that the optimum conditions are as follows:70% ethanol as the solvent, material-water ratio 1:4(g/mL), ultrasonic power 270W, ultrasonic time 6min, the extraction temperature 60℃, extraction time 40min and the extraction times 2. Under these conditions, the extraction rate of flavonoids is 0.0297%. The content of flavonoids from Actnidia arguta is 29.7mg/100g fresh fruit.2. ZrOCl2 colorimetry, AlCl3 colorimetry, NaNO2-Al(NO3)3 colorimetry and HPLC determination method are compared to determine the content of flavonoids from Actinidia arguta. Wavelength scanning is performed in the range of 200～600nm on the reaction product produced by the flavonoids crude extract and lutin standard with different colour reagents from the three colorimetries. According to the scanning result, the detection wavelength is determined. The results show that NaNO2-Al(NO3)3 colorimetry and AlCl3 colorimetry are not suitable for determining the content of flavonoids from Actinidia arguta whose results are inaccurate with big error. While the result of ZrOCl2 colorimetry is similar to that of HPLC method. At 415nm, the standard curve drew by A415nm as abscissa and concentration of lutin as ordinate is Y284=0.0114X-0.0001, R2=0.9991. The average recovery is 99.4% with RSD of 1.61% (n=5). ZrOCl2 colorimetry is a quick and accurate method for determining the content of flavonoids from Actinidia arguta.3. Among 9 kinds of macroporous absorbing resins, HPD-600 resin has better absorbing and desorbing ability to purify flavonoids crude extract. The optimum conditions are as follows:flavonoids concentration 0.5mg/mL, pH 4～5, ratio of sample solution and macroporous resins 8:1(mL/g), sample solution 3BV (bed volume), sample flow rate 2mL/min, water 3BV and the elution solvent is 4BV of 80% ethanol, the desorption flow rate 2mL/min. Under these conditions, the average recovery rate and purity of flavonoids are 99.4% and 37.2% respectively. Flavonoids from Actinidia arguta are purified in the way of gradient elution by polyamide column chromatography after HPD600 resin purifying. The results show that among the 20 gradients, separation is well done by water:methanol 6:4 and 0:10 as elution solvent. The collection of the two gradients is concentrated and froze drying for next preparation respectively.4. Flavonoids from Actinidia arguta are prepared by semi-preparative reversed phase liquid chromatography (RPLC) whose purity is over 99%. The separation and preparation conditions of eluate by water:methanol 6:4 are as follows:flavonoids concentration 30mg/mL, water:methanol as mobile phase with the ratio of 55:45(V/V), flow rate 5.0mL/min, sample solution 400μL, column temperature 30℃, detection wavelength 340nm, preparation time 60min. The other separation and preparation conditions of eluate by water:methanol 0:10 are as follows:flavonoids concentration 30mg/mL, water:methanol as mobile phase with the ratio of 20:80(V/V), flow rate 5.0mL/min, sample solution 600μL, column temperature 30℃, detection wavelength 340nm, preparation time 60min. Under these conditions, five compounds are obtained.5. The structures of the three samples are identified by NMR and thin-layer chromatography (TLC). The samples are isoquercitrin, quercetin and rutin. The most important is that isoquercitrin is the first found in the variety of Actinidia arguta.6. To study the in vitro antioxidant activity of flavonoids from Actinidia arguta, spectrophotometry is used to detect and analyze compared with ascorbic acid. Flavonoids from Actinidia arguta are found to have strong DPPH radical scavenging activity and effective lipid peroxidation inhibitory activity with a 50% inhibition concentration (IC50) of 16.09μg/mL and 0.46mg/mL respectively and better than that of ascorbic acid. The extract shows strong reduction ability and it is 0.86 times as much as that of ascorbic acid. The extract also has certain superoxide anion radical and hydroxyl radical scavenging activities with IC50 of 7.33mg/mL and 9.16mg/mL respectively and lower than that of ascorbic acid, in a dose dependent manner. These results suggest that flavonoids from Actinidia arguta have potent in vitro antioxidant activity.7. Flavonoids from Actinidia arguta has obvious effect of growth inhibition on bacteria and yeast, but unconspicuous on mould. Flavonoids from Actinidia arguta have certain antimicrobial effect.