Extraction, Purification And Antioxidant Activities Of Flavonoids From Alfalfa

The thesis included three parts. Firstly, the study was designed to optimize the refluxing and ultrasonic-assisted extraction parameters of the alfalfa total flavonoids using orthogonal design and central composite rotatable response surface design. Furthermore, the study was designed to purify crude extracts using macroporous resin and further purified by polyamide resin. Moreover, the antioxidant activities of flavonoids from alfalfa were evaluated using free radical production systems in vitro. Exp. 1 Study on the extraction technology of alfalfa flavonoidsThe study was divided into two parts. The first part was designed to develop an efficient extraction technology of alfalfa flavonoids through optimizing the parameters of the refluxing and ultrasonic-assisted extraction method. Firstly, the single-factor experiments were used to determined the effects of extraction factors on the yield of flavonoids. Based on the results of single factor extractions, the extraction technologies were optimized by orthogonal test and central composite rotatable response surface. Finally, we determined the best extraction technology of alfalfa flavonoids using ultrasonic-assisted extraction. The optimized conditions were: ratio of liquid to solid 51.16:1(mL/g), extraction temperature 62.33 oC, extraction time 57.08 min and ethanol concentration 52.14%. Under this optimal conditions, the experiment extraction yield of alfalfa flavonoids was 6.40 mg/g, which was well matched with the value predicated by the model. The second part was designed to develop an efficient extraction technology of antioxidants from alfalfa. The optimal extraction parameters of extracts for the highest antioxidant activity by DPPH method was: ratio of liquid to solid 60.3:1(mL/g), extraction temperature 54.56 °C, extraction time 45.59 min, and ethanol concentration 46.67%; and by ABTS assay was: ratio of liquid to solid 47.29:1(mL/g), extraction temperature 63.73 °C, extraction time 51.62 min, and ethanol concentration 60%. Exp. 2 Study on the purification and separation of total flavonoids from alfalfaThe study was designed to obtain high purity of alfalfa flavonoids part through purifing by resins. First, five kinds of macroporous resins with different physical properties(NKA-9, AB-8, HPD-100, HPD-600 and D101) were employed for the separation of the flavonoids from alfalfa extracts. They were compared through static adsorption and desorption tests. And then to develop the best purification parameters. The resuts showed that the nonpolar resin labeled AB-8 showing better adsorption/desorption properties for total flavonoids among the test resins. The best purification parameters were: the concentration of the sample was 0.6 mg/mL, the pH of the sample was 4 and the range volume of loading sample was 1~4 BV. After one run treatment with AB-8 resin, the purify of the total flavonoids in the product increased to 33.03%. And then the product was further purificated using polyamide resin. Finally, we got the product with high purity of 79.62%. Thus, a simple and efficient method for enrichement and purification of total flavonoids from alfalfa with reisn were obtained. Exp. 3 In vitro study on the antioxidant activities of flavonoids from alfalfaThe study was designed to envaluate the antioxidant activities of total flavonoids and different polarity parts from alfalfa. They were tested using free radical production in vitro. 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid)(ABTS), 2,2’-diphenyl-1-picrylhydrazyl(DPPH) and hydroxyl radical were used in the study to envaluate the antioxidant activity of the total flavonoid compared with Vc. The results showed that the total flavonoids have a good ability to scavenge DPPH radical and the maximum value was 88.01% while only 75.25% capacity to scavenge ABTS radical. Compared with that of ascorbic acid, the scavenging activity of hydroxyl radical of flavonoids was higher at 0.1mg/mL of the 75% ethanol elution part. The 75% ethanol eluation part has the highest total antioxidant ability and the value was equal to 55.19 ± 3.37 mg Vc/g.