Studies On Extraction, Purification, Structrue And Antioxidant Activities Of Flavonoids From Ramie Leaves

Ramie (Boehmeria nivea(L.)Gaud) is an Urticaceae perennial herb of Boehmeria, which is an important export crops. Ramie leaves were discarded as industrial waste products which wasted resources and polluted the environment seriously. Ramie leaves have some medicinal value. In this paper, we studied the extraction, purification, structure and antioxidant activities of flavonoids which are active ingredients from Ramie leaves as raw material.1 Optimization for extraction condition of flavonoids in Ramie leavesIn this paper, flavonoids were extracted from leaves of "Hua Zhu 4" Ramie with two methods. One was the ethanol refluxing method. The extraction condition was optimized by orthogonal experiment on four simple factors such as ethanol concentration, the ratio of material to liquid, extraction time and extraction temperature. The optimum conditions were as follows:ethanol concentration was 70%, the ratio of material to liquid was 1:60, extraction time was 1.5 h and extraction temperature was 80℃, one extraction time. Under the optimized condition, the yield of flavonoids from leaves of "HuaZhu 4" was 3.76%. The other method was ultrasonic assisted extraction. The optimum extraction condition of flavonoids from Ramie leaves was obtained by single factor and orthogonal experiments. The result was as follows:liquid to solid ratio 30:1,70% ethanol concentration, ultrasonic power 60 W, ultrasonic time of 30 min, ultrasound temperature 60℃, one extraction time. Under this condition, the yield of flavonoids from Ramie leaves was 4.94%.flavonoids in different varieties of Ramie leaves under the first optimum extraction conditions were measured. The results showed that the highest flavonoid content was "Lu Zhuqing", which yield of flavonoids was 6.62%; the lowest was "HuaZhu NO.5", which only was 2.74%.By comparing the two methods, the yield of flavonoids from Ramie leaves with ultrasonic assisted extraction was 1.18%, higher than that of the ethanol extraction.2 Separation and purification of flavonoids from Ramie leavesThe adsorption effects of four macro-porous resins (AB-8, D101, S-8 and polyamide) on flavonoids from Ramie leaves were studied. Through static adsorption and desorption experiment, AB-8 was selected as the adsorption resin because of its higher adsorption and desorption rate. Through the dynamic adsorption-desorption by single factor experiment of macro porous resin AB-8, the optimum dynamic elution condition was:the pH value of solution was adjusted to 4, the concentration of extracts was 1.508 mg/mL, eluted with distilled water and 2 BV 70%ethanol at the flow rate of 1 mL/min.50% ethanol elution was collected, concentrated and freeze-dried to obtain a preliminary purified flavonoids of Ramie leaves and the concentration of it reached 29.85%, which was 5.3 times as high as the crude extracts.Sephadex LH-20 was used to further separate flavonoids from Ramie leaves. The preliminary purified flavonoids with AB-8 resin were applied to a Sephadex LH-20 column after ethyl acetate extracted. Eluted with different concentrations of methanol repeatedly,50%and 90%methanol eluting solution were collected and obtained two kinds of components after freeze-dried called RF1 and RF2. Their purity reached above 90%by HPLC analysis.3 Structure identification of flavonoids from Ramie leavesThe samples of refined flavonoids were identified as flavonols and isoflavones by using color reaction and UV. The characteristic group of flavonoids was found by using infrared spectra analysis. The suitable mobile phase was:water (pH=3)-methanol (0.45:0.55), which was selected with HPLC tested among six kinds of mobile phases. The sample one (RF1) was identified as rutin by HPLC and GC. The other component (RF2) was needed to be further examined.4 Antioxidant activities of flavonoids from Ramie leavesThe capacity of crude flavonoids and RF1 sample was studied under chemical model system experiments in this paper. The results showed that the IC50Vc RFl>crude flavonoids. With the concentration of flavonoids extracts increased, the antioxidant capacity was gradually enhanced, which showed a good dose-effect relationship with them. But antioxidant of flavonoids extracts tended balance after reaching a certain concentration. It was also showed that the antioxidant activity of RF1 was higher than crude flavonoids extracts. The results showed that flavonoids extracts of Ramie leaves had good antioxidant capacity on superoxide, hydroxyl radical, hydrogen peroxide etc.