Study On The Extraction And Separation Of The Flavonoids From Apocynum Venetum Leaves And Its Anti-oxidant And Anti–depressant Activity Evaluations

Apocynum Venetum L. was the famous traditional Chinese medicine in the literature, which was perennial herb of apocynaceae. Many compositions with kinds biological activities exist in it, and the flavonoid was the primary compounds. Flavonoids have pharmacological and biological activities variously after a mounts of pharmacological tests being examined. The extraction of Apocynum Venetum L. showed anti-depressant activity obviously in investigations during recent years, but systematic researches were not carried out for single composition. Therefore, study on isolation of the active components and single compounds from extraction and evaluations of anti–oxidant and anti–depressant activities plays a positive role in extending its application range.Firstly, this paper adopted reflux and ultrasonic and immersion methods to extract flavonoids from Apocynum Venetum L., Hyperoside was used as standard compound and extraction rate of total flavonoid was evaluation standard, comparision among above extract methods. The result showed that the reflux had the highest extraction rate, and extraction processes were optimized, the perfect extract conditions were as follows: 60% ethanol in water was used as the extraction solvent at the ratio of solid to liquid of 18 in 1h, 3 times were repeated. Examination on the extract conditions were also tested for Accelerated solvent extraction (ASE), and the 70% ethanol in water was used as the extraction solvent, static time of 10 min and 3 times were repeated at the 70℃. By comparison of the extraction rate of total flavonoids, the ASE was much better, but the adopted method in this study was reflux, since the reflux is suit for large mounts of medicinal materials. Othter wise, ASE was fit for extracting biological compounds from tradition medicine materials for which the advantages of rapidly and security and simple operation.Secondly, High performance counter–current chromatography (HPCCC) method was used for separation and isolation of the biological compounds and single flavonoid from Apocynum venetum leaves. The optimized operation conditions were that the solvent system electing was ethyl acetate–acetonitrile–water–acetic acid at the volume rations of 5:0.8:5:0.02(v:v:v:v), the upper phase was used as the stationary phase, then the lower phase was used as the mobile phase, the rate of mobile phase was 2.5 mL·min^-1, and the detection wavelength was set at 360 nm, the rotation speed was 1500 r·min^-1, and the column temperature was 30℃and 150 mg sample was injected. The results of the separation demonstrated that six obvious chromatographic peaks were obtained. The fractions from the HSCCC was analyzed and identified by HPLC–MS,ESI–MS/MS, then four single compounds and two mixtures with high purification were separated and isolated. The four single flavonoids were Quercetin–3–O–maltoside, Kaempferol–3–O–galactoside, Astragalin, Acetylated quetcitin–3–O–galactoside and the two mixtures were Hyperforin and Isoquercetin, Quercetin and Kaempferol. HPLC technology was used for testing the purities of fractions, the purities of single compound were all above of 95% with area normalization. The experimental results demonstrated that HPCCC was an effective and rapid method for separation and purification of the compounds from natural plants.Study on the anti–oxidant activities of target compounds which were collected from HPCCC and total flavonoid employed elimination hydroxyl radical of DPPH? method. The results showed that all of target compounds have great anti–oxidant activities and the sequence of them was total flavonoid > Hyperforin and Isoquercetin >Quercetin–3–O–maltoside > Kaempferol–3–O–galactoside > Astragalin > Acetylated quetcitin–3–O–galactoside. The qualitative and quantitative measurement and the protection on the cultured pc–12 cell lesioned by corticoeron of the constituents and the forced swimming test were also taken for the research on the anti-depressed activities of the target compounds, and the results showed that all of the target compounds have perfect anti-depressed activities and the sequence of them was Hyperforin and Isoquercetin>Isoquercetin>Hyperforin>total flavonoids>Astragalin>Kaempferol–3–O–galactoside>Acetylated quetcitin–3–O–galactoside > Quercetin–3–O–maltoside > Quercetin and Kaempferol.