Application Of High-Speed Counter-Current Chromatography In The Research Of Natural Products And Development Of New Health Product In The Citrus

This study was mainly consisted of two different parts. The first part studyed of the application of high-speed counter-current chromatography (HSCCC) in the research of natural products. HSCCC was mainly used in the separation of cucurbitacin in Pedicellus Melo and polymethoxylated flavones (PMFs) in Orange Peel. First, a preparative HSCCC was used to isolate cucurbitacins from Pedicellus Melo. A successful isolation and purification was achieved at the following conditions:the two-phase solvent system was composed of n-hexane, ethyl acetate, methanol and water at a volume ratio of 1.2:0.8:0.8:1.2 (v/v/v/v) and the lower phase of the system was used as the mobile phase at the flow rate of 2.0 ml/min, the isolation temperature and revolution speed were set at 25℃ and 800 rpm. In 4.0 hours,33.5 mg cucurbitacin B,21.7 mg cucurbitacin E, and 14.9 mg cucurbitacin I with purities of 99.1%,98.3% and 97.5% by high performance liquid chromatography (HPLC) from 200 mg crude extract.The chemical structure was determined by 1H- NMR and 13C-NMR, and the result was the same with the literatures. Second, a preparative HSCCC was used to isolate PMFs from Orange Peel. A successful isolation and purification was achieved at the following conditions:the two-phase solvent system was composed of petroleum ether, ethyl acetate, methanol and water at a volume ratio of 1:0.8:1:1.2 (v/v/v/v) and the lower phase of the system was used as the mobile phase at the flow rate of 2.0 ml/min, the isolation temperature and revolution speed were set at 25℃ and 800 rpm. In 6.0 hours,32.8 mg nobiletin, 10.9 mg 3,5,6,7,8,3’,4’-heptamethoxyflavone,43.5 mg tangeretin, and 18.7 mg 5-hydroxy-6,7,8,3,4’-pentamethoxyflavone with purities of 98.9%,97.8%, 989.2% and 99.8% was obtained by high performance liquid chromatography (HPLC) from 200 mg crude extract. The chemical structure was examined by MS and 1H-NMR, and the result was consistent with the literatures. It was proved that HSCCC was very broad and excellent in the research of natural productsThe second part studied of the extraction and purification of PMFs from Orange Peel and the related prescription research with soft capsule. First, To establish a method for the quantification of nobiletin and tangeretin in Orange Peel by HPLC. The Aglient Extend C18 column(250 mm ×4.6 mm,5 μm)was adopted with the mobile phase of acetonitrile-water (0～10 min:acetonitrile 40%; 10～15 min:acetonitrile 40%-60%; 15-20 min:acetonitrile 40%) at 25 ℃; The flow rate was 1.0 mL/min, and the detection wavelength was 330 nm. Second, To study the optimal process for nobiletin and tangeretin from orange peel by microwave assisted extraction. The optimal technology condition for microwave assisted extraction was determinaed as follows:the ethanol concentration was 75%, the microwave power was 850 W, the microwave treating was 5 min and the material-liquid ratio was 1:15. Then the extraction yield of nobiletin and tangeretin were 90.21% and 84.25%, respectively. And then compared with the traditional reflux and ultrasonic extraction, the microwave-assisted extraction could not only shorten the extraction time greatly, but also increase the yield of nobiletin and tangeretin. Third, macroporous resin was used to purify PMFs. D101, AB-8, HZ816 and HZ841 resin was investigated. And AB-8 resin PMFs was the best to purify PMFs. At last, to study the formulation and the prescription of soft capsules with PMFs, the best prescription was determinaed as follows (The total contents was 1 g):PMFs powder content was 20%(200 mg); Suspending agents was 1.5% beeswax (15 mg); Wetting agent was 2% lecithin (20 mg); Dispersion medium was 76.5% soybean oil (765 mg). The basic experimental data was provided from the above research for the development of new health care products.