| Extraction and purification of natural products from Chinese traditional medicine are not only key steps of rational combination, pharmacology and molecule structure identification, but also important for Chinese medicine development. In this study, we first established a reliable method to search a suitable two-phase solvent system. And then an efficient new model for extraction by supercritical CO2/ ultrasonic-assisted, purification by HSCCC, and analysis and identification by HPLC, MS and NMR was constructed. This model could be used for preparation of natural products, construction of chromatography fingerprint and quality control of Chinese herbal medicine. By using this model, nine bioactive compounds were isolated and identified from peony flowers, a potential herb material. The main results are as follows:1. A reliable method to search a suitable two-phase solvent system for HSCCC was established. First, the polarity of target compounds was hypothesized and several solvent systems were chosen. To obtain satisfactory retention of the stationary phase, the settling time of the solvent system should be shorter than 60 seconds. To avoid excessive waste of the solvent, the mixture should contained nearly equal volumes of each phase. Second, TLC technique was used to examine the partition of target compounds between the two solvent phases to minimize the range of selected solvent systems. Third, after sample partitioning between the two solvent phases, aliquots of the upper and lower layers were analyzed byHPLC. From these two chromatograms, the K value of each component was determined by calculating the ratio of the peak heights (or areas) between the corresponding peaks. The K value should be close to 1 and the separation factor a, which is the ratio of K between two components, should be great than 1.5. Fourth, using determined solvent system above, small sample size and high flow rate were used to primary separation, and the preparative separation was then performed by adjusting the flow rate.Based on the above procedure, honokiol and magnolol were isolated and purified from Cortex Magnoliae Officinalis by HSCCC. A crude sample, 500 mg, was successfully separated with a two-phase solvent system composed of rc-hexane, ethyl acetate, methanol and water (1:0.4:1:0.4, v/v), and produced 265 and 150 mg of honokiol and magnolol with purities of 99.2 and 98.2%, respectively, in 2.5 h. In addition, following an initial clean-up step on the AB-8 resin, HSCCC was used to purify an arctiin from an extract of the Arctium lappa L. The two-phase solvent system used was composed of ethyl acetate-rt-butanol-ethanol -water at an optimized volume ratio of 5:0.5:l:5(v/v/v/v). A total amount of 159 mg of arctiin at 98% purity was obtained from 350 mg of the crude extract (containing 49.2% arctiin) with 91% recovery in 5 hr in a separation. Identification of the target compounds were further performed by MS and NMR.2. An efficient new method for extraction, separation and purification of natural products by supercritical fluid extraction and high-speed counter-current chromatography. Psoralen and isopsoralen were extracted from Fructus Psoraleae (Psoralea corylitolia L.) by supercritical CO2. The optimum extracting conditions of pressure (26 MPa), temperature (60°C) and a sample particle size of 40-60 mesh were got by using orthogonal experimental design. After cleaning with petroleum ether, the extract were separated and purified by HSCCC with a two-phase solvent system composed of rc-hexane-ethyl acetate-methanol-water (1:0.7:1:0.8, v/v), and obtained 39 mg and 40mg psoralen and isopsoralen with purities of 99.2%and 99.0%, respectively. The optimum extracting conditions of extracting imperatorin and isoimperatorin from Angelica anomala L. by supercritical CO2 were investigated by using orthogonal experimental design. The results showed that pressure (25 MPa), temperature (60°C) and a sample particle size of 40-60 mesh were optimal. The crude extract (500mg) was purified by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.4:1.1:0.5, v/v/v/v) in 5h. The separation produced 296 mg and 40 mg of imperatorin and isoimperatorin with purities of 89.0% and 99%, respectively. Evodiamine and rutecarpine were extracted by supercritical CO2 from Evodiarutacearpa (Juss.) benth at conditions of pressure (28 MPa), temperature (40°C) and a sample particle size of 40-60 mesh. The crude extract (160mg) was purified by HSCCC with a two-phase solvent system composed of tetrachloromethane-methanol -water (4:4:1, v/v/v) in 4.5 h and produced 13 mg and 8.4 mg of imperatorin and isoimperatorin.3. An efficient new method for extraction, separation and purification of natural products by ultrasonic-assisted extraction and high-speed counter-current chromatography. The optimum extraction conditions of flavonoids from male Inflorescence of Populus tomentosa Carr. were investigated by using orthogonal experimental design. The results showed that 150W ultrasonic wave, 30:1 of 70% ethanol to male Inflorescence weight, extraction for 40min were optimal. The extract was combined and evaporated to get rid of ethanol, then was extracted by petroleum, ethyl acetate successively. The ethyl acetate extract was further subjected to silica gel chromatography by eluting stepwise with chloroform-methanol (97:3, 90:10, 80:20 and 70:30, v/v) to obtain four fractions. The fractions were isolated and purified by HSCCC with the two-phase solvent systems composed of petroleum-ethyl acetate-methanol-water (1:0.8:0.8:0.9, v/v/v/v), chloroform-methanol-water (5:3:2,v/v/v), chloroform-methanol-water- n-butanol (5:3:2:0.5,v/v/v/v) and ethyl acetate- n-butanol methanol-water (2:1:3, v/v/v), respectively. Seven compounds were obtained and five of them were identified on basis of spectral methods and HPLC with authentic samples. Tectoridin was extracted from Belamcanda chinensis (L.) DC. Using ultrasound-assisted extraction. The extract was cleaned up by AB-8 resin. Portion of 20% ethanol extract was subjected to HSCCC with the two-phase solvent system composed of ethyl acetate-ethanol-methanol-water (5:0.5:0.5:5, v/v/v/v). 30 mg tectoridin at 99.0% purity was obtained from 100 mg of the crude extract in a separation.4. Bioactive compound identification and pharmacological analysis of peony flower extract. Our results showed that peony flower extract had no influence on blood pressure, respiration, heart rate and electrocardiogram (EGC) in mice and rabbits, indicating its safety to animals. However, chemiluminescence and spectrophotometry analysis revealed that the extract of peony flowers could result in the removal of active oxygen species in some modified chemical systems. The inhibition of DNA damaging induced by hydroxyl radical in the peony flowers extract was also observed. The results showed that the extract could efficiently remove DPPH', O2', 'OH, H2O2 and the 50% inhibition concentration (IC50) are 94.29, 199.0, 193.18 and 50.85 ug/ml, respectively. A novel extraction procedure offlavonoids from paeonia suffruticosa Andr. flowers by ultrasonic was established. The ratios of sample quantity to solvent volume and extraction time were optimized. The flavonoids could be quantitatively extracted from Paeonia suffruticosa Andr. with 20:1 liquid to flower sample and with a 22Hz ultrasonic-assisted treatment for 10 min. The extract was cleaned by the polyamide and then was isolated by HSCCC and silica gel. Nine compounds were obtained and their structures were identified by the physical and chemical characters especially by the spectral analysis. They were apigenin-7-O-D-lutinoside, luteolin-7-O-D-glucoside, apigenin-7-O-D-glucoside, kaempferol-7-O-D-glucoside, apigenin, kaempferol, luteolin, garlic acid and benzoic acid. To our knowledge, it is the first time that the nine compounds are obtained and identified from this important plant species. Our results will accelerate the exploration and application of peony flowers.
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