| Kunlun Chrysanthemum is also known as Coreopsis tinctoria Nuff., Tianshan Coreopsis tinctoria Nuff., Coreopsis basalis, Coreopsis tinctoria, and ―snow chrysanthemum‖. The scientific name of Kunlun Chrysanthemum from Xinjiang, China, is Coreopsis tinctoria, annual herb, family Compositae. Kunlun Chrysanthemum naturally grows in the Kunlun Mountains region at an elevation of 3000 meters in Hetian region of Xinjiang, and its Uighur name is ―guriqiayi‖. Its flowering period is short, wild scarce. It is the rare alpine wild plant with unique effect and high medicinal value par with the snow lotuses in Xinjiang. Coreopsis tinctoria flowering tops infusion that is a kind of ancestral natural herbs is traditionally used for keeping fit by Uighur nationality. It was taken for granted that it is a special plants used not only as a tea-like beverage but also as a folk medicine with functions of Lipid-lowing, bating blood-vessel, and retarding bacterial growth applied in treatment for coronary heart disease by domestic cardiovascular experts in China. As people have come to realize the health benefits of Kunlun Chrysanthemum, researches on the active ingredients and biological activityof Kunlun Chrysanthemum gradually become the focus of numerous studies. However, currently, the main product of Kunlun Chrysanthemum on market is dry tea, with almost no refinery processed products. Therefore, to enhance the level of deep processing of Kunlun Chrysanthemum, we aimed to examine the extraction, isolation, purification technology and bioactivities of Kunlun Chrysanthemum polysaccharide(KCCP), Kunlun Chrysanthemum total flavonoids(KCTF) and Kunlun Chrysanthemum proanthocyanidins(KCPC) with tops of Coreopsis tinctoria Nutt.as raw material, and simultaneously analyzed and compared. These studies results could induce important theoretical and practical significance in terms of regulating the market, developing high value-added approach and promoting the application of Kunlun Chrysanthemum in food or pharmacology.The extraction technology of ultrasound- assisted combined enzymolysis method(USCE) applied for KCCP, ultra high pressure(UHP) extraction method used for KCTF and ultrasoound- assisted method(US) utilized for KCPC were optimized, repectively. In the present study, the scavenging activities against DPPH?, ?OH, and total antioxidant capacity(TAC) were selected to evaluate the antioxidant activity in vitro of KCCP, KCTF and KCPC. With healthy Kunming male mice as test animals, the antioxidant activity in vivo of KCCP, KCTF and KCPC were evaluated by assaying the value of superoxide dismutase(SOD), malondialdehyde(MDA) and total antioxidant capacity(T-AOC) from serum, liver and kidney. The antimicrobial effect against Eshrichia.coil, Staphylococcus aureus, Bacillus.subtilis, Penicillium citri, Aspergillus nige and Saccharomyces cerevisiae of KCCP, KCTF and KCPC were monitored by the disc diffusion method, and the minimum inhibitory concentration(MIC) values were determined using the broth microdilution method. In vitro antiproliferative activity of KCCP, KCTF and KCPC were determined using the colorimetric methyl thiazolyl tetrazolium(MTT) method concerning four kinds of tumor cells(Cervical cancer He La cells, esophagus cancer Eca-109 cells, Hepatoma BEL-7404 cells and mouse increased(p<0.01), which sμggested that the experimenal model was successfully established. Moreover, the levels of ALT and AST were reduced in the administration group by high dose(HD) of KCPC when compared to CCL4 control(p<0.05), its ability to reduce levels of ALT and AST are 1.5-fold and 2.8-fold of positive control(Bifendate), respectively. KCPC can inhibit the increase of transaminase activity induced by CCl4 hepatic fibrosis, thereby demonstrating hepatoprotective effect, which could not be observed by KCTF and KCPC intake. ascites hepatomas H22), together with a Vero cell(normal cell) as control. The IC50 value was determined as the concentration that caused a 50 % inhibition of cell proliferation and was applied for comparing the tumor suppressor activity. To evaluate the protective effect on liver of KCCP, KCTF and KCPC, CCl4-induced acute liver injury model was established, and serum alanine aminotransferase(ALT) levels and serum aspartate aminotransferase(AST) levels were used as index. In additional, this paper focused on the research about isolation, identification and screening of polysaccharide component from KCCP, analysis of monosaccharide composition and molecular weight distribution, assay of bioactivity of KCCP and its fractions. According the results of antioxidant experiments, the anti-aging effect and mechanism of KCPC was observed by using the model system Drosophila.The optimal extraction process conditions of KCCP by USCE was optimized by orthogonal experiment, and the optimal extraction conditions were as follows: firstly, sonicated under the conditions of 40 ℃, 15 min, 36 k Hz, then hydrolyzed under compound enzyme 2.5%(cellulase 2%, acid protease 0.5%), enzymolysis temperature 55℃, p H4.5, and enzymolysis time 2.0h, the yield of crude polysaccharide was up to 9.83%, purity was of 28.58%. Thus, its extraction rate and purity with USCE were increased 188.3% and 216.2% respectively, compared with traditional reflux extraction method. Based on the content of carbohydrate and antioxidant activity test, two components of PI(1mg) and PⅡ(3.7mg) were obtained by DEAE-52 chromatography from samples of a content of KCCP(100mg), then the main KCCP fraction PⅡ(content of carbohydrate 2%,g/m L) was further separated into three fractions by Sephadex G-100 gel chromatography column, that are fraction PⅠ’(from 9 to19 tube)contained 0.2%(g/m L) carbohydrate, fraction PⅡ’(from 20 to 35) contained 0.4%(g/m L) carbohydrate, and fraction PⅢ’(from 40 to 60) contained 0.2%(g/m L) carbohydrate. KCCP could be purified well by DEAE-52 under the conditions of material concentration 20mg/m L, sample flow velocity 0.5 m L/min, sample quantity 5m L. The monosaccharide composition analysis of KCCP fraction PⅡwas analysed and quantified by HPAEC-PAD compared the retention time of peaks from samples and standards monosaccharides, the monosaccharide composition of KCCP fraction PⅡ is as the following: fucose(Fuc), 1.84%; rhamnose(Rham), 14.99%; arabinose(Ara), 14.39%; galactose(Gal), 35.08%; glucose(Glc), 15.92%; xylopyranose(Xyl), 2.21%; mannose(Man), 6.31%; galacturonic acid(Gal UA), 4.17%; glucuronic acid(Glc UA), 2.87%. The determination of the molecular mass(MW) of KCCP fraction PⅡwas carried out on a waters 600 high performance liquid chromatography system(HPLC) by comparison with the retention time of a glucose standard, and the molecular mass of KCCP fraction PⅡ mainly distributes in the range of 1.7×104~4.4×106.The bioactivity of KCCP and fraction PⅡwas studied with in vitro antioxidant activity and inhibition of tumor cell proliferation as index. The results showed that the reducing power of all KCCP samples correlated well with their increasing concentrations. The scavenging capability of each fraction on DPPH? free radical was both significant and in a concentration-dependent manner. KCCP, KCCP fraction PⅡ, and ascorbic acid had an IC50 value of 0.08557±0.1593mg/m L, 1.941±0.1728mg/m L and 0.02884±0.07601mg/m L, respectively. The scavenging activity on DPPH? free radical for both KCCP and its fraction PⅡ was less than that of ascorbic acid. The scavenging capacity of each fraction on ·OH radical was both significant and in a concentration-dependent manner. KCCP, fraction PⅡ, and ascorbic acid had an IC50 value of 0.2701±0.05723mg/m L, 6.74±0.07375mg/m L and 0.07107±0.07437mg/m L, respectively. The scavenging activity on ·OH radical for both KCCP and its fraction PⅡ was weaker than that of ascorbic acid. KCCP and KCCP fraction PⅡ induced a significant decrease in proliferation rate of Hela cells, Eca-109 cells and H22 cells in both dose- and time-dependent manner. The IC50 value of KCCP against Hela cells, Eca-109 cells and H22 cells were found to be 212.8±0.05834μg/m L, 497.6±0.05472μg/m L and 884.2±0.06069μg/m L, respectively, while that of KCCP fraction PⅡ was 1215±0.4279μg/m L, 2157±0.1444μg/m L and 2211±0.1554μg/m L, respectively. Therefore, both KCCP and fraction PⅡshowed remarkable antiproliferative effect. Moreover, both of them also showed stronger inhibitory proliferation effect on Hela cells.The extraction conditions of UHP method was optimized by response surface methodology(RSM), and the regression model was established, the equation analog effect was good. The optimal conditions for KCTF by UHP method was as following: with 70% ethanol as extraction solvent, extraction temperature 25 ℃, ratio of solid to liquid l:16(g/m L), pressure 340 MPa, dwell time 3 min, material particles 80 mesh, and under this condition, the yeild of KCTF was up to 12.35%. In addition, compared with traditional reflux extraction, extraction rate increased by 21.6%, purity improved 24.3%. The optimum isolation and purification technological conditions by AB-8 macroporous resin were as follows: material concentration 1.04mg/m L, sample flow velocity 3BV/h, and the highest purification was obtained eluted at alcohol concentration 70% with sample quantity 5 BV and velocity 2BV/h, and the purity of flavonoids was up to 52.43% while that of 28.12% without treatment by AB-8 macroporous resin. The purity was increased 86.5%. Moreover, it was found that the antioxidant activity of KCTF extracted by ultra high pressure extraction was stronger than that of reflux extraction and ultrasonic extraction. These results sμggested that KCTF showed considerable antioxidant activity in vitro.The key impact factors of US method was optimized by response surface RSM, experimental validation was consistent with mathematical description of the model which confirmed that the model could be used for optimization of US of different groups of KCPC. The optimal conditions for KCPC by US method was as following: ethanol concentration 60%, ultrasonic power 240 W, material-liquid ratio 1:20, extraction 33 min, temperature 44℃, and under this condition, the yeild of KCTF was up to 13.39%. In addition, compared with traditional reflux extraction, extraction rate increased by 29.8%. The optimum conditions for isolating and purifying KCPC was obtained throμgh the static and dynamic experiments on the AB-8 macroporous adsorption resin and as follows: material concentration 1.06 mg/m L, p H 6, sample flow velocity 2BV/h, and the highest purification was obtained eluted at alcohol concentration 70% with sample quantity 4 BV and velocity 2BV/h, and the purity of flavonoids was up to 63.76±0.34%(w/w) while that of 22.68±1.02%(w/w)without isolation and purification. The purity was increased 181%.The anti-aging of PKCF were evaluated by the indexes of the survival test in vivo of drosophila melanogaster, such as median lethal time(LT50), average life-span, and average maximum life span biochemical. The results showed that the average life-span of male and female drosophila groups at the dosage of 0.0067% were significantly longer than that of control group(P<0.01), the average rate of life extension was 15.19% and 16.77%, respectively. Thus, PKCF could prolong Drosophila Melanogaster’s life-pan. The mechanisms underlying delayed aging by PKCF in the flies was preliminarily studied throμgh measuring the content of SOD and the MDA levels in female and male drosophila group, and the results demonstrated that the activity of SOD in drosophila group was significantly higher than the other four groups while the content of MDA was remarkably lower at the concentration of PKCF 0.0201%. We can reason that PKCF enhances the antioxidant enzymes activity in vivo, free radicals are cleaned in a time-dependent manner leading to life-span extension in fruit flies.The IC50 of KCCP, KCPC and KCTF for scavenging hydroxyl radical were 0.08557±0.1593 mg/m L, 0.01851±0.2434 mg/m L and 1.369±0.05184 mg/m L while the IC50 for scavenging DPPH? free radical were 0.02701±0.05723mg/m L, 0.03560±0.06335mg/m L and 1.126±0.3418mg/m L, respectively. Hence, the scavenging activity of KCPC on DPPH? free radical was the strongest, and the scavenging activity of KCCP on hydroxyl radicals was the most powerful.KCCP, KCPC and KCTF could significantly enhance the SOD activity in test animals and further decrease the MDA level in mice serum, liver and kidney tissue, and all of them showed remarkable antioxidant activity in vivo. However, different antioxidant capacity could be found, and the ranking of antioxidant activity in vivo was KCPC >KCTF >KCCP.The inhibitory effects on Eshrichia.coil, Staphylococcus aureus and Penicillium was as the followig: KCTF > KCPC > KCCP from strong to weak. The inhibition against Bacillus.subtilis and Aspergillus of KCPC and KCTF with the same MIC value were stronger than that of KCCP. The MIC value of KCCP against bacteria and yeast were bigger than that of KCPC and KCTF, which meaned the inhibitory effect of KCCP was weaker than that of KCPC and KCTF. These results indicated that KCTF possed the strongest antimicrobial potential.The IC50 values against Hela cells for KCCP, KCTF and KCPC were 82.23± 0.0376μg/m L, 133.6±0.1885μg/m L and 113.3±0. 0862μg/m L, respectively, while the respaective values against Eca-109 cells were 220.8±0.06542μg/m L, 192.0±0.07719μg/m L and 260.4±0.06887μg/m L. Moreover, the values for the three compounds against H22 cells were 169.8±0.05409μg/m L, 84.50±0.1245μg/m L and 70.96±0.05409μg/m L, respectively. The IC50 values regarding the growth of the cancer cells presented above demonstrated that the antiproliferative effect on Hela cells of KCCP was stronger than that on Eca-109 cells and H22 cells while antiproliferative effect on H22 cells of KCPC and KCTF were stronger than that on Eca-109 cells and Hela cells.The ranking of antiproliferative effect on Hela cells was KCCP > KCPC >KCTF, and on H22 cells was KCPC >KCTF>KCCP. It proved that KCCP and KCTF could induce the change of apoptosis of He La cell.The results of hepatoprotective effects of KCCP, KCTF and KCPC against CCl4 –induced liver injury in mice revealed that the levels of ALT and AST for model group... |
PDF Full Text Request