Studies On The Purification, Structure And Bioactivities Of Polysaccharides From Cyclocarya Paliurus (Batal.) Iljinskaja

Cyclocarya paliurus (Batal.) Iljinskaja as a Chinese native plant belongs to the genus Cyclocarya Iljinskaja (Juglangdaceae), which is the sole species in its genus, and is mainly found at 420-2500 m elevation in the mountainous regions of Anhui, Fujian, Hubei, Hunan, Jiangsu, Jiangxi, Sichuan, Guizhou, and Zhejiang provinces. Cyclocarya paliurus (Batal.) Iljinskaja commonly known as "sweet tea tree" is a medicinal plant, the leaves of Cyclocarya paliurus (Batal.) Iljinskaja have traditionally been used in China both as drug formulations in traditional Chinese medicine (TCM) and for treating various diseases, such as diabetes, hypertensive, hyperliposis and so on. Significant attention has recently been drawn to the use of Cyclocarya paliurus (Batal.) Iljinskaja for developing functional food, as Cyclocarya paliurus (Batal.) Iljinskaja produce a great variety of nutrients that are essential for human health. Cyclocarya paliurus (Batal.) Iljinskaja health tea, the aqueous extract of Cyclocarya paliurus (Batal.) Iljinskaja leaves has been become the first FDA approved health tea of China in 1999. Therefore, the aim of the present research was to establish a more reasonable content determination method and purification procedure, determination on molecule weight, determination on composition of the polysaccharides present in this dried leaves of Cyclocarya paliurus (Batal.) Iljinskaja, identification on primary structure and bioactivities tests based on the polysaccharides from the dried leaves of Cyclocarya paliurus (Batal.) Iljinskaja by using a series of modern food science, organic chemistry, natural product chemistry, analytical chemistry and modern instrument analysis techniques. The present research, which will provide scientific functions for their further developments as active ingredients in functional food, or new medicine. The main studies and results in the paper are mentioned as follows:1. An anthrone-H₂SO₄ colorimetry method was developed for the determination of polysaccharides in Cyclocarya paliurus (Batal.) Iljinskaja samples. Purified polysaccharide in Cyclocarya paliurus (Batal.) Iljinskaja was prepared by extracting with water and removing the interference components such as lipid, monosaccharide, disaccharide, oligosaccharide, amino acids and protein. The optimum absorption wavelength was confirmed by UV-Vis scanning after the reaction of purified polysaccharide and anthrone-H₂SO₄ A corrected factor was confirmed by comparing purified polysaccharide and glucose in order to minimize the error of determination. The results showed that the method is simple and available, and the color of the treated samples could be stable within 8 h. The average value of the recovery for the determination of the polysaccharides was 100.8% with 1.90% of RSD(n=3). The contents of polysaccharides in the Cyclocarya paliurus(Batal.) Iljinskaja samples of Xiu-Shui county determined are 4.387%.2. Two kinds of extraction technologies on plant active components were applied in extracting of Cyclocarya paliurus(Batal.) Iljinskaja polysaccharides. Polysaccharides in Cyclocarya paliurus(Batal.) Iljinskaja samples of Xiu-Shui County were obtained from dried Cyclocarya paliurus(Batal.) Iljinskaja powder by hot water extraction. The effects of extraction times, extraction time and the powder-to-water ratio were studied.A L₉(3³) orthogonal experiment was carried out by taking extraction times, extraction time and the powder-to-water ratio as the parameters to determine at extraction temperature of 90℃. The optimal comdition of the extraction three times, extraction time 150 mintues and the powder-to-water ratio 1:8. Under this condition, the yield of Cyclocarya paliurus(Batal.) Iljinskaja polysaccharides was 6.54%. On the basis of single-factor experiments, the optimum conditions for the extraction of polysaccharides from Cyclocarya paliurus(Batal.) Iljinskaja were obtained through Box-Benhnken center-united experiment design and response surface methodology. The optimum extraction condition were as follows: powder-to-water ratio: 1:8, extraction temperature: 58℃, time: 59 min. The yield of polysaccharides in Cyclocarya paliurus(Batal.) Iljinskaja was 4.96%.3. The color of polysaccharide from the lesves of Cyclocarya paliurus(Batal.) Iljinskaja, black and brown is major obstacles for isolation and purification of polysaccharides and research of chemical structure mechanism and structure-activity relationship. In this study, the effect of decolorization on crude polysaccharides solution of Cyclocarya paliurus(Batal.) Iljinsk was studied by activated carbons adsorption and the feasibility of decolorization of the crude polysaccharide solutions of Cyclocarya paliurus(Batal.) Iljinsk with activated carbons adsorption was also explored. Taking decolorization efficiency and polysaccharide yield ratio as indexes and adopting orthogonal experimental design to select the best condition for the discoloration of crude polysaccharide solutions. Results showed tha activated carbons adsorption is effective to decolor the crude polysaccharide solutions of Cyclocarya paliurus(Batal.) Iljinsk. The best technological process of decolorization is as follows: put 1.0% active carbons to crude polysaccharide solutions at 40℃, pH 3.5 with 20 minutes process time. Conclusion: activated carbons adsorption is an effective and simple process on the decolorization of crude polysaccharide solutions of Cyclocarya paliurus(Batal.) Iljinsk.4. In this study, a polysaccharide was isolated and purified from Cyclocarya paliurus(Batal.) Iljinskaja which is a well-known native medicinal plant in China for the first time by using macromolecular purifying system by column gel permeation. The process of isolating and purifying the polysaccharide was achieved through hot water extraction, filtration, ethanol precipitation, deproteination, decolourization and a combination of anion-exchange chromatography on DEAE-Sephadex A-25 and gel permeation chromatography on Sephacryl S-400. The homogeneity of the polysaccharide CPC-1 was proved by gel filtration chromatography on Sepharose CL-6B, with a molecular weight of about 1105859 Da determined by high-performance Liquid chromatography(HPLC) against the calibration curve of Dextran standards(T-2000, T-500, T-70, T-40 and T-10).; 900295 Da determined by high-performance gel permeation chromatography(HPGPC) against the calibration curve of Dextran standards(T-2000, T-500, T-70, T-40 and T-10). The data obtained indicated that the CPC-1 contains 8.44% of protein and 17 general amino acids and it is rich in glutamic acid, asparagic acid, leucine, glycine, arginine, tyrosine and alanine. The analysis of monosaccharide composition in the polysaccharide by gas chromatography and GC-MS revealed that it was composed of L-rhamnose, L-arabinose, D-xylose, D-mannose, D-glucose and D-galactose in the molar ratios of 10.64: 31.23: 3.23: 8.72: 30.16: 16.02. Infrared spectrum of CPC-1 indicates that they have typical characteristic absorption peaks of polysaccharides. The polysaccharide was concluded to be first time isolated from Cyclocarya paliurus(Batal.) Iljinskaja.5. The bioactivities properties of polysaccharides from Cyclocarya paliurus (Batal.) Iljinskaja, including anti-oxidation in vitro experiments and inhibition a-amylase experiments and as well as the antifungal activities were investigated. The effect of remove OH·and O₂·is relatively low, while it can remove bitterness of diphenyl-radical(DPPH), and the linoleic acid oxidation was determined by thiobarbituric acid(TBA). The data of inhibits a-amylas showed that the water-soluble polysaccharide CP-1 isolated from the leaves of Cyclocarya paliurus(Batal.) Iljinskaja inhibits a-amylase(a disaccharide-degrading enzyme), the inhibitory activity were about 13% at the concentration of 1.0 mg/mL, which leading to a decrease in the absorption of glucose into the blood and a subsequent lowering of the blood glucose level. The bacteriostasis functions of polysaccharides from Cyclocarya paliurus(Batal.) Iljinskaja in the partial bacteria were explored by inhibitory zone with filter paper disk method. The results showed that polysaccharides from Cyclocarya paliurus(Batal.) Iljinskaja have significant inhibitant activity against the Saccharomy cescerevisiae and Candida sp., but have little inhibitant activity against Escherichia coli, Staphylococcus aureus, Bacillus subtili, have no inhibitant activity against Aerobacter aerogenus, Proteus vulgaris and Aspergillus niger.