The Analysis Of Saccharides In Chinese Yam And The Separation, Purification And Structure Identification Of The Bioactive Compounds

As an important medicine edible tuber plant, Chinese yam（Dioscorea opposita） has a long history in our country. The previous project was to analysis the gelatinization properties of macromoleclar polysaccharide starch isolated from Chinese yam tubers, elaborate the change of starch crystalline structure through the system research of different media influence on the hot pasting process. Extract, separate and do structure identification of bioactive polysaccharides, and compare the mice hypoglycemic effect of the polysaccharides with different average molecular weight. Separation, purification of the skin, flesh of mature Chinese yam and immature Chinese yam tubers with solvent impregnated method and active tracking method. The active ingredient content change during growth process was compared and the bioactive-structure relationship was researched system. This research helps clarify Chinese yam as the theoretical basis for medicine and food material, laying the theoretical foundation and applying scientific methods for the development of additional merchandise drugs.In order to analyze the influence of different media on the gelatinization process of Chinese yam, the digital image analysis, integral optical density（IOD） method, was applied to dynamically analyze the influence of different salts and sugars on the gelatinization of Chinese yam starch. ―The model of response difference of crystallite change（MRDCC）‖ was combined to verify two peaks of C-type Chinese yam starch corresponding to the pasting process of A- and B- type structure. IOD method as the latest technique, is able to characterize the partially gelatinized starch granules, in the mean time, it is a real-time monitor without pre- and after treatment of various starch samples. Thus real situation of the gelatinization process can be obtained.This new method（IOD） and MRDCC were employed to dynamically detect the influence of different concentrations（NaCl） and various salts（NaCl, KCl, CaCl2, MgCl2, FeCl3, NaNO3, Na2CO3, Na2SO4, CuSO4） on the gelatinization process of Chinese yam starch. The result demonstrated: With the increase of NaCl concentration from 0 to 4 mol/L（M） the gelatinization degree（DG） of B-type allomorph increased at lower concentration to a maximum value and then decreased with increasing concentration, however all of the concentrations（1⁴ M） had inhibition effect on A-type polymorph. The inhibition effect of low NaCl concentration contributed to the dominated water structure-making effect of Na+, while in high concentration the electrostatic interaction between starch e-OH groups and Na+ ions was significant. The influence of various neutral anions was in accordance with the Hofmeister series while the situation of cations was far more complicated. Anions with higher charge density had water structure making effect to reduce water activity, and repeled starch e-OH groups to stabilize starch granules at the same time; however, the higher charge density of cations increased the water structure on the one hand, while attracted starch e-OH groups and destabilize starch granules with generated heat on the other hand.The influence of different concentration of sucrose and various sugars on the gelatinization process of Chinese yam starch was analyzed. All of the saccharides（ribose, fructose, glucose, sucrose, lactose, trehalose, maltose, raffinose and stachyose） proposed in our research showed protection effect on starch crystalline structure during gelatinization. The protection effect increased with the increase of sucrose concentration（0²0%）. Trisaccharide and tetrasaccharide were more effective in inhibiting the gelatinization process than disaccharide; and the protection effect of disaccharides on starch was bigger than that of monosaccharide during gelatinization. The gelatinization inhibition effect had good relationship with nDHN（dynamic hydration number）, and the increase of the equatorial OH(e_OH) group number of saccharides might increase the inhibition effect on starch gelatinization. However, in addition to the e_OH groups, the combination ability of sugar with water molecules might be also related to the size of the sugar molecules and their three-dimensional structure. We believed that owing to the helical structure, which formed though hydrogen bonds, tetrasaccharide tended to decrease the hydration ability of saccharide and destabilized the water structure, thus the inhibitory effect of stachyose was smaller than raffinose.To further investigate the health-benefits of Chinese yam, separation, purification, structure identification and bioactive analysis were carried out on the skin and flesh of mature yam together with the immature yam. With active tracking method, 18 type of compounds were identified including Phenanthrene（1, 2, 3, 4）, soyacerebroside（5）, sitosterols（6, 7, 13）, glycolipids（8, 14）, fatty acids（9, 10, 11, 12, 15） and sugars（16, 17, 18）. Phenanthrene 1（2,6-dimethyl-4,7-dihydroxy-phenanthrene） is a novel analogue, and C4（6,7-dimethyl-2,4-dihydroxy-phenanthrene） was isolated for the first time from D. opposite. The extracts/ pure compounds were tested for MTT antioxidant activity, LPO antioxidant activity, COX-1 and-2 enzyme inhibition activity, and antitumor activity. The results showed that the four phenanthrene compound isolated from the skin of this yam showed higher antiinflammatory activity than the counter nonsteroidal anti-inflammatory drugs（NSAIDs） aspirin, ibuprofen and naproxen. This is the first report of the antiinflammatory activity of phenanthrenes 1, 2 and 4. The structure-activity relationship of phenanthrenes, based on the COX enzyme inhibitory assay, suggested that free hydroxyl group in compounds 1, 3 and 4 contributed to the enhanced inhibitory activity of COX-1 and-2 enzymes. The immature yam has higher medicinal activity value for the majority of the active compounds complete the accumulation in the initial stage of growth.Using the method of gradient alcohol sink, combined with the tangential flow ultrafiltration membrane system, three fragment molecular weight of yam polysaccharides were isolated: HSY-Ⅰ（> 50 kDa）, HSY-Ⅱ（10 50 kDa）, HSY-Ⅲ（< 10 kDa） and HSY mixed polysaccharides（700 kDa 4 kDa）. The monosaccharide composition of HSY-â… was glucuronic acid and galactose with the ratio of 1.86 ∶ 5.19, mainly bonded with 1→4 glycosidic bond; HSY-â…¡was comprised of glucuronic acid, rhamnose, arabinose, glucose and galactose with the mole ratio of about 0.81 ∶ 2.35 ∶ 1.24 ∶ 66.79 ∶ 28.81. Some of the galactose was bonded with type-â…¢ glycosidec bond（like 1→3） and other monosaccharaides were bonded with type-Ⅰ（1→2, 1→4） or type-Ⅱ（1→6, 1→）. The composition of HSY-â…¢ was mannose, glucose and galactose with the ratio of 13.20 ∶ 12.79 ∶ 74.0. Part of mannose was bonded with type-â…¢ glycosidec bond and other monosaccharaides were bonded with type-â… or type-â…¡. Dexamethasone-induced insulin resistance sugar/ lipid metabolism diabetic mice model was established to evaluate the hypoglycemic effect of different concentration of HSY and different molecular weights polysaccharide HSY-â… , HSY-â…¡ and HSY-â…¢.Fasting blood glucose level, glucose tolerance, triglycerides, cholesterol and fasting insulin level were used as determination index. The results indicated that the HSY, HSY-â…  and HSY-â…¡ had auxiliary hypoglycemic effect. And the effectiveness and their structure-effect relationship were further discussed.