| The emblica, which is the fruit of the plant Phyllanthus emblica L., in the genus Phyllanthus of Euphorbiaceae, is the traditional ethnic medicine of China. Not only is the emblica designated one of the three health protection plants by WHO, but also is the appointed plant both of medicine and food by Chinese ministry of health in 2002. The major active component in emblica fruits is emblica polysaccharides (EPS), which has been discovered to have biological activities of anti-oxidation and anti-tumor. For now, the studies of EPS are focused mostly on pharmacology. The functional groups and structure of EPS, however, remain poorly understood. In this paper, the extraction, isolation and purification, structure and anti-oxidation activity of EPS were studied systematically. The results were summarized as follows:The crude EPS was firstly extracted containing a sugar content of 31.13%. After freezing and thawing fractionation, depigmentation, deproteinization, dialysis and gel column chromatography, the crude EPS was purified into refined EPS exhibiting white filamentous structure, with a yield efficiency of 10.33%. The chemical composition of EPS consisted of 61.67% of neutral sugar which contained Gal, Rha, Ara, Glc, Xyl as well as few Man, 26.04% of GalA-rich uronic acid and 1.98% of protein.Ultrasonic intensification and microwave-assisted methods for ESP extraction were compared. The optimal parameters of ultrasonic intensification extraction were 80℃for extraction temperature, 70 min for ultrasonic treatment, 475 W for ultrasonic power and 1:20 for solid– liquid ratio. The optimal parameters of microwave pretreatment– hot water extraction were 480 W for microwave pretreatment power, 60 s for microwave treatment and 90℃for 4 h for extraction temperature.The stepwise purification procedure and chemical composition of water-extracted refined ESP were studied. Two fractions of EPS1 and EPS2 were obtained after fractionating through DEAE-Sepharose CL-6B column. The neutral polysaccharide EPS1 could be divided into two fractions of EPS1-1 and EPS1-2 after fractionating through HPGFC, while EPS2 was an acid polysaccharide having narrow molecular weight distribution. The rates of EPS1-1, EPS1-2 and EPS2 in elution amount were 3.39%, 19.60% and 77.01%, respectively, and the total yield efficiency was 92.32%. The neutral polysaccharide EPS1-1, 98.53% in purity and 95.09% in total sugar content, consisted mostly of Gal and Glc, with a relative molecular weight of 123 KDa. The other neutral polysaccharide EPS1-2, 98.91% in purity and 96.66% in total sugar content, consisted mostly of Gal, Glc and Xyl, with a relative molecular weight of 15 KDa. The IR spectrum showed that both of EPS1-1 and EPS1-2 wereβ-pyran aldoses.The acid polysaccharide EPS2 containing 79.01% galacturonic acid, 98.96% in purity and 97.53% in total sugar content, consisted mostly of GalA, Gal and Rha as well as few Ara, with a relative molecular weight of 140 KDa. Combination of monosaccharide composition analysis and IR spectrum revealed that EPS2 was a pectin polysaccharide polymerized majorly byα-glycosidic bonds as well as a certain amount ofβ- bonds.By using technology of NMR, GC-MS, FT-IR, GC and HPLC and cooperating with classical chemical methods of partial acid hydrolysis and methylation analysis, etc. the chemical structure of EPS1-2 and EPS2 were studied.The major configuration of glycoside residues of EPS1-2 were 1, 4-, 1, 6-β-D-Galp, T-, 1, 4-, 1, 3, 6-β-D-Glcp, and 1, 3, 6- and 1, 2-β-D-Manp. The galacto-glucan of main chain, of which the non-reducing terminal residue was (1→)-β-D-Glcp, was formed through the connection between Galp and Glcp. The branch, which was formed through the connection between 1,6-β-D-Glcp, at the position of O-3, and neutral sugar, such as 1, 3, 6- and 1, 2-β-D-Manp, appeared in every five glycoside residues. The residue of Manp located at the side chain, with 1, 3, 6-Manp at the core region. EPS1-2 was majorly composed ofβ-configuration as well as fewα- structure, parts of glycosyl residues on which were replaced by acetyl. The possible repeat units of EPS1-2 were:The EPS2 was a typical pectic polysaccharide, with a galacturonoglycan main chain formed through linear connection of 1, 4-GalpA. The branched rhamnogalacturonan of main chain was formed through the alternative connection between 1, 4-GalpA at position of O-4 and 1, 2, 4-Rhap at O-2. Parts of carboxyls on the polysaccharide were methylated, while some were replaced by acetyl at the position of O-2 or O-3. The side chain of EPS2, linking to the main chain through Rhap residues at the position of O-4, consisted of arabinogalactan and galactan which were polymerized by T-, 1, 6- and 1, 3, 6-α-Galp and T-, 1, 5-α-Araf residues. T-α-GalpA, T-α-L-Araf and T-β-Galp were located at the non-reducing terminal. The possible molecular configuration of EPS2 was:In vitro activities of anti-oxidation of refined EPS, EPS1, EPS1-2 and EPS2 were determined according to their scavenging capacities on the free-radicals of hydroxyl, DPPH and superoxide anion, and their anti-lipid peroxidation and reduction efficacy, using BHT as positive control. The results showed that both of EPS1-2 and EPS2 exhibited strong anti-oxidation activity. The degrees of different fractions of EPS on the anti-oxidation activity were in order of EPS2, EPS1-2, EPS1 and refined EPS, indicating the bioactivity of homopolysaccharide was higher than that of heteropolysaccharide. In addition, the reduction activity of EPS2 was higher than that of BHT.Compared with neutral polysaccharide, EPS1-2 and EPS1, the higher anti-oxidation activity of EPS2 was likely related to the abundance of negative electricity-carrying uronic acid on this acid polysaccharide. The higher anti-oxidation activity of EPS1-2 over EPS1 suggested the molecular weight of polysaccharide influenced the corresponding bioactivity. Finally, the structural factors such as types of sugar units, configuration of glycosidic bond, substituent group and spatial structure of polysaccharide also play an important role in anti-oxidation activity.
|
PDF Full Text Request