| Curcuma phaeocaulis Valeton, a perennial herbaceous plant of the family Zingiberaceae, is widely spreads in the southwest of China, termed as Ezhu in Chinese. Ezhu are recorded officially in the Chinese pharmacopoeia (2010) because of their medicinal properties. They are one of the most common folk herbal remedies known by the popular name Rhizoma Curcumae, which is prescribed in the BenCaoGangMu. It has been used to reduce pain and help digestion in Traditional Chinese Medicine (or TCM) for thousands of years.The objective of this study was to optimize the extraction process of polysaccharides from C. phaeocaulis. RSM was designed to systematically analyze the effects of extraction parameters, employing a Box-Behnken design (BBD) to assess polysaccharide yields. Then the purified polysaccharides were characterized by chemical analysis, infrared spectroscopy analysis (FT-IR), gas chromatography-mass spectrometry (GC-MS) and gel column chromatography. Finally, the antioxidant activity (scavenging ability of DPPH radical, hydroxyl radical, superoxide anion radical, and reducing power) of purified polysaccharides were investigated in vitro assays. The main results were as follows:1. Polysaccharides extracts from the rhizomes of C. phaeocaulis were conducted by hot-water extraction method. RSM was the point on determine the key variables of extraction process. The optimum operational conditions were determined as follows: extraction temperature was 93℃, extraction time was 5.2 h, and ratio of water to raw material was 33:1. Under optimal conditions, the experimental yield was 57.13 ± 0.08%, which close to the predicted value (57.47%).2. An ultrasonic-powered technique was employed to extract polysaccharides from the rhizomes of C. phaeocaulis and RSM was used to determine the main and interaction effects of the key variables that influence the extraction process. The optimal extraction conditions were determined to be a temperature of 78.8℃, duration of 35 min, ratio of water to raw material of 47:1 and ultrasonic power of 295 W. Under those conditions, the experimental yield of polysaccharides was 17.12±0.19%, close to the predicted value of 17.22%.3. The crude polysaccharides were obtained using the optimize conditions. And the small molecular impurity and protein were removed by aether, acetone and Savage assay, and the polysaccharide was obtained after concentrating and drying. The poilsaccharide was further purified by chromatography of DEAE-52, and the characterization of purified fraction (CPP-1 CPP-2) was detected. The chemical composition and functional groups of two polysaccharide fraction were different. The chemical composition indicated that CPP-2 were acidic polysaccharides with a high uronic acid (16.18%). Furthermore, the structural characterization of purified fraction was detected by FT-IR spectra, which showed that CPP-1 was β-dominating configuration in pyranose form; CPP-2 was α-pyranose form with a few of β-pyranose. The relative molecular weight of CPP-1 was 84,664Da, which was mainly composed of Glucose (77.64%), Galactose (12.38%), Mannose (1.6%), Arabinose (5.8%) and Ribose (2.58%); The relative molecular weight of CPP-2 was 22,464Da, which was mainly composed of Glucose (61.55%), Galactose (21.69%), Mannose (2.78%), Xylose (11.62%) and Ribose (2.36%).4. In addition, antioxidant capacities of purified fraction in vitro were evaluated by four indicators, and presented strong capacity to scavenge DPPH radical, hydroxyl radical, superoxide anion radical, and along with strong reducing power. Results indicated that all of the three fractions exhibited significant antioxidant ability in a dose-dependent manner. CPP-2 exhibited the highest DPPH radical scavenging ability, hydroxyl radical scavenging ability and superoxide anion radical scavenging ability than CPP-1. |
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