| Increasingly serious air pollution in China has gradually raised the morbidity of diseases related to immune and cardiovascular system. American ginseng, as a medicinal and edible plant, has a variety of physiological activity, such as immunostimulating and hypoglycemic activity. It has great significance for prevention of the diseases.In the paper, American ginseng of high quality was screened from different origins. Polysaccharides in the American ginseng were extracted, separated, purified and structurally identified by using a variety of modern techniques. The biological activities of them were evaluated by in vitro and animal experiments which could provide information and basic data for the development of polysaccharides related functional food. The main results are as follows:1. Analysis of heavy metals, nutritional and functional components of American ginseng:American ginseng, collected from 24 locations both in China and Canada, were determined of 5 heavy metals,5 nutritional and functional components. The content of heavy metals (Cu, Pb, As, Cd, and Hg) ranged from 9.79 to 14.23 mg/kg. The lowest heavy metal content was found in the sample from Fusong County, Baishan City, Jilin Province. The average content of heavy metals in Chinese ginseng samples was very close to that of the Canadian. However, the difference of heavy metal contents among different locations in China was relatively larger than that of Canada. The ranges of crude protein, crude fat, crude starch, total saponins, and crude polysaccharides content were 10.44-14.98%,0.45-0.78%,21.77-31.59%, 3.28-4.66%, and 8.78-10.16%, respectively. The highest protein content was found in the ginseng from Dongning County, Mudanjiang City, Heilongjiang Province, which also showed relatively higher starch content (29.65%). The highest total saponins and polysaccharides content were both found in the sample from Fusong, Baishan, Jilin. The average contents of nutritional and functional components in China were slightly differed from Canada, but the differences of them among different locations in China were relatively larger than that of Canada. Overall, American ginseng from Fusong, Baishan, Jilin, with lowest heavy metals and highest saponins and polysaccharides, could be used as a raw material for related product development.2. Ultrasonic assisted extraction of polysaccharides from American ginseng:Ultrasonic assisted hot water extraction was used to extract polysaccharides from American ginseng. Single factor experiments were performed to identify the effects of extraction times, ultrasonic power, extraction temperature, extraction time and ratio of water to raw material (W/M) on the yield of polysaccharides. Response surface method was used to optimize the extraction process. Results showed that the five factors could all affect the yield of AGP, but ultrasonic power, extraction time and W/M were the three main impactors. A three-factors-three-levels Box-Behnken design was used to optimize the extraction process and a regression model was gained. The analysis of variance showed that the model was significant and the experimental method is reliable. The predicted optimal conditions were adjusted according to the actual operating conditions and the final optimum conditions were as follows:ultrasonic power 400W, extraction time 71min, W/M 33mL/g. Under this condition, the actual yield of AGP was 8.09%, which was close to the predicted value (8.13%). Compared with the hot water extraction (extraction yield of 7.85%), ultrasonic assisted extraction was short-time, low-temperature, and high-yield.3. Separation, purification, and composition analysis of polysaccharides from American ginseng:American ginseng polysaccharides (AGP), extracted by ultrasonic assisted extraction, was preliminarily separated by ultrafiltration membrane and further purified by ion exchange and gel chromatography. Gel permeation chromatography, gas chromatography, and infrared spectroscopy were used to characterize the polysaccharides fractions. Results showed that four polysaccharide fractions in different molecular weight range were obtained by ultrafiltration and named as AGP1 (>100ku), AGP2 (30-100ku), AGP3 (10-30ku), and AGP4 (<10ku), respectively. The yield of them was 29.79%,2.10%,7.78%, and 24.23%, respectively. AGP1 was further separated by DEAE Sepharose Fast Flow anion exchange chromatography and another four fractions (AGP1-1, AGP1-2, AGP1-3, and AGP1-4) were obtained with the yield of 3.29%,10.37%,32.83%, and 3.85%, respectively. AGP 1-2 and AGP 1-3 were purified by Sephacryl S-400 HR and only one elution peak was found for each of them, indicating that both of them were relatively homogeneous polysaccharide. Composition analysis showed that AGP 1-2 and AGP 1-3 are free from protein and nucleic acid. The purity and molecular weight of AGP 1-2 was 98.45% and 117.25ku. And the monosaccharide composition was arabinose, rhamnose, mannose, galactose, glucose, and glucuronic acid, in the ratio of 9.54:16.75:19.66:21.45:14.97: 17.63. AGP 1-3 had a purity of 96.27% and molecular weight of 956.48ku, with the monosaccharide composition of arabinose, rhamnose, xylose, mannose, galactose, glucose, galacturonic acid, and glucuronic acid, in the ratio of 6.94:8.87:14.21:20.15:16.34:11.15:9.66:12.68.4. Immunomodulatory activity of polysaccharides from American ginseng:RAW264.7 was used as the cell model and enzyme linked immunosorbent assay was used to determine the cytokines content. Effect of different polysaccharides on NO and cytokines production of macrophages was investigated in vitro. The immunomodulatory activity of AGP was also evaluated by cyclophosphamide-induced immunosuppressed mice. The results showed that AGP, AGP1, AGP2, AGP1-2, and AGP 1-3, with higher molecular weight, could increase the production of NO, TNF-α, IL-6, and IL-10. However, AGP3 and AGP4, with smaller molecular weight, showed no immune-enhancing activity. AGP could significantly increase the spleen index, thymus index, adjusted carbon clearance index, serum hemolysin content, ear swelling level, and NK cell killing activity of the immunosuppressed mice. It indicated that AGP could promote the growth of immune organs and enhance the monocyte-macrophage function, humoral immunity function, cellular immunity function, and NK cell activity, showing good immune-enhancing activity.5. Hypoglycemic activity of polysaccharides from American ginseng: Inhibitory activity of AGP and AGP1 on a-glucosidase was studied by in vitro assay. KK-Ay mouse was used as the diabetes model animal. Glucometer and microplate reader were used to determine the biochemical index of blood. Effect of AGP on the blood glucose and biochemical index of diabetes model mouce was investigated in vivo. The results showed that AGP and AGP1 could inhibit the activity of α-glucosidase. The IC5o of them was 1.26 and 0.82 mg/mL, which was approximately 3 and 2 times of that of the acarbose (positive control), respectively. AGP could significantly reduce the content of blood glucose, serum C-peptide, and glucagon of model mice, as well as improving the level of oral glucose tolerance. It suggested that AGP could regulate the function of pancreas islet A and B cells, showing good hypoglycemic activity. |
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