莪术细胞悬浮培养，挥发油、多糖分离及其多糖生物学活性研究
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摘要
摘要: 本研究建立了高产细胞悬浮系，是以莪术黄色、疏松愈伤组织细小颗粒为实验材料，将其接种于不同种类培养基，在不同碳源、氮源、激素种类及浓度、pH值、摇床转速、接种量、继代周期、光暗培养周期等不同条件下，培养一定时期后离心收获细胞，利用水蒸汽法测挥发油含量，苯酚—硫酸法测多糖含量。在挥发油的合成调控中，进行了乙酸铵、乙酸钾、泛酸钙等前体物质及能量物质ATPNa2添加的调控研究。确立了有利于莪术细胞生长及多糖和挥发油合成的最佳培养条件。结果表明，在最佳培养条件下，莪术细胞生长量是未优化培养对照组的1.82倍；悬浮细胞中多糖和挥发油含量大大提高了，分别可达45.53%和2.62%。其中多糖含量是饮片多糖含量的1.37倍，挥发油含量是未优化培养对照组的1.71倍。这方面的研究未见报道。本研究为工业化生产提供有意义的理论依据。
在莪术多糖的生物学活性研究中，采用热水浸提法从莪术饮片粉末浸提得粗多糖，经醇析、浓缩、DEAE-52纤维素柱层析，经蒸馏水、NaCl溶液梯度洗脱纯化后，利用琼脂糖凝胶电泳进行纯度检测，得一水洗脱组分，用该组分进行动物活体内抗肿瘤、免疫及体内外抗氧化研究。实验结果表明：莪术饮片多糖含量为33.12%。莪术多糖具有体内抗H22腹水癌的实体瘤作用，剂量为500mg?kg-1?day-1的抑瘤率高达54.87%，并能提高小鼠的淋巴细胞转化率、脾指数及血液中SOD酶活力；体外具有抗羟自由基氧化的能力，多糖浓度为100mg/mL时，对羟自由基的清除率达36.56%。莪术多糖可能是通过提高小鼠的免疫力及抗氧化能力的途径来抑制H22肿瘤细胞的生长。
利用正交实验设计确立了从莪术饮片中同时提取多糖及挥发油的最佳工艺。结果表明：粉碎程度为200目、水浸提４h、1∶20的浸提比的条件下，可以同时获得莪术多糖和挥发油的最大提取量，分别为24.8%和2.21%。
The high-yield cell suspension line of Curcuma Zedoaria (Berg.) Rosc was established with yellow and loose small callus inoculated into different liquid medium. These conditions for cell growth and volatile oil and polysaccharides synthesis well were selected, such as carbon source, nitrogen source, hormone, pH, rotation speed of table, inoculation concentration, subculture period, light/dark circle and so on. The cells were collected after a certain culture period. The volatile oil content was determined by steam distillation method. The polysaccharide content was determined with PhenyIhydrate-Sulfuric acid. The precursors of Ammonium acetate and Potassium acetate and Calcium Pantothenate and energy substances of ATPNa2 were added into the volatile oil synthesis-controlling condition.The results showed that the cell growth enhanced 1.82 times contrast to the CK group. The polysaccharides content was 45.53% which was 1.37 times of it in herbal slice .The volatile oil content was 2.62% and it's 1.71 times of CK group. These studies were not reported and provided theoretical base for industrializing.
The crude polysaccharides was extracted from herbal slice of Curcuma zedoaria Rosc with hot water ,and precipitated by ethanol after concentration. The constituents were isolated by DEAD-52 column chromatography. The water effluent was isolated. Its purification was detected by Sepharose electrophoresis. The bioactivities of immunology, anti-oxidative function and antitumor activity of the polysaccharides in vivo or vitro were studied. The results showed that the content of total polysaccharides was 33.12%. A higher antitumor effect of the polysaccharides by injection was observed, and the effect was dose-dependent. The inhibitive rate was 54.78% when the dose at 500mg.·kg-1·day-1. The polysaccharides could raise the spleen cell proliferation and transformation rate of lymphocyte and the activity of SOD in blood of mice. The polysaccharides could scavenge ?OH . The ?OH scavenging ratio was 36.56% at the density of 100mg/mL. The polysaccharides could raise the immune regulation and the activities of SOD, having the anti-oxidation activity had a higher antitumor effect on H22 tumor .
The orthogonal design method was used to establish the optimum condition for extracting polysaccharide and volatile oil at the same time. The result showed that the herbal slices were pulverized in 200 orders, and was extracted 4 hours and with solution 1∶20 could obtain the highest extraction amount, which was 24.8%and 2.21%.
在莪术多糖的生物学活性研究中，采用热水浸提法从莪术饮片粉末浸提得粗多糖，经醇析、浓缩、DEAE-52纤维素柱层析，经蒸馏水、NaCl溶液梯度洗脱纯化后，利用琼脂糖凝胶电泳进行纯度检测，得一水洗脱组分，用该组分进行动物活体内抗肿瘤、免疫及体内外抗氧化研究。实验结果表明：莪术饮片多糖含量为33.12%。莪术多糖具有体内抗H22腹水癌的实体瘤作用，剂量为500mg?kg-1?day-1的抑瘤率高达54.87%，并能提高小鼠的淋巴细胞转化率、脾指数及血液中SOD酶活力；体外具有抗羟自由基氧化的能力，多糖浓度为100mg/mL时，对羟自由基的清除率达36.56%。莪术多糖可能是通过提高小鼠的免疫力及抗氧化能力的途径来抑制H22肿瘤细胞的生长。
利用正交实验设计确立了从莪术饮片中同时提取多糖及挥发油的最佳工艺。结果表明：粉碎程度为200目、水浸提４h、1∶20的浸提比的条件下，可以同时获得莪术多糖和挥发油的最大提取量，分别为24.8%和2.21%。
The high-yield cell suspension line of Curcuma Zedoaria (Berg.) Rosc was established with yellow and loose small callus inoculated into different liquid medium. These conditions for cell growth and volatile oil and polysaccharides synthesis well were selected, such as carbon source, nitrogen source, hormone, pH, rotation speed of table, inoculation concentration, subculture period, light/dark circle and so on. The cells were collected after a certain culture period. The volatile oil content was determined by steam distillation method. The polysaccharide content was determined with PhenyIhydrate-Sulfuric acid. The precursors of Ammonium acetate and Potassium acetate and Calcium Pantothenate and energy substances of ATPNa2 were added into the volatile oil synthesis-controlling condition.The results showed that the cell growth enhanced 1.82 times contrast to the CK group. The polysaccharides content was 45.53% which was 1.37 times of it in herbal slice .The volatile oil content was 2.62% and it's 1.71 times of CK group. These studies were not reported and provided theoretical base for industrializing.
The crude polysaccharides was extracted from herbal slice of Curcuma zedoaria Rosc with hot water ,and precipitated by ethanol after concentration. The constituents were isolated by DEAD-52 column chromatography. The water effluent was isolated. Its purification was detected by Sepharose electrophoresis. The bioactivities of immunology, anti-oxidative function and antitumor activity of the polysaccharides in vivo or vitro were studied. The results showed that the content of total polysaccharides was 33.12%. A higher antitumor effect of the polysaccharides by injection was observed, and the effect was dose-dependent. The inhibitive rate was 54.78% when the dose at 500mg.·kg-1·day-1. The polysaccharides could raise the spleen cell proliferation and transformation rate of lymphocyte and the activity of SOD in blood of mice. The polysaccharides could scavenge ?OH . The ?OH scavenging ratio was 36.56% at the density of 100mg/mL. The polysaccharides could raise the immune regulation and the activities of SOD, having the anti-oxidation activity had a higher antitumor effect on H22 tumor .
The orthogonal design method was used to establish the optimum condition for extracting polysaccharide and volatile oil at the same time. The result showed that the herbal slices were pulverized in 200 orders, and was extracted 4 hours and with solution 1∶20 could obtain the highest extraction amount, which was 24.8%and 2.21%.
引文
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