楮实和淫羊藿多糖的分离纯化、理化性质及活性研究
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摘要
中药楮实为桑科植物构树的成熟果实,资源丰富,具有悠久的药用历史,我国历版药典均有收载,具有清肝明目、补肺益肾功效。本草记载并具有滋补强壮、延年益寿之功效;淫羊藿具有补肾阳、强筋骨、祛风湿、抗衰老、提高免疫功能、抑制肿瘤等多种功效,为最具开发潜力的中药之一,一直是国内外学者研究的重点。但关于褚实多糖和淫羊藿多糖的现代研究较为匮乏。本课题对两种植物的多糖成分进行分离纯化,并对其理化性质及生物活性进行研究,主要内容如下:
(一)响应面分析法优化楮实多糖提取工艺
在单因素试验基础上选取试验因素与水平,根据中心组合(Box-Benhnken)试验设计原理采用四因素五水平的响应面分析法,依据回归分析确定各工艺条件的影响因子,以楮实多糖提取率为响应值作响应面和等高线,并分析各个因素的显著性和交互作用。楮实多糖水浸提的最佳工艺条件为:料液比1:5.3,提取时间3h,提取次数4次,提取温度96.5℃。在最佳工艺条件下,楮实多糖的实际提取率为6.03%。
(二)楮实多糖和淫羊藿多糖的分离纯化及理化性质
粗多糖经DEAE-纤维素柱和Sephacryl S-300柱分离、纯化出CS-1～CS-5和YYH-1～YYH-5共10个组分。经鉴定,CS-1、CS-2和YYH-1为分子量分布均一组分。红外分析,它们具有多糖特征吸收峰。凝胶渗透色谱法测定相对分子质量,CS-1、CS-2和YYH-1相对分子质量分别为306 310、333 732和6 898。并运用气相色谱分析得出CS-1、CS-2和YYH-1的单糖组成及摩尔比。
(三)楮实多糖和淫羊藿多糖的生物活性
楮实多糖和淫羊藿多糖粗品及纯品对羟基自由基体系和超氧阴离子自由基体系均有不同程度的清除作用,呈现一定的量效关系,且多糖粗品对两种自由基的清除效果均大于同浓度时的纯品;楮实粗多糖的10,20,50,80mg/kg·d剂量组均能显示出具有提高小鼠在运动前后体内肌糖原和肝糖原储备量、乳酸脱氢酶活力及降低尿素含量的功效,50mg/kg·d剂量组即达到最佳效果,继续增大给药量未显示出更强作用效果;楮实多糖和淫羊藿多糖粗品及纯品组分能刺激PC12细胞产生神经纤维样突起,显示出NGF样神经营养活性。
Broussonetia is the nature fruit of Broussonetia papyrifera (L.) Vent. that oringinal resources are very abundant in China. For more than one thousand years it has been used as a traditional medicine which was collected in every edition of the Chinese Pharmacopoeia on account of its great effects of cleaning hepatic-heat, treating ophthalmic disorders and invgorating lung and kidney. Moreover Broussonetia also has the effects of strengthening with tonics and lengthening life in Chinese material medica. Epimedium has multiple positive effects, including invigorate the kidney, made bones and muscles powerful, cure beriberoid disease, effectiveness of anti-aging, boost immunological function, inhibit tumor. It was one of the developed potential traditional Chinese medicine which was widely studied by domestic and foreign scholars. Till today, the main active components of polysaccharides from plants keep out of our knowledge. In this paper, polysaccharides were extracted and isolated from above two plants, their physicochemical characteristics and biological activities were studied. The details were showed below:
(1) Optimization of the technology for extracting polysaccharide by response surface methodology from Broussonetia
On the basis of single factor experiments, the optimum conditions for the extraction of polysaccharide were obtained through Box-Benhnken center-united experiment design and response surface methodology with 4 factors and 5 levels. Then the factors influencing the technological parameters were determined by meaxls of regression analysis. Response surface and contour were finally graphed with the extraction rate as the response value. The optimum conditions of Broussonetia polysaccharide extraction were as follows: the ratio of material to solvent of 1:5.3 (w/v), extraction time of 3h, 4 times extraction and extraction temperature of 96.5℃. Conclusion: The yield of polysacchande is 6.03 % under the optimum conditions.
(2) Purification and characteristics of polysaccharides from Broussonetia and Epimedium
CS-1～CS-5 and YYH-1～YYH-5 were obtained by DEAE-cellulose and Sephacryl S-300 column chromatography from the two plants. CS-1, CS-2 and YYH-1 were homogeneous composition and their IR spectrum show typical characteristic absorption peaks of polysacchande . The average molecular weight of CS-1, CS-2 and YYH-1 were measured by gel filtration for 306 310, 333 732 and 6 898. By GC analysis, obtained the monosaccharide composition and the molar ratio of CS-1, CS-2 and YYH-1.
(3) The biological activities of polysaccharides from Broussonetia and Epimedium
Crude and pure polysaccharides from Broussonetia and Epimedium had different scavenging effect in anion free radical system, hydroxyl radical system, and the antioxidetive activities become stronger with the increase of the polysaccharide concentration. But the scavenging capability of the crude polysaccharides were stronger than those of pure polysaccharide on the two free radical system.
10, 20, 50, 80 mg/kg·d dose of polysaccharides from Broussonetia were applied to mice to study their effects on antifatigue and the relationship between dose and response. The results showed the effect on enhancing the reserves of muscle and liver glycogen, increasing latic dehydrogenase (LDH) activity, decreasing the content of rise in blood urea nitrogin (BUN) before and after exercise. 50mg/kg·d had the best effect among of the dose.
The screening results showed that the crude polysaccharides and the pure fractions from Broussonetia and Epimedium can induce differentiation of PC12 cell and had obvious neurotrophic activity.
(一)响应面分析法优化楮实多糖提取工艺
在单因素试验基础上选取试验因素与水平,根据中心组合(Box-Benhnken)试验设计原理采用四因素五水平的响应面分析法,依据回归分析确定各工艺条件的影响因子,以楮实多糖提取率为响应值作响应面和等高线,并分析各个因素的显著性和交互作用。楮实多糖水浸提的最佳工艺条件为:料液比1:5.3,提取时间3h,提取次数4次,提取温度96.5℃。在最佳工艺条件下,楮实多糖的实际提取率为6.03%。
(二)楮实多糖和淫羊藿多糖的分离纯化及理化性质
粗多糖经DEAE-纤维素柱和Sephacryl S-300柱分离、纯化出CS-1～CS-5和YYH-1～YYH-5共10个组分。经鉴定,CS-1、CS-2和YYH-1为分子量分布均一组分。红外分析,它们具有多糖特征吸收峰。凝胶渗透色谱法测定相对分子质量,CS-1、CS-2和YYH-1相对分子质量分别为306 310、333 732和6 898。并运用气相色谱分析得出CS-1、CS-2和YYH-1的单糖组成及摩尔比。
(三)楮实多糖和淫羊藿多糖的生物活性
楮实多糖和淫羊藿多糖粗品及纯品对羟基自由基体系和超氧阴离子自由基体系均有不同程度的清除作用,呈现一定的量效关系,且多糖粗品对两种自由基的清除效果均大于同浓度时的纯品;楮实粗多糖的10,20,50,80mg/kg·d剂量组均能显示出具有提高小鼠在运动前后体内肌糖原和肝糖原储备量、乳酸脱氢酶活力及降低尿素含量的功效,50mg/kg·d剂量组即达到最佳效果,继续增大给药量未显示出更强作用效果;楮实多糖和淫羊藿多糖粗品及纯品组分能刺激PC12细胞产生神经纤维样突起,显示出NGF样神经营养活性。
Broussonetia is the nature fruit of Broussonetia papyrifera (L.) Vent. that oringinal resources are very abundant in China. For more than one thousand years it has been used as a traditional medicine which was collected in every edition of the Chinese Pharmacopoeia on account of its great effects of cleaning hepatic-heat, treating ophthalmic disorders and invgorating lung and kidney. Moreover Broussonetia also has the effects of strengthening with tonics and lengthening life in Chinese material medica. Epimedium has multiple positive effects, including invigorate the kidney, made bones and muscles powerful, cure beriberoid disease, effectiveness of anti-aging, boost immunological function, inhibit tumor. It was one of the developed potential traditional Chinese medicine which was widely studied by domestic and foreign scholars. Till today, the main active components of polysaccharides from plants keep out of our knowledge. In this paper, polysaccharides were extracted and isolated from above two plants, their physicochemical characteristics and biological activities were studied. The details were showed below:
(1) Optimization of the technology for extracting polysaccharide by response surface methodology from Broussonetia
On the basis of single factor experiments, the optimum conditions for the extraction of polysaccharide were obtained through Box-Benhnken center-united experiment design and response surface methodology with 4 factors and 5 levels. Then the factors influencing the technological parameters were determined by meaxls of regression analysis. Response surface and contour were finally graphed with the extraction rate as the response value. The optimum conditions of Broussonetia polysaccharide extraction were as follows: the ratio of material to solvent of 1:5.3 (w/v), extraction time of 3h, 4 times extraction and extraction temperature of 96.5℃. Conclusion: The yield of polysacchande is 6.03 % under the optimum conditions.
(2) Purification and characteristics of polysaccharides from Broussonetia and Epimedium
CS-1～CS-5 and YYH-1～YYH-5 were obtained by DEAE-cellulose and Sephacryl S-300 column chromatography from the two plants. CS-1, CS-2 and YYH-1 were homogeneous composition and their IR spectrum show typical characteristic absorption peaks of polysacchande . The average molecular weight of CS-1, CS-2 and YYH-1 were measured by gel filtration for 306 310, 333 732 and 6 898. By GC analysis, obtained the monosaccharide composition and the molar ratio of CS-1, CS-2 and YYH-1.
(3) The biological activities of polysaccharides from Broussonetia and Epimedium
Crude and pure polysaccharides from Broussonetia and Epimedium had different scavenging effect in anion free radical system, hydroxyl radical system, and the antioxidetive activities become stronger with the increase of the polysaccharide concentration. But the scavenging capability of the crude polysaccharides were stronger than those of pure polysaccharide on the two free radical system.
10, 20, 50, 80 mg/kg·d dose of polysaccharides from Broussonetia were applied to mice to study their effects on antifatigue and the relationship between dose and response. The results showed the effect on enhancing the reserves of muscle and liver glycogen, increasing latic dehydrogenase (LDH) activity, decreasing the content of rise in blood urea nitrogin (BUN) before and after exercise. 50mg/kg·d had the best effect among of the dose.
The screening results showed that the crude polysaccharides and the pure fractions from Broussonetia and Epimedium can induce differentiation of PC12 cell and had obvious neurotrophic activity.
引文
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