栀子组织和悬浮细胞培养及其多糖的分离纯化和生物学活性的研究
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摘要
多糖是生物化学领域中的一个重要课题，并正在成为新的研究热点。到目前为止，已有很多关于植物多糖，特别是中草药多糖的报道。人们发现多糖具有提高免疫能力，抗病毒，抗菌，抗氧化等多种生物学活性。本论文研究了栀子多糖的分离纯化及部分生物学活性，并探讨了通过生物技术生产栀子多糖的可行性。
栀子是传统中药，其多糖在抑制肿瘤生长和抗氧化过程中起重要作用。100ug／ml的栀子多糖在体外可以抑制S-180肉瘤细胞(58％)、Hca-f腹水肝癌细胞(51％)、K562人红白血病细胞(63％)。栀子多糖在活体中对Hca-f肝癌实体瘤的抑制率为49％，口服给药的效果好于注射给药。
栀子多糖对超氧阴离子自由基有很好的清除效果，清除率高达83.48％，而对羟自由基的清除率却只有22.31％。一些金属离子的存在可以改变栀子多糖对自由基的清除能力。Zn~(2+)、Fe~(2+)和Ca~(2+)能不同程度地削弱栀子多糖对超氧阴离子的清除能力，只有Cu~(2+)可以稍稍提高栀子多糖对超氧阴离子的清除能力。而这四种金属离子都能促进栀子多糖对羟自由基的清除作用。在实验动物体内，栀子多糖能增加SOD酶的活力，降低MDA的含量，同时这种改变与肿瘤的生长有密切的关联。SOD酶活性强的小鼠，MDA含量低，肿瘤轻；SOD酶活性弱的小鼠，MDA含量高，肿瘤重。因此栀子多糖可能是通过提高小鼠体内SOD等抗氧化酶的活性，来清除多余的自由基，从而达到抑制肿瘤生长的目的。
在利用悬浮细胞生产栀子多糖的研究中，通过选择栀子多糖的高产细胞株系以及改变培养基和培养条件来增加细胞的生长和多糖的百分含量。结果表明，细胞聚集体大小并不影响栀子多糖的合成，细胞团内部存在着物质传递结构；5～10天的继代周期有利于细胞保持快速分裂和多糖合成的能力；80g／L的接种量则大大缩短了细胞生物量达到最大值的时间；在悬浮培养中，蔗糖可迅速分解成葡萄糖和果糖，栀子细胞优先利用葡萄糖，培养后期多糖合成量的下降主要与培养基碳源供应不足有关；氮源浓度过高不利于栀子多糖的合成；在激素的使用上，生长素对栀子细胞生长和多糖合成有促进作用，2，4-D效果优于NAA，NAA又优于IAA；栀子细胞对剪切力不是很敏感，在80～130r／min是较宽范围内都能正常
大连理工大学博士学位论文
生长；红色光和蓝色光是柜子悬浮细胞生长和多糖合成的两个重要的单色光；而
温度和PH值等物理因素的影响与其他植物悬浮培养细胞无大大差异。
尽管桅子悬浮细胞培养有许多富于个性的特点，但是有一个总的规律，即利
于细胞生长的条件很可能不利于桅子多糖的合成和积累。板子多糖一般在悬浮细
胞培养的初期形成，板子黄色素则最早在培养的第12天开始合成，因此通过培
养条件的调整以及收获时间的改变，能有效地排除桅子黄色素对桅子多糖分离提
取的干扰，即通过改变培养条件抑制板子黄色素的合成，并在桅子黄色素产生之
前收获细胞，提取桅子多糖。
我们采用了正交实验设计确立了板子多糖的提取和Saveqe法除蛋白的耕，
结果发现，用50倍体积的碱水在80℃下提取2小时，再经过一系列醇析、脱色
和脱蛋白等处理，桅子果实多糖的得率为1．74％，是文献报道的3倍。DEAE纤
维素柱层析可以将桅子多糖分成分子量分别为14，000和10，000的两种，它们
在结构上只是略有差异，分子量为10，000的柜子多糖含有糖醛酸，但是它们在
生物学活性上没有太大的差别。柜子多糖主要分布在果实中，其次是叶，茎和根
中最少。在悬浮细胞中桅子粗多糖的百分含量约为2．5％左右，纯化后为0．65％，
并且证实与板子果实中的多糖有相同的生物学功能，可以用于工业化生产，以缓
解有限的资源问题。
The research on Polysaccharide is very important in the field of biochemistry, and it is becoming a new hot point. There are many reports on plant polysaccharide recently, especially those of traditional Chinese medicine. The authors found polysaccharide has many biological activities, such as anti-tumor, anti-virus, anti-bacteria and anti-oxidize etc. Part of biological activities, isolation, purification and feasibility of producing GJEP by biotechnology were studied here.
Gardenia jasminoides Bills is a kind of traditional Chinese medicine. The compositions used of traditional Chinese medicine are those that can dissolve in water. GJEP is one of them, it is important to restrain the growth of tumors and anti-oxidation .In vitro, 100ug/ml GJEP can restrain sarcoma 180 cell (58%), Hca-f ascites hepatoma cell (51%) and man erythroleukemia K562 cancer cell (63%). The inhibition ratio of GJEP on Hca-f ascites hepatoma in mice achieved 49%, and the results of ig are better than those of ip.
GJEP can scavenge super-oxygen negative ion free radical, and the scavenging ratio is 83.48%. However, scavenging ratio of GJEP on hydroxyl free radical is 22.31%. Some medal ion can influence the scavenging ratio of GJEP on free radical. Zn2+,Fe2+ and Ca2+ can decrease the capacity of GJEP on scavenging ?O2- in varying degrees, Cu2+ can increase a litter the capacity of GJEP on scavenging ?O2- .However, these four medal ions all can increase the effect on scavenging ?OH. GJEP can increase SOD enzyme activities and decrease MDA content of mice, the changes are related with the growth of tumor. The mice that have Dowerful SOD activity, have low MDA content, their tumors are heavy; On the contrary, the mice that have weak SOD activity, have high
MDA content, their tumors are light. So we infer that GJEP can restrain the growth of tumor by increasing SOD enzyme activities of mice to scavenge unnecessary free radical.
The production of GJEP was studied by suspension cell culture. The results show that:(l) The size of cell aggregate does not affect the production of GJEP. There are some structure for transferring material in cell aggregate; (2) The subculture periods of 5-10 days are good for keeping the capacities of cell growth and polysaccharide production; (3) The inoculum concentration of 80g/L cell can shorten the time, which the cell biomass achieve the biggest value needed; (4) Sucrose can be decomposed into glucose and fructose in suspension culture. The cell of Gardenia jasminoides Bills has priority to using glucose; (5) The capacity of GJEP production was decreased in later stage, one of the important reason was that the carbon source in the medium was used up; (6) Excessive nitrogen source was not good for producing GJEP; (7) Auxins have more affective on cell growth and polysaccharide production. The result of 2,4-D is priority to that of NAA and the result of NAA is priority to that of IAA; (8) Gardenia jasminoides Bills cell is not sensitive to shearing power, cell can grow normally in 80~130r/min rotational speed of table; (9) Red light and blue light are two important lights on cell growth and polysaccharide production in suspension culture of Gardenia jasminoides Bills; (10) The effects of temperature and PH value on cell growth and polysaccharide production of GJEP are the same as other plants.
By selecting high production cell of GJEP and changing medium and cultural condition, growth of cell and polysaccharide content were increased. The suspension culture of Gardenia jasminoides Bills has some special character, but there is a regular that if the condition is good for cell growth, then it is probably not good for biological synthesis of GJEP.
Abstract
Usually, GJEP is produced at the early stage of suspension cell culture, and the yellow pigment of Gardenia jasminoides Bills is produced from the 12th day of suspension cell culture, so we can remove the disturbance of yellow pigment from GJEP by controlling the culture conditions and harvest time of cell, that is, inhibiting the synt
栀子是传统中药，其多糖在抑制肿瘤生长和抗氧化过程中起重要作用。100ug／ml的栀子多糖在体外可以抑制S-180肉瘤细胞(58％)、Hca-f腹水肝癌细胞(51％)、K562人红白血病细胞(63％)。栀子多糖在活体中对Hca-f肝癌实体瘤的抑制率为49％，口服给药的效果好于注射给药。
栀子多糖对超氧阴离子自由基有很好的清除效果，清除率高达83.48％，而对羟自由基的清除率却只有22.31％。一些金属离子的存在可以改变栀子多糖对自由基的清除能力。Zn~(2+)、Fe~(2+)和Ca~(2+)能不同程度地削弱栀子多糖对超氧阴离子的清除能力，只有Cu~(2+)可以稍稍提高栀子多糖对超氧阴离子的清除能力。而这四种金属离子都能促进栀子多糖对羟自由基的清除作用。在实验动物体内，栀子多糖能增加SOD酶的活力，降低MDA的含量，同时这种改变与肿瘤的生长有密切的关联。SOD酶活性强的小鼠，MDA含量低，肿瘤轻；SOD酶活性弱的小鼠，MDA含量高，肿瘤重。因此栀子多糖可能是通过提高小鼠体内SOD等抗氧化酶的活性，来清除多余的自由基，从而达到抑制肿瘤生长的目的。
在利用悬浮细胞生产栀子多糖的研究中，通过选择栀子多糖的高产细胞株系以及改变培养基和培养条件来增加细胞的生长和多糖的百分含量。结果表明，细胞聚集体大小并不影响栀子多糖的合成，细胞团内部存在着物质传递结构；5～10天的继代周期有利于细胞保持快速分裂和多糖合成的能力；80g／L的接种量则大大缩短了细胞生物量达到最大值的时间；在悬浮培养中，蔗糖可迅速分解成葡萄糖和果糖，栀子细胞优先利用葡萄糖，培养后期多糖合成量的下降主要与培养基碳源供应不足有关；氮源浓度过高不利于栀子多糖的合成；在激素的使用上，生长素对栀子细胞生长和多糖合成有促进作用，2，4-D效果优于NAA，NAA又优于IAA；栀子细胞对剪切力不是很敏感，在80～130r／min是较宽范围内都能正常
大连理工大学博士学位论文
生长；红色光和蓝色光是柜子悬浮细胞生长和多糖合成的两个重要的单色光；而
温度和PH值等物理因素的影响与其他植物悬浮培养细胞无大大差异。
尽管桅子悬浮细胞培养有许多富于个性的特点，但是有一个总的规律，即利
于细胞生长的条件很可能不利于桅子多糖的合成和积累。板子多糖一般在悬浮细
胞培养的初期形成，板子黄色素则最早在培养的第12天开始合成，因此通过培
养条件的调整以及收获时间的改变，能有效地排除桅子黄色素对桅子多糖分离提
取的干扰，即通过改变培养条件抑制板子黄色素的合成，并在桅子黄色素产生之
前收获细胞，提取桅子多糖。
我们采用了正交实验设计确立了板子多糖的提取和Saveqe法除蛋白的耕，
结果发现，用50倍体积的碱水在80℃下提取2小时，再经过一系列醇析、脱色
和脱蛋白等处理，桅子果实多糖的得率为1．74％，是文献报道的3倍。DEAE纤
维素柱层析可以将桅子多糖分成分子量分别为14，000和10，000的两种，它们
在结构上只是略有差异，分子量为10，000的柜子多糖含有糖醛酸，但是它们在
生物学活性上没有太大的差别。柜子多糖主要分布在果实中，其次是叶，茎和根
中最少。在悬浮细胞中桅子粗多糖的百分含量约为2．5％左右，纯化后为0．65％，
并且证实与板子果实中的多糖有相同的生物学功能，可以用于工业化生产，以缓
解有限的资源问题。
The research on Polysaccharide is very important in the field of biochemistry, and it is becoming a new hot point. There are many reports on plant polysaccharide recently, especially those of traditional Chinese medicine. The authors found polysaccharide has many biological activities, such as anti-tumor, anti-virus, anti-bacteria and anti-oxidize etc. Part of biological activities, isolation, purification and feasibility of producing GJEP by biotechnology were studied here.
Gardenia jasminoides Bills is a kind of traditional Chinese medicine. The compositions used of traditional Chinese medicine are those that can dissolve in water. GJEP is one of them, it is important to restrain the growth of tumors and anti-oxidation .In vitro, 100ug/ml GJEP can restrain sarcoma 180 cell (58%), Hca-f ascites hepatoma cell (51%) and man erythroleukemia K562 cancer cell (63%). The inhibition ratio of GJEP on Hca-f ascites hepatoma in mice achieved 49%, and the results of ig are better than those of ip.
GJEP can scavenge super-oxygen negative ion free radical, and the scavenging ratio is 83.48%. However, scavenging ratio of GJEP on hydroxyl free radical is 22.31%. Some medal ion can influence the scavenging ratio of GJEP on free radical. Zn2+,Fe2+ and Ca2+ can decrease the capacity of GJEP on scavenging ?O2- in varying degrees, Cu2+ can increase a litter the capacity of GJEP on scavenging ?O2- .However, these four medal ions all can increase the effect on scavenging ?OH. GJEP can increase SOD enzyme activities and decrease MDA content of mice, the changes are related with the growth of tumor. The mice that have Dowerful SOD activity, have low MDA content, their tumors are heavy; On the contrary, the mice that have weak SOD activity, have high
MDA content, their tumors are light. So we infer that GJEP can restrain the growth of tumor by increasing SOD enzyme activities of mice to scavenge unnecessary free radical.
The production of GJEP was studied by suspension cell culture. The results show that:(l) The size of cell aggregate does not affect the production of GJEP. There are some structure for transferring material in cell aggregate; (2) The subculture periods of 5-10 days are good for keeping the capacities of cell growth and polysaccharide production; (3) The inoculum concentration of 80g/L cell can shorten the time, which the cell biomass achieve the biggest value needed; (4) Sucrose can be decomposed into glucose and fructose in suspension culture. The cell of Gardenia jasminoides Bills has priority to using glucose; (5) The capacity of GJEP production was decreased in later stage, one of the important reason was that the carbon source in the medium was used up; (6) Excessive nitrogen source was not good for producing GJEP; (7) Auxins have more affective on cell growth and polysaccharide production. The result of 2,4-D is priority to that of NAA and the result of NAA is priority to that of IAA; (8) Gardenia jasminoides Bills cell is not sensitive to shearing power, cell can grow normally in 80~130r/min rotational speed of table; (9) Red light and blue light are two important lights on cell growth and polysaccharide production in suspension culture of Gardenia jasminoides Bills; (10) The effects of temperature and PH value on cell growth and polysaccharide production of GJEP are the same as other plants.
By selecting high production cell of GJEP and changing medium and cultural condition, growth of cell and polysaccharide content were increased. The suspension culture of Gardenia jasminoides Bills has some special character, but there is a regular that if the condition is good for cell growth, then it is probably not good for biological synthesis of GJEP.
Abstract
Usually, GJEP is produced at the early stage of suspension cell culture, and the yellow pigment of Gardenia jasminoides Bills is produced from the 12th day of suspension cell culture, so we can remove the disturbance of yellow pigment from GJEP by controlling the culture conditions and harvest time of cell, that is, inhibiting the synt
引文
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