苦瓜多糖的纯化及其免疫和抗氧化活性作用
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摘要
苦瓜是人们生活中的一种常用蔬菜,同时也是重要的功能食品原料。现代研究表明,苦瓜具有提高免疫力、降血糖、抑菌和抗氧化等作用,并明确苦瓜多糖(Momordica charantia polysaccharide,简称MCP)是其重要生物活性成分之一。目前国内外对MCP的研究主要集中在降血糖方面,对其抗氧化和免疫活性方面的研究较少,并且所用多糖样品多为未经纯化的粗多糖,无法全面了解苦瓜多糖的作用机理、多糖活性组分与其活性作用之间的量效、构效关系。本文对MCP体内外的抗氧化和免疫活性进行了深入研究,用抗氧化和免疫指标对分离组分进行了活性追踪,并对获得的活性组分进行结构分析,旨在为MCP药物及功能性食品开发提供一定的理论依据。主要结果如下：
1 MCP提取工艺的优化：在单因素实验基础上通过二次旋转回归组合设计实验,对MCP的纤维素酶协同超声波法提取工艺条件进行了优化。结果表明：MCP最优工艺条件为超声波功率390W、纤维素酶量3500 U/g(酶活≥200 U/mg)、料液比1：38、时间40min、pH值5、温度56℃。在此工艺条件下,苦瓜多糖的提取率为21.1%,比热水浸提法、超声波法、纤维素酶法分别提高了7.8%、13.5%和7.7%。
2 MCP对免疫低下小鼠免疫活性的影响：从正常小鼠脾脏中获取脾淋巴细胞,采取体外与多糖共同培养的方式,分析MCP对脾淋巴细胞体外增殖的影响,结果表明：MCP浓度在60～100μg/mL范围时能直接和协同ConA促进体外培养的脾淋巴细胞增殖(P<0.01),并具有双向调节作用；采用环磷酰胺(CY)造模法建立免疫低下小鼠模型,灌胃不同浓度的苦瓜多糖,分析MCP对免疫抑制小鼠的巨噬细胞吞噬功能、血清溶血素含量、脾脏淋巴细胞转化能力、NK细胞活性、脾脏指数和胸腺指数的影响,结果表明：150mg/(kg·d)和300 mg/(kg·d)剂量MCP均能明显提高免疫低下小鼠的巨噬细胞吞噬功能、血清溶血素含量、脾脏淋巴细胞转化能力、NK细胞活性、脾脏指数和胸腺指数(P<0.05),300 mg/(kg·d)剂量MCP能使免疫抑制小鼠的以上各项免疫指标均达到正常小鼠的水平(P<0.05)。
3 MCP的抗氧化活性研究：通过测定DPPH·、ABTS·+自由基清除能力和Fe3+还原能力(FRAP),考察了MCP体外的抗氧化能力,结果表明：MCP清除DPPH和ABTS·+的IC50分别为571.8μg/mL、737μg/mL,浓度在1.6mg/mL时,MCP对DPPH·和ABTS·+的清除率达90%以上；MCP的FRAP值为0.187μmolFeSO4/mg,其还原能力与溶液呈显著正相关(P<0.01)。通过CY建立免疫低下小鼠模型,考察了MCP对体内产自由基酶及自由基水平的影响,结果表明：CY使脾脏中XOD、NOS活力,胸腺中XOD、MPO、NOS活力显著升高(P<0.05),使小鼠血清中T-AOC、T-SOD活力、胸腺中GSH含量显著降低(P<0.05)；MCP对CY引起的脾脏和胸腺中XOD、MPO的上升有显著的拮抗作用(P<0.05),能显著增加免疫低下小鼠的T-AOC、T-SOD、GSH水平以及降低MDA含量,并能使以上各测试指标达正常水平(P<0.05),但对NOS的活性无显著影响。
4 MCP的分离纯化及活性组分的结构分析：采用DEAE-52离子交换柱对MCP进行分级纯化,得到6个单一多糖组分；将各组分进行ABTS自由基清除实验和脾淋巴细胞增殖实验,筛选得出MCP3、MCP6具有较好的抗氧化和免疫活性。对这两个活性组分进行结构分析,结果表明：MCP3和MCP6均是不含蛋白和核酸的杂多糖。MCP3主要是由葡萄糖、半乳糖、阿拉伯糖、甘露糖、木糖、鼠李糖6种单糖组成,分子量为2.00×104；MCP6主要是由葡萄糖、甘露糖、半乳糖、木糖、阿拉伯糖5种单糖组成,分子量为1.35×104。MCP3和MCP6具有多糖的特征吸收峰,其糖环为吡喃环,MCP3和MCP6糖环均有两种连结方式,即α-型糖苷键和β-型糖苷键。
本文的创新点在于：(1)建立了苦瓜多糖的纤维素酶协同超声波法高效提取苦瓜多糖的方法和条件；(2)确证了苦瓜多糖具有较强的免疫调节和抗氧化作用,(3)从苦瓜多糖中分离得到的2个具有免疫调节和抗氧化活性的组分,并鉴定其均为低分子量杂多糖,其糖环为吡喃环。
Momordica charantia is a common vegetable in people living and also an important functional food ingredients. Studies have shown that Momordica charantia could improve immunity, reduce blood sugar, antimicrobial and antioxidant effects, and clearly pointed out that the momordica charantia polysaccharide (MCP) is one of the main functional active components. Previous reports on MCP mainly focuses on lowering blood glucose, while the active reseach of antioxidant and immune aspects were weak, especially in the preparation of homogeneous fraetions, so it was impossible to learned their mechanism, and dose-effect relationship, structure-activity relationship between active component and its pharmacological effects. This dissertation is focused on the study of antioxidant and immune activity of MCP in vivo and in vitro, track of antioxidant and immune active fraction isolated from crude polyaccharide, structural analysis of active fraction. Aimed to provide a theoretical for the further development of drugs and functional foods of MCP. The main findings are as follows:
1. Optimization of extraction condition of MCP:Based on the single factors experiment, the optimum parameters of cellulase assisted ultrasonic wave of momordica charantia polysaccharide (MCP) were ensured by the method of orthogonally rotational combination design and the regression equation was established. The results indicated that the optimal extraction technology was to extract at ultrasound power 390W, cellulase amount 3500 U/g (enzyme activity≥200 U/mg), ratio of solid to liquid 1:38, extracting time 40 min, pH value 5, temperature 56℃,then the yield of crude momordica charantia polysaccharide is 21.1%, respectively higher 7.8%,13.5% and 7.7% comparaing the specific heat of flooding,ultrasonic wave and cellulase.
2. Evaluation of the immunomodulatory activity of MCP:lymphocytes were obtained from healthy mice spleen and cultivated with different concentrate MCP in 96 wells for improved lymphopoiesis test, reslutes showed that MCP ranging from 60μg/mL to 100μg/mL could remarkly promot the spleen cell proliferation in vitro by rejecting or assisting ConA treated in a dose-dependent manner (P<0.01); Immunosuppressed mouse models were established by intraperitoneal injections with cyclophosphamide (CY) followed by daily intragastric administration of different dose MCP. After the treatments, the mice were examined for phagocytotic function of the macrophages, serum IgM level following exposure to chicken red blood cells, the lymphocyte transformation rate, natural killer cells activity and immune organ weight index. Reslutes showed that treatment of the immunosuppressed mice with MCP at the daily dose of 150 mg/(kg-d) and 300 mg/(kg-d) was able to antagonize CY ide-induced immunosuppression and significantly increased their phagocytotic function of the macrophages, serum IgM level following exposure to chicken red blood cells, the lymphocyte transformation rate, natural killer cells activity and immune organ weight index (P<0.05); the later increased all the test level above of immuno suppressive mice to the same level of health mice (P<0.05).
3 Evaluation of the immunomodulatory activity of MCP:The antioxidant activity of extracts from MCP was assayed by three methods, DPPH, ABTS radical scavenging and ferric reducing (FRAP) assay, the reslutes showed that IC50 value of DPPH and ABTS radical scavenging activity is respectively 571.8μg/mL,737μg/mL, and MCP could eliminate radical at a higher percentage of 90% with content 1.6mg/mL; FRAP value of MCP is 0.187μmolFeSO4/mg, the positive correlation (P<0.01) existed between reductive capacity and concentration. The antioxidant activity Immunosuppressive mouse model was established by CY and the level of oxygen radical genesis enzyme and free radical in vivo was observed, results showed that a single treatment of CY could increase the level of XOD, NOS in spleen and the level of XOD, MPO, NOS in thymus (P<0.05), decrease activity of T-AOC, T-SOD activity in serum, content of GSH in the thymus (P<0.05); while MCP could antagonize prominently the decreasing of the activity of XOD, MPO in spleen and thymus induced by CY, significantly increase the level of T-AOC, T-SOD, GSH, decrease the content of MDA and make all the test index above up to normal levels (P<0.05), but had no effect on NOS.
4 Purification and structural analysis of active components of MCP:6 purified polysaccharides were isolated from crude polyaccharide by deproteinization, dialysis and column chromatography on DEAE-52 cellulose, The mouse spleen cells proliferation stimulating effects and the activity of ABTS radical scavenging in vitro of each fraction at various concentrations were assayed, and two active fraction of immunomodulation and antioxidant, MCP3, MCP6,were obtained and structural analysised. The results showed that:the two active fraction were all heteropolysaccharide and didn't contain protein and starch; MCP3 consisted of glucose, galactose, arabinose, mannose, xylose, rhamnose and its relative molecular weight was 2.00×104;MCP6 consisted of glucose, mannose, galactose, xylose, arabinose and its relative molecular weight was 1.35×104; MCP3, MCP6 had adsorption bands characteric of polysaccharides, contained pyranoid rings and two kinds of link methods of a-glycoside linkage andβ-glycoside linkage.
Innovation:(1) To establish the extraction technology of cellulase assisted ultrasonic wave of MCP; (2) To confirm that MCP have strong immunomodulation and antioxidant activity; (3) There were two components of immunomodulation and antioxidant activity that were solated from the MCP and identified that they were all heteropolysaccharide with low molecular weight and had adsorption bands characteric of polysaccharides with pyranoid rings.
1 MCP提取工艺的优化：在单因素实验基础上通过二次旋转回归组合设计实验,对MCP的纤维素酶协同超声波法提取工艺条件进行了优化。结果表明：MCP最优工艺条件为超声波功率390W、纤维素酶量3500 U/g(酶活≥200 U/mg)、料液比1：38、时间40min、pH值5、温度56℃。在此工艺条件下,苦瓜多糖的提取率为21.1%,比热水浸提法、超声波法、纤维素酶法分别提高了7.8%、13.5%和7.7%。
2 MCP对免疫低下小鼠免疫活性的影响：从正常小鼠脾脏中获取脾淋巴细胞,采取体外与多糖共同培养的方式,分析MCP对脾淋巴细胞体外增殖的影响,结果表明：MCP浓度在60～100μg/mL范围时能直接和协同ConA促进体外培养的脾淋巴细胞增殖(P<0.01),并具有双向调节作用；采用环磷酰胺(CY)造模法建立免疫低下小鼠模型,灌胃不同浓度的苦瓜多糖,分析MCP对免疫抑制小鼠的巨噬细胞吞噬功能、血清溶血素含量、脾脏淋巴细胞转化能力、NK细胞活性、脾脏指数和胸腺指数的影响,结果表明：150mg/(kg·d)和300 mg/(kg·d)剂量MCP均能明显提高免疫低下小鼠的巨噬细胞吞噬功能、血清溶血素含量、脾脏淋巴细胞转化能力、NK细胞活性、脾脏指数和胸腺指数(P<0.05),300 mg/(kg·d)剂量MCP能使免疫抑制小鼠的以上各项免疫指标均达到正常小鼠的水平(P<0.05)。
3 MCP的抗氧化活性研究：通过测定DPPH·、ABTS·+自由基清除能力和Fe3+还原能力(FRAP),考察了MCP体外的抗氧化能力,结果表明：MCP清除DPPH和ABTS·+的IC50分别为571.8μg/mL、737μg/mL,浓度在1.6mg/mL时,MCP对DPPH·和ABTS·+的清除率达90%以上；MCP的FRAP值为0.187μmolFeSO4/mg,其还原能力与溶液呈显著正相关(P<0.01)。通过CY建立免疫低下小鼠模型,考察了MCP对体内产自由基酶及自由基水平的影响,结果表明：CY使脾脏中XOD、NOS活力,胸腺中XOD、MPO、NOS活力显著升高(P<0.05),使小鼠血清中T-AOC、T-SOD活力、胸腺中GSH含量显著降低(P<0.05)；MCP对CY引起的脾脏和胸腺中XOD、MPO的上升有显著的拮抗作用(P<0.05),能显著增加免疫低下小鼠的T-AOC、T-SOD、GSH水平以及降低MDA含量,并能使以上各测试指标达正常水平(P<0.05),但对NOS的活性无显著影响。
4 MCP的分离纯化及活性组分的结构分析：采用DEAE-52离子交换柱对MCP进行分级纯化,得到6个单一多糖组分；将各组分进行ABTS自由基清除实验和脾淋巴细胞增殖实验,筛选得出MCP3、MCP6具有较好的抗氧化和免疫活性。对这两个活性组分进行结构分析,结果表明：MCP3和MCP6均是不含蛋白和核酸的杂多糖。MCP3主要是由葡萄糖、半乳糖、阿拉伯糖、甘露糖、木糖、鼠李糖6种单糖组成,分子量为2.00×104；MCP6主要是由葡萄糖、甘露糖、半乳糖、木糖、阿拉伯糖5种单糖组成,分子量为1.35×104。MCP3和MCP6具有多糖的特征吸收峰,其糖环为吡喃环,MCP3和MCP6糖环均有两种连结方式,即α-型糖苷键和β-型糖苷键。
本文的创新点在于：(1)建立了苦瓜多糖的纤维素酶协同超声波法高效提取苦瓜多糖的方法和条件；(2)确证了苦瓜多糖具有较强的免疫调节和抗氧化作用,(3)从苦瓜多糖中分离得到的2个具有免疫调节和抗氧化活性的组分,并鉴定其均为低分子量杂多糖,其糖环为吡喃环。
Momordica charantia is a common vegetable in people living and also an important functional food ingredients. Studies have shown that Momordica charantia could improve immunity, reduce blood sugar, antimicrobial and antioxidant effects, and clearly pointed out that the momordica charantia polysaccharide (MCP) is one of the main functional active components. Previous reports on MCP mainly focuses on lowering blood glucose, while the active reseach of antioxidant and immune aspects were weak, especially in the preparation of homogeneous fraetions, so it was impossible to learned their mechanism, and dose-effect relationship, structure-activity relationship between active component and its pharmacological effects. This dissertation is focused on the study of antioxidant and immune activity of MCP in vivo and in vitro, track of antioxidant and immune active fraction isolated from crude polyaccharide, structural analysis of active fraction. Aimed to provide a theoretical for the further development of drugs and functional foods of MCP. The main findings are as follows:
1. Optimization of extraction condition of MCP:Based on the single factors experiment, the optimum parameters of cellulase assisted ultrasonic wave of momordica charantia polysaccharide (MCP) were ensured by the method of orthogonally rotational combination design and the regression equation was established. The results indicated that the optimal extraction technology was to extract at ultrasound power 390W, cellulase amount 3500 U/g (enzyme activity≥200 U/mg), ratio of solid to liquid 1:38, extracting time 40 min, pH value 5, temperature 56℃,then the yield of crude momordica charantia polysaccharide is 21.1%, respectively higher 7.8%,13.5% and 7.7% comparaing the specific heat of flooding,ultrasonic wave and cellulase.
2. Evaluation of the immunomodulatory activity of MCP:lymphocytes were obtained from healthy mice spleen and cultivated with different concentrate MCP in 96 wells for improved lymphopoiesis test, reslutes showed that MCP ranging from 60μg/mL to 100μg/mL could remarkly promot the spleen cell proliferation in vitro by rejecting or assisting ConA treated in a dose-dependent manner (P<0.01); Immunosuppressed mouse models were established by intraperitoneal injections with cyclophosphamide (CY) followed by daily intragastric administration of different dose MCP. After the treatments, the mice were examined for phagocytotic function of the macrophages, serum IgM level following exposure to chicken red blood cells, the lymphocyte transformation rate, natural killer cells activity and immune organ weight index. Reslutes showed that treatment of the immunosuppressed mice with MCP at the daily dose of 150 mg/(kg-d) and 300 mg/(kg-d) was able to antagonize CY ide-induced immunosuppression and significantly increased their phagocytotic function of the macrophages, serum IgM level following exposure to chicken red blood cells, the lymphocyte transformation rate, natural killer cells activity and immune organ weight index (P<0.05); the later increased all the test level above of immuno suppressive mice to the same level of health mice (P<0.05).
3 Evaluation of the immunomodulatory activity of MCP:The antioxidant activity of extracts from MCP was assayed by three methods, DPPH, ABTS radical scavenging and ferric reducing (FRAP) assay, the reslutes showed that IC50 value of DPPH and ABTS radical scavenging activity is respectively 571.8μg/mL,737μg/mL, and MCP could eliminate radical at a higher percentage of 90% with content 1.6mg/mL; FRAP value of MCP is 0.187μmolFeSO4/mg, the positive correlation (P<0.01) existed between reductive capacity and concentration. The antioxidant activity Immunosuppressive mouse model was established by CY and the level of oxygen radical genesis enzyme and free radical in vivo was observed, results showed that a single treatment of CY could increase the level of XOD, NOS in spleen and the level of XOD, MPO, NOS in thymus (P<0.05), decrease activity of T-AOC, T-SOD activity in serum, content of GSH in the thymus (P<0.05); while MCP could antagonize prominently the decreasing of the activity of XOD, MPO in spleen and thymus induced by CY, significantly increase the level of T-AOC, T-SOD, GSH, decrease the content of MDA and make all the test index above up to normal levels (P<0.05), but had no effect on NOS.
4 Purification and structural analysis of active components of MCP:6 purified polysaccharides were isolated from crude polyaccharide by deproteinization, dialysis and column chromatography on DEAE-52 cellulose, The mouse spleen cells proliferation stimulating effects and the activity of ABTS radical scavenging in vitro of each fraction at various concentrations were assayed, and two active fraction of immunomodulation and antioxidant, MCP3, MCP6,were obtained and structural analysised. The results showed that:the two active fraction were all heteropolysaccharide and didn't contain protein and starch; MCP3 consisted of glucose, galactose, arabinose, mannose, xylose, rhamnose and its relative molecular weight was 2.00×104;MCP6 consisted of glucose, mannose, galactose, xylose, arabinose and its relative molecular weight was 1.35×104; MCP3, MCP6 had adsorption bands characteric of polysaccharides, contained pyranoid rings and two kinds of link methods of a-glycoside linkage andβ-glycoside linkage.
Innovation:(1) To establish the extraction technology of cellulase assisted ultrasonic wave of MCP; (2) To confirm that MCP have strong immunomodulation and antioxidant activity; (3) There were two components of immunomodulation and antioxidant activity that were solated from the MCP and identified that they were all heteropolysaccharide with low molecular weight and had adsorption bands characteric of polysaccharides with pyranoid rings.
引文
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