藻酸双酯钠的分级制备及其生物活性研究
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摘要
多糖的生物活性、化学结构以及构效关系的研究已成为多糖领域的前沿阵地,并且取得了很大的进展。其中,关于一些相对分子量在几千以上、强生物活性多糖的研究日益受到重视。
本文以我国自主知识产权的海洋多糖药物藻酸双酯钠(PSS)作为研究对象,采用超滤及低压柱层析方法对其分级制备,分离得到具有相似骨架、磺化度及不同相对分子量(Mw)的分级产物;通过对各分级产物进行抗凝血活性、免疫调解作用、保护神经元细胞凋亡、抗菌、降糖等生物活性的考察,探讨PSS的链长与活性的关系,为PSS的广泛应用及多糖构效关系的深入研究进行有益的探索。
研究结果如下:
1、低压柱层析法以Sephadex G100作为分离介质,对PSS(Mw:17.19 kDa)进行分离,高效凝胶渗透色谱法测定产物重均分子量(Mw),得到Mw分别为51.95,25.62,11.76,5.41 kDa 4个分级产物(Z-PSS:1-4);以截留分子量分别为30,10,5,3,1 kDa的超滤膜分级PSS,得到Mw分别为21.97,20.25,18.27,11.35,4.93,3.33 kDa 6个分级产物(C-PSS:1-6)。氧瓶燃烧法(Oxygen Flask Method)测定所有分级产物磺化度多在1.2-1.3之间;红外光谱(FT-IR)、核磁共振碳谱(13C-NMR)证明分级产物磺化取代发生在糖环的C-2,C-3,酯化取代发生在C-6。
2、分级产物生物活性比较研究:
2.1、PSS及柱层析分级产物Z-PSS:1-4抗凝血实验(凝血三项:凝血酶原时间PT,活化部分凝血活酶时间APTT,凝血酶时间TT)显示:PSS及4个分级产物延长TT时间作用最强,其次为APTT,延长PT时间作用最弱,以上结果通过与对照组比较,t-test,显示统计学差异(p<0.05)。4个分级产物抗凝活性比较显示,抗凝血作用与分子量显著相关,不同的凝血指标对分子量的要求显著不同,APTT显著随PSS分级产物分子量的降低而降低。
2.2、PSS及超滤分级产物C-PSS:1-5抗凝血实验(PT,APTT,TT)进一步证实了柱层析分级产物凝血三项实验结果:样品对TT作用最强,APTT次之,对PT作用较弱,而且活性大小与样品的分子量显著相关。
2.3、在凝血三项实验的基础上,进一步验证了PSS及柱层析分级产物Z-PSS:1-4对凝血因子FIIa和FXa的作用。结果显示PSS主要通过作用于FIIa因子发挥抗凝血作用,强度与低分子肝素类似;对FXa作用较弱。各分级产物抗凝血因子作用比较显示,作用强度与分子量显著相关,随着分级产物Z-PSS1→4分子量的降低,抗凝血因子作用显著降低,Z-PSS4失去抗FXa作用。
2.4、抗蜂毒肽(Mastoparan)诱导的大鼠原代神经元细胞凋亡实验结果显示:PSS对Mastoparan诱导的大鼠子鼠原代神经元细胞凋亡有显著的保护作用,其分级产物Z-PSS1-3也表现出对凋亡神经元细胞的保护作用,而Z-PSS4没有这种作用,证明该作用有最低糖链片断要求,但并不会随糖链长度的延长而保护作用增强。采用Fluo-3/AM荧光探针技术标记胞内钙离子,测定胞内钙离子浓度变化,推测PSS保护神经元细胞的凋亡作用机制是抑制胞内钙释放。
2.5、本课题对PSS及Z-PSS1-4的免疫调节作用进行了细胞水平的研究。主要考察样品对小鼠脾脏细胞、小鼠腹腔巨噬细胞、刀豆蛋白A(Concanavalin A,Con A)诱导的免疫T-Cell、脂多糖(Lipopolysaccharide,LPS)诱导的B-Cell的作用,探讨PSS及分级产物的免疫调节作用。实验结果显示,PSS能增强脾脏细胞的增殖作用,抑制Con A诱导的T-Cell和LPS诱导的B-Cell增殖,同时能够促进巨噬细胞的吞噬功能。对于脾脏细胞的作用,PSS分子量在10-20 kDa之间较显著;对于促进巨噬细胞吞噬功能,显示PSS分子量最大分级产物作用最强。
2.6、以革兰氏阳性菌-金黄色葡萄球菌、革兰氏阴性菌-大肠杆菌作为目标菌株,检验PSS及其层析产物Z-PSS:1-4,C-PSS1-5的抑菌作用。结果显示:PSS及Z-PSS:1-3,C-PSS1-4对金黄色葡萄球菌有良好的抑制效果,且Z-PSS2-3,C-PSS4效果强于Z-PSS1,C-PSS1-3,证明分子量较小PSS片断具有较强的抑菌作用;同时Z-PSS4及C-PSS5失去抑菌作用,显示PSS抑菌活性与糖链长度的关系。
2.7、在胰岛细胞筛选PSS超滤分级产物促进胰岛素分泌作用的基础上,进行C-PSS4的降低四氧嘧啶高血糖小鼠血糖作用研究。结果显示:C-PSS4有降低高血糖小鼠血糖的趋势,但未见统计学差异(p>0.05)。鉴于细胞水平研究的结果,整体药效学实验有待于进一步研究。
Polysaccharides and their derivatives have attracted considerable attention in biomedicine and have been used as a variety of biomaterials. Currently, the study of polysaccharides is mainly focused on their structure-activity relationships. And more and more studies have been concentrated on these polysaccharides, which molecular weight (Mw) are several thousand Daltons and have the outstanding bioactivities.
Sulfated alginate derivatives is a kind of heterogeneous and highly dispersed sulfated polysaccharide, and it had various bioactivities, e.g. anticoagulation, anti-oxidation, anti-HIV, heparanase inhibition, anti-inflammation and preventing calcium phosphate crystals formation in urine tract. However, little is known about the structure-activity relationships of sulfated alginate derivatives.
Propylene Glycol Alginate Sodium Sulfate (PSS), which has been used as a marine derived drug for lowering blood lipid in China for nearly 20 years, is one of the most important sulfated alginate derivatives, it is necessary to make an intensive study of structure-activity relationships of PSS. In this study, the relationship between molecular mass of PSS and its several activities was investigated. The structural characteristics and activities of PSS and its fractions were studied in detail.
The main results and conclusions of the research were as follows:
1、PSS was fractionated by low-pressure gel permeation chromatography and ultrafiltration. Four column fractions (Z-PSS1-4) and five ultrafiltration fractions (C-PSS1-5) with different weight-averaged molecular mass (Z-PSS1-4: 51.95, 25.62, 11.76 and 5.41 kDa, C-PSS1-5: 21.97, 20.25, 18.27, 11.35, 4.93 kDa, respectively) were obtained, and their structural characteristics were determined and compared by Fourier transform infrared and nuclear magnetic resonance spectroscopes. The degrees of sulfate substitutions of most fractions were at 1.2-1.3 analyzed by oxygen flask method. The FT-IR data provided the information of backbone and sulfation of PSS and its fractions. That meant that the backbones of PSS and its fractions was made up ofα-1, 4-guluronic acids andβ-1, 4-mannuronic acids, respectively, and the sulfation was really occurred. The NMR data indicated that the propylene glycol group was linked to the C-6 position of the hexauronic acid residues of alginate and sulfate group was located at C-2 and C-3 positions of PSS and its fractions.
The results of chemical analysis showed that PSS and PSS1-4 have similar structure characteristics except for the molecular mass.
2、The comparative study of bioactivities of PSS and its fractions can be summarized as below:
2.1、The anticoagulant activity of the column fractions was studied by evaluating their influence on the prothrombin time, activated partial thromboplastin time and thrombin time. The results showed that the highest activity of PSS and its column fractions was found in the TT, followed by that in the APTT, the moderate activity was found in the PT. And that the anticoagulant increased with the ascending molecular weight of its fractions. That proved that the anticoagulant of PSS was molecular weight dependence.
2.2、The results of anticoagulant experiments of the ultrafiltration fractions were same as the results of the column fraction anticoagulant experiments. That showed that PSS and its fractions had the highest activity of TT, followed by that in the APTT, the moderate PT activity. At the same time, the molecular weight dependence effect of anticoagulant of PSS was also observed.
2.3、Based on the above results of anticoagulant 3 items, their anticoagulant activities were studied by evaluating their influence on anti-thrombin and anti-FXa activities mediated by antithrombin III and heparin cofactor II furthermore. The results demonstrated that Z-PSS1 and Z-PSS2 strongly inhibited the activity of thrombin mediated by heparin cofactor II and antithrombin III, whereas PSS4 only showed very weak anti-thrombin activity. Z-PSS1-3 weakly inhibited the activity of coagulation factor Xa mediated only by antithrombin III while Z-PSS4 lacked the anti-Xa activity.
2.4、The results of the protection of PSS on mastoparan-induced apoptosis of cultured cerebellar granule neurons showed that PSS can protect the mastoparan- induced apoptosis of cultured cerebral granule neurons. Measurements of intracellular free calcium concentration ([Ca2+]i) with Fluo-3/AM proved that PSS and Z-PSS1-3 debased the elevated [Ca2+]i induced by mastoparan. That clued on the protection of PSS on apoptosis neuron was through the inhibition on elevated [Ca2+]i induced by mastoparan. Z-PSS1-3 also showed the inhibition effect on apoptosis of cerebellar granule neurons, but Z-PSS4 was not. This meant the protection effect had the limit of molecular weight. But the molecular weight dependence was not observed as the effect did not increase with the ascending molecular weight of fractions.
2.5、The immunoregulation activity of PSS and its fractions was investigated using immunocyte cultivation technique in vitro. The structure-activity relationship was analyzed on the basis of the structure studies of PSS’fractions. The experimental results showed that PSS could improve spleen cell proliferation, increase macrophage phagocytic function and inhibit the proliferation of T-cell induced by Concanavalin A (Con A) and B-cell induced by lipopolysaccharide (LPS). That proved that PSS possesses the significant immunoregulation effect, whilst the immunocompetence comparison of PSS’column fractions proved that the different immunocyte had different requirement for saccharides length. For spleen cell proliferation, the best molecular weight was about 10-20 kDa. For macrophage phagocytic function, the bigger Mw fraction of PSS had the stronger effect.
2.6、In-vitro antibacterial activities of PSS and all fractions were compared and analyzed in the studies of the inhibition effect on Gram-Negative Bacteria (G+) (S. Aureus) and Gram-Positive Bacteria (G-) (E. Coli). The results showed that PSS, Z-PSS:1-3, C-PSS1-4 possessed the favorable antibacterial activity. Z-PSS2-3 and C-PSS4 showed the stronger effect than that of Z-PSS1 and C-PSS1-3, whilst Z-PSS4 and C-PSS lose the effect. This meant that the smaller molecular weight had the stronger antibacterial activities. To some extent, PSS had the molecular weight dependence of antibacterial activity.
2.7、Based on the screen assay of PSS’promotion on insulin secretion in vitro, hypoglycemic effect of C-PSS4 was studied. C-PSS4 showed the possibility of decreasing blood glucose, but the statistical significant difference was not displayed (p>0.05). The effect needs the further investigation.
本文以我国自主知识产权的海洋多糖药物藻酸双酯钠(PSS)作为研究对象,采用超滤及低压柱层析方法对其分级制备,分离得到具有相似骨架、磺化度及不同相对分子量(Mw)的分级产物;通过对各分级产物进行抗凝血活性、免疫调解作用、保护神经元细胞凋亡、抗菌、降糖等生物活性的考察,探讨PSS的链长与活性的关系,为PSS的广泛应用及多糖构效关系的深入研究进行有益的探索。
研究结果如下:
1、低压柱层析法以Sephadex G100作为分离介质,对PSS(Mw:17.19 kDa)进行分离,高效凝胶渗透色谱法测定产物重均分子量(Mw),得到Mw分别为51.95,25.62,11.76,5.41 kDa 4个分级产物(Z-PSS:1-4);以截留分子量分别为30,10,5,3,1 kDa的超滤膜分级PSS,得到Mw分别为21.97,20.25,18.27,11.35,4.93,3.33 kDa 6个分级产物(C-PSS:1-6)。氧瓶燃烧法(Oxygen Flask Method)测定所有分级产物磺化度多在1.2-1.3之间;红外光谱(FT-IR)、核磁共振碳谱(13C-NMR)证明分级产物磺化取代发生在糖环的C-2,C-3,酯化取代发生在C-6。
2、分级产物生物活性比较研究:
2.1、PSS及柱层析分级产物Z-PSS:1-4抗凝血实验(凝血三项:凝血酶原时间PT,活化部分凝血活酶时间APTT,凝血酶时间TT)显示:PSS及4个分级产物延长TT时间作用最强,其次为APTT,延长PT时间作用最弱,以上结果通过与对照组比较,t-test,显示统计学差异(p<0.05)。4个分级产物抗凝活性比较显示,抗凝血作用与分子量显著相关,不同的凝血指标对分子量的要求显著不同,APTT显著随PSS分级产物分子量的降低而降低。
2.2、PSS及超滤分级产物C-PSS:1-5抗凝血实验(PT,APTT,TT)进一步证实了柱层析分级产物凝血三项实验结果:样品对TT作用最强,APTT次之,对PT作用较弱,而且活性大小与样品的分子量显著相关。
2.3、在凝血三项实验的基础上,进一步验证了PSS及柱层析分级产物Z-PSS:1-4对凝血因子FIIa和FXa的作用。结果显示PSS主要通过作用于FIIa因子发挥抗凝血作用,强度与低分子肝素类似;对FXa作用较弱。各分级产物抗凝血因子作用比较显示,作用强度与分子量显著相关,随着分级产物Z-PSS1→4分子量的降低,抗凝血因子作用显著降低,Z-PSS4失去抗FXa作用。
2.4、抗蜂毒肽(Mastoparan)诱导的大鼠原代神经元细胞凋亡实验结果显示:PSS对Mastoparan诱导的大鼠子鼠原代神经元细胞凋亡有显著的保护作用,其分级产物Z-PSS1-3也表现出对凋亡神经元细胞的保护作用,而Z-PSS4没有这种作用,证明该作用有最低糖链片断要求,但并不会随糖链长度的延长而保护作用增强。采用Fluo-3/AM荧光探针技术标记胞内钙离子,测定胞内钙离子浓度变化,推测PSS保护神经元细胞的凋亡作用机制是抑制胞内钙释放。
2.5、本课题对PSS及Z-PSS1-4的免疫调节作用进行了细胞水平的研究。主要考察样品对小鼠脾脏细胞、小鼠腹腔巨噬细胞、刀豆蛋白A(Concanavalin A,Con A)诱导的免疫T-Cell、脂多糖(Lipopolysaccharide,LPS)诱导的B-Cell的作用,探讨PSS及分级产物的免疫调节作用。实验结果显示,PSS能增强脾脏细胞的增殖作用,抑制Con A诱导的T-Cell和LPS诱导的B-Cell增殖,同时能够促进巨噬细胞的吞噬功能。对于脾脏细胞的作用,PSS分子量在10-20 kDa之间较显著;对于促进巨噬细胞吞噬功能,显示PSS分子量最大分级产物作用最强。
2.6、以革兰氏阳性菌-金黄色葡萄球菌、革兰氏阴性菌-大肠杆菌作为目标菌株,检验PSS及其层析产物Z-PSS:1-4,C-PSS1-5的抑菌作用。结果显示:PSS及Z-PSS:1-3,C-PSS1-4对金黄色葡萄球菌有良好的抑制效果,且Z-PSS2-3,C-PSS4效果强于Z-PSS1,C-PSS1-3,证明分子量较小PSS片断具有较强的抑菌作用;同时Z-PSS4及C-PSS5失去抑菌作用,显示PSS抑菌活性与糖链长度的关系。
2.7、在胰岛细胞筛选PSS超滤分级产物促进胰岛素分泌作用的基础上,进行C-PSS4的降低四氧嘧啶高血糖小鼠血糖作用研究。结果显示:C-PSS4有降低高血糖小鼠血糖的趋势,但未见统计学差异(p>0.05)。鉴于细胞水平研究的结果,整体药效学实验有待于进一步研究。
Polysaccharides and their derivatives have attracted considerable attention in biomedicine and have been used as a variety of biomaterials. Currently, the study of polysaccharides is mainly focused on their structure-activity relationships. And more and more studies have been concentrated on these polysaccharides, which molecular weight (Mw) are several thousand Daltons and have the outstanding bioactivities.
Sulfated alginate derivatives is a kind of heterogeneous and highly dispersed sulfated polysaccharide, and it had various bioactivities, e.g. anticoagulation, anti-oxidation, anti-HIV, heparanase inhibition, anti-inflammation and preventing calcium phosphate crystals formation in urine tract. However, little is known about the structure-activity relationships of sulfated alginate derivatives.
Propylene Glycol Alginate Sodium Sulfate (PSS), which has been used as a marine derived drug for lowering blood lipid in China for nearly 20 years, is one of the most important sulfated alginate derivatives, it is necessary to make an intensive study of structure-activity relationships of PSS. In this study, the relationship between molecular mass of PSS and its several activities was investigated. The structural characteristics and activities of PSS and its fractions were studied in detail.
The main results and conclusions of the research were as follows:
1、PSS was fractionated by low-pressure gel permeation chromatography and ultrafiltration. Four column fractions (Z-PSS1-4) and five ultrafiltration fractions (C-PSS1-5) with different weight-averaged molecular mass (Z-PSS1-4: 51.95, 25.62, 11.76 and 5.41 kDa, C-PSS1-5: 21.97, 20.25, 18.27, 11.35, 4.93 kDa, respectively) were obtained, and their structural characteristics were determined and compared by Fourier transform infrared and nuclear magnetic resonance spectroscopes. The degrees of sulfate substitutions of most fractions were at 1.2-1.3 analyzed by oxygen flask method. The FT-IR data provided the information of backbone and sulfation of PSS and its fractions. That meant that the backbones of PSS and its fractions was made up ofα-1, 4-guluronic acids andβ-1, 4-mannuronic acids, respectively, and the sulfation was really occurred. The NMR data indicated that the propylene glycol group was linked to the C-6 position of the hexauronic acid residues of alginate and sulfate group was located at C-2 and C-3 positions of PSS and its fractions.
The results of chemical analysis showed that PSS and PSS1-4 have similar structure characteristics except for the molecular mass.
2、The comparative study of bioactivities of PSS and its fractions can be summarized as below:
2.1、The anticoagulant activity of the column fractions was studied by evaluating their influence on the prothrombin time, activated partial thromboplastin time and thrombin time. The results showed that the highest activity of PSS and its column fractions was found in the TT, followed by that in the APTT, the moderate activity was found in the PT. And that the anticoagulant increased with the ascending molecular weight of its fractions. That proved that the anticoagulant of PSS was molecular weight dependence.
2.2、The results of anticoagulant experiments of the ultrafiltration fractions were same as the results of the column fraction anticoagulant experiments. That showed that PSS and its fractions had the highest activity of TT, followed by that in the APTT, the moderate PT activity. At the same time, the molecular weight dependence effect of anticoagulant of PSS was also observed.
2.3、Based on the above results of anticoagulant 3 items, their anticoagulant activities were studied by evaluating their influence on anti-thrombin and anti-FXa activities mediated by antithrombin III and heparin cofactor II furthermore. The results demonstrated that Z-PSS1 and Z-PSS2 strongly inhibited the activity of thrombin mediated by heparin cofactor II and antithrombin III, whereas PSS4 only showed very weak anti-thrombin activity. Z-PSS1-3 weakly inhibited the activity of coagulation factor Xa mediated only by antithrombin III while Z-PSS4 lacked the anti-Xa activity.
2.4、The results of the protection of PSS on mastoparan-induced apoptosis of cultured cerebellar granule neurons showed that PSS can protect the mastoparan- induced apoptosis of cultured cerebral granule neurons. Measurements of intracellular free calcium concentration ([Ca2+]i) with Fluo-3/AM proved that PSS and Z-PSS1-3 debased the elevated [Ca2+]i induced by mastoparan. That clued on the protection of PSS on apoptosis neuron was through the inhibition on elevated [Ca2+]i induced by mastoparan. Z-PSS1-3 also showed the inhibition effect on apoptosis of cerebellar granule neurons, but Z-PSS4 was not. This meant the protection effect had the limit of molecular weight. But the molecular weight dependence was not observed as the effect did not increase with the ascending molecular weight of fractions.
2.5、The immunoregulation activity of PSS and its fractions was investigated using immunocyte cultivation technique in vitro. The structure-activity relationship was analyzed on the basis of the structure studies of PSS’fractions. The experimental results showed that PSS could improve spleen cell proliferation, increase macrophage phagocytic function and inhibit the proliferation of T-cell induced by Concanavalin A (Con A) and B-cell induced by lipopolysaccharide (LPS). That proved that PSS possesses the significant immunoregulation effect, whilst the immunocompetence comparison of PSS’column fractions proved that the different immunocyte had different requirement for saccharides length. For spleen cell proliferation, the best molecular weight was about 10-20 kDa. For macrophage phagocytic function, the bigger Mw fraction of PSS had the stronger effect.
2.6、In-vitro antibacterial activities of PSS and all fractions were compared and analyzed in the studies of the inhibition effect on Gram-Negative Bacteria (G+) (S. Aureus) and Gram-Positive Bacteria (G-) (E. Coli). The results showed that PSS, Z-PSS:1-3, C-PSS1-4 possessed the favorable antibacterial activity. Z-PSS2-3 and C-PSS4 showed the stronger effect than that of Z-PSS1 and C-PSS1-3, whilst Z-PSS4 and C-PSS lose the effect. This meant that the smaller molecular weight had the stronger antibacterial activities. To some extent, PSS had the molecular weight dependence of antibacterial activity.
2.7、Based on the screen assay of PSS’promotion on insulin secretion in vitro, hypoglycemic effect of C-PSS4 was studied. C-PSS4 showed the possibility of decreasing blood glucose, but the statistical significant difference was not displayed (p>0.05). The effect needs the further investigation.
引文
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