沙葱多糖的分离、纯化和结构鉴定及其生物学活性的研究
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摘要
沙葱(Allium mongolicum Regel)是内蒙古草原生长量大且为羊喜食的牧草。天然植物多糖具有抗菌、抗病毒、增强免疫力和促进动物生长等作用,是确保饲料和动物性食品安全以及人体健康的理想添加剂。论文通过四部分内容研究了沙葱多糖的提取、纯化和结构,结合体内和体外试验对沙葱多糖的生物学活性进行了较为系统的研究,并从分子水平初步探讨了其免疫调节机制。为开发沙葱资源和绿色、安全的动物免疫佐剂或饲料添加剂提供理论和科学依据。
第一部分沙葱多糖的提取、纯化和结构鉴定。
采用热水浸提法提取了沙葱多糖,优化了沙葱多糖活性炭脱色的工艺。研究了活性炭添加量、脱色时间和脱色温度对沙葱多糖脱色效果的影响,通过正交试验确定了沙葱多糖活性炭脱色的最佳工艺参数,即脱色温度80℃,活性炭添加量1.0%,吸附时间40 min,在此条件下,脱色率为93.25%,多糖损失率为18.79%。
对脱色后的沙葱多糖进行SephadexG-100柱层析纯化并测定其分子量,采用紫外光谱、纸层析、比旋光度法和凝胶柱层析法检验纯度;硅胶薄层层析(TLC)、高效液相色谱(HPLC)鉴定单糖组成,红外光谱(IR)和核磁共振(NMR)进行结构解析。结果表明,纯化后的沙葱多糖(SCSP)是一种白色疏松、易溶于水、分子量为5.89×104 D的均一组分中性多糖,是由L-Rha、D-Glc、D-Gal三种吡喃糖构成的杂多糖,糖链的主要连接方式为α-Glc(1→4)、[α-Glc(1→4)-]n和端基α-Rha。
第二部分SCSP的体外抑菌试验。
采用琼脂扩散法比较了沙葱多糖(SCSP)、沙葱鲜汁及黄芪多糖(HQSP)在体外对大肠杆菌、痢疾杆菌、金黄色葡萄球菌、白色葡萄球菌、假单胞绿脓杆菌、变形杆菌的抑菌作用。结果表明,20 mg/mL和40 mg/mL的SCSP对以上6种细菌均有一定的抑制作用,尤其对大肠杆菌和金黄色葡萄球菌的抑菌效果较为明显,最大抑菌圈直径分别为13.3 mm、9.9 mm;HQSP对以上细菌均无任何抑制作用;沙葱鲜汁的抑菌效果更为显著,对6种细菌均有较强的抑制作用,抑菌圈直径均≥8 mm,尤其对肠道细菌痢疾杆菌的抑菌效果最为显著,最大抑菌圈直径为21.2 mm。
第三部分SCSP的抗肿瘤作用。
体外采用MTT法,研究了不同浓度的SCSP对S180、L1210、YAC-1、K562和A549 5种细胞株的抑制作用;通过体内饲养试验研究了SCSP对S180腹水瘤小鼠肿瘤的抑制作用和免疫调节作用。结果表明,SCSP高、中剂量组对S180、YAC-1、K562细胞株均有显著抑制作用(P<0.05),SCSP各剂量组对L1210和A549细胞株无显著抑制作用(P>0.05);高、中、低三个剂量组的SCSP均能够改善S180腹水瘤小鼠的生存状态,提高其生存质量,抑制腹水的形成,延长存活期,此外,还可以提高小鼠的胸腺、脾脏指数及血清IL-2、TNF-α和IFN-γ的含量,尤其以高剂量组的作用效果最为显著(P<0.05)。
第四部分SCSP对正常小鼠的免疫调节作用及机理。
研究饲喂SCSP对正常小鼠各项免疫指标和免疫细胞分泌细胞因子及基因表达的影响。结果表明,1.25 mg/mL的SCSP能显著提高正常小鼠的免疫器官指数(P<0.05),12.5 mg/mL的SCSP可显著提高小鼠血清IgM、IgG的含量(P<0.05);100~200μg/mL的SCSP作用72 h,能显著促进小鼠脾淋巴细胞的增殖(P<0.05),50~150μg/mL的SCSP作用72 h,可显著增强小鼠腹腔巨噬细胞的吞噬功能(P<0.05),50~200μg/mL的SCSP能显著提高小鼠NK细胞对YAC-1的杀伤作用(P<0.05),但SCSP各剂量组对正常小鼠骨髓细胞和胸腺淋巴细胞的增殖无显著影响(P>0.05);100、150μg/mL的SCSP能显著增强ConA对正常小鼠脾淋巴细胞分泌IL-2的诱导作用及LPS对正常小鼠腹腔巨噬细胞分泌TNF-α的诱导作用(P<0.05);经RT-PCR技术分析,这两个浓度的SCSP体外能显著促进小鼠脾淋巴细胞IL-2和腹腔巨噬细胞TNF-αmRNA的表达(P<0.05)。
整体试验证明,提取的沙葱多糖较纯,含量达87.23%。沙葱多糖体外具有较广普的抗菌活性,对S180腹水瘤有明显的抑制作用,能够促进正常小鼠的特异性和非特异性免疫功能,也能通过诱导免疫细胞产生相关细胞因子及促进细胞因子在mRNA水平的表达来调节动物的免疫机能。
Allium mongolicum Regel, a widespread herb on Inner Mongolia grassland, is sheep’s favorite. Natural polysaccharides in plant have such function as antibacteria, antivirus, immune enhancement and promotion of animal growth, so Allium mongolicum Regel polysaccharide is an ideal, and safe cattle food additive. This paper consists of four parts, namely, the methods of extraction, purification and structure identification of Allium mongolicum Regel polysaccharide, the systematical study on the biological activity of it in both vivo and vitro, and the approach to investage its mechanism of immune regulation on molecular level . The purpose of the study is to provide theoretical and experimental data for the development of Allium mongolicum Regel resources, a green and safe animal immune adjuvant or food additive.
PartⅠExtraction, purification and identification of Allium mongolicum Regel polysaccharide.
The Hot-water extraction was used to extract Allium mongolicum Regel polysaccharide, thus Allium mongolicum Regel polysaccharide’s active carbon decoloring process was optimized. The impact of the quantity of active carbon, decoloring time and decoloring temperature on the decoloring effect of Allium mongolicum Regel polysaccharide were studied. The optimal process parameters were determined over orthogonal test: decoloring temperature 80℃, the quantity of active carbon 1.0%, adsorption time 40 min. Under this condition, the decoloring rate was 93.25% and the loss rate of polysaccharide was 18.79%.
Decolored Allium mongolicum Regel polysaccharide was purified with SephadexG-100 column chromatography, and its molecular weight was measured. Its purity was examined by means of UV spectra, paper chromatography, specific rotation and gel column chromatography. Furthermore, monosaccharide composition was identified with silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), and its structure with infra-red spectrum (IR) and nuclear magnetic resonance (NMR). The result shows that purified Allium mongolicum Regel polysaccharide (SCSP) is a homogeneous neutral polysaccharide which is loose, white and easily soluble in water, and with a molecular weight of 5.89×104 D. It is a heteropolysaccharide consisting of three kinds of pyranose—L-Rha, D-Glc and D-Gal. The main connecting ways of the sugar chain areα-Glc(1→4), [α-Glc(1→4)-]n and terminalα-Rha.
PartⅡThe antibacterial effect of SCSP in vitro.
Agar diffusion technique was adopted to compare their antibacterial ability in vitro to E. coli, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus albus, Pseudomonas aeruginosa, Proteus between SCSP, fresh juice of Allium mongolicum Regel and Astragalus polyose (HQSP). The result shows that SCSP had a definite inhibitory effect to all 6 tested bacteria above, especially to E. coli and Staphylococcus aureus, and the diameter of the biggest inhibition zone is 13.3 mm and 9.8 mm respectively; but HQSP did not show any inhibitory effect; whereas inhibitory effect of fresh juice of Allium mongolicum Regel are more remarkable (the diameter of the inhibition zone are all≥8 mm), especially to intestinal flora Shigella dysenteriae, and the diameter of the biggest inhibition zone reached 21.2 mm.
PartⅢThe effect of SCSP on anti-tumor.
MTT method was employed in vitro to explore the inhibitory effect of different concentrations of SCSP on 5 cell strains—S180、L1210、YAC-1、K562 and A549, and the inhibitory effect of SCSP on S180 ascites tumor in mice and its immune regulatory effect were also studied in vivo by feeding test. The result showed that high and medium dosage of SCSP had notable inhibitory effect on all cell strains of S180, YAC-1 and K562 (P<0.05), that all different dosage group of SCSP had no notable inhibitory effect on the cell strains of L1210 and A549 (P>0.05), that high, medium and low dosage of SCSP were able to improve the living condition of mice suffering from S180 ascites tumor, to lift their quality of life, to inhibit the formation of ascites and to extend the survival time of the mice with ascites tumor. Furthermore, SCSP can also enhance the thymus and spleen index and IL-2, TNF-αand IFN-γlevels in serum, in particular, the high dosage group of SCSP was the most effective (P<0.05).
Part IV Immune regulation effect and mechanism of SCSP on normal mice。
The effects of SCSP on immune indexes, cytokines secreted by immune cells and their gene expressions of normal mice were studied over feeding experiment. The result showed that: medium dosage of SCSP could enhance immune indexes of normal mice significantly (P<0.05), and high dosage of SCSP could lift notably the content of IgM、IgG in serum of mice (P<0.05); SCSP could boost remarkably the transformation of lymphocyte on mice at 100~200μg/mL dosage acting 72 h(P<0.05), and the metabolism of mouse peritoneal macrophage could be strengthened evidently by SCSP at 50~150μg/mL dosage acting 72 h (P<0.05), SCSP at each dosage group could significantly increase the cytotoxity of NK cells to YAC-1 on mice (P<0.05), but the effect of SCSP on the proliferation of normal mouse bone marrow cells and thymocytes had no significant difference at each dosage (P>0.05). SCSP could strengthen the induction of ConA to normal mouse spleen lymphocyte IL-2, and the induction of LPS to TNF-αsecretion of normal mouse peritoneal macrophages (P<0.05). Analyzed by RT-PCR technique, SCSP is proved to increase the expression of mouse spleen lymphocyte IL-2 and peritoneal macrophages TNF-αmRNA in vitro.
The whole experiment showed that the content of purified SCSP reached at 87.23%. SCSP had a relatively wide spectrum of antibacterial activity in vitro, and its inhibition to S180 ascites tumor was evident, it could also enhance the specific and nonspecific immunity of normal mice, it was able to regulate animal immune function as well through the induction of immune cells to produce relevant cytokine and the promotion of the expression of cytokine at mRNA level.
第一部分沙葱多糖的提取、纯化和结构鉴定。
采用热水浸提法提取了沙葱多糖,优化了沙葱多糖活性炭脱色的工艺。研究了活性炭添加量、脱色时间和脱色温度对沙葱多糖脱色效果的影响,通过正交试验确定了沙葱多糖活性炭脱色的最佳工艺参数,即脱色温度80℃,活性炭添加量1.0%,吸附时间40 min,在此条件下,脱色率为93.25%,多糖损失率为18.79%。
对脱色后的沙葱多糖进行SephadexG-100柱层析纯化并测定其分子量,采用紫外光谱、纸层析、比旋光度法和凝胶柱层析法检验纯度;硅胶薄层层析(TLC)、高效液相色谱(HPLC)鉴定单糖组成,红外光谱(IR)和核磁共振(NMR)进行结构解析。结果表明,纯化后的沙葱多糖(SCSP)是一种白色疏松、易溶于水、分子量为5.89×104 D的均一组分中性多糖,是由L-Rha、D-Glc、D-Gal三种吡喃糖构成的杂多糖,糖链的主要连接方式为α-Glc(1→4)、[α-Glc(1→4)-]n和端基α-Rha。
第二部分SCSP的体外抑菌试验。
采用琼脂扩散法比较了沙葱多糖(SCSP)、沙葱鲜汁及黄芪多糖(HQSP)在体外对大肠杆菌、痢疾杆菌、金黄色葡萄球菌、白色葡萄球菌、假单胞绿脓杆菌、变形杆菌的抑菌作用。结果表明,20 mg/mL和40 mg/mL的SCSP对以上6种细菌均有一定的抑制作用,尤其对大肠杆菌和金黄色葡萄球菌的抑菌效果较为明显,最大抑菌圈直径分别为13.3 mm、9.9 mm;HQSP对以上细菌均无任何抑制作用;沙葱鲜汁的抑菌效果更为显著,对6种细菌均有较强的抑制作用,抑菌圈直径均≥8 mm,尤其对肠道细菌痢疾杆菌的抑菌效果最为显著,最大抑菌圈直径为21.2 mm。
第三部分SCSP的抗肿瘤作用。
体外采用MTT法,研究了不同浓度的SCSP对S180、L1210、YAC-1、K562和A549 5种细胞株的抑制作用;通过体内饲养试验研究了SCSP对S180腹水瘤小鼠肿瘤的抑制作用和免疫调节作用。结果表明,SCSP高、中剂量组对S180、YAC-1、K562细胞株均有显著抑制作用(P<0.05),SCSP各剂量组对L1210和A549细胞株无显著抑制作用(P>0.05);高、中、低三个剂量组的SCSP均能够改善S180腹水瘤小鼠的生存状态,提高其生存质量,抑制腹水的形成,延长存活期,此外,还可以提高小鼠的胸腺、脾脏指数及血清IL-2、TNF-α和IFN-γ的含量,尤其以高剂量组的作用效果最为显著(P<0.05)。
第四部分SCSP对正常小鼠的免疫调节作用及机理。
研究饲喂SCSP对正常小鼠各项免疫指标和免疫细胞分泌细胞因子及基因表达的影响。结果表明,1.25 mg/mL的SCSP能显著提高正常小鼠的免疫器官指数(P<0.05),12.5 mg/mL的SCSP可显著提高小鼠血清IgM、IgG的含量(P<0.05);100~200μg/mL的SCSP作用72 h,能显著促进小鼠脾淋巴细胞的增殖(P<0.05),50~150μg/mL的SCSP作用72 h,可显著增强小鼠腹腔巨噬细胞的吞噬功能(P<0.05),50~200μg/mL的SCSP能显著提高小鼠NK细胞对YAC-1的杀伤作用(P<0.05),但SCSP各剂量组对正常小鼠骨髓细胞和胸腺淋巴细胞的增殖无显著影响(P>0.05);100、150μg/mL的SCSP能显著增强ConA对正常小鼠脾淋巴细胞分泌IL-2的诱导作用及LPS对正常小鼠腹腔巨噬细胞分泌TNF-α的诱导作用(P<0.05);经RT-PCR技术分析,这两个浓度的SCSP体外能显著促进小鼠脾淋巴细胞IL-2和腹腔巨噬细胞TNF-αmRNA的表达(P<0.05)。
整体试验证明,提取的沙葱多糖较纯,含量达87.23%。沙葱多糖体外具有较广普的抗菌活性,对S180腹水瘤有明显的抑制作用,能够促进正常小鼠的特异性和非特异性免疫功能,也能通过诱导免疫细胞产生相关细胞因子及促进细胞因子在mRNA水平的表达来调节动物的免疫机能。
Allium mongolicum Regel, a widespread herb on Inner Mongolia grassland, is sheep’s favorite. Natural polysaccharides in plant have such function as antibacteria, antivirus, immune enhancement and promotion of animal growth, so Allium mongolicum Regel polysaccharide is an ideal, and safe cattle food additive. This paper consists of four parts, namely, the methods of extraction, purification and structure identification of Allium mongolicum Regel polysaccharide, the systematical study on the biological activity of it in both vivo and vitro, and the approach to investage its mechanism of immune regulation on molecular level . The purpose of the study is to provide theoretical and experimental data for the development of Allium mongolicum Regel resources, a green and safe animal immune adjuvant or food additive.
PartⅠExtraction, purification and identification of Allium mongolicum Regel polysaccharide.
The Hot-water extraction was used to extract Allium mongolicum Regel polysaccharide, thus Allium mongolicum Regel polysaccharide’s active carbon decoloring process was optimized. The impact of the quantity of active carbon, decoloring time and decoloring temperature on the decoloring effect of Allium mongolicum Regel polysaccharide were studied. The optimal process parameters were determined over orthogonal test: decoloring temperature 80℃, the quantity of active carbon 1.0%, adsorption time 40 min. Under this condition, the decoloring rate was 93.25% and the loss rate of polysaccharide was 18.79%.
Decolored Allium mongolicum Regel polysaccharide was purified with SephadexG-100 column chromatography, and its molecular weight was measured. Its purity was examined by means of UV spectra, paper chromatography, specific rotation and gel column chromatography. Furthermore, monosaccharide composition was identified with silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), and its structure with infra-red spectrum (IR) and nuclear magnetic resonance (NMR). The result shows that purified Allium mongolicum Regel polysaccharide (SCSP) is a homogeneous neutral polysaccharide which is loose, white and easily soluble in water, and with a molecular weight of 5.89×104 D. It is a heteropolysaccharide consisting of three kinds of pyranose—L-Rha, D-Glc and D-Gal. The main connecting ways of the sugar chain areα-Glc(1→4), [α-Glc(1→4)-]n and terminalα-Rha.
PartⅡThe antibacterial effect of SCSP in vitro.
Agar diffusion technique was adopted to compare their antibacterial ability in vitro to E. coli, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus albus, Pseudomonas aeruginosa, Proteus between SCSP, fresh juice of Allium mongolicum Regel and Astragalus polyose (HQSP). The result shows that SCSP had a definite inhibitory effect to all 6 tested bacteria above, especially to E. coli and Staphylococcus aureus, and the diameter of the biggest inhibition zone is 13.3 mm and 9.8 mm respectively; but HQSP did not show any inhibitory effect; whereas inhibitory effect of fresh juice of Allium mongolicum Regel are more remarkable (the diameter of the inhibition zone are all≥8 mm), especially to intestinal flora Shigella dysenteriae, and the diameter of the biggest inhibition zone reached 21.2 mm.
PartⅢThe effect of SCSP on anti-tumor.
MTT method was employed in vitro to explore the inhibitory effect of different concentrations of SCSP on 5 cell strains—S180、L1210、YAC-1、K562 and A549, and the inhibitory effect of SCSP on S180 ascites tumor in mice and its immune regulatory effect were also studied in vivo by feeding test. The result showed that high and medium dosage of SCSP had notable inhibitory effect on all cell strains of S180, YAC-1 and K562 (P<0.05), that all different dosage group of SCSP had no notable inhibitory effect on the cell strains of L1210 and A549 (P>0.05), that high, medium and low dosage of SCSP were able to improve the living condition of mice suffering from S180 ascites tumor, to lift their quality of life, to inhibit the formation of ascites and to extend the survival time of the mice with ascites tumor. Furthermore, SCSP can also enhance the thymus and spleen index and IL-2, TNF-αand IFN-γlevels in serum, in particular, the high dosage group of SCSP was the most effective (P<0.05).
Part IV Immune regulation effect and mechanism of SCSP on normal mice。
The effects of SCSP on immune indexes, cytokines secreted by immune cells and their gene expressions of normal mice were studied over feeding experiment. The result showed that: medium dosage of SCSP could enhance immune indexes of normal mice significantly (P<0.05), and high dosage of SCSP could lift notably the content of IgM、IgG in serum of mice (P<0.05); SCSP could boost remarkably the transformation of lymphocyte on mice at 100~200μg/mL dosage acting 72 h(P<0.05), and the metabolism of mouse peritoneal macrophage could be strengthened evidently by SCSP at 50~150μg/mL dosage acting 72 h (P<0.05), SCSP at each dosage group could significantly increase the cytotoxity of NK cells to YAC-1 on mice (P<0.05), but the effect of SCSP on the proliferation of normal mouse bone marrow cells and thymocytes had no significant difference at each dosage (P>0.05). SCSP could strengthen the induction of ConA to normal mouse spleen lymphocyte IL-2, and the induction of LPS to TNF-αsecretion of normal mouse peritoneal macrophages (P<0.05). Analyzed by RT-PCR technique, SCSP is proved to increase the expression of mouse spleen lymphocyte IL-2 and peritoneal macrophages TNF-αmRNA in vitro.
The whole experiment showed that the content of purified SCSP reached at 87.23%. SCSP had a relatively wide spectrum of antibacterial activity in vitro, and its inhibition to S180 ascites tumor was evident, it could also enhance the specific and nonspecific immunity of normal mice, it was able to regulate animal immune function as well through the induction of immune cells to produce relevant cytokine and the promotion of the expression of cytokine at mRNA level.
引文
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