CN106046191B - 一种羊栖菜多糖在制备用于抗炎的片剂、胶囊或功能性食品中的应用 - Google Patents
一种羊栖菜多糖在制备用于抗炎的片剂、胶囊或功能性食品中的应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
本申请公开了一种羊栖菜多糖在制备用于抗炎的片剂、胶囊或功能性食品中的应用，本方法制备的水溶性羊栖菜多糖无细胞毒性，安全可靠，表现出明显的抗炎作用，因此可以成为治疗和预防炎症的功能性食品。且本发明方法操作简单，适宜规模化工业生产。
Description
技术领域
本发明涉及一种用于具有抗炎作用的羊栖菜多糖，还涉及该羊栖菜多糖的制备方法以及该羊栖菜多糖在保健品中的应用。
背景技术
炎症(inflanunation)是具有血管系统的活体组织对损伤因子所产生的防御性反应。它包括致炎因子引起的损伤、机体防御性反应和组织损伤恢复三个方面。适度的炎症反应是机体保护自我的一种免疫反应，过度的炎症会导致机体的免疫损伤，诱发各种病症，如类风湿关节炎、系统性红斑狼疮、自身免疫性溶血性贫血等。患病机体的血液中检测到大量的炎症相关因子(如TNF-α、IL-1β、IL-6、iNOS和COX-2等)，这些因子浸润机体，引发各种不良反应，严重威胁到机体健康。因此预防和辅助治疗炎症显得尤为重要。
在炎症治疗和预防研究中，已经开发出许多抗炎药物，如肾上腺皮质激素类制剂：强的松、氢化可的松、地塞米松等激素类药物。但这类药物容易产生依赖性，并且对过敏性的重度炎症的疗效还不尽人意。而抗炎效果较强的环磷酰胺、氨甲喋呤等药物的毒性较大，在发挥免疫作用的同时，对机体的正常免疫功能也产生较大的伤害。因此，开发有效、安全、依赖性及毒副作用低的抗炎药物显得日益重要。相比之下，生产出具有抗炎作用的功能性食品，长期服用，预防急性炎症，辅助治疗慢性炎症具有更大的市场价值。
在长期的研究中发现，许多多糖类物质作为一种天然产物，在炎症抑制方面表现出良好的效果。如银杏多糖(刘春明等，东北师范大学，2007：1-29)在小鼠体内急性腹腔炎症实验中表现出较强的抑制作用，在体外则能明显抑制中性粒细胞与稳定转染P-选择(P-selectin)的CHO(中国仓鼠卵巢细胞)细胞的粘附。LPS是产生内毒素血症的重要原因，可刺激机体产生TNF-α、IL-6等促炎症细胞因子，后者触发瀑布式炎症级联反应，进一步激活炎症细胞(吴金峰等，中国中西医结合杂志，2009，29(4)：330-334)。灵芝多糖(Md.AbulHasnat，et.Phytochemistry，phytochem.2014.10.019)和来自墨角藻的褐藻多糖硫酸酯(Hye Young Park，et.Food and Chemical Toxicology 49(2011)1745-1752)能显著抑制LPS诱导的炎症异常表达的NO、PGE2、IL-1β、iNOS和COX-2等炎症因子，并且通过抑制IκBα的磷酸化来抑制NF-κB经典免疫调节通路的活化，同时下调ERK、JUK、P38和MAPK等相关通路来达到有效抑制炎症的作用。这些天然产物给我们提供了丰富的资源。
发明内容
本发明公开了一种羊栖菜多糖的制备方法，该方法制备的羊栖菜多糖有较好的抗炎功效，调节机体免疫，保护机体免受免疫反应过激带来的损伤。
本发明还公开了将上述羊栖菜多糖作为一种用于抗炎的片剂、胶囊等功能性食品。
针对羊栖菜多糖本身，一种抗炎作用的羊栖菜多糖的制备方法，其特征在于包括有以下步骤：
(1)干燥粉碎；新鲜羊栖菜放入鼓风干燥箱中，40～60℃干燥48h，干燥藻体剪碎成段，放入粉碎机中研磨至10-40目，得到羊栖菜藻粉；
(2)回流脱脂：按照羊栖菜藻粉∶95％乙醇＝1∶4(m∶v)，回流脱脂3次，每次3h，得到脱脂羊栖菜藻粉；
(3)提取浓缩液：按照脱脂羊栖菜藻粉∶蒸馏水＝1∶30(m∶v)，60～90℃煮提3次，时间依次为4、3、2h，合并3次煮提液，减压浓缩体积至1/10，得到浓缩液；
(4)乙醇沉淀：在浓缩液中加入95％乙醇至乙醇终浓度为80％，室温醇沉过夜，弃上清，离心收集沉淀；
(5)干燥：将得到的沉淀进行溶剂干燥，依次使用95％乙醇、无水乙醇、丙酮洗涤沉淀，得到羊栖菜多糖；
(6)烘干：将上述羊栖菜多糖放入真空干燥箱中，40～60℃减压烘干；
(7)脱色及洗脱：烘干后的羊栖菜多糖经DEAE-纤维素柱脱色，用2mol/L NaCl洗脱，洗脱液浓缩后，冷冻干燥所得样品为SPFs；
(8)SPFs再次经DEAE-琼脂糖凝胶CL-6B，依次经过0.5和1.0mol/L NaCl洗脱分级，得到的第一个峰和第二个峰分别命名为F1和F2；
(9)F2再用丙烯葡聚糖凝胶S-400HR凝胶柱按分子量大小进行分级，得到两个级分，截取峰型均一且含量较多的峰二，透析冻干后得产物羊栖菜多糖SFPF-2，产率3％-1.5％。
将上述羊栖菜多糖作为原料，将上述制备得到羊栖菜多糖作为原料，按照药学领域的常规生产方法，制备用于抗炎的片剂、胶囊或功能性食品。
本方法制备的水溶性羊栖菜多糖无细胞毒性，安全可靠，表现出明显的抗炎作用，因此可以成为治疗和预防炎症的功能性食品。且本发明方法操作简单，适宜规模化工业生产。
附图说明
图1 SFPF-2对RAW264.7细胞活性的影响；
图2不同浓度SFPF-2对RAW264.7巨噬细胞吞噬能力的影响；
图3-1、3-2、3-3分别为不同浓度SFPF-2对LPS诱导下RAW264.7细胞因子分泌量的影响；
图4 SFPF-2对iNOS mRNA转录水平的影响；
图5-1、5-2、5-3、5-4及图5-5分别为SFPF-2对NF-κB免疫调节信号通路的相关蛋白的影响及灰度分析。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
一种羊栖菜多糖的制备方法，将清洗干净的新鲜羊栖菜放入烘箱中，40～60℃烘烤48h，剪碎成段，放入粉碎机中研磨1～2min，得到羊栖菜藻粉。称量500g羊栖菜藻粉，按照羊栖菜藻粉∶95％乙醇＝1∶4(m∶v)，60～80℃回流脱脂3次，时间依次为4、3、2h，，得到脱脂羊栖菜藻粉。按照脱脂羊栖菜藻粉∶蒸馏水＝1∶30(m∶v)，80℃煮提3次，时间依次为4h，3h，2h，合并3次煮提液，减压浓缩体积至1/12。浓缩液中加入乙醇至终浓度为80％乙醇，室温醇沉过夜，弃上清，离心收集沉淀。将得到的沉淀常规干燥，依次使用95％乙醇、无水乙醇、丙酮洗涤沉淀，得到羊栖菜多糖。将上述羊栖菜多糖放入真空干燥箱中，50℃减压烘干，得到奶黄色粉末，羊栖菜多糖SFPs得率为18～25％，经测定SFPs(羊栖菜多糖)成分含量为75～85％。再次经DEAE-Sephaose CL-6B，经过0.5和1.0mol/L NaCl分级，，得到级分F1和F2。F2经Sephacryl S-400HR凝胶柱，按分子量大小进行分级，得到两个级分，截取峰型均一且含量较多的峰，透析冻干后得最终的产物SFPF-2。该多糖主要由褐藻多糖硫酸酯组成，其单糖组成为甘露糖、半乳糖醛酸、半乳糖、木糖和岩藻糖(2.4∶0.7∶13.3∶3.1∶80.6)等构成，其分子量为2.4×104Da。其得率为1.76％。
按本发明方法制得的羊栖菜多糖(SFPF-2)进行抗炎活性实验，结果如下：
SFPF-2对RAW264.7细胞毒性实验结果，参考图1；
SFPF-2对细胞吞噬能力的影响，参考图2；
SFPF-2减少LPS诱导下细胞因子的表达量，参考图3；
SFPF-2对LPS诱导下免疫因子反转录水平的影响，参考图4；
SFPF-2对NF-κB免疫调节信号通路的相关蛋白的影响，参考图5；
上述实验结果显示SFPF-2对RAW264.7巨噬细胞无细胞毒性。在LPS诱导构造的炎症细胞模型中，与LPS组比较，细胞的吞噬能力显著降低，趋向于control组。在ELISA检测试验中，与LPS组相比，多糖共处理组中TNF-α、IL-1β和IL-6的分泌量明显下降，其中TNF-α和IL-1β的分泌量随着多糖浓度的增加，分泌量随之减少。iNOS——诱导型一氧化氮合成酶，是机体NOS活性氮自由基产生的催化酶。其含量的高低可做为一种机体内炎症程度的指标。与LPS组相比，SFPF-2处理组的iNOS在mRNA转录水平明显降低。在WB相关蛋白检测实验中，多糖处理组中总P-65和IκBα明显呈现浓度负相关作用，同时P-65和IκBα蛋白的磷酸化也受到明显抑制。NF-κB信号通路的激活明显受到了抑制，从而缓解了LPS诱导的炎症反应带来的损伤。
综上所述，本方法制备的水溶性羊栖菜多糖SFPF-2无细胞毒性，安全可靠，SFPF-2表现出明显的抗炎作用，因此可以成为治疗和预防炎症的功能性食品。且本发明方法操作简单，适宜规模化工业生产。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，上述假设的这些改进和变型同应视为本发明的保护范围。
Claims (1)
1.一种羊栖菜多糖在制备抗炎作用的片剂、胶囊或功能性食品中的应用，其特征在于包括有以下步骤：
(1)干燥粉碎；新鲜羊栖菜放入鼓风干燥箱中，40～60℃干燥48h，干燥藻体剪碎成段，放入粉碎机中研磨至10-40目，得到羊栖菜藻粉；
(2)回流脱脂：按照羊栖菜藻粉∶95％乙醇＝1∶4(m∶v)，回流脱脂3次，每次3h，得到脱脂羊栖菜藻粉；
(3)提取浓缩液：按照脱脂羊栖菜藻粉∶蒸馏水＝1∶30(m∶v)，60～90℃煮提3次，时间依次为4、3、2h，合并3次煮提液，减压浓缩体积至1/10，得到浓缩液；
(4)乙醇沉淀：在浓缩液中加入95％乙醇至乙醇终浓度为80％，室温醇沉过夜，弃上清，离心收集沉淀；
(5)干燥：将得到的沉淀进行溶剂干燥，依次使用95％乙醇、无水乙醇、丙酮洗涤沉淀，得到羊栖菜多糖；
(6)烘干：将上述羊栖菜多糖放入真空干燥箱中，40～60℃减压烘干；
(7)脱色及洗脱：烘干后的羊栖菜多糖经DEAE-纤维素柱脱色，用2mol/L NaCl洗脱，洗脱液浓缩后，冷冻干燥所得样品为SPFs；
(8)SPFs再次经DEAE-琼脂糖凝胶CL-6B，依次经过0.5和1.0mo l/L NaCl洗脱分级，得到的第一个峰和第二个峰分别命名为F1和F2；
(9)F2再用丙烯葡聚糖凝胶S-400HR凝胶柱按分子量大小进行分级，得到两个级分，截取峰型均一且含量较多的峰二，透析冻干后得产物羊栖菜多糖SFPF-2，产率3％-1.5％；
(10)将步骤9中制备得到羊栖菜多糖作为原料，按照药学领域的常规生产方法，制备用于抗炎的片剂、胶囊或功能性食品。
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