CN111514160A - 五味子多糖在制备治疗炎症性肠病药物或保健品中的应用 - Google Patents
五味子多糖在制备治疗炎症性肠病药物或保健品中的应用 Download PDFInfo
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- CN111514160A CN111514160A CN202010428961.8A CN202010428961A CN111514160A CN 111514160 A CN111514160 A CN 111514160A CN 202010428961 A CN202010428961 A CN 202010428961A CN 111514160 A CN111514160 A CN 111514160A
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- polysaccharide
- schisandra chinensis
- water
- schisandra
- fructus schisandrae
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Abstract
本发明涉及五味子总多糖在制备治疗炎症性肠病的药物或保健品中的新用途，具体如在制备治疗溃疡性结肠炎(UC)、克罗恩病(CD)及急慢性肠道疾病的药物或保健品中的新用途。实验表明五味子总多糖能显著改善结肠的炎性损伤，降低脾脏指数，升高胸腺指数，并有效降低结肠中MPO、NO、MDA、ROS的含量(活性)，同时升高GSH和SOD的含量。同时，五味子多糖调节了结肠炎小鼠肠道微生物组成和多样性，显著提高了厚壁菌门、糖菌门、Enterorhabdus等的相对丰度，降低了Firmicutes、Turicibacter、Akkermansia等肠道菌的相对丰度，并且显著升高了结肠中丙酸、丁酸和异丁酸含量，提示五味子总多糖可用于制备治疗或改善炎症性肠病的药物。
Description
技术领域
本发明属于生物制药及分子生物学技术领域，具体涉及一种五味子总多糖提取物及其在改善炎症性肠病的应用。
背景技术
炎症性肠病(inflammatory bowel disease,IBD)主要包括炎症性肠病(ulcerative colitis,UC)和克罗恩病(Crohn's disease,CD)两种，是以肠道炎症和上皮损伤为病理特征的慢性复发性疾病，难以根治，对人体健康构成严重威胁。近年来IBD发病率呈逐年上升趋势，逐渐演变为全球性疾病。此外，流行病学资料显示，慢性结肠炎的持续时间和严重程度是诱发结肠炎相关性结直肠癌的重要危险因素。炎症性肠病的主要病理机制尚不十分明确，其发病原因是多方面的，如遗传因素，环境因素，这些因素都可能导致肠道先天和适应性黏膜免疫反应。近年，肠道菌群失衡被认为是IBD的主要病理机制。
近年来，大量研究表明摄入膳食纤维(dietary fiber,DF)可以影响肠道微生物的组成。DF为肠道微生物菌群的细菌活性提供了重要能量来源，可以直接或间接影响肠道粘膜免疫应答。肠道微生物在维持结肠稳态和局部以及全身免疫中也发挥至关重要的作用，可以简单分为“有益”细菌和“有害”细菌。“有益”细菌能够抑制“有害”细菌的生长，刺激免疫系统产生对机体有利的免疫、诱导产生短链脂肪酸(SCFA)、帮助消化和吸收营养物质并合成维生素；“有害”细菌诱导蛋白质纯化、抑制SCFA产生并促进促炎症因子和有害物质的产生。有研究表明，紧密粘附在肠上皮上的分段丝状杆菌能诱导Th17反应，并增加结肠中Treg细胞数量，同时单一定植的脆弱拟杆菌可促进Treg细胞增殖并诱导IL-10产生，两者共同作用抑制化学诱导的结肠炎。这表明微生物菌群正在驱动宿主免疫反应并减弱疾病易感性。
研究表明复合碳水化合物会增加有益菌双歧杆菌的水平，并能调节肠道微环境，降低肠道pH值，刺激肠道中巨噬细胞和淋巴细胞，对抗炎症性疾病。肠道菌群与生化因子的相关分析表明，丁酸菌、螺旋菌、乳酸菌和双歧杆菌等保护性细菌相对丰度与IL-10等抗炎症细胞因子呈显著正相关；而普雷沃氏菌(Prevotella)，瘤胃球菌(Ruminococcus)，拟杆菌属(Bacteroides)，埃希氏菌属(Escherichia-Shigella)相对丰度与IL-23，TNF-α，IL-1β，IL-6和IFN-γ等促炎症细胞因子呈正相关。有研究人员利用从猴头菌中提取的粗多糖干预DSS诱导的小鼠结肠炎模型，发现粗多糖能够提高结肠炎小鼠细菌群落α-多样性指数，一定程度上抑制DSS诱导的结肠炎中细菌生长抑制和受损；并且粗多糖降低了变形菌数量，有效缓解DSS诱导的肠道菌群紊乱，表明猴头菌中的粗多糖能够减少致病菌，对稳定并改善肠道微生态环境有积极作用，为炎症性疾病提供良好的预防和治疗作用。据研究发现灵芝菌丝体中提取的高分子量(＞300KDa)多糖能显著降低小鼠中Escherichia fergusonii，肠球菌等菌群的丰度，表明多糖类物质能降低肠道微生物有害菌群比例，减少有害菌群对结肠黏液屏障的损伤。
此外，微生物发酵多糖类物质产生的短链脂肪酸(short chain fatty acid,SCFA)，例如乙酸盐、丙酸盐和丁酸盐，有助于调节肠内稳态并可能通过表观遗传调控基因表达，降低人脂肪组织中促炎症因子的产生。球形梭菌(Clostridium Coccoides)是SCFA的主要生产者，并能发酵多糖产生丁酸盐作为结肠细胞主要能量来源，已被证明可以保护结肠免受炎症损伤。有研究发现淀粉包埋的微球(含多糖类物质)在IBD患者的粪便微生物体外发酵的过程中能调节SCFA含量，减少潜在有害菌的丰度，如Bacteroides vulgatus和Veillonella等，其具体机制可能包括：淀粉包埋的微球显示出缓慢发酵的特征，有利于增加结肠远端中有益发酵产物并改善结肠健康；产生SCFA有助于维持相对低的结肠pH值，防止潜在有害菌生长并降低葡萄糖醛酸酶、糖苷酶和7α-羟化酶等共致癌酶的活性；产生丁酸盐作为结肠上皮细胞的主要能量来源；作为抗炎剂抑制转录因子NF-κB的激活抑制免疫炎症反应和下游产物如促炎细胞因子IL-12和TNF；并上调抗炎细胞因子IL-10产生量。有学者在研究桑葚多糖体外发酵对肠道微生物群组成及代谢产物影响时发现，随着发酵时间的延长，pH降低，总SCFA产量显著增加，并且桑葚多糖中的半乳糖和半乳糖醛酸发酵导致乙酸和丁酸产量增加，阿拉伯糖和葡萄糖发酵导致丙酸产量增加。这些研究表明多糖类物质能够影响肠道微生物及其代谢产物。因此，调控肠道菌群组成或利用肠道菌群发酵多糖类物质的代谢产物可有效预防IBD及防止IBD患者在疾病发作期间肠道炎症症状。五味子为药食两用中药，可作为食品或营养补充剂使用，表明其与化学药物相比具有较高的安全性，适合长期服用。因此，将其开发成为改善炎症性肠病(克罗恩病，炎症性肠病等)的药物，具有较好的应用前景。
发明内容
本发明目的是针对现有改善炎症性肠病药物多副作用且疗效不显著的问题，提供了一种五味子多糖提取物及其在改善炎症性肠病中的应用。具体为应用提取物影响肠道菌群结构和功能，进而提高机体免疫力，为其在制备改善结肠炎药物中的应用前景提供依据，具体阐述如下：
本发明提供五味子多糖在制备治疗炎症性肠病药物或保健品中的应用。
所述炎症性肠病为急性或慢性炎症性肠病。
所述五味子多糖提取自北五味子的生品或醋炮制品，植物来源为木兰科植物五味子Schisandra chinensis(Turcz.)Baill.的干燥果实。
作为进一步改进，所述五味子多糖的制备过程包括以下步骤：五味子饮片水提，醇沉，脱蛋白，脱色除杂，冻干，得五味子多糖提取物。
具体制备过程如下：
1)水提五味子，收集液相部分；
2)将步骤1)中的液相部分或其浓缩液进行醇沉；
3)步骤2)得到的沉淀，用水溶解后采用Sevag法去除蛋白质，收集上清液；
4)取步骤3)得到的上清液，转移到装有AB-8大孔树脂的层析柱中进行脱色除杂，收集洗脱液脱色除杂；
5)取步骤4)脱色得到的五味子多糖溶液，冻干，得精制五味子多糖提取物。
五味子多糖制备的优化方案为：
1)提取：加入饮片重量8倍量的纯水于100℃条件下加热回流，提取2次，每次3小时，合并水提液；
2)醇沉：浓缩步骤1)中水提取液，加入无水乙醇，使含醇量达80％，静置过夜，离心得上清滤液和沉淀，沉淀即为1次醇沉五味子粗多糖提取物；将第一次醇沉的五味子多糖加适量水溶解(使其浓度达到以生药量计约为0.5g/ml)，继续加入无水乙醇，使含醇量达80％，静置过夜，离心得上清滤液和沉淀，沉淀即为2次醇沉五味子粗多糖；
3)Savage法脱蛋白：将步骤2)中五味子粗多糖加纯水溶解，因氯仿中蛋白质可变形析出，将氯仿、正丁醇、多糖溶液按25:5:1的体积配比加入分液漏斗，剧烈振摇使混合均匀，且蛋白质变性成凝胶状析出，静置使沉淀分层，最上层水溶液为多糖，变性蛋白存在于水相与溶剂相的交界面上，最下层为溶剂相。离心，分离变性蛋白，取多糖水溶液，重复5次；
4)AB-8大孔树脂脱色除杂：A.大孔树脂预处理：大孔树脂经超纯水浸泡后，纯水冲去细碎的树脂颗粒，无水乙醇多次浸泡洗涤，加入超纯水未浑浊，超纯水再反复洗涤，备用；B.脱色除杂：将步骤3)中脱蛋白之后的多糖溶液进行吸附脱色，上柱流速为1.0mL/min，洗脱浓度为75％乙醇，洗脱体积为3倍柱体积；
5)冻干：将步骤4)脱色除杂后的多糖溶液冻干，得到精制后的五味子多糖提取物。
作为进一步改进，所述五味子多糖的制备过程还包括在水提之前的石油醚脱脂步骤。具体如下：称取五味子干燥样品，三倍量石油醚回流4小时脱脂，弃去石油醚并挥干至无石油醚味。
作为五味子多糖提取物的质量控制，五味子多糖提取物中总多糖的含量控制在80～95％，即每1克五味子多糖提取物中总多糖的含量为800-950毫克。
优化方案为五味子多糖提取物中总多糖的含量控制在90±5％，即每1克五味子多糖提取物中总多糖的含量为900±50毫克。
有益效果
本发明所提供的五味子总多糖提取物能够改善炎症性肠病；经研究发现，该提取物作用的靶位点为肠道微生物，通过在体影响肠道菌群的结构、功能及代谢产物，影响机体免疫，抑制炎症反应，从而达到改善结肠炎的目的。提取物可制备成一种改善炎症性肠病的药物尤其是口服药物。我们将具有抗炎，增强免疫的五味子多糖进行研究开发，发现其改善炎症性肠病的新用途及新机制。
附图说明
以下将结合附图对本发明作进一步说明：
图1表示小鼠结肠组织病理切片图(200╳)，A：空白对照组，B：模型组，C：阳性药组，D：五味子多糖低剂量组，E：五味子多糖高剂量组，F：病理学评分
图2表示提取物对结肠炎小鼠肠道微生物结构的影响(n＝6)，肠道微生物群的组成。(A)门水平，(B)属水平，(C)门水平热图分析，(D)属水平热图分析，(E)门水平菌群差异，(F)属水平菌群差异。
图3表示提取物对结肠炎小鼠粪便中短链脂肪酸(SCFAs)含量的影响(X±SD，n＝6)，*0.01<p≤0.05，**0.001<p≤0.01，**p≤0.001。
具体实施方式
下面结合具体实施例进一步说明本发明的技术解决方案，不是对本技术方案的限制。
本发明中的生物材料来源如下：
五味子饮片购于江西省南城县建洪中药饮片厂，产地：辽宁，批号：1804126。
实施例1一种能改善炎症性肠病的提取物的制备方法
称取生醋五味子干燥样品各400g，三倍量(1200mL)石油醚回流4小时脱脂，脱脂后除去石油醚。再加入饮片重量8倍量的纯水(3200mL)于100℃条件下，加热回流，提取2次，每次3小时，合并提取液。浓缩水提取液至200mL，加入无水乙醇，使含醇量达到80％，静置过夜，离心得上清滤液和沉淀，沉淀即为五味子粗多糖。
Savage法脱蛋白：将五味子粗多糖加纯水溶解至400mL，因氯仿中蛋白质可变形析出，将氯仿及正丁醇以多糖溶液:氯仿:正丁醇＝25:5:1的比例加入1000mL分液漏斗，剧烈振摇使混合均匀，且蛋白质变性成凝胶状析出，静置使沉淀分层，存在于水相与溶剂相的交界面上，离心，分离变性蛋白，取多糖水溶液，重复5次。
AB-8大孔树脂脱色除杂：大孔树脂的预处理超纯水浸泡后，纯水冲去细碎的树脂颗粒，无水乙醇多次浸泡洗涤，加入超纯水未浑浊，超纯水再反复洗涤，备用。将脱蛋白之后的多糖溶液进行吸附脱色。上柱流速为1.0mL/min，用纯水洗脱，洗脱体积约为3倍柱体积。洗脱后的多糖溶液冻干，得到五味子精制多糖。
实施例2五味子多糖提取物中总多糖含量测定
取实施例1中精制得到的五味子多糖冻干粉1mg，加蒸馏水定容至10mL，即样品溶液浓度0.1mg/mL。以葡萄糖为对照，采用苯酚-硫酸法和紫外分光光度计法于490nm处测定总多糖含量。经计算，精制得到的五味子多糖提取物中总多糖含量为85～95％，如表1所示。
表1各批次五味子多糖提取物样品中的多糖含量(X±SD，n＝3)
实施例3五味子多糖提取物对结肠炎小鼠作用的研究
用实施例1中提取方法获得五味子多糖提取物，配制得不同浓度的提取物溶液2份(即高、低剂量)，低剂量：0.2g/mL，高剂量：0.4g/mL。
C57BL6小鼠，体重20.0+2.0g。所有动物饲养条件均为(SPF)级，适应性饲养一周。利用灭过菌的蒸馏水将DSS配成2％DSS和3.5％DSS水溶液，替代饮用水，让实验C57BL6小鼠自由饮用诱导结肠炎。第1-3天给予2％DSS水溶液,第4-7天换上3.5％DSS水溶液。
实验用小鼠适应性饲养一周，随机分为8组，正常组(Normal):饮用不含DSS的饮用水21天。模型组(DSS):小鼠通过饮用含DSS的饮用水7天获得急性结肠炎，并在第8天开始饮用低浓度DSS(2％)的饮用水，共14天。DSS+SASP组:小鼠通过饮用含DSS的饮用水，7天获得急性结肠炎，并在第8天开始经口灌胃SASP(200mg/kg)，连续14天。DSS+SCP高剂量组：小鼠通过饮用含DSS的饮用水，7天获得急性结肠炎，并在第8天开始经口灌胃SCP高剂量(8g/kg)，连续14天。DSS+SCP低剂量：小鼠通过饮用含DSS的饮用水，7天获得急性结肠炎，并在第8天开始经口灌胃SCP低剂量(4g/kg)，连续14天。以上药物干预每天一次，每组实验小鼠6只，第22天将所有实验动物眼眶采血后麻醉，采取结肠组织、肠道内容物等生物样本，以备后续研究。
取结肠福尔马林固定，做病理切片，并做苏木素-伊红(HE)染色。结果显示(图1)：正常组小鼠，粘膜上皮排列整齐，隐窝腺体明显完整，杯状细胞丰富，无炎性细胞浸润；DSS模型组小鼠，粘膜上皮出现较大溃瘍，隐窝结构局部消失或变形，杯状细胞丢失，大量炎性细胞浸润。造模期间给予SCP各个剂量，小鼠结肠组织损伤程度显著改善，说明SCP对炎症的发生有一定的缓解和保护作用。
血清生化指标检测实验结果显示，模型组与正常对照组相比较，MPO、ROS、MDA及NO水平显著升高，表明造模成功。与模型组比较，提取物在给药14天后，血清中MPO、ROS、MDA及NO水平显著降低，SOD、GSH水平显著升高(见表2)。
表2提取物对结肠炎小鼠MPO、ROS、MDA、SOD、GSH及NO的影响(X±SD，n＝6)
DSS vs NC,*P<0.05，**P<0.01，***P<0.001；SASP,L-SCP or H-SCP vs DSS,#P<0.05，##P<0.01，###P<0.001；L-SCP vs H-SCP,&P<0.05，&&P<0.01,&&&P<0.001
实施例4五味子多糖提取物对结肠炎小鼠肠道菌群结构影响
基于细菌16S V3-V4可变区，利用Illumina/Miseq二代测序技术平台进行高通量测序技术及生物信息学分析方法，研究五味子多糖对DSS诱导结肠炎小鼠肠道菌群结构的影响(见图2)。结果表明，与正常组相比，模型组大鼠肠道菌群组成发生明显变化，其中放线菌门、糖菌门、另枝菌属、肠杆菌属及假单胞菌属物种丰度显著降低，而疣菌门、绿脓杆菌属、梭杆菌属、阿克曼菌属、Faecalibaculum及Romboutsia物种丰度均显著升高；而经五味子多糖的给药干预明显改善了这种趋势，绝大多数菌群的丰度都回归正常水平，基本维持了肠道菌群的生态平衡。此外，我们对五味子多糖高、低剂量给药干预的效果进行比较，结果发现：高、低剂量五味子多糖组对物种多样性、物种丰度等的影响无显著性差异。
实施例5五味子多糖提取物对结肠炎小鼠短链脂肪酸含量的影响
采用GC-MS分析方法，测定了结肠炎小鼠肠道内容物中乙酸(acetic acid)、丙酸(propionic acid)、丁酸(butyric acid)等6种短链脂肪酸(SCFAs)的含量变化(图3)，结果表明，模型组小鼠的丙酸、丁酸、异丁酸(isobutyric acid)和戊酸(valeric acid)的含量均显著下调。而经不同剂量五味子多糖干预后，均在不同程度上上调了结肠炎小鼠肠道内SCFAs的含量，其中L-SCP显著上调了乙酸、丙酸、异丁酸、丁酸和戊酸的含量，而H-SCP对6种SCFAs均呈现显著上调，且效果较L-SCP更佳；L-SCP和H-SCP组相比，丙酸、丁酸、异丁酸、丁酸、戊酸和异戊酸(isovaleric acid)上调趋势呈显著性差异。该结果提示：五味子多糖可能通过改善结肠炎大鼠肠道菌群的失衡，恢复肠道菌群的代谢功能，増强肠道菌群产生SCFAs的能力，从而缓解和改善结肠炎的症状。
Claims (10)
1.五味子多糖在制备治疗炎症性肠病药物或保健品中的应用。
2.如权利要求1所述的应用，其特征在于：所述炎症性肠病为急性或慢性炎症性肠病。
3.如权利要求1或2所述的应用，其特征在于：所述五味子多糖提取自北五味子的生品或醋炮制品，植物来源为木兰科植物五味子Schisandra chinensis
(Turcz.)Baill.的干燥果实。
4.如权利要求3所述的应用，其特征在于：所述五味子多糖的制备过程包括以下步骤：五味子饮片水提，醇沉，脱蛋白，脱色除杂，冻干，得五味子多糖提取物。
5.如权利要求3所述的应用，其特征在于：所述五味子多糖的制备过程包括以下步骤：
1)水提五味子，收集液相部分；
2)将步骤1)中的液相部分或其浓缩液进行醇沉；
3)步骤2)得到的沉淀，用水溶解后采用Sevag法去除蛋白质，收集上清液；
4)取步骤3)得到的上清液，转移到装有AB-8大孔树脂的层析柱中进行脱色除杂，收集洗脱液脱色除杂；
5)取步骤4)脱色得到的五味子多糖溶液，冻干，得精制五味子多糖提取物。
6.如权利要求4或5所述的应用，其特征在于所述五味子多糖的制备过程还包括在水提之前的石油醚脱脂步骤。
7.如权利要求3所述的应用，其特征在于：所述五味子多糖的制备过程包括以下步骤：
1)提取：加入饮片重量8倍量的纯水于100℃条件下加热回流，提取2次，每次3小时，合并水提液；
2)醇沉：浓缩步骤1)中水提取液，加入无水乙醇，使含醇量达80％，静置过夜，离心得上清滤液和沉淀，沉淀即为1次醇沉五味子粗多糖提取物；将第一次醇沉的五味子多糖加适量水溶解(使其浓度达到以生药量计约为0.5g/ml)，继续加入无水乙醇，使含醇量达80％，静置过夜，离心得上清滤液和沉淀，沉淀即为2次醇沉五味子粗多糖；
3)Savage法脱蛋白：将步骤2)中五味子粗多糖加纯水溶解，因氯仿中蛋白质可变形析出，将氯仿、正丁醇、多糖溶液按25:5:1的体积配比加入分液漏斗，剧烈振摇使混合均匀，且蛋白质变性成凝胶状析出，静置使沉淀分层，最上层水溶液为多糖，变性蛋白存在于水相与溶剂相的交界面上，最下层为溶剂相。离心，分离变性蛋白，取多糖水溶液，重复5次；
4)AB-8大孔树脂脱色除杂：A.大孔树脂预处理：大孔树脂经超纯水浸泡后，纯水冲去细碎的树脂颗粒，无水乙醇多次浸泡洗涤，加入超纯水未浑浊，超纯水再反复洗涤，备用；B.脱色除杂：将步骤3)中脱蛋白之后的多糖溶液进行吸附脱色，上柱流速为1.0mL/min，洗脱浓度为75％乙醇，洗脱体积为3倍柱体积；
5)冻干：将步骤4)脱色除杂后的多糖溶液冻干，得到精制后的五味子多糖提取物。
8.如权利要求7所述的应用，其特征在于所述五味子多糖的制备过程还包括在水提之前的石油醚脱脂步骤，具体操作为：称取五味子干燥样品，三倍量石油醚回流4小时脱脂，弃去石油醚并挥干至无石油醚味。
9.如权利要求1～8中任一项所述的应用，其特征在于：所述五味子多糖提取物中总多糖的含量控制在80～95％，即每1克五味子多糖提取物中总多糖的含量为800-950毫克。
10.如权利要求9中所述的应用，其特征在于：所述五味子多糖提取物中总多糖的含量控制在90±5％，即每1克五味子多糖提取物中总多糖的含量为900±50毫克。
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