CN108553480A - 麦冬多糖提取物在制备具有抗炎作用的药物中的应用 - Google Patents
麦冬多糖提取物在制备具有抗炎作用的药物中的应用 Download PDFInfo
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Abstract
本发明公开了麦冬多糖提取物(峰尖分子量在3000‑6500Da范围内)在制备具有抗炎功能的药物或保健食品中的应用。本发明利用腹型肥胖小鼠模型考察麦冬多糖提取物抑制小鼠血清内炎性因子分泌及组织炎性因子mRNA表达水平的实验，评价麦冬多糖提取物的抗炎作用，实施例及其动物结果证实麦冬多糖提取物具有显著的抗炎作用，因此，麦冬多糖提取物可用于制备具有抗炎作用的药物或保健食品。
Description
技术领域
本发明涉及医药保健品领域，特别是涉及一种麦冬多糖提取物的医药用途。
背景技术
炎症性疾病或有炎症性机制参与的疾病涵盖了人体多个***器官，包括呼吸***、心血管***、神经***、消化***和泌尿***等，部分慢性炎症还容易诱发形成恶性肿瘤。加强药物治疗炎症疾病的开发具有重要的意义，有利于扩展临床治疗方案的选择范围。
细胞因子是一类重要的炎症介质，根据其对炎症反应的影响与作用分为2类，及促进炎症反应的致炎因子，如肿瘤坏死因子-α(TNF-α)和白细胞介素(IL-1β、IL-2、IL-6、IL8、IL-12、IL-17等)，其中TNF-α、IL-1、IL-6是介导炎症的主要细胞因子；减轻或抑制炎症反应的抗炎因子(如IL-4、IL-10等)。
养阴类中药具有生津润燥、滋阴养液等功效，以治疗阴虚证为主，它们的毒性一般较小，作用比较温和，以滋补作用为主，对提高人体免疫力，防病治病具有重要的意义。随着人们对多糖药理作用的认识，多糖的研究也愈来愈受到重视，具有养阴作用的中药常含有大量的多糖，它们的生理活性作用分别体现在：免疫调节、抗肿瘤、降血糖、降血脂、抗氧化、抗衰老、保肝、抗病毒、抗疲劳作用等各方面。
现有技术中尚无分子量3000-6500Da范围内的麦冬多糖提取物具有抗炎用途的文献报道。
发明内容
本发明的目的旨在提供一种麦冬多糖提取物在制备具有抗炎作用的药物或保健食品中的应用。
本发明所述的麦冬多糖提取物的峰尖分子量在3000-6500Da范围内，且是从麦冬总多糖中分离获得的，其分离方法包括如下步骤：
1)麦冬总多糖的提取
取干燥麦冬块根，用5-6倍重量70％-95％乙醇回流3次，弃之，药渣水提2-3次，水提液减压浓缩至0.5-1g/ml，加入75％-95％乙醇至含醇量达60％-80％，静置过夜，倾去上清液，沉淀重复醇沉1次，干燥，得淡黄色麦冬总多糖粉末；
2)麦冬多糖的纯化：
取麦冬总多糖，加水溶解，离心去沉淀；上清液加入1-2倍体积70％-95％乙醇沉淀，静置过夜，去除沉淀；上清液再加入1-2倍体积70％-95％乙醇沉淀，静置过夜，收集所得沉淀，即为峰尖分子量在5000-6500Da范围的麦冬多糖提取物；剩余上清液再加入4-5倍体积70％-95％乙醇沉淀，静置过夜，收集所得的沉淀，即为峰尖分子量在3000-5000Da范围的麦冬多糖提取物。
本发明利用腹型肥胖小鼠模型考察本发明的麦冬多糖提取物抑制小鼠炎性因子增加的实验，评价麦冬多糖提取物的抗炎作用。实验表明，口服麦冬多糖提取物12周后，可显著下调小鼠结肠部位IL-1、IL-6、IL-17A、TNF-α等致炎因子，显著上调抑炎因子IL-10的mRNA表达水平。同时，对肝组织开展qPCR分析，也发现口服麦冬多糖提取物可以抑制IL-1、IL-17A、TNF-α，并提升IL-10的mRNA表达水平。血清炎性因子含量测定进一步证实，口服麦冬多糖提取物可以抑制IL-1，并提高IL-10的分泌水平。这些实验结果提示：本发明麦冬多糖提取物具有显著的抗炎作用，可用于制备具有抗炎作用的药物或保健食品。
作为具有抗炎作用的药物或保健食品，本发明的麦冬多糖提取物可制成各种形式的口服制剂包括片剂、胶囊剂、颗粒剂或口服液等。所述的口服制剂优选由重量百分比为5％-60％的麦冬多糖提取物和95％-40％的药用辅料或附加剂组成。
本发明各个方面的细节将在随后的章节中得以详尽描述。通过下文以及权利要求的描述，本发明的特点、目的和优势将更为明显。
附图说明
图1是本发明各组动物结肠炎性因子mRNA表达水平示意图；
图2是本发明各组动物肝脏炎性因子mRNA表达水平示意图。
具体实施方式
以下通过实施例对本发明作进一步的阐述
实施例1麦冬多糖提取物(峰尖分子量5000-6500Da)的制备
1)麦冬总多糖的提取：
取干燥麦冬块根，用5-6倍重量70％-95％乙醇回流3次，弃之；药渣水提2-3次。水提液减压浓缩至0.5-1g/ml，加入75％-95％乙醇至含醇量达60％-80％，静置过夜，倾去上清液，沉淀重复醇沉1次，冷冻干燥，得淡黄色麦冬总多糖粉末；
2)麦冬多糖的纯化：
取麦冬总多糖，加水溶解，离心去沉淀；上清液加入1-2倍体积70％-95％乙醇沉淀，静置过夜，去除沉淀。上清液再加入1-2倍体积70％-95％乙醇沉淀，静置过夜，收集所得沉淀，即为峰尖分子量在5000-6500Da范围的麦冬多糖提取物。
实施例2麦冬多糖提取物(峰尖分子量3000-5000Da)的制备
1)麦冬总多糖的提取：
取干燥麦冬块根，用5-6倍重量70％-95％乙醇回流3次，弃之；药渣水提2-3次。水提液减压浓缩至0.5-1g/ml，加入75％-95％乙醇至含醇量达60％-80％，静置过夜，倾去上清液，沉淀重复醇沉1次，冷冻干燥，得淡黄色麦冬总多糖粉末；
2)麦冬多糖的纯化：
取麦冬总多糖，加水溶解，离心去沉淀；上清液加入1-2倍体积70％-95％乙醇沉淀，静置过夜，去除沉淀。上清液再加入1-2倍体积70％-95％乙醇沉淀，静置过夜，收集上清液。上清液中再加入4-5倍体积70％-95％乙醇沉淀，静置过夜，收集所得的沉淀，即为峰尖分子量在3000-5000Da范围的麦冬多糖提取物。
实施例3含麦冬多糖提取物的片剂的制备
处方(1000片)：
按处方量将麦冬多糖提取物加入微晶纤维素、微粉硅胶、硬脂酸、乳糖混匀，制成颗粒，加入硬脂酸镁，混匀，压片，1000片(每片含麦冬多糖提取物0.1g)。成人推荐口服用量1-4片/次，每日2-3次。
实施例4含麦冬多糖提取物的颗粒剂的制备
处方(1000袋)：
按处方量将麦冬多糖提取物加入微晶纤维素、微粉硅胶、硬脂酸、乳糖混匀，制成颗粒，装袋，1000袋(每袋含麦冬多糖提取物0.1g)。成人推荐口服用量1-4袋/次，每日2-3次。
下面通过试验例以本发明麦冬多糖提取物的药理活性试验及结果来进一步说明本发明。
试验例1：麦冬多糖提取物对膳食诱导肥胖小鼠结肠及肝脏部位炎性因子基因表达水平及血清炎性因子含量的影响。
1、动物
选用6-8周的C57BL/6小鼠，雄性，48只。饲养条件：中央空调调控温度和湿度，在鼠专用笼具内饲养，每盒5只。温度22±1℃，40％-60％相对湿度，控制光照明12小时，暗12小时，动物自由摄食及饮水。
2、药品与试剂
麦冬多糖提取物I(峰尖分子量5000-6500Da，由实施例1制得)；麦冬多糖提取物II(峰尖分子量3000-5000Da，由实施例2制得)；低脂饲料(10％含脂肪量,D12450B，购自美国Research Diet公司)；高脂饲料(60％含脂肪量,D12492，购自美国Research Diet公司)。
3、实验方法
3.1腹型肥胖小鼠模型
适应性喂养一周后，小鼠根据体重随机分为6组，每组8只(详见表1)。试验分组如下：空白组，模型组，1％麦冬提取物I组，8％麦冬提取物I组，1％麦冬提取物II组，8％麦冬提取物II组。空白组小鼠喂以低脂饲料，其他各组均喂以高脂饲料。其中，各麦冬提取物给药组以麦冬多糖混入高脂饲料后压制制得，1％含量为每99g高脂饲料中均匀混入1g麦冬多糖提取物，8％含量为每92g高脂饲料中均匀混入8g麦冬多糖提取物。动物实验持续12周，期间动物自由进食、饮水。
表1试验动物分组情况
3.2组织样本收集及处理
在实验终点，小鼠禁食10h，称取小鼠体重，根据各组小鼠体重用20％乌拉坦溶液按0.1mL/10g剂量麻醉。待小鼠麻醉后，心脏取血，收集于1.5mL EP管中。4℃静置4h后，3500rpm离心10min，取上清，分装于新的1.5mL EP管中，-80℃保存。
取下肝脏，生理盐水冲洗，滤纸印干。在右外叶位置剪取一小块组织，按200mg左右重量分装于2mL离心管中，液氮速冻，之后转至-80℃保存。
分离结肠，生理盐水冲洗干净，分装于1.5mL的离心管中，冻存于液氮中，之后转移-80℃保存。
3.3炎性因子相关基因表达量测定
组织总RNA提取：用分析天平称量从-80℃冰箱中取出的组织样本，按100mg标准加入预冷的2mL EP管中，每100mg组织加1mL Trizol溶液。每管加2颗钢珠，用组织研磨仪，设定65Hz，60s程序，研磨2次。移取Trizol匀浆至新的预冷EP管，按比例加入氯仿，上下震荡混匀，室温静置5min。4℃，12,000rpm离心15min，转移上层水相至新的EP管中，加入等体积的异丙醇。上下剧烈颠倒15s，室温静置10min。4℃，12,000rpm离心15min沉淀RNA。弃上清，沉淀加预冷的75％乙醇1mL洗涤2次。4℃，12,000rpm离心5min。弃上清，超净台干燥5-10min，再加20μL DEPC水溶解混匀，用Biophotometer Plus测RNA浓度，取OD260/OD280在1.8-2.0的RNA样本，进一步调浓度至1ug/μL，保存于-80℃。
cDNA的合成：使用High capacity cDNA Reverse Transcription Kit试剂盒，反应体系在冰上配置。为保证准确性，应按反应数×1.05的量配置Mix液，混匀后再分装20μL到每个反应管中，以备后续反转录研究(详见表2)。
表2反转录反应体系
将反应体系混匀，并离心，25℃孵育10min，37℃孵育120min，85℃孵育5min，得到cDNA，-20℃保存。
以β-actin为内参，使用ABI公司的Power Green PCR Master Mix试剂盒，运用实时荧光定量PCR仪检测基因的相对表达量，建立10μL Real-time PCR反应体系，具体如下：
表3 Real-time PCR反应体系
反应体系在冰上配置，第一步：50℃，2min；第二步：95℃预变性10min；第三步：95℃，15s；第四步：60℃，1min，重复40个循环；以融解曲线判断产物特异性，相对定量法进行数据统计(其中：-△△CT＝(CT,Target-CT,β-actin)试验组-(CT,Target-CT,β-actin)对照组)。
PCR反应所用引物信息见表4。
表4 RT-PCR引物序列表
3.4血清炎性因子含量测定
各组小鼠血清中IL-1以及IL-10的含量采用酶联免疫吸附法方法，并严格按照试剂盒操作步骤进行。主要方法如下：在包被过的酶标板中加入标准品或待测样品，混匀后，37℃孵育40min；用洗涤液洗涤5次，滤纸印干；每孔加入ddH2O和一抗工作液，混匀后，37℃孵育20min；用洗涤液洗5次，印干；每孔加酶标抗体工作液，混匀后，37℃孵育10min；用洗涤液洗板5次，滤纸印干；每孔加入底物工作液，置37℃暗处反应15min；每孔加入终止液，混匀后，用酶标仪在450nm处测OD值。绘制标准曲线，拟合回归方程。所有待测样品孔OD值减去空白孔OD值后再代入标准曲线方程，计算相应浓度。
3.5数据处理
实验数据结果以均数±标准误(Mean±SEM)形式表示，使用SPSS 17.0软件进行统计分析。采用One-way ANOVA进行多组数据间的差异比较。*P<0.05表示与模型组比较具有显著性差异，**P<0.01表示具有极显著差异。
4实验结果
各组小鼠肝组织和结肠组织炎性因子mRNA表达水平如说明书附图1、2所示。连续12周，口服1％或8％的麦冬提取物I、II，相比模型组，均可显著调低IL-1、IL-6、IL-17等致炎因子的基因表达水平，并提高抑炎因子IL-10的mRNA表达水平，表明口服麦冬多糖提取物可以干预模型小鼠肝组织、结肠组织炎性因子的基因表达。同时，分离各组小鼠血清，检测炎性因子分泌水平(详见表5)，也发现麦冬多糖提取物可以显著抑制IL-1，并提高IL-10的含量。表明麦冬多糖的抗炎作用不仅表现在上游基因层面，在下游血生化指标上也有确切的体现。
这些结果表明，从麦冬中提取分离制得的麦冬多糖提取物(峰尖分子量3000-6500Da)可显著降低模型动物炎性因子表达量，具有明显的抗炎作用，可用于制备具有抗炎作用的药物或保健食品。
表5麦冬多糖提取物对小鼠血清炎性因子含量的影响(n＝8,Mean±SEM)
本发明所涉及的多个方面已做如上阐述。然而，应理解的是，在不偏离本发明精神之前提下，本领域专业人员可对其进行等同改变和修饰，所述改变和修饰同样落入本申请所附权利要求的覆盖范围。
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Claims (3)
1.一种麦冬多糖提取物在制备具有抗炎作用的药物或保健食品中的应用，其特征在于，所述的麦冬多糖提取物的峰尖分子量在3000-6500Da之间，并由下述方法获得：
1)麦冬总多糖的提取：
取干燥麦冬块根，用5-6倍重量70％-95％乙醇回流3次，弃之，药渣水提2-3次，水提液减压浓缩至0.5-1g/ml，加入75％-95％乙醇至含醇量达60％-80％，静置过夜，倾去上清液，沉淀重复醇沉1次，干燥，得淡黄色麦冬总多糖粉末；
2)麦冬多糖的纯化：
取麦冬总多糖，加水溶解，离心去沉淀；上清液加入1-2倍体积70％-95％乙醇沉淀，静置过夜，去除沉淀；上清液再加入1-2倍体积70％-95％乙醇沉淀，静置过夜，收集所得沉淀，即为峰尖分子量在5000-6500Da范围的麦冬多糖提取物；剩余上清液再加入4-5倍体积70％-95％乙醇沉淀，静置过夜，收集所得的沉淀，即为峰尖分子量在3000-5000Da范围的麦冬多糖提取物。
2.如权利要求1所述的应用，其特征在于，所述的药物或保健食品为口服制剂。
3.如权利要求2所述的应用，其特征在于，所述口服制剂中麦冬多糖提取物的重量百分比为5％-60％，余量为药用辅料或附加剂。
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| CN112791037A (zh) * | 2020-03-05 | 2021-05-14 | 上海友仁生物科技有限公司 | 一种组合活性物及其制备方法和其在制备舒缓皮肤过敏的产品或药物中的应用 |
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| CN101062262A (zh) * | 2006-04-24 | 2007-10-31 | 黄振华 | 麦冬和细辛脑的药物组合物 |
| CN102757508A (zh) * | 2011-04-25 | 2012-10-31 | 上海张江中药现代制剂技术工程研究中心 | 一种麦冬多糖提取物在制备具有减肥功能的食品添加剂、保健品或药物中的应用 |
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| CN110846259A (zh) * | 2019-12-16 | 2020-02-28 | 湖北文理学院 | 一种湖北麦冬多糖的高产细菌菌株及其应用 |
| CN110846259B (zh) * | 2019-12-16 | 2021-03-02 | 湖北文理学院 | 一种湖北麦冬多糖的高产细菌菌株及其应用 |
| CN112791037A (zh) * | 2020-03-05 | 2021-05-14 | 上海友仁生物科技有限公司 | 一种组合活性物及其制备方法和其在制备舒缓皮肤过敏的产品或药物中的应用 |
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