CN116672390A - 马蔺子提取物在制备抗溃疡性结肠炎药物中的应用 - Google Patents
马蔺子提取物在制备抗溃疡性结肠炎药物中的应用 Download PDFInfo
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Abstract
本发明属于中药技术领域，具体涉及一种马蔺子提取物在制备抗溃疡性结肠炎药物中的应用，该马蔺子提取物是中药马蔺子的水提物，其提取方法包括以下步骤：将马蔺子浸泡50～70min，加入10倍量的水回流提取110～130min，过滤，然后再加入8倍量的水回流提取50～70min，过滤；重复回流提取2～4次，合并滤液，浓缩，冷冻干燥制得粉末，即为马蔺子提取物。本发明通过体内外药效实验，探究马蔺子提取物的抗溃疡性结肠炎药理活性，多角度阐释并验证了马蔺子提取物对溃疡性结肠炎的药效，具有较好的治疗效果。
Description
技术领域
本发明属于中药技术领域，涉及中药马蔺子的提取与应用，具体涉及一种马蔺子提取物在制备抗溃疡性结肠炎药物中的应用。
背景技术
溃疡性结肠炎(UC)又称非特异性溃疡性结肠炎，是一种主要累及结直肠黏膜层和黏膜下层的难治性复发性消化道疾病，具有急性暴发型病死率高、癌变率高、慢性持续型、易反复发作等特点；其发病机制是多因素的，涉及上皮屏障缺陷，遗传易感性，失调的免疫应答等。溃疡性结肠炎已成为一种全球性疾病，尤其在亚洲国家发病率加速上升。
目前，溃疡性结肠炎的治疗主要以西医治疗为主，其治疗类型包括四种：氨基水杨酸、糖皮质激素、免疫调节药物和生物药物。在临床上，使用氨基水杨酸和糖皮质激素是轻度至中度UC患者的主要治疗方法，而中度至重度溃疡性结肠炎患者通常使用免疫抑制剂、生物制剂或这些药物的联合使用。然而，采用上述治疗方法所产生的诸如激素依赖、不良反应以及对所有这些治疗方法的耐药性等问题仍有待解决，并且，现有的治疗方案还存在无法根治且治疗费用高的问题，给患者造成较大的经济负担。
中医药治疗溃疡性结肠炎内外合用，辨证论治，不良反应小，属于安全有效的治疗方法。在中医诊疗意见中指出溃疡性结肠炎用药治疗以清热解毒，凉血化瘀为主，这与马蔺子的主要功效相符合。马蔺子为被子植物门鸢尾科鸢尾属马蔺的成熟干燥的种子，选自《中华海洋本草》，始载于《神农本草经》，其味甘、辛，归胃、肺经。马蔺子主要功效为清热利湿，解毒散结，止血，杀虫；主治黄疸，淋浊，小便不利，泻痢，风湿痹痛，喉痹，肠痈，牙痛，瘰疬，疝气，疮疖肿毒，痔疮，吐血，衄血，便血，崩漏，外伤出血，烫伤，蛇伤，虫积，疟疾。从马蔺子中分离鉴定的化合物共有30个，主要含黄酮类、苯醌类和茋类等，其代表成分有马蔺子甲素、马蔺子乙素和马蔺子丙素。现代药理研究发现马蔺子具有抗炎、抗肿瘤、抗氧化、放射增敏，改善糖脂代谢等生理活性。马蔺子主治中的泻痢、便血等也与溃疡性结肠炎中医诊疗症状脓血便、泄泻、腹痛症状相吻合，但目前还未有关于马蔺子治疗溃疡性结肠炎的研究。
发明内容
本发明的目的是为了解决现有的溃疡性结肠炎治疗方案所存在的不良反应、耐药性以及无法根治且费用高等问题，提出一种马蔺子提取物在制备抗溃疡性结肠炎药物中的应用，采用水提法从中药马蔺子中提取制备一种马蔺子提取物，该马蔺子提取物对溃疡性结肠炎具有较好的治疗作用。
本发明的技术方案是：
本发明提供了马蔺子提取物在制备抗溃疡性结肠炎药物中的应用。
进一步的，所述马蔺子提取物是中药马蔺子的水提物，其提取方法包括以下步骤：
将马蔺子浸泡50～70min，加入10倍量的水回流提取110～130min，过滤，然后再加入8倍量的水回流提取50～70min，过滤；重复回流提取2～4次，合并滤液，浓缩，冷冻干燥制得粉末。
上述浸泡时间可以采用50～70min范围内的任一时间，例如可以是50min、55min、58min、60min、62min、65min或70min等，也可以是该范围内的其他任意时间；
上述回流提取110～130min可以为该范围内的任一时间，例如可以是110min、115min、118min、120min、122min、125min或130min等，也可以为该范围内的其他任意时间；
上述回流提取50～70min可以为该范围内的任一时间，例如可以是50min、55min、58min、60min、62min、65min或70min等，也可以为该范围内的其他任意时间；
回流提取次数优选2次，也可以为3次或4次。
进一步的，所述提取方法包括以下步骤：
马蔺子浸泡60min，加入10倍量的水回流提取120min，过滤，然后再加入8倍量的水回流提取60min，过滤，合并两次滤液，浓缩，冷冻干燥制得粉末。
进一步的，所述马蔺子提取物用于制备治疗溃疡性结肠炎的药物，所述药物的剂型包括溶液剂、散剂、颗粒剂、片剂、胶囊剂、气雾剂、软膏剂或膜剂中的任意一种。
进一步的，所述药物还包括药学上可接受的辅料。
进一步的，所述药物的活性成分包括马蔺子提取物。
本发明的有益效果：
(1)本发明通过体内实验中马蔺子提取物治疗TNBS诱导的溃疡性模型大鼠，综合分析DAI指数、脾脏指数、结肠病理状况、结肠长度和炎症因子等指标，判定马蔺子提取物对溃疡性结肠炎具有较好的治疗效果；通过在体外实验中马蔺子提取物治疗脂多糖诱导的人正常结肠上皮细胞，对细胞增殖、形态、炎症因子释放的检测，分析并验证马蔺子提取物具有较好的治疗溃疡性结肠炎的药效。
(2)本发明提出一种马蔺子提取物在制备抗溃疡性结肠炎药物中的应用，并提供提取方法制备马蔺子提取物，探究并验证了其抗溃疡性结肠炎药理活性，同时，通过体内外药效实验多角度阐释马蔺子提取物对溃疡性结肠炎的治疗作用及较好的治疗结果。
附图说明
图1为各组大鼠给药后体征变化；其中，图1A为DAI指数，图1B为脾脏指数，图1C为结肠长度，图1D为结肠整体图片(*P<0.05与治疗组相比，#P<0.05与正常对照组相比)；
图2为炎症因子检测结果；其中，图2A为结肠中促炎细胞因子TNFα的表达，图2B为结肠损伤的组织病理学评分，图2C为每组的代表性组织学切片(HE染色，×100)(*P<0.05与治疗组相比，#P<0.05与正常对照组相比)；
图3为为马蔺子提取物体外药效研究结果；其中，图3A为用LPS造模和马蔺子处理后NCM460细胞的相对细胞存活率，图3B为促炎细胞因子TNFα在细胞上清液中的表达图，图3C为使用倒置显微镜观察细胞形态(Bar＝1000μm)(*P<0.05与治疗组相比。#P<0.05与正常对照组相比)。
具体实施方式
为了进一步理解本发明，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，对本发明作进一步的说明；显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1马蔺子提取物的制备
本实施例提供了一种马蔺子提取物的制备方法，即采用水提法从中药马蔺子中提取制得马蔺子提取物，提取方法包括以下步骤：
马蔺子浸泡60min后，第一次用10倍量的水，回流提取120min，过滤，然后再加入8倍量的水，回流提取60min，过滤，合并两次滤液，滤液用旋转蒸发仪浓缩，用冷冻干燥机制得粉末；
该粉末即为马蔺子提取物，将粉末溶解在蒸馏水中制成溶液剂后给药。
实施例2马蔺子提取物的制备
本实施例同样是采用水提法从中药马蔺子中提取制备马蔺子提取物，提取方法包括以下步骤：
马蔺子浸泡50min后，加入10倍量的水回流提取110min，过滤，然后再加入8倍量的水回流提取50min，过滤；重复回流提取3次后合并三次的滤液，滤液用旋转蒸发仪浓缩，用冷冻干燥机制得粉末；
该粉末即为马蔺子提取物，该粉末可以溶解在蒸馏水中制成溶液剂给药，也可以制成散剂、颗粒剂、片剂或胶囊剂。
实施例3马蔺子提取物的制备
本实施例同样是采用水提法从中药马蔺子中提取制备马蔺子提取物，提取方法包括以下步骤：
马蔺子浸泡70min后，加入10倍量的水回流提取130min，过滤，然后再加入8倍量的水回流提取70min，过滤；重复回流提取4次后合并四次的滤液，滤液用旋转蒸发仪浓缩，用冷冻干燥机制得粉末；
该粉末即为马蔺子提取物，该粉末可以制成气雾剂、软膏剂或膜剂。
试验例1马蔺子提取物对溃疡性结肠炎模型大鼠的药效研究
(1)动物
48只SD大鼠(200±20g)饲养在温度21～25℃、相对湿度50％～60％、光照/黑暗周期为12h的环境中；大鼠每笼四只适应性喂养7天，可自由饮水和食物。适应后随机分为6组(n＝8)：正常对照组、模型对照组、阳性药物组(SASP)、低剂量治疗组(LM)、中剂量治疗组(MM)和高剂量治疗组(HM)。模型对照组、阳性药物组和治疗组(包括LM、MM和HM组)禁食1天后用水合氯醛溶液麻醉，然后将直径2mm的硅胶软管插入大鼠肛门8cm处到达结肠，注射0.2mL/100g结肠炎诱导剂(TNBS)溶液(50mgTNBS溶于50％乙醇)后，捏紧肛门，倒置大鼠1min；当出现便血或潜血阳性时即为造模成功。正常对照组经直肠灌注0.9％生理盐水2mL。
TNBS处理24h后，LM、MM和HM组分别给予马蔺子提取物溶液18.9mg/(kg·天)、56.7mg/(kg·天)和170.1mg/(kg·天)，其中，低、中、高剂量分别相当于马蔺子标准剂量的1/3、1、3倍；阳性药物组给予柳氮磺吡啶(SASP)溶液670mg/(kg·天)通过灌胃给药。第7天，大鼠禁食12h，麻醉处死。
分离结肠并用盐水冲洗，测量结肠长度作为炎症的间接标志；肉眼观察炎症和溃疡情况；对于组织学评估，结肠样本固定在4％多聚甲醛中，其他样本保持在-80℃以进行进一步评估；称重每组大鼠的胸腺并计算器官指数；通过腹主动脉穿刺收集血液并离心以获得血清直至进一步使用。
(2)疾病活动指数(DAI)测定
根据体质量、大便特征和大便中的血液对大鼠进行评分。0分表示无体质量下降，大便正常，大便无血；1分表示体质量下降1％～5％，大便疏松，大便隐血；2分表示体质量下降5％～10％，大便疏松，大便隐血；3分表示体质量下降10％～15％，腹泻，便血；4分表示体质量下降15％以上，腹泻，便血。DAI＝(体质量下降分数+大便性状分数+肠出血程度分数)/3。
DAI评分越高，结肠损伤程度越高。
结果：如图1A所示，与正常对照组(图1中Control)相比，模型对照组DAI评分显着升高(P<0.01)。同时，结肠长度显着缩短(P<0.01)。与模型对照组(图1中Model)相比，每个治疗组(SASP、LM、MM和HM)的DAI评分显着恢复(P<0.05)。
(3)病理组织观察
收集结肠组织，截取结肠远端部分并纵行剖开后用冰生理盐水冲洗肠道内容物。使用滤纸吸收多余水分，纵行一分为二，一部分用4％多聚甲醛固定，石蜡包埋切片、HE染色，显微镜下观察结肠组织形态；一部分-80℃低温冰箱保存，用于检测结肠中基因表达。结肠黏膜损伤指数评分标准(表1)对结肠黏膜病理学改变进行分级评分，多维度对疾病进行判定。
表1HE病理评分标准
| 分数 | 炎症程度 | 病变深度 | 隐窝损伤 | 病变范围(％) |
| 0 | 无 | 无 | 无 | 无 |
| 1 | 轻度 | 黏膜层 | 基底1/3隐窝损伤 | 1-25 |
| 2 | 中度 | 黏膜下层 | 基底2/3隐宽损伤 | 26-50 |
| 3 | 重度 | 黏膜全层 | 隐窝损伤，黏膜上皮表面无缺损 | 51-75 |
| 4 | 重度 | 黏膜全层 | 隐窝损伤，黏膜上皮表面脱落 | 76-100 |
结果：如图1C和1D所示，结肠长度有不同程度的增加，说明各组药物均有一定程度的治疗作用。如图1B所示，脾指数结果显示，与模型对照组相比，LM、MM、HM组有显着增加(P<0.01)。光镜下观察炎症浸润及黏膜病变，结肠腺体排列紊乱、隐窝萎缩、炎性细胞大量浸润、杯状结构脓肿，与模型组结肠组织学损伤指标一致。
(4)炎症因子检测
对结肠匀浆进行直接测试。结肠组织用PBS匀浆，然后以3000rpm/min离心20分钟，按ELISA试剂盒(购买所得)的说明确定TNF-α的浓度。
结果：如图2A所示，用马蔺子提取物治疗显示TNBS诱导的UC大鼠的组织损伤和炎症评分明显减少。组织病理学表明，如图2B所示，造模增加了组织学评分，而马蔺子提取液治疗后显着降低了组织学评分。如图2C所示，马蔺子提取物显着降低了UC大鼠的TNF-α，表明马蔺子提取物在UC过程中具有抗炎活性。
(5)结论
上述结果表明马蔺子可以改善TNBS诱导的结肠炎模型大鼠的病理体征状态，减轻结肠组织损伤和炎症反应。
试验例2马蔺子提取物对溃疡性结肠炎模型细胞的药效研究
(1)细胞培养
脂多糖(LPS)诱导的NCM460细胞用作人正常结肠上皮细胞模型，细胞在DMEM培养基中培养，与10％胎牛血清、1％含青霉素和链霉素的抗生素混合；细胞在5％CO2、37℃潮湿环境中生长。各组设计为正常对照组、模型组和马蔺子治疗组。8h后，除正常对照组外，细胞用LPS(10μg/mL)处理24h，除模型组外，用不同浓度的马蔺子提取物稀释液给药24h。马蔺子提取物先用超纯水溶解，再用培养基稀释至试验浓度；在100μL或1mL培养系统的每个孔中，马蔺子给药的最终浓度分别为100μg/mL、50μg/mL、25μg/mL和12.5μg/mL。
(2)MTT法检测细胞活力
NCM460细胞体外增殖通过MTT测定法测定。吸去培养液，每孔加入100μLMTT，于37℃培养箱中培养4h后，每孔加入150μL二甲基亚砜，于室温摇床震荡10min，使用酶标仪对各孔在490nm波长处的吸光度进行测定，扣除空白对照组吸光度值，计算细胞存活率，细胞活力(％)＝(OD实验-OD空白)/(OD正常-OD空白)*100％，实验重复3次，最终结果用三次实验的平均值代表。
结果：如图3A所示，培养48小时后LPS诱导的细胞存活率明显低于正常对照组。
(3)细胞形态学观察
处于对数生长期的NCM460细胞在24孔板中以5×104/孔培养。细胞培养、造模和给药方法与细胞增殖实验相同。使用倒置显微镜观察并拍摄了NCM460细胞的形态变化。
如图3C所示，与MTT结果一致的是，LPS诱导的细胞在细胞形态上受到严重影响并显著抑制细胞增殖。
(4)细胞炎症因子检测
对收集的细胞上清液进行检测。按照ELISA试剂盒的说明确定TNF-α的含量。
结果：如图3B所示，酶标仪检测细胞上清液中的炎症因子显示，LPS诱导的细胞中TNFα的释放显著增加，而马蔺子提取物处理的细胞中TNFα的含量出现不同程度的降低。
(5)结论
体外实验结果表明，马蔺子能够抑制LPS诱导的NCM460细胞凋亡、细胞形态改变和促炎细胞因子的释放。
上述说明仅为本发明的优选实施例，并非是对本发明的限制，尽管参照前述实施例对本发明进行了详细的说明，对于本领域技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改型等，均应包含在本发明的保护范围之内。
Claims (6)
1.马蔺子提取物在制备抗溃疡性结肠炎药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述马蔺子提取物是中药马蔺子的水提物，其提取方法包括以下步骤：
将马蔺子浸泡50～70min，加入10倍量的水回流提取110～130min，过滤，然后再加入8倍量的水回流提取50～70min，过滤；重复回流提取2～4次，合并滤液，浓缩，冷冻干燥制得粉末。
3.根据权利要求2所述的应用，其特征在于，所述提取方法包括以下步骤：
马蔺子浸泡60min，加入10倍量的水回流提取120min，过滤，然后再加入8倍量的水回流提取60min，过滤，合并两次滤液，浓缩，冷冻干燥制得粉末。
4.根据权利要求1所述的应用，其特征在于，所述马蔺子提取物用于制备治疗溃疡性结肠炎的药物，所述药物的剂型包括溶液剂、散剂、颗粒剂、片剂、胶囊剂、气雾剂、软膏剂或膜剂中的任意一种。
5.根据权利要求4所述的应用，其特征在于，所述药物还包括药学上可接受的辅料。
6.根据权利要求1所述的应用，其特征在于，所述药物的活性成分包括马蔺子提取物。
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| 吴茜等: "《备急千金要方》以寒热为纲治痢的方药特点探析", 中华中医药杂志, vol. 35, no. 11, 30 November 2020 (2020-11-30), pages 5427 - 543 * |
| 张艳红: "肠澼（溃疡性结肠炎）的古代文献研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技》, 15 December 2004 (2004-12-15), pages 056 - 211 * |
| 韩涛;殷胜骏;薛新丽;尹晓娟;李宏;: "溃疡性结肠炎临床用药频数分析", 中华中医药学刊, vol. 26, no. 10, 10 October 2008 (2008-10-10), pages 2176 - 2177 * |
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