CN108743732A - 用于防治炎症性肠病的含植物成分混合物及其制备方法与应用 - Google Patents
用于防治炎症性肠病的含植物成分混合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供用于防治炎症性肠病的含植物成分混合物及其制备方法和应用。该混合物包括茶茎微粉或者茶梗/茶茎提取物，茶梗/茶茎富有多种多酚类化合物，能够有效改善和防治炎症性肠病，成本低廉、安全可靠，且能够通过抗炎、抗菌作用发挥预防和治疗炎症性肠病的临床症状。该混合物的制备工艺简单、耗能低、无污染。
Description
技术领域
本发明属于防治炎症性肠病植物提取物技术领域，具体涉及茶梗/茶茎的提取物。
背景技术
炎症性肠病(inflammatory bowel disease)是一种发生于直肠和结肠黏膜与黏膜下层的，同时伴有器官损害的肠道非特异性慢性复发性炎症。炎症性肠病包括溃疡性结肠炎(ulcerative colitis)和克罗恩病(Crohn’s disease)。研究表明，在炎症性肠病发生时，巨噬细胞浸润到肠黏膜间质、破坏肠黏膜屏障、导致炎症，引发包括体重减轻、腹泻、血便、发热、胃动力障碍等临床症状，并增加结肠炎发病的风险，严重危害人们的健康，备受关注。
炎症性肠病可能与遗传、饮食结构、环境、免疫反应以及肠道菌群有关，但炎症性肠病的具体病因及发病机制目前尚未明确，且缺乏特异有效的药物，因此，迫切需要开发或寻找治疗炎症性肠病的有效药物。中药及天然来源的保健食品在我国的应用历史悠久，为保证中华民族的健康和繁衍生息做出了巨大贡献。由于中药及天然来源的保健食品在抗炎抗菌方面具有作用维持时间长、副作用小及作用靶点多、细菌不易产生耐药等诸多作用特点，从天然产物中获取有效抗炎活性成分治疗炎症性肠病已成为目前研究的热点。
至今为止的研究表明，茶叶具有较好的抗菌、抗炎、抗氧化等多种生理活性，已被广泛应用。然而，具有大部分相同活性成分的茶梗、茶茎尚未用于防治炎症性肠病。茶茎是冬季修剪茶树的废料，粉碎可作牲畜饲料添加剂；茶梗是茶叶制作后的废料，具有一定的保健价值。
本说明书中，茶梗/茶茎是指茶梗或茶茎。
发明内容
本发明提供用于防治炎症性肠病的含植物成分混合物及其制备方法与应用，旨在解决将茶梗/茶茎用于防治炎症性肠病的技术问题。
本发明技术方案之一是一种用于防治炎症性肠病的含植物成分混合物，包括茶梗/茶茎微粉或者茶梗/茶茎提取物。茶梗/茶茎含有表没食子酸儿茶素没食子酸酯、表没食子酸儿茶素、表儿茶素没食子酸酯、表儿茶素等多酚类化合物。
进一步地，还包括抗氧化剂、防腐剂和甜味剂，所述抗氧化剂是亚硫酸盐或抗坏血酸或以上二者的混合物，所述防腐剂是二羟基乙酸或氯丁醇或以上二者的混合物，所述甜味剂是蔗糖或乳糖或D-甘露醇或以上三者的混合物。
进一步地，还包括赋形剂和润滑剂，所述赋形剂是淀粉或结晶纤维素或以上二者的混合物，所述润滑剂是硬脂酸镁或硬脂酸钙或滑石粉或以上三者的混合物。
进一步地，采用的剂型是颗粒剂、片剂、胶囊剂或丸剂。
本发明提供的用于防治炎症性肠病的含植物成分混合物包括茶茎微粉或者茶梗/茶茎提取物，茶梗/茶茎富有多种多酚类化合物，能够有效改善和防治炎症性肠病，成本低廉、安全可靠，且能够通过抗炎、抗菌作用发挥预防和治疗炎症性肠病的临床症状。
本发明技术方案之二是上述用于防治炎症性肠病的含植物成分混合物的制备方法，包括以下三个步骤：S110，将修剪茶树获得的茶梗/茶茎机械粉碎成平均粒径小于0.85mm(20目)的粉末，从而获得所述茶梗/茶茎微粉；S120，步骤 S110中获得的所述茶梗/茶茎微粉与所述添加剂混合；S130，将步骤S120中获得混合物制备成预定的剂型。所述剂型是颗粒剂、片剂、胶囊剂等临床用药剂型。
本发明技术方案之三是上述用于防治炎症性肠病的含植物成分混合物的制备方法，包括以下三个步骤：S210，将修剪茶树获得的茶梗/茶茎机械粉碎成粉末；S220，将步骤S210获得的原料粉末与溶剂混合，所述溶剂为水、乙醇或含水乙醇，所述的原料粉末与溶剂的重量比在1∶(10～20)，然后通过加热提取、微波提取或超声提取获得所述茶梗/茶茎提取物；S230，步骤S220获得的所述茶梗/茶茎提取物与所述添加剂混合，然后制备成预定的剂型。
进一步地，步骤S220包括以下三个分步骤：S221，步骤S210获得的原料粉末与水混合，加热提取后过滤得到一次滤液和一次滤渣；S222，将所述一次滤渣与水再次混合，加热第二次提取后过滤得到二次滤液；S223，合并所述一次滤液和所述二次滤液，浓缩得到所述茶梗/茶茎提取物。
本发明提供的用于防治炎症性肠病的含植物成分混合物的以上两个制备方法，其一是先制备平均粒径小于0.85mm的茶梗/茶茎微粉，再与添加剂混合，然后制备成预定的剂型；其二是先采用水、乙醇或含水乙醇作溶剂，制备茶梗/茶茎提取物，再与添加剂混合，然后制备成预定的剂型。采用粒径小于0.85mm的微粉或溶剂提取物的形式，便于消化吸收，而且制备工艺简单、耗能低、无污染。
本发明技术方案之四是上述用于防治炎症性肠病的含植物成分混合物在制备防治炎症性肠病保健品中的应用。
本发明技术方案之五是上述用于防治炎症性肠病的含植物成分混合物在制备防治炎症性肠病药品中的应用。
本发明技术方案之六是上述用于防治炎症性肠病的含植物成分混合物在制备防治炎症性肠病食品中的应用。
下面为一个实施例的茶梗/茶茎提取物及茶叶提取物的成分分析：
实验采用高效液相色谱法进行分析。DIONEX液相分析仪为Ultimate 3000 型，电子天平为Sartorius BP211D十万分之一型，恒温干燥箱为德国Memmert 型，超纯水仪为Merck millipore公司，小型粉碎机为杭州熊火科技有限公司，乙腈为Fisher(色谱纯)，甲酸为广州化学试剂厂(分析纯)。对照品咖啡因 (Caffeine，Caff)，可可碱(Theobromine，Tb)，没食子儿茶素没食子酸酯((-) -Gallocatechin gallate，GCG)，表没食子儿茶素没食子酸酯((-) -Epigallocatechin gallate，EGCG)购自日本和光纯药株式会社；没食子酸(Gallic acid，GA)，儿茶素((+)-Catechin，C)购自美国Sigma公司；没食子儿茶素((-)-Gallocatechin，GC)，表没食子儿茶素(Epigallocatechin，EGC)，表儿茶素(Epicatechin，EC)购自日本栗田工业株式会社；表儿茶素没食子酸酯 ((-)-Epicatechin-3-gallate，ECG)购自上海晶纯生化科技股份有限公司。
色谱条件，色谱柱COSMOSIL 5C18-AR-II(4.6*250mm，5μm，Nacalai tesque.Inc.，Japan)，流速为1ml/min，定量检测波长为231nm，柱温为35℃，进样量为20μl。流动相A：H2O(含0.1％甲酸)；流动相B：CH3CN(含0.1％甲酸)，梯度洗脱程序见表一。供试样品经蒸馏水溶解后离心，经0.45μm水膜过滤后进HPLC分析，样品中各成分含量见表二。
由表二可以看出，在茶梗/茶茎及茶叶的提取物中，茶叶提取物中的咖啡因(Caffeine，Caff)含量要远远高于茶梗/茶茎的提取物，可能会导致失眠、兴奋以及心悸等不良反应；而茶梗/茶茎的提取物富含儿茶素等多酚类抗菌/抗病毒活性成分，有利于炎症的预防治疗。
表一，HPLC梯度洗脱程序
表二，茶梗/茶茎及芽茶提取物中生物碱及多酚类化合物含量(％)
| 提取物成分 | 茶叶 | 茶茎 | 茶梗 |
| GA | 0.15 | 0.17 | 0.20 |
| Tb | 0.26 | 0.05 | 0.03 |
| GC | 0.71 | 0.17 | 0.06 |
| EGC | 2.60 | 0.53 | - |
| C | 0.27 | 0.24 | 0.16 |
| Caff | 3.31 | 0.91 | 0.25 |
| EC | 0.85 | 0.58 | 0.17 |
| EGCG | 4.58 | 1.29 | - |
| GCG | 1.55 | 0.08 | - |
| ECG | 1.76 | 1.04 | 0.06 |
注：-表示未检测出
下面是茶梗/茶茎提取物对葡聚糖硫酸钠诱发的溃疡性结肠炎的改善作用。
葡聚糖硫酸钠诱发的实验性溃疡结肠炎特异性强、重复性高、持续时间较长，且同样存在与人类炎症性肠病相似的肠道菌群紊乱等病理特征，因而广泛应用于炎症性肠病的药效评价。因而本发明采用葡聚糖硫酸钠诱发的溃疡性结肠炎模型评价实施例2中制备的茶梗、茶茎提取物的药效作用。
实验动物采用体重18～20g的SPF级雌性8周龄BALB/c小鼠(购自广东省医学实验动物中心，动物许可证编号为SCXK(粤)2013-0002)。葡聚糖硫酸钠购自MP Biomedicals公司，柳氮磺胺砒啶购自上海三维制药有限公司。
实验动物在清洁级层流架中饲养，环境温度为23±2℃，相对湿度为75±10％，照明、时间为12小时/天(7：00～19：00)。实验动物适应性喂养一周后，进行药效活性评价实验。
实验动物随机分为正常对照组、葡聚糖硫酸钠组、500mg/kg茶茎提取物+葡聚糖硫酸钠组、250mg/kg茶茎提取物+葡聚糖硫酸钠组、500mg/kg茶梗提取物+ 葡聚糖硫酸钠组、250mg/kg茶梗提取物+葡聚糖硫酸钠组、以及柳氮磺胺砒啶+ 葡聚糖硫酸钠组，共7组，每组10只小鼠。除正常对照组外，其余各组均给予 3.5％葡聚糖硫酸钠水溶液代替饮用水给药1周，隔日更换临时配制的葡聚糖硫酸钠水溶液。柳氮磺胺砒啶、茶梗、茶茎提取物组每日灌胃给药，实验期间观察小鼠体重、饮食、精神状态等，实验结束后颈椎脱位处死小鼠，剪取结肠，测量长度，清理内容物，用预冷生理盐水清洗，滤纸除去多余水分后称重，计算结肠重量/长度比值，并观察药效作用。
如实验结果表三及表四所示。给予葡聚糖硫酸钠3天后，小鼠即出现血便， 5天后体重下降，饮食明显减少。解剖小鼠发现，与正常对照组相比，葡聚糖硫酸钠组结肠壁肿胀，充血水肿，肠壁皱缩，形成溃疡，长度明显下降及单位(长度)结肠重量增加。与葡聚糖硫酸钠对照组相比，茶梗、茶茎提取物给药小鼠有明显的改善作用。
表三 茶梗、茶茎对葡聚糖硫酸钠负荷小鼠体重的影响
| 实验组别 | 动物数 | 小鼠体重(g) |
| 水溶剂对照组 | 10 | 18.9±0.6 |
| 葡聚糖硫酸钠模型组 | 10 | 16.3±0.7 |
| 柳氮磺胺砒啶 | 10 | 18.2±0.9* |
| 500mg/kg茶茎提取物组 | 10 | 18.3±0.7* |
| 250mg/kg茶茎提取物组 | 10 | 18.2±0.6* |
| 500mg/kg茶梗提取物 | 10 | 18.1±0.5* |
| 250mg/kg茶梗提取物 | 10 | 18.0±0.6* |
与葡聚糖硫酸钠给药模型组相比，*P＜0.001
表四 茶梗、茶茎对葡聚糖硫酸钠负荷小鼠结肠长度和重量的影响
| 实验组别 | 结肠长度(em) | 结肠重量(g/cm) |
| 溶剂对照组 | 6.4±0.3 | 0.023±0.003 |
| 葡聚糖硫酸钠模型组 | 5.3±0.5 | 0.036±0.002 |
| 柳氮磺胺砒啶 | 6.0±0.5 | 0.027±0.004* |
| 500mg/kg茶茎提取物组 | 5.9±0.8 | 0.026±0.005* |
| 250mg/kg茶茎提取物组 | 5.8±0.3 | 0.027±0.003* |
| 500mg/kg茶梗提取物 | 5.8±0.5 | 0.027±0.004* |
| 250mg/kg茶梗提取物 | 5.6±0.4 | 0.028+0.002* |
与葡聚糖硫酸钠给药模型组相比，*P＜0.005
下面是茶梗、茶茎提取物对甲氨蝶呤诱发溃疡性结肠炎的改善作用。
化疗药物甲氨蝶呤具有较强的细胞毒性作用在临床广泛使用，但也会导致消化道黏膜损伤等毒副作用。因此，人们利用甲氨蝶呤这种作用特点制备实验动物模型来评价防治炎症性肠炎药物的药效活性。本发明采用甲氨蝶呤致小肠炎症模型来观察茶梗、茶茎提取物的保护作用。实验动物采用7周龄雄性昆明种小鼠(购自广东省医学实验动物中心，动物许可证编号为SCXK(粤)2013-0002)，体重 18～22g；甲氨蝶呤购于上海信帆生物有限公司；氯化钠购自国药集团化学试剂有限公司。实验动物在清洁级层流架中饲养，环境温度为23±2℃，相对湿度为75±10％，照明时间为12小时/天(7：00～19：00)，实验动物在适应性喂养一周后，进行药效活性评价实验。
将小鼠随机分为正常对照组、甲氨蝶呤组、500mg/kg茶茎提取物+甲氨蝶呤组、250mg/kg茶茎提取物+甲氨蝶呤组、500mg/kg茶梗提取物+甲氨蝶呤组、 250mg/kg茶梗提取物+甲氨蝶呤组，共6组，每组10只。除等容量水溶液对照组腹腔注射1ml生理盐水外，其余各组均连续2天腹腔注射给予20mg/kg/1ml 甲氨蝶呤生理盐水溶液。茶梗及茶茎提取物组每日灌胃给药，正常对照组及甲氨蝶呤组给予等量蒸馏水，实验期间观察小鼠体重、饮食、精神状态等。甲氨蝶呤给药一周后，各组小鼠禁食12h后颈椎脱位处死小鼠，剪取结肠，测量长度，清理内容物，用预冷生理盐水清洗，滤纸除去多余水分后称重，计算结肠重量/ 长度比值，并观察药效作用。
实验结果如表五及表六所示。给予甲氨蝶呤后，小鼠出现血便，体重下降，饮食明显减少。解剖小鼠发现，与正常对照组相比，甲氨蝶呤导致结肠壁肿胀，充血水肿，肠壁皱缩，形成溃疡，长度明显下降及单位(长度)结肠重量增加。与甲氨蝶呤组相比，茶梗、茶茎提取物给药小鼠显示明显的改善作用。
表五，茶梗、茶茎对甲氨蝶呤负荷小鼠体重的影响
| 实验组别 | 动物数 | 小鼠体重(g) |
| 水溶剂对照组 | 10 | 23.5±0.8 |
| 甲氨蝶呤模型组 | 10 | 17.8±0.8 |
| 500mg/kg茶茎提取物 | 10 | 21.3±0.6* |
| 250mg/kg茶茎提取物 | 10 | 20.4±0.9* |
| 500mg/kg茶梗提取物 | 10 | 20.8±0.5* |
| 250mg/kg茶梗提取物 | 10 | 20.2±0.4* |
与甲氨蝶呤给药模型组相比，*P＜0.05
表六，茶梗、茶茎对甲氨蝶呤负荷小鼠结肠长度和重量的影响
| 实验组别 | 结肠长度(cm) | 结肠重量(g/cm) |
| 溶剂对照组 | 6.6±0.6 | 0.023±0.002 |
| 甲氨蝶呤模型组 | 5.3±0.7 | 0.039±0.005 |
| 500mg/kg茶茎提取物 | 6.1±0.8 | 0.028±0.006* |
| 250mg/kg茶茎提取物 | 6.2±0.5 | 0.029±0.004* |
| 500mg/kg茶梗提取物 | 6.2±0.7 | 0.029±0.008 |
| 250mg/kg茶梗提取物 | 5.9±0.5 | 0.030±0.002 |
与甲氨蝶呤给药模型组相比，*P＜0.05
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
以下实施例所用茶茎和茶梗，由贵州普安宏鑫茶厂提供，已除杂和初步粉碎。
实施例1，用于制作防治炎症性肠病食品的含茶梗/茶茎微粉的混合物
茶茎和茶梗原料，粉碎成平均粒径小于0.85mm(20目)的粉末；每100重量份上述茶梗/茶茎微粉中加入抗坏血酸1份、二羟基乙酸0.2份、D-甘露醇2份、玉米淀粉20份、硬脂酸钙3份，水100份，混合均匀后干燥；
干燥物粉碎为平均粒径小于0.85mm的颗粒，即得该混合物。该混合物作为面包添加剂使用时，每100克面包专用面粉中加入1～5g。
实施例2，作防治炎症性肠病保健品的含茶梗/茶茎微粉的混合物
茶茎和茶梗原料，粉碎成平均粒径小于0.85mm(20目)的粉末；每100重量份上述茶梗/茶茎微粉中加入抗坏血酸1份、二羟基乙酸0.2份、D-甘露醇2份、结晶纤维素10份、硬脂酸钙3份、水90份，混合均匀后干燥。
干燥物压制成片剂或丸剂，或灌制为胶囊。该片剂/丸剂/胶囊作防治炎症性肠病保健品，温水送服口服，餐后服用1～5g。
实施例3，茶梗/茶茎提取物的制备
将原料粉碎成粗粉，取500g粗粉，置提取罐中，加20倍量水回流提取，保持沸腾8小时，过滤，滤渣再加入20倍量水回流提取，保持沸腾1小时，过滤，合并两次滤液。滤液置浓缩罐中60℃减压浓缩至相对密度为1.2左右的流浸膏，流浸膏取出装入托盘，经冷冻干燥得提取物约50g。
实施例4，茶梗/茶茎提取物的制备
将原料粉碎成粗粉，取500g粗粉，置提取罐中，加10倍量50％乙醇水溶液回流提取，保持沸腾2小时，过滤，滤渣再加入10倍量50％乙醇回流提取，保持沸腾1小时，过滤，合并两次滤液。滤液置浓缩罐中60℃减压浓缩至相对密度为1.0左右的流浸膏，经冷冻干燥得提取物约50g。
该提取物、用上述方法制备的茶叶提取物，用于以上的测试分析。
实施例5，用于制备防治炎症性肠病药品的含茶梗/茶茎提取物的混合物
以上茶梗/茶茎提取物制备成多种药物制剂，添加常规的赋形剂、润滑剂、抗氧化剂、防腐剂、着色剂、甜味剂等添加剂；其中，优选的赋形剂有乳糖、白糖、D-甘露醇、淀粉、结晶纤维素等；优选的润滑剂有硬脂酸镁、硬脂酸钙、滑石粉等；优选的抗氧化剂有亚硫酸盐、抗坏血酸等；优选的防腐剂有二羟基乙酸、氯丁醇等。茶梗/茶茎提取物与添加剂混和均匀后，制成颗粒剂、片剂、胶囊剂或丸剂等。上述药物制剂的服用方式为餐后口服，每次服用剂量中茶梗/茶茎提取物地含量为0.2～0.6g。
以上对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。
Claims (10)
1.一种用于防治炎症性肠病的含植物成分混合物，其特征是，包括茶梗/茶茎微粉，或茶梗/茶茎提取物。
2.根据权利要求1所述的用于防治炎症性肠病的含植物成分混合物，其特征是，还包括抗氧化剂、防腐剂和甜味剂，所述抗氧化剂是亚硫酸盐或抗坏血酸或以上二者的混合物，所述防腐剂是二羟基乙酸或氯丁醇或以上二者的混合物，所述甜味剂是蔗糖或乳糖或D-甘露醇或以上三者的混合物。
3.根据权利要求2所述的用于防治炎症性肠病的含植物成分混合物，其特征是，还包括赋形剂和润滑剂，所述赋形剂是淀粉或结晶纤维素或以上二者的混合物，所述润滑剂是硬脂酸镁或硬脂酸钙或滑石粉或以上三者的混合物。
4.根据权利要求2或3所述的用于防治炎症性肠病的含植物成分混合物，其特征是，采用的剂型是颗粒剂、片剂、胶囊剂或丸剂。
5.一种制备权利要求1所述的用于防治炎症性肠病的含植物成分混合物的方法，其特征是，包括以下三个步骤：S110，将茶梗/茶茎机械粉碎成平均粒径小于0.85mm的粉末，从而获得所述茶梗/茶茎微粉；S120，步骤S110中获得的所述茶梗/茶茎微粉与所述添加剂混合；S130，将步骤S120中获得混合物制备成预定的剂型。
6.一种制备权利要求1所述的用于防治炎症性肠病的含植物成分混合物的方法，其特征是，包括以下三个步骤：
S210，将修剪茶树获得的茶梗/茶茎机械粉碎成粉末；
S220，将步骤S210获得的原料粉末与溶剂混合，所述溶剂为水、乙醇或含水乙醇，所述的原料粉末与溶剂的重量比在1∶(1～20)，然后通过加热提取、微波提取或超声提取获得所述茶梗/茶茎提取物；
S230，步骤S220获得的所述茶梗/茶茎提取物与所述添加剂混合，然后制备成预定的剂型。
7.根据权利要求6所述的用于防治炎症性肠病的含植物成分混合物的制备方法，其特征是，步骤S220包括以下三个分步骤：S221，步骤S210获得的粉末与水混合，加热提取后过滤得到一次滤液和一次滤渣；S222，将所述一次滤渣与水再混合，加热第二次提取后过滤得到二次滤液；S223，合并所述一次滤液和二次滤液，浓缩得到所述茶梗/茶茎提取物。
8.根据权利要求1所述的用于防治炎症性肠病的含植物成分混合物在制备防治炎症性肠病保健品中的应用。
9.根据权利要求1所述的用于防治炎症性肠病的含植物成分混合物在制备防治炎症性肠病药品中的应用。
10.根据权利要求1所述的用于防治炎症性肠病的含植物成分混合物在制备防治炎症性肠病食品中的应用。
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