CN111202789A - 以五种果蔬为原料的通便组方增加肠道水分、润滑肠道的新应用 - Google Patents
以五种果蔬为原料的通便组方增加肠道水分、润滑肠道的新应用 Download PDFInfo
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- CN111202789A CN111202789A CN201911109614.2A CN201911109614A CN111202789A CN 111202789 A CN111202789 A CN 111202789A CN 201911109614 A CN201911109614 A CN 201911109614A CN 111202789 A CN111202789 A CN 111202789A
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Abstract
五种果蔬为原料的通便组方的新用途。本发明提供了五种果蔬为原料的通便组方在制备增加肠道水分、润滑肠道的食品或者药品中的用途：所述的通便组方是由5‑20μm细度的南瓜、芦荟、茯苓、银耳、低聚木糖组成的变通牌天天胶囊。本发明研究发现，该通便组方会通过激活鸟苷酸环化酶(GC‑C)来刺激肠液的分泌，由于肠液分泌的增加，导致AQP3的表达量增加，将肠液从肠腔传输到血管中。同时该组方通过刺激AQP9的表达，增加粘液的分泌。为增加肠道水分、润滑肠道提供了一种新的手段，具有广阔的临床应用前景。
Description
技术领域
本发明涉及通便组方及其新用途，属于中药领域。
背景技术
一、水通道蛋白
水通道蛋白(Aquaporin)，又名水孔蛋白，是一种位于细胞膜上的蛋白质(内在膜蛋白)，在细胞膜上组成"孔道"，可控制水在细胞的进出，就像是"细胞的水泵"一样。
水通道是由约翰霍普金斯大学医学院的美国科学家彼得·阿格雷所发现，他与通过X射线晶体学技术确认钾离子通道结构的洛克斐勒大学霍华休斯医学研究中心的罗德里克·麦金农共同荣获了2003年诺贝尔化学奖。
Agre等在分离纯化红细胞膜上的Rh多肽时，发现了一个28kD的疏水性跨膜蛋白，称为形成通道的整合膜蛋白28(channel-forminginte-gralmembraneprotein，CHIP28)，1991年完成了其cDNA克隆(Verkman，2003)。但当时并不知道该蛋白的功能，在进行功能鉴定时，将体外转录合成的CHIP28mDNA注入非洲爪蟾的***中，发现在低渗溶液中，***迅速膨胀，并于5min内破裂。为进一步确定其功能，又将其构于蛋白磷脂体内，通过活化能及渗透系数的测定及后来的抑制剂敏感性等研究，证实其为水通道蛋白。从此确定了细胞膜上存在转运水的特异性通道蛋白，并称CHIP28为Aquaporinl(AQPl)。水分子经过Aquaporin时会形成单一纵列，进入弯曲狭窄的通道内，内部的偶极力与极性会帮助水分子旋转，以适当角度穿越狭窄的通道，因此Aquaporin的蛋白构形为仅能使水分子通过之原因。水通道蛋白 (AQPs)广泛的分布于不同的组织细胞中，其作用主要是选择性地运输水和甘油通过水通道。目前AQPs有13种类型的，其中，AQP3在结肠粘膜上皮细胞中表达量有较高的表达。在生理条件下，水通过AQP3从渗透压较低的肠腔输送到渗透压较高的血管侧。当出现慢传输型便秘时，AQP3的表达量降低。而用刺激性泻剂进行干预时，AQP3的表达量升高。AQP9特异性地存在于粘液分泌的杯状细胞，并参与某种粘液的合成和/或分泌，这种粘液保护肠表面并使肠内容物的流动平滑。
据研究报道GC-C/cGMP信号轴已经成为调节肠道正常液体和电解质稳态的关键机制，激活结肠上皮细胞上的GC-C，细胞内第二信使环磷酸鸟苷(cGMP)的浓度增加，细胞内cGMP 的水平升高激活cGMP依赖性蛋白激酶II(PKG-II)，导致磷酸化和激活囊性纤维化跨膜传导调节因子(CFTR)，CFTR活化导致氯离子和碳酸氢根离子的分泌和钠吸收的抑制，导致水流量增加，肠和胃肠道转运的加速。
二、腹泻
肠道吸收水分增加是指小肠吸收不良，而导致肠道对水分的吸收增多，从而引起身体的不适。肠道吸收水分增加会引起腹泻，腹泻(diarrhea)是一种常见症状，是指排便次数明显超过平日习惯的频率，粪质稀薄，水分增加，每日排便量超过200g，或含未消化食物或脓血、粘液。腹泻常伴有排便急迫感、***不适、失禁等症状。腹泻分急性和慢性两类。急性腹泻发病急剧，病程在2～3周之内。慢性腹泻指病程在两个月以上或间歇期在2～4周内的复发性腹泻。
腹泻的原发疾病或病因诊断主要须从病史、症状、体征、常规化验特别是粪便检验中获得依据。许多病例通过仔细分析病史和上述检查的初步结果，往往可以得出正确诊断。如诊断仍不清楚，可进一步作X线钡灌肠和钡餐检查，和(或)直、结肠镜检查。如仍无明确结论，则须根据不同情况选用超声、CT、内镜逆行胆胰管造影(ERCP)等影象诊断方法以检查胆、胰疾病，或进行小肠吸收功能试验、呼气试验、小肠粘膜活检以检查小肠吸收不良。高度怀疑肠结核、肠阿米巴病等有特效治疗的疾病，经过努力都不能确诊时，可在一定限期内进行治疗试验。
1.婴幼儿急疹：多见于1岁以下的小儿，6个月以下的小儿更多见，皮疹出现前常有发热，有些伴有腹泻，但这种腹泻经过治疗易缓解，皮疹出现后，腹泻症状和发热一样，也消失了。
2.胃肠型感冒：多见于挑食的小儿，这样的小儿看上去就是有点消瘦，舌苔也常有地图舌和花剥苔，如果仔细问病史，家长会告诉你，以前发热感冒都会呕吐和腹泻，胃肠型感冒，以呕吐症状较常见，腹泻较轻，常不被重视。
3.细菌性肠炎，尤其是侵袭性肠炎早期，在脓血便出现之前，可以仅有呕吐或稀便，有些有轻度发热，此时就诊，极易误诊，常是治疗后不久家长又来找你了：小孩又发热了，拉脓血便了，有腹痛了。总的来说：鉴别诊断也有取巧的办法：对于鉴别细菌性和病毒性腹泻，年龄是个关键，一般1岁以下的小儿，以母乳和奶粉喂养为主，接触污染食物的机会不大，所以以病毒性腹泻多见。而年长儿童，进食种类复杂，较易食入细菌污染的食物，尤其是夏秋季，以细菌性腹泻为主。
腹泻的治疗
腹泻是症状，根本治疗要针对病因。认识腹泻的发病机理有助于掌握治疗原则。
(一)病因治疗不言而喻，肠道感染引起的腹泻必需抗感染治疗，以针对病原体的抗菌治疗最为理想。复方新诺明、氟哌酸(诺氟沙星)、环丙氟哌酸(环丙沙星)、氟嗪酸(氧氟沙星)对菌痢，沙门菌或产毒性大肠杆菌，螺杆菌感染有效，甲硝唑对溶组织阿米巴、梨形鞭毛虫感染有效，因此，这数种药物常用于急性感染性腹泻，包括预防和治疗所谓旅行者腹泻。治疗乳糖不耐受症和麦胶性乳糜泻所致的腹泻在饮食中分别剔除乳糖或麦胶类成发。高渗性腹泻的的治疗原则是停食或停用造成高渗的食物或药物。分泌性腹泻易致严重脱水和电解质丢失，除消除病因，还应积极由口服和静脉补充盐类和葡萄糖溶液，纠正脱水。胆盐重吸收障碍引起的结肠腹泻可用消胆胺吸附胆汁酸而止泻。治疗胆汁酸缺乏所致的脂肪泻，可用中链脂肪代替日常食用的长链脂肪，因前者不需经结合胆盐水解和微胶粒形成等过程而直接经门静脉***吸收。
(二)对症治疗选择药物时，应避免成瘾性药物，必要时也只能短暂使用。病因治疗是主要的，凡病因不明者，尽管经对症治疗后症状已有好转，绝不可放松或取消应有的检查步骤，对尚未排除恶性疾病的病例尤其如此。
1.止泻药常用的有活性炭、鞣酸蛋白、次碳酸铋、氢氧化铝凝胶等，日服3～4次。药效较强的复方樟脑酊(3～5ml)和可待因(0.03g)，每日2～3次。因久用可成瘾，故只短期适用于腹泻过频的病倒。复方苯乙哌酊(每片含苯乙哌啶2.5mg和阿托品0.025mg)，每次1～2片，2～ 4/d，此药有加强中枢抑制的作用，不宜与巴比妥类、阿片类药物合用。氯苯哌酰胺(咯派丁胺， loperamide)的药效较复方苯乙哌啶更强且持久，不含阿托品，较少中枢反应。初服4mg，以后调整剂量至大便次数减至1～2次/d，日量不宜超过8mg。培菲康可调节肠道功能。
2.解痉止痛剂可选用阿托品、普鲁本辛、山莨菪碱、普鲁卡因等药。
3.镇静药可选用安定、利眠宁、***类药物。
4.调节肠道植物神经紊乱药可选解郁抗虑胶囊等药物。
(三)考虑到吃西药副作用很大，可以考虑服用中成药进行治疗。优点是中药制剂没有毒副作用，治疗效果比较理想，缺点是服用时间比较长一般的患者都需要按疗程进行服用。
(四)蒙脱石散
抗生素、微生态、中成药等方法的治疗都会或多或少的具有毒性，可能伤害到肠道内的健康菌群。而新型治疗药物蒙脱石散，可以利用蒙脱石的层纹状结构及非均匀性电荷分布，对消化道内的病毒、病菌及其产生的毒素有固定、一直作用对消化道粘膜有覆盖能力，并通过与粘液糖蛋白相互结合，从质和量两方面修复、提高粘膜屏障对攻击因子的防御功能。并且药物成分不进入血液循环***，并连同所固定的供给因子随消化道自身蠕动排出体外。不改变正常的肠蠕动。可选择药物：司迈特蒙脱石散(3g成人儿童装、1g婴幼儿装)。
三、便秘是临床常见的一种症状，而不是一种具体的疾病，主要是指排便次数减少、粪便量减少、粪便干结及排便费力等。上述症状同时存在2种以上时，可诊断为症状性便秘。便秘通常以排便频率减少为主，一般每2～3天或更长时间排便1次(或每周<3次)。食物被消化吸收后，食物残渣留在大肠内，其中一部分水分被大肠粘膜吸收，同时经过细菌的发酵和腐败变为粪便，再通过肠道的蠕动讲粪便排出体外。一旦肠道健康出了问题，肠道无法正常工作，就容易出现便秘。
肠道就是人体***中最大、最主要的排毒器官。人体90％以上的毒素经由肠道排出体外，肠道排毒最关键、最有效的途径就是排便。人体内每天产生的有毒、有害物质多达1000多种，人体只有不断将这些危害健康的毒素排出体外，才能保持身体各种机能的正常运行。如果人不能做到每天排便，人体内脂肪、糖、蛋白质等物质新陈代谢产生的废物和肠道内的食物残渣，就会积存在肠道内，形成腐败的陈便，我们将之称为宿便。中老年习惯性便秘的人，最容易患直肠癌。由于胃肠蠕动减慢，消化能力减弱，干燥的粪便可在肠道内滞留较长时间，这样以来，加重了粪便内致癌物对肠道黏膜的刺激，诱发直肠癌。
1、以单纯通便为目的，属于“头痛医头脚痛医脚”，不能调节肠道健康，并没有彻底的解决产生便秘的根本原因，所以说出现“用就通，不用就不通”的现象也就不足为奇了。
2、很多长期便秘的患者，单纯的追求通便的速度快，从而选择一些含有番泻叶、大黄、决明子等泻药成分的刺激性导泻药。这些导泻药在我们肠道内的作用就像是高压水枪一样，虽然能够带走宿便，但是同时也会将肠道内的益生菌一同带走破坏了肠道内的菌群结构，一旦停用，便秘会更加严重，而且长期服用，还会引发更多的肠道疾病。
3、而以中药为组方的产品，由于中药材使用人群的局限性，并不适合所有类型的便秘。
本发明通便组方是金建文教授研制发明的果蔬组合物，针对便秘的发病机理，首创运用“通调养”理论获得国家食品药品监督管理局保健食品批准文号(国食健字G20040762)：组方中的芦荟起到通的作用，因为芦荟中中含有丰富的芦荟甙，芦荟甙被称为肠道清道夫，帮助排出肠道内毒素，净化肠道血液，促进肠道蠕动，快速排出肠道内的宿便。南瓜、茯苓、银耳，均富含粗多糖，可以润滑肠道，软化干硬的宿便，同时修复受损的肠黏膜，恢复肠道的自主排便能力。其中南瓜独有的保健功能因子，还可以能改善肠道微循环，血液循环旺盛了，肠蠕动就正常了。茯苓和银耳具有滋阴、健脾、扶正固本、保护大肠的作用。低聚木糖可以大量增殖肠道内的双歧杆菌等有益菌，同时抑制有害菌的繁殖，使肠道内菌群结构更加合理。大量的有益菌可以形成健康的肠道内环境，使肠道始终处于健康状态。
发明内容
本发明的目的是在现有技术的基础上提供以五种果蔬为原料的通便组方的几种新临床用途。
下面对本发明涉及的内容逐一进行详细说明
我们经过动物实验和临床研究，发现本发明组合物具有以下的临床新用途，并最终通过科学试验得到证明，从而完成本发明，其具体的新用途包括以下几种：
1、本发明组合物在制备调节水通道蛋白的药物或食品中的应用。
2、本发明组合物在制备增加肠道水分、润滑肠道的药物或食品中的应用。
附图说明
图1通便组方(以下皆称作TTC)对LOP诱导的便秘型大鼠水液代谢的影响；正常组A，模型组B，阳性药组C，低剂量组D，高剂量组E
具体实施方式
下面通过具体的药理和临床实施例对本发明所提供的新用途予以进一步的说明
实施例1水通道蛋白药理实验
为了研究TTC是否对是否水液代谢有影响。本实验利用IHC检测结肠组织中AQP9的表达和WB检测结肠组织中AQP3、AQP9以及GC-C/cGMP的表达变化。
1样本采集与保存
血清样本：实验3周后，大鼠禁食12h，称重，然后用10％水合氯醛(0.3mL/100g)麻醉，进行腹主动脉采血。取出的血液自然凝固后，3000rpm离心10min得到血清样本，置于-80℃保存待测；
组织样本：实验3周后，大鼠禁食12h，称重，然后用10％水合氯醛(0.3mL/100g)麻醉，立即取出结肠组织，取出的结肠组织用生理盐水漂洗并用医用纱布吸干水分，然后置于-80℃保存待测。
2生化指标测定
血清中胃动素(MTL)、内皮素(ET-1)、生长抑素(SS)、血管活性肠肽(VIP)、P物质(SP)水平按试剂盒说明书要求进行检测。
3结肠病理学检查
取结肠组织，用10％的中性***液固定48h，石蜡包埋，切成5μm厚的切片，进行 H&E染色，在荧光倒置显微镜下观察。
4免疫组化检测(c-kit、AQP9)
1)脱蜡、水化：将石蜡切片先在室温放置60min,用甲苯脱蜡；再用不同梯度的酒精浸泡将其水化。
2)阻断：3％双氧水室温孵育10-30min，以灭活过氧化物酶的活性。蒸馏水冲洗，PBS 浸泡5min×2次。
3)抗原修复：切片放入盛有0.01％枸橼酸钠溶液中，在95℃煮沸15-20min，冷却20min 以上，再用冷水冲洗缸子，冷却至室温，进行抗原修复。PBS浸泡，5min×2次，甩干。
4)血清封闭：在室温条件下，用血清工作液孵育15-30min，倾去，勿洗。
5)一抗孵育：以抗体说明书为参考稀释抗体(c-kit按照1:500的比例用一抗稀释液进行稀释；AQP9按照1:100的比例用一抗稀释液进行稀释)，4℃过夜。PBS冲洗，3min×5次。
6)二抗孵育：孵育生物标记素二抗，室温孵育60min；PBS冲洗，3min×5次；滴加SP，室温或37℃孵育30min-60min；PBS冲洗5min×3次；
7)DAB显色：DAB显色5-10min，在显微镜下掌握染色程度(显色剂的配制：在1ml水中加1滴显色剂A，摇匀，然后加1滴显色剂B，摇匀，再加1滴显色剂C，摇匀)；自来水冲洗10min终止反应。
8)复染：放入苏木素染液复染2-10min，自来水冲洗10min-15min终止反应.
9)常规脱水、透明、封片。
10)光学显微镜下观察组织细胞中蛋白的表达情况，取3个区域拍照保存。
5Westernblot检测结肠组织的蛋白表达
5.1组织中蛋白的提取
1)取冻存的结肠组织50mg置入1.5mlEP管中，在EP管中加入500ul的强裂解液，并在自动研磨机中研磨2min。
2)研磨结束后，12000rpm，4℃离心30min，吸取上清，采用BCA法进行蛋白浓度测定
5.2BCA法检测蛋白浓度
1)将浓度为0.5mg/ml的蛋白标准品按0、1、2、4、8、12、16和20μl加到96孔板的标准品孔中，并用PBS将每孔补足到20μl(标准蛋白浓度分别为0、0.025、0.05、0.1、0.2、 0.3、0.4和0.5mg/ml)，作为标准曲线。
2)用PBS或裂解液将蛋白上清液稀释40倍(5μl蛋白上清加入到195μl的PBS或裂解液中)，涡旋离心，以每孔20μl加入到96孔板中，每组设置三个复孔。
3)按50体积BCA试剂A加1体积BCA试剂B(50:1)比例配制适量BCA工作液(现配现用)，涡旋混匀；每孔加入200μlBCA工作液，37℃孵育30min。
4)用酶标仪测定562nm波长下的吸光度值，并根据标准曲线和稀释倍数计算样品的蛋白浓度。
5.3蛋白样品的制备
1)用PBS将各组蛋白稀释成相同浓度，加入1/4总体积的SDS-PAGE蛋白上样缓冲液(5 ×)。
2)在金属加热器中，100o C加热5min，以充分变性蛋白。
3)室温冷却后，-80℃储存备用
5.4Westernblot操作过程
1)试漏：将大小玻璃板对齐后放入夹中夹紧，垂直卡在架子上，用双蒸水将玻璃板灌满，放置15min检漏。检漏结束后，将水倒掉并用吸水滤纸将残余水分吸干。
2)配胶：根据蛋白分子量的大小选择合适的分离胶胶浓度(6％gel，>200kD；7.5％gel， 120-200kD；8-10％gel，40-120kD；12％gel，15-40kD；15％gel，<20kD)进行配置。
3)灌胶：首先灌入分离胶至适宜高度，加水封胶，放置至分离胶凝固，倒去上层的蒸馏水并用吸水滤纸将残余水分吸干。用浓缩胶灌满玻璃板的剩余空间，立即***梳子(切勿产生气泡)。待浓缩胶凝固后，轻轻拔出梳子，装入电泳***。
4)上样电泳：将制备好的蛋白样品约50μg加入上样孔中。恒压80V，待样品刚好跑出浓缩胶后，将电压调为120V，恒压电泳至分离胶底部即可。
5)裁胶：按照Marker指示的分子量大小选取裁胶范围，裁剪厚滤纸和NC膜，并将三者的大小裁剪的尽可能相近。
6)转膜：将胶、滤纸和NC膜在转膜液中平衡5min，按照滤纸-膜-胶-滤纸从下到上在半干转仪上依次叠放好，根据蛋白分子量选择合适的转膜条件。
7)封闭：将NC膜取出，在TBST中洗涤2次，每次30s(去除残留在NC膜上的转膜液)，洗涤结束后，5％BSA室温封闭1-2h。
8)一抗孵育：封闭结束后，将NC膜在TBST中洗涤10s，参照抗体说明书以合适的比例稀释抗体(SCF、AQP3、和GC-C按照1:1000的比例用一抗稀释液进行稀释；c-kit和AQP9 按照1:500的比例用一抗稀释液进行稀释)，然后将NC膜放置在稀释好的一抗中，室温孵育 2h或4℃摇床孵育过夜。
9)二抗孵育：一抗孵育结束后，回收抗体，TBST洗涤NC膜，洗涤3次，每次5min，吸去NC膜上残留的洗膜液，将NC膜放入稀释好的二抗中(按1:10000-1:30000稀释)，室温摇床孵育1-2h。
10)扫描：二抗孵育结束后，避光条件下，洗膜3次，每次5min，吸去NC膜上残留的洗膜液，将NC膜置于Odyssey红外荧光扫描成像***中扫描。
11)分析：ImageJ软件进行定量分析。
1.3统计分析
数据以平均值±标准差(Mean±SD)表示，使用SPSS22.0软件进行统计分析。两组间比较采用非配对的双边t检验(two-tailedunpairedStudent’sttest)；多组间比较采用完全随机设计的单因素方差分析(one-wayANOVA)，方差齐性采用LSD法或SNK法检验，方差不齐采用非参数检验(Kruskal-WallisH检验)方法。假设检验水准按α＝0.05判定。P<0.05表示差异有统计学意义。
IHC(图1，(A))实验结果显示，AQP9在结肠组织中有表达；
WB实验结果(图1)表明:
与正常组相比，模型组中AQP3的表达量显著降低；与模型组相比，阳性药组和TTC低、高剂量组中AQP3的表达量显著性增加。
与正常组相比，模型组中AQP9的表达量显著降低；与模型组相比，阳性药组和TTC低剂量组中AQP9的表达量显著性增加，TTC高剂量组中AQP9的表达量呈增长趋势，但并没有显著性差异。
与正常组相比，模型组中GC-C的表达量显著降低；与模型组相比，TTC低、高剂量组中 GC-C的表达量显著性加,而模型组中GC-C的表达量没有显著性的差异。
上述数据表明：TTC会通过激活鸟苷酸环化酶(GC-C)来刺激肠液的分泌，由于肠液分泌的增加，导致AQP3的表达量增加，将肠液从肠腔传输到血管中。同时TTC通过刺激AQP9的表达，增加粘液的分泌。
实施例2
1、实验动物：NIH小鼠，SPF级，体质量18～23g，雌雄兼用。
2、药物：TTC药粉；复方地酚诺酯。
3、试剂：石蜡油，***，番泻叶水。
4、仪器：FA1104电子分析天平，X102血压计，医用导尿管(8号)。
5、TTC对番泻叶致小鼠腹泻模型的止泻作用：将NIH小鼠(雌雄各半)随机分为模型组， TTC低、高剂量组，复方地酚诺酯组(剂量为2.6mg·kg/d)。
TTC组预先给药3d，每日1次，复方地酚诺酯组在第3天给药1次。末次给药前禁食12h，给药1h后，小鼠灌服1kg/L番泻叶煎剂0.5mL/只。然后将小鼠放在垫有滤纸的铁丝网上，每只小鼠上罩一大玻璃漏斗，每隔2h换1次滤纸。分别计2h、4h、6h3个时间段内腹泻的次数 (软便、水样便、粘液便称为腹泻便)，比较给药组与模型对照组的统计学差异。
7、统计学方法采用SPSS11.0软件包进行单因素方差分析，实验结果以均数±标准差(x ±s)表示。
8、结果：
TTC对番泻叶致小鼠腹泻模型的止泻作用(见表1)结果显示：TTC低剂量组4h、6h累计排便次数较模型组减少，TTC高剂量组4h、6h累计排便次数较模型组显著减少(P＜0.05或P＜0.01)，提示TTC有一定的止泻作用。
表1各组腹泻小鼠模型的止泻作用比较(x±s)
统计方法:单因素方差分析；①P＜0.05，②P＜0.01，与模型组比较。
Claims (3)
1.以五种果蔬为原料的通便组方增加肠道水分、润滑肠道的新应用，所述通便组方是由5-20μm细度的南瓜、芦荟、茯苓、银耳、低聚木糖组成。
2.按权利要求1所述的通便组方在制备调节水通道蛋白的药物或食品中的应用。
3.按权利要求1所述的通便组方在制备增加肠道水分、润滑肠道的药物或食品中的应用。
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