CN116251132B - 一种九蒸九制白术提取物及应用 - Google Patents
一种九蒸九制白术提取物及应用 Download PDFInfo
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- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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Abstract
本发明公开了一种九蒸九制白术提取物及应用,属于中药特色炮制技术领域。所述九蒸九制白术提取物的制备方法为:(1)将九蒸九制白术加入提取容器中,随后加入甲醇溶液,回流提取两次,合并两次提取液,减压浓缩,冷冻真空干燥即得粗提取物。(2)将所述粗提取物与硅胶混合,后加入70%vol甲醇溶解充分混匀,真空干燥后装填进分离柱,随后经20%vol甲醇洗脱,得到所述九蒸九制白术提取物。本发明的九蒸九制白术提取物能有效改善体重减轻、血便及结肠缩短等症状;抑制炎症并恢复结肠上皮紧密连接屏障,适用于气滞血瘀,脾胃虚弱,湿热内蕴证所致腹痛、腹泻、脓血便和里急后重等,方法简单,易于推广。
Description
技术领域
本发明涉及中药特色炮制技术领域,特别是涉及一种九蒸九制白术提取物及应用。
背景技术
溃疡性结肠炎(Ulcerative colitis,UC)是一种主要累及直肠、结肠粘膜的反复发作的慢性终身性疾病,属于炎症性肠病(Inflammatory bowel disease,IBD)之一,有“绿色癌症”之称。目前,国内外学者普遍认为UC发病机制与基因易感性、环境、微生物群失调、肠道屏障受损及免疫功能的过度激活高度相关。UC临床治疗主要包括升阶和降阶两大手段,代表性药物有柳氮磺胺吡啶(SASP),糖皮质激素,6-巯基嘌呤或甲氨蝶呤,维多珠单抗/乌司奴单抗/阿达木单抗/英夫利昔单抗等;此外,多菌种耐药益生菌复合产品合用益生元作为临床轻中度UC辅助治疗。然而,临床UC外科治疗用的磺胺类、激素类、免疫抑制剂类及生物制剂类药物普遍存在肠外并发症不良反应,如艰难梭状芽胞杆菌感染、肺结核、骨质疏松、腹部绞痛、过敏、菌血症等。因此,寻找一种安全、有效、稳定的可用于UC中西医联合诊疗的药物至关重要。
中医认为UC属于“久痢”、“休息痢”、“肠风”等范畴,病位在大肠,与脾肾不足、情志内伤、外感时邪、饮食不节(洁)等因素有关,病理因素主要有脾虚、肝郁、湿热、肾亏、气滞、血瘀、痰浊等。其中,土虚木乘,肝脾不和,脾失健运为UC主要病因病机。中医临床治疗UC经验方—白术芍药散来源于清·朱丹溪《丹溪心法》卷七“洞泻要方”,《医方考》记载:“泻责之脾,痛责之肝;肝则之实,脾则之虚,脾虚肝实,故令痛泻”,方中白术、白芍为君药和臣药,主以补脾柔肝,祛湿止泻为主。
越来越多的研究表明中医药治疗UC具有高效、副作用小等独特优势,被广泛地应用于临床,已显示了极具吸引力的应用前景,将为研究预防日趋严重的UC疾病提供新途径和新方法。已证实具有抗UC作用的中药有白术、白芍、陈皮、防风、茯苓等。其中,现有白术抗结肠炎公开或授权专利多集中在其中药复方抗急、慢和溃疡性结肠炎研究。主要炮制品种涵盖生白术、炒白术、麸炒白术、焦白术、土炒白术品种,尚未见有九蒸九制白术报道。
白术是“益气健脾第一要药”,为菊科植物白术的干燥根茎,主产于浙江台州、安徽亳州、河北安国等地。白术味苦,甘,性温。归脾、胃经。具有健脾益气,燥湿利水,止汗,安胎功效。近年来,白术化学成分及药理作用研究表明白术内酯类化学成分是其主要抗炎活性物质。白术炮制品包括生白术、清炒白术、米泔水制白术、土炒白术、糠炒白术、蜜糠炒白术、麸炒白术、蜜麸炒白术及九蒸九制白术等,是多种经典中药复方的重要组成药味,如白术芍药散(又名“痛泻要方”)、参苓白术散、半夏白术天麻汤等。白术不同炮制方法临床功效不同,以适应临床辨证诊治处理错综复杂的主病症。白术生用长于健脾燥湿,经蒸制后药性缓和,长于健脾益气。传统临床用于脾虚食少,腹胀泄泻,水肿等症的治疗。
目前有关九蒸九制白术特色炮制品制备,九蒸九制白术粗提取物、有效部位和活性物质在治疗溃疡性结肠炎中应用均无报道。主要原因是研究不够系统,同时提取、分离、纯化工艺上还存在不少困难。本专利前期通过体内动物和体外细胞实验研究发现,九蒸九制白术粗提取物具有显著抗UC作用,且抗UC疗效强于白术生品及其它炮制品,并产生了活性物质;此外,体外生物学等效评价实验表明九蒸九制白术提取物和九蒸九制白术活性物质同样具有显著抗UC作用。
发明内容
本发明的目的是提供一种九蒸九制白术提取物及应用,以解决上述现有技术存在的问题,该提取物适用于气滞血瘀,脾胃虚弱,湿热内蕴证所致腹痛、腹泻、脓血便和里急后重等,具有显著的抗溃疡性结肠炎作用。
为实现上述目的,本发明提供了如下方案:
本发明提供一种九蒸九制白术提取物,所述九蒸九制白术提取物的制备方法包括:
(1)将九蒸九制白术加入提取容器中,随后加入甲醇溶液,回流提取两次,合并两次提取液,减压浓缩,冷冻真空干燥即得粗提取物;
(2)将所述粗提取物与硅胶混合,后加入甲醇溶液溶解充分混匀,真空干燥后装填进分离柱,随后经甲醇溶液洗脱,得到所述九蒸九制白术提取物。
进一步的,在步骤(1)中,所述甲醇溶液的体积分数为70%。
进一步的,在步骤(1)中,所述回流提取的时间为1h,温度为80℃。
进一步的,在步骤(2)中,所述粗提取物与硅胶混合的质量比为1:3。
进一步的,在步骤(1)中,所述九蒸九制白术的制备方法包括:
将白术与米泔水混合浸泡,取出,沥干后加入山黄土包裹,而后加水蒸制,蒸制完成后取出,晒干,反复蒸晒九次,即得所述九蒸九制白术。
本发明还提供一种九蒸九制白术活性物质,所述九蒸九制白术活性物质的制备方法包括:
(1)所述的九蒸九制白术提取物经反向ODS柱分离,等度洗脱,收样,减压浓缩,真空冷冻干燥,得九蒸九制白术活性物质粗组分;
(2)将所述九蒸九制白术活性物质粗组分经液相色谱分离,梯度洗脱,收样,减压浓缩,真空冷冻干燥,得九蒸九制白术活性物质。
进一步的,在步骤(1)中,所述等度洗脱时采用的洗脱液为5%vol甲醇溶液。
进一步的,步骤(2)中所述梯度洗脱过程中的参数为:5min,10%A;10min,17%A;13min,17%A;18min,20%A;21min,20%A;25min,25%A;30min,25%A;35min,10%A;40min,10%A;所述A为无水乙醇,流动相B为正己烷,流速1.0mL/min,检测波长276nm和320nm。
本发明还提供一种利用所述的九蒸九制白术提取物或所述的九蒸九制白术活性物质在制备治疗溃疡性结肠炎的药物中的应用。
本发明还提供一种治疗溃疡性结肠炎的药物,所述药物包括九蒸九制白术提取物或所述的九蒸九制白术活性物质。
本发明公开了以下技术效果:
本发明的中药白术特色炮制品——九蒸九制白术传承古法炮制工艺(明·缪希雍《炮制大法》),并以中医药理论为基础研制而成(清·朱丹溪《丹溪心法》之“痛泻要方”),科学合理,工艺简单,安全稳定有效。能有效改善体重减轻、粘液样便、血便及结肠缩短等症状;抑制炎症并恢复结肠上皮紧密连接屏障。适用于气滞血瘀,脾胃虚弱,湿热内蕴证所致腹痛、腹泻、脓血便和里急后重等,具有显著的抗溃疡性结肠炎作用。本发明的九蒸九制白术粗提取物、九蒸九制白术提取物和九蒸九制白术活性物质具有治疗溃疡性结肠炎作用,适用于气滞血瘀,脾胃虚弱,湿热内蕴证所致腹痛,腹泻,脓血便,里急后重等,方法简单,易于推广。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为不同样品对UC小鼠体重的影响;
图2为不同样品对UC小鼠疾病活动指数评分(DAI)的影响;
图3为不同样品对UC小鼠结肠长度变化的影响;
图4为不同样品对UC小鼠结肠组织中MPO的影响;
图5为不同样品对UC小鼠结肠组织中H&E病理变化的影响;
图6为不同样品对UC小鼠结肠组织中Occludin,Claudin-1,ZO-1蛋白变化的影响,其中标尺均为50μm;
图7为不同浓度的九蒸九制白术粗提取物对结肠上皮细胞毒性评价的OD值;
图8为不同浓度的九蒸九制白术活性物质对结肠上皮细胞毒性评价的OD值;
图9为不同浓度的九蒸九制白术粗提取物抗DSS诱导的结肠上皮细胞损伤的活性评价;
图10为九蒸九制白术粗提取物、九蒸九制白术提取物和九蒸九制白术活性物质对DSS诱导的结肠上皮细胞损伤的生物等效性评价;其中Z-BZ-JZJZ-20%-Unknown-1是九蒸九制白术粗提取物中20%甲醇提取物部分所含对DSS诱导NCM460细胞损伤无保护作用的物质。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1
1.九蒸九制白术的制备:
净选粉碎后的米粉按照1:50的重量体积百分比(g:mL)加水配制米泔水溶液,称取白术按照1:9的重量体积百分比(kg:L)与米泔水混合静置,室温25℃左右、浸泡55小时且每16小时置换新鲜米泔水,既得米泔水漂白术,取出,沥干,按质量百分比(g:g)1:0.085用净选过筛后的山黄土包裹;将上述山黄土包裹的米泔水漂白术置于蒸药笼中,按质量体积百分比(kg:L)1:0.6加水,武火煮沸、文火保沸,蒸制2小时,2次,将蒸制残留药液经药材重吸收,取出,晒干,反复蒸晒九次,既得九蒸九制白术。
2.九蒸九制白术粗提取物的制备:
称取上述九蒸九制白术,将它们投入提取容器中,按照重量体积百分比(g:mL)1:10加入70%vol甲醇溶液,80℃,回流1小时,2次,合并两次提取液,减压浓缩,真空冷冻干燥既得粗提取物。
3.九蒸九制白术20%、40%、80%和100%甲醇提取物的制备:将上述所得粗提取物按质量百分比(g:g)1:3与键合相C18硅胶混合,加入70%甲醇溶解充分混匀,真空干燥后装填进iLOK快速分离柱,并串联键合相球形C18快速分离柱,然后分别通过20%vol甲醇、40%vol甲醇、80%vol甲醇和100%vol甲醇洗脱收集,减压浓缩,真空冷冻干燥既得九蒸九制白术20%甲醇提取物、九蒸九制白术40%甲醇提取物、九蒸九制白术80%甲醇提取物和九蒸九制白术100%甲醇提取物。其中,九蒸九制白术20%甲醇提取物即为九蒸九制白术提取物。
4.九蒸九制白术活性物质的制备:选取九蒸九制白术提取物,通过反向ODS柱经5%vol甲醇等度洗脱、收样,减压浓缩,真空冷冻干燥得九蒸九制白术活性物质粗组分,后经CHIRALPAKAD-H正向手性柱梯度洗脱(0-5min,10%A,90%B;5-10min,17%A,83%B;10-13min,17%A,83%B;13-18min,20%A,80%B;18-21min,20%A,80%B;21-25min,25%A,75%B;25-30min,25%A,75%B;30-35min,10%A,90%B;35-40min,10%A,90%B。流动相A为无水乙醇,流动相B为正己烷,流速1.0mL/min,检测波长276nm和320nm),收样,减压浓缩,真空冷冻干燥得九蒸九制白术活性物质。
实施例2九蒸九制白术粗提取物体内对溃疡性结肠炎的影响
1、样品性状:样品为棕褐色干浸膏。
2、剂量设计:本样品经查阅古籍名方记载、结合现代临床医生处方常用量并根据前期预实验,推荐C57BL/6小鼠给药剂量为每日4g/kg体重(折合饮片剂量为18g/kg),
具体采用各种分组组别如表1所示:
分组组别 | 分组简称 | 用量g/kg |
正常对照组 | Control | —— |
模型对照组 | 3%DSS | —— |
阳性药组 | 3%DSS+SASP | 0.4 |
生白术组 | 3%DSS+S-BZYP | 18 |
清炒白术组 | 3%DSS+QC-Z-BZ | 18 |
麸炒白术组 | 3%DSS+FC-Z-BZ | 18 |
蜜麸炒白术组 | 3%DSS+MFC-Z-BZ | 18 |
糠炒白术组 | 3%DSS+KC-Z-BZ | 18 |
蜜糠炒白术组 | 3%DSS+MKC-Z-BZ | 18 |
土炒白术组 | 3%DSS+TC-Z-BZ | 18 |
米泔水漂白术组 | 3%DSS+MGSZ-Z-BZ | 18 |
一蒸一制白术组 | 3%DSS+YZYS-1-Z-BZ | 18 |
二蒸二制白术组 | 3%DSS+EZES-2-Z-BZ | 18 |
三蒸三制白术组 | 3%DSS+SZSS-3-Z-BZ | 18 |
四蒸四制白术组 | 3%DSS+SZSS-4-Z-BZ | 18 |
五蒸五制白术组 | 3%DSS+WZWS-5-Z-BZ | 18 |
六蒸六制白术组 | 3%DSS+LZLS-6-Z-BZ | 18 |
七蒸七制白术组 | 3%DSS+QZQS-7-Z-BZ | 18 |
八蒸八制白术组 | 3%DSS+BZBS-8-Z-BZ | 18 |
九蒸九制白术组 | 3%DSS+JZJS-9-Z-BZ | 18 |
其它白术不同炮制品常规制备方法如下(各组粗提取物方法同实施例1中步骤2):
清炒白术组:取生白术饮片,中火加热翻炒,至白术呈焦黄色,取出放凉。
麸炒白术组:取1/10量麸皮撒入热锅,待冒烟加入生白术饮片,不断翻炒至焦黄色,筛去麸皮,取出放凉。
蜜麸炒白术组:取1/10量蜜炙麸皮(麸皮:熟蜜=5:1,熟蜜先加1/2量开水稀释)撒入热锅,待冒烟加入生白术饮片,不断翻炒至黄棕色,筛去蜜炙麸皮,取出放凉。
糠炒白术组:先武火将锅底烧至微红,加适量干糠,待冒青烟,加入生白术饮片,不断翻炒至微黄或黄色,逸出香气,筛糠,取出放凉。
蜜糠炒白术组:先武火将锅底烧至微红,加适量蜜糠,待冒青烟,将蜜糠铺平锅底和四周,加入生白术饮片并覆盖,盖上锅盖,密封20秒,揭盖炒至微黄或黄色,趁热筛糠,转入密闭容器转为橘黄色。
土炒白术组:取1/5量鲜黄土,与生白术饮片拌匀,小火炒至焦香味时,筛土,取出,晾晒。
米泔水漂白术组:净选粉碎后的米粉按照1:50的重量体积百分比(g:mL)加水配制米泔水溶液,称取白术按照1:9的重量体积百分比(kg:L)与米泔水混合静置,室温25℃左右、浸泡55小时且每16小时置换新鲜米泔水,既得米泔水漂白术,取出,沥干。
实验采取3%(g/mL)DSS溶液自由饮水造模,阳性药组以柳氮磺胺吡啶(SASP)灌胃,正常和模型对照组均以蒸馏水灌胃,其它各给药组除九蒸九制白术组按实施例1中步骤1九蒸九制白术的制备方法制得,其余给药组中药物均通过常规制备方法制得,将除阳性药组以及正常和模型对照组以外的其它各给药组经口连续给予相应样品9天。
3、实验方法和结果:
(1)体重:C57BL/6雄性小鼠157只,称重,按体重随机分组,分为20组。其中,正常对照组、模型对照组和阳性药组每组7只,其余各组每组8只。适应一周后,按剂量设计连续喂养9天,在实验周期中每天称重一次,体重变化如图1。
如图1所示,九蒸九制白术粗提取物组(3%DSS+JZJZ-9-Z-BZ)可显著恢复UC小鼠体重减轻,且效果优于其它白术不同炮制品。
(2)疾病活动指数(DAI):各组给药方法同上述3(1)项下,在实验周期中每天观察各组小鼠排便黏稠程度及血便程度,最终得到不同分组处理疾病活动指数如图2所示。
由图2可知,九蒸九制白术粗提取物组(3%DSS+JZJZ-9-Z-BZ)可显著降低UC小鼠DAI评分,且效果优于其它白术不同炮制品。
(3)结肠长度:各组给药方法同上述3(1)项下,于实验周期第9d后,取各组小鼠结肠。观察不同分组处理对小鼠结肠长度的影响,如图3所示。
由图3可知,九蒸九制白术粗提取物组(JZJZ)可有效延长UC小鼠结肠长度。
(4)髓过氧化物酶(MPO)ELISA试验:各组处理方法同上述3(1)项下,取各组小鼠相同位置段1cm结肠组织,按1:9质量体积比(mg/μL)加入pH=7.3PBS,按照MEIMIAN小鼠髓过氧化物酶(MPO)ELISA试剂盒操作说明处理制备待测样本,用酶标仪在450nm下检测吸光度OD值,最终得到不同分组对UC小鼠结肠组织中MPO的影响如图4所示。
由图4可知,九蒸九制白术粗提取物组(JZJZ)可显著降低UC小鼠结肠组织中性粒细胞浸润的MPO炎性指标水平,且效果优于其它白术不同炮制品。
(5)H&E病理试验:各组处理方法同上述3(1)项下,取各组小鼠结肠近端1cm结肠组织,10%福尔马林固定、石蜡包埋、切片5μm,苏木精-伊红染色,脱水、封片后显微镜下观察。最终得到不同分组对UC小鼠结肠组织中H&E病理变化的影响如图5所示。
由图5可知,九蒸九制白术粗提取物组(JZJZ-Z-BZ)可一定程度恢复UC小鼠结肠隐窝结构并减轻炎症细胞浸润水平,且效果优于生白术组。
(6)免疫荧光(IFC)染色试验:各组处理方法同上述3(1)项下,取各组小鼠结肠近端1cm往后的1cm结肠组织,OCT包埋,冷冻切片4μm,室温放置15min,预冷甲醇:丙酮(1:1)浸泡10min,室温挥干10min,1X PBS洗涤5min X 2次,5%山羊血清封闭30min,加入相应紧密连接蛋白Occludin、Claudin-1、ZO-1一抗,4℃过夜,1X PBS洗涤5min X 3次,加入相应比例荧光二抗,1X PBS洗涤5min X 3次,加入抗淬灭封片液(含DAPI),于蔡司荧光显微镜下观察。最终得到不同样品对UC小鼠结肠组织中Occludin,Claudin-1,ZO-1蛋白变化的影响如图6所示。
由图6可知,九蒸九制白术粗提取物组(JZJZ-Z-BZ)能够增加UC小鼠结肠上皮组织中紧密连接蛋白Occludin,Claudin-1,ZO-1表达,维持结肠上皮屏障功能,且效果优于生白术组。
总结:九蒸九制白术粗提取物组能有效恢复UC小鼠体重下降、减轻疾病活动期(DAI)评分、延长结肠长度、降低结肠组织中MPO炎症水平,并增加结肠组织紧密连接蛋白Occludin,Claudin-1,ZO-1表达,维持结肠组织屏障功能,且效果优于其它白术不同炮制品。
实施例3不同九蒸九制白术提取样品体外对结肠上皮细胞活性影响试验:
1、样品性状:九蒸九制白术粗提取物样品为棕褐色干浸膏、九蒸九制白术提取物为棕黄色干浸膏、九蒸九制白术活性物质为淡黄色油状物。
2、剂量设计:本样品根据相关文献报道并结合前期药物提取率和MTT药物浓度摸索预实验,推荐结肠上皮细胞(NCM460)经DSS损伤造模给药浓度为0.6μg/mL。本次实验设立正常对照组、模型对照组、九蒸九制白术粗提取物不同浓度组(6.25μg/mL、12.5μg/mL、25μg/mL、50μg/mL、100μg/mL、200μg/mL)、九蒸九制白术提取物组(0.66μg/mL),正常和模型对照组均以蒸馏水灌胃。
3、实验方法和结果:
(1)毒性评价:正常结肠上皮细胞(NCM460)培养于RPMI 1640培养基中,细胞密度调整为105个/mL,以100μL/孔接种于96孔板中,待细胞贴壁后给予不含血清的培养基配置的相应浓度药物,37℃,5%CO2培养,24h后,避光加入10μL/孔MTT溶液,3h后加入150μL/孔的DMSO溶液,酶标仪490nm下检测吸光度OD值。不同浓度的九蒸九制白术粗提取物的OD值如图7所示,不同浓度的九蒸九制白术活性物质的OD值如图8所示。
由图7可知,九蒸九制白术粗提取物(Z-BZ-JZJZ)在200μg/mL剂量以下对正常结肠上皮细胞(NCM460)无明显毒性作用,400μg/mL时显著影响NCM460细胞活力。由图8可知,九蒸九制白术活性物质(Z-BZ-JZJZ-20%-Unknown-2)在10.56μg/mL剂量以下对NCM460无明显毒性作用,21.12μg/mL时显著影响NCM460细胞活力。
(2)活性评价:正常结肠上皮细胞(NCM460)损伤模型采用DSS(0.6μg/mL)诱导,正常结肠上皮细胞(NCM460)以5×104个/mL密度接种于96孔板,24h后,给予含血清的培养基配置的DSS造模剂(0.6μg/mL)诱导刺激,给药及检测方法同上述3(1)项下。最终得到不同浓度的九蒸九制白术粗提取物抗DSS诱导的结肠上皮细胞损伤的活性评价如图9所示。
由图9可知,九蒸九制白术粗提取物(Z-BZ-JZJZ)在低浓度6.25μg/mL剂量下可显著改善DSS(0.6μg/mL)诱导的结肠上皮细胞(NCM460)损伤,且呈一定的剂量依赖性。
(3)生物等效评价:根据上述九蒸九制白术粗提取物毒性和活性评价,选取100μg/mL给药剂量的九蒸九制白术粗提取物(JZJZ-9)进行后续相关生物等效评价实验,给药及检测方法同上述步骤3(1)项。最终得到九蒸九制白术粗提取物、九蒸九制白术提取物和九蒸九制白术活性物质对DSS诱导的结肠上皮细胞损伤的生物等效性评价如图10所示。
由图10可知,九蒸九制白术粗提取物(JZJZ-9)具有明显的抗DSS(0.6μg/mL)诱导的结肠上皮细胞(NCM460)损伤作用;且根据提取率由该剂量(100μg/mL)折算得到的九蒸九制白术提取物(JZJZ-9-20%)及九蒸九制白术活性物质(JZJZ-9-20%-Unknown-2)给药组,体外均呈显著的抗DSS(0.6μg/mL)诱导的结肠上皮细胞(NCM460)损伤作用,且两者效果相近并优于九蒸九制白术粗提取物(JZJZ-9)。
总结:九蒸九制白术粗提取物、九蒸九制白术提取物及九蒸九制白术活性物质体外均能有效的抗DSS(0.6μg/mL)诱导的结肠上皮细胞(NCM460)损伤,且九蒸九制白术提取物和九蒸九制白术活性物质效果优于九蒸九制白术粗提取物。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (5)
1.一种治疗溃疡性结肠炎的九蒸九制白术提取物,其特征在于,所述九蒸九制白术提取物的制备方法包括:
(1)将九蒸九制白术加入提取容器中,随后加入70%甲醇溶液,回流提取两次,合并两次提取液,减压浓缩,冷冻真空干燥,得到粗提取物;
所述回流提取的时间为1h,温度为80℃;
所述九蒸九制白术的制备方法为:将白术与米泔水按照1:9的重量体积百分比混合浸泡,室温25℃左右、浸泡55小时且每16小时置换新鲜米泔水,取出,沥干后按质量百分比1:0.085加入山黄土包裹,而后按质量体积百分比1:0.6加水蒸制,武火煮沸、文火保沸,蒸制2小时,2次,将蒸制残留药液经药材重吸收,取出,晒干,反复蒸晒九次,即得所述九蒸九制白术;
(2)将所述粗提取物与硅胶混合,后加入70%甲醇溶液溶解充分混匀,真空干燥后装填进分离柱,随后经20%vol甲醇溶液洗脱,得到所述九蒸九制白术提取物。
2.根据权利要求1所述的九蒸九制白术提取物,其特征在于,在步骤(2)中,所述粗提取物与硅胶混合的质量比为1:3。
3.一种治疗溃疡性结肠炎的九蒸九制白术活性物质,其特征在于,所述九蒸九制白术活性物质的制备方法包括:
(1)权利要求1-2任一项所述的九蒸九制白术提取物经反向ODS柱分离,等度洗脱,收样,减压浓缩,真空冷冻干燥,得九蒸九制白术活性物质粗组分;
所述等度洗脱时采用的洗脱液为5%vol甲醇溶液;
(2)将所述九蒸九制白术活性物质粗组分经液相色谱分离,梯度洗脱,收样,减压浓缩,真空冷冻干燥,得九蒸九制白术活性物质;
所述梯度洗脱过程中的参数为:0-5min,10%A,90%B;5-10min,17%A,83%B;10-13min,17%A,83%B;13-18min,20%A,80%B;18-21min,20%A,80%B;21-25min,25%A,75%B;25-30min,25%A,75%B;30-35min,10%A,90%B;35-40min,10%A,90%B;流动相A为无水乙醇,流动相B为正己烷,流速1.0mL/min,检测波长276nm和320nm。
4.一种利用权利要求1-2任一项所述的九蒸九制白术提取物或权利要求3所述的九蒸九制白术活性物质在制备治疗溃疡性结肠炎的药物中的应用。
5.一种治疗溃疡性结肠炎的药物,其特征在于,所述药物为权利要求1-2任一项所述的九蒸九制白术提取物或权利要求3所述的九蒸九制白术活性物质。
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