CN116889574B - 白扁豆多糖在制备预防和/或治疗溃疡性结肠炎的药物中的应用 - Google Patents
白扁豆多糖在制备预防和/或治疗溃疡性结肠炎的药物中的应用 Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- 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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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
本发明公开了白扁豆多糖在制备预防和/或治疗溃疡性结肠炎的药物中的应用。白扁豆多糖由如下步骤制备得到:(1)向白扁豆果实粗粉中加入乙醇提取,提取液过滤得滤渣,取滤渣重复提取,滤渣烘干得白扁豆果实脱脂粉末;(2)取白扁豆果实脱脂粉末,加入水进行提取得到提取液,收集上清液,滤渣重复提取,合并提取液,提取液真空浓缩得到浓缩液;(3)向浓缩液中加入无水乙醇,搅拌均匀后静置离心后的沉淀物复溶于水中,加入Sevage试剂,离心处理后合并上清液,继续加入Sevage试剂离心处理,真空冷冻干燥后得白扁豆多糖。本发明为白扁豆多糖用于预防和治疗UC提供了科学依据,也为以后相关的产业转化(比如:茶包)提供参考。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及白扁豆多糖在制备预防和/或治疗溃疡性结肠炎的药物中的应用。
背景技术
溃疡性结肠炎(ulcerative colitis,UC)是一种病因不明的世界性疾病,青壮年患病率高,易癌变,难治愈,有反复发作的特征,对社会的经济稳定和人群健康构成重大威胁。在我国,UC的发病率逐年上升,预计到2025年UC患者达到150万。据统计UC经过***治疗仍然有50%的患者临床症状未取得好转。现代医学治疗UC效果欠佳的原因可能是靶点单一,而中医药在治疗UC方面是多靶点治疗,有一定的优势(提高肠道黏膜愈合率,减少复发,减轻激素依赖等)。UC发的病因病机主要是内因脾虚,外受六淫,情志,饮食环境等因素诱发,病位主要位于大肠也与脾,肾,肝诸脏有关。病理性质为本虚标实。病理因素主要有:①湿邪(热);②瘀热;③热毒;④痰浊;⑤气滞;⑥血瘀等。病理特征表现:活动期多属实证,主要病机为湿热蕴肠,气血不调,而重度以热毒、瘀热为主,反复难愈者应考虑痰浊血瘀的因素。缓解期多属虚实夹杂,主要病机为脾虚湿恋,运化失健。健脾祛湿是中医药治疗UC的基本治法。白扁豆多糖在提高人体免疫功能、抗肿瘤、保护酒精性肝损伤、抗氧化及降血糖等过程中起着重要作用,但是是否能够在UC中发挥治疗作用尚未有文献报道,亟待解决该问题。
发明内容
本发明的目的是提供一种白扁豆多糖在制备预防和/或治疗溃疡性结肠炎的药物中的应用,本发明为白扁豆多糖用于预防和治疗UC提供了科学依据,也为以后相关的产业转化(比如:茶包)提供参考。
本发明是通过以下技术方案予以实现的:
本发明的第一个目的是提供白扁豆多糖在制备预防和/或治疗溃疡性结肠炎的药物中的应用。经过实验发现,白扁豆多糖能够长期进行饮食调理用于减轻UC的炎症反应和改善肠道菌群分布。
优选地,所述的白扁豆多糖由如下步骤制备得到:
(1)向过30-50目筛的白扁豆果实粗粉中加入乙醇提取,提取液过滤得滤渣,取滤渣重复提取若干次,滤渣烘干得白扁豆果实脱脂粉末;
(2)取步骤(1)得到的白扁豆果实脱脂粉末,加入水进行提取得到提取液,提取液过滤后收集上清液,滤渣重复提取若干次,合并提取液,提取液真空浓缩得到浓缩液;
(3)向步骤(2)得到的浓缩液中加入无水乙醇,搅拌均匀后静置,离心后的沉淀物复溶于水中,加入Sevage试剂,离心处理后合并上清液,继续加入 Sevage 试剂震荡并离心处理,直至溶液中无变性蛋白质出现为止,真空浓缩除去溶液中的有机试剂,真空冷冻干燥后得白扁豆多糖。
进一步优选,步骤(1)具体步骤为:向过30-50目筛的白扁豆果实粗粉中加入体积分数为95%的乙醇溶液提取,白扁豆果实粗粉与乙醇溶液的质量体积比为1:15-25 g/mL,75°C-85°C提取2.5-3.5 h,提取液过滤得滤渣,取滤渣重复提取若干次,最后将滤渣烘干得白扁豆果实脱脂粉末。
进一步优选,步骤(2)中提取条件为:白扁豆果实脱脂粉末与水的质量体积比为1:15-25 g/mL,90°C-100°C提取2.5-3.5 h。
进一步优选,步骤(3)向步骤(2)得到的浓缩液中加入无水乙醇,搅拌均匀后静置,离心后的沉淀物复溶于水中,加入Sevage试剂,离心处理后合并上清液,继续加入 Sevage试剂震荡并离心处理的具体步骤为:向步骤(2)得到的浓缩液中加入无水乙醇,浓缩液与无水乙醇的体积比为1:3-5,搅拌均匀后静置,离心后的沉淀物复溶于水中,加入Sevage试剂,水与Sevage试剂的体积比为4-6:1,Sevage试剂是体积比为4:1的氯仿和正丁醇的混合物,离心处理后合并上清液,向合并的上清液中继续加入 Sevage 试剂震荡并离心处理,合并的上清液与Sevage试剂的体积比为4-6:1。
本发明第二个目的是提供一种预防和/或治疗溃疡性结肠炎的药物,含有所述的白扁豆多糖作为活性成分。以目标对象的体重为准,所述的白扁豆多糖的用量为100-200mg/kg。
与现有技术相比,本发明的有益效果是:本发明提出白扁豆多糖能够减轻UC小鼠模型肠道炎症反应并且机制与抑制肠道炎症反应有关。鉴于白扁豆是药食同源的属性,临床用药安全性较高,本研究结果为白扁豆多糖用于预防和治疗UC提供了科学依据,也为以后相关的产业转化提供参考。
附图说明
图1为白扁豆多糖治疗UC小鼠模型的影响,其中,图1A和1B代表各组结肠长度;图1C代表各组便血程度;图1D代表各组疾病活动指数;图1E代表各组病理结构;图1F代表各组肠道组织IL-1β的mRNA含量;图1G代表各组肠道组织TNF-α的mRNA含量;图1中,control:对照组;3%DSS:模型组;Mesalazine:阳性受试药美沙拉秦组;BM:白扁豆多糖中剂量组;BH:白扁豆多糖高剂量组。(#:与正常组,P<0.05;##:与正常组相比,P<0.01;*:与模型组相比,P<0.05;**:与模型组相比,P<0.01)。
图2为白扁豆多糖对UC小鼠外周血清的影响,图2A代表各组外周血清TNF-α的相对含量;图2B代表各组外周血清IL-1β的相对含量;图2C代表各组外周血清IL-6的相对含量;图2D代表各组外周血清IL-10的相对含量;图2E代表各组外周血清IL-37的相对含量;图2F代表各组外周血清TGF-β的相对含量;图2中,control:对照组;3%DSS:模型组;Mesalazine:阳性受试药美沙拉秦组;BM:白扁豆多糖中剂量组;BH:白扁豆多糖高剂量组。(#:与正常组,P<0.05;##:与正常组相比,P<0.01;*:与模型组相比,P<0.05;**:与模型组相比,P<0.01)。
图3为白扁豆多糖对UC小鼠肠道菌群的影响,其中,control:对照组;model:模型组;mesalazine:阳性受试药美沙拉秦组;BM:白扁豆多糖中剂量组;BH:白扁豆多糖高剂量组。
具体实施方式
下面结合实施例对本发明做进一步详细的描述。这些实施例仅用于说明本发明而不用于限制本发明的范围。以下实施例中未注明具体条件的实验方法,通常按照本领域常规条件或按照制造厂商建议的条件;所使用的原料、试剂等,如无特殊说明,视为可以通过常规市场等商业途径得到的原料和试剂。
下述实施例中所用的实验仪器和试剂:离心机(TDL-5-A,上海安亭科学仪器厂),移液器(10/100/200/1000μL,Eppendorf),低温冰箱(BCD-539WT,海尔股份有限公司),超低温冰箱(Forma 900 Series,ThermoFisher Scientific),超纯水*** (Milli-QAdvantage A10,Millipore),电子天平(ME104,瑞士 Mettler-Toledo),水浴锅 (SB-1300,东京理化器械株式会社),旋转蒸发仪(N-1300,东京理化器械株式会社),循环水多用真空泵,(CA-1116A,东京理化器械株式会社),中药粉碎机(GX-03,浙江高鑫工贸有限公司),恒流泵,(HL-2D,上海沪西分析仪器厂有限公司),自动馏分收集器(BSZ-100,上海沪西分析仪器厂有限公司),真空冷冻干燥机(SCIENZT-10N,宁波新芝生物科技股份有限公司),C57BL/6J雄性小鼠,葡聚糖硫酸钠(MP,货号9011-18-1),ELISA试剂盒(江苏酶免)。
实施例1
白扁豆多糖的提取
白扁豆果实购自广州康美药业有限公司(批号:220503661),采收年份为 2022年,鉴定为白扁豆(Lablab purpureus (Linn.) Sweet)果实。白扁豆恒温干燥后粉碎,过40目筛,得白扁豆果实粗粉。取200 g白扁豆,加入4 L体积分数为95%的乙醇溶液,80°C提取3h,过滤后取滤渣重复提取3次,除去色素和小分子物质。最后将滤渣烘干得白扁豆果实脱脂粉末。取200 g 白扁豆果实脱脂粉末,加入4 L去离子水,95°C提取3 h,过滤后收集上清液,滤渣重复提取 3 次。合并提取液,60°C真空浓缩至一定体积。向浓缩液中加入4倍浓缩液体积的无水乙醇,大力搅拌后于4°C冰箱中静置过夜。离心取沉淀物,复溶于400 mL去离子水中,加入 80 mL Sevage试剂(氯仿:正丁醇=4:1),充分震荡后离心(5000 rpm,5 min),离心后合并上清液,继续加入Sevage试剂震荡并离心,合并的上清液与Sevage试剂的体积比为5:1,直至溶液中无变性蛋白质出现为止。真空浓缩除去溶液中的有机试剂,真空冷冻干燥后得白扁豆多糖,备用。
实施例2
与实施例1相同,不同之处在于,白扁豆多糖由如下步骤制备得到:
(1)向过30目筛的白扁豆果实粗粉中加入体积分数为95%的乙醇溶液提取,白扁豆果实粗粉与乙醇溶液的质量体积比为1:15 g/mL,75°C提取3.5 h,提取液过滤得滤渣,取滤渣重复提取3次,最后将滤渣烘干得白扁豆果实脱脂粉末;
(2)取步骤(1)得到的白扁豆果实脱脂粉末,加入去离子水进行提取得到提取液,白扁豆果实脱脂粉末与水的质量体积比为1:15 g/mL,90°C提取3.5 h,提取液过滤后收集上清液,滤渣重复提取3次,合并提取液,提取液真空浓缩得到浓缩液;
(3)向步骤(2)得到的浓缩液中加入无水乙醇,浓缩液与无水乙醇的体积比为1:3,搅拌均匀后静置,离心后的沉淀物复溶于去离子水中,加入Sevage试剂,去离子水与Sevage试剂的体积比为4:1,Sevage试剂是体积比为4:1的氯仿和正丁醇的混合物,离心处理后合并上清液,向合并的上清液中继续加入 Sevage 试剂震荡并离心处理,合并的上清液与Sevage试剂的体积比为4:1,直至溶液中无变性蛋白质出现为止,真空浓缩除去溶液中的有机试剂,真空冷冻干燥后得白扁豆多糖。
实施例3
白扁豆多糖由如下步骤制备得到:
(1)向过30-50目筛的白扁豆果实粗粉中加入体积分数为95%的乙醇溶液提取,白扁豆果实粗粉与乙醇溶液的质量体积比为1:25 g/mL,85°C提取2.5 h,提取液过滤得滤渣,取滤渣重复提取3次,最后将滤渣烘干得白扁豆果实脱脂粉末;
(2)取步骤(1)得到的白扁豆果实脱脂粉末,加入去离子水进行提取得到提取液,白扁豆果实脱脂粉末与水的质量体积比为1:25 g/mL,100°C提取2.5 h,提取液过滤后收集上清液,滤渣重复提取3次,合并提取液,提取液真空浓缩得到浓缩液;
(3)向步骤(2)得到的浓缩液中加入无水乙醇,浓缩液与无水乙醇的体积比为1:5,搅拌均匀后静置,离心后的沉淀物复溶于去离子水中,加入Sevage试剂,去离子水与Sevage试剂的体积比为6:1,Sevage试剂是体积比为4:1的氯仿和正丁醇的混合物,离心处理后合并上清液,向合并的上清液中继续加入 Sevage 试剂震荡并离心处理,合并的上清液与Sevage试剂的体积比为6:1,直至溶液中无变性蛋白质出现为止,真空浓缩除去溶液中的有机试剂,真空冷冻干燥后得白扁豆多糖。
实施例4
急性溃疡性结肠炎小鼠模型(UC模型)的建立。动物来源:广东省医学实验动物中心,动物种类:C57BL/6小鼠,雄性的小鼠;动物年龄及质量:8周龄,体重大约为20~22 g,动物饲养于广州中医药大学SPF级动物房(伦理审查编号:20210903008),采用12 h/12 h的明暗交替环境,且温度和湿度适宜,自由摄食和饮水,适应性喂养2周后进行实验。
通过自由饮用3%(质量体积百分比浓度) DSS构建UC模型,将实施例1得到的白扁豆多糖做如下实验。用美沙拉秦(德国福克制药股份有限公司)作为阳性受试药,参考《中药药理研究方法学》,按成年人 60 kg 的标准换算,按照实际用法将白扁豆多糖换算成小鼠剂量为100 mg/kg。同样的将白扁豆多糖换算成小鼠的剂量为200 mg/kg。所以参照这个将小鼠分为以下5个组别:对照组(n=10),自由饮用纯水;模型组(n=15),DSS模型组,自由饮用3%DSS;DSS模型+白扁豆多糖中剂量组(200 mg/kg,BM)(n=15),DSS模型+白扁豆多糖高剂量组(400 mg/kg,BH)(n=15),DSS模型+美沙拉秦组(100 mg/kg)(n=15),让小鼠自由饮用7天。边造模边灌胃给药,灌胃体积为0.2 mL,对照组和模型组予以生理盐水进行灌胃。整个实验期间记录疾病活动指数(DAI=(体重指数+大便形状+出血情况)/3),于第7天禁食不禁水,第8天处死,取肠道组织,量取整段结肠长度,收集结肠黏膜组织,4%多聚甲醛,收集外周血清。
小鼠疾病活动评分指数及炎症因子检测分析
1、HE染色
4%多聚甲醛固定后的肠道组织,于二甲苯中进行10 min中的脱蜡处理。再依次置于体积分数95%、85%、70%的梯度乙醇中各浸泡5 min进行样本水化。水化后的样本置于PBS中清洗3次,每次5 min。添加苏木素染液,染色10 min。随后使用质量分数1%的盐酸乙醇进行分化。紧接着加入伊红染液,染色3 min。再将组织切片用乙醇溶液进行梯度脱水,样本封片风干后进行拍照。
2、qPCR检测
用1 mL Trizol提取肠道组织中的RNA,将提取好的RNA用PrimeScript™ RTreagent Kit with gDNA Eraser反转录成cDNA。将TB Green® Premix Ex Taq™用于检测cDNA靶基因的相对浓度,反应体系是:TB Green® Premix Ex Taq™ 10 μL,TNF-a (前引物 :CCCTCACACTCAGATCATCTTCT 后引物: GCTACGACGTGGGCTACAG)IL-1Beta(前引物 :GCAACTGTTCCTGAACTCAACT,后引物:ATCTTTTGGGGTCCGTCAACT)前后引物均为0.4 μL(浓度为0.2 μM),cDNA 2 μL(浓度为50 ng/μL),DEPC水7.2 μL,总共20 μL。cDNA上机扩增后,以GAPDH为内参采取2-ΔΔCT方法计算目标基因的相对表达水平。
3、ELISA检测外周血清炎症因子浓度
将待测样品和标准品,使用以下试剂盒:小鼠肿瘤坏死因子α(TNF-α)ELISA科研试剂盒(江苏酶免,货号# MM-0132M2),小鼠白细胞介素1β(IL-1β)ELISA科研试剂盒(江苏酶免,货号#MM-0040M2),小鼠白细胞介素6(IL-6)ELISA科研试剂盒(江苏酶免,货号#MM-0163M2),小鼠白细胞介素10(IL-10)ELISA科研试剂盒(江苏酶免,货号#MM-0176M1),小鼠白细胞介素37(IL-37)ELISA科研试剂盒(江苏酶免,货号#MM-44906M2),小鼠转化生长因子-β(TGF-β)ELISA科研试剂盒(江苏酶免,货号#MM-0689M2)。按照试剂盒说明书进行以下操作。加入孔板中,37℃反应30分钟;洗板5次,加入酶标试剂,37℃反应30分钟;洗板5次,加入显色液A、B,37℃显色10分钟;加入终止液,15分钟内读OD值,计算样品浓度。
4、菌群分析
在清洁环境中采集各组小鼠的新鲜粪便,置于无菌采样管中送至实验室,-80℃保存待检。使用 E.Z.N.A.StoolDNA Kit提取各组小鼠粪便中微生物DNA.DNA浓度和质量检测合格后,对DNA样本 进行16S核糖体 RNA 基因V3-V4 (341F-806R) 区域的 PCR扩增引物341F:5-CCTAYGGGRB-GCASCAG-3,806R:5-GGACTACNNGGG-TATCTAAT-3。PCR 反 应 体 系 包含 4 mL 5×FastPfuBuffer、2 mL 2.5 mmol/LdNTPs、0.8 μL 上 游 引 物 (5 μmol/L)、0.8 μL 下 游 引 物 (5 μmol/L)、0.4 μL FastPfuDNA Polymerase和 10 ng模板 DNA。PCR反应程序如下:95℃初始变性5min,95℃变性30s,55℃退火30 s,72℃延伸 45 s,27个循环;72℃ 10 min,10℃终止。扩增产物使用 AxyPrepDNA Gelextractionkit纯化,纯化后的PCR产物进行IIIuminaPE250文库构建、IllumiGminaPE250测序,对测序后的原始数据进行分析。以上工作委托华大基因有限公司完成.IlluGminaPE250测序获得的序列,对Reads的质量进行质控过滤,对质控拼接好的序列按照97%相似性进行OTU聚类分析,基于 OTU 聚类分析结果进行多样性指数分析、测序深度的检测,基于分类学信息在各个分类水平上进行群落结构的统计分析。
如图1和图2所示,模型组与正常相比:模型组的疾病活动指数(DAI)、便血程度、促炎炎症因子(TNF-α、IL-1β、IL-6)含量明显高于正常组(P﹤0.01);模型组肠道HE染色病理结果显示肠道黏膜正常结构破坏,黏膜下层有大量的炎症浸润;抑制炎症因子(IL-10,IL-37,TGF-β)较正常组显著降低(P﹤0.01);模型组结肠长度低于正常组(P﹤0.01)。这些结果说明UC小鼠模型造模成功。白扁豆多糖组与模型组相比(图1和图2):白扁豆多糖组的疾病活动指数(DAI)、便血程度、促炎症因子(TNF-α、IL-1β、IL-6)含量明显低于模型组(P﹤0.01);白扁豆多糖组肠道HE染色病理结果显示肠道黏膜结构破坏较轻,炎症浸润较少;抑制炎症因子(IL-10,IL-37,TGF-β)较模型组显著升高(P﹤0.01);白扁豆多糖结肠长度高于模型组(P﹤0.01)。这些结果提示白扁豆多糖能够减轻UC小鼠模型肠道炎症反应并且机制与抑制肠道炎症反应有关。阳性受试药美沙拉嗪与白扁豆多糖药效相似。
图3中可以看出白扁豆多糖组(BM和BH)较小鼠溃疡性结肠炎模型组(3%DSS)能够增加有益菌双歧杆菌属(Bifidobacterium)的丰度,减少条件致病菌的梭菌属(Clostridium)的丰度,但是临床成熟用药美沙拉嗪却无此类功效。此结果可以说明白扁豆多糖有益生元的作用,可以通过肠道菌群的分布来治疗溃疡性结肠炎。
以上实施例的说明只是用于帮助理解本发明的技术方案及其核心思想,应当指出,对于本技术领域的技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求的保护范围内。
Claims (1)
1.白扁豆多糖在制备预防和/或治疗溃疡性结肠炎的药物中的应用,其特征在于,所述的白扁豆多糖由如下步骤制备得到:
(1)向过30-50目筛的白扁豆果实粗粉中加入体积分数为95%的乙醇溶液提取,白扁豆果实粗粉与乙醇溶液的质量体积比为1:15-25 g/mL,75°C-85°C提取2.5-3.5 h,提取液过滤得滤渣,取滤渣重复提取若干次,最后将滤渣烘干得白扁豆果实脱脂粉末;
(2)取步骤(1)得到的白扁豆果实脱脂粉末,加入水进行提取得到提取液,提取液过滤后收集上清液,滤渣重复提取若干次,合并提取液,提取液真空浓缩得到浓缩液,提取条件为:白扁豆果实脱脂粉末与水的质量体积比为1:15-25 g/mL,90°C-100°C提取2.5-3.5 h;
(3)向步骤(2)得到的浓缩液中加入无水乙醇,浓缩液与无水乙醇的体积比为1:3-5,搅拌均匀后静置,离心后的沉淀物复溶于水中,加入Sevage试剂,水与Sevage试剂的体积比为4-6:1,Sevage试剂是体积比为4:1的氯仿和正丁醇的混合物,离心处理后合并上清液,向合并的上清液中继续加入 Sevage 试剂震荡并离心处理,合并的上清液与Sevage试剂的体积比为4-6:1,直至溶液中无变性蛋白质出现为止,真空浓缩除去溶液中的有机试剂,真空冷冻干燥后得白扁豆多糖。
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