CN113940945A - 鱼腥草多糖在制备防治炎症性肠病药物中的用途 - Google Patents
鱼腥草多糖在制备防治炎症性肠病药物中的用途 Download PDFInfo
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- CN113940945A CN113940945A CN202010684649.5A CN202010684649A CN113940945A CN 113940945 A CN113940945 A CN 113940945A CN 202010684649 A CN202010684649 A CN 202010684649A CN 113940945 A CN113940945 A CN 113940945A
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- polysaccharide
- houttuynia
- inflammatory
- houttuynia cordata
- colon
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Abstract
本发明属中药技术领域,涉及鱼腥草多糖新的制药用途,具体涉及鱼腥草多糖在制备防治炎症性肠病(IBD)药物中的用途。本发明从中药鱼腥草中分离提取得到总多糖提取物,平均产物收率为3.65%,多糖含量超过70%。所述鱼腥草总多糖提取物经整体实验证实,对2.5%DSS诱导的小鼠结肠损伤有显著的治疗作用。鱼腥草多糖可能通过其抗炎机制,抑制炎性细胞的浸润,降低炎性细胞因子的分泌,抑制炎症转录因子NFκB的表达,从而减少肠道损伤,保护肠道粘膜,缓解结肠缩短与体重下降。可用于制备防治溃疡性结肠炎的药物。
Description
技术领域
本发明属中药技术领域,涉及鱼腥草多糖新的制药用途,具体涉及鱼腥草多糖在制备防治炎症性肠病(IBD)药物中的用途。
背景技术
现有技术公开了炎症性肠病由基因、环境、免疫等多种原因引起。所述炎症性肠病常见的类型有溃疡性结肠炎(uclerative colitis,UC)和克罗恩病(CD);其中,溃疡性结肠炎主要好发在直肠、结肠的粘膜层,以溃疡、糜烂为主,常累及整个结肠,呈现连续的大面积病灶[1],UC临床表现为反复发作的腹泻、腹痛、恶心、呕吐、血便、发热和营养不良,可伴有皮肤、黏膜、肝胆、关节和眼等肠外表现[2]。
对于所述的UC和CD,具体的病因还有待深入研究,目前研究表明其发病与基因、环境(生活方式、环境污染、药物及其它)和粘膜免疫等因素关系密切[3];在健康人群中,肠道上皮细胞,免疫细胞和肠道共生菌相互作用,维持肠道稳态,而溃疡性结肠炎患者体内,肠道稳态被打破,肠道屏障受到破坏,导致肠腔内微生物能够进入固有层,进而引起体内免疫细胞激活,发生炎症,免疫系统紊乱同时,肠道菌群也发生变化;因此,保护肠道屏障以及调控肠道菌将可能是治疗溃疡性结肠炎的有效策略[4]。
对于上述UC的治疗,目前无有效方法,据统计,大约15%的溃疡性结肠炎患者在确诊后20年内需要进行结肠切除手术。目前治疗首选药物是氨基水杨酸和糖皮质激素,前者主要用于治疗轻、中度活动期UC,疗效显著,也用于UC缓解期的维持治疗[5];糖皮质激素是UC急性发作最有效的药物,适用于重度UC患者或氨基水杨酸疗效不佳的情况使用,疗效明确,但能阻断UC患者的T淋巴细胞增殖、活化,出现免疫抑制,常诱发白细胞减少等骨髓抑制表现、增加机会性感染的发生率的副作用[6-7]。其它治疗药物还包括抗菌药物、生物制剂以及中药。应用中药治疗UC的研究显示在动物模型上已取得了理想的结果,研究证实:姜黄提取物能够通过抑制炎症反应和氧化活性从而保护DSS造成的小鼠肠炎[8];槐果碱通过调节促炎和抗炎细胞因子的产生改善在小鼠中结肠炎的发生[9];菊花多糖通过调节肠道微生物群落改善大鼠的结肠炎症[10];有关部分中药制剂已进入临床研究,如治疗溃疡性结肠炎的五味苦参肠溶胶囊已进行到四期临床试验。越来越多的中药成分被证实能缓解结肠炎症状,但机制尚不清,亟需进一步研究与挖掘。
目前已建立的多种小鼠结肠炎模型模拟人类IBD病理过程,其中由化学药物葡聚糖硫酸钠(Dextran Sulfate Sodium Salt,DSS)诱导的结肠炎模型应用最为广泛;DSS在常温下极易溶于水,造模时只要将DSS粉末溶于蒸馏水即可配置成相应浓度的DSS溶液,让动物进行自由摄取或定量灌胃即可建模;该种模型具有以下几个优点:方法简单、成本低、成功率高、便于重复;临床表现、病变部位、病理学与人UC基本一致;可以控制条件建立急性或慢性模型。DSS诱导结肠炎的具体机制主要涉及到三方面:DSS能够直接侵蚀肠道,造成肠道损伤,并且DSS具有抗凝特性,能够造成肠道持续出血;同时,DSS能够促进免疫细胞的浸润,影响了炎症细胞因子的分泌;摄取DSS进入肠道后,它会破坏肠道微环境,影响肠道菌群及其代谢产物。
鱼腥草是我国的传统中药,药食两用,临床应用广泛。鱼腥草(HouttuyniacordataThunb.)为三白科植物蕺茶属蕺菜的全草,性味辛、寒,具有清热解毒、消肿排脓、利尿通淋之功效,主要治疗肿痈化脓、痰热喘咳、热痢热淋、痈肿疮毒等[11]。有研究证实:鱼腥草全株均有一定的抑菌作用,包括金黄色葡萄球菌、大肠杆菌、酵母菌[12];鱼腥草挥发油对流感病毒有抑制作用;鱼腥草注射液(全草水提物)肌注能显著降低甲型流感病毒H1N1感染小鼠死亡率,降低肺指数和抑制肺内病毒的增殖[13];鱼腥草全草也可以抗单纯疱疹病毒(HSV)、SARS病毒。本研究团队前期研究结果已证实:鱼腥草多糖(HCP)能显著减轻LPS诱导的肺损伤[14],体外的抗溶血实验也证实其对补体的过度激活有明显抑制作用[15],说明HCP具有显著的抗炎活性。借助甲流病毒诱导的肺炎模型,证实HCP能显著治疗病毒诱导的肺损伤,并对流感病毒诱导的继发肠道损伤也有保护作用[16]。
综观国内外的研究,对鱼腥草的研究多见于针对肺病的研究,未见见鱼腥草多糖治疗炎症性肠病的药效及其药物研究报道。基于现有技术的现状和基础,本申请的发明人拟提供鱼腥草多糖新的制药用途,具体涉及鱼腥草多糖在制备防治炎症性肠病(IBD)药物中的用途。
与本申请相关的参考文献有:
[1]葛均波,徐永健.内科学[M].8版.北京:人民卫生出版社,2013:385-393.
[2]屈冬冬,金世禄.溃疡性结肠炎发病机制研究进展[J].实用临床医药杂志,2016,20(3):184-186.
[3]樊慧丽,陈玉梅.溃疡性结肠炎的发病机制和治疗进展[J].中国全科医学,2012,15(1B):228-230.
[4]MichaelEisenstein.GutReaction.[J].Nature,2018,563
[5]Prefontaine E,Macdonald JK,et al.Azathioprine or 6-mercaptopurinefor induction of remission in Crohn's disease.[J].Cochrane Database SystRev,2010,16(6):CD000545.
[6]戎兰.免疫抑制剂在炎症性肠病治疗中的应用[J].上海医药,2016,37(1):11-14.
[7]Ma FL,Zhang XL,et al.Research progress of gastrointestinal mucosalprotective effect of Chinese medicine.[J].MedPlant,2014,5:1-4
[8]RuiqiongWang,Guotai Wu,et al.Semi-bionic extraction of compoundturmeric protects against dextran sulfate sodium-induced acute enteritis inrats.[J].Ethnopharmacol.2016,190:288-300.
[9]XiaojuanWang,Hongzhu Deng,et al.The natural plant productsophocarpine ameliorates dextran sodium sulfate-induced colitis in mice byregulating cytokine balance.[J].Colorectal Dis.2012,27(5):575-81.
[10]Jinhua Tao,Jinao Duan,et al.Polysaccharides from Chrysanthemummorifolium Ramat ameliorate colitis rats by modulating the intestinalmicrobiota community.[J].Oncotarget,2017,46,80790-8080
[11]中华人民共和国卫生部药典委员会.中国药典.一部.北京:中国医药科技出版社,2010.
[12]江苏新医学院.中药大辞典[M].上海:上海人民出版社,1977:1439.
[13]杨慧,李剑琦等.鱼腥草抗甲1型流感病毒诱导细胞程序化死亡的初步研究.[J].江西医药,2006,41(12):960-961.
[14]Yanyan Xu,Yunyi Zhang,et al.Houttuynia cordatathumb.polysaccharides ameliorates lipopolysaccharide-induced acute lunginjury in mice.[J].Ethnopharmacol,2015,173:81-90.
[15]姜韵.鱼腥草的抗补体活性成分及其药理作用.[D].上海:复旦大学,2011.
[16]Haiyan Zhu,Xiaoxiao Lu,et al.Houttuynia cordata polysaccharidesameliorates pneumonia severity and intestinal injury in mice with influenzavirus infection.[J].Ethnopharmacol,2018,218:90-99.。
发明内容
本发明的目的是基于现有技术的现状和基础,提供鱼腥草多糖新的制药用途,具体涉及鱼腥草多糖在制备防治炎症性肠病(IBD)药物中的用途。
本发明从中药鱼腥草(Houttuynia cordata Thunb.)中分离提取得到总多糖提取物,平均产物收率为3.65%,多糖含量超过70%。所述鱼腥草总多糖提取物经整体实验证实,对2.5%DSS诱导的小鼠结肠损伤有显著的治疗作用。
本发明通过下述方法制得鱼腥草多糖(HCP):
取鱼腥草(Houttuynia cordata Thunb.)药材粉碎,以95%乙醇冷浸提取,过滤,合并提取液,浓缩,离心,上清液以三氯醋酸去游离蛋白,离心,上清液透析,透析液浓缩加乙醇至含醇量80%,离心,沉淀冷冻干燥,制得总多糖,产物收率达3%以上;鱼腥草总多糖的糖含量超过70%;鱼腥草总多糖的糖醛酸含量超过25%;鱼腥草总多糖的蛋白质含量不超过10%。
本发明采用所述的鱼腥草多糖进行了对DSS诱导的小鼠溃疡性结肠炎的治疗作用实验,实验结果显示,本发明从中药鱼腥草中分离提取得到总多糖提取物经整体动物模型试验证实其针对DSS诱导的溃疡性结肠炎,具有显著治疗肠道损伤的作用。
本发明实验结果表明:所述的鱼腥草多糖(HCP)对DSS诱导的小鼠溃疡性结肠炎具有显著治疗作用,所述HCP可显著改善溃疡性结肠炎小鼠的体重、改善小鼠结肠缩短、抑制肠道炎症发生、保护肠道粘膜屏障完整性、抑制肠道匀浆中细胞因子(包括IFN-α、IL-1β、IL-6)的释放,抑制炎症核转录因子NFκB的表达。所述的鱼腥草多糖通过其抗炎机制,抑制炎性细胞的浸润,降低炎性细胞因子的分泌,抑制炎症转录因子NFκB的表达,从而减少肠道损伤,保护肠道粘膜,缓解结肠缩短与体重下降。
本发明所述的鱼腥草多糖可用于制备防治炎症性肠病(IBD)的药物。
附图说明
图1,鱼腥草多糖对DSS诱导结肠炎小鼠体重的影响,其中,*P<0.05,Normal.vs.2.5%DSS。
图2,鱼腥草多糖对DSS诱导的结肠炎小鼠结肠长度的影响,其中,*P<0.05,***P<0.001。
图3,正常对照组小鼠结肠组织HE染色(x200),
其中显示,结肠组织层次分明,粘膜层完整,腺体排列整齐,无炎性细胞的浸润,无隐窝缺失病变。
图4,DSS模型组小鼠结肠组织HE染色(x200),其中显示,炎性细胞大量浸润,粘膜层破坏严重,腺体结构紊乱,粘膜严重损伤,隐窝大量缺失。
图5,80mg/kgHCP治疗组小鼠结肠组织HE染色(x200),其中显示,局部炎性细胞浸润,粘膜层整体完整,层次结构清晰,隐窝无明显缺失或病变。
图6,正常对照组小鼠结肠组织PAS染色(x200),其中显示,正常组结肠中杯状细胞呈紫红色着色,表达量高。
图7,DSS模型组小鼠结肠组织PAS染色(x200),其中显示,模型组结肠结构破坏,杯状细胞表达量少,与正常组相比,模型组杯状细胞显著减少。
图8,80mg/kgHCP治疗组小鼠结肠组织PAS染色(x200),其中显示,给药组结肠结构受到保护,杯状细胞数量与正常组接近,显著高于模型组。
图9,正常对照组小鼠结肠组织中MUC2的表达(免疫组化法)(x200),其中显示,粘液素MUC2,由杯状细胞分泌分布于肠粘膜上皮细胞上,免疫组化染色后呈棕黄色,在正常组中高表达。
图10,DSS模型组小鼠结肠组织中MUC2的表达(免疫组化)(x200),其中显示,MUC2在模型组中的表达减少,棕黄色的染色面积明显少于正常组。
图11,80mg/kgHCP治疗组小鼠结肠组织中MUC2的表达(免疫组化)(x200),其中显示,HCP治疗组MUC2表达量与正常组接近,有大量特异性染色的部分。
图12,正常对照组小鼠结肠组织中ZO-1的表达(免疫组化)(x200),其中显示,细胞间紧密连接蛋白ZO-1,在细胞膜上表达并发挥作用,主要位于上皮细胞膜上,免疫组化染色后呈棕色,在正常组中高表达。
图13,DSS模型组小鼠结肠组织中ZO-1的表达(免疫组化)(x200),其中显示,ZO-1在模型组中的表达减少,特异性染色面积明显少于正常组。
图14,80mg/kgHCP治疗组小鼠结肠组织中ZO-1的表达(免疫组化)(x200),其中显示,HCP治疗组ZO-1表达量与正常组接近,有大量特异性染色的部分。
图15,鱼腥草多糖对DSS模型小鼠结肠匀浆中细胞因子IL-1β的作用,其中,***P<0.001。
图16,鱼腥草多糖对DSS模型小鼠结肠匀浆中细胞因子TNF-α的作用,其中,**P<0.01,***P<0.001。
图17,鱼腥草多糖对DSS模型小鼠结肠匀浆中细胞因子IL-6的作用,其中,*P<0.05。
图18,鱼腥草多糖对DSS模型小鼠结肠紧密连接蛋白Z0-1表达的影响,其中显示,模型组ZO-1蛋白表达量与正常组相比显著下降,而HCP治疗组增加了ZO-1的表达水平。
图19,鱼腥草多糖对DSS模型小鼠结肠NF-κB信号通路相关蛋白表达的影响,其中显示,与正常组相比,模型组NF-κB蛋白表达量上升,IKBα蛋白表达量下降。而HCP治疗组抑制了NF-κB的表达水平,同时增加了IKBα蛋白表达量,与正常组接近。
具体实施方式
实施例1:鱼腥草多糖的制备
取鱼腥草(Houttuynia cordata Thunb.)药材100g,粉碎,以95%乙醇冷浸提取3次,药渣于室温下置通风处晾干,然后用热水提取3次,过滤,合并提取液,浓缩,离心,上清液以三氯醋酸去游离蛋白,离心,上清液用水透析3天,透析液浓缩至小体积加乙醇至含醇量80%,离心,沉淀冷冻干燥即得总多糖,产物收率达3%以上。采用硫酸-苯酚法测得鱼腥草总多糖的糖含量超过70%;采用间羟联苯法测得鱼腥草总多糖的糖醛酸含量超过25%;采用考马斯亮蓝法测得鱼腥草总多糖的蛋白质含量不超过10%,结果如表1所示。
表1三批鱼腥草多糖的收率及含量
实施例2:鱼腥草多糖缓解溃疡性结肠炎的药效试验
2.1实验材料
2.1.1实验动物及药品
C57BL/6雄性小鼠24只,体重16-18g,购于上海灵畅有限公司,动物合格证号:SCXK(沪)2013-0018。实验动物分笼饲养,每3天更换一次垫料,适应性饲养2天,实验期间自由饮水和进食。饲养环境温度为20±2℃,相对湿度为60%,每天12小时光照和12小时黑夜循环;
鱼腥草多糖,即由实施例1从鱼腥草中提取出的总多糖(批号JY2011);2.1.2实验试剂和仪器
化学试剂:DSS(葡聚糖硫酸钠,MW36000-50000,MP Biomedicals,LOT NO:Q6182),PBS缓冲液,分析纯甲醛溶液,10%福尔马林。氯化钠,氯化钾,磷酸氢二钠,磷酸二氢钾(由国药集团化学试剂有限公司提供);
试剂盒:BCA蛋白浓度检测试剂盒(碧云天生物科技有限公司,批号P0012),小鼠白介素-1β酶联免疫试剂盒(上海船夫生物科技有限公司,M4000),小鼠白介素-6酶联免疫试剂盒(上海船夫生物科技有限公司,M1390),小鼠肿瘤坏死因子-α酶联免疫试剂盒(上海船夫生物科技有限公司,M1900);
实验仪器:FA2204电子天平(上海力辰邦西仪器科技有限公司),微孔板分光光度计(bio-tek),Allegra64R台式高速冷冻离心机(Beckman库尔特商贸中国有限公司),Tissuelyser-24多样品组织研磨机(上海净信实验科技研究部),TS-2脱色摇床(海门市其林贝尔仪器制造有限公司),DL-120J智能超声波清洗器(上海之信仪器有限公司),AF-100制冰机(Scotsman),移液枪,涡旋仪,迷你微量离心机;
实验器械:眼科镊,手术剪,烧杯,量筒,EP管,离心管;
2.1.3药品配置
2.1.3.1鱼腥草多糖混悬液制备
用电子天平称量100mg鱼腥草多糖粉末倒入15ml离心管中,加入双蒸水,定容至12.5ml;超声溶解,配制成终浓度为8mg/ml工作液,置于4℃冰箱中,溶胀过夜,每次使用前恢复室温,涡旋混匀;
2.1.3.2DSS溶液配制
称量12.5gDSS粉末,倒入1000ml烧杯中,加双蒸水至500ml,搅拌至彻底溶解,配置成终浓度为2.5%的DSS溶液,每两天重新配置,给动物换上新配的溶液;
2.1.3.3PBS缓冲液配制
称量8gNaCl,0.2gKCl,1.44gNa2HPO4,0.24g KH2PO4,加入2升烧杯中,加800ml双蒸水,调节PH至7.4,加双蒸水定容至1L。过滤除菌;
2.1.3.4组织固定液配制
用量筒量取50ml分析纯甲醛溶液至广口瓶,再加入配制的PBS缓冲液450ml,得500ml4%甲醛溶液;
2.2实验方法
2.2.1动物分组及模型建立
C57BL/6小鼠24只(16-18g),按体重随机分为4组(A、B、C、D):A组为正常对照组;B组为DSS模型组;C组为鱼腥草多糖低剂量组(40mg/kg);D组为鱼腥草多糖高剂量组(80mg/kg);每组6只;除A组外的所有组动物的饮用水换成含有2.5%DSS的水溶液,C、D组灌胃给药,剂量分别为40mg/kg、80mg/mg;同时A组与B组给予灌胃等量三蒸水,一天给药一次,连续给药七天;2.2.2实验过程
每天观察记录小鼠活动、进食、精神状态、体重、粪便状态和死亡等情况;造模10天后后,称量体重,摘眼球取血,剪下盲肠与结肠,结肠记录下长度并拍照保存,剪取连接盲肠一侧的结肠约0.5厘米置于10%福尔马林中;将剩下的结肠置于-80℃保存;冻存的肠组织之后使用预冷的PBS,用匀浆器在冰浴中匀浆,收获匀浆液,离心,收集上清,分装,-80℃保存,用于检测肠道细胞因子等指标;
2.2.3结肠组织HE染色、PAS染色与免疫组化检测
小鼠解剖后选取靠近盲肠一侧的结肠,剪下大约0.5厘米长度的组织,浸泡于10%福尔马林固定,固定组织切片脱水,石蜡包埋。HE染色:先将样品水化,然后用苏木精与伊红染色,最后再对样品脱水;PAS染色:将切片水化,用过碘酸与雪夫氏液染色,最后脱水;免疫组化:在提取抗原后,在4℃下分别用ZO-1,MUC2孵育肠切片,37℃水浴用HRP-结合抗体孵育30分钟,切片用显色底物溶液显色,在倒置显微镜下观察标本并拍照,放大200倍;
2.2.4肠匀浆制备及结肠IL-1β、TNF-α、IL-6测定
称量约50mg结肠组织于2mlEP管中,加500ulPBS缓冲液,加入小钢珠;用匀浆机匀浆,频率30赫兹,每次10秒,共三次,每次间隔约一分钟;将匀浆液吸出到新的1.5mlEP管中,进行离心,条件为3500rpm,10min,离心后的上清分装到0.6mlEP管中,每管装100ul,做好标记,置于-80℃贮存;
每个样品取出一管肠匀浆液,(应用ELISA法,参照IL-1β、TNF-α、IL-6试剂盒说明书测定肠匀浆上清中细胞因子的含量;
本实施例中以下表所示的IL-1β为例:
2.2.5肠组织NF-κB信号通路相关蛋白WB检测
小鼠结肠组织用RIPA裂解液裂解提取蛋白,蛋白质经过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离,转到聚偏氟乙烯膜(PVDF膜);用碧云天封闭液封闭1h,室温下用相应抗体孵育过夜,用HRP标记的二抗孵育1h,用显色剂在Bio-Rad凝胶成像仪下显色;
2.2.6数据统计
计量资料均用均数±标准差(mean±SD),各组间差异比较用方差分析检验,两两比较用单因素方差分析(one-way ANOVA),以P<0.05判断统计学意义,“ns”表示p>0.05,即无显著性差异;“*”表示p<0.05;“**”表示p<0.01;“***”表示p<0.001,用Graphpad Prism5.0进行统计。
2.3实验结果显示:
2.3.1鱼腥草多糖对DSS诱导结肠炎小鼠模型体重的影响
研究结果如图1所示:正常组动物的体重每天缓慢增加,而DSS模型组和给药组都经历了先增加后下降两个阶段的变化,从D3天体重缓慢下降,并且最终模型组下降的程度更加明显,D10天体重统计显示,高剂量鱼腥草多糖组(80mg/kg)能够显著缓解DSS模型造成的体重下降(p<0.05),低剂量组也能够缓解体重降低,但与模型组比较无显著性差异;
2.3.2鱼腥草多糖对DSS诱导结肠炎小鼠模型结肠长度的影响
研究结果(如图2所示)表明:D10取材后,DSS模型组小鼠的结肠长度与正常组比较显著缩短,与模型组比较,鱼腥草多糖高低治疗组均能显著恢复结肠长度(P<0.05),高剂量组效果最为显著(P<0.001);
2.3.3鱼腥草多糖对DSS诱导结肠炎小鼠结肠组织形态的影响
HE染色结果(如图3,图4,图5所示)显示:正常组小鼠结肠组织层次分明,粘膜层完整,腺体排列整齐,无炎性细胞的浸润,无隐窝缺失病变;模型组小鼠炎性细胞大量浸润,粘膜层破坏严重,腺体结构紊乱,粘膜严重损伤,隐窝大量缺失;而给药组小鼠结肠组织仅有局部炎性细胞浸润,粘膜层整体完整,层次结构清晰,隐窝无明显缺失或病变;
PAS染色结果显示:正常组结肠中杯状细胞呈紫红色着色,表达量高;而模型组结肠结构破坏,杯状细胞表达量少,与正常组相比杯状细胞数量显著减少;给药组结肠结构受到保护,杯状细胞数量与正常组接近,与模型组相比显著增加(如图6,图7,图8所示);
2.3.4鱼腥草多糖对DSS诱导结肠炎小鼠的结肠黏膜屏障的影响
细胞间紧密连接蛋白(ZO-1)与粘液素(MUC2)可以反映小鼠肠道屏障的完整性,ZO-1表达在细胞膜上,与紧密连接密切相关,紧密连接位于上皮细胞间的顶端,是维持粘膜通透性的重要组成部分,粘液素由杯状细胞分泌,是覆盖肠粘膜上皮细胞的黏液胶质,该两种蛋白用免疫组化特异性染色后,被染为棕色;免疫组化的结果显示:正常组小鼠中ZO-1与MUC2呈高表达,而模型组小鼠这两种蛋白表达量均下降,特异性染色面积减少;给药组阳性着色面积与正常组接近,呈高表达量,高于模型组;ZO-1的结果如图9,图10,图11所示;MUC2的结果如图12,图13,图14所示;
2.3.5鱼腥草多糖对DSS诱导结肠炎小鼠结肠匀浆细胞因子分泌水平的影响
用ELISA法,将匀浆上清按IL-1β、TNF-α、IL-6试剂盒说明书测定,结果显示:与模型组相比,鱼腥草多糖可显著降低小鼠肠中IL-1β(P<0.001)、TNF-α(P<0.01)、IL-6(P<0.05)水平的升高,结果如图15,图16,图17所示;
2.3.6鱼腥草多糖对DSS诱导结肠炎小鼠结肠中相关蛋白表达的影响
研究结果表明:与正常组相比,模型组小鼠肠道细胞间紧密连接蛋白ZO-1的表达量下降,NF-κB蛋白表达量上升,IKBα蛋白表达量下降;所述的鱼腥草多糖能抑制炎症转录因子NF-κB的表达,提高肠道屏障的ZO-1蛋白的表达水平,如图18,图19所示。
Claims (5)
1.鱼腥草多糖在制备防治炎症性肠病药物制备中的用途。
2.按权利要求所述的用途,其特征在于,所述的鱼腥草多糖按下述方法制备,
取鱼腥草药材粉碎,以95%乙醇冷浸提取,过滤,合并提取液,浓缩,离心,上清液以三氯醋酸去游离蛋白,离心,上清液透析,透析液浓缩加乙醇至含醇量80%,离心,沉淀冷冻干燥,制得总多糖,产物收率达3%以上;鱼腥草总多糖的糖含量超过70%;鱼腥草总多糖的糖醛酸含量超过25%;鱼腥草总多糖的蛋白质含量不超过10%。
3.按权利要求1所述的用途,其特征在于,所述的炎症性肠病是溃疡性结肠炎。
4.按权利要求1所述的用途,其特征在于,所述的鱼腥草多糖抑制炎性细胞的浸润,降低炎性细胞因子的分泌,抑制炎症转录因子NFκB的表达,减少肠道损伤,保护肠道粘膜,缓解结肠缩短与体重下降。
5.按权利要求4所述的用途,其特征在于,所述的炎性细胞因子包括IFN-α、IL-1β、IL-6。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114288388A (zh) * | 2022-01-25 | 2022-04-08 | 华中科技大学同济医学院附属协和医院 | 网膜素在制备炎症性肠病治疗药物中的应用 |
CN115089641A (zh) * | 2022-05-25 | 2022-09-23 | 西安交通大学医学院第一附属医院 | 一种难治型溃疡性结肠炎灌肠剂及其制备方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101721534A (zh) * | 2010-01-15 | 2010-06-09 | 复旦大学 | 鱼腥草总多糖及其制备方法和在制药中的用途 |
CN103070967A (zh) * | 2013-01-25 | 2013-05-01 | 南京大学 | 一种鱼腥草水提液及其制备方法和应用 |
CN105311048A (zh) * | 2014-06-30 | 2016-02-10 | 复旦大学 | 鱼腥草多糖在制备防治甲型流感与病毒性肺炎的药物中的用途 |
CN106421715A (zh) * | 2016-11-24 | 2017-02-22 | 蒋玉明 | 一种治疗慢性结肠炎的中药组合物及其制备方法 |
-
2020
- 2020-07-16 CN CN202010684649.5A patent/CN113940945A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101721534A (zh) * | 2010-01-15 | 2010-06-09 | 复旦大学 | 鱼腥草总多糖及其制备方法和在制药中的用途 |
CN103070967A (zh) * | 2013-01-25 | 2013-05-01 | 南京大学 | 一种鱼腥草水提液及其制备方法和应用 |
CN105311048A (zh) * | 2014-06-30 | 2016-02-10 | 复旦大学 | 鱼腥草多糖在制备防治甲型流感与病毒性肺炎的药物中的用途 |
CN106421715A (zh) * | 2016-11-24 | 2017-02-22 | 蒋玉明 | 一种治疗慢性结肠炎的中药组合物及其制备方法 |
Non-Patent Citations (3)
Title |
---|
CHEN等: "Houttuynia cordata polysaccharide alleviated intestinal injury and modulated intestinal microbiota in H1N1 virus infected mice", CHINESE JOURNAL OF NATURAL MEDICINES, vol. 17, no. 3, 20 March 2019 (2019-03-20), pages 187 * |
XU等: "Houttuynia cordata Thunb. polysaccharides ameliorates lipopolysaccharide-induced acute lung injury in mice", JOURNAL OF ETHNOPHARMACOLOGY, vol. 173, 17 July 2015 (2015-07-17), pages 71 - 90 * |
ZHU等: "Houttuynia cordata polysaccharides ameliorate pneumonia severity and intestinal injury in mice with influenza virus infection", JOURNAL OF ETHNOPHARMACOLOGY, vol. 218, 19 February 2018 (2018-02-19), pages 90 - 99, XP018531686 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114288388A (zh) * | 2022-01-25 | 2022-04-08 | 华中科技大学同济医学院附属协和医院 | 网膜素在制备炎症性肠病治疗药物中的应用 |
CN114288388B (zh) * | 2022-01-25 | 2023-11-03 | 华中科技大学同济医学院附属协和医院 | 网膜素在制备炎症性肠病治疗药物中的应用 |
CN115089641A (zh) * | 2022-05-25 | 2022-09-23 | 西安交通大学医学院第一附属医院 | 一种难治型溃疡性结肠炎灌肠剂及其制备方法 |
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