CN110403012A - 一种鱼针草内酯用于制备抑制胃幽门螺旋杆菌的蛋白质合成的组合物的用途 - Google Patents
一种鱼针草内酯用于制备抑制胃幽门螺旋杆菌的蛋白质合成的组合物的用途 Download PDFInfo
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Abstract
本发明涉及一种鱼针草内酯用于制备抑制胃幽门螺旋杆菌的蛋白质合成的组合物的用途,其中该鱼针草内酯通过抑制胃幽门螺旋杆菌的30S核醣体RpsB的表达,进而抑制其蛋白质合成,从而达到抑制胃幽门杆菌的功效。
Description
【技术领域】
本发明涉及鱼针草内酯的用途,特别涉及抑制抗药性胃幽门螺旋杆菌的用途。
【背景技术】
幽门螺旋杆菌属于微需氧的革兰氏阴性菌,而人类是幽门螺旋杆菌的唯一宿主,其感染在患者的胃和十二指肠的黏膜层上,是最常见的细菌感染之一,其中流行病学研究发现幽门螺旋杆菌与胃癌有高达70%相关性(Marshall B.2002.)。许多研究指出,幽门螺旋杆菌感染会造成肠胃疾病,例如:胃溃疡(gastric ulcer)、慢性胃炎(chronicgastritis)甚至胃腺癌(gastric adenocarcinoma)(Marshall BJ,Warren JR.1984.,NIHConsensus Conference 1994,Schistosomes 1994.)。因此,若能根除幽门螺旋杆菌即可降低罹患胃癌的机率。
目前根除幽门螺旋杆菌的主要方法为三联疗法(triple therapy),另外还有单一疗法(mono therapy)、二联疗法(dual therapy)及四联疗法(quadruple therapy)。单一疗法使用单一药剂治疗,如抗生素、铋盐等。二联疗法使用铋盐或质子帮浦抑制剂(protonpump inhibitor,PPI)加上一种抗生素,需时2周。三联疗法使用铋盐或质子帮浦抑制剂加上两种抗生素,需时1-2周。四联疗法使用铋盐、质子帮浦抑制剂加上两种抗生素,需时1周,但是药剂使用复杂,彼此之间容易发生交互作用,以及产生新的副作用等。
目前市面上常用于根除幽门螺旋杆菌的抗生素包括克拉霉素(clarithromycin)、阿莫西林(amoxicillin)、氟红霉素(flurithromycin)、四环素(tetracycline)以及甲硝唑(metronidazole)等。质子帮浦抑制剂对幽门螺旋杆菌的作用主要是抑制胃酸的形成和静菌作用,常用的质子帮浦抑制剂有奥美拉唑(omeprazole)和兰索拉唑(lansporazole)等。铋盐(bismuth)具有杀死幽门螺旋杆菌和保护胃壁细胞的作用,常用的铋盐有bismuth(III)和bismuth(V)等。抗生素是目前最有效的根除幽门螺旋杆菌感染的药物,然而,抗生素的不当使用所带来的抗药性问题,成为幽门螺旋杆菌根除失败的主要原因。为了解决抗药性导致治疗困难的问题,近几年开始研究从天然药草中萃取可以有效治疗幽门螺旋杆菌的化合物(Lee SY,Shin YW,Hahm KB.2008.),发展出一种非抗生素的治疗方法。
鱼针草(Anisomeles indica,又称金剑草)为唇形科多年生草本植物,生长于亚热带地区,常用于治疗肠胃炎和免疫系统不全等相关疾病,先前发现其萃取物能有效抑制发炎反应以及肿瘤增生(Rao YK,Fang SH,Hsieh SC,Yeh TH,Tzeng YM.2009.,Hsieh SC,Fang SH,Rao YK,Tzeng YM.2008.)。此外,有文献(Lien HM,Wang CY,Chang HY,Huang CL,Peng MT,Sing YT,Chen CC,Lai CH.2013.,Rao YK,Lien HM,Lin YH,Hsu YM,Yeh CT,ChenCC,Lai CH,Tzeng YM.2012.)揭示鱼针草萃取物具有抑制幽门螺旋杆菌的功效,由于其抑制机制以及是否具有抑制抗药性幽门螺旋杆菌的功效仍不清楚,因此需要提供鱼针草抑制幽门螺旋杆菌的作用机制以解决本领域存在的问题。
【发明内容】
鉴于此,本发明提供一种鱼针草内酯(ovatodiolide)用于抑制胃幽门杆菌的蛋白质合成的用途。
为达到上述目的,本发明提供一种鱼针草内酯用于制备抑制胃幽门螺旋杆菌的蛋白质合成的组合物的用途,其中该鱼针草内酯通过抑制蛋白质合成从而达到抑制胃幽门杆菌的功效。
其中,胃幽门螺旋杆菌是指多重抗药性胃幽门螺旋杆菌或幽门杆菌标准菌株,且多重抗药性胃幽门螺旋杆菌是指HP v633或HP v1354,幽门杆菌标准菌株为H.pylori26695。
其中,该蛋白质合成是通过30S核醣体RpsB的作用完成的。
其中,该鱼针草内酯抑制胃幽门杆菌的有效剂量为16毫克~1642毫克;优选剂量为82毫克~821毫克;最优选剂量为164毫克~329毫克。
其中,该组合物可进一步包括药学上可接受的载剂、赋形剂、稀释剂;剂型选自溶液、悬浮液、乳剂、粉末、锭剂、丸剂、糖浆、口含锭、片剂、咀嚼胶、浓浆、和胶囊。
其中,该组合物可进一步制成流体乳制品、浓缩牛奶、酸奶、酸乳、冷冻优格、乳杆菌发酵饮料、奶粉、冰淇淋、奶酪、干酪、豆浆、发酵豆浆、蔬果汁、果汁、运动饮料、甜点、果冻、糖果、婴儿食品、健康食品、动物饲料、中草药组合物、膳食补充品。
【附图说明】
图1为鱼针草抑制幽门螺旋杆菌的蛋白质合成的图;
图2为鱼针草抑制幽门螺旋杆菌的核醣体组装的图。
【具体实施方式】
本说明书中所述的所有技术性及科学术语,除非另有定义,均为本领域技术人员可共同理解的含义。
本说明书术语“载剂(carrier)”、“赋形剂(vehicle)”,是指不具毒性的化合物或药剂,其具有协助细胞或组织吸收药物的功能。
前述载剂或赋形剂可以为芳香剂(aromatics)、缓冲剂、黏着剂(binders)、着色剂(colorants)、崩解剂(disintegrants)、稀释剂、乳化剂(emulsifiers)、增量剂(extenders)、气味改善剂(flavor-improving agents)、胶凝剂(gellants)、助流剂(glidants)、防腐剂、皮肤渗透促进剂(skin-penetration enhancers)、助溶剂(solubilizers)、安定剂(stabilizers)、悬浮剂(suspending agents)、甜味剂(sweeteners)、张度剂(tonicity agents)、黏度增强剂(viscosity-increasing agents)或上述任意组合。
本说明书术语“有效剂量(effective amount)”或“治疗(therapeutically)有效剂量”,是指相关化合物或药物的足够剂量,其可使服用者服药后减轻一种或多种疾病症状或生理状况;其结果为降低和/或缓和迹象(sign)、症状(symptom)、或病因,或有意图地改变其他生理系统。举例而言,治疗的「有效剂量」包括本发明提供的化合物可在临床上显著降低疾病症状的剂量。适当的有效量的有效值取决于普通药学技术,例如药物增量方法(dose escalation methods)。
本发明所使用的组合物,可进一步添加一种可食性材料,以制备一种食品产品或保健产品。其中该可食性材料包含,但不限于:水(water)、流体乳制品(fluid milkproducts)、牛奶(milk)、浓缩牛奶(concentrated milk);发酵乳制品(fermented milk),诸如酸奶(yogurt)、酸乳(sour milk)、冷冻优格(frozen yogurt)、乳杆菌发酵饮料(lactic acid bacteria-fermented beverages);奶粉(milk powder);冰淇淋(icecream);奶酪(cream cheeses);干酪(dry cheeses);豆奶(soybean milk);发酵豆奶(fermented soybean milk);蔬果汁(vegetable-fruit juices);果汁(juices);运动饮料(sports drinks);甜点(confectionery);果冻(jellys);糖果(candies);婴儿食品(infant formulas);健康食品(health foods);动物饲料(animal feeds);中草药材(Chinese herbals);膳食补充品(dietary supplements)等。
本发明所使用的前述组合物可为膳食补充品,可以通过下列方式给予服用者:与合适的可饮用液体混合,例如水、酸奶酪、牛奶或果汁;或可与固体或液体食品进行混合。在本说明书中,膳食补充品的形式可为锭剂、丸剂、胶囊、糖锭(lozenge)、颗粒、粉剂、悬浮剂、小药囊、软锭剂、糖果、棒、糖浆及相应的投与形式,通常以单位剂量的形式,并通过膳食补充品的常规制备方法来制造。
以下实施例仅作为例示,剂量可依据需要而作出改变,而不限于所使用的化合物的活性、治疗疾病或生理状态、给药方式、个体需求、疾病严重性以及医生的判断。
实施例1:鱼针草内酯抗幽门螺旋杆菌的最小抑菌浓度
用不同浓度的鱼针草内酯(ovatodiolide)纸锭,分别与幽门螺旋杆菌培养两天,通过琼酯孔洞扩散法(agar-well diffusion)测定鱼针草内酯抑制幽门螺旋杆菌的最小浓度,由此分析鱼针草内酯抑制幽门螺旋杆菌标准菌株(H.pylori 26695)生长的最低浓度。表一为抑制幽门螺旋杆菌标准菌株(H.pylori 26695)的最小抑菌浓度,由实验结果可知10μM及20μM的鱼针草内酯均有抑制环,而其抑制环直径分别为10±0.5mm和19±0mm,这证明鱼针草内酯抑制幽门螺旋杆菌的最低浓度为10μM。
表一、抑制幽门螺旋杆菌标准菌株(H.pylori 26695)的最小抑制浓度
实施例2:抗幽门螺旋杆菌标准菌株及其多重抗药性菌株的最小杀菌浓度
利用系列稀释的方式配制不同浓度的鱼针草内酯,并分别与从实验室分离的幽门螺旋杆菌标准菌株和多重抗药性幽门螺旋杆菌(v633,v1354)培养六小时,再以涂布平板法(spread plate method)观察并分析菌落生长数目,分析可完全杀死幽门螺旋杆菌标准菌株及多重抗药性菌株的鱼针草内酯的最低杀菌浓度。由表二结果可知,鱼针草内酯对幽门螺旋杆菌标准菌株的最小杀菌浓度为200μM,而对多重抗药性菌株v633和v1354的最小杀菌浓度均为100μM。
表二、鱼针草内酯对多重抗药螺旋杆菌的最小杀菌浓度
实施例3:鱼针草内酯抑制幽门螺旋杆菌的蛋白质合成
由于鱼针草内酯的结构与抗生素中抑制蛋白质生成的药物相似,为了解鱼针草内酯抑制幽门螺旋杆菌的机制是否与蛋白质合成有关,利用体外转录-转译系统(in vitroTranscription/Translation system),将鱼针草内酯与大肠杆菌萃取物(E.coli S30)作用10分钟后,加入带有荧光素酶(luciferase)的pGL3进行转录和转译反应2小时,最后利用双荧光素酶报告基因检测系统(Dual-Luciferase Reporter Assay System),将反应完成的样品加入荧光素(luciferin)使其呈色,再通过微孔板光度计(microplateluminometer)测定吸光值,即可得知鱼针草内酯是否会抑制蛋白质合成。
其中,以抑制30S亚单元(subunit)的卡那霉素(kanamycin)和抑制50S亚单元的红霉素(erythromycin)作为对照组。图1结果显示10μM的鱼针草内酯对蛋白质合成的抑制为50%以上,而1μM的卡那霉素和红霉素抑制蛋白质合成的能力为75%以上,由此可知鱼针草内酯的药物机制与抑制蛋白质合成有关。
实施例4:鱼针草内酯抗幽门螺旋杆菌的核醣体组装
在另一实施例中,蛋白质转译的过程分三个步骤,分别为核糖体起始(Initiation)、延长(Elongation)、终止(Termination),而为了进一步分析鱼针草内酯是否作用于核糖体导致蛋白质合成无法顺利进行,以蛋白质印迹分析法(western blotanalysis)测定核醣体30S亚单元中的RpsB蛋白质含量,将幽门螺旋杆菌与不同浓度的鱼针草内酯一起培养6小时并萃取总细胞溶解产物(total cell lysates)。再利用10%SDS-PAGE分离总细胞溶解产物,并用PVDF膜转渍蛋白质,将RpsB抗体与蛋白质进行杂合(hybridization),再用抗兔(anti-rabbit)抗体侦测RpsB抗体,最后加入呈色剂,利用仪器Azure C400Biosystems观察结果。
图2为利用蛋白质印迹分析法分析核醣体30S亚单元的RpsB表达量,其中卡那霉素(400μM)和红霉素(400μM)为对照组,结果显示RpsB蛋白质与鱼针草内酯作用后,其蛋白质表达量明显下降,这与对照组结果相似。
鱼针草内酯在体外实验对于细菌抑制的有效浓度为10μM~1mM,鱼针草内酯分子量为328.4,假设65公斤的成人体内约有血液5公升,由此推算出人体每日有效剂量为16毫克~1642毫克;优选药物浓度为50μM~500μM,则推算出每日优选剂量为82毫克~821毫克;最优选浓度为100μM~200μM,则推算出最优选剂量为每日164毫克~329毫克。
综上所述,本发明的鱼针草内酯通过抑制胃幽门螺旋杆菌的30S核醣体RpsB的表达,从而抑制蛋白质合成,并且进一步有效抑制幽门螺旋杆菌标准菌株及多重抗药菌株的生长,该鱼针草内酯抑制胃幽门杆菌的有效剂量为16毫克~1642毫克;优选剂量为82毫克~821毫克;最优选剂量为164毫克~329毫克。
以上所述的实施例仅用于说明本发明的技术思想及特点,其目的在于使本领域技术人员能够了解本发明的内容并予以实施,而不能因此限定本发明的专利范围,即,凡是按照本发明所揭示的精神所作出的等同变化或修改,仍应涵盖在本发明的专利范围内。
Claims (10)
1.一种鱼针草内酯用于制备抑制胃幽门螺旋杆菌的蛋白质合成的组合物的用途,其特征在于,所述鱼针草内酯通过抑制胃幽门螺旋杆菌的蛋白质合成而达到抑制胃幽门杆菌的功效。
2.如权利要求1所述的用途,其特征在于,所述胃幽门螺旋杆菌为多重抗药性胃幽门螺旋杆菌或幽门杆菌标准菌株。
3.如权利要求2所述的用途,其特征在于,所述多重抗药性胃幽门螺旋杆菌为HP v633或HP v1354,所述幽门杆菌标准菌株为H.pylori 26695。
4.如权利要求1所述的用途,其特征在于,所述蛋白质合成是通过30S核醣体RpsB的作用完成的。
5.如权利要求1所述的用途,其特征在于,所述鱼针草内酯抑制所述胃幽门杆菌的有效剂量为16毫克~1642毫克。
6.如权利要求1所述的用途,其特征在于,所述鱼针草内酯抑制所述胃幽门杆菌的剂量为82毫克~821毫克。
7.如权利要求1所述的用途,其特征在于,所述鱼针草内酯抑制所述胃幽门杆菌的剂量为164毫克~329毫克。
8.如权利要求1所述的用途,其特征在于,所述组合物包括药学上可接受的载剂、赋形剂、稀释剂。
9.如权利要求1所述的用途,其特征在于,所述组合物的剂型选自溶液、悬浮液、乳剂、粉末、锭剂、丸剂、糖浆、口含锭、片剂、咀嚼胶、浓浆和胶囊。
10.如权利要求1所述的用途,其特征在于,所述组合物制成流体乳制品、浓缩牛奶、酸奶、酸乳、冷冻优格、乳杆菌发酵饮料、奶粉、冰淇淋、奶酪、干酪、豆浆、发酵豆浆、蔬果汁、果汁、运动饮料、甜点、果冻、糖果、婴儿食品、健康食品、动物饲料、中草药组合物、膳食补充品。
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