CN101181249A - 姜黄素在制备尿酸转运子urat1抑制药物中的应用 - Google Patents
姜黄素在制备尿酸转运子urat1抑制药物中的应用 Download PDFInfo
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Abstract
本发明涉及黄酮类单体化合物姜黄素的新用途,具体是涉及姜黄素在制备尿酸转运子URAT1抑制药物中的应用。经动物试验结果表明:姜黄素对高尿酸血模型动物表达异常增高的尿酸转运子URAT1具有确切的抑制作用,而对正常动物的正常水平的尿酸转运子URAT1表达无显著抑制作用,具有很好的安全性,利用姜黄素配以相关的药用辅料,用常规的制剂方法可制成姜黄素口服液、胶囊剂、片剂、颗粒剂等尿酸转运子URAT1的抑制药物,可用于治疗与尿酸转运子URAT1异常高表达相关的疾病。
Description
技术领域:本发明涉及黄酮类单体化合物姜黄素的新用途,具体是涉及姜黄素在制备尿酸转运子URAT1抑制药物中的应用。
背景技术:尿酸排出减少或生成增加,可导致血液中尿酸浓度增高,直接或间接引起相关疾病。尿酸的排泄是一复杂过程,尿酸随血液循环流入肾小球时,游离型的尿酸将全部滤过;在近端肾小管有约99%尿酸盐被重吸收(Diamond HS,Meisel AD.Postsecretory reabsorption of urate in man.Arthritis AndRheumatism,1975,18(6):805-809.)。位于近曲小管的尿酸转运子(蛋白)URAT1参与尿酸重吸收过程,通过与阴离子的交换将尿酸盐由管腔转运入细胞。URAT1蛋白主要分布于肾皮质近曲小管上皮细胞向腔膜,是一个电中性的尿酸转运蛋白。URAT1基因由Enomoto等于2002年首先从人肾脏克隆出来,位于染色体11q13,由SLC22A12基因编码,由10个内含子9个外显子组成,拥有12个跨膜结构域,其cDNA全长2642bp,编码区1659bp,编码含555个氨基酸的蛋白质。原发性肾低尿酸血症(hypouricemia)病人的SLC22A12基因缺陷病例进一步证明人的URAT1(hURAT1)参与尿酸的重吸收(Atsushi Enomoto,Hiroaki Kimura,Arthit Chairoungdua,Yasuhiro Shigeta,et al.Molecular identificationof a renal urate-anion exchanger that regulates blood urate levels.Nature,2002,417(6887):447-452)。
姜黄素结构式、分子式和美国化学文摘收录号(CAS No.)见下:
姜黄素可以由天然产物中分离得到,例如有文献报道了采用渗漉法提取及大孔树脂分离正交试验,确定了提取姜黄素用9倍体积的70%乙醇,以3mL/min的速度渗漉的最佳提取工艺和利用D101大孔树脂纯化姜黄素工序中调节上样料液以及pH7,80%乙醇以4ml/min的速率洗脱的最佳分离工艺,由姜黄中分离姜黄素(尤本明,王忠壮,胡晋红.姜黄中姜黄素的提取及分离工艺研究.药学服务与研究2006;6(4):277-279)。也有报道由化学改构/合成得到姜黄素(Venkateswarlu,Somepalli;Ramachandra,Marellapudi S.;Subbaraju,Gottumukkala V.Synthesis and biological evaluation ofpolyhydroxycurcuminoids.Bioorganic & Medicinal Chemistry(2005),13(23),6374-6380.)。
姜黄素已见有多种生理和药理活性的报道,包括抗氧化作用、抗肿瘤作用、抗炎作用、抗病毒作用等(许东晖,王胜,金晶,梅雪婷,许实波.姜黄素的药理作用研究进展.中草药2005;36(11):1737-1740)。但单体化合物姜黄素用于制备针对尿酸转运子URAT1的抑制药物,则未见报道。
发明内容:
本发明的目的是提供黄酮类单体化合物姜黄素的新用途,具体是姜黄素在制备尿酸转运子URAT1抑制药物中的应用。
本发明的技术解决方案之一是:提供黄酮类单体化合物姜黄素在制备尿酸转运子URAT1抑制药物的新用途。该新用途包括了以本发明所述的姜黄素,配以本领域的技术人员所熟知的药用辅料(赋形剂、助溶剂、控释剂等),制成本领域的技术人员所熟知的剂型(包括口服液、注射剂、胶囊剂、片剂、颗粒剂、微囊剂等),用于制备尿酸转运子URAT1抑制药物。
本发明的优点是,提供了对尿酸转运子URAT1具有确切抑制调节作用的姜黄素,用于制备尿酸转运子URAT1抑制药物,可用于治疗与尿酸转运子URAT1异常高表达相关疾病。与针对痛风或者高尿酸血病症的发明比较,本发明提供的单体化合物姜黄素制备尿酸转运子URAT1抑制药物新用途具有3个明显的进步和优点:1、有效成分确切(单体化合物);2、作用靶点明确(尿酸排泄过程中重吸收作用的尿酸转运子URAT1);3、调节作用明确(抑制调节)。
本发明利用高尿酸血动物模型在基因转录水平和蛋白表达水平上科学地评价并确定了单体化合物姜黄素对尿酸转运子URAT1的抑制调控作用。
本发明所涉及的单体化合物姜黄素对模型动物表达异常增高的尿酸转运子URAT1具有确切的抑制作用;而对正常动物的正常水平的尿酸转运子表达无显著抑制作用,具有很好的安全性。
为了更好的理解本发明的实质,下面将用单体化合物姜黄素的的药理实验及结果来说明其在制备尿酸转运子URAT1抑制药物中的应用。
具体实施方式:以下实施例仅作为进一步阐述发明之用,不能用来限制本发明。
实施例1:黄酮类单体化合物姜黄素对尿酸转运子URAT1的抑制调节作用
实验动物:昆明种小鼠,15-20克,雄性
药物配制:姜黄素均匀分散于生理盐水中,用于灌胃给药,给药剂量为25mg/kg
实验材料:Trizol Reagment(Invigen),氯仿,异丙醇,无水乙醇,DEPC,M-MLV反转录酶,PCR试剂盒,RIPA裂解液等
实验仪器:高速冷冻离心机,高速匀浆机,Bio-rad垂直电泳槽,MBI-PCR仪、水平电泳槽等
实验模型:小鼠高尿酸血症模型。
实验方法:
1、模型的建立:动物适应环境1周后,灌胃给予氧嗪酸钾盐250mg/kg;对照给予等体积生理盐水,造模周期10天。
2、给药:造模期间同时给药,在给予氧嗪酸钾盐造模/生理盐水对照1h后,灌胃给予单体化合物姜黄素25mg/kg;对照给予生理盐水。
3、小鼠处死及组织的保存:小鼠于灌胃给予姜黄素/生理盐水后1小时处死,冰台上分离肾脏组织,肾组织经液氮冷冻后保存于-80℃。
4、蛋白质印迹法(Western-blotting):取组织约100mg加入1ml的RIPA裂解液匀浆提取,12000g 4℃离心15分钟,得肾脏组织总蛋白提取液,Bradford法测蛋白浓度,稀释蛋白样品至终浓度为5ug/ul。混合上样缓冲液变性上样,SDS-PAGE凝胶电泳后PVDF转膜,封闭液封闭1小时后加入mURAT1抗体(根据NCBI数据库mURAT1蛋白序列制备,使用浓度1∶4000)4℃孵育过夜,二抗(1∶4000)室温摇床孵育1小时,HRP-ECL发光,暗室曝光显影。胶片扫描后对图片进行灰度分析。
5、逆转录聚合酶链反应(RT-PCR):提取小鼠肾脏总RNA,逆转录得到cDNA模板,根据NCBI数据库的mURAT1基因设计引物,进行mURAT1目的基因扩增。扩增产物经琼脂糖凝胶电泳后UV成像,对图片进行灰度分析。
实验结果:
a.蛋白质印迹法Western-Blotting
Groupn=5 | 空白+生理盐水 | 空白+姜黄素 | 模型+生理盐水 | 模型+姜黄素 |
灰度值 | 44.9±14.2 | 41.2±13.3 | 84.3±18.7++ | 51.7±12.5** |
++P<0.01与空白+生理盐水组比较 **P<0.01与模型+生理盐水组比较连续10天灌胃给予氧嗪酸钾盐250mg/kg造模造成小鼠肾组织中URAT1蛋白高水平表达,灰度值达到84.3,显著高于空白对照44.9。灌胃给予25mg/kg的单体化合物姜黄素可显著抑制模型小鼠肾组织中URAT1蛋白的高水平表达,灰度值为51.7,接近空白对照水平。说明单体化合物姜黄素显著抑制模型动物异常增高的尿酸转运子URAT1蛋白表达。灌胃给予单体化合物姜黄素对空白小鼠肾组织中URAT1蛋白表达水平无显著影响,说明单体化合物姜黄素对小鼠肾组织正常水平的URAT1蛋白无显著抑制作用,具有较好的安全性。
b.逆转录聚合酶链反应RT-PCR
Groupn=5 | 空白+生理盐水 | 空白+姜黄素 | 模型+生理盐水 | 模型+姜黄素 |
灰度值 | 33.0±9.1 | 35.7±10.2 | 68.7±18.0++ | 38.9±10.4** |
++P<0.01与空白+生理盐水组比较 **P<0.01 与模型+生理盐水组比较连续10天灌胃给予氧嗪酸钾盐250mg/kg造模造成小鼠肾组织中URAT1 mRNA高水平表达,灰度值达到68.7,显著高于空白对照33.0。灌胃给予25mg/kg的单体化合物姜黄素可显著抑制模型小鼠肾组织中URAT1 mRNA的高水平表达,灰度值为38.9,接近空白对照水平。说明单体化合物姜黄素可在转录水平上抑制异常增高的尿酸转运子URAT1 mRNA表达。灌胃给予单体化合物姜黄素的空白小鼠肾组织中URAT1 mRNA表达水平为35.7,相对于空白对照组无显著变化,说明单体化合物姜黄素对小鼠肾组织正常水平的URAT1 mRNA无显著抑制作用,具有较好的安全性。
实施例2:
将单体化合物姜黄素按照常规制剂方法,加入水及适量增溶剂(聚乙二醇400)溶解,分装,灭菌,制备成规格为20mg/ml的姜黄素口服液;
将单体化合物姜黄素按照常规制剂方法,软胶囊材质选用明胶和山梨醇,制备成规格为20mg/粒的姜黄素胶囊;
将单体化合物姜黄素按照常规制剂方法,加入赋形剂环糊精,混合均匀,制粒,压片,制备成规格为20mg/片的姜黄素片剂。
Claims (1)
1.黄酮类单体化合物姜黄素在制备尿酸转运子URAT1抑制药物中的应用。
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CN108653539A (zh) * | 2018-07-19 | 2018-10-16 | 东莞市广易中医药发展有限公司 | 一种降低尿酸药食同源压片及其制备方法 |
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US8808768B2 (en) * | 2008-07-21 | 2014-08-19 | Kailash Chandra Agarwal | Development of biochemically standardized extracts from fresh rhizomes of turmeric (Curcuma longa) for treatment of diseases caused by hyperuricemia |
CN108653539A (zh) * | 2018-07-19 | 2018-10-16 | 东莞市广易中医药发展有限公司 | 一种降低尿酸药食同源压片及其制备方法 |
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