CN106317178B - 一种具有降尿酸活性的多肽及其应用 - Google Patents
一种具有降尿酸活性的多肽及其应用 Download PDFInfo
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- CN106317178B CN106317178B CN201610752731.0A CN201610752731A CN106317178B CN 106317178 B CN106317178 B CN 106317178B CN 201610752731 A CN201610752731 A CN 201610752731A CN 106317178 B CN106317178 B CN 106317178B
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- polypeptide
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
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- C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
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Abstract
本发明涉及一种具有降尿酸活性的多肽及其应用。一种具有降尿酸活性的多肽,氨基酸序列为:Tyr‑Glu‑Gly。本发明的多肽使用多肽合成仪,采用固相合成法合成。本发明的多肽通过HPLC法进行体外降尿酸活性检测,结果表明本发明的多肽对黄嘌呤氧化酶的抑制效果好,具有良好的降尿酸活性。本发明的多肽可以作为痛风疾病的辅助治疗药物或者用于具有降尿酸作用的功能性保健食品或药品。
Description
技术领域
本发明涉及多肽技术领域,具体涉及一种具有降尿酸活性的多肽及其应用。
背景技术
近年来,随着人们饮食结构的改善,国内外痛风的患病率逐年增长,且有年轻化的趋势。高尿酸血症是痛风的生化基础,主要是指长期嘌呤代谢紊乱,致使尿酸产生过多或尿酸排泄过少而引起的一种疾病。越来越多的研究表明,高尿酸血症已经不是一个单纯的尿酸含量超标的病症,它与多种心脑血管疾病及肾脏疾病紧密相关,除此之外,与Ⅱ型糖尿病及胰岛素抵抗综合征的关系也密不可分。
目前,治疗高尿酸血症主要有降低尿酸生成、促进尿酸排泄和促进尿酸分解等方法。降低尿酸生成的药物主要包括黄嘌呤氧化酶(XO)抑制剂:别嘌呤醇(Allopurinol),非布索坦(Febuxostat),托匹司他(Topiroxostat)等;促进尿酸排泄的药物:丙磺舒(Probenecid),苯溴马隆(Benzbromarone)等;促进尿酸分解的药物:拉布立酶((ELITEK,Rasburicase),聚乙二醇尿酸酶(PEG-uricase)等。这些药物对人体脏器具有一定的毒副作用,易引起严重的过敏及炎症反应,且价格昂贵。因此,开发一种高效安全的活性物质用于高尿酸血症的预防及治疗具有一定的经济及社会意义。
黄嘌呤氧化酶是目前研究高尿酸血症的重要靶点。有研究报道,中草药制剂可用于治疗高尿酸血症,对其功效成分进行分离提纯,发现一些多酚黄酮类物质能有效抑制黄嘌呤氧化酶的活性。张珞研究了鼠尾藻各组分对黄嘌呤氧化酶的作用,发现乙酸乙酯组分对XO无抑制作用,其水溶性组分对XO具有反竞争性抑制作用,抑制常数为Ki=0.11mg/ml,IC50=0.42mg/mL。王晓从核桃壳提取物中分离得到的对香豆醛、β-D-胡萝卜苷、对羟基苯甲醛和氢化胡桃醌等对黄嘌呤氧化酶具有较高的抑制活性,以166mg/kg核桃壳提取物的剂量灌胃大鼠30d,在30d时提取物组大鼠的血尿酸含量降低到模型组的76.41%,同时大鼠血清中的尿素氮和肌酐含量显著降低,说明核桃壳提取物具有体内降尿酸活性和肾脏保护作用。但是该多酚黄酮类物质的提取得率低,且成本较高,其安全性有待考证。
据报道,生物活性肽具有抗病毒、抗肿瘤、降血压、降血糖、抗真菌、抗氧化、抗衰老等生理功能,较蛋白和氨基酸更易被人体吸收,且无毒副作用,可用于医药、食品、化妆品等领域。目前,对于具有降尿酸活性的生物活性肽的研究尚处于萌芽阶段。专利公开号为CN101417119的专利涉及一种用于快速降低个体血液中的尿酸含量的医药组合物,其有效物质为鹅肌肽,主要通过调节生理pH值及清除体内乳酸的含量从而促进尿酸的排泄,达到降低血液中尿酸含量的目的。AliceB.Nongonierma等人研究发现,Trp及含Trp的二肽(Arg-Trp,Trp-Val,Val-Trp,Lys-Trp和Ile-Trp)能够抑制黄嘌呤氧化酶的活性。通过双倒数作图及分子对接模拟对其抑制机理进行分析得知,Trp及含Trp的二肽能和XO的至少6个疏水性氨基酸残基发生相互作用,占据其疏水通道,并未阻碍底物黄嘌呤进入XO的活性中心,属于非竞争性抑制剂。
然而,三肽用于降低体外或体内尿酸含量相关的文献尚未见报道。本发明提供了一种体外检测降尿酸活性的方法并对Tyr-Glu-Gly的降尿酸功效及机理进行了研究,对于开发具有以降尿酸肽为功能因子的保健食品及药品具有重要的指导意义。
发明内容
本发明的首要目的在于提供一种具有降尿酸功效的生物活性肽,可用于开发具有预防或治疗痛风类疾病功效的保健品及药品。
一种具有降尿酸活性的多肽,氨基酸序列为:Tyr-Glu-Gly(YEG)。
进一步地,所述Tyr为酪氨酸的氨基酸残基,所述Glu为谷氨酸的氨基酸残基,所述Gly为甘氨酸的氨基酸残基。
进一步地,所述的氨基酸序列采用已有的成熟技术,通过树脂的筛选,获取合理的多肽合成方法:采用标准Fmoc方案,选用Wang树脂进行固相合成。
进一步地,所述多肽在体外降尿酸活性检测中表现出抑制黄嘌呤氧化酶(XO)的活性。
进一步地,本发明采用了HPLC法检测Tyr-Glu-Gly(YEG)的体外降尿酸活性,分子对接结果显示:Tyr-Glu-Gly与XO产生的相互作用主要由疏水作用及氢键作用主导。
进一步地,所述多肽对黄嘌呤氧化酶抑制的IC50值为13.43±0.73mg/ml,即36.59±2.00mmol/L。
所述多肽用作包括痛风疾病的辅助治疗药物或者用于开发具有以降尿酸为功能因子的保健食品及药品。
附图说明
图1为各实施例生成尿酸含量的色谱图;
图2为Tyr-Glu-Gly对XO的抑制曲线。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
固相合成多肽:
1、树脂选型
采用标准Fomc方案,选用0.0125mmol,2-chlorotritylchlorideresin树脂(天津市南开合成科技有限公司),按照氨基酸序列Tyr-Glu-Gly的序列特征,加入0.3mol第一个9-芴甲氧羰基(Fmoc)保护氨基酸,将N,N'-二环己基碳二亚胺(DCC)和5wt%4-二甲氨基吡啶(DMAP)加入到反应器振荡反应,用N-甲基吡咯烷酮(NMP)冲洗树脂除去多余保护氨基酸。测定第一个氨基酸与树脂的连接效率即偶合率,偶合率如表所示。
采用标准Fomc方案,选用Wang树脂,按照氨基酸序列Tyr-Glu-Gly的序列特征,加入0.3mol第一个Fmoc保护氨基酸,将DCC和5wt%DMAP加入到反应器振荡反应,用NMP冲洗树脂除去多余保护氨基酸。测定第一个氨基酸与树脂的连接效率即偶合率,偶合率如表所示。
表1第一个氨基酸与树脂的偶合率
2、合成过程
采用标准Fomc方案,选用偶合率较高的树脂,按照氨基酸序列Tyr-Glu-Gly的序列特征,使肽链从C端逐个向N端延伸,各氨基酸的用量为0.1mol,加入0.3mol Fmoc保护氨基酸,每步缩合都加入1-羟基苯并三氮唑(HOBT)活化保护氨基酸的羧基,每步缩合采用20%哌啶/二甲基甲酰胺(DMF)溶液(15ml/g),处理20min,去除Fmoc保护基。肽侧链合成后,将含有树脂的肽链加入到如下反应液中:二氯甲烷(99%)三氟乙酸(1%)(体积分数),将肽链从树脂上切割下来。再次将多肽加入到反应液:三氟乙酸(94.5%)、酒石酸乙二胺(2.5%)、蒸馏水(2%)、TIS(1%)(体积分数)中反应2h,脱去侧链保护基。以上过程均在SYMPHONY型12通道多肽合成仪中完成,所合成的多肽经SHIMADZU高效液相色谱仪纯化,纯度达到95%以上,并经ESI-MS鉴定结构。
反相高效液相色谱法(RP-HPLC法)测定尿酸含量的方法如下:
(1)实验样品及试剂制备
50mmol/L(pH8.0)Tris-HCl缓冲液:准确称取6.0572g Tris,用超纯水溶解,以1mol/L HCl调节pH至8.0,定容至1000mL,备用。
Tyr-Glu-Gly梯度溶液:准确称取20mg Tyr-Glu-Gly,用pH8.0的Tris-HCl缓冲液配成20mg/ml溶液,分别稀释配制成10、12、14、16、18mg/ml的Tyr-Glu-Gly溶液,备用。
黄嘌呤标准储备液:称取0.0307g黄嘌呤,用1mL1M NaOH溶解,用pH8.0的Tris-HCl缓冲液定容至100ml,震荡均匀,备用。
尿酸标准储备液:称取0.0084g尿酸,用1mL 1M NaOH溶解,用pH8.0的Tris-HCl缓冲液定容至100ml,震荡均匀,分别稀释至100、200、300、400、500μmol/L,备用。
流动相:95%(V/V)0.015mol/L的NH4H2PO4+5%(V/V)色谱级甲醇,超纯水定容至1000ml,过0.22μm有机膜,超声,备用。
(2)样品预处理
将所述Tyr-Glu-Gly溶液分别取100μl于10ml离心管中,加入50μl黄嘌呤氧化酶,30℃下水浴保温10min;加入2.9ml所述pH8.0的Tris-HCl缓冲液,400μl黄嘌呤标准储备液,30℃下水浴保温20min。反应结束后,加入150μl 1M HCl,冷却后过0.22μm水系膜,以pH8.0的Tris-HCl缓冲液代替多肽样品溶液作为空白对照,待测。
色谱柱:ZorbaxEClipsePlusC18(4.6×250mm,5μm,安捷伦)
液相条件:洗脱液为5%(V/V)甲醇+95%(V/V)NH4H2PO4,进样体积为20μl,流速lml/min,检测波长为290nm,运行时间15min。
黄嘌呤氧化酶抑制率IXO(%)表达式:
式中:Ccontrol为空白组中尿酸的生成量,μmol/L;Csample为样品组中尿酸的生成量,μmol/L。
实施例1
取100μl 18mg/ml的Tyr-Glu-Gly溶液于10ml离心管中,加入50μl黄嘌呤氧化酶,30℃下水浴保温10min;加入2.9ml所述pH8.0的Tris-HCl缓冲液,400μl黄嘌呤标准储备液,30℃下水浴保温20min;反应结束后,加入150μl 1M HCl,冷却后过0.22μm水系膜,以pH8.0的Tris-HCl缓冲液代替多肽样品作为空白对照,待测。
实施例2
取100μl 16mg/ml Tyr-Glu-Gly溶液于10ml离心管中,加入50μl黄嘌呤氧化酶,30℃下水浴保温10min;加入2.9ml所述pH8.0的Tris-HCl缓冲液,400μl黄嘌呤标准储备液,30℃下水浴保温20min;反应结束后,加入150μl 1M HCl,冷却后过0.22μm水系膜,以pH8.0的Tris-HCl缓冲液代替多肽样品作为空白对照,待测。
实施例3
取100μl 14mg/ml Tyr-Glu-Gly溶液于10ml离心管中,加入50μl黄嘌呤氧化酶,30℃下水浴保温10min;加入2.9ml所述pH8.0的Tris-HCl缓冲液,400μl黄嘌呤标准储备液,30℃下水浴保温20min;反应结束后,加入150μl 1M HCl,冷却后过0.22μm水系膜,以pH8.0的Tris-HCl缓冲液代替多肽样品作为空白对照,待测。
实施例4
取100μl 12mg/ml Tyr-Glu-Gly溶液于10ml离心管中,加入50μl黄嘌呤氧化酶,30℃下水浴保温10min;加入2.9ml所述pH8.0的Tris-HCl缓冲液,400μl黄嘌呤标准储备液,30℃下水浴保温20min;反应结束后,加入150μl 1M HCl,冷却后过0.22μm水系膜,以pH8.0的Tris-HCl缓冲液代替多肽样品作为空白对照,待测。
实施例5
取100μl 10mg/ml Tyr-Glu-Gly溶液于10ml离心管中,加入50μl黄嘌呤氧化酶,30℃下水浴保温10min;加入2.9ml所述pH8.0的Tris-HCl缓冲液,400μl黄嘌呤标准储备液,30℃下水浴保温20min;反应结束后,加入150μl 1M HCl,冷却后过0.22μm水系膜,以pH8.0的Tris-HCl缓冲液代替多肽样品作为空白对照,待测。
测得实施例1~5中生成尿酸含量的色谱图如图1所示,Tyr-Glu-Gly对XO的抑制率如图2所示。由图2可知,所述多肽对黄嘌呤氧化酶抑制的IC50值为13.73mg/ml,即37.41mmol/L。
使用MGL tools 1.5.6、AutoGrid 4和AutoDock 4.0对Tyr-Glu-Gly和黄嘌呤氧化酶进行分子对接。Tyr-Glu-Gly与黄嘌呤氧化酶分子对接的结果显示:Tyr-Glu-Gly分别与黄嘌呤氧化酶的GLU802、ARG880、LYS771形成氢键作用,此外,进入由LEU1014、PHE1009、THR1010、PHE914、LEU873、PRO1076、ASN768形成的活性口袋。分子对接的结果表明,Tyr-Glu-Gly与XO产生的相互作用主要由疏水作用及氢键作用主导。
Claims (4)
1.一种具有降尿酸活性的多肽的应用,其氨基酸序列为Tyr-Glu-Gly,其特征在于,所述多肽在制作痛风疾病的辅助治疗药物或者在制备具有降尿酸功能的药品中的应用;所述多肽在体外降尿酸活性检测中表现出抑制黄嘌呤氧化酶的活性;所述多肽对黄嘌呤氧化酶产生抑制时的相互作用主要由疏水作用及氢键作用主导;所述多肽对黄嘌呤氧化酶抑制的IC50值为13.43±0.73mg/ml,即36.59±2.00mmol/L。
2.根据权利要求1所述的一种具有降尿酸活性的多肽的应用,其特征在于,所述Tyr为酪氨酸的氨基酸残基,所述Glu为谷氨酸的氨基酸残基,所述Gly为甘氨酸的氨基酸残基。
3.根据权利要求1所述的一种具有降尿酸活性的多肽的应用,其特征在于,所述的氨基酸序列采用已有的成熟技术,通过树脂的筛选,获取合理的多肽合成方法:采用标准Fmoc方案,选用Wang树脂进行固相合成。
4.根据权利要求1所述的一种具有降尿酸活性的多肽的应用,其特征在于,所述体外降尿酸活性检测采用HPLC法。
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