CN111574586B - 一种中华冀土鳖虫来源的具有降血脂功能的活性肽及其制备方法和应用 - Google Patents
一种中华冀土鳖虫来源的具有降血脂功能的活性肽及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一种中华冀土鳖虫来源的具有降血脂功能的活性肽及其制备方法和应用。本发明所述的活性肽氨基酸序列为LAPAPGTL。本发明提供的活性肽具有降血脂功能,可以开发成药物或保健品。
Description
技术领域
本发明涉及活性肽技术领域。更具体地,涉及一种中华冀土鳖虫来源的具有降血脂作用的活性肽及其制备方法和应用。
背景技术
高血脂是目前常见的心血管疾病,随着人民生活水平的提高,高热量与高脂肪饮食使得高血脂的发病率持续增高。高血脂存在很多并发症,例如脂肪肝、动脉粥样硬化、糖尿病等,因此对高血脂的防治与治疗是目前研究的热点之一。
土鳖虫为传统中药,具有破血逐瘀,续筋接骨之功效,用于跌打损伤,筋伤骨折,血瘀经闭,产后瘀阻腹痛,瘾瘕痞块。现代研究得出土鳖虫具有治疗血脑血管疾病的作用,例如动脉粥样硬化、高血脂、脑血栓等。
目前对于中药动物药研究是中药基础研究的一大热点,而且取得了很大的进展,例如在水蛭中发现具有防凝血的水蛭素,在地龙体内发现具有溶栓作用的蚓激酶,这些研究发现中药动物药发挥药理作用的物质基本为蛋白质,对土鳖虫蛋白质进行研究得出土鳖虫蛋白具有促进血栓溶解、扩张血管、消除自由基的作用。
蛋白质多为100K或更大分子量的物质,口服后多经过胃蛋白酶、胰蛋白酶等胃肠道蛋白酶作用成小分子多肽被吸收进入人体,发挥药理作用。所以蛋白质存在吸收效率低、过敏等缺点。
本发明采用胃蛋白酶和胰蛋白酶在体外对土鳖虫进行仿生酶解来模拟蛋白质在体内的消化,这样使得大分子蛋白在体外既可以被裂解为多肽,增加吸收效率。分解后的多肽采用超滤、纳滤、大孔树脂、凝胶分子筛色谱进行分离,得到具有活血化瘀功能的多肽单体,多肽单体纯度高,安全有效;消除了服用异蛋白等引起的免疫原反应,减小了不良反应发生率。
发明内容
本发明的第一个目的在于提供一种中华冀土鳖虫来源的活性肽。
本发明的第二个目的在于提供上述活性肽制备方法
本发明的第三个目的在于提供上述活性肽在制备预防或治疗降血脂的药物或保健品中的应用。
为达到上述目的,本发明采用下述技术方案:
第一方面,本发明提供一种中华冀土鳖虫来源的活性肽,所述活性肽的氨基酸序列为LAPAPGTL,如SEQ ID NO:1所示。
可选的,所述活性肽还可以是与上述活性肽的氨基酸序列具有80%及以上同源性的活性肽,该活性肽和上述活性肽的功能相同或相似。例如,所述活性肽的氨基酸序列可以是与上述活性肽的氨基酸序列具有85%,90%,95%或97%同源性的序列。
本发明还提供了编码上述活性肽的多核苷酸。
在已知活性肽的氨基酸序列的情况下,本领域技术人员可以根据对于活性肽表达的需要,基于密码子的简并性原则和不同物种对于密码子的使用偏好性,设计具有不同核苷酸序列的活性肽的编码基因。
在本发明中,示例性的,上述活性肽的核苷酸序列为ATGCTGGCACCAGCGCCGGGTACCCTG,如序列表SEQ ID NO:2所示。
第二方面,本发明提供了一种上述中华冀土鳖虫来源的活性肽的制备方法,包括如下步骤:
(1)将中华冀土鳖鲜虫体加入8-10倍重量份的去离子水或者蒸馏水进行剪切,制成土鳖虫浆液,剪切时间为10-20min,剪切速率为5000转/分钟,剪切温度为室温;
(2)将土鳖虫浆液加热至100℃杀菌15min,然后放冷至37℃,调节浆液pH值至2.5,加入胃蛋白酶进行酶解,酶解时间为45min-80min,酶解温度为37℃;然后再调节浆液的pH值至8.5,加入胰蛋白酶进行酶解,酶解时间为180min-300min,酶解温度为37℃,制得土鳖虫仿生酶解液;其中,按土鳖虫浆液重量计,胃蛋白酶的加入量为1.5%-2.5%,胰蛋白酶的加入量为1.5%-2.5%;
(3)用DA201-C树脂柱对步骤(2)制得的土鳖虫仿生酶解液进行吸附层析,DA201-C树脂重量与土鳖虫仿生酶解液体积比例为5:1-10:1,吸附完毕后采用25%乙醇进行洗脱;制得洗脱液A;
(4)用葡聚糖G25凝胶柱对步骤(3)制得的洗脱液A进行分子筛层析,每次洗脱液A的加入量为0.5-2ml,用去离子水洗脱,制得洗脱液B;
(5)将步骤(4)制得的洗脱液B经过RP-HPLC进行组分分离,采用的色谱条件为色谱柱:waters ZOBRX-300SB C18色谱柱,150mm X 4.6um,检测器为紫外检测器,检测波长:220nm、280nm,流动相:A相0.1%三氟乙酸去离子水溶液(TFA),B相0.1%三氟乙酸(TFA)乙腈溶液;采用梯度洗脱方法进行分析,梯度顺序:0-55min 90%A-30%A;55-60min 30%A-60%A;60-75min60%A-90%A;
(6)经RP-HPLC分离后的各组分样品进行降血脂活性筛选,对活性最强的组分样品进行氨基酸序列分析,即得。
上述方法中,优选的,所述胃蛋白酶的酶活力不低于1500U/g,胰蛋白酶的酶活力不低于2500U/mg,胰蛋白酶的酪蛋白转化力不低于30.0。
在本发明的具体实施方式中,经RP-HPLC分离后的样品采用LC-TOF-MS进行分析,得到活性肽质谱,得出活性肽结构为LAPAPGTL,命名为LL-8,相对分子质量为738Da。
第三方面,本发明提供所述活性肽在制备预防或治疗降血脂的药物或保健品中的应用。进而,本发明进一步涉及一种药物或一种保健品,其中含有本发明的活性肽LAPAPGTL。
本发明的药物和保健品可以制成本领域常见的各种形式,包括但不限于,适于口服的散剂、片剂(包括各种包衣片剂、缓释或控释片剂)、锭剂、胶囊剂(包括软胶囊和硬胶囊)、颗粒剂、丸剂、可分散粉末、水性或油性混悬剂、水性或油性溶液剂、乳剂、酏剂、糖浆剂等等,适于经胃肠外给药的静脉内、皮下或肌内注射用无菌水性或油性的注射剂或冻干粉针剂等等。
本发明的药物和保健品中可以进一步含有常规的各种辅料和/或其他活性成分。合适的辅料包括但不限于赋形剂、润滑剂、粘合剂、崩解剂、水溶性聚合物、无机盐、溶剂、溶解助剂、悬浮剂、等渗剂、缓冲液、防腐剂、抗氧剂等等。
根据本发明的具体实施方案,本发明的活性肽优选被制成注射剂或粉针剂,其中溶剂为无菌用水;防腐剂为三氯叔丁醇;盐酸普鲁卡因的一种或两种的组合。
本发明的有益效果如下:
本发明采用胃蛋白酶和胰蛋白酶在体外对土鳖虫进行仿生酶解来模拟蛋白质在体内的消化,这样使得大分子蛋白在体外既可以被裂解为多肽,增加吸收效率。分解后的多肽采用吸附层析、凝胶分子筛色谱、RP-HPLC、LC-TOF-MS等进行分离,得到具有降血脂功能的多肽单体,多肽单体纯度高,安全有效;消除了服用异蛋白等引起的免疫原反应,减小了不良反应发生率。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出G25凝胶色谱分离图。
图2示出thrmeo半制备HPLC色谱图。
图3示出LC-TOF-MS一级质谱图。
图4示出LC-TOF-MS二级质谱图。
具体实施方式
为了更清楚地说明本发明,下面结合优选实施例和附图对本发明做进一步的说明。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
实施例1活性肽制备
(1)土鳖虫药材处理
中华冀土鳖鲜虫体加入10倍重量份的去离子水或者蒸馏水,然后进行剪切15min,剪切速率为5000转/分钟,剪切温度为室温,制成土鳖虫浆液。
(2)蛋白酶酶解
将土鳖虫浆液加热至100℃杀菌15min,然后放冷至37℃,采用0.1mol/L HCl调整溶液pH至2.5,加入1.5%重量份的胃蛋白酶置恒温酶解仪进行酶解,酶解时间为60min,酶解温度为37℃。然后再采用2.0%氢氧化钠溶液调整pH至8.5,加1.5%重量份的胰蛋白酶置于恒温酶解仪进行酶解,酶解时间为180min,酶解温度为37℃,制得土鳖虫仿生酶解液。
(3)吸附层析
取DA201-C树脂,采用无水乙醇、去离子水洗涤后装入玻璃层析柱内,土鳖虫仿生酶解液由上端加入树脂柱内,DA201-C树脂重量与土鳖虫仿生酶解液体积比例为10:1,吸附完毕后采用25%乙醇进行洗脱,制得洗脱液A。
(4)分子筛层析
取葡聚糖G25,无水乙醇、去离子水洗涤完毕后,装入玻璃柱内,然后将土鳖虫25%乙醇洗脱液加入,每次加入量0.5ml,去离子水洗脱,制得洗脱液B。
(5)RP-HPLC分离
将洗脱液B经过RP-HPLC进行组分分离,采用的色谱条件为色谱柱:waters ZOBRX-300SB C18色谱柱,150mm X 4.6um,检测器为紫外检测器,检测波长:220nm、280nm,流动相:A相0.1%三氟乙酸去离子水溶液(TFA),B相0.1%三氟乙酸(TFA)乙腈溶液;采用梯度洗脱方法进行分析,梯度顺序:0-55min90%A—30%A;55-60min 30%A-60%A;60-75min 60%A-90%A。
f,LC-TOF-MS分离
经RP-HPLC分离后的各组分样品进行降血脂活性筛选,筛选出2个具有降血脂活性的组分,分别命名为LL-8、LM-9。
对于组分样品LL-8、LM-9采用LC-TOF-MS进行分析,得到活性肽质谱,得出活性肽结构为LAPAPGTL,LHRIGAVPM。
实施例2
基于实施例1鉴定得到的LL-8和LM-9活性肽的氨基酸序列,通过固相合成方法获得实验样品,进行降血脂活性验证。固相合成肽公司:上海吉尔生物技术有限公司。
SD大鼠80只,雄性,适应性饲养7天,然后分为九组,即空白组,模型组、血栓通胶囊组、土鳖虫药材组、LL-8高剂量组(0.5g/kg)、LL-8低剂量组(0.25g/kg)、LM-9高剂量组(0.5g/kg),LM-9低剂量组(0.25g/kg)、除空白组以外,其他组每只老鼠每天定量给予50g高脂饲料,同时除空白组大鼠,每天上午9:00给予每只大鼠高脂乳剂4.0mL,高脂乳剂饲养方式:灌胃口服,空白组大鼠持续给予普通饲料,连续45天,给予高脂饲料、高脂乳剂45天后。先将5.0mLEP管采用预选配置后的肝素钠溶液润洗后,备用。给药15天后,于第16天末次给药后皮下注射给药5.0%戊巴本妥钠溶液进行麻醉,待大鼠被完全麻醉后,先在腹主动脉取血3.0mL,注入未加抗凝剂的玻璃硅化管内。离心;取血清;采用ELISA酶联检测方法检测各组血液内血脂四项(LDL、HDL、TG、TC)含量。
表1调节血脂实验结果
观察除空白组、模型组以外的其他实验组血脂四项数据,所有组别的血脂四项数据和空白组进行对比,都具有显著性或者极显著性差异(P<0.05、P<0.01),说明造模良好,给药45天后,在持续给予高脂饲料的同时,与模型组,LM9高低剂量组可明显降低高脂血症大鼠血清内TG、TC和LDL的含量,存在显著性差异,而与LM9高低剂量组,模型组相比,LL8高低剂量组则可更加明显降低模型组大鼠血清中血脂四项(P<0.01),说明LL8较LM9具有更好地降低血脂功能。
实施例3
基于实施例1鉴定得到的LL-8活性肽的氨基酸序列,通过固相合成方法获得实验样品,进行安全性实验验证。固相合成肽公司:上海吉尔生物技术有限公司。
健康SD大鼠,250~300g,40只,适应性饲养7天,然后设置为空白组、生理盐水组、LL-8组,LL-8组分别给予20.0g/200g的大剂量药物,给药方式:皮下注射给药;生理盐水组同样采用皮下注射给药方式给予等体积的生理盐水,持续给药12天,每天测定大鼠的饮食量、饮水量,同时每隔3天测定大鼠的体重、体温。同时测定各组大鼠胸腺,脾脏等免疫器官重量,衡量LL-8的安全性。
表2短期安全性评价各组大鼠体温变化结果
表3各组大鼠器官重量结果
组别 | 肝重(g) | 肾重(g) | 脑重(g) | 脾脏重(g) | 胸腺重(g) |
空白组 | 15.364±1.4428 | 3.6479±0.3637 | 1.8096±0.0911 | 0.6827±0.0149 | 0.2496±0.0394 |
生理盐水组 | 15.793±3.3575 | 3.6458±0.2549 | 1.9007±0.0762 | 0.6024±0.0771 | 0.2245±0.0339 |
LL-8组 | 15.821±0.5968 | 3.6225±0.1913 | 1.9916±0.0733 | 0.7690±0.0217 | 0.2774±0.0419 |
表4免疫器官指数实验结果
组别 | 脾脏指数(mg/g) | 胸腺指数(mg/g) |
空白组 | 1.8512±0.4219 | 0.4071±0.0743 |
生理盐水组 | 1.7084±0.8575 | 0.3982±0.0537 |
LL-8组 | 1.9831±0.8701 | 0.4361±0.0757 |
以上实验结果表明:LL-8并未造成正常大鼠体温的明显变化,这也说明LL-8不会引起大鼠的异常免疫反应,同时LL-8也不会引起大鼠正常器官的萎缩,同时也不会使大鼠免疫器官出现增殖,因此LL-8在注射给药条件下是安全的。
显然,本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定,对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动,这里无法对所有的实施方式予以穷举,凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
SEQUENCE LISTING
<110> 滨州医学院
<120> 一种中华冀土鳖虫来源的具有降血脂功能的活性肽及其制备方法和应用
<130> JLP20I0221
<160> 2
<170> PatentIn version 3.5
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Leu Ala Pro Ala Pro Gly Thr Leu
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atgctggcac cagcgccggg taccctg 27
Claims (7)
1.一种中华冀土鳖虫来源的活性肽,其特征在于,所述活性肽的氨基酸序列为LAPAPGTL。
2.一种多核苷酸,其特征在于,编码权利要求1所述的活性肽。
3.如权利要求1所述的中华冀土鳖虫来源的活性肽的制备方法,其特征在于,该方法包括如下步骤:
(1)将中华冀土鳖鲜虫体加入8-10倍重量份的去离子水或者蒸馏水进行剪切,制成土鳖虫浆液,剪切时间为10-20min,剪切速率为5000转/分钟,剪切温度为室温;
(2)将土鳖虫浆液加热至100℃杀菌15min,然后放冷至37℃,调节浆液pH值至2.5,加入胃蛋白酶进行酶解,酶解时间为45min-80min,酶解温度为37℃;然后再调节浆液的pH值至8.5,加入胰蛋白酶进行酶解,酶解时间为180min-300min,酶解温度为37℃,制得土鳖虫仿生酶解液;其中,按土鳖虫浆液重量计,胃蛋白酶的加入量为1.5%-2.5%,胰蛋白酶的加入量为1.5%-2.5%;
(3)用DA201-C树脂柱对步骤(2)制得的土鳖虫仿生酶解液进行吸附层析,DA201-C树脂重量与土鳖虫仿生酶解液体积比例为5:1-10:1,吸附完毕后采用25%乙醇进行洗脱;制得洗脱液A;
(4)用葡聚糖G25凝胶柱对步骤(3)制得的洗脱液A进行分子筛层析,每次洗脱液A的加入量为0.5-2ml,用去离子水洗脱,制得洗脱液B;
(5)将步骤(4)制得的洗脱液B经过RP-HPLC进行组分分离,采用的色谱条件为色谱柱:waters ZOBRX-300SB C18色谱柱,150mm X 4.6um,检测器为紫外检测器,检测波长:220nm、280nm,流动相:A相0.1%三氟乙酸去离子水溶液,B相0.1%三氟乙酸乙腈溶液;采用梯度洗脱方法进行分析,梯度顺序:0-55min 90%A-30%A;55-60min 30%A-60%A;60-75min60%A-90%A;
(6)经RP-HPLC分离后的各组分样品进行降血脂活性筛选,对活性最强的组分样品进行氨基酸序列分析,即得。
4.根据权利要求3所述的制备方法,其特征在于,所述胃蛋白酶的酶活力不低于1500U/g,胰蛋白酶的酶活力不低于2500U/mg,胰蛋白酶的酪蛋白转化力不低于30.0。
5.权利要求1所述的中华冀土鳖虫来源的活性肽在制备预防或治疗降血脂的药物中的应用。
6.一种药物,其特征在于,包含权利要求1所述的中华冀土鳖虫来源的活性肽。
7.根据权利要求6所述的药物,其特征在于,所述药物为注射剂或粉针剂。
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