CN114891067B - 一种蒲公英抗炎活性肽及其制备方法与应用 - Google Patents
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- 229960002477 riboflavin Drugs 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000001845 taraxacum officinale leaf extract Substances 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000019164 vitamin B2 Nutrition 0.000 description 1
- 239000011716 vitamin B2 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
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Abstract
本发明属于生物技术领域,具体涉及一种蒲公英抗炎肽及其在药物和功能性食品中的应用。本发明以蒲公英为原料,使用木瓜蛋白酶对蒲公英进行酶解,酶解后的多肽经凝胶层析和反相色谱分离获得序列为QLFSQPF的多肽。研究表明,该多肽具有显著的抗炎活性,且没有明显的细胞毒性。本发明提供了一种制备抗炎肽的方法,并成功鉴定到一个高活性的低聚肽,具有良好的开发利用前景。
Description
技术领域
本发明属于生物活性肽技术领域,涉及一种蒲公英抗炎肽的制备及其在制备抗炎活性药物中的中的应用。
背景技术
炎症是机体对于刺激的一种防御反应,表现为红、肿、热、痛和功能障碍。炎症可分为感染性炎症和非感染性炎症。通常情况下,炎症是有益的,是人体的自动的防御反应,但是有的时候,炎症也是有害的,例如对人体自身组织的攻击、发生在透明组织的炎症等等。目前临床上主要采用抗生素类药物治疗感染性炎症,采用激素类或非甾体类抗炎药物治疗非感染性炎症。上述这些临床药物虽然能很好地治疗各类炎症,但也存在不同程度的副反应,例如,机体对抗生素产生耐药性;激素类存在水钠潴留,变虚胖,骨质疏松等副作用;非甾体类造成胃肠道不适、过敏反应、神经系统和心血管疾病。传统中医药对于清热解毒药材的发掘利用有悠久的历史,从清热解毒药材中发掘新型抗炎成分是抗炎药物开发的重要途经。
蒲公英别名黄花地丁、婆婆丁、华花郎等,是菊科多年生草本植物。蒲公英是传统中药材,可生吃、炒食、做汤,是药食兼用的植物。蒲公英性味甘,微苦,寒。归肝、胃经。具有清热解毒,利尿散结等功效。在中医药中用于急性乳腺炎,淋巴腺炎,瘰疬,疔毒疮肿,急性结膜炎,感冒发热,急性扁桃体炎,急性支气管炎,胃炎,肝炎,胆囊炎,尿路感染等疾病的防治。
生物活性肽(Bioactive peptides),是由2-20个氨基酸单位组成的具有一定生物活性的肽段。从大豆、谷蛋白、酪蛋白、水产品蛋白中都已分离得到了具有抗肿瘤、抗菌、抗炎、降糖、抗病毒及降血压等活性的多肽。截至2021年9月,BIOPEP数据库收录的具有各种生物活性的多肽多达4300余个。与大分子的蛋白相比,生物活性肽具有分子量小,易于吸收,抗原性低等特点,基于此,生物活性肽被广泛应用于化妆品和药物等领域。
过去数十年间,从牛奶、大豆、鱼类蛋白的酶解物中都分离得到了具有降血压、降血糖、抗菌、免疫调节等活性的天然多肽。这些天然来源的活性肽具有相较化学合成类药物更高的安全性,目前虽然有一部分来源于动物植物的抗炎活性肽的报道,但是活性更好,安全性更高的多肽还有待发现。
对蒲公英化学成分研究表明蒲公英含有蒲公英醇、蒲公英素、胆碱、有机酸、菊糖等多种健康营养成分,富含维生素A、维生素C及钾,也含有铁、钙、维生素B2、维生素B1、镁、维生素B6、叶酸及铜。但是对于蒲公英抗炎活性肽的发现尚属空白。
发明内容
本发明的目的是提供一种蒲公英抗炎肽及其在药品中的应用。
为实现上述目的,本发明采用的技术方案为:
所述抗炎肽以蒲公英为原料,经过酶解和进一步的分离得,其序列为Gln-Leu-Phe-Ser-Gln-Pro-Phe(QLFSQPF)。
该蒲公英抗炎肽制备方法为:将蒲公英粉加水混匀,料水质量比为1:10~1:20,混悬液以200-800w的功率使用超声细胞破碎仪处理料液,以蒲公英加入质量1%-10%的酶底比加入木瓜蛋白酶,酶解条件为:pH5-8,温度25-65℃,酶解1-12h后,80-100℃加热10-100min灭酶;离心去除沉淀,上清液分别使用Sephadex G-15凝胶柱和Eclipse XDB-C18柱分离,组分冻干得降压肽。
一种蒲公英抗炎肽的应用,所述抗炎活性肽在制备预防和治疗炎症相关的药物制剂中的应用。
以蒲公英抗炎肽为基础,配以任何符合药品生产允许的辅料制成预防和治疗炎症相关的药物制剂。
蒲公英抗炎肽抑制多种炎症因子(NO、IL-6、IL-1β和TNF-α等)和环氧化酶-2(COX-2)表达。
本发明所具有的优点:
本发明获得的多肽具有优良的抗炎活性,下调细胞COX-2的表达量,同时抑制NO、IL-6、IL-1β和TNF-α等炎症因子的产生,从而对炎症的发生和发展有一定的预防和治疗作用。在细胞水平实验中无明显细胞毒性。该多肽为七肽,分子量小易于吸收,且具有一定的胃肠道消化酶稳定性。在具有抗炎活性的药物等领域具有良好的应用前景。
研究表明,该多肽具有显著的抗炎活性,且没有明显的细胞毒性。本发明提供了一种制备抗炎肽的方法,并成功鉴定到一个高活性的低聚肽,具有良好的开发利用前景。
附图说明
图1为QLFSQPF纯度鉴定液相色谱图;
图2为不同浓度QLFSQPF对小鼠巨噬细胞RAW264.7增殖能力影响;
图3为不同浓度QLFSQPF对LPS刺激的小鼠RAW264.7巨噬细胞NO产生的作用;
图4为QLFSQPF抑制LPS刺激的小鼠巨噬细胞RAW264.7炎症因子表达。
具体实施方式
下面结合附图和实施例对本发明做进一步的解释说明。本发明的目的是以蒲公英为原料,经过蛋白酶解,分离并筛选出具有明确序列的,应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本发明所限定的范围。
实施例1
蒲公英抗炎活性肽的制备
称取1000g蒲公英干粉(包括根茎叶全株的通过200目筛的干燥粉末)分散于10L去离子水中,搅拌混匀,成蒲公英混悬液,超声细胞破碎仪参数设置为工作15s,间隔15s,循环120次,550W功率。超声时容器置于冰上防止液体因超声升温(控制液体温度在0-30℃之间)。以上循环重复3次后,调整pH到7(采用1M氢氧化钠或质量浓度35-37%浓盐酸)后加入木瓜蛋白酶20g于55℃酶解反应4h后加热至100℃搅拌1小时灭酶,12000g离心10min取上清冻干得蒲公英酶解物。
以葡聚糖凝胶(Sephadex G15,Cytiva)柱层析(20×100cm)对蒲公英酶解物进行分离纯化,以去离子水作为流动相,流速控制在1mL/min,在280nm波长下检测洗脱液吸光度,依据洗脱曲线收集富含多肽组分,对应的洗脱时间为82-89min,浓缩并冻干。然后以Zorbax SB-Aq C18柱(4.6mm×150mm×5μm,Agilent)对Sephadex G-15分离得到的组分进行分离,洗脱程序为:1-5min:5%乙腈(体积浓度);5-55min:5%-95%乙腈(线性梯度);55-60min:95%乙腈(体积浓度);流速为0.8mL/min。依据洗脱曲线进行收集并经过适当合并得到15组分。依据实施例2方法分别对15个组分进行抗炎活性筛选,其中组分8对LPS诱导的NO产生抑制率为89%,对该组分进行质谱分析,测定其多肽序列为Gln-Leu-Phe-Ser-Gln-Pro-Phe(QLFSQPF)。
多肽纯度采用HPLC法进行分析,称取0.5mg多肽QLFSQPF样品以0.5mL超纯水溶解后以配有NanoChrom Chromcore TM120C18(4.6mm*250mm*5μm)色谱柱的高效液相色谱系统对样品进行分析。上样量为40μL,流动相分别为含有体积浓度0.1%三氟乙酸的乙腈和超纯水,洗脱程序为1-5min:5%乙腈(体积浓度);5-55min:5%-95%乙腈(线性梯度);55-60min:95%乙腈(体积浓度)。流速为1.0mL/min,在214nm下检测出峰情况,色谱图见图1。以归一化方法对多肽色谱峰进行分析,多肽的纯度≥95%。
SEQ ID No.1的信息
(a)序列特征
*长度:7氨基酸残基
*类型:氨基酸
*链型:单链
*拓扑结构:线性
(b)分子类型:蛋白
序列描述:SEQ ID No.1
Gln-Leu-Phe-Ser-Gln-Pro-Phe
实施例2
QLFSQPF体外给药对RAW264.7细胞增殖活性的影响
取生长对数期的RAW264.7细胞,吹打制成单细胞悬液,经细胞计数后,将RAW264.7细胞按照5×105个/mL细胞密度接于96孔板中培养,每孔200μL,培养基为含有体积浓度10%胎牛血清的DMEM高糖培养基,待细胞生长至50%左右时,用100μL不含胎牛血清的培养基(DMEM高糖培养基)代替原培养基,饥饿处理12h。然后用100μL空白(空白对照组)或含不同终浓度的QLFSQPF(实验组)的不含胎牛血清的DMEM高糖培养基(DMEM高糖培养基)代替原培养基,继续培养24h。向每孔中加入20μL浓度为5mg/mL的MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,Thiazolyl Blue TetrazoliumBromide,汉语化学名为3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐,商品名:噻唑蓝)溶液,继续孵育4h,弃去上清,每孔加入150μL DMSO(二甲基亚砜),室温轻轻震摇10min使其结晶充分溶解,在490nm波长下测定吸光度。
细胞存活率=(实验组OD值/空白对照组OD值)×100%。
结果如图2所示,结果表明QLFSQPF对RAW264.7细胞单独孵育时,在浓度为50~200μg/mL时对细胞增殖无明显抑制作用。
实施例3
体外给药抑制RAW264.7细胞分泌NO的分泌
小鼠单核巨噬细胞系RAW264.7细胞于含有体积浓度10%胎牛血清的DMEM高糖培养基,放置于37℃,含体积浓度5%CO2空气的饱和湿度的培养箱中培养。每隔一天换一次新鲜的同样培养基,保证足够的营养。每天观察细胞密度,当细胞贴壁生长达到80%左右进行传代或其他操作,一般需要2天-3天。传代时,小心弃去原培养液,用PBS缓冲液润洗瓶壁5分钟,使细胞易于吹落,将新培养液移入培养瓶,用移液枪吸取培养液进行小心吹打,使细胞脱落,至瓶壁上无明显细胞,吸取适量细胞悬液转至新鲜的同样培养基中,放入培养箱继续培养。
QLFSQPF对RAW264.7细胞分泌NO的影响
将RAW264.7细胞按照5×105个/mL细胞密度接于96孔板,每孔200μL,置于恒温培养箱中培养(37℃,培养基为含有体积浓度10%胎牛血清的DMEM高糖培养基,含体积浓度5%CO2空气),待细胞生长至50%左右时,用100μL不含胎牛血清的培养基(DMEM高糖培养基)代替原培养基,饥饿处理12h。用100μL不含胎牛血清的DMEM高糖培养基(DMEM高糖培养基)代替原培养基,按照以下分组加样:(1)空白对照组;(2)LPS(脂多糖)处理组(LPS终浓度1μg/mL);(3)阳性药NO合酶抑制剂L-NMMA(终浓度50μM)及阿司匹林(终浓度200μg/mL)与LPS(LPS终浓度1μg/mL)联合处理组;4)QLFSQPF(终浓度分别分别为50、100和200μg/mL)与LPS(LPS终浓度1μg/mL)联合处理组;每组6复孔,置于培养箱中培养24h。收集上清,采用Griess法测定NO浓度:取上清液50μL依次各加入50μL Griess试剂Ⅰ、Ⅱ液,混匀后室温放置10min,使其充分反应,在540nm波长下测定吸光度,以亚硝酸钠溶液为标准溶液建立NO标准曲线,确定个实验组细胞培养上清中的NO水平。
NO抑制率=(LPS处理组-实验组)/LPS处理组×100%,此处实验组是指上述阳性药NO合酶抑制剂L-NMMA及阿司匹林与LPS联合处理组和QLFSQPF与LPS联合处理组
图3结果表明QLFSQPF单独作用于RAW264.7细胞时,在6.25μg/mL~200μg/mL浓度时对细胞NO生成无明显的影响;QLFSQPF作用于LPS诱导的RAW264.7细胞时,在6.25μg/mL~200μg/mL浓度区间,NO生成量随药物浓度增加而降低,浓度为100μg/mL时其NO生成量与阳性药与LPS联合处理组相当。说明QLFSQPF能显著抑制LPS诱导的RAW264.7细胞NO分泌,具有较强的抗炎作用。
实施例4
QLFSQPF体外给药抑制环氧化酶-2(COX-2)的表达量
将RAW264.7细胞按照5×105个/mL细胞密度接于12孔板,每孔200μL,培养基为含有体积浓度10%胎牛血清的DMEM高糖培养基,置于恒温培养箱中培养(37℃,含体积浓度5%CO2空气),待细胞生长至50%左右时,用100μL不含胎牛血清的DMEM高糖培养基(DMEM高糖培养基)代替原培养基,饥饿处理12h后用100μL不含胎牛血清的DMEM高糖培养基(DMEM高糖培养基)代替原培养基,分为四组:(1)空白对照组:不加多肽QLFSQPF和LPS;(2)LPS组(LPS终浓度1μg/mL);(3)阳性药对照组:加入阿司匹林(终浓度200μg/mL)和LPS(LPS终浓度1μg/mL);(4)实验组:加入不同浓度(终浓度分别为50、100和200μg/mL)的多肽QLFSQPF和LPS(LPS终浓度1μg/mL)。培养24h后,吸去培养板内的培养基,用预冷至0-4℃的PBS缓冲液润洗3次,加入10μL细胞裂解液,于冰上震荡裂解10min。裂解完后用移液枪吹打细胞,将裂解液转移至离心管中,于4℃下12000rpm离心5min。对离心后的上清分别检测COX-2(环氧化酶-2)、IL-6(白介素6)、IL-1β(白介素1β)和TNF-α(肿瘤坏死因子α)的表达水平。
图4结果表明,QLFSQPF作用于LPS诱导的RAW264.7细胞时,浓度为200μg/mL时,COX-2的表达量与200μg/mL阿司匹林相当,说明QLFSQPF能有效抑制COX-2的表达,具有抗炎作用。QLFSQPF作用于LPS诱导的RAW264.7细胞,IL-6、IL-1β和TNF-α等多种炎症因子的表达量随着药物浓度增加而降低,当浓度为100μg/mL时,各炎症因子表达水平与200μg/mL阿司匹林相当。说明QLFSQPF能抑制炎症因子的表达,具有抗炎作用。
Claims (3)
1.一种蒲公英抗炎肽,其特征在于:所述抗炎肽的序列为Gln-Leu-Phe-Ser-Gln-Pro-Phe(QLFSQPF)。
2.一种权利要求1所述蒲公英抗炎肽的应用,其特征在于:所述抗炎肽在制备预防和/或治疗炎症相关疾病的药物中的应用。
3.按权利要求2所述的应用,其特征在于:以权利要求1所述抗炎肽配以任何符合药品或药物学生产允许或可接受的辅料或载体制备。
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Patent Citations (2)
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---|---|---|---|---|
CN105601707A (zh) * | 2016-02-05 | 2016-05-25 | 福州大学 | 一种蒲公英籽抗氧化四肽及其制备方法 |
CN108653487A (zh) * | 2018-06-27 | 2018-10-16 | 张家口万全区科泰生物科技有限公司 | 一种增强奶牛免疫力的活性肽及其应用 |
Non-Patent Citations (4)
Title |
---|
Isolation, identification and in vivo antihypertensive effect of novel angiotensin I-converting enzyme (ACE) inhibitory peptides from Spirulina protein hydrolysate;Qishan Suo et al.;《Food Funct》;第9108–9118页 * |
Purification of novel anti-inflammatory peptides from enzymatic hydrolysate of the edible microalgal Spirulina maxima;Thanh-Sang Vo et al.;《journal of functional foods》;第5卷;摘要,第1337-1338页第2.2-2.4节,第1339-1340页 第3.2-3.3节,第1336页 右栏第1段-第1337页左栏第1段,第1337页左栏最后一段 * |
The antioxidant, immunomodulatory, and anti-inflammatory activities of Spirulina: an overview;Qinghua Wu et al.;《Archives of Toxicology》;第1817–1840页 * |
蒲公英酶解活性肽的研究;林贺;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;第E057-267页 * |
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