CN110950925A - 具有修复营养激活细胞功能的ace抑制肽及制备方法 - Google Patents
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Abstract
本发明提供一种具有修复营养激活细胞功能的ACE抑制肽及制备方法,是黑色食品中分离提取的小分子肽,发现其本身是ACE(抗高血压)抑制剂,并且经实验证明,它对由黑色素瘤细胞诱导的TIDCs起到激活作用,对过氧化氢导致的A549细胞损伤起到修复作用;利用小分子肽制成的药物具有临床效果显著,毒副作用小的特点;本发明提取的食物小分子肽制备方法简单实用。
Description
技术领域
本发明提供一种具有修复营养激活细胞功能的ACE抑制肽,具有细胞修复、细胞激活的小分子肽,同时进一步公开了该小分子肽的制备方法,属于涉及药物技术领域。
背景技术
目前,黑色食品的保健养生作用越来越受广大人们认可和喜爱,本发明发明所用的黑色食品包含黑花生、黑芝麻、黑米、黑豆、黑枸杞等众多食物种类,并且各个具有不同功效和食用方法;本发明的任务是提供一种从以上这些食品中提取的小分子肽,对细胞损伤起到修复作用和细胞激活等作用。
肽是介于氨基酸与蛋白质之间的一种生化物质,它比蛋白质分子量小,比氨基酸分子量大。两个以上的氨基酸之间以肽键相连,形成的“氨基酸链”就叫做肽。按照氨基酸的组成分为多肽、寡肽和小分子肽。多肽是指由 10 个以上氨基酸组成的肽,分子量大于10000 道尔顿,比如大豆蛋白,分子量几万;胶原蛋白,分子量十几万到三十万等等。寡肽是指由2至9 个氨基酸组成的肽,分子量小于 1000 道尔顿。与多肽和寡肽比较而言,小分子肽(O C O)是超低分子量寡肽,分子量一般在 180 ~ 480 道尔顿,是仅由2~4 个氨基酸构成的高活性肽。小分子肽的胶原蛋白,是指平均分子量在300道尔顿的二肽、三肽、四肽的小肽胶原蛋白。众所周知的小分子玻尿酸 ( 透明质酸 )也是小肽胶原蛋白的一种。
肽是人体必需的基础的活性物质。肽的发现破译了人类生老病死的密码,也开辟了疾病治疗的新纪元。根据目前的研究发现,肽对人体有着抑制、激活、修复、促进等多种积极作用。小分子肽更是即可以补充营养需求,修复缺损,又可以改善功能状态。小肽具有优先被人体吸收的特点,并且小肽在被人体吸收时,可保护氨基酸不被破坏,小肽与氨基酸的混合物是人体吸收蛋白质的最佳吸收机制。小肽在人体中还承担着载体的使命,可将人平常所摄入的营养物质,特别是钙这类对人体有益的微量元素,吸附、粘贴、装载在本体上。然后小肽又发挥其运输工具的职责,将营养物质运送到人体各个细胞、器官、组织,同本体一起被人体吸收利用,发挥各自的功能作用。在被人体吸收后,小肽作为神经递质传递信息,让人体各个器官、系统、组织发挥各自和整体的作用。较多肽和寡肽而言,小分子肽对人体的作用更有优势,它注重于修复人体的变性细胞,解决了人体吸收和利用的难题。对于像高血压、糖尿病、痛风、心脑血管疾病、甲亢、关节炎、胃炎、失眠、癌症等常见的慢性疾病,药物的作用只能是将这类病症控制在一定范围内,而小分子肽因其具备双向调节功能,既能营养保健又能预防疾病。
在细胞修复领域,小分子肽同样起着至关重要的作用。研究表明高氧可导致机体细胞的损伤,引发急性肺损伤、肺炎、肺癌等疾病。市面上一些普遍的抗氧化剂如维生素 E等,虽然能够清除活性氧,但对已造成的损伤修复效果较弱且会产生副作用。经实验证明,本发明研发的小肽在抗氧化方面有着极强的功效。
发明内容
本发明提供了一种具有修复营养激活细胞功能的ACE抑制肽,是黑色食品中分离提取的小分子肽,在抗氧化方面有着极强的功效,对细胞损伤的修复作用以及细胞激活等作用。
本发明所述的一种具有修复营养激活细胞功能的ACE抑制肽的制备方法,包括以下步骤:
按重量份数比称量黑豆(200~300):黑花生(200~300):黑芝麻(100~150):黑米(100~150):黑燕麦(100~150):枸杞(80~125):黑桑葚(100~150):黑莓(30~80):黑糖(80~125);
A.发酵罐使用酒精充分消毒,沸水冲烫灭菌;
B.将上述物料,水清洗干净,烘干去除水分并使用破壁机破壁粉碎,随后将物料倒入发酵桶罐;
C.向发酵罐中加沸水,物料:水体积比=1: 4,搅拌均匀;
D.待发酵罐内温度降至室温左右,向发酵罐内加入发酵菌粉,酵母菌:乳酸菌=1:1 ,密封7天进行发酵;
E.水解:发酵液置于混合酶解发酵罐,使温度升至53℃,加入纤维素酶0.1%W,反应温度45-65℃之间,pH控制在6.0附近,反应1h;
F.将纤维素酶高温灭活处理后的酵素加入木瓜蛋白酶0.1%W;
G.反应条件:pH 6.0左右,温度53℃,用量0.2%w;
H.反应时间:30-60min;
I.装入离心机,5000r/min,20min,将上清和沉淀分别保存,上清过滤,沉淀于干燥箱中烘干备用;得到一种食物小分子肽,命名为:ACE抗高血压抑制肽(参见图1)。
发明所述的ACE抑制肽在制备修复营养激活细胞功能药物及保健品中的用途。
【用法用量】口服;2次/日;50ml/次。
本发明的药理药性如下:
黑花生是内含钙、钾、铜、锌、铁、硒、锰和8种维生素及19种人体所需的氨基酸等营养成分,还富含硒、铁、锌等微量元素和黑色素的新品种。黑花生富含18种氨基酸,氨基酸总量为27.57%,仅次于黑大豆,必需氨基酸比例占22.90%。
黑豆为豆科植物大豆的黑色种子。黑豆蛋白质含量36%,易于消化,对满足人体对蛋白质的需要具有重要意义; 脂肪含量16%,主要含不饱和脂肪酸,吸收率高达95%,除满足人体对脂肪的需要外,还有降低血液中胆固醇的作用; 黑豆含有丰富的维生素、蛋黄素、黑色素及卵磷脂等物质,其中B 族维生素和维生素E 含量很高,具有营养保健作用,黑豆中还含有丰富的微量元素,对保持机体功能完整,延缓机体衰老、降低血液粘度、满足大脑对微量物质需求都是必不可少的。但黑豆本身食用有一定弊端,比如
(1)黑豆入肾阴,冬天怕冷、肾阳虚的人最好不要过多食用。
(2)黑豆炒熟后食用会引起便秘。
所以本发明加入黑米。黑米营养丰富,食、药用价值高,同时黑米可以解决花生和黑豆在破壁后多油的现象,调和黑豆性阴少阳的特点,达到发挥各个成分的优点而消除各种缺点的目的;对于黑豆易引起便秘的弊端,本发明加入黑芝麻,其含有大量的脂肪和蛋白质,可以达到润肠通便的功效,还含有糖类、维生素A、维生素E、卵磷脂、钙、铁、铬等营养成分。有健胃、保肝、促进红细胞生长的作用,同时可以增加体内黑色素,有利于头发生长。
除了上述几种黑色食物,本发明还加入了黑枸杞、枸杞子、黑燕麦、黑桑葚、黑莓和黑糖。黑枸杞含有大量花青素,具有延缓衰老、美容养颜、生津明目、提高皮肤吸收氧分的能力、保肝功能、补肾功能、提高免疫力、抗脂肪肝、防癌抗癌、抗疲劳等作用;枸杞子,为茄科植物枸杞的成熟果实,具有多种保健功效,是卫生部批准的药食两用食物。适量食用有益健康,主要食用功效
益精明目,滋补肝肾;适宜人群:虚劳精亏,腰膝酸痛,视力功能下降,眩晕耳鸣;
小分子肽在细胞激活领域起到了关键作用。本发明发现了从黑色食品中提取的蛋白中含量较高的一种有效抑制剂小分子肽:ACE抑制肽(抗高血压),根据这一发现我们继续研究了其对黑色素瘤细胞诱导的TIDCs的激活作用。树突状细胞( DCs) 是专职抗原递呈细胞,具有捕获、加工和递呈抗原的能力,成熟的 DCs 能够诱导 T 细胞活化为肿瘤特异性细胞毒性 T 细胞( CTL) ,能分泌共刺激分子( CD80、CD86) 、主要组织相容性复合体Ⅱ (MHCⅡ) 和细胞因子 IL-12 等,发挥免疫调节作用。因 CD80、CD86、MHCⅡ、IL-12 等细胞因子只能在成熟 DCs 中高表达,又被称为 DCs 的成熟标志分子。肿瘤组织中浸润有大量的DCs,即肿瘤浸润树突状细胞( TIDCs) ,然而,肿瘤细胞及肿瘤微环境释放因子会抑制或逆转 DCs 的成熟及正常功能,导致黑色素瘤组织中的 TIDCs 处于未成熟状态,只能表达低水平的 CD80、CD86、IL-12和跨膜糖蛋CD11c。DCs 也能分泌具有免疫抑制作用的细胞因子IL-10。肿瘤发生免疫逃逸主要归因于肿瘤特异性 MHC-Ⅰ限制性 CTL 的活化不足,而不成熟 TIDCs 上低表达的共刺激分子可能是 CTL 激活失败的主要原因。因此,TIDCs 已成为控制肿瘤逃逸最有希望的靶标之一。本发明公开的黑色食品中分离提取的小分子肽,能增强化疗或放疗患者受抑制的免疫功能,研究发现,该小肽能诱导骨髓来源树突状细胞(BMDCs) 的成熟,促进 BMDCs 表面成熟标志分子的表达。
本发明的积极效果在于:
提供了一种黑色食物小分子肽,这种小分子肽经过成分鉴定,发现其本身是ACE(抗高血压)抑制剂,并且经实验证明,它对由黑色素瘤细胞诱导的TIDCs起到激活作用,对过氧化氢导致的A549 细胞损伤起到修复作用;利用小分子肽制成的药物具有临床效果显著,毒副作用小的特点;本发明提取的食物小分子肽制备方法简单实用。
附图说明
图1为本发明ACE抑制肽成分鉴定分析质谱图;
图2为本发明黑色食品蛋白中活性肽出现的频率;
图3为本发明选取五种酶,对黑色食品蛋白序列进行单酶/酶组合切,从而获得ACE 抑制肽频率,其中proteinase K+papain 双酶切组合获得ACE抑制肽最高。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明实施方式作进一步地详细描述。
实施例1
按重量份数比称量黑豆200g:黑花生200g:黑芝麻100g:黑米100g:黑燕麦100g:枸杞80g:黑桑葚100g:黑莓30g:黑糖80g;
A.发酵罐使用酒精充分消毒,沸水冲烫灭菌;
B.将上述物料,水清洗干净,烘干去除水分并使用破壁机破壁粉碎,随后将物料倒入发酵桶罐;
C.向发酵罐中加沸水,物料:水体积比=1: 4,搅拌均匀;
D.待发酵罐内温度降至室温左右,向发酵罐内加入发酵菌粉,酵母菌:乳酸菌=1:1 ,密封7天进行发酵;
E.水解:发酵液置于混合酶解发酵罐,使温度升至53℃,加入纤维素酶0.1%W,反应温度45-65℃之间,pH控制在6.0附近,反应1h;
F.将纤维素酶高温灭活处理后的酵素加入木瓜蛋白酶0.1%W;
G.反应条件:pH 6.0左右,温度53℃,用量0.2%w;
H.反应时间:30-60min;
I.装入离心机,5000r/min,20min,将上清和沉淀分别保存,上清过滤,沉淀于干燥箱中烘干备用;得一种食物小分子肽,根据其主要成分和作用机制命名为:ACE抗高血压抑制肽
实施例2
按重量份数比称量黑豆300g:黑花生300g:黑芝麻150g:黑米150g:黑燕麦150g:枸杞125g:黑桑葚150g:黑莓80g:黑糖125g;
A.发酵罐使用酒精充分消毒,沸水冲烫灭菌;
B.将上述物料,水清洗干净,烘干去除水分并使用破壁机破壁粉碎,随后将物料倒入发酵桶罐;
C.向发酵罐中加沸水,物料:水体积比=1: 4,搅拌均匀;
D.待发酵罐内温度降至室温左右,向发酵罐内加入发酵菌粉,酵母菌:乳酸菌=1:1 ,密封7天进行发酵;
E.水解:发酵液置于混合酶解发酵罐,使温度升至53℃,加入纤维素酶0.1%W,反应温度45-65℃之间,pH控制在6.0附近,反应1h;
F.将纤维素酶高温灭活处理后的酵素加入木瓜蛋白酶0.1%W;
G.反应条件:pH 6.0左右,温度53℃,用量0.2%w;
H.反应时间:30-60min;
I.装入离心机,5000r/min,20min,将上清和沉淀分别保存,上清过滤,沉淀于干燥箱中烘干备用;得一种食物小分子肽,命名为:ACE抗高血压抑制肽。
实施例3
按重量份数比称量黑豆250g:黑花生250g:黑芝麻125g:黑米125g:黑燕麦125g:枸杞100g:黑桑葚125g:黑莓50g:黑糖100g;
A.发酵罐使用酒精充分消毒,沸水冲烫灭菌;
B.将上述物料,水清洗干净,烘干去除水分并使用破壁机破壁粉碎,随后将物料倒入发酵桶罐;
C.向发酵罐中加沸水,物料:水体积比=1: 4,搅拌均匀;
D.待发酵罐内温度降至室温左右,向发酵罐内加入发酵菌粉,酵母菌:乳酸菌=1:1 ,密封7天进行发酵;
E.水解:发酵液置于混合酶解发酵罐,使温度升至53℃,加入纤维素酶0.1%W,反应温度45-65℃之间,pH控制在6.0附近,反应1h;
F.将纤维素酶高温灭活处理后的酵素加入木瓜蛋白酶0.1%W;
G.反应条件:pH 6.0左右,温度53℃,用量0.2%w;
H.反应时间:30-60min;
I.装入离心机,5000r/min,20min,将上清和沉淀分别保存,上清过滤,沉淀于干燥箱中烘干备用;得一种食物小分子肽,命名为:ACE抗高血压抑制肽。
试验例
对实施例1~实施例3制备的黑色食品进行蛋白提取,首先对提取的小分子肽进行成分鉴定,参见图1及表1:
表1,从黑色食品中经分离提纯得到的有效小分子肽-ACE抑制肽
其次用BIOPEP测量黑色食品蛋白中活性肽出现的频率,图表参照图2。然后选取五种酶,对提取的蛋白序列进行单酶/酶组合切,从而获得第一种小分子抑制肽频率(ACE抑制肽),参见图3。
实验例1
对提取的小分子肽进行成分鉴定,获得抑制肽分布频率。
参见图1、图2、图3,从本发明小分子肽成分鉴定分析及本发明黑色食品蛋白中活性肽出现的频率;本发明选取五种酶,对黑色食品蛋白序列进行单酶/酶组合切,从而获得ACE 抑制肽频率,其中proteinase K+papain 双酶切组合获得ACE抑制肽最高。
实验例2
黑色食物中的小分子肽对细胞的激活作用
BALB /c 小鼠购自北京维通利华实验动物有限公司,在室温以及安静的环境下进行喂养,日照时间为每天 7: 00 ~ 19: 00,由专业动物饲养人员每日给小鼠提供足够的食物和水。
所用试剂DMSO 及胰蛋白酶、血清、培养基、ELISA 试剂盒、 PE-CD86、 APC-MHCⅡ、FITC-CD11c 、IL-4、重组鼠粒细胞/巨噬细胞集落刺激因子 ( GM-CSF)、小鼠 IL-12 及IL-10 ELISA 试剂盒均购自南京建成生物工程公司。
1.TIDCs 的诱导培养及鉴定
将对数生长期的黑色素瘤细胞 B16 在无血清培养基中培养 24 h,收集上清即得 B16细胞条件培养基,经过0.22 μm 滤膜过滤后分装并于-80 ℃保存备用。BALB /c 小鼠脱颈处死,剥离出完整的股骨,从股骨中收集细胞悬液培养于 RPMI1640 培养基中,加入 IL-4和重组鼠 GM-CSF,37 ℃培养24h 后去除非贴壁细胞,重新加入含有 IL-4 及重组鼠 GM-CSF 的 RPMI1640 培养基,隔天换液,诱导培养至第 4天时,加入 50% B16 细胞条件培养基继续诱导培养3 d,用倒置相差显微镜拍照鉴定 TIDCs。
2.TIDCs 中 CD11c、CD86、MHCⅡ和 IL-12、IL-10 的检测方法
将培养鉴定后的 TIDCs 接种于 96 孔板中,分别将 0、15、25、50μmol /L 的ACE加入TIDCs 中,记为 0 μmol /L 组、15 μmol /L 组、25 μmol /L 组、50 μmol /L 组,37 ℃共同孵育 48 h,分离出 TIDCs 细胞和上清液。取收集到的 TIDCs 细胞, PBS 洗涤 2 遍后加入冰预冷的 PBS 100 μL 与大鼠 ChaoXing血清 10 μL,4 ℃封闭 40 min,加入荧光标记的 CD11c、CD86 及 MHCⅡ抗体,4 ℃孵育 40 min,PBS 洗涤 2 遍,用流式细胞仪进行检测,以平均荧光强度值表示 TIDCs 表面 CD11c、CD86、MHCⅡ的表达量。取 TIDCs 细胞上清液,按 ELISA 试剂盒说明书测定 TIDCs 中 IL-2 和 IL-10。
3. TP5 与 TIDCs 的结合情况观察向 TIDCs 细胞培养皿中加入 FITC 标记1μg/mL TP5 溶液 200 μL,37 ℃孵育3 h,PBS 洗涤后用 4%中性多聚甲醛溶液固定,然后加入细胞核染料 Hoechst 33342,室温染色 10 min,PBS 洗涤后加入抗荧光淬灭剂 200 μL,激光共聚焦显微镜下观察ACE与 TIDCs 的结合情况。
4.统计学方法采用 SPSS 20. 0 统计学软件。计量资料用 x± s 表示,组间比较用单因素方差分析或者重复测量的方差分析。P < 0. 05 为差异有统计学意义。
结果参见表2:
表2,TIDCs 中 CD11c、CD86、MHCⅡ和 IL-12、IL-10 的表达量比较( x ± s)
注: 与 0 μmol /L 组相比,﹡ P < 0. 05; 与 10 μmol /L 组相比,﹟ P < 0. 05;与20μmol/L相比,△ P < 0. 05。
结论:
TIDCs 经ACE作用后表达共刺激分子的能力显著提高,IL-12 分泌量逐渐提高,说明TIDCs 被 ACE活化。相反,IL-10 的分泌量却随 ACE浓度的升高而逐渐减少,可能原因是ACE促进了 IL-12的分泌,增加的 IL-12 进而抑制了 IL-10 的分泌。
结果显示,ACE可促进 TIDCs 表面成熟标志分子 CD11c、CD86 及 MHC Ⅱ的表达,促进 TIDCs 分泌 IL-12 及抑制 IL-10 的分泌,表明ACE 能够激活免疫活性受抑制的TIDCs 的抗原递呈功能,在特异性免疫应答中发挥重要作用。
实验例3
黑色食品中的小肽对细胞修复作用
培养A549 细胞株,复苏后培养于改良 Eagle(DMEM)高糖培养液中,其中含小牛血清体积分数 10%,青霉素(100 mg/L)和链霉素(100 mg/L),37℃下置于体积分数为 5% 的 CO2培养箱中无菌培养,消化传代用体积分数为 0.25% 的胰蛋白酶。
以500μmol/L的H2O2刺激细胞产生损伤。将细胞分为空白对照组,H2O2 组(500 μmol/L 的 H2O2 损伤 24 h),ACE抑制肽前处理组(10、20、30、50 mg/mLACE孵育 24 h,再加500 μmol/L H2O2损伤 24 h)和 ACE 后处理组(500 μmol/L H2O2 损伤 24 h,再加 10、20、30、50 mg/mL ACE孵育 24 h)。阳性对照组用抗氧化剂 N- 乙酰半胱氨酸(NAC)处理细胞,即阳性对照前处理组用6mmol/L NAC 孵育 24 h,再加500μmol/L H2O2损伤24h;阳性对照组后处理组用500μmol/L H2O2损伤 24h,再加6mmol/L NAC 孵育 24 h。
用直径 10 cm 培养皿培养细胞,药物处理后收集细胞,用 4℃磷酸缓冲盐溶液(PBS)洗涤3遍。沉淀用细胞裂解液裂解,4℃ 1 600×g离心 15 min,取上清作为待测样品。用 MTT 比色法测细胞活力,严格按照说明书测定 MDA 含量、SOD 活性、CAT 活性T-AOC,
使用蛋白浓度试剂盒测定不同组细胞蛋白浓度,校正细胞内 MDA、SOD、CAT、T-AOC 水平。
实验数据均以x±s进行表示。采用 SPSS 19.0 软件进行统计学分析,组间均数的比较采用单因素方差分析,两两比较采用 Dunnett t检验。检验水准α=0.05。
本发明所述的小分子肽对由黑色素瘤细胞诱导的TIDCs起到激活作用,对过氧化氢致 A549 细胞损伤起到修复作用;利用小分子肽制成的药物具有临床效果显著,毒副作用小的特点;本发明提取的食物小分子肽制备方法简单实用。
上述本发明实施例序号仅仅为了描述,不代表实施例的优劣。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
1.一种具有修复营养激活细胞功能的ACE抑制肽,其抑制肽成分如鉴定分析质谱图图1所示。
2.权利要求1所述的一种具有修复营养激活细胞功能的ACE抑制肽的制备方法,包括以下步骤:
按重量份数比称量黑豆(200~300):黑花生(200~300):黑芝麻(100~150):黑米(100~150):黑燕麦(100~150):枸杞(80~125):黑桑葚(100~150):黑莓(30~80):黑糖(80~125);
A.发酵罐使用酒精充分消毒,沸水冲烫灭菌;
B.将上述物料,水清洗干净,烘干去除水分并使用破壁机破壁粉碎,随后将物料倒入发酵桶罐;
C.向发酵罐中加沸水,物料:水体积比=1: 4,搅拌均匀;
D.待发酵罐内温度降至室温左右,向发酵罐内加入发酵菌粉,酵母菌:乳酸菌=1:1 ,密封7天进行发酵;
E.水解:发酵液置于混合酶解发酵罐,使温度升至53℃,加入纤维素酶0.1%W,反应温度45-65℃之间,pH控制在6.0,反应1h;
F.将纤维素酶高温灭活处理后的酵素加入木瓜蛋白酶0.1%W;
G.反应条件:pH 6.0,温度53℃,用量0.2%w;
H.反应时间:30-60min;
I.装入离心机,5000r/min,20min,将上清和沉淀分别保存,上清过滤,沉淀于干燥箱中烘干备用;得到一种食物小分子肽。
3.权利要求1所述的ACE抑制肽在制备修复营养激活细胞功能药物及保健品中的用途。
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