CN111034997A - 具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物 - Google Patents
具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物 Download PDFInfo
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- CN111034997A CN111034997A CN201911344308.7A CN201911344308A CN111034997A CN 111034997 A CN111034997 A CN 111034997A CN 201911344308 A CN201911344308 A CN 201911344308A CN 111034997 A CN111034997 A CN 111034997A
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Abstract
本发明公开一种具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物，由以下质量百分比的原料混合制备而成：果葡糖浆0.1~10%、异麦芽酮糖醇0.1~1%、大鲵活性肽0.1~5%、猕猴桃汁0.5~10%、山楂汁0.5~10%、牡蛎多糖0.1~10%、蓝莓浓缩汁0.5~10%、DL‑苹果酸0.01~0.1%、柠檬酸钠0.01~0.1%、果胶0.01~0.1%、黄原胶0.01~0.1%及水余量。
Description
技术领域
本发明属于功能食品领域，尤其涉及一种具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物。
背景技术
衰老是生物体生长成熟之后所发生的一个复杂的生理过程，在衰老的过程中往往产生很多代谢疾病，如高血压、高血脂等。目前已有大量关于通过干预延缓衰老的相关报道，例如增加机体里的抗氧化活性物质，及时清除机体内过量的自由基，保持细胞、组织的正常功能，从而达到延缓衰老的目的。
缺氧是指生物体生命活动所需要的氧不能得到充分的供给，可以由外界环境、机体本身等原因造成。缺氧可使机体的代谢、机能发生异常变化，长期缺氧会给机体带来严重危害，甚至死亡。
中国发明专利号ZL201610314751.X公开了一种“大鲵活性肽及用途”，其大鲵活性肽是按照如下步骤制备：
a . 将大鲵肉匀浆，向匀浆中加入1~3倍体积pH 7~8的0 .01~0 .1M磷酸缓冲液及与匀浆重量比为0 .1~1%的复合酶，酶解12~48 h；所述复合酶为海洋碱性蛋白酶和木瓜蛋白酶按活力单位比为7:3比例混合；
b . 离心取上清，采用截留分子量为4000 Da以下用物理吸附法制备的胰蛋白酶固定超滤膜分离器分离，取透过超滤膜的液体；
c . 通过Sephadex LH-20分子筛层析柱，按每管1ml收集层析产物，取第30~36管层析产物进行冷冻干燥；
d．将c步骤所得到的冷冻干燥物制备成10~50mg/ml的去离子水溶液，8000 rpm、4℃条件下离心10 min，用0 .22 μm水膜过滤，得到上清液；
e . 使用ddH2O 配制5%甲醇缓冲溶液，通过孔径0 .45 μm水膜抽滤，通过超声波除气作为高效液相色谱柱流动相A，使用ddH2O 配制95%甲醇缓冲溶液，通过孔径0 .45 μm油膜抽滤，通过超声波除气作为高效液相色谱柱流动相B；使用流动相A和流动相B进行梯度洗脱，洗脱条件：流速为1ml/min，洗脱梯度分配：0~20min，流动相A100%；20 .5~40min，流动相A50%，流动相B50%；40 .5~60min，流动相B100%；将上清液注入C18柱高效液相色谱仪进样口，收集紫外检测波长280 nm的吸收峰；
f . 挥发收集物中的甲醇后，真空冷冻干燥，得到大鲵肽制品。
所制备的大鲵活性肽具有促进皮肤纤维原细胞增殖的作用。
迄今为止，尚没有关于具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物的相关报道。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物。
本发明的技术解决方案是：一种具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物，其特征在于由以下质量百分比的原料混合制备而成：果葡糖浆0.1~10%、异麦芽酮糖醇0.1~1%、大鲵活性肽0.1~5%、猕猴桃汁0.5~10%、山楂汁0.5~10%、牡蛎多糖0.1~10%、蓝莓浓缩汁0.5~10%、DL-苹果酸0.01~0.1%、柠檬酸钠0.01~0.1%、果胶0.01~0.1%、黄原胶0.01~0.1%及水余量。
本发明各组分协同作用，同时具有抗衰老、调节血压及抗缺氧作用。
附图说明
图1 是本发明实施例血清样品SDS-PAGE检测结果示意图
图2 是本发明实施例血清样品质谱检测图。
具体实施方式
实施例1：
本发明的具有抗衰老、调节血压、抗缺氧作用的大鲵活性肽组合物，由以下质量百分比的原料混合制备而成：果葡糖浆8%、异麦芽酮糖醇0.1%、大鲵活性肽0.3%、猕猴桃汁7%、山楂汁1.5%、牡蛎多糖0.2%、蓝莓浓缩汁3%、DL-苹果酸0.1%、柠檬酸钠0.1%、果胶0.05%、黄原胶0.08%及水余量。
实施例2：
本发明的具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物，由以下质量百分比的原料混合制备而成：果葡糖浆0.1%、异麦芽酮糖醇0.1%、大鲵活性肽5%、猕猴桃汁0.5%、山楂汁10%、牡蛎多糖5%、蓝莓浓缩汁0.5%、DL-苹果酸0.01%、柠檬酸钠0.01%、果胶0.01%、黄原胶0.01%及水余量。
实施例3：
本发明的具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物，由以下质量百分比的原料混合制备而成：果葡糖浆10%、异麦芽酮糖醇1%、大鲵活性肽0.1%、猕猴桃汁10%、山楂汁0.5%、牡蛎多糖10%、蓝莓浓缩汁10%、DL-苹果酸0.05%、柠檬酸钠0.05%、果胶0.1%、黄原胶0.1%及水余量。
实施例1、2、3所用原料来源如下：
大鲵活性肽按照中国发明专利号ZL201610314751.X“大鲵活性肽及用途”所公开的方法制备。
猕猴桃汁、山楂汁按照文献方法制备（尚宏丽等. 猕猴桃山楂复合饮料的研制.粮油加工，2009，（12）：150-152）。蓝莓浓缩汁按照文献方法制备（王鑫等. 蓝莓果汁贮藏中非酶褐变影响因素评价. 食品工业科技，2019,40（14）：94-99）。
牡蛎多糖按照中国发明专利号ZL2016106037668“牡蛎多糖的制备方法”所公开的方法制备。
果葡糖浆、异麦芽酮糖醇、DL-苹果酸、柠檬酸钠、果胶、黄原胶购自阿里巴巴。
实验例
一．利用非标记蛋白质组技术分析本发明实施例对D-半乳糖致衰老小鼠的作用
将本发明实施例1冻干制成干粉。
将20只SPF级昆明小鼠（雄性）分为2组，分别为衰老模型组、本发明实施例组。各组每天都皮下组织注射0.5 mL/d（生理盐水配置）5% D-半乳糖，本发明实施例组按200mg /（kg•d）灌胃一次本发明实施例1冻干粉，每次灌胃样品溶于0.1ml水中，衰老模型组给予同体积的生理盐水。连续灌胃6周后，称重，摘取眼球取血，立即离心（3000 r/min 10 min），取上层血清，-20℃冰箱冻存备用。
分别取50μl本发明实施例组和衰老模型组小鼠血清（本发明实施例组小鼠血清样品标记为ADB-T，衰老模型组小鼠血清样品标记为ADB-M）。均采用去血清高丰度亲和色谱柱Agilent Multiple Affinity Removal LC Column-Mouse 3，按照Agilent对应protocol中的操作方法获得小鼠血清中低丰度蛋白组分。各样品血清低丰度蛋白组分经过超滤浓缩后加入等体积SDT裂解液，沸水浴裂解15 min，于14000×g条件下离心20 min。采用BCA法对上清液进行定量并分别取裂解后的各样品20μg，分别加入上样缓冲液，沸水浴5 min，进行12.5% SDS-PAGE电泳后，考马斯亮蓝染色，判断样品裂解情况。结果如图1所示，图1表明，样品平行性较好，可以进行质谱检测。
采用Filter aided proteome preparation (FASP) 方法对各样品进行酶解。分别取样品ADB-T及ADB-M各30μL蛋白质溶液，分别加入0.003 mmol DTT，沸水浴5 min后冷却至室温。加入200μL尿素缓冲液（8 mol/L尿素，150 mmol/L Tris-HCl，pH8.0）混匀后，在10kD超滤离心管中于14000×g离心15 min，弃滤液（重复该步骤一次）。加入100μL 100 mMIAA缓冲液（以尿素缓冲液溶解），在600 rpm条件下振荡1 min，室温避光30 min后于相同离心条件下进行离心。加入100μL尿素缓冲液，相同离心条件下离心，该步骤重复两次。加入100 μL 25 mM NH4HCO3溶液，相同离心条件下离心两次。加入40μL 0.1 g/L胰蛋白酶缓冲液（以100 mM NH4HCO3溶液配制），600 rpm振荡反应1 min，37℃静置16 h。换新收集管，相同离心条件下离心；再加入40μL 25 mM NH4HCO3，在相同离心条件下离心并收集滤液。通过C18Cartridge脱盐冻干后以40μL 0.1%甲酸溶液复溶，测定280 nm吸光值。
根据FASP酶解后OD280定量结果，各取2μg酶解后的ADB-T及ADB-M进行LC-MS/MS分析。样品先经过RP-C18毛细管高效液相色谱柱（Thermo EASY column SC200 150 μm×100mm）的纳升流速HPLC液相系统EASY-nLC1000，以甲酸乙腈水溶液进行梯度分离。预柱为RP-C18 Thermo EASY column SC001 traps 150 μm×20 mm。A液为0.1%甲酸乙腈水溶液（乙腈为2%），B液为0.1%甲酸乙腈水溶液（乙腈为84%）。梯度条件为：0～100 min，B液线性梯度0%～45%；100～108 min，B液线性梯度45%～100%；108～120 min，B液维持在100%。分离后用Q-Exactive质谱仪进行质谱分析，结果如图2所示。预分析时长60min，正式分析时长120min。
质谱分析原始数据为RAW文件，采用MaxQuant软件（版本号1.3.0.5）进行查库鉴定，并根据Label free算法进行定量分析。差异表达蛋白质鉴定结果如表1。
表1
| Protein | Protein Name | GeneName | MolWeight | ADB-T2 | ADB-M | ADB-T2/ADB-M | t test pvalue |
| P07628 | Kallikrein 1-relatedpeptidase b8 | Klk1b8 | 28.531 | 766580000 | 170593333.3 | 4.493611 | 0.000143024 |
| Q9JM71 | Kallikrein 1-relatedpeptidase b27 | Klk1b27 | 28.742 | 692376666.7 | 165830000 | 4.17522 | 2.7462E-05 |
| Q61759 | Kallikrein 1-relatedpeptidase b21 | Klk1b21 | 28.69 | 1834766667 | 627263333.3 | 2.925034 | 0.000180122 |
| Q922D8 | C-1-tetrahydrofolatesynthase, cytoplasmic | Mthfd1 | 101.2 | 50219666.67 | 18048000 | 2.782561 | 0.001235332 |
| P15946 | Kallikrein 1-relatedpeptidase b11 | Klk1b11 | 28.727 | 4327433333 | 1600533333 | 2.703745 | 2.14663E-05 |
| P00756 | Kallikrein 1-relatedpeptidase b3 | Klk1b3 | 28.998 | 1160366667 | 456480000 | 2.541988 | 0.000100133 |
| P15948 | Kallikrein 1-relatedpeptidase b22 | Klk1b22 | 28.384 | 8513233333 | 3375300000 | 2.522215 | 5.15232E-05 |
| P15949 | Kallikrein 1-relatedpeptidase b9 | Klk1b9 | 28.9 | 9910900000 | 4046000000 | 2.449555 | 0.000153609 |
| P04071 | Kallikrein 1-relatedpeptidase b16 | Klk1b16 | 28.722 | 2548166667 | 1170633333 | 2.176742 | 2.53521E-06 |
| P36369 | Kallikrein 1-relatedpeptidase b26 | Klk1b26 | 28.463 | 17401666667 | 8201733333 | 2.121706 | 0.00020584 |
| P28666 | Murinoglobulin-2 | Mug2 | 162.38 | 20192666667 | 9539633333 | 2.116713 | 8.11766E-07 |
| Q9WUB3 | Glycogen phosphorylase,muscle form | Pygm | 97.285 | 834436666.7 | 406900000 | 2.050717 | 3.36532E-05 |
| P32261 | Antithrombin-III | Serpinc1 | 52.003 | 22722666667 | 46184333333 | 0.491999 | 0.000116282 |
| Q61805 | Lipopolysaccharide-binding protein | Lbp | 53.055 | 268280000 | 554823333.3 | 0.483541 | 0.001460697 |
| P35441 | Thrombospondin-1 | Thbs1 | 129.65 | 2653500000 | 5516966667 | 0.480971 | 4.09351E-06 |
| P13634 | Carbonic anhydrase 1 | Ca1 | 28.33 | 120761333.3 | 252620000 | 0.478036 | 0.009123136 |
| P68373 | Tubulin alpha-1Cchain | Tuba1c | 49.909 | 214340000 | 483050000 | 0.443722 | 0.000526163 |
| P11499 | Heat shock proteinHSP 90-beta | Hsp90ab1 | 83.28 | 40434500 | 96523000 | 0.418911 | 0.04460069 |
| P07743 | BPI fold-containingfamily A member 2 | Bpifa2 | 24.753 | 199116666.7 | 596136666.7 | 0.334012 | 9.05666E-05 |
| Q71KU9 | Fibrinogen-likeprotein 1 | Fgl1 | 36.439 | 41525666.67 | 150836666.7 | 0.275302 | 0.00043986 |
| P31725 | Protein S100-A9 | S100a9 | 13.049 | 42477000 | 159603333.3 | 0.266141 | 0.004491928 |
| P05366 | Serum amyloid A-1protein | Saa1 | 13.77 | 466780000 | 7019033333 | 0.066502 | 2.30186E-05 |
| P05367 | Serum amyloid A-2protein | Saa2 | 13.622 | 126827000 | 6132366667 | 0.020682 | 8.21174E-05 |
| P02089 | Hemoglobin subunitbeta-2 | Hbb-b2 | 15.878 | 30119000 | 8486200000 | 0.003549 | 9.1173E-06 |
结果表明，本发明实施例组血管舒缓素1相关肽酶b8、b27、b21、b11、b3、b22、b9、b16、b26以及糖原磷酸化酶表达上调。血管舒缓素1相关肽酶b8、b27、b21、b11、b3、b22、b9、b16、b26的上调，可以调节血压，使血压处于正常状态，有助于减缓衰老现象。糖原磷酸化酶表达上调表明糖代谢能力增强，是适应低氧环境的重要标志。
二．本发明实施例对大鼠血压的影响
分别将本发明实施例1、大鲵活性肽（对比例1）以及本发明实施例1中不加入大鲵活性肽的产品（对比例2）冻干制成干粉。高血压模型Wistar大鼠随机分为5组，每组6只。分别为高血压模型组、卡托普利（20 mg/kg）组、对比例1（5 mg/kg）组、对比例2（5 mg/kg）和本发明实施例组（5 mg/kg），灌胃1次/天，每次样品均溶于2 mL水中，共23d，通过ALC-NIBP无创血压测量分析系统（上海奥尔科特生物科技有限公司）监测大鼠给药前后血压的变化情况。卡托普利购于安徽城市药业有限责任公司。
数据处理采用SPSS 19.0软件进行单因素方差分析，结果表示为平均值±标准差（表2、表3）。
表2
表3
从试验结果来看，经过灌胃23 d后，本发明实施例组高血压大鼠的收缩压和舒张压均明显低于对比例组，本发明实施例组的降压效果与20 mg/kg的卡托普利相当。
三．小鼠封闭氧耐受能力实验
分别将本发明实施例1、大鲵活性肽（对比例1）以及本发明实施例1中不加入大鲵活性肽的产品（对比例2）冻干制成干粉。
取SPF级昆明小鼠（雄性）50只，适应性饲养3d后随机分成5组：缺氧模型组、乙酰唑胺组（20mg/kg）、对比例1（5mg/kg）组、本发明实施例组（5mg/kg），对比例2组（5mg/kg），每组10只。口服灌胃给药5d，每次样品溶于0.2ml水中，缺氧模型组给等体积的生理盐水。给药50min后，将小鼠分别放入盛有5g钠石灰的250ml广口瓶内，每瓶放1只小鼠，用凡士林涂抹瓶口，密封，使之不漏气，立即计时，直至小鼠停止呼吸，结果如表4。结果表明，与缺氧模型组相比，本发明实施例组能够延长小鼠常压密闭缺氧环境中的存活时间，延长率达到33.28%。
表4
| 组别 | 存活时间 | 延长率% |
| 缺氧模型组 | 26.89±2.59 | - |
| 乙酰唑胺组（20mg/kg） | 32.16±3.14 | 19.60% |
| 对比例1组 | 33.71±7.23 | 25.36% |
| 本发明实施例组 | 35.84±4.65 | 33.28% |
| 对比例2组 | 28.86±5.82 | 7.33% |
Claims (1)
1.一种具有抗衰老、调节血压及抗缺氧作用的大鲵活性肽组合物，其特征在于由以下质量百分比的原料混合制备而成：果葡糖浆0.1~10%、异麦芽酮糖醇0.1~1%、大鲵活性肽0.1~5%、猕猴桃汁0.5~10%、山楂汁0.5~10%、牡蛎多糖0.1~10%、蓝莓浓缩汁0.5~10%、DL-苹果酸0.01~0.1%、柠檬酸钠0.01~0.1%、果胶0.01~0.1%、黄原胶0.01~0.1%及水余量。
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| 尚宏丽，等: "猕猴桃山楂复合饮料的研制", 《粮食加工》 * |
| 徐阳等: "大鲵胶原蛋白多肽口服液的开发研究", 《食品工业》 * |
| 王鑫，等: "蓝莓果汁贮藏中非酶褐变影响因素评价", 《食品工业科技》 * |
| 胡代花: "大鲵生物活性肽和酶解产物研究及应用进展", 《黑龙江畜牧兽医》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534390A (zh) * | 2020-05-25 | 2020-08-14 | 浙江爱鲵生物科技有限公司 | 大鲵活性肽葡萄酒 |
| CN111534390B (zh) * | 2020-05-25 | 2023-10-27 | 浙江水肌蕾生物科技集团有限公司 | 大鲵活性肽葡萄酒 |
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| CN111034997B (zh) | 2022-11-22 |
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