CN116836239A - 一种具有促进创伤愈合作用的鹿茸多肽 - Google Patents
一种具有促进创伤愈合作用的鹿茸多肽 Download PDFInfo
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Abstract
本发明提供一种具有促进创伤愈合作用的鹿茸多肽，是将鹿茸粉碎加水匀浆制成匀浆液，然后将匀浆液调节pH值为6.0～7.5，再加入蛋白酶进行酶解，获得的酶解液进行离心，取上层酶解液经过超滤膜，截取3kDa分子量以下的酶解液，再采用Sephadex G25进行纯化，得到鹿茸多肽。本发明提供的鹿茸多肽提取物在促进伤口愈合及皮肤修护方面功效显著。可诱导创伤部位产生蛋白肽Pro‑Hyp，以及多种炎性细胞因子，促进伤口处胶原合成，预防瘢痕生成，具有免疫调节以及促表皮再上皮化等多重功效，快速形成封闭空间，防止伤口感染。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种具有促进创伤愈合作用的鹿茸多肽，及其制备方法和应用。
背景技术
柳叶刀杂志统计数据显示，全球每年大约开展的各类型手术达2.34亿例。在发达国家，1％～2％的人群面临着皮肤创伤问题，仅在美国，每年大约有50万烧伤患者住院。目前我国就慢性伤口而言，患者数就高达1,300万。典型的慢性伤口主要由糖尿病、下肢静脉溃疡及造口等疾病诱发。其中，糖尿病足的典型特征为局部皮肤及深层组织破坏，伴随着伤口溃烂并反复发作，约20％的糖尿病患者甚至面临着截肢风险，因此，安全、高效的创伤修复产品具有极大的市场需求。
皮肤损伤的愈合是包括细胞、体液和分子机制在内的一个动态的、可高度调控的过程，伤口愈合过程分为四期:凝血期、炎症期、修复期、成熟期；涉及免疫细胞、角质形成细胞、成纤维细胞、毛囊干细胞等，各种细胞功能异常或修复细胞数量不足常常导致伤口延迟愈合或慢性伤口。目前市面促进伤口愈合药物和保健品都是根据伤口愈合四个时期来设计，不能同时兼顾多个时期共同发挥作用。
鹿茸中粗蛋白含量占干重的60％以上，特别是胶原蛋白、IGF-1、EGF和NGF等含量丰富，具有显著促进骨骼发育、免疫调节、促进表皮细胞增殖、神经再生等生物活性。相较于鹿茸蛋白，鹿茸活性肽具有组织透过性好、免疫原性弱等优点，而且由于其分子小可以直接作用于关键位点，对于皮肤损伤有显著治疗作用，开发潜力较大。研究表明使用鹿茸多肽对大鼠皮肤创伤分别采用单纯注射、单纯外用、肌肉注射与外用协同的方式处理后发现，无论注射还是外用，鹿茸多肽都有显著促进创面愈合的能力，验证了鹿茸多肽的促进创伤愈合的能力。因此，以鹿茸粗蛋白制备多肽在食品和医疗领域中受到人们的认可。然而，针对鹿茸多天活性的而研究多为抗氧化、骨愈合活性，而制备具有促进伤口愈合活性的多肽的方法鲜有报道，因此，确定一种具有促进伤口愈合活性的鹿茸多肽的制备方法是行业发展的攻坚方向。
发明内容
本发明的目的是提供一种具有促进创伤愈合作用的鹿茸多肽，及其制备方法和应用，所提供的鹿茸多具有伤口修复的效果，其主要治疗作用包括抗炎、调节细胞因子、促进伤口愈合和抑制瘢痕生成等活性。
本发明所提供的鹿茸多肽，其制备方法如下：
将鹿茸粉碎加水匀浆制成匀浆液，然后将匀浆液调节pH值为6.0～7.5，再加入蛋白酶进行酶解，获得的酶解液进行离心，取上层酶解液经过超滤膜，截取3kDa分子量以下的酶解液，再采用Sephadex G25进行纯化，得到鹿茸多肽。
所述的匀浆制成匀浆液，其一种具体条件是采用高速剪切匀浆，剪切速度为8000-10000rpm，剪切时间为60-90s。
所述的酶解，是采用胰蛋白酶和中性蛋白酶对匀浆液进行酶解；
作为实施例的一种具体记载，所述的酶解，是在50℃酶解5h；
更进一步的，所述的鹿茸多肽中，其一种多肽的氨基酸序列为
GGYDESMPDPLPEFTE(SEQ ID NO:1)。
本发明制得的促进伤口愈合的鹿茸多肽，其多肽分子量主要在500-2000Da之间，由脯氨酸、天冬氨酸、甘氨酸、丝氨酸、酪氨酸、苯丙氨酸、精氨酸、异亮氨酸、蛋氨酸、谷氨酸、缬氨酸组成，能有效提高伤口愈合速度、及伤口处炎症因子及胶原蛋白分泌。
本发明再一个方面还保护所提供的鹿茸多肽在制备皮肤浅皮层或皮肤真皮层损伤修复相关的药物、保健品、化妆品中的应用。
本发明还提供一种药物组合物，其包含有所述的鹿茸多肽以及药学上可接受的辅料。
本发明的药物组合物可以制成本领域常规剂型，包括但不限于口服地散剂、片剂、胶囊剂、颗粒剂、丸剂、可分散粉末、水性或有幸混悬剂、水性或油性溶液剂、乳剂、糖浆剂等，适用于局部使用的霜剂、软膏剂、凝胶、水性或油性溶液剂、水性或油性混悬剂等。
本发明提供的鹿茸多肽提取物在促进伤口愈合及皮肤修护方面功效显著。可诱导创伤部位产生蛋白肽Pro-Hyp，以及多种炎性细胞因子，促进伤口处胶原合成，预防瘢痕生成，具有免疫调节以及促表皮再上皮化等多重功效，快速形成封闭空间，防止伤口感染。
附图说明
图1：伤口皮肤组织HE染色照片图。
具体实施方式
在前期实验中，直接使用鹿茸粉具有一定促进伤口愈合的作用，但但使用量较大，因此采用本发明分离纯化鹿茸粉，得到效果更优的鹿茸多肽提取物，用于皮肤修护，可以更快的使伤口愈合，从而减少伤口感染的几率。
下面结合实施例对本发明进行详细的描述。
实施例1：鹿茸多肽提取物制备
取1kg鹿茸剁成小块，粉碎，过100目筛，按重量比1:10加入蒸馏水，采用高速剪切匀浆，剪切速度为8000-10000rpm，剪切时间为60-90s，剪切3次打碎鹿茸使得细胞内容物流出。调节pH 6.8，加入胰蛋白酶：中性蛋白酶，质量比为1:1，蛋白酶添加量为5％；在50℃酶解5h，酶解结束后于沸水浴(90℃)灭酶10min，冷却，8000rpm离心10min，将上层酶解液经过超滤膜，截取3kDa分子量以下酶解液，减压浓缩，冻干。
取鹿茸蛋白酶解产物用蒸馏水溶解至100mg/mL溶液，Sephadex G25凝胶色谱柱层析，2.0cm×100cm进行分离纯化，流动相为蒸馏水，洗脱速度为0.5mL/min，收集洗脱组分。将上述洗脱液进行真空浓缩后，再进行冷冻干燥，从而得到具有促进伤口愈合功效的鹿茸多肽，得到鹿茸多肽提取物0.11kg，得率为11％。
采用凝胶渗透色谱法检测多肽相对分子量分布，鹿茸多肽分子量小于2000Da的多肽占92％，分布在500-2000Da之间。采用全自动氨基酸分析仪对鹿茸多肽氨基酸组成进行测定。结果显示，鹿茸多肽包含有脯氨酸、天冬氨酸、甘氨酸、丝氨酸、酪氨酸、苯丙氨酸、精氨酸、异亮氨酸、蛋氨酸、谷氨酸和缬氨酸。
实施例2：鹿茸多肽提取物制备
取5kg鹿茸剁成小块，粉碎，过100目筛，按重量比1:10加入蒸馏水，采用高速剪切匀浆，剪切速度为8000-10000rpm，剪切时间为60-90s，剪切3次打碎鹿茸使得细胞内容物流出。调节pH 6.8，加入胰蛋白酶：中性蛋白酶1:1(含量为5％)在50℃酶解5h，酶解结束后于沸水浴(90℃)灭酶10min，冷却，8000rpm离心10min，将上层酶解液经过超滤膜，截取3kDa分子量以下酶解液，减压浓缩，冻干。
取鹿茸蛋白酶解产物用蒸馏水溶解至100mg/mL溶液，Sephadex G25凝胶色谱柱层析，2.0cm×100cm进行分离纯化，流动相为蒸馏水，洗脱速度为0.5mL/mL，收集洗脱组分。将上述洗脱液进行真空浓缩后，再进行冷冻干燥，从而得到具有促进伤口愈合功效的鹿茸多肽，得到鹿茸多肽提取物0.45kg，得率为9.0％。
采用凝胶渗透色谱法检测多肽相对分子量分布，鹿茸多肽分子量小于2000Da的多肽占88％，分布在500-2000Da之间。
对比例3：
取1kg鹿茸剁成小块，粉碎，过100目筛，按重量比1:10加入蒸馏水，采用高速剪切匀浆，剪切速度为8000-10000rpm，剪切时间为60-90s，剪切3次打碎鹿茸使得细胞内容物流出。调节pH 6.8，加入碱性蛋白酶(含量为5％)在50℃酶解5h，酶解结束后于沸水浴(90℃)灭酶10min，冷却，8000rpm离心10min，将上层酶解液经过超滤膜，截取3kDa分子量以下酶解液，减压浓缩，冻干。得到鹿茸多肽提取物0.26kg。
实施例4：小鼠创伤愈合效果研究
采用3％戊巴比妥钠1mL/kg静脉麻醉。小鼠背部备皮后，75％乙醇消毒手术区域；在背部术区制备6mm直径大小、圆形全层皮肤缺损模型；成功造模后，在圆形全层皮肤缺损的圆心和创面边缘的3、6、9、12等体积的20μL实施例1中所得鹿茸多肽提取物、实施例3中所得鹿茸多肽提取物和生理盐水，给药成功后，3M辅料覆盖创面，隔天换药；在造模后第10d，沿伤口外周3mm，取全层皮肤，从正中间分成两半，分别用4％多聚甲醛固定过夜，经脱水、包埋，制成石蜡切片，用于HE染色，以Image J软件测量伤口面积，愈合率＝(未给药时创口面积-每天创口面积)/未给药时创口面积。
结果表明，鹿茸多肽组(实施例1)的伤口愈合率为(76.9±10.1)％，明显高于空白对照组(33.3±4.5)％，伤口已基本完全愈合。由此可见，本发明的鹿茸多肽能够促进伤口愈合，缩短伤口愈合的时间，对大面积创伤的治疗具有很好的治疗效果。
小鼠创伤处组织HE染色结果见图1。由图可知，3d空白组仍有大量炎症细胞浸润，而鹿茸多肽组已有新的毛细血管生成；9d空白对照组仅有较少疏松的肉芽组织和毛细血管生成，而鹿茸多肽组(实施例1)的肉芽组织已经排列较为有序且紧密；该染色结果说明鹿茸多肽能够促进毛细血管和肉芽组织生成从而促进伤口愈合。
此外，与实施例1所得鹿茸多肽相比，用碱性蛋白酶水解所得鹿茸多肽伤口愈合率仅为58.0±9.50％，远低于实施例1所得鹿茸多肽，表明胰蛋白酶和中性蛋白酶酶解鹿茸多肽具有显著促愈合能力。
表1：伤口愈合率表
与空白对照相比，*P＜0.05，**P＜0.05。
实施例5：小鼠创伤免疫调节因子及胶原生成影响
羟脯氨酸是胶原蛋白特异性成分，可用于胶原蛋白的定量，测定羟脯氨酸含量，参照南京建成的羟脯氨酸试剂盒(碱水解法)说明书进行测定。采用ELISA试剂盒比较小鼠炎症指标：白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)的水平。
结果如表2所示，与空白对照组相比，实施例1的鹿茸多肽给药后伤口处羟脯氨酸含量明显提高(P<0.05)，表明鹿茸多肽可促进伤口处胶原蛋白合成。此外，空白对照组血清中TNF-α、IL-1β水平明显升高，在给予鹿茸多肽后，与模型组相比TNF-α、IL-1β水平减少且具有统计学差异(P<0.05)。以上结果表明，在伤口愈合后期鹿茸多肽能够抑制促炎细胞因子的释放。与实施例1所得鹿茸多肽相比，对比例3所得鹿茸多肽羟脯氨酸含量(5.37±0.15)相对较低，伤口处炎症因子IL-1β、TNF-α明显偏高，表明胰蛋白酶和中性蛋白酶酶解鹿茸多肽具有显著促进伤口闭合，缓解炎症发生能力。
表2：鹿茸多肽对创伤免疫调节因子及胶原生成影响表
与空白对照相比，*P＜0.05，**P＜0.05。
因此，本发明提供的鹿茸多肽提取物在促进伤口愈合及皮肤修护方面功效显著。可诱导创伤部位产生蛋白肽Pro-Hyp，以及多种炎性细胞因子，促进伤口处胶原合成，预防瘢痕生成，具有免疫调节以及促表皮再上皮化等多重功效，快速形成封闭空间，防止伤口感染。
实施例6：液相色谱分离纯化鹿茸活性肽
采用反相液相色谱对鹿茸多肽进行分离纯化，采用ZORBAX Eclipse XDB-C18色谱柱(4.6mm×250mm，5μm)，流动相A：水(0.1％ TFA)，流动相B：乙腈，进行线性洗脱，洗脱条件：乙腈(0％→75％，0～60min)，流速0.8mL/min，检测波长214nm。根据监测图谱收集多肽并鉴定多肽序列。
通过对液相色谱分离得到组分进行质谱鉴定，具有促进伤口愈合的氨基酸序列为GGYDESMPDPLPEFTE(GE-16)。
表3：伤口愈合率表
与空白对照相比，*P＜0.05，**P＜0.05。
该多肽促进伤口愈合能力增强，伤口愈合率达到89.2％(表3)，相对于实施例1的鹿茸多肽组具有显著性差异(P＜0.05)，表明GE-16是鹿茸多肽中发挥促进伤口愈合能力的主要成分。
Claims (10)
1.一种鹿茸多肽，其特征在于，所述的鹿茸多肽，其制备方法如下：
将鹿茸粉碎加水匀浆制成匀浆液，然后将匀浆液调节pH值为6.0～7.5，再加入蛋白酶进行酶解，获得的酶解液进行离心，取上层酶解液经过超滤膜，截取3kDa分子量以下的酶解液，再采用Sephadex G25进行纯化，得到鹿茸多肽。
2.如权利要求1所述的鹿茸多肽，其特征在于，所述的匀浆制成匀浆液，是采用高速剪切匀浆，剪切速度为8000-10000rpm，剪切时间为60-90s。
3.如权利要求1所述的鹿茸多肽，其特征在于，所述的酶解，是采用胰蛋白酶和中性蛋白酶对匀浆液进行酶解。
4.如权利要求3所述的鹿茸多肽，其特征在于，所述胰蛋白酶和中性蛋白酶的质量比为1:1。
5.如权利要求3所述的鹿茸多肽，其特征在于，所述的酶解，是在50℃酶解5h。
6.如权利要求1所述的鹿茸多肽，其特征在于，所述的鹿茸多肽的分子量为500-2000Da。
7.如权利要求1所述的鹿茸多肽，其特征在于，所述的鹿茸多肽中，其一种多肽的氨基酸序列为SEQ ID NO:1。
8.权利要求1所述的鹿茸多肽在制备皮肤浅皮层或皮肤真皮层损伤修复相关的制品中的应用。
9.权利要求7所述的鹿茸多肽在制备皮肤浅皮层或皮肤真皮层损伤修复相关的制品中的应用。
10.一种药物组合物，其特征在于，所述的药物组合物中包含有权利要求1所述的鹿茸多肽和/或权利要求7所述的鹿茸多肽，以及药学上可接受的辅料。
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