CN117024525B - 一种白细胞提取物的制备以及在化妆品和药品中的应用 - Google Patents
一种白细胞提取物的制备以及在化妆品和药品中的应用 Download PDFInfo
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Abstract
本发明涉及一种白细胞提取物的制备以及在化妆品和药品中的应用。本发明提供了一种白细胞提取物，分离自哺乳动物细胞，所述提取物具有较好的抗菌及抗氧化特性。将所述的白细胞提取物与抗菌肽一起使用后，能够有效的抑制病原菌的增殖，具有较好的治疗效果。可以将所述的白细胞提取物与抗菌肽制备成为相应的药物或者化妆品。
Description
技术领域
本申请涉及生物领域，具体的涉及一种白细胞提取物的制备以及在化妆品和药品中的应用。
背景技术
白细胞提取物(LEUKOCYTE EXTRACT)是从白细胞中提取的一种细胞分泌的复合物。白细胞提取物中含有大量细胞特异性蛋白(PDGF、VEGF、HGF、PDGF、bFGF、TGF-β、EGF等几百种修复因子)、脂质、核酸(DNA、RNA)等，可直接作用于粘膜层，其透皮吸收率高，对成纤维细胞、上皮细胞、内皮细胞等都具有促进增殖分裂、促进血管新生及粘膜修复和细胞再生等作用。这些成分可以有效激活衰老细胞、促进细胞新生、修复炎症损伤、提高皮肤免疫、减少色素沉着等等，帮助身体中的细胞恢复正常的功能和活力。因此，白细胞提取物常被作为抗氧化剂的药物或者加入到护肤品中。
由于夏季紫外线强烈，紫外线辐射导致的炎症因子会刺激黑色素细胞，黑色素细胞受到刺激便会产生黑色素。许多人皮肤会生长出斑点、甚至出现晒红、晒伤。在紫外线辐射下，皮肤细胞内会产生一种自由基。夏季日晒后的肌肤护理，抗氧化是重中之重，选择白细胞提取物护肤品，帮助我们快速走出日晒损伤，恢复肌肤透亮。体内自由基过多正是人老化、体力衰退、皮肤失去光泽和弹性的主要原因。当自由基的生成量，超过皮肤清除自由基极限时，皮肤发生自由基的积聚，就会对皮肤产生严重的影响，比如胶原蛋白和弹力蛋白的受损，你会感受到自己的皮肤变得干燥，失去光泽和弹性。而白细胞提取物作为抗氧化剂，它具有再生、增加细胞活力，抑制酪氨酸酶的活性的效果。酪氨酸酶是一种氧化酶，具有调控黑色素生成的作用，能够有效抑制黑色素异常合成，起到淡化色素、色斑的作用。此外，它还能刺激细胞外一些大分子，如透明质酸和糖蛋白等的合成和分泌，有助于滋润皮肤，促进皮肤新陈代谢，可以将日晒后过量的色素沉积代谢出去，加快皮肤愈合。
防腐体系是化妆品中必不可少的重要组成部分。由于化妆品本身含有大量的营养成分，如油脂和功效提取物等，这些成分极有利于微生物的生长和繁殖，产品一旦被微生物污染则很容易腐败变质，所以为了使化妆品在其保质期内及消费者使用期间不被微生物污染，化妆品中必然要添加一定量的防腐剂。随着崇尚自然的消费理念逐步得到人们的认可，天然防腐剂的研究与开发也成为防腐剂领域的研究热点。国内外研究者纷纷从不同植物中得到一些抑菌物质，并试图将其开发成防腐剂。嗜热链球菌发酵产物主要是由嗜热链球菌发酵并提纯得到的多肽结构化合物，其分子中的羧基和氨基形成酰胺键连接成多聚体，是具有20～30个赖氨酸单体组成的化合物，和盐酸进一步反应后得到一种分子量介于3500～4500之间的高纯度水溶性化合物。其可与微生物表面带负电的位点结合，并破坏微生物的细胞膜结构，从而引起细胞膜间物质传递的中断，导致微生物细胞死亡。曲霉发酵产物主要由曲霉发酵并提纯得到。其作用机理是：霉菌和酵母菌的细胞壁结构特殊，存在一种甾醇特殊结构的化合物，AF与甾醇发生化学反应，使其结构发生改变，迫使细胞内的物质发生渗漏，导致霉菌和酵母菌死亡，故其针对霉菌具有较好的抑菌作用。因此，微生物源天然防腐剂取代合成类防腐剂用于化妆品配方抑菌。
但是，目前有效的抑菌肽种类还不够多，特别是具有广谱特性的抑菌肽的开发还有待进一步的提高。
发明内容
本发明一方面，从牛血液中分离制备得到特异性的牛白细胞-NK细胞及其提取物，所述提取物本身具有较好的抑菌特性。
研究表明，细菌感染过程中，宿主NK细胞(natural killer cell，自然杀伤细胞)体内含有颗粒酶A(granzyme A，GZMA)，能够介导宿主上皮细胞中GSDMB的活化。而活化的N端GSDMB能够靶向到革兰氏阴性菌内膜上的心磷脂(CL)，从而杀伤并清除细菌。因此，本发明的牛白细胞-NK细胞提取物能够抑制小鼠模型中的细菌增殖。
此外，本发明还从牛血液中分离获得了天然的具有抑菌特性的活性肽，所述活性肽具有较好的抑制病原菌的特性，同时还具有抗氧化特性。
进一步的，所述的活性肽序列为SEQ ID No：1所示，所述的病原菌是金黄色葡萄球菌ATCC25923、大肠杆菌ATCC35218、副溶血弧菌ATCC 33845。
进一步的，本发明提供一种抑菌的组合物，其中含有NK细胞提取物和序列为SEQID No：1所示的活性肽。
进一步的，本发明提供一种抑菌的组合物，其中含有SEQ ID No：1所示的活性肽。
进一步的，所述组合物针对的离体的细菌。
进一步的，所述组合物针对的是动物。
本发明的组合物还可以与常用赋形剂混合而使用，所述的常用赋形剂即药物上可接受的适合于非肠道、肠道(例如口服或吸入)或局部施用的有机或无机载体物质，它们不与活性组合物发生有害反应。合适的药物上可接受的载体包括但不限于：水；盐溶液；醇类；阿拉伯树胶；植物油；苄醇类；聚乙二醇类；明胶；碳水化合物，诸如乳糖、直链淀粉或淀粉；硬脂酸镁；滑石；硅酸；粘性石蜡；芳香油；脂肪酸酯类；羟甲基纤维素；聚乙烯吡咯烷酮等。可以对药物制剂灭菌且如果需要，将其与助剂混合，例如润滑剂、防腐剂、稳定剂、湿润剂、乳化剂、影响渗透压的盐、缓冲剂、着色剂、调味剂和/或芳香物质等，它们不与活性组合物发生有害反应。如果需要，还可以将它们与其它活性剂合并，例如维生素。在具体的实施方案中，所述的液体载体还可以含有增稠剂，例如蜂蜡、硬石蜡或鲸蜡醇。作为可选的添加剂注意到的还有苄醇类、聚乙二醇类、粘性石蜡、芳香油和脂肪酸酯类。
适于本发明的赋形剂是药学上可接受的并且与聚原酸酯相容的物质。它们在室温下为液体，并且容易与聚原酸酯溶混。合适的赋形剂包括：具有200-4000的分子量的聚(乙二醇)醚衍生物，如聚(乙二醇)单-或二-烷基醚、优选聚(乙二醇)单甲基醚550或聚(乙二醇)二甲基醚250；具有400-4000的分子量的聚(乙二醇)共聚物，如聚(乙二醇-共-聚丙二醇)；C2-19脂肪族羧酸或这样的酸的混合物的丙二醇单-或二-酯，如丙二醇二辛酸酯或二癸酸酯；C2-19脂肪族羧酸或这样的酸的混合物的单-、二-或三-甘油酯，如辛酸甘油酯、癸酸甘油酯、辛酸甘油酯/癸酸甘油酯、辛酸甘油酯/癸酸甘油酯/月桂酸甘油酯、聚氧乙烯(2)四氢糠醚和类似的乙氧基化的四氢糠醇和它们的C1-4烷基醚和C2-19脂肪族羧酸酯；以及生物相容的油，如葵花油、芝麻油以及其它非氢化或部分氢化的植物油。这些物质中的大多数是商业上可获得的，
本发明的药物组合物优选施用于哺乳动物，如狗、猫、马、猪、大鼠，特别是人。
优选本发明的药物组合物以胶囊、片剂、口服液、粉剂等口服，或者作为用于肌内或静脉内给药的肠胃外组合物液体。
在一个优选的实施方案中，本发明提供一种适于口服的形式，特别是口服单元剂型的组合物，如其剂型为片剂、胶囊、饮用溶液或用于重使用前配制的干粉；或者由本领域中已知的标准技术如通过在沉积时喷雾包衣而制备的sohxlet形式。
特别适宜口服的单元剂型包括胶囊形式，如软或硬明胶胶囊形式，它是优选的口服剂型。患者的药物剂量和给药次数依赖于多种因素、患者的年龄、患者病症的严重程度、过去的服药史而变，并由内科医师合理考虑来决定。
当本发明的组合物制成软或硬胶囊的形式时，该组合物可以装入包含任何常规增塑剂的明胶胶囊壳内。由于在明胶囊壳中可以包含增塑剂，可以不受限制地使用一种或多种选自以下的增塑剂：甘油、山梨醇、己三醇碳酸异丙烯酯、己二醇、山梨聚糖、四氢呋喃基醇醚、二乙二醇一乙基醚、1，3-三甲基-2-咪唑啉酮、二甲基异山梨醇等。
但是，应该理解本发明中可以使用的增塑剂不限于上述这些。本发明的胶囊制剂可以在常规机器中，通过胶囊化所得的本发明乳液的预浓缩物，如含有或不含有上述药学上可接受的添加剂的微乳液预浓缩物而制备。
进一步的，本发明的组合物还用于抑菌的化妆品中的用途。所述化妆品一方面能够抑制本身的细菌生长，另外一方面，也能够抑制皮肤表面细菌的生长，提高皮肤的抗氧化特性。
有益效果
本发明提供了一种白细胞提取物，分离自哺乳动物细胞，所述提取物具有较好的抗菌特性。将所述的白细胞提取物与抗菌肽一起使用后，能够有效的抑制病原菌的增殖，具有较好的治疗效果。可以将所述的白细胞提取物与抗菌肽制备成为相应的药物或者化妆品。
附图说明
图1抗菌肽的抗氧化特性结果图
具体实施方式
本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。以下实施案例中的方法、设备、材料，如果未进行特别说明，均为本领域常规方法、设备和材料，均可由市场购得。
实施例1白细胞提取物的制备
将牛外周血50ml肝素抗凝，用PBS等体积稀释后，用淋巴细胞分离液以密度梯度离心法分离得到单个核细胞PBMNC，PBS洗涤2次。将上述PBMNC用溶红素处理，然后用MACSbuffer洗涤1次，血细胞计数板计数。每107个细胞重悬于40μl MACS buffer，加入10μlNK cell biotin antibody cocktail，4℃孵育15分钟，然后依次加入30μlMACS buffer、20μlanti-biotin microbeads，4℃20r/min摇床孵育20分钟，再次用MACS buffer洗涤标记好的细胞1次，重悬于500μlMACSbuffer中。选用MS分离柱，安放于miniMACS分离器磁场中，使用前润湿分离柱，将分离柱放置于合适的细胞收集管上。将细胞悬液上柱，用4×500μlMACSbuffer洗柱，收集细胞。纯化前后计数单个核细胞，并以CD3、CD56荧光标记抗体分析NK细胞数，结果显示，NK细胞CD3-CD56+含量由分选前的不到12％提高到(95.3±0.7)％，获得了较为纯净的NK细胞。将纯化的NK细胞加入含有10％人AB血清、2mmol/L左旋谷氨酰胺、青、链霉素(各100U/ml)及细胞因子IL2(250U/ml)、IL15(15ng/ml)、IL12(5ng/ml)的NKMACS培养基内，调至细胞终浓度1×105/ml，然后接种于24孔板，每孔终体积为1ml，置37℃、5％CO2、饱和温度培养箱中培养，每3天半量换液并全量补充上述细胞因子。于培养第15天通过台盼蓝染色法计数并检测细胞活性。免疫表型分析将取自不同来源的细胞用荧光抗体CD3FITC、CD56PE标记，流式细胞仪检测发现，NK细胞纯度CD3-CD56+NK细胞纯度大于95％与开始培养时0天NK细胞纯度无显著差异(P<0.05)。细胞的扩增倍数为73倍。
将培养的NK细胞通过5000r/min离心10min，获得沉淀NK细胞，取200mg NK细胞用PBS缓冲液溶解，液氮冻融3次后，用超声破碎仪以10秒/次破碎，镜检破碎完毕后，4℃10000r/min离心30分钟，取上清，冷冻冻干即得到牛白细胞提取物。
实施例2抗菌肽的筛选分离
取牛血液2mL，加入抗凝剂，充分混匀，过滤后，用质量分数为0.83％的NH4Cl裂解红细胞，4℃下2500r/min离心10min，以沉淀白细胞。重复上述操作2次，并染色镜检，以保证沉淀中不含红细胞。将上述白细胞用高速组织捣碎机匀浆，以释放白细胞颗粒，经离心4℃，4000r/min15min收集细胞颗粒，悬浮于体积分数为10％的醋酸中，然后于4℃下搅拌过夜，次日于4℃15000r/min离心取上清，在旋转蒸发仪器蒸发除酸，冻干，然后用体积分数为0.01％的醋酸溶解，即为抗菌肽粗提物。将粗提物采用离子交换柱和反向高效液相色谱分离有活性的阳离子峰，采用琼脂糖弥散法测定离子交换柱和反向高效液相色谱分离的肽的抗微生物活性，所述微生物为大肠杆菌、金黄色葡萄球菌、副溶血弧菌。结果显示S3F8号多肽具有最好的抗菌活性，将该组分通过质谱鉴定，获得氨基酸序列如SEQ ID NO：1所示。
实施例3S3F8多肽的抗菌特性鉴定
培养基的制备：牛肉膏蛋白胨琼脂：牛肉膏3g，蛋白胨10g，NaC15g，琼脂20g，水1000mL，pH7.2，该培养基用于金黄色葡萄球菌ATCC25923和大肠杆菌ATCC35218的培养。
2216E琼脂：蛋白胨5g、酵母膏1g、柠檬酸铁0.1g、琼脂15g和过滤陈海水1000mL，调pH值为7.6，该培养基用于副溶血弧菌ATCC 33845的培养。液体培养基为相应的不加琼脂的培养基。
供试菌种的活化及菌悬液的制备：预先将3种供试菌种接入相对应的斜面培养基上进行活化，细菌置37℃恒温培养箱中培养24h，副溶血弧菌置28度恒温培养箱中培养48h，然后分别挑取一环已活化好的菌种放入9mL无菌生理盐水中，振荡摇匀，制成一系列菌悬液，浓度约为107cfu/mL，备用。
比浊法测定多肽抑菌活性：
取多肽于带塞的试管中，分别加入液体培养基，配制成含多肽终浓度分别为100μg/mL和200μg/mL的培养基各5.0mL，再分别加入50μL供试菌悬液，金黄色葡萄球菌和大肠杆菌置于37℃振荡培养24h；副溶血弧菌置于28℃振荡培养48h，用紫外分光光度计测量OD560值，并计算抑菌率。抑菌率(％)＝(对照组0D值-试验组OD值)/对照组OD值×lO0％。以100μg/mL的brevilaterin肽作为阳性对照，结果如表1所示。
表1 S3F8多肽的抑菌率
从表1的抑菌率显示本发明的多肽对细菌的抑制率随着浓度的增加而增加，对大肠杆菌的作用最强。在多肽浓度为200μg/mL时，对大肠杆菌和金黄色葡萄球菌抑制作用最强，对副溶血弧菌的抑制效果也较好，比阳性对照的抑制效果要好。
实施例4多肽的抗氧化性能实验
将多肽样品浓度调节为0.2mg/mL，以同浓度的Vc作为阳性对照。清除超氧阴离子自由基的测定：在比色管中分别加入0.2mL、0.4mL、0.6mL、0.8mL、1.OmL样品，加蒸馏水至lmL。加入Tris-HC1溶液(pH8.2)5.0mL和50mmol/L邻苯三酚溶液lmL，迅速摇匀，以蒸馏水为空白对照。每30s在波长325nm处测定吸光度值。按下式计算清除率。清除率＝(F0-Fs)×100％。式中：F0为邻苯三酚的自氧化速率；Fs为加样品的反应溶液吸光度值变化率。结果如图1所示。
本发明的多肽样品对超氧阴离子自由基的清除率测定结果见图1，由图1可知，多肽样品均具有较好的超氧阴离子自由基清除能力，且优于阳性对照组。同时清除率随其加入量的增大而增加。在浓度为0.5mg/mL时，清除率达到最大值(98.1±1.9)％。
实施例5白细胞提取物和抗菌肽的动物实验
将50只小鼠随机分成5组，每组1O只。实验组(1～3)及感染对照组(4)小鼠以腹腔注射金黄色葡萄球菌ATCC25923 100μL/只(5×107CFU/mL)；不感染对照组(5)小鼠仅肌肉注射0.1％乙酸。实验组(1)在注射金黄色葡萄球菌后，右腿肌肉注射实施例1制备的白细胞提取物浓度为1mg/mL的提取物100μL/只；实验组(2)在注射金黄色葡萄球菌后，右腿肌肉注射实施例2制备的多肽浓度为1mg/mL的提取物100μL/只；实验组(3)在注射金黄色葡萄球菌后，右腿肌肉注射实施例2制备的多肽浓度为1mg/mL的提取物100μL/只，左腿肌肉注射实施例1制备的白细胞提取物浓度为1mg/mL的提取物100μL/只；
各组小鼠分别置于不同的饲养箱中，自由饮水和采食饲料，每天观察2次临床表现，测量1次体重，记录临床症状和死亡情况。如小鼠死亡，则取其大脑组织进行涂片，并断尾取血制成血液涂片，革兰氏染色镜检，确定死因是否为菌感染。统计7d内各组小鼠的死亡情况，计算相对保护率(RPS)。结果如表2所示。
表2白细胞提取物和抗菌肽对小鼠感染的保护效果
| 组别 | 保护率(％) |
| 实验组1 | 70 |
| 实验组2 | 90 |
| 实验组3 | 100 |
小鼠在感染金黄色葡萄球菌时，模型组死亡率达到90％。而通过给小鼠注射血白细胞提取物和多肽，对小鼠抗金黄色葡萄球菌都有一定的效果。从小鼠的死亡情况看，各实验组小鼠死亡数无显著差异(P>0.05)，都获得了明显的保护，而实验组与感染对照组则差异显著(P<0.01)，感染对照组出现严重死亡，死亡率达到90％。实验结果表明，实验组1小鼠分别获得了70％的相对保护率；实验组2小鼠分别获得了90％的相对保护率；实验组3小鼠获得了100％的相对保护率。
实施例6白细胞提取物和本发明的多肽安全性鉴定
安全性检测：取小鼠1O只，以白细胞提取物和本发明的多肽(浓度均为1×103μg/mL)腹腔注射5只小鼠(200μL/只)，另设对照组以生理盐水腹腔注射5只小鼠(200μL/只)，观察7d内小鼠的健康状况及存活情况。
结果显示，小鼠未产生任何不良反应，其摄食、运动均正常，说明高浓度的白细胞提取物和多肽的安全性均达到了机体注射的要求，具有较好的安全性。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。
提供前述内容主要是为了解释的目的。本领域的技术人员可以容易地认识到：递送载体或药物组合物中的各种组分的分子结构以及比例、本文所述的本发明的制造方法和其他参数可以以各种方式进一步加以改变或替换，而不偏离本发明的精神和范围。例如，对于上面示出的本发明的化合物的适应症的任何一种，由于被治疗的哺乳动物的反应有差异，所以可以应用不同于本文上面所述的特定剂量的有效剂量。同样地，所观察的特定的药理学反应可以根据并依赖于下列因素变化：所选择的特定活性化合物或者是否存在药物载体，以及制剂的类型和所用的施加方式，并且结果中这样的预期的变化或差异可以根据本发明的目的和实践来预期。因此，应该理解，本发明限于所附权利要求的范围并且这样的权利要求解释为合理宽的范围。
Claims (4)
1.一种具备抗菌和抗氧化特性的S3F8肽，其特征在于氨基酸序列如SEQ ID NO：1所示。
2.权利要求1所述的具备抗菌和抗氧化特性的S3F8肽在制备抑制细菌增殖的药物中的用途，其中所述的细菌是金黄色葡萄球菌ATCC25923、大肠杆菌ATCC35218和副溶血弧菌ATCC 33845。
3.权利要求1所述的具备抗菌和抗氧化特性的S3F8肽和白细胞提取物在制备抑制细菌增殖的药物中的用途；其中白细胞提取物是将来自牛的NK细胞裂解后的上清液冻干制备获得的，其中所述的细菌是金黄色葡萄球菌ATCC25923、大肠杆菌ATCC35218和副溶血弧菌ATCC 33845。
4.权利要求1所述的具备抗菌和抗氧化特性的S3F8肽和白细胞提取物在制备化妆品中的用途，其中白细胞提取物是将来自牛的NK细胞裂解后的上清液冻干制备获得的。
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