CN113520909A - 促进皮肤创伤后修复的组合物及其制备方法和应用 - Google Patents
促进皮肤创伤后修复的组合物及其制备方法和应用 Download PDFInfo
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- CN113520909A CN113520909A CN202110959333.7A CN202110959333A CN113520909A CN 113520909 A CN113520909 A CN 113520909A CN 202110959333 A CN202110959333 A CN 202110959333A CN 113520909 A CN113520909 A CN 113520909A
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Abstract
本发明属于医疗美容领域，具体涉及促进皮肤创伤后修复的组合物及其制备方法和应用。本发明的促进皮肤创伤后修复的组合物，包括溶媒液和干粉，溶媒液包括：甘油、丁二醇、甘油聚醚‑26、精氨酸/赖氨酸多肽、透明质酸钠、烟酰胺、灵芝多糖、黄原胶、对羟基苯乙酮、1，2‑己二醇、乙基己基甘油，干粉包括：甘露糖醇、三肽‑1铜、乙酰基六肽‑8、九肽‑1。本发明的组合物安全、有效、温和、低致敏，通过三肽‑1铜与灵芝多糖配伍，协同重建肌肤、修复皮肤屏障，抑制致敏因素的发生。
Description
技术领域
本发明属于医疗美容领域，具体涉及促进皮肤创伤后修复的组合物及其制备方法和应用。
背景技术
随着人们生活水平的提高,人们在满足基本的衣食住行需求后，开始关注其他方面，尤其是形体、容貌的美观等方面,其中让皮肤保持年轻更是许多人梦寐以求的事，因此，医疗美容也越来越受到人们的关注。激光热玛吉是目前最重要的医美手段之一，由于激光的光热效应、压强作用等对皮肤造成不同程度的损伤，术后皮肤通常出现干燥、红斑、紫瘢、水疱、易感染、结痂、瘢痕色素沉着等症状，此时皮肤处于高敏感状态，对外界环境变化及轻微的刺激均不耐受。其中，红斑是由于皮肤表面受热而发生毛细血管扩张引起的，也是最常见的不良反应；紫癱是由于血红蛋白受热氧化变成高铁血红蛋白，血管壁受热凝固变性，皮肤呈现紫色；水疱是由于表皮积聚的热量过多，也可能是由于激光能置过大、术中的冷却做得不够，或者术后冰敷不到位造成的；易感染是由于表皮损伤或变薄，对外界微生物防御能力差；结痂是皮肤愈合过程中的必经环节，此时保持皮损处的水活度，更有助于皮肤的修复与更新；如果感染的层面深及真皮深层，胶原重塑过程中可能遗留瘢痕；干燥是由于热效应使角质层中的角蛋白变性，角质层的吸收和保湿功能下降，经皮水分散失埔多造成的；另外，皮肤经历了水疱、感染、结痂这样的破皮过程之后，易形成炎症后色素沉着(PIH)。
因此，可以加快恢复期的后处理策略将有利于皮肤恢复。当前的后处理程序包括使用半封闭片材和各种皮肤护理产品。半闭塞膜在口腔周围区域经常会移位，因此覆盖范围不一致。大多数皮肤护理产品均以凡士林为基础，并可能富含维生素，矿物质或有机物质，以帮助治愈。
发明内容
本发明的目的在于提供一种促进皮肤创伤后修复的组合物。
本发明的再一目的在于提供上述组合物的制备方法。
本发明的再一目的在于提供促进皮肤创伤后修改的组合物的应用。
根据本发明具体实施方式的促进皮肤创伤后修复的组合物，所述组合物包括溶媒液和干粉，所述干粉与所述溶媒液的比例为1：(30～50)mg/mL；其中，
按照质量百分比，所述溶媒液包括以下组分：甘油1～3％、丁二醇2～5％、甘油聚醚-26 0.5～2％、精氨酸/赖氨酸多肽1～4％、透明质酸钠0.1～0.5％、烟酰胺1～5％、灵芝多糖1～3％、黄原胶0.02～1％、对羟基苯乙酮0.1～1％、1，2-己二醇0.1～1％、乙基己基甘油0.1～1％，余量为水；
按照重量份数，所述干粉包括以下组分：甘露糖醇15～20份、三肽-1铜0.5～2份、乙酰基六肽-8 1～4份、九肽-1 1～5份。
根据本发明具体实施方式的促进皮肤创伤后修复的组合物，所述组合物包括溶媒液和干粉，所述干粉与所述溶媒液的比例为1：40mg/mL；其中，
按照质量百分比，所述溶媒液包括以下组分：甘油2％、丁二醇3％、甘油聚醚-261％、精氨酸/赖氨酸多肽2％、透明质酸钠0.15％、烟酰胺2％、灵芝多糖2％、黄原胶0.05％、对羟基苯乙酮0.5％、1，2-己二醇0.5％、乙基己基甘油0.1％，余量为水；
按照重量份数，所述干粉包括以下组分：甘露糖醇18份、三肽-1铜1份、乙酰基六肽-8 2份、九肽-1 2份。
本发明的组合物配方中含有封闭剂、保湿剂和润肤剂(柔肤剂)等成分。其中，保湿剂包括聚醚多元醇类保湿剂、纯天然保湿补水类、碳水化合物类保湿剂、分子结构生物化学类保湿剂。
聚醚多元醇类保湿剂包括丙三醇、丁二醇、聚乙二醇、丙二醇、已二醇、麦芽糖醇、聚丙二醇、山梨糖醇等。
分子结构生物化学类保湿剂包括胶原蛋白粉、粘多糖体、玻尿酸、糖蛋白及盐酸软骨素等。
封闭剂主要分为油脂类，合成酯类、硅类、神经酰胺、胆甾醇、角鲨烷(烯)等。油脂类封闭剂包括橄榄果油、米糠油、鳄梨油、可可籽脂、羊毛脂、棕榈酸异丙酯等。硅类封闭剂包括环己硅氧烷、聚二甲基硅氧烷等。
润肤剂包括多元醇类、天然保湿类、氨基酸类。多元醇类润肤剂包括丙三醇、丁二醇、聚乙二醇、丙二醇、已二醇、木糖醇、聚丙二醇、山梨糖醇等。天然保湿类润肤剂包括氨基酸、PCA、乳酸钠、尿素等。氨基酸类润肤剂包括有蛋白类，例如植物蛋白、大豆蛋白、动物蛋白、水解蛋白等。
根据本发明具体实施方式的促进皮肤创伤后修复的组合物，所述灵芝多糖由包括以下步骤的方法制备而成：
(1)取灵芝粉，进行水提；
(2)将步骤(1)所得水提物进行离心，抽滤，浓缩；
(3)将步骤(2)所得的浓缩液进行醇沉；
(4)离心，收集沉淀，得到灵芝多糖粗品；
(5)纯化灵芝多糖粗品，得到灵芝多糖。
根据本发明具体实施方式的促进皮肤创伤后修复的组合物，步骤(1)中，按照料液比20：1mg/L的比例将灵芝粉与水混合，在85～95℃条件下，浸提2～4h，冷却至室温。
根据本发明具体实施方式的促进皮肤创伤后修复的组合物，步骤(5)中，纯化灵芝多糖粗品的步骤包括除蛋白、活性炭脱色。
根据本发明具体实施方式的促进皮肤创伤后修复的组合物，步骤(5)中，用Sevag法去除灵芝多糖中的蛋白。
根据本发明具体实施方式的促进皮肤创伤后修复的组合物的制备方法，所述制备方法包括以下步骤：
制备干粉：将甘露糖醇、三肽-1铜、乙酰基六肽-8、九肽-1溶解后，进行冷冻干燥，得到干粉；
制备溶媒液：
(1)将透明质酸、黄原酸在丁二醇中分散，加入热水溶解后，依次加入甘油、甘油聚醚-26，75～80℃保温搅拌溶解30min；
(2)降温至60～70℃，加入对羟基本乙酮搅拌溶解；
(3)降温至35～45℃，依次加入烟酰胺、精氨酸/赖氨酸多肽、灵芝多糖、1，2-已二醇、乙基乙基甘油搅拌溶解；
(4)降温至35～40℃时，出料，得到溶媒液；
将干粉、溶媒液分别分装。
根据本发明具体实施方式的促进皮肤创伤后修复的组合物的制备方法，所述灵芝多糖由包括以下步骤的方法制备而成：
(1)取灵芝粉，进行水提；
(2)将步骤(1)所得水提物进行离心，抽滤，浓缩；
(3)将步骤(2)所得的浓缩液进行醇沉；
(4)离心，收集沉淀，得到灵芝多糖粗品；
(5)纯化灵芝多糖粗品，得到灵芝多糖。
根据本发明具体实施方式的促进皮肤创伤后修复的组合物的制备方法，步骤(5)中，纯化灵芝多糖粗品的步骤包括除蛋白、活性炭脱色。
本发明的有益效果：
1.本发明通过除蛋白及脱色两个简单步骤去除了灵芝多糖的细胞毒性，得到安全性显著提高的灵芝多糖；
2.本发明的组合物安全、有效、温和、低致敏，通过三肽-1铜与灵芝多糖配伍，协同重建肌肤、修复皮肤屏障，抑制致敏因素的发生，具体效果为：
针对激光美容后出现的皮肤干燥问题，本发明采用甘油、甘油聚醚-26、1,2-戊二醇、透明质酸钠、丁二醇、乙基己基甘油进行配合，保湿剂一方面具有从真皮吸取水分并传输到角质层的作用，另一方面，具有当周囤湿度大于70％时，保湿剂将从环境中吸取和吸收水分的作用。封闭剂和保湿剂共同作用从而保证角质层的水分和屏障功能的正常运行。润肤剂成分具有油性物质“填充脱落的角质细胞碎片间裂缝”等作用，可使皮肤表面纹理光滑、柔软；
针对激光美容后皮肤出现的活性表皮层和真皮层损伤问题，本发明采用三肽-1铜与灵芝多糖复合，可以极大地促进γ-射线辐照后人皮肤成纤维细胞的存活和清除活性氧，能够显著降低γ-射线辐照后DNA的损伤水平。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是灵芝多糖处理HFF-1细胞24h的细胞存活率；
图2是三肽-1铜及其与精制灵芝多糖的混合物以及本发明组合物处理HFF-1细胞3天的细胞存活率；
图3是三肽-1铜及其与灵芝多糖的混合物以及本发明组合物对HFF-1细胞γ-射线辐照后增殖能力影响(*p<0.05，**p<0.01，***p<0.001)；
图4为三肽-1铜及其与灵芝多糖的混合物以及修复组合物对HFF-1细胞γ-射线辐照后活性氧水平影响；
图5显示三肽-1铜及其与灵芝多糖的混合物以及修复组合物对HFF-1细胞γ-射线辐照后DNA损伤的修复能力。
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，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
实施例1制备灵芝多糖提取物
1.1制备灵芝多糖粗品
准确称取9g灵芝粉，分三个实验组，每组3g；按照液料比20：1(mg/L)的比例溶解于超纯水中，混匀，静置3min；
在90℃条件下，浸提3h，冷却至室温；
2740×g离心，20min后，抽滤；
浓缩至原体积的1/5；
酒精醇沉至80％，4℃冰箱内过夜；
2740×g离心，20min后，收集沉淀，冷冻干燥后，得到灵芝多糖粗品。
浓缩后，分别用30％、50％、80％乙醇醇提，其中，30％乙醇醇提粗品得率为0.73％；50％乙醇醇提粗品得率为0.93％；80％乙醇醇提粗品得率为2.10％。
1.2纯化灵芝多糖
(1)Sevag法除蛋白
将得到的灵芝多糖粗品500mg，溶解于80mL超纯水中，向溶液中加入其体积1/3的Sevag试剂(氯仿：正丁醇＝4:1)混合震荡后，静置30min，离心(4000rpm，5min)，取得水层，去除交界处变性蛋白质。重复多次，直到离心后试剂层交界处没有白色沉淀，出现说明蛋白质去除干净，得到脱蛋白灵芝多糖。
(2)活性炭脱色
向步骤(1)得到的脱蛋白灵芝多糖溶液中，加入5％(w/v)的活性炭，在70℃水浴环境中90min，不断搅拌，抽滤，醇沉过夜，离心，然后再用无水乙醇洗涤沉淀，冷冻干燥，得到精制灵芝多糖。
此过程，灵芝多糖粗品得到精制灵芝多糖的产率为10％，由灵芝子实体得到精制灵芝多糖产率约为0.2％。
实施例2制备促进皮肤创伤后修复的组合物
2.1本发明的促进皮肤创伤后修复的组合物，包括溶媒液和干粉，所述干粉与所述溶媒液的比例为1：30mg/mL；其中，
按照质量百分比，所述溶媒液包括以下组分：甘油1％、丁二醇2％、甘油聚醚-260.5％、精氨酸/赖氨酸多肽1％、透明质酸钠0.1％、烟酰胺1％、灵芝多糖1％、黄原胶0.02％、对羟基苯乙酮0.1％、1，2-己二醇0.1％、乙基己基甘油0.1％，余量为水；
按照质量百分比，所述干粉的原料包括：包括甘露糖醇15％、三肽-1铜0.5％、乙酰基六肽-8 1％、九肽-1 1％，余量为水。
2.2本发明的促进皮肤创伤后修复的组合物，包括溶媒液和干粉，所述干粉与所述溶媒液的比例为1：40mg/mL；其中，
按照质量百分比，所述溶媒液包括以下组分：甘油2％、丁二醇3％、甘油聚醚-261％、精氨酸/赖氨酸多肽2％、透明质酸钠0.15％、烟酰胺2％、灵芝多糖2％、黄原胶0.05％、对羟基苯乙酮0.5％、1，2-己二醇0.5％、乙基己基甘油0.1％，余量为水；
按照质量百分比，所述干粉的原料包括：包括甘露糖醇18％、三肽-1铜1％、乙酰基六肽-8 2％、九肽-1 2％，余量为水。
2.3本发明的促进皮肤创伤后修复的组合物，包括溶媒液和干粉，所述干粉与所述溶媒液的比例为1：50mg/mL；其中，
按照质量百分比，所述溶媒液包括以下组分：甘油3％、丁二醇5％、甘油聚醚-262％、精氨酸/赖氨酸多肽4％、透明质酸钠0.5％、烟酰胺5％、灵芝多糖3％、黄原胶1％、对羟基苯乙酮1％、1，2-己二醇1％、乙基己基甘油1％，余量为水；
按照质量百分比，所述干粉的原料包括：包括甘露糖醇20％、三肽-1铜2％、乙酰基六肽-8 4％、九肽-1 5％，余量为水。
本发明的促进皮肤创伤后修复的组合物的制备方法：
(1)制备干粉：把各成分的水溶液混合，同时加入一定体积的超纯水，混合溶解均匀，-20℃冷冻12-24h后，转入真空冷冻干燥机中冻干24-48h，待冰消失，样品全部转为干燥粉末；
(2)制备溶媒液：
(2-1)将透明质酸、黄原酸在丁二醇中分散，加入热水溶解后，依次加入甘油、甘油聚醚-26，75～80℃保温搅拌溶解30min；
(2-2)降温至60～70℃，加入对羟基本乙酮搅拌溶解；
(2-3)降温至35～45℃，依次加入烟酰胺、精氨酸/赖氨酸多肽、灵芝多糖、1，2-已二醇、乙基乙基甘油搅拌溶解；
(2-4)降温至35～40℃时，出料，得到溶媒液；
(3)将干粉、溶媒液分别分装。
本发明的使用方法：将冻干粉与溶媒液充分混合，使冻干粉充分溶于冻干粉。当溶媒和冻干粉融合之后，冻干粉里面的高生物活性物质，能够快速恢复其生物活性，施用后，能够快速渗入基底层进行修复。
实施例3生物安全性研究
将人皮肤成纤维细胞HFF-1细胞按照3000个/孔的密度铺于96孔细胞培养板，培养24h后，分别加入灵芝多糖粗品、精制灵芝多糖、三肽-1铜以及精制灵芝多糖、三肽-1铜肽按照一定比例复合物与细胞共孵育，一定时间后进行CCK8实验检测细胞存活率。
由图1可以看出，没有经过去蛋白、脱色等纯化步骤的灵芝多糖粗品具有浓度依赖的细胞毒性，39μg/mL的粗多糖培养HFF-1细胞24h的细胞存活率为71.2％，浓度大于312μg/mL时细胞存活率降低为41％以下；经过纯化处理的灵芝多糖在浓度为1250μg/mL时细胞存活率为100％，没有表现出细胞毒性，说明本发明的灵芝多糖经除蛋白、活性炭脱色步骤纯化后，其生物安全性得到了显著提高。
以5nM的三肽-1铜、三肽-1铜与浓度由6.25至50μg/mL的多糖复合物以及修复组合物培养HFF-1细胞3天，考察三肽-1铜与灵芝多糖的混合物、本发明的修复组合物的细胞毒性。
如图2所示，三肽-1铜以及三肽-1铜、灵芝多糖混合物以及修复组合物培养细胞3天，细胞存活率均为97％以上，未表现出明显的细胞毒性，说明本发明的修复组合物具有良好的生物安全性。
实施例4考察本发明的辐射损伤防护作用
4.1考察细胞增殖能力
经一定浓度三肽-1铜和灵芝多糖复合物孵育12h的HFF-1细胞进行6Gyγ-射线辐照，继续培养3天后通过CCK8实验检测细胞存活率。各组及其浓度设置如下：三肽-1铜(5nM)、多糖(50μg/mL)、三肽-1铜(5nM)+多糖(50μg/mL)以及含同浓度三肽-1铜和灵芝多糖的修复组合物。
如图3所示，6Gyγ-射线辐照后HFF-1细胞增殖能力明显减弱，细胞存活率不足50％，三肽-1铜、灵芝多糖单组分提高细胞存活率有限，本申请的组合物相较于单组份(三肽-1铜、灵芝多糖)更显著地提高了细胞的存活率和增殖能力(**p<0.01)，细胞存活率恢复到了未照射对照组的80％以上。
4.2考察活性氧自由基清除能力
利用DCFHDA法对灵芝多糖及三肽-1铜的活性氧自由基的清除能力进行研究。
各组浓度设置如下：三肽-1铜(5nM)、灵芝多糖(50μg/mL)、三肽-1铜+多糖(5nM+50μg/mL)以及本发明的组合物。
如图4所示，灵芝多糖具有一定的活性氧清除能力，且单独增加灵芝多糖至800μg/mL并没有增强其活性氧的清除效果；类似地，三肽-1铜也具有一定的活性氧清除能力，将三肽-1铜浓度增加至80nM，其对活性氧的清除效率并未显著增强；但是，本发明能够显著地增强活性氧清除能力(***p<0.001)。
4.3考察DNA损伤修复能力
利用γ-H2AX染色实验进一步研究本发明组合物的DNA辐射损伤修复能力。各组浓度设置如下：三肽-1铜(5nM)、灵芝多糖(50μg/mL)、三肽-1铜+多糖(5nM+50μg/mL)以及含同浓度三肽-1铜和灵芝多糖的组合物。
在DNA彗星电泳图像中，尾巴越大说明DNA损伤越严重。如图5所示，经6Gyγ-射线照射后，HFF-1细胞出现大量尾巴，产生了明显的DNA损伤，使用三肽-1铜、灵芝多糖单组份处理组彗星尾巴并无明显改善，灵芝多糖/三肽-1铜复合物以及本发明的组合物预处理12h后无明显尾巴出现，说明本发明可明显缓解DNA断裂的水平。
本发明的组合物可以极大地促进γ-射线辐照后人皮肤成纤维细胞的存活和清除活性氧，能够显著降低γ-射线辐照后DNA的损伤水平，表明，本发明具有显著强于单组份的生物活性，协同增效的效果，可用于细胞辐射防护、皮肤损伤修复以及化妆品添加剂。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
Claims (10)
1.促进皮肤创伤后修复的组合物，其特征在于，所述组合物包括溶媒液和干粉，所述干粉与所述溶媒液的比例为1：(30～50)mg/mL；其中，
按照质量百分比，所述溶媒液包括以下组分：甘油1～3％、丁二醇2～5％、甘油聚醚-260.5～2％、精氨酸/赖氨酸多肽1～4％、透明质酸钠0.1～0.5％、烟酰胺1～5％、灵芝多糖1～3％、黄原胶0.02～1％、对羟基苯乙酮0.1～1％、1，2-己二醇0.1～1％、乙基己基甘油0.1～1％，余量为水；
按照重量份数，所述干粉包括以下组分：甘露糖醇15～20份、三肽-1铜0.5～2份、乙酰基六肽-8 1～4份、九肽-1 1～5份。
2.根据权利要求1所述的促进皮肤创伤后修复的组合物，其特征在于，所述组合物包括溶媒液和干粉，所述干粉与所述溶媒液的比例为1：40mg/mL；其中，
按照质量百分比，所述溶媒液包括以下组分：甘油2％、丁二醇3％、甘油聚醚-26 1％、精氨酸/赖氨酸多肽2％、透明质酸钠0.15％、烟酰胺2％、灵芝多糖2％、黄原胶0.05％、对羟基苯乙酮0.5％、1，2-己二醇0.5％、乙基己基甘油0.1％，余量为水；
按照重量份数，所述干粉包括以下组分：甘露糖醇18份、三肽-1铜1份、乙酰基六肽-8 2份、九肽-1 2份。
3.根据权利要求1所述的促进皮肤创伤后修复的组合物，其特征在于，所述灵芝多糖由包括以下步骤的方法制备而成：
(1)取灵芝粉，进行水提；
(2)将步骤(1)所得水提物进行离心，抽滤，浓缩；
(3)将步骤(2)所得的浓缩液进行醇沉；
(4)离心，收集沉淀，得到灵芝多糖粗品；
(5)纯化灵芝多糖粗品，得到灵芝多糖。
4.根据权利要求3所述的促进皮肤创伤后修复的组合物，其特征在于，步骤(1)中，按照料液比20：1mg/L的比例将灵芝粉与水混合，在85～95℃条件下，浸提2～4h，冷却至室温。
5.根据权利要求3所述的促进皮肤创伤后修复的组合物，其特征在于，步骤(5)中，纯化灵芝多糖粗品的步骤包括除蛋白、活性炭脱色。
6.根据权利要求5所述的促进皮肤创伤后修复的组合物，其特征在于，步骤(5)中，用Sevag法去除灵芝多糖粗品中的蛋白。
7.根据权利要求1所述的促进皮肤创伤后修复的组合物的制备方法，其特征在于，所述制备方法包括以下步骤：
制备干粉：将甘露糖醇、三肽-1铜、乙酰基六肽-8、九肽-1溶解后，进行冷冻干燥，得到干粉；
制备溶媒液：
(1)将透明质酸、黄原酸在丁二醇中分散，加入热水溶解后，依次加入甘油、甘油聚醚-26，75～80℃保温搅拌溶解30min；
(2)降温至60～70℃，加入对羟基本乙酮搅拌溶解；
(3)降温至35～45℃，依次加入烟酰胺、精氨酸/赖氨酸多肽、灵芝多糖、1，2-已二醇、乙基乙基甘油搅拌溶解；
(4)降温至35～40℃时，出料，得到溶媒液；
将干粉、溶媒液分别分装。
8.根据权利要求7所述的促进皮肤创伤后修复的组合物的制备方法，其特征在于，所述灵芝多糖由包括以下步骤的方法制备而成：
(1)取灵芝粉，进行水提；
(2)将步骤(1)所得水提物进行离心，抽滤，浓缩；
(3)将步骤(2)所得的浓缩液进行醇沉；
(4)离心，收集沉淀，得到灵芝多糖粗品；
(5)纯化灵芝多糖粗品，得到灵芝多糖。
9.根据权利要求8所述的促进皮肤创伤后修复的组合物的制备方法，其特征在于，步骤(5)中，纯化灵芝多糖粗品的步骤包括除蛋白、活性炭脱色。
10.权利要求1所述的促进皮肤创伤后修复的组合物的应用。
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