CN108969415A - 黄芪总苷在抗皮肤光老化和光损伤中的应用 - Google Patents
黄芪总苷在抗皮肤光老化和光损伤中的应用 Download PDFInfo
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Abstract
本发明涉及黄芪总苷在抗皮肤光老化和光损伤中的应用,所述黄芪总苷能改善UVB长期反复照射下造成的光老化,通过细胞水平的实验发现,黄芪总苷在抗氧化,抗光损伤和光老化,保护线粒体以及抑制UVB引起的凋亡等均有良好的效果,同时,黄芪总苷亦能逆转光老化过程中皮肤细胞的胶原的流失过程,促进胶原蛋白的积累。另一方面可上调光老化细胞中自噬活力,维持正常的细胞稳态过程,从而更好地抵抗光老化过程。本发明证明了黄芪总苷在抗皮肤光老化过程中有良好的效果,可应用于制药、保健品、化妆品等领域。
Description
技术领域
本发明属于医药技术、保健品及化妆品技术领域,具体涉及黄芪总苷在抗皮肤光老化的用途。
背景技术
皮肤是人体最大的暴露于外界的器官,因而人类衰老首先表现在了皮肤的衰老,比如皮肤皱纹增多,皮肤松弛下垂等。皮肤光老化是由于外源性的因素如紫外照射、环境毒素接触、吹风以及吸烟等持续的暴露导致的DNA改变和皮肤破坏导致的老化,其中,日光中的紫外线长期反复照射是环境中最主要的影响皮肤老化的因素。
光老化的发生机制目前尚未完全阐明,但自由基被认为是光老化发生发展中最为重要的因素。自由基的过度积累会导致细胞内DNA、蛋白质、脂质以及糖类的氧化损伤。UV照射会直接或者间接地通过多种途径产生过量的ROS,如UV照射会损伤线粒体,导致线粒体电子传递链以及氧化磷酸化功能的异常,因而在代谢中产生大量的ROS,打破了细胞原有的稳态平衡则会引起氧化应激导致细胞的凋亡。
目前,预防和防治光老化的手段包括物理手段、化学手段以及目前研究较为热门的中草药治疗手段。由于从天然产物中获取的天然活性物质一般毒性较小,安全性高,人们的接受度高,具有一定的优势。
黄芪是多年生草本植物,在我国常作为滋补益气的中药材,可增强身体功能比如免疫系统、代谢系统、呼吸系统以及排泄系统。其主要化学成分有黄芪多糖、黄芪黄酮、黄芪总皂苷。黄芪总皂苷中成分较为复杂,161种皂苷中有142种为环菠萝烷样皂苷,余下19种为齐墩果烷样皂苷。黄芪皂苷I、II、IV以及异黄芪皂苷I、II占了所有皂苷的80%以上。但至今未见黄芪总苷在改善皮肤光老化方面的研究报道。并且目前应用的一些预防和治疗光老化的产品伴随着较大的毒副作用,因而各种研究机构以及公司都在积极研发新产品。
发明内容
本发明的目的是针对现有技术的不足,提供一种黄芪总苷抵抗皮肤光老化的用途。
针对上述目的,本发明采用的技术方案为:
黄芪总苷在抗皮肤光老化和光损伤中的应用。
所述黄芪总苷在制备预防和治疗皮肤光老化和光损伤的药物中的应用。
所述药物包括有效量的黄芪总苷及药学上所接受的辅料。
所述药物的剂型包括乳剂、膏剂、口服液、颗粒剂、片剂、丸剂、散剂、胶囊剂或滴丸剂。
所述黄芪总苷在制备抗皮肤光老化和光损伤的化妆品中的应用。
所述黄芪总苷在制备抗皮肤光老化和光损伤的保健品中的应用。
本发明还提供了一种预防和治疗光老化的药物制剂,包括了有效量的黄芪总苷和药学上可接受的辅料。本领域内的技术人员可不用多加学习地将所述黄芪总苷直接或间接地加入制备不同剂型所需的药学上普遍运用的辅料,如崩解剂、润滑剂、乳化剂、粘合剂等,以通用的药剂制备方法,制成预防和治疗皮肤光老化的注射液、口服液、乳膏、颗粒剂、片剂、丸剂、散剂、胶囊剂或滴丸剂等。
本发明与现有技术相比,所具有的优点有:
本发明首次提出了黄芪总苷可以明显抵抗紫外线导致的光老化作用,能改善UVB长期反复照射下造成的光老化,通过细胞水平的实验发现,黄芪总苷在抗氧化,抗光损伤和光老化,保护线粒体以及抑制UVB引起的凋亡等均有良好的效果。
同时,黄芪总苷亦能逆转光老化过程中皮肤细胞的胶原的流失过程,促进光老化细胞中胶原积累。另一方面可上调光老化细胞中自噬活力,维持正常的细胞稳态过程,从而更好地抵抗光老化过程。实验结果表明,黄芪总苷对紫外线导致的皮肤光老化和光损伤具有保护作用。本发明提出的黄芪总苷应用于预防和治疗光老化和光损伤的医药领域,同时由于原料来源丰富,可应用于制药、保健品、化妆品等。
附图说明
图1是SD大鼠真皮成纤维细胞的免疫荧光染色图,A、B、C分别为Vimentin、Hoechst33342、Merge。
图2是UVB对大鼠真皮成纤维细胞的存活率影响示意图。
图3是UVB对蛋白p53、p21表达的影响Western Blot检测图(A)及p53、p21蛋白表达量统计结果图(B、C)。
图4是正常成纤维细胞(A)与UVB照射后成纤维细胞(B)的SA-β-Gal衰老染色图及衰老细胞比例统计结果图(C)。
图5中A图是正常成纤维细胞(CM)与UVB照射后成纤维细胞以及UVB+AST、UVB+ASI、UVB+ASF、UVB+APS处理后成纤维细胞的SA-β-Gal染色图;B图是A图中衰老细胞比例统计结果图。
图6中A图是正常成纤维细胞(CM)与UVB照射后成纤维细胞以及UVB+AST、UVB+ASI、UVB+ASF、UVB+APS处理后对ROS水平影响的荧光图;B图是A图中荧光信号强弱的统计结果图。
图7中A图是正常成纤维细胞(CM)与UVB照射后成纤维细胞以及UVB+AST、UVB+ASI、UVB+ASF、UVB+APS处理后对线粒体膜电位影响的荧光图;B图是A图中红色及绿色荧光比值的统计结果图。
图8是黄芪总苷(AST)对光损伤细胞(UVB)及正常细胞(Non-UVB)的增殖的影响示意图。
图9中A图是黄芪总苷(AST)影响光老化细胞p21,IL-1β,IL-6,TNF-αmRNA表达的统计结果图;B图是黄芪总苷(AST)影响光老化细胞MMP1,MMP3,MMP9,ICAM mRNA表达的统计结果图。
图10是黄芪总苷(AST)抑制UVB导致的凋亡效应蛋白cleaved caspase3,p53蛋白表达上调的Western Blot图(A)及cleaved caspase 3蛋白表达量的统计结果图(B)。
图11中A图是UVB影响成纤维细胞胶原蛋白I表达量的Western Blot图及统计结果图;B图是正常成纤维细胞(CM)与UVB照射后成纤维细胞以及UVB+AST、UVB+ASI、UVB+ASF、UVB+APS处理后影响成纤维细胞胶原蛋白I表达量的Western Blot图及统计结果图;C图是不同浓度黄芪总苷(AST)影响光老化成纤维细胞胶原蛋白I表达量的Western Blot图及统计结果图。
图12中A图是UVB影响成纤维细胞自噬标记物LC3-II蛋白表达量的Western Blot图及统计结果图;B图是正常成纤维细胞(CM)与UVB照射后成纤维细胞以及UVB+AST、UVB+ASI、UVB+ASF、UVB+APS处理后影响成纤维细胞LC3-II蛋白含量的Western Blot图及统计结果图;C图是不同浓度黄芪总苷(AST)影响光老化成纤维细胞LC3-II,p62蛋白含量的Western Blot图及LC3-II蛋白表达量统计结果图;D图是CQ阻断光老化细胞自噬流后p62蛋白和LC3-II蛋白含量的Western Blot图及对应的统计结果图。
具体实施方式
下面结合具体的实施例对本发明作进一步的说明,但本发明的保护内容不仅仅限于以下实施例。
本实施例是以大鼠真皮成纤维细胞为主要研究对象,探讨黄芪提取物中黄芪总苷(Astragaloside,AST)对大鼠皮肤成纤维细胞在UVB反复照射下诱导的光老化影响。
一实验材料与方法
1实验材料
试验所用黄芪总苷(Astragaloside,AST)、黄芪多糖(Astragaluspolysaccharides,APS)、黄芪甲苷(Astragaloside IV,ASI)、黄芪黄酮(astragalusflavonoids,ASF)均购自国家标准物质中心,取20mg粉末溶于1ml二甲基亚砜(DMSO)中,配成20mg/ml的储存液于-20℃中保存,临用时溶解,并用含血清(FBS)的完全培养基稀释至100μg/ml;Vimentin,p53,p21等抗体购自CST公司,Tubulin,LC3B等抗体购自Sigma,Collagen I抗体购自武汉博士德公司,其他常规细胞培养操作参照常规细胞培养规范。
实施例1
大鼠真皮成纤维细胞(RDFs)的分离培养
选取出生3天以内Sprague-Dawley乳鼠(SD乳鼠),雌雄不限,用于原代皮肤成纤维细胞的提取和培养。
(1)取1-3天的SD乳鼠,置于培养皿中,培养皿置于冰上,将处死的小鼠浸入碘伏中2分钟,重复2次。再将乳鼠浸入去离子水中除去碘伏,之后继续将乳鼠浸入70%的乙醇中2分钟,重复2次,随后培养皿置于冰上。
(2)用镊子及剪刀先将乳鼠四肢及尾巴剪去,随后沿尾巴缺口顺着脊椎方向用剪刀缓慢仔细的剪开皮肤,注意不要用力过猛剪到内脏引起大出血,剪至头部后,用镊子分别夹着两边的皮肤沿相反方向撕开,用剪刀剪去过程中一些黏连的组织,最后将整张皮撕下,除去头部含有胡须部分的皮肤。
(3)将剥下的皮肤摊开放置于冰上的培养皿中,用镊子缓慢的剔除皮下结缔组织及血管和一些脂肪层,随后置于预冷的PBS(加有双抗,青霉素和链霉素)溶液中浸泡30分钟,随后将皮肤薄片置于另一无菌的培养皿中,真皮层朝下,尽量铺开摊平,加入预冷的0.1%的胰酶溶液,小心缓慢添加,使皮肤漂浮于胰酶溶液之上,盖上盖子,将培养皿小心转移至4度冰箱消化过夜。
(4)用镊子剥去皮肤的表皮组织,将剩下的真皮组织在超净工作台中用眼科剪剪碎成1mm3的小块,用吸管将真皮组织吸入含有搅拌子的锥形瓶中,加入0.1%I型胶原酶5ml后置于冰上消化20分钟,中间每隔5分钟轻轻摇动一下锥形瓶。
(5)将锥形瓶置于恒温(37℃左右)磁力搅拌器上水浴消化30分钟,随后收集细胞上清,将上清液转移至15ml离心管中,离心弃去上清,用新鲜培养基重悬细胞收集到新的15ml离心管中。
(6)锥形瓶中的组织块继续加入5ml的0.06%的胰酶溶液,置于磁力搅拌器上消化5分钟,置于冰上吸取上清,该步骤重复3次。
(7)含有胰酶溶液的上清中加入3ml左右含10%FBS的DMEM培养基,终止消化,1500rpm常温离心10分钟,细胞沉淀用1-2ml含10%FBS的DMEM培养基重悬收集到新的15ml离心管中。
(8)重复上述(5)(6)(7)步骤,直至皮肤组织消失。
(9)将所有重悬收集的细胞1500rpm离心8分钟。
(10)弃去上清,细胞沉淀用新鲜培养基重悬后,接种到T25培养瓶中,在37℃的细胞培养箱中培养1小时。
(11)差速贴壁后,弃去上清中的细胞,加入新鲜的含10%FBS的DMEM培养基继续培养。由于3-8代细胞活性较好,因而优选8代以内的细胞进行后续实验。
实施例2
大鼠真皮成纤维细胞的鉴定
将原代真皮成纤维细胞接种于激光共聚焦专用小皿中,加入含10%FBS的DMEM置于5%的CO2细胞培养箱中培养24小时。待细胞生长至约70%-80%融合时,弃去培养基,常温PBS缓冲液轻柔冲洗2-3次。吸尽PBS,在培养皿中加入适量的4%的多聚甲醛,至完全覆盖细胞即可,室温固定细胞30分钟。弃去多聚甲醛,常温PBS缓冲液轻柔冲洗2-3次,培养皿置于脱色摇床上振荡,每次5分钟。吸尽PBS液体,加入适量的完全覆盖细胞的1%Triton-X100PBS溶液,室温破膜30分钟。弃去1%Triton-X 100PBS溶液,常温PBS缓冲液轻柔冲洗2-3次,培养皿置于脱色摇床上震荡,每次5分钟。吸尽PBS液体,加入至覆盖完全细胞的量的10%山羊血清,室温封闭1小时。弃去山羊血清,无须常温PBS洗涤,培养皿中加入适量的完全覆盖细胞的Vimentin一抗(山羊血清稀释,1:200),4℃孵育过夜。回收一抗,常温PBS缓冲液轻柔冲洗2-3次,培养皿置于脱色摇床上震荡,每次5分钟。吸尽培养皿中液体,加入适量的荧光二抗(1::500,山羊血清稀释),室温避光孵育30分钟,此后全程需避光操作,防止荧光淬灭。弃去荧光二抗,常温PBS缓冲液轻柔冲洗2-3次,培养皿置于脱色摇床上震荡,每次5分钟。加入1×的hoechst33342(PBS稀释)复染10分钟,常温PBS冲洗3次,每次5分钟。荧光显微镜观察荧光分布特征。结果显示:成纤维细胞的特异性标志物Vimentin存在于绝大多数的细胞上(如图1),表明成功分离了RDFs,可进行下一步实验。
实施例3
UVB照射
将细胞计数后按照一定的数量接种到6孔板或者10mm小培养皿中,待细胞在5%CO2、37℃的细胞培养箱中培养12小时以上,细胞密度大约在30%左右时,取出培养皿或培养板,置于超净工作台中,提前将便携式UVB辐照灯放入紫外杀菌,调整好紫外灯的高度,打开光源,使其输出光强保持稳定,在UVB灯的正下方,使用UVB辐照仪在培养皿的高度处检测其辐射强度,按照照射剂量(mJ)=照射强度(mW)×时间(s)来计算不同剂量的照射时间,随后将培养皿或培养板盖子拿开,置于UVB光源下,照射剂量分别为0mJ,1mJ,2mJ,3mJ,4mJ,5mJ,若不在照射中的孔或皿则用铝箔进行UVB的屏蔽,照射完后,放入细胞培养箱中继续培养,按如上的剂量,一天照射2次,每次隔12小时,照射3天后,换新的完全培养基后细胞继续在培养箱中培养48小时,随后进行衰老表型检测。
1)UVB对RDFs的毒性呈剂量依赖性
不同的剂量照射RDFs后,换新的完全培养基后继续培养24h,避光加入10μlWST-8染色工作液,在37℃中孵育30min,多功能酶标仪检测其在450nm处的吸光度值,以等量的无细胞培养液与WST-8溶液混合的孔作为空白对照,并计算细胞存活率,结果如图2所示,细胞增殖速度随着剂量增加而下降,在照射剂量大于4mJ/cm2时,细胞生长速度受到明显的阻滞,由此我们选定4mJ/cm2作为诱导光老化模型的剂量。
2)UVB照射上调衰老相关蛋白p53,p21的表达水平
p53,p21都是在细胞周期调控过程中发挥重要作用的蛋白,由于衰老本身意味着生长的阻滞,也必然带来细胞周期的停滞,因而在衰老过程中,p53,p21等细胞周期调控蛋白会出现异常的变化,可作为衰老发生的分子标志。为进一步验证UVB诱导的真皮成纤维细胞是否发生了衰老,我们检测了p53以及p21的蛋白含量变化,发现p53、p21蛋白水平均随着UVB照射剂量的增加而剂量依赖性升高,在我们选择的实验剂量4mJ下均有明显的上调(图3)。
3)UVB照射导致RDFs发生光老化
于衰老细胞中β-半乳糖苷酶的活性会显著升高,因而β-半乳糖苷酶的活性成为衰老的经典标志物。我们使用了测定β-半乳糖苷酶活性的试剂盒检测在4mJ剂量下长期反复照射后真皮成纤维细胞的衰老情况。末次照射后细胞换新的完全培养基后继续培养48h,弃去培养基,用PBS洗涤三次,加入100μl的β-半乳糖苷酶染色固定液,室温下固定10min,吸走残留的固定液,加入PBS洗涤,每次5min,共三次,按照试剂盒说明书的具体步骤,避光配置好β-半乳糖苷酶染色工作液,盖上盖子,用保鲜膜包裹好培养板,防止液体蒸发,同时需要用铝箔包裹以避光,在37℃无CO2的环境中孵育过夜,在显微镜下对被染色的阳性细胞进行拍照和计数,可以发现在UVB照射下,视野中阳性细胞数明显增多,表明在此剂量的UVB照射下细胞发生了显著的衰老(图4)。
上述实验结果表明,在4mJ的UVB照射剂量下,通过反复照射可诱导RDFs光老化,表现相应的衰老表型,类似于人皮肤光老化,因而该模型可用于筛选和研究治疗和预防光老化的药物及其作用机制。
实施例4
AST改善UVB导致的RDFs光老化程度
按照实施例3中所述对原代分离培养的RDFs诱导其发生光老化,在末次照射后,更换含有100μg/ml的AST及黄芪其余对照成分APS,ASF,ASI的新鲜完全培养基,于细胞培养箱中继续培养48h后,弃去培养液,进行衰老相关β-Gal染色,如图5所见,黄芪总苷(AST)具有明显的降低UV导致的衰老染色阳性细胞的数量的作用,同时APS以及ASF也具有改善光老化的效果。
实施例5
AST抑制UVB导致的ROS的增加
将传代消化重悬后的RDFs计数后,按4×10^4的数量接种于96孔中,将细胞置于5%CO2、37℃细胞培养箱中贴壁生长12小时后,换新的培养基,且UVB组以及未受辐射组加入等量DMSO,药物干预组分别加入100μg/ml的黄芪黄酮、黄芪甲苷、黄芪多糖、黄芪总苷,后继续培养24小时后。UVB处理组细胞置于UVB光源下接受照射,照射剂量为10mJ,照射强度有UVB辐照仪测定,照射完毕后,按照1:1000用无血清培养液稀释DCFH-DA,使终浓度为10μM/L。去除细胞培养液,加入适当体积稀释好的DCFH-DA。37℃细胞培养箱内孵育20分钟。用无血清细胞培养液洗涤细胞三次,以充分去除未进入细胞内的DCFH-DA。洗涤完毕后,换上常温的PBS使用高内涵成像系统观察拍照,并使用内置的统计软件进行统计分析。如图6所示,AST能显著抑制UVB导致的ROS的产生,且ASI,APS,ASF等均有良好的抗ROS效果。
实施例6
AST逆转UVB导致的线粒体膜电位下降
将传代消化重悬后的RDFs计数后,按4×10^5的数量接种于6孔中,将细胞置于5%CO2、37℃细胞培养箱中贴壁生长12小时,UVB处理组细胞置于UVB光源下接受照射,照射剂量为10mJ,照射强度有UVB辐照仪测定,照射完毕后,换新的培养基,且UVB组以及未受辐射组加入等量DMSO,药物干预组分别加入黄芪黄酮、黄芪甲苷、黄芪多糖、黄芪总苷,后继续培养24小时后进行线粒体膜电位(JC-1)检测,按照说明书进行染色后使用高内涵成像系统观察拍照,并使用内置的统计软件进行统计分析。如图7所示,AST可明显逆转UVB导致的线粒体膜电位的下降,进而保护线粒体,使得线粒体功能有所改善。
综合实施例4到6,我们发现黄芪总苷(AST)在抵抗光老化方面较ASI,ASF,APS具有更好的效果。
实施例7
AST对UVB导致的光老化细胞的保护作用
如图8所示,在原代分离的RDFs中加入100μg/ml的AST后再细胞培养箱中培养24h,随后使用MTT/CCK8等检测方法,检测AST对其增殖的影响;同样的如实施例六中进行相同的UVB照射操作后,加入AST孵育24h,使用CCK8检测细胞活力,可以看到AST对正常细胞并没有促进生长作用,但是却可以抑制UVB导致的RDFs的死亡。通过进一步的实验证明,AST可显著抑制UVB导致的细胞凋亡效应蛋白cleaved caspase 3的蛋白表达水平的升高(如图9),表明AST可显著抑制UVB导致的细胞凋亡,对皮肤成纤维细胞起到保护作用,抵抗皮肤光老化。同时在RNA水平,我们检测了光老化过程中表达升高的衰老相关分泌表型因子的mRNA表达水平,如图10所示,AST可明显抑制促炎因子IL-1β、IL-6的表达,抑制UVB导致的金属蛋白酶MMP1、MMP9、MMP13的上调,以上结果表明,AST可在多方面体现了对UVB导致的光老化细胞的保护作用。
实施例8
AST改善UVB导致的光老化细胞的胶原降解
如实施例3中所述按照实施例3中所述对原代分离培养的RDFs诱导其发生光老化,末次照射后,换新鲜培养基或含有100μg/ml的药物或不同浓度梯度的AST进行给药48小时后,提取细胞总蛋白,并进行BSA蛋白定量,分装成每份30μg蛋白样品后上样进行Westernblot检测胶原蛋白I(Col 1)的表达变化,如图11所示,Col1的蛋白表达水平随着UVB照射剂量的增加而剂量依赖性减少,在我们造模剂量下,呈显著减少趋势(图11-A)。而AST给药48h后,相比于其余的黄芪提取物ASF,APS,ASI,AST可显著逆转UVB导致的Col1的降解(图11-B),进一步使用不同梯度浓度的AST处理48h发现AST随着浓度增加剂量依赖性促进Col1蛋白的积累,浓度达到100μg/ml后具有显着的改善效果(图11-C),而光老化过程中,皮肤的结构和功能的改变与胶原蛋白I的降解有着密切的联系,而AST表现出了较好的促进胶原蛋白I的积累的效果,进一步证实AST抵抗光老化的潜在应用价值。
实施例9
AST逆转UVB导致光老化细胞的自噬活力的下降
衰老皮肤中自噬功能和活性的下降,导致了皮肤新陈代谢以及清除能力的下降。因而与实施例8相似的,我们在蛋白水平检测了AST对自噬的影响。如图12所示,在我们建立UVB诱导的光老化模型中,细胞自噬随着UVB剂量的增加而不断下降(图12-A),说明长期UVB照射可抑制自噬的产生,而这必将影响细胞的正常功能,同样地,我们考察了四种黄芪提取物对UVB导致的自噬抑制的影响时,发现黄芪总苷同样能促进自噬标志分子LC3-II的上调(图12-B),而这种作用呈现一种剂量依赖性(图12-C)。LC3-II型位于自噬体膜上,即可能是由于自噬诱导的增加也可能是后期与溶酶体融合降解通路阻滞导致的增加,因而使用了溶酶体抑制剂CQ阻断自噬体的降解后发现,AST+CQ组较单独的CQ组和AST组能进一步增加LC3-II型的含量。P62是自噬降解过程中重要的底物蛋白,其含量一般代表了自噬活力,可以看出黄芪总苷同样可以下调p62的水平,表明自噬活动的增强(图12-D)。
以上结果从多方面证实了黄芪总苷在黄芪提取物中具有较优的抵抗光老化的作用效果。在UVB诱导的RDFs光老化模型中,黄芪总苷在抵抗UVB致皮肤成纤维细胞的氧化,抗UVB诱导的皮肤光老化,保护线粒体免受光损伤以及抑制UVB诱导的凋亡等均有良好的效果,同时黄芪总苷能显著的逆转UVB导致的光老化细胞的胶原降解,并且可促进光老化细胞的自噬活力,从而起到抵抗光老化,保护皮肤细胞免受损伤的效果。
本发明的上述实施例并不是对本发明的限制,在本领域内的技术人员,在不脱离本发明的前提下,所做的常规的改进与补充,也应视为本发明的保护范围。
Claims (6)
1.黄芪总苷在抗皮肤光老化和光损伤中的应用。
2.根据权利要求1所述的应用,其特征在于:所述黄芪总苷在制备预防和治疗皮肤光老化和光损伤的药物中的应用。
3.根据权利要求2所述的应用,其特征在于:所述药物包括有效量的黄芪总苷及药学上所接受的辅料。
4.根据权利要求2或3所述的应用,其特征在于:所述药物的剂型包括乳剂、膏剂、口服液、颗粒剂、片剂、丸剂、散剂、胶囊剂或滴丸剂。
5.根据权利要求1所述的应用,其特征在于:所述黄芪总苷在制备抗皮肤光老化和光损伤的化妆品中的应用。
6.根据权利要求1所述的应用,其特征在于:所述黄芪总苷在制备抗皮肤光老化和光损伤的保健品中的应用。
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