CN108685808B - 使用兰花愈伤组织萃取物于制备一护肤组合物的用途 - Google Patents
使用兰花愈伤组织萃取物于制备一护肤组合物的用途 Download PDFInfo
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Abstract
一种使用兰花愈伤组织萃取物于制备一组合物的用途,其中该萃取物是一兰花的叶片愈伤组织的极性溶剂萃取物,且该组合物用于护肤,尤其是用于保湿、提升皮肤柔润度、提升皮肤饱满度、提升皮肤弹性、紧致皮肤、减少皮肤纹理、推迟皮肤老化、帮助提升皮肤胶原蛋白含量、修复皮肤组织、预防皮肤病变、及/或促进伤口愈合。
Description
技术领域
本发明关于兰花愈伤组织萃取物于护肤的应用,尤其是关于兰花叶片的愈伤组织的萃取物于护肤的应用。
背景技术
皮肤是人体最大的组织,也是抵抗外物侵入及外在环境刺激的第一道防线,具有保水、保暖及感觉等重要功能。当皮肤受到紫外线、辐射、环境中的微生物及悬浮粒子等的影响时,皆可能使皮肤的含水量降低并加速皮肤细胞老化,导致皮肤发生角质增厚、皮肤干燥脱屑、产生细纹及斑点、皮肤松弛、及蛋白质的分解与流失等现象,甚至会损害皮肤细胞中DNA,而引发细胞毒性、造成皮肤细胞变异而导致皮肤病变。
胶原蛋白及透明质酸皆为增加皮肤含水量的重要物质,其中,胶原蛋白除具有保水功能以外,还可赋予皮肤韧性及弹性;皮肤真皮层中的弹力蛋白则具有支撑皮肤细胞结构的功能。因此,若可提升上述三种于皮肤中的含量,即可达到提升肌肤延展性及弹性、紧致皮肤、以及抚平细纹的效果,并可改善皮肤凹陷及松弛的问题。
近年来,人们越来越重视皮肤保养的问题,且讲究使用天然且安全的原料于皮肤保养上,因此,业界仍持续开发可通过天然且安全的原料的使用以有效保养皮肤的手段。本申请发明人研究发现,兰花愈伤组织萃取物可促进胶原蛋白、弹力蛋白、及透明质酸的合成,且具有帮助修复皮肤细胞中DNA的效果,故可用于保湿、提升皮肤柔润度、提升皮肤饱满度、提升皮肤弹性、紧致皮肤、减少皮肤纹理、推迟皮肤老化、帮助提升皮肤胶原蛋白含量、修复皮肤组织、预防皮肤病变、及/或促进伤口愈合。
发明内容
本发明的一目的,在于提供一种使用兰花愈伤组织萃取物于制备一组合物的用途,其中该萃取物是一兰花叶片的愈伤组织的极性溶剂萃取物,且该组合物用于护肤。较佳地,该兰花是白花蝴蝶兰(Phalaenopsis amabilis)。较佳地,该极性溶剂是水、C1-C4醇类、或前述的组合。较佳地,该组合物是一保养品组合物、食品组合物、或医药组合物。
本发明的另一目的,在于提供一种护肤组合物,其包括一有效量的兰花愈伤组织萃取物,其中该萃取物是一兰花叶片的愈伤组织的极性溶剂萃取物。较佳地,该极性溶剂是水、C1-C4醇类、或前述的组合。较佳地,该组合物是一保养品组合物、食品组合物、或医药组合物。
根据本发明所提供的保养品组合物可用于以下的至少一种:保湿、提升皮肤柔润度、提升皮肤饱满度、提升皮肤弹性、紧致皮肤、减少皮肤纹理、及推迟皮肤老化。较佳地,该保养品组合物以精华液、乳液或化妆水的形式提供。
根据本发明所提供的食品组合物可用于帮助提升皮肤胶原蛋白含量,尤其是帮助提升皮肤第一型胶原蛋白含量。较佳地,该食品组合物以美容饮品的形式提供。
根据本发明所提供的医药组合物可用于以下的至少一种:修复皮肤组织、预防皮肤病变、及促进伤口愈合。该医药组合物可呈一选自以下群组的剂型:经皮投予、口服及皮下注射。
本发明的又一目的,在于提供一种护肤的方法,其包括对一有需要的个体投予一有效量的兰花愈伤组织萃取物。本发明方法可用于以下的至少一种:保湿、提升皮肤柔润度、提升皮肤饱满度、提升皮肤弹性、紧致皮肤、减少皮肤纹理、推迟皮肤老化、帮助提升皮肤胶原蛋白含量、修复皮肤组织、预防皮肤病变、及促进伤口愈合。于根据本发明的方法中,所采用的兰花愈伤组织萃取物可以上述的组合物的形式投予至该有需要的个体。
本发明的详细技术内容及部分具体实施方案,将描述于以下内容中,以供本领域技术人员据以了解本发明的特征。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1至图4显示本发明兰花愈伤组织萃取物于提升皮肤胶原蛋白、弹力蛋白与透明质酸含量、及提升皮肤抗氧化能力的效果,其中,图1显示COL1A1基因及COL1A2基因的相对表现量(倍)(*表示p<0.05、**表示p<0.01),图2显示ELN基因的相对表现量(倍)(**表示p<0.01),图3显示HAS3基因的相对表现量(倍),图4则显示CAT基因及SOD2基因的相对表现量(倍)(***表示p<0.001),且其中,控制组以不含兰花愈伤组织萃取物的培养基培养24小时,“萃取物”组则以含有兰花愈伤组织萃取物的培养基培养24小时;以及
图5至图7显示本发明兰花愈伤组织萃取物于帮助修复皮肤细胞中受损DNA的效果,其中,图5显示MSH2基因、MLH1基因及MSH6基因(前述三种属于碱基错误配对的修复基因)的相对表现量(倍)(**表示p<0.01、***表示p<0.001),图6显示ERCC1基因(属于核苷酸切除的修复基因)的相对表现量(倍)(**表示p<0.01),图7则显示UNG基因、OGG1基因及APE1基因(前述三种属于碱基切除的修复基因)的相对表现量(倍)(**表示p<0.01、***表示p<0.001)。
具体实施方式
以下将描述根据本发明的部分具体实施方案;但是,在不背离本发明精神下,本发明还可以多种不同形式的方案来实践,不应将本发明保护范围解释为限于说明书所陈述的内容。此外,除非文中有另外说明,于本说明书中(尤其是在权利要求书中)所使用的“一”、“该”及类似用语应理解为包括单数及复数形式;所谓“个体”是指人类或非人的哺乳动物。另外,于本发明中,除非特别说明,否则所称“兰花愈伤组织”指兰花的叶片的愈伤组织。
本申请发明人研究发现,兰花植株的叶片在受伤后所产生的愈伤组织具有类似哺乳类全能干细胞的特性,该愈伤组织萃取物可有效提升皮肤细胞的抗氧化能力、帮助皮肤细胞的DNA修复、促进胶原蛋白合成、促进弹力蛋白合成、及促进透明质酸合成。
因此,本发明关于使用兰花愈伤组织萃取物于护肤的应用,此包括:提供一包括兰花愈伤组织萃取物的组合物、使用兰花愈伤组织萃取物于制造一组合物的用途、以及使用该包括兰花愈伤组织萃取物的组合物的方法,该组合物用于护肤,尤其用以保湿、提升皮肤柔润度、提升皮肤饱满度、提升皮肤弹性、紧致皮肤、减少皮肤纹理、推迟皮肤老化、帮助提升皮肤胶原蛋白含量、修复皮肤组织、预防皮肤病变、及/或促进伤口愈合。其中,该兰花愈伤组织尤其是指兰花的叶片的愈伤组织。
根据本发明所采用的兰花愈伤组织萃取物,可通过以极性溶剂萃取兰花的叶片的愈伤组织而提供,其中,该极性溶剂可以为水、醇(例如C1-C4醇类)、或其组合。此外,萃取溶剂的用量并无特殊限制,通常采用可以分散原料的用量。举例言之,可于萃取步骤中采用萃取溶剂:兰花愈伤组织=100-300:1的重量比用量。视需要地,可于超音波震荡操作下进行萃取,以提升萃取效果。较佳地,可以在进行萃取之前,先进行一干燥处理及/或一破碎处理。
于本发明的部分实施方案中,于进行萃取之前,先冷冻干燥兰花愈伤组织。举例言之,可以100:1(水:经冷冻干燥的兰花愈伤组织)的重量比混合水与经冷冻干燥的兰花愈伤组织,并于70℃下对所得到的混合物进行超音波震荡历时45分钟以完成萃取。其中,该兰花愈伤组织可通过包括如下处理的操作而提供:
I.以6%次氯酸钠溶液冲洗兰花植株,其后以灭菌水冲洗兰花植株,视需要地,可重复前述冲洗操作;
II.在兰花植株的叶片的表面上切出伤口,以诱导产生愈伤组织(历时1至3个月);
III.在25℃、50%至60%湿度下,以MS培养基(Murashige and Skoog BasalMedium)培养步骤II所得的愈伤组织(历时1至1.5个月)。
根据本发明所采用的兰花愈伤组织萃取物,可以萃取所得的萃取原液直接使用,或视需要对该萃取原液进行例如过滤、灭菌、浓缩、稀释等一或多个步骤,以提升萃取液的使用便利性。举例言之,通过例如浓缩干燥、喷雾干燥、或冷冻干燥等操作,可由萃取液得到方便携带或方便储存的浓缩液或粉末。
根据本发明所提供的组合物是一保养品组合物、食品组合物、或医药组合物。其中,根据本发明所提供的保养品组合物可用于以下的至少一种:保湿、提升皮肤柔润度、提升皮肤饱满度、提升皮肤弹性、紧致皮肤、减少皮肤纹理、及推迟皮肤老化。根据本发明所提供的保养品组合物可呈任何适宜的形式,并无特殊的限制。举例言之,该保养品组合物可呈供直接外用的乳液、乳霜、凝胶(例如水凝胶)、或溶液(例如精华液、化妆水)等形式,但不以此为限。
根据本发明所提供的食品组合物用于帮助提升皮肤胶原蛋白含量,尤其是帮助提升皮肤第一型胶原蛋白含量。根据本发明所提供的食品组合物可呈任何适宜的形式,并无特殊的限制。举例言之,可将本发明的食品组合物制备成可供食用或饮用的形式,例如健康食品、美容饮品等,但不以此为限。
视需要地,可于根据本发明所提供的保养品组合物、食品组合物、或医药组合物中另外含有适宜用量的添加剂,例如可提高该食品组合物或医药组合物于服用时的口适感及视觉感受的调味剂、调色剂、着色剂等,以及可改善该保养品组合物、食品组合物、或医药组合物的稳定性及储存性的缓冲剂、保存剂、防腐剂、抗菌剂、抗真菌剂等。
根据本发明所提供的医药组合物可呈任何适宜的型式,并无特殊限制,端视所欲采取的投药方式而呈对应的适宜剂型。举例言之,但不以此为限,该医药组合物可以口服或非经口服(例如:经皮投予、皮下注射)的投药方式施用至有需要的个体上。其中,视使用形式及用途而定,可选用适宜的载剂以提供该医药组合物,其中,该载剂包括赋形剂、稀释剂、辅助剂、安定剂、吸收延迟剂、崩散剂、增溶剂、乳化剂、抗氧化剂、黏合剂、结合剂、增黏剂、分散剂、悬浮化剂、润滑剂、吸湿剂等。
以适于口服的剂型为例,于根据本发明所提供的医药组合物中可含有任何不会不利影响活性成分(即,兰花愈伤组织萃取物)的所欲效益的医药上可接受的载剂,例如:水、食盐水、葡萄糖(dextrose)、甘油、乙醇或其类似物、纤维素、淀粉、糖膨润土(sugarbentonite)、及前述的组合。可利用任何适宜的方法,将该医药组合物以适于口服投药的剂型提供,例如:锭剂(例如糖衣锭)、丸剂、胶囊剂、颗粒剂、散剂、流浸膏剂、溶液剂、糖浆剂、悬液剂、酊剂等。
以适于经皮投予的剂型为例,根据本发明所提供的医药组合物可呈供直接外用的贴布、乳液、乳霜、凝胶(例如水凝胶)、膏状物(例如分散膏、软膏)、喷雾剂、或溶液(例如悬浮液)等形式,但不以此为限。
至于适于皮下注射的针剂或点滴剂型,则可于该医药组合物中含有一或多种例如等张溶液、盐类缓冲液(如磷酸盐缓冲液或柠檬酸盐缓冲液)、增溶剂、乳化剂、5%糖溶液、以及其他载剂等成分。或者,将该医药组合物制备成一注射前固体,以可溶于其他溶液或悬浮液中的剂型、或可乳化的剂型提供该注射前固体,并于投予至有需要的个体之前,将该注射前固体溶于其他溶液或悬浮液中或将其乳化,提供所需要的注射剂。
根据本发明的应用所提供的组合物可以一日一次、一日多次、或数日一次等不同频率施用,端视投予个体的需求、年龄、体重、健康状况、及施用目的而异。
本发明亦提供一种护肤的方法,其包括对一有需要的个体投予一有效量的兰花愈伤组织萃取物。于根据本发明的方法中,所采用的兰花愈伤组织萃取物可以一组合物的形式投予至该有需要的个体。有关该组合物的投予方案、投予途径、投予形式、施用频率、以及相关的应用,均如上述的说明。
现以下列实施例进一步例示说明本发明。其中这些实施例仅提供作为说明,而非用以限制本发明的保护范围。本发明保护范围如权利要求书内容所示。
实施例
[制备实施例]
A.兰花愈伤组织萃取物的制备
对购自中国台湾地区国光花市232兰园的白花蝴蝶兰(Phalaenopsis amabilis)植株施以如下处理,以提供兰花愈伤组织:
I.以6%次氯酸钠溶液冲洗兰花植株,其后以灭菌水冲洗兰花植株,视需要地,可重复前述冲洗操作;
II.在兰花植株的叶片的表面上切出伤口,以诱导产生愈伤组织(历时1至3个月);
III.在25℃、50%至60%湿度下,以MS培养基(Murashige and Skoog BasalMedium;购自SIGMA公司;产品编号:M5519)培养步骤II所得的愈伤组织(历时1至1.5个月)。
以下列步骤处理上述兰花愈伤组织,以制备兰花愈伤组织萃取物:
(1)兰花愈伤组织置于-22℃下进行冷冻干燥,历时12小时,接着粉碎前述经干燥的兰花愈伤组织,以提供兰花愈伤组织粉末;
(2)将水与步骤(1)的兰花愈伤组织粉末以100:1(水:兰花愈伤组织粉末)的重量比混合,在70℃下,对所获得的混合物进行超音波震荡,历时45分钟;
(3)以滤膜过滤步骤(2)所得的混合物,以提供一滤液;
(4)加热由步骤(3)所获得的滤液至95℃,并维持20分钟,以进行灭菌;
(5)待步骤(4)的滤液冷却后,将其分装并冷藏保存以供后续实验使用;以及
(6)对步骤(5)的滤液进行冷冻干燥,以提供一干燥物(即,本发明的兰花愈伤组织萃取物)。
B.细胞处理
于MEM培养基(Minimum essential medium,购自Gibco公司;产品编号:61100-061)中培养人类皮肤纤维母细胞(CCD-966SK;购自ATCC)历时24小时,接着,将细胞分成两组,并进行以下处理:
1.控制组:将细胞置于MEM培养基中培养24小时(即,将细胞培养于不含兰花愈伤组织萃取物的培养液中)。
2.“萃取物”组:将细胞置于含有1重量%获得自[制备实施例A]的兰花愈伤组织萃取物(以干重计)的MEM培养基中培养24小时。
C.cDNA的制备
以RNA萃取套组(RNA extraction kit,购自GENEmark公司)对经[制备实施例B]处理的各组细胞进行RNA萃取,再以反转录酶将各组RNA反转录为cDNA,供后续的基因表现量检测使用。
实施例1:兰花愈伤组织萃取物于提升皮肤胶原蛋白、弹力蛋白与透明质酸含量的效果
研究已知,皮肤细胞的COL1A1、COL1A2、ELN、HAS3等基因的表现量提升有助于促进胶原蛋白、弹力蛋白、及透明质酸的合成,可参见例如:Modulation of collagensynthesis and its gene expression in human skin fibroblasts by tocotrienol-rich fraction.Arch Med Sci.7,5:889-895(2011)、The complexity if elastic fiberbiogenesis in the skin-a perspective to the clinical heterogeneity of cutislaxa.Exp Dermatol.22(2):88-92(2013)、Hyaluronan Synthase 3Regulates HyaluronanSynthesis in Cultured Human Keratinocytes.The Journal of InvestigativeDermatology.118:43-48(2002)、及An overview of the Beneficial Effects ofHydrolysed Collagen as a Nutraceutical on Skin Properties:ScientificBackground and Clinical Studies.The Open Nutraceuticals Journal.8:29-42(2015),这些文献的全文并于此处以供参考。为确认本发明兰花愈伤组织萃取物可以提升皮肤的胶原蛋白的含量、弹力蛋白含量、及透明质酸含量,使用ABI Step One Plus仪器及KAPA SYBR FAST qPCR套组对[制备实施例C]所提供的各组cDNA进行qPCR(quantitativepolymerase chain reaction),以检测各组细胞的COL1A1、COL1A2、ELN、HAS3等基因的表现量。接着,以学生t检验(Student t-test)进行统计分析,并以控制组的结果为基准(即,将控制组的基因表现设定为1倍),计算“萃取物”组的基因相对表现量。结果示于图1至3。
由图1至3可知,相较于控制组,萃取物组的COL1A1、COL1A2、ELN、及HAS3基因表现量皆明显提升。前述结果显示,本发明兰花愈伤组织萃取物确实可以提升COL1A1、COL1A2、ELN、HAS3等基因的表现量,故可有效促进胶原蛋白、弹力蛋白及透明质酸的合成与分泌,从而达到保湿、提升皮肤柔润度、提升皮肤饱满度、提升皮肤弹性、紧致皮肤、减少皮肤纹理、推迟皮肤老化、帮助提升皮肤胶原蛋白含量、及/或促进伤口愈合的效果。
实施例2:兰花愈伤组织萃取物于提升皮肤抗氧化能力的效果
研究已知,皮肤细胞的CAT及SOD2基因的表现量提升有助于皮肤细胞抗氧化能力的提升,可参见例如:Anti-oxidant gene expression imbalance,aging and Downsyndrome.Life Sciences.76:1407-1426(2005),该文献的全文并于此处以供参考。为确认本发明兰花愈伤组织萃取物的抗氧化力,使用ABI Step One Plus仪器及KAPA SYBR FASTqPCR套组对[制备实施例C]所提供的各组cDNA进行qPCR,以检测各组细胞的CAT及SOD2等抗氧化基因的表现量。接着,以学生t检验(Student t-test)进行统计分析,并以控制组的结果为基准(即,将控制组的基因表现设定为1倍),计算“萃取物”组的基因相对表现量。结果示于图4。
由图4可知,相较于控制组,萃取物组的CAT及SOD2基因表现量皆明显提升。前述结果显示,本发明兰花愈伤组织萃取物确实可以提升抗氧化基因的表现量,故可有效提升皮肤细胞抵抗过氧化物质伤害的能力,从而达到推迟皮肤老化、修复皮肤组织、及/或预防皮肤病变的效果。
实施例3:兰花愈伤组织萃取物于帮助修复皮肤细胞中受损DNA的效果
研究已知,生物体中的碱基错误配对修复(mismatch repair,MMR)、核苷酸切除修复(nucleotide excision repair,NER)、及碱基切除修复(base excision repair,BER)等DNA修复机制可避免因DNA突变或断裂所造成的伤害,若可提升皮肤细胞的MMR相关基因、NER相关基因、及BER相关基因的表现量,则可有助于皮肤细胞中受损DNA的修复,可参见例如:DNA repair mechanisms in dividing and non-dividing cells.DNA repair(Amst).12(8):620-636(2013),该文献的全文并于此处以供参考。为确认本发明兰花愈伤组织萃取物可以帮助修复皮肤细胞中受损的DNA,使用ABI Step One Plus仪器及KAPA SYBR FASTqPCR套组对[制备实施例C]所提供的各组cDNA进行qPCR,以检测各组细胞中MMR相关基因(包括MSH2、MLH1及MSH6)、NER相关基因(包括ERCC1)、及BER相关基因(包括UNG、OGG1及APE1)的表现量。接着,以学生t检验(Student t-test)进行统计分析,并以控制组的结果为基准(即,将控制组的基因表现设定为1倍),计算“萃取物”组的基因相对表现量。结果分别示于图5至7。
由图5至7可知,相较于控制组,萃取物组的MMR相关基因(包括MSH2、MLH1及MSH6)、NER相关基因(包括ERCC1)、及BER相关基因(包括UNG、OGG1及APE1)的表现量皆明显提升。前述结果显示,本发明兰花愈伤组织萃取物确实可以提升DNA修复相关基因的表现量,故可有效修复皮肤细胞中受损DNA,从而达到推迟皮肤老化、修复皮肤组织、及/或预防皮肤病变的效果。
如上述实施例所示,本发明兰花愈伤组织萃取物确实可促进皮肤中胶原蛋白、弹力蛋白、与透明质酸的合成、提升皮肤细胞抗氧化能力、及帮助修复皮肤细胞中受损DNA,故可用于保湿、提升皮肤柔润度、提升皮肤饱满度、提升皮肤弹性、紧致皮肤、减少皮肤纹理、推迟皮肤老化、帮助提升皮肤胶原蛋白含量、修复皮肤组织、预防皮肤病变、及/或促进伤口愈合。
Claims (8)
1.一种使用白花蝴蝶兰(Phalaenopsis amabilis)愈伤组织萃取物于制备一组合物的用途,其特征在于:其中该萃取物是白花蝴蝶兰的叶片愈伤组织的极性溶剂萃取物,且该组合物通过所述萃取物促进皮肤中胶原蛋白、弹力蛋白与透明质酸的合成、提升皮肤细胞抗氧化能力、及/或帮助修复皮肤细胞中受损DNA而用于护肤,且该极性溶剂选自以下群组:水、C1-C4醇类、及前述的组合。
2.如权利要求1所述的用途,其特征在于:其中该组合物是一保养品组合物、食品组合物、或医药组合物。
3.如权利要求2所述的用途,其特征在于:其中该组合物是保养品组合物,用于以下的至少一种:保湿、提升皮肤柔润度、提升皮肤饱满度、提升皮肤弹性、紧致皮肤、减少皮肤纹理、及推迟皮肤老化。
4.如权利要求3所述的用途,其特征在于:其中该保养品组合物以精华液、乳液或化妆水的形式提供。
5.如权利要求2所述的用途,其特征在于:其中该组合物是食品组合物,用于帮助提升皮肤胶原蛋白含量。
6.如权利要求5所述的用途,其特征在于:其中该胶原蛋白是第一型胶原蛋白。
7.如权利要求5所述的用途,其特征在于:其中该食品组合物以美容饮品的形式提供。
8.如权利要求2所述的用途,其特征在于:其中该组合物是医药组合物,用于以下的至少一种:修复皮肤组织、预防皮肤病变、及促进伤口愈合。
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