竹子外表皮中护肤功能因子的研究
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摘要
皮肤是维护人体生命和健康的重要部分,直接与外界接触,是机体的第一道屏障,极易受到环境因素的影响而导致损伤。同时,伴随着“回归自然”、“崇尚天然”的大趋势,植物来源的活性产物作为在功能性化妆品中的应用已成为世界各国化妆品行业追逐的热点。竹子外表皮中医上又称竹茹,《中药辞海》中记载,竹茹为禾本科刚竹属、箣竹属和牡竹属中一些竹种的茎秆所刮下的外皮层或其次一层,是临床常用中药之一,又是卫生部批准的“可用于保健食品的物品”。但长期以来中医药对竹茹成分和药理的研究十分贫乏。本课题组于2002年间发明的、采用超临界CO2流体萃取技术从刚竹属竹子的茎秆所刮下的外皮层中提取的“竹子三萜皂甙元”,是一种由木栓酮、羽扇豆烯酮及其同系物为主的五环三萜类化合物组成的标准化制剂(EZR2002)。由于超临界C02流体萃取得到的主要是低极性部位,得率偏低,并伴随大量萃余物的产出。传统中医炮制竹茹的方式为酒灸、炒干后水煎,得到的药用成分主要是其强极性部位,故竹茹的超临界CO2流体萃余物应该具有良好的研究和开发价值。本课题正是基于这种背景提出的,以竹茹超临界萃余物为原料,进一步提取、分离得到了竹子精华素(一种代码为EZR2005的标准化制剂)。本文研究探讨了EZR2005的化学组成、生物活性和安全性,现将主要的研究结果归纳如下：
(1)EZR2005制剂的总酚含量为42.1%(其中含总黄酮15.9%)、总糖29.0%(其中多糖含量9.4%)、三萜29.0%、蛋白质9.40%、灰分9.1%和水分5.0%。钾元素极为丰富(2.6%),是一种富钾制剂；在微量元素中,锰的含量最高,达808 mg/kg,其次是锌和铁,分别为81.6和80.7 mg/kg,同时还含有一定量的铜和硒。
(2)采用制备型HPLC从EZR2005乙醚相中得到两个特征性化合物单体,经UV、MS、NMR等手段鉴定为对香豆酸和苜蓿素。借鉴竹叶提取物质量研究的成果,对EZR200s及其不同分级相中的荭草苷、异荭草苷、牡荆苷、异牡荆苷、绿原酸、咖啡酸、对香豆酸和阿魏酸进行分析,确认了EZR2005中同样含有这八种特征性成分。同时,发现了EZR2005中腺苷的存在,并采用分析型HPLC对EZR2005及其分级部位中的腺苷进行定量分析。
(3)综合考虑腺苷提取率及其在提取物中含量的高低,确定超声波辅助提取竹子外表皮中腺苷的优化工艺为：水提(乙醇浓度0%)、固液比15：1、提取温度50℃、超声处理时间45 min。在该工艺条件下,竹子外表皮中腺苷的提取率为96.97±0.25%,提取物中腺苷含量为1.42士0.11 mg/g。
(4)EZR2005及其四个分级部位(乙醚相、乙酸乙酯相、正丁醇相和水分级相)中,四种碳苷黄酮(异荭草苷、荭草苷、牡荆苷和异牡荆苷)和四种酚酸(对香豆酸、咖啡酸、对香豆酸和绿原酸)在ABTS法、DPPH法、FRAP法和总抗氧化能力法等四种不同的抗氧化体系中均表现出优良的抗自由基活性和还原能力。
(5)EZR2005的美容保健功能研究表明,其具有显著的保湿效果,略逊于透明质酸；并能有效地保持皮肤弹性,作用优于透明质酸；对黑色素B16细胞生成黑色素的抑制作用明显,美白效果接近熊果苷。EZR2005及其不同分级部位和对香豆酸单体对体外无细胞系蘑菇酪氨酸酶均表现出抑制作用,并呈剂量依赖关系,EZR2005对蘑菇酪氨酸酶的抑制作用属于混合型抑制。
(6)EZR2005的安全性毒理学评价试验表明,对动物皮肤和眼粘膜无刺激作用；小鼠的经口MTD均大于20 g/kg体重,属实际无毒类；小鼠微核试验、小鼠精子畸形试验、Ames试验结果均呈阴性,未见致突变作用；大鼠30 d喂养试验各项指标均未见明显毒性反应,对高剂量组经解剖及组织学观察也未见异常病理改变,对大鼠体重、进食量、食品利用率、血常规和血生化均无明显影响。得出其最大无作用剂量为10 g/kg体重。表明EZR2005具有良好的外用和食用安全性。
研究表明,来自于竹子外表皮的有效成分“EZR2005”是一种安全、高效的皮肤美容因子,具有抗自由基、抗氧化、保湿、美白和延缓皮肤衰老的功能,可广泛应用于日用化妆品领域。
Skin is an important barrier to keep human body from environmental injury. Along with the global tendency of "Back to Nature", more and more attentions have focused on the R&D of "cosmeceuticals",i e., cosmetic functional factors with multiple bioactivities such as anti-free radical, anti-oxidation, anti-irradiation and skin- whiting; moisture-holding; wrinkle-shrinking. Bamboo bark, also called Zhuru, usually refers to the out-layer and next-layer of Bambusa tuldoides Munro, Sinocalamus beecheyana var. pubescens P.F. Li or Phyllostachys nigra (Lodd.) Munrom Var. henonis (Mitf.) Stapf ex Rendle, and all of them belong to the Bambusoideae of Gramineae family. Zhuru is not only a kind of traditional Chinese medicine, but also natural goods that can be used as the raw material of health food approved by the Health Ministry of China. However, the research and investigation on the effective compounds and their pharmacologic activities about Zhuru is rather seldom. The unique works have been conducted by our group including a triterpenoids-rich standard preparation made of Zhuru using by SFE (supercritical CO2 fluid extraction) technology and first created in 2002, so named as EZR2002;, and some publications on its functionality and safety. As SFE method mainly extracted the Zhuru's low-polar position at lower yield, and followed by a staple side-product residue. As we known, the traditional processing course of Zhuru is as follows, dipped in wine, fried drying, and then water decoction. The main medicinal ingredients should be due to polar position in Zhuru. It could be expected that there are abundant effective compounds still remain in the side-product. Therefore, we got another polyphenols-rich standard preparation from the SFE residue, named as EZR2005. This present study is figured out to research the chemistry, functionality and safety of EZR2005 at the view of novel cosmeceuticals. Critical investigating results were summarized as follows:
a) EZR2005 consists of 42.1% total polyphenols (including 15.9% total flavonoids), 29.0% total sugar 9.0%(including 9.4% polysaccharides)、29.0% triterpenoids, 9.40% protein,9.1% ash and 5.0% moisture; EZR2005 is a potassium-rich preparation (2.6%); The highest content of microelements was manganese, up to 808 mg/kg, followed by zinc and iron, respectively,81.6 and 80.7 mg/kg. It also contains a certain amount of copper and selenium.
b) The two compounds were isolated and purified by preparative HPLC and were quantified by HPLC and identified by UV, MS, NMR. The two compounds were p-coumaric acid and tricin. Quality of bamboo leaf extract reference results, the contents of homoorientin, orientin, isovitexin, vitexin, chlorogenic acid,p-coumaric acid, caffeic acid and ferulic acid were quantified by HPLC, in different polar fractions, respectively. At the same time, adenosine has been discovered and quantified in EZR2005, using analytical HPLC.
c) Considering the extraction rate and content of adenosine, The optimal extraction condition were confirmed as follows:water extraction (the concentration of ethanol is 0%), ration of solvent to material is 15:1, the extract temperature is 50℃the extract time is 45min. On best experimental conditions, the extraction of adenosine was 96.97±0.25%, the content of adenosine 1.42±0.11 mg/g.
d) The antioxidant activities of EZR2005, DE, EA, BU, WT, homoorientin, orientin, isovitexin, vitexin, chlorogenic acid, p-coumaric acid, caffeic acid and ferulic acid were in different antioxidant system has shown excellent anti-free radical activity and reducing power. It has been proved that EZR2005 is a kind of powerful bio-antioxidant.
e) EZR2005 skin care functional tests showed that it had significant moisturizing effect, which slightly lower than the positive control, hyaluronic acid, and the significant effect of maintaining flexibility, higher than hyaluronic acid. Melanin was significantly inhibited by EZR2005 on B16 melanoma cells, which was close to the positive control arbutin. EZR2005, DE, EA, BU, WT, and p- coumaric acid in vitro mushroom tyrosinase inhibition were shown and was concentration- dependent manner; The study also shows that EZR2005 on mushrooms inhibitory type of mixed-type inhibition.
f) Toxicological evaluation of the EZR2005 showed that the animals skin and eye irritation test results were negative; oral MTD of mice were greater than 20 g/kg body weight, which can be regarded as virtually non-toxic; Mouse micronucleus test, Mouse sperm abnormality test, Ames test results were negative, and no mutagenic effects; 30 d feeding study in rats showed no obvious toxicity of the indicator reaction, the anatomy of the high dose group, and also no abnormal histological pathology changes in rat body weight, food intake, food utilization, blood and blood biochemistry were not affected. The experiment showed no effect on the maximum dose of 10 g/kg body weight. That EZR2005 has a good food security.
Research showed that bamboo essence, EZR2005, is a novel cosmeceuticals with multiple bioactivities such as anti-free radical, anti-oxidation, anti-irradiation and skin- whiting; moisture-holding; wrinkle-shrinking. It could be widely used as skin-care factor in cosmetics.
(1)EZR2005制剂的总酚含量为42.1%(其中含总黄酮15.9%)、总糖29.0%(其中多糖含量9.4%)、三萜29.0%、蛋白质9.40%、灰分9.1%和水分5.0%。钾元素极为丰富(2.6%),是一种富钾制剂；在微量元素中,锰的含量最高,达808 mg/kg,其次是锌和铁,分别为81.6和80.7 mg/kg,同时还含有一定量的铜和硒。
(2)采用制备型HPLC从EZR2005乙醚相中得到两个特征性化合物单体,经UV、MS、NMR等手段鉴定为对香豆酸和苜蓿素。借鉴竹叶提取物质量研究的成果,对EZR200s及其不同分级相中的荭草苷、异荭草苷、牡荆苷、异牡荆苷、绿原酸、咖啡酸、对香豆酸和阿魏酸进行分析,确认了EZR2005中同样含有这八种特征性成分。同时,发现了EZR2005中腺苷的存在,并采用分析型HPLC对EZR2005及其分级部位中的腺苷进行定量分析。
(3)综合考虑腺苷提取率及其在提取物中含量的高低,确定超声波辅助提取竹子外表皮中腺苷的优化工艺为：水提(乙醇浓度0%)、固液比15：1、提取温度50℃、超声处理时间45 min。在该工艺条件下,竹子外表皮中腺苷的提取率为96.97±0.25%,提取物中腺苷含量为1.42士0.11 mg/g。
(4)EZR2005及其四个分级部位(乙醚相、乙酸乙酯相、正丁醇相和水分级相)中,四种碳苷黄酮(异荭草苷、荭草苷、牡荆苷和异牡荆苷)和四种酚酸(对香豆酸、咖啡酸、对香豆酸和绿原酸)在ABTS法、DPPH法、FRAP法和总抗氧化能力法等四种不同的抗氧化体系中均表现出优良的抗自由基活性和还原能力。
(5)EZR2005的美容保健功能研究表明,其具有显著的保湿效果,略逊于透明质酸；并能有效地保持皮肤弹性,作用优于透明质酸；对黑色素B16细胞生成黑色素的抑制作用明显,美白效果接近熊果苷。EZR2005及其不同分级部位和对香豆酸单体对体外无细胞系蘑菇酪氨酸酶均表现出抑制作用,并呈剂量依赖关系,EZR2005对蘑菇酪氨酸酶的抑制作用属于混合型抑制。
(6)EZR2005的安全性毒理学评价试验表明,对动物皮肤和眼粘膜无刺激作用；小鼠的经口MTD均大于20 g/kg体重,属实际无毒类；小鼠微核试验、小鼠精子畸形试验、Ames试验结果均呈阴性,未见致突变作用；大鼠30 d喂养试验各项指标均未见明显毒性反应,对高剂量组经解剖及组织学观察也未见异常病理改变,对大鼠体重、进食量、食品利用率、血常规和血生化均无明显影响。得出其最大无作用剂量为10 g/kg体重。表明EZR2005具有良好的外用和食用安全性。
研究表明,来自于竹子外表皮的有效成分“EZR2005”是一种安全、高效的皮肤美容因子,具有抗自由基、抗氧化、保湿、美白和延缓皮肤衰老的功能,可广泛应用于日用化妆品领域。
Skin is an important barrier to keep human body from environmental injury. Along with the global tendency of "Back to Nature", more and more attentions have focused on the R&D of "cosmeceuticals",i e., cosmetic functional factors with multiple bioactivities such as anti-free radical, anti-oxidation, anti-irradiation and skin- whiting; moisture-holding; wrinkle-shrinking. Bamboo bark, also called Zhuru, usually refers to the out-layer and next-layer of Bambusa tuldoides Munro, Sinocalamus beecheyana var. pubescens P.F. Li or Phyllostachys nigra (Lodd.) Munrom Var. henonis (Mitf.) Stapf ex Rendle, and all of them belong to the Bambusoideae of Gramineae family. Zhuru is not only a kind of traditional Chinese medicine, but also natural goods that can be used as the raw material of health food approved by the Health Ministry of China. However, the research and investigation on the effective compounds and their pharmacologic activities about Zhuru is rather seldom. The unique works have been conducted by our group including a triterpenoids-rich standard preparation made of Zhuru using by SFE (supercritical CO2 fluid extraction) technology and first created in 2002, so named as EZR2002;, and some publications on its functionality and safety. As SFE method mainly extracted the Zhuru's low-polar position at lower yield, and followed by a staple side-product residue. As we known, the traditional processing course of Zhuru is as follows, dipped in wine, fried drying, and then water decoction. The main medicinal ingredients should be due to polar position in Zhuru. It could be expected that there are abundant effective compounds still remain in the side-product. Therefore, we got another polyphenols-rich standard preparation from the SFE residue, named as EZR2005. This present study is figured out to research the chemistry, functionality and safety of EZR2005 at the view of novel cosmeceuticals. Critical investigating results were summarized as follows:
a) EZR2005 consists of 42.1% total polyphenols (including 15.9% total flavonoids), 29.0% total sugar 9.0%(including 9.4% polysaccharides)、29.0% triterpenoids, 9.40% protein,9.1% ash and 5.0% moisture; EZR2005 is a potassium-rich preparation (2.6%); The highest content of microelements was manganese, up to 808 mg/kg, followed by zinc and iron, respectively,81.6 and 80.7 mg/kg. It also contains a certain amount of copper and selenium.
b) The two compounds were isolated and purified by preparative HPLC and were quantified by HPLC and identified by UV, MS, NMR. The two compounds were p-coumaric acid and tricin. Quality of bamboo leaf extract reference results, the contents of homoorientin, orientin, isovitexin, vitexin, chlorogenic acid,p-coumaric acid, caffeic acid and ferulic acid were quantified by HPLC, in different polar fractions, respectively. At the same time, adenosine has been discovered and quantified in EZR2005, using analytical HPLC.
c) Considering the extraction rate and content of adenosine, The optimal extraction condition were confirmed as follows:water extraction (the concentration of ethanol is 0%), ration of solvent to material is 15:1, the extract temperature is 50℃the extract time is 45min. On best experimental conditions, the extraction of adenosine was 96.97±0.25%, the content of adenosine 1.42±0.11 mg/g.
d) The antioxidant activities of EZR2005, DE, EA, BU, WT, homoorientin, orientin, isovitexin, vitexin, chlorogenic acid, p-coumaric acid, caffeic acid and ferulic acid were in different antioxidant system has shown excellent anti-free radical activity and reducing power. It has been proved that EZR2005 is a kind of powerful bio-antioxidant.
e) EZR2005 skin care functional tests showed that it had significant moisturizing effect, which slightly lower than the positive control, hyaluronic acid, and the significant effect of maintaining flexibility, higher than hyaluronic acid. Melanin was significantly inhibited by EZR2005 on B16 melanoma cells, which was close to the positive control arbutin. EZR2005, DE, EA, BU, WT, and p- coumaric acid in vitro mushroom tyrosinase inhibition were shown and was concentration- dependent manner; The study also shows that EZR2005 on mushrooms inhibitory type of mixed-type inhibition.
f) Toxicological evaluation of the EZR2005 showed that the animals skin and eye irritation test results were negative; oral MTD of mice were greater than 20 g/kg body weight, which can be regarded as virtually non-toxic; Mouse micronucleus test, Mouse sperm abnormality test, Ames test results were negative, and no mutagenic effects; 30 d feeding study in rats showed no obvious toxicity of the indicator reaction, the anatomy of the high dose group, and also no abnormal histological pathology changes in rat body weight, food intake, food utilization, blood and blood biochemistry were not affected. The experiment showed no effect on the maximum dose of 10 g/kg body weight. That EZR2005 has a good food security.
Research showed that bamboo essence, EZR2005, is a novel cosmeceuticals with multiple bioactivities such as anti-free radical, anti-oxidation, anti-irradiation and skin- whiting; moisture-holding; wrinkle-shrinking. It could be widely used as skin-care factor in cosmetics.
引文
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