酪氨酸酶抑制剂和激活剂的研究
摘要
本论文主要研究了酪氨酸酶激活剂补骨脂素及其复配物对酪氨酸酶的协同激活性能,以及酪氨酸酶抑制剂芒柄花苷的合成、优化及其对酪氨酸酶抑制性能和抗氧化性能测试。
酪氨酸酶是产生黑色素的主要限速酶,通过激活或抑制酪氨酸酶的活性可促进毛发和皮肤黑素的生成或抑制,达到乌发或皮肤增白的目的。在化妆品原料的研究过程中,酪氨酸酶作为性能测试试剂起着重要的作用。
中药补骨脂细粉经75 %的乙醇提取得到补骨脂素,其HPLC纯度为99 %。将补骨脂素配制成不同的浓度,用紫外-可见光分光光度法进行酪氨酸酶激活性能的检测,当浓度为0.03 mg/mL时,最高激活率为44.9 %。选择对酪氨酸酶都有一定激活作用的不同添加助剂Cu~(2+)、Fe~(2+)、Mn~(2+)、Zn~(2+)和半胱氨酸、咖啡因、人参皂苷等与补骨脂素复配,结果显示Cu~(2+)、Mn~(2+)、Zn~(2+)和半胱氨酸与补骨脂素都有不同程度协同激活效应。
采用单因素法将Cu~(2+)、Mn~(2+)、Zn~(2+)和半胱氨酸同时与补骨脂素复配,测定其对酪氨酸酶的激活率,当Cu~(2+)浓度为0.1 mg/mL、Mn~(2+)浓度为0.05 mg/mL、Zn~(2+)浓度为0.1 mg/mL、半胱氨酸浓度为0.05 mg/mL,加入量均为0.3 mL时,对酪氨酸酶激活率最高为104.3 %。因此,补骨脂素及其复配物可以作为促进黑色素生成的物质在化妆品或药物中使用。
采用脱氧安息香路线两步法合成芒柄花素,以芒柄花素和四乙酰溴代葡萄糖为原料,采用固-液和液-液两种不同的相转移催化合成芒柄花苷。通过对比产率等因素,最终确定采用液-液相转移催化体系。并采用正交试验进行优化,收率可达64.8 %。分离得到的中间体四乙酰芒柄花苷及产物芒柄花苷的HPLC纯度分别为98.27 %和98.36 %。
采用紫外-可见光分光光度法测定芒柄花苷对酪氨酸酶活性的抑制性能。结果表明:0.05 mg/mL时,芒柄花苷对酪氨酸酶最大抑制率为51.7 %。同时采用紫外-可见光分光光度法,测定了芒柄花苷对超氧自由基的抑制率。结果表明:在1 mg/mL时,芒柄花苷对超氧自由基的最大抑制率为45.2 %。因此芒柄花苷可以作为美白剂和抗衰老成分在化妆品中使用。
The synergistic activation on tyrosinase of psoralen cooperated with substrates, and the synthesis and optimization of ononin, as well as inhibition activity on tyrosinase and antioxidant activity were primarily studied in this thesis .
Tyrosinase is a major enzyme, which controls the speed of melanin biosynthesis, the generating of hair and skin melanin, which is the key factor to black hair and whiten skin, can be promoted or prohibited by activating or inhibiting the tyrosinase. Tyrosinase plays a vital role in selecting cosmetic materials as a reagent.
Psoralen was extacted by 75% ethanol water solution from Chinese traditional medicine psoralen . UV-Vis spectrophotometry method was selected to study the tyrosinase avtivity of psoralen which was in different concentration. The highest activation can be 44.9% when the concentration is 0.03 mg/mL . Different additives which can activate the tyrosinae , such as Cu~(2+)、Fe~(2+)、Mn~(2+)、Zn~(2+)and cysteine、affein、panaxcoside were selected to cooperate with psoralen to study the synergistic activation on tyrosinase . It indicated that Cu~(2+)、Mn~(2+)、Zn~(2+)and cysteine were the right substances.
Single factor method was selected to study the synergistic activation on tyrosinase of psoralen cooperated with all the right substances together, the highest activation can be 104.3 % , when the the concentration of Cu~(2+)、Mn~(2+)、Zn~(2+)and cysteine was 0.1 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.05 mg/mL respectively . Psoralen and the complex were proved to be a good additive to promote melanin biosynthesis in cosmetics and medicines .
α-D-acetobromoglucose was used as sugar donor and formononetin,which was prepared by Deoxybenzoin route ,was used as nucleophilic reagent to synthesize ononin. Two Phase Transfer Catalysis (PTC) systems-solid-liquid PTC (SLPTC) system and liquid-liquid PTC (LLPTC) system were used to synthesize ononin. By comparing the yields, LLPTC system was selected and the highest yield can be 64.8 % by orthogonal experimentation. acetyl-ononin and ononin was isolated the HPLC purity can be 98.27 % and 98.36 % respectively.
By using UV-Vis spectrophotometry method to determine the inhibitory rates of ononin and formononetin against tyrosinase and superoxide radical, it was found that the highest inhibitory rate of ononin against superoxide radical is lower than this of formononetin. The inhibitory rate can reach 45.2 % at the concentration of 1 mg/mL. And the inhibitory rate of ononin against tyrosinase can reach 51.7 % when the concentration is 0.05 mg/mL,. So ononin can be used in cosmetics as natural safe skin-lightening and anti-aging agent.
酪氨酸酶是产生黑色素的主要限速酶,通过激活或抑制酪氨酸酶的活性可促进毛发和皮肤黑素的生成或抑制,达到乌发或皮肤增白的目的。在化妆品原料的研究过程中,酪氨酸酶作为性能测试试剂起着重要的作用。
中药补骨脂细粉经75 %的乙醇提取得到补骨脂素,其HPLC纯度为99 %。将补骨脂素配制成不同的浓度,用紫外-可见光分光光度法进行酪氨酸酶激活性能的检测,当浓度为0.03 mg/mL时,最高激活率为44.9 %。选择对酪氨酸酶都有一定激活作用的不同添加助剂Cu~(2+)、Fe~(2+)、Mn~(2+)、Zn~(2+)和半胱氨酸、咖啡因、人参皂苷等与补骨脂素复配,结果显示Cu~(2+)、Mn~(2+)、Zn~(2+)和半胱氨酸与补骨脂素都有不同程度协同激活效应。
采用单因素法将Cu~(2+)、Mn~(2+)、Zn~(2+)和半胱氨酸同时与补骨脂素复配,测定其对酪氨酸酶的激活率,当Cu~(2+)浓度为0.1 mg/mL、Mn~(2+)浓度为0.05 mg/mL、Zn~(2+)浓度为0.1 mg/mL、半胱氨酸浓度为0.05 mg/mL,加入量均为0.3 mL时,对酪氨酸酶激活率最高为104.3 %。因此,补骨脂素及其复配物可以作为促进黑色素生成的物质在化妆品或药物中使用。
采用脱氧安息香路线两步法合成芒柄花素,以芒柄花素和四乙酰溴代葡萄糖为原料,采用固-液和液-液两种不同的相转移催化合成芒柄花苷。通过对比产率等因素,最终确定采用液-液相转移催化体系。并采用正交试验进行优化,收率可达64.8 %。分离得到的中间体四乙酰芒柄花苷及产物芒柄花苷的HPLC纯度分别为98.27 %和98.36 %。
采用紫外-可见光分光光度法测定芒柄花苷对酪氨酸酶活性的抑制性能。结果表明:0.05 mg/mL时,芒柄花苷对酪氨酸酶最大抑制率为51.7 %。同时采用紫外-可见光分光光度法,测定了芒柄花苷对超氧自由基的抑制率。结果表明:在1 mg/mL时,芒柄花苷对超氧自由基的最大抑制率为45.2 %。因此芒柄花苷可以作为美白剂和抗衰老成分在化妆品中使用。
The synergistic activation on tyrosinase of psoralen cooperated with substrates, and the synthesis and optimization of ononin, as well as inhibition activity on tyrosinase and antioxidant activity were primarily studied in this thesis .
Tyrosinase is a major enzyme, which controls the speed of melanin biosynthesis, the generating of hair and skin melanin, which is the key factor to black hair and whiten skin, can be promoted or prohibited by activating or inhibiting the tyrosinase. Tyrosinase plays a vital role in selecting cosmetic materials as a reagent.
Psoralen was extacted by 75% ethanol water solution from Chinese traditional medicine psoralen . UV-Vis spectrophotometry method was selected to study the tyrosinase avtivity of psoralen which was in different concentration. The highest activation can be 44.9% when the concentration is 0.03 mg/mL . Different additives which can activate the tyrosinae , such as Cu~(2+)、Fe~(2+)、Mn~(2+)、Zn~(2+)and cysteine、affein、panaxcoside were selected to cooperate with psoralen to study the synergistic activation on tyrosinase . It indicated that Cu~(2+)、Mn~(2+)、Zn~(2+)and cysteine were the right substances.
Single factor method was selected to study the synergistic activation on tyrosinase of psoralen cooperated with all the right substances together, the highest activation can be 104.3 % , when the the concentration of Cu~(2+)、Mn~(2+)、Zn~(2+)and cysteine was 0.1 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.05 mg/mL respectively . Psoralen and the complex were proved to be a good additive to promote melanin biosynthesis in cosmetics and medicines .
α-D-acetobromoglucose was used as sugar donor and formononetin,which was prepared by Deoxybenzoin route ,was used as nucleophilic reagent to synthesize ononin. Two Phase Transfer Catalysis (PTC) systems-solid-liquid PTC (SLPTC) system and liquid-liquid PTC (LLPTC) system were used to synthesize ononin. By comparing the yields, LLPTC system was selected and the highest yield can be 64.8 % by orthogonal experimentation. acetyl-ononin and ononin was isolated the HPLC purity can be 98.27 % and 98.36 % respectively.
By using UV-Vis spectrophotometry method to determine the inhibitory rates of ononin and formononetin against tyrosinase and superoxide radical, it was found that the highest inhibitory rate of ononin against superoxide radical is lower than this of formononetin. The inhibitory rate can reach 45.2 % at the concentration of 1 mg/mL. And the inhibitory rate of ononin against tyrosinase can reach 51.7 % when the concentration is 0.05 mg/mL,. So ononin can be used in cosmetics as natural safe skin-lightening and anti-aging agent.
引文
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[2]陈晓英,楼向荣.浅论功能性化妆品的开发[J].福建轻纺, 2006, 3: 1-5.
[3] Nanninga PB et al. Pigment Cell Res, 1991, 4: 193-198.
[4]张彩华,徐国清.化妆品美白剂[J].萍乡高等专科学校学报, 2004, 4: 16-18.
[5]邹先伟,蒋志胜.植物源酪氨酸酶抑制剂研究进展[J].中草药, 2004, 35(6): 702-705.
[6]李航,赵国华等.天然产物对酪氨酸酶的抑制及抑制机理的研究进展[J].日用化学工业, 2003, 33(6): 383-386
[7] Alvaro Sanchez-Ferrer, Jose NeptunoRodriguez-Lopez , etc. Tyrosinase: A comprehensive review of its mechanism[J]. Biochimica et Biophysica Acta, 1995, 50(14): 1-11.
[8]刘秋均,段春燕.影响酪氨酸酶活性的药物及其作用机理[J].中华实用中西医杂志, 2003, 3(16) : 1002-1003
[9]阮博,孔令义.补骨脂化学成分的研究[J].中药研究与信息, 2005, 7(4): 7-9.
[10]朱铁君.常见色素性皮肤病的中西医结合治疗[J].中国医刊, 1999, 34(6): 6-9.
[11]王春娥.大豆异黄酮的成分、含量及特性[J].食品科学, 1998, 19(4): 39-43.
[12]井乐刚.大豆异黄酮的物理化学性质[J].中国农学通报, 2006, 22(1): 85-87.
[13]崔洪斌.大豆异黄酮—活性研究与应用[M].北京:科学出版社, 2005:113-126.
[14]朱庆磊.葛根素的病理学和临床应用研究进展[J].中草药, 1997, 28(11): 693-69.
[15]周耘.芒柄花素氨基醚衍生物的合成及体外促兔成骨细胞生长的作用[J].中国医药工业杂志, 2001, 32(1): 9-12.
[16]卢田静男.异黄酮对活性氧引起的脂质过氧化的抑制作用[M].国外医学中医中药分册, 1999, 21(3): 58-59
[17] Chung Hung Lee , Lin Yang , Jin Ze Xu ,etc. Relative antioxidant activity of soybean iso.avones and their glycosides[J]. Food Chemistry, 2005(Vol.90)(No.4): 735-741
[18]刘文等.染料木素对猪血小板聚集的影响[J].中国药理学报, 1998, 13(6): 540-542.
[19]颜春洪.异黄酮对小鼠恶性肿瘤转移的实验性治疗[J].药学学报, 1999, 34(11): 14-17.
[20]黄芸.异黄酮类化合物在植物界的分布及药理作用[J].中国野生植物资源, 2001, 20(1):5-7.
[21]刘丽,金宏.大豆异黄酮抗氧化作用的研究进展[J].中华放射医学和防护杂志, 2003, 23(2): 132-134.
[22]黄芸.异黄酮类化合物在植物界的分布及药理作用[J].中国野生植物资源, 2001, 20(1):5-7.
[23]旺海波,刘大川等.大豆异黄酮类物质的提取、抗氧化性及稳定性研究[J].食品科学, 2004, 25(1): 111-114
[24] Ishida Hitoshi, Tsuji Kunnio, etc. Skin stress-decreasing compositions containing isofalavone or flavone derivatives[P]. JP, 2001213775. 2001-08-27.
[25] Gorbach,Sherwood L. Isoflavonoids for treatment and prevention of aging skin and wrikless [P]. USA, 9856373 A1. 1998-10-17.
[26] Zerykier Sevi. Phytohormones of red clover extract used as additives for cosmetics collagen formation[P]. Ger, DE 20214684 U1.2003-01-30.
[27]韩正康.异黄酮植物雌性激素调控动物神经内分泌及生产性能的研究[J].中国农业科技导报, 1999, 1: 61-66.
[28]王克军等.天然黄酮苷的合成研究进展[J].化学通报, 2005, 4(7): 490-496.
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