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学校编码：10384 分类号________密级________
学号：20120051403142 UDC________
博 士 学 位 论 文
天然植物活性物质对酪氨酸
酶作用机理的研究
Study on the inhibition and activation kinetics to
tyrosinase by active molecules from plants
邱 凌
指导教师姓名： 陈清西 教授
专 业 名 称 ：生物化学与分子生物学
论文提交日期：2008 年 4 月 28 日
论文答辩时间：2008 年 5 月 30 日
学位授予日期：2008 年 月 日
答辩委员会主席：
评 阅 人：
2008 年 5 月
厦门大学博硕士论文摘要库
厦门大学博硕士论文摘要库
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厦门大学博硕士论文摘要库
厦门大学博硕士论文摘要库
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作者签名： 日期：2008 年 5 月 20 日
导师签名： 日期：2008 年 5 月 20 日
厦门大学博硕士论文摘要库
厦门大学博硕士论文摘要库
I
目录
中文摘要 ...........................................................................................................................1
英文摘要 ...........................................................................................................................2
1. 前言 ............................................................................................................................3
1.1 酪氨酸酶的研究概况 ........................................................................................ 3
1.2 酪氨酸酶的结构和理化性质 ............................................................................ 3
1.3 酪氨酸酶基因的结构和表达 ............................................................................ 4
1.4 酪氨酸酶的活性中心和催化机理 .................................................................... 6
1.5 酪氨酸酶的应用 ................................................................................................ 7
1.5.1 肌体中黑色素代谢及调控...................................................................... 7
1.5.2 植物中的酪氨酸酶.................................................................................. 9
1.5.3 微生物中的酪氨酸酶............................................................................ 10
1.5.4 药物合成、生物传感器与酶的固定化................................................ 10
1.6 酪氨酸酶抑制剂的研究概况 ...........................................................................11
1.6.1 果酸........................................................................................................ 13
1.6.2 维生素.................................................................................................... 13
1.6.3 内皮素拮抗剂........................................................................................ 14
1.6.4 汞............................................................................................................ 14
1.6.5 铜离子的络合物.................................................................................... 14
1.6.6 蛋白质和多肽........................................................................................ 15
1.6.7 氨基酸及其衍生物................................................................................ 15
1.6.8 黄酮类化合物........................................................................................ 15
1.6.9 含酚羟基的化合物................................................................................ 16
1.6.10 芪类及其类似物.................................................................................... 17
1.6.11 醛类化合物............................................................................................ 17
1.6.12 羧基化合物............................................................................................ 18
2. 实验材料与仪器 ......................................................................................................21
2.1 实验原料 .......................................................................................................... 21
2.2 实验试剂 .......................................................................................................... 23
厦门大学博硕士论文摘要库
天然植物活性物质对酪氨酸酶作用机理的研究
II
2.3 实验仪器 .......................................................................................................... 23
3. 实验方法 ..................................................................................................................25
3.1 样品的制备方法 .............................................................................................. 25
3.1.1 少量样品的提取.................................................................................... 25
3.1.2 大量样品的提取.................................................................................... 25
3.1.3 大量样品的分离.................................................................................... 25
3.1.4 提取后的样品的制备............................................................................ 25
3.2 效应物对酪氨酸酶的抑制作用机理的测定 .................................................. 26
3.2.1 效应物对蘑菇酪氨酸酶单酚酶活力的影响........................................ 26
3.2.2 效应物对蘑菇酪氨酸酶二酚酶作用的研究........................................ 26
3.2.3 效应物对蘑菇酪氨酸酶二酚酶的抑制类型和抑制常数的测定........ 26
3.2.4 效应物对酶抑制作用动力学常数的测定............................................ 27
3.3 采用 DPPH 法测定化合物清除氧自由基的能力 ......................................... 27
3.4 效应物对小鼠 B16 黑素瘤细胞产黑色素相关指标的影响 ......................... 27
3.4.1 小鼠 B16 黑素瘤细胞增殖率的测定方法 ........................................... 27
3.4.2 小鼠 B16 黑色素瘤细胞中酪氨酸酶活性的测定方法 ....................... 28
3.4.3 小鼠 B16 黑色素瘤细胞中黑色素生成量的测定方法 ....................... 28
3.4.4 小鼠 B16 黑色素瘤细胞中 IC50 的计算方法....................................... 28
4. 实验结果 ..................................................................................................................29
4.1 海生植物中有效成分的筛选 .......................................................................... 29
4.1.1 海带（Laminaria japonica Aresch）.................................................... 29
4.1.2 坛紫菜(Porphyra haitanensis) .............................................................. 30
4.1.3 海桑(Sonneratia caseolaris) .................................................................. 31
4.1.4 红海榄(Rhizophora stylosa ) ................................................................. 31
4.1.5 秋茄(Kandelia candel (L.) Druce)......................................................... 32
4.2 热带、亚热带果树植物中有效成分的筛选 .................................................. 36
4.2.1 仙蜜果（Hylocereus undatus (Haw. )Britt. et. Rose）......................... 36
4.2.2 枇杷（Eriobotrya japonica (Thunb.) L.）............................................ 40
4.2.3 苏拉维西凤果........................................................................................ 43
4.3 杀虫植物中有效成分的筛选 .......................................................................... 47
厦门大学博硕士论文摘要库
III
4.3.1 木贼（Equisetum arvense （Radix Allcklandiae））........................... 47
4.3.2 荆芥（Herba Schizonepetae ）............................................................ 50
4.3.3 菖蒲（Acorus calamus） ...................................................................... 51
4.3.4 半夏（Pinellia ternata） ...................................................................... 54
4.3.5 马齿苋（Portulaca oleracea）............................................................. 55
4.3.6 牛蒡子（Arctium lappa）..................................................................... 56
4.3.7 马鞭草（verbena officinalis）.............................................................. 56
4.3.8 川楝（Melia toosendan） ..................................................................... 58
4.3.9 苍耳（Xanthium sibiricum） ................................................................ 59
4.4 南药植物中有效成分的筛选 .......................................................................... 60
4.4.1 金粉蕨（Onychium japonicum (Thunb.) Kunze）............................... 60
4.4.2 金毛狗脊(Cibotium barometz) .............................................................. 60
4.4.3 鸭跖草(Commelina communis L.) ......................................................... 61
4.4.4 野慈姑（Sagittaria trifolia L.） ........................................................... 62
4.4.5 阔叶丰花草(Borreria latifolia (Aubl.) K. Schum.)............................... 62
4.4.6 了哥王(Radix Wikstroemia indica (Linn.) C. A. Mey.) ......................... 63
4.4.7 使君子(Rangoon creeper) ..................................................................... 63
4.4.8 毛叶蝴蝶草(Torenia benthamiana Hance)............................................ 65
4.4.9 毛麝香(Adenosma glutinosum (Linn.) Druce)....................................... 66
4.4.10 爵床(Rostellularia procumbens (L.))..................................................... 66
4.4.11 疏花马蓝(Serobilanthes divaricatus (Nees) A. Anders) ....................... 68
4.4.12 翅柄马蓝(Pteracanthus alatus (Wall. ex Nees) Bremek) ..................... 68
4.4.13 扭序花（Clinacanthus nutans (Burm. f.) Lindan） ............................. 68
4.4.14 倒地铃（Cardiospermum halicacabum L.）........................................ 70
4.4.15 金酸枣(Spondias cytherea Sonn.) ......................................................... 70
4.4.16 鸦胆子(Brucea javanica (L.) Merr)....................................................... 71
4.4.17 三叶崖爬藤(Tetrastigma hemsleyanum Diels et Gilg).......................... 72
4.4.18 细风轮菜(Clinopodium gracile (Benth.) Matsum.) .............................. 73
4.4.19 溪黄草(Rabdosia serra (Maxim.)) ........................................................ 73
4.4.20 绞股蓝(Gynostemma pentaphyllum (Thunb.) Mak.) ............................. 74
厦门大学博硕士论文摘要库
天然植物活性物质对酪氨酸酶作用机理的研究
IV
4.4.21 斑鸠菊（Vernonia solanifolia Benth.）................................................ 75
4.4.22 千里光(Senecio scandens Buch) ........................................................... 76
4.4.23 亚贡（Polymuia edulis）...................................................................... 77
4.4.24 白面风（Lnula cappa DC.） ................................................................ 78
4.4.25 毛冬青(Ilex pubescens Hook. et Arn.)................................................... 79
4.4.26 冬青(Ilex Purpurea Hassk (I. Chinensis Sims)) .................................... 79
4.4.27 秤星树(Ilex asprella (Hook. et Arn.) Champ. ex Benth.)...................... 80
4.4.28 牛白藤(Oldenlandia hedyotidea (DC.) Hand)....................................... 81
4.4.29 伞房花儿草............................................................................................ 81
4.4.30 腰骨藤(Ichnocarpus frutescens (L.) W. T. Aiton) ................................. 83
4.4.31 长春花(Catharanthus roseus (L.))......................................................... 85
4.4.32 大青（Clerodendrum cyrtophyllum Turcz.） ....................................... 85
4.4.33 木芙蓉(Hibiscus mutabilis L.)............................................................... 86
4.4.34 猫爪藤(Macfadyena unguis-cati) .......................................................... 86
4.4.35 叶下珠（Phyllanthus urinaria L.）...................................................... 89
4.4.36 珠仔草（Phyllanthus niruri L.）.......................................................... 95
4.4.37 余甘子（Phyllanthus Emblica L. ）.................................................... 96
4.4.38 三加皮（Eleutherococcus trifoliatus (L.) S.y. Hu） ............................ 97
4.4.39 铁包金(Berchemia floribun-da (Wall.) Brongn) ................................... 97
4.4.40 两面针（Zanthoxylum nitidum（Raxb.）DC）................................... 99
4.4.41 瘤茎藤（Tinospora crispa） ................................................................ 99
4.4.42 中国芦荟（Aloe vera L.）.................................................................. 101
4.5 裸子植物银杏（Ginkgo Biloba L.） ........................................................... 105
4.5.1 银杏叶.................................................................................................. 105
4.5.2 银杏提取的标准品.............................................................................. 108
4.5.3 银杏外种皮.......................................................................................... 109
4.6 天然植物标准品对酪氨酸酶的作用机理 .................................................... 122
4.6.1 芦丁...................................................................................................... 122
4.6.2 芦荟甙.................................................................................................. 122
4.6.3 土大黄素.............................................................................................. 123
厦门大学博硕士论文摘要库
V
4.6.4 大黄素.................................................................................................. 123
4.6.5 芦荟大黄素.......................................................................................... 124
4.6.6 大黄酸.................................................................................................. 124
4.6.7 大黄甲醚.............................................................................................. 125
4.6.8 儿茶素.................................................................................................. 125
4.6.9 表儿茶素.............................................................................................. 126
4.6.10 原儿茶酚.............................................................................................. 126
4.6.11 绿原酸.................................................................................................. 127
4.6.12 葛根素.................................................................................................. 127
4.6.13 木樨草素.............................................................................................. 128
4.6.14 槲皮素.................................................................................................. 128
4.6.15 山萘素.................................................................................................. 129
4.7 基于母核分析的抑制剂设计及机理研究 .................................................... 129
4.7.1 α-氰基-4-羟基肉桂酸.......................................................................... 129
4.7.2 桧木醇.................................................................................................. 133
4.7.3 槲皮素与 3,5-二羟苯基癸酸酯 .......................................................... 137
4.7.4 曲酸对小鼠黑色素瘤细胞 B16 细胞的作用 ..................................... 143
5. 讨论 ........................................................................................................................145
5.1 酪氨酸酶激活剂的机理分析 ........................................................................ 145
5.2 HCCA 对酪氨酸酶的作用机理 ................................................................... 146
5.3 DPD 的对酪氨酸酶的抑制机理................................................................... 148
5.4 2-羟基-4-异丙基结构类似物与桧木醇的抑制机理 .................................... 149
5.5 仙蜜果提取物对酪氨酸酶的作用机理 ........................................................ 152
5.6 叶下珠属植物提取物对酪氨酸酶的作用机理 ............................................ 153
5.7 芦荟表皮提取物对酪氨酸酶的作用机理 .................................................... 153
5.8 银杏外种皮提取物 GA1 和 GA2 对酪氨酸酶的作用机理 .......................... 154
5.9 效应物清除氧自由基效应与抑制酪氨酸酶活力的关系............................ 156
5.10 效应物在胞内和胞外水平上抑制酪氨酸酶活力的比较............................ 157
6. 结论 ........................................................................................................................159
6.1 天然植物中酪氨酸酶抑制剂的筛选 ............................................................ 159
厦门大学博硕士论文摘要库
天然植物活性物质对酪氨酸酶作用机理的研究
VI
6.2 酪氨酸酶抑制剂的母核分析 ........................................................................ 159
6.3 酪氨酸酶抑制剂的机理探讨 ........................................................................ 159
6.4 抑制酪氨酸酶活力与抗氧化之间的联系 .................................................... 159
6.5 胞外酪氨酸酶检测与细胞生物学效应的关系 ............................................ 159
6.6 酪氨酸酶抑制剂的应用前景 ........................................................................ 160
7. 参考文献 ................................................................................................................161
厦门大学博硕士论文摘要库
Contents
VII
Contents Chinese Abstract..............................................................................................................1
English Abstract...............................................................................................................2
1. Introduction ..............................................................................................................3
1.1 Background of tyrosinase................................................................................. 3
1.2 Structure and physicochemical properties of tyrosinase .............................. 3
1.3 Structure and expression of tyrosinase gene .................................................. 4
1.4 Active center and catalytic mechanism of tyrosinase.................................... 6
1.5 Applications of tyrosinase ................................................................................ 7
1.5.1 Melanin metabolism and regulation in human body................................ 7
1.5.2 Tyrosinases in plants ................................................................................ 9
1.5.3 Tyrosinases in microorganism................................................................ 10
1.5.4 Drug synthesis, biosensor and immobilization of tyrosinae .................. 10
1.6 酪氨酸酶抑制剂的研究概况 ...........................................................................11
1.6.1 Fruit Acid ............................................................................................... 13
1.6.2 Vitamin................................................................................................... 13
1.6.3 Endothelin antagonist............................................................................. 14
1.6.4 Mercury.................................................................................................. 14
1.6.5 Complexes of copper ............................................................................. 14
1.6.6 Protein and peptides ............................................................................... 15
1.6.7 Amino acids and their derivatives.......................................................... 15
1.6.8 Flavonoids.............................................................................................. 15
1.6.9 Comprounds with phenol hydroxy groups............................................. 16
1.6.10 Stilbene and its derivatives .................................................................... 17
1.6.11 Aldehydes............................................................................................... 17
1.6.12 Carboxyl comprounds ............................................................................ 18
2. Experimental materials and equipment ...............................................................21
2.1 Experimental materials.................................................................................. 21
2.2 Experimental reagents ................................................................................... 23
2.3 Experimental equipment................................................................................ 23
3. Methods ...................................................................................................................25
3.1 Preparation for samples ................................................................................. 25
3.1.1 Extraction of small amount of samples.................................................. 25
厦门大学博硕士论文摘要库
天然植物活性物质对酪氨酸酶作用机理的研究
VIII
3.1.2 Extraction of muchness samples ............................................................ 25
3.1.3 Saparation of muchness samples............................................................ 25
3.1.4 Preparation of Samples .......................................................................... 25
3.2 Assay of inhibitory mechanism of effects on mushroom tyrosinase .......... 26
3.2.1 Assay of effects on the monophenolase activity by compounds............ 26
3.2.2 Assay of effects on the diphenolase activity by compounds.................. 26
3.2.3 Assay of effects on the inhibition mechanism by componds................. 26
3.2.4 Assay of effects on the inhibition constant by compounds.................... 27
3.3 Anti-oxidant assay through DPPH method.................................................. 27
3.4 Effect of compounds on melonagenesis in B16 cell...................................... 27
3.4.1 Assay the change of cell vability in B16 cell by compounds ................ 27
3.4.2 Assay the change of tyrosinase activity in B16 cell by compounds ...... 28
3.4.3 Assay the change of melanin content in B16 cell by compounds.......... 28
3.4.4 Calculation of IC50 in the cell experiments........................................... 28
4. Results......................................................................................................................29
4.1 Plants from the sea ......................................................................................... 29
4.1.1 Laminaria japonica Aresch.................................................................... 29
4.1.2 Porphyra haitanensis ............................................................................. 30
4.1.3 Sonneratia caseolaris............................................................................. 31
4.1.4 Rhizophora stylosa ................................................................................. 31
4.1.5 Kandelia candel (L.) Druce ................................................................... 32
4.2 Fruits plant...................................................................................................... 36
4.2.1 Hylocereus undatus (Haw. )Britt. et. Rose............................................. 36
4.2.2 Eriobotrya japonica (Thunb.) L............................................................. 40
4.3 Insecticidal plant............................................................................................. 47
4.3.1 Equisetum arvense （Radix Allcklandiae）......................................... 47
4.3.2 Herba Schizonepetae.............................................................................. 50
4.3.3 Acorus calamus ...................................................................................... 51
4.3.4 Pinellia ternata ...................................................................................... 54
4.3.5 Portulaca oleracea................................................................................. 55
4.3.6 Arctium lappa ......................................................................................... 56
4.3.7 verbena officinalis .................................................................................. 56
4.3.8 Melia toosendan ..................................................................................... 58
4.3.9 Xanthium sibiricum ................................................................................ 59
厦门大学博硕士论文摘要库
Contents
IX
4.4 Chinese traditional medicine ......................................................................... 60
4.4.1 Onychium japonicum (Thunb.) Kunze................................................... 60
4.4.2 Cibotium barometz ................................................................................. 60
4.4.3 Commelina communis L......................................................................... 61
4.4.4 Sagittaria trifolia L. ............................................................................... 62
4.4.5 Borreria latifolia (Aubl.) K. Schum. ..................................................... 62
4.4.6 Radix Wikstroemia indica (Linn.) C. A. Mey. ........................................ 63
4.4.7 Rangoon creeper .................................................................................... 63
4.4.8 Torenia benthamiana Hance .................................................................. 65
4.4.9 Adenosma glutinosum (Linn.) Druce ..................................................... 66
4.4.10 Rostellularia procumbens (L.) ............................................................... 66
4.4.11 Serobilanthes divaricatus (Nees) A. Anders .......................................... 68
4.4.12 Pteracanthus alatus (Wall. ex Nees) Bremek ........................................ 68
4.4.13 Clinacanthus nutans (Burm. f.) Lindan ................................................. 68
4.4.14 Cardiospermum halicacabum L............................................................. 70
4.4.15 Spondias cytherea Sonn. ........................................................................ 70
4.4.16 Brucea javanica (L.) Merr ..................................................................... 71
4.4.17 Tetrastigma hemsleyanum Diels et Gilg ................................................ 72
4.4.18 Clinopodium gracile (Benth.) Matsum. ................................................. 73
4.4.19 Rabdosia serra (Maxim.)....................................................................... 73
4.4.20 Gynostemma pentaphyllum (Thunb.) Mak............................................. 74
4.4.21 Vernonia solanifolia Benth..................................................................... 75
4.4.22 Senecio scandens Buch .......................................................................... 76
4.4.23 Polymuia edulis...................................................................................... 77
4.4.24 Lnula cappa DC. .................................................................................... 78
4.4.25 Ilex pubescens Hook. et Arn. ................................................................. 79
4.4.26 Ilex Purpurea Hassk (I. Chinensis Sims) ............................................... 79
4.4.27 Ilex asprella (Hook. et Arn.) Champ. ex Benth. .................................... 80
4.4.28 Oldenlandia hedyotidea (DC.) Hand ..................................................... 81
4.4.30 Ichnocarpus frutescens (L.) W. T. Aiton ................................................ 83
4.4.31 Catharanthus roseus (L.) ....................................................................... 85
4.4.32 Clerodendrum cyrtophyllum Turcz. ....................................................... 85
4.4.33 Hibiscus mutabilis L. ............................................................................. 86
4.4.34 Macfadyena unguis-cati ......................................................................... 86
厦门大学博硕士论文摘要库
天然植物活性物质对酪氨酸酶作用机理的研究
X
4.4.35 Phyllanthus urinaria L........................................................................... 89
4.4.36 Phyllanthus niruri L............................................................................... 95
4.4.37 Phyllanthus Emblica L. ........................................................................ 96
4.4.38 Eleutherococcus trifoliatus (L.) S.y. Hu ................................................ 97
4.4.39 Berchemia floribun-da (Wall.) Brongn .................................................. 97
4.4.40 Zanthoxylum nitidum（Raxb.）DC....................................................... 99
4.4.41 Tinospora crispa .................................................................................... 99
4.4.42 Aloe vera L........................................................................................... 101
4.5 Ginkgo Biloba L. ........................................................................................... 105
4.5.1 The Leaf extractions of Ginkgo Biloba L. ........................................... 105
4.5.2 Standard substances of Ginkgo Biloba L. ............................................ 108
4.5.3 Sarcotesta of Ginkgo biloba L ............................................................. 109
4.6 The inhibtion mechanism of known standards from plants ..................... 122
4.6.1 Putin ..................................................................................................... 122
4.6.2 Aloin..................................................................................................... 122
4.6.3 Rhaponticin .......................................................................................... 123
4.6.4 Emodin ................................................................................................. 123
4.6.5 Aloe - emodin....................................................................................... 124
4.6.6 Phein..................................................................................................... 124
4.6.7 Physcion ............................................................................................... 125
4.6.8 Catechin ............................................................................................... 125
4.6.9 Epicatechin........................................................................................... 126
4.6.10 Protocatechnic Acid ............................................................................. 126
4.6.11 Chlorogenic.......................................................................................... 127
4.6.12 Puerarin ................................................................................................ 127
4.6.13 Luteolin ................................................................................................ 128
4.6.14 Quercetinum......................................................................................... 128
4.6.15 Kaempferol........................................................................................... 129
4.7 The inhibition mechanism of Some inhibitors ........................................... 129
4.7.1 HCCA................................................................................................... 129
4.7.2 Hinokitiol ............................................................................................. 133
4.7.3 Quercetin and 3,5- dihydroxyphenyl decanoate .................................. 137
4.7.4 The inhibiton of Kojic acid in B16 cell................................................ 143
5. Discussion ..............................................................................................................145
厦门大学博硕士论文摘要库
Degree papers are in the “Xiamen University Electronic Theses and Dissertations Database”. Fulltexts are available in the following ways: 1. If your library is a CALIS member libraries, please log on http://etd.calis.edu.cn/ and submitrequests online, or consult the interlibrary loan department in your library. 2. For users of non-CALIS member libraries, please mail to [email protected] for delivery details.
厦门大学博硕士论文摘要库