黑色素生成是為了保護皮膚細胞免受紫外光傷害的一個生化過程。在這過程中，黑色素在人類表皮底層的黑色素細胞的黑色素體內產生。該過程可以被基因，荷爾蒙或環境因素所影響。黑色素的製造量是依賴速度限制酶酪氨酸酶的活性，黑色素體的數量和大小，黑色素體通過黑色素細胞的偽足傳送致角質細胞的速度及黑色素體在角質細胞內的分佈。這些細胞過程會受皮膚顏色或紫外光曝光量的變化而影響。當黑色素的產生超過黑色素的降解，黑色素沉著毛病便出現。根據不同的皮膚類型，年齡組別及累積紫外光曝光程度而引發雀斑或黃褐斑的形成。很多治療方法市面上能夠提供，它們包括人工合成化粧品，激光，整容手術等。這些治療方法通常會產生副作用及蘊藏高風險。因此從天然物質裏尋找治療藥物便成了美容學的一個新的研究方向。在這研究裏，十種草本植物就從自古以來用作治療黑色素沉著毛病的傅統中藥中被挑選出來。那些草本植物被四種擁有不同極性的溶劑提取。小鼠黑色素細胞被用以篩選提取物的降黑色素能力。結果發現當歸的正己烷及二氯甲烷的提取物有正面效用。當歸的化學成份4-乙基間苯二酚、4-乙基苯酚及1-十四烷醇也能降低小鼠黑色素細胞的黑色素量。數種生化技術繼而被應用作研究有效化學物的藥理。他們包括西方墨點法、環磷酸腺苷測試、蛋白激酶A活性測試及酪氨酸酶活性測試。 / Melanogenesis is a biochemical process designated for protecting skin cells from ultraviolet (UV)-induced damage. During the process, melanin is produced in the melanosomes of the melanocytes located at the basal epidermis of human. The process could be affected by genetic, hormonal or environmental factors. Amount of melanin synthesized depending on the activity of the rate-limiting enzyme tyrosinase, number and size of melanosomes, the transfer rate of melanosomes to keratinocytes through the melanocyte dendritic projections and the distribution pattern of melanosomes within keratinocytes. These cellular processes are influenced by variations in skin color or UV exposure amount. When melanin synthesis exceeds melanin degradation, hyperpigmentation disorder arises. This lead to the formation of freckles or chloasma according to different skin types, age groups and degree of cumulative UV exposure. A number of treatments are commercially available, they include applying synthetic cosmetics, laser, plastic surgery, etc. These treatments usually produce side-effects and possess high risk. Therefore, searching for therapeutic agents from natural compounds has become a new research direction in cosmetology. In this study, ten herbs were chosen from traditional Chinese medicine (TCM) which had been applied for treating hyperpigmentation. The herbs were extracted by four solvents with different polarity. The extracts were screened for their hypopigmentary ability by using melan-a cells. It was found that the hexane and dichloromethane extracts of Angelica sinensis possessed positive effects. 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol, the chemical constituents of A. sinensis, also attenuated melanin amount in melan-a cells. Moreover, several biochemical techniques were utilized to study the pharmaceutical mechanisms of the potent compounds. They include Western blot, cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay and tyrosinase activity assay. / Detailed summary in vernacular field only. / Lam, Rosanna Yen Yen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 127-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Chinese Abstract --- p.iii / Acknowledgements --- p.iv / List of Publications --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xii / List of Figures --- p.xv / List of Tables --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Demand of cosmetics --- p.1 / Chapter 1.2 --- Skin structures and functions --- p.1 / Chapter 1.2.1 --- Epidermis --- p.3 / Chapter 1.2.1.1 --- Stratum corneum --- p.4 / Chapter 1.2.1.2 --- Stratum granulosum --- p.4 / Chapter 1.2.1.3 --- Stratum spinosum --- p.4 / Chapter 1.2.1.4 --- Stratum basale --- p.4 / Chapter 1.2.2 --- Dermis --- p.5 / Chapter 1.2.3 --- Hypodermis --- p.5 / Chapter 1.3 --- Sun irradiation --- p.5 / Chapter 1.4 --- Variety of skin types --- p.6 / Chapter 1.5 --- Biochemical reactions within melanocyte --- p.7 / Chapter 1.6 --- Pigmentation disorder --- p.14 / Chapter 1.7 --- From the view of traditional Chinese medicine --- p.16 / Chapter 1.8 --- Treatments available for hyperpigmentation --- p.18 / Chapter 1.9 --- Aims of study and application of strategies --- p.19 / Chapter Chapter 2 --- Investigation of the inhibitory effect of herbal extracts and their constituent compounds on melanin synthesis --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and methods --- p.21 / Chapter 2.2.1 --- Materials --- p.21 / Chapter 2.2.2 --- Herbal extraction --- p.23 / Chapter 2.2.3 --- Cell culture --- p.25 / Chapter 2.2.4 --- Growth curve and melanin production curve --- p.25 / Chapter 2.2.5 --- SRB assay --- p.26 / Chapter 2.2.6 --- Calibration curve for SRB assay --- p.27 / Chapter 2.2.7 --- Measurement of melanin production --- p.27 / Chapter 2.2.8 --- Calibration curve for melanin production assay --- p.28 / Chapter 2.2.9 --- Statistical analysis --- p.28 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- Growth curve and melanin production curve for assay development --- p.29 / Chapter 2.3.2 --- Calibration curves of SRB assay and melanin production assay --- p.32 / Chapter 2.3.3 --- Hypopigmentary effect of 40 herbal extracts --- p.35 / Chapter 2.3.4 --- Hypopigmentary effects of chemical components of A. sinensis --- p.41 / Chapter 2.4 --- Discussion --- p.49 / Chapter Chapter 3 --- Study of the effect of potential compounds on melanogenic protein expression by Western blot --- p.54 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and methods --- p.56 / Chapter 3.2.1 --- Materials --- p.56 / Chapter 3.2.2 --- Cell culture --- p.56 / Chapter 3.2.3 --- Preparation of cell lysates --- p.57 / Chapter 3.2.4 --- Protein assay --- p.57 / Chapter 3.2.5 --- SDS-PAGE and membrane transfer --- p.58 / Chapter 3.2.6 --- Washing of blotted antibodies and film exposure --- p.59 / Chapter 3.3 --- Results --- p.61 / Chapter 3.4 --- Discussion --- p.70 / Chapter Chapter 4 --- Study of the effect of potential compounds on melanogenic gene expression by RT-PCR --- p.76 / Chapter 4.1 --- Introduction --- p.76 / Chapter 4.2 --- Materials and methods --- p.76 / Chapter 4.2.1 --- Materials --- p.77 / Chapter 4.2.2 --- Cell culture --- p.77 / Chapter 4.2.3 --- RNA extraction --- p.78 / Chapter 4.2.4 --- cDNA synthesis --- p.78 / Chapter 4.2.5 --- PCR --- p.80 / Chapter 4.3 --- Results --- p.83 / Chapter 4.4 --- Discussion --- p.85 / Chapter Chapter 5 --- Study of the effect of potential compounds on cAMP level by EIA --- p.85 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Materials and methods --- p.86 / Chapter 5.2.1 --- Materials --- p.86 / Chapter 5.2.2 --- Cell culture --- p.86 / Chapter 5.2.3 --- Preparation of cell lysates --- p.86 / Chapter 5.2.4 --- Protein assay --- p.87 / Chapter 5.2.5 --- The cAMP assay --- p.88 / Chapter 5.2.6 --- Preparation of cAMP calibration curve --- p.88 / Chapter 5.2.7 --- Calculation --- p.89 / Chapter 5.2.8 --- Statistical analysis --- p.89 / Chapter 5.3 --- Results --- p.90 / Chapter 5.4 --- Discussion --- p.94 / Chapter Chapter 6 --- Study of the effect of potential compounds on PKA activity by PKA activity assay --- p.96 / Chapter 6.1 --- Introduction --- p.96 / Chapter 6.2 --- Materials and methods --- p.96 / Chapter 6.2.1 --- Materials --- p.97 / Chapter 6.2.2 --- Cell culture --- p.97 / Chapter 6.2.3 --- Preparation of cell lysates --- p.98 / Chapter 6.2.4 --- Protein assay --- p.98 / Chapter 6.2.5 --- The PKA kinase activity assay --- p.100 / Chapter 6.2.6 --- Calculation --- p.100 / Chapter 6.2.7 --- Statistical analysis --- p.100 / Chapter 6.3 --- Results --- p.101 / Chapter 6.4 --- Discussion --- p.104 / Chapter Chapter 7 --- Study of the effect of potential compounds on tyrosinase activity by enzyme inhibition assay --- p.107 / Chapter 7.1 --- Introduction --- p.107 / Chapter 7.2 --- Materials and methods --- p.108 / Chapter 7.2.1 --- Materials --- p.108 / Chapter 7.2.2 --- Assay development for mushroom tyrosinase --- p.109 / Chapter 7.2.3 --- Mushroom tyrosinase inhibition assay --- p.109 / Chapter 7.2.4 --- Cell culture --- p.110 / Chapter 7.2.5 --- Preparation of cellular tyrosinase --- p.110 / Chapter 7.2.6 --- Protein assay --- p.111 / Chapter 7.2.7 --- Cellular tyrosinase inhibition assay --- p.111 / Chapter 7.2.8 --- Calculation --- p.112 / Chapter 7.2.9 --- Statistical analysis --- p.112 / Chapter 7.3 --- Results --- p.113 / Chapter 7.4 --- Discussion --- p.120 / Chapter Chapter 8 --- General discussion --- p.123 / References --- p.127

Greyia radlkoferi is a South African indigenous tree, which has recently been discovered to be a source of extracts that have a potential in the development of cosmeceutical herbal products having the ability to treat hyperpigmentation disorders. For product development however, G. radlkoferi would need to be available in a commercial scale. Greyia radlkoferi grows naturally in the wild and is often available for cultivation as an ornamental plant. In order to establish this plant into cultivation, suitable propagation techniques must be established for rapid multiplication of trees and thus a sustainable leaf production. For consistent and improved leaf supply to the market, agronomic practices that will enhance leaf production were investigated in the current study. Furthermore, in order to meet market demand in terms of good quality extracts with guaranteed therapeutic efficiency, pre-harvest and post-harvest factors that affect the chemical composition of the extracts were investigated. Recently developed biotechnology techniques such as metabolomics using 1H-NMR and multivariate data analysis offered a platform to study the chemical variation of extracts. Therefore, the current study was aimed at understanding the requirements for propagation and optimum leaf production as well as conditions that favour optimum production of secondary metabolite of G. radlkoferi plant material (at pre and post-harvest) and thus assess its commercial viability. To understand the effects of temperature on seed germination of G. radlkoferi, seeds were exposed to five temperatures (10°C, 15°C, 20°C, 25°C and 30°C) in the incubators in the laboratory. Germination of G. radlkoferi by seeds was discovered to be temperature dependent. The optimum germination temperature of 81% was obtained at 25°C. In the case of vegetative propagation by stem cuttings, the effect of cutting position (basal or apical), exogenous rooting hormone (Seradix1, Seradix 2, 0.1% IBA, 0.3% IBA and 0.8% IBA) and cutting position were investigated in the glasshouse. The cutting position had a significant effect on rooting of G. radlkoferi cuttings with basal cuttings exhibiting 35% rooting as compared to 6% rooting attained for the apical cuttings. A clear trend in rooting response to application of rooting hormones was observed, with 0.1% Indole butyric acid (IBA) showing the highest rooting percentage of 63%. Considering the outcomes of the propagation studies as well as the limited material for vegetative propagation, seed propagation appears to be the most suitable technique for large-scale multiplication of G. radlkoferi. The effect of different pruning techniques as well as harvesting frequencies on fresh and dry weights of G. radlkoferi leaves were evaluated. Factors considered were four pruning treatments (‘pruned but not tipped’, ‘tipped but not pruned’, ‘not pruned nor tipped’ as well as ‘pruned and tipped’) and three harvesting periods (monthly, bimonthly and once–off). Bimonthly harvests highly increased leaf production compared to trees that were harvested monthly and once-off with higher leaf fresh weight yield of 238 g per tree or 2.38 tons/per hectare as well as dry weight yield of 83 g per tree or 0.830 tons/hectare. This outcomes of this study further suggested that a suitable pruning practice for G. radlkoferi would be to either ‘prune only’ or ‘cut back the main stem’ rather than a combination of the two treatments. The influence of seasons (summer, autumn, winter and spring) on the anti-tyrosinase activity and metabolomics profile of G. radlkoferi leaf extracts were investigated. Seasons significantly influenced the chemical composition and the efficacy of the plant extracts. Tyrosinase enzyme inhibition was investigated against monophenolase (tyrosine) with kojic acid as positive control. The highest tyrosinase inhibition concentration with IC50 (50% tyrosinase inhibition concentration) value of 30.3±1.8 μg/ml were obtained in winter harvested leaves compared to the other seasons. The lowest IC50 values were obtained in spring. Metabolomics analysis using orthogonal partial least square discriminant analysis (OPLS-DA) provided a clear class separation according to the harvest season. Extracts from winter harvested leaves contained sucrose, acetamide, alanine and a compound of the catechin group (gallocatechin-(4 alpha->8)-epigallocatechin) as revealed by 1H-NMR metabolomics with assistance of LC-MS. Since compounds of the catechin group are well-known tyrosinase inhibitors, the high tyrosinase activity exhibited in extracts of winter harvested G. radlkoferi leaves could be ascribed to the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Based on the outcomes of the seasonal study, we suggest that in order to obtain extracts with high bioactivity, the best suitable time for harvesting leaf samples is in late autumn-early winter. Processing leaf material using three different drying methods (sun, oven and air drying) significantly influenced chemical composition and the efficacy of the plant extracts. Extracts prepared from air-dried leaf material showed the highest tyrosinase inhibition with IC50 value of 17.80 μg/ml compared to extracts of the other drying methods. Extracts of leaves processed with air drying preserved most metabolites during processing while extracts of sun-dried and oven-dried leaves clearly depleted some metabolites especially amino acids and some aromatic compounds. 1H-NMR metabolomics approach with the assistance of LC-MS data successfully determined a positive association of alanine, acetamide, sucrose and gallocatechin-(4 alpha->8)-epigallocatechin as the chemical constituents contributing to the variation in the air-dried leaves compared to the oven-dried leaves. A positive association of valine, alanine, leucine, isoleucine, gallocatechin-(4 alpha->8)-epigallocatechin and glucose contributed to the variation in air-dried group, compared to the sun-dried group. The highest tyrosinase inhibitory activity exhibited in air-dried samples compared to the other drying methods was associated with the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Because air drying preserved most leaf metabolites compared to sun and oven drying, it was regarded as the most suitable method for processing G. radlkoferi leaf material. This study is the first scientific account that provides guidelines and recommendations to (1) establish G. radlkoferi as a cultivated plant for commercialization, (2) optimize leaf production for sustainable supply to the commercial markets and (3) optimize medicinal content of G. radlkoferi related to harvesting time and post-harvest processing (drying), for enhanced quality of extracts and its products / Agriculture, Animal Health and Human Ecology / Ph. D. (Agriculture)