Tyrosinase Inhibitors from Traditional Chinese Medicine As Potential Anti-Hyperpigmentation Agents
Total Page：16
File Type：pdf, Size：1020Kb
Tyrosinase Inhibitors from Traditional Chinese Medicine as Potential Anti-hyperpigmentation agents Author Shi, Gengxuan Published 2019 Thesis Type Thesis (Masters) School School of Environment and Sc DOI https://doi.org/10.25904/1912/1780 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/387690 Griffith Research Online https://research-repository.griffith.edu.au Tyrosinase Inhibitors from Traditional Chinese Medicine as Potential Anti-hyperpigmentation agents Gengxuan SHI School of Environment and Science Griffith Science Griffith University Submitted in fulfilment of the requirements of the degree of Master of Science July 2019 i Abstract Several treatments for skin pigmentation are available today, however, many have unwanted side effects. Classic drugs like hydroquinone, arbutin, mequinol and kojic acid have been considered strongly carcinogenic, with related adverse effects. Traditional Chinese medicines (TCMs) have long been documented for their skin- lightening properties with little known negative effects. However, little is known about precisely how these herbal medicines work and what are the chemical basis for their activity. In this project, a 96-well plate based tyrosinase assay was established and used to test 44 TCM with known skin-lightening properties. Out of 44, 17 TCM extracts showed over 60% inhibition against tyrosinase at the concentration of 0.5 mg/mL. One of the TCM extracts, Xanthium strumarium L. extract, possessed 81.7% inhibition at 0.5 mg/mL. Further bioassay-guided isolation of the crude extract resulted in 10 compounds. Three of the compounds showed moderate activity against tyrosinase with IC50 values of 0.18 mM (cytidine), 0.29 mM (1,4-dicaffeoylquinic acid) and 2.47 mM (xanthiside). Chapter 1 contained the introduction of this project as well as the literature review. Chapter 2 was mainly about the screening of TCM extracts against mushroom tyrosinase. Chapter 3 contained the bioassay-guided extraction and isolation of Xanthium strumarium L. Additionally, the IC50 values of the active compounds were included in chapter 3 as well. ii Statement of Originality This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. (Signed)_ iii Table of contents Chapter 1. Introduction and Literature Review ......................................................... 1 1.1 Hyperpigmentation ............................................................................................................. 1 1.2 Melanin Biosynthesis ........................................................................................................... 4 1.3 Tyrosinase .............................................................................................................................. 7 1.4 Tyrosinase inhibitors from natural products ................................................................ 8 1.4.1 Flavonoids .................................................................................................................. 9 1.4.2 Organic acids .......................................................................................................... 10 1.4.3 Glycoside ................................................................................................................. 11 1.4.4 Terpenes .................................................................................................................. 12 1.4.5 Esters ........................................................................................................................ 14 1.4.6 Other compounds ................................................................................................. 15 1.5 Traditional Chinese Medicine (TCM) ............................................................................ 16 1.6 Project aims ......................................................................................................................... 18 Chapter 2. Tyrosinase assay and the screening of TCM extracts against tyrosinase .. 23 2,1 Mushroom tyrosinase assay ........................................................................................... 23 2.2 Positive controls ................................................................................................................. 26 2.3 selection of TCM against hyperpigmentation ........................................................... 27 2.4 Anti-tyrosinase activity ..................................................................................................... 30 2.5 Experimental ....................................................................................................................... 32 Protein sequence alignments....................................................................................... 32 Chemicals ........................................................................................................................... 32 Tyrosinase assay ............................................................................................................... 32 Crude extracts preparations ......................................................................................... 33 Chapter 3 Chemical investigation of plant Xanthium Strumarium L. ........................ 35 3.1 Xanthium Strumarium L. .................................................................................................. 35 3.2 Bioassay-guided extraction and isolation ................................................................... 40 3.2.1 Further purification for fraction B .................................................................... 42 3.2.2 Further purification for fraction C .................................................................... 49 3.2.3 Further purification for fraction D ................................................................... 51 3.2.4 Further purification for fraction E .................................................................... 57 3.3 Inhibitory activity of the pure compounds against tyrosinase ............................. 60 3.4 Conclusions and discussions ........................................................................................... 61 3.5 Experimental ....................................................................................................................... 64 General instrument ......................................................................................................... 64 Bioassay-guided extraction and isolation ................................................................. 64 Appendix ................................................................................................................ 72 iv List of figures Figure 1.1 Atomic force microscopy performed on epidermal melanocytes. .............................. 2 Figure 1.2 Hypopigmentation (left) verse hyperpigmentation (right) .......................................... 2 Figure 1.3 Symptom of three kinds of hyperpigmentation. .......................................................... 3 Figure 1.4 Biosynthesis of melanin ............................................................................................... 5 Figure 1.5 Signaling pathways of series synthesis of eumelanin and pheomelanin.[34] ................ 7 Figure 1.6 Representitive tyrosinase inhibitory active flavonoids .............................................. 10 Figure 1.7 Representative reported natural organic acids with inhibitory activity against tyrosinase .................................................................................................................................... 11 Figure 1.8 Representative natural glycoside with inhibitory activity against tyrosinase ............ 12 Figure 1.9 representative terpenes reported as tyrosinase inhibitors. ...................................... 14 Figure 1.10 Representative esters as natural tyrosinase inhibitors ............................................ 15 Figure 1.11 Other compounds reported as natural tyrosinase inhibitors .................................. 16 Figure 1.12 Representative TCM traditionally used for skin-lightening purpose ....................... 17 Figure 2.1 Sequence alignment of human tyrosinase ...................................................................... 24 Figure 2.2 Sequence alignment of mushroom tyrosinase (C7FF04) and TYRP1(P17643). .......... 25 Figure 2.3 The metal binding sites of TYRP1 (A) and mushroom tyrosinase (B) ......................... 26 Figure 2.4 The inhibition of arbutin against tyrosinase. ............................................................. 26 Figure 2.5 The inhibition of kojic acid against tyrosinase. .......................................................... 27 Figure 2.7 HTS tyrosinase assay results ....................................................................................... 31 Figure 3.1A Photographs of Xanthium Strumarium L. ................................................................ 35 Figure 3.1B Isolated compounds with anti-cancer effects from Xanthium ................................ 36 Figure 3.1C Representative isolated compounds from Xanthium .............................................. 37 Scheme 3.2A Bioassay-guided isolation of 10 compounds from Xanthium. .............................. 38 Figure 3.2B All 10 compounds