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Anti-melanogenenic and anti-inflammatory effect of Hibiscus syriacus anthocyanin-rich extracts

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Abstract
Hibiscus syriacus is the national flower of South Korea and grows in a wide climatic range from mild to tropical temperature. The bark and roots of H. syriacus have been used to cure diarrhea and bacterial infection, and its seed are effective to fever and cold. Recently, native flowers of H. syriacus possesses promising potential as a new edible source and colorants with various anthocyanins. However, the function of anthocyanins isolated from H. syriacus has been determined. In the current study, we, for the first time, evaluated whether H. syriacus varieties, Pulsae and Paetanshim, anthocyanin-rich extract (PS and PTS, respectively) inhibits melanin biogenesis. Our results showed that PS and PTS did not strongly downregulate mushroom tyrosinase activity in vitro; but significantly decreased the extracellular and intracellular melanin production in B16F10 cells accompanied by the inhibition of α-melanocyte-stimulating hormone (α-MSH)-induced microphthalmia-associated transcription factor (MITF) and tyrosinase expression. We also found that PS and PTS attenuated pigmentation in α-MSH-stimulated zebrafish larva without any severe toxicity. Furthermore, PS and PTS activated the phosphorylation of extracellular signal-regulated kinase (ERK) and a specific ERK inhibitor, PD98059, resulted in the recovery of intracellular and extracellular melanin downregulation induced by PS and PTS in B16F10 cells and of melanogenesis in zebrafish larvae. PS and PTS possess 17 specific anthocyanins, which mostly target dual specificity protein phosphastase 7 (DUSP7) according to molecular docking prediction. These findings suggest that anthocyanins from PS and PTS inhibit melanogenesis in vitro and in vivo by activating the ERK signaling pathway.
Author(s)
Wisurumini Arachchilage Hasitha Maduranga Karunarathne
Issued Date
2019
Awarded Date
2019. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009121
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Kim, Gi Young
Table Of Contents
Acknowledgments . ii
Table of Content . iii
List of Figures vi
List of Tables viii
PART 01 ix
Abstract . x
1. Introduction . 1
2. Material and Methods 4
2.1 Plant material and sample preparation . 4
2.2 Regents and antibodies 4
2.3 Cell culture and Cell viability assay 5
2.4 Flow cytometry Analysis. 5
2.5 In vitro mushroom tyrosinase assay . 6
2.6 Measurement of extracellular and intracellular melanin content 6
2.7 Real-time reverse transcription-polymerase chain reaction (RT-PCR) . 6
2.8 Western blot analysis 7
2.9 Maintenance and phenotype based evaluation of zebrafish 8
2.10 Determination of melanogenic inhibitors effects on zebrafish toxicity . 8
2.11 UPLCQT of MS for flavonoid analysis . 9
.12 Statistical Analysis 9
3. Results . 10
3.1 PS and PTS do not alter cell viability at the concentrations used in this study . 10
3.2 PS and PTS decrease extracellular and intracellular melanin production in B16F10 cells stimulated by α-MSH. 12
3.3 PS and PTS does not downregulate mushroom tyrosinase activity 14
3.4 PS and PTS inhibits the expression of MITF and tyrosinase in α-MSHstimulated B16F10 cells 15
3.5 PS and PTS inhibit melanin synthesis in zebrafish larvae 17
3.6 PS- and PTS-induced ERK phosphorylation regulates MITF expression . 19
3.7 The ERK signaling pathway regulates melanogenesis in PS- and PTS-treated B16F10 cells and zebrafish larvae . 21
3.8 Tentative identification of metabolites in PS . 24
4. Discussion . 32
5. References. 36
PART 02 . 41
Abstract . 42
1. Introduction . 43
2. Material and Methods 45
2.1 Plant material and sample preparation . 45
2.2 Reagents and antibodies 45
2.3 Cell culture and MTT assay 46
2.4 Flow cytometry analysis 46
2.5 NO Assay . 46
2.6 Isolation of total RNA and Reverse transcriptase polymerase chain reactions (RT-PCR) . 47
2.7 Western blotting assay 48
2.8 Enzyme immunosorbent assay (ELISA) 48
2.9 Zebrafish maintenance and toxicity evaluation 49
2.10 Exposure of LPS by microinjection to zebrafish and Sudan black and Neutral Red staining 49
2.11 Statistical analysis . 50
3. Results . 50
3.1 Effect of PS on the viability of RAW264.7 macrophage cells 50
3.2 Effect of PS on iNOS, COX-2 expression and NO, PGE 2 Production 52
3.3 Effect of PS on LPS-induced TNF-α, IL-6, IL-12 production and mRNA expression . 54
3.4 Effect of PS on LPS-induced NF-κb nuclear translocation . 56
3.5 Effect of PS on Morphological changes and toxicity of zebrafish 57
3.6 Effect of PS on LPS-induced recruitment of neutrophil and macrophages in zebrafish . 59
4. Discussion . 61
5. References. 65
Degree
Master
Publisher
제주대학교 대학원
Citation
Wisurumini Arachchilage Hasitha Maduranga Karunarathne. (2019). Anti-melanogenenic and anti-inflammatory effect of Hibiscus syriacus anthocyanin-rich extracts
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General Graduate School > Marine Life Sciences
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