Hypopigmenting Mechanisms of Ethanolic Extract from Sargassum serratifolium and Its Active Components
- Alternative Title
- 톱니모자반 주정추출물과 활성성분의 색소침착 억제 기전
- Abstract
- Hyperpigmentation is an increased deposition of melanin in the skin, which adversely influence the quality of life requiring topical treatment with depigmenting agents. This study aims to investigate the effects of ethanolic extract from the brown alga Sargassum serratifolium (ESS) and its active components sargahydroquinoic acid (SHQA) and sargaquinoic acid (SQA) on melanogenic protein expressions in alpha-melanocyte stimulating hormone (α-MSH)-treated B16F10 mouse melanoma cells and to elucidate their mechanisms of hypopigmenting action.
ESS remarkably reduced tyrosinase (TYR) activity and melanin synthesis in α-MSH-stimulated B16F10 cells in a dose-dependent manner. Western blot analysis revealed that ESS attenuated the expression of melanogenic enzymes, TYR and tyrosinase related protein 1 (TRP1), by cyclic adenosine monophosphate (cAMP)-responsive element-binding protein (CREB)-mediated downregulation of microphthalmia-associated transcription factor (MITF). ESS inhibited accumulation of cellular cAMP that leads to inhibition of CREB phosphorylation. Moreover, ESS activated extracellular signal regulated kinase (ERK), but not Akt and other mitogen-activated protein kinases, which is responsible for post-translational downregulation of MITF. Therefore, ESS attenuated α-MSH-stimulated hyperpigmentation in B16F10 cells through modulation of CREB/ERK signaling pathways. Two anti-melanogenic compounds such as sargahydroquinoic acid and sargaquinoic acid were identified in ESS depending on inhibition of melanin synthesis. These findings suggest that ESS could be a potential agent in the treatment of hyperpigmentation-related skin disorders.
SHQA was the major active compound in the ethanolic extract from Sargassum serratifolium, which exhibited potent hypopigmenting activity. SHQA reduced cellular TYR activity and melanin content in a dose-dependent manner and attenuated the expression of TYR and TRP1, along with their transcriptional regulator, MITF. SHQA also suppressed α-MSH-induced cellular production of cAMP which inhibited protein kinase A (PKA)-dependent CREB activation. Docking simulation data showed strong binding affinity of SHQA to the regulatory subunit RIIβ of PKA, which may also adversely affect PKA and CREB activation. Moreover, SHQA activated ERK1/2 signaling in B16F10 cells, leading to the proteasomal degradation of MITF. SHQA ameliorated hyperpigmentation in α-MSH-stimulated B16F10 cells by downregulating MITF via PKA inactivation and ERK1/2 phosphorylation, indicating that SHQA is a potent therapeutic agent against skin hyperpigmentation disorders.
SQA was identified as another active component in the extract exhibited promising hypopigmenting activity. SQA attenuated cellular TYR activity and melanin production by inhibiting the expression of the melanogenic enzymes TYR, TRP1, and TRP2. SQA markedly reduced intracellular cAMP accumulation in α-MSH-stimulated B16F10 cells, which, consequently, inhibited the PKA-dependent phosphorylation of CREB, leading to the downregulation of MITF. Moreover, SQA increased the phosphorylation of ERK1/2, therefore inhibiting the production of TYR via the proteasome-mediated degradation of MITF. SQA also showed high binding affinity to the cAMP binding domain of PKA; the direct binding of SQA to PKA may exert an additional inhibitory effect on the PKA-dependent CREB activation.
Data in this study reveal that ESS, SHQA, and SQA ameliorate hyperpigmentation in B16F10 cells through the cAMP/CREB- and ERK1/2-mediated downregulation of MITF. These findings suggest that ESS, SHQA, and SQA are candidate depigmenting agents for the treatment of skin hyperpigmentation disorders.
- Issued Date
- 2018
- Awarded Date
- 2018. 8
- Type
- Dissertation
- Keyword
- Hyperpigmentation MITF Melanin Sargassum serratifolium Sargahydroquinoic acid Sargaquinoic acid Tyrosinase
- Publisher
- 부경대학교
- Alternative Author(s)
- 모하메드 샤리풀 아잠
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 식품생명과학과
- Advisor
- Hyeung-Rak Kim
- Table Of Contents
- 요 약 xi
Abstract xiv
Chapter I: General Introduction 1
1. The brown algae Sargassum spp. and their therapeutic potential 2
2. Melanogenesis and related signaling pathways 7
2.1. cAMP-CREB signaling pathway 11
2.2. MITF: the master regulator of melanogenesis 12
2.3. The MAPK signaling pathways 15
2.4. PI3K/Akt signaling pathway 16
2.5. Wnt/β-catenin signaling pathway 17
2.6. Autophagy and other mechanistic targets of hypopigmenting agents 18
3. Hyperpigmentation disorders of the skin 22
4. Current status of commercial hypopigmenting agents 25
5. Purpose of the study 27
Chapter II: Ethanolic extract from Sargassum serratifolium attenuates hyperpigmentation through CREB/ERK signaling pathways in α-MSH-stimulated B16F10 melanoma cells 28
1. Introduction 29
2. Materials and methods 32
2.1. Materials 32
2.2. Preparation of ESS 33
2.3. Isolation of active compounds from ESS 33
2.4. Cell culture and treatment with ESS 35
2.5. Cell viability assay 35
2.6. Cellular TYR activity assay 35
2.7. Measurement of melanin content 36
2.8. Intracellular cAMP assay 36
2.9. Preparation of cytosolic and nuclear extract 37
2.10. Western blot analysis 38
2.11. Statistical analysis 38
3. Results 39
3.1. ESS attenuated cellular TYR activity and melanin synthesis 39
3.2. ESS suppressed the α-MSH-stimulated expression of melanogenic enzymes 39
3.3. ESS downregulated MITF expression through inhibition of CREB 42
3.4. ESS suppressed intracellular cAMP 42
3.5. ESS attenuated MITF through ERK signaling pathway 45
3.6. Identification of hypopigmenting compounds in EES 45
4. Discussion 53
5. Conclusion 56
Chapter III: cAMP and ERK-1/2 mediated downregulation of MITF in B16F10 cells: a therapeutic target of sargahydroquinoic acid against skin hyperpigmentation 57
1. Introduction 58
2.Materials and methods 61
2.1. Materials 61
2.2. Plant material, extraction and isolation of SHQA 61
2.3. Cell culture, treatment and viability assay 62
2.4. Melanin content assay 63
2.5. Cellular TYR activity assay 63
2.6. Western blot analysis 64
2.7. Intracellular cAMP assay 64
2.8. Immunoprecipitation 65
2.9. Molecular docking simulation 65
2.10. Data and Statistical analysis 66
3. Results 67
3.1. SHQA inhibits cellular TYR activity and melanin production 67
3.2. SHQA suppresses the production of melanogenic enzymes TYR and TRP1 67
3.3. SHQA inhibits CREB activation in B16F10 cells 71
3.4. SHQA suppresses cellular cAMP induction and PKA activation 71
3.5. SHQA has high binding affinity to PKA (RIIβ) 73
3.6. SHQA induces proteasome-mediated degradation of MITF via ERK1/2 77
4. Discussion 84
5. Conclusion 88
Chapter IV: Sargaquinoic acid ameliorates hyperpigmentation through cAMP and ERK-mediated downregulation of MITF in α-MSH-stimulated B16F10 cells 89
1. Introduction 90
2. Materials and methods 92
2.1. Materials 92
2.2. Cell culture and viability assay 92
2.3. Melanin content assay 93
2.4. Cellular TYR activity assay 94
2.5. Isolation of cytosolic and nuclear fractions of B16F10 cells 94
2.6. Western blot analysis 95
2.7. Intracellular cAMP immunoassay 95
2.8. Immunoprecipitation 96
2.9. Molecular docking simulation 96
2.10. Data and Statistical analysis 97
3. Results 98
3.1. SQA attenuates the intracellular TYR activity and melanin content 98
3.2. SQA inhibits the expression of melanogenic enzymes 98
3.3. SQA attenuates hyperpigmentation through the CREB-mediated 99
3.4. SQA inhibits intracellular cAMP induction and PKA activation 103
3.5. SQA leads to proteasomal degradation of MITF through ERK1/2 109
4. Discussion 117
5. Conclusion 120
References 121
- Degree
- Doctor
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