Anti-inflammatory and anti-melanogenic effects of pinostrobin
- Abstract
- Pinostrobin, an edible bioflavonoid was discovered more than 60 years ago in the heartwood of pine trees (Pinus strobus) andBoesenbergia rotunda(L.), a perennial herb in the ginger family commonly known as fingerroot. Isolated pinostrobin compounds have shown many valuable pharmacological activities, including anticancer, antiviral, antioxidant, antileukemic, antiulcer, anti-leptospira and anti-aromatase effects. In addition, the finger roots of medicinal plants are mainly used for healing and anti-inflammatory in Korean medicine, and the anti-inflammatory effect of extracted resin ester compounds is unclear. Therefore, we investigated the anti-inflammatory result of pinostrobin on the expression of pro-inflammatory lipopolysaccharide (LPS) mediators in RAW264.7 macrophages and the molecular mechanism of this activity. We observed that the highest concentration of pinostrobin (20μg/mL) had no significant impact on cell viability and cell morphological modification. Furthermore, pinostrobin suppressed LPS-induced nitric oxide and prostaglandin E2 and reduced the expression of iNOS and COX-2 in a concentration-dependent manner. In addition, pinostrobin inhibited the production of pro-inflammatory cytokines, containing IL-12 and TNF-α in LPS-stimulated RAW264.7 cells. Other studies have shown that pinostrobin significantly reduces LPS-influenced NF-κB, p50 and p65 subunits. The inhibitory effect of pinostrobin was confirmed in LPS-microinjected zebrafish larvae due to decreased neutrophil and macrophage recruitment. Taken together, our results indicate that pinostrobin attenuates inflammation bothin vitroandin vivo, by obstructing the activation of NF-κB activity. It could be used as an effective medicine for inflammatory-related diseases.
In the Paroral study, we observed that pinostrobin inhibits melanin biogenesis bothin vitroandin vivo. The highest concentration of pinostrobin (50 µg / mL) inhibited melanogenesis in B16F10 melanoma cells without a significant effect on cell viability and cell morphological modification. Our experimental results showed that pinostrobin reduced mushroom tyrosinase activityin vitroand significantly reduced extracellular and intracellular melanin production in melanoma cells, which was associated with inhibition of α-MSH-influenced tyrosinase expression. We also found that pinostrobin inhibits pigment formulation in α-MSH-stimulated zebrafish larvae without severe toxicity. These results suggest that pinostrobin is a potent inhibitor of melanogenic activityin vitroandin vivo.
Key words:Pinostrobin; Flavonoid; Inflammation; NO, COX-2; IL-12, TNF-α; NF-κB; Neutrophil; Macrophages; α-MSH; Melanin; Tyrosinase; Zebrafish
- Issued Date
- 2022
- Awarded Date
- 2022-08
- Type
- Dissertation
- Affiliation
- 제주대학교 대학원
- Department
- 대학원 해양생명과학과
- Advisor
- 김기영
- Table Of Contents
- PART 01 1
Abstract 2
1. Introduction. 4
2. Material and methods 6
2.1 Reagents and antibodies 6
2.2 Cell culture and viability 6
2.3 Flow cytometric analysis 7
2.4 Isolation of total RNA and Reverse transcriptase-polymerase chain reactions (RT-PCR) 7
2.5 Western blotting 8
2.6 NO assay . 9
2.7 Enzyme-linked immunosorbent assay (ELISA) 9
2.8 Immunofluorescence staining . 9
2.9 Molecular docking 10
2.10 Maintenance of zebrafish 10
2.11 Maintenance of zebrafishHeart rate, abnormality, and mortality in LPS microinjected zebrafish larvae. 10
2.12 Macrophage and neutrophil staining in LPS-microinjected zebrafish 11
2.13 Isolation of total zebrafish mRNA and RT- PCR 11
2.14 Statistical analysis. 12
3. Results. 13
3.1 Pinostrobin at concentrations less than 20 μM is not toxic to RAW 264. 7 macrophages 13
3.2 Pinostrobin inhibits NO and PGE2 release along with downregulation of iNOS and COX-2 expression in LPS-stimulated RAW 264.7 macrophages 16
3.3 Pinostrobin decreases LPS-induced IL-12 and TNF-α production in LPS- stimulated RAW 264.7 macrophages 18
3.4 Pinostrobin downregulates nuclear translocation of NF-κB in LPS-stimulated RAW 264.7 macrophages 20
3.5 Pinostrobin potently binds to the TLR4/MD2 complex . 22
3.6 Pinostrobin decreases mortality and abnormalities in LPS-microinjected zebrafish larvae 28
3.7 Pinostrobin attenuates recruitment macrophages and neutrophils in LPS- microinjected zebrafish larvae accompanied by downregulation of proinflammatory genes 31
4. Discussion. 33
5. References. 36
PART 02 41
Abstract 42
1. Introduction. 43
2. Material and methods 46
2.1 Reagents and antibodies. 46
2.2 In vitro mushroom tyrosinase assay. 46
2.3 Molecular docking . 47
2.4 Cell culture and cell viability assay . 47
2.5 Flow cytometry analysis 48
2.6 Measurement of extracellular and intracellular melanin content 48
2.7 Measurement of Intracellular tyrosinase activity. 48
2.8 Enzyme-linked immunosorbent assay (ELISA) for cAMP . 49
2.9 Reverse transcription-polymerase chain reaction (RT-PCR) 49
2.10 Western blot analysis . 50
2.11 Maintenance of zebrafish. 50
2.12 Melanogenesis and heart rate in zebrafish . 50
2.13 Statistical analysis 51
3. Results. 52
3.1 Pinostrobin inhibits in vitro mushroom tyrosinase activity . 52
3.2 Pinostrobin non-competitively binds to tyrosinase 54
3.3 Pinostrobin concentrations above 100 μM are cytotoxic. 56
3.4 Pinostrobin decreases melanin production and intracellular tyrosinase activity 58
3.5 Pinostrobin inhibits cAMP, p-CREB, MITF, and tyrosinase expression in α- MSH-stimulated B16F10 melanoma cells. 60
3.5 Pinostrobin inhibits melanin biosynthesis in zebrafish larvae. 62
4. Discussion. 64
5. References. 67
- Degree
- Master
- Publisher
- 제주대학교 대학원
- Appears in Collections:
- General Graduate School > Marine Life Sciences
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