Antioxidant Potential-Rich Betel Leaves (Piper betle L.) Exert Depigmenting Action by Triggering Autophagy and Downregulating MITF/Tyrosinase In Vitro and In Vivo

Abstract

Each individual has a unique skin tone based on the types and quantities of melanin pigment, and oxidative stress is a key element in melanogenesis regulation. This research sought to understand the in vitro and in vivo antioxidant and depigmenting properties of betel leaves (Piper betle L.) extract (PBL) and the underlying mechanism. Ethyl acetate fractions of PBL (PBLA) demonstrated excellent phenolic content (342 ± 4.02 mgGAE/g) and strong DPPH, ABTS radicals, and nitric oxide (NO) scavenging activity with an IC₅₀ value of 41.52 ± 1.02 μg/mL, 45.60 ± 0.56 μg/mL, and 51.42 ± 1.25 μg/mL, respectively. Contrarily, ethanolic extract of PBL (PBLE) showed potent mushroom, mice, and human tyrosinase inhibition activity (IC₅₀ = 7.72 ± 0.98 μg/mL, 20.59 ± 0.83 μg/mL and 24.78 ± 0.56 μg/mL, respectively). According to gas chromatography-mass spectrometry, PBL is abundant in caryophyllene, eugenol, O-eugenol, 3-Allyl-6-methoxyphenyl acetate, and chavicol. An in vitro and in vivo investigation showed that PBLE suppressed tyrosinase (Tyr), tyrosinase-related protein-1 and -2 (Trp-1 and Trp-2), and microphthalmia-associated transcription factors (MITF), decreasing the formation of melanin in contrast to the untreated control. PBLE reduced the cyclic adenosine monophosphate (cAMP) response to an element-binding protein (CREB) phosphorylation by preventing the synthesis of cAMP. Additionally, it activates c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (p38), destroying Tyr and MITF and avoiding melanin production. Higher levels of microtubule-associated protein-light chain 3 (LC3-II), autophagy-related protein 5 (Atg5), Beclin 1, and lower levels of p62 demonstrate that PBLE exhibits significant anti-melanogenic effects via an autophagy-induction mechanism, both in vitro and in vivo. Additionally, PBLE significantly reduced the amount of lipid peroxidation while increasing the activity of several antioxidant enzymes in vivo, such as catalase, glutathione, superoxide dismutase, and thioredoxin. PBLE can therefore be employed in topical formulations as a potent skin-whitening agent.

Keywords: Piper betle; antioxidant; autophagy; cAMP; melanogenesis.

Figure 1

Antioxidant effects of extract/factions of betel leaves extract (PBL). (A) Total phenol (TPC) and flavonoid contents (TFC),. (B) Radical scavenging activities of PBL extract/fractions. (C) Principal component analysis. (D) Hierarchical clustering analysis with heat map. mgGAE/g: mg gallic acid equivalent/g extract; mgCE/g: mg catechin equivalent/g extract; PBLE: ethanolic extract of PBL; PBLH: hexane fraction of PBL; PBLA: ethyl acetate fraction of PBL; and PBLW: aqueous fraction of PBL. Numerical no. represents the compounds described in Table 1.

Figure 2

Tyrosinase inhibitory effects of PBL extract/fractions. (A) Effect of PBL extract/fractions on mushroom tyrosinase. (B) The inhibitory effect of PBLE on mushroom tyrosinase, mice, and human cellular tyrosinase. Mean ± SD (n = 3). ** p < 0.05 vs. non-treated (NT) control. PBLE: ethanolic extract of PBL; PBLH: hexane fraction of PBL; PBLA: ethyl acetate fraction of PBL; PBLW: aqueous fraction of PBL; and ARB: arbutin.

Figure 3

The depigmenting effects of PBLE on MNT-1 cells. Effect of PBLE on (A) cell viability, (B) melanin production, (C) Tyrosinase (Tyr) inhibition activity measured by zymography, (D) proteins involved in melanogenesis, including microphthalmia-associated transcription factor (MITF), Tyr, tyrosinase-related protein-1 (Trp-1), and -2 (Trp-2), as determined by Western blotting. Densitometric analysis of relative protein expression matching to Figure 3D. Mean ± SD (n = 3). ** p < 0.05 vs. non-treated (NT) control. NS: non-significant compared to NT. ARB: arbutin.

Figure 4

The effect of PBLE on mitogen-activated protein kinase (MAPK) phosphorylation in MNT-1 cells. (A) Effects of PBLE concentration on the activation of MAPK proteins. (B) Effects of various MAPK protein inhibitors on the expression of MITF and Tyr, with or without PBLE. Densitometric analysis of relative protein expression are presented in adjacent figure (C) Effects of different MAPK protein inhibitors on melanin production, with or without PBLE. Mean ± SD (n = 3). ** p < 0.05 against the untreated (NT) control. NS: non-significant compared to equivalent inhibitor alone. U0126: extracellular signal-regulated protein kinases (ERKs) inhibitor; SB239063: p38 inhibitor; and SP600125: c-jun N-terminal kinases (JNKs) inhibitor.

Figure 5

The depigmenting impact of PBLE on isobutylmethylxanthine (IBMX)-induced MNT-1 cells. (A) PBLE prevents the overproduction of melanin caused by IBMX. (B) The effect of PBLE on IBMX-induced cellular cyclic adenosine monophosphate (cAMP) production. (C) PBLE inhibits IBMX-stimulated causes cAMP response to element-binding protein (CREB) protein phosphorylation. Densitometric analysis of relative protein expression are presented in adjacent figure. (D) Effect of PBLE on proteins associated in melanogenesis, such as MITF, Tyr, Trp-1, and Trp-2, in IBMX-stimulated cells, as measured by Western blotting. (E) Densitometric analysis of relative protein expression, as depicted in Figure 5D. Mean ± SD (n = 3). # p < 0.05 vs. non-treated (NT) control. ** p < 0.05 vs. IBMX-treated control. ARB: arbutin.

Figure 6

The effect of PBLE on the activation of autophagy in MNT-1 cells. (A) Effect of PBLE on proteins associated with autophagy, such as microtubule-associated protein-light chain 3 (LC3), Atg5, Beclin 1, and p62, in MNT-1 cells, as measured by Western blotting. (B) Effects of autophagy inhibitors on the expression of LC3, with or without PBLE. (C) Effects of autophagosome fusion with lysosome inhibitor on the expression of LC3 and p62, with or without PBLE. (D) Effects of autophagy inhibitors on the expression of Tyr, with or without PBLE. (E) Effects of autophagy inhibitors on the melanin production, with or without PBLE. Mean ± SD (n = 3). ** p < 0.05 against the untreated (NT) control. NS: non-significant compared to equivalent inhibitor alone. 3MA: 3-Methyladenine (autophagy inhibitor), WTM: wortmannin, (PI3K inhibitor), and CQ: chloroquine (autophagosome fusion inhibitor).

Figure 7

The depigmenting effects of PBLE on UVB-induced HRM-2 mice. (A) Representative images demonstrate the dorsal skin’s variations in pigmentation in the studied animals. (B) The average values following the final UVB exposure were subtracted from the average baseline values before treatment to determine the ΔL* and Δa* values displayed in the upper and lower panels, respectively. (C) The dorsal skin slices of mice stained with Fontana–Masson dye. (D) Western blot analysis of p-CREB, tyr, Trp-1, Trp-2, and MITF. Quantification and statistical analysis of the band intensities are in adjacent figure. (E) Tissue malonaldehyde (MDA) levels. (F) Antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels in mice skin tissue. (G) Tissue thioredoxin activity. (H) Autophagy biomarkers such as LC3, Atg5 and p62 were confirmed by Western blot analysis. (I) Quantification and statistical analysis of the band intensities of represented Figure 7G. NNFE. Results are presented as the means ± SDs (n =3). # p < 0.05 vs. non-treated (NT) control. ** p < 0.05 vs. UVB-treated control. ARB: arbutin.

Figure 8

The potentially effective depigmenting mechanism of betel leaves (Piper betle L.). Green arrows: activation by PBLE; red arrows: inhibition by PBLE.