Amorphigenin from Amorpha fruticosa L. Root Extract Induces Autophagy-Mediated Melanosome Degradation in mTOR-Independent- and AMPK-Dependent Manner

In this study, we investigated the depigmentation effect of Amorpha fruticosa L. root extract (RE), an herbal medicine. A. fruticosa RE significantly induced depigmentation in α-MSH-treated B16F10 cells at noncytotoxic concentrations. Further, the RE decreased the protein levels of the melanosomal proteins Tyr and Pmel without decreasing their transcript levels. We found that MG132, a proteasome complex inhibitor, was unable to rescue the protein levels, but PepA/E-64D (a lysosomal enzyme inhibitor), 3-MA (a representative autophagy inhibitor), and ATG5 knockdown effectively rescued the protein levels and inhibited the depigmentation effect following RE treatment. Among rotenoids, amorphigenin composed in the RE was identified as a functional chemical that could induce depigmentation; whereas rapamycin, an mTOR inhibitor and a nonselective autophagy inducer, could not induce depigmentation, and amorphigenin effectively induced depigmentation through the degradation of melanosomal proteins. Amorphigenin activated AMPK without affecting mTOR, and knockdown of AMPK offset the whitening effect through degradation of melanosome proteins by amorphigenin. Results from this study suggested that amorphigenin can induce degradation of the melanosome through an AMPK-dependent autophagy process, and has the potential to be used as a depigmentation agent for the treatment of hyperpigmentation.

Recent work has reported that autophagy is related to skin color [15]. Autophagy is a destruction mechanism that naturally degrades unnecessary or nonfunctional cellular components under cellular physiological stress conditions [16]. Autophagy is classified as either nonselective autophagy or selective autophagy, depending on the specificity of target molecules or organelles (cargo). Several studies have reported on the selective autophagy of several organelles, including the endoplasmic reticulum, lysosome, mitochondria, nucleus, and peroxisome. In both human [17,18] and rodent [19], melanomas are often characterized by the accumulation of autophagosomes that contain preformed melanosomes (Bomirski et al. 1987), perhaps suggesting a role for autophagosomes in melanosomal degradation. Although the molecular mechanisms underlying melanosome autophagy remain unclear, several chemicals have been proposed that could induce skin depigmentation through autophagy [20][21][22][23][24].
In the present study, we investigated the effects of the root extract (RE) of Amorpha fruticosa L. on autophagy-mediated melanosome degradation in B16F10 cells, and determined that an active ingredient, amorphigenin, can induce depigmentation by inducing autophagy in B16F10 cells.

Cell Viability Assay
B16F10 cells were seeded in 96-well plates and incubated overnight. Following incubation, the cells were treated with the indicated concentrations of A. fruticosa RE for 48 h. Next, MTT was added, and the cells were incubated for another 3 h at 37 • C in a 5% CO 2 humidified incubator. After incubation, the cell culture medium was discarded, and dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added for 20 min at room temperature (RT). Cell viability was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 595 nm.

Melanin Assay
B16F10 cells were seeded in 6-well plates and incubated overnight. Following incubation, the cells were treated with α-MSH in the presence or absence of A. fruticosa RE for 48 h, harvested by trypsinization, and dissolved in 1 N NaOH containing 10% DMSO at 65 • C for 24 h. The melanin content of cells was measured using a microplate reader (Bio-Rad Laboratories) at a wavelength of 415 nm.

L-DOPA Oxidation
B16F10 cells were seeded in 6-well plates and incubated overnight. Following incubation, the cells were treated with α-MSH in the presence or absence of A. fruticosa RE for 48 h, harvested by trypsinization, and lysed with 1% Triton X-100 in phosphate-buffered saline (PBS) for 1 h. A total of 40 µg of proteins in solution were incubated with 2 mg/mL of L-3,4-dihydroxyphenylalanine (L-DOPA) (Sigma-Aldrich) at 37 • C for 4 h. Following incubation, absorbance was measured on a microplate reader (Bio-Rad Laboratories) at a wavelength of 490 nm.

Preparation of Root Bark Extract and Isolation of Amorphigenin
Root bark extract (RE) was prepared by adding 20 mL ethanol per 1 g dried and chopped root bark of A. fruticosa and then sonicating the sample at RT for 4 h. Metabolite analysis of the RE was performed using the Vion UPLC-ESI-Q-TOF/MS system (Waters, Milford, MA, USA). For purification of amorphigenin from RE, the dried root bark of A. fruticose (0.5 kg) was extracted with ethanol (10 L) for one week at RT. The solution was then evaporated to obtain the concentrate (75 g). The concentrate was dissolved in water and portioned with n-hexane, ethyl acetate, and butanol in order to achieve polarity. Among the solvent fractions, the ethyl acetate (8 g) was fractionated on a silica-gel column (10 × 40 cm, 230~400 mesh, 50 g) using the n-hexane-ethyl acetate mixture to yield 8 fractions (A-H). Fraction D (1.8 g) was subjected to a reversed silica gel column (250 × 30 mm, S-5 µm, 12 nm, YMC) under prep-HPLC (Forte-R, YMC CO., LTD., Kyoto, Japan) and eluted with an isocratic of 80% MeOH at a rate of 10 mL/min to give 10 subfractions (Fr. D1-D10). Subfraction D5 (280 mg), which was enriched with amorphigenin, was further purified by recycling LC (LC 9110 NEXT, JAI Co., Ltd., Tokyo, Japan) on reversed silica-gel (250 × 30 mm, S-5 µm, 12 nm, Thermo Fisher Scientific) with a gradient condition of 0-100% acetonitrile to obtain amorphigenin as a single compound (160 mg). The amorphigenin was characterized and identified using spectroscopic methods, in addition to comparisons with published data [25].

Confocal Microscopy
B16F10 cells were seeded on a coverslip and incubated overnight. Following incubation, the cells were treated with RE, fixed in 4% paraformaldehyde for 15 min at RT, and permeabilized using 0.1% Triton X-100 for 10 min at RT. Following permeabilization, we used 1% bovine serum albumin (BSA) for blocking and then incubated the cells with anti-Trp-1 primary antibody (Santa Cruz Biotechnology, CA, USA) overnight at 4 • C. The cells were treated with anti-mouse-fluorescein isothiocyanate (FITC; Sigma-Aldrich) for 1 h at RT and then treated with 20 µg/mL of 4 ,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 10 min at RT. The cells were mounted on a glass slide with VECTASHIELD ® mounting media (Vector Laboratories, CA, USA), and images were acquired on an Olympus FluoView™ FV1000 confocal microscope (Olympus, Tokyo, Japan).

A. fruticosa RE Induces Depigmentation in α-MSH-Stimulated B16F10 Cells
To determine the cytotoxicity of A. fruticosa RE in B16F10 cells, we treated B16F10 cells with various concentrations of A. fruticosa RE for 48 h and then measured cell viability using an MTT assay. We found that A. fruticosa RE-mediated cytotoxicity was significantly increased from 12.5 ppm ( Figure 1A). To determine whether A. fruticosa RE affects melanogenesis, B16F10 cells were stimulated with α-MSH in the presence or absence of the indicated concentrations of A. fruticosa RE for 48 h. Kojic acid (200 ppm), a Tyr inhibitor, was used as a positive control for depigmentation. A. fruticosa RE effectively inhibited α-MSHmediated accumulation of melanin and cellular L-DOPA oxidation ( Figure 1B,C). This result indicated that A. fruticosa RE could inhibit melanogenesis through a decrease in intracellular tyrosinase activity. We found that α-MSH upregulated MITF, Tyr, and Pmel expression, while A. fruticosa RE effectively decreased levels of Tyr and Pmel, but not MITF, proteins ( Figure 1D). A. fruticosa RE did not affect expression at the messenger RNA level for each gene ( Figure 1E). This observation suggested that A. fruticosa RE-mediated depigmentation may be due to the degradation of melanosomal proteins involved in melanin synthesis. We inhibited the ubiquitin-proteasome system (UPS) that induces cellular protein degradation by treating α-MSH-treated B16F10 cells with a proteasome inhibitor, MG132, along with A. fruticosa RE. We found that MG132 failed to rescue the reduction in melanosomal protein levels induced by A. fruticosa RE ( Figure 1F), and did not inhibit depigmentation induced by A. fruticosa RE treatment ( Figure 1G). This result indicated that A. fruticosa RE inhibits melanogenesis through a mechanism in which melanosome-related proteins are degraded in a UPS-independent manner. not MITF, proteins ( Figure 1D). A. fruticosa RE did not affect expression at the messenger RNA level for each gene ( Figure 1E). This observation suggested that A. fruticosa RE-mediated depigmentation may be due to the degradation of melanosomal proteins involved in melanin synthesis. We inhibited the ubiquitin-proteasome system (UPS) that induces cellular protein degradation by treating α-MSH-treated B16F10 cells with a proteasome inhibitor, MG132, along with A. fruticosa RE. We found that MG132 failed to rescue the reduction in melanosomal protein levels induced by A. fruticosa RE ( Figure 1F), and did not inhibit depigmentation induced by A. fruticosa RE treatment ( Figure 1G). This result indicated that A. fruticosa RE inhibits melanogenesis through a mechanism in which melanosome-related proteins are degraded in a UPS-independent manner. fruticosa RE in the presence or absence of 120 nM MG132, the indicated protein levels (F) and melanin content (G) were analyzed. Data were obtained from at least three independent experiments, and the values represent the mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001 according to t-tests.

A. fruticosa RE-Mediated Depigmentation Depends on Autophagy
To determine whether lysosome-dependent proteolysis is involved in A. fruticosa REmediated depigmentation, B16F10 cells were stimulated with α-MSH in the presence of A. fruticosa RE and pepstatin A1 (PepA)/E-64d, an inhibitor of lysosomal proteinase. The degradation of melanosomal proteins by A. fruticosa RE was inhibited by PepA/E-64d (Figure 2A). We also investigated whether the autophagy pathway was involved in the A. fruticosa RE-mediated depigmentation by inhibiting autophagy in B16F10 cells through fruticosa RE in the presence or absence of 120 nM MG132, the indicated protein levels (F) and melanin content (G) were analyzed. Data were obtained from at least three independent experiments, and the values represent the mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001 according to t-tests.

A. fruticosa RE-Mediated Depigmentation Depends on Autophagy
To determine whether lysosome-dependent proteolysis is involved in A. fruticosa RE-mediated depigmentation, B16F10 cells were stimulated with α-MSH in the presence of A. fruticosa RE and pepstatin A1 (PepA)/E-64d, an inhibitor of lysosomal proteinase. The degradation of melanosomal proteins by A. fruticosa RE was inhibited by PepA/E-64d (Figure 2A). We also investigated whether the autophagy pathway was involved in the A. fruticosa RE-mediated depigmentation by inhibiting autophagy in B16F10 cells through treatment with 3-MA or ATG5 knockdown. The autophagy inhibitor 3-MA not only restored the level of melanosomal proteins in the A. fruticosa RE-treated B16F10 cells, but also counteracted the A. fruticosa RE-mediated depigmentation effect ( Figure 2B,C). Knockdown of the expression of the ATG5 gene, an essential gene for autophagy [27], also inhibited the degradation of melanosomal proteins and depigmentation by A. fruticosa RE ( Figure 2D,E). The Trp-1-associated dotty subcellular pattern, representing melanosomes, decreased significantly following A. fruticosa RE treatment, while ATG5 knockdown res-cued the decrease in melanosomes ( Figure 2F). Together, these results suggested that an autophagy process was involved in A. fruticosa RE-mediated degradation of melanosomal proteins and depigmentation in the α-MSH-stimulated B16F10 cells. treatment with 3-MA or ATG5 knockdown. The autophagy inhibitor 3-MA not only restored the level of melanosomal proteins in the A. fruticosa RE-treated B16F10 cells, but also counteracted the A. fruticosa RE-mediated depigmentation effect ( Figure 2B,C). Knockdown of the expression of the ATG5 gene, an essential gene for autophagy [27], also inhibited the degradation of melanosomal proteins and depigmentation by A. fruticosa RE ( Figure 2D,E). The Trp-1-associated dotty subcellular pattern, representing melanosomes, decreased significantly following A. fruticosa RE treatment, while ATG5 knockdown rescued the decrease in melanosomes ( Figure 2F). Together, these results suggested that an autophagy process was involved in A. fruticosa RE-mediated degradation of melanosomal proteins and depigmentation in the α-MSH-stimulated B16F10 cells.

Identification of Amorphigenin as a Functional Metabolite in A. fruticosa RE
A previous study [25] identified several flavones and rotenoids in A. fructicosa roots. Amorphigenin was a major rotenoid ( Figure 3A) and showed a depigmentation effect in B16F10 cells. Amorphigenin showed significant cytotoxicity above 0.098 ppm in B16F10 , and confocal microscopy images (F) were performed. Data were obtained from at least three independent experiments, and the values represent the mean ± SEM. Note: ns, not significant; ** p < 0.01 and *** p < 0.001 according to t-tests. Scale bar = 10 µm.

Identification of Amorphigenin as a Functional Metabolite in A. fruticosa RE
A previous study [25] identified several flavones and rotenoids in A. fructicosa roots. Amorphigenin was a major rotenoid ( Figure 3A) and showed a depigmentation effect in B16F10 cells. Amorphigenin showed significant cytotoxicity above 0.098 ppm in B16F10 cells. For this reason, we selected 0.049 ppm as the maximum concentration in our experiments ( Figure 3B). Amorphigenin effectively induced depigmentation ( Figure 3C) and decreased Tyr and Pmel levels ( Figure 3D) without affecting mRNA ( Figure 3E) levels, as observed for A. fruticosa RE. These result suggested that amorphigenin is a functional metabolite that can induce depigmentation in A. fruticosa RE through degradation of melanosomal proteins. cells. For this reason, we selected 0.049 ppm as the maximum concentration in our experiments ( Figure 3B). Amorphigenin effectively induced depigmentation ( Figure 3C) and decreased Tyr and Pmel levels (Figure3D) without affecting mRNA ( Figure 3E) levels, as observed for A. fruticosa RE. These result suggested that amorphigenin is a functional metabolite that can induce depigmentation in A. fruticosa RE through degradation of melanosomal proteins.

Amorphigenin-Mediated Depigmentation Depends on Autophagy
To determine whether an autophagy process is involved in amorphigenin-mediated depigmentation, α-MSH-stimulated B16F10 cells were treated with amorphigenin in the presence or absence of PepA/E-64d. PepA/E-64d effectively inhibited the amorphigenininduced decrease in Tyr and Pmel expression levels ( Figure 4A). The amorphigenin-mediated degradation of melanosomal proteins and depigmentation effect also disappeared in ATG5-knockdown cells ( Figure 4B,C). We reasoned that if amorphigenin showed a depigmentation effect via autophagy, it should show the same depigmentation effect in B16F10 cells in which melanosomes were already formed, because autophagy can degrade preformed cellular components. To test whether amorphigenin has a depigmentation effect on preformed pigments, we treated B16F10 cells with amorphigenin on day 2 following α-MSH stimulation. We found that amorphigenin induced a significant loss of the preformed melanin in a time-dependent manner ( Figure 4D), and 3-MA could inhibit the depigmentation effect induced by amorphigenin ( Figure 4E). These results indicated that

Amorphigenin-Mediated Depigmentation Depends on Autophagy
To determine whether an autophagy process is involved in amorphigenin-mediated depigmentation, α-MSH-stimulated B16F10 cells were treated with amorphigenin in the presence or absence of PepA/E-64d. PepA/E-64d effectively inhibited the amorphigenininduced decrease in Tyr and Pmel expression levels ( Figure 4A). The amorphigeninmediated degradation of melanosomal proteins and depigmentation effect also disappeared in ATG5-knockdown cells ( Figure 4B,C). We reasoned that if amorphigenin showed a depigmentation effect via autophagy, it should show the same depigmentation effect in B16F10 cells in which melanosomes were already formed, because autophagy can degrade preformed cellular components. To test whether amorphigenin has a depigmentation effect on preformed pigments, we treated B16F10 cells with amorphigenin on day 2 following α-MSH stimulation. We found that amorphigenin induced a significant loss of the preformed melanin in a time-dependent manner ( Figure 4D), and 3-MA could inhibit the depigmentation effect induced by amorphigenin ( Figure 4E). These results indicated that amorphigenin is a functional chemical in A. fruticosa RE that can induce depigmentation through autophagy-mediated melanosome degradation in α-MSH-treated B16F10 cells.
Curr. Issues Mol. Biol. 2022, 2, FOR PEER REVIEW 8 amorphigenin is a functional chemical in A. fruticosa RE that can induce depigmentation through autophagy-mediated melanosome degradation in α-MSH-treated B16F10 cells. After experiments were performed according to the schematic, the melanin content was measured (D,E). Data were obtained from at least three independent experiments, and the values represent the mean ± SEM. Note: ns, not significant; ** p < 0.01 and *** p < 0.001 according to t-tests.

Amorphigenin Induces Degradation of Melanosomes in an mTOR-Independent and AMPK-Dependent Manner
To test whether autophagy-mediated depigmentation by amorphigenin was dependent on the mTOR/AMPK pathway, the phosphorylations of mammalian target of rapamycin (pmTOR) and AMP-activated protein kinase (AMPK) were analyzed following amorphigenin or rapamycin treatment. Whereas rapamycin induced mTOR inhibition and but depigmentation, amorphigenin induced AMPK activation and depigmentation but not mTOR inhibition ( Figure 5A,B). To confirm whether amorphigenin-induced AMPK activation was essential for depigmentation, levels of melanosomal proteins and melanin content were analyzed in B16F10 cells transfected with siAMPK. Amorphigenin-mediated downregulation of the melanosomal proteins Tyr and cleaved PMEL was effectively rescued following AMPK knockdown ( Figure 5C), and AMPK-dependent melanosome degradation was induced by amorphigenin, as confirmed by confocal microscopy analysis ( Figure 5D). The depigmentation effect of amorphigenin was also inhibited by AMPK knockdown ( Figure 5E). After experiments were performed according to the schematic, the melanin content was measured (D,E). Data were obtained from at least three independent experiments, and the values represent the mean ± SEM. Note: ns, not significant; ** p < 0.01 and *** p < 0.001 according to t-tests.

Amorphigenin Induces Degradation of Melanosomes in an mTOR-Independent and AMPK-Dependent Manner
To test whether autophagy-mediated depigmentation by amorphigenin was dependent on the mTOR/AMPK pathway, the phosphorylations of mammalian target of rapamycin (pmTOR) and AMP-activated protein kinase (AMPK) were analyzed following amorphigenin or rapamycin treatment. Whereas rapamycin induced mTOR inhibition and but depigmentation, amorphigenin induced AMPK activation and depigmentation but not mTOR inhibition ( Figure 5A,B). To confirm whether amorphigenin-induced AMPK activation was essential for depigmentation, levels of melanosomal proteins and melanin content were analyzed in B16F10 cells transfected with siAMPK. Amorphigenin-mediated downregulation of the melanosomal proteins Tyr and cleaved PMEL was effectively rescued following AMPK knockdown ( Figure 5C), and AMPK-dependent melanosome degradation was induced by amorphigenin, as confirmed by confocal microscopy analysis ( Figure 5D). The depigmentation effect of amorphigenin was also inhibited by AMPK knockdown ( Figure 5E).

Discussion
A. fruticosa roots contain various polyphenols, such as rotenoids, prenylated flavanones, isoflavones, and stilbenes [28], and may play a role in several biological properties, including antitumor [29], anti-inflammatory [30], and antidiabetic activities [31]. In this study, we investigated autophagy-mediated depigmentation by A. fruticosa RE in B16F10 cells, and identified amorphigenin as a functional compound.
Melanosomes are specific organelles in which melanogenesis occurs in melanocytes [32]. Following melanogenesis, melanosomes are transferred from melanocytes to keratinocytes [33]. Skin color is determined by the melanin content of melanosomes, so targeting melanosomes is important for skin-whitening applications [34,35]. Many skin depigmentation agents have been identified on the basis of tyrosinase inhibition. A. fruticosa RE also inhibits L-DOPA oxidation, which correlates with a decrease in tyrosinase activity [36]. The A. fruticosa RE-induced decrease in intracellular melanin content may be due to the direct inhibition of Tyr activity [37][38][39], inhibition of Tyr modification [40,41], or a decrease in Tyr expression [42,43]. The decrease in melanosomal protein levels by RE was due to a decrease in protein stability and not a decrease in their transcript levels. Generally, the stability of intracellular proteins is mainly regulated through the UPS [44]. The A. fruticosa RE-mediated decrease in melanosomal proteins levels might be due to the UPS, because A. fruticosa RE cannot decrease the mRNA levels for the proteins. However, MG132, a representative cell-permeable proteasome inhibitor that works against the UPS [45], could not rescue A. fruticosa RE-mediated protein decrease. Another protein degradation process is the lysosome-dependent mechanism [46]. Autophagy in particular is a type of homeostatic mechanism that degrades cellular proteins or cellular organelles in a To inhibit AMPK activity, validated AMPK siRNA, as described in the Materials and Methods section, was transfected into B16F10 cells, and then the cells were treated with amorphigenin for 48 h after α-MSH stimulation (C-E). To analyze melanosome degradation, TRP-1 spot formation was analyzed in the indicated experimental groups (D). Data were obtained from at least three independent experiments, and the values represent the mean ± SEM. Note: ns, not significant; *** p < 0.005 and ** p < 0.01 according to t-tests. Scale bar = 10 µm.

Discussion
A. fruticosa roots contain various polyphenols, such as rotenoids, prenylated flavanones, isoflavones, and stilbenes [28], and may play a role in several biological properties, including antitumor [29], anti-inflammatory [30], and antidiabetic activities [31]. In this study, we investigated autophagy-mediated depigmentation by A. fruticosa RE in B16F10 cells, and identified amorphigenin as a functional compound.
Melanosomes are specific organelles in which melanogenesis occurs in melanocytes [32]. Following melanogenesis, melanosomes are transferred from melanocytes to keratinocytes [33]. Skin color is determined by the melanin content of melanosomes, so targeting melanosomes is important for skin-whitening applications [34,35]. Many skin depigmentation agents have been identified on the basis of tyrosinase inhibition. A. fruticosa RE also inhibits L-DOPA oxidation, which correlates with a decrease in tyrosinase activity [36]. The A. fruticosa RE-induced decrease in intracellular melanin content may be due to the direct inhibition of Tyr activity [37][38][39], inhibition of Tyr modification [40,41], or a decrease in Tyr expression [42,43]. The decrease in melanosomal protein levels by RE was due to a decrease in protein stability and not a decrease in their transcript levels. Generally, the stability of intracellular proteins is mainly regulated through the UPS [44]. The A. fruticosa RE-mediated decrease in melanosomal proteins levels might be due to the UPS, because A. fruticosa RE cannot decrease the mRNA levels for the proteins. However, MG132, a representative cell-permeable proteasome inhibitor that works against the UPS [45], could not rescue A. fruticosa RE-mediated protein decrease. Another protein degradation process is the lysosome-dependent mechanism [46]. Autophagy in particular is a type of homeostatic mechanism that degrades cellular proteins or cellular organelles in a lysosome-dependent manner [47]. Autophagy can capture cytosolic contents in double-membrane vesicles called autophagosomes. ATG5 forms a protein complex with ATG12 and ATG16L during autophagosome membrane elongation [27]. In the final step of autophagy, autophagosomes can fuse with lysosomes to form autolysosomes, which in turn degrade the captured cytosolic contents through lysosomal protease. Inhibition of both autophagosome formation through ATG5 knockdown or autophagy-mediated degradation using lysosomal protease inhibitors can block the degradation of Tyr and Pmel induced by A. fruticosa RE or its component, amorphignin. These results suggest that amorphigenin induces the degradation of melanosomes through an autophagy process. Interconnection among mTOR, AMPK, and unc-51-like 1/2 (ULK1/2) play an essential role in macroautophagy [48]. Rapamycin, a representative nonselective autophagy inducer and mTOR inhibitor, did not induce depigmentation in α-MSH-stimulated B16F10 cells. Amorphigenin-mediated melanosome degradation was effectively inhibited by AMPK knockdown, regardless of mTOR. AMPK has been reported to play an important role in selective autophagy. AMPK deficiency results in defective mitophagy [49], and AMPK-mediated ULK1 activation is essential for the initiation of mitophagy under hypoxia conditions [50]. AMPK is localized to the mitochondrial outer membrane, and the inhibition of the AMPK activity attenuates mitophagy in skeletal muscle [51].
Until now, there have been suggestions regarding the possibility of selective autophagy for melanosomes, but few reports have focused on the molecular mechanisms. This study suggested that amorphigenin-induced melanosome degradation may be an AMPKdependent selective autophagy on melanosomes.

Data Availability Statement:
The data used to support the findings of this study are included in the article.

Conflicts of Interest:
The authors declare no conflict of interest.