Abstract

The interest of consumers in cosmetic industry products with skin-lightening effects has increased the demand for preparations that reduce melanogenesis. A number of anti-melanogenic drugs are known for their side effects, such as contact dermatitis and high toxicity, and poor skin penetration. Considerable recent research has focused on plant-derived products as alternatives to chemotherapeutic medications with fewer side consequences.

In the instant study, we examined the anti-melanogenic action of extracellular vesicles (EVs) extracted from the foliage and stems of Dendropanax pathogenic. Working with spectrophotometric and biochemical methods, we found that leaf-derived extracellular vesicles (LEVs) and stem-derived extracellular vesicles (SEVs) reduced melanin content and tyrosinase (TYR) activity in a concentration-dependent manner in a B16BL6 mouse melanoma cell line. Electron microscopic analysis further demonstrated that LEVs and SEVs induced a concentration-dependent decrease in melanin content in melanoma cells. Compared to arbutin as a positive control, LEVs and SEVs showed a stronger whitening effect on melanoma cells, and the whitening effect of LEVs was stronger. Notably, neither LEVs nor SEVs induced significant cytotoxicity. We also examined the effects of plant-derived EVs on the expression of tyrosinase-related proteins (TRPs) in melanoma cells. LEVs inhibited the expression of melanogenesis-related genes and proteins, including microphthalmia-associated transcription factor (MITF), TYR, TRP-1, and TRP-2. In a human epidermal model, LEVs inhibited melanogenesis more strongly than arbutin. Taken together, our data suggest that lev from D. pathogens may be a new candidate natural substance for use as an anti-melanogenic agent in pharmaceutical preparations.

keywords: Plant-derived EVs; LEVs and SEVs; anti-melanogenic; TYR activity; melanin content and Cistanche extract benefits

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Introduction

Melanin, a critical part of the human hair, eye, and skin pigmentation system, is produced by melanocytes through a procedure called melanogenesis. Aberrant melanin accumulation can result in skin disorders such as freckles, sun freckles, and melasma, and can also cause cancer and vitiligo. Regulating melanogenesis is therefore a vital strategy in the treatment of hyperpigmented disorders. For example, hydroquinone, a hydroxyphenyl compound that interferes with TYR activity, is used as a skin-bleaching agent in the cosmetic industry. Nevertheless, hydroquinone may cause side effects such as contact dermatitis and exogenous browning disease. Va-acid is another synthetic agent that inhibits TYR activity, but its use is associated with a high frequency of edema or irritation.

There has been an increasing interest in identifying alternative drugs from natural sources against melanogenesis, given the constraints of existing chemical compounds, which reflects the fact that cosmetic products made from plants and herbs tend to be milder, more biodegradable, and demonstrate lower toxicity than synthetic compounds. Dendrobium disease leaf extracts have been shown to inhibit melanin production by interacting directly with intracellular TYR activation and the expression of enzymes involved in melanin biosynthesis. In a similar manner, Croton officinalis leaf extract suppressed melanin content and cellular TYR activity by inhibiting melanogenesis-related transcription factor (MITF) and melanogenic enzymes. In addition, mulberry leaves had an inhibitory effect on TYR activity and melanin formation in melan-A cells. P-coumaric acid from ginseng leaves was identified as the major TYR inhibitor.

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Despite the fact that a wide range of botanical compounds has been used in medicinal cosmetic formulations, their low solubility, low target affinity, and moderate skin-lightening action have hindered progress in improving the therapeutic effects of botanical cosmetics. This has motivated the search for new and progressive techniques to enhance the effectiveness of medicinal and bioactive compounds and improve their efficiency of delivery to the skin. For example, a number of nano-delivery technologies have been successfully developed, including nano-lovely for effective skin care, nano-quercetin for delaying cell damage caused by ultraviolet (UV) radiation, nano-fullerenes for collagen regeneration and prevention of skin aging, nano-luteolin for maintaining antioxidant activity and nano-resveratrol for protecting the skin from UV radiation.

In the present study, we concentrate on the role of plant-derived extracellular vesicles (EVs). Recent studies have shown that plant-derived EVs have a structure similar to that of mammalian isolated exosomes and act as extracellular messengers mediating intercellular communication. In addition, these vesicles are capable of translocating mRNA, micro-RNA (miRNA), bioactive lipids, and proteins into animal cells.

In the meantime, we have studied the inhibitory effect of EVs derived from leaves and stems of diseased legumes on melanogenesis. We characterized the size and properties of leaf-derived extracellular vesicles (LEVs) and stem-derived extracellular vesicles (SEVs) extracted from leaves and stems of diseased legumes and showed that these EVs were readily taken up by melanoma cells and were not cytotoxic. To demonstrate the anti-melanogenic effects of LEVs and SUVs, we examined melanin content and TYR activity in melanoma cells. We further assessed the effect of EVs on the complex process of melanin synthesis by monitoring changes in the levels of various proteins and enzymes.

Alpha-melanocyte stabilizing hormone (α-MSH) binds to the melanocortin 1 receptor (MC1R) on the cell surface and activates adenylate cyclase, leading to increased intracellular levels of cyclic adenosine monophosphate (cAMP). cAMP is mediated through cAMP-dependent protein kinase A, leading to phosphorylation of cAMP response element binding protein (CREB). Activated CREB induces MITF, which is expressed in melanocytes and plays a key role in melanocyte differentiation and development. -TRP1 is essential for the correct translocation of TYR to melanin synthesis and TRP2 plays an important role in the catalytic activity of TRP in the early stages of melanin synthesis. These three interact in melanoma cells (Supplementary Fig. 1).

We found reduced expression of MITF in LEVs-treated melanoma cells, followed by reduced expression of TYR, TRP-1, and TRP-2, and confirmed by electron microscopy that melanin synthesis was reduced within these cells at the ultrastructural level. We further confirmed the anti-melanogenic effect of LEVs using a reconstructed human epidermal model. To quantitatively assess the inhibitory effect of LEVs on cellular melanin synthesis, we prepared standard solutions from tissues and measured melanin content using a colorimeter. melanin spots were reduced in Fontana-Masson stained tissue sections. the LEVs inhibited melanin production more effectively than the TYR inhibitor arbutin, which was used as a positive control.

In summary, these findings indicate that the use of natural substance-derived EVs for the management of hyperpigmentation is a feasible future approach for the pharmaceutical industry. Furthermore, with the advantages of small size, low toxicity, high uptake, and environmental safety, plant-derived EVs are expected to be the next generation of therapeutic delivery systems for the treatment of other diseases. Notably, plant-derived EVs have good anti-melanogenic effects on reconstructed human skin tissue (similar to the human epidermis), setting the stage for future clinical trials.

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Materials and methods

1. Isolation of D. morbifera LEVs and SUVs

Fresh leaves and stems were collected from Poge Island, Guandao-gun, and Jeollanam-do. EVs were isolated from 50 g of leaves and stem, respectively, by grinding using an extractor and passing the resulting juice through filter paper, and centrifuging at 10,000 × g for 10 min. Large debris was removed by filtering the supernatant through a 0.22 μm membrane, followed by centrifugation using an Amicon Ultra-4 PL 100 K centrifuge filter (Merck Millipore. Darmstadt, Germany) to concentrate the EVs by centrifuging the samples at 5000 × g for 10 min at 4°C. After centrifugation, the protein concentration of EVs was determined using a bis quinolinic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

2. Size characterization of isolated EVs

The hydrodynamic size of the isolated EVs was measured by dynamic light scattering (DLS), a technique used to determine the size distribution of small particles in suspension, using a Zetasizer Nano ZS90 system (Malvern Instruments, Malvern, UK). The collected EVs were placed in a constant temperature cell at 20°C. The particle size distribution and the z-average used to determine the hydrodynamic particle size distribution were determined by measuring the scattering intensity autocorrelation function. Isolated EVs were diluted by vesicle-free bubble water and then subjected to nanoparticle tracking analysis (NTA) (Nanosight; using a 488 nm laser at 25°C).

3. Transmission electron microscopy analysis of EVs

5 μL of sample solution was loaded onto a copper grid-coated carbon film for transmission electron microscopy (TEM) analysis. After sample adsorption for 1 min, the grids were washed with a drop of pure water and then negatively stained with 1% uranyl acetate for 1 min. Excess stain was removed with filter paper and the grids were air dried. Samples were imaged in focus between 0.8 - 1.5 μm using a JEM- 1400 Plus transmission electron microscope (JEOL Ltd., Tokyo, Japan) equipped with a Lab6 gun running at 120 kV. Images were recorded using an UltraScan OneView CMOS camera (Gatan, Pleasanton, CA, USA).

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4. Preparation of liposomes

The liposome blends were prepared using a 95:5 (mol/mol) ratio of DMPC (1,2-stearoyl-sn-glycerol-3-phosphocholine) (Avanti Polar Lipids, Alabaster, AL, USA) with DSPE-mPEG (1,2-stearoyl-sn-glycerol-3-phosphoethanolamine-[methoxy(polyethylene glycol)- 2000] (Avanti polar lipids) to prepare the liposome blends as lipid membranes. The hydrophobic fluorescent dye 1,1-dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindocarbocyanine perchlorate (DiI, Invitrogen, Waltham, MA, USA) was mixed with EVs, 725.49 μg DMPC, 151.64 μg DSPE-PEG and 15 μg DiI. After evaporation of the organic solvent, the membrane containing the lipid and DiI mixture was hydrated with 1 mL of phosphate-buffered saline (PBS). Next, liposomes of 100 nm in size were prepared using an extruder (Avanti Polar Lipids).

5. Cell culture and viability assays

B16BL6 melanoma cells were cultured in alpha-minimal essential medium (alpha-MEM) containing 10% fetal bovine serum (Rocky Mountain Biologicals, Missoula, MT, USA) and 1% penicillin/streptomycin (Lonza, Basel, Switzerland) (Gibco, Thermo Fisher Scientific) in culture. Cells were incubated at 37°C in a humidified 5% CO2 atmosphere. 100 μL of B16BL6 melanoma cells were inoculated in 96-well plates (5 × 104 cells/well) for cell viability assays. After incubation for 24 h, cells were treated with LEVs and SEVs at concentrations of 1, 5, and 10 µg/mL, respectively, for 24 h. The concentrations of liposomes and arbutin were 10 µ g/mL and 70 µ g/mL, respectively, for all experiments. 10 µL of EZ-Cytox reagent (Daeil Lab Service, Seoul, Korea) was subsequently added to each well The plates were incubated for 1 h. The plates were then gently shaken and the absorbance was then measured at 450 nm using an enzyme marker (BioTek, Winooski, VT, USA).

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