Experiments were conducted in compliance with the Narcotics Control Act of the Republic of Korea. The plant species used is not classified as protected or endangered.

Stems of 2-month-old CS variety Cheungsam were collected in August 2022 from an open field at Jay Hemp Korea. Initially, stems were washed three times with sterile distilled water. After cutting into 2-cm pieces, 200 g CSS was mixed with 1 l PBS and ground in a mixer grinder at a 30-sec for a total of three cycles. The isolation of CSS-NPs involved centrifugation at 1,800, 3,500 and 10,000 × g for 20 min each at 4°C. Supernatants were filtered using a 0.45-µm membrane filter (GVS S.p.A.). Filtered supernatants were first concentrated to a 15-ml using a tangential flow filtration (TFF) system equipped with a 500-kDa ultrafilter membrane (Pall Life Sciences). For purification, sucrose cushioned ultracentrifugation was performed using Optiprep solution (60% iodixanol; Stemcell Technologies). Specifically, 15 ml concentrated supernatant was transferred to a 17 ml ultracentrifuge tube (Beckman Coulter, Inc.) with 1 ml Optiprep using a Pasteur pipette (MilliporeSigma). The tubes were ultracentrifuged at 120,000 × g for 60 min at 4°C (Optima XE-100; Beckman Coulter Inc.). The highly purified CSS-NPs were collected and dialyzed with 5 ml cold PBS. Finally, CSS-NPs were filtered using a 0.45-µm syringe filter (Advantec) and stored at −80°C until further use. The protein concentration of CSS-NPs was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.).

Size distribution and ζ potential of CSS-NPs were confirmed using qNano-Exoid (Izon Science Limited) according to the manufacturer's instructions using the NP200 nanopore (size; 85–500 nm). To visualize the morphology of CSS-NPs, scanning electron microscopy (SEM) was used. Samples were first mixed at 20°C for 60 min with 4% paraformaldehyde solution for fixation. CSS-NPs were washed twice with PBS and dehydrated in 70% ethanol before the evaporation of ethanol. Following dehydration, the samples were coated with gold-palladium sputtering and assessed using a S-4700 scanning electron microscope (Hitachi, Ltd.). To evaluate the stability of CSS-NPs against external stimuli, CSS-NPs were exposed to different pH concentrations (pH 2.0 or 12.1) for 30 min at 37°C. Additionally, CSS-NPs were incubated with 3 DNase (Thermo Fisher Scientific, Inc.), 6 RNase and 100 µg/ml proteinase K (both BioLabs) at 37°C for 30 min, after which changes in size and ζ potential were measured.

Biochemical composition of CSS-NPs was evaluated using quadrupole time-of-flight mass spectrometry (Q-TOF-MS) and ultra-high-performance liquid chromatography (UPLC; both Waters Corporation). Briefly, 1 mg CSS-NPs was dried using a freezing/vacuum-drying system (VD-800F; TAITEC CORPORATION) and resuspended with 100 µl 80% methanol. UPLC was conducted by injecting 10 µl reconstituted sample onto an Acquity UPLC BEH C18 column (Waters Corporation). Q-TOF-MS and tandem MS spectra were acquired as previously described (24). For gas chromatography (GC)-MS analysis, 1 mg CSS-NPs were mixed with 70 µl hydroxymethyl amine solution, incubated at 37°C for 90 min and then methylated with 70 µl N,O-bis(trimethylsilyl)trifluoroacetamide at 90°C for 30 min. After cooling, 1.5 saturated NaCl solution and 1.0 ml n-hexane were added. Next, the mixture was centrifugated at 180 × g at 20°C for 2 min, and the upper organic phase was transferred to a GC vial. Analysis was performed on a Shimadzu GC-2010 Plus GC-MS-TQ 8030 (Shimadzu) device with a DB-5 MS column (30 m × 0.25 mm; film thickness; 0.25 µm). GC-MS spectra were acquired as previously described (25).

Mouse skin-derived B16F10 melanoma (Korean Cell Line Bank; Korean Cell Line Research Foundation) and the HaCaT human keratinocyte cell line (Cytion) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (all Gibco; Thermo Fisher Scientific, Inc.). Cells were maintained at 37°C in a 5% CO₂ atmosphere and sub-cultured every 3 days by seeding 1×10⁶ cells/T-75 flask. All experiments were conducted using cells at passages 5–8. Mycoplasma contamination testing was performed regularly to ensure cell line integrity.

Bone marrow-derived dendritic cells (BMDCs) were generated from C57BL/6 mice (Orient Bio Inc.). Female mice (n=4; age 7 weeks, weight, ~18 g) were acclimated for 1 week at the Korea Research Institute of Bioscience and Biotechnology animal facility under specific pathogen-free conditions (22±2°C; 12/12 h light/dark cycle; 55±5% humidity), with ad libitum access to food and water. Mice were anesthetized with 2–3 % isoflurane (Hana Pharm Co., Ltd.) and euthanized by cervical dislocation. Bone marrow cells were isolated from femurs and tibias and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 2.5 ng/ml IL-4 and 20 ng/ml granulocyte-macrophage colony-stimulating factor (both JW CreaGene). Cells were incubated at 37°C with 5% CO₂ for 8 days to induce BMDC differentiation.

B16F10 and HaCaT cells and BMDCs (1×10⁴ cells/well) were placed in a 96-well plate, incubated for 24 h, then treated with CSS-NPs (5–100 µg/ml) for 24 h at 37°C in a 5% CO₂ incubator. Subsequently, the effect of CSS-NPs on cell viability was assessed using an EZ-Cytox assay kit (Dogenbio Co., Ltd.) according to the manufacturer's instructions. The EZ-Cytox solution (1X) was added to each well, and the plates were incubated at 37°C for 30 min. Absorbance was measured at 450 nm using a SpectraMax iD5 microplate reader (Molecular Devices, LLC). To evaluate the cytotoxicity of CSS-NPs, B16F10 and HaCaT cells and BMDCs were seeded in a 12-well plate (1×10⁶ cells/well) and treated with CSS-NPs at concentrations of 50 and 100 µg/ml, respectively, for 24 h at 37°C. Cells were stained using an apoptosis and necrosis detection kit (Thermo Fisher Scientific, Inc.), which included Annexin V and propidium iodide, according to the manufacturer's instructions. Live and dead cells were counted using a Life Launch Attune Nxt Flow Cytometer (Thermo Fisher Scientific, Inc.) and FlowJo software (Version 10; Tree Star, Inc.).

B16F10 cells were seeded at a density of 1×10⁵ cells/well in a six-well plate for 24 h. Then, the cells were incubated with 5, 25, 50 and 100 µg/ml CSS-NPs at 37°C for 3 days in the presence of α-melanocyte stimulating hormone (α-MSH; 100 nM). Cells were collected via trypsin solution (Gibco; Thermo Fisher Scientific, Inc.) and centrifuged at 8,000 × g at 20°C for 3 min. The supernatant was removed, and cell pellets were washed twice with PBS. Cell pellets were lysed with 100 µl mixed lysis solution (1 M NaOH and 10% DMSO) and heated to 100°C for 1 h. The solubilized melanin was transferred to a 96-well plate and the absorbance at 405 nm was measured using a SpectraMax iD5 microplate reader. The amount of solubilized melanin was normalized to the total protein content of the lysate using Pierce™ BCA Protein Assay kit.

B16F10 cells (1.5×10⁵/well in a six-well plate) were treated with 25 µg/ml CSS-NPs for 2 days at 37°C in the presence of α-MSH (100 nM), harvested and washed twice in PBS. TYR activity was measured using the TYR assay kit (cat. no. MAK550-1KT; MilliporeSigma) according to the manufacturer's instructions.

B16F10 cells (0.5×10⁵/per well in a six-well plate) were incubated with 5 or 25 µg/ml CSS-NPs for 2 days at 37°C in the presence of α-MSH (100 nM). Cells were lysed in M-PER Mammalian Protein Extraction Reagent and Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The protein was quantified using the BCA kit, and protein samples were denatured for 10 min at 70°C. Subsequently, 25 µg/lane protein was separated using 4–12% SDS-PAGE, transferred onto PVDF membranes (Thermo Fisher Scientific, Inc.) and blocked with EveryBlot Blocking Buffer (cat. no. #12010020; Bio-Rad Laboratories, Inc.) at 25°C for 1 h. The membrane was then incubated with primary antibodies at 4°C for 24 h (Table I). The membrane was incubated with anti-mouse (1:10,000; cat. no. 7076S) or rabbit IgG (1:10,000; Cat. no. 7074S; both Cell signaling technology, Inc.) HRP-linked secondary antibodies at 25°C for 1 h. Protein bands were detected using enhanced SuperSignal West Pico PLUS chemiluminescence substrates (Thermo Fisher Scientific, Inc.) and visualized using Amersham ImageQuant™ 800 (Cytiva, Amersham, England). Densitometry was quantified using the ImageQuant TL analysis software (Cytiva, version 10.0.261).

B16F10 cells (1×10⁵/well in a 12-well plate) were incubated with CSS-NPs (5 and 25 µg/ml) and α-MSH (100 nM) for 24 h at 37°C. Cells were washed twice in PBS, and total RNA was isolated using a RNeasy Mini kit (Cat no. 74106; Qiagen GmbH). For reverse transcription-quantitative (RT-q)PCR, 10 µl RNA was reverse-transcribed into cDNA using the iScript Adv cDNA kit for RT-qPCR (Cat. no. #1725038; Bio-Rad Laboratories, Inc.), according to the manufacturer's instructions. Next, target cDNA levels were quantified by RT-qPCR using the CFX96 Touch RT-PCR detection system and SYBR green (Bio-Rad Laboratories, Inc.). The PCR steps for all genes were as follows: For a hot start, 40 cycles of 95°C for 5 min, 95°C for 20 sec, 60°C for 20 sec and 72°C for 30 s, melting curve at 95°C for 1 min, 55°C for 1 min, and 30°C for 1 min. The quantification of gene expression was performed using the 2^-ΔΔCq method (26). Specific forward and reverse primers are presented in Table II.

B16F10 cells were seeded (0.5×10⁵/per well in a six-well plate) and pretreated with PD98059 (ERK inhibitor, 20 µM) or LY294002 (PI3K inhibitor, 10 µM) at 37°C for 1 h before treatment with 25 µg/ml CSS-NPs and α-MSH (100 nM) at 37°C. After 2 days, cells were harvested for subsequent experiments.

DPPH radical scavenging activity of CSS-NPs was determined as previously described (27). The samples, diluted to specific concentrations (0.1, 0.2, 0.5 and 1 mg/ml) with distilled water, were mixed with 0.2 mM DPPH solution (MilliporeSigma) at a 1:1 ratio (100 µl each). The mixture was stirred and left to stand in the dark for 30 min at 20°C, after which the absorbance was measured at 517 nm using a SpectraMax iD5 microplate reader.

ABTS radical scavenging activity of CSS-NPs was determined as previously described (28). To prepare the ABTS solution, 88 µl 140 mM potassium persulfate (Samchun Pure Chemical Co., Ltd.) and two ABTS diammonium salt tablets (MilliporeSigma) were added to 5 ml distilled water, mixed thoroughly, and allowed to react in the dark for 12–16 h at 20°C. ABTS solution was diluted with 95% ethanol at a ratio of 1:88 to achieve an absorbance of 0.7±0.02 at 734 nm. A total of 50 µl sample, diluted to specific concentrations (0.1, 0.2, 0.5 and 1.0 mg/ml) with distilled water, was mixed with 1 ml ABTS solution. Finally, the mixture was stirred and allowed to react in the dark for 5 min at 20°C, and the absorbance was measured at 734 nm.

B16F10 cells (1/10⁵/well) were seeded in 12-well plates and incubated with CSS-NPs and vitamin C (Vit C; both 50 µg/ml) in the presence of α-MSH (100 nM) for 24 h at 37°C. Following cell harvesting, intracellular ROS levels were measured using a CellROX kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Total antioxidant capacity (TAC) and catalase (CAT) enzyme activity in sonicated (40 kHz at 25°C for 5 min) lysate of B16F10 cells treated with CSS-NPs and Vit C (both 50 µg/ml) for 24 h at 37°C in the presence of α-MSH (100 nM) were measured using EZ-Total Antioxidant Capacity (Dogenbio Co., Ltd.) and Catalase Colorimetric Assay (Thermo Fisher Scientific, Inc.). kits, respectively, according to the manufacturers' protocols.

All data are expressed as the mean ± standard deviation of three independent experiments. Significance of groups was evaluated using one-way ANOVA followed by Tukey's multiple comparison test. Data were analyzed using GraphPad Prism 9 (Dotmatics), P<0.05 was considered to indicate a statistically significant difference.

CSS-NPs were isolated from CSS using TFF and sucrose cushioned ultracentrifugation, resulting in highly purified nanoparticles (Fig. 1A). The purified CSS-NPs were characterized by size, ζ potential and morphological features using qNano-Exoid (Fig. 1B) and SEM (Fig. 1C). CSS-NPs, when resuspended in PBS at a pH of 7.0, had a mean diameter of 120±7 nm. The ζ potential value was −29.4 mV (Fig. 1B), which falls within the stable range (−30-30 mV) for nanoparticles (29). SEM imaging confirmed that CSS-NPs possessed typical nanovesicle structure characterized by spherical morphology (Fig. 1C). Notably, CSS-NPs maintained consistent particle size and ζ potential values within the stable range across acidic (pH 2.0) and alkaline (pH 12.1) conditions, compared with those in the neutral condition (pH 7.0), and following exposure to DNase, RNase and proteinase K (Fig 1D). This demonstrated that CSS-NPs exhibit stability under external stress conditions.

Next, the biochemical composition of CSS-NPs was analyzed using UPLC/Q-TOF-MS (Fig. 2A and Table III) and GC-MS (Fig. 2B and Table IV). A total of 48 metabolites were detected within the CSS-NPs. UPLC/Q-TOF-MS analysis identified four nucleosides, three amino acids, two flavonoids and 20 lipids and chlorophyll derivatives (Fig. 2A; Table II). The nucleosides were guanine, adenosine, guanosine and 1-methyladenosine; the amino acids were N-(1-deoxy-1-fructosyl) valine, L-phenylalanine and L-tryptophan and the flavonoids were pterosin E and pterosin K. Furthermore, CSS-NPs contained pheophorbide A, a chlorophyll derivative. CSS-NPs contained various lipid compositions including dehydrophytosphingosine, 3-ketosphingosine, phytosphingosine, stearoylethanolamide, lysophosphatidylcholine 16:0 (lysoPC 16:0), 2-O-protocatechuoylalphitolic acid, PC[18:1(9Z)/2:0], methyl 4,8-decadienoate, lysoPC(18:0), lysoPC[16:1(9Z)], PC[16:1(9Z)/17:1(9Z)], lysoPC[18:1(9Z)], PC[17:1(9Z)/17:1(9Z)], lysoPC[22:6(4Z,7Z,10Z,13Z,16Z,19Z)], PC(18:0/2:0), 13-docosenamide, octadecanamide, oleoylethanolamide, stigmastane-3,6-dione and PC[18:3(9Z,12Z,15Z)/16:0]. In GC-MS analysis, an additional 18 metabolites were identified, including glycerol, ribose, threose, xylose, lyxose, arabinose, rhamnose, fructofuranose, psicofuranose, tagatose, pinitol, fructose, talose, glucose, galactose, palmitic acid, myo-inositol and stearic acid (Fig. 2B and Table IV).

Cytotoxic effects of CSS-NPs were observed in B16F10 and HaCaT cells and BMDCs. CSS-NPs treatment did not affect cellular cytotoxicity, and B16F10 cell viability remained unchanged at concentrations of 5–50 µg/ml but revealed a significant increase at a concentration of 100 µg/ml. By contrast, HaCaT cells and BMDCs exhibited no significant changes in viability up to 100 µg/ml (Fig. 3A). Annexin V and PI staining confirmed that no death occurred in any of the cells treated with CSS-NPs at 50 or 100 µg/ml (Fig. 3B). These findings demonstrate that CSS-NPs did not exhibit cytotoxicity and that the selected concentrations were appropriate for assessing the whitening and antioxidant activity of CSS-NPs in melanoma cells.

CSS-NPs inhibited melanin formation in a dose-dependent manner (Fig. 3C). Melanin synthesis is catalyzed by three enzymes: TYR and tyrosinase-related protein (TRP)-1 and TRP-2, with TYR being an enzyme that initiates melanin synthesis (30,31). Therefore, TYR activity was measured in B16F10 cells treated with CSS-NPs. Compared with the control (α-MSH-treated B16F10 cells), low-dose CSS-NPs (25 µg/ml) resulted in decrease TYR activity (Fig. 3D), suggesting that CSS-NPs regulate melanin synthesis by modulating TYR activity.

Microphthalmia-associated transcription factor (MITF) is a key transcription factor that controls the activity levels of TYR, TRP-1 and TRP-2 in melanogenesis (32). Thus, to explore how CSS-NPs affect the regulatory mechanisms of melanin synthesis induced by α-MSH in B16F10 cells, western blot analysis was performed out. Protein expression levels of MITF, TYR, TRP-1 and TRP-2 decreased in cells treated with 5 and 25 µg/ml CSS-NPs compared with those treated with α-MSH alone (Fig. 4A). CSS-NPs significantly inhibited the expression of melanin synthesis-associated genes including MITF, TYR, TRP-1 and TRP-2 (Fig. 4B). These results suggested that the anti-melanogenic effects of CSS-NPs in B16F10 cells were induced by downregulation of melanogenic genes and protein including MITF, TYR, TRP-1 and TRP-2.

MAPK signaling regulates cellular processes by transducing external signals into intracellular responses (33). Therefore, whether MAPK signaling regulated the anti-melanogenic activity induced by CSS-NPs was evaluated. CSS-NPs dose-dependently increased the phosphorylation of ERK, but not JNK and p38 (Fig. 5A). To elucidate the functional role of CSS-NPs in activating ERK signaling in α-MSH-stimulated B16F10 cells, melanin content and biosynthesis proteins in the presence of PD98059 (an ERK inhibitor) were assessed. Melanin content (Fig 5B) and biosynthesis protein levels (MITF, TYR, TRP-1 and TRP-2; Fig. 5C), which were decreased by CSS-NPs in α-MSH-treated B16F10 cells, increased when ERK signaling (PD98059-treated condition) was inhibited. Subsequently, the PI3K/Akt signaling pathway was investigated because this signaling pathway regulates melanin synthesis by promoting the degradation of MITF (34). CSS-NPs promoted an increase of Akt phosphorylation and consequent decrease of GSK3β phosphorylation in α-MSH-treated B16F10 cells (Fig. 6A). Following inhibition of Akt signaling (LY294002-treated condition), CSS-NPs restored the melanin content (Fig. 6B). Additionally, expression of melanin biosynthesis proteins increased following α-MSH treatment (Fig. 6C). These results indicate that CSS-NPs suppressed melanin synthesis by activating the ERK and Akt signaling pathway.

Synthesis of melanin leads to an increase in ROS within skin cells, which is associated with skin aging, pigmentation alteration and the onset of inflammatory reactions (35–37). To determine whether CSS-NPs exert antioxidant effects by controlling ROS generated during melanin synthesis, the indirect antioxidant potential of CSS-NPs was assessed using DPPH and ABTS radical scavenging assays, with Vit C serving as a positive control. CSS-NPs exhibited lower direct radical scavenging activity than Vit C; however, the activity of CSS-NPs increased in a concentration-dependent manner; these results were not significant (Fig. 7A). Whether CSS-NPs could provide protection in α-MSH-stimulated B16F10 cells at non-toxic concentrations was evaluated. CSS-NPs restored cell viability in a dose-dependent manner (Fig. 7B). The antioxidant functionality of CSS-NPs was explored by comparing their effects on cellular toxicity induced during melanin synthesis with those of Vit C. CSS-NPs effectively reduced α-MSH-induced cytotoxicity, demonstrating effects comparable with treatment with Vit C (Fig. 7C). Finally, to investigate the underlying mechanism, intracellular ROS levels (Fig. 7D) and antioxidant enzyme activity (TAC and CAT; Fig. 7E) were measured. In α-MSH-stimulated B16F10 cells treated with CSS-NPs, intracellular ROS levels were significantly decreased, while TAC and CAT activities were increased compared with α-MSH-stimulated B16F10 cells. These effects were similar to those observed with Vit C treatment. These results suggest that CSS-NPs preserved melanocyte survival by suppressing free radicals generated during excessive melanin formation and alleviating impaired antioxidant activity.