Abstract Deer antler velvet (DAV) is rich in bioactive compounds with potential applications in regenerative medicine, particularly as inducers of melanogenesis. A combination of chitosan-liposomes (C-LI) and microspicule (MS) effectively delivered DAV extract through hair follicles. This study aimed to investigate the melanogenesis stimulation of DAV extract and its loaded C-LI and MS formulation for hair graying treatment. In vitro biological studies of DAV extract were conducted using B16F10 melanoma cells, focusing on stimulating tyrosinase activity and melanin content. DAV extract was loaded into C-LI and MS formulation. In vivo human study evaluated changes in the melanin after applying the formulation. For the result, the DAV extract demonstrated non-toxicity to B16F10 melanoma cells at concentrations below 1,000 µg/mL. At a 100 µg/mL concentration, DAV extract exhibited significantly higher tyrosinase activity in both mushroom tyrosinase activity assay and cultured B16F10 cells compared to other concentrations. This resulted in a significantly higher melanin content than the control and kojic acid (a tyrosinase inhibitor). After applying DAV extract loaded C-LI MS gel to the scalp skin for four weeks, the percent change of melanin was higher than the control (untreated scalp skin), suggesting  that bioactive compounds from DAV extract may contribute to increased melanin content in hair. In conclusion, DAV extract plays a crucial role in stimulating melanogenesis, thereby increasing melanin in aging hair. This research provides valuable insights into the potential use of DAV and its formulations for addressing hair graying concerns.

Keywords: Deer antler velvet, Melanogenesis stimulation, Chitosan liposomes, Microspicules, Hair aging

Funding: This work (Grant No. RGNS 64-234) was supported by the Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI), Thailand Science Research and Innovation (TSRI).

Citation: Rangsimawong, W., Duangjit, S., Sritananuwat, P., Samseethong, T., Ngawhirunpat, T., and Opanasopit, P. 2025. Melanogenesis stimulation of deer antler velvet extract and its loaded chitosan liposomes and microspicule formulation: In vitro biological study and in vivo human study. Natural and Life Sciences Communications. 24(2): e2025026.

INTRODUCTION

The aging process significantly impacts various aspects of hair, including the production, color, and structural characteristics of the hair fiber. Researchers have extensively focused on studying hair growth and pigmentation in the hair follicle as a readily accessible model for investigating age-related effects (Trüeb et al., 2018). Melanogenesis in hair closely correlates with the stages of the hair cycle. Hair is actively pigmented during the anagen phase but becomes inactive during the catagen phase and absent during telogen (Slominski and Paus, 1993). The pigmentary unit, a pear-shaped black structure at the tip of the dermal papilla in pigmented hair, undergoes changes in gray hair, appearing fuzzy, with fewer and rounded melanocytes, and lightly pigmented oligodendritic melanocytes becoming visible in the proximal hair bulb (Arck et al., 2006; Peters et al., 2011). During anagen, there is a significant reduction in the number of melanocytes in hair follicles due to autophagolysosomal degeneration, resulting in pigment loss (Horikawa et al., 1996; Kumar et al., 2018).

Numerous active compounds have been investigated to address age-related hair graying. For melanocyte activity, bioactive compounds extracted from animal sources, including pro-opiomelanocortin peptides, corticotrophin-releasing factor, and various growth factors, have been identified as effective stimulators of melanogenesis (Slominski et al., 2005; Rousseau et al., 2007). Additionally, fibroblast growth factor-2 (FGF-2) emerges as a potent mitogen for human melanocytes (Halaban et al., 1988; Sarkar et al., 2006).

Deer antler velvet is rich in essential bioactive components, notably amino acids, polypeptides, and proteins. Among these, some of the most prominent bioactive components are growth factors, including epidermal growth factor (EGF), FGF, insulin-like growth factor-1 (IGF-1), and transforming growth factor-β1 (TGF-β1) (Tansathien et al., 2019, Rangsimawong et al., 2024). The presence of these growth factors is noteworthy, considering that exogenous growth factors have been successfully derived from natural products through extraction methods and applied in regenerative medicine to replace or repair damaged cells, tissues, and organs (Sui et al., 2014). Given the abundance of these bioactive compounds in DAV extract, there is a compelling basis to explore their potential role in stimulating hair melanogenesis.

To enhance the transfollicular delivery of bioactive compounds from DAV extract, liposomes are potential nanocarriers for delivering various therapeutic molecules into the skin and follicular region (Rangsimawong et al., 2018). Chitosan-coated liposomes (C-LI) were chosen as nanocarriers due to their superior physicochemical properties, which enhance the entrapment and delivery of hydrophilic macromolecules compared to conventional liposomes. However, hydrophilic macromolecular proteins and growth factors face limitations in passive skin penetration. Therefore, combining C-LI with minimally invasive microspicules (MS) plays a crucial role as a transdermal and transfollicular delivery system for hydrophilic macromolecules (Rangsimawong et al., 2024). Therefore, the aim of this study was to evaluate the melanogenesis stimulation of DAV extract and its loaded C-LI and MS formulation for hair graying treatment. The in vitro biological study was investigated in B16F10 melanoma cells, which the tyrosinase activity and melanin content were determined. DAV extract was loaded into C-LI and MS formulation and applied into scalp skin to compare the melanin result of hair with untreated skin (in vivo).

MATERIALS AND METHODS

Materials

Fresh DAV was from Karakada 2011 Co., Ltd, Ratchaburi, Thailand. B16F10 melanoma cells were obtained from Cell Applications Inc., USA. The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was procured from Sigma-Aldrich, Co., MO, USA. Dulbecco’s modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid, L-alanyl-L-glutamine dipeptide (Glutamax^TM), non-essential amino acids, and penicillin–streptomycin were obtained from Gibco BRL, Rockville, MD, USA. Dimethyl sulfoxide (DMSO) was purchased from Merck, Darmstadt, Germany. Egg phosphatidylcholine (PC) was generously provided by Lipoid GmbH, Ludwigshafen, Germany. Cholesterol was purchased from Carlo Erba Reagent, Ronado, Italy. Polysorbate 20 (Tween 20) was sourced from Namsiang Group in Bangkok, Thailand. Chitosan (MW 8000 g/mol) was procured from OliZac Technologies Co., Ltd., Bangkok, Thailand. MS from Spongilla Lacustris extract powder (98% plus spicule; 100-180 mesh) was purchased from Hunan Sunshine Bio-Tech Co., Ltd., Hunan, China.

Extraction of DAV

The fresh DAV extract was prepared using water and probe sonication methods. In brief, DAV was mashed and mixed with distilled water at a 1:20 ratio, followed by probe sonication at a frequency of 40 kHz and 40% amplitude for 30 minutes in an ice bath. Subsequently, centrifugation was performed at 4000 rpm for 10 minutes to collect the supernatant. The extract was then freeze-dried at −49°C for 72 hours. The total protein and growth factor contents in the extract were determined using a bicinchoninic acid (BCA) protein assay kit (Novagen^®, EMD Millipore Corp., USA) and FGF-2 enzyme-linked immunosorbent assay (ELISA) kits (Abcam, USA), yielding 548.00 ± 17.09 mg/g and 28.36 ± 6.40 ng/g, respectively (Rangsimawong et al., 2024).

Cytotoxicity against Melanoma Cells B16F10

B16F10 cells (10⁴ cells per well) were cultured in 100 µL of DMEM supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 μg/mL amphotericin B. The cells were incubated in a humidified atmosphere (5% CO₂, 95% air, 37°C) until reaching confluence, typically within 24–48 h post-plating. Subsequent to removing the cell medium from the plates, each concentration of the diluted DAV extract in DMEM was added, and the cells were incubated for 24 h. For the MTT assay, the diluted extract was aspirated, and the cells were treated with 100 µL of MTT-containing medium (0.5 mg/mL) for 4 h. The medium was then removed. Following dissolution of the formazan crystals with DMSO, the absorbance was measured at 550 nm using a microplate reader (VICTOR Nivo^TM Multimode Plate Reader, PerkinElmer, Germany). The percentage of cell viability was calculated using the following equation:

Tyrosinase activity assay

The 60 µL of DAV extract at different concentrations (0, 1, 10, 100, 500, and 1,000 µg/mL) dissolved in DMEM was mixed with 20 µL of L-3,4-dihydroxyphenylalanine (L-DOPA) (0.9 mg/mL) in a 96-well plate. This mixture was then incubated with mushroom tyrosinase enzyme (500 units/mL in PBS, pH 6.8), adding 10 µL, at room temperature for 10 min. Subsequently, the absorbance of each sample was measured at a wavelength of 450 nm using a microplate reader (VICTOR Nivo™ Multimode Plate Reader, PerkinElmer, Germany). In this study, kojic acid (at a concentration of 100 µg/mL) served as the negative control, and the percentage of tyrosinase activity was calculated based on the obtained absorbance readings (equation 2).

For tyrosinase activity in B16F10 melanoma cells, 5×10⁴ cells/well were cultured in 24-well plates at 37°C for 24 h. The different concentrations of samples were added into B16F10 cells and incubated for 48 h. Afterward, the treated cells were washed, and 1% triton X-100 was added to disrupt the cell membranes. Upon centrifugation at 10,000 rpm for 10 minutes, the supernatant was collected to assess tyrosinase enzyme activity using the dopachrome method. The 60 µL of samples were mixed with 20 µL of L-DOPA (0.9mg/mL) in 96 well plate. Then, samples were incubated at room temperature for 10 min. The absorbance of each sample was measured at a wavelength of 450 nm using a microplate reader. The percentage of tyrosinase activity was calculated as described above. 

Melanin content in B16F10-melanoma cells

B16F10 cells (1×10⁴ cells/well) were cultured in 24-well plates at 37°C for 24 h. Different concentrations of DAV extract dissolved in DMEM were added to the B16F10 cells, followed by incubation with 200 nM α-MSH (melanotropin) for 48 h. Subsequently, the treated cells were washed with PBS, and 0.25% trypsin was added, incubating at 37°C for 5-8 min until cell detachment. The cell suspension was transferred into a 1.5 mL microtube. After centrifugation at 3,000xg for 10 min, the pellets were mixed with 1N NaOH and incubated at 80°C for 1 h. The absorbance at a wavelength of 405 nm was then measured using a microplate reader. Melanin contents were determined by employing a standard curve for accurate quantification.

Preparation of DAV extract loaded C-LI MS gel

DAV extract loaded C-LIs was prepared using thin film hydration and sonication method. Briefly, a mixture of PC and cholesterol at a ratio of 10:2 mM was dissolved in a solution of chloroform and methanol (2:1, v/v). The solvent was evaporated using a stream of nitrogen, forming a dry lipid film. The lipid film was rehydrated by adding the DAV extract dissolved in distilled water and polysorbate 20 (2% w/v). The hydrated liposome vesicles were reduced in size by a probe-sonicator in an ice bath for 30 min to eliminate excess lipid components. The resulting supernatant was then combined with 10 mM chitosan (dissolved in 1% HCl) and stirred using a magnetic stirrer for 60 min. The DAV extract-loaded C-LIs were then combined with MSs at a concentration of 1.0% w/v. Finally, the prepared DAV extract-loaded C-LIs with MSs were mixed with a gel base containing hydroxypropyl methyl cellulose, glycerin, microcare PHC, and ethylene diamine tetraacetic acid (Rangsimawong et al., 2024). The total protein content of the formulation was 4.06 ± 0.03 mg/ml.

In vivo human study

This study received approval from the Investigational Review Board (No. UBU-REC-05/2566, Human Studies Ethics Committee, Ubon Ratchathani University), which the sample size was calculated from G* Power program (Means: Wilcoxon signed-rank test (one sample case), effect size d = 0.95, α err prob = 0.05, power (1-β err prob) = 0.95, total sample size = 15). Healthy volunteers meeting inclusion criteria (male and female, aged between 30 and 60, experiencing hair loss and greying) willingly participated in the clinical trial. The marked position, a 2 cm x 2 cm area at the occipital head, was designated using a medical scissor to remove the hair shaft. Participants applied the appropriate amount of DAV extract loaded C-LI MS gel twice a day, every day, continuously. Following the application, the marked skin area underwent a gentle massage (~20 rubbing times) using the forefinger (Rangsimawong et al., 2024). After 4 weeks, the marked skin was examined using the API-100 (Aram Huvis Co., Ltd., Gyeonggi-do, South Korea) and reported as the percent change of treatment compared to before treatment. The percent change in melanin was calculated from the equation (3).

Statistical analysis

The presentation of each dataset was expressed as mean ± standard deviation (S.D.). A one-way analysis of variance (ANOVA) was employed to assess statistical significance, followed by Tukey’s post hoc test for multiple comparisons. However, for the in vivo human study, the Wilcoxon signed-rank test was applied. The predetermined threshold for significance was set at a P-value less than 0.05.

RESULTS

Cytotoxicity of DAV extract on B16F10 cells

As shown in Figure 1, DAV extract exhibited non-toxic effects on B16F10 melanoma cells within the 1–1,000 µg/mL concentration range. Notably, at concentrations of 2,000 µg/mL, the extract significantly decreased the cell viability of B16F10 cells (P < 0.05). However, it's crucial to emphasize that even at this concentration, the percentage of cell viability remained higher than 80%, indicating an absence of cytotoxicity on B16F10 cells.

Figure 1. B16F10 cell viability after being treated with DAV extract for 24 h. Data presents mean ± S.D. (N=3). * indicates a significant difference from the control (Untreated cells) (P < 0.05).

Tyrosinase activity

Mushroom tyrosinase is a widely utilized target enzyme for screening potential tyrosinase regulators (Guan et al., 2018). As shown in Figure 2 (A), the tyrosinase activity levels of DAV extract at the concentration of 100-1,000 µg/mL were higher than control, while the tyrosinase inhibitory effect of kojic acid, known as a tyrosinase inhibitor (Tangyuenyongwatana and Gritsanapan, 2022), was significantly lower than the control. Among the concentrations tested, 100 µg/mL of DAV extract exhibited the highest tyrosinase activity. Furthermore, in assessing tyrosinase activity in cultured B16F10 cells (Figure 2 (B)), DAV extract at concentrations of 100 and 500 µg/mL displayed significantly higher activity compared to both kojic acid and the control.

Figure 2. The percent tyrosinase activity of DAV extract and kojic acid on mushroom tyrosinase activity assay (A) and in cultured B16F10 cells (B). Data represents mean ± S.D. (N=3). * indicates significant difference than control (P < 0.05).

Melanin content in B16F10 melanoma cells

In Figure 3, DAV extract showed a significant stimulatory effect on melanocyte melanogenesis at concentrations of 100 and 500 µg/mL. Notably, the results indicated the highest melanin content when treated with 100 µg/mL of DAV extract. In contrast, the application of kojic acid resulted in a reduction in melanin content in B16F10 cells.

Figure 3. Melanin content from B16F10 cell after treated with DAV extract and kojic acid for 48 h. Data presents mean ± S.D. (N=3). * indicates a significant difference from the control (Untreated cells) (P < 0.05).

In vivo human study

Melanin content serves as an indicator of pigmentation level, with melanin playing a significant role in determining overall skin and hair color (Tobin 2009). The application of DAV extract-loaded nanocarriers and MS formulation onto the scalp skin of human volunteers exhibited the growth of hair and increment of melanin contents (Tansathien et al., 2021; Rangsimawong et al., 2024). During the study, three participants were excluded due to factors, including non-compliance with study protocols and adverse events unrelated to the study intervention. Twelve human volunteers fully participated in the study conducted between June and July 2023, yielding preliminary results on the effect of formulation on hair melanin. For the result, all volunteers had dark brown or black hair, with some experiencing hair graying. After applying the DAV extract-loaded C-LI MS gel for 4 weeks, a significantly higher change in melanin levels than untreated skin (control) was found (Figure 4). However, a larger sample size was continuously studied in subsequent research.

Figure 4. The percent change in melanin of the scalp skin treated with DAV extract-loaded C-LI MS gel (), compared with control (untreated skin; ●). The data shows the mean ± S.D. of the skin treated with DAV extract-loaded C-LI MS gel (), compared with control (untreated skin; ○) (N = 12). * indicates a significant difference from the control (Untreated skin) (P < 0.05).

DISCUSSION

DAV extract was found to contain water soluble and high molecular weight bioactive compounds, including proteins, growth factors (EGF, FGF-2, IGF-1,TGF-β1) and amino acids (Tansathien et al., 2019; Rangsimawong et al., 2024), demonstrating  the bioactivity in stimulating melanogenesis without exhibiting cytotoxic effects on B16F10 melanoma cells. The increase of tyrosinase activity in mushroom tyrosine test and B16F10 cells when treated with a lower concentration of DAV extract, with attenuated results at higher concentrations, may be attributed to its amino acid components. Several amino acids were found in DAV, for example: 55.54 mg/g of cysteine, 12.10 mg/g of lysine, 10.45 mg/g of glutamic acid, 3.44 mg/g of histidine, 0.56 mg/g of valine, etc (Rangsimawong et al., 2024). The effect of amino acids on tyrosinase activity was observed in plant enzyme extracts. Histidine, aspartic acid, glycine, and β-alanine gradually induced tyrosinase activity from 2.5 to 10 mM, after which enzyme activity declined (El-Shora and Rania, 2014). This concentration-dependent stimulation of tyrosinase activity is consistent with the effects observed in our study with DAV extract. Consequently, the promotion of tyrosinase activity and melanin content by DAV extract is evident at specific concentrations of 100 and 500 µg/mL. The potential of DAV extract to modulate tyrosinase activity, both in isolated enzyme assays and in the cellular context, highlights its significance in regulating melanogenesis. Moreover, the measurement of melanin content serves as an indicator of melanocyte melanogenesis activity (Guan et al., 2018). This suggested that the increase of tyrosinase activity and melanin content of DAV extract on B16F10 melanoma cells may have the potential to stimulate melanogenesis in hair follicles.

Previous studies have reported that delivering DAV extract into hair follicles can be achieved by utilizing nanocarriers such as positively charged liposomes, which are homogeneously mixed with a gel base containing MS. C-LIs loadingDAV extract have demonstrated favorable physicochemical properties, including a nanometer size of 71.67 nm, a low polydispersity index (PDI) of 0.11, a positive zeta potential of +16.70 mV, and a high entrapment efficiency of 71.45%. For MS, microscopic images revealed a sharp tip from two opposite sides, with an average middle width of 5.15 μm and spicule lengths of 83.40 μm, respectively (Rangsimawong et al., 2024). The C-LIs exhibited good stability at 4°C for 6 months and at 25°C for 3 months, while the C-LI MS gel showed stability at 4°C for 3 months and at 25°C for 1 month (data not shown). Moreover, the C-LI MS gel effectively overcame the skin barrier, facilitating the transfollicular delivery of bioactive macromolecules from DAV extract (Rangsimawong et al., 2024).

Melanocyte stem cells in the bulge area of hair follicles are responsible for hair pigmentation, and defects in them cause hair graying (Nishimura et al., 2002, 2010). The aging process induces hair graying through distinct mechanisms that contribute to the decline in the melanocyte population within hair follicles. The primary cause of graying hair is the reduction in the number of pigmented melanocytes (Si et al., 2018).

Melanin, produced within the melanosome – an organelle enclosed by the membrane of melanocytes, is transferred to the hair shaft-forming keratinocytes, imparting the hair its inherent color (Peters et al., 2011; Kim et al., 2020; Le et al., 2021). The biosynthesis of melanin is a highly complex phenomenon, regulated by various melanogenic enzymes within the melanosome. Tyrosinase is a glycoprotein situated in the melanosome membrane, comprising internal, transmembrane, and cytoplasmic domains. It serves as the rate-limiting step in melanin synthesis by catalyzing the conversion of the essential amino acid L-tyrosine to L-DOPA, followed by its oxidation to L-dopaquinone. This process culminates in the formation of highly polymerized melanin pigment (Slominski et al., 2004; Sarkar et al., 2006; Schallreuter et al., 2008; Park et al., 2009; Videira et al., 2013). FGF-2 is an important keratinocyte-derived factor in mlenaocytes and essential for their proliferation (Halaban 2000).  Utilizing FGF-2 to regulate melanocyte number and melanin level represents an alternative strategy for inducing pigmentation (Dong et al., 2012). In addition, TGF-β also promotes melanocyte immaturity (Murakami et al., 2009; Nishimura et al., 2010; Feng et al., 2023). Hence, exogenous proteins and growth factors from DAV extract may play a crucial role in increasing melanin content, suggesting a potential use in hair aging treatment.

CONCLUSION

In this study, DAV extract stimulated melanogenesis by increasing tyrosinase activity and producing melanin in B16F10 melanoma cells, which DAV extract at the concentration of 100 µg/mL provided the highest melanogenesis effect. To improve the transfollicular delivery, DAV extract was successfully loaded into chitosan-modified liposomes (C-LI) and minimally invasive MS gel. Application of DAV extract loaded C-LI MS gel onto the scalp skin for 4 weeks exhibited the higher change in melanin level than control (untreated skin), suggesting the melanogenesis stimulation of DAV extract. Therefore, DAV extract plays an importance role to stimulate melanogenesis, in which its loaded C-LI MS transfollicular delivery system might be a potential formulation for stimulating melanin pigment in hair aging.

ACKNOWLEDGEMENTS

The authors thank the Faculty of Pharmaceutical Sciences, Ubon Ratchathani University and Faculty of Pharmacy, Silpakorn University for facility support.

AUTHOR CONTRIBUTIONS

Worranan Rangsimawong: conceptualization, methodology, visualization, funding acquisition, writing—original draft preparation; Sureewan Duangjit, Phaijit Sritananuwat, and Tipada Samseethong: methodology, writing—review and editing; Tanasait Ngawhirunpat and Praneet Opanasopit: supervision.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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