Characterization of Phenethyl Cinnamamide Compounds from Hemp Seed and Determination of Their Melanogenesis Inhibitory Activity

Inhibitory Potential of Hemp Seed Solvent Fractions against Melanogenesis

To assess the anti-melanogenesis activity of the four solvent fractions, we conducted anti-melanogenesis tests in melanoma cells. First, the effects of the hemp seed solvent fractions on melanoma cell proliferation were investigated by treating B16F10 cells with 20 μg/mL of each different fractions or arbutin in the presence of α-melanocyte stimulating hormone (α-MSH), a hormone known to promote melanogenesis through microphthalmia-associated transcription factor induction.¹³ The results showed that none of the fractions exhibited significant effect on melanoma cell proliferation (Figure​Figure11C). We then investigated the inhibitory effect of the solvent fractions on melanogenesis stimulated by α-MSH in melanoma cells. Treatment with α-MSH induced an approximately 1.7-fold increase in the intracellular melanin content of B16F10 cells (Figure​Figure11D). Findings showed that α-MSH-mediated melanogenesis was completely inhibited by the EtOAc fraction (p < 0.001) but partially inhibited by n-Hex (75%, p < 0.001), CH₂Cl₂ (33%, p < 0.05), and n-BuOH (50%, p < 0.01) fractions (Figure​Figure11D). Furthermore, since tyrosinase plays a key role in melanogenesis, the inhibitory effects of the solvent fractions on tyrosinase activity mediated by α-MSH were also determined. The EtOAc (61%, p < 0.001), n-Hex (28%, p < 0.05), and n-BuOH (22%, p < 0.05) fractions reversed tyrosinase activation mediated by α-MSH, whereas CH₂Cl₂ fractions did not show significant inhibitory property, as shown in Figure​Figure11E. To identify functional ingredients in hemp seed extracts that could be responsible for the inhibition of melanogenesis in melanoma cells, we determined the total phenolic content in each solvent fraction. Results demonstrated that phenolic compounds were most abundant in the EtOAc fraction [192.3 ± 4.5 mg of the gallic acid equivalents (GAE)/g extract], followed by n-Hex (95.8 ± 2.4 mg of the GAE/g extract), n-BuOH (51.4 ± 3.5 mg of the GAE/g extract), and CH₂Cl₂ (44.5 ± 2.7 mg of the GAE/g extract), as shown in Figure​Figure11F. These results indicated that the inhibition rate of each solvent fraction on melanogenesis followed the same order as that of total phenolic content (i.e., EtOAc > n-Hex > n-BuOH > CH₂C_l2). Phenolic compounds are known to have antioxidant, anti-tyrosinase, and anti-inflammatory effects. Taken together, these results suggested that the bioactive ingredients inhibiting melanin synthesis were mainly found in the EtOAc fraction, one of which are phenolic compounds.

Identification and Characterization of Bioactive Compounds of Hemp Seed Extracts

In order to identify and characterize compounds inhibiting melanogenesis, we established an in-house library isolated from hemp seed extracts (up to 2020). Previous studies reported the presence of various compounds such as lignanamides and phenolic acids in hemp seed extracts.¹⁴ Herein, the coupled ultra-performance liquid chromatography to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) tentatively identified 26 compounds in the hemp seed extract including phenolic compounds within 18 min based on their negative ion electrospray ionization (ESI) mass spectra (Figure​Figure22). Compounds were identified based on the m/z values of the molecular ion [M – H]⁻, and the identifications were supported by comparing obtained mass spectra with mass spectra available in the MassLynx V4.1 Library (Waters Corporation, Milford, MA, USA). However, three major peaks (peaks 16, 20, and 22) could not be identified using the in-house library. Details on their observed masses and calculated molecular formula are given in Table 1. In accordance with previous investigations of chemical components of hemp seeds, compounds identified by MS/MS were predominantly phenylpropionamides, namely, phenolic amides and lignanamides.

To identify compounds inhibiting melanogenesis, out of the 26 identified compounds, we prepared three phenethyl cinnamamides such as N–trans-caffeoyltyramine, N–trans-coumaroyltyramine, and N–trans-feruloyltyramine which were expected to be associated with melanogenesis. N–trans-caffeoyltyramine was obtained from the hemp seed EtOAc fraction using prep-high-performance liquid chromatography, while N–trans-coumaroyltyramine and N–trans-feruloyltyramine were obtained by chemical synthesis. The three compounds were then identified by comparing their ¹H and ¹³C NMR data with the literature and were found to be in agreement with the proposed structures (Figure​Figure33).¹⁵ Compound 1 (yield, 2.1 + 0.1 mg/50 mg EtOAc fraction) was obtained as a white powder. ¹H NMR (400 MHz, MeOD): δ 7.37 (d, J = 15.69 Hz, 1H), 7.05 (d, J = 8.52 Hz, 2H), 6.99 (d, J = 1.88 Hz, 1H), 6.89 (dd, J = 8.20, 1.92 Hz, 1H), 6.75 (d, J = 8.12 Hz, 1H), 6.71 (dd, J = 6.52, 4.92 Hz, 2H), 6.33 (d, J = 15.66 Hz, 1H), 3.45 (t, J = 7.40 Hz, 2H), 2.74 (t, J = 7.40 Hz, 2H); ¹³C NMR (100 MHz, MeOD-d₄): δ (169.3, 156.9, 148.7, 146.7, 142.2, 131.3, 130.7, 128.3, 122.1, 118.4, 116.5, 116.3, 115.1, 42.6, 35.8). Compound 1 was identified as N–trans-caffeoyltyramine by NMR analysis and by comparison with the previous literature (Figure​Figure33A,B).¹⁵ Synthetic N–trans-feruloyltyramine (compound 2) (overall yield, 17%) was obtained as a brown crystalline and identified by NMR analysis (Figure​Figure33C,D). ¹H NMR (400 MHz, MeOD): δ 7.45 (d, J = 15.68 Hz, 1H), 7.14 (d, J = 1.91 Hz, 1H), 7.08 (d, J = 8.56 Hz, 2H), 7.04 (dd, J = 8.44, 2.04 Hz, 1H), 6.81 (d, J = 8.12 Hz, 1H), 6.74 (d, J = 8.56 Hz, 2H), 6.42 (d, J = 15.68 Hz, 1H), 3.90 (s, 3H), 3.48 (t, J = 7.37 Hz, 2H), 2.77 (t, J = 7.37 Hz, 2H); ¹³C NMR (100 MHz, MeOD-d₄): δ (169.2, 156.9, 149.9, 149.3, 142.0, 131.3, 130.8, 128.3, 123.2, 118.8, 116.5, 116.3, 56.4, 42.6, 35.8). Synthetic N–trans-coumaroyltyramine (compound 3) (overall yield, 32%) was obtained as a white powder and identified by NMR analysis (Figure​Figure33E,F). ¹H NMR (400 MHz, MeOD): δ 7.44 (d, J = 15.74 Hz, 1H), 7.39 (d, J = 8.56 Hz, 2H), 7.05 (d, J = 8.56 Hz, 2H), 6.78 (d, J = 8.56 Hz, 2H), 6.71 (d, J = 8.56 Hz, 2H), 6.38 (d, J = 15.70 Hz, 1H), 3.45 (t, J = 7.40 Hz, 2H), 2.75 (t, J = 7.39 Hz, 2H); ¹³C NMR (100 MHz, MeOD-d₄): δ (169.3, 160.5, 156.9, 141.8, 131.3, 130.7, 130.6, 127.8, 118.5, 116.7, 116.3, 42.6, 35.8).

Inhibitory Potential of Phenethyl Cinnamamide Compounds Identified in Hemp Seed Extracts against Melanogenesis

To assess the inhibitory activity of the three phenethyl cinnamamide derivatives identified in hemp seed extracts, we conducted anti-melanogenesis tests in melanoma cells. First, the effects of the three compounds on melanoma cell proliferation were investigated by treating B16F10 cells with 50 μM of each compound in the presence of α-MSH. The results showed that N–trans-caffeoyltyramine and N–trans-feruloyltyramine exhibited no significant effect on melanoma cell proliferation, while N–trans-coumaroyltyramine inhibited melanoma proliferation by more than 50% of control (Figure​Figure44A). Furthermore, we investigated the inhibitory effect of the three derivatives on α-MSH-induced melanin synthesis in B16F10 cells. Treatment with α-MSH induced approximately a 1.7-fold increase in the intracellular melanin content of B16F10 cells (Figure​Figure44B). It is observed that compound 2 (N–trans-feruloyltyramine) partially inhibited α-MSH-induced melanogenesis (45%, p < 0.05), whereas compounds 1 (N–trans-caffeoyltyramine) and 3 (N–trans-coumaroyltyramine) completely reversed α-MSH-induced melanogenesis (Figure​Figure44B, p < 0.001). We also investigated the effect of the three compounds on tyrosinase activity in B16F10 cells. Results showed that tyrosinase activity mediated by α-MSH was partially reversed by N–trans-caffeoyltyramine (68%, p < 0.001) and N–trans-feruloyltyramine (36%, p < 0.05). IC₅₀ values of N–trans-caffeoyltyramine, N–trans-feruloyltyramine, and N–trans-coumaroyltyramine for inhibition of α-MSH-mediated tyrosinase activation were 0.8, 20.2, and 6.3 μM, respectively (data not shown). Interestingly, N–trans-coumaroyltyramine showed the strongest inhibitory activity on melanogenesis at 50 μM concentration and demonstrated anti-proliferative activity on cancer cells, which is consistent with a previous report.¹⁶ Thus, N–trans-coumaroyltyramine could be more appropriate for the development of future anticancer agents rather than functional cosmetic materials. Furthermore, our results suggested that the three phenethyl cinnamamide derivatives with similar structures but different substitution groups on the same positions showed different biological activities on melanogenesis and cell proliferation. Among the three phenethyl cinnamamide derivatives under investigation, N–trans-caffeoyltyramine exhibited potent inhibitory activity against melanogenesis and lowest IC₅₀ values for inhibition of α-MSH-mediated tyrosinase activation in melanoma cells.

To predict interaction between the three phenethyl cinnamamides and tyrosinase, a computational molecular docking study was performed using the Flare V4.0 program. The best poses for the three compounds, docked to tyrosinase, are depicted in Figure​Figure55. The molecular docking study revealed that N–trans-caffeoyltyramine and N–trans-coumaroyltyramine established a π–π stacking with His 263 and H-bond with Glu 322, while N–trans-feruloyltyramine established a π–π stacking with Phe 264 and a series of H-bond with Met 280 and Thr 261. Furthermore, binding energies between tyrosinase and the three compounds, including N–trans-caffeoyltyramine, N–trans-feruloyltyramine, and N–trans-coumaroyltyramine, were −11.44, −10.35, and −10.64 kJ/mol, respectively. These results indicated that binding energy between tyrosinase and N–trans-caffeoyltyramine was higher than the other two molecules, which is consistent with the cell-based evaluation. In addition, we observed that the hydroxyl group at the meta position of the benzene ring helped the hydroxyl group at the para position of the benzene ring to interact with tyrosinase.

Extraction and Fractionation of Hemp Seeds

Dried hemp seeds (960 g) were pulverized using a pulverizer (HMF-3250s, Hanil Electric, Korea), then defatted with petroleum ether (9.6 L) at room temperature (RT) for 2 h, as described previously.^8a Defatted hemp seeds were added to 70% EtOH (9.6 L), and the mixture was sonicated three times for 15 min at 20% amplitude (Bandelin, HD 4200, Germany). The resulting extract was vacuum filtered using Advantec filter papers no. 1 and no. 2 (Toyo Roshi Kaisha, Ltd, Tokyo, Japan) and concentrated using a Hei-VAP Advantage rotary evaporator (Heidolph, Germany) to obtain 27 g of the EtOH crude extract. The crude extract was suspended in dH₂O (270 mL) and successively portioned with n-Hex, CH₂Cl₂, EtOAc, and n-BuOH to yield n-Hex (1.44 g, 5.3%), CH₂Cl₂ (4.50 g, 16.7%), EtOAc (0.85 g, 3.1%), and n-BuOH (1.01 g, 3.7%) fractions, as described previously.¹⁷ The crude extracts and fractions were freeze-dried and kept at −80 °C prior to use.

Determination of ABTS Radical Scavenging Activity

In order to evaluate antioxidant potential of hemp seed solvent fractions, the ABTS radical scavenging activity was assessed, according to the previously described method.¹⁸ To prepare fresh ABTS radical solution, 10 mL of 7 mM ABTS was mixed with 176 μL of 140 mM potassium peroxydisulfate in dH₂O and reacted in the dark at RT for 16 h. The ABTS radical solution was then diluted with methanol to obtain an absorbance of approximately 0.7 at 732 nm. Aliquots of 100 μL of each fraction in the concentration range of 2–200 μg/mL were mixed with 100 μL of the diluted ABTS radical solution and incubated for 10 min in the dark at RT. The absorbance was then taken at 732 nm using a SpectraMax M5 Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). The percentage of ABTS radical scavenging activity was calculated as follows

1

In Vitro Tyrosinase Inhibition

The inhibitory potential of hemp seed solvent fractions on tyrosinase activity was assessed by measuring the total amount of dopachrome generated during the catalytic enzyme reaction of tyrosinase.¹⁹ Briefly, 50 μL of each fraction with the concentration range of 1–100 μg/mL was added to mushroom tyrosinase solution (50 U/mL) prepared in 50 mM phosphate-buffered saline (PBS; 8.1 mM Na₂HPO₄, 1.2 mM KH₂PO₄, pH 6.8, 2.7 mM KCl, 138 mM NaCl) in a 96-well plate and incubated for 30 min at RT. Then, equal quantities of 1 mM L-DOPA were added to the reaction mixtures, followed by further incubation for an additional 10 min at 37 °C. The absorbance of the resulting solution was measured at 475 nm, using a SpectraMax M5 Multi-Mode microplate reader.

Determination of the Total Phenolic Content

The total phenolic content of each solvent fraction was measured via the Folin-Ciocalteu method adopted in the previous report with slight modifications.²⁰ Briefly, diluted crude extracts or solvent fractions of hemp seeds (25 μL) were mixed with 100 μL of the diluted Folin-Ciocalteu reagent (1:4) and incubated for 5 min. Then, 75 μL of sodium carbonate solution (100 g/L) was added to the reaction mixture and further incubated for 2 h. The absorbance was measured at 765 nm using a microplate reader. Gallic acid dilutions (10–200 mg/L) were used as standards for calibration. The total phenolic content was expressed as mg GAE per g of the extract or fraction.

Cell Culture

B16F10 murine melanoma cells were cultured in Dulbecco’s modified eagle medium (Gibco, Gaithersburg, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37 °C in a humidified atmosphere of 5% carbon dioxide (CO₂).

Cell Viability

The viability of B16F10 cells was determined using an MTT assay, as previously described.²¹ Briefly, B16F10 cells were seeded in 24-well plates (1 × 10⁴ cells/well) and incubated for 24 h. The cells were treated with hemp seed solvent fractions or chemical compounds at concentrations of 20 μg/mL or 50 μM, respectively. After being incubated for 48 h, the cells were then incubated with MTT solution for 4 h, and the generated formazan crystals were dissolved in dimethyl sulfoxide (DMSO). 200 μL of the resulting solution was transferred to 96-well plates, and the absorbance was measured at 540 nm using a SpectraMax M5 Multi-Mode microplate reader.

Melanin Content Determination

The melanin content was determined, as previously described, with some modifications.²² The melanoma cells were seeded in a 6-well plate and cultured for 24 h. They were treated with hemp seed solvent fractions or chemical compounds at concentrations of 20 μg/mL or 50 μM, respectively, for a further 48 h in the presence of 100 nM α-MSH. After washing twice with chilled Dulbecco’s PBS supplemented with calcium chloride and magnesium chloride (D-PBS, Gibco), the resulting cells were collected via trypsinization. After centrifugation at 1000 rpm for 3 min, the cell pellet was dissolved in 150 μL of 1 N NaOH containing 10% DMSO for 1 h at 60 °C. The melanin content was determined by the absorbance at 405 nm, using a microplate reader.

Determination of Cellular Tyrosinase Activity in Melanoma Cells

Tyrosinase activity in B16F10 cells was examined based on the amount of dopachrome produced from the catalytic reaction of intracellular tyrosinase.²³ Briefly, melanoma cells were cultured in a 6-well plate for 24 h, followed by treatment with different concentrations of hemp seed solvent fractions or chemical compounds for a further 48 h in the presence of 100 nM α-MSH. After washing twice with ice-cold D-PBS, the cells were lysed in 200 μL of radio-immunoprecipitation assay buffer (Sigma-Aldrich) containing protease and phosphatase inhibitors. The cell lysate from each well was collected after being centrifuged at 15,000g for 15 min. 100 μL of the supernatant was mixed with 100 μL of 1 mM L-DOPA in PBS (pH 6.8), followed by incubation for 30 min at 37 °C. The absorbance of dopachrome was measured at 475 nm using a microplate reader. Data were normalized with protein concentration determined by bicinchoninic acid assay.

Characterization of Bioactive Compounds Using UPLC-QTOF-Mass Spectrometry/Mass Spectrometry

The EtOAc fraction was analyzed using the Waters Acquity UPLC I-Class system (Waters Co., Milford, MA, USA) coupled with mass spectrometry, as previously described.²⁴ The EtOAc fraction was dissolved in 70% methanol at a concentration of 100 ppm and filtered through a 0.2 μm PTFE syringe filter. The Waters Acquity UPLC BEH C18 column (150 mm × 2.1 mm, 1.7 μm) was used for chromatographic separation. The separation conditions were as follows: mobile phases: 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), and the following gradient was used: 0–5 min, 10–20% B; 5–25 min, 20–22% B; 25–45 min, 22–25% B; 45–65 min, 25–30% B; and 65–85 min, 30% B. The injection volume was 2 μL. The mass spectrometry (MS) experiments were performed on a Waters Xevo G2 QTOF mass spectrometer (Waters MS Technologies, Manchester, UK) equipped with an ESI interface. The MS/MS ion patterns were obtained using a collision energy ramp from 15 to 45 eV in the MSE mode. The ESI parameters were set as follows: in the negative ion mode in a continuum format, a capillary voltage of 2.5 kV, a cone voltage of 45 V, a source temperature of 120 °C, a desolvation temperature of 350 °C, a cone gas flow of 50 L/h, and a desolvation gas flow of 800 L/h. The ion acquisition rate was 0.25 s with resolution in excess of 20,000 fwhm, and the inter-scan delay time was 0.014 s. The mass range was from m/z 100 to 1500. The instrument was calibrated using a sodium formate solution as the calibration standard. Leucine encephalin (m/z 554.2615 in the negative mode) was used as a reference or lock mass at a concentration of 200 pg/μL and a flow rate of 5 μL/min and was sprayed into the MS instrument every 10 s to ensure accuracy and reproducibility. Data acquisition was controlled by MassLynx V4.1 software (Waters Corporation, Milford, USA), and identification of components was performed by UNIFI.

Tentative Identification by UNIFI and Search of an In-House Library

Compound identifications were achieved by UNIFI 1.8 (Waters, Milford, USA). The Traditional Chinese Medicine (TCM) library in UNIFI consisted of more than 7500 natural product compounds. The library was updated with compounds that were either isolated or analyzed from hemp seed (up to 2020) by searching databases such as Dictionary of Natural Product (CRC, 2009), SciFinder Scholar of the American Chemical Society, and Natural Product Activity & Species Source Database. The database and structure files that were prepared using ChemBioDraw 15.0 were saved as a mol file individually into UNIFI. Key parameters within UNIFI were set as follows: peak detection time, 1–85 min; intensity threshold, 3000 and 300 counts for low-energy and high-energy acquisitions, respectively; and mass accuracy, ±5 ppm. Both the TCM library and the in-house library were retrieved in identification.

Molecular Modeling

The crystal structure of Agaricus bisporus mushroom tyrosinase (PDB dose: 2Y9X) for molecular modeling was obtained from Protein Data Bank (PDB). The enzyme was prepared using the protein wizard preparation workflow embedded in the Flare V4.0 program (Cresset Software Inc., England). Water and all the other molecules present in the pdb files were removed. Molecular docking was performed using the standard docking mode and the extra precision mode.

This study was partially supported by grants from the Research and Development of Chuncheon Hemp (Chuncheon city, Korea), Presidential Committee for Balanced National Development (PCBND), and the Ministry of Land, Infrastructure and Transport (MOLIT) through the pilot project for regional development investment agreement (B0070128000223). HR-ESI-MS spectra were obtained from Korea Basic Science Institute (KBSI, Chuncheon center).