Human skin color is primarily determined by the content of melanin, a pigment that is produced by dermal melanocytes through melanogenesis. Melanin plays an important role in photoprotection and preventing skin cancer caused by UV radiation [1
]. However, hyperpigmentation disorders like melasma and actinic lichen planus are considered dermatological problems. Controlling melanogenesis is important for preventing aberrant pigmentation. Melanogenesis is regulated by tyrosinase (TYR), tyrosinase-related protein-1 and 2 (TRP-1 and TRP-2), and the microphthalmia-associated transcription factor (MITF) [2
]. TYR is the pivotal regulator of melanin production, catalyzing the hydroxylation of tyrosine to form l
-3, 4-dihydroxyphenylalanine (l
-DOPA), followed by oxidation of l
-DOPA to produce DOPA-quinone [4
]. TRP-1 and TRP-2 function in the biosynthesis of melanin downstream of TYR. TRP-2 catalyzes the production of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) from dopa-chrome, and TRP-1 oxidizes DHICA to produce indole-5,6-quinone carboxylic acid, ultimately resulting in melanin formation [5
]. The tyrosinase family genes, including TYR, TRP-1, and TRP-2 are regulated by MITF [8
]. MITF strongly regulates TYR gene expression and this transcription factor plays a key role in melanogenesis [5
]. Inhibition of melanin synthesis, through disruption of melanogenesis, could improve or prevent hyperpigmentation disorders. Numerous substances have been reported as tyrosinase inhibitors and mediate their effects either as competitive and noncompetitive inhibitors [12
]. Tyrosinase inhibitors include l
-ascorbic acid [13
], kojic acid [16
], ellagic acid [18
], tranexamic acid [19
], arbutin [22
], and hydroquinone [23
]. These compounds have been proposed as skin-whitening agents for the topical treatment of hyperpigmentation disorders. However, several side effects from these skin-whitening agents have been reported. 1,4-dihydroquinone can cause reversible inhibition of cellular metabolism, which is attributed to induce melanocyte cytotoxicity and mutation [25
]. In addition, kojic acid can trigger contact dermatitis [26
] and has carcinogenic potential [27
]. Therefore, there is considerable interest in finding alternative, herbal depigmenting agents.
(Al) Roxb. (Moraceae), also known as Artocarpus lacucha
, is a tropical tree found in South and Southeast Asia, including Southern China, India, Sri Lanka, Myanmar, Thailand, Malaysia, and Indonesia [29
]. In Thailand, Ma-Haad is the common name for Al and the aqueous extract from this plant, Puag-Haad, has been traditionally used as an antihelmintic drug for treatment of tapeworm infections [30
]. Ethanolic extracts of Al heartwood have shown potential antioxidant and tyrosinase inhibitory activities [32
]. The active components in the Al heartwood extract with tyrosinase inhibitory activity are oxyresveratrol and resveratrol, which have potential use as skin whitening agents [31
]. Hydroglycolic extracts of Al heartwood also exhibit antioxidant and in vitro tyrosinase inhibitory activities, as well as in vivo melanin-reducing effects in human volunteers. These reports indicate that Al extracts may have potential for skin-whitening agents [29
(Gg) from the family Fabaceae, is commonly known as liquorice, is cultivated in several regions, including China, Turkey, Syria, Italy, Pakistan, and Uzbekistan [36
]. Liquorice is widely utilized as a natural flavor extract in food and pharmaceutical products. In addition, it has been traditionally proposed for the treatment of upper and lower respiratory diseases, skin disorder, diabetes, gastrointestinal ulcers, stomach ache, and cardiovascular diseases [38
]. The hydrophobic fraction from extracts of Gg contained flavonoids and exhibited inhibitory effects on melanogenesis [39
] and extracts from Gg have been used as a depigmentation agent in cosmetic products [41
]. Glabridin, a polyphenolic flavonoid compound found in Gg extract, has been reported to inhibit the tyrosinase activity of melanocytes [42
] and may be the active component of Gg extracts.
Loss of effectiveness and side effects, like skin irritation, may appear when using a mono-extract as a long-term treatment. In this study, the combination of Al and Gg extracts was investigated for cellular cytotoxicity, tyrosinase inhibitory activities, and reduction of melanin contents in melanoma B16 cells. The expression of MITF, TYR, TRP-1, and TRP-2 in B16 cells was also evaluated. Enhancement of tyrosinase inhibitory activity and reduction of melanin content using a combination of Al and Gg extracts appears to be an alternative approach for treatment of hyperpigmentation disorders. This combination of Al and Gg extracts also has potential use as a whitening agent in skin care products.
In this study, a combination of Al and Gg extracts was investigated for cellular tyrosinase inhibitory activity, reduction of melanin contents and downregulated expression of melanogenesis-related genes MITF, TYR, TRP-1 and TRP-2 in melanoma B16 cells. This is the first report exhibiting additive activity of the combination of Al and Gg extracts to inhibit tyrosinase and reduce melanin pigments by suppressing the expression of MITF, TYR and TRP-2 in B16 cells.
Previously, Artocarpus lakoocha
(Al) extracted with 95% ethanol was shown to contain higher total phenolic content and exhibit higher tyrosinase inhibitory activity compared to Al extracted with propylene glycol [29
]. A high total phenolic content was also reported for Al extracted with 80% ethanol [33
]. Extraction of Al using water, methanol, ethanol, propanol and butanol demonstrated that ethanol was the most effective solvent for isolating oxyresveratrol and resveratrol [43
]. Similarly, Glycyrrhiza glabra
(Gg) extracted with ethanol had higher total phenolic and flavonoid contents than water extraction [45
]. Glabribin and glycyrrhizic were more abundant from Gg extracted in ethanol compared to extracted that with water, methanol, acetonitrile, and chloroform [46
]. In this study, Al and Gg were macerated in 70% and 95% ethanol and higher antioxidant and tyrosinase inhibitory activity was found when using 95% ethanol. These results were consistent with the levels of total phenolic and flavonoid contents found in Al95 and Gg95 extracts. In particular, oxyresveratrol and glabridin were more abundant in Al95 and Gg95 extracts, respectively, compared to extracts prepared using 70% ethanol. Al and Gg extracted with ethanol are expected to contain oxyresveratrol and glabridin, respectively, and these compounds appear to possess tyrosinase inhibitory activity. The mushroom tyrosinase test is a simple analytical method to assess inhibitory activity of compounds or extracts. However, mushroom tyrosinase is different from mammalian tyrosinase due to its unique requirements for substrate and cofactors. In addition, the sensitivity of mushroom tyrosinase to inhibitors is distinct compared to the mammalian enzyme [47
]. Another difference arises from protein modifications to these tyrosinase proteins. Mushroom tyrosinase is secreted, while mammalian tyrosinase is present in an inactive form that requires glycosylation in the Golgi apparatus and phosphoactivation by protein kinase C beta (PKCβ) [48
]. Many plant extracts that show in vitro inhibitory activity against mushroom tyrosinase are unable to reduce pigmentation in cells [51
]. Previously, crude extracts isolated from A. lakoocha
and G. glabra
have been found to have tyrosinase inhibitory activities and the ability to reduce melanin pigmentation as well as containing antioxidant and antimicrobial activities [34
]. Similarly, Al and Gg extracts in this study were able to inhibit both mushroom and cellular tyrosinase activities and reduce melanin pigments in B16 cells.
Mixtures of compounds can often lead to more pronounced effects than the individual products [54
]. Inhibition of melanin production by a combination of plant extracts had been reported previously. A combination of Siberian larch and pomegranate extracts exhibited a two-fold reduction in melanin content compared to S. larch or pomegranate extracts alone with no cytotoxicity to the cell. These combined extracts reduced expression of melanocyte specific genes, TYR, MITF, and melanosome structural proteins (melanocyte-specific glycoprotein (Pmel17) and melanoma antigen recognized by T cells 1 (Mart1)); however, inhibition of the tyrosinase enzyme was not observed [56
]. A combination of chia seed and pomegranate fruit extracts exhibited an additive effect on the inhibition of melanin biosynthesis also with no corresponding effect on tyrosinase activity. The combination of chia seed and pomegranate fruit extracts is likely due to decreased expression of melanogenesis-related genes (TYR, TRP1, and melanocortin 1 receptor (MC1R)) [57
]. In our study, a combination of Al95 and Gg95 extracts, at concentrations that did not reduce the viability of B16 cells, was able to decrease melanin pigments as well as inhibit tyrosinase activity. These results indicate that a combination between Al95 and Gg95 extracts additively effect the reduction of cellular melanin pigments. Investigation into the possible mechanism of action revealed that Al95 extract alone downregulated expression of melanogenesis-related genes MITF, TYR, and TRP-1. While a combination of Al95 with Gg95 extracts more significantly reduced expression of MITF, TYR, and TRP-2. The hypopigmentation effect of a combination of Al95 and Gg95 extracts may potentially be the result of down-regulation of MITF gene expression. Reduced MITF levels may affect in turn repress the expression of tyrosinase and TRP-2, as MITF is a major regulator of the synthesis of tyrosinase-related proteins (TRPs), including TYR, TRP-1 and TRP2 [58
Numerous topical products for skin whitening are available and contain active ingredients to reduce melanin production. Ascorbic acid and its derivatives, as active ingredients with anti-tyrosinase properties have been used in some commercial lightening products [60
]. Ascorbic acid acts as a reducer to block the chain of oxidations transforming tyrosine into melanin and interacts with copper, an essential cofactor in tyrosinase activity [61
]. This active ingredient is not used alone but always used in combination with another ingredient. A. lakoocha
extract was reported to exhibit in vitro tyrosinase inhibitory activity as well as the ability to reduce in vivo melanin content in a human volunteer. It was suggested based on these findings that A. lakoocha
extract has been proposed as a potential skin-whitening agent [34
]. However, another report raised the concern that at high concentrations the A. lakoocha
ethanolic extract may be toxic in the blood system [33
]. Glabridin and glabrene from G. glabra
are known to inhibit tyrosinase activity and have been used as depigmentation agents in cosmetics [25
]. Induction of melanin pigments by UV-B was reduced by topical application of 0.5% glabridin [42
]. The main drawbacks of glabridin are its poor skin-penetrating ability and instability in formulations [25
]. The combination of Al95 and Gg95 extracts has an additive effect on the reduction of melanin pigments, an alternative way to reduce the cytotoxicity of compounds in Al95 extracts and improve skin-penetration of compound Gg95 extracts; however, this will be further investigated. Finally, this is the first study suggesting that combination between A. lakoocha
and G. glabra
extracts is useful in development of skin care products for skin hyperpigmentation disorders.
4. Materials and Methods
Tyrosinase from mushroom (SLBJ5647, activity of 5771 unit/mg), 3,4-dihydroxy-l-phenylalanine (l-DOPA), dimethyl sulfoxide (DMSO), Folin and Ciocalteu’s Phenol reagent, 2,2-diphenyl-l-picrylhydrazyl (DPPH), sodium dihydrogen phosphate, 3-(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), kojic acid, gallic acid, quercetin, oxyresveratrol, resveratrol, and glabridin were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Disodium hydrogen phosphate and disodium carbonate were supplied by Merck Millipore (Temecula, California, USA). l-ascorbic acid and aluminium choride were purchased from Ajax Finechem (Taren Point, Australia).
4.2. Plant Materials and Extraction
Dried powder of A. lakoocha
(Al) heartwood and G. glabra
(Gg) root was purchased from Vejpongosot Pharmacy (Bangkok, Thailand). The extraction methods of Al and Gg were modified from those previously described [43
]. Briefly, one kilogram of dried plant material was macerated overnight in 5 L of 70% or 95% ethanol at room temperature for Al and at 48 °C for Gg. After three rounds of maceration, the alcoholic extracts were pooled, filtered, and evaporated under reduced pressure below 45 °C. The yields of crude extracts for Al70, Al95, Gg70 and Gg95 were 9.703%, 8.675%, 8.46% and 8.153%, respectively.
4.3. Determination of Total Phenolic Content
The total phenolic compounds in Al and Gg extracts were detected using the Folin–Ciocalteu reagent, [63
]. In 96-well plates, 4.5 μL of 1 mg/mL extracts was diluted in 126 μL of deionize water, the samples were mixed with 90 μL of 2% Na2CO3 for 3 min and then 4.5 μL of 50% Folin–Ciocalteu reagent was added. The samples were incubated at room temperature for 30 min and the resulting blue molybdenum–tungsten complex was monitored at 750 nm using a microplate reader (Biochrom EZ Read 2000, Cambridge, UK). The total phenolic content in each sample was calculated by comparison to a gallic acid standard and results are presented as milligrams of gallic acid equivalent to one gram of extract (mg GAE/g extract).
4.4. Determination of Flavonoid Content
The flavonoid compounds of Al and Gg extracts were determined using the aluminum chloride assay [64
]. In 96-well plates, 100 μL of 1 mg/mL extracts were mixed with 100 μL of 2% AlCl3. Following incubation at room temperature for 10 min, the absorbance was monitored at 415 nm with a microplate reader (Biochrom EZ Read 2000, Cambridge, UK). The total flavonoid content of samples was analyzed by comparison to quercetin standard and the results were presented as quercetin equivalent (mg QE/g extract).
4.5. Phytochemical Screening Assay
Chemical composition was determined using Ultra High Performance Liquid Chromatography (UHPLC) modified as previously described [31
]. UHPLC analysis was performed using a Thermo Scientific UHPLC UltiMate 3000 (Waltham, MA, USA) with a Hypersil GOLD™ a Q column (100 × 2.1 mm i.d., 1.9 µm, Thermo Scientific™) and results were analyzed with Thermo Scientific™ LCQUAN™ software. Crude extracts of Al and Gg and standard solutions (oxyresveratrol, resveratrol, gallic acid and glabridin) were dissolved in methanol at concentrations of 1 and 10 mg/mL. Samples were filtered (0.20 mm, Millipore) and 1 μL was directly injected. Solvents for HPLC analysis were formic acid (0.1% v
) in water as solvent A and formic acid (0.1% v
) in methanol as solvent B, at a flow rate of 0.5 mL/min. These experiments used the following gradient: 30% B linear (0–4 min), 30–50% B linear (4–5 min), 50–70% B linear (5–8 min), 70–100% B linear (8–12 min), 100% B (12–15 min), and 30% B linear (15–18 min). An equilibrium period of 5 min was performed prior to the injection of subsequent samples. Chromatograms were recorded at 280 nm (glabridin and gallic acid), 305 nm (resveratrol), and 326 nm (oxyresveratrol), using the photodiode detector. Quantitative determination of compounds was performed using peak area with an external standard.
4.6. 2,2-di-phenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay
Antioxidant activity of Al and Gg extracts was determined based on DPPH scavenging ability [65
]. Seventy-five microliters of the extracts at various concentrations (0.015–1 mg/mL) were mixed with 150 μL of 0.2 mM DPPH, incubated for 30 min, and the absorbance at 515 nm was monitored using a microplate reader (Biochrom EZ Read 2000, UK). Ethanol was used as the blank solution and L-ascorbic acid was the positive control. The scavenging activity of DPPH radicals was calculated and expressed as a percentage of the blank. The IC50
value represents the concentration of extract capable of reducing DPPH by 50%, calculated using a linear regression graph.
4.7. Mushroom Tyrosinase Assays
Inhibition of mushroom tyrosinase activity was detected using L-3,4-dihydroxyphenylalanine (l
-DOPA) as a substrate [66
]. Briefly, 20 μL of extracts at various concentrations (0.0024–1.25 mg/mL), 140 μL of 20 mM phosphate buffer (pH 6.8), and 20 μL of 461.68 unit/mL of mushroom tyrosinase were added to each well of a 96-well plate. Samples were mixed for 10 min, 20 μL of 4 mM l
-DOPA was then added and this was followed by incubation at 37 °C for 30 min. The relative amount of dopachrome formed in the mixture was monitored by measuring the absorbance at 475 nm using a microplate reader (Biochrom EZ Read 2000, UK). Tyrosinase and l
-DOPA only solutions were used as a negative control. Kojic acid at concentrations ranging from 0.0024 to 1.25 mg/mL was a positive control. The inhibition of tyrosinase activity was calculated and presented as a percentage of the control. The IC50 for tyrosinase inhibition was calculated using a linear regression graph and is the concentration of extract capable of reducing tyrosinase activity to 50%.
4.8. Cell Viability Assay
Cell viability of B16 cells was determined with the MTT assay to establish the non-cytotoxic concentrations of Al and Gg extracts [67
]. B16 melanoma cells (RIKEN Cell Bank, Tsukuba, Japan) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco®
/Thermo Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS) and penicillin/streptomycin (50 μg/mL) in an incubator with 5% CO2 at 37 °C. Briefly, 3.0 × 104
cells were plated into 96-well plates and incubated at 37 °C overnight. After removal of the culture medium, cells were treated for 72 h wth 200 μL of extracts, diluted with medium at various concentrations of 0.02–0.12 mg/mL and 0.1–1 mg/mL for the Al and Gg extracts, respectively. The culture media with extracts was removed and treated cells were then added with 100 μL of 0.5 mg/mL MTT solution. After additional 2 h, the formazan crystals were dissolved with 100 μL of dimethyl sulfoxide (DMSO). The absorbance was monitored at 530 nm using a microplate reader (1420 Victor 2, Wallac, Ramsey, MN, USA). The results were presented as the percentage of viable cells (% cell viability) by comparing with untreated cells as a control.
4.9. Melanin Content Assay
Melanin content in B16 cells was evaluated as described previously [68
]. In brief, 1.5 × 105
B16 cells were plated into 24-well plates and incubated overnight to allow cells to adhere. Cells were treated for 48 h with various concentrations of 0.02–0.1 mg/mL and 0.1–0.8 mg/mL for Al and Gg extracts, respectively. After cells were harvested using trypsin, the cell pellet was resuspended with 100 μL of 1 N NaOH, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride (PMSF) and incubated at 60 °C for 1 h to allow cell lysis. Melanin content of lysates was monitored at 405 nm using a microplate reader (1420 Victor 2, Wallac, USA). Hydroquinone at 20 mM was used as a positive melanin inhibitor. Melanin content was presented as a percentage of the value obtained for untreated control cells.
Accumulation of melanin pigments and cellular morphology of B16 cells were observed with a Masson-Fontana stain using a procedure modified from that previously described [69
]. In brief, 5.0 × 105
B16 cells/mL were plated into 6-well plates for 24 h. Cells were treated with non-toxic doses of extracts and incubated for 3 days. Treated cells were fixed with absolute ethanol and rehydrated with distilled water. Cells were then stained with an ammoniacal silver solution for 24 h, sodium thiosulfate for 5 min and Mayer’s Carmalum for 10 min. Melanin pigments and cellular morphology were observed under a light microscope at a magnification of 100×. The number of melanin containing cells were calculated from a total of 1000 cells counted.
4.10. Cellular Tyrosinase Activity Assay
Tyrosinase activity from B16 cells was determined using l
]. B16 cells, 1.0 × 105
cell/well were plated into a 24-well plate and incubated overnight to allow cells to adhere. Cells were treated for 48 h with various concentrations of 0.02–0.1 mg/mL and 0.1–0.8 mg/mL for Al and Gg extracts, respectively, and harvested. Cell pellets were lysed with 100 µL of 0.1% Triton X-100 and 0.1 mM PMSF (pH 7.5) in phosphate buffer saline (PBS). Lysates were clarified by centrifugation at 12,000× g
at 4 °C for 15 min to obtain the supernatant. Reaction mixtures consisting of 80 μL of supernatant and 20 μL of 20 mM l
-DOPA were determined after incubation at 37 °C for 60 min. The optical densities were measured at 492 nm using a microplate reader (1420 Victor 2, Wallac, Ramsey, MN, USA). Sodium l
-lactate (SLL) at a concentration of 20 mM was used as a positive control. The cellular tyrosinase activity was calculated and presented as a percentage of the control (100% for untreated cells).
4.11. Evaluation of Additive Effects of Al and Gg Extracts on Cellular Melanin Content and Tyrosinase Activity
Combinations between Al95 and Gg95 extracts were evaluated for inhibition of tyrosinase activity and melanin production in B16 cells. Mixtures of Al and Gg extracts was prepared at ratios of 9:1, 7:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:7 and 1:9. The final concentration of all combined extracts of Al and Gg was 0.1 mg/mL. These combined extracts were evaluated for cytotoxicity, inhibition of tyrosinase activity, and effects on melanin pigments in melanoma B16 cells.
4.12. Western Blot Analysis
The abundance of proteins involved in melanogenesis was determined using Western blot analysis, modified from the procedure previously described [71
]. Briefly, 1.0 × 105
cells were plated into the 6 well plates and incubated at 37 °C, 5% CO2 for 24 h. Following removal of the old media, 2 mL of conditioned media containing combined Al and Gg extracts at ratios of 1:1 and 9:1 and single extract in final concentration at 0.1 mg/mL was added and samples were incubated at 37 °C, 5% CO2 for 48 h. Treated cells were harvested and lysed in 100 μL radioimmunoprecipitation assay buffer (RIPA) buffer. Protein content was determined using a protein quantitation kit (Merck, Darmstadt, Germany). Total protein, 20 μg, from each sample was separated using 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were treated separately with the following primary antibodies at the indicated dilutions at room temperature for 1 h with gentle agitation: rabbit anti-mouse tyrosinase (immunoglobulin G (IgG) (1:100), rabbit anti-mouse TRP-1 IgG (1:200), rabbit anti-mouse TRP-2 IgG (1:100), goat anti-mouse MITF IgG (1:200) and rabbit anti-mouse β-actin IgG (1:500). The membranes were treated with secondary anti-mouse or goat IgG conjugated with horseradish peroxides (1:2000). Protein bands were visualized with enhanced chemiluminescence (ECL) with a Gel documentation system and quantitated using Image J software.
4.13. Statistical Analysis
All grouped data were evaluated with SPSS version 17. Data were expressed as the mean ± SD of three independent experiments and analyzed for statistical significance using t-test and one-way ANOVA. Differences were considered statistically significant at p < 0.01.