2013 Volume 36 Issue 8 Pages 1341-1347
Rhamnetin is a naturally occurring polyphenolic compound. In this report, experimental evidence is presented on the suppression of melanogenesis by rhamnetin using B16 murine melanoma cells (B16 cells). To document the underlying anti-melanogenic action of rhamnetin, several key biochemical mediators were quantified: superoxide (O2•−), nitric oxide (·NO) and peroxynitrite (ONOO−) in vitro, and total reactive species (RS) generation, O2•−, ·NO and ONOO−, reduced glutathione (GSH)/GSH-to-oxidized glutathione (GSSG) ratio, prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) in B16 cells. Results revealed that rhamnetin inhibited murine tyrosinase activity, suppressed melanin content and oxidative stress, reducing O2•−,·NO and ONOO− in vitro and total RS generation, O2•−, ·NO and ONOO− in B16 cells, while maintaining a well-balanced GSH/GSSG ratio in B16 cells. Results further revealed that rhamnetin suppressed key pro-inflammatory mediators such as PGE2 and TXB2. Thus, these results strongly indicate that rhamnetin has powerful anti-melanogenic properties through its anti-oxidative and anti-inflammatory actions.
Melanin is synthesized by melanocytes that are derived from melanoblasts during embryogenesis and migrate to the skin’s basal layer of the epidermis. Melanocytes are dendritic cells that produce melanin within vesicles, termed melanosomes, that then are later transferred to neighbouring keratinocytes1,2) Tyrosinase is known to catalyze the first two steps in melanin generation: the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the subsequent oxidation of this o-diphenol to the corresponding quinone, L-dopaquinone.1–3)
Melanogenesis plays a significant role in preventing skin damage.4) However, altered, excessive production of melanin and the abnormal hyperpigmentation from overexposure to UV rays may cause excessive generation of reactive species (RS) that can lead to various skin injuries, including inflammation, age spots, melasma and freckles.4)
Skin injuries also can be elicited by both exogenous and endogenous reactive species (RS), including reactive oxygen species (ROS) and reactive nitrogen species (RNS) that cause oxidative stress.5) Especially potent is peroxynitrite (ONOO−), an RNS formed by the reaction of nitric oxide (·NO) with superoxide (O2•−), which recently has been defined as a strong oxidant and potential mediator of skin injury.6) Reduced glutathione (GSH), the most abundant non-protein thiol, plays significant roles in cellular defenses against oxidant aggression from excess RS, and the depletion of reduced GSH, which leads to a shift in cellular GSH-to-oxidized glutathione (GSSG) redox balance, is indicative of oxidative insult to the skin.7)
Oxidative stress due to excessive production of RS and a disturbed redox balance are known to be implicated in skin aging and abnormal pigmentation.4) Recently, we reported a causal relation between oxidative stress and inflammation,8) and more recently, Tatsuno et al. reported a possible connection between the inhibition of melanognesis and anti-inflammatory properties in skin.9) Therefore, it seems that enhancing the skin’s anti-oxidative and anti-inflammatory capacities would be a suitable strategy for developing skin-lightening reagents. This strategy has gained further experimental support from our previous work in which we found that modulating oxidative insult by the suppression of RS generation is an important factor in melanogenesis inhibition.10,11)
Many natural phenolic compounds have been reported as melanogenesis inhibitors.10–13) Although rhamnetin, a phenolic flavonoid compound, is known to function as an antioxidant14) and alkylperoxyl radical-scavenging,15) anti-inflammatory,16) xanthine oxidase inhibitory17) and antiviral agent,18) its protective action against melanogenesis, oxidative stress, and inflammatory mediators has not been fully elucidated to date.
The mechanisms underlying melanogenesis inhibition are not fully discovered at present, but several possibilities are suggested. Among these mechanisms are tyrosinase inhibition, antioxidant action, desquamation stimulation, adrenergic and glutaminergic signaling regulation, and the ability to control tetrahydrobiopterins in the human skin.1,2)
The purpose of the current study is to investigate whether rhamnetin has melanogenesis inhibitory effects, and if so, to elucidate the underlying actions of its ability to inhibit melanogenesis. To explore the antioxidative and anti-inflammatory actions of rhamnetin, our investigation utilized B16 murine melanoma cells (B16 cells).
The focus of present work was to determine the effect of rhamnetin on melanin production and the biological mechanisms underlying its anti-melanogenesis property in B16 cells by quantifying cell viability and cellular melanin content as well as murine tyrosinase activity as an estimate for rhamnetin’s anti-melanogic action.
To better understand the biological mechanism of anti-melanogenesis by rhamnetin, oxidative insult parameters, including O2•−, ·NO and ONOO− in vitro, and total RS generation, O2•−, ·NO, ONOO− and GSH/GSSG ratio in B16 cells, were measured. Additionally, to evaluate the anti-inflammatory action associated with anti-melanogenic action, activity of the key pro-inflammatory mediators, prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) were determined.
L-Tyrosine, α-melanocyte stimulating hormone (MSH), and other chemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
Cell Lines and CulturesB16 cells (from Korean Cell Line Bank, Seoul, Korea) were maintained in a Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS, Gibco, NY, U.S.A.), containing penicillin/streptomycin (100 IU/50 g/mL), at 37°C in a humidified atmosphere of 5% CO2 and 95% air. These B16 cells were cultured in 24-well plates for each assay. All the experiments were carried out in triplicate and repeated three times to ensure reproducibility.
Measurement of O2•− LevelsO2•− scavenging activity was assessed according to the method of Ewing and Janero.19) Briefly, samples were pipetted into microplate wells containing 200 µL freshly prepared, 125 µM ethylenediamine tetraacetic acid (EDTA), 62 µM nitro blue tetrazolium (NBT) and 98 µM β-reduced nicotinamide adenine dinucleotide (NADH) in 50 mM phosphate-buffered saline (PBS, pH 7.4). The reaction was initiated by adding 25 µL freshly prepared 33 µM phenazine methosulfatein, 50 mM phosphate buffer (pH 7.4). After 5 min, the absorbance at 550 nm, as an index of NBT reduction, was estimated using a microplate reader, Tecan SPECTRAFluor (Tecan, U.K., Goring-on-Thames, U.K.).
Estimation of ·NO LevelsThe assay was carried out as described previously by Sreejayan Rao.20) In brief, 5 mM sodium nitroprusside (SNP) in PBS (pH 7.4) was mixed with various concentrations of samples and incubated at 25°C for 150 min in a test tube. The amount of ·NO produced by SNP was assayed by evaluating the accumulation of nitrite, using a microplate assay method based on the Griess reaction.21) The absorbance at 550 nm, as an index of produced nitrite, was determined after 5 min after transferring the reacted solution to 96-well plates.
Determination of ONOO− Scavenging ActivityONOO− scavenging activity was assessed using method of Kooy et al.22) In brief, sample solutions and 10 µM 3-morpholinosydnomine were added to 5 mM dihydrorhodamine 123 (DHR 123) solution, left to stand for 20 min at 37°C, and then the fluorescence of rhodamine 123 (the reduced form of DHR) was determined at 485 nm excitation and 535 nm emission with a microplate fluorescence reader, Tecan SPECTRAFluor (Tecan, U.K., Goring-on-Thames, U.K.).
Cell Proliferation AssayCell viability was determined according to the method by Tada et al.23) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO, U.S.A.). 5×104 cells/well were plated in a 24-well plate. After cells were exposed rhamnetin at concentrations of 5–100 µM for 24 h, MTT solutions were added and the insoluble derivative formed by cellular dehydrogenase was solubilized with EtOH–dimethyl sulfoxide (DMSO) (1 : 1 mixture solution); the absorbance of each well was determined at 560 nm using a microplate reader.
Estimation of Murine Tyrosinase ActivityTyrosinase activity in B16 cells was estimated by examining the rate of oxidation of l-DOPA.1) Cells were plated in 24-well dishes at a density of 5×104 cells/mL. B16 cells were incubated in the presence or absence of 100 nM α-MSH and then treated for 24 h at various concentrations (0–40 µM) of rhamnetin. The cells were lysed in 100 µL of 50 mM sodium phosphate buffer (pH 6.8) containing 1% Triton X-100 and 0.1 mM phenylmethylsulfonyl fluoride and then frozen at −80°C for 30 min. After thawing and mixing, cellular extracts were clarified by centrifugation at 12000×g for 30 min at 4°C. The supernatant (80 µL) and 20 µL of L-DOPA (2 mg/mL) were placed in a 96-well plate, and the absorbance at 492 nm was estimated every 10 min for 1 h at 37°C using an enzyme-linked immunosorbent assay (ELISA) plate reader.
Determination of MelanogenesisIn this present study, the amount of melanin content was used as an index of melanogenesis. The amount of melanin content was assayed using a modified method of Bilodeau et al.24) Briefly, B16 cells (5×104) were plated on 24-well, multi-dishes and incubated in the presence or absence of 100 nM α-MSH. Cells were then incubated for 24 h with or without rhamnetin at concentrations ranging from 5 to 40 µM. After washing twice with PBS, samples were dissolved in 100 µL of 1 N NaOH. The samples were incubated at 60°C for 1 h and mixed to solubilize the melanin. The absorbance was measured at 405 nm, and compared with a standard curve of synthetic melanin.
Measurement of Total RS GenerationTotal RS production was estimated in culture supernatant.25) Twenty-five millimolar 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was added to incubation media, and changes in fluorescence were estimated at an excitation wavelength of 486 nm and emission wavelength of 530 nm for 30 min.
Determination of O2•− LevelsO2•− levels were examined following the method as previously described.18) The O2•− scavenging activity was estimated by assessing the decrease in the ratio of the decrease of nitro blue tetrazolium (NBT). The culture supernatant was added to the reaction buffer (50 mM PBS with 125 µM EDTA, 62 µM NBT and 98 µM NADH) containing 33 µM 5-methylphenazium methyl sulfate. The absorbance at 540 nm, as an index of NBT reduction, was determined after 5 min.
Estimation of ·NO Generation·NO level was estimated by measuring the accumulation of nitrite in the conditioned medium by the Griess assay. In short, 100 µL of culture supernatant was allowed to react with 100 µL of Griess reagent20) and then was incubated at room temperature for 5 min. The optical density of the samples at 540 nm was read using a microplate reader.
Assessment of ONOO−ONOO−-dependent oxidation of dihydrorhodamine 123 (DHR 123) to rhodamine 123 was examined based on the method as described previously.21) Samples were added to the rhodamine buffer (pH 7.4) containing 6.25 µM DHR 123 and 125 µM diethylenetriaminepentaacetic acid (DTPA) and incubated 5 min at 37°C. The absorbance was measured at 500 nm, which is the absorbance of rhodamine 123.
Measurement of Pro-inflammatory MediatorsPGE2 and TXB2 content in supernatant from each culture well was determined in triplicate using a competitive enzyme immunoassay kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, U.S.A.) according to the manufacturer’s instructions.26)
Determination of GSH and GSSG LevelsGSH levels were examined according to the method of Pandey and Katiyar.27) Twenty-five percent of the meta-phosphoric acid-added cell pellets were centrifuged at 12000×g for 10 min, and then the supernatant was taken for assay. One millimolar EDTA–50 mM phosphate buffer was added to the supernatant followed by o-phthalaldehyde. After 20 min at room temperature, the fluorescence was estimated at excitation wavelength of 360 nm and emission wavelength of 460 nm. GSSG levels were assayed after preincubation with N-ethylmaleimide for 20 min, and 0.1 M NaOH was replaced for 1 mM EDTA–50 mM phosphate buffer.
Protein AssayThe concentration of protein was examined by a bicinchoninic acid protein assay.28) All samples were assayed in triplicate.
Statistical AnalysisData were analyzed as mean±standard error (n=5), and the biological significance p<0.05 was determined by the Student’s t-test.
To determine the radical scavenging activities of rhamnetin in vitro, suppressed levels of O2•−, ·NO and ONOO− were quantified. As shown in Table 1, rhmanetin reduced O2•− by 25.1, 51.4 and 69.2% at concentrations of 2.5, 5 and 10 µg/mL, respectively. Rhamnetin also suppressed ·NO levels by 38.6, 61.4, 74.8% at concentrations of 2.5, 5 and 10 µg/mL, respectively. In addition, rhamnetin reduced ONOO− levels by 34.6, 56.9 and 86.8% at concentrations of 2.5, 5 and 10 µg/mL, respectively. These data suggest that rhamnetin decreased O2•−, ·NO and ONOO− levels in vitro, indicating that rhamnetin has O2•−, ·NO and ONOO− scavenging actions.
| Group | O2•− level (%) | NO level (%) | ONOO− level (%) |
|---|---|---|---|
| Control | 100.8±0.69 | 101.3±0.002 | 100.1±0.17 |
| Rhamnetin (2.5 µg/mL) | 75.7±9.33** | 62.7±3.10 | 65.5±5.60***,## |
| Rhamnetin (5 µg/mL) | 49.4±9.50**,# | 39.9±4.34***,# | 43.2±5.72***,### |
| Rhamnetin (10 µg/mL) | 30.8±2.12***,# | 26.5±2.04***,# | 13.3±4.45 ** |
| Troloxa) (10 µg/mL) | 28.3±1.55***,### | ||
| Curcumina) (10 µg/mL) | 21.4±2.00***,### | ||
| Penicillaminea) (10 µmg/mL) | 10.8±1.09***,### |
a) Trolox, curcumin and penicillamine are included for comparison purpose. Each value represents the mean±S.E.M. of three determinations. Statistically significant differences were compared with control group (** p<0.01, *** p<0.001), and rhamnetin (2.5 µg/mL) treated group (# p<0.05, ## p<0.01, ### p<0.001).
The results from the cell viability assay using MTT for B16 cells are revealed in Fig. 1B. At growth doses of 5, 10, 50 and 100 µM of rhamnetin, cell viability registered at 99.9, 98.4, 92.4 and 89.2%, respectively, with 24 h of treatment. These data indicate that rhamnetinin is relatively non-cytotoxic to cells under the experimental conditions used.
(A) (B) Cells treated with various doses of the rhamnetin were evaluated by the MTT assay. Data are expressed as % of cell viability. (C) Results are expressed as the mean±S.E.M. of at least three determinations. Each value represents the mean±S.E.M. of at least three determinations that are significantly different from the control group (* p<0.05, ** p<0.01, *** p<0.001), and rhamnetin (5 µM) treated group (# p<0.05, ## p<0.01). Mefenamic acid (40 µM) was used as a positive control.
As the inhibition of tyrosinase is associated with anti-melanogenic action, the effect of rhamnetin on tyrosinase activity in B16 cells was monitored. Table 2 shows that after 24 h incubation with rhamnetin, murine tyrosinase activities were suppressed by 30.6% at 5 µM, 63.3% at 20 µM and 75.5% at 40 µM, thereby indicating rhamnetin’s anti-melanogic property in B16 cells.
| Group | Tyrosinase activity (%) |
|---|---|
| Control | 100.01±0.40 |
| Rhamnetin (5 µM) | 69.4±13.4* |
| Rhamnetin (20 µM) | 36.7±8.68***,# |
| Rhamnetin (40 µM) | 24.5±3.87***,## |
| Kojic acida) (40 µM) | 41.93±10.4***,# |
a) Kojic acid used as a positive control. Each value represents the mean±S.E.M. of three determinations. Statistically significant differences were compared with the control group (* p<0.05, *** p<0.001), and rhamnetin (5 µM) treated group (# p<0.05, ## p<0.01).
In the current study, melanin content was used for determining melanogenesis status. Rhamnetin was added to the culture medium at various concentrations (5, 20, 40 µM) to investigate its melanogenesis inhibitory effect. As Fig. 1C displays, rhamnetin down-regulated melanin content in a concentration-dependent manner. The melanin content was decreased by 20.9% at 5 µM, 52.9% at 20 µM, and 78.5% at 40 µM, as compared against the non-treated control group. These results suggest that rhamnetin possesses an anti-melanogenesis effect.
Effect of Rhamnetin on Total RS Generation, O2•−, ·NO and ONOO− Levels in B16 CellsTo elucidate the underlying action of rhamnetin’s anti-melanogenesis property, total RS generation was examined in B16 cells. As revealed in Fig. 2A, total RS production decrease by 14.7% at 5 µM, 31.5% at 20 µM and 55.1% at 40 µM when compared to the control group. Based on these results, rhamnetin effectively reduces intracellular total RS generation in B16 cells, indicating that rhamnetin shows its anti-oxidative effect by suppressing total RS production. In our previous work, we found that the modulation of O2•−, ·NO and ONOO− critically contribute to anti-melanogenic action; therefore, O2•−, ·NO and ONOO− levels were measured in B16 cells. As displayed in Fig. 2B, rhamnetin inhibited O2•− by 13.7, 35.2, 56.3%, at concentrations of 5, 20 and 40 µM, respectively. Figure 2C displays that rhamnetin suppressed ·NO levels by 23.4, 50.3, 72.7%, at concentrations of 5, 20 and 40 µM, respectively. As shown in Fig. 2D, rhamnetin down-regulated ONOO− levels by 32.8, 58.9, 78.8%, at concentrations of 5, 20 and 40 µM, respectively. These results confirm that rhamnetin effectively reduced O2•−, ·NO and ONOO− in B16 cells, showing that the suppression of O2•−, ·NO and ONOO− is an important factor in the anti-oxidative property of rhamnetin.
Each value represents the mean±S.E.M. of at least three determinations that are significantly different from the control group (* p<0.05, ** p<0.01, *** p<0.001), and rhamnetin (5 µM) treated group (# p<0.05, ## p<0.01, ### p<0.001). Trolox (40 µM), curcumin (40 µM) and penicillamin (40 µM) were used as positive controls.
To clarify the association between anti-inflammatoy and the anti-melanogenic effects of rhamnetin, key pro-inflammatory mediators, such as PGE2 and TXB2 were determined in B16 cells. Figure 3A shows that rhamnetin suppressed PGE2 by 66.7, 108.3 and 139%, at concentrations of 5, 20 and 40 µM, respectively. Figure 3B reveals that rhamnetin reduced TXB2 by 19.7, 28.3 and 30.7% at concentrations of 5, 20 and 40 µM, respectively. These results indicate that rhamnetin down-regulated PGE2 and TXB2, showing that not only does rhamnetin have an anti-inflammatory property, but that its anti-inflammatory action is a significant factor in melanogenesis regulation.
Cells were incubated with 10 µM/L H2O2 for 30 min after pretreatment with rhamnetin. The levels of PGE2 and TXB2 in cell supernatants were detected by ELISA. Each value represents the mean±S.E.M. of at least three determinations that are significantly different from the control group (* p<0.05, ** p<0.01, *** p<0.001), and rhamnetin (5 µM) treated group (# p<0.05, ## p<0.01). Mefenamic acid (40 µM) was used as a positive control.
As the GSH/GSSG ratio is a significant oxidative stress marker, the effect of rhamnetin on GSH, GSSG levels and their ratio were evaluated in B16 cells. Table 3 reveals that GSH levels increased by 229% at 5 µM, 366% at 20 µM and 898% at 40 µM, respectively. However, GSSG levels decreased by 25.9% at 5 µM, 70.3% at 20 µM and 92.9% at 40 µM, respectively. As a result, GSH/GSSG ratio was enhanced by rhamnetin treatment. These results confirm that rhamnetin boosted intracellular GSH levels in B16 cells, while its treatment reduced GSSG levels. Finally, the GSH/GSSG ratio was augmented by rhamnetin, showing that rhamnetin maintained a well-regulated redox balance. These data indicate rhamnetin exerts its anti-oxidative action not only by suppressing RS, but also by enhancing the GSH/GSSG ratio.
| Group | GSH level (%) | GSSG level (%) | GSH/GSSG ratio |
|---|---|---|---|
| Control | 100.8±0.40 | 100.0±0.002 | 1.01±0.01 |
| Rhamnetin (5 µM) | 330.0±32.1** | 74.11±3.10** | 4.50±1.01** |
| Rhamnetin (20 µM) | 466.7±20.3***,# | 29.7±4.34***,### | 19.9±6.38*,# |
| Rhamnetin (40 µM) | 998.3±3.29***,### | 7.14±2.04***,# | 175.8±111.8 |
| α-Tocopherola) (40 µM) | 836.7±46.6***,### | 76.7±5.13**,## | 10.9±1.75***,## |
a) α-Tocopherol used as a positive control. The cells were cultured to sub-confluence then incubated for 24 h in the presence of rhamnetin, then were assayed for GSH and GSSG levels as detailed in Materials and Methods. Each value represents the mean±S.E.M. of three determinations. Statistically significant differences were compared with the control group (* p<0.05, ** p<0.01, *** p<0.001) and rhamnetin (5 µM) treated group (# p<0.05, ## p<0.01, ### p<0.001).
Many phenolic compounds have recently attracted much attention because of the possible beneficial roles they play in melanogenesis.10–13,29) To my knowledge, this is the first report showing rhamnetin’s potency on melanogenesis and its effects against diverse oxidative stresses and inflammatory mediators.
Because a compound’s potential toxicity and safety are first and foremost considerations when developing skin-lightening ingredients, the effect of rhamnetin on cell cytotoxicity was examined. As Fig. 1B reveals, rhamnetin showed no cytotoxicity at the tested concentrations.
Melanogenesis is known to be implicated in the generation of several RS, including ONOO−, which play a critical role in the induction of melanogenesis.30) Thus, my first assumption was that the anti-melanogenic action of rhamnetin might be due to its potent anti-oxidative property. Therefore, the effects of rhamnetin were examined on O2•−, ·NO and ONOO− in vitro and total RS production, O2•−, ·NO and ONOO− in B16 cells, with the finding that rhamnetin suppressed all these RS. These results are in agreement with our previous work indicating the scavenging activities of RS is critical for an anti-melanogenic action,31) and the associated report showing that the existence of ·NO is sufficient to induce melanogenesis in UV-irradiated cultures of human melanocytes.32) Moreover, in B16 cells, rhamnetin was shown to strongly enhance the GSH and GSSG ratio, a known marker of oxidative stress, through a well-maintained oxidative status, indicating that sustaining redox balance is significant for melanogenesis suppression.
Several studies, including ours have shown the interrelationship between oxidative stress and inflammation,8,33–35) and others have reported on the possible connection between oxidative stress and inflammatory pigmentation in skin.9) Among RS, O2•− is produced under inflammatory conditions that acts with ·NO to generate toxic ONOO−, which is known to be a significant substrate for cyclooxygenase activity. Therefore, it is suspected that rhamnetin’s melanogenic inhibitory action might be attributed to its anti-inflammatory action. In order to substantiate this possible relationship, the effect of rhamnetin on PGE2 and TXB2 was assessed with the finding that rhamnetin suppressed the pro-inflammatory mediators, PGE2 and TXB2. The data on PGE2 and TXB2 indicate that the anti-inflammatory action of rhamnetin is one of the critical factors in its anti-melanogenic property (Figs. 3A, B). Based on these data, ·NO may directly act with cyclooxygenase activity; and thus the suppression of ·NO by rhamnetin might influence cyclooxygenase activity, causing the suppression of pro-inflammatory PGE2 and TXB2. It is therefore noteworthy to point out that these results support the notion that rhamnetin has anti-inflammatory effects and may affect melanogenic inhibitory actions.
One of the novel findings of the current work is that rhamnetin shows diverse protective effects in exerting its anti-melanogenic property by enhancing various antioxidant activities in vitro and in B16 cells while simultaneously having anti-inflammatory effects in B16 cells. It also is interesting to note the possible structure–activity relationship of rhamnetin. As illustrated in Fig. 1A, the structure of rhamnetin contains a catechol moiety, and the 3-OH group of rhamnetin may play a role as a diverse free radical scavenger, characteristics that are in agreement with a recent study showing that a catechol moiety and the 3-OH group are important for the elimination of different types of free radicals.36) Also, this structure suggests the possibility that rhamnetin’s hydroxyl group on the 3 position of ring C and the o-dihydroxyl structure in the B ring might contribute to rhamnetin’s potent antioxidant effect, which is consistent with another report indicating that a hydroxyl group on the 3 position of ring C and the o-dihydroxyl structure in the B ring are significant in the inhibition of free radical scavenging activity.37) Moreover, a multiple hydroxyl group and hydroxyl groups at C-3′ and C-4′ might be important factors for anti-melanogenic actions, findings that are associated with our previous work that indicate the significance of the hydroxyl group in deterring melanogensis.10)
In summary, the present studies demonstrate that rhamnetin has anti-melanogenic activity by its ability to modulate diverse oxidative stress mediators, such as O2•−, ·NO and ONOO− in vitro, and total RS, O2•−, ·NO and ONOO− and the GSH/GSSG ratio in B16 cells. Moreover, rhamnetin showed anti-inflammatory action by its ability to suppress pro-inflammatory mediators such as PGE2 and TXB2. In conclusion, rhamnetin inhibits melanogenesis, and the plausible mechanism of rhamnetin’s anti-melanogenic action might be the result of its diverse anti-oxidative and anti-inflammatory properties. Additionally, the structural characteristics of rhamnetin might play a role in its beneficial efficacy. Further detailed molecular investigations of the anti-inflammatory action of rhamnetin and its role in anti-melanogenesis are warranted.
This work was supported by the Busan Women’s College Research Grant of 2013 Coll-45.
