SEARCH

SEARCH BY CITATION

Keywords:

  • melanin;
  • natural extracts;
  • skin;
  • tyrosinase;
  • whitening

Synopsis

  1. Top of page
  2. SynopsisRésumé
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Several treatments for skin whitening are available today, but few of them are completely adequate, especially owing to the carcinogenic potential attributed to classical drugs like hydroquinone, arbutin and kojic acid. To provide an alternative and safer technology for whitening, we developed two botanical compounds originated from Brazilian biodiversity, an extract of Schinus terebinthifolius Raddi and a linoleic acid fraction isolated from Passiflora edulis oil. The whitening effect of these compounds was assessed using biochemical assays and in vitro models including cellular assays and equivalent skin. The results showed that S. terebinthifolius Raddi extract is able to reduce the tyrosinase activity in vitro, and the combination of this extract with linoleic acid is able to decrease the level of melanin produced by B16 cells cultured with melanocyte-stimulating hormone. Furthermore, melanin was also reduced in human reconstituted epidermis (containing melanocytes) treated with the compounds. The combination of the compounds may provide a synergistic positive whitening effect rather than their isolated use. Finally, we demonstrated that the performance of these mixed compounds is comparable to classical molecules used for skin whitening, as kojic acid. This new natural mixture could be considered an alternative therapeutic agent for treating hyperpigmentation and an effective component in whitening cosmetics.

Résumé

On trouve un bon nombre de produits proposés pour éclaircir la peau, mais peu parmi eux s'avèrent acceptables, en particulier à cause du potentiel carcinogène attribué aux ingrédients classiques tels que l'hydroquinone, l'arbutine et l'acide kojique. Pour trouver une alternative plus sure, nous avons développé deux composés d'origine végétale issus de la biodiversité Brésilienne, à savoir un extrait de Schinus terebinthifolius Raddi et une fraction d'acide linoléique isolée de l'huile de Passiflora edulis. L'effet éclaircissant de ces composes a été évalué à l'aide de tests biochimiques et de modèles in vitro, y compris de cellules et de peaux équivalentes. Les résultats montrent que l'extrait de Schinus terebinthifolius Raddi est capable de réduire l'activité de la tyrosinase in vitro, et que la combinaison de cet extrait avec l'acide linoléique permet de diminuer la quantité de mélanine produite par les cellules B16 cultivées en présence de l'hormone de stimulation des mélanocytes (MSH). La quantité de mélanine produite diminue également dans la peau humaine reconstituée contenant des mélanocytes quand elle est traitée avec ces composés. L'utilisation conjointe de ces composés pourrait apporter un effet blanchissant synergique comparé à l'utilisation isolé de chacun. Nous montrons que l'activité des ces composés est comparable à celles des substances utilisées classiquement, tel que l'acide kojique. Ce nouveau mélange pourrait représenter une alternative thérapeutique pour traiter l'hyperpigmentation et s'avérer être un composé efficace dans les produits cosmétiques éclaircissants.


Introduction

  1. Top of page
  2. SynopsisRésumé
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The skin pigmentation is a physiological biochemical process that provides, in addition to the tanned aspect, an efficient skin protection against the damage caused by solar irradiation, such as DNA strand breaks, DNA mutation or exceeding reactive oxygen species production [1-3].

The skin ability to produce pigment is thus essential for maintaining skin health and protection. However, there are situations in which the process of pigmentation can become uncontrolled, such as after continuous exposure to ultraviolet radiation. In fact, chronological ageing and solar exposition (photoageing), including some resultant processes like microinflammation, can affect the regular course of the pigmentation process, often leading to hyperpigmentation and the appearance of irregular dark spots on skin [4-8]. Knowing that conserving a good appearance has important socio-psychological impact in the human relationships, contributing to the well-being, the treatments providing a more uniform appearance of the skin have shown growing importance [9].

Several treatments for punctual skin hyperpigmentation are available today, but few of them are completely satisfactory [10-13]. Classical drugs like hydroquinone, arbutin, mequinol and kojic acid have been considered strongly carcinogenic, with related evidences for leukaemia mononuclear, kidney tubular adenoma and hepatic neoplasia [14, 15]. Recent studies suggest that topical application of hydroquinone can also disrupt fibres in extracellular matrix (especially collagen and elastin), leading to abnormalities in the adrenal glands and thyroid physiology, as well as loss of skin firmness [16, 17]. Furthermore, despite the initial whitening effect, hydroquinone and arbutin can cause ochronosis, a condition in which the skin becomes more hyperpigmented than it was at the beginning of the treatment [18]. Kojic acid, an aromatic acid of fungal origin that acts as a chelating agent, is reported to lead to liver and thyroid cancer in mice [19]. Contact dermatitis, cutaneous rash, burning sensation and increased susceptibility of the skin to the effects of UV irradiation are common effects attributed to the application of these classical pharmacological substances [13]. Hydroquinone and kojic acid were banned in Europe and Japan, respectively, for cosmetic formulations [12, 13]. In this context, natural products and botanical extracts with non-toxic and environmentally friendly properties provide a good opportunity to develop therapeutic agents for treating hyperpigmentation; furthermore, they could be applied in cosmetic formulation as effective whitening compounds [20, 21].

Melanin biosynthesis is a complex biochemical pathway that takes place in the melanocyte, a highly specialized cell type which is found within the basal epidermis. After UV exposure, the melanocytes increase the expression of proopiomelanocortin, the precursor of the melanocyte-stimulating hormone (MSH), as well as its melanocortin 1 receptor, the tyrosinase enzyme and other signalling factors [22]. Tyrosinase catalyses the rate-limiting reaction of the melanogenic process – the conversion of the amino acid l-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). Thus, melanin production is considered to be regulated mainly by the expression and activation of tyrosinase [7, 23, 24]. The subsequent oxidation steps of L-DOPA yield some intermediary compounds and finally melanin. The spreading of melanin throughout keratinocytes and sometimes dermal cells involves melanosomes (membrane-bound organelles) dispersion and results in skin darkening [21, 25].

In this study, we developed two whitening compounds coming from the Brazilian botanical biodiversity. Schinus terebinthifolius Raddi is a Brazilian native plant with some described medicinal properties such as wound healing and antiemetic. We had also used leaves of a widely distributed Brazilian specie with palatable fruits, Passiflora edulis, to isolate a pure fraction of linoleic acid. In this study, the depigmenting effect of both compounds on melanin synthesis was explored in murine B16F1 melanoma (B16) cells and in human reconstituted epidermis containing melanocytes, as well as through in vitro tyrosinase activity assay. Both compounds were proven to have depigmenting effects, and the combination of S. terebinthifolius extract and linoleic acid at specific concentrations leads to a synergistic positive effect in the whitening properties. Once the effects of this new natural mixture were comparable to some pharmacological compounds, and it was shown to be non-irritant on skin, the new natural mixture would be useful as a therapeutic agent in whitening cosmetics and therapies.

Materials and methods

  1. Top of page
  2. SynopsisRésumé
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Natural extracts, reagents and cells

Schinus terebinthifolius Raddi and Passiflora edulis were collected in Brazil under the current National legislation and their extracts were produced at Natura Cosméticos (Brazil) in pilot scale. For that, fresh leaves of S. terebinthifolius were triturated and submitted to aqueous extraction under pressure and high temperature, followed by filtration, evaporation, ethanolic precipitation and spray dryer steps. The final extract contains 10.0–17.0% of gallic acid, 35.0–40.0 of total tannins and 15% of fructosis. Linoleic acid was obtained from P. edulis oil through enzymatic hydrolysis. The conversion rate using the Lipozyme TL 100L : CALB (Novozymes, Araucaria, Brazil) 9 : 1 was 95.9%. After that, two steps of molecular distillation were performed resulting in a raw material containing 70% of linoleic acid.

Gallic acid, kojic acid, mequinol, arbutin, hydroquinone, mushroom tyrosinase solution, L-DOPA, a-MSH, synthetic melanin, neutral red, [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sodium dodecyl sulphate (SDS) were purchased from Sigma-Aldrich (St Louis, MA, U.S.A.). Solvable solution to perform melanin extraction was purchased from PerkinElmer (Waltham, MA, U.S.A.). All histological reagents were purchased from Klinipath, Olen, Belgium.

B16F1 melanoma cell line was obtained from the European Collection of Cells Cultures (code 92101203). Human reconstituted epidermis containing melanocytes was obtained from StratiCELL® (Gembloux, Belgium). Reconstituted epidermal tissues (Episkin) were acquired from SkinEthic laboratory (Lyon, France).

In vitro cytotoxicity and phototoxicity assays

Schinus terebinthifolius Raddi extract and linoleic acid were assayed for cytotoxicity and phototoxicity potential according to international validated methods (BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test – European Centre for the Validation of Alternative Methods – and 3T3 NRU Phototoxicity Test – OECD 432). Cytotoxicity index corresponds to the sample concentration that causes 50% of cell death considering the total cell population used in the test (IC50 value). The phototoxic potential is estimated by the photo-irritancy factor (PIF), which is calculated by the ratio of the IC50 value reached in non-exposed cultures and the IC50 value reached in UVA exposed cultures.

Evaluation of genotoxicity

Bacterial reversal mutation test (AMES) was performed according to the OECD Guideline, protocol 471 (1977). For this proposal, S. terebinthifolius extract was incorporated in plates of five different lineage of mutant bacteria Salmonella typhimurium, whose genetic characteristics prone them to be more sensitive for detection of mutations.

In vitro cutaneous irritation assay

Cutaneous irritation was assessed on reconstituted epidermis (Episkin). Four tissues (0.38 cm2) were used for each treatment (controls and S. terebinthifolius extract). Tissues were exposed to not solubilized compounds (10 mg) for 15 min and washed with PBS. After additional 42 h under standard conditions (37°C, 5% CO2), one tissue per condition was sent to histological analysis and a viability test (MTT) was performed on the remaining tissues. The reduction of cell viability in treated tissues was compared to negative control and expressed as percentage (%). A sample is considered irritant if the cells viability is <50% compared to negative control. SDS (10 μL, 5%) and PBS were used as positive and negative control, respectively.

Compatibility test (PC5)

A clinical evaluation for detecting skin tolerance to repeated application of samples was carried out to verify the skin compatibility and the presence/absence of cutaneous discomfort sensations. For this proposal, 12 volunteers of both sexes were selected under approbation of the appropriate Medical Ethics Research Committee and in conformity with the principles of Declaration of Helsinki. All volunteers gave their written informed consent to this research. The extract was applied for 45 min on the cutaneous antecubital region (40 cm2), twice a day during four consecutive days and one time at the fifth day. After 24, 48, 72 and 96 h, the volunteers were submitted to a clinical evaluation by dermatologists, and discomfort sensations related by the volunteers were collected. Results were determined by the ratio of the total reactive volunteers and the volunteers who completed the study. Skin compatibility was classified as excellent, good, moderate or poor. The less the extract causes discomfort sensations, the better is the compatibility of the extract with the skin as well as its classification.

Tyrosinase activity in vitro assay

Tyrosinase activity was determined as described previously with minor modifications [25]. Briefly, gallic acid, kojic acid, hydroquinone and S. terebinthifolius extract were dissolved in phosphate buffer solution (PBS) pH 6.8. Aqueous solution of tyrosinase (20 µL, 20 units) was mixed to test samples solutions (40 µL) and supplemented with 100 µL of 2 mg mL−1 L-DOPA in PBS. The final concentrations of S. terebinthifolius extract were 0.001, 0.005, 0.01, 0.05, 0.1, 0.5 and 1.0 mg mL−1. The assay mixture was incubated at 37°C for 40 min in the dark. Following incubation, the amount of dopachrome was determined by spectrophotometry at 475 nm (Spectra Max; Molecular Devices, Wokingham, U. K.). The inhibitory activity was expressed as the concentration that inhibited 50% of the tyrosinase activity (IC50), as determined by the optical density. Kojic acid was used as positive control [26]. The linoleic acid fraction was not assayed owing to its lipophilic feature, which is incompatible with the aqueous solution of tyrosinase used in this test to measure the effect of inhibitory compounds.

Determination of melanogenesis in B16 cells

The B16 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Paisley, U.K.) at 37°C in a CO2 incubator. The cells were grown in 12-well dishes and then treated with 500 µM α-MSH alone or with the same amount of α-MSH supplemented with test compounds solutions at different concentrations: 0.025, 0.05 and 0.075 mg mL−1 of S. terebinthifolius extract; 0.025, 0.05 and 0.075 mg mL−1 of linoleic acid; and 3 different mixtures (0.05 + 0.05 mg mL−1, 0.025 + 0.075 mg mL−1 and 0.075 + 0.025 mg mL−1 of S. terebinthifolius extract and linoleic acid, respectively). After 48 h, the cells were washed twice with PBS and dissolved in 500 μL 3 N NaOH. Suspensions were homogenized by sequential pipetting to solubilize melanin. The amount of produced melanin was used as the index of melanogenesis. The optical density of each solution was detected at 400 nm. Data are expressed as inhibition index (%) compared with untreated control cells. Kojic acid at 0.05 mg mL−1 was used as positive control [27].

Skin whitening activity on human reconstituted epidermis with melanocytes

Skin whitening assay was carried out using StratiCELL® in vitro reconstituted 3D human epidermis containing melanocytes from phototype VI donor. Keratinocytes and melanocytes were obtained from one single donor. Tissues were cultivated at the air–liquid interface in DMEM. 250 µM (35 µg mL−1) Kojic acid was used as whitening control. Three tissues were treated during 6 days with each test sample or controls. S. terebinthifolius extract, linoleic acid and the mixture of S. terebinthifolius extract, and linoleic acid were assayed at 0.025, 0.075 and 0.025 + 0.075 mg mL−1, respectively. The medium change and the treatments were performed every 2 days. The epidermal tissues were used for measurement of melanin content using the solvable extraction method. Briefly, tissues were removed from the insert by cutting out the polycarbonate filter, immersed into 400 μL solvable solution and heated at 100°C for 45 min. Afterwards, the optical density of the supernatants was measured at 490 nm and the melanin content was quantified by comparison with the absorbance calibration curve made from synthetic melanin.

Statistical analysis

All results are expressed as the mean ± SE. Statistical analysis was performed for each test and is indicated on each figure legend. Values were determined to be significant when P < 0.05.

Results

  1. Top of page
  2. SynopsisRésumé
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Safety assays

Schinus terebinthifolius Raddi extract and linoleic acid were assayed for cytotoxicity and phototoxicity potential. Linoleic acid was considered non-cytotoxic (IC50 was not reached). The higher value of IC50 estimated to the S. terebinthifolius Raddi extract was 0.3 mg mL−1, which corresponds to a LD50 of 882.04 mg kg−1. Both samples were considered to be non-phototoxic (PIF value was not reached).

Schinus terebinthifolius was also evaluated for genotoxic potential (AMES test) and cutaneous irritation. The extract showed negative results to mutation induction in the bacterial reversal mutation test. In the cutaneous irritation assay, the cell viability of the tissues exposed to the S. terebinthifolius extract was 96% compared to the negative control, and the sample was considered to be non-irritant.

In the skin compatibility test, none of the 12 volunteers treated with S. terebinthifolius extract showed any clinical irritancy signal on skin after sequential application of the extract. Only one volunteer (8.3%) related a discomfort sensation during the sample application, but no clinical signals were observed. The skin compatibility was classified as good.

Linoleic acid was not assayed for genotoxicity, cutaneous irritation and skin tolerance once medium- and long-chain triacylglycerols are well known to be inert to the skin [28, 29].

In vitro tyrosinase activity assay

The S. terebinthifolius extract was tested at seven concentrations (0.001; 0.005; 0.01; 0.05; 0.1; 0.5; and 1.0 mg mL−1) and showed a significant and dose-dependent potential to inhibit tyrosinase activity, with a IC50 value of 0.44 ± 0.13 mg mL−1. This value was smaller than IC50 value showed by hydroquinone (2.07 ± 0.23 mg mL−1) and near to the IC50 value observed to gallic acid (0.22 ± 0.02 mg mL−1). A small IC50 value was also found for kojic acid (0.19 ± 0.02 mg mL−1). When all samples were assayed at 1 mg mL−1, the S. terebinthifolius extract showed 89.8 ± 1.0% of tyrosinase inhibition, which was comparable to the inhibition of 90.7 ± 2.11% and 85.8 ± 1.31% provided by kojic and gallic acids, respectively (Kruskal–Wallis test, P < 0.0001, following by Dunn test with Bonferroni′s correction). In the same assay, hydroquinone showed only 18.3 ± 4.14% of inhibition, which was a significantly smaller inhibition than the one was provided by the S. terebinthifolius extract (Kruskal–Wallis test, P < 0.0001, following by Dunn test with Bonferroni′s correction). All results are summarized in the Table 1.

Table 1. Summary of results for in vitro tyrosinase assays. Linoleic acid was not assayed owing to its lipophilic feature, which is incompatible with the aqueous tyrosinase solution
 Schinus terebinthifolius RaddiKojic acidGalic acidHydroquinone
  1. Line 1, percentage of tyrosinase inhibition when samples were tested at 1.0 mg mL−1. Line 2, IC50 value for each sample. The data are expressed as ‘value ± standard deviation’. Kruskal–Wallis followed by Dunn test with Bonferroni′s correction was employed using the absorbance corresponding to the effect observed at 1 mg mL−1.

  2. a

    Sample considered different from S. terebinthifolius extract, P < 0.001.

Inhibition (%) at 1.0 mg mL−189.9 ± 1.090.7 ± 2.1185.8 ± 1.3118.3 ± 4.14a
IC50 for tyrosinase inhibition (mg mL−1)0.44 ± 0.6 0.19 ± 0.020.22 ± 0.022.07 ± 0.23)

Melanin production in B16 cells

To determine if the samples could inhibit melanogenesis, B16 cells were treated with S. terebinthifolius extract and/or linoleic acid after a-MSH stimulation. The isolated compounds did not show a significant reduction in melanin content, but mixed compounds, especially mixtures 1 and 2, significantly reduced the melanin content compared to the control (Fig. 1, Kruskal–Wallis test, P < 0.0001, following by Dunn test with Bonferroni′s correction). The combination of 0.025 mg mL−1 S. terebinthifolius extract and 0.075 mg mL−1 linoleic acid (mixture 2) provided the better reduction in melanin content (38.2 ± 1.2%), showing 13.13% of extra inhibition when compared to the expected effect by the sum of the isolated effects.

image

Figure 1. Percentage of reduction on melanin content in B16 cells after treatment with isolated or combined compounds. Mixtures 1 and 2 (StR 0.05 mg/mL + LA 0.05 mg/mL and StR 0.025 mg/mL + LA 0.075 mg/mL) are significantly different from control and the mixture 2 showed the best inhibition result (Kruskal–Wallis test, P < 0.001, following by Dunn test with Bonferroni′s correction, performed with raw absorbance data, P < 0.05). All mixtures showed an additional effect (top bars) to those expected by the sum of the individual performances. StR, S. terebinthifolius Raddi extract; LA, linoleic acid.

Download figure to PowerPoint

Skin whitening activity on human reconstituted epidermis

Human reconstituted epidermis containing melanocytes from phototype VI donor was used to assess the effect of samples on the melanin production. Kojic acid was used as whitening control and significantly decreased (21.02%) the melanin content compared to untreated controls. When 25 µg mL−1 S. terebinthifolius extract and 75 µg mL−1 linoleic acid were tested separately, they provided 15.9% and 19.3% of reduction in the melanin content, respectively (not significant compared to untreated controls). The mixture of both samples provided 23.2% of reduction in the melanin content, which was significantly higher than the untreated control and than the compounds on their own (Fisher test, P < 0.01) and comparable to the effect provided by kojic acid.

Discussion

  1. Top of page
  2. SynopsisRésumé
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There are a number of different factors involved in the skin pigmentation. Apart from minor contributors coming from microvascularization pattern and skin thickness, the production and distribution of melanin is the main responsible for the skin colour, tanned aspect and spots appearance [3]. Despite of the biological programmed melanogenesis, different external aggressors, including skin exposition to solar radiation and inflammation conditions (i.e. caused by depilation), can accelerate this phenomenon [6-8]. One of the major tanning consequences is the cutaneous hyperpigmentation, a dermatological condition characterized by an overproduction of melanin and by the increased transfer of melanosomes to the keratinocytes, sometimes including even an overflow to the dermal compartment [24]. The clinical result is the appearance of spots of several intensities, often strongly dark and in a disorderly pattern. Several treatments for skin pigmentation are available today, but the classical pharmaceutical drugs like hydroquinone, arbutin, mequinol and kojic acid have been considered severely carcinogenic as well as able to damage the structural architecture of several different tissues [15, 16, 18, 19]. Contact dermatitis, cutaneous rash, burning sensation and increased susceptibility of skin to the effects of UV irradiation are other common consequences attributed to the application of these classical pharmacological substances, which have been gradually banned from cosmetic formulations [13].

In this study, we investigated the whitening potential of two different compounds originated from the botanical Brazilian biodiversity, a S. terebinthifolius Raddi extract and a linoleic acid fraction isolated from P. edulis oil. For this proposal, the depigmenting effect of both compounds on melanin synthesis was explored in murine B16F1 melanoma (B16) cells and in human reconstituted epidermis, as well as using also an in vitro tyrosinase activity assay.

Both linoleic and gallic acids, the last one being the main active compound of the Schinus terebinthifolius Raddi extract, are described owing to their capacity in reducing the tyrosinase activity [30, 31]. In addition, linoleic acid could accelerate the spontaneous degradation of tyrosinase [32]. Based on the fact that these compounds may have complementary mechanism of action on melanogenesis (reduce enzymatic activity and enzymatic degradation), we had also attempted to assess if there was any performance advantage on mixing these compounds. Our results showed that those mixed compounds were more effective than each one alone, once it was possible to reach a higher and significant melanin reduction using the mixture than each counterpart individually. Therefore, the whitening effect provided by the mixture in specific concentrations may represent a positive synergistic pattern, leading to an over, unexpected and significant effect on B16 cells. These findings could be particularly interesting concerning the application of the mixture as a whitening compound in cosmetic or therapeutic formulations, once low concentrations of each compound may be used without compromising their effectiveness.

Once knowing that the mixture of compounds had a higher potential to reduce the melanin content in MSH-activated B16 cells, we considered to be important to evaluate the whitening effect in cells obtained from human tissues. For this proposal, human reconstituted epidermis containing melanocytes was used to assess the effect of the botanical samples on the melanin production. Once more, the results showed that despite the significant melanin reduction provided by each compound alone, the mixture of the two samples provided even a higher reduction in the melanin content. Using this complex 3D model, we had also demonstrated that the whitening effect provided by the mixture was comparable to the effect reached by kojic acid, one of the most widely and toxic depigmenting substances currently applied for whitening skin.

In addition to the whitening effect, in this work the S. terebinthifolius Raddi extract and the linoleic acid fraction showed no evidence of toxic, irritant, mutagenic or genotoxic risk. In that way, both substances could replace kojic acid on topical whitening formulations, ensuring a similar performance but in a safer way. Kojic acid and hydroquinone were longer reported to be mutagenic on bacterial systems (AMES test) [33-35], whereas S. terebinthifolius extract was demonstrated to be inert on this model, as well as reported to linoleic acid [28, 29].

Finally, in this study we demonstrated that S. terebinthifolius extract had a significant potential to inhibit the tyrosinase activity. This mechanism of action is particularly interesting for cosmetical application as it could provide a gradual whitening effect without abruptly disrupting the existing melanin, leading the keratinocytes devoid of their natural UV protection. Additional experiments to those described here demonstrate that the mixture of compounds can be easily applied in different cosmetic formulations such as gel, emulsions o/w and deodorant with good stability (data not shown).

Irregular melanogenesis caused by chronological ageing, photoageing or several external aggressors leads to cutaneous hyperpigmentation condition, but the classical available treatments have been considered harmful not only to the skin but also to the whole human organism. We described here a new potent whitening alternative comprising a mixture of two new depigmenting compounds originated from the Brazilian botanical biodiversity, a S. terebinthifolius Raddi extract and a linoleic acid fraction isolated from P. edulis oil. The combination of both compounds may provide a synergistic positive effect on their isolated whitening properties, evidenced in B16 cells culture and also in human reconstructed epidermis model. Finally, we demonstrated that the performance of the mixed compounds in reducing melanin on human epidermis is comparable to the kojic acid, a classical and harmful molecule for whitening the skin. Taken together, the obtained results allow us to conclude that the new natural mixture proposed in this study would be useful as a therapeutic agent to address skin hyperpigmentation problems, as well as an effective component in whitening long-term topical skin care solutions, with the clear advantage of being devoid of known toxic, irritant or genotoxic effects. Moreover, UV filters should be frequently topically applied, including treated areas, preventing new damages and ageing spots caused by exposition to UVB.

Acknowledgements

  1. Top of page
  2. SynopsisRésumé
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was totally funded by a grant from Natura Innovation and Product Technology Ltda. The authors state no conflict of interest.

References

  1. Top of page
  2. SynopsisRésumé
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Hashimoto, M.W., Ishigaki, Y., Ohnishi, T. and Mori, T. Supranuclear melanin caps reduce ultraviolet induced DNA photoproducts in human epidermis. J. Invest. Dermatol. 110, 806810 (1998).
  • 2
    Duval, C., Regnier, M. and Schmidt, R. Distinct melanogenic response of human melanocytes in mono-culture, in co-culture with keratinocytes and in reconstructed epidermis to UV exposure. Pigment Cell Res. 14, 348355 (2001).
  • 3
    Slominski, A., Tobin, D.J., Shibahara, S. and Wortsman, J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol. Rev. 84, 11561228 (2004).
  • 4
    Funasaka, Y., Chakraborty, A.K., Hayashi, Y. et al. Modulation of melanocyte-stimulating hormone receptor expression on normal human melanocytes: evidence for a regulatory role of ultraviolet B, interleukin-1, interleukin-1, endothelin-1 and tumour necrosis factor. Br. J. Dermatol. 139, 216224 (1998).
  • 5
    Lacz, N.L., Vafaie, J., Kihiczak, N.I. and Schwartz, R.A. Postinflammatory hyperpigmentation: a common but troubling condition. Int. J. Dermatol. 43, 362365 (2004).
  • 6
    Okazaki, M., Yoshimura, K., Uchida, G. and Harii, K. Correlation between age and the secretion of melanocyte-stimulating cytokines in cultured keratinocytes and fibroblasts. Br. J. Dermatol. 153, 2329 (2005).
  • 7
    Eves, P.C., MacNeil, S. and Haycock, J.W. A melanocyte stimulating hormone, inflammation and human melanoma. Peptides 27, 444452 (2006).
  • 8
    Ortonne, J.P. and Bissett, D.L. Latest insights into skin hyperpigmentation. J. Investig. Dermatol. Symp. Proc. 13, 1014 (2008).
  • 9
    Draelos, Z.D. Skin care and cosmetic use in elderly patients. J. Geriatr. Dermatol. 5, 6066 (1997).
  • 10
    Katsambas, A.D. and Stratigos, A.J. Depigmenting and blenching agents: coping with hyperpigmentation. Clin. Dermatol. 19, 483488 (2001).
  • 11
    Cayce, K.A., Feldman, S.R. and McMichael, A.J. Hyperpigmentation: a review of common treatment options. J. Drugs Dermatol. 3, 668673 (2004).
  • 12
    Ortonne, J.P. and Passeron, T. Melanin pigmentary disorders: treatment update. Dermatol. Clin. 23, 209226 (2005).
  • 13
    Solano, F., Briganti, S., Picardo, M. and Ghanem, G. Hypopigmenting agents: an updated review on biological, chemical and clinical aspects. Pigment Cell Res. 19, 550571 (2006).
  • 14
    Westerhof, W. and Kooyers, T.J. Hydroquinone and its analogues in dermatology – a potential health risk. J. Cosmet. Dermatol. 4, 5559 (2005).
  • 15
    McGregor, D. Hydroquinone: an evaluation of the human risks from its carcinogenic and mutagenic properties. Crit. Rev. Toxicol. 37, 887914 (2007).
  • 16
    Philips, N., Burchill, D., O'Donoghue, D., Keller, T. and Gonzalez, S. Identification of benzene metabolites in dermal fibroblasts as nonphenolic: regulation of cell viability, apoptosis, lipid peroxidation and expression of matrix metalloproteinase 1 and elastin by benzene metabolites. Skin Pharmacol. Physiol. 17, 147152 (2004).
  • 17
    Alcazar, O., Cousins, S.W. and Marin-Castaño, M.E. MMP-14 and TIMP-2 overexpression protects against hydroquinone-induced oxidant injury in RPE: implications for extracellular matrix turnover. Invest. Ophthalmol. Vis. Sci. 48, 56625670 (2007).
  • 18
    Charlín, R., Barcaui, C.B., Kac, B.K., Soares, D.B., Rabello-Fonseca, R. and Azulay-Abulafia, L. Hydroquinone-induced exogenous ochronosis: a report of four cases and usefulness of dermoscopy. Int. J. Dermatol. 47, 1923 (2008).
  • 19
    Fujimoto, N., Onodera, H., Mitsumori, K., Tamura, T., Maruyama, S. and Ito, A. Changes in thyroid function during development of thyroid hyperplasia induced by kojic acid in F344 rats. Carcinogenesis 20, 15671571 (1999).
  • 20
    Zhu, W. and Gao, J. The use of botanical extracts as topical skin-lightening agents for the improvement of skin pigmentation disorders. J. Investig. Dermatol. Symp. Proc. 13, 2024 (2008).
  • 21
    Chen, L.G., Chang, W.L., Lee, C.J., Lee, L.T., Shih, C.M. and Wang, C.C. Melanogenesis inhibition by gallotannins from Chinese galls in B16 mouse melanoma cells. Biol. Pharm. Bull. 32, 14471452 (2009).
  • 22
    Rousseau, K., Kauser, S., Pritchard, L.E. et al. Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis. FASEB J. 21, 18441856 (2007).
  • 23
    Chakraborty, A.K., Funasaka, Y., Slominski, A., Ermak, G., Hwang, J., Pawelek, J.M. and Ichihashi, M. Production and release of proopiomelanocortin (POMC) derived peptides by human melanocytes and keratinocytes in culture: regulation by ultraviolet B. Biochim. Biophys. Acta 1313, 130138 (1996).
  • 24
    Costin, G.E. and Hearing, V.J. Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J. 21, 976994 (2007).
  • 25
    Cardinali, G., Ceccarelli, S., Kovacs, D., Aspite, N., Lotti, L.V., Torrisi, M.R. and Picardo, M. Keratinocyte growth factor promotes melanosome transfer to keratinocytes. J. Invest. Dermatol. 125, 11901199 (2005).
  • 26
    Curto, E.V., Kwong, C., Hermersdorfer, H. et al. Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors. Biochem. Pharmacol. 57, 663672 (1999).
  • 27
    Jang, J.Y., Lee, J.H., Kang, B.W., Chung, K.T., Choi, Y.H. and Choi, B.T. Dichloromethane fraction of Cimicifuga heracleifolia decreases the level of melanin synthesis by activating the ERK or AKT signaling pathway in B16F10 cells. Exp. Dermatol. 18, 232237 (2008).
  • 28
    Schürer, N.Y. Implementation of fatty acid carriers to skin irritation and the epidermal barrier. Contact Dermatitis. 47, 199205 (2002).
  • 29
    Matulka, R.A., Noguchi, O. and Nosaka, N. Safety evaluation of a medium- and long-chain triacylglycerol oil produced from medium-chain triacylglycerols and edible vegetable oil. Food Chem. Toxicol. 44, 15301538 (2006).
  • 30
    Wehner, F.C., Thiel, P.G., van Rensburg, S.J. and Demasius, I.P. Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicillium mycotoxins. Mutat. Res. 58, 193203 (1978).
  • 31
    Shimogaki, H., Tanaka, Y., Tamai, H. and Masuda, M. In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibition. Int. J. Cosmet. Sci. 22, 291303 (2000).
  • 32
    Ando, H., Watabe, H., Valencia, J.C. et al. Fatty acids regulate pigmentation via proteosomal degradation of tyrosinase: a new aspect of ubiquitin-proteasome function. J. Biol. Chem. 279, 1542715433 (2004).
  • 33
    Shibuya, T., Murota, T., Sakamoto, K., Iwahara, S. and Ikeno, M. Mutagenicity and dominant lethal test of kojic acid Ames test, forward mutation test in cultured Chinese hamster cells and dominant lethal test in mice. J. Toxicol. Sci. 7, 255262 (1982).
  • 34
    Wei, C.I., Huang, T.S., Fernando, S.Y. and Chung, K.T. Mutagenicity studies of kojic acid. Toxicol. Lett. 59, 213220 (1991).
  • 35
    Hakura, A., Tsutsui, Y., Mochida, H., Sugihara, Y., Mikami, T. and Sagami, F. Mutagenicity of dihydroxybenzenes and dihydroxynaphthalenes for Ames Salmonella tester strains. Mutat. Res. 20, 293299 (1996).