Galloyl-RGD as a new cosmetic ingredient
© Park et al.; licensee BioMed Central Ltd. 2014
Received: 6 May 2014
Accepted: 31 July 2014
Published: 8 August 2014
The cosmetics market has rapidly increased over the last years. For example, in 2011 it reached 242.8 billion US dollars, which was a 3.9% increase compared to 2010. There have been many recent trials aimed at finding the functional ingredients for new cosmetics. Gallic acid is a phytochemical derived from various herbs, and has anti-fungal, anti-viral, and antioxidant properties. Although phytochemicals are useful as cosmetic ingredients, they have a number of drawbacks, such as thermal stability, residence time in the skin, and permeability through the dermal layer. To overcome these problems, we considered conjugation of gallic acid with a peptide.
We synthesized galloyl-RGD, which represents a conjugate of gallic acid and the peptide RGD, purified it by HPLC and characterized by MALDI-TOF with the aim of using it as a new cosmetic ingredient. Thermal stability of galloyl-RGD was tested at alternating temperatures (consecutive 4°C, 20°C, or 40°C for 8 h each) on days 2, 21, 41, and 61. Galloyl-RGD was relatively safe to HaCaT keratinocytes, as their viability after 48 h incubation with 500 ppm galloyl-RGD was 93.53%. In the group treated with 50 ppm galloyl-RGD, 85.0% of free radicals were removed, whereas 1000 ppm galloyl-RGD suppressed not only L-DOPA formation (43.8%) but also L-DOPA oxidation (54.4%).
Galloyl-RGD is a promising candidate for a cosmetic ingredient.
The worldwide cosmetics market reached 242.8 billion US dollars in 2011, which was a 3.9% increase compared to 2010 . There are many compounds used as cosmetic ingredients, such as phycobiliprotein from natural sources, which is used as a colorant , polysaccharides used as emulsifiers , and a polymer used for mascara .
Much research has been conducted to develop appropriate materials for cosmetic ingredients. In particular, these studies focused not only on beauty care, but also on functional aspects. Many trials are underway that aim to find ingredients for functional cosmetics, which would have whitening , anti-oxidant  or anti-ageing  effects. Any cosmetic ingredient has to satisfy several requirements, such as thermal stability, high dermal absorption rate, and perfume.
Gallic acid (3,4,5-trihydroxybenzoic acid) is a phenolic acid and a phytochemical derived from herbs. It is found in gallnuts, sumac, witch hazel, tea leaves, oak bark, and other plants , and has anti-fungal , anti-viral , and antioxidant  properties, which are useful for a cosmetic ingredient.
With the development of biotechnology, peptides can be now produced on a large scale massively produced, and are used in cosmetic industry as ingredients. Peptides used in topical anti-ageing products are classified into 4 categories: carrier peptides, signaling peptides, enzyme inhibitors, and neurotransmitter inhibitors . Carrier peptides can deliver other components of cosmetic preparations when these are applied topically.
In this study, we explored the possibility of synthesis of a phytochemical (gallic acid) and a peptide for use as a cosmetic ingredient. We assessed 3 aspects of the novel compound (galloyl-RGD): its safety, stability, functionality as a cosmetic ingredient. To evaluate the safety of galloyl-RGD to the skin, we measured the viability of HaCaT keratinocytes. To assess its stability, we measured its thermal stability. We also analysed its free radical–scavenging effect, and its ability to inhibit L-DOPA formation and L-DOPA oxidation.
Galloyl-RGD purification using a C18 preparative column
Confirmation of galloyl-RGD structure by MALDI-TOF
Galloyl-RGD is stable for 60 days at alternating temperatures
Storage conditions of cosmetics may change from low temperature (in a refrigerator) to room temperature (although most of them are kept at room temperature), i.e. they may be subjected to temperature changes of ca. 20°C. For this reason, thermal stability is an important characteristic of cosmetic materials.
Stability of galloyl-RGD during 60-day storage
99 ± 2%,
99 ± 2%,
81 ± 4%,
94 ± 4%,
74 ± 6%,
92 ± 8%,
66 ± 2%
87 ± 2%
Galloyl-RGD is safe to HaCaT keratinocytes
Galloyl-RGD scavenges free radicals produced by DPPH and reactive oxygen species by UV irradiation
Galloyl-RGD efficiently inhibits L-DOPA formation and L-DOPA oxidation
Galloyl-RGD is a synthetic conjugate of gallic acid and a peptide, synthesized for intended use as a new cosmetic ingredient, purified using HPLC and validated by MALDI-TOF mass spectrometry. Thermal stability of galloyl-RGD was tested at alternating temperatures (consecutive 4°C, 20°C and 40°C [8 h each] on days 2, 21, 41, and 61). It was relatively safe to HaCaT keratinocytes, as their viability after 48 h incubation was 93.53% in the presence of 500 ppm galloyl-RGD. In the 50 ppm galloyl-RGD–treated group, 85.0% of free radical was removed, ROS was scavenged more effectively more than by 20 ppm NAC-treated group in HaCaT cells, and treatment with 1000 ppm galloyl-RGD suppressed not only activation-DOPA formation (43.8%) but also L-DOPA oxidation (54.4%).
To pursue the beauty is a basic human instinct, and as people were looking for ways to make themselves more beautiful the cosmetic industry has significantly grown. Recently, the number of trials aimed at finding more effective ingredients for cosmetics has increased, especially search for the functional ingredients effective for whitening , smoothing the creases , scavenging free radicals , or anti-ageing .
For a long time, natural products have been used for multiple purposes such as remedies, cosmetics, culinary materials, or additives; these various functions are due to multiple phytochemicals in the natural products. A number of recent studies have found that the functions of natural products depend on constituent phytochemicals [17–22].
Gallic acid is a phytochemical, which has been isolated from several plants such as Emblica officinalis Gaertn , Phaleria macrocarpa Boerl , Quercus robur , and Castanea sativa L.  and has anti-fungal , anti-viral , antioxidant  properties. However, gallic acid is thermally unstable , which makes its use as cosmetic ingredient difficult.
To overcome this weakness, we considered gallic acid conjugation with a peptide. Recently, many peptides have been introduced, as they not only have important functions in specific fields, but are also able to improve something more effective. For example, peptides have been used in cosmetic industry to increase skin permeability and duration .
We conclude that galloyl-RGD is a promising candidate for a cosmetic ingredient.
Galloyl-RGD synthesis and purification
Galloyl-RGD was synthesized with 9-fluorenylmethoxycarbonyl (as an amino acid protector against aminolysis) by solid-phase peptide synthesis, linked with amino acid residues using N-hydroxybenzotriazol-N,N-di-cyclohexylcarbodiimide, and then purified by reverse-phase HPLC (Waters, MA, USA; column: Gemini C18 110 Å 250 × 21.2 mm) (Figure 1a & b) in a gradient of acetonitrile in 0.1% trifluoroacetic acid. A MALDI-TOF mass spectrometry assay (linear mode, α-cyano-4-hydroxy-cinnamic acid matrix) was performed to ensure the synthetic quality of galloyl-RGD (molecular weight and chemical structure). Briefly, Axima CFR™ (Kratos Analytical Ltd., Japan) was used to conduct MALDI-TOF assay with 8.0 × 10-4 Pascal Gauge Pressure, Linear mode, and 96 square well sample plate. The purified galloyl-RGD was combined with an equal volume of alpha-cyano-4-hydroxycinnamic acid solution (10 mg/ml CHCA in 50:50 water/Acetonitrile solution) and spotted onto a MALDI target. MALDI-MS data was acquired using Axima-CFR™ Plus-mass spectrometer in positive ion reflectron mode.
Stability at alternating temperatures
Galloyl-RGD was exposed consecutively to 4°C, 20°C, and 40°C (8 h each) on days 2, 21, 41, and 61, and quantified by HPLC.
Cell safety assessment
HaCaT keratinocytes were seeded in 24-well plates (5 × 103 cells/well) in triplicate and treated with 10, 50, 100, or 1000 ppm galloyl-RGD for 48 h; control cells were treated with DMEM with 10% FBS. Cell proliferation was then analysed later using the MTT method.
Analysis of the free radical–scavenging effect
Galloyl-RGD (0–1000 ppm) was mixed with 0.1 mM 1,1-diphenyl-2-picryl hydrazyl (DPPH; Sigma-Aldrich, St. Louis, MO, USA.) in ethanol, stirred vigorously with a vortex mixer for 10 s, and then incubated for 30 min in a refrigerator. The results of DPPH scavenging were determined using an ELISA reader (Biochrom Ltd., Cambridge, UK). In order to analyze ROS scavenging effect of galloyl-RGD, HaCaT keratinocytes were used and intracellular ROS levels were investigated using with the oxidation-sensitive fluorescent probe, 2',7'-dichlorofluorescein diacetate (DCF-DA, Sigma-Aldrich). Cells (1 × 104 cells/well) were cultured in 24-well plate for 12 h and then treated with 20 ppm N-Acetyl-L-cysteine (NAC, Sigma-Aldrich) or with various concentrations of galloyl-RGD (0, 25, 50, and 100 ppm) for 24 h. In order to generate intracellular ROS in cells they were exposed to 40 mJ/cm2 ultraviolet radiations for 60 min using with ultraviolet radiation (Crosslinker Model BLX-254, VILBER Lourmat, France). After irradiation they were washed with PBS and incubated with 10 μM 2′,7′-Dichlorofluorescin diacetate (Sigma-Aldrich) for 30 min at 37°C. The cellular fluorescent images were obtained using with Axiovert 40 cfl (Carl Zeiss, Göttingen, Germany).
L-DOPA formation analysis
The inhibitory effect of galloyl-RGD on tyrosinase activity was analysed using mushroom tyrosinase according to Vallisuta et al. . The sample (20 μL), 0.1 M phosphate buffered saline (220 μL) and tyrosinase (20 μL; 1500–2000 U/mL) were mixed, 1.5 mM tyrosine solution (40 μL) was added, and the mixture was incubated for 15 min at 37°C. Tyrosinase activity was measured as absorbance at 492 nm using an ELISA reader. Phosphate buffered saline was used as a substrate and control.
The percentage of tyrosinase inhibition was calculated using the following equation:
Tyrosinase Inhibition Activity (%) = [1 – A/B] × 100, where ‘A’ is the difference between the optical density (OD) value of the sample and that of the substrate, and ‘B’ is the difference between the OD value of the mixture of mushroom tyrosinase and the substrate and that of the substrate alone.
Analysis of L-DOPA oxidation inhibition
Inhibition of L-DOPA oxidation by galloyl-RGD was assessed based on a modification of the tyrosinase inhibition assay. The sample (50 μL), 0.1 M phosphate buffered saline (850 μL) and mushroom tyrosinase (50 μL; 1500–2000 U/mL) were mixed, 1.5 mM tyrosine solution (40 μL) was added, and the mixture was incubated for 6 min at 37°C. L-DOPA (50 μL; 0.06 mM) was then added, and the mixture was incubated for 1 min at 37°C and measured using ELISA (wavelength: 475 nm). Phosphate buffer saline was used as a substrate and control.
The percentage of L-DOPA oxidation inhibition effect was calculated using the following equation:
L-DOPA Oxidation Inhibition Effect (%) = [1 – A/B] × 100, where ‘A’ is the difference between the OD value of the sample and that of the substrate, and ‘B’ is the difference between the OD value of the mixture without L-DOPA and that of the substrate.
Differences between groups were evaluated by one-way analysis of variance followed by Dunnett’s multiple comparison test; p < 0.01 was considered significant.
Specially thank Ji-Hye Seo and Ha-Neul Kim to assist to get the result of galloylgalloyl-RGD’s ROS scavenging effect in HaCaT keratinocytes.
- MarketWatch Personal Care. 2012, London, UK: DatamonitorGoogle Scholar
- Kuddus M, Singh P, Thomas G, Al-Hazimi A: Recent developments in production and biotechnological applications of C-phycocyanin. BioMed Res Inter. 2012, 2013: 1-9.View ArticleGoogle Scholar
- Gilbert L, Loisel V, Savary G, Grisel M, Picard C: Stretching properties of xanthan, carob, modified guar and celluloses in cosmetic emulsions. Carbohydr Polym. 2013, 93 (2): 644-650. 10.1016/j.carbpol.2012.12.028.PubMedView ArticleGoogle Scholar
- Loginova Y, Shah V, Allen G, Macchio R, Farer A: Approaches to polymer selection for mascara formulation. J Cosmet Sci. 2009, 60: 125-133.PubMedGoogle Scholar
- Kim HJ, Lee JH, Shin MK, Hyun Leem K, Kim YJ, Lee MH: Inhibitory effect of Gastrodia elata extract on melanogenesis in HM3KO melanoma cells. J Cosmet Sci. 2013, 64 (2): 89-98.PubMedGoogle Scholar
- Wang HM, Chen CY, Chen CY, Ho ML, Chou YT, Chang HC, Lee CH, Wang CZ, Chu IM: (-)-N-Formylanonaine from Michelia alba as a human tyrosinase inhibitor and antioxidant. Bioorg Med Chem. 2010, 18 (14): 5241-5247. 10.1016/j.bmc.2010.05.045.PubMedView ArticleGoogle Scholar
- Binic I, Lazarevic V, Ljubenovic M, Mojsa J, Sokolovic D: Skin ageing: natural weapons and strategies. Evid Based Complement Alternat Med. 2013, 2013: 10-View ArticleGoogle Scholar
- Reynolds LD, Wilson NG: Scribes and Scholars. 1991, Oxford University Press, 193-194. 3Google Scholar
- Nguyen DM, Seo DJ, Lee HB, Kim IS, Kim KY, Park RD, Jung WJ: Antifungal activity of gallic acid purified from Terminalia nigrovenulosa bark against Fusarium solani. Microb Pathog. 2013, 56: 8-15.PubMedView ArticleGoogle Scholar
- Choi HJ, Song JH, Park KS, Baek SH: In vitro anti-enterovirus 71 activity of gallic acid from Woodfordia fruticosa flowers. Lett Appl Microbiol. 2010, 50: 438-440. 10.1111/j.1472-765X.2010.02805.x.PubMedView ArticleGoogle Scholar
- Liu Z, Ma LP, Zhou B, Yang L, Liu ZL: Antioxidative effects of green tea polyphenols on free radical initiated and photosensitized peroxidation of human low density lipoprotein. Chem Phys Lipids. 2000, 106 (1): 53-63.PubMedView ArticleGoogle Scholar
- Gorouhi F, Maibach HI: Role of topical peptides in preventing or treating aged skin. Int J Cosmet Sci. 2009, 31 (5): 327-345. 10.1111/j.1468-2494.2009.00490.x.PubMedView ArticleGoogle Scholar
- Bangert C, Brunner PM, Stingl G: Immune functions of the skin. Clin Dermatol. 2011, 29: 360-376.PubMedView ArticleGoogle Scholar
- Pacher A, Beckman JS, Liaudet L: Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007, 87 (1): 315-424.PubMedPubMed CentralView ArticleGoogle Scholar
- Hearing VJ, Tsukamoto K: Enzymatic control of pigmentation in mammals. FASEB J. 1991, 5 (14): 2902-2909.PubMedGoogle Scholar
- Schmidt C: FDA approves first cell therapy for wrinkle-free visage. Nature Biotech. 2011, 29: 674-675.View ArticleGoogle Scholar
- Kanlayavattanakul M, Kitsiripaisarn S, Lourith N: Aroma profiles and preferences of Jasminum sambac L. flowers grown in Thailand. J Cosmet Sci. 2013, 64 (6): 483-493.PubMedGoogle Scholar
- Rana J, Diwakar G, Saito L, Scholten JD, Mulder T: Inhibition of melanin content by Punicalagins in the super fruit pomegranate (Punica granatum). J Cosmet Sci. 2013, 64 (6): 445-453.PubMedGoogle Scholar
- Lee J, Park EY, Lee J: Non-toxic nanoparticles from phytochemicals: preparation and biomedical application. Bioprocess Biosyst Eng. 2014, 37 (6): 983-989.PubMedView ArticleGoogle Scholar
- Fontana AR, Antoniolli A, Bottini R: Grape pomace as a sustainable source of bioactive compounds: extraction, characterization, and biotechnological applicantions of phenolics. A Agric Food Chem. 2013, 61 (38): 8987-9003.View ArticleGoogle Scholar
- Epifano F, Fiorito S, Genovese S: Phytochemistry and Pharmacognosy of the genus Acronychia. Phytochemistry. 2013, 95: 12-18.PubMedView ArticleGoogle Scholar
- Simmler C, Pauli GF, Chen S-N: Phytochemistry and biological properties of glabridin. Fitoterapia. 2013, 90: 160-184.PubMedView ArticleGoogle Scholar
- D’souza JJ, D’souza PP, Fazal F, Kumar A, Bhat HP, Baliga MS: Anti-diabetic effects of the Indian indigenous fruit Emblica officinalis Gaertn: active constitutents and modes of action. Food Funct. 2014, 5 (4): 635-644.PubMedView ArticleGoogle Scholar
- Altaf R, Asmawi MZ, Dewa A, Sadikun A, Umar MI: Phytochemistry and medicinal properties of Phaleria macrocarpa (Scheff.) Boerl extracts. Pharmacogn Rev. 2013, 7 (13): 73-80.PubMedPubMed CentralView ArticleGoogle Scholar
- Eyles A, Davies NW, Mitsunaga T, Mihara R, Mohammed C: Role of Eucalyptus globulus wound wood extractives: evidence of superoxide dismutase-like activity. Forest Pathol. 2004, 34: 225-232.View ArticleGoogle Scholar
- Murugananthan M, Bhaskar RG, Prabhakar S: Removal of tannins and polyhydroxy phenols by electro-chemical techniques. J Chem Technol Biotechnol. 2005, 80: 1188-1197.View ArticleGoogle Scholar
- Reblova Z: Effect of temperature on the antioxidant activity of phenolic acids. Czech J Food Sci. 2012, 30 (2): 171-177.Google Scholar
- Vallisuta O, Nukoolkarn V, Mitrevej A, Sarisuta N, Leelapornpisid P, Phrutivorapongkul A, Sinchaipanid N: In vitro studies on the cytotoxicity, and elastase and tyrosinase inhibitory activities of marigold (Tagetes erecta L.) flower extracts. Exp Ther Med. 2014, 7: 246-250.PubMedPubMed CentralGoogle Scholar
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