Cell viability assay of active-free formulations and formulations containing ascorbyl glucoside to HaCaT and HDF cell lines

Considering the premise that any product for human use should be evaluated in advance for its safety, the cell viability assay of formulations containing different clay minerals active free and also formulations containing different clay minerals and active ascorbyl glucoside to HaCaT and HDF cells was studied. HaCaT was selected as it is the first permanent epithelial cell line from adult human skin that exhibits normal proliferation and differentiation behavior. HDF was selected as it is the major cellular component of dermis and are responsible for producing the extracellular matrix forming the connective with skin tissue (Brugè et al., 2015; Kilfoyle et al., 2001). Alamar Blue® assay has been used to quantitatively analyze the cell viability conversion of fluorescent Resazurin in the Alamar Blue® to Resorufin occurring in response to cell metabolic activity (Nakayama et al., 1997).

Figure 20 shows the cell viability of formulations containing different clay minerals active free and formulations containing different clay minerals plus active ascorbyl glucoside to HaCaTs and HDFs. The HaCaTs and HDFs treated with formulations containing different clay minerals and active free (concentration range 500–31.25 μg/mL) showed no significant decrease in metabolic activity (cell viability between 79 and 87% for HaCaTs, and 99 and 104% for HDFs ). The HaCaTs and HDFs treated with formulations containing different clay minerals and ascorbyl glucoside active (concentration range 500–31.25 μg/mL) showed no significant decrease in metabolic activity (cell viability between 84 and 94% for HaCaTs, and 98 and 103% for HDFs), confirming that clay minerals and ascorbyl glucoside active presence do not induce any short-term cytotoxicity.

The difference in the cytotoxicity of formulations containing different clay minerals active free and formulations containing different clay minerals and ascorbyl glucoside active to HaCaTs was more evident than to HDFs: applied at the same clay mineral and/or active concentration, formulations containing different clay minerals active free resulted in lower viability of HaCaTs as compared to formulations containing different clay minerals and ascorbyl glucoside active. To confirm the significance and validation of the data, two independent experiments were performed

and to reach the IC50 of formulations containing different clay minerals and ascorbyl glucoside active to HaCaTs and HDFs (1.2 mg/mL and 6.74 mg/mL) were 55 % and 93% higher than the highest concentrations of ascorbyl glucoside active tested, as well as to reach the IC50 of formulations containing different clay minerals and active free to HaCaTs and HDFs (1.1 mg/mL and 3.4 mg/mL) were 54% and 85% higher than the highest concentration of each clay mineral tested. These results suggest that all clay minerals and also the active ascorbyl glucoside tested are non-toxic by in vitro assay performed, thus all the formulations are safety for topical application.

NC F- Base F- SGY F- SP F- SW F- W4 PC and 10% AlamarBlue® containing medium were applied as negative control, positive control, and blank, respectively. Cell viability data were plotted and the error bars represent the SD of the replicates. The p-value > 0.05 was considered to be not statistically significant between the cell viability of HaCat cell line_active versus HaCat cell line_active free and HDF cell line_active versus HDF cell line_active free.

5 Conclusion

Kaolin and smectite clay demonstrated to extend ascorbyl glucoside release

which favored the longer-acting effect of active on the skin ie a slower release, which allows better efficacy of the product, once the antioxidant activity from the active ascorbyl glucoside can be exercised . Moreover, the study showed that smectite clay presents an advantage of retaining more amount of ascorbyl glucoside between the skin layers than kaolin clay does, which favors a target effect. Considering that the research on different types of clay minerals have been able to attract great interest of the pharmaceutical and cosmetic industries and to make room for greater use of this type of material in various applications, particularly when the goal is dermal topical application. More research that focus on the development of new clay-based dermocosmetic formulations along with detailed analyses of their physicochemical stability are required, specially due to the chemical interactions between active and clay mineral into the formulation.

6 Acknowledgements

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superio – Brasil (CAPES) – - Finance Code 0001

7 References

Ambrogi, V.; Fardella, G.; Grandolini, G.; Perioli, L. Intercalation compounds of hydrotalcite-like anionic clays with antiinflammatory agents — I. Intercalation and in vitro release of ibuprofen. Int. J. Pharm. 2001, 220, 23–32.

ANVISA Cosmetics Products Stability Guide. Quality in Cosmetic Series, Vol.1, 2004.

Arco, M. D.; Fernández, A.; Martín, C.; Rives, V. Release studies of different NSAIDs encapsulated in Mg, Al, Fe-hydrotalcites. Appl Clay Sci. 2009, 42, 538-544.

Ascenso, A.; Salgado, A.; Euletério, C.; Praça, F. G.; Bentley, M. V. L. B.; Marques, H. C.; Oliveira, H.; Santos, C.; Simões, S. In vitro and in vivo topical delivery studies of tretinoin-loaded ultradeformable vesicles. Eur. J. Pharm. Biopharm. 2014, 88(1), 48-55.

Bastianini, M.; Faffa, C.; Sisani, M.; Petracci, A. Caffeic Acid-layered Double Hydroxide Hybrid: A New Raw Material for Cosmetic Applications. Cosmetics. 2018, 5(3), 51.

Bauselinck, L. A.; Goverbs, G.; Poesen, J.; Degraer, G.; Froyen L. Grain-size analysis by laser diffractometry: comparison with the sieve-pipette method. Catena, 1998, 32, 193-208.

Bretzke, P. E. Caracterização e disponibilidade biológica in vitro e ex vivo de argilominerais utilizados como insumos farmacêuticos e cosméticos. Dissertação de Mestrado, Itajaí-SC, Brazil, 2015; pp 100-110.

Brindley, G.; Brown, G. Crystal structure of clay minerals and their X-Ray identification. Mineralogical Society, London, 1980.

F.; Damiani, E.; Brugè, F.; Offerta, A.; Puglia, C.; Tiano, L. A comparative study on the possible cytotoxic effects of different nanostructured lipid carrier (NLC) compositions in human dermal fibroblasts. Int. J. Pharm. 2015, 495(2), 879-885.

Byrn, S. R.; Pfeiffer, R. R.; Towell, J. G. Solid-State Chemistry of Drugs. 2nd ed., Academic Press, New York, 1999.

Carretero, M. I.; Gomes, C. S. F.; Tateo, F, Clays and Human Health. In: Bergaya, F.;

Benny, K.G.T.; Lagaly, G (Eds.) Handbook of Clay Science., Elsevier. 2006, 1, pp 717-741.

Colipa/CTFA Guidelines on Stability Testing of Cosmetic Products, March 2004.

Davis, H. M. Analysis Of Creams And Lotion. In: Newburguer, S. H., Senzel, A.J (Eds.) Newburguer’s Manual Of Cosmetic Analysis; Association Of Official Analytical Chemists, Washington, 1977; pp 32.

Dinnebier, R. E.; Billinge, S. J. L. Powder Diffraction: Theory and Practice. Cambrige:

The Royal Society of Chemistry, UK, 2008.

Fabbri, B.; Gualtieri, S.; Leonardi, C. Modifications induced by the thermal treatment of kaolin and determination of reactivity of metakaolin. Appl Clay Sci. 2013, 73(1), 2-10.

Godic, A.; Poljsak, B.; Adamic, M.; Dahmane, R. The role of antioxidants in skin cancer prevention and treatment. Oxid. Med. Cell. Longev. 2014, 1-6.

Hakozaki, T.; Takiwaki, H.; Miyamoto, K.; Sato, Y.; Arase, S. Ultrasound enhanced skin-lightening effect of vitamin C and niacinamide. Skin Res Technol. 2006, 12, 105-113.

Hanaor, D. A. H.; Michelazzi, M.; Leonelli, C.; Sorrell, C. C. The effects of carboxylic acids on the aqueous dispersion and electrophoretic deposition of ZrO2. J. Eur.

Ceram. Soc. 2012, 32, 235-244.

Heber, G. K.; Markovic, B.; Hayes, A. An immunohistological study of anhydrous topical ascorbic acid compositions on ex vivo human skin. J. Cosmet. Dermatol.

2006, 5, 150-156.

Hsiao, Y. C.; Huang, H. C.; Hu, S.; Ko, Y. S.; Sung, H. C.; Chen, C. C.; Huang, S. Y.

Fractional Carbon Dioxide Laser Treatment to Enhance Skin Permeation of Ascorbic Acid 2-Glucoside with Minimal Skin Disruption. Dermatol Surg. 2012, 38, 1284–1293.

Huang, S. C.; Lin, C. C.; Huang, M. C.; Wen, K. C. Simultaneous Determination of Magnesium Ascorbyl Phosphate, Ascorbyl Glucoside, Kojic Acid, Arbutin and Hydroquinone in Skin Whitening Cosmetics by HPLC. J. Food Drug Anal. 2004, 12 (1), 13-18.

Idson, B., Stability Testing of Emulsions, Drug & Cosmetic Industry, Part I, Jan. 1993;

Part II, Feb. 1993. The Cosmetic, Toiletry, and Fragrance Association, CTFA Microbiology Guidelines, 1993 (or current edition), www.ctfa.org

Jenke, D. R. Chromatographic Method Validation: A Review of Current Practices and Procedures. III. Ruggedness, Re-Validation and System Suitability. J. Liq.

Chromatogr. Relat. Technol. 1996, 19(12), 1873-1891.

Kilfoyle, B. E.; Sheihet, L.; Zhang, Z.; Laohoo, M.; Kohn, J.; Michniak-Kohn. B.B.

Development of paclitaxel-TyroSpheres for topical skin treatment. J. Control.

Release. 2012, 163, 18-24.

Lin, C. C.; Lin, Y. C.; Gao, M. Y.; Fang, Y. P. In Vitro Evaluation of Permeation Ability

and In Vivo Whitening of Ascorbic Acid 2-Glucoside in Microemulsion. Int. J. Pharma.

Sci. Res. 2016, 3(118), 2-6.

Maderuelo, C.; Zarzuelo, A.; Lanao, J. Critical factors in the release of drugs from sustained release hydrophilic matrices. J. Control. Rel. 2011, 154(1), 2-19.

Maia, A. M.; Baby, A. R.; Yasaka, W. J.; Suenaga, E.; Kaneko, T. M.; Velasco, M. V.

R. Validation of HPLC stability-indicating method for Vitamin C in semisolid pharmaceutical/cosmetic preparations with glutathione and sodium metabisulfite, as antioxidants. Talanta. 2007, 71, 639–643.

Madan, J.R.; Patil, S.; Mathure, D.; Bahirat, P.S.; Awasthi, R.; Dua, K. Improving dissolution profile of poorly water-soluble drug using non-ordered mesoporous silica.

Marmara Pharm J. 22(2): 249-258, 2018.

Messabeb-Ouali, A. E.; Benna-Zayani, M.; Ayadi-Trabelsi, M.; Sauvé, S. Morphology, Structure, Thermal Stability, XR-Diffraction, and Infrared Study of Hexadecyltrim ethylammonium Bromide–Modified Smectite. Int. J. Chem. 2013, 5(2), 12.

Monteiro, L. M.; Souza, A. E.; Gianotto, E. A. S.; Nery, M. M. F.; Duarte, J. C.;

Freitas, O.; Casagrande, R.; Braracat, M. M. Comprimidos matriciais preparados com hidroxipropilmetilcelulose e pectina contendo quercetina para liberação cólon-específica. Lat. Am. J. Pharm. 2007, 26(2), 179-184.

Moore, D. M.; Reynolds, R. C. J. X-Ray Diffraction and the Identification and Analysis of Clay Minerals, 2nd ed., Oxford, New York, 1997.

Moraes, J. D. D.; Bertolino, S. R. A.; Cuffini, S. L.; Ducart, D. F.; Bretzk, P. E.;

Leonardi, G. R. Clay minerals: properties and applications to dermocosmetic products and perspectives of natural raw materials for therapeutic purposes – a review. Int. J. Pharm. 2017, 534, 213-219.

Moribe, K.; Limwikrant, W.; Higashi, K.; Yamamoto, K. Drug Nanoparticle Formulation Using Ascorbic Acid Derivatives. J. Drug Deliv. 2011, 1-9.

Nakayama, G. R.; Caton, M. C.; Nova, M. P.; Parandoosh, Z. Assessment of the Alamar Blue assay for cellular growth and viability in vitro. J. Immunol. Methods.

1997, 204, 205-208.

Paiva, L. B.; Morales, A. R.; Valenzuela, F. R. D. Organoclays: properties, preparation and applications. Appl. Clay Sci. 2008, 42, 8–24.

Pastore, L. S.; Bahniuk, A.; Vasconcellos, E.; Bretzke, P. E.; Reis, J. M.

Characterization of cosmetic materials. In: Congresso Brasileiro de Geologia, Salvador – Bahia, Brazil, 2014 presentation 47.

Perioli, L.; Pagano, C. Inorganic-Organic Hybrids to Improve the Performances of Anti-Inflammatory Topical Formulations. Int. J. Pharm. Rev. Res. 2016, 5, 1–18.

Rodrigues, L. A. S.; Figueiras, A.; Veiga, F.; Freitas, R. M.;Nunes, L. C. C.; Filho, E.

C. S.; Leite, C. M. S. The systems containing clays and clay minerals from modified drug release: A review. Colloids Surf B Biointerfaces. 2013, 103, 642–651.

Shah P.; Rajput, S.J. Amine decorated 2d hexagonal and 3d cubic mesoporous silica nanoparticles: A comprehensive dissolution kinetic study in simulated and biorelevant media. J. Disper. Sci.Technol. 2018, vol. 40, 55-73.

Serrano, G.; Almudéver, P.; Serrano, M. J.; Milara, J.; Torrens, A.; Expósito, I.;

Cortijo, J. Phosphatidylcholine liposomes as carriers to improve topical ascorbic acid treatment of skin disorders. Clin. Cosmet. Investig. Dermatol. 2015, 8, 591-599.

Simo, A.; Kawal, N.; Paliyath, G.; Bakovic, M. Botanical Antioxidants for Skin Health in the World of Cosmeceuticals. Int. J. Adv. Nutr. Health Sci. 2014, 2(1), 67-88.

Yang, J. H.; Lee, J. H.; Ryu, H. J.; Elzatahry, A. A.; Alothman, Z. A.; Choy, J. H.

Drug–clay nanohybrids as sustained delivery systems. Appl. Clay Sci. 2016, 130, 20–32.

Zhang, X.; Cresswell, M, Alternative Inorganic Systems for Controlled Release Applications, in: Inorganic Controlled Release Technology, 1st ed., Butterworth Heinemann, USA, 2016, pp 189-219.

Capítulo 3

ARTIGO 3

Rheology and stability study of cosmetic creams containing skin whitening AA2G (ascorbyl glucoside) associated with different Clay mineral kaolinites and sodium smectite.

Jemima D. Shultz^1* Bozena Michniak-Kohn² Gislaine Ricci Leonardi³

1,3Medicine Department, Graduate Program in Translational Medicine, Federal University of Sao Paulo, Rua Pedro de Toledo, 720 - 2º floor, zip code: 01039032, São Paulo (SP), Brazil;

1,2Center for Dermal Research, The State University of New Jersey - Rutgers, 145 Bevier Rd, Piscataway, NJ 08854, USA;

3Faculty of Pharmaceutical Science, University of Campinas - Unicamp, Rua Cândido Portinari, 200, zip code: 13083-859, Campinas (SP), Brazil, e-mail:

Abstract: Clay minerals can be found in several skin care products. In particular, they are presented in many semisolid preparations with different functions, including stabilization of suspensions and emulsions, thickener and other special rheological tasks. These minerals are widely used in cosmetic formulations such as creams, lotions, make-ups, and also in spas as treatment to minimize skin disorders such as boils, acne and ulcers. The aim of this study was development of cosmetic formulation containing vitamin C derivative (ascorbyl glycoside - AA2G) associated with different natural clays such as kaolinites and sodium smectite, in a study involving the development of cosmetic cream formulations, rheology assays, quantification of the active through HPLC technique, pH evaluation and organoleptic evaluation of cosmetic cream. The results showed that Formulation F_Base (without mineral clay) and formulation F_SW (with kaolinite SW) were the most stable formulations during the entire stability period, indicating that the most stable mineral clay in the cosmetic formulation was SW, while the least stable was W4 clay (sodium smectite), the pseudo-plastic rheological behavior with thixotropy was observed for

all formulations studied.

Keywords: clay minerals, rheology study, ascorbyl glucoside, stability study, topical emulsion formulations.

1 INTRODUCTION

Clay minerals are widely used in cosmetics products such as creams, powders, bar soaps, emulsions, etc., as antiperspirants and to give the skin opacity, remove shine and cover blemishes. Moreover, clays are used in spas such as face masks, due to their high adsorbency level of substances such as greases, toxins, etc.(Moraes et al., 2017).

The types of clay used are varied within the selected group of clay minerals used in cosmetics formulations, therefore the clay minerals kaolinites and smectites are mostly used and recommended for use only in semi-solid formulations (creams, emulsions, etc.). The unique properties of these clay minerals which make them useful in cosmetic products and spas are: their absorption/adsorption capacity, their high cation exchange capacity, plastic properties, rheological properties, grain size, cooling index, high specific area and sorptive capacity, chemical inertness and low or null toxicity for the consumers (Carretero, 2002; Moraes et al., 2017).

Emulsions (liquid-in-liquid formulations) are known as disperse systems and there is a fact that they are thermodynamically unstable and the phases tend to separate with time, requiring rheological behavior and stability study. Clay minerals are frequently used in semisolid health care formulations for such purposes, but they are also included with other specific functions, or play more than one role in a formulation. Finally, some semisolid clay formulations include other substances (clays, polymers, etc.) thought to complement the mineral activity (Viseras et al., 2007).

In the cosmetic industry, one of the most common application of clay minerals is as an excipient to adjust the rheological properties and to stabilize emulsions by avoiding coalescence of oil and water droplets. Clay minerals act as a barrier in between these droplets, ensuring the physical-chemical stability of the system (Viseras et al., 2007).

Smectite, also known as bentonite clay has been used as a suspending and emulsion agent for a long time and it is the most widely used in the cosmetic industry for stabilization of emulsions. Besides acting as a barrier that

prevents coalescence, it can also modify the rheological behavior of the emulsion, resulting in a final product with features that are both acceptable and desirable to consumers (Viseras et al., 2007; Teixeira-Neto and Teixeira-Neto, 2009; Soleymani et al., 2016). Because of its thixotropic properties it is included in corn and callus emulsions, allowing easy application of the preparation under stress, and permanence in contact with the skin. Moreover, when it used in hand cream, increases the source of moisture to skin, leaving the skin soft but not sticky or greasy (Viseras et al., 2007).

The vitamin C derivative, known as ascorbyl glycoside (AA2G) was studied by Hakozaki et al., 2006, Hsiao et al., 2012 and Lin et al., 2016. The AA2G molecule is hydrolyzed by the cellular enzyme α-glycosylase and is converted to L-ascorbic acid (vitamin C) in the skin layers once applied topically, exerting antioxidant activity. In addition, vitamin C has been used topically to treat aged photo skin by increasing collagen synthesis and also decreasing melanin synthesis, yet this active agent has been used topically for skin whitening treatment.

The, the aim of this study is to evaluate topical formulations containing ascorbyl glucoside associated with clay mineral kaolinites and sodium smectite regarding to accelerated stability study and rheological profile.