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   Molecules 2012 ,  17  ,   2219-2230; doi:10.3390/molecules17022219 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules  Article Benefits of Combinations of Vitamin A, C and E Derivatives in the Stability of Cosmetic Formulations Mirela Donato Gianeti, Lorena Rigo Gaspar, Flávio Bueno de Camargo Júnior and Patrícia Maria Berardo Gonçalves Maia Campos * Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Av do Cafe s/n, Monte Alegre, Ribeirao Preto 14040-903, SP, Brazil; E-Mails: mikadg@yahoo.fr (M.D.G.); lorena@fcfrp.usp.br (L.R.G.); flaviobcjr@yahoo.com.br (F.B.C.J.) *  Author to whom correspondence should be addressed; E-Mail:pmcampos@usp.br; Tel./Fax: +55-16-3602-4197.  Received: 19 December 2011; in revised form: 1 February 2012 / Accepted: 16 February 2012 /  Published: 22 February 2012 Abstract:  Chemically stable ester derivatives of vitamins A, C and E have become a focus of interest for their role in the satisfactory results in skin aging treatments. Accordingly, the aim of this study was to evaluate the physical and chemical stability of a cosmetic formulation containing 1% retinyl palmitate, ascorbyl tetraisopalmitate and tocopheryl acetate, alone or in combination. In the studies of physical stability, a Brookfield rheometer was used to determine rheological behavior of formulations containing the vitamins. Chemical stability was determined by HPLC on a Shimadzu system with UV detection. Results showed that formulations had pseudoplastic behavior and that vitamins did not alter their apparent viscosity and thixotropy. In the chemical stability studies, first-order reaction equations were used for determinations of the shelf-life of vitamins derivatives considering a remaining concentration of 85%. Combined vitamins in a single formulation had a slightly lower degradation rate as compared to different preparations containing only one of the vitamins. Considering that many cosmetic formulations contain vitamin combinations it is suggested that the present study may contribute to the development of more stable formulations containing liposoluble vitamins. Keywords:  stability studies; cosmetics; vitamins; rheology; shelf-life OPEN ACCESS   Molecules 2012 , 17    2220   1. Introduction A special interest in vitamins A, C and E has been observed in recent years due to their useful role in the treatment of skin aging and protection of human skin against UV-induced damage [1]. However, esterified vitamin derivatives (retinyl palmitate, ascorbyl tetraisopalmitate and tocopheryl acetate) having improved stability have been preferentially used in topical formulations [2]. The greater stability of ester forms of vitamins A, C and E led to studies that demonstrated their effectiveness in cosmetic formulations. Retinyl palmitate acts on the epithelization of dry and rough skin, as well as on the improvement of abnormal keratinization [3]. Ascorbyl tetraisopalmitate, a precursor of Vitamin C reduces cell damage triggered by UVB and effectively suppresses UVB-induced pigmentation [4]. Maia Campos [5] showed a significant effect of a vitamin C derivative in the improvement of skin hydration, which is very important for normal skin metabolism, prevention of skin alterations and early aging. Tocopheryl acetate is a free radical scavenger that may reduce DNA damage and the rate of death in keratinocytes [6]. In addition, it can enhance stratum corneum hydration [7] and reduce skin roughness. Vitamins may also act synergistically when used in combination [8]. Some studies suggest the importance of combining topical vitamins C and E to protect the skin against UV irradiation and maximize  photoprotection [9,10]. Seité [11] showed that topical application of a combination of vitamins A and C is able to reverse, at least in part, skin changes induced by both chronologic and photoaging. However, vitamin supplemented formulations, even in the ester form, may present low shelf-life, as shown by Guaratini et al  . [12]. The authors evaluated a gel-cream formulation containing vitamin A  palmitate and vitamin E acetate and obtained a shelf-life of only 77 days (at 45, 37 and 25 °C and 75% relative humidity), characterized by a first-order degradation reaction of the vitamin A derivative. Beyond the characteristics of the active substance, physical and chemical properties of the vehicle such as pH and water content and the combination of active substances should be considered to assure the stability of cosmetic formulations containing vitamins. The vehicle raw materials in cosmetic formulations must be compatible with the active substances selected to fulfill the indicated use of the product [13]. Physical and chemical stability of cosmetic formulations containing vitamins A, C and E derivatives must be determined to evaluate quality, since physical characteristics of the formulation and vitamin integrity must be kept constant during the formulation shelf-life. The physical stability of a cosmetic formulation can be evaluated by studying rheological behavior under thermal stress. In this condition, prediction of instability processes may be possible since some  parameters such as viscosity, solubility, creaming facilitation, coalescence, melting of waxes or hydration of polymers are altered [12]. Therefore, it is important to understand the rheological behavior when formulating, handling, mixing, processing, transporting, and storing such formulations [14]. Furthermore, the performance of dermatological products depends to a great extent on rheological behavior. HPLC measurements are currently used to obtain chemical stability data about formulations over time [14]. To predict formulation shelf-life the kinetics of chemical degradation may be mathematically treated by using the Arrhenius equation [15]; however physical stability is not considered in this case. Prediction based only on chemical parameters cannot be totally trusted especially for cosmetic formulations where physical stability is of utmost importance and other  parameters should also be analyzed.   Molecules 2012 , 17    2221  Clinical studies performed by our research group showed the importance of cosmetic formulations containing combinations of liposoluble derivatives of vitamins A, C and E in the improvement of skin conditions [16]. Considering that there are no studies that evaluate the stability of a cosmetic formulation containing these liposoluble derivatives in combination, it is very important to understand the influence of the vehicle on the degradation rate of the components as well as on the shelf-life of the formulation. The aim of this study was to evaluate the physical and chemical stability of a cosmetic formulation containing 1% of retinyl palmitate, ascorbyl tetraisopalmitate and tocopheryl acetate, alone or in combination. The results obtained should contribute to a better understanding of the stability of vitamin derivatives added to cosmetic formulations and of the physical and chemical stability of the proper formulations and to predict their shelf-life as well. 2. Results and Discussion 2.1. Physical Stability Rheological properties are the most important parameters to be measured in the development of a topical formulation. They characterize features of spreadability, i.e. , the consumer's first contact with the formulation. In addition the rheological characteristics may also be an indication of changes caused  by the addition of some active substances [17]. In this study, rheological parameters indicated that addition of the vitamins derivatives under study to the vehicle formulation did not compromise its structure, since no peaks or other alterations in the rheogram curves were observed (Figure 1). Data on the apparent viscosity, flow index, consistency index and thixotropy during the experimental period is shown in Figure 2. Figure 1.  Rheograms of formulations supplemented ( b ) or not ( a ) with the combination of vitamins under study, when stored at 25 °C, during 0, 7, 14, 21 and 28 days after preparation. ( a ) formulation with vitamins in combination stored at 25 °C; ( b ) formulation without vitamins stored at 25 °C. 0510152025303540456008001000120014001600180020002200240026002800300032003400  F8ASS.T0 F8ASS.T07 F8ASS.T14 F8ASS.T21 F8ASS.T28    S   h  e  a  r   S   t  r  e  s  s   (   D   /  c  m    2    ) Shear Rate (1/Sec)   T0T7 daysT14 daysT21 daysT28 days 0510152025303540456008001000120014001600180020002200240026002800300032003400    F8ASS.T0 F8ASS.T07 F8ASS.T14 F8ASS.T21 F8ASS.T28    S   h  e  a  r   S   t  r  e  s  s   (   D   /  c  m    2    ) Shear Rate (1/Sec)   T0T7 daysT14 daysT21 daysT28 days 0510152025303540456008001000120014001600180020002200240026002800300032003400  F8T0 F8T7 F8T14 F8T21 F8T28    S   h  e  a  r   S   t  r  e  s  s   (   D   /  c  m    2    ) Shear Rate (1/Sec)   T0T7 daysT14 daysT21 daysT28 days 0510152025303540456008001000120014001600180020002200240026002800300032003400    F8T0 F8T7 F8T14 F8T21 F8T28    S   h  e  a  r   S   t  r  e  s  s   (   D   /  c  m    2    ) Shear Rate (1/Sec)   T0T7 daysT14 daysT21 daysT28 days ( a ) ( b )   Molecules 2012 , 17    2222   Figure 2.  Alterations in the apparent viscosity ( a ), flow index ( b ), consistency index ( c ) and thixotropy ( d ) of vehicle (F V), or the vehicle supplemented with vitamin A (FA), vitamin C (FC), vitamin E (FE) or with the combination of these vitamins (F ACE) initially (0), 7, 14, 21 and 28 days after preparation, at room temperature. 010203001000200030004000500060007000 F AF CF EF ACEF V time (days)    V   i  s  c  o  s   i   t  y   (  c   P   ) 01020300.050.150.250.350.450.550.65 F ACEF VF AF CF E time (days)    F   l  o  w   i  n   d  e  x   01020304000070500101000131500162000192500223000 F CF EF ACEF VF A time (days)    C  o  n  s   i  s   t  e  n  c  y   i  n   d  e  x 102030-15000-11000-7000-300010005000900013000 F VF AF CF EF ACE time (days)    T   h   i  x  o   t  r  o  p  y   (   d   i  n  e   /  c  m    2  .  s   )  It indicates that the addition of vitamins derivatives did not produce any alterations in the apparent viscosity or on the consistency index of the formulations. Furthermore, no alterations were detected by storage at 37 and 45 °C (Figure 2A,C). The results showed that the formulations had pseudoplastic behavior, with a flow index below 1, which was not altered by the addition of vitamins to the vehicle formulation (Figure 2B). Pseudoplasticity is a desirable rheological property in cosmetic formulations. It improves application and spreading providing a pleasant sensory feeling. Newtonian materials do not behave in this way,  because when spread on the skin they run very quickly [18]. The consistency index of the formulations during the study was not changed by the addition of the vitamin derivatives. Similarly, hysteresis areas (thixotropy), a pseudoplastic natural characteristic observed in the rheograms (Figure 1), were not changed by vitamins (Figure 2D). Antithixotropic behavior (negative thixotropy values) observed during the experiment could be due to the raw material employed and does not indicate instability since some authors suggest that high molecular weight polymer solutions present anomalous behavior under shear flow [19]. The present study showed that, over time, physical alterations are not prominent in formulations with added single vitamins or combined (Figure 2). ( a ) ( b ) ( d ) ( c )

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