In-vitro Assessment of Effectiveness and Photostability Avobenzone in Cream Formulations by Combination Ethyl Ascorbic acid and alpha Tocopherol Acetate

Article history: Received on: 22/03/2015 Revised on: 19/04/2015 Accepted on: 22/05/2015 Available online: 27/06/2015 Avobenzone is UVA sunscreen active substances, which are unstable when exposed to UV radiation, especially UVA. Theaim of this study is to determine the effectiveness and photo stability avobenzone when combined with ethyl ascorbic acid and alpha tocopherol acetate with various concentrations of ethyl ascorbic acid and alpha tocopherol by in vitro using spectrophotometric method. The photostability study of the eight variations of formula showed that the formula3, 4, 5, 6, 7 and 8 had significant differences with Formula 1 (p<0,05) and the best photostability represented by the formula 4 (avobenzone 2%, ethyl ascorbic acid2%) with avobenzone concentrations decrease by 11,82% for 15 hour using UVA lamp irradiation 4,7 mW/cm. In effectiveness of SPF (Sun Protection Factor) value determination showed moderate protection category (SPF ±6). The percent transmission of erythema and pigmentation test showed that cream had effectiveness as sunscreen by showing the category as sun block on the area pigmentation and not to erythema areas at concentration 1000 μg/ml.


INTRODUCTION
The sunlight that reaches the Earth's surface is composed of ultraviolet (200-400 nm), visible (400-800 nm) and infrared (> 800 nm) radiation.Ultraviolet light emitted is divided into three regions, namely the UVC (200-290 nm), UVB (290-320 nm) and UVA I region (340-400 nm) and UVA II region (320-340 nm) (Gonzalez et al., 2007;Bonda and Marinelli, 1999;Chaudhuri et al., 2006).The skin need protect from ultraviolet radiation hazard.Sunscreen is one effort to minimize of ultraviolet radiation penetration into the skin.Indonesia is a tropical country where the sun exposure is high to require the preparation of cosmetics that acts as a sunscreen (WHO, 2013).The Environmental Working Group (EWG) that founded in 1993 by Ken Cook and Richard Wiles reviewed over 1800 sunscreens and more than 257 brands and found morethan75% of sunscreens contain toxic chemicals that can increase the risk of cancer and other health problems.Chemical sunscreens identified hazards .include PABA (Para amino benzoic acid), menthylanthranilate, oxybenzone, 4-metilbenzilidin camphor, 3-benzilidin camphor, Octylmethoxycinnamate, homosalate, octisalate, and octocrylene.FDA announced to use sunscreen safety and agreed that avobenzone can be used as chemical sunscreens (EWG's, 2013).The good sunscreen should have photo-stability.Photostability is defined as the ability of a molecule to remain intact with irradiation filter or sunscreen (Gonzalez et al., 2007;Hojerova et al., 2011), because they are deliberately selected as the molecules that absorb UV radiation (Nash and Tanner, 2014).
The effectiveness of sun-screen preparations can be determined in-vitro by calculating the value of SPF (Sun Protection Factor), percent transmission of erythema (%Te) and percent transmission of pigmentation (%Tp) (Pelizzo et al., 2012;Gonzalez et al., 2007;Mishra and Chattopadhyay, 2012;Hupel et al., 2011) .The aim of this study was to determine avobenzone photostability that combined with ethyl ascorbic acid and alphatocopherol acetate to assess avobenzone levels before and after UVA lamp exposure, and determine effectiveness include the SPF value, percent transmission of erythema and pigmentation.

Chemicals
Avobenzone, ethyl ascorbic acid, alpha tocopherol acetate were purchased from Sigma (St Louis, USA), ethanol 96% (Merck), cream bases were purchased from local industry in Bandung, Indonesia.

Stock Solution
20 mg avobenzone was weighed, transferred to a 100 ml volumetric flask, diluted to volume with ethanol 96% to give 200 µg/ml solution.

Maximum wavelength determination of avobenzone ( max)
The maximum wavelength is determined at a concentration of 6 µg/ml of avobenzone diluted with ethanol 96%.The measurements were taken in the range of 200 to 400 nm

Preparation of Calibration Curve
Appropriate dilutions of the stock solution were done separately to get 2, 3, 4, 6, 8 and 10 µg/ml.The absorbances were measured at 356 nm ( max of Avobenzone), and the calibration curves were plotted.

Evaluation of Sunscreen Cream Organoleptic observations
Organoleptic testing was done by looking at changes in color, odor (rancidity), and the occurrence of phase separation or rupture of cream.

Homogeneity test
Homogeneity testing was observed by checking the particles size between two object glasses to determine the formation of coarse particles.

pH Measurements
The pH measurements were carried out with pH strip acid indicator.

Viscosity Measurements
The viscosity cream measurements were carried out on rhion viscometer using spindle number 2 and the measurement were carried out in triplicate and calculated the average (Kumar, et al., 2011).

Centrifuge test
Centrifuge testing has been done by centrifuging a cream preparation at a speed of 3800 rpm for 5 hour, and phase separation was observed (Lachman et al., 1994;Elya et al., 2013).

Photostability Testing of Sunscreen Cream Sample Irradiation
Cream weighed of 0.1 g and placed on object, irradiated by UVA 4,7mW/cm 2 for 15 hour and radiation dose gave 846 KJ/m2, 1.692 KJ/m2 and 2.538 KJ/m2.This correspond to UVA dose that reaches the earth's surface during 2-5 at sunny day.For a comparison of each formula are treated without irradiation.Every time each formula is taken and stored in a place protected from sunlight (Venditti et al., 2008).

Initial Absorbance Measurements
Cream (0.1 g) was weighed, dissolved on 96% ethanol and the absorbances was measured by UV spectrophotometerat 356 nm.Avobenzone levels were calculated using the regression line equation of calibration curve.

Absorbance Measurements after UVA irradiation
A cream (0.1 g) were UVA irradiation dissolved in 96% ethanol and absorbance was measured with UV spectrophotometer at 356 nm ( max of Avobenzone), and avobenzone levels were calculated using the regression line equation of calibration curve.

Evaluation
The Data of photostability testing of avobenzone cream was performed between the level of avobenzone and length of exposure time to UV light and the significance of the differences between mean values (where p < 0.05) was analyzed by the block method of variance (ANAVA) from SPSS version 18.0.

Effectiveness Assessment of Sunscreen Cream SPF (Sun Protection Factor)
The cream (0.1 g) was weighed, further diluted in ethanol 96% to obtain 1000 µg/ml solution.The solution was diluted to obtain 100, 200, and 500 µg / ml.The absorption data were obtained in the range of 290-320 nm, every 5 nm.The SPF could be calculated followed by the application of Mandur equation (Gonzalez et al., 2007;Pelizzo et al., 2012;Sayre et al., 1980): Where :CF = correction factor = 10; EE (λ) = erythemal effect spectrum; I (λ) = solar intensity spectrum; Abs = absorbance of sunscreen product.The values of EE (λ) x I (λ) are constants.It is determined by Sayre et al (1980).SPF assessments refer to the FDA regulations that categorize the effectiveness of sunscreen based on SPF values (Diffey and Robson, 1989).SPF assessments refer to the FDA regulations that categorize the effectiveness of sunscreen based on SPF values (Diffey and Robson, 1989).In evaluating SPF, sample was treated at thickness of 2 mg/cm 2 , the SPF was calculated as the ratio of MED (Minimal Erythema Dose) of sunscreenprotected skin compare to the MED of unprotected skin (Haywood et al., 2003;Stephens et al., 2011).

CONCLUSIONS
The results of analysis of variance showed that the addition of ethyl ascorbic acid and alpha tocopherol acetate in cream formula gave the significant difference photostability with the formula 1 (avobenzone 2%) with 95% confidence level (p≤0,05).The formula 4 (avobenzone 2% and 2% ethyl ascorbic acid) was the best formula to stabilize avobenzone in preparation.The addition of ethyl ascorbic acid and alpha tocopherol acetate might improved the SPF value, but have been not able to improve the effectiveness of protection against erythema/UVB.While, the value of percent transmission of erythema (Te) and percent transmission of pigmentation (Tp) decreased.

Table 1 :
Normalized product function used in the calculation of SPF.

Table 2 :
Prediction of SPF value.

Table 6 :
The results of measurement of cream absorbances before and after UVA irradiation

Table 7 :
The Results of Effectiveness -SPF (Sun Protection Factor) Testing.

Table 8 :
The Results of Effectiveness -Percent Transmission of Erythema and Pigmentation at 100 µg/ml.

Table 9 :
The Results of Effectiveness -Percent Transmission of Erythema and Pigmentation at 200 µg/ml

Table 10 :
The Results of Effectiveness -Percent Transmission of Erythema and Pigmentation at 500 µg/ml.

Table 11 :
The Results of Effectiveness -Percent Transmission of Erythema and Pigmentation at 1000 µg/ml.
= Percent transmission of erythema value., %Tp = Percent transmission of pigmentation value