UV protection test of the ethanol fraction of papaya cream (Carica papaya L.) added with titanium dioxide

Cream containing papaya fruit (10% of the 70% ethanol fraction) has been reported to effectively protect skins from ultraviolet (UV) light, though with a minimum protection ability. This study was aimed to determine the effects of adding titanium dioxide to sunscreen composed of 70% ethanol fraction of papaya flesh on the cream effectiveness, as measured by SPF values and physical properties. The ethanol fraction was obtained by fractionating the 70% ethanol extract of papaya fruit using n-hexane and ethyl acetate. Then, it was added with three different concentrations of titanium dioxide, namely, 1% (FI), 3% (FII), and 5% (FIII), to form cream preparations. These sunscreens were tested for their effectiveness in UV protection by in vitro spectrophotometry and based on the resultant SPFs. Based on the results of the study, the cream prepared with no titanium dioxide had SPF= 1.1283, while the SPFs of cream preparations added with 1%, 3%, and 5% titanium dioxide were 2.0572, 2.5708, and 2.8832, respectively. At these three concentrations, titanium dioxide increased the SPFs by 45%, 56%, and 61%, respectively. The cream preparations were found to have excellent physical properties. Based on the results of the statistical Kruskal-Wallis test, there are significant differences (p<0.05) between FI, FII, and FIII.


INTRODUCTION
All living organisms require sunlight for survival. Although human needs it as a source of energy and for healthy skin and bones, its radiations also pose a negative impact on the skin, especially, UVA, UVB, and UVC. When the epidermal tissue of the skin is overexposed to these UV lights, it can develop into erythema, sunburn, degeneration of the skin, and skin cancer (Ismail et al., 2014).
Sunscreen potentially reduces the effects of exposure to UV rays because it can protect the skin from UV radiation. Although sunscreens that contain chemicals are widely developed, natural materials that are considered safer and more affordable are preferred. Papaya is an example of natural ingredients reportedly effective in UV protection (Islamiyati, 2018).
According to Islamiyati (2018), cream containing 10% of the 70% ethanol fraction of papaya flesh has a protective activity similar to that of sunscreen, though less optimal (SPF= 2.2570). With a higher SPF, sunscreens can provide better skin protection. In response to this, various substances have been added to increase SPFs, including the addition of titanium dioxide to papaya cream. Based on the mechanism of action, there are two categories of sunscreens: chemical sunscreen (absorbing radiation) and physical sunscreen (reflecting radiation) (Gustiani et al., 2015).
Titanium dioxide is one of the physical sunscreens that can protect the skin against UV rays (Zulkarnain et al., 2013). Its inorganic compounds have a better effect compared to their organic counterparts, which can only absorb UV radiation (Smijs and Pavel, 2011). Titanium dioxide is a very efficient sunscreen with low unfavorable effects like irritation and sensitization (Antoniou et al., 2008). With the addition of titanium dioxide to cream containing 70% ethanol fraction of papaya flesh, the effectiveness of these sunscreens is expected to increase.

MATERIALS AND METHODS Materials
The materials used in this research were papaya fruits collected from Karang Kemiri Village, pharmaceutical-degree titanium dioxide, 70% Ethanol, N-hexane, Ethyl acetate, and the ingredients of the cream with pharmaceutical degrees (i.e., stearic acid, Cera alba, TEA, Vaseline alba, propylene glycol, nipagin, and aquadest).

Simplisia preparation
The papaya fruits were sorted out from impurities and washed with running water until clean. Afterward, they were mashed, placed on a black cloth, and dried in the sun. The resultant simplisia was ground using a blender until powdered simplisia was produced. It was then stored in a tightly closed dry container in a room protected from sunlight (Rahayu et al., 2009).

Preparation of papaya fraction
A total of 3.5 kg of the powdered simplisia was extracted by maceration using 70% ethanol solvent. After filtration, the filtrate was concentrated in a water bath at 40 o C until a dense extract was obtained. The dense extract was fractionated twice with 100 mL of n-hexane using a separating funnel. Then, the 70% ethanol fraction was collected and fractionated with 50 mL of ethyl acetate using a separating funnel, and this procedure was repeated four (4) times to obtain an optimal 70% ethanol fraction (Eka, 2014).

Cream formulation
The sunscreen cream was prepared from the 70% ethanol fraction of papaya fruit by mixing oil-phase with water-phase ingredients that had been prepared separately in a warm state. The oil phase (Cera alba, Vaseline alba, stearic acid, and titanium dioxide) was formed over a water bath at Pharmaciana ISSN: 2088 4559; e-ISSN: 2477 0256

Development of Antioxidant …(Priani et al.,)
63 70 o C until it melted, while the water phase was made by dissolving the ingredients (nipagin, propylene glycol, and TEA) with warm water. Both phases were mixed until homogeneous, added with the ethanol fraction of papaya fruit, and stirred until a homogeneous state and room temperature were achieved (Anief, 2006). The composition of the papaya sunscreen cream is presented in Table I.

Testing of the physical characteristics of the cream Organoleptic observation
The organoleptic test of the papaya cream included observations of color, texture, and odor (Faradiba, 2013).

Homogeneity test
The papaya cream was examined for its homogeneity by applying it on a glass object and observing the absence or presence of coarse grains (Suhery et al., 2016).

pH measurement
A homogeneous solution made by diluting the papaya cream in a water solvent was measured for its pH with a pH meter. The readings of the pH meter were then recorded (Puspitasari et al., 2017).

Spreadability test
Five hundred mg of the papaya cream was applied on a round glass scale and pressed with another round glass with a known weight for 5 minutes. Afterward, weights of 50 and 100 g were placed consecutively on top of the round glass for 1 minute. This procedure was replicated five (5) times (Miranti, 2009), and the diameter of the cream spread was measured.

Adhesion test
Five hundred mg of the papaya cream was placed on a glass object with a known area, covered by another glass object, and pressed with a weight of 1 kg for 5 minutes. The glass object was mounted on a test instrument and released with an 80-gram load, then the time needed to separate the two glass objects was recorded. This procedure was replicated five (5) times and reproduced for all cream formulas (Susilowati et al., 2014).

Determination of the SPFs of the cream
The effectiveness of the papaya sunscreen cream was determined from its SPF, which was measured in vitro by spectrophotometry.

Sample preparation
One gram of the papaya cream was weighed, transferred to a 100 mL volumetric flask, and then diluted with 70% ethanol. After that, the cream was cultivated for 5 minutes then filtered with a filter paper. Ten mL of the first filtrate was removed. A total of 5 mL of the solution was pipetted out into a 25 mL volumetric flask and then diluted with ethanol (Setiawan, 2010).

SPF calculation
The absorption spectrum of the test solution in cuvettes was obtained using a UV-Vis spectrophotometer at wavelengths of 290-320 nm with 70% ethanol as blank. The uptake of the test solution was measured at 290-320 nm, with an interval of 5 nm. Finally, the SPF of the cream preparations was analyzed by Mansur's method.
The mathematical equation for in vitro SPF calculation in a spectrophotometer is as follows: .  Table II) Abs : Absorbance of the sunscreen sample CF : Correction factor (10) (Mansur et al., 1986).

RESULTS AND DISCUSSION Physical characteristics of the cream
The sample determination revealed that the papaya fruit used in this study was Carica papaya L., a species of the Caricaceae family. From 3.5 kg of papaya fruit, 280 grams of powdered simplisia were obtained. The maceration of this simplisia (using 70% ethanol) produced 60 grams of papaya extract, with a yield of 21.43%. Afterward, this ethanol extract was fractionated with nhexane and ethyl acetate, producing 30 grams of papaya fraction with a 50% yield.
The four creams prepared previously (FI, FII, FIII, and FIV) were each subjected to organoleptic observation and homogeneity, pH, spreadability, and adhesion tests. The organoleptic test results showed that the four formulas produced cream preparations with a cream color, a distinctive smell of the papaya fraction, and a smooth texture. Meanwhile, the cream added with titanium dioxide had a white color. Based on the homogeneity test results, all of the four formulas created homogeneous creams. The pH measured proved that the four formulas (FI, FII, FIII, and FIV) fulfilled the requirements for a safe-to-use sunscreen, namely, 4.5-8.0 (SNI 16-4399-1996). The higher the concentration of titanium dioxide added to the cream, the higher the pH value (Anggraini et al., 2013). The Kruskal-Wallis test revealed a significant difference (p˂0.05) between FI, FII, FIII, and FIV, meaning that differences in the concentration of titanium dioxide affect the pH of the cream. It is possible because titanium dioxide has a pH of 7.5 or tends to be alkaline (National Library of Medicine, 2019).
The spreadability test determines the ability of a dosage form to spread over a particular area from the diameter of the spread. Based on the results of this test (Table III), FI and FIV created a categorically good spread because FI had the least amount of titanium dioxide, and FIV did not contain titanium dioxide. Titanium dioxide has a high specific gravity (3.9-4.3 g/cm 3 ), which in this study was a solid powder that could reduce the spread of the cream (National Library of Medicine, 2019). The independent samples t-test further confirmed the significant difference (p˂0.05) between FI and FIV.
For topical preparations, the excellent adhesion is when the debonding time is more than 4 seconds (Ulaen et al., 2012). The test results showed that the adhesive strength of FI, FII, and FIII met the requirements of good adhesion (i.e., more than 4 seconds), but that of FIV did not (i.e., less than 4 seconds). In conclusion, a higher concentration of titanium dioxide can prolong the debonding of two glass objects adhered to each other using the papaya cream or, in other words, strengthen the adhesion. Titanium dioxide has a high specific gravity (3.9-4.3 g/cm 3 ), which can increase the adhesion of the cream (National Library of Medicine, 2019). Based on the Kruskal-Wallis test, there is a statistically significant difference (p˂0.05) between FI, FII, and FIII, which is believed due to differences in the concentration of the added titanium dioxide that potentially affect the stickiness of the cream. The results of the adhesion test can be seen in Table III.

Sun Protection Factor (SPF) of the Cream
The Sun Protection Factor (SPF) of the papaya cream added with titanium dioxide was measured in vitro by spectrophotometry (using a UV-Visible spectrophotometer). The tests were reproduced five times at wavelengths of 290-320 nm, with an interval of 5 nm. The SPF was determined by preparing the cream samples (FI, FII, FIII, FIV, and positive control) and measuring their absorbances. FI = formulated with 1% titanium dioxide FII = formulated with 3% titanium dioxide FIII = formulated with 5% titanium dioxide FIV = formulated without titanium dioxide C (+) = positive control = market products with scientifically proven SPFs (i.e., Garnier) When added with titanium dioxide, cream containing 10% of the 70% ethanol fraction of papaya flesh can increase the SPF of the cream preparations. It is higher than FIV (SPF= 1.1283) but lower than the cream in the market. The results showed that the higher the concentration of the added titanium dioxide, the higher the SPF of the cream produced. Titanium dioxide is an active substance known to function as a sunscreen, whose physical protection mechanism involves reflecting UV lights (Anggraini et al., 2013). It is composed of inorganic particles that spread microparticles in the upper skin layer, increasing the protection factor against sunlight (Latha, 2013).
As presented in Table V, the effectiveness of the cream can be categorized based on its SPF. The Kruskal-Wallis test of the SPFs of FI, FII, FIII, FIV, and positive control resulted in p<0.05, indicating a statistically significant difference between the five cream preparations.

CONCLUSION
The addition of titanium dioxide to cream prepared with 70% ethanol fraction of papaya (Carica papaya L.) flesh has been proven to increase the effectiveness, pH value, and adhesive strength of the sunscreen creams, although with SPFs lower than the ones available in the market.