Java plum (Syzygium cumini) extract cream effect on decreasing the number of sunburn cell on the dorsal skin of male Wistar rat (Rattus norvegicus) exposed to UV-radiation

Background: The primary acute damage due to UV radiation is keratinocyte apoptosis or sunburn cell. Previous studies showed that antioxidants inhibit UV damage. Java plum (Syzygium cumini) which is found in Indonesia, was known to have a high concentration of antioxidants. This study was aimed to assess the Java plum ethanol extract cream effects on the number of sunburn cells on the dorsal skin of male Wistar rats that exposed to UV-B. Methods: This experimental research was a post-test only control group design that was conducted on 30 male rats. Groups were divided into control, placebo, 5%, 10%, and 20% ethanol extract cream concentrations (P1, P2, P3, P4, and P5, respectively). The number of sunburn cell were then observed and recorded based on visual evaluation of the skin biopsy under a light microscope. Results: The mean results of P1, P2, P3, P4, and P5 were 132.67, 98.67, 118.67, 104.00, and 54.67 respectively. The control, placebo, 5%, and 10% ethanol extract cream showed no significant result (p>0.05). In contrast, 20% ethanol extract cream showed a protective effect (p<0.05). Conclusion: Java plum ethanol extract cream with 20% dose could decrease the number of sunburn cells in the skin.


Introduction
Aging is a process that causes physical and functional changes in the body. 1 Since Indonesia has a tropical climate, Indonesian people are exposed to sunlight almost 10 to 12 hours a day.
This shows that their skin is in high risk of aging due to Ultraviolet (UV) irradiation also known as photo aging. 2 Two types of ultraviolet rays are emitted through sunlight and contact the human skin, which are the UV-A and UV-B which cause acute and chronic effects on the skin. The acute effects includes erythema, sunburn, rising of skin temperature, thickening of the skin, pigmentation prob-sunburn cells, that result in skin redness and pain. 4 Human skin has antioxidants which protect it against reactive oxygen species. Antioxidants are molecules that inhibit the oxidation of other atoms by sharing its electron to other atoms. However, over-exposure of the skin towards UV radiation leads to a significant reduction of the antioxidant, thus increasing oxidative stress. 5 Java plum (Syzygium cumini) or also known as jambolan is a purple colored oblong fruit. The fruit and seeds have been reported to be rich in anthocyanin which belongs to a parent class of molecules called flavonoids, a well-known antioxidant, which accounts for the scavenging of free radicals and protective effect of antioxidant enzymes. 6 Flavonoids are divided into five subgroups according to their different chemical structures which are the anthoxanthins, flavanones, flavanonols, flavans, and anthocyanidins. Java plum is found to be rich in anthocyanidins which are also aglycones of anthocyanin.
Anthocyanin that is found in Java plum is in the form of delphinidin-3-gentiobioside, malvidin-3-laminaribioside, petunidin-3-gentiobioside, cyanidin diglycoside, petunidin, and malvidin. 7 Previous study shows that anthocyanin prevents acute and chronic effects of UV irradiation. 8 Considering the activity of anthocyanin in oxidative stress, further research is needed to understand the effect of Java plum on keratinocyte apoptosis due to UV irradiation.

Method
The methods of the research are, preparation of extract, 5 kg of Java plum was sorted and processed to get 60 g of extract. Java plum ethanol extract was then processed to form 5%, 10%, and 20% of Java plum ethanol extract cream. Next, a total of 30 samples were Descriptive analysis was conducted to obtain a central tendency value (mean and median) and standard deviation (SD) of the dependent variable. Statistical analysis was conducted to test the normality of the data using the Shapiro-Wilk test, homogeneity of the data by Levene's test and proceeded with test parametric statistics with one-way ANOVA. Least Significance Difference (LSD) test was performed to analyze the difference between each group.

Result
The average number of sunburn cell on the dorsal skin of the Wistar rat in Group P1, P2, P3, P4, and P5 were 132.67, 98.67, 118.67, 104.00, and 54.67, respectively. These results were analyzed by using one way ANOVA method with a significance value of p <0.05. Table 2 showed that there was no significant difference between the number of sunburn cells (p=0.225). Statistical analysis of the number of sunburn cells in experimental animals was continued using the Least Significant Difference (LSD) test. From the LSD results, it was found that cell assessment data differed significantly (p<0.05) between group P1 (Control) and P5 (20%). Based on the post hoc test results, the mean difference between P1 and P5 is significant at the 0.05 level (Table 3).

Table 3. Post-Hoc result of number of sunburn cells on the dorsal skin of Wistar rat analysed by using the Least Significant Difference (LSD) test.
Tanabbal et al.

Neurologico Spinale Medico Chirurgico
The number of sunburn cells were observed under light microscope (400x magnification) as shown in Figure 1. The results of this study are supported by other researches. A human study showed that flavonoids called catechins found in green tea decreased the number of sunburn cells and proved that it is suitable for protection from ultraviolet radiaton and sunburn. 10 Bolfa et al., 11 conducted an animal study of Romanian propolis and its photoprotec- and 8-dihydro-2'-deoxyguanosine (8-OHdG) suggesting the protective effects against the oxidative damage to proteins and DNA. 13 Anthocyanin also protects from cell damage which results in apoptosis of keratinocytes by strongly absorbing the visible and UV spectrums, with maximum absorbance in the ranges of 500nm-600nm. 14

Conclusion
Based on the results of the research that has been done, it can be concluded that cream with the concentration of 20% Java plum ethanol decreases the number of sunburn cells on the dorsal skin of the male Wistar rat. Further research is needed to understand other protective effects and its mechanism.