Chemical Screening and Antioxidant Activity of Ethyl Acetate Fraction and Residue from Lansium domesticum Ethanolic Extract

3 . Previous studies reported that it has antimalarial, antibacterial, antioxidant, and antipyretic activities 4-6


INTRODUCTION
Meanwhile, ultraviolet radiation that reaches the earth's surface is about 5%. In 2019, there were reportedly more than 287,723 cases of malignant melanoma (MM) associated with UV radiation, with a mortality ratio of 21%, as well as 1,042,056 cases of non-melanoma skin cancer (NMSC) with a mortality ratio of 6%. The development of photoprotector-based cosmetic products can protect against sunburn, which causes suppression of the immune system and health problems caused by damage to intracellular DNA, increased production of reactive oxygen species (ROS), and oxidative stress due to premature skin aging 7 . Previous study 2 on developing photoprotector products stated that dry hydroethanolic extract of L. domesticum fruit could be used as cosmetics. The extract is dissolved in propylene glycol and used as a skincare product for skin depigmentation and moisturizing. Furthermore, clinical trials conducted on 30 women aged 32-52 years for four weeks found that L. domesticum extract can increase skin moisture and the melanin index. The use of bioactive compounds found in plant parts in cosmetic formulations to meet skincare needs is increasing in line with the development of raw materials from new sustainable sources 8 . Plant extracts have been the subject of several studies in the beauty industry sector 9 . However, the potential use of tropical plants' bark in formulating cosmetic preparations has yet to be explored adequately. Therefore, this study aims to determine the chemical compounds contained in the ethyl acetate fraction and the water fraction of L. domesticum stem bark using gas chromatography-mass spectrophotometry (GC-MS), as well as to measure the antioxidant activity using the DPPH and FRAP methods, and the SPF value.

Extraction and fractionation
Lansium domesticum bark was taken and determined in the Laboratorium of Biology, Faculty of Mathematics and Natural Sciences, Universitas Tanjungpura, on 27 th September 2016. Simplicia was made using stem bark and sifted using a 40-mesh sieve (Figure 1). Simplicia was macerated with ethanol 86% and evaporated with a rotary evaporator, and the thick L. domesticum bark extract was obtained. The ethanol extract was fractionated using ethyl acetate and water solvent to get the ethyl acetate fraction and residue (water fraction).

Phytochemical screening
Phytochemical screening was carried out on the ethyl acetate fraction and residue of the ethanol extract of L. domesticum stem bark 4 . 1. In the alkaloid test, chloroform ammonia was added to the extract and fraction, shaken, and filtered, then 1 mL of 2 N sulfuric acid was added. The acid layer (top layer) was pipetted and put into a test tube containing Mayer's, Dragendorff's, and Wagner's reagents. 2. For the phenol test, the extract and fraction were dissolved in ethanol, then a few drops of 1% FeCl3 were added. 3. In the flavonoid test, the extract and fraction were dissolved in hot water, 0.5 mg of Mg powder was added, three drops of concentrated HCl were added, then five drops of amyl alcohol were added. 4. For terpenoid test, extract and fraction were dissolved with n-hexane, and Liebermann-Burchard reagent was added. 5. For the anthraquinone test, 1 g of the extract and fraction was boiled for 2 minutes with 2 mL of 0.5 N KOH and three drops of hydrogen peroxide. After cooling, the suspension was filtered, the filtrate was added with acetic acid to a pH of 5, and 3 mL of benzene was added. The top layer was separated with a pipette and put into a test tube, then 0.5 N potassium hydroxide was added. 6. In the tannin test, 1 g of extract and fraction was heated with 10 mL of water for 30 minutes in a water bath. The solution was filtered, and the filtrate was added with 5 mL of 1% gelatin solution. 7. In the saponin test or foam test, 1 g of extract and fraction in a test tube was added to 10 ml of distilled water, then closed and shaken vigorously for 30 minutes. The tube was then left in an upright position for 30 minutes.

GC-MS analysis
The chemical compounds were examined using GC-MS analysis based on Giri and Rajbhandari 10 methods with slight modifications. The sample fraction used was ethyl acetate, and the residue from the ethanol extract was dissolved using 3 ml of chloroform. The solution was sonicated for a minute, and chloroform was added to 5 mL in a measuring flask. GC-MS analysis was performed using an Agilent J&W GC column DB-5MS type (30 m x 0.25 mm). A total of 1 µL of sample solution was injected into Agilent Gas Chromatography GC:7890A (G3440A) for 40 minutes, with an injection port temperature of 230°C, while helium gas was used as a carrier. The mass spectroscopy was operated in electron collision mode with an ionization energy of 70 eV, while mass spectrum scans were performed at 30 to 380 m/z. The detected compounds were identified by processing the raw GC/MS data with ChemStation software and comparing the results with a mass spectral database.

Antioxidant activity with DPPH assay
Antioxidant activity was tested using a DPPH assay to identify the sample's free-radical-scavenging activity. The technique was based on previous studies 4, 11 , with slight modifications. About 1 mL of ethyl acetate and residue fraction, each with various concentrations of 100-500 ppm and 1-300 ppm, was added to 3 mL of 1 mM DPPH and 1 mL methanol. It was incubated in the dark for 15 minutes at room temperature, and absorbance was measured at 515.5 nm, in which the experiments were carried out in triplicate.

Antioxidant activity with FRAP assay
The antioxidant activity test with FRAP was performed according to Nur et al. 12 with slight modifications. About 30 µL each of ethyl acetate and residue fraction with various concentrations of 100-500 ppm and 1-300 ppm was added with 30 µL FeCl3 solution, i.e., 3 mM in 5 mM citric acid, and 240 µL 1 mM TPTZ in 0.05 M HCL in 96 microplate wells. Incubation was performed for 20 minutes at room temperature, and absorbance was measured at 615 nm. The results were presented in triplicate.

SPF value
The ethyl acetate fraction and residue were dissolved in ethanol at 250 µg/mL concentration and scanned across the 290 to 320 nm range at 5 nm intervals. Screening of sun protection activity was measured by determination in vitro of SPF, based on the equation proposed by Mansur et al. 13 The absorbance sample was measured three times and used for SPF calculation, as shown in Equation 1.

Statistical analysis
Data were presented as means±standard deviation, while statistical analysis was performed using SPSS software, with a value of p <0.05 considered significant.

RESULTS AND DISCUSSION
A qualitative examination of the chemical content contained in the langsat bark is presented in Table I. It is widely known that the ethanol extract of L. domesticum barks contains flavonoids, alkaloids, terpenoids, and tannins. The ethyl acetate fraction was confirmed to have terpenoids, while the residue contains phenolics, flavonoids, tannins, and saponins. A study conducted by Worang et al. 5 on the L. domesticum bark stems found alkaloids, flavonoids, phenols, and terpenoids in methanol extract, while the chloroform and n-butanol fractions contain alkaloids, terpenoids, and flavonoids. In this study, testing the ethyl acetate fraction and the residue was focused on preparing it as an active ingredient in cosmetic formulas; knowing the functional content of chemical compounds can be the basis for making cosmetic formulas. The chemical compounds detected by GC-MS in the ethyl acetate fraction and residues from the ethanol extract of L. domesticum bark are shown in Tables II and III. Forty-seven chemical compounds were found in the ethyl acetate fraction, including terpenoids and fatty acids. Meanwhile, four types of chemical compounds were confirmed in the residue, especially the terpenoid and fatty acid groups. The results of chromatogram separation using GC-MS are shown in Figures 2 and 3, with 35-and 60-minutes separation times for the ethyl acetate fraction and residue.   The most significant chemical compound, according to the percentage of the highest peak value, was germacrene D. This is in line with previous studies 3, 14 , which stated that germacrene D is the most abundant compound obtained from L. domesticum. Germacrene is a terpenoid compound, and the sesquiterpene group is volatile. There are two types: germacrene A and D. Moreover, it is a biogenetic compound and the precursor of cadinenes and muurolenes 15 . The results also showed γ and α muurolenes compounds with peak percentages of 2.25% and 73.65%. In previous studies 3, 16 , germacrene D was found in the fruit and had antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, Trichophyton mentagrophytes, and Aedes aegypti with a bond energy of -9.5 kcal/mol. γ muurolene compound produced by germacrene biosynthesis have larvicidal activity against Anopheles stephensi. It was also reported that L. domesticum bark has antibacterial and antimalarial activities 5,17,18 . Studies related to antioxidant activity in medicinal plants have been widely reported. Oxidation in lipids, proteins, and DNA caused by ROS (reactive oxygen species) induces degenerative and neurogenerative diseases, wrinkled skin, DNA damage, cardiovascular disease, inflammatory conditions, and carcinogenesis 19 . Therefore, further studies are needed to produce antioxidants sourced from natural ingredients to replace synthetic types. Previous studies 4,6,20 reported that L. domesticum has antioxidant activity. This is further demonstrated in this study, as presented in Table IV. The antioxidant activity was tested by measuring the IC50 value after reacting the sample with the DPPH and FRAP. DPPH, as a radical compound, predicts antioxidant activity through a reduction event that occurs due to the acceptance of electrons or hydrogen radicals by antiradical. Meanwhile, the FRAP method is based on the ability of antiradical compounds to reduce Fe 3+ to Fe 2+ in an acidic environment. This method uses TPTZ reagents, with tripyridyltriazine acting as an iron-linking ligand and ferrozine compounds, to assess the reduction ability 21, 22 . In terpenoid compounds, antioxidant activity occurs when a hydrogen atom is donated in the framework and based on its ability to inhibit lipid oxidation 23 .  26 . This is presumably due to the compound responsible for the sample with the FRAP test, which is classified as a secondary antioxidant having a mechanism to stabilize hydroperoxidase by inhibiting the breakdown of hydroperoxides into free radicals. Combinations that can chelate metals are also included in this class of antioxidants. Secondary antioxidants bind metal ion mechanisms, capture oxygen, convert hydrogen peroxide to nonradical species, and deactivate singlet oxygen, unlike DPPH testing, where the compound responsible has a mechanism for scavenging free radicals through breaking the chain of radical reactions by giving or donating hydrogen radicals quickly 27 . The SPF value was determined in the ethyl acetate fraction and residue. The test was conducted at 250 g/mL concentration using the Mansur formula 13 with UV/Vis spectrophotometry in the 290-320 nm wavelength range while performing absorbance measurements at every 5 nm. The measurement results are demonstrated in Table V. The mechanism of sunscreen as a photoprotector can be explained through molecules capable of absorbing energy from UV rays, then experiencing excitation events to a higher energy level followed by the release of energy and the return to a lower level. The UV rays absorbed by these molecules can absorb energy and function as a sunscreen because they have low power, thereby reducing the effect of UV exposure 28 . The higher the SPF value, the higher the protective effect against UV rays 29 .
The measurement results show that the ethyl acetate and residual fractions have SPF values of 2.87 and 3.9, indicating a nonsignificant difference with the minimal protection category. The minimal protection offered was due to predominant chemical compounds: terpenoids and followed by fatty acids. According to previous studies, optimal protection against UV rays is given by the group of phenolics and flavonoids. This occurs through a conjugated double bond mechanism in the benzene nucleus which will experience resonance due to electron transfer when exposed to UV light and have a chromophore group that acts as an aromatic conjugated double bond, absorbing light at UV A and B wavelengths.

DATA AVAILABILITY
None.