Determination of Antiradical Activity, Total Phenolic, and Total Flavonoid Contents of Extracts and Fractions of Langsat (Lansium domesticum Coor.) Seeds

Lansium domesticum Coor. is a fruit species from the Meliaceae family, which is a tropical plant native to Southeast Asia. Local citizens call it langsat, longkong, or duku and have used it as traditional medicine. The seeds of L. domesticum are used as a fever medicine, its bark is used to treat scorpion sting, and its leaves are used to repel mosquitoes. Because of its various uses, it is necessary to explore the antiradical potential of L. domesticum seeds. This study aims to determine the antiradical potential of L. domesticum seeds extract and fractions by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and to discover compounds that act as antiradical. Lansium domesticum seed powder was macerated with methanol, and then the extract was concentrated using a rotary evaporator and fractionated by n-hexane and ethyl acetate. The antiradical assay was conducted on extract and fractions by using DPPH radicals. Phenolic and flavonoid contents from extract and fractions were also tested. The ethyl acetate fraction obtained strong antiradical potential with an IC50 value of 8.938 ± 0.031 μg/mL. Total phenolic and flavonoid contents of ethyl acetate fraction were higher with values of 58.25 ± 0.501 mgGAE/g sample and 75.123 ± 0.175 mgQE/g sample, respectively. Correlation of phenolic and flavonoid contents, which inhibited radicals had R2 values of 0.9182 and 0.7658. Ethyl acetate fraction of L. domesticum seeds had very strong antiradical activity. Further isolation is expected to be conducted to discover which compounds are the most responsible as antiradical.

These polyphenols' antioxidant properties are due to the polyphenol compound's redox properties, which acts as a reducing agent by donating its hydrogen (Piluzza & Bullitta, 2011).
The body needs antioxidants because they can delay substrate oxidation by inhibiting initiation and propagation (Pham-Huy et al., 2008;Widodo et al., 2020).
Synthetic antioxidants, such as beta-hydroxy acid (BHA), tert-butyl hydroquinone (TBHQ), propyl gallate (PG), and butylated hydroxytoluene (BHT), have been used extensively in the world. However, these synthetic antioxidants have side effects, such as carcinogenic and cytotoxic effects on the heart and lungs (de Oliveira et al., 2010;Sulastri et al., 2018). Additionally, BHA and BHT also have low solubility and moderate antioxidant activity (Sannigrahi et al., 2010). Therefore, current research focused on discovering new antioxidant compounds from natural products that exhibit high activity and lower toxic effects than synthetic compounds (Rohman et al., 2010).
domesticum showed antimalarial activity against Plasmodium falciparum (Klungsupya et al., 2015;Saewan et al., 2006). Extracts and fractions from Persea americana peel have also shown antimalarial activity. This research aims to determine the potential of antiradical activity from extracts and fractions of L. domesticum seeds.

Extraction
Lansium domesticum seeds were obtained from fruit traders in Fruit Market Wua-Wua, Kendari, Southeast Sulawesi, Indonesia. The seeds of L. domesticum are then crushed into a powder and dried. Furthermore, L.
domesticum seed powder was macerated using methanol for 3 x 24 hours. Every 24 hours, the macerate was filtered, and the solvent was replaced. Then, the macerate was concentrated using a rotary evaporator at 40°C to obtain crude extract.

Fractionation
As much as 40 g of L. domesticum seed crude extract was partitioned using the gradient elution liquid-liquid fractionation method. First, the seed extract was partitioned using a separatory funnel with n-hexane, followed with ethyl acetate, and water fraction as the remaining fraction. Each fraction was evaporated with a rotary evaporator into crude fractions. The working method in a diagram is presented in Figure 1.

Phytochemical screening
Phytochemical screening was conducted to determine the profile of secondary metabolites in L. domesticum seeds extracts and fractions. Phytochemical screening methods were performed based on previous research by Yamin et al. (2020).

Alkaloid test
Lansium domesticum seeds' extract and fractions were inserted separately into 1 mL test tubes and added three drops of Dragendorff's reagent. The formation of brown precipitate indicated the presence of alkaloid.

Flavonoid test
The extract and fractions of L. domesticum seeds were inserted separately into test 1 mL tubes and added with 0.2 g of magnesium powder and 2 mL of concentrated HCl. The formation of red, orange, and green solutions indicated the presence of flavonoid.

Terpenoid test
The extract and fractions of L. domesticum seeds were inserted separately into 1 mL test tubes and added with 0.5 mL of acetic acid anhydride and 2 mL of concentrated sulfuric acid. The formation of green, bluish, and brown solutions indicated the presence of terpenoid.

Tannin test
The extract and fractions of L. domesticum seeds were inserted separately into 1 mL test tubes and added with 1 mL of 1% ferric chloride solution. The formation of blue to black solution indicated the presence of tannin.

Saponin test
The extract and fractions of L. domesticum seeds were inserted separately into 1 mL test tubes and added with 2 mL of hot water, then cooled and shaken for ten seconds.
It was declared positive for saponin if the fume generated stabilized in less than ten minutes.

Determination of antioxidant activity with DPPH
The antioxidant activity was method according by (Rohman et al., 2010) with modified. As much as 1 mL from each sample solution was briefly taken and added with 3 mL methanol p.a., then 1 mL of 0.6 mM DPPH.
Then, the samples were shaken until homogeneous.
After that, the samples were incubated for 30 minutes in a dark room at room temperature. The absorbance of each solution was measured at 515 nm wavelength. The following equation calculated the power of antioxidants: Abscontrol is absorbance of control Abssample is absorbance of sample Therefore, the percentage of inhibition was plotted with the concentration (µg/mL) to obtain the linear regression equation of y = bx + a. The IC50 value was obtained by replacing y with 50 and calculated the x value. The IC50 is defined as the concentration of the sample that is needed to inhibit 50% of DPPH radical.

Determination of total phenolic content
Phenolic contents in extract and fractions of L. domesticum seeds were determined using a spectrophotometric method according to Parthasarathi & Park (2015) with modified. Briefly, 1 mL from each sample concentration series was taken, then 0.4 mL of Folin-Ciocalteu reagent was added into the samples. The mixture was shaken and allowed to stand for eight minutes. As much as 4 mL of 7% Na2CO3 solution was added and shaken until homogeneous. Then, water was added until the volume reached 10 mL. Absorbance was measured using a UV-Vis spectrophotometer at 647 nm wavelength with three replications. Phenolic content was expressed as g gallic acid equivalent (GAE)/100 g sample.

RESULTS AND DISCUSSION
The phytochemical screening results of L. domesticum seeds showed that the methanol extract, n-hexane, ethyl acetate, and water fraction positively contained flavonoids, alkaloids, tannins, and terpenoids.
Meanwhile, the results of the saponin test on the extract and fractions were negative. These results are consistent with those reported by Nur et al. (2017). The result of phytochemical screening is shown in Table I.  (Olugbami et al., 2014). The smaller IC50 value indicated the potent antioxidant in extract or fractions (Maisuthisakul et al., 2007). The standard antioxidant used in this study was vitamin C.  Molyneux (2004). According to the data of IC50 shown in  (Sannigrahi et al., 2010), Pandanus conoideus Lam (Rohman et al., 2010), Oroxylum indicum Linn (Trang et al., 2014), Polygala sabulosa, Cyathea phalerata (Brighente et al., 2008), as well as the stem bark from Dracontomelon dao(Blanco) Merr (Yamin et al., 2020). The DPPH is a free radical widely used to examine radical scavenging activity of plant extracts (Jamuna et al., 2012), pure compounds, food ingredients, and others (Koleva et al., 2002). Besides, this method is fast, reliable, reproducible, requires less energy, does not require sophisticated instruments, the reagents needed in this method are inexpensive (Jamuna et al., 2012, Koleva et al., 2002. An antioxidant compound's intrinsic ability to donate hydrogen atoms or electrons to homogeneously reactive radical compounds can be determined. This method is based on a decrease in the solubility of methanolic DPPH, which is caused by antioxidant compounds that donate their hydrogen (Rohman et al., 2010).  (Yokozawa et al., 1998).  Phenolic and flavonoid compounds are the most responsible for antioxidants activity. This is due to the hydroxy groups present in phenolic and flavonoid compounds in free radical scavenging (Saxena et al., 2012;Aryal et al., 2019). Based on the IC50 values in Table II, the total phenolic and flavonoid contents in the L. domesticum seeds are presented in Table III. It is known that the antioxidant activity of a material correlated with the phenolic and flavonoid contents in that material. The higher the total phenolic and flavonoid levels in the sample, the stronger the sample will be as an antioxidant.

The antioxidant activity of extract and fractions is
Correlation of total phenolic and flavonoid contents to radical activity (IC50 values) in L. domesticum seeds is showed in Figure 2 and Figure 3, respectively. The relationship between radical activity (y) with total phenol (x) revealed a coefficient of determination (R 2 ) of 0.9182, whereas total flavonoid content (x) has an R 2 of 0.7658.
The results suggested that phenolic compounds and flavonoids compounds contributed to 91.82% and 76.38% to free DPPH radical scavenging of extract and fraction of L. domesticum seeds. Also, it can be stated that the scavenging effect of extracts/fractions is not limited to phenolic and flavonoid compounds. The activity may also come from other antioxidant secondary metabolites in the extracts such as volatile oils, carotenoids, and vitamins (Javanmardi et al., 2003;Rohman et al., 2010;Mistriyani et al., 2018). Ethyl acetate fraction can be further isolated to find out which compounds are most responsible as antioxidants.