Phytochemicals Screening, GC/MS Characterization, and Antioxidant Activity of Falcataria moluccana Miq. Barneby and J. W. Grimes Methanolic Extract

Plants are very rich in secondary metabolite components with specific bioactivity for human needs in the health sector. The factor determining the pharmacological activity of plants is the content of secondary metabolites. Isolated secondary metabolites from plants have interesting bioactivity. Several studies found that secondary plant metabolites have bioactivity as antidiabetic1, anti-inflammatory2, antimicrobial3, anticancer4, anthelmintic5, and antioxidants.6


INTRODUCTION
Plants are very rich in secondary metabolite components with specific bioactivity for human needs in the health sector. The factor determining the pharmacological activity of plants is the content of secondary metabolites. Isolated secondary metabolites from plants have interesting bioactivity. Several studies found that secondary plant metabolites have bioactivity as antidiabetic 1 , anti-inflammatory 2 , antimicrobial 3 , anticancer 4 , anthelmintic 5 , and antioxidants. 6 Antioxidants can be produced both synthetically and naturally, but synthetic antioxidants have a toxic effect compared to natural antioxidants. 7 Natural antioxidants can be sourced from plants containing bioactive compounds in the form of secondary metabolites such as alkaloids, flavonoids, steroids, terpenoids, and others. Currently, a lot of biological and pharmaceutical exploration is carried out on plants. This is based on the fact that plants contain antioxidants and other bioactive components. Therefore, studies to replace synthetic antioxidants with natural antioxidants are necessary.
One of the potential plants as a source of natural antioxidant compounds is F. moluccana which is a multipurpose species in Southeast Asian plantations, especially Indonesia and Malaysia. 8 The purpose of this study was to find out phytochemical content, characterization of secondary metabolites by GC/MS analysis, and antioxidant activity of F. moluccana methanolic extract.

Extraction
The extraction of F. moluccana twig was carried out with a maceration technique using a methanol solvent. 9 Dried branches were crushed to a powder. 50 g of simplicia was macerated using 200 mL of methanol for 72 hours while shaking it in a shaker. Then, the solution was filtered using Whatman No. 42 filter paper. Then, the filtrate was evaporated using a rotary evaporator until obtaining a concentrated extract.
Alkaloids 50 mg was added to 5 mL of chloroform and 3 drops of ammonia. The chloroform fraction was separated, then 45 drops of 2 M H 2 SO 4 were added. The acid fraction (above) was taken and Dragendorf, Mayer, and Wagner reagents were added. The positive result was characterized by the formation of red deposit on the addition of Dragendorf reagent, white deposit in Meyer reagent, and brown deposit in Wagner reagent.
Steroid and terpenoid 5 mg of extract was added to 5 mL of hot ethanol. The solution was filtered and evaporated until dry. Then, 1 mL of diethyl ether was added and homogenized. A drop of concentrated H 2 SO 4 and anhydrous acetic acid were added to the solution. Green or blue color indicated steroid content, while red or purple color indicated terpenoid content.
Phenolic 5 mg of sample was dissolved in 2 mL of methanol. The solution was filtered then the filtrate was mixed with 10% NaOH then heated. The red color indicated phenolic compounds.

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis
The F. moluccana methanolic extract was analyzed using GC/MS Shimadzu QP 2010 (Shimadzu Corp., Kyoto, Japan), RTx5MS (Restek Corp.) columns of 30 m length, 250 °C injectors and detectors, and 50-300 °C operating temperature. The temperature rise at 50-120 °C was regulated with a temperature rise rate of 40 °C/minute and held for 1 minute then a temperature increase of 120-300 °C was adjusted with a temperature rise rate of 60 °C/minute and held for 5 minutes in total retention time (Rt) of 60 minutes. The carrier gas was helium with a molecular weight range of 50-500. The compound was identified by the Wiley/NIST Library software. 11

Total phenolic content
The total phenolic content was calculated to determine the amount of phenol in the sample. The extract solution was produced at a concentration of 100 mg/L in the form of the extract dissolved in absolute methanol. After that, 0.2 mL of the extract was added with 2.5 mL of 10% Folin Ciocalteu reagent dissolved in water, then added with 2 mL 7.5% Na 2 CO 3 . The sample was left in a water bath at 45 °C for 45 minutes. This procedure was carried out three times and the absorbance measurement was carried out at a wavelength of 765 nm. Gallic acid was used as standard with concentrations of 10, 20, 30, 40, and 50 ppm. The total phenolic content was expressed as GAE = Gallic Acid Equivalent milligrams per gram of extract. 12

Total flavonoid content
The total flavonoid content was calculated using the colorimetric method. The extract solution was produced at a concentration of 500 mg/L in the form of the extract dissolved in methanol, then 5 mL extract was added with 0.3 mL 5% NaNO 2 . Then, it was added with 0.3 mL 10% AlCl 3 , dissolved with methanol, and rested at room temperature for 5 minutes. Then, it was added with 2 mL 1 M NaOH and the solution volume was added up to 10 mL with distilled water. The absorbance was measured with a UV-Visible spectrophotometer at a wavelength of 510 nm. The standard used was quercetin with a concentration of 10, 20, 30, 40, and 50 ppm. The flavonoid content was expressed by Quercetin Equivalent (QE) mg per gram of dry extract (Quercetin Equivalent (QE) mg/g extract). 13 Antioxidant activity 1 mL of F. moluccana methanolic extract with 100, 200, 300, 400, and 500 ppm concentrations were mixed with 2 mL of 0.002% DPPH solution and rested for 30 minutes. The absorbance was measured at a wavelength of 517 nm. The positive control used ascorbic acid (5,10,15,20, and 25 ppm concentrations). The inhibitory power was calculated using the following formula: Inhibitory power (%) = Blank absorbance -Sample Absorbance x 100% Blank absorbance IC 50 (inhibitory concentration) was determined by calculating the concentration with 50% inhibition from linear regression equation.

Phytochemical Screening of F. moluccana Methanolic Extract
Phytochemical screening was carried out to test the alkaloids, flavonoids, saponins, tannins, steroids, terpenoids, and phenolics. The phytochemical components of F. moluccana methanolic extract can be seen in Table 1. The results of phytochemical testing showed that the F. moluccana methanolic extract had flavonoids, steroids, terpenoids, phenolics, saponins, and tannins, except alkaloid.
Phenolic and flavonoid compounds showed high antioxidant activity. Flavonoids have the ability to be antiviral, antibacterial, anti-tumor, anti-allergic, anti-inflammatory, and anti-carcinogen. 15 Several studies found that secondary metabolites from plants that have many roles as antioxidants and antibacterials are phenolic compounds in the form of flavonoids. 16 F. moluccana methanolic extract contains active compounds in the form of steroids and terpenoids. Steroid and terpenoid compounds have a carbon structure derived from six isoprene units and are biosynthetically derived from acyclic C30 hydrocarbons such as squalene. Squalene is a natural antioxidant, classified as triterpene compounds, and intermediates in plant and animal sterol biosynthesis. Steroids are natural antioxidant as an anti-radical and antioxidant. 17

GC/MS analysis
The results of GC/MS analysis showed that 20 compounds were identified in F. moluccana methanolic extract. This analysis showed that the highest abundance was α-terpinolenic from the terpenoid group with a retention time of 6.776 minutes and a percentage area of 25.85%. α-terpinolene is a terpenoid group (monoterpenes) with a molecular formula of C 10 H 16 and a molecular weight of 136.24 g/ mol which functions as a food additive or flavoring agent. 19 The F. moluccana methanolic extract contains different types of triterpene such as α-pinen, α-terpinolene, α-terpineol, and dl-limonene.

Total phenolic content of F. moluccana methanolic extract
The total phenolic content was calculated with the Folin-Ciocalteau reagent to form a complex solution with phenolic compounds. The total phenolic content was expressed by gallic acid equivalent milligrams per gram of extract. This study used gallic acid as a standard. This study used gallic acid with 10, 20, 30, 40, and 50 ppm concentrations. The standard curve of gallic acid had linear regression with y = 0.046x +0.647 and R 2 = 0.963. Based on the results, the total phenolic content of F. moluccana methanolic extract was 145.21 mg GAE/g. The total phenolic content of F. moluccana methanolic extract was higher than the total phenolic content of Averrhoa bilimbi (79 mg GAE/g) methanolic extract 26 and Moringa oliefera (44.03 mg GAE/g) methanolic extract. 27 Phenolic compounds are known to have beneficial antioxidant activity for the body as an antidote to free radicals. Phenolic compounds reduce free radicals by binding to metal ions and inhibiting enzymatic systems that play a role in the formation of free radicals such as cyclo-oxygenase, mono-oxygenase, or xanthine oxidase. The antioxidant effect on phenolic compounds is due to the ability to reduce and allow phenolic compounds to have free radical scavenging activity mechanisms, metal chelating intermediate activity, and singlet oxygen-reducing activity.
In addition, phenolic compounds play an important role in stabilizing lipid peroxidases and inhibiting the oxidation of various enzymes. 28 Total flavonoid content of F. moluccana methanolic extract The total flavonoid content of F. moluccana methanolic extract was calculated by using the AlCl 3 staining method which can be seen from the complex formation between AlCl 3 and the keto group. The total flavonoid content of F. moluccana methanolic extract was calculated with a standard quercetin curve, namely y = 0.134x -0.3 with R 2 = 0.945. The total flavonoid content was determined by substituting the absorbance values into the standard curve equation. Flavonoid content was expressed by Quercetin Equivalent (QE) mg per gram of dry extract. Based on the results, the total flavonoid content of F. moluccana methanolic extract was 95.39 mg QE/g. The total flavonoid content of F. moluccana methanolic extract was higher than the total flavonoid content of Averrhoa bilimbi (15 mg GAE/g) ethanolic extract 26 and Moringa oliefera (28.33 mg GAE/g) methanolic extract. 27 Flavonoids are polyphenolic compounds in plants. Flavonoids are divided into six subgroups, namely flavones, flavanols, flavanones, isoflavones, and anthocyanins. Flavonoids can prevent cancer and have an important effect on cancer chemoprevention and chemotherapy.
There are many mechanisms of flavonoids including carcinogen inactivation, antiproliferation, cell cycle arrest, induction of apoptosis and differentiation, inhibition of angiogenesis, antioxidants, and a   31 This study used ascorbic acid as a standard measure of antioxidant activity.
The antioxidant activity of F. moluccana methanolic extract and ascorbic acid as positive control can be seen in Table 3. IC 50 of F. moluccana methanolic extract was 12.6 ppm (below 50 ppm) so that the secondary metabolite of F. moluccana methanolic extract can be expressed as a very strong antioxidant. 31 IC 50 of ascorbic acid was 2.17 ppm. The antioxidant activity of F. moluccana methanolic extract was lower when compared with ascorbic acid as positive control. Ascorbic acid is easily oxidized by donating hydrogen atoms and forms relatively stable ascorbyl free radicals.
IC 50 value of F. moluccana methanolic extract was lower than IC 50 of Averrhoa bilimbi (4.27 ppm) methanolic extract. 27 The high antioxidant activity of F. moluccana methanolic extract is related to the content of compounds in the extract that play an important role in antioxidant activity. The results of phytochemical screening showed that F. moluccana methanolic extract has phenols, steroids, terpenoids, flavonoids, saponins, and tannins. The results of GC/MS analysis showed the methanolic extract of F. moluccana branches which affect radical inhibiting properties such as phenols and triterpenes.

CONCLUSION
The phytochemical screening of F. moluccana methanolic extract showed the presence of phenolics, flavonoids, steroids, terpenoids, saponins, and tannins. The results of GC/MS analysis showed the presence of compounds with antioxidant activity. F. moluccana methanolic extract had a very strong antioxidant activity with an IC 50 value was 12.6 ppm. F. moluccana methanolic extract has the potential as a source of bioactive antioxidant compounds.