Phytochemical investigation and antioxidant activity of ethanolic extract, n-hexane and ethyl acetate fraction of salaon leaves (Indigofera tinctoria Linn.)

Plants known as salaon (Indigofera tinctoria L.) are commonly employed in conventional medicine. Salaon leaves have secondary metabolites sunch as alkaloid, flavonoid, tannin, glycoside, phenol, saponin and triterpenoid levels have benefit as antioxidants in counteracting free radicals. The aimed of this research was to investigate the Antioxidant properties of The EESL, NHFSL and EAFSL. Phytochemical investigation process was used on extracts and fractions. Antioxidant properties was determined using the DPPH technique and a UV/Vis spectrophotometer at 515 nm. The water level of salaon leaf simplicia was around 9.32%; water-soluble extract of 34.70%, ethanol-soluble extract of 23.94%, total ash of 8.57% and acid insoluble ash of 1.38%. Alkaloids, flavonoids, glycosides, tannins, saponins, steroids, and triterpenoids were found in the phytochemical investigation of EESL. The EESL, NHFSL and EAFSL which were tested for antioxidant properties revealed IC50 values of (70.5046 ± 0.0409; 77.2190 ± 0.0021; 56.5593 ± 0.0120) µg/mL. IC50 value of quercetin in the extremely strong category is 2.5424± 0.0010 µg/mL. The results of this investigation indicate that salaon leaf ethanol extract, n-hexane, and ethyl acetate fraction have potent antioxidant properties.


Introduction
Antioxidants are the body's initial defense against the impacts of free radicals.Antioxidants are compounds that can stabilize free radicals.Antioxidants are divided into endogenous and exogenous antioxidants [2].When the accumulation of endogenous antioxidants is insufficient to counteract free radicals, it will cause a condition called oxidative stress which causes cell damage and over time leads to degenerative diseases such as cancer and atherosclerosis.Therefore, in an effort to prevent the adverse effects of free radicals, antioxidant intake is needed from outside the body (exogenous) [3].Exogenous antioxidants can be natural antioxidants as well as synthetic antioxidants.However, the use of synthetic antioxidants has begun to be limited because several studies have shown that these antioxidants are toxic and carcinogenic.In addition, synthetic antioxidants are also sold at high prices, even though the antioxidant components are found in nature in abundance such as in plants [4].Medicinal plants are very potential plants to obtain new sources of antioxidants [5].
One that is quite widely used to treat various diseases is plants of the Indigofera genus.Based on several studies, plants of this genus have pharmacological activities such as anthelmintic, anticancer, antidiabetic, antidiarrheal, antibacterial, antioxidant, anti-inflammatory, antimicrobial and others.One of the plant species of this genus is Indigofera tinctoria L. [6].Salaon plant (Indigofera tinctoria L.), which is widely found in North Tapanuli Regency, North Sumatra Province, is a plant known by the common name nila or tarum.Salaon can grow easily in nature and is widely found in Indonesia.In addition, salaon is also used in traditional medicine such as curing apoplexy, nervous disorders, diarrhea, wounds and ulcers [7].In India and ChinaTraditional medicine also makes extensive use of Indigofera tinctoria L., namely for treating tooth and gum discomfort, mouth ulcers, hair rejuvenation, dog bites, and other skin-related conditions.Based on phytochemical investigation was conducted by Mishra et al. (2018), the ethanol extract of the leaves contains alkaloids, flavonoids, tannins, phenols, saponins, glycosides and triterpenoids, some of which are known to have antioxidant activity [8].
One of the methods used in testing antioxidant properties is the DPPH (1,1-diphenyl-2picrylhydrazyl) method.The DPPH method is a fast, simple, accurate method, does not require many reagents and is suitable for testing antioxidant activity of either foodstuffs or plant extracts.The parameter to measure antioxidant activity is determined by the concentration of antioxidants that give a % inhibition of 50% against DPPH [9].Previous research explained that ethanol extracts and water extracts of salaon (I.tinctoria) leaves provide moderate antioxidant properties with IC50 values of 182.77 µg/mL and 204.66 µg/mL compared to vitamin C with IC50 values of 6.01 µg/mL [10].The sustainable development goals of this research number 3 were might to contribute improvement healthy live by developing natural compounds as antioxidants that can prevent the emergence of various diseases.However, the research is still limited to extracts so that based on the description above, fractionation is carried out by liquid search extraction method using solvents with different levels of polarity, namely n-hexane solvent which is a non-polar solvent and ethyl acetate as a semipolar solvent.Fractionation is used to extract more pure components from the ethanol extract of salaon leaves and separate the compounds that have a lower concentration of active ingredients for purposes of isolation.

Research site and materials
This research uses the DPPH technique to experimentally assess the antioxidant properties of the salaon leaves ethanol extract, n-hexane fraction, and ethyl acetate fraction.This research includes the stages of collecting and processing salaon leaves, making simplicia, examining the characteristics of simplicia, making ethanol extracts and fractions of salaon leaves, phytochemical screening and testing antioxidant activity with the DPPH (1,1-diphenyl-2-picrylhydrazil) method using a UV-Visible spectrophotometer.This study was conducted at the Phytochemistry Laboratory and Research Laboratory of the Faculty of Pharmacy, University of North Sumatera.

Simplicia preparation
The leaves of the salaon plant obtained from Desa Lontung, Kecamatan Muara, North Tapanuli Regency, North Sumatera Province were wet sorted, then cleaned from dirt attached with running water, then drained and weighed so that the wet weight was obtained, then put into the drying cabinet with a temperature of ± 40 o C. Salaon leaves are considered dry when they are brittle.The dried leaves were sorted again.Furthermore, the simplicia is pollinated using a blender and stored in a plastic container that is tightly closed with silica gel and protected from sunlight [11].

Ethanolic extract and fraction preparation
500 grams of powdered salaon leaf were extracted with 3750 milliliters of 96% ethanol (1: 7.5) then placed in a tight container.After that, it was covered with aluminum foil, kept out of the sun, and constantly stirred for five days.To create a filtrate 1, the macerate was filtered via filter paper.After being dried, the residue was extracted once again using 1250 mL of 96% ethanol for five days while being shielded from light and constantly mixed.After filtering the macerate, filtrate 2 was produced.Next, To create thick extracts, filtrates 1 and 2 were collected, evaporated at 40° C in a rotary evaporator, and then dried in a water bath (ethanolic extract of salaon leaves; EESL) [12].
Fractions were prepared by liquid-liquid extraction using n-hexane and ethyl acetate solvents.A total of 10 g of EESL was dissolved with ethanol little by little and added distilled water up to 50 mL, then put into a separatory funnel.Then added 50 mL of n-hexane, shaken and allowed to stand until 2 layers were obtained, n-hexane (top layer) was separated.Fractionation was conducted until the color of the n-hexane layer was clear and didn't give positive result of the Liebermann-Bouchard reagent (NHFSL).The remaining (lower layer) was added 50 mL of ethyl acetate, shaken and then allowed to stand until there were 2 layers, the ethyl acetate layer (lower layer) was separated and fractionation was conducted until the color of the ethyl acetate layer was clear.The fractions obtained were evaporated until a thick extract was obtained (EAFSL).[12] 2.4.Simplicia characterization Simplicia, which may be classified into five parameters: Finding the water level, total ash level, insoluble acid total ash level, water soluble extract level, and ethanol soluble extract level [14,15].

Phytochemicals screening
By using phytochemical screening, each extract and fraction is examined.Several established techniques, including those involving flavonoids, alkaloids, tannins, saponins, and steroids/triterpenoids, are used to conduct the test.The findings show which kinds of chemicals are present or absent in the extract and fractions, denoted by the symbols + or -. [14,15].

Preparing the sample solution's raw standard
Methanol was used to dissolve 10 mg of EESL, 10 mg of NHFSL, and 10 mg of EAFSL.The resulting mixture was then poured to each 10 mL volumetric flask until the mark was reached (concentration = 1000 μg/mL).
Methanol was used to dissolve 5 mg of quercetin powder, which was then added to a volumetric flask (5 mL) along with methanol (concentration = 1000 μg/mL) to mark the line.Subsequently, the solution (concentration = 1000 μg/mL (LIB I)) was pitted 1 mL, placed in a 10-mL volumetric flask, and methanol (concentration = 100 μg/mL (LIB II)) was added to the mark line [14,15].

Preparation of ethanol extract of salaon leaves (EESL) and Ethyl Acetate Fraction Salaon
Leaves (EAFSL) Solutions.The solutions of raw standard of EESL and EAFSL were pipetted as much as 0.10; 0.20; 0.30; 0.40 mL and transferred into a 5 mL of volumetric flask, then into each volumetric flask was added 1 mL of DPPH solution (concentration = 200 μg/mL and then methanol was then added to the volume to the mark line, homogenized, and left in the dark for 26 minutes.A UV-Visible spectrophotometer operating at a wavelength of 515 nm was used to measure the absorbance.[16].

Preparation of n-hexane fraction salaon leaves (NHFSL) Solutions.
The solutions of raw standard of NHFSL were pipetted as much as 0.15; 30; 0.45 and 0.60 mL, and transferred into a 5 mL of volumetric flask (concentration = 30, 60, 90 and 120 μg/mL.Subsequently, 1 milliliter of DPPH solution (concentration = 200 μg/mL) was added to each volumetric flask.The volume was then filled with methanol to the mark line, homogenized, and left in the dark for 26 minutes.The absorbance was measured with a UV-Visible spectrophotometer at a wavelength of 515 nm [16].

Determination of percent reduction (percent inhibition)
The test solution's antioxidant activity was measured as the amount that the addition of the test solution reduced the absorbance of the DPPH solution (DPPH purple color attenuation).The percentage of inhibition was calculated using the absorbance value of the DPPH solution both before and after the test solution was added [16].

Determination of IC50 value
The IC50 is a value that represents the concentration of the test sample (g/mL) that inhibits DPPH by 50% (capable of reducing DPPH oxidation by 50%).The computed results are entered into a regression equation where the ordinate (Y) is the % inhibition (antioxidant) value and the abscissa (X) is the test solution concentration [16].The lower the IC50 value of a sample, the greater the ability as an antioxidant.The formula for determining the IC50 is as follows [16]: Description: Y= % value of DPPH silencing x = concentration (μg/mL) or IC value50 a = slope b = intercept / regression coefficient

Simplicia characterization
The water level, ethanol-soluble extract, water-soluble extract, total ash, and acid insoluble ash of salaon leaves were all determined.The result of characterization analysis of salaon leaves showed on bellow Table 1.
Table 1.The characterization of salaon leaves simplicial.
Acid insoluble ash level 1.38

Phytochemical investigation result
EESL, NHFSL and EAFSL were subjected to phytochemical investigation to obtain an overview of the chemical level contained in the samples.Table 2 shows the findings of phytochemical screening.
Based on the Table 2 The phytochemical investigation test revealed that alkaloids, flavonoids, tannins, saponins, glycosides, and steroids were present in the simplicia and EESL; the NHFSL contained alkaloids, flavonoids, and steroids; and the EAFSL contained alkaloids, flavonoids, glycosides, and tannins.These phytochemical substances are known to promote bioactivity and are thus responsible for antioxidant activity [15,16,17].The antioxidant activity of salaon leaves ethanol extracts and fractions was determined by measuring DPPH absorbance at the 26th minute with the addition of ethanol extract test solution and comparing each fraction to DPPH control (without the addition of test solution).The antioxidant properties examination of EESL and it fractions, as well as quercetin, revealed a decrease in DPPH absorbance when the test solution concentration was increased.From the Table 3 shows the results of antioxidant properties testing as measured by the decrease in absorbance and the inhibition percentage.Based on the Table 3 that the calculation of the IC50 it was found that the EAFSL provided better antioxidant activity than the EESL and NHFSL.This is probably because most of the secondary metabolites that act as antioxidants are more semi-polar so that more is extracted in ethyl acetate solvent [18].Flavonoids are antioxidants because they contain hydroxyl groups that may release protons as hydrogen ions.Because hydrogen ions have one proton and no electrons, they can link to nitrogen atoms in DPPH molecules to form reduced DPPH [19].This is characterized by a change in solution from dark purple to pale yellow and its absorbance at the maximum wavelength decreases as the concentration increases [16].The main flavonoid-derived compound components found in salaon are flavonoid aglycones (apigenin and luteolin) and quercetin, where these three compounds include flavonoids with low polarity.The antioxidant activity of EESL, NHFSL, EAFSL which is classified as strong in this study, is expected to be developed and has the potential as a new natural antioxidant to meet antioxidant needs in the body, which can stabilize free radicals and prevent stress oxidative stress, which causes degenerative diseases, and reduces the use of synthetic antioxidants [6,20,21].

Conclusions and suggestion
The results of this investigation indicate that EESL, NHFSL and EAFSL have potent antioxidant properties.The novelty of research on salaon leaves is still limited in Indonesia and still needs to be developed.The suggestion in this study to continue testing as antidiabetic and anticholesterol activity

Table 2 .
Result of phytochemical investigation.Antioxidant activity test was conducted by DPPH method using UV/Vis spectrophotometer.Based on the determination of the maximum absorption wavelength, the result is 515 nm.The level of antioxidant properties is defined by the IC50, which is the sample concentration that offers 50% inhibition against DPPH free radicals.

Table 3 .
Antioxidant activity test salaon leaves extract and its fractions.