Phytochemical and Antioxidant Test of Binahong (Anredera cordifolia (Tenore) Steenis) Leaves Ethanol Extract

Diterima 06 Maret 2021 Disetujui 26 April 2021 Binahong Anredera cordifolia (Tenore) Steenis leaves contain chemical compounds that can be used as antioxidants. This study aims to examine the phytochemical and antioxidant activity of the ethanol extract of binahong leaves. Binahong leaves were extracted using the maceration method with an ethanol solvent. The phytochemical test showed that the ethanol extract of binahong leaves contained phenolic compounds, flavonoids, alkaloids, and tannins to have potential antioxidants. The antioxidant activity test was carried out using the DPPH method and absorbance measurement with a UV-Vis spectrophotometer at a wavelength of 517 nm. The results showed that the ethanol extract of binahong leaves had strong antioxidant activity, as evidenced by the IC50 value of 87.423 μg/mL. Key word: Antioxidant Phytochemical Binahong Anredera cordifolia DPPH Kata kunci: Antioksidan Fitokimia Binahong Anredera cordifolia DPPH *fensia@fmipa.unpatti.ac.id Introduction Binahong is cultivated as herbal medicine, planted in pots, in the yard, or garden. Binahong leaves are often used as a traditional medicine to heal burns, rheumatism, gout, lack of appetite, nosebleeds, inflammation of the kidneys, colitis, and cancer. However, the binahong plant's benefits have not been widely known in Indonesian society [1]. Binahong leaves contain saponins, tannins, flavonoids, alkaloids, and polyphenols with antioxidant properties [2]. Antioxidants have an important role to play in maintaining health. It is due to the ability of antioxidants to scavenge free radicals. Free radicals are highly reactive because they have one or more unpaired electrons. Reactive oxygen compounds are produced continuously in the human body due to normal metabolic processes [3]. Synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have side effects on our health [4]. The side effect has spurred researchers to explore natural materials as a source of antioxidants. Generally, natural antioxidants are phenolic compounds scattered throughout the plant, both in the roots, stems, leaves, seeds, fruits, and flowers. Natural antioxidants from plants are polyphenols (phenolic acids, flavonoids, anthocyanins, lignans, and stilbene), carotenoids (xanthophyll and carotene), and vitamins (vitamins E and C) [5]. Several natural-based antioxidants have been studied. The methanol extract of banyan fruit has an IC50 of 40.36 μg/mL against DPPH [6]. The IC50 of the methanol extract of Kesumba Keling seeds was 69.425 ppm [7]. Ethanolic extract of binahong possesses total antioxidants of 4.25 mmol/100 g (fresh) and 3.68 mmol/100 g (dry) [3]. Binahong leaf ethyl acetate extract had an IC50 68.07 μg/mL [8]. EC50 of binahong tuber flavonoid extract was 178.60 mg/L, and 70% ethanol extract was 298.10 mg/L [9]. This study aimed to determine the chemical content and antioxidant power of binahong leaf ethanol extract. Binahong leaf Souhoka,F.A., Kapelle,I. B. D., Sihasale, E., 2021 29 extract was obtained by maceration using ethanol. The antioxidant activity test was carried out in-vitro with the DPPH (1,1diphenyl-2-picrylhydrazil) method. The DPPH method is often used for testing the antioxidant activity of several natural compounds because this method is relatively easy and simple compared to other methods [10]. Materials and Methods Materials The materials used in the study were binahong (Anredera cordifolia (Tenore) Steenis) leaves, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 96% of ethanol, FeCl3, HCl, Mg powder, distilled water, Mayer's reagent, and quercetin. All the chemicals were purchased from Merck with pro analysis grade. Sample preparation Binahong leaves were taken, washed, sliced into small pieces, dried in an oven at 105 C for 5 hours until a constant weight was calculated for the moisture content. The dry sample was mashed and sieved until a powder was obtained. Procedure Sample Extraction The 10 g of binahong leaf powder was put into an Erlenmeyer and added 200 mL of 96% ethanol until the sample was immersed and then macerated for 24 hours. The filtrate is filtered and evaporated using a rotary evaporator. The binahong leaf ethanol extract obtained was weighed and performed a phytochemical test. Phytochemical test The phytochemical test is a preliminary test for the ethanol extract of binahong leaves, including phenolic, flavonoid, tannin, and alkaloid tests. Preparation of test solutions The ethanol extract of binahong leaves as much as 0.5 g was dissolved in ethanol 96% in a 10 mL volumetric flask. Phenolic test The 2 mL sample extract was put into a test tube added 5 mL of ethanol, and two drops of 2% FeCl3 were added (positive if it produces a dark blue color) [11]. Flavonoid test A total of 2 mL of binahong leaf extract was added with 1 mL of 1% HCl into a test tube, then added a little Mg powder (positive if a yellow color is formed in the presence of flavonoids) [12]. Tannin test Ethanol extract 2 mL was dissolved in 1 mL of distilled water and brought to a boil. Then filtered and the filtrate is added 2 drops of FeCl3 1% and shaken (positive if it produces a brown color) [13]. Alkaloid test 2 mL of the sample extract was added with 5 mL of ethanol. Then added with Mayer's reagent drop by drop (positive for pink color) [14]. Preparation of 40 ppm DPPH solution A total of 0.01 g of DPPH is put into a 250 mL measuring flask, then ethanol is added to the limit mark. The solution is used immediately and is kept at a low temperature and protected from light [8]. Determination of the maximum DPPH wavelength A total of 5 mL of DPPH solution were observed for their absorption in the wavelength range of 400–600 nm using ethanol blanks. Determination of DPPH free radical scavenging activity. A total of 2 mg of ethanol extract of binahong leaf was made into a solution with 10, 20, 30, 40, 50, 60, 70, and 80 ppm. Quercetin comparison solution was made with a concentration of 2.5; 5.0; 10; 15; 20; and 25 ppm. Each test solution of 1 mL was put into a test tube, and 2 mL of 40 ppm DPPH solution were added, then left to stand for 30 minutes at room temperature. The absorbance measurement was carried out at a wavelength of 517 nm using a UV-Vis spectrophotometer [15]. The test was carried out with twice measurements. Furthermore, the IC50 value is calculated based Souhoka,F.A., Kapelle,I. B. D., Sihasale, E., 2021 30 on the regression equation obtained. Results and Discussions Sample Preparation The moisture content of the binahong leaf samples was 54.805%. The high and low water content will affect the maceration process. The lower the water content, the easier it is to withdraw the sample's active substance because the solvent easily penetrates the sample cell wall without interference from water molecules. Sample Extraction Binahong leaf antioxidant test begins with preparing a sample extract with 96% ethanol using the maceration method. The extraction results obtained dark green extract. The extract was filtered, then the filtrate was evaporated using a rotary evaporator to remove the solvent to obtain a thick extract weighing 0.94 g (1.063%). The amount of the active compound content of a sample is related to the yield value. If the yield obtained is large, the active compound content will also increase. The high bioactive compounds in a sample are indicated by the high yield value produced. Phytochemical Test Phenolic test A positive result is indicated in phenol testing by forming a dark blue color when reacted with 1% FeCl3. Binahong leaf ethanol extract showed positive results indicated by a change in color from yellow to dark blue. The test results showed that the ethanol extract of binahong leaves contained phenolic compounds. The hydroxyl groups in phenolic compounds can react with FeCl3 to form dark blue complexes [11]. Flavonoid test To determine the presence or absence of flavonoid compounds presence in the ethanol extract of binahong leaves, testing was carried out using the Shinoda test, namely by reacting the extract with 1% HCl powder and Mg powder. The formation of yellow color indicates a positive test [13]. The test results showed that the ethanol extract of binahong leaves contained flavonoids. Tannin test In the tannin test, the ethanol extract of binahong leaves reacted with 1% FeCl3 showed positive results, marked by a change in color from green to brown and frothy. The test results showed that the ethanol extract of binahong leaves contained tannin compounds. Tannins will form complex compounds with the Fe3+ ion as the central atom. Tannins contain O atoms which have lone pairs that can coordinate with the central atom. The Fe3+ ion in the above reaction binds three tannins with two donor atoms, namely the O atoms in the 4ʼ and 5ʼ dihydroxy positions, so that six lone pairs can be coordinated to the central atom. Atoms O at 4ʼ and 5ʼ dihydroxy positions have the lowest energy in forming complex compounds [16]. Alkaloid test Testing for the presence of alkaloid compounds in a sample was carried out using Mayer's reagent. The ethanol extract of binahong leaves showed positive results, indicated by a color change from white to pink. Mayer's reagent contains potassium iodide and mercuric chloride, producing alkaloid potassium, a white precipitate. The white precipitate is thought to be a complex alkaloid potassium compound. Potassium iodide, which is added excess, will form potassium tetraiodomercurate (II) [17]. The test results showed that the ethanol extract of binahong leaves contained alkaloid compounds. Determination of the maximum DDPH wavelength Determination of the maximum wavelength of DDPH is carried out to determine the wavelength required by the DPPH solution to achieve maximum absorption. The determination of wavelength was carried out using 96% ethanol blank. The maximum absorbance obtained was 0.736 in the 517 nm wavelength range. The wavelength corresponds to the maximum wavelength of the DPPH, which is 400–600 nm. Furthermore, the maximum wavelength of DPPH was used in determining the antioxidant activity of binahong leaf ethanol extract. Souhoka,F.A., Kapelle,I. B. D., Sihasale, E., 2021 31 Determination of DPPH Free Radical Counter Activity Calibration curve Before determining the activity of the DPPH free radical scavenger of binahong leaf ethanol extract, a calibration curve was made to test the linearity of the sample. Based on the calculation results, the data presented in Table 1 and Figure 1 are obtained. Table 1. The activity of DPPH free radical scavenger of quercetin No Quercetin concentration (ppm) Absorbance % Inhibition 1 2.5 0.451 27.84 2 5 0.342 45.28 3 10 0.256 59.04 4 15 0.122 80.48 5 20 0.025 96.00 6 25 DPPH + Ethanol 0.020 0.625 96.80 Figure 1. Standard curve of quercetin DPPH free radical scavenging activity Determination of DPPH free radical scavenging activity was expressed by IC50 value using quercetin control. The measurements using UV-Vis spectroscopy at a wavelength of 517 nm obtained each concentration's absorbance value. The absorbance value was used to calculate % inhibition and IC50 quercetin. Based on Table 1 and Figure 1, it can be seen that the increase in quercetin concentration means the higher the ability to ward off DPPH free radicals. From the calculation, the IC50 quercetin value was 46.88 μg/mL. Results of determining DPPH free radical scavenging activity The antioxidant activity of binahong leaf ethanol extract was tested using the DPPH method. DPPH radical is an organic compound containing unstable nitrogen with strong absorbance at a wavelength of 517 nm. The presence of antioxidant activity from the sample results in a color change from purple to yellow. This change occurs when DPPH free radicals are captured by antioxidants and release hydrogen atoms to capture stable DPPH-H. Qualitatively, the ethanol extract of binahong leaves contains phenolic compounds, flavonoids, alkaloids, and tannins which can donate electrons to ward off free radicals. The quantitative antioxidant activity test was carried out by measuring the free radical scavenger of a compound with an antioxidant activity using UV-Vis spectrophotometry. Thus will be known the activity of reducing free radicals is expressed by the value of IC50. The IC50 value is defined as the test compound's concentration that can reduce free radicals by 50%. The smaller the IC50 value, the higher the free radical scavenging activity. Specifically, a compound is a powerful antioxidant if the IC50 value is <50 ppm, 50-100 ppm strong, 100-150 ppm medium, 150-200 ppm weak, and very weak >200 ppm. Binahong leaf ethanol extract showed an increase in % inhibition per concentration. The percentage of inhibition was obtained from the difference in the absorbance of the control and the sample's absorbance. The activity of the DPPH free radical scavenger of binahong leaf ethanol extract is presented in Table 2. Table 2. The activity of DPPH free radical scavenger of binahong leaf ethanol extract No Sampel Concentration (ppm) Average Absorbance % Inhibition 1 10 0.615 1.75 2 20 0.606 3.27 3 30 0.588 6.06 4 40 0.574 8.38 5 50 0.562 10.29 6 60 0.530 15.20 7 70 0.487 23.52 8 80 0.371 40.78 y = 3.1474x + 26.919 R2 = 0.9469


Introduction
Binahong is cultivated as herbal medicine, planted in pots, in the yard, or garden. Binahong leaves are often used as a traditional medicine to heal burns, rheumatism, gout, lack of appetite, nosebleeds, inflammation of the kidneys, colitis, and cancer. However, the binahong plant's benefits have not been widely known in Indonesian society [1]. Binahong leaves contain saponins, tannins, flavonoids, alkaloids, and polyphenols with antioxidant properties [2].
Antioxidants have an important role to play in maintaining health. It is due to the ability of antioxidants to scavenge free radicals. Free radicals are highly reactive because they have one or more unpaired electrons. Reactive oxygen compounds are produced continuously in the human body due to normal metabolic processes [3].
Synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have side effects on our health [4]. The side effect has spurred researchers to explore natural materials as a source of antioxidants. Generally, natural antioxidants are phenolic compounds scattered throughout the plant, both in the roots, stems, leaves, seeds, fruits, and flowers. Natural antioxidants from plants are polyphenols (phenolic acids, flavonoids, anthocyanins, lignans, and stilbene), carotenoids (xanthophyll and carotene), and vitamins (vitamins E and C) [5].
This study aimed to determine the chemical content and antioxidant power of binahong leaf ethanol extract. Binahong leaf extract was obtained by maceration using ethanol. The antioxidant activity test was carried out in-vitro with the DPPH (1,1diphenyl-2-picrylhydrazil) method. The DPPH method is often used for testing the antioxidant activity of several natural compounds because this method is relatively easy and simple compared to other methods [10].

Sample preparation
Binahong leaves were taken, washed, sliced into small pieces, dried in an oven at 105 C for 5 hours until a constant weight was calculated for the moisture content. The dry sample was mashed and sieved until a powder was obtained.

Procedure Sample Extraction
The 10 g of binahong leaf powder was put into an Erlenmeyer and added 200 mL of 96% ethanol until the sample was immersed and then macerated for 24 hours. The filtrate is filtered and evaporated using a rotary evaporator. The binahong leaf ethanol extract obtained was weighed and performed a phytochemical test.

Phytochemical test
The phytochemical test is a preliminary test for the ethanol extract of binahong leaves, including phenolic, flavonoid, tannin, and alkaloid tests.

Preparation of test solutions
The ethanol extract of binahong leaves as much as 0.5 g was dissolved in ethanol 96% in a 10 mL volumetric flask.

Phenolic test
The 2 mL sample extract was put into a test tube added 5 mL of ethanol, and two drops of 2% FeCl3 were added (positive if it produces a dark blue color) [11].

Flavonoid test
A total of 2 mL of binahong leaf extract was added with 1 mL of 1% HCl into a test tube, then added a little Mg powder (positive if a yellow color is formed in the presence of flavonoids) [12].

Tannin test
Ethanol extract 2 mL was dissolved in 1 mL of distilled water and brought to a boil. Then filtered and the filtrate is added 2 drops of FeCl3 1% and shaken (positive if it produces a brown color) [13].

Alkaloid test
2 mL of the sample extract was added with 5 mL of ethanol. Then added with Mayer's reagent drop by drop (positive for pink color) [14].

Preparation of 40 ppm DPPH solution
A total of 0.01 g of DPPH is put into a 250 mL measuring flask, then ethanol is added to the limit mark. The solution is used immediately and is kept at a low temperature and protected from light [8].

Determination of the maximum DPPH wavelength
A total of 5 mL of DPPH solution were observed for their absorption in the wavelength range of 400-600 nm using ethanol blanks.

Determination of DPPH free radical scavenging activity.
A total of 2 mg of ethanol extract of binahong leaf was made into a solution with 10, 20, 30, 40, 50, 60, 70, and 80 ppm. Quercetin comparison solution was made with a concentration of 2.5; 5.0; 10; 15; 20; and 25 ppm. Each test solution of 1 mL was put into a test tube, and 2 mL of 40 ppm DPPH solution were added, then left to stand for 30 minutes at room temperature. The absorbance measurement was carried out at a wavelength of 517 nm using a UV-Vis spectrophotometer [15]. The test was carried out with twice measurements. Furthermore, the IC50 value is calculated based on the regression equation obtained.

Results and Discussions Sample Preparation
The moisture content of the binahong leaf samples was 54.805%. The high and low water content will affect the maceration process. The lower the water content, the easier it is to withdraw the sample's active substance because the solvent easily penetrates the sample cell wall without interference from water molecules.

Sample Extraction
Binahong leaf antioxidant test begins with preparing a sample extract with 96% ethanol using the maceration method. The extraction results obtained dark green extract. The extract was filtered, then the filtrate was evaporated using a rotary evaporator to remove the solvent to obtain a thick extract weighing 0.94 g (1.063%). The amount of the active compound content of a sample is related to the yield value. If the yield obtained is large, the active compound content will also increase. The high bioactive compounds in a sample are indicated by the high yield value produced.

Phytochemical Test Phenolic test
A positive result is indicated in phenol testing by forming a dark blue color when reacted with 1% FeCl3. Binahong leaf ethanol extract showed positive results indicated by a change in color from yellow to dark blue. The test results showed that the ethanol extract of binahong leaves contained phenolic compounds. The hydroxyl groups in phenolic compounds can react with FeCl3 to form dark blue complexes [11].

Flavonoid test
To determine the presence or absence of flavonoid compounds presence in the ethanol extract of binahong leaves, testing was carried out using the Shinoda test, namely by reacting the extract with 1% HCl powder and Mg powder. The formation of yellow color indicates a positive test [13]. The test results showed that the ethanol extract of binahong leaves contained flavonoids.

Tannin test
In the tannin test, the ethanol extract of binahong leaves reacted with 1% FeCl3 showed positive results, marked by a change in color from green to brown and frothy. The test results showed that the ethanol extract of binahong leaves contained tannin compounds. Tannins will form complex compounds with the Fe 3+ ion as the central atom. Tannins contain O atoms which have lone pairs that can coordinate with the central atom. The Fe 3+ ion in the above reaction binds three tannins with two donor atoms, namely the O atoms in the 4ʼ and 5ʼ dihydroxy positions, so that six lone pairs can be coordinated to the central atom. Atoms O at 4ʼ and 5ʼ dihydroxy positions have the lowest energy in forming complex compounds [16].

Alkaloid test
Testing for the presence of alkaloid compounds in a sample was carried out using Mayer's reagent. The ethanol extract of binahong leaves showed positive results, indicated by a color change from white to pink. Mayer's reagent contains potassium iodide and mercuric chloride, producing alkaloid potassium, a white precipitate. The white precipitate is thought to be a complex alkaloid potassium compound. Potassium iodide, which is added excess, will form potassium tetraiodomercurate (II) [17]. The test results showed that the ethanol extract of binahong leaves contained alkaloid compounds.

Determination of the maximum DDPH wavelength
Determination of the maximum wavelength of DDPH is carried out to determine the wavelength required by the DPPH solution to achieve maximum absorption. The determination of wavelength was carried out using 96% ethanol blank. The maximum absorbance obtained was 0.736 in the 517 nm wavelength range. The wavelength corresponds to the maximum wavelength of the DPPH, which is 400-600 nm. Furthermore, the maximum wavelength of DPPH was used in determining the antioxidant activity of binahong leaf ethanol extract.

Determination of DPPH Free Radical Counter Activity Calibration curve
Before determining the activity of the DPPH free radical scavenger of binahong leaf ethanol extract, a calibration curve was made to test the linearity of the sample. Based on the calculation results, the data presented in Table 1 and Figure 1 are obtained.  Determination of DPPH free radical scavenging activity was expressed by IC50 value using quercetin control. The measurements using UV-Vis spectroscopy at a wavelength of 517 nm obtained each concentration's absorbance value. The absorbance value was used to calculate % inhibition and IC50 quercetin. Based on Table 1 and Figure 1, it can be seen that the increase in quercetin concentration means the higher the ability to ward off DPPH free radicals. From the calculation, the IC50 quercetin value was 46.88 µg/mL.

Results of determining DPPH free radical scavenging activity
The antioxidant activity of binahong leaf ethanol extract was tested using the DPPH method. DPPH radical is an organic compound containing unstable nitrogen with strong absorbance at a wavelength of 517 nm. The presence of antioxidant activity from the sample results in a color change from purple to yellow. This change occurs when DPPH free radicals are captured by antioxidants and release hydrogen atoms to capture stable DPPH-H. Qualitatively, the ethanol extract of binahong leaves contains phenolic compounds, flavonoids, alkaloids, and tannins which can donate electrons to ward off free radicals.
The quantitative antioxidant activity test was carried out by measuring the free radical scavenger of a compound with an antioxidant activity using UV-Vis spectrophotometry. Thus will be known the activity of reducing free radicals is expressed by the value of IC50. The IC50 value is defined as the test compound's concentration that can reduce free radicals by 50%. The smaller the IC50 value, the higher the free radical scavenging activity. Specifically, a compound is a powerful antioxidant if the IC50 value is <50 ppm, 50-100 ppm strong, 100-150 ppm medium, 150-200 ppm weak, and very weak >200 ppm.
Binahong leaf ethanol extract showed an increase in % inhibition per concentration. The percentage of inhibition was obtained from the difference in the absorbance of the control and the sample's absorbance. The activity of the DPPH free radical scavenger of binahong leaf ethanol extract is presented in Table 2.  Based on Figure 2, the regression equation Y=0.4807x-7.9754 and the IC50 value is 87.423 µg/mL, which is classified as a strong antioxidant. Compared with the results of a study regarding the antioxidant activity test of the ether fraction from the hydrolysis of binahong leaf infusion, an IC50 was obtained of 249.31 ppm, which means that the antioxidants are fragile [18]. Meanwhile, the antioxidant activity test of binahong leaf ethanol extract with the maceration method obtained an IC50 of 40.27 ppm, which is classified as a powerful antioxidant [14].

Conclusions
Based on the research results, it can be concluded that the phytochemical test showed that the ethanol extract of binahong leaves contained phenolic compounds, flavonoids, alkaloids, and tannins. The ethanol extract of binahong leaves has a strong antioxidant activity with an IC50 value of 87.423 µg/mL.