Analysis of Phytochemical Content and Antioxidant Activity in Phyllanthus Niruri Linn. by Different Methods

The main materials used in this study were distilled water (aquadest), filler bags, and all parts of Phyllanthus niruri Linn. which was harvested in August 2020 in Ciawi, Tasikmalaya, West Java, Indonesia. The results of research by Septhi proved that all parts of plant Phyllanthus niruri Linn. that can be used are leaves, stems, flowers, fruits, and roots wich are generally called Phyllanthus niruri Linn. herb [11].

Phyllanthus niruri Linn. is an annual herbaceous plant, grows upright with a stem height of 30 – 50 cm. Phyllanthus niruri Linn. has a top root system, roots are deep and strong, the roots are yellowish white with few root branches. Stem of Phyllanthus niruri Linn, round, not gummy, with a hard stem base and woody, and forming branches. On the stems the leaves grow opposite and in the axils of the leaves grow shoots that form branches. Phyllanthus niruri Linn. has small ovoid leaves with 5 mm long and 3 mm wide. Each stalk consists pf compound leaves, opposite leaf arrangement, pinnate leaf bones, flat, and smooth leaf edges. Phyllanthus niruri Linn. has small flowers, greenish white, short flowers stalks, with a size of 2 mm. stamens and pistils sticking out. Phyllanthus niruri Linn. fruits flat round shape, 2 – 2,5 mm diameter and slippery texture. The seeds are kidney-shaped, hard, and brown [12].

In addition, the chemicals used to analyze the antioxidant activity and phytochemical content were brewing water from Phyllanthus niruri Linn., FeCl₃, HCl, H₂SO₄, NaOH, CH₃COOH, and 2, 2-diphenyl-1-picrylhydrazil. Furthermore, the tools utilized in this study were tools for brewing Phyllanthus niruri Linn., tools for solutes, and the separation of the brewing product with solid material. This is strongly influenced by temperature and brewing time (water contact with the herbs) [13]. During brewing, all volatile and non-volatile compounds are extracted and dissolved in water and will affect the antioxidant activity of the herbal water.

Previous studies showed that the method by brewing the herbs with boiling water for 5 minutes and then being cooled for 30 seconds (decoction brew method) and the method by brewing the herbs with distilled water and then being stored in the refrigerator at 0 – 5℃ for 8 hours (cold brew method) can provide significant results, in which the total obtained flavonoids and phenolics are higher than other methods and temperatures applied to green coffee [14]. The results of another study regarding the brewing method reported that the decoction of brewing water using the decoction brew, cold brew, and true brew methods produced significantly different amounts of phenolic compounds [15]. Therefore, this study aimed to examine the effect of the decoction brew, cold brew, and true brew methods with modification in the use of filters and non-filters on the phytochemical content and antioxidant activity of Phyllanthus niruri Linn. analyzing antioxidant activity, storage racks, volumetric flasks, vortices, cuvettes, test tubes, test tube racks, pipettes, and vials.

2.1 Sample preparation

The preparation process starts with cleaning, dryng, and grinding [16]. All parts of Phyllanthus niruri Linn. were rinsed and sorted to remove dirt and damaged parts. All parts of the plant were then dried at room temperature (80,6 – 84,2^°F) without sunlight and then crushed to a size of 0.5 mesh [17].

2.2 Brewing methods

For the brewing methods as the independent variable, the researchers applied modified methods developed by Muller, namely Decoction Brew (DB), Cold Brew (CB), and True Brew (TB) methods with the utilization of filter (F) and non-filter (NF). The method developed by Muller adequately describes the brewing method commonly used in the community. These methods are also different in each step, temperature, and processing time, so it will be expected to provide a different picture of the total yield of phytochemical compounds and their antioxidant activity [15]. The Decoction Brew (DB) method was conducted by preparing 1.25 g of mashed Phyllanthus niruri Linn. added with 100 ml of distilled water. After that, it was heated to boiling for 5 minutes, and then being stirred for 10 seconds every 1 minute. The Cold Brew (CB) method was carried out by pouring 100 ml of room temperature distilled water into 1.25 g of finely dried Phyllanthus niruri Linn. then stored in the refrigerator for 8 hours at a temperature of 0 – 5℃ in a closed container. Every 1 hour the container is rotated 360^° 3 times, so that the brewing process is evenly distributed in each ingredient used. Furthermore, the True Brew (TB) method is a brewing method using 1.25 grams of mashed dry Phyllanthus niruri Linn. brewed with distilled water at a temperature of 212^°F, stirred with a stirrer 10 times in a clockwise direction, to flatten the surface of the material in contact with water, then let stand for 10 minutes. After that separate the water with Phyllanthus niruri Linn. Modification of the filler (F) method was carried out by putting Phyllanthus niruri Linn. in a dip bag before being brewed, while the modification of the non-filler (NF) was implemented by letting Phyllanthus niruri Linn. have direct contact with distilled water. The use of the filter aims to provide practical value and make it easier when you want to separate Phyllantus niruri Linn. from brewed water. However, using a filter there is a risk that the total yield of phytochemical compounds will be trapped in the filter, so two treatments are carried out to see the effect. This study used six treatments with four replications.

2.3 Phytochemical analysis

Phytochemical analysis was carried out based on the method proposed by Chigozie [18]. The identification of phenolic compounds and tannins was conducted by adding 5% and 1% FeCl₃ compounds. The brewing water of Phyllanthus niruri Linn. was put on a drip plate and then added with 5% FeCl₃ compound. After that, the color change was observed. The darker the color of the sample is, the higher the presence of phenolic compounds will be. In addition, the identification of tannin compounds was performed by putting the brewing water of Phyllanthus niruri Linn. into a test tube and reacting it with a 1% FeCl₃ solution. If the extract contains tannins, a blackish green or dark blue color will be formed.

The qualitative test of flavonoid compounds was executed by adding Mg metal and 4 – 5 drops of concentrated HCl to the brewing water of Phyllanthus niruri Linn. in a test tube. The positive result is indicated by the formation of a red or orange solution. Moreover, the presence of saponins was identified by shaking the brewing water of Phyllanthus niruri Linn. for 1 minute in a water bath. If foam appears, it must be added with N₁HC_l. Furthermore, if the foam persists for 10 minutes, it means that the sample is positive containing saponins. On top of that, triterpenoids and steroids were tested by putting 1 ml of the brewing water of Phyllanthus niruri Linn. in a test tube and then added with anhydrous acetic acid and 1 – 2 ml of concentrated H₂SO₄ through the wall of the test tube. If a brown or purple ring is formed at the boundary of the two solvents, it means that the water contains triterpenoids. Furthermore, if the results obtained has a bluish-green color, it indicates the presence of steroids.

2.4 Antioxidant activity test

Antioxidant activity was measured with the DPPH free radical scavenging method (2,2-diphenyl-1- picrylhydrazyl) [16]. Analysis of the radical scavenging activity of DPPH (antioxidant activity) was carried out by preparing a sample stock solution. The DPPH assay is a very simple assay system that provides the first indication of the radical scavenging potential of the 2, 2-diphenyl-1-picrylhydrazyl radical. This method is used to test compounds that act as free radical scavengers or hydrogen donors and evaluate their antioxidant activity [19]. Samples were made in five concentration series, namely 100, 150, 200, 250, and 300 ppm. Determination of antioxidant activity was carried out by adding 1 ml of sample from each concentration into a different test tube and then adding 4 ml of 50 M DPPH solution. The mixture was homogenized and left for 30 minutes in a dark place. After that, absorption was measured using a UV-VIS spectrometer at a wavelength of 517 nm.

The parameter used for examining antioxidant activity with this DPPH radical scavenging method as IC₅₀, which is the concentration of the test compound required to capture 50% of DPPH free radicals. The smaller the IC₅₀ value is, the stronger the test compound as a DPPH radical scavenger will be.

2.5 Statistical analysis

In the statistical analysis for the different test on antioxidant activity with six treatments, the researchers applied ANOVA with a single factor by excel program. One factor analysis of variance is a special case of analysis of variance (ANOVA), for one factor of interest, and a generalization of two sample t-test.

Phyllanthus niruri Linn. is a plant that possesses many health benefits. The results of the previous studies on the extract of Phyllanthus niruri Linn. showed that it can improve insulin resistance by reducing blood glucose levels, improving dyslipidemia, and inhibiting inflammation [2]. Other previous studies also had shown that this plant contains compounds that provide high antioxidant activity, such as flavonoids, alkaloids, terpenoids, lignans, polyphenols, tannins, coumarins, and saponins [20-22]. In this study, the results of the response analysis to phytochemical contents for all brewing methods indicated the presence of flavonoids, triterpenoids, and alkaloids. The brewing water of Phyllanthus niruri Linn. that contained the most abundant compounds covering all phytochemical attributes was obtained by the DBF method. This is in line with a previous study that showed positive values for the presence of phenolics, flavonoids, steroids, saponins, and tannins in the extract of Phyllanthus niruri Linn [23]. In addition, the results of this study indicated that the abundance of phytochemical contents from the brewing water of Phyllanthus niruri Linn. using the Cold Brew (CB) and True Brew (TB) methods was low. This is consistent with the previous studies which showed positive values for the presence of phenolics, flavonoids, steroids, saponins, and tannins in the extract of Phyllanthus niruri Linn [20-22].

The brewing methods applied to Phyllanthus niruri Linn. may affect the antioxidant activity of the brewing water. This is due to the type and abundance of compounds extracted [24]. The type and abundance of extracted compounds are strongly influenced by the time and temperature during the brewing process. Therefore, different brewing methods will affect the value of the antioxidant activity of the brewing water of Phyllanthus niruri Linn [25, 26].

The DPPH method can be used to determine the ability of the sample to capture free radicals as an antioxidant activity feature. The principle of the DPPH method is the donation of hydrogen atoms from the sample substance to the DPPH compound which makes DPPH non-radical with a color change as the indicator. Changes in color and intensity from purple to yellow are directly proportional to the antioxidant activity to capture the free radicals.

The measurement of the antioxidant activity value can be carried out by observing the intensity of the yellow color in the solution which is measured using a UV-Vis spectrophotometer at a wavelength of 514 nm. In this study, the measurement was carried out on 5 series of comparison stock solution concentrations and the samples of the brewing water of Phyllanthus niruri Linn. The absorbance value of the UV-Vis spectrophotometer was used to calculate the percent inhibition value (%). The percent value obtained was then regressed between percent and concentration. The result was a curve with the equation Y = a + bX. Furthermore, it was then continued by finding the IC50 value.

The data presented in Table 2 showed that the antioxidant activity of the brewing water of Phyllanthus niruri Linn. had a value between 2240.37 and 2569.90 ppm. The highest antioxidant activity of the brewing water of Phyllanthus niruri Linn. was obtained from the CBNF method with an average IC50 value of 2240.37 ± 93.79 ppm. This Hantioxidant activity was lower than that possessed by the brewing water of Phyllanthus niruri Linn. extracted using 96% ethanol, which was 237.33 ± 17.26 ppm [21, 25]. This was influenced by the type and abundance of phytochemicals that can be extracted by certain brewing methods. The optimization value of the brewing water of Phyllanthus niruri Linn. was also influenced by water temperature and contact time. Based on a previous study conducted by Nikniaz [27] on brewing black tea, the best temperature for brewing tea was 80℃ with a contact time of 5 minutes. This combination between temperature and time gave higher antioxidant activity compared to the brewing water of black tea with the time under 5 minutes. It will be different if the tea is processed by being not fermented like green tea. Furthermore, the temperature applied and the optimal time for brewing tea, which is 95℃ in no more than 30 minutes, has better antioxidant content compared to those more than 30 minutes.