Comparison of different methods for evaluating the antioxidant activity of instant Torbangun (Coleus amboinicus L.) drinks

Torbangun (Coleus amboinicus L.) is a natural breast milk stimulant with antioxidant activity. This study aimed to compare the antioxidant activity of instant Torbangun drinks (ITD) ethanolic extract using DPPH (2,2-diphenyl-1-picrylhydrazil), ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), and FRAP (Ferric Reducing Antioxidant Power) methods. Ethanolic extract of commercial Torbangun tea (CTT) and lactation booster milk (CLM) was also included in all analyses as a product reference. At the same time, vitamin C was used as an antioxidant standard. The antioxidant activity was expressed as EC50 value. Among breastmilk booster drinks, the sample with the highest antioxidant activity obtained from ABTS and FRAP methods was ITD (EC50 18.26 mg/mL) and CLM (EC50 0.187 mg/mL), respectively. Assessing with the DPPH method, the activity of ITD and CLM was not significantly different (EC50 35.200 mg/mL and 33.035 mg/mL, respectively), and those were higher than that of CTT (EC50 52.22 mg/mL). However, the antioxidant activity of all products was significantly lower than vitamin C in all analysis methods (EC50 < 0.05 mg/mL). This finding was proposed due to different antioxidant compounds and antioxidant mechanisms among breast milk booster products. This study also revealed that ABTS and FRAP were the most suitable methods for determining antioxidant properties in all samples.


Introduction
Torbangun (Coleus amboinicus L.) leaf has been known as a natural breast milk stimulant (galactagogue) that can increase the production and secretion rates of breast milk [1,2].In addition, torbangun leaf contains immunoglobulin, vitamin C, vitamin B12, and antioxidant, antihypertensive, and anti-inflammatory compounds that may also support breastfeeding mothers' health [3].Torbangun leaves are commonly consumed after being processed with different domestic cooking methods (stewing, cooking with coconut milk, stir-frying); however, this practice is not convenient.
The making of instant drinks facilitates the ease of Torbangun consumption.This product can be made using the foam mat drying technique because it is relatively simple and inexpensive.This method uses a foaming agent (such as Tween80) for foaming formation, which helps to accelerate the water evaporation.The process undergoes at a lower temperature; thus, it can preserve the heat-sensitive compounds and product color [4].
Various results may occur when a specific sample is evaluated using different antioxidant evaluation methods.Kusumorini et al. [8] compared the potential antioxidant activity of isolated piperine using DPPH, ABTS, and FRAP tests.From this study, the isolated piperine only showed an antioxidant activity in the ABTS and FRAP test, with the IC50 value of 4.35 ± 0.004 mg/mL and 10.53 ± 0.06 mol TE/g sample, respectively.The isolated piperine showed no free radical activity against the DPPH reagent.Another study by Antasionasti et al. [7] reported that the antioxidant activity of nutmeg was found to be higher when determined using the ABTS method than that of the DPPH test, as indicated by their IC50 value (89.980±0.480µg/mL in ABTS and 105.669±0.102µg/mL in DPPH).Those results can be due to the influence of the chemical structure of antioxidants, the source of free radicals, and the physicochemical properties of the sample.Therefore, selecting the appropriate antioxidant activity methods for a particular type of sample is necessary.Different solvents also have different abilities in dissolving plant bioactive compounds, thus affect the expression of antioxidant activity from a particular sample.Garmus et al. [9] found that ethanol had a better performance for phenolic and flavonoid extraction when compared to water.
This result indicated that ethanol has a greater chemical affinity for the compounds of interest and favors the extraction process.Another study by Suryowati et al. [10] suggests that ethanol is more effective in extracting the antioxidant-contributing compounds, such as phenolics and flavonoids, in Torbangun than water.The nature of ethanol, which has polar and non-polar groups, makes it easier to dissolve the polar and non-polar compounds.Thus, the higher activity is proposed due to the presence of the various type of bioactive compounds that can be extracted by ethanol [11].
To the best of our knowledge, assessment of Torbangun instant drinks using different antioxidant evaluation methods had never been conducted.In this study, the antioxidant activity of instant Torbangun drinks (ITD) ethanolic extract was assessed using DPPH, ABTS, and FRAP methods.Commercial Torbangun tea (CTT) and lactation booster milk (CLM) were used as references for breast milk booster drinks, while vitamin C was also used as a standard [12].The findings of this study are expected to determine the antioxidant activity among samples and to reveal the optimum antioxidant evaluating method for each sample.

The ITD preparation
Table 1 shows the ITD formulation.The sample was prepared by foam mat drying [13][14][15].The process was initialized by extracting 150 g of blanched Torbangun leaf with 300 ml of water.The extract was mixed with maltodextrin and Tween80 at medium speed for 5 minutes.A very thin of foamy mixture was spread evenly in stainless steel baking sheet and dried in an oven at 60 °C for 4 hours.Then, the thin dried sheet was ground and sieved.The procedure of ITD making can be seen in Figure 1.

The DPPH assay
The DPPH assay was conducted according to Youn et al. [16] with slight modification in the maximum wavelength of the UV-Vis spectrophotometry.The DPPH crystal was diluted in ethanol, and the concentration of the DPPH solution was 0.1 mM.The serial concentration for the sample was 10, 20, 30, 40, 50, 60 mg/mL, and for ascorbic acid was 10, 20, 30, 40, and 50 ppm.The reaction mixture consisted of 1 mL sample and 3 mL DPPH; then, it was incubated for 30 minutes in dark conditions before measuring the absorbance at 517 nm.Blank solution was made by replacing sample portion with ethanol in reaction mixture.Radical scavenging activity was expressed as a percentage of inhibition, which was determined according to the following formula: DPPH radical scavenging activity (%) =

The ABTS assay
The procedure for ABTS assay followed the method from Shalaby and Shanab [17] with slight modification in the ratio of sample and solution mixtures.The ABTS solution was prepared by mixing 5 mL of 7 mM ABTS stock solution was mixed with 5 mL of 2.45 mM potassium persulfate solution, then continuing with 16 hours incubation.After that, the ABTS solution was diluted using 70% ethanol to obtain an absorbance value of 0.700 ± 0.05 at a wavelength of 734 nm.Analysis was conducted by mixing 0.9 mL sample and 4.5 mL ABTS solution, then incubating it in a dark condition for 6 minutes before measuring the absorbance at a wavelength of 734 nm.Blank solution was made by replacing sample portion with ethanol in reaction mixture.The serial concentration for the sample was 10, 20, 30, 40, 50, 60 mg/mL, and for ascorbic acid was 2, 4, 6, 8, 10, and 12 ppm.Determination of ABTS reduction activity percentage (%) used the following formula: ABTS reduction activity (%) =

The FRAP assay
Evaluation of antioxidant activity using FRAP assay was conducted according to Youn et al. ( 2019) [16] with slight modification in ratio of the sample mixture.The first step was the preparation of FRAP working solution by mixing 25 mL of 300 mM acetate buffer (pH 3.6), 2.5 mL of 10 mM TPTZ in 40 mM HCl, and 2.5 mL of 20 mM FeCl3.6H2O, then making the solution into 100 mL by adding distilled water.Before being used, the solution was incubated at 37 °C for 10 minutes.After that, 1 mL sample and 3 mL FRAP solution were mixed and then incubated in a dark condition for 30 minutes.Sample absorbance was then measured at 593 nm.Blank solution was made by replacing sample portion with ethanol in reaction mixture.Serial concentration for the sample was 20, 4, 0.8, 0.16, 0.032, 0.0064 mg/mL, and for ascorbic acid was 2, 4, 6, 8, 10, and 12 ppm.The FRAP value was determined with the following formula: Reducing Power (%) =

The EC50 calculation
The EC50 value from each antioxidant activity evaluation method was determined out by plotting the sample concentrations and % activity as x and y axis, respectively, to obtain the regression equation (y = ax + b).Then, the EC50 value can be calculated using the following formula: EC50 =

Statistical analysis
The data obtained from the above assays were analyzed using analysis of variance (ANOVA) and post hoc Tukey's HSD test to determine the significant difference between mean values at a confidence level of 95% (p < 0.05).Statistical analyses were carried out using IBM Statistics SPSS 16.0.

Results and Discussion
In this study, antioxidant activity of sample assessed using DPPH, ABTS, and FRAP methods was expressed as EC50 value.This value indicates the efficient sample concentration necessary to obtain 50% antioxidant activity.The lower EC50 value indicates the higher antioxidant activity, since lower amount sample concentration required to achieve 50% activity.Based on the EC50 value, the antioxidant activity can be classified into following groups: very strong (< 0.05 mg/mL), strong (0.05 -0.10 mg/mL), moderate (0.101-0.15 mg/mL), weak (0.15 -0.20 mg/mL), and very weak (> 0.20 mg/mL) [18].

Antioxidant activity evaluation using DPPH Assay
The DPPH assay is a test that measures the activity of antioxidant compounds in reducing the free radicals of 2,2-diphenyl-1-picrylhydrazyl.Discoloration of DPPH solution, from deep purple to yellow, occurs when a sample containing antioxidant compounds reacts with DPPH.Higher discoloration indicates potent activity of the sample [19].
The antioxidant activity of ethanolic extract of ITD compared to the product references and standard evaluated using this assay is presented in Table 2.The significantly highest EC50 value sample was ITD, followed by ITD and CLM.However, there was no statistical difference between ITD and CLM.Vitamin C, as an antioxidant standard, exhibited the significantly lowest EC50 value.From the result, all breast milk booster drinks can be considered to have a very weak antioxidant activity.The ITD and CLM samples were more potent than CTT, as indicated by the significantly lower EC50 value.Vitamin C as an antioxidant standard showed the greatest performance by having the lowest EC50 value which can be considered as a very strong antioxidant.Essential oils, terpenes, phenolics, flavonoids, esters, tannins, alkaloids, and steroids are antioxidant compounds reportedly found in Torbangun leaves [20,21].The making of instant drinks using foam mat drying method successfully showed a better antioxidant compounds preservation compared to the tea processing method.The foam mat drying was conducted at 60 o C for 4 hours and this drying condition was somewhat similar to Torbangun tea process.Based on Girsang et al. [22], Torbangun tea can be made by drying the leaves at 50 o C for 3 hours.Both methods employ a relatively short duration and low temperature.However, the presence of foam in ITD making was expected to protect the leaves from direct contact with heat.This reason was proposed as an underlying reason of the higher antioxidant activity of ITD sample.
The ethanolic extract of fresh Torbangun leaves had an EC50 value of 247,942 ppm obtained from the DPPH result [10].This value is higher than the EC50 value of Torbangun-based products, indicating oven drying employed in ITD and CTT making may increase the antioxidant activity.A similar result was reported by Sadek and Hamidah [26], showing an increase of % inhibition against DPPH radicals in the oven drying step of sorghum-moringa substituted tapioca noodle production.This increment was positively correlated with an increase in the total phenolic compound.
Lutfiani and Nasrulloh [27] reported the EC50 value of Torbangun and Katuk flours was 39.8 mg/mL and 308 ppm, respectively, by DPPH free radical scavenging assay.Oat cultivars were reportedly to have DPPH scavenging activity with the EC50 value of 0.013 to 0.019 ppm [28].The aqueous extract of moringa leaves also exhibited free radical scavenging activity with an EC50 value of 0.217 ppm in the DPPH assay [29].From these reports, oat and aqueous moringa extract were expected to have higher antioxidant activity than Torbangun leaves.However, the small amount of oat powder and moringa extract used in CLM formulation was proposed as an underlying reason for the insignificant different EC50 values of CLM and ITD.

Antioxidant activity evaluation using ABTS Assay
The ABTS reduction test is measured using the free radical assay of diammonium salt ABTS+ 2,2azinobis-(3-ethylbenzothiazoline-6-ssulfonate).The ABTS radical is reduced by hydrogen-donating antioxidants, as indicated by the blue-green-colored solution that turns colorless.The higher ABTS discoloration is proportional to the higher the rate of the ABTS salt reduction, which indicates the higher antioxidant activity of the sample [18].
The antioxidant activity of ethanolic extract of ITD compared to the product references and standard evaluated using this assay can be seen in Table 3.In this analysis, the EC50 value was statistically different among testing samples.Breast milk booster drinks showed significantly higher EC50 values compared to vitamin C. The CLM sample was found to have a significantly highest EC50 value, followed by CTT and ITD in the second and third positions, respectively.
Compared to the DPPH analysis, the EC50 value of breast milk booster drinks evaluated using ABTS assay was lower.Vitamin C, as a standard compound, also exhibited a higher antioxidant activity when assessed using this method than DPPH, as shown by the lower EC50 value.This finding was probably due to the different characteristics of DPPH and ABTS testing.The DPPH radical is soluble in methanol, ethanol, or their aqueous mixtures but not in water.Modification DPPH analysis with water IOP Publishing doi:10.1088/1755-1315/1324/1/0121246 incorporation should limit the water content should not exceed 60% to ensure the radical is more readily soluble [30].Meanwhile, the ABTS radical can be dissolved in water and organic solvents.The solvent used for dissolving radicals may affect the solubility of antioxidant compounds in the testing sample.If the components are less soluble in the reaction medium, they can show no optimal expression of antioxidant abilities [31].It was reflected in the findings of this research, revealing the higher ABTS-antioxidant activity rather than the DPPH assay.The nature of the ABTS assay allows for determining the antioxidant activity obtained from both lipophilic and hydrophilic compounds from ITD and product.However, the antioxidant activity of all breast milk booster drinks samples was still categorized as very weak.
Evaluation with ABTS assay exhibited a different trend than the DPPH result.The Torbangun-based products (ITD and CTT) had a significantly higher antioxidant activity than the CLM sample.The ITD sample had the highest antioxidant activity among breast milk booster drinks, followed by CTT and CLM in the second and third positions, respectively.This result indicated the sum of hydrophilic and hydrophobic antioxidant compounds in Torbangun-based products was higher than that of the CLM sample.However, the lower EC50 value of ITD than CTT was proposed due to the protective effect of the foaming agent on bioactive compounds during the ITD-making process.Interestingly, CLM had a lower EC50 value than CTT in the DPPH evaluation, but the opposite result was found in the ABTS result.This phenomenon probably indicated that the portion of hydrophilic antioxidant compound was higher in Torbangun-based products than CLM sample.
The results were supported by Krol et al. [32] which stated the ABTS assay showed a higher antioxidant capacity, likely due to its ability to assess the antioxidant both hydrophilic and lipophilic compounds.In contrast, the DPPH exclusively measured the capacity of hydrophobic compounds.These findings indicate that when applied to a variety of plant foods containing hydrophilic, lipophilic, and high-pigmented antioxidant compounds, the ABTS assay is superior to the DPPH assay.However, no reports on antioxidant activity evaluation using ABTS assay in Torbangun leaves and its derivative products.

Antioxidant activity evaluation using FRAP Assay
The FRAP method measures the reduction of the colorless complex of ferric ions (Fe 3+ )-ligand to the intensely blue ferrous complex (Fe 2+ ) by electron donating antioxidants.This reaction is conducted in an acidic environment to maintain iron solubility [33].As previously mentioned, the environment largely affects the reduction activity of tested compounds due to their solubility in the measured mixture.The FRAP reaction environment can be considered as hydrophilic.However, the use of ethanolic extract of testing samples used in this study may gave no adverse effect since their high solubility in water [34].
The ABTS EC50 value of ethanolic extract of ITD compared to the product references and standard is presented in Table 4.The results showed statistical differences among samples.Breast milk booster drinks showed EC50 values ranging from 0.187 to 1.813 mg/mL, which were significantly lower when compared to the antioxidant standard of vitamin C. The CLM performed a significantly lowest EC50 value among breast milk booster drinks, followed by CTT and ITD in the second and third ranks, respectively.The FRAP test resulted in the lowest EC50 among all antioxidant activity evaluation methods.This result indicated that antioxidant compounds in breast milk booster drinks were more efficient in reducing Fe 3+ as prooxidants rather than reducing organic DPPH and ABTS radicals.The expression of antioxidant activity of Vitamin C as a standard compound was also highest among all assessment methods.Based on EC50 characterization, the ITD and CTT can be classified as very weak, while CLM is a weak antioxidant.
Interestingly, the FRAP test also gave a different result to ITD sample when compared to the two previous assessment methods.The highest EC50 value of ITD sample reflected the lowest antioxidant activity among breast milk booster drinks.In this analysis, the activity of CLM sample was significantly higher than that of Torbangun-based products (ITD and CTT).This result indicated the presence of higher concentration of ferric ions reducing antioxidant in CLM sample, which was proposed due to phenolic compounds.Pokorna et al. [35] reported a high correlation between FRAP and total phenolic compounds.Phenolic compounds serve as an excellent electron donor due to their hydroxyl groups which can directly contribute to antioxidant action.
Based on the DPPH and ABTS results, it was expected that CLM would have a higher proportion of hydrophobic antioxidants.Thus, the highest antioxidant activity of CLM among breast milk booster drinks was considered due to hydrophobic phenolics as abundant compounds in CLM.Based on phenolic compounds' hydrophilic and hydrophobic area, ferulic and sinapic acids are the least water soluble [36].As one of the CLM compositions, oat is subjected to ferulic acids as the primary phenolic compound found in oat products [37].The amount of ferulic acid in 22 commercial oat products ranged from 24 to 58.6 ppm as free and conjugated forms and 1153.6 -247.2 ppm as bound form.
Among Torbangun-based products, the CTT showed a more significant antioxidant activity than ITD, as the lower EC50 value shows.This data is supported by Panyatip et al. [38], which reported that tea processing increases the total flavonoid content (TFC) in the mulberry leaf extract sample.It was found that thermal exposure causes flavonoid degradation and transformation.The transformation generated the flavonoid-derived products and increased TFC, which might contribute to the higher FRAP antioxidant activity since flavonoid reportedly has a positive correlation with FRAP result [39].

Comparison of DPPH, ABTS, and FRAP results
This study showed a difference in the performance of the antioxidant activity of ITD and its product references when they were tested using various antioxidant activity test methods: DPPH, ABTS, and FRAP.Different results may also reflect a component's different antioxidant mechanisms of action [7].Based on the principle of each method, DPPH and ABTS reflect the radical scavenging activity, while FRAP represents the ferric-reducing power of antioxidant compounds found in samples.The lower EC50 value of the FRAP result compared to the DPPH and ABTS indicated that all samples had more potent activity in ferric reducing than radical scavenging.Different results from those methods can be used to predict the type of antioxidant compounds in the sample since their solubility in the reaction mixture significantly affects the antioxidant activity.From the result, the ITD and CTT were considered to have a higher proportion of hydrophilic antioxidants, while CLM contained a more hydrophobic antioxidant portion.

Conclusion
It can be concluded that various method resulted in different antioxidant activity when subjected to a particulate sample.The highest activity of ABTS and FRAP methods was achieved by ITD and CLM, respectively.However, both samples shared the first rank in DPPH result.The antioxidant activity of all samples assessed with all methods was classified as a very weak, except FRAP activity of CLM that may be considered as weak.The activity of all samples was also significantly lower when compared to Vitamin C as a standard compound.The ITD sample developed in this study was proven to have comparable activity when compared to CLM as the commercial breast milk booster drinks.However, they may have a different mechanism of reaction in performing their activities.The ABTS and FRAP were proposed as the more suitable method in determining antioxidant activity of all samples.

Table 2 .
Antioxidant activity evaluation of ethanolic-extracted samples using DPPH assay Note: *Standard compound.Data is presented as mean ± SD of EC50 (effective concentration 50, n=3).Number following with different superscript indicates significance differences (p < 0.05).

Table 3 .
Antioxidant activity evaluation of ethanolic-extracted samples using ABTS assay

Table 4 .
Antioxidant activity evaluation of ethanolic-extracted samples using FRAP assay