Xanthine, xanthine oxidase, 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, gallic acid, cupric chloride, and neocuproine were obtained from Sigma-Aldrich Chemicals (St. Louise, MO, USA). The ascorbic acid was obtained from Merck (Darmstadt, Germany). Allopurinol was purchased from TCI (Tokyo, Japan), and all other reagents were analytical grade.

Fresh aerial parts of five selected medicinal plants, Myrmecodia beccarii Hook.f, Villebrunea rubescens (Bl), Breynia cernua Muel. Arg, Bridelia spp. and Dodonaea viscosa Jacq. were collected from five different locations in Papua Province-Indonesia and determined in the Herbarium of Research Center for Biology-Indonesian Institute of Sciences listed in Table 1. Samples were washed, dried, and milled into powder.

Extraction was conducted using Soxhlet with increasing polarity solvent, ranging from nonpolar (n-hexane), semi-polar (ethyl acetate), and polar solvent (96% ethanol). Crude drug powder 100 grams of five medicinal plants from Papua, Myrmecodia beccarii Hook.f, Villebrunea rubescens (Bl), Breynia cernua Muel. Arg, Bridelia spp., and Dodonaea viscosa Jacq. were extracted with n-hexane, and the filtrate and residue were then separated. The residue was dried and extracted with ethyl acetate. Finally, the residue was extracted using 96% ethanol. All extracts were evaporated using a rotary vacuum evaporator with a temperature of 50 °C to produce fifteen extracts. There were five n-hexane extracts (M. beccarii (MBH), V. rubescens (VRH), B. cernua (BCH Bridelia spp. (BSH), and n-hexane extract of D. viscosa (DVH)); five ethyl acetate extract (M. beccarii (MEA), V. rubescens (VEA), Breynia cernua (BEA), Bridelia spp. (BSE) and ethyl acetate extract of D. viscosa (DEA)) and five ethanol extract (M. beccarii (MET); V. rubescens (VET), B. cernua (BCE), Bridelia spp. (BET) and ethanolic extract of D. viscosa (DET)). The phytochemical test was performed on crude drugs and extracts using the method described in Farnsworth and Sarker’s modified method (Farnsworth 1966; Sarker 2013).

The total phenolic content was evaluated with a Folin-Ciolcalteu reagent adapted to Pourmorad’s method (Pourmorad et al. 2006). Each extract of 0.5 mL was piped into 5 mL Folin-Ciolcalteu reagent 10% and 4 mL of sodium carbonate 1M and incubated the mixtures for 15 minutes. The absorbance was measured at wavelength 765 nm. For each extract, the analysis was performed in triplicate. The results were reported as g gallic acid equivalents (GAE) per 100 g extract (g GAE/100 g) according to the calibration curve of gallic acid 25–150 µg/mL as standard.

The total content of flavonoids was measured using an adapted method from Chang (Chang et al. 2002). Each extract of 0.5 mL was piped into 0.1 mL aluminum chloride 10%, 0.1 mL sodium acetate 1M and 2.8 mL distilled water. The mixture was diluted with 1.5 mL ethanol and incubated for 30 minutes at wavelength 415 nm, and the absorbance was read. For each extract, the analysis was carried out in triplicate. The results were presented as g equivalents of quercetin (QE) per 100 g extract (g QE /100 g) according to the calibration curve of quercetin 5–100 µg/mL as standard.

Determination of the AAI (Antioxidant Activity Index) based on Blois’s method (Blois 1958) with some modification was applied to determine antioxidative activity by DPPH. Each extract was prepared in various concentrations and mixed with the DPPH solution of 50 µg/mL (volume 1:1). The DPPH solution was dissolved in methanol. The absorbance was evaluated at 517 nm by UV-vis spectrophotometry after 30 minutes of incubation. The standard was ascorbic acid. Analysis was investigated in triplicate. The purple color of the DPPH solution will be shifted when the antioxidant scavenges the free radical, so an inhibitory concentration of 50% (IC₅₀) can be determined from the sample. A calibration curve was plotted between % (the percentage) of DPPH scavenging activity versus concentration to obtain IC₅₀. After that, the AAI of each extract was determined by dividing the final concentration of DPPH with IC₅₀ at each extract (Scherer and Godoy 2009).

The AAI (Antioxidant Activity Index) CUPRAC was determined based on Apak’s method (Apak et al. 2013). Several extract concentrations were added to the CUPRAC solution of 100 µg/mL (volume 1:1). After incubation for 30 minutes, the absorbance was read at 450 nm. The CUPRAC solution was prepared by mixing CuCl₂ in distilled water and neocuproine in ethanol, and then the mixture was dissolved in ammonium acetate buffer pH 7. Ascorbic acid was applied as standard. Analysis was performed in tri-replication. If the sample acts as an antioxidant agent, Cu (II) will reduce to Cu (I). Neocuproine’s function is to make a chromophore with Cu (I) and show a yellow color, and then the exhibitory concentration of 50% (EC₅₀) was obtained from the calibration curve. Afterward, the AAI of each extract can be calculated by dividing the final concentration of CUPRAC with EC₅₀.

The inhibitory activity of xanthine oxidase was measured by spectrophotometer in 96-well plates (Corning, UV-Transparent Clear Microplates) below the aerobic conditions, following the method reported by Owen and Duong with some minor modification (Owen and John 1999; Duong et al. 2017). The extracts were dissolved in dimethyl sulfoxide (DMSO) and diluted with phosphate buffer pH 7.5. The final DMSO concentration was not higher than 0.5%. Allopurinol was used as a standard. The test solution consisted of 50 µl of the sample of extract (100 µg/mL), 69 µL of phosphate buffer, 15 µL of freshly made solution for the enzyme (0.2 unit/mL xanthine oxidase in phosphate buffer, then 66 µL of substrate solution (0.15 mM of xanthine in phosphate buffer). The test solution was incubated at 25 °C for 15 minutes. The absorbance was measured at 15 minutes using a microplate reader (Tecan infinite M200 pro) at λ 290 nm. In the same procedure, a blank was prepared, but the enzyme was substituted by phosphate buffer. The analysis for allopurinol and each extract was performed in triplicate. The inhibitory activity of xanthine oxidase was determined using the formula: I% = [(A – B)/A] × 100, where A = absorbance of enzyme xanthine oxidase without test extract – blank of A (absorbance without XO and test extract), B = absorbance of the test extract – blank of B (absorbance without XO).

TLC-densitometric studies were carried out using the standard method by Wagner (Wagner et.al 1996). 25 mg extract was dissolved in 0.5 mL methanol and centrifuged at 3000 rpm for 5 minutes. These solutions were used as test solutions for phenolics and flavonoid. 2 µL of test solutions and standart solution (caffeic acid, quercetin, and naringenin) were loaded as 5 mm band length in the aluminium silica gel 60 F₂₅₄ TLC plates (5 × 10 cm). The linear ascending development was eluted in a saturated mode condition with twin through chamber room using the eluent system toluene: ethyl acetate: formic acid (4:2:0.2). The plate was dried at room temperature before being scanned using densitometry (CAMAG TLC Scanner 3 with winCATS 1.4.2 software) at 254 nm and 366 nm UV light.

The results were the means ± SD of at least three independent tests, using MS Excel Software to evaluate the IC₅₀ and EC₅₀ values. Analysis of the statistic (one-way ANOVA – post hoc Tukey) was carried out by SPSS 23. Using Pearson’s method, correlations were made between the total phenolic and flavonoid content with antioxidant and xanthine oxidase inhibitory activities.

In the present investigation, antioxidative activity, total phenolic and flavonoid content, xanthine oxidase inhibitory activities of five medicinal plants from Papua, M. beccarii, V. rubescens, B. cernua, Bridelia spp., and D. viscosa were evaluated. Extraction was carried out by Soxhlet with different polarity solvents, three solvents n-hexane, ethyl acetate, and ethanol. Three other polarities solvents, such as n-hexane, ethyl acetate, and ethanol, were used to separate based on the polarity of compounds in crude drugs. n-hexane and ethyl acetate were selected solvents. Therefore, the nonpolar compounds will be mainly extracted in n-hexane and semipolar compounds in ethyl acetate. Meanwhile, ethanol will find most of the polar compounds. The extracts’ preliminary qualitative investigation revealed phytochemical compounds such as alkaloids, flavonoids, triterpenoids/steroids, quinone, saponin, tannin, and phenols. The phytochemical screening results were carried out from each extract of the Soxhlet extraction method using increasing polarity solvents from five plants M. beccarii, V. rubescens, B. cernua, Bridelia spp., and D. viscosa were displayed in Table 2. Based on phytochemical screening results, all crude drugs and extracts detected flavonoids, phenol, tannin, saponin, and steroid/triterpenoid. Meanwhile, alkaloid was not seen in M. beccarii, and quinone was not detected in V. rubescens, B. cernua, and D. viscosa. Secondary metabolite compounds from plants that contain flavonoid and phenolic compounds have the potential to be antioxidant and xanthine oxidase inhibitor agents from natural resources (Ryu et al. 2012; Petkova et al. 2017).

Density is a crucial assay to compare its activity and phytochemistry compound levels. Higher density will result in stronger activity and phytochemical content. The measurement of the concentrated extract was carried out using a 1% extract solution in a pycnometer. In this study, the density of each extract showed values in the range of 0.661–0.895 g/mL.

In determining the TPC in each extract based on Table 3, gallic acid was used as standard, and a linear regression equation of gallic acid was applied y = 0.0068× - 0.0577, R² = 0.9872. The TPC in all Papua medicinal plant extracts presented different results from 2.40 to 9.01 g GAE 100/ g. Ethanolic extract of B. cernua (BCE) showed the highest TPC (9.01 g GAE/100 g). Meanwhile, quercetin was used as standard for determining TFC with the liner regression y = 0.0105× – 0.0078, R² = 0.9907. The TFC in all Papua medicinal plant extracts presented various results from 0.89 to 8.03 g QE/100 g. Ethyl acetate extract of Bridelia spp. gave the highest TFC (8.03 g QE/100 g). Phenolic compounds generally have antioxidative activity due to the ability to donate electrons or as chelating metals. The antioxidative activity of flavonoids was influenced by the degree of hydroxylation and other substituents found in flavonoids (Heim et al. 2002). Flavonoid compounds will give high antioxidant power through the transfer hydrogen mechanism when it has ortho di-OH in C3’-C4’, C2-C3 double bond, OH in C3, and oxo in C4. Ortho di-OH in C3’-C4’ had the highest effect on antioxidative activity. Flavonoids with OH substitution on rings A and B belong to the phenolic group (Fidrianny et al. 2013). Phenolic compounds and flavonoids have xanthine oxidase inhibitory activity like apigenin, luteolin, quercetin, myricetin, morin, and kaempferol (Nagao et al. 1999; Dong et al. 2013). A high antioxidant capacity in a flavonoid will be presented when the flavonoid has a hydroxyl group in C-3´-C-4´, OH at C-3, oxo function at C-4, double bond at C-2 and C-3 (Rice-Evans et al. 1996).

Based on Table 4, the higher antioxidative activity was demonstrated by lower IC₅₀ DPPH and EC₅₀ CUPRAC or higher AAI of DPPH and AAI CUPRAC. The ethyl acetate extract of M. beccarii (MEA) with an AAI value of 3.22 for AAI DPPH and 4.27 for AAI CUPRAC were classified in the category of very strong antioxidants (AAI > 2). Meanwhile, the AAI value for standard ascorbic acid was 10.20 for DPPH and 11.92 for CUPRAC (Scherer and Godoy 2009).

A recent study (Dirgantara et al. 2013) confirmed that the methanol extract of M. beccarii showed antioxidative activity with IC₅₀ 8.18 µg/mL. Many species of the Myrmecodia genus showed potential antioxidative activity with the DPPH method. The free radical scavenging activity (antioxidative activity) of the ethanol extract of hypocotyl Myrmecodia pendens was evaluated using DPPH radical. The IC₅₀ value occurred at 96.21 µg/mL of extract and contained total phenol and flavonoid contents 330.61 mg GAE/ g and 63.28 mg QE/ g of dry extract, respectively (Engida et al. 2013). The ethyl acetate fraction of this extract contained procyanidin B1 dimer (3.236 mg/g dry sample) and rosmarinic acid (20.688 mg/g dry sample) significant free radical scavenging capacities with IC₅₀ values of 27.59 µg/mL and 35.80 µg/mL, respectively (Engida et al. 2015). Ethanolic extract of stem bark, leaves, and tuber of Myrmecodia tuberosa showed antioxidative activity with 95.17%, 94.55%, and 93.42% DPPH scavenging activities, respectively (Rasemi et al. 2014). The previous study investigated ethyl acetate fractions of the hypocotyl of Myrmecodia platytyrea and showed antioxidative activity with IC₅₀ 21.57 µg/mL (Mohamad Haris et al. 2016). Polyphenols and flavonoids of ethanol and ethyl acetate extracts expressed the highest antioxidative activity compared to dichloromethane and methanol extracts of M. platytyrea with high-performance thin-layer chromatography (HPTLC)-DPPH bioautographic method (Agatonovic-Kustrin and Morton 2018b; Agatonovic-Kustrin et al. 2018a).

Based on Table 5, it can be seen there were significant and positive correlation between TPC in VR extract with AAI DPPH (r = 0.636, p<0.05); TPC and TFC in MB, BC and BS extract with AAI DPPH (r = 0.880, p<0.01; r = 0.986, p<0.01; r = 0.889, p<0.01; r = 0.850, p<0.01; r = 0.976, p<0.01; r = 0.959, p<0.01, respectively). Meanwhile, TPC and TFC in MB extracts with AAI CUPRAC (r = 0.952, p<0.01; r = 0.961, p<0.01, respectively). Based on this result, it can be concluded that phenolic and flavonoid compounds in MB, BC, and BS extract were the main compound for antioxidative activity using DPPH and CUPRAC assays.

The correlation between two antioxidant testing DPPH and CUPRAC methods has also been exposed in Table 6. The different antioxidant capacities assays will give different results. Two methods with different antioxidant mechanisms might give no linear result. Pearson’s correlation could analyze whether the AAI of each method gave a linear result or not. DPPH is a combination of hydrogen transfer and electron transfer. Meanwhile, CUPRAC electron transfer only. Two methods will give linear results when they show positive and linear correlation. The result demonstrated that AAI DPPH of MB and BC extract positively and significantly correlated with their AAI CUPRAC (r = 0.973, p<0.01; 0.883, p<0.01, respectively). TPC in ethyl acetate of B. cernua extracts (BEA) 7.14 ± 0.18 g GAE 100/g was in line with ethyl acetate of M. beccarii extract (MEA) 7.92 ± 0.24 g GAE 100/ g, but AAI DPPH BEA (0.28) was lower than AAI MEA (3.22). It can be suggested that most phenolic constituents in MEA showed higher potent antioxidative activity, which had many hydroxyl substituents in their structure. In the CUPRAC assay, a sample will act as an antioxidant when it has a reduction potential smaller than Cu²⁺/Cu+ (1.59V). TFC in n-hexane of V. rubescens extract (VRH) 2.93 ± 0.02 g QE/100 g was in line with ethyl acetate of M. beccarii extract (MEA) 2.80 ± 0.04 g QE 100/g, but AAI CUPRAC MEA (4.27) was higher than AAI VRH (0.22). So, the MEA can be categorized as a remarkably potent antioxidant, while VRH is a poor antioxidant. It can be suggested that most of the flavonoids in MEA had reduction potentials smaller than 1.59 V, while only a few flavonoids in VRH were lower than 1.59 V.

Based on Fig. 1, the ethyl acetate extract with a concentration of 100 µg/mL of M. beccarii extract (MEA) showed the highest potential XOI activity with a percentage of inhibition of 66.42 ± 2.89%. Meanwhile, allopurinol, a standard with a concentration of five µg/mL, showed 80.84 ± 0.88%. Based on this research, ethyl acetate extract of M. beccarii extract (MEA) showed the highest potential xanthine oxidase inhibitory and antioxidative activity in line with phytochemical total phenolic content. The previous study by Simanjuntak et al. (2010) investigated the other species, M. pendens showed the highest inhibition at 61.99% from n-butanol fraction, and the isolated compound showed the xanthine oxidase inhibitory activity at 79.77%.

Figure 1. 

Percentage of XOI fifteen Papua medicinal plant extracts.

TLC-densitometric analysis showed that ethyl acetate extract of M. beccarii extract (MEA) revealed the presence of various phytochemical contents, as illustrated the Fig. 2 and Table 7. The chromatograms were obtained upon scanning at UV 254 nm, and peak tables were generated. All phytochemical compound peaks were identified based on Rf values of MEA extract compared with standard caffeic acid, naringenin, and quercetin. MEA extract exerted eight prominent bands, of which peak number 7 was identified as caffeic acid compared with the Rf values and blue fluorescent band color of the caffeic acid standard.

Figure 2. 

TLC profile of MEA extract with standard caffeic acid (CA), naringenin (N) and quercetine (Q). a) UV light 254 nm, b) UV light 366 nm, c) densitogram MEA extract, d) caffeic acid, e) naringenin, f) quercetin.

The research results showed that each extract of the Papua medicinal plants plant gave a different pattern of xanthine oxidase inhibitory and antioxidative activity depending on the solvent used. This can be due to the other chemical content in every fifteen extracts. According to the result, it can be presumed that the total phenolic content of extracts contributed to its antioxidative activity and xanthine oxidase inhibitory activity.