Antihyperuricemia, Antioxidant, and Antibacterial Activities of Tridax procumbens L.

Tridax procumbens L. is a medicinal plant and used as a drink to treat bronchial catarrh, diarrhea, dysentery and liver diseases. In this study, we evaluated the potential use of T. procumbens to treat hyperuricemia, oxidative stress, and bacterial infection. Ethyl acetate extract of this plant was separated to different fractions by column chromatography (CC) using chloroform and methanol as eluents and subjected to xanthine oxidase (XO) inhibitory, antioxidant, and antibacterial assays. The results showed that the F45–47 fraction exhibited the strongest XO inhibitory activity (IC50 = 133.17 µg/mL), while the F48–50 fraction possessed maximum antioxidant activity assessed by DPPH (2,2-diphenyl-2-picrylhydrazyl) and ABTS (2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) assays (IC50 = 0.51 and 1.04 mg/mL, respectively). In addition, the F4–5 fraction presented the most effective inhibition on the growth of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Proteus mirabilis. Gas chromatography-mass spectrophotometry (GS-MS) and liquid chromatography-electrospray ionization-mass spectrophotometry (LC-ESI-MS) results revealed that fatty acids, glycerides, and flavonoids were the major compounds of the F45–47 fraction. Glycerides, triose sugar alcohols, and fatty acids were dominant compounds of the F48–50 fraction, while sterols were principal components of the F4–5 fraction. This study indicated that T. procumbens had potent inhibitory effects on XO inhibitory, antioxidant, and antibacterial activities. These biological activities may be attributed to the presence of fatty acids, flavonoids, and sterols in this plant. It is suggested that T. procumbens can be utilized as a healthy source to develop beverages and foods to treat antihyperuricemia, oxidative stress, and bacterial infection.


Preparation of Plant Extract
An amount of 1.2 kg of T. procumbens powder was immersed in 16 L of methanol for seven days at room temperature. After filtration, the filtrate was then evaporated under vacuum at 45 • C using a rotary evaporator (SB-350-EYELA, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) to obtain 62.95 g of crude extract. This crude extract was then subsequently extracted with hexane, chloroform, EtOAc, and water to produce 10.37, 14.12, 5.42, 23.54 g extracts, respectively. The EtOAc extract, the extracts active on XO inhibitory and antioxidant activities were subsequently fractionated by column chromatography.

Preparation of Plant Extract
An amount of 1.2 kg of T. procumbens powder was immersed in 16 L of methanol for seven days at room temperature. After filtration, the filtrate was then evaporated under vacuum at 45 °C using a rotary evaporator (SB-350-EYELA, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) to obtain 62.95 g of crude extract. This crude extract was then subsequently extracted with hexane, chloroform, EtOAc, and water to produce 10.37, 14.12, 5.42, 23.54 g extracts, respectively. The EtOAc extract, the extracts active on XO inhibitory and antioxidant activities were subsequently fractionated by column chromatography.

Fractionation of Ethyl Acetate Extract
The EtOAc extract (5.42 g) was subjected to a normal-phase of column chromatography (20 mm diameter × 500 mm height, Climbing G2, Tokyo, Japan) over 30 g of silica gel (size Ǻ 60, 200-400 mesh particle size, Sigma-Aldrich). This process yielded 15 fractions ( All of these fractions were tested for XO inhibitory, antioxidant, and antibacterial activities. The most active fractions were analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-electrospray ionizationmass spectrometry (LC-ESI-MS) to determine their chemical components.

Xanthine Oxidase (XO) Inhibitory Activity
The XO inhibitory activity was assayed spectrofotometrically in vitro under aerobic condition at 290 nm based on a method reported previousy [17,24]. Briefly, a volume of 50 µL of tests solution dissolved in in phosphate buffer (pH = 7.5) contained <0.1% DMSO was mixed with 35 µL of 70 mM phosphate buffer (pH = 7.5), and 30 µL of fresh enzyme solution (0.01 units/mL in 70 mM phosphate buffer, pH = 7.5). Reaction was initiated by adding 60 µL of substrate solution (150 µM xanthine in the same buffer) after pre-incubation at 25 °C for 15 min. The assay mixture was then incubated at 25 °C for 30 min. A volume of 25 µL HCl (1 M) was added to stop the reaction and the absorbance was measured at 290 nm with a microplate reader (MultiskanTM Microplate Spectrophotometer, Thermo Fisher Scientific, Osaka, Japan). For the blank, the assay mixture was prepared in its present condition, but the enzyme solution was added after adding HCl. One unit of XO was defined as the amount of enzyme required to produce 1 µmol of uric acid/min at 25 °C. The XO inhibitory activity

Xanthine Oxidase (XO) Inhibitory Activity
The XO inhibitory activity was assayed spectrofotometrically in vitro under aerobic condition at 290 nm based on a method reported previousy [17,24]. Briefly, a volume of 50 µL of tests solution dissolved in in phosphate buffer (pH = 7.5) contained <0.1% DMSO was mixed with 35 µL of 70 mM phosphate buffer (pH = 7.5), and 30 µL of fresh enzyme solution (0.01 units/mL in 70 mM phosphate buffer, pH = 7.5). Reaction was initiated by adding 60 µL of substrate solution (150 µM xanthine in the same buffer) after pre-incubation at 25 • C for 15 min. The assay mixture was then incubated at 25 • C for 30 min. A volume of 25 µL HCl (1 M) was added to stop the reaction and the absorbance was measured at 290 nm with a microplate reader (MultiskanTM Microplate Spectrophotometer, Thermo Fisher Scientific, Osaka, Japan). For the blank, the assay mixture was prepared in its present condition, but the enzyme solution was added after adding HCl. One unit of XO was defined as the amount of enzyme required to produce 1 µmol of uric acid/min at 25 • C. The XO inhibitory activity was expressed as the percentage inhibition of XO in the above assay system and calculated by the following formula: where A was the activity of the enzyme without either test extract or fraction, B was the control of A without either test extract or fraction and enzyme, C and D were the activities of either the test extract or fraction with and without XO. Allopurinol (10-100 µg/mL) was used as a positive control. The IC 50 values were calculated from the mean values of percentage inhibition data.

DPPH Radical Scavenging Activity
The 1,1-diphenyl-2-picryhydrazyl (DPPH) was used to evaluated radical scavenging activity as described previously [25]. The mixture assay consisted of 100 µL sample dissolved in MeOH, 50 µL of 0.2 mM DPPH solution, and 100 µL of 0.1 M acetate buffer (pH 5.5) were put in a 96-wells microplate and incubated at room temperature in the dark condition for 30 min. The absorbance was recorded at 517 nm using a microplate reader (Multiskan TM Microplate Spectrophotometer, Thermo Fisher Scientific, Osaka, Japan) and butylated hydroxytoluene (BHT) (10-100 µg/mL) was used as a positive control. Percentage of inhibition was calculated according to the formula: The Abs control was the absorbance of reaction without sample and Abs sample was the absorbance of reaction with the sample. The IC 50 values were the concentrations required to give 50% DPPH radical scavenging activity, were also calculated.

ABTS Radical Scavenging Activity
The 2,2 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS) solution was used to examine the radical scavenging activity followed a method previously described [25] with slight modifications. The ABTS solution was generated by a reaction of 7 mM ABTS and 2.45 mM potassium persulfate solution after incubation at room temperature in the dark for 16 h. The mixture was then diluted with methanol to obtain an absorbance of 0.70 ± 0.05 at 734 nm. In brief, a volume of 24 µL sample dissolved in MeOH was mixed with 120 µL of ABTS solution, and the mixture was left in the dark at room temperature for 30 min. The absorbance was recorded at 734 nm using microplate a reader (Multiskan TM Microplate Spectrophotometer, Thermo Fisher Scientific, Osaka, Japan). BHT standard (5-125 µg/mL) was used as a positive control. The ABTS radical scavenging activity was calculated by the following equation: The Abs control was the absorbance of reaction without sample and Abs sample was the absorbance of reaction with the sample. The IC 50 values were determined as the inhibitory concentration of the samples necessary to reduce the ABTS radical cation concentration by 50% and were expressed in mg/mL.

Antibacterial Activity
The disk diffusion method described previously [26] was used for antibacterial assay. Four bacteria strains, including Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Bacillus subtilis, were grown in Luria-Bertani (LB) broth medium by incubated at 37 • C for 24 h. The final population was standardized to be 1.45 × 10 6 CFU/mL (E. coli), 2.87 × 10 6 CFU/mL (P. mirabilis), 1.29 × 10 6 CFU/mL (S. aureus), and 1.63× 10 6 CFU/mL (B. subtilis). A volume of 100 µL of bacteria culture was covered evenly on agar-LB broth medium in a Petri dish (diameter = 9 cm). Afterward, a volume of 20 µL of sample dissolved in MeOH was applied into filter paper (6 mm diameter) and placed on the surface of LB agar plates. After 24 h incubation at 37 • C, the inhibition zones were measured. Streptomycin and ampicillin were used as the positive controls in this experiment. The concentrations of the samples ranged from 1.25 to 40 mg/mL (40,30,25,20, 10, 5, 2.5, 1.5, 1.25 mg/mL). The lowest concentration that inhibited the visible bacterial growth was determined as minimal inhibitory concentration (MIC). Streptomycin and ampicillin (1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.0195, 0.0097, 0.0048, 0.0024, 0.0012, 0.0006 mg/mL) were used as positive control in this experiment. Subsequently, MeOH was used as a negative control.

Determination of Total Phenolic Contents
The total phenolic contents of the samples were measured by the Folin Ciocalteu (FC) reagent following a method reported previously [27] with some modifications. Briefly, a volume of 20 µL of either sample solution (1.0 mg/mL), or gallic acid standard solution (5-25 µg/mL) was pipetted into separate wells of a 96-well microplate. Then a volume of 100 µL of the FC reagent (10% v/v in water) was added to each well, thoroughly mixed, and an aliquot of 80 µL sodium carbonate (5% w/v in water) was then added. The reaction was carried out for 30 min at room temperature. The absorbance was read at 765 nm using a microplate reader (Multiskan TM Microplate Spectrophotometer, Thermo Fisher Scientific, Osaka, Japan). The total phenolic contents were expressed as mg gallic acid equivalent (GAE) per gram of extract or fraction (r 2 = 0.996).

Determination of Total Flavonoid Contents
The total flavonoid contents were assessed by a colorimetric assay as described previously [28], with some modifications. Briefly, a volume of either 100 µL sample (1 mg/mL) or quercetin standard (5-25 µg/mL) was mixed with 100 µL aluminum (III) chloride hexahydrate (2% w/v in water) in a 96-well microplate. After a 15-min incubation at room temperature, the absorbance of the reaction mixture was measured at 430 nm using a microplate reader (Multiskan TM Microplate Spectrophotometer, Thermo Fisher Scientific, Osaka, Japan). The total flavonoid contents were expressed as mg quercetin equivalent (QE) per gram of extract or fraction (r 2 = 0.999).

Identification of Chemical Constituents by Gas Chromatography-Mass Spectrometry (GC-MS)
A volume of 1 µL of sample was injected into a GC-MS system (JMS-T100 GCV, JEOL Ltd., Tokyo, Japan). The column was DB-5MS with 30 m in length, 0.25 mm internal diameter, and 0.25 µm in thickness (Agilent Technologies, J&W Scientific Products, Folsom, CA, USA). Helium was chosen as the carrier gas, and the split ratio was 5.0/1.0. The operating condition of GC oven temperature was maintained as follows: The initial temperature = 50 • C without hold time, the programmed rate = 10 • C/min up to a final temperature of 300 • C with 20 min for hold time. The injector and detector temperatures were set at 300 • C and 320 • C, respectively. The mass range scanned from 29-800 amu. The control of the GC-MS system and the data peak processing were carried out using the JEOL's GC-MS Mass Center System version 2.65a software (JEOL Ltd., Tokyo, Japan).

Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) Analysis
Identification of the most active fraction was performed by LC-ESI-MS system (Thermo Fisher Scientific TM , LTQ XL TM , Ion Trap Mass Spectrometer, Tokyo, Japan). The column for LC system was JASCO J-Pak Symphonia C18 (250 mm × 4.6 mm × 5 µm). The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The proportion of solvents A:B was 30:70 and the flowrate was 0.4 mL/min. Operation time of this analysis was 30 min and the volume of sample injection was 5 µL. For ESI-MS conditions, the ionization method was electrospray (ESI). The flow rate of sheath gas was 60, while auxiliary gas was 20 arbitrary units of sheath gas. The spray voltage was 4.5 kV. The measurements were performed in the positive mode. For positive polarity, fourier transform mass spectrometry (FTMS)/orbitrap with 60,000 resolution and scan range 100-1000 m/z was applied for mass analyzer. For negative polarity, ion trap mass spectrometer (ITMS)/linear ion trap with scan range 115-1000 m/z was employed [29]. Peak processing was conducted using Thermo Xcalibur Qual Browser software (Thermoscientific TM , Tokyo, Japan) equipped with NIST MS Library.

Statistical Analysis
The statistical analysis was performed by one-way ANOVA using Minitab ® 16.2.3 (copyright© 2012 Minitab Inc., Philadelphia, PA, USA). The results were presented as means ± standard deviation (SD) values. Differences among treatments, controls and standard data were considered significant at p < 0.05 using Fisher's test.

XO Inhibitory and Antioxidant Activities, Total Phenolic and Flavonoid Contents of T. procumbens Extracts
The XO inhibitory and antioxidant activities of T. procumbens extracts were shown in Table 1. The results indicated that EtOAc extract exhibited the strongest inhibitory activity in both XO and antioxidant assays. At 0.1 µg/mL dose, the EtOAc extract inhibited 19.44% of XO, while the others showed negligible inhibition. In antioxidant activity, the EtOAc extract also possessed maximum inhibition on antioxidant activity. The IC 50 values of EtOAc extracts of both DPPH and ABTS radical scavenging assays were the lowest (0.13 and 0.45 mg/mL, respectively). It was observed that XO inhibitory and antioxidant activity of T. procumbens extracts were proportional with total phenolic and flavonoids contents.

Antibacterial Activity of T. procumbens Extracts
In the antibacterial activity assay, different extracts of T. procumbens were tested against four bacteria strain, including E. coli, P. mirabilis, S. auereus, B. subtilis. The EtOAc extract had the strongest inhibitory effects on all bacteria strain (minimum inhibitory activity = 25-10 mg/mL). (Table 2).
Hexane extract gave inhibition on E. coli and S. auereus, while chloroform extract only inhibited S. auereus.

XO Inhibitory and Antioxidant Activities of T. procumbens Fractions
All of the 15 fractions separated from the EtOAc extract of T. procumbens were tested for XO inhibitory and antioxidant activities (Table 3). In the XO inhibitory activity, the F 45-47 fraction showed the most potential inhibition (IC 50 = 133.17 µg/mL) followed by the F 27-33 and F 22-26 fractions (IC 50 = 150.71 and 188.04 µg/mL, respectively). Other fractions possessed trivial inhibitory activities which were not considerable enough to calculate IC 50 values. Compared with allopurinol, all fractions presented lower inhibitory levels. In antioxidant activity, most of the fractions possessed both DPPH and ABTS radical scavenging activities. The F 48-50 fraction showed the most effective antioxidant potential (IC 50 DPPH and ABTS were 0.51 and 1.04 mg/mL, respectively).

Antibacterial Activity of T. procumbens Fractions
Antibacterial activity of T. procumbens was assayed on Gram positive (E. coli and P. mirabilis) and negative (S. auereus and B. subtilis) bacteria. The inhibitory effects of T. procumbens fractions on tested bacteria were illustrated in Table 4. The inhibition levels of tested fractions on four bacteria strain were varied. The F 4-5 fraction was the best candidate to inhibit the growth of all tested bacteria (MIC = 15-25 mg/mL). All fractions showed lower inhibitory levels than ampicillin and streptomycin which were used as the positive controls. Ampicillin and streptomycin gave MIC values of 0.0012-0.039 and 0.156 mg/mL respectively.

Total Phenolic Contents (TPC) and Total Flavonoid Contents (TFC) of T. procumbens Fractions
The TPC and TFC of all fractions were illustrated in Figure 2. The F 27-33 fraction showed the highest phenolic contents, while F 16-17 fraction exhibited the maximal flavonoids contents. Nevertheless, neither F 27-33 nor F 16-17 fractions provided maximum inhibition on XO inhibitory, antioxidant, and antibacterial activities. Besides phenolic and flavonoid compounds, constituents belonging to other chemical classes probably related to these inhibitory activities.

Compounds Identification by Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS)
The identification of the most active fraction in XO inhibitory (F45-47 fraction), antioxidant (F48-50 fraction), and antibacterial (F4-5 fraction) activities was conducted by using GC-MS and LC-ESI-MS (Supplementary Materials Figures S1-S15). The results were summarized in Table 4. Chemicals belonging to fatty acids, glycerides, and flavonoid groups were detected in the F48-50 fraction. In this

Discussion
In this study, we screened bioactive compounds from T. procumbens using extracting solvents with different polarities by column chromatography. The EtOAc extract of this plant gave the most inhibitory effects in XO, antibacterial, and antioxidant assays. This extract was subsequently selected and fractionated by column chromatography eluting by chloroform and methanol (10:0 to 0:10 v/v) to yield 15 fractions. The levels of biological activities as mentioned above of each fraction were then evaluated in vitro. It was found that T. procumbens fractions showed potent XO inhibitory antioxidant, and antibacterial activities. To date, XO inhibitors, such as flavonoids [30], deoxyadenosines for example cordycepin [31], lanostanoids [32], and phenolics [33], have been reported. In this study, we detected n-hexadecanoid acid, 2-monopalmitin, and centaureidin in the F 45-47 fraction (Table 4) involved in XO inhibitory activity.
Centaureidin is one type of flavonoid compounds identified from T. procumbens [5]. In line with this study, Nagao et al., [30] reported that various dietary flavonoids to have inhibitory activity on XO. From chemical structure and binding affinities of flavonoids acting as XO inhibitors, Lin et al., [34] reported that generally, hydrophobic interaction was essential in the binding of flavonoids to XO inhibition. The planar structure and double bond C 2 = C 3 of flavonoids are advantageous for XO suppressive potential [34]. In this study, due to the inhibitory effects of the F 45-47 fraction was potent on XO, the interaction of fatty acids, glycerides, and flavonoids responsible XO inhibition were also needed to clarify.
Regarding antioxidant activity, the F 48-50 fraction showed the highest radical scavenging activities both in DPPH and ABTS assays. Of which, 2-monopalmitin, glycerin, and methyl palmitate were the dominant compounds detected. Similar to this result, Mohadjerani et al. [35] reported that fatty acids and their derivatives have been reported to have antioxidant activity. Methyl palmitate found in this fraction (Table 4) may also play a role in the antioxidant activity of the plant.
In antibacterial activity, the F 4-5 fraction presented the maximal inhibitory effects on Gram positive (E. coli and P. mirabilis) and negative (S. auereus and B. subtilis) bacteria. The GC-MS and ESI-MS results revealed that stigmasterol, β-sitosterol, and n-hexadecanoid acid were the principal compounds of this fraction. In a previous report, Sharma [36], noted that stigmasterol and β-sitosterol had a wide spectrum of antibacterial activity. These sterols showed effective inhibition on S. aureus and S. albus (Gram positive), and E. coli and Pseudonomas pyocyeanea (Gram negative). In addition, Desbois and Smith [37], reported that n-hexadecanoid acid inhibited both Gram positive and negative bacteria. The insertion of fatty acid into the bacterial inner membrane increased the permeability of the membrane, causes internal contents to leak from the cell, and induces growth inhibition or even death [38]. It has been revealed that bioactive compounds from medicinal plants appeared as promising sources for natural beverages and foods [39][40][41][42][43]. Observations of this study highlighted that T. procumbens possessed potent constituents and may be served as a healthy source to develop healthy drinks and foods, especially in developing countries, where this plant is abundantly available.

Conclusions
Gout is a problematic issue of health in both developed and developing countries, of which utilization of natural products with high effectiveness and less undesirable effects to treat this problematic disease is required. In this study, we found that T. procumbens was exhibited strong inhibitory effects on XO, antioxidant and growth of four bacteria strains E. coli, P. mirabilis, S. aureus, and B. subtilis. The GC-MS and LC-ESI-MS results revealed that fatty acids, glycerides, and flavonoids may contribute to XO inhibitory activity, while sterols and fatty acids may play a role in the antibacterial property. In antioxidant activity, glycerides, triose sugar alcohols, terpenes, and fatty acids may be active on the radical scavenging inhibitory assay. Findings of this study suggested that T. procumbens is a promising source to develop beverages and foods to treat hyperuricemia, oxidative stress, and bacterial infection.