Antioxidant and Xanthine Oxidase Inhibitory Activities of Persicaria hydropiper

Abstract

Five different polarity fractions of methanolic extract from Persicaria hydropiper, which are consumed as vegetables, were evaluated for its total phenolic content, and antioxidant activities by 1,1-diphenyl-2-picrylhydrazyl radical scavenging, ferric thiocyanate, and xanthine oxidase inhibition assays. Particularly, higher phenolic content was exhibited by butanol and ethyl acetate fractions with the values of 224.38 and 68.95 mg GAE/100 g dry extract, respectively. Both butanol and ethyl acetate fractions exhibited higher 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with IC₅₀ values of 28.61 and 25.55 μg/ml. Meanwhile, both fractions also were shown to inhibit xanthine oxidase activity compared to other fractions with IC₅₀ values of 28.72 and 165.25 μg/ml. As for the ferric thiocyanate method, all the fractions except hexane fraction showed similar activity against lipid peroxidation and were comparable to butylated hydroxyl toluene, with percentage of inhibition from 95 to 98%.

Keywords:

• Persicaria hydropiper
• Antioxidant activity
• 1,1-Diphenyl-2-picrylhidrazyl (DPPH)
• Ferric thiocyanate (FTC)
• Xanthine oxidase

INTRODUCTION

Persicaria hydropiper (Polygonum minus or Polygonum hydropiper) is a popular herb from the family Polygonaceae, locally known as ‘kesum.’ The plant is well known for its use as a flavoring for food and for medicinal uses.[1] In traditional medicine, the juice of the leaves is used for headache, pain, toothache, gastric ulcer, dysentery, loss of appetite, and dismenorrhea.[2] In Japan, the sprout of P. hydropiper is a well-known traditional vegetable.[3] Results of a previous study reported a diverse array of compounds, which included flavonoids, chalcones, sesquiterpenoids, coumarins, and stilbene glycosides.[1,4–9]

Antioxidants are vital substances that protect the body from damage caused by free radical induced oxidative stress. Free radicals or reactive oxygen species have been shown to be the causative agents in aging and several degenerative diseases, such as cancer, atherogenesis, heart, and neurodegenerative diseases.[10] For protection against free radical, humans rely on antioxidants produced by the body and those obtained from their diet. Thus, the consumption of high sources of antioxidants, such as vegetables and fruits as well as vitamin A, C, E, carotenoids, polyphenolic compounds, and flavonoids, would prevent free radical damage, reducing risk of chronic diseases.[10] The use of synthetic antioxidants, such as BHA (butylated hydroxyl anisole) and BHT (butylated hydroxyl toluene), are very effective but they may possess some side effects and toxic properties to human health.[11]

Over the past decade, evidence has shown that plant polyphenols, especially flavonoids, play an essential role in various biochemical and physiological processes.[11] The activity of flavonoids towards anticancer activity, anticonceptive, anti-inflammatory effects, especially free radicals and reactive oxygen species, draw attention to the health-promoting effects.[12] The presence of flavonoids, which have been reported for antioxidant properties, could be the basis to observe the antioxidant activity.[13] Previously it was reported that the crude methanolic extract of P. hydropiper exhibited strong antioxidant activity.[14] Hence, the present work was carried out to explore the total phenolic contents and antioxidant potential of crude methanolic extracts as well as the different polarity fractions of this plant.

MATERIALS AND METHODS

Plant Material

Persicaria hydropiper was obtained from the medicinal plant nursery of the Laboratory of Natural Products, Universiti Putra Malaysia and was identified by Mr. Shamsul Khamis. A voucher specimen (SK154/02) was deposited at the herbarium of the Laboratory of Natural Products.

Extraction and Fractionation of MeOH Extract

The fresh plant material (800 g) was cut into small pieces and air-dried under the shade. The samples were then ground into fine powder and extracted three times with methanol, each time by soaking in 1.5 L of solvent overnight. The plants extracts were filtered through Whatman No. 1 filter paper (Whatman Ltd., Maidstone, England) and evaporated under reduced pressure to give 220 g of methanolic extract. A portion of the extract (78 g) was redissolved in 200 ml of water: MeOH (3:1) mixture and fractionated with hexane, dichloromethane, ethyl acetate, and butanol (3 × 200 ml each fraction). The fractionation afforded five different polarity fractions (hexane, dichloromethane, ethyl acetate, butanol, and aqueous), which were then subjected to bioassays.

Chemicals

Ethanol was purchased from Scharlau (Barcelona, Spain). Water was purified by a MiliQ system (Milipore, Bedford, MA, USA). Linoleic acid, α-tocopherol, DPPH, ammonium thiocyanate, and ferrous chloride were purchased from Sigma (St. Louis, MO, USA). Gallic acid was purchased from Acros Organics (Fair Lawn, NJ, USA). Anhydrous sodium carbonate (Na₂CO₃), Folin-Ciocalteu phenol reagent, and hydrochloric acid (HCl) were obtained from Merck (Darmstadt, Germany). Xanthine oxidase (EC 1.1.3.22) from cow's milk (1.3 units/mL), Xanthine, and allopurinol was obtained from Sigma (St. Louis, MO, USA). The buffer used was 1/15 M potassium phosphate-sodium phosphate buffer, pH 7.5 (Sigma, St. Louis, MO, USA). The substrate solution, 0.15 mM xanthine in water, and the enzyme solution, 0.28 units/mL in 1/5 M phosphate buffer (pH 7.5) were prepared immediately before use.

Total Phenolic Contents

Total phenolic content (TPC) was determined using the Folin-Ciocalteu method.[15] Briefly, an aliquot of 0.5 ml of an extract was mixed with 0.5 ml of Folin-Ciocalteu phenol reagent and allowed to react for 5 min. Then 10 ml of 7% of Na₂CO₃ solution was added and allowed to react for 1 h before the absorbance of the reaction mixture was read at 725 nm. A standard curve of Gallic acid was constructed (0–100 ppm) and TPC content was expressed as mg gallic acid equivalent per 100 grams of dry extract.

Antioxidant Using Ferric Thiocyanate and Free Radical Scavenging Activity (DPPH) Activity

The potential antioxidant activities of methanolic extract and fractions of P. hydropiper were assessed using ferric thiocyanate lipid peroxidation inhibition and the scavenging activity of the stable 1,1-diphenyl-2-picrylhidrazyl (DPPH) free radical based on the previously described protocols.[14] Different concentrations of test samples were prepared in 96 microtiter plates for determination on radical scavenging. Reaction mixtures consist of 100 μl of test sample (various samples dissolved in methanol) and 5 μl of DPPH in methanol (300 μM). These reaction mixtures were incubated for 30 min, and absorbance was measured at 517 nm. Percent inhibition by sample treatment was determined by comparison with the methanol treated control group. The IC₅₀ values denote the concentration of each sample required to give 50% of the optical density shown by the control. All test analyses were run in triplicate and averaged. Quercetin was used as positive controls.

For ferric thiocyanate assay, a mixture of 4 mg of samples (final concentration 0.02% w/v) in 4 ml of 99.5% ethanol, 4.1 ml of 2.51% linoleic acid in 99.5% ethanol, 8.0 ml of 0.02 M phosphate buffer (pH 7.0), and 3.9 ml of distilled water contained in a screw-cap vial (Ø 38 × 75 mm) was placed in an oven at 40°C in the dark. To measure the extent of antioxidant activity, 0.1 ml of the reaction mixture was transferred to a test tube (Ø 13 × 150 mm) and to it, 9.7 ml of 75% (v/v) aqueous ethanol followed by 0.1 ml of 30% aqueous ammonium thiocyanate and 0.1 ml of 0.02 M ferrous chloride in 3.5% hydrochloric acid was added. Three minutes after the addition of ferrous chloride to the reaction mixture, the absorbance was measured at 500 nm. The measurement was taken every 24 h until 1 day after absorbance of the control reached its maximum value. BHT, quercetin, and α-tocopherol were used as standard antioxidants. Results are reported as mean ± SEM values. All in vitro experiments were conducted three times, each time with three or more independent observations.

Xanthine Oxidase Inhibition Activity

The xanthine oxidase inhibition activity with xanthine as the substrate was measured spectrometrically based on the procedure reported with slight modification.[16] The assay mixture consisted of 0.036 ml of extract solution, 0.055 ml of 1/15 M phosphate buffer (pH 7.5), and 0.014 ml of enzyme solution. To ensure that there was no absorbance change due to the plant material, after preincubation of the mixture at 25°C for 1 min, the absorbance (295 nm) was measured spectrophotometrically every 12 s for 2 min. Then, the reaction was initiated by adding 0.107 ml of substrate solution (0.6 mM in water). The assay mixture was incubated at 25°C with the absorbance of 295 nm measured spectrophotometrically every 6 s, using a SpectraMax spectrophotometer (SpectraMax Plus, Molecular Devices, CA, USA) at a temperature control unit and software. All test analyses were run in triplicate and averaged. A negative control (blank; 0% XO inhibition activity) was prepared containing methanol solution (1%, v/v/0.1%, w/v in assay mixture) without extract solution. Allopurinol, a known inhibitor of XO, was used as a positive control (final concentration, 10 μg/ml). XO inhibitory activity was expressed as the percentage inhibition of XO in the above assay mixture system, and is calculated as:

where test inclination is the linear change in absorbance per minute of test material, and blank inclination is the linear change in absorbance per minute of blank.

Statistical Analysis

Data were analyzed using the Statistical Package for Social Science (SPSS™) software for Windows, Version 16.0 (SPSS Inc., Chicago, IL, USA). Differences in means were determined using ANOVA. Results are expressed as a mean of three determinations ± SD. Significance level was set as p < 0.05.

RESULTS AND DISCUSSION

Total Phenolic Content

It is well known that plant polyphenols are widely distributed in the plant kingdom and play an important role in stabilizing lipid oxidation associated with antioxidant activity.[10] There was a wide range of phenolic concentrations in the crude methanol and fractions as shown in Table 1. A linear calibration curve of gallic acid, in the range of 20–100 μg/ml with R ² value of 0.9943, was constructed. As the yield of extract varied significantly, the amount of total phenolics in fractions of P. hydropiper also varied widely and ranged from 0.00 to 224.38 mg gallic acid equivalents (GAE)/100 g dry extract as measured by the Folin-Ciocalteu method. Among those fractions, low levels of phenolics were found in hexane, aqueous, and dichloromethane fractions (4.34–8.74 mg GAE/100 g dry extract), whereas ethyl acetate fraction contains relatively high amounts of phenolic (68.95 mg GAE/100 g dry extract). Crude methanolic extract and butanol fraction had considerably high contents of phenolic substances (229.38–419.86 mg GAE/100 g dry extract).

Table 1  Total phenolic content and antioxidant activities of methanolic extract and fractions of P. hydropiper

Sample	Yield (w/w) (%)	Total phenolic content (mg GAE/100 g dry extract)^y,z	Ferric thiocyanate (% inhibition)^y,z	DPPH radical scavenging activity (IC50 value, μg/ml)^y,z	Xanthine oxidase inhibitory effect (IC50 value, μg/ml)^y,z
Crude methanol	78.00	419.86 ± 1.34^a	87.60^b	25.89 ± 0.13^a	29.27 ± 5.43^a
Hexane	9.63	4.34 ± 0.90^b	95.67^a	378.11 ± 0.15^b	NA
Dichloromethane	6.05	8.74 ± 1.53^c	98.18^a	94.2 ± 0.37^c	NA
Ethyl acetate	13.24	68.95 ± 3.93^d	98.49^a	25.55 ± 0.13^a	165.25 ± 2.97^b
Butanol	50.80	224.38 ± 3.38^e	98.98^a	28.61 ± 0.3^d	28.72 ± 7.61^a
Aqueous	20.22	6.39 ± 1.50^c	98.87^a	104.56 ± 0.11^e	NA
Quercetin	ND	ND	ND	6.11 ± 0.11^f	ND
Allopurinol	ND	ND	ND	ND	6.76 ± 0.11^c
BHT	ND	ND	99.85^a	ND	ND
Vitamin E	ND	ND	85.56^b	ND	ND
^zEach experiment was performed in triplicates.
^yValues with the same lowercase letters are not significantly different (p < 0.05) according to Duncan Test, SPSS Version 16 (n = 3 ± SD).
ND: Not determined.

Previous reports on different extracts of P. hydropiper confirmed that this plant had high phenolic contents.[17–20] However, the amount of TPC in crude methanolic extracts in this study is not comparable to that reported in a previous study. The differences in TPC levels may be attributed to different plants extracts, procedures, and standards used to express the TPCs.[18] Besides polyphenol, there could be other substances that react and are oxidized with Folin reagent.[11] The responses of phenolic compounds to the Folin-Ciocalteu reagent depend on the number of phenolic groups.[11,17] Phenolic compounds have been reported to possess antioxidant effects, including chelating metals, scavenging radicals, and reducing capability.[21] Therefore, a higher content of phenolic compounds in the fractions may imply a stronger antioxidant capacity.

Antioxidant Using Ferric Thiocyanate (FTC)

The FTC method measures the amount of peroxide at the primary stage in linoleic acid peroxidation. From the analysis, it shows that all samples effectively inhibit lipid oxidation when compared to vitamin E (α-tocopherol). As shown in Table 1, the percentage inhibition ranged from 95.6–98.9%. The percentage inhibition observed for vitamin E and BHT was 85.6 and 99.8%, respectively. Except for the hexane fraction, which exhibited a percent of inhibition of 95.6%, all other fractions showed percent inhibition of more than 98% in this assay and were comparable to butylated hydroxytoluene (BHT), a synthetic antioxidant. During the oxidation process, the concentration of the peroxide decreased as the antioxidant activity increased, the intensity of the reddish pigment will reduce, leading to lower absorbance value.

DPPH Free Radical Scavenging Activity

The DPPH free radical scavenging activity was widely used in the determination of antioxidant. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable molecule.[22] The radical scavenging activity of the fractions was determined from the reduction in the optical absorbance at 517 nm due to the scavenging of the stable diphenyl-p-picrylhydrazyl (DPPH) radical. The rapid reduction of absorbance caused the antioxidant activity to become more potent in terms of hydrogen donating.

The crude extract of P. hydropiper showed strong free radical scavengers with an IC₅₀ value of 25.8 μg/ml. Among the fractions, ethyl acetate and butanol fractions of P. hydropiper were the most active fractions with the IC₅₀ values of 25.5 and 28.6 μg/ml, respectively, while hexane fraction showed the lowest activity (Table 1). Besides, it has been reported that phenolic and flavonoids compounds are the most ubiquitous antioxidant phytochemicals in the plant kingdom and possess both singlet oxygen quenching activity and radical scavenging activity.[10,23] The scavenging activity was reported to depend on the structure of a C ring in the flavonoids skeleton. Most researchers agree that 2,3-double bond with both 4 keto group and 3-hydroxyl group in the C ring and ortho 3′,4′-dihydroxy moiety in B ring enhance the scavenging activity.[21]

Inhibition of Xanthine Oxidase Activity

Xanthine oxidase (XO) is a flavoprotein, which has been reported to increase its activity during oxidative stress, catalyzes the oxidation of hypoxanthine to xanthine, and generates superoxide and uric acid. Inhibition of this enzyme is measured by decreased uric acid production. The generation of uric acid and superoxide anion radical in excess can cause hyperuricemia, associated with gout. The XO inhibitor, allopurinol, may be useful for the treatment of chronic gout. However, allopurinol exhibits some negative effects, such as hepatitis, nephropathy, and allergic reaction.[24] Hence, there is importance to search for a new XO inhibitor.

The investigation on the inhibitory activity of the crude methanol showed that the crude extract inhibits the xanthine oxidase at 100 μg/ml. Based on the result of crude extract, the fractions of P. hydropiper were assayed. Of the fractions assayed, the butanol and ethyl acetate fractions demonstrated xanthine oxidase activity with inhibitory effects greater than 50%. The IC₅₀ values of ethyl acetate and butanol was 166.9 and 32.3 μg/ml, respectively, as shown in Table 1. However, the positive control, allopurinol exhibits a higher activity compared to butanol and ethyl acetate. The other fractions were considered to have minimal or no XO inhibitory activity.

The effects of XO to the flavonoids, polyphenols, tannins, and coumarins have been reported to be potent XO inhibitors.[25] Studies on XO inhibition on flavonoids revealed that the inhibitory activity depends on the location of hydroxyl moiety in the skeleton as well as the presence of a single and double bond. Accordingly, the hydroxyl groups at C-5 and C-7 and the double bond between C-2 and C-3 are important, which increased the inhibition activity. However, the presence of a hydroxyl group at C-3, C-2, and C-8 decreases the inhibitory activity. These indicate that flavones showed slightly higher inhibitory activity than flavonols. The number and position of glycosyl groups on a flavonoid also decreases inhibition, resulting in reduced contact of the glycosidic flavonoid with the enzyme.[26] Therefore, phenolic contents of the fractions made an important contribution of XO inhibition.

Correlation Between Antioxidant Activity and Total Phenolic Content

The results obtained showed that there were weak correlations between TPC and the antioxidant activity based on the DPPH (R ² = 0.263), FTC (R ² = 0.216), and xanthine oxidase inhibitory activity (R ² = 0.688). The difference in correlations between TPCs and antioxidant activity might be due to different methods of antioxidant activity with different mechanisms.[11] In addition, the Folin-Ciocalteu provides a crude estimate of the phenolic content present in fractions, whereas free radical scavenging assay is not only specific to polyphenols.[27] The synergism effects among the antioxidant compounds in the mixture may have contributed to the antioxidant activity.[11] Besides, the number of hydroxyl groups in phenolic compounds respond differently in DPPH assay.[15] The phenolic compounds may also donate the hydrogen and terminate the free radical reaction chain by converting to stable compounds.[28]

CONCLUSION

As a conclusion, it was found that the methanolic extract, ethyl acetate, and butanol fractions of P. hydropiper demonstrated antioxidant activities. However, there were weak correlations between TPC and antioxidant activities (R ² = 0.263–0.688). Since the methanolic extract and fractions of P. hydropiper have shown potential as a source of antioxidant, further studies need to be carried out to isolate and characterize the active compounds that are responsible for the antioxidant activities, especially from ethyl acetate and butanol fractions. The evidence from this study could justify that the consumption of P. hydropiper can be a cheap and practical approach to the prevention of disease, especially those related to aging.

ACKNOWLEDGMENTS

The authors wish to thank Universiti Putra Malaysia and Ministry of Science, Technology and Innovation (MOSTI) for the grant provided (Grant no: 5450255 to Faridah Abas). Noor Haslinda Noor Hashim also gratefully acknowledges support by the MOSTI for a NSF scholarship.