International Journal of Food Properties

ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: https://tandfonline.com/loi/ljfp20

Antioxidant and Xanthine Oxidase Inhibitory

Activities of Persicaria hydropiper

Noor Haslinda Noor Hashim , Faridah Abas , Khozirah Shaari & Nordin H.
Lajis

To cite this article: Noor Haslinda Noor Hashim , Faridah Abas , Khozirah Shaari & Nordin H. Lajis
(2013) Antioxidant and Xanthine Oxidase Inhibitory Activities of Persicaria�hydropiper , International
Journal of Food Properties, 16:5, 1028-1036, DOI: 10.1080/10942912.2011.575497

To link to this article: https://doi.org/10.1080/10942912.2011.575497

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International Journal of Food Properties, 16:1028–1036, 2013
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942912.2011.575497

ANTIOXIDANT AND XANTHINE OXIDASE INHIBITORY

ACTIVITIES OF PERSICARIA HYDROPIPER

Noor Haslinda Noor Hashim1 , Faridah Abas1,2 ,

Khozirah Shaari1 , and Nordin H. Lajis1
1
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra
Malaysia, Serdang, Selangor, Malaysia
2
Department of Food Science, Faculty of Food Science and Technology, Universiti
Putra Malaysia, Serdang, Selangor, Malaysia

Five different polarity fractions of methanolic extract from Persicaria hydropiper, which are
consumed as vegetables, were evaluated for its total phenolic content, and antioxidant activ-
ities 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 per-
oxidation 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,

Received 28 December 2010; accepted 23 March 2011.

Address correspondence to Faridah Abas, Department of Food Science, Faculty of Food Science
and Technology, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia. E-mail: faridah_abas@putra.
upm.edu.my

1028
 OXIDASE INHIBITORY ACTIVITIES OF PERSICARIA HYDROPIPER 1029

atherogenesis, heart, and neurodegenerative diseases.[10] For protection against free radi-
cal, 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 con-
tents 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 fraction-
ated 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, ammo-
nium 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 (Na2 CO3 ), 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,
1030 NOOR HASHIM ET AL.

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 Na2 CO3 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 pre-
pared 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 com-
parison 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
 OXIDASE INHIBITORY ACTIVITIES OF PERSICARIA HYDROPIPER 1031

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 con-
taining 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:

% inhibition = (1 - (test inclination/blank inclination)) × 100,

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 (SPSSTM ) soft-
ware 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 king-
dom 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 R2 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).
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
 Table 1 Total phenolic content and antioxidant activities of methanolic extract and fractions of P. hydropiper.

Yield Total phenolic content Ferric thiocyanate DPPH radical scavenging Xanthine oxidase inhibitory
Sample (w/w) (%) (mg GAE/100 g dry extract)y,z (% inhibition)y,z activity (IC50 value, μg/ml)y,z effect (IC50 value, μg/ml)y,z

Crude methanol 78.00 419.86 ± 1.34a 87.60b 25.89 ± 0.13a 29.27 ± 5.43a
Hexane 9.63 4.34 ± 0.90b 95.67a 378.11 ± 0.15b NA
Dichloromethane 6.05 8.74 ± 1.53c 98.18a 94.2 ± 0.37c NA
Ethyl acetate 13.24 68.95 ± 3.93d 98.49a 25.55 ± 0.13a 165.25 ± 2.97b
Butanol 50.80 224.38 ± 3.38e 98.98a 28.61 ± 0.3d 28.72 ± 7.61a

1032
Aqueous 20.22 6.39 ± 1.50c 98.87a 104.56 ± 0.11e NA
Quercetin ND ND ND 6.11 ± 0.11f ND
Allopurinol ND ND ND ND 6.76 ± 0.11c
BHT ND ND 99.85a ND ND
Vitamin E ND ND 85.56b ND ND
z Each
experiment was performed in triplicates.
y Values
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.
 OXIDASE INHIBITORY ACTIVITIES OF PERSICARIA HYDROPIPER 1033

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 oxi-
dation 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 deter-
mined 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
1034 NOOR HASHIM ET AL.

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 inhibi-
tion 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 (R2 = 0.263), FTC (R2 = 0.216), and xanthine
oxidase inhibitory activity (R2 = 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 (R2 = 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.
 OXIDASE INHIBITORY ACTIVITIES OF PERSICARIA HYDROPIPER 1035

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