| |

Received: 22 April 2021    Revised: 15 July 2021    Accepted: 11 August 2021

DOI: 10.1111/jfbc.13909

ORIGINAL ARTICLE

Polygonum odoratum leaf extract attenuates oxidative stress

and cell death of Raw 264.7 cells exposed to low dose ionizing
radiation

Supannika Kawvised1  | Thawatchai Prabsattroo2,3  | Waranon Munkong2,3  |

Panuwat Pattum2,3  | Sittichai Iamsaard3,4  | Kanokporn Boonsirichai5  |
Pimporn Uttayarat5  | Lamai Maikaeo5  | Waraporn Sudchai5  |
Woranan Kirisattayakul2,3

1
Radiological Technology School, Faculty of
Health Science Technology, HRH Princess Abstract
Chulabhorn College of Medical Science, This study aims to investigate the effect of Polygonum odoratum leaf extract (POE) on
Chulabhorn Royal Academy, Bangkok,
Thailand oxidative stress markers and cell death induced by low dose ionizing radiation (LDIR)
2
Department of Radiology, Faculty of in Raw 264.7 cells. The biological activities, chromatographic fingerprint, and cyto-
Medicine, Khon Kaen University, Khon
toxicity of POE were investigated. To determine the radioprotective effect of POE,
Kaen, Thailand
3
Neurovascular Radiology and
Raw 264.7 cells were incubated with POE for 1 hr prior to 100 mGy x-­irradiation.
Neurointervention Research Group, Faculty The cell viability, oxidative stress damage marker (malondialdehyde level; MDA), and
of Medicine, Khon Kaen University, Khon
Kaen, Thailand
endogenous antioxidant markers (superoxide dismutase: SOD, catalase: CAT, and
4
Department of Anatomy, Faculty of glutathione peroxidase: GSH-­P x) were also determined. The results showed that
Medicine, Khon Kaen University, Khon POE contained 8 essential substances and exhibited a potent antioxidant without
Kaen, Thailand
5 any cytotoxicity. It was found that POE significantly decreased the MDA level and
Thailand Institute of Nuclear Technology
(Public Organization), Nakhon Nayok, activated cell viability, SOD, CAT, and GSH-­P x activities. The results from this study
Thailand
indicate that POE is a potent antioxidant, which can be developed as a radioprotector
Correspondence for diagnostic procedures.
Woranan Kirisattayakul, Department of
Radiology, Faculty of Medicine, Khon Kaen Practical applications
University, Khon Kaen 40002, Thailand. Polygonum odoratum leaf extract (POE) is a potent antioxidant that attenuates oxida-
Email: woraki@kku.ac.th
tive stress and cell death induced by low dose ionizing radiation (LDIR). POE might
Funding information protect against cell damage from LDIR, particularly in diagnostic radiology proce-
Research Institute for Human High
Performance and Health Promotion, Khon dures. Therefore, the development of functional food containing POE might be
Kaen University, Thailand beneficial for patients who plan to undergo the diagnostic radiology procedure. The
functional food containing POE might prevent stochastic and deterministic effects
for these patients.

KEYWORDS

antioxidant, low dose ionizing radiation, oxidative stress, Polygonum odoratum

J Food Biochem. 2021;00:e13909. wileyonlinelibrary.com/journal/jfbc |

© 2021 Wiley Periodicals LLC.     1 of 11
https://doi.org/10.1111/jfbc.13909
 |
2 of 11       KAWVISED et al.

1 |  I NTRO D U C TI O N effects of Polygonum odoratum leaf extract (POE) on oxidative stress
markers and cell death induced by LDIR in Raw 264.7 cells.
Nowadays, medical imaging technology is extensively used for di-
agnostic and therapeutic purposes by operating low dose x-­ray ap-
paratuses including radiography, computed tomography (CT), and 2 | M ATE R I A L S A N D M E TH O DS
fluoroscopy. Such procedures are a common source of radiation
exposure to the population (Ruano-­Ravina & Wakeford, 2020). The 2.1 | Chemicals and reagents
low dose ionizing radiation (LDIR) is defined as radiation dose which
is less than or equal to 100 mGy (UNSCEAR, 2015), such as natural Cell culture media (Dulbecco’s Modified Eagle Medium; DMEM),
radiation, radiological accident, and medical radiation. The effects of fetal bovine serum, penicillin, streptomycin, trypsin EDTA, and 3-­(
LDIR are primarily stochastic such as gene mutation and cancers (Ali 4,5-­dimethylthiazol-­2-­yl)-­2,5-­diphenyl tetrazolium bromide (MTT)
et al., 2020; Miller et al., 2013). While LDIR effects occur in various bi- were purchased from Gibco (Invitrogen, USA). All chemicals for bio-
ological mechanisms of living organisms, some low-­dose techniques logical activities, chromatogram, and oxidative stress markers were
with multiple image acquisition or long exposure time may increase purchased from Sigma-­Aldrich (St. Louis, MO, USA).
deterministic risks such as hair loss (Imanishi et  al.,  2005), skin in-
jury (Mooney et al., 2000), and cataracts (National Research Council,
2006). The production of reactive oxygen species (ROS) is the most 2.2 | Plant material and plant extraction
common effect of LDIR, which in turn increased the radiation-­
induced biomolecules and cellular damages (Kawamura et al., 2018). P. odoratum leaves were collected from Khon Kaen Province in June,
The massive production of ROS induces numerous cellular damages 2019. The plant specimen (KKU No. 25,553) was prepared and kept
particularly cell membrane destruction leading to the increase in at KKU herbarium, Department of Biology, Faculty of Science, Khon
lipid peroxidation such as malondialdehyde (MDA) which is a toxic Kaen University, Khon Kaen, Thailand. Leaves were cleaned, dried
molecule (Draper & Hadley, 1990; Li et al., 2006; Lykkesfeldt, 2007; in an oven for 24 hr and blended into powder. Two-­hundred grams
Niedernhofer et al., 2003). Moreover, the radiation-­induced oxida- of dried P. odoratum leaves were extracted twice by boiling with
tive stress also alters the function of endogenous antioxidant en- distilled water at 100℃ for 30 min. Then, the filtrate was filtered
zymes, including superoxide dismutase (SOD), catalase (CAT), and through a filter cloth and Whatman paper (Whatman®), then dried
glutathione peroxidase (GSH-­P x) (Birben et al., 2012). It has been re- by using Mini Spray dryer (BUCHI/B-­290). The percent yield of the
ported that the increase in MDA after exposure to ionizing radiation POE was 6.21. POE was freshly dissolved and diluted in distilled
is associated with decreasing SOD, catalase, and GSH-­P x activities water before use on the day of experiment.
(Motallebzadeh et al., 2020).
Recently, it has been reported that the natural active com-
pounds exhibited the radioprotective effect and protected against 2.3 | Biological activities evaluation
excessive oxidative stress and cell death caused by ionizing radia-
tion (Jagetia,  2007; Smith et  al.,  2017). In addition, several lines The biological activities of POE were evaluated by using following
of evidence have been reported for the radioprotective effect of indicators: total phenolic compound as well as antioxidant activ-
polyphenol-­rich extract in living organisms by promoting cell viabil- ity using DPPH radical scavenging assay and FRAP assay. All as-
ity and preventing cell apoptosis (Kim et al., 2008; Saliev et al., 2020). says were performed following the previously reported method
The possible underlying mechanisms might be exhibited via the inhi- (Kirisattayakul et al., 2017).
bition of ROS production (Materska et al., 2015), lipid peroxidation
(Uma Devi et al., 2000), enhancement of scavenging enzymes activ-
ity (Georgieva et al., 2013), protection from DNA damage (Cinkilic 2.4 | Total phenolic compound
et al., 2013), and increment of immunomodulatory potential (El-­
Desouky et al., 2017). To determine the total phenolic compound of POE, 20 μl of POE,
Polygonum odoratum Lour or Pak Paew is a traditional herb that 1.58 ml of distilled water, and 1 ml of Folin-­Ciocalteu phenol reagent
has been widely consumed in Thailand. It consists of many bioactive (50% vol/vol) were mixed and incubated at room temperature for
compounds such as phenolic acids and flavonoids that can exhibit po- 8  min. Then, 0.3  ml of sodium carbonate (20% wt/vol) was added
tent antioxidant and anti-­inflammatory activities (Ahongshangbam to the mixture and shaken well before incubation in a dark room at
et al., 2014; Chansiw et al., 2019). In addition, it has been demon- room temperature for 2 hr. The optical density (OD) for absorbance
strated that the functional food containing combined extracts of the blue color presented in the mixture was observed at 765 nm
between Morus alba and P. odoratum leaf could decrease the oxi- using UV-­spectrophotometer (Pharmacia LKB-­Biochrom4060). The
dative damage and exhibit antiosteoporotic effects (Sungkamanee total phenolic compound of POE was determined using the standard
et al., 2014). However, there was no scientific evidence to support its calibration curve from gallic acid (0–­500  mg/ml) and expressed as
radioprotective effect. Therefore, this study was set up to reveal the mg/ml gallic acid equivalent per mg extract (mg/ml GAE/mg extract).
KAWVISED et al. |
      3 of 11

2.5 | DPPH radical scavenging activity 370 nm). Subsequently, the area under the curve (AUC) of each
chromatogram was analyzed and the detected active ingredients
The DPPH radical scavenging assay was performed based on the were expressed as mg/g extract.
ability of POE to turn the stable free radical (pink color) into neu-
tral state (pale yellow). The DPPH solution (0.15  mM in methanol)
at the volume of 0.4 ml was added to 1 ml of POE at various con- 2.8 | Cell culture
centrations (5, 10, 25  μg/ml) and incubated at room temperature
for 30  min. The absorbance (Abs) was measured at 517  nm using The mouse monocyte macrophage cell line, Raw 267.4 cells
UV-­
spectrophotometer (Pharmacia LKB-­
Biochrom4060). The ab- (ATCC® TIB-­
71™), had been kindly provided by Professor Dr.
sorbance of methanol (0.8 ml) mixed with DPPH solution (0.4 ml) Jintanaporn Wattanathorn. The cells were cultured in culture
was used as control. The percentage of inhibition of POE in each media (DMEM with 10% FBS, 100 units/ml of penicillin and 100
concentration was calculated and plotted against the concentration. µg/ml of streptomycin) with 6 wells plate at the condition of 37°,
The half maximal inhibitory concentration (IC50) of POE on DPPH 5% CO2 for 4 days. The culture media was changed daily.
radical scavenging was obtained from the equation of the graph and
expressed as µg/ml.
2.9 | Cytotoxicity test

2.6 | Ferric Reducing Antioxidant Power The cytotoxicity test of POE was conducted following a previ-
(FRAP) assay ous study (Wattanathorn et al., 2019). In brief, 10 4 cells/well were
subcultured within 96 wells plate and further incubated with
The ferric reducing antioxidant power (FRAP) assay was assessed culture media for 24  hr at the condition of 37°, 5% CO2 . After
based on the capability of POE to change ferric tripyridyl triazine washing with phosphate buffer saline (pH 7.2), cells were incu-
of Fe III TPTZ complex (light brown color) to ferrous form (intense bated with POE at various concentrations ranging from 5,000 to
blue color) at low pH. The FRAP solution was freshly prepared on 156.25  µg/ml. The vehicle and the positive control were DMEM
the day of experiment by mixing 10 ml of 300 mM acetate buffer and 1% Tween20, respectively. All treatments were performed in
(pH 3.6), 1 ml of 10 mM 2,4,6-­t ripyridyl-­s triazine (TPTZ) in 40 mM duplicate and incubated for 24 hr at the condition of 37°, 5% CO2 .
HCl and 1 ml of 20 mM ferric chloride on hot plate at 37℃. The After incubation, the cells were washed with phosphate buffer
POE solution (50 μl) and FRAP solution (1.45 ml) were mixed and saline (pH7.2), and thiazolyl blue tetrazolium blue or MTT solu-
incubated in water bath at 37℃ for 10 min. The absorbance was tion (0.5 mg/ml) was added to all wells and incubated for 4 hr. The
determined at 593  nm using UV-­s pectrophotometer (Pharmacia viable cells could turn MTT solution (yellow color) into formazan
LKB-­B iochrom4060). The standard calibration curve was created crystal (purple color). After the remaining MTT solution was re-
using ferrous ammonium sulfate (0‒­2 50  μM). The FRAP activity moved, dimethyl sulfoxide (DMSO, 100 µl) was added to all wells
of POE was expressed as µM Fe(II)/mg extract. and kept in a dark room for 30 min. The optical density was meas-
ured at 540  nm using a microplate reader (Tecan®, Sunrise™,
USA). The percentage of cytotoxicity was calculated using the
2.7 | Chromatographic fingerprint evaluation equation below.

The chromatographic fingerprint of POE was performed using

( ( ))
Percentage of cytotoxicity = 1 − ODtreatment ∕ODvechicle × 100
gradient high performance liquid chromatography (HPLC) sys-
tem, which consists of in-­line degasser AF, a Waters 515 HPLC 2.10 | Cell preparation and treatment
pump (pump control module II), a Rheodyne injector with a sam-
ple loop of 20 µl, and a Waters 2,998 photodiode array detector On the experiment day, 105 cells/well were seeded into 24 wells
(Water Company, USA). The separation of sample chromatogram plate and incubated with the assigned substances as follow;
was performed using Poroshell 120 EC-­C18 column (250 × 4.6 mm
id, 4.0 µm) and guard column of Poroshell 120 EC-­C18 column Group 1 Cells + DMEM with no radiation exposure
(5 × 4.6 mm id, 4.0 µm; Agilent Technologies, USA). The Acetonitrile which served as control group
(A), 0.1% Formic acid (B) were used as mobile phase and followed
through a gradient with the flow rate of 1.0 ml/min: 0 min, 90%B; Group 2 Cells +DMEM + x-­irradiation which served
10 min, 75%B; 20 min, 40%B; 30 min, 30%B; 35 min, 20%B. POE as negative control.
at the concentration of 0.1 g/ml was filtered through a 0.22 nylon
membrane and the injection volume was 20 µl. Chromatograms Group 3 Cells + POE solution (the final concentration
were detected at various wavelengths (254, 275, 280, 320, and was 10 µg/ml) + x-­irradiation
 |
4 of 11       KAWVISED et al.

Group 4 Cells + L-­ascorbic acid (the final concentra- TA B L E 1   The biological activities parameters of Polygonum
tion was 2 µg/ml) + x-­irradiation which served as pos- odoratum leaf extract

itive control Biological activities

parameters POE
Three wells per group were conducted and all wells were incubated Total phenolic compound 223.00 ± 9.70 mg/ml GAE/mg extract
for 1 hr prior to the irradiation, which had been reported as the optimal DPPH antioxidant activity 11.51 ± 0.15 µg/ml
time for radioprotective effect of L-­ascorbic acid (Brand et al., 2015). (IC50)
The control group (non-­irradiated group) was incubated with DMEM in FRAP activity 279.18 ± 2.49 mM Fe(II)/mg extract
a separated plate and kept in the same condition as the experimental
Note: Data are presented as mean ± SEM.
plate except for irradiation.

TA B L E 2   The essential substances in Polygonum odoratum leaf

2.11 | Cell irradiation extract analyzed by high performance liquid chromatography
technique

Irradiation was performed using bi-­plane digital subtraction angi- Detected value
ography (DSA) unit (Artis Zee, Siemens, Germany) at Srinagarind Essential substances (mg/g extract)
Hospital, Khon Kaen University, which was annually quality control Gallic acid 7.645 ± 0.300
checked by the Department of Medical Sciences. Dosimetry was Rutin 0.396 ± 0.019
performed by using a dosimeter (Piranha 657, Sweden) before the Quercetin 0.075 ± 0.004
experiment to confirm the radiation technique and radiation dose.
Kaempferol 0.025 ± 0.001
For 100 mGy irradiation to the cells, the irradiation techniques were
Caffeic acid 0.072 ± 0.007
set as 102  kV, 359  mA, 1 frame per second for 10  s at room tem-
Ferulic acid 0.021 ± 0.002
perature, where the source-­to-­object distance was 60 cm. During
Catechin 1.767 ± 0.098
irradiation, the plate was placed at the center of the radiation field
Ellagic acid 3.310 ± 0.135
while the dosimeter was placed over the plate to confirm the radia-
tion dose during irradiation. Total duration of the cell irradiation pro- Note: Data are presented as mean ± SEM.
cedure was 5 min and all cells were brought to the cell culture room
for further investigation. method previously described (Kirisattayakul et al., 2017). Protein of
the supernatant was evaluated by using NanoDrop UV-­Vis spectro-
photometers (Thermo Scientific, USA) and expressed as mg protein.
2.12 | Cell viability assay All tests were performed in duplicate.

A portion of Raw 267.4 cells was harvested and stained with

0.4%Trypan blue, then counted on a hemacytometer (Uttayarat 2.14 | Statistical analysis
et al., 2015). The dead cells were stained with blue dye, while the
viable cells were not stained and appeared transparent. The evalu- All data were presented as mean ± SEM and analyzed by using SPSS
ation of cell viability was conducted by a well-­trained research as- Statistics 19.0 for Windows. The normality of data was tested by
sistant who was blinded to the treatment. Data was expressed as using the Shapiro–­Wilk test prior to the one-­way ANOVA test (nor-
relative cell viability to the control group. mal distribution data set) followed by the post hoc test. The signifi-
cance was regarded when p < .05.

2.13 | Oxidative stress damage and endogenous

antioxidant markers evaluation 3 | R E S U LT S

The remaining portion of Raw 267.4 cells was harvested and manu- 3.1 | The biological activities and chromatographic
ally homogenized for 2 min. The homogenates were centrifuged fingerprint of POE
at 10,000 rpm 4℃ for 10 min. The supernatant was collected for
further oxidative stress markers investigation including oxidative The biological activities of POE are shown in Table 1. The total phe-
stress damage and endogenous antioxidant activities. MDA level nolic compound, DPPH antioxidant activity, and FRAP activity of
was used as oxidative stress damage marker, while SOD activity, POE were 223.00 ± 9.70 mg/ml GAE/mg extract, 11.51 ± 0.15 µg/ml,
catalase (CAT) activity, and glutathione peroxidase (GSH-­P x) activity and FRAP activity 279.18 ± 2.49 mM Fe(II)/mg extract, respectively.
were used as endogenous antioxidant activity markers. The evalu- The chromatographic fingerprint and amounts of essen-
ation of oxidative stress markers was performed according to the tial substances of POE are shown in Figure 1 and Table 2. There
KAWVISED et al. |
      5 of 11

F I G U R E 1   The chromatogram of Polygonum odoratum leaf extract (black line) and the standard substances (red line) at various
wavelength; A: Gallic acid (a) 60 µg/ml at 275 nm, B: Rutin (b), Quercetin (c) and Kaempferol (d) 20 µg/ml at 370 nm, C: Chlorogenic acid (e),
Caffeic acid (f) and Ferulic acid (g) 50 µg/ml at 320 nm, D: Catechin (h) 100 µg/ml at 280 nm, E: Ellagic acid (i) 100 µg/ml at 254 nm

were 8 essential substances including gallic acid, rutin, quer-

cetin, kaempferol, caffeic acid, ferulic acid, catechin, and ellagic 3.2 | The cytotoxicity of POE
acid found in POE. Gallic acid was presented with the high-
est value (7.645  ± 0.300 mg/g extract), followed by ellagic acid The cytotoxicity of various concentrations of POE on Raw 267.4
(3.310  ±  0.135  mg/g extract), and catechin (1.767  ± 0.098 mg/g cells is shown in Table 3. The result showed that after incuba-
extract), respectively tion for 24  hr, POE at the concentration of 5,000–­312.5  µg/ml
 |
6 of 11       KAWVISED et al.

exerted cytotoxicity at the percentage of 85.48 ± 1.7, 38.72 ± 3.1,

156.25 µg/ml
27.01 ± 2.9, 27.93 ± 5.1, and 20.06 ± 4.7, respectively. Interestingly,

0.00 ± 3.7
POE at the concentration of 156.25 µg/ml failed to produce cytotox-
icity. These results showed that the selected concentration of POE
did not produce any toxicity to Raw 267.4 cells.

312.5 µg/ml 3.3 | The effect of POE on cell viability

20.06 ± 4.7 after x-­irradiation

The result of POE on cell viability after x-­irradiation is shown in

Figure 2. It was found that x-­irradiation significantly decreased cell
viability to approximately 50% (p < .01), while the prior incubation
of cells with POE and L-­ascorbic acid could significantly elevate
27.93 ± 5.1

the relative cell viability to 0.900 ± 0.032 and 0.782 ± 0.086, re-

625 µg/ml

spectively. Although the relative cell viability of the POE group was
slightly greater than that of the L-­ascorbic acid group, there was no
significant difference between these 2 groups.
1,250 µg/ml

27.01 ± 2.9

3.4 | The effect of POE on oxidative stress markers

TA B L E 3   The percentage cytotoxicity of various concentration of Polygonum odoratum leaf extract on Raw 267.4 cell

The effect of POE on oxidative stress is shown in Figures 3–­4. The

result in Figure 3 shows that x-­irradiation has significantly increased
MDA level (p < .01 compared with DMEM Nonirradiation group),
while pre-­incubation of cells with POE and L-­ascorbic acid has signif-
2,500 µg/ml

38.72 ± 3.1

icantly attenuated x-­irradiation-­induced oxidative stress damage (p

< .01 all, compared to DMEM + 100 mGy x-­irradiation). Interestingly,
the elevation of endogenous antioxidant activities including SOD,
CAT, and GSH-­P x were significantly observed in POE pre-­treated
POE concentration

group (p < .001 all), while pre-­incubation with L-­ascorbic acid also
5,000 µg/ml

showed a similar trend as POE with a significant difference from

85.48 ± 1.7

DMEM + 100 mGy x-­irradiation-­treated group (p < .001, .01, and

.05, respectively) as shown in Figure 4.

4 | D I S CU S S I O N
0.00 ± 2.1

The results gained from this preliminary study indicated that POE
Vehicle

could protect against low dose x-­irradiation-­induced cell death in

Raw 267.4 cells without any cytotoxicity. This effect might occur
partly via the attenuation of free radicals by POE antioxidant activ-
ity and the activation of endogenous antioxidant activities.
Note: Data are presented as mean ± SEM.
1% Tween20

95.73 ± 0.5

It has been reported that the ability of plant phenolic compounds

on scavenging of free radicals depends on the number and location
of the phenol OH group and the C2-­C3 double bond in the aromatic
ring of phenol (Seyoum et al., 2006). Since POE provided a high phe-
nolic compound and contained numerous active substances which
are presented in the form of phenol OH, POE exerted a high antioxi-
Growth control

dant activity in both DPPH radical scavenging assay and FRAP assay.
0.00 ± 0.0

Although P. odoratum leaves have been widely consumed as a

vegetable, the evaluation of cytotoxicity to the tested cell was also
important. The maceration extraction with water of P. odoratum
KAWVISED et al. |
      7 of 11

F I G U R E 2   The effect of Polygonum odoratum leaf extract on the relative cell viability. aap < .01, compared with Dulbecco’s modified eagle
medium (DMEM) Nonirradiation group. *,**p < .05 and .01, respectively, compared with DMEM + 100 mGy x-­irradiation

F I G U R E 3   The effect of Polygonum odoratum leaf extract on the alteration of malondialdehyde (MDA) aap < .01 compared with
Dulbecco’s modified eagle medium (DMEM) Nonirradiation group. ** p < .01, compared with DMEM + 100 mGy x-­irradiation

leaves has no cytotoxicity to RAW 264.7 cells with cell viability more Numerous markers have been used to evaluate the oxidative dam-
than 70% at the concentration of 200 µg/ml (Chansiw et al., 2019). age (Ho et al., 2013). MDA is one of the famous oxidative damage
Interestingly, POE provided the same trend of cell viability, but up to markers which is the product of lipid peroxidation, an interaction
1,250 µg/ml which is higher than reported in the previous study. This of free radicals with polyunsaturated fatty acid (Ayala et al., 2014).
suggests that extraction with decoction technique or boiling might Several lines of evidence have reported that irradiation produces
eliminate toxic substances and provide less cytotoxicity effects to elevation of MDA level even with high or low dose radiation (Karimi
RAW 264.7 cells. et al.,2017; Puthran et al., 2009; Rezaeyan et al., 2016; Shaban
The main mechanism for radiation-­induced cell injury is the et al., 2017). In accordance with previous reports, the result of
indirect effect which produces several radical species (Alizadeh this study showed that the elevation of MDA was observed in cells
et al., 2013; Michaels & Hunt, 1978; von Sonntag, 1991), this with x-­irradiation. It has been reported that polyphenol rich sub-
in turn increases oxidative damage to the particular cell DNA. stances could inhibit the production of lipid peroxidation (Abraham
 |
8 of 11       KAWVISED et al.

F I G U R E 4   The effect of Polygonum odoratum leaf extract (POE) on endogenous antioxidant activities including superoxide dismutase
(SOD), catalase (CAT) and glutathione peroxidase (GSH-­P x). aaap < .001 compared with Dulbecco’s Modified Eagle Medium (DMEM)
Nonirradiation group. *,**,***p < .05, .01, and .001, respectively, compared with DMEM + 100 mGy x-­irradiation

et al., 1993; El-­Habit et al., 2000; Nagpal & Abraham, 2017). The The endogenous antioxidants are the physiological defense mech-
incubation of cells with POE or L-­ascorbic acid, which are poly- anisms that protect against cellular damage from both endogenous and
phenol rich substances, could decrease MDA level as in agreement exogenous sources of reactive species (Rizzo et al., 2010). Endogenous
with previous studies. This result indicates the ability of POE and protein antioxidants or enzymatic antioxidants including SOD, CAT,
L-­ascorbic on scavenging free radical along with inhibiting lipid and GSH-­Px are the first line of defense against free radicals (Nimse &
peroxidation. Pal, 2015). Therefore, the role of these enzymatic antioxidants has been
KAWVISED et al. |
      9 of 11

extensively discussed. Previous studies have reported irradiated tissue AU T H O R C O N T R I B U T I O N S

showing reduction of these enzymatic antioxidants (Karimi et al.,2017; Methodology; Writing-­
original draft; Writing-­
review & editing:
Rezaeyan et al., 2016; Shaban et al., 2017). In addition, other patholog- Supannika Kawvised. Methodology: Thawatchai Prabsattroo.
ical disease models also suggested that POE or the product contain- Methodology: Waranon Munkong. Methodology: Panuwat Pattum.
ing POE could improve the endogenous antioxidants (Sungkamanee Writing-­ review & editing: Sittichai Iamsaard.
original draft; Writing-­
et al., 2014; Thongra-­ar et al., 2021). The irradiated cell in this study Methodology: Kanokporn Boonsirichai. Methodology: Pimporn
showed the reduction of SOD, CAT, and GSH-­Px which is consistent Uttayarat. Methodology: Lamai Maikaeo. Methodology: Waraporn
with these research. The pretreatment with POE and L-­ascorbic acid Sudchai. Data curation; Formal analysis; Investigation; Methodology;
could attenuate the effect of radiation on these parameters. Project administration; Writing-­original draft; Writing-­review & editing:
The identification of active compounds of POE was also evaluated Woranan Kirisattayakul.
in this study which resembled that of a previous study (Somananda
et al., 2014). Gallic acid, ellagic acid, and catechin are the main active DATA AVA I L A B I L I T Y S TAT E M E N T
compounds of POE in this study which have been reported as a potent Data available on request from the authors.
antioxidant and protection against oxidative stress (Bernatoniene and
Kopustinskiene, 2018; Grzesik et al., 2018; Priyadarsini et al., 2002; ORCID
Velderrain-­Rodríguez et al., 2018). In addition, several lines of evidence Supannika Kawvised  https://orcid.org/0000-0002-5868-3196
also demonstrate that these compounds produce the radioprotective Thawatchai Prabsattroo 
effect against gamma or x-­ray irradiation-­induced cell damage (Emiko https://orcid.org/0000-0001-6292-9040
et al., 2018; Nemavarkar et al., 2004; Richi et al., 2012). Therefore, the Waranon Munkong  https://orcid.org/0000-0002-1912-3903
expression of antioxidant and radioprotective effects of POE might Panuwat Pattum  https://orcid.org/0000-0001-6442-5225
contribute from these main compounds. Sittichai Iamsaard  https://orcid.org/0000-0002-6793-2879
Taking all data into account, the radioprotective effect of POE Kanokporn Boonsirichai 
might be exerted partly via the ability of POE on scavenging free https://orcid.org/0000-0002-5706-3665
radicals, the activation of enzymatic antioxidants production and the Pimporn Uttayarat  https://orcid.org/0000-0002-7993-2526
radioprotective effect of the main active compounds. Finally, POE Lamai Maikaeo  https://orcid.org/0000-0003-4125-3699
could inhibit the cell injury and cell death induced by x-­radiation. Waraporn Sudchai  https://orcid.org/0000-0002-7626-8877
Since the mechanism of cell damage after irradiation is also contrib- Woranan Kirisattayakul  https://orcid.org/0000-0001-5330-9421
uted by the direct damage of radiation and free radicals to the DNA,
the ability of DNA damage recovery should be focused. Moreover, REFERENCES
the defense mechanism of the living organism also depends on other Abraham, S. K., Sarma, L., & Kesavan, P. C. (1993). Protective effects of
factors such as nonenzymatic antioxidants. Therefore, further inves- chlorogenic acid, curcumin and beta-­carotene against gamma-­radiation-­
tigation of the other related factors including DNA recovery-­related induced in vivo chromosomal damage. Mutation Research, 303(3),
protein and nonenzymatic antioxidants should be evaluated. v109–­v112. https://doi.org/10.1016/0165-­7992(93)90022​-­n
Ahongshangbam, S. K., Devi, G. S., & Chattopadhyay, S. (2014). Bioactive
compounds and antioxidant activity of Polygonum odoratum Lour.
International Journal of Basic and Applied Biology, 1, 94–­97.
5 | CO N C LU S I O N Ali, Y. F., Cucinotta, F. A., Ning-­Ang, L., & Zhou, G. (2020). Cancer risk of
low dose ionizing radiation. Frontiers in Physics, 8, 234. https://doi.
org/10.3389/fphy.2020.00234
This study revealed that POE attenuates oxidative stress damage
Alizadeh, E., Sanz, A. G., Garcia, G., & Sanche, L. (2013). Radiation damage to
and cell death without any cytotoxicity in Raw 267.4 cells. The pos- DNA: The indirect effect of low-­energy electrons. The Journal of Physical
sible mechanism might be contributed from the free radical scav- Chemistry Letters, 4(5), 820–­825. https://doi.org/10.1021/jz400​0998
enging ability of POE and the activation of endogenous antioxidants Ayala, A., Muñoz, M. F., & Argüelles, S. (2014). Lipid peroxidation:
Production, metabolism, and signaling mechanisms of malondial-
including SOD, CAT, and GSH-­P x. POE has effective antioxidant
dehyde and 4-­hydroxy-­2-­nonenal. Oxidative Medicine and Cellular
capacity which can be developed as a potential radioprotector in di- Longevity, 2014, 360438. https://doi.org/10.1155/2014/360438
agnostic procedure. Bernatoniene, J., & Kopustinskiene, D. M. (2018). The role of catechins in
cellular responses to oxidative stress. Molecules (Basel, Switzerland),
23(4), 965. https://doi.org/10.3390/molec​ules2​3 040965
AC K N OW L E D G E M E N T
Birben, E., Sahiner, U. M., Sackesen, C., Erzurum, S., & Kalayci, O. (2012).
This research was supported by a research grant (2019) from the Oxidative stress and antioxidant defense. World Allergy Organization
Research Institute for Human High Performance and Health Journal, 5(1), 9–­19. https://doi.org/10.1097/WOX.0b013​e3182​439613
Promotion (HHP & HP), Khon Kaen University, Thailand. Brand, M., Sommer, M., Ellmann, S., Wuest, W., May, M. S., Eller, A.,
Vogt, S., Lell, M. M., Kuefner, M. A., & Uder, M. (2015). Influence of
different antioxidants on X-­ray induced DNA double-­strand breaks
C O N FL I C T O F I N T E R E S T (DSBs) using γ-­H2AX immunofluorescence microscopy in a prelim-
The author declares that there is no conflict of interest that could be inary study. PLoS One, 10(5), e0127142. https://doi.org/10.1371/
perceived as prejudicing the impartiality of the research reported. journ​al.pone.0127142
 |
10 of 11       KAWVISED et al.

Chansiw, N., Chotinantakul, K., & Srichairatanakool, S. (2019). Anti-­ Kirisattayakul, W., Wattanathorn, J., Iamsaard, S., Jittiwat, J., Suriharn,
inflammatory and antioxidant activities of the extracts from leaves B., & Lertrat, K. (2017). Neuroprotective and memory-­enhancing ef-
and stems of Polygonum odoratum Lour. Anti-­Inflammatory & Anti-­ fect of the combined extract of purple waxy corn cob and pandan in
Allergy Agents in Medicinal Chemistry (Formerly Current Medicinal ovariectomized rats. Oxidative Medicine and Cellular Longevity, 2017,
Chemistry-­Anti-­Inflammatory and Anti-­Allergy Agents), 18(1), 45–­54. 5187102. https://doi.org/10.1155/2017/5187102
https://doi.org/10.2174/18715​23017​66618​11091​4 4548 Li, G., Li, H., Wang, B., & Yin, D. (2006). Effects of malondialdehyde on
Cinkilic, N., Cetintas, S. K., Zorlu, T., Vatan, O., Yilmaz, D., Cavas, T., Tunc, growth and proliferation of human bone marrow mesenchymal stem
S., Ozkan, L., & Bilaloglu, R. (2013). Radioprotection by two pheno- cells in vitro. Frontiers of Biology in China, 1(2), 131–­136. https://doi.
lic compounds: Chlorogenic and quinic acid, on X-­ray induced DNA org/10.1007/s1151​5-­0 06-­0 021-­z
damage in human blood lymphocytes in vitro. Food and Chemical Lykkesfeldt, J. (2007). Malondialdehyde as biomarker of oxidative dam-
Toxicology, 53, 359–­363. https://doi.org/10.1016/j.fct.2012.12.008 age to lipids caused by smoking. Clinica Chimica Acta, 380(1–­2), 50–­
Draper, H. H., & Hadley, M. (1990). Malondialdehyde determination as 58. https://doi.org/10.1016/j.cca.2007.01.028
index of lipid peroxidation. Methods in Enzymology, 186, 421–­431. Materska, M., Konopacka, M., Rogoliński, J., & Ślosarek, K. (2015).
https://doi.org/10.1016/0076-­6879(90)86135​-­I Antioxidant activity and protective effects against oxidative damage
El-­Desouky, W., Hanafi, A., & Abbas, M. M. (2017). Radioprotective effect of human cells induced by X-­radiation of phenolic glycosides isolated
of green tea and grape seed extracts mixture on gamma irradiation from pepper fruits Capsicum annuum L. Food Chemistry, 168, 546–­
induced immune suppression in male albino rats. International Journal 553. https://doi.org/10.1016/j.foodc​hem.2014.07.023
of Radiation Biology, 93(4), 433–­439. https://doi.org/10.1080/09553​ Michaels, H. B., & Hunt, J. W. (1978). A model for radiation damage
002.2016.1254834 in cells by direct effect and by indirect effect: A radiation chem-
El-­Habit, O. H., Saada, H. N., Azab, K. S., Abdel-­Rahman, M., & El-­Malah, istry approach. Radiation Research, 74(1), 23–­ 3 4. https://doi.
D. F. (2000). The modifying effect of beta-­ c arotene on gamma org/10.2307/3574754
radiation-­induced elevation of oxidative reactions and genotoxic- Miller, M. S., Moore, J. E., Walb, M. C., Kock, N. D., Attia, A., Isom, S., McBride,
ity in male rats. Mutation Research, 23, 466(2), 179‒­186. https://doi. J. E., & Munley, M. T. (2013). Chemoprevention by N-­acetylcysteine of
org/10.1016/s1383​-­5718(00)00010​-­3. PMID: 10727905 low-­dose CT-­induced murine lung tumorigenesis. Carcinogenesis, 34(2),
Emiko, S., Ikuo, N., Kohei, I., Megumi, U., Takashi, S., Ken-­I chiro, M., 319–­324. https://doi.org/10.1093/carci​n/bgs332
& Kiyoshi, F. (2018). Efficient protective activity of a planar cat- Mooney, R. B., McKinstry, C. S., & Kamel, H. A. (2000). Absorbed dose
echin analogue against radiation-­i nduced apoptosis in rat thymo- and deterministic effects to patients from interventional neurora-
cytes. RSC Advances, 8, 10158–­10162. https://doi.org/10.1039/ diology. The British Journal of Radiology, 73(871), 745–­751. https://
C7RA1​3111A doi.org/10.1259/bjr.73.871.11089467
Georgieva, S., Popov, B., & Bonev, G. (2013). Radioprotective effect of Motallebzadeh, E., Tameh, A. A., Zavareh, S. A. T., Farhood, B.,
Haberlea rhodopensis (Friv.) leaf extract on-­radiation-­induced DNA Aliasgharzedeh, A., & Mohseni, M. (2020). Neuroprotective effect of
damage, lipid peroxidation and antioxidant levels in rabbit blood. melatonin on radiation-­induced oxidative stress and apoptosis in the
Indian Journal of Experimental Biology, 51(1), 29–­36. brainstem of rats. Journal of Cellular Physiology, 235(11), 8791–­8798.
Grzesik, M., Naparło, K., Bartosz, G., & Sadowska-­Bartosz, I. (2018). https://doi.org/10.1002/jcp.29722
Antioxidant properties of catechins: Comparison with other antioxi- Nagpal, I., & Abraham, S. K. (2017). Protective effects of tea polyphenols
dants. Food Chemistry, 15(241), 480–­492. https://doi.org/10.1016/j. and β-­c arotene against γ-­radiation induced mutation and oxidative
foodc​hem.2017.08.117 stress in Drosophila melanogaster. Genes and Environment, 39, 24.
Ho, E., Galougahi, K. K., Liu, C. C., Bhindi, R., & Figtree, G. A. (2013). https://doi.org/10.1186/s4102​1-­017-­0 084-­x
Biological markers of oxidative stress: Applications to cardiovascu- National Research Council. (2006). Health risks from exposure to low levels
lar research and practice. Redox Biology, 1(1), 483–­491. https://doi. of ionizing radiation: BEIR VII phase 2.
org/10.1016/j.redox.2013.07.006 Nemavarkar, P., Chourasia, B. K., & Pasupathy, K. (2004). Evaluation of
Imanishi, Y., Fukui, A., Niimi, H., Itoh, D., Nozaki, K., Nakaji, S., Ishizuka, radioprotective action of compounds using Saccharomyces cerevisiae.
K., Tabata, H., Furuya, Y., Uzura, M., Takahama, H., Hashizume, S., Journal of Environmental Pathology, Toxicology, and Oncology, 23(2),
Arima, S., & Nakajima, Y. (2005). Radiation-­induced temporary hair 145–­151. https://doi.org/10.1615/jenvp​athto​xoncol.v23.i2.70
loss as a radiation damage only occurring in patients who had the Niedernhofer, L. J., Daniels, J. S., Rouzer, C. A., Greene, R. E., & Marnett,
combination of MDCT and DSA. European Radiology, 15(1), 41–­46. L. J. (2003). Malondialdehyde, a product of lipid peroxidation, is
https://doi.org/10.1007/s0033​0 -­0 04-­2459-­1 mutagenic in human cells. Journal of Biological Chemistry, 278(33),
Jagetia, G. C. (2007). Radioprotective potential of plants and herbs against 31426–­31433. https://doi.org/10.1074/jbc.M2125​49200
the effects of ionizing radiation. Journal of Clinical Biochemistry and Nimse, S. B., & Pal, D. (2015). Free radicals, natural antioxidants, and their
Nutrition, 40(2), 74–­81. https://doi.org/10.3164/jcbn.40.74 reaction mechanisms. RSC Advances, 5(35), 27986–­28006. https://
Karimi, N., Monfared, A. S., Haddadi, G. H., Soleymani, A., Mohammadi, doi.org/10.1039/C4RA1​3315C
E., Hajian-­Tilaki, K., & Borzoueisileh, S. (2017). Radioprotective ef- Priyadarsini, K. I., Khopde, S. M., Kumar, S. S., & Mohan, H. (2002). Free
fect of hesperidin on reducing oxidative stress in the lens tissue of radical studies of ellagic acid, a natural phenolic antioxidant. Journal
rats. International Journal of Pharmaceutical Investigation, 7(3), 149–­ of Agricultural and Food Chemistry, 27, 50(7), 2200‒­2206. https://doi.
154. https://doi.org/10.4103/jphi.JPHI_60_17 org/10.1021/jf011​275g. PMID: 11902978
Kawamura, K., Qi, F., & Kobayashi, J. (2018). Potential relationship be- Puthran, S. S., Sudha, K., Rao, G. M., & Shetty, B. V. (2009). Oxidative
tween the biological effects of low-­dose irradiation and mitochon- stress and low dose ionizing radiation. The Indian Journal of Physiology
drial ROS production. Journal of Radiation Research, 59(suppl_2), ii91‒­ and Pharmacology, 53(2), 181–­184.
ii97. https://doi.org/10.1093/jrr/rrx091 Rezaeyan, A., Haddadi, G. H., Hosseinzadeh, M., Moradi, M., & Najafi, M.
Kim, J., Moon, C., Kim, H., Jeong, J., Lee, J., Kim, J., Hyun, J. W., Park, (2016). Radioprotective effects of hesperidin on oxidative damages
J. W., Moon, M. Y., Lee, N. H., Kim, S. H., Jee, Y., & Shin, T. (2008). and histopathological changes induced by X-­irradiation in rats heart
The radioprotective effects of the hexane and ethyl acetate extracts tissue. Journal of Medical Physics/Association of Medical Physicists of
of Callophyllis japonica in mice that undergo whole body irradiation. India, 41(3), 182–­191. https://doi.org/10.4103/0971-­6203.189482
Journal of Veterinary Science, 9(3), 281–­284. https://doi.org/10.4142/ Richi, B., Kale, R. K., & Tiku, A. B. (2012). Radio-­ modulatory ef-
jvs.2008.9.3.281 fects of green tea catechin EGCG on pBR322 plasmid DNA and
KAWVISED et al. |
      11 of 11

murine splenocytes against gamma-­ radiation induced damage. Uma Devi, P., Ganasoundari, A., Vrinda, B., Srinivasan, K. K., &
Mutation Research, 747(1), 62–­ 70. https://doi.org/10.1016/j.mrgen​ Unnikrishnan, M. K. (2000). Radiation protection by the ocimum
tox.2012.04.002 flavonoids orientin and vicenin: Mechanisms of action. Radiation
Rizzo, A. M., Berselli, P., Zava, S., Montorfano, G., Negroni, M., Corsetto, Research, 154(4), 455–­460.
P., & Berra, B. (2010). Endogenous antioxidants and radical scaven- UNSCEAR (2015). Sources, effects and risks of ionizing radiation. Report
gers. In M.T. Giardi, G. Rea & B. Berra (Eds.), Bio-­farms for nutraceuticals to the General Assembly and Scientific Annexes A and B. UNSCEAR
(pp. 52–­67). Springer. https://doi.org/10.1007/978-­1-­4 419-­7347-­4_5 2012 Report. United Nations Scientific Committee on the Effects of
Ruano-­Ravina, A., & Wakeford, R. (2020). The increasing exposure of the Atomic Radiation. United Nations sales publication E.16.IX.1. United
global population to ionizing radiation. Epidemiology, 31(2), 155–­159. Nations, New York.
https://doi.org/10.1097/EDE.00000​0 0000​0 01148 Uttayarat, P., Tangtong, T., Sukapirom, K., & Boonsirichai, K. (2015).
Saliev, T., Baiskhanova, D., Beznosko, D., Begimbetova, D., Umbayev, B., Gamma irradiation induces DNA double-­strand breaks in fibroblasts:
Nurgozhin, T., Fakhradiyev, I., Tanabayev, B., & Pavalkis, D. (2020). A new A model study for the development of biodosimetry. In Journal of
insight on the radioprotective potential of epsilon-­aminocaproic acid. Physics: Conference Series (Vol. 611, pp. 012030). IOP Publishing.
Medicina, 56(12), 663. https://doi.org/10.3390/medic​ina56​120663 Velderrain-­
Rodríguez, G. R., Torres-­ Moreno, H., Villegas-­ Ochoa, M.
Seyoum, A., Asres, K., & El-­Fiky, F. K. (2006). Structure–­radical scaveng- A., Ayala-­Zavala, J. F., Robles-­Zepeda, R. E., Wall-­Medrano, A., &
ing activity relationships of flavonoids. Phytochemistry, 67(18), 2058–­ González-­A guilar, G. A. (2018). Gallic acid content and an antioxi-
2070. https://doi.org/10.1016/j.phyto​chem.2006.07.002 dant mechanism are responsible for the antiproliferative activity of
Shaban, N. Z., Zahran, A. M. A., El-­Rashidy, F. H., & Kodous, A. S. A. “Ataulfo” mango peel on LS180 cells. Molecules (Basel, Switzerland),
(2017). Protective role of hesperidin against γ-­radiation-­induced 23(3), 695. https://doi.org/10.3390/molec​ules2​3 030695
oxidative stress and apoptosis in rat testis. Journal of Biological Von Sonntag, C. (1991). The chemistry of free-­radical-­mediated DNA dam-
Research-­Thessaloniki, 24(1), 1–­ 11. https://doi.org/10.1186/s4070​ age. Physical and Chemical Mechanisms in Molecular Radiation Biology,
9-­017-­0 059-­x 58, 287–­321. https://doi.org/10.1007/978-­1-­4684-­7627-­9_10
Smith, T. A., Kirkpatrick, D. R., Smith, S., Smith, T. K., Pearson, T., Wattanathorn, J., Wannanon, P., Muchimapura, S., Thukham-­Mee, W.,
Kailasam, A., Herrmann, K. Z., Schubert, J., & Agrawal, D. K. (2017). Tong-­Un, T., & Polyiam, P. (2019). Toxicity evaluation of Anacardium
Radioprotective agents to prevent cellular damage due to ionizing occidentale, the potential aphrodisiac herb. BioMed Research
radiation. Journal of Translational Medicine, 15(1), 1–­18. https://doi. International, 2019, 1459141. https://doi.org/10.1155/2019/1459141
org/10.1186/s1296​7-­017-­1338-­x
Somananda, K., Ahongshangbam, G., & Chattopadhyay, S. (2014).
Bioactive compounds and antioxidant activity of Polygonum odora-
How to cite this article: Kawvised, S., Prabsattroo, T.,
tum Lour. International Journal of Applied Biology, 2(1), 94–­97.
Munkong, W., Pattum, P., Iamsaard, S., Boonsirichai, K.,
Sungkamanee, S., Wattanathorn, J., Muchimapura, S., & Thukham-­mee,
W. (2014). Antiosteoporotic effect of combined extract of Morus alba Uttayarat, P., Maikaeo, L., Sudchai, W., & Kirisattayakul, W.
and Polygonum odoratum. Oxidative Medicine and Cellular Longevity, (2021). Polygonum odoratum leaf extract attenuates oxidative
2014, 579305. https://doi.org/10.1667/RR1399.1 stress and cell death of Raw 264.7 cells exposed to low dose
Thongra-­ar, K., Rojsanga, P., Chewchinda, S., Mangmool, S., & Sithisarn, P.
ionizing radiation. Journal of Food Biochemistry, 00, e13909.
(2021). Antioxidant, α-­glucosidases and α-­amylase inhibitory activities
of Persicaria odorata. Chiang Mai University Journal of Natural Sciences, https://doi.org/10.1111/jfbc.13909
20(3), e2021051. https://doi.org/10.12982/​CMUJNS.2021.051