Food Bioscience 25 (2018) 44–51

Contents lists available at ScienceDirect

Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Antioxidant properties of Etlingera pubescens, an edible ginger plant endemic T

to Borneo
Delhousie Daniel-Jambuna, Kuan Shion Onga,d, Yau Yan Lima, Joash Ban Lee Tana, Wai Leng Leea,
⁎
Azira Muhamadb, Sau Wai Yapc, Sui Mae Leea,d,
a
School of Science, Monash University Malaysia, Selangor, Malaysia
b
Structural Biology and Biophysics Facility, Malaysia Genome Institute, National Institutes of Biotechnology Malaysia (MOSTI), Selangor, Malaysia
c
Conservation and Environmental Management Division, Yayasan Sabah Group, Sabah, Malaysia
d
Tropical Medicine and Biology, Monash University Malaysia, Selangor, Malaysia

A R T I C LE I N FO A B S T R A C T

Keywords: Zingiberaceae is a family that consists of many edible ginger plants. Etlingera pubescens (B.L.Burtt & R.M.Sm.)
Zingiberaceae R.M.Sm. was studied for its antioxidant properties. Preliminary screening of the methanol extracts of its leaves,
Etlingera pubescens stems, rhizomes showed that the fresh leaves had the highest antioxidant activity, with total phenolic content
Ginger (TPC), ascorbic acid equivalent antioxidant capacity (AEAC), and oxygen radical absorbance capacity (ORAC)
Curcumin
values of 1020 ± 20 mg GAE/100 g, 1130 ± 30 mg AAE/100 g, and 140 ± 30 µmol TE/g, respectively.
Etlingerin
Subsequently, a diarylheptanoid, etlingerin, was isolated from the leaf methanol extract. When contrasted with
1,7-bis(3,4-dihydroxyphenyl)heptan-3-yl
acetate curcumin (a well-known diarylheptanoid), etlingerin showed comparable free radical scavenging properties.
However, using a cell-based antioxidant assay, etlingerin showed significantly better protective effects (with
cellular antioxidant activity values as high as 84 ± 4%) than curcumin at all concentrations tested. In the
cytoprotective study, only etlingerin could protect cells that were challenged with hydrogen peroxide. These
effects might be explained by etlingerin's high lipophilicity and good free radical scavenging properties.
Etlingerin was also found to be significantly less cytotoxic than curcumin (up to 10-fold, as determined using a
MTT cell viability assay), thus suggesting its safety. The work showed that etlingerin has better antioxidant
properties, hence highlighting its potential as a functional food component that could reduce the risks of oxi-
dative stress-related diseases.

1. Introduction
Antioxidants have numerous beneficial effects for human health,
including anti-inflammatory, anti-carcinogenic, and anti-athero-
sclerotic activities, as a result of their antioxidant activities (Krishnaiah,
Sarbatly, & Nithyanandam, 2011). Plants are excellent sources of nat-
ural antioxidants as they produce these compounds in significant
amounts as secondary metabolites to protect themselves from free ra-
dical damage (Kasote, Katyare, Hegde, & Bae, 2015).
In the Asian countries, ginger plants (Zingiberaceae) are often
consumed, while also being used as a dye or medicine (Sirirugsa, 1999).
The Etlingera plants are primarily found in Southeast Asia. They can be
found in Borneo, and are not only consumed as vegetables, but are also
used in traditional medicine practices (Chan, Lim, & Wong, 2013;
Poulsen, 2006). E. elatior, or torch ginger, is the most well-known
species of this genus. It is traditionally used as an ingredient in local
delicacies and used as a remedy for headaches (Chan et al., 2013;
Poulsen, 2006). A previous study showed that its extracts have high
total phenolic content (TPC) and ascorbic acid equivalent antioxidant
capacity (AEAC) as high as 3600 ± 300 mg GAE/100 g and
3800 ± 600 mg AAE/100 g, respectively (Chan, Lim, & Omar, 2007).
It has also been shown to have a powerful antioxidant effect against
lead-induced hepatotoxicity in rats (Haleagrahara, Jackie,
Chakravarthi, Rao, & Kulur, 2010). Apart from E. elatior, other species
of Etlingera, including E. brevilabrum, E. fimbriobracteata, E. linguiformis,
E. littoralis, and E. philippinensis have also been reported to have bene-
ficial effects (Barbosa, Peteros, & Inutan, 2016; Chan et al., 2007;
Hossan et al., 2013; Mahdavi, Yaacob, Din, Heng, & Ibrahim, 2016;
Shahid-Ud-Daula et al., 2016). This study focuses on another ginger
species, E. pubescens.
According to Poulsen (2006), E. pubescens is a ‘Near-Threatened’
species endemic to Borneo. It is commonly known by its various local

⁎
Corresponding author at: School of Science, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia.
E-mail address: lee.sui.mae@monash.edu (S.M. Lee).

https://doi.org/10.1016/j.fbio.2018.07.007
Received 26 February 2018; Received in revised form 13 July 2018; Accepted 13 July 2018
Available online 17 July 2018
2212-4292/ © 2018 Published by Elsevier Ltd.
D. Daniel-Jambun et al.
names including ‘tubu apo’, ‘tubu bunga’, ‘tubu kerinang’, and ‘tubu
labo’. The leaves of E. pubescens are edible, and also often used as
roofing by the locals (Poulsen, 2006).
Oxidative stress is a health issue that plays an important role in the
development of degenerative diseases and metabolic syndromes. Many
of these health complications arise due to imbalance in reactive oxygen
species (ROS) levels (Khansari, Shakiba, & Mahmoudi, 2009). At ap-
propriate amounts, ROS are critical for normal cellular functions as they
act as signaling molecules (Buonocore, Perrone, & Tataranno, 2010).
However, excessive amounts of ROS can cause damage to DNA, pro-
teins, and lipids, hence impairing cellular functions (Buonocore et al.,
2010). Studies have correlated the benefits of phenolic compounds with
the reduction in risks of chronic diseases (Colarusso et al., 2017; Zhang
et al., 2015). Therefore, it may be beneficial to identify phytochemicals
that can be used to prevent oxidative stress-related diseases.
2. Materials and methods
2.1. Chemicals and reagents
Acetonitrile, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA)
(≥ 94%), dichloromethane, ethyl acetate, hydrogen peroxide (30%),
methanol, quercetin (> 95%), 0.25% trypsin and 1 mM ethylenedia-
minetetraacetic acid (EDTA) in Hank's balanced salt solution (HBSS)
and trolox (≥ 98%) were purchased from Merck (Kenilworth, NJ,
USA). Ascorbic acid (99%), 2,2′-azobis(2-amidinopropane) dihy-
drochloride (AAPH) (97%), caffeic acid (≥ 98%), catechin (≥98%),
1,1-diphenyl-1-picryl-hydrazyl (DPPH), fluorescein and gallic acid
(97.5–102.5% according to the manufacturer) were obtained from
Sigma Aldrich (St. Louis, MO, USA). Curcumin (98%) and 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (≥ 98%)
were purchased from Nacalai Tesque (Kyoto, Japan). Dulbecco's mod-
ified eagle medium (DMEM, containing 4500 mg/L D-glucose, 584 mg/L
L-glutamine, and 110 mg/L sodium pyruvate) culture media powder,
fetal bovine serum (FBS), Folin's reagent, penicillin-streptomycin
(containing 10,000 units/mL of penicillin and 10,000 µg/mL of strep-
tomycin and Roswell Park Memorial Institute (RPMI 1640, containing
300 mg/L L-glutamine) cell culture media powder were purchased from
Thermo Fisher Scientific (Waltham, MA, USA).
2.2. Plant materials
Specimens of E. pubescens were sampled from the Danum Valley,
Sabah (GPS coordinate: 04°57′59.3″N, 117°47′29.8″E), with permission
from the Sabah Biodiversity Centre (SaBC) and Conservation and
Environmental Management Division, Yayasan Sabah (CEMD). Plant
identification was done by Mr. Bernadus Bala Ola from the Danum
Valley Conservation Area (DVCA) and John Sugau from the Forest
Research Centre (FRC), Sabah. The leaves, stems, and rhizomes were
sampled randomly from plants that grow in the wild with heights ap-
proximately between 1 and 2 m. Then, the specimens in large black
plastic bags and placed inside foam boxes were immediately trans-
ported to the laboratory and processed within 24 h. Voucher specimens
were deposited at the Forest Department Herbarium Sandakan, Sepilok,
90000 Sandakan, Sabah, Malaysia, with the reference number 157375.
2.3. Extraction of plant specimens
Phytochemical extraction (for antimicrobial activity screening) was
done as outlined by Daniel-Jambun et al. (2017), with methanol as the
extraction solvent. Briefly, the specimens were crushed with the aid of
liquid nitrogen, added to methanol, subjected to sonication (35 kHz
frequency, Elmasonic S130H, Elma, Singen, Germany) for 15 min, fol-
lowed by filtration of the solution using filter papers (Type 1 Whatman,
Buckinghamshire, UK) and a Buchner funnel. Then, the solutions were
pooled, concentrated under reduced pressure using a rotary evaporator
45
Food Bioscience 25 (2018) 44–51
(N-1110, Eyela, Tokyo, Japan), and dried using a freeze-drier (Freezone
4.5 Plus, Labconco, Kansas City, MO, USA). For antioxidant activity
testing, approximately 1 g of fresh plant materials were grounded using
a mortar and a pestle and added to 50 mL of various concentrations of
methanol (50%, 70%, and 100%). After sonication for 15 min, the so-
lutions were filtered again and kept at −20 °C until further use, up to a
maximum of one wk.
2.4. In vitro chemical-based antioxidant assays
2.4.1. Folin Ciocalteu (FC) assay
TPC was determined using the Folin Ciocalteu (FC) assay as de-
scribed by Zhang et al. (2006), with modifications. In 96-well microtiter
plates (SPL 30196, Biolab, Singapore), 30 µL of plant extract was mixed
with 150 µL of 10% Folin's reagent and 120 µL of 7.5% sodium carbo-
nate. After 10 min of incubation in the dark, absorbance was measured
at 765 nm using a microtiter plate reader (Infinite M200, Tecan, Man-
nedorf, Switzerland). Gallic acid was used to construct a standard curve
and TPC was reported as mg GAE/100 g fresh sample. The calibration
equation was y = 0.008 × ± 0.0086 (R2 = 0.9985).
2.4.2. DPPH free radical scavenging
DPPH free radical scavenging (FRS) assay was done using the
method of Cheng, Moore, and Yu (2006), with modifications. In the 96-
well microtiter plates, plant extracts/pure compounds were serially
diluted (with final volumes of 100 µL) and 200 µL of DPPH solution was
added to each well (6.0 mg/100 mL methanol). After 30 min of in-
cubation, absorbance was measured at 517 nm using the microtiter
plate reader. Ascorbic acid solution was used as a reference and the
ascorbic acid equivalent antioxidant capacity (AEAC) was reported as
mg ascorbic acid equivalent (AAE)/100 g fresh sample. The IC50 value
(concentration that resulted in 50% change in the DPPH color) de-
termined for ascorbic acid was 3.9 ± 0.2 µg/mL. For comparison be-
tween pure compounds, the IC50 values are reported as µM.
2.4.3. Oxygen radical absorbance capacity (ORAC)
The ORAC assay was done using the method of Asensio, Grosso, and
Juliani (2015). In 96-well black microtiter plates (SPL 30296, Biolab),
25 µL of serially diluted plant extracts were added to 150 µL of 84 nM
fluorescein, followed by 15 min incubation at 37 °C. Then, 25 µL of
153 mM AAPH was added into each well. Immediately after this, the
fluorescence values were measured using the microtiter plate reader for
90 min with 2 min intervals, with excitation and emission wavelengths
of 485 and 528 nm, respectively. The antioxidant, trolox, was used as a
reference and the ORAC values were reported as µmol trolox equivalent
(TE)/g fresh weight. The calibration equation was
y = 0.3694 × ± 1.309 (R2 = 0.9906). For comparison between pure
compounds, the ORAC EC50 (concentration that provides 50% protec-
tion) values are reported as µM.
2.5. Isolation and identification of bioactive compound
2.5.1. Isolation of bioactive compound
Sequential solvent extraction (SSE) was carried out using the
method of Daniel‐Jambun et al. (2017) on the dried methanol extract of
E. pubescens leaves to separate out the non-polar compounds. Hexane,
dichloromethane, and ethyl acetate were used as the solvents. Briefly,
the extract was added to hexane and sonicated. The solution (200 mg/
mL) was then filtered, concentrated under reduced pressure, and dried.
These steps were repeated on the undissolved extract using di-
chloromethane and ethyl acetate, sequentially. C18 gravity column
chromatography was done on the ethyl acetate fraction to further re-
move non-polar compounds. The following parameters were used: di-
mension of packed beads: 3 cm diameter × 20 cm height; stationary
phase: LiChroprep RP-18 (40–63 µm) (Merck); mobile phase: 90%
acetonitrile. The crude extract (100 mg/mL) was introduced into the
D. Daniel-Jambun et al.
column and eluted with three column volumes of 90% acetonitrile. The
eluent was collected, filtered, concentrated under reduced pressure and
finally dried using the freeze-drier. Preparative HPLC (Agilent 1200
Infinity, Agilent, Santa Clara, CA, USA) was used to purify bioactive
compounds. Pure 1,7-bis(3,4-dihydroxyphenyl)heptan-3-yl acetate
(etlingerin) was obtained using the following conditions: Cosmosil C18
preparative column, 20 × 250 mm, 5-micron particles (Nacalai
Tesque); solvent system: A-water, B-acetonitrile; 30–50% B in 20 min,
95% B for 5 min; flow rate: 20 mL/min; injection volume: 100 µL;
concentration: 100 mg/mL. The compound was observed at tR
= 17.00 min (yield: 165 mg dry etlingerin weight per 1 kg of dried E.
pubescens leaves).
2.5.2. Identification of bioactive compound
The accurate mass of etlingerin was determined using negative ion
mode through electrospray ionization – mass spectrometry (ESI-MS).
The following conditions were used: Agilent 1290 Infinity LC instru-
ment coupled to an Agilent 6520 Accurate-Mass Q-TOF, Agilent
Poroshell EC-C18 column, 2.1 × 100 mm, 2.7-micron particles; solvent
system: A-0.1% formic acid, B-methanol with 0.1% formic acid;
40–100% B in 13 min, 100% B for 2 min; flow rate: 0.5 mL/min; in-
jection volume: 1 µL; concentration of pure etlingerin: 1 mg/mL. The
compound was observed at tR = 4.96 min 1H NMR (700 MHz) and 13C
NMR (176 MHz) analyses were done using a Bruker Ascend instrument
(Bruker, Karlsruhe, Germany) to aid structure elucidation. Data for 1H
NMR are reported in terms of chemical shift (δ ppm), integration,
multiplicity, coupling constant (Hz). Data for 13C NMR are reported as
chemical shift (δ ppm) and multiplicity (C, CH, CH2 or CH3). The fol-
lowing lists the spectroscopic data for etlingerin.
2.5.2.1. 1,7-bis(3,4-dihydroxyphenyl)heptan-3-yl acetate (etlingerin). oil-
like; 1H NMR (CD3OD, 700 MHz) δ 6.67 (d, J = 8.0 Hz, 1H, H-5a), 6.67
(d, J = 8.0 Hz, 1H, H-5b), 6.61 (s, broad, 2H, H-2a, H-2b), 6.48 (d,
broad, J = 8.0 Hz, 2H, H-6a, H-6b), 4.86 (quintet, J = 6.3 Hz, 1H, H-3),
2.50 (m, 2H, H-1), 2.44 (m, 2H, H-7), 2.01 (s, 3H, NCOOCH3), 1.78 (m,
2H, H-2), 1.59 (m, 2H, H-4), 1.53 (m, 2H, H-6), 1.31 (m, 2H, H-5); 13C
NMR (176 MHz, MeOD) δ 171.56 (NCOOCH3), 144.75 (C, C-3a),
144.63 (C, C-3b), 142.93 (C, C-4a), 142.72 (C, C-4b), 133.97 (C, C-
1a), 133.15 (C, C-1b), 119.27 (CH, C-6a), 119.19 (CH, C-6b), 115.13
(CH, C-2a), 115.06 (CH, C-2b), 114.92 (CH, C-5a), 114.81 (CH, C-5b),
73.90 (CH, C-3), 35.78 (CH2, C-2), 34.60 (CH2, C-7), 33.56 (CH2, C-4),
31.10 (CH2, C-6), 30.73 (CH2, C-1), 24.29 (CH2, C-5), 19.72 (CH3,
NCOOCH3); LCESIMS m/z 374.1769 (calculated MW for C21H26O6,
374.1730).
2.6. Mammalian cell cultures
The HaCaT (human keratinocyte, 300493, CLS GmbH, Eppelheim,
Germany) cell line was cultured in high glucose DMEM supplemented
with 10% FBS and 1% penicillin-streptomycin. HT-29 (human colon
carcinoma, HTB-38, ATCC), SW-480 (human colon adenocarcinoma,
CCL-228, ATCC) and CCD-841 (human colon normal epithelial, CRL-
1790, ATCC) cells were cultured in RPMI 1640 media supplemented
with 10% FBS and 1% penicillin-streptomycin. All cell lines were cul-
tured in T75 tissue culture flasks (SPL 70075, Biolab) at 37 °C with 5%
CO2 supplied in a biological incubator (Galaxy R+, RSBiotech, Irvine,
UK). The cells were sub-cultured/harvested when they reach about 80%
confluency (determined by visual inspection). Quantitation of cells was
done using the trypan blue exclusion method (Strober, 2015). Briefly,
10 µL of the harvested cell suspension was added to 10 µL of trypan blue
(0.4%, Amresco, Dallas, TX, USA) and mixed well. Then, 10 µL of the
mixture was placed onto the hemocytometer and the number of cells in
the corner squares were enumerated.
46
Food Bioscience 25 (2018) 44–51
2.7. Cell-based antioxidant assay
The cell-based antioxidant assay was done using the method of
Bender and Graziano (2015), with modifications. Approximately
50,000 HaCaT cells were loaded into wells of 96-well black microtiter
plates. After 24 h incubation, the cells were pre-treated with various
concentrations of the compounds, as well as 25 µM DCFH-DA. Fol-
lowing 1 h incubation, the wells were washed and 600 µM AAPH was
added. Fluorescence kinetics was measured every 5 min for 1 h using
the microtiter plate reader (Spark 10 M, Tecan, Mannedorf, Switzer-
land) with excitation and emission wavelengths of 485 and 540 nm,
respectively. Quercetin was used as a standard. A graph of percentage
protection (%) against concentration of compound (µg/mL) was plotted
to enable comparison of antioxidant activity across various concentra-
tions.
2.8. MTT cell viability assay
The MTT assay was done using the method of Sylvester (2011), with
modifications. Approximately 5000 cells in 100 µL culture media were
loaded into the wells of a 96-well microtiter plate and incubated for
24 h. After that, the cells were treated with various concentrations of
compounds for an additional 24 h. Then, the cells were exposed to
5 mg/mL MTT solution for 2 h and the purple formazan crystals were
eluted with DMSO. Absorbance values were measured at 590 nm using
the microtiter plate reader and dose response curves obtained. Cyto-
toxicity was determined as the concentration of compounds that in-
hibited 50% cell growth (IC50).
2.9. Cytoprotective effect
Determination of cellular protective effects on HaCaT cell line was
done using the method of Chen, Zhong, Xu, Chen, and Wang (2011),
with modifications. Approximately 5000 cells of HaCaT cells in 100 µL
culture media were loaded into the wells of a 96-well microtiter plate.
After 24 h incubation, the cells were treated with various concentra-
tions of compounds for 1 h. Then, the wells were washed to remove
extracellular compounds, followed by addition of 500 µM hydrogen
peroxide. After an additional 24 h incubation, the MTT assay was done
to determine cell viability. Determination of cell viability using trypan
blue exclusion was done using the method of Strober (2015) with the
following modifications: Approximately 30,000 cells in 600 µL culture
media were seeded in wells of a 24-well microtiter plate (NunC 150687,
ThermoFisher Scientific). After treatment, the cells were washed with
PBS, detached using 0.25% trypsin and 1 mM EDTA in HBSS, mixed
with equal volume of FBS, and enumerated on a hemocytometer (As-
sistent, Sondheim, Germany). Visual assessment of cellular morphology
(of 3 replicates) was done using a Nikon TS-100 (Nikon, Tokyo, Japan)
inverted phase-contrast microscope.
2.10. Determination of log P values
The log P values (which represents the predicted lipophilicity of
compounds) were calculated using computational methods using the
SwissADME web tool (which can be accessed at http://www.
swissadme.ch), as described by Daina, Michielin, and Zoete (2017).
According to the authors, a common practice to obtain accurate log P
values is to use multiple predictors. This is because different predictors
were developed based on different principles. Five predictive models
(XLOGP3, WLOGP, MLOGP, SILICOS-IT, and iLOGP) were used based
on the authors’ recommendation. The consensus log P represents the
arithmetic mean of the values predicted by the 5 methods. Briefly, on
the use of the web tool, the chemical structures of the compounds were
drawn in the given space. The software will then analyse the structures
and all log P values were automatically calculated and displayed.
D. Daniel-Jambun et al. Food Bioscience 25 (2018) 44–51

2.11. Statistical analysis

Statistical analysis was done using IBM SPSS Statistics Version 21.0
(Armonk, NY, USA). The significance of results for different assays were
done using independent sample t-test or one-way analysis of variance
(ANOVA) followed by Tukey's Honest Significant Difference (Tukey's
HSD) test at P < 0.05. Independent sample t-test was done to compare
the means of two groups while ANOVA compares the means of multiple
(more than two) groups.

3. Results and discussion

3.1. Antioxidant activity screening

Antioxidant activity screening was done to determine the free ra- Fig. 1. The chemical structures of 1,7-bis(3,4-dihydroxyphenyl)heptan-3-yl
dical scavenging abilities of the crude plant extracts. TPC was quanti- acetate (etlingerin, top), and (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
fied to estimate the amount of phenolic compounds present, as well as heptadiene-3,5-dione (curcumin, bottom).
to assess the extracts’ reducing capabilities (Tan & Lim, 2015). DPPH
FRS and ORAC tests were used to evaluate the extracts’ antioxidant group of compounds containing two aromatic rings joined by a heptane
activity using different mechanisms (combination of single electron chain. They can be found in various plants, including the ginger plant
transfer and hydrogen atom transfer, and hydrogen atom transfer, re- genera: Zingiber, Curcuma, Alpinia, Betula, Alnus, and Etlingera (Chan
spectively) (Tan & Lim, 2015). The outcomes of these experiments are et al., 2013; Novaković, Novaković, Cvetković, Sladić, & Tešević, 2015).
summarised in Table 1. These compounds are known to have good antioxidant properties
In general, the crude extracts obtained using 100% methanol (Kuroyanagi et al., 2005; Tung et al., 2010). Like etlingerin, curcumin
showed higher TPC and AEAC values, as compared to 70% and 50% (see Fig. 1 for its chemical structure) is also a diarylheptanoid. This
methanol (see Table 1), hence suggesting that the antioxidant com- justifies the use of curcumin as a reference compound in determining
pounds present in E. pubescens are relatively non-polar in nature. This the antioxidant potential of etlingerin. Curcumin is known to have
was desirable as compounds that are more soluble in organic solvents various beneficial effects due to its ability to modulate ROS levels
tend to have higher lipophilicity, and thus may pass through the cell (Sandur et al., 2007).
membrane and exert their effects from within the cells (Wolfe & Liu, The yield of etlingerin was determined as 1.7 × 10−2% of dried
2008). When the different plant parts were compared, the leaf extract of leaves weight. When compared to the yield reported by Giang et al.
E. pubescens showed significantly better antioxidant activity than the (2012) which was about 12.8 mg per 0.84 kg of dried rhizomes weight
stems and rhizomes, as suggested by the former's high TPC, AEAC, and (1.5 × 10−3%), etlingerin was found to have a higher abundance in E.
ORAC values. Therefore, the leaf methanol extract of E. pubescens was pubescens. This suggests that the leaves of E. pubescens are a good source
used for isolating its compounds, and subsequently, 1,7-bis(3,4-dihy- of etlingerin.
droxyphenyl)heptan-3-yl acetate was purified and identified. In the
subsequent sections, this compound will be referred to as etlingerin.
3.2. Antioxidant activities of etlingerin
Etlingerin (see Fig. 1 for its chemical structure) is a diarylheptanoid
that was once isolated from Amomum muricarpum (Giang, Son,
The antioxidant activity of etlingerin was first tested using in vitro
Matsunami, & Otsuka, 2012). The spectroscopic data reported by Giang
chemical-based assays to evaluate its ability to react with free radicals.
et al. (2012) match those from this study. Diarylheptanoids are a small
Curcumin, as well as ascorbic acid and trolox, were used as reference
compounds. The results are shown in Table 2.
Table 1
Etlingerin showed a significantly lower DPPH FRS IC50 value as
The total phenolic content (TPC), ascorbic acid equivalent antioxidant capacity
(AEAC), and oxygen radical absorbance capacity (ORAC) values determined for
compared to curcumin, suggesting that the former is a better free ra-
E. pubescens samples extracted using various percentages of methanol. dical scavenger than the latter. This could be due to etlingerin having 4
phenolic groups while curcumin has only two phenolic groups (see
Part of plant Percentage TPC AEAC ORAC
Fig. 1). A compound with more phenolic groups is able to transfer more
methanol (mg GAE/100 g (mg AAE/100 g (µmol TE/g
fresh weight) fresh weight) fresh weight) hydrogen ions to the free radicals (Priyadarsini et al., 2003). However,
etlingerin and curcumin had similar ORAC EC50 values. This
Leaves 50 250 ± 10c 380 ± 20c ND
70 429 ± 8b 500 ± 10b ND
Table 2
100 1020 ± 20aA 1130 ± 30aA 150 ± 30A
The DPPH FRS IC50 and ORAC EC50 values of etlingerin in comparison with
Stems 50 59.0 ± 0.6b 58 ± 1b ND
70 68 ± 2b 81 ± 2b ND curcumin, ascorbic acid, and trolox.
100 109 ± 4B 137 ± 9B 14 ± 2B Compound DPPH FRS IC50 (µM) ORAC EC50 (µM)
Rhizomes 50 49.6 ± 0.7c 55 ± 1c ND
70 59 ± 2b 79 ± 2b ND Curcumin 26.1 ± 0.2bc 2.59 ± 0.03a
100 92 ± 2aB 115 ± 3aB 8.0 ± 0.2B Etlingerin 9.91 ± 0.06a 2.73 ± 0.02a
Ascorbic acid 21.6 ± 0.2b ND
Assays were done with three replicates and values are reported as mean ± Trolox ND 14.3 ± 0.1b
standard error. ND indicates that the experiment was not done as ORAC values
were only determined for the extracts with highest TPC and AEAC values. The Assays were done with three replicates and values are reported as mean ±
significance of the results was assessed using ANOVA with Tukey's post-hoc test standard error. The significance of the results was assessed using the in-
at P < 0.05. Small letters compare statistical differences between means of the dependent samples t-test at P < 0.05. Different letters indicate significant
three different percentages of methanol (50%, 70%, and 100%) for the same difference between mean values within the same assay. DPPH FRS = DPPH free
part of the plant while capital letters compare highest mean values between the radical scavenging; IC50 = concentration that scavenges 50% of free radical;
three plant parts (leaves, stems, and rhizomes). GAE = gallic acid equivalent; ORAC = oxygen radical absorbance capacity; EC50 = concentration that pro-
AAE = ascorbic acid equivalent; TE = trolox equivalent; ND = not determined. vides 50% protection; ND = not determined.

47
D. Daniel-Jambun et al.
contradiction with the results with the DPPH radical scavenging assay
might be explained several ways. First, the two assays evaluate anti-
oxidant activities using different mechanisms, as previously mentioned.
Second, in the ORAC assay, the solutions were buffered using phosphate
buffer (pH 7.4), whereas the DPPH radical scavenging assay used un-
buffered methanol. It is known that pH can affect a phenolic com-
pound's antioxidant activity. A previous study found that the increase in
pH was associated with stabilization of phenolic species, leading to
polymerization reactions, which subsequently results in new oxidizable
hydroxyl moieties being formed (Altunkaya, Gökmen, & Skibsted,
2016). Nonetheless, it can be inferred that etlingerin has comparable
free radical scavenging activity with curcumin. When contrasted with
ascorbic acid and trolox, etlingerin showed significantly lower DPPH
FRS IC50 and ORAC EC50 values (see Table 2), hence implying that
etlingerin is a better free radical scavenger than ascorbic acid and
trolox.
Though it is important for an antioxidant to be able to effectively
scavenge free radicals, it is also important that the compound can exert
this effect from within cells. This warrants the use of the cell-based
antioxidant assay. In this assay, DCFH-DA enters the cells, where it
becomes membrane-impermeable after being cleaved by intracellular
esterases (yielding DCFH). Therefore it stays in the cells after the
washing step. If the antioxidant compounds can enter the cells, they
could protect the DCFH from being attacked by free radicals generated
by AAPH. For this reason, the antioxidant compounds’ chemical
structures have a significant role in their ability to cross the cell
membrane. A previous study by Wolfe and Liu (2007) showed that
antioxidant compounds such as gallic acid, ascorbic acid and caffeic
acid have low CAA as these compounds might only loosely adsorb on
the cell membrane and/or are taken up less readily compared to other
compounds that have high CAA such as quercetin, kaempferol and
epigallocatechin gallate (EGCG). In the present work, the ability of
etlingerin to protect intracellular DCFH was evaluated in comparison
with curcumin and quercetin. Fig. 2 shows the percentage protection
values determined for various concentrations of these compounds.
At the highest concentration tested (20 µg/mL), quercetin and
etlingerin showed the highest percentage protection values (87 ± 7
and 84 ± 4%, respectively). Curcumin also showed a positive
Fig. 2. The percentage protection (%) of quercetin, etlingerin, and curcumin,
on DCFH, at various concentrations (µg/mL). Four independent replicates were
done, and error bars represent mean ± standard error. The significance of the
results was assessed using ANOVA with Tukey's post-hoc test at P < 0.05.
Different letters indicate significant differences between the mean percentage
protection values of the different compounds within the same concentration.
Asterisks indicate significant differences between pre-treatment and untreated
cells for each compound, at P < 0.05 (as determined using the independent
samples t-test).
48
Food Bioscience 25 (2018) 44–51
protective effect, although at a significantly lower percentage
(66 ± 6%). These results suggested that all three compounds could
cross the cell membrane and protect the intracellular DCFH. Based on
Fig. 2, dose-dependent decreases in percentage protection values were
seen with decreasing concentrations of the three compounds. However,
the protective effect of etlingerin became more prominent than quer-
cetin and curcumin, hence implying that etlingerin can still be an ef-
fective antioxidant, even at low concentrations. This is highly desirable
as beneficial effects could still be achieved while having low cell toxi-
city. Further work is required to evaluate this hypothesis. Hence, the
cytoprotective and cytotoxicity studies were done, and are presented in
the next sections.
3.3. Cytoprotective effect of etlingerin on the HaCaT cell line
As previously mentioned, the cell-based antioxidant assay measures
a compound's ability to protect the intracellular DCFH. However, it
does not provide any information on the consequences of the treat-
ments to the cells. Additionally, the prolonged exposure to free radicals
is also not considered, as the assay only proceeds for 1 h. Therefore, the
cytoprotective study was done to address these limitations. Hydrogen
peroxide was chosen as the free radical generator as it is one of the most
prominent ROS in mammalian cells. Other well-known antioxidants
(curcumin, quercetin, trolox, catechin, and caffeic acid) were also
tested for comparison purposes, and the results are shown in Fig. 3.
As seen in Fig. 3, treatment with hydrogen peroxide caused an ap-
proximately 40% drop in cell viability as compared to untreated cells
(100%), thereby validating the detrimental effects of hydrogen per-
oxide. When the cells were pre-treated with the antioxidant com-
pounds, varying degrees of change in cell viability were observed: at
50 µg/mL, quercetin and etlingerin pre-treatments resulted in sig-
nificantly higher cell viability values than hydrogen peroxide treatment
only, while lower cell viability was associated with curcumin pre-
treatment. No significant differences were observed between the cell
viability values linked to trolox, catechin, and caffeic acid pre-treat-
ments, with hydrogen peroxide treatment only. One plausible reason
for this might be associated with the lipophilicity of these compounds.
Therefore, to assess this hypothesis, the log P values were determined
(see Table 3).
Based on the predicted log P values, quercetin, trolox, catechin, and
caffeic acid have low lipophilicity, as compared to curcumin and
etlingerin, consensus log P values of 1.23, 2.47, 0.85, 0.93, 3.03, and
3.42, respectively. Therefore, these compounds might not readily pe-
netrate the cell membrane during the 1 h pre-treatment period, hence
are unable to effectively protect the cells from hydrogen peroxide-in-
duced damage. Conversely, etlingerin has the highest lipophilicity
among the antioxidants tested, as suggested by its consensus predicted
log P value. A lipophilic antioxidant is advantageous as it can modulate
intracellular free radicals (Wolfe & Liu, 2008). This, together with the
fact that etlingerin is a good free radical scavenger, could account for
the results shown in Fig. 3, whereby etlingerin showed the highest
percentage cell viability as compared to the other antioxidants, across
various concentrations. Although etlingerin has a good protective effect
at low concentrations, it was noted that cytotoxicity was observed at a
high concentration (200 µg/mL).
In general, etlingerin showed better protective effects than the other
antioxidants. However, at 10–100 µg/mL, its associated cell viability
values exceeded 100%. This could be due to the possible interaction
between etlingerin and the MTT dye, as some antioxidant compounds
can reduce MTT in the absence of cells (Talorete, Bouaziz, Sayadi, &
Isoda, 2006). This means that the observed increase in cell viability
might be caused by 1) its true antioxidant (protective) effect, and/or 2)
etlingerin-MTT interactions. Therefore, direct cell enumeration (via
trypan blue exclusion) was done to validate the results seen in Fig. 3.
The concentration of 50 µg/mL was chosen as varying degrees of
change in cell viability could be observed. Fig. 4 summarises the cell
D. Daniel-Jambun et al. Food Bioscience 25 (2018) 44–51

Fig. 3. HaCaT cell viability (as determined using a MTT

cell viability assay) following treatment with hydrogen
peroxide alone and in combination with either etlingerin,
quercetin or curcumin. Three independent replicates were
done, and values are reported as mean cell viability ±
standard error. The significance of the results was assessed
using ANOVA with Tukey's post-hoc test at P < 0.05.
Different letters indicate significant differences between
pre-treatments within the same concentrations. Asterisks
indicate significant differences between pre-treatments
(10, 25, 50, 100, and 200 µg/mL) with hydrogen peroxide
treatment only (0 µg/mL) for the same compound, at P <
0.05 (using ANOVA with Tukey's post-hoc test).

counts following pre-treatment with the antioxidants, and subsequent

treatment with hydrogen peroxide.
In Fig. 4, hydrogen peroxide treatment caused a significant reduc-
tion in cell viability compared to the untreated cells,
0.56 × 105 ± 0.04 × 105 cells against 1.8 × 105 ± 0.2 × 105 cells,
respectively. However, when pre-treated with etlingerin, the number of
viable cells were significantly higher than cells treated with hydrogen
peroxide only. This supports etlingerin's cytoprotective effect. On the
other hand, no protective effect was found for curcumin pre-treatment;
instead, a significant reduction in cell viability was observed (as also
seen in Fig. 3). This was not surprising as a previous study reported that
short curcumin pre-treatments has no obvious protective effect on hy-
drogen peroxide induced cell injury (Chen et al., 2011). Fig. 4 also
showed that quercetin pre-treatment did not significantly change the
number of viable cells as compared to hydrogen peroxide treated cells.
This contradicts the results from the previous experiments where 1)
quercetin protected DCFH from degradation with percentage protection
up to 69 ± 3% (in cell-based antioxidant assay), and 2) pre-treatment
with 50 µg/mL quercetin resulted in increased MTT cell viability (81%).
The following reasons might explain these contradictions. First, the cell-
based antioxidant assay primarily depends on a compound's ability to
protect DCFH from free radical damage. As previously mentioned, this Fig. 4. HaCaT cell viability (as determined using trypan blue exclusion) fol-
assay does not consider the consequences to the cells. AAPH and hy- lowing treatment with hydrogen peroxide alone and in combination with
drogen peroxide can generate free radicals that not only cause damage etlingerin, quercetin or curcumin. Three independent replicates were done, and
to DCFH, but also to cellular components, which in turn can lead to cell values are reported as mean cell count ± standard error. The significance of the
death (Minotti & Aust, 1987; Terao, Piskula, & Yao, 1994; Wijeratne, results was assessed using the independent samples t-test at P < 0.05.
Cuppett, & Schlegel, 2005). Second, the assay only proceeded for Asterisks indicate significant difference compared to HaCaT cells treated with
90 min whereas the treatments in the cytoprotective study were for hydrogen peroxide alone.
24 h. Hence, depletion of antioxidant compounds (in this case, quer-
cetin) might affect their ability to protect cells for a longer period.

Table 3
Lipophilicity of curcumin, etlingerin, quercetin, trolox, catechin, and caffeic acid, as determined using computational methods.
Computational method Curcumin Etlingerin Quercetin Trolox Catechin Caffeic acid

iLOGP 3.27 2.58 1.63 2.11 1.47 0.97

XLOGP3 3.20 4.29 1.54 2.84 0.36 1.15
WLOGP 3.15 3.79 1.99 2.49 1.22 1.09
MLOGP 1.47 2.51 − 0.56 1.81 0.24 0.70
SILICOS-IT 4.04 3.92 1.54 3.11 0.98 0.75
Consensus 3.03 3.42 1.23 2.47 0.85 0.93

iLOGP, XLOGP3, WLOGP, MLOGP, and SILICOS-IT are 5 different predictive models used to determine the log P values. The consensus log P values are the arithmetic
mean values of the 5 models. All values were obtained from the SwissADME web tool (http://www.swissadme.ch).

49
D. Daniel-Jambun et al.
Table 4
Cytotoxicity of curcumin and etlingerin with various cell lines.
Compound IC50 (µg/mL)
HaCaT HT-29 SW-480 CCD-841
Curcumin 4.2 ± 0.9b 14 ± 2a 10.5 ± 0.5a 31 ± 2a
Etlingerin 48 ± 4a 60 ± 6b 46 ± 4b 144 ± 3b
Assays were done with three independent replicates, and values are reported as
mean ± standard error. The significance of the results was assessed using the
independent samples t-test at P < 0.05. Different letters indicate significant
differences between curcumin and etlingerin for the same cell line.
Third, quercetin might react with the MTT, leading to a false positive.
3.4. Cytotoxicity of etlingerin
Cytotoxicity studies were done to assess the effects of prolonged
treatment with etlingerin on the viability of various cell lines. The IC50
values determined are summarised in Table 4.
In general, etlingerin was found to be less cytotoxic (about 4–10-
fold) compared to curcumin. It is noteworthy that curcumin itself is not
considered toxic when used in vivo as clinical trials had proven that it
was safe even when consumed at a daily dose of 12 g for 3 months
(Gupta, Patchva, & Aggarwal, 2013). Therefore, these results suggest
that etlingerin could be safe when consumed in high doses. The reason
for curcumin's low toxicity in humans is mainly due to its low bioa-
vailability. This is because curcumin is unstable at physiological pH,
susceptible to autoxidation, has poor absorption through the gastro-
intestinal tract, is rapidly metabolised and rapidly eliminated, thus
resulting in low bioavailability (Anand, Kunnumakkara, Newman, &
Aggarwal, 2007; Metzler, Pfeiffer, Schulz, & Dempe, 2013).
4. Conclusion
The study showed that etlingerin isolated from E. pubescens has good
antioxidant properties, comparable to curcumin, and better than those
of other antioxidants such as ascorbic acid, trolox, and quercetin, as
shown using DPPH radical scavenging, ORAC, cell-based antioxidant
assay, and cytoprotective studies. Furthermore, etlingerin was sig-
nificantly less cytotoxic than curcumin, hence suggesting its safety. The
present work highlights etlingerin's potential as a functional food
component that could reduce the risks of oxidative stress-related dis-
eases.
Acknowledgements
The authors are grateful to Monash University Malaysia for aiding
the research financially. We would like to acknowledge Ms. Rita Stuel
Galid (Conservation and Environmental Management Division, Yayasan
Sabah Group), for the reports and information regarding medicinal
plants of Sabah as well as aiding the access to the Danum Valley. Also,
we are thankful to Mr. Bernadus Bala Ola (DVCA local botanist), Mr.
Jaludin Abun (Manager of INFAPRO), Mr. Asni bin Bulot (INFAPRO
Senior Forest Ranger), and Mr. John Sugau (FRC research officer) for
assisting in collection and identification of the plant specimens. The
Sabah Biodiversity Centre (SaBC) is greatly acknowledged for the access
and export licenses (JKM/MBS.1000-2/2 (394) and JKM/MBS.1000-2/
3 JLD.2 (18), respectively). The work was funded by Monash University
Malaysia.
Conflict of interest declaration
The authors declare no conflict of interest.
50
Food Bioscience 25 (2018) 44–51
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