Food Chemistry 253 (2018) 5–12

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

The antioxidant activity and genotoxicity of isogarcinol T

a,b c a c a,⁎ a,⁎
Zijin Liu , Gang Li , Cheng Long , Jing Xu , Juren Cen , Xiaobo Yang
a
Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, People’s Republic of China
b
Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing 100875, People’s Republic of China
c
College of Materials and Chemical Engineering, Hainan University, Haikou 570228, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: The antioxidant activity and genotoxicity of isogarcinol were assessed by several in vitro tests. Its IC50 values for
Isogarcinol DPPH% and ABTS% were 36.3 ± 3.35 µM and 16.6 ± 3.98 µM, respectively, which were all lower than those of
Antioxidant activity VC and BHT. Isogarcinol had no cyctotoxic or promotional activities at 1–10 µM in the CCK-8 assay, and neg-
Cytotoxicity ligible genotoxic effects at 50–500 µM on HepG2 cells by the single-cell gel electrophoresis assay. Pre-incubation
Genotoxicity
of the cells with 0.5–1.5 µM isogarcinol, before exposure to H2O2, significantly increased cell viability in a
HepG2 cells
concentration-dependent manner. Isogarcinol also reduced intercellular reactive oxygen species accumulation,
Oxidative stress
lactate dehydrogenase release and malondialdehyde levels, and increased superoxide dismutase activity and
glutathione levels. Western-blot analysis revealed that it up-regulated pro-caspase-3 and reduced cleaved cas-
pase-3 during H2O2-induced oxidative stress. All the above results indicate that isogarcinol promises to be useful
as a natural antioxidant.

1. Introduction effective antioxidants containing natural compounds from food, fruit or

plants are needed. Many phenolics, flavonoids and benzophenones from
Free radicals produced by normal oxidative metabolism in the fruit, vegetables and plants are good exogenous antioxidants with
human body can cause chain oxidation of food, accelerate food dete- chemopreventive effects (Hua & Rong, 2016; Wu, Long, & Kennelly,
rioration and cause serious problems of food safety (Choe & Min, 2006). 2014). In addition, these bioactive compounds can reinforce biological
Excess free radicals can lead to cancer, atherosclerosis, cardiovascular defence systems by regulating the activities of antioxidant enzymes
disease, Parkinson’s disease and diabetes mellitus (Fearon & Faux, such as superoxide dismutase, catalase and glutathione peroxidase
2009). Reactive oxygen species (ROS) and reactive nitrogen species (Kong et al., 2016).
(RNS) play critical roles in modulating numerous biochemical processes The purple mangosteen Garcinia. mangostana L. is a promising
(Tsukagoshi, Busch, & Benfey, 2010). High levels of ROS or RNS can source of both novel biologically active substances and compounds
damage DNA, proteins and lipids, generating liver disorders, coronary essential for human nutrition (Zarena, Sachindra, & Sankar, 2012;
heart disease, and many other health issues (Ward, Prosser, & Lederer, Zadernowski, Czaplicki, & Naczk, 2009). Isogarcinol (Fig. S1) is a
2014). ROS are highly reactive non-specific molecules, including hy- bioactive polyisoprenylated benzophenone derivative isolated from G.
drogen peroxide and other species, such as oxygen free radicals and mangostana L. and exists in other Guttiferae plants that are widespread
singlet oxygen derived from the metabolism of oxygen (Liu et al., in tropical rain forests (Liu et al., 2015). The review showed that the
2014). Oxidative stress ensues when ROS and free radicals overwhelm prenylation of aromatic compounds and a variety of subsequent tai-
the regulatory ability of the body (Sies, 2015), and such a dynamic loring reactions, including oxidation, reduction and cyclisation, give
imbalance between ROS production and elimination can be counter- rise to a plethora of ‘hybrid’ natural products that exhibit diverse bio-
acted by various enzymes and antioxidants. logical properties. In particular, the introduction of prenyl moieties
Nowadays, synthetic antioxidants are widely used in food and often increases the lipophilicity of these compounds, enhancing their
pharmaceutical products, but their use is often accompanied by toxic interactions with biological membranes (Chen et al., 2017). Simulta-
and other side effects. For example, butylated hydroxytoluene (BHT), neously, many reports have also demonstrated that natural benzophe-
butyl hydroxy anisole (BHA) and tertiary butylhydroquinone (TBHQ) nones, most with polyisoprenylated benzophenone skeletons, exhibit a
are commonly used in the food industry, and all have some toxic and range of biological activities, including antifungal, anti-HIV, anti-
carcinogenetic effects (Carocho & Ferreira, 2012). Thus, safer and more microbial, antioxidant, antiviral and cytotoxic activities (Wu et al.,

⁎
Corresponding authors.
E-mail addresses: cenjr@hainu.edu.cn (J. Cen), yanfengxb@hainu.edu.cn (X. Yang).

https://doi.org/10.1016/j.foodchem.2018.01.074
Received 9 August 2017; Received in revised form 7 December 2017; Accepted 9 January 2018
Available online 12 January 2018
0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
Z. Liu et al. Food Chemistry 253 (2018) 5–12

2014). Additionally, the hydroxylation pattern on the phenyl rings is an

DPPH % −scavenging activity (%) = [(A blank-A sample)/A blank] ×100 %
important structural feature for their antioxidant activity, with greater
numbers of hydroxyl groups resulting in more antioxidant activity (Wu
et al., 2014). It has been proved that isogarcinol has a variety of bio- where Ablank is the absorbance of the control reaction mixture (con-
logical activities, including anti-bacterial and anti-plasmodial activities taining all reagents except the test compound), and Asample is the ab-
(Marti, Eparvier, Litaudon, Grellier, & Guéritte, 2010). It also has sorbance of the test compound.
therapeutic effects in experimental autoimmune encephalomyelitis
(Wang et al., 2016) and is a powerful and low-toxicity im-
munosuppressant (Cen et al., 2013; Li et al., 2015) with desirable anti- 2.3. ABTS radical-scavenging capacity
inflammatory effects (Fu, Zhou, Wang, Cen, & Wei, 2014). Moreover it
is less toxic to liver and kidney function than is cyclosporin A (CsA) in The antiradical activities of samples were compared to those of
experimental animals (Cen et al., 2013). Stark, Salger, and Frank (2015) known antioxidants, such as BHT (butylated hydroxytoluene) and VC
reported that isogarcinol had some anti-oxidative activity, but obtained (vitamin C) with 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic
acid) (ABTS% ) as substrate, using the method of Zhou et al. (2013) The
+
no evidence of its biological relevance in their study, which only in-
volved chemical experiments. ABTS radical cation was prepared by mixing equal volumes of 7 mM
Therefore, we have further investigated the anti-oxidant activity of ABTS solution and 2.45 mM potassium persulphate. The mixture was
isogarcinol in vitro by measuring the scavenging of 2,2′-diphenyl-1- incubated for 12–16 h at room temperature in the dark to yield a dark-
1picrylhydrazyl (DPPH%) and 2,2′-azino-bia (3-ethylben-zothiazoline-6- coloured solution containing ABTS radicals and then diluted with
sulphonicacid) (ABTS% ), and determining its reducing power and
+
ethanol to an absorbance of 0.7 ± 0.05 units at 734 nm. Extracts
ability to prevent lipid peroxidation. We also assessed the cytotoxicity (0.1 ml) or a reference substance (0.1 mL) were allowed to react with
and genotoxicity of isogarcinol, and observed its effect on cell viability, 3.9 mL of the ABTS solution for 30 min in the dark until a stable ab-
ROS, lactate dehydrogenase (LDH) release, malondialdehyde (MDA) sorbance was obtained. The decrease of absorbance at 734 nm was
levels, superoxide dismutase (SOD) activity and glutathione (GSH) le- measured by UV-2100 spectrophotometer against a blank (ethanol).
vels, as well as its cytoprotective effect on mitochondria-dependent Data for each assay were recorded in triplicate. VC and BHT were used
apoptosis. as positive controls. Antioxidant activity, as ABTS radical-scavenging
capacity, was estimated, based on the percentage, by the following
2. Materials and methods formula:

2.1. Materials ABTS % −scavenging activity (%) = [(A blank-A sample)/A blank] ×100 %

Isogarcinol (Iso) with a purity of 95.55% was extracted and purified where Ablank is the absorbance of the control reaction (containing all
by our research team (Cen et al., 2013). DPPH was purchased from reagents except the test compound), and Asample was the absorbance of
Sigma-Aldrich (St. Louis, MO USA), ABTS and butylated hydro- the test compound.
xytoluene (BHT) were from Tokyo Chemical Industry (TCL) Develop-
ment Co. Ltd. (Shanghai, China) and lecithin from Shanghai Macklin
Biochemical Co. Ltd. (China). Fetal bovine serum (FBS) was from 2.4. Determination of reducing power
Zhejiang Tianhang Biotechnology Co. Ltd. (China) and the Cell
Counting kit-8 was bought from BBI Life Sciences Corporation The reducing power of samples was measured by a modification of
(Shanghai, China). Vitamin C (VC) and vitamin E (VE) were purchased the method of Roy et al. (2011) 1.0 mL aliquots of solutions containing
from Beijing solarbio science & technology Co. Ltd. (Beijing, China). various concentrations of the samples (50–350 µM) were mixed in a
Glatathione (GSH), superoxide dismutase (SOD), lactate dehydrogenase tube with 2.5 mL of 0.2 mM phosphate buffer (PBS, pH 6.6) and po-
(LDH) and the protein detection kits were obtained from Nanjing tassium ferricyanide (K3Fe(CN)6, 1%). The mixtures were incubated at
Jiancheng Bioengineering Institute (Nanjing, China). Caspase-3 (human 50 °C for 30 min. After addition of 2.5 mL of 10% trichloroacetic acid,
specific) monoclonal antibody was bought from Proteintech Group Inc. each mixture was centrifuged at 3000 r/min for 20 min. The upper layer
(Wuhan, China); dimethyl sulfoxide (DMSO), minimum essential of solution (2.5 mL) was collected and mixed with 2.5 mL of deionized
medium (MEM), polyvinylidene difluoride (PVDF) membranes, β-actin water containing 0.5 mL of 0.1% ferric chloride. The absorbance was
antibody, HRP goat anti-mouse IgG and 2′,7′-dichlorofluorescein dia- read at 700 nm in a UV-2100 spectrophotometer against a blank
cetate (DCFH-DA) kit were purchased from Boster Biological Tech- sample. Higher absorbance indicated greater reducing power.
nology Co. Ltd. (Wuhan, China). Other chemical reagents were pur-
chased from the Guangzhou chemical reagent factory (Guangzhou,
China). The benchtop system (SW-CJ-2FD) was purchased from Suzhou 2.5. Anti-lipid peroxidation activity
Antai Airtech Co. Ltd. (Suzhou, China). The HF90 CO2 incubator was
purchased from Heal Force Bio-Meditech Co. Ltd. (Shanghai, China). Anti-lipid peroxidation activity was measured as described, with
The micro-plate reader XMARK and ultraviolet and visible spectro- minor modifications (Zheng et al., 2015). 1 mL aliquots of a 10 mg/mL
photometer 2100 were purchased from BIORAD Ltd. (Japan) and lecithin solution, 0.4 mM FeSO4 and sample were mixed and incubated
Shanghai Unico Spectrotech Instruments Co., Ltd. (China), respectively. for 60 min at 37 °C. The mixed liquor was heated for 15 min at 95 °C
after adding 2 mL of TCA-TBA-HCl. The tubes were centrifuged at
2.2. DPPH radical-scavenging activity 3000 r/min for 10 min and the absorbance of the upper layer was read
at 535 nm in a UV-2100 spectrophotometer. Data for each assay was
DPPH%-scavenging capacity was measured according to Zhou et al. recorded in triplicate. VC and BHT were used as positive controls.
(2013) Briefly, different concentrations of samples to be tested (0.1 mL)
were added to 0.39 mL of ethanolic DPPH (60 µM). The reaction mix- Inhibition rate(%) = [(A blank−A sample)/A blank] × 100%
tures were shaken vigorously and left at indoor temperature for 30 min,
avoiding light. Absorbance was determined at 517 nm in a UV-2100 where Ablank was the absorbance of the control reaction (replacing the
spectrophotometer. All determinations were performed in triplicate. sample with distilled water), and Asample was the absorbance of the test
The percentage reduction of DPPH radical was calculated as: compound.

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Z. Liu et al. Food Chemistry 253 (2018) 5–12

2.6. Cell lines and culture percentage of the control.

The human hepatocellular carcinoma (HepG2) cell line was pro- 2.11. Measurement of intracellular ROS
vided by the Type Culture Collection of the Chinese Academy of
Sciences, Shanghai, China. Cells were cultured in MEM with 10% fetal Intracellular ROS were monitored using the DCFH-DA fluorescence
bovine serum, penicillin (100 units/mL), and streptomycin (100 µg/mL) probe assay (Liu et al., 2014). HepG2 cells were seeded on 12-well
at 37 °C in a humidified atmosphere containing 5% CO2. plates at 1 × 105 cells/well and incubated at 37° C in 5% CO2 for 24 h.
The cells were pretreated with 0.5, 1 or 1.5 µM concentrations of iso-
2.7. Determination of cytotoxicity garcinol (final concentration) for 30 min before exposure to 500 µM
H2O2 for a further 3 h. Then DCFH-DA was added to the culture plates
Cytotoxicity was tested, using the tetrazolium salt-based CCK-8 at a final concentration of 10 µM and incubation continued for 30 min.
assay. Stock solutions of isogarcinol and BHT were made in di- The cells were washed three times in serum-free medium, and sus-
methylsulfoxide (DMSO) and diluted in MEM before use. Cells were pended in PBS. Fluorescence intensity was immediately measured with
seeded in 96-well plates at 5 × 104 cells/mL in 100 µL of complete a multi-mode microplate reader at an excitation wavelength of 488 nm
medium and various concentrations of isogarcinol or BHT were added. and emission wavelength of 525 nm.
The final concentrations of isogarcinol were 1, 3, 5, 7, 10, 13, 15 µM.
The highest content of DMSO was 0.2%. After 24 h of incubation, 10 µL 2.12. Determination of LDH release, MDA level, GSH level and SOD activity
of CCK-8 reagent was added to the wells and incubation continued for
1 h. Cytotoxicity was measured as described in the CCK-8 kit. Each HepG2 cells in 6-well plates (3 × 105 cells/well) were cultured and
experiment was repeated five times. treated according to the procedures described above. LDH release, MDA
level, GSH level, SOD activity and protein content were measured,
2.8. SCGE assay using appropriate assay kits (Nanjing Jiancheng Bioengineering
Institute (Nanjing, China).
The single cell gel electrophoresis (SCGE) assay, also referred to as
the comet assay, was performed as described by Zhang, Jia, Hao, Cen, 2.13. Western blotting analysis
and Li (2011) with slight modifications. After exposure to isogarcinol or
BHT, 100 µL of each cell suspension was mixed with 100 µL of 1% low- Western blotting analysis was carried out as described by Liu et al.
melting-point agarose (LMA, prepared in phosphate buffer) placed on (2014) with slight modifications. HepG2 cells were cultured in 6-well
frosted glass microscope slides pre-coated with 100 µL of solidified plates (3 × 105 cells/well) and treated as described above. Afterwards,
normal-melting-point agarose (NMA, 1% in PBS). The slides were the cells were washed twice with PBS (0.1 M, pH 7.4, 4 °C), centrifuged
placed on ice for 5 min and freshly prepared cold lysis solution (10 mM at 2000 rpm for 5 min and lysed by incubation in 20 µL RIPA buffer for
Tris, 0.1 M EDTA, 2.5 M NaCl, 1% Triton X-100, and 10% DMSO, pH 15 min on ice. Lysates were centrifuged at 12,000 rpm for 10 min at
10.0) added and incubated at 4 °C for 1 h. Then the slides were placed in 4 °C, and supernatant protein concentrations were determined with the
freshly prepared electrophoresis buffer (1 mM Na2EDTA and 300 mM BCA assay.
NaOH, pH > 13) for 15 min to allow the DNA to denature and to re- Equal amounts of protein were fractionated by 12% sodium dodecyl
solve alkali-labile sites. They were then electrophoresed at 0.75 V cm−1 sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
(26 V, 300 mA) at 4 °C for 15 min, washed three times in 0.4 M Tris onto polyvinylidene difluoride (PVDF) membranes, which were in-
buffer (pH 7.5) at 4 °C for 10 min each time and dehydrated by im- cubated with 5% skimmed milk in TBST buffer for 2 h at room tem-
mersion in cold absolute ethanol for 10 min. They were then dried at perature in a bio-shaker. Then the membranes were incubated with
room temperature, stained with 70 µL of 20 mg/mL ethidium bromide primary antibody (monoclonal anti-caspase-3 (1:1000) and anti-β-actin
solution for 30 min and analyzed at 400× magnification with a fluor- (1:1000)) overnight at 4 °C. After three washes with TBST buffer, for
escence microscope (BX51-32P01, Olympus, Japan), equipped with an 5 min each, the membranes were incubated with appropriate secondary
excitation filter of BP 546/10 nm and a barrier filter of 590 nm. antibody (1:10,000) for 1 h at room temperature and again washed
three times in TBST buffer. Bands were visualized with an ECL system
2.9. The oxidative stress model of HepG2 cells and band densities were optically scanned with ImageJ software.

Cells in logarithmic growth were transferred to 96-well plates at a 2.14. Statistical analysis
density of 5 × 104 cells/ml in 100 µL and cultured for 24 h. Then, the
medium was replaced with fresh medium containing various final All experiments were performed in triplicate and data were re-
concentrations (200, 300, 400, 500, 600 and 700 µM) of H2O2 for 3 h. corded as means ± standard error (SD). One way ANOVA was used to
10 µL of CCK-8 reagent were added to the wells and they were in- assess differences between the means using SAS 9.1 software. Student’s
cubated for 1 h. Absorption was measured at 450 nm with a micro-plate t-test was used for paired samples, and differences were deemed sig-
reader, as described in the CCK-8 kit. nificant at p < 0.01 (∗∗, ##), and p < 0.05 (∗, #).

2.10. Protective effect of isogarcinol against H2O2-induced oxidative stress 3. Results

The CCK-8 method was used to determine the viability of HepG2 3.1. Antioxidant activity of isogarcinol in vitro
cells, as described above. Cells were seeded in 96-well plates
(5 × 104 cells/mL, 100 µL) and incubated at 37 °C in 5% CO2 for 24 h. The DPPH and ABTS radical-scavenging capacities, ferric reducing
They were pretreated with various concentrations of isogarcinol (final antioxidative potentials and anti-lipid peroxidation activities of iso-
concentrations of 0.5, 1, 1.5 µM) and cultured for 30 min. The control garcinol, VC and BHT were examined in a series of antioxidant ex-
was treated with 0.2% (v/v) DMSO under the same conditions. periments, as described in Materials and Methods. The results are
Thereafter 500 µM H2O2 was added to the mixtures and they were in- shown in Fig. 1 and Table 1. Isogarcinol displayed higher antioxidant
cubated for 3 h. The isogarcinol was not removed after the addition of activity than did VC and BHT in both the DPPH and the ABTS assays
H2O2. Finally, 10 µL of CCK-8 reagent were added to the wells and (Fig. 1A and B). The antioxidant activity is proportional to the con-
incubation continued for 1 h. The cell viability was expressed as a centration of isogarcinol; the IC50 of isogarcinol was 36.3 ± 3.35 µM,

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Z. Liu et al. Food Chemistry 253 (2018) 5–12

Fig. 1. Antioxidant activities of isogarcinol: (A) DPPH radical-scavenging activity; (B) ABTS radical-scavenging capacity; (C) Reducing power; (D) Anti-lipid peroxidation activity. All
values are expressed as means ± SD, n = 3.

Table 1 3.3. Genotoxicity of isogarcinol

DPPH%- and ABTS%+-scavenging activities.
To evaluate the genotoxicity of isogarcinol, we assessed the extent
IC50 (µM)
of damage to HepG2 cells by fluorescence microscopy. BHT was chosen
Iso VC BHT as the negative control. As illustrated in Fig. 3, isogarcinol (Fig. 3: B–D)
had negligible genotoxicity, whereas BHT induced obvious trailing at
**
DPPH (µM) 36.3 ± 3.35 66.0 ± 1.90 116 ± 2.55 100 µM and 500 µM (Fig. 3: F–G). Thus, isogarcinol was less genotoxic
ABTS (µM) 16.6 ± 3.98** 59.4 ± 3.45 183 ± 3.89
than was BHT.
Data are expressed as means ± SD, n = 3; ** p < 0.01 Isogarcinol versus VC and BHT,
IC50: concentration corresponding to 50% inhibition. Iso-isogarcinol, VC-vitamin C, BHT- 3.4. Oxidative stress model of HepG2 cells
butylated hydroxytoluene.

H2O2 induction of oxidative damage to cells has been widely used to

almost 2-fold and 3-fold lower than those of VC (66.0 ± 1.90 µM) and study the ability of many kinds of bioactive material to inhibit oxidative
BHT (116 ± 2.55 µM), respectively, in the DPPH assay (Table 1). In stress (Liu et al., 2014). We used HepG2 cells for such assays. The data
addition, isogarcinol was a more effective scavenger of the ABTS ra- in Fig. 2B show concentration-dependent reduction of HepG2 cell via-
dical, with an IC50 of 16.6 ± 3.98 µM (compared with VC bility by H2O2. We found that 500 µM H2O2 caused cell viability to
59.4 ± 3.45 µM) and BHT (183 ± 3.89 µM) (Table 1). When the decrease by about 50%; hence this concentration was used in sub-
concentration of the treatment ranged from 50 to 350 mM, the optical sequent experiments.
density of the isogarcinol-treated group was 0.2–0.6, which was lower
than VC (0.4–1.2) and VE (0.4–1.5) (Fig. 1C). Fig. 1D shows that the
dose response curves for the anti-lipid peroxidation activities of iso- 3.5. Isogarcinol protects HepG2 cells from H2O2-induced oxidative stress
garcinol and VE (20–260 µM) were equivalent. However, the con-
centrations of isogarcinol and BHT were both lower than that of VC in The results in Fig. 2C show that preincubation with isogarcinol re-
the same anti-lipid peroxidation activity assay (Fig. 1D). These results duced the viability of HepG2 cells exposed to 500 µM H2O2 for 3 h. At
indicated that the anti-lipid peroxidation capacity of isogarcinol was the same concentration of 1.5 µM, isogarcinol promoted higher cell
weaker than that of VC. viability than did BHT against H2O2-induced oxidative stress. These
data demonstrate that isogarcinol possesses remarkable HepG2 pro-
tective capacities.
3.2. Cytotoxicity of isogarcinol
3.6. Isogarcinol prevents H2O2-induced ROS generation
The cytotoxic effect of isogarcinol on HepG2 cells is shown in
Fig. 2A. After 24 h treatment with isogarcinol at concentrations be- H2O2 treatment increased intracellular ROS approximately three-
tween 1 and 10 µM, there was no significant difference in cell viability fold compared with the control group. However, isogarcinol pretreat-
compared to the control group. However, cell viability dropped slightly ment (0.5–1.5 µM) significantly reduced the fluorescence density
at 13 and 15 µM isogarcinol. compared with the H2O2-treated group in HepG2 cells (Fig. 2D). The
reduced level of ROS was proportional to the concentration of iso-
garcinol. The accumulation of ROS in the isogarcinol-treated group was

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Z. Liu et al. Food Chemistry 253 (2018) 5–12

Fig. 2. Effect of isogarcinol on HepG2 cells. (A) Cytotoxicity of isogarcinol on HepG2 cells. Cells were treated with the indicated concentrations of isogarcinol (1–15 µM), and the control
cells received 0.2% DMSO (v/v). Cell viability was determined by the CCK-8 assay after 24 h, Values are means ± SD. (B) Effect of H2O2 on HepG2 cell viability. Cells were exposed to
H2O2 at concentrations from 200 to 700 µM for 3 h, and viability was measured by the CCK-8 assay. (C) The protective effect of isogarcinol on H2O2-induced oxidative damage. (D) Effect
of isogarcinol on HepG2 intracellular ROS. Cells were pretreated with 0.5–1.5 µM isogarcinol or 1.5 µM VE for 30 min and then exposed to 500 µM H2O2 for 3 h. The control group was
incubated with 0.2% DMSO (v/v). Data represent means ± SD of three independent experiments (n = 3). **p < 0.01 versus H2O2-treated cells, ##p < 0.01 versus the control group.

3.7. Isogarcinol reduces H2O2-induced release of LDH, increased level of

MDA and the loss of GSH and SOD activities

Exposure of HepG2 cells to 500 µM H2O2 dramatically increased the

release of LDH and levels of MDA, and pre-treatment with 0.5–1.5 µM
isogarcinol or 1.5 µM VE for 30 min markedly attenuated these effects
(Fig. 4A and B). LDH release and MDA level were significantly reduced
in cells pretreated with 1.0, 1.5 µM isogarcinol (p < 0.05 or 0.01). And
LDH release was more decreased by isogarcinol than VE at 1.5 µM. To
further determine the likely transcriptional mechanism underlying the
protective effects of isogarcinol, we measured GSH levels and SOD ac-
tivity. As shown in Fig. 4C and D pre-incubation of cells with
0.5–1.5 µM progressively reduced GSH levels and SOD activity.

3.8. Isogarcinol inhibits H2O2-induced HepG2 cell apoptosis

Fig. 3. DNA damage induced by isogarcinol in HepG2 cells. The cells were treated with Treatment with H2O2 (500 µM) significantly reduced the level of
different concentrations of isogarcinol and BHT for 3 h. (A) Control; (B-D) isogarcinol: pro-caspase-3 and increased that of caspase-3, and pretreatment with
50 µM, 100 µM, 500 µM; (E-G) BHT: 50 µM; 100 µM; 500 µM. (B-G) shows typical comet 0.5, 1.0 and 1.5 µM isogarcinol dose-dependently opposed these effects
migration induced by BHT (magnification 400×).
(Fig. 5A and B).

similar to that in the VE-treated group when the concentration was

4. Discussion
15 µM. The results showed that isogarcinol dose-dependently reduced
the overproduction of ROS.
It is widely believed that free radicals are responsible, at least in
part, for many degenerative diseases such as brain dysfunction, cancer
and heart diseases (Kozics et al., 2013). Antioxidants can prevent oxi-
dative-related disorders and in some cases help in their treatment
(Pisoschi & Pop, 2015). Nowadays, phytochemical or antioxidant

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Z. Liu et al. Food Chemistry 253 (2018) 5–12

Fig. 4. Effects of isogarcinol on LDH release (A), MDA level (B), GSH levels (C) and SOD activity (D) in HepG2 cells. Cells were pretreated with 0.5–1.5 µM isogarcinol or 1.5 µM VE for
30 min and then exposed to 500 µM H2O2 for 3 h. The control group was incubated with 0.2% DMSO (v/v). Data represent means ± SD of three independent experiments (n = 3).
*
P < 0.05 and **p < 0.01 versus H2O2-treated cells, #p < 0.05 and ##p < 0.01 versus control group.

therapy is regarded as a promising strategy to protect cells from oxi- activity (Fig. 1), which furthermore confirmed the results of Stark et al.
dative damage and natural products of plant origin are also important (2015). In particular, the IC50 of isogarcinol in the DPPH assay was
sources of antioxidants (Miremadi, Ayyash, Sherkat, & Stojanovska, almost 2-fold and 3-fold lower than those of VC and BHT, respectively
2014). Isogarcinol is a benzophenone with a variety of biological (Table 1). Ito et al. (2003) also found that the DPPH radical-scavenging
properties, such as antioxidant, antimicrobial, antifungal, anti-in- activity of isogarcinol (IC50 = 13.3 ± 1.3 µM) was about 2-fold lower
flammatory, and anti-HIV activities (Baggett, Mazzola, & Kennelly, than that of VE (IC50 = 22.8 ± 0.2 µM). The reducing power and anti-
2005). It also has antiplasmodial activity against P. Falciparum (Marti lipid peroxidation assays revealed that isogarcinol had antioxidant ac-
et al., 2010), acetylcholinesterase and butyrylcholinesterase inhibitory tivity, but the results of both assays were lower than those obtained for
activities, antiprotozoal activity against Leishmania donovani (Lenta VC. A different chemical reaction mechanism, leading to a different
et al., 2007) and antibacterial activities (Deachathai, Mahabusarakam, ranking order of antioxidant activity for isogarcinol, BHT and VC, in
Phongpaichit, & Taylor, 2005). In the present work, we confirmed the this context, could provide an explanation for the obtained results. In
radical-scavenging activity, reducing power and anti-lipid peroxidation addition, we observed, in FRAP experiments, that isogarcinol possesses
capacity of isogarcinol, and showed that it has significant antioxidant some reducing power (data not shown). Hence, our results confirmed

Fig. 5. Effect of isogarcinol on the expression of caspase-3 levels in HepG2 cells. Cells were pretreated with 0.5–1.5 µM isogarcinol or 1.5 µM VE for 30 min and then exposed to 500 µM
H2O2 for 3 h. The control group was incubated with 0.2% DMSO (v/v). (A) The expressions of pro-caspase-3, cleaved caspase-3 and β-actin were used for normalization and verification of
protein loading; (B) Quantitative pro-caspase-3 and cleaved caspase-3 expression after normalization to β-actin. Data represent means ± SD of three independent experiments (n = 3).
**
p < 0.01 versus H2O2-treated cells, ##p < 0.01 versus control group.

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that isogarcinol has strong antioxidant activity in vitro, so we explored 5. Conclusion

its biological activity further.
High levels of dietary compounds can be toxic and mutagenic in cell Isogarcinol has strong antioxidant activity and reduces oxidative
culture systems (Alía et al., 2006). Cytotoxic effects of isogarcinol have damage induced by H2O2 in HepG2 cells. It is not cytotoxic and geno-
been reported against human diploid embryonic lung MRC-5 cells (IC50 toxic to HepG2 cells in the range of concentrations tested. It alleviates
value of 3.5 µM) (Marti et al., 2010) and human leukemia H-L60 cells oxidative stress by reducing ROS generation and blocking the ROS-in-
(Matsumoto et al., 2003). However, isogarcinol showed toxicity at more duced mitochondrial apoptosis pathway. At the same time, isogarcinol
than 10 µM and did not have any cytotoxic or promotional effects on decreases LDH release, MDA level and increases GSH level and SOD
HepG2 cells at 1–10 µM in our experiments (Fig. 2A). We also assessed activity in a concentration-dependent manner. It may be of use for
the genotoxic activity of isogarcinol, using the SCGE assay, which is a treating a variety of diseases associated with free radical and oxidative
trustworthy and rapid method for the quantification of DNA damage. damage.
The images clearly indicated that BHT was genotoxic at 100 and
500 µM in HepG2 cells (Fig. 3: E–G); in contrast isogarcinol had neg- Acknowledgements
ligible genotoxic effects at 50, 100 and 500 µM (Fig. 3: B–D).
HepG2 cells are a well-established model for studying the anti- Thanks for Youth Foundation Project of National Natural Science
oxidant effects of dietary compounds (Wen et al., 2015; Kong et al., Foundation of Hainan province, China and the National Natural Science
2016). They retain many of the functions of normal liver cells (Knowles, Foundation of China (31460120, 31760170, 81660584).Notes
Howe, & Aden, 1980) and their morphological features and cell shapes
are also similar to those of liver parenchymal cells (Zhou, Jiang, Geng, All authors declare no conflicts of interest.
Cao, & Zhong, 2009). In the present study, treatment of HepG2 cells
with 500 µM H2O2 for 3 h resulted in about 50% death (Fig. 2B) and Appendix A. Supplementary data
preincubation with isogarcinol attenuated this death. These results
imply that isogarcinol protected the HepG2 cells from H2O2-induced Supplementary data associated with this article can be found, in the
damage. Accumulating evidence has revealed that H2O2 triggers oxi- online version, at http://dx.doi.org/10.1016/j.foodchem.2018.01.074.
dative damage by elevating intracellular ROS (Parthasarathi et al.,
2015). In this study, we found that H2O2 (500 µM) elevated in- References
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