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The antioxidant activity of curcumin extract against HepG2 ... · The aim of the present study was...
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The antioxidant activity of curcumin extract against HepG2 cells
Abeer A Khamis (1), Amira H Sharshar* (1), Amal H Mahmoud (2), Tarek M Mohamed (1)
(1) Biochemistry Division, Chemistry Department, Faculty of Science, Tanta University
(2) Head Researcher of Special Food and Nutrition Department, Food Technology Research
Institute, Agricultural Research Center, Giza
Correspondence:
Amira H Sharshar
Researcher Assistant at Food and Nutrition Department
Food Technology Research Institute
Agricultural Research Center
Tel: +201006135205
Abstract
Oxidative stress plays a major role in the pathogenesis of various diseases including
neurodegenerative diseases, myocardial ischemia-reperfusion injury and cancer. The
aim of this work was to extract curcumin from curcuma longa then evaluated its
antioxidant activity against HepG2 cells. FTIR was used to confirme the structure of
the extracted curcumin. Antioxidant activity was assessed by DPPH method. Results
showed that curcumin had antioxidant activity with Ic50 value of 3.7±0.20 μg/ml.
Moreover treatment of HepG 2 cells with curcumin resulted in decreasing the
concentration of L-malondialdehyde (L-MDA) and increasing the activity of the
antioxidant enzymes superoxide ismutase (SOD) and catalase. In conclusion, the
present study showed the effect of curcumin as a natural antioxidant compound that
enhanced the antioxidant defense system in HepG2 cells by increasing the activity
levels of antioxidant enzymes SOD and catalase and decreasing the concentration of
L-MDA.
Key words: Antioxidant activity, curcumin, catalase, superoxide dismutase
1. Introduction
Oxidative stress occurs in cells and tissues of organisms wherever oxidation
and reduction processes abound. This has been attributed to production of reactive
oxygen species (ROS) beyond the ability of the system to neutralize them (Halliwell,
1996). Oxidative stress has been incriminated in the pathogenesis of several disease
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conditions such as cancer, cardiovascular, liver injury, myocardial infarction,
Alzheimer’s disease, Parkinsonism as well as in the ageing process (Grzegorczyk et
al., 2007).
Continuous exposure to synthetic compounds, which is characteristic of
orthodox medicine, could lead to increase in the amount of free radicals in the body
and hence cause different tissue and/or biochemical lesions. A lot of scientific
researches has focused on screening plants and herbs for their potential antioxidant
properties because active principles from these plants are thought to play important
roles in preventing diseases caused by oxidative stress (Bulus et al., 2017).
Curcumin is extracted from Curcuma longa (turmeric) rhizomes is the main
ingredient in turmeric and curry (Tayyem et al., 2006). Turmeric was used in China
and India many years ago as a traditional medicine (Teiten et al., 2010). Several
studies have shown that curcumin exerts antioxidant, anti-inflammatory, anti-
carcinogenic and chemopreventive activities on many tumor cells.
The essential structure of curcumin is the feruloylmethane skeleton. . It
consists of two benzene rings, one having a phenol hydroxy and the other having a
phenol methoxy showing the ability of curcumin to act as a hydrogen donor
eliminating oxygen free radicals (Lv et al., 2016). Curcumin influences cell death by
multiple, interdependent processes. It exerts its anticancer and antioxidant activity by
modulating the expression level of several target genes. (Chiu and Su, 2009;
Choudhuri et al., 2002; Perrone et al., 2015).
2. Materials and methods
2.1. Extraction of curcumin from Curcuma Longa
Extraction of curcumin was carried out according to the method explained by
(Kulkarni et al., 2012) using soxhlet extraction followed by column chromatography.
2.2. Fourier transforms infrared spectroscopy
A JNS-CO Spectrum System 4100 LE FTIR spectrometer (Japan) was used for the
analysis of purified extract of Curcuma Longa. The spectral FTIR are measured at
mid infrared region corresponding to wavenumbers of 4000-650 cm-1 with resolution
of 4 cm-1. (Lestari et al., 2017)
2.3. Determination of antioxidant activities of curcumin
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Total antioxidant activity was estimated according to the free radical
diphenylpicrylhydrazyl (DPPH) method was described by (Molyneux, 2004).
2.4. Cell line and cell culture
HepG2 cells (ATCC, Manassas, Virginia, USA; Cat.no. HB 8065) were cultured in
DMEM medium (GIBCO, New York, USA; Cat.no.11995073) supplemented with
10% heat-inactivated fetal bovine serum (GIBCO, Grand Island, New York, USA;
Cat.no.10099133), 1% penicillin/streptomycin (Thermo Fisher Scientific; Waltham,
MA, USA; Cat.no. SV30082) and 2% L-glutamine (Invitrogen, Grand Island, New
York, USA; Cat.no. 25030024) in a humidified atmosphere of 95% air with 5% CO2
at 37˚C (Jiang et al., 2013a).
2.5. Determination of antioxidant activity of curcumin in HepG-2 cells:
L-Malondialdhyde (L-MDA) was determined according to the method of (Macotpet
et al., 2013). The activity of superoxide dismutase (SOD) was determined according
to the method of (Weydert and Cullen, 2010). One unit of enzyme activity is defined
as the amount of enzyme that gave 50% inhibition of NBT reduction in one minute.
SOD Activity: IU/ml = % inhibition x 3.75. Catalase activity was determined
according to the method described by (Aebi, 1984).
3. Results
3.1. Determination of curcumin in Curcuma Longa
Curcumin was extracted from Curcuma Longa as described previously. The identity
of isolated curcumin was further confirmed by FTIR (Fig.1).
FTIR spectrum of curcumin showed a characteristic stretching band of O-H at 3512
cm–1
. The peak at 3014 cm–1
represents C-H Streching and 1602 cm–1
peak was
assigned to C=C symetric aromatic ring streching. The peak at 1506 cm–1
represents
C=O, while enol C-O peak was obtained at 1280 cm–1
and benzoate trans-C-H
vibration was at 962 cm–1
.
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Fig. (1): (a) FTIR spectrum of extracted curcumin from Curcuma Longa, (b) FTIR
spectrum of standard curcumin.
3.2. Antioxidant activity of curcumin
By using DPPH free radical scavenging activity the antioxidant activity of the
curcumin was evaluated by comparing the IC50 of the ascorbic acid. The IC50 of the
curcumin was found to be 3.7±0.20 μg/ml which is closer to the ascorbic acid
2.2±0.23 μg/ml (Fig. 2).
a
b
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Fig. (2): Percentage of radical scavenging activity of curcumin
3.3. Assessment of antioxidant effect of curcumin
Our results revealed a significant (P≤0.05) decrease in the level of L-MDA in
HepG2 cells of control groups (G1) (Fig. 3). This reduced level was significantly
(P≤0.05) elevated after administration of standard curcumin at a dose of 10 ug/ml
when applied for 24h (G2). Similarly, the extracted curcumin (at dose of 10 and 20
ug/ml) resulted in a significant increase in L-MDA concentration when applied for
24h (G3 and G4) as compared to control group. In general, the standard curcumin
showed a significant higher L-MDA concentration than that of the extracted curcumin
with no significant difference in the L-MDA concentration was shown between
treatment by low dose (10 ug/ml) and high dose (20 ug/ml).
In case of detecting SOD activity, our results revealed a significant (P≤0.05)
decrease in the level of SOD in HepG2 cells of control groups (G1) (Fig. 4). This
reduced level was significantly (P≤0.05) elevated after administration of standard
curcumin at a dose of 10 ug/ml when applied for 24h (G2). Similarly, the extracted
curcumin (at dose of 10 and 20 ug/ml) resulted in a significant increase in SOD level
when applied for 24h (G3 and G4) as compared to control group. In general, the
standard curcumin showed a significant higher levels of SOD than that of the
extracted curcumin with no significant difference in the SOD concentration was
shown between treatment by low dose (10 ug/ml) and high dose (20 ug/ml).
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Also our results revealed a significant (P≤0.05) decrease in the level of CAT
in HepG2 cells of control groups (G1) (Fig. 5). This reduced level was significantly
(P≤0.05) elevated after administration of standard curcumin at a dose of 10 ug/ml
when applied for 24h (G2). Similarly, the extracted curcumin (at dose of 10 and 20
ug/ml) resulted in a significant increase in CAT level when applied for 24h (G3 and
G4) as compared to control group. In general, the standard curcumin showed a
significant higher CAT level than that of the extracted curcumin with no significant
difference was shown between treatment by low dose (10 ug/ml) and high dose (20
ug/ml).
Fig. (3): Effect of curcumin on L-MDA concentration in HepG2 cells, Means within
the same column carrying different superscript letters are significantly different (P≤
0.05).
b
0
10
20
30
40
50
60
70
80
G1 G2 G3 G4
MD
A (
nM
/ml)
b
c
a
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Fig. (4): Effect of curcumin on SOD activity in HepG2 cells, Means within the same
column carrying different superscript letters are significantly different (P≤ 0.05).
Fig. (5): Effect of curcumin on catalase activity in HepG2 cells, Means within the
same column carrying different superscript letters are significantly different (P≤ 0.05).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
G1 G2 G3 G4
SO
D (
IU/m
l)
c
a
b b
0
1
2
3
4
5
6
7
8
G1 G2 G3 G4
CA
T (
IU/
ml)
c
a
b b
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4. Discussion
In recent few years, several natural products extracted from medicinal plants
have been studied for their antioxidant and anti-cancer effects by inhibiting the
proliferation, invasion, and metastasis of cancer cells. However, several types of
research have revealed the mechanisms of their action, much research is still needed.
(Chang et al., 2017) Curcumin which is extracted from the rhizomes of Curcuma
longa sp. has received attention as a potential agent in cancer therapy. (Shariati et al.,
2017) Accumulating evidence shows that curcumin has a diverse range of molecular
targets, and therefore acts upon numerous molecular and biochemical cascades.
(Gallardo and Calaf, 2016)
The aim of the present study was to extract curcumin from curcuma longa and
evaluate the antioxidant activity of curcumin against human cancer in vitro.
In the present study curcumin was extracted from curcuma longa and the structure of
the extracted curcumin was confirmed by using FTIR
The FTIR spectrum revealed the presence of stretching band of O-H at 3512
cm–1
, C-H Stretching band at 3014 cm–1
, a peak at 1602 cm–1
peak which was
assigned to C=C symmetric aromatic ring stretching, a peak at 1506 cm–1
represents
C=O, while enol C-O peak was obtained at 1280 cm–1
and benzoate trans-C-H
vibration was at 962 cm–1
.
This FTIR spectrum of the extracted curcumin was matching with the FTIR spectrum
of the standard curcumin. Also some studies use FTIR spectroscopy for identification
of curcumin isolated from different curcuma species for instance, (Lestari et al.,
2017) performed FTIR spectroscopy to determine the curcuminoid content isolated
from curcuma xanthorrhiza.
In this study, the antioxidant activity of curcumin was evaluated using DPPH
scavenging assay. 50% of inhibitory concentration (IC 50) value was 3.7±0.20 μg/ml.
The curcumin structure contains a variety of functional groups including the β-diketo
group, carbon–carbon double bonds and phenyl rings containing varying amounts of
hydroxyl and methoxy substituents which may be responsible for its antioxidant
activity against DPPH (Wright, 2002).
Oxidative stress is defined as an excess of reactive oxygen species (ROS) due
to imbalance between the rate of ROS production and ROS removal. Overproduction
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of free radicals and reactive oxygen species (ROS) interact with important biological
molecules such as DNA, lipid or protein leading to many degenerative diseases, such
as cancer. The antioxidant activity of curcumin makes it able to eliminate oxygen free
radicals serving as a hydrogen donor or adjust other mediators involved in the
oxidation reaction against antioxidant and anti-lipid peroxidation (Lv et al., 2016).
Since oxidative stress triggers various antioxidant mechanisms in the body biomarkers
such as lipid peroxidation products and endogenous enzymes with antioxidant
properties have been used to assess oxidative stress (Trujillo et al., 2013).
Malondialdehyde (MDA) one of the end products of lipid peroxidation has
been widely used as a biomarker of oxidative stress (Macotpet et al., 2013). In this
work L-MDA was used to monitor the oxidative stress status in curcumin treated
HepG 2 cells. The results obtained in this work demonstrated that curcumin decreased
the concentration of L-MDA in HepG 2 cells. The result of (Yeni et al., 2017) also
showed that curcumin decreased MDA level in preeclampsia-induced cell.
Superoxide dismutases (SODs) are essential enzymes that catalyze the
conversion of superoxide into oxygen and hydrogen peroxide. Through their activity,
SOD enzymes control the levels of a variety of reactive oxygen species (ROS) and
reactive nitrogen species, thus both limiting the potential toxicity of these molecules
and controlling broad aspects of cellular life that are regulated by their signaling
functions (Wang et al., 2018). This study showed that curcumin treated cells showed
increased activity of SOD than untreated cells. This result is similar to the results of
(Aziza et al., 2014) that showed that curcumin treatment enhanced the activity of
SOD in colon cancer. Also the results of (Jagetia and Rajanikant, 2015) indicated
that curcumin stimulated the antioxidant mechanisms in mouse skin exposed to
fractionated γ-irradiation resulting in a significant rise in the activity superoxide
dismutase enzymes in mouse skin.
Catalase is an enzyme present in most of the aerobic cells, it protects them
from oxidative stress by catalyzing the rapid decomposition of hydrogen peroxide
(H2O2) in two types of reactions depending on its peroxidatic and catalatic activities
(Al-Abrash et al., 2000). Our results indicated that curcumin increase catalase levels
in treated cells than untreated cells this (El‐ Bahr, 2015) result is in accordance with
the results of that showed that the levels of antioxidant enzymes SOD and catalase
were significantly increased in AFB1-intoxicated rats treated with curcumin
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compared to untreated AFB1- intoxicated. Also the results of (Desai et al., 2011)
showed significant increase in activities of SOD, catalase and GPx in presence of
curcumin on studying the antioxidant effects of curcumin on rat peripheral blood
lymphocytes (RPL) in culture. These results showed that curcumin possesses
antioxidant which may be explained by the presence of two benzene rings, with one
ring having a phenol hydroxy and the other ring having a phenol methoxy.
In conclusion, the present study showed the effect of curcumin as a natural
antioxidant compound that enhanced the antioxidant defense system in HepG2 cells
by increasing the activity levels of antioxidant enzymes SOD and catalase and
decreasing the concentration of L-MDA suggesting that curcumin may be developed
as anti-cancer drug in treatment of human hepatocellular carcinoma.
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