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Food Science and Human Wellness 6 (2017) 60–69

Potential antioxidant and cytoprotective effects of essential oil extracted

from Cymbopogon citratus on OxLDL and H2O2 LDL induced Human
Peripheral Blood Mononuclear Cells (PBMC)
S. Jamuna a , Sakeena Sadullah M.S. a , R. Ashokkumar a , Gokul Shanmuganathan b ,
Senguttuvan Sivan Mozhi b , Niranjali Devaraj S. a,∗
a Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India
b Department of Biotechnology, Prist University, Puducherry, India

Received 16 July 2016; received in revised form 12 January 2017; accepted 6 February 2017
Available online 24 February 2017

Abstract
Cymbopogon citratus (lemon grass) is commonly used in traditional folk medicine. The essential oil extracted from C. citratus has been reported
as a potential anti-oxidant and anti-inflammatory agent. This study has been designed to explore the protective effect of C. citratus (lemon grass)
against modified LDL (OxLDL and H2 O2 LDL) induced cytotoxicity in Peripheral Blood Mononuclear Cells (PBMC). The essential oil extracted
from C. citratus (EOC) was subjected to FT-IR spectroscopic analysis for the identification of functional groups. In vitro antioxidant assays were
carried out to assess the electron donating capability of EOC as compared with a known standard L-ascorbic acid. The cytoprotective effects of
EOC were determined in PBMC induced with modified LDL. Spectra obtained from FT-IR analysis showed the presence of functional groups
in EOC such as H-bonded, O H stretching, N H stretching, aldehyde C H stretching, aldehyde/ketone C O stretching, C C-stretching,
CH3 bending, C H in plane bending. EOC has greater antioxidant property when compared with the standard L-ascorbic acid. EOC at all
test concentrations demonstrated free radical scavenging activity and cytoprotective effect when challenged against modified LDL in PBMC. The
above results show EOC as a promising antioxidant and cytoprotective agent.
© 2017 Beijing Academy of Food Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Cymbopogon citratus; Antioxidants; Cytoprotective; PBMC; Modified LDL

1. Introduction lized as antimicrobial [1], free radical scavenging, antioxidant

Cymbopogon citratus (lemon grass), a native herb from India,
is also cultivated in other tropical and subtropical countries. It
is consumed as an aromatic drink and used in traditional cui-
sine for its lemon flavour, and is also employed in popular
medicine. Infusion or decoctions of dry leaves have been uti-
∗ Corresponding author.
E-mail address: niranjali@yahoo.com (N.D. S.).
Peer review under responsibility of Beijing Academy of Food Sciences.
[2,3], anti-inflammatory [4], antihepatotoxic [5] and antihyper-
tensive agents. In many countries, it is used to treat fever as a
relaxant and sleeping aid. Studies on C. citratus leaf extract have
demonstrated its anti-inflammatory, hypotensive, hypocholes-
terolemic [6], vasorelaxing and diuretic effects. Its efficiency
against oxidative damage and cancer chemopreventive proper-
ties have also been reported by Sharma et al. [7] and Ramar
Thangam et al. [8].
The phytochemical compounds identified in C. citratus are
mainly terpenes, alcohols, ketones, aldehyde and esters. Some of
the reported phytoconstituents are essential oils that contain Cit-
ral ␣, Citral ␤, Nerol, Geraniol, Citronellal, Terpinolene, Geranyl
acetate, Myrecene and Terpinol Methyl heptenone [9,10,11].

http://dx.doi.org/10.1016/j.fshw.2017.02.001
2213-4530/© 2017 Beijing Academy of Food Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
 J. S. et al. / Food Science and Human Wellness 6 (2017) 60–69
Atherosclerosis is defined as the loss of elasticity of the arte-
rial wall due to accumulation of LDL beneath the endothelial
cells [12]. This leads to migration of smooth muscle cells by sup-
pressing NO levels. The current research scenario focuses on the
restraint in the modification of LDL and subsequent formation
of foam cells which impaired lipase activity and accumulation
of ROS [13].
In the early phase, the native LDL coming from the liver
gets accumulated in the sub-endothelial space of the artery;
where they may be minimally oxidized by the enzymes phos-
pholipases, sphingomyelinase and 12/15-lipoxygenase to give
rise to products that are atherogenic Levitan et al. [42]. Mono-
cytes generally flow in the blood stream and when there is
an inflammatory response in the artery walls they get differ-
entiated into macrophages. The accumulation of macrophages,
chemokines, cytokines and metalloproteinases and continuous
presence of modified lipoproteins result in the formation of com-
plicated atherosclerotic lesions. These lesions are characterized
by migration of smooth muscle cells from the middle layer of
artery into the sub endothelial layer [14,15].
As suggested by Khalid Rahman [16], the oxidation of LDL
begins once the endogenous lipophilic anti-oxidants such as
␣-tocopherol, ␣-carotene and ubiquinol levels get completely
exhausted. Therefore, additional sources of natural antioxidants
would be required. Thus, we derived the main objectives of this
study as: (1) extraction of essential oil from C. citratus (2) FT-IR
analysis to find the presence of functional groups in the essential
oil (3) in vitro anti-oxidant assays and (4) cytoprotective effect
of essential oil from C. citratus in PBMC induced with modified
LDL (OxLDL and H2 O2 LDL).
2. Materials and methods
2.1. Essential oil extraction from C. citratus (EOC)
Essential oil of C. citratus (EOC) was prepared by the
reported method of Uzama [17]. Briefly, shade dried leaves were
powdered and extracted using 70% (v/v) ethanol for 48 h at room
temperature. After vacuum filtration, the extract was evaporated
under vacuum to yield an oily residue. The residue was resus-
pended and dissolved in sterile water. The aliquots were stored
at −20 ◦ C and used for further study.
2.2. Fourier transform infrared (FT-IR) spectrometry
FT-IR spectroscopic analysis of EOC was done using Thermo
scientific Nicolet IS-5, spectral range of 400–4000 cm−1 . EOC
samples were prepared by making a pellet in potassium bromide
(KBr). Four scans were performed at a resolution of 4.00 cm−1
at a scan speed of 0.20 cm/s. Spectral data obtained were plotted
on a graph of transmittance (%) versus wave number/cm [18].
2.3. In vitro antioxidant assays
2.3.1. DPPH assay
The antioxidant capacity of the EOC was estimated as
reported by Sahoo et al. [43] and compared with L-ascorbic
61
acid (positive control) using the stable DPPH reagent. Different
concentrations of the extract (25–100 ␮g/mL) were dissolved
and made upto 1 mL with methanol. 1 mL of DPPH reagent was
added. 1 mL of methanol was then added and the tubes were
incubated for 10 min in dark. Standard concentrations of ascor-
bic acid were also treated in the same way. The absorbances
were measured at 517 nm in Shimadzu UV–vis spectrophotome-
ter. The free radical scavenging activities of the extracts were
calculated as a percentage of radical reduction. Radical scav-
enging activity % = [1 − (absorbance of control/absorbance of
sample)] × 100.
2.3.2. Total antioxidant assay
The total antioxidant activity of the EOC was evaluated by
the phosphomolybdenum assay as described previously [19].
Briefly, 0.3 mL of EOC ranging between 25–100 ␮g/mL was
added to 3 mL reagent containing 0.6 mol/L H2 SO4 , 28 mmol/L
of sodium phosphate, 4 mM of ammonium molybdate and incu-
bated at 95 ◦ C for 90 min. The absorbance was measured at
695 nm using a Shimadzu UV–vis spectrophotometer.
2.3.3. Nitric oxide (NO) scavenging activity
Nitric oxide (NO) scavenging activity of the extracts was
determined using Griess reaction [20]. Sodium nitroprusside
(5 mM) in phosphate-buffered saline was mixed with different
concentrations of EOC and incubated at 25 ◦ C for 150 min. The
samples from the above were reacted with Greiss reagent (1%
sulphanilamide, 2% H3 PO4 and 1% N-(1-naphthyl) ethylene-
diamine dihydrochloride). The absorbance of the chromophore
formed during the diazotization of nitrite with sulphanilamide
and subsequent coupling with napthylethylenediamine was read
at 546 nm and referred to the absorbance of standard solution of
Sodium nitrite treated the same way with Griess reagent.
2.3.4. Superoxide anion scavenging assay
The superoxide scavenging activity of the extract was deter-
mined by light induced superoxide generation with riboflavin
and subsequent reduction of nitro blue tetrazolium. Different
concentrations of EOC ranging from 25 to 100 ␮g/mL were
added to the reaction mixture containing 3 mg of NaCN in
0.1 mol EDTA, 0.12 mmol/L riboflavin and 0.6 mol phosphate
buffer (pH 7.8) in a final volume of 3 mL. The tubes containing
the reaction mixture were continuously illuminated with incan-
descent lamp for 15 min. The optical density was read at 530 nm
before and after illumination [21].
2.3.5. Reducing power assay
EOC at different concentrations was mixed with phosphate
buffer (0.2 mol, pH 6.6) and 30 mM of K3 FE (CN)6 . The mixture
was incubated at 50 ◦ C for 20 min. To this, 10% TCA was added
to stop the reaction and centrifuged at 3000 rpm for 10 min. The
upper layer was mixed with equal volume of distilled water and
0.1% FeCl3 and the absorbance was measured at 700 nm [22].
2.3.6. Hydrogen peroxide scavenging assay
According to Ruch et al. [23], a solution of hydrogen peroxide
(40 mM) was prepared in phosphate buffer (pH 7.4). EOC at
62 J. S. et al. / Food Science and Human Wellness 6 (2017) 60–69
different concentrations (25–100 ␮g/mL) was added to 0.6 mL
of H2 O2 solution. The total volume was made up to 3 mL with
phosphate buffer. The absorbance of the reaction mixture was
recorded at 230 nm against the blank solution which contained
phosphate buffer without H2 O2 .
2.3.7. Anti-hemolytic activity
Human whole blood was washed three times in 9 volumes of
sterile, 0.9% saline solution. Different concentrations of EOC
and oxidative stress reagent (H2 O2 ) were added to the iso-
lated erythrocytes. 100 ␮L of 5% erythrocytes were diluted in
PBS and 50 ␮L of EOC containing different concentrations
(25–100 ␮g/mL PBS, pH 7.4) were added. The reaction mix-
ture was diluted with 3 mL of PBS and centrifuged at 2000 × g
for 10 min. The absorbance from the resultant supernatant was
read at 540 nm [24].
2.4. Isolation of LDL + VLDL from human plasma
When 2 g of sucrose was added to 2 mL of plasma, the volume
increased to 3.2 mL.
To this solution, 32 ␮L of 5% heparin and 160 ␮L of 2 mol/L
MgCl2 were added. LDL + VLDL precipitated immediately.
After 15 min at room temperature, the mixture was centrifuged
in stoppered tubes at 6000 × g for 30 min. The high density
of the sucrose solution causes the precipitated lipoproteins to
float to the top and form a pellicle on the surface of the clear
supernatant. The supernatant was removed. The tubes were cen-
trifuged for 1 min to sediment the pellicle and the, precipitate was
dissolved in 40 ␮L of 5% NaCl by incubation for 30 min at 37 ◦ C.
Lipoprotein was precipitated again by adding 2 mL Tris–HCL
buffer and 50 ␮L of 2 mol MgCl2 to remove contaminating
serum proteins. The precipitate sedimented upon centrifugation
(10 min, 6000 × g) and was dissolved in 40 ␮L of 5% NaCl.
The washed precipitate was then dissolved in 10% sodium cit-
rate and dialysed against Tris–HCl–NaCl buffer for 24 h at 4 ◦ C.
After 24 h, the dialysate was centrifuged in cold condition with
heparin–barium salt. The precipitate was again dialyzed against
Tris–HCl–NaCl buffer to remove the barium ions. The resultant
clear yellow solution of lipoprotein was LDL + VLDL [25]. The
obtained lipoproteins were used for further study.
2.4.1. Preparation of oxidized LDL
Copper sulphate and H2 O2 were used as catalysts for oxida-
tion based on the method of Witting et al. [26] and Lopes-Virella
et al. [27]. Briefly, 5.0 mL of CuSO4 and 30 mmol/L of H2 O2
were added to 1 mL of LDL + VLDL (200 mg/mL) followed by
incubation at 37 ◦ C for 24 h and 3 h in dark. At the end of the
incubation period, the oxidation of sample was stopped by the
addition of 1 mL of 10% TCA.
2.5. Cytotoxicity assay
2.5.1. Isolation of Peripheral Blood Mononuclear Cell
(PBMC)
The ficoll density gradient separation of whole blood is the
most commonly used procedure for separation of mononuclear
cells [28]. Briefly, 0.5 mL of heparin was added to 5 mL of blood
in a falcon tube and mixed. To 2.5 mL of ficoll, 1 mL of PBS was
added in a sterile falcon tube. 2.5 mL of blood was layered onto
the ficoll slowly and centrifuged at 1700 rpm for 20 min. The
buffy coat was isolated, completely washed with PBS and cen-
trifuged. Cell viability test was assessed by trypan blue method
[29]. The cell count was done by counting of cells in haematocy-
tometer. The isolated monocytes were maintained as suspension
culture under 5% CO2 incubator at 37 ◦ C, for further study.
2.5.2. MTT assay
Cells were seeded in a 96 wells plate, at 1 × 106 cells/well and
incubated overnight at 37 ◦ C and 5% CO2 . Complete medium
was then replaced with 100 ␮L incomplete medium containing
different concentrations of the drug dissolved in sterile water
and incubated for 24 and 48 h, respectively. After incubation,
the medium was discarded, 100 ␮L of incomplete medium and
10 ␮L of MTT were added in each well. The plates were incu-
bated for 4 h in dark. After incubation, the medium was discarded
and 100 ␮L of DMSO was added. The absorbance was measured
at 630 and 492 nm in a plate reader [30].
2.5.3. Experimental grouping
To assess the oxidative stress induced by OxLDL and H2 O2
LDL and the cytoprotective effect of EOC on PBMC, the cells
were grouped as:
Control: PBMC (5 × 106 cells) were maintained for 48 h
under 5% CO2 at 37 ◦ C.
Induced: PBMC (5 × 106 cells) were induced with
OxLDL (10 ␮g/mL)/H2 O2 LDL (5 ␮g/mL) for 48 h for mono-
cyte/macrophage differentiation.
Treated: PBMC (5 × 106 cells) were induced with
OxLDL (10 ␮g/mL)/H2 O2 LDL (5 ␮g/mL) and co-treated with
100 ␮g/mL of EOC for 48 h.
After 48 h,cells were harvested and used for further studies.
2.5.4. Cytotoxicity assay
2.5.4.1. Trypan blue dye exclusion method. According to
Strober [29], Trypan blue (0.4% solution) staining was used to
document the viability of suspension and adherent PBMC with
100 ␮g/mL of EOC. After 48 h at 37 ◦ C, the adherent PBMC
were washed by centrifugation and fresh medium was added.
10 ␮l of cells from each experimental group were stained with
trypan blue dye (0.4% w/v) and the numbers of viable and
dead cells were scored under a light microscope with Neubauer
hemocytometer.
2.5.4.2. LDH leakage assay. To evaluate the potential cytotoxic
effect of OxLDL and H2 O2 LDL on PBMC, lactate dehydro-
genase (LDH) released in the supernatant was determined as
described by King (1965). The percentage of cellular LDH
 J. S. et al. / Food Science and Human Wellness 6 (2017) 60–69
release was calculated as the portion of LDH detected in the
conditioned medium divided by total LDH from conditioned
medium plus LDH from the cell lysate.
2.5.4.3. Quantification of nitric oxide. Nitrite production, an
indicator of NO production, was measured in the conditioned
culture medium by the Griess reaction Schulz et al. (1999).
The supernatants were collected for the quantification of nitrite.
Briefly, 100 ␮l of supernatants were mixed with 100 ␮l of Griess
reagent (equal volumes of 1% (w/v) sulphanilic acid in 30% (v/v)
acetic acid and 0.1% (w/v) N-(1-naphthyl) ethylenediamine in
60% (v/v) acetic acid) and incubated at room temperature for
30 min in the dark. The absorbance was measured at 540 nm
spectrophotometrically.
2.5.4.4. Quantification of intracellular ROS generation. Intra-
cellular ROS generation by PBMC was assessed by the nitroblue
tetrazolium (NBT) reduction assay Rook et al. (1985). This assay
is based on the ability of phagocytic cells to produce superoxide
upon stimulation with OxLDL and H2 O2 LDL. Cell suspension
was incubated with an equal volume of 1% NBT dissolved in
PBS at 37 ◦ C, for 30 min in the dark. After the incubation time, it
was examined under a light microscope for blue–black nitroblue
diformazan deposits, indicative of OxLDL and H2 O2 LDL stim-
ulated respiratory burst. At least 200 cells were assessed for each
experiment. After that, blue–black nitroblue diformazan was
solubilized with 2 mol KOH and DMSO and the absorbance at
630 nm was measured spectrophotometrically. A standard curve
was prepared by adding KOH and DMSO to a known concentra-
tion of NBT and 100 ␮M H2 O2 was added to the cells as positive
control.
2.5.4.5. Lipid peroxidation. The TBARS assay is a modified
method of Buege and Aust [31]. 0.5 mL of lysates were mixed
thoroughly with 1 mL of a solution containing 15% (w/v)
trichloroacetic acid, 0.375% (w/v) TBA, and 0.25 N HCl, and
heated for 15 min. After cooling, the flocculent precipitates were
removed by centrifugation. The absorbance of the sample was
determined at 535 nm against a blank containing all reagents
except samples. The level of lipid peroxides is expressed as ng
of MDA released/mg protein.
2.5.5. Statistical analysis
All the experiments were carried out in triplicate and results
are given as the mean ± standard deviation. The data were ana-
lyzed (Microsoft Excel 2007) for statistical significance using
Students t-test and differences were considered significant at
p < 0.05.
3. Results
3.1. FT-IR spectrum of essential oil extracted from C.
citratus (EOC)
The infrared spectra of the EOC were recorded by Thermo
scientific Nicolet IS-5 and run under infrared region of
63
400–4000 cm−1 range. Stretching of CH3 was observed cor-
responding to an alkyl saturated aliphatic group and at 2925.75
and 2854 cm−1 symmetric and asymmetric stretching of CH2
were observed. The intense band observed at 1724.83 cm−1 is
due to vibrations of C C (cis and trans), confirming the pres-
ence of conjugated double bonds (C C CHO) in citral which
are common in acyclic monoterpenes. The peak at 1629 cm−1
indicated the stretching of C O of the aldehyde group. At the
1383.65 cm−1 peak, bending of the CH2 group was observed.
At 1229.02 cm−1 bending of CH3 group was observed. From
1194 to 1048.64 cm−1 , stretching of C O and vibrations of
the CH skeleton were observed in Fig. 1 and Table 1. Simi-
lar peaks were reported by Wany et al. [18] for Geranial from
another species of Cymbopogon.
3.2. Antioxidant potential of essential oil extracted from C.
citratus
The scavenging activity of the EOC against DPPH was
concentration-dependent (Table 2). The highest concentration
(100 ␮g/mL) of EOC showed highest DPPH scavenging activity
when compared to the other concentrations of EOC. Interest-
ingly, EOC was as effective as the positive control, L-ascorbic
acid, in scavenging 50% of DPPH radicals present in the reaction
mixture.
EOC was found to possess significant reducing power and
antioxidant activity (Table 2). 100 ␮g/mL concentration was
found to be more active when compared to the lower concen-
trations. Reducing power of EOC and at antioxidant activity
100 ␮g/mL were comparable with standard L-ascorbic acid. This
was due to the presence of various phytochemicals in higher
proportions.
EOC was found to exhibit better inhibition of superoxide rad-
ical (Table 2) at concentrations of 75 ␮g/mL and 100 ␮g/mL.
The lower concentration (25 ␮g/mL) did not show much inhi-
bition when compared to higher concentrations. 100 ␮g/mL of
EOC showed comparable inhibition of superoxide radical with
L-ascorbic acid.
H2 O2 scavenging activity was evaluated by a decrease in
the formation of chromogen in the Fenton reaction (Table 2).
The H2 O2 scavenging activity was highest at 100 ␮g/mL,
when compared with L-ascorbic acid. The percentage NO
inhibition by various concentrations of EOC (100 ␮g/mL,
75 ␮g/ml,50 ␮g/mL and 25 ␮g/mL) respectively was found to be
62%, 50%, 35% and 16%. From the obtained results, 100 ␮g/mL
and 75 ␮g/mL of EOC showed 50% inhibition of nitric oxide,
whereas the lower concentration (25 ␮g/mL) showed only 16%
of inhibition. Higher concentration (100 ␮g/mL) was found to
be effective in nitric oxide scavenging compared with L-ascorbic
acid.
The results obtained show the inhibition of hemolysis at each
concentration of the EOC. As shown in Fig. 2, the protective
effect of EOC against hemolysis was directly proportional to
the concentration of the extract. The degree of protection was of
the order 100 > 75 > 50 > 25 ␮g/mL. However, the level of pro-
64 J. S. et al. / Food Science and Human Wellness 6 (2017) 60–69

Fig. 1. FT-IR spectrum of essential oil of Cymbopogon citratus. Region I may correspond to the presence of cycloalkanes, region II to the presence of terpenoids
and flavonoids and region III to that of methyl group.

Table 1
FT-IR spectra of essential oil of Cymbopogon citratus.
Sl. no. Region (cm−1 ) IR γ max (cm−1 ) Functional groups Possible Phytochemicals present in
(vibration mode) essential oil of Cymbopogon citratus

1. I (4000–2500) 3854.70–2854.48 (H-bonded, O H stretching; Polymeric hydroxyl compound,

N H stretching) cycloalkanes cyclobutane
Aldehyde C H stretching
2. II (2000–1500) 1724.83–1629.64 Aldehyde/Ketone C O Presence of terpenoids and
stretching, C C-stretching flavonoids, carbonyl compound
ketone
3. III (1500–400) 1383.65–1048.64 CH3 bending, C H in Methyl group, sulphonic acid ester
plane bending (SO3 ), aryl conjugates.

Table 2
In vitro antioxidant potential of essential oil extracted from Cymbopogon citratus.
Sl. no. Essential oil DPPH Radical Reducing Phosphomoly- Superoxide Hydrogen Nitric oxide
concentration scavenging activity power bdenum anion peroxide scavenging
(␮g/mL) % of Absorbance antioxidant scavenging scavenging activity (%)
inhibition ± SEM ± SEM (%) activity (%) activity (%)

1 25 13 ± 2.05 0.330 ± 0.0015 16.1 ± 0.249 19.5 ± 1.607 26.6 ± 0.378 16.7 ± 0.568
2 50 35 ± 2.51 0.460 ± 0.0005 41.2 ± 1.4 33.6 ± 2.400 39.1 ± 1.374 32.6 ± 1.106
3 75 57 ± 3.51 0.679 ± 0.0047 57.3 ± 1.123 48.7 ± 0.723 52.4 ± 1.001 59.2 ± 1.078
4 100 69 ± 2.64 0.730 ± 0.004 63 ± 3.055 63.5 ± 1.664 79.6 ± 0.680 66.1 ± 1.903
5 Ascorbic acid 83 ± 2.51 1.456 ± 0.055 69.5 ± 2.753 78.7 ± 1.609 83.2 ± 1.429 73.0 ± 0.404
(100 ␮g/mL)

Experiment was performed in triplicate and data show the mean ± sd. The data show a dose dependent increase in the antioxidant potential of EOC with the highest
being at 100 ␮g/ml concentration. In all cases, the antioxidant potential of EOC at this concentration was comparable to that of the standard l-ascorbic acid.
 J. S. et al. / Food Science and Human Wellness 6 (2017) 60–69
Fig. 2. The percentage of inhibition of hemolysis by EOC at different concentration (25–100 ␮g/mL) compared to L-ascorbic acid (100 ␮g/mL) as standard.
Experiment was performed in triplicate and data shows the mean ± SD (n = 3).
tection afforded by the extracts was less than that of L-ascorbic
acid.
3.3. Cytoprotective effect of essential oil extracted from C.
citratus
The percentage of PBMC viability was tested for different
concentrations of EOC, OxLDL and H2 O2 LDL. As shown
in Fig. 3(A and B), there was no cytotoxicity at any of the
concentrations of EOC. Hence we found 100 ␮g/mL of EOC
was a suitable concentration for further study because of the
presence of phytochemicals at higher concentration. PBMC
incubated with OxLDL and H2 O2 LDL concentrations ranging
between 5–25 (␮g/mL of protein) showed maximum viability
at 10 ␮g/mL for OxLDL and 5 ␮g/mL for H2 O2 LDL.
As shown in Fig. 4(A) the cytoprotective effect of EOC on
PBMC induced with OxLDL and H2 O2 LDL for 48 h, showed
cellular toxicity by decreasing cell viability which was assessed
by trypan blue exclusion assay. EOC exhibits cytoprotection
against OxLDL and H2 O2 LDL as observed by the signifi-
cant reduction in cell death. We assessed cellular integrity using
LDH leakage assay as depicted in Fig. 4(B). The PBMC treated
with various concentrations of EOC showed significant reduc-
tion in LDH leakage. Therefore, results from MTT, trypan blue
exclusion assay and LDH leakage assay can be correlated and
concluded that EOC could efficiently reduce the cytotoxicity
produced by oxidative stress which was induced by modified
LDL (H2 O2 LDL and OxLDL).
PBMC treated with EOC induced with OxLDL and H2 O2
LDL demonstrated significant reduction in ROS generation
(Fig. 5A). The effect of C. citratus extract on the production
of the proinflammatory mediator NO was analysed by mea-
suring the nitrite released into the medium. Fig. 5(B) shows
increased NO production in the OxLDL and H2 O2 LDL induced
PBMC cells when compared to the EOC treated groups. The
above results suggest the potent antioxidant and cytoprotective
activity of C. citratus. The inhibition of lipid peroxidation by
the essential oil of C. citratus on PBMC cells induced with
OxLDL and H2 O2 LDL was measured by the color intensity
of the MDA–TBA complex. The result demonstrates inhibition
of lipid peroxidation by EOC (Fig. 6).
65
4. Discussion
The utilization of medicinal plants has relied largely on long-
term clinical experience with little or no scientific data on their
efficacy or safety after long-term treatment (Zhu et al., 2002;
Ernst, 2004). As is the case in relation to many other medicinal
plant species, there are few toxicological/toxicogenetic studies
on the safety of C. citratus.
Degenerative disorders, such as cancer, cardiovascular dis-
eases and neurodegenerative diseases progress due to high levels
of ROS or free radicals Yen et al. (2002). Plant based antioxi-
dants can elicit increased synthesis of endogenous antioxidant
defenses or by themselves act directly as antioxidants [32].
The cardioprotective effect of C. citratus in isoproterenol-
induced cardiotoxicity has been reported by Gayathri et al.
(2010) who showed that lemon grass administration decreases
the toxic effects of lipid peroxidation in both serum and heart
tissue by increasing the levels of antioxidants. Costa et al. (2011)
showed that oral intake of C. citratus essential oil for 21 days
showed reduction in cholesterol and genotoxicity.
DPPH radical scavenging ability of EOC was comparable
with standard L-ascorbic acid suggesting the presence of pro-
ton donating ability of EOC that could serve as free radical
inhibitor. The reducing power of EOC suggested that the strong
antioxidant activity of EOC might be due to the presence of phe-
nolic compounds which absorb and neutralize free radicals and
quench peroxides, due to their redox properties (Gordan, 1990;
Osawa et al., 1994).
The total antioxidant capacity of EOC was assessed by phos-
phomolybdenum method. Luximon-Ramma et al. (2005). In the
present study, the obtained results demonstrate that the highest
antioxidant activity was at the highest concentration of EOC
used (100 ␮g/mL). The difference in the antioxidant capacity of
EOC is because of the presence of phytochemicals such as tan-
nins, triterpenoids, steroids and flavonoids [33,34]. Our results
indicate that EOC has good antioxidant potential.
Superoxide and hydroxyl radical are the two major ROS
produced during the reduction of oxygen to water. Superox-
ide radicals are formed by one electron reduction of molecular
oxygen [35,36]. Superoxide scavenging activity of EOC was
comparable with the known antioxidant L-ascorbic acid, thus
EOC also possesses superoxide scavenging activity.
66 J. S. et al. / Food Science and Human Wellness 6 (2017) 60–69

Fig. 3. Effect of the EOC (A) and (B) OxLDL and H2 O2 LDL on cell viability of PBMC assessed by MTT assay. Experiment was performed in triplicate and data
shows the mean ± SD (n = 3).

Fig. 4. Cytotoxic effect of OxLDL and H2 O2 LDL. (A) Trypan blue exclusion test (B) LDH leakage assay * p < 0.05 compared to control group; $ p < 0.05 in
comparison with the OxLDL induced group.** p < 0.05 compared to control group; $$ p < 0.05 in comparison with the H2 O2 LDL induced group.

Fig. 5. Inhibition of ROS (A) and nitric oxide production (B) in PBMC induced with OxLDL and H2 O2 LDL and treated with EOC. * p < 0.05 compared to control
group; $ p < 0.05 in comparison with the OxLDL induced group.** p < 0.05 compared to control group; $$ p < 0.05 in comparison with the H2O2 LDL induced group.

Nitric oxide can be described as a pleiotropic inhibitor of
physiological process because it plays a vital role in smooth
muscle relaxation, neural signalling, inhibition of platelet aggre-
gation and regulation of cell mediated toxicity [37]. Our results
exhibit that EOC could increase the demand for NO under oxida-
tive stress and since heart diseases can occur in a background of
oxidative stress, the EOC may be considered to contain cardio-
protective property.
Lee et al. [38] have stated that H2 O2 is poorly reactive because
of its weaker oxidizing and reducing capabilities. Hence, bio-
logically it can be a cytotoxic agent by converting itself into
hydroxyl radical in the presence of metal ions and superoxide
anion and produces singlet oxygen through reaction with super-
oxide anion or with hypochlorous acid. In this study, EOC was
less effective at lower concentration, whereas at 100 ␮g/mL the
H2 O2 scavenging activity was more as compared to L-ascorbic
acid.
H2 O2 can degrade haemoglobin, to release Fe2+ ions that
can lead to erythrolysis. Azizova et al. [39] showed that ery-
throcyte membranes during atherosclerotic conditions are prone
to lysis in the presence of oxidized LDL. We found that EOC
could efficiently inhibit lipid peroxidation, which supports its
 J. S. et al. / Food Science and Human Wellness 6 (2017) 60–69
Fig. 6. Formation of MDA indicates the lipid peroxide levels in PBMC induced with OxLDL and H2 O2 LDL and treated with EOC * p < 0.05 compared to control
group; $ p < 0.05 in comparison with the OxLDL induced group.** p < 0.05 compared to control group; $$ p < 0.05 in comparison with the H2 O2 LDL induced group.
potential to stabilize erythrocyte membranes thereby acts as an
anti-inflammatory agent. The erythrocyte membrane resembles
the lysosomal membrane and, as such, the effect of drugs on
the stabilization of erythrocyte membrane could be extrapolated
to the stabilization of lysosomal membrane [44]. Thus, EOC
exerts its protective effect on the lysosomal membrane which
could otherwise lead to oxidative stress induced cell death.
OxLDL is the product of oxidation of their polyunsaturated
fatty acid component due to the increased concentration of
ROS at the subendothelial level. Two aldehydes with strong
immunogenic properties are formed: malondialdehyde and 4-
hydroxynonenal with anionic valence. Uptake of oxidized LDL
renders the macrophages less mobile, thereby promoting the
accumulation of these lipid-laden foam cells in the intima.
The foam cells retain their metabolic activity and secrete a
variety of cytokines and inflammatory mediators. The out-
come of their activation include recruitment and proliferation of
smooth muscle cells which in turn elaborate additional locally
active cytokines. Subsequent LDL oxidation causes recruitment
of additional monocyte/foam cells and further impairment of
endothelial function.
OxLDL and H2 O2 LDL induction lead to macrophage
conversion and glycation of collagen which enhances the entrap-
ment of LDL and its subsequent oxidative modification. Thus,
there is a release of reactive oxygen intermediates associated
with the formation. The increased LDH leakage induced by
OxLDL and H2 O2 LDL cytotoxicity increased the permeabil-
ity of the cell. Arterial smooth muscle cells modify LDL so that
macrophages will take it up faster. The mechanism of LDL mod-
ification by cells involves lipid peroxidation. Lipid peroxidation
was found to be increased upon OxLDL and H2 O2 LDL induc-
tion. On treatment with C. citratus the levels were significantly
reduced. This proves that C. citratus possesses strong anti-lipid
peroxidation property [45].
The polarity features of the phytochemical components is
a key factor that confers their solubility and ability to access
the lipid phase where the lipid peroxidation process is taking
place, thus influencing their capacity to break chain reactions.
Antioxidant compounds with high partition coefficients are
67
preferentially distributed to hydrophobic compartments, thus
protecting lipids from free radical attack.
Peroxidation of lipid is probably one of the important inter-
mediary events in oxidative stress-induced cellular damage
through the perturbation of cellular redox balance. It has been
reported that lipid peroxidation is a persistent process in cells,
maintained at basal level, by protective enzymes and antiox-
idants which prevent it from entering the autocatalytic phase
[40].
Inhibition of oxidation of LDL due to the antioxidant
potential of the EOC could prevent lipid accumulation within
macrophage which in turn could inhibit atherosclerosis.
Hishikawa et al. [41] suggested that the antioxidant activities
of many polyphenols are responsible for their protection against
atherosclerosis.
Phagocytes are crucial in the host defence against invad-
ing microorganisms through reactive oxygen species (ROS)
production. Measurement of ROS production by phagocytes
is of critical importance to investigate the physiological con-
sequences resulting from cellular mechanisms that lead to
oxidative burst. ROS production occurs when macrophages were
stimulated by modified LDL.
The NBT reduction assay demonstrates the production of
superoxide anion by accepting electrons from a variety of donor
substances, such as superoxide, and thus is converted into
a reduced form which precipitates as a blue-black insoluble
material (formazan) in the cytoplasm of phagocytes. Reduced
respiratory burst activity when treated with EOC could be
due to the blocking of potassium channels of macrophages, as
described.
Figueirinha et al. [4], suggested that the anti-inflammatory
activity of C. citratus extract is due to its inhibition of NO pro-
duction and iNOS expression in dendritic cells. Nitric oxide
production was independent of the concentration of exogenous
L-arginine. In the present study, nitric oxide production and res-
piratory burst activity were inhibited by EOC. This supports the
subsequent depolarization of the plasma membrane, which may
lead to the impairment of intracellular signalling steps required
for macrophage activation.
68 J. S. et al. / Food Science and Human Wellness 6 (2017) 60–69
To conclude, our finding has excavated knowledge on drug
formulation of C. citratus which exhibits negligible cytotoxic
effect on normal cells and demonstrates antioxidant proper-
ties as evidenced from in vitro antioxidant assessment, has
anti-inflammatory properties as evidenced by the inhibition of
hemolysis of erythrocytes and exhibited cytoprotective proper-
ties, when added to PBMC induced with OxLDL and H2 O2
LDL. Thus, EOC showed differential response against dele-
terious action of ROS, suggesting that this can be validated
as a therapeutic and preventive agent against coronary artery
diseases.
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