Food Chemistry 151 (2014) 175181

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

Lemon grass (Cymbopogon citratus (D.C) Stapf) polyphenols protect

human umbilical vein endothelial cell (HUVECs) from oxidative damage
induced by high glucose, hydrogen peroxide and oxidised low-density
lipoprotein
J. Campos a,c,1, G. Schmeda-Hirschmann b, E. Leiva c, L. Guzmn c, R. Orrego c, P. Fernndez a, M. Gonzlez d,
C. Radojkovic a, F.A. Zuiga a, L. Lamperti a, E. Pastene e, C. Aguayo a,
a

Departamento de Bioqumica Clnica e Inmunologa, Facultad de Farmacia, Universidad de Concepcin, Concepcin, Chile
Instituto de Qumica de Recursos Naturales, Universidad de Talca, Talca, Chile
c
Departamento de Bioqumica Clnica e Immunohematologa, Facultad de Ciencias de la Salud, Universidad de Talca, Talca, Chile
d
Vascular Physiology Laboratory, Departamento de Fisiologa, Universidad de Concepcin, Concepcin, Chile
e
Laboratorio de Farmacognosia, Departamento de Farmacia, Facultad de Farmacia, Universidad de Concepcin, Concepcin, Chile
b

a r t i c l e

i n f o

Article history:
Received 19 March 2013
Received in revised form 13 September 2013
Accepted 4 November 2013
Available online 14 November 2013
Keywords:
Cymbopogon citratus
Oxidative stress
Nitric oxide
Atherosclerosis
Endothelial dysfunction

a b s t r a c t
The aromatic herb Cymbopogon citratus Stapf is widely used in tropical and subtropical countries in
cooking, as a herbal tea, and in traditional medicine for hypertension and diabetes. Some of its properties
have been associated with the in vitro antioxidant effect of polyphenols isolated from their aerial parts.
However, little is known about C. citratus effects on endothelial cells oxidative injury. Using chromatographic procedures, a polyphenol-rich fraction was obtained from C. citratus (CCF) and their antioxidant
properties were assessed by cooper-induced LDL oxidation assay. The main constituents of the active CCF,
identied by high-performance liquid chromatography with diode-array detection and mass spectrometry (HPLC-DADMS), were chlorogenic acid, isoorientin and swertiajaponin. CCF 10 and 100 lg/ml diminishes reactive oxidative species (ROS) production in human umbilical vein endothelial cell (HUVECs),
challenged with high D-glucose (60% inhibition), hydrogen peroxide (80% inhibition) or oxidised low-density lipoprotein (55% inhibition). CCF 10 or 100 lg/ml did not change nitric oxide (NO) production. However, CCF was able to inhibit vasoconstriction induced by the thromboxane A2 receptor agonist U46619,
which suggest a NO-independent vasodilatador effect on blood vessels. Our results suggest that lemon
grass antioxidant properties might prevent endothelial dysfunction associated to an oxidative imbalance
promoted by different oxidative stimuli.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Reactive oxygen species (ROS) are involved in several cardiovascular conditions, including atherosclerosis, heart failure, hypertension and endothelial dysfunction. The endothelial dysfunction is
caused by an imbalance between vasoconstrictor and vasodilator
molecules, and between pro-atherogenic and pro-coagulant states.
Importantly, chronic exposure to harmful physical and chemical
stimuli can lead to endothelial dysfunction (Caballero, 2003),
among which, oxidised low-density lipoprotein (oxLDL) and

Corresponding author. Address: Bioqumica Clnica e Inmunologa, Facultad de

Farmacia, Universidad de Concepcin, P.O. Box 237, Concepcin, Chile. Tel.: +56 41
2207196; fax: +56 41 2207086.
E-mail address: caguayo@udec.cl (C. Aguayo).
1
Present address: Facultad de Ciencias de la Salud, Universidad San Sebastin,
Concepcin, Chile, P.O. Box 3427.
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.11.018

hyperglycemia are the most relevant factors (Booth, Stalker, Lefer,

& Scalia, 2002). The hallmark of endothelial dysfunction is the inhibition of nitric oxide (NO)-mediated vasodilatation, the increase of
ROS production and the enhanced expression of endothelial cell
adhesion molecules (Cominacini et al., 2001). In this context, compounds able to scavenge ROS or suppress their formation may offer
therapeutic benets by inhibiting both the LDL oxidation and the
endothelium oxidative lesion, and they could, therefore, reduce
atherosclerosis progression (Halliwell, 2008; Kaliora, Dedoussis,
& Schmidt, 2006).
According to an earlier report, vascular injury progression could
be slowed using antioxidants such as probucol, vitamin E and butylhydroxytoluene (BHT) (Manach, Scalbert, Morand, Remesy, &
Jimenez, 2004). Moreover, antioxidant properties displayed by
some plant constituents have a potential application in human
healthcare and in cardiovascular diseases prevention. It has been
shown that the regular intake of fresh fruits, vegetables or herbal

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J. Campos et al. / Food Chemistry 151 (2014) 175181

teas, rich in natural antioxidants (phenolic compounds), can reduce the relative risk of cardiovascular illness (Hertog, Feskens,
Hollman, Katan, & Kromhout, 1993; Mink et al., 2007).
The perennial grass Cymbopogon citratus Stapf is widespread in
tropical and subtropical countries. Due to its pleasant aroma and
good taste, this herb is used for cooking and for preparing beverages and teas. Chemical studies of C. citratus showed the presence
of essential oils, triterpenes, and polyphenols in the aerial parts of
the plant (Cheel, Theoduloz, Rodriguez, & Schmeda-Hirschmann,
2005; Figueirinha, Cruz, Francisco, Lopes, & Batista, 2010). Experiments with C. citratus extracts in vitro have demonstrated its natural antioxidant and anti-inammatory properties in macrophages
(Tiwari, Dwivedi, & Kakkar, 2010), where it reduces interleukin-1
beta (IL-1b) and interleukin-6 production (Bachiega & Sforcin,
2011; Sforcin, Amaral, Fernandes, Sousa, & Bastos, 2009). Also, in
mouse skin dendritic cells, C. citratus displays anti-inammatory
effects, by inhibiting both nitric oxide (NO) production and inducible NO synthase expression generated by lipopolysaccharide. On
the other hand, C. citratus has demonstrated some vascular effects.
For instance, citronellol, an essential oil of C. citratus, lowers blood
pressure in rats by a direct effect on vascular smooth muscles (Bastos et al., 2010). Also, citral (3,7 dimethyl-2,6-octadienal), a volatile
compound identied in aerial parts of C. citratus, has a smooth
muscle relaxant effect on isolated thoracic rat aorta (Devi, Sim, &
Ismail, 2012). However, the antioxidant capacity of compounds extracted from C. citratus, has not been evaluated in human endothelial cells, which could improve their function. In this work we
evaluated the effect of the most antioxidant fraction of a polar C.
citratus extract (CCF) on copper-induced LDL oxidation. Main compounds found in this fraction were identied by HPLCESI-MS.
Then, the protective effects of CCF on endothelial function were
evaluated by measuring NO bioavailability and oxidative stress
promoted by several oxidants (oxLDL, D-glucose and H2O2) in human umbilical vein endothelial cells (HUVEC). Finally, the effect
of CCF on NO-mediated vasodilatation was evaluated in segments
of umbilical vein rings.
2. Materials and methods
2.1. Chemicals
Gelatin, H2O2, D-glucose, U46619, 2,7-dichlorouorescein
diacetate (DCF) and 20 -dichlorouorescin diacetate (DAF-DA) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). M199
medium and newborn and foetal calf serum were purchased from
GIBCO Co. (Grand Island, NY, USA). HPLC grade acetonitrile and
methanol, formic acid and acetic acid were from Merck
(Darmstadt, Germany).
2.2. Plant material
The aerial parts of C. citratus were obtained from plants grown
at the Botanical Garden from the Universidad de Talca. The airdried and powdered plant material (586 g) was successively extracted under reux with methanol (MeOH) (2  10l) and
MeOH:H2O (70:30 v/v, 2  5l). The extract was ltered and dried
under reduced pressure and then lyophilized to obtain 83.13 g
from the MeOH-extract and 32.76 g from the MeOH:H2O extract.
The lyophilized extracts were resuspended in water and partitioned with dichloromethane (DCM) to obtain a lipophilic, DCMsoluble fraction (58.8 g), while the polar constituents remained in
the aqueous phase (57.05 g). A representative sample from the
aqueous phase (47.4 g) was separated in a Sephadex LH-20 column
(Pharmacia, Sweden) (200 cm length, 7.5 cm internal diameter)
with MeOH:H2O 7:3, to obtain ten fractions after TLC analysis

(Silica gel, EtOAc:acetic acid:formic acid:water 20:1:1:3, upper

phase, detection under UV light at 254 and 365 nm). Flavonoids
were detected after spraying with a diphenylboric acid ethanolamine solution in MeOH. The fraction 5 (1.23 g), which presented
the highest lag time on LDL oxidation, was used for the study
and the constituents identication was carried out by high-performance liquid chromatography coupled to diode-array detection
and mass spectrometry (HPLC-DADMS/MS), high eld NMR spectroscopy and comparison with standard compounds previously
isolated from the plant (Cheel et al., 2005; Figueirinha et al., 2010).
2.3. Isolation and oxidation of LDL
Human LDL was isolated from the plasma of healthy volunteers
by differential centrifugation as described previously (Carru et al.,
2004; Searle et al., 2011). The LDL obtained was dialyzed against
Ca+2Mg+2-free phosphate-buffered saline (PBS) (without EDTA),
then sterilized by ltration through a 0.2 lm pore size lter and
stored in dark at 4 C. Further LDL oxidation was prevented by
the addition of 45 mM BHT and 2 mM EDTA. OxLDL was obtained
by incubating antioxidant-free native LDL (50 lg/ml) with CuSO4
(5 lM) in PBS for 34 h at 37 C. In copper-induced LDL oxidation
experiments, the oxidation degree was estimated by conjugated
dienes formation: Briey, LDL oxidation was followed by measuring
the increase in absorbance at 243 nm, due to conjugated dienes
formation, in a controlled temperature (37 C) plate reader (Sinergy 2, BioteK 311). Native LDL (50 lg/ml) was oxidised with 5 lM
CuSO4 in the absence (control) or presence of CCF (0.1 and
0.3 lg/ml) dissolved in 0.15 M NaCl, for 210 min at 37 C. The lag
time was determined using the software GEN 5 by Biotek instruments. Data were expressed as means SE (n = 6). Ascorbic acid
(0.25 mM) was used as a reference antioxidant.
2.4. High-performance liquid chromatography with diode-array
detection and mass spectrometry (HPLC-DADMS) analyses
The composition of the most antioxidant fraction of C. citratus
polar extract, as determined by LDL oxidation, was assessed by
HPLC-DADMS. HPLC-DAD analyses was performed using a
Merck-Hitachi diode array detector (Merck-Hitachi L-7455) with
a L-7100 pump and a D-7000 chromatointegrator. A
254  4.6 mm i.d., 5 lm C18-RP column (Kromasil 100 C18) was
used. The compounds were monitored between 220 and 600 nm.
Two different chromatographic systems were used as follows. System 1: Solvents: 0.1% formic acid in water (solvent A) and 20% solvent A in 80% acetonitrile (solvent B). Gradient: 0100% B in A
(t = 010 min), 08% B in A (t = 1015 min), 820% B in A (t = 15
35 min), 2030% B in A (t = 3540 min), 3040% B in A (t = 40
45 min), 4020% B in A (4550 min), 20% B in A (t = 5055 min),
0% B in A (t = 5560 min). System 2: Solvents: 2.5% acetic acid in
water (A), 2.5% acetic acid in water: acetonitrile 90:10 (B) and acetonitrile (C) as mobile phase. Gradient: 0100% B in A (t = 05 min),
015% C in B (t = 530 min), 1550% C in B (t = 3035 min) and 50%
C in B (t = 3540 min), isocratic, ow rate: 0.5 ml/min. Mass spectra were recorded using an Agilent 1100 LC system connected
through a split to an Esquire 4000 Ion Trap LC/MS system (Bruker
Daltoniks, Germany). The extract was dissolved in MeOH-formic
acid (99:1) (approx. 3 mg/ml), and submitted to LC-MS. The volume injected was 20 ll. Full scan mass spectra were measured between m/z 150 and 2000 u in the negative ion mode. Nitrogen was
used as nebulizer gas at 27.5 psi, 350 C and at a ow rate of 8 l/
min. The mass spectrometric conditions for negative ion mode
were as follows: electrospray needle, 4000 V; end plate offset,
500 V; skimmer 1, 56.0 V; skimmer 2, 6.0 V; capillary exit offset,
84.6 V; Collision induced dissociation (CID) spectra were obtained

J. Campos et al. / Food Chemistry 151 (2014) 175181

with a fragmentation amplitude of 1.00 V (MS/MS) using helium as

the collision gas.
2.5. Isolation and culture of HUVECs
Endothelial cells were obtained from human umbilical cord
veins by digestion with collagenase (0.2 lg/ml for 10 min at
37 C). These protocols were in agreement with the principles outlined in the Declaration of Helsinki, and the informed consent, approved by the Ethics Committee from the Universidad of
Concepcin, was signed by every volunteer participating in this research. HUVECs were cultivated to reach conuence in M-199
medium supplemented with 15% foetal bovine serum and 100 UI/
ml penicillin/streptomycin, at 37 C in a humidied 5% CO2 atmosphere, as described previously (Montecinos et al., 2000; Vsquez
et al., 2007).
2.6. ROS formation
ROS formation was assessed by using the probe 2,7-dichlorouorescein diacetate (DCF). HUVECs were exposed to CCF (24 h, 10 or
100 lg/ml) and then to oxLDL (50 lg/ml, 1 h), D-glucose (25 mM,
6 h) or H2O2 (1.0 mM, 10 min). Cells were washed and loaded with
0.5 lM DCF in M199 medium for 30 min at 37 C. ROS formation
was measured by evaluating DCF uorescence (kex: 495 nm and
kem: 510 nm) with a Sinergy 2, Biotek 311 microplate reader. Results were expressed as relative uorescence units (RFU) per cell
protein content (Takaishi, Taniguchi, Takahashi, Ishikawa, &
Yokoyama, 2003; Zmijewski et al., 2005).
2.7. NO measurements
NO detection was performed with the uorescent probe 4,5diaminouorescein diacetate (DAF-2DA). HUVECs were cultured
in 96 well plates (105 cells/well) and treated with CCF (10
100 lg/ml) for 24 h. Then, DAF-2DA was added (0.4 lM, 60 min)
0.1 mM histamine (endothelial nitric oxide synthase (eNOS) activator) or 2 mM L-NAME (eNOS inhibitor) were used as controls.
Fluorescence was measured with a Sinergy 2, Biotek 311 plate
reader (kex: 495 nm and kem: 510 nm). Results were expressed
as relative uorescence units (RFU) per cell protein content
(Kamiyama, Kishimoto, Tani, Utsunomiya, & Kondo, 2009; Kojima
et al., 1998).

177

2.9. Statistical analyses

Statistical analyses were performed with GraphPad Prism
(GraphPAD, San Diego, CA). All data were expressed as mean SE.
Comparisons between groups were carried out by one-way ANOVA
Test. Differences were considered signicant at P < 0.05. Correlations between all variables were carried out by the Pearson test.
3. Results
3.1. Effect of CCF on Cu+2-induced human LDL oxidation
As expected, native low-density lipoprotein (nLDL) incubated
during 210 min with Cu+2 (5 lM) was oxidised faster that nLDL
without Cu+2. The lag time for Cu+2-induced LDL oxidation was
57 1.5 min and non nLDL oxidation was observed in a copper-free
solution. When CCF (0.1 lg/ml) was added, this lag time increased
signicantly up to 109 10 min (Fig. 1) (P < 0.05). A similar effect
was observed when we used vitamin C (0.25 mM) as antioxidant
control (73 6 min). At a higher CCF concentration (0.3 lg/ml)
the copper-induced LDL oxidation was completely inhibited.
3.2. CCF analyses by high-performance liquid chromatography with
diode-array detection and mass spectrometry (HPLC-DADMS)
The main constituents of CCF were identied by spectroscopic
and spectrometric methods, and comparison with standard (reference) compounds. According to LC-MS data, CCF consisted in a
mixture of three main constituents identied as chlorogenic acid,
isoorientin and swertiajaponin. Two other minor compounds were
recognised as 6-C-pentosyl-8-C-hexosyl apigenin and luteolin
C-rhamnosyl rhamnoside (Cheel et al., 2005; Figueirinha, Cruz,
Francisco, Lopes, & Batista, 2010; Kite et al., 2006). The information
is summarised in Table 1.
3.3. Effect of CCF on the protection of HUVECs from ROS production
To demonstrate the protective effect of CCF on ROS production,
we cultured HUVECs cells with different oxidant compounds

2.8. Vascular reactivity of umbilical vein vessels

Human umbilical cords were obtained from full-term normal
healthy pregnants. These protocols were in agreement with the
principles outlined in the Declaration of Helsinki, and the informed
consent, approved by the Ethics Committee from the Universidad
of Concepcin, was signed by every volunteer participating in this
research. Umbilical veins were carefully isolated, cleaned and immersed in a beaker containing Krebs solution at 4 C. Venous segments were rapidly sliced into rings (2.5 mm length) and
suspended for the measurement of isometric force in organ chambers (5 ml) bubbled continuously with a mixture of 95% O2 and 5%
CO2. Isometric tension changes were recorded as previously described (Gonzalez, Cruz, Sepulveda, & Rudolph, 1990) through a
forcedisplacement transducer (Grass FT03), which was connected
to a Grass polygraph. Vein rings were suspended under a tension of
1.0 g and equilibrated during 60 min. After washes, U46619 (9,11dideoxy-11a,9a-epoxymethanoprostaglandin F2a), a potent
vasoconstrictor (thromboxane A2 receptor agonist), was added to
induce maximal contraction. Then, CCF concentrationresponse
curves (10101  106 M) were cumulatively obtained and compared with U4661-precontracted rings (without CCF).

Fig. 1. Effect of C. citratus extract (CCF) on LDL oxidation. Native LDL was diluted to
standard concentration (50 lg/ml total cholesterol) and oxidation initiated in
presence (d) or absence (s) of copper sulphate (5 lM), CCF extract 0.1 lg/ml (h),
0.3 lg/ml (h) or vitamin C (0.25 lM) (4). Absorbance at 234 nm was measured
during 210 min in 5 min intervals at 37 C in a spectrophotometer to obtain a
typical conjugated diene-formation (CD) curve. From the CD-formation curve, the
lag time dened as end of the cross point of the time axis and the curve slope was
estimated.

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J. Campos et al. / Food Chemistry 151 (2014) 175181

Table 1
Identication of the main phenolics in CCF by HPLCMS/MS.
Compound
1
2
3
3a
4 (minor)
a

Rt (min) (S1)

Rt (min) (S2)

UV maxima

28.43
35.36
36.35

25.43
34.84
36.02

325, 295 sh, 242

349, 285 sh, 269, 255 sh
346, 285 sh, 269, 260 sh

43.52

349, 285 sh, 269, 260 sh

M-1
353
447
461
563
577

MS/MS

Compound identication

191,
429,
443,
545,
431

173,
357,
371,
503,

127, 85
327
341
473, 443, 353

Caffeoylquinic acid (chlorogenic acid)

Isoorientin
Swertiajaponin
6-C-pentosyl-8-C-hexosyl apigenin
Luteolin C-rhamnosyl rhamnoside

a
a

Identied by Rt, UV data, fragmentation pattern and comparison with a reference compound.

(H2O2, glucose or oxLDL), and ROS production was detected by a

uorescent probe.
Regarding HUVECs treated with H2O2 (1.0 mM, 10 min), ROS
production increased signicantly, when compared with the control (non-treated cells, P < 0.001) (Fig. 2). The co-incubation of
H2O2 with CCF (10 or 100 lg/ml) inhibited signicantly the ROS
synthesis in H2O2-exposed HUVECs (about 30 and 60%, respectively). There was no statistical difference between the two concentrations of CCF assayed.
In relation to D-glucose and oxLDL, HUVECs were pretreated with
CCF and then incubated with high D-glucose (25 mM, 6 h) or oxLDL
(50 lg/ml, 1 h). ROS production was signicantly increased with Dglucose, compared with the basal ROS synthesis (P < 0.01) (Fig. 3).
CCF (10 lg/ml) inhibited this effect by 45% (P < 0.01) and there
was no further effect when using CCF 100 lg/ml. On the other hand,
oxLDL increased signicantly ROS production in comparison with
the control (P < 0.01) (Fig. 3). This effect was reduced signicantly
(P < 0.01) in the presence of CCF, showing a stronger anti-oxidant effect at 10 lg/ml than at 100 lg/ml. However, there was no statistical
difference between these two concentrations. CCF alone did not
change basal ROS production and did not affect cell viability at the
concentrations used in these experiments (data not shown).
3.4. Effect of CCF on NO production and vasodilator responses
To assess the effect on NO bioavailability, HUVECs were treated
with CCF 10 or 100 lg/ml for 24 h and NO production was detected
by a uorescent probe. CCF did not produce any change in NO bioavailability, as compared with control. In contrast, histamine, a
well known stimulus for NO production in HUVEC, increased
uorescence by 30%, and this was completely abrogated by
L-NAME (P < 0.001) (Fig. 4A).

Fig. 2. Effect of C. citratus extract on H2O2-induced ROS production in HUVECs.

Serum-starved HUVECs were incubated with hydrogen peroxide (H2O2, 1.0 mM,
10 min) in presence of C. citratus extract (CCF, 10 or 100 lg/ml, 24 h). Reactive
oxygen species generation was determinate using 2,7-dichlorouorescein diacetate
probe (DCF, 100 lM). P < 0.05 vs. control; P < 0.05 vs. HUVECs cultured with CCF.

P < 0.001 vs. control; P < 0.01 vs. HUVECs cultured without CCF.

Fig. 3. Effect of C. citratus extract oxLDL or D-glucose induced ROS production in

HUVECs. Serum-starved HUVECs were incubated with oxLDL (50 lg/ml, 1 h) and Dglucose (25 mM, 6 h) in presence of C. citratus extract (CCF, 10 or 100 lg/ml, 24 h).
Reactive oxygen species generation was determined using 2,7-dichlorouorescein
diacetate probe (DCF, 100 lM). P < 0.01 vs. control; P < 0.01 vs. HUVECs cultured
without CCF.

Interestingly, when we evaluated the effect of CCF on pre-contracted umbilical vein rings with U46619, a thromboxane A2 receptor agonist, we observed a vasodilator dose-dependent response
(Fig. 4B).
4. Discussion
Initial stage of atherosclerosis has been associated with changes
in endothelial function, mainly a reduction in the NO synthesis and
an increased production of reactive oxygen species (ROS). Regarding the latter, multiple studies have focused on the potential use of
natural antioxidants as alternative medicine for preventing or
treating atherosclerosis (Li & Forstermann, 2000). However, little
is known about the possible effect of chemical molecules found
in selected foods and medicinal plants. Since plant extracts are
complex mixtures, chemical characterisation represents a mandatory step previous to pharmacological investigation. In the present
study, an antioxidant polar fraction of C. citratus was investigated
for vascular effects in vitro, for which we studied the LDL oxidation
an initial step for endothelial dysfunction and cardiovascular disease development , the endothelial ROS and NO production, and
the vasodilator response of venous rings.
The protocol used to obtain CCF eliminates volatile constituents,
sugars and inorganic salts. Therefore, only polar compounds, like
polyphenols, are concentrated in CCF. The same compounds could
be extracted with hot water and are present in infusions and
decoctions of this herbal tea (Hertog et al., 1993; Mink et al., 2007).
4.1. Protective effect of CCF on LDL oxidation
Hypercholesterolemia is associated with high levels of LDL,
which is considered a major risk factor for the development of

J. Campos et al. / Food Chemistry 151 (2014) 175181

179

MS showed that chlorogenic acid, isoorientin and swertiajaponin

are the major constituents of this mixture. Previous studies
showed that C-glycosylavonoids including isoorientin, swertiajaponin and isoorientin 200 -O-rhamnoside, isolated from C. citratus
inhibit oxidation of human LDL, and suggest that isoorientin is
an effective inhibitor of LDL oxidation in vitro (Orrego, Leiva, &
Cheel, 2009). However, we cannot exclude that minor components,
such as 6-C-pentosyl-8-C-hexosyl apigenin and luteolin C-rhamnosyl rhamnoside, could also account for the antioxidant properties
of CCF extracts.

4.2. CCF and reactive oxygen species (ROS)

Fig. 4. Effect of C. citratus extract in NO synthesis in HUVECs and vasodilation of

human umbilical veins. A, Serum-starved HUVECs were incubated with C. citratus
extract (CCF, 10 or 100 lg/ml, 20 h), histamine (1 mM, 5 min) or NG-nitro-Larginine methyl ester (L-NAME, 10 lM, 30 min). NO was determined using 4,5diaminouorescein diacetate probe (DAF-2DA, 1 lM, 30 min) as described in
methods. P < 0.05 vs. HUVECs treated with histamine. B. CCF concentration
response curves in human umbilical veins from normal gestation. U46619
contractile responses are expressed as percent of the contractile response to KCl
(124 mM) in presence (d) or absence (s) of CCF. Each point represents the
mean S.E.M. of seven to nine experiments.

endothelial dysfunction and atherosclerosis. In the plasma of normal individuals, most lipoproteins are found in a native form
(9099%) and only a minor fraction is modied by oxidation. However, in hypercholesterolemia and other pathological conditions,
lipoproteins oxidation is increased (Yla-Herttuala, 1999; Koller
et al., 2012). High levels of oxLDL generate injury and inammation
on the vascular tissue, which is related directly with the progression of the atherogenic process. Thus, molecules able to inhibit or
slow LDL oxidation, i.e. antioxidants, have been proposed to disminish atherosclerosis progression. In this context, our results
show that CCF protects LDL oxidation induced by Cu+2, as the lag
time is signicantly higher, compared with the condition without
CCF. When using a higher concentration of CCF, LDL oxidation is
completely abolished. Interestingly, CCF was more efcient than
ascorbic acid, a well known antioxidant molecule, to reduce LDL
oxidation (Fig. 1). Further characterisation of CCF by HPLC-DAD

Our results showed that CCF (10 and 100 lg/ml) inhibited the
synthesis of ROS in HUVECs exposed to H2O2 (1.0 mM), D-glucose
(25 mM) or oxLDL (50 lg/ml) (Fig. 3). These results are the rst evidence that CCF inhibits ROS generation in human endothelial cells
and are consistent with other ndings suggesting that CCF have
antioxidant and anti-inammatory properties (Lotito & Frei,
2006; Orrego et al., 2009; Rao et al., 2009; Tiwari et al., 2010).
Among the biomolecules that generate ROS and endothelial dysfunction, oxLDL plays a central role. In the early steps of atherosclerosis, oxLDL promote superoxide anion (O
2 ) formation in
endothelial cells, inducing cell death (Galle, Heinloth, Wanner, &
Heermeier, 2001). Moreover, ROS can react with biological molecules, such as NO, which diminishes NO bioavailability and vasodilatory responses. It has been shown that hypercholesterolemia,
Diabetes Mellitus and obesity are closely linked to hypertension
and stroke, and obesity is one of the major risk factors contributing
to the overall burden of cardiovascular diseases worldwide (Choi,
Benzie, Ma, Strain, & Hannigan, 2008; Lavie, Milani, & Ventura,
2009). As shown in the present study, CCF has ROS scavenging
activity and inhibits the ROS generation induced by H2O2, oxLDL
and D-glucose. However, in the presence of H2O2 and oxLDL, a high
concentration (100 lg/ml) of CCF seems to be less effective (Figs. 2
and 3). This can be explain by the presence of polyphenols, which
are easily oxidised in solution (Akagawa, Shigemitsu, & Suyama,
2003; Aoshima & Ayabe, 2007), in cell culture media, and even in
the oral cavity, to generate high levels of H2O2 (Lambert, Sang, &
Yang, 2007). Pro-oxidant effects of polyphenols involve interactions with metal ions (Otero, Viana, Herrera, & Bonet, 1997), generating O
2 , H2O2 and a quinones mixture which are potentially
cytotoxic (Sang, Lee, Hou, Ho, & Yang, 2005; Suh et al., 2010). Likewise, studies by Bellion et al. (2009), demonstrate that high concentrations of an apple extract rich in polyphenols increases the
formation of ROS in HT-29 cells. Based on these evidences, the lower antioxidant capacity of CCF at 100 lg/ml, compared to 10 lg/ml,
could be due to an increase in ROS formation because of a higher
content of free polyphenols that can be oxidised in the cell culture.
This is not the case of oxLDL, in which both concentrations of CCF
have the same antioxidant effect.
The main constituents identied in CCF are chlorogenic acid, a
caffeoylquinic acid derivative as well as the C-glycosylavonoids
isoorientin and swertiajaponin. Chlorogenic acid is a well known
natural antioxidant and presents an IC50 of 13.8 lM in the 1,1-diphenyl-2-picrylhydrazylz (DPPH) bleaching assay and 54.2 lM in
the superoxide scavenging test (NBT). At 100 lg/ml the compound
inhibits lipoperoxidation by 33.8 % (Cheel et al., 2005). According
to DPPH and NBT assays, isoorientin show strong antioxidant effect
with IC50 values of 9.1 and 52.9 lM, respectively. Furthermore,
isoorientin inhibits lipid peroxidation by 71.3% at 100 lg/ml (Cheel
et al., 2005). In line with our results, the inhibitory effect of some
C-glycosylavonids from C. citratus aerial parts on LDL oxidation
was reported (Orrego et al., 2009). Interestingly, isoorientin was
isolated as the active constituent of Gentiana olivieri showing both

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J. Campos et al. / Food Chemistry 151 (2014) 175181

hypoglycemic and hypolipidemic effects in diabetic rats (Sezik,

Aslan, Yesilada, & Ito, 2005). Isoorientin and chlorogenic acid were
associated with the hypoglycemic effects of a Cecropia obtusifolia
extract (Andrade-Cetto & Wiedenfeld, 2001). Our results agree
with the published evidence and demonstrate that CCF has antioxidant properties (Cheel et al., 2005). Therefore, C. citratus infusions
could be used for a complementary treatment of cardiovascular
diseases.
4.3. CCF and nitric oxide (NO) in HUVECs
NO is a potent endothelial-derived vasodilator molecule that
also displays anti-inammatory and anti-thrombotic effects. Due
to its properties, NO can be regarded as an anti-atherosclerotic
agent and its measurement is an indicator of endothelial function
(Li & Forstermann, 2000). In our experiments, CCF 10 and
100 lg/ml did not change NO bioavailability in HUVECs. In contrast, other plant extracts, such as green tea, signicantly decrease
NO production in HUVECs (Ahn & Kim, 2011). These differences
can be associated to the metabolites found in each species. For instance, tannins and catechin represent the most abundant polyphenols in green tea, whereas C-glycosylavonoids and
chlorogenic acid are the most important in CCF. Also, the cell model used for experiments, and the nitric oxide synthase (NOS) isoform expressed by each cell type, can account for these
differences. For instance, macrophages and murine macrophage
cell lines express inducible nitric oxide synthase (iNOS) (Ahn &
Kim, 2011). While iNOS is activated by inammatory stimuli in
various cell types and participates in immune defense against
exogenous pathogens (Mayer & Hemmens, 1997), eNOS responds
to inammatory mediators and produces low basal levels of NO.
Thus, results obtained on NO bioavailability in HUVECs incubated
with CCF should not be extrapolated to other cell type or to other
kinds of plant extract. However, although CCF did not generate
changes in NO bioavailability in HUVECs, it induced vasorelaxation
in human umbilical vein rings pre-contracted with U41669. These
results are consistent with some works performed on rabbit and
rat vessels (Devi et al., 2012, 2011) suggesting that C. citratus has
a NO-independent vasodilator effect, probably by inactivating calcium channels in smooth muscle cells. However, further experiments are needed to conrm this hypothesis.
In conclusion, CCF inhibits oxidation of native LDL by Cu+2, scavenges ROS in HUVECs treated with H2O2, oxLDL or high D-glucose
and does not change NO bioavailability, but generates a vasodilator
response in pre-contracted vein rings. These results suggest that
the vasodilatory response induced by CCF is independent of NO
synthesis; these are the rst results using human umbilical vein
from normal pregnancies showing the direct effect of CCF on vascular tone control. These effects could be explained by the ROS
scavenger properties of CCF, which could improve vessel relaxation
by increasing vasodilator molecules bioavailability or by blocking
ROS effects on gene expression. However, we cannot exclude that
CCF could regulate the synthesis of other vasoactive molecules
not assessed in this study (i.e. increased expression of vasodilator
agents or inhibition of vasoconstrictor molecules).
In consequence, additional studies are needed to determine the
mechanisms by which CCF generates vasodilatation and to evaluate the CCF effects in animal models of atherosclerosis.
Acknowledgements
J. Campos thank Programa de Magister en Ciencias Biomdicas, Facultad de Ciencias de la Salud, Universidad de Talca for partial funding. This study was supported by Fondo Nacional de
Desarrollo Cientco y Tecnolgico (FONDECYT 11070035) Chile,

Direccin de Investigacin, Universidad de Concepcin (DIUC

205-072.031-1.0), Chile and Programa de Investigacin en Productos Bioactivos, Universidad de Talca. We also thank the midwives
of Hospital Clnico Guillermo Grant Benavente and Clnica Sanatorio Alemn, Concepcin labour ward for the supply of umbilical
cords.
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