Food Chemistry 151 (2014) 175–181

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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. Guzmán c, R. Orrego c, P. Fernández a, M. González d, C. Radojkovic a, F.A. Zuñiga a, L. Lamperti a, E. Pastene e, C. Aguayo a,⇑ a

Departamento de Bioquímica Clínica e Inmunología, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile Instituto de Química de Recursos Naturales, Universidad de Talca, Talca, Chile c Departamento de Bioquímica Clínica e Immunohematología, Facultad de Ciencias de la Salud, Universidad de Talca, Talca, Chile d Vascular Physiology Laboratory, Departamento de Fisiología, Universidad de Concepción, Concepción, Chile e Laboratorio de Farmacognosia, Departamento de Farmacia, Facultad de Farmacia, Universidad de Concepción, Concepción, 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, identified by high-performance liquid chromatography with diode-array detection and mass spectrometry (HPLC-DAD–MS), 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. Ó 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: Bioquímica Clínica e Inmunología, Facultad de Farmacia, Universidad de Concepción, P.O. Box 237, Concepción, Chile. Tel.: +56 41 2207196; fax: +56 41 2207086. E-mail address: [email protected] (C. Aguayo). 1 Present address: Facultad de Ciencias de la Salud, Universidad San Sebastián, Concepción, Chile, P.O. Box 3427. 0308-8146/$ - see front matter Ó 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 benefits 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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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-inflammatory 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-inflammatory 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 identified 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 identified by HPLC–ESI-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-dichlorofluorescein diacetate (DCF) and 20 -dichlorofluorescin 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 reflux with methanol (MeOH) (2  10l) and MeOH:H2O (70:30 v/v, 2  5l). The extract was filtered 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 identification was carried out by high-performance liquid chromatography coupled to diode-array detection and mass spectrometry (HPLC-DAD–MS/MS), high field 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+2–Mg+2-free phosphate-buffered saline (PBS) (without EDTA), then sterilized by filtration through a 0.2 lm pore size filter 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 3–4 h at 37 °C. In copper-induced LDL oxidation experiments, the oxidation degree was estimated by conjugated dienes formation: Briefly, 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 5Ò 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-DAD–MS) analyses The composition of the most antioxidant fraction of C. citratus polar extract, as determined by LDL oxidation, was assessed by HPLC-DAD–MS. 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: 0–100% B in A (t = 0–10 min), 0–8% B in A (t = 10–15 min), 8–20% B in A (t = 15– 35 min), 20–30% B in A (t = 35–40 min), 30–40% B in A (t = 40– 45 min), 40–20% B in A (45–50 min), 20% B in A (t = 50–55 min), 0% B in A (t = 55–60 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: 0–100% B in A (t = 0–5 min), 0–15% C in B (t = 5–30 min), 15–50% C in B (t = 30–35 min) and 50% C in B (t = 35–40 min), isocratic, flow 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 flow 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

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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 Concepción, was signed by every volunteer participating in this research. HUVECs were cultivated to reach confluence in M-199 medium supplemented with 15% foetal bovine serum and 100 UI/ ml penicillin/streptomycin, at 37 °C in a humidified 5% CO2 atmosphere, as described previously (Montecinos et al., 2000; Vásquez et al., 2007). 2.6. ROS formation ROS formation was assessed by using the probe 2,7-dichlorofluorescein 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 fluorescence (kex: 495 nm and kem: 510 nm) with a Sinergy 2, Biotek 311 microplate reader. Results were expressed as relative fluorescence 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 fluorescent probe 4,5diaminofluorescein 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 fluorescence units (RFU) per cell protein content (Kamiyama, Kishimoto, Tani, Utsunomiya, & Kondo, 2009; Kojima et al., 1998).

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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 significant 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 significantly 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-DAD–MS) The main constituents of CCF were identified 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 identified 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 Concepción, 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 force–displacement 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 concentration–response curves (1010–1  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 defined as end of the cross point of the time axis and the curve slope was estimated.

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Table 1 Identification of the main phenolics in CCF by HPLC–MS/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 identification

191, 429, 443, 545, 431

a

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

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

(H2O2, glucose or oxLDL), and ROS production was detected by a fluorescent probe. Regarding HUVECs treated with H2O2 (1.0 mM, 10 min), ROS production increased significantly, 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 significantly 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 significantly 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 significantly ROS production in comparison with the control (P < 0.01) (Fig. 3). This effect was reduced significantly (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 fluorescent 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 fluorescence 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-dichlorofluorescein 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-dichlorofluorescein 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

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MS showed that chlorogenic acid, isoorientin and swertiajaponin are the major constituents of this mixture. Previous studies showed that C-glycosylflavonoids 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 4,5diaminofluorescein 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 (90–99%) and only a minor fraction is modified 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 inflammation 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 significantly higher, compared with the condition without CCF. When using a higher concentration of CCF, LDL oxidation is completely abolished. Interestingly, CCF was more efficient 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 first evidence that CCF inhibits ROS generation in human endothelial cells and are consistent with other findings suggesting that CCF have antioxidant and anti-inflammatory 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 identified in CCF are chlorogenic acid, a caffeoylquinic acid derivative as well as the C-glycosylflavonoids 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-glycosylflavonids 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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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-inflammatory 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, significantly 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-glycosylflavonoids 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 inflammatory stimuli in various cell types and participates in immune defense against exogenous pathogens (Mayer & Hemmens, 1997), eNOS responds to inflammatory 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 confirm 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 first 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 Biomédicas’’, Facultad de Ciencias de la Salud, Universidad de Talca for partial funding. This study was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 11070035) Chile,

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