journal of functional foods 27 (2016) 189–200

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In vitro studies of an aged black extract enriched in S-allylcysteine and polyphenols with cardioprotective effects

A.L. García-Villalón a, S. Amor a, L. Monge a, N. Fernández a, M. Prodanov b, M. Muñoz c, A.M. Inarejos-García c, M. Granado a,* a Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain b Departamento de Química Física Aplicada, Facultad de Ciencias, CIAL (CEI, CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain c Pharmactive Biotech Products SL, Parque Científico de Madrid, Madrid, Spain

ARTICLE INFO ABSTRACT

Article history: Aged black garlic (ABG) exerts metabolic and cardiovascular beneficial effects. The aim of Received 1 April 2016 this work was to analyse the in vitro cardiovascular effects of an ABG extract enriched in Received in revised form 22 August S-allyl-cysteine and polyphenols (ABG10+) in Sprague-Dawley rats. Hearts were pre- 2016 treated either with ABG10+ or vehicle and subjected to 30 min ischaemia followed by 45 min Accepted 29 August 2016 reperfusion (IR) using the Langendorff technique. Segments of aorta and tail artery were Available online used for inflammation/oxidative stress and vascular reactivity experiments respectively in presence/absence of ABG10+. ABG10+ induced a nitric oxide (NO) dependent vasodilating Keywords: effect in tail artery segments and directly increased the release of NO in aorta segments. + Aged black garlic In the heart, ABG10 induced a relaxing effect on coronary arteries before and after IR and Ischaemia–reperfusion prevented the IR induced decrease in myocardial contractility. Functional changes were as- Nitric oxide sociated with increased expression of both pro- and antioxidant and pro- and anti- Antioxidant inflammatory markers in the myocardium and in aorta. Anti-inflammatory © 2016 Elsevier Ltd. All rights reserved.

Allicin is highly unstable at pH close to neutral values, at high 1. Introduction temperatures or in the presence of oils and rapidly degrades during processing or during storage, limiting its bioaccessibility Raw garlic has long been used as traditional medicine for treat- (Kim, Nam, Rico, & Kang, 2012). ing a diverse range of human diseases, due to the presence Concerning toxicity, raw garlic must be consumed in mod- of several bioactive components with proven beneficial effects eration because it might be toxic at high doses (Bae, Cho, Won, (Bautista et al., 2005; Rees, Minney, Plummer, Slater, & Skyrme, Lee, & Park, 2014; Kodera et al., 2002). In addition to toxicity, 1993). Among them, the most studied and relevant is the consumption of unprocessed raw garlic is limited due to and its derivatives; allicin is the major thiosulfinate in fresh its characteristic odour, taste and tendency to cause an upset crushed garlic, which is responsible for its characteristic taste stomach (Kodera et al., 2002). Therefore, in recent years, various and aroma (Rosin, Tuorila, & Uutela, 1992; Salazar et al., 2008). processing methods such as heat treatment, ageing and

* Corresponding author. Department of Physiology, Faculty of Medicine, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo n°2, 28029 Madrid, Spain. Fax: +34 91 497 5478. E-mail address: [email protected] (M. Granado García). http://dx.doi.org/10.1016/j.jff.2016.08.062 1756-4646/© 2016 Elsevier Ltd. All rights reserved. 190 journal of functional foods 27 (2016) 189–200

fermentation have been used to eliminate the offensive odour 1982) and hypertension (Zhang et al., 2001); therefore these dis- and improve garlic palatability. Heat treatment is the most fre- eases may be improved and/or prevented by garlic products. quently used processing method to improve the taste and Raw garlic extract reduces infarct size in isolated rabbit flavour of garlic. During heating, non-enzymatic browning hearts (Sharma et al., 2012) and rat hearts (Banerjee, Dinda, (Maillard reaction) produces some physical–chemical changes Manchanda, & Maulik, 2002), although high doses were car- such as colour, flavour, texture, macronutrients and minor com- diotoxic and improved oxidative myocardial damage (Ku et al., pounds content (Nencini, Menchiari, Franchi, & Micheli, 2011), 2002). Moreover, it produces arterial dilatation mediated by nitric which results in the production of aged black garlic. oxide (Benavides et al., 2007; Grman et al., 2011) or hydrogen The general procedure to obtain black garlic is by heating sulfide (Chuah, Moore, & Zhu, 2007). These beneficial effects the whole raw garlic bulb at a high temperature and con- might be enhanced in aged garlic, as SAC present in ABG10+ trolled humidity for approximately 1 month. Black garlic is reported to induce myocardial protection during isch- has a rubbery texture and during the browning process the aemia (Numagami, Sato, & Ohnishi, 1996). Indeed, aged garlic soluble solids content (˚Brix) increases and the taste becomes has been shown to protect against ischaemia–reperfusion injury sweet. The pH of the product also decreases from approxi- in rat brain (Augusti, 1996), but the effects of this particular mately 6 in raw garlic to less than 3.8 in aged black garlic; preparation have not been studied during cardiac ischaemia. this effect together with the high processing temperature The preventive and/or therapeutic use of garlic products on explains the longer shelf life of the product (Nencini et al., the cardiovascular system needs to be improved by a better 2011; Toledano-Medina, Perez-Aparicio, Moreno-Rojas, & characterization of their effects as there is evidence that Merinas-Amo, 2016). Another important change during the there may be undesirable effects at high doses (Banerjee et al., ageing process is the increase in polyphenols content (Kim et al., 2001; Egen-Schwind, Eckard, & Kemper, 1992; Joseph, Rao, & 2011; Park, Park, & Park, 2009), and consequently also in its an- Sundaresh, 1989; Nakagawa, Masamoto, Sumiyoshi, Kunihiro, tioxidant capacity (Nencini et al., 2011). & Fuwa, 1980; Robards, Prenzler, Tucker, Swatsitang, & Glover, On the other hand, the increased antioxidant capacity is 1999). also due to the transformation of some of the unstable and The chemical characterization of SAC and its derivatives is odorous components of raw garlic into stable and odourless still a challenge to be overcome in aged black garlic, mainly due compounds during ageing process, mostly organosulfur com- to its complexity and the very limited absorption of ultraviolet pounds such as S-allylcysteine (SAC) (Lee et al., 2009). SAC is (UV) light of these compounds, 205–210 nm. This is the range, a water-soluble bioactive compound known for its high anti- where the most common solvents used in reversed phase HPLC, oxidant capacity (Bae, Cho, Won, Lee, & Park, 2012) formed acetonitrile and/or methanol, also absorb. In addition, fine sepa- during the enzymatic hydrolysis of γ-glutamyl-S-allylcysteine, ration of these compounds requires the use of salt-containing catalyzed by γ-glutamyl transpeptidase (γ-GTP, EC 2.3.2.2). Its mobile phases, which is a serious problem in HPLC mainte- content is about 20–30 μg/g in raw garlic, and rises up to six nance and mass detection (Beato, Sanchez, de Castro, & Montano, times after the ageing process (Bae et al., 2014; Hanum, Sinha, 2012). This is why most of the analytical techniques developed Guyer, & Cash, 1995). Nevertheless, the γ-GTP activity is af- for determination of amino acids are based on their derivatization fected by heat (Munday, James, Fray, Kirkwood, & Thompson, to chromophores (o-phthaldialdehyde, OPA) or fluorophores (9- 1999), and therefore high temperatures can limit its forma- fluorenylmethyl chloroformate, FMOC or 6-aminoquinolyl-N- tion during ageing. hydroxysuccinimidyl carbamate, AQC) (Bae et al., 2012; Cronin, Several studies have reported that SAC has cardioprotective Pizzarello, & Gandy, 1979; Gartenmann & Kochhar, 1999). effects (Banerjee, Mukherjee, & Maulik, 2003; Ried, Frank, & However, while they considerably improve the detection Stocks, 2010; Steiner, Khan, Holbert, & Lin, 1996) although the selectivity and sensitivity, they are also time-consuming, labo- mechanisms involved are not completely understood. As re- rious and/or require specific instrumentation for carrying out active oxygen species (ROS) play a central role in these a pre- or post-column derivatization, which often leads to low alterations, research has focused on the antioxidant proper- reproducibility. Consequently, it is necessary to develop a more ties of garlic products (Yamasaki, Li, & Lau, 1994). simple and efficient methodology for the assessment of these The wide range of beneficial effects of aged black garlic molecules. include the protection of vascular endothelial cells against hy- Therefore, in this study we have used a newly developed drogen peroxide induced injury (Reeve, Bosnic, Rozinova, & fast methodology for the determination of SAC and its main Boehm-Wilcox, 1993), protection against damage caused by ion- derivatives in an aged black garlic extract ABG10+ and analysed izing radiation (Kojima, Toyama, & Ohnishi, 1994)ortoxic its in vitro effects on isolated rat arteries and isolated per- substances (Alkreathy et al., 2010; Lee et al., 2011), inhibition fused rat hearts during coronary ischaemia–reperfusion. of oxidative and inflammatory markers (Weiss, Papatheodorou, Morihara, Hilge, & Ide, 2013) and an increase in nitric oxide availability in endothelial cells (Munday et al., 1999) are re- 2. Materials and methods ported. In addition, aged garlic has enhanced metabolic beneficial effects compared to row garlic, for example, de- Aged Black Garlic Extract ABG10+ was obtained from creasing LDL oxidation and thus preventing atherosclerosis Pharmactive Biotech Products SL (Madrid, Spain); eight samples (Arora, Arora, & Gupta, 1981; Ide & Lau, 1999). from different batches during the year 2015 were used in this Oxidative damage is involved in many cardiovascular dis- study. eases, including heart disease (Kiesewetter et al., 1993), Folin–Ciocalteu reagent, gallic acid, sodium carbonate, peripheral arterial occlusive disease (Foushee, Ruffin, & Banerjee, S-allylcysteine, U46619 (9,11-dideoxy-1a,9a-epoxymethanopros- journal of functional foods 27 (2016) 189–200 191

taglandin F2α) and L-NAME (N-omega-nitro-L-arginine methyl 2.2. Methods to test the in vitro cardiovascular effects of ester) were purchased from Sigma-Aldrich (Madrid, Spain); ABG10+ in experimental animals heptanesulfonic acid sodium salt and orthophosphoric acid were from Fisher Scientific (Santa Clara, CA, USA). HPLC sol- In the present study male Sprague-Dawley rats with a body vents were from Merck (VWR International, Spain). Ultrapure weight of 300–350 g were used. All the experiments were con- water for chromatographic use was obtained from a MilliQ ducted in accordance with the US National Institutes of Health system (Millipore Corp., Bedford, MA, USA). U46619 (9,11- Guide for the Care and Use of Laboratory Animals and in com- dideoxy-1a,9a-epoxymethanoprostaglandin F2α) and L-NAME pliance with all relevant laws and regulations. The use of these (N-omega-nitro-L-arginine methyl ester) were obtained from animals was also approved by the Animal Care and Use Com- Sigma-Aldrich (Madrid, Spain). mittee of the Universidad Autónoma de Madrid. After anaesthesia with sodium pentobarbital (100 mg/kg i.p.) and fol- lowing i.v. injection of heparin (1000 UI), the heart, the aorta 2.1. Analytical characterization of ABG10+ extract and the tail artery were collected.

2.1.1. Determination of total phenol content Total phenol content of ABG10+ was ascertained by colorim- 2.2.1. Perfused hearts etry employing the Folin–Ciocalteu reagent (Singleton & Rossi, Immediately after removal of the heart, the ascending aorta 1965). Briefly, ABG10+ was dissolved in water and mixed with was cannulated and the heart was subjected to retrograde per- Folin–Ciocalteu reagent. After 3 min a sodium carbonate so- fusion with Krebs–Henseleit buffer (115 mM NaCl, 4.6 mM KCl, lution was added. Total phenols were determined after a 2 h 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3 incubation at room temperature. The absorbance of the re- and 11 mM glucose) equilibrated with 95% oxygen and 5% sulting blue colour solution was measured at 765 nm with a carbon dioxide to a pH of 7.3–7.4. Perfusion was initiated in a Beckman Coulter DU Series 730 Life Science UV/Vis Scanning non-recirculating Langendorff heart perfusion apparatus at a Spectrophotometer (Fullerton, CA, USA). The quantification of constant flow rate of 11–15 mL/min to provide a basal perfu- total phenol content was expressed as grams of gallic acid per sion pressure of approximately 70 mmHg. Both the perfusion 100 grams of ABG10+ (dry basis). solution and the heart were maintained at 37 °C. Coronary per- fusion pressure was measured through a lateral connection in the perfusion cannula and left ventricular pressure was mea- 2.1.2. HPLC analysis (SAC quantification) sured using a latex balloon inflated to a diastolic pressure of The exclusive quantification of SAC in ABG10+ by HPLC was 5–10 mmHg, both connected to Statham transducers. Left ven- initially performed according to Bae et al. (2012) employing an tricular developed pressure (systolic left ventricular pressure Agilent Technologies 1220 Infinity series system (Palo Alto, CA, minus diastolic left ventricular pressure), left ventricular end- USA) equipped with autosampler and photo-diode array de- diastolic pressure, the first derivative of the left ventricular tector; the separation of SAC was carried out on a C18-PFP Ultra- pressure curve (dP/dt) and heart rate were calculated from the Inert HPLC Column (250 × 4.6 mm × 5 μm; ACE, Scotland). left ventricular pressure curve. These parameters were re- corded on a computer using the PowerLab/8e data acquisition system (AD instruments). 2.1.3. HPLC-PAD-MS assessment After a 30 min equilibration period with constant flow per- For the identification and quantitative assessment of SAC to- fusion, ABG10+ at concentrations of 50–500 mg/L was added gether with its derivatives, a simple HPLC-MS method with to the perfusion solution, and the hearts were perfused for a direct injection of the sample (without any purification and/ further 30 min. After 30 min perfusion with ABG10+, the hearts or derivatization step) was developed. The chromatographic were exposed to global zero-flow ischaemia for 30 min, and system was an Agilent 1200 series, (binary pump, autosampler, reperfused for 45 min at the same flow rate used before photodiode array detector), coupled to an electrospray ioniza- ischaemia. Coronary perfusion pressure, left developed ven- tion (ESI) quadrupole mass analyser. The stationary phase was tricular pressure, dP/dt and heart rate were measured before a Liquid Purple ODS column from Análisis vinicos, S.L. and after ischaemia–reperfusion, in the absence and in the pres- (Tomelloso, Spain) (250 × 4.6 mm and 5 μm particle size), at ence of ABG10+. After the experiment, the hearts were removed ambient temperature. The mobile phase was pumped at a flow from the perfusion system and stored at −80 °C for further rate of 0.5 mL/min and was a linear gradient of component A analysis. (0.1% (v/v) formic acid in water) and component B (acetoni- trile), as follows: from 0 to 20 min, 10 to 90% of B; for 10 min, 90% of B; from 90 to 10% of B at 1 min and 10 min at 90% B 2.2.2. Tail arteries for conditioning the column for the next analysis. The PAD was After collection, tail arteries were cut into 2 mm long seg- set at 208 nm and the injection volume was 20 μL. The total ments and each segment was prepared for isometric tension run time was 41 min. ESI(MS) was tuned as follows: mass recording in a 4-mL organ bath containing modified Krebs– range (SCAN): from 50 to 1500 umas, ionization mode: ESI+, Henseleit solution at 37 °C (mM): NaCl, 115; KCl, 4.6; KH2PO4, drying gas flow: 9 L/min, nebulizer pressure: 60 psig, drying gas 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25; glucose, 11. The solu- temperature: 250 °C, vaporizer temperature: 150 °C, capillary tion was equilibrated with 95% oxygen and 5% carbon dioxide tension: 2000 V, charging tension: 2000 V, fragmentator tension: to a pH of 7.3–7.4. Briefly, two fine 100 μm diameter steel wires 40 V. were passed through the lumen of the vascular segment; one 192 journal of functional foods 27 (2016) 189–200

wire was fixed to the organ bath wall and the other was con- Rn01525859_g1), interleukin 10 (IL-10; Rn01483988_g1), nected to a strain gauge for isometric tension recording NADPH oxidase 1 (NOX1; Rn00586652_m1), NADPH oxidase (Universal Transducing Cell UC3 and Statham Microscale Ac- 4 (NOX4; Rn00585380_m1), glutathione peroxidase 3 (GPX3; cessory UL5; Statham Instruments, Inc, Oxnard, CA, USA). This Rn00574703_m1), glutathione reductase (GSR; Rn01482159_m1) arrangement enables the application of passive tension in a and superoxide dismutase-1 (SOD1; Rn00566938_m1) was plane perpendicular to the long axis of the vascular cylinder. assessed in myocardial and arterial tissue by quantitative The changes in isometric force were recorded using a PowerLab real-time PCR. Quantitative real-time PCR was performed data acquisition system (ADInstruments). An optimal passive using assay-on-demand kits (Applied Biosystems) for each tension of 1 g was applied to the vascular segments and then gene. TaqMan Universal PCR Master Mix (Applied Biosystems) they were allowed to equilibrate for 60–90 min. Before begin- was used for amplification according to the manufacturer’s ning the experiment, the vascular segments were stimulated protocol in a Step One machine (Applied Biosystems). Values with potassium chloride (100 mM) to determine the contrac- were normalized to the housekeeping gene 18S (Rn01428915_g1). tility of smooth muscle, and any segments which failed to The ΔΔCT method was used to determine relative expression contract at least 0.5 g were discarded. levels. Statistics were performed using ΔΔCT values (Livak & After equilibration, the relaxation to ABG10+ (10–1000 mg/L) Schmittgen, 2001). was recorded in the segments precontracted with the throm- boxane A2 analogue U46619. First, 10-8 M U46619 was added 2.3. Statistical analysis to the bath, and when the contraction reached a stable level, + ABG10 (10–1000 mg/L) was added cumulatively, and the re- The parameters measured are expressed as means ± S.E.M., and + laxation recorded. The relaxation to ABG10 was also recorded compared in the absence and in the presence of ABG10+ using in the presence of an inhibitor of nitric oxide synthase L-NAME the unpaired Student’s t test. A P value of <0.05 was consid- (10-4 M), which was added to the bath 20 min before the ered statistically significant. concentration–response curve.

2.2.3. Incubation of aorta segments Aorta was carefully dissected from each animal and cut in 2 mm 3. Results segments. Segments were placed in 24 well plates (1 segment 3.1. Bioactive components analysis per well) and incubated for 24 hours (37 °C, 5% CO2) either with culture medium DMEM-F12 or with ABG10+ at two different + concentrations: 50 mg/L and 500 mg/L. After 24-hour incuba- The total phenol content (TPC) of the ABG10 samples analysed tion both culture medium and segments were collected and by Folin–Ciocalteu (as gallic acid, dry basis) was homoge- immediately frozen and stored at −80 °C to further analyse ni- neous (Table 1). The analysis was performed on eight different trites and nitrates concentrations and the gene expression of batches to establish the corresponding percentiles distribu- different genes in arterial tissue. tion (P25, P50, P75), the minimum (P0) and the maximum (P100) with 3.41 and 3.65 mg/100 mg ABG10+ respectively (Table 1). 2.2.4. Nitrite and nitrate determination in the During ageing the TPC increases due to the higher levels of culture medium complex polyphenols from the later phase of the browning re- Nitrite and nitrate concentrations were measured in the culture action as suggested by Robards et al. (1999), which together with medium by a modified Griess assay, described by Miranda, the SAC content are responsible for the antioxidant capacity Espey, and Wink (2001). Briefly, 100 μL of vanadium chloride was (Lee et al., 2009). added to 100 μL of culture medium in 96-well plate. Immedi- Likewise SAC concentration throughout the different batches ately after Griess reagent (1:1 mixture of 1% sulfanilamide, and analysed by HPLC was also homogeneous (Table 1); the per- 0.1% naphthylethylenediamine dihydrochloride) was added to centile distribution shows that the SAC content was within 0.15 + each well and incubated at 37 °C for 30 min. The absorbance and 0.17 mg/100 mg ABG10 . The increase in SAC and TPC in was measured at 540 nm. Nitrite and nitrate concentration was aged black garlic when compared to raw garlic agrees with the findings of other authors (Park et al., 2009; Toledano-Medina calculated using a NaNO2 standard curve and expressed as μM. et al., 2016). 2.2.5. RNA preparation and purification In order to confirm the presence of SA and its derivatives, Total RNA was extracted from myocardial and arterial tissue and to perform the corresponding quantitative assessment, a according to the Tri-Reagent protocol (Chomczynski, 1993). cDNA was then synthesized from 1 μg of total RNA using a high ca- pacity cDNA reverse transcription kit (Applied Biosystems, Foster Table1–Total phenol content (TPC) by Folin–Ciocalteu City, CA, USA). (mg GA/100 mg, dry basis) of ABG10+ and S-allyl cysteine (SAC) by HPLC analysis (mg/100 mg, dry basis) 2.2.6. Quantitative real-time PCR of ABG10+ analysed according to Bae et al. (2012). The gene expression of inducible nitric oxide synthase (iNOS; Average P0 P25 P50 P75 P100 Rn00561646_m1), endothelial nitric oxide synthase (eNOS; TPC 3.41 ± 0.21 3.09 3.17 3.46 3.59 3.65 Rn02132634_s1), cyclooxygenase-2 (COX-2; Rn01483828_m1), SAC 0.15 ± 0.03 0.11 0.12 0.14 0.15 0.17 interleukin 1β (IL-1β; Rn00580432_m1), interleukin 6 (IL-6; GA: gallic acid. Rn01489669_m1), tumoral necrosis factor alpha (TNF-α; journal of functional foods 27 (2016) 189–200 193

Fig. 1 – Ultraviolet (UV) chromatogram of the aged black garlic extract, registered at 208 nm, the total ion current and extractions of the ion current, corresponding to the ions of m/z 162, 291 and 265.

new simple HPLC-MS method was developed. This included a Furthermore, the SAC reference solution was diluted to common octadecylsilane stationary phase, a binary water/ obtain different concentrations and a calibration curve was acetonitrile mobile phase and 1% formic acid, as an ionization plotted according to the previously described HPLC-MS/ESI agent. The SAC reference solution was injected directly into the method. The results showed a concentration between ESI source and the ionization conditions were tuned as de- 0.26 and 0.34 mg SAC per 100 mg ABG10+. The other com- scribed above. PAD was used only for the qualitative control of pounds detected such as iso-SAC, GSAC and GSMC had the separation. The identification and quantification of SAC and concentrations below the quantification limit of the detector its derivatives was carried out on the basis of their MS spec- (1.1 ppm). tral characteristics. The ability of the MS detector to extract specific ions enabled their selective identification. Fig. 1 shows the UV chromatogram of ABG10+ analysed by HPLC-PAD-MS, reg- 3.2. Effects of ABG10+ in perfused hearts istered at 208 nm, the total ion current, and extractions of the ion current, corresponding to the ions of the most important Treatment of the perfused rat heart with ABG10+ at 50 mg/L sulfur-containing amino acids (S-allyl-L-cysteine (SAC) (m/z 162.7), or 500 mg/L induced coronary vasodilatation with a reduc- γ-L-glutamyl-S-allyl-L-cysteine (GSAC) (m/z 291.0) and γ-L-glutamyl- tion in perfusion pressure, and an increase in the left ventricle S-methyl-L-cysteine (GSMC) (m/z 265)). This figure illustrates that developed pressure and dP/dt (Table 2), without modifying the the extraction of ion m/z 162 showed responses at 7.4 and heart rate. The changes in the left ventricle developed pres- 8.8 min, corresponding to SAC and its isomer iso-S-allyl-L- sure and dP/dt were transitory and returned to basal values cysteine (iso-SAC), respectively. The extraction of ion m/z 291 after 30 min of treatment, whereas the reduction in the coro- also revealed the presence of two peaks at 11.1 and 11.6 min, nary perfusion pressure persisted. After ischaemia–reperfusion, which correspond to GSAC and its isomer γ-L-glutamyl-S-(trans- coronary perfusion pressure increased in untreated hearts, but 1-propenyl)-L-cysteine (GSPC), respectively.The extraction of ion not in the hearts treated with ABG10+ at a dose of 50 mg/L m/z 265 showed no detectible response for GSMC. (P < 0.05). The left intraventricular developed pressure and dP/dt 194 journal of functional foods 27 (2016) 189–200

Table 2 – Haemodynamic parameters of perfused rat hearts before and after ischaemia–reperfusion treated with vehicle (n = 6) or with ABG10 50 mg/L (n = 6) or 500 mg/L (n = 4). Control Treated Ischaemia–reperfusion 15 min 30 min 45 min

Coronary perfusion pressure (mmHg) Vehicle 70 ± 373± 569± 479± 4* 83 ± 5* ABG10 50 mg/L 75 ± 247± 3**†† 63 ± 563± 5† 64 ± 6† ABG10 500 mg/L 75 ± 353± 4*† 82 ± 684± 685± 7 Left ventricle developed pressure (mmHg) Vehicle 114 ± 7 106 ± 761± 14* 89 ± 8* 93 ± 6* ABG10 50 mg/L 108 ± 9 103 ± 673± 11 101 ± 3 105 ± 4 ABG10 500 mg/L 107 ± 11 163 ± 3*†† 48 ± 16 94 ± 15 112 ± 10 dP/dt (mmHg/s) Vehicle 2670 ± 167 2625 ± 173 1196 ± 293** 1850 ± 222* 2100 ± 178* ABG10 50 mg/L 2811 ± 233 2789 ± 123 1369 ± 247* 2352 ± 247 2664 ± 140† ABG10 500 mg/L 2812 ± 197 3687 ± 400† 702 ± 287* 1637 ± 423 2375 ± 233 Values are means ± S.E.M. *P < 0.05 and **P < 0.01 compared with control, †P < 0.05, ††P < 0.01 compared with vehicle.

were reduced after ischaemia–reperfusion in both treated and treated with ABG10+ at a dose of 500 mg/L, coronary perfu- untreated hearts, but dP/dt was significantly higher (P < 0.05) sion pressure, the left intraventricular developed pressure and in the hearts treated with ABG10+ 50 mg/L after 45 min of dP/dt after ischaemia–reperfusion were not improved com- reperfusion, compared with untreated hearts. In the hearts pared with the untreated hearts.

A 400 B C 200 200 ** * 300 150 150

200 100 100

100 50 50 iNOS/18S (% overiNOS/18S (% control values) COX-2/18S (% over (% control values)COX-2/18S eNOS/18S (% overeNOS/18S (% control values) 0 0 0

IR /L) IR L) IR g / 0mg/L) (50m (5 (50mg 10 G10 AB IR+ABG IR+ IR+ABG10 DFE 200 * 200 ** 200

150 150 150

100 100 100

50 50 50 /18S (% over control values) control over (% /18S /18S (% over control values) control over (% /18S IL-6/18S (% overIL-6/18S (% control values) IL-1 TNF- 0 0 0 ) ) IR L IR L IR

(50mg/ (50mg/ 0 10 (50mg/L) G10 G ABG1 AB AB IR+ IR+ IR+

Fig.2–Myocardial gene expression of inducible nitric oxide synthase (iNOS) (A), endothelial nitric oxide synthase (eNOS) (B), cyclooxygenase-2 (C), interleukin 1β (IL-1β) (D), interleukin 6 (IL-6) (E) and tumoral necrosis factor alpha (TNF-α) (F) in non-treated hearts (Control; n = 6) and ABG10+ (50 mg/L) pre-treated hearts (n = 6) after ischaemia–reperfusion (IR). Values are represented as mean ± SEM.* P < 0.05 vs control; **P < 0.01 vs control. journal of functional foods 27 (2016) 189–200 195

3.3. Cell death in myocardial tissue ABG10+ treated hearts compared to untreated hearts (P < 0.05 and P < 0.01 respectively). Likewise the myocardial mRNA levels ABG10+ (50 mg/L) administered before ischaemia and during of IL-1β and IL-6 were also increased in ABG10+ treated hearts 45 min of reperfusion significantly decreased ischaemia– compared to non-treated ones (P < 0.05 and P < 0.01 respec- reperfusion induced cell death in the myocardium (IR = 100 ± 18; tively), whereas the gene expression of COX-2 and TNF-α was IR − ABG10+=50 ± 9); P < 0.05). unchanged between groups.

3.4. Gene expression of cytokines and enzymes involved 3.5. Gene expression of pro-oxidative and anti-oxidative in the production of vasoactive substances and inflammatory enzymes and cytokines in myocardial tissue factors in myocardial tissue The mRNA levels of pro-oxidative enzymes such as NADPH The gene expression of inducible nitric oxide synthase (iNOS) oxidase 1 (NOX1) and NADPH oxidase 4 (NOX4) and anti- and endothelial nitric oxide synthase (eNOS) as producers of oxidative enzymes such as glutathione peroxidase 3 (GPX3), nitric oxide, and cyclooxygenase-2 (COX-2) as a producer of glutathione reductase (GSR) and superoxide dismutase-1 (SOD1) prostanoids was measured in myocardial tissue after IR both were analysed in hearts treated and untreated with ABG10+ in non-treated hearts and in hearts treated with ABG10+ before before ischaemia and during reperfusion (Fig. 3A, 3B, 3C, 3D ischaemia and during reperfusion (Fig. 2A, 2B and 2C respec- and 3F respectively). In addition the gene expression of the anti- tively). In addition the gene expression of different pro- inflammatory cytokine interleukin 10 (IL-10) was also measured inflammatory cytokines such as interleukin 1β (IL-1β), (Fig. 3E). Treatment with ABG10+ induced an overexpression interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) in the mRNA levels of NOX-1, NOX-4, GSR and SOD1 (P < 0.05 was also measured (Fig. 2D, 2E and 2F respectively). There was for all), whereas the gene expression of GPX3 and IL-10 was an up-regulation of both iNOS and eNOS gene expression in not modified.

A B C 500 250 * 150 * 400 200

100 300 150

200 100 50

100 50 GPX3/18S (% over control values) control over (% GPX3/18S NOX-4/18S (% overNOX-4/18S (% control values) NOX-1/18S (% overNOX-1/18S (% control values) 0 0 0

IR IR IR /L) g/L) 50mg/L) (50m (50mg 0 1 G

+ABG10 IR IR+ABG10 ( IR+AB

DF200 E 150 200 * 150 150 * 100

100 100

50 50 50 IL-10/18S (% overIL-10/18S (% control values GSR/18S (% overGSR/18S (% control velues) SOD1/18S (% overSOD1/18S (% control values) 0 0 0 ) ) IR IR L IR /L

0mg/ 0mg 5 (50mg/L) ( (5 0 G1 ABG10 IR+ IR+ABG10 IR+AB

Fig.3–Myocardial gene expression of NADPH oxidase 1 (NOX1) (A), NADPH oxidase 4 (NOX4) (B), glutathione peroxidase 3 (GPX3) (C), glutathione reductase (GSR) (D), interleukin 10 (IL-10) (E) and superoxide dismutase (SOD) (F) in non-treated hearts (Control; n = 6) and ABG10+ (50 mg/L) pre-treated hearts (n = 6) after ischaemia–reperfusion (IR). Values are represented as mean ± SEM. * P < 0.05 vs control. 196 journal of functional foods 27 (2016) 189–200

A B

0 * 20

25 * *** 15 *** (µM)

50 3 10 +NO 2

75 NO 5 Control Relaxation initial (% tone) L-NAME 100 310301003001000 0 ) ABG10 (mg/L) /L) trol g Con (50 m 0

ABG1 ABG10 (500 mg/L

Fig. 4 – (A) Relaxation of tail artery segments to ABG10+ (3–1000 mg/L) in presence/absence of N-ω-Nitro-L-arginine methyl ester (L-NAME) 10-4 M and (B) nitrites release from aorta segments to culture medium in presence/absence of ABG10+ (50 and 500 mg/L). (n = 6 segments from 6 different rats.) Values are represented as mean ± SEM.* P < 0.05 vs control; ***P < 0.001 vs control.

3.6. Vasoactive response to ABG10+ in tail arteries and 3.8. Gene expression of pro-oxidative and anti-oxidative release of nitrites and nitrates enzymes and cytokines in arterial tissue

ABG10+ (30–1000 mg/L) induced a concentration-dependent re- The mRNA levels of NOX-1, NOX-4, GPX3 and GSR were un- laxation in isolated rat tail arteries, which was reduced by the changed between groups (Fig. 6A, 6B, 6C and 6D respectively). inhibition of nitric oxide synthesis with L-NAME (Fig. 4A; However IL-10 and SOD1 gene expression was significantly in- P < 0.05). In addition two different doses of ABG10+ (50 and creased in aorta segments incubated with ABG10+ 500 (Fig. 6E; 500 mg/L) significantly increased the release of nitrites and ni- P < 0.01) and ABG10+ 50 mg/L respectively (Fig. 6F;P< 0.05). trates when incubated with aorta segments for 24 hours (Fig. 4B; P < 0.001 for both). 4. Discussion 3.7. Gene expression of cytokines and enzymes involved in the production of vasoactive substances and inflammatory The results of the present study suggest that the aged garlic factors in arterial tissue extract ABG10+ exerts beneficial effects on the cardiovascu- lar system. In the systemic circulation, ABG10+ increased the Fig. 5 shows the gene expression of iNOS, eNOS, COX-2, IL-1β, production of nitric oxide in the aorta and produced relax- IL-6 and TNF-α in rat aorta segments incubated either with ation of tail arteries mediated, at least in part, by nitric oxide. vehicle (control) or either with two different doses of ABG10+ This agrees with previous studies showing that garlic induces (50 and 500 mg/L) for 24 h. The gene expression of eNOS and nitric oxide release (Kim et al., 2001; Weiss et al., 2013) and TNF-α was not modified in segments incubated with ABG10+ endothelium-dependent relaxation in rat pulmonary arteries compared to controls. On the contrary iNOS (Fig. 5A) and COX-2 (Ku et al., 2002) and aorta (Grman et al., 2011). Nitric oxide exerts (Fig. 5C) gene expression was significantly increased in arte- different beneficial effects on the vascular system protecting rial tissue after incubation with ABG10+ at both doses (P < 0.05 against atherosclerosis, hypertension and other pathologies and P < 0.001, and P < 0.05 for both). In addition IL-1β and IL-6 (Desjardins & Balligand, 2006). were also upregulated when incubated with ABG10+ 500 and Since myocardial ischaemia is the most important cause ABG10+ 50 mg/L respectively (Fig. 5D and 5E;P< 0.001 for both). of cardiovascular disease in developed countries, we have journal of functional foods 27 (2016) 189–200 197

A B C 500 150 200 * *

400 *** 150 100 300

* 100

200 50 50 100 iNOS/18S (% over control values) control over (% iNOS/18S eNOS/18S (% overeNOS/18S (% control values) COX-2/18S (% over (% control values)COX-2/18S 0 0 0

ol rol mg/L) mg/L) ontr mg/L) mg/L) Control C Cont 0 00 00 mg/L) (50 (5 0 (500 mg/L) 1 0 G10 (50 10 (5 ABG AB ABG10 ABG10 (5 ABG1 ABG DFE 250 150 ** 200

200 ** 150 100 150

100

100 50 50

50 values) control over (% /18S IL-6/18S (% over control values) control over (% IL-6/18S IL-1 /18S (% values)over control TNF- 0 0 0 l ) ) L /L) g mg/L) mg/L) mg/L mg/L) Contro Control 0 Control 0 00 00 (5 (5 (5 0 (500 m 0 (5 1 0 0 G 1 G1 ABG10 (50 mg/ AB ABG1 ABG10 ABG AB

Fig. 5 – Gene expression of inducible nitric oxide synthase (iNOS) (A), endothelial nitric oxide synthase (eNOS) (B), cyclooxygenase-2 (C), interleukin 1β (IL-1β) (D), interleukin 6 (IL-6) (E) and tumoral necrosis factor alpha (TNF-α) (F) in aorta segments in presence/absence of ABG10+ (50 and 500 mg/L). (n = 6 segments from 6 different rats.) Values are represented as mean ± SEM.* P < 0.05 vs control; ***P < 0.001 vs control.

studied the effect of two different doses of ABG10+ on the iso- is not beneficial or even toxic at higher doses (Banerjee et al., lated heart during ischaemia–reperfusion. After ischaemia– 2001; Egen-Schwind et al., 1992). reperfusion a concentration of 50 mg/L ABG10+ improved The cardioprotective effect of ABG10+ may be related, at least myocardial contractility as assessed by dP/dt. This concentra- in part, to an improvement in coronary blood flow before and tion of ABG10+ also reduced cellular death in the myocardium after ischaemia–reperfusion, as coronary vascular resistance after ischaemia–reperfusion. The cardioprotective effect of garlic in perfused hearts was lower in hearts treated with ABG10+ has previously been reported. Garlic extract reduced infarct size compared with untreated hearts. One of the pathophysiologi- and enhanced the protective effect of preconditioning in per- cal mechanisms of heart damage after ischaemia is the fused rat or rabbit (Zhang et al., 2001) hearts, although higher phenomenon of “no-reflow”, by which coronary blood flow doses were toxic (Sharma et al., 2012). Similarly our results show remains reduced during reperfusion (Bouleti, Mewton, & that the effects of ABG10+ in the perfused heart were concen- Germain, 2015). This was observed in the present study as an tration dependent, as no improvement in coronary perfusion increase in coronary vascular resistance after ischaemia– and myocardial contractility after ischaemia–reperfusion was reperfusion, which was abrogated by ABG10+ treatment. The observed with a higher concentration (500 mg/L). This sug- vasodilator effect of ABG10+ in coronary arteries is most likely gests that the beneficial effect of ABG10+ ameliorating due to an increase in nitric oxide production as ABG10+ ischaemia–reperfusion induced alterations in the myocardium (50 mg/L) significantly increased the gene expression of eNOS may be present at an intermediate range of concentrations. and iNOS in myocardial tissue after ischaemia–reperfusion, pos- A similar biphasic dose–response curve has been described for sibly improving coronary perfusion and myocardial protection several chemicals and agents (Mattson, 2008), including garlic in this condition. In addition it is important to point out that that is reported to exert beneficial effects at low doses but it the vasodilator effect of ABG10+ may be different depending 198 journal of functional foods 27 (2016) 189–200

A 150 B 150 C 150

100 100 100

50 50 50 GPX3/18S (% over control values) control over (% GPX3/18S NOX-1/18S (% over (% control values)NOX-1/18S NOX-4/18S (% over (% control values)NOX-4/18S 0 0 0 ) ) ol L rol L) /L) tr g/ ontrol m C Con 0 mg/ Cont 0 0 mg (5 0 (50 G1 ABG10 (50 mg/L) ABG10 (5 AB ABG10 (500 mg/L ABG10 (500 mg/L) ABG10

DF150 E 400 200 * **

300 150 100

200 100

50 100 50 GSR/18S (% over (% control values)GSR/18S IL-10/18S (% over control values) (% IL-10/18S SOD1/18S (% over (% SOD1/18S control values) 0 0 0

ol r /L) trol g ntrol g/L) m mg/L) on mg/L) mg/L) m mg/L) Cont 0 C Co 00 (50 0 (5 (500 G1 G10 B G10 (5 G10 (500 B G10 A B ABG10 (50 B A A A AB

Fig. 6 – Gene expression of NADPH oxidase 1 (NOX1) (A), NADPH oxidase 4 (NOX4) (B), glutathione peroxidase 3 (GPX3) (C), glutathione reductase (GSR) (D), interleukin 10 (IL-10) (E) and superoxide dismutase (SOD) (F) in aorta segments in presence/absence of ABG10+ (50 and 500 mg/L). (n = 6 segments from 6 different rats.) Values are represented as mean ± SEM.* P < 0.05 vs control; **P < 0.01 vs control.

on the vascular bed. Our results show that ABG10+ improved ischaemia–reperfusion injury, because raw garlic has also been coronary blood flow when administered in a low concentra- reported to exert protection against this type of injury (Banerjee tion (50 mg/L) but it only induced vasodilation in tail arteries et al., 2002; Rietz, Isensee, Strobach, Makdessi, & Jacob, 1993). when administered at high concentrations (300 and 1000 mg/L). Our results are in agreement with these previous studies This fact possibly suggests that coronary arteries are more sen- as we found that hearts treated with ABG10+ (50 mg/L) before sitive than systemic arteries to low doses of ABG10+. This may ischaemia and during 45 min of reperfusion showed in- be beneficial as therapeutic doses for the heart in terms of va- creased gene expression of the antioxidant enzymes GSR and sodilation do not produce vasodilation in systemic arteries SOD-1. However, contrary to what was expected, we also found which could induce hypotension. increased in the myocardium of ABG10+ treated hearts the As ABG10+ contains multiple compounds it is difficult to mRNA levels of different genes related to inflammation such assess which one is responsible and in what extent for its ben- as IL-1β, IL-6 and oxidative stress such as NOX-1 and NOX-4. eficial effects in the myocardium. However the protective effects Similarly aorta segments incubated for 24 hours with ABG10+ of ABG10+ increasing heart contractility after ischaemia– also presented not only increased gene expression in both anti- reperfusion are most likely mediated by SAC as this molecule inflammatory and anti-oxidative markers such as IL-10 and has been previously described as protecting against acute SOD-1 but also an upregulation of pro-inflammatory cytokines myocardial infarction (Chuah et al., 2007) by reducing the such as IL-1β and IL-6 and enzymes such as iNOS and COX-2. level of ROS and increasing the activity of antioxidant enzymes These observations may be understood by the concept of such as SOD (Xue et al., 2011). In addition, the quantity of hormesis. This term describes the phenomenon by which low SAC is much higher compared to the quantity of other levels of stressor agents may induce adaptive responses which compounds, as shown by the HPLC analysis. However we cannot have overall beneficial effects (Gems & Partridge, 2008; Mattson, exclude the possibility that compounds other than SAC could 2008). This theory might explain the favourable effects of caloric contribute to the protective effect of ABG10+ against restriction, physical exercise or ischaemic preconditioning. Also, journal of functional foods 27 (2016) 189–200 199

the phytochemicals present in fruits and vegetables activate Bautista, D. M., Movahed, P., Hinman, A., Axelsson, H. E., Sterner, stress response pathways, which may be related to the health O., Hogestatt, E. D., Julius, D., Jordt, S. E., & Zygmunt, P. M. benefits provided by a diet rich in these foods. Accordingly, al- (2005). Pungent products from garlic activate the sensory ion channel TRPA1. Proceedings of the National Academy of Sciences though in this study we have found that treatment with ABG10+ of the United States of America, 102(34), 12248–12252. increases the expression of inflammatory and oxidative doi:10.1073/pnas.0505356102. markers, it also increases antioxidant mechanisms, which may Beato, V. M., Sanchez, A. H., de Castro, A., & Montano, A. (2012). explain its protective effect during ischaemia–reperfusion. Effect of processing and storage time on the contents of organosulfur compounds in pickled blanched garlic. [Comparative Study Research Support, Non-U.S. Gov’t]. Journal of Agricultural and Food Chemistry, 60(13), 3485–3491. 5. Conclusion doi:10.1021/jf3002075. Benavides, G. A., Squadrito, G. L., Mills, R. W., Patel, H. D., Isbell, T. S., Patel, R. P., Darley-Usmar, V. M., Doeller, J. E., & Kraus, D. W. + ABG10 exerts a protective effect on the heart during ischaemia– (2007). Hydrogen sulfide mediates the vasoactivity of garlic. reperfusion, which could be due to the induction of adaptive Proceedings of the National Academy of Sciences of the United antioxidant mechanisms in the tissue. In our study ABG10+ was States of America, 104(46), 17977–17982. applied before ischaemia, suggesting that it may be useful for Bouleti, C., Mewton, N., & Germain, S. (2015). The no-reflow prevention of ischaemia–reperfusion in subjects with coro- phenomenon: State of the art. Archives of Cardiovascular nary risk factors. In addition, our results also emphasize the Diseases, 108(12), 661–674. doi:10.1016/j.acvd.2015.09.006. Chomczynski, P. (1993). 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