*2.3. Extraction and Identification of CocoaVia*® *Contents*

Total phenolic compounds of the cocoa extract supplement (Cocoavia; Mars Inc., Hackettstown, NJ, USA) made by patented process (Cocoapro; Mars Inc., Hackettstown, NJ, USA) were extracted with three volumes of ice-cold methanol (*w*/*v*)). Samples were mixed, sonicated (15 min, 4 ◦C), centrifuged (16,000× *g* rpm, 10 min, 4 ◦C), and filtered through 0.2 μm pore filters. The supernatant samples were diluted 1:100 and 1:10 (*v*/*v*) with LC-MS grade methanol for HPLC-ESI-MS. The samples were further diluted 1:2 with LC-MS grade water and passed through Minisart 0.2 μm filters following protocol [19]. Each sample was analyzed in two technical and three biological replicates, with 20 μL injection volumes. The unknown metabolites of samples were analyzed using HPLC-ESI-MS (Esquire 6000, Bruker Daltonics, Billerica, USA), followed by ESI (Electrospray Ionization)-base peaks, MS/MS, and MS3 fragmentation at its retention time and mass to charge ratio (*m*/*z*). For this purpose, an "in house" library of commercial standard spectra, scientific literature, and online databases such as Mass bank (www.massbank.jp) were used. The quantification of the metabolites in the methanolic extracts was carried out through HPLC-DAD (Beckman Coulter Gold 126 Solvent Module coupled with a Gold 168 Diode Array Detector), relying on the calibration curves of authentic standard compounds.

#### *2.4. Quantification of Polyphenols Levels in Blood Samples*

Thirty rats were divided into six groups of five rats each. Among them, group 1 served as control, fed with normal rat diet, while the animals of other groups were administrated oral gavage with 5, 10, 15, 20, and 25 mg/kg body weight of cocoa extract dissolved in water, five times per week for 15 days, respectively. All animals were kept at a temperature of 22–24 ◦C and fed with a regular pellet diet ad libitum.

A total of 1 mL of blood was collected, 1 hour after oral administration of cocoa extract powder from each rat, which showed a higher concentration of flavonoids in plasma between 30 and 60 min through lateral tail vein [19]. Blood was deposited in clean heparinized glass tubes, centrifuged (3500 rpm for 15 min), and stored at −80 ◦C for the HPLC-ESI-MS analysis. The total amount of flavonoid in blood sera from each sample were calculated by HPLC-ESI-MS [19,20].

#### *2.5. Induction of Ischemia*/*Reperfusion Injury and Tissue Collection*

Twenty rats were divided into two groups, control and treated, with ten (10) in each group. The selected dose of 15 mg/kg of cocoa from our previous results was given to the treated group (*n* = 10) once a day for 15 days. The control group remained untreated. Rats (*n* = 20) were anesthetized with 5% isoflurane in 50% O2 administered through a facial mask and maintained anesthesia using 2% isoflurane throughout procedure. These anesthetized animals were subjected to ischemia for 30 min by left anterior descending (LAD) coronary artery ligation, followed by 120 min reperfusion, as previously described [21]. The onset of ischemia was confirmed when cyanosis developed on the wall of the ischemic myocardium, as evidenced by saddleback-type (ST) segment elevation and a significant T-wave increase recorded on the electrocardiograph Power Lab data acquisition system (model ML866, AD Instruments, Colorado Springs, CO) and Animal Bio Amp (model ML136, AD Instruments)). Then, the ligation was opened to allow reperfusion to the ischemic part of the myocardium for 120 min. Hearts were excised as demonstrated in Figure 1, the ischemic part of the left ventricle was split into two: one portion was stored in 4% formalin for immunohistochemistry and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) analysis, the second portion was stored in liquid nitrogen for of nitro-oxidative stress measurements. Experimental design is illustrated in Figure 1.

**Figure 1.** Schematic experimental design.

#### *2.6. Immunohistochemistry Staining*

To access the inflammation and nitrative stress and signaling pathways activation in myocardial tissue, samples (*n* = 10 each group) were embedded in paraffin. Fixed tissues were sectioned 3 μm, deparaffinized, and dehydrated with two grades of xylene and four grades of ethanol, following the method in [22]. After antigen retrieval and endogenous peroxidase activity procedure, all sections were incubated with primary antibodies, IL-6 (1:100), NF-κB2 (1:500), Nitrotyrosine (1:300), p-Erk (1:200), and p-Akt (1:200) (Sigma–Aldrich, United Kingdom), diluted in antibody diluent and kept at 4 ◦C for overnight. Following the incubation with primary antibodies, tissue sections were rinsed, and incubated with the biotinylated anti-rabbit secondary antibody (1:400), avidin–biotin complex substrate, and diaminobenzidine (Dako Corp., Carpinteria, CA, USA). Sections were rinsed two times and mounted after dehydration in three grades of ethanol and cleared in two grades of xylene. The negative control was used to confirm and check the absence of the signal or specificity of staining. Image acquisition of all the sections was done under Olympus System BX51 Universal research microscopy (Olympus corporation, Tokyo, Japan). The images were analyzed by using ImageJ software (NIH, Bethesda, MD, USA) to quantify the strength of immune-peroxidase staining in heart tissue.

#### *2.7. Oxidative Stress Measurement*

The analysis of malondialdehyde (MDA) levels in myocardial tissue samples collected from the affected area was performed as described by Ben Maunsour et al. [23].

The reactive oxygen species (ROS) in myocardial tissue was measured using a spectrophotometry and the absorbance determined (peak at 505 nm max) value is expressed as Carr. Units, as explained by Rizzo et al. [24].
