*2.8. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay*

TUNEL assay is a method that labels 3 -hydroxyl termini in the double-stranded DNA fragments generated as a result of apoptosis. Paraffin-mounted heart sections from control and treated groups (*n* = 10) were deparaffinized with xylene and descending concentrations of ethanol. Apoptotic cells were stained following the protocol provided by the In Situ Cell Death Detection Kit, AP (ref. 11684809910, Version 11, Roche Diagnostics, Indianapolis, IN, USA). Hoechst 33342 solution was used for staining nuclei and examined under confocal microscope. The images were analyzed by ImageJ software (NIH, Bethesda, MD, USA), average mean fluorescent intensities (MFIs) were measured by multiplying the intensities of each image [25].

## *2.9. Statistical Analysis*

Analyses were performed using SPSS software version 21 (SPSS Inc., Chicago, IL, USA). Comparison between control and experimental groups was statistically evaluated by the Students t-test, and *p*-value < 0.05 was considered statistically significant. All the results were expressed as mean ± SD.

#### **3. Results**

#### *3.1. Identification of Phenolic Compounds*

A total of 250 mg of flavan-3-ols (procyanidin) was identified from 1 g of cocoa extract. From this, 19 different phenolic compounds were determined (Table 1 and Figure 2a).


**Table 1.** Phenolic molecules identified from the cocoa extract samples analyzed by chromatographic mass spectra in negative ion mode.

#### *3.2. Dose Response and Dose Adjustment*

Oral administration of cocoa extract in incremental doses from 5 mg/kg up to 25 mg/kg daily for 15 days produced a corresponding increase in blood serum polyphenols and became constant after 15 mg/kg (Figure 2B).

#### *3.3. Anti-Inflammatory E*ff*ect of Cocoa*

The extent of inflammation after I/R was measured by the expression of IL-6 and NF-kB in the myocardium. Immunohistochemistry revealed a reduced expression of IL-6 (Figure 3A,B) was significantly lowered after cocoa extract treatment 15 mg/kg (*p* < 0.01) in the ischemia-reperfusion injury model. The levels of NF-kB were remarkably low in the treated group as compared to control (Figure 3C,D).

**Figure 2.** Cocoa Extract: (**A**) shows HPLC-ESI-MS graph with various peaks correlating with 19 different phenolic compounds found in cocoa extract. (**B**) shows a curve correlating different oral doses (mg/kg) of cocoa extract and blood levels of phenolic compounds in rats. It shows an optimal dose of 15 mg/kg to be the optimal dose of cocoa extract (six groups (*n* = 30) and each group (*n* = 5)).

**Figure 3.** Immunohistochemical analysis showing expression (representative images, 20× magnification) of IL-6 and NF-kB2 in myocardial tissue of cocoa treated rats (*n* = 10) compared to control rats (*n* = 10), (\**p* = 0.0032, \*\**p* = 0.0001 respectively). presented as mean ± SD and scale bar is 100 μm. (**A**,**B**): IL-6, (**C**,**D**): NF-kB.

#### *3.4. Nitro-Oxidative Stress Attenuation with Cocoa*

Oxidative stress was studied by identifying the levels of the "lipid peroxidation index, Thiobarbituric acid reactive substances (TBARS)". TBARS expressed as malondialdehyde (MDA) levels was lower in the treated group as compared to control (Figure 4A). The concentration of ROS was also attenuated in the treated group as compared to control (Figure 4B). Nitrosative stress was assessed by measuring nitrotyrosine levels in myocardial tissue. The nitrotyrosine was reduced by cocoa treatment (*p* < 0.05) (Figure 4C,D).

**Figure 4.** Effects of cocoa on oxidative stress on myocardial tissue rats: Lower oxidative stress was recorded in cocoa-treated rats (*n* = 10) myocardial tissue as compared to control (*n* = 10). (**A**) Lipid peroxidation index (TBARS) expressed as malondialdehyde (MDA) concentration measured in nmol/μg (*p* = 0.031), \* *p* < 0.05. (**B**) Reactive oxygen species (ROS) measured as Carr. Unit (Carratelli Unit) (*p* = 0.0001), \*\* *p* < 0.001. (**C**,**D**) Expression of nitrotyrosine staining in myocardial tissue in the control and treated groups (20×) (*p* = 0.04), \* *p* < 0.05. Data illustrated in the graphs are presented as mean ± SD and scale bar is 100 μm.

#### *3.5. Akt and Erk1*/*2 Signaling Pathways Activation*

Immuno-peroxidase analysis of p-Akt and p-ERK1/2 in myocardial tissue from rats treated with cocoa extract and control group was performed. Enhanced phosphorylation of Akt (Figure 5A,B,E) and ERK1/2 (Figure 5C,D,F) was observed as compared to control group (*p* < 0.05 in both cases).

**Figure 5.** Immunohistochemical labeling of p-Akt (**A**,**B**,**E**) and p-Erk1/2 (**C**,**D**,**F**) in cocoa-treated rat myocardial tissue compared to control. The graphs show a significant elevation in the activation of p-Akt and p-Erk1/2 in the myocardial tissue of cocoa-treated rats (*n* = 10) compared to control (*n* = 10). Representative images (20×). Data illustrated in the bar graph are presented as mean ± SD. *p*-value less than 0.05 considered statistically significant. P-ERK1/2, phosphorylated extracellular signal regulated kinases <sup>1</sup> <sup>2</sup> ; p-Akt, phosphorylated serine-threonine protein kinase, (\* *p* < 0.05, \*\* *p* < 0.001).

#### *3.6. TUNEL Assay*

TUNEL-positive nuclei were less in number in the treated group (A) as compared to the control group (B). Yellow arrows indicate apoptotic nuclei (Figure 6A–C). There was a significant reduction in apoptosis in treated samples as compared to control (*p* < 0.001).

**Figure 6.** TUNEL Assay: (**A**,**B**,**C**): Representative images (*n* = 10 for each group) immunofluorescent staining for TUNEL-positive nuclei in control, treated, and negative control groups (20× magnification). TUNEL-positive myocytes were less in number in the treated group (**A**) as compared to control group (**B**). Yellow arrows indicate apoptotic nuclei and (**C**) negative control. *p*-value = 0.002 considered significant (\*\* *p* < 0.001, control versus treated), scale bar is 100 μm. (**D**): Graphical presentation of apoptotic nuclei in control vs treated groups.

#### **4. Discussion**

Cocoa has the highest flavonol contents of all foodstuffs and its extract contains a considerable concentration of proanthocyanidins [16]. Flavonoids characterize a main division of phenolic compounds, and they are greatly active scavengers of most oxidizing molecules and free radicals involved in several diseases [17,18], including cardiovascular diseases.

In the present study, a total of 250 mg of procyanidins was determined in 1 gram of cocoa extract. From this, 19 different phenolic compounds were identified (Figure 1 and Table 1). Many studies have shown the protective effect of cocoa in myocardial ischemia-reperfusion injury and in improving post-ischemic functional recovery [13,17]. Thus, it was observed that cocoa extract has anti-inflammatory properties [26] and it can protect against myocardial injury [27]. Even though flavonoids have many health-related benefits, their bioavailability is a major concern [28]. We hypothesized that these phenolic compounds, introduced daily with cocoa extract supplement, might contribute to reducing inflammatory markers, oxidative stress, myocardial apoptosis, and activating pro-survival pathways in the heart exposed to I/R injury. In the present study, different concentrations of a commercially available cocoa extract were investigated in a dose-response assessment in order to establish the optimal dose in a daily administration regimen in order to have maximal plasma concentration of polyphenols. An optimal dose of 15 mg/kg/BW was administered daily to investigate the possible cardioprotective effects during acute myocardial I/R.

The expression of inflammatory mediators, which recruits leukocytes into inflammatory sites, was reduced in the cocoa-treated rat heart (Figure 3). During the inflammatory process at tissue, the usual trend is the elevation of IL-6 and NF-kB levels [29]. These mediators were significantly reduced in cocoa extract-treated rat hearts. In vitro reduction in other inflammatory meditators, such as IL-1 mRNA expression and IL-2 secretion by T-lymphocyte treated with cocoa liquor was also reported [17]. Thus, reduction in inflammatory markers in cocoa-treated rat hearts suggests an anti-inflammatory and cardioprotective role of cocoa extract flavonoids.

Malondialdehyde (MDA), a marker of oxidative stress, was attenuated in cocoa-treated heart tissue (Figure 4A). The levels of reactive oxygen species were found to be significantly higher in cocoa-treated rat myocardium as compared to the control group after the induction of I/R (Figure 4B). Similarly, Akinmoladun et al. explored the protective effects of cocoa and kola nut tree extracts, and also reported a reduction in oxidative stress and post I/R injury in an in-vitro model [13]. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) constitutively is reduced in ischemia but inducible NOS is activated by I/R, thus increasing nitrosative stress. Nitrotyrosine (peroxynitrite) levels, which indicate nitrosative stress [30] in myocardial tissue due to I/R, were lower in cocoa-treated rat heart in our study (Figure 4C,D).

The ultimate step of myocardial injury in reperfusion injury is apoptosis, and inhibition of apoptotic pathways demonstrated significant cardioprotection. Reduced apoptosis leads to reduced myocardial injury [31]. Apoptosis could be due to mitochondrial dysfunction, which results in low energy for myocyte contraction. Additionally, there may be an increase in oxidative stress that directly damages and induces apoptosis in myocytes [32–34]. During I/R, the fuel preference switches to glucose. This alteration safeguards the heart, in part, because "free acids waste more oxygen to be oxidized". However, over time, while not oxidized, fatty acids hoard inside the myocytes, leading to cardiac lipotoxicity [35,36]. Nitrite reacts with critical thiols to form nitrosothiols, which act as antioxidants that prevent the irreversible oxidation of proteins and lipids during the early oxidative burst of reperfusion" [37,38].

Despite the pharmacological effectiveness of cardioprotective drugs, novel products which inhibit ischemic organ damage are required and care is being given to discovering novel pharmacological agents from plants [39]. Infact, the medicinal plants' antioxidant content may contribute to protection from diseases. Commonly found in plants, phenolic compounds are major antioxidant phytochemicals [27]. When the plants are consumed, these phytochemicals contribute to the intake of natural antioxidants in the diets of animals as well as humans.

The reduced levels of myocardial apoptosis observed in cocoa-treated rats demonstrate potential effect of flavonoids containing cocoa in reducing myocardial apoptosis. The toxicological study also indicated the contribution of epicatechin and catechin in cocoa in the reduction of apoptosis via the inhibition of amyloid-β protein [40]. The reduced myocardial apoptosis could be due to the antioxidant content of cocoa, which might contribute to the protection against oxidative stress produced by I/R.

In our study, immuno-peroxidase analysis of myocardial tissue from rats treated with cocoa extract showed elevation in the activity of p-Erk1/2 and p-Akt (Figure 5). Pro-survival pathways proteins (Erk1/2 and Akt) are known for their contribution to cell survival during ischemia-reperfusion injury. Enhanced phosphorylation of Erk1/2 and Akt were observed with cocoa treatment, which leads to cardioprotection [41]. The TUNEL assay, which is good indicator to measure apoptosis, demonstrated reduced apoptosis in cocoa-treated heart tissues as compared to control (Figure 6).

Therefore, this study suggests that treating rats with cocoa extract significantly attenuates inflammation, oxidative stress, and apoptosis in the myocardium and the process can be modulated by the activation of Erk1/2 and Akt pathways.

The present study investigated only one commercially available cocoa extract. Other products could give different results. Additionally, we investigated healthy rats; in order to translate the results to humans they should be confirmed in a randomized clinical study in patients with ischemic cardiomyopathy taking different medications.
