*Article* **Cocoa Flavonoids Reduce Inflammation and Oxidative Stress in a Myocardial Ischemia-Reperfusion Experimental Model**

**Sajeela Ahmed 1,**†**, Naseer Ahmed 1,2,**†**, Alessio Rungatscher 1,\*, Daniele Linardi 1, Bibi Kulsoom 3, Giulio Innamorati 1, Sultan Ayoub Meo 4, Mebratu Alebachew Gebrie 1, Romel Mani 1, Flavia Merigo 5, Flavia Guzzo <sup>6</sup> and Giuseppe Faggian <sup>1</sup>**


Received: 10 January 2020; Accepted: 16 February 2020; Published: 18 February 2020

**Abstract:** Consumption of flavonoid-rich nutraceuticals has been associated with a reduction in coronary events. The present study analyzed the effects of cocoa flavonols on myocardial injury following acute coronary ischemia-reperfusion (I/R). A commercially available cocoa extract was identified by chromatographic mass spectrometry. Nineteen different phenolic compounds were identified and 250 mg of flavan-3-ols (procyanidin) were isolated in 1 g of extract. Oral administration of cocoa extract in incremental doses from 5 mg/kg up to 25 mg/kg daily for 15 days in Sprague Dawley rats (*n* = 30) produced a corresponding increase of blood serum polyphenols and become constant after 15 mg/kg. Consequently, the selected dose (15 mg/kg) of cocoa extract was administered orally daily for 15 days in a treated group (*n* = 10) and an untreated group served as control (*n* = 10). Both groups underwent surgical occlusion of the left anterior descending coronary artery and reperfusion. Cocoa extract treatment significantly reversed membrane peroxidation, nitro-oxidative stress, and decreased inflammatory markers (IL-6 and NF-kB) caused by myocardial I/R injury and enhanced activation of both p-Akt and p-Erk1/2. Daily administration of cocoa extract in rats is protective against myocardial I/R injury and attenuate nitro-oxidative stress, inflammation, and mitigates myocardial apoptosis.

**Keywords:** flavonoids; cocoa extract; ischemia-reperfusion injury; oxidative stress; apoptosis; inflammatory markers

#### **1. Introduction**

In United States, about one million people suffer from myocardial infarction per year [1]. Ischemic heart disease provoked by limited blood supply to cardiomyocytes upon occlusion of coronary vessels is the main precursor of myocardial infarction. Prevention of tissue damage in patients with ischemic heart disease can be effectively accomplished through reperfusion of the ischemic myocardial

tissue [2,3]. However, reperfusion itself is responsible for inducing injury to cardiomyocytes via multiple mechanisms, leading to heart failure [4,5]. Oxidative stress and myocardial inflammatory pathways play a leading role in the pathogenesis of myocardial ischemia-reperfusion (I/R) injury [6]. Oxidative stress is caused by an intracellular redox imbalance between pro- and anti-oxidants [7–9]. Exogenous antioxidants might influence the course of the ischemic heart disease by providing therapeutic substances which help in restoring and maintaining a balanced system [10]. Therefore, plant resources with anti-oxidant activity would be worthy natural substances for protection against ischemic heart disease. The progression of reperfusion, thus, is accompanied by the development of oxidative stress with the generation of free radicals and leukocyte activation that lead to myocytes apoptosis [11]. Exogenous antioxidants might influence the course of the ischemic heart disease by contributing to restoring a balanced system.

Many scientists have been working to explore antioxidant-rich natural sources to protect against myocardial I/R injury [12]. Many studies have reported plants as the main reservoir of natural antioxidants and anti-inflammatory compounds, thus pointing towards the protective role of plant products against inflammatory and reperfusion injury [12–15].

Cocoa and chocolate products contribute high levels of flavonoids among commonly consumed foods and have been historically used as a medicine to cure inflammation, pain, and numerous other diseases [13,16]. It is observed that adipose tissue inflammation can be reduced by long-term cocoa supplementation. In a recent study reported by Akinmoladun et al., antioxidant-containing extracts of cocoa and the kola nut tree have shown a protective effect against myocardial I/R injury using Langerdorff-perfused rat hearts [13]. In another study, flavonoids (5-hydroxy derivatives: 5-hydroxy flavone, apigenin, chrysin, and naringenin) lowered myocardial tissue injury and improved post-ischemic functional recovery [17]. Earlier, an in vitro study reported reduced IL-1 mRNA expression and IL-2 secretion by T-cells in polyphenol containing cocoa liquor [18]. However, previous studies lack biochemical analysis of inflammatory markers and signaling protein activities, and evaluation of myocardial nuclei apoptotic levels in rat hearts after I/R injury. Moreover, to the best of our knowledge, dose optimization of the cocoa extract in blood sera in rats has not been documented previously in literature.

In this study, we investigate the effects of a commercially available cocoa extract on oxidative stress, inflammation, and apoptosis to ascertain whether it can protect myocardium in an in vivo experimental model of I/R injury.

#### **2. Methods and Materials**

#### *2.1. Chemicals and Materials Used*

CocoaVia® was purchased from Mars Inc., (Hackettstown, NJ, USA). Primary antibodies Nitro-tyrosine, IL-6, NFkB2, P-Erk, P-akt antibody were obtained from Bioss antibodies, Novusbio, and Cell Signaling Technology (Danvers, MA USA). In Situ Cell Death Detection Kit, AP was from Roche (Basel, Switzerland).

#### *2.2. Animals*

Thirty healthy Sprague Dawley (SD) male rats of average weight 300–350g were used for the dose response experiment and 20 rats were used to assess the effect of cocoa extract on ischemia reperfusion injury. All animal experiments were done per the ethical guidelines reviewed and approved by "University of Verona Ethical Committee and the Italian Ministry of Health (341/2016-PR) at C.I.R.S.A.L. (Interdepartmental Research Centre for Laboratory Animals) of the Biological Institutes, University of Verona, and Verona, Italy".
