Effects of Punica granatum L. peel extract supplementation on body weight, cardiac function, and haematological and biochemical parameters in an animal model of metabolic syndrome

Metabolic syndrome (MetS) is a pathological condition defined as a set of several metabolic disorders comprising visceral obesity, insulin resistance, dyslipidaemia and hypertension (5). It is defined by diagnostic criteria with parametric cut-off points varying as appropriate for particular target populations. Any three out of the five following risk factors must be present to make a diagnosis: central obesity (abnormal waist circumference or body mass index), insulin resistance (impaired glucose tolerance, impaired fasting glucose or type 2 diabetes requiring treatment), dyslipidaemia (hypertriglyceridaemia, low concentration of high-density lipoprotein cholesterol (HDL) or hyperlipidaemia requiring treatment) and hypertension (high resting blood pressure or need for antihypertensive drugs) (39). Additionally, apart from the main components, MetS is also associated with impaired kidney function, polycystic ovary syndrome, hyperuricaemia, fatty liver disease, obstructive sleep apnoea and heart failure with preserved ejection fraction (EF) (47). The pathogenesis is complex and still not completely elucidated. However, it seems that the primary roles are played by insulin resistance, chronic inflammation and neurohormonal activity (45). Early diagnosis and treatment are vital to prevent the development of more severe conditions such as atherosclerotic cardiovascular disease and to modify the risk factors. The first line treatment of MetS is based on lifestyle changes promoting physical activity, sleep hygiene, reduced alcohol consumption, and dietary intervention (12, 47). Drugs such as antihypertensives, statins and metformin are used when non-medical strategies prove ineffective. Nonetheless, their application is limited by drug-related adverse effects emerging over the course of long-term therapy (12).

In veterinary clinics, MetS is well described in horses but still requires further investigation in obese dogs and cats and in individuals with hypercortisolaemia. Some reports have already shown the connection between obesity, hyperlipidaemia and insulin resistance. The occurrence of these disorders in humans escalates the risk of cardiovascular diseases such as atherosclerosis, a fatal condition well known in humans but unusual in small animals or horses. Establishing diagnostic criteria for MetS in veterinary medicine will facilitate its diagnosis and help in the early implementation of appropriate treatment.

A novel approach to the management of MetS involves plant food supplements. Nutraceuticals are natural dietary components with proven health benefits. Studies show that compounds derived from plants display potentially propitious features in MetS (48). Polyphenols in particular, which are biomolecules present in flowers, seeds, juice, arils, roots and leaves, are believed to be integral to the future of the food industry as natural food additives (48). Studies have shown that polyphenols exhibit antioxidative, anti-inflammatory, antihypertensive, antimicrobial, antiatherogenic, antiaging and antimutagenic effects. Favourable medical features also include the promotion of weight loss, antidiabetic properties, improvement of lipid profile, and cardioprotective, hepatoprotective and nephroprotective activity (13, 48). Therefore, they are considered potential therapeutic agents in MetS. Consumption of functional food enriched with phenolic compounds could improve public well-being and help prevent diseases of affluence such as cardiovascular diseases, type 2 diabetes and obesity (22). Additionally, incorporating phenolic compounds in animal food may promote health and serve as a component of pre-emptive veterinary medicine.

Pomegranate (Punica granatum L.) is a rich source of phenolic compounds. Numerous studies highlight its high concentration of health-promoting phytochemicals and promising pharmaceutical properties in dietary supplementation (1, 21). It is predominantly consumed fresh or in the form of juice. Health benefits are bestowed not only by juice and the flesh of the fresh fruit but also by the uneatable parts, which contain a wide range of bioactive compounds (19, 37). Pomegranate peel, a by-product of industrial juice processing, contains a high concentration of polyphenol fractions such as phenolic acids (ellagic acid, caffeic acids, hydroxybenzoic acid, hydroxycinnamic acid and gallic acid), hydrolysable tannins (catechin, ellagitannins, gallotannins and gallagyl esters), flavonols (epicatechin and gallocatechin) and anthocyanins (9). The bioactivity of pomegranate peel is anti-inflammatory, antioxidant, antimutagenic, anticancerogenic, antidiabetic and antimicrobial (4, 7). Pomegranate peel’s content of phytochemicals with nutraceutical and medical significance make it an auspicious natural source in the development of functional food products.

The main objective of the present study was to assess the potential health benefits from pomegranate peel extract (EPP) supplementation in an animal MetS model.

Pomegranate (Punica granatum L.) is a promising source of polyphenols that could help prevent or mitigate the course of MetS. The flesh excluded, pomegranates contain a substantial amount of active phenolic compounds. These compounds scavenge various reactive oxygen species (ROS) (14). For many years juice was believed to contain the most bioactive particles that could be utilised as supplements and nutraceuticals in miscellaneous diseases (2). However, studies investigating the health benefits of pomegranate peel showed that polyphenols in this material possess even higher antioxidant activity than the pulp itself (14, 24). Its nutritional value has been explored in many in vivo studies on animal models, and these proved that pomegranate peel extract reduces obesity (38), dyslipidaemia (34), hypertension (11), and hyperinsulinaemia and insulin resistance (49), and also ameliorates chronic inflammatory status (30). In the presented study, we aimed to evaluate the health benefits of pomegranate peel extract supplementation in a model of MetS and healthy control rats maintained on a high-calorie diet.

The role of pomegranate peel extract in preventing obesity has been previously reported (30). Our result consonantly indicates that phenolic compounds derived from pomegranate peel possess bioactivity that could be helpful in the prevention or mitigation of body weight gain. The ability of pomegranate peel to prevent obesity depends on the impact of polyphenol metabolites in the expression of four proteins: an adipose-formulation–related one such as adiponectin, peroxisome proliferator–activated receptor γ, glucose transporter type 4, and fatty acid–binding protein 4 (23). It has also been proposed that oral supplementation with polyphenols modulates gut microbiota, which elicits an obesity control effect (30, 38). In vivo studies have shown that phenolic compounds could promote beneficial bacterial species, such as Bifidobacterium spp. and Lactobacillus spp., as well as suppress potentially pathogenic bacteria (46). Targeting microbiota with the pomegranate by-product phenolic compounds which have probiotic properties encourages the proliferation of Bacteroides spp. and significantly reduces the abundance of Firmicutes spp. and Proteobacteria spp. (38). Inverse proportionality of these species of bacteria was reported to be a likely diagnostic biomarker of gut dysbiosis correlated with the risk of metabolic diseases (36). One benefit of phenolic compounds is that they promote the abundance of beneficial bacterial genera, and a separate positive effect is that the fermentation of polyphenols by bacteria contributes to the generation of metabolite products, especially urolithin A, that inhibit triglyceride accumulation and suppress the gene expression involved in adipogenesis (23, 41). The relationship between the consumption of polyphenols and gut microbiota is complex and certainly requires further investigation.

Obesity is associated with many pathological conditions, such as insulin resistance, atherosclerosis and lipid metabolism disorders (18). Pomegranate peel extract also improves the glycaemic and lipid profiles. It has been proved that EPP lowers the serum concentration of triglycerides, total cholesterol and LDL-cholesterol in obese hamsters and an obese mice model (25, 30, 38). The mechanisms lying behind this propitious impact on lipid metabolism were partially revealed. The outcomes elucidated how phenolic compounds derived from pomegranate peel upregulated liver X receptor α, peroxisome proliferator-activated receptor α, peroxisome proliferator-activated receptor γ and gene ATP-binding cassette transporter A1, downregulated fatty acid synthase through inhibition of the keto-acetyl synthase and acetyl/malonyl transferase domains, and supported cholesterol removal by enhancing faecal bile acid (25, 26). Interestingly, in the study on hamsters, the effect of pomegranate ellagic acid extracted from peel on lipid metabolism was stronger than that of simvastatin alone in raising HDL and lowering LDL (25). Contrary to these findings, our research indicated no change in lipid profile in individuals treated with pomegranate peel extract. However, some other in vivo studies with results consistent with ours also reported no difference in serum levels of triglycerides and cholesterol fractions between the control group and pomegranate-treated groups on a high-fat, high-sucrose diet (16).

Metabolic syndrome also leads to improper insulin utilisation and production (47). As evidenced by recent studies, many natural plant derivatives classified as flavonoids, alkaloids, terpenoids and phenolics display antidiabetic properties (35). Phenolic components from pomegranate peels, of which punicalagin, gallic acid and ellagic acid are examples, have been proved to exhibit antidiabetic, antihyperglycemic and antiglycation effects (28, 29). Pomegranate peel diminishes fasting blood glucose concentration and improves insulin sensitivity through various mechanisms. Research findings highlighted the crucial involvement of pomegranate phenolics in carbohydrate regulation (44). The principal mechanisms are the inhibition of α-glucosidases and α-amylases (20), inhibition of advanced glycation end-product formation (8), and mitigation of hyperglycaemic-induced oxidative stress (10). Pomegranate seed oil extract improved insulin tolerance and reduced serum fasting blood glucose in diet-induced obese mice (16). However, in the same study, extracts from pomegranate flowers and peel did not invigorate carbohydrate metabolism and in this were inferior to rosiglitazone; the extracts did nevertheless decrease the plasma level of proinflammatory cytokines IL-6 and TNF-α, which also protects against dysregulation of glucose metabolism (10, 16). Furthermore, anthocyanins extracted from pomegranate peel favourably altered the insulin signalling pathway. These findings implied that the supplementation with polyphenols from pomegranate peel might alleviate insulin resistance (38). In the present study, EPP did not lower the glucose level in the serum. The data from other research on animal models are also at some variance on this point.

The cardiovascular consequences of MetS also comprise an alteration in cardiac function associated with obesity. This cardiac failure in MetS was described as “cardiomyopathy of obesity” (43). It is characterised in humans by the development of concentric left ventricular hypertrophy and mild diastolic or systolic dysfunction with normal or elevated EF (3). Impairment in myocardial contractility also has been reported in animal models of obesity (27). Furthermore, changes occurring in MetS disrupt the equilibrium between coronary blood flow and myocardial metabolism, significantly increasing the risk of myocardial infarction and mortality (42). The pleiotropic effect of phenolic compounds on cardiovascular diseases comprises vasodilatative activity and anti-inflammatory, antithrombotic and antiatherogenic effects (32). Cardiovascular protection is also provided with polyphenols from pomegranate peel. A study in a spontaneously hypertensive rat model found the consumption of EPP to reduce systolic blood pressure, coronary angiotensin-converting enzyme activity and oxidative stress level, and prevent vascular remodelling (11). Evidence suggests that phenolics from peel may find application in mitigating coronary heart disease by attenuation of electrocardiographic changes, myocardial tissue damage and heart weight increase. Notably, the phenolics’ role as a cardioprotective agent arises from their upregulating of endothelial nitric oxide synthase expression, leading to intensification of antioxidant mechanisms and inhibition of apoptosis (15). In addition, punicalagin suppresses cardiac fat accumulation by stimulating the cardiac adenosine monophosphate–activated protein kinase signalling pathway. Concomitantly, it prevents mitochondrial loss by enhancing mitochondrial biogenesis and ameliorating oxidative stress (6). In our study, we also noted a cardioprotective effect of the largest EPP supplementation in the relative decrease in heart rate. Likewise, EPP improves mFS, representing myocardial contractility.

Blood morphological and haematological indices represent the general state of health of individuals. The assessment of these parameters could indicate metabolic abnormalities or the noxious effect of xenobiotics (31). Changes in blood morphology and haematology may also suggest pathological conditions such as anaemia, infection, thrombotic state and bone marrow impairment. In this context, red blood cell morphology is a very sensitive marker of exposure to ROS (17). Polyphenols are characterised by low toxicity and, in recent years, have gained attention as natural radioprotective and cytoprotective agents (40). Our findings demonstrated that no toxicity levels manifested by haematological changes were observed in individuals treated with EPP. In the blood smears, the only alterations were the increased number of acanthocytes and schistocytes in rats without genetically programmed MetS, which may have resulted from diet-induced obesity and its consequences. No blast cells nor anaemic state were observed.

The present study suggests that phenolic compounds from pomegranate peel have a potentially beneficial effect in dietary intervention in metabolic syndrome. Together these results confirm the promise of pomegranate peel as a nutrient, especially in restricting body weight gain. However, studies on humans and animals suffering from MetS are needed in order to determine the bioaccessibility of bioactive constituents and metabolites and to indicate their actual effectiveness in ameliorating individual abnormalities involved in the pathogenesis of MetS. Polyphenols incorporated into the diet of humans and animals could help maintain health and counteract metabolic syndrome.