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
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.
The study was carried out on Zucker diabetic fatty rats with missense mutations in the
The polyphenolic extract was obtained from
Identification and quantification of polyphenolic compounds were performed on an Acquity ultra-performance liquid chromatography (UPLC) system, coupled with a Synapt quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) instrument (Waters Corp., Milford, MA, USA), with an electrospray ionisation source and photodiode array detector. Separation was obtained on the Acquity bridged ethylene hybrid C18 column (100 mm × 2.1 mm i.d., 1.7 m, Waters Corp.). The water phase was a mixture of 0.1 % (v/v) aqueous formic acid (A) and acetonitrile (B). The gradient programme was as follows: the initial conditions were 1% B in A, 12 min of 25% B in A, 12.5 min of 100% B in A and 13.5 min of 1% B in A. The flow rate was 0.45 mL/min, and the injection volume was 10 μL. The column was operated at 30°C. Ultraviolet-visible absorption spectra were recorded online during UPLC analysis, and the spectral measurements were made in the wavelength range of 200–600 nm in steps of 2 nm. The primary operating parameters for the Q-TOF MS were set as follows: capillary voltage of 2.5 kV, cone voltage of 30 V, cone gas flow of 11 L/h, collision energy 28–30 eV, source temperature of 100°C, desolvation temperature of 250°C, argon collision gas, nitrogen desolvation gas flowing at 300 L/h, data acquisition range m/z 100–2000 Da, and negative ionisation mode. The data were collected with MassLynx v. 4.1 software (Waters Corp). The results of the analysis are presented in Table 1.
Mass spectrum characteristic and content of phenolic compounds in pomegranate peel extract
Rt (min) | MS (M-H)− |
MS/MS (M-H)− |
Name of compounds | Polyphenol content |
---|---|---|---|---|
1.67 | 331 | 271/169 | Galloyl-glucose | 2.00 ± 0.03 |
1.73 | 781 | 721/601 | Punicalin α/A | 3.11 ± 0.06 |
2.02 | 1083 | 611/331/146 | HHDP-galloyl-hexoside (punicalagin) | 4.20 ± 0.09 |
2.12 | 1083 | 781/622/301 | Punicalagin isomer | 14.82 ± 1.04 |
2.33 | 933 | 631/450/301 | Ellagitannin | 4.71 ± 0.40 |
2.87 | 1083 | 781/301 | HHDP-gallagyl-hexoside (punicalagin) | 93.91 ± 2.05 |
3.12 | 1085 | 907/783/301 | Ellagic acid derivative | 2.49 ± 0.53 |
3.69 | 1083 | 781/301 | HHDP-gallagyl-hexoside (punicalagin) | 157.00 ± 2.65 |
3.89 | 799 | 301 | Granatin A | 4.74 ± 0.32 |
5.08 | 783 | 481/301 | Ellagitannin | 25.86 ± 1.53 |
6.20 | 1085 | 933/301 | Digalloyl-gallagyl-hexoside | 10.37 ± 0.65 |
6.25 | 783 | 481/301 | Ellagitannin | 13.51 ± 0.99 |
6.38 | 463 | 301 | Ellagic acid-hexoside | 33.63 ± 1.23 |
6.89 | 951 | 907/635/301 | Galloyl-HHDP-DHHDP-hex (granatin B) | 2.68 ± 0.11 |
Total (mg/g dry weight) | 373.05 |
Rt – retention time; MS – mass spectrometry; (M-H)− – deprotonated molecule;
Body weight was recorded twice per week. Measurements for each individual in a single survey point were taken three times, and the mean was calculated and subsequently incorporated into the statistical analysis. The weight of individuals was measured using an SW-II certified calibrated scale (CAS Poland Sp. z.o.o., Warsaw, Poland).
The blood samples were obtained at three time points: before starting EPP administration, after four weeks, and after eight weeks of study. Blood was drawn from the lateral tail vein after warming the tail to increase the obtainable blood volume, and was collected into microtubes containing 1.6 mg/mL liquid ethylenediaminetetraacetic acid (EDTA) and serum tubes. Serum samples were separated by centrifugation (4,000 ×
The leukocyte quantities were determined by a Schilling differential cell count. Erythrocyte abnormalities of size and shape were recorded and reticulocytes were counted in a microscopic examination. After staining EDTA blood with 1% new methylene blue with 1.6% potassium oxalate anticoagulant and 1% brilliant cresol blue in saline (ANALAB Sp. z o.o., Warsaw, Poland), the percentage of reticulocytes per 1,000 non-nucleated red blood cells was calculated. Blood biochemical indices were investigated with an Epoll 300 analyser (Alpha Diagnostic Intl. Inc., San Antonio, TX, USA). Serum levels of glucose and the lipid panel (total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglycerides) were established.
Heart size and function assessment was performed before EPP administration and eight weeks after it. Echocardiography measurements were performed in the Department of Internal Medicine and Clinic of Diseases of Horses, Dogs and Cats in the Faculty of Veterinary Medicine at Wroclaw University of Environmental and Life Sciences. The echocardiographic measurements were taken by the same researcher over at least three consecutive cardiac cycles using an Arietta echocardiograph (Aloka Company, Tokyo, Japan) and a 7.5–10 MHz transducer according to the guidelines of the American Society for Echocardiography. The following cardiac dimensions were determined: the relative left atrial size (from the left atrial diameter to aortic root diameter ratio (LA/Ao), end-diastolic and end-systolic thickness of the interventricular septum, left ventricle posterior wall, and the internal left ventricular dimensions at end diastole and end systole (LVIDd and LVIDs). Estimates of left ventricular systolic function were obtained from the index of circumferential myocardial contraction and fractional shortening (FS) using the Teicholz formula ((LVIDd-LVIDs/LVIDd) × 100%). Estimates of left ventricular end-diastolic volume, end-systolic volume, stroke volume, and EF were calculated by the echocardiographic software.
The results are displayed as a mean ± standard deviation. All statistical analyses were performed using the R statistical computing environment (version 4.1.1.; 32). If the data followed a normal distribution, parametrical tests were used. The non-normal data were analysed with the use of nonparametric tests. For three or more groups of variables with normal distribution, one-way analysis of variance was applied, and to compare two groups of normal data, Welch’s
Body weight assessments were analysed at 16 time points. No significant differences were noted at the last time point between the control group fa/fa H2O (mean = 407.14 g, standard deviation (SD) = 32.68 g) and the experimental group fa/+ 200 (mean = 337.69 g, SD = 29.34g), FWelch = 4.56 using the value 4 as adjustment for degrees of freedom, 11.68 as adjustment for error degrees of freedom, and 0.02 as the rectified p-value according to the Welch formula. Similarly, no significant effect of pomegranate peel extract on body weight in other groups was observed (Table 2 and Fig. 1).
Mean body weights for individual groups at the final 16th time point
group | fa/fa H2O | fa/fa 100 | fa/fa 200 | fa/+ 100 | fa/+ 200 |
---|---|---|---|---|---|
n | 6 | 6 | 6 | 6 | 6 |
Mean | 407.14 | 382.08 | 392.75 | 358.97 | 337.69 |
Standard deviation | 32.68 | 35.48 | 36.28 | 12.90 | 29.34 |
fa/fa H2O – control group of Zucker diabetic fatty (ZDF) rats with missense mutation in the
The dynamic of body weight increase was registered at 16 time points. Graphic representations of data are presented in Fig. 2. The data show clear tendencies, despite slight differences between individuals at the beginning of the study. Body weights in all groups trended upwards. However, in groups without MetS (fa/+), the increase was more pronounced than in rats predisposed to the syndrome (fa/fa), and the gain in these groups was considerably quicker (Fig. 3). Experimental groups with MetS were characterised by smaller weight gains than the group of control rats with MetS administered only water. The body weight gain at each time point in group fa/fa 100 was 1.37 g less and in group fa/fa 200 was 1.22 g less than that in group fa/fa H2O. In groups of rats without MetS, the body mass increase was higher at each time point in comparison to the increase in group fa/fa H2O, in group fa/+ 100 being so by about 3.09 g, and in group fa/+200 by about 2.25 g.
Echocardiographic parameters were determined to evaluate the influence of EPP administration on cardiac function. The measurements were made twice: before the beginning of the study and after its termination (Table 3). The analysis was based on the design control group pretest-posttest approach. The results indicate that there were no significant differences between groups with EPP supplementation and the group receiving only water in most of the analysed parameters. However, some of them showed changes versus the fa/fa H2O group. At the end of the study, the heart rate in experimental groups was significantly lower – by 54.67 bpm (95% CI: –88.19––21.15, P = 0.002) – than at the beginning. Moreover, the heart rate in the fa/fa 200 group was markedly decreased – by 43.83 bpm – compared to the control group (CI: –79.29––8.38, P = 0.017). The value of end-diastolic volume in the fa/fa 200 group was significantly altered when compared with the fa/fa H2O group (95% CI: 0.03–0.45, P = 0.026). Cardiac output was diminished in all groups after eight weeks by 0.03 L/min (95% CI –0.06––0.01, P = 0.015). Also LA/Ao was lower in all groups by 0.11 without regard to EPP supplementation (95% CI: –0.19––0.04, P = 0.005). Mid-wall fractional shortening (mFS) representing myocardial contractility was better by 7.65% in the fa/fa 200 group in comparison to the control group (95% CI: 0.69%–14.61%, P = 0.032).
Echocardiographic parameters at two time points: at the beginning of the study (t1) and after eight weeks of pomegranate peel supplementation (t2)
Index | fa/fa H2O | fa/fa 100 | fa/fa 200 | fa/+ 100 | fa/+ 200 | |||||
---|---|---|---|---|---|---|---|---|---|---|
t1 | t2 | t1 | t2 | t1 | t2 | t1 | t2 | t1 | t2 | |
289.00 ± 28.08 | 234.33 ± 37.03 | 265.00 ± 21.02 | 232.25 ± 45.85 | 245.17 ± 21.34 | 226.83 ± 32.62 | 264.60 ± 20.40 | 230.60 ± 46.55 | 275.20 ± 11.82 | 245.80 ± 31.40 | |
2.52 ± 0.54 | 2.65 ± 1.03 | 3.35 ± 1.61 | 2.65 ± 1.03 | 3.35 ± 1.23 | 2.90 ± 1.01 | 3.36 ± 0.69 | 2.36 ± 1.19 | 2.80 ± 0.70 | 2.28 ± 0.42 | |
6.48 ± 0.51 | 6.08 ± 1.15 | 6.65 ± 0.83 | 5.82 ± 0.74 | 6.87 ± 1.25 | 6.52 ± 0.95 | 6.08 ± 1.13 | 5.56 ± 1.39 | 5.84 ± 0.77 | 5.68 ± 0.69 | |
2.93 ± 0.32 | 3.00 ± 0.39 | 2.72 ± 0.43 | 3.00 ± 0.55 | 2.62 ± 0.39 | 2.77 ± 0.67 | 2.68 ± 0.49 | 3.32 ± 0.72 | 2.38 ± 0.49 | 3.18 ± 0.72 | |
1.80 ± 0.39 | 1.95 ± 0.42 | 1.78 ± 0.39 | 2.22 ± 0.38 | 1.88 ± 0.28 | 2.03 ± 0.43 | 1.98 ± 0.56 | 1.86 ± 0.19 | 1.62 ± 0.30 | 1.94 ± 0.42 | |
3.32 ± 0.38 | 3.15 ± 0.62 | 3.10 ± 0.55 | 3.67 ± 0.71 | 3.68 ± 0.55 | 3.22 ± 0.41 | 3.04 ± 1.10 | 3.24 ± 0.58 | 3.30 ± 0.71 | 3.38 ± 0.36 | |
1.77 ± 0.16 | 1.77 ± 0.29 | 1.73 ± 0.13 | 2.00 ± 0.27 | 1.92 ± 0.23 | 1.80 ± 0.43 | 1.96 ± 0.55 | 1.62 ± 0.46 | 1.78 ± 0.19 | 1.86 ± 0.30 | |
0.63 ± 0.15 | 0.55 ± 0.26 | 0.68 ± 0.22 | 0.48 ± 0.22 | 0.80 ± 0.38 | 0.65 ± 0.26 | 0.54 ± 0.26 | 0.44 ± 0.37 | 0.50 ± 0.16 | 0.44 ± 0.15 | |
0.05 ± 0.05 | 0.07 ± 0.05 | 0.15 ± 0.17 | 0.05 ± 0.10 | 0.13 ± 0.12 | 0.08 ± 0.08 | 0.10 ± 0.07 | 0.04 ± 0.09 | 0.06 ± 0.09 | 0.02 ± 0.04 | |
0.60 ± 0.14 | 0.50 ± 0.24 | 0.58 ± 0.10 | 0.40 ± 0.22 | 0.65 ± 0.31 | 0.57 ± 0.20 | 0.44 ± 0.21 | 0.40 ± 0.28 | 0.40 ± 0.16 | 0.38 ± 0.13 | |
0.17 ± 0.04 | 0.12 ± 0.07 | 0.15 ± 0.02 | 0.09 ± 0.04 | 0.16 ± 0.07 | 0.14 ± 0.06 | 0.12 ± 0.06 | 0.09 ± 0.04 | 0.11 ± 0.04 | 0.10 ± 0.03 | |
92.85 ± 3.36 | 89.83 ± 6.38 | 83.50 ± 14.01 | 84.12 ± 18.64 | 85.80 ± 8.92 | 89.55 ± 5.71 | 80.68 ± 7.18 | 90.22 ± 6.40 | 87.00 ± 7.26 | 92.64 ± 1.78 | |
61.25 ± 6.78 | 57.72 ± 12.38 | 51.52 ± 18.55 | 53.75 ± 21.09 | 52.07 ± 12.09 | 56.62 ± 10.47 | 44.80 ± 8.12 | 58.58 ± 13.68 | 52.18 ± 8.29 | 59.94 ± 3.22 | |
25.40 ± 3.67 | 22.05 ± 3.24 | 21.50 ± 7.14 | 18.10 ± 12.31 | 20.95 ± 4.67 | 25.25 ± 3.25 | 18.84 ± 3.43 | 15.22 ± 3.37 | 19.92 ± 1.76 | 19.80 ± 7.22 | |
4.22 ± 0.71 | 3.57 ± 0.47 | 4.34 ± 1.08 | 3.82 ± 0.36 | 4.87 ± 0.53 | 3.83 ± 0.69 | 4.30 ± 0.58 | 4.15 ± 0.42 | 4.58 ± 0.42 | 3.76 ± 0.29 | |
3.37 ± 0.38 | 3.23 ± 0.35 | 3.34 ± 0.73 | 3.44 ± 0.25 | 3.85 ± 0.23 | 3.12 ± 0.51 | 3.28 ± 0.39 | 3.65 ± 0.83 | 3.44 ± 0.22 | 3.12 ± 0.33 | |
1.25 ± 0.19 | 1.10 ± 0.08 | 1.30 ± 0.11 | 1.12 ± 0.13 | 1.26 ± 0.11 | 1.24 ± 0.17 | 1.31 ± 0.08 | 1.17 ± 0.18 | 1.34 ± 0.18 | 1.21 ± 0.13 |
fa/fa H2O – control group of Zucker diabetic fatty (ZDF) rats with missense mutation in the
Regarding the results of blood morphology, no explicit differences between tested groups were found (Table 4). However, in all groups, some tendencies were revealed. During the course of the study, the WBC, HGB, PLT, MCV, MCH, MCHC and lymphocytes decreased, in contrast to the RBC and neutrophil count, which increased. The only marked difference noted in blood morphology between the control and experimental groups was the eosinophilic granulocyte count, which was relevantly elevated in individuals obtaining EPP. The results of blood smears and determination of abnormal cells showed a higher number of acanthocytes and schistocytes in rats without MetS (fa/+ 100 and fa/+ 200) than in individuals with genetically programmed MetS (fa/fa H2O, fa/fa 100 and fa/fa 200) regardless of EPP supplementation. The findings are presented in Figs 4 and 5. Images of peripheral blood smears containing acanthocytes and schistocytes are shown in Fig. 6. No blast cells or other abnormalities were reported.
Blood cell counts at two time points: at the beginning of the study (t1) and after eight weeks of EPP supplementation (t2)
Index | fa/fa H2O | fa/fa 100 | fa/fa 200 | fa/+ 100 | fa/+ 200 | |||||
---|---|---|---|---|---|---|---|---|---|---|
t1 | t2 | t1 | t2 | t1 | t2 | t1 | t2 | t1 | t2 | |
10.40 ± 1.63 | 7.05 ± 1.96 | 10.50 ± 1.26 | 6.02 ± 2.33 | 11.26 ± 2.56 | 6.40 ± 1.29 | 8.52 ± 0.86 | 5.00 ± 1.41 | 9.38 ± 0.99 | 3.73 ± 1.25 | |
7.49 ± 0.21 | 8.36 ± 0.69 | 7.63 ± 0.23 | 8.59 ± 0.44 | 7.60 ± 0.13 | 8.19 ± 0.53 | 7.53 ± 0.64 | 8.07 ± 0.54 | 7.69 ± 0.24 | 7.82 ± 0.63 | |
16.13 ± 0.37 | 14.53 ± 0.99 | 16.10 ± 0.37 | 14.80 ± 0.65 | 15.90 ± 0.34 | 14.32 ± 0.73 | 15.70 ± 0.73 | 13.88 ± 0.88 | 16.05 ± 0.42 | 13.90 ± 0.35 | |
43.47 ± 1.25 | 43.23 ± 3.31 | 39.18 ± 12.49 | 44.52 ± 2.32 | 44.12 ± 0.92 | 42.22 ± 2.66 | 41.88 ± 3.76 | 41.86 ± 2.61 | 42.75 ± 1.48 | 39.75 ± 3.18 | |
1,069.33 ± 207.16 | 748.00 ± 76.58 | 1033.83 ± 74.09 | 658.67 ± 67.82 | 1078.80 ± 76.59 | 718.20 ± 52.11 | 905.80 ± 53.27 | 656.60 ± 244.39 | 911.67 ± 60.07 | 738.00 ± 39.33 | |
58.00 ± 1.10 | 51.83 ± 0.75 | 57.67 ± 0.52 | 51.83 ± 0.41 | 58.00 ± 1.00 | 51.60 ± 0.55 | 55.60 ± 1.14 | 51.80 ± 0.84 | 55.50 ± 0.55 | 51.00 ± 0.00 | |
21.55 ± 0.58 | 17.43 ± 0.63 | 21.15 ± 0.22 | 17.25 ± 0.29 | 20.88 ± 0.40 | 17.50 ± 0.42 | 20.90 ± 1.07 | 17.18 ± 0.26 | 20.90 ± 0.19 | 17.87 ± 1.11 | |
37.15 ± 0.69 | 33.65 ± 1.13 | 36.50 ± 0.49 | 33.30 ± 0.62 | 36.02 ± 0.29 | 33.92 ± 0.73 | 37.62 ± 2.00 | 33.20 ± 0.17 | 37.58 ± 0.42 | 35.08 ± 2.23 | |
2.17 ± 0.87 | 1.85 ± 1.56 | 2.72 ± 0.31 | 1.15 ± 0.82 | 3.60 ± 0.67 | 0.92 ± 0.33 | 2.34 ± 0.61 | 1.12 ± 1.11 | 1.48 ± 0.69 | 0.53 ± 0.30 | |
13.33 ± 6.92 | 25.50 ± 4.76 | 19.33 ± 8.04 | 21.50 ± 4.59 | 20.40 ± 5.08 | 33.20 ± 5.72 | 16.25 ± 7.37 | 20.75 ± 3.95 | 12.83 ± 5.53 | 18.67 ± 4.50 | |
81.17 ± 5.00 | 71.50 ± 5.68 | 76.83 ± 10.26 | 74.83 ± 6.11 | 78.40 ± 5.13 | 61.80 ± 4.76 | 81.00 ± 8.68 | 75.75 ± 5.68 | 84.67 ± 4.63 | 79.50 ± 4.46 | |
2.50 ± 3.51 | 0.83 ± 0.98 | 1.00 ± 0.89 | 0.33 ± 0.52 | 0.20 ± 0.45 | 2.00 ± 1.58 | 0.50 ± 1.00 | 0.25 ± 0.50 | 1.83 ± 1.33 | 0.17 ± 0.41 | |
1.83 ± 0.75 | 0.67 ± 0.52 | 1.50 ± 1.38 | 2.33 ± 2.42 | 0.60 ± 0.55 | 1.60 ± 1.34 | 2.00 ± 1.15 | 2.50 ± 1.29 | 0.33 ± 0.52 | 1.33 ± 1.03 |
fa/fa H2O – control group of Zucker diabetic fatty (ZDF) rats with missense mutation in the
The levels of fasting blood glucose and serum lipidic indices are presented in Table 5. Regarding the glucose level, the EPP supplementation did not improve the glycaemic status of rats. The glucose concentration in individuals with MetS (fa/fa H2O, fa/fa 100 and fa/fa 200) was significantly elevated compared to rats without MetS (fa/+ 100 and fa/+ 200), as was to be expected (Fig. 7). The same pattern was observed in the lipid profiles. No significant differences (P > 0.05) were found in the total cholesterol (CHOL), LDL, HDL or triglyceride concentrations among the tested groups (Fig. 8).
Glucose and lipid profile at the beginning of the study (t1) and after eight weeks of pomegranate supplementation (t2)
Index | fa/fa H2O | fa/fa 100 | fa/fa 200 | fa/+ 100 | fa/+ 200 | |||||
---|---|---|---|---|---|---|---|---|---|---|
t1 | t2 | t1 | t2 | t1 | t2 | t1 | t2 | t1 | t2 | |
17.50 ± 5.90 | 59.40 ± 11.30 | 21.40 ± 6.40 | 63.00 ± 12.80 | 21.10 ± 8.50 | 57.80 ± 8.50 | 7.60 ± 0.60 | 23.00 ± 3.70 | 7.70 ± 1.20 | 22.90 ± 2.80 | |
4.30 ± 0.30 | 5.10 ± 0.70 | 4.30 ± 0.20 | 4.80 ± 0.50 | 4.10 ± 0.10 | 5.10 ± 1.00 | 3.40 ± 0.20 | 2.20 ± 0.30 | 3.60 ± 0.10 | 2.40 ± 0.10 | |
2.50 ± 0.10 | 2.90 ± 0.40 | 2.40 ± 0.20 | 3.00 ± 0.40 | 2.40 ± 0.20 | 3.20 ± 0.60 | 1.70 ± 0.10 | 1.30 ± 0.20 | 1.80 ± 0.10 | 1.40 ± 0.10 | |
2.20 ± 0.10 | 2.80 ± 0.20 | 1.90 ± 0.10 | 2.80 ± 0.20 | 1.90 ± 0.20 | 2.80 ± 0.40 | 1.20 ± 0.20 | 1.20 ± 0.20 | 1.20 ± 0.10 | 1.10 ± 0.10 | |
11.50 ± 2.70 | 5.60 ± 5.60 | 12.70 ± 3.40 | 5.70 ± 2.90 | 12.50 ± 2.80 | 5.90 ± 1.30 | 1.30 ± 0.30 | 1.20 ± 0.60 | 1.30 ± 0.30 | 0.90 ± 0.60 |
fa/fa H2O – control group of Zucker diabetic fatty (ZDF) rats with missense mutation in the
Pomegranate
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).
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
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.