EFFECTS OF POMEGRANATE (PUNICA GRANATUM L.) FRESH JUICE AND PEEL EXTRACT ON DIABETIC MALE ALBINO RATS

EFFECTS OF POMEGRANATE (PUNICA GRANATUM L.) FRESH JUICE AND PEEL EXTRACT ON DIABETIC MALE ALBINO RATS

By

Nageh Mabrouk Gabr

Department of Medical Physiology, Faculty of Medicine,

Al-Azhar University, Cairo, Egypt

ABSTRACT

Background: Pomegranate is used in traditional medicine for its therapeutic properties.

Objective: Evaluation of the effectiveness of pomegranate fresh juice and peel extract on diabetes mellitus.

Material and Methods: Sixty adult male albino rats of local strain were distributed randomly into 6 equal groups: normal control group, control group received Pomegranate fresh juice (500 mg/kg body weight per rat orally), control group received Pomegranate peel extract (500 mg/kg body weight per rat orally), diabetic group received alloxan (140 mg/kg body weight intraperitoneally), diabetic group received Pomegranate fresh juice (500 mg/kg body weight per rat orally), and diabetic group received Pomegranate peel extract (500 mg /kg body weight per rat orally).

Results: Oral administration of Pomegranate juice and peel extract to diabetic rats for 4 weeks significantly decreased serum levels of TC, TG, low density lipoproteins cholesterol (LDL-c), and liver enzymes when compared to the control group. Levels of high density lipoprotein cholesterol (HDL-c) and antioxidant enzymes significantly increased as compared to the control group. Histopathological examination of liver and pancreas of Pomegranate juice-treated groups showed amelioration of histological changes caused by high level of cholesterol in diabetic group. These results were more prominent by Pomegranate fresh juice than peel extract.

Conclusion: Pomegranate has antioxidant and lipid lowering effects and improves the health of pancreatic islets of Langerhans in diabetic rats.

Key words: Pomegranate, alloxan, diabetogenic, antiatherogenic, antioxidants, dyslipidemia.

INTRODUCTION

    Many metabolic disturbances accom-pany diabetes mellitus including hyper-glycemia, hyperlipidemia, relative or absolute deficiency of insulin, and increased oxidative stress. These abnormalities represent the backbone in the pathogenesis of diabetic complications (American Diabetes Association, 2016).

    Pomegranate (Punica granatum L.) is a member of the family of Punicaceae, one of the most ancient edible fruits and they are widely grown in Mediterranean regions including Iran, Egypt, Iraq and India, but sparsely cultivated in the USA, China, Japan and Russia (Matthaeus and Ozcan, 2016). The Pomegranate fruit has valuable compounds in different parts of the fruit. These parts can be divided into several anatomical origins: peel, seeds, and arils (Zhang et al., 2010).

    An important product obtained from Pomegranate fruit is the juice that can be obtained from arils or from whole fruit. The edible part of Pomegranate fruit represents 52% of total fruit weight, comprising 78% juice and 22% seeds. (Amri et al., 20017).  Both pomegranate juice and Pomegranate peel extract (PPE) are rich in bioactive compounds such as polyphenols, anthocyanidins, tannic acid, gallic acid, and ellagic acid that exert anti-oxidant activities and prevent oxidative stress (Faghihimani et al., 2017).

    Dietary intake of antioxidants can inhibit or delay the oxidation of susceptible cellular substrates so prevent oxidative stress. Therefore, it is important to enrich our diet with antioxidants to protect against many chronic diseases (Rouhi et al., 2017).

    The aim of the present work was to evaluate the effects of Pomegranate fresh juice and peel extract on diabetic adult male albino rats.

MATERIALS AND METHODS

- Pomegranate fresh juice: The fruits of fresh Pomegranate were washed and manually peeled, without separating the seeds. Pomegranate juice was obtained using a commercial blender (Moulinex) and filtrated with a Buchner funnel to remove water insoluble materials and immediately diluted with distilled water to volume of (1:10 water/volume). The juice was stored at -20°C for further use in a dose of 500 mg/kg body weight per rat by nasogastric tube (Al-Olayan et al., 2014).

- Pomegranate peel extraction (PPE): Pomegranate peels were separated from the fruit manually and were cut into small pieces (2 cm×2 cm). The cut pieces were dried for 5 days. The dried samples were ground into fine powder by a grinder (Moulinex). 500 grams of pomegranate peel powders were separately extracted in water (1:10 water/volume).  PPE were stored in a deep freezer (-18 °C) until use and administered orally at a dose of 500 mg/kg body weight per rat by nasogastric tube (Mesgari et al., 2016).

- Animals: This experimental study was performed at Medical Physiology department, Al-Azhar Faculty of medicine, Cairo. A total of sixty adult male albino rats of local strains were used in this study ranging in weight from 155-170 grams at the time of the research. The animals were housed under similar standard environmental conditions in suitable cages (20 x 32 x 20 cm for every 3 rats) with wide meshed raised floors to prevent coprophagia. They were kept ten days on basal diet before starting experimental diet for adaptation. They were also kept at room temperature and normal light/dark cycle. Animals were divided randomly and equally into 6 groups as follows:

Group I (Control Group): Rats fed on normal standard rat chow with free water supply and served as a control group.

Group II (Pomegranate fresh juice -treated group): Rats received 500 mg /kg body weight per rat for 4 weeks orally.

 Group III (Pomegranate peel extract -treated group): Rats received 500 mg /kg body weight per rat for 4 weeks orally.

Group IV (Alloxan-treated diabetic control Group): Rats were subjected to induction of diabetes by a single intraperitoneal injection of alloxan (140 mg/kg body weight) in normal saline and fed on normal standard rat chow diet.

Group V (Diabetic pomegranate fresh juice-treated group): Rats were subjected to induction of diabetes and received pomegranate fresh juice 500 mg /kg body weight per rat for 4 weeks orally.

Group VI (Diabetic Pomegranate peel extract -treated group): Rats were subjected to induction of diabetes and received 500 mg /kg body weight per rat for 4 weeks orally.

- Determination of body weight gain percentage (BWG %): The biological values of diets were assessed by the determination of body weight gain percent (BWG %) which was calculated at the end of the experimental period. It was calculated using the equation ofLei et al. (2007):

- Induction of diabetes mellitus: Alloxan (Sigma Pharmaceuticals Company) was dissolved in 0.9% NaCl and injected intraperitoneally at a dose of 140 mg/kg body weight after overnight fasting. Just before alloxan injection, 2 ml of glucose (5%) were given orally.  Blood samples was collected on the 3rd day ensure production of diabetes. Rats with blood sugar higher than 200 mg/dl were considered diabetic (Ezazul et al., 2012).

- Collection of Blood Samples: Blood was collected (4 ml of blood each) from the retro-orbital plexus using heparinized capillary tube (0.75 - 1.0 mm internal diameter) inserted in the medial canthus. To obtain serum, the blood was collected into a dry clean graduated glass centrifuge tube. It was rapidly set to centrifuge at 3000 r.p.m. for 15 minutes. About half of the supernatant serum was sucked out into Eppendorf tubes and stored frozen at -20ºC (Margoni et al., 2011).

- Histopathological studies: At the end of the 4th week and under ether anesthesia, abdomen of the animal was opened after reaching the stage of surgical anesthesia, as evident by loss of withdrawal reflex. Pancreas and the liver were excised for histopathological studies. Specimens from both tissues were taken immediately and stained with Hematoxylin and eosin (H and E) and examined microscopically according to William (2010).

- Statistical Analysis: Data input and analysis were done using SPSS computer program. All results were expressed as the mean ± standard deviation (SD). Mean values of the different groups were compared using a one way analysis of variance (ANOVA). Least significant difference (LSD) post hoc analysis was used to identify significantly different mean values. P Value < 0.05 was accepted to denote a significant difference.

RESULTS

     In this study, body weight gain percen-tage (BWG %) was 13.29 ± 1.15, 13.30 ± 1.15, 13.31 ± 1.15, 28.16 ± 1.30, 16.76 ± 1.9, and 17.01 ± 3.05 in groups I, II, III, IV, V and VI respectively.  Groups II and III showed insignificant changes in respect to the control group I.  There was a significant increase in this parameter in diabetic group IV when compared with the control group. Pomegranate (fresh juice and peel extract)-treated groups showed a significant decrease in body weight gain percent (BWG %) in relation to group IV. There were no significant differences in body weight between group II and III when compared to group I. there were no significant changes also between group V and VI when compared with diabetic group (Table 1).

    The mean ± standard deviation of blood glucose was 95.3 ± 8.9, 92.3 ± 11.47, 90.35 ± 11.3, 379.2 ± 23.9, 287.4 ± 12.58 and 293.9 ± 19.4 mg/dl in in groups I, II, III, IV, V and VI respectively. Groups II and III showed insignificant changes in respect to the control group I. Diabetes induced by alloxan resulted in a significant elevation in the levels of fasting blood glucose (FBG) in group IV (diabetic group) in respect to control group I. while the treatment with Pomegranate fresh juice and peel extract reduced the elevated fasting blood glucose in groups V and VI respectively in respect to untreated alloxan-induced diabetic group, but still significantly higher than that of  groups I, II and III. Also, there was no significant difference in fasting blood glucose levels between group V and group VI (Table 2).

     It is noted that Pomegranate fresh juice has no significant influence on plasma glucose level and lipid profile of normal rats. Also, these results were prominent changes in treatment with Pomegranate juice than treatment with Pomegranate peel extracts (Table 2). 

     The mean ± standard deviation of total serum cholesterol was 110.4 ± 10.3, 107.74 ± 7.98, 197.1± 13.7, 190.5 ± 7.15, and 186.6 ± 11.5 mg/dl in groups I, II, III, IV, V and VI respectively. Whereas the mean ± standard deviation of triglycerides (TG) was 160.2 ± 16.7, 159.3 ± 32.4, 146.9 ± 15.2, 269.2 ± 35, 149.2 ± 23.5 and 164.2 ± 35.2 mg/dl in groups I, II, III, IV, V and VI respectively. Groups II and III showed insignificant changes in both total cholesterol and triglycerides in respect to the control group I. Treatment with Pomegranate fresh juice and peel extract significantly decreased the total serum cholesterol and triglycerides levels when compared to group IV (Table 2).

    As regard LDL levels, the mean ± standard deviation was 4.3 ± 0.9, 5.9 ± 1.5, 4.8 ± 1.2, 76.8 ± 8.7, 60.7 ± 14.5 and 69.9 ± 8.1  mg/dl in groups I, II, III, IV, V and VI respectively. Groups II and III showed insignificant changes in LDL level in respect to the control group I. Treatment with Pomegranate fresh juice and peel extract significantly decreased LDL levels when compared to group IV. Also, there was no significant difference in LDL levels between group V and group VI (Table 2).

    The mean ± standard deviation of HDL was 74.6± 4.2, 75± 6.2, 76.3± 9.3, 63.9± 7.2, 94.7± 9.4 and 92.3± 17.1 mg/dl in groups I, II, III, IV, V and VI respectively. Groups II and III showed insignificant changes in HDL in respect to the control group I. There was a significant decrease in the level of HDL levels in diabetic group (group1V) when compared to the normal control group I.  Treatment with Pomegranate fresh juice and peel extract significantly increased HDL levels when compared to group IV. Also, there was no significant difference in HDL levels between group V and group VI (Table 2).

     It is noted that Pomegranate fresh juice has no significant influence on plasma glucose level and lipid profile of normal rats. Also, these results were prominent changes in treatment with Pomegranate juice than treatment with Pomegranate peel extracts (Table 2). 

    The mean ± standard deviation of AST was 55.24 ± 2.33, 54.24 ± 1.22, 55.94 ± 3.03, 106.38 ± 4.33, 90.02 ± 0.22 and 92.72. ± 1.59 U/L in groups I, II, III, IV, V and VI respectively. Whereas the mean ± standard deviation of ALT was 26.74 ± 0.88, 25.34 ± 0.55, 24.74 ± 0.72, 50.00 ± 1.01, 38.50 ± 1.10 and 40.50 ± 1.1 U/L in groups I, II, III, IV, V and VI respectively. Groups II and III showed insignificant changes in both AST and ALT in respect to the control group I. there were significant increase in both AST and ALT levels in diabetic groups in respect to control group. Results of liver function tests of diabetic rats orally given Pomegranate fresh juice and peel extract showed significant decreases of AST and ALT levels in respect to control group. These results were also more significant in ALT than AST and in treatment with Pomegranate fresh juice than treatment with Pomegranate peel extracts (Table 2).

Table (1):Effects of diabetes and pomegranate on initial body weight, final body weight and body weight gain %( BWG %) in different groups..

Number of rats in each group = 10.               @ Groups I, II, and III were compared to control group IV.

*All groups were compared to control group1.                Ω Groups II was compared to control group III

# Groups V and VI was compared to control group        ¶ Groups V and VI were compared to group IV..

Table (2): Effects of diabetes and Pomegranate in different groups (Mean ±SD)

Number of rats in each group = 10.                        @ Groups I, II, and III were compared to control group IV. *All groups were compared to control group1.                        Ω Groups II was compared to control group III

# Groups V and VI was compared to control group1.              ¶ Groups V and VI were compared to group IV.

     There was a significant decrease in the level of catalase as an antioxidant (21.7 ± 2.83 U/ml) and a significant increase in the level of MDA as an oxidant (23.73 ± 3.29 µmol/l) in diabetic group in comparison to control group (Table 2). On the other hand, after treatment of alloxan-induced diabetic rats with Pomegranate juice, the level of catalase was significantly increased (39.5 ± 2.27 U/ml), and MDA level significantly decreased (16.37 ± 2.77 µmol/l) in respect to untreated alloxan-induced diabetic group. After treatment of alloxan-induced diabetic rats with Pomegranate peel extract, the level of catalase was significantly increased (38.20 ± 3.08 U/ml), and MDA level significantly decreased (14.1 ± 2.54 µmol/l) in respect to untreated alloxan-induced diabetic group (Table 2).

    These results were also more significant in treatment with Pomegranate fresh juice than treatment with Pomegranate peel extracts.  

    Histopathological examination of the pancreas showed normal pancreatic islets, pancreatic acini and normal blood vessels in groups I, II and III (Figure 1). On the other hand, pancreatic islets decreased in number and size, normal pancreatic acini and thick wall of the blood vessels, in diabetic group (Figure 2). The pancreas in diabetic group received Pomegranate juice and that received Pomegranate peel extracts showed normalized number of pancreatic islets and decreased size, some normal pancreatic acini and partial improvement of the thick walled blood vessels (thinner than diabetic group but thicker than normal group) (Figure 3).

    Histological examination of liver of the control groups I,II and III demonstrated normal histological pattern where hepatic lobules appeared as hexagonal masses of hepatocytes radiating form a central vein. Blood sinusoids appeared between cords of hepatocytes. The hepatocytes had a hexagonal outline with central rounded nucleus. The cytoplasm showed some vacuoles (Figure 4). Diabetic group revealed marked impairment of the normal structure of hepatic lobules in many areas and deposition of large lipid droplet in cells, vacuolar degeneration and swollen cells (Figures 5a and 5b). Diabetic rats treated by Pomegranate juice showed more improvement in histological structures comparing with sections of diabetic rats that orally given Pomegranate peel extract. The examined sections showed almost normal structure with regular arrangement of hepatocyte cell cords and exhibited a reduction in fat accumulation. The hepatocytes around the central vein (CV) showed rounded nuclei and vesicular indicating active cells. Blood sinusoids between the cells had also normal appearance (Figure 6).

Figure (1): Section in the pancreas of group I, II and III (normal control groups) showing some normalized pancreatic islets (A), some normal pancreatic acini (B) and normal blood vessels (C). Hx&E, 400X.

Figure (2): Section in the pancreas of group IV (diabetic-group) showing pancreatic islets decreased in number and size (A), normal pancreatic acini (B) and thick wall of the blood vessels (C). Hx&E, 400X.

Figure (3): Section in the pancreas of group V and VI- diabetic-received Pomegranate juice and Pomegranate peel extracts respectively showing normalized number of pancreatic islets and decreased size (A), normal pancreatic acini (B) and improvement of the thick walled blood vessels (C). Hx&E, 400X.

Figure (4): Section in the liver (Hx&E, 400X) of group I, II and III (normal control groups) showing Normal hepatocytes arranged in plates.

                                     a                                                                  b

Figure (5): Section in the liver of group IV (diabetic-group) showing vacuolar degenerated hepatocytes, swollen and vacuolated cells with lymphocytic infiltration (a). Degenerated hepatocytes showing ballooning (b). Hx&E, 400X.

Figure (6): Section in the liver of group V and VI- diabetic-received Pomegranate juice and Pomegranate peel extracts respectively showing almost normal structure with regular arrangement of hepatic cell cords (black thin arrows) around the central vein. Hepatic sinusoids between the cells showed normal appearance (white arrows). Hx&E, 400X.

DISCUSSION

     In spite of the presence of known antidiabetic medicines in the pharma-ceutical market, remedies from medicinal plants are used with success to treat diabetes and its complications.

    Results of the present study revealed incidence of significantincreases in BWG % of diabetic rats when compared to thecontrol rats. These findings were in agreement with those obtained by Amin et al. (2011) and Nwozo et al. (2011) who confirmed our results. The increase in body weight of diabeticrats might be due to the increase of feed and caloric intake by rats.

    In the present study, there was a significant increase in blood glucose level in diabetic group (IV) when compared with the control group (I). In treated groups with Pomegranate fresh juice and peel extract (V and VI), there were significant decrease in blood glucose levels when compared with diabetic group (IV). The mechanisms by which alloxan brought about its diabetic state included selective destruction of pancreatic insulin secreting ß-cells, which make cells less active and lead to poor glucose utilization by tissues (Papaccio et al., 2017). Amri et al. (2017) attributed the anti-diabetic effect of pomegranate contents as phenolic compounds, tannic acids, gallic acid, ellagic acids and flavonoides which have hypoglycemic effects.

    The result of this study revealed that treatment of alloxan-induced diabetic rats with pomegranate (fresh juice and peel extract) significantly reduced blood glucose level, and this triggered the liver to revert to its normal homeostasis during experimental diabetes (Rouhi et al., 2017). The anti-hyperglycemic activity of pomegranate may be through a stimulatory effect on insulin secretion or through improvement of insulin action. Also, pomegranate may have extra pancreatic mechanism of action which improves pancreatic ß-cell function, and thus enhance insulin secretion (Papaccio et al., 2017).

    Results of the present study revealed that diabetic rats showed significant increases in serum concentrations of TC, TG and LDL-c and the reduction in serum HDL-c. The present findings were in the same line as with those reported by Frantz et al. (2012) who demonstrated that lipid metabolic disorders and levels of serum TC and TG increased significantly when compared with control group.

    Concerning serum TG level, the present findings agreed with the study of Rouhi et al. (2017) who demonstrated that plasma TG level increased significantly in diabetic rats.

    Regarding to serum LDL-c and HDL-c levels in diabetic group, the current results were in agreement with those of Kumar et al. (2010) who concluded that oxidation of LDL-c resulted in formation of a wide range of biologically active products, including peroxides and malondialdehyde. Moreover, Sezer et al. (2011) demons-trated that the oxidative modified lipids and their degradation products are believed to have adverse effects such as pro-inflammatory, immunogenic and cytotoxic activities which contribute to both the initiation and progression of atherosclerotic lesions.

    The present study showed that serum HDL-c level was decreased significantly in diabetic group in respect to the control group. These results were well documented by the study of Farideh et al. (2017). It has been reported that cholesterol transport to extra-hepatic tissues is primarily ensured by LDL-c (bad cholesterol); while HDL-c (good cholesterol) has an important role in reversing the cholesterol transport process (Faghihimani et al., 2017).

    The present study showed that oral administration of Pomegranate juice significantly decreased serum level of TC, TG and LDL-c but increased HDL-c as compared to the control group. These findings correlated with those obtained by Tezcan et al. (2016) who reported that Pomegranate juice consumption by diabetic rats significantly reduced cholesterol accumulation and foam cell formation in tissues. Pomegranate juice treatment significantly inhibited the progression of atherosclerotic lesions by inhibition of atherogenic modifications of LDL-c including its retention, oxidation, and aggregation.

    Rouhi et al. (2017) reported that diabetic rats with elevated blood lipids treated with Pomegranate juice experien-ced significant reductions in their TC and LDL-c.

     The results of Farideh et al. (2017) demonstrated that Pomegranate juice can inhibit LDL-c oxidation by polyphenols content that inhibit copper ion-induced LDL-c oxidation, and thus reduce the oxidized LDL content. Pomegranate juice polyphenols also increase the activity of serum HDL-c associated paraoxonase 1 (PON1) which hydrolyze lipid peroxides and convert them to a less atherogenic LDL-c thus causing further reduction in LDL content (Czerska et al., 2015).

    Plasma AST and ALT, alone or in combination are primarily recommended for the assessment of hepatocellular injury. They are sensitive markers for drug-induced liver damage, and the elevated activities of these marker enzymes in plasma are indicative of cellular leakage and loss of the functional integrity of cell membranes in the liver (Gurbet et al., 2013).

    Results of the present study showed that there were significant decreases in serum levels of AST and ALT enzymes in diabetic rats orally given Pomegranate juice compared to the control group. The present results agreed with the results obtained by Osman et al. (2012) who examined the antioxidant effect of Pomegranate juice and peel on diabetes mellitus induced by alloxan in Female Rats. The results showed that AST and ALT were significantly increased in diabetic group, but after treatment with peel and juice, AST and ALT levels decreased and become near to the control level especially ALT value. This effect is due to antioxidant content of Pomegranate juice and peel extract. ROS stimulate glutathione and superoxide dismutase enzymes depletion, accumulation of lipid peroxides and oxidative damage of different organelles in liver tissue (Daftardar et al., 2014). Antioxidant supplements may have a role in preventing or treating hepatic lesion in patients with diabetes (Siham et al., 2017).

    The ameliorative effect as anti-oxidant was in agreement with Messarah et al. (2012) who revealed that stimulation of anti-oxidant defense system reversed hepatic stress.

    The biochemical results of our study were confirmed by histopathological findings, which seen in liver sections. The histological findings of liver of the treated rats showed almost completely normal structure with regular arrangement of hepatocyte cell cords and exhibited reduction in fat accumulation. The nuclei of hepatocytes around the central vein (CV) were rounded and vesicular indicating active cells. Blood sinusoids between the cells had also normal appearance when compared to the positive control group. These histological findings agreed with the study of Fyiad et al. (2012) who investigated the effect of Pomegranate juice on nucleic acids alterations and oxidative stress in experimentally hepatitis rats.

    As indicated in the present study, the untreated diabetic rats had significant decrease in the level of antioxidant enzyme system as catalase enzyme. These findings agreed with Illana et al. (2014) who reported that diabetes mellitus enhanced the free radical generation in various ways. Several studies suggested that disorders of lipid metabolism, hyperlipidemia and obesity are associated with overproduction of oxygen free radicals (Issaoui et al., 2010). The enhanced accumulation of these free radicals and dysfunction of antioxidant defense system resulted in oxidative stress (Gouda et al., 2016).

    Pomegranate juice and peel extract were shown to have significant higher levels of antioxidants in comparison to commonly consumed fruit juices, such as grape, cranberry, grapefruit or orange juice (Tapias et al., 2014). The principal antioxidant polyphenols in Pomegranate juice extract include tannins and anthocyanins which have been shown to be the antioxidant responsible for the free radicals scavenging ability of Pomegranate juice (Illana et al., 2014). Farideh et al. (2017) concluded that Pomegranate has also been shown to protect the antioxidant enzymes CAT from the effects of toxic chemicals.

    Turk et al. (2008) reported that there was a significant decrease in malondialdehyde (MDA) level and marked increase in catalase (CAT) activities in rats treated with Pomegranate seeds juice.

     In the present study, Oral adminis-tration of Pomegranate juice and peel extract caused a significant increase in the activity of CAT enzymes when compared to the control group. The improvement of CAT enzyme activities could be explained by antioxidant properties of Pomegranate seeds extract due to presence of bioactive polyphenolic compounds which play a role in scavenging free radicals and also prevent DNA damage (Rom et al., 2016).

     Valadares et al. (2010) confirmed the ability of Pomegranate to protectDNA and prevent chromosomal damage in mice. In addition, Rouhi et al. (2017) demonstrated that Pomegranate afforded up to 60 % protection against hepaticlipid peroxidation due to maintenance of the GSH and serum levels and activities ofCAT, GPx and glutathione reductase (GR) enzymes.

ACKNOWLEDGEMENT

     I would like to express my deep appreciation and utmost thanks to Assist. Prof. Dr. Amr El-Karef, Assistant Professor of Medical Pathology, Faculty of Medicine, Mansoura University, for his great help in this work

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