Antioxidant and Antidiabetic Potentials of Punica granatum Peel Extracts in Alloxan-Induced Diabetic Albino Rats

Aamir Khan Awan1, Nighat Sultana1*, Rahmat Ali Khan2, Rifhat Sultana3,

Umm-e-Kalsoom1, Fayaz Ahmed Sahibzada4, Rifat Ullah Khan5, Naimat Ullah Khan6, Nazir Ahmad Khan7, Syed Haider Zaman8, Mir Sadiq Shah9 and Assar Ali Shah10*

1Department of Biochemistry, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan.

2Department of Biotechnology, University of Science and Technology, Bannu, Khyber Pakhtunkhwa Pakistan.

3Department of Chemistry, Hazara University Mansehra, Khyber Pakhtunkhwa, Pakistan.

4Department of Clinical Nutrition AIMS, Hospital, Muzaffarabad, Pakistan.

5College of Veterinary Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan.

6College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University, Mardan, Pakistan.

7Department of Animal Nutrition, The University of Agriculture Peshawar, 25130, KP, Pakistan.

8Department of Clinical sciences, KBCMA, College of Veterinary and Animal sciences, Narowal sub-campus University of Veterinary and Animal sciences Lahore, Pakistan.

9Department of Zoology, University of Science and Technology, Bannu, Khyber Pakhtunkhwa, Pakistan.

10Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand.

ABSTRACT

The peel/rind of Punica granatum is approximately 60% of the total fruit weight which is generally wasted every year, although, it is known to have a variety of medicinal benefits. The present study was conducted to find the antioxidant activity and antidiabetic potentials of P. granatum peels extracts in alloxan-induced diabetic albino rats. The diabetes was induced in albino rats using alloxan at the concentration of 150 mg kg-1 body weight. After 15 days, the treated groups with glabinclemide and different varieties of methanolic and aqueous extracts showed a significant (P<0.05) decrease in the blood glucose level as compared to diabetic control while slight differences were seen in body weight. The total cholesterol, triglycerides, and low-density lipoprotein, total bilirubin, alkaline phosphatase, alanine aminotransferase, urea, serum creatinine and total proteins were significantly (P<0.05) decreased in response to oral dosing of three varieties of P. granatum peel methanolic and aqueous extracts which were comparable to healthy control. Moreover, the aqueous extracts and wild P. grantaum were more effective than methanolic extracts and other species against hyperglycemia and biochemical profiles of treated rats. It was concluded that the extracts of peels of P. granatum possess significant antihyperglycemic and antihyperlipidemic potentials due to presence of phytochemical and have no side effects. Based on the outcome of the study it can be recommended that the peel of P. granatum can be further used for the development of antidiabetic drugs or in folk medicines traditionally to treat the diabetes.

Article Information

Received 15 May 2022

Revised 05 November 2022

Accepted 23 November 2022

Available online 22 May 2023

(early access)

Published 12 October 2024

Authors’ Contribution

AKA, NS and RAK conceived and designed the experiments. RS, NAK, NUK UK, and FAS performed the experiments. SHZ, MSS, RUK and AAS analyzed the data computationally. AKA, NS and AAS wrote the manuscript.

Key words

Antioxidant activity, Alkaline phosphatase, Alanine aminotransferase, Diabetes mellitus, Punica granatum

DOI: https://dx.doi.org/10.17582/journal.pjz/20220515110519

* Corresponding author: nighat.sultana@hu.edu.pk, assaralishah@yahoo.com

0030-9923/2024/0006-2685 $ 9.00/0

Copyright 2024 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Diabetes mellitus (DM) is a chronic metabolic disorder, affecting millions of people all over the world each year with an increasing rate of mortality which results in imposing a major socio-economic challenge (Middha et al., 2013; Akhtar et al., 2017; Cho, 2018). According to the International Diabetes Federation (IDF) 451 million (M) people were affected all over the world with diabetes in 2017 which could be reckoned to be 693 M by 2045 if effective stratigies are not developed to control it (Abeeleh et al., 2009). Among the other non-insulin dependent diabetes mellitus (NIDDM) is the most common type which is due to insulin resistance and dysfunctioning of insulin receptor and ultimately β-cell in results lead to hyperglycemia (Zeb et al., 2015). The commercially available antidiabetic drugs including insulin, thiazolidinediones biguanides, and sulfonylureas are less stable and have many other side effects on the body. Albeit, the rising rate of diabetes is consistently increasing with time but no effective treatment with minimum side effects are available so far (Singh and Gupta, 2015). Previously, the antidiabetic potentials of raw extracts of many plants and herbs were determined in albino mice or rats due to their close resemblance with human DM (Thanh et al., 2019).

Plant materials have been reported as a major source of natural antidiabetic drugs for the last many years. According to WHO, around 80 % of the population from all over the world rely on traditional medicine (Uttra et al., 2018). In the subcontinent, the plants oriented medicine used extensively for the last many decades. Pakistan is also one of the countries which extensively use medicinal plants as traditional medicines (Ismail and Yahyeh, 2009; Uttra et al., 2018). Plants posseses the medicinal properties due to the presence of wide range of biochemical constitute (natural drugs) which contributes in many biological activities including antidiuretic, antimicrobial and antioxidant properties (Goncalves and Romeiro, 2019; Shah et al., 2019, 2020). Due to significant biological properties of plants they can be used as safest and richest source of natural drugs. Currently, natural products have been reported as a valuable source of drugs, especially in the era of cancer and infectious diseases (Huang et al., 2017). Recent decades have witnessed a renewed enhancement in the demand for herb-based medicinal and nutraceutical products because of their cost-effectiveness and fewer side effects (Sagbo et al., 2018). These herb-based medicinal and nutraceutical products cannot be prepared through synthetic procedures due to their complex structures. So, therefore the idea of extraction and purification of these biomolecules has been developed and new advancement with time was seen.

The Punica granatum (family of Punicaceae) commonly known as pomegranate is the oldest fruit of tropical and subtropical regions and has been used as food for the last many decades (Anwar et al., 2007). With diverse varietal distribution between wild and cultivated genotypes the worldwide production of pomegranate is around 1.5 tons (Ay et al., 2012). Red pomegranate (locally called Q and harianar) is considered the best variety in Pakistan due to its size and taste, though white and wild pomegranates are also grown in their different parts. Wild pomegranate is sour in taste and small in size, contrary to the cultivated ones, which are sweet and delicious, indicating different biochemical properties. Medicinal properties of P. granatum against diarrhea, ulcers, acidosis, hemorrhage, anemia, cardiac and respiratory diseases were reported previously (Ay et al., 2012; Middha et al., 2012; Shaikh and Bhandary, 2021). Moreover, it also showed immune-boosting effects against bacterial, viral, and fungal infections (Middha et al., 2013). The health benefits of P. granatum could be attributed to its chemical constituents including phenolics, flavonoids, terpenoids, phytosterols, glycosides, and tannins present in its different parts.

The peel/rind of pomegranate is approximately 60 % of the total fruit weight (Middha et al., 2012). While the fruit has a variety of applications, its peel is generally wasted, in spite of having a variety of medicinal benefits. Need arises to explore the chemical constituents and medicinal importance of the P. granatum rind. Therefore, the rationale of the proposed study was to decipher the antidiabetic and anti-hyperlipedemic potentials of peels of three varieties of P. granatum cultivated in Pakistan. Moreover, the antioxidant potential and the effects of peels extracts on body weight liver functions test (LFTs) and renal functions test (RFTs) were also measured.

MATERIALS AND METHODS

Plant collection and extracts preparation

All the three varieties of Punica granatum including red, white, and wild fresh and healthy fruits were collected from Mardan, the local market of Khyber Pakhtunkhwa (PK), Pakistan as shown in Figure 1. The plant specimens were identified by Botanist at the Department of Botany, Hazara University, Mansehra, KP, Pakistan, and then deposited in the departmental herbarium. The peels of all three varieties were separated carefully, washed with dH2O, and dried at room temp (25-30oC) under shadow for two weeks, and ground into fine powdered. Then, 250 ml of commercial methanol (95%) was added in 50 g peel’s powder and mixed properly. Then, the mixture was incubated at 25-30oC for 2 weeks with a regular shaking. After the incubation the homogenate was filtered with double-layer cheesecloth followed by Whatman filter paper (Circles: 10 mm to 150 cm (available prepleated) Sheets: 26 x 31 mm to 600 x 600 mm). The methanol from the filtrate was then evaporated to dry mass using a rotary evaporator under reduced pressure at 45oC. Then, 3 g of dried mass was re-suspended in each 15 mL of methanol and dH2O separately to prepare aqeous and methanolic extracts and stored at 4 °C for further analysis (Das and Barman, 2012).

 

DPPH radical scavinging activity

Antioxidant activity of methanolic and aqueous fractions of all varieties P. granatum peel was determined based on the scavenging potentials of 2, 2- diphenyl-2-picrylhydrazyl (DPPH) free radical as reported previously (Brand et al., 1995). The three dilutions of each extract 1, 2, and 3mg mL-1 were prepared. 1 mM DPPH was prepared and mixed with each extract (n =3) in 4:1 ratio and incubated for 30 min at room temperature and then the absorbance was measured at 517 nm. Ascorbic acid was used as a positive standard. The inhibition (%) of DPPH was calculated as; Inhibition of DPPH (%) = Abs of sample- Abs of control/ Abs of sample ×100.

In vitro antidiabetic activity

For the determination of activity against α-amylase, each extract was prepared separately. Then, 1.4 % starch solution (Sigma, Aldrich) and 0.05 % α- amylase were prepared in dH2O. The commercially available antidiabetic drug, Glucophage (metformin-500 mg) (Martin dow) was used as a reference to determine the inhibition of α-amylase (Merck pharma, Karachi Pakistan). The assay mixture consists of 200 µl of 0.02M sodium phosphate buffer, 20 µl of 0.05 % α- amylase enzyme, and 300 µl of three different concentrations (1mg/ml to 3 mg/ml) of each extract were incubated for 10 min at room temperature. Then, 200 µl of the starch solution was added to initiate the reaction by following the addition of 400 µl DNS and placed in a boiling water bath for 5 min to terminate the reaction. The mixture was then cooled at room temp and the absorbance was measured at 540 nm. The mixture without extract was used as a control. The % inhibition was calculated according to the following formula (Lewis and Liu, 2012).

Experimental animals and induction of diabetes

A total of 54, approximately three months old female albino rats in a weight range of 185–200 g-1 were used to evaluate the antidiabetic and hypolipedemic potentials of peels extracts of P. granatum. The rats were purchased from the Department of Pharmacy, the University of Sargodha and were maintained in steel cages at room temp (24-26°C) with 12 h light/darkness for 7-21 days in the laboratory. After one week of adaptation, diabetes was induced in albino rats through a single intraperitoneal injection of alloxan, 150 mg kg-1 b.w, in the peripheral abdomen. After 8 h of fasting all the rats were given 5% glucose for 6 h and then 20% glucose and foods for 10 h. The blood glucose level through glucometer strips using SD glucometer (G110 Germany) was measured to find the hyperglycemic condition after 48 h of alloxan administration. Rats having fasting blood glucose levels > 200 mg/dl were considered as diabetic and selected for further study. All the rats were grouped into nine different groups (n=6) and given different treatments, provided free food and water, and maintained in specific laboratory conditions. The rats were randomly divided into 9 groups (G1 to G9) each of six. G1, Normal control; G2, Diabetic control; G3, Glibinclamide (10 mg kg-1) (Alliance pharma); G4, wild (daroona) pomegranate peel aqueous extract (DPPw), (150 mg kg-1); G5, red pomegranate peel methanolic extract (RPPm) (150 mg kg-1); G6, white pomegranate peel aqueous extract (WPPw) (150 mg kg-1); G7, wild pomegranate peel methanolic extract (DPPm) (150 mg kg-1); G8, white pomegranate peel methanolic extract (WPPm) (150 mg kg-1); G9, pomegranate peel aqueous extract (RPPw) (150 mg kg-1).

Collection of blood samples and biochemical analysis

For the determination of glucose, blood sample was collected from the tail of each rat at different intervals (1st -15th day) during the treatment. The glucose level was measured with glucometer strips using SD glucometer (G110, Germany) at 1st, 5th, 10th, and 15th days of treatment. After 15 days all rats were anesthetized by chloroform and blood samples were taken through cardiac puncture for biochemical analysis including Liver functioning tests (LFTs) such as serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bilirubin, renal function tests (RFTs) such as urea, serum creatinine, and total proteins and lipid profile including total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL) and low-density lipoproteins (LDL) (Khan et al., 2019; Shah et al., 2019).

Statistical analysis

The statistical analyses were done in a completely randomized design. The analysis of variance (One way ANOVA) was performed using SPSS (version 18.0). Means of the significantly different parameters were separated by Tukey’s test. A P value < 0.05 was considered statistically significant.

RESULTS

DPPH scavenging activity of P. granatum extracts

The in-vitro antioxidant activity of three different concentrations (1 mg mL-1, 2 mg mL-1, and 3 mg mL-1) of each peels extracts showed DPPH scavenging potentials. The peel extract of wild variety showed the highest activity in both methanolic and aqueous extract which was comparable to standard ascorbic acid as shown in Figure 2.

 

In-vitro antidiabetic activity (α-amylase inhibition) of P. granatum extract

The in-vitro antidiabetic potentials of peel extracts of all three varieties of P. granatum showed significant inhibition against α-amylase in both methanolic and aqueous extract which was comparable with commercially available drug Glucophage (Martin Dow) as shown in Figure 3. The acute toxicity study revealed no mortality or any physical or behavioral changes in rats after oral dosing of peels extract at a dose of 150 mg kg-1 b.w. Hence, the same dose was selected further to evaluate the antidiabetic potentials and biochemical profiling of all rats.

 

Effect of peel extract of P. granatum on blood glucose level

The effects of orally administered methanolic and aqueous peels fractions of all varieties of P. granatum on blood glucose levels in alloxan-induced diabetic albino rats are shown in Table I. The fasting blood glucose level in all untreated diabetic rats (G2-G9) was significantly high compared to control (healthy rats). After oral administration of the methanolic and aqueous extracts of three varieties of P. grantum peels at 150mg/kg b.w concentration showed significant (P<0.05) reduction in the blood glucose level at 1st, 5th, 10th, and 15th days of treatment.

Effect of peel extracts of P. granatum on body weight

A slight variation in the body weight was seen in treated diabetic rats compared to normal control after the treatment but no significant differences were observed as shown in Table II.

 

Table I. Effect of peel extract of P. granatum on blood glucose level in alloxan induced diabetic albino rats

Samples	Groups	Dose (mg/kg body weight)	Glucose level (mg/dl) at different days interval
			1st day	5th day	10th day	15th day
Healthy (Untreated)	Normal control	--	86.2±0.4	88.6±0.5	88.4±0.5	86.8±0.2
Diabetic (untreated)	Diabetic control	--	227.1±6.6**	236.2±6.4**	248.2±6.4**	267.2±6.0**
Standard	Glibenclamide	10	211.3±4.5	207.4±5.6+	202.3±5.2+	193.5±4.4+
Aqueous extract	RPPw	150	284.7±5.5	264.7±6.6	197.5±6.6+	148.7±5.4++
	WPPw	150	215.3±4.3	142.3±4.7++	119.4±5.8++	92.6±6.6++
	DPPw	150	288.2±6.2	239.5±4.4	197.2±3.8+	143.5±5.5++
Methanolic extracts	RPPm	150	291.6±4.4	273.6±4.4	239.6±4.6	113.3±5.2++
	WPPm	150	265.2±5.2	223.7±4.3	213.3±5.7	128.4±5.2++
	DPPm	150	262.5±3.7	233.3±4.4	196.4±5.2+	157.3±4.8++

 

Mean ± SEM (n=6)** indicate significance from the diabetic control group at P<0.05 and P<0.01 probability level. +, ++ indicates significance from the Punica granatum peels group at P<0.05and P<0.01probability level, respectively. RPP, red pomegranate peel; WPP, white pomegrants peel; DPP, wild pomegranate peel.

 

Table II. Effect of peel extracts of P. granatum on body weight in alloxan induced diabetic albino rats.

Samples	Groups	Dose mg (kg body weight)	Body weight (mg) at different time interval
			1st day	5th day	10th day	15th day
Healthy (Untreated)	Normal control	-----	189.3±5.4	191.2±4.2	191.5±5.3	192.6±5.3
Diabetic (untreated)	Diabetic control	-----	189.5±3.7	187.5±5.3	184.2±6.3	182.5±5.7
Standard	Glibenclamide	10	180.6±4.9	180.2±4.5	182.5±6.5	185.5±7.3
Aqueous extracts	RPP	150	180.3±6.6	180.3±6.6	184.2±6.4	188.3±6.4
	WPP	150	186.2±5.5	186.6±5.5	191.6±6.6	193.3±4.7
	DPP	150	181.6±6.6	185.5±6.5	189.5±4.2	196.2±3.4
Methanolic extracts	RPP	150	190.2±4.6	193.1±4.6	193.5±3.3	196.7±5.5
	WPP	150	186.3±6.4	186.6±6.2	188.3±6.2	193.3±6.3
	DPP	150	182.5±5.6	189.3±5.4	191.6±6.6	198.2±6.6

 

Mean ± SEM (n=6). Each value indicated the body weight (mg). No significant differences (P>0.05) has been found. For abbreviation, see Table I.

 

Table III. Effect of peel extracts of P. granatum on liver function test (LFTs) in alloxan induced diabetic albino rats.

Samples	Groups	Dose (mg/kg body weight)	Total bilirubin (mg/dl)	ALP (mg/dl)	ALT (mg/dl)
Healthy (Untreated)	Normal control	--	0.78±0.3	53.2±2.2	99.3±2.8
Diabetic (untreated)	Diabetic control	--	1.24±0.2**	121.5±2.2**	187.5±0.1**
Standard	Glebinclamide	10	0.81±0.3++	110.5±3.1++	147.1±2.2++
Aqueous extract	RPP	150	0.87±0.3++	76.2±2.6++	137.5±3.5++
	WPP	150	1.00±0.1++	69.2±0.1++	109.7±4.6++
	DPP	150	0.87±0.2++	73.3±2.7++	115.5±3.5++
Methanolic extracts	RPP	150	0.83±0.3++	88.3±3.3++	141.5±3.2++
	WPP	150	0.80±0.2++	63.4±4.3++	131.5±3.2++
	DPP	150	0.99±0.1++	68.5±0.2++	148.3±3.3++

 

Mean ± SEM (n=6)** indicate significance from the diabetic control group at P<0.05 and P<0.01 probability level. +, ++ indicates significance from the Punica granatum peels group at P<0.05and P<0.01probability level respectively. ALP, Alkaline phosphatase; ALT, Alanine amino transferase. For abbreviation, see Table I.

 

Effect of peel extracts of P. granatum on liver function test

The effect aqueous and methanolic peels fractions of Punica granatum on liver profile such as total bilirubin, ALT and ALP in alloxan-induced diabetic and control rats are shown in Table III. A significant increase was observed in serum total bilirubin, ALT, and ALP in alloxan-induced diabetic groups. The daily oral effect of various fractions of peel extracts of P. granatum at the rate of 150 mg/kg b.w in alloxan-induced diabetes rats showed a significant (**P<0.05) decrease in total bilirubin, ALT, and ALP as compared to diabetic control which was comparable to normal control group.

Effect of peel extracts of P. granatum on renal function test

The effects of peels fractions at a dose of 150 mg/kg b.w on RFTs including urea, creatinine, and total protein are presented in Table IV. After induction, significant (**P<0.05) increase were seen in RFTs in diabetes-induced rats as compered to healthy rats (control). The daily dose effect of both type of peels extracts (aqueous and methanolic) of P. granatum showed a significant (**P<0.05) reduction in treated diabetic groups compared to diabetic control which was compareable to healthy control.

Effect of peel extracts of P. granatum on lipid profile

The effect of different varieties of peel extracts of Punica granatum on lipid profile in diabetic and control rats are shown in Table V. After induction of diabetes, an increase in TC, TGs, and LDL, and a decrease in high-density lipoprotein (HDL) was observed in diabetic groups as compared to normal control. The daily oral effect of three different varieties of peel fractions of Punica granatum at 150 mg kg-1 b.w concentration in diabetic rats showed a significant (**P<0.05) decrease in TC, TGs, and LDL and increase in HDL compared to the control groups (diabetic and normal).

 

Table IV. Effect of peel extracts of P. granatum on renal function test (RFTs) in alloxan induced diabetic albino rats.

Samples	Groups	Dose (mg/kg bodyweight)	Urea (mg/dl)	Creatinine (mg/dl)	Total proteins (mg/dl)
Healthy (Untreated)	Normal control	---	30.5±5.5++	0.85±3.5++	6.6±0.1++
Diabetic (untreated)	Diabetic control	150	55.5±2.2**	1.11±2.2**	8.3±0.2**
Standard	Glebinclamide	10	42.5±2.2++	0.61±1.6++	6.3±0.3++
Aqueous extract	RPPw	150	34.3±4.2++	0.95±3.2++	5.3±0.2++
	WPPw	150	38.2±3.2++	1.05±3.3++	5.8±0.2++
	DPPw	150	37.5±2.1++	0.95±0.1++	5.2±0.2++
Methanolic extracts	RPPm	150	39.5±2.5++	0.82±5.1++	5.0±0.2++
	WPPm	150	33.2±3.6++	0.95±0.1++	5.2±0.3++
	DPPm	150	33.5±2.2++	0.93±2.0++	5.1±0.1++

 

Mean ± SEM (n=6)** indicate significance from the diabetic control group at P<0.05 and P<0.01 probability level. +, ++ indicates significance from the Punica granatum peels group at P<0.05and P<0.01probability level respectively. For abbreviation, see Table I.

 

Table V. Effect of peel extracts of P. granatum on lipid profile in alloxan induced diabetic albino rats

Samples	Groups	Dose (mg/kg bodyweight)	TCH (mg/dl)	TGs (mg/dl)	HDL (mg/dl)	LDL (mg/dl)
Healthy (Untreated)	Normal control	--	72.4±4.2++	108.3±3.3++	65.5±5.2++	35.5±01++
Diabetic (untreated)	Diabetic control	--	92.5±2.2**	197.5±2.0**	46.2±3.6**	83.5±01**
Standard	Glibenclamide	10	68.4±2.2++	154.7±3.7++	49.3±2.1++	71.5±03++
Aqeous extract	RPP	150	73.1±0.1++	186.2±1.2++	48.7±4.6++	47.3±5.0++
	WPP	150	64.5±4.2++	174.5±2.2++	53.7±2.l++	51.5±01++
	DPP	150	73.2±2.1++	124.6±6.3++	59.2±4.l++	42.5±03++
Methanolic extracts	RPP	150	70.3±2.2++	187.7±3.5++	50.5±3.3++	63.5±02++
	WPP	150	72.6±0.1++	114.7±5.5++	60.7±3.3++	56.1±00++
	DPP	150	63.5±3.4++	147.4±6.5++	46.8±4.4++	57.5±02++

 

Mean ± SEM (n=6)** indicate significance from the diabetic control group at P<0.05 and P<0.01 probability level. +, ++ indicates significance from the Punica granatum peels group at P<0.05and P<0.01probability level respectively. TCH, Total cholesterol; HDL, High density lipoprotein; TGs, Triglycerides; LDL, Low density lipoprotein. For abbreviation, see Table I.

 

DISCUSSION

DM is a metabolic disorder in which secretion and action of insulin are affected which in result causes the changes not only in carbohydrates but also effects the lipids, and proteins. Insulin hormone is involved in the signal transduction and utilization of carbohydrates by the activation of different signaling molecules, and enzymes involved in the metabolic pathways including glycolysis, glycogen synthesis and lipogenesis (Patterson, 2019). The β cells dysfunctioning or defects in the insulin-stimulated pathway causes impaired carbohydrates metabolism and lead to persistent hyperglycemia and lipedemia (Wang et al., 2017). Alloxan is an oxygenated pyrimidine and an analog of glucose is very toxic which is taken by pancreatic β-cells via Glut-2 receptors and led to insulin deficiency (Dwyer-Lindgren et al., 2016). After the injection of Alloxan monohydrate elevated level of blood glucose were observed in rats. In this study the methanolic and aqueous extracts of three varieties of P. granatum from Mardan, Pakistan were evaluated for in vitro and in vivo antidiabetic potentials. It was found that both types of extracts from all varieties showed substantial inhibition of α-amylase and in the reduction of blood glucose level in alloxan-induced diabetic albino rats. Furthermore, the effects of peels extracts of all varieties were also measured on lipid profile, and other biochemical parameters related to liver and kidney functioning and found very effective in the reduction of elevated levels of TCs, TGs, LDL, ALT, ALP, bilirubin, and serum creatinine. The methanolic and aqueous peel extracts of all three varieties also showed remarkable antioxidant activity.

It has been previously reported that all parts of P. granatum possess varieties of secondary metabolites including polyphenols, flavonoids, and alkaloids (Aragaw et al., 2020). The reduction in blood glucose level may be due to the suppression of absorption of glucose due to high concentrations of flavonoids, which could contribute to the control of hyperglycemic conditions (Bloonogarden, 2016). Many studies reported that dietary intake of flavonoids played an effective role in the enhancement of β-cell and revival of insulin, and reduction of glucose which indicated their role in the prevention of T2D (Fang et al., 2019; Aragaw et al., 2020). The peels extracts reduces the blood glucose may be due to enhanced functions of β-cell by secondary compounds presents in the peels. In line with this study, several studies have been reported the effect of plants extracts containing many secondary metabolites on the regulation of plasma insulin levels (Uttra et al., 2018; Pengelly and Bone, 2020; Gurrero-Solano et al., 2020). Few studies reported the oral administration of secondary metabolites such as gallic acid, ellagic acid, quercetin other polyphenolic compounds in diabetic mice models and found their pronounced effects in the regeneration of the pancreas, regulation of insulin, and reduction of blood glucose (Singh and Gupta, 2018; Nafees et al., 2018; Uhlenbrock et al., 2018).

In the current study derangement of ALT, ALP and bilirubin were found in diabetes-induced rats (control) compared to healthy rats (normal control) indicating the abnormal liver function in diabetes. In diabetic rats, the elevation of the serum enzymes is directly associated with changes in related metabolic pathways. The elevated levels of ALT may be due to increased proteolysis and the availability of amino acids in diabetes (Goncalves and Romeiro, 2019). The free availability of amino acids may be responsible for the increased gluconeogenesis and ketogenesis in diabetes. Interestingly, the oral dosing of P. granatum peels extracts significantly reduced the level of ALT, ALP, and total bilirubin in the diabetic rats which was comparable with the diabetes-induced rats treated with standard drug glibenclamide (Alliance pharma) and normal control. The restoration of total bilirubin, ALT, and ALP levels in treated rats also indicates a revival of insulin secretion and normal function of the liver. Similarly, the elevated level of creatinine, urea, and decreased level of total protein in diabetic rats indicating renal injuries. It was previously reported that serum creatinine level increases when 40-50% of kidney nephrons were damages (Calin-Sanchez et al., 2013; Shah et al., 2018, 2019). Our results showed the regeneration in renal function after oral dosing of methanolic and aqueous extracts of peels of P. granatum which significantly (P<0.05) reduced urea, creatinine, and serum total proteins. The level of serum lipids usually rises in diabetes due to relative or absolute deficiency of insulin and increased lipogenesis and such elevation represents a risk factor for coronary heart disease. In diabetics, the concentration of LDL cholesterol, and TG was significantly increased and HDL cholesterol was decreased from that seen in diabetic control compared to normal controls. The oral administration of aqueous and methanolic peel extract of P. granatum at a dose of 150 mg/kg body weight efficiently reduced total cholesterol, TGs, and LDL and increased the HDL diabetes-induced rats. The results were consistent with the previous study that reported significant changes in lipid metabolism in diabetics (Calin-Sanchez et al., 2013; Hanani et al., 2019; Shah et al., 2019). After the treatment, the decrease in TG level may be due to increased insulin release from the β-cells of the pancreas which result favors lipogenesis and may activate a lipoprotein-lipase enzyme that hydrolyse TGs (Hanani et al., 2019; Khan et al., 2019). Oral administration of various fractions of P. granatum peels was found to be safe because no apparent toxicity was observed under the experimental conditions as well as no organ toxicity (liver, heart, and kidney) was seen in healthy rats. Although, to confirm the adverse effect of various fractions of P. granatum peels extract analysis were also performed in healthy rats which indicated no side effects of orally administered extracts at a dose of up to 2500 mg kg-1 b.w. The current study confirms that peel extracts of P. granatum can be contributory to reduce the complications of diabetics including hyperlipidemia and nephropathy as it lowers the lipids contents and recovers the liver and kidney function parameters.

CONCULSION

It was concluded that the extracts of peels of Punica granatum possess significant antihyperglycemic and antihyperlipidemic potentials due to antidiabetic compounds. The methanolic extract of pomegranate peel was found more effective than aqeous extract. The glucose level, lipid and liver profile was signficantlty affected in diabetic compared compared to normal control. It was significantly improved after oral doing of Punica granatum peel extract in alloxan induced diabetic rats which was compareable to normal control. It is recommended that peel of Punica granatum can be used for antidiabetic drugs development or in folk medicines to treat diabetes.

ACKNOWLEDGEMENT

This work was supported by the Department of Biochemistry, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan.

Funding

There was no funding available for the project.

Ethical and IRB approval

Approval to conduct the study was obtained from bioethical committee of Hazara University, Mansehra. The research project was approved by IRB of Hazara university, Mansehra.

Satement of conflict of interest

The authors have declared no conflict of interest.

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