# Isolation of  Antidiabetic Principle from Fruit Rinds ofPunica granatum

Authors: Vishal Jain; G. L. Viswanatha; D. Manohar; H. N. Shivaprasad
Journal: Evidence-Based Complementary and Alternative Medicine
 (2012)

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## Abstract

Present study was aimed to isolate and evaluate the antidiabetic activity of phytoconstituents from fruit rinds ofPunica granatum. With the above objectives Valoneic acid dilactone (VAD) was isolated from methanolic fruit rind extracts of  Punica granatum (MEPG) and confirmed by   1H-NMR, 13C-NMR, and mass spectral data. Antidiabetic activity was evaluated by Aldose reductase,  α-amylase  and PTP1B inhibition assays in in vitro and Alloxan-induced diabetes in rats was used as an in vivo model. In bioactivity studies, MEPG and VAD have showed potent antidiabetic activity in α-amylase, aldose reductase, and PTP1B inhibition assays with IC50 values of 1.02, 2.050, 26.25 μg/mL and 0.284, 0.788, 12.41 μg/mL, respectively. Furthermore, in alloxan-induced diabetes model MEPG (200 and 400 mg/kg, p.o.) and VAD (10, 25, and 50 mg/kg, p.o.) have showed significant and dose dependent antidiabetic activity by maintaining the blood glucose levels within the normal limits. Inline with the biochemical findings histopathology of MEPG  (200 and 400 mg/kg, p.o.), VAD  (10, 25, and 50 mg/kg, p.o.), and glibenclamide  (10 mg/kg, p.o.) treated animals showed significant protection against alloxan-induced pancreatic tissue damage. These findings suggest that MEPG and VAD possess significant antidiabetic activity in both in vitro and in vivo models.


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## Body

## 1. Introduction

Diabetes mellitus is a metabolic disorder characterized by hyperglycaemia and alterations in carbohydrate,  lipid, and protein metabolism, associated with absolute or relative deficiencies in insulin secretion and/or insulin action [1].The World Health Organization (WHO) has estimated that major burden of diabetes will occur in developing countries, and by 2025, there will be 42% increase (from 51 to 72 million) in the developed countries and 170% increase (from 84 to 228 million) in the developing countries [2]. It is estimated that, by 2025, India, China, and the United States will be the top three countries to have large number of diabetic patients [3].The high prevalence of diabetes as well as its long-term complications has led to an ongoing search for hypoglycaemic agents; over the years, various medicinal plants and their extracts have been reported to be effective in the treatment of diabetes [4]. Plants are rich sources of antidiabetic, antihyperlipidemic, and antioxidant agents such as flavonoids, gallotannins, amino acids, and other related polyphenols [5].Pomegranate (Punica granatumL., Punicaceae) is used in the traditional system of medicine in different Asian cultures for the treatment of variety of ailments. In Ayurvedic medicine, the plant is described under its Sanskrit name “dalima” (fruit) as a “blood purifier” and used to cure  parasitic infections, aphthae (mouth ulcers), diarrhoea, and ulcers [6].  Punica granatum is a deciduous tree belongs to the family Punicaceae; it is known for its edible fruit. During the last two decades, various parts of the plant had been subjected to extensive phytochemical, pharmacological, and clinical investigations; many interesting findings have been reported in various fields [7].Punica granatumis found to contain hydrolysable tannins as  major active chemical constituents and phytoconstituents, namely, corilagin, ellagic acid, kaempferol, luteolin, myricetin, quercetin, quercimetrine, and quercetin-3-o-rutinoside which were previously isolated from the fruits of Punica granatum[8].In the literature, various parts of the plant are reported for various pharmacological activities such as antioxidant [9], antidiarrheal [10], anti-inflammatory [11], antimalarial [12], antimutagenic [13], immunomodulatory [14], hepatoprotective [15], antiulcer [16], atherosclerosis and thyroid dysfunction [17], memory enhancing [18], wound healing [19], and anticancer [20] activities. Apart from these medicinal uses, various parts of the plant have been scientifically proved for antidiabetic activity [21]. In our previous studies, we have isolated three components from methanolic extracts of Punica granatum[8]. In continuation, the present study was undertaken to isolate the antidiabetic principle from the methanolic extracts of fruit rinds of Punica granatum.


## 2. Materials and Methods

### 2.1. Drugs and Chemicals

Human recombinant PTP1B (BIOMOL International LP (Plymouth Meeting, PA)),α-amylase (Sigma Aldrich, Bangalore), acarbose (Glenmark, Mumbai), glibenclamide (Cipla, Mumbai), and biochemical kits (ERBA diagnostic Mannheim GMBH, Germany) were used, and all the solvents used for the extraction and isolation were of analytical grade and purchased from local firms.


### 2.2. Plant Material

The fruit rinds ofPunica granatumwere collected locally and authenticated by Dr. K. P. Srinath, Department of Botany, Bangalore University, Bangalore; a voucher specimen was deposited in the department (PESCP/2009-10/PG-06).


### 2.3. Isolation and Identification of the Compound

Fruit rinds ofPunica granatum were collected, dried, and powdered; the powder (3.0 Kg) was successively extracted with methanol in a static extractor and concentrated under vacuum at 60°C and dried in vacuum tray dryer. The percentage yield of the methanolic extract was found to be 26.67% w/w (800 g per 3.0 kg of methanolic extract).Vacuum-dried methanolic extract (800 g) was macerated with ethyl acetate in the solvent : solute ratio of 4 : 1 for 72 h with frequent shaking. The fraction was dried using vacuum tray dryer. The yield of ethyl acetate fraction was found to be 27%w/w. The major phytochemical constituents present in the ethyl acetate fraction were identified by thin-layer chromatography (TLC). TLC plates were developed using water : acetic acid solvent system (3 : 2) and scanned at 254 and 366 nm. The 5% FeCl3 reagent was sprayed, and the chromatogram was observed.The fraction was subjected to HPLC for the identification and quantification of the active components; based on HPLC data, ethyl acetate fraction was subjected to column chromatography for the isolation of phytochemical constituent using a glass column and Diaion resin as stationary phase. Totally 12 subfractions (FR-1 to FR-12) were obtained from the ethyl acetate fraction, and all the sub fractions were subjected to HPLC for the identification of the desired compound. Scheme of isolation process is given in Figure1. In our previous study, the FR-1 and FR-8 were identified as gallic acid (95% pure) and ellagic acid (98% pure), respectively, based on spectral data. With this background information, FR-7 was further taken forward for characterization by NMR and Mass spectroscopy at Radiant Research and Training Institute and IISc, Bangalore.Figure 1
Scheme of isolation process.


### 2.4. Experimental Animals

Male Wistar rats (200–250 g) were purchased from Bioneeds, Nelamangala, Tumkur, India, and they were maintained in polypropylene cages at a temperature of25±1°C and relative humidity of 45–55% in clean environment under 12:12 h light-dark cycle. The animals had free access to food pellets (Pranav Agro Industries, Bangalore, India) and purified water ad libitum.All the experimental protocols were approved by Institutional Animal Ethics Committee (IAEC),  and all the animal experiments were conducted according to the principles and guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experimentation on Animals), India.


### 2.5. Experimental Protocol

#### 2.5.1.In Vitro  α-Amylase Inhibition Assay

In vitro α-amylase inhibition assay was carried out based on the spectrophotometric assay using acarbose as the reference compound [4]. 2 mg of MEPG and VAD was dissolved in 500 μL of DMSO, and further dilutions were prepared using deionised water. The enzyme α-amylase solution (0.5 unit/mL) was prepared by mixing 3.246 mg of α-amylase (EC 3.2.1.1) in 100 mL of 40 mM phosphate buffer pH 6.9. In short, 60 μL of 40 mM phosphate buffer/acarbose/test drug and 30 μL of α-amylase enzyme were added, mixed, and preincubated at 37°C for 10 min, and then 120 μL of 2-chloro-p-nitrophenyl-α-D-maltotrioside (CNPG3) was added, mixed, and incubated at 37°C for 8 min. The absorbance was measured at 405 nm, and control reaction was carried out without the extract. Percentage inhibition was calculated by expression:
(1)%Inhibition=Absorbance of control-Absorbance of testAbsorbance of control×100.


#### 2.5.2.In Vitro Aldose Reductase Inhibition Assay

(a) Preparation of Aldose Reductase
One-gram of eye lenses were pooled and homogenised in 12 volumes of 135 mM sodium phosphate buffer (pH 7.0) containing 0.5 mM phenylmethylsulfonyl fluoride and 10 mM 2-mercaptoethanol. The homogenate was centrifuged at 10,000 g for 30 minutes, and the resultant supernatant was retained as an enzyme preparation. All the procedures were carried out at 4°C. The activity of this preparation was determined by measuring the amount of NADP released per unit time at 37°C and pH 7.0. One unit (U) of activity is defined as the amount of the enzyme catalysed the oxidation of 1μmol of NADPH per minute under our experimental conditions [22]:
(2)ActivityU/mL=ChangeinODoftest/min⁡×Totalvolumeoftheassay6.2×Volumeofenzymetakenforanalysis,
where 6.2 = micromolar extinction coefficient of NADPH at 340 nm.(b) Assay Procedure
Aldose reductase inhibition activity was carried out by photometric method [22]. The procedure was miniature and carried out using 96-well plate. The drug solutions were prepared by dissolving in buffer solution (67 mM of sodium phosphate buffer, pH 7.0) containing 10% DMSO solution. The stock concentration of 1 mg/mL solution was prepared, and it was serially diluted in a 96-well plate to obtain varying dilutions. Similarly without the drug, a positive control was also run. Each well of the plate contained 50 μL of drug solution (buffer in case of control), 50 μL of NADPH (0.04 mM), and 100 μL of aldose reductase enzyme preparation. The enzymatic reaction was initiated by the addition of 75 μL of substrate, DL-glyceraldehyde (5×10-4 M). The absorbance was recorded every minute for duration of 20 minutes at 340 nm. The decrease in the absorbance due to oxidation of NADPH to NADP was recorded at different time points. The concentrations of the inhibitors producing 50% inhibition of the enzyme activity (IC50) were calculated from the percentage inhibition [23].


#### 2.5.3. Protein Tyrosine Phosphatase 1B Inhibition Assay

The phosphatase activity was assayed usingpNPP (Para-Nitro Phenyl Phosphate) as substrate and carried out essentially as described previously [24]. The assay buffer (pH 7.4), consisting of 50 mM 3,3-dimethyl glutarate, 1 mM EDTA, 5 mM glutathione, and 0.5% FCS (not heat inactivated) was adjusted to an ionic strength of 0.15 M by the addition of NaCl. In brief, appropriately diluted inhibitors (undiluted and 50 μM compound) were added to the reaction mixture containing 0 or 2.5 mM pNPP (final assay concentration, total volume of 100 μL). The reaction was initiated by the addition of the enzyme (i.e., recombinant PTP1B) and allowed to proceed for 5 min before the inhibitor was added and the time was recorded, that is, it continued incubation for 5–60 min at 37°C. The reaction was stopped by the addition of 20 μL 0.5 M NaOH in 50% ethanol. The enzyme activity was determined by measuring the absorbance at 405 nm using Tecan ELISA reader with appropriate corrections for absorbance of substrate, compounds, and nonenzymatic hydrolysis of substrate.


#### 2.5.4. Alloxan-Induced Diabetes in Rats

Alloxan monohydrate was dissolved in saline and administered intravenously to fasted rats      (200–250 g) at a dose of 120 mg/kg. The solution should be fresh and prepared just prior to the administration. The rats were given 5% (w/v) glucose solution in feeding bottles for next 24 h in their cages to prevent hypoglycaemia after alloxan injection. After 72 h, rats with blood glucose levels greater than 200 mg/dL and less than 400 mg/dL were selected and observed for consistent hyperglycaemia (fasting blood glucose level greater than 200 mg/dL and lesser then 400 mg/dL) up to 7 days. Those animals were divided into eight groups (G-I to G-VIII), each group consisting of eight animals (8×8=64 animals), G-I served as normal control, G-II served as diabetic control received 3% v/v Tween 80 in water (10 mL/kg, p.o.), G-III treated with reference standard glibenclamide (10 mg/kg, p.o.) and G-IV and G-V treated with MEPG 200 and 400 mg/kg, p.o.,respectively, while G-VI, G-VII, and G-VIII were treated with 10, 25, and 50 mg/kg, p.o. of VAD. All the treatments were given for 21 days.On day-1 of drug treatment, blood samples were collected by retro-orbital puncture at 0, 1, 2, 4, and 8 h after the administration, while on 4th, 7th, 14th, and 21st days blood samples were collected after 1 h of respective drug treatments. Blood glucose levels were estimated by using GOD/POD kit. Oral glucose tolerance test (OGTT) was carried out on 21st day. Body weight of all animals was recorded on the 0, 4th, 7th, 14th, and 21st days before vehicle/glibenclamide/test drug administration. The percentage change of body weight was calculated from its initial weight.(a) Assessment of Mortality Rate in Alloxan-Induced Diabetic Rats
Alloxan may cause severe ketoacidosis and may lead to death of the animal. In view of this, the mortality rate was monitored throughout the study. The % of mortality was calculated on 7th, 14th, and 21st days of the study.(b) Histopathology
At the end of the study, all the animals are sacrificed, and pancreas was excised and rinsed in ice-cold normal saline. A portion of the tissue was fixed in 10% formalin, cut into 5μm thick sections, and stained using hematoxylin-eosin, and histopathological observations were made.


### 2.6. Statistical Analysis

The values were expressed as mean ± S.E.M. Data was statistically analyzed using one-way ANOVA followed by Tukey’s multiple comparison test.P<0.05 was considered as statistically significant.


## 2.1. Drugs and Chemicals

Human recombinant PTP1B (BIOMOL International LP (Plymouth Meeting, PA)),α-amylase (Sigma Aldrich, Bangalore), acarbose (Glenmark, Mumbai), glibenclamide (Cipla, Mumbai), and biochemical kits (ERBA diagnostic Mannheim GMBH, Germany) were used, and all the solvents used for the extraction and isolation were of analytical grade and purchased from local firms.


## 2.2. Plant Material

The fruit rinds ofPunica granatumwere collected locally and authenticated by Dr. K. P. Srinath, Department of Botany, Bangalore University, Bangalore; a voucher specimen was deposited in the department (PESCP/2009-10/PG-06).


## 2.3. Isolation and Identification of the Compound

Fruit rinds ofPunica granatum were collected, dried, and powdered; the powder (3.0 Kg) was successively extracted with methanol in a static extractor and concentrated under vacuum at 60°C and dried in vacuum tray dryer. The percentage yield of the methanolic extract was found to be 26.67% w/w (800 g per 3.0 kg of methanolic extract).Vacuum-dried methanolic extract (800 g) was macerated with ethyl acetate in the solvent : solute ratio of 4 : 1 for 72 h with frequent shaking. The fraction was dried using vacuum tray dryer. The yield of ethyl acetate fraction was found to be 27%w/w. The major phytochemical constituents present in the ethyl acetate fraction were identified by thin-layer chromatography (TLC). TLC plates were developed using water : acetic acid solvent system (3 : 2) and scanned at 254 and 366 nm. The 5% FeCl3 reagent was sprayed, and the chromatogram was observed.The fraction was subjected to HPLC for the identification and quantification of the active components; based on HPLC data, ethyl acetate fraction was subjected to column chromatography for the isolation of phytochemical constituent using a glass column and Diaion resin as stationary phase. Totally 12 subfractions (FR-1 to FR-12) were obtained from the ethyl acetate fraction, and all the sub fractions were subjected to HPLC for the identification of the desired compound. Scheme of isolation process is given in Figure1. In our previous study, the FR-1 and FR-8 were identified as gallic acid (95% pure) and ellagic acid (98% pure), respectively, based on spectral data. With this background information, FR-7 was further taken forward for characterization by NMR and Mass spectroscopy at Radiant Research and Training Institute and IISc, Bangalore.Figure 1
Scheme of isolation process.


## 2.4. Experimental Animals

Male Wistar rats (200–250 g) were purchased from Bioneeds, Nelamangala, Tumkur, India, and they were maintained in polypropylene cages at a temperature of25±1°C and relative humidity of 45–55% in clean environment under 12:12 h light-dark cycle. The animals had free access to food pellets (Pranav Agro Industries, Bangalore, India) and purified water ad libitum.All the experimental protocols were approved by Institutional Animal Ethics Committee (IAEC),  and all the animal experiments were conducted according to the principles and guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experimentation on Animals), India.


## 2.5. Experimental Protocol

### 2.5.1.In Vitro  α-Amylase Inhibition Assay

In vitro α-amylase inhibition assay was carried out based on the spectrophotometric assay using acarbose as the reference compound [4]. 2 mg of MEPG and VAD was dissolved in 500 μL of DMSO, and further dilutions were prepared using deionised water. The enzyme α-amylase solution (0.5 unit/mL) was prepared by mixing 3.246 mg of α-amylase (EC 3.2.1.1) in 100 mL of 40 mM phosphate buffer pH 6.9. In short, 60 μL of 40 mM phosphate buffer/acarbose/test drug and 30 μL of α-amylase enzyme were added, mixed, and preincubated at 37°C for 10 min, and then 120 μL of 2-chloro-p-nitrophenyl-α-D-maltotrioside (CNPG3) was added, mixed, and incubated at 37°C for 8 min. The absorbance was measured at 405 nm, and control reaction was carried out without the extract. Percentage inhibition was calculated by expression:
(1)%Inhibition=Absorbance of control-Absorbance of testAbsorbance of control×100.


### 2.5.2.In Vitro Aldose Reductase Inhibition Assay

(a) Preparation of Aldose Reductase
One-gram of eye lenses were pooled and homogenised in 12 volumes of 135 mM sodium phosphate buffer (pH 7.0) containing 0.5 mM phenylmethylsulfonyl fluoride and 10 mM 2-mercaptoethanol. The homogenate was centrifuged at 10,000 g for 30 minutes, and the resultant supernatant was retained as an enzyme preparation. All the procedures were carried out at 4°C. The activity of this preparation was determined by measuring the amount of NADP released per unit time at 37°C and pH 7.0. One unit (U) of activity is defined as the amount of the enzyme catalysed the oxidation of 1μmol of NADPH per minute under our experimental conditions [22]:
(2)ActivityU/mL=ChangeinODoftest/min⁡×Totalvolumeoftheassay6.2×Volumeofenzymetakenforanalysis,
where 6.2 = micromolar extinction coefficient of NADPH at 340 nm.(b) Assay Procedure
Aldose reductase inhibition activity was carried out by photometric method [22]. The procedure was miniature and carried out using 96-well plate. The drug solutions were prepared by dissolving in buffer solution (67 mM of sodium phosphate buffer, pH 7.0) containing 10% DMSO solution. The stock concentration of 1 mg/mL solution was prepared, and it was serially diluted in a 96-well plate to obtain varying dilutions. Similarly without the drug, a positive control was also run. Each well of the plate contained 50 μL of drug solution (buffer in case of control), 50 μL of NADPH (0.04 mM), and 100 μL of aldose reductase enzyme preparation. The enzymatic reaction was initiated by the addition of 75 μL of substrate, DL-glyceraldehyde (5×10-4 M). The absorbance was recorded every minute for duration of 20 minutes at 340 nm. The decrease in the absorbance due to oxidation of NADPH to NADP was recorded at different time points. The concentrations of the inhibitors producing 50% inhibition of the enzyme activity (IC50) were calculated from the percentage inhibition [23].


### 2.5.3. Protein Tyrosine Phosphatase 1B Inhibition Assay

The phosphatase activity was assayed usingpNPP (Para-Nitro Phenyl Phosphate) as substrate and carried out essentially as described previously [24]. The assay buffer (pH 7.4), consisting of 50 mM 3,3-dimethyl glutarate, 1 mM EDTA, 5 mM glutathione, and 0.5% FCS (not heat inactivated) was adjusted to an ionic strength of 0.15 M by the addition of NaCl. In brief, appropriately diluted inhibitors (undiluted and 50 μM compound) were added to the reaction mixture containing 0 or 2.5 mM pNPP (final assay concentration, total volume of 100 μL). The reaction was initiated by the addition of the enzyme (i.e., recombinant PTP1B) and allowed to proceed for 5 min before the inhibitor was added and the time was recorded, that is, it continued incubation for 5–60 min at 37°C. The reaction was stopped by the addition of 20 μL 0.5 M NaOH in 50% ethanol. The enzyme activity was determined by measuring the absorbance at 405 nm using Tecan ELISA reader with appropriate corrections for absorbance of substrate, compounds, and nonenzymatic hydrolysis of substrate.


### 2.5.4. Alloxan-Induced Diabetes in Rats

Alloxan monohydrate was dissolved in saline and administered intravenously to fasted rats      (200–250 g) at a dose of 120 mg/kg. The solution should be fresh and prepared just prior to the administration. The rats were given 5% (w/v) glucose solution in feeding bottles for next 24 h in their cages to prevent hypoglycaemia after alloxan injection. After 72 h, rats with blood glucose levels greater than 200 mg/dL and less than 400 mg/dL were selected and observed for consistent hyperglycaemia (fasting blood glucose level greater than 200 mg/dL and lesser then 400 mg/dL) up to 7 days. Those animals were divided into eight groups (G-I to G-VIII), each group consisting of eight animals (8×8=64 animals), G-I served as normal control, G-II served as diabetic control received 3% v/v Tween 80 in water (10 mL/kg, p.o.), G-III treated with reference standard glibenclamide (10 mg/kg, p.o.) and G-IV and G-V treated with MEPG 200 and 400 mg/kg, p.o.,respectively, while G-VI, G-VII, and G-VIII were treated with 10, 25, and 50 mg/kg, p.o. of VAD. All the treatments were given for 21 days.On day-1 of drug treatment, blood samples were collected by retro-orbital puncture at 0, 1, 2, 4, and 8 h after the administration, while on 4th, 7th, 14th, and 21st days blood samples were collected after 1 h of respective drug treatments. Blood glucose levels were estimated by using GOD/POD kit. Oral glucose tolerance test (OGTT) was carried out on 21st day. Body weight of all animals was recorded on the 0, 4th, 7th, 14th, and 21st days before vehicle/glibenclamide/test drug administration. The percentage change of body weight was calculated from its initial weight.(a) Assessment of Mortality Rate in Alloxan-Induced Diabetic Rats
Alloxan may cause severe ketoacidosis and may lead to death of the animal. In view of this, the mortality rate was monitored throughout the study. The % of mortality was calculated on 7th, 14th, and 21st days of the study.(b) Histopathology
At the end of the study, all the animals are sacrificed, and pancreas was excised and rinsed in ice-cold normal saline. A portion of the tissue was fixed in 10% formalin, cut into 5μm thick sections, and stained using hematoxylin-eosin, and histopathological observations were made.


## 2.5.1.In Vitro  α-Amylase Inhibition Assay

In vitro α-amylase inhibition assay was carried out based on the spectrophotometric assay using acarbose as the reference compound [4]. 2 mg of MEPG and VAD was dissolved in 500 μL of DMSO, and further dilutions were prepared using deionised water. The enzyme α-amylase solution (0.5 unit/mL) was prepared by mixing 3.246 mg of α-amylase (EC 3.2.1.1) in 100 mL of 40 mM phosphate buffer pH 6.9. In short, 60 μL of 40 mM phosphate buffer/acarbose/test drug and 30 μL of α-amylase enzyme were added, mixed, and preincubated at 37°C for 10 min, and then 120 μL of 2-chloro-p-nitrophenyl-α-D-maltotrioside (CNPG3) was added, mixed, and incubated at 37°C for 8 min. The absorbance was measured at 405 nm, and control reaction was carried out without the extract. Percentage inhibition was calculated by expression:
(1)%Inhibition=Absorbance of control-Absorbance of testAbsorbance of control×100.


## 2.5.2.In Vitro Aldose Reductase Inhibition Assay

(a) Preparation of Aldose Reductase
One-gram of eye lenses were pooled and homogenised in 12 volumes of 135 mM sodium phosphate buffer (pH 7.0) containing 0.5 mM phenylmethylsulfonyl fluoride and 10 mM 2-mercaptoethanol. The homogenate was centrifuged at 10,000 g for 30 minutes, and the resultant supernatant was retained as an enzyme preparation. All the procedures were carried out at 4°C. The activity of this preparation was determined by measuring the amount of NADP released per unit time at 37°C and pH 7.0. One unit (U) of activity is defined as the amount of the enzyme catalysed the oxidation of 1μmol of NADPH per minute under our experimental conditions [22]:
(2)ActivityU/mL=ChangeinODoftest/min⁡×Totalvolumeoftheassay6.2×Volumeofenzymetakenforanalysis,
where 6.2 = micromolar extinction coefficient of NADPH at 340 nm.(b) Assay Procedure
Aldose reductase inhibition activity was carried out by photometric method [22]. The procedure was miniature and carried out using 96-well plate. The drug solutions were prepared by dissolving in buffer solution (67 mM of sodium phosphate buffer, pH 7.0) containing 10% DMSO solution. The stock concentration of 1 mg/mL solution was prepared, and it was serially diluted in a 96-well plate to obtain varying dilutions. Similarly without the drug, a positive control was also run. Each well of the plate contained 50 μL of drug solution (buffer in case of control), 50 μL of NADPH (0.04 mM), and 100 μL of aldose reductase enzyme preparation. The enzymatic reaction was initiated by the addition of 75 μL of substrate, DL-glyceraldehyde (5×10-4 M). The absorbance was recorded every minute for duration of 20 minutes at 340 nm. The decrease in the absorbance due to oxidation of NADPH to NADP was recorded at different time points. The concentrations of the inhibitors producing 50% inhibition of the enzyme activity (IC50) were calculated from the percentage inhibition [23].


## 2.5.3. Protein Tyrosine Phosphatase 1B Inhibition Assay

The phosphatase activity was assayed usingpNPP (Para-Nitro Phenyl Phosphate) as substrate and carried out essentially as described previously [24]. The assay buffer (pH 7.4), consisting of 50 mM 3,3-dimethyl glutarate, 1 mM EDTA, 5 mM glutathione, and 0.5% FCS (not heat inactivated) was adjusted to an ionic strength of 0.15 M by the addition of NaCl. In brief, appropriately diluted inhibitors (undiluted and 50 μM compound) were added to the reaction mixture containing 0 or 2.5 mM pNPP (final assay concentration, total volume of 100 μL). The reaction was initiated by the addition of the enzyme (i.e., recombinant PTP1B) and allowed to proceed for 5 min before the inhibitor was added and the time was recorded, that is, it continued incubation for 5–60 min at 37°C. The reaction was stopped by the addition of 20 μL 0.5 M NaOH in 50% ethanol. The enzyme activity was determined by measuring the absorbance at 405 nm using Tecan ELISA reader with appropriate corrections for absorbance of substrate, compounds, and nonenzymatic hydrolysis of substrate.


## 2.5.4. Alloxan-Induced Diabetes in Rats

Alloxan monohydrate was dissolved in saline and administered intravenously to fasted rats      (200–250 g) at a dose of 120 mg/kg. The solution should be fresh and prepared just prior to the administration. The rats were given 5% (w/v) glucose solution in feeding bottles for next 24 h in their cages to prevent hypoglycaemia after alloxan injection. After 72 h, rats with blood glucose levels greater than 200 mg/dL and less than 400 mg/dL were selected and observed for consistent hyperglycaemia (fasting blood glucose level greater than 200 mg/dL and lesser then 400 mg/dL) up to 7 days. Those animals were divided into eight groups (G-I to G-VIII), each group consisting of eight animals (8×8=64 animals), G-I served as normal control, G-II served as diabetic control received 3% v/v Tween 80 in water (10 mL/kg, p.o.), G-III treated with reference standard glibenclamide (10 mg/kg, p.o.) and G-IV and G-V treated with MEPG 200 and 400 mg/kg, p.o.,respectively, while G-VI, G-VII, and G-VIII were treated with 10, 25, and 50 mg/kg, p.o. of VAD. All the treatments were given for 21 days.On day-1 of drug treatment, blood samples were collected by retro-orbital puncture at 0, 1, 2, 4, and 8 h after the administration, while on 4th, 7th, 14th, and 21st days blood samples were collected after 1 h of respective drug treatments. Blood glucose levels were estimated by using GOD/POD kit. Oral glucose tolerance test (OGTT) was carried out on 21st day. Body weight of all animals was recorded on the 0, 4th, 7th, 14th, and 21st days before vehicle/glibenclamide/test drug administration. The percentage change of body weight was calculated from its initial weight.(a) Assessment of Mortality Rate in Alloxan-Induced Diabetic Rats
Alloxan may cause severe ketoacidosis and may lead to death of the animal. In view of this, the mortality rate was monitored throughout the study. The % of mortality was calculated on 7th, 14th, and 21st days of the study.(b) Histopathology
At the end of the study, all the animals are sacrificed, and pancreas was excised and rinsed in ice-cold normal saline. A portion of the tissue was fixed in 10% formalin, cut into 5μm thick sections, and stained using hematoxylin-eosin, and histopathological observations were made.


## 2.6. Statistical Analysis

The values were expressed as mean ± S.E.M. Data was statistically analyzed using one-way ANOVA followed by Tukey’s multiple comparison test.P<0.05 was considered as statistically significant.


## 3. Results

The present study was undertaken to isolate and evaluate the antidiabetic activity of bioactive constituents present in the fruit rind extracts ofPunica granatumin in vitroand in vivomodels. In continuation of our previous studies, we have shortlisted some of the fractions for isolation of active constituents and subsequent evaluation of antidiabetic activity of the actives in various in vitroand in vivomodels.


### 3.1. Identification and Quantification of the Isolated Compound

Fraction 7 was subjected to NMR and Mass spectroscopy and by comparing the obtained spectral data of the Fraction-7 with the literature reports; it was identified as a well known polyphenolic compound “Valoneic acid dilactone.” The HPLC chromatogram of Methanolic extract, Fraction-7 (Valoneic acid dilactone) and structure of Valoneic acid dilactone  are given in Figures2, 3, and 4, respectively.Figure 2
Analytical HPLC for methanolic extract.Figure 3
Analytical HPLC for fraction 7 (valoneic acid dilactone).Figure 4
Structure of valoneic acid dilactone.Spectral Data
Chemical formula: C21H10O13; molecular weight: 470.
1H NMR (δ values ppm): 6.97 (s, 1H), 6.93 (s, 1H), 7.47 (s, 1H).
13C NMR (δ values ppm): 113.9, 112.0, 108.3, 108.5, 110.4, 106.7, 108.1, 114.7, 139.1, 136.2, 136.7, 135.2, 139.6, 139.6, 140.8, 142.9, 148.6, 149.5, 159.2, 159.2, 165.9.
LC-MS [m/zvalue]: 469.03 [M-H] (molecular ion peak).The Valoneic acid dilactone is a off white amorphous powder, soluble in methanol, purity was found to be greater than 95% and melting point was less than 300°C.


### 3.2.In Vitro  α-Amylase Inhibition Assay

In the present study, MEPG and VAD isolated from fruit rinds ofPunica granatumhave significantly and dose dependently inhibited the α-amylase enzyme activity. Acarbose was used as a reference standard to validate the assay procedure; the findings of the present study are in accordance with the literature reports. Acarbose, MEPG, and VAD have evolved as potent inhibitors of α-amylase enzyme activity with IC50 values of 0.378, 1.02, and 0.284 μg/mL, respectively (Table 1).Table 1
Effect of MEPG and VAD isolated from fruit rinds ofPunica granatum on α-amylase enzyme activity in in vitro.
Sl. no
Sample
Concentration (μg/mL)
% Inhibition#
IC50 (μg/mL)*
0.15
10.21
±
0.54
0.3
29.41
±
0.17
(1)
Methanolic extract
0.6
44.28
±
0.83
1.02
1.25
70.21
±
0.54
2.5
85.88
±
0.52
0.15
29.65
±
0.85
0.3
49.78
±
0.46
(2)
Valoneic acid dilactone
0.6
74.25
±
0.29
0.284
1.25
82.85
±
0.31
2.5
99.52
±
0.71
0.1
33.53
±
0.46
(3)
Acarbose
0.5
62.41
±
0.87
0.378
1
74.25
±
0.29
2
99.21
±
0.34
Values are expressed as mean ± SEM (% inhibition#) and mean (IC50*) of three trials.


### 3.3.In Vitro Aldose Reductase Inhibition Assay

Aldose reductase is the key enzyme involved in the polyol pathway; drugs which inhibit aldose reductase enzyme activity are believed to be beneficial in the prevention of long-term diabetic complications. With this background, MEPG and VAD were evaluated for aldose reductase inhibition activity, and the findings ofin vitroaldose reductase inhibition assay revealed that MEPG and VAD isolated from fruit rinds of Punica granatum showed significant and dose-dependent inhibition of aldose reductase enzyme activity; the IC50 values were found to be 2.050 and 0.788 μg/mL, respectively (Table 2).Table 2
Effect of MEPG and VAD isolated from fruit rinds ofPunica granatum on aldose reductase enzyme activity in in vitro.
Sl. no
Sample
Concentration (μg/mL)
% Inhibition#
IC50 (μg/mL)*
0.3
22.95
±
0.25
0.6
35.38
±
0.47
(1)
Methanolic extract
1.2
45.77
±
0.14
2.050
2.5
65.64
±
0.74
5
84.49
±
0.21
10
99.62
±
0.33
0.15
9.31
±
0.84
0.3
26.63
±
0.18
(2)
Valoneic acid  dilactone
0.6
48.50
±
0.54
0.788
1.2
67.80
±
0.71
2.5
79.19
±
0.21
5
89.43
±
0.34
0.5
8.03
±
0.62
(3)
Quercetin
1.0
24.23
±
0.51
2.807
2.0
42.48
±
0.91
5.0
82.45
±
0.27
Values are expressed as mean ± SEM (% inhibition#) and mean (IC50*) of three trials.


### 3.4. Protein Tyrosine Phosphatase 1B Inhibition (PTP1B) Assay

Inin vitroPTP1B assay, the MEPG and VAD isolated from fruit rinds of Punica granatum showed significant dose-dependent inhibition of PTP1B enzyme activity; the IC50 values were found to be 26.25 and 12.41 μg/mL, respectively (Table 3).Table 3
Effect of MEPG and VAD isolated from fruit rinds ofPunica granatum on protein tyrosine phosphatase-1B (PTP1B) enzyme activity in  in vitro.
Sl. no
Sample
Concentration (μg/mL)
% Inhibition#
IC50 (μg/mL)*
5
21.6
±
0.24
10
39.6
±
0.15
(1)
Methanolic extract
25
55.4
±
0.22
26.25
50
78.7
±
0.34
100
98.9
±
0.22
5
33.3
±
0.35
10
46.7
±
0.54
(2)
Valoneic acid dilactone
25
68.5
±
0.64
12.41
50
84.0
±
0.11
100
98.5
±
0.75
Values are expressed as mean ± SEM (% inhibition#) and mean (IC50*) of three trials.


### 3.5. Alloxan-Induced Diabetes in Rats

Alloxan-induced diabetes is a well-known and commonly used animal model to screen the antidiabetic activity of the herbal and synthetic drugs. In the present study, the intravenous administration of alloxan (120 mg/kg, i.v.) had increased the fasting blood glucose level more than 250 mg/dL after 6 days, while normal rats have showed 90–100 mg/dL. Animals treated with single dose of MEPG (200 and 400 mg/kg) and VAD (10 and 25 mg/kg) did not significantly reduce the blood glucose levels in alloxan-induced diabetic rats. In contrast, VAD (50 mg/kg, p.o.) and glibenclamide (10 mg/kg, p.o.) have significantly reduced the BG levels at 1st, 2nd, and 4th hours after single-dose administration in alloxan-induced diabetic rats (Table4).Table 4
Effect of single-dose oral administration of MEPG and VAD on blood glucose levels in alloxan-induced diabetic rats.
Group
Treatment
Blood glucose (mg/dL)
0 hour
1 hour
2 hours
4 hours
G-I
Normal control
91.23
±
5.2
92.45
±
2.8
92.15
±
4.5
91.54
±
3.1
G-II
Vehicle control (3% Tween 80) 10 mL/kg, p.o.
272.84
±
3.9
#
274.41
±
5.1
#
277.65
±
5.1
#
268.94
±
4.6
#
G-III
Glibenclamide-10 mg/kg, p.o.
271.94
±
4.8
234.15
±
5.3
197.14
±
6.2
*
164.85
±
6.5
*
G-IV
MEPG-200 mg/kg, p.o.
268.51
±
6.2
271.15
±
4.8
255.41
±
7.9
264.12
±
6.1
G-V
MEPG-400 mg/kg, p.o.
272.84
±
5.2
269.64
±
6.5
242.12
±
4.8
*
235.15
±
5.8
*
G-VI
VAD-10 mg/kg, p.o.
259.87
±
5.5
265.32
±
7.3
264.85
±
3.7
261.25
±
7.3
G-VII
VAD-25 mg/kg, p.o.
264.51
±
7.1
261.25
±
3.5
*
258.65
±
5.7
*
256.52
±
4.9
*
G-VIII
VAD-50 mg/kg, p.o.
274.56
±
4.5
241.85
±
6.1
*
220.12
±
6.7
*
198.56
±
5.3
*
All the values are expressed as mean± SEM, P#<0.05 compared to normal control, and P*<0.05 compared to vehicle control (3% Tween 80).Exceptionally, repeated dose administration of MEPG (200 and 400 mg/kg, p.o.) for 21 days had progressively reduced the blood glucose levels significantly (P<0.05) and dose dependently (Table 5); similarly, animals treated with VAD (10, 25, and 50 mg/kg, p.o.) also showed significant (P<0.01) decrease in blood glucose levels compared to diabetic control group (G-II).Table 5
Effect of repeated-dose oral administration of MEPG and VAD on blood glucose levels in alloxan-induced diabetic rats.
Group
Treatment
Blood glucose (mg/dL)
0th day
7th day
14th day
21st day
G-I
Normal control
91.23
±
5.2
92.45
±
2.8
92.15
±
4.5
91.54
±
3.1
G-II
Vehicle control (3% Tween 80) 10 mL/kg, p.o.
272.84
±
3.9
#
279.22
±
7.4
#
281.25
±
6.2
#
274.84
±
5.7
#
G-III
Glibenclamide-10 mg/kg, p.o.
271.94
±
4.8
124.98
±
8.1
*
104.28
±
4.9
*
94.47
±
2.9
*
G-IV
MEPG-200 mg/kg, p.o.
268.51
±
6.2
198.64
±
2.8
*
172.94
±
23
*
132.44
±
7.2
*
G-V
MEPG-400 mg/kg, p.o.
272.84
±
5.2
165.22
±
5.2
*
152.94
±
5.1
*
115.27
±
4.9
*
G-VI
VAD-10 mg/kg, p.o.
259.87
±
5.5
169.68
±
2.7
*
159.32
±
4.8
*
124.66
±
4.1
*
G-VII
VAD-25 mg/kg, p.o.
264.51
±
7.1
148.25
±
4.6
*
129.80
±
3.8
*
98.26
±
2.15
*
G-VIII
VAD-50 mg/kg, p.o.
274.56
±
4.5
132.58
±
3.1
*
109.85
±
2.2
*
95.12
±
3.85
*
All the values are expressed as mean ± SEM,P#<0.05 compared to normal control, and P*<0.05 compared to vehicle control (3% Tween 80).Furthermore, repeated dose administration of glibenclamide (10 mg/kg), MEPG (200 and 400 mg/kg, p.o.), and VAD (10, 25, and 50 mg/kg, p.o.) for 21 days had significantly improved the glucose tolerance as compared to diabetic control animals (G-II) (Table6).Table 6
Effect of repeated-dose oral administration of MEPG and VAD on oral glucose tolerance test (OGTT) in alloxan-induced diabetic rats on the 21st day.
Group
Treatment
Blood glucose (mg/dL)
0 min
30 min
60 min
120 min
180 min
G-I
Normal control
91.54
±
3.1
135.45
±
3.5
175.68
±
4.8
124.56
±
4.9
94.556
±
2.8
G-II
Vehicle control (3% Tween 80) 10 mL/kg, p.o.
274.84
±
5.7
#
384.11
±
4.75
#
471.25
±
6.7
#
352.12
±
7.4
#
330.23
±
8.5
#
G-III
Glibenclamide-10 mg/kg, p.o.
94.47
±
2.9
115.46
±
3.5
*
124.56
±
3.4
*
102.56
±
4.1
*
96.56
±
4.2
*
G-IV
MEPG-200 mg/kg, p.o.
132.44
±
7.2
164.56
±
7.2
*
186.35
±
6.2
*
148.90
±
5.2
*
135.65
±
3.1
*
G-V
MEPG-400 mg/kg, p.o.
115.27
±
4.9
139.25
±
6.3
*
162.30
±
4.2
*
129.34
±
5.2
*
113.25
±
6.2
*
G-VI
VAD-10 mg/kg, p.o.
124.66
±
4.1
147.65
±
3.2
*
172.31
±
6.8
*
138.56
±
3.9
*
127.65
±
4.7
*
G-VII
VAD-25 mg/kg, p.o.
98.26
±
2.15
138.69
±
6.4
*
151.26
±
4.1
*
119.87
±
6.3
*
102.65
±
5.8
*
G-VIII
VAD-50 mg/kg, p.o.
95.12. ±3.85
118.65
±
6.2
*
121.45
±
2.3
*
99.56
±
5.9
*
92.65
±
3.7
*
All the values are expressed as mean ± SEM,P#<0.05 compared to normal control, and P*<0.05 compared to vehicle control (3% Tween 80).Alloxan administration is known to reduce the body weight in experimental animals; in present study, administration of alloxan (120 mg/kg, i.v.) had significantly decreased the body weight gradually from day 1 to day 21 of the study period as compared to normal animals. In contrast, repeated administration of VAD (25 and 50 mg/kg) and glibenclamide (10 mg/kg) have prevented the reduction in body weight from the 4th day onwards,  while MEPG (400 mg/kg) and VAD (10 mg/kg) have prevented the decrease in body weight from the 14th day onwards, whereas with MEPG 200 mg/kg they had shown negligible inhibition against decrease in body weight (Table7).Table 7
Effect of repeated-dose oral administration of MEPG and VAD on body weight in alloxan-induced diabetic rats.
Group
Treatment
Body weight (g)
Day 1
Day 7
Day 14
Day 21
G-I
Normal control
238.45
±
6.5
246.85
±
7.1
251.63
±
4.9
254.85
±
7.3
G-II
Vehicle control (3% Tween 80) 10 mL/kg, p.o.
243.56
±
6.4
186.23
±
8.7
#
169.74
±
7.0
#
151.65
±
6.9
#
G-III
Glibenclamide-10 mg/kg, p.o.
241.58
±
8.2
236.56
±
4.5
*
233.84
±
5.2
*
229.56
±
8.2
*
G-IV
MEPG-200 mg/kg, p.o.
240.65
±
6.9
199.23
±
4.9
*
172.94
±
9.1
*
162.75
±
7.9
*
G-V
MEPG-400 mg/kg, p.o.
247.23
±
6.1
232.12
±
7.2
*
224.23
±
7.3
*
204.11
±
10.5
*
G-VI
VAD-10 mg/kg, p.o.
236.52
±
8.4
210.41
±
5.9
*
189.68±7.7*
174.55
±
9.5
*
G-VII
VAD-25 mg/kg, p.o.
242.95
±
7.6
219.64
±
6.6
*
212.47
±
5.9
*
205.56
±
7.8
*
G-VIII
VAD-50 mg/kg, p.o.
243.57
±
6.3
234.85
±
8.4
*
226.18
±
6.3
*
210.38
±
9.4
*
All the values are expressed as mean ± SEM,P#<0.05 compared to normal control, and P*<0.05 compared to vehicle control (3% Tween 80) on corresponding day.Furthermore, single-dose intravenous administration of alloxan (120 mg/kg) caused 48% mortality of animals over a period of 21 days. Exceptionally, administration of MEPG (400 mg/kg), VAD (25 and 50 mg/kg), and glibenclamide (10 mg/kg) for 21 days showed significant protection against alloxan-induced mortality in rats, while MEPG (200 mg/kg) and VAD (10 mg/kg) have showed maximum mortality of 34% and 27%, respectively among all the treated groups at the end of 21 days. These results suggest that MEPG and VAD at higher doses and glibenclamide (10 mg/kg) could protect the animals against alloxan-induced mortality.


#### 3.5.1. Histopathology

In the present study, histopathology of pancreatic tissue collected from normal control (G-I) animals showed normal cytoarchitecture of the pancreas, while alloxanper se treated diabetic control (G-II) rats showed destruction of pancreatic β-cell mass associated with severe acinar cell damage. In contrast, 21-days treatment with MEPG (400 mg/kg, p.o.), VAD (25 and 50 mg/kg), and glibenclamide (10 mg/kg) showed significant protection against alloxan-induced pancreatic tissue damage. However, MEPG (200 mg/kg, p.o.) and VAD (10 mg/kg) treatments showed very minimal protection against alloxan-induced cellular damage (Figure 5).Effect of MEPG and VAD on alloxan-induced cellular damage of pancreas. (a) Normal control (normal cytoarchitecture of pancreas), (b) diabetic control (significant loss of islets and sever acinar damage), (c) glibenclamide (near normal structure of pancreas), (d) MEPG (200 mg/kg) (moderate acinar damage and very minimal number of pancreatic islets), (e) MEPG (400 mg/kg) (mild acinar damage and pancreatic islets), (f) VAD (10 mg/kg) (moderate acinar damage and minimal number of pancreatic islets), (g) VAD (25 mg/kg) (mild acinar damage and sufficient number of pancreatic islets), and (h) VAD (50 mg/kg) (very mild acinar damage and nearly normal pancreatic islets).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.1. Identification and Quantification of the Isolated Compound

Fraction 7 was subjected to NMR and Mass spectroscopy and by comparing the obtained spectral data of the Fraction-7 with the literature reports; it was identified as a well known polyphenolic compound “Valoneic acid dilactone.” The HPLC chromatogram of Methanolic extract, Fraction-7 (Valoneic acid dilactone) and structure of Valoneic acid dilactone  are given in Figures2, 3, and 4, respectively.Figure 2
Analytical HPLC for methanolic extract.Figure 3
Analytical HPLC for fraction 7 (valoneic acid dilactone).Figure 4
Structure of valoneic acid dilactone.Spectral Data
Chemical formula: C21H10O13; molecular weight: 470.
1H NMR (δ values ppm): 6.97 (s, 1H), 6.93 (s, 1H), 7.47 (s, 1H).
13C NMR (δ values ppm): 113.9, 112.0, 108.3, 108.5, 110.4, 106.7, 108.1, 114.7, 139.1, 136.2, 136.7, 135.2, 139.6, 139.6, 140.8, 142.9, 148.6, 149.5, 159.2, 159.2, 165.9.
LC-MS [m/zvalue]: 469.03 [M-H] (molecular ion peak).The Valoneic acid dilactone is a off white amorphous powder, soluble in methanol, purity was found to be greater than 95% and melting point was less than 300°C.


## 3.2.In Vitro  α-Amylase Inhibition Assay

In the present study, MEPG and VAD isolated from fruit rinds ofPunica granatumhave significantly and dose dependently inhibited the α-amylase enzyme activity. Acarbose was used as a reference standard to validate the assay procedure; the findings of the present study are in accordance with the literature reports. Acarbose, MEPG, and VAD have evolved as potent inhibitors of α-amylase enzyme activity with IC50 values of 0.378, 1.02, and 0.284 μg/mL, respectively (Table 1).Table 1
Effect of MEPG and VAD isolated from fruit rinds ofPunica granatum on α-amylase enzyme activity in in vitro.
Sl. no
Sample
Concentration (μg/mL)
% Inhibition#
IC50 (μg/mL)*
0.15
10.21
±
0.54
0.3
29.41
±
0.17
(1)
Methanolic extract
0.6
44.28
±
0.83
1.02
1.25
70.21
±
0.54
2.5
85.88
±
0.52
0.15
29.65
±
0.85
0.3
49.78
±
0.46
(2)
Valoneic acid dilactone
0.6
74.25
±
0.29
0.284
1.25
82.85
±
0.31
2.5
99.52
±
0.71
0.1
33.53
±
0.46
(3)
Acarbose
0.5
62.41
±
0.87
0.378
1
74.25
±
0.29
2
99.21
±
0.34
Values are expressed as mean ± SEM (% inhibition#) and mean (IC50*) of three trials.


## 3.3.In Vitro Aldose Reductase Inhibition Assay

Aldose reductase is the key enzyme involved in the polyol pathway; drugs which inhibit aldose reductase enzyme activity are believed to be beneficial in the prevention of long-term diabetic complications. With this background, MEPG and VAD were evaluated for aldose reductase inhibition activity, and the findings ofin vitroaldose reductase inhibition assay revealed that MEPG and VAD isolated from fruit rinds of Punica granatum showed significant and dose-dependent inhibition of aldose reductase enzyme activity; the IC50 values were found to be 2.050 and 0.788 μg/mL, respectively (Table 2).Table 2
Effect of MEPG and VAD isolated from fruit rinds ofPunica granatum on aldose reductase enzyme activity in in vitro.
Sl. no
Sample
Concentration (μg/mL)
% Inhibition#
IC50 (μg/mL)*
0.3
22.95
±
0.25
0.6
35.38
±
0.47
(1)
Methanolic extract
1.2
45.77
±
0.14
2.050
2.5
65.64
±
0.74
5
84.49
±
0.21
10
99.62
±
0.33
0.15
9.31
±
0.84
0.3
26.63
±
0.18
(2)
Valoneic acid  dilactone
0.6
48.50
±
0.54
0.788
1.2
67.80
±
0.71
2.5
79.19
±
0.21
5
89.43
±
0.34
0.5
8.03
±
0.62
(3)
Quercetin
1.0
24.23
±
0.51
2.807
2.0
42.48
±
0.91
5.0
82.45
±
0.27
Values are expressed as mean ± SEM (% inhibition#) and mean (IC50*) of three trials.


## 3.4. Protein Tyrosine Phosphatase 1B Inhibition (PTP1B) Assay

Inin vitroPTP1B assay, the MEPG and VAD isolated from fruit rinds of Punica granatum showed significant dose-dependent inhibition of PTP1B enzyme activity; the IC50 values were found to be 26.25 and 12.41 μg/mL, respectively (Table 3).Table 3
Effect of MEPG and VAD isolated from fruit rinds ofPunica granatum on protein tyrosine phosphatase-1B (PTP1B) enzyme activity in  in vitro.
Sl. no
Sample
Concentration (μg/mL)
% Inhibition#
IC50 (μg/mL)*
5
21.6
±
0.24
10
39.6
±
0.15
(1)
Methanolic extract
25
55.4
±
0.22
26.25
50
78.7
±
0.34
100
98.9
±
0.22
5
33.3
±
0.35
10
46.7
±
0.54
(2)
Valoneic acid dilactone
25
68.5
±
0.64
12.41
50
84.0
±
0.11
100
98.5
±
0.75
Values are expressed as mean ± SEM (% inhibition#) and mean (IC50*) of three trials.


## 3.5. Alloxan-Induced Diabetes in Rats

Alloxan-induced diabetes is a well-known and commonly used animal model to screen the antidiabetic activity of the herbal and synthetic drugs. In the present study, the intravenous administration of alloxan (120 mg/kg, i.v.) had increased the fasting blood glucose level more than 250 mg/dL after 6 days, while normal rats have showed 90–100 mg/dL. Animals treated with single dose of MEPG (200 and 400 mg/kg) and VAD (10 and 25 mg/kg) did not significantly reduce the blood glucose levels in alloxan-induced diabetic rats. In contrast, VAD (50 mg/kg, p.o.) and glibenclamide (10 mg/kg, p.o.) have significantly reduced the BG levels at 1st, 2nd, and 4th hours after single-dose administration in alloxan-induced diabetic rats (Table4).Table 4
Effect of single-dose oral administration of MEPG and VAD on blood glucose levels in alloxan-induced diabetic rats.
Group
Treatment
Blood glucose (mg/dL)
0 hour
1 hour
2 hours
4 hours
G-I
Normal control
91.23
±
5.2
92.45
±
2.8
92.15
±
4.5
91.54
±
3.1
G-II
Vehicle control (3% Tween 80) 10 mL/kg, p.o.
272.84
±
3.9
#
274.41
±
5.1
#
277.65
±
5.1
#
268.94
±
4.6
#
G-III
Glibenclamide-10 mg/kg, p.o.
271.94
±
4.8
234.15
±
5.3
197.14
±
6.2
*
164.85
±
6.5
*
G-IV
MEPG-200 mg/kg, p.o.
268.51
±
6.2
271.15
±
4.8
255.41
±
7.9
264.12
±
6.1
G-V
MEPG-400 mg/kg, p.o.
272.84
±
5.2
269.64
±
6.5
242.12
±
4.8
*
235.15
±
5.8
*
G-VI
VAD-10 mg/kg, p.o.
259.87
±
5.5
265.32
±
7.3
264.85
±
3.7
261.25
±
7.3
G-VII
VAD-25 mg/kg, p.o.
264.51
±
7.1
261.25
±
3.5
*
258.65
±
5.7
*
256.52
±
4.9
*
G-VIII
VAD-50 mg/kg, p.o.
274.56
±
4.5
241.85
±
6.1
*
220.12
±
6.7
*
198.56
±
5.3
*
All the values are expressed as mean± SEM, P#<0.05 compared to normal control, and P*<0.05 compared to vehicle control (3% Tween 80).Exceptionally, repeated dose administration of MEPG (200 and 400 mg/kg, p.o.) for 21 days had progressively reduced the blood glucose levels significantly (P<0.05) and dose dependently (Table 5); similarly, animals treated with VAD (10, 25, and 50 mg/kg, p.o.) also showed significant (P<0.01) decrease in blood glucose levels compared to diabetic control group (G-II).Table 5
Effect of repeated-dose oral administration of MEPG and VAD on blood glucose levels in alloxan-induced diabetic rats.
Group
Treatment
Blood glucose (mg/dL)
0th day
7th day
14th day
21st day
G-I
Normal control
91.23
±
5.2
92.45
±
2.8
92.15
±
4.5
91.54
±
3.1
G-II
Vehicle control (3% Tween 80) 10 mL/kg, p.o.
272.84
±
3.9
#
279.22
±
7.4
#
281.25
±
6.2
#
274.84
±
5.7
#
G-III
Glibenclamide-10 mg/kg, p.o.
271.94
±
4.8
124.98
±
8.1
*
104.28
±
4.9
*
94.47
±
2.9
*
G-IV
MEPG-200 mg/kg, p.o.
268.51
±
6.2
198.64
±
2.8
*
172.94
±
23
*
132.44
±
7.2
*
G-V
MEPG-400 mg/kg, p.o.
272.84
±
5.2
165.22
±
5.2
*
152.94
±
5.1
*
115.27
±
4.9
*
G-VI
VAD-10 mg/kg, p.o.
259.87
±
5.5
169.68
±
2.7
*
159.32
±
4.8
*
124.66
±
4.1
*
G-VII
VAD-25 mg/kg, p.o.
264.51
±
7.1
148.25
±
4.6
*
129.80
±
3.8
*
98.26
±
2.15
*
G-VIII
VAD-50 mg/kg, p.o.
274.56
±
4.5
132.58
±
3.1
*
109.85
±
2.2
*
95.12
±
3.85
*
All the values are expressed as mean ± SEM,P#<0.05 compared to normal control, and P*<0.05 compared to vehicle control (3% Tween 80).Furthermore, repeated dose administration of glibenclamide (10 mg/kg), MEPG (200 and 400 mg/kg, p.o.), and VAD (10, 25, and 50 mg/kg, p.o.) for 21 days had significantly improved the glucose tolerance as compared to diabetic control animals (G-II) (Table6).Table 6
Effect of repeated-dose oral administration of MEPG and VAD on oral glucose tolerance test (OGTT) in alloxan-induced diabetic rats on the 21st day.
Group
Treatment
Blood glucose (mg/dL)
0 min
30 min
60 min
120 min
180 min
G-I
Normal control
91.54
±
3.1
135.45
±
3.5
175.68
±
4.8
124.56
±
4.9
94.556
±
2.8
G-II
Vehicle control (3% Tween 80) 10 mL/kg, p.o.
274.84
±
5.7
#
384.11
±
4.75
#
471.25
±
6.7
#
352.12
±
7.4
#
330.23
±
8.5
#
G-III
Glibenclamide-10 mg/kg, p.o.
94.47
±
2.9
115.46
±
3.5
*
124.56
±
3.4
*
102.56
±
4.1
*
96.56
±
4.2
*
G-IV
MEPG-200 mg/kg, p.o.
132.44
±
7.2
164.56
±
7.2
*
186.35
±
6.2
*
148.90
±
5.2
*
135.65
±
3.1
*
G-V
MEPG-400 mg/kg, p.o.
115.27
±
4.9
139.25
±
6.3
*
162.30
±
4.2
*
129.34
±
5.2
*
113.25
±
6.2
*
G-VI
VAD-10 mg/kg, p.o.
124.66
±
4.1
147.65
±
3.2
*
172.31
±
6.8
*
138.56
±
3.9
*
127.65
±
4.7
*
G-VII
VAD-25 mg/kg, p.o.
98.26
±
2.15
138.69
±
6.4
*
151.26
±
4.1
*
119.87
±
6.3
*
102.65
±
5.8
*
G-VIII
VAD-50 mg/kg, p.o.
95.12. ±3.85
118.65
±
6.2
*
121.45
±
2.3
*
99.56
±
5.9
*
92.65
±
3.7
*
All the values are expressed as mean ± SEM,P#<0.05 compared to normal control, and P*<0.05 compared to vehicle control (3% Tween 80).Alloxan administration is known to reduce the body weight in experimental animals; in present study, administration of alloxan (120 mg/kg, i.v.) had significantly decreased the body weight gradually from day 1 to day 21 of the study period as compared to normal animals. In contrast, repeated administration of VAD (25 and 50 mg/kg) and glibenclamide (10 mg/kg) have prevented the reduction in body weight from the 4th day onwards,  while MEPG (400 mg/kg) and VAD (10 mg/kg) have prevented the decrease in body weight from the 14th day onwards, whereas with MEPG 200 mg/kg they had shown negligible inhibition against decrease in body weight (Table7).Table 7
Effect of repeated-dose oral administration of MEPG and VAD on body weight in alloxan-induced diabetic rats.
Group
Treatment
Body weight (g)
Day 1
Day 7
Day 14
Day 21
G-I
Normal control
238.45
±
6.5
246.85
±
7.1
251.63
±
4.9
254.85
±
7.3
G-II
Vehicle control (3% Tween 80) 10 mL/kg, p.o.
243.56
±
6.4
186.23
±
8.7
#
169.74
±
7.0
#
151.65
±
6.9
#
G-III
Glibenclamide-10 mg/kg, p.o.
241.58
±
8.2
236.56
±
4.5
*
233.84
±
5.2
*
229.56
±
8.2
*
G-IV
MEPG-200 mg/kg, p.o.
240.65
±
6.9
199.23
±
4.9
*
172.94
±
9.1
*
162.75
±
7.9
*
G-V
MEPG-400 mg/kg, p.o.
247.23
±
6.1
232.12
±
7.2
*
224.23
±
7.3
*
204.11
±
10.5
*
G-VI
VAD-10 mg/kg, p.o.
236.52
±
8.4
210.41
±
5.9
*
189.68±7.7*
174.55
±
9.5
*
G-VII
VAD-25 mg/kg, p.o.
242.95
±
7.6
219.64
±
6.6
*
212.47
±
5.9
*
205.56
±
7.8
*
G-VIII
VAD-50 mg/kg, p.o.
243.57
±
6.3
234.85
±
8.4
*
226.18
±
6.3
*
210.38
±
9.4
*
All the values are expressed as mean ± SEM,P#<0.05 compared to normal control, and P*<0.05 compared to vehicle control (3% Tween 80) on corresponding day.Furthermore, single-dose intravenous administration of alloxan (120 mg/kg) caused 48% mortality of animals over a period of 21 days. Exceptionally, administration of MEPG (400 mg/kg), VAD (25 and 50 mg/kg), and glibenclamide (10 mg/kg) for 21 days showed significant protection against alloxan-induced mortality in rats, while MEPG (200 mg/kg) and VAD (10 mg/kg) have showed maximum mortality of 34% and 27%, respectively among all the treated groups at the end of 21 days. These results suggest that MEPG and VAD at higher doses and glibenclamide (10 mg/kg) could protect the animals against alloxan-induced mortality.


### 3.5.1. Histopathology

In the present study, histopathology of pancreatic tissue collected from normal control (G-I) animals showed normal cytoarchitecture of the pancreas, while alloxanper se treated diabetic control (G-II) rats showed destruction of pancreatic β-cell mass associated with severe acinar cell damage. In contrast, 21-days treatment with MEPG (400 mg/kg, p.o.), VAD (25 and 50 mg/kg), and glibenclamide (10 mg/kg) showed significant protection against alloxan-induced pancreatic tissue damage. However, MEPG (200 mg/kg, p.o.) and VAD (10 mg/kg) treatments showed very minimal protection against alloxan-induced cellular damage (Figure 5).Effect of MEPG and VAD on alloxan-induced cellular damage of pancreas. (a) Normal control (normal cytoarchitecture of pancreas), (b) diabetic control (significant loss of islets and sever acinar damage), (c) glibenclamide (near normal structure of pancreas), (d) MEPG (200 mg/kg) (moderate acinar damage and very minimal number of pancreatic islets), (e) MEPG (400 mg/kg) (mild acinar damage and pancreatic islets), (f) VAD (10 mg/kg) (moderate acinar damage and minimal number of pancreatic islets), (g) VAD (25 mg/kg) (mild acinar damage and sufficient number of pancreatic islets), and (h) VAD (50 mg/kg) (very mild acinar damage and nearly normal pancreatic islets).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.5.1. Histopathology

In the present study, histopathology of pancreatic tissue collected from normal control (G-I) animals showed normal cytoarchitecture of the pancreas, while alloxanper se treated diabetic control (G-II) rats showed destruction of pancreatic β-cell mass associated with severe acinar cell damage. In contrast, 21-days treatment with MEPG (400 mg/kg, p.o.), VAD (25 and 50 mg/kg), and glibenclamide (10 mg/kg) showed significant protection against alloxan-induced pancreatic tissue damage. However, MEPG (200 mg/kg, p.o.) and VAD (10 mg/kg) treatments showed very minimal protection against alloxan-induced cellular damage (Figure 5).Effect of MEPG and VAD on alloxan-induced cellular damage of pancreas. (a) Normal control (normal cytoarchitecture of pancreas), (b) diabetic control (significant loss of islets and sever acinar damage), (c) glibenclamide (near normal structure of pancreas), (d) MEPG (200 mg/kg) (moderate acinar damage and very minimal number of pancreatic islets), (e) MEPG (400 mg/kg) (mild acinar damage and pancreatic islets), (f) VAD (10 mg/kg) (moderate acinar damage and minimal number of pancreatic islets), (g) VAD (25 mg/kg) (mild acinar damage and sufficient number of pancreatic islets), and (h) VAD (50 mg/kg) (very mild acinar damage and nearly normal pancreatic islets).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 4. Discussion

In the traditional system of medicine, several medicinal plants have been widely used for the treatment of diabetes mellitus, andPunica granatumis one of such plants which has been used as a key ingredient in many ayurvedic antidiabetic formulations [21]. In line with the ayurvedic literature, various parts of the plant have been scientifically proved for antidiabetic activity [21]. In our earlier studies, we have isolated some of the antioxidant principles from methanolic extracts of fruit rinds of Punica granatum[8]. In continuation of our previous work, the present work is been undertaken to isolate and evaluate the bioactive constituents relevant to antidiabetic activity.In context of the mentioned objectives, a bioactive phytochemical constituent has been isolated from methanolic extracts ofPunica granatum and identified as a well-known polyphenolic compound valoneic acid dilactone (VAD) by comparing the obtained spectral data with the literature [25, 26].The isolated compound (VAD) and the crude  methanolic extract (MEPG) had been evaluated for antidiabetic activity; aldose reductase inhibition assay, PTP1B assay, andα-amylase inhibition assays were used for in vitroevaluation, and alloxan-induced diabetes in rats was used as an in vivomodel.Aldose reductase is an NADPH-dependent oxidoreductase enzyme involved in polyol pathway; it plays a central role in the conversion of aldose to polyol. Under diabetic condition, excessive influx of glucose into the tissues results in the increased aldose reductase activity, and thereby increased levels of polyol [27]. Furthermore, evidences suggest that aldose reductase plays pivotal role in the pathogenesis of diabetic complications such as neuropathy, nephropathy, cataract, retinopathy, and microangiopathy [28]. Available preclinical and preliminary clinical evidence suggests that inhibition of aldose reductase enzyme activity may be useful in the prevention of onset of diabetic complications [28]. In present study, the MEPG and VAD have showed significant and dose-dependent inhibition of aldose reductase enzyme activity with IC50 values of 2.050 and 0.788 μg/mL, respectively. Quercetin used as a reference standard showed dose-dependent inhibition of aldose reductase activity with IC50 value of 2.807 μg/mL. The phytochemicals were evolved as very potent inhibitors of aldose reductase enzyme, especially the plant derivatives rich in polyphenols, flavonoids, and sugar derivatives are well known for their aldose reductase inhibition property [27]. In present study, VAD was evolved as highly potent inhibitor of aldose reductase may be due to its polyphenolic nature.Drugs that inhibit carbohydrate-hydrolyzing enzymes have been demonstrated to decrease postprandial hyperglycemia and improve impaired glucose metabolism without promoting insulin secretion in type II diabetic patients [29]. α-amylase is one of the key enzymes that hydrolyse alpha-bonds of alpha-linked large polysaccharides, such as starch and glycogen yielding glucose and maltose [30]. The results of in vitro studies showed that MEPG and VAD inhibit α-amylase activity significantly and dose dependently with IC50 values of 1.02 and 0.284 μg/mL, respectively.Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of insulin action. Overexpression of PTP1B protein has been observed in insulin-resistant states associated with obesity and diabetes [31]. PTP1B directly catalyzes the dephosphorylation of cellular substrates of the insulin receptor kinase results in the downregulation of insulin action [31].In a recent study, it has been reported that mice lacking functional PTP1B gene showed enhanced tyrosine kinase activity and increased insulin sensitivity [32]. The levels of PTP1B expression in muscle and adipose tissues in humans were strongly correlated to insulin resistance states. In another animal-based approach, treatment with antisense oligonucleotide specific for PTP1B resulted in normalization of blood glucose and insulin levels in animal models of type II diabetes [32]. Accordingly, PTP1B inhibitors could increase insulin receptor tyrosine phosphorylation and mimic the cellular and in vivo actions of insulin and thereby decrease plasma glucose levels in diabetic animals [33].In the present study, the MEPG and VAD have inhibited the PTP1B enzyme activity inin vitro conditions significantly and dose dependently with IC50  values of 26.25 and 12.41 μg/mL, respectively. All the in vitrostudies have demonstrated the antidiabetic effect of MEPG and VAD. The VAD was evolved as potent inhibitor of aldose reductase, α-amylase, and PTP1B enzymes and hence thought to be beneficial in the treatment of diabetes.Based on thein vitro results, the MEPG and VAD were further studied in in vivo against alloxan-induced diabetes in rats.Alloxan is a hydrophilic and chemically unstable pyrimidine derivative; with its toxic nature it can cause a massive reduction in insulin release by the destruction of the pancreaticβ cells, inducing hyperglycaemia [4]. The diabetic rats (induced by alloxan) showed a persistent rise in blood glucose levels after 7 days with the characteristic features of diabetes mellitus.Traditional plant medicines are used throughout the world for a range of diabetic presentations, and the study of such medicines might offer a natural key to unlock a diabetologist’s pharmacy for the future [1]. In light of this, we made an attempt to study the effect of MEPG (200 and 400 mg/kg, p.o.) and VAD (10, 25, and 50 mg/kg, p.o.) against alloxan-induced diabetic rats based on the obtained positive results in in vitrostudies. Oral glucose tolerance test (OGTT) carried out after single-dose administration of MEPG (400 mg/kg, p.o.) showed marginal decrease in elevated blood glucose levels after alloxan treatment, exceptionally glibenclamide (10 mg/kg) and VAD (25 and 50 mg/kg, p.o.) have showed marked decrease in blood glucose levels as compared to diabetic control animals (G-II). In contrast, MEPG (200 mg/kg, p.o.) and VAD (10 mg/kg, p.o.) were failed to decrease the blood glucose levels in alloxan-induced diabetic rats.On repeated administration, the MEPG (200 and 400 mg/kg, p.o.) and VAD (10, 25, and 50 mg/kg, p.o.) have showed significant antihyperglycemic activity from day 7 onwards by controlling the elevated blood glucose levels compared with vehicle control group. All the doses of MEPG (200 and 400 mg/kg, p.o.) and VAD (10, 25, and 50 mg/kg, p.o.) have showed potent antihyperglycemic activity in OGTT carried out on day 21 of the study. In contrast, impaired glucose tolerance is observed in alloxan-induced diabetic rats due to destructing the pancreaticβ cells [23]. These findings suggest that the MEPG and VAD may show insulin mimetic activity or improved glucose utilization mechanism.The possible mechanism by which the MEPG and VAD bring about their antihyperglycemic action may be by increasing the secretion of insulin from pancreaticβ cells or its release from the bound form [34].In context of understanding the mechanism, it is well established that sulfonylurea class of drugs produce hypoglycaemia by increasing the secretion of insulin from pancreas, and hence, these compounds are active in mild alloxan-induced diabetes, whereas they are not effective against intense alloxan-induced diabetes in animals (nearly all the beta cells have been destroyed) [1]. In our work, histopathology of alloxan per se treated animals showed significant but not complete loss of pancreatic islets associated with sever acinar damage. However, glibenclamide- (10 mg/kg, p.o.) treated animals showed normal architecture of the pancreas; MEPG (400 mg/kg, p.o.) and VAD (25 and 50 mg/kg, p.o.) treated groups have showed very minimal acinar damage and sufficient number of pancreatic islets (Figure 1). These findings suggest that some pancreatic β cells are still surviving to exert their insulin releasing effect, and hence, it can be concluded that the anti-hyperglycemic effect of MEPG and VAD was probably mediated by enhanced secretion of insulin-like sulfonylureas.One of the major complications of type I diabetes is weight loss. It arises due to the impairment in insulin action in the conversion of glucose into glycogen and catabolism of fats and inhibition of lipolysis due to its unavailability because of destruction in beta cells [35]. Due to this, there will be a decrease in the body weight of the animals and finally leads to death.Treatment with glibenclamide, MEPG, and VAD has substantially prevented the body weight loss and mortality produced by the alloxan. The findings of the present study such as blood glucose levels, oral glucose tolerance test, body weight, mortality, and histopathology findings are in correlation with each other and indicate that MEPG and VAD could be beneficial in management of diabetes and associated complications.


## 5. Conclusion

In present study, valoneic acid dilactone (VAD) was isolated and confirmed by comparing the obtained spectral data with the literature values. In preliminary antidiabetic studies, the methanolic extracts (MEPG) and VAD isolated from fruit rinds ofPunica granatumpossessed potent antidiabetic activity in both in vitroand in vivomodels.



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*Source: 147202-2012-07-31.xml*