Isolation of Antidiabetic Principle from Fruit Rinds of Punica Granatum

Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 147202, 11 pages doi:10.1155/2012/147202

Research Article Isolation of Antidiabetic Principle from Fruit Rinds of Punica granatum

Vishal Jain,1 G. L. Viswanatha,2 D. Manohar,1 and H. N. Shivaprasad1

1 Research and Development, Olive Lifesciences Pvt. Ltd., Karnataka, Tumkur 572 106, India 2 Department of Pharmacology, PES College of Pharmacy, Karnataka, Bangalore 560 050, India

Correspondence should be addressed to Vishal Jain, [email protected]

Received 26 February 2012; Accepted 26 April 2012

Academic Editor: I-Min Liu

Copyright © 2012 Vishal Jain et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Present study was aimed to isolate and evaluate the antidiabetic activity of phytoconstituents from fruit rinds of Punica 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.

1. Introduction such as flavonoids, gallotannins, amino acids, and other related polyphenols [5]. Diabetes mellitus is a metabolic disorder characterized by Pomegranate (Punica granatum L., Punicaceae) is used hyperglycaemia and alterations in carbohydrate, lipid, and in the traditional system of medicine in different Asian protein metabolism, associated with absolute or relative cultures for the treatment of variety of ailments. In Ayurvedic deficiencies in insulin secretion and/or insulin action [1]. medicine, the plant is described under its Sanskrit name The World Health Organization (WHO) has estimated “dalima” (fruit) as a “blood purifier” and used to cure that major burden of diabetes will occur in developing parasitic infections, aphthae (mouth ulcers), diarrhoea, and countries, and by 2025, there will be 42% increase (from 51 ulcers [6]. Punica granatum is a deciduous tree belongs to to 72 million) in the developed countries and 170% increase the family Punicaceae; it is known for its edible fruit. During (from 84 to 228 million) in the developing countries [2]. the last two decades, various parts of the plant had been It is estimated that, by 2025, India, China, and the United subjected to extensive phytochemical, pharmacological, and States will be the top three countries to have large number of clinical investigations; many interesting findings have been diabetic patients [3]. reported in various fields [7]. The high prevalence of diabetes as well as its long- Punica granatum is found to contain hydrolysable tan- term complications has led to an ongoing search for nins as major active chemical constituents and phyto- hypoglycaemic agents; over the years, various medicinal constituents, namely, corilagin, ellagic acid, kaempferol, plants and their extracts have been reported to be effective luteolin, myricetin, quercetin, quercimetrine, and quercetin- in the treatment of diabetes [4]. Plants are rich sources 3-o-rutinoside which were previously isolated from the fruits of antidiabetic, antihyperlipidemic, and antioxidant agents of Punica granatum [8]. 2 Evidence-Based Complementary and Alternative Medicine

In the literature, various parts of the plant are reported Dried fruit rind powder of pomegranate 3 kg 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 800 g methanolic extract dysfunction [17], memory enhancing [18], wound healing [19], and anticancer [20] activities. Apart from these medic- Partitioned with ethyl acetate inal uses, various parts of the plant have been scientifically proved for antidiabetic activity [21]. In our previous studies, Ethyl acetate fraction (137.3 g) we have isolated three components from methanolic extracts Fractionation of ethyl acetate fraction 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. Fr-1 Fr-(2-6) Fr-7 Fr-8 Fr-(9-12) 2. Materials and Methods 2.1. Drugs and Chemicals. Human recombinant PTP1B (BIOMOL International LP (Plymouth Meeting, PA)), α- Valoneic acid dilactone amylase (Sigma Aldrich, Bangalore), acarbose (Glenmark, Mumbai), glibenclamide (Cipla, Mumbai), and biochemical Figure 1: Scheme of isolation process. 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. (98% pure), respectively, based on spectral data. With this 2.2. Plant Material. The fruit rinds of Punica granatum were background information, FR-7 was further taken forward for collected locally and authenticated by Dr. K. P. Srinath, characterization by NMR and Mass spectroscopy at Radiant Department of Botany, Bangalore University, Bangalore; Research and Training Institute and IISc, Bangalore. a voucher specimen was deposited in the department (PESCP/2009-10/PG-06). 2.4. Experimental Animals. Male Wistar rats (200–250 g) were purchased from Bioneeds, Nelamangala, Tumkur, 2.3. Isolation and Identification of the Compound. Fruit rinds India, and they were maintained in polypropylene cages at ± ◦ of Punica granatum were collected, dried, and powdered; the atemperatureof25 1 C and relative humidity of 45– powder (3.0 Kg) was successively extracted with methanol in 55% in clean environment under 12:12 h light-dark cycle. a static extractor and concentrated under vacuum at 60◦C Theanimalshadfreeaccesstofoodpellets(PranavAgro and dried in vacuum tray dryer. The percentage yield of the Industries, Bangalore, India) and purified water ad libitum. methanolic extract was found to be 26.67% w/w (800 g per AlltheexperimentalprotocolswereapprovedbyInstitu- 3.0 kg of methanolic extract). tional Animal Ethics Committee (IAEC), and all the animal Vacuum-dried methanolic extract (800 g) was macerated experiments were conducted according to the principles with ethyl acetate in the solvent : solute ratio of 4 : 1 for 72 h and guidelines of CPCSEA (Committee for the Purpose of with frequent shaking. The fraction was dried using vacuum Control and Supervision of Experimentation on Animals), tray dryer. The yield of ethyl acetate fraction was found to India. be 27% w/w. The major phytochemical constituents present in the ethyl acetate fraction were identified by thin-layer 2.5. Experimental Protocol chromatography (TLC). TLC plates were developed using water : acetic acid solvent system (3 : 2) and scanned at 254 2.5.1. In Vitro α-Amylase Inhibition Assay. In vitro α-amylase and 366 nm. The 5% FeCl3 reagent was sprayed, and the inhibition assay was carried out based on the spectrophoto- chromatogram was observed. metric assay using acarbose as the reference compound [4]. The fraction was subjected to HPLC for the identifi- 2 mg of MEPG and VAD was dissolved in 500 μLofDMSO, cation and quantification of the active components; based and further dilutions were prepared using deionised water. on HPLC data, ethyl acetate fraction was subjected to The enzyme α-amylase solution (0.5 unit/mL) was prepared column chromatography for the isolation of phytochemical by mixing 3.246 mg of α-amylase (EC 3.2.1.1) in 100 mL of constituent using a glass column and Diaion resin as 40 mM phosphate buffer pH 6.9. In short, 60 μLof40mM stationary phase. Totally 12 subfractions (FR-1 to FR-12) phosphate buffer/acarbose/test drug and 30 μLofα-amylase were obtained from the ethyl acetate fraction, and all the enzyme were added, mixed, and preincubated at 37◦Cfor sub fractions were subjected to HPLC for the identification 10 min, and then 120 μL of 2-chloro-p-nitrophenyl-α-D- of the desired compound. Scheme of isolation process is maltotrioside (CNPG3) was added, mixed, and incubated at given in Figure 1. In our previous study, the FR-1 and FR- 37◦C for 8 min. The absorbance was measured at 405 nm, 8 were identified as gallic acid (95% pure) and ellagic acid and control reaction was carried out without the extract. Evidence-Based Complementary and Alternative Medicine 3

Percentage inhibition was calculated by expression: adjusted to an ionic strength of 0.15 M by the addition of NaCl. In brief, appropriately diluted inhibitors (undiluted % Inhibition and 50 μM compound) were added to the reaction mixture containing 0 or 2.5 mM pNPP (final assay concentration, Absorbance of control − Absorbance of test μ = × 100. total volume of 100 L). The reaction was initiated by the Absorbance of control addition of the enzyme (i.e., recombinant PTP1B) and (1) allowed to proceed for 5 min before the inhibitor was added and the time was recorded, that is, it continued 2.5.2. In Vitro Aldose Reductase Inhibition Assay 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. (a) Preparation of Aldose Reductase. One-gram of eye The enzyme activity was determined by measuring the lenses were pooled and homogenised in 12 volumes absorbance at 405 nm using Tecan ELISA reader with appro- ff of 135 mM sodium phosphate bu er (pH 7.0) contain- priate corrections for absorbance of substrate, compounds, ing 0.5 mM phenylmethylsulfonyl fluoride and 10 mM and nonenzymatic hydrolysis of substrate. 2-mercaptoethanol. The homogenate was centrifuged at 10,000 g for 30 minutes, and the resultant supernatant was 2.5.4. Alloxan-Induced Diabetes in Rats. Alloxan monohy- retained as an enzyme preparation. All the procedures were ◦ drate was dissolved in saline and administered intravenously carried out at 4 C. The activity of this preparation was to fasted rats (200–250 g) at a dose of 120 mg/kg. The determined by measuring the amount of NADP released per ◦ solution should be fresh and prepared just prior to the unit time at 37 C and pH 7.0. One unit (U) of activity is administration. The rats were given 5% (w/v) glucose defined as the amount of the enzyme catalysed the oxidation solution in feeding bottles for next 24 h in their cages to μ of 1 mol of NADPH per minute under our experimental prevent hypoglycaemia after alloxan injection. After 72 h, rats conditions [22]: with blood glucose levels greater than 200 mg/dL and less / than 400 mg/dL were selected and observed for consistent Activity U mL hyperglycaemia (fasting blood glucose level greater than / × 200 mg/dL and lesser then 400 mg/dL) up to 7 days. Those = Change in OD of test min Total volume of the assay . × , animals were divided into eight groups (G-I to G-VIII), each 6 2 Volume of enzyme taken for analysis × = (2) group consisting of eight animals (8 8 64 animals), G- Iservedasnormalcontrol,G-IIservedasdiabeticcontrol where 6.2 = micromolar extinction coefficientofNADPHat received 3% v/v Tween 80 in water (10 mL/kg, p.o.), G- 340 nm. 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 (b) Assay Procedure. Aldose reductase inhibition activity was were treated with 10, 25, and 50 mg/kg, p.o. of VAD. All the carried out by photometric method [22]. The procedure was treatments were given for 21 days. miniature and carried out using 96-well plate. The drug On day-1 of drug treatment, blood samples were col- ff solutions were prepared by dissolving in bu er solution lectedbyretro-orbitalpunctureat0,1,2,4,and8h ff (67 mM of sodium phosphate bu er, pH 7.0) containing after the administration, while on 4th, 7th, 14th, and 21st 10% DMSO solution. The stock concentration of 1 mg/mL days blood samples were collected after 1 h of respective solution was prepared, and it was serially diluted in a 96- drug treatments. Blood glucose levels were estimated by well plate to obtain varying dilutions. Similarly without the using GOD/POD kit. Oral glucose tolerance test (OGTT) drug, a positive control was also run. Each well of the plate was carried out on 21st day. Body weight of all animals μ ff contained 50 Lofdrugsolution(bu er in case of control), was recorded on the 0, 4th, 7th, 14th, and 21st days μ μ 50 L of NADPH (0.04 mM), and 100 L of aldose reductase before vehicle/glibenclamide/test drug administration. The enzyme preparation. The enzymatic reaction was initiated percentage change of body weight was calculated from its μ by the addition of 75 L of substrate, DL-glyceraldehyde initial weight. (5 × 10−4 M). The absorbance was recorded every minute for duration of 20 minutes at 340 nm. The decrease in (a) Assessment of Mortality Rate in Alloxan-Induced Diabetic the absorbance due to oxidation of NADPH to NADP was Rats. Alloxan may cause severe ketoacidosis and may lead to ff recorded at di erent time points. The concentrations of the death of the animal. In view of this, the mortality rate was inhibitors producing 50% inhibition of the enzyme activity monitored throughout the study. The % of mortality was (IC50) were calculated from the percentage inhibition [23]. calculated on 7th, 14th, and 21st days of the study.

2.5.3. Protein Tyrosine Phosphatase 1B Inhibition Assay. The (b) Histopathology. At the end of the study, all the animals phosphatase activity was assayed using pNPP (Para-Nitro are sacrificed, and pancreas was excised and rinsed in ice- Phenyl Phosphate) as substrate and carried out essentially cold normal saline. A portion of the tissue was fixed in 10% as described previously [24]. The assay buffer (pH 7.4), formalin, cut into 5 μm thick sections, and stained using consisting of 50 mM 3,3-dimethyl glutarate, 1 mM EDTA, hematoxylin-eosin, and histopathological observations were 5 mM glutathione, and 0.5% FCS (not heat inactivated) was made. 4 Evidence-Based Complementary and Alternative Medicine

DetA Ch1 DetA Ch1

1500 8.514 6.762 1000 1000 (mV) 6.117 500 (mV) 500 10.076 8.491 10.121 11.192 9.180 11.459 0 0 5 10 15 20 25 30 0 (min) 0 5 10 15 20 25 30 (min) 1DetA Ch1/254 nm Peak table Figure 2: Analytical HPLC for methanolic extract. Detector A Ch1 254 nm Peak #no. Ret. time Area Height Area % Height % 1 8.514 17773454 1641965 95.389 93.922 2.6. Statistical Analysis. Thevalueswereexpressedasmean± 2 9.180 60708 8350 0.326 0.478 S.E.M. Data was statistically analyzed using one-way ANOVA 3 10.121 379681 45676 2.038 2.613 followed by Tukey’s multiple comparison test. P<0.05 was 4 11.192 351827 43582 1.888 2.493 considered as statistically significant. 5 11.459 66965 8644 0.359 2.494 Total 18632635 1748216 100.000 100.000 3. Results Figure 3: Analytical HPLC for fraction 7 (valoneic acid dilactone). The present study was undertaken to isolate and evaluate the antidiabetic activity of bioactive constituents present in O OH the fruit rind extracts of Punica granatum in in vitro and OH in vivo models. In continuation of our previous studies, 7 1 we have shortlisted some of the fractions for isolation of O O  O  2 2  active constituents and subsequent evaluation of antidiabetic 7 6 activity of the actives in various in vitro and in vivo models.  1 5 1 3 3.1. Identification and Quantification of the Isolated Com- 5 HO OH pound. Fraction 7 was subjected to NMR and Mass spec- 7 troscopy and by comparing the obtained spectral data of 2 OH O the Fraction-7 with the literature reports; it was identi- HO O fied as a well known polyphenolic compound “Valoneic acid dilactone.” The HPLC chromatogram of Methanolic OH extract, Fraction-7 (Valoneic acid dilactone) and structure Figure 4: Structure of valoneic acid dilactone. of Valoneic acid dilactone are given in Figures 2, 3,and4, respectively. 3.2. In Vitro α-Amylase Inhibition Assay. In the present Spectral Data study, MEPG and VAD isolated from fruit rinds of Punica granatum have significantly and dose dependently inhibited Chemical formula: C21H10O13; molecular weight: the α-amylase enzyme activity. Acarbose was used as a 470. reference standard to validate the assay procedure; the 1HNMR(δ values ppm): 6.97 (s, 1H), 6.93 (s, 1H), findings of the present study are in accordance with the 7.47 (s, 1H). literature reports. Acarbose, MEPG, and VAD have evolved α 13CNMR(δ values ppm): 113.9, 112.0, 108.3, 108.5, as potent inhibitors of -amylase enzyme activity with IC50 μ 110.4, 106.7, 108.1, 114.7, 139.1, 136.2, 136.7, 135.2, values of 0.378, 1.02, and 0.284 g/mL, respectively (Table 1). 139.6, 139.6, 140.8, 142.9, 148.6, 149.5, 159.2, 159.2, 165.9. 3.3. In Vitro Aldose Reductase Inhibition Assay. Aldose reduc- LC-MS [m/z value]: 469.03 [M-H] (molecular ion tase is the key enzyme involved in the polyol pathway; drugs peak). which inhibit aldose reductase enzyme activity are believed to be beneficial in the prevention of long-term diabetic The Valoneic acid dilactone is a off white amorphous complications. With this background, MEPG and VAD were powder, soluble in methanol, purity was found to be greater evaluated for aldose reductase inhibition activity, and the than 95% and melting point was less than 300◦C. findings of in vitro aldose reductase inhibition assay revealed Evidence-Based Complementary and Alternative Medicine 5

Table 1: Eﬀect of MEPG and VAD isolated from fruit rinds of Punica 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 0.378 174.25 ± 0.29 299.21 ± 0.34 # ∗ Values are expressed as mean ± SEM (% inhibition )andmean(IC50 ) of three trials.

that MEPG and VAD isolated from fruit rinds of Punica improved the glucose tolerance as compared to diabetic granatum showed significant and dose-dependent inhibition control animals (G-II) (Table 6). of aldose reductase enzyme activity; the IC50 values were Alloxan administration is known to reduce the body found to be 2.050 and 0.788 μg/mL, respectively (Table 2). weight in experimental animals; in present study, administration of alloxan (120 mg/kg, i.v.) had significantly 3.4. Protein Tyrosine Phosphatase 1B Inhibition (PTP1B) decreased the body weight gradually from day 1 to day 21 Assay. In in vitro PTP1B assay, the MEPG and VAD isolated of the study period as compared to normal animals. In from fruit rinds of Punica granatum showed significant dose- contrast, repeated administration of VAD (25 and 50 mg/kg) dependent inhibition of PTP1B enzyme activity; the IC50 and glibenclamide (10 mg/kg) have prevented the reduc- values were found to be 26.25 and 12.41 μg/mL, respectively tion in body weight from the 4th day onwards, while (Table 3). MEPG (400 mg/kg) and VAD (10 mg/kg) have prevented the decrease in body weight from the 14th day onwards, whereas 3.5. Alloxan-Induced Diabetes in Rats. Alloxan-induced dia- with MEPG 200 mg/kg they had shown negligible inhibition betes is a well-known and commonly used animal model to against decrease in body weight (Table 7). screen the antidiabetic activity of the herbal and synthetic Furthermore, single-dose intravenous administration of drugs. In the present study, the intravenous administration alloxan (120 mg/kg) caused 48% mortality of animals over of alloxan (120 mg/kg, i.v.) had increased the fasting blood a period of 21 days. Exceptionally, administration of MEPG glucose level more than 250 mg/dL after 6 days, while normal (400 mg/kg), VAD (25 and 50 mg/kg), and glibenclamide rats have showed 90–100 mg/dL. Animals treated with single (10 mg/kg) for 21 days showed significant protection against dose of MEPG (200 and 400 mg/kg) and VAD (10 and alloxan-induced mortality in rats, while MEPG (200 mg/kg) 25 mg/kg) did not significantly reduce the blood glucose and VAD (10 mg/kg) have showed maximum mortality of levels in alloxan-induced diabetic rats. In contrast, VAD 34% and 27%, respectively among all the treated groups at (50 mg/kg, p.o.) and glibenclamide (10 mg/kg, p.o.) have the end of 21 days. These results suggest that MEPG and VAD significantly reduced the BG levels at 1st, 2nd, and 4th hours at higher doses and glibenclamide (10 mg/kg) could protect after single-dose administration in alloxan-induced diabetic the animals against alloxan-induced mortality. rats (Table 4). Exceptionally, repeated dose administration of MEPG 3.5.1. Histopathology. In the present study, histopathology (200 and 400 mg/kg, p.o.) for 21 days had progressively of pancreatic tissue collected from normal control (G-I) reduced the blood glucose levels significantly (P<0.05) and animals showed normal cytoarchitecture of the pancreas, dose dependently (Table 5); similarly, animals treated with while alloxan per se treated diabetic control (G-II) rats VAD (10, 25, and 50 mg/kg, p.o.) also showed significant showed destruction of pancreatic β-cell mass associated with (P<0.01) decrease in blood glucose levels compared to severe acinar cell damage. In contrast, 21-days treatment diabetic control group (G-II). with MEPG (400 mg/kg, p.o.), VAD (25 and 50 mg/kg), Furthermore, repeated dose administration of gliben- and glibenclamide (10 mg/kg) showed significant protection clamide (10 mg/kg), MEPG (200 and 400 mg/kg, p.o.), and against alloxan-induced pancreatic tissue damage. However, VAD (10, 25, and 50 mg/kg, p.o.) for 21 days had significantly MEPG (200 mg/kg, p.o.) and VAD (10 mg/kg) treatments 6 Evidence-Based Complementary and Alternative Medicine

Table 2: Eﬀect of MEPG and VAD isolated from fruit rinds of Punica 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 ± 0.74 584.49 ± 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 ± 0.21 589.43 ± 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 )andmean(IC50 ) of three trials.

Table 3: Eﬀect of MEPG and VADisolated from fruit rinds of Punica granatum on protein tyrosine phosphatase-1B (PTP1B) enzyme activity in in vitro.

# ∗ Sl. no Sample Concentration (μg/mL) % Inhibition IC50 (μg/mL) 521.6 ± 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 ± 0.22 533.3 ± 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 )andmean(IC50 ) of three trials.

Table 4: Eﬀect of single-dose oral administration of MEPG and VAD on blood glucose levels in alloxan-induced diabetic rats.

Blood glucose (mg/dL) Group Treatment 0 hour 1 hour 2 hours 4 hours G-I Normal control 91.23 ± 5.292.45 ± 2.892.15 ± 4.591.54 ± 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-10mg/kg, p.o. 259.87 ± 5.5 265.32 ± 7.3 264.85 ± 3.7 261.25 ± 7.3 G-VII VAD-25mg/kg, p.o. 264.51 ± 7.1 261.25 ± 3.5∗ 258.65 ± 5.7∗ 256.52 ± 4.9∗ G-VIII VAD-50mg/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). Evidence-Based Complementary and Alternative Medicine 7

Table 5: Eﬀect of repeated-dose oral administration of MEPG and VAD on blood glucose levels in alloxan-induced diabetic rats.

Blood glucose (mg/dL) Group Treatment 0th day 7th day 14th day 21st day G-I Normal control 91.23 ± 5.292.45 ± 2.892.15 ± 4.591.54 ± 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-10mg/kg, p.o. 259.87 ± 5.5 169.68 ± 2.7∗ 159.32 ± 4.8∗ 124.66 ± 4.1∗ G-VII VAD-25mg/kg, p.o. 264.51 ± 7.1 148.25 ± 4.6∗ 129.80 ± 3.8∗ 98.26 ± 2.15∗ G-VIII VAD-50mg/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).

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.

Blood glucose (mg/dL) Group Treatment 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 ± 4.994.556 ± 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-10mg/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-25mg/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-50mg/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).

showed very minimal protection against alloxan-induced aldose reductase inhibition assay, PTP1B assay, and α- cellular damage (Figure 5). amylase inhibition assays were used for in vitro evaluation, and alloxan-induced diabetes in rats was used as an in vivo 4. Discussion model. Aldose reductase is an NADPH-dependent oxidoreduc- In the traditional system of medicine, several medicinal tase enzyme involved in polyol pathway; it plays a central role plants have been widely used for the treatment of diabetes in the conversion of aldose to polyol. Under diabetic con- mellitus, and Punica granatum is one of such plants which dition, excessive influx of glucose into the tissues results in has been used as a key ingredient in many ayurvedic the increased aldose reductase activity, and thereby increased antidiabetic formulations [21]. In line with the ayurvedic levels of polyol [27]. Furthermore, evidences suggest that literature, various parts of the plant have been scientifically aldose reductase plays pivotal role in the pathogenesis of proved for antidiabetic activity [21]. In our earlier studies, diabetic complications such as neuropathy, nephropathy, we have isolated some of the antioxidant principles from cataract, retinopathy, and microangiopathy [28]. Available methanolic extracts of fruit rinds of Punica granatum [8]. In preclinical and preliminary clinical evidence suggests that continuation of our previous work, the present work is been inhibition of aldose reductase enzyme activity may be useful undertaken to isolate and evaluate the bioactive constituents in the prevention of onset of diabetic complications [28]. In relevant to antidiabetic activity. present study, the MEPG and VAD have showed significant In context of the mentioned objectives, a bioactive phy- and dose-dependent inhibition of aldose reductase enzyme tochemical constituent has been isolated from methanolic activity with IC50 values of 2.050 and 0.788 μg/mL, respec- extracts of Punica granatum and identified as a well-known tively. Quercetin used as a reference standard showed dose- polyphenolic compound valoneic acid dilactone (VAD) by dependent inhibition of aldose reductase activity with IC50 comparing the obtained spectral data with the literature value of 2.807 μg/mL. The phytochemicals were evolved as [25, 26]. very potent inhibitors of aldose reductase enzyme, especially The isolated compound (VAD) and the crude methanolic the plant derivatives rich in polyphenols, flavonoids, and extract (MEPG) had been evaluated for antidiabetic activity; sugar derivatives are well known for their aldose reductase 8 Evidence-Based Complementary and Alternative Medicine

Table 7: Eﬀect of repeated-dose oral administration of MEPG and VAD on body weight in alloxan-induced diabetic rats.

Body weight (g) Group Treatment 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-10mg/kg, p.o. 236.52 ± 8.4 210.41 ± 5.9∗ 189.68 ±7.7∗ 174.55 ± 9.5∗ G-VII VAD-25mg/kg, p.o. 242.95 ± 7.6 219.64 ± 6.6∗ 212.47 ± 5.9∗ 205.56 ± 7.8∗ G-VIII VAD-50mg/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.

(a) (b) (c) (d)

(e) (f) (g) (h)

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). inhibition property [27]. In present study, VAD was evolved results of in vitro studies showed that MEPG and VAD inhibit as highly potent inhibitor of aldose reductase may be due to α-amylase activity significantly and dose dependently with its polyphenolic nature. IC50 values of 1.02 and 0.284 μg/mL, respectively. Drugs that inhibit carbohydrate-hydrolyzing enzymes Protein tyrosine phosphatase 1B (PTP1B) has been have been demonstrated to decrease postprandial hyper- implicated as a negative regulator of insulin action. Over- glycemia and improve impaired glucose metabolism without expression of PTP1B protein has been observed in insulin- promoting insulin secretion in type II diabetic patients resistant states associated with obesity and diabetes [31]. [29]. α-amylase is one of the key enzymes that hydrolyse PTP1B directly catalyzes the dephosphorylation of cellular alpha-bonds of alpha-linked large polysaccharides, such as substrates of the insulin receptor kinase results in the starch and glycogen yielding glucose and maltose [30]. The downregulation of insulin action [31]. Evidence-Based Complementary and Alternative Medicine 9

In a recent study, it has been reported that mice lacking insulin mimetic activity or improved glucose utilization functional PTP1B gene showed enhanced tyrosine kinase mechanism. activity and increased insulin sensitivity [32]. The levels The possible mechanism by which the MEPG and VAD of PTP1B expression in muscle and adipose tissues in bring about their antihyperglycemic action may be by humans were strongly correlated to insulin resistance states. increasing the secretion of insulin from pancreatic β cells or In another animal-based approach, treatment with antisense its release from the bound form [34]. oligonucleotide specific for PTP1B resulted in normalization In context of understanding the mechanism, it is well of blood glucose and insulin levels in animal models established that sulfonylurea class of drugs produce hypogly- of type II diabetes [32]. Accordingly, PTP1B inhibitors caemia by increasing the secretion of insulin from pancreas, could increase insulin receptor tyrosine phosphorylation and and hence, these compounds are active in mild alloxan- mimic the cellular and in vivo actions of insulin and thereby induced diabetes, whereas they are not effective against decrease plasma glucose levels in diabetic animals [33]. intense alloxan-induced diabetes in animals (nearly all the In the present study, the MEPG and VAD have inhibited beta cells have been destroyed) [1]. In our work, histopathol- the PTP1B enzyme activity in in vitro conditions signif- ogy of alloxan per se treated animals showed significant but not complete loss of pancreatic islets associated with sever icantly and dose dependently with IC50 values of 26.25 and 12.41 μg/mL, respectively. All the in vitro studies have acinar damage. However, glibenclamide- (10 mg/kg, p.o.) demonstrated the antidiabetic effect of MEPG and VAD. The treated animals showed normal architecture of the pancreas; VAD was evolved as potent inhibitor of aldose reductase, MEPG (400 mg/kg, p.o.) and VAD (25 and 50 mg/kg, p.o.) α-amylase, and PTP1B enzymes and hence thought to be treated groups have showed very minimal acinar damage ffi beneficial in the treatment of diabetes. and su cient number of pancreatic islets (Figure 1). These findings suggest that some pancreatic β cells are still Based on the in vitro results, the MEPG and VAD were surviving to exert their insulin releasing effect, and hence, it further studied in in vivo against alloxan-induced diabetes can be concluded that the anti-hyperglycemic effect of MEPG in rats. and VAD was probably mediated by enhanced secretion of Alloxan is a hydrophilic and chemically unstable pyrim- insulin-like sulfonylureas. idine derivative; with its toxic nature it can cause a massive One of the major complications of type I diabetes is reduction in insulin release by the destruction of the weight loss. It arises due to the impairment in insulin action β pancreatic cells, inducing hyperglycaemia [4]. The diabetic in the conversion of glucose into glycogen and catabolism rats (induced by alloxan) showed a persistent rise in blood of fats and inhibition of lipolysis due to its unavailability glucoselevelsafter7dayswiththecharacteristicfeaturesof because of destruction in beta cells [35]. Due to this, there diabetes mellitus. will be a decrease in the body weight of the animals and Traditional plant medicines are used throughout the finally leads to death. world for a range of diabetic presentations, and the study Treatment with glibenclamide, MEPG, and VAD has of such medicines might offer a natural key to unlock a substantially prevented the body weight loss and mortality diabetologist’s pharmacy for the future [1]. In light of this, produced by the alloxan. The findings of the present study we made an attempt to study the effect of MEPG (200 such as blood glucose levels, oral glucose tolerance test, and 400 mg/kg, p.o.) and VAD (10, 25, and 50 mg/kg, p.o.) body weight, mortality, and histopathology findings are in against alloxan-induced diabetic rats based on the obtained correlation with each other and indicate that MEPG and positive results in in vitro studies. Oral glucose tolerance VAD could be beneficial in management of diabetes and test (OGTT) carried out after single-dose administration associated complications. of MEPG (400 mg/kg, p.o.) showed marginal decrease in elevated blood glucose levels after alloxan treatment, 5. Conclusion exceptionally glibenclamide (10 mg/kg) and VAD (25 and 50 mg/kg, p.o.) have showed marked decrease in blood In present study, valoneic acid dilactone (VAD) was isolated glucose levels as compared to diabetic control animals (G- and confirmed by comparing the obtained spectral data II). In contrast, MEPG (200 mg/kg, p.o.) and VAD(10 mg/kg, with the literature values. In preliminary antidiabetic studies, p.o.) were failed to decrease the blood glucose levels in the methanolic extracts (MEPG) and VAD isolated from alloxan-induced diabetic rats. fruit rinds of Punica granatum possessed potent antidiabetic On repeated administration, the MEPG (200 and activity in both in vitro and in vivo models. 400 mg/kg, p.o.) and VAD (10, 25, and 50 mg/kg, p.o.) have showed significant antihyperglycemic activity from day 7 Conflict of Interests onwards by controlling the elevated blood glucose levels compared with vehicle control group. All the doses of MEPG The authors do not have any conflict of Interests. (200 and 400 mg/kg, p.o.) and VAD (10, 25, and 50 mg/kg, p.o.) have showed potent antihyperglycemic activity in Acknowledgment OGTT carried out on day 21 of the study. In contrast, impaired glucose tolerance is observed in alloxan-induced The authors are thankful to Principal and Management, diabetic rats due to destructing the pancreatic β cells [23]. PES College of Pharmacy, Bangalore, for providing all the These findings suggest that the MEPG and VAD may show necessary facilities to carry out the research work. 10 Evidence-Based Complementary and Alternative Medicine

References (pomegranate) methanolic extract,” Journal of Ethnopharma- cology, vol. 96, no. 1-2, pp. 171–176, 2005. [1] A. Yashwant Kumar, K. Nandakumar, M. Handral, S. Tal- [17] H. S. Parmar and A. Kar, “Protective role of Citrus sinensis, war, and D. Dhayabaran, “Hypoglycaemic and anti-diabetic Musa paradisiaca,andPunica granatum peels against diet- activity of stem bark extracts Erythrina indica in normal and induced atherosclerosis and thyroid dysfunctions in rats,” alloxan-induced diabetic rats,” Saudi Pharmaceutical Journal, Nutrition Research, vol. 27, no. 11, pp. 710–718, 2007. vol. 19, no. 1, pp. 35–42, 2011. [18] S. Adiga, P. Trivedi, V. Ravichandra, D. Deb, and F. Mehta, [2] S. Wild, G. Roglic, A. Green, R. Sicree, and H. King, “Global “Effect of Punica granatum peel extract on learning and prevalence of diabetes: estimates for the year 2000 and memory in rats,” Asian Pacific Journal of Tropical Medicine, vol. projection for 2030,” Diabetes Care, vol. 27, no. 5, pp. 1047– 3, no. 9, pp. 687–690, 2010. 1053, 2004. [19] E. A. Hayouni, K. Miled, S. Boubaker et al., “Hydroalcoholic [3] K. Poongothai, P. Ponmurugan, K. S. Z. Ahmed, B. S. Kumar, extract based-ointment from Punica granatum L. peels with ff ff andS.A.Sheri , “Antihyperglycemic and antioxidant e ects enhanced in vivo healing potential on dermal wounds,” of Solanum xanthocarpum leaves (field grown & in vitro raised) Phytomedicine, vol. 18, no. 11, pp. 976–984, 2011. extracts on alloxan induced diabetic rats,” Asian Pacific Journal [20] E. P. Lansky and R. A. Newman, “Punica granatum of Tropical Medicine, vol. 4, no. 10, pp. 778–785, 2011. (pomegranate) and its potential for prevention and treatment [4]B.S.AshokKumar,K.Lakshman,K.N.Jayaveeaetal., of inflammation and cancer,” Journal of Ethnopharmacology, “Antidiabetic, antihyperlipidemic and antioxidant activities vol. 109, no. 2, pp. 177–206, 2007. of methanolic extract of Amaranthus viridis Linn in alloxan [21] O. Y. Althunibat, A. H. Al-Mustafa, K. Tarawneh, K. M. induced diabetic rats,” Experimental and Toxicologic Pathology, Khleifat, B. H. Ridzwan, and H. N. Qaralleh, “Protective role vol. 64, no. 1-2, pp. 75–79, 2012. of Punica granatum L. peel extract against oxidative damage in [5] Y. Miyake, E. Suzuki, S. Ohya et al., “Lipid-lowering effect of experimental diabetic rats,” Process Biochemistry, vol. 45, no. eriocitrin, the main flavonoid in lemon fruit, in rats on a high- 4, pp. 581–585, 2010. fat and high-cholesterol diet,” Journal of Food Science, vol. 71, [22] S. Chethan, S. M. Dharmesh, and N. G. Malleshi, “Inhibition no. 9, pp. S633–S637, 2006. of aldose reductase from cataracted eye lenses by finger millet [6] J. Jurenka, “Therapeutic applications of pomegranate (Punica (Eleusine coracana) polyphenols,” Bioorganic and Medicinal granatum L.): a review,” Alternative Medicine Review, vol. 13, Chemistry, vol. 16, no. 23, pp. 10085–10090, 2008. no. 2, pp. 128–144, 2008. [7]N.Artik,B.Ceremroglu,H.Murakami,andT.Mori,“Deter- [23] H. S. Lee, “Inhibitory activity of Cinnamomum cassia bark- mination of phenolic compounds in pomegranate juice by derived component against rat lens aldose reductase,” Journal HPLC,” Fruit Processing, vol. 8, pp. 492–499, 1998. of Pharmacy and Pharmaceutical Sciences,vol.5,no.3,pp. [8] V. Jain, G. Murugananthan, M. Deepak, G. L. Viswanatha, and 226–230, 2002. D. Manohar, “Isolation and standardization of various phyto- [24] I. K. Lund, H. S. Andersen, L. F. Iversen et al., “Structure-based chemical constituents from methanolic extracts of fruit rinds design of selective and potent inhibitors of protein-tyrosine β of Punica granatum,” Chinese Journal of Natural Medicines, vol. phosphatase ,” The Journal of Biological Chemistry, vol. 279, 9, no. 6, pp. 414–420, 2011. no. 23, pp. 24226–24235, 2004. [9] L. Zhang, X. Yang, Y. Zhang, L. Wang, and R. Zhang, “In [25] R. S. Hilris, C. M. S. Carmem, B. C. N. Laurentino, A. D. L. vitro antioxidant properties of different parts of pomegranate Jose,´ M. G. L. Antonia, and H. C. Mariana, “Constituintes flowers,” Food and Bioproducts Processing,vol.89,no.3,pp. Qu´ımicos Das Cascas Do Caule De Cenostigma Macrophyl- 234–240, 2011. lum: Ocorrenciaˆ de Colesterol,” Qu´ımica Nova, vol. 30, no. 8, [10] A. K. Das, S. C. Mandal, S. K. Banerjee et al., “Studies on pp. 1877–1881, 2007. antidiarrhoeal activity of Punica granatum seed extract in [26] T. Unno, A. Sugimoto, and T. Kakuda, “Xanthine oxidase rats,” Journal of Ethnopharmacology, vol. 68, no. 1-3, pp. 205– inhibitors from the leaves of Lagerstroemia speciosa (L.) Pers,” 208, 1999. Journal of Ethnopharmacology, vol. 93, no. 2-3, pp. 391–395, [11] C. J. Lee, L. G. Chen, W. L. Liang, and C. C. Wang, “Anti- 2004. inflammatory effects of Punica granatum Linne in vitro and [27] R. Kumar, D. K. Patel, D. Laloo, K. Sairam, and S. Hemalatha, in vivo,” Food Chemistry, vol. 118, no. 2, pp. 315–322, 2010. “Inhibitory effect of two Indian medicinal plants on aldose [12] M. Dell’Agli, G. V. Galli, Y. Corbett et al., “Antiplas- reductase of rat lens in vitro,” Asian Pacific Journal of Tropical modial activity of Punica granatum L. fruit rind,” Journal of Medicine, vol. 4, no. 9, pp. 694–697, 2011. Ethnopharmacology, vol. 125, no. 2, pp. 279–285, 2009. [28] L. Scotti, M. B. Fernandes, E. Muramatsu et al., “Self- [13] M. Zahin, F. Aqil, and I. Ahmad, “Broad spectrum antimuta- organizing maps and VolSurf approach to predict aldose genic activity of antioxidant active fraction of Punica granatum reductase inhibition by flavonoid compounds,” Brazilian L. peel extracts,” Mutation Research, vol. 703, no. 2, pp. 99– Journal of Pharmacognosy, vol. 21, no. 1, pp. 170–180, 2011. 107, 2010. [29] R. Punitha and S. Manoharan, “Antihyperglycemic and [14] R. G. Ross, S. Selvasubramanian, and S. Jayasundar, antilipidperoxidative effects of Pongamia pinnata (Linn.) “Immunomodulatory activity of Punica granatum in Pierre flowers in alloxan induced diabetic rats,” Journal of rabbits—a preliminary study,” Journal of Ethnopharmacology, Ethnopharmacology, vol. 105, no. 1-2, pp. 39–46, 2006. vol. 78, no. 1, pp. 85–87, 2001. [30] G. H. Perry, N. J. Dominy, K. G. Claw et al., “Diet and the [15] I. Celik, A. Temur, and I. Isik, “Hepatoprotective role and evolution of human amylase gene copy number variation,” antioxidant capacity of pomegranate (Punica granatum)flow- Nature Genetics, vol. 39, no. 10, pp. 1256–1260, 2007. ers infusion against trichloroacetic acid-exposed in rats,” Food [31] B. J. Goldstein, A. Bittner-Kowalczyk, M. F. White, and M. and Chemical Toxicology, vol. 47, no. 1, pp. 145–149, 2009. Harbeck, “Tyrosine dephosphorylation and deactivation of [16] K. B. Ajaikumar, M. Asheef, B. H. Babu, and J. Padikkala, “The insulin receptor substrate-1 by protein-tyrosine phosphatase inhibition of gastric mucosal injury by Punica granatum L. 1B. Possible facilitation by the formation of a ternary complex Evidence-Based Complementary and Alternative Medicine 11

with the GRB2 adaptor protein,” The Journal of Biological Chemistry, vol. 275, no. 6, pp. 4283–4289, 2000. [32] M. Elchebly, P.Payette, E. Michaliszyn et al., “Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene,” Science, vol. 283, no. 5407, pp. 1544–1548, 1999. [33] M. S. Malamas, J. Sredy, C. Moxham et al., “Novel benzo- furan and benzothiophene biphenyls as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties,” Journal of Medicinal Chemistry, vol. 43, no. 7, pp. 1293–1310, 2000. [34] P. S. M. Prince, N. Kamalakkannan, and V. P. Menon, “Antidi- abetic and antihyperlipidaemic eﬀect of alcoholic Syzigium cumini seeds in alloxan induced diabetic albino rats,” Journal of Ethnopharmacology, vol. 91, no. 2-3, pp. 209–213, 2004. [35] K. M. Gillespie, “Type 1 diabetes: pathogenesis and prevention,” Canadian Medical Association Journal, vol. 175, no. 2, pp. 165–170, 2006. Copyright of Evidence-based Complementary & Alternative Medicine (eCAM) is the property of Hindawi Publishing Corporation and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.