Isolation of Antidiabetic Principle from Fruit Rinds of Punica granatum
Identifieur interne : 000E88 ( Pmc/Corpus ); précédent : 000E87; suivant : 000E89Isolation of Antidiabetic Principle from Fruit Rinds of Punica granatum
Auteurs : Vishal Jain ; G. L. Viswanatha ; D. Manohar ; H. N. ShivaprasadSource :
- Evidence-based Complementary and Alternative Medicine : eCAM [ 1741-427X ] ; 2012.
Abstract
Present study was aimed to isolate and evaluate the antidiabetic activity of phytoconstituents from fruit rinds of
Url:
DOI: 10.1155/2012/147202
PubMed: 22919408
PubMed Central: 3419443
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Isolation of Antidiabetic Principle from Fruit Rinds of <italic>Punica granatum</italic></title><author><name sortKey="Jain, Vishal" sort="Jain, Vishal" uniqKey="Jain V" first="Vishal" last="Jain">Vishal Jain</name><affiliation><nlm:aff id="I1">Research and Development, Olive Lifesciences Pvt. Ltd., Karnataka, Tumkur 572 106, India</nlm:aff></affiliation></author><author><name sortKey="Viswanatha, G L" sort="Viswanatha, G L" uniqKey="Viswanatha G" first="G. L." last="Viswanatha">G. L. Viswanatha</name><affiliation><nlm:aff id="I2">Department of Pharmacology, PES College of Pharmacy, Karnataka, Bangalore 560 050, India</nlm:aff></affiliation></author><author><name sortKey="Manohar, D" sort="Manohar, D" uniqKey="Manohar D" first="D." last="Manohar">D. Manohar</name><affiliation><nlm:aff id="I1">Research and Development, Olive Lifesciences Pvt. Ltd., Karnataka, Tumkur 572 106, India</nlm:aff></affiliation></author><author><name sortKey="Shivaprasad, H N" sort="Shivaprasad, H N" uniqKey="Shivaprasad H" first="H. N." last="Shivaprasad">H. N. Shivaprasad</name><affiliation><nlm:aff id="I1">Research and Development, Olive Lifesciences Pvt. Ltd., Karnataka, Tumkur 572 106, India</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22919408</idno><idno type="pmc">3419443</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419443</idno><idno type="RBID">PMC:3419443</idno><idno type="doi">10.1155/2012/147202</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000E88</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Isolation of Antidiabetic Principle from Fruit Rinds of <italic>Punica granatum</italic></title><author><name sortKey="Jain, Vishal" sort="Jain, Vishal" uniqKey="Jain V" first="Vishal" last="Jain">Vishal Jain</name><affiliation><nlm:aff id="I1">Research and Development, Olive Lifesciences Pvt. Ltd., Karnataka, Tumkur 572 106, India</nlm:aff></affiliation></author><author><name sortKey="Viswanatha, G L" sort="Viswanatha, G L" uniqKey="Viswanatha G" first="G. L." last="Viswanatha">G. L. Viswanatha</name><affiliation><nlm:aff id="I2">Department of Pharmacology, PES College of Pharmacy, Karnataka, Bangalore 560 050, India</nlm:aff></affiliation></author><author><name sortKey="Manohar, D" sort="Manohar, D" uniqKey="Manohar D" first="D." last="Manohar">D. Manohar</name><affiliation><nlm:aff id="I1">Research and Development, Olive Lifesciences Pvt. Ltd., Karnataka, Tumkur 572 106, India</nlm:aff></affiliation></author><author><name sortKey="Shivaprasad, H N" sort="Shivaprasad, H N" uniqKey="Shivaprasad H" first="H. N." last="Shivaprasad">H. N. Shivaprasad</name><affiliation><nlm:aff id="I1">Research and Development, Olive Lifesciences Pvt. Ltd., Karnataka, Tumkur 572 106, India</nlm:aff></affiliation></author></analytic><series><title level="j">Evidence-based Complementary and Alternative Medicine : eCAM</title><idno type="ISSN">1741-427X</idno><idno type="eISSN">1741-4288</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Present study was aimed to isolate and evaluate the antidiabetic activity of phytoconstituents from fruit rinds of <italic>Punica granatum</italic>. With the above objectives Valoneic acid dilactone (VAD) was isolated from methanolic fruit rind extracts of <italic>Punica granatum</italic> (MEPG) and confirmed by <sup>1</sup>H-NMR, <sup>13</sup>C-NMR, and mass spectral data. Antidiabetic activity was evaluated by Aldose reductase, <italic><italic>α</italic></italic>-amylase and PTP1B inhibition assays in <italic>in vitro</italic> and Alloxan-induced diabetes in rats was used as an <italic>in vivo</italic> model. In bioactivity studies, MEPG and VAD have showed potent antidiabetic activity in <italic><italic>α</italic></italic>-amylase, aldose reductase, and PTP1B inhibition assays with IC<sub>50</sub> values of 1.02, 2.050, 26.25 <italic><italic>μ</italic></italic>g/mL and 0.284, 0.788, 12.41 <italic><italic>μ</italic></italic>g/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. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Evid Based Complement Alternat Med</journal-id><journal-id journal-id-type="iso-abbrev">Evid Based Complement Alternat Med</journal-id><journal-id journal-id-type="publisher-id">ECAM</journal-id><journal-title-group><journal-title>Evidence-based Complementary and Alternative Medicine : eCAM</journal-title></journal-title-group><issn pub-type="ppub">1741-427X</issn><issn pub-type="epub">1741-4288</issn><publisher><publisher-name>Hindawi Publishing Corporation</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22919408</article-id><article-id pub-id-type="pmc">3419443</article-id><article-id pub-id-type="doi">10.1155/2012/147202</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Isolation of Antidiabetic Principle from Fruit Rinds of <italic>Punica granatum</italic></article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Jain</surname><given-names>Vishal</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Viswanatha</surname><given-names>G. L.</given-names></name><xref ref-type="aff" rid="I2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Manohar</surname><given-names>D.</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Shivaprasad</surname><given-names>H. N.</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib></contrib-group><aff id="I1"><sup>1</sup>Research and Development, Olive Lifesciences Pvt. Ltd., Karnataka, Tumkur 572 106, India</aff><aff id="I2"><sup>2</sup>Department of Pharmacology, PES College of Pharmacy, Karnataka, Bangalore 560 050, India</aff><author-notes><corresp id="cor1">*Vishal Jain: <email>julian.vishal@gmail.com</email></corresp><fn fn-type="other"><p>Academic Editor: I-Min Liu</p></fn></author-notes><pub-date pub-type="ppub"><year>2012</year></pub-date><pub-date pub-type="epub"><day>31</day><month>7</month><year>2012</year></pub-date><pub-date pub-type="pmc-release"><day>31</day><month>7</month><year>2012</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>2012</volume><elocation-id>147202</elocation-id><history><date date-type="received"><day>26</day><month>2</month><year>2012</year></date><date date-type="accepted"><day>26</day><month>4</month><year>2012</year></date></history><permissions><copyright-statement>Copyright © 2012 Vishal Jain et al.</copyright-statement><copyright-year>2012</copyright-year><license license-type="open-access"><license-p>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.</license-p></license></permissions><abstract><p>Present study was aimed to isolate and evaluate the antidiabetic activity of phytoconstituents from fruit rinds of <italic>Punica granatum</italic>. With the above objectives Valoneic acid dilactone (VAD) was isolated from methanolic fruit rind extracts of <italic>Punica granatum</italic> (MEPG) and confirmed by <sup>1</sup>H-NMR, <sup>13</sup>C-NMR, and mass spectral data. Antidiabetic activity was evaluated by Aldose reductase, <italic><italic>α</italic></italic>-amylase and PTP1B inhibition assays in <italic>in vitro</italic> and Alloxan-induced diabetes in rats was used as an <italic>in vivo</italic> model. In bioactivity studies, MEPG and VAD have showed potent antidiabetic activity in <italic><italic>α</italic></italic>-amylase, aldose reductase, and PTP1B inhibition assays with IC<sub>50</sub> values of 1.02, 2.050, 26.25 <italic><italic>μ</italic></italic>g/mL and 0.284, 0.788, 12.41 <italic><italic>μ</italic></italic>g/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 <italic>in vitro</italic> and <italic>in vivo</italic> models.</p></abstract></article-meta></front><body><sec id="sec1"><title>1. Introduction</title><p>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 [<xref ref-type="bibr" rid="B1">1</xref>].</p><p>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 [<xref ref-type="bibr" rid="B2">2</xref>]. 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 [<xref ref-type="bibr" rid="B3">3</xref>].</p><p>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 [<xref ref-type="bibr" rid="B4">4</xref>]. Plants are rich sources of antidiabetic, antihyperlipidemic, and antioxidant agents such as flavonoids, gallotannins, amino acids, and other related polyphenols [<xref ref-type="bibr" rid="B5">5</xref>].</p><p>Pomegranate (<italic>Punica granatum </italic>L., 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 [<xref ref-type="bibr" rid="B6">6</xref>]. <italic>Punica granatum</italic> 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 [<xref ref-type="bibr" rid="B7">7</xref>].</p><p><italic>Punica granatum </italic>is 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 <italic>Punica granatum </italic>[<xref ref-type="bibr" rid="B8">8</xref>].</p><p>In the literature, various parts of the plant are reported for various pharmacological activities such as antioxidant [<xref ref-type="bibr" rid="B9">9</xref>], antidiarrheal [<xref ref-type="bibr" rid="B10">10</xref>], anti-inflammatory [<xref ref-type="bibr" rid="B11">11</xref>], antimalarial [<xref ref-type="bibr" rid="B12">12</xref>], antimutagenic [<xref ref-type="bibr" rid="B13">13</xref>], immunomodulatory [<xref ref-type="bibr" rid="B14">14</xref>], hepatoprotective [<xref ref-type="bibr" rid="B15">15</xref>], antiulcer [<xref ref-type="bibr" rid="B16">16</xref>], atherosclerosis and thyroid dysfunction [<xref ref-type="bibr" rid="B17">17</xref>], memory enhancing [<xref ref-type="bibr" rid="B18">18</xref>], wound healing [<xref ref-type="bibr" rid="B19">19</xref>], and anticancer [<xref ref-type="bibr" rid="B20">20</xref>] activities. Apart from these medicinal uses, various parts of the plant have been scientifically proved for antidiabetic activity [<xref ref-type="bibr" rid="B21">21</xref>]. In our previous studies, we have isolated three components from methanolic extracts of <italic>Punica granatum </italic>[<xref ref-type="bibr" rid="B8">8</xref>]. In continuation, the present study was undertaken to isolate the antidiabetic principle from the methanolic extracts of fruit rinds of <italic>Punica granatum</italic>.</p></sec><sec id="sec2"><title>2. Materials and Methods</title><sec id="sec2.1"><title>2.1. Drugs and Chemicals</title><p>Human recombinant PTP1B (BIOMOL International LP (Plymouth Meeting, PA)), <italic>α</italic>-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. </p></sec><sec id="sec2.2"><title>2.2. Plant Material</title><p>The fruit rinds of <italic>Punica granatum </italic>were 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).</p></sec><sec id="sec2.3"><title>2.3. Isolation and Identification of the Compound</title><p>Fruit rinds of <italic>Punica granatum</italic> 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% <italic>w</italic>/<italic>w</italic> (800 g per 3.0 kg of methanolic extract).</p><p>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% <italic>w/w</italic>. 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% FeCl<sub>3</sub> reagent was sprayed, and the chromatogram was observed. </p><p>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 <xref ref-type="fig" rid="fig1">Figure 1</xref>. 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.</p></sec><sec id="sec2.4"><title>2.4. Experimental Animals</title><p>Male Wistar rats (200–250 g) were purchased from Bioneeds, Nelamangala, Tumkur, India, and they were maintained in polypropylene cages at a temperature of 25 ± 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 <italic>ad libitum</italic>.</p><p>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.</p></sec><sec id="sec2.5"><title>2.5. Experimental Protocol</title><sec id="sec2.5.1"><title>2.5.1. <italic>In Vitro</italic> <italic>α</italic>-Amylase Inhibition Assay</title><p><italic>In vitro α</italic>-amylase inhibition assay was carried out based on the spectrophotometric assay using acarbose as the reference compound [<xref ref-type="bibr" rid="B4">4</xref>]. 2 mg of MEPG and VAD was dissolved in 500 <italic>μ</italic>L of DMSO, and further dilutions were prepared using deionised water. The enzyme <italic>α</italic>-amylase solution (0.5 unit/mL) was prepared by mixing 3.246 mg of <italic>α</italic>-amylase (EC 3.2.1.1) in 100 mL of 40 mM phosphate buffer pH 6.9. In short, 60 <italic>μ</italic>L of 40 mM phosphate buffer/acarbose/test drug and 30 <italic>μ</italic>L of <italic>α</italic>-amylase enzyme were added, mixed, and preincubated at 37°C for 10 min, and then 120 <italic>μ</italic>L of 2-chloro-p-nitrophenyl-<italic>α</italic>-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:
<disp-formula id="eq1"><label>(1)</label><mml:math id="M1"><mml:mtable><mml:mtr><mml:mtd><mml:malignmark></mml:malignmark><mml:mi>%</mml:mi><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>Inhibition</mml:mtext></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:maligngroup></mml:maligngroup><mml:malignmark></mml:malignmark><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mtext>Absorbance of control</mml:mtext><mml:mo>−</mml:mo><mml:mtext>Absorbance of test</mml:mtext></mml:mrow><mml:mrow><mml:mtext>Absorbance of control</mml:mtext><mml:mi> </mml:mi></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:mn>100</mml:mn><mml:mo>.</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula></p></sec><sec id="sec2.5.2"><title>2.5.2. <italic>In Vitro</italic> Aldose Reductase Inhibition Assay</title><p><statement id="head1"><title>(a) Preparation of Aldose Reductase</title><p>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 1 <italic>μ</italic>mol of NADPH per minute under our experimental conditions [<xref ref-type="bibr" rid="B22">22</xref>]:
<disp-formula id="eq2"><label>(2)</label><mml:math id="M2"><mml:mtable><mml:mtr><mml:mtd><mml:malignmark></mml:malignmark><mml:mtext>Activity</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>U</mml:mtext><mml:mo>/</mml:mo><mml:mtext>mL</mml:mtext></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:maligngroup></mml:maligngroup><mml:malignmark></mml:malignmark><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mtext>Change</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>in</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>OD</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>of</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>test</mml:mtext><mml:mo>/</mml:mo><mml:mi>min</mml:mi><mml:mo></mml:mo><mml:mo>×</mml:mo><mml:mtext>Total</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>volume</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>of</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>the</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>assay</mml:mtext></mml:mrow><mml:mrow><mml:mn mathvariant="normal">6.2</mml:mn><mml:mo>×</mml:mo><mml:mtext>Volume</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>of</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>enzyme</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>taken</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>for</mml:mtext><mml:mo> </mml:mo><mml:mo> </mml:mo><mml:mtext>analysis</mml:mtext></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula>where 6.2 = micromolar extinction coefficient of NADPH at 340 nm.</p></statement></p><p><statement id="head2"><title>(b) Assay Procedure</title><p>Aldose reductase inhibition activity was carried out by photometric method [<xref ref-type="bibr" rid="B22">22</xref>]. 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 <italic>μ</italic>L of drug solution (buffer in case of control), 50 <italic>μ</italic>L of NADPH (0.04 mM), and 100 <italic>μ</italic>L of aldose reductase enzyme preparation. The enzymatic reaction was initiated by the addition of 75 <italic>μ</italic>L of substrate, DL-glyceraldehyde (5 × 10<sup>−4</sup> 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 (IC<sub>50</sub>) were calculated from the percentage inhibition [<xref ref-type="bibr" rid="B23">23</xref>].</p></statement></p></sec><sec id="sec2.5.3"><title>2.5.3. Protein Tyrosine Phosphatase 1B Inhibition Assay</title><p>The phosphatase activity was assayed using <italic>p</italic>NPP (Para-Nitro Phenyl Phosphate) as substrate and carried out essentially as described previously [<xref ref-type="bibr" rid="B24">24</xref>]. 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 <italic>μ</italic>M compound) were added to the reaction mixture containing 0 or 2.5 mM <italic>p</italic>NPP (final assay concentration, total volume of 100 <italic>μ</italic>L). 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 <italic>μ</italic>L 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.</p></sec><sec id="sec2.5.4"><title>2.5.4. Alloxan-Induced Diabetes in Rats</title><p>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.</p><p>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.</p><p><statement id="head3"><title>(a) Assessment of Mortality Rate in Alloxan-Induced Diabetic Rats</title><p>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.</p></statement></p><p><statement id="head4"><title>(b) Histopathology</title><p>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 5 <italic>μ</italic>m thick sections, and stained using hematoxylin-eosin, and histopathological observations were made.</p></statement></p></sec></sec><sec id="sec2.6"><title>2.6. Statistical Analysis</title><p>The values were expressed as mean ± S.E.M. Data was statistically analyzed using one-way ANOVA followed by Tukey's multiple comparison test. <italic>P</italic> < 0.05 was considered as statistically significant.</p></sec></sec><sec id="sec3"><title>3. Results</title><p>The present study was undertaken to isolate and evaluate the antidiabetic activity of bioactive constituents present in the fruit rind extracts of <italic>Punica granatum </italic>in <italic>in vitro </italic>and <italic>in vivo </italic>models. 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 <italic>in vitro </italic>and <italic>in vivo </italic>models.</p><sec id="sec3.1"><title>3.1. Identification and Quantification of the Isolated Compound</title><p>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 Figures <xref ref-type="fig" rid="fig2">2</xref>, <xref ref-type="fig" rid="fig3">3</xref>, and <xref ref-type="fig" rid="fig4">4</xref>, respectively.</p><p><statement id="head5"><title>Spectral Data</title><p><list list-type="simple"><list-item><label> </label><p>Chemical formula: C<sub>21</sub>H<sub>10</sub>O<sub>13</sub>; molecular weight: 470.</p></list-item><list-item><label> </label><p><sup>1</sup>H NMR (<italic>δ</italic> values ppm): 6.97 (s, 1H), 6.93 (s, 1H), 7.47 (s, 1H).</p></list-item><list-item><label> </label><p><sup>13</sup>C NMR (<italic>δ</italic> 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.</p></list-item><list-item><label> </label><p>LC-MS [<italic>m/z </italic>value]: 469.03 [M-H] (molecular ion peak).</p></list-item></list></p></statement></p><p>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.</p></sec><sec id="sec3.2"><title>3.2. <italic>In Vitro</italic> <italic>α</italic>-Amylase Inhibition Assay</title><p>In the present study, MEPG and VAD isolated from fruit rinds of <italic>Punica granatum </italic>have significantly and dose dependently inhibited the <italic>α</italic>-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 <italic>α</italic>-amylase enzyme activity with IC<sub>50</sub> values of 0.378, 1.02, and 0.284 <italic>μ</italic>g/mL, respectively (<xref ref-type="table" rid="tab1">Table 1</xref>).</p></sec><sec id="sec3.3"><title>3.3. <italic>In Vitro</italic> Aldose Reductase Inhibition Assay</title><p>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 of <italic>in vitro </italic>aldose reductase inhibition assay revealed that MEPG and VAD isolated from fruit rinds of <italic>Punica granatum</italic> showed significant and dose-dependent inhibition of aldose reductase enzyme activity; the IC<sub>50</sub> values were found to be 2.050 and 0.788 <italic>μ</italic>g/mL, respectively (<xref ref-type="table" rid="tab2">Table 2</xref>).</p></sec><sec id="sec3.4"><title>3.4. Protein Tyrosine Phosphatase 1B Inhibition (PTP1B) Assay</title><p>In <italic>in vitro </italic>PTP1B assay, the MEPG and VAD isolated from fruit rinds of <italic>Punica granatum</italic> showed significant dose-dependent inhibition of PTP1B enzyme activity; the IC<sub>50</sub> values were found to be 26.25 and 12.41 <italic>μ</italic>g/mL, respectively (<xref ref-type="table" rid="tab3">Table 3</xref>).</p></sec><sec id="sec3.5"><title>3.5. Alloxan-Induced Diabetes in Rats</title><p>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 (<xref ref-type="table" rid="tab4">Table 4</xref>).</p><p>Exceptionally, repeated dose administration of MEPG (200 and 400 mg/kg, p.o.) for 21 days had progressively reduced the blood glucose levels significantly (<italic>P</italic> < 0.05) and dose dependently (<xref ref-type="table" rid="tab5">Table 5</xref>); similarly, animals treated with VAD (10, 25, and 50 mg/kg, p.o.) also showed significant (<italic>P</italic> < 0.01) decrease in blood glucose levels compared to diabetic control group (G-II).</p><p>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) (<xref ref-type="table" rid="tab6">Table 6</xref>).</p><p>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 (<xref ref-type="table" rid="tab7">Table 7</xref>).</p><p>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.</p><sec id="sec3.5.1"><title>3.5.1. Histopathology</title><p>In the present study, histopathology of pancreatic tissue collected from normal control (G-I) animals showed normal cytoarchitecture of the pancreas, while alloxan <italic>per se</italic> treated diabetic control (G-II) rats showed destruction of pancreatic <italic>β</italic>-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 (<xref ref-type="fig" rid="fig5">Figure 5</xref>).</p></sec></sec></sec><sec id="sec4"><title>4. Discussion</title><p>In the traditional system of medicine, several medicinal plants have been widely used for the treatment of diabetes mellitus, and <italic>Punica granatum </italic>is one of such plants which has been used as a key ingredient in many ayurvedic antidiabetic formulations [<xref ref-type="bibr" rid="B21">21</xref>]. In line with the ayurvedic literature, various parts of the plant have been scientifically proved for antidiabetic activity [<xref ref-type="bibr" rid="B21">21</xref>]. In our earlier studies, we have isolated some of the antioxidant principles from methanolic extracts of fruit rinds of <italic>Punica granatum </italic>[<xref ref-type="bibr" rid="B8">8</xref>]. In continuation of our previous work, the present work is been undertaken to isolate and evaluate the bioactive constituents relevant to antidiabetic activity.</p><p>In context of the mentioned objectives, a bioactive phytochemical constituent has been isolated from methanolic extracts of <italic>Punica granatum</italic> and identified as a well-known polyphenolic compound valoneic acid dilactone (VAD) by comparing the obtained spectral data with the literature [<xref ref-type="bibr" rid="B25">25</xref>, <xref ref-type="bibr" rid="B26">26</xref>].</p><p>The isolated compound (VAD) and the crude methanolic extract (MEPG) had been evaluated for antidiabetic activity; aldose reductase inhibition assay, PTP1B assay, and <italic>α</italic>-amylase inhibition assays were used for <italic>in vitro </italic>evaluation, and alloxan-induced diabetes in rats was used as an <italic>in vivo </italic>model.</p><p>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 [<xref ref-type="bibr" rid="B27">27</xref>]. Furthermore, evidences suggest that aldose reductase plays pivotal role in the pathogenesis of diabetic complications such as neuropathy, nephropathy, cataract, retinopathy, and microangiopathy [<xref ref-type="bibr" rid="B28">28</xref>]. 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 [<xref ref-type="bibr" rid="B28">28</xref>]. In present study, the MEPG and VAD have showed significant and dose-dependent inhibition of aldose reductase enzyme activity with IC<sub>50</sub> values of 2.050 and 0.788 <italic>μ</italic>g/mL, respectively. Quercetin used as a reference standard showed dose-dependent inhibition of aldose reductase activity with IC<sub>50</sub> value of 2.807 <italic>μ</italic>g/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 [<xref ref-type="bibr" rid="B27">27</xref>]. In present study, VAD was evolved as highly potent inhibitor of aldose reductase may be due to its polyphenolic nature.</p><p>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 [<xref ref-type="bibr" rid="B29">29</xref>]. <italic>α</italic>-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 [<xref ref-type="bibr" rid="B30">30</xref>]. The results of <italic>in vitro</italic> studies showed that MEPG and VAD inhibit <italic>α</italic>-amylase activity significantly and dose dependently with IC<sub>50</sub> values of 1.02 and 0.284 <italic>μ</italic>g/mL, respectively.</p><p>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 [<xref ref-type="bibr" rid="B31">31</xref>]. PTP1B directly catalyzes the dephosphorylation of cellular substrates of the insulin receptor kinase results in the downregulation of insulin action [<xref ref-type="bibr" rid="B31">31</xref>].</p><p>In a recent study, it has been reported that mice lacking functional PTP1B gene showed enhanced tyrosine kinase activity and increased insulin sensitivity [<xref ref-type="bibr" rid="B32">32</xref>]. 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 [<xref ref-type="bibr" rid="B32">32</xref>]. Accordingly, PTP1B inhibitors could increase insulin receptor tyrosine phosphorylation and mimic the cellular and <italic>in vivo</italic> actions of insulin and thereby decrease plasma glucose levels in diabetic animals [<xref ref-type="bibr" rid="B33">33</xref>].</p><p>In the present study, the MEPG and VAD have inhibited the PTP1B enzyme activity in <italic>in vitro</italic> conditions significantly and dose dependently with IC<sub>50</sub> values of 26.25 and 12.41 <italic>μ</italic>g/mL, respectively. All the <italic>in vitro </italic>studies have demonstrated the antidiabetic effect of MEPG and VAD. The VAD was evolved as potent inhibitor of aldose reductase, <italic>α</italic>-amylase, and PTP1B enzymes and hence thought to be beneficial in the treatment of diabetes.</p><p>Based on the <italic>in vitro</italic> results, the MEPG and VAD were further studied in <italic>in vivo</italic> against alloxan-induced diabetes in rats.</p><p>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 pancreatic <italic>β</italic> cells, inducing hyperglycaemia [<xref ref-type="bibr" rid="B4">4</xref>]. The diabetic rats (induced by alloxan) showed a persistent rise in blood glucose levels after 7 days with the characteristic features of diabetes mellitus.</p><p>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 [<xref ref-type="bibr" rid="B1">1</xref>]. 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 <italic>in vitro </italic>studies. 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.</p><p>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 pancreatic <italic>β</italic> cells [<xref ref-type="bibr" rid="B23">23</xref>]. These findings suggest that the MEPG and VAD may show insulin mimetic activity or improved glucose utilization mechanism.</p><p>The possible mechanism by which the MEPG and VAD bring about their antihyperglycemic action may be by increasing the secretion of insulin from pancreatic <italic>β</italic> cells or its release from the bound form [<xref ref-type="bibr" rid="B34">34</xref>].</p><p>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) [<xref ref-type="bibr" rid="B1">1</xref>]. In our work, histopathology of alloxan <italic>per se</italic> 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 (<xref ref-type="fig" rid="fig1">Figure 1</xref>). These findings suggest that some pancreatic <italic>β</italic> 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.</p><p>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 [<xref ref-type="bibr" rid="B35">35</xref>]. Due to this, there will be a decrease in the body weight of the animals and finally leads to death.</p><p>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.</p></sec><sec id="sec5"><title>5. Conclusion</title><p>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 of <italic>Punica granatum </italic>possessed potent antidiabetic activity in both <italic>in vitro </italic>and <italic>in vivo </italic>models.</p></sec><sec sec-type="supplementary-material" id="supplementary-material-sec"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="f1"><caption><p>In present study, 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, <italic>α</italic>-amylase and PTP1B inhibition assays <italic>in vitro</italic> and Alloxan-induced diabetes in rats was used as an <italic>in vivo</italic> model. In bioactivity studies, MEPG (200 and 400 mg/kg, p.o.), VAD (10, 25, and 50 mg/kg, p.o.) have showed potent antidiabetic activity in <italic>α</italic>-amylase, aldose reductase, and PTP1B inhibition assays. 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. These findings suggest that MEPG and VAD possess significant antidiabetic activity in both <italic>in vitro</italic> and <italic>in vivo</italic> models.</p></caption><media xlink:href="147202.f1.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><title>Conflict of Interests</title><p>The authors do not have any conflict of Interests.</p></ack><ack><title>Acknowledgment</title><p>The authors are thankful to Principal and Management, PES College of Pharmacy, Bangalore, for providing all the necessary facilities to carry out the research work.</p></ack><ref-list><ref id="B1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yashwant 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(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).</p></caption><graphic xlink:href="ECAM2012-147202.005"></graphic></fig><table-wrap id="tab1" orientation="portrait" position="float"><label>Table 1</label><caption><p>Effect of MEPG and VAD isolated from fruit rinds of <italic>Punica granatum</italic> on <italic>α</italic>-amylase enzyme activity in <italic>in vitro</italic>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Sl. no</th><th align="center" rowspan="1" colspan="1">Sample</th><th align="center" rowspan="1" colspan="1">Concentration (<italic>μ</italic>g/mL)</th><th align="center" rowspan="1" colspan="1">% Inhibition<sup>#</sup></th><th align="center" rowspan="1" colspan="1">IC<sub>50</sub> (<italic>μ</italic>g/mL)<sup>∗</sup></th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.15</td><td align="center" rowspan="1" colspan="1">10.21 ± 0.54</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">29.41 ± 0.17</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">(1)</td><td align="center" rowspan="1" colspan="1">Methanolic extract</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">44.28 ± 0.83</td><td align="center" rowspan="1" colspan="1">1.02</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">1.25</td><td align="center" rowspan="1" colspan="1">70.21 ± 0.54</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">85.88 ± 0.52</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" colspan="5" rowspan="1"><hr></hr></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.15</td><td align="center" rowspan="1" colspan="1">29.65 ± 0.85</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">49.78 ± 0.46</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">(2)</td><td align="center" rowspan="1" colspan="1">Valoneic acid dilactone</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">74.25 ± 0.29</td><td align="center" rowspan="1" colspan="1">0.284</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">1.25</td><td align="center" rowspan="1" colspan="1">82.85 ± 0.31</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">99.52 ± 0.71</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" colspan="5" rowspan="1"><hr></hr></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">33.53 ± 0.46</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">(3)</td><td align="center" rowspan="1" colspan="1">Acarbose</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">62.41 ± 0.87</td><td align="center" rowspan="1" colspan="1">0.378</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">74.25 ± 0.29</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">99.21 ± 0.34</td><td align="center" rowspan="1" colspan="1"></td></tr></tbody></table><table-wrap-foot><fn><p>Values are expressed as mean ± SEM (% inhibition<sup>#</sup>) and mean (IC<sub>50</sub>*) of three trials.</p></fn></table-wrap-foot></table-wrap><table-wrap id="tab2" orientation="portrait" position="float"><label>Table 2</label><caption><p>Effect of MEPG and VAD isolated from fruit rinds of <italic>Punica granatum</italic> on aldose reductase enzyme activity in <italic>in vitro</italic>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Sl. no</th><th align="center" rowspan="1" colspan="1">Sample</th><th align="center" rowspan="1" colspan="1">Concentration (<italic>μ</italic>g/mL)</th><th align="center" rowspan="1" colspan="1">% Inhibition<sup>#</sup></th><th align="center" rowspan="1" colspan="1">IC<sub>50</sub> (<italic>μ</italic>g/mL)<sup>∗</sup></th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">22.95 ± 0.25</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">35.38 ± 0.47</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">(1)</td><td align="center" rowspan="1" colspan="1">Methanolic extract</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">45.77 ± 0.14</td><td align="center" rowspan="1" colspan="1">2.050</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">65.64 ± 0.74</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">84.49 ± 0.21</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">99.62 ± 0.33</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" colspan="5" rowspan="1"><hr></hr></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.15</td><td align="center" rowspan="1" colspan="1">9.31 ± 0.84</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">26.63 ± 0.18</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">(2)</td><td align="center" rowspan="1" colspan="1">Valoneic acid dilactone</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">48.50 ± 0.54</td><td align="center" rowspan="1" colspan="1">0.788</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">67.80 ± 0.71</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">79.19 ± 0.21</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">89.43 ± 0.34</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" colspan="5" rowspan="1"><hr></hr></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">8.03 ± 0.62</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">(3)</td><td align="center" rowspan="1" colspan="1">Quercetin</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">24.23 ± 0.51</td><td align="center" rowspan="1" colspan="1">2.807</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">42.48 ± 0.91</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">5.0</td><td align="center" rowspan="1" colspan="1">82.45 ± 0.27</td><td align="center" rowspan="1" colspan="1"></td></tr></tbody></table><table-wrap-foot><fn><p>Values are expressed as mean ± SEM (% inhibition<sup>#</sup>) and mean (IC<sub>50</sub>*) of three trials.</p></fn></table-wrap-foot></table-wrap><table-wrap id="tab3" orientation="portrait" position="float"><label>Table 3</label><caption><p>Effect of MEPG and VAD isolated from fruit rinds of <italic>Punica granatum</italic> on protein tyrosine phosphatase-1B (PTP1B) enzyme activity in <italic>in vitro</italic>. </p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Sl. no</th><th align="center" rowspan="1" colspan="1">Sample</th><th align="center" rowspan="1" colspan="1">Concentration (<italic>μ</italic>g/mL)</th><th align="center" rowspan="1" colspan="1">% Inhibition<sup>#</sup></th><th align="center" rowspan="1" colspan="1">IC<sub>50</sub> (<italic>μ</italic>g/mL)<sup>∗</sup></th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">21.6 ± 0.24</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">39.6 ± 0.15</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">(1)</td><td align="center" rowspan="1" colspan="1">Methanolic extract</td><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">55.4 ± 0.22</td><td align="center" rowspan="1" colspan="1">26.25</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">50</td><td align="center" rowspan="1" colspan="1">78.7 ± 0.34</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">98.9 ± 0.22</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" colspan="5" rowspan="1"><hr></hr></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">33.3 ± 0.35</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">46.7 ± 0.54</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">(2)</td><td align="center" rowspan="1" colspan="1">Valoneic acid dilactone</td><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">68.5 ± 0.64</td><td align="center" rowspan="1" colspan="1">12.41</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">50</td><td align="center" rowspan="1" colspan="1">84.0 ± 0.11</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">98.5 ± 0.75</td><td align="center" rowspan="1" colspan="1"></td></tr></tbody></table><table-wrap-foot><fn><p>Values are expressed as mean ± SEM (% inhibition<sup>#</sup>) and mean (IC<sub>50</sub>*) of three trials.</p></fn></table-wrap-foot></table-wrap><table-wrap id="tab4" orientation="portrait" position="float"><label>Table 4</label><caption><p>Effect of single-dose oral administration of MEPG and VAD on blood glucose levels in alloxan-induced diabetic rats.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1">Group</th><th align="center" rowspan="2" colspan="1">Treatment</th><th align="center" colspan="4" rowspan="1">Blood glucose (mg/dL)</th></tr><tr><th align="center" rowspan="1" colspan="1">0 hour</th><th align="center" rowspan="1" colspan="1">1 hour</th><th align="center" rowspan="1" colspan="1">2 hours</th><th align="center" rowspan="1" colspan="1">4 hours</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">G-I</td><td align="center" rowspan="1" colspan="1">Normal control</td><td align="center" rowspan="1" colspan="1">91.23 ± 5.2</td><td align="center" rowspan="1" colspan="1">92.45 ± 2.8</td><td align="center" rowspan="1" colspan="1">92.15 ± 4.5</td><td align="center" rowspan="1" colspan="1">91.54 ± 3.1</td></tr><tr><td align="left" rowspan="1" colspan="1">G-II</td><td align="center" rowspan="1" colspan="1">Vehicle control (3% Tween 80) 10 mL/kg, p.o.</td><td align="center" rowspan="1" colspan="1">272.84 ± 3.9<sup>#</sup></td><td align="center" rowspan="1" colspan="1">274.41 ± 5.1<sup>#</sup></td><td align="center" rowspan="1" colspan="1">277.65 ± 5.1<sup>#</sup></td><td align="center" rowspan="1" colspan="1">268.94 ± 4.6<sup>#</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">G-III</td><td align="center" rowspan="1" colspan="1">Glibenclamide-10 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">271.94 ± 4.8</td><td align="center" rowspan="1" colspan="1">234.15 ± 5.3</td><td align="center" rowspan="1" colspan="1">197.14 ± 6.2*</td><td align="center" rowspan="1" colspan="1">164.85 ± 6.5*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-IV</td><td align="center" rowspan="1" colspan="1">MEPG-200 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">268.51 ± 6.2</td><td align="center" rowspan="1" colspan="1">271.15 ± 4.8</td><td align="center" rowspan="1" colspan="1">255.41 ± 7.9</td><td align="center" rowspan="1" colspan="1">264.12 ± 6.1</td></tr><tr><td align="left" rowspan="1" colspan="1">G-V</td><td align="center" rowspan="1" colspan="1">MEPG-400 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">272.84 ± 5.2</td><td align="center" rowspan="1" colspan="1">269.64 ± 6.5</td><td align="center" rowspan="1" colspan="1">242.12 ± 4.8*</td><td align="center" rowspan="1" colspan="1">235.15 ± 5.8*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VI</td><td align="center" rowspan="1" colspan="1">VAD-10 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">259.87 ± 5.5</td><td align="center" rowspan="1" colspan="1">265.32 ± 7.3</td><td align="center" rowspan="1" colspan="1">264.85 ± 3.7</td><td align="center" rowspan="1" colspan="1">261.25 ± 7.3</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VII</td><td align="center" rowspan="1" colspan="1">VAD-25 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">264.51 ± 7.1</td><td align="center" rowspan="1" colspan="1">261.25 ± 3.5*</td><td align="center" rowspan="1" colspan="1">258.65 ± 5.7*</td><td align="center" rowspan="1" colspan="1">256.52 ± 4.9*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VIII</td><td align="center" rowspan="1" colspan="1">VAD-50 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">274.56 ± 4.5</td><td align="center" rowspan="1" colspan="1">241.85 ± 6.1*</td><td align="center" rowspan="1" colspan="1">220.12 ± 6.7*</td><td align="center" rowspan="1" colspan="1">198.56 ± 5.3*</td></tr></tbody></table><table-wrap-foot><fn><p>All the values are expressed as mean ± SEM, <sup>#</sup><italic>P</italic> < 0.05 compared to normal control, and *<italic>P</italic> < 0.05 compared to vehicle control (3% Tween 80).</p></fn></table-wrap-foot></table-wrap><table-wrap id="tab5" orientation="portrait" position="float"><label>Table 5</label><caption><p>Effect of repeated-dose oral administration of MEPG and VAD on blood glucose levels in alloxan-induced diabetic rats.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1">Group</th><th align="center" rowspan="2" colspan="1">Treatment</th><th align="center" colspan="4" rowspan="1">Blood glucose (mg/dL)</th></tr><tr><th align="center" rowspan="1" colspan="1">0th day</th><th align="center" rowspan="1" colspan="1">7th day</th><th align="center" rowspan="1" colspan="1">14th day</th><th align="center" rowspan="1" colspan="1">21st day</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">G-I</td><td align="center" rowspan="1" colspan="1">Normal control</td><td align="center" rowspan="1" colspan="1">91.23 ± 5.2</td><td align="center" rowspan="1" colspan="1">92.45 ± 2.8</td><td align="center" rowspan="1" colspan="1">92.15 ± 4.5</td><td align="center" rowspan="1" colspan="1">91.54 ± 3.1</td></tr><tr><td align="left" rowspan="1" colspan="1">G-II</td><td align="center" rowspan="1" colspan="1">Vehicle control (3% Tween 80) 10 mL/kg, p.o.</td><td align="center" rowspan="1" colspan="1">272.84 ± 3.9<sup>#</sup></td><td align="center" rowspan="1" colspan="1">279.22 ± 7.4<sup>#</sup></td><td align="center" rowspan="1" colspan="1">281.25 ± 6.2<sup>#</sup></td><td align="center" rowspan="1" colspan="1">274.84 ± 5.7<sup>#</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">G-III</td><td align="center" rowspan="1" colspan="1">Glibenclamide-10 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">271.94 ± 4.8</td><td align="center" rowspan="1" colspan="1">124.98 ± 8.1*</td><td align="center" rowspan="1" colspan="1">104.28 ± 4.9*</td><td align="center" rowspan="1" colspan="1">94.47 ± 2.9*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-IV</td><td align="center" rowspan="1" colspan="1">MEPG-200 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">268.51 ± 6.2</td><td align="center" rowspan="1" colspan="1">198.64 ± 2.8*</td><td align="center" rowspan="1" colspan="1">172.94 ± 23*</td><td align="center" rowspan="1" colspan="1">132.44 ± 7.2*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-V</td><td align="center" rowspan="1" colspan="1">MEPG-400 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">272.84 ± 5.2</td><td align="center" rowspan="1" colspan="1">165.22 ± 5.2*</td><td align="center" rowspan="1" colspan="1">152.94 ± 5.1*</td><td align="center" rowspan="1" colspan="1">115.27 ± 4.9*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VI</td><td align="center" rowspan="1" colspan="1">VAD-10 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">259.87 ± 5.5</td><td align="center" rowspan="1" colspan="1">169.68 ± 2.7*</td><td align="center" rowspan="1" colspan="1">159.32 ± 4.8*</td><td align="center" rowspan="1" colspan="1">124.66 ± 4.1*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VII</td><td align="center" rowspan="1" colspan="1">VAD-25 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">264.51 ± 7.1</td><td align="center" rowspan="1" colspan="1">148.25 ± 4.6*</td><td align="center" rowspan="1" colspan="1">129.80 ± 3.8*</td><td align="center" rowspan="1" colspan="1">98.26 ± 2.15*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VIII</td><td align="center" rowspan="1" colspan="1">VAD-50 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">274.56 ± 4.5</td><td align="center" rowspan="1" colspan="1">132.58 ± 3.1*</td><td align="center" rowspan="1" colspan="1">109.85 ± 2.2*</td><td align="center" rowspan="1" colspan="1">95.12 ± 3.85*</td></tr></tbody></table><table-wrap-foot><fn><p>All the values are expressed as mean ± SEM, <sup>#</sup><italic>P</italic> < 0.05 compared to normal control, and *<italic>P</italic> < 0.05 compared to vehicle control (3% Tween 80).</p></fn></table-wrap-foot></table-wrap><table-wrap id="tab6" orientation="portrait" position="float"><label>Table 6</label><caption><p>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.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1">Group</th><th align="center" rowspan="2" colspan="1">Treatment</th><th align="center" colspan="5" rowspan="1">Blood glucose (mg/dL)</th></tr><tr><th align="center" rowspan="1" colspan="1">0 min</th><th align="center" rowspan="1" colspan="1">30 min</th><th align="center" rowspan="1" colspan="1">60 min</th><th align="center" rowspan="1" colspan="1">120 min</th><th align="center" rowspan="1" colspan="1">180 min</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">G-I</td><td align="center" rowspan="1" colspan="1">Normal control</td><td align="center" rowspan="1" colspan="1">91.54 ± 3.1</td><td align="center" rowspan="1" colspan="1">135.45 ± 3.5</td><td align="center" rowspan="1" colspan="1">175.68 ± 4.8</td><td align="center" rowspan="1" colspan="1">124.56 ± 4.9</td><td align="center" rowspan="1" colspan="1">94.556 ± 2.8</td></tr><tr><td align="left" rowspan="1" colspan="1">G-II</td><td align="center" rowspan="1" colspan="1">Vehicle control (3% Tween 80) 10 mL/kg, p.o.</td><td align="center" rowspan="1" colspan="1">274.84 ± 5.7<sup>#</sup></td><td align="center" rowspan="1" colspan="1">384.11 ± 4.75<sup>#</sup></td><td align="center" rowspan="1" colspan="1">471.25 ± 6.7<sup>#</sup></td><td align="center" rowspan="1" colspan="1">352.12 ± 7.4<sup>#</sup></td><td align="center" rowspan="1" colspan="1">330.23 ± 8.5<sup>#</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">G-III</td><td align="center" rowspan="1" colspan="1">Glibenclamide-10 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">94.47 ± 2.9</td><td align="center" rowspan="1" colspan="1">115.46 ± 3.5*</td><td align="center" rowspan="1" colspan="1">124.56 ± 3.4*</td><td align="center" rowspan="1" colspan="1">102.56 ± 4.1*</td><td align="center" rowspan="1" colspan="1">96.56 ± 4.2*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-IV</td><td align="center" rowspan="1" colspan="1">MEPG-200 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">132.44 ± 7.2</td><td align="center" rowspan="1" colspan="1">164.56 ± 7.2*</td><td align="center" rowspan="1" colspan="1">186.35 ± 6.2*</td><td align="center" rowspan="1" colspan="1">148.90 ± 5.2*</td><td align="center" rowspan="1" colspan="1">135.65 ± 3.1*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-V</td><td align="center" rowspan="1" colspan="1">MEPG-400 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">115.27 ± 4.9</td><td align="center" rowspan="1" colspan="1">139.25 ± 6.3*</td><td align="center" rowspan="1" colspan="1">162.30 ± 4.2*</td><td align="center" rowspan="1" colspan="1">129.34 ± 5.2*</td><td align="center" rowspan="1" colspan="1">113.25 ± 6.2*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VI</td><td align="center" rowspan="1" colspan="1">VAD-10 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">124.66 ± 4.1</td><td align="center" rowspan="1" colspan="1">147.65 ± 3.2*</td><td align="center" rowspan="1" colspan="1">172.31 ± 6.8*</td><td align="center" rowspan="1" colspan="1">138.56 ± 3.9*</td><td align="center" rowspan="1" colspan="1">127.65 ± 4.7*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VII</td><td align="center" rowspan="1" colspan="1">VAD-25 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">98.26 ± 2.15</td><td align="center" rowspan="1" colspan="1">138.69 ± 6.4*</td><td align="center" rowspan="1" colspan="1">151.26 ± 4.1*</td><td align="center" rowspan="1" colspan="1">119.87 ± 6.3*</td><td align="center" rowspan="1" colspan="1">102.65 ± 5.8*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VIII</td><td align="center" rowspan="1" colspan="1">VAD-50 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">95.12. ±3.85</td><td align="center" rowspan="1" colspan="1">118.65 ± 6.2*</td><td align="center" rowspan="1" colspan="1">121.45 ± 2.3*</td><td align="center" rowspan="1" colspan="1">99.56 ± 5.9*</td><td align="center" rowspan="1" colspan="1">92.65 ± 3.7*</td></tr></tbody></table><table-wrap-foot><fn><p>All the values are expressed as mean ± SEM, <sup>#</sup><italic>P</italic> < 0.05 compared to normal control, and *<italic>P</italic> < 0.05 compared to vehicle control (3% Tween 80).</p></fn></table-wrap-foot></table-wrap><table-wrap id="tab7" orientation="portrait" position="float"><label>Table 7</label><caption><p>Effect of repeated-dose oral administration of MEPG and VAD on body weight in alloxan-induced diabetic rats.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1">Group</th><th align="center" rowspan="2" colspan="1">Treatment</th><th align="center" colspan="4" rowspan="1">Body weight (g)</th></tr><tr><th align="center" rowspan="1" colspan="1">Day 1</th><th align="center" rowspan="1" colspan="1">Day 7</th><th align="center" rowspan="1" colspan="1">Day 14</th><th align="center" rowspan="1" colspan="1">Day 21</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">G-I</td><td align="center" rowspan="1" colspan="1">Normal control</td><td align="center" rowspan="1" colspan="1">238.45 ± 6.5</td><td align="center" rowspan="1" colspan="1">246.85 ± 7.1</td><td align="center" rowspan="1" colspan="1">251.63 ± 4.9</td><td align="center" rowspan="1" colspan="1">254.85 ± 7.3</td></tr><tr><td align="left" rowspan="1" colspan="1">G-II</td><td align="center" rowspan="1" colspan="1">Vehicle control (3% Tween 80) 10 mL/kg, p.o.</td><td align="center" rowspan="1" colspan="1">243.56 ± 6.4</td><td align="center" rowspan="1" colspan="1">186.23 ± 8.7<sup>#</sup></td><td align="center" rowspan="1" colspan="1">169.74 ± 7.0<sup>#</sup></td><td align="center" rowspan="1" colspan="1">151.65 ± 6.9<sup>#</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">G-III</td><td align="center" rowspan="1" colspan="1">Glibenclamide-10 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">241.58 ± 8.2</td><td align="center" rowspan="1" colspan="1">236.56 ± 4.5*</td><td align="center" rowspan="1" colspan="1">233.84 ± 5.2*</td><td align="center" rowspan="1" colspan="1">229.56 ± 8.2*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-IV</td><td align="center" rowspan="1" colspan="1">MEPG-200 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">240.65 ± 6.9</td><td align="center" rowspan="1" colspan="1">199.23 ± 4.9*</td><td align="center" rowspan="1" colspan="1">172.94 ± 9.1*</td><td align="center" rowspan="1" colspan="1">162.75 ± 7.9*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-V</td><td align="center" rowspan="1" colspan="1">MEPG-400 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">247.23 ± 6.1</td><td align="center" rowspan="1" colspan="1">232.12 ± 7.2*</td><td align="center" rowspan="1" colspan="1">224.23 ± 7.3*</td><td align="center" rowspan="1" colspan="1">204.11 ± 10.5*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VI</td><td align="center" rowspan="1" colspan="1">VAD-10 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">236.52 ± 8.4</td><td align="center" rowspan="1" colspan="1">210.41 ± 5.9*</td><td align="center" rowspan="1" colspan="1">189.68
±7.7*</td><td align="center" rowspan="1" colspan="1">174.55 ± 9.5*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VII</td><td align="center" rowspan="1" colspan="1">VAD-25 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">242.95 ± 7.6</td><td align="center" rowspan="1" colspan="1">219.64 ± 6.6*</td><td align="center" rowspan="1" colspan="1">212.47 ± 5.9*</td><td align="center" rowspan="1" colspan="1">205.56 ± 7.8*</td></tr><tr><td align="left" rowspan="1" colspan="1">G-VIII</td><td align="center" rowspan="1" colspan="1">VAD-50 mg/kg, p.o.</td><td align="center" rowspan="1" colspan="1">243.57 ± 6.3</td><td align="center" rowspan="1" colspan="1">234.85 ± 8.4*</td><td align="center" rowspan="1" colspan="1">226.18 ± 6.3*</td><td align="center" rowspan="1" colspan="1">210.38 ± 9.4*</td></tr></tbody></table><table-wrap-foot><fn><p>All the values are expressed as mean ± SEM, <sup>#</sup><italic>P</italic> < 0.05 compared to normal control, and *<italic>P</italic> < 0.05 compared to vehicle control (3% Tween 80) on corresponding day.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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