Research Journal of Recent Sciences ______ ______________________________ ______ ____ ___ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 22 - 29 (201 5 ) Res. J. Recent . Sci. International Science C ongress Association 22 A Study on the Inhibitory potential of DPP - IV Enzyme by apigenin through in silico and in vivo approaches Kalivarathan Jagan, Mutulur Krishnamoorthy Radika, Emayavaramban Priyadarshini and Carani Venkatraman Anuradha* Department of Biochemistry and Biotech nology, Annamalai University, Chidambaram, Tamil Nadu, INDIA Available online at: www.isca.in , www.isca.me Received 30 th July 201 5 , revised 21 st August 20 1 5 , accepted 8 th September 20 1 5 Abstract Dipeptidyl peptidase - IV (DPP - IV) inhibitors are incretin enhancers used in the treatment of diabetes. Due to their undesirable side effects, development of new agents preferably from natural sources with less adverse effects is needed. Apigeni n (API), a well known insulin - secretagogue and insulin - mimetic agent is abundantly present in fruits, nuts and plant derived beverages. The present study investigates the DPP - IV inhibitory potential and antidiabetic effects of API using various approaches. Through in silico study, we showed that API has binding efficacy at Glu206 in the active site of DPP - IV. Plasma glucose and insulin levels were decreased when API (1.5 mg/kg b.w. for every alternate for last 30 days) was administered to high fat, high fru ctose diet (HFFD) fed rats. Further, the activity of DPP - IV in plasma and hippocampal homogenate was inhibited more in the API - treated group than in the sitagliptin (STG) - treated group. API has a strong inhibitory effect towards DPP - IV enzyme in vivo and in silico and therefore can be a promising compound for type 2 diabetes (T2D) treatment. Keywords : Apigenin, Docking, DPP - IV inhibition, HFFD, Insulin resistance . Introduction Incretins are hormones which play a vital role in glucose homeostasis by p ancreatic and extrapancreatic glucoregulatory effects. Dipeptidyl peptidase - IV (DPP - IV) is a protease which rapidly inactivates the incretin hormones like glucagon like peptidase - 1 (GLP - 1) and glucose - dependent insulinotropic polypeptide (GIP) 1 . Both hormo nes have very short half - lives (approximately 2 min) due to the rapid degradation by DPP - IV enzyme 2 . DPP - IV inhibitors, gliptins act as incretin enhancers through the inhibition of DPP - IV enzyme 3 . DPP - IV resistant incretin analogues/mimetics are therefore used in type 2 diabetes (T2D) treatment. Insulin resistance is a pathophysiol jh ogical state that begins with excess weight gain and obesity and is believed to be the major underlying factor for the development of T2D 4 . Numerous studies have observed perip heral and neuronal insulin resistance associated with cognitive disturbances in diet - induced resistant rats 5 . Previous studies suggest that hippocampus is sensitive to changes in diet and high - calorie diet can impair hippocampal synaptic plasticity and neu rogenesis thereby reducing learning and memory 6 . Findings from our laboratory have firmly established that high fat, fructose diet (HFFD) feeding produces adverse changes in skeletal muscle, liver and heart 7,8 . Protein - ligand docking study is a tool to i dentify the binding modes of protein - ligand interaction. Docking studies are employed in drug development because of its high accuracy. Computational docking analyses have been commonly used for designing inhibitors 9 , screening of potential inhibitors 10 , a nd explaining the differences in activity of drugs with different structures 11 . Apigenin (API, 4’,5’, 7’ - trihydroxy flavone) is a non - mutagenic, non - toxic flavone shown to have antidiabetic, anti - inflammatory , anti - oxidative and anti - carcinogenic propertie s 12 - 14 . API is shown to be neuroprotective improving the blood - brain barrier integrity and learning and memory and to offer neurovascular protection in mice injected with A β25 – 35 15 . Our present study is to focus on targeting DPP - IV enzyme through API as an incretin enhancer and to analyze its antidiabetic potential and DPP - IV inhibitory property by in silico and in vivo approaches. Material and Methods Chemicals: API and sitagliptin (STG) (Januvia) were purchased from Cayman Chemical Company, USA and Merck Pvt. Ltd., USA respectively. Gly - pro - p - nitroanilide (Gly - pro - p NA) was obtained from Sigma – Aldrich, USA. Glucose and insulin assay kits were purchased from Agappe diagnostics Pvt. Ltd., Kerala, India and Accubind, Monobind Inc., CA, USA respectively. Chemi cals and reagents used in the study were acquired from Himedia Laboratories Pvt. Ltd., Mumbai, India or Sisco Research Laboratories Pvt. Ltd., Mumbai, India. In silico : Molecular modeling: The DPP - IV inhibitory potential of API was assessed by molecular d ocking with DPP - IV crystal structure. The docking score of API was compared Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 22 - 29 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 23 with those of the derivatives of API (apigetrin, isorhoifolin, isovitexin, rhoifolin, vicenin, vitexin) using GLIDE software (Maestro, version 8.5, Schrödinger, LLC, 2008). Prepar ation of proteins and ligand: Docking was performed using GLIDE software 16 . DPP - IV (PDB: 1X70) was retrieved from the data base. The crystal structures were cleaned by deleting the ligand and cofactor. The protein structures were pre - processed, refined and the geometries were optimized with OPLS - 2005 force field using the standard protocol in GLIDE software. The structure of ligands (apigetrin, isorhoifolin, isovitexin, rhoifolin, vicenin, vitexin and API ) were manipulated and adjusted with LigPrep 2.5 (Sch rödinger LLC, Portland, USA;). First, all the protonation states at pH 2.0 – 7.0 were generated using Epik software 17 and for the generation of ligand binding site for metalloproteins the “Add metal binding site” was selected. Tautomers if any were gener ated at the respective pH. All combinations were generated to determine chiralities and to maintain stereochemistry. The geometries of the ligand are minimized using Schrodinger’s Optimization Potential for Ligand Simulation (OPLS - 2005) force field. Rece ptor Grid Generation: The shape of API and its derivatives, and their binding properties with DPP - IV enzyme were represented by the different sets of fields on a grid. Previous information reported in the literature on crystal structure and docking and the Q - Site finder software were used. Ligand Docking: The protein - ligand docking was performed in Glide XP extra precision mode 18 . The ligand structure was kept flexible while the protein structure was rigid. The best docked pose was saved. The results of t he best poses were analyzed using the G score and Dock score and number of H - bonds. Post docking calculation like the estimation of binding energy of the ligands with receptor was performed by automated Multi ligand Biomolecular Association with Energetics (MBAE). The ligand binding energy was calculated using: Ligand Binding energy, ∆E = E complex – E ligand – E protein The negative binding score indicates higher affinity of the ligand to the protein. Maintenance of animals: Male albino Wistar rats of body weight 100 to 120 g were purchased from Pranav Agro Indust ries Ltd., Bangalore, India and maintained in polypropylene cages under hygienic conditions with a standard light (12 h light/ 12 h dark cycle) and temperature (22 - 24 ï‚° C). The animals were given free access to standard pellet and water ad libitum and mainta ined in the Central Animal House, Rajah Muthiah Medical College and Hospital (RMMC and H), Annamalai Nagar. The experimental procedures were approved by the Institutional Animal Ethics Committee of Animal Care (IAEC), Annamalai University (No.160/1999/ CPC SEA/1101). Composition of HFFD: The HFFD prepared in the laboratory had the following composition (g/100 g): fructose 45.0, ground nut oil 10.0, beef tallow 10.0, casein 22.5, DL - methionine 0.3, wheat bran 5.5, vitamin mixture 1.2 and mineral mixture 5.5 . The standard diet, commercially obtained from Pranav Agro Industries Ltd., Bangalore, India contained 60 % (w/w) starch, 22.08 % (w/w) protein and 4.38 % (w/w) fat, normal chow provided 382.61 cal/100 g while HFFD provided 471.25 cal/100 g. Animal Groups : The animals were grouped into five with six animals in each group (Group I - Control, Group II - HFFD, Group III - HFFD + API, Group IV - HFFD + STG, Group V - Control + API). The experimental duration was 60 days. For treatment, 1.0 ml of API dissolved in 0. 1 % dimethyl sulfoxide (DMSO) was injected intraperitoneally (i.p.) at a dose of 1.5 mg/kg b.w. every alternate day for last 30 days of the experimental period 19 . For the comparative study, 1.0 ml of STG ( 30 mg/kg b.w. ) dissolved in 0.9 % saline 20 was give n orally for the last 30 days of the experimental duration . Sample preparation: At the end of 60 days, the animals were denied food overnight and sacrificed the next day by cervical dislocation. Blood was collected by sino - ocular puncture. Hippocampus wa s removed and immediately cleaned with ice - cold saline (0.89 % sodium chloride). Tissue sample was homogenized and plasma was collected for the assay of DPP - IV enzyme, glucose and insulin. In vivo DPP - IV inhibition assay: DPP - IV activity in plasma and hi ppocampal homogenate was measured according to the method of Kreisel et al.. A total volume of 0.20 ml assay mixture contained 0.1 M Tris - HCl buffer (pH 8) and 2×10 - 3 M of Gly - Pro - p - NA, a chromogenic substrate. After incubation for 30 min at 37 ° C, the react ion was stopped by adding 0.80 ml of sodium acetate (1M, pH 4.5). The amount of p - nitroaniline released was arrived using the molar extinction co - efficient of pNA (9600 M - 1 ·L·cm - 1 ) and the absorbance of the samples was measured in a UV - Vis spectrophotometer at 405 nm. Activity of the enzyme was expressed in unit per litre (U/L) for plasma and unit per mg protein (U/mg protein) for homogenate. Measurement of glucose, insulin and insulin sensitivity indices: Glucose and insulin in plasma were assayed using kits and insulin sensitivity indices like homeostatic model assessment (HOMA) and quantitative insulin sensitivity check index (QUICKI) 22 were calculated as shown below. HO MA= [Insulin (µU/ml) × glucose (mM)]/22.5 QUICKI = 1/[log(insulin µU/ml) × log (glucose mg/dL)] Statistical analysis: The data obtained was evaluated for statistical significance was done by one way analysis of variance (ANOVA) followed by Tukey’s Multipl e Range Test for multiple comparisons of groups. SPSS statistical software (version 16.0; SPSS, Chicago, IL, USA) was used. A value of P 0.05 was considered significant for all the cases. Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 22 - 29 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 24 Results and Discussion In silico study : Molecular docking was perfo rmed to find the interaction between API and its derivatives with DPP - IV enzyme using Schrödinger software. Docking score, glide score and hydrogen bond interaction were analyzed. The binding affinity of API and its derivatives (apigetrin, isorhoifolin, is ovitexin, rhoifolin, vicenin and vitexin) with DPP - IV enzyme are tabulated in table - 1 while figure - 1 depicts the pictorial representation of the same. The docking score and the formation of hydrogen bonds of the docked proteins are also tabulated in Table 1. Of all these compounds tested API binds at Glu206, Tyr662, Arg358 and Phe357 of DPP - IV enzyme ( figures - 2a and 2b). In vivo DPP - IV inhibition study: Based on the binding efficiency of API with DPP - IV enzyme, we analyzed the in vivo DPP - IV inhibition po tential of API. The results showed that API administrated animals significantly greater inhibition of DPP - IV by API than STG both in plasma and hippocampal homogenate ( table - 3). Glucose, insulin and insulin sensitivity : Table 2 shows the plasma glucose a nd insulin levels in experimental rats. Compared to all other groups HFFD - fed group, registered a rise in glucose and insulin levels in plasma, HOMA and QUICKI were also significantly altered in HFFD group when compared to control suggesting insulin resist ance in HFFD group. API administration significantly decreased glucose and insulin levels and improved insulin sensitivity. Discussion: DPP - IV is a proteolytic enzyme that specifically inactivates proteins that have an amino acid sequence with proline or alanine at the N - terminal penultimate position 23 . DPP - IV has three different binding pockets/active sites namely S1, S2 and S3. The S1 active site (Ser630, Asn710, and His740) is composed of side chains of catalytic triad involved in strong hydrophobic in teractions 24 and the S2 active site (Glu205, Glu206 and Tyr662) is present near the cavity of DPP - IV. The S3 active site (Ser209, Arg358, and Phe357) allows larger groups outside the pocket and the inside position favors smaller groups 25 . DPP - 4 inhibitors interact with the S2 pocket at Glu205 and Glu206 by forming salt bridges. This interaction plays an important role in inhibiting the enzyme 26 . Interestingly, docking analysis showed that API could dock into S2 and S3 pockets with residues Glu206, Tyr662, A rg358 and Phe357 (Figures 2a and 2b) at the active sites of DPP - IV. A study conducted by Fan et al., (2013) 27 reported that resveratrol, luteolin, API and flavones had low Ki values to inhibit DPP - IV activity and thereby they have high affinity to the acti ve site of DPP - IV. API derivatives, apigetrin binds at S2 pocket, isorhoifolin and isovitexin at S3 pocket and rhoifolin, vicenin and vitexin had no DPP - IV inhibitory effect. We presume that the binding of API with the enzyme leads to conformational change s in DPP - IV. In the present docking study, API showed lower binding energy than its derivatives indicating more potent inhibitory effect of API. Assay of DPP - IV enzyme in vivo showed significant inhibition by API and STG, but API has better efficacy in ra t plasma (38% and 25% inhibition by API and STG respectively) ( table - 3) and also in hippocampal homogenate (24% and 16% inhibition by API and STG respectively) ( table - 3). The differences in percentage inhibition of enzyme show that API has more inhibitory potential than STG in HFFD - fed insulin resistant rats. The HFFD - fed rat form an excellent model to study obesity and insulin resistance as the diet simulates ‘‘Western type’’ high - calorie diet 28 . Consumption of high fat with high quantities of fructose p romotes weight gain, obesity, insulin resistance and subsequently T2D 29 . HFFD feeding impairs insulin signaling and glucose uptake and utilization with 15 days of feeding 7 . In our present study, rats fed with high calorie diet for 60 days displayed altered HOMA and QUICKI values, insulin resistance, hyperglycemia and hyperinsulinemia. These findings are consistent with our earlier reports 5,30 . Since DPP - IV inhibitors (STG, vildagliptin and saxagliptin) have several side effects like headache, nausea, weight gain, swelling low blood sugar levels especially when taken in excess 31 , API which is non - toxic 12 can be a good therapeutic agent for T2D. Table - 1 Docking score, glide score and H - bond score of API and its derivative with DPP4 enzyme. Name of the compou nd and structure Amino acid residues of showing hydrogen bond with flavonoids Dockin g score Glide score Hydrogen bond score Apigetrin Lys554, Asp545, Gln553, Tyr547, Glu205, Trp627,Gly628 - 8.2 - 8.2 - 4.5 Isorhoifolin Ser630, Gln553, Tyr547, Val207, Arg358 , Trp629 - 10.8 - 10.8 - 7.4 Isovitexin Gln553, Arg358, Val207,Tyr547, Lys554, Ser552 - 7.8 - 7.8 - 4.8 Rhoifolin Tyr666, Tyr456, Arg429, Tyr547, Tyr585 - 8.5 - 8.5 - 4.1 Vicenin Arg125, Hie126 - 9.1 - 9.1 - 6.2 Vitexin Gln553, Tyr547, Arg125, Ser552, Ser630, Lys5 54 - 7.1 - 7.1 - 3.5 API Glu206 , Tyr662, Phe357, Arg358, Val207 - 2.8 - 2.8 - 2.9 Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 22 - 29 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 25 Table - 2 Plasma glucose, insulin, HOMA and QUICKI values of experimental animals. PARAMETERS CON HFFD HFFD + API HFFD + STG CON + API Glucose(mg/dL) 100.90±3.02 158.44±12.67 a 116 .04±4.64 a,b 134.19±6.70 a,b,c 94.38±2.83 Insulin (µU/mL) 16.8±0.50 31.0±2.48 a 23.3±0.93 a,b 26.7±1.33 a,b,c 16.5±0.49 HOMA 0.70±0.02 2.02±0.16 a 1.12±0.05 a,b 1.47±0.08 a,b,c 0.64±0.02 QUICKI 0.41±0.01 0.31±0.02 a 0.35±0.02 a,b 0.33±0.02 a,b,c 0.42±0.01 Values are means ± SD of 6 rats from each group. CON - Control rats; HFFD - High fat - high fructose diet - fed rats; HFFD + API - High fat - high fructose diet - fed rats treated with API; HFFD + STG - High fat - high fructose diet - fed rats treated with STG; CON + API - Con trol rats treated with API. a - Significant as compared to CON (P 0.05; ANOVA followed by Tukey’s test) b - Significant as compared to HFFD (P 0.05; ANOVA followed by Tukey’s test) c - Significant as compared to HFFD+API (P 0.05; ANOVA followed by Tukey ’s test) Table - 3 In vivo DPP - IV enzyme activity in plasma and hippocampal homogenate of experimental animals PARAMETERS CON HFFD HFFD + API HFFD + STG CON + API Plasma (U/L) 294.79 ±16.1 424.32 ±31.7 a 263.81 ±20.5 a,b 319.97 ±22.3 a,b,c 107.25 ±7.5 d Hippocampu s (U/mg protein × 10 - 5 ) 3.4 ±0.08 5.6±0.35 a 4.26 ±0.23 a,b 4.72 ±0.31 a,b,c 3.7 ±0.12 d Values are means ± SD of 6 rats from each group. CON - Control rat; HFFD, High fat - high fructose diet - fed rat; HFFD + API, High fat - high fructose diet - fed rat treated with AP I; HFFD + STG, High fat - high fructose diet - fed rat treated with STG; CON + API - Control rat treated with API. a - Significant as compared to CON (P 0.05; ANOVA followed by Tukey’s test) b - Significant as compared to HFFD (P 0.05; ANOVA followed by Tukey ’s test) c - Significant as compared to HFFD+API (P 0.05; ANOVA followed by Tukey’s test) d - Significant as compared to CON (P 0.05; ANOVA followed by Tukey’s test) Apigetrin Isorhoifolin Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 22 - 29 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 26 Isovitexin Vicenin Rhoifolin Vitexin Figure - 1 D ocking images of API derivatives with DPP - IV enzyme Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 22 - 29 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 27 Figures - 2a and 2b Docking images of API to DPP - IV protein active site formed H - bonds with residues Glu206, Tyr662, Phe357, Arg358 Conclusion API has a strong inhibitory action on DPP - IV enzyme thr ough in silico and in vivo studies and thus may be used as DPP - IV inhibitor. API also has a potent antihyperglycemic activity. The results observed in this study are the first step in exploring an incretin enhancer which is specific and efficacious in impr oving T2D symptoms in humans. Further studies are warranted using this compound. Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 22 - 29 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 28 References 1. Ansorge S., Nordhoff K., Bank U., Heimburg A., Julius H., Breyer D., Thielitz A., Reinhold D. and Tager M., Novel aspects of cellular action of dipeptidyl peptidas e IV/CD26 , Biol.Chem ., 392 , 153 – 168 (2011) 2. Palalau A. I., Tahrani A. A., Piya M. K. and Barnett A.H., DPP - 4 inhibitors in clinical practice, Postgrad. Med ., 121 , 70 – 100 (2009) 3. Brian D.G., Clifford J.B. and Peter R.F., Gliptin Therapies for Inhibiting Dipep tidyl Peptidase - 4 in Type 2 Diabetes, European Endocrinology , 6(2) , 19 – 25 (2010) 4. Reaven G. M., Role of insulin resistance in human disease, Diabetes ., 37(12) , 1595 - 1607 (1988) 5. Pratchayasakul W., Kerdphoo S., Petsophonsakul P., Pongchaidecha A., Chattipako rn N. and Chattipakorn S.C., Effects of high - fat diet on insulin receptor function in rat hippocampus and the level of neuronal corticosterone, Life Sciences ., 88(3 - 14) , 88619 – 88627 (2011) 6. Lindqvist A., Mohapel P., Bouter B., Frielingsdorf H., Pizzo D., Br undin P. and Erlanson - Albertsson C., High - fat diet impairs hippocampal neurogenesis in male rats, Eur. J. Neurol ., 13 , 1385 – 1388 (2006) 7. Geetha R., Yogalakshmi B., Sreeja S., Bhavani K., Anuradha C. V., Troxerutin suppresses lipid abnormalities in the heart of high - fat – high - fructose diet - fed mice, Mol. Cell Biochem ., 387(1 - 2) ,123 - 34 (2013) 8. Bhuvaneswari S., Yogalakshmi B., Sreeja S., Anuradha C. V., Astaxanthin reduces hepatic endoplasmic reticulum stress and nuclear factor - κB - mediated inflammation in high fructose and high fat diet - fed mice, Cell Stress and Chaperones ., 19 , 183 – 191 (2014) 9. Semighini E.P., Resende J.A., De Andrade P. , Morais P.A . , Carvalho I . , Taft C.A . , Silva C.H ., Using computer - aided drug design and medicinal chemistry strategies in the fight against diabete s, Journal of Biomolecular Structure and Dynamics. , 28(5) , 787 – 796 (2011) 10. Jadav P., Bahekar R., Shah S. R., Patel D . , Joharapurkar A . , Kshirsagar S . , Jain M . , Shaikh M . and Sairam K.V ., Long - acting peptidomimetics based DPP - IV inhibitors, Bioorganic and Medicinal Chemistry Letters. , 22(10) , 3516 – 3521 (2012) 11. Janardhan S., Homology modeling and molecular docking studies of human DPP8 and DPP9, International Journal of Pharma Research and Development , 2(12) , 131 – 146 (2011) 12. Cao X., Liu B., Cao W., Zhang W., Zhang F. , Zhao H . , Meng R . , Zhang L . , Niu R . , Hao X . and Zhang B ., Autophagy inhibition enhances apigenin induced apoptosis in human breast cancer cells, Chin. J. Cancer. Res., 25 , 212 - 222 (2013) 13. Liu Q., Chen X., Yang G., Min X. and Deng M., Apigenin inhibits cell migration through MAPK pathways in human bladder smooth muscle cells, Biocell ., 35 , 71 - 79 (2011) 14. Chowdhury M. H., Miltu K. G., Bhabani S. S., Niladri S. D., Biswajit M., Apigenin causes biochemical modulation, GLUT 4 and CD38 alterations to improve diabetes and to protect damages of some vital organs in experimental diabetes, American Journal of Pharmacology and Toxicology , 9(1) , 39 - 52 (2014) 15. Liu R., Zhang T., Yang H., Lan X., Ying J. and Du G., The flavonoid apigenin protects brain neurovascular coupling against amyloid - beta25 - 35 - induced toxicity in mice, J. Alzheimers Dis ., 24 , 85 – 100 (2011) 16. Friesner R.A., Banks J.L., Murphy R.B., Beard H.S., Frye L.L., Halgren T.A., Klicic J.J., Mainz D .T., Repasky M. P., Knoll E. H., Shelley M., Perry J. K., Shaw D. E., Francis P., Shenkin P. S., Glide: a new approach for rapid, accurate docking and scoring method and assessment of docking accuracy, J. Med. Chem ., 7 , 1739 – 1749 (2004) 17. Shelley J.C., Choll eti A., Frye L.L., Greenwood J.R., Timlin M.R. and Uchimaya M ., Epik: a software program for pK(a) prediction and protonation state generation for drug - like molecules, J. Comput. Aided Mol. Des. , 12 , 681 – 691 (2007) 18. Friesner R.A., Murphy R.B., Repasky M.P., Frye L.L., Greenwood J.R., Ha lgren T.A . , Sanschagrin P.C . and Mainz D.T ., Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein - ligand complexes, J. Med. Chem ., 21 , 6177 – 6196 (2006) 19. Ohno M., Shibata C., Kishikawa T., Yoshikawa T., Takata A., Kojima K., Akanuma M ., Kang Y.J., Yoshida H., Otsuka M. and Koike., The flavonoid apigenin improves glucose tolerance through inhibition of microRNA maturation in miRNA103 transgenic mice, Sci. Rep ., 3 , 2553 - 2559 (2013) 20. Hiranya P., Nattayaporn A., Nipon C. and Siriporn C.C., DPP4 inhibitors improve cognition and brain mitochondrial functions of insulin - resistant rats, S ociety for Endocrinology., 218(1), 1 - 11 (2013) 21. Kreisel W., Heussner R., Volk B., Buchsel R., Reutter W. and Gerok W., Identification of the 110 000 M r glycopro tein isolated from rat liver plasma membrane as dipeptidylaminopeptidase IV., FEBS. Lett ., 147 , 85 – 88 (1982) 22. Gutt M., Davis C. L., Spitzer S. B., L labre M.M., Kumar M., Czarnecki E.M., Schneiderman N . , Skyler J.S . and Marks J.B ., Validation of the insulin sensitivity index (ISI 0 , 120 ): comparison with other measures, Diabetes Res. Clin. Pract., 47(3) , 177 - 184 (2000) Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 22 - 29 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 29 23. Mentlein R., Dipeptidyl - peptidase IV (CD26) - role in the i nactivation of regulatory peptides, Regul. Pept ., 85 , 9 – 24 (1999) 24. Jadav P., Bahekar R., Shah S. R., Patel D . , Joharapurkar A . , Kshirsagar S . , Jain M . , Shaikh M . and Sairam K.V ., Long - acting peptidomimetics based DPP - IV inhibitors, Bioorganic and Medicinal Chemistry Letters. , 22(10) , 3516 – 3521 (2012) 25. Pissurlenkar R.R.S., Shaikh M.S., Coutinho E.C., 3DQSAR studies of Dipeptidyl peptidase IV inhibitors using a docking based alignment, Journal of Molecular Modeling ., 13(10), 1047 – 1071 (2007) 26. Kimata H., Fukuda - Tsuru S., Yoshida K., Iwasaki S., Kiuchi S., Akahoshi F. and Ishii S., Teneligliptin, a n ovel selective dipeptidyl peptidase - 4 inhibitor, improves postprandial hyperglycemia in a model of type 2 diabetes, zucker diabetic fatty rats, Med. Consult. New Remedies ., 49 , 9 (2012) 27. Fan J., Johnson M.H., Lila M.A., Yousef G. and Mejia E.G., Berry and C itrus Phenolic Compounds Inhibit Dipeptidyl Peptidase IV: Implications in Diabetes Management, Complementary and Alternative Medicine., 2013 , 1 - 13 (2013) 28. Reddy S. S., Karuna R., Baskar R. and Saralakumari D., Prevention of insulin resistance by ingesting a queous extract of Ocimum sanctum to fructose - fed rats, Horm. Metab. Res ., 40 , 44 - 49 (2008) 29. Samane S., Christon R., Dombrowski L., Turcotte S., Charrouf Z., Lavigne C., Levy E., Bachelard H., Amarouch H., Marette A. and Haddad P.S., Fish oil and argan oil i ntake differently modulate insulin resistance and glucose intolerance in a rat model of dietary - induced obesity, Metab. Clin. Exp ., 58 , 909 – 919 (2009) 30. Thirunavukkarasu V. and Anuradha C.V., Influence of lipoic acid on lipid peroxidation and antioxidant defence system in blood of insulin resistant rats, Diabetes Obes. Metab ., 6(3) , 200 – 207 (2004) 31. Chakrabarti R., Bhavtaran S., Narendra P., Varghese N., Vanchhawng L., Shihabudeen M. S. and Thirumurgan K., Dipeptidyl Peptidase - IV Inhibitory Activity of Ber beris aristata, Journal of Natural Products. , 4 , 158 - 163 (2011) 32. www.rcsb.org , (2015) 33. http://www.schrodinger.com , (2015) 34. www.bioinformatics.leeds.ac.uk/qsitefinder , (2015)