VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY INTERNATIONAL UNIVERSITY AN INVESTIGATION OF ANTIDIABETIC ACTIVITIES OF BIOACTIVE COMPOUNDS IN EUPHORBIA HIRTA LINN USING MOLECULAR DOCKING AND PHARMACOPHORE A thesis submitted to The School of Biotechnology, International University In partial fulfillment of the requirements for the degree of B.S. in Biotechnology Student name: Trinh Xuan Quy – ID.No BTIU08133. Supervisor: Dr Le Thi Ly. 03/2013 ACKNOWLEDGEMENT. I take this opportunity to express my very great appreciation to my supervisor, Dr Le Thi Ly, who gave me the valuable guidance and advice. By this way, she inspired me greatly to work in this thesis. I also would like to thank her for giving me permission to work at Institute for Computational Science and Technology (ICST). This Institute supported me a good environment, wide variety of information and computer work experiences. I also would like to thank Dr Gay Marsden, lecturer of The University of Queensland, Australia for her diligence shown in proof-reading of this manuscript so that this manuscript would have been made possible for publication. Another special thank goes to BSc Nguyen Thanh Hieu, a staff of ICST, he always gave me great ideas and advise for my work. Without his knowledge and assistance, this study would not have been successful. My grateful thank is also extended to MSc Vo Thi Minh Thu who willing helped me edit the data and my thesis. AN INVESTIGATION OF ANTIDIABETIC ACTIVITIES OF BIOACTIVE COMPOUNDS IN EUPHORBIA HIRTA LINN USING MOLECULAR DOCKING AND PHARMACOPHORE Quy Trinh1, Ly Le1 1 School of Biotechnology, International University – Vietnam National University in HCMC Corresponding author: [email protected] ABSTRACT. Herbal remedies have been considered as potential medication for diabetes type 2 treatment.Bitter melons, onions, or Goryeong Ginsengs arepopular herbals and their functions in diabetes patientshave been well documented. Recently, the Euphorbia hirta has been shown to have strong affects on diabetes in mice, however, there has been no research clearly indicatingwhat the active compound is. The main purpose of the current study was thereforeto evaluate whether a relationship exists between various bioactive compounds in Euphorbia Hirta Linn and targeted protein relating diabetes type 2 in human. In view of this,extraction from E.Hirtawas tested if they contained the bioactive compounds. This process involved the docking of3D structures of those substances (ligand) into targeted proteins: 11-β hydroxysteroid dehydrogenase type 1 (11β-HSD1), Glutamine: fructose-6-phosphate amidotransferase (GFPT or GFAT), Protein Phosphatase (PPM1B) and Mono-ADP-ribosyltransferase sirtuin-6 (SIRT6). Then, LigandScout was applied to evaluate the bond formed between ligand and the binding pocket in the protein.These test identified ineight substances with high binding affinity (<-8.0 kcal/mol) to all four interested proteins of this article. The substances are quercetrin, rutin, myricitrin, cyanidin 3,5-O-diglucoside, pelargonium 3,5- diglucose in “flavonoid family” and alphaamyrine, beta-amyrine, taraxerol in “terpenes group”. The result can be explained by the 2D picture which showedhydrophobic interaction, hydrogen bond acceptor and hydrogen bond donor forming between carbonyl oxygen molecules of ligand with free residues in the protein. These pictures of the bonding provide evidence thatEuphorbia Hirta Linnmay prove to be an effective treatment for diabetes type 2. Keywords: Diabetes type 2, ligand, receptor, docking. 1 I. INTRODUCTION. Diabetes, one of the metabolic diseases that have high blood sugar as a pathognomonic symptom, is spreading like an epidemic. Worldwide, the number of patients climbed steeply from 171 million in 2000 to 366 million in 2030 (Wild et al., 2004) and approximately, 90% are of type 2 (International Diabetes Federation (IDF)., 2006). A person with this type of diabetes suffers a combination of insulin resistance and a weakness in insulin production. Insulin resistance is considered as stage one in diabetes type 2. In this phase, the glucose, energy molecule of the cell cannot cross the cell membrane due to blocking of the insulin receptor at the cell surface. This result is a high glucose concentration in the blood stream. To solve the problem, the pancreatic beta cells produce extra insulin to maintain glucose in the normal range. However, this process is only effective in the short term as burnout beta cell occurs. The failure for beta cell to produce the extra insulin is the second stage of diabetes type 2. Determine the best treatment for diabetes type 2 is complicated because this is a progressive disease. Currently, insulin combined with other drugs is the preferred treatment method. However, some natural herbal medicinesare also applied. Euphorbia hirta (E.hirta) is one of these recognized herbal medicine and is a member in Euphorbiaceae family. List bio-active compounds have been shown in vitro to be effective in reducing diabetes in mice (Anup et al., 2012, Sunil et al., 2010). When, the ethanol extracted compound from the leaves, stems and flowers of E.hirta were applied to mice which had induced diabetes by a single intraperitoneal injection of streptozotocin (150mg/kg), the result revealed that compounds displayed antihyperglycemic activity in the diabetic mice. To further understand this result, the current study focuses on identifying the bioactivity of the anti-diabetes components of the ethanol extracts of E.hirtaby using them as ligand molecules for fourtargeted proteins to determine which compound are effective binder. E.hirtacontains three families of bio-molecular such as tannin, flavonoid and terpenes (Mohammad et al., 2010, Sandeep et al.; 2011).Tannin and flavonoid are strong antioxidant(Pietta et al., 2000, Rield et al., 2001). Products of oxidation have been shown to play an essential role in the pathogenesis of diabetes type 1 and 2 (Maritim et al., 2003). In addition, the combination of high level of free radicals and inactivation of antioxidant defense has been shown to cause damage in cellular organelles and to the production of insulin (Maritim et al., 2003). Therefore antioxidants such as tannin and flavonoid areconsidered to 2 have potential as therapeutic drugs for diabetes treatment. Both flavonoid and terpenesfrom medicinal plants have already been shown to have strong effectson diabetes (Mankil et al, 2006). In light of this evidence, the current study will screen a range of bioactive compounds from all three families to determine if and how they interact with proteins important to human diabetes type 2”. II. MATERIAL and METHODOLOGY. 1. Molecular docking. 1.1 Receptor. 11-beta HSD1, GFAT, PPM1B, SIRT6 are the proteins relating to diabetes type 2 in humans (Hasan et al., 2002, Trang and Ly., 2012,Vogel et al., 2002, Yigong., 2009).The 3D structures of these molecules were taken from Protein Data Bank as following, 11β-HSD1 (code 1XU7),GFAT (code2ZJ4), PPM1B (code2P8E) and SIRT6 (code 3K35). All these structures were tested again at the binding site to verify the capacity of the model in reproducing experimental observations with new ligand. In view of this, 11β-HSD1 (1XU7) was tested again with molecule: NADPH dihydro-nicotinamide-adenine-dinucleotide phosphate (NDP), GFAT (2ZJ4) was tested with 2-deoxy-2-amino glucitol-6phosphate (AGP), SIRT6 (3K35) with adenosine-5-diphosphoribose (APR) and PPM1B (2P8E) with cysteinesulfonic acid (OCS). This work was done by Autodock vina and VMDmade viewing easier. (Humphrey et al., 1996). 1.2 Bioactive compound in E.hirta. Most of the 3D structures of drug molecules in E.hirta were downloaded from PubChem Compound section of National Center for Biotechnology Information (NCBI). For molecules with unknown structure, the 3D models were built based on 2D picture by GaussView 5.0, optimized by Gaussian with HatreeFock method and the basis-set 6-31G* to increase reliability of structure.The 2D structures of 27 ligands are illustrated in Table 1. 1.3 Docking simulations. The docking process was done usingAutodock Vina (Oleg et al., 2009). Autodocktool, one section in Molecular Graphic Laboratory was applied to build a complete pdbqt file name of ligands and receptors. Receptor preparation was carried out by four major sub-steps: (i) adding polar hydrogen, (ii) removing water molecule (iii) computation of Gasteiger charges, and (iv) 3 location of Grid box. The site of Grid Box is illustrated in Table 2.For setting theligands, the 3D structure in pdb file-type was loaded into Autodocktool to detect the root and convert it to pdbqt. Before switching on the Autodock Vina, one configure file was built to encode information for starting this program. The content of configure file was determined as position of receptor file, ligand file, data of Grid-box’s three coordinates (Table 2), the size of Gridbox which was set up in 30x30x30 points, number of modes which were 10 and the energy range which was set up at 9 kcal/mol. 2. Pharmacophore modeling. This part of process wascarried out using the pharmacophore tool included in LigandScout. The program showed us the 2D and 3D structure with the position and interaction of ligand inthe binding pocket of the receptor. From these 2D pictures, some types of bondwere identified by color and symbol. Four features namely hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), negative ionizable area (NIA), hydrophobic interaction werelabeled as red arrow, green arrow, red star and orange bubble (supporting information) respectively. III. RESULT AND DISCUSSION. 1. Free energy binding of bioactive compound to targeted protein related to diabetes type 2. In order to investigate the binding capacity of bioactive compounds in Euphorbia Hirta Linn on proteins related to diabetes type 2 in humans, we docked thecompounds to the proteins. Results showed thatthe absolute value of binding energyranged from 7.0 to 12.8 kcal/mol(Figure 2). The group of terpenes including alpha amyrine, beta amyrine,friedelin, taraxerol, taraxerone and cycloartenol showedthe best results. All receptor forterpenes group had particularly high binding affinities with the highest at 11β-HSD1 which being100% larger than 11 (kcal/mol). The nexthighest positions were SIRT6, GFAT and PPM1B (Figure 1). For the terpenes group, the line for 11β-HSD1 stayed at the upper level when compared to the other three receptors.For the ligands tested, terpenes were therefore considered to be the best drug candidate for diabetes type 2and the three compounds that hadgreater than 8kcal/mol in terms of absolute value in binding affinity were chosen for pharmacophore modeling.These werealpha amyrine, beta amyrine and taraxerol. The high binding efficiency is thought to be due to the multiple methyl groups in the 4 structure as these functional groups have a strong ability to construct hydrophobic bonds with the free residue of the receptor. The flavonoid family had thelargest number of ligands and some of these also had high binding affinity to all four receptors.Five of these quercitrin, rutin, myricitrin, cyanidin 3,5-O-diglucoside, pelargonium 3,5-diglucose wereselected for pharmacophore modeling step. Unsurprisingly, the five molecules hadmultiple aromatic phenol rings in their structure which is characteristic of polyphenol family.This structure contains a high number of hydroxyl groups which serve to facilitate ligands in forming hydrogen bonds with free residue of receptor. In addition, to containing a high number of ligands with high binding capacities, the flavonoid family also contained three compounds (quercitol, rhamnose,camphol) which hadthe lowest binding affinity. The absolute value for these three ligands is shown sequentiallyin Table 1. They all share a simple structure with only one ring and few hydroxyl groups outside which may explain their low binding affinity. Thus, these molecules appear to have a low capacity to form a complex withthe four target proteins. The tannin family also hadmolecules which bound well to thereceptors, but there was no representative molecule for pharmacophore docking. However, they displayed strong interaction with 11β-HSD1, GFAT1, SIRT6, and low interaction with PPM1B. Neuchlogenic acid and 3,4 dio galloy-quinic acid are illustrated in Table 3. From the results of this section, we determined thateight compounds showed strong binding capacity (|binding energy|>8.0 kcal/mol) to all four 11βHSD1, SIRT6, GFAT and PPM1B receptors. Three of them belong to terpenes group (alpha-amyrine, beta-amyrine, taraxerol), the other five are members of flavonoid family (quercitrin, rutin, myricitrin, cyanidin 3,5-O-diglucoside, pelargonium 3,5-diglucose). Five of them have structure of polyphenolfamily which had previously considered as potential drug candidate for diabetes type 2 patients (Kati et al., 2010). Besides that, overall viewing Figure 1, the line of 11β-HSD1 stayed in highest level in most of the case. It means that there is stronger interaction of ligand on this protein, compared to other three receptors. Figure 2 shows 24 of the 27 tested (89%) were higher than 8kcal/mol and the friedelin molecule inthe terpenes group had better binding capacity than thecontrols. Thus the results provide strong evidences that 11beta-HSD1 is a suitable receptor for diabetes type 2 patients being treated with bioactive compounds derived fromE.hirta. 5 Figure 1: Absolute value of binding energy of 27 ligands to 4 receptors. The short name of these ligands were written as Quercetin = QTin, quercitrin = QTrin, quercitol = QTol, Rhamnose = RhNose. Rutin = RTn, Leucocyanidin = LDin, Myricitrin = MTrin, cyanidin -3,5-diglucose = CyGlu, kaemferon = KRon, pelargonium-3,5-diglucose = PeGlu, camphol = CPhol, Neuchlogenic acid = Ngenic, 3,4 dio galloy-quinic acid = GQnic, Benzyl gallate = BGlate, Betasitosterol = BSrol, Campesterol = CSrol, Stigmasterol = SSrol, 12 deoxyphor-13 dodecanoate-20 acetate = DodeAte, 12 deoxyphor-13 phenylacetate-20 acetate = phenylAte, Ingenol triacetate = InTate, Resiniferonol = RNol, Alphaamyrine = ARine, Beta amyrine = BRine, Friedelin = Flin, Taraxerol = TRol, Taraxerone = TRone, Cycloartenol = CyNol. 6 Figure 2: Absolute value of binding energy between E.hirta’s ligand and 11β-HSD1 protein. 2. Pharmacophoremodeling. 2.1 11β-HSD1 High binding affinity of the ligand to the receptor (Figure 2) wasexplained clearly by interaction analysisin Figure 3. Five molecules (cyanidin 3,5-odiglucose, myricitrin, pelargonium 3,5-diglucose, quercitrin and rutin)werefrequently within hydrogen contact with residues Tyr 183, Thr 124, Ala 172.From this observation, three residues seemedto play a critical role in catalytic activity of 11β-HSD1. This conclusion is strongly supported by studies on crystal structures and biochemical of 11beta-HSD1 (Malin et al., 2006, David et al., 2005). In Figure 3.4, 3.5, 3.6, 3.7 and 3.8 theTyr 183 subunit has an important function in the bonding to the hydroxyl hydrogen of all five ligands whereas Thr 124 could form close vicinity to the ligand surface, and from there, the hydrogen bond could be set up between them. The same kind of interaction also happenedin case of Ala 172 but this residue was also within hydrophobic contact with hydrophobes part on ligand (Figure 3.4, 3.6). Moreover, cyanidin 3,5-O-diglucose, pelargonium 3,5-diglucose and rutin could link to the receptor with a high number of hydrogen bonds compared to myricitrin and quercitrin. This action can be explained by the affinity of each steroidal hydroxyl group for the receptor. For example, this functional group in cyanidin 3,5-o-diglucose could donate two or three hydrogen bonds with different residue such as Ser 169, Ser 170, Tyr 183, Leu 215. Along with hydrogen bond, hydrophobic interactions were also displayed. α amyrine, β amyrine and taraxerol seemed to be rich on hydrophobic contact at position of the methyl group which is non-polar. The compounds cyanidin 3,5-o- 7 diglucose, pelargonium 3,5-diglucose, quercitrin were also in contact with this receptor because of thepresence of the benzene ring. Previous studies using crystal structure analysis have reported, Ser 261 and Arg 269 are reported as largely hydrophobic residues in previous study involving crystal structure analysis (Malin et al., 2006)but in the figures from the our study, these hydrophobic interactions were not present. Ile 46, Ile 121, Leu 217, Leu 126, Thr 220, Thr 222… was frequently observed in ligand-receptor interactions between, so they can be a critical part in binding pocket. 8 Figure 3: Binding modes of selective compounds with 11β-HSD1 1:α amyrine, 2:β amyrine, 3:taraxerol, 4:myricitrin, 5: pelargonium 3,5diglucose, 6:quercitrin, 7:rutin, 8:cyanidin 3,5-O-diglucose. 2.2 GFAT There were similarities in the binding mode of 11beta-HSD1 and the steroidal hydroxyl group of cyanidin 3,5-o-diglucose, 9 myricitrin, pelargonium 3,5- diglucose, quercitrin and rutin. All establisheda hydrogen bond with GFAT1 at position of Ser 420, Ser 376, Gln 421, Thr 375, Ser 422 in the binding pocket. This result was validated in previous studies (Kuo-Chen et al., 2004, Vedantham et al., 2007, Yuichiro et al., 2009). In particular, pelargonium 3,5-diglucose was seen to have a similar binding mode to the Glc6P which is a strong inhibitor of GFAT1(Vedantham et al., 2007). Besides that, Figure 4.1, 4.2, 4.6, 4.7, 4.3 displayed Thr 425 which was closed to not only methyl groups but also the hydroxyl groups of alpha amyrine, beta amyrine, quercitrin, rutin, taraxerol. In addition, all of these ligandshad hydrophobic interactions with receptors at positions of residue Leu 673, Val 677, Leu 556, and Thr 425. The mechanism of these interactions however, differed amongst the ligands. Alpha amyrine, beta amyrine, myricitrin, amd taraxerol developed hydrophobic bonds with the hydrophobic receptor from methyl group. Meanwhile, the link between the benzene ring and interested part of receptor was decisive tendency in cyanidin 3,5-o-diglucose, pelargonium 3,5-diglucose, quercitrin, rutin. Figure 4: Binding modes of selective compounds with GFAT1 1:α amyrine, 2:β amyrine, 3: taraxerol, 4:myricitrin 10 Figure 4: Binding modes of selective compounds with GFAT1 5: pelargonium 3,5-diglucose, 6:quercitrin, 7:rutin, 8: cyanidin 3,5-o-diglucose 2.3 PPM1B and SIRT6. PPM1B had low binding affinity to ligand when compared to 11β-HSD1, GFAT, and SIRT6 but not compared to rutin. This could be explained firstly by a low number of bonds between the ligand and the receptor. Alpha amyrine, beta amyrine, cyanidin 3,5-o-diglucoseandpelargonium 3,5-diglucose are good illustrations. The binding energy ofalpha amyrineto 11β-HSD1, GFAT, SIRt6 and PPM1B was-11.5, -9.6, -10.4, -8.6 (kcal/mol) respectively and the numbers of bonds for their ligand interaction with the receptor were 23, 12, 11 and 8, respectively. Moreover, the number of hydrophobic and hydrogen bondswas also significantly reduced in the arrangement from 11β-HSD1 to PPM1B. For rutin, the total number of bonds in PPM was lower than GFAT but higher than 11βHSD1 and SIRT6. However, binding affinity did not follow this pattern and to understand this finding required a molecular dynamic (MD) and hydrogen bond analysis step to show. The duration time of the interaction between ligand and receptor is high frequency of residues Ala 197, Leu 196, Asp 286, Asp 60, Asn 287 seemed to play an important role in binding at mode of PPM1B (Figure 5).This result differed to Yigong (2009) result which showed Asp 119, Asp 231, 11 Asp 34, Asp 18, Arg 13 and Gly 35 as the key residue in binding site. This difference can be explained due to different in chain we tested on. By describing the crystal structure of SIRT6, Figure 6 revealed the differentposition of each ligand in the binding pocket of 8. Pictures6.5 and 6.7,6.8 supported thisfinding. Although there is similarity in the structure of the molecules, three compounds bound to different residues with different mechanisms. The benzene ring in cyanidin 3,5-o-diglucose and rutin contacted Trp 255 and Ala 56 through hydrophobic interaction, but in pelargonium 3,5diglucose, the Trp 186 had this function. The hydroxyl group of the benzene ring in picture 6.8 was the hydrogen bond donor to Thr 55, in contrast with hydrogen bond acceptor of Tyr 255 in Figure 6.7. From this, SIRT6 is seen to have a high number of residue which could form interactions with the functional group of the ligand. However, most of ligand could link with Trp 186 and Leu 184 which waspreviously found by Patricia et al (2011) in their study of the structure and biochemical function of SIRT6. In SIRT6, the total number of bondsdid not used to explain the differences in binding affinity amongst the three other receptors in most of situation. For example, there were 11 bonds between rutin and SIRT6, this number was lower than 16 bonds in GFAT and 13 bonds in PPM1B but rutin had a stronger binding affinity to SIRT6 with -10 (kcal/mol) in binding affinity which was lower than -8.6 (kcal/mol) in GFAT and -8.6 (kcal/mol) in PPM1B. This result for rutin can however be explained by MD and hydrogen bond analysis in PPM1B. These analysis will figure out stable hydrogen bond and hydrophobic interaction between ligands and receptors 12 Figure 5: Binding modes of selective compounds with PPM. 1:α amyrine, 2:β amyrine, 3:taraxerol, 4:myricitrin, 5: pelargonium 3,5diglucose, 6:quercitrin, 7:rutin, 8:cyanidin 3,5-o-diglucose. 13 Figure 6: Binding modes of selective compounds with SIRT6 1:α amyrine, 2:β amyrine, 3:taraxerol, 4:myricitrin, 5: pelargonium 3,5diglucose, 6:quercitrin, 7:rutin, 8:cyanidin 3,5-o-diglucose. IV. CONCLUSION. Docking simulation of 27 drug candidates extracted from Euphorbia Hirta, showed thatthe flavonoid and terpenes 14 families includingcyanidin 3,5-o- diglucose, myricitrin, pelargonium 3,5-diglucose, quercitrin, rutin, α amyrine, β amyrine and taraxerol have high binding affinity to all four interested receptors which are strongly relevant to diabetes type 2 in humans. These binding results were shown by LigandScout to consist of a high number of hydrogen bond and hydrophobic interactions. The binding pocket of each receptor: Tyr 183, Thr 124, Ala 172, Ile 46, Ile 121, Leu 217, Leu 126, Thr 220, Thr 222 in 11β-HSD1, Ser 420, Ser 376, Gln 421, Thr 375, Ser 422, Leu 673, Val 677, Leu 556, Thr 425 in GFAT1, Ala 197, Leu 196, Asp 286, Asp 60, Asn 287 in PPM1B and Trp 186 and Leu 184 in SIRT6 is in agreement with the previous research. Moreover, five molecules from the flavonoid family have the polyphenol structure indirectly confirming the strong capacity of the polyphenol family as a treatment for diabetes type 2(Kati et al., 2010).Also the binding affinity of three of the terpenes compounds also suggest that this family is also a good prospect for the treatment of type 2 diabetes.Finally, the comparison of the binding affinity amongst the four receptors indicates that 11β-HSD1 is the best receptor for accepting of these bioactive compounds derived fromE.hirta. Thus the results of this study have partially demonstrated the effect of E.hirta on some proteins relating to diabetes type 2.However, further research is required using, the molecular dynamic (MD) and hydrogen bond analysis to clearly determined the stability of the hydrogen bonds and hydrophobic interactions between ligands and receptors. V. REFERENCES. Anup, K. M., Smriti, T., Zabeer, A., Ram, K, Sahu., 2012. 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Wild, S., Roglic, G., Green, A., Sicree, R., King, H., 2004. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27:1047–1053. Yigong Shi., 2009. Serine/Threonine Phosphatases: Mechanism through structure. Cell 139,1016. Yuichiro, N., Masahiko, B., Hiroshi, S., Kenji, W., Fumitaka, G., Hideaki, T., Kazumi, K., Makoto, K., 2009. Structural analysis of human glutamine: fructose-6-phosphate amidotransferase, a key regulator in type 2 diabetes. FEBS letters 583, 163-167. 17 Appendix. Table 1: 2D structures of 27 drug candidates suggested from NCBI. Quercetin Quercitrin Quercitol Rhamnose Cycloartenol Rutin Leucocyanidin Myricitrin Cyanidin 3,5-O- Pelargonium 3,5- Kaemferon Camphol diglucoside diglucose Neuchlogenic acid 3.4-di-O- Benzyl gallate Campesterol 12-deoxy- 12-Deoxy- phorbol-13- phorbol-13- galloyquinic acid Beta sitosterol Stigmasterol 18 phenylacetate- dodecanoate-20- 20- acetate acetate Resiniferonol Ingenol triacetate Beta-amyrine Taraxerol Taraxerone Friedelin Alpha-amyrine Table 2: Position of the Grid box center in four protein molecules. Protein Protein code molecule X,Y,Z coordination (Angstroms) X Y Z 11β-HSD1 1XU7 18.125 -27.72 -0.34 GFAT 2ZJ4 8.27 4.54 -7.67 PPM1B 2P8E -11.72 -18.53 9.86 SIRT6 3K35 14.5 -18.02 17.04 19 Table 3: Binding energy (kcal/mol) of bio-molecule in E.hirta to 11β-HSD1, PPM1B, GFAT and SIRT6. Ligand’s Ligand’s name family Binding energy ( kcal/mol ) 11β-HSD1 SIRT6 GFAT PPM1B (1XU7) (3K35) (2ZJ4) (2P8E) NDP: - APR: - AGP: -7.0 OCS: -7.1 12.5 11.0 Quercetin -9.7 -8.3 -7.7 -7.5 Quercitrin -9.4 -9.3 -9.0 -8.5 Quercitol -5.6 -5.4 -6.3 -5.5 Rhamnose -6.0 -6.0 -6.2 -5.6 Rutin -10.5 -10 -8.6 -8.6 Leucocyanidin -9.1 -8.3 -7.5 -7.9 Myricitrin -9.5 -8.7 -9.3 -8.6 Cyanidin 3,5- -10.1 -9.1 -8.5 -8.6 Kaemferon -9.1 -8.1 -7.8 -7.2 Pelargonium -10.3 -9.2 -9.7 -8.3 Camphol -6.2 -5.7 -5.9 -4.7 Neuchlogenic -9.1 -9.6 -8.3 -7.5 -9.2 -8.9 -8.6 -7.9 Benzyl gallate -7.8 -8.8 -7.9 -7.4 Betasitosterol -10.3 -9.3 -7.9 -6.2 Campesterol -10.1 -9.4 -8.2 -6.6 Stigmasterol -11.0 -9.7 -8.2 -6.7 12 deoxyphor- -8.5 -9.3 -6.7 -6.1 -9.7 -9.4 -8.5 -7.4 Control Sample Flavonoid O-diglucoside 3,5 diglucose Tannin acid 3,4 dio galloyquinic acid Terpenes 13 dodecanoate20 acetate 12 deoxyphor13 phenylacetate- 20 20 acetate Ingenol -9.5 -9.7 -7.0 -6.5 Resiniferonol -8.5 -8.4 -7.6 -6.5 Alphaamyrine -11.5 -10.4 -9.6 -8.6 Beta amyrine -11.6 -10.9 -9.2 -8.5 Friedelin -12.8 -11.4 -9.1 -8.0 Taraxerol -12.1 -11.0 -9.1 -8.2 Taraxerone -12.4 -11.4 -9.1 -8.0 Cycloartenol -11.6 -9.1 -9.1 7.3 triacetate 21