DSpace at VNU: An investigation of antidiabetic activities of bioactive compounds in Euphorbia hirta Linn using molecular docking and pharmacophore

DSpace at VNU: An investigation of antidiabetic activities of bioactive compounds in Euphorbia hirta Linn using molecula...

O R I G I N A L R E S E A R C H

An investigation of antidiabetic activities of bioactive compounds

in Euphorbia hirta Linn using molecular docking

and pharmacophore

Quy Trinh• Ly Le

Received: 20 May 2013 / Accepted: 12 September 2013 / Published online: 2 October 2013

 Springer Science+Business Media New York 2013

Abstract Herbal remedies have been considered as

potential medication for diabetes type 2 treatment Bitter

melons, onions, or Goryeong Ginsengs are popular herbals

and their functions in diabetes patients have been well

documented Recently, the Euphorbia hirta has been

shown to have strong effects on diabetes in mice, however,

there has been no research clearly indicating what the

active compound is The main purpose of the current study

was therefore to evaluate whether a relationship exists

between various bioactive compounds in E hirta Linn and

targeted protein relating diabetes type 2 in human In view

of this, extraction from E hirta Linn was tested if they

contained the bioactive compounds This process involved

the docking of 3D structures of those substances (ligand)

into targeted proteins: 11-b hydroxysteroid dehydrogenase

type 1, glutamine: fructose-6-phosphate amidotransferase,

protein phosphatase, and mono-ADP-ribosyltransferase

sirtuin-6 Then, LigandScout was applied to evaluate the

bond formed between ligand and the binding pocket in the

protein These test identified in eight substances with high

binding affinity (\-8.0 kcal/mol) to all four interested

proteins of this article The substances are quercetrin, rutin,

myricitrin, cyanidin O-diglucoside, pelargonium

3,5-diglucose in ‘‘flavonoid family’’ and a-amyrine, b-amyrine,

taraxerol in ‘‘terpenes group.’’ The result can be explained

by the 2D picture which showed hydrophobic interaction, hydrogen bond acceptor, and hydrogen bond donor form-ing between carbonyl oxygen molecules of ligand with free residues in the protein These pictures of the bonding provide evidence that E hirta Linn may prove to be an effective treatment for diabetes type 2

Keywords Diabetes type 2
Euphorbia hirta Linn
Molecular docking
Pharmacophore analysis

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 *90 % are of type 2 (Inter-national Diabetes Federation, 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 nor-mal 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

Determination of the best treatment for diabetes type 2

is complicated because this is a progressive disease Cur-rently, insulin combined with other drugs is the preferred

Electronic supplementary material The online version of this

article (doi: 10.1007/s00044-013-0794-y ) contains supplementary

material, which is available to authorized users.

Q Trinh
L Le

School of Biotechnology, International University–Vietnam

National University, Ho Chi Minh City, Vietnam

L Le ( &)

Life Science Laboratory, Institute of Computational Science

and Technology, Ho Chi Minh City, Vietnam

e-mail: ly.le@hcmiu.edu.vn

RESEARCH

treatment method Recently, natural herbal medicines are

preferable options A study by Modak and coworkers have

provided a list of several medicinal plants used for diabetes

treatment (Modak et al.,2007) Several of them, such as

Caesalpinia bonducella (L) Roxb (Chandramohan et al.,

2008), Allium cepa (Onion) (G B Kavishankar et al.,

2011), Vitis vinifera and Euonymus alatus (Chan et al.,

2012), share a high concentration of Flavonoid compounds

including Quercetin, Kaemferol, Cyanidin, and

Pelargo-nium Our study focuses on Euphorbia hirta, one member

of Euphorbiaceae family which has high concentration of

these bioactive compounds in their extraction E hirta has

been reported to be effective in reducing diabetes in mice

in vitro studies (Anup et al., 2012; Sunil and Rashmi,

2010) When, the ethanol extracted compound from the

leaves, stems, and flowers of E hirta was applied to mice

which had induced diabetes by a single intraperitoneal

injection of streptozotocin (150 mg/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

antidiabetes components of the ethanol extracts of E hirta

by using them as ligand molecules for four targeted

pro-teins to determine which compound is an effective binder

E hirta contains three families of biomolecular

com-pounds such as tannin, flavonoid, and terpenes

(Moham-mad et al.,2010; Sandeep and Chandrakant,2011) Tannin

and flavonoid are strong antioxidants (Pietta,2000; Rield

and Hagerman, 2001) Quercitrin, one compound in

Fla-vonoid group, was good illustration In the thiobarbituric

acid (TBA) experiment quercitrin showed strong

antioxi-dant activity, giving 92.5 % inhibition and the IC50 was

calculated to 23.40 lM (Basma et al.,2011) 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 have 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 are considered to

have potential as therapeutic drugs for diabetes treatment

Both flavonoid and terpenes from medicinal plants have

already been shown to have strong effects on diabetes

(Mankil et al.,2006) In light of this evidence, the current

study will screen a range of bioactive compounds from all

the three families to determine if and how they interact

with proteins important to human diabetes type 2

Several proteins which were involved in glucose

metabolism consequently related to diabetes type 2 From

our intensive review on targeted proteins for antidiabetic

drug development (Trang and Ly, 2012), these important

target proteins including 11-b hydroxysteroid

dehydroge-nase type 1 (11b-HSD1), Glutamine fructose-6-phosphate

amidotransferase (GFPT or GFAT), protein phosphatase (PPM1B), and Mono-ADP-ribosyltransferase sirtuin-6 (SIRT6) were selected as receptors in this study

Material and methodology Molecular docking

Receptor 11-b HSD1, GFAT, PPM1B, and SIRT6 are the proteins relating to diabetes type 2 in humans (Hasan et al.,2002; Trang and Ly, 2012; Vogel, 2002; Shi, 2009; Nerlich

et al., 1998) The 3D structures of these molecules taken from Protein Data Bank are as follows: 11b-HSD1 (PDB code 1XU7), GFAT (PDB code 2ZJ4), PPM1B (PDB code 2P8E), and SIRT6 (PDB code 3K35) All these structures were tested again at the binding site to verify the capacity of the model in reproducing experimental obser-vations with new ligand In view of this, 11b-HSD1 (PDB code 1XU7) was tested again with molecule: NADPH dihydro-nicotinamide-adenine-dinucleotide phos-phate; GFAT (PDB code 2ZJ4) was tested with 2-deoxy-2-amino glucitol-6-phosphate; SIRT6 (PDB code 3K35) with adenosine-5-diphosphoribose; and PPM1B (PDB code 2P8E) with cysteine sulfonic acid They served as control docking models illustrated in supplementary Table 4 This work was done by Autodock vina in molecular docking experiment and VMD in visualization (Humphrey et al.,

1996)

Bioactive compounds 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, opti-mized by Gaussian with Hatree-Fock method, and the basis-set 6-31G* to increase reliability of structure The 2D structures of 27 ligands are illustrated in Table1

Docking simulations The docking process was done using Autodock Vina (Oleg and Arthur, 2009)

Autodocktool, one section in Molecular Graphic Labo-ratory, 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) Location of Grid box (supplementary

Table

Table

Fig 7) The site of Grid Box is illustrated in Table2 For setting the ligands, 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 pro-gram The content of configure file was determined as position of receptor file, ligand file, data of Grid-box’s three coordinates (Table2), the size of Gridbox which was set up in 30 9 30 9 30 points, number of modes which were ten, and the energy range which was set up at 9 kcal/ mol

Pharmacophore modeling This part of process was carried out using the pharmaco-phore tool included in LigandScout The program showed

us the 2D and 3D structure with the position and interaction

of ligand in the binding pocket of the receptor From these 2D pictures, some types of bond were identified by color and symbol Four features namely hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), negative ionizable area, hydrophobic interaction were labeled as red arrow, green arrow, red star, and orange bubble (supporting information), respectively

Result and discussion 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 E hirta Linn on proteins related to diabetes type 2 in humans, we docked the compounds to the pro-teins Results showed that the absolute value of binding energy ranged from 7.0 to 12.8 kcal/mol (Fig.2) The group of terpenes including a-amyrine, b-amyrine, friede-lin, taraxerol, taraxerone, and cycloartenol showed the best results All receptor for terpenes group had particularly

Table 2 Position of the Grid box center in four protein molecules Protein molecule PDB code X, Y, Z coordination (A ˚ )

11b-HSD1 1XU7 18.125 -27.72 -0.34

high binding affinities with the highest at 11b-HSD1 (PDB

code 1XU7) which being 100 % larger than 11 (kcal/mol)

The next highest positions were SIRT6, GFAT, and

PPM1B (Fig.1) For the terpenes group, the line for

11b-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 2 and the three compounds that had [8 kcal/

mol in terms of absolute value in binding affinity were

chosen for pharmacophore modeling They were

a-amy-rine, b-amya-amy-rine, and taraxerol The high binding efficiency

is thought to be due to the multiple methyl groups in the

structure as these functional groups have a strong ability to

construct hydrophobic bonds with the free residue of the

receptor

The flavonoid family had the largest number of ligands

and some of these also had high binding affinity to all four

receptors Five of these quercitrin, rutin, myricitrin,

cy-anidin 3,5-O-diglucoside, pelargonium 3,5-diglucose were

selected for pharmacophore modeling step Unsurprisingly,

the five molecules had multiple 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

(querci-tol, rhamnose, and camphol) which had the lowest binding

affinity The absolute value for these three ligands is shown

sequentially in Table1 They all share a simple structure

with only one ring and few hydroxyl groups outside which may explain their low binding affinity Thus, these mole-cules appear to have a low capacity to form a complex with the four target proteins

The tannin family also had molecules which bound well

to the receptors, but there was no representative molecule for pharmacophore docking However, they displayed strong interaction with 11b-HSD1, GFAT1, SIRT6, and low interaction with PPM1B Neuchlogenic acid and 3,4 dio galloy-quinic acid are illustrated in supplementary Table 3 From the results of this section, we determined that eight compounds showed strong binding capacity (|binding energy| [8.0 kcal/mol) to all four 11b-HSD1, SIRT6, GFAT, and PPM1B receptors Three of them belong to terpenes group (a-amyrine, b-amyrine, and taraxerol), the other five are members of flavonoid family (quercitrin, rutin, myricitrin, cyanidin 3,5-O-diglucoside, and pelargo-nium 3,5-diglucose) Five of them have structure of poly-phenol family which had previously considered as potential drug candidate for diabetes type 2 patients (Kati et al.,

2010) Besides that, overall viewing Fig.1, the line of 11b-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 Figure2shows 24 of the

27 tested (89 %) were higher than 8 kcal/mol and the friedelin molecule in the terpenes group had better binding capacity than the controls Thus the results provide strong evidences that 11b-HSD1 is a suitable receptor for diabetes type 2 patients being treated with bioactive compounds derived from E hirta

Fig 1 Absolute value of binding energy of 27 ligands to 4 receptors.

The short name of these ligands was written as QTin Quercetin, QTrin

quercitrin, QTol quercitol, RhNose Rhamnose RTn Rutin, LDin

Leucocyanidin, MTrin Myricitrin, CyGlu cyanidin-3,5-diglucose,

KRon kaemferon, PeGlu pelargonium-3,5-diglucose, CPhol camphol,

Ngenic Neuchlogenic acid, GQnic 3,4 dio galloy-quinic acid, BGlate

Benzyl gallate, BSrol Betasitosterol, CSrol Campesterol, SSrol Stigmasterol, DodeAte 12 deoxyphor-13 dodecanoate-20 acetate, phenylAte 12 deoxyphor-13 phenylacetate-20 acetate, InTate Ingenol triacetate, RNol Resiniferonol, ARine a-amyrine, BRine b amyrine, Flin Friedelin, TRol Taraxerol, TRone Taraxerone, CyNol Cycloartenol

Pharmacophore modeling

11b-HSD1

High binding affinity of the ligand to the receptor (Fig.2)

was explained clearly by interaction analysis in Fig.3

According to the molecular framework, there is a tenable

pharmacophore identified between flavonoid family and

non-flavonoid family (terpenes group) Structure of

flavo-noid contained high number of hydroxyl group which can

form strong hydrogen bonds with receptors Five molecules

(cyanidin O-diglucose, myricitrin, pelargonium

3,5-diglucose, quercitrin, and rutin) were frequently within

hydrogen contact with residues Tyr 183, Thr 124, and Ala

172 From this observation, three residues seemed to play a

critical role in catalytic activity of 11b-HSD1 (PDB code

1XU7) This conclusion is strongly supported by studies on

crystal structures and biochemical of 11b-HSD1 (Malin

et al.,2006; David et al.,2005) In Fig.3d–h, the Tyr 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 happened in case of Ala

172 but this residue was also within hydrophobic contact

with hydrophobes part on ligand (Fig.3d, f) 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

ste-roidal 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 residues such as

Ser 169, Ser 170, Tyr 183, and Leu 215

In case of terpenes group which has many hydrophobic

components (CH3group, benzene ring) Thus, terpenes can

form many hydrophobic interactions with other

hydro-phobic residues in receptors’ active site a-amyrine,

b-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-diglucose, pelargonium 3,5-diglucose, and quercitrin were also in contact with this receptor because of the presence 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 our study, these hydrophobic interactions were not present Ile 46, Ile 121, Leu 217, Leu 126, Thr 220, Thr 222… were frequently observed in ligand–receptor inter-actions between, so they can be a critical part in binding pocket

GFAT There were similarities in the binding mode of 11b-HSD1 and the steroidal hydroxyl group of cyanidin 3,5-O-diglucose, myricitrin, pelargonium 3,5-diglucose, quercitrin, and rutin All established a hydrogen bond with GFAT1 (PDB code 2ZJ4) at position of Ser 420, Ser 376, Gln 421, Thr 375, and Ser 422 in the binding pocket This result was validated in previous studies (Kuo-Chen 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, Fig 4a–c, f, g displayed Thr 425 which was close to not only methyl groups but also to the hydroxyl groups of a-amyrine, b-amyrine, quercitrin, rutin, and taraxerol

In addition, all of these ligands had hydrophobic inter-actions with receptors at positions of residue Leu 673, Val

677, Leu 556, and Thr 425 The mechanism of these interactions, however, differed among the ligands a-am-yrine, b-ama-am-yrine, myricitrin, and 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

Fig 2 Absolute value of

binding energy between E.

hirta’s ligand and 11b-HSD1

protein

Fig 3 Binding modes of selective compounds with 11b-HSD1 a a amyrine,

b b-amyrine, c taraxerol, d myricitrin, e pelargonium 3,5-diglucose, f

querci-trin, g rutin, and h cyanidin 3,5-O-diglucose a–c belong to terpenes family

and the rest are members of Flavonoid family Hydrogen Bond Acceptor (HBA) was shown asgreen vectors, Hydrogen Bond Donor (HBD) was drawn as red vectors Hydrophobic (H) was illustrated as yellow spheres

Fig 4 Binding modes of selective compounds with GFAT a a amyrine,

b b-amyrine, c taraxerol, d myricitrin, e pelargonium 3,5-diglucose, f

querci-trin, g rutin, and h cyanidin 3,5-O-diglucose a–c belong to terpenes family

and the rest are members of Flavonoid family Hydrogen Bond Acceptor (HBA) was shown as green vectors, Hydrogen Bond Donor (HBD) was drawn as red vectors Hydrophobic (H) was illustrated as yellow spheres

Fig 5 Binding modes of selective compounds with PPM1B a

a-amyrine, b b-a-amyrine, c taraxerol, d myricitrin, e pelargonium

3,5-diglucose, f quercitrin, g rutin, and h cyanidin 3,5-O-diglucose a–c belong

to terpenes family and the rest are members of Flavonoid family Hydrogen

Bond Acceptor (HBA) was shown as green vectors, Hydrogen Bond Donor (HBD) was drawn as red vectors Hydrophobic (H) was illustrated

as yellow spheres