1

Anti-diabetic activities of bioactive compounds

in Euphorbia Hirta Linn to receptors for
diabetes type 2 drug development
Authors: Tram Nguyen, Nghi Nguyen, Luan Vu, Thanh Nguyen, Huong Nguyen
School of Biotechnology, International University Vietnam National University in HCMC
Abstract
Objectives: Euphorbia hirta has been lately studied as a potential therapeutic herbal used in
Diabetes type 2 patients. In this research, we examine the antidiabetic activities of bioactive
compounds in Euphorbia hirta Linn to five receptors (11HSD1, PTP1B, -glucosidase,
PPARy, DDP4) to establish treatment for diabetes type 2.
Methods: Firstly, the 3D structures of 29 major bioactive compounds of Euphorbia hirta
were sketched by Gaussview. Secondly, Autodock Tools was applied to build a complete set
of ligands and receptors. Subsequently, a ligand based pharmacophore approach has been
generated for those 29antidiabetic compounds with significance for the development of new
drugs by using LigandScout software.
Result and conclusion:Three pharmacophore features: hydrophobic domain, hydrogen bond
acceptor and hydrogen bond donor were obtained. Also, the top nine affinity binding
compounds (1,3,4-tetra-O-galloyl--D-glucose; -amyrine; campesterol; myricitrin,
quercitrin, pelargonium-3,5-diglucoside; friedelin; taraxerone, taraxerol) give strong
evidence to be good candidates for drug development to diabetes type 2 treatment. Besides,
the pharmacophore models applied the Lipinskis rule to determine if a chemical compound
with a certain pharmacological or biological activity has similar properties that would make
it a likely orally active drug in humans. Moreover, molecular docking is highly
recommended to use to reach the optimum results.
Key words: Diabetes type 2, Euphorbia Hirta Linn, drug design, docking, pharmacophore
modeling.
2

Introduction
In normal body, the production of insulin stimulates cells to take up glucose from the
bloodstream to manufacture energy for the whole body metabolism. However, when a body
produces little or no insulin, the blood glucose (blood sugar) will increase uncontrollably and
causes serve damage through whole body (heart, eyes, kidney, nerve, teeth, etc.)
i
. There are
two types of diabetes: type 1 and type 2. Diabetes type 2 is far more common than diabetes
type 1. About 90% of adults with diabetes have type 2, and about 10% have type 1
ii
. Type 2
diabetes (non-insulin-dependent diabetes) is called insulin resistance the pancreas does
not make enough insulin or the body cannot use the insulin well
iii
.
Herbal drugs are studied recently due to their less side effects, low cost and high
effectiveness. Therefore, numerous of researches had been processed to test the bioactivities
of some potential herbal candidates. In diabetic treatment, Euphorbia hirta a member of
Euphorbiaceae and genus Euphorbia has been lately studied as a potential therapeutic herbal
used in Type 2 diabetic patients. E. hirta is commonly found in garden paths and wastelands.
Phytochemical analysis of Euphorbia hirta (E. hirta) revealed the presence of alkaloids,
flavonoids, sterols, tannins and triterpenoids in the whole plant
iv
. Some chemical
components extracted from E. hirta performed biological acitivities, such as: antimalarial,
anti-diarrhoeic, anti-inflammatory, antimicrobial, antibacterial, diuretic, anti-allergic
activities, etc.
v,vi,vii,viii,ix,x,xi,xii,xiii,xiv
In this research, we examine the antidiabeticactivities of 29
bioactive compounds (belongs to Flavonoids, polyphenols, tannins, triterpenes and
phytosterols)
xv
in Euphorbia hirta Linn to five receptors: 11HSD1, PTP1B, -glucosidase,
PPARy, DDP4 (directly related to type 2 diabetes) by molecular docking tools and
pharmacophore analysis to2 to determine which compounds are the best candidates for
potential drug design.
xvi

3

Material and Method
1. Data set collection and receptor-compound preparation
The most important process in pharmacophore model generation is the selection of test set
compounds. Over the last few years, a number of anti-diabetic compounds have been
identified and the Euphorbia Hirta showed a potential bioactivity in anti-diabetes type
2
xvii
.Therefore, we collect 28 major bioactive compounds in Euphorbia hirta in this current
research. Table 7 shows the 2D structures of 28 bioactive compounds candidates from the
ncbi and Chemspider. The 3D structures of these 28 compounds were sketched by the
Gaussview 5.0
xviii
and save in mol2 format. Subsequently, they were imported to Autodock
and ready for docking.
Receptor
11 beta hydroxysteroid dehydrogenase type 1, PTP1B, Alpha-glucosidase, PPARy,
DDP4 are the proteins relating to Diabetes type 2 in humans.
xix


11 BETA HYDROXYSTEROID DEHYDROGENASE TYPE 1(11B HSD 1)
11 Hydroxysteroid dehydrogenase - a microsomal glycoprotein enzyme - belongs
to SDRs (short-chain dehydrogenase /reductase) protein family. 11HSD1 composes
of 282 amino acid and weights 38kDa. It is expressed predominantly in peripheral
tissues such as liver, adipose tissues, skeletal muscles and central nervous system.The
main function of 11 HSD1 is catalysis of cortisone to cortisol active glucocortisoid
conversion process in human. Glucocorticoids have been shown to inhibit a number
of steps in the insulin signaling network through several different mechanisms
xx
. Due
to the fact that cortisol plays a critical role in diabetes, 11HSD1 has a potential
therapeutic target for type II diabetes. Many studies have indicated that the high
circulating levels of the active glucorticoid cortisol can also lead to other syndrome
such as central obesity, dyslipidemia and hypertension
xxi
. 11 -HSD 1 has both dimer
and tetramer organization. In this study,3D structure of this protein was taken from
4

Protein Data Bank as the accession number is 1XU7 (tetramer). The further step
(docking and modeling) is worked on chain A of 11 -HSD 1only.

PROTEIN-TYROSINE PHOSPHATASE 1B
Tyrosine-protein phosphatase non-receptor type 1 also known as protein-tyrosine
phosphatase 1B (PTP1B) is an enzyme that is the founding member of the protein
tyrosine phosphatase (PTP) family. In humans it is encoded by the
PTPN1 gene.
xxii
PTP1B is a negative regulator of the insulin signaling pathway and is
considered a promising potential therapeutic target, in particular for treatment of type
2 diabetes.
xxiii

PTP1B was the first member of the protein tyrosine phosphatase (PTP) superfamily to
be identified and was purified to homogeneity from human placenta as a catalytic
domain of 37 kDa (Tonks et al., 1988). Later, it was characterized as an 50 kDa
protein (435 amino acids), consisting of an N-terminal catalytic domain followed by a
C-terminal segment that serves a regulatory function and anchors the protein at the
cytoplasmic face of the endoplasmic reticulum (ER) membrane (Tonks, 2003).
PTP1B can dephosphorylate the phosphotyrosine residues of the activated insulin
receptor kinase. The phosphatase activity of PTP1B occurs via a two-step mechanism
(Figure 3). In the first step: pTyr substrate is dephosphorylated (a nucleophilic attack
at the phosphocenter by the reduced Cys215 residue, followed by subsequent
protonation by Asp181 to yield the neutral tyrosine phenol). In the second step: The
enzyme intermediates are broken down. The active enzyme is regenerated after the
thiophosphate intermediate is hydrolyzed, which is facilitated by the hydrogen
bonding interactions of Gln262 and Asp181 that help to position in the water
molecule at the desired site of nucleophillic attack.
xxiv

In this study, 3D structure of this protein was taken from Protein Data Bank as the
accession number is 3CWE. The further step (docking and modeling) is worked on
chain A of 3CWE only.

5

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR-)
Peroxisome proliferator-activated receptor gamma (PPAR- or PPARG), or glitazone
receptor, or NR1C3 (nuclear receptor subfamily 1, group C, member 3) is a type
II nuclear receptor that in humans is encoded by the PPARG gene. It belongs to
the nuclear hormone receptor family.
xxvxxvixxvii

In human and mouse, PPARG has been found in 2 isoforms: PPAR-1 (found in
nearly all tissues except muscle) and PPAR-2 (mostly found in adipose tissue and the
intestine).
xxviii
Defects in PPARG can lead to type 2 insulin-resistant diabetes and
hypertension. Nuclear receptor that binds peroxisome proliferators such as
hypolipidemic drugs and fatty acids. Once activated by a ligand, the nuclear receptor
binds to DNA specific PPAR response elements (PPRE) and modulates the
transcription of its target genes. Therefore control the regulation of fatty acid storage
and glucose metabolism. Many insulin sensitizing drugs (namely,
the thiazolidinediones) used in the treatment of diabetes target PPARG as a means to
lower serum glucose without increasing pancreatic insulin secretion.
xxix


DIPEPTIDYL PEPTIDASE-4 (DPP4)
DPPIV adenosine deaminasecomplexing protein 2 or CD26 - belongs to the
exopeptidase class of proteolytic enzymes. This antigenic enzyme expressed on the
surface of most cell types and associated with immune regulation, signal
transduction and apoptosis. Exopeptidases that cleave N- terminal and C- terminal
amino acid residues from peptide and protein. DPP4 plays a major role
in glucose metabolism. It is responsible for the degradation of incretions such as GLP-
1 which stimulates insulin release and inhibits glucagon release to lower of blood
glucose
xxx
.
In this study, 3D structure of this protein was taken from Protein Data Bank as the
accession number is 4J3J (dimer).
ALPHA- GLUCOSIDASE
6

Alpha-glucosidase is a glucosidase that acts upon 1,4-alpha-glucosidase bonds
(Figure 5).
xxxi
They are in contrast to beta-glucosidase. The main function of alpha-
glucosidase is breaking down starch and disaccharides to glucose. Maltase belongs to
this family.

Figure 5: The effect of alpha-glucosidase on 1,4-alpha bonds

Maltase glucoamylase, the proteinconsist of 875 amino acids, is one of the four
intestinal glycoside hydrolase 31 enzyme activities which respond for the hydrolysis
of terminal starch products into glucose. As the result, an inhibition of the N-terminal
catalytic domain of maltase-glucoamylase (ntMGAM) is necessary for controlling
blood glucose levels in individuals with type 2 diabetes
xxxii
. In this research, 3L4T (a
monomer) was taken from Protein Data Bank (PDB) like the representative ofmaltase
glucoamylase.
Bioactive compound in E.hirta
Bioactive compounds in E.hirta is classified into three main families including tannin,
flavonoid and terpenes
xxxiii
. Tannin and flavonoid are strong antioxidant whose products
have been shown as a key in pathogenesis of diabetes type 1 and 2. Therefore
antioxidants such as tannin and flavonoid are considered to have potential ability for
therapeutic drugs for diabetes treatmnent,. The current study will investigate 28 of
bioactive compounds from all three families to determine how they interact with target
proteins in diabetes type 2.In this research, we will focus on 3 families that mainly
contribute in E. hirta: Flavonoid family (Afzelin, quercetin, quercitrin, quercitol,
rhamnose, rutin, leucocyanidin, myricitrin, cyanidin 3,5- O-diglucoside, kaemferon,
7

pelargonium 3,5- diglucoside). Tannin family (gallic acid, 3,4-di-O-galloyl-quinic acid,
3,4,5-tri-O-galloylquinic acid, 1,2,3,4,6 tetraOgalloyl-b-D-glucose). Triterpenes and
phytosterols family (2,4-methylenecycloartenol, betasitosterol, campesterol, -
stigmasterol, ingenol triacetate, resiniferonol, alpha-amyrine, beta-amyrine, friedelin,
taraxerol, taraxerone, cycloartenol, protocatechuic acid).
xxxiv

xxxv

Many bioactive compounds in E.hirta were found to perform several functions:
antioxidant, anti-inflammatory, antimicrobial, anticancer, cardioprotective,
neuroprotective, antidiabetic, antiosteoporotic, estrogenic/antiestrogenic, anxiolytic,
analgesic,antiallergic activities, etc. (Table 11). However, in this research, we will only
analyze the anti-diabetic activities of those compounds to the target protein receptors
which directly related to diabetes type 2.
Most of the 3D structures of 28 bioactive compounds in E.hirta were built based on 2D
picture by GaussView 5.0. The 2D and 3D structures of 29 ligands are illustrated in
Table7.
Docking simulation
The docking process was done using Autodock Tools 1.5.6
xxxvi

Autodock Tools was applied to build a complete set of ligands and receptors with the file
name pdbqt. Receptor fixation was done by following steps: (1) adding polar hydrogen,
(2) removing water molecule (3) computating of Gasteiger charges and adding charges to
receptors, and (4) locating of Grid box by using Center on ligand (with number of points
in x-dimension/ y-dimension and z-dimension are 40x40x40, spacing is 1 and
exhaustedness is 100). The site of Grid Box is illustrated in Table 9.
The 3D structures of all 29 compounds in pdb or mol2 file type was converted into pdbqt
file type after detect the root to set up the appropriated ligands.

8

2. Pharmacophore modeling
LigandScout 3.12
xxxvii
will be used to derive the pharmacophore models. LigandScout
software efficiently allows rapidly and transparently generation of 2D and 3D
pharmacophore of data set. It creates the pharmacophore, aligned pharmacophore and
features, aligning of merge pharmacophore of compounds and molecules by reference
points. This tool is scientifically published and based on several years of experience in
pharmacophore generation.
xxxviii

In this study, the program was applied to show 3D structure of the receptors, both 2D and
3D structure of ligand in the binding pocket of that receptor with the position, interaction
(types and residues of interaction) as well as ligand properties such as molecular weights,
number of atoms, rings, etc. After identifying pharmacophore of ligands and receptors,
types of bonds were colored and symbol as red arrow, green arrow, red star and orange
bubble are hydrogen bond acceptor, hydrogen bond donor, and negative ionization and
hydrophobic interaction, respectively.
Then in order to evaluate their drugs likeness property, rule of five (Lipinski's rule) was
used, it is a popular rule to evaluate drug like properties or determine if a chemical
compound with a certain pharmacological or biological activity has similar properties
that would make it a likely orally active drug in humans
xxxix

The rule is as follows:
There should be less than 5 H-bond donors.
Molecular weight should be less than 500 Daltons.
Partition coefficient (LogP) not over 5 (or MLogP is over 4.15).
There should be less than 10 H-bond acceptors.
9

Result and discussion
1. Free energy binding of bioactive compound to receptor related to diabetes
type 2
The results of docking process showed that the absolute value of binding energy ranged
from 4.4 to 11.3 kcal/mol (Table 6). From the observation, there are eight bioactive
compounds shows the strong binding affinity (|binding affinity| >7.0 kcal/mol) to 5
receptors (11HSD1, PTP1B, -glucosidase, PPARy, DDP4). The tannin family had
high binding affinity such as 1,3,4,6 tetraOgalloyl-b-D-glucose. The group of terpenes
including alpha amyrine, beta amyrine, friedelin, taraxerone, campesterol, and
cycloarterol also yield good result. In flavonoid family, three of these members
quercitin, pelargonium 3,5-diglucose and myricitrin were selected for pharmacophore
modeling step. The receptor DDP4 showed highest binding affinities of all bioactive
compounds to the others (20 out of 29 compounds (~70.%) have |binding affinity| >7.0
kcal/mol). Therefore, DDP4 is considered to be the good receptor for diabetes type 2 for
patients that were treated with bioactive compounds in E. hirta.
The absolute value for all of 29 bioactive compounds with five target proteins are
revealed in Graph 1 and Table 10. Top binding affinity includes: Friedelin, alpha-
amyrine, pelargonium-3,5-diglucoside, taraxerone, 1,3,4-tetra-O-galloyl-b-Dglucose with
the absolute binding affinity is higher than 7.5 kcal/mol.

10


Graph 1:The absolute value for all of 29 bioactive compounds with 5 target proteins
1 = 1,3,4-tetra-O-galloyl-b-Dglucose, 2 = 2,4_methylenecycloartenol, 3 = 3,4 diOgalloyquinic acid, 4=
afzelin, 5 = alpha-amyrin, 6 = beta-amyrin, 7 = beta-sitosterol, 8 = campesterol, 9 = cyaniding-3,5-
diglucoside, 10 = cycloartenol, 11 = friedelin, 12 = gallic acid, 13 = galloylquinic acid, 14 = ingenol
triacetate, 15 = kaempferol, 16 = leucocyanidin, 17 = L-rhamnose, 18 = myricitrin, 19 = tinyatoxin, 20 =
pelargonium-3,5-diglucoside, 21 = protocatechuic acid, 22 = quercetin, 23 = quercitol, 24 = quercitrin, 25 =
resiniferonol, 26 = rutin, 27 = stigmasterol, 28 = taraxerol, 29 = taraxerone.

2. Pharmacophore modeling
11 -HSD 1
High binding energy of the ligands to the receptor was explained clearly by interaction
analysis (Table 1). Five molecules (1,3,4-tetra-O-galloyl-b-Dglucose,
24_methylenecycloartenol, alpha amyrin, friedelin, taraxerone) were frequently within
hydrophobic interaction with residues Val 227A, Val 231A, Tyr 177A, Met 179A, Leu
126A and Hydrogen bond donor with residue Tyr 257A. Hydrophobic contacts at
position of methyl group which is non-polar whereas hydrogen bonds contact at steroidal
hydroxyl group of the protein. From this observation, six listed residues seemed to play
an essential role in catalytic activity of 11 -HSD 1. Moreover, 1,3,4-tetra-O-galloyl-b-
Dglucose, 24_methylenecycloartenol, alpha amyrin link to the receptor with a high
number of hydrogen bonds and hydrophobic interaction compared with friedelin and
taraxerone. 1,3,4-tetra-O-galloyl-b-Dglucose has all hydrogen bonds at residues Arg
269A, Glu 254A, Asn 270A, Lys 274A, Leu 266A. 24_methylenecycloartenol, alpha
4
6
8
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
11betaHSD1
A-glucosidase
PTP1B
PPARy
DDP4
11

amyrin, friedelin have all hydrophobic interactions with many identical interactions with
each other. Based on the analysis of ligands and target receptor interaction, the
conclusion of this process is all five selected compounds have good interaction with
receptor. However, 1,3,4-tetra-O-galloyl-b-Dglucose has the molecular weight higher
than 500kDa which will be excluded by Lipinski's rule of five
xl
.1,3,4-tetra-O-galloyl-b-
Dglucose needs to be modified to reduce molecular weight for using as a tempting
compounds for design diabetes type 2 treatment.
PTP1B
The binding affinity of the ligands to the PTP1B receptor is described in the Figure 5.
The top 5 molecules (24_methylenecycloartenol, alpha amyrin, cycloartenol, friedelin,
taraxerone), which show the highest binding affinity to PTP1B receptor (|Binding
affinity| > 7.5 kcal/mol), performed the hydrophobic interaction (yellow bubble) with
residues Thr763A, Tyr520A, Tyr546A, Phe682A, Leu588A, Hydrogen bond donor with
(green arrow) residue Gln762A, Phe862A, Lys541A and Hydrogen bond acceptor (red
arrow) with residue Gln521A. (Table 2.) Those nine named residues worked as emergent
residue in the activity of PTP1B.
Based on the analysis of ligands and target receptor interaction, the conclusion of this
process is all five selected compounds have good interaction with PTP1B receptor.
Therefore, these bioactive compounds are potential candidates for drug development to
diabetes type 2 treatments.

ALPHA-GLUCOSIDASE (MALTASE-GLUCOAMYLASE)
The molecular docking (Autodockvina)s result provided that there are 5 molecules:
1,3,4-tetra-O-galloyl-b-Dglucose, Alpha amyrin, Friedelin, Taraxerol, Taraxerone which
have highest affinities with the target protein 3L4T (Table 10). Moreover, hydrophobic is
considered as a main interaction between those molecules with Maltase-Glucoamylase
(3L4T) at PHE450A, TRP406A, THR204A, exclusive of 1,3,4-tetra-O-galloyl-b-
Dglucose (Table 5) when it links with 3L4T at ASP542A, ASP203A,
ARG202A,BJ11001A, TYR605A, GLN603A, TYR299A,ARG334A by hydrogen bond
donor or aceptor. It follows that hydrogen bonds keep molecules tightly bound to proteins
as well as enclosed hydrophobic interactions strengthen the binding of ligands and
proteins, therefore hydrogen bonds are important in forming a stable link between
molecules and target protein. In that case, 1,3,4-tetra-O-galloyl-b-Dglucose is best
candidate for drug development among all five selected molecules. However 1,3,4-tetra-
O-galloyl-b-Dglucose weights nearly 795 kDa which violates Lipinski's rule of five. As
consequence, 1,3,4-tetra-O-galloyl-b-Dglucose needs to be remodeled for the purpose of
12

decreasing molecule weight or be treated as a template for designing a new compounds in
diabetes type 2 treatment.
DPP4
These compounds - 1,3,4-tetra-O-galloyl-b-Dglucose, 24-methylenecycloartenol, beta -
amyrin, cycloartenol, taraxeronehave highest binding affinity. Hydrophobic interactions
were showed (in Ligand scout) at TYR547A, TRP627, TRP629A, especially at
TYR547A and hydrogen bond at VAL546A, ASP545A frequently. This analysis proved
that these residues play critical role in DPP4s catalytic activity. These interactions show
compounds ability to interact with receptor DPP4. After comparison (number of
hydrophobic interactions, H bonds), 1,3,4,6-tetra-O-galloyl-b-D-glucose showed strong
interaction to DPP4 receptor. Therefore, its considered to be a potential factor for drug
development.
PPAR
Five compounds that have highest binding affinity are 1,3,4-tetra-O-galloyl-b-Dglucose,
alpha -amyrin, beta - amyrin, cycloartenol, taraxerone are. Hydrophobic interactions were
showed (in Ligand scout) repeatedly at ILE262A, PHE287A . On the other hand,
hydrogen bondsincludeH bond acceptors: GLU259A, ARG280A, SER464A and H bond
donors: LYS275A, OH group, HIS466A. The pharmacoporesshowed that these residues
play critical role in PPARsregulation activity. These interactions show compounds
ability to interact with receptorPPAR. Thus, all the top binding compound showed a
stable and consistent interaction with PPAR. Therefore, its considered to be a potential
factor for drug development. One thing noticed is 1,3,4-tetra-O-galloyl-b-D-glucose
which has hydrogen bonds to keep molecules tightly bound to proteins.
13

Table 1: Binding modes of selective compounds with 11-HSD1
NAME OF
COMPOUND
IMAGE INTERACTION LIGAND DETAIL
24-
methylene
cycloartenol

Hydrophobic:Val231
A, Leu126A,
Met179A, Tyr177A
Formula: C
33
H
60
O
1

Molweight: 472.842

Size & Flexibility
Atoms/Bonds: 94 / 97
Rings: 4
Rotatable Bonds: 17
Aromatic Atoms: 0

Polarity & Chemical Features
cLogP: 9.521
TPSA: 20.230
Acceptors: 0
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0

14

Alpha-
amyrine

Hydrophobic:
Val231A, Ile230A,
Val227A, Leu126A,
Met179A, Tyr177A.
Formula: C
30
H
52
O
1

Molweight: 428.745
Size & Flexibility
Atoms/Bonds: 83 / 87
Rings: 5
Rotatable Bonds: 9
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 8.105
TPSA: 20.230
Acceptors: 0
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0

1,3,4-tetra-
O-galloyl-b-
D-glucose

H bond
donor:Asn270A,
Tyr257A, Leu266A.
H bond acceptor:
Glu254A, Arg269A,
Tyr257A, Lys274A.
Formula: C
34
H
36
O
22

Molweight: 796.640
Size & Flexibility
Atoms/Bonds: 92 / 96
Rings: 5
Rotatable Bonds: 31
Aromatic Atoms: 24
Polarity & Chemical Features
cLogP: -2.469
TPSA: 390.060
Acceptors: 5
Donors: 4
Neg. Ionizable: 0
Pos. Ionizable: 0

15

Friedelin

Hydrophobic:
Thr265A, Leu262A.
H bond
acceptor:Tyr257A
Formula: C
30
H
50
O
1

Molweight: 426.729
Size & Flexibility
Atoms/Bonds: 81 / 85
Rings: 5
Rotatable Bonds: 8
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 8.457
TPSA: 17.070
Acceptors: 1
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0

Taraxerone

Hydrophobic:
Tyr177A, Val175A,
Leu126A, Val227A,
Val231A
H donor bond:
Ser228A
Formula: C
30
H
52
O
1

Molweight: 428.745
Size & Flexibility
Atoms/Bonds: 83 / 87
Rings: 5
Rotatable Bonds: 9
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 8.249
TPSA: 20.230
Acceptors: 1
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0


16

Table2: Binding modes of selective compounds with PTP1B
NAME OF
COMPOUND
IMAGE INTERACTION LIGAND DETAIL
24-methylene
cycloartenol

Hydrophobic:
Phe682A, Thr763A,
Tyr520A

H bond donor:
Gln762A
Formula: C
30
H
50
O
1

Molweight: 426.729

Size & Flexibility
Atoms/Bonds: 81 / 84
Rings: 4
Rotatable Bonds: 13
Aromatic Atoms: 0

Polarity & Chemical Features
cLogP: 8.818
TPSA: 20.230
Acceptors: 0
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0
17

Alpha-amyrine

Hydrophobic:
Phe682A, Thr763A

H bond donor:
Phe682A
Formula: C
29
H
48
O
1

Molweight: 412.702

Size & Flexibility
Atoms/Bonds: 78 / 82
Rings: 5
Rotatable Bonds: 8
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 7.635
TPSA: 20.230
Acceptors: 0
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0
Cycloartenol

Hydrophobic:
Tyr546A, Leu588A

H bond donor:
Lys541A
Formula: C
30
H
54
O
1

Molweight: 430.761

Size & Flexibility
Atoms/Bonds: 85 / 88
Rings: 4
Rotatable Bonds: 14
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 8.495
TPSA: 20.230
Acceptors: 0
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0
18

Friedelin

Hydrophobic: Phe682A Formula: C
30
H
50
O
1

Molweight: 426.729

Size & Flexibility
Atoms/Bonds: 81 / 85
Rings: 5
Rotatable Bonds: 8
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 8.457
TPSA: 17.070
Acceptors: 0
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0
Taraxerone

Hydrophobic:
Phe682A, Tyr520A,
Thr763A

H bond acceptor:
Gln521A
Formula: C
30
H
52
O
1

Molweight: 428.745

Size & Flexibility
Atoms/Bonds: 83 / 87
Rings: 5
Rotatable Bonds: 9
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 8.249
TPSA: 20.230
Acceptors: 1
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0


19

Table4: Binding modes of selective compounds with DPP4
NAME OF
COMPOUND
IMAGE INTERACTION LIGAND DETAIL
1,3,4,6-tetra-O-
galloyl-b-D-
glucose

H bond acceptors:
GLN553A, TYR547A,
LYS122A, ARG125A,
ASN710A, TRP629A

H bond donors:
TYR547A, VAL546A,
ASP545A, TRP629A,
HIS740A, TRP124A,
ASP709A

Formula: C
34
H
36
O
22

Molweight: 796.640

Size & Flexibility
Atoms/Bonds: 92 / 96
Rings: 5
Rotatable Bonds: 30
Aromatic Atoms: 24
Polarity & Chemical Features
cLogP: -2.470
TPSA: 390.060
Acceptors: 7
Donors: 11
Neg. Ionizable: 0
Pos. Ionizable: 0
24-
methylenecyclo
artenol

Hydrophobic
interactions :
TYR547A
TRP627A
TRP629A

H bond donor:
VAL546A
Formula: C
29
H
52
O
1

Molweight: 416.734

Size & Flexibility
Atoms/Bonds: 82 / 85
Rings: 4
Rotatable Bonds: 13
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 7.961
TPSA: 20.230
Acceptors: 0
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0
20

beta-amyrin

H bond donor :
TYR752A
Formula: C
22
H
36
O
1

Molweight: 316.529

Size & Flexibility
Atoms/Bonds: 59 / 63
Rings: 5
Rotatable Bonds: 1
Aromatic Atoms: 0

Polarity & Chemical Features
cLogP: 5.416
TPSA: 20.230
Acceptors: 0
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0
Cycloartenol

Hydrophobic
interactions:
TYR547A
TRP627A
TRP629A
Formula: C
29
H
52
O
1

Molweight: 416.734

Size & Flexibility
Atoms/Bonds: 82 / 85
Rings: 4
Rotatable Bonds: 13
Aromatic Atoms: 0

Polarity & Chemical Features
cLogP: 8.105
TPSA: 20.230
Acceptors: 0
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0
21



Taraxerone

Hydrophobic
interaction:
TYR547A

H bond acceptors:
D3C801A
ARG560A
LYS554A
ASN562A

H bond donors:
VAL546A
LYS554A
ASP545A
Formula: C
26
H
30
O
16

Molweight: 598.510

Size & Flexibility
Atoms/Bonds: 72 / 76
Rings: 5
Rotatable Bonds: 17
Aromatic Atoms: 16

Polarity & Chemical Features
cLogP: -1.774
TPSA: 272.590
Acceptors: 5
Donors: 3
Neg. Ionizable: 0
Pos. Ionizable: 0
22

Table5: Binding modes of selective compounds with Alpha-Glucosidase
NAME OF
COMPOUND
IMAGE INTERACTION LIGAND DETAIL
Alpha-amyrin

Hydrophobic
interaction: PHE450A,
THR204A, TRP406A
Formula: C
30
H
52
O
1

Molweight: 428.745

Size & Flexibility
Atoms/Bonds: 83 / 87
Rings: 5
Rotatable Bonds: 9
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 8.105
TPSA: 20.230
Acceptors: 0
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0

1, 3, 4-Tetra-O-
galloyl-b-
Dglucose.

Hydrogen bond donor:
TYR605A, ASP203A,
ASP542A, GLN603A,
TYR299A, BJ110011A

Hydrogen bond
acceptor:
TYR605A, ARG202A,
GLN603A, TYR299A,
BJ11001A, ARG334A
Formula: C
34
H
34
O
22

Molweight: 794.624

Size & Flexibility
Atoms/Bonds: 90 / 95
Rings: 6
Rotatable Bonds: 30
Aromatic Atoms: 24
Polarity & Chemical Features
cLogP: -2.716
TPSA: 390.060
Acceptors: 22
Donors: 17
Neg. Ionizable: 0
Pos. Ionizable: 0
23

Taraxerol

Hydrophobic
interaction: TRP406A,
PHE450A
Formula: C
30
H
52
O
1

Molweight: 428.745

Size & Flexibility
Atoms/Bonds: 83 / 87
Rings: 5
Rotatable Bonds: 9
Aromatic Atoms: 0

Polarity & Chemical Features
cLogP: 8.249
TPSA: 20.230
Acceptors: 0
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0
Friedelin

Hydrophobic
interaction: PHE450A
Formula: C
30
H
50
O
1

Molweight: 426.729

Size & Flexibility
Atoms/Bonds: 81 / 85
Rings: 5
Rotatable Bonds: 8
Aromatic Atoms: 0

Polarity & Chemical Features
cLogP: 8.457
TPSA: 17.070
Acceptors: 0
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0
24

Taraxerone

Hydrophobic
interaction: PHE450A,
TRP406A
Formula: C
30
H
52
O
1

Molweight: 428.745

Size & Flexibility
Atoms/Bonds: 83 / 87
Rings: 5
Rotatable Bonds: 9
Aromatic Atoms: 0

Polarity & Chemical Features
cLogP: 8.249
TPSA: 20.230
Acceptors: 0
Donors: 0
Neg. Ionizable: 0
Pos. Ionizable: 0

25


Table3: Binding modes of selective compounds with PPAR
NAME OF
COMPOUND
IMAGE INTERACTION LIGAND DETAIL
1,3,4,6-tetra-O-
galloyl-b-D-
glucose

H bond acceptor:
GLU259A, ARG280A,
SER464A

H bond donor:
LYS275A, OH group,
HIS466A
Formula: C
34
H
34
O
22

Molweight: 794.624

Size & Flexibility
Atoms/Bonds: 90 / 95
Rings: 6
Rotatable Bonds: 30
Aromatic Atoms: 24
Polarity & Chemical Features
cLogP: -2.716
TPSA: 390.060
Acceptors: 22
Donors: 17
Neg. Ionizable: 0
Pos. Ionizable: 0
26

Alpha-amyrine

Hydrophobic: ILE262A,
PHE287A
Formula: C
30
H
52
O
1

Molweight: 428.745

Size & Flexibility
Atoms/Bonds: 83 / 87
Rings: 5
Rotatable Bonds: 9
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 8.105
TPSA: 20.230
Acceptors: 1
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0
beta-amyrin

Hydrophobic: ILE262A,
PHE287A
Formula: C
30
H
50
O
1

Molweight: 426.729

Size & Flexibility
Atoms/Bonds: 81 / 86
Rings: 6
Rotatable Bonds: 8
Aromatic Atoms: 0

Polarity & Chemical Features
cLogP: 7.859
TPSA: 20.230
Acceptors: 1
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0
27

Friedelin

Hydrophobic: ILE262A,
PHE287A
Formula: C
30
H
52
O
1

Molweight: 428.745

Size & Flexibility
Atoms/Bonds: 83 / 87
Rings: 5
Rotatable Bonds: 9
Aromatic Atoms: 0
Polarity & Chemical Features
cLogP: 8.249

TPSA: 20.230
Acceptors: 1
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0
Taraxerone

Hydrophobic: ILE262A Formula: C
30
H
50
O
1

Molweight: 426.729

Size & Flexibility
Atoms/Bonds: 81 / 86
Rings: 6
Rotatable Bonds: 8
Aromatic Atoms: 0

Polarity & Chemical Features
cLogP: 7.859
TPSA: 20.230
Acceptors: 1
Donors: 1
Neg. Ionizable: 0
Pos. Ionizable: 0
28

Conclusion
Docking simulation and pharmacophore analysis of 28 bioactive compounds extracted from
Euphorbia hirta has successfully performed the binding modes and gave strong evidence of
molecular interactions to all five receptors (11HSD1, PTP1B, -glucosidase, PPARy,
DDP4)which directly related to diabetes type 2. In detail, this study showed that flavonoid
and terpenes families including 2,4_methylenecycloartenol, 1,3,4-tetra-O-galloyl-b-
Dglucose, friedelin, amyrine, amyrine and taraxerol, taraxeronehave high binding
affinity to all 5 interested receptors.These binding results consist of a high number of
hydrogen bond and hydrophobic interactions at some similar specific position of each
receptor (showed from table 1 to 5). Moreover, the terpenes families (alpha-amyrine, beta-
amyrine, friedelin, taraxerol, taraxerone, cycloartenol) and the 3,4 di-O-galloyquinicacid of
tannin family show high binding affinity compared to others. However, based on the
Lipinski's rule of five, themolecular weight of 1,3,4-tetra-O-galloyl-b-Dglucose is greater
than 500Da (~700Da) so that a modification should be applied to decrease molecule weight
or be treated as a template for designing a new compounds in diabetes type 2 treatment.
Since normal docking process (AutodockVina was used in this research) docks thousands of
compounds from free chemical databases which are in freezes, it against the rigid structure
of receptors inaccurate binding affinity between drugs and proteins. Therefore, Molecular
dynamic simulation (MD simulation) is highly recommended to use for further research in
order to reach the optimum and accurate results of hydrophobic interaction and H bond
between ligands and receptors.







29

Appendix
Table 6. 3D structure of 5 target proteins from NCBI
Species
Protein PDB
ID
3D structure Method
Resolution
(A
0
)
References
Human
(Homos
apien)
11 HSD 1XU7

X-ray
diffraction
1.80 Hosfield
et al, 2005
Human
(Homos
apien)
PTP1B 3CWE

X-ray
diffraction
1.60

Bioorg.
Med.
Chem.Lett
(2008)
Human
(Homos
apien)
PPAR- 4A4V

X-ray
diffraction
2.00 Journal:
(2013)
J.Med.Che
m
30

Human
(Homos
apien)
DPP4 4J3J

X-ray
diffraction
3.20 Chemedch
em ,2013
Human
(Homos
apien)
Alpha-
glucosida
se
3L4T

X-ray
diffraction
1.90 Sim,, L
2010

31

Table 7: 2D structures of 28 bioactive compounds candidates suggested from
Chemspider.
1,3,4,6-tetra-O-galloyl-
b-Dglucose

24_methylenecycloarte
nol

3_4 diOgalloyquinic
acid

Afzelin

Alpha amyrin

Beta amyrin

Beta sitosterol

Campesterol

cyanidin 3,5-
diglucoside

Cycloarterol

Friedelin

Gallic acid

3,4,5-tri-O-
galloylquinic acid

Ingenoltriacetate

Kaempferol

Leucocyanidin

L_Rhamnose

Myricitrin

Pelargonium 3_5_
diglucoside
32




Protocatechuic acid

Quercetin

Quercetol

Quercetrin

Resiniferonol

Rutin

Stigmasterol

Taraxerone

Taraxerol




33

Table 8: 3D structure of 28 bioactive compounds building from GaussView
1,3,4-tetra-O-galloyl-b-D-
glucose

24_methylenecycloartenol


3_4 diOgalloyquinic acid


Afzelin


Alpha-amyrin

Beta-amyrin

Beta sitosterol

Campesterol

cyanidin 3,5-diglucoside

Cycloarterol

Friedelin

Gallic acid

Galloylquinic acid

Ingenol triacetate

Kaempferol

Leucocyanidin

34

Potocatechuic acid

Quercetin

Quercetol

Resiniferonol

Rutin

Stigmasterol

Taraxerone



35

Table 9: Position of Grid box center of five target proteins
Protein molecule Protein code
X,Y,Z coordination (Angstroms)
X Y Z
11b HSD1 1XU7 -64.809 -74.388 -13.559
PTP1B 3CWE 23.677 22.779 1.835
PPAR 4A4V -16.666 21.568 -47.791
DPP4 4J3J 5.418 15.755 -23.517
-glucosidase 3L4T 44.964 90.541 34.242

Table 10: Absolute binding energy (kcal/mol) of bioactive compounds in E.hirta
to five receptors.

11betaHSD1 A-glucosidase PTP1B PPARy DDP4
1,3,4-tetra-O-galloyl-b-Dglucose 8.3 7.9
6.9
7.9
9.4
24_methylenecycloartenol 6.4 6.8
6.4
7.4
6.9
3_4 diOgalloyquinic acid 6.9 7.3
7.5
7.5
8
Afzelin 6.5 7.6
7.1
6.9
8
Alpha amyrin 8.4 7.7
8
10
8.6
Beta amyrin 7.4 6.6
7.8
8.6
7.3
Beta sitosterol 6.5 6.4
7
7.3
8.7
Campesterol 7.6 6.8
7
7.2
9.9
cyanidin 3,5-diglucoside 6.4 7.2
7.5
7.9
8.3
cycloarterol 6.9 7.1
7.8
7.9
8
friedelin 8 7.7
8.3
8.8
9.7
gallic acid 5.5 6.4
5.2
5.2
5.7
galloylquinic acid 7.5 7
6.8
7.4
8.6
Ingenol triacetat 6.5 6.2
7.3
7
7.1
kaempferol 7.4 6.2
7
7
7.3
Leucocyanidin 7.5 6.4
6.9
6.9
6.9
L_Rhamnose 5.3 5.5
4.5
5.2
5
myricitrin 7.4 7.8
7.1
7
7.9
tinyatoxin 7.1




pelargonium 3_5_ diglucoside 8.3 7.6
7.9
8.3
9
protocatechuic acid 5.3 6.5
5
5.2
5.8
Quercetin 7.6 6.7
6.8
7.3
7.4
Quercitol 5.3 5.6
4.7
4.9
5.1
Quercitrin 6 7.9
6.9
7.4
7.7
36

Resiniferono 6.1 6
7.2
6.5
6.7
rutin 7.4 7.4
7.4
7.5
8.1
Stigmasterol 6.4 6.8
7
7.7
8.4
taraxerol 8.4 7.6
7.5
8.8
8.5
taraxerone 8.5 7.6
8.6
8.9
9.9

Table 11. Function of bioactive compounds in Euphorbia hirta
Family Bioactive
compounds
Biological
functions
References
Flavonoids Afzelin, quercetin,
quercitrin, quercitol,
rhamnose, rutin,
leucocyanidin,
myricitrin, cyanidin
3,5- O-diglucoside,
kaemferon,
pelargonium 3,5-
diglucoside
Antioxidation, Anti-
allergy,
antibacterial,
molluscicidal, anti-
diarrheal activity.
Mei Fen Shih1 and
Jong Yuh Cherng2
Taiwan 2012; Quy
Trinh, Ly Le, 2013.
Tannin gallic acid, 3,4-di-O-
galloyl-quinic acid,
3,4,5-tri-O-
galloylquinic acid,
1,2,3,4,6
tetraOgalloyl-b-D-
glucose
Antioxidation, anti-
inflammatory
activity.
Sunil Kumar, Rashmi
Malhotra, and Dinesh
Kumar 2010, Yoshida
et al, 1990; Chen
1991.
Triterpenes and
phytosterols
2,4-
methylenecycloartenol,
betasitosterol,
campesterol, -
stigmasterol, ingenol
triacetate,
resiniferonol, alpha-
amyrine, beta-amyrine,
friedelin, taraxerol,
taraxerone,
cycloartenol,
tinyatoxin,
protocatechuic acid
Anti-inflammatory,
antiplasmodial
activity.
Mei Fen Shih1 and
Jong Yuh Cherng,
2012; Sandeep
b.Patil, Nilofar
S.Naikwade,
Shandrakant
S.Magdum, 2009;
Quy Trinh, Ly Le,
2013.

37

Reference

i
Curb JD, Rodriguez BL, Burchfiel CM, Abbott RD, Chiu D, Yano K: Sudden death,
impaired glucose tolerance, and diabetes in Japanese American men. Circulation
91:2591-2595,1995
ii
National Diabetes Data Group: Classification and Diagnosis of Diabetes Mellitus and
Other Categories of Glucose Intolerance.
doi:10.2337/diab.28.12.1039Diabetes December 1979vol. 28 no. 12 1039-1057
iii
Sanford burnham medical research institute: diabetes type 2
iv
Mei Fen Shih and Jong YuhCherng: Potential Applications of Euphorbiahirta in
Pharmacology. Department of Pharmacy, Chia-Nan University of Pharmacy & Science,
Tainan. Department of Chemistry & Biochemistry, National Chung Cheng University,
Chia-Yi, Taiwan
v
Liu, Y. et al. 2007. Antimalarial Flavonol Glycosides from Euphorbia hirta.
PharmaceuticalBiology. 45:278-81. Abstract
vi
Tona, L., et al. 1999. Antimalarial activity of 20 crude extracts from nine African
medicinal plants used in Kinshasa, Congo. Journal of Ethnopharmacology 68:193-203
vii
Galvez, J. 1993. Antidiarrhoeic activity of Euphorbia hirta extract and isolation of an
active flavonoid constituent.Planta Med. 59: 333-6. Abstract
viii
Galvez, J. 1993. Antidiarrhoeic activity of quercitrin in mice and rats. J. Pharm.
Pharmacol. 45:157-9. Abstract
ix
Lanhers, M.C. et al. 1991.Analgesic, antipyretic and anti-inflammatory properties of
Euphorbia hirta.Planta Med. 57(3):225-31 Abstract
x
Sudhakar, M., et al., 2006, Antimicrobial activity of Caesalpiniapulcherrima, Euphorbia
hirta and Asystasiagangeticum.Fitoterapia, 77: 37880
xi
Vijaya K., Ananthan S., &Nalini R. 1995.Antibacterial effect of theaflavin, polyphenon
60 (Camellia sinensis) and Euphorbia hirta on Shigella spp. -- a cell culture study.J.
Ethanopharmacol, 49(2): 115-8
xii
Johnson, P.B. et al. 1999. Euphorbia hirta leaf extracts increase urine output and
electrolytes in rats. J. Ethnopharmacol. 65(1):63-9.
xiii
Singh, G. D., et al. 2006. Inhibition of early and late phase allergic reactions by
Euphorbia hirta L. PhytotherapyRes.20(4): 316-21.
xiv
Martinez-Vazquez, M et al. 1999. Anti-inflammatory Active Compounds from the n-
Hexane Extract of Euphorbia hirta. Revista de la SociedadQuimica de Mxico.
43(3,4):103-5
38


xv
Goldie Uppal, Vijay Nigam, Anil Kumar: Antidiabetic activity of ethanolic extract of
Euphorbia hirta Linn SRET College of Pharmacy, Barsar, Hamirpur, India
xvi
Zhang ZY, Lee SY - PTP1B inhibitors as potential therapeutics in the treatment of type
2 diabetes and obesity - 2003 Feb;12(2):223-33.
xvii
Antihyperglycemic, antihyperlipidemic and antioxidant activities of Euphorbia hirta
extract - Sunil Kumar, RashmiMalhotra, Dinesh Kumar. International Research Journal
of Pharmacy (01/2010)
xviii
http://www.gaussian.com/g_prod/gv5.htm
xix
Review on targeted proteins for Diabetes Drug Desgin Trang D. Ngoc Nguyen, LyT.
Le.
xx
New mechanisms of glucocorticoid-induced insulin resistance
xxi
Role of glucocorticoids in the physiopathology of excessive fat deposition and insulin
resistance - C Asensio1,2, P Muzzin2 and F Rohner-Jeanrenaud
xxii
Tyrosine-protein phosphatase non-receptor type 1 also known as protein-tyrosine
phosphatase 1B (PTP1B) is anenzyme that is the founding member of the protein
tyrosine phosphatase (PTP) family. In humans it is encoded by thePTPN1 gene.
xxiii
Combs AP (March 2010). "Recent advances in the discovery of competitive protein
tyrosine phosphatase 1B inhibitors for the treatment of diabetes, obesity, and cancer". J.
Med. Chem. 53 (6): 233344.
xxiv
Tonks, NK (2003 Jul 3). "PTP1B: from the sidelines to the front lines!". FEBS
letters 546 (1): 1408.
xxv
Greene ME et al (1995). "Isolation of the human peroxisome proliferator activated
receptor gamma cDNA: expression in hematopoietic cells and chromosomal
mapping". Gene Expr. 4 (45): 28199
xxvi
Elbrecht A et al (July 1996). "Molecular cloning, expression and characterization of
human peroxisome proliferator activated receptors gamma 1 and gamma
2". Biochem.Biophys. Res. Commun. 224 (2): 4317
xxvii
Michalik L et al. (December 2006). "International Union of Pharmacology.LXI.
Peroxisome proliferator-activated receptors". Pharmacol. Rev. 58 (4): 72641.
xxviii
Fajas L et al (July 1997)."The organization, promoter analysis, and expression of the
human PPARgamma gene". J. Biol. Chem. 272 (30): 1877989.
xxix
Atanasov AG et al "Honokiol: a non-adipogenicPPAR agonist from
nature".Biochim.Biophys.Acta 1830 (10):48139.
xxx
P.Lalitha* and Shubashini K Sripathi -In silico ligand receptor docking of few
cyclitols for type II diabetes using hex-Department of Chemistry, Avinashilingam
Deemed University for Women, Coimbatore-641043.
39


xxxi
Alpha-Glucosidases at the US National Library of Medicine Medical Subject Headings
(MeSH)
xxxii
http://www.rcsb.org/pdb/explore/explore.do?structureId=3L4T , abstraction
xxxiii
Mohammad et al., 2010, Sandeep et al.; 2011
xxxiv
REVIEW ON PHYTOCHEMISTRY AND PHARMACOLOGICAL ASPECTS OF EUPHORBIA HIRTA LINN.
SANDEEP B. PATIL*, MRS. NILOFAR S. NAIKWADE, CHANDRAKANT S. MAGDUM
xxxv
Potential Applications of Euphorbia hirta in Pharmacology Mei Fen Shih1 and Jong Yuh
Cherng2*
xxxvi
Downloaded at http://vina.scripps.edu/
xxxvii
Downloaded at
http://www.rsc.org/chemistryworld/Issues/2006/September/LigandScout.asp
xxxviii
Wolber G, Langer T. LigandScout: 3-D pharmacophore derived from protein bound
ligans and their use as virtual screening filters. J Chem Info Model. 2005: 45:160-169
xxxix
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ Experimental and computational
approaches to estimate solubility and permeability in drug discovery and development
settings. Adv Drug Delivery Rev. 1997; 23:325.
xl
Lipinski's rule of five http://www.pharmainformatic.com/html/rule_of_5.html