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Molecular Docking and ADME studies of Natural

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Current Computer-Aided Drug Design, 2013, 9, 360-370

360

Molecular Docking and ADME Studies of Natural Compounds of

Agarwood Oil for Topical Anti-Inflammatory Activity
Dharmendra K. Yadav1, Vipin Mudgal2, Jyoti Agrawal2, Anil K. Maurya2,
Dnyaneshwar U. Bawankule*,2, Chandan S. Chanotiya3, Feroz Khan*,1 and Sanjog T. Thul4,
1
4

Metabolic and Structural Biology Department, 2Molecular Bioprospection Department, 3Analytical Chemistry Division,
Plant Biotechnology Department, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow - 226 015, India
Abstract: Aquilaria agallocha Roxb. family, Thymelaeaceae, is an evergreen plant of South-East Asia, commonly
described as aloe wood or agarwood. Traditionally, the bark, root and heartwood are used for their medicinal properties as
a folk medicine for hundreds of years. Chemical analyses revealed that the bulk of the oil is constituted by agarospirol
(12.5%), jinkoh-eremol (11.8%) and hinesol (8.9%) as major contributor. In the present work, a QSAR model for antiinflammatory activity of 10-epi--Eudesmol, jinkoh-eremol, agarospirol and other compounds has been developed by
multiple linear regression method. The r2 and rCV2 of a model were 0.89 and 0.81 respectively. In silico molecular
docking study suggests that compound 10-epi--Eudesmol, jinkoh-eremol and agarospirol are preferentially more active
than other identified compounds with strong binding affinity to major anti-inflammatory and immunomodulatory
receptors. The oil displayed a significant and dose dependent reduction of 12-O-tetradecanoylphorobol-13 acetate (TPA)induced ear edema and MDA activity when compared with vehicle treated mice. Pro-inflammatory cytokines (IL-1, IL-6
and TNF-) were also reduced significantly in a dose dependent manner in all the TPA treated groups as compared to
control. The present study indicates that agarwood oil significantly reduced the skin thickness, ear weight, oxidative stress
and pro-inflammatory cytokines production in TPA-induced mouse ear inflammation model and contributed towards
validation of its traditional use to treat inflammation related ailments.

Keywords: Aquilaria, GC/MS analysis, agarospirol, in silico molecular modelling, ear oedema, docking, QSAR, ADME/T.
INTRODUCTION
Agar, a valuable aromatic oleoresinous deposit found in
the stems of Aquilaria agallocha Roxb. (syn. Acquilaria
malaccensis Lamk., family: Thymelaeaceae) is available in
North-East region of India, Bangladesh, and other parts of
South East Asia [1]. It is also known as Agarwood, Eagle
wood or Aloewood, in several countries. It has been widely
used in the treatment of various kinds of pain, cough and
anaphylaxis for hundreds of years in South-East Asia [2, 3].
The different parts of plant extract were found to possess
analgesic, anti-inflammatory and hypersensitivity activities
[4, 5], which support its folkloric use for some diseases
related with painful and inflammatory conditions such as
trauma.
The essential oil of the tree has been considered to be a
pathological product produced by fungal invasion of the host
[6, 7]. Its heartwood is fine, black or brown in color and
*Address correspondence to these authors at the (Feroz Khan) Department of
Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and
Aromatic Plants, P.O.-CIMAP, Kukrail Picnic Spot Road, Lucknow - 226
015 (U.P.), India; Tel: +91 +91 522 2718668; Fax: +91 522 2342666;
E-mail: [Link]@[Link] and
(Dnyaneshwar U. Bawankule) Department of Molecular Bioprospection,
CSIR-Central Institute of Medicinal and Aromatic Plants, P.O.-CIMAP,
Kukrail Picnic Spot Road, Lucknow-226 015 (U.P.), India; Tel: +91 522
2717529; Fax: +91 522 2342666; E-mail: [Link]@[Link]

Present address: Environmental Biotechnology Division, CSIR-National

Environmental Engineering Research Institute, Nagpur 440020, India

1875-6697/13 $58.00+.00

fragrant. Phytochemical analyses of commercial agarwood

and its oil have revealed sesquiterpenes [8-10] as their
constituents. Meier et al. (2003) reported agarospirol
(12.1%) and jinkoh-eremol (10.0%) from Aquilaria
agallocha from Assam, India. Naf et al. (1992, 1995)
described -agarofuran, vetispira-2(11), 6(14)-dien-7-ol,
dihydrokaranone and valerianol as major constituents of
Indian agarwood oil [11-13]. Natural compounds of
agarwood oil are also among such biologically active
compounds which have shown promising anti-inflammatory
and immunomodulatory activity.
It was noticed that too many compounds were terminated
in clinical development because of unsatisfactory
pharmacokinetics or in silico ADME studies, therefore
computational tools were needed to assess the relationship
between chemical structure and ADME properties [14, 15].
Chen and Ung (2002) demonstrated the results of
computational approach on three Chinese medicinal plants
compounds (allicin, catechin and camptotecin) that show
60% of computer-identified potential therapeutic protein
targets and 27% of computer-identified potential toxicity
targets have been implicated or confirmed by experiments.
The skin plays a central role in host immunological
defences; however, the regulation of these defense
mechanisms is also crucial, as inappropriate or misdirected
immune activity is implicated in the pathogenesis of a large
variety of acquired inflammatory skin disorders [16]. High
levels of inflammatory cytokines and reactive oxygen
species are proposed contributors to the pathophysiologic
mechanisms associated with various inflammatory
2013 Bentham Science Publishers

Molecular Docking and ADME Studies of Natural Compounds

dermatoses [17]. It is widely recognized that the secretions

of cytokines by keratinocytes in response to injury,
particularly tumor necrosis factor alpha (TNF-) and
interleukin (IL-1), are key mediators of the cutaneous
inflammatory response. Although the currently used
steroidal anti-inflammatory drugs (SAID) and non-steroidal
anti-inflammatory drugs (NSAID) treat acute inflammatory
disorders, these conventional drugs have not been successful
to cure chronic inflammatory disorders. Since the critical
etiology and exacerbating mechanisms are not completely
understood, it is difficult to develop a magic bullet for
chronic inflammatory disorders [18]. Therefore, there is a
need for new and safe anti-inflammatory agents derived from
plant origin.
Topical application of TPA has been used to screen for
topically applied anti-inflammatory steroids and nonsteroid
agents and events of the inflammatory processes such as
oedema, cell infiltration, and proliferation. Present study
highlighted the validation of in silico based prediction of
biologically active compounds of the agarwood oil for
topical anti-inflammatory and immunomodulatory properties
in the TPA-induced mouse ear inflammation.
MATERIALS AND METHODS
Materials
Agarwood oil samples were procured from local market
of Assam, India. Quality of the oils was ascertained to be
more than 98% pure and identification of the active chemical
compounds was carried out using GC/GC-MS, NMR
analysis. All other chemicals and reagents were of the
highest grade available.
Capillary GC and GC/MS
For capillary GC, a PerkinElmer AutoSystem XL gas
chromatograph was used fitted with an Equity-5 column (60
m x 0.32 mm i.d., film thickness 0.25
m; Supelco
Bellefonte, PA, USA). The oven column temperature ranged
from 70250 C, programmed at 3 C/min, with initial and
final hold time of 2 min, using H2 as carrier gas at 10 psi
constant pressure, a split ratio of 1:30, an injection size of
0.03
L neat, and injector and detector (FID) temperatures
were 250 C and 280 C, respectively. GC/MS utilized a
PerkinElmer AutoSystem XL GC interfaced with a
Turbomass Quadrupole mass spectrometer fitted with an
Equity-5 fused silica capillary column (60 m x 0.32 mm i.d.,
film thickness 0.25
m; Supelco Bellefonte, PA, USA). The
oven temperature program was the same as described in
capillary GC; injector, transfer line and source temperatures
were 250 C; injection size 0.03
L neat; split ratio 1:30;
carrier gas He at 10 psi constant pressure; ionization energy
70 eV; mass scan range 40-450 amu. Characterization was
achieved on the basis of retention time, relative retention
index using a homologous series of n-alkanes (C8-C25
hydrocarbons, Polyscience Corp. Niles IL), coinjection with
standards in GC-FID capillary column (Aldrich and Fluka),
mass spectra library search (NIST/EPA/NIH version 2.1 and
Wiley registry of mass spectral data 7th edition) and by
comparing with the mass spectral literature data [19]. The

Current Computer-Aided Drug Design, 2013, Vol. 9, No. 3

361

relative amounts of individual components were calculated

based on GC peak areas without using correction factors.
NMR Experiment
For NMR experiment, Bruker Avance-300 was utilized
for 13CNMR experiments with tetramethylsilane (TMS) as
internal standard. About 20 mg of the essential oil was
dissolved in CDCl3 and spectral data are reported to support
the GC-MS analysis.
Geometry Cleaning, Energy Optimization and Parameterization of Compounds
Drawing and geometry cleaning of the series of 33
compounds were performed through ChemBioDraw-Ultrav12.0 ([Link] The 2D structures
were transformed into 3D structures using converter module
of ChemBioDraw. The 3D structures were then subjected to
energy minimization, which was performed in two steps, in
the first step energy minimized using molecular mechanics-2
(MM2) until the root mean square (RMS) gradient value
became smaller than 0.100 kcal/mol and then in second
step minimized MM2 (dynamics) compounds were subjected
to re-optimization through MOPAC (Molecular Orbital
Package) method until the RMS gradient attained a value
smaller than 0.0001 kcal/mol .
Structure Retrieval and Molecular Docking Parameters
The chemical structures of small molecules and drugs
were retrieved from PubChem compound database (NCBI)
([Link]
Crystallographic
structure of IL-1, IL-6, TNF- and COX-I protein was
taken from PDB database ([Link] The valency
and hydrogen bonding of compounds and target protein were
checked through Scigress Explorer v.[Link] (previously,
CAChe) software (Fujitsu Ltd., Poland; [Link]
Hydrogen atoms were added to protein to achieve the correct
ionization and tautomeric states of amino acid residues such
as His, Asp, Ser, and Glu. Molecular docking of drugs and
the agarospirol, jinkoh-eremol and hinesol against antiinflammatory target were achieved by using FastDock
Manager and FastDock Compute engines of Scigress
Explorer software. To perform the automated docking of
compounds into the protein active sites, we used a genetic
algorithm with a fast and simplified potential of mean force
(PMF) scoring scheme [20-22]. PMF uses atom types that
are similar to the empirical force fields used in mechanics
and dynamics. To adapt individuals to the surrounding
environment, a minimization is performed by the FastDock
engine of Scigress Explorer, which uses a Lamarkian genetic
algorithm (LGA). The best fits are sustained by analyzing
the PMF scores of all chromosomes and assigning more
reproductive opportunities to those with lower scores. This
process was repeated for 3000 generations with 500
individuals and 100,000 energy evaluations. Other
parameters were left as their default values. Structure-based
screening involves docking of compounds into protein and
then applying a PMF scoring function to estimate the
likelihood that the compound will bind to the protein with
high affinity or not [23-25].

362 Current Computer-Aided Drug Design, 2013, Vol. 9, No. 3

Parameters for QSAR Model Development

Initially, a total of 33 anti-inflammatory compounds/
drugs with reported anti-inflammatory activities were used
as training data set compounds while developing the QSAR
model (Supplementary Table 1). The anti-inflammatory
activity was in lethal dose concentration (LD50 in mg/kg)
form. Total 52 chemical descriptors (physico-chemical
properties) were calculated for each compounds (Supplementary Tables 2 and 3). While selecting the best subset of
chemical descriptors, highly correlated descriptors were
excluded by using correlation matrix approach or covariance analysis (Supplementary Table 4). Finally 10
chemical descriptors were used for QSAR modeling
(Supplementary Table 4). Various descriptors like electronic,
steric, and thermodynamic were calculated by the Scigress
Explorer software. On the basis of small molecules structural
similarity, compounds selection was performed so that to
ensure diverse training data set. Finally, robust model was
developed by using forward stepwise MLR statistical
method. The resulting QSAR model showed a high
regression coefficient. The models were evaluated for the
robustness of their predictions through cross-validation
coefficient. Leave-one-out (LOO) method was used for
validating QSAR models [20-25]. The best model was
selected on the basis of various statistical parameters such as
correlation coefficient (r), square of correlation coefficient
(r2), quality of the each model was estimated from the crossvalidated squared correlation coefficient (rCV2), which
confirmed the robustness and applicability of QSAR
equation (Table 1).
Screening Through Pharmacokinetic Properties
As it is well known that most of drugs in discovery
process fail to cross clinical trials because of poor PK. The
properties of PK depend on chemical descriptors of
compounds such as absorption, distribution, metabolism,
excretion, and toxicity (ADMET) which are important
descriptors for human therapeutic use of any compounds.
Some descriptors correlate well with PK properties e.g.,
primary determinant of fractional absorption referred as
polar surface area (PSA) and low molecular weight (MW;
for oral absorption). The compound distribution in human
depends on factors e.g., bloodbrain barrier (log BB),
Table 1.

Yadav et al.

permeability (apparent Caco-2 and MDCK permeability,

logKp for skin permeability), the volume of distribution and
plasma protein binding referred by logKhsa for serum
protein binding. These ADMET descriptors were calculated
and checked for compliance with their standard ranges. The
octanolwater partition coefficient (logP) has been
implicated in logBB penetration and permeability studies.
Process of excreting the compound from human body
depends on MW and logP. Likewise, rapid renal clearance is
associated with small and hydrophilic compounds.
Metabolism of most drugs is associated with large and
hydrophobic compounds in liver [20-22]. Higher compound
lipophilicity leads to increased metabolism and poor
absorption. The descriptors values of 90% orally active
compounds follow Lipinskis rule. The bioavailability of
compounds was evaluated by topological polar surface area
(TPSA) value, calculated through ChemAxons MarvinView
5.2.6: PSA plugin software. This descriptor has been shown
to correlate well with passive molecular transport through
membranes. The number of rotatable bonds is also a
topological parameter as a measure of molecular flexibility
(cut-off
10) and oral bioavailability. Some researchers use
the sum of H-bond donors and acceptors (cut-off
12) as a
secondary determinant of fractional absorption. The primary
determinant of fractional absorption is PSA (cut-off
1402)
[23-25].
Experimental Animals
Female Swiss mice (2535 g), housed at 222oC (60
80% humidity) under a 12h light/12h dark cycle and with
access to food and water ad libitum, were used in these
experiments that were performed during the light phase of
the cycle. The animals were allowed to adapt to the
laboratory for 24h before testing and were used only once.
Experiments were performed according to the ethical
guidelines suggested by the Institutional Animal Ethics
Committee (IAEC) and Committee for the Purpose of
Control and Supervision of Experiments on Animals,
Government of India.
TPA-Induced Mouse Ear Inflammation
Topical inflammation was induced in right ears of female
Swiss mice by the topical application of TPA (2g)

Major Constituents Separated and Identified in Aquilaria agallocha Roxb. Essential Oil Using GC-FID and GC-MS
KI*
Constituents

-Agarofuran
-Agarofuran
10-epi--Eudesmol
Agarospirol
Hinesol
Jinkoh-eremol
Total identified

FID (%)

Mode of Identification

14771

4.5

KI, MS

1553

1.0

KI, MS

1618

2.8

KI, MS

1631

12.5

KI, MS

1638

8.9

KI, MS

1659

11.8

KI, MS

Calculated

Reported

1484
1546
1622
1631
1643
1654

41.5

*KI on Equity-5 capillary columns using a homologous series of n-alkanes (C9-C28 hydrocarbons, Polyscience Corp. Niles IL); KI=Kovats Index; 1 Nor Azah et al. 2008, 12(2), 338340; 2Adams, 1995; MS=mass spectrum on a) Mass Finder version 4.0, b)NIST/EPA/NIH version 2.1 and Wiley registry of mass spectral data 7th edition and c) Adams, 1995.

Molecular Docking and ADME Studies of Natural Compounds

dissolved in acetone (20L). A volume of 10L was

delivered to both the inner and outer surface of the ear.
Agarwood oil was diluted in acetone in the ratio of 0.1%,
0.3% and 1.0% for topical anti-inflammatory study.
Agarwood oil was applied to the right ears at the dose of
20L/ear/time 30 min after TPA administration for three
times till 24h after the TPA administration. For comparison,
two other groups were treated with TPA 1) the vehicle
control and 2) indomethacin (200g/ear).

Current Computer-Aided Drug Design, 2013, Vol. 9, No. 3

363

agarofuran (1%) were the characteristics constituents. The

calculated retention index values (RI) relative to homologous
series of saturated hydrocarbons were in close similarity to
the reported values. The essential oil was also analyzed as
preliminary support to GC-MS analysis using 13C NMR
technique. Based on the carbon resonances in 13C NMR
spectrum, the presence agarospirol and hinesol isomers were
confirmed (Supplementary Table 5). Hinesol, which was
absent in the several previous reports [26-28] was the only
new compound characterized in this oil.

Ear Edema and Tissue Weight Measurement

Edema was expressed as the increase in ear thickness due
to the inflammatory challenge. Ear thickness was measured
before and after induction of the inflammatory response by
using a screw gauge micrometer (Mitutoyo Series 293). The
micrometer was applied near the tip of the ear just distal to
the cartilaginous ridges and the thickness was recorded in
10.00AM. To minimize variation due to technique, a single
investigator performed the measurements throughout any
one experiment. At 24h, when TPA-induced inflammation
was maximal, animals were euthanized by ether asphyxiation
and 1cm diameter punch of ear tissue wet weight was taken
for quantification of inflammatory mediators from tissue
homogenate. These tissues were quickly placed in a beaker
containing ice-cold Tris- HCl buffer (pH 7.4) and minced
into small pieces on ice and homogenized immediately in
tissue homogenizer (Pro Scientific Inc, USA). The
homogenate (5%) was frozen and stored at -80C before
using it for biochemical estimation. The tissue homogenate
was processed for the estimation of malondialdehyde (MDA)
and pro-inflammatory cytokines.
Quantification of Oxidative Stress and Inflammatory
Cytokines
The quantitative measurement of lipid peroxidation was
performed following the thiobarbituric acid (TBA) test. The
amount of MDA formed was quantitated by reaction with
TBA and used as an index of lipid peroxidation. The results
were expressed as
mol MDA/ml tissue homogenate.
Expression pro-inflammatory cytokines (IL-1, IL-6 and
TNF-) were quantified from tissue homogenate using
commercially available mouse specific enzyme immune
assay (EIA) kits (Endogen-Pierce, USA) as per the
manufactures instruction.

Chemical Structure-Activity Relationship

In the present work, physico-chemical properties of
known
anti-inflammatory
compounds/drugs
having
experimental anticancer activity against pro-inflammatory
cytokines (IL-1, IL-6 and TNF-) were calculated for the
training set. Further, forward stepwise multiple linear
regression study was carried out to identify the highly
correlated properties responsible for the anti-inflammatory
activity. For validation of the derived QSAR model, leaveone-out (LOO) approach was used and evaluated the QSAR
model through test data set, which also showed significant
accuracy of QSAR model.
Predicting Anti-Inflammatory Activity with the QSAR
Model
In the present work, the activity of compound 10-epi-Eudesmol, jinkoh-eremol and agarospirol was predicted
through QSAR model. The structureactivity relationship
denoted by the QSAR model yielded a very high activity
descriptors relationship accuracy of 88% referred by
regression coefficient (r2= 0.889608) and a high activity
prediction accuracy of 82% (rCV2= 0.808469) (Fig. 1). Six
chemical descriptors were found to be applicable to the antiinflammatory activity. The QSAR equation indicated that
chemical descriptors namely, dipole vector X (debye), dipole
vector Y (debye), steric energy (kcal/mole), size of smallest
ring, size of largest ring and carboxyl group count correlated
well with activity. The QSAR model equation is given
below, showing the relationship between experimental
activity in vivo [i.e., the lethal dose concentration to 50% of
the population (LD50)] as the dependent variable and five
independent variables (chemical descriptors):
QSAR model equation:

Statistical Analysis for In Vivo Experiments

Predicted log LD50 (mg/kg) = + 0.22 x Dipole vector X

(debye)

Data were expressed as Means SEM. For statistical

analysis, one-way ANOVA followed by Bonferroni test was
used. Probability (P) values less than 0.05 were considered
significant.

- 0.09 x Dipole vector Y (debye)

RESULTS

+ 0.02 x Steric energy (kcal/mole)

+ 1.33 x Size of smallest ring
+ 0.58 x Size of largest ring
- 0.65 x Carboxyl group count

Essential Oil Chemistry

The agarwood essential oil was analyzed using GC-FID,
GC/qMS and NMR techniques. The identified major
constituents are enlisted (Table 1). Agarospirol (12.5%)
followed by jinkoh-eremol (11.8%), hinesol (8.9%), agarofuran (4.5%), 10-epi--Eudesmol (2.8%) and -

- 8.03
Multiple Linear Regression coefficient (r2) = 0.89
Cross validation coefficient (rCV2) = 0.81

364 Current Computer-Aided Drug Design, 2013, Vol. 9, No. 3

Yadav et al.

Here, rCV2 (the cross-validation regression coefficient)

=0.80, which indicates that the newly derived QSAR model
has a prediction accuracy of 80%, and r2 (regression
coefficient)=0.88, which indicates that the correlation
between the activity (dependent variable) and the descriptors
(independent variables) for the training data set compounds
was 88% (Table 1 and Fig. 1).

Virtual Screening of Natural Compounds of Agarwood

Oil for Anti-Inflammatory Activity
After developing a QSAR model, we screened a total of
six compound Agarospirol, -Agarofuran, -Agarofuran, 10epi--Eudesmol, Hinesol and Jinkoh-eremol which are
summarized in the Table 2, which showed that all the
derivatives were predicted active against the proinflammatory cytokines (IL-1, IL-6 and TNF-). QSAR
results showed that out of six compounds, three compounds
were more active than others. Further, careful analysis
showed that 10-epi--Eudesmol, jinkoh-eremol and
agarospirol were most active and possessed almost
equivalent anti-inflammatory activity as of diclofenac.
Binding Affinity and ADME Studies
In the present study, potential anti-inflammatory and
immunomodulatory natural active compounds from the
agarwood oil have been identified through predictive QSAR
models and molecular docking. All the compounds showed
compliance with Lipinskis Rule of Five [20-22] and
Jorgensens rule of three [23-25] (Table 3).

Fig. (1). Graphical plot of multiple linear regression analysis

indicating linear relationship between experimental and predicted
log LD50 with r2= 0.88 for anti-inflammatory activity.

Thus, study indicates successful development of a

predictive QSAR model for in vivo anti-inflammatory
activity. A multiple linear regression QSAR mathematical
model was developed for activity prediction that successfully
and accurately (noting the corresponding experimental
activities) predicted the anti-inflamatory activities of some
anti-inflamatory compounds such as 10-epi--Eudesmol,
Alpha-Agarofuran, -Agarofuran, Hinesol, Jinkoh-eremol
and Agarospirol that had the basic pharmacophore. The
QSAR model quantified the activity-dependent chemical
descriptors and predicted the inhibitory concentration (log
LD50) of each compound, thus indicating their potential
range of inhibition. Results showed that the predicted
activities were comparable with those obtained
experimentally. Results showed that the predicted activities
of compounds 10-epi--Eudesmol, jinkoh-eremol and
agarospirol are preferentially more active than than
diclofenac. Later, predicted activity of all the compound
validated experimentally by in vivo testing mice.
Table 2.

On the basis of binding affinity energy, possible antiinflammatory and immunomodulatory targets were
identified. Quantitative data of chemical descriptors showed
similarity with 95% range of known drugs and study
revealed that compound 10-epi--Eudesmol, Jinkoh-eremol
and agarospirol are found to be preferentially more active
than other identified compounds with strong binding affinity
to most of the immunomodulatory receptors. However, 10epi--Eudesmol seems lipophilic and can be used for local
treatment of infection & inflammation. Surprisingly, both and -agarofuran did not show any binding affinity to antiinflammatory and immunomodulatory receptors such as, IL1, IL-6, TNF- and COX-I (Table 4).
In addition, the bioavailability of all compounds was
assessed through topological polar surface area (TPSA).
Calculations were carried out for the polar surface area
(PSA) by using method, termed topological PSA, based on
the summation of tabulated surface contributions of polar
fragments. PSA is formed by polar atoms of a molecule. This
descriptor was shown to correlate well with passive
molecular transport through membranes and therefore,
allows prediction of transport properties of drugs and has
been linked to drug bioavailability. Generally, it has been

Predicted Chemical Descriptor and In Vivo activity LD50 (nM) Data Calculated Through Derived QSAR Model Equation

Compound Name

Dipole Vector X
(Debye)

Dipole Vector Y
(Debye)

Steric Energy
(kcal/Mole)

Size of
Smallest Ring

Size of
Largest Ring

Group Count
(Carboxyl)

Pred. Log
LD50 mg/kg

Diclofenac

-1.440

1.517

-6.660

2.253

10-epi- -Eudesmol

-0.961

-0.207

26.36

3.682

Alpha-Agarofuran

-0.382

-0.588

31.601

2.596

Hinesol

1.258

-0.925

27.025

2.908

Jinkoh-eremol

1.102

0.54

22.315

3.995

-Agarofuran

0.169

1.007

30.617

2.552

Agarospirol

-1.285

0.873

27.097

2.19

Molecular Docking and ADME Studies of Natural Compounds

Table 3.

Current Computer-Aided Drug Design, 2013, Vol. 9, No. 3

365

Compliance of Compounds to Computational Parameters of Drug Likeness. TPSA Correlate Well with Passive Molecular
Transport Through Membranes

S. No.

Compound

Mol. Formula

TPSA (2)

Mol. Weight

Log P

H-Bond Donors

H-Bond Acceptors

Rule of 5 Violation

1.

Diclofenac

C14H 11NO 2Cl2

49.33

296.152

3.9

2.

Indomethacin

C19H 16 ClNO4

68.5

357.787

4.3

3.

10-epi--Eudesmol

C15H 26O

20.2

222.366

3.4

4.

 -Agarofuran

C15H 24O

9.2

220.350

3.3

5.

Hinesol

C15H 26O

20.2

222.366

3.7

6.

-Agarofuran

C15H 24O

9.2

220.350

3.0

7.

Jinkoh-eremol

C15H26O

20.2

222.366

3.5

8.

Agarospirol

C15H 26O

20.2

222.366

3.7

Table 4.

Molecular Docking Results of Studied Compounds on Anti-Inflammatory and Immunomodulatory Targets. Low Binding
Affinity of Compound Indicated by Nil Docking Score
Docking Score (kcal/mol)
Compounds

COX-I
IL-1

IL-6

TNF-

10-epi--Eudesmol

-11.92

-48.24

-70.247

-59.71

-Agarofuran

Nil

Nil

-65.181

Nil

Hinesol

8.39

-39.27

-65.335

-47.84

-Agarofuran

Nil

Nil

-64.419

Nil

Jinkoh-eremol

-8.72

-55.84

-66.541

-64.80

Agarospirol

-7.78

-48.13

-64.421

-61.13

Indomethacin

-69.537

-51.184

-111.563

-98.745

Declofenac

-60.984

-47.026

-92.109

-88.452

seen that passively absorbed molecules with a PSA>140 2

are thought to have low oral bioavailability [29]. Calculation
of absorption, distribution, metabolism, excretion and
toxicity (ADMET) properties of studied compounds was
performed through QikProp-v3.2 software (Schrdinger,
LLC, NY, 2009). All the studied compounds satisfied these
theoretical QSAR properties and thus are considered as
potent drug like phytomolecules (Table 5).
Toxicity Risks Assessment
It is now possible to predict activity and toxicity risks of
compounds through reliable bioinformatics tools e.g., Osiris
calculator [22-24]. In the present study, we calculated
toxicity risks parameters such as mutagenicity,
tumorogenicity, irritation and reproductive or developmental
toxicity of all the Agarospirol compound 10-epi--Eudesmol,
Jinkoh-eremol, Agarospirol, -Agarofuran, Hinesol and Agarofuran (Table 6). The toxicity risk predictor locates
fragments within a molecule which indicates a potential
toxicity risk. Toxicity screening results showed that all the
compound possess no risk of mutagenicity, tumorogenicity
and reproductive toxicity. On the other hand compounds
Hinesol and -Agarofuran indicate medium risk of irritation.
To judge the active compounds overall potential to qualify
for a drug, we calculated overall drug score which combines

drug-likeness, hydrophilicity (CLogP), aqueous solubility

(LogS), molecular weight (MW), and toxicity risks
parameters. Predicted active compounds showed there
calculated parameters within acceptable limit. The results of
toxicity risk assessment screening showed overall drug score
of all the predicted active compounds namely, compound 10epi--Eudesmol, Jinkoh-eremol, Agarospirol, -Agarofuran,
Hinesol and -Agarofuran moderate to good, as compared to
standard anticancer compounds Diclofenac.
Ear Thickness and Ear Weight
Ear edema was observed in all TPA-treated animals at
24h after treatment. In animals treated with vehicle
(Acetone), initial ear thickness was 0.224 0.015 mm. Ear
thickness increased to 0.506 0.023 mm by 24h after TPA
treatment. Agarwood oil treated experimental mice showed
significant reduction of ear edema when compared with
vehicle treated mice. Agarwood oil at 1.0% and
indomethacin treatment exhibited the significant reduction in
ear edema when compared with the mice treated with vehicle
(Fig. 2A). Edema was also indicated by changes in ear punch
masses at 24h and the treatment effects were similar to the
changes in ear thickness. Ear punch biopsy weights at 24h
were significantly lower in 1.0% agarwood oil and
indomethacin treated mice groups compared to the TPA
treated vehicle control group (Fig. 2B). Increased skin

366 Current Computer-Aided Drug Design, 2013, Vol. 9, No. 3

Table 5.

Yadav et al.

Compliance of ADMET Properties of Compounds with Anti-Inflammatory Drug

Predictions for
Properties

Diclofenac

-Agarofuran

-Agarofuran

10-epi-Eudesmol

Agarospirol

Hinesol

Jinkoheremol

Standard
Range

log S for aqueous

solubility

-5.176

-5.361

-5.485

-4.251

-4.25

-4.188

-4.2

-6.5/ 0.5

log K hsa Serum

Protein Binding

-0.032

0.701

0.736

0.683

0.657

0.663

0.656

-1.5 /1.5

log BB for brain/

blood

-0.05

0.667

0.699

0.17

0.156

0.223

0.17

-3.0 /1.2

No. of Primary
Metabolites

1.0 / 8.0

Predicted CNS
Activity

-1

2 (inactive)
/+2 (active)

log HERG for K+

Channel Blockage

-2.547

-2.605

-2.78

-2.937

-3.175

-3.047

-3.087

concern below
-5

Apparent
Caco-2
Permeability
(nm/sec

483.646

9906.038

9906.038

4701.016

4571.153

5415.923

4708.863

<25 poor, >500

great

Apparent MDCK
Permeability
(nm/sec)

1001.69

5899.293

5899.293

2635.783

2557.171

3071.589

2640.539

<25 poor, >500

great

QP log Kp for skin

permeability

-1.589

-1.417

-1.438

-1.948

-1.87

-1.729

-1.853

Kp in cm/hr

Jm, max transdermal

transport rate

2.140

0.037

0.026

0.141

0.169

0.269

0.197

micrograms/cm
^2-hr

Lipinski Rule of 5
Violations

max. 4

Jorgensen Rule of 3
Violations

max. 3

% Human Oral
Absorption in GI (+20%)

100

100

100

100

100

100

100

<25% is poor

Note: * indicate violation of the quantitative parameters of 95% of drugs; M indicate molecular weight outside the training range.

Table 6.

Compliance of Active Compounds to Computational Toxicity Risks Parameters i.e., Mutagenicity, Tumorogenicity,
Irritation and Reproduction
Toxicity Risk Parameters

Drug Likeness Parameters (Osiris)

Compound
MUT

TUMO

IRRI

REP

CLP

MW

DL

DS

Diclofenac

No risk

No risk

No risk

High risk

4.4

-4.64

295

2.06

0.36

10-epi--Eudesmol

No risk

No risk

No risk

No risk

3.75

-3.27

222

-4.04

0.41

Jinkoh-eremol

No risk

No risk

No risk

No risk

3.55

-3.31

222

-3.64

0.42

Agarospirol

No risk

No risk

No risk

No risk

3.55

-3.31

222

-2.82

0.35

-Agarofuran

No risk

No risk

No risk

No risk

3.16

-3.22

206

-8.35

0.43

Hinesol

No risk

No risk

Medium risk

No risk

3.83

-3.49

236

-2.33

0.34

-Agarofuran

No risk

No risk

Medium risk

No risk

3.55

-3.34

220

-4.98

0.33

Note: MUT= Mutagenicity, TUMO= Tumorogenicity, IRRI= Irritation, REP= Reproduction, MW = Molecular weight, CLP= ClogP, S= Solubility, DL= Drug-likeness, DS= Drugscore.

thickening is often the first hallmark of skin irritation and

local inflammation. This parameter is indicative of a number
of processes that occur during skin inflammation, including
increased vascular permeability, edema and swelling within
the dermis, and proliferation of the epidermal keratinocytes.

Effect of Agarwood Oil on Lipid Peroxidation

MDA was measured from the homogenate of ear punch
biopsies taken 24h after TPA administration as an index of
lipid peroxidation. The ears treated with 0.3%, 1.0% and

Molecular Docking and ADME Studies of Natural Compounds

Current Computer-Aided Drug Design, 2013, Vol. 9, No. 3

367

indomethacin doses had significantly reduced the MDA

activity (Fig. 3).

Fig. (3). Dose-response effect of agarwood oil on TPA-induced

oxidative stress mediators quantification from ear tissue
homogenate, MDA formation expressed in mol/ml tissue
homogenate. Results are Mean SEM of 5 mice. * P<0.05 (Vehicle
vs Treatment).

DISCUSSION

Fig. (2). Dose-response effect of agarwood oil on TPA-induced

inflammatory ear swelling in Swiss albino mice (A) Change in ear
thickness (B) Tissue wet weight. Results are Mean SEM of 5
mice.* P<0.05 (Vehicle vs Treatment).

Effect of Agarwood Oil on Pro-Inflammatory Cytokines

The mice treated with vehicle in TPA-induced ear
inflammation showed the significant (P<0.05) increase in
pro-inflammatory cytokines (IL-1, IL-6 and TNF-)
production when compared with the normal mice treated
with acetone alone whereas agarwood oil showed the
inhibition of pro-inflammatory cytokines levels. Significant
(P<0.05) inhibition of pro-inflammatory cytokines
production was observed in 1.0% agarwood oil and
Indomethacin treated mice (Table 7).

The identified constituents in oil in present study have a

similar compositional structure except hinesol, however, a
sharp quantitative variation is evident as compared to the
earlier report [27]. Except, 2-phenyl-2-butanone, the present
oil composition is similar to the collections taken from
Pahang from Peninsular Malaysia. In addition to this, other
collections from Selangor, Kelantan and Terengganu regions
were also quite similar. Moreover, constituents such as oxoagarosirol,
2-phenyl-2-butanone,
-guaiene,
norketoagarofuran, kusunol and jinkohol were completely
absent in our oil sample [26, 27]. Agarospirol (12.5 %) and
jinkoh-eremol (11.8 %) identified as major contributors in
present analysis of oil are in congruent with the earlier report
[11] and have been previously reported to be responsible for
prolonged effect on the hexobarbital-induced sleeping time,
and hypothermic effects in terms of rectal temperature, a
suppressive effect on acetic acid-writhing, and a reduction of
the spontaneous motility in mice [28]. Further, the third
major contributor hinesol (8.9 %) was reported to possess
liver-protective effects [30] and might be due to these
compounds, oil exhibits significant activity against
inflammation and reduction of oxidative stress.
To examine the activity specially focused on antiinflammatory and immunomodulatory action of the earlier
reports, a computational approach was followed for the
identified compounds in present study. A predictive model
could provide an important step in the discovery of multitarget drugs by identifying the group of proteins targeted by
a particular ligand, leading to innovation in pharmaceutical
research [31]. The obtained results indicate that all studied
compounds possess significant anti-inflammatory and
immunomodulatory activity. Molecular docking results
revealed that compound jinkoh-eremol possessed higher
anti-inflammatory activity with docking energy score -55.84,

368 Current Computer-Aided Drug Design, 2013, Vol. 9, No. 3

Table 7.

Yadav et al.

Effect of Agarwood Oil on TPA-Induce Pro-Inflammatory Mediators from Ear Tissue Homogenate Quantified Using
ELISA for (A) Mouse IL-1 (B) Mouse IL-6 (C) Mouse TNF-
Pro-Inflammatory Cytokines
Treatments
IL-1 (pg/ml)

IL-6 (pg/ml)

TNF- (pg/ml)

Normal

117.5219.66

117.8719.25

84.024.28

Vehicle Control

1141.75236.08

1468.12193.15

206.6710.80

Agar wood oil (0.1 %)

834.1879.36

1074.17116.09

156.686.92

Agar wood oil (0.3 %)

831.1255.33

1041.09151.85

133.4532.86

Agar wood oil (1.0 %)

695.0281.11*

741.8349.13*

121.6622.14*

Indomethacin

619.9028.67*

797.31106.79*

130.496.27*

All the values are expressed as a meanSEM of 5 mice. *P<0.05 (Vehicle vs Treatment).

-66.541 and -64.80 kcal/mol against IL-6 (PDB: 1N26),

TNF- (PDB: 1A8M) and COX-I (PDB: 2OYE) receptors,
respectively (Fig. 4A-C) whereas, 10-epi--Eudesmol
possessed -11.92, -48.24 and -70.247 kcal/mol against IL-1
(PDB: 3O4O), IL-6 (PDB: 1N26), TNF- (PDB: 1A8M)
receptors, respectively (Fig. 5A-C) which were comparable
to standard drug (Table 4). Beside this, all the studied
compounds showed compliance with standard parameters of
drug likeness and ADME and are thus considered as potent
drug like active natural molecules (Table 6).
Okugawa et al. (1996), reported the effect of jinkoheremol and agarospirol from agarwood on the central
nervous system in experimental mice. Zuniga et al. (2005)
has demonstrated the anti-inflammatory properties of
Bursera lancifolia, wherein the plant possessed 6.1 %
agarospirol, 14.4 % eudesmol along with other constituents

and exhibited a 16.71 % inhibition in TPA-induced edema in

mice, which is in line and supports the computational
predictions made in present study.
This study provides first assessed evidence that agarwood
oil when applied topically has anti-inflammatory activity in
TPA-induced mouse ear inflammation model. The limited
bioavailability of agarwood oil, however, makes it best
suited for clinical indications that can be treated with topical
therapeutics. Although the skin is an easily accessible tissue
for topical administration, its barrier functions pose
enormous challenges for most therapeutics. The induction of
ear skin inflammation in mice by TPA represents a
promising animal model for elucidating the mechanism of
clinical dysfunction and for evaluating the efficacy of topical
anti-inflammatory agents [32, 33]. The results of the study
exhibit that the topical application of TPA increased the ear

Fig. (4). Docked jinkoh-eremol on (A) IL-6 (PDB: 1N26), (B) TNF- (PDB: 1A8M) and (C) COX-I (PDB: 2OYE).

Fig. (5). Docked 10-epi--Eudesmol on (A) IL-1 (PDB: 3O4O), (B) IL-6 (PDB: 1N26) and (C) TNF- (PDB: 1A8M).

Molecular Docking and ADME Studies of Natural Compounds

thickness and ear weight significantly when compared to the

normal mice treated with acetone alone. Increased skin
thickening is often the first hallmark of skin irritation and
local inflammation. This parameter is indicative of a number
of processes that occur during skin inflammation, including
increased vascular permeability, edema and swelling within
the dermis, and proliferation of the epidermal keratinocytes.
The mice treated with agarwood oil exhibited the reduction
of oxidative stress and pro-inflammatory markers level [34].
Oxidative stress is well recognized to be a key step in the
pathogenesis of liver injury. An imbalance between oxidants
and antioxidants can lead to oxidative stress, characterized
by escalating cell damage [35]. Oxidative stress is
characterized by increased lipid peroxidation and/or altered
nonenzymatic and enzymatic antioxidant systems [36]. TPA
treatment caused a significant increase in lipid peroxidation
compared to acetone alone treated mice. It is widely
recognized that the secretions of cytokines by keratinocytes
in response to injury are key mediators of the cutaneous
inflammatory response [37, 38]. In this study, it has been
clearly demonstrated that the topical treatment with
agarwood oil inhibits the secretion of IL-1 and IL-6 in the
dermatitis model of inflammation.
CONCLUSION

Current Computer-Aided Drug Design, 2013, Vol. 9, No. 3

369

Council of Scientific and Industrial Research, New Delhi for

providing financial support. The researchers indebted to Mr.
Mohd. Anwar Hussain, Hojai, Assam, India who gifted one
sample of Agarwood oil for the study. Authors were also
thankful to Dr. J. Kotesh Kumar, CIMAP for preliminary
help in NMR analysis.
SUPPLEMENTARY MATERIAL
Supplementary material is available on the publishers
web site along with the published article.
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CONFLICT OF INTEREST

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The authors confirm that this article content has no

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Revised: June 5, 2012

Accepted: January 25, 2013