ORIGINAL RESEARCH

Effect of dehydroabietylamine in angiogenesis and GSK3-binhibition during wound healing activity in rats

Mahadevappa Paramesha • Chapeyil Kumaran Ramesh •

Venkatarangaiah Krishna • H. Malleshappa Kumar Swamy •

S. J. Aditya Rao • Joy Hoskerri

Received: 28 March 2014 / Accepted: 16 June 2014

� Springer Science+Business Media New York 2014

Abstract The present research evaluates the wound heal-

ing capacity of dehydroabietylamine, a diterpenoid isolated

from methanolic leaves extract of Carthamus tinctorius L.

(Asteraceae). The wound healing activity was screened by

excision, incision, and dead space wound models on albino

rats and for the angiogenic property using chick chorioa-

llantoic membrane (CAM) assay. Significant increase in

wound contraction rate, skin breaking strength, granuloma

strength, and dry granuloma weights was observed as

compared to control. The pro-healing action may be due to

increasing in collagen deposition as well better alignment

and maturation. In CAM assay, the dehydroabietylamine

showed increased angiogenic zone formation in the devel-

oping embryos compared to normal embryos and evidenced

pro-angiogenic activity of the active constituent. Further, in

silico comparative docking of dehydroabietylamine, stan-

dard drug nitrofurazone and positive control CHIR_98014 to

ATP-binding active pocket of glycogen synthase kinase3-b(GSK3-b) protein that consisted of Tyr216 as a catalytic

residue, with residues Asn64, Gly65, Ser66, Phe67, Gly68,

Val70, Lys85, Leu132, Val135, Asp181, Lys183, Gln185,

Asn186, and Asp200 in the active pocket docked with de-

hydroabietylamine at the torsional degree of freedom 0.5 U

with Lamarckian genetic algorithm showed the inhibition

constant of 315.6 9 10-9. But the inhibition constant of

nitrofurazone and CHIR_98014 was 34.76 9 10-6 and

4.64 9 10-6 respectively. Docking of dehydroabietylamine

to GSK3-b protein involved in Wnt signaling pathway

supported the research on wound healing property. The

above said studies explore the role of dehydroabietylamine

in wound healing activity.

Keywords Carthamus tinctorius L. �Dehydroabietylamine � CAM assay � GSK3-b protein �In silico analysis � Wound healing

Introduction

Wound healing disorders are medical and public health

problem of high magnitude with diseases such as diabetes,

hypertension, and obesity (Pietro et al., 2003). The wound

healing animal model viz., excision, incision, dead space

wound models and histopathological studies are exten-

sively used to determine the healing property of natural and

chemical products. The excision wounding method repre-

sents an animal model that provides access to investigate

complex tissue movements associated with repairs such as

hemorrhage, granulation, tissue formation, re-epitheliali-

zation and angiogenic processes (Wetzler et al., 2000).

Measurement of wound strength (incision wound model)

and granulation of tissue (produced in dead space wound

model) provides highly quantifiable estimate of the healing

process. Mechanism of healing processes can be studied by

determination of several individual components of healing

phases (Wetzler et al., 2000).

M. Paramesha (&)

Plant Cell Biotechnology Department, CSIR-Central Food

Technological Research Institute, Mysore 570020, Karnataka,

India

e-mail: parameshbt@gmail.com; paramesham@cftri.res.in

C. K. Ramesh � S. J. Aditya Rao

Department of P.G. Studies and Research in Biotechnology,

Sahyadri Science College (Autonomous), Kuvempu University,

Shivamogga 577203, Karnataka, India

V. Krishna � H. M. Kumar Swamy � J. Hoskerri

Department of P.G. Studies in Biotechnology and

Bioinformatics, Kuvempu University, Jnana Sahyadri,

Shankaraghatta 577451, Karnataka, India

123

Med Chem Res

DOI 10.1007/s00044-014-1110-1

MEDICINALCHEMISTRYRESEARCH

The development of strategies for the treatment of

angiogenesis-dependent diseases has been greatly aided by

the development of in vivo models of angiogenesis

(Auerbach et al., 1991; Folkman, 1995; Pietro et al., 2003).

These bioassays provide investigators with tools to visu-

alize vessel architecture and function and also to analyze

and manipulate several steps in the angiogenic response

(Pietro et al., 2003). The widely used model systems are

iris and avascular cornea of the rodent eye, the chick

chorioallantoic membrane (CAM), the hamster cheek

pouch, the dorsal skin and air sac with several non-invasive

imaging techniques to visualize living vascularized tissues.

These techniques afford the opportunity to study a number

of essential steps in the angiogenic response (Auerbach

et al., 1974). In the present research, the chick CAM assay

was adopted along with the excision, incision, and dead

space wound models to find the efficacy of dehydroabie-

tylamine, a diterpenoid isolated from the methanolic leaves

extract of Carthamus tinctorius L., var. Annigeri-2, an oil-

yielding crop (Paramesha et al., 2011). Wnts constitute a

family of secreted glycoproteins with distinct expression

patterns in the embryo and the adult organism. Wnts appear

to be involved in differentiation processes by controlling

the polarity of cell division, cell growth, and cell fate. It is

clear that the genes encoding for Wnts and other compo-

nents of the pathway are expressed during regeneration of

the skin (Harish et al., 2008; Vidya et al., 2012).

Carthamus tinctorius L. (Asteraceae) is an annual oil-

yielding herb and it is commonly known as ‘‘safflower.’’ It

is traditionally cultivated in Northwest India, Iran and

Northern Africa, Eastern and Northern America for its

seeds which are used for the extraction of edible oil and

also for its flower and the latter is used for coloring and

flavoring the food stuffs (Li and Mundel, 1996; Asgarpa-

nah and Kazemivash, 2013). Ayurveda suggests that the

leaves are laxative, appetizer and diuretic also useful in

urorrhea and ophthalmopathy (Duke, 2010; Asgarpanah

and Kazemivash, 2013). Phytochemical studies of the

species revealed the presence of sterols and triterpenes

(Mitova et al., 2003), sesquiterpene glycosides (Lahloub

et al., 1993), aromatic acids and serotonins (El-Shaer et al.,

1998) and flavonoids (Mitova et al., 2003), and a diterpe-

noid, dehydroabietylamine (Paramesha et al., 2011). The

pharmacological properties of safflower have been evalu-

ated for anti-tumor, sedative (Benedi et al., 1986), anti-

microbial (Taskova et al., 2003; Paramesha et al., 2009a),

anti-inflammatory and analgesic effects (Bocheva et al.,

2003) antihyperglycemic effects (Paramesha et al., 2009b)

and hepatoprotective and in vitro antioxidant properties

(Paramesha et al., 2011).

Literature survey indicated that these plant species have

been subjected to rigorous phytochemical and pharmaco-

logical research. However, an investigation of wound

healing activity has not been reported anywhere and hence

this paper reports the screening of wound healing property

of its isolated constituent, dehydroabietylamine of leaves

on albino rats and CAM assay using Chicken egg. The

mode of action of the dehydroabietylamine molecule was

hypothesized in silico by docking the molecule to glycogen

synthase kinase3-b (GSK3-b) protein, an important regu-

latory enzyme whose inhibition promotes wound healing

through b-catenin dependent Wnt signaling pathway.

Materials and methods

Animals used

Studies were carried out using albino rats (Wistar Strain)

of sex weighing 150–200 g. The animals were main-

tained under standard laboratory conditions (temperature

27 ± 2 �C, relative humidity 55 ± 10 % and 12 h light and

dark cycles). The animals were fed with standard dry pellet

diet (Hindustan Lever, Kolkata, India) and water ad libitum.

The animals were acclimatized to laboratory condition for

7 days prior to the experiments. The study was permitted

by the institutional animal ethical committee (Reg No.

144/1999/CPCSEA/NCP/IAEC/CLEAR/P. COL./06/06/

2007-08).

Drug formulation

Dehydroabietylamine is a light yellow viscous molecule

with a melting point of 113 �C; chemically it has an aromatic

diterpene structure with three rings and a reactive amino

group with four methyl groups. The isolation and charac-

terization of dehydroabietylamine from the Carthamus

tinctorius L. var. Annigere-2 were described in our earlier

publication (Paramesha et al., 2011), and the same was used

in the present work. For the topical administration, 100 mg

of dehydroabietylamine was homogenized with sodium

alginate (50 g) to get 0.2 % (w/w) ointment gel. For oral

administration, suspension of 5 mg/ml of dehydroabietyl-

amine was homogenized with Tween 80 (1 %). The drug

formulations were prepared every fourth day and were

administered by intraperitoneal injection. For CAM assay,

the dehydroabietylamine was dissolved in DMSO solution.

Experimental design for wound healing activity

Three groups of animals containing six each were used for

each of the excision and incision wound models and the

application of the drug was topical. The animals of group I

was considered as a control and received 5 % sodium

alginate ointment base. The animals of group II served as

the reference standard and treated with 0.2 % w/w

Med Chem Res

123

nitrofurazone ointment (Furacin, Smithkline Beecham,

Mumbai, India). The III group received 0.2 % w/w oint-

ment gel of the dehydroabietylamine. However, in dead

space wound model, the animals were divided into two

groups containing six animals each. The first group of

animals served as a control and was treated with 1 ml/kg of

1 % Tween 80 I.P, whereas the second group of animals

was treated with the dehydroabietylamine. The methods of

Morton and Malone (1972), Ehrlich and Hunt (1969), Lee

and Tong (1968) and Galigher and Kayloff (1971) were

used to evaluate the wound healing activity for excision,

incision and dead space wound model, respectively. The

results of these experiments were expressed as mean ± SE

of six animals in each group, and the data were analyzed by

one-way ANOVA followed by Tukey’s pair-wise com-

parison test. The values of P \ 0.01 were considered sta-

tistically significant.

CAM assay

CAM assay was performed according to the detailed pro-

cedure of Gururaj et al. (2002). The fertilized chicken eggs

were incubated at 37 �C in a humidified incubator. On the

11th day of development, a rectangular window was

opened in the egg shell and glass cover slip (6-mm diam-

eter) with either vehicle or test sample was placed on the

CAM and the window was closed using sterile vegetable

wrap. The windows were opened after 48 h of incubation

and inspected for changes in the microvessel density below

the cover slip and photographed using a Nikon digital

camera (Prasanna Kumar et al., 2009).

In silico docking

Auto Dock 4.2 was used for performing the in silico

docking studies to determine the binding of the dehyd-

roabietylamine, nitrofurazone (a standard drug) and a

ATP-competitive inhibitor (CHIR-98014) as a positive

control into the active site of GSK3-b with binding ori-

entation of inhibitor ligands. A genetic algorithm method

implemented in the program Auto Dock 4.2 was

employed (Bhat et al., 2003). The ligand molecules de-

hydroabietylamine, nitrofurazone and CHIR-98014 were

initially designed, and the obtained structure was energy

minimized using Chem Draw Ultra 6.0. 3D structure

coordinates file for dehydroabietylamine, nitrofurazone,

and CHIR-98014 were prepared to use PRODRG server

(Ghose and Crippen, 1987). GSK3-b protein was studied

for its involvement in Wnt signaling pathway by Kegg

pathway database (www.genome.jp/kegg). The protein

structure file was collected from PDB (www.rcsb.org/

pdb). 1Q5K.pdb was considered for docking studies,

using the online CASTp server in support with PDBSum;

the activation domain in the protein under study was

identified, which resulted in the following residues in the

ATP-Binding active site of GSK3-b viz., Val61, Ile62,

Asn64, Gly65, Ser66, Phe67, Gly68, Val70, Lys85,

Leu132, Val135, Pro136, Asp181, Lys183, Gln185,

Asn186, and Asp200. This protein file was edited by

removing the hetero-atoms such as water molecules and

other bound ligands, and further C terminal oxygen was

added (Binkowski et al., 2003). For docking calculations,

Gasteiger–Marsili partial charges (Gasteiger and Marsili,

1980) were assigned to the ligands, and non polar

hydrogen atoms were merged using Auto Dock 4.2. (By

providing six degrees of freedom, all torsions were

allowed for the ligand to rotate during docking.) The grid

was set in such a way that the box was covering the

active pocket residues found in the ATP-Binding active

site of the protein (Reya and Clevers, 2005). Grids were

generated with Auto Grid. The Lamarckian genetic

algorithm and the pseudo-Solis and Wets methods were

used, by applying default parameters. Using Lamarckian

genetic algorithm, number of ligand docked orientation

was 50 obtained by runs proportional to the orientations;

the population in the genetic algorithm was 250 with

100,000 energy evaluations, and the maximum number of

iterations was 10,000.

Results

Wound healing

In the present investigation, the dehydroabietylamine-

treated animals showed the significant promotion of wound

healing in the entire three wound models viz., excision,

incision, and dead space. In excision wound model, the

mean percentage closure of wound area was calculated on

the 4, 8, 12, and 16th post wounding days, respectively,

(P \ 0.05) as shown in Table 1. The dehydroabietylamine-

treated animals showed the closure of wound area

84.79 ± 4.68 and 97.78 ± 2.15 on the 12th and 16th days,

respectively; and results were comparable with the refer-

ence standard nitrofurozon. In regard to epithelization, the

treated animals showed epithelization in 17.67 ± 2.62 days

against 23.17 ± 1.14 days for control.

The promotion of wound healing activity assessed by its

tensile strength of the incision wound model had shown

significant (P \ 0.05) increase in breaking strength

425.67 ± 10.03 and 460.33 ± 13.75 for the animals trea-

ted with topical application of dehydroabietylamine and

nitrofurazone respectively when compared to control

(277.00 ± 9.39) (Table 2). The effect of oral administra-

tion of dehydroabietylamine on dead space wound model

was observed for the increase in the dry weight of

Med Chem Res

123

granulation tissue and increase in its tensile strength

(Table 3). Compared to the control (49.83 ± 0.87 for dry

weight of granulation and 493.33 ± 54.87 for tensile

strength), the dehydroabietylamine showed significant

(P \ 0.05) increase in dry weight (62.67 ± 0.49) and

breaking strength (778.33 ± 47.55). Further, the estima-

tion of hydroxyproline content in the granulation tissue

revealed that the treated animal groups had high

hydroxyproline content (2106.50 ± 2.62) when compared

to the control group which showed a lesser amount of

hydroxyproline content (1369.67 ± 10.54) and the varia-

tions are statistically significant (P \ 0.05).

The histological studies of granulation tissue of the

control animals showed more aggregation of macrophages

or inflammatory cells, fibroblastic connective tissue, and

very little number of blood vessels (Fig. 1a). The lesser

epithelialization and lesser collagen formation indicated

incomplete healing of the wound in control animals.

Whereas, dehydroabietylamine-treated animals showed

complete healing with more of fibroblasts, increase in

collagen, and number of blood vessels (Fig. 1b).

CAM assay

The chick CAM performed manifested increased vascu-

lature zone in the developing embryos, in dehy-

droabietylamine-treated compared to normal embryos

(Fig. 2c). Further, newly formed micro-vessels were

increased around the area of dehydroabietylamine in a

concentration-dependent manner. However, in the control,

only a few number of vasculature zones were noticed

(Fig. 2a, b).

In silico docking

In parallel to in vitro wound healing activity and CAM

assay, the In silico molecular docking study was considered

to support the efficiency of dehydroabietylamine in wound

healing. The results of comparative docking of GSK3-b by

different molecules were depicted in Table 4 and showed

in Fig. 3A–C. The results reveal that the docking of GSK3-bwith dehydroabietylamine exhibited binding energy

with -8.87 kJ/mol and estimated inhibition constant of

315.6 9 10-9 with the intermolecular energy -9.76 kJ/mol

when compared to standard drug and positive control

Table 1 Effect of topical application of dehydroabietylamine on excision wound model

Treatment Day 4 Day 8 Day 12 Day 16 Epithelialization in days

Group I control 25.41 ± 2.19 46.97 ± 5.88 60.65 ± 5.04 82.92 ± 1.83** 23.17 ± 1.14

Group II nitrofurozon (0.2 %) 40.51 ± 3.31** 57.37 ± 3.71** 86.24 ± 5.36** 99.73 ± 3.01** 15.50 ± 2.59**

Group III dehydroabietylamine 38.40 ± 2.01** 56.09 ± 1.65* 84.79 ± 4.68** 97.78 ± 2.15** 17.67 ± 2.62**

One-way, F 13.5 7.5 38.5 10.4 3.4

df 3 3 3 3 3

Values are mean ± SE; n = 6 in each group

* P \ 0.05 and ** P \ 0.01 when compared to control

Table 2 Effect of topical application of dehydroabietylamine on

Incision wound model

Group (N) Breaking strength (g)

Group I control 277.00 ± 9.39

Group II nitrofurazone (0.2 %) 460.33 ± 13.75**

Group III dehydroabietylamine 425.67 ± 10.03**

F value 36.2

df 3

Values are mean ± SE; n = 6 in each group

** P \ 0.01 when compared to control

Table 3 Effect of oral administration of dehydroabietylamine on dead space wound model

Group (N) Granulation tissue dry

weight (mg/100 g)

Tensile strength (g) Hydroxyproline

content (lg/100 g)

Group I control 49.83 ± 0.87 493.33 ± 54.87 1369.67 ± 10.54

Group II dehydroabietylamine 62.67 ± 0.49** 778.33 ± 47.55** 2106.50 ± 2.62*

F value 60.7 15.2 78.9

df 3 3 3

Values are mean ± SE; n = 6 in each group

* P \ 0.05 and ** P \ 0.01 when compared to control

Med Chem Res

123

(Table 4; Fig. 3A–C). The dehydroabietylamine was com-

pletely enfolded in the entire ATP-binding pocket of

GSK3-b (Fig. 3A) as compared to nitrofurazone (Fig. 3B)

and CHIR-98014 (Fig. 3C). The topology of the active

site of GSK3-b was similar to all the three molecules

which are lined by interacting amino acids as predicted

from the CASTp server (Fig. 3A–C). The dehydroabietyl-

amine (ball and stick indicated gray) was found to sit in the

proper orientation complementary to the topology of the

ATP-binding site (residues shown in MSMS-MOL)

(Fig. 3A(c)). However, orientation of dehydroabietylamine

molecule was perpendicular to the plane made by Val135,

Pro136, and Asp137 (Fig. 3A(d)). The reactive group

oxygen (OAI) of dehydroabietylamine formed hydrogen

bonded with the backbone hydrogen of ASP 264 with a

bond length of 1.659 and bond energy of -0.068 A.

Therefore, the dehydroabietylamine was considered as a

better inhibitor of GSK3-b and one of the potent wound

healing agents that have been shown to elicit the cutaneous

wound healing comparable to the reference drug

nitrofurazone.

Discussion

Wound healing is the interaction of a complex series of

phenomena that eventuate in the resurfacing, reconstitu-

tion, and proportionate restoration of tensile strength of

wounded skin (Pietro et al., 2003; Harish et al., 2008;

Vidya et al., 2012). Many research works on the animal

model have been showed that wound healing process

involves the four phases viz., Hemostasis, Inflammation,

Proliferation or Granulation, and Remodeling or Matura-

tion (Hukkeri et al., 2006; Kumara Swamy et al., 2007;

Umachigi et al., 2008). In the present study, three different

models were used to evaluate the wound healing effect of

dehydroabietylamine of C. tinctorius L., var. Annigeri-2 on

various phases of wound healing, which run concurrently

but independent of each other. The standard drug nitrofu-

razone was used as a standard reference to evaluate the

healing potency of the constituent. In excision wound

model, significant decrease in the period of epithelializa-

tion and increase in wound contraction rate were observed

in dehydroabietylamine-treated animals. In dehydroabie-

tylamine and the standard nitrofurazone-treated animals,

epithelialization was completed on 17th and 15th post

wounding day, respectively. While in control animals, the

rate of wound contraction was slow, and the complete

epithelization of the excision wound was extended up to

23rd post wound day. A critical outcome of the wound

repair process is the restoration of the mechanical proper-

ties of tissue strength. Measurement of wound strength

provides highly quantifiable estimates of the efficacy of the

aggregate healing process. Determination of various indi-

vidual components of the phases of healing can provide

important insights about events operative during repair

(Kumara Swamy et al., 2007). Breaking strength can be

defined as the load required to break a wound. The

breaking strength increases rapidly as collagen deposition

increases, and cross linkages are formed between the col-

lagen fibers. In the incision wound model, the animals

treated with dehydroabietylamine showed a significant

increase in the breaking strength when compared to the

control. The dead space wound model is a highly repro-

ducible and biologically valid model for studying acute

healing responses because it begins to elicit foreign body

reaction with giant cell accumulation and fibrosis after

about 2 weeks in the rat. Dead space wound model pro-

vides to assess reparative wound collagen accumulation by

measuring hydroxyproline content in granule tissue. Fur-

ther, measured collagen deposition correlates well with the

breaking strength of incisional cutaneous wounds. The

healing effectiveness of dehydroabietylamine on the dead

space wound models was evaluated by assessing the weight

of granuloma tissue by estimating its breaking strength

and hydroxyproline content of the granuloma tissue.

Fig. 1 Wound healing activity. a Section of the granulation tissue of

the control animal showing more number of macrophages (arrow

head) and lesser collagen fibers (arrow) (H & E 9100). b Section of

the granulation tissue of the animal treated with dehydroabietylamine

showing increased collagenation (arrows) and lesser macrophages

(arrow head) (H & E 9100)

Med Chem Res

123

Dehydroabietylamine-treated animals showed significantly

enhanced granuloma tissue tensile strength, weight and

hydroxyproline content which was harvasted on the 8th day

of the treatment. This may be due to the enhanced collagen

maturation by increased cross-linking of collagen fibers.

The increased weight of both wet and dry granuloma tissue

also revealed the presence of higher hydroxyproline con-

tent (Harish et al., 2008; Vidya et al., 2012). The results of

this investigation were compared with the works of Leite

et al. (2002) in Veronica scorpioides; Shirwaikar et al.

(2003) in Hyptis suaveolens; Khan et al. (2004) in Eclipta

alba; Hukkeri et al. (2006) in Moringa oleifera, Kumara

Swamy et al. (2007) in Embelia ribes and Umachigi et al.

(2008) in Quercus infectoria.

Histological examination of the granuloma tissue

showed that the infiltration of fibroblasts and monocytes in

the subcutis was significantly greater in the untreated ani-

mals. Microscopically, these granules possess newly

Effect of dehydroabietylamine on CAM

0

5

10

15

20

25

A B

Control and dehydroabietylamine

Nu

mb

er o

f n

ew

blo

od

ves

sels

a b

c

Fig. 2 CAM assay. a Control

CAM. b Effect of

dehydroabietylamine showing

increased vasculature. c Effect

of dehydroabietylamine on

CAM

Table 4 Molecular docking results with GSK3-b

Molecule Docking

energy

(kJ/mol)

Inhibition

constant (M)

Ligand

efficiency

Intermolecular

energy

Torsional

energy

RMS Hbond Bandings Bond

length

Dehydroabietyl-

amine

-8.87 315.6 9 10-9 0.42 -9.76 0.89 0.0 01 Dehydroabietylamine::LIG1:HN1:

1Q5K:B:ASP264:OD2

1.659

CHIR_98014 -7.28 4.64 9 10-6 0.220 -9.96 2.68 0.0 02 CHIR_98014::LIG1:HN: 2.039

2.0721Q5K:A:VAL135

CHIR_98014::LIG1:HN:

1Q5K:A:ASN186

Nitrofurazone -6.08 34.76 9 10-6 0.43 -6.98 0.89 0.0 03 Nitrofurazone::LIG1:HN:

1Q5K:A:ASP200:OD2

2.152

2.232

2.169Nitrofurazone::LIG1:H:

1Q5K:B:ASP264:OD1

Nitrofurazone::LIG1:H:

1Q5K:A:SER203:OG

Med Chem Res

123

Fig. 3 In silico models. A:

(a) Ribbon model of GSK3-b

showing the interaction of

dehydroabietylamine (MSMS-

MOL) with the amino acids in

the binding pocket predicted by

CASTp server. (b) Molecular

surface model of GSK3-b with

the bound dehydroabietylamine

(purple and blue stick). (c) The

dehydroabietylamine molecule

enfolded in the ATP-binding

pocket of GSK 3-b showing

interacting amino acids: Val61,

Ile62, Gly63, Asn64, Val70,

Lys85, Glu97, Leu132, Asp133,

Tyr134, Val135, Pro136,

Glu137, Asp181, Asp183,

Asp186, Cys199, and Asp200

(MSMS-MOL). (d) Stick and

ball model of

dehydroabietylamine molecule

hydrogen bonded with the GSK

3-b with ASP264 and showing

the residues in the linker/hinge

region and those involved in the

important ionpair interaction are

shown. B: (a) Ribbon model of

GSK3-b showing the interaction

of nitrofurazone (MSMS-MOL)

with the amino acids in the

binding pocket predicted by

CASTp server. (b) Molecular

surface model of GSK3-b with

the bound nitrofurazone (purple

and blue stick). (c) The

molecule nitrofurazone

enfolded in the ATP-binding

pocket of GSK 3-b showing

interacting amino acids: Val61,

Ile62, Gly63, Asn64, Val70,

Lys85, Glu97, Leu132, Asp133,

Tyr134, Val135, Pro136,

Glu137, Asp181, Asp183,

Asp186, Cys199, and Asp200

(MSMS-MOL). (d) Hydrogen

bond between the nitrofurazone

(stick and ball colored by green)

and the ASP-200, SER-203 and

ASP264 residues of GSK 3-b.

C: (a) Molecular surface model

of GSK3-b with the bound

CHIR_98014 (purple and blue

stick). (b) Hydrogen bond

between the CHIR_98014

(purple and blue stick) and the

VAL-135 and ASN-186

residues of GSK 3-b (Color

figure online)

Med Chem Res

123

formed capillaries, fibroblasts, and leukocytes. As more

and more collagen fibers laid down, vascularization tissue

decreases. The breaking strength of the granulation tissue

increases proportionately with the collagen deposition. Due

to oral administration of the dehydroabietylamine, the

breaking strength of the granulation tissue was increased

(Hukkeri et al., 2006; Kumara Swamy et al., 2007; Harish

et al., 2008; Vidya et al., 2012; Ahamed et al., 2013).

Angiogenesis is the term used for processes leading to

the generation of new blood vessels through sprouting from

already existing blood vessels in a process involving the

migration and proliferation of endothelial cells from pre-

existing vessels. Blood vessel growth occurs in the embryo

and rarely in the adult with exceptions such as the female

reproductive system, wound healing, and pathological

processes such as cancer (Carmeliet, 2000; Carmeliet and

Jain, 2000; Patan, 2004).

A plethora of experiments has suggested that the

angiogenic factors secreted by tissues will promote angio-

genesis under conditions of poor blood supply during nor-

mal and pathological angiogenesis processes. Angiogenic

molecules are generated by tumor, inflammatory (due to

wound formation), and connective tissue cells in response to

hypoxia and other as yet ill-defined stimuli. Angiogenic

activities include a number of other compounds such as

prostaglandins E1 and E2, steroids, heparin, 1-butyryl

glycerol (monobutyrin) secreted by adipocytes, and many

undefined derivatives of the arachidonic acid metabolism.

The biologically active principle extracted from plant sec-

ondary metabolites and factors such as nicotinic amide are

also a potent angiogenic compound in several bioassays,

although its mechanism of action remains to be elucidated.

These factors and compounds differ in cell specificity and

also in the mechanisms by which they induce the growth of

new blood vessels (Patan, 2004).

In the present investigation, the CAM assay was per-

formed in order to evaluate the effect of dehydroabietyl-

amine along with control on angiogenesis process using

chick CAM, and it has exhibited a significant increase in

neoangiogenesis, in the studied groups. Angiogenesis plays

a crucial role in a wide range of physiological events such

as wound healing, embryonic development and placental

implantation for the delivery of oxygen and nutrients as

well as removal of waste products. (Auerbach et al., 1974;

Ausprunk et al., 1974; Ausprunk and Folkman, 1977;

Richardson and Singh, 2003). The present study supports

the wound healing efficacy of the tested samples. Further,

by in silico analysis, it seems that the dehydroabietylamine

is promoting the cutaneous wound healing through the

elicitation of beta catenin-dependant Wnt pathway through

the inhibition of GSK3b (Harish et al., 2008; Vidya et al.,

2012; Ahamed et al., 2013).

In several reports, it has been documented that the plants

having antioxidant property would also enhance wound

healing activity (Shirwaikar et al., 2003; Ahamed et al.,

2013). In our earlier report, it has been proved that the

dehydroabietylamine, a diterpene compound, is a good

antioxidant agent (Paramesha et al., 2011). Therefore, the

dehydroabietylamine was promoted a faster wound healing

in all the three models viz, excision, incision, and dead

space models in rats. CAM assay also provide strong evi-

dence of wound healing capacity of dehydroabietylamine,

and the in silico analyses boost up the findings of in vivo

studies.

Acknowledgments The authors express their gratitude to The

Principal, Sahyadri Science College (Autonomous), Shivamogga, for

providing lab facility and encouragement. Thanks to Dr. Goutham

Chandra, Indian Institute of Science Bangalore, for rendering help in

animal work and support during the experimentation.

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