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Wound healing activity and docking of glycogen-synthase-kinase-3-[beta]-protein with isolated triterpenoid lupeol in rats.

Abstract

A triterpene compound lupeol isolated from petroleum ether extract of leaves of Celastrus paniculatus was screened for wound healing activity (8 mg/ml of 0.2% sodium alginate gel) by excision, incision and dead space wound models on Swiss Albino rats (175-225g). In lupeol treated groups wound healing activity was more significant (17.83[+ or -]0.48) than the standard skin ointment nitrofurazone (18.33[+ or -]0.42). Epithelialization of the incision wound was faster with a high rate of wound contraction (571.50[+ or -]5.07) as compared with the control group. In dead space wound model also the weight of the granulation tissue of the lupeol treated animal was increased indicating increase of collagenation and absence of monocytes.

The comparative docking of isolated lupeol molecule and standard drug nitrofurazone to glycogen synthase kinase3-[beta] protein by Wnt signaling pathway also supported the wound healing property of lupeol. The activation domain of GSK 3-[beta] consisted of Tyr216, with residues Asn64, Gly65, Ser66, Phe67, Gly68, Val70, Lys85, Leu132, Val135, Asp181 in the active pocket docked with lupeol at the torsional degree of freedom 0.5 units with Lamarckian genetic algorithm showed the inhibition constant of 1.38 x [10.sup.-7]. The inhibition constant of nitrofurazone was only 1.35 x [10.sup.-4].

[C] 2007 Elsevier GmbH. All rights reserved.

Keywords: Celastrus paniculatus; Lupeol; Wound healing; GSK3-[beta]; Wnt signaling; Docked

Introduction

Wound healing is a highly ordered and well coordinated process that involves inflammation, cell proliferation, matrix deposition, tissue remodeling, collagenation and epithelialization. Many investigators evaluated the wound healing properties of many of the medicinal herbs, clinically on animal models using excision, incision and dead space models (Angela et al., 2001; Kumara Swamy et al., 2007).

Wnts constitute a family of secreted glycoproteins with distinct expression patterns in the embryo and in the adult organism. Wnts appear to be involved in differentiation processes by controlling polarity of cell division, cell growth and cell fate. It is clear that the genes encoding for Wnts and other components of the pathway are expressed during regeneration of the skin (Fathke et al., 2004).

In this paper we have made an attempt to screen the wound healing property of the constituent lupeol isolated from the leaves of Celastrus paniculatus Willd. (Celastraceae) a woody climbing shrub, sparsely distributed in the hilly regions of India was screened in vivo on Swiss albino rats employing excision, incision and dead space models. The mode of action of the lupeol molecule was hypothesized in silico by docking the molecule to GSK3-[beta] protein an important regulatory enzyme whose inhibition promotes wound healing through [beta]-catanin dependent Wnt signalling pathway.

Materials and methods

Drug formulations

Two types of drug formulations were prepared for the compound lupeol. For topical administration 100 mg of lupeol was prepared in 20% sodium alginate to get 0.2% w/v gel. For oral administration, suspensions of 8 mg/ml isolated compound lupeol was incorporated with 1% w/v gum tragacanth. The drug formulations were prepared every third day and the drug was administered orally by a feeding tube. The study was permitted by the Institutional Animal Ethical Committee (Reg. No. 144/1999/CPCSEA/SMG).

Wound healing activity

Three groups of animals containing six each were used for each of the excision and incision wound models. The animals of group I were considered as the control, the animals of group II served as the reference standard and treated with 0.2% w/w nitrofurazone (Furacin, Smithklin Beecham, Bombay, India) ointment. The animals of group III were treated with the isolated constituent lupeol. Wound healing activity of the test drug lupeol was evaluated concomitantly following the methods of Morton and Malone (1972), Ehrlich and Hunt, (1968) and Lee and Tong (1968) for excision, incision and dead space wound models respectively. The results of the in vivo experiments are expressed as mean [+ or -] SE of six animals in each group. The data were evaluated by one-way ANOVA followed by Tukey's pair-wise comparison test. The values of p < 0.01 and p < 0.05 were considered as statistically significant.

Molecular docking

Automated docking was used to determine the orientation of inhibitors bound in the active site of GSK3-[beta]. A genetic algorithm method, implemented in the program AutoDock 3.0, was employed (Bhat et al., 2003). The ligand molecules, lupeol and nitrofurazone were designed and the structure was analyzed by using ChemDraw Ultra 6.0. 3D coordinates were prepared using PRODRG server (Ghose and Crippen, 1987) and preADMET server was used for drug likeliness prediction. The protein structure file 1Q5K was taken from PDB (www.rcsb.org/pdb) was edited by removing the heteroatoms, adding C terminal oxygen (Binkowski et al., 2003). For docking calculations, Gasteigere-Marsili partial charges (Gasteiger and Marsili, 1980) were assigned to the ligands and nonpolar hydrogen atoms were merged. All torsions were allowed to rotate during docking. The grid map, which was centered at the following residues of the protein (Val 61, He 62, Asn64, Gly65, Ser66, Phe67, Gly68, Val 70, Lys 85, Leu 132, Val 135, Pro 136, Asp 181, and Asp 200) were predicted from the CASTp server (Reya and Clevers, 2005) were generated with AutoGrid. The Lamarckian genetic algorithm and the pseudo-Solis and Wets methods were applied for minimization, using default parameters. The number of docking runs was 50, the population in the genetic algorithm was 250, the number of energy evaluations was 100,000, and the maximum number of iterations 10,000.

Results and discussion

Mammalian skin serves for a number of vital physiological functions to maintain homeostasis. The functional properties of skin are often underappreciated until substantial loss of the skin occurs. The existence of undifferentiated cells in the skin suggests that skin has the potential to regenerate, but the context of molecular signals after tissue injury promotes scar repair. In the present study the three different wound-healing models were employed to assess the wound healing potency of the constituent lupeol concurrently with the standard drug nitrofurazone. Many investigators used nitrofur-azone as a reference standard to assess the wound healing potency of the extracts and the constituents isolated from the plant source (Jaswanth et al., 2001; Singh et al., 2005). In all the three models studied the isolated constituent lupeol exhibited significant wound healing potency as compared to reference standard nitrofurazone and the control.

The excision wound model was employed to asses the potency of drug to promote the wound healing in Trauma types of wound which will be assesses by the rate of wound contraction and number of days required for complete epithelialization of the wound area. The period of epithelialization and percentage of wound contraction due to effect of drug molecule is shown in Table 1. In both these groups complete epithelialization was observed on the day 16. While in the control animals it was delayed up to the day 20. The mean time taken for complete epithelialization of the excision wound in lupeol treated group was less, than the animals treated with the reference drug nitrofurazone. The incision wound was made on the paravertebral region of the rat and the important parameters assed in the employment of incision wound is the measurement of skin breaking strength on 10th post wound day using tcnsiometer. A significant increase in the skin breaking strength of the wound was noticed in the animals treated with the constituent lupeol (571.50 0 [+ or -] 5.07) and the standard drug nitrofurazone (544.17 [+ or -] 8.70) and it was insignificant in control groups (363.67 [+ or -] 5.48) Table 2.
Table 1. Effect of topical application of isolated constituent
 lupeol on excision wound model

Treatment Percentage of closure of excision wound area
 (original wound area 250 m[m.sup.2])

 Day 4 Day 8 Day 12 Day 16

Control 246.33 193.33 67.33 67.33
 [+ or -] [+ or -] [+ or -] [+ or -]
 0.49 0.80 0.95 1.52

Nitrolurazone 244.50 187.50 94.83 44.00
 [+ or -] [+ or -] [+ or -] [+ or -]
 0.76 0.76 ** 1.11 ** 1.32 **

Lupcol 244.17 176.83 89.50 38.83
 [+ or -] [+ or -] [+ or -] [+ or -]
 0.91 0.60 ** 0.76 ** 0.87 *

F-value 2.5 132.2 186.8 143.9

Treatment Epithelialization
 in days

Control 19.67 [+ or -] 0.42
Nitrolurazone 18.33 [+ or -] 0.42*
Lupcol 17.83 [+ or -] 0.48*
F-value 4.6

Values are mean [+ or -] SE; n = 6 in each group.
** Significant at p 0.01 and * significant at p < 0.05 are
compared tocontrol.

Table 2. Effect of oral administration of isolated
constituent lupeol on incision wound model Group (n)
Breaking strength (g)

Control 363.67 [+ or -] 5.48
Nitrofurazone 544.17 [+ or -] 8.70 **
Lupeol 571.50 [+ or -] 5.07 *
F-value 291.1

Values are mean [+ or -] SE; n = 6 in each group.
** Significant at p < 0.01 and * significant at
p < 0.05 are compared to control.


In the dead space wound model the dry weight of the granulation tissue (82.17 [+ or -] 1.14) of lupeol treated group was significantly increased due to collagen maturation. The animals treated with nitrofurazone showed moderate collagenation and its dry weight is 62.17 [+ or -] 1.92 (Table 3). The histological sections of control animals showed the presence of macrophages and lesser collagen fibers (Fig. la). The increased collagenation of granulation tissue and lesser macrophages was observed in the animals treated with the lupeol (Fig. 1b).

[FIGURE 1 OMITTED]
Table 3. Effect or oral administration of isolated constituent lupeol
on dead space wound model

 Granulation
 tissue dry weight
Treatment (mg/100 g) Breaking strength (g)

Control 62.17 [+ or -] 1.92 ** 369.17 [+ or -] 2.74 **
Lupeol 82.17 [+ or -] 1.14 ** 561.50 [+ or -] 6.04 **
F-value 80.2 840.5

Values are mean [+ or -] SE; n = 6 in each group.
** Significant at p < 0.01 and * significant at p < 0.05 are
compared to control.


The earlier reports also indicated that Wnts are necessary for normal skin development (Reya and Clevers, 2005). Comparative docking of GSK3-[beta] with the lupeol and the standard drug nitrofurazone revealed that the docked energy for the lupeol was -9.48 with an estimated inhibition constant of 1.38 x [10.sup.-7], intermolecular energy -9.67. The docked energy of the nitrofurazone was only -6.37 with an inhibition constant of 1.35 x [10.sup.-4], with an intermolecular energy of -6.28. The lupeol was completely enfolded in the entire ATP binding pocket of GSK-3 [beta] (Fig. 2a) as compared to nitrofurazone. The topology of the active site of GSK-3 [beta] was similar in both lupeol and nitrofurazone, which is lined by interacting amino acids as predicted from the CASTp server (Figs. 2b, e). The lupeol (CPK indicated blue) found to sit in the proper orientation complementary to the topology of the ATP binding site (residues shown in MSMS-MOL) (Fig. 2c). However, orientation of lupeol molecule was perpendicular to the plane made by Val 135, Pro 136, and Asp 137 (Fig. 2d). The reactive group oxygen (OAI) of lupeol hydrogen bonded with the backbone hydrogen of Val 135 with a bond distance of 1.93 A units. Where as in nitrofurazone docking part of the molecule was oriented at this site and the rest was inclined exhibiting hydrogen bond between terminal oxygen and Asp200 (atoms OAM and OAG) (Fig. 2f). The bond distance of backbone hydrogen of Val 135 and oxygen of nitrofurazone radical was 1.94 A units. The earlier investigator (Bhat et al., 2003) also noticed that the backbone hydrogen of Val 135 was involved in hydrogen bonding with the ligands.

[FIGURE 2 OMITTED]

Lupeol has been proved to be one of the potent wound healing agent which has been shown to elicit the cutaneous wound healing better than the reference drugnitrofurazone. By Insilco analysis, it seems that lupeol is promoting the cutaneous wound healing through the elicitation of beta catanin dependant wnt pathway through the inhibition of GSK3bata.

Acknowledgements

The authors are grateful to the Registrar, Kuvempu University, Shimoga, Karnataka, Dr. B. Abdul Rahiman, Professor, and Dr.Riaz Mahmood, Professor, Department of Biotechnology and Bioinformatics, Kuvempu University, Dr. Satish B.G., Dr. Sanjay, Adithya Pathological Laboratory, Shimoga, Karnataka, and Dr. Madhusudhan, Sophisticated Analytical Instrument Facility, CDRI, Lucknow, India.

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B.G. Harish, V. Krishna *, H.S. SantoshKumar, B.M. Khadeer Ahamed, R. Sharath, H.M. Kumara Swamy

Department of Biotechnology and Bioinformatics, Kuvempu University, Shimoga 577451, Karnataka, India

* Corresponding author. Tel.: +91 8282256235; fax: +918282 256255.

E-mail address: krishnabiotech2003@gmail.com (V. Krishna).
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Author:Harish, B.G.; Krishna, V.; Kumar, H.S. Santosh; Ahamed, B.M. Khadeer; Sharath, R.; Swamy, H.M. Kumar
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Sep 1, 2008
Words:2468
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