DOCKING STUDIES ON XANTHONES OF MANGOSTEEN AS COX-2 …shodhganga.inflibnet.ac.in/bitstream/10603/6719/13/... · The inhibitor binding positions and affinity were evaluated using

Volume: 2: Issue-3: July-Sept -2011 ISSN 0976-4550

DOCKING STUDIES ON XANTHONES OF MANGOSTEEN AS COX-2 INHIBITORS

Navya. A1, Jayasimha Rayalu. D 2 and Uma Maheswari Devi. P1*

1, 1*Department of Applied Microbiology, Sri Padmavati Mahila Visvavidyalayam, Tirupati, Andhra Pradesh, India.

2Department of Bioinformatics, Global institute of Biotechnology, Hyderabad, Andhra Pradesh, India.

ABSTRACT : The prostaglandins found in most of the tissues and organs are synthesized by sequential oxidation of cyclooxygenases (COX-1 and COX-2). Prostaglandins synthesized by COX-1 are responsible for the protection of gastrointestinal tract and by COX-2 are responsible for inflammation and pain. The objective of this investigation was to characterize and determine the effect of α-mangostin, β-mangostin and γ-mangostin on COX-1 and COX-2. We have carried out the docking of α, β and γ-mangostin inhibitors into the three dimensional structure of COX-1 and COX-2 enzymes using GOLD software. The inhibitor binding positions and affinity were evaluated using GOLD scoring fitness functions. We identified that amino acid residues Leu52, Arg49, Val33 in COX-1 and Ala18, Ser23, Asp38, Cys22 in COX-2 are important for inhibitor recognition via hydrogen bonding interactions. These hydrogen bonding interactions play an important role for stability of the complex. This information can be exploited to design Mangostin based inhibitors. Our results may be helpful for further experimental investigations.Key words: Cyclooxygenases, Xanthones, Mangosteen, GOLD software and COX-2 inhibitors.

INTRODUCTIONProstaglandins (PGs) are the arachidonic acid (AA) metabolites of cyclooxygenase (COX) pathway and are major mediators in the regulation of inflammation and immune function (Smith et al., 2000). Cyclooxygenase (COX), also known as Prostaglandin endoperoxide H synthase (PGHS, EC.1.14.99.1), catalyzes the conversion of arachidonic acid to prostaglandins. This enzyme exists in two isoforms; PGHS-1 (COX-1) and PGHS-2 (COX-2), which has same enzymatic activities (Smith et al., 2000), (Smith et al., 1996), (Marnett et al., 1999). COX-1 and COX-2 enzymes are homodimers that are widely distributed heme proteins (Alex et al., 2011). Both enzymes are associated primarily with cell membrane structures; COX-1 primarily associated with the endoplasmic reticulum where as COX-2 on the nuclear envelope (Morita et al., 1995). In terms of amino acid composition, these enzymes are approximately 60% identical, and their catalytic regions are widely conserved (Picot et al., 1994), (Luong et al., 1996), (Kurumbail et al., 1996). Moreover, the two active sites of these isoforms differ only by two amino acids, at positions 513 (His for COX-1 and Arg for COX-2) and 523 (Ile for COX-1 and Val for COX-2) (Zhang et al., 1996).

International Journal of Applied Biology and Pharmaceutical Technology Page:263 Available online at www.ijabpt.com

http://www.ijabpt.com/

Devi et al ISSN 0976-4550

COX-1 is expressed constitutively in most mammalian tissues and is thought to be responsible for housekeeping functions of prostaglandins such as regulation of gastric response (Seibert et al., 1995), (Masferrer et al, 1994). COX-2 is an inducible enzyme that is thought to give rise to the increased prostaglandin levels produced during inflammation (Seibert et al., 1994). COX-2 gene is an early inducible gene in response to many inflammatory cytokines, including IL-1, TNF-α and Lipopolysaccharide (LPS). COX-2 gene expression is controlled at the transcriptional and post-transcriptional levels (Dixon et al., 2000). Because COX-2 isozyme was found to be over expressed during inflammation, drug investigation was focused on selective COX-2 inhibition, hoping to prevent inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) block the production of prostaglandins by inhibiting both COX-1 and COX-2. Most of these drugs are associated with well-known side effects at the gastrointestrial level and less frequently at the renal level. NSAIDs appear to produce at least some of their beneficial effects by inhibiting COX-2 and with lethal side effects by inhibiting COX-1 (Singh et al., 2009). Thus, there is a need to design new compounds with optimum COX-1 and COX-2 inhibition by docking. With our long standing interest in the transcriptional regulation based control of inflammation, we are particularly interested in xanthones derived from mangosteen of Garcinia mangostana, Mangosteen has been used as traditional medicine for the treatment of skin infection, wounds and diarrhea in south East Asia. (Nakatani et al., 2002). The main objective of the present study is to perform the docking analysis of xanthones of mangosteen.

METHODOLOGYThe molecular structures of α-mangostin (Figure-1), β-mangostin (Figure-2) and γ-mangostin (Figure-3) were generated and optimized using chemsketch software. The ligands were docked into Cyclooxygenase-1 (COX-1, PDB_ID: 3N8V) and Cycloxygenase-2 (COX-2, PDB_ID: 3NTG) using docking program GOLD 3.0.1. Hetero atoms were removed from the binding site and the chain A was selected for docking studies. Hydrogen atoms were added to COX-1 and COX-2 enzymes. The binding sites of the target enzymes were identified using CASTp server (Joe Dundas et al., 2006) based on precise computational geometry methods, including alpha shape and discrete flow theory. The chain A was selected for docking studies, hetero atoms were removed and hydrogen atoms were added to the binding site of COX-1 and COX-2 enzymes CASTp automatically locates and measures the volume and area of protein pockets and cavities. In addition CASTp provides information about the atoms lining pockets, pocket openings, and buried cavities; circumference of mouth openings.




Docking with GOLD 3.0.1GOLD (Genetic Optimization of Ligand Docking) a genetic algorithm (GA) based software, mainly utilizes an evolutionary strategy involving 3 genetic operators; cross overs, mutations and migrations (Jones et al., 1997). GOLD imports the partial flexibility to proteins and full flexibility to inhibitors. The compounds are docked into the active site of COX-1 and COX-2 and the interaction of these ligands with the active site residues are thoroughly studied using calculations of molecular mechanics. The parameters used for GA were population size (100), selection pressure (1.1), number of operations (10,000), number of island (1) and niche size. Operator parameters for crossover, mutation and migration were set to 100, 100 and 10 respectively. Default cutoff values of 3.0A° (dH-X) for hydrogen bonds and 6.0A° for vanderwaals were employed. The default algorithm speed was selected and the inhibitor binding site in the COX-1 and COX-2 was defined within a 10A° radius with the centroid as HH atom of PHE220 and ARG170 respectively. The number of poses for each inhibitor was set 100, and early termination was allowed if the top three bound conformations of inhibitors were within 1.5A° RMSD. After docking, the individual binding poses of each inhibitor were observed and their interactions with the protein were studied. The best and most energetically favorable conformation of each inhibitor was selected. GOLD Score fitness functionThe four components vig, Protein-ligand hydrogen bond energy (external H-bond); Protein-ligand vanderwaals energy (external vdw); Ligand internal vanderwaals energy (internal vdw); and Ligand intramolecular hydrogen bond energy (internal- H- bond) were considered for calculating the fitness function of GOLD score. The protein-ligand hydrophobic contact was encouraged by making an empirical correction by multiplying external vdw score with 1.375. The fitness function has been optimized for the prediction of ligand binding positions.

Gold Score = S (hb_ext) + S (vdw_ext) + S (hb_int) + S (vdw_int)Where S (hb_ext) is the protein-ligand hydrogen bond score, S (vdw_ext) is the protein-ligand vanderwaals score, S (hb_int) is the score from intramolecular hydrogen bond in the ligand and S (vdw_int) is the score from intramolecular strain in the ligand.RESULTS AND DISCUSSIONThe concept of docking is important to determine the properties associated with protein-ligand interactions such as binding energy, electron distribution, hydrogen bond donor acceptor properties and hydrophobicity. In the present study, CASTp server was used to found the possible binding site of COX-1 (Figure-4) and COX-2 (Figure-5). From the binding site analysis it was observed that binding pockets are identical both in COX-1 and COX-2 and the largest binding pocket was selected for the docking studies. Due to similar crystal structures, 3N8V and 3 NTG were used as representative structures for COX-1 and COX-2 respectively. The xanthone ligands were docked into COX-1 and COX-2 using GOLD 3.0.1 and all docking solutions for COX-1 and COX-2 were ranked according to the GOLD fitness function. The docking results showed that all the xanthone derivatives of mangosteen are active COX inhibitors with a significant preference for COX-2.




Among the three xanthones, α-mangostin and β-mangostin showed common hydrogen bond interactions with Leu52, Arg49 of COX-1. α-mangostin showed a bond length of 1.817A° and 2.656 A° (Figure-6); β-mangostin showed a bond length of 1.806 A° and 2.180 A° (Figure-7), and γ-mangostin showed a different pattern of hydrogen bonding with Val33 of COX-1 (Figure-8). On the other hand α-Mangostin extended O11 and O13 of its oxygen atoms to form two hydrogen bonds with Ala18 of COX-2 with a bond length of 2.019A° and 2.556A° respectively (Figure-9). Similarly, β-mangostin exhibited two hydrogen bonds with Asp38, Ser23 of COX-2; one bond is between oxygen atom of Asp38 with H39 and another bond is seen between hydrogen atom of Ser23 with O20 (Figure-10). As shown in Figure-11 γ-mangostin showed strong hydrogen bond interactions with Ala18, Cys22 and Asp38 of COX-2 between hydroxyl group of Ala18 and O22; other bondings observed between oxygen atom of Asp38 and H37, hydrogen atom of Cys22 and oxygen atom O21.

International Journal of Applied Biology and Pharmaceutical Technology Page: 266 Available online at www.ijabpt.com



The atoms involved in hydrogen bonding, their bond lengths and docking energies of all the three ligands based on GOLD fitness score were indicated for COX-1 (Table-1) and COX-2 (Table-2). The results deduce that all the three ligands were potential against COX-2 and they are ranked as γ-mangostin > α-mangostin >β-mangostin.Table 1: Docking score and bonding of mangostins with COX-1 using GOLD 3.0.1

Molecule No. of Hydrogen

bonds

Atoms Bond length (Aº)

Docking score (kcal/mol)Protein molecule

α-mangostin 2 Arg49(HH2) Leu52(O)

O(21)H(41)

2.6561.817

14.77

β-mangostin 2 Arg49(HH2)Leu52(O)

O(21)H(41)

2.1801.806

10.20

γ-mangostin 1 Val33(O) H(50) 2.488 16.20Table 2: Docking score and bonding of mangostins with COX-2 using GOLD 3.0.1

Molecule No. of Hydrogen

bonds

Atoms Bond length (Aº)

Docking score (kcal/mol)Protein molecule

α-mangostin 2 Ala18(H2) Ala18(H1)

O(11) O(13)

2.019 2.556

22.33

β-mangostin 2 Asp38(O) Ser23(HG)

H(39) O(20)

1.894 2.122

21.34

γ-mangostin 3 Ala18(H1)Cys22(H)

Asp38(OD1)

O(22)O(21)H(37)

1.6672.6301.980

22.91

ACKNOWLEDGEMENTWe gratefully acknowledge the Department of Science and Technology for the financial support.

REFERENCES

1. Alex JV and Michael GM., The structural basis of endocannabnoid oxygenation by Cyclooxygenase 2. J. Biol. Chem. 2011; 286(26): 1-20.

2. Dixon DA, Kaplan CD and McIntyre TM., Post-transcriptional control of cyclooxygenase-2 gene expression. The role of the 3′-untranslated region. J. Biol. Chem. 2000; 275: 11750-11757.

3. Joe Dundas, Zheng Ouyang, Jeffery Tseng, Andrew Binkowski, Yaron Turpaz, and Jie Liang., CASTp: computed atas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucl. Acids Res. 2006; 34: W116-W118.

4. Jones G, Willett P, Glen RC, Leach AR and Taylor R., Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 1997; 267: 727-748.

5. Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ, Stegeman RA, Pak JY, Gildehaus D, Miyashiro JM, Penning TD, Seibert K, Isakson PC and Stallings WC., Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature.1996; 384: 644-648.

6. Luong C, Miller A, Barnett J, Chow J, Ramesha C and Browner MF., Flexibility of the NSAID binding site in the structure of human cyclooxygenase-2. Nat. Struct. Biol. 1996; 3: 927-933.

7. Marnett LJ, Rowlinson SW, Goodwin DC, Kalgutkar AS and Lanzo CA., Arachidonic acid oxygenation by COX-1 and COX-2. Mechanisms of catalysis and inhibition. J. Biol. Chem. 1999; 274(33): 22903-22906.




8. Morita I, Schindler M, Regier MK, Otto JC, Hori T and DeWitt DL., Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J.Biol. Chem. 1995; 270: 10902-10908.

9. Masferrer JL, Zweifel BS, Manning PT, Hauser SD, Leahy KM, Smith WG, Isakson PC and Seibert K., Selective inhibition of inducible cyclooxygenase-2 in vivo is antiinflammatory and nonulcerogenic. Proc. Natl. Acad. Sci. USA. 1994; 91: 3228-3232.

10.Nakatani K, Nakahata N, Arakawa T, Yasuda H and Ohizumi Y., Inhibition of cyclooxgenase and prostaglandia E2 synthesis by γ-mangostin, a xanthone derivative in mangosteen, in C6 rat glioma cells. Biochem Pharmacol. 2002; 63: 73–79

11.Picot D, Loll PJ and Garavito RM., The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature.1994; 367: 243-249.

12. Smith WL, Dewitt DL and Garavito RM., Cyclooxygenase: structural, cellular and molecular biology. Annu. Rev. Biochem. 2000; 69: 145-182.

13. Smith WL and DeWitt DL., Prostaglandin endoperoxide H synthases-1 and -2. Adv. Immunol. 1996; 62: 167-215.

14. Seibert K, Masferrer J, Zhang Y, Leahy K, Hauser S, Gierse J, Koboldt C, Anderson G, Bremer M, Gregory S and Isakson P., Expression and selective inhibition of constitutive and inducible forms of cyclooxygenase. Adv. Prostaglandin Thromboxane Leukotriene Res. 1995; 23: 125-127.

15. Seibert K, Zhang Y, Leahy K, Hauser S, Masferrer J, Perkins W, Lee L and Isakson P., Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc. Natl. Acad. Sci. USA.1994; 91: 12013-12017.

16. Singh R, Kumar R and Singh DP., Nitric oxide-releasing nonsteroidal anti-inflammatory drugs: gastrointestinal-sparing potential drugs. J.Med. Food. 2009; 12(1): 208–218.

17. Zhang V, O’Sullivan M, Hussain H, Roswit WT and Holtzman MJ., Molecular cloning, functional expression, and selective regulation of ovine prostaglandin H synthase-2. Biochem. Biophys. Res. Commun. 1996; 227: 499-506.



International Journal of Applied Biotechnology and Biochemistry. ISSN 2248-9886 Volume 2, Number 1 (2012) pp. 69-80 © Research India Publications http://www.ripublication.com/ijabb.htm

In vivo and in silico Analysis Divulges the Anti-Inflammatory Activity of α-Mangostin

Navya A.1, Nanda Kumar Y.2, Hari Prasad O.2, Santhrani T.3 and Uma Maheswari Devi P.1*

1,1*Department of Applied Microbiology,

Sri Padmavati Mahila Visvavidyalayam, Tirupati-517502, Andhra Pradesh, India. 2Division of Animal Biotechnology, Department of Zoology, S. V. University,

Tirupati-517502, Andhra Pradesh, India. 3Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam,

Tirupati-517502, Andhra Pradesh, India. 1*Corresponding Author E-mail: [email protected]

Abstract

α-Mangostin, a bioactive natural product derived from the pericarp of Mangosteen fruit belongs to Garcinia mangostana, showed potential anti-inflammatory activity against the paw oedema experimentally induced by carrageenan. In this study, α-Mangostin with known action on inflammation has been examined in silico as a ligand against Cyclooxygenase-2 (COX-2) and inducible Nitric Oxide Synthase (iNOS), utilizing AutoDock 3.0 as docking tool. Comparative docking of Indomethacin and α-Mangostin with COX-2 suggested a similar hydrogen bond interaction with Ser353. In addition, Indomethacin showed hydrogen bonds with Arg120 and Trp387, and these residues lie at the junction of the anchoring site of the COX-2 active site. The lack of selectivity towards Arg120 may be a significant component of α-Mangostin selectivity towards COX-2. The docking energies of α-Mangostin and Indomethacin with iNOS were found to be -12.20kcal/mol and -2.75 kcal/mol respectively. In the present study, the predicted Pharmacokinetic (PK) values of α-Mangostin and Indomethacin (NSAID) deduce that α-Mangostin satisfies all PK parameters and has qualified as best lead candidate as an anti-inflammatory agent compared to Indomethacin. Keywords: Mangosteen, Paw oedema, Comparative docking, Cyclooxygenase-2, Indomethacin, Hydrogen bonds, Pharmacokinetic properties and Anti-inflammatory agent.

70 Navya A. et al

Introduction A major challenge of modern medicine is to design compounds that modulate specific enzymes while leaving related isozymes unaffected. The two notable enzymes namely Cyclooxygenase-2 (COX-2) and inducible Nitric Oxide Synthase (iNOS) are important mediators of an inflammatory process. Nonsteroidal Anti-inflammatory drugs (NSAIDs) like Indomethacin act via inhibition of COX enzyme, COX catalyzes the first step of the biosynthesis of prostaglandins [1]. Prostaglandins (PGs), found in most of the tissues and organs, are the arachidonic acid metabolites of the Cyclooxygenase (COX) pathway and are major mediators in the regulation of the inflammation and immune function [2]. It has been shown that the COX enzyme exists in two isoforms COX-1 and COX-2 [3]. In terms of amino acid composition, these enzymes are approximately 60% identical, and their catalytic regions are widely conserved [4,5,6]. COX-1 enzyme is responsible for maintaining gastric and renal integrity and COX-2 is an inducible enzyme responsible for the production of pro-inflammatory PGs causing inflammation and pain [7]. The COX-2 inhibitors are effective for the relief of chronic pain in elderly patients with osteoarthritis and rheumatoid arthritis [8]. Inducible Nitric Oxide Synthase (iNOS), is another inducible enzyme, that plays a significant role in the over production of nitric oxide (NO) and has been implicated in several pathophysiological states, for example; various inflammation, septic shock, vascular dysfunction in diabetes and cancer patients [9,10]. Three homologous NOS isozymes [inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS)] catalyze the five-electron, two- step oxidation of L-arginine (L-Arg) to form nitric oxide which is an important biological signaling molecule and cellular cytotoxin [11]. The constitutive isozymes, eNOS and nNOS, function to produce low levels of NO predominantly for blood pressure regulation and nerve function respectively. In contrast, iNOS is induced by microbial products, such as lipopolysaccharide (LPS) and inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α) and interferon-γ (INF-γ) in macrophages and some other cells [12]. COX-2 and iNOS over expression has been observed in many human invasive malignant tumors, e.g. breast, lung, prostate, bladder, colorectal cancer and malignant melanoma [13,14,15,16]. Therefore, the modulation of iNOS and COX-2 can be a good strategy for the management of diseases accompanying the overproduction of NO and PGs. With our long standing interest in the transcriptional regulation based control of inflammation, we are particularly interested in α-Mangostin, a Xanthone derivative from the pericarp of Mangosteen fruit belongs to Garcinia mangostana tree of South East Asia with renowned medicinal usage. The objectives of the present study are; i) to obtain binding and inhibitory parameters of α-Mangostin and Indomethacin (NSAID) on COX-2 and iNOS by means of AutoDock, ii) prediction of their absorption and distribution properties. Materials and Methods Carrageenan induced paw oedema in rats The anti-inflammatory activity of α-Mangostin was determined by inducing acute

In vivo and in silico Analysis Divulges the Anti-Inflammatory Activity 71

inflammation by carrageenan in rats [17]. The Institutional Animal Ethical Committee approved protocols were followed for experimental analysis. All the animals were acclimatized for a week before use and were grouped in polyacrylic cages and maintained under standard laboratory conditions. The room temperature was maintained at 25 ± 2ºC with dark and light cycle of 14/10h. They were fed on commercial diet and water ad libitum. The rats were divided into five groups of six animals each. The first group, referred as control received normal saline (0.9% w/v, 3ml/kg/p.o). Second group with Indomethacin (10mg/kg/p.o) served as standard where as third, fourth and fifth groups were orally administered with 0.5mg/kg/p.o, 5mg/kg/p.o and 10mg/kg/p.o of 40% HPLC purified α-Mangostin (Indfrag Company, Bangalore, India) respectively with the help of an oral catheter. Food was withdrawn overnight but adequate supply of water was given to rats before the experiments. After 1h of drug treatment, a subplantar injection of 1% solution of carrageenan was administered in the left hind paw of all five groups. The volume of paw oedema was measured with Plethysmometer (UGO Basile, USA) after 3h of injections. The average paw volume was calculated and compared with control and standard to determine the anti-inflammatory activity of α-Mangostin. The percentage of paw oedema inhibition was calculated using the formula; Inhibition of oedema (%) = (Oc-Ot / Oc) x 100 Where, ‘Oc’ is oedema volume of control group and ‘Ot’ is oedema volume of treated groups. Preparation of protein structures and prediction of binding sites The experimental coordinates of COX-2 (PDB_ID:1CX2) and iNOS (PDB_ID:1NS1) structures were taken from PDB (rcsb.org/pdb/). The active sites of COX-2 and iNOS were identified using CASTp server [18] based on precise computational geometry methods, including alpha shape and discrete flow theory. Among the active site residues, the important residues were selected compared to previous data [19, 20]. Ligands were removed from the binding sites and the chain A was selected for COX-2 and chain B for iNOS docking studies. Hetero atoms were removed and polar hydrogen atoms were added to protein structures and partial atomic charges were assigned. The proteins were saved in MOL2 format; atomic solvation parameters were assigned and converted finally into PDBQS format. The Molecular displays were created by Swiss-PdbViewer (ca.expasy.org/spdbv/) and RasMol (openrasmol.org/). Preparation of ligand structures The molecular structures of α-Mangostin and Indomethacin were generated and optimized using ACD/ChemSketch software (acdlabs.com/download/sda). The ligands were saved in PDBQ format using Deftors to define torsions during docking analysis. Molecular docking Docking of α-Mangostin and Indomethacin was carried out against COX-2 and iNOS using AutoDock 3.0 (scripps.edu/pub/olson-web/doc/autodock/). AutoDock is widely

72 Navya A. et al

distributed public domain molecular docking software [21]. It consists of the components like AutoGrid and AutoTors and uses Monte Carlo simulated annealing and Lamarckian genetic algorithm to create a set of possible conformations. This program addresses automatically the flexible docking of the ligands into a known protein structure. The proteins for each docking were kept rigid and torsional flexibility was permitted to the ligands. The rotatable bonds in the ligands were defined using AutoTors and grid maps were calculated using AutoGrid. The search was conducted in a grid points of 48x54x62 for COX-2 and 52x56x48 for iNOS in three dimensions built in x, y, and z directions with 0.375A˚ spacing centered on the binding site of macromolecules. Each docking experiment consisted of 10 docking runs with 150 individuals. The default settings were used for all other parameters. The AutoDock results gives the binding energy and bound conformations of docked structures. The resultant structure files were analyzed using RasMol visualization programs. Pharmacokinetic Properties The bioavailability of α-Mangostin and Indomethacin was determined using PK/DB Database (PK/DB:http://miro.ifsc.usp.br/pkdb/). The ligand structures were manually edited using PK/DB sketcher, then converted into SMILES format and searched for pharmacokinetic properties like human intestinal absorption (%HIA), human oral bioavailability (%F), plasma protein binding (%PPB), blood brain barrier (logBB) and water solubility (logS) by Hologram QSAR Technique [22]. Results The paw oedema experimentally induced by carrageenan is the most commonly employed method for the evaluation of anti-inflammatory activity. The α-Mangostin showed significant anti-inflammatory activity in dose dependant manner (Table-1) by restricting the paw oedema volume to 0.883±0.012 with 40.21% oedema inhibition at a dose of 10mg/kg after 3h of treatment where as the standard drug Indomethacin (10mg/kg) showed 0.910±0.070 volume of paw oedema with 38.38% oedema inhibition. To identify a potential anti-inflammatory lead compound between α-Mangostin and Indomethacin individual docking studies were performed using AutoDock 3.0 against COX-2 (Figure-1A) and iNOS (Figure-2A) enzymes. The active sites of target enzymes were predicted by CASTp (Figure-1B, Figure-2B) and key binding site residues were used for docking studies (Table-2). The α-Mangostin (Figure-3) and Indomethacin (Figure-4) structures were generated using ChemSketch software. Among the various binding poses in the active site, the best pose for both the ligands and most stable conformation was selected based on docking energy. The α-Mangostin with COX-2 (Figure-5) showed a docking energy of -12.01kcal/mol which is two folds greater compared to the docking energy -5.96kcal/ mol of Indomethacin with COX-2 (Table-3). The docking of Indomethacin with COX-2 demonstrates that the inhibitor makes hydrogen bonds with three residues Arg120, Ser353 and Trp387 and these residues lie at the junction of the anchoring site of the COX-2 active site


(Figure-6). But the complex of α-Mangostin with COX-2 showed two hydrogen bonds with Ser530 and Ser353 (Figure-5) with a bond length of 1.927A˚ and 1.724A˚ respectively (Table-3). The docking energies of α-Mangostin and Indomethacin with iNOS were found to be -12.20kcal/mol and -2.75kcal/mol respectively (Table-3). However, the hydrogen bonds were observed at different positions with different residues of iNOS. α-Mangostin showed a single hydrogen bond of 2.012A˚ with Tyr489 (Figure-7, Table-3) and Indomethacin showed 2 hydrogen bonds of 2.013A˚ and 2.076A˚ with Trp346 and Gln263 respectively (Figure-8, Table-3). Computational methods have emerged as a powerful strategy to predict human pharmacokinetic properties earlier in the investigation of lead candidates to reduce the failures in late stages of drug development. The Pharmacokinetic properties (PK) of α-Mangostin and Indomethacin were determined using PK/DB Database. It is the measure of the rate/kinetics of absorption, distribution, metabolism and excretion (ADME). The obtained Pharmacokinetic (PK) values of α-Mangostin and Indomethacin were tabulated in Table-4. Discussion A systematic approach was made to find out the efficacy of α-Mangostin against inflammation so as to exploit it as herbal anti-inflammatory agent. It is well known that carrageenan induced oedema is characterized by biphasic response with the involvement of different inflammatory mediators. The late phase (3-4h) is mediated with the release of prostaglandins [23]. Our results indicate that the administration of α-Mangostin inhibited the oedema during all phases of inflammation probably by inhibiting the chemical mediators of inflammation. A drug intended for use in humans should have an ideal balance of efficacy and safety, as well as good Pharmacokinetic (PK) properties [24]. Absorption and distribution, the part of pharmacokinetics, were considered as important parameters to choose compounds as drug candidates. Prediction of in silico ADME properties has been developed to reduce the probability of the failure at the development stage of drug candidates. The values for HIA (%), F (%) and PPB (%) must be ≤ 100; BBB (logBB) value must be ≤ 3 and Solubility (logS) was measured at 20-25ºC, pH 7.5 [22]. In the present study the predicted PK values of α-Mangostin and Indomethacin deduce that α-Mangostin satisfies all PK parameters and has qualified as best lead candidate compared to Indomethacin. By using the AutoDock 3.0, we investigated the molecular interaction mechanisms of α-Mangostin and Indomethacin with the two enzymes namely COX-2 and iNOS, which are involved in chronic inflammation. Docking of Indomethacin and α-Mangostin exhibited one hydrogen bond with Ser353 even though they are not at all structurally similar. Indomethacin, a classical nonselective COX inhibitor, binds deeply within the COX [6]. The crystal structure of a complex of Indomethacin with COX-2 (PDB_ID:1CX2) demonstrates that the inhibitor makes hydrogen bonds with residues Arg120 and Trp387 and these residues lie at the junction of the anchoring site of the COX-2 active site. Arg120, the guanidinium group of which stabilizes the carboxylate of classical NSAIDs, is one the few charged residues in the hydrophobic COX [6]. Indomethacin causes a slow, time dependent inhibition of both COX-1 and COX-2. The time dependence of inhibition may result from the formation of salt

74 Navya A. et al

bridge between the carboxylate of Indomethacin and Arg120 from helix D of both COX-1 and COX-2 [25]. But the complex of α-Mangostin with COX-2 (PDB_ID:1CX2) showed two hydrogen bonds with Ser530 and Ser353 and importantly, neither Tyr355 nor Arg120 contact the α-Mangostin. The lack of selectivity towards Arg120, similar to SC-558, may be a significant component of its selectivity towards COX-2. Another feature that clearly indicates the selectivity of α-Mangostin towards COX-2 from the other classical NSAIDs is the absence of carboxylate group in α-Mangostin. The active site of iNOS is more or less in an inverted pear-shape with the guanidine moiety of substrate occupying the farther end of the site above the heme and its tail part protruding into the middle pocket, substrate access channel [26]. The R-amino function of L-Arg is a key factor for binding at the NOS active sites and that a highly conserved crucial Glu residue (Glu377 in human iNOS) forms a salt bridge with the guanidine moiety of L-Arg and also hydrogen bonds with its R-NH2 group [27]. But, in the present study the iNOS docking showed different pattern of hydrogen bond interaction; α-Mangostin formed a single hydrogen bond with Tyr489 in contrast Indomethacin formed two hydrogen bonds with Trp346 and Gln263. On the basis of the bioavailability score, docking energies and in vivo anti-inflammatory results we deduce that α-Mangostin has potential anti-inflammatory activity compared to Indomethacin. Conclusion The in silico pharmacokinetic properties, docking energies and in vivo studies reveals the anti-inflammatory activity of α-Mangostin and it can be recommended as a potent anti-inflammatory agent compared to Indomethacin. Acknowledgement We are immensely thankful to UGC Non-SAP for the financial assistance by awarding RFSMS (Research Fellowships in Sciences, Medical Sciences and Engineering Sciences for Meritorious Students) JRF to A. Navya to carry out this investigation.

Table 1: Effect of α-Mangostin on carrageenan induced paw oedema in rats

Treatment Oedema volume

(ml) ± SEM Percentage of

oedema inhibition Control 1.477±0.117 - Indomethacin (10mg/kg) 0.910±0.070* 38.38 α-Mangostin (0.5mg/kg) 1.157±0.042* 21.66 α-Mangostin (5mg/kg) 1.097±0.038* 25.72 α-Mangostin (10mg/kg) 0.883±0.012* 40.21

N=6, Data given are mean of three replicates ± SEM (Standard Error of Mean)

*p<0.001 when compared to control based on a Student’s‘t’ test.


Table 2: Binding site residues of COX-2 and iNOS

Protein Binding site residues COX-2 His90, Leu117, Arg120, Gln192, Val349, Leu352, Ser353, Tyr355,

Leu359, Trp387, Arg513, Ala516, Phe518, Met522, Val523, Gly526, Ala527, Ser530 and Leu534.

iNOS Trp194, Cys200, Gln263, Trp346, Pro350, Val352, Phe369, Gly371, Trp372, Tyr373, Met374, Glu377, Asp382, Ile462, Trp463, Tyr489.

Table 3: Docking energies and bond lengths of docked ligands using AutoDock 3.0

Ligand Protein No. of

Hydrogen Bonds

Bond lengths

(Ao)

Aminoacids involved in Hydrogen Bonding

Docking Energy

(kcal/mol)

α-Mangostin

COX-2 2 1.927 Ser530 -12.01 1.724 Ser353

iNOS 1 2.012 Tyr489 -12.20

Indomethacin

COX-2

3

1.361 Trp387 -5.96 3.095 Arg120

2.149 Ser353 iNOS 2 2.013 Trp346 -2.75

2.076 Gln263

Table 4: Pharmacokinetic (PK) Properties of α-Mangostin and Indomethacin

Compound HIA (%)

F (%)

PPB (%)

BBB (logBB)

Solubility (logS)

α-Mangostin 60.65 48.66 84.80 -0.05 -5.97 Indomethacin 104.31 92.28 102.46 -1.05 -5.31

Figure 1: A) COX-2 protein structure and B) COX-2 active site

76

Figure 2: A)

F

F

) iNOS protein structure and B) iNOS active s

Figure 3: Structure of α-Mangostin

Figure 4: Structure of Indomethacin

Navya A. et al

site

In vivo and in silico Analy

Figure 5: Dockin

Figure 6: Dockin

ysis Divulges the Anti-Inflammatory Activity

ng of α-Mangostin with COX-2 using AutoDo

g of Indomethacin with COX-2 using AutoD

77

ock 3.0

ock 3.0

78 Navya A. et al

Figure 7: Docking of α-Mangostin with iNOS using AutoDock 3.0

Figure 8: Docking of Indomethacin with iNOS using AutoDock 3.0


References

[1] Dannhardt, G., and Kiefer, W., 2001, “Cyclooxygenase inhibitors - current status and future prospects,” Eur J Med Chem., 36, pp.109-126.

[2] Smith, W.L., Dewitt, D.L., and Garavito, R.M., 2000, “Cyclooxygenase: structural, cellular and molecular biology,” Annu Rev Biochem., 69, pp.145-182.

[3] Marnett, L.J., Rowlinson, S.W., Goodwin, D.C., Kalgutkar, A.S., and Lanzo, C.A., 1999, “Arachidonic acid oxygenation by COX-1 and COX-2. Mechanisms of catalysis and inhibition,” J Biol Chem., 274(33), pp. 22903-22906.

[4] Picot, D., Loll, P.J., and Garavito, R.M., 1994, “The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1,” Nature., 367, pp. 243-249.

[5] Luong, C., Miller, A., Barnett, J., Chow, J., Ramesha, C., and Browner, M.F., 1996, “Flexibility of the NSAID binding site in the structure of human cyclooxygenase-2,” Nat Struct Biol., 3, pp. 927- 933.

[6] Kurumbail, R.G., Stevens, A.M., Gierse, J.K., McDonald, J.J., Stegeman, R.A., Pak, J.Y., Gildehaus, D., Miyashiro, J.M., Penning, T.D., and Seibert, K., 1996, “Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents,” Nature., 384, pp. 644-648.

[7] Seibert, K., Zhang, Y., Leahy, K., Hauser, S., Masferrer, J., Perkins, W., Lee, L., and Isakson, P., 1994, “Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain,” Proc Natl Acad Sci., 91, pp. 12013-12017.

[8] Savage, R., 2005, “Cyclo-oxygenase-2 inhibitors: when should they be used in the elderly?,” Drugs Aging., 22(3), pp. 185-200.

[9] Thiemermann, C., Wu, C.C., Szabo, C., Perretti, M., and Vane, J.R., 1993, “Role of tumour necrosis factor in the induction of nitric oxide synthase in a rat model of endotoxin shock,” Br J Pharmacol., 110(1), pp. 177-182.

[10] Halliwell, B., 1994, “Free radicals, antioxidants, and human disease: curiosity, cause, or consequence?,” Lancet., 344, pp. 721-724.

[11] Griffith, O.W., and Stuehr, D.J., 1995, “Nitric oxide synthases: properties and catalytic mechanism,” Annu Rev Physiol., 57, pp. 707-736.

[12] Mari, H., Riina, N., Pia, V., Marina, H., and Eeva, M., 2007, “Anti-Inflammatory Effects of Flavonoids: Genistein, Kaempferol, Quercetin, and Daidzein Inhibit STAT-1 and NF-κB Activations, Where as Flavone, Isorhamnetin, Naringenin, and Pelargonidin inhibit only NF-κB Activation along with their Inhibitory Effect on iNOS Expression and NO Production in Activated Macrophages,” Mediators Inflamm., 45673, pp. 1-10.

[13] Soslow, R.A., Dannenberg, A.J., Rush, D., Woerner, B.M., Khan, K.N., Masferrer, J., and Koki, A.T., 2000, “COX-2 is expressed in human pulmonary, colonic, and mammary tumors,” Cancer., 89, pp. 2637-2645.

[14] Ermert, L., Dierkers, C., and Ermert, M., 2003, “Immunohistochemical expression of cyclo-oxygenase isoenzymes and downstream enzymes in human lung tumors,” Clin Cancer Res., 9, pp. 1604-1610.

80 Navya A. et al

[15] Koki, A.T., and Masferrer, J.L., 2002, “Celecoxib: a specific COX-2 inhibitor with anticancer properties,” Cancer Control., 9(2), pp. 28-35.

[16] Lirk, P., Hoffmann, G., and Rieder, J., 2002, “Inducible nitric oxide synthase-time for reappraisal,” Curr Drug Targets Inflamm Allergy., 1(1), pp. 89-108.

[17] Winter, C.A., Risky, E. A., and Nuss, G.W., 1962, “Carrageenan induced oedema in hind paw of the rat as an assay for anti-inflammatory drugs,” Proc Soc Exp Biol Med., 11I, pp. 544-547.

[18] Joe, D., Zheng, O., Jeffery, T., Andrew, B., Yaron, T., and Jie, L., 2006, “CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues,” Nucl Acids Res., 34, pp. W116-W118.

[19] Souhila, B.T., Amel, T.M., Boubekeur, M., Safia., and Tairi.K., 2010, “Modeling the binding modes of stilbene analogs to cyclooxygenase-2: a molecular docking study,” J Mol Mod., 16, pp. 1919-1929.

[20] Marialuigia, F., Cristina, M., Fabio, L., Antonia, P., Simona, M., Mirko, P., Lorenza, S., Alessandra, A., Barbara, D.F., Letizia, G., Nazzareno., and Rosa, A., 2011, “Selective Inhibition of iNOS by Benzyl- and Dibenzyl Derivatives of N-(3-Aminobenzyl) acetamidine,” Chem Med Chem., 6, pp. 1203-1206.

[21] Thomas, J.L., Mack, V.L., Glow, J.A., Moshkelani, D., Terrell, J.R., and Bucholtz, K.M., 2008, “Structure/function of the inhibition of human 3beta-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane,” J Steroid Biochem Mol Biol., 111(1-2), pp. 66-73.

[22] Tiago, L.M., Leonardo, G.T., Alexandre, E.C., and Adriano, D.A., 2008, “PK/DB:database for pharmacokinetic properties and predictive in silico ADME models” Bioinformatics., 24(19), pp. 2270-2271.

[23] Hernandez, P.M., and Rabanal Gallego, R.M., 2002, “Evaluation of the anti- inflammatory and analgesic activity of Sideritis canariensis var. pannosa in mice,” J Ethanopharmacol., 81, pp. 43-47.

[24] Tiago, L.M., Montanari, C.A., and Andricopulo, A.D., 2007, “Hologram QSAR model for the prediction of human oral bioavailability,” Bioorg Med Chem., 15, pp. 7738-7745.

[25] Callan, O.H., So, O.Y., and Swinney, D.C., 1996, “The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen,” J Biol Chem., 271(7), pp. 3548-3554.

[26] Sandrea, M.F., Amit, M., Manishika, S., and Prasad, V.B., 2008, “Design of Benzene-1,2- diamines as selective inducible nitric oxide synthase inhibitors: a combined de novo design and docking analysis,” J Mol Mod., 14(3), pp. 215-224.

[27] Crane, B.R., Arvai, A.S., Ghosh, D.K., Wu, C., Getzoff, E.D., Stuehr, D.J., and Tainer, J.A., 1998, “Structure of nitric oxide oxygenase dimer with pterin and substrate,” Science., 279, pp. 2121-2126.

356Current Trends in Biotechnology and PharmacyVol. 6 (3) 356-363 July 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)

AbstractThe anti-inflammatory activity was

evaluated using acute and chronic inflammatorymodels like carrageenan induced paw oedemaand cotton pellet induced granuloma modelsrespectively. Oral administration of α -Mangostinshowed dose-dependent and significant anti-inflammatory activity both in acute and chronicphases of inflammation. Antioxidant propertiesof α-Mangostin analyzed by DPPH, nitric oxide,superoxide radical scavenging assays showedenmarked free radical scavenging activity of α-Mangostin. Lipid peroxidation was also drasticallyinhibited by α-Mangostin in concentrationdependent manner and showed an IC

50 value of

900μg/ml. The present findings clearly validatesthe traditional use of α -Mangostin by confirmingthe anti-inflammatory and antioxidant potentialof α-Mangostin.

Keywords: α-Mangostin, Xanthone, Anti-inflam-mation, Lipid peroxidation and Mangosteen.

Introductionα-Mangostin, a xanthone derivative, is a

major bioactive compound found in the fruit hullof Mangosteen and belongs to Garciniamangostana tree of South East Asia. The wholeMangosteen fruit especially the xanthone packedpericarp has been used traditionally to treat avariety of health disorders. Medicinal propertiesof α-Mangostin include usage against trauma,diarrhea, gonorrhea, bladder infections and skininfections (1).

Inflammation is considered as a primaryphysiological defense mechanism that helps thebody to protect against infection. The mechanismof inflammation is linked to release of reactiveoxygen species (ROS) such as superoxide (O

2),

hydroxyl (OH-) and peroxy radicals (ROO-) fromactivated neutrophils and macrophages. TheROS play an important role in the pathogenesisof various diseases and in the propagation ofinflammation by stimulating release of cytokinesand interferon-γ. Thus free radicals are importantmediators that provoke or sustain inflammatoryprocesses and conse-quently their neutralizationby antioxidants and radical scavengers canattenuate inflammation (2).

The inflammatory response occurs in threedistinct temporal phases, each apparentlymediated by different mechanisms: i) an acutephase characterized by transient localvasodilation and increased capillary permeability,ii) a delayed sub-acute phase characterized byinfiltration of leucocytes and phagocytic cells andiii) a chronic proliferative phase, in which tissuedegeneration and fibrosis occur. However,chronic inflammation can also lead to a numberof diseases, such as hay fever, rheumatoidarthritis, arteriosclerosis, myocarditis and cancer(3). The goal of present study is the validation ofthe traditional use of α-Mangostin and todetermine the in vivo anti-inflammatory and invitro antioxidant potential of 40% HPLC purifiedα-Mangostin.

Anti-inflammatory and Antioxidant Potential ofααααα -Mangostin

Navya A.1, Santhrani T.2 and Uma Maheswari Devi P.1*

1 Department of Applied Microbiology, Sri Padmavati Mahila Visvavidyalayam,Tirupati-517502, Andhra Pradesh, India.

2 Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam,Tirupati-517502, Andhra Pradesh, India.

*For Correspondence - [email protected]

Anti-inflammatory and Antioxidant potential of α-Mangostin


Materials and Methods

Plant material: The 40% HPLC purified α-Mangostin was procured from INDFRAGCompany, Bangalore, India; for analyzing anti-inflammatory and antioxidant properties.

Animals: Adult male albino rats (Wistar Strain)weighing between 150-200g were obtained fromBros Scientifics, Tirupati, India, and used for anti-inflammatory studies. All the animals wereacclimatized for a week before use and weregrouped in polyacrylic cages and maintainedunder standard laboratory conditions. The roomtemperature was maintained at 25 ± 2ºC with darkand light cycle of 14/10h. They were fed oncommercial diet and water ad libitum. TheInstitutional Animal Ethical Committee approvedprotocols were followed for experimentalanalysis.

Acute toxicity test: Healthy rats, starvedovernight, were divided into six groups of sixanimals in each group and fed with increasingdoses (10,100, 250, 500, 1000, and 1500 mg/kg, b.w, p.o) of 40% HPLC purified α-Mangostinup to 14 days.

Anti-inflammatory activity of ααααα-Mangostin

Carrageenan induced paw oedema in rats:The anti-inflammatory activity of α-Mangostinwas determined by inducing acute inflammationby carrageenan in rats (4). For the experimentalanalysis, the rats were divided into five groupsof six animals each. The first group (control)received normal saline (0.9% w/v, 3ml/kg, b.w,p.o). Second group with Indomethacin (10mg/kg) served as standard where as third, fourth andfifth groups were orally administered with lowdose, mild dose and high doses (0.5, 5 and 10mg/kg) of α-Mangostin respectively with the help ofan oral catheter. After 1h of drug treatment, asubplantar injection of 1% Carrageenan solutionwas administered in the left hind paw of rats. Thevolume of paw oedema was measured withPlethysmometer (UGO Basile, USA) after 3h ofinjections. The average paw volume wasmeasured and compared with control and

standard groups. The percentage of paw oedemainhibition was calculated using the formula;

Inhibition of oedema (%) = Oc-Ot / Oc x 100.

Where, ‘Oc’ is oedema volume of controlgroup and ‘Ot’ is oedema volume of treatedgroups.

Cotton pellet induced granuloma in rats: Thecotton pellet method is frequently used toevaluate the chronic phase of inflammation. Therats were divided into five groups of six animalsin each group. The rats were anaesthetized byether and sterile cotton pellets weighing 10mgwere implanted subcutaneously into the groinregion of each rat. The first group referred ascontrol received normal saline (0.9% w/v, 3ml/kg, b.w, p.o), Second group served as standard,received Indomethacin (10mg/kg), where asthird, fourth and fifth groups received low dose(0.5mg/kg), mild dose (5mg/kg) and high dose(10mg/kg) of α-Mangostin respectively with anoral catheter. The α-Mangostin treatment wascontinued for seven consecutive days from theday of cotton pellet implantation (5). After thecompletion of 7 days, i.e., on the beginning of 8th

day, the animals were anaesthetized with etherand the pellets along with the granuloma tissueformed around were removed carefully and freedfrom extraneous tissue. Then the wet pellets weredried in an oven at 60oC for 24h to constantweight for measuring the granuloma formation.The percent inhibition of granuloma formationwas calculated by using the formula;

Inhibition of granuloma (%) =Gc-Gt / Gc x 100.

Where, ‘Gc’ is granuloma tissue weight incontrol group and ‘Gt’ is granuloma tissue weightin treated groups.

In vitro antioxidant study of ααααα-Mangostin

DPPH radical scavenging assay: Theantioxidant activity of α-Mangostin was analyzedbased on the scavenging activity of stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical (6).All experiments were repeated thrice. Differentconcentrations (62.5-1000μg/ml) of α-Mangostin

Navya et al


in ethanol (0.05ml) were added to methanolicsolution of DPPH (200μM). The mixture wasshaken and allowed to stand at room temperaturefor 30min and the absorbance was measured at517nm using methanol as blank on UV-VISspectrophotometer (Shimadzu, Germany).Ascorbic acid was used as the standard. Thescavenging activity is expressed as thepercentage of inhibition at differentconcentrations calculated by using the formulaand IC

50 was determined by linear regression

analysis.

Inhibition (%) = Absorption of control-Absorption of test / Absorption of control × 100.

Nitric oxide radical scavenging assay:Sodium nitroprusside in an aqueous solution atphysiological pH, spontaneously generates nitricoxide which interacts with oxygen to producenitrite ions that were measured at 546nm usingGriess reagent (7). Scavengers of nitric oxidecompete with oxygen leading to reducedproduction of nitrite ions. In order to determinethe scavenging activity of α-Mangostin, sodiumnitroprusside (5mM) in standard phosphate buffersolution was mixed with different concentrationsof α-Mangostin (62.5-1000μg/ml) and the tubeswere incubated at 25 ±2oC for 5h. Control wasmaintained by adding an equal amount ofphosphate buffer. About 0.5ml of incubatedsolution was mixed with equal amount of Griessreagent and the color developed was measuredat 546nm. The percentage inhibition wascalculated by using the same formula as givenabove and IC50 was determined.

Scavenging of superoxide radical: Thescavenging activity towards the superoxideradical (O

2--) was measured in terms of inhibition

of generation of O2

- (8) using alkaline dimethylsulphoxide (DMSO) method (9). Potassiumsuperoxide and dry DMSO were allowed to standin contact for 24h and the solution was filteredjust before use. The filtrate (200μl) was added to2.8ml of an aqueous solution containing nitrobluetetrazolium (56μl), ethylene diamine tetra aceticacid (10μl) and potassium phosphate buffer

(10mM). Then various concentrations (62.5-1000μg/ml) of α-Mangostin in ethanol (1ml) wereadded and absorbance was recorded at 560nmagainst a blank. The percentage inhibition wascalculated by using the same formula as givenabove and then IC

50 was determined.

Lipid peroxidation assay: The extent of Lipidperoxidation in rat brain homogenate wasmeasured in vitro in terms of formation ofthiobarbituric acid reactive substances (TBARS)(10). Different concentrations of α-Mangostin(62.5-1000μg/ml) in ethanol were individuallyadded to the brain homogenate (0.5ml). Thismixture was incubated with 0.15M KCl (100μl).Lipid peroxidation was initiated by adding 100μlof 15mM FeSO

4 solution and the reaction mixture

was incubated at 37ºC for 30 min. Afterincubation, the mixture was added to 1ml ofsolution containing equal volume of TBA(thiobarbituric acid): TCA (trichloroacetic acid).Then the mixture was heated at 80ºC for 20 minafter the addition of 1ml of butyrated hydroxyltoluene and was centrifuged after cooling to roomtemperature. The absorbance of supernatant wasread at 532nm against blank. The percentage ofLipid peroxidation inhibition was measured byusing the same formula as given above and thenIC

50 was calculated.

Statistical analysis: All the data are triplicatesof independent experiments reported as Mean ±SEM (Standard Error of Mean). Statisticalsignificance was assessed by t-test using InStatsoftware.

ResultsAcute toxicity test: The oral doses of α-Mangostin up to 1500mg/kg did not produce anyevident sign of toxicity and mortality in rats whenobserved up to 14 days since administration.Thus, the median lethal dose (LD50) wasdetermined to be higher than the maximum(1500mg/kg) dose tested.

Anti-inflammatory activity of ααααα-Mangostin: Inexperimentally induced paw oedema bycarrageenan, α-Mangostin showed significant



anti-inflammatory activity in dose dependantmanner by restricting the paw oedema volume to0.883±0.012 at 10mg/kg b.w, p.o after 3h oftreatment compared to control group (Table 1).The inhibition of paw oedema was clearlyobserved in α-Mangostin (10mg/kg) treated groupcompared to control group (Fig.1). The maximumpercentage of paw oedema inhibition of 40.21%was observed at 10mg/kg of α-Mangostin (Fig.2) in comparison with standard Indomethacin(10mg/kg).

In cotton pellet induced granuloma methoddry weight of the cotton pellets were taken asmeasure of granuloma formation, α-Mangostin

significantly reduced the granuloma formation at10mg/kg b.w, p.o (Table 2). The formation ofgranuloma around subcutaneously implantedcotton pellets in the groin region of rats wasobserved in control group whereas it was notobserved in 10mg/kg of α-Mangostin treatedgroup (Fig. 3). The maximum inhibition of 66.71%at 10mg/kg of α-Mangostin was observed whereas Indomethacin showed a maximum of 50.57%inhibition of granuloma at 10mg/kg (Fig. 4).

Antioxidant potential of ααααα-Mangostin: TheDPPH was widely used as a model system toinvestigate the scavenging activities of naturalcompounds. As shown in Table 3 the percentageof inhibition was found to be 95% at 1000μg/ml

Fig. 2. Anti-inflammatory activity of α-Mangostinevaluated by carrageenan induced acuteinflammation in rats.

Fig. 4. Anti-inflammatory activity of α-Mangostinevaluated by cotton pellet induced chronicinflammation in rats.

Navya et al

Fig. 3. The anti-inflammatory activity of α-Mangostinwas evaluated in chronic phase of inflammation in rats.The chronic inflammation was induced by cotton pelletshowing formation of granuloma aroundsubcutaneously implanted cotton pellets in the groinregion of rats in control group (a) but not in 10mg/kgb.w of α-Mangostin treated group (b) after 7 days ofinduction.

Fig. 1. The anti-inflammatory activity of α-Mangostin was evaluated in acute phase ofinflammation in rats. The acute inflammation wasexperimentally induced by carrageenan showingpaw oedema in control group (a) and inhibition ofpaw oedema in 10mg/kg b.w of α-Mangostintreated group (b) after 3h of induction.


and 40% at a concentration of 62.5μg/ml. TheIC50 value of α-Mangostin was found to be at100μg/ml. This study showed that the α-Mangostin has the proton-donating ability andcould serve as free radical scavenger.

Nitric oxide scavenging effect of α-Mangostin was found to be 12% and 49% at62.5μg/ml and 1000ìg/ml respectively. α-Mangostin showed an IC50 value at 1000μg/ml(Table 4). Superoxide scavenging effect of α-Mangostin was found to be 27% and 89% at62.5μg/ml and 1000μg/ml respectively with an IC50

of 250μg/ml (Table 5).

In vitro induction of lipid peroxidation is atool for measuring antioxidant potential of á-Mangostin. Dose-dependent protection againstlipid peroxidation (Table 6) exhibiting the IC50 valueat 900μg/ml was observed with α-Mangostin. Thisactivity may be related to the H+ ion donating

capability of the α-Mangostin which can scavengethe peroxyl radical.

DiscussionIn the present study, systematic approach

was made to find out the efficacy of α-Mangostinagainst inflammation so as to exploit it as herbalanti-inflammatory agent. Carrageenan inducedrat paw oedema is commonly employedexperimental animal model for evaluating theanti-inflammatory activity of natural compounds(11). It is well known that carrageenan inducedoedema is multimediated phenomenoncharacterized by biphasic response with theinvolvement of different inflammatory mediators.The early phase (1-2h) is mediated by histamineand serotonin and late phase (3-4h) is mediatedwith the release of prostaglandins (12). Ourresults indicate that the administration of α-Mangostin inhibited the oedema during acute

Table 1. Effect of α-Mangostin on paw oedema induced by carrageenan in rats

Group / Treatment Dose (mg/kg,p.o) Mean paw oedemavolume (ml) ± SEM

Group 1 / Control — 1.477±0.117Group 2 / Indomethacin 10 0.910±0.070*

Group 3 / α-Mangostin 0.5 1.157±0.042*

Group 4 / α-Mangostin 5 1.097±0.038*


Data given are mean of three replicates ± SEM *p<0.001 when compared to control based on a Student’s‘t’ test.

Table 2. Effect of α-Mangostin on cotton pellet induced granuloma in rats

Group / Treatment Dose Dry weight of cotton(mg/kg,p.o) pellet (mg) ± SEM

Group 1 / Control — 70.0±0.13Group 2 / Indomethacin 10 34.6±0.03*

Group 3 / α-Mangostin 0.5 55.3±0.09*



Data given are mean of three replicates ± SEM*p<0.01 when compared to control based on a Student’s‘t’ test.



phase of inflammation probably by inhibiting thechemical mediators of inflammation.

The cotton pellet method is widely used toevaluate the proliferative components of chronicinflammation (13). Cytokines, such as IL-1 andTNFα, as well as growth factors influenceproliferation of smooth muscle cells andfibroblasts and production of granuloma (14, 15).The weight of the wet cotton pellets correlateswith transude material and the weight of dry pelletcorrelates with the amount of granuloma tissueformation. The Nonsteroidal anti-inflammatorydrugs (NSAIDs) reduce the size of granulomawhich results from cellular reaction by inhibitinggranulocyte infiltration, preventing generation ofcollagen fibers and suppressingmucopolyssaccharides (16). In the present study,oral administration of α-Mangostin has beenobserved to inhibit the wet weight of cotton pelletin a dose dependent manner and the higher doseof α-Mangostin exhibited more inhibition ofinflammation compared with the standard NSAIDIndomethacin, which indicates that theproliferative phase was effectively suppressedby α-Mangostin. Several chronic humandiseases associated with inflammation arecharacterized by over production of ROS (17).DPPH radical is scavenged by antioxidantsthrough the donation of proton by forming thereduced DPPH. The color changes from purpleto yellow after reduction can be quantified at517nm (6). DPPH was used to determine theproton radical scavenging action of α-Mangostin,because it possess a proton free radical andshowed a characteristic absorbance at 517nm.From the present study, it was found that α-Mangostin reduces the radical to correspondinghydrazine when it reacts with hydrogen donors.

Nitric oxide (NO) is an importantchemical mediator generated by endothelial cells,macrophages, neurons etc., and is involved inthe regulation of various physiological processes(18). The nitric oxide generated from sodiumnitroprusside reacts with oxygen to form nitrite.The α-Mangostin inhibited the nitrite formationeither by competing with oxygen or with its

Table 3. DPPH radical scavenging activity of α-Mangostin

α-Mangostin Percentage of IC50

(μg/ml) inhibition* (μg/ml)

62.5 40125 64250 68 100500 70

1000 95

*Values are means (n=3)

Table 4. Scavenging effect of α-Mangostin on Nitricoxide radical



62.5 12125 15250 20 1000500 30

1000 49


Table 5. Scavenging activity α-Mangostin onSuperoxide radical



62.5 27125 39250 54 250500 59

1000 89


Table 6. Inhibition of Lipid peroxidation by α-Mangostin



62.5 12125 19250 26 900500 32

1000 54


Navya et al


synthesis. Scavengers of nitric oxide competewith oxygen leading to reduced production ofnitric oxide (19). Nitric oxide radical inhibitionassay proved that α-Mangostin is a potentscavenger of nitric oxide.

Superoxide dismutase (SOD) continues tobe an important link in the biological defensemechanism through dismutation of endogenouscytotoxic superoxide radicals to H

2O

2 (20). The

findings proved the strongest superoxidescavenging activity of α-Mangostin.

Lipid peroxidation is the oxidativedegradation of polyunsaturated fatty acids andinvolves in the formation of lipid radicals leadingto membrane damage (21). Ferrous ions caninitiate lipid peroxidation by the Fenton reactionas well as accelerating peroxidation bydecomposing lipid hydroperoxides into peroxyland alkoxyl radicals which eventually yieldnumerous carbonyl products such asmalondialdehyde (MDA). The inhibition could becaused by the absence of ferrule-perferrylcomplex or by scavenging the hydroxyl radicalor the superoxide radicals or by changing theFe3+/Fe2+ or by reducing the rate of conversionof ferrous to ferric or by chelating iron itself. Theantioxidant activity of α-Mangostin was furtherconfirmed by decreased production of MDA inthe biomembrane of rat brain homogenate.

ConclusionThe administration of α-mangostin inhibited

the oedema and granuloma formation duringacute and chronic inflammatory conditions. Thepresent study reveals that α-mangostin hassignificant anti-inflammatory and free radicalscavenging activity. Further studies are neededto analyze the therapeutic potential of α-mangostin against chronic inflammatorydiseases.

AcknowledgementWe gratefully acknowledge the support by

DST-SERC programme (SR/SO/HS/007/2008)for providing the financial assistance to carry outthis investigation.

References1. Nakatani, K., Nakahata, N., Arakawa, T.,

Yasuda, H. and Ohizumi, Y. (2002).Inhibition of cyclooxgenase andprostaglandia E2 synthesis by α-mangostin,a xanthone derivative in mangosteen, in C6rat glioma cells. Biochem. Pharmacol.,63:73-79.

2. Delaporte, R.H., Sanchez, G.M., Cuellar,A.C., Giuliani, A. and Palazzodemella, J.C.(2002). Anti-inflammatory activity and lipidperoxidation inhibition of iridoid lamiideisolated from Bouchea fluminensis (vell)Mold (verbenaceae). J. Ethnopharmacol.,82:127-130.

3. Nandini, V., Subhash, K., Tripathi, Debasis,S., Hasi, R.D. and Rakha, H.D. (2009).Evaluation of inhibitory activities of plantextracts on production of LPS-stimulatedpro-inflammatory mediators in J774 murinemacrophages. Mol. Cell. Biochem., 36(1-2):127-135.

4. Winter, C.A., Risky, E.A. and Nuss, G.W.(1962). Carrageenan induced oedema inhind paw of the rat as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol.Med., 11I:544-547.

5. D’ Arcy, P.F., Howard, E.M., Muggleton,P.W. and Townsend, S.B. (1960). The anti-inflammatory action of Griseofulvin inexperimental animals. J. Pharm.Pharmacol., 12:659-665.

6. Panchawat, S. and Sisodia, S.S. (2010). Invitro antioxidant activity of Saraca asocaRoxb. De Wilde stem bark extracts fromvarious extraction processes. Asi. J. Pharm.Clin. Res., 3(3):231-233.

7. Sreejayan, N. and Rao, M.N.A. (1997).Nitric oxide scavenging by curcuminoids.J. Pharm. Pharmacol., 49:105-107.

8. Sanchez-Mareno, C. (2002). Review:Methods used to evaluate the free radical



scavenging activity in foods and biologicalsystems. Food. Sci. Technol. Intern., 8:121-137.

9. Henry, L.E.A., Halliwell, B. and Hall, D.O.(1976). The superoxide dismutase activityof various photosynthetic organismsmeasured by a new and rapid assaytechnique. FEBS Lett., 66:303-306.

10. Prashanth kumar, V., Shashidhara, S.,Kumar, M.M. and Sridhara, B.Y. (2000).Effect of Luffa echinta on lipid peroxidationand free radical scavenging activity. J.Pharm. Pharmacol., 52:891.

11. Sharma. U.S., Sharma, U.K., Sutar, N.,Singh, A. and Shukla, D.K. (2010). Anti-inflammatory activity of Cordia dichotomaforst f. seeds extracts. Int. J. Pharm. Ana.,2(1):1-4.

12. Hernandez, P.M. and Rabanal Gallego,R.M. (2002). Evaluation of the anti-inflammatory and analgesic activity ofSideritis canariensis var pannosa in mice.J. Ethnopharmacol., 81:43-47.

13. Ghosh, M.N. (2008). Fundamentals ofExperimental Pharmacology. 4th ed. Hilton& Company, Kolkata, 164-166.

14. Mitchell, R.N. and Cotran, R.S. (2000). In;Robinsons Basic Pathology, 7th ed.,Harcourt (India) Pvt Ltd., New Delhi, 33-42.

15. Joanna, M.K., Ken, I.O., Naomi, W.,Sigridur, A.A., Jan, A.A.M.K., Robbert, J.K.and Grietje, M. (2005). Molecular Pathwaysof Endothelial Cell Activation for (Targeted)Pharmacological Intervention of Chronic

Inflammatory Diseases. Cur. Vas.Pharmacol., 3:11-39.

16. Mahesh, S.P., Patil, M.B., Ravi K. and Patil,S.R. (2009). Evaluation of anti-inflammatoryactivity of ethanolic extract of Borassusflabellifer L. male flowers (inflorescences)in experimental animals. J. Med. Plants.Res., 3:49-54.

17. Edwina, N. and Vishva, M.D. (2011).Mitochondrial reactive oxygen species driveproinflammatory cytokine production. J.Exp. Med., 208(3):417-420.

18. Baskar, R., Rajeswari, V. and Satish kumarT. (2007). In vitro antioxidant studies in theleaves of Annona species. Ind. J. Exp. Biol.,45:480-485.

19. Ganesh, C.J., Shaival, K.R., Manjeshwar,S.B. and Kiran, S.B. (2004). The Evaluationof Nitric Oxide Scavenging Activity ofCertain Herbal Formulations in vitro: APreliminary Study. Phytother. Res., 18:561-565.

20. Hasan, S. M., Hossain, M. M., Faruque, A.,Mazumder, M.E.H., Rana, M. S., Akter, R.and Alam, M. A. (2008). Comparison ofantioxidant potential of different fractions ofCommelina benghalensis Linn. Bang. J.Life. Sci., 20(2):9-16.

21. Devasagayam, T.P.A., Boloor, K.K. andRamasarma, T. (2003). Methods forestimating lipid peroxidation: An analysis ofmerits and demerits. Ind. J. Biochem.Biophy., 40:300-308.

Navya et al

Documents

DOCKING STUDIES ON XANTHONES OF MANGOSTEEN AS COX-2 …shodhganga.inflibnet.ac.in/bitstream/10603/6719/13/... · The inhibitor binding positions and affinity were evaluated using