|Year : 2012 | Volume
| Issue : 2 | Page : 197-203
Molecular docking and ex vivo pharmacological evaluation of constituents of the leaves of Cleistanthus collinus (Roxb.) (Euphorbiaceae)
Subramani Parasuraman1, Ramasamy Raveendran1, Balakrishnan Vijayakumar2, Devadasan Velmurugan3, Subramani Balamurugan4
1 Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India
2 Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Maraimalai (Guindy) Campus, India
3 Centre of Advanced Study in Crystallography and Biophysics and Bioinformatics Infrastructure Facility (BIF), University of Madras, Maraimalai (Guindy) Campus, India
4 College of Pharmacy, Madras Medical College, Chennai, India
|Date of Submission||24-May-2011|
|Date of Decision||30-Nov-2011|
|Date of Acceptance||14-Dec-2011|
|Date of Web Publication||16-Mar-2012|
Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry
Source of Support: None, Conflict of Interest: None
Objective: To investigate the involvement of alpha adrenergic receptors in hypotension induced by cleistanthin A and cleistanthin B.
Materials and Methods: Cleistanthins A and B were isolated from the leaves of Cleistanthus collinus using a column chromatographic method and purified. Structures were confirmed by spectroscopic analysis. The compounds were prepared for molecular docking studies using Ligprep 2.3 module and Induced Fit Docking was carried out against α-1 adrenergic receptors using Glide. The ex vivo experiments were carried out on male Wistar rats. Under anaesthesia, the femoral vein and carotid artery were cannulated for drug administration and for monitoring the blood pressure, respectively. The effect of epinephrine, norepinephrine, acetylcholine, histamine and dopamine were recorded before and after the administration of cleistanthin A or cleistanthin B. The molecular docking studies showed favorable molecular interactions, glide score, energy and emodel.
Result: Cleistanthins A and B per se reduced the mean blood pressure and the effect was dose dependent. Both the compounds reduced the effect of epinephrine, norepinephrine and α-1 receptor activity of dopamine. Cleistanthin B significantly increased the duration of action of acetylcholine on mean blood pressure.
Conclusion: The molecular docking and ex vivo studies conclude that cleistanthin A and cleistanthin B have significant α-1 adrenergic receptor antagonist effect on the peripheral vascular system.
Keywords: Blood pressure, cleistanthin A, cleistanthin B, dopamine, homology modeling, norepinephrine, Induced fit docking
|How to cite this article:|
Parasuraman S, Raveendran R, Vijayakumar B, Velmurugan D, Balamurugan S. Molecular docking and ex vivo pharmacological evaluation of constituents of the leaves of Cleistanthus collinus (Roxb.) (Euphorbiaceae). Indian J Pharmacol 2012;44:197-203
|How to cite this URL:|
Parasuraman S, Raveendran R, Vijayakumar B, Velmurugan D, Balamurugan S. Molecular docking and ex vivo pharmacological evaluation of constituents of the leaves of Cleistanthus collinus (Roxb.) (Euphorbiaceae). Indian J Pharmacol [serial online] 2012 [cited 2022 Sep 29];44:197-203. Available from: https://www.ijp-online.com/text.asp?2012/44/2/197/93848
| » Introduction|| |
Cleistanthin A and cleistanthin B are glycosides present in the leaves of Cleistanthus collinus (Roxb.) (Euphorbiaceae) which is a small tree found in Africa, India, Sri Lanka and Malaysia. , Cleistanthus collinus poisoning causes cardiovascular abnormalities such as hypotension, nonspecific ST-T changes and QTc prolongation. ,, The studies with the crude aqueous extract of the Cleistanthus collinus leaves show a direct inhibition of the α-adrenergic receptors present in the guinea pig vas deferens.  The in vitro isolated tissue experiments and receptor-ligand interaction studies using ArgusLab molecular modelling and drug docking software demonstrated the nicotinic cholinergic and the α-adrenergic receptor antagonism by cleistanthins A and B.  In our earlier studies both cleistanthin A and cleistanthin B showed dose-dependent fall in blood pressure in Wistar rats.  No study has been carried out to explore the mechanism of hypotension induced by Cleistanthus collinus and its constituents cleistanthin A and B. It was hypothesised that hypotension is mediated through α-adrenergic receptors in the peripheral vascular system. Hence the present study was planned to find out the involvement of α-adrenergic receptors in hypotension caused by cleistanthin A and B.
| » Materials and Methods|| |
The taxonomically identified Cleistanthus collinus (Roxb.) (Euphorbiaceae) plant parts were collected in the regions of Pondicherry, India, rural parts of Villupuram, Cuddalore districts of Tamil Nadu, India and certified by the Botanical Survey of India (BSI), Coimbatore (BSI/SC/5/23/08-09/Tech.241). Leaves of Cleistanthus collinus were collected in the months of February-April every year. Voucher specimen of the plant is kept in the Department of Pharmacology, JIPMER, Pondicherry, for further reference.
Isolation, Spectroscopic Analysis of Cleistanthin A and B from Cleistanthus collinus Leaves
Freshly collected Cleistanthus collinus leaves were used for extraction. The shadow, air dried leaves were powdered and defatted with n-hexane by cold maceration process for 24 h. The marc of the n-hexane was extracted with acetone by cold maceration process for the duration of 36 h. The acetone extract was then concentrated. The constituents of the plant extract were identified with primary qualitative analysis and thin layer chromatography (TLC) method for the presence of glycosides. Cleistanthins A and B were isolated from the acetone extract using column chromatography. They were isolated using mobile phase benzene:ethyl acetate (1:1) and methanol:chloroform (9:1) solvent system respectively. ,, The fraction of cleistanthin A and B were purified using preparative TLC and crystallization method, respectively. The functional groups and facial arrangement of atoms in cleistanthin A and B molecules were confirmed by Fourier Transform Infra Red (FT-IR) spectroscopy (Avatar FT-IR 330) and Nuclear Magnetic Resonance (NMR) spectroscopy (Bruker 300 MHz). 
Molecular Modeling Calculations
All computational works were performed on Red Hat Enterprise Linux EL-5 workstation using the molecular modeling software Maestro (Schrodinger LLC 2009, USA). GLIDE-5.5 (Grid-based Ligand Docking with Energetics) searches were made for favorable docking interactions between one or more ligand molecules with a macromolecule, usually a protein. All the molecular modeling simulations were carried out using OPLS-AA (Optimized Potential liquid simulation for All Atom) force field [Glide 2009].  PyMOL  and Chimera software were used for graphical visualization, analyzing hydrogen bond interactions and producing quality images. Hydrophobic contacts were observed between protein and ligand using Ligplot software. 
a. Ligand preparation and biological activity prediction
Cleistanthin A and Cleistanthin B are natural compounds [Figure 1], which were built using builder panel in Maestro. The compounds were taken for ligand preparation by Ligprep 2.3 module (Schrödinger, USA)  which performs addition of hydrogens, 2D to 3D conversion, realistic bond lengths and bond angles, low energy structure with correct chiralities, ionization states, tautomers, stereochemistries and ring conformations. Prazosin, a selective α1 adrenergic receptor blocker used to treat hypertension,  was taken to docking studies to validate the binding affinity for natural compounds. The prazosin 3D structure was obtained from drug bank [http://www.drugbank.ca/] (DB00457).
|Figure 1: Schematic representation of a natural compounds (a) Cleistanthin A. (b) Cleistanthin B|
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b. Homology modeling and validations
Since 3D structure of human α1A adrenergic receptor (ADRA1A) was not available (as of January, 2011) in the Protein Data Bank (PDB; http://www.pdb.org), sequence (UniProt: P35348) similarity search was employed using BLASTp. However, no suitable template was obtained for ADRA1A. Therefore SWISS-MODEL automatic mode for homology modeling was performed to get a valid 3D structure of the target receptor which was also unsuccessful. Hence, 3D-JIGSAW software was used to obtain a reliable 3D structure of the ADRA1A. ,, The modeled structure was validated in Dali server  for structural similarity analysis (Z-score) against PDB database. Phi/Psi dihedral angle for the predicted model was validated using Ramachandran plot from PROCHECK. 
c. Protein preparation and active site prediction
For the docking study modifications were carried out in the ADRA1A structure. Missing hydrogen atoms were added and correct bond orders were assigned, then formal charges and orientation of various groups were fixed. Following this, optimization of the amino acid orientation of hydroxyl groups, amide groups of Asn and Gln was carried out. All amino acid flips were assigned and H-bonds were optimized. Nonhydrogen atoms were minimized until the average root mean square deviation reached default value of 0.3 Å. Sitemap 2.3 was used to explore binding site in the ADRA1A for docking studies. 
d. Induced fit docking
Induced fit docking (IFD) is one of the main complicating factor in docking studies which predicts accurate ligand-binding modes and concomitant structural movements in the receptor using Glide and Prime modules. In IFD, when ligand binds to the receptor, it undergoes side-chain or backbone conformational changes or both in many proteins. These conformational changes allow the receptor for better binding according to the shape and binding mode of the ligand.  Here, the prepared protein was loaded in the workspace and the sitemap predicted active site was specified for IFD. Grid was calculated about 20 Å to cover all the active site residues defined by the sitemap. The van der Waal's radii of nonpolar receptor and ligand atoms were scaled by a default factor of 0.50. IFD calculations were carried out for the prazosin, cleistanthin A and cleistanthin B with the ADRA1A receptor. Following this, 20 conformational poses were calculated where the best conformational pose was selected based on the docking score, glide energy, glide emodel values and non-bonded interactions.
Prime MM-GBSA Binding Free Energy
The binding free energy of receptor and a set of ligands was predicted using the approach of Prime MM-GBSA (Molecular Mechanics, The Generalized Born model and Solvent Accessibility) calculation available with Prime 2.1 module.  MM-GBSA is a contraction for a method that combines OPLS-AA (Optimized Potential for Liquid Stimulations- All Atom) molecular mechanics energies (EMM), an SGB solvation model for polar solvation (GSGB), and a nonpolar solvation term (GNP) composed of the nonpolar solvent accessible surface area and van der Waals interactions. The properly prepared ligands and receptors were used to calculate ligand binding and ligand strain energies for a set of ligands and a receptor according to the following formula: The expression of the total binding free energy
ΔG bind=Gcomplex-(Gprotein+Gligand), where G=EMM+GSGB+GNP
Healthy, adult, male albino rats of Wistar strain, weighing 170 - 190 g were obtained from JIPMER Animal House, Pondicherry, India. The animals were housed under 25 ± 2°C temperature, 40-60% humidity and 12-12 ± 1 h light-dark cycle. The animals were fed with water and rat pellets ad libitum. The rat pellets were supplied by M/s. Hindustan Lever Ltd., Bangalore, India. The study protocol was approved by the Institutional Animal Ethics Committee and all the animal experiments were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.
Cleistanthin A and cleistanthin B were insoluble in water and soluble in benzene, acetone and ethyl alcohol. So, cleistanthin A and cleistanthin B (1 mg/ml stock solution) were dissolved using the minimum volume of ethyl alcohol and the required volume was made up with distilled water qs. Acetylcholine (ACh) (Titan) and histamine (Sigma) were dissolved in distilled water and the concentration was adjusted to 1 mg/ml. Norepinephrine (1 mg/ml) was dissolved in 1% w/v ascorbic acid solution and further dilution was made with distilled water. Commercially available epinephrine (1 mg/ml), heparin (5000 I.U.) and dopamine (40 mg/5 ml) injections were used for the experiment. All the drug solutions were prepared afresh and used for the experiment.
Effect of Cleistanthin A and B on Rat Blood Pressure
Totally eighteen healthy, adult male Wistar rats were used for the experiment. The animals were anaesthetized with urethane (1200 mg/kg, i.p.) and the femoral vein was cannulated for drug administration. Tracheostomy was carried out and the carotid artery was cannulated with a 27G hypodermic needle. The carotid cannula was pre-filled with 5 I.U. heparinised saline (5 IU/ml) and connected to a bridge amp transducer (Model: ML T844, SP 844 physiological pressure transducer) through a three-way stopper. The transducer was connected to the bridge amplifier (Model: ML 221) of data acquisition system (Model: ML 866; PowerLab 4/30; AD instruments, Australia) to monitor mean blood pressure of the animal. After cannulation 0.1 ml of heparinised saline was injected through femoral vein. The volume of injected saline, heparinised saline and drug solution did not exceed 0.1 ml+0.1 ml per injection. A temperature of 37 ± 1°C was maintained during the entire duration of the experiment. After the cannulation, the animals were allowed to stabilize for 15-20 min to adapt the experimental conditions.  The effect on mean blood pressure of epinephrine, norepinephrine, ACh and histamine were recorded before and after the administration of cleistanthin A or cleistanthin B.
The blood pressure readings were expressed as mean±SD and the difference in mean blood pressure before and after administering cleistanthin A or cleistanthin B were analyzed using paired "t " test. P<0.05 was considered significant.
| » Result|| |
The spectroscopic results support the already elucidated structure of cleistanthins A and B. There was a clear TLC separation of cleistanthin at an Rf value of 0.37-0.42. The purified cleistanthins A and B were identified using UV-visible spectrophotometer, FT-IR, and NMR spectroscopy. The percentage yield of isolated cleistanthins A and B were 0.9-1.0% w/w and 0.6-0.8% w/w, respectively. The glycone part of the cleistanthins A and B were differing in structure and the remaining aglycone part was same for both cleistanthins A and B [Figure 1]. ,,
ADRA1A model was successfully obtained using 3D-JIGSAW which was found to be 372 amino acids in length (23-394). Turkey β-1 adrenergic receptor (ADRB1) (2VT4-A chain) was used as a template for homology model of ADRA1A in 3D-JIGSAW. The template and model were superimposed using chimera software that showed 0.304 Å rms deviation [Figure 2]. The ADRA1A was submitted in the Dali server which has shown list of structural similarities against adrenergic receptors. The top ranked structural similarity is ADRB1 2VT4-A (which was a template used by 3D-JIGSAW) with a Z- score of 37.4. Procheck analysis has shown 94.4% residue in most favored region where only 4% residue is generally allowed and 1.6% residues in disallowed region for the ADRA1A model. Sitemap 2.3  explored the binding site residues which are follows: Gln 191, Glu 195, Gln 201-Glu 204, Ser 212, Gly 319, Phe 321, Asp 324, Phe 325, Glu 329, Phe 332, Lys 333, Phe 336 and Tyr 340.
Binding of Prazosin
Prazosin was docked at the active site of the ADRA1A which shows a docking score -9.48, Glide energy -52.68 and Glide emodel −79.42 [Table 1]. The ADRA1A complex has four hydrogen bond interactions between the ligand and the active site residues. O3 and O4 atoms of ligand were involved in two hydrogen bond interactions with Gln 191 (NE2), 3.18 Å and 2.96 Å. O2 and N5 atoms were found hydrogen bonded with Gln 191 (NE2), 3.13 Å and Gly 319 (O), 2.96 Å, respectively [Table 2] and [Figure 3]a. Eight hydrophobic contacts were observed between ligand and active site residues (Ile 202, Asn 203, Glu 204, Val 209, Phe 325, Phe 332, Lys 333, and Phe 336).
|Figure 3: (a) Prazosin docked at the active site (green). (b) Cleistanthin A docked at the active site (orange). (c) Cleistanthin B docked at the active (purple)|
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|Table 1: Induced fi t docking score, glide energy and glide emodel calculations|
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|Table 2: ADRA1A active site residues maintained hydrogen bond interactions with prazosin, cleistanthin A and cleistanthin B|
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Binding of Cleistanthin A
Docked orientation of the cleistanthin A with the active site residues showed the docking score −8.68, Glide energy −49.83 and Glide emodel -73.62 [Table 1]. O1, O2 and O4 ligand atoms had three hydrogen bond interactions with the active site of the receptor (Gln 201 (NE2); 3.24 Å, Ile 202 (N), 2.94 Å, and Glu 329 (OE1), 2.52 Å), respectively [Table 2] and [Figure 3]b. The ligand and active site residues have maintained eight hydrophobic interactions (Asn 203, Glu 204, Gly 319, Phe 321, Asp 324, Phe 325, Phe 332, and Lys 333).
Binding of Cleistanthin B
Cleistanthin B showed docking score −10.32, Glide energy −63.06 and Glide emodel −101.68 at the active site of ADRA1A [Table 1]. Five hydrogen bond interactions were observed between ligand and active site residues. O4, O2 and O8 ligand atoms have shown hydrogen bond interactions with Gln 201 (NE2) 2.93 Å; Ile 202 (N) 3.07Å; and Gly 319 (O) 2.77 Å, receptively. Asp 324 (OD1) residue has maintained two hydrogen bond interactions with O8 and O10 ligand atoms in distance 2.64 Å and 2.74 Å, respectively [Table 2] and [Figure 3]c. Eight hydrophobic interactions were observed between ligand and active site residues of Gln 191, Glu 195, Asn 203, Glu 204, Phe 325, Glu 329, Phe 332 and Phe 336.
Prime MM-GBSA approach was used to predict ligand binding energy (ΔG bind ) docked compounds. The ligand binding energies were observed for prazosin (−45.55), cleistanthin A (−41.79) and cleistanthin B (−61.36) [Table 3]. Both cleistanthin A and B have showed favorable binding free energy. However, cleistanthin B has shown the best binding free energy.
Effect of Cleistanthin A and Cleistanthin B on the Rat Mean Blood Pressure
Cleistanthin A and cleistanthin B per se reduced the mean blood pressure and the effect was dose-dependent. These compounds caused death in 80% of the animals at 64 μg (cleisthanthin A) and 128 μg (cleistanthin B). Normal saline and solvent (used to dissolve cleistanthins A and B) did not have a direct effect on blood pressure.
Effect of Cleistanthin A and Cleistanthin B on the Blood Pressure Alterations Induced by Cholinergic and Adrenergic Agents
The mean resting blood pressure of animals administered with cleistanthin A and cleistanthin B was 66.23 ± 10.1 and 65.44 ± 6.2 mmHg, respectively. In general, cleistanthins A and B when administered alone showed a dose-dependent fall in mean blood pressure in rats. Cleistanthins A and B reduced the mean blood pressure and the change from basal observation was calculated to be 49.6 ± 4.9 and 40.2 ± 7.8%, respectively. Cleistanthins A and B significantly reduced the change (increase) in the mean blood pressure induced by norepinephrine and dopamine [Table 4] and [Table 5]; [Figure 4] and [Figure 5]. Cleistanthin A (1 μg) significantly increased the response time of ACh on mean blood pressure (P<0.01). No significant change was observed in the action of histamine.
| » Discussion|| |
IFD studies were carried for prazosin, cleistanthin A and cleistanthin B against the ADRA1A target. The docking results of cleistanthins A and B were compared with that of prazosin based on the various parameters such as the glide score, energy, emodel, hydrogen bond interactions, hydrophobic interactions and binding free energy. Cleistanthins A and B have favorable interactions with active site residues and also have favorable glide energy, emodel and glide scores. However, clesitanthin B has good glide score, energy and emodel as compared to prazosin. The MM-GBSA binding free energy (ΔG) calculation also promises these natural compounds to be potent inhibitors of α1A adrenergic receptor.
Cleistanthins A and B antagonized the effect of norepinephrine and dopamine and but not that of acetylcholine, histamine, isoprenaline and dobutamine. Dopamine reduces the mean blood pressure by acting on renal D 1 receptor but the higher concentrations of the drug raise the mean blood pressure by activating vascular α1 receptors.  Cleistanthins A and B selectively reduced the rise and not the initial fall in blood pressure due to dopamine. This finding and the reduction in the rise of blood pressure by norepinephrine indicate that the test compounds act by blocking the α- adrenoceptors. However, these compounds did not show significant antagonism of epinephrine. Though the rise in blood pressure is reduced by cleistanthins A and B, the effect was not statistically significant in case of epinephrine. The rise in blood pressure induced by epinephrine is less than that by norepinephrine and hence the blocking effect of cleistanthin A and cleistanthin B was more pronounced with norepinephrine and dopamine. At the same time, 100 ng of prazosin did not have a significant effect on α1 receptors but 200 ng of the drug blocked the effects of epinephrine, norepinephrine, and dopamine. The effects of cleistanthins A and B on mean blood pressure are similar to those of prazosin.
The increase in the duration of action of ACh can be attributed to the blockade of the α- adrenoceptors by cleistanthin A and B. ACh action is said to be evanescent and in the normal animal the blood pressure is brought back to basal level by the normal body mechanism as soon as ACh is destroyed. Since cleistanthin A and B block α- receptors, the recovery by the natural mechanisms is delayed leading to an increase in the duration of action of ACh. While this seems to be a plausible explanation, the possibility that the test compounds inhibit the plasma cholinesterase partially to prolong the action of ACh remains to be ruled out.
Cleistanthin A, cleistanthin B, diphyllin and collinusin are the major phytoconstituents present in the plant.  Cleistanthin A and B are arylnapththalide lignan glycoside compounds and it has been reported that these compounds are responsible for the toxicity, but there is no scientific data available for toxicity of cleistanthins A and B.  Hypotension has been commonly observed in patients of Cleistanthus collinus poisoning. ,, It is reported that Cleistanthus collinus poisoning causes refractive hypotension which cannot be reversed by inotropic agents such as dopamine and dobutamine. , Our observation in animals is in conformity with this clinical finding. The inability of inotropic agents to bring blood pressure back to normal could be attributed to the strong α1 receptor blocking action of cleistanthin A and B.
| » Conclusions|| |
Molecular docking studies show that the naturally occurring cleistanthin A and cleistanthin B have favorable interactions with α1 adrenergic residues at the active site and also have favorable Glide energy, emodel and Glide scores. The ex vivo study results suggest that cleistanthin A and B per se have hypotensive effect in rat and inhibit the hypertensive effect of adrenaline, noradrenaline and dopamine. This study suggests that the fall in mean blood pressure caused by cleistanthin A and cleistanthin B is mediated by α- adrenergic receptors present in the peripheral vascular system.
| » Acknowledgments|| |
The authors are grateful to Botanical Survey of India, Southern Circle, Coimbatore for identification of the plant. The authors (BV and DV) also thank the University Grant Commission (UGC) and the Department of Biotechnology (DBT-BIF), Government of India for financial support to carry out molecular simulation studies.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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