INTRODUCTION:

The histone deacetylase inhibitor belinostat is a drug designed for the treatment of hematological malignancies and solid tumors. It can be used to treat ovarian cancer when combined with carboplatin and paclitaxel for relapsed ovarian cancer^[^1].

Pharmacodynamics analyses have been conducted to describe the effect of belinostat on the acetylation of histones 3 and 4 (H3 and H4), the acetylation activity of both of these histones after belinostat exposure showed an increase in the level of acetylation both in vivo and in vitro ^[2-4]. Growth inhibition and apoptosis of the malignant cells were associated with these increased levels of acetylation, and no noticeable toxicity was seen in mice^[^5]. Belinostat has shown encouraging effect against peripheral T-cell lymphoma^[^6-
8]. This medication is available for intravenous administration only^[^9].

Local charges such as Mulliken charges and ZDO charges are also generated from arguslab using the AM1 parameterized method. In the zero deferential overlap (ZDO) approximation, the product of two deferent atomic orbitals is set to zero. The integra which survives the ZDO approximation was partly computed using the uniform charge sphere and the rest parameterized. The result produced is the integrated form of Hückel Theory which takes into account electron repulsion. Mulliken charges arise from the Mulliken population analysis ^{[10, 11]}and provide a means of estimating partial atomic charges from calculations carried out by the methods of computational chemistry, particularly those based on the linear combination of atomic orbitals molecular orbital method, and are routinely used as variables in linear regression (QSAR) procedures^[12,13].The energies computed by molecular mechanics are usually conformational energies. This means that the energy computed is meant to be an energy that will reliably predict the diference in energy from one conformation to the next. The effect of strained bond lengths or angles is also included in this energy. This is not the same as the total energies obtained from ab initio programs or the heat of formation from semiempirical programs. Molecular mechanics methods are not generally applicable to structures very far from equilibrium, such as transition structures. Arguslab is the electronic structure program that is based on the quantum mechanics, it predicts the potential energies, molecular structures; geometry optimization of structure, vibration frequencies of coordinates of atoms, bond length, bond angle and reactions pathway^[^14]. Conformational analysis of molecule is based on molecular mechanics, it is a method for the calculation of molecular structures, conformational energies and other molecular properties using concept from classical mechanics. The energy (E) of the molecule is calculated as a sum of terms as in equation (1).

E = E_stretching + E_bending + E_torsion + E_Vander_Waals+ E_{electrostatic} + E_hydrogen_bond + cross term (Equation 1)

These terms are of importance for the accurate calculation of geometric properties of molecules. The set of energy functions and the corresponding parameters are called force field ^[15]
.

MATERIALS AND METHODS:

All conformational analysis (geometry optimization) study was performed on a window based computer using Arguslab^[^16] and ACDlab Chemsketch software’s^[17]. Belinostat molecule was built with ACDlab and saved as mol file. The saved mol file of belinostat was generated by Arguslab, and minimization was performed with the semi-empirical Austin Model 1 (AM1) parameterization^[^18,19]. The minimum potential energy was calculated by using geometry convergence function in Arguslab software. In order to determine the allowed conformation, the contact distance between the atoms in adjacent residues is examined using criteria for minimum Vander Waal contact distance ^{[20
– 23]}. Surfaces created to visualize ground state properties as well as excited state properties such as orbital, electron densities, electrostatic potentials (ESP) spin densities generated the grid data used to make molecular orbital surfaces. The minimum potential energy was calculated for drug receptor interaction through the geometry convergence map. Retrieval of ebola virus envelop glycoprotein Crystal structure of ebola virus envelop glycoprotein from the organism Homo sapiens with the PDB ID 2EBO was retrieved from the Protein Data Bank (PDB).

Molecular docking:

Molecular docking was performed using patchdock online server^[^25]. Patchdock is a molecular docking algorithm based on shape complementarity principles. The Receptor (ebola virus) and ligand molecule (belinostat) were uploaded in PDB format in Patchdock server, an automatic server for molecular docking. Clustering RMSD was chosen as 1.5 Å. E-mail address to retrieve the result was given. Complex type was chosen as enzyme – inhibitor type. The docking job was submitted to the Patchdock server and refined in firedock online server^[^26,27]. The best conformation result was processed using Swiss PDB viewer software^[^28]. At the end of each docking run, interactions are shown in the form of "poses" with the energy values given in kcal/mol for each pose. Global energy and atomic contact energy scoring function of the firedock result were used to evaluate the stable interactions as indicated by lowest global energy (the binding energy of the ligand and the protein target). The lowest energy poses indicate the highest binding affinity as high energy produces unstable conformations.

RESULTS AND DISCUSSION:

Prospective view and calculated properties of belinostat molecule are shown in Figure1. The active conformation and electron density mapped of belinostat by ACD labs-3D viewer software are shown in Figure 2 and 3 respectively. Figures 4 and 5 shows the highest occupied molecular orbital’s and lowest unoccupied molecular orbital’s of belinostat. Figure 6 shows electrostatic potential mapped density of belinostat calculated with the AM1 (NDDO) RHF SCF type . The potential energy convergence map of belinostat is shown in Figure 7. UFF (molecular mechanics) geometry optimized atomic coordinates of belinostat is given in Table 1. Bond length and bond angles are given in Tables 2 and 3 respectively, which were calculated after geometry optimization of molecule from Arguslab by using molecular mechanics calculation. Tables 4 and 5 shows the dihedral angles and improper torsion angles of belinostat respectively. Table 6 shows calculated Final energy evaluation of belinostat molecule. The Ground State Dipole (debye) of belinostat is presented in Table 7. List of Mulliken atomic charges and ZDO atomic charges of belinostat is shown in Table 8 while the relative interaction energy values of belinostat docked unto ebola virus is presented in Table 9.

ArgusLab was used to see what happened to the electrons in molecules when it absorbed light. Surfaces were made to explore this fascinating phenomenon. Molecules absorbsed energy in the form of UV/visible light, it made a transition from the ground electronic state to an excited electronic state. The excited and ground states have different distributions of electron density. This property is often valuable and sought after by chemists who are interested in molecules that are useful as dyes, sunscreens, etc^[^16]. The HOMO is localized to the plane of the molecule and is a non-bonding molecular orbital. The LUMO is perpendicular to the plane of the molecule and is a combination of the p_z atomic orbitals. The n -> π^* transition is dominated by the excitation from the HOMO to the LUMO. The positive and negative phases of the orbital are represented by the two colors, the red regions represent an increase in electron density and the blue regions a decrease in electron density. However, these calculations were examined in the ground state and also in vacuum ^[16]. The electrostatic potential is a physical property of a molecule that relates to how a molecule is first “seen” or “felt” by a positive "test" charge at a particular point in space. A distribution of electric charge creates an electric potential in the surrounding space. A positive electric potential means that a positive charge will be repelled in that region of space. A negative electric potential means that a positive charge will be attracted. A portion of a molecule that has a negative electrostatic potential will be susceptible to electrophilic attack – the more negative the better^[^16]. QuickPlot ESP mapped density generates an electrostatic potential map on the total electron density contour of the molecule. The electron density surface depicts locations around the molecule where the electron probability density is equal^[^14]. This gives an idea of the size of the molecule and its susceptibility to electrophilic attack. Electron density surface shows the complete surface with the color map. The surface color reflects the magnitude and polarity of the electrostatic potential. The color map shows the ESP energy (in hartrees) for the various colors. The red end of the spectrum shows regions of highest stability for a positive test charge, magenta/ blue show the regions of least stability for a positive test charge^[^16]. These images show that the triple and double bonded end of the molecule is electron rich relative to the single bonded end^[^16]. The self-consistent field (SCF) energy is the average interaction between a given particle and other particles of a quantum-mechanical system consisting of many particles. Because the problem of many interacting particles is very complex and has no exact solution; calculations are done by approximate methods. One of the most often used approximated methods of quantum mechanics is based on the interaction of a self-consistent field, which permits the many-particle problem to be reduced to the problem of a single particle moving in the average self-consistent field produced by the other particles^[24]. The final SCF energy of belinostat was found to be -126.3659168682 au (-79295.8815 kcal/mol) as calculated by RHF/PM3 method using ArgusLab 4.0.1 suite. It should be noted that the Mulliken charges do not reproduce the electrostatic potentials of a molecule very well. Mulliken charges were calculed by determining the electron population of each atom as defined by the basis functions^[^21]. The standard heat of formation of a compound is the enthalpy change for the formation of 1mole of the compound from its constituent elements in their standard states at 1 atmosphere. Its symbol is ΔH_f^θ^.The most energetically favourable conformation of belinostat was found to have a heat of formation of 581.1137 kcal/mol via PM3 (NDDO) RHF SCF Type. The steric energy calculated for belinostat was 0.64665673 a.u. (405.78359283 kcal/mol). The docked poses of the derivatives were ranked according to the docking scores i.e. the lowest global energy values was the top ranked and best fitted conformations of the docked pose were analyzed for the study. The complex models generated after successful docking of the belinostat and ebola was based on the parameters such as hydrogen bond interactions, global energy, atractive Vander Waals energy, repulsive Vander Waals energy, and atomic contact energy of the docked compound within the active site. These parameters play important roles in the biological activity of a compound. The molecular docking result predicted the most feasible position for belinostat to inhibit ebola virus glycoprotein. This was found to be -28.87 kcal/mol.

Table 1: Geometry optimised atomic coordinates of belinostat.

Atoms	X	Y	Z	Atomic No.
C	13.57317552	-12.00779349	0.00000000	6
C	13.21151122	-13.44904618	0.00000000	6
C	12.57270712	-11.10713415	0.00000000	6
C	11.93206519	-13.84091147	0.00000000	6
C	11.15804759	-11.53396071	0.00000000	6
C	10.85335140	-12.83488810	0.00000000	6
N	14.86409519	-11.55954248	0.00000000	7
S	16.39167960	-12.48565335	0.00000000	16
O	17.22478053	-11.18014456	0.00000000	8
O	15.77692566	-13.90436742	0.00000000	8
C	18.09236292	-13.31137288	0.00000000	6
C	20.56974473	-13.12559106	0.00000000	6
C	20.64708363	-14.60208976	0.00000000	6
C	19.37191193	-12.52967171	0.00000000	6
C	19.52798805	-15.32807618	0.00000000	6
C	18.21278750	-14.65778439	0.00000000	6
C	21.77993020	-12.28996022	0.00000000	6
C	23.00952118	-12.81216498	0.00000000	6
C	24.21756830	-11.96550048	0.00000000	6
N	25.44123886	-12.54390926	0.00000000	7
O	24.11241288	-10.70543889	0.00000000	8
O	26.54007519	-11.80284723	0.00000000	8
H	14.98310302	-10.51320864	0.00000000	1
H	25.53413263	-13.58974246	0.00000000	1

Table 2: Bond length of belinostat

Atoms	Length
(C1)-(C2)	1.458000
(C1)-(C3)	1.323387
(C1)-(N7)	1.343384
(C2)-(C4)	1.323387
(C3)-(C5)	1.458000
(C4)-(C6)	1.458000
(C5)-(C6)	1.323387
(N7)-(S8)	1.749002
(S8)-(O9)	1.546726
(S8)-(O10)	1.546726
(S8)-(C11)	1.800077
(C11)-(C14)	1.458000
(C11)-(C16)	1.323387
(C12)-(C13)	1.458000
(C14)-(C14)	1.323387
(C12)-(C17)	1.461000
(C13)-(C15)	1.323387
(C15)-(C16)	1.458000
(C17)-(C18)	1.328833
(C18)-(C19)	1.464000
(C19)-(N20)	1.346235
(C19)-(O22)	1.260307
(N20)-(O21)	1.323604

Table 3: Bond angles of belinostat

Atoms	Angles	Alternate angles
(C2)-(C1)-(C3)	120.000000	216.488007
(C2)-(C1)-(N7)	120.000000	282.167276
(C1)-(C2)-(C4)	120.000000	216.488007
(C3)-(C1)-(N7)	120.000000	327.778708
(C1)-(C3)-(C5)	120.000000	216.488007
(C1)-(N7)-(S8)	120.000000	218.427741
(C2)-(C4)-(C6)	120.000000	216.488007
(C3)-(C5)-(C6)	120.000000	216.488007
(C4)-(C6)-(C5)	120.000000	216.488007
(N7)-(S8)-(O9)	92.100000	425.732935
(N7)-(S8)-(O10)	92.100000	425.732935
(N7)-(S8)-(C11)	92.100000	286.724350
(O9)-(S8)-(O10)	92.100000	471.223100
(O9)-(S8)-(C11)	92.100000	303.587174
(O10)-(S8)-(C11)	92.100000	303.587174
(S8)-(C11)-(C14)	120.000000	188.274860
(S8)-(C11)-(C16)	120.000000	210.303144
(C14)-(C11)-(C16)	120.000000	216.488007
(C11)-(C14)-(C12)	120.000000	216.488007
(C11)-(C16)-(C15)	120.000000	216.488007
(C13)-(C12)-(C14)	120.000000	216.488007
(C13)-(C12)-(C17)	120.000000	187.861407
(C12)-(C13)-(C15)	120.000000	216.488007
(C14)-(C12)-(C17)	120.000000	215.760874
(C12)-(C17)-(C18)	120.000000	214.555074
(C13)-(C15)-(C16)	120.000000	216.488007
(C17)-(C18)-(C19)	120.000000	213.837163
(C18)-(C19)-(N20)	120.000000	279.479738
(C18)-(C19)-(O22)	120.000000	275.966448
(N20)-(C19)-(O22)	120.000000	421.698151
(C19)-(N20)-(O21)	120.000000	295.314382

Table 4: Dihedral angles of belinostat.

Atoms	Dihedral Angles
(C4)-(C2)-(C1)-(C3)	5.000000 2
(C2)-(C1)-(C3)-(C5)	19.486776 2
(C4)-(C2)-(C1)-(N7)	5.000000 2
(C2)-(C1)-(N7)-(S8)	13.474221 2
(C1)-(C2)-(C4)-(C6)	38.973552 2
(C5)-(C3)-(C1)-(N7)	19.486776 2
(C3)-(C1)-(N7)-(S8)	13.474221 2
(C1)-(C3)-(C5)-(C6)	10.000000 2
(C1)-(7N)-(S8)-(O9)	2.635231 2
(C1)-(N7)-(S8)-(O10)	2.635231 2
(C1)-(N7)-(S8)-(C11)	2.635231 2
(C2)-(C4)-(C6)-(C5)	10.000000 2
(C3)-(C5)-(C6)-(C4)	38.973552 2
(N7)-(S8)-(C11)-(C14)	1.317616 2
(N7)-(S8)-(C11)-(C16)	1.317616 2
(O9)-(S8)-(C11)-(C14)	1.317616 2
(O9)-(S8)-(C11)-(C16)	1.317616 2
(O10)-(S8)-(C11)-(C14)	1.317616 2
(O10)-(S8)-(C11)-(C16)	1.317616 2
(S8)-(C11)-(C14)-(C12)	5.000000 2
(S8)-(C11)-(C16)-(C15)	19.486776 2
(C12)-(C14)-(C11)-(C16)	5.000000 2
(C14)-(C11)-(C16)-(C15)	19.486776 2
(C11)-(C14)-(C12)-(C13)	19.486776 2
(C11)-(C14)-(C12)-(C17)	19.486776 2
(C11)-(C16)-(C15)-(C13)	10.000000 2
(C15)-(C13)-(C12)-(C14)	5.000000 2
(C15)-(C13)-(C12)-(C17)	5.000000 2
(C13)-(C12)-(C17)-(C18)	5.000000 2
(C12)-(C13)-(C15)-(C16)	38.973552 2
(C14)-(C12)-(C17)-(C18)	5.000000 2
(C12)-(C17)-(C18)-(C19)	38.973552 2
(C17)-(C18)-(C19)-(N20)	5.000000 2
(C17)-(C18)-(C19)-(O22)	5.000000 2
(C18)-(C19)-(N20)-(O21)	13.474221 2
(O21)-(N20)-(C19)-(O22)	13.474221 2

Table 5: Improper torsions of belinostat

Atoms	Improper torsions
3 7 1 2 (C)-(N)-(C)-(C)	2.000000
8 23 7 1 (S)-(H)-(N)-(C)	2.000000
14 16 11 8 (C)-(C)-(C)-(S)	2.000000
14 17 12 13 (C)-(C)-(C)-(C)	2.000000
20 21 19 1 ( N)-(O)-(C)-(C)	16.666667
22 24 20 19 (O)-(H)-(N)-(C)	2.000000

Table 6: Final energy evaluation.

S.No.	Force field	Energy components (au)
1	Molecular mechanics bond (Estr)	0.00453750
2	Molecular mechanics angle (Ebend)+ (Estr‑bend)	0.57603306
3	Molecular mechanics dihedral (Etor)	0.02519703
4	Molecular mechanics ImpTor (Eoop)	0.00000000
5	Molecular mechanics vdW (EVdW)	0.04088915
6	Molecular mechanics coulomb (Eqq)	0.00000000
Total		0.64665673a.u. (405.78359283 kcal/mol)

Table 7 : Ground State Dipole (debye)

X	Y	Z	Length
-4.83698520	-5.04353016	-0.00000000	6.98810577

Table 8: List of Mulliken atomic charges and ZDO atomic charges of belinostat

S/NO	Atoms	ZDO Atomic Charges	Mulliken Atomic Charges
1	C	0.1477	0.1604
2	C	0.2007	0.1952
3	C	-0.0041	-0.0204
4	C	-0.1060	-0.0791
5	C	-0.0040	-0.0338
6	C	0.0580	0.0459
7	N	-1.0977	-1.3370
8	S	3.382	4.2811
9	O	-0.9370	-0.8867
10	O	-0.8667	-0.9706
11	C	-1.2220	-1.8037
12	C	-0.1185	-0.1673
13	C	0.2448	0.2833
14	C	0.2815	0.3526
15	C	-0.2352	-0.2464
16	C	0.2651	0.2591
17	C	0.1380	0.1564
18	C	-0.0962	-0.0989
19	C	0.1896	0.2325
20	N	0.1035	0.0772
21	O	-0.1212	-0.1243
22	O	-0.2533	-0.2661

Table 9 : Relative interaction energy values of belinostat docked unto Ebola Virus

Rank	Solution Number	Global Energy	Attractive VdW	Repulsive VdW	ACE	HB
		Kcal/mol
1	1	-28.87	-12.55	6.57	-10.85	0.00
2	6	-28.42	-12.46	4.98	-9.65	0.00
3	5	-28.02	-12.37	6.51	-10.53	0.00
4	8	-27.89	-12.60	8.85	-10.31	0.00
5	9	-26.53	-12.66	12.37	-11.16	0.00
6	10	-26.34	-13.64	15.39	-12.07	0.00
7	4	-25.28	-12.80	12.47	-10.90	0.00
8	2	-21.85	-12.01	12.96	-10.01	0.00
9	3	-16.55	-12.63	21.22	-10.78	0.00
10	7	-13.52	-6.91	3.68	-5.47	0.00

CONCLUSION:

Geometry optimization of (2E)-N-hydroxy-3-[3-(phenylsulfamoyl) phenyl]prop-2-enamide (belinostat) using Argus lab software was performed. Molecular mechanics calculations were based on specific interactions within the molecule. These interactions included stretching or compressing of bond beyond their equilibrium lengths and angles. The excited states of belinostat were created. The final self- consistent field (SCF) energy was found to be be -126.3659168682 au (-79295.8815 kcal/mol). This is the average interaction between a given belinostat particle and other belinostat particles of a quantum-mechanical system consisting of

many particles. The most energetically favourable conformation of belinostat was found to have a heat of formation of 581.1137 kcal/mol via PM3 (NDDO) RHF SCF Type. The steric energy calculated for belinostat was 0.64665673 a.u.(405.78359283 kcal/mol). Molecular docking result revealed the binding free energy. The global binding energy value -28.87 Kcal/mole was ranked first because it had the least energy. The most feasible position for belinostat to inhibit ebola virus glycoprotein was predicted to be -28.87 kcal/mol.

REFERENCES:

1. Plumb JA, Finn PW, Williams RJ, Bandara MJ, Romero MR, Watkins CJ, La T, Nicholas B, Brown R. Pharmacodynamic Response and Inhibition of Growth of Human Tumor Xenografts by the Novel Histone Deacetylase Inhibitor PXD101".Molecular Cancer Therapeutics 2 (8); 2003: 721–728.

2. Available from: URL: http://www.curagen.com//.

3. Brad P, Ranjana A, Madeleine D, Kenneth B.H, Tanin I, Arnuparp L, Ofer S, Adam L, Dina BY, Marie BBarry, Uwe H, Jan F and Francine MF. Final Results of a Phase II Trial of Belinostat (PXD101) in Patients with Recurrent or Refractory Peripheral or Cutaneous T-Cell Lymphoma, 51^st ash annual meeting and exposition of American society of Hematology, 2009 ; 260 – 262.

4. John C. Spectrum adds to cancer pipeline with $350M deal. Available from: http://www.fiercebiotech.com/story /spectrum-adds-cancer-pipeline-350m-deal/2010-02-02

5. Spreitzer H. Neue Wirkstoffe – Belinostat. Österreichische Apothekerzeitung, 2014: 27.

6. Hamadani M, Abukar SM, Usmani SZ, Savani BN, Ayala E, Kharfan-Dabaja MA. Management of relapses after hematopoietic cell transplantation in T-cell non-Hodgkin lymphomas. Semin Hematol. 51(1); 2014:73–86.

7. Reddy NM, Savani BN. Management of T-cell lymphomas: overcoming challenges and choosing the best treatment. Semin Hematol. 51(1); 2014:1 – 4.

8. Reddy NM, Evens AM. Chemotherapeutic advancements in peripheral T-cell lymphoma. Semin Hematol. 51(1); 2014:17 – 24.

9. Henderson NV .FDA Grants Spectrum Pharmaceuticals Accelerated Approval of Beleodaq™ (belinostat) for Injection. Available from: URL: http://investor.sppirx.com/releasedetail

10. Mulliken RS. Electronic Population Analysis on LCAO-MO Molecular Wave Functions. I. The Journal of Chemical Physics 23(10); 1955: 1833–1840.

11. Csizmadia IG and Enriz RD . Peptide and protein folding. J. Mol. Struct.-Theochem., 543; 2001: 319 - 361.

12. Leach AR. Molecular modelling: principles and applications. Englewood Cliffs, N.J: Prentice Hall. 2001: 12

13. Ohlinger WS, Philip EK, Bernard JD, Warren JH. Efficient Calculation of Heats of Formation . The Journal of Physical Chemistry A (ACS Publications), 113(10); 2009 : 2165–2175.

14. Peng C, Ayali PY, Schlegel HB and Frisch MJ. Using redundant internal coordinates to optimize equilibrium geometries and transition states. J.Comp.Chem., 16; 1995 : 49-51.

15. Cramer CJ and Truhlar DG . AM1-SM2 and PM3-SM3 parameterized SCF solvation models for free energies in aqueous solution. Computer-Aided Mol. Design, 6; 1992 : 629-666.

16. Mark AT, ArgusLab 4.0, Planaria Software LLC, Seattle, WA. 2003. http://www.arguslab.com.

17. http://www.acdlab.com.

18. Dewar MJS, Zoobisch EG, Healy EF and Stewart JJP. AM1: A new general purpose quantum mechanical molecular model. J. Am. Chem. Soc. 107; 1985: 3902 - 3910.

19. Loew GH, Villar HO and Alkorta I. Strategies for indirect computer aided drug design. Pharmaceut. Res., 10; 1998: 475- 486.

20. Simons J, Jorgens P, Taylor H and Ozment J. Walking on potential energy surfaces". J. Phys. Chem., 87; 1983: 2745-2753.

21. Martin YC. Perspective in drug discovery and design. Springer Publisher, USA, 12; 1988: 3- 23.

22. Cruciani G, Clementi S and Pastor M (1998). GOLPEguided region selection. Perspectives in Drug Discovery and Design, 12-14(16); 1998 : 71 - 86.

23. Dunn III and Hopfinger AJ (1998). Drug Discovery, Kluwer Academic Publishers. Chapter 12; 1998: 167-182.

24. http://www.thefreedictionary.com.

25. Duhovny D, Nussinov R and Wolfson H.J .Efficient Unbound Docking of Rigid Molecules. In Gusfield et al., Ed. Proceedings of the 2'nd Workshop on Algorithms in Bioinformatics(WABI), Springer Verlag, Rome, Italy, Lecture Notes in Computer Science , 2452; 2002: 185-200.

26. Mashiach E. Schneidman-Duhovny D, Andrusier N, Nussinov R and Wolfson H.J. FireDock a webserver for fast interaction refinement in molecular docking, Nucleic Acid Res, 36(web server issue); 2008: 229 – 292.

27. Andrusier N, Nussinov R and Wolfson H.J. FireDock: fast interaction refinement in molecular docking proteins, 69(1); 2007: 139 – 159.

28. http://www.expasy.org/spdbv/.

Received on 10.08.2015 Accepted on 05.09.2015

Asian J. Pharm. Res. 5(3): July- Sept., 2015; Page 131-137

DOI: 10.5958/2231-5691.2015.00020.9

*Corresponding Author E-mail: ifeanyiotuokere@gmail.com