In Silico Study of secondary metabolites from leaf extract of Plumbago zeylanica

 

D. Roselin Jenifer^1*, Dr. B. R. Malathy², Dr. Jemmy Christy. H³

¹Department of Bioinformatics, Faculty of Bio and Chemical Engineering,

Sathyabama Institute of Science and Technology, Chennai – 119.

²Department of Microbiology, Reader, Sathyabama Dental College and Hospital, Chennai – 119.

³Assistant Professor, Department of Bioinformatics, Sathyabama Institute of Science and Technology,

Chennai – 119.

 

ABSTRACT:

The anticancer activity of secondary metabolites identified from the medicinal plant Plumbago zeylanica was studied using In silico analysis to interpret the inhibitory activity of Matrix metalloproteinases (PDB ID: 3C7X). Polymorphism in Matrix metalloproteinases (MMPs) has been linked to different neoplaplasias, including oral squamous cell carcinoma (OSCC). Docking effect of the 14 secondary metabolites identified from Plumbago zeylanica with receptors was explored. Among the 14 secondary metabolites studied, five of them (Tetradecanal, Sulfurous acid 2-ethylhexyl isohexyl ester, 7, 9 -Di-tert-butyl-1-oxaspiro(4, 5)deca, 1-Fluorononane, Nonadecanol) bind to protein receptors effectively. This was proved by computational modeling based on highest docking score and hydrogen bond interaction.

 

 

INTRODUCTION:

Use of medicinal plants in allopathy and herbal medicine is in great demand due to increased success in validating the safety and efficacy of herbal remedies, the interest shown by drug companies in isolating useful compounds from plants; search for new drugs for treatment of serious and drug-resistant diseases; marketing strategies by the companies dealing in herbal medicine.

 

Plumbago zeylanica is one of the important medicinal plant in China and other Asian countries used for the treatment of rheumatoid arthritis, cancer, dysmenorrhea and contusion of extremities¹. The phytochemical screening of Plumbago zeylanica confirmed the existence of various bioactive compounds such as linoleic acid, palmitic acid, plumbagin, nonylnonanoate, lupeol acetate, stigmasterol acetate, lupanone, friedelinol stigmasterol, sitosterone.

 

Plumbagin is a stable naphtha quinine, which exhibit anticancer^2,3, Insecticidal and antimutagenic activity⁴, antifertile⁵, antimicrobial activity⁶ and leishmanicidal activity⁷.

 

Aberrant activation of intracellular signaling pathways induces malignant properties of a healthy cells in humans. Gene expression profiling and the differentially expressed genes were targeted for therapeutic purposes⁸. Oral squamous cell carcinoma (OSCC) is the widespread malignant cancer in the oral cavity⁹. Matrix metalloproteinases (MMPs) were a group of enzymes, which are involved in the breakdown of extracellular matrix protein during the time of organogenesis, growth and normal tissue replacement. Polymorphic property of proteinases is one of the main highlights OSCC. The activity of MMPs in adult tissues is quite low, but its activity is increased in several pathological conditions such as inflammatory diseases, tumor growth and metastasis¹⁰. MMPs is major enzymes that play a role in extracellular matrix (ECM) degradation and are called as matrixins. There are 23 MMPs enzymes in humans. In normal steady state tissues the activity of MMPs is low or negligible. Their expression is controlled by cytokines, growth factors, hormones, cell –cell and cell – matrix interaction¹¹. MMPs are also regulated by tissue inhibitors of metalloproteinases (TIMPs).

 

Balance between MMPs and TIMPs are required for the extracellular matrix remodeling. Large number of MMPs inhibitors were designed and synthesized and were clinically tested for the treatment cancer or arthritis, which showed little efficacy so far. Hence a study was undertaken to explore the secondary metabolites (Nonane 5-(2-methylpropyl ), Tetradecanal, Sulfurous acid 2-ethylhexyl isohexyl ester, Undecane 3, 8-dimethyl, Hydrazine tetra phenyl, Nonane 3, 7-dimethyl, Nonane 5-methyl-5-propyl, Hexadecane 1-iodo, 7, 9-Di-tert-butyl-1-oxaspiro(4, 5)deca, Undecane 2, 9-dimethyl, Heptadecane 2, 6, 10, 15-tetramethyl, 1-Fluorononane, Bromo dodecane and Nonadecanol) from medicinal plant Plumbago zeylanica and evaluate its anticancer activity by inhibiting MMPs. Mutation in MMp gene causes oral cancer. This computational modelling proved the effect of phytochemical from Plumbago zeylanica against oral cancer target protein Hemopexin (HPX) -3c7x. Docking results of 14 bioactive compounds tested showed, only five secondary metabolites effectively bind with receptor and inhibit the protein action.

 

MATERIAL AND METHODS:

Plumbago zeylanica plant compounds as ligands:

The structure of secondary metabolites (Nonane 5-(2-methylpropyl), Tetradecanal, Sulfurous acid 2-ethylhexyl isohexyl ester, Undecane 3, 8-dimethyl, Hydrazine tetra phenyl, Nonane 3, 7-dimethyl, Nonane 5-methyl-5-propyl, Hexadecane 1-iodo, 7, 9-Di-tert-butyl-1-oxaspiro(4, 5) deca, Undecane 2, 9-dimethyl, Heptadecane 2, 6, 10, 15-tetramethyl, 1-Fluorononane, Bromo dodecane and Nonadecanol) were retrieved from PubChem database in ‘.sdf’ format (Table-1). Doxycycline structure was also down loaded in order to do a comparative analysis of their effectiveness against the phytochemical of our interest.

 

Table:1-GC-MS analysis of chemical constituents from Plumbago zeylanica and its PubChem compound ID.

S. No	Name of the chemical constituents	R. Time	Pub Chem ID
1.	Nonane 5-(2-methylpropyl)	5.892	545936
2.	Tetradecanal	7.374	31291
3.	Sulfurous acid 2-ethylhexyl isohexyl ester	8.539	6420722
4.	Undecane 3, 8-dimethyl	9.232	86540
5.	Hydrazine tetra phenyl	10.966	96041
6.	Nonane 3, 7-dimethyl	12.207	28458
7.	Nonane 5-methyl-5-propyl	13.415	551397
8.	Hexadecane 1-iodo	17.593	11007
9.	7, 9-Di-tert-butyl-1-oxaspiro (4, 5)deca	17.697	545303
10.	Undecane 2, 9-dimethyl	18.842	519385
11.	Heptadecane 2, 6, 10, 15-tetramethyl	19.593	41209
12.	1-Fluorononane	23.805	10029
13.	Bromo dodecane Nonadecanol	54.982	8919
14.	Nonadecanol	30.136	80281

 

Preparation of protein receptor:

The X-ray crystallographic structure of MMPs was found from protein data bank (PDB). The hemopexin domain of protein matrix metalloproteinases (PDB ID: 3C7X)¹², consists of 196 amino acid and its structural resolution in 1.70 A⁰ was analyzed by the X-Ray diffraction method. The characterization of structure was analyzed by Ramachandran plot technique¹³. The required hydrogen bond was added to prepare target by applying CHARMforcefield¹⁴ and the docking study was carried out by discovery studio based on Ligand fit module¹⁵ for all secondary metabolites and doxycycline.

 

RESULTS:

Binding site specification and molecular are docking:

A binding site in the target was identified by mapping the grid points in them. Among the three binding sites, site 1 is the largest with 462 points and 57.750 A⁰ in size, site 2 is with 188 points and 23.500 A⁰ in size followed by the smallest site 3 of 120 points and 15.000 A⁰ in size. The molecular docking study revealed the possible conformation by the interaction of the ligand with the binding site of the target protein. Interaction between ligand with the target was calculated depending on the different binding scores which are represented in table: 2.

Table 2: The name of each chemical constituent along with their dock scores in comparison with the already available drugs for treating OSCC.

S. No	Compound Name	Ligand Score1	Ligand Score2	PLP1	PLP2	Jain	Dock Score
1	Nonane 5-(2-methylpropyl)	0.62	2.56	67.38	71.99	4.97	65.08
2	Tetradecanal	2.02	3.55	75.18	74.84	3.02	67.754
3	Sulfurous acid 2-ethylhexyl isohexyl ester	-999.9	-999.9	70.78	69.33	2.85	69.082
4	Undecane 3, 8-dimethyl	0.78	2.95	64.16	65.72	3.91	63.486
5	Hydrazine tetra phenyl	-1.55	-1.36	35.3	53.4	5.04	47.292
6	Nonane 3, 7-dimethyl	0.36	2.24	54.2	58.59	3.77	58.142
7	Nonane 5-methyl-5-propyl	-999.9	-999.9	64.08	69.11	2.88	68.566
8	Hexadecane 1-iodo	-999.9	999.9	65.2	71.37	5.25	55.371
9	7, 9-Di-tert-butyl-1-oxaspiro (4, 5) deca	-0.27	1.04	64.31	71.42	5.46	64.86
10	Undecane 2, 9-dimethyl	0.95	3.21	60.63	61.24	3.25	62.569
11	Heptadecane2, 6, 10, 15-tetramethyl	-999.9	-999.9	72.91	77.26	4.16	67.753
12	1-Fluorononane	0.71	2.68	46.53	50.17	2.31	57.571
13	Bromododecane Nonadecanol	0.73	3.04	59.01	57.53	3.73	62.581
14	Nonadecanol	-999.9	-999.9	75.26	90.4	6.1	76.486

Interaction between target and ligand:

The intermolecular connection of the ligand with the target protein was calculated to decide the type of bond present between ligand and the target and the distance was calculated. The intermolecular interaction formed between the atom in the target with the atom of the ligand is represented in Table-3. Figure 1 to 6 shows the two dimensional and three-dimensional structure of the intermolecular interaction of each ligand with the amino acids in the target.

 

Table: 3 The intermolecular interaction of the atoms in ligand with the atoms of the target in the binding site.

S. No	Compound name	Interacting atom	Distance	Category of interaction	Type of interaction	No.of Hydrogen
1	Nonane 5-(2-methylpropyl)	545936:C8 - A: LEU419	5.2415	Hydrophobic	Alkyl	Nil
		545936:C9 - A: LEU419	5.35594	Hydrophobic	Alky
		545936:C9 - A: MET422	4.88187	Hydrophobic	Alky
		545936:C9 - A: MET468	5.03175	Hydrophobic	Alky
		545936:C13 - A: MET422	4.79483	Hydrophobic	Alky
2	Tetradecanal	A: GLY331:HN - 31291: O1	1.92148	Hydrogen Bond	Conventional Hydrogen Bond	1
3	Sulfurous acid 2-ethylhexyl isohexyl ester	A: GLY331:HA1 - 6420722: O4	3.08176	Hydrogen Bond	Carbon Hydrogen Bond	10
		6420722:H41 - A: MET328:O	2.6986	Hydrogen Bond	Carbon Hydrogen Bond
		6420722:H41 - A: GLU373:OE1	2.5664	Hydrogen Bond	Carbon Hydrogen Bond
		6420722:H42 - A: GLU373:OE1	2.42848	Hydrogen Bond	Carbon Hydrogen Bond
		6420722:C17 - A: LEU419	5.33491	Hydrophobic	Alkyl
		6420722:C17 - A: MET422	5.01816	Hydrophobic	Alkyl
		6420722:C17 - A: MET468	5.19848	Hydrophobic	Alkyl
		A: PHE420 - 6420722:C15	4.3156	Hydrophobic	Pi-Alkyl
		A: MET422:HN - A:PHE420:O	2.60338	Hydrogen Bond	Conventional Hydrogen Bond
		A:MET422:HN - A:MET468:SD	2.89892	Hydrogen Bond	Conventional Hydrogen Bond
		A:GLY331:HA1 - 6420722:O4	3.08176	Hydrogen Bond	Conventional Hydrogen Bond
		6420722:H41 - A:MET328:O	2.6986	Hydrogen Bond	Conventional Hydrogen Bond
		6420722:H41 - A:GLU373:OE1	2.5664	Hydrogen Bond	Conventional Hydrogen Bond
		6420722:H42 - A:GLU373:OE1	2.42848	Hydrogen Bond	Conventional Hydrogen Bond
4	Undecane 3, 8-dimethyl	86540:C9 - A:MET328	4.97869	Hydrophobic	Alkyl	Nil
		86540:C11 - A:MET328	5.27044	Hydrophobic	Alkyl
		86540:C12 - A:LEU419	5.07398	Hydrophobic	Alkyl
		96041 - A:MET422	5.07711	Hydrophobic	Pi-Alkyl
		96041 - A:MET328	3.88377	Hydrophobic	Pi-Alkyl
		96041 - A:MET328	5.46753	Hydrophobic	Pi-Alkyl
	Nonane 3, 7-dimethyl	28458:C8 - A:MET422	5.14016	Hydrophobic	Alkyl	Nil
		28458:C9 - A:LEU419	5.45146	Hydrophobic	Alkyl
		28458:C9 - A:MET422	4.83024	Hydrophobic	Alkyl
		28458:C9 - A:MET468	5.04321	Hydrophobic	Alkyl
		28458:C11 - A:LEU419	5.06472	Hydrophobic	Alkyl
6	Nonane 5-methyl-5-propyl	551397:C5 - A:MET422	4.8198	Hydrophobic	Alkyl	2
		551397:C5 - A:MET468	5.1229	Hydrophobic	Alkyl
		551397:C11 - A:MET422	4.63045	Hydrophobic	Alkyl
		551397:C13 - A:MET328	4.33093	Hydrophobic	Alkyl
		A:PHE420 - 551397:C11	4.67566	Hydrophobic	Pi-Alkyl
		A:MET422:HN - A:PHE420:O	2.60338	Hydrogen Bond	Conventional Hydrogen Bond
		A:MET422:HN - A:MET468:SD	2.89892	Hydrogen Bond	Conventional Hydrogen Bond
7	Hexadecane 1-iodo	A:ALA371:O - 11007:I1	2.81511	Halogen	Halogen (Cl, Br, I)	NIl
8	7, 9-Di-tert-butyl-1-oxaspiro(4, 5)deca	A:MET468:HN - 545303:O3	2.02424	Hydrogen Bond	Conventional Hydrogen Bond	6
		A:PHE467:HA - 545303:O3	2.66261	Hydrogen Bond	Carbon Hydrogen Bond
		545303:C15 - A:MET328	4.06984	Hydrophobic	Alkyl
		545303:C16 - A:MET422	3.89841	Hydrophobic	Alky
		545303:C19 - A:MET328	4.17122	Hydrophobic	Alkyl l
		545303:C19 – A:MET422	5.35166	Hydrophobic	Alkyl
		A:PHE420 - 545303:C16	5.04243	Hydrophobic	Pi-Alkyl
		A:PHE420 - 545303:C17	4.31636	Hydrophobic	Pi-Alkyl
		A:MET422:HN - A:PHE420:O	2.60338	Hydrogen Bond	Conventional Hydrogen Bond
		A:MET422:HN - A:MET468:SD	2.89892	Hydrogen Bond	Conventional Hydrogen Bond
		A:MET468:HN - 545303:O3	2.02424	Hydrogen Bond	Conventional Hydrogen Bond
		A:PHE467:HA - 545303:O3	2.66261	Hydrogen Bond	Conventional Hydrogen Bond
9	Nonane 5-methyl-5-propyl	551397:C5 - A:MET422	4.8198	Hydrophobic	Alkyl	2
		551397:C5 - A:MET468	5.1229	Hydrophobic	Alkyl
		551397:C11 - A:MET422	4.63045	Hydrophobic	Alkyl
		551397:C13 - A:MET328	4.33093	Hydrophobic	Alkyl
		A:PHE420 - 551397:C11	4.67566	Hydrophobic	Pi-Alkyl
		A:MET422:HN - A:PHE420:O	2.60338	Hydrogen Bond	Conventional Hydrogen Bond
		A:MET422:HN - A:MET468:SD	2.89892	Hydrogen Bond	Conventional Hydrogen Bond
10	Undecane 2, 9-dimethyl	519385:C10 - A:MET422	4.73984	Hydrophobic	Alkyl	Nil
		519385:C10 - A:MET468	5.07456	Hydrophobic	Alkyl
11	Heptadecane 2, 6, 10, 15-tetramethyl	41209:C14 - A:MET422	5.21503	Hydrophobic	Alkyl	Nil
		41209:C19 - A:MET328	5.16836	Hydrophobic	Alkyl
		A:PHE420 - 41209:C12	4.93307	Hydrophobic	Pi-Alkyl
12	1-Fluorononane	A:TYR372:HA - 10029:F1	2.40349	Hydrogen Bond; Halogen	Carbon Hydrogen Bond; Halogen (Fluorine)	1
		A:MET328:O - 10029:F1	3.16044	Halogen	Halogen (Fluorine)
		A:ALA371:O - 10029:F1	3.67746	Halogen	Halogen (Fluorine)
		A:SER466:O - 10029:F1	3.68951	Halogen	Halogen (Fluorine)
		10029:C10 - A:MET422	4.90784	Hydrophobic	Alkyl
13	Bromo dodecane Nonadecanol	A:ALA371:O - 8919:Br1	3.32016	Halogen	Halogen (Cl, Br, I)	Nil
		8919:C12 - A:MET422	4.82414	Hydrophobic	Alkyl
14	Nonadecanol	80281:H60 - A:ALA418:O	2.01843	Hydrogen Bond	Conventional Hydrogen Bond	4
		80281:H60 - A:SER466:O	1.85929	Hydrogen Bond	Conventional Hydrogen Bond
		80281:H55 - A:ALA371:O	2.39015	Hydrogen Bond	Carbon Hydrogen Bond
		80281:H56 - A:SER466:O	2.68578	Hydrogen Bond	Carbon Hydrogen Bond
		80281:C20 - A:MET422	3.25873	Hydrophobic	Alkyl

 

Figure 1: Intermolecular interaction of Tetradecanal with the target protein Hemopexin (HPX) -3c7x.

 

Figure 2: Intermolecular interaction of Sulfurous acid 2-ethylhexyl isohexyl ester with the target protein Hemopexin (HPX) -3c7x.

Figure 3: Intermolecular interaction of 7, 9-Di-tert-butyl-1-oxaspiro(4, 5)deca with the target protein Hemopexin (HPX) -3c7x.

 

Figure 4: Intermolecular interaction of 1-Fluorononane with the target protein Hemopexin (HPX) -3c7x.

 

Figure 5: Intermolecular interaction of Nonadecanol with the target protein Hemopexin (HPX) -3c7x.

DISCUSSION:

MMP accumulates in low level in normal cells and are involved in remodeling of connective tissue. In pathological conditions, the level of MMPs dysfunction leads to initiate the connective tissue destruction. High level of MMPs production is related to diseases such as tumor invasion/metastasis, atherosclerosis, periodontitis and arthritis^16,17,18. The research involved in exploring MMPs inhibitors are on rise and lead to the discovery of drugs such as Batimastat, Marimastat, Prinomastat, CGS27023A, Tanomastat and Doxycycline. Among these, the use of doxycycline for treatment of ovarian cancer is under clinical trials¹⁹, whereas clinical trials were stopped for the rest due to their toxicity. Insilico study conducted by us proved that Tetradecanal, Sulfurous acid 2-ethylhexyl isohexyl ester, 7, 9 -Di-tert-butyl-1-oxaspiro(4, 5)deca, 1-Fluorononane and Nonadecanol have high docking score compared to doxycycline. Among the fourteen secondary metabolites studied, Tetradecanal-31291, Sulfurous acid 2-ethylhexyl isohexyl ester-6420722, 7, 9 -Di-tert-butyl-1-oxaspiro (4, 5) deca-545303, 1-Fluorononane-10029 and Nonadecaneol-80281 possess efficient interaction with receptor based on high docking score and hydrogen bonding interaction. Hence it is evident that these compounds can outperform the efficacy of doxycycline in inhibiting MMPs.

 

CONCLUSION:

Discovery of new drugs is laborious, time consuming and expensive. With the help of newer applications like Insilco approaches, disadvantages can be minimized. Insilico study based drug design and development has contributed significantly to the modern pharmacology. Thus the high docking score and hydrogen bond interaction obtained for five secondary metabolites from P. zeylanica i.e. Tetradecanal - 31291 which possess one hydrogen bond, Sulfurous acid 2-ethylhexyl isohexyl ester - 6420722 which possess ten hydrogen bonds, 7, 9 -Di-tert-butyl-1-oxaspiro(4, 5)deca- 545303 which possess six hydrogen bonds, 1-Fluorononane – 10029 which possess one hydrogen bond, Nonadecaneol-80281which possess four hydrogen bonds, conclude that they can serve as better MMPs inhibitors. MMPs inhibiting compounds (31291, 6420722, 545303, 10029 and 8028) extracted from Plumbago zeylanica need to be experimentally validated.

 

ACKNOWLEDGMENT:

The authors thank to Sathyabama Institute of Science and Technology to provide facilities to finish this work successfully.

 

ETHICAL CLEARANCE:

Since the study does not involve clinical sample ethical clearance was not obtained.

CONFLICT OF INTEREST:

Nil.

 

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Received on 06.06.2019           Modified on 10.07.2019

Accepted on 30.08.2019         © RJPT All right reserved