Nalini Namasivayam & Chinniagounder Theivarasu. Int. Res. J. Pharm. 2016, 7 (9)

4

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com

ISSN 2230 – 8407

Research Article SPECTRAL CHARACTERIZATION AND BIOCIDAL STUDIES OF 2, 2’,6, 6’-TETRAMETHYLPIPERIDIN-4-ONE AND THEIR PALLADIUM (II) COMPLEX Nalini Namasivayam 1 and Chinniagounder Theivarasu 2* 1Department of Chemistry, Sri Krishna College of Technology, Coimbatore, India 2Department of Chemistry, PSG College of Technology, Coimbatore, India *Corresponding Author Email: theivarasu@yahoo.co.in Article Received on: 23/07/16 Revised on: 12/08/16 Approved for publication: 03/09/16 DOI: 10.7897/2230-8407.079105 ABSTRACT A binuclear palladium (II) complex, of the type [Pd2(μ-Cl)2(L)4]Cl2 (where L-2,2’,6,6’-tetramethylpiperidin-4-one) have been synthesised and characterised by elemental analysis, FT-IR, UV-Vis, mass,1H and 13C NMR spectra. The ligand containing two coordinating sites, namely ring nitrogen and carbonyl group. The spectral studies revealed that the ligand is monodentate in which the ring nitrogen coordinated to palladium (II) but not the carbonyl group. Electronic spectral data reveal that the complex has square planar geometry. The anticancer studies against the cervical HeLa cell line and antimicrobial activity against Escherichia coli, Staphylococcus aureus, Aspergillus niger and Candida albicans were screened for Palladium(II) complex(C), Dichloro(1,5-cyclooctadiene)palladium(II)(M) and 2,2’,6,6’-tetramethylpiperidin-4-one(L). Keywords: palladium, 2,2’,6,6’-tetramethylpiperidin-4-one, HeLa cell line, Antimicrobial INTRODUCTION The chemistry of noble metal complexes derived from nitrogen and oxygen donor ligands has a wide spectrum, ranging from synthesis to application in many diverse fields. Over a decade, piperidin-4-ones have a dramatic growth of interest in inorganic complexes based on their special properties such as rigidity, co-planarity, reactivity, ion selectivity, stereochemistry and biological activities1. The chemistry of VIII group elements in the periodic table exhibits common features, but wide variations are also frequent2. In this group, cisplatin is the parent compound and widely used for the treatment of tumors3, 4. The importance of heterocyclic chemistry made interest in exhibiting coordination modes and biochemical analysis. Palladium (II) complex and piperidin-4-ones has shown potential in biological properties. To synthesise palladium complex with a suitable precursor is a challenging domain5-12. MATERIALS AND METHODS All synthetic materials are procured from Merck and Aldrich companies and used without further purification. The purity of the synthesised compounds was monitored by thin layer chromatography (TLC) on silica gel coated plates. Melting points of the synthesised compounds were determined in open glass capillaries and uncorrected. The IR (KBr) spectra (ν, cm-1) were recorded by using KBr pellets on Shimadzu FT/IR-330E instrument (4000-400 cm-1). Nuclear Magnetic spectroscopy (NMR) measurements (1H-NMR and 13C-NMR) spectra was carried out by using Joel GSX 4OOMB NMR spectrometer (500.13 MHz and 125.76 MHz) with DMSO d6 as solvent and tetramethylsilane (TMS) as internal standard and the mass spectra (EI-MS) was recorded on Jeol GC mate instrument at 70 eV at Sophisticated Analytical Instrumentation Facility (SAIF), IIT Madras, Chennai.

Synthesis of palladium (II) complex13

The title compound was synthesized by using stoichiometric quantities of dichloro(1,5-cyclooctadiene)palladium(II) (0.5 mmol) (Sigma-Aldrich) and 2,2’,6,6’-tetramethylpiperidin-4-one (0.5 mmol) (Sigma-Aldrich) in a ratio of 1:2 was suspended in 25ml of dichloromethane(Merck) solution. The reaction mixture was stirred for 30 minutes at room temperature. The resulting brownish yellow colour solution was condensed under reduced pressure. The precipitate was dried in vacuo in the air was repeatedly washed with hot ethanol, acetone, and ether to remove the untreated piperidin-4-one and then dried. The yield of the isolated product was 70%. In-vitro cytotoxicity test (Cell lines and growth conditions)

14-18

The human cervical cancer cell line (HeLa) was obtained from the National Centre for Cell Science (NCCS), Pune, and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS).The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspensions and viable cells were counted using hemocytometer and diluted with medium with 5% FBS to give a final density of 1x105 cells/ml. One hundred microliters per well of the cell suspension were seeded into 96-well plates at a plating density of 10,000 cells/well and incubated to allow for cell attachment at 37ºC, 5% CO2, 95% air, and 100% relative humidity. After 24 hours, the cells were treated with serial concentrations of the extracts and fractions. They were initially dissolved in dimethyl sulfoxide (DMSO) and further diluted in serum-free medium to produce five concentrations. One hundred microliters per well of each concentration were added to plates to obtain final concentrations of 1000, 500, 250, 125 and 62.5 µM. The final volume in each well was 200 µl and the plates were incubated at 37°C, 5% CO2, 95% air and 100% relative humidity for 48 hours. The medium containing without samples

Nalini Namasivayam & Chinniagounder Theivarasu. Int. Res. J. Pharm. 2016, 7 (9)

5

were served as control. Triplicate was maintained for all concentrations. In vitro MTT assay on HeLa cancer cell lines The dichloro(1,5-cyclooctadiene)palladium(II), 2,2’,6,6’-tetramethylpiperidin-4-one and palladium (II) complex were subjected to the evaluation of anticancer activity against cervical cancer cell lines (HeLa) by MTT (yellow water-soluble tetrazolium salt) assay. Cells were seeded in 96-well plates in triplicate. Following a 24 hours incubation of cultures at 37°C, the medium was replaced with fresh medium, with various concentrations (1000, 500, 250, 125 and 62.5 µM) of the ligand, metal precursor, and complex in a final volume of 200 µl. At the same time, drug-free medium negative control well and solvent control well with the same volume of DMSO was examined as well. The cells were incubated at 37°C for 4 hours. Then 15 µL of MTT (5 mg/ml) in a phosphate buffered saline (PBS) was added to each well and the plates were incubated for an additional 4 hours. The medium with MTT was then flicked off and formed formazan crystals were solubilized in 100 µl of DMSO and then measured the absorbance at 570 nm using a microplate reader. The % cell inhibition was determined using the following formula.

% Growth inhibition = [100- {Abs (sample) /Abs (control)}] x100.

Nonlinear regression graph was plotted between % Growth inhibition and log10 concentration and IC50 were determined using Graph pad Prism software. Antimicrobial Screening19,20

The dichloro(1,5-cyclooctadiene)palladium(II), 2,2’,6,6’-tetramethylpiperidin-4-one and palladium (II) complex were screened for their antimicrobial activity against bacterial species like Escherichia coli, Staphylococcus aureus and fungal species like Candida albicans and Aspergillus niger by using disc diffusion method. The standardised inoculums were prepared in Petri plates. Then treated with nutrient agar medium and the suspension of microbial cultures were standardised by adjusting the turbidity of the culture according to McFarland standards. The turbidity of the culture can be adjusted by the addition of sterile saline. The standardised inoculum was prepared in the Petri plates at 37°C for 24 hours. Antimicrobials for ciprofloxacin and clotrimazole were used as positive control. The metal precursor, ligand and palladium (II) complex were retained in the refrigerator for an hour for diffusion. The zone of inhibition is measured after incubation of inoculum at 37°C.Also, the minimum inhibitory concentration (MIC) were evaluated.

Table 1: Analytical data of ligand and complex

Compound Molecular formula M.pt

(ºC) Elemental analysis (%)

calculated found C H N Cl Pd C H N Cl Pd

L C9H17NO 34-36 69.63 11.04 9.02 - - 69.64 11.02 8.99 - - C [Pd2(μ-Cl)2(L)4]Cl2 172-174 44.50 6.64 5.77 14.60 21.91 44.46 6.63 5.72 14.57 21.89

Figure 1: Concentration versus Vs % Cell viability

Table 2: Antimicrobial activity of ligand, metal precursor and complex

Organism Complex Ligand Metal Standard MIC

(µg/Ml) Zone Of

Inhibition (Mm)

MIC (µg/Ml)

Zone Of Inhibition

(Mm)

MIC (µg/Ml)

Zone Of Inhibition

(Mm)

MIC (µg/Ml)

Zone Of Inhibition

(Mm) Bacteria

Staphylococcus aureus

62.5 13 125 00 250 18 - 40

Escherichia coli 125 11 250 08 250 20 - 30 Fungi

Aspergillus niger 500 09 500 00 250 24 - 10 Candida albicans 500 09 500 08 250 13 - 12

0

20

40

60

80

100

6.25 12.5 25 50 100

% Cell viability

Co n cen tratio n (u M )

Nalini Namasivayam & Chinniagounder Theivarasu. Int. Res. J. Pharm. 2016, 7 (9)

6

RESULTS AND DISCUSSION The present investigation involves the preparation of palladium (II) chloride complex containing the ligand 2,2’,6,6’-tetramethylpiperidin-4-one in dichloromethane solvent. The complex results in dark brown coloured amorphous solid, non-hygroscopic, stable in air and the melting point is 276° C. Palladium complex is soluble in dichloromethane, dimethylsulphoxide, and ethanol and insoluble in water. The analytical and conductance data of palladium (II) complex are given in Table 1. The molar conductivity of palladium (II) complex in dichloromethane is found to be 114.57 v-1 cm2 mol-1 indicating that the complex is 1:1 electrolyte. Anal. [Pd2(C9H17NO)4Cl2]Cl2. Molar mass m/z 979.7991. Infrared spectral studies21-22

The FT-IR spectral data of the 2,2’,6,6’-tetramethylpiperidin-4-one ligand showed bands due ٧ (C=O) and ٧( H -N ) which were observed at 1705 cm-1 and 3579 cm -1 respectively. The band around 3579 cm-1 due to N-H stretching for the ligand has been shifted to high frequency in the palladium (II) complex. A series of the Bolhmann bands in the region 2800-2600 cm-1 were found to be absent in palladium (II) complex. The missing band in the spectrum of the complex reveals that there may be a conformational change in 2,2’,6,6’-tetramethylpiperidin-4-one ligand due to complexation with palladium (II) ion through the lone pair of electrons of ring nitrogen. The carbonyl stretching frequency of the 2, 2’, 6, 6’-tetramethylpiperidin-4-one appears at 1705 cm -1. This absorption is shifted to a higher frequency in the corresponding palladium (II) complex. The higher frequency shift in carbonyl group is attributed to conformational change and not for complexation. For Pd-N, the band is observed at 532 cm -1. UV-Visible spectral studies23 ,24

The electronic spectrum of the complex shows two bands in the UV region due to the carbonyl group of the ligand. A strong band around 210nm and 234 nm in the spectrum of palladium (II) complex are due to π→π* and n→π* transitions, respectively. Mostly no significant band is observed in the visible region. 1H and 13C NMR spectral analysis25 The spectrum of 1H and 13C NMR data in DMSO-d6 as a solvent for the 2,2’,6,6’-tetramethyl piperidin-4-one ligand and its diamagnetic palladium complex. The proton NMR spectral data analysis for N-H at 4.8 ppm, H(3) and H(5) at 2.4-2.5 ppm and methyl protons appeared at 1.3 ppm. On complexation the proton signals in position 3 and 5 are found to be slightly upfield, the lone pair of electrons on N-H proton acquires a partial positive charge by involving coordination and thus electron withdrawing nature of nitrogen is increased in the downfield shift. In 13C NMR data of palladium complex in piperidin-4-one ring C (2) and C (6) carbons are observed at 51.58 ppm. The methyl carbons appeared in the range of 27.97 ppm and the carbonyl carbon observed at 128.87ppm. Anticancer activity26,27

The human cervical HeLa cell line, at 6.25 to 100 µM concentration values of ligand, metal precursor and palladium (II) complex were evaluated. In palladium (II) complex, HeLa cell line was found to be active to observe the percentage of cell viability. By increasing the concentration, % of cell viability decreases. From concentration versus % of cell viability, it is

impossible to calculate with IC50. On the HeLa cell line, at 6.25, 12.5,25,50 and 100 concentrations, the ligand and metal precursor shows no significant anticancer activity. Antimicrobial activity28,29

The antimicrobial activity of the ligand, metal precursor and palladium (II) complex were investigated with Escherichia coli, Staphylococcus aureus, Aspergillus niger and Candida albicans. The zone of inhibition and minimum inhibitory concentration are expressed in mm and µg/ml. Table 4 shows the mean of inhibition zone of ligand, metal precursor and complex, which tested for above organisms by using standard ciprofloxacin and clotrimazole. The zone of inhibition for ligand shows poor activity against staphylococcus aureus and aspergillus niger and the metal precursor exhibit superior activity in bacterial and fungal activity. In palladium (II) complex, the zone of inhibition is greater in bacterial activity and less in fungal activity. It is suggested that in chelated complex, the positive charge of the metal ion is partially shared with donor atoms and there is delocalization over the complex. This moderate activity suspected by solubility and cell mechanism influenced by the presence of metal precursor. The minimum inhibitory concentration (MIC) values have ranged from 62.5 to 500 μg/mL for the ligand, metal precursor and palladium (II) complex. In the bacterial activity, ligand and complex show minimum inhibitory concentration at 62.5 and 125 μg/mL against Staphylococcus aureus and in fungal activity, metal precursor exhibit at 250 μg/mL against Aspergillus niger and Candida albicans. CONCLUSION In conclusion, we have accomplished a convenient synthesis of palladium (II) complex. The synthesized complex was analyzed by spectral studies, anticancer and antimicrobial activities. The spectral characteristics act as monodentate ligands and confirm that the palladium metal is coordinated to nitrogen. The anticancer activity in 2,2’,6,6’-tetramethylpiperidin-4-one and dichloro(1,5-cyclooctadienepalladium(II) remained passive. On the combination of ligand and metal precursor the formation of palladium (II) complex shows cell viability in moderate to marginal activity and it is difficult to calculate for IC50 due to the substituents of the methyl group of the ligand. The presence electron releasing substituent particularly methyl group at position-2, 2’,6 and 6’ of piperidin-4-one ring found to turn off the anticancer activity. A possible explanation for this lack of activity might be their high reactivity: they may be inactivated before reaching their target. Thus, substituents of piperidin-4-one is unable to enhance the anticancer activity. In the antimicrobial studies for ligand, metal and complex, the zone of inhibition is more active to Staphylococcus aureus and Aspergillus niger organisms and minimum inhibitory concentrations exhibit a lower value for Staphylococcus aureus in palladium (II) complex. The novel skeleton of ligand seems to be further development towards a new potential with other metals. REFERENCES 1. Bartecki A, Kurzak K. Colour of 3d Transition Metal

Compounds. Part 1. Experimental Quantitative Studies and Ligand-Field Based Predictions, d4, d6 and d9 Systems.

Nalini Namasivayam & Chinniagounder Theivarasu. Int. Res. J. Pharm. 2016, 7 (9)

7

Reviews in Inorganic Chemistry 2006; 26(4):389-442. https:// www. researchgate.net/.../233990023_

2. Agarwal RK, Prasad S. Synthesis and spectral investigations of some platinum metals ions coordination compounds of 4 [N-(furan-2'-carboxalidene) amino] antipyrine thiosemicarbazone and 4 [N-(3', 4', 5'-trimethoxybenzalidene) amino] antipyrine thio semicarbazone. Turkish Journal of Chemistry May 2005; 29(3):289-297. http://journals.tubitak.gov.tr/chem/ issues/kim-05-29-3/kim-29-3-8-0405-1.pdf.

3. Rosenberg B, Van Camp L, Krigas T. Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. Nature 1965; 205(4972):698-699. http://www.oalib.com/references/14305986.

4. Jamieson ER, Lippard SJ. Structure, recognition, and processing of cisplatin-DNA adducts.Chemical reviews 1999; 99(9): 2467-2498. http:// lem.ch. unito.it /didattica /infochimica/Antitumorale_CisPt_2006/molecole_banche_dati/Chem.Rew.1999.pdf.

5. Desoize B. Metals and metal compounds in cancer treatment. Anticancer Research. 2004; 24(3A):1529-1544. http://ar.iiarjournals.org/content/24/3A/1529.full.pdf

6. Urquiola C,Vieites M, Torre MH,Cabrera M, Lavaggi ML, Cerecetto H, Garat B. Cytotoxic palladium complexes of bioreductive quinoxaline N 1, N 4-dioxide prodrugs. Bioorganic & medicinal chemistry 2009; 17(4):1623-1629. http://riquim.fq.edu.uy/archive/files/436052c6e65547f3294704f76c22fcca.pdf

7. Das U, Alcorn J, Shrivastav A, Sharma RK, De Clercq E, Balzarini J,Dimmock JR. Design, Synthesis and cytotoxic properties of novel 1-[4-(2-alkylaminoethoxy) phenylcarbonyl]-3, 5-bis (arylidene)-4-piperidones and related compounds. European journal of medicinal chemistry 2007; 42(1): 71-80.doi: 10.1016 /j.ejmech. 2006. 08. 002

8. Cohen SM, Lippard SJ.Cisplatin from DNA damage to cancer chemotherapy. Progress in nucleic acid research and molecular biology 2001; 67: 93-130. doi:10.1016/S0079-6603(01)67026-0

9. Chen HH, Zhou HJ, Fang X. Inhibition of human cancer cell line growth and human umbilical vein endothelial cell angiogenesis by artemisinin derivatives in vitro. Pharmacological Research 2003;48(3): 231-236.http://dx.doi.org/10.1016/S1043-6618(03)00107-5

10. Wang X, He L, He Y, Zhang J, Su CY.Solvent-free synthesis of a Pd (II) coordination networked complex as reusable catalyst based on 3, 5-bis (diphenylphosphino) benzoic acid. Inorganica Chimica Acta 2009; 362(10):3513-3518. http:// dx.doi. org /10.1016/j.ica.2009.03.039

11. Aridoss G, Amirthaganesan S, Kumar NA, Kim JT, Lim KT, Kabilan S, Jeong YT. A facile synthesis, antibacterial, and antitubercular studies of some piperidin-4-one and tetrahydropyridine derivatives. Bioorganic & medicinal chemistry letters 2008; 18(24): 6542-6548.http://dx.doi.org/10.1016/j.bmcl.2008.10.045

12. Solimana HA, Yousifa MN, Saidb MM, Hassana NA, Ali MM, Awad HM, Abdel-Megeid F M. 2014. Synthesis of novel 1, 6-naphthyridines, pyrano [3, 2-c] pyridines and pyrido [4, 3-d] pyrimidines derived from 2, 2, 6, 6-tetramethylpiperidin-4-one for in vitro anticancer and antioxidant evaluation. Der Pharma Chemica 2014; 6(3): 394-410. derpharmachemica.com/vol6-iss3/DPC-2014-6-3-394-410.pdf

13. Selvaraj K, Theivarasu C. Synthesis and Characterization of Samarium (III) Complexes with Piperidin-4-ones. Synthesis and Reactivity in Inorganic and Metal-Organic Chemistry 2000; 30(6): 1113-1128. http: // dx. doi.org /10.1080 /00945710 009351823

14. Pati HN, Das U, Das S, Bandy B, De Clercq E, Balzarini J, Dimmock JR. The cytotoxic properties and preferential toxicity to tumour cells displayed by some 2, 4-bis (benzylidene)-8-methyl-8-azabicyclo [3.2.1] octan-3-ones and 3,5-bis (benzylidene)-1-methyl-4-piperidones. European journal of medicinal chemistry 2009; 44(1):54-62. http://dx.doi.org/10.1016/j.ejmech.2008.03.015

15. Bauer AW, Kirby WMM. Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Microbiology: A Centenary Perspective. Washington, DC, USA: ASM Press;1999.p 40-45.

16. Bauer AW. The significance of bacterial inhibition zone diameters. A study of the factors influencing antibiotic disc sensitivity tests. Proc. 3rd Int. Congr. Chemo; 1964.

17. Gillespie SH. Medical microbiology illustrated. Butterworth-Heinemann; 2014.

18. Boyd MR, Paull KD. Some practical considerations and applications of the National Cancer Institute in vitro anticancer drug discovery screen. Drug Development Research February1995; 34(2): 91-109.doi: 10.1002/ ddr.430340203

19. Petersdorf RG, Sherris JC. Methods and significance of in vitro testing of bacterial sensitivity to drugs. The American journal of medicine 1965; 39(5):766-779.http://dx.doi.org/10.1016/0002-9343(65)90096-3

20. Gillespie SH. Synthesis, Characterization of a nt imicro bia l evaluation of thiazolidine.Bentley’s textbook of pharmaceutical chemistry book, Butterworth Heine mann: London; 1994.

21. Pop F, Branzea DG, Cauchy T, Avarvari N. Bimetallic neutral palladium (II) bis (dithiolene) complex: Unusual synthesis, structural and theoretical study. Comptes Rendus Chimie 2012; 15(10):904-910.http://dx.doi.org/10.1016/j.crci.2012.03.007

22. Asma M, Badshah A, Ali S, Sohail M, Fettouhi M, Ahmad S,Malik A. Synthesis, Characterization of Mixed Ligand Palladium (II) Complexes of Triphenylphosphine and Anilines and their Enzyme Inhibition Studies against β-glucuronidase. The Crystal Structure of trans-dichloro-(m-chloroaniline)(triphenylphosphine) palladium (II). Transition metal chemistry 2006; 31(4): 556-559.doi: 10.1007/s11243-006-0027-z

23. Kavitha P,Reddy KL. Pd (II) complexes bearing chromone based Schiff bases: synthesis, characterisation and biological activity studies. Arabian Journal of Chemistry March 2013:http://dx.doi.org/10.1016/j.arabjc.2013.06.018

24. Selvaraj K, Theivarasu C. Thermal and spectroscopic studies of terbium (III) and dysprosium (III) complexes with piperidin-4-ones. Thermochimica acta 2003; 401(2): 187-197. http://dx.doi.org/10.1016/S0040-6031(02)00563-4

25. Urquiola C, Vieites M, Torre MH, Cabrera M, Lavaggi ML, Cerecetto H, Garat B. Cytotoxic palladium complexes of bioreductive quinoxaline N 1, N 4-dioxide prodrugs. Bioorganic & medicinal chemistry 2009; 17(4): 1623-1629.http://dx.doi.org/10.1016/j.bmc.2008.12.064

26. Utku S, Gümüş F, Guer S, Özkul A. Synthesis and cytotoxic activity of platinum (II) and platinum (IV) complexes with 2-hydroxymethylbenzimidazole or 5(6)-chloro-2-hydroxymethylbenzimidazole ligands against MCF-7 and HeLa cell lines. Turkish Journal of Chemistry Febuary 2007; 31(4):503-514.

27. Theivarasu C,Sivaprakash K. Characterization and Antibacterial Studies on Copper (II) Binuclear Complexes with Substituted Piperidin-4-ones.Oriental journal of Chemistry August 2012;28(3):1379-1387.orientjchem.org /...THEIVARASU-and SIVAPRAKASH /OJCV028I0 3P1379-1387.pdf

Nalini Namasivayam & Chinniagounder Theivarasu. Int. Res. J. Pharm. 2016, 7 (9)

8

28. Suresh T, Dhanabal T, Kumar RN, Mohan PS. Synthesis, characterization and antimicrobial activities of fused 1, 6-naphthyridines. Indian Journal of Chemistry Section B 2005; 44(11): 2375-2379 http:// nopr. niscair. res.in/ bitstream/ 123456789/ 9227/1/IJCB%2044B(11)%202375-2379.pdf

29. Bhakiaraj D, Elavarasan T,Gopalakrishnan M. Synthesis, spectral analysis, antimicrobial evaluation and molecular docking studies of some novel 3, 5-dichloro-2,6-diarylpiperidin-4-ones.Der Pharma Chemica 2014;6(5):243-

250.der pharma chemica.com/vol6-iss5/DPC-2014-6-5-243-250.pdf

Cite this article as: Nalini Namasivayam and Chinniagounder Theivarasu. Spectral characterization and biocidal studies of 2, 2’,6, 6’-tetramethylpiperidin-4-one and their palladium (II) complex. Int. Res. J. Pharm. 2016;7(9):4-8 http://dx.doi.org/10.7897/2230-8407.079105

Source of support: Nil, Conflict of interest: None Declared

Disclaimer: IRJP is solely owned by Moksha Publishing House - A non-profit publishing house, dedicated to publish quality research, while every effort has been taken to verify the accuracy of the content published in our Journal. IRJP cannot accept any responsibility or liability for the site content and articles published. The views expressed in articles by our contributing authors are not necessarily those of IRJP editor or editorial board members.