Synthesis, Spectral, Antibacterial, Antifungal and Anticancer activity Studies of Schiff bases Derived from O-Vanillin and Aminoquinolines

 

S. N. Battin1, A. H. Manikshete1*, S. K. Sarasamkar2, M. R. Asabe1, D. J. Sathe3

1Department of Chemistry, Walchand College of Arts and Science, Solapur, MH, India.

2Department of Chemistry, Walchand Institute of  Technology, Solapur, MH, India.

3Department of Chemistry, Kolhapur Institute of Thechnology, Kolhapur, MH, India

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

 

ABSTRACT:

In this work, five varieties of o-vanillin Schiff bases viz. 2–methoxy-6-(3/5/6/8- iminoquinolinyl methyl) phenol and 2–methoxy-6-(4-iminoquinaldinyl methyl) phenol have been synthesized with the aid of direct reflux technique in ethanol solution. The synthesized compounds were characterized through CHN evalution, UV- Visible, FTIR, 1H-NMR and Mass spectral studies. The in vitro antimicrobial activities of the synthesized Schiff bases were tested using bacterial species such as Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa and Escherichia coli. These compounds were also tested for antifungal activities against Candida albicans and Aspergillus niger. All the Schiff bases were also screened for their anticancer activities against breast cancer MCF-7 cell line and colon cancer HT-29 cell line.

 

KEYWORDS: Synthesis; o-vanillin; Schiff base; spectral studies; biological activities.

 

1. INTRODUCTION:

Chemistry and bioactivities of o-vanillin and various aminoquinolines prompted us to synthesize a series of new potentially active Schiff bases bearing imine group at various positions in their condensation products. Aromatic Schiff bases constitute a large class of organic compounds, obtained by condensation reaction of aromatic aldehyde and amine [1-3]. The special plane structure of the azomethine –HC=N– group and strong binding ability of the aromatic Schiff bases have the function of antibacterial, antifungal, antitumor and antiviral activities [4-9]. o- vanillin is found to have biological activities such as analgesic, anti-inflammatory, antibacterial, sterilizing and antiviral activities [10-15]. o- vanillin is an useful herbicide, pesticide and bactericide [16-19]. It plays an important role in the process of chemical engineering as a medicine and as a reactant in organic synthesis [20-23].

 

Hence, o-vanillin is used to synthesize various aromatic Schiff bases with biological properties. Quinoline is an azaheterocyclic aromatic weak tertiary base. The quinoline moiety is nontoxic to humans on oral absorption and inhalation. It is found in many compounds which are pharmacologically active and hence display a wide range of biological activities. In particular, quinoline derivatives are found to exhibit antimalarial, antibacterial, antiprotozoal, anti-HIV, anticancer and antifungal activities [24-29].

 

In this paper, we report the synthesis of five varieties (SB-1 to SB-5) of Schiff bases by means of condensation of o-vanillin with 3/5/6/8-aminoquinoline and 4-aminoquinaldine. These Schiff bases were synthesized by direct reflux reaction of basic precursors viz. o-vanillin with 3/5/6/8-aminoquinoline and 4-aminoquinaldine in ethanolic medium as solvent. The synthesized Schiff bases were characterized by CHN analysis, IR, 1H-NMR UV-Vis, Mass spectral studies and their biological (antibacterial, antifungal and anticancer) activities.

 

2. EXPERIMENTAL DETAILS:

All the chemicals / reagents used were of analytical grade (A.R). Sigma- Aldrich o-vanillin, 3-aminoquinoline, 5-aminoquinoline, 6-aminoquinoline, 8-aminoquinoline and 4-aminoquinaldine were used. Solvents were dried and distilled before use according to the standard procedures [30-31]. The purity of the compounds was confirmed by TLC using Merck silica gel 60-F254 coated alumina plates and visualized by exposure of iodine vapors.

 

For synthesis of Schiff bases, 20 mmol (3.04g) of o-vanillin was dissolved in ethanol. Then it was introduced drop wise into 20 mmol (2.88g) of ethanolic solution of 3-aminoquinoline (1:1 molar proportion). The mixture was refluxed for 2-hours and cooled followed by filtration of the formed and coloured solid Schiff base products. Further, it was washed with ethanol and dried over anhydrous CaCl2. The procedure was repeated using o-vanillin and 5/6/8-aminoquinoline and 4-aminoquinaldine in 1:1 molar proportion to obtain their respective Schiff bases. The purity of the products was checked by TLC. Reactions involved in the synthesis are shown in scheme 1.

 

SCHEME-1: Synthesis of Schiff bases

Sr. No

Schiff Base

Schiff Base Abbreviations

Mol. formula

1

2-methoxy-6-(3-iminoquinal methyl) phenol

SB-1

C17H14O2N2

2

2-methoxy-6-(5-iminoquinal methyl) phenol

SB-2

C17H14O2N2

3

2-methoxy-6-(6-iminoquinal methyl) phenol

SB-3

C17H14O2N2

4

2-methoxy-6-(8-iminoquinal methyl) phenol

SB-4

C17H14O2N2

5

2-methoxy-6-(4-iminoquinal methyl) phenol

SB-5

C18H16O2N2

 

 

3. RESULTS AND DISCUSSION:

Electronic spectra were recorded on Shimadzu VU-VIS-NIR-3600 spectrophotometer using ethanol. IR spectra were scanned on Bruker, Germany Model 3000 Hyperion Microscope with Vertex 80FTIR system range 400-4000 cm-1 (KBr discs) at IIT  Bombay. 1H-NMR spectra were recorded in CDCl3 on Bruker-400 MHz using TMS as internal standard. Mass spectra were recorded on Hewlet Packard 5989B while the CHN elemental analysis was recorded on thermo Finnigan, Italy, Model Flash EA1112 series.

 

3.1 Elemental and physical constant data of Schiff bases

Theoretical and experimentally observed values of elemental analysis of compounds are in good agreement with each other confirming the stoichiometry of synthesized Schiff bases. The elemental and physical constant data of Schiff bases is listed in Table 1.

 

Table 1:  Elemental and Physical constant data of Schiff bases

Sr. No

Schiff base

Mol. formula

Melting Point

(⁰C )

Mol. Weight

Color

Elemental Analysis

C% Observed

(Theoretical)

H% Observed

(Theoretical)

N% Observed

(Theoretical)

1

SB-1

C17H14O2N2

210

278

Red Orange

72.79

(73.78)

4.81

(5.03)

9.99

 (10.07)

2

SB-2

C17H14O2N2

162

278

Yellow Orange

72.46

(73.38)

4.83

(5.03)

9.94

(10.07)

3

SB-3

C17H14O2N2

90

278

Orange

72.82

 (73.38)

4.85

(5.03)

9.99

(10.07)

4

SB-4

C17H14O2N2

103

278

Red

72.91

(73.38)

4.87

(5.03)

9.98

(10.07)

5

SB-5

C18H16O2N2

119

292

Yellow

73.39

(73.97)

5.28

(5.48)

9.45

(9.58)

 

 

 

Fig.1: The UV spectra of Schiff bases of SB-1 to SB-5

 

3.2 Electronic spectral analysis

Electronic spectra of all Schiff bases are characterized by two bands in the UV- Visible region. The UV spectra, as shown in Fig. 1 of Schiff bases, reveal that the bands occurring in the range of 220-280 nm are due to low or medium energy  transition within aromatic moieties. These transitions may originate due to perturbed local excitation of the phenyl group [30-32]. Another intense band in the lower energy region of the spectra between 280-380 nm is assigned to the  transition of azomethine group. Electronic spectral data of Schiff bases is as shown in Table 2.

 

Table2:Electronic absorption spectral data () values in nm

Sr.

No

Schiff Base

Mol. formula

1

SB-1

C17H14O2N2

243

348

2

SB-2

C17H14O2N2

272

337

3

SB-3

C17H14O2N2

270

335

4

SB-4

C17H14O2N2

241

346

5

SB-5

C18H16O2N2

265

327

 

 

3.3 FTIR Spectroscopic studies:

The selected vibrational frequencies for the Schiff base ligands are presented in Table 3. From the FTIR spectra represented in Fig. 2, a very strong band in the range 1589-1636 cm-1 is characteristic of the azomethine nitrogen (C=N) present in the Schiff base ligands SB-1 to SB-5 [34]. The ligands showed broad absorption bands at 3297-3433 cm-1 which is characteristic of (Ar-OH). The absorption bands corresponding to (Ar-O) are seen at 1217-1255 cm-1, while the strong bands for (C-N) stretching at 1457-1479 cm-1 and for (O-CH3) at 2929-3001 cm-1 [35]. The appearance of the characteristic infrared absorption peak (C=N) at 1589-1636 cm-1 for the ligands indicates the formation of Schiff bases.

 

Table 3: Relevant infrared frequencies (cm-1) of the Schiff base

Schiff Base

(O-H)

(C=N)

(Ar-O)

(C-N)

(O-CH3)

SB-1

3426

1622

1250

1465

2929

SB-2

3427

1614

1246

1457

2949

SB-3

3433

1589

1256

1468

2999

SB-4

3428

1618

1244

1459

3001

SB-5

3297

1636

1218

1479

2994

 

(a)

 

(b)

 

(c)

 

(d)

 

(e)

 

Fig.2: The FTIR spectra of the synthesized Schiff bases of  (a) SB-1; (b) SB-2; (c) SB-3, (d) SB-4,and (e) SB-5 samples.

 

3.4  1H–NMR spectroscopic studies

The 1H–NMR spectra of the five synthesized Schiff bases were determined using CDCl3 as a solvent. The corresponding data is listed in Table No. 4 and the spectrum is shown in Fig.No.3. The 1H–NMR spectrum of the Schiff base ligands, showing D2O exchangeable singlet in the range 13.258-14.544 ppm, integrating for one proton, is assigned to –OH [36]. From table it can be seen that the single peaks in the range 8.966-9.945 ppm should be attributed to the proton peaks of –CH=N– bond of Schiff bases. The results indicate that the imino bond (–CH=N–) has been formed between o-vanillin and aminoquinolines. The multiple signals of the complexes in the range 6.541-9.002 ppm are due to quinoline –H protons and aromatic –H protons. The O-CH3 group proton is assigned at 3.947-4.008 ppm in all the Schiff base ligands [37].

 

Table 4:1H –NMR(δin ppm) spectral data of Schiff bases in CDCl3

Sr.

No

Schiff Base

NMR signals in

1

SB-1

13.258 (s,1H, Ar-OH); 6.947-8.960 (m,9H, Ar-H);

8.966(s,1H,-CH=N); 3.988 (s,3H, -OCH3)

2

SB-2

13.664 (s,1H, Ar-OH); 6.958-9.002 (m,9H, Ar-H);

9.006(s,1H, -CH=N); 4.008 (s,3H, -OCH3)

3

SB-3

13.554 (s,1H, Ar-OH); 6.932-8.937 (m,9H, Ar-H);

8.941(s,1H,-CH=N); 3.986 (s,3H, -OCH3)

4

SB-4

14.544 (s,1H, Ar-OH); 6.856-8.994 (m,9H, Ar-H);

8.998(s,1H,-CH=N); 3.977 (s,3H, -OCH3)

5

SB-5

13.711 (s,1H, Ar-OH); 6.541-7.942 (m,8H, Ar-H);

9.945 (s,1H, -CH=N); 3.947 (s,3H, -OCH3); 2.616 (s, 3H, -CH3)

 

(a)

 

(b)

 

(a)

 

(b)

 

(c)

 

(d)

 

(e)

 

Fig.3: The 1H–NMR spectra of the synthesized Schiff bases of (a) SB-1; (b) SB-2; (c) SB-3, SB-4, and (e)  SB-5 samples.

 

(c)

 

(d)

 

(e)

 

Fig.4: The mass spectra of the synthesized Schiff bases of (a) SB-1; (b) SB-2; (c) SB-3, (d)  SB-4, and (e) SB-5 samples.

 

3.5 Mass spectroscopic studies (GCMS):

The mass spectra of all the synthesized Schiff base ligands were recorded and are as shown in Fig.No.4. All the spectra exhibited parent peak due to molecular ion (M+). The proposed molecular formula of these compounds was confirmed by comparing their molecular formula weights with the m/z values. The m/z values for molecular ion peaks obtained are as follows- 279 for SB-1 to SB-4 and 293 for SB-5, which correspond with the proposed molecular formula for these Schiff base ligands. In addition to the peaks due to molecular ions, the spectra exhibited peaks assignable to various fragments arising from the thermal cleavage of the compounds [38].

 

4. ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY:

The synthesized Schiff bases were studied for their antibacterial and antifungal activities using two Gram positive bacteria Staphylococcus aureus and Bacillus cereus, two Gram negative bacteria Pseudomonas auruginosa and Escherchia coli and two fungi Candida albicans and Aspergillus niger. Agar well diffusion method was used to evaluate the antibacterial activity [39,40] while the agar ditch method was used for studying the antifungal activity [41,42]. The stock solutions of 1000 μg/ml concentration were prepared using DMSO as solvent which were further used to prepare various concentrations like 100, 200, 300, 400 and 500 μg/ml. The bacteria and fungi were inoculated on the surface of nutrient agar and Sabouraud’s agar respectively. The various concentrations of the ligands were inoculated in the wells and ditches prepared on the agar plates. The plates were incubated at room temperature for 24 hours for bacteria and 48 hours for fungi. In order to clarify the effect of DMSO for its antimicrobial activity by agar diffusion assay, separate studies were carried out with DMSO which showed no activity against any bacteria or fungi. The antimicrobial activity was determined by measuring the diameter of the zone of inhibition (mm) and thereby they were classified as inactive, weakly active, moderately active and highly active. The results are summarized in Table 5.

 

Table No. 5: Antibacterial and antifungal activity of Schiff base

Schiff

Base

Concentration

µg/ml

Staphylococcus aureus

Bacillus cereus

Pseudomonas aeruginosa

Escherichia coli

Candida albicans

Aspergillus niger

SB-1

100

-

-

+

+

-

-

200

+

-

++

++

-

-

300

+

-

++

++

+

-

400

++

+

++

++

+

-

500

++

+

+++

++

+

-

SB-2

100

-

-

-

-

-

-

200

-

-

+

+

-

-

300

+

+

+

+

+

-

400

+

+

++

++

+

-

500

++

++

++

++

+

-

SB-3

100

++

+++

++

+

-

-

200

++

+++

++

++

-

-

300

++

+++

++

++

+

-

400

++

+++

++

++

+

-

500

++

+++

+++

++

+

-

SB-4

100

+

+

++

++

-

-

200

+

+

++

++

-

-

300

+

++

++

++

+

-

400

++

++

+++

++

+

-

500

++

++

+++

++

+

+

SB-5

100

+

++

++

++

-

-

200

+

++

++

++

-

-

300

++

++

++

++

+

-

400

++

++

++

++

+

-

500

++

++

++

+++

+

-

Activity Scale

‘-‘Inactive (Zone of inhibition < 5 mm)

‘+’ Weakly active (5 mm ≤ Zone of inhibition < 10mm)

 

 

In general, Schiff base ligands exhibited better antibacterial activity than antifungal activity. They are active against both Gram positive and Gram negative bacteria. The Schiff base SB-3(2-methoxy-6-(6-iminoquinal methyl) phenol) is highly active against Bacillus cereus at lowest concentration of 100 μg/ml.

 

6. Anticancer activity:

The anticancer activity of the five Schiff base ligands was determined by sulforhodamine B (SRB) assay on human malignant breast cancer cell line MCF-7 and colon cancer cell line HT-29. The cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 millimolar L-glutamine at 37 in a humidified atmosphere of 5% CO2. About 3cells/well were seeded in 96 well micro titer plate using a culture medium. After 24 hours, each drug (Schiff base) was added to the wells at 4 levels of doses (10, 20, 40 and 80 g/ml) and incubated for 48 hours. After incubation, in-vitro testing was done using SRB assay protocols [43, 44]. Adriamycin, a positive control drug was also run in each experiment and each experiment was repeated thrice. The results are given in terms of GI5O, TGI and LC50 values. Schiff bases SB-1, SB-4 and SB-5 with GI50 <10 g/ml were super active on human breast cancer cell line MCF-7 in the SRB assay while the Schiff base SB-2 (GI5O = 19 g/ml) was weakly active and the Schiff base SB-3 (GI5O = 15.3 g/ml) moderately active on the breast cancer cell line MCF-7.

 

All the Schiff base samples except SB-5 were inactive on human colon cancer cell line HT-29 in the SRB assay system. However SB-5 sample was weakly active on colon cancer cell line HT-29 in the assay system used. The results of the cytotoxicity of Schiff bases on human breast cancer cell line MCF-7 and colon cancer cell line HT-29 are represented in Table 6.

 

Table-6: Cytotoxicity of Schiff bases on Human breast cancer MCF-7 cell line and colon cancer               HT-29 cell line

Sr.

No

Schiff

Base

Concentrations (µg/ml) calculated from graph*

LC50

TGI

GI50

MCF-7 Cell line

HT-29 Cell line

MCF-7 Cell line

HT-29 Cell line

MCF-7 Cell line

HT-29 Cell line

1

SB-1

>80

>80

76.5

>80

<10

64.3

2

SB-2

>80

>80

>80

>80

19.0

>80

3

SB-3

>80

>80

98.7

>80

15.3

59.1

4

SB-4

>80

>80

53.35

71.2

<10

45.5

5

SB-5

>80

>80

56.2

58.0

<10

27.9

6

ADR

>80

NE

30.11

<10

<10

<10

 

Value GI50 if 1)< 10µg/ml–Superactive2) 10-15 µg/ml – Moderately active

3) 15-30 µg/ml – Weakly active4) 30-80 µg/ml – Resistant

GI50 = Concentration of drug causing 50% inhibition of cell growth

TGI= Concentration of drug causing total inhibition of cell growth

LC50= Concentration of drug causing 50% inhibition of cell kill

ADR = Adriamycin (Doxorubicin, positive control drug)

*= Concentrations (µg/ml) calculated from graphs represented in Fig. 5 and Fig. 6

 

 

Fig. 5: Cytotoxicity of all the synthesized Schiff bases on human breast cancer MCF-7 cell line.

 

 

Fig. 6: Cytotoxicity of all the synthesized Schiff bases on human colon cancer HT-29 cell line.

 

In vitro testing based on GI50 values indicate that in general Schiff base compounds are more active on human breast cancer MCF-7 cell line than on human colon cancer HT-29 cell line. Cytotoxicity of the five Schiff bases on MCF-7 cell lines and HT-29 cell lines is represented in Fig. No. 5 and 6 respectively.

 

7. CONCLUSION:

The structure of each synthesized Schiff base was confirmed by CHN analysis, UV-Vis, FTIR, 1H-NMR and mass spectral data. All the synthesized Schiff bases are stable in air and soluble in methanol, ethanol and DMSO while insoluble in water. The antimicrobial studies show that the Schiff bases possess better antibacterial activity than antifungal activity. They are active against Staphylococcus aureus, Bacillus cereus, Pseudomonas auruginosa and Escherchia coli while inactive against Aspergillus niger and weakly active against Candida albicans. The in-vitro anticancer activity shows that synthesized Schiff bases are more active on human breast cancer MCF-7 cell line than on human colon cancer HT-29 cell line.

 

8. ACKNOWLEDGEMENT:

The authors wish to acknowledge the Department of Microbiology, WCBT, Walchand College of Arts and Science, Solapur for technical assistance in the studies of antimicrobial activity. We are also thankful to ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai, India for assistance in the study of anticancer activity.

 

9. REFERENCES:

1.       M. Valiollah, T. Shahram, M. Majid, M. Maryam Bioorgan. Med.Chem.12 (2004) 4673-4677.

2.       C.T. Supuran, A.Scozzafava,A. Popescu, R.Bobes, Tureac, A.Banciu, A.Creanga, G.Bobes-Tureac, M.D.Banciu, Eur.J.med.Chem.32(1997) 445-452.

3.       M.Ziorek, J.kubicki, A. Maciejewski, R.Naskrecki, A.Grabowska, Chem.Phys.Lett. 369 (2003) 80-89.

4.       S.K.Bharti, G.Nath, R.Tilak, S.K.Singh,Eur.J.Med.Chem.45(2010) 651-660.

5.       P.M.Ronad, M.N.Noolvi, S.Sapkal, S.Dharbhamulla, V.S.Maddi, Eur.J.Med.Chem. 45(2010), 85-89.

6.       Z.H.Huang, S.L.Lin, J.L.Huang, Eur.J.Med.Chem.36(2001) 863-872.

7.       M.S.Nair, R.S.Joseyphus, Spectrochim.Acta A 70 (2008) 749-754.

8.       Y.L.Xiang, F.Y.Wu, Spectrochim.Acta A 77(2010) 430-436.

9.       B.Tang, L.Zhang, J.X.Hu, P.Li, H.Zhang, Y.X.Zhao, Anal.Chim.Acta 502(2004)125-131.

10.     F.Beaudry, A.Ross, P.P.Lema, P.Vachon, Phytother, Res.24(2010)525-530.

11.     H.J.Jung, Y.S.Song,K.Kim, C.J.Lim, E.H.Park, Arch.Phar.Res,33(2010)309-316.

12.     E.J.Lim, H.J.Kang, H.J.Jung, Y.S.Song, C.J.Lim, E.H.Park,Biomol.Ther.16(2008)132-136.

13.     A.G.Xie, Y.Qu, M.M.Wang, G.Q.Gan, H.Chem, Z.D.Lin, D.Zhen, J.Coord.Chem.62(2009), 2268-2275.

14.     G.Mazzanti, L.Baffinelli, C.Pompeo, A.M.Serrilli, R.Rossi, I.Sauzullo, F.Mengoni, V.Vullo, Nat.Prod.Res. 22(2008)1433-1440.

15.     C.Queffelec, F.Bailly, G.M.bemba, J.F.Mouscadel, S.Hayes, Z.Debyser, M.Witvrouw, P.Cotelle,  Bioorg.Med.Chem, Lett.18(2008)4736-4740.

16.     F.A.Macias, A.Oliveros, Bastidas, D.Marin, C.Carrera, N.Chinchilla, J.M.F.Molinillo, Phytochem.Res.7(2008) 179-194.

17.     D.X.Tran, T.Tsuneaki, F.Masakazu, D.K.Tran, T.Shinkichi, J.Agr.Food.Chem.57 (2009) 9448-9453.

18.     S.Phogat, B.S.SeemaYadav, Ann.Biol.24(2008) 63-66.

19.     V.S.Ashalakshmi, K.Mohanan, Asian.J.Chem.20(2008) 623-628.

20.     B.Tang, L.L.Zang, J.Zang, X.Wang, ,Z.Z.Chen, H.J.Wang, C.J.Qu, Acta.Chim.Sin. 62(2004)399-404.

21.     A.Barik, K.I, Priyadarsini, H.Mohan, Radiat,Phys, Chem.70(2004)687-696.

22.     I.Kaya, A.Bilici, M.Gul, Polym.Adv.Tech. 19(2008)1154-1163.

23.     E.A.Velcheva, B.A.Stamboliyska, J.Mole.Stru. , 963(2010),57-62.

24.     K.Kaur, M.Jain, R.P.Reddy, R.Jain, Eur.J.Med.Chem.45(2010),3245-3264.

25.     V.Krishnakumar, F.R.N.Khan, B.k.Mandal, S.Mitta, R.Dhasmondhca, V.N.Govindan, Res.Chem.Intermed. 38(2012)1819-1826.

26.     S.Bongarzone, M.L.Biolognesi, Expert.Opin.Drug.Dis.6(2011) 251-268.

27.     Z.G.Luo, C.C.Zeng, F.Wang, H.Q.He, C.X.Wang, H.G.Du, L.M.Hu, Chem.Res.Chin.Univ. 25(2009)841-845.

28.     V.R.Solomon, H.Lee, Curr.Med.Chem. 18(2011) 1488-1508.

29.     R.Musiol, M.Serda, S.Hensel-Bielowka, CurrentMed.Chem. 17 (2010)1960-1973.

30.     J.R.Reid, N.D.Heindel, J.Heterocycl.Chem.13(1976) 925-926.

31.     S.Durairaja, S.Srinivasan, P.L.Perumalsamy,Electran.J.Biol. 5(1) (2009) 5-10.

32.     E.Conpolt, M.Kaya, J.Coord.Chem. 57(14)(2004)1217-1223.

33.     Kamat,M.Ibrahim, et.al.TransitionMet.Chem 10(1985)175.

34.     Bo.Tang, TaixingYue, Junsen Wu, Yuming Dong, Yi Ding, Hongjian Wang, Talanta 64 (2004)955-960.

35.     M.B.Fugu, N.P.Ndahi, B.B.Paul and A.N.Mustapha, J.Chem.Pharm.Res., 5(4) (2013)22-28.

36.     Wang Z, Gao J, Wang J, Jin X, Zou M, Li K, Kang P, Spectrochimica Acta. Part A, 83(1) (2011) 511-517.

37.     SartajTabassum, ShipraYadav, IqbalYadav, J.Org.Chem.752 (2014) 17-24.

38.     G.Zengin, A.Muayad, Nafea Al Kawaz, Huseyinzengin, AdemMert, BediaKucuk, J Mol.Stru.1103 (2016) 45-55.

39.     N.Thilagavathi, A.Manimaran et.al. Appl.Organo-Metallic Chem. 24(2010) 301.

40.     E.F.Bowrsand, L.R.Jeffries, J.Clin.Pathol. 8(1995) 58-61.

41.     C.H.Collins, P.M.Lyne, J.M.Grange Editors Cdlins and Lynes Microbiological Methods , Butterworth Heinemann , Oxford 1995,320.

42.     A.H.Manikshethe, S.K. Sarsamkar, S.A. Deodware, V.N. Kamble Inorg. Chem. Commun. 14(2011)618-621.

43.     A.H. Manikshethe, M.M. Awatade, S.K.Sarsamkar, M.R.Asabe, Int.J.Engg.Sci.Inv. 4(1)(2015) 22-29.

44.     Bao-dui Wang, Zheng-Yin Yang ,J.Photo.chem.Photobiol.A.,194 (2008)49-53.

45.     Peter Houghton, Rui Fang, Isariya Techatanawat, C. C. Lee, Methods,42(2007) 377-387.

 

 

 

 

Received on 22.07.2017         Modified on 20.08.2017

Accepted on 11.09.2017         © AJRC All right reserved

Asian J. Research Chem. 2017; 10(5): 660-668.

DOI: 10.5958/0974-4150.2017.00112.2