1 s2.0 S0022328X19303791 mmc1

Synthesis and characterization of metallophthalocyanine with
morpholine containing Schiff base and determination of their

antimicrobial and antioxidant activities
Dilek Unluera , Ayşe Aktas Kamiloglub*, Sahin Direkelc, Ersan Bektasd, Halit
Kantekina, Kemal Sancaka

a
Department of Chemistry, Faculty of Science, Karadeniz Technical University, 61080
Trabzon, Turkey
b*
Artvin Vocational School, Artvin Çoruh University, 08100, Artvin, Turkey.
c
Department of Medical Microbiology, Faculty of Medicine, Giresun University, 28100 Giresun, Turkey
d
Giresun University, Espiye Vocational School, Espiye, Giresun, 28600 Turkey
1. Experimental
1.1. Materials and Equipment
All reagents and solvents were of reagent-grade quality and were obtained from commercial
suppliers. All solvents were dried and purified as described by Perrin and Armarego [1]. 4-
nitrophthalonitrile was obtained from commercial suppliers. Schiff base compound (1) was
prepared according to literature [2]. All reactions were carried out under dry and oxygen free
nitrogen atmosphere using schlenk system. 1H-NMR, 13C-NMR spectra were recorded on a
Bruker AVANCE III 400 MHz NMR spectrophotometer in CDCl 3, and chemical shifts were
reported (δ) relative to Me4Si as internal standard. IR spectra were recorded on a Perkin–
Elmer Spectrum one FT-IR spectrometer. Matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry (MALDI-TOF-MS) measurements were performed on a BRUKER
Microflex. Melting point was recorded on a GallenKamp melting point apparatus. DPPH was
purchased from Sigma-Aldrich and trolox was supplied by Sigma- Aldrich Appli Chem
(Darmstadt, Germany). Optical spectra in the UV-vis region were recorded with a Perkin
Elmer Lambda 25 spectrophotometer.
*
1.2. Determination of Antioxidant Capacity
The antioxidant activities of the compounds (Co, Cu and Zn) were analyzed by two
commonly preferred methods (FRAP and DPPH tests), which are considered as a good
indicator of the antioxidant capability of various compounds. The FRAP is an antioxidant
activity determination method based on the reduction of Fe3+ -TPTZ complex to the Fe2+
-TPTZ complex in the presence of antioxidants [3]. A volume of 100 µl of the compound
dissolved in DMSO was mixed with 3 mL of freshly prepared FRAP reagent. The reaction
mixture was then incubated for 4 min at 37 °C. The absorbances of the substances were
determined at 593 nm against the blank. A calibration curve was prepared using absorbances
from the FeSO4.7H20 solution in the range of 31.25-1000 μM. FRAP values were stated as
µM FeSO4.7H2O equivalent/g sample.
The scavenging effects of the compounds against the 2,2-diphenyl-1-picrylhydrazyl (DPPH)

radical were examined according to the method of Brand-Williams et al. (1995) with some
alterations [4]. In the presence of an antioxidant, it is based on the decolorization of purple
color of DPPH, and the change in absorbance is measured spectrophotometrically at 517 nm.
A volume of 0.75 mL of 0.1 mM DPPH in methanol was mixed with an equal volume of
dissolved compound solution in DMSO (at various concentrations), shaken well, kept in the
dark for 50 minutes, and activity measured at 517 nm using Trolox as standard and values
were revealed as SC50 (µg sample per mL).
1.3. Preparation of standart bacteria isolates

Antibacterial activity of the compounds were evaluated against the selected six different
standart bacteria. The synthesized compound has been screened in vitro against Escherichia
coli ATCC 25922, Staphylococcus aureus (Methicillin Resistance) ATCC 43300, Salmonella
typhimurium ATCC 14028, Yersinia enterocolitica ATCC 9610, Shigella flexneri ATCC
12022, Pseudomonas aeruginosa ATCC 27853. Standard bacteria isolates were obtained from
the American Type Culture Collection. Firstly, bacterial isolates which been kept in deep
freezer at -80°C were allowed to stand for about one hour to warm to room temperature and
vitalized by inoculate into Blood Agar medium (Merck) and Eosin Methylene Blue (EMB,
Merck). Colonies growing in bacterial culture performed purity controls for contamination.
Growing bacteria colonies were taken into sterile glass tubes which contain 2 ml Mueller–
Hinton Broth (MHB, Merck) medium and for bacterial growth were incubated by keeping
overnight in incubator at 37°C. In the preparation of standart bacterial suspensions was used
McFarland 0,5 turbidity standards (CLSI M100-S25) which in the range of 10 7 –108 cfu per
mL in MHB.
1.4. Preparation of the test compounds
For the preparation of the compounds; it was dissolved inside 10% dimethyl sulphoxide
(DMSO, Sigma–Aldrich, USA) that did not show inhibition against the tested
microorganisms to obtain 20 mg/mL stock solution. This stock solution was filtered by 0.45
diametered steril membrane filter and the compound was collected into a sterile tube.
1.5. Preparation of the Alamar Blue Dye
Alamar Blue Dye (Sigma–Aldrich, USA) was used as indicator stain for detection of
antimicrobial activity. The Alamar Blue Dye stock solution (270 mg alamar blue dye
dissolved in 40 mL sterile distilled water) was filtered by 0.45 diametered membrane filter
and collected into a sterile tube.
1.6. Determination of MIC of the compounds
The antibacterial activity of the synthesized compounds were determined by microdilution
broth assay with Alamar Blue Dye. Antibacterial activity test was carried out by repeating the
procedures of our previous study [5]. Briefly: The in vitro antimicrobial activities of the
compounds were tested in MHB for bacteria by the two fold serial dilution method (CLSI
M100-S25). Determine to the minimum inhibitory concentration (MIC) by two fold serial
dilution of the compounds at the descending concentration ranging from 10.000 to 312 µg/ml
were prepared in MHB medium in a sterile 96-well round bottomed microplate, final volume
of 100 µl per well. After a 100 µl volume of final bacteria inoculum suspension was added to
each well containing diluted compounds. Last two well used as negative (the compound and
alamar blue dye) and positive control (standart bacterial isolates and alamar blue dye),
respectively. About 20 µl Alamar Blue Dye was added to each well, after 20 h of incubation
at 37°C. Then, the plate was incubated again with Alamar Blue Dye for 4 h at 37°C. In
determination of antibacterial activities of compounds, that changing the color from blue to
pink in well was evaluated as proliferation of bacteria, whereas the unchanged color wasn't
proliferation and the unchanged last well was described as the minimum inhibitory
concentration (MIC) value. The MICs were recorded observing visually after 24 h incubation
of bacteria. Amikacin was used as standard control for bacteria studies. All tests were carried
out in duplicate.
References
[1] D. D. Perrin, W. L. F.: Armarego, 2nd ed. Pergamon Press, Oxford, (1989).
[2] P. Panneerselvam, R.R. Nair, G. Vijayalakshmi, E.H. Subramanian, S.S. Sridhar, Eur. J.
Med. Chem. 40 (2005) 225-229.
[3] I.F.F. Benzie, Y.T. Szeto, Journal of Agricultural and Food Chemistry, 47 (1999) 633-636.
[4] W. B.Williams, M.E. Cuvelier, C. Berset, LWT – Food Sci Technol, 28 (1995) 25-30.
[5] N.O. İskeleli, Y.B. Alpaslan, Ş. Direkel, A.G. Ertürk, N. Süleymanoğlu, R. Ustabaş,
Spectrochim Acta A Mol Biomol Spectrosc., 15 (2015) 356-66.

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Synthesis and characterization of metallophthalocyanine with

morpholine containing Schiff base and determination of their

Kantekina, Kemal Sancaka

1.1. Materials and Equipment

The scavenging effects of the compounds against the 2,2-diphenyl-1-picrylhydrazyl (DPPH)

1.3. Preparation of standart bacteria isolates

1.4. Preparation of the test compounds

1.5. Preparation of the Alamar Blue Dye

1.6. Determination of MIC of the compounds

The antibacterial activity of the synthesized compounds were determined by microdilution

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