A Perkin-Elmer spectrum-2000 Fourier transform IR spectrophotometer (USA) was used to obtain the IR spectra between 400 and 4000 cm⁻¹ CDRI Lucknow U.P. India. The samples were prepared in pellet form using spectroscopic grade KBr. ¹H-NMR spectra were recorded by multinuclear FTNMR spectrometer Model Advance-II (Bruker). The instrument was equipped with a cryo-magnet of field strength 9.4 T. Its 1H frequency was 400 MHz at CDRI Lucknow U.P. India. The ESR spectra of the complexes were recorded at X-Band at a modulation frequency of 100KHZ at liquid nitrogen temperature. TCNE was used as the field marker at RSIC, IIT. Powai, Mumbai, India. The g values for ESR signal are calculated by the formula-(Eq 1).

(1)

Where υ = Frequencies of Band in KHz

B = Bohr magneton

H = Magnetic field, h = Planks constant.

On the basis of g_ll and g _┴, the g_av and G values were calculated using the following (Eq 2 and 3).

(2)

(3)

The general laboratory techniques followed in the course of this investigation are as suggested by Boothand Hawks worth [15Booth, C. “Methods in Microbiology” AcadPress.N.Y. Vol-4, 795P, (1971).] as follows:

For a biological activity, the glassware were thoroughly washed and cleaned with chromic acid, followed by washing with distilled water. Now they were sterilized by keeping them in a hot air oven at 160 ⁰C for 24h. All operations concerning inoculation are done in a completely sterilized chamber.

The artificial induction of micro-organism into a medium is called inoculation. The latter is the most fundamental technique for studying the growth characteristics of micro-organisms and for the transfer and maintenance of culture under aseptic condition.

Agar slants were prepared to inoculate microbial culture. To prepare agar slant, the required number of culture tubes were taken and about 12 to 15 ml of liquefied agar medium was poured in each of them. The tubes were now cotton-plugged and sterilized in an autoclave. After the sterilization was over, the tubes were taken out and were placed in slanting (stopping) position for some time, the tubes got cooled and the medium in them was solidified resulting in a sloppy surface.

In preparing a Culture medium for any micro-organism, the primary goal is to provide a balanced mixture of the nutrient that will permit good growth. Additionally, the culturing of micro-organisms requires Careful Control of various environmental factors which normally are maintained within narrow culture media.

The culture medium used for the growth of the organism in the present study was natural media Potato Dextrose Agar (Abbreviated as ‘PDA”).For the preparation of culture media standard reported procedure [16Kumar, A.; Rajput, C.S. Synthesis and anti-inflammatory activity of newer quinazolin-4-one derivatives. Eur. J. Med. Chem., 2009, 44(1), 83-90.
[http://dx.doi.org/10.1016/j.ejmech.2008.03.018] [PMID: 18501478] ].

The test organisms used in the present study were Alternaria alternate, Aspergillus fumigatus and Aspergillus niger which were isolated from their natural habitat (plants, debris) and then purified, characterized and identified.

The antifungal activity of the copper soaps and their complexes derived from saturated fatty acid under study was checked by the agar plate technique as reported in the literature [17Sharma, A.K.; Sharma, R.; Gangwal, A.K. Antifungal activities and characterization of some new environmentally safe Cu (II) surfactants substituted 2-amino-6-methyl benzothiazole. Open Phar Sci. J. , 2018, 1-11 3-12 https://DOI:10.2174/187484490180501]. The growth of fungus was measured by recording the total area of the fungal colony

The data were statistically analyzed according to the following formula [18Mathur, N.; Bargotya, S. DNA binding and cleavage activities of macro cyclic metal complexes containing heteroatomic ligand Chem. Sci. Trans., 2016, 5(1), 117-124.].

Fungal Growth Formula = (2)

(% Inhibition)

C = Total area of the fungal colony in control plats after 2 days.

T = Total area of the fungal colony in the test tube after 2 days.

The absorption bands observed at 2926 cm^-1 and 2859 cm^-1 are assigned to the antisymmetric and symmetric stretching for –CH₂ group (methylene) of the soap segment present in the complex. The absorption bands observed at 2961 cm^-1 are due to –CH₃ antisymmetric stretching of lauric acid. A strong absorption band at 1551 cm^-1 is due to carboxylate ion COO^-, C-O antisymmetric respectively. The peaks corresponding to –CH₃ and –CH₂ have been seen at 1118 cm^-1 and 726 cm^-1respectively. Cu- O stretching bands have been shown in the region of 476.4 cm^-1 similar observation was reported by Khan et al., [19Khan, S.; Sharma, R.; Sharma, A.K. Antifungal activities of copper surfactants derived from Neem (AzadirectaIndica) and Karanj (Pongamiapinnata) Oils: A pharmaceutical application. Glob. J. Pharmaceu. Sci., 2017, 3(4) GJPPS.MS.ID.555616]. The absorption bands observed at 3333 cm^-1 and 3180 cm^-1 are due to the N-H antisymmetric stretching and N-H symmetric stretching of NH₂ group. Other strong peaks in the region of 1479 cm^-1 are due to the CH₂ bending group. The absorption band 1714 cm^-1 was found to be representative of amide group of >C=O. The C-N stretching band of primary amide was observed at 1413 cm^-1. Apart from these absorption corresponding to the ligand moiety, a peak in the region of 1266 cm^-1 N=C=S stretching of ligand thiourea in the complex was observed. Due to the C=S stretching group in an absorption in the region of 624 cm^-1(Table 2). On the basis of the above observations, it can be safely assumed that complexation of copper laurate soap has been done with thiourea ligand (Fig. 3).

Table 2
Infrared absorption spectral frequencies (cm^-1) of complex (CL_rtT).

Fig. (3) IR Spectra of complex (CLrtT).

NMR spectra of Cu (II) laurate thiourea complex show a signal of aliphatic –CH₃ proton attached -CH₂-R group at nearly δ-0.979 and – CH₂ proton attached to -CH₂-R group which shows a signal at δ-1.256. Other signal observed is co-responding to the –CH₂ proton attached to –CH₂(COO)₂Cu group and is observed at δ-2.354. All these peaks are due to the saturated fatty acid content of the soap in the complex. A broad peak is observed in the spectra of the complex at δ-2.354 due to the presence of –NH₂ protons (Table 3). This peak indicated co-ordination through the -NH₂ group of thiourea segment to the metal atom of the soap segment. A very weak signal is observed at δ-7.84 in the spectra, which may be due to tautomerism [20Mahajan, k.; Swami, M.; Singh, R.V. Microwave synthesis, spectral studies, antimicrobial approach, and coordination behavior of antimony(III) and bismuth(III) compounds with benzothiazoline. Russ. J. Coord. Chem., 2009, 35, 179-180.
[http://dx.doi.org/10.1134/S1070328409030038] ] present in the complex (Fig. 4).

Table 3
NMR spectral signals (δ) of complex (CL_rtT).

Fig. (4) NMR Spectra of complex (CLrtT).

The value of ESR parameters for the complex is given in the Table 4. A perusal of Table 4 shows that the values of g₁ g₁₁, g are greater than the value of g i.e. 2.0027. This indicates that the distortion from the regular octahedron has taken place in the shape of the complex. Also the trend g_11>g for complex indicates that the unpaired electron is most likely in the d_x2-y2 orbit of Cu (II) giving the ground state. This fact supports that complex possesses elongated octahedral geometry. It is well known that g₁₁ is a moderately sensitive function for indicating co-valency [21Garg, B.S.; Kumar, D.N.; Singh, R.V. Spectral studies of complexes of nickel (II) with tetradentate schiff bases having N2O2 donor groups. Spectrochim. Acta., 2003, 59A, 229-234.
[http://dx.doi.org/10.1016/S1386-1425(02)00142-7] ]. Thus the values of g₁₁ are of the covalent character of the metal-ligand bond. Earlier magnetic studies about Cu (II) soap confirmed the binuclear configuration in solid state [22Sharma, A.K.; Saxena, M.; Sharma, R. Synthesis, spectroscopic and fungicidal studies of Cu (II) soaps derived from groundnut and sesame oils and their urea complexes Bulletin of Pure and Applied Sciences 2017, 36(2), 26-37.
[http://dx.doi.org/10.5958/2320-320X.2017.00004.8] ] (Fig. 5).

Table 4
ESR spectral values of CL_rtT.

Fig. (5) ESR Spectra of complex (CLrtT).

A perusal of Figs. (6-8) reveals that the complex shows higher activity than pure soap suggesting that complex is more powerful antifungal agent. Thiourea and other N,S,O etc. containing compounds are able to enhance the performance of copper soap. These results show that the Cu (II) soap complex of ligands is much toxic than the copper-soap themselves. The enhanced activity of synthesis of the complex as compared to copper soap can possibly be explained on the basis of chelate formation in the presence of donor atoms, basicity as well as the structural compatibility with molecular nature of the toxic moiety [23Neha Mathur and SonlataBargotya. A facile synthesis and biological evalution of some macrocyclic copper complexes. IJPSR, 2015, 6(6), 2538-2545., 24 Sharma, A, K. Saxena, M. Sharma R.: Synthesis, Spectroscopic and Biocidal activities of environmentally safe Agrochemicals, J. Biochem. tech., 2018, 7(3), 1139-1147.]. The final conclusion suggests that the appearance of enhanced activity may be due to a synergistic mechanism i.e. the soap is less active but on complexation shows more activity in combination with thiourea. The studies suggest that the Cu (II) ions in soaps may be responsible for the enhancement of the activity against fungi. The evaluation of anti-fungal studies further revealed that fungitoxicity of the complex also depends on the nature of metal ions [25 Sujamol, M.S., Athira, C.J., Sindhu, Y., Mohanan, K.:Synthesis, spectroscopic characterization, electrochemical behaviour and thermal decomposition studies of some transition metal complexes with an azo derivative,Spectrochim. Acta. 75A(2010) 106-112; https://DOI:10.1016/j.saa.2009.09.050]. The chelating reduces the polarity of central metal ion mainly because of the partial attaining of its positive charge with the donor groups and possible ring. Such chelating increases the lipophilic character of the central atom, which subsequently favors its permeation through the lipid layer of the cell membrane [26Bhutra, R.; Sharma, R.; Sharma, A.K. Synthesis, Characterization and fungicidal activities of Cu (II) surfactants derived from groundnut and mustard oils treated at high temperatures. J. Inst. Chemists (India), 2018, 90(3), 66-80.]. Their efficiency increases with their concentration. Thus, it is evident that concentration plays a vital role in increasing the degree of inhibition So, fungicidal screening data revealed that at a lower concentration of the inhibition of growth is less as compared to higher concentration [27 Mishra, A.P., Mishra, R.K., Shrivastava, S.P.:Structural and antimicrobial studies of coordination compounds of VO(II), Co(II), Ni(II) and Cu(II) with some Schiff bases involving 2-amino-4-chlorophenol, J. Serb. Chem. Soc., 74(2009) 523-535; https://DOI: 10.2298/JSC0905523M-29Mathur, N.; Ahmad, I.; Kasana, A. SonlataBargotya, Biplab Manna, Biological Activities of Some New Environmentally Safe 2 Aminobenzothiazole Complexes of Copper (II). Derived Under Microwave Irradiation, IAASCA, 2013, 5(1), 37-42.]. On the basis of fungicidal screening data, it was revealed that the complex shows lower activities for Alternaria alternate and Aspergillus fumigatus in comparison with Aspergillus niger (Figs. 9a-c). For Aspergillus niger, the complex’s efficiency increases with its concentration. Thus it is evident that the concentration plays a vital role in the degree of inhibition. The study suggests that copper laurate soap is the least fungi toxic (% inhibition lowest) whereas its thiourea complex which shows the highest inhibition. The activity of copper soap and complex derived from lauric acid is found to increase in the order.

It has also been observed that, in general, copper complex derived from lauric acid shows higher % inhibition for Aspergillus niger as compared to Aspergillus fumigatus and Alternaria alternata.

Higher concentration shows higher % inhibition when compared to the lower concentration.

The results of ANOVA for the antifungal activities for all sops complexes are shown in Table 5. The predicted R² is in reasonable agreement and closer to 1.0. This confirms that the experimental data are well satisfactory. The descriptive statistics results of CL_rtS and Cl_rtT showed in Tables 6-8 confirm satisfactory results in triplet for all the fungi studied earlier in our laboratory and other studies conducted by scientists [30Sharma, A.K.; Sharma, R.; Saxena, M. 2018. Biomedical and antifungal application of Cu(II) soaps and its urea complexes derived from various oils. Open access J Trans Med res 2(2) 40-43. https://Doi:10.15406/oajtmr.2018.02.00033]. The findings are similar to our studies on different systems [31Sharma, A.K.; Sharma, R.; Gangwal, A. Biomedical and fungicidal application of copper surfactants derived from pure fatty acid, Organic & Medicinal Chem., IJ 5(5): OMCIJ.MS.ID.555680 (2018) 1-4 https://DOI:10.19080/OMCIJ.2018.05.555680.]. The result is statistically significant, by the standards of the study, due to p < F.

Fig. (6) Antifungal activity of ClrtS and CLrtT for fungi Aspergillus Niger.

Fig. (7) Antifungal activity of ClrtS and CLrtT for fungi Aspergillus fumigatus.

Fig. (8) Antifungal activity of ClrtS and CLrtT for fungi Alternaria Alternata.

Table 5
ANOVA results of Cu (II) laurate soap and thiourea complex.

Fig. (9a) Presence of Aspergillus fumigatus on papaya fruit.

Fig. (9b) Presence of Alternaria Alternata on tomato.

Fig. (9c) Presence of Aspergillus Niger on bread.

Table 6
Descriptive Statics results of Cu (II) laurate soap and thiourea complex for Aspergillus Niger fungi.

Table 7
Descriptive Statics results of Cu (II) laurate soap and thiourea complex for Aspergillus Fumigatus fungi.

Table 8
Descriptive Statics results of Cu (II) laurate soap and thiourea complex for Alternata.