Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 534–544

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Preparation, spectroscopic, thermal, antihepatotoxicity, hematological parameters and liver antioxidant capacity characterizations of Cd(II), Hg(II), and Pb(II) mononuclear complexes of paracetamol anti-inflammatory drug Samy M. El-Megharbel a,b, Reham Z. Hamza c, Moamen S. Refat a,d,⇑ a

Department of Chemistry, Faculty of Science, Taif University, Al-Haweiah, P.O. Box 888, Zip Code 21974, Taif, Saudi Arabia Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt c Department of Zoology, Faculty of Science, Zagazig University, Zagazig, Egypt d Department of Chemistry, Faculty of Science, Port Said University, Port Said, Egypt b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The Cd(II), Hg(II), and Pb(II)

complexes of paracetamol have been synthesized.  The general formula of complexes is [M(Para)2(H2O)2]nH2O.  In vivo the antihepatotoxicity effect were measured. 2+  The Cd + Para complex has succeeded in improvement of the antioxidant capacities.

a r t i c l e

i n f o

Article history: Received 28 January 2014 Received in revised form 11 April 2014 Accepted 21 April 2014 Available online 30 April 2014 Keywords: Paracetamol Heavy metal Spectroscopic Antihepatotoxicity Liver function parameters Antioxidant capacities

a b s t r a c t Keeping in view that some metal complexes are found to be more potent than their parent drugs, therefore, our present paper aimed to synthesized Cd(II), Hg(II) and Pb(II) complexes of paracetamol (Para) anti-inflammatory drug. Paracetamol complexes with general formula [M(Para)2(H2O)2]nH2O have been synthesized and characterized on the basis of elemental analysis, conductivity, IR and thermal (TG/DTG), 1 H NMR, electronic spectral studies. The conductivity data of these complexes have non-electrolytic nature. Comparative antimicrobial (bacteria and fungi) behaviors and molecular weights of paracetamol with their complexes have been studied. In vivo the antihepatotoxicity effect and some liver function parameters levels (serum total protein, ALT, AST, and LDH) were measured. Hematological parameters and liver antioxidant capacities of both Para and their complexes were performed. The Cd2+ + Para complex was recorded amelioration of antioxidant capacities in liver homogenates compared to other Para complexes treated groups. Ó 2014 Elsevier B.V. All rights reserved.

Introduction ⇑ Corresponding author at: Department of Chemistry, Faculty of Science, Taif University, Al-Haweiah, P.O. Box 888, Zip Code 21974, Taif, Saudi Arabia. Tel.: +966 551352497. E-mail address: [email protected] (M.S. Refat). http://dx.doi.org/10.1016/j.saa.2014.04.108 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

Paracetamol (Para; Fig. 1) was a widely used as analgesic and antipyretic drug [1–6], also, it was known to be hepatotoxic in

S.M. El-Megharbel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 534–544

OH O

N H Fig. 1. Paracetamol (Para) drug structure.

man and various experimental animals upon overdose [7–9]. Taking the presumed molecular mechanisms of analgesic activity as well as of the hepatotoxicity of paracetamol into consideration, there have been several efforts to improve its analgesic activity while preventing its toxicity by modifying its structure [10–13]. In an attempt to improve the analgesic activity of paracetamol by mono-substitution ortho to the hydroxyl group, Harvison et al. [11] showed that 3-methyl paracetamol was equipotent to paracetamol with respect to analgesic activity in mice. Unfortunately however, hepatotoxicity was also equal, the hepatotoxicity of paracetamol was decreased by 2-methyl substitution (meta to the hydroxyl group), however, the analgesic activity was also decreased. N-methyl paracetamol was found to be completely devoid of hepatotoxicity but also of analgesic activity [14]. In addition to mono-substitution, it has been shown that dialkylsubstitution at the 3- and 5-positions of the aromatic nucleus of paracetamol did not reduce the analgesic activity [15]. A toxicological study showed that the in vivo hepatotoxicity of the 3,5-dialkylated analogs was reduced almost completely [16]. Recently, it was reported that aromatic ring-substitution by one or two fluorines decreased the analgesic activity of paracetamolacetamol in mice [17]. It was also shown that these modifications decreased the in vivo toxicity [18]. The formation of complexes of paracetamol and Zn(II) was studied in aqueous media at pH 7.2 by polarography and spectroscopy [19]. The stoichiometry of the Zn(II)-paracetamol complex was 1:1. Analgesic studies on the drug and its metal complex have been performed in albino mice. Revealing the complex to be more potent in analgesic activity compared to the paracetamol alone drug. The present work was built on the study of the interactions between paracetamol drug and some of heavy metal ions like Cd(II), Hg(II), and Pb(II). The elemental analysis, conductivity, IR and, thermal (TG/DTG), 1H NMR, electronic spectral studies of these complexes were discussed and deduced the suggested structure which associated via deprotonation of AOH hydroxyl group. The antihepatotoxicity, hematological parameters and antioxidant effects of the Para complexes were investigated on the treated rats upon the calculations of liver function parameters levels (serum total protein, ALT, AST, and LDH), SOD, GST and TAC.

Experimental Materials and preparations Analytical grade of chemicals used were purchased from Aldrich and Merck chemical companies. Paracetamol drug was received from Egyptian International Pharmaceutical Industrial Company (EIPICo.). The Para complexes were prepared by mixing twice amount of Para (2 mmol) and 1 mmol of metal(II) nitrates (Cd(II), Hg(II) and Pb(II)) in MeOH/H2O (50/50, w/w; 40 cm3) solvent, then pH of the mixtures was adjusted to 7–8 using 5% alcoholic ammonia solution. The reaction mixtures were stirred at 60 °C for 2 h and left to stand overnight. The precipitated complexes were filtered off, washed with MeOH/H2O and dried in vacuum at room temperature over anhydrous CaCl2.

535

Physical measurements Carbon, hydrogen and nitrogen contents were determined using a Perkin–Elmer CHN 2400. The metal content was determined by atomic absorption spectrometer model PYE-UNICAM SP 1900 and the corresponding lamps were used for this purpose. Infrared spectra were recorded on Bruker FTIR Spectrophotometer (4000– 400 cm1) in KBr pellets. The UV–Vis spectra were studied in the DMSO solvent with a concentration of 1.0  103 M for the Para and their complexes using Jenway 6405 Spectrophotometer with 1 cm quartz cell, in the range 800–200 nm. Molar conductance’s of the freshly prepared solutions of the Para complexes with 1.0  103 M in DMSO were measured using Jenway 4010 conductivity meter. 1H NMR spectra were recorded on a Varian Gemini 200 MHz spectrometer using DMSO-d6 as solvent. Thermogravimetric analyses (TG/DTG) were carried out in a dynamic nitrogen atmosphere (30 mL/min) with a heating rate of 10 °C/min using a Shimadzu TGA-50H thermal analyzer. Antimicrobial activities According to Gupta et al. [20], the antimicrobial tests were done. The investigated isolates of bacteria were seeded in tubes with nutrient broth (NB). The seeded NB (1 cm3) was homogenized in the tubes with 9 cm3 of melted (45 °C) nutrient agar (NA). The homogeneous suspensions were poured into Petri dishes. The holes (diameter, 4 mm) were done in the cool medium. After cooling, 2  103 dm3 of the investigated compounds were applied using a micropipette. After incubation for 24 h in a thermostat at 25– 27 °C, the inhibition (sterile) zone diameters (including disc) were measured and expressed in mm. An inhibition zone diameter of over 7 mm indicates that the tested compound is active against the bacteria under investigation. The antibacterial activities of the investigated compounds were tested against Escherichia coli (Gram, ve), Bacillus subtilis (Gram, +ve) and antifungal (Asperagillus oryzae, Asperagillus niger, and Asperagillus flavus). Experimental animals Antihepatotoxicity effect in male albino rats The present study was carried out at Zoology Department, Faculty of Science-Zagazig University, Egypt using fifty clinically healthy mature adult male albino rats. The animals were obtained from the animal House of Faculty of Veterinary Medicine, Zagazig University, Egypt. Their weights ranged from (200–250 g). The animals were housed in standard conditions, where the animals were housed in metal cages and bedded with wood shavings and kept under standard laboratory conditions of aeration and room temperature at about 25 °C. The animals were allowed to free access of standard diet and water ad-libtum, We have followed the European community Directive (86/609/EEC) and national rules on animal care. The animals were accommodated to the laboratory conditions for 2 weeks before being experimented, adaptation and 10 rats were placed into each cage. Six groups were established in the study as follows. The present study was undertaken to assess the effects of single or multiple-dose administration of Para and it is complexes in normal treated rats. All experiments were performed during the same time of day, between 10 am and 1 pm to avoid variations due to diurnal rhythms. Experimental design – Test compounds; Para, Cd2+/Para, Hg2+/Para, and Pb2+/Para. Animal groups Treatment schedule (each group comprises 10 rats): In order to optimize Para drug absorption, all animals were starved overnight

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prior to Para exposure and administration of its complexes. Paracetamol and its complexes were administered to rats following 12 h of fasting and allowed free access to food and water 1 h after vehicle/Para and its complexes administration. i. The 1st control group Animals received 1 ml of distilled water orally daily for 4 weeks, and served as the control group. ii. The 2nd Para treated group Animals were daily received orally Para, the Para was dissolved in DMSO (650 mg/kg b.w) [21] for 30 successive days using metallic stomach tube. iii. The 3rd Cd2+/Para treated group Animals were received orally Cd2+/Para (650 mg/kg b.w) for 30 successive days using metallic stomach tube. iv. The 4th Hg2+/Para treated group Animals were received orally Hg2+/Para (650 mg/kg b.w) for 30 successive days using metallic stomach tube.

Lipid profile Triglycerides, cholesterol and high density lipoprotein-cholesterol (HDL-c) were determined using the commercial kits. Low density lipoprotein-cholesterol (LDL-c) levels were calculated by using the following formula of Muruganandan et al. [26]: LDL-c = total cholesterol  (HDL-c + triglycerides)/5. Volatile low density lipoprotein-cholesterol (VLDL-c) levels were calculated by using the following formula of Prakasam et al. [27]: VLDLc = triglyceride/5. The risk ratio was calculation by dividing the total cholesterol by HDL-c. Tissue homogenate preparation Liver was immediately removed; weighed and washed using chilled saline solution. Tissues were minced and homogenized (10% w/v), separately, in ice-cold sodium, potassium phosphate buffer (0.01 M, pH 7.4) containing 1.15% KCl in a Potter–Elvehjem type homogenizer. The homogenate was centrifuged at 10,000g for 20 min at 4 °C, and the resultant supernatant was used for the determination of antioxidant enzyme assays, (liver only). Assessment of antioxidants capacities

v. The 5th (Pb2+/Para treated group Animal were given orally Pb2+/Para (650 mg/kg b.w) for 30 successive days using metallic stomach tube. The rats included in the groups that were given Para and its complexes were weighed daily, and accordingly the dose that was given per body weight was calculated and administration by metallic tube was considered to be more accurate and safety. Collection and processing of samples Twenty-four hours after the last administration of the indicated substances, blood samples were collected from the retro-orbital vein, which is a simple, convenient and successful procedure that allows bleeding of the same animal more than one time with minimal stress [22]. Individual blood was drawn by orbital puncture (from eye plexus) using microhematocrit capillary tubes into dry tubes from 10 rats in each group, Serum was harvested from blood without EDTA. And their Kidney tissue was excised. Blood samples were centrifuged at 336 rpm for 10 min for the separation of sera. The serum samples obtained were transferred into eppendorf tubes and were preserved in a deep freezer at 80 °C. Part of the Kidney tissue was transferred into 10% buffered formalin for histopathological examination. Determination of key liver functions biochemical markers Serum total protein, ALT, AST, and LDH levels were measured. Some Liver function parameters:

The principle of SOD activity assay was based on the inhibition of nitro blue tetrazolium (NBT) reduction. Illumination of riboflavin in the presence of O2 and electron donor like methionine generates superoxide anions and this has been used as the basis of assay of SOD. The reduction of NBT by superoxide radicals to blue colored formazan was followed at 480 nm [28]. GlutathioneS-transferase (GST) was determined according to the method of Habig et al. [29]. While Glutathione level (GSH) as non-enzymatic antioxidant was estimated based on the method of Beutler et al. [30] who reported that the 5,50-dithiobis-(2-nitrobenzoic acid) is reduced by SH group to form 1 mol of 2-nitro-5-mercaptobenzoic acid per mole of SH. Total antioxidant capacity of hepatocytes was determined using ferric reducing antioxidant power assay according to the method of Prieto et al. [31]. FRAP reagent (300 mM acetate buffer, pH 3.6, 10 mM 2,4,6-tri(Z-pyridy)-S(riazine, 99%) (TPTZ) in 40 mM HCl and 20 mM FeCl36H2O in the ratio of 10:1:1 was prepared. 1.5 mL FRAP reagent was added to 50 L of homogenized liver tissues and incubated at 37 °C for exactly 5 min. The change in absorbance was measured at 593 nm due to the formation of a blue colored Fe(II)-tripyridyltriazine complex from the colorless oxidized Fe(III) form by the action of electron donating antioxidants. The absorbance of the sample was read against reagent blank (1.5 mL FRAP reagent and 50 L distilled water) at 593 nm. The data was expressed as percentage of the control value. Hematological parameters

i. Determination activities

of

serum

aminotransferase

enzymes

Activities of AST and ALT in the serum were determined calorimetrically by using bio Merieux kit (France), using method adopted by [23]. ii. Determination of serum total protein concentration Colorimetric determination of the total protein based on the principle of Biuret reaction (copper salts in an alkaline medium). Protein in plasma or serum forms a blue colored complex when treated with cupric icons in alkaline solution. The intensity of the blue color is proportional to the protein concentration [24]. iii. Determination of LDH level The reaction velocity is determined by a decrease in absorbance at 340 nm resulting from the oxidation of NADH. One unit causes the oxidation of one micromole of NADH per minute at 25 °C and pH 7.3, under the specified conditions [25] by using Diamond kit.

Hematological parameters A complete blood count includes five major measurements were determined using cell counter (Sysmex, model KX21N). White blood cells (WBC) were measured in thousands per cubic milliliter of blood. Red blood cells (RBCs) were measured in millions per cubic millimeter of blood. Hemoglobin (Hb) was measured in grams per deciliter (g/dL) of blood. The hematocrit value (PCV) is the percentage of red blood cells in relation to total blood volume was also determined. Mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were also calculated. Platelets were measured in thousands per cubic millimeter of blood. Preparation of tissues for histopathological examination Tissue samples were taken from the liver of animals and fixed in 10% formalin neutral buffer solution. The trimmed tissues were

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first washed with tap water followed by dehydration through a graded alcohol series and then passed though xylol and paraffin series before finally blocked in paraffin. The paraffin blocks were cut into 5–6 mm sections using a The trimmed tissues were first washed with tap water followed by dehydration through a graded alcohol series and then passed through xylol and paraffin series before finally blocked in paraffin. The paraffin blocks were cut into 5–6 lm sections using a microtome stained using hematoxylin and eosin and examined under a light microscope microtome stained using hematoxylin and eosin and examined under a light microscope [32]. Statistical analysis Data were collected, arranged and reported as mean ± standard error of mean (S.E.M) of nine groups (each group was considered as one experimental unit), summarized and then analyzed using the computer program SPSS/version 15.0) The statistical method was one way analyzes of variance ANOVA test (F-test), and if significant differences between means were found, Duncan’s multiple range test (whose significant level was defined as P < 0.05) was used according to [33] to estimate the effect of different treated groups.

Fig. 2. Infrared spectrum of Cd2+/Para complex.

Table 2 IR frequencies (cm1) of Para and its metal complexes. Assignments

Compounds

Results and discussion t(OAH) t(NAH)

Molar conductivities

t(C@O) t(CAO) t(CAN) t(MAO)

The molar conductance values for the Cd(II), Hg(II) and Pb(II) complexes of Para in DMSO solvent (1.00  103 M) were found to be in the range 40–52 X1 cm2 mol1 at 25 °C, suggesting them to be non-electrolytes (Table 1). These data matched with the calculated elemental analysis. Infrared spectra The infrared spectra of Para free ligand and its complexes were recorded in Fig. 2 and Table 2. The spectra of Para complexes are similar; this is due to the same place of coordination toward Cd(II), Hg(II) and Pb(II) ions. The most interesting feature in the infrared spectra of these complexes is the shift and decreasing in the intensities of bands in the 3360–3500 cm1 region. However, the ligand exhibited four bands at 3785, 3694, 3184 and 3162 cm1. This fact is hard to reconcile with either structure proposed by both Medvedovskii [34,35] and Fazakerley et al. [36]. Ionization of the OH groups with subsequent ligation through oxygen atoms seems a plausible explanation. Weak bands at 3184 and 3162 cm1 can be assigned to NH which is not shifted; we can conclude that the NH group is not involved in coordination process. The band at 1653 cm1, assigned to C@O vibration, is not shifted in the complex but has decreased intensity. Band at 1324 cm1 in the free ligand is not changed in the complexes. Nakamoto [37] assigned the band at 1324 cm1 in the metal oxamido complexes to the CAN vibration. If we accept that the 1324 cm1 band is due to CAN vibration, we conclude that the amido group is not involved in coordination. The free ligand has a band in the CAO region, namely at 1256 cm1. On complexation the maxima are significant

Para

I

3785 3694 3184 3162 1650 1256 1324 –

3626 3323 3626 3323 1653 1013 1324 611 511

II

III

3434 3434

3535 3535

1653 1022 1324 666 567

1653 1047 1324 605 412

shifted to 1047, 1022 and 1013 cm1 with a marked change of intensity. If we assume that the bands arise from CAO vibration, we may conclude that the CAO group is probably involved in coordination, therefore, the metal M(II) chelate was assigned through hydroxyl group. 1

H NMR spectrum of Cd(II) complex

The 1H NMR data of Para and its Cd(II) complex, as an example are listed in Table 3 and shown in Fig. 3. The 1H NMR spectrum of Para show the signal at d = 9.27 ppm, which is assigned to the protons of the amide group which is the same in the Cd(II) complex and confirm that the amide group does not contribute in the complexation between Para and Cd(II) metal ions. The proton NMR spectrum for [Cd (Para)2(H2O)2]6H2O show a singlet at peak at 3.38 ppm. This singlet peak is not observed in the free Para ligand spectrum and can be assigned due to protons of H2O molecules, supporting the complexes formula. The disappearance of the signal of the proton of the hydroxyl group in the 1H NMR spectrum of the complex confirms that the hydroxyl group contribute in the complexation between Para and Cd(II) ion, therefore, the hydroxyl group is disappear in the complex of Para moiety. So, the complexation between two molecules of Para with Cd(II) ion has been

Table 1 Elemental analysis and physical data for Para complexes. A (S cm2 mol1)

Complexes

Mwt.

Color

Content (found) calculated %C

%H

%N

%M

[Cd(Para)2(H2O)2]6H2O (I) [Hg(Para)2(H2O)2]4H2O (II) [Pb(Para)2(H2O)2]4H2O (III)

558 611 617

White White White

34.40 (34.33) 31.42 (31.53) 31.11 (31.04)

6.09 (6.31) 4.90 (4.87) 4.86 (4.75)

5.01 (4.95) 4.58 (4.70) 4.53 (4.62)

20.16 (20.21) 32.89 (32.76) 33.54 (33.63)

40 47 52

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formed by the chelation of the metal ion with the hydroxyl group to form the complex 2-Para-Cd(II) including the oxygen metal [38]. Electronic absorption spectra The formation of the Cd(II), Hg(II) and Pb(II) complexes of Para was also confirmed by UV–Vis spectra. The electronic absorption spectra of the M(II) complexes in DMSO in the 200–600 nm range were discusses. It can see that free Para has two distinct absorption bands. The first one at 300 nm may be attributed to p ? p⁄ intra-ligand transition of the aromatic ring. The second band observed at 390 nm is attributed to n ? p⁄ electronic transition. In the spectra of the M(II) complexes, the two bands are hypsochromically affected clearly, suggesting the ligand is deprotonated and the lone pair of electrons on nitrogen and carbonyl-oxygen atoms of the amide group is not participated in the complexation. These results are clearly in accordance with the results of the FTIR and 1H NMR spectra. Thermal analysis of Para complexes The heating rates were controlled at 10 °C/min under nitrogen atmosphere and the weight loss was measured from ambient temperature up to ffi136 °C. The data are listed in Table 4 and shown in Fig. 4. The weight losses for each chelat calculated within the corresponding temperature ranges. [Cd(Para)2(H2O)2]6H2O The thermal decomposition of [Cd(Para)2(H2O)2]6H2O complex occurs at three steps. The first degradation step takes place in the range of 50–240 °C and they corresponds to the elimination of 2(H2O) and C5H5NO2 (organic moiety) with an observed weight loss of (obs. = 45.92%, calc. = 45.69%). The second step falls in the range of 250–440 °C which is assigned to the loss of C4H3NO (organic moiety) with a weight loss (obs. = 14.28%, calc. = 14.51%). The final decomposition step within the temperature range 450– 575 °C is accompanied by mass loss of 16.62% (calc. = 16.84%), which are assigned to the loss of C7H10 (organic moiety). The CdO is the final product remains stable till 800 °C. [Hg(Para)2(H2O)2]4H2O The Hg(II) Para complex is decomposed in three steps. The first step is exhibited at 50–160 °C and corresponding to the loss of 6(H2O) and C5H10NO (organic moiety), representing a weight loss of (obs. = 34.12%, calc. = 34.04%). The second step takes place Table 3 H NMR spectral data of Para and its Cd(II) complex.

1

Compounds

Para Cd(II) complex

d (ppm) of hydrogen H; CH3

H; H2O

H; ArH

H; NH

1.96 1.96

– 3.38

6.57–7.28 6.63–7.34

9.57 9.64

within the temperature range 170–290 °C and can assigned to the loss of C5H6NO3 (organic moiety) and Hg vapor and the mass loss due to this step was (obs. = 54.17%, calc. = 53.84%). The last step is occurring at 300–500 °C and corresponding to the evolution of 3(C4H2) (organic moiety) representing a weight loss of 7.93% and its calculated value is 8.18%. The final products resulted at 800 °C contain 2 carbon atoms. [Pb(Para)2(H2O)2]4H2O The Pb(II) complex is decomposed in three steps. The first step is occurring at 50–175 °C and corresponding to the evolution of 5(H2O) molecules, representing a weight loss of 14.38% and its calculated value is 14.58%. While, the second decomposition step is occurring at 180–250 °C and corresponding to the loss of H2O and C9H10N (organic moiety), representing a weight loss of 25.42% and its calculated value is 25.93%. The horizontal bump in Fig. 4C, it is meaning to the start of melting point of lead metal which is completely melting up to 320 °C. This confirms the confused between logically dissociation of paracetamol ligand and melting point of lead metal. So the non-horizontal bump with mass loss refers to decomposition of ligand and horizontal bump with non-mass loss refer to melting of lead metal. The final decomposition step takes place in the range of 290–430 °C and corresponding to the elimination of C7H8NO3with an observed weight loss of 25.94% (calc. = 26.58%). The final product resulted at 800 °C contain PbO. Kinetic and thermodynamic parameters The kinetic and thermodynamic parameters were determined by non-isothermal methods. The non-isothermal kinetic analysis for the thermal decomposition of all ligands and complexes in this work was carried out by the application of the Coats–Redfern [39] method. The kinetic parameters were evaluated for all decomposition stages. Kinetic studies were applied for the decomposition stages that occur within a temperature range, resulting in a TG curve providing enough data to be collected. The kinetic parameters (Ti, Tf, Dm, and Ea) were calculated according to the Coats–Redfern method [39]. Methods From the TGA curves (TG, DTG) recorded for the successive steps in the decomposition process of ligands and their complexes, it was possible to determine the following characteristic thermal parameters for each reaction step as follows. Initial point temperature of decomposition (Ti): the point at which DTG curve starts deviating from its base line. Final point temperature of decomposition (Tf): the point at which DTG curve returns to its base line. Peak temperature, i.e. temperature of maximum rate of mass loss (TDTG): the point obtained from the intersection of tangents to the peak of DTG curve. Mass loss at the decomposition step (Dm): it is the amount of mass that extends from the point Ti up to the point Tf on the TG curve. The material

Fig. 3. 1H NMR spectrum of Cd2+/Para complex.

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Found

45.69 14.51 16.84

45.92 14.28 16.62

8H2O + C5H5NO2 C4H3NO C7H10

CdO

II

160 290 500

34.04 53.84 8.18

34.12 54.17 7.73

6H2O + C5H10NO C5H6NO3 + Hg vabour C4H2

2C

5H2O H2O + C9H10N C7H8NO3

PbO

14.58 25.93 26.58

14.36 25.42 25.94

0.00E+00

100

240 445 575

175 240 430

2.00E+00

(A)

Residue

-2.00E+00 80 -4.00E+00 60 -6.00E+00 40

-8.00E+00

20

-1.00E+01 -1.20E+01 1000

0

released at each step of the decomposition is identified by attributing the mass loss (Dm) at a given step to the component of similar weight calculated from the molecular formula of the investigated compounds, comparing that with literatures of relevant compounds considering their temperature. This may assist identifying the mechanism of reaction in the decomposition steps taking place in the compounds under study.

log

h

1ð1aÞ T 2 ð1nÞ

logð1aÞ T2

i

i

h

i

Ea ZR 1  2RT ¼ log qE  2:303RT Ea a

h i Ea ZR 1  2RT ¼ log qE  2:303RT Ea a

400

Weight % (%)

600

800

Derivative Weight % (%/m)

(B)

-2 -4 -6

60

-8

40

-10 20

for n–1

-12 -14 600

0 0

for n ¼ 1

where a is the fraction of weight loss, T the temperature (K), n the order of reaction, Z the pre-exponential factor, R the molar gas constant, Ea the activation energy and q is the heating rate. The activation energies (Ea) are calculated from the slopes of the best fit straight lines (r  1) obtained when the plots of the Coats–Redfern equation [40] are used for the best values of reaction order (n). Order of reaction (n): it is the one for which a plot of the Coats– Redfern expression gives the best straight line among various trial values of n that are examined, i.e., by trial and error for various trial values of n, estimated by the Horowitz–Metzger method [40]. The thermodynamic parameters: entropy change (DS ), enthalpy change (DH ) and free energy of activation change (DG ) were calculated using the following equations:

2 0

100

100

200

300

Weight % (%)

400

500

Derivative Weight % (%/m)

120

20

(C) 100

Weight % (%)

log

1n

200

120

Weight % (%)

Activation energy (Ea) of the decomposition step: the integral method used is the Coats–Redfern equation [40] for reaction order n – 1, which when linearized for a correctly chosen n yields the activation energy from the slope;

h

0

80

Coats–Redfern method

Derivative weight % (%/m)

Calc.

Assignments

Derivative weight % (%/m)

TG weight loss (%)

I

III

Derivative Weight % (%/m)

0

80

-20

60

-40

40

-60

20

-80

Derivative weight % (%/m)

DTG peak/(°C)

Weight % (%)

Compounds

Weight % (%)

120



DS ¼ R½lnðZh=KTÞ

-100

0

DH ¼ Ea  RT s

0

100

200

300

400

500

600

700

DG ¼ DH  T s DS

Fig. 4. TG/DTG curves of (A): Cd2+/Para, (B): Hg2+/Para and (C): Pb2+/Para complexes.

where Z, K and h are the pre-exponential factor, Boltzman and Plank constants, respectively [42]. The negative DS values indicate that the activated complexes have more ordered structure than the reactants and the reactions are slower than normal [41,42]. The positive values of DG indicate the non-spontaneous character for the reactions at the transitionstate. The positive DH values show endothermic transition-state reactions [43]. From the abnormal values of Z, the reactions of the complexes at the transition-state can be classified as a slow reaction [44]. It can be concluded that the thermal decomposition of these three complexes is of a several of degrees of similarity (Table 5). The variation in the thermal stability of these complexes

may be attributed to the differences of the electronegativities of the central metal ions and the ionic size. This can be discussed in terms of the associated repulsion between the non-bonded electrons (on the donating oxygen) and the bonding electron pairs in the valance shell of the metal ions. The extent of covalent character in the metal–ligand bond influences the stabilities of the complexes [45]. It is noticeable that as the electronegativities of the central metal increases the thermal stability increases because the covalent bond character increases, giving a strong interaction between the metal and the ligand [46]. The higher stabilities of

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Table 5 Kinetic and thermodynamic parameters of the thermal decomposition of Para complexes. Complexes

Z (s1)

r

I II III

0.9950 0.9992 0.9991

9

1.54  10 1.32  106 7.21  102

Tmax k

E (kJ mol1)

DS⁄ (J k1 mol1)

DH⁄ (kJ mol1)

DG⁄ (kJ mol1)

240 160 175

72 86 84

70 130 199

69 83 77

72 121 182

r = correlation coefficient of the linear plot, n = order of reaction, Z = pre-exponential factor.

the metal complexes than that of the ligand may be due to the molecular symmetry [47].

Table 7 Effect of Para, Cd2+/Para, Hg2+/Para, and Pb2+/Para complexes on liver functions in male albino rats.

Antimicrobial activity

Groups

Total protein

AST

ALT

LDH

The results of antimicrobial activities (bacteria and fungi) in vitro of the Para ligand and their Cd(II), Hg(II) and Pb(II) complexes, Table 6, and Fig. 1S, show that, the [Hg(Para)2(H2O)2]4H2O test complex has high activities against A. flavus > E. coli > A. oryzae = A. niger > B. subtilis. On the other hand, the cadmium(II) and lead(II) Para complexes have antimicrobial activities against E. coli, A. oryzae and A. niger, A. flavus and B. subtilis, respectively. These results clearly obviously that, some metal ions after complexation give the sensitive nature for the ligand against some bacteria and fungi, also, we found that the antimicrobial activities increasing with higher molecular weights (Fig. 1S).

Control Para Cd2+/Para Hg2+/Para Pb2+/Para

8.18 ± 0.07a 6.64 ± 0.71c 7.98 ± 0.41b 3.88 ± 0.20d 1.88 ± 0.30e

12.20 ± 0.54d 55.40 ± 1.12c 87.80 ± 6.02b 177.80 ± 7.39a 175.80 ± 7.39a

13.00 ± 0.56e 20.40 ± 0.30d 78.80 ± 1.82bc 91.00 ± 1.18a 70.45 ± 1.18c

310.66 ± 20.63d 345.69 ± 17.33cd 341.30 ± 23.10cd 1922.40 ± 25.37b 2200.40 ± 25.37a

Hepatotoxicity effect and histopathological changes of liver tissues Effect on serum total protein The administration of Para in it is recommended doses for successive 30 days into normal rats elicited significant decrease (P < 0.05) in serum total protein level compared with normal control group but the effect was less intense as well as group treated with Cd2+/Para. Whereas, a highly significant decrease (P < 0.05) were observed in the groups treated with Hg2+/Para and Pb2+/Para complexes after the end of the experiment when compared with normal control group (Table 7 and Fig. 2S). Our results were supported with Ekam and Ebong [48] who reported significant decrease in serum total protein after treatment of male rats with paracetamol for 14 days as a result of hepatic damage induced by paracetamol administration and this assure our findings that paracetamol cause significant damage in liver and this level was increased significantly in Cd2+/Para complex treated group which showed improvement in liver functions parameters due to complexation between paracetamol and Cadmium metal ion as compared to Para treated group. Effect on serum GOT and GPT (AST&ALT) Serum transferases (AST&ALT) levels were slightly elevated after 30 days post Para administration to normal rats when compared with normal control group. Meanwhile highly significant increase (P < 0.05) in serum AST&ALT were recorded in normal rats in response to administration of Pb2+/Para or Hg2+/Para but the administration of Cd2+/Para has revealed significant increase in

Table 6 Antimicrobial data of Cd+2/Para, Hg+2/Para and Pb+2/Para complexes. Complexes

Control I II III

Inhibition zone (cm) E. coli

B. subtilis

Asperagillus oryzae

Asperagillus niger

Asperagillus flavus

0 0.7 4 4

0 1.5 1.3 1.3

0 1.5 3.5 3.5

0 1 1 3.5

0 0 5 2

Means within the same column in each category carrying different letters are significant at (P 6 0.05) using Duncan’s multiple range tests, where the highest mean value has symbol (a) and decreasing in value were assigned alphabetically.

serum ALT when compared with normal control group but with significant decrease when compared with other two complexes (Hg2+ or Pb2+) (Table 7 and Fig. 3S). Our results come in parallel with Galal et al. [49] who reported that administration of paracetamol to adult rats caused significant elevation in ALT and AST levels after treatment for 7 successive days, several researchers have reported significant elevations in serum ALT and AST following administration of toxic doses of paracetamol in rats [50]. Owing to their high concentrations and ease of liberation from the hepatocyte cytoplasm, ALT and AST are indicators which are very sensitive of necrotic lesions within the liver [51]. Hence, the marked release of transaminases into the circulation is indicative of severe damage to hepatic tissue membranes during paracetamol intoxication and this damage was treated partially by Cd2+/Para and also, approve the deleterious effect of Pb(Lead) exposure on hepatic functions parameters. Effect on serum lactic dehydrogenase enzyme (LDH) activity Treatment of normal rats with Para and Cd2+ + Para for successive 30 days in their recommended doses elicited non-significant changes in serum LDH activity after the end of the study when compared with normal control group. While the other treatments with Para complexes (Hg2+ and Pb2+) elicited a marked elevation in serum LDH activity when compared with normal control group (Table 7 and Fig. 4S). The significant increases of LDH are mainly due to leakage of these enzymes into the blood because of Pb2+, HG2+ toxicity in liver [52] and we consider the first author to demonstrate these results on Para and these complexes. Effect on lipid profile in male albino rats Normal healthy control was found to be stable in their cholesterol values (Table 8). Total cholesterol in the Para treated group was significantly higher when compared with the other groups except the group treated with Hg2+ + Para as it was increased significantly when compared with other groups and control group. The Cd2+ + Para treated group showed non-significant change when compared with control group while the group treated with Pb2+/Para was significantly increased when compared with the control group but not as Hg2+ + Para treated group. Treatment the animals with Para induced highly significant increase in triglycerides contents when compared with normal control group while treatment of animals with Cd2+ + Para afforded

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S.M. El-Megharbel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 534–544 Table 8 Effect of Para, Cd2+/Para, Hg2+/Para, and Pb2+/Para complexes on lipid profile in male albino rats. Groups Control Para Cd2+/Para Hg2+/Para Pb2+/Para

Cholesterol (mg/dl)

Triglycerides (mg/dl)

e

e

68.22 ± 4.57 87.52 ± 3.54c 70.11 ± 2.15de 255.14 ± 22.1a 99.41 ± 6.75bc

HDL-c (mg/dl)

LDL-c (mg/dl)

ab

98.99 ± 4.77 149.24 ± 9.45bc 110.23 ± 8.65d 173.62 ± 9.54a 142.32 ± 10.25c

VLDL-c (mg/dl)

e

36.78 ± 3.62 25.25 ± 3.24c 31.20 ± 5.24b 17.28 ± 3.21d 7.25 ± 0.42e

d

33.24 ± 2.52 59.21 ± 4.12b 39.20 ± 5.42de 49.52 ± 3.52c 210.23 ± 12.45a

19.79 ± 2.45 29.84 ± 4.21b 22.04 ± 3.52c 34.72 ± 5.75a 28.46 ± 6.75b

Risk ratio (%) 1.85 ± 0.24e 3.46 ± 0.52c 2.24 ± 0.53d 14.76 ± 2.11a 13.71 ± 1.63b

Means within the same column in each category carrying different letters are significant at (P 6 0.05) using Duncan’s multiple range tests, where the highest mean value has symbol (a) and decreasing in value were assigned alphabetically. Low density lipoprotein cholesterol (LDL-c) = Total cholesterol  (HDL-c + triglyceride)/5, volatile. Low density lipoprotein cholesterol (VLDL-c) = Triglycrid/5, risk ratio = total cholesterol/high density lipoprotein cholesterol (HDL-c).

significant decrease in TG when compared with Para group but induced significant increase when compared with control group. Pb2+ and Hg2+ + Para groups did not improve the TG contents as compared to the normal control group but the higher increase was noticed in Hg2+ + Para treated group after 30 days of treatment. Table 8 shows the levels of HDL-c for all treatment groups with Para and it is complexes to be lower than control group except the Cd2+ + Para treated group showed non-significant change in HDL-c when compared with normal control group. Table 8 and (Fig. 5S) revealed that all groups treated with Para and it is complexes afforded significant increase in LDL-c except Cd2+ + Para which showed non-significant changes when compared with control group but with less increase in LDL-c content, on the other hand, VLDL-c was significantly increased in group treated with Hg2+ + Para when compared with control group while Cd2+ + Para group showed the less increase in VLDL-c when compared with control group and it was the best treated group. So, as a result of the previous data, the risk factor will be high in Para treated group and it is complexes with Hg2+ and Pb2+ but the risk ratio was more less in Cd2+ treated group with Para. Effect on SOD, GST and total antioxidant capacities (TAC) in male albino rats Table 9 and (Fig. 6S) showed that all groups treated with Para complexes showed significant decrease in SOD, GSH and TCA capacities when compared with control group unless the group treated with Cd2+/Para complex which showed improvement in the results of antioxidant capacities when compared Table 9 Effect of Para, Cd+2/Para, Hg+2/Para, and Pb+2/Para complexes on SOD, Glutathione– S-transferase (GST) and total antioxidant capacity (TAC) in male albino rats. Groups

SOD (U/mg)

GST (U/mg)

TAC (%)

Control Para Cd2+/Para Hg2+/Para Pb2+/Para

1.66 ± 0.20a 1.23 ± 0.32b 1.53 ± 0.42a 1.10 ± 0.53c 0.89 ± 0.33d

97.23 ± 3.11a 77.21 ± 5.32c 80.32 ± 6.21b 57.25 ± 5.20d 50.02 ± 4.20d

100.20 ± 6.32a 76.42 ± 4.42c 83.10 ± 4.65b 54.21 ± 3.61d 45.32 ± 3.54e

Means within the same column in each category carrying different letters are significant at (P 6 0.05) using Duncan’s multiple range tests, where the highest mean value has symbol (a) and decreasing in value were assigned alphabetically.

with normal control group as it ameliorate the total antioxidant capacity when compared with Para treated group and other complexes. LPO is supposed to cause the destruction and damage of cell membranes leading to changes in membrane permeability and fluidity, enhancing the protein degradation in mice [53]. In the present study, the levels of LPO were increased indicating an increased in the generation of free radicals in the liver tissues of Para treated animals and Hg2+, Pb2+ Para complexes [54]. After administration of Cd2+ complex with Para for 30 days, the LPO were decreased in the liver tissues (Fig. 6S) indicating decreased in the generation of free radicals. It is possible that Cd2+ + Para complex act as a scavenger of oxyradicals and thus could prevent liver damage. According to the present data, Cd2+ + Para complex has a protective effect by decreasing LPO. Paracetamol is mainly metabolized in liver to excretable glucuronide and sulfate conjugates. However, the hepatotoxicity of paracetamol has been attributed to the formation of toxic metabolites when a part of paracetamol is activated by hepatic cytochrome P-450, to a highly reactive metabolite N-acetyl-P-benzoquinoneimine (NAPQI). NAPQI which is believed to play an important role in paracetamol mediated toxicity is initially detoxified by conjugation with reduced glutathione (GSH) to form mercapturic acid. However, when the rate of NAPQI formation exceeds the rate of detoxification by GSH, it oxidizes tissue macromolecules such as lipid or ASH group of protein and alters the homeostasis of calcium after depleting GSH [55]. In the present study the Cd2+/Para complex competes with oxygen thus inhibits the generation of the anions. Effect on hematological parameters in male albino rats Table 10 and (Fig. 7S) represents effect of Para and it is complexes on the hematological parameters in normal and rats during the 30 days exposure. The level of RBCs, Hb, HCT, MCH and WBCs decreases in Para complexes treated groups as compared to the control group. However, the level of RBCs, Hb, HCT, MCH and WBCs were non-significantly changed when compared with control group with less decrease in platelets counts. The major pathological consequences of free radical induced membrane lipid peroxidation include increased membrane rigidity, decreased cellular

Table 10 Effect of Para, Cd2+/Para, Hg2+/Para, and Pb2+/Para complexes on the hematological parameters in male albino rats. Groups Control Para Cd2+/Para Hg2+/Para Pb2+/Para

RBCs (106/mm3) a

8.99 ± 1.90 7.57 ± 1.35c 8.56 ± 1.75b 5.20 ± 1.68d 5.34 ± 1.45d

Hb (g/dl)

WBCs (103/mm3)

HCT (%) a

14.43 ± 3.20 13.20 ± 2.85b 13.43 ± 3.61b 10.47 ± 2.10c 9.63 ± 1.95c

a

47.30 ± 3.65 39.07 ± 3.22c 44.42 ± 2.85b 34.25 ± 2.75d 33.20 ± 4.21d

ab

8.52 ± 2.10 5.68 ± 2.35c 7.69 ± 1.98b 2.98 ± 1.77d 3.20 ± 1.65d

Platelets (103/mm3) 665.32 ± 7.85a 534.33 ± 9.52c 580.95 ± 15.32b 342.35 ± 8.52d 336.10 ± 7.12d

Means within the same column in each category carrying different letters are significant at (P 6 0.05) using Duncan’s multiple range tests, where the highest mean value has symbol (a) and decreasing in value were assigned alphabetically.

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deformability, reduced erythrocyte survival, and lipid fluidity [56] and thus this explain the significant decrease in the number of R.B.Cs and other blood parameters in groups treated with Para and it is complexes with Hg2+ and Pb2+. Histopathology of liver

– Para group Microscopically, severe congestion of the hepatic blood vessels was seen (Fig. 6). – Cd2+/Para group

– Control group Microscopically: Normal liver tissues formed of small central vein surrounded by cords of hepatocytes showing central vesicular nuclei and eosinophilic cytoplasm (Fig. 5).

Liver tissue showing severe fatty change in the hepatocytes (Fig. 7) and liver tissue showing necrosis in liver cells in the form of pyknotic nucleic (") and more eosinophilia of the cytoplasm was seen in (Fig. 8).

Fig. 5. Cross section of control rat liver formed of small central vein surrounded by cords of hepatocytes showing central vesicular nuclei and eosinophilic cytoplasm (H and E  200) where CV: central vein, EN: eosinophilic nuclei.

Fig. 8. Cross section of rat liver tissues of Cd+2/Para treated group showing some necrosis in liver cells in the form of pyknotic nuclei (") and more eosinophilia of the cytoplasm (H and E  200) (PN: Pyknotic nuclei).

Fig. 6. Cross section of rat liver tissues of Para treated group showing severe congestion of the hepatic blood vessels (H & E stain  150) where S: Severe congestion.

Fig. 9. Cross section of rat liver tissues of Hg+2/Para treated group showing severe fatty change of hepatocytes and markedly congested central vein (H & E stain  400) (F: Fatty change , CCV: Congested central vein).

Fig. 7. Cross section of rat liver tissues of Cd+2/Para treated group showing severe fatty change in the hepatocytes (H and E  200) (SFC: Severe fatty change).

Fig. 10. Cross section of rat liver tissues of Pb+2/Para treated group showing severe congestion of the hepatic blood vessels) H & E stain  400) (SC: Severe congestion).

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– Hg2+/Para group Microscopically, severe fatty change of hepatocytes and markedly congested central vein was shown (Fig. 9). – Pb2+/Para group

Fig. 11. Cross section of rat liver tissues of Pb+2/Para treated group showing marked necrosis of hepatocytes with congested blood sinusoids (H & E stain  400) (N :Necrosis).

Microscopically, variable degrees of congestion of the hepatic blood vessels, severe congestion were seen (Fig. 10) and also marked necrosis of hepatocytes with congested blood sinusoids (Fig. 11). Liver tissue showing markedly dilated central vein filled by large number of red blood cells and surrounded by hepatic cords (Fig. 12). Our results come in harmony with Galal et al. [49] who reported that histological examinations of liver sections of rats that was given paracetamol proved the presence of hepatotoxic markers which revealed degenerative changes that involved the hepatocytes and cells that line the blood sinusoids. The damage related to the majority of the hepatic lobule with marked loss of its normal pattern. These changes positively correlated with the noted increases in transaminase activities and this is greatly agreed with our recorded results. Conclusion

Fig. 12. Cross section of rat liver tissues of Pb+2/Para treated group showing markedly dilated central vein filled by large number of red blood cells and surrounded by hepatic cords (DCCV: dilated congested central vein).

The complexation between metal ions like (Cd(II), Hg(II) and Pb(II) with Para produced 1:2 M ratio (metal: Para) as a monodentate via hydroxyl group and give general formula: [M(Para) (H2O)2]nH2O, where n = 6 or 4 (Fig. 13). The resulted Para compounds were assigned by infrared and electronic spectra. Thermogravimetric analyses have proved the thermal stability feature of Para complexes. The antimicrobial activities of the metal complexes of Para recorded a significant effect against some bacteria and fungi. The Para/Hg(II) and Para/Pb(II) complexes duplicate

Fig. 13. The mode of chelation of the Para complexes where, M = Cd (II), Hg (II) and Pb(II).

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