*3.8. Thermal Analysis*

*3.8. Thermal Analysis*  Thermal analysis is an essential tool used for the characterization of metal complexes where, when heating a compound, its weight loss increases. In addition, DTG differential thermogravimetric analysis (DTGA) is used to study the thermal stability of compounds and the weight loss at different temperatures. TGA confirmed the successful chelation of CFX with different metal ions. Thermogravimetric analysis (TGA) and differential (DTGA) analysis for Mg (II), Cu (II), Fe (III), Zn (II) and Se (IV) CFX complexes were carried out in a temperature range of 30–800 °C under an N2 atmosphere. The first decomposition endothermic step occurred in a temperature range 30–130 °C and corre-Thermal analysis is an essential tool used for the characterization of metal complexes where, when heating a compound, its weight loss increases. In addition, DTG differential thermogravimetric analysis (DTGA) is used to study the thermal stability of compounds and the weight loss at different temperatures. TGA confirmed the successful chelation of CFX with different metal ions. Thermogravimetric analysis (TGA) and differential (DTGA) analysis for Mg (II), Cu (II), Fe (III), Zn (II) and Se (IV) CFX complexes were carried out in a temperature range of 30–800 ◦C under an N<sup>2</sup> atmosphere. The first decomposition endothermic step occurred in a temperature range 30–130 ◦C and corresponded to a loss of molecules of crystalline water. The second cracking step was carried out between 130–380 ◦C owing

sponded to a loss of molecules of crystalline water. The second cracking step was carried

to a loss of ceftriaxone ligand. The third stage of decomposition occurred in temperature ranges of 380–530 ◦C and 530–620 ◦C, representing the evolution of NH3, CO<sup>2</sup> and HNCO. The final residual products for all ceftriaxone metal complexes are metal oxides.

#### *3.9. Ceftriaxone Metal Complexes Alleviate Hepatic Injury in Male Rats*

Excessive CFX exposure for 30 consecutive days afforded a significant increment in serum activities of ALT and AST in male rats. However, in contrast, the supplementation of CFX complexes induced significant improvements in liver enzymes, and the best ameliorations were recorded in CFX/Mg, CFX/Zn and CFX/Se, respectively, as compared to the CFX treated group alone, as shown in Table 5.

### *3.10. Ceftriaxone Metal Complexes Alleviate Oxidative Injury in the Hepatic Tissues and Structural Alterations of Male Rats Exposed to Ceftriaxone*

Reactive oxygen species levels manifested a marked elevation in the hepatic tissues of CFX-exposed male rats. Supplementation of CFX metal complexes reduced the excessive hepatic generation of free radicals, as recorded in the CFX-treated group. Hepatic MDA levels were significantly elevated in the CFX-treated group only. These changes were mainly inverted in the group to which CFX metal complexes were administered, especially CFX/Zn and CFX/Mg, respectively.

On the contrary, male rats exposed to CFX exhibited a high decline in hepatic antioxidant enzyme (GSH, SOD and CAT) activities. GSH, SOD activity and CAT declined markedly as compared with the control group. Oral supplementation of CFX metal complexes elevated the hepatic antioxidant enzymes as compared to CFX alone, especially for CFX/Mg, CFX/Zn and CFX/se, CFX/Cu and CFX/Fe, in the same order as shown in Table 6.

**Table 6.** Antioxidant enzyme activities in groups treated with either ceftriaxone or ceftriaxone metal complexes.


Values are means ± standard error. Mean values with different letters in the same row show significance at *<sup>p</sup>* <sup>≤</sup> 0.05, where the highest mean value has the symbol <sup>a</sup> and <sup>b</sup>−<sup>g</sup> : Decreases in value were assigned alphabetically, with similar letters implying partial or complete non-significance and different letters implying significance. CFX = group supplemented with ceftriaxone; CFX/Mg = group supplemented with ceftriaxone/Mg complex; CFX/Zn = group supplemented with ceftriaxone/Zn complex; CFX/Se = group supplemented with ceftriaxone/Se complex; CFX/Cu = group supplemented with ceftriaxone/Cu complex.

Histological sections of hepatic tissues of different treated groups as shown in (Figure 8) which showed marked hepatic alterations and some atrophy in CFX treated group only, while there was marked improvement recorded in hepatic tissues of other treated groups with either CFX/Mg, CFX/Zn, CFX/Se, CFX/Cu and CFX/Fe, Meanwhile the marked improvement was recorded in CFX/Mg, CFX/Zn and CFX/Cu treated groups respectively.

**Figure 8.** Photomicrographs showing histological sections of the hepatic tissues. (**A**) Control group showing normal hepatic structure and normal central vein (CV) (H&EX400). (**B**) CFX-treated group showing severe toxicity in the form of the hypertrophy of hepatocytes (orange arrow) with the appearance of binucleated hepatocytes and increased eosinophilia, granular cytoplasm and vesic-**Figure 8.** Photomicrographs showing histological sections of the hepatic tissues. (**A**) Control group showing normal hepatic structure and normal central vein (CV) (H&EX400). (**B**) CFX-treated group showing severe toxicity in the form of the hypertrophy of hepatocytes (orange arrow) with the appearance

ular nuclei (\*\*\*); the central vein is dilated and filled with hemorrhage and necrotic tissue (black arrow), with focal necrosis in some hepatocytes with increased eosinophilia and nuclear disap-

of binucleated hepatocytes and increased eosinophilia, granular cytoplasm and vesicular nuclei (\*\*\*); the central vein is dilated and filled with hemorrhage and necrotic tissue (black arrow), with focal necrosis in some hepatocytes with increased eosinophilia and nuclear disappearance and the accumulation of a few mononuclear inflammatory cells in blood sinusoids (H&EX400). (**C**) CFX/Mg-treated group showing an almost normal hepatic structure (green arrow) with a normal sized central vein (CV) (H&EX400). (**D**) CFX/Zn-treated group showing very mild toxicity in the form of the hypertrophy of hepatocytes with granular eosinophilic cytoplasm and vesicular nuclei and the appearance of some binucleated cells (orange arrow), with a mild congested central vein which contains (CV) mild brown particles of bilirubin and single hepatocyte necrosis (H&EX400). (**E**) CFX/Se-treated group showing an almost normal hepatic structure with normal hepatocytes with a moderately enlarged central vein (CV) filled with some red blood cells (red arrow) with some mild detachment (\*\*\*) (H&EX400). (**F**) CFX/Cu-treated group showing an almost normal hepatic structure with ballooning cytoplasm (blue arrow) and a central vein (CV) filled with some red blood cells (H&EX400). (**G**) CFX/Fe-treated group showing mild toxicity in the form of the hypertrophy of hepatocytes with the. appearance of binucleated hepatocytes and increased eosinophilia, granular cytoplasm and vesicular nuclei (red arrow); the central vein is normal in diameter with mild congestion (CV) and ballooning degeneration in some hepatocytes (\*\*\*), with increased eosinophilia around the central vein indicating the beginning of necrosis (H&EX400). pearance and the accumulation of a few mononuclear inflammatory cells in blood sinusoids (H&EX400). (**C**) CFX/Mg-treated group showing an almost normal hepatic structure (green arrow) with a normal sized central vein (CV) (H&EX400). (**D**) CFX/Zn-treated group showing very mild toxicity in the form of the hypertrophy of hepatocytes with granular eosinophilic cytoplasm and vesicular nuclei and the appearance of some binucleated cells (orange arrow), with a mild congested central vein which contains (CV) mild brown particles of bilirubin and single hepatocyte necrosis (H&EX400). (**E**) CFX/Se-treated group showing an almost normal hepatic structure with normal hepatocytes with a moderately enlarged central vein (CV) filled with some red blood cells (red arrow) with some mild detachment (\*\*\*) (H&EX400). (**F**) CFX/Cu-treated group showing an almost normal hepatic structure with ballooning cytoplasm (blue arrow) and a central vein (CV) filled with some red blood cells (H&EX400). (**G**) CFX/Fe-treated group showing mild toxicity in the form of the hypertrophy of hepatocytes with the. appearance of binucleated hepatocytes and increased eosinophilia, granular cytoplasm and vesicular nuclei (red arrow); the central vein is normal in diameter with mild congestion (CV) and ballooning degeneration in some hepatocytes (\*\*\*), with increased eosinophilia around the central vein indicating the beginning of necrosis (H&EX400). *3.11. Antibacterial Activity Evaluation* 

#### *3.11. Antibacterial Activity Evaluation* (*Bacillus subtilis*, *Streptococcus pneumoniae* and *Staphylococcus aureus*) and Gram-negative

Biological evaluations were performed for CFX complexes against Gram-positive (*Bacillus subtilis*, *Streptococcus pneumoniae* and *Staphylococcus aureus*) and Gram-negative (*Escherichia coli* and *Pseudomonas aeruginosa*) bacteria. Results from the agar disc diffusion tests for the antimicrobial activities are presented in Table 6 and demonstrated in Figure 9. The diameters of the zone of inhibition (in mm) of the standard drug Amikacin (Aminoglycoside) (C22H43N5O13) against Gram-positive bacteria *B. subtilis* and *S.aureus* and Gram-negative bacteria *E. coli* and *P. aeruginosa* were found to be 36, 30, 31 and 35 mm, respectively. Under identical conditions, Table 7 and Figure 9 show that all complexes were found to be efficient, with a high antimicrobial activity that exceeded CFX itself. (*Escherichia coli* and *Pseudomonas aeruginosa*) bacteria. Results from the agar disc diffusion tests for the antimicrobial activities are presented in Table 6 and demonstrated in Figure 9. The diameters of the zone of inhibition (in mm) of the standard drug Amikacin (Aminoglycoside) (C22H43N5O13) against Gram-positive bacteria *B. subtilis* and *S.aureus* and Gram-negative bacteria *E. coli* and *P. aeruginosa* were found to be 36, 30, 31 and 35 mm, respectively. Under identical conditions, Table 7 and Figure 9 show that all complexes were found to be efficient, with a high antimicrobial activity that exceeded CFX itself.

Biological evaluations were performed for CFX complexes against Gram-positive

**Figure 9.** Antibacterial activity of CFX and Zn (II) and Se (IV) metal complexes**. Figure 9.** Antibacterial activity of CFX and Zn (II) and Se (IV) metal complexes.


**Table 7.** Inhibition zone diameter (mm/mg sample) of CFX and CFX metal complexes.

Means within the same column (mean ± SE) carrying different letters are significant at *p* ≤ 0.05 using Duncan's multiple range test, where the highest mean value has the symbol <sup>a</sup> and <sup>b</sup>−<sup>e</sup> those decreasing in value are assigned alphabetically.

#### **4. Discussion**

A serious problem now facing humanity is oxidative damage and its dangerous consequences for human health and hepatic vitality. The world now needs powerful antibiotics with potent antioxidant activities and fewer side effects for the liver tissues to reinforce the immune system and fight against resistant microbes and microorganism. Thus, the current study was designed to synthesize novel metal complexes of ceftriaxone (CFX) with Mg (II), Fe (III), Cu (II), Zn (II) and Se (IV) to investigate the potency of the hepatoprotective effects and antioxidant capacities of CFX complexes and determine if they succeed in the elevation of antioxidant capacities to investigate novel compounds with high antioxidant capacities.

The oxidative injury of biomolecules is the major concern in the pathogenesis of a large number of diseases such as cancer, degenerative diseases, metabolic diseases and even dangerous instances of SARS-CoV-2. Thus, it is very important to investigate the role of potent novel metal complexes to prevent oxidative stress and the treatment of side effects and diseases induced by such oxidative stress.

Liver injury caused by drugs ranges from mild biochemical abnormalities to chronic liver failure. The majority of adverse liver reactions occur in most antibiotic treatments [23].

Some antibiotics are considered to be a common cause of liver injury. The hepatotoxicity that occurs is usually associated with hepatic impairment [40]. CFX is a broad-spectrum antibiotic with potent activity against Gram-positive and Gram-negative bacteria [41].

The hepatotoxicity caused by CFX appears after an average of 9–11 days [42,43]. Previous studies have reported high aspartate aminotransferase (ALT) and alanine aminotransferase (AST) activities with the administration of CFX [44,45], and this concept coincides with the current findings that confirmed that CFX causes hepatic damage as a result of elevations in some biochemical parameters such as AST, ALT and low-density lipoprotein (LDL) as well as a decline in high-density lipoprotein (HDL) concentrations [46–48].

Based on the previous background, researchers have demonstrated that CFX is widely used as a third-generation cephalosporin antibiotic that has a broad spectrum of bactericidal activity. However, previous evidence has indicated that CFX carries a risk of the elevation of liver enzymes, with liver injury as an adverse effect [48].

In agreement with previous findings [23] that reported that the oxidative stress mediated by oxygen free radicals (ROS) has been implicated as a common link between chronic liver damage and hepatic fibrosis, the current study demonstrated that the administration of CFX resulted in a significant decline in hepatic GSH. Conversely, a significant increment in the level of hepatic MDA (a marker of lipid peroxidation) was shown. The increase in MDA level was more pronounced in rats in CFX-treated groups.

The novel finding of our current study is the improvement effects of CFX metal complexes, especially in groups treated with CFX with Mg, Zn, Se and Cu complexes, respectively, as they improved hepatic function serum levels and ameliorated the hepatic structure greatly.

Our explanation depends on the functional activity of the used metals, as previous researchers demonstrated the importance of Se metal in enhancing the glutathione levels of hepatic tissues, leading to enhancements of the whole antioxidant defense system [49–52] and a decline in the excessive generation of reactive oxygen species.

Metal ions are required to keep the human body healthy because several critical biological functions in humans depend upon their presence, and their absence or scarcity may lead to diseases. Magnesium (Mg) belongs to the main group of elements and is required mainly for fat and carbohydrate metabolism. Meanwhile, zinc (Zn) belongs to the transition metal group of elements. Zn is a vital element due to its strong binding to proteins. Zn also is exceptionally stable with respect to oxidation and reduction, and thus it does not participate in redox reactions. Additionally, Zn2+ shows a strong preference in enzymes for a tetrahedral coordination over an octahedral one [53], and all these previous aspects confirm the findings of the current study and the great improvement activities of CFX metal complexes, especially with Zn2+, in the alleviation of the hepatic side effects induced by CFX alone.

A deficiency of Fe leads to anemia, as it is known that Fe is a part of hemoglobin; besides this, a deficiency of Cu leads to heart diseases and anemia. The importance of all of these metals and especially in conjugation with CFX has been proved chemically in alleviating oxidative stress and providing high hepatic protection. The Gram-negative strains are exposed to various stress conditions during pathogenesis, of which the stress of acids serves as a major defense mechanism in the host [54]. Such environments are encountered, and this supports the main hypothesis of the study and proved the hepatoprotective and antioxidant capacities of two synthesized CFX complexes with either Zn (II) or Se (IV); in addition, the two complexes showed antibacterial activity besides their abilities to reduce free radicals' production.

#### **5. Conclusions**

The current study aimed to synthesize five ceftriaxone complexes by the reaction of ceftriaxone sodium salt with (Mg2+, Zn2+, Fe3+, Cu2+ and Se4+) ions. The structures of the CFX complexes have been explained using microanalytical, molar conductance, IR, <sup>1</sup>HNMR, UV–Vis, magnetic, SEM, TEM and X-ray diffraction analyses. Mg (II), Cu (II), Zn (II), Fe (III) and Se (VI) complexes form six-coordinate systems with a distorted octahedral geometry. The obtained results clarified that ceftriaxone metal complexes, especially (CFX/Mg, CFX/Zn and CFX/Se), greatly ameliorated hepatic enzyme functions and enhanced the antioxidant activities of hepatic tissues while reducing the excessive triggering of excessive reactive oxygen species (ROS) and the protection of hepatic structures as compared with ceftriaxone-treated groups alone. For antibacterial activity, the CFX/Zn complex was highly effective against *Streptococcus pneumoniae*, while CFX/Se was highly effective against *Staphylococcus aureus* and *Escherichia coli*. These results are very promising in providing protection for the hepatic tissues and reducing damaging effects and the severe oxidative stress induced by antibiotics on liver tissues, especially during the COVID-19 pandemic; in addition, it brings the recent research that correlated the damage of hepatic functions and severe instances of SARS-CoV-2 on the health of the body up to date.

**Author Contributions:** Conceptualization, S.M.E.-M., S.H.Q., F.S.A. and R.Z.H.; methodology, S.M.E.-M., S.H.Q. and R.Z.H.; validation, S.M.E.-M., S.H.Q., F.S.A. and R.Z.H.; formal analysis, S.M.E.-M., S.H.Q., F.S.A. and R.Z.H.; investigation, S.M.E.-M., S.H.Q., F.S.A. and R.Z.H.; resources, S.M.E.-M., S.H.Q., F.S.A. and R.Z.H.; data curation, S.M.E.-M. and R.Z.H.; writing—original draft preparation, S.M.E.-M., S.H.Q., F.S.A. and R.Z.H.; writing—review and editing, S.M.E.-M., S.H.Q., F.S.A. and R.Z.H.; supervision, S.M.E.-M., F.S.A., S.H.Q. and R.Z.H.; project administration, S.M.E.-M. and R.Z.H.; funding acquisition, S.M.E.-M., S.H.Q., F.S.A. and R.Z.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work received no external funding.

**Institutional Review Board Statement:** The animal study was conducted in accordance with the Declaration of Helsinki, and approved by Ethics Committee of Taif University.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All the data are available inside the text.

**Conflicts of Interest:** The authors declare no conflict of interest.
