Research Article

 

Ameliorative Effect of Vitamin E and Panax ginseng Against Some Adverse Effects of Levofloxacin in Male Rats

 

Hosny A. E. Ibrahim1, Nevertyty M. Mahmoud2, Dalia Mohamed Abd El-Mottleb2, Heba Ismail Khatab3*

1Department of Pharmacology, Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig, Egypt; 2Department of Pharmacology, Faculty of Medicine, Zagazig University, Egypt; 3Al-Ahrar Teaching Hospital, Ministry of Health, Zagazig, Egypt.

 

Received | May 01, 2021; Accepted | May 21, 2021; Published | November 15, 2021

*Correspondence | Heba Ismail Khatab, Al-Ahrar Teaching Hospital, Ministry of Health, Zagazig, Egypt; Email: h[email protected]

Citation | Ibrahim HAE, Mahmoud NM, Abd El-Mottleb DM, Khatab HI (2021). Ameliorative effect of vitamin e and panax ginseng against some adverse effects of levofloxacin in male rats. J. Anim. Health Prod. 9(4): 512-523.

DOI | http://dx.doi.org/10.17582/journal.jahp/2021/9.4.512.523

ISSN | 2308-2801

Copyright © 2021 Khatab et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

Introduction

 

Levofloxacin is a third-generation, strictly synthetic flouroquinolone antibiotic. It has bactericidal activity against a wide variety of bacteria that are gram positive (G+ve) and gram negative (G-ve) (Al-Soufi and Al-Rekabi, 2019). The principal target of quinolones is DNA gyrase, an essential bacterial enzyme (Abdel-Alim et al., 2017). Several studies have recorded that the most common serious effects associated with fluoroquinolones are headache, diarrhea, oxidative stress, hemolytic anemia, liver and kidney cellular damages (Owens and Ambrose, 2005; Talla and Veerareddy, 2011; Afolabi and Oyewo, 2014). Levofloxacin elevates malondialdehyde (MDA) and nitric oxide (NO) levels in addition to decrease in the level of glutathione in brain areas (Gupta et al., 2009). Also, it raises the activity of liver enzymes due to hepatic diseases (Eyrisofla et al., 2015).

 

There is a great need for finding protective substances to decrease levofloxacin side effects. Vitamin E is a powerful exogenous antioxidant can protect cell membranes against free radical damage (Bharrhan et al., 2010). Also, it is well known as free radical scavenger and protects cell membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction (Wang et al., 2010). Examples of endogenous antioxidants with a high catalytic effect are SOD and CAT, which are greatly concentrated in all tissues to protect cells from oxidative stress (Morikawa et al., 2000). Generally, vitamin E is commonly used as a medication for the treatment of some health conditions such as muscular dystrophies and certain nervous disorders, male infertility (Keskes-Ammar et al., 2003; Muller, 2010), cardiovascular disease (Lee et al., 2005), enhanced action of insulin in diabetic patients (Pavithra et al., 2018), platelet adhesion inhibition (Gonzalez-Correa et al., 2005) and ageing (Gonzalez-Correa et al., 2005).

 

Several investigations have been performed on the beneficial effects of medicinal plants (El-Karim et al., 2017; Hashem et al., 2020c). Panax ginseng is also a known plant with a strong antioxidant properties and a wide range of actions such as anti-aging, immune enhancing, anti-stress and anti-cancerous (Cheng et al., 2006). This has re-inforced it’s as a potential antioxidant supplement (Kim et al., 2011). Panax ginseng alleviates oxidative stress by scavenging of free radicals and induction of SOD and CAT activity, thus preventing lipid peroxidation (Chang et al., 1999). Also, it raises SOD, CAT, glutathione peroxidase, and GSH activities in the aged rat liver (Ramesh et al., 2012) and hepatotoxin-induced injury to rat’s liver (Abdul-Hamid et al., 2020).

 

The existing work was carried out to study the possible ameliorative effects of vitamin E and Panax ginseng on the adverse effects of levofloxacin.

 

Materials and methods

 

Drugs

Levofloxacin (TAVANIC®) tablets were obtained from Sanofi Aventis Company, Egypt. Each tablet contains 500 mg levofloxacin, dissolved in DW (distilled water) and taken by oral route in a dosage of 10 mg/kg b.wt. (Olayinka et al., 2015). Vitamin E (VITAMIN E®) capsules were obtained from Pharco Pharmaceuticals Company, Egypt. Each capsule contains 1000 mg vitamin E, dissolved in a vehicle, olive oil and orally administered (100 mg/kg b.wt.) as standard antioxidant according to Ambali et al. (2011). Panax ginseng (GINSANA®) capsules were obtained from EIPICO Pharmaceuticals Company, Egypt, dissolved in DW and administered orally at a dosage of 200 mg/kg b.wt. according to Esawe (2017). All medications were administered orally once a day for 14 consecutive days.

 

Ethical statements

The care and revival of the rats used was consistent with the strategies in Research Committee at Zagazig University, Egypt.

 

Experimental animals

Twenty five adult apparently healthy male albino rats (weighing 200±10g) were attained from the Laboratory House of Experimental Animal, Faculty of Veterinary Medicine, Zagazig University, Egypt. Rats were accustomed prior to the experiment for two weeks. In well ventilated plastic cages containing hard wood bedding and good aeration, the animals were housed at a temperature (25 ± 5°C) normal dark/light cycle of 12h, humidity (55±%). During the investigation, the rats were supplied with a known basal diet composition and water ad-libitum.

 

Experimental design

Rats were divided into five equal groups of 5 rats each; group 1 received 0.2 ml/kg b.wt. of distilled water (normal control group), group 2 received 0.2 ml/kg b.wt. of olive oil (vehicle control), group 3 received 10 mg/kg b.wt. of levofloxacin, group 4 received 100 ml/kg b.wt. of vitamin E and 10 mg/kg b.wt. of levofloxacin while group 5 received Panax ginseng (200 ml/kg b.wt.) and levofloxacin (10 mg/kg b.wt.). All doses were taken once a day via oral gavage for 14 successive days to all the groups. Vitamin E or Panax ginseng was provided 2 hours prior to the administration of levofloxacin.

 

Sampling

At the experiment termination, 5ml sample blood obtained from each rat using heparinized microhematocrite tubes through the retro-orbital venous plexus was separated into two portions (Hashem et al., 2018). The 1st part was put in tubes containing EDTA as anticoagulant for hematological studies using Hemascreen 18 Automatic Cell Counter (Hospitex Diagnostics, Sesto Fiorentino, Italy) and 2nd part into plain tubes without anticoagulant to obtain clear serum for biochemical analysis. Serum was analyzed for estimation of liver enzymes (AST, ALT and ALP), total proteins, albumin, globulin and albumin/globulin (A/G) ratio, lipids profile (cholesterol, TG, LDL, VLDL and HDL) and kidney function markers (urea and creatinine) utilizing a entirely automated analyzer for chemistry (New Delhi, India). In addition, serum antioxidants (SOD and CAT) and MDA as oxidative stress were determined on UV-Vis Spectrophotometer (Apple 303S, Japan), while TNF-α, proinflammatory marker, was measured by ELISA using ELISA kit Catalog number: MBS702888. All biochemical parameters were evaluated by means of viable test kits obtained from Biodiagnostic Co., Cairo, Egypt as instructed by the manufacturer.

 

Tissue samples were taken from the liver and kidneys of euthanized rats by manual cervical dislocation and fixed in neutral formalin of 10 %. The samples preserved with formalin are dehydrated and embedded in paraffin. Five- micron thick paraffin slices were set and tainted with haematoxyline and eosin (H& E) and inspected microscopically (Suvarna et al., 2018).

 

Statistical analysis

After obtaining the data, it was statistically analyzed by ANOVA using SPSS computer program (Tamhane and Dunlop, 2000) for windows version16.

 

Results

 

Hematological results

Erythrogram: Existing study showed significantly (P<0.05) reduction in erythrocyte counts, Hb concentration, PCV and PLT count in rats administered levofloxacin in related to control group. However, erythrocyte indices (MCV and MCHC) revealed non-significant alterations. Oral administration of vitamin E or Panax ginseng before administration of levofloxacin by 2hours (groups 4&5) produced a significant rise and improvement for the erythrocyte parameters and PLT count if matched with levofloxacin group (Table 1).

 

Leukogram: The present study revealed that levofloxacin elicited a significant (P < 0.05) intensification in WBCs, lymphocytes, neutrophils and monocytes counts in relation to group 1. Administration of vitamin E or Panax ginseng before levofloxacin by 2hours (groups 4&5) in male rats displayed a significant decreases in the aforementioned parameters in consideration with levofloxacin group (Table 2).

 

Serum biochemistry

Liver enzymes: The present investigation indicated that administration of levofloxacin in male rats displayed an elevation significantly (P < 0.05) in ALT, AST and ALP activities when compared with control group while administration of vitamin E or Panax ginseng with levofloxacin (groups 4&5) displayed a noticeable decrease in ALT, AST and ALP activities in comparison to levofloxacin group (Table 3).

 

Serum proteins: Oral administration of levofloxacin in male rats produced a significant (P<0.05) hypoproteinemia with a diminution in albumin, globulins and A/G ratio. These values were significantly improved after supplementation of vitamin E or Panax ginseng before levofloxacin by 2 hours (groups 4&5) compared to the levofloxacin group (Table 4).

 

Lipid profile: Levofloxacin given orally in male rats elicited a significant (P < 0.05) rise in all estimated lipid fractions except HDL levels which decreased when compared to control (group1). Vitamin E or Panax ginseng administration before levofloxacin by 2hours (groups 4&5) led to discernible falling in lipogram with a significant high HDL levels compared with levofloxacin group (Table 5).

 

Kidney function markers: Oral administration of levofloxacin in male rats produced a significant (P < 0.05) upsurge in urea and creatinine levels when compared to control (group1). However, supplementation of vitamin E or Panax ginseng before levofloxacin by 2hours (groups 4&5) in male rats displayed a statistical decline in urea and creatinine levels when compared to the levofloxacin group (Table 6).

 

Oxidant/Antioxidant status: Rats taken levofloxacin (group3) exhibited at probability <0.05 intensification in MDA concentration, with lessening in the activities of CAT and SOD related to control. Vitamin E- or Panax ginseng- levofloxacin supplementation (groups 4&5) displayed declines in MDA concentration with elevated CAT and SOD activities associated with levofloxacin group (Table 7).

 

Tumor necrosis factor-α: As illustrated in Table (8) levofloxacin exposure produced a significant rise in serum TNF-α in assessment with group (1). Oral administration of vitamin E or Panax ginseng before administration of levofloxacin by 2hours (groups 4&5) elicited a significant drop in TNF-α matching with 3rd group. Non-significant variation in TNF-α were recorded post administration of Panax ginseng and levofloxacin (group5).

 

Histopathological results

Sections from liver of control (group1) exhibited typical liver parenchyma, such as normal hepatocytes and sinusoids morphology, as well as usual hepatic lobules and ducts (Figure 1 A&B). Kidney sections (group1) revealed normal Bowman’s capsules, glomeruli and tubules structure (Figure 1 B&C).

 

Liver sections from rats given olive oil (group 2) showed ordinary hepatic structure with preserved lobular pattern, vascular tree, kupffur cells and stromal component (Figure 2 A&B). Kidney sections (group2) revealed normal

 

Table 1: Effect of oral administration of vitamin E (100 mg/kg b.wt.) or Panax ginseng (200 mg/kg b.wt) on erythrogram of rats exposed to levofloxacin (10 mg/kg b.wt.) for 14 successive days. (Mean ± SE) (n = 5)

 

Groups	Erythrogram
	RBCs (x106 /µl)	Hb (g/dl)	PCV (%)	MCV (Fl)	MCHC (pg)	PLT (x103 /µl)
Control	8.07a ± 0.12	15.10a 0.31	56.66a 1.20	70.23a ± 0.69	28.41a ± 0.35	336.00a ± 8.19
Olive oil	8.00a 0.03	15.56a 0.34	54.66a 1.20	68.32a ± 1.2	28.47a ± 0.39	331.00a ± 15.09
Levofloxacin	4.91d ± 0.05	9.67d ± 0.36	34.75d ± 1.11	70.68a ± 1.56	27.98a ± 0.57	258.75c ± 10.63
Vitamin E+ Levofloxacin	6.89b 0.04	13.62b 0.17	46.50b 1.65	67.49a ± 1.15	24.31a ± 0.59	282.50b ±10.91
Panax ginseng + Levofloxacin	5.71c 0.04	11.40c 0.18	40.75c 0.85	71.35a ± 1.34	28.00a ± 0.63	293.25b ± 5.85

Means within the same column carrying different superscript letters are significant at P < 0.05

RBCs: Red blood cells count, Hb: Hemoglobin, PCV: packed cell volume, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, PLT: platelet count

 

Table 2: Effect of oral administration of vitamin E (100 mg/kg b.wt.) or Panax ginseng (200 mg/kg b.wt.) on leukogram of rats exposed to levofloxacin (10 mg/kg b.wt.) for 14 successive days. (Mean ± SE) (n = 5)

 

Groups	Leukogram
	WBCs (x103/µl)	Lymphocytes (x103 /µl)	Neutrophils ( x103 /µl)	Eosinophils ( x103 /µl)	Monocytes ( x103 /µl)
Control	11.08d ± 0.24	8.59c ± 0.24	1.34d ± 0.05	0.44a ± 0.018	0.72d ± 0.02
Olive oil	11.01d ± 0.03	8.51c ± 0.49	1.35d ± 0.03	0.44a ± 0.03	0.71d ± 0.03
Levofloxacin	18.49a ± 0.59	11.89a ± 0.48	4.44a ± 0.20	0.42a ± 0.07	1.75a ± 0.09
Vitamin E+Levofloxacin	13.33c ± 0.45	8.47b ± 0.39	2.51c ± 0.22	0.44a ± 0.03	0.92c ± 0.01
Panax ginseng +Levofloxacin	15.16b ± 0.22	9.37b ± 0.22	3.29b ± 0.28	0.42a 0.05	1.09b ± 0.02

Means within the same column carrying different superscript letters are significant at P < 0.05

WBCs: White blood cells count

 

Table 3: Effect of oral administration of vitamin E (100 mg/kg b.wt.) or Panax ginseng (200 mg/kg b.wt.) on serum liver enzymes activity of rats exposed to levofloxacin (10 mg/kg b.wt.) for 14 successive days. (Mean ± SE) (n = 5)

 

Groups	Liver enzymes
	ALT (U/l)	AST (U/l)	ALP (U/l)
Control	13.67d ± 1.20	14.67d ± 0.88	72.10d ± 0.05
Olive oil	13.00d ± 1.54	14.00d ± 1.73	71.87d ± 1.47
Levofloxacin	69.25a ± 2.14	57.50a ± 1.55	147.22a ± 2.25
Vitamin E + Levofloxacin	40.00c ± 1.29	34.50c ± 2.63	99.95c 1.8268
Panax ginseng + Levofloxacin	57.00b ± 1.58	43.75b ± 1.38	127.65b ± 2.62

Means within the same column carrying different superscript letters are significant at P < 0.05.

ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, ALP: Alkaline phosphatase.

 

Table 4: Effect of oral administration of vitamin E (100 mg/kg b.wt.) or Panax ginseng (200 mg/kg b.wt.) on serum proteins profile of rats exposed to levofloxacin (10 mg/kg b.wt.) for 14 successive days. (Mean ± SE) (n = 5)

 

Groups	Serum proteins
	Total Proteins (g/dl)	Albumin (g/dl)	Globulins (g/dl)	A/G ratio
Control	7.30a ± 0.11	5.15a ± 0.06	2.15a ± 0.09	2.39ab ± 0.12
Olive oil	7.18a ± 0.09	5.19a ± 0.16	1.99a ± 0.25	2.72a 0.47
Levofloxacin	5.19d ± 0.09	2.97d ± 0.11	2.23a 0.11	1.34c ± 0.11
Vitamin E+ Levofloxacin	6.36b ± 0.07	4.19b ± 0.05	2.16a 0.09	1.96bc ± 0.11
Panax ginseng + Levofloxacin	6.01c ±0.05	3.52c ± 0.16	2.15a ±0.19	1.787bc ± 0.22

 

Means within the same column carrying different superscript letters are significant at P < 0.05

A/G ratio: Albumin/ globulin ratio

 

Table 5: Effect of oral administration of vitamin E (100 mg/kg b.wt.) or Panax ginseng (200 mg/kg b.wt.) on serum lipids profile of rats exposed to levofloxacin (10 mg/kg b.wt.) for 14 successive days. (Mean ± SE) (n = 5)

 

Groups	Lipid profile
	Total cholesterol (mg/dl)	Triglycerides (mg/dl)	HDL (mg/dl)	LDL (mg/dl)	VLDL (mg/dl)
Control	92.00d ± 1.53	146.33d ± 4.05	68.33a ± 4.91	54.21d ± 4.81	29.27d ± 0.82
Olive oil	93.00d ± 2.08	147.00d ± 2.65	66.67a ± 3.76	56.91d ± 5.37	24.40d ± 0.53
Levofloxacin	193.75a ± 1.49	259.25a ± 3.22	23.500d ± 1.443	239.90a ± 1.27	51.85a ± 0.64
Vitamin E+ Levofloxacin	139.25c ± 2.29	199.50c ± 4.41	45.500b 1.5545	142.65c ± 2.75	39.90c ± 0.88
Panax ginseng + Levofloxacin	164.75b ± 2.14	236.00b ± 2.27	33.250c ± 1.7017	194.43b ± 3.93	47.20b ± 0.45

Means within the same column carrying different superscript letters are significant at P < 0.05

HDL: High density lipoprotein, LDL: Low density lipoprotein, VLDL: Very low density lipoprotein.

 

Table 6: Effect of oral administration of vitamin E (100 mg/kg b.wt.) or Panax ginseng (200 mg/kg b.wt.) on serum kidney function parameters of rats exposed to levofloxacin (10 mg/kg b.wt.) for 14 successive days. (Mean ± SE) (n = 5)

 

Groups	Kidney functions
	Urea (mg/dl)	Creatinine (mg/dl)
Control	27.47d± 0.99	0.79d± 0.03
Olive oil	27.33d± 0.49	0.79d± 0.02
Levofloxacin	72.75a± 1.15	2.03a± 0.05
Vitamin E + Levofloxacin	39.85c± 1.27	1.01c± 0.04
Panax ginseng + Levofloxacin	51.03b± 0.52	1.23b± 0.03

Means within the same column carrying different superscript letters are significant at P < 0.05

TNF-α: Tumor necrosis factor-alpha.

 

Table 7: Effect of oral administration of vitamin E (100 mg/kg b.wt.) or Panax ginseng (200 mg/kg b.wt.) on oxidant/antioxidant status of rats exposed to levofloxacin (10 mg/kg b.wt.) for 14 successive days. (Mean ± SE) (n = 5)

 

Groups	Oxidant/antioxidant status
	MDA (mmol/ml)	CAT (u/l)	SOD (u/ml)
Control	11.47d ± 0.49	501.32a ± 9.43	3.84a ± 0.08
Olive oil	12.43d ± 0.33	506.25a ± 7.29	3.82a ± 0.08
Levofloxacin	73.75a ± 1.59	311.09c ± 4.08	1.01d ± 0.04
Vitamin E + Levofloxacin	35.93c ± 1.83	410.75b ± 6.79	1.48b ± 0.03
Panax ginseng + Levofloxacin	56.24b ± 1.61	362.62c ± 3.95	1.42c ± 0.05

Means within the same column carrying different superscript letters are significant at P < 0.05.

MDA: Malondialdehyde, CAT: Catalase, SOD: Superoxide dismutase.

 

nephron units with preserved glomerular and tubular structures (Figure 2 B&C).

 

Exposure to levofloxacin (group3) produced slight to reasonable congestion of portal veins, central veins and hepatic sinusoids. Moderate biliary proliferation and round aggregations of cells have been seen. Some hepatocytes showed degenerative changes mainly vacuolations and few

 

Table 8: Effect of oral administration of vitamin E (100 mg/kg b.wt.) or Panax ginseng (200 mg/kg b.wt.) on serum TNF-α of rats exposed to levofloxacin (10 mg/kg b.wt.) for 14 successive days. (Mean ± S.E) (n = 5)

 

Groups	TNF-α (pg/ml )
Control	19.23d± 0.90
Olive oil	20.03d± 0.55
Levofloxacin	90.80a± 1.45
Vitamin E + Levofloxacin	51.30c± 1.01
Panax ginseng + Levofloxacin	71.80b± 0.63

Means within the same column carrying different superscript letters are significant at P < 0.05. TNF-α: Tumor necrosis factor-alpha.

 

 

 

 

apoptotic cells (Figure 3 A&B). Kidney sections (group3) revealed renal lesions such as interstitial, perivascular and periglomerular lymphocytic aggregations, with dilated renal tubules in both cortex and medulla with hyaline casts (Figure 3 C-H).

 

Liver sections from rats administered vitamin E before the administration of levofloxacin by 2 hours (group4) revealed an enhancing vitamin E result which appeared as discount in number of lymphocytic aggregation in the portal area. Most of hepatocytes appeared normal (Figure 4 A- E). Kidney sections (group4) showed apparently normal renal tubules with few renal tubular epithelial cells showing vacuolar degeneration (Figure 4 F- H).

 

 

Liver sections from rats administered Panax ginseng before the administration of levofloxacin by 2 hours (group5) revealed apparently normal hepatocytes with some binucleated hepatocytes, beside mild lymphocytic inflammatory reaction (Figure 5 A&B). Kidney sections (group5) showed mildly congested renal blood vessels and normal medulla. Some renal tubules in both cortex and medulla were dilated (Figure 5 C&D).

 

 

Discussion

 

Levofloxacin is a flouroquinolone antibiotic of 3rd generation and an optical S-(-) isomer of the racemic drug substance ofloxacin. It has a wide range of action against G+ve and G-ve bacteria, as well as other pathogens, for example Mycoplasma, Legionella, Chlamydia, and Mycobacteria species (Riahifard et al., 2017).

 

The existing work revealed that levofloxacin exposure for 14 successive days in male rats caused leukocytosis as seen in the significant increase in WBCs, lymphocytes, neutrophils and monocytes counts with associated anemia due to diminution in values of RBCs, Hb and percentage levels of PCV, with decreased PLT count (thrombocytopenia) matching with control (group1). This alteration in the erythrocytic parameters may be due to inhibitory influence of levofloxacin on the erythropoiesis in bone marrow (Hashem et al., 2020a). The observed thrombocytopenia may be attributed to either reduced bone-marrow depltion platelet production or increased drug-mediated destruction (Shih et al., 2018). In particular, levofloxacin may have minor blood clotting defects, which have reduced the platelets number in dogs during the treatment course (Psarros et al., 2014). The results of leukogram may be due to some drugs as levofloxacin specifically stimulate truthful autoantibodies production as those seen in autoimmune hemolytic anemia with high formation of lymphocytes (Psarros et al., 2014). The increases in neutrophil and monocyte counts are as a defense mechanism against inflammation (Weiss and Wardrop, 2011). Similar observations were reported following oral administration of levofloxacin in rabbits (Khan et al., 2017) or in male rats (Tohamy, 2017).

 

Oral administration of vitamin E or Panax ginseng before levofloxacin by 2 hours (groups 4&5) for 14 successive days ameliorated these adverse effects caused by levofloxacin as observed in the noticeable elevation in erythrocyte count, Hb content, and PCV % and PLT count compared to levofloxacin- exposed rats (group 1). The enhanced erythrogram and leukogram post-administration of Panax ginseng in rats treated with levofloxacin may be endorsed to the Panax ginseng anti-oxidants activity (Kim et al., 2011). In addition, vitamin E has contributed to increase in the number of erythroid precursor units forming colonies, preventing polyunsaturated fatty acid oxidation in the erythrocyte membrane, inhibiting premature erythrocyte lysis and improving erythropoiesis. Vitamin E can thus enhance post-supplemental Hb content and percentage levels of PCV (Jilani and Iqbal, 2011). The obtained results were supported by earlier reports (Mahran, 2013; Tohamy, 2017), in which vitamin E supplementation with levofloxacin in rats resulted in a significant increase in total erythrocyte counts, Hb content and PCV % and insignificant drop in TLC is counted on the 1st and 10th days after administration of the drug and restored to almost normal levels on the 20th day following administration.

 

A significant increase was detected in the activities of ALT, AST and ALP enzymes after administration of levofloxacin in male rats for 14 successive days. The elevation in the enzymatic activities may be related to hepatic malfunction (Ara et al., 2020) or due to increased breakdown of hepatocytes (Hashem et al., 2020a,b). The pathological changes, round cells aggregation with degenerative changes in hepatocytes, in the liver have long-established this results. Our findings was clearly supported by Olayinka et al. (2015) and Tohamy (2017) who mentioned that oral administration of levofloxacin (7.5 mg/kg b.wt.) in rats displayed rise in enzymes of the liver on the 1st and 10th days and return nearly to normal levels on 20th day post administration in parallel to control (group1). Moreover, Farid and Hegazy (2019) stated that levofloxacin- administered rats for 2 weeks (40 mg / kg b. wt.) daily revealed hepatic dysfunction with increased in AST &ALT activities. The current study indicated that co-administration of vitamin E or Panax ginseng with levofloxacin in male rats declined the previous results for enzymes in consistent with the levofloxacin group. Our results come to an agreement with findings of Liu et al. (2018) who reported that administration of Panax ginseng in alcohol treated rats improved liver functions (ameliorated the elevated AST, ALT and ALP activities), and Hashem et al. (2019) who exposed that dosing antioxidants like vit. E to diet with cadmium significantly restored the activities of serum AST, ALT to normal values. Similar results were recorded by George and Adegoke (2011) who found that vitamin E displayed a reduction in liver transaminases, GGT &ALP activity in albino rats, and Tohamy (2017) who stated the same findings after administration of vitamin E in male rats. The obtained results in vitamin E or Panax ginseng- supplemented rats were supported by histopathological findings.

 

In this experiment, oral administration of levofloxacin in male albino rats produced a hypoproteinemia, hypoalbuminemia, and reduction in the ratio of A/G, with insignificant globulins changes compared to the normal control. The hypoproteinemia and hypoalbuminemia may be attributed to liver and kidney damage caused by high dose of levofloxacin. Moreover, hypoalbuminemia may be a result of decreased production of albumin from damaged liver or increased loss of albumin via from damaged kidneys (Braun and Lefebvre, 2008). The increased A/G ratio in this study is due to hypoalbuminaemia caused by levofloxacin - exposure with normal total globulins level. Liver disease is the cause of reduction in plasma albumin, α- and ß-globulins with a rise in the level of γ–globulins (David Eckersall, 2008), this explains the unchanged total globulins levels as end result in rats with hepatic damage. The results in proteinogram were significantly improved after supplementation of vitamin E or Panax ginseng matched to the levofloxacin group. These results confirm the hepatoprotective role of vitamin E or Panax ginseng against levofloxacin- induced liver damage. Similar findings were reported by Abdelfattah-Hassan et al. (2019).

 

Levofloxacin administration causes dyslipidemia evidenced by numerical augmentation in all measured lipid profile in this work, with drop in HDL comparatively with control group. This may be attributed to liver damage and nephrotoxic effect induced by levofloxacin (Oda et al., 2014). Moreover, the alterations in lipids profile indicate lipoproteins and lipids metabolism disorders and might contribute to the development of phospholipidosis and cholesterogenesis in tissues which might represent additional adverse effects of levofloxacin (Owoade et al., 2018). Our results are consistent with Olayinka et al. (2015), described a numerical rise in serum lipogram with levofloxacin administration.

 

The current study revealed co-administration of vitamin E or Panax ginseng with levofloxacin resulted in alleviation in lipid profile compared to administration of levofloxacin alone. This can be due to a decline in the damaging effects of ROS and nitrogen molecules produced by levofloxacin administration (Talla and Veerareddy, 2011).

 

Serum urea and creatinine levels are widely used as markers for renal function screening as they are the most sensitive parameters in diagnosis of renal disease (Ferguson and Waikar, 2012). Levofloxacin administartion produced a remarkable upsurge in urea and creatinine levels. The increase in estimated renal bio-markers might be attributed to a reduction in the filtration rate of glomeruli or subordinate to the increase of ROS induced-renal damage and decreased renal excretory function (Noori and Mahboob, 2010). Improved protein catabolism along with accelerated gluconeogenesis amino acid deamination is possibly an appropriate propose for the understanding of elevated urea levels (Hashem and El-Sharkawy, 2009). The obtained outcomes correspond to those obtained by Mouton and Holder (2006), Afolabi and Oyewo (2014) who stated that levofloxacin produces ROS that can cause oxidative stress, with liver and kidney cellular damage. Our findings were supported by the pathological examination kidneys sections of rats exposed to levofloxacin only, which showed interstitial lymphocytic aggregations, perivascular edema with mild hemorrhages as well as, dilated renal tubules with intratubular hyaline cast. Similar findings were obtained by Oda et al. (2014) who reported that levofloxacin treated rabbits showed diffuse lymphocytic interstitial nephritis, tubular necrosis and some hyalinized and necrotic tubules. Moreover, Ara et al. (2020) showed that the administration of levofloxacin in mice induced renal damage, as evidenced by glomerulonephritis, degenerations of epithelium and tubules degeneration with tubule dilation. However, co-administration of vitamin E or Panax ginseng with levofloxacin displayed diminution in kidney function parameters in correlated to the group treated with levofloxacin only. The same result was also obtained by George and Adegoke (2011) who reported that rats fed diet containing vitamin E showed reduction in urea and creatinine levels. The reduction in kidney function tests after supplementation of vitamin E with levofloxacin was due to its antioxidant effect, the scavenging activity of free radicals, and the defense of protein thiols from the deleterious effect of levofloxacin in the kidney (Hashem et al., 2019). While Panax ginseng containing phenolic acids, flavonoids and saponins are the cause of improvement in the function of kidney through dissimilar antioxidant characteristics corresponding to scavenging action of free radical or oxidized product formation inhibition (Kalkan et al., 2012).

 

There was also a significant rise in MDA concentrations with a significant decline in CAT & SOD enzymes in rats administered levofloxacin alone in this study, indicative of induction of oxidative stress by the drug. A biomarker of tissue damage has been identified as an elevation in the MDA level (Gutteridge, 1995). Antioxidant enzymes are inactivated by MDA as one of lipid peroxidation products resulting in augmented ROS addition and worsening macromolecular damage (Noeman et al., 2011). The depletion of antioxidant enzymes such as SOD and CAT was attributed to levofloxacin-induced oxidative cell damage as a consequence of the production of reactive free radicals that overpower the antioxidant protection (Talla and Veerareddy, 2011). Our findings were consistent with previous results in levofloxacin- exposed rabbits (Khan et al., 2017) or immature albino rats (Abd Elfadil et al., 2019). Moreover, the observed data in this study corroborates with the work of Farid and Hegazy (2019) who reported that levofloxacin administration in rats displayed oxidative- stress represented by lessening in antioxidant enzymes (CAT & SOD) with high MDA concentrations.

 

Co-administration of vitamin E or Panax ginseng with levofloxacin ameliorated this effect as seen in the decline in MDA concentrations with a concurrent upsurge in CAT and SOD enzyme activities comparing to the levofloxacin group. This suggests the ability of the antioxidant constituents of vitamin E or plants (Panax ginseng) to break lipoperoxidation chain reaction and facilitate the removal of reactive oxygen species generated by levofloxacin over dose (Hashem et al., 2015). The improved antioxidant enzymes (SOD and CAT) activity induced by vitamin E could be validated to the well-established potential or capacity of vitamin E as antioxidant (Helen et al., 2000), as it inhibits toxic effect to the reactive membrane lipids by preventing creation of hydroperoxide. It is reported that vitamin E- selenium administration is recorded to protect cell membranes and organelles-containing lipids from peroxidative destruction (Gupta et al., 2005; Hashem et al., 2019). Administration of Panax ginseng (3g for 12 weeks) was also reported to have produced intensification in SOD level, with a drop in MDA concentration (Seo et al., 2014). Analogous outcomes reported that administration of Panax ginseng in alcohol- treated rats caused statistical elevation in SOD activity and lessening in MDA concentration (Liu et al., 2018).

 

The present investigation indicated that oral administration of levofloxacin in rats produced a numerical upsurge of TNF-α in the serum in comparison to control group. Tumor necrosis factor is a potent pro- inflammatory cytokine that is high in systemic and local inflammation and is considered essential for the recruitment of neutrophils (Diez-Pina et al., 2009). Similar results were also previously reported (Sharma et al., 2015). Fischer and Marier (2015) found a strong association between the expression of cytokines like TNF-α and oxidative stress in rats. This study may explain the significant reduction in TNF-α level in groups which were treated with vitamin E or Panax ginseng 2 hours before levofloxacin when compared to levofloxacin administration only, as they had powerful antioxidant properties which strongly counteracted any adverse effect produced by oxidative stress such as elevation of TNF-α level in levofloxacin group. Vitamin E significantly decreased serum TNF-α level equated to rats group- treated with vancomycin only in the exploration of Blesa et al. (2003) and attributed that to its direct antioxidant effect, additionally it can protect indirectly via decreasing neutrophil recruitment.

 

Conclusion

 

The results obtained from this work showed that levofloxacin administration induced hematological and biochemical alterations which were ameliorated by the vitamin E and Panax ginseng. The Panax ginseng administration exhibited superior antioxidant and antihepato-renal toxic activities compared with vitamin E, that may be attributed to its’ radical scavenging activity.

 

Conflict of interest

 

The authors pronounce that they have no conflict of interest.

 

Availability of data and materials

All author stated that all information produced or analyzed during this work is included.

 

Funding

 

This study does not have a funding source.

 

Acknowledgments

 

The authors are grateful to all stuff members and workers of Pharmacology Department, Faculty of Veterinary Medicine, Zagazig University, for their valuable assistance during this work.

 

authors contribution

 

All authors contributed equally.

 

References 

 

• Abd Elfadil H, Mohamed A, Behairy A, Ghany H, Amaym E (2019). Protective role of curcumin, glucosamine and vitamin c on oxidative stress, biochemical and histopathological alterations induced by levofloxacin. Alex J. Vet. Sc. 62:182. https://doi.org/10.5455/ajvs.51618

• Abdel-Alim A, Kamel M, Elsayed R (2017). Some pharmacological studies of ciprofloxacin and levofloxacin in rats. Zag Vet. J. 45:172-180.

• Abdelfattah-Hassan A, Shalaby SI, Khater SI, El-Shetry ES, Abd El Fadil H, Elsayed SA (2019). Panax ginseng is superior to vitamin E as a hepatoprotector against cyclophosphamide-induced liver damage. Complement Ther. Med. 46:95-102. https://doi.org/10.1016/j.ctim.2019.08.005

• Abdul-Hamid M, Mohamed HM, Abd El-Twab SM, Zaied K (2020). Histological, ultrastructural, and biochemical study on the possible role of Panax ginseng in ameliorating liver injury induced by Lambda cyhalotherin Beni-Suef University. J. Bas. App. Sc. 9:52. https://doi.org/10.1186/s43088-020-00076-6

• Afolabi DK, Oyewo EB (2014). Effects of ciprofloxacin and levofloxacin administration on some oxidative stress markers in the rat. Inter. J. Bio. Vet. Life Sc. Eng. 8(1): 38-42. https://doi.org/10.5281/zenodo.1090856

• Al-Soufi WF, Al-Rekabi FMK (2019). The cytogenetic effects of levofloxacin in male rats. Adv. Anim. Vet. Sci.  7(3): 138-150. https://doi.org/10.17582/journal.aavs/2019/7.3.138.150

• Ambali SF, Akanbi DO, Oladipo OO, Yaqub LS, Kawu MU (2011). Subchronic chlorpyrifos-induced clinical, hematological and biochemical changes in swiss albino mice: Protective effect of Vitamin E. Int. J. Biol. Med. Res. 2(2): 497-503.

• Ara C, Asmatullah A, Kanwal S, Chaudhary A, Siddiqua A (2020). Haematological and Histopathological Analyses of Levofloxacin Induced Toxicity in Mammals. Punjab Univ. J. Zool., 35(1): 01-06. https://doi.org/10.17582/journal.pujz/2020.35.1.01.06

• Bharrhan S, Chopra K, Rishi P (2010). Vitamin E supplementation modulates endotoxin-induced liver damage in a rat model. Am. J. Biom. Sci. 2(1): 51-62.

• Blesa S, Cortijo J, Mata M, Serrano A, Closa D, Santagelo F, Morcillo EJ (2003). Oral N-acetylcysteine attenuates the rat pulmonary inflammatory response to antigen. Eur. Resp. J. 21(3), 94-400. https://doi: 10.1183/09031936.03.00039602.

• Braun J-P, Lefebvre HP (2008). Chapter 16 - Kidney Function and Damage A2 - Kaneko, J. Jerry. In: Harvey JW, Bruss ML (eds) Clinical Biochemistry of Domestic Animals (Sixth Edition). Academic Press, San Diego, pp 485-528.

• Chang, MS Lee, SG Rho, HM (1999). Transcriptional activation of Cu/Zn superoxide dismutase and catalase genes by panaxadiol ginsenosides extracted from Panax ginseng. Phyt Res: PTR, 13(8): 641-644. https://doi: 10.1002/(sici)1099-1573(199912)13:8<641::aid-ptr527>3.0.co;2-z.

• Cheng, LQ Kim, MK Lee, JW Lee, YJ Yang, DC (2006). Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia. Biotechnol. Lett., 28 (14): 1121-1127. https://doi.org/10.1007/s10529-006-9059-x

• David Eckersall P (2008). Chapter 5 - Proteins, Proteomics, and the Dysproteinemias A2 - Kaneko, J. Jerry. In: Harvey JW, Bruss ML (eds) Clinical Biochemistry of Domestic Animals (Sixth Edition). Academic Press, San Diego, pp 117-155.

• Diez-Pina JM, Fernandez-Aceñero MJ, Llorente-Alonso MJ, Diaz-Lobato S, Mayoralas S, Florez A (2009).Tumor necrosis factor alpha as a marker of systemic and local inflammation in “healthy” smokers. Int. J. Gen. Med., 2:9-14. https://doi.org/10.2147/IJGM.S4723

• El-Karim DRG, Oda SS, Tohamy HG, Hashem MA (2017): The Effect of Metformin and Ginseng on Alloxan-Induced Diabetic Rats: Hematological, Biochemical and Histopathological Studies. Alex J. Vet. Sc. 55: 60-66.

• Esawe EEA (2017). Ameliorative effect of Ginseng and Vitamin E on Male Fertility in Cyclophosphamide Treated Rats. Master thesis, Pharm Dept., Fac. Vet. Med., Zagazig Univ, Egypt.

• Eyrisofla N, Ahmadifar M, Eini A-M, Kalami A (2015). The Study of Levofloxacin Effects on Liver Tissue in Wistar Rat.. J. Liver 04.

• Farid AS, Hegazy AM (2019). Ameliorative effects of Moringa oleifera leaf extract on levofloxacin-induced hepatic toxicity in rats. Drug Chem. Toxicol. 1-7. https://doi.org/10.1080/01480545.2019.1574811

• Ferguson MA, Waikar SS (2012). Established and Emerging Markers of Kidney Function. Clin. Chem. 58 (4) 680-689. https://doi.org/10.1373/clinchem.2011.167494

• Fischer R, Marier O (2015). Interrelation of Oxidative Stress and Inflammation in Neurodegenerative Disease: Role of TNF. OxideMed Cell Longev. 610813. https://doi.org/10.1155/2015/610813

• George MI, Adegoke OA (2011). Effect of vitamin e on biochemical parameters in albino rats treated with gasoline. J. Sci Res., 4(2)37-39. https://doi.org/10.3329/jsr.v4i2.7670

• Gonzalez-Correa J, Arrebola M, Guerrero AT, Muñoz-Marín J, Ruiz-Villafranca D, Cuesta F, De La Cruz JP (2005). Influence of vitamin E on the antiplatelet effect of acetylsalicylic acid in human blood. Platelets. 16:171-179. https://doi.org/10.1080/09537100400016797

• Gupta S, Gupta HK, Soni J (2005). Effect of Vitamin E and selenium supplementation on concentrations of plasma cortisol and erythrocyte lipid peroxides and the incidence of retained fetal membranes in crossbred dairy cattle. Theriogeno. 64(6): 1273-86. https://doi: 10.1016/j.theriogenology.2005.03.008.

• Gupta YK, Briyal S, Sharma M (2009). Protective effect of curcumin kainic acid induced seizures and oxidative stress in rats. Ind. J. Physio. Pharma., 5 (1): 39-46.

• Gutteridge JM (1995). Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 41:1819-1828.

• Hashem M A, El-Mandrawy SA, El-Diasty MM, Zidan AZ (2020b). Hematological, Biochemical and Immunological Studies on Brucellosis in Cows and Ewes in Dakahlia and Damietta Governorates, Egypt. Zag Vet. J. 48(1): 23-35. 

• Hashem M A, Gamal El-Din I M, Eltahawy S NA (2019). Clinicopathological Studies on the Ameliorative Effects of Selenium and Vitamin E against Cadmium Toxicity in Chickens. Zag Vet. J. 47(3):277-287.

• Hashem M A, Mahmoud EA, Abd-Allah NA (2020a). Alterations in hematologic and biochemical parameters and DNA status in mice bearing Ehrlich ascites carcinoma cells and treated with cisplatin and cyclophosphamide. Comp. Clin. Path. 29: 517–524. https://doi.org/10.1007/s00580-019-03089-5

• Hashem MA, El-Sharkawy NI (2009). The effects of low electromagnetic field and lead acetate combination on some hemato-biochemical and immunotoxicological parameters in mice. Turk. J. Haematol., 26(4):181-9.

• Hashem MA, Mahmoud EA, Abd El-Rahman GI (2015). Evaluation of immunostimulatory and antioxidant activities of ginger and moringa extracts against paracetamol- induced hepatic damage in rats. SCVMJ. 20(2): 303-316.

• Hashem MA, Mohamed WA M, Attia ESM (2018). Assessment of protective potential of Nigella sativa oil against carbendazim- and/or mancozeb-induced hematotoxicity, hepatotoxicity, and genotoxicity. Environ. Sci. Pollut. Res., 25:1270–1282. https://doi.org/10.1007/s11356-017-0542-9

• Hashem MA, Neamat-Allah ANF, Hammza HEE, Abou-Elnaga HM (2020c). Impact of dietary supplementation with Echinacea purpurea on growth performance, immunological, biochemical, and pathological findings in broiler chickens infected by pathogenic E. coli. J. Anim. Health Prod. 52:1599-1607. https://doi.org/10.1007/s11250-019-02162-z

• Helen A, Krishnakumar K, Augusti K (2000). Antioxidant effect of onion oil on damages induced by nicotine in rats as compared to alpha-tocopherol. Toxico. Lett., 116(1-2): 61-68. https://doi.org/10.1016/S0378-4274(00)00208-3

• Jilani T, Iqbal M (2011). Does vitamin E have a role in treatment and prevention of Anemia’s? Pak. J. Pharm. Sci., 24:237-242.

• Kalkan Y, Kapakin KA, Kara A, Atabay T, Karadeniz A, Simsek N, Karakus E, Can I, Yildirim S, Ozkanlar S, Sengul E (2012). Protective effect of Panax ginseng against serum biochemical changes and apoptosis in kidney of rats treated with gentamicin sulphate. J. Mol. Histol.; 43(5):603-13. https://doi.org/10.1007/s10735-012-9412-4

• Keskes-Ammar L, Feki-Chakroun N, Rebai T, Sahnoun Z, Ghozzi H, Hammami S, Zghal K, Fki H, Damak J, Bahloul A (2003). Sperm oxidative stress and the effect of an oral vitamin E and selenium supplement on semen quality in infertile men. Arch. Androl. 49(2):83-94. https://doi.org/10.1080/01485010390129269

• Khan AM, Rampal S, Sood NK (2017). Effect of repeated oral administration of levofloxacin, enrofloxacin, and meloxicam on antioxidant parameters and lipid peroxidation in rabbits. Hum. Exp. Toxicol. 36:42-50. https://doi.org/10.1177%2F0960327116637111

• Kim H J, Lee S G, Chae I G, Kim M J, Im N K, Yu M H, Lee E J, Lee I S (2011). Antioxidant Effects of Fermented Red Ginseng Extracts in Streptozotocin-Induced Diabetic Rats. J. Ginseng. Res. 35: 129-137. https://doi.org/10.5142/jgr.2011.35.2.129

• Kurutas EB (2016). The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutr. J., 15: 71. https://doi.org/10.1186/s12937-016-0186-5

• Lee IM et al. (2005). Vitamin E in the primary prevention of cardiovascular disease and cancer: the Women’s Health Study: a randomized controlled trial. JAMA. 294:56-65. https://doi.org/10.1001/jama.294.1.56

• Liu R, Chen Q-H, Ren J-W, Sun B, Cai X-X, Li D, Mao R-X, Wu X, Li Y (2018). Ginseng (Panax ginseng Meyer) Oligopeptides Protect Against Binge Drinking-Induced Liver Damage through Inhibiting Oxidative Stress and Inflammation in Rats. Nutrients. 10(11): 1665. https://doi.org/10.3390/nu10111665

• Mahran KM (2013). Clinicopathological Studies on Endosulfan-Induced Oxidative Stress and the Protective Role of Vitamin E. Global Vet., 11 (2): 258-265. 

• Mocchegiani E, Costarelli L, Giacconi R, Malavolta M, Basso A, Piacenza F, Ostan R, Cevenini E, Gonos ES, Franceschi C, Monti D (2014). Vitamin E-gene interactions in aging and inflammatory age-related diseases: implications for treatment. A systematic review. Ageing Res Rev.14:81-101. . https://doi.org/10.1016/j.arr.2014.01.001

• Morikawa M, Hirata Y, Imanaka T (2000). A study on the structure-function relationship of lipopeptide biosurfactants. Biochimica et Biophysica. Acta. 1488 (3): 211-218. https://doi.org/10.1016/S1388-1981(00)00124-4

• Mouton R, Holder K (2006). Laboratory tests of renal function, Anesthesia and Intensive Care Medicine, 7(7): 240-243. https://doi.org/10.1053/j.mpaic.2006.04.003   

• Muller DP (2010). Vitamin E and neurological function. Mol Nutr. Food Res. 54(5):710-718. https://doi.org/10.1002/mnfr.200900460

• Noeman SA, Hamooda HE, Baalash AA (2011). Biochemical study of oxidative stress markers in the liver, kidney and heart of high fat diet induced obesity in rats. Diabetol. Metab. Syndr. 3(1):17. https://doi.org/10.1186/1758-5996-3-17

• Noori S, Mahboob T (2010). Antioxidant effect of carnosine pretreatment on cisplatin-induced renal oxidative stress in rats. Ind. J. Clin. Biochem. 25: 86-91. https://doi.org/10.1007/s12291-010-0018-x

• Oda SS, Hashem MA, El-Karim DRG (2014). A comparative study of levofloxacin- and gentamicin-induced nephrotoxicity in rabbits. Glob Vet. 13 (5): 898-905.

• Olayinka ET, Ore A, Ola OS (2015). Influence of different doses of levofloxacin on antioxidant defense systems and markers of renal and hepatic dysfunctions in rats. Adv. Toxic. 20: 1-7. https://doi.org/10.1155/2015/385023

• Owens RC, Jr., Ambrose PG (2005). Antimicrobial safety: focus on fluoroquinolones. Clin. Inf. Dis. 41 Suppl. 2:S144-157. https://doi.org/10.1086/428055

• Owoade O, Airaodion A, Adetutu A, Akinyomi O (2018). Levofloxacin induced dyslipidemia in male albino rats. Asi. J. Phar. Phar. 4: 620-629.

• Pavithra D, Devanandan P, Chowdary P R, Muthukumar VA (2018). A review on role of Vitamin E supplementation in type 2 diabetes mellitus. Drug Invent. Today. 10.

• Psarros T, Trammell T, Morrill K, Giller C, Morgan H, Allen B (2004). Abnormal coagulation studies associated with levofloxacin. Report of three cases. J. Neur. 100:710712.https://doi.org/10.3171/jns.2004.100.4.0710

• Ramesh T, Kim S-W, Sung J-H, Hwang S-Y, Sohn S-H, Yoo S-K, Kim S-K (2012). Effect of fermented Panax ginseng extract (GINST) on oxidative stress and antioxidant activities in major organs of aged rats. Experimen. Gerontol. 47:77-84. https://doi.org/10.1016/j.exger.2011.10.007

• Riahifard N, Tavakoli K, Yamaki J, Parang K, Tiwari R (2017). Synthesis and Evaluation of Antimicrobial Activity of [R4W4K]-Levofloxacin and [R4W4K]-Levofloxacin-Q Conjugates. Mol. 22: 957. https://doi.org/10.3390/molecules22060957

• Seo SK, Hong Y, Yun BY, Chon S J, Jung YS, Park JH, Cho S, Choi YS, Lee BK (2014). Antioxidative effects of Korean red ginseng in postmenopausal women: A double-blind randomized controlled trial. J. Ethnopharmacol. 154: 753-757. https://doi.org/10.1016/j.jep.2014.04.051

• Sharma S, Purohit S, Sharma A, Hopkins D, Steed L, Bode B, Anderson SW, Caldwell R, She J X (2015). Elevated Serum Levels of Soluble TNF Receptors and Adhesion Molecules Are Associated with Diabetic Retinopathy in Patients with Type-1 Diabetes. Mediators Inflamm. 2015:279393.   https://doi.org/10.1155/2015/279393   

• Shih AW, Lam AS, Warkentin TE (2018). Levofloxacin-Induced Acute Immune-Mediated Thrombocytopenia of Rapid-Onset. J. Pharm. Pract. 31(2):234-237. https://doi.org/10.1177/0897190017702306

• Suvarna KS, Layton C, Bancroft JD (2018). Bancroft’s theory and practice of histopathological techniques, 8th edition. Hardcover ISBN: 9780702068645. eBook ISBN: 9780702068874

• Talla V, Veerareddy PR (2011). Oxidative Stress Induced by Fluoroquinolones on Treatment for Complicated Urinary Tract Infections in Indian patients. J. Young Pharm. 3(4): 304-309. https://doi.org/10.4103/0975-1483.90242

• Tamhane AC, Dunlop DD (2000). Statistics and Data Analysis from Elementary to Intermediate. Prentic Hall Ajitc.

• Tohamy AA (2017). Protective Effect of Some Antioxidants (Vitamin E and Selenium) on Adverse Effects of Levofloxacin. Master thesis, Pharm Dept., Fac. of Vet. Med., Zagazig Univ, Egypt.

• Wang QL, Yuan JL, Tao YY, Zhang Y, Liu P, Liu CH (2010). Fuzheng Huayu recipe and vitamin E reverse renal interstitial fibrosis through counteracting TGF-beta1-induced epithelial-to-mesenchymal transition. J. Ethnopharmacol. 127:631-640. https://doi.org/10.1016/j.jep.2009.12.011

• Weiss DJ, Wardrop KJ (2011). Schalm’s Veterinary Hematology 6th Edition, Kindle Edition edn. Wiley-Blackwell, Hoboken, New Jersey. https://doi.org/10.1111/j.1939-165X.2011.00324.x