Serum and Liver Tissue Bio-Element Levels,and Antioxidant Enzyme Activities in Carbon Tetrachloride-Induced Hepatotoxicity: Protective Effects of Royal Jelly

Abstract

The liver is a vital organ, and its function is generally impaired by chemicals. Some natural compounds have a protective role against liver diseases such as royal jelly (RJ). To our knowledge, there are no data available on the effect of RJ therapy on the levels of bio-element metabolisms and antioxidant enzyme activities in the carbon tetrachloride (CCl₄)-induced liver damage. Therefore, in the present study, we have investigated the role of RJ therapy in the trace and major elements and antioxidant enzymes in CCl₄-induced hepatotoxicity in rats. Antioxidant enzyme activities decreased in the CCl₄-treated group more than they did in the sham and RJ-administered groups. Many bio-element levels were also reduced in only the CCl₄-treated group. This showed that the depletion of trace elements was related to erythrocyte antioxidant enzyme activities. RJ administration clearly increased the trace and major element levels and antioxidant enzyme activities in RJ groups. RJ may be used as functional foods because of their naturally high antioxidant potential and rich element content.

Introduction

T he liver is a vital organ, and it plays a major role in metabolism. It has a wide range of functions in the body, such as detoxification, protein and enzyme synthesis, hormone balances, vitamins and minerals storage, and production of the biochemicals that are necessary for digestion. Acute and chronic liver diseases are commonly observed with exposure to viruses, alcohol, drugs, and toxic chemicals.¹ Thus, liver diseases are one of the worldwide problems. Synthetic drugs that are used in the treatment of liver diseases are sometimes inadequate and can have serious adverse effects.² Therefore, natural compounds and food supplements could be used as an alternative treatment in liver diseases.

Carbon tetrachloride (CCl₄) intoxication is a well-known model that is widely used to induce oxidative stress and hepatic injury in experimental animals. CCl₄-induced hepatotoxicity has been shown to be superficially similar to human cirrhosis of the liver.³

Both the essential trace bio-elements and major elements in food are important for the development and maintenance of life functions and, protection against diseases in humans, animals, or plants.⁴ Furthermore, bio-elements affect all aspects of growth, development, physiology, health, and reproduction, starting from the formation of cells, tissues, bones, and organs to the initiation and development of host defense by the immune system in response to foreign microorganisms.⁵ Additionally, however, bio-elements participate in the control of various metabolic and signaling pathways in biological processes. They are also related to many enzymes, hormones, and vitamin systems.⁶

Royal jelly (RJ) is secreted by the hypopharyngeal and mandibular glands of worker honeybees (Apis mellifera) as an essential food for the queen bee larva and for the queen herself.⁷ RJ contains many important compounds with biological activity, such as free amino acids, proteins, sugars, fatty acids, trace and major bio-elements, vitamins, aspartic acid, gelatin, sterols, phosphorous compounds, acetylcholine, nucleic acids, and numerous trace ingredients, and has been used for medical and nutritional purposes in folk medicine.^8,9 RJ has been shown as possessing several pharmacological effects, such as antioxidant.^10,11 antiallergic,¹² antitumor,¹³ and antihypercholesterolemic properties.¹⁴ Furthermore, the effects of RJ on biological molecules was also demonstrated, such as hormones,¹⁵ vitamins and sialic acid,^10,11 lipoprotein and cholesterol,⁹ fatty acids,^16,17 peptides,¹⁸ glycoproteins,¹² and DNA¹⁹ in in vitro and in vivo studies. However, no available study was published on the effect of RJ on the bio-element metabolisms and antioxidant enzyme activities in hepatotoxicity in rats. Thus, in the present study, we investigated the possible protective effects of RJ on bio-elements and antioxidant enzymes in CCl₄-induced hepatotoxicity in experimental animals.

Materials And Methods

Chemicals

Potassium phosphate, sodium phosphate dibasic dihydrate, hydrogen peroxide (H₂O₂), ethanol, sodium chloride, nitric acid (HNO₃), perchloric acid (HClO₄), suprapur inductively coupled plasma (ICP), multielement standard solutions, and CCl₄ were purchased from Merck. Commercial kits of glutathione peroxidase (GPx; Ransel) and superoxide dismutase (SOD; Ransod) were obtained from Randox Laboratories. All other chemicals and reagents used in this study were of analytical grade. Ultra-distilled water was used as the solvent.

Royal jelly

Fresh RJ was obtained in the same year (2009) and provided by same beekeepers (Erdemli, Mersin, Turkey). Samples of RJ were collected when larvae of queen honey bees were 3 days old, and kept frozen at −20°C until they were used.

Animals

Forty-eight male Sprague-Dawley rats with a weight of 190–200 g were used for the experiment. The rats were fed with standard laboratory feed and given tap water during the experiment. They were divided into six equal groups (n=8) and housed in cages. The study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 85–23, revised 1996), and approval was received from our institutional Animal Ethics Committee.

Experimental design

RJ was diluted with distilled water and given orally by gavage. CCl₄ was diluted with sunflower oil (1:1, v:v) and subcutaneously administered. Group I rats (Sham) did not receive any substance. Group II rats (CCl₄) received CCl₄ 0.8 mL/kg, every other day, for 20 days. Group III rats (100 mg/kg RJ) received only 100 mg/kg RJ every other day, for 20 days. Group IV rats (CCl₄+50 mg/kg RJ) received CCl₄ 0.8 mL/kg and 50 mg/kg RJ, every other day, for 20 days. Group V rats (CCl₄+100 mg/kg RJ) received CCl₄ 0.8 mL/kg and 100 mg/kg RJ, every other day, for 20 days. Group VI rats (CCl₄+200 mg/kg RJ) received CCl₄ 0.8 mL/kg and 200 mg/kg RJ, every other day, for 20 days.

Twenty-four hours after final treatment, all rats were anesthetized with ketamine hydrochloride (100 mg/kg, i.p.), and blood samples were obtained by cardiac puncture and placed in heparinized and normal tubes. Then, the animals were sacrificed under anesthesia; liver tissues were removed and rapidly placed in a deep freezer at −70°C until the time of biochemical analysis.

Biochemical analysis

Blood samples for the biochemical analysis were collected in heparinized and normal polystyrene tubes. The heparinized blood samples were centrifuged at 1000 g for 10 min at +4°C, and the plasma was removed using EDTA-washed Pasteur pipettes. Heparinized red blood cells (erythrocytes) were washed thrice with phosphate-buffered saline, pH 7.4. They were collected in a polystyrene microtube blood, after clotting, centrifuged at 1000 g for 10 min at +4°C, and the serum was removed using EDTA-washed Pasteur pipettes. The erythrocyte and serum samples were stored in a polystyrene plastic tube at −70°C until the time of analysis. Erythrocyte SOD, GPx, and catalase (CAT) activities were studied by a spectrophotometer (Jenway 6305 UV/VIS). CAT activity was measured according to the method of Aebi.²⁰ The principle of the assay is based on the determination of the rate constant [k (s⁻¹)] of H₂O₂ decomposition by the CAT enzyme. The rate constant was calculated from the following formula: k=(2.3/Δt)(a/b) log(A ₁/A ₂). According to this formula, A ₁ and A ₂ are the absorbance values of H₂O₂ at t ₁ (0 s) and t₂ (15 s) times, respectively; a is the dilution factor; and b is the hemoglobin content of erythrocytes.²⁰ Erythrocyte SOD and GPx activities were studied in hemolysates by using commercial kits (Randox Laboratories).^21,22

Determination of serum and liver tissue concentrations of trace and major elements

All the plastic containers were previously washed in 10% HNO₃ (ultrapure grade) and then repeatedly rinsed with ultra water. Decomposition of the organic matrix in the tissue samples was performed by using a microwave oven. A Milestone Start D (Italy) microwave oven equipped with a Pro 24 High Throughput Rotor and a temperature control program were used to simultaneously digest 24 samples of tissue in one cycle. The remaining elements were determined after mineralization of the samples. The serum or liver tissue samples of exactly 0.1 g were weighed (wet weight), placed in high-pressure Teflon vessels, and added with 3 mL of concentrated HNO₃, 1 mL of H₂O₂, and 0.5 mL of HClO₄ (ultrapure; Merck). The Teflon vessels were then sealed with a teflon lid and placed in the steel bombs, which were sealed with exactly the same momentum. The mixture in the bombs was heated in a microwave oven according to the following sequence (temperature/time): 90°C/15 min, 120°C/15 min, 140°C/60 min, and 150°C/60 min. After cooling to room temperature, this solution was quantitatively transferred and adjusted in a flask to 10 mL with 18.2 MΩ·cm ultra water (Milipore DirectQ UV). Trace and major element concentrations in the digest were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES; Spectro Genesis). Nebulizer cross flow, cross flow 1380 W, coolnat flow 14.00 L/min, auxiliary flow 1.00 L/min, nebulizer flow 1.05 L/min, optic flush normal, and measure strategy best signal noise ratio were used for the operating conditions of the ICP-OES. Accuracy of the analysis was verified by the determination of the mineral content of the ICP multielement standard obtained from Merck.

Statistical analysis

A statistical analysis of the sham and the five experimental groups was compared using a one-way analysis of variance and Tukeys post test. A value of P<.05 was considered statistically significant. All values were expressed as mean+standard deviation. Statistical tests were performed using SPSS version 12.0 PL for Windows.

Results

Trace and major element compositions of the RJ, used in the present study, are depicted in Table 1.

Table 1.

Composition (mg/kg) of the Royal Jelly in Trace and Major Elements

Aluminum (Al)	5.665±0.21	Potassium (K)	> 2517.08
Boron (B)	1.088±0.05	Lithium (Li)	0.139±0.02
Barium (Ba)	0.131±0.02	Magnesium (Mg)	217.493±4.35
Beryllium (Be)	0.263±0.03	Manganese (Mn)	0.547±0.36
Calcium (Ca)	215.006±3.42	Sodium (Na)	228.29±4.78
Copper (Cu)	6.390±0.55	Selenium (Se)	0.556±0.03
Iron (Fe)	20.054±0.74	Strontium (Sr)	0.302±0.01
Gallium (Ga)	0.501±0.04	Zinc (Zn)	33.203±0.85

The experiment was performed thrice.

The activities of antioxidant enzymes (GPx, SOD, and CAT) in the erythrocytes of sham and experimental groups are presented in Table 2. We found that GPx activities of the CCl₄ group (P<.001) and 100 mg/kg RJ (P<.05) were lower than those of the sham group. In addition, GPx activity in the CCl₄+50 mg/kg RJ group decreased when compared with the sham and 100 mg/kg RJ group (P<.001). The SOD level in the CCl₄ group exhibited a decrease when compared with the sham and 100 mg/kg RJ group (P<.001). However, the SOD level in the CCl₄+50 mg/kg RJ group was lower than that in the sham and 100 mg/kg RJ group (P<.01 and P<.001, respectively); there was an increase when compared with the CCl₄ group (P<.05). Although the CAT enzyme activity of the CCl₄ group exhibited a decrease when compared with the sham group (P<.01), the CAT levels in the 100 mg/kg RJ and CCl₄+200 mg/kg RJ groups were higher than the level in the CCl₄ group (P<.01). The extent of CCl₄-induced hepatic injury was evidenced by the determination of alanine transaminase (ALT) and aspartate transaminase (AST) levels in our previous study.^10,11 After CCl₄ treatment, serum ALT and AST activities increased four and six times, respectively, when compared with the sham group (P<.001).

Table 2.

Erythrocyte Antioxidant Enzyme Activities in the Study Groups

Enzymes	Sham	CCl₄	100 mg/kg RJ	CCl₄+50 mg/kg RJ	CCl₄+100 mg/kg RJ	CCl₄+200 mg/kg RJ
GPx (U/L)	8079.17±256.6	6126.60±334.8^***	7455.20±383.9^*,†††	6429.51±310.7^*** ^,###	7583.75±281.3^†††	7720.17±317.2^†††
SOD (U/mL)	217.17±7.9	181.01±10.9^*** ^,###	221.41±6.5^†††	195.50±6.9^**,†,##	202.01±7.3^††,#	205.02±8.5^††,#
CAT (kU/L)	5342.98±363.9	4417.52±453.9^**	5432.28±213.7^††	5060.72±348.2	4972.80±257.7	5394.63±444.4^††

Data are the mean±standard deviation. P values were calculated using a one-way analysis of variance and Tukeys post test. A value of P<.05 was considered statistically significant.

P<.05, ^** P<.01, ^*** P<.001 with regard to sham.

†

P<.05, ^†† P<.01, ^††† P<.001 with regard to CCl₄.

P<.05, ^## P<.01, ^### P<.001 with regard to 100 mg/kg RJ.

GPx, glutathion peroxidase; SOD, superoxide dismutase; CAT, catalase; RJ, royal jelly; CCl₄, carbon tetrachloride.

Serum trace and major element concentrations in the study groups are shown in Table 3. Aluminum concentration of the CCl₄+100 mg/kg RJ group increased when compared with the 100 mg/kg RJ group (P<.05). There were some differences between beryllium and gallium concentrations in the CCl₄+100 mg/kg RJ group and in the sham, CCl₄, and 100 mg/kg RJ groups (P<.001). While potassium (K) concentration in the CCl₄ group was higher than that in the sham group (P<.001), K concentrations in the 100 mg/kg RJ group were lower than those in the CCl₄ group (P<.001). The K concentrations in the CCl₄+50 mg/kg RJ, CCl₄+100 mg/kg RJ, and CCl₄+200 mg/kg RJ groups were significantly different from those in the sham and 100 mg/kg RJ groups (P<.001). In spite of the fact that the lithium level of the CCl₄+100 mg/kg RJ group increased when compared with the sham and 100 mg/kg RJ group (P<.05), the magnesium (Mg) level of the 100 mg/kg RJ group decreased compared with the CCl₄ group (P<.05). Selenium (Se) concentrations in the sham and CCl₄+200 mg/kg RJ group were below those in the 100 mg/kg RJ group (P<.01). Strontium (Sr) levels in the 100 mg/kg RJ and CCl₄+200 mg/kg groups decreased when compared with those in the sham group (P<.05), and there were some differences in Sr level between the CCl₄+100 mg/kg RJ group and the sham and 100 mg/kg RJ groups (P<.05 and P<.01 respectively). Zinc (Zn) concentration of the CCl₄ group was the highest among the other study groups. In addition, there were no differences in the copper (Cu) and iron (Fe) concentrations of all the study groups.

Table 3.

Serum Trace and Major Element Concentrations (mg/L) in the Study Groups

Elements	Sham	CCl₄	100 mg/kg RJ	CCl₄+50 mg/kg RJ	CCl₄+100 mg/kg RJ	CCl₄+200 mg/kg RJ
Al	1.04±0.3	1.12±0.4	0.83±0.2	0.93±0.2	1.39±0.3^#	1.25±0.1
Be	0.26±0.08	0.26±0.05	0.26±0.02	0.26±0.07	0.31±0.02^{**,†††,###}	0.26±0.08
Ca	128.14±11.3	148.24±21.2	111.11±5.8^††	116.18±15.5^†	150.38±11.9^##	118.47±9.6^†
Cu	1.13±0.2	1.15±0.2	1.00±0.1	1.34±0.5	1.18±0.4	0.16±0.1
Fe	5.52±1.3	8.08±1.9	6.09±2.4	5.10±0.9	5.84±2.1	5.99±2.7
Ga	0.49±0.01	0.49±0.07	0.48±0.05	0.49±0.01	0.59±0.02^{***,†††,###}	0.49±0.08
K	225.43±11.9	302.59±13.9^***	204.08±9.3^†††	282.49±22.3^***,###	283.85±28.7^*** ^,###	298.84±21.6^*** ^,###
Li	0.09±0.01	0.12±0.03	0.09±0.01	0.10±0.02	0.13±0.04^* ^,#	0.11±0.03
Mg	25.01±4.1	33.06±9.1	22.71±2.1^†	25.34±4.4	30.96±3.4	24.71±3.6
Se	0.69±0.11^##	0.82±0.20	1.01±0.07	0.84±0.15	0.79±0.06	0.68±0.10^##
Sr	0.29±0.09	0.39±0.13	0.24±0.02^†	0.27±0.05	0.47±0.08^*,##	0.25±0.04^†
Zn	2.59±0.6^†††	4.68±1.4	2.41±0.2^†††	2.53±0.5^†††	3.13±0.4^†	2.91±0.2^††

P<.05, ^** P<.01, ^*** P<.001 with regard to sham.

†

P<.05, ^†† P<.01, ^††† P<.001 with regard to CCl₄.

P<.05, ^## P<.01, ^### P<.001 with regard to 100 mg/kg RJ.

Liver tissue trace and major element concentrations of all the groups are presented in Table 4. The Cu levels in the sham and 100 mg/kg RJ groups were higher than those in the CCl₄ group (P<.01). The Fe concentration in the sham was below that in the 100 mg/kg RJ group (P<.05) when the Fe concentration in the CCl₄ group was higher than that in the sham group (P<.05). In addition, the Fe concentrations in the CCl₄+100 mg/kg RJ and CCl₄+200 mg/kg RJ groups were higher than those in the sham group (P<.01). The Mg levels in the sham, 100 mg/kg RJ, and CCl₄+100 mg/kg RJ groups increased when compared with the CCl₄ group (P<.05). The Se concentrations in the sham (P<.05), CCl₄+100 mg/kg RJ (P<.01), and CCl₄+200 mg/kg RJ groups (P<.001) were higher than those in the CCl₄ group, even though the Se concentration in the CCl₄ group was lower than that in the 100 mg/kg RJ group (P<.01). On the contrary, the Zn concentration in the CCl₄ group decreased when compared with the sham and 100 mg/kg RJ groups; there were also some increases between the sham and all-treatment groups (P<.01 and P<.001).

Table 4.

Liver Tissue Trace and Major Elements Concentrations (μg/g Tissue) in the Study Groups

Elements	Sham	CCl₄	100 mg/kg RJ	CCl₄+50 mg/kg RJ	CCl₄+100 mg/kg RJ	CCl₄+200 mg/kg RJ
Al	4.133±1.79	5.349±2.76	6.161±1.02	5.585±2.21	6.404±1.193	4.475±1.92
B	1.103±0.83	2.678±0.16^*	2.509±0.96^*	2.958±0.09^**	3.547±0.63^**	1.679±0.88
Ba	1.491±0.48	1.188±0.24	1.471±0.46	1.652±0.59	1.764±0.27	1.618±0.53
Be	0.218±0.01	0.234±0.06	0.246±0.01	0.265±0.04	0.279±0.06	0.284±0.04
Ca	413.35±78.08	374.69±52.15	415.77±98.67	386.44±40.97	486.27±31.82	394.66±63.12
Cu	4.183±0.46^††	3.152±0.52	4.483±0.30^††	3.854±0.54	3.689±0.41	3.786±0.31
Fe	152.29±20.63^#	194.021±19.73^*	201.34±13.85	171.07±12.87	203.26±16.66^**	205.84±18.54^**
Ga	0.439±0.02	0.456±0.02	0.438±0.02	0.458±0.01	0.458±0.02	0.451±0.01
Li	0.106±0.03	0.092±0.01	0.096±0.02	0.092±0.01	0.087±0.01	0.082±0.01
Mg	210.792±2.72^†	191.00±17.31	212.04±8.62^††	206.53±10.06	215.46±10.14^†	205.78±3.37
Mn	1.047±0.12	1.193±0.08	1.043±0.09	1.157±0.18	1.056±0.12	1.018±0.15
Se	2.605±0.27^†	2.201±0.29^##	2.883±0.17	2.481±0.07	2.894±0.06^††	2.881±0.21^†††
Sr	0.264±0.05	0.221±0.03	0.283±0.07	0.234±0.03	0.235±0.05	0.206±0.03
Zn	31.468±1.56	22.896±1.23^*** ^,###	33.066±1.88	29.958±3.21^†††	31.066±2.91^†††	28.434±1.53^††

P<.05, ^** P<.01, ^*** P<.001 with regard to sham.

†

P<.05, ^†† P<.01, ^††† P<.001 with regard to CCl₄.

P<.05, ^## P<.01, ^### P<.001 with regard to 100 mg/kg RJ.

Discussion

The liver plays an important role in the metabolism of endogenous and exogenous substances, and liver diseases are considered a serious health problem all over the world. Liver diseases cause abnormality in the metabolism.^1,2 The use of naturally originated agents or dietary food supplements might help prevent those conditions and maintain human health.⁷ In the present study, we investigated the levels of serum and liver bio-elements and antioxidant enzyme activities in CCl₄-induced hepatotoxicity and the protective effects of RJ. The results of this study showed that the CCl₄-treated rat's trace and major element levels and antioxidant enzyme activities were affected, and RJ normalized the altered levels of the bio-elements and antioxidant enzymes.

Liver injury induced by CCl₄ is a common model that is used for screening the hepatoprotective activity of drugs, because this chemical is a potent hepatotoxin, and a single exposure can rapidly lead to severe hepatic necrosis and steatosis.³ CCl₄ toxicity is derived from its biotransformation products. These metabolites, such as trichloromethyl free radicals and proxy trichloromethyl free radicals, are metabolized products derived from CCl₄ by cytochrome P450; are capable of binding to DNA, lipids, proteins, or carbohydrates; and, eventually, lead to membrane-lipid peroxidation, oxidative stress, and cell death.²³ Antioxidants may protect liver injury by oxidative stress, which is defined, in general, as the excess formation of reactive oxygen species (ROS) and/or an imbalance between the production of ROS and the biological systems, which are capable of detoxification of highly reactive intermediates.⁶ Aerobic organisms are protected against free radicals and oxidative stress by enzymatic antioxidants (GPx, SOD, and CAT).²⁴ In a previous study, we investigated the possible antioxidant activities of RJ in CCl₄-induced hepatotoxicity. Therefore, we studied nonenzymatic antioxidants such as reduced glutathione, ceruloplasmin, ascorbic acid, retinol, and β-carotene levels in the study groups. It was demonstrated that treatment with different doses of RJ significantly reduced oxidative stress, and augmented the efficiency of the various nonenzymatic antioxidant systems.^10,11 In addition, several researchers^7,8 reported that RJ has powerful antioxidant effects with rich content.

Enzymatic antioxidant activities or the inhibition of free radical generation are important in terms of protecting the liver from CCl₄-induced damage.²⁵ Many previous in vivo studies have shown the influence of CCl₄ on antioxidant enzyme activities.^26,27 These researchers reported that GPx, SOD, and CAT activities decreased in CCl₄-induced hepatic injury. In the present study, antioxidant enzyme activities were significantly decreased in the CCl₄ group when compared with the sham group. Our result in this study is in agreement with previous reports about CCl₄-induced liver injury. The decreases in the antioxidant enzyme activities might be peripheral responses of the organism to increased free radical generation in CCl₄ toxicity. Increased free radicals might also be the oxidative inactivation of antioxidant enzyme proteins. However, pretreatment with RJ resulted in a statistically significant increase in the antioxidant enzyme activities in RJ groups. These results showed that the RJ has highly antioxidant potential.

The functions of bio-elements in cells are generally complex. Enzymes have a protein structure and contain essential trace elements such as manganese (Mn), Zn, Cu, Fe, and Se. Among the trace elements, Se is an essential component of the GPx as a family of primary antioxidant enzymes; while Mn, Cu, and Zn are structural components of both Mn SOD and Cu-Zn SOD, with Fe also being a part of CAT.²⁵

Low erythrocyte SOD activity was reported in Cu-deficient rats.²⁸ In the present study, there was no statistically significant difference between the groups for Mn element level in liver tissue. However, we found that the Cu and Zn element levels in the liver tissue of the CCl₄ group were lower than those in the sham and RJ study groups. Moreover, RJ administration increased the Cu and Zn element concentrations of the liver tissues in the treatment groups compared with the CCl₄ group. Furthermore, erythrocyte SOD activities significantly decreased in the CCl₄ group. This study also showed that the depletion of Cu and Zn was related to erythrocyte SOD activity. In addition, SOD enzyme activities were significantly elevated in the RJ-administered groups.

Se is predominantly incorporated into selenoproteins, which are vital for normal health and reproduction. It is necessary for immunocompetence, testosterone, and thyroid hormone metabolism. Se also plays structural and enzymatic roles. It is an essential part of >30 selenoenzymes. The best known biological activity of Se is in the selenoenzyme GPx. Sub-clinical deficiency predisposes to cardiovascular disease, mood disorders, and cancer, as well as has an impact on immune and antioxidant function.²⁹ In the previous study, we studied Se levels in organophosphate toxicity and heroin addiction or heroin withdrawal in rats. We found statistically significant decreased differences between the sham and study groups.^10,11,30 In the current study, we found decreases in the Se element concentrations in the liver tissues of rats administered with CCl₄. Parallel to this, it was observed that there was a significant decrease in GPx levels in the CCl₄ group, when compared with those in the sham and RJ groups. RJ administration clearly increased the Se element concentrations and GPx enzyme activities in RJ groups.

Fe is essential for normal physiological function. It is a constituent of hemoglobin, myoglobin, and a number of metalloenzymes such as CAT; as much as 30% of the body Fe is found in storage forms such as ferritin and hemosiderin, in the spleen, liver, and bone marrow; and a small amount is associated with the blood transport protein transferrin. Clinical studies have shown a relation among elevated Fe stores, intravenous Fe administration, and increased risk of bacterial infection. Excess Fe can enhance bacterial growth and virulence by impairing the chemotactic and phagocytic properties of neutrophils.⁴ In the present study, we found that the Fe levels of serum and liver tissue in the CCl₄-administered whole groups were higher than those in the sham group. These increases might be due to CCl₄-induced liver injury. However, erythrocyte CAT activity significantly decreased in the CCl₄ group.

In conclusion, the present study demonstrates that CCl₄ intoxication caused liver damage and oxidative stress by decreasing GPx, SOD, and CAT activities. Many trace element levels are also reduced in only CCl₄-administered rats. Our results show that RJ has increased both antioxidant enzymes activities and essential bio-element levels. It can be speculated that it might increase the gene expression of antioxidant enzymes after RJ administration. It has also been shown that RJ has a rich bio-elemental composition.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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