Antioxidant and chelating effects of Citrus sinensis peel extract on wistar rats administered with lead and cadmium

Heavy metals possess toxic effects that leads to serious health and environmental problems, anthropogenic activities are chiefly responsible for metal exposure, and they manifest toxic effects on a biological cell. The peels of Citrus sinensis have been reported to have medicinal effect. However, these benefits have not been adequately investigated against metals toxicity. This study evaluated the antioxidant and chelating effect of Citrus sinensis peel extract (CSPE) on wistar rats administered with lead and cadmium. Forty-five (45) rats were separated into nine (9) groups of 5 rats each. The group under control was given distilled water daily, group 2 and 3 received 8mg/kg of cadmium and 15 mg/kg of lead per body weight respectively. Animals in group 4 received 8 mg/kg of cadmium and 100 mg/kg of EDTA treatment per body weight. Group 5 were given 15 mg/kg of lead and treated with 100 mg/kg of EDTA. Group 6 and 7 were respectively treated with CSPE of 250 and 500 mg/kg and administered cadmium-8 mg/kg. Lastly, group 8 and 9 received lead and 250 and 500 mg/kg of CSPE respectively. Administration lasted for 28 days afterwards the rats were sacrificed. The whole blood was separated for analysis of haematological parameters and the liver and kidney tissues harvested for metal analysis. Glutathione Peroxidase (GPx), Malondialdehyde (MDA), Superoxide dismutase (SOD) and Catalase (CAT) were also estimated. Results showed a statistically significant increase (p<0.05) of MDA levels in lead and cadmium and a significant reduction in SOD, CAT and GPx values compared to the control and treated group. The level of cadmium and lead in blood, kidney, and liver tissues of 500 mg/kg CSPE were significantly reduced. CSPE possesses antioxidant properties and exhibits chelating effects on wistar rats administered with lead and cadmium.


INTRODUCTION
Heavy metals have been known to possess toxic ef f ects that are detrimental to humans and the environment at large (Balali-mood et al., 2021). A study conducted by the World Health Organisation (WHO) states that approximately 2.2 million people die in Af rica yearly as a result of environmental risk f actors (WHO, 2016). Although lead and cadmium are two major toxic metals present in the environment, anthropogenic activities are mainly responsible f or their exposure (Tchounwou et al., 2012). Lead has no physiological benef it to the body, it manif ests its toxic actions on the blood, liver and kidney immediately it enters the body. In addition, research has shown that exposure to lead can result to a statistically signif icant reduction in red blood cells count and haematocrit in the blood (Terayama, 1993). Similarly, cadmium is an environmental and industrial pollutant that has been proven to cause injury to a biological cell (Massanyi et al., 2005). For the vast majority, the primary sources of cadmium exposure are f ood and tobacco smoking due to the increased cadmium uptake by crops (Jarup and Akesson, 2009). Af ter penetrating the body, cadmium is transported into the blood via blood protein albumin and red blood cells and cumulates in the liver and kidneys (Goyer, 1991). Cadmium exposure leads to renal dysf unction, damage to hepatic system, digestive and respiratory system disorders, testicular atrophy and anemia (Schwartz and Reis, 2000). Report by Salazar-Flores et al., 2020 revealed that toxic metals such as cadmium and lead has been implicated in oxidative stress which results in tissues and membranes damages in a biological entity. Thus, reducing the antioxidant activities of enzymes such as Catalase (CAT), Superoxide dismutase (SOD), and Glutathione Peroxidase (GPx). Although the potential of new drugs discovery f rom higher plants sources is still largely unexplored, about 80 percent of people in the developing countries depend on phytomedicine f or primary healthcare f or man and domestic animals (Oke and Hamburger, 2002).
Citrus sinensis is an essential commercial f ruit grown globally due to its diversif ied use (Manthey and Grohmann, 2001). Citrus sinensis is good source of vitamin C, it is a strong natural antioxidant and builds the body's immune system (Etebu and Nwauzoma, 2014). Constipation, cramps, colic, diarrhea, bronchitis, TB, cough, cold, obesity, menstruation disorders, angina, hypertension, anxiety, depression, and stress are just a f ew of the conditions it has been used to treat locally (Milind and Chaturvede, 2012).  (Hegazy and Ibrahim, 2012), larvicidal, pupicidal, repellent, and adulticidal pharmacological activities (Murugan et al., 2012). Although, studies have reported that C. sinensis peels may possess a protective potency against cadmium induced liver toxicity, its ef f ect on lead induced nephrotoxicity and hepatotoxicity has not been investigated (Nwaf or et al., 2020). Hence, this study was designed to evaluate the antioxidant and chelating ef f ects of Citrus sinensis peel extract (CSPE) on administered lead and cadmium to wistar rats.

MATERIALS AND METHODS
All chemicals used in the present study were of analytical grade. 100 g of cadmium chloride (99.9% pure) and 100g of Ethylenediamine tetracetic acid (EDTA) manuf actured by Cartivalue Chemical Limited, Mumbai, India was obtained f rom chemical store in Benin City, alongside 100 g of lead acetate, manuf actured by BDH Chemical Limited, Poole, England. Equipment used f or analysis are: Centrif uge and UV-VIS Spectrophotometer (Hitachi F 7000).

Extract preparation and extraction
Fresh Citrus sinensis f ruits were purchased f rom Oba Market, Oredo Local Government Area of Edo state. The zest of the f ruit (peels) was authenticated by Dr. H. Akinnibosun of the Department of Plant Biology and Biotechnology, Faculty of Lif e Sciences, University of Benin, Benin City, Edo State, Nigeria. The peels of the f resh Citrus sinensis f ruits were manually separated f rom its f ruit, washed with distilled water to remove adhering dirt and air dried f or a period of three weeks until they turned crisp. The crisp leaves were dried in an incubator at 40-45°C f or about two hours then pulverized to a f ine powder using a mechanical grinder. The dry weight of the peel was recorded, af ter which was soaked in analytical grade ethanol f or 72hours, with periodic agitation to ensure proper extraction. The resultant mixture was f iltered using a cheese cloth and the f iltrate obtained was f urther subjected to f iltration by Whatman f ilter paper (number 1). The f iltrate was concentrated in a thermostatically regulated water bath at a controlled temperature of 44°C f or about 40mins, af ter which it was placed in an air tight container and stored in the ref rigerator.

Experimental animals and design
Forty-f ive adult wistar rats with weights ranging f rom 180-220g were purchased f rom pharmacology animal house and housed in plastic cages f or experimental purposes. They were f ed with standard rats f eed (growers mash), distilled water and acclimatized f or a period of 14 days with a constant temperature of 26 ± 2 o C and 12 hours' alternates dark and light cycles. The animals were handled in accordance with the guidelines of the Institutional Animal Ethics Committee of the Department of Science Laboratory Technology, University of Benin. The wistar rats were randomly selected and divided into nine (9) groups of f ive (5) rats in each group as f ollows: Group 1 (control): Animals received distilled water daily. Group 2: Animals received 8 mg/kg of Cadmium. Group 3: Animals received 15 mg/kg of Lead. Group 4: Animals received 8 mg/kg of Cadmium + 100 mg/kg of ethylenediamine tetracetic acid (EDTA) Group 5: Animals received 15 mg/kg of Lead + 100 mg/kg of ethylenediamine tetracetic acid (EDTA) Group 6: Animals received 8 mg/kg of cadmium + 250 mg/kg of CSPE. Group 7: Animals received 8 mg/kg of cadmium + 500 mg/kg of CSPE. Group 8: Animals received 15 mg/kg of lead + 250 mg/kg of CSPE. Group 9: Animals received 15 mg/kg of Lead + 500 mg/kg of CSPE.
The body weight of the animals was recorded at seven-day intervals. EDTA was dissolved in water and administered to the rats with the aid of an oral f eeding gavage. At the end of the 28 days' experimental period, rats were f asted overnight and sacrif iced af ter anesthetization by chlorof orm inhalation, then their blood was collected and liver and kidney excised.

Blood collection and haematological analysis
The Wistar rats were weighed bef ore sacrif ice and blood collection. To avoid variations due to circadian rhythm, all samples were collected between 7 and 9 am. Using the traditional technique, whole blood was drawn f rom an incision of the retro-orbital sinus. By mixing blood samples with the anticoagulant in the tube, blood samples collected in EDTA anticoagulant tubes (8.5%) were immediately returned. All blood samples had labels applied bef ore being sent right away to the lab for examination. red blood cells (RBC), White blood cell count (WBC), haemoglobin concentration (HGB), monocyte (MON), granulocyte (GRAN), platelet count (PLT), haematocrit (HCT), and the number of lymphocytes were the haematological parameters analyzed (LYM). All haematological parameters were analysed in the "Haematology Unit, University of Benin Teaching Hospital (UBTH)" using the automated method with the automatic analyzer "Haematology auto analyzer Sysmex KX-21N".

Heavy metal analysis
For the determination of the various metals, the wet chemical digestion method was used to decompose the samples collected. The kidney and liver samples were separately weighed at one gram and placed into the digesting f lask. In a ratio of 3:1, 15 ml of 0.1 N concentrated HNO3 and 5 ml of perchloric acid were added to each of the sample's portions in the f lask bef ore being heated on an electric plate until the sample was clear. Following digestion, the content was given 5 ml of 20% HCl (0.1 N). In order to assess the amount of metal present, the contents of the f lask were f iltered using Whatman f ilter paper (number 42) into a 100 ml volumetric f lask, f illed to the appropriate level with distilled water, and then stored in a plastic reagent bottle.

Measurement of malondialdehyde level
Malondialdehyde (MDA) levels, as a measure of lipid peroxidation was determined in the supernatant of the homogenate tissues by the thiobarbiturate acid (TBA) method. The concentration of MDA was measured spectrophotometrically at 532 nm and the coef f icient of absorption of MDA-TBA complex was measured (Kheradmand et al., 2009).

Measurement of antioxidant enzymes
Superoxide dismutase (SOD): Using a SOD detection kit in accordance with the manuf acturer's instructions, the activity of SOD was assessed. The level of inhibition of this process served as a proxy f or measuring SOD activity. Using a standard curve, the concentration of SOD was determined at 505 nm and represented as U/mg protein (Rasyidah et al., 2014).
Glutathione Peroxidase (GPx): The activity of GPx, which catalyzes the destruction of GSH by hydrogen peroxide, was measured and expressed as U/mg protein. GSH consumption will be assessed at 420 nm using a spectrophotometer (Flohe and Gunzler, 1984).
Catalase (CAT): Using the technique of Aebi et al the CAT activity was assessed (1984). The direct measurement of catalase's H2O2 degradation is the reduction in absorbance at 240 nm. 650 ml of 50 mm phosphate buf f er with a pH of 7.0 will be mixed with 50 ml of organ homogenate that has been diluted 50 times. 300 ml of 54 Mm H2O2 were added to start the reaction, and the absorbance chang e was monitored f or 1 minute at 25 o C. A unit of catalase activity was established as the quantity of the enzyme required to break down 1 mol of H2O2 every minute. CAT was expressed in terms of U/mg protein

Statistical analysis
All data were presented as mean ± standard error of mean (SEM) and 'n' represents the number of wistar rats per experimental group. Data were subjected to one-way analysis of variance (ANOVA) f ollowed by Turkey's multiple comparisons test. All data were analysed using Graph Pad Prism (UK) sof tware version 6.0. p<0.05 indicates signif icance dif f erence between compared data.

The effect of CSPE on haematological parameters of wistar rat
The haematological ef f ects of Citrus sinensis peels on the wistar rats exposed to lead and cadmium heavy metals are shown in Tables 3  and 4 respectively. It was discovered that the level of WBC, LYM, MON and GRAN increased in the lead group compared to other treated groups. However, the values of RBC and HCT in the lead group, though not signif icant, decreased when compared to other treated groups. These reductions were ameliorated by CSPE treated groups. In addition, a signif icant increase (p > 0.05) in LYM level was observed in the cadmium group when compared to the control.

Results of heavy metal analysis
In Fig. 3 below, the lead level in the blood showed a signif icant dif f erence between the CSPE treated groups, the EDTA group and the lead group (p<0.05). In addition, a signif icant dif f erence was observed in both CSPE treated groups, this shows that the ef f ect of CSPE was dose dependent. The control group had minute concentration of lead, this can be attributed to the presence of trace and heavy metals in the f eed.
Results of lead concentration in the Liver tissue showed high signif icant (p<0.05) dif f erence between the lead group and the treated groups of EDTA, 250 and 500 mg/kg CSPE group.
The lead level in the kidney showed a signif icant decrease (p<0.05) in lead in the 500 mg/kg CSPE group compared to the lead group and other treated groups (Fig. 3). The 500 mg/kg CSPE was observed to have the lowest lead concentration in all treated groups, and there was no signif icant dif f erence when compared to the control group.   Cadmium concentration in the blood showed a signif icant decrease in the CSPE treated group in comparison with the cadmium group. (p<0.05). There was also a signif icant dif f erence in both CSPE treated groups, this shows that the ef f ect of CSPE was dose dependent (Fig. 4). However, there was no signif icant dif f erence between the cadmium group and the EDTA group. The control group had minute concentration of cadmium, this can be attributed to the presence of trace and heavy metals in the f eed and water.
Results of cadmium concentration showed no signif icant (p>0.05) dif ference between the cadmium group and the treated groups of EDTA, 250 and 500 mg/kg CSPE. Although, reduction in cadmium concentration was observed in all treated groups, especially that of 500 mg/kg CSPE.
The cadmium level in the kidney tissue showed a signif icant decrease (p<0.05) in cadmium in the 500 mg/kg CSPE group compared to the cadmium group and other treated groups. The 500 mg/kg CSPE was observed to have the lowest cadmium concentration in all treated groups, and there was no signif icant dif ference when compared to the control group.

DISCUSSION
The results obtained in this study shows the antioxidant protective and chelating properties of Citrus sinensis peel extract against lead and cadmium induced toxicity in wistar rats. The biochemical mechanisms involved in the toxicity was studied by measuring the levels of Malondialdehyde (MDA) and by testing the activities of basal antioxidant enzymes such as Superoxide dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx). MDA is used as an indicator of oxidative stress f ormed by lipid peroxidation of polyunsaturated f atty acids. Results obtained showed that the administered lead and cadmium caused a signif icant increase of MDA level in the blood as shown in Figures 1 and 2 respectively. Bahrami et al., (2016) suggested that the increase production of Reactive Oxygen Species (ROS) during toxicity can present a threat to biomolecules by oxidation of proteins, impairment to nucleic acids, and causing peroxidation of lipids. Studies conducted by Ndubisi et al., (2020) as well as Selmi et al., (2017) showed that CSPE inhibited lipid peroxidation in heavy metals induced rats. This present study was in agreement with f indings of studies stated above, as our results showed that the CSPE was able to decrease the value of MDA to a level comparable to that of the control group in both lead and cadmium treated groups. The signif icant reduction in MDA levels between the CSPE treatment groups indicates that the f ree radical reduction and lipid peroxidation inhibitory ef f ect is dose dependent.
Enzymatic antioxidant def ence is one of the mechanism the body deploys to help guard the host cells against excess f ree radicals such as ROS. Examples of these antioxidants are SOD, CAT and GPx. SOD is a major enzyme that appears to act as the f irst line of def ence against ROS, it breaks down superoxide radicals (O2 -) to produce hydrogen peroxide (Sudjarwo et al., 2017). The two primary enzymes involved in H2O2 detoxif ication are Glutathione peroxidase and Catalase. Hydrogen peroxide (H2O2) pose to be a signif icant ROS that results in oxidative stress. These enzymes, breakdown hydrogen peroxides and hydroperoxides to harmless molecules. According to our results in this study, there was no signif icant changes of Catalase in the lead administered group. However, in the groups administered with cadmium, expressions of catalase were signif icantly decreased (p<0.05). This is in correlation with studies conducted by Jun et al., (2003) who suggested that cadmium-mediated oxidative stress by hydrogen peroxide may trigger an increase in the reduction of the activity of antioxidant enzymes in conjunction with increased lipid peroxidation. The chelating ef f ects of EDTA and CSPE were able to sustain the catalase expression in the groups they were administered. In addition, a signif icant dif f erence (p<0.05) was observed in the 500 mg/kg group of CSPE, which not only sustained catalase activity in the blood, but also resulted in an increase beyond that of the control group. This indicates that the antioxidant activity of CSPE is dose dependent. Similar occurrence was observed f or the GPx expressions in both lead and cadmium groups, where the levels of GPx decreased in contrast to the control group. CSPE was able to ameliorate this ef f ect in the treatment groups, studies suggest that this might be due to its ability to reduce f ree radical accumulation and ROS in the blood. Bearing on the ef f ects of toxic metals on oxidative stress condition, the level of SOD in lead and cadmium were decreased drastically to 2.22±0.03 U/mg and 3.15±0.77 U/mg respectively. CSPE treatment signif icantly and dose-dependently decreased f ree radical generation in lead induced wistar rats, thus, increasing the SOD expression in the treated groups. The same was the case f or the SOD tests carried out on cadmium induce rats. Newairy and Abdou (2009) reported that lead toxicity causes numerous deleterious ef f ects in organisms and also inf luences haematological parameters. Studies conducted by Bersenyi et al., (2003) shows that lead exposure in rabbits caused a signif icant reduction in red blood cells (RBC) and haematocrit (HCT) values. These f indings suggest that red blood cells are susceptible to oxidative injury and lipid peroxidation that occurs in the membrane of RBC than other types of cells. This study revealed that HCT level decreased signif icantly (p<0.05) to approximately 30 % compared to the control group. The values of WBC in the blood, increased in the group administered lead . Similarly, there was an increase in the LYM, MON, and GRAN level in the lead and EDTA treatment groups as shown in Table 3. In the case of rats administered with cadmium, the number of WBC increased signif icantly in the cadmium and CSPE treatment groups.
Although there was a reduction in the levels of MON and GRAN of the cadmium group, it was ameliorated by the 500 mg/kg dose of CSPE. RBC, Hb, HCT and PLT values were similar in the control and 250 mg/kg CSPE groups, however, the levels were higher in the 500 mg/kg CSPE group. This f inding correlates with studies conducted by Nazima et al., (2016) suggesting that some antioxidants show protective activity against breakdown of RBC by cadmium. Findings f rom this study revealed the phytochelating ef f ect of CSPE o n lead and cadmium as evidence by the signif icant decreased levels of these metals in blood, liver and kidney in the treated groups compared to the toxic group.

CONCLUSION
The results f rom this study have shown that Citrus sinensis peel extract, even at low doses, possesses chelating and antioxidant properties capable of reducing the deleterious ef f ects of lead and cadmium induced toxicity in blood, kidney, and liver. Further studies are needed to evaluate long term usage of Citrus sinensis peel in metal detoxif ication as well as understanding its mechanism of action.

Declaration of interest
The authors declare that they have no conf lict of interests to declare. All authors read and approved the manuscript.

Author contribution
EOC conceived and designed the study, wrote the original draf t, and revised the paper. OEP was involved in material preparation, data collection, analysis and writing of the paper, AAB provided reagent and laboratory analysis, EJU, AON, OPO and ECM were involved in the laboratory analysis and interpretation of the data. All authors read and approved the f inal manuscript.