Amelioration of Lipid Peroxidation and Antioxidant Enzymes Status in the Serum and Erythrocytes of Phenylhydrazine-Induced Anemic Male Rats: The Protective Role of Artichoke Extract (Cynara scolymus L.)

Introduction

Anemia is defined as a condition in which the number of red blood cells (RBCs) or their oxygen-carrying capacity is inadequate to meet the physiological needs of the body (Prasad et al., 2018). There are several kinds of anemia classified by a variety of under- lying causes. Hemolytic anemia is the most frequent form of anemia (Lee et al., 2012). Hemolytic anemia is categorized as acquired or hereditary. Common acquired causes of hemolytic anemia are autoimmunity, micro- angiopathy, infection and drug (Dhaliwai et al., 2004; Paul et al., 2018).

Phenylhydrazine (PHZ) is a strong oxi- dant agent which has toxicity on various tis- sues at various levels. It is known to shorten life-span of erythrocytes resulting in hemo- lytic anemia, increased erythropoietic activ- ity, enhanced iron absorption and tissue iron overload (Luangaram et al,. 2007). PHZ has been used experimentally for the induction of hemolytic anemia in animal models. The auto-oxidation of PHZ leads to generation of ROS and PHZ-derived radicals, which causes a wide variety of deleterious cellular response including hemolytic anemia (Sung et al., 2013). The observed haematotoxicity is a result of the reaction of PHZ with oxy- genated hemoglobin to form oxygen radicals and methemoglobin (Shetlar and Hill, 1985). Medicinal plants and natural  products have been used as traditional treatments for numerous diseases particularly in devel- oping countries because of several reasons including therapeutic effects, affordability, accessibility and fewer side effects (Asase et al., 2008). Several studies have demonstrat- ed that natural medicinal plants with potent antioxidant activity and their potential pro- tective effects can alleviate the damage of oxidative stress‐associated diseases through

inhibition of ROS generation and improve- ment of antioxidant defense mechanisms (Forni et al.,2019; Banerjee et al., 2018).

Cynara scolymus L. (Artichoke), a mem- ber of Asteraceae family, is an ancient her- baceous perennial plant, originating from the Mediterranean area, which today is widely cultivated all over the world because of its nutritional benefits and medicinal purposes (Salekzamani et al., 2019). Phytochemicals analysis of Cynara scolymus has been found to contain powerful polyphenolic compounds which have therapeutic options including re- markable antioxidant activity against ROS and preventing the formation of free radi- cals (El-Boshy et al., 2017). Previous stud- ies have reported that artichoke extract has important activities such as hepatoprotective (Gebhardt and Fausel, 1997), hypoglycemic (Salem et al., 2017), antibacterial (Shimoda et al. 2003), antioxidant (Salekzamani et al., 2019), anti-inflammatory and immunomod- ulatory (El-Boshy et al., 2017).

There is little evidence to indicate arti- choke is useful to alleviate hemolytic anemia. Therefore, this study was undertaken to eval- uate the putative antioxidant action of Cynara scolymus extract in an experimental model of PHZ-induced toxicity in Wistar rats.

Materials and methods

Chemicals and preparation of extract

The ethanolic extract of artichoke that was used in this study was purchased from Dineh Iran Industries Complex (Pharmaceutical Company, Tehran, Iran). Phenylhydrazine (PHZ) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Animals housing conditions

This study was carried out on 30 mature male Wistar rats, weighing approximate-

ly 210-220 gr that were obtained from the Animal Care Unit of Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran. The rats were acclimatized for approx- imately one week before use. The animals were housed in stainless steel cages under controlled environmental conditions with temperature (22±2 °C), relative humidity of 55±5% and lighting (12-h light/12-h dark cycle). All rats were fed with a standard lab- oratory pelleted chow diet and fresh  water ad libitum. Animal experiments used in this study were approved by the Animal Ethics Committee of Razi University following the Guidelines for the Care and Use of Labora- tory Animals in Research (Animal Ethical Approval Number: 397-2-008).

Experimental procedure

After one week of acclimatization, the rats were randomly assigned into five groups with six animals per group. In group 1, animals were orally and daily injected with normal saline and served as the normal control. In groups 2 to 5, anemia was induced by Intraperioneal (IP) in- jection of PHZ at 40 mg/kg for 2 consecutive days. Rats that developed anemia with hemo- globin (Hb) concentration lower than 14 g/dl were used for the study. The anemic rats were randomly divided into four groups and treated as follows: In group 2, rats were given normal saline orally and served as the anemic control. Groups 3, 4 and 5, anemic rats daily received the hydroalcoholic extract of C. scolymus at doses of 100, 200, 400 mg/kg body weight by oral gavage, respectively. The normal saline and hydroalcoholic extract of C. scolymus was administered from days 2 to 15 after PHZ in- jection. The experimental period was 14 days. In this study, dose of PHZ to induce anemia and

C. scolymus doses were determined according to previous studies (Diallo et al., 2008; Lee et al., 2012; Salem et al., 2017).

Sample collection

At the end of the study period, all rats were weighed and then anesthetized using dieth- yl ether. Blood samples were taken from the heart to determine the hematological and an- tioxidant parameters. For erythrocyte prepa- ration, the erythrocytes were sedimented by centrifugation at 500 g for 10 min at 4 °C. The erythrocytes were washed  three  times (5 ml, each) with cold isotonic saline then the buffy coat was discarded. RBCs and sera were stored at -20 °C until assayed.

Determination of hematological parameters

For hematological analyses, blood was ex- amined using an automatic hematology ana- lyzer (Celltac, Alpha Vet MEK-6550; Nihon Kohden Co, Tokyo, Japan).

Measurement of total antioxidant capacity (TAC) levels in serum and erythrocytes

Spectrophotometer analysis with the aid of colorimetric assay kit (Naxifer™,  Navand- salamat Co., Iran) was used to estimate the concentrations of TAC in RBCs and serum by the ferric reducing ability (FRAP) meth- od. This procedure is based on the ability of serum or RBC lysis to reduce iron III (Fe3+) to iron II (Fe2+) in the presence of 2,4,6-Tripyr- idyl-S-triazine (TPTZ). A complex with blue color and maximum absorbance appeared at 593 nm with reaction of Fe2+ and TPTZ. The serum level of TAC was expressed in nano- moles per milliliter (nmol/ml) and nmol per g of hemoglobin (nmol/gHb) for RBC lysis.

Measurement of malondialdehyde (MDA) levels in serum and erythrocytes

The levels of lipid peroxidation in the serum and RBCs lysis were measured as TBARS using a Nalondi™ assay kit (Na- vandsalamat Co., Iran). Sera were assayed directly using the kit. The RBCs were first Lysed in deionized water containing butylat- ed hydroxytoluene (BHT) provided in the kit,

and were subsequently centrifuged  at 10,000 g for 5 min to collect the supernatant. Serum MDA level was expressed as nmol/ml and it is also expressed in nmol/g Hb in RBCs.

Measurement of superoxide dismutase (SOD) levels in serum and erythrocytes

Copper, zincsuperoxide dismutase (SOD) was assessed by the pyrogallol assay of Nas- dox™ kit (Navandsalamat Co., Iran). The method depends on the spontaneous autoxi- dation of pyrogallol at alkaline pH, resulting in the production of superoxide anion radi- cals which cause enhanced autoxidation of pyrogallol. Autoxidation is manifested as an increase in absorbance at 420 nm. Specific activity of SOD was calculated as units/ml (unit/g Hb for RBCs), in which one unit of enzyme gave 50% inhibition of pyrogallol autoxidation.

Statistical analysis

The obtained data were analyzed by the One-way Analysis of Variance (ANOVA) and Tukey’s HSD post-hoc test using IBM SPSS software version 21.0 for Windows. The results were expressed as mean val-

ues±SD. The level of statistical significance was set at P≤ 0.05.

Results

Effect of cynara scolymus extract on hematological parameters

The effects of Cynara scolymus extract on hematological parameters in anemic rats are presented in Table 1. The PHZ group showed significant decrease in PCV, Hb, RBC, MCV and MCHC levels compared with the control group (P≤ 0.05). Administration of HECS (100-400 mg/kg) significantly improved the decreased levels of above mentioned pa- rameters (mainly at doses of 200 and 400 mg/kg) compared to PHZ-treated rats (P≤ 0.05). There was also a significant increase in levels of WBC and lymphocyte in PHZ group when compared with control group (P≤ 0.05). Treatment with artichoke extract only at 400 mg/kg has an increasing impact on WBC level compared to PHZ-exposure group (P≤ 0.05). Furthermore, we did not observe significant differences in neutrophil levels among all groups (P>0.05).

               
Table 1. Effect of Cynara scolymus Extract on Hematological Parameters in PHZ-Induced Anemic Rats

Hematological Parameters              Control                     PHZ                  PHZ+HECS

PHZ+HECS

PHZ+HECS

Data are expressed as mean±SD (n=6). * P≤0.05 as compared with control group within each row; # P≤0.05 as compared with PHZ‌-treated group within each row. PHZ: Phenylhydrazine; HECS: Hydroethanolic Extract of Cynara scolymus; PCV: Packed Cell Volume; Hb: Hemoglobin; RBC: Red Blood Cell; MCV: Mean Corpuscular Volume; MCHC: Mean Corpuscular Hemoglobin Concentration; WBC: White Blood Cell.

Effect of cynara scolymus extract on TAC levels in serum and erythrocytes

PHZ-induced anemic rats showed a sig- nificant reduction of serum and erythro- cytes TAC level as compared to the healthy control group (P≤ 0.05). Administration of Cynara scolymus extract at doses of 200

mg/kg and 400 mg/kg considerably in- creased these parameters toward the control value (P≤ 0.05). Significant difference in the TAC level between the groups receiving the HECS in anemic rats induced with PHZ was also observed in the current study (P≤ 0.05) (Figure 1 and Figure 2).

Figure 1. Effect of Cynara scolymus extract at different doses on serum TAC concen- tration in PHZ-induced anemic rats. Data are expressed as mean±SD (n=6). * P≤ 0.05.

PHZ: Phenylhydrazine; HECS: Hydroethanolic Extract of Cynara scolymus

Figure 2. Effect of Cynara scolymus extract at different doses on TAC concentration in eryth- rocyte in PHZ-induced anemic rats. Data are expressed as mean±SD (n=6). * P≤ 0.05.

PHZ: Phenylhydrazine; HECS: Hydroethanolic Extract of Cynara scolymus

Effect of cynara scolymus extract on MDA levels in serum and erythrocytes

A marked increase in the MDA  level  was found in the serum and erythrocytes   of PHZ-exposed rats relative to  normal  rats (P≤ 0.05). The treatment of anemic  rats with increasing concentrations of Cy- nara scolymus extract (100-400 mg/kg)

was significantly effective in decreasing  the elevated level of MDA (P≤ 0.05). No significant difference was observed in se- rum MDA level between HECS treated groups (P>0.05) but a decline in efficacy   of HECS in MDA concentration in RBCs was in a dose-dependent manner (P≤ 0.05) (Figure 3 and Figure 4).

Figure 3. Effect of Cynara scolymus extract at different doses on serum MDA concen- tration in PHZ-induced anemic rats. Data are expressed as mean±SD (n=6). * P≤ 0.05.

PHZ: Phenylhydrazine; HECS: Hydroethanolic Extract of Cynara scolymus

Figure 4. Effect of Cynara scolymus extract at different doses on MDA concentration in eryth- rocyte in PHZ-induced anemic rats. Data are expressed as mean±SD (n=6). * P≤ 0.05.

PHZ: Phenylhydrazine; HECS: Hydroethanolic Extract of Cynara scolymus

Effect of cynara scolymus extract on SOD levels in serum and erythrocytes

The SOD activity in serum and eryth- rocytes has significantly decreased in PHZ group when compared with the normal control group (P≤ 0.05). Sig- nificant increase of serum and erythro- cytes SOD level was determined in rats

treated with Cynara scolymus extract at different doses, especially at 200 mg/kg and 400 mg/kg, as compared to untreat- ed anemic rats. Also, statistically signif- icant difference in the SOD level was ascertained between the rats in the vari- ous groups treated with HECS (P≤ 0.05) (Figure 5 and Figure 6).

Figure 5. Effect of Cynara scolymus extract at different doses on serum SOD concen- tration in PHZ-induced anemic rats. Data are expressed as mean±SD (n=6). * P≤ 0.05.

PHZ: Phenylhydrazine; HECS: Hydroethanolic Extract of Cynara scolymus

Figure 6. Effect of Cynara scolymus extract at different doses on SOD concentration in eryth- rocyte in phenylhydrazine-induced anemic rats. Data are expressed as mean±SD (n=6). * P≤

0.05. PHZ: Phenylhydrazine; HECS: Hydroethanolic Extract of Cynara scolymus

Discussion

The purpose of the study was to verify  the antioxidant effects of artichoke  extract on serum and erythrocytes antioxidant ca- pacity following hemolytic anemia induced by PHZ injection. Our results indicated that the injection of PHZ significantly decreased Hb and PCV levels in comparison with the control group. In PHZ-induced anemia, ROS-mediated damage of RBCs membrane components is thought to increase lipid per- oxidation, erythrocyte membrane  rigidity and fragility. In this regard, intravascular hemolysis was increased and higher levels  of hemoglobin released into the serum (Pan- dey et al.,  2014).  Extract-treated  animals  in this study have enhanced Hb, PCV lev- els and RBCs numbers in comparison with PHZ group. Therefore, the beneficial impact of ethanol extract of artichoke in improving hematinic profile in PHZ-induced anemia may be due to reducing ROS which leads to establishing fragility of RBCs and restoring the total Hb concentration in the blood (Avcı et al., 2006; Rezazadeh et al., 2012, Salek- zamani et al., 2019). On the other hand, it was reported that Cynara scolymus is rich in phenolic compounds belonging to different classes such as benzoic and cinnamic deriv- atives, flavonoids and tannins (Lattanzio et al., 2009). Salem et al. (2017) indicated that flavonoids could induce secretion of eryth- ropoietin, which stimulates stem cells in the bone marrow to produce RBCs.

The present study demonstrated that ad- ministration of PHZ to rats results in a marked leukocytosis in comparison with control group. This leukocyte response is  due to an increase in circulating mononu- clear cells, primarily lymphocytes. The re- sults of present study are in line with the re- sults of Borley et al. (2010) and Xia et al.

(2014) which indicated that leukocytosis in PHZ-treated animals occurred by increasing oxidative and nitrosative stress. The use of high doses of artichoke extract increased the number of leukocytes compared to the PHZ group, which may be due to its antioxidant capacity and its ability to stimulate the pro- duction of leucocytes and could serve as immune booster (Pérez-García et al., 2000; Zapolska-Downar et al., 2002).

The antioxidant capacity of whole  blood is driven by the circulating RBCs that act as mobile scavengers and exert their antioxi- dant properties (Siems et al., 2000). If oxi- dative stress persists, the production of free radicals is greater than the antioxidant capac- ity of erythrocytes or serum. Thus, incidence of hemolytic anemia or sickle cell anemia is imminent (Chan et al., 1999). Measurement of antioxidant molecules is difficult and re- quires time and cost. Therefore, the total an- tioxidant capacity of erythrocytes can be one of the reliable indices to determining oxida- tive stress in erythrocytes and serum (Singh et al., 2018). Administration of PHZ causes ROS production which leads to the hemo- lytic anemia. There are several ways to re- duce the effects of ROS on haematotoxicity, the most common of which are medicinal plants. Natural antioxidants found in foods and vegetables are one of several factors that increase the antioxidant capacity of erythro- cytes (Jimenez-Escrig et al., 2003). The col- lection data of the present study revealed that TAC level of serum and erythrocytes in PHZ group decreased in comparison with control group, which is attributed to increased oxi- dative stress and ROS generation following PHZ administration (Shukla et al., 2012). Administration of Cynara scolymus extract considerably increased TAC level as com- pared to the PHZ group. This effect of arti-

choke extract might be dependent on its fla- vonoids. According to previous research, it was demonstrated that phenolics and flavo- noids compounds have a highly antioxidant effect by reducing ROS (Petropoulos et al., 2017; Sahebkar et al., 2018).

Evaluation of MDA concentrations in serum and erythrocytes showed a significant increase in PHZ group compared to the control group. which in line with the results of Mozafari et al.(Mozafari et al., 2016). The level of lipid peroxide is a measure of membrane damage and alterations in the structure and function of cellular membranes (Salem et al., 2017).

One of the important oxidation products is MDA, which is considered as the main mark- er of lipid peroxidation in oxidative stress, inflammation and various health disorders (Singh et al., 2014). Recent studies have sug- gested that increase in MDA concentrations is one of the most important mechanisms of PHZ in development of hemolytic anemia (Gaschler et al., 2017; Gheith et al., 2018). Erythrocytes and erythrocyte membrane are more vulnerable to lipid peroxidation and ox- ygen radical formation because of constant exposure to high oxygen tension and richness in polyunsaturated fatty acid. Alterations in lipid composition of RBC membranes result in morphologically abnormal erythrocytes with decreased life span (Revin et al., 2016). It has been suggested that the membrane lip- ids play crucial roles in cellular homeostasis including the maintenance of erythrocytes structure, cellular function and viability. In- creased ROS, as stated, increases lipid per- oxidation and produces substances that are toxic to cells and disrupt cells structure (i.e., hemolysis) (Pandey et al., 2014; Mladenov et al., 2015). The present work showed that Cynara scolymus extract resulted in a signif- icant reduction of the MDA concentrations in

PHZ-exposed rats. Hence, it is possible that the mechanism of this protection may be due to its antioxidant activity. In this regard, recent study has shown that Bidens pilosa extract with its antioxidant properties, could prevent oxidative damage and reduce MDA level in erythrocytes (Yang et al., 2006).

Our results revealed that SOD activity in serum and erythrocytes has significantly de- creased in PHZ group when compared with the control group. The reduction in the SOD activity may be due to the oxidation of cys- teine in this enzyme by superoxide anion during its transformation to hydrogen per- oxide. In other words, the decrease in SOD activity in PHZ-induced anemic rats may be due to the use of this enzyme in converting the superoxide anion (O2•−) to hydrogen per-

oxide  (H2O2)  (Plestina-Borjan  et al., 2015).

In addition, PHZ generates free radicals and ROS such as superoxide anion and hydrogen peroxide (Kamisah et al., 2014). Moreover, increased lipid peroxidation induced by PHZ in the serum and erythrocytes was accompa- nied by a decrease in SOD activity, which was also reported by recent studies (Maity et al., 2013; Anbara et al., 2015). Erythrocytes contain several biological defense mecha- nisms against free radical-induced injury, including many antioxidant enzymes  such  as SOD (Kolanjiappan et al., 2002). Further- more, SOD could inhibit the accumulation of membrane proteins, which might prevent the disruption or destruction of erythrocyte wall proteins. Also, SOD activity in the cytosol  of erythrocytes is about 3 times more than serum, so it could help modulate the oxidant effects of PHZ in erythrocytes. Given these points, measuring SOD can be an important criterion for the prognosis of diseases that cause hemolysis or damage to erythrocytes (Valenzuela  et al.,  1977;  Pandey  and Rizvi,