Evaluation of Cardioprotective Potential of Hydroalcoholic Leaf Extract of Citrullus colocynthis against Doxorubicin Induced Oxidative Stress in Rats

1Fatima-Tu-Zahra, Department of Life Sciences Muhammad Institute of Medical and Allied Sciences, Multan, Pakistan 2Department of Pharmacology, The Islamia University of Bahawalpur, Bahawalpur, Pakistan 3Ali-Ul-Murtaza, Department of Rehabilitation Sciences, Muhammad Institute of Medical and Allied Sciences, Multan, Pakistan 4Department of Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, Ocean University of China, China 5Department of Pharmacology and Toxicology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan Article Information Received 15 July 2021 Revised 05 October 2021 Accepted 27 October 2021 Available online 21 February 2022 (early access)


D oxorubicin (DOX) was first isolated in 1960 from
bacteria Streptomyces peucetius and it belongs to the family of anthracyclines. DOX is very useful for treating lymphoma, leukemia and solid cancer (Slingerland et al., 2012). It is used as an anticancer drug and DOX (hydrochloride) liposomal injection became the first liposomal-encapsulated anticancer agent that received clinical approval (Slingerland et al., 2012). In 1967, it was O n l i n e

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acute phase occurs in less than one percent patients and it is reversible if the therapy stopped (Xiong et al., 2006), while within one week, subacute form appears (Curigliano et al., 2010). The chronic phase occurs within one year due to decreased activity of left ventricular portion of heart and leads to myocardial infarction (Gharib and Burnett, 2002). Several pathways have been linked to DOX-induced cardiomyopathy, such as stimulation and inactivation of enzymatic pathways, oxidative stress, exaggerated immune response and functional down regulation of proteins present in mitochondria and cardiac muscles. These multiple pathological events altered the signaling pathways and activates the apoptotic cascade that results in the reduction of nucleic acid, loss of myofibril, and disruption of sarcomere structure (Abushouk et al., 2019). So, the enhanced mitochondria-to-myocyte ratio increased the susceptibility of cardio myocytesto oxidative stress and proposed a main factor in pathogenesis that can be compared with the aforementioned mechanisms. DOX-semiquinone is an unstable DOX metabolite which combines with Oxygen (O 2 ) molecule to produce H 2 O 2 and O 2− (superoxide) . DOX also increases the quantity of extra mitochondrial oxidative enzymes e.g., xanthine and NADPH oxidase. It also inhibits mitochondrial iron export, that results in the production of Reactive Oxygen Species (ROS). DOX also prevents the functions of endogenous non-enzymatic and enzymatic antioxidants. So, an imbalance occurs between the production of ROS and neutralization that causes oxidative stress and cause a great damage to heart compared with other organs including kidney and liver (Abushouk et al., 2017). Importance of herbal medicines in the cure of different diseases is accepted worldwide. Herbal medicines always provide essential therapeutic agents not only in traditional but also in modern medicines and also because they are less toxic than synthetic medicines (Vakiloddin et al., 2015). Citrullus colocynthis is a medicinal plant belongs to the family Cucurbitaceae. It has a lot of environmental benefits, such as mosquito repellent and larvicidal potential (Rahuman et al., 2008). Its oils are also used as botanical insecticides for the prevention of smoke-dried fish from D. maculatus larvae attack (Akpotu et al., 2015). Its shoots are used as bio-indicators and soil pollution accumulator (Khattak et al., 2015). In Arab, it was used to deal with the skin eruption in camels and for insect bites (Thangavel and Ramasamy, 2019). Traditionally, it was used to treat asthma, cough, edema, constipation (Delazar et al., 2006). Its fruit is used as hepatoprotective (Vakiloddin et al., 2015), and its pulp and seeds as an anti-inflammatory and diabetes (Nmila et al., 2000;Marzouk et al., 2010). Till now there is no recorded experimental foundation for its utilization as acardio protective agent. The current research was performed to evaluate the cardio protective potential of hydro alcoholic leaf extract of C. colocynthis against DOX-induced oxidative stress.

MATERIALS AND METHODS
Authentication and preparation of plant extract C. colocynthis leaves were taken from the botanical garden of Muhammad Institute of Medical and Allied Sciences, Multan. It was authenticated by an expert taxonomist at the Department of Botany, Bahauddin Zakariya University, Multan. The voucher specimen (R.R. Stewart F.W. Pak.702/20) had been placed. The fresh plant was subjected to shade dry. All the foreign adulterants and vegetative wastes were eliminated through manual picking before grinding the leaf part of the plant into a coarse powder with the assistance of a special herbal grinder. C. colocynthis 1kg leaf powder was soaked in a hydroalcoholic solvent (70:30 v/v) for 9 days in amber colored air-tight bottles. Then, it was filtered and the filtrate was evaporated at 37°C under reduced pressure on a rotatory evaporator (specification of rotatory) to obtain a thick paste-like consistency (Bashyal and Guha, 2018).

Animals
Male Wister albino rats, weighing 350-400g, obtained from animal house of Muhammad Institute of Medical and Allied Sciences, Multan. The rats were kept at Fatima-Tu-Zahra, Department of Life Sciences, Muhammad Institute of Medical and Allied Sciences, Multan under 12 h dark and light cycle. All the rats were given standard pallet diet and with water ad libitum. The standard temperature 25°C and humidity 50-60% conditions were maintained. The experiments were performed according to the guidelines issued by the National Institute of Health guide for the care and use of laboratory animals (NIH, 1985) and approved by the Ethical Review Committee of Muhammad Institute of Medical and Allied Science Multan, Pakistan (MIMAS/03/ Biochem/315/20).

Chemicals
Doxorubicin, dexrazoxane, ethanol, distilled water was obtained from M/s. Sigma Chemical Company, St. Louis. MO USA. The rest of the chemicals employed in the research project were of analytical quality. AST, ALP, ALT, LDH, CRPK it's were obtained from GS Chemicals and diagnostics, Multan.

Acute oral toxicity dose test
The acute oral toxicity of the extract was evaluated in 15 albino rats weighing 350-450g. They were divided into O n l i n e

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3 groups and each group contained 5 albino rats and they were kept on fasting for 24 h and dosed in the following manner 2000mg/kg, 4000mg/kg, 6000mg/kg body weight. After the dosing, the rats were noticed for 14 days for lethargy, jerkiness and death (Thippeswamy et al., 2011).

Preliminary phytochemical screening
The absence or presence of various phytochemical classes in C. colocynthis leaf extract was determined using standard phytochemical screening procedures (Thippeswamy et al., 2011).

Determination of DPPH radial scavenging activity
The antioxidant activity of hydro alcoholic leaf extract of C. colocynthis was performed on the base of scavenging activity of stable DPPH (1, 1-diphenyl-1picrylhydrazyl) radical. 0.5ml plant extract was taken in a sample cavity that contained 3.5ml of methanol solution of DPPH (0.004g100ml -1 ). The mixture was incubated for 30 min in the darkness and room temperature and absorbance was measured at 517 nm. The percent inhibition DPPH (I%) was measured by decline in absorbance using the following formula. Low absorbance value represents higher free radical scavenging activity (Ahmed et al., 2019).

Determination of reactive oxygen species
The reactive oxygen species (NO and H 2 O 2 ) were detected by standard methods described by Garrat (1964).

Assay of nitric oxide radical scavenging
Nitric oxide produced from aqueous sodium nitroprusside (SNP) reacts with oxygen at physiological pH to create nitrite ions measured by the Illosvoy Griess reaction. 10 mM SNP, buffered saline (pH 7.4) and varied amounts of test solution were all mixed in a 3-mL reaction mixture. Sulfanilamide (0.33% in 20% glacial acetic acid) was added to 0.5mL of the incubated solution, which was allowed to stand for 5 min before 0.5mL of 0.1M triethanolamine was added and the mixture incubated for a further 75 min at 25°C.0.1% w/v of NED solution was added and the mixture was incubated for 30 min at 25 degrees Celsius.

Hydrogen peroxide scavenging assay
According to the approach of Ruch et al. (1989) the capability of the extract to scavenge hydrogen peroxide (H 2 O 2 ) was examined.0.1 microliters of each of the samples was put into a sterile Eppendorf tube, and the tube was half-filled with 50 mM phosphate buffer (pH 7.4) to make the total volume up to 0.4 mL. This was followed by the addition of 0.6 microliters of H 2 O 2 solution (2 mM). After 10 min of reaction time, the liquid was vortexed and the absorbance was measured at 532 nm. Ascorbic acid was selected as the positive control, since it has antiscorbutic properties. The results of the test and standard sample were compared to calculate percentage inhibition.

Experimental design
The animals had been divided into 5 major groups each group contained 8 rats. Group-1 rats were given saline for 2 weeks and on 11 th day DOX 18mg/kg was injected intraperitoneally after 16h fast. Group-2 rats were pre-treated with 250mg/kg of extract for a time of 2 weeks, and besides administrated DOX 18mg/kg, on the 11 th day after 16 h fast. The rats of Group-3 were pre-treated with 500mg/kg of extract for 2 weeks consecutively and on the 11 th , day DOX 18mg/kg was administrated intraperitoneally after 16 h fast. In preliminary screening we found 500mg/ kg dose significant so that's why we used upper and lower limit. Group-4 rats were pre-treated with extract in an amount of 750 mg/kg for 2 weeks orally and on the 11 th , day DOX 18mg/kg was inoculated intraperitoneally after 16 h fast. Group-5 was the positive control and they were given 30 min before the inoculation of DOX 18mg/kg dexrazoxane (DZR) was administrated intraperitoneally 180 mg/kg after 16 h fast (Sandamali et al., 2020).

Histopathology of heart
After the final dose of DOX, rats had been anesthetized by ketamine and the blood samples of rats had been taken from the retroorbital vein to assess the biochemical parameters including CK-MB, LDH, troponin-I, CRP, ALT, AST and ALP levels in serum were measured by commercially available Kits. Rats had been killed under intense anesthesia by Ketamine and the heart was isolated and for histological examination, then the heart swiftly transferred to a10 % formalin solution. After that tissue had been submerged in the paraffin. A 5μm thick segment had been cut and stained with a hematoxylin-eosin dye then mounted in the diphenyl xyline. The histopathology of cardiac muscle had been determined beneath a compound microscope and micro-images had been taken (Sandamali et al., 2020).

Statistical analysis
The experimental statistics had been demonstrated as mean ± SD. Statistical analysis had been accomplished through one-way ANOVA. The statistical value with p˂0.05 was taken as significant.
Cardioprotective Potential of C. colocynthis O n l i n e

Acute oral toxicity
The acute oral toxicity test showed that any dose of hydro alcoholic leaf extract of C. colocynthis up to 6000 mg/kg did not yield any mortality.

Phytochemical analysis
Phytochemical analysis indicated presence of steroids, flavonoids, saponins, tannins, phenols, alkaloids and glycosides in extract while, terpenoids and flavones were absent in the extract.

DPPH activity
C. colocynthis showed outstanding antioxidant activity when compared with standard Ascorbic acid shown in Figure 1.

Estimation of biochemical parameters
Biochemical estimations showed a highly significant (p<0.001) rise in the level of AST, ALP, ALT, CKMB, CRP, troponin-I, LDH in DOX treated group in comparison with extract-treated Group-2, Group-3, Group-4 and DZR treated Group-5. As shown in Figure 2. Group-2 rats pretreated with 250mg/kg for 14 successive days did not show any cardioprotective activity (p>0.05) and there was a very milddecrease in the levels of biochemical parameters. Group-3 rats pretreated with the extract 500mg/kg for 14 successive days showed a significant decrease (p<0.05) in the level of biochemical parameters. There was a mild to moderate reduction in the biochemical parameters. Group-4 rats pretreated with extract 750mg/ kg for 14 successive days and Group-5 which was given DZR showed a highly significant (p<0.001) reduction in diagnostic biomarkers and cardiac parameters of myocardial injury and maintained the biochemical parameters near to normal level. The positive control was dexrazoxane, which is the only protective drug utilized in medical practice for the treatment of DOX-induced oxidative damage. Although there was a highly significant difference (p <0.001) among the DOX-induced group and the positive control and the extract pre-treated groups at various doses.

Determination of reactive oxygen species
The findings of the nitric oxide-enhancing properties of C. colocynthis revealed significant increases in the nitric oxide scavenging capability of the extract. Maximum activity was seen at 6000μg/ml of the plant extract. When the plant extract concentration increases, the activity of the hydrogen peroxide scavenging increases, the highest concentration at 6000μg/ml has been reported, while the lowest concentration at 2000μg/ml has been recorded as shown in Table I.

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Cardioprotective Potential of C. colocynthis

Histopathological observation of heart tissues
Microscopic observation of the heart tissues from different groups was used to evaluate the DOX injection effect on the cardiac cells structure as well as its variation by the drug test inoculation. Microscopic analysis of heart tissues in the DOX-induced group showed a remarkable change in cardiac cell structure. Such as mono-nucleate cellular infiltration, interstitial edema, disintegration tear of muscular fibers, vacuolar disintegration, distention of capillaries, mottled staining, hemorrhage, and obstruction of the myocardium were observed as shown in Figure  3A. Severe necrotic lesions were noticed in all the rats while, Plant extract treated groups observed decreased histopathological variations in dose dependent fashion as shown in Figure 3B, D. The DZR treated group also showed reduced histopathological variations as shown in Figure 3E (Thippeswamy et al., 2011). Effect of hydroalcoholic leaf extract on histological structure of rats. Heart section from DOXinduced group (B) shows images from heart sections of Extract 250mg/kg + DOX 18mg/kg (C) shows images from heart sections of extract 500 mg/kg + DOX 18mg/kg (D) Shows heart sections from Extract 750mg/kg + DOX 18mg/kg (E)shows images from heart sections of DZR 180mg/kg+ DOX 18mg/kg.

DISCUSSION
Phospholipids that are present in the membranes of mitochondria have high affinity for DOX and when DOX induces it accumulates in the heart cells and causes several changes in function and structure of cardiac tissues (El-Sayed et al., 2011). The enhanced mitochondria to myocyte ratio increased the susceptibility of cardiomyocytes to oxidative stress and proposed a main factor in pathogenesis compared with the aforementioned mechanisms. DOXsemiquinone is an unstable DOX metabolite which combines with oxygen (O 2 ) molecule to produce H 2 O 2 and O 2− (superoxide). DOX also increases the quantity of extra mitochondrial oxidative enzymes e.g., xanthine and NADPH oxidase. It also inhibits mitochondrial iron export that results in the production of Reactive Oxygen Species (ROS). DOX also prevents the functions of endogenous non-enzymatic and enzymatic antioxidants. So, an imbalance occurs between the production of ROS and neutralization that causes oxidative stress and cause a great damage to heart compared with other organs including kidney and liver (Abushouk et al., 2017). Phytochemical screening and antioxidant activity of leaf extract of C. colocynthis showed that it contains glycosides, flavonoids, saponins, alkaloids, steroids, phenols and tannins. These phytochemicals might be responsible for reducing the oxidative stress caused by DOX which is a major contributor to change in cardiac tissue physiology.
The whole concentration of the marker enzyme LDH and CKMB of rats pre-treated with the DOX showed a highly significantly increase (p<0.001). Groups pretreated with C. colocynthis leaf extract decreased the LDH and CKMB level in a dose-dependent fashion, parallel to group pretreated with DZR showed a highly significant reduction in the level of LDH and CKMB as compared to Group-1 (as shown in Fig. 2A, B). This indicates that C. colocynthis pretreated groups showed restriction against the myocardial damage due to oxidative stress caused by DOX which results in less leakage of CKMB and LDH from the myocardium similar to previous studies published against same topic.
The whole concentration of the marker enzyme troponin-I (as shown in Fig. 2C) that is considered as a specific biomarker in the dysfunction of left ventricular portion of heart (as shown in Fig. 2C) of rats were pretreated with the DOX showed a highly significant (p<0.001) increased value, in contrast C. colocynthis extract pretreated groups decreases the troponin-I level in a dose-dependent way. Which confirms its cardioprotective potential by resisting the oxidative stress and tissue damage caused by DOX (Table I). Pretreatment with DZR showed a highly significant (p<0.001) reduction in the troponin-I level as compared to Group-1. The mean concentration of the serum parameter AST (as shown in Fig. 2D) which is considered as a specific marker indicates the damage of cardiomyocytes cell membrane damage (as shown in Fig. 2D) of rats pretreated with the DOX showed a highly significant (p<0.001) increased value in Group-1. While C. colocynthis extract pretreated groups showed marked resistance against the DOX toxicity which is evident through decreased AST level in dose-dependent O n l i n e

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A. Manzoor et al. way. Pretreatment with DZR showed a highly significant (p<0.001) reduction in the AST level as compared to Group-1. The mean concentration of the parameter ALT of rats pretreated with the DOX showed a highly significant (p<0.001) increase (as shown in Fig. 2E), in contrast, C. colocynthis extract decreased the level in dose-dependent way in all three treated groups parallel to DZR pretreated group which showed a highly significant (p<0.001) reduction in the ALT level.
The mean concentration of parameter ALP of rats pretreated with the DOX showed a highly significant increased value in contrast to this, C. colocynthis extract pretreated groups showed decreased ALP level in a dosedependent fashion, similar to group pretreatment with DZR showed a highly significant (p<0.001) reduction in ALP level (Fig. 2F). So many previous studies showed that plant with antioxidant potential produced cardioprotection by decreasing the level of ALP, AST and ALT in DOX induced toxicity (Momin et al., 2012).
The mean concentration of parameter CRP of rats pretreated with the DOX showed a highly significant increased value in contrast to this, C. colocynthis extract pretreated groups showed decreased CRP level in a dosedependent fashion, similar to group pretreated with DZR showed a highly significant (p<0.001) reduction in CRP level (Fig. 2G). As the oxidative damage caused by DOX results in the inflammatory process in the myocardial tissue damage and cell necrosis. It is well documented in so many previous studies that the stressed area undergoes local necrosis, generation of free radicals which ends in apoptosis of cardio myocytes (Wang et al., 2019). This is followed by infiltration of neutrophils and macrophages into the stressed area, initiating myocardial cell damage by releasing the cytokines and proteolytic enzymes. Pretreatment with C. colocynthis extract showed marked decrease in the serum levels of CRP which advocates its cardio protective potential. C. colocynthis leaf extract reported for good anti-inflammatory activity (Rahuman et al., 2008), which further strengthen this claim.
Histological variation resembled the rise in serum enzyme level. Histopathology of the ventricular portion of the heart from DOX treated group showed mitochondrial swelling, dilation of the sarcotubular system, disruption of several sub-cellular elements including formation of lysosomal bodies, vacuolization of the cytoplasm, loss of myofibrils. In Group-4, treatment with the hydroalcoholic leaf extract of C. colocynthis showed less vacuolization of the cytoplasm and disruption of the myofibrils. This further confirms the membrane stabilizing effect of the extract. C. colocynthis offered cardio protectionby decreasing oxidative stress-induced in experimental myocardial infarction by preventing the free radical-mediated damage of DOX assault (Wang et al., 2019).

CONCLUSIONS
Hydroalcoholic leaf extract of Citrullus colocynthis showed cardioprotective potential by decreasing the DOX induced oxidative stress in rats may be due to its strong antioxidant potential that is evident from the amelioration of histological variations as well as by the estimation of various cardiac biomarkers.

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Cardioprotective Potential of C. colocynthis