Irvingia gabonensis Seed Extract: An Effective Attenuator of Doxorubicin-Mediated Cardiotoxicity in Wistar Rats

Cardiotoxicity as an off-target effect of doxorubicin therapy is a major limiting factor for its clinical use as a choice cytotoxic agent. Seeds of Irvingia gabonensis have been reported to possess both nutritional and medicinal values which include antidiabetic, weight losing, antihyperlipidemic, and antioxidative effects. Protective effects of Irvingia gabonensis ethanol seed extract (IGESE) was investigated in doxorubicin (DOX)-mediated cardiotoxicity induced with single intraperitoneal injection of 15 mg/kg of DOX following the oral pretreatments of Wistar rats with 100-400 mg/kg/day of IGESE for 10 days, using serum cardiac enzyme markers (cardiac troponin I (cTI) and lactate dehydrogenase (LDH)), cardiac tissue oxidative stress markers (catalase (CAT), malonyldialdehyde (MDA), superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione peroxidase (GSH-Px), and reduced glutathione (GSH)), and cardiac histopathology endpoints. In addition, both qualitative and quantitative analyses to determine IGESE's secondary metabolites profile and its in vitro antioxidant activities were also conducted. Results revealed that serum cTnI and LDH were significantly elevated by the DOX treatment. Similarly, activities of tissue SOD, CAT, GST, and GSH levels were profoundly reduced, while GPx activity and MDA levels were profoundly increased by DOX treatment. These biochemical changes were associated with microthrombi formation in the DOX-treated cardiac tissues on histological examination. However, oral pretreatments with 100-400 mg/kg/day of IGESE dissolved in 5% DMSO in distilled water significantly attenuated increases in the serum cTnI and LDH, prevented significant alterations in the serum lipid profile and the tissue activities and levels of oxidative stress markers while improving cardiovascular disease risk indices and DOX-induced histopathological lesions. The in vitro antioxidant studies showed IGESE to have good antioxidant profile and contained 56 major secondary metabolites prominent among which are γ-sitosterol, Phytol, neophytadiene, stigmasterol, vitamin E, hexadecanoic acid and its ethyl ester, Phytyl palmitate, campesterol, lupeol, and squalene. Overall, both the in vitro and in vivo findings indicate that IGESE may be a promising prophylactic cardioprotective agent against DOX-induced cardiotoxicity, at least in part mediated via IGESE's antioxidant and free radical scavenging and antithrombotic mechanisms.


Introduction
Doxorubicin (otherwise known as Adriamycin) is one of the antibiotic cytotoxic agent belonging to the anthracycline class of anticancer agents [1]. Doxorubicin is known to bind to and intercalate with DNA, thereby inhibiting the resealing action of topoisomerase II during normal DNA replication needed for cancer cell division and growth [2][3][4][5]. Doxorubicin is often used in clinical setting in combination with other classes of anticancer agents as "chemo cocktail" in the management of various types of solid and blood cancers such as breast and ovarian, leukemia (acute myelogenous leukemia (AML) and acute lymphoblastic leukemia), Hodgkin lymphoma, non-Hodgkin lymphoma, Wilm's tumor, neuroblastoma, and sarcoma [6][7][8]. For example, for breast cancer management, doxorubicin is typically combined and given with cyclophosphamide; for lymphomas and leukemias, it is combined with other cytotoxic agents to make regimens like CHOP (cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, and prednisone), R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, and prednisone), and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) [9][10][11][12]. However, the clinical use of doxorubicin have been reported to be associated with major common side effects such as pain at the injection site, anorexia, fever, nausea and vomiting, stomatitis, dyspnea, nose bleeding, alopecia, immunosuppression, weight gain, hepatic and renal injuries, and severe cardiotoxicity [3,13], while its occasional side effects include hyperuricemia, heart failure, pericardial effusion, cardiomyopathy, conjunctivitis, and skin rashes [14,15]. Of these side effects, cumulative and dose-related cardiomyopathy and heart failure are of grave concerns to cancer patients and managing physicians alike, thus, limiting its clinical use [16][17][18]. Although the pathogenesis of doxorubicin-induced cardiotoxicity has been reported to be complex and fuzzy, the pivotal role of ironmediated formation of reactive oxygen species (ROS) cannot be underscored [19].
In preventing the development of doxorubicin-induced cardiotoxicity, chemocurative and chemopreventive strategies involving the use of flavonoids, especially monoHER, have been advocated [20,21]. MonoHER has been reported to elicit potent antioxidant, iron chelating, and carbonyl reductase inhibiting effects while still protecting the antitumor activity of anthracycline anticancer agents [22]. Similarly, the effectiveness of dexrazoxane (an iron chelating agent) [5,23], dextromethionine [24,25], and angiotensinconverting enzyme inhibitors-zofenopril and lisinopril [26,27]-in ameliorating doxorubicin-related cardiotoxicity have also been reported. These agents, especially dexrazoxane, are known to mitigate oxidative stress by chelating iron and catalytically inhibiting topoisomerase II, thus preventing doxorubicin-induced double strand DNA breaks [28,29]. However, these chemopreventive agents are expensive and not readily accessible to patients, therefore, necessitating the need for the discovery and development of more effective but cheaper and more readily accessible alternatives especially ones of medicinal plant origin. One of these is the Irvingia gabonensis seed extract.
Irvingia gabonensis (Aubry-Le Comte ex O'Rorke) Bail belonging to the family, Irvingiaceae, is known as African Mango (in English). Its other common names include bread tree, African wild mango, wild mango, and bush mango [30,31], and its local names include Apon (in Yoruba, Southwest Nigeria), Ogbono (in Igbo, Southeast Nigeria), and Goron or biri (in Hausa, Northern Nigeria) [32,33]. Irvingia gabonensis is widely cultivated in West African countries including southwest and southeast Nigeria, southern Cameroon, Côte d'Ivoire, Ghana, Togo, and Benin, to produce its edible fruit whose seed is used in the preparation of local delicious viscous soup for swallowing yam and cassava puddings [34]. Fat extracted from its seeds is commonly known as dika fat and majorly consists of C12 and C14 fatty acids, alongside with smaller quantities of C10, C16, and C18, glycerides and proteins [34]. Irvingia gabonensis seeds are also a good source of nutrients including a variety of vitamins and minerals such as sodium, calcium, magnesium, phosphorus, and iron. It is also a rich source of flavonoids (quercetin and kaempferol), ellagic acid, mono-, di-, and tri-O-methylellagic acids, and their glycosides which are potent antioxidants [35,36].
In view of the above, the current study was designed at evaluating the possible protective effect of the crude nondefatted ethanol seed extract of Irvingia gabonensis against doxorubicin-mediated cardiotoxicity in rats using cardiac injury markers, oxidative stress markers, and histopathology results as endpoint outcomes.

Extraction Process and Calculation of Percentage Yield.
For Irvingia gabonensis seed extraction, 3 kg of pulverized Irvingia gabonensis dried seeds was macerated in 12 L of absolute ethanol for 72 hours after which it was continuously stirred for 1 hour before it was filtered using 180 mm of filter paper. The filtrate was then concentrated at 40°C to complete dryness using rotary evaporator. The dark-colored, oily paste-like residue left behind was weighed, stored in airand water-proof container which was kept in a refrigerator at 4°C. This extraction process was repeated for two more times. From the stock, fresh solutions were made whenever required.
% yield was calculated as fweight of crude extract obtained ðgÞ ÷ weight of pulverized dry seed extracted ðgÞg × 100:

Preliminary Quantitative Determination of Secondary
Metabolites in and Phytoscan of IGESE. Preliminary quantitative analysis of the secondary metabolites (including phenol, flavonoids, tannin, terpenoids, steroids, reducing sugars, saponin, and phlobatannin) in IGESE was done using methods earlier described by Olorundare et al. [42]. Similarly, using gas chromatography-mass spectrophotometer (GC-MS) for phytoscan, the relative abundance of the secondary metabolites in IGESE was done using the procedures earlier described by Olorundare et al. [42].  [43]. The rats were maintained on standard rat feed (Ladokun Feeds, Ibadan, Oyo State, Nigeria) and potable water which were made available ad libitum. The rats were maintained at an ambient temperature between 28 and 30°C, humidity of 55 ± 5%, and standard (natural) photoperiod of approximately 12/12 hours of alternating light and dark periodicity.

In
2.6. Measurement of Body Weight. The rat body weights were taken at the beginning and last of the experiment using a digital rodent weighing scale (®Virgo Electronic Compact Scale, New Delhi, India). The obtained values were expressed in grams (g).

Induction of DOX-Induced Cardiotoxicity and Treatment of Rats.
Prior to commencement of the experiment, rats were randomly allotted into 7 groups of 7 rats per group such that the weight difference between and within groups was not more than ±20% of the average weight of the sample population of rats used for the study. However, the choice of the therapeutic dose range of 100, 200, and 400 mg/kg/day of IGESE was made based on the result of the orientation studies conducted. Treatments of rats with distilled water, 100-400 mg/kg/day of IGESE in 5% DMSO distilled water, 20 mg/kg/day of vitamin C (standard antioxidant drug) for 10 days, and subsequent treatment with single intraperito-neal dose (15 mg/kg) doxorubicin in 0.9% normal saline on day 11 are as indicated in Table 1. 2.8. Collection of Blood Samples. 72 hours postdoxorubicin injection, overnight fasted rats were humanely sacrificed under light inhaled diethyl ether anesthesia, and whole blood samples were collected directly from the heart with fine 21G injectable needle and 5 ml syringe without causing damage to the heart tissues. The rat heart, liver, kidneys, and testes were carefully identified, harvested, and weighed.
2.9. Bioassays. Blood samples collected into 10 ml plain sample bottles were allowed to clot at room temperature for 6 hours and then centrifuged at 5000 rpm to separate clear sera from the clotted blood samples. The clear samples were obtained for assays of the following biochemical parameters: serum cardiac troponin I, LDH, TG, TC, and cholesterol fractions (HDL-c, LDL-c) using estimated standard bioassay procedures and commercial kits.

Determination of Cardiac Tissue Antioxidant Profile.
After the rats were sacrificed humanely under inhaled diethyl ether, the heart was harvested en bloc. The heart was gently and carefully divided into two halves (each consisting of the atrium and ventricle) using a new surgical blade. The left half of the heart was briskly rinsed in ice-cold 1.15% KCl solution in order to preserve the oxidative enzyme activities of the heart before being placed in a clean sample bottle which itself was in an ice-pack filled cooler. This is to prevent the breakdown of the oxidative stress enzymes in these organs.
Activities of cardiac tissue oxidative stress markers such as SOD, CAT, MAD, GSH, GPx, and GST were assays using methods earlier described by Olorundare et al. [42].
2.12. Histopathological Studies. The right halves of the seven randomly selected rats from each treatment and control groups were subjected to histopathological examinations; the choice of the right ventricle was based on its reported most susceptibility to doxorubicin toxicity of the four heart chambers. The dissected right heart half was briskly rinsed in normal saline and then preserved in 10% formo-saline. It was then completely dehydrated in 100% ethanol before it was embedded in routine paraffin blocks. 4-5 μm thick sections of the cardiac tissue were prepared from these paraffin blocks and stained with hematoxylin-eosin. These were examined under a photomicroscope connected to a host computer for any associated histopathological lesions.
2.13. Statistical Analysis. Data were presented as mean ± S:E :M: of four observations for the in vitro studies and mean ± S:D: of seven observations for the in vivo studies, respectively. Statistical analysis was done using a two-way analysis of variance followed by the Student-Newman-Keuls test on GraphPad Prism Version 5. Statistical significance was considered at p < 0:05, p < 0:001, and p < 0:0001.

% Yield.
Complete extraction of Irvingia gabonensis ethanol seed extract in absolute ethanol resulted in an average yield of 4.31%, which was a very dark brown, oily, and sweet-smelling paste-like residue that was soluble in methanol and ethanol but not in water.

Effect of Oral IGESE Pretreatment on Cardiovascular Risk
Indices (AI and CRI) of DOX-Treated Rats. Acute intraperitoneal injections with DOX resulted in significant (p < 0:001) increases in the AI and CRI values when compared to Groups I and II values (Table 10). However, with oral pretreatment with 100-400 mg/kg/day of IGESE, there were significant (p < 0:05, p < 0:001, and p < 0:0001) dose-related decreases in the AI and CRI values with similar effect induced by oral pretreatments with 20 mg/kg/day of Vit. C (Table 10).

Histopathological Studies of the Effect of IGESE Oral
Pretreatment on DOX-Intoxicated Treated Heart. Figure 2 is a photomicrograph of a cross-sectional representative of DOX-only treated heart showing myocyte congestion and antemortem coronary microthrombi when compared to untreated normal ( Figure 3) and IGESE-only treated heart tissues with normal cardiac architecture ( Figure 4). However, pretreatment with varying doses of IGESE resulted in doserelated improvements in the histological distortions induced by DOX especially at 200 mg/kg/day ( Figure 5) and 400 mg/kg/day of IGESE ( Figure 6); although, histological features of vascular congestion were still seen with 100 mg/kg/day of IGESE oral pretreatment (Figure 7). On the contrary, there were histological features of persistent coronary microthrombi in rat heart pretreated with 20 mg/kg/day of Vit. C, indicating the lingering DOX-induced histological lesions, even with the standard antioxidant drug (Figure 8).

Discussion
The clinical use of doxorubicin in the management of solid and hematological cancers has been widely limited by its off-target severe cardiotoxicity which manifests biochemically by elevation of serum enzyme markers of cardiotoxicity. The diagnostic serum marker enzymes of cardiotoxicity are    7 Oxidative Medicine and Cellular Longevity AST, ALT, CK-MB, LDH, and cTnI which leak from cardiac tissue damage to the bloodstream due to their tissue specificity and serum catalytic activity [46]. DOX administration may result in the damage to the myocardial cell membrane or make myocytes more permeable, resulting in the leakage of the diagnostic cardiac enzyme markers cardiac AST, ALT, CK-MB, LDH, and cTnI into the bloodstream and their high circulating levels. In the present study, DOX-mediated cardiotoxicity was fully established as evidenced by the profound elevations in the serum cTnI and LDH levels which is in complete agreement with previous studies [47][48][49][50][51][52]. With oral IGESE pretreatments, the serum levels of cTnI and LDH were profoundly attenuated toward normal serum level indicating the ameliorative potential of IGESE in DOX-mediated cardiotoxicity. These effects were probably mediated through high antioxidant and/or free radical scavenging activities of IGESE on the myocardium, thus reducing the damaging effects of DOX to the cardiac muscle fibers, subsequently minimizing the leakage of such enzymes in the serum. Similarly, ROS-mediated mechanism is one of the proposed DOXmediated cardiotoxicity mechanisms, leading to oxidative stress that causes cardiomyopathy [53]. Oxidative stress has been reported to increase lipid peroxidation as indicated by an increase in MDA levels and altered enzymatic and nonenzymatic antioxidant systems [54,55]. In this study, MDA level was profoundly increased by DOX treatment, while DOX treatment also suppressed the cardiac tissue activities of SOD, CAT, GPx, GST, and GSH levels in the treated rats in agreement with other studies. These altered biochemical alterations were supported by histological lesions characterized by myocyte congestion and coronary intravascular microthrombi formation. DOX has been previously reported to profoundly reduce vascular blood flow, disintegrate vascular endothelium, and promote GPIIb/IIIa-mediated platelet adhesion and aggregation, all resulting in microthrombi formation [56][57][58]. The fact that IGESE prevented microthrombi formation in DOX-treated coronary vasculature as evidenced by histopathological results of this study highlighted the possible inherent antithrombotic potential of IGESE; although, further studies are still needed in this respect in order to validate this hypothesis. However, IGESE profoundly attenuated      Oxidative Medicine and Cellular Longevity significant alterations in the cardiac tissue oxidative markers whose activities were significantly suppressed by DOX intoxication. IGESE has the tendency to neutralize ROS like superoxide radicals, singlet oxygen, nitric oxide, and peroxynitrite, thereby reducing the damage to lipid membranes [39]. Similarly, oral IGESE pretreatments profoundly improved and reversed the DOX-induced histological lesions especially at 200 mg/kg/day and 400 mg/kg/day of IGESE pretreatments. The effects of DOX on serum lipids are also significant. DOX has been reported to cause hyperlipidemia (which include increased serum cholesterol, triglyceride, LDL-c, and FFAs) [59][60][61][62][63][64] and increases cardiovascular disease risk [65]. This hyperlipidemia is thought to be mediated via downregulation of PPAR-γ and subsequently affect GLUT4 and FAT/CD36 expression resulting in glucose and fatty acid transporters expression and causing hyperglycemia and hyperlipidemia [65]. Irvingia gabonensis seeds have been reported to induce weight loss, antihyperlipidemia, and reduced cardiovascular disease risk factors in both animal [59][60][61][62][63][64] and human studies [66][67][68][69][70][71][72] which were reportedly mediated via downregulation of the PPAR-γ and leptin genes and upregulation of the adiponectin gene mechanisms [67]. Thus, the results of this study are in tandem with those of earlier studies.

Conclusion
Overall, results of this study showed that IGESE effectively attenuated DOX-mediated cardiotoxicity and its cardioprotective activities were mediated via antioxidant, free radical scavenging, antilipoperoxidation, and antithrombotic mechanisms.

Data Availability
Answer: Yes. Comment

Conflicts of Interest
The authors have none to declare.

Authors' Contributions
Olufunke Olorundare designed the experimental protocol for this study and was involved in the manuscript writing; Adejuwon Adeneye supervised the research, analyzed data, and wrote the manuscript; Akinyele Akinsola and Olalekan Agede are postgraduate students in Olufunke Olorundare's laboratory that performed the laboratory research under supervision; Phillip Kolo was part of the protocol design and read through the manuscript; Ikechukwu Okoye prepared the cardiac tissue slides; Sunday Soyemi and Alban Mgbehoma independently read and interpreted the cardiac tissue slides; Ralph Albrecht and Hasan Mukhtar are our collaborators in the U.S.A. who read through the manuscript.