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Doxorubicin is a standard medication utilized in therapy in cancer and its related form.
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Cumulative and dose-dependent cardiac toxicity of doxorubicin (DOX) has been a major challenge in its usage.
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Paucity of scientific information on medicinal value of some plants is a major concern.
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Antioxidant properties of A. digitata confirm its outstanding protective role.
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Properties of Adansonia digitata is based on some bioactive compounds present.
Summary
Scope
Doxorubicin (DOX), for decades, is one of the most widely used and successful chemotherapeutic drug but its cumulative and dose-dependent cardiac toxicity has been the major concern of oncologists in cancer therapeutic practice.
Aim
In this study, the antioxidative properties of Adansonia digitata leaf extract to protect heart against doxorubicin-induced cardiotoxicity in wistar rats was evaluated.
Material and Methods
Thirty male wistar rats (100±0.35 g) grouped into five of six (6) each: group1 (normal control) received 0.5 mL of distilled water; group 2 received 20 mg/kg of DOX only; group3 treated with 20 mg/kg of DOX +100 mg/kg of A. digitata leaf extract; group4 received 20 mg/kg of DOX + 200 mg/kg of A. digitata leaf extract and group5 was administered 20 mg/kg of DOX+400 mg/kg of A. digitata DOX was administered subcutaneously weekly while extract was fed orally for three weeks respectively.
Result
Quantitative analysis revealed some bioactive compounds: phenols, flavonoids, saponin, alkaloid and phenols (168.26±40, 26.07±0.15, 16.18±0.31 and 10.03±0.10 mg/100ml). Significant increase (P< 0.05) in Troponin T concentration was observed in group 2 against experimental groups administered A. digitata. Similarly, LDH and CK activities increased in all test groups against normal control. Decreased Troponin T, CK, and LDH concentrations were observed in rats fed 400 mg/kg against the groups pretreated with 100 mg/kg and 200 mg/kg bwt of A. digitata. MDA levels decreased significantly (P>.0.05) in the groups orally fed with the extract compared with group2. Variations of activities of SOD, CAT, and GPX were observed in the groups treated with the extracts compared with DOX induced untreated rats. Conversely, Protein concentration increased in all the test groups administered extract as against group2. Histopathological revealed infiltration of inflammatory cells and congestion in the blood vessels. Administration of the extract showed predominantly normal structure without inflammatory cell infiltration.
Conclusion
It has been demonstrated that A. digitata leaf extract exert protective effects on DOX-induced cardio toxicity in wistar rats.
Doxorubicin (DOX) induced cardiomyopathy is considered an extremely serious adverse effect of oncologic treatment. The quality of patient’s life who survived cancer, especially children is significantly affected by this disease. Doxorubicin (DOX) is a standard medication utilized in therapy of sicknesses like intense lymphocytic leukaemia, bosom malignant growth, intense myeloblastic leukaemia, kidney disease, liver malignant growth, lymphoma and bladder disease [
]. However, there is limitation to use of these drugs due to their adverse reactions as well as the loss of beneficial cardiac effects years after the end of the treatment. Enlarged cardiomyopathy is because of DOX prompted cardiomyopathy that makes every one of the four heart chambers to become enlarged [
]. This drug is famous for attaching to and inhibiting DNA topoisomerase II, resulting in DNA damage and membrane damage as well as production of oxidants. The side effect of this helpful agent (DOX) equally includes balding, marrow concealment, weakness, low blood tallies, and obscuring of skin. Attainable unfavourable impacts are hypersensitive responses like anaphylaxis, as well as myocardial dead tissue.
Years ago, diverse molecular mechanisms have been proposed to understand the pathogenesis of acute and chronic DOX-induced cardiotoxicity (DIC). The proposed molecular invents include: oxidative stress, iron metabolism, disruption of Ca2+ homeostasis, sarcomeric structure alterations, gene expression modulation, and apoptosis. Reactive oxygen species (ROS) play a pivotal role in DOX-induced cardiotoxicity and several other mechanisms [
In line with these processes, several approaches have been developed in order to safeguard the heart during the treatment/management of cancer. Applications of iron-chelating compounds and adrenergic receptor agonists have been the possible approach to avert the condition. Scientific community has been challenged with the development of new therapies. A new emergent strategy should have been a cell therapy. But every therapy has toxic and therapeutic windows, and defining the side effects of any new therapeutic modality is the first order of business in the development of a treatment. With transformative therapies such as cell-based approaches, treatment side effects can be unpredictable and unanticipated. Due to unfriendly impacts of these medications, the cardio protective properties of some restorative plants have been researched [
Cardioprotective effect of HPLC standardized ethanolic extract of Terminaliapallida fruits against isoproterenol-induced myocardial infarction in albino rats.
Cardioprotective potential of hydroalcoholic fruit extract of Ananascosmosus against isoproterenol-induced myocardial infarction in Winstar Albino rats.
Andonsonia digitata L. (Baobab) plants are tropical trees, native to Africa, Australia and Madagascar but dispersed widely by humans. A. digitata is commonly found in the thorn woodlands of African savannah, it is a very long-lived tree with multipurpose use. Baobab has numerous biological properties including antimicrobial, anti-malarial, diarrhoea, anaemia, asthma, antiviral, anti-oxidant and anti-inflammatory activities amongst others. Phytochemical investigation revealed the presence of flavonoids, phytosterols, amino acids, fatty acids, vitamins and minerals, glycosides, saponin, steroids [
Phytochemical, Elemental and proximate analyses of stored sun-dried and shade dried BaobabAdansonia digitata) leaves. Preprint Converting enzyme inhibitors and angiotensin II receptor blockers on all cause, mortality cardiovascular deaths and cardiovascular events in patients with diabetes mellitus:Ameta analyses.
]. In reality, because it was found to show interesting pharmacological properties, the whole plant is used for the multiple medicinal purposes in many parts of Africa as well as other states (Jitin et al., 2015). The medicinal property of A. digitata is based on some bioactive compounds that produce a definite physiological action on the human body, these chemical substances have a potential or established biological activity that has been identified and they are known as phytochemicals [
]. Scientific evidence on cardioprotective properties of A. digitata leaf extract has been limited in scientific community. In this context, the research was aimed to investigating protective potential exerts by ethanol leaf extract of A. digitata against doxorubicin-induced cardiac toxicity in wistar rats.
Material and methods
Plant material
Fresh leaves of A. digitata were collected from Kaduna State, Northern part of Nigeria. The plant was identified and authenticated by the a plant taxonomist in the Department of Plant Biology and Biotechnology Herbarium Unit, Faculty of Life Sciences, University of Benin, Benin City, Edo State in collaboration with Department of Plant Science and Biotechnology, Michael Okpara University of Agriculture, Umudike, where voucher specimen (UBH-A473) was kept for referral purposes in the departmental herbarium. The leaves obtained were carefully rinsed under running water, dried under room temperature (250C) in the laboratory. It was milled and weighed before extraction.
Assay kits and chemicals
Most of the chemical materials utilized in the study came from Sigma Chemicals Co. (St Louis, Mo, USA). Doxorubicin was provided by Pfizer Global Pharmaceutical Limited in Nigeria. Reagents for assay of Creatine kinase, Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPX) and Lactate dehydrogenases (LDH) were procured commercially. TroponinT research kits was supplied by Glory Science Co Ltd, www.glorybio.com.
Experimental animals
Thirty (30) male wistar rats of mean weight 100±0.35 g were obtained from the animal house of Department of Zoology and Environmental Science, University of Nigeria, Nsukka. Clean iron cages were used to house the animals under standard environmental conditions of temperature (24±10C) and relative humidity (45–50%) under a 12 h dark-light cycle. Rats were acclimatized for 7 days before dosing and allowed free access to drinking water and standard pellets feed. The research protocols were approved by the Animal Ethics Committee, College of Natural Science, Michael Okpara University of Agriculture, Abia State according to the Guide for the Care and Use of Laboratory Animals.
Preparation of extract
Five hundred grams (500 g) of milled plant material was extracted in 2.0 L ethanol for 72 h at 300C on an orbital shaker (Stuart Scientific Orbital Shaker, UK). This was centrifuged at 1500 rpm for 5 min and the filtrate further filtered with Whatman No. 4 filter paper. It was concentrated using rotary evaporator at 400C and the yield was 21.60g (4.32%). Reconstitution of the sample in distilled water was done to give the required concentrations of, 100, 200 and 400 mg/Kg body weight used in the study.
Design of experimental
A total of thirty male wistar rats were grouped into five of six (6) each: (1) Normal control, received 0.5 mL of distilled water (2) received 20 mg/kg of DOX only (3) received 20 mg/kg of DOX +100 mg/kg of A. digitata leaf extract (4) received 20 mg/kg of DOX + 200 mg/kg of A. digitata leaf extract and (5) received 20 mg/kg of DOX+400 mg/kg of A. digitata.
DOX was administered subcutaneously weekly while extract was fed orally per day for three weeks.
Quantitative determination of phytochemical of A. digitata
Methodologies for the determination of phytochemical used in this research were adapted from those reported by Keay et al. [
Rats were sacrificed under anaesthesia by cervical dislocation. The blood sample was aseptically collected through cardiac puncture and transferred into sample labeled bottles, while the heart was still beating. Whole blood was used for enzyme assays while other part was allowed to stand for 2 hours to perfect clotting and centrifuged (Model SM800B, Surgifriend Medicals, Essex, England) at 1000 rpm. Sera were removed with Pasteur pipette for determination of other parameters. Rats were quickly dissected and their heart were excised and transferred into ice-cold 0.25 M sucrose solution for histopathological examination.
] methods were used to determine the activity of creatine kinase (CK). The activity of lactate dehydrogenase (LDH) was determined using the Witt and Trendelenburg method. TroponinT was evaluated by routine method using research kit in line with the manufacturer’s instruction in the manual.
Cardiac antioxidant markers determination
The activity of superoxide dismutase (SOD) was measured using Misra and Fridovich's [
]. Briefly, 0.1 ml of sample, 0.9 ml of distilled water, 0.5 ml of 25 % trichloroacetic acid (TCA) and 0.5 ml of 17 % TBA in 0.3 % NaOH were pipetted into test tubes. The mixture was incubated at 95°C for 40 minutes and cooled in water after incubation. Afterwards, 0.1 ml of 20 % sodium dodecyl sulphate was added to the mixture. The absorbance of the mixture was determined at 532 and 600 nm against a blank.
Histopathological evaluation
Heart tissues were fixed in buffered 10% formalin and processed for histopathological examination as described by Abdel-Raheem [
]. Briefly, four micrometer-thick paraffin sections were prepared and stained with haematoxylin and eosin for light microscope examination by pathologist.
Statistical analysis
Data collected were analysed using one-way analysis of variance (ANOVA). Level of significance was used to assess significant difference between the control and treated group at P<0.05. Results were expressed as mean ±SEM.
Phytochemical screening of A. digitata
Quantitative screening of the leaves revealed some bioactive compounds; phenols, flavonoids, saponin, alkaloid and phenols (168.26±40, 26.07±0.15, 16.18±0.31 and 10.03±0.10 mg/100ml) among others with high concentration of phenolic compounds (Table1).
Effect of the extract on troponin T level of DOX-induced cardiotoxociy in rats
Fig 1. Shows decreased in Troponin T, concentrations in group administered DOX + 400 mg/Kg against the group treated with 100 mg/kg and 200 mg/kg bwt of the extract respectively.
Significant (P< 0.05) increase of this maker was recorded in group 2 which received DOX only compare with normal control and other test groups.
Effects of the extract on creatinine kinasse activity of DOX-induced cardiotoxociy in rats
The activity of creatine kinse is shown in fig. 2. Significant (P<0.05) increase in the craetine kinase activity was observed in group 2 in reference to group 1, 4 and 5. Observable difference in the activity was not recorded by comparing group2 and group3. Dose dependent decrease in the activity exists among group 3, 4 and 5.
Fig. 1Mean of TroponinT (mg/ml) by group. Values are mean ± SEM of six determinations (n=6). Bars with different letter superscript are significantly different (P<0.05).
Fig. 2Mean of Ck (u/L) by group. Values are mean ± SEM of six determinations (n=6). Bars with different letter superscript are significantly different (P<0.05).
Effects of the extract on lactate dehydrogenase activity of DOX-induced cardiotoxociy in rats
Decrease in the activity of LDH was obtained as shown in fig. 3 below. The activity increased significantly (P<0.05) across the test groups compared with the normal control. The activity varies in dose dependent manner but non significant within the test groups 2,3 and 4. Significant reduction of LDH activity was recorded in group 5 compared with other test groups and normal control.
Fig. 3Mean of LDH (u/L) by group. Values are mean ± SEM of six determinations (n=6). Bars with different letter superscript are significantly different (P<0.05).
Effects of the extract on total protein level of DOX-induced cardiotoxociy in rats
Result of total protein concentration was depicted in fig. 4. Significant (P<0.05) decrease in protein level was obtained in group 2 (Dox–induced without pre-treatment) as against normal control and across the test groups. Total protein level was high in group 5 which received 400 mg/Kg b. wt of the extract compared with those treated with 100 mg/Kg and 200 mg/Kg of the extract respectively.
Fig. 4Mean of total Protein (g/dL) by group. Values are mean ± SEM of six determinations (n=6). Bars with different letter superscript are significantly different (P<0.05).
Effects of the extract on malondialdehyde level of DOX-induced cardiotoxociy in rats
High elevation of MDA was observed in rats given DOX without pre-treatment of the extract compared with the normal control and the test groups. (Fig. 5). Dose dependent decrease exist among groups 3, 4 and 5.
Fig. 5Mean of MDA (mg/ml) by group. Values are mean ± SEM of six determinations (n=6). Bars with different letter superscript are significantly different (P<0.05).
Effects of the extract on GPx activity of DOX-induced cardiotoxociy in rats
The activity GPx of DOX-induced cardio toxicity in rats is shown in fig. 6. GPx activity decreased significantly (P<0.05) as shown in group2 in comparison with normal. Dose depended increase of the enzyme activity was obtained among grou3, 4 and 5.
Fig. 6Mean of GPX (u/mg) by group. Values are mean ± SEM of six determinations (n=6). Bars with different letter superscript are significantly different (P<0.05).
Effects of the extract on SOD activity of DOX-induced cardiotoxociy in rats
Fig. 7 shows the activity of SOD of DOX-induced cardiotoxicity in rats. Group2 recorded significant (P<0.05) decrease of SOD activity compared with other test groups. Across group, the activity of SOD varied non significantly.
Fig. 7Mean of SOD (u/mg) by group. Values are mean ± SEM of six determinations (n=6). Bars with different letter superscript are significantly different (P<0.05).
Effects of the extract on CAT activity of DOX-induced cardiotoxociy in rats
The activity of CAT was high in group 5 pre-treated with 400 mg/Kg b. wt of Adansonia digitata extract as shown in fig. 8 below. Dose dependent increase was observed in group3, 4 and 5. Decrease activity of enzyme in group2 (DOX-induced without pretreatment) was recorded against normal and other test groups. Generally, the activity of the enzyme decreased in all the test groups when compared with normal control (group 1).
Fig. 8Mean of CAT (u/mg) by group. Values are mean ± SEM of six determinations (n=6). Bars with different letter superscript are significantly different (P<0.05).
The myocardium of the control group animals had regular architecture as indicated in Plate 1. Heart section of doxorubicin induced rats revealed remarkable necrosis, oedema and infiltration of chronic inflammatory cells with haemorrhage (Plate 2, double white arrow). In Plates 3, and 4, the myocardium showed normal elongated myocytes arranged in overlapping bundles, surrounded by a rich network of blood vessels and capillaries embedded in a connective tissue matrix with mild haemorrhagic architecture (single white arrow). The section of the heart (DOX+400 mg/kg of the extract) showed moderate damage, very mild oedema at the pericardial layer while other areas of epicardial layer appear normal (Plate 5). Fibrocytes of the connective tissue matrix appear as spindle shaped cells with spindle shaped hyperchromatic nuclei (black arrow).
PlateHaematoxylin and eosin staining shows the effect of a methanol leaves extract of Adansonia digitata on the histological morphology of the rat heart Plate1: Control (Normal saline); Plate2: DOX-induced without pre-treatment; Plate3: DOX + 100 mg/Kg b. wt of extract; Plate4: DOX + 200 mg/kg b. wt of extract; Plate5: DOX+ 400 mg/kg b. wt. extract.
Oncologic treatment with doxorubicin (DOX) result to a cardiomyopathy considered as an extremely side effect of the drug. Quality of patients’ life who survived cancer, especially children is significantly affected. Years ago, diverse molecular mechanisms have been proposed to understand the pathogenesis of acute and chronic DOX-induced cardiotoxicity (DIC). The proposed molecular invents include: oxidative stress, iron metabolism, Ca2+ homeostasis dysregulation, sarcomeric structure alterations, gene expression modulation, and apoptosis. The most significant cardiovascular disorders (CVDs) are elevated blood pressure, coronary heart attack, myocardial infarction, congenital heart abnormalities, cardiac arrhythmias, heart failure, and stroke [
], in his projection and analysis, in 2012, almost 17.5 million people died of CVDs, representing 31% of all worldwide deaths. Should the current trends persist, a projected 23.6 million people would die from cardiovascular diseases by 2030. As such, some current medications, such as organic nitrate, calcium channel antagonist, and - blocker, are useful in preventing heart disease, their usage is also restricted due to side effects. The current medications are useful in preventing heart disease but their usage is also restricted due to side effects. Doxorubicin, an anthracyline compound, is a commonly prescribed chemotherapeutic agent for a variety of cancers. The cause of doxorubicin cardiotoxicity is thought to be elevated oxidative stress, which leads to increased amounts of reactive oxygen species and lipid peroxidation [
In the present research, there was a significant (P<0.05) increase in levels of cardiac markers (Troponin T, Creatine kinase, and LDH) in doxorubicin -induced rats untreated as against the normal control. Reduction of Troponin T, Creatine kinase, and LDH were observed in Group 3, 4, and 5 administered 100,200 and 400 mg/kg body weight of A. digitata respectively compared with doxorubicin–induced untreated rats as shown in Fig. 2, Fig. 3 and Fig. 4. The potency of A. digitata to ameliorate cardiotoxicity in rat could be attributed to the phytochemicals in the leaf extract [
]. The a Adansonia digitata leaf extract display cardioprotective effects in the current study which can be attributed to their active constituents of polyphenols such as proanthocyanidins and flavonoids, saponins, and glycosides [
]. It was in line with the decrease in concentration of the cardiac markers in the text groups in reference to DOX-induced untreated experimental control. Increase serum LDH activity due to tissue injury was in line with the report of Danborno et al., [
]. Malondialdehyde (MDA) is a lipid peroxidation product, a significant marker of the degree of cellular damage caused by reaction of oxidants with lipids, thereby causing peroxidation and release of products including hydrogen peroxide. Orally administration of A. digitata ethanol extract significantly reduced the elevated level of MDA in Group 5, Group4 and Group3 respectively compared to the untreated Group2. The results were in agreement with the report of Ogunleye et al. [
Effects of aqueous extract of fruit pulp of Adansonia digitata L. on the oxidative stress profile against Trypanosoma brucei brucei infection in albino rats.
]. The possible mechanism of the extract to delay lipid peroxidation can be attributed to its ability to donate electrons to abort the rampaging effect of the lipid radical. Lipid peroxidation is a predisposing factor to cardiovascular events including coronary heart diseases, myocardial infarction among others. It is therefore, proposed that the mechanism of inhibition of the extract to cardiotoxicity induced by DOX is through radical scavenging activity.
Lipid peroxidation, an evidence-based myocardial injury, occurs as a result of the increase reactive oxygen species (ROS) production, including superoxide (O2−˙) and hydroxyl radicals (OH˙) as well as other non-radicals such as hydrogen peroxide (H2O2), singlet oxygen (O2), [
Baobab (Adansonia digitata L.) a review of traditional uses, phytochemistry and pharmacology.
in: Juhani R.H. Simon J.E. Ho C.T. African natural plant productise: new discoveries and challenges in chemistry and quality. American Chemical Society,
Washington D.C2009: 51-84
]. Furthermore, the chemical structure of quinone groups in DOX can be reduced to a semiquinone, an unstable metabolite which can react with molecular oxygen (an electron acceptor) and rapidly revert to the parent compound. This redox cycle leads to the formation of superoxide anion radicals within mitochondria, causing cardiotoxicity [
]. Glutathione peroxidase (GPX) is an antioxidant enzyme class with the capacity to scavenge free radicals. Catalase (CAT) is an enzyme that catalyzes the conversion of hydrogen peroxide to water and molecular oxygen, and as a result, protects the body from oxidative insult [
]. Superoxide dismutase (SOD) scavenges superoxide ion to either molecular oxygen or hydrogen peroxide. Heart tissue is extremely prone to oxidative damage unlike other tissues, partly due to lower levels of antioxidant enzymes such as peroxidase, catalase, and superoxide dismutase. The activities of GPX, CAT and SOD were significantly reduced (P<0.05) in group2 (Fig. 4) compared with normal. The reduction was reversed following the pre-treatment of the A. digitata (Baobab) extract in dose dependent manner. Similarly, high degree of reversal was recorded in group5 which received 400mg/kg body weight of the extract. The bioactive compounds present in this plant possibly protect the heart from DOX toxicity in the present study. Full mechanism of this process is yet to be ascertained. Increase level of TroponinTand Creatine kinase in DOX-induced untreated group was supportive of oxidative damage. Invariably, oxidative damage was protected by baobab extract due to its antioxidant potentials supported by the data obtained. A digitata extract reversed the oxidative damage initiated by doxorubicin through free radical generation mechanism of the phytochemicals present in the leaf extract.
Histopathological examination of heart tissue of DOX–induced cardiotoxicity model rat showed infiltration of inflammatory cells and congestion in the blood vessels. However, treatment with A. digitata leaf extract at various concentrations (100,200 and 400 mg/kg body weight) showed predominantly minor myocardial structure and minor inflammatory cell infiltration (Plate 3, 4 and 5) in reference to Plate 2 with various degree of myocardial damage and inflammatory cell infiltration. The extract ameliorated haemorrhage indicating its significant cardio protective effects and it also maintained myocardial membrane.
Conclusion
Adansonia digitata (BAOBAB) leaf extract can protect doxorubicin induced cardiotoxicity, oxidative stress and tissue damage based on the findings of this study. The significant reductions in cardiac and oxidative biomarkers have given credence to protective potential of ethanol leaf extract of A. digitata in doxorubicin-induced myocardial injury.
Conflict of interest
Authors declare non existing conflict of interest.
Acknowledgement
The authors sincerely wish to acknowledge all staff of Biochemistry Laboratory unit, Michael Okpara University of Agriculture, Umudike, Nigeria and Shalom Laboratory Nig. Limited for providing some of the instrument used in this study.
References
Brayfield A.
ed. “Doxorubicin”. Martindale: The Complete Drug Reference.
Cardioprotective effect of HPLC standardized ethanolic extract of Terminaliapallida fruits against isoproterenol-induced myocardial infarction in albino rats.
Cardioprotective potential of hydroalcoholic fruit extract of Ananascosmosus against isoproterenol-induced myocardial infarction in Winstar Albino rats.
Phytochemical, Elemental and proximate analyses of stored sun-dried and shade dried BaobabAdansonia digitata) leaves. Preprint Converting enzyme inhibitors and angiotensin II receptor blockers on all cause, mortality cardiovascular deaths and cardiovascular events in patients with diabetes mellitus:Ameta analyses.
Effects of aqueous extract of fruit pulp of Adansonia digitata L. on the oxidative stress profile against Trypanosoma brucei brucei infection in albino rats.
Baobab (Adansonia digitata L.) a review of traditional uses, phytochemistry and pharmacology.
in: Juhani R.H. Simon J.E. Ho C.T. African natural plant productise: new discoveries and challenges in chemistry and quality. American Chemical Society,
Washington D.C2009: 51-84
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