SQU Journal for Science, 2023, 28(1), 1-9 DOI:10.53539/squjs.vol28iss1pp1-9 Sultan Qaboos University 1 Toxicological Evaluation of the Leaves of Mangifera indica L. (Mango) on Albino Rats (Rattus norvegicus) Tajudeen Olanrewaju Yahaya 1 *, Titilola F. Salisu 2 , Esther O. Oladele 3 , Clement Yaro 4 , Nura Iliyazu 1 , Adam Tukur 1 , Abdulrazaq Izuafa 1 and Muhammad A. Yari 1 . 1 Department of Biological Sciences, Federal University Birnin Kebbi, PMB 1157, Kebbi State, Nigeria; 2 Department of Zoology and Environmental Biology, Olabisi Onabanjo University, Ago- Iwoye, Ogun State; 3 Biology Unit, Distance Learning Institute, University of Lagos, Nigeria; 4 Department of Animal and Environmental Biology, University of Uyo, Akwa Ibom State, Nigeria *Email: yahaya.tajudeen@fubk.edu.ng. ABSTRACT: The increasing use of plant-based medicines necessitates safety evaluations of all medicinal plants. This study evaluated the effects of mango (Mangifera indica) leaf extracts on albino rats (Rattus norvegicus). The phytochemistry of the plant’s extract was evaluated, followed by a determination of its acute toxicity using 24 rats. Thereafter, the chronic toxicity of the extract was determined using another set of 24 rats, separated into four groups of six rats each. Rats in group 1 (the control) were administered distilled water, while groups 2, 3, and 4 received daily doses of 1000, 2000, and 3000 mg kg -1 body weight, respectively. The rats’ body weights and reactions were monitored for 90 days before blood, liver, and kidney samples were collected for hematological and histopathological examinations. The phytochemistry revealed phenols, alkaloids, tannins, flavonoids, steroids, glycosides, and saponins. The acute toxicity test recorded no mortality at doses up to 5000 mg kg -1 , and all the rats behaved normally, except for one that was sluggish. The chronic toxicity test revealed no significant (p>0.05) weight difference between the control and treated rats. The packed cell volume, hemoglobin, and red and white blood cells of the rats fed 2000 and 3000 mg were significantly altered (p<0.05), while lymphocytes exhibited no significant alterations in any of the groups. The treated rats’ livers revealed dose-dependent necrosis, whereas their kidneys showed atrophy and epithelial cell degeneration. The results obtained suggest that a single dose of the plant’s extract is not harmful, but repeated high-concentration dosing for a long time may result in toxicity. Keywords: Acute toxicity; Livers; Medicinal plants; Necrosis; Red blood cells. تقييم السموم في أوراق مانغيفيرا إنديكال مانجو )على جرذان ألبينو( راتوس نورفيغيكوس تاج الدين أوالنريواجو يحيى ، تيتيال فوزية ساليسو ،إستر أو. أوالدي، كليمنت يارو ، نورا إليازو، عبدالرزاق إيزوافا، محمد أ. ياري مانجيفيرا )المانجو أوراق مستخلصات آثار الدراسة هذه قيمت .الطبية النباتات جميع في لألمان تقييمات إجراء النباتية لألدوية المتزايد االستخدام يستلزم الملخص: تحديد تم ، ذلك وبعد را.فأ 24باستخدام حادة سمية ذلك وتلت النبات، الستخراج النباتية الكيمياء تقييم تم وقد .(نورفيجيكوس راتوس )البرص فئران على (إنديكا ) 1 المجموعة في الجرذان إعطاء تم .فئران ستة مع منها كل مجموعات، أربع إلى مقسمة فأرا، 24من أخرى مجموعة باستخدام للمستخلص المزمنة السمية مراقبة تم .التوالي على الجسم، وزن من كغم - 1ملغم 3000و 2000و 1000من يومية جرعات 4و 3و 2المجموعات تلقت بينما المقطر، الماء (المكافحة عن النباتية الكيمياء كشفت .النسج أمراض وعلم الدم فحوصات إلجراء والكلى والكبد الدم من عينات جمع قبل يوما 90لمدة وتفاعالتها الجرذان جسم أوزان ملغم 5000إلى تصل بجرعات وفاة أي الحادة السمية إختبار يسجل ولم .والصابونات والجليكوزيدات والستيرويدات والفالفونويدات والتانينات والقلويات الفينوالت عنصر بين الوزن في (p>0.05)كبير فرق عن المزمنة السمية إختبار يكشف لم .بطيئة كانت واحدة باستثناء طبيعي، بشكل الجرذان جميع وتتصرف ،-1الكغم من الخاليا تظهر لم بينما ، (p<0.05)كبير بشكل للجرذان والبيضاء الحمراء الدم وخاليا والهيموغلوبين، المعبأة، الخاليا حجم تغيير تم فقد .المعالجة والجرذان التحكم الخاليا وتنكس ضمورا الكلى أظهرت حين في الجرعة، على يعتمد نخر عن المعالجة الجرذان أكباد كشفت .كبيرة تغييرات أي المجموعات جميع في اللمفاوية يؤدي قد طويلة لفترة التركيز عالية الجرعات تكرار ولكن ضارة، ليست النبات مستخلص من واحدة جرعة أن إلى عليها الحصول تم التي النتائج تشير .الظهارية .السمية إلى .الحمراء الدم خاليا النخر، الطبية، النباتات الكبد، الحادة، السمية :الكلمات المفتاحية mailto:yahaya.tajudeen@fubk.edu.ng. TOXICOLOGICAL EVALUATION OF THE LEAVES OF MANGIFERA INDICA L. (MANGO) 2 1. Introduction here has been a global renaissance in plant-based medicines since the middle of the 19 th century because they are affordable, potent, and simple to prepare. Worldwide, approximately 21,000 medicinal plants have been documented for disease treatment [1]. The renaissance is partly because plants contain many bioactive compounds used to produce pharmaceutical drugs [2]. At the minimum, 30% of pharmaceutical drugs are derived directly or indirectly from medicinal plants [3]. Plant-based medicines are used to treat ailments by at least 80% of the world’s population [4]. Mango (Mangifera indica L.), which belongs to the family Anacardiaceae, is a cosmopolitan tree, whose parts are often used to make plant-based medicines [5]. Extracts of M. indica are used to treat diabetes, bronchitis, diarrhea, asthma, kidney diseases, scabies, respiratory problems, syphilis, and urinary disorders [6-8]. The plant is antimicrobial, antioxidant, anti- diabetic, and immunomodulatory, and it combats tumors [4]. M. indica contains abundant minerals, such as nitrogen and potassium, and also contains protein [9, 4]. The plant is also rich in phytochemicals, including mangiferin, phenolic acids, benzophenones, flavonoids, carotenoids, quercetin, isoquercetin, ascorbic acid, and tocopherols [10-12]. The oil from M. indica contains monoterpenes, sesquiterpenes, non-terpenoid hydrocarbons, oxygenated hydrocarbons, α-gurjunene, trans- caryophyllene, α-humulene, α-selinene, α-glucosidase, and camphor [10, 13]. In recent times, however, there has been a growing concern about the safety of plant-based medicines. Studies have shown that some plant-based medicines are toxic due to their constituents, while others induce toxicity due to interactions and contamination during production [2]. The toxic effects of plant-based medicines on humans depend on the chemical composition of the medicine and the extraction methods used, as well as on the state of the consumer’s cell membrane, cell surface, tissues, and extracellular matrix [10, 12]. Thus, there is a need for toxicological studies of all plants used in making plant medicines [8]. Although there are several studies on the health-promoting efficacy of M. indica, there is a dearth of studies on the toxicity of the plant [10, 13]. Consequently, this study evaluated the toxicity of M. indica leaf extracts in albino rats (Rattus norvegicus). 2. Materials and Methods 2.1 Management of experimental animals Twenty-four (24) male and female albino rats (Rattus norvegicus) with a mean weight of 185±10 g and aged 50 days were used for this study. The rats were housed in well-ventilated metallic cages (six rats per cage) in a well-ventilated animal house at a room temperature of about 32 °C. Before beginning the experiment, the rats were given 14 days to acclimate to their new environment. Water and pellet feeds from Premier Feed Mills in Ibadan, Nigeria, were freely available to the rats. 2.2 Collection of plant materials and identification Fresh leaves of M. indica were plucked within the Kebbi metropolis in February 2021. The leaves were identified by a botanist in the Department of Biological Sciences, Federal University Birnin Kebbi, Kebbi State, Nigeria. A sample of the authenticated materials was deposited in the department’s herbarium with voucher number FUBK/a/6. 2.3 Preparation and extraction The plant’s leaves were gently washed with distilled water, dried in the shade, pulverized into powder, and sieved through a stainless-steel mesh with a diameter of 200 mm to ensure size consistency. To extract the bioactive components, 150 g of the powdered material was soaked in 900 ml of 98% methanol for 48 hours. The extract obtained was filtered using a muslin cloth, and the methanol was evaporated using a rotary evaporator at 45 °C until a constant dry weight of the extract was achieved. The dried extract was kept in a desiccator. 2.4 Qualitative screening of the extracts The qualitative screening was conducted using conventional procedures as detailed by Yahaya et al. [2]. Test for flavonoids A small quantity of 10% ferric chloride solution was mixed with 0.5 g of the extract. The presence of flavonoids was indicated by the appearance of a green or blue color. Test for tannins Five milliliters (5 ml) of water were added to 0.5 g of the extract, followed by a few drops of 10% ferric chloride. The presence of tannins was indicated by a blue-black, green, or blue-green precipitate. Test for alkaloids For 2 minutes, 0.2 g of the extract was boiled with 2% H2SO4. A few drops of Dragendorff’s reagent were added after the liquid was filtered. The presence of alkaloids was revealed by an orange-red precipitate. T TAJUDEEN OLANREWAJU YAHAYA ET AL. 3 Test for steroids One milliliter (1 ml) of the extract and 5 ml of anhydrous acetic acid were mixed thoroughly. Four (4) drops of the above mixture were placed in a porcelain dish, and one drop of concentrated H2SO4 was added. A change in color from rose, through red, violet, and blue, to green showed the extract contained steroids. Test for glycosides One milliliter (1 ml) of the extract was mixed with 1 ml of glacial acetic acid containing one drop of ferric chloride solution. Exactly 1 ml of concentrated H2SO4 was added to the liquid, which resulted in the development of two layers. The presence of glycosides was shown by the development of a brown ring at the interface of the two layers. Test for saponins In a graduated cylinder, 1 ml of the extract was diluted to 20 ml with distilled water, and wa s shaken for 15 seconds. The presence of saponins was shown by the formation of a foam layer after 15 minutes. Test for phenols Exactly 0.5 g of the extract was dissolved in 2 ml of distilled water, and a small quantity of ferric chloride was added. The presence of phenols was determined by the appearance of a red, purple, or green color. 2.5 Acute toxicity test The acute toxicity of M. indica extracts was determined using the "classical LD50" approach as outlined by Trevan [15]. Twenty-four (24) rats (males and females) were randomly divided into four groups, each of six rats. Group 1 was the control, while groups 2, 3, and 4 were orally administered 1000, 3000, and 5000 mg of M. indica leaf extract per kg of body weight of the rats, respectively. The rats were monitored for 72 hours to establish the general toxicity of the extracts based on observation of behavioral change. 2.6 Chronic toxicity test A new batch of 24 rats was separated into four groups, each containing six rats. M. indica leaf extracts were administered to groups 2, 3, and 4 on a daily basis in doses of 1000, 2000, and 3000 mg kg -1 body weight of the rats, respectively. Group 1 was designated as the control group, with rats receiving only standard feed and water. The rats’ weights were tracked and general observations recorded for 90 days before the animals were sacrificed via cervical dislocation. Hematological tests were performed on blood samples, and histological examinations were performed on the liver and kidneys. 2.7 Body weight measurement The rats’ body weights were measured at the start of the experiment using a digital weighing scale manufactured by KERN, Germany. Throughout the rest of the experiment, the measurements were taken every other day in the morning. 2.8 Hematological examination Each rat was held firmly to a workbench using office pins. About 2.5 ml of blood was drawn from the rats’ tails into ethylenediamine tetra acetic acid (EDTA) bottles using a 5 ml syringe and a 20-gauge needle. The Sysmex auto-analyzer was used to assess the amounts of blood parameters, including packed cell volume (PCV), hemoglobin (Hb), white blood cells (WBC), red blood cells (RBC), and lymphocytes (LYM). 2.9 Histopathological analysis The rats’ liver and kidney tissues were processed for histological analysis, as described by Yahaya et al. [16]. The rats’ chests were cut open in a dorsal-ventral orientation with a surgical blade. The livers and kidneys were collected, and about 5 mm of each tissue was stored in 10% neutral buffered formalin. The tissues were dehydrated in increasing volumes of alcohol before being embedded in paraffin wax (65, 80, and 100%). With a rotary microtome (model YR421), the embedded tissues were sectioned at 5 μm, placed on glass slides, and air-dried. Hematoxylin and eosin dyes were used to stain the slides, which were then examined under a light microscope for histological abnormalities. 2.10 Data analysis All of the analyses were performed using the Statistical Package for Social Sciences (SPSS) version 20 for Windows. The analysis of variance (ANOVA) was used to compare data between the test and control groups. The statistical significance level was set at p<0.05. TOXICOLOGICAL EVALUATION OF THE LEAVES OF MANGIFERA INDICA L. (MANGO) 4 3. Results 3.1 Phytochemicals in the extract Table 1 below shows the phytochemicals detected in the crude extracts of M. indica leaves. Phenols and flavonoids were abundant, while glycosides, alkaloids, tannins, saponins, and steroids were present in moderate amounts. Table 1. Phytochemicals detected in the crude extracts of M. indica leaves. ++ indicates abundantly available ; + indicates moderately available 3.2 Acute toxicity of the extracts Table 2 shows the reactions of the rats treated with different doses of M. indica leaf extracts. No deaths were recorded, and all the rats behaved normally, except for one in the group fed 5000 mg kg -1 , which was sluggish. Table 2. Reactions of the treated rats (n = 6 per group) to different doses of M. indica leaf extracts. Group Dosage (mg kg -1 body weight) Observation period (hour) Behavioral change Mortality 1 Distilled water 72 None 0 2 1000 72 None 0 3 3000 72 None 0 4 5000 72 Sluggishness (n=1) 0 3.3 Effects of the extracts on body weight The body weights of the rats treated with M. indica leaf extracts are shown in Table 3. Compared with the control, no significant differences (p>0.05) were observed. Table 3. Change in body weights of the treated rats (n = 6 per group) to different doses of M. indica leaf extracts. Dosage (mg kg -1 body weight) Initial body weight (g) Final body weight (g) Control (Distilled water) 197.33±5.32 a 210.34±6.31 a 1000 ml 200.01±2.31 a 205.39±6.09 a 2000 ml 198.35±1.31 a 212.00±3.22 a 3000 ml 201.33±3.34 a 206.15±3.54 a Values were expressed as mean ± SD (n = 6); values with the same subscript "a" are not significantly different from control at p> 0.05 (ANOVA). 3.4 Effects of the extracts on hematological parameters Table 4 compares the hematological parameters of rats fed M. indica leaf extracts to those of the control rats. The packed cell volume (PCV), hemoglobin (Hb), and red blood cells (RBC) of the treated rats were reduced, and this reduction was significant (p<0.05) in the rats fed 2000 and 3000 mg of the extracts. The white blood cells (WBC) of the treated rats were increased, but significantly only in the group fed 3000 mg. There was no noticeable change in the lymphocytes (LYM) of the treated rats. Alkaloids Inference Tannins + Saponins + Phenols ++ Steroids + Flavonoids ++ Glycoside + TAJUDEEN OLANREWAJU YAHAYA ET AL. 5 Table 4. Hematological parameters of rats treated with M. indica leaf extracts. Parameters Control 1000 mg 2000 mg 3000 mg PCV (L L-1) 28.18±2.83 a 26.63±3.49 a 25.90±2.50 b 22.67±2.7 b Hb (g dL-1) 11.63±1.83 a 11.00±0.89 a 0.9.37±0.58 b 6.82±0.22 b WBC (mc mm-3) 1.28±0.87 a 1.31±0.55 a 1.59±0.40 a 2.99±1.6 b RBC (mc mm-3) 5.06±0.78 a 4.94±0.60 a 3.91±0.18 b 3.64±0.67 b LYM (c µL-1) 82.83±6.49 a 82.17±1.73 a 82.27±0.99 a 82.63±0.95 a Values were expressed as mean ± SD (n = 6); mean values with different subscripts "a" and "b" along the same row are statistically different from control at p< 0.05 (ANOVA). PVC = packed cell volume; Hb = hemoglobin; WBC = white blood cells; LYM = lymphocytes. 3.5 Histopathological effects of the extracts Figures 1a–d show the liver tissues of the control and treated rats. Normal hepatocytes were observed in the liver of the control rats, while dose-dependent necrosis was observed in the treated rats. The kidney tissues of the control and treated rats are shown in Figures 2a–2d. Normal hepatocytes were observed in the control group, while the rats fed 1000, 2000, and 3000 mg kg -1 of the extracts revealed mild atrophic glomerulus, tubular atrophy, and epithelial cell degeneration. a b c d Figure 1. Photomicrography of the liver tissues of the control and treated rats. a = control rats showing normal hepatocytes. b = rats treated with 1000 mg kg -1 M. indica extract showing mild necrosis. c = rats treated with 2000 mg kg -1 M. indica extract showing moderate necrosis d = rats treated with 3000 mg kg -1 M. indica extract showing moderate necrosis. TOXICOLOGICAL EVALUATION OF THE LEAVES OF MANGIFERA INDICA L. (MANGO) 6 a b c d Figure 2. Photomicrography of the kidney tissues of the control and treated rats. a = control rats showing normal glomerulus b = rats treated with 1000 mg kg -1 M. indica extract showing mild atrophic glomerulus c = rats treated with 2000 mg kg -1 M. indica extract showing tubular atrophy d = rats treated with 3000 mg kg -1 M. indica extract showing epithelia cell degeneration (outer layer) 4. Discussion The toxicity of Mangifera indica leaves was evaluated in this study. The study was conceived to determine the safe doses of the plant extract to prevent or minimize its health hazards among users worldwide, particularly in Nigeria. Table 1 shows that the plant contains bioactive substances, including phenolic acids, alkaloids, tannins, flavonoids, steroids, glycosides, and saponins. These substances are well known for their health-boosting activities, but some of them could induce toxicity at certain doses. In the current study, the acute toxicity test of the plant’s extract revealed no mortality nor any abnormality at doses up to 5000 mg kg -1 (Table 2), which is the upper limit at which a single dose of an extract is considered non-toxic [2, 17]. Table 3 further demonstrates the plant’s non-toxicity, as there was no significant difference in body weight between the treated and control rats. These results are consistent with those of Zhang et al. [18], Ahomadegbe et al. [19], EASL [20], and Reddeman et al. [21], all of whom reported the non-toxicity of the plant. Table 4, however, shows that long-term dosing (90 days) at 2000 and 3000 mg kg -1 body weight significantly altered the hematological parameters of the treated rats. The packed cell volume (PCV), hemoglobin (Hb), and red blood cells (RBC) were reduced, while the white blood cells (WBC) were increased. This result contradicts that of Ogbe et al. [22] and Abidakun et al. [23], who observed an increase in PCV, Hb, and RBC in experimental animals treated with mango extract. The inconsistency of the current study with the previous ones might be due to the longer duration and higher concentrations used in this study. In contrast to the current study’s 90-day treatment with 1000, 2000, and 3000 mg kg -1 body weight, Ogbe et al. [22] and Abidakun et al. [23] gave experimental animals 20 mg for 14 days and 1 mg for 40 days, respectively. The plant contains alkaloids and saponins, which, at high concentrations and duration, can accumulate to toxic levels and TAJUDEEN OLANREWAJU YAHAYA ET AL. 7 destroy blood cells [24, 25]. The plant also contains tannins, which have been implicated in iron deficiency, resulting in anemia [26]. Figures 1a-d and 2a-d further show that M. indica extracts can be toxic at high concentrations and over long periods of time. The treated rats’ livers developed dose-dependent necrosis, while their kidneys developed atrophic glomerulus, tubular atrophy, and epithelial cell degeneration. Atiba et al. [27] reported abnormal liver morphology, such as an enlarged central vein and reduced sinusoids, in rats treated with M. indica leaf extracts for a long time. Zhang et al. [18] also reported enlargement of liver and kidney tissues in rats dosed with M. indica for 90 days. Amien et al. [28] and Nadella and Kumar [29], on the other hand, found that M. indica has chemo-protective effects on the kidneys and livers. However, the studies that reported chemoprotective effects used lower concentrations and shorter durations. For instance, Amien et al. [28] dosed experimental animals with 300 mg kg -1 for 14 days, while Nadella and Kumar [29] dosed them with 150 mg kg -1 for 10 days. The reported histopathological effects could be due to potentially toxic phytochemicals in the extracts, primarily alkaloids, saponins, and tannins. High doses and prolonged use of an alkaloid-containing substance can result in hepatocyte necrosis and renal failure [30, 31]. Tannins have also been linked to liver necrosis and kidney problems [32]. Additionally, liver and kidney damage due to necrosis of liver cells and renal tubules has been reported among mice treated with high concentrations of saponins [33, 34]. Toxicants primarily target the kidneys and liver because these organs process all foreign substances in the body [2, 35]. 5. Conclusion The results showed that M. indica leaves contain pharmacologically important phytochemicals such as mangiferin, phenolic acids, flavonoids, carotenoids, alkaloids, tannins, quercetin, isoquercetin, ascorbic acid, and tocopherols. However, some of these compounds, especially tannins, saponins, and alkaloids, could be toxic at certain doses. A single dose of the plant’s extract is not expected to be toxic, as it is non-toxic at doses up to 5000 mg kg - 1 , which is the maximum dose at which an extract can be considered non-toxic. However, repeated high-concentration and long-duration dosing may induce toxicity, as evidenced by the alteration of the hematological parameters and tissues of the treated rats. Conflict of interest The authors declare no conflict of interest. Acknowledgment Not applicable Ethical statement This study was conducted in accordance with the ethical standards of the European and German Animal Welfare legislation, declaration principles set out by Helsinki and the National Institutes of Health guidelines for care and use of animals in research. All protocols were approved by the local ethics committee of the Federal University Birnin Kebbi, Nigeria (regulation CEE 86/609). References 1. Gururaja, G.M., Mundkinajeddu, D., Kumar, A.S., Dethe, S.M., Allan, J.J., Agarwal, A. Evaluation of cholesterol-lowering activity of standardized extract of Mangifera indica in albino wistar rats. Pharmacognosy Research, 2017, 9(1), 21-26. https://doi.org/10.4103/0974-8490.199770. 2. Yahaya, O.T., Oladele, E.O., Bunza, M.D.A., Yusuf, A.B., Izuafa, A., Danjuma, J.B. Hematotoxicity and nephrotoxicity of long-term administration of Guiera senegalensis (J.F. Gme), Cassia occidentalis (Linn), and Ziziphus mauritiana (Lam) leaves obtained in Birnin Kebbi, Nigeria. Journal of Herbmed Pharmacology, 2022, 11(3), 367-374. https://doi.org/10.34172/jhp.2022.42. 3. Ramírez-Atehortúa, A.M., Morales-Agudelo, L., Osorio, E., Lara-Guzmán, O.J. The Traditional Medicinal Plants Cuphea calophylla, Tibouchina kingii, and Pseudelephantopu spiralis Attenuate Inflammatory and Oxidative Mediators. Evidence- Based Complementary and Alternative Medicine, 2018. https://doi.org/10.1155/2018/1953726. 4. Pan, J., Yi, X., Wang, Y., Chen, G., He, X. Benzophenones from mango leaves exhibit alpha glucosidase and NO inhibitory activities. Journal of Agricultural and Food Chemistry, 2016, 64(40), 74757480. https://doi.org/10.1021/acs.jafc.6b02404. 5. Guo, L., Gong, S., Wang, Y., Sun, Q., Duo, K., Fei, P. Antibacterial Activity of Olive Oil Polyphenol Extract Against Salmonella typhimurium and Staphylococcus aureus: Possible Mechanisms. Foodborne Pathogens and Diseases, 2020, 17, 396-403. https://doi.org/10.1089/fpd.2019.2713. 6. Kulkarni, V.M., Rathod, V.K. Extraction of mangiferin from Mangifera indica leaves using three phase partitioning coupled with ultrasound. Industrial Crop and Product, 2014, 52, 292-297. https://doi.org/10.1016/j.indcrop.2013.10.032. 7. Kumar, Y., Kumar, V., Sangeeta, C. Comparative antioxidant capacity of plant leaves and herbs with their anti-oxidative https://doi.org/10.4103/0974-8490.199770 https://doi.org/10.34172/jhp.2022.42 https://doi.org/10.1155/2018/1953726 https://doi.org/10.1021/acs.jafc.6b02404 https://doi.org/10.1089/fpd.2019.2713 http://dx.doi.org/10.1016/j.indcrop.2013.10.032 TOXICOLOGICAL EVALUATION OF THE LEAVES OF MANGIFERA INDICA L. (MANGO) 8 potential in meat system under accelerated oxidation conditions. Journal of Food Measurement and Characterization, 2020, 14, 3250-3262. 8. Shah, K.A., Patel, M.B., Patel, R.J., Parmar, P.K. Mangifera indica (Mango). Pharmacognosy Reviews, 2010, 4, 42-48. https://doi.org/10.4103/0973-7847.65325. 9. Bhuvaneshwari, J., Khanam, S., Devi, K. In-vitro enzyme inhibition studies for antidiabetic activity of mature and tender leaves of Mangifera indica var. Totapuri. Journal of Microbiology and Biotechnology, 2014, 3(3), 36-41. 10. Ediriweera, M.K., Tennekoon, K.H., Samarakoon, S.R. A review on ethnopharmacological applications, pharmacological activities, and bioactive compounds of Mangifera indica (Mango). Evidence-Based Complementary and Alternative Medicine, 2017. https://doi.org/10.1155/2017/6949835. 11. Kabir, Y., Shekhar, H.U., Sidhu, J.S. Phytochemical Compounds in Functional Properties of Mangoes. In Handbook of Mango Fruit, Wiley: Hoboken, NJ, USA, 2017, pp. 237-254. 12. Kumar, M., Saurabh, V., Tomar, M., Hasan, M., Changan, S., Sasi, M. Mango (Mangifera indica L.) Leaves: Nutritional Composition, Phytochemical Profile, and Health- Promoting Bioactivities. Antioxidants, 2021; 10(2), 299. https://doi.org/10.3390/antiox10020299. 13. Ribeiro, S.M.R., Schieber, A. Bioactive Compounds in Mango (Mangifera indica L.). In Bioactive Foods in Promoting Health; Elsevier BV: Oxford, UK, 2010, pp. 507-523. 14. Batool, N., Ilyas, N., Shabir, S., Saeed, M., Mazhar, R.A. Mini-review of therapeutic potential of Mangifera indica L. Pakistan Journal Pharmaceutical Science, 2018, 31, 1441-1448. https://europepmc.org/article/med/30033432. 15. Trevan, J.W. (1927). The Error of Determination of Toxicity. Proceedings Royal Society London, 1927, 101 (712), 483-514. https://doi.org/10.1098/rspb.1927.0030. 16. Yahaya, T., Oladele, E., Obaroh, I., Bala, J., Jubril, A., Muhammad, A. Effects of Telecommunication Mast Electromagnetic Radiation (EMR) on Exposed Rats (Rattus norvegicus). East African Journal of Science, 2020, 14(2), 131-140. 17. OECD (Organization for Economic Co-operation and Development). Guidelines for the Testing of Chemical, No. 425. Acute Oral Toxicity- Up and down Procedure October 2008. 18. Zhang, Y., Li, J., Wu, Z., Liu, E., Shi, P., Han, L., Guo, L., Gao, X., Wang, T. Acute and Long -Term Toxicity of Mango Leaves Extract in Mice and Rats. Evidence-Based Complementary and Alternative Medicine, 2014, Article ID 691574. https://doi.org/10.1155/2014/691574. 19. Ahomadegbe, M.A., Ladekan, E.Y., Togbenou, N., Assogba, F., Agbonon, A., Gbenou, J (2019). Phytochemical and toxicity studies of the leaves of Mangifera indica, Cajanus cajan and of Piliostigma thonningii, acclimated in Benin, used against diarrheal disease. Journal of Pharmacognosy and Phytochemistry, 2019, 7(2), 2971-2978. https://www.phytojournal.com/archives/2018/vol7issue2/PartAP/7-2-316-293.pdf. 20. EASL (European Association for the Study of the Liver). Clinical Practice Guidelines: Drug-induced liver injury. Journal of Hepatology, 2019, 70, 1222-1261. https://doi.org/10.1016/j.jhep.2019.02.014. 21. Reddeman, R.A., Glávits, R., Endres, J.R., Clewell, A.E., Hirka, G., Vértesi, A., Béres, E., Szakonyiné, I.P. A Toxicological Evaluation of Mango Leaf Extract (Mangifera indica) Containing 60% Mangiferin. Journal of Toxicology, 2019, Article ID 4763015. https://doi.org/10.1155/2019/4763015. 22. Ogbe, R.J., Adoga, G.I., Abu, A.H. Antianaemic potentials of some plant extracts on phenyl hydrazine -induced anaemia in rabbits. Journal of Medicinal Plants Research, 2010, 4(8), 680-684. https://doi.org/10.5897/JMPR09.487. 23. Abidakun, O.M., Modupe, O., Oladiji, A.T. Mangifera indica Solvent Fractions: A Treatment for Iron Deficiency Anaemia Induced by Diet in Albino Rats. Journal of Complementary Medicine and Alternative Healthcare, 2018, 6(5), 555698. https://doi.org/10.19080/JCMAH.2018.07.555698. 24. Yeh, S.P., Liu, C.H., Sung, T.G., Lee, P.P., Cheng, W. Effect of saponin on hematological and immunological parameters of the giant freshwater prawn, Macrobrachium rosenbergii. Aquaculture, 2006, 261(4), 1432-1439. https://doi.org/10.1016/j.aquaculture.2006.08.051. 25. Matsuura, H.N., Fett-Neto, A.G. Plant Alkaloids: Main Features, Toxicity, and Mechanisms of Action. In: Gopalakrishnakone P., Carlini C., Ligabue-Braun R. (eds) Plant Toxins. Toxinology. Springer, Dordrecht. 2015. https://doi.org/10.1007/978-94-007-6728-7_2-1. 26. Delimont, N.M., Haub, M.D., Lindshield, B.L. The Impact of Tannin Consumption on Iron Bioavailability and Status: A Narrative Review. Current Developments in Nutrition, 2017, 1(2), 1–12. https://doi.org/10.3945/cdn.116.000042. 27. Atiba, P.M.S., Ajibade, A.J., Olaitan, P.B. Some effects of simultaneous administration of crude aqueous extracts of Mangifera indica leaves and tetracyline on the liver of adults wistar rats. Anatomy Journal of Africa, 2017, 6(2), 934-942. https://doi.org/10.4314/aja.v6i2.160463. 28. Amien, A.I., Fahmy, S.R., Abd-Elgleel, F.M., Elaskalany, S.M. Renoprotective effect of Mangifera indica polysaccharides and silymarin against cyclophosphamide toxicity in rats. The Journal of Basic and Applied Zoology, 2015, 72, 154-162. https://doi.org/10.1016/j.jobaz.2015.09.006. 29. Nadella, S.K., Kumar, N.R. Mangifera indica Leaves Extract Effect on Liver Function in Experimental Animal Studies. Biomedical and Pharmacological Journal, 2016, 9(3), 1-5. https://dx.doi.org/10.13005/bpj/1041. 30. Diaz, G.J. Toxicosis by Plant Alkaloids in Humans and Animals in Colombia. Toxins, 2015, 7(12), 5408-5416. https://doi.org/10.3390/toxins7124892. 31. Solofomalala, A.H.D., Rajemiarimoelisoa, C.F., Judicael, R.L., Randrianarivo, H.R., Rakoto, D.A.D., Jeannoda, V.L., Boumendjel, A. Pyrrolizidine-Derived Alkaloids: Highly Toxic Components in the Seeds of Crotalaria cleomifolia Used in Popular Beverages in Madagascar. Molecules, 2021, 26, 3464. https://doi.org/10.3390/molecules26113464. https://doi.org/10.4103/0973-7847.65325 https://doi.org/10.1155/2017/6949835 https://doi.org/10.3390/antiox10020299 https://europepmc.org/article/med/30033432 https://doi.org/10.1098/rspb.1927.0030 https://doi.org/10.1155/2014/691574 https://www.phytojournal.com/archives/2018/vol7issue2/PartAP/7-2-316-293.pdf https://doi.org/10.1016/j.jhep.2019.02.014 https://doi.org/10.1155/2019/4763015 https://doi.org/10.5897/JMPR09.487 https://doi.org/10.19080/JCMAH.2018.07.555698 https://doi.org/10.1016/j.aquaculture.2006.08.051 https://doi.org/10.1007/978-94-007-6728-7_2-1 https://doi.org/10.3945/cdn.116.000042 https://doi.org/10.4314/aja.v6i2.160463 https://doi.org/10.1016/j.jobaz.2015.09.006 https://dx.doi.org/10.13005/bpj/1041 https://doi.org/10.3390/toxins7124892 https://doi.org/10.3390/molecules26113464 TAJUDEEN OLANREWAJU YAHAYA ET AL. 9 32. Chung, K.T., Wong, T.Y., Wei, C.I., Huang, Y.W., Lin, Y. Tannins and human health: a review. Critical Reviews in Food Science and Nutrition, 1998, 38(6), 421-464. https://doi.org/10.1080/10408699891274273. 33. Diwan, F.H., Abdel-Hassan, I.A., Mohammed, S.T. Effect of saponin on mortality and histopathological changes in mice. Eastern Mediterranean Health Journal, 2000, 6(2-3), 345-351. 34. Obaroh, I.O., Keta, J.N., Nwaogu, J., Abdullahi, A. Effects of Moringa oleifera Saponins Extract on Histology of Liver and Intestine of Clarias gariepinus. Asian Journal of Biological Sciences, 2020, 13, 335-340. https://scialert.net/abstract/?doi=ajbs.2020.335.340. 35. Shelton, L.M., Park, B.K., Copple, I.M. Role of Nrf2 in protection against acute kidney injury. Kidney International, 2013, 84(6), 1090-1095. https://doi.org/10.1038/ki.2013.248. Received 6 December 2022 Accepted 21 February 2023 https://doi.org/10.1080/10408699891274273 https://scialert.net/abstract/?doi=ajbs.2020.335.340 https://doi.org/10.1038/ki.2013.248