Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 12, Number 2, October 2023 | Pages: 477-484 | DOI: 10.14421/biomedich.2023.122.477-484 ISSN 2540-9328 (online) Nephroprotective Activities of Ethanol Root Extract and Fractions of Hippocratea africana Against Doxorubicin-Induced Kidney Toxicity Kufre U. Noah1, John A. Udobang2, Jude E. Okokon1,*, Martin O. Anagboso3, Nwakaego Omonigho Ebong4 1Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria. 2Department of Clinical Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, University of Uyo, Uyo, Nigeria. 3Department o Microbiology, Madonna University, Elele, Nigeria. 4Department of Pharmacology and Toxicology, Faculty of Pharmacy, Madonna University, Elele, Nigeria. Corresponding author* judeefiom@yahoo.com, Tel.no. +234-8023453678 Abstract Hippocratea africana root used locally in the treatment of poisoning was investigated to confirm its antidotal potential in rats. The root extract (200-600 mg/kg) and fractions; dichloromethane (DCM) and aqueous, 400 mg/kg) were evaluated for nephroprotective activity against doxorubicin-induced kidney injury in rats. Kidney function parameters, kidney oxidative stress markers and kidney histology were used to assess the kidney protective effect of the extract. The root extract and fractions (200-600 mg/kg) significantly (p<0.05-0.01) reduced the levels of creatinine, urea and electrolytes that were elevated by doxorubicin. Also, the MDA level elevated by doxorubicin was reduced by the extract and fractions co-administration, while the levels of GSH, GST, SOD, GPx, and CAT that were decreased by doxorubicin were significantly (p<0.01) elevated by the root extract/fractions. Histology of the kidney sections of extract/fractions - treated animals showed reductions in the pathological features compared to the organotoxic-treated animals. The chemical pathological changes were consistent with histopathological observations suggesting marked nephroprotective potential. The anti-toxic effect of this plant may in part be mediated through the chemical constituents of the plant. The plant, Hippocratea africana possesses anti-toxicant properties which can be exploited in the treatment of doxorubicin related toxicities. Keywords: Renoprotective; Hippocratea africana; doxorubicin; oxidative stress. Abbreviations: Dichloromethane (DCM), Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), Reduced glutathione (GSH), Malondialdehyde (MDA), haematotoxylin and eosin (H&E), Reactive oxygen species (ROS), reduced nicotinamide adenine dinucleotide phosphate (NADPH). INTRODUCTION Hippocratea africana (Willd.) Loes. ex Engl. (Celastraceae) known in English as ‘African paddle-pod’ and ‘Eba enang enang’ in Ibibio language in Nigeria, is a climber perennial plant distributed widely in tropical Africa (Hutchison and Dalziel, 1973). Traditionally, the plant root has been variously utilized in herbal preparations to treat diseases like malaria and diabetes (Okokon et al., 2006), as well as liver diseases (Ajibesin et al., 2008). Previous reports showed that the root extract possess antimalarial (Okokon et al., 2006; Okokon et al., 2021), antioedema and antinociceptive (Okokon et al., 2008), antidiabetic and hypolipidemic (Okokon et al., 2010; 2022), antidiarrhoeal and antiulcer (Okokon et al., 2011), hepatoprotective (Okokon et al., 2013a), antileishmanial, cytotoxicity and cellular antioxidant (Okokon et al., 2013b), antibacterial, anticonvulsant and depressant (Okokon et al., 2014). Also, earlier studies had reported the presence of ᵟ-3- Carene and α-terpineol (Okokon et al., 2017), isolation of 1,3,7-trihydroxy-6-methoxyxanthone [isoathyriol] and 1,3,6,7-tetrahydroxyxanthone [norathyriol] (Umoh et al., 2021) from ethyl acetate fraction. Monoterpenes and sesquiterpenes have been identified in the n-hexane fraction (Okokon et al., 2013a). We report nephroprotective and antioxidative stress effects of the root extract and fractions of H. africana against doxorubicin-induced nephrotoxicity in rats. MATERIALS AND METHODS Plants Collection Fresh roots of Hippocratea africana were collected in bushes in Uruan area, Akwa Ibom State, Nigeria in November, 2021. The plant was identified and authenticated by a taxonomist in the Department of Manuscript received: 28 June, 2023. Revision accepted: 17 August, 2023. Published: 23 August, 2023. https://doi.org/10.14421/biomedich.2023.122.477-484 478 Biology, Medicine, & Natural Product Chemistry 12 (2), 2023: 477-484 Botany and Ecological Studies, University of Uyo, Uyo, Nigeria. Herbarium specimen was deposited at Department of Pharmacognosy and Natural Medicine Herbarium, University of Uyo. Preparation of extract and fractions Fresh root of H. africana were washed, cut into smaller pieces and dried under shade for two weeks. They were powdered using electric grinder. The pulverised root of H. africana (HAE) was soaked in ethanol (50%) for 72 hours. The liquid filtrate obtained was concentrated in a rotary evaporator at 40˚C. The crude extract (20 g) was dissolved in 500 mL of distilled water and partitioned with equal volume of dichloromethane (DCM, 5 x 500 mL) till no colour change was observed, to obtain DCM and aqueous fractions. The extract and fractions were stored at 4˚C in a refrigerator until used for the experiment. Animals In this study, male albino Wistar rats were used. The animals were sourced from University of Uyo Animal house and sheltered in plastic cages. The rats were fed with pelleted standard Feed (Guinea feed) and given unlimited access to water. The study was approved by College of Health Sciences Animal Ethics Committee, University of Uyo. Experimental design In this study, repeated dose model earlier described by Raskovi et al. (2011) and Olorundare et al., (2020), which lasted for 14 days was used. Groups I rats which served as the untreated control were orally pretreated with 10 mL/kg/day of distilled water. Group 2 rats were given normal saline (10 mL/kg/day) but equally treated on alternate days with 1.66 mg/kg of doxorubicin hydrochloride dissolved in 0.9% normal saline administered on alternate days for 14 days. Groups’ 3-5 rats were orally pretreated with 200 mg/kg/day, 400 mg/kg/day, and 600 mg/kg/day of Hippocratea africana dissolved in distilled water 2 hours before treatment with 1.66 mg/kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days, respectively. Groups 6 and 7 were pretreated with 400 mg/kg of DCM and aqueous fractions respectively. Group 8 rats which served as the positive control group were equally pretreated with 100 mg/kg/day of silymarin two hours before treatment with 1.66 mg/kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days. Collection of blood samples and organs After 14 days of treatment (24 hours after the last administration) the rats were weighed again and sacrificed under light diethyl ether vapour. Blood samples were collected by cardiac puncture and used immediately. Blood samples were collected into plain centrifuge tubes. The blood in the centrifuge tubes were centrifuged at 1500 rpm for 15 minutes to separate of serum at room temperature used for biochemical assays. The rats’ kidneys were identified, harvested, and weighed. Kidney function test The following biochemical parameters such as levels of electrolytes (Na, K, Cl, and HCO3), creatinine and blood urea were assayed as markers of kidney function using diagnostic kits at the Chemical Pathology Department of University of Uyo Teaching Hospital. Oxidative Stress Markers The antioxidant enzymes assays were performed on kidney homogenates of rats that were used in this study. These oxidative stress markers were used to assess antioxidative stress potentials of the extract. Preparation of Renal Homogenate In each rat, the kidneys were removed and one kidney was fixed in 10% formaldehyde for histological processes, while the other kidney was dissected free from the surrounding fat and connective tissue and used for assays of oxidative markers. The kidneys were longitudinally sectioned, and renal cortex was separated and kept at -8°C. Subsequently, renal cortex was homogenized in cold potassium phosphate buffer (0.05M, Ph 7.4). The renal cortical homogenates were centrifuged at 5000 rpm for 10 min at 4°C. The resulting supernatant was used for the determination of superoxide dismutase (SOD) (Marklund and Marklund, 1974), catalase (CAT) (Sinha, 1972), glutathione peroxidase (GPx) (Lawrence and Burk, 1976), reduced glutathione (GSH) (Ellman, 1959) and malondialdehyde (MDA) content (Esterbauer and Cheeseman, 1990). Histopathological studies The kidneys of the animals that were surgically removed and fixed in 10% formaldehyde were processed and stained with haematotoxylin and eosin (H&E) (Drury and Wallington, 1980), according to standard procedures at Department of Chemical Pathology, University of Port Harcourt Teaching Hospital, Port Harcourt. Morphological changes observed and recorded in the excised organs of the sacrificed animals. Histologic pictures were taken as micrographs. Statistical Analysis and Data Evaluation Data obtained from this work were analysed statistically using ANOVA (one –way) followed by a post test (Tukey-Kramer multiple comparison test). Differences between means were considered significant at 5% level of significance ie p≤ 0.05. Noah et al. – Nephroprotective Activities of Ethanol Root Extract and Fractions … 479 RESULTS AND DISCUSSION Effect of root extract and fractions of H. africana on body and organs weights of rats with doxorubicin- induced toxicity Administration of H. africana root extract and fractions to rats with doxorubicin-induced organs toxicities caused considerable improvement of the body weights compared to the organotoxic group. The crude extract caused a pronounced dose-dependent effect (7.14 -8.09%) when compared to the organotoxic group with the dichloromethane fraction treated group exerting the highest effect (9.30%). Silymarin also improved the weight of the treated animals considerably (7.99%). The weights of kidneys of the group treated with doxorubicin only were found to be reduced when compared to those of the normal control group though not statistically significant (p>0.05). However, treatment of rats with doxorubicin-induced toxicities with the root extract and fractions of H. africana improved the organs weights though insignificantly (p>0.05) except in the group treated with aqueous fraction (Table 1). Evaluation of effect of root extract and fractions of H.africana on kidney function parameters of doxorubicin-induced kidney injury in rats. Table 2 shows the effect of root extract/fractions of H. africana on kidney function parameters of rats. Administration of doxorubicin (1.66 mg/kg) to the rats caused significant (p<0.05-0.001) elevation of serum urea, creatinine and electrolytes (K+, Na+ and HCO-3) except Cl- when compared to normal control. These increased levels of serum urea, creatinine and electrolytes (K+, Na+ and HCO-3) were significantly (p<0.05 - 0.001) reduced when compared to organotoxic group following pretreatment of the rats with silymarin and root extract/fractions (200 – 600 mg/kg), with the DCM fraction having the highest effect in most cases. These reductions were non dose-dependent with the lower doses (200 and 400 mg/kg) exerting more significant effects. However, the Cl- level was not affected significantly (p>0.05) with the root extract/fraction treatment (Table 2). Effect of root extract and fraction on kidney oxidative stress markers of doxorubicin-induced kidney toxicity in rats. The effect of H. africana root extract/fractions on kidney oxidative stress markers of the rats is as shown in Table 3. Administration of doxorubicin (1.66 mg/kg i.p) on alternate days for 14 days caused significant (p<0.05- 0.001) decreases of kidney antioxidant enzymes activities (SOD, GPx, GST, CAT) and GSH levels when compared to control. The MDA level was also elevated by doxorubicin treatment significantly (p<0.001) when compared to control. However, repeated administration of root extract/fractions of H. africana (200- 600 mg/kg) concomitantly with doxorubicin for 14 days caused non dose-dependent elevations of the enzymatic and non- enzymatic endogenous antioxidants in the treated rats groups which were mostly significant (p<0.05-0.001) in the higher doses (400 and 600 mg/kg) of the root extract when compared to the organotoxic groups with DCM fraction being the most active. Dose-dependent and significant (p<0.05-0.001) decreases in MDA levels of the extract/fractions treated groups were recorded when compared to control with aqueous fraction as the most active fraction. Similar decreases were also observed in the silymarin-treated group when compared to organotoxic control (Table 3). Effect of root extract and fractions of H. africana on histology of rat kidney in doxorubicin-induced nephrotoxicity Histological sections of kidneys of rats receiving various treatments at magnification (x400) stained with H&E method revealed that group 1 (A) treated with distilled water (10 mL/kg) showed normal renal tubules and glomeruli. No evidence of pathology was seen. The organotoxic group (Group 2, B) treated with doxorubicin (1.66 mg/kg) showed focal non-specific inflammation, normal renal tubules and glomeruli (GM) and few congested blood vessels were seen. Rats in group 3 (C ) treated with 200 mg/kg of H. africana root extract and doxorubicin (1.66 mg/kg), group 4 (D) treated with 400 mg/kg of H. africana root extract and doxorubicin (1.66 mg/kg), group 6 (F) treated with 400 mg/kg of aqueous fraction of H. africana root and doxorubicin (1.66 mg/kg), group 7 (G) treated with 400 mg/kg of dichloromethane fraction of H. africana root and doxorubicin (1.66 mg/kg) and group 8 (H) treated with 100 mg/kg of silymarin of H. africana root and doxorubicin (1.66 mg/kg) had kidney sections showing normal renal tubules and glomeruli with no evidence of pathology seen. Group 5 (E) rat treated with 600 mg/kg of H. africana root extract and doxorubicin (1.66 mg/kg) showed distorted parenchyma with both normal and dilated renal tubules. The dilated tubular epithelium appears flattened. There were also multifocal interstitial inflammatory infiltrates (figures A-H). 480 Biology, Medicine, & Natural Product Chemistry 12 (2), 2023: 477-484 Table 1. Effect of H. africana root extract on body and kidney weights of rats with doxorubicin-induced toxicity. Parameters/ Treatment Dose mg/kg Kidney Body weight Before After % increase in body weight Normal control - 1.24±0.10 135.6±18.34 151.0± 12.33 11.35 Doxorubicin 1.66 0.94±0.10 130.0 ± 9.45 126.3± 10.43 -2.84 Silymarin+DOX 100 1.23±0.17 132.6± 14.55 143.2 ± 3.15 7.99 Extract+DOX 200 1.20±0.05 142.8± 10.56 153.0± 5.29 7.14 400 1.14±0.02 140.3± 7.36 151.6 ± 6.22 8.05 600 1.11±0.06 138.4 ± 8.54 149.6 ± 8.48 8.09 Aqueous fraction 400 1.05±0.11 138.4 ± 6.26 144.6± 10.22 4.47 DCM fraction 400 1.17±0.09 134.3± 8.50 146.8± 13.20 9.30 Data were expressed as mean ±SEM. significant at dp<0.001 when compared to normal control; ap< 0.05, bp< 0.01, cp< 0.001 when compared to organotoxic control. (n = 6). Table 2. Effect of H.africana root extract and fractions on kidney function parameters of rats with doxorubicin-induced toxicity. Treatment Dose mg/kg Urea (mMol/L) Creatinine (µmol/L) Chloride (mMol/L) Potassium (mMol/L) Sodium (mMol/L) Bicarbonate (mMol/L) Control 10 4.50± 0.30 94.5±5.86 45.75±1.93 3.47± 0.19 111.0± 5.11 22.00± 0.81 Doxorubicin 1.66 10.50± 0.40c 171.6±2.02c 46.66±0.88 6.53± 0.08c 165.0± 1.73c 30.66± 0.66a Crude extract 200 7.13±0.31e 143.0± 6.35a 57.0±2.51 4.13± 0.78f 125.0±16.37d 22.66± 1.66d 400 5.50±0.30f 112.3±11.34e 48.33±0.88 4.63± 0.28f 138.6± 6.76 25.0± 1.00 600 6.16±0.97f 125.6± 16.33d 48.66±1.20 3.40± 0.05f 107.3±1.45f 24.0± 2.00 Aqueous Fraction 400 4.80±0.50f 108.5±2.72f 49.50±1.70 4.17± 0.55b 118.7± 6.27b 22.33± 1.33d DCM fraction 400 5.00±0.40f 101.0±8.62f 44.0±1.15 3.10± 0.15f 104.6± 1.45f 25.66± 2.33 Silymarin 100 3.60±0.05f 75.6±2.33f 44.60±1.45 3.13± 0.17f 105.0± 2.51f 22.33± 2.60d Data is expressed as mean ± SEM, Significant at ap<0.05, bp<0.01, cp<0.001, when compared to control; Significant at dp<0.05, ep<0.01, fp<0.001 compared to organotoxic group. (n=6). Table 3. Effect of H.africana root extract and fractions on kidney oxidative stress markers of rats with doxorubicin-induced toxicity. Treatment Dose mg/kg SOD (U/ml) CAT (U/g of protein) GPx (µg/ml) GSH (µg/ml) GST MDA (µMol/ml) Control 10 0.61±0.02 1.51±0.06 0.090±0.002 1.18±0.02 0.40±0.01 0.22±0.02 Doxorubicin 1.66 0.17±0.01c 0.46±0.01a 0.040±0.00a 0.31±0.01b 0.22±0.01c 0.63±0.01c Crude extract 200 0.30±0.01b 1.04± 0.15c 0.051±0.001 0.85± 0.15 0.25±0.01b 0.50±0.01b 400 0.39±0.01a,d 1.11±0.12c 0.067±0.001e 1.16±0.01d 0.28±0.01b 0.46±0.03b 600 0.51±0.02e 1.26± 0.05c,e 0.064±0.004 1.12± 0.08 0.34±0.01e 0.31±0.02f Aqueous Fraction 400 0.23±0.06c 1.15±0.02f 0.045±0.003a 1.02± 0.07 0.31±0.001d 0.39± 0.01e DCM fraction 400 0.48±0.04a,d 1.36±0.02c,d 0.069±0.002e 1.15± 0.14 0.33±0.01e 0.30± 0.01f Silymarin 100 0.52±0.02f 1.15±0.06c 0.076±0.002e 1.13±0.01d 0.36±0.01f 0.34±0.02a,d Data is expressed as MEAN ± SEM, Significant at ap<0.05, bp<0.01, cp<0.001, when compared to control; Significant at dp<0.05, ep<0.01, fp<0.001 compared to organotoxic group. (n=6). Figure A. Photomicrograph of kidney section of rat treated with distilled water (10mL/kg) showing normal renal tubules (RT) and glomeruli (GM), no evidence of pathology seen. H&E Stain, x400 magnification Figure B. Photomicrograph of kidney section of rat treated with doxorubicin (1.66mg/kg) showing focal non-specific inflammation (red arrowhead) with lower magnification, normal renal tubules (RT), and glomeruli (GM). There are few congested blood vessels (V). H&E stain x400 Noah et al. – Nephroprotective Activities of Ethanol Root Extract and Fractions … 481 Figure C. Photomicrograph of kidney section of rat treated with 200 mg/kg of H. africana root extract and doxorubicin (1.66mg/kg) showing normal renal tubules (RT) and glomeruli (GM), no evidence of pathology seen. H&E Stain, x400 magnification Figure D. Photomicrograph of kidney section of rat treated with 400 mg/kg of H. africana root extract and doxorubicin (1.66mg/kg) showing normal parenchymal with renal tubules (RT) and glomeruli (GM). No lesion seen. H&E Stain, x400 magnification Figure E. Photomicrograph of kidney section of rat treated with 600 mg/kg of H. africana root extract and doxorubicin (1.66 mg/kg) showing distorted parenchyma with both normal (RT) and dilated renal tubules (). The dilated tubular epithelium appears flattened. There are also multifocal interstitial inflammatory infiltrates (red arrowhead). H&E Stain, x400 magnification Figure F. Photomicrograph of kidney section of rat treated with 400 mg/kg of aqueous fraction of H. africana root and doxorubicin (1.66 mg/kg) showing normal parenchyma with renal tubules (RT), glomeruli (GM) and a focal congested blood vessel (V). No degenerative changes seen. H&E Stain, x400 magnification Figure G. Photomicrograph of kidney section of rat treated with 400 mg/kg of dichloromethane fraction of H. africana root and doxorubicin (1.66mg/kg) showing normal renal tubules (RT) and glomeruli (GM), no evidence of pathology seen. H&E Stain, x400 magnification Figure H. Photomicrograph of kidney section of rat treated with 100 mg/kg of silymarin and doxorubicin (1.66mg/kg) showing normal renal tubules (RT) and glomeruli (GM), no evidence of pathology seen. H&E Stain, x400 magnification Discussion This work was designed to investigate the effect of root extract and fractions of Hippocratea africana on doxorubicin-induced kidney toxicity in rats in a bid to confirm the folkloric claim of its antidotal activity. Doxorubicin is an anthracycline glycoside antibiotic that 482 Biology, Medicine, & Natural Product Chemistry 12 (2), 2023: 477-484 possesses a potent and broad spectrum antitumour activity against a variety of human solid tumours and haematological malignancies (Calabresi and Chamber, 1990). However its use in chemotherapy has been limited largely due to its diverse toxicities, including cardiac, hepatic, hematological and testicular toxicity (Yilmaz et al., 2006). The semiquinone form of doxorubicin is a toxic short-lived metabolite which interacts with molecular oxygen and initiates a cascade of reactions, producing reactive oxygen species (ROS). ROS generation, inflammatory processes and lipid peroxidation have been suggested to be responsible for doxorubicin-induced cardio-, hepato- and nephrotoxicity (Injac et al., 2009; Kalender et al., 2005). It has been proposed that DOX-semiquinone, an unstable metabolite of DOX, reacts with O2, producing H2O2 and O2 − (superoxide). In addition, DOX enhances the activity of extramitochondrial oxidative enzymes such as xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and also interferes with mitochondrial iron export, resulting in formation of ROS (reactive oxygen species) (Bachur et al., 1979). In this study, doxorubicin administration was found to have caused elevation of serum urea, creatinine and electrolytes (K+, Na+, Cl- and HCO-3) levels when compared to normal control, which is an indication of a serious injury to the kidney. This finding is consistent with earlier report of Rajasekaran (2019), which similar elevations were reported. It is well documented that kidney injury are indicated by increase in serum level of creatinine and urea (Laskshmi and Sudhakar, 2010) as well as increase serum levels of Na, K, Cl and bicarbonate (James and Mitchel, 2006). However, these increases were reduced significantly by the co- administration of root extract and fractions of H. africana. Doxorubicin is reported to cause nephrotoxicity via oxidative stress as free radicals formed caused tubular atrophy and increased glomerular capillary permeability. Nephrotoxicity by doxorubicin can also result from lipid peroxidation and biological macromolecules damage by iron-dependent oxidative damage (Mohan et al., 2010). Degenerative changes in kidney depend on cumulative dose and duration of treatment as doxorubicin metabolites are partly excreted from the kidney. Another mechanism for renal injury is the conversion of DOX to semiquinone free radical by NADPH-cytochrome P-450 which generates hydroxyl radical and superoxide anion which causes lipid peroxidation (Rashid et al., 2013). The reduction of the levels of urea, creatinine and electrolytes by the root extract and fractions in this study is as a result of their free radical scavenging potentials, thereby protecting the kidney against oxidative stress by free radicals generated by doxorubicin. The antioxidative burst and antioxidant activities of the root extract and fractions of H. africana had previously been reported (Okokon et al., 2013a; 2022; Umoh et al., 2021). Moreso, the antioxidative stress activities of the root extract and fractions observed in this study further support the antioxidant potentials of the plant. These activities may have contributed to the observed protective effects in this study. The findings of this study show that administration of doxorubicin (2.5 mg/kg, i.p) on alternate days for 14 days to rats caused significant decreases (p<0.05) in levels of enzymatic and non-enzymatic endogenous antioxidants (GSH,SOD, CAT, GPX and GSH) when compared to control, while the MDA level was elevated, Lipid peroxidation is a marker of oxidative stress and elevations in the amount of MDA, a lipid peroxidation product, have been reported following doxorubicin treatment (Rashid et al., 2013, Rehman et al., 2014, Khames et al., 2019). This trend was observed in this study. Concomitant administration of root extract H.africana (200-600 mg/kg) with doxorubicin caused significant (p<0.05-0.001) non dose- dependent elevation in the levels of the antioxidant enzymes (SOD, CAT, GPX) when compared to control. Similarly, GSH level was significantly (p<0.001) elevated following treatment with the extract when compared to control. Also, there were significant (p<0.05-0.01) reductions in the level of MDA of the extract-treated rats. It has been documented that DOX inhibits the activities of endogenous enzymatic and non-enzymatic antioxidants as was the case in this study. So, an imbalance between ROS generation and neutralization leads to oxidative stress and injury to the kidney (Abushouk et al., 2017; Abdel-Daim et al., 2017; Aboushouk et al., 2019). The reduced MDA level caused by the administration of the root extract and fractions may have resulted from reduction in lipid peroxidation and generation of free radicals which might have been scavenged by the phytoconstituents present in the root extract and fractions, revealing the antioxidative stress potentials of the root extract and hence the protective effect on the kidney as was observed. Histological findings in this study revealed that kidneys of rats treated with doxorubicin (2.5 mg/kg) alone showed pathological signs of injury which were seen as degenerated microvesicles in the tubular lining cells among others. However, co-administration of H. africana root extract/fractions and doxorubicin reduced the toxic effects of the doxorubicin as normal glomeruli which were devoid of pathological signs were seen in the kidney sections of the extract-treated rats examined. This further confirms the nephroprotective potential of the root extract which may have been exerted through the antioxidant and antioxidative stress activities of its phytochemical constituents. CONCLUSIONS The findings of this study show that the root extract and fractions of Hippocratea africana have the potential to Noah et al. – Nephroprotective Activities of Ethanol Root Extract and Fractions … 483 counteract the injurious effect of doxorubicin on the kidney. This activity can be attributed to the antioxidant and antioxidative stress activities of their phytochemical constituents. Thus, the root extract can be used to alleviate and/or prevent doxorubicin-induced renotoxicity. Acknowledgements: The technical support from Mr Nsikan Malachy of Pharmacology and Toxicology Department was tremendously acknowledged. Authors’ Contributions: KN, JAU and JEO initiated and designed this study. JEO, KN and JAU carried out the experiments and drafted the manuscript. NOE and MOA performed the statistical analysis, edited and reviewed the manuscript. KN, JAU, MOA, JEO and NOE read and approved the final manuscript. Competing Interests: The authors declare that there are no competing interests. REFERENCES Abdel-Daim MM, kilany OE, Khalifa HA, Ahmed AAM. (2017). Allicin ameliorates doxorubicin-induced cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Cancer Chemotherapy and Pharmacology 80(4): 745–753. Abushouk AI, Ismail A, Salem AMA, Afifi AM, Abdel-Daim MM. (2017). Cardioprotective mechanisms of phytochemicals against doxorubicin-induced cardiotoxicity. Biomedicine & Pharmacotherapy 90: 935–946. Abushouk AI, Salem AMA, Saad A. (2019). Mesenchymal stem cell therapy for doxorubicin-induced cardiomyopathy: potential mechanisms, governing factors, and implications of the heart stem cell debate. Frontiers in Pharmacology 10: 635. Ajibesin K K, Ekpo BA, Bala D N, Essien E E, Adesanya SA.2008. Ethnobotanical survey of Akwa Ibom State of Nigeria. J Ethnopharm 115: 387 – 408. Bachur NR, Gordon SL, Gee MV, Kon H. (1979). NADPH- cytochrome P450 reductase activation of quinone anticancer agents to free radicals. Proceedings of the National Academy of Sciences USA 76: 954-957. Burkill HM. 2000. The useful plants of West Tropical Africa. 2nd Edition. Volume 5, Families S–Z, Addenda. Royal Botanic Gardens, Kew, United Kingdom. 686 pp. Calabresi P, Chabner BA. (1990) Chemotherapy of neoplastic diseases, In: Gilman AG, Rall TW, Nies AS, Taylor P. (eds.). The Pharmacological Basis of Therapeutics. NY: Pergamon Press Inc. pp. 1203-1263. Drury RA, Wallington EA. (1980) Carleton’s Histological Techniques. 5th Edition, Oxford University Press, New York, 195. Ellman GL. (1959). Tissue sulfhydryl groups. Archieves of Biochemistry and Biophysics. 82: 70-77. Esterbauer H, Cheeseman KH. (1990). Determination of aldehydic lipid peroxidation products: malonaldehyde and 4- hydroxynonenal. Methods in Enzymology 186: 407–421. Gunes FE. 2013. Medical Use of squalene as a natural antioxidant. Journal of Marmara University Institute of Health Sciences 3(4):220-228. Gupta R, Sharma AK, Dobhal MP, Sharma MC and Gupta RS. (2011). Antidiabetic and antioxidant potential of β-sitosterol in streptozotocin-induced experimental hyperglycemia. Journal of Diabetes 3(1):29-37. Gupta RK, Hussain T, Panigrahi G, Das A, Singh GN, Sweety K, Faiyazuddin MD and Rao CV. (2011a). Hepatoprotective effect of Solanum xanthocarpum fruit extract against CCl4 induced acute liver toxicity in experimental animals. Asian Pacific Journal of Tropical Medicine 6: 964-968. Hutchinson J, Dalziel JM. 1973. Flora of West Tropical Africa. 2nd edition. Crown Agents for Overseas Government and Administration, Vol.1, Part 2, p.638 Injac R, Perse M, Cerne M, Potocnik N, Radic N, Govedarica B, Djordjevic A, Cerar A, Strukelj B. (2009). Protective effects of fullerenol C60 (OH)24 against doxorubicin-induced cardiotoxicity and hepatotoxicity in rats with colorectal cancer. Biomaterials 30: 1184-1196. Kalender Y, Yel M, Kalender S. (2005). Doxorubicin hepatotoxicity and hepatic free radical metabolism in rats. The effects of vitamin E and catechin. Toxicology 209: 39-45. Khames A, Khalaf MM, Gad AM, Abd El-Raouf OM, Kandeil MA. (2019). Nicorandil combats doxorubicin-induced nephrotoxicity via amendment of TLR4/P38 MAPK/NFj-B signaling pathway. Chemico-Biological Interactions 311, 108777. Lawrence RA, Burk RF. (1976). Glutathione peroxidase activity in selenium- deficient rat liver. Biochemical and Biophysical Research Communications 71: 952-958. Leng J, Li X, Tian H, Liu C, Guo Y, Zhang S, Chu Y, Li J, Wang Y, Zhang L. (2020). Neuroprotective effect of diosgenin in a mouse model of diabetic peripheral neuropathy involves the Nrf2/HO-1 pathway. BMC Complementary Medicine and Therapies 20:126. Marklund S, Marklund G. (1974). Involvement of superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry. 47: 469 - 474. Micera M, Botto A, Geddo F, Antoniotti S, Bertea CM, Levi R, Gallo MP, Querio G (2020). Squalene: More than a Step toward Sterols. Antioxidants 9: 688. Mohan M, Kamble S, Gadhi P, Kasture S. (2010). Protective effect of Solanum torvum on doxorubicin-induced nephrotoxicity in rats,. Food and Chemical Toxicology 48(1): 436–440, 2010. Okokon J E, Ita BN, Udokpoh A.E. 2006. The in vivo antimalarial activities of Uvaria chamae and Hippocratea africana. Annals Trop Med Parasitol 100:585-590. Okokon JE, Antia BS, Umoh EE, Etim EI. 2010. Antidiabetic and hypolipidaemic activities of Hippocratea africana. Int J Drug Dev Res 2: 501 -506. Okokon JE, Nwafor PA, Charles U, Dar A, Choudhary MI. 2013a. The antioxidative burst and hepatoprotective effects of ethanolic root extract of Hippocratea africana against paracetamol-induced liver injury. Pharm Biol 51 (7):872 - 880. Okokon JE, Dar A, Choudhary MI. 2013b. Immunomodulatory, cytotoxic and antileishmanial activities of Hippocratea africana. J Nat Pharmaceut 4 (2):81 – 85. Okokon JE, Davies K, Okokon PJ, Antia BS. 2014. Depressant, anticonvulsant and antibacterial activities of Hippocratea africana. Int J Phytother 4 (3):144 – 153. 484 Biology, Medicine, & Natural Product Chemistry 12 (2), 2023: 477-484 Okokon JE, Akpan HD, Ekaidem I, Umoh EE. 2011. Antiulcer and antidiarrheal activity of Hippocratea africana. Pak J Pharm Sci 24: 201- 205. Okokon JE, Okokon PJ, Sahal D. 2017. In vitro antiplasmodial activity of some medicinal plants from Nigeria. Int J Herbal Med 5 (5):102-109. Okokon JE, Chinyere CP, Bassey AL, Udobang JA. 2021. In vivo alpha amylase and alpha glucosidase activities of ethanol root extract and fractions of Hippocratea africana. South Asian J Parasitol 5(4): 42-48. Okokon JE, Chinyere PC, Amaechi P, Bassey AL, Thomas PS (2022). Antioxidant, antidiabetic and hypolipidemic activities of ethanol root extract and fractions of Hippocratea africana. Tropical Journal of Natural Product Research. 6(3):446-453. Olorundare OE, Adeneye AA, Akinsola AO, Sanni DA, Koketsu M, Mukhtar H. (2020). Clerodendrum volubile ethanol leaf extract: A potential antidote to doxorubicin-induced cardiotoxicity in rats. Journal of Toxicology 2020:n8859716. Rajasekaran M. (2019). Nephroprotective effect of Costus pictus extract against doxorubicin-induced toxicity on Wistar rat. Bangladesh Journal of Pharmacology 14:93-100. Raškovi A, Stilinovi N, Kolarovi J, Vasovi V, Vukmirovi S. (2011). The protective effects of silymarin against doxorubicin-induced cardiotoxicity and hepatotoxicity in rats Molecules 16: 8601-8613 Rashid S, Ali N, Nafees S, Ahmad ST, Arjumand W, Hasan SK, Sultana S. (2013). Alleviation of doxorubicin-induced nephrotoxicity and hepatotoxicity by chrysin in Wistar rats. Toxicology Mechanisms and Methods 23: 337–345. Rehman MU, Tahir M, Khan AQ, Khan R, Oday OH, Lateef A, Hassan SK, Rashid S, Ali N, Zeeshan M, Sultana S. (2014). D- limonene suppresses doxorubicin-induced oxidative stress and inflammation via repression of COX- 2, iNOS, and NFjB in kidneys of Wistar rats. Experimental Biology and Medicine (Maywood) 239:465–476. Rhetso T, Shubharani R, Roopa MS, Sivaram V. (2020). Chemical constituents, antioxidant, and antimicrobial activity of Allium chinense G. Don. Future Journal of Pharmaceutical Sciences 6:102. Sengupta S, Nandi I, Bhattacharyya DK, Ghosh M. (2018). Anti- Oxidant and anti-bacterial properties of 1-Octacosanol isolated from rice Bran Wax. Journal of Plant Biochemistry and Physiology 6: 206. Sinha AK. (1972). Colorimetric assay of catalase. Analytical Biochemistry, 47: 389 - 94. Tietz WW. (1990). Clinical Guide to Laboratory tests. 2nd edition. Sanders Company. Philadelphia, PA. pp. 554-556. Umoh UF, Thomas PS, Essien EE, Okokon JE, De Leo M, Ajibesin KK, Flamini G, Eseyin OA. (2021). Isolation and characterization of bioactive xanthones from Hippocratea africana (Willd.) Loes.ex Engl. (Celastraceae). Journal of Ethnopharmacol. 280:114031. Yilmaz S, Atessahin A, Sahna E, Karahan I, Ozer S. (2006) Protective effect of lycopene on adriamycin- induced nephrotoxicity and nephrotoxicity. Toxicology 218: 164-171.