Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 13, Number 2, October 2024 | Pages: 397-406 | DOI: 10.14421/biomedich.2024.132.397-406 ISSN 2540-9328 (online) Hippocratea africana Ethanol Root Extract and Fractions Attenuate Doxorubicin-Induced Testicular Toxicity and Oxidative Stress Kufre U. Noah, Jude E. Okokon* Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria. Corresponding author* judeefiom@yahoo.com Manuscript received: 22 June, 2024. Revision accepted: 30 September, 2024. Published: 04 October, 2024. Abstract Hippocratea africana root, used locally in treating 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 hepatoprotective activity against doxorubicin-induced testicular toxicity in male rats. Testosterone level, lipid profile indices, testicular oxidative stress markers, and testis histology were used to assess the testicular protective effect of the extract. The root extract (200-600 mg/kg) and fractions, dichloromethane (DCM) and aqueous, 400 mg/kg reduced the serum levels of total cholesterol, triglycerides, LDL, and VLDL that were elevated by doxorubicin. In contrast, the high-density lipoprotein (HDL) reduced by doxorubicin was increased by the extract and fractions co-administration. Testosterone level, which was significantly (p<0.05) reduced by doxorubicin, was significantly (p<0.05-0.01) elevated by the root extract and fractions co-administration. The levels of GSH, GST, SOD, GPx, and CAT that were decreased by doxorubicin were significantly (p<0.01) elevated, and raised MDA level was reduced by the root extract and fractions. Histology of the testes sections of extract/fractions -treated animals showed absent/or reductions in the pathological features compared to the organotoxic- treated animals. The chemical pathological changes were consistent with histopathological observations, suggesting marked testicular protective potential. The anti-toxic effect of this plant may in part be mediated through the chemical constituents of the plant. The plant, H. africana possesses anti-toxicant properties which can be exploited in the treatment of doxorubicin-related toxicities. Keywords: Hippocratea africana; anti-oxidant; oxidative stress; testicular-protective; antioxidant. INTRODUCTION Doxorubicin, an anthracycline glycoside antibiotic with a broad spectrum of activity against various human solid tumors and hematological malignancies (Calabresi & Chamber, 1990) but limited clinical usefulness due to its diverse toxicities, including cardiac, hepatic, hematological, and testicular toxicity (Yilmaz et al., 2006). The toxic, short-lived metabolite, semiquinone form of doxorubicin, triggers the generation of reactive oxygen species (ROS) through cascades of events. Doxorubicin-induced cardio, hepatic, testicular, and nephrotoxicities have been attributed to ROS generation, inflammatory processes, and lipid peroxidation (Injac et al., 2009; Kalender et al., 2005). Doxorubicin has also been proposed to promote free radical generation by enhancing the activities of extra-mitochondrial oxidative enzymes such as NADPH and xanthine oxidases and interfering with mitochondrial iron export (Bachur et al., 1979). These free radicals cause distortion of the cell's membranes and cause organ dysfunction. Research on plants that can counteract the toxic effects of doxorubicin has been ongoing, especially on H. africana (Noah et al., 2023c). Hippocratea africana (Willd.) Loes. Ex Engl. (Celastraceae) known in English as African paddle-pod and Eba enang enang’ in the Ibibio language in Nigeria, is a climber perennial plant distributed widely in tropical Africa (Hutchison & Dalziel, 1973). Traditionally, the plant root has been utilized in various 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;2021), angioedema and antinociceptive (Okokon et al., 2008), antidiabetic and hypolipidemic (Okokon et al., 2010; 2022), antidiarrhoeal and antiulcer (Okokon et al., 2011), nephroprotective (Noah et al., 2023a; 2023b) hepatoprotective (Okokon et al., 2013a;Noah et al., 2023c, Noah et al., 2023d), antileishmanial, cytotoxicity and cellular antioxidant (Okokon et al., 2013b), antibacterial, anticonvulsant and depressant (Okokon et al., 2014), genotoxic and cytotoxic (Jonhnny et al., 2023). 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 https://doi.org/10.14421/biomedich.2024.132.397-406 398 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 397-406 fraction. Monoterpenes and sesquiterpenes have been identified in the n-hexane fraction (Okokon et al., 2013a). We report testicular protective and antioxidative stress potentials of the root extract and fractions of H. africana against doxorubicin-induced testicular toxicity in male rats. MATERIALS AND METHODS Plants collection Fresh root of Hippocratea africana were collected in bushes in the Uruan area, Akwa Ibom State, Nigeria in November 2021. The plant was identified and authenticated by a taxonomist in the Department of Botany and Ecological Studies, University of Uyo, Uyo, Nigeria. Hebarium 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 an electric grinder. The pulverised root of H. africana (HAE) was soaked in ethanol (50%) for 72 h. 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 This study used the repeated dose model earlier described by Raskovi et al. (2011) and Olorundare et al. (2020), which lasted for 14 days. Group 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 was centrifuged immediately at 1500 rpm for 15 mins to separate the serum at room temperature to avoid haemolysis and used for biochemical assays. The testes of the rats were surgically removed, weighed. One testis was fixed in 10 % formaldehyde for histological processes, while the other testis was briskly rinsed in ice cold 1.15% KCl solution and put in a clean sample bottle. These were stored in ice cold 0.9% NaCl. Evaluation of progressive motility, viability, count, and the structural abnormality of sperm The caudal piece of epididymis was isolated to retrieve the sperm samples. Initially, the epididymal part was finely minced in 5 mL of physiological-saline and was incubated for 30 min at 37◦C for spermatozoa releasing of the epididymal ducts. Sperm progressive motility percentage was noted through the phase-contrast microscope at 400X (Kenjale, 2008). Sperm viability was assessed, by eosin or nigrosin staining and accompanied by microscopic evaluation. Moreover, a hemocytometer was employed to count epididymal sperm in the suspension (Yokoi et al., 2003). Furthermore, morphological anomalies of the head, tail, and mid piece of sperm were determined in percentage using the method of Filler (1993). The apparent abnormal characteristics included (i) the size and shape of spermatozoa heads (bigor small heads) with lighter and emphasized curvature;(ii) intermediary pieces’ defects that result in untied heads; and (iii) defects of tails (short, multiple, folded, and broken tails). Biochemical Assays. Lipid Profile Blood samples from the heart chamber were allowed to clot and then centrifuged at 5000 rpm to separate clear sera from the clotted blood samples. The clear samples were obtained for assays of the following biochemical parameters: serum testosterone, serum cholesterol, triglyceride and high density lipoprotein (HDL) levels of the treated rats were measured using standard colorimetric methods (Tietz, 1995). These lipid parameter determinations were done Noah & Okokon – Hippocratea africana Ethanol Root Extract and Fractions Attenuate … 399 spectrophotometrically using Fortress Diagnostic Kits® according to standard procedures of manufacturer’s protocols. Serum testosterone levels were measured using the corresponding rat enzyme-linked immunosorbent assay kits (Testosterone, Cambridge, UK). Effect of the root extract and fractions on testis oxidative stress markers The oxidative marker assays were performed on the testes homogenates of rats that were used in this study. These oxidative stress markers were used to assess the antioxidant stress potentials of the extract. Preparation of testis Homogenate After the rats were sacrificed humanely under inhaled diethyl ether, the testes of the rats were surgically removed and weighed. They were briskly rinsed in ice cold 1.15% KCl solution and put in a clean sample bottle. These were stored in ice cold 0.9% NaCl. Homogenates were made in a ratio of 1 g of wet tissue to 9 ml of 1.25% KCl by using motor driven Teflon-pestle. The homogenates were centrifuged at 7000 rpm for 10 min at 4˚C and the supernatants were used for the assays of superoxide dismutase (SOD) (Marklund & Marklund, 1974), catalase (CAT) (Sinha,1972), glutathione peroxidase (GPx) (Lawrence & Burk,1976), reduced gluthathione (GSH) (Ellman,1959) & malondialdehyde (MDA) content (Esterbauer & Cheeseman, 1990). The assays were performed on testes homogenates of rats used in this study. These oxidative stress markers were used to assess the antioxidative stress potentials of the extract. Histopathological studies The excised testes fixed in 10 % buffered formalin were used for histological processes. They were processed and stained with haematotoxylin and eosin (H&E) (Drury & 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 The Data collected were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-test (Graph pad prism software Inc. La Jolla, CA, USA). Values were expressed as mean ± SEM and significance relative to control were considered at p˂0.001 and p˂0.05. RESULTS 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 organ toxicities caused considerable improvement in the body weights compared to the organotoxic group. The crude extract caused a significant (p<0.01) dose-dependent effect compared to the organotoxic group, with the dichloromethane fraction treated group exerting the highest effect. The testis weight of the group treated with doxorubicin was only reduced when compared to that of the standart control group although it was 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 testis weights insignificantly (p>0.05) except in the group treated with aqueous fraction (Table1). Effect of leaf extract of Hippocratea africana on Seminal analysis Table 2 shows the seminal analysis of semen from rats with doxorubicin-induced testicular toxicity. The semen appearances in all treatment groups were observed to be milky. However, the semen volume in the group administered with doxorubicin alone was found to be small (0.01 mL). There was a considerable dose- dependent increment in the semen volume of rats treated with the leaf extract (0.02 -0.04 ml). The PH of the semen samples from all the groups was 8.0. The extract/fractions-treated groups were found to have a higher percentage of viable sperm cells (75-90%) compared to the organotoxic group (55%). Silymarin- treated group had 90% viable cells while control had 80%. The viscosity of semen in all the groups was normal. At the same time, the percentage of active sperm cells in the extract/fractions-treated groups ranged from 70-90% dose-dependently compared to 55% recorded in the organotoxic group. The percentage of dead sperm cells in the organotoxic group was found to be about 40% compared to 30% in the low dose (200 mg/kg) group, 15% in the middle dose (400 mg/kg) and aqueous fraction- treated groups and 10% in the high dose (600 mg/kg) and 5% in DCM fraction and silymarin treated- groups. The sperm count for the organotoxic group was 200 cells, while 400, 450 and 600 cells were respectively recorded from 200, 400 and 600 mg/kg treated groups. The control and aqueous fraction-treated groups had sperm count of 500 cells,while silymarin, DCM and aqueous fractions-treated groups had sperm count of 600 cells (Table 2). Effect of root extract and fraction on testes oxidative stress markers of doxorubicin-induced testes toxicity. Table 3 shows the effect of H. africana root extract/fractions on testes oxidative stress markers of the 400 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 397-406 rats. Administration of doxorubicin (1.66 mg/kg i.p) on alternate days for 14 days caused significant (p<0.05- 0.001) decreases in liver antioxidant enzymes activities (SOD, GPx, GST, CAT) and GSH levels compared to control. The MDA level was also significantly (p<0.05) elevated by doxorubicin treatment when compared to standart control. However, concomitant administration of root extract/fractions of H. africana (200 - 600 mg/kg) with doxorubicin for 14 days caused significant (p<0.05- 0.001) and non-dose-dependent elevations of the enzymatic and non enzymatic endogenous antioxidants in the treated rats groups when compared to the organotoxic groups, with the DCM fraction exerting the highest effect. Dose-dependent and significant (p<0.05) decreases in MDA levels of the extract/fractions treated groups were recorded with DCM having the highest effect. A Similar decrease was also observed in the silymarin-treated group compared to organotoxic control (Table 3). Effect of the root extract and fractions of H. africana on serum testosterone level of rats with doxorubicin- induced testicular toxicity Administration of doxorubicin (1.66 mg/kg i.p) on alternate days for 14 days was found to significantly (p<0.001) caused decreases of serum testosterone levels of rats when compared to control. However, concomitant administration of root extract and fractions of H africana (200-400 mg/kg) and silymarin with doxorubicin for 14 days caused significant (p<005-0.01) dose-dependent elevation of the testosterone levels of the treated rats when compared to the organotoxic groups with the DCM fraction having the highest effect (Figure 1). Effect of root extract and fraction of H. africana on lipid profile of rats with doxorubicin –induced organs toxicities Administration of doxorubicin (1.66 mg/kg) was observed to caused significant (p<0.05-0.001) elevation in levels of total cholesterol, triglyceride, low density lipoprotein, and very low density lipoprotein, while the high density lipoprotein level was not significantly (p>0.05) elevated. The total cholesterol and low density lipoprotein levels were significantly (p<0.05-0.001) reduced compared to organotoxic group following concommitant treatment with root extract and fractions of H. africana and silymarin. Similarly, triglyceride and very low density lipoprotein levels were also reduced but the reductions were only significant (p<0.05) at the highest dose (600 mg/kg) of the extract. However, the high density lipoprotein level was not affected by the concommitant treatment of the doxorubicin intoxicated rats with the root extract/fractions as well as silymarin (Table 4). Effect of root extract and fractions of H. africana on histology of rat testis in doxorubicin-induced testicular toxicity Histological sections of testes of rats receiving various treatments at magnification (x400) stained with the H&E method revealed that Group 1 (standard control, A) rat treated with distilled water (10 mL/kg) showed a well preserved cellular and connective tissue architecture, multiple stages of seminiferous tubules with lumen and developing germinal cell layers, normal tunica albuginea and no evidence of pathological changes were seen. The organotoxic group (Group 2, B) treated with doxorubicin (1.66 mg/kg) showed multiple seminiferous tubules with ceased germinal cell maturation, focal dilated congested blood vessels were seen. The basal seminiferous tubule showed germinal epithelium degeneration (Figure 2). 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 5 (E) treated with 600 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 testis sections that showed well preserved cellular and connective tissue architectures, multiple stages of seminiferous tubules with lumen, developing germinal cell layers, normal tunica albuginea and no evidence of pathologic changes were found except focal germinal cell vacuolation that was seen in group 6 (Figure 2 A-H). Table 1. Effect of H. africana root extract on body and testis weights of rats with doxorubicin-induced toxicity. PARAMETERS/ TREATMENT Dose mg/kg Testis(g) Body weight(g) Before After % increase in body weight Normal control - 2.42±0.29 135.6±18.34 151.0± 12.33 11.35 Doxorubicin 1.66 1.78±0.5 130.0 ± 9.45 126.3± 10.43 -2.84 Silymarin+DOX 100 2.39±0.35 132.6± 14.55 143.2 ± 3.15 7.99 Extract+DOX 200 2.20±0.35 142.8± 10.56 153.0± 5.29 7.14 400 2.42±0.03 140.3± 7.36 151.6 ± 6.22 8.05 600 2.41±0.19 138.4 ± 8.54 149.6 ± 8.48 8.09 Aqueous fraction 400 1.87±0.24 138.4 ± 6.26 144.6± 10.22 4.47 DCM fraction 400 2.50±0.14 134.3± 8.50 146.8± 13.20 9.30 Data are expressed as mean ±SEM. Not significant at p>0.05 compared to normal control and organotoxic control. n = 6. Noah & Okokon – Hippocratea africana Ethanol Root Extract and Fractions Attenuate … 401 Table 2. Effect of root extract of H. africana on seminal analysis of rats with doxorubicin-induced testicular toxicity. Parameters Control Doxorubicin Silymarin Extract 200 mg/kg Extract 400 mg/kg Extract 600 mg/kg DCM fraction Aqueous fraction App Milky Milky milky milky milky milky milky milky Volume 0.03mL 0.01mL 0.02mL 0.02mL 0.03mL 0.03mL 0.043mL 0.02mL PH 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 Viability 80% 55% 90% 78% 80% 90% 90% 80% Viscosity Normal Normal Normal Normal Normal Normal Normal Normal Normal 80% 55% 90% 70% 80% 80% 90% 80% Abnormal 20% 45% 10% 32% 20% 10% 10% 20% Active 80% 50% 90% 70% 75% 80% 90% 75% Sluggish 10% 10% 5% 10% 10% 10% 5% 10% Dead 10% 40% 5% 30% 15% 10% 5% 15% Sperm 500 200 600 400 450 600 600 500 Table 3. Effect of H.africana root extract and fractions on testes 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 0.55± 0.02 1.32±0.01 0.071±0.002 1.41±0.11 0.46±0.02 0.24±0.01 Doxorubicin 1.66 0.20±0.01c 0.50± 0.01a 0.030±0.001a 0.46±0.01c 0.28±0.02b 0.66±0.02c Crude extract 200 0.35±0.04a,d 1.34± 0.15c 0.061±0.001 1.02±0.15a,e 0.59±0.01f 0.51±0.01b 400 0.33±0.04a,d 1.18±0.10c 0.069±0.001f 1.19± 0.06f 0.52±0.01e 0.45±0.02b,e 600 0.42±0.02e 1.26±0.22c,e 0.058±0.003 1.28± 0.12f 0.43±0.02e 0.42±0.01a,d Aqueous fraction 400 0.29±0.03c 1.05±0.36f 0.045±0.005c 0.74±0.15b,d 0.48±0.03e 0.50±0.01b,d DCM fraction 400 0.48±0.02e 1.12±0.42c,d 0.070±0.002f 1.25± 0.11f 0.59±0.05f 0.40±0.01a,e Silymarin 100 0.50±0.04f 1.14±0.31c 0.057±0.002c 1.37±0.11f 0.45±0.02f 0.35±0.02f Data are 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 4. Effect of H.africana root extract and fractions on lipid profile parameters of rats with doxorubicin-induced toxicity. Treatment Dose mg/kg TOTAL CHOLESTEROL (mMol/L) TRIGLYCERIDE (mMol/L) HDL-C (mMol/L) LDL-C (mMol/L) VLDL (mMol/L) Control 10 2.25± 0.09 0.75±0.07 1.25±0.03 1.34± 0.11 0.34± 0.03 Doxorubicin 1.66 3.63± 0.20c 1.45±0.04c 1.51±0.11 2.77± 0.18c 0.66± 0.01c Crude extract 200 2.86±0.20d 1.29± 0.11b 1.38±0.10 2.03± 0.25 0.58±0.05b 400 3.03±0.13a 1.31±0.03b 1.33±0.06 2.43± 0.18 0.59±0.01b 600 2.46±0.14f 0.97±0.10e 1.38±0.06 1.52± 0.16e 0.43±0.04a Aqueous Fraction 400 2.85±0.06f 1.39±0.04c 1.31±0.09 1.37± 0.12f 0.63± 0.02c DCM fraction 400 2.33±0.12f 1.32±0.15b 1.36±0.08 1.57± 0.12e 0.60± 0.07b Silymarin 100 2.30±0.15f 1.37±0.04c 1.41±0.03 1.30± 0.13f 0.62± 0.01b Data are 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) 402 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 397-406 Figure 1. Effect of root extract and fractions of H. africana on serum testosterone levels of rats with doxorubicin-induced testicular toxicity. Data are expressed as MEAN ± SEM, Significant at bp<0.01; cp<0.001 compared to standart control; ep< 0.01, fp< 0.001, compared to organotoxic control. (n=6). Figure 2. Photomicrograph of testis section of rat treated with (A) distilled water (10mL/kg),(B) doxorubicin (1.66 mg/kg), (C) 200 mg/kg of H. africana root extract and doxorubicin (1.66 mg/kg), (D) 400 mg/kg of H. africana root extract and doxorubicin (1.66 mg/kg) (D) showing a well preserved cellular and connective tissue (arrowhead) architecture, multiple stages of seminiferous tubules (double end arrow) with lumen (L), developing germinal cell layers (Red double end arrows), normal tunica albuginea (thin arrow), multiple seminiferous tubule with with ceased germinal cell maturation ($) , focal dilated congested blood vessel (V), basal seminiferous tubule showing germinal epithelium degeneration (#).H&E stain x100 magnification. DISCUSSION Doxorubicin, an anthracycline used in the treatment of tumours (Konopa et al., 1988; Thorn et al., 2012), exhibits undesirable severe effects on the male reproductive system (Sridevi et al., 2012), such as disruption of normal reproductive functions via the induction of oxidative stress. Investigation of agents with antagonistic potentials to counteract the toxic effects of doxorubicin is ongoing. This study was carried out to Noah & Okokon – Hippocratea africana Ethanol Root Extract and Fractions Attenuate … 403 assess the effects of root extract of H. africana on doxorubicin-induced male reproductive system toxicity. The exact mechanisms through which doxorubicin induces toxicity in the male reproductive system, though reported widely, have not been adequately elucidated. The drug reportedly retards testicular growth, suppresses spermatogenesis, leading to male infertility through imposing oxidative stress and cellular apoptosis (Yeh et al., 2007). It also causes double-strand DNA breaks and cell death by intercalating into DNA strands (Konopa et al., 1988; Gewirtz et al., 1999) in meiotically dividing spermatocytes and spermatogonia (Cabral et al., 2014). Spermatogenesis suppression, sperm motility impairment, increased percentage of abnormal spermatozoa, decreased body and testicular weights, reduced testosterone levels, and testicular failure through oxidative stress and cell apoptosis in testicular tissue have also been reported (Howell & Shalet, 2005). In this study, doxorubicin was found to cause a significant increase in the percentages of dead and abnormal cells. Also, decreased seminal volume and sperm count were observed in the doxorubicin alone treated group. Similarly, testes from rats treated with doxorubicin alone were found to have marked degenerated germ cells and spermatozoa, supporting previous reports (Howell & Shalet, 2005). However, all these toxic effects of doxorubicin were alleviated by concomitant administration of root extract and fractions of Hippocratea africana, as animals treated with the root extract/fractions had high sperm cell count, greater seminal volume, low level of dead cells as well as standart testicular architecture, portraying the extract’s potentials in preventing doxorubicin-induced testicular toxicity probably through the antioxidative stress activity of its phytochemical components. 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). Moreover, the antioxidative stress activities of the root extract and fractions observed in this study further support the antioxidant potential of the plant. These activities may have contributed to the observed protective effects in this study. The membranes of the male germ cells have a high amount of polyunsaturated fatty acids, one of the targets of reactive oxygen species (Robinson et al., 1992). Thus, spermatozoa are vulnerable to oxidative damage because of the high amount of lipids in their membranes, making them lose their integrity and become less motile (Robinson et al., 1992). This effect could have contributed to the low sperm count, high percentages of dead and abnormal cells observed in the group treated with doxorubicin only in this study. Doxorubicin has been shown to impair male fertility by causing germ cell oxidative stress and apoptosis (El-Maddawy and El- Naby, 2019., Aksu et al., 2019). It has also been demonstrated to impair spermatogenesis (Kato et al., 2001) and steroidogenesis (Rizk et al., 2014; Olusoji et al., 2017). Exposure to doxorubicin appears to affect testicular integrity at both prepubertal and post-pubertal stages of development. In vitro studies with prepubertal mouse testis have demonstrated significant loss in germ cell number following exposure to doxorubicin at concentrations that were equivalent to human therapeutic doses (Smart et al., 2018). Further, studies have demonstrated early testicular developmental arrest and long-term germ cell DNA damage following prepubertal doxorubicin exposure (Vendramini et al., 2010). The study observed these effects as marked degenerated germ cells and spermatozoa were seen in the histological sections of the testes. However, concomitant administration of the root extract/fractions protected the testes as the toxic effects observed in the doxorubicin alone treated group were absent or mild compared to that group treated doxorubicin alone. The high susceptibility of testes and sperm to doxorubicin -induced testicular oxidative stress may be due to a weak anti-oxidant defence system in testicular tissue and semen (Nowrouzi et al., 2019). Besides, earlier studies demonstrated that antioxidant supplementation improved the quality of the semen profile in infertile men (Talevi et al., 2013). Lipids are an essential part of the reproductive system. Cholesterol is considered to be the precursor of steroid hormones. Steroidogenesis plays an essential role in the synthesis of spermatogenesis hormones. The biosynthesis of testosterone from pregnenolone is carried out by steroidogenesis enzymes including 17β hydroxysteroid dehydrogenase (17β-HSD) 3β and hydroxysteroid dehydrogenase (3β- HSD). It has been reported that doxorubicin results in the downregulation of these enzymes (Prahalathan et al., 2006; Takashima, et al., 2009). Adipocytes are the primary sites for triacylglycerol storage. It has been found that DOX downregulates adipogenesis in vitro by decreasing the expression of PPARγ (Arunachalam et al., 2012). Doxorubicin inhibits spermatogenesis by causing defects in epididymal adipose tissue (Tirupathi Pichiah et al., 2012), which is crusial for normal spermatogenesis (Chu et al., 2010). Also, studies using adult rat models of DOX exposure have reported decreased testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels, decreased sperm count, motility and viability, and increased abnormally formed spermatozoa (Das et al., 2012). The results of this study showed that testosterone level was significantly reduced in the group treated with doxorubicin alone compared to standart control. However, the root extract and fractions- treated groups had improved levels of testosterone which further point to the protective effect of the extract against doxorubicin-induced toxicity. This result agrees with an earlier report by Ndem and Johnson (2017) in which improved testosterone concentration was reported following H. africana root administration. Oxidative stress plays an essential role in doxorubicin-induced toxicity by forming reactive oxygen species (ROS) (Arunachalam et al., 2021). The results of 404 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 397-406 the doxorubicin alone treated group in this study revealed a significant elevation of testicular MDA levels and a significant decreases in testicular SOD, CAT, GST, GPx and GSH when compared with the DOX group. These results are from previous reported studies (Khodir et al., 2021). However, co-treatment of root extract/fractions and DOX elevated the levels of these endogenous antioxidants revealing the free radicals scavenging potentials of the root extract/fractions and its antioxidative stress activity which is due to the activities of its phytochemical constituents such as monoterpenes, sesquiterpenes and xanthones earlier reported by Okokon et al.(2013a) and Umoh et al., (2021) to be present in this root extract. CONCLUSION The findings of this study showed that the root extract and fractions of Hippocratea africana possess testicular protective potentials against doxorubicin-induced testicular toxicity. These properties can be attributed to its phytochemical constituents’ antioxidant and antioxidative stress activities. Thus, the root can be used to alleviate and/or prevent doxorubicin-induced male reproductive system toxicity. Acknowledgements: The authors are grateful to the staff of the Animal house, Pharmacology and Toxicology Department of University of Uyo for providing technical assistance. Authors’ Contributions: JEO, KUN - Research concept and design; JEO, KUN Animal studies, JEO, Data analysis and interpretation; JEO, Writing the article. JEO and KUN read and approved the final manuscript. Competing Interests: The authors have not declared any conflict of interests. REFERENCES 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. Aksu EH, Kandemir FM, Yıldırım S, Küçükler S, Dörtbudak MB, Çağlayan C, Benzer F. (2019). Palliative effect of curcumin on doxorubicin-induced testicular damage in male rats. Journal of Biochemical and Molecular Toxicology, 33(10): e22384. Arunachalam S, Meeran MF, Azimullah S, Sharma C, Goyal SN, Ojha S. (2021). Nerolidol attenuates oxidative stress, inflammation, and apoptosis by modulating Nrf2/MAPK signaling pathways in doxorubicin-induced acute cardiotoxicity in rats. Antioxidants. 10(6):984. Arunachalam S, Kim SY, Kim MS, Yi HK, Yun BS, Lee DY, Hwang PH. (2012). Adriamycin inhibits adipogenesis through the modulation of PPARγ and restoration of adriamycin- mediated inhibition of adipogenesis by PPARγ over- expression. Toxicol. Mech. Methods. 22:540–546. 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. 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. Cabral REL, Okada FK, Stumpp T, Vendramini V, Miraglia SM. (2014). Carnitine partially protects the rat testis against the late damage produced by doxorubicin administered during pre- puberty. Andrology 2(6): 931–942. Chu Y, Huddleston GG, Clancy AN, Harris RBS, Bartness TJ. (2010). Epididymal fat is necessary for spermatogenesis, but not testosterone production or copulatory behavior, Endocrinology. 151:5669–5679 Das J, Ghosh J, Manna P, Sil PC. (2012). Taurine protects rat testes against doxorubicin-induced oxidative stress as well as p53, Fas and caspase 12-mediated apoptosis. Amino acids, 42(5), 1839–1855. 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. El-Maddawy ZK, AbdEl Naby WSH. (2019). Protective effects of zinc oxide nanoparticles against doxorubicin induced testicular toxicity and DNA damage in male rats. Toxicology Research, 8(5): 654–662. Esterbauer H, Cheeseman KH. (1990). Determination of aldehydic lipid peroxidation products: malonaldehyde and 4- hydroxynonenal. Methods in Enzymology. 186: 407–421. Filler R. (1993). Methods for evaluation of rat epididymal sperm morphology. Methods in Toxicol. 3:334–343. Friedewald WT, Levy RI, Fredrickson DS. (1972). Estimation of the concentration of low density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem., 18(6): 499-502. Gewirtz DA. (1999). A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochemical Pharmacology 57(7): 727–741. Howell SJ, Shalet SM. (2001). Testicular function following chemotherapy. Human Reproduction Update, 7(4): 363–369. 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. Johnny II, Okokon JE, Ochigbo EB, Udo IJ, Adefabi AM. (2023). Genotoxic and cytotoxicity potentials of Hippocratea africana. Asian Journal of Biochemistry, Genetics and Molecular Biology. 15(2): 38-45. 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. Noah & Okokon – Hippocratea africana Ethanol Root Extract and Fractions Attenuate … 405 Khodir S, Alafify A, Omar E, Al-Gholam M. (2021). Protective potential of ginseng and/or coenzyme Q10 on doxorubicin- induced testicular and hepatic toxicity in rats. Macedonian Journal of Medical Sciences. 9(A):993-1005. Konopa J. (1988). G2 block induced by DNA crosslinking agents and its possible consequences. Biochem Pharmacol 37(12): 2303–2309. Lawrence RA, Burk RF. (1976). Glutathione peroxidase activity in selenium- deficient rat liver. Biochem Biophys Res Comm 71: 952-958. 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. Ndem, JI, Johnson VE (2017). Testosterone Concentration and Testicular Histomorphology of Rats Exposed to Hippocratea africana Root Bark Extract.IOSR Journal of Pharmacy and Biological Sciences 12 (2): 13-17. Noah K, Anagboso MO, Iyanyi l, Ajaghaku D, Okokon JE. (2023d). Hippocratea africana root extract and fractions amelioriate paracetamol-induced oxidative stress and liver injury in rats. Asian Journal of Biochemistry, Genetics and Molecular Biology. 15(3):114-122. Noah K,Udobang JA, Oyepata SJ, Okokon JE, (2023c). Hepatoprotective activities of ethanol root extract and fractions of Hippocratea africana against doxorubicin-induced liver toxicity. Journal of Current Biomedical Research 3(4):1132- 1158. Noah K,Edem UA,Okokon JE, Ebong NO (2023a). Hippocratea africana root extract and fractions attenuate paracetamol- induced oxidative stress and kidney injuries in rats. Journal of Current Biomedical Research. 3(4):1067-1083. Noah K, Udobang JA, Okokon JE, Anagboso MO, Ebong NO. (2023b). Nephroprotective activities of ethanol root extract and fractions of hippocratea africana against doxorubicin-induced kidney toxicity. Biology Medicine and Natural Product Chemistry. 12(2):475-482. Nowrouzi F, Azadbakht M, Kalehoei E, Modarresi M. (2019). Effect of Rosa canina on testicular toxicity. Brazilian Archives of Biology and Technology. Vol.62: e19180017, 2019 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, Akpan HD, Ekaidem I, Umoh EE. (2011). Antiulcer and antidiarrheal activity of Hippocratea africana. Pak J Pharm Sci 24: 201- 205. 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, Antia BS, Umoh EE. (2008). Analgesic and antinflammatory effects of ethanolic root extract of Hippocratea africana. Int J Pharmacol l4 (1):51-55. 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. 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. 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, Okokon PJ, Sahal D. (2017). In vitro antiplasmodial activity of some medicinal plants from Nigeria. Int J Herbal Med 5 (5):102-109. 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, Volume 2020, Article ID 8859716, 17 pages. Olusoji MJ, Oyeyemi OM, Asenuga ER, Omobowale TO, Ajayi OL, Oyagbemi AA. (2017). Protective effect of gallic acid on doxorubicin-induced testicular and epididymal toxicity. Andrologia. 49(4): e12635. Prahalathan C, Selvakumar E, Varalakshmi P. (2006). Lipoic acid modulates adriamycin-induced testicular toxicity, Reprod. Toxicol. 21:54–59. 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. Rizk SM, Zaki HF, Mina MA. (2014). Propolis attenuates doxorubicin-induced testicular toxicity in rats. Food and Chemical Toxicology 67: 176–186. Robinson BS, Johnson DW, Poulos A. (1992). Novel molecular species of sphingomyelin containing 2-hydroxylated polyenoic very-long-chain fatty acids in mammalian testes and spermatozoa. The Journal of Biological Chemistry, 267(3): 1746–1751. Sinha AK. (1972). Colorimetric assay of catalase. Analytical Biochemistry, 47: 389 - 94. Smart E, Lopes F, Rice S, Nagy B, Anderson RA, Mitchell RT, Spears N. (2018). Chemotherapy drugs cyclophosphamide, cisplatin and doxorubicin induce germ cell loss in an in vitro model of the prepubertal testis. Sci Rep. 29;8(1):1773. Sridevi T, Nisha PV, Appavu Arulnathan G. (2012). Effect of Doxorubicin on the morphology, histology and karyology of male reproductive system of white mice, Mus musculus. Indian J Sci Technol 5: 2614–2618. Takashima S M, Takahashi M, Lee J, Chuma S, Okano M, Hata K, Suetake I, Nakatsuji N, Miyoshi H, Tajima S, Tanaka Y, Toyokuni S, Sasaki H, Komatsu-Shinohara M, Shinohara T. (2009). Abnormal DNA methyltransferase expression in mouse germline stem cells results in spermatogenic defects, Biol. Reprod. 81: 155–164. Talevi R, Barbato V, Fiorentino I, Braun S, Longobardi S, Gualtieri R. (2014). Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on human sperm motility, lipid peroxidation and DNA fragmentation. Reprod Biol Endocrinol 2013, 11, 81. Thorn CF, Oshiro C, Marsh S, Hernandez-Boussard T, McLeod H, Klein TE, Altman RB. (2011). Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenetics and Genomics, 21(7), 440–446. Tietz WW. (1995) Clinical Guide to Laboratory tests. 2nd edn. Sanders Company. Philadelphia, PA. pp. 554-556. Tirupathi Pichiah PB, Sankarganesh A, Kalaiselvi S, Indirani K, Kamalakkannan S, SankarGanesh D, Hwang PH, Cha YS, 406 Biology, Medicine, & Natural Product Chemistry 13 (2), 2024: 397-406 Achiraman S. (2012). Adriamycin induced spermatogenesis defect is due to the reduction in epididymal adipose tissue mass: A possible hypothesis, Med. Hypotheses. 78:218–220. 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. Vendramini V, Sasso-Cerri E, Miraglia SM. (2010). Amifostine reduces the seminiferous epithelium damage in doxorubicin- treated prepubertal rats without improving the fertility status. Reproductive Biology and Endocrinology: RB&E, 8, 3. Yeh YC, Lai HC, Ting CT, Lee WL, Wang LC, Wang KY. (2007). Protection by doxycycline against doxorubicin-induced oxidative stress and apoptosis in mouse testes. Biochem Pharmacol 74(7): 969–980. Yilmaz S, Atessahin A, Sahna E, Karahan I, Ozer S. (2006). Protective effect of lycopene on adriamycin- induced nephrotoxicity and nephrotoxicity. Toxicol. 218: 164-171. Yokoi K, Uthus EO, Nielsen FH. (2003). Nickel deficiency diminishes sperm quantity and movement in rats.Biol Trace Elem Res.93:141–153.