Seed Extract and Fractions of Telfairia OccidentalisAttenuated Doxorubicin-Induced Testiculotoxicity in Rats

Ugochi Queenette Nwosu¹ , Chinyelu C. Osigwe² , Kenneth Chidi Opara³ , Ugonma F. Uwaeme² , Unyime A. Fabian⁴ , Jude Efiom Okokon ¹

¹Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria

² Federal Teaching Hospital, Owerri, Imo State, Nigeria

³3. Department of Pharmacology and Toxicology, Faculty of Pharmacy, Madonna University Nigeria, Elele campus, Rivers State, Nigeria

⁴4. University of Uyo, Faculty of Allied Health sciences, Department of Medical Laboratory Sciences Uyo, Nigeria

Corresponding Author Email: judeefiom@yahoo.com

DOI : https://doi.org/10.51470/ABP.2025.04.03.42

Abstract

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1. Introduction

Doxorubicin, an anthracycline glycoside antibiotic with broad-spectrum of activity against various human solid tumors and hematological malignancies [7], but limited clinical usefulness due to its diverse toxicities, including cardiac, hepatic, hematological, and testicular toxicity [58]. 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 [22,23]. Doxorubicin has also been proposed to promote free radical generation by enhancing the activities of extramitochondrial oxidative enzymes such as NADPH and xanthine oxidases and also interferes with mitochondrial iron export [5]. 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 have been ongoing, especially on T. occidentalis.

Telfairia occidentalis Hook is a fluted pumpkin of the Cucurbitaceae family widely consumed as food in Nigeria [35]. It is a popular vegetable all over Nigeria, especially in the Niger-Delta region and the Eastern part of the country; varieties of meals are prepared from the leaves, stem, and seeds of the plant [53]. The seeds are very nutritious and are eaten roasted or boiled.  The seed extract has been reported to exert antidiabetic [16], cellular antioxidant, immunodulatory, anticancer, antiinflammatory [36], antiplasmodial  [35], antioxidant [40], analgesic [37,40], genotoxic and cytotoxic [30], in vivo inhibitory effect on alpha amylase and alpha glucosidase [15], genotoxic and cytotoxic [30], antiulcer [53] and antiprostatic [18] activities.   Phytochemical studies of the extract have shown the presence of alkaloid, flavonoid, tannins, terpenes, saponin, and cardiac glycosides [12].  Phytochemicals  such as pentadecanoic acid, hexadecanoic acid; 16-octadecenoic acid methyl ester; 9, 12-octadecadienoyl chloride (Z,Z); 9- octadecadienoic acid (Z)-, 2, 3-dihydroxypropyl ester; octadecanoic acid; hexadecanoic acid,2,3-is[(trimethylsilyl) oxy] propyl ester, 2,4-heptadien-6-ynal,(E,E); benzoic acid ; dodecanoic acid ; linoleic acid ethyl ester ; hexadecanoic acid, methyl ester ; α-phellandrene ; α-campholene aldehyde; terpinen-4-ol ; trans-β-ocimene ; borneol and stigmastan-3- ol, have been reported in the seed extract from GCMS analysis [36] and  HPLC characterisation of the seed extract and fractions revealed the presence of  eleven flavonoids; kaempferol, catechin, epicatechin, anthocyanidin, narigenin, flavonones, flavones, narigenin,rutin, naringin, and resveratol in the seed extract and fractions with butanol fraction having the highest concentration. Also, alkaloids such as ribalinidine, ammodendrine, spartein and lunamarin were also found in the seed extract and fractions [53].

The present study was designed to evaluate the activities of seed extract and fractions of T.occidentalis against doxorubicin-induced testicular toxicity in rats.

2. Materials and methods

Plant collection

Fresh  seeds of Telfairia occidentalis were purchased from Itam market in Itu L. G. A, Akwa Ibom State, Nigeria, in June, 2023. The seeds were previously identified and authenticated by a taxonomist in the Department of Botany, University of Uyo, Uyo, Nigeria. Herbarium specimens (UUPH 1(b)) were deposited at the Department of Pharmacognosy and Natural Medicine Herbarium, University of Uyo.

 The fresh seeds of the plant were dried on a laboratory table for 2 weeks and reduced to powder. The seed powder (1 kg) were separately macerated in 50% ethanol (5000 mL) for 72 hours. The liquid filtrates obtained were concentrated at 40˚C and all the ethanol was completely removed. 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 (150-200 g) were used. The animals were sourced from the 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, the repeated dose model earlier described [38,43] was used, 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 for 14 days.  Groups 3 – 5 rats were orally pretreated, respectively with 138 mg/kg/day, 276 mg/kg/day, and 553 mg/kg/day of Telfairia occidentalis dissolved in 10% Tween 80, one hour before treatment with 1.66 mg/kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days. Groups 6 and 7 were pretreated with 276 mg/kg of DCM and aqueous fractions respectively, and also treated with 1.66 mg/kg of doxorubicin in 0.9% normal saline intraperitoneally on alternate days for 14 days. Group 8 rats which served as the positive control group were equally pretreated with 100 mg/kg/day of silymarin one hour 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 into plain centrifuge tubes and left for one hour. The blood samples were then centrifuged at 1500 rpm for 15 mins to separate of serum at room temperature and used for biochemical assays.  The testes of the rats were surgically removed and 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 stored in ice cold 0.9% NaCl  in a clean sample bottle.    

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 release of the epididymal ducts. Sperm progressive motility percentage was noted through the phase-contrast microscope at 400 X [25]. Sperm viability was assessed, by eosin or nigrosin staining, accompanied by microscopic evaluation. Moreover, a hemocytometer was employed to count epididymal sperm in the suspension [59]. Furthermore, morphological anomalies of the head, tail, and mid piece of sperm were determined in percentage using the method of Filler [19]. 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.

Effect of the seed extract and fractions on testis oxidative stress markers

The oxidative markers assays were performed on testes homogenates of rats that were used in this study in order to assess antioxidative stress potentials of the extract and fractions. Homogenates of the stored testes samples 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) [31], catalase (CAT) [46], glutathione peroxidase (GPx) [29], reduced gluthathione (GSH) [13], and malondialdehyde (MDA) content [17].

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) [11], according to standard procedures at the Department of Chemical Pathology, University of Uyo Teaching Hospital, Uyo, Nigeria. Morphological changes observed and recorded in the excised organs of the sacrificed animals. Histologic pictures were taken as micrographs.

 Statistical analysis

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 was considered at p˂0.001 and p˂0.05.

3. Results

Effect of seed extract and fractions of T. occidentalis on body and  testes weights of rats with doxorubicin-induced toxicity

Administration ofT.occidentalis seed extract and fractions to rats with doxorubicin-induced organs toxicities caused considerable improvement of body weights compared to the organotoxic group. The crude extract caused a significant (p<0.01) dose-dependent effect when compared to the organotoxic group, with the dichloromethane fraction-treated group exerting the highest effect. The testis weight of the group treated with doxorubicin only was found to be reduced when compared to that of the normal control group though not statistically significant (p>0.05).  However, treatment of rats with doxorubicin-induced toxicities with the seed extract and fractions of T.occidentalis improved the testis weights though insignificantly (p>0.05), except in the group treated with aqueous fraction (Table  1).

Effect of T. occidentalis seed extract and fraction on testes oxidative stress markers of doxorubicin-induced testes toxicity:

Table 2 shows the effect of T.occidentalis seed extract/fractions on testes oxidative stress markers of the rats. Administration of doxorubicin (1.66 mg/kg i.p) on alternate days for 14 days caused significant (p<0.05-0.001) decreases of CAT activity and GSH levels when compared to control, while SOD and GPx were insignificantly decreased. The MDA level was also significantly (p<0.01) elevated by doxorubicin treatment when compared to the normal control. However, concomitant administration of seed extract/fractions of T. occidentalis (138 – 553 mg/kg) with doxorubicin for 14 days caused significant (p<0.05-0.001) and non-dose-dependent elevations of GPx and GSH activities in the treated rats groups when compared to the organotoxic groups, with the  DCM fraction exerting the highest effect. Dose-dependent and significant (p<0.05-0.001) increase in SOD levels of the extract/fractions treated groups were recorded with DCM having the highest effect. Similarly, CAT activity was elevated in extract/fractions treated groups non dose-dependently with the middle dose (126 mg/kg) and silymarin treated groups having the highest effect. MDA levels in various extract/fractions treated groups were non-dose-dependently decreased with the most significant effect (p<0.001) recorded in DCM fraction treated group when compared to organotoxic control. (Table 2).

Effect of the seed extract and fractions of T. occidentalis 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) cause decreases of serum testosterone levels of rats when compared to controls.  However, concomitant administration of seed extract and fractions of T.occidentalis (138-553 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 seed extract and fraction of on Seminal analysis of rats with doxorubicin –induced organs toxicities

Table 3 shows the seminal analysis of semen from rats with doxorubicin–induced organs toxicities. The semen in all treatment groups was found to be milky in appearance, while the semen volume in the group administered with doxorubicin alone was found to be small (0.01 mL). There was a marked improvement in the semen volume of rats treated with the seed extract and fractions (0.02 -0.04 mL) with the DCM fraction-treated group having the highest volume (0.043 mL). The PH of the semen samples from all the groups was 8.0. The extract/fraction-treated groups were found to have a higher percentage of viable sperm cells (78-90%) compared to the doxorubicin only-treated group (55%). silymarin treated group had 90% viable cells while control had 80%. Viscosity of semen in all the groups was normal. The average number of cells was 33.25 x 10⁶ in the doxorubicin only treated group, but non-dose dependent improvements were observed in extract / fractions treated groups with high dose (553 mg/kg) having 130.75 x 10⁶ cells, while DCM fraction treated group recorded average cell of 126.75 x 10⁶ cells and silymarin group 91.0 x 10⁶ cells. The percentage of active sperm cells in the extract-treated groups ranged from 55-80% non non-dose-dependent compared to 50 % recorded in the testosterone only group. The percentage of dead sperm cells in the doxorubicin only group was found to be about 10% compared to 5% in the middle dose (276 mg/kg) and high dose (553 mg/kg) treated groups as well as the groups treated with the various fractions. The standard drug (silymarin) group had 5 % dead sperm cell (Table 3).

 Effect of seed extract and fractions of T. occidentalis on histology of rat testis  in doxorubicin-induced testicular toxicity

 Histological sections of testes of rats receiving various treatments at magnification  (x100) stained with H&E method revealed that Group 1 (normal control, CONT)  rat treated with distilled water (10 mL/kg) showed A normal histo-architecture with well-presented seminiferous tubules having well-defined basement layer, well-lined spermatogenic cells and arrays of spermatozoa within the tubular lumen, and well-presented Leydig cells and blood vessels within the interstitial connective tissue  and no evidence of pathological changes were seen. The organotoxic group (Group 2, T+CONT) treated with doxorubicin (1.66 mg/kg) showed an artrophying histo-architectural, with areas of spermatogenic cells degeneration and altered spermatogenic processes, with widened tubular lumen, vacuolated and degenerating leydig cells within the interstitial space and degenerating spermatogenic cells within the seminiferous tubules (Figure 2). Rats in group 3 (T+STD ) treated with 100 mg/kg of silymarin and doxorubicin (1.66 mg/kg) showed mildly affected testicular tissue, demonstrating a mild histo-architectural alteration, with area of vacuolated and degenerating leydig cells, within the interstitial connective tubule.  Group 4 (T+LDE)  treated with 138 mg/kg of T.occidentalis seed extract and doxorubicin (1.66 mg/kg) showed a moderately affected testicular tissue demonstrating moderate histo-architectural alteration, with area of altered spermatogenic processes, with widened tubular lumen, within the seminiferous tubules. Group 5 (T+MDE ) treated with 276 mg/kg of T.occidentalis seed extract and doxorubicin (1.66 mg/kg) showed a moderately affected testicular tissue, demonstrating moderate histo-architectural alteration, with an area of altered spermatogenic processes having a widened tubular lumen, within the seminiferous tubules. Group 6 (T+HDE) treated with 553 mg/kg of T.occidentalis seed extract and doxorubicin (1.66 mg/kg) showed testicular tissue demonstrating moderate histo-architectural alteration, with area of altered spermatogenic processes, with widened tubular lumen, within the seminiferous tubules. Group 7 (T+AQE) treated with 276 mg/kg of aqueous fraction of T.occidentalis seed and doxorubicin (1.66 mg/kg) showed a normal histo-architecture with well-presented seminiferous tubules having well-defined basement layer, well-lined spermatogenic cells and arrays of spermatozoa within the tubular lumen and well-presented Leydig cells within the interstitial connective tissue.  Group 8 (T+DCME) treated with 276 mg/kg of dichloromethane fraction of  T.occidentalis seed and doxorubicin (1.66 mg/kg) showed a moderate histo-architectural alteration, with an area of altered spermatogenic processes having widened tubular lumen within the seminiferous tubules (Figure 2).

 Effect of seed extract and fraction of T.occidentalis 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, high density lipoprotein, low density lipoprotein, and very low density lipoprotein. These raised levels of total cholesterol, triglyceride, high-density lipoprotein, low density lipoprotein, and very low density lipoprotein were significantly (p<0.05-0.001) reduced when compared to organotoxic group following concomitant treatment with seed extract and fractions of T. occidentalis and silymarin. However, thereductions in total cholesterol and low density lipoprotein were dose-dependent, while non dose-dependent reductions were observed in triglyceride, high density lipoprotein and very low density lipoprotein levels   (Table 4).

Photomicrographs of  sections of testes of rats treated with distilled water (CONT), doxorubicin only,1.66 mg/kg (T.CONT), Sylimarin,100 mg/kg and DOX(T+STD), T occidentalis, 138 mg/kg and DOX (T+LDE),T.occidentalis, 276 mg/kg and DOX (T+MDE), T.occidentalis, 553 mg/kg and DOX (T+HDE), Aqueous fraction,276 mg/kg and DOX (T+AQE) and DCM fraction, 276 mg/kg  and DOX (T+DCME) showing well-defined basement layer (Bl), well-lined spermatogenic cells (Sp) and arrays of spermatozoa (Sz) within the tubular lumen, and well-presented Leydig cells (Lc) and blood vessels (Bv), spermatogenic cells degeneration and altered spermatogenic processes (red arrow), with widened tubular lumen (black star), vacuolated and degenerating leydig cells (red arrow) within the interstitial space and degenerating spermatogenic cells (black arrow) within the interstitial connective tissue (H&E x100)

4.Discussion

Doxorubicin, an anthracycline use in the treatment of tumors [27,51], exhibits serious undesirable effects on the male reproductive system [48], such as disruption of normal reproductive functions via the induction of oxidative stress. Investigations of agents with antagonistic potentials to counteract the toxic effects of doxorubicin are ongoing. This study was carried out to assess effects of seed extract of T. occidentalis on doxorubicin-induced male reproductive system toxicity.

         The exact mechanisms through which doxorubicin induces toxicity in male reproductive system, though reported widely, have not been properly elucidated. The drug reportedly retards testicular growth, suppresses spermatogenesis, leading to male infertility through imposing oxidative stress and cellular apoptosis [57]. It also causes double-strand DNA breaks and cell death by intercalating into DNA strands [20,27]  in meiotically dividing spermatocytes and spermatogonia [6].  Spermatogenesis suppression,  sperm motility impairment, increase percentage of abnormal spermatozoa, decreased body and testicular weights, reduced testosterone level and testicular failure through oxidative stress and cell apoptosis in testicular tissue have also been reported [21]. In this study, doxorubicin was found to cause 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 [21]. However, all these toxic effects of doxorubicin were alleviated by concomitant administration of seed extract and fractions of T.occidentalis, as animals treated with the seed extract/fractions had high sperm cell count, greater seminal volume, low level of dead cells as well as normal 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 seed extract and fractions of T.occidentalis  previously  reported [34,36,40] may be responsible for these activities. Moreso, the findings 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 membranes of the male germ cells have a high amount of polyunsaturated fatty acids which is one of the targets of reactive oxygen species [45]. Thus, spermatozoa are vulnerable to oxidative damage because of the high amount of lipids in their membranes, and this makes them lose their integrity and become less motile [45]. 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 [1,14]. It has also been demonstrated to impair spermatogenesis [24] and steroidogenesis [31,39,44]. 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 [47]. Further, studies have demonstrated early testicular developmental arrest and long-term germ cell DNA damage following prepubertal doxorubicin exposure [54].  These effects were observed in the study as marked degenerated germ cells and spermatozoa were seen in the histological sections of the testes.  However, concomitant administration of the seed extract/fractions protected the testes as the toxic effects observed in the doxorubicin alone treated group were absent or mild compared to the group treated with doxorubicin alone. The high susceptibility of testes and sperm to doxorubicin -induced testicular oxidative stress may be due to a weak anti-oxidant defense system in testicular tissue and semen [33]. Besides, earlier studies demonstrated that antioxidant supplementation improved the quality of the semen profile in infertile men [50].

The levels of lipid profile indices such as  total cholesterol, triglyceride, HDL, LDL and VLDL were significantly elevated in doxorubicin only treated group when compared to normal control group. These observations are in accordance with the previous reports  [2,42]. However, these increases  in lipid profile parameters were reduced significantly when compared to organotoxic group following concomitant treatment with seed extract and fractions of T. occidentalis.

 Lipids are a very important part of the reproductive system. Cholesterol is considered to be the precursor of steroid hormones. Steroidogenesis plays an important 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 result in the downregulation of these enzymes [41,49]. Adipocytes are the main sites for triacylglycerol storage. It has been found that DOX downregulates adipogenesis in vitro by decreasing the expression of PPARγ [3]. Doxorubicin inhibits spermatogenesis by causing defects in epididymal adipose tissue [52], which is very important for normal spermatogenesis [9].  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 [10]. The findings of this investigation show that the seed extract and fractions were able to prevent the toxic effects of doxorubicin on the male reproductive system as evident in the reduced lipid profile indices and improved testosterone levels and sperm densities in the extract/fractions-treated groups. This suggests that the seed extract and fractions may have enhanced steriodogenesis as well as spermatogenesis, which are inhibited by doxorubicin. This result further confirms the testicular protective potential of the T.occidentalis seed extract.

Oxidative stress plays an essential role in doxorubicin-induced toxicity through the formation of reactive oxygen species (ROS) [4]. The results of the doxorubicin alone treated group in this study revealed significant elevation of testicular MDA levels and a significant decrease in testicular SOD, CAT,  GPx and GSH when compared with the DOX group. These results are in accordance with previous reported studies [26]. However, co-treatment of seed extract/fractions and DOX elevated the levels of these endogenous antioxidants revealing the free radicals scavenging potentials of the seed extract/fractions and its antioxidative stress activity, which is due to the activities of its phytochemical constituents, such as flavonoids, monoterpenes and polyunsaturated fatty acids previously reported [36,53]  to be present in this seed extract.

The dichloromethane and butanol fractions of the seed extract has been found to contain some pharmacological active compounds especially flavonoids like kaempferol, catechin, epicatechin, anthocyanidin, rutin flavones, flavonones, narigenin, naringin among others which are potent antioxidant compounds [53] and are likely to contribute to the observed activities in this study. Kumar et al. [28] had reported on the activities of some phyto-components with compound nature of flavonoids; palmitic acid (hexadecanoic acid ester and n-hexadecanoic acid), unsaturated fatty acid and linolenic (docosatetraenoic acid and octadecatrienoic acid) as anti-inflammatory, antioxidant, hypocholesterolemic, cancer preventive, hepatoprotective, among others. These compounds could have contributed to the observed  anti-inflammatory, antioxidant, hypocholesterolemic, cardioprotective, renoprotective, testiculoprotective and hepatoprotective activities of the seed extract observed in this study. Besides, antioxidant compounds such as borneol and terpen-4-ol[8,56], present in the extract may have played a role in the antioxidant/antioxidative stress activity observed in this study. Similarly, phytosterols such as Stigmastan-3-ol, 5-chloro-, acetate, (3a, 5a)-, present in this extract have been reported to have preventive effects on the development of diseases due to reactive oxygen species [55]. Moreover, Yoshida and Niki [60] showed the antioxidant effects of the phytosterols against lipid peroxidation.  This radical scavenging activity of the phytochemical components of this extract could have accounted for all the activities observed in this study and may be the mechanism of action  of the seed extract.

• Conclusion

The findings of this study showed that the root extract and fractions of Telfairia occidentalis possess testicular protective potentials against doxorubicin-induced testicular toxicity. These properties can be attributed to the antioxidant and antioxidative stress activities of its phytochemical constituents. Thus, the root can be use to alleviate and/or prevent doxorubicin-induced male reproductive system toxicity.

Acknowledgements

The authors are grateful to staff of the Animal house, Pharmacology and Toxicology Department of the University of Uyo for providing technical assistance.

Conflicts of interest

There is no conflict of interest        

References

1.Aksu, E. H., Kandemir, F. M., Yıldırım, S., Küçükler, S., Dörtbudak, M. B., 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.

2.Akter, H., Rashid, M.M., Islam, M.S., Hossen, M.A., Rahman,M.A., Algheshairy, R.M. and Alnajeebi, A.M. (2022).Biometabolites of Tamarindus indica play a remarkable cardioprotective role as a functional food in doxorubicin-induced cardiotoxicity models. J. Funct. Foods, 96: 105212.

3.Arunachalam, S., Kim, S.Y., Kim, M. S., Yi, H. K., Yun, B. S., Lee, D.Y.,  Hwang, P. H. (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.

4.Arunachalam, S., Meeran, M. F., Azimullah, S., Sharma, C., Goyal, S. N., 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.

5.Bachur, N. R., Gordon, S. L., Gee, M. V., 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.

6.Cabral, R. E. L., Okada, F. K., Stumpp, T., Vendramini, V., Miraglia, S. M. (2014). Carnitine partially protects the rat testis against the late damage produced by doxorubicin administered during pre-puberty. Andrology 2(6): 931-942.

7.Calabresi, P., Chabner, B. A. (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.

8.Chen, L., Su, J., Li, L., Li, B., and Li, W. (2011). A new source of natural D-borneol and its characteristic. Journal of Medicinal. Plants Research. 5: 3440-3447.

9.Chu, Y., Huddleston, G. G, Clancy, A. N., Harris, R. B. S., Bartness, T. J. (2010). Epididymal fat is necessary for spermatogenesis, but not testosterone production or copulatory behavior, Endocrinology. 151:5669–5679

10.Das J., Ghosh, J., Manna, P., Sil, P. C. (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.

11.Drury, R. A., Wallington, E. A. (1980) Carleton’s Histological Techniques. 5th Edition, Oxford University Press, New York, 195.

12.Ebong, A. S., Eseyin, O. A., Etim, E. I., Okokon, J. E. (2020). Telfairia occidentalis potentiates antiplasmodial activity of artemisinin and amodiaquine combination therapy. Anti-Infective Agents. 18(2): 152-159.

13. Ellman, G. L.(1959). Tissue sulfhydryl groups. Archieves of Biochemistry and Biophysics. 82: 70-77.

14.El-Maddawy, Z. K., Abdel-Naby, W.S.H. (2019). Protective effects of zinc oxide nanoparticles against doxorubicin induced testicular toxicity and DNA damage in male rats. Toxicology Research, 8(5): 654-662.

15.Enin, G.N., Okokon, J.E., Odokwo, B.O., Antia, B. S. (2023).Preliminary phytochemical screening and in vivo Inhibitory study of Telfaira occidentalis Hook f. seeds extract on alpha amylase and alpha glucosidase of rats. Journal of Science and Technology Research 5(4) : 26-35.

16.Eseyin, O. A., Ebong, P., Ekpo, A., Igboasoiyi, A., Oforah, E.(2007). Hypoglycemic effect of the seed extract of Telfairia occidentalis in rat. Pak J Biol Sci  10(3): 498- 501.

1. Esterbauer, H., Cheeseman, K. H. (1990). Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods in Enzymology.  186: 407-421.

18.Fabian, U. A., Anagboso, M. O., Samuel, A. E., Okokon, J. E. (2025). Effect of seed extract and fractions of Telfairia occidentalis on liver and kidney functions and histologies of rats with testosterone-induced benign prostatic hyperplasia in rats. Journal of Complementary and Alternative Medical Research. 26(6): 1 -18.

19.Filler, R. (1993). Methods for evaluation of rat epididymal sperm morphology. Methods in Toxicol. 3:334-343.

20.Gewirtz, D. A. (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. 

21.Howell, S. J., Shalet, S. M. (2001). Testicular function following chemotherapy. Human Reproduction Update, 7(4): 363-369.

22.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.

23.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.

24. Kato, M., Makino, S., Kimura, H., Ota, T., Furuhashi, T., & Nagamura, Y. (2001). Sperm motion analysis in rats treated with adriamycin and its applicability to male reproductive toxicity studies. The Journal of Toxicological Sciences, 26(1), 51-59.

25.Kenjale,  R., Shah, R., Sathaye, S.(2008). Effects of Chlorophytum borivilianumon sexual behaviour and sperm count in male rats. Phytotherapy Research.:22:796-801.

26.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.

27.Konopa, J. (1988). G2 block induced by DNA crosslinking agents and its possible consequences. Biochem Pharmacol 37(12): 2303-2309.

28.Kumar, P. P., Kumaravel, S., and Lalitha, C. (2010). Screening of antioxidant activity, total phenolics and GC-MS study of Vitex negundo. African Journal of Biochemical Research, 4: 191-195.

29.Lawrence, R. A, Burk, R. F. (1976). Glutathione peroxidase activity in selenium- deficient rat liver. Biochem Biophys Res Comm 71: 952-958.

30.Magnus, S. P., Anagboso, M. O., Johnny, I. I., Ise, U. P.,  Okokon, J. E. (2024). Evaluation of genotoxic and cytotoxic  activities of leaf and seed extracts of Telfairia occidentalis. Journal of Complementary and Alternative Medicine Research. 25(3):7-16.

31.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.

32.Mohan, U. P., Tirupathi, P. P.B.,  Akhtar Iqbal,  S. T., Arunachalam, S.(2021). Mechanisms of doxorubicin-mediated reproductive toxicity, A review, Reproductive Toxicology, 102:80-89.

33.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

34.Oboh, G., Nwanna, E., Elusiyan, C. (2010). Antioxidant and antimicrobial properties of Telfairia  occidentalis (Fluted pumpkin) leaf extracts. J Pharmacol Toxicol  5(8): 539-547.

35.Okokon, J.E.,  Ekpo, A. J., Eseyin, O. A. (2009). Evaluation of in vivo antimalarial activities of ethanolic leaf and seed extracts of Telfairia occidentalis. Journal of Medicinal Food,  12(3):649-653.

36.Okokon, J. E., Antia, B. S., Dar, A., Choudhary, M. I. (2012a). Immunomodulatory, anticancer and antiinflammatory activities of Telfairia occidentalis seed extract and fractions. International Journal of Food Nutrition and Safety 2(2): 72 – 85.

37.Okokon, J. E., Dar, A., Choudhary, M. I. (2012b).Chemical constituents and analgesic activity of Telfairia occidentalis. Phytopharmacology 3(2): 359 – 366.

38.Olorundare, O. E., Adeneye, A. A.,  Akinsola, A. O., Sanni, D. A, 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.

39.Olusoji, M. J., Oyeyemi, O. M., Asenuga, E. R., Omobowale, T. O., Ajayi, O. L., Oyagbemi, A. A. (2017). Protective effect of gallic acid on doxorubicin-induced testicular and epididymal toxicity. Andrologia. 49(4): e12635.

40.Osukoya, O. A., Adegbenro, D., Onikanni, S. A., Ojo, O. A., Onasanya, A.(2016). Antinociceptive and antioxidant activities of the methanolic extract of Telfairia occidentalis Seeds. Anc Sci Life.  36(2):98-103.

41.Prahalathan, C., Selvakumar, E., Varalakshmi, P.(2006).  Lipoic acid modulates adriamycin-induced testicular toxicity, Reprod. Toxicol. 21:54-59.

42.Ramavtar Kondampati, K. D., Matukumalli, U. R., Borr, S., Beesam, S., Debbarma, B. (2024). Hematological and serum biochemical evaluation of doxorubicin induced toxicity in wistar male rats and its amelioration with Hesperidin. J. Anim. Res., 14(02): 115-123.

43.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.

44.Rizk,  S. M., Zaki,  H. F., Mina, M. A. (2014). Propolis attenuates doxorubicin-induced testicular toxicity in rats. Food and Chemical Toxicology 67: 176-186.

45.Robinson, B. S., Johnson, D. W., 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.

46.Sinha, A. K.(1972). Colorimetric assay of catalase. Analytical Biochemistry, 47: 389 – 94.

47.Smart, E., Lopes, F., Rice, S., Nagy, B., Anderson, R. A., Mitchell, R.T., 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.

48.Sridevi, T., Nisha, P.V., 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.

49.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. 

50.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. 

51.Thorn, C. F., Oshiro, C., Marsh, S., Hernandez-Boussard, T., McLeod, H., Klein, T. E., Altman, R. B. (2011). Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenetics and Genomics, 21(7), 440-446.

52.Tirupathi Pichiah, P. B., Sankarganesh, A., Kalaiselvi, S., Indirani, K., Kamalakkannan, S., SankarGanesh, D., Hwang, P. H., Cha, Y. S., 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, Ubengama EE, Udofia  EU, Obasi OI,  Okonna UK,  Umanah ES, Etefia JE, Okokon JE (2025).HPLC characterization and anti-ulcer effects of methanol seed extract and fractionated components of Telfairia occidentalis in rodents.Nigerian Journal of Pharmaceutical and Applied Science Research 14 (2): 111-117.https://doi.org/10.60787/nijophasr-v14-i2-622

54.Usunomena, U., Okpiabhele, A.(2023). Telfairia occidentalis Hook f. mitigates Carbon Tetrachloride induced Nephrotoxicity in Rat. Journal of Research in Applied and Basic Medical Sciences  9 (3) :130-137.

55.Vendramini, V., Sasso-Cerri, E., Miraglia, S.M. (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.

56.Vivancos, M., and Moreno, J. J. (2005). β-sitosterol modulates antioxidant enzyme response in RAW264.7 macrophages. Free Radical Biological Medicine., 39: 91-97.

57.Wu, C., Chen, Y., Chen, J., Shieh, J., Huang, C., Lin, P., Chang, G., Chang, J. H., and Lin, C.             (2012). Terpinen-4-ol induces apoptosis in human nonsmall cell lung cancer in vitro         and in vivo. Evidence Based International Journal of Food Nutrition and Safety.            2(2): 72-85.

58.Yeh, Y. C., Lai, H. C., Ting, C. T., Lee, W. L., Wang, L. C., Wang, K.Y.(2007). Protection by doxycycline against doxorubicin-induced oxidative stress and apoptosis in mouse testes. Biochem Pharmacol 74(7): 969-980.

59.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.

60.Yokoi, K., Uthus, E. O., Nielsen, F. H. (2003). Nickel deficiency diminishes sperm quantity and movement in rats. Biological Trace Elements Resources. 93:141-153.

61.Yoshida, Y., and Niki, E. (2003). Antioxidant effects of phytosterol and its components.         Journal of Nutrition and Science Vitam., 49: 277-280.