Introduction
Chemotherapy remains one of the most effective treatment modalities for various malignancies, including myeloma, sarcoma, and breast cancer. This involves the administration of chemical agents designed to inhibit or halt the proliferation of tumor cells [1]. CYP is among the most widely used chemotherapeutic agents [2].
However, despite its clinical efficacy, CYP exhibits cytotoxicity toward normal cells, particularly those with high mitotic activity, such as reproductive [3] and hematopoietic cells [4]. In male animal models, CYP administration has been shown to impair sperm quality, including reductions in sperm count, motility, and morphology [5-7]. Furthermore, CYP decreases testosterone levels and induces structural damage to the seminiferous tubules [8,9].
Hematologically, the rapid turnover of blood cells renders hematopoiesis particularly vulnerable to CYP-induced toxicity. Several studies have documented hematological disturbances following CYP exposure, characterized by the suppression of hematopoietic activity and significant reductions in WBC and RBC counts [10,11].
CYP exerts its anticancer effects primarily through DNA alkylation [12]. Upon metabolic activation, CYP forms cytotoxic compounds that inhibit protein synthesis by cross-linking DNA and RNA strands [13]. In addition to its cytostatic action, CYP metabolism generates free radicals, contributing to oxidative stress as a secondary pathological mechanism [14].
Prolonged or excessive exposure to CYP may intensify its cytotoxicity by increasing the production of reactive oxygen species (ROS) [15]. Emerging evidence that CYP elevates ROS levels, at least in part, through increased intracellular iron accumulation. Although iron is essential for the proliferation and differentiation of hematopoietic stem and progenitor cells, excess iron promotes ROS generation via the Fenton reaction, leading to oxidative stress, disrupted hematopoiesis, and reduced blood cell counts [16].
In the context of male fertility, spermatozoa are particularly susceptible to oxidative damage because of the high content of polyunsaturated fatty acids (PUFAs) in their membranes. These lipid compositions render cells susceptible to lipid peroxidation, leading to membrane disruption and cell death under oxidative stress conditions [17].
Antioxidants are bioactive compounds that neutralize ROS, thereby protecting cells against oxidative damage [18]. Numerous studies have demonstrated that natural antioxidant-rich compounds can mitigate the detrimental effects of ROS, thereby preserving both reproductive function [19-21] and hematological parameters in male rats [22]. These compounds act by directly scavenging ROS, thereby preventing their interaction with critical intracellular targets [23].
Additionally, the administration of natural antioxidants has been shown to enhance endogenous enzymatic antioxidant systems [24,25], thereby augmenting the overall ROS-neutralizing capacity and contributing to the maintenance of redox homeostasis [26].
Coffea arabica L, commonly known as Arabica coffee, is one of the most widely consumed coffee varieties in the world. This high consumption generates substantial quantities of coffee pulp, a major agro-industrial by-product [27]. Although typically regarded as an environmental pollutant, coffee pulp exhibits promising health-promoting properties under certain conditions [28].
Recent studies have identified Arabica coffee pulp as a rich source of antioxidant compounds, particularly phenolic acids [27-29]. These phenolic acids are well-documented for their potent antioxidant activity, including their ability to scavenge ROS and reduce oxidative stress [30]. Given its favorable phytochemical profile and limited number of studies examining its pharmacological potential, this investigation aimed to evaluate the modulatory effects of Arabica coffee pulp extract on reproductive function and hematological parameters in male Wistar rats exposed to cyclophosphamide-induced toxicity.
Materials and Methods
Methanolic CPE preparation
Arabica coffee pulp was collected from coffee plantations in Klaten and Kulon Progo, Indonesia, thoroughly washed under running water to remove surface impurities, and mechanically separated from the seeds using a coffee peeler. The cleaned pulp was dried, ground into a fine powder, and 800 g of the dried material was extracted with methanol at a 1:6 (w/v) ratio by maceration at room temperature for four days. The extract was filtered, and the resulting filtrate was concentrated under reduced pressure using a rotary evaporator at 40 °C, followed by oven drying at 36 °C until a constant weight was obtained to minimize thermal degradation of thermolabile compounds, yielding 48 g of dried methanolic extract (6% w/w).
CPE Dose determination
As specific sub-acute data on coffee pulp remain limited, references from other coffee by-products and acute toxicity studies were used as the initial safety rationale. The treatment doses were set at 250, 1000, and 1750 mg/kg body weight. The highest dose (1750 mg/kg) was selected based on acute toxicity studies of coffee by-products (silverskin, parchment, husk), which reported no toxic effects at 2000 mg/kg [85].
To account for potential cumulative effects of repeated administration, the dose was slightly reduced to 1750 mg/kg. The intermediate dose (1000 mg/kg) was chosen because it had previously been shown to be safe with respect to hematological parameters in silverskin studies [86]. The lowest dose (250 mg/kg) was selected from literature reporting pharmacological and biological effects on reproductive and hematological parameters in rats at this level [87,88].
Animal model and research design
This study received ethical approval from the Faculty of Veterinary Medicine, Universitas Gadjah Mada (No. 00135/EC-FKH/Eks./2021). Twenty adult male Wistar rats (2 months old, 250 g) were obtained from the Integrated Research and Testing Laboratory (LPPT), Universitas Gadjah Mada.
Rats were acclimated for 7 days under standard conditions (±23 °C, 50–60% humidity, 12-hour light/dark cycle) with ad libitum access to reverse osmosis water and Biorat feed. The animals were randomly divided into five groups (n = 4): control (plain water), CYP (single oral dose of cyclophosphamide, 150 mg/kgbw), T1 (CYP + CPE 250 mg/kgbw), T2 (CYP + CPE 1000 mg/kg bw), and T3 (CYP + CPE 1750 mg/kg bw). CPE was administered once daily for 16 days, while CYP was administered only on day 1.
Aquabidest was used as the vehicle at a volume of 10 ml/kg bw. Accordingly, 2.5 ml of aquabidest was mixed with the respective doses of CPE and administered orally.
Phenolic and saponin measurement
Phytochemical separation was performed using thin-layer chromatography, while quantitative analysis was carried out using UV-Vis spectrophotometry. Total phenolic content was determined by the Folin–Ciocalteu method and expressed as gallic acid equivalents (GAE), and total saponin content was determined by the vanillin–sulfuric acid method and expressed as quillaja saponin equivalents. Values are expressed as % w/w of dry weight.
Assessment was conducted at LPPT, Universitas Gadjah Mada.
CYP preparation
CYP was purchased from the Oncology Clinic in Yogyakarta, Indonesia. The CYP dose of 150 mg/kgbw was adopted based on the study by Akomolafe (2019) [8], and the maximum tolerated dose was selected according to Aston et al. (2017) [31].
Anesthesia and euthanasia procedure
Rats were anesthetized with a ketamine–xylazine cocktail prepared in a 2:1:1 (v/v) ratio of ketamine (100 mg/mL), xylazine (20 mg/mL), and 0.9% NaCl. The mixture was administered intraperitoneally at 0.1 mL/100 g body weight (1 mL/kg), corresponding to 50 mg/kg ketamine and 5 mg/kg xylazine, which provided adequate short-term anesthesia for blood collection. On day 17, a subset of animals were euthanized under the same anesthetic protocol, followed by exsanguination via cardiac puncture and transcardial perfusion with physiological saline (0.9% NaCl) [89].
Biochemicals and blood analysis
Serum total testosterone levels were measured using a Competitive Enzyme-Linked Immunosorbent Assay (ELISA) kit (CALBIOTECH, Catalog No. TE373S), and malondialdehyde (MDA) levels were measured using a CheKineTM Lipid Peroxidation (MDA) Assay kit (Cat#: KTB1050, Abbkine). A Sysmex KX-21 Hematology Analyzer was used for blood profile determination at the LPPT, Universitas Gadjah Mada.
Sperm count analysis
To evaluate the sperm count, a 10 μL sperm suspension was obtained using a micropipette and transferred to a hemocytometer. Furthermore, the hemocytometer was placed under a light microscope, and the sperm were counted in the counting grid. The total sperm count was determined as follows:
Sperm motility, viability, and morphology analysis
Sperm motility was determined using previously reported methods [32]. The sperm suspension was incubated at 37 °C for 5 minutes before sperm motility measurement. A 10 μl aliquot of the sperm suspension was transferred with a micropipette to the hemocytometer.
Sperm motility was assessed only in the counting grid and categorized according to motility grades: grade 1 (immotile sperm), grade 2 (non-progressive), grade 3 (non-linear), and grade 4 (progressive). The sperm motility percentage was evaluated using the following equation:
Sperm viability and abnormal morphology were determined using the smear method with 10% Giemsa staining [32]. A glass slide was filled with three drops of the sperm suspension and fixed with 96% methanol as a fixative solution. Subsequently, 10% Giemsa stain was applied, and the smear method was performed.
Percentage sperm viability was measured according to the following method:
Histopathology of seminiferous tubules
The middle of the testis was minced and fixed with a 10% Neutral Buffered Formalin (NBF) solution. The paraffin method was used for histological analysis of the seminiferous tubules, followed by Hematoxylin-Eosin staining. A pathologist from the Department of Pathology Anatomy, Faculty of Medicine, Public Health and Nursing (FK-KMK), Universitas Gadjah Mada conducted this process.
For each animal, five seminiferous tubules were randomly selected in cross-section at 400× magnification. Tubules were included if they displayed an intact basement membrane, clear lumen, and absence of cutting artifacts. Tubular diameter, epithelial thickness, and lumen diameter were measured on digital images using Image Raster software, and the values from five tubules were averaged to obtain one mean value per animal for statistical analysis.
Germ cell loss was assessed manually by a blinded observer.
Statistical analysis
All data were normalized and homogeneously tested. Then, the data were analyzed using a one-way ANOVA test, followed by a Duncan post hoc test to determine the significance levels between treatments.
Results
Phenolic and saponin
The phytochemical analysis of the extract revealed the presence of both phenolic compounds and saponins (Table 1). Quantitative determination showed that the total phenolic content, expressed as GAE, was 6.5% w/w, while the total saponin content, expressed as quillaja saponin equivalents, was 1.09% w/w of dry weight.
Malondialdehyde
Figure 1A shows the MDA levels across all groups. On day 0, no significant differences were observed, indicating baseline homogeneity. By day 8, MDA levels had significantly increased in both the CYP and CPE groups (p ≤ 0.05), with the highest level recorded in the T2 group (7.67 nmol/mL).
On day 16, MDA levels in the CYP group further increased to 7.16 nmol/mL, whereas all CPE-treated groups showed a significant reduction compared with day 8 (p ≤ 0.05). Notably, all CPE groups differed significantly from the CYP group (p ≤ 0.05), with T2 showing the most significant reduction in MDA levels.
Testosterone level
Figure 1B illustrates serum testosterone levels measured on days 0, 8, and 16. No significant differences were observed on day 0 (p ≤ 0.05), indicating baseline uniformity. In the CYP group, testosterone levels declined significantly by day 8 (3.65 ng/mL) and continued to decrease by day 16 (2.23 ng/mL), reaching the lowest level among all groups.
CPE-treated groups also showed a decrease on day 8, with significant reductions in T2 and T3 compared to T1 (p ≤ 0.05). By day 16, testosterone levels increased in T2 (4.32 ng/mL) and T3 (3.45 ng/mL), whereas T1 showed a slight decline (3.72 ng/mL) compared with day 8. These results demonstrate that CPE has a modulatory effect on CYP-induced testosterone suppression.
Body and testis weight
Figure 2A summarizes body and testes weight data across the treatment period. On day 8, the CYP group exhibited a significant reduction in body weight (p ≤ 0.05), followed by partial recovery by day 16. Similar patterns were observed in the T1 and T2 groups, with significant reductions in day 8 and subsequent increases by day 16.
The T3 group also exhibited significant weight loss by day 8 (p ≤ 0.05), but recovery by day 16 was not statistically significant. Notably, the CYP group had the lowest body weight on both days 8 and 16, significantly different from the other groups (p ≤ 0.05). The T1 group consistently showed the highest body weight on both days, which was significantly higher than that in the T2 and T3 (p ≤ 0.05).
According to Figure 2B, testis weight in the CYP group was significantly reduced compared to the control group (p ≤ 0.05). In contrast, the CPE-treated groups showed improved testis weights, with T1 and T2 exhibiting significantly higher values than the CYP group (p ≤ 0.05).
Blood profile
Table 2 presents the effects of CYP and CPE on blood profile on day 8. CPE administration in the T1 and T2 groups resulted in increased WBC counts compared to the CYP group, whereas the T3 group exhibited a further reduction in WBC count, significantly lower than both the control and T1 groups (p ≤ 0.05). RBC counts in the CYP group were slightly reduced compared to the control.
Interestingly, RBC counts in all CPE-treated groups (T1, T2, and T3) were lower than in the CYP group. Hb levels were significantly lower in the CYP group than in the control (p ≤ 0.05), with only the T1 group showing a modest improvement over CYP following CPE treatment. PLT counts in the CYP group were significantly lower than those in the control (p ≤ 0.05).
Furthermore, T1 led to a significant improvement compared with CYP, although the values remained below the control (p ≤ 0.05). In contrast, T2 and T3 showed a significant reduction compared with both the control and T1 (p ≤ 0.05). HCT levels showed a marked decline in the CYP group compared to control, and this reduction persisted in the CPE-treated groups, with T2 and T3 exhibiting the lowest values (p ≤ 0.05).
PDW values were significantly decreased in the CYP group compared to the control, with the T1 group showing the best recovery, significantly higher than the CYP, T2, and T3 groups (p ≤ 0.05). MCH values showed no significant differences between CYP compared to T1, T2 and T3. MCHC in the T1, T2, and T3 groups differed significantly from the CYP group (p ≤ 0.05) but showed no difference compared with the control group.
Table 3 summarizes the effects of CYP and CPE on blood profile on day 16. The CYP group showed significant reductions in WBC, RBC, Hb, PDW, and MCHC levels compared with the control (p ≤ 0.05). In the CPE-treated groups, T1 exhibited the highest WBC count, which was significantly higher than that of CYP and T3 (p ≤ 0.05).
RBC counts improved in all CPE groups, with T1 showing a significant increase over CYP (p ≤ 0.05). Similarly, T1 also exhibited significantly higher Hb levels than the CYP group, whereas T2 and T3 exhibited lower Hb levels than the CYP group. PLT counts in the CYP group were significantly higher than control (p ≤ 0.05).
Moreover, T1 and T2 also resulted in significant increases relative to the control (p ≤ 0.05), but these levels were still significantly lower than CYP alone. Interestingly, T3 produced a significant elevation of PLT compared with both the control and T1 (p ≤ 0.05). HCT values show reductions in the CYP and T3 groups, while T1 maintained levels closer to the control (p ≤ 0.05).
PDW was reduced in the CYP group compared with the control, but partially recovered in CPE-treated groups, particularly in T1. MCH values did not differ significantly across groups, remaining within a narrow range. MCHC in all CP-treated groups was significantly different from the CYP group, with T1 showing the highest value (p ≤ 0.05).
Sperm quality and seminiferous tubules histology
Figure 3A shows the sperm analysis results after 16 days. The CYP group showed a significantly reduced sperm count (156 × 10⁶) compared with the control (p ≤ 0.05). CPE treatment improved sperm counts, with the T1, T2, and T3 groups all showing significantly higher values than the CYP group (p ≤ 0.05).
Notably, T1 and T2 had significantly higher counts than T3 (p ≤ 0.05).
Sperm motility, assessed by movement percentage, was significantly reduced in the CYP group (42%) compared with the control (p ≤ 0.05). CPE treatment improved motility, with all treated groups showing significantly higher values than the CYP group (p ≤ 0.05).
Sperm viability, assessed by plasma membrane integrity[33], was significantly reduced in the CYP group (56%) compared with the control (p ≤ 0.05). In Figure 3B, viable and non-viable sperm are indicated by green and blue arrows, respectively. CPE treatment significantly improved viability in the T1, T2, and T3 groups compared with CYP (p ≤ 0.05), with T2 showing the highest viability among them (p ≤ 0.05).
Normal sperm morphology consists of head, midpiece, and tail [34]. Abnormalities result from defects in any of these parts. In Figure 3B, yellow, black, and orange arrows indicate short sperm, bent tails, and abnormal heads, respectively.
The CYP group showed 19% abnormal sperm, which was significantly higher than that of the control group (p ≤ 0.05). CPE treatment reduced abnormalities, with T1, T2, and T3 showing significantly lower percentages than the CYP group (p ≤ 0.05). T2 demonstrated the most significant reduction, significantly lower than both T1 and T3 (p ≤ 0.05).
Figure 4 shows the measurements of the seminiferous tubule structure. Tubule diameters remained consistent across groups. However, the CYP group exhibited significantly reduced epithelial thickness and enlarged lumen diameter compared with the control (p ≤ 0.05).
CPE treatment significantly increased epithelial thickness and reduced lumen diameter relative to the CYP group (p ≤ 0.05).
Figure 5 illustrates the histopathological features of the seminiferous tubules across different treatment groups. In the control group (A), the seminiferous epithelium appeared thick with densely packed germ cells and minimal luminal space, reflecting normal testicular architecture. In contrast, CYP-treated rats (B) exhibited pronounced histological alterations, including germ cell depletion, tubular shrinkage, and markedly dilated lumina.
The T1 group (C; CYP + CPE 250 mg/kg bw) demonstrated partial protection, with improved epithelial integrity and reduced germ cell loss compared to the CYP group. The T2 group (D; CYP + CPE 1000 mg/kg bw) showed the most preserved morphology, characterized by thick seminiferous epithelium and well-maintained tubular structure, indicating the strongest protective effect. Meanwhile, the T3 group (E; CYP + CPE 1750 mg/kg bw) displayed moderate improvements, with seminiferous architecture resembling that of the T1 group but less optimal than T2.
Overall, these observations suggest that CPE supplementation, particularly at 1000 mg/kg bw, confers a dose-dependent protective effect against CYP-induced testicular damage.
Multivariate analysis
The multivariate analysis presented in the Figure 6 highlights the relationships among physiological, hematological, and reproductive parameters. The principal component analysis (PCA) biplot shows clear clustering of groups with variables such as sperm count, testosterone, and testis weight contributing significantly to variation. The scree plot further indicates that the first two principal components explain the majority of the variance.
Meanwhile, the correlation heatmap reveals strong positive and negative associations, particularly between reproductive indices (sperm count, motility, viability) and hematological parameters, suggesting interconnected effects on male reproductive health.
Discussion
During its metabolism, CYP produces two stable toxic intermediates, namely phosphoramide mustard and acrolein [35]. Phosphoramide mustard inhibits cell division by forming cross-links with DNA at guanine bases, whereas acrolein contributes to CYP-induced side effects by generating ROS and triggering oxidative stress [36,37]. Oxidative stress leads to lipid peroxidation [38] and disrupts antioxidant enzyme activity [39].
In this study, MDA levels were significantly elevated in the CYP group on day 8 compared with the control group, and this increase continued by day 16. These findings are consistent with previous reports [25, 40]. Elevated MDA levels reflect lipid peroxidation within cells [41].
This process affects various cell types, including blood and sperm cells, because of the presence of PUFAs in their membranes [42,43]. Lipid peroxidation is initiated when ROS oxidize unsaturated lipid chains, generating hydroperoxides and alkyl radicals, which disrupt membrane structure, fluidity, and integrity [44].
In this study, CYP administration led to a reduction in body weight on both days 8 and 16, which consistent with previous findings [40,45,46]. This effect may result from CYP targeting adipogenic precursor cells, which impair fat storage and lipid metabolism [47]. In contrast, CPE reduced body weight on day 8, followed by partial recovery by day 16.
The T1 group had the highest body weight among the T2 and T3 groups. This condition may be attributed to the increased caffeine content associated with higher CPE doses, which can directly influence adipose tissue or suppress appetite, resulting in a caloric deficit [48,49].
Hematological analysis revealed that all hematological parameters were significantly decreased in the CYP-treated group compared to the control, except for RBC and PLT on day 8, and PLT, HCT and MCH on day 16. This result is consistent with earlier studies [16,50]. These effects may be linked to lipid peroxidation, as indicated in Figure 2A.
Given that RBCs lack nuclei, the reduction in their count likely results from ROS-induced oxidative damage to PUFA-rich plasma membranes. The decrease in Hb further reflects oxidative stress, as ROS oxidizes heme iron from Fe²⁺ to Fe³⁺, forming methemoglobin with diminished oxygen-binding capacity [51,52]. In WBCs, which possess nuclei [53], CYP may induce DNA alkylation, leading to base damage and ultimately triggering cell death [54].
However, on day 16 the PLT profile appeared distinct, with T3 showing levels comparable to CYP and representing the highest among the CPE-treated groups. Interestingly, the data revealed a biphasic pattern. On day 8, CYP induced significant thrombocytopenia, which was partially attenuated by low-dose CPE but exacerbated at higher doses.
Conversely, by day 16, CYP triggered a pronounced rebound thrombocytosis, and while CPE moderated this effect at moderate doses, the highest dose further amplified platelet overproduction. This biphasic pattern likely reflects an interplay between early toxic suppression and subsequent stimulatory effects on thrombopoietin-mediated recovery [92,93].
Sperm quality analysis revealed that CYP administration significantly reduced sperm count, motility, and viability while increasing abnormal morphology, consistent with previous studies [3, 55]. The high PUFA content in sperm membranes renders them susceptible to lipid peroxidation, resulting in structural damage and cell death [17]. Histological analysis (Figure 3) shows that the seminiferous tubules in the CYP group had a thinner epithelial layer and a wider lumen compared with the controls, indicating germ cell depletion.
Figure 4b, which contains green and black asterisks, further supports this observation. These findings are further supported by the reduced testicular weight observed in the CYP group (Table 1), consistent with previous reports showing CYP-induced structural damage to seminiferous tubules and decreased testicular mass [56,57]. This indicates impaired spermatogenesis, a process regulated by testosterone [58].
The combination of low testosterone levels and elevated MDA levels indicates that lipid peroxidation contributes to hormonal disruption [59]. The decline in testosterone is likely due to CYP-induced damage to Leydig cells, which are essential for testosterone production [60,61]. Additionally, sperm have limited antioxidant defenses, making them highly susceptible to oxidative stress [62,63].
Our findings indicated that Arabica CPE administration resulted in elevated MDA levels on day 8, particularly in the T2 and T3 groups. However, MDA levels decreased in all CPE-treated groups by day 16. This indicates that the antioxidant effects of CPE had not yet reached full efficacy by day 8, which is consistent with the pharmacodynamic profile of antioxidants that typically require sustained exposure for optimal activity [7,64].
Notably, the T2 group exhibited the most significant reduction in MDA levels, supporting the notion that an increased antioxidant dosage may enhance ROS-scavenging efficacy [65-67]. Our results show that CP contains a total phenolic content of 6.55% w/w (Table 1), which is relatively high and may play an important role in counteracting ROS.
Hematological data indicated that all hematological levels were lower on day 8 than on day 16, supporting the notion that the therapeutic efficacy is time-dependent [68,69]. Interestingly, the lowest CPE dose produced the most favorable outcomes in terms of all hematologicals levels, suggesting a dose-dependent effect on hematological recovery [70,71]. These findings align with previous studies [24,72], which demonstrated that antioxidant compounds, such as Naringin and Bergenin, can mitigate CYP-induced hematotoxicity and improve blood profiles.
Regarding reproductive parameters, testosterone levels were initially lower in the T1 group than in the CYP group on day 8. However, they increased in all groups by day 16. This trend agreed with improvements in sperm quality and seminiferous tubule histology, with the T2 group showing the most pronounced recovery.
Similar results were reported [73,74], who found enhanced fertility outcomes in male rats treated with various natural compounds rich in antioxidants.
This study demonstrated that the therapeutic effects of CPE are influenced by cell type, dosage, and duration of exposure, underscoring the pleiotropic nature of its antioxidant activity. CPE contains several bioactive compounds, including phenolic acids, tannins, and caffeine [75,76]. Among these, chlorogenic acids, which are key phenolic constituents, exert potent antioxidant effects by scavenging ROS, thereby reducing oxidative stress and preventing cellular damage [77,78].
Tannins also contribute to the antioxidant potential of CPE by directly inhibiting lipid peroxidation through their ability to scavenge ROS [79]. The lowest dose of CPE produced the most favorable hematological outcomes. This indicates that increasing the dose elevates the concentration of bioactive compounds.
Tannins, which are recognized for their antioxidant properties, are likely contributors to the decline in blood cell counts at higher concentrations [80,81]. Excessive tannin levels are linked to morphological alterations in blood cells, ultimately leading to cell lysis [81]. In addition, Table 1 shows that CPE contains a total saponin content of 1.09% w/w, and several studies have reported the negative effects of saponins on erythrocyte [90,91].
Studies reveal that saponins can cause hemolysis. Saponins are amphipathic glycosides that interact strongly with membrane cholesterol. By forming saponin–cholesterol complexes, they disrupt the structural integrity of the erythrocyte membrane, increase its permeability, and induce pore formation.
This disturbance leads to osmotic imbalance, cellular swelling, and ultimately erythrocyte lysis (hemolysis).
A different trend was observed for sperm parameters, with the intermediate CPE dose (T2) exhibiting the most pronounced protective effect. Although T1 also showed improvement, T2 yielded superior outcomes. Overall, parameters improved within the 250–1000 mg/kg bw range but declined at higher doses.
This suggests that optimal antioxidant efficacy may not be fully achieved at 250 mg/kg bw, potentially due to insufficient concentrations and activation of enzymatic antioxidants such as superoxide dismutase (SOD) and catalase (CAT). The coordinated activity of enzymatic and nonenzymatic antioxidants are essential for effective ROS neutralization [23]. The concentration of bioactive compounds, including antioxidants, also increases with increasing CPE dosage.
However, excessive antioxidant levels may overwhelm cellular redox homeostasis [82]. When the adaptive antioxidant response is exhausted, high concentrations can induce toxicity [83]. Reductive stress caused by antioxidant overload impairs sperm quality.
The polyphenols in CPE may undergo autooxidation at high concentrations, generating ROS and promoting cellular damage [83,84]. Although this study highlighted the beneficial effects of CPE on hematological parameters and sperm quality, future investigations should evaluate its toxic and lethal doses, enzymatic antioxidant activity, and DNA integrity to better elucidate its therapeutic profile.
Conclusions
This study demonstrated that CYP administration has an adverse effect on hematological parameters and sperm quality in male Wistar rats. MDA levels increased in the CYP group, with a significant rise by day 16 (p ≤ 0.05). All hematological levels declined from day 8 to 16, except PLT on day 16.
In addition, CYP significantly reduced testosterone levels, impaired sperm quality, and caused structural damage to the seminiferous tubules compared with the control (p ≤ 0.05).
CPE administration initially increased the MDA level on day 8, particularly in the T2 group. However, levels declined by day 16, with the most significant reduction observed in the T2 group. Hematological parameters improved with CPE, with the T1 group showing the most significant increases in WBC, RBC, Hb, PDW, and MHCH by day 16 (p ≤ 0.05).
Meanwhile, on day 16, the T3 group showed the highest PLT count. CPE also enhanced testosterone levels, sperm quality, and seminiferous tubule structure compared with the CYP group (p ≤ 0.05), with T2 yielding the most favorable outcomes in all sperm parameters.
To the best of our knowledge, studies investigating the therapeutic potential of CPE remain scarce, and available toxicity data are also limited. In the present study, we provide evidence that CPE may exert therapeutic effects; however, these outcomes appear to depend on both the administered dose and the duration of treatment. Therefore, our findings should be considered preliminary and somewhat speculative, warranting cautious interpretation.
Future investigations are required to examine a wider range of CPE doses and to incorporate mechanistic endpoints, including antioxidant capacity, apoptotic pathways, and inflammatory markers, in order to obtain a more comprehensive understanding of its biological effects.