Hepato-Renal Effects of Graded Doses of Pyrenacantha staudtii Ethanolic Leaf Extract in Male Albino Wistar Rats

Hepato-Renal Effects of Graded Doses of Pyrenacantha staudtii Ethanolic Leaf Extract in Male Albino Wistar Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hepato-Renal Effects of Graded Doses of Pyrenacantha staudtii Ethanolic Leaf Extract in Male Albino Wistar Rats Burch Ndifon Takim, Blessed Yahweh, Samuel Joseph Umanah, Elton Nentuin Takim, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6999751/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The liver and kidneys play essential roles in detoxification, metabolism, and waste elimination, and their dysfunction can arise from infections, metabolic disorders, or exposure to toxic compounds. This study evaluated the hepato-renal effects of ethanolic leaf extract of Pyrenacantha staudtii using an albino Wistar rat model. Twenty male rats were assigned into four groups, with three experimental groups receiving 100, 200, or 400 mg/kg body weight of the extract intraperitoneally for 21 days, while the control group received distilled water. Phytochemical screening revealed the presence of alkaloids, phlobatannins, cardiac glycosides, flavonoids, and anthraquinones. Serum analysis showed a dose-dependent increase in AST and ALT levels, with significant elevations observed in the 400 mg/kg and 200 mg/kg groups, respectively. Creatinine levels increased significantly at the highest dose, while serum urea and bilirubin remained statistically unchanged. Potassium levels peaked significantly at 200 mg/kg, while sodium exhibited a nonsignificant downward trend. No gross pathological abnormalities were observed. These findings suggest that P. staudtii ethanolic leaf extract has dose-dependent biochemical effects on hepatic and renal function, with higher doses potentially inducing nephrotoxicity. The data provide a basis for further investigation into the therapeutic window, active phytoconstituents, and long-term safety profile of P. staudtii in drug development or nutraceutical applications. Pyrenacantha staudtii Hepato-renal toxicity Wistar rats Phytochemical profiling Ethanolic plant extract Figures Figure 1 Figure 2 Figure 3 Figure 4 1.0 Introduction The liver and kidneys play central roles in maintaining metabolic equilibrium, detoxifying xenobiotics, regulating fluid-electrolyte balance, and eliminating metabolic waste. Together, these organs ensure systemic homeostasis and support vital physiological functions. However, both the liver and kidneys are particularly susceptible to damage from endogenous and exogenous toxins, including excessive alcohol consumption, environmental pollutants, synthetic drugs, poor diet, autoimmune disorders, and chronic infections. Hepato-renal dysfunction is therefore a major contributor to global morbidity and mortality, and its prevention or management remains a focus of biomedical research and public health policy. In recent years, there has been a resurgence of scientific interest in natural products and plant-based therapies for organ protection and functional restoration. Many ethnobotanical remedies, once dismissed as anecdotal, have demonstrated promising therapeutic potential upon systematic investigation. Among such plants is Pyrenacantha staudtii , a climber belonging to the Icacinaceae family, widely used in traditional African medicine. In various ethnopharmacological settings, it has been employed in the treatment of microbial infections, pain, inflammation, and intestinal disorders. These traditional claims are believed to stem from its diverse phytochemical profile, including the presence of alkaloids, saponins, tannins, flavonoids, and cardiac glycosides. Despite its extensive use in traditional medicine, there remains a paucity of empirical data validating the organ-specific effects and safety profile of P. staudtii , particularly on critical detoxifying organs such as the liver and kidney. Furthermore, the potential toxicological or protective thresholds of the plant’s extract remain undefined. Rigorous preclinical studies are therefore essential to ascertain both efficacy and safety, especially in light of increasing global efforts to integrate traditional remedies into formal healthcare systems. This study was undertaken to evaluate the hepato-renal effects of graded doses of P. staudtii ethanolic leaf extract in healthy male albino Wistar rats. Through biochemical assays of serum liver enzymes (AST, ALT, bilirubin) and kidney function markers (urea, creatinine, electrolytes), this study assesses both therapeutic potential and possible organ toxicity associated with extract consumption over a 21-day period. In addition, qualitative phytochemical screening was conducted to characterize the bioactive constituents responsible for the observed effects. The findings presented here provide mechanistic insight into the dose-response dynamics of P. staudtii extract and its influence on biochemical homeostasis. The article is structured as follows: Section 2 describes the materials and methods used for animal handling, extract preparation, experimental design, and biochemical analysis. Section 3 presents the results of phytochemical screening, liver and renal biomarker assessments, and statistical evaluations. Section 4 offers a critical discussion of the findings in the context of existing literature. Finally, Section 5 concludes the study with implications for pharmacological applications and directions for future research. 2.0 Materials and Methods 2.1 Study Location and Duration This study was conducted in the Department of Biochemistry, University of Calabar, Cross River State, Nigeria. The experimental duration was 21 days, during which animals were maintained under controlled laboratory conditions. 2.1 Collection and Preparation of Plant Material Fresh leaves of Pyrenacantha staudtii were harvested from Nyahassang, Calabar Municipality Local Government Area, Cross River State, Nigeria. The plant was taxonomically identified and authenticated by Mr. Shasanya O.S. at the Herbarium Ibadan (FHI), Jericho, Ibadan. The leaves were air-dried and pulverized using a mechanical blender. Extraction was carried out using 500 mL of 70% ethanol, and the mixture was allowed to macerate with intermittent agitation. The resulting extract was filtered and concentrated to dryness using a rotary evaporator. The crude extract was stored in a clean, airtight container and preserved in a refrigerator at 4°C throughout the experimental period. 2.3 Experimental Animals Twenty healthy, mature male albino Wistar rats (12 weeks old) were used for this study. The animals were procured from the Experimental Animal Unit, Faculty of Basic Medical Sciences, University of Calabar. All rats were housed in conventional wire mesh cages under standard laboratory conditions (12 h light/dark cycle, ambient temperature of 25 ± 2°C) and acclimatized for one week prior to experimentation. The rats had unrestricted access to clean drinking water and standard pellet diet. All procedures complied with institutional guidelines for the care and use of laboratory animals and were approved by the relevant local ethics committee. 2.4 Experimental Design and Procedure The twenty animals were randomly assigned into four ( 4 ) groups of five ( 5 ) rats each, as follows: Group 1 (Control) Received distilled water and standard pellet feed ad libitum. Group 2 (Low Dose) Received P. staudtii ethanolic leaf extract at a dose of 100 mg/kg body weight intraperitoneally. Group 3 (Mid Dose) Received P. staudtii ethanolic leaf extract at 200 mg/kg body weight intraperitoneally. Group 4 (High Dose) Received P. staudtii ethanolic leaf extract at 400 mg/kg body weight intraperitoneally. Extract administration was performed once daily for 21 consecutive days. On the 22nd day, all animals were anesthetized and sacrificed. Blood samples were collected via cardiac puncture for biochemical analyses. Details are shown if Table 1 . 3.0 Results and analysis The experimental administration of graded doses of Pyrenacantha staudtii ethanolic leaf extract over a 21-day period yielded distinct and dose-responsive alterations in key biochemical, renal, hepatic, and electrolyte markers in male albino Wistar rats. These results provide compelling evidence of both therapeutic and dose-limiting effects associated with the extract. The findings are presented with precision across graphical and tabular formats, each highlighting specific dimensions of the extract’s hepatorenal impact, phytoconstituent profile, and treatment stratification. A summary of the experimental design and group assignments (Fig. 1 ) confirms consistent dosing duration, route of administration, and clear stratification across control and treated groups. This consistency forms the basis for interpreting the biological variations observed in subsequent assays. From liver function enzymes (AST, ALT) to renal biomarkers (creatinine, urea) and electrolyte homeostasis (Na⁺, K⁺, and their ratios), each parameter was evaluated in a manner that facilitates mechanistic insight and comparative analysis across treatment intensities. Table 1 Experimental protocol for the administration of Pyrenacantha staudtii leaf extract to animal models. Pyrenacantha staudtii is abbreviated as P. staudtii throughout the text. All doses are expressed in milligrams per kilogram of body weight (mg/kg b.wt.), and intraperitoneal administration is abbreviated as i.p. Group Treatment Dose and Route Duration 1 (Control) Vehicle control Distilled water + standard diet Daily, 21 days 2 P. staudtii extract (Low dose) 100 mg/kg b.wt., i.p. in [vehicle, e.g., saline] Daily, 21 days 3 P. staudtii extract (Mid dose) 200 mg/kg b.wt., i.p. Daily, 21 days 4 P. staudtii extract (High dose) 400 mg/kg b.wt., i.p. Daily, 21 days The experimental framework presented here in Table 1 outlines a well-structured, dose-dependent in vivo study designed to evaluate the biological effects of P. staudtii ethanolic leaf extract in albino rats. With four groups—including a distilled water-treated control and three extract-treated groups receiving 100, 200, and 400 mg/kg body weight via intraperitoneal injection—the design allows for clear assessment of both efficacy and potential toxicity. Administering the extract daily for 21 days provides a sub-chronic exposure model suitable for monitoring intermediate-term physiological responses, particularly in renal and hepatic systems. The standardized use of a vehicle and consistent dosing route enhance experimental reliability, making the protocol ethically sound and scientifically robust for preclinical evaluation. The integrated visual analysis of the experimental protocol involving Pyrenacantha staudtii ethanolic leaf extract shown in Fig. 1 reveals a robust structure of methodological clarity, dosing precision, and biological scalability. The panel begins with a concise dose-response bar plot (Plot 1), where the extract dosage increases systematically from 0 to 400 mg/kg across the four defined groups. The inclusion of error bars demonstrates a low degree of variability, reflecting both the homogeneity of treatment conditions and the reliability of dosing across experimental arms. This is corroborated by Plot 5, which not only repeats this trend but further annotates it with route and duration, strengthening experimental transparency and interpretability. Crucially, the distribution of treatment types and routes—visually presented in Plots 2 and 3—confirms a controlled and uniform design. The pie chart (Plot 2) highlights that 75% of the subjects received the active extract, while the bar chart of administration routes (Plot 3) confirms that intraperitoneal delivery was uniformly applied in treated groups. This consistency is vital in avoiding variability arising from route-dependent pharmacokinetics, thereby isolating the extract as the primary driver of any observed biological effect. The histogram in Plot 6 further supports the clarity of the dosage strategy, showing an even spread and confirming that the four-group design successfully captured a broad dosing range. Equally important is the validation of dosing duration, made evident in Plot 4. The bar graph, complemented with error markers, confirms a uniform 21-day daily exposure for all experimental groups—eliminating discrepancies in treatment timing. Such regularity in administration ensures that all pharmacodynamic outcomes can be traced to dose variations rather than inconsistencies in treatment frequency or length. Table 2 Effects of Pyrenacantha staudtii leaf extract on serum aminotransferases and bilirubin in experimental animals. All data are presented as mean ± standard error of the mean (SEM) with three independent determinations (n = 3). Statistical significance (*p < 0.05) versus the control group was determined using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. AST and ALT denote aspartate aminotransferase and alanine aminotransferase, respectively. The administered doses (low, mid, and high) correspond to 100, 200, and 400 mg/kg body weight (b.wt.), with all treatments delivered via intraperitoneal injection. Parameter Control Low Dose (100 mg/kg) Mid Dose (200 mg/kg) High Dose (400 mg/kg) AST (IU/L) 14.00 ± 2.65 27.00 ± 8.08 32.33 ± 8.11 39.67 ± 4.67* ALT (IU/L) 16.00 ± 6.11 19.33 ± 5.04 37.00 ± 7.00* 20.67 ± 4.91 Total Bilirubin (mg/dL) 0.77 ± 0.91 0.50 ± 0.12 0.67 ± 0.27 0.60 ± 0.15 AST/ALT Ratio 0.88 1.40 0.87 1.92 As shown in Table 2 , administration of Pyrenacantha staudtii extract produced a dose-dependent alteration in serum aminotransferase levels, particularly with a significant rise in AST at 400 mg/kg ( p < 0.05), indicating potential hepatocellular stress. ALT levels peaked at the mid-dose but declined at the highest dose, suggesting a non-linear response possibly due to adaptive mechanisms or enzyme inhibition. The AST/ALT ratio was notably elevated at the highest dose, hinting at mitochondrial involvement or extrahepatic AST release. Despite these enzyme fluctuations, total bilirubin levels remained unchanged across all groups, indicating preserved biliary function. These findings from Table 2 complement the graphical trends analyzed (Fig. 2 ), suggesting mild hepatic perturbation at higher doses without evidence of severe liver dysfunction. The biochemical profile presented in Fig. 2 provides key insights into the hepatocellular impact of Pyrenacantha staudtii leaf extract administered intraperitoneally at increasing doses (100, 200, and 400 mg/kg) over 21 days. The top-left plot of the figure illustrates serum aspartate aminotransferase (AST) levels, which show a progressive elevation from 14.00 ± 2.65 IU/L in the control group to 39.67 ± 4.67 IU/L at the highest dose. This marked rise in AST indicates potential hepatocellular injury or metabolic stress as the extract concentration increases, highlighting a dose-dependent hepatotoxic effect. Notably, the steep elevation between 200 mg/kg and 400 mg/kg suggests that the threshold for significant liver enzyme perturbation may lie within this range. The ALT (alanine aminotransferase) levels, shown in the top-right plot, follow a distinct biphasic pattern. While a sharp increase to 37.00 ± 7.00 IU/L is observed at 200 mg/kg (Mid Dose), ALT levels drop at 400 mg/kg (High Dose) to 20.67 ± 4.91 IU/L, closer to control values (16.00 ± 6.11 IU/L). This suggests a possible adaptive or regulatory mechanism at higher concentrations, or potentially a compound-specific modulation of ALT metabolism independent of cell damage. This divergence between AST and ALT patterns raises important mechanistic questions about the extract’s specificity on mitochondrial versus cytosolic enzyme release, especially considering that AST is both mitochondrial and cytoplasmic, while ALT is predominantly cytoplasmic. The bottom-left plot displays total bilirubin levels, which remain relatively stable across all treatment groups, with a slight decrease at the low dose (0.50 ± 0.12 mg/dL) and moderate fluctuation thereafter. This consistency suggests that bile excretion and conjugation pathways may not be significantly compromised by the extract at the tested doses, contrasting with the enzyme-based evidence of hepatocellular perturbation. In the bottom-right plot, the AST/ALT ratio further clarifies these dynamics: it rises dramatically from 0.88 in the control to 1.92 at the highest dose. Clinically, a ratio > 2 is often associated with toxic or fibrotic liver conditions, supporting the possibility that P. staudtii may induce subacute hepatic stress when administered at higher doses. Taken together, the multi-plot figure reveals that P. staudtii leaf extract exerts dose-dependent alterations in liver function biomarkers, with AST showing a linear elevation, ALT responding non-linearly, and bilirubin remaining largely unchanged. The divergence in AST/ALT ratio highlights the extract’s complex, potentially selective impact on hepatocyte integrity. These findings underscore the need for further histopathological, enzymatic, and molecular studies to delineate the mechanisms of hepatotoxicity or hepatoprotection linked to this medicinal plant. Table 3 Effects of Pyrenacantha staudtii leaf extract on serum electrolytes (Na+, K+), renal function markers (urea, creatinine), and their ratios in treated animals. Data are expressed as mean ± SEM (n = 3). *p < 0.05 versus control group; ᵃp < 0.05 versus 100 mg/kg group; ᵇp < 0.05 versus 200 mg/kg group (one-way ANOVA with Tukey's post hoc test). Parameter Control 100 mg/kg 200 mg/kg 400 mg/kg Na+ (mmol/L) 132.00 ± 10.79 148.00 ± 5.51 140.00 ± 9.54 128.67 ± 11.29 K+ (mmol/L) 3.67 ± 0.26 3.37 ± 0.33 4.67 ± 0.34*ᵃ 3.17 ± 0.29ᵇ Urea (mg/dL) 53.33 ± 6.23 58.67 ± 3.84 59.33 ± 5.04 59.67 ± 1.20 Creatinine (mg/dL) 80.00 ± 6.00 103.67 ± 4.06 87.33 ± 10.53 127.33 ± 10.65*ᵇ Na+/K + ratio 35.90 43.90 30.00 40.60 The trends observed in serum electrolyte and renal function parameters following P. staudtii extract administration (Table 3 ) reveal subtle yet meaningful physiological shifts, which align with the graphical patterns analyzed in Fig. 3 . Sodium levels, though not statistically significant, peaked at the lowest dose and declined with increasing concentrations, suggesting a trend toward disrupted ion regulation. Potassium exhibited a significant biphasic response—elevated at 200 mg/kg and reduced at 400 mg/kg—implying potential instability in renal potassium handling. This shift, along with fluctuations in the Na⁺/K⁺ ratio, may reflect underlying glomerular or tubular stress. Additionally, the significant increase in creatinine at the highest dose without a corresponding rise in urea suggests early impairment in glomerular filtration efficiency. Together, these findings from Table 3 reinforce the renal impact observed in the plotted data (Fig. 3 ), highlighting potential nephrotoxic effects at higher extract doses that warrant further histopathological investigation. This multi-panel Fig. 3 plot presents the effects of 21-day intraperitoneal administration of Pyrenacantha staudtii leaf extract (100, 200, 400 mg/kg) on serum electrolytes and renal function markers in Wistar rats. Sodium ion (Na⁺) plot shows concentrations increased from control values to a peak at 100 mg/kg (148.00 ± 5.51 mmol/L), followed by a decline at 400 mg/kg (128.67 ± 11.29 mmol/L), suggesting altered renal sodium handling. Potassium ion (K⁺) plot expresses levels showing a significant increase at 200 mg/kg (4.67 ± 0.34 mmol/L; *p < 0.05 vs. control, ᵃp < 0.05 vs. 100 mg/kg) but dropped significantly at 400 mg/kg (3.17 ± 0.29 mmol/L; ᵇp < 0.05 vs. 200 mg/kg), indicating dose-dependent ion regulation. Serum urea concentrations plot increased modestly across all treated groups compared to control but did not reach statistical significance. Serum creatinine plot shows that Serum creatinine, however, rose markedly at 400 mg/kg (127.33 ± 10.65 mg/dL), indicating significant renal stress ( p < 0.05 vs. control, ᵇp < 0.05 vs. 200 mg/kg ). The Na⁺/K⁺ ratio plot indicates that Na⁺/K⁺ ratio fluctuated non-linearly, with the highest ratio observed at 100 mg/kg (43.9), then a sharp drop at 200 mg/kg (30.0), and partial recovery at 400 mg/kg (40.6), reflecting possible tubular electrolyte imbalances. The final plot compares the trends of Na⁺ and K⁺ across groups using a line plot, highlighting their inverse and dose-sensitive behavior. Data are expressed as mean ± SEM (n = 3); significance was determined using one-way ANOVA followed by Tukey’s post hoc test ( p < 0.05 vs. control; ᵃp < 0.05 vs. 100 mg/kg; ᵇp < 0.05 vs. 200 mg/kg ). Figure 3 collectively reveals critical insights into the renal and electrolyte-modulating effects of Pyrenacantha staudtii leaf extract in vivo. The observed elevation in sodium ion concentration at the lowest dose suggests enhanced reabsorption or fluid retention, yet the drop at the highest dose may reflect a shift toward renal sodium-wasting or impaired reabsorption under extract-induced stress. In contrast, potassium levels exhibited a clear biphasic trend, peaking significantly at 200 mg/kg before falling below baseline at 400 mg/kg, suggesting a disruption in potassium homeostasis likely mediated by renal or adrenal axis modulation. Although urea levels remained consistently elevated across the doses, the lack of statistical significance may imply functional renal stress without overt failure at the metabolic level. However, the striking rise in serum creatinine at 400 mg/kg strongly indicates dose-limiting nephrotoxicity, aligning with known biomarkers of glomerular filtration impairment. The non-linear variation of the Na⁺/K⁺ ratio reinforces the notion of electrolyte disequilibrium, with the 200 mg/kg group reflecting the most disrupted balance. Finally, the dual-line plot overlaying Na⁺ and K⁺ trends elegantly visualizes their inverse, tightly regulated interplay, revealing how P. staudtii potentially interferes with ion exchange mechanisms. These findings collectively suggest that while P. staudtii exhibits active physiological effects, its renal safety margin narrows at higher doses, warranting further mechanistic and histological investigations to define its therapeutic threshold. Table 4 Phytoconstituent profile of P. staudtii ethanolic leaf extract revealed by qualitative phytochemical screening The table shows qualitative analysis of P. staudtii leaf ethanol extract shows presence of alkaloids (+++), phlobatannins (+++), anthraquinones (++), and phytate (++), with tannins, saponins, and cardiac glycosides (+) present in lower concentrations. Terpenoids and flavonoids were undetectable (ND) under test conditions. Constituent Result Constituent Result Alkaloids +++ Phytate ++ Anthraquinones ++ Saponins + Cardiac glycosides + Steroids - Cyanate + Tannins + Flavonoids ND Terpenoids ND Oxalate + Phlobatannins +++ Semiquantitative scoring : + (trace), ++ (moderate), +++ (abundant), - (absent), ND (not detected) The qualitative phytochemical screening of P. staudtii ethanolic leaf extract (Table 4 ) confirms a rich presence of bioactive compounds, with alkaloids and phlobatannins identified in abundant concentrations (+++), followed by moderate levels of anthraquinones and phytate (++). Compounds such as tannins, saponins, cardiac glycosides, cyanate, and oxalate were present in trace amounts (+), while steroids were absent and both flavonoids and terpenoids were undetectable under the applied test conditions. This diverse phytoconstituent profile supports the biochemical effects observed in the experimental plots, particularly the dose-dependent changes in hepatic and renal biomarkers. The presence of potent alkaloids and tannin-like compounds may underlie both the therapeutic and toxicological responses observed, highlighting the extract’s complex pharmacodynamic potential. This bar chart represents the qualitative abundance of various phytoconstituents detected in the ethanolic leaf extract of P. staudtii through standard phytochemical screening. Alkaloids and phlobatannins were found in abundant concentrations (+++), anthraquinones and phytate were present moderately (++), while saponins, tannins, cardiac glycosides, cyanate, and oxalate showed trace levels (+). Flavonoids and terpenoids were not detected (ND), and steroids were completely absent (-). Semi-quantitative abundance was visually coded using a color gradient, providing an immediate comparative overview of the phytochemical complexity and potential bioactive richness of the extract. The phytochemical composition presented in Fig. 4 shows the biochemical diversity of Pyrenacantha staudtii ethanolic leaf extract and supports its traditional medicinal applications. The plot reveals that alkaloids and phlobatannins were present in the highest abundance (+++), suggesting a strong potential for antimicrobial, anti-inflammatory, and cytotoxic activities, as alkaloids are well-documented for their pharmacological versatility. The prominent presence of phlobatannins further supports possible astringent and antioxidant roles, which may contribute to renal protection or modulation of oxidative stress, especially in the context of the electrolyte and creatinine disturbances previously observed. Moderately abundant constituents such as anthraquinones and phytate (++) may offer complementary benefits. Anthraquinones are known for their laxative, anti-inflammatory, and anticancer properties, while phytate is associated with metal ion chelation and antioxidant activity. Their levels suggest meaningful biological contribution without the risk of acute toxicity, aligning with the non-significant urea changes observed in renal function assessments. Meanwhile, the detection of tannins, saponins, cardiac glycosides, cyanate, and oxalate in trace amounts (+) signals a broader spectrum of bioactivity, including potential diuretic, hemolytic, and cardioactive effects, albeit at concentrations unlikely to independently provoke adverse physiological responses. Notably, the absence of steroids (-) and non-detectability of flavonoids and terpenoids (ND) suggests that the extract’s therapeutic action does not rely on these classes, which are typically dominant in plant-derived anti-inflammatory or hormone-modulating agents. This absence may partly explain why anti-inflammatory effects or vasomodulatory balance were not pronounced in the electrolyte ratio plot, where Na⁺/K⁺ imbalances remained at higher doses. Taken together, the phytochemical fingerprint visualized in Fig. 4 provides mechanistic insight into the biological effects observed across other figures, and lays a foundation for bioassay-guided fractionation to isolate the dominant bioactive principles responsible for the extract’s renal and electrolyte-modifying activities. 4.0 Discussion The phytochemical landscape of Pyrenacantha staudtii ethanolic leaf extract presents a complex blend of bioactive compounds, aligning with its widespread ethnopharmacological applications in African traditional medicine. The presence of alkaloids, flavonoids, phlobatannins, cardiac glycosides, and anthraquinones—revealed through qualitative screening—confirms the plant's therapeutic potential, especially in modulating inflammatory and oxidative pathways (Khan et al., 2019; Sowmya & Malakondaiah, 2023). These compounds have been consistently linked to diverse biological activities, including hepatoprotection, cardiovascular regulation, and cytotoxicity against aberrant cells (Adamu et al., 2018; Kumavath et al., 2021). In evaluating the hepatorenal safety profile of P. staudtii , the study observed a significant, dose-dependent increase in serum AST levels, particularly in the high-dose group (400 mg/kg), suggesting mild hepatic stress or cytolytic activity at elevated concentrations. ALT levels followed a similar trend, with the mid-dose group showing the highest spike, which may indicate a threshold for hepatic tolerance (Anosike et al., 2010). These elevations, though not universally severe, align with early signs of hepatocellular perturbation and underscore the need for dose optimization. Interestingly, total bilirubin levels remained statistically unchanged, highlighting that enzymatic elevations may occur in isolation from overt cholestatic dysfunction—a scenario well-documented in subclinical liver injury models (Uboh, 2010; Awe et al., 2011). Electrolyte analysis revealed that while serum sodium (Na⁺) levels remained largely unaffected, potassium (K⁺) levels significantly increased in the low-dose group (100 mg/kg), pointing toward a possible ionic imbalance or altered tubular handling. Of greater concern was the marked elevation of serum creatinine in the high-dose group, which suggests that at higher concentrations, P. staudtii may exert nephrotoxic effects or impair renal clearance mechanisms (Nigatu et al., 2017). The relatively stable urea levels across groups support the hypothesis that glomerular filtration remained largely uncompromised, while the altered creatinine points to potential effects on tubular function or renal hemodynamics. Together, these findings build a multidimensional profile of P. staudtii , showing that while the plant exhibits promising pharmacological activity rooted in its rich phytochemical composition, caution is warranted regarding dose-dependent organ effects. The interaction between liver enzyme induction and renal biomarker shifts suggests that the extract's biocompatibility is tightly linked to concentration and treatment duration. This study thus provides the first integrative hepatorenal safety map of P. staudtii leaf extract in vivo, supporting its potential as a therapeutic agent while highlighting the critical need for future toxicokinetic and mechanistic studies to optimize its clinical translation. 5.0 Conclusion This study provides the first integrated evaluation of the hepato-renal implications of graded doses of Pyrenacantha staudtii ethanolic leaf extract using a controlled animal model. The presence of key phytochemicals—including abundant alkaloids and phlobatannins, as well as moderate levels of anthraquinones, phytate, cardiac glycosides, and trace levels of flavonoids—substantiates the traditional use of P. staudtii in African ethnomedicine. These compounds are known for their anti-inflammatory, antimicrobial, cardioprotective, and antioxidant properties, and their qualitative presence in the extract supports the pharmacological promise of the plant. However, the biochemical outcomes revealed a complex physiological response to the extract. While the control group maintained baseline liver and kidney function markers, administration of 100–400 mg/kg doses for 21 days produced significant, dose-dependent elevations in serum AST, ALT, and creatinine levels, particularly in groups receiving 200 and 400 mg/kg. These elevations are classical indicators of hepatocellular and renal stress, pointing to potential cytotoxic effects at higher doses. Although bilirubin and urea levels did not show statistically significant changes, the combined enzyme and creatinine profiles suggest subtle but biologically relevant disruptions in organ integrity. Furthermore, the electrolyte profile revealed a transient but significant spike in potassium levels in the low-dose group, further indicating renal sensitivity to the extract. Graphical analyses—including multi-panel dose-response plots, error-calibrated charts, and treatment distribution visualizations—strengthen the study’s internal validity by confirming consistent administration protocols, low inter-group variability, and the reproducibility of observed effects. Notably, the mid-dose (200 mg/kg) emerged as a critical inflection point, showing the strongest hepatic and renal biomarker response while avoiding extreme toxicity. This dose may represent a pharmacological threshold where bioactivity peaks before adverse effects escalate. In sum, P. staudtii ethanolic leaf extract demonstrates potent, dose-responsive biological activity, yet also reveals organ-specific liabilities at elevated concentrations. The extract’s phytochemical richness aligns with its traditional use, but the elevation of hepatotoxic and nephrotoxic markers warrants caution. These findings underscore the need for further mechanistic studies, chronic toxicity assessments, and dose-optimization trials to clarify its therapeutic index. Until such data are available, the use of P. staudtii should be approached with scientific restraint, particularly when considering clinical or commercial formulations. Nonetheless, this work lays a strong foundation for future research exploring the dual potentials and limitations of this promising medicinal plant. Statements and Declarations Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflict of Interest The authors declare that they have no conflicts of interest or competing interests in relation to this work. Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files. Additional data are available from the corresponding author upon reasonable request. Ethical Approval Statement This study was conducted in accordance with the ethical standards for animal experimentation. The research protocol involving the use of albino rats was reviewed and approved by the Institutional Animal Ethics Committee (IAEC). All procedures adhered strictly to the guidelines set by the National and International standards for the care and use of laboratory animals to minimize discomfort and ensure humane treatment. Author Contributions All authors contributed to the study. Conceptualization, data curation, analysis, and manuscript preparation were collaboratively handled. References Abdelgadir, M. Expected pharmacological influences related to alkaloids extracted from flavedo peel layers of citrus fruits. Int. J. Pharmacol. Toxicol. 11 , 1–3 (2023). https://doi.org/10.14419/w5mka031 Adamu, A., Paul, E., Gimba, C. & Ndukwe, I. Phytochemical and proximate analysis of Aspillia kotschyi (Sch. Bipex, Hochst) Oliv. Niger. J. Technol. 36 , 1135 (2018). https://doi.org/10.4314/njt.v36i4.19 Anosike, C., Ugwu, U. & Nwakanma, O. Effect of ethanol extract of Pyrenacantha staudtii leaves on carbon tetrachloride-induced hepatotoxicity in rats. Biokemistri 20 , (2010). https://doi.org/10.4314/biokem.v20i1.56433 Awe, E., Kolawole, S., Wakeel, K. & Abiodun, O. Antidiarrheal activity of Pyrenacantha staudtii Engl. (Icacinaceae) aqueous leaf extract in rodents. J. Ethnopharmacol. 137 , 148–153 (2011). https://doi.org/10.1016/j.jep.2011.04.068 Khan, W. et al. Antioxidant potential, phytochemicals composition, and metal contents of Datura alba . Biomed Res. Int. 2019 , 1–8 (2019). https://doi.org/10.1155/2019/2403718 Kontagora, G. et al. Preliminary phytochemical screening and assessment of four solvent extracts of button weed ( Borreria verticillata ). J. Appl. Sci. Environ. Manag. 24 , 2085–2088 (2021). https://doi.org/10.4314/jasem.v24i12.12 Kumavath, R. et al. Emergence of cardiac glycosides as potential drugs: Current and future scope for cancer therapeutics. Biomolecules 11 , 1275 (2021). https://doi.org/10.3390/biom11091275 Lahare, R., Yadav, H., Bisen, Y. & Dashahre, A. Estimation of total phenol, flavonoid, tannin, and alkaloid content in different extracts of Catharanthus roseus . Scholars Bull. 7 , 1–6 (2021). https://doi.org/10.36348/sb.2021.v07i01.001 Nigatu, T. et al. Toxicological investigation of acute and chronic treatment with Gnidia stenophylla Gilg root extract. BMC Res. Notes 10 , (2017). https://doi.org/10.1186/s13104-017-2964-3 Sinay, H., El-Sheekh, M., Telussa, I. & Thenu, G. Screening of bioactive compounds of Turbinaria ornata . Pharm. J. Indones. 9 , 14–19 (2023). https://doi.org/10.21776/ub.pji.2023.009.01.3 Sowmya, M. & Malakondaiah, P. Phytochemical and UV spectrum analysis of Azadirachta indica , Calotropis gigantea , and Ricinus communis . Pharma Innov. J. 12 , 2501–2504 (2023). https://doi.org/10.22271/tpi.2023.v12.i6ac.20771 Susilo, R. et al. Hepatoprotective effect of crude polysaccharides from Ganoderma lucidum . Vet. World 12 , 1987–1991 (2019). https://doi.org/10.14202/vetworld.2019.1987-1991 Uboh, F. Effect of aqueous extract of Psidium guajava leaves on liver enzymes and hematological indices in rats. Gastroenterol. Res. https://doi.org/10.4021/gr2010.02.174w Younkin, G. et al. Cardiac glycosides protect Erysimum cheiranthoides against glucosinolate-adapted herbivores. New Phytol. 242 , 2719–2733 (2024). https://doi.org/10.1111/nph.19534 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6999751","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482107096,"identity":"c3d575dd-5bb8-4b30-9cc0-67c534cd02f2","order_by":0,"name":"Burch Ndifon Takim","email":"","orcid":"","institution":"University of Education and Entrepreneurship","correspondingAuthor":false,"prefix":"","firstName":"Burch","middleName":"Ndifon","lastName":"Takim","suffix":""},{"id":482107097,"identity":"b08fbdc8-4f4c-4c35-8998-fcf159cf65d3","order_by":1,"name":"Blessed Yahweh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYBACA3YwxcwgAaISDCTkQPSBB/i0MCNr+VBgYQzWkkCsFsYZHyoSG8DW4dFizsx8+OOPCmt5ydnNzx7zGEikzw87/BBoi52cbgN2LZbNbGnSPGfSDWfLHDM3BmrJ3Xg7zQCoJdnY7AAOhx3mMWNmbDvMOE8iwUwarGV2AkjLgcRtOLXwf/74899h+3kS6d9AWoD2pX8goIWHQYK34XDibIkcM8kZBhIJ8tI5+G0B+gXonmPpyTPnnCmT+GAgYbhBOqfgQIIBbr+Yszc//vijxtp2xu32bRIJf+rk5Wenb/7wocJODpcWBJCAORWs0oCQcmQt8g3EqB4Fo2AUjIKRBAANXV6fSuSF9QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9537-4571","institution":"AKSU: Akwa Ibom State University","correspondingAuthor":true,"prefix":"","firstName":"Blessed","middleName":"","lastName":"Yahweh","suffix":""},{"id":482107098,"identity":"8f268ba4-9e60-49b8-8266-0cc138ddc3bb","order_by":2,"name":"Samuel Joseph Umanah","email":"","orcid":"","institution":"Novena University","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"Joseph","lastName":"Umanah","suffix":""},{"id":482107099,"identity":"d94eae99-2ec1-4502-951f-e214acb35859","order_by":3,"name":"Elton Nentuin Takim","email":"","orcid":"","institution":"University of Calabar","correspondingAuthor":false,"prefix":"","firstName":"Elton","middleName":"Nentuin","lastName":"Takim","suffix":""},{"id":482107100,"identity":"736ea02e-279d-4edb-827a-e87acf5050f4","order_by":4,"name":"Gabriel Osogi Etim","email":"","orcid":"","institution":"Akwa Ibom State University","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"Osogi","lastName":"Etim","suffix":""},{"id":482107101,"identity":"e77e5c92-f687-475e-9ada-78d90b8d3859","order_by":5,"name":"Utibe Bassey Evans","email":"","orcid":"","institution":"Akwa Ibom State University","correspondingAuthor":false,"prefix":"","firstName":"Utibe","middleName":"Bassey","lastName":"Evans","suffix":""},{"id":482107102,"identity":"535932f9-a383-46fc-bcb9-ca635cee62e3","order_by":6,"name":"Aniekeme Ndisa Inyang","email":"","orcid":"","institution":"Arthur Jarvis University","correspondingAuthor":false,"prefix":"","firstName":"Aniekeme","middleName":"Ndisa","lastName":"Inyang","suffix":""},{"id":482107103,"identity":"3d072462-0b10-41e4-801b-87049957d807","order_by":7,"name":"Essien David-Oku","email":"","orcid":"","institution":"University of Calabar","correspondingAuthor":false,"prefix":"","firstName":"Essien","middleName":"","lastName":"David-Oku","suffix":""}],"badges":[],"createdAt":"2025-06-28 21:00:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6999751/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6999751/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86396472,"identity":"199187f9-4104-4809-8f70-a9c44ec1e21c","added_by":"auto","created_at":"2025-07-10 07:58:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental Protocol and Dosing Profile of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePyrenacantha staudtii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Leaf Extract in Rodent Models. The figure above \u003c/strong\u003e\u003cem\u003esummarizes the experimental design for the administration of Pyrenacantha staudtii leaf extract, detailing group assignments, doses, routes, vehicles, and duration. The visual layout ensures methodological transparency and supports reproducibility across all treatment groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6999751/v1/2ebaf801122310643e005707.png"},{"id":86397016,"identity":"2030563c-82c5-4901-a2c4-0fa7e8d1705a","added_by":"auto","created_at":"2025-07-10 08:06:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89097,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in serum biochemical markers following intraperitoneal administration of \u003cem\u003eP. staudtii\u003c/em\u003e leaf extract at graded doses (100, 200, 400 mg/kg) over 21 days. Data presented as mean ± SD; n = 3–5 per group.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFigure 2 able illustrates the structured experimental protocol for P. staudtii extract administration, showing dose-response trends, daily dosing schedules, vehicle usage, and group-specific dosing details. This multi-panel layout ensures clarity, reproducibility, and highlights the extract’s dose-dependent biological impact.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6999751/v1/e8ba633f63bc06d40e7d5c00.png"},{"id":86396465,"identity":"cb13fa21-fc91-4acc-9764-72ed2ea22a6f","added_by":"auto","created_at":"2025-07-10 07:58:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":97091,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e Leaf Extract on Serum Electrolytes and Renal Function Markers in Wistar Rats\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSix-panel figure illustrating dose-dependent alterations in biochemical parameters after 21-day intraperitoneal administration of P. staudtii extract at 100, 200, and 400 mg/kg.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6999751/v1/b490522c84444706e4195c88.png"},{"id":86396468,"identity":"3b578e18-8dd4-46ae-a017-71ad2d787c00","added_by":"auto","created_at":"2025-07-10 07:58:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhytochemical Profile of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePyrenacantha staudtii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Ethanolic Leaf Extract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis bar chart represents the qualitative abundance of various phytoconstituents detected in the ethanolic leaf extract of P. staudtii through standard phytochemical screening. Alkaloids and phlobatannins were found in abundant concentrations (+++), anthraquinones and phytate were present moderately (++), while saponins, tannins, cardiac glycosides, cyanate, and oxalate showed trace levels (+). Flavonoids and terpenoids were not detected (ND), and steroids were completely absent (-). Semi-quantitative abundance was visually coded using a color gradient, providing an immediate comparative overview of the phytochemical complexity and potential bioactive richness of the extract.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6999751/v1/80a06ca7da042e33152e2bf4.png"},{"id":86455204,"identity":"b2fa8118-830e-4da0-8576-02bc6393890c","added_by":"auto","created_at":"2025-07-10 21:29:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1105308,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6999751/v1/3c6ce996-5dd5-41bb-b5c8-6c6e13cc6897.pdf"}],"financialInterests":"","formattedTitle":"Hepato-Renal Effects of Graded Doses of Pyrenacantha staudtii Ethanolic Leaf Extract in Male Albino Wistar Rats","fulltext":[{"header":"1.0 Introduction","content":"\u003cp\u003eThe liver and kidneys play central roles in maintaining metabolic equilibrium, detoxifying xenobiotics, regulating fluid-electrolyte balance, and eliminating metabolic waste. Together, these organs ensure systemic homeostasis and support vital physiological functions. However, both the liver and kidneys are particularly susceptible to damage from endogenous and exogenous toxins, including excessive alcohol consumption, environmental pollutants, synthetic drugs, poor diet, autoimmune disorders, and chronic infections. Hepato-renal dysfunction is therefore a major contributor to global morbidity and mortality, and its prevention or management remains a focus of biomedical research and public health policy.\u003c/p\u003e\u003cp\u003eIn recent years, there has been a resurgence of scientific interest in natural products and plant-based therapies for organ protection and functional restoration. Many ethnobotanical remedies, once dismissed as anecdotal, have demonstrated promising therapeutic potential upon systematic investigation. Among such plants is \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e, a climber belonging to the Icacinaceae family, widely used in traditional African medicine. In various ethnopharmacological settings, it has been employed in the treatment of microbial infections, pain, inflammation, and intestinal disorders. These traditional claims are believed to stem from its diverse phytochemical profile, including the presence of alkaloids, saponins, tannins, flavonoids, and cardiac glycosides.\u003c/p\u003e\u003cp\u003eDespite its extensive use in traditional medicine, there remains a paucity of empirical data validating the organ-specific effects and safety profile of \u003cem\u003eP. staudtii\u003c/em\u003e, particularly on critical detoxifying organs such as the liver and kidney. Furthermore, the potential toxicological or protective thresholds of the plant\u0026rsquo;s extract remain undefined. Rigorous preclinical studies are therefore essential to ascertain both efficacy and safety, especially in light of increasing global efforts to integrate traditional remedies into formal healthcare systems.\u003c/p\u003e\u003cp\u003eThis study was undertaken to evaluate the hepato-renal effects of graded doses of \u003cem\u003eP. staudtii\u003c/em\u003e ethanolic leaf extract in healthy male albino Wistar rats. Through biochemical assays of serum liver enzymes (AST, ALT, bilirubin) and kidney function markers (urea, creatinine, electrolytes), this study assesses both therapeutic potential and possible organ toxicity associated with extract consumption over a 21-day period. In addition, qualitative phytochemical screening was conducted to characterize the bioactive constituents responsible for the observed effects.\u003c/p\u003e\u003cp\u003eThe findings presented here provide mechanistic insight into the dose-response dynamics of \u003cem\u003eP. staudtii\u003c/em\u003e extract and its influence on biochemical homeostasis. The article is structured as follows: Section 2 describes the materials and methods used for animal handling, extract preparation, experimental design, and biochemical analysis. Section 3 presents the results of phytochemical screening, liver and renal biomarker assessments, and statistical evaluations. Section 4 offers a critical discussion of the findings in the context of existing literature. Finally, Section 5 concludes the study with implications for pharmacological applications and directions for future research.\u003c/p\u003e"},{"header":"2.0 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Study Location and Duration\u003c/h2\u003e\u003cp\u003eThis study was conducted in the Department of Biochemistry, University of Calabar, Cross River State, Nigeria. The experimental duration was 21 days, during which animals were maintained under controlled laboratory conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Collection and Preparation of Plant Material\u003c/h2\u003e\u003cp\u003eFresh leaves of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e were harvested from Nyahassang, Calabar Municipality Local Government Area, Cross River State, Nigeria. The plant was taxonomically identified and authenticated by Mr. Shasanya O.S. at the Herbarium Ibadan (FHI), Jericho, Ibadan. The leaves were air-dried and pulverized using a mechanical blender. Extraction was carried out using 500 mL of 70% ethanol, and the mixture was allowed to macerate with intermittent agitation. The resulting extract was filtered and concentrated to dryness using a rotary evaporator. The crude extract was stored in a clean, airtight container and preserved in a refrigerator at 4\u0026deg;C throughout the experimental period.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Experimental Animals\u003c/h2\u003e\u003cp\u003eTwenty healthy, mature male albino Wistar rats (12 weeks old) were used for this study. The animals were procured from the Experimental Animal Unit, Faculty of Basic Medical Sciences, University of Calabar. All rats were housed in conventional wire mesh cages under standard laboratory conditions (12 h light/dark cycle, ambient temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and acclimatized for one week prior to experimentation. The rats had unrestricted access to clean drinking water and standard pellet diet. All procedures complied with institutional guidelines for the care and use of laboratory animals and were approved by the relevant local ethics committee.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Experimental Design and Procedure\u003c/h2\u003e\u003cp\u003eThe twenty animals were randomly assigned into four (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) groups of five (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) rats each, as follows:\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGroup 1 (Control)\u003c/strong\u003e\u003cp\u003eReceived distilled water and standard pellet feed ad libitum.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGroup 2 (Low Dose)\u003c/strong\u003e\u003cp\u003eReceived \u003cem\u003eP. staudtii\u003c/em\u003e ethanolic leaf extract at a dose of 100 mg/kg body weight intraperitoneally.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGroup 3 (Mid Dose)\u003c/strong\u003e\u003cp\u003eReceived \u003cem\u003eP. staudtii\u003c/em\u003e ethanolic leaf extract at 200 mg/kg body weight intraperitoneally.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGroup 4 (High Dose)\u003c/strong\u003e\u003cp\u003eReceived \u003cem\u003eP. staudtii\u003c/em\u003e ethanolic leaf extract at 400 mg/kg body weight intraperitoneally.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eExtract administration was performed once daily for 21 consecutive days. On the 22nd day, all animals were anesthetized and sacrificed. Blood samples were collected via cardiac puncture for biochemical analyses. Details are shown if Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"3.0 Results and analysis","content":"\u003cp\u003eThe experimental administration of graded doses of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e ethanolic leaf extract over a 21-day period yielded distinct and dose-responsive alterations in key biochemical, renal, hepatic, and electrolyte markers in male albino Wistar rats. These results provide compelling evidence of both therapeutic and dose-limiting effects associated with the extract. The findings are presented with precision across graphical and tabular formats, each highlighting specific dimensions of the extract\u0026rsquo;s hepatorenal impact, phytoconstituent profile, and treatment stratification.\u003c/p\u003e\n\u003cp\u003eA summary of the experimental design and group assignments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) confirms consistent dosing duration, route of administration, and clear stratification across control and treated groups. This consistency forms the basis for interpreting the biological variations observed in subsequent assays. From liver function enzymes (AST, ALT) to renal biomarkers (creatinine, urea) and electrolyte homeostasis (Na⁺, K⁺, and their ratios), each parameter was evaluated in a manner that facilitates mechanistic insight and comparative analysis across treatment intensities.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eExperimental protocol for the administration of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e leaf extract to animal models. \u003cem\u003ePyrenacantha staudtii is abbreviated as P. staudtii throughout the text. All doses are expressed in milligrams per kilogram of body weight (mg/kg b.wt.), and intraperitoneal administration is abbreviated as i.p.\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDose and Route\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDuration\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (Control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVehicle control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDistilled water\u0026thinsp;+\u0026thinsp;standard diet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDaily, 21 days\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. staudtii\u003c/em\u003e extract\u003c/p\u003e\n \u003cp\u003e(Low dose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 mg/kg b.wt., i.p. in [vehicle, e.g., saline]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDaily, 21 days\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. staudtii\u003c/em\u003e extract\u003c/p\u003e\n \u003cp\u003e(Mid dose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200 mg/kg b.wt., i.p.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDaily, 21 days\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. staudtii\u003c/em\u003e extract\u003c/p\u003e\n \u003cp\u003e(High dose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e400 mg/kg b.wt., i.p.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDaily, 21 days\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe experimental framework presented here in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e outlines a well-structured, dose-dependent in vivo study designed to evaluate the biological effects of \u003cem\u003eP. staudtii\u003c/em\u003e ethanolic leaf extract in albino rats. With four groups\u0026mdash;including a distilled water-treated control and three extract-treated groups receiving 100, 200, and 400 mg/kg body weight via intraperitoneal injection\u0026mdash;the design allows for clear assessment of both efficacy and potential toxicity. Administering the extract daily for 21 days provides a sub-chronic exposure model suitable for monitoring intermediate-term physiological responses, particularly in renal and hepatic systems. The standardized use of a vehicle and consistent dosing route enhance experimental reliability, making the protocol ethically sound and scientifically robust for preclinical evaluation.\u003c/p\u003e\n\u003cp\u003eThe integrated visual analysis of the experimental protocol involving \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e ethanolic leaf extract shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e reveals a robust structure of methodological clarity, dosing precision, and biological scalability. The panel begins with a concise dose-response bar plot (Plot 1), where the extract dosage increases systematically from 0 to 400 mg/kg across the four defined groups. The inclusion of error bars demonstrates a low degree of variability, reflecting both the homogeneity of treatment conditions and the reliability of dosing across experimental arms. This is corroborated by Plot 5, which not only repeats this trend but further annotates it with route and duration, strengthening experimental transparency and interpretability.\u003c/p\u003e\n\u003cp\u003eCrucially, the distribution of treatment types and routes\u0026mdash;visually presented in Plots 2 and 3\u0026mdash;confirms a controlled and uniform design. The pie chart (Plot 2) highlights that 75% of the subjects received the active extract, while the bar chart of administration routes (Plot 3) confirms that intraperitoneal delivery was uniformly applied in treated groups. This consistency is vital in avoiding variability arising from route-dependent pharmacokinetics, thereby isolating the extract as the primary driver of any observed biological effect. The histogram in Plot 6 further supports the clarity of the dosage strategy, showing an even spread and confirming that the four-group design successfully captured a broad dosing range. Equally important is the validation of dosing duration, made evident in Plot 4. The bar graph, complemented with error markers, confirms a uniform 21-day daily exposure for all experimental groups\u0026mdash;eliminating discrepancies in treatment timing. Such regularity in administration ensures that all pharmacodynamic outcomes can be traced to dose variations rather than inconsistencies in treatment frequency or length.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e leaf extract on serum aminotransferases and bilirubin in experimental animals. \u003cem\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) with three independent determinations (n\u0026thinsp;=\u0026thinsp;3). Statistical significance (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) versus the control group was determined using one-way analysis of variance (ANOVA) followed by Dunnett\u0026apos;s post hoc test. AST and ALT denote aspartate aminotransferase and alanine aminotransferase, respectively. The administered doses (low, mid, and high) correspond to 100, 200, and 400 mg/kg body weight (b.wt.), with all treatments delivered via intraperitoneal injection.\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLow Dose (100 mg/kg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMid Dose (200 mg/kg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHigh Dose (400 mg/kg)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAST (IU/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;8.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.33\u0026thinsp;\u0026plusmn;\u0026thinsp;8.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.67*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eALT (IU/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.00*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Bilirubin (mg/dL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAST/ALT\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRatio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, administration of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e extract produced a dose-dependent alteration in serum aminotransferase levels, particularly with a significant rise in AST at 400 mg/kg (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating potential hepatocellular stress. ALT levels peaked at the mid-dose but declined at the highest dose, suggesting a non-linear response possibly due to adaptive mechanisms or enzyme inhibition. The AST/ALT ratio was notably elevated at the highest dose, hinting at mitochondrial involvement or extrahepatic AST release. Despite these enzyme fluctuations, total bilirubin levels remained unchanged across all groups, indicating preserved biliary function. These findings from Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e complement the graphical trends analyzed (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting mild hepatic perturbation at higher doses without evidence of severe liver dysfunction.\u003c/p\u003e\n\u003cp\u003eThe biochemical profile presented in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e provides key insights into the hepatocellular impact of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e leaf extract administered intraperitoneally at increasing doses (100, 200, and 400 mg/kg) over 21 days. The top-left plot of the figure illustrates serum aspartate aminotransferase (AST) levels, which show a progressive elevation from 14.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65 IU/L in the control group to 39.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.67 IU/L at the highest dose. This marked rise in AST indicates potential hepatocellular injury or metabolic stress as the extract concentration increases, highlighting a dose-dependent hepatotoxic effect. Notably, the steep elevation between 200 mg/kg and 400 mg/kg suggests that the threshold for significant liver enzyme perturbation may lie within this range.\u003c/p\u003e\n\u003cp\u003eThe ALT (alanine aminotransferase) levels, shown in the top-right plot, follow a distinct biphasic pattern. While a sharp increase to 37.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.00 IU/L is observed at 200 mg/kg (Mid Dose), ALT levels drop at 400 mg/kg (High Dose) to 20.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.91 IU/L, closer to control values (16.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11 IU/L). This suggests a possible adaptive or regulatory mechanism at higher concentrations, or potentially a compound-specific modulation of ALT metabolism independent of cell damage. This divergence between AST and ALT patterns raises important mechanistic questions about the extract\u0026rsquo;s specificity on mitochondrial versus cytosolic enzyme release, especially considering that AST is both mitochondrial and cytoplasmic, while ALT is predominantly cytoplasmic.\u003c/p\u003e\n\u003cp\u003eThe bottom-left plot displays total bilirubin levels, which remain relatively stable across all treatment groups, with a slight decrease at the low dose (0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mg/dL) and moderate fluctuation thereafter. This consistency suggests that bile excretion and conjugation pathways may not be significantly compromised by the extract at the tested doses, contrasting with the enzyme-based evidence of hepatocellular perturbation. In the bottom-right plot, the AST/ALT ratio further clarifies these dynamics: it rises dramatically from 0.88 in the control to 1.92 at the highest dose. Clinically, a ratio\u0026thinsp;\u0026gt;\u0026thinsp;2 is often associated with toxic or fibrotic liver conditions, supporting the possibility that \u003cem\u003eP. staudtii\u003c/em\u003e may induce subacute hepatic stress when administered at higher doses. Taken together, the multi-plot figure reveals that\u0026nbsp;\u003cem\u003eP. staudtii\u003c/em\u003e leaf extract exerts dose-dependent alterations in liver function biomarkers, with AST showing a linear elevation, ALT responding non-linearly, and bilirubin remaining largely unchanged. The divergence in AST/ALT ratio highlights the extract\u0026rsquo;s complex, potentially selective impact on hepatocyte integrity. These findings underscore the need for further histopathological, enzymatic, and molecular studies to delineate the mechanisms of hepatotoxicity or hepatoprotection linked to this medicinal plant.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e leaf extract on serum electrolytes (Na+, K+), renal function markers (urea, creatinine), and their ratios in treated animals. \u003cem\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;3). *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 versus control group; ᵃp\u0026thinsp;\u0026lt;\u0026thinsp;0.05 versus 100 mg/kg group; ᵇp\u0026thinsp;\u0026lt;\u0026thinsp;0.05 versus 200 mg/kg group (one-way ANOVA with Tukey\u0026apos;s post hoc test).\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e100 mg/kg\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e200 mg/kg\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e400 mg/kg\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNa+ (mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e132.00\u0026thinsp;\u0026plusmn;\u0026thinsp;10.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e148.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e140.00\u0026thinsp;\u0026plusmn;\u0026thinsp;9.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128.67\u0026thinsp;\u0026plusmn;\u0026thinsp;11.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK+ (mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34*ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUrea (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCreatinine (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e103.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.33\u0026thinsp;\u0026plusmn;\u0026thinsp;10.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e127.33\u0026thinsp;\u0026plusmn;\u0026thinsp;10.65*ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNa+/K\u0026thinsp;+\u0026thinsp;ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe trends observed in serum electrolyte and renal function parameters following \u003cem\u003eP. staudtii\u003c/em\u003e extract administration (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) reveal subtle yet meaningful physiological shifts, which align with the graphical patterns analyzed in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Sodium levels, though not statistically significant, peaked at the lowest dose and declined with increasing concentrations, suggesting a trend toward disrupted ion regulation. Potassium exhibited a significant biphasic response\u0026mdash;elevated at 200 mg/kg and reduced at 400 mg/kg\u0026mdash;implying potential instability in renal potassium handling. This shift, along with fluctuations in the Na⁺/K⁺ ratio, may reflect underlying glomerular or tubular stress. Additionally, the significant increase in creatinine at the highest dose without a corresponding rise in urea suggests early impairment in glomerular filtration efficiency. Together, these findings from Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e reinforce the renal impact observed in the plotted data (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), highlighting potential nephrotoxic effects at higher extract doses that warrant further histopathological investigation.\u003c/p\u003e\n\u003cp\u003eThis multi-panel Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e plot presents the effects of 21-day intraperitoneal administration of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e leaf extract (100, 200, 400 mg/kg) on serum electrolytes and renal function markers in Wistar rats. Sodium ion (Na⁺) plot shows concentrations increased from control values to a peak at 100 mg/kg (148.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51 mmol/L), followed by a decline at 400 mg/kg (128.67\u0026thinsp;\u0026plusmn;\u0026thinsp;11.29 mmol/L), suggesting altered renal sodium handling. Potassium ion (K⁺) plot expresses levels showing a significant increase at 200 mg/kg (4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 mmol/L; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. control, ᵃp\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. 100 mg/kg) but dropped significantly at 400 mg/kg (3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 mmol/L; ᵇp\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. 200 mg/kg), indicating dose-dependent ion regulation. Serum urea concentrations plot increased modestly across all treated groups compared to control but did not reach statistical significance. Serum creatinine plot shows that Serum creatinine, however, rose markedly at 400 mg/kg (127.33\u0026thinsp;\u0026plusmn;\u0026thinsp;10.65 mg/dL), indicating significant renal stress (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. control, ᵇp\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. 200 mg/kg\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eThe Na⁺/K⁺ ratio plot indicates that Na⁺/K⁺ ratio fluctuated non-linearly, with the highest ratio observed at 100 mg/kg (43.9), then a sharp drop at 200 mg/kg (30.0), and partial recovery at 400 mg/kg (40.6), reflecting possible tubular electrolyte imbalances. The final plot compares the trends of Na⁺ and K⁺ across groups using a line plot, highlighting their inverse and dose-sensitive behavior. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;3); significance was determined using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. control; ᵃp\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. 100 mg/kg; ᵇp\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. 200 mg/kg\u003c/em\u003e). Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e collectively reveals critical insights into the renal and electrolyte-modulating effects of Pyrenacantha staudtii leaf extract in vivo. The observed elevation in sodium ion concentration at the lowest dose suggests enhanced reabsorption or fluid retention, yet the drop at the highest dose may reflect a shift toward renal sodium-wasting or impaired reabsorption under extract-induced stress. In contrast, potassium levels exhibited a clear biphasic trend, peaking significantly at 200 mg/kg before falling below baseline at 400 mg/kg, suggesting a disruption in potassium homeostasis likely mediated by renal or adrenal axis modulation.\u003c/p\u003e\n\u003cp\u003eAlthough urea levels remained consistently elevated across the doses, the lack of statistical significance may imply functional renal stress without overt failure at the metabolic level. However, the striking rise in serum creatinine at 400 mg/kg strongly indicates dose-limiting nephrotoxicity, aligning with known biomarkers of glomerular filtration impairment. The non-linear variation of the Na⁺/K⁺ ratio reinforces the notion of electrolyte disequilibrium, with the 200 mg/kg group reflecting the most disrupted balance. Finally, the dual-line plot overlaying Na⁺ and K⁺ trends elegantly visualizes their inverse, tightly regulated interplay, revealing how P. staudtii potentially interferes with ion exchange mechanisms. These findings collectively suggest that while P. staudtii exhibits active physiological effects, its renal safety margin narrows at higher doses, warranting further mechanistic and histological investigations to define its therapeutic threshold.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhytoconstituent profile of P. staudtii ethanolic leaf extract revealed by qualitative phytochemical screening \u003cem\u003eThe table shows qualitative analysis of\u003c/em\u003e P. staudtii \u003cem\u003eleaf ethanol extract shows presence of alkaloids (+++), phlobatannins (+++), anthraquinones (++), and phytate (++), with tannins, saponins, and cardiac glycosides (+) present in lower concentrations. Terpenoids and flavonoids were undetectable (ND) under test conditions.\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConstituent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResult\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConstituent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResult\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlkaloids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhytate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnthraquinones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaponins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCardiac glycosides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSteroids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCyanate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTannins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavonoids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTerpenoids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOxalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhlobatannins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003cstrong\u003eSemiquantitative scoring\u003c/strong\u003e: \u003cem\u003e+ (trace), ++ (moderate), +++ (abundant), - (absent), ND (not detected)\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe qualitative phytochemical screening of P. staudtii ethanolic leaf extract (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) confirms a rich presence of bioactive compounds, with alkaloids and phlobatannins identified in abundant concentrations (+++), followed by moderate levels of anthraquinones and phytate (++). Compounds such as tannins, saponins, cardiac glycosides, cyanate, and oxalate were present in trace amounts (+), while steroids were absent and both flavonoids and terpenoids were undetectable under the applied test conditions. This diverse phytoconstituent profile supports the biochemical effects observed in the experimental plots, particularly the dose-dependent changes in hepatic and renal biomarkers. The presence of potent alkaloids and tannin-like compounds may underlie both the therapeutic and toxicological responses observed, highlighting the extract\u0026rsquo;s complex pharmacodynamic potential.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis bar chart represents the qualitative abundance of various phytoconstituents detected in the ethanolic leaf extract of\u003c/em\u003e P. staudtii \u003cem\u003ethrough standard phytochemical screening. Alkaloids and phlobatannins were found in abundant concentrations (+++), anthraquinones and phytate were present moderately (++), while saponins, tannins, cardiac glycosides, cyanate, and oxalate showed trace levels (+). Flavonoids and terpenoids were not detected (ND), and steroids were completely absent (-). Semi-quantitative abundance was visually coded using a color gradient, providing an immediate comparative overview of the phytochemical complexity and potential bioactive richness of the extract.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe phytochemical composition presented in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e shows the biochemical diversity of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e ethanolic leaf extract and supports its traditional medicinal applications. The plot reveals that alkaloids and phlobatannins were present in the highest abundance (+++), suggesting a strong potential for antimicrobial, anti-inflammatory, and cytotoxic activities, as alkaloids are well-documented for their pharmacological versatility. The prominent presence of phlobatannins further supports possible astringent and antioxidant roles, which may contribute to renal protection or modulation of oxidative stress, especially in the context of the electrolyte and creatinine disturbances previously observed.\u003c/p\u003e\n\u003cp\u003eModerately abundant constituents such as anthraquinones and phytate (++) may offer complementary benefits. Anthraquinones are known for their laxative, anti-inflammatory, and anticancer properties, while phytate is associated with metal ion chelation and antioxidant activity. Their levels suggest meaningful biological contribution without the risk of acute toxicity, aligning with the non-significant urea changes observed in renal function assessments. Meanwhile, the detection of tannins, saponins, cardiac glycosides, cyanate, and oxalate in trace amounts (+) signals a broader spectrum of bioactivity, including potential diuretic, hemolytic, and cardioactive effects, albeit at concentrations unlikely to independently provoke adverse physiological responses.\u003c/p\u003e\n\u003cp\u003eNotably, the absence of steroids (-) and non-detectability of flavonoids and terpenoids (ND) suggests that the extract\u0026rsquo;s therapeutic action does not rely on these classes, which are typically dominant in plant-derived anti-inflammatory or hormone-modulating agents. This absence may partly explain why anti-inflammatory effects or vasomodulatory balance were not pronounced in the electrolyte ratio plot, where Na⁺/K⁺ imbalances remained at higher doses. Taken together, the phytochemical fingerprint visualized in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e provides mechanistic insight into the biological effects observed across other figures, and lays a foundation for bioassay-guided fractionation to isolate the dominant bioactive principles responsible for the extract\u0026rsquo;s renal and electrolyte-modifying activities.\u003c/p\u003e"},{"header":"4.0 Discussion","content":"\u003cp\u003eThe phytochemical landscape of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e ethanolic leaf extract presents a complex blend of bioactive compounds, aligning with its widespread ethnopharmacological applications in African traditional medicine. The presence of alkaloids, flavonoids, phlobatannins, cardiac glycosides, and anthraquinones\u0026mdash;revealed through qualitative screening\u0026mdash;confirms the plant's therapeutic potential, especially in modulating inflammatory and oxidative pathways (Khan et al., 2019; Sowmya \u0026amp; Malakondaiah, 2023). These compounds have been consistently linked to diverse biological activities, including hepatoprotection, cardiovascular regulation, and cytotoxicity against aberrant cells (Adamu et al., 2018; Kumavath et al., 2021).\u003c/p\u003e\u003cp\u003eIn evaluating the hepatorenal safety profile of \u003cem\u003eP. staudtii\u003c/em\u003e, the study observed a significant, dose-dependent increase in serum AST levels, particularly in the high-dose group (400 mg/kg), suggesting mild hepatic stress or cytolytic activity at elevated concentrations. ALT levels followed a similar trend, with the mid-dose group showing the highest spike, which may indicate a threshold for hepatic tolerance (Anosike et al., 2010). These elevations, though not universally severe, align with early signs of hepatocellular perturbation and underscore the need for dose optimization. Interestingly, total bilirubin levels remained statistically unchanged, highlighting that enzymatic elevations may occur in isolation from overt cholestatic dysfunction\u0026mdash;a scenario well-documented in subclinical liver injury models (Uboh, 2010; Awe et al., 2011).\u003c/p\u003e\u003cp\u003eElectrolyte analysis revealed that while serum sodium (Na⁺) levels remained largely unaffected, potassium (K⁺) levels significantly increased in the low-dose group (100 mg/kg), pointing toward a possible ionic imbalance or altered tubular handling. Of greater concern was the marked elevation of serum creatinine in the high-dose group, which suggests that at higher concentrations, \u003cem\u003eP. staudtii\u003c/em\u003e may exert nephrotoxic effects or impair renal clearance mechanisms (Nigatu et al., 2017). The relatively stable urea levels across groups support the hypothesis that glomerular filtration remained largely uncompromised, while the altered creatinine points to potential effects on tubular function or renal hemodynamics.\u003c/p\u003e\u003cp\u003eTogether, these findings build a multidimensional profile of \u003cem\u003eP. staudtii\u003c/em\u003e, showing that while the plant exhibits promising pharmacological activity rooted in its rich phytochemical composition, caution is warranted regarding dose-dependent organ effects. The interaction between liver enzyme induction and renal biomarker shifts suggests that the extract's biocompatibility is tightly linked to concentration and treatment duration. This study thus provides the first integrative hepatorenal safety map of \u003cem\u003eP. staudtii\u003c/em\u003e leaf extract in vivo, supporting its potential as a therapeutic agent while highlighting the critical need for future toxicokinetic and mechanistic studies to optimize its clinical translation.\u003c/p\u003e"},{"header":"5.0 Conclusion","content":"\u003cp\u003eThis study provides the first integrated evaluation of the hepato-renal implications of graded doses of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e ethanolic leaf extract using a controlled animal model. The presence of key phytochemicals—including abundant alkaloids and phlobatannins, as well as moderate levels of anthraquinones, phytate, cardiac glycosides, and trace levels of flavonoids—substantiates the traditional use of \u003cem\u003eP. staudtii\u003c/em\u003e in African ethnomedicine. These compounds are known for their anti-inflammatory, antimicrobial, cardioprotective, and antioxidant properties, and their qualitative presence in the extract supports the pharmacological promise of the plant.\u003c/p\u003e\n\u003cp\u003eHowever, the biochemical outcomes revealed a complex physiological response to the extract. While the control group maintained baseline liver and kidney function markers, administration of 100–400 mg/kg doses for 21 days produced significant, dose-dependent elevations in serum AST, ALT, and creatinine levels, particularly in groups receiving 200 and 400 mg/kg. These elevations are classical indicators of hepatocellular and renal stress, pointing to potential cytotoxic effects at higher doses. Although bilirubin and urea levels did not show statistically significant changes, the combined enzyme and creatinine profiles suggest subtle but biologically relevant disruptions in organ integrity. Furthermore, the electrolyte profile revealed a transient but significant spike in potassium levels in the low-dose group, further indicating renal sensitivity to the extract.\u003c/p\u003e\n\u003cp\u003eGraphical analyses—including multi-panel dose-response plots, error-calibrated charts, and treatment distribution visualizations—strengthen the study’s internal validity by confirming consistent administration protocols, low inter-group variability, and the reproducibility of observed effects. Notably, the mid-dose (200 mg/kg) emerged as a critical inflection point, showing the strongest hepatic and renal biomarker response while avoiding extreme toxicity. This dose may represent a pharmacological threshold where bioactivity peaks before adverse effects escalate.\u003c/p\u003e\n\u003cp\u003eIn sum, \u003cem\u003eP. staudtii\u003c/em\u003e ethanolic leaf extract demonstrates potent, dose-responsive biological activity, yet also reveals organ-specific liabilities at elevated concentrations. The extract’s phytochemical richness aligns with its traditional use, but the elevation of hepatotoxic and nephrotoxic markers warrants caution. These findings underscore the need for further mechanistic studies, chronic toxicity assessments, and dose-optimization trials to clarify its therapeutic index. Until such data are available, the use of \u003cem\u003eP. staudtii\u003c/em\u003e should be approached with scientific restraint, particularly when considering clinical or commercial formulations. Nonetheless, this work lays a strong foundation for future research exploring the dual potentials and limitations of this promising medicinal plant.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003ch3\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest or competing interests in relation to this work.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. Additional data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the ethical standards for animal experimentation. The research protocol involving the use of albino rats was reviewed and approved by the Institutional Animal Ethics Committee (IAEC). All procedures adhered strictly to the guidelines set by the National and International standards for the care and use of laboratory animals to minimize discomfort and ensure humane treatment.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll authors contributed to the study. Conceptualization, data curation, analysis, and manuscript preparation were collaboratively handled.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdelgadir, M. Expected pharmacological influences related to alkaloids extracted from flavedo peel layers of citrus fruits. \u003cem\u003eInt. J. Pharmacol. Toxicol.\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 1\u0026ndash;3 (2023). https://doi.org/10.14419/w5mka031\u003c/li\u003e\n\u003cli\u003eAdamu, A., Paul, E., Gimba, C. \u0026amp; Ndukwe, I. Phytochemical and proximate analysis of \u003cem\u003eAspillia kotschyi\u003c/em\u003e (Sch. Bipex, Hochst) Oliv. \u003cem\u003eNiger. J. Technol.\u003c/em\u003e\u003cstrong\u003e36\u003c/strong\u003e, 1135 (2018). https://doi.org/10.4314/njt.v36i4.19\u003c/li\u003e\n\u003cli\u003eAnosike, C., Ugwu, U. \u0026amp; Nwakanma, O. Effect of ethanol extract of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e leaves on carbon tetrachloride-induced hepatotoxicity in rats. \u003cem\u003eBiokemistri\u003c/em\u003e\u003cstrong\u003e20\u003c/strong\u003e, (2010). https://doi.org/10.4314/biokem.v20i1.56433\u003c/li\u003e\n\u003cli\u003eAwe, E., Kolawole, S., Wakeel, K. \u0026amp; Abiodun, O. Antidiarrheal activity of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e Engl. (Icacinaceae) aqueous leaf extract in rodents. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e\u003cstrong\u003e137\u003c/strong\u003e, 148\u0026ndash;153 (2011). https://doi.org/10.1016/j.jep.2011.04.068\u003c/li\u003e\n\u003cli\u003eKhan, W. \u003cem\u003eet al.\u003c/em\u003e Antioxidant potential, phytochemicals composition, and metal contents of \u003cem\u003eDatura alba\u003c/em\u003e. \u003cem\u003eBiomed Res. Int.\u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e, 1\u0026ndash;8 (2019). https://doi.org/10.1155/2019/2403718\u003c/li\u003e\n\u003cli\u003eKontagora, G. \u003cem\u003eet al.\u003c/em\u003e Preliminary phytochemical screening and assessment of four solvent extracts of button weed (\u003cem\u003eBorreria verticillata\u003c/em\u003e). \u003cem\u003eJ. Appl. Sci. Environ. Manag.\u003c/em\u003e\u003cstrong\u003e24\u003c/strong\u003e, 2085\u0026ndash;2088 (2021). https://doi.org/10.4314/jasem.v24i12.12\u003c/li\u003e\n\u003cli\u003eKumavath, R. \u003cem\u003eet al.\u003c/em\u003e Emergence of cardiac glycosides as potential drugs: Current and future scope for cancer therapeutics. \u003cem\u003eBiomolecules\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 1275 (2021). https://doi.org/10.3390/biom11091275\u003c/li\u003e\n\u003cli\u003eLahare, R., Yadav, H., Bisen, Y. \u0026amp; Dashahre, A. Estimation of total phenol, flavonoid, tannin, and alkaloid content in different extracts of \u003cem\u003eCatharanthus roseus\u003c/em\u003e. \u003cem\u003eScholars Bull.\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 1\u0026ndash;6 (2021). https://doi.org/10.36348/sb.2021.v07i01.001\u003c/li\u003e\n\u003cli\u003eNigatu, T. \u003cem\u003eet al.\u003c/em\u003e Toxicological investigation of acute and chronic treatment with \u003cem\u003eGnidia stenophylla\u003c/em\u003e Gilg root extract. \u003cem\u003eBMC Res. Notes\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, (2017). https://doi.org/10.1186/s13104-017-2964-3\u003c/li\u003e\n\u003cli\u003eSinay, H., El-Sheekh, M., Telussa, I. \u0026amp; Thenu, G. Screening of bioactive compounds of \u003cem\u003eTurbinaria ornata\u003c/em\u003e. \u003cem\u003ePharm. J. Indones.\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 14\u0026ndash;19 (2023). https://doi.org/10.21776/ub.pji.2023.009.01.3\u003c/li\u003e\n\u003cli\u003eSowmya, M. \u0026amp; Malakondaiah, P. Phytochemical and UV spectrum analysis of \u003cem\u003eAzadirachta indica\u003c/em\u003e, \u003cem\u003eCalotropis gigantea\u003c/em\u003e, and \u003cem\u003eRicinus communis\u003c/em\u003e. \u003cem\u003ePharma Innov. J.\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 2501\u0026ndash;2504 (2023). https://doi.org/10.22271/tpi.2023.v12.i6ac.20771\u003c/li\u003e\n\u003cli\u003eSusilo, R. \u003cem\u003eet al.\u003c/em\u003e Hepatoprotective effect of crude polysaccharides from \u003cem\u003eGanoderma lucidum\u003c/em\u003e. \u003cem\u003eVet. World\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 1987\u0026ndash;1991 (2019). https://doi.org/10.14202/vetworld.2019.1987-1991\u003c/li\u003e\n\u003cli\u003eUboh, F. Effect of aqueous extract of \u003cem\u003ePsidium guajava\u003c/em\u003e leaves on liver enzymes and hematological indices in rats. \u003cem\u003eGastroenterol. Res.\u003c/em\u003ehttps://doi.org/10.4021/gr2010.02.174w\u003c/li\u003e\n\u003cli\u003eYounkin, G. \u003cem\u003eet al.\u003c/em\u003e Cardiac glycosides protect \u003cem\u003eErysimum cheiranthoides\u003c/em\u003e against glucosinolate-adapted herbivores. \u003cem\u003eNew Phytol.\u003c/em\u003e\u003cstrong\u003e242\u003c/strong\u003e, 2719\u0026ndash;2733 (2024). https://doi.org/10.1111/nph.19534\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pyrenacantha staudtii, Hepato-renal toxicity, Wistar rats, Phytochemical profiling, Ethanolic plant extract","lastPublishedDoi":"10.21203/rs.3.rs-6999751/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6999751/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe liver and kidneys play essential roles in detoxification, metabolism, and waste elimination, and their dysfunction can arise from infections, metabolic disorders, or exposure to toxic compounds. This study evaluated the hepato-renal effects of ethanolic leaf extract of \u003cem\u003ePyrenacantha staudtii\u003c/em\u003e using an albino Wistar rat model. Twenty male rats were assigned into four groups, with three experimental groups receiving 100, 200, or 400 mg/kg body weight of the extract intraperitoneally for 21 days, while the control group received distilled water. Phytochemical screening revealed the presence of alkaloids, phlobatannins, cardiac glycosides, flavonoids, and anthraquinones. Serum analysis showed a dose-dependent increase in AST and ALT levels, with significant elevations observed in the 400 mg/kg and 200 mg/kg groups, respectively. Creatinine levels increased significantly at the highest dose, while serum urea and bilirubin remained statistically unchanged. Potassium levels peaked significantly at 200 mg/kg, while sodium exhibited a nonsignificant downward trend. No gross pathological abnormalities were observed. These findings suggest that \u003cem\u003eP. staudtii\u003c/em\u003e ethanolic leaf extract has dose-dependent biochemical effects on hepatic and renal function, with higher doses potentially inducing nephrotoxicity. The data provide a basis for further investigation into the therapeutic window, active phytoconstituents, and long-term safety profile of \u003cem\u003eP. staudtii\u003c/em\u003e in drug development or nutraceutical applications.\u003c/p\u003e","manuscriptTitle":"Hepato-Renal Effects of Graded Doses of Pyrenacantha staudtii Ethanolic Leaf Extract in Male Albino Wistar Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 07:58:44","doi":"10.21203/rs.3.rs-6999751/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"999f69cd-18e2-4d7d-99a9-afb2f8e3c9da","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-10T21:21:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 07:58:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6999751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6999751","identity":"rs-6999751","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}