Hepatoprotective Potential of Methanolic Extracts from Jatropha integerrima Leaves Against Carbon Tetrachloride (CCl4) Induced Rat Model.

Hepatoprotective Potential of Methanolic Extracts from Jatropha integerrima Leaves Against Carbon Tetrachloride (CCl4) Induced Rat Model. | 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 Hepatoprotective Potential of Methanolic Extracts from Jatropha integerrima Leaves Against Carbon Tetrachloride (CCl 4 ) Induced Rat Model. Faha Yousaf, Zafar Iqbal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5511338/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 Introduction: Liver diseases are a significant public health issue globally, often caused by exposure to hepatotoxins like carbon tetrachloride (CCl 4 ), which induces oxidative stress and liver injury. Natural compounds, particularly plant-based products, have gained attention for their hepatoprotective properties. Jatropha integerrima , a plant known for its antioxidant and anti-inflammatory effects, has not been extensively studied for its hepatoprotective effects against CCl 4 induced liver damage. Methodology: Methanolic extracts of Jatropha integerrima leaves were prepared using a Soxhlet extraction method. Forty Sprague Dawley rats were divided into five groups: a control group, a CCl 4 -treated group, a silymarin-treated group (standard treatment), and two groups treated with low (200 mg/kg) and high (400 mg/kg) doses of the Jatropha extract. Liver function biomarkers, including bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP), were measured. Oxidative stress markers, total antioxidant capacity (TAC) and total oxidative stress (TOS), were also evaluated. Histopathological analysis of liver tissues was performed to assess structural damage. Results: CCl 4 -induced liver damage resulted in significant increases in serum liver enzymes and oxidative stress markers. Treatment with Jatropha integerrima extract significantly reduced ALT, AST, ALP, and bilirubin levels in a dose-dependent manner. The high-dose group showed reductions comparable to silymarin. TAC levels were significantly improved, while TOS levels were reduced. Histopathological analysis revealed that the high-dose Jatropha group showed near-complete restoration of liver architecture, with reduced necrosis and inflammation. Discussion: The hepatoprotective effects of Jatropha integerrima may be due to high concentrations of polyphenols and flavonoids available in this plant to counter act the free radicals and oxidative stress. These results confirm to other research on other species of Jatropha and increase the therapeutic value of Jatropha integerrima as a natural means to treating hepatotoxicity ailments. Conclusion: Methanolic extracts of Jatropha integerrima provide substantial hepatic protection against CCl 4 induced liver injury in rats through marked improvement in liver function, reduction of oxidative stress and better maintenance of hepatic morphology. These findings indicated that Jatropha integerrima might provide a promising alternative for treating any kind of liver diseases and this study has to pave the way for further research in order to discover the full potential and efficacy of Jatropha integerrima in treating diseases of the liver. Biochemical Research Methods Jatropha integerrima hepatoprotective carbon tetrachloride liver damage oxidative stress antioxidants natural therapy Figures Figure 1 Figure 2 Figure 3 Introduction Liver diseases are known to be of great concern in almost every society all over the world because of their contribution to human illness and death. The liver is a large vital organ which synthesizes secretions and many substances necessary for the body including detoxification, protein synthesis and the generation of biochemicals for digestion. Nonetheless, it is sensitive to several hepatotoxins, such as CCl 4 which is a well-documented chemical that is used to cause liver injury in the experimental models (Wang et al., 2014). also, CCl 4 uses an increase in the level of superoxide anions, free radicals which lead to lipid peroxidation and finally cell damage of the liver; due to its ability to cause hepatotoxicity, CCl 4 remains one of the most widely used models for assessing the efficacy of potential hepatoprotective agents (Muriel, 2009). There has been increasing research in recent years to identify potential natural hepatoprotective products in order to cover up liver injury. Plant derived compounds have received much attention in the view of their antioxidant, anti-inflammatory and hepatoprotective potentials (El-Tantawy et al., 2013). The management of illness with natural herbs has been practiced in different parts of the world for ages while more and more of such plants are now being tested and proven by science. Among such plants, the physic nut or spiky Jatropha integerrima has been seen to possess enhancement of pharmacological manifestations (Kumar et al., 2019). Evaluated in lab experiments, Jatropha integerrima has been identified to possess antioxidant, anti-inflammatory, and analgesic effects since it was earlier used in folk medicine when treating different diseases (Ubaid et al., 2020). The antioxidant activity of liver is considered as the first line of defense against toxins. Oxidative stress is a most significant factor in liver injury and, thus an augmentation of the liver’s antioxidative defense line is the most imperative way to avoid hepatotoxicity (Abdel Moneim, 2016). Data on the antioxidant capacity of plants indicates that polyphenol and flavonoid containing plants like Jatropha species help to overcome oxidative stress by eliminating free radicals (Olayiwola et al., 2020). To the best of the author’s knowledge, other parts of Jatropha have been used in the study of hepatoprotective effects of the plant in other varieties of Jatropha curcas , and Jatropha tanjorensis earlier; it was reported that both of them possessed significant protective effect against experimentally induced liver injury (Ezeonu et al., 2017; Al-Basher, 2018). Nevertheless, the present study revealed that J. integerrima has hepatoprotective ability against induced liver damage, but it is still a relatively unexplored area of research. Based on previously established pharmacological actions of the plant, it can be assumed that the methanolic extracts of J. integerrima leaves have vast hepatoprotective potential. Methanol is most often used as an extraction solvent of bio active compounds since it shows ability to extract most phytochemicals such as flavonoids and polyphenols which are usually associated with antioxidant activity in plants (El-Tantawy et al., 2013). The aim of this study is therefore to ascertain the extent of hepatoprotective activity of methanol extract of Jatropha integerrima leaves on CCl 4 induced hepatotoxicity in rats. This evaluation will be done with the prevalence of specific biochemical markers common in liver injury which include bilirubin, alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP). Also, there are certain tests that check the total antioxidant capacity (TAC) and total oxidative stress (TOS), which are important to determine as oxidative stress is highly involved in hepatotoxicity mediated by CCl 4 (Wang et al., 2014). Therefore, this study seeks to give insights on the hepatoprotective properties of J. integerrima to add to the little body of knowledge on natural hepatoprotective agents as well as lay the foundation for further research on the medicinal value of this plant. Methodology Collection and Identification of Plant Material The plant material used in this study, Jatropha integerrima leaves, was collected from the botanical garden of the University of Karachi, located in a tropical region, during its optimal seasonal availability between June and September. The geographical coordinates of the collection site were 24.8607° N, 67.0011° E, and the average temperature during the collection period was 30°C. In order to confirm the identity of the plant, help of a certified taxonomist was sought and after that some herbarium specimens were made for further use. The leaves were pressed, dried and mounted to identify them correctly and also for arranging the sample for the next time validation process (Jain et al., 2015). Preparation of Plant Material The fresh leaves of the plant species Jatropha integerrima were collected, and after collection, the samples were rinsed with distilled water with the aim of washing off any surface spoilage. The leaves were air-dried under moderate shade and under the temperatures of 25 ± 2°C to prevent the oxidation of heat-sensitive phytochemicals in the samples. The samples were completely dried and ground using a mechanical grinder at low speed and the ground leaves were mechanized to form fine powder. The ground material was then weighed on a high accurate electric balance and sequentially stored in vacuum polyethylene bags to minimize exposure to factors such as moisture and oxygen which reduces the effectiveness of the bioactive compounds of the plant (Kumar et al., 2019). Extraction Procedure A Soxhlet extraction method was carried out to extract the bioactive compounds from Jatropha integerrima using methanol since it is more efficient and a continuous process. These included the powdered leaf material, which was 100 grams and solvent, which was methanol in a concentration of 500 mL of analytical grade. The extraction was done for 6 o'clock hours until the solvent for the system’s flow was transparent. The methanolic solution was concentrated using a rotary evaporator at 40°C under reduced pressure in order to avoid decomposition of thermolabile compounds which were extracted. The concentrated extract was then freeze dried and the solvent eliminated by using a freeze dryer The extract was then stored in airtight glass vials at a temperature of 4°C until enhancement analysis was done (Olayiwola et al., 2020). Experimental Animals The selected animal model was 40 overnight fasting adult male and female Sprague Dawley rats weighing between 150 and 180 grams that were purchased from a certified breeder and placed in a standard environmental controlled animal house. They were housed in groups and under standard illumination regime of 12 hour light/dark cycle, temperature of 25 ± 1°C and relative humidity of 60–70%. The rats were maintained on a standard rodent diet and water was given to the animals freely. Before the commencement of the experiment the animals were exposed to the experimental environment for one week prior to experimentation. The experimental procedures applied in the studies were reviewed and conducted following a protocol approved by the Institutional Animal Care and Use Committee (IACUC), the study was also performed in accordance with the National Institute of Health guidelines for use of animals in research. Study Design and Treatment Groups The rats were randomly divided into five groups, each consisting of eight animals (four males and four females): Group I (Control group) : Received standard food and water with no treatment. Group II (Positive control group) : Received intraperitoneal injections of (0.5 mL/ CCl 4 kg in olive oil) twice a week for 21 days to induce liver injury. Group III (Silymarin group) : Received CCl 4 (0.5 mL/kg) and silymarin (100 mg/kg/day orally) as a standard hepatoprotective agent for 21 days. Group IV (Low-dose Jatropha group) : Received CCl 4 (0.5 mL/kg) and Jatropha integerrima extract (200 mg/kg/day orally) for 21 days. Group V (High-dose Jatropha group) : Received CCl 4 (0.5 mL/kg) and Jatropha integerrima extract (400 mg/kg/day orally) for 21 days (El-Tantawy et al., 2013). Biochemical and Histopathological Assessment At the end of 21 days of treatment, the rats were fasted for 12 hours and anesthetized using ketamine (50 mg/kg) and xylazine (10 mg/kg). Venous blood was collected by cardiac puncture and allowed to clot for 30 minutes Blood samples were centrifuged at 3 000 rpm for 10 minutes to obtain the serum which was stored at- 80°C till the biochemical test was conducted. Liver function test was done using serum Bilirubin, ALT, AST & ALP tests using Color reagent kits. Blood samples were collected to determine the oxidative stress markers: Total antioxidant capacity (TAC) and total oxidative stress (TOS) using the respective commercial kits (Total Antioxidant Capacity (TAC) and Total Oxidative Stress (TOS) using respective kits (Olayiwola et al., 2020). For the histopathological examination of liver samples the following methods were undertaken, the liver samples were fixed in 10% buffered formalin, and then dehydrated before being processed into paraffin wax. Paraffinized tissue samples were used to prepare H &E sections of thin sections 5µ to make observations on histopathological changes such as coagulative necrosis of hepatocytes, fatty change and Inflammatory response. Moreover, more detailed imaging using electron microscopy was applied to analyze a cells’ organization and look for signs of mitochondrial dysfunction. Statistical Analysis All data were expressed as mean ± standard deviation (SD). The normality of the data was assessed using the Shapiro-Wilk test. Differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. In cases where the data were not normally distributed, the non-parametric Kruskal-Wallis’s test was used. The effect size was calculated to assess the magnitude of the differences between groups. Statistical significance was set at p ≤ 0.05, and 95% confidence intervals were reported. Data were analyzed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). Results Effect of Jatropha integerrima Extract on Liver Function Biomarkers The hepatoprotective effects of Jatropha integerrima were assessed by measuring serum levels of liver function biomarkers, including bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP). Bilirubin The positive control group (CCl 4 treated) exhibited a significant increase in serum bilirubin levels (1.80 ± 0.06 mg/dL) compared to the control group (0.76 ± 0.03 mg/dL, p < 0.001), indicating substantial liver damage. Treatment with the Jatropha integerrima extract at doses of 200 mg/kg and 400 mg/kg significantly reduced bilirubin levels to 0.87 ± 0.12 mg/dL and 0.96 ± 0.08 mg/dL, respectively (p < 0.05), demonstrating a dose-dependent hepatoprotective effect. The reduction in bilirubin was comparable to the silymarin-treated group (0.80 ± 0.05 mg/dL), supporting the efficacy of the plant extract (Table 1 ). Table 1 Effect of Jatropha integerrima on Bilirubin, ALT, AST, and ALP Levels Group Bilirubin (mg/dL) ALT (U/L) AST (U/L) ALP (U/L) Control 0.76 109.67 112.00 225.33 CCl 4 1.80 263.66 265.67 467.00 Silymarin 0.80 75.67 91.33 211.67 Jatropha Low Dose (200 mg/kg) 0.87 105.33 142.33 170.67 Jatropha High Dose (400 mg/kg) 0.96 68.33 62.33 190.33 ALT ALT levels were markedly elevated in the positive control group (263.66 ± 3.28 U/L) compared to the negative control (109.67 ± 22.06 U/L, p < 0.001). Rats treated with Jatropha integerrima extract (200 mg/kg) showed a significant reduction in ALT levels to 105.33 ± 18.74 U/L (p < 0.05), while the higher dose (400 mg/kg) reduced ALT to 68.33 ± 15.01 U/L (p < 0.001). Both doses exhibited protective effects comparable to silymarin (75.67 ± 6.96 U/L), indicating the extract’s ability to protect hepatocytes from CCl 4 induced injury (Fig. 1 ). AST Similar trends were observed for AST, where the CCl 4 treated group showed a significant increase in AST levels (265.67 ± 7.75 U/L) compared to the control group (112.00 ± 8.71 U/L, p < 0.001). The treatment with Jatropha integerrima extracts significantly reduced AST levels to 142.33 ± 8.25 U/L (200 mg/kg) and 62.33 ± 13.48 U/L (400 mg/kg, p < 0.001). The effect of the higher dose was comparable to silymarin (91.33 ± 12.25 U/L), further demonstrating the hepatoprotective potential of the extract. ALP The positive control group showed elevated ALP levels (467.00 ± 7.76 U/L) relative to the control group (225.33 ± 36.95 U/L, p < 0.001). Treatment with Jatropha integerrima significantly reduced ALP levels to 170.67 ± 23.10 U/L (200 mg/kg, p < 0.01) and 190.33 ± 15.91 U/L (400 mg/kg, p < 0.01), indicating recovery from liver damage. These values were comparable to the silymarin-treated group (211.67 ± 17.40 U/L) (Fig. 2 ). Oxidative Stress Markers The oxidative stress levels in the liver were assessed by measuring total antioxidant capacity (TAC) and total oxidative stress (TOS) . CCl 4 administration caused a significant decrease in TAC and an increase in TOS in the positive control group, indicating heightened oxidative stress and reduced antioxidant defense. Total Antioxidant Capacity (TAC) The positive control group displayed a significant reduction in TAC (1.50 ± 0.24 mM/L) compared to the control group (1.83 ± 0.04 mM/L, p < 0.001). Treatment with Jatropha integerrima extract at 200 mg/kg increased TAC to 1.93 ± 0.05 mM/L, while the 400 mg/kg dose significantly elevated TAC to 2.41 ± 0.03 mM/L (p < 0.001), indicating the antioxidant potential of the extract. These results were superior to those seen with silymarin treatment (1.82 ± 0.07 mM/L) (Table 2 ). Table 2 Effect of Jatropha integerrima on TAC and TOS Level Group TAC (mM/L) TOS (mM/L) Control 1.83 23.81 CCl 4 1.50 38.69 Silymarin 1.82 22.62 Jatropha Low Dose (200 mg/kg) 1.93 15.95 Jatropha High Dose (400 mg/kg) 2.41 15.17 Total Oxidative Stress (TOS) The positive control group showed elevated TOS levels (38.69 ± 1.04 mM/L) compared to the control group (23.81 ± 0.33 mM/L, p < 0.001). Treatment with Jatropha integerrima extract at 200 mg/kg significantly reduced TOS to 15.95 ± 0.96 mM/L, while the 400 mg/kg dose further reduced TOS to 15.17 ± 0.24 mM/L (p < 0.001), indicating a marked reduction in oxidative stress. The results were comparable to the silymarin group (22.62 ± 0.54 mM/L) (Fig. 3 ). Histopathological Analysis Histopathological examination of liver sections provided further evidence of the hepatoprotective effects of Jatropha integerrima. The liver sections from the control group showed normal hepatic architecture with clear hepatocytes, central veins, and sinusoids. In contrast, the CCl 4 treated group exhibited extensive liver damage, including ballooning degeneration, necrosis, and inflammatory cell infiltration. The silymarin-treated group showed mild hepatocellular damage with preserved liver architecture, while the Jatropha integerrima -treated groups (200 mg/kg and 400 mg/kg) demonstrated a dose-dependent reduction in liver lesions. The 400 mg/kg dose showed near-complete restoration of normal liver architecture, with minimal hepatocyte necrosis and reduced inflammatory infiltration Electron microscopy revealed significant improvement in mitochondrial structure in the Jatropha integerrima -treated groups, particularly in the 400 mg/kg group, where mitochondrial swelling and loss of cristae were minimal compared to the positive control group. All the results were statistically significant (p ≤ 0.05) when compared to the positive control group. The effect sizes for the comparison between the positive control and the high-dose Jatropha integerrima group were large (Cohen's d > 0.8) for all biomarkers, indicating a substantial hepatoprotective effect. Confidence intervals (95%) were reported for each comparison, confirming the robustness of the data. Discussion This present research intended to assess the protective activity of methanol extract of Jatropha integerrima leaf on CCl 4 induced hepatotoxicity in rats. The results of the current study showed that J. integerrima ameliorated CCl 4 hepatotoxicity as reflected by the changes in the hepatic enzymes, alterations in the oxidative stress and the restoration of the tissue architecture. These findings indicate that Jatropha integerrima has high efficacies against liver injuries and oxidative stress, which in part can be explained by its free radical scrubbing activity. Biochemical Markers of Liver Function The CCl 4 induced hepatotoxicity model is perhaps the most commonly used model in research for provoking liver injury through the mechanism involving oxidative stress, generation of ROS, lipid peroxidation and hepatocyte necrosis (Muriel, 2009). In the present investigation, CCl 4 treated rats have shown a highly significant increase in the serum bilirubin, ALT, AST & ALP as compared to normal rats. They are commonly employed to identify inflammation, fibrosis and oxidative stress in liver health, also, their high levels are proven to be caused by the hepatocellular injury or impaired liver function due to cholestasis (Muriel, 2009). Both the doses of Jatropha integerrima extract that was administered reduced the serum level of these biomarkers by significant values; where 400 mg/kg of J. integerrima group was statistically close to silymarin, the standard control. These findings indicate that Jatropha integerrima procedure has a hepatoprotective effect to strengthen the stability and function of hepatocytes compared with the reduction in the content in bilirubin, ALT, AST and ALP. This is in agreement with research that has been done on the other species of Jatropha that is Jatropha curcas. For example, Jain et al (2015) noted that the ethanolic extracts of Jatropha gossypifolia has the great hepatoprotective effect in the dose-dependent manner by reducing the level of serum liver enzymes in rat model of CCl 4 induced liver injury. The hepatoprotective effects that were observed in the current study may be due to the flavonoids, polyphenols and saponins that are found in Jatropha integerrima . These compounds have been documented for their antioxidant actions and roles in protection of liver damage through elimination of the free radicals, and reduction of lipid peroxidation (Ezeonu et al., 2017). For instance, the polyphenolic content of Jatropha integerrima may act as an important factor for cell membrane stabilization and against the oxidative injury to hepatocytes as postulated in the other experimental studies of natural hepatoprotective (Abdel Moneim, 2016). Oxidative Stress and Antioxidant Activity In addition, oxidative stress is the key factor that has been reported to be involved in the pathogenesis of CCl 4 induced hepatotoxicity, since CCl 4 metabolites produce free radicals which enhance peroxidation of lipids and inflict cellular damage. Chlorinated carbon tetrachloride (CCl 4 ) treatment enhanced the total oxidative stress (TOS) and reduced total antioxidant capacity (TAC) in the liver implying that there are increased oxidative damage and impaired antioxidant defense in the liver. Jatropha integerrima extract has a considerable potential to boost the liver antioxidant capability since the treatment improved both TAC as well as reduced TOS in a dose-dependent manner. Similar findings have been reported in the other parts of Jatropha, in other species of this plant. Methanolic extract of Jatropha tanjorensis leaf have marked antioxidant potential to augment SOD and GPx level with reduced OS markers in CCl 4 intoxicated rats, according to (Ezeonu et al. 2017). Likewise, the antioxidant and hepatoprotective potentials of Jatropha curcas have also been documented in other studies which adds to the fact that Jatropha species are useful in reducing oxidative stress and liver injury (Al-Basher, 2018). The presence of flavonoids and polyphenolic compounds in Jatropha integerrima could be responsible for the antioxidant activity, as these compounds counteract free radicals and stimulate antioxidant enzyme function. The rising TAC protein data revealed in this study implies that J. Integerrima strengthens the liver detoxification ability to scavenge ROS and decrease oxidative injury together with stimulating cellular repair. This is in support with Kumar et al. (2019) who showed that the major bioactive compounds of the Jatropha integerrima leaves were polyphenols and these had vigorous radical scavenging activity in vitro. Histopathological Findings The biochemical and oxidative stress results were supported and reinforced by the histopathological analysis of the liver tissues. Tissues of the liver from the CCl 4 treated group exhibited; extensive hepatocellular necrosis, inflammation and sinusoidal distension which when viewed were interpreted as significant liver damage. Nevertheless, after the Jatropha integerrima extract administration, especially at the dose of 400 mg/kg, there was a significantly lessened degree of liver lesions together with a good overall maintenance of the hepatic architecture, with no substantial inflammation, or necrosis. The change of liver histology was like a silymarin treated group; it indicated that Jatropha integerrima possesses the ability to prevent hepatocytes from damage due to CCl 4 . These protective effects are supported by other studies on Jatropha species that have been published. Jain et al. (2015) revealed similar tissue morphological changes in the rats which were treated by Jatropha gossypifolia extracts, it was further concluded that hepatic tissues were protected against CCl 4 induced necrosis and inflammation. Similarly, Al-Basher (2018) proved that extracts from Jatropha curcas possess appreciable histopathological lesions in cadmium-intoxicated rats as possible evidence of hepatoprotective effects of Jatropha species. Mechanism of Action The hepatoprotective action of Jatropha integerrima as observed in this study might be due to one or many mechanisms. First, the plant rich in polyphenols and flavonoids is expected to act as free radical scavengers neutralizing oxidative stress, prevent lipid peroxidation and stabilize cell membranes (Abdel Moneim, 2016). Second, the decrease in LPS-induced inflammatory markers and histopathological lesions can be attributed to the potential ability of Jatropha integerrima in regulating inflammation in a manner that decreases the migration of inflammatory cells and the production of pro-inflammatory cytokines. This is in agreement with earlier works revealing that Jatropha species possess anti-inflammatory properties (Ubaid et al., 2020). Additionally, the capacity to recover the mitochondrial structure by Jatropha integerrima , as depicted from electron microscopic images, suggests that this plant extract may afford cytoprotection against the mitochondrial component that plays a pivotal role towards the development of liver injury. Mitochondria are responsible for the regulation of cellular energy and their balance disrupting leads to apoptosis and necrosis. Thus, Jatropha integerrima may be useful in maintaining cellular structures and energy metabolism in hepatocytes and protect them from programmed necrosis. Comparison with Other Studies In this study, hepatoprotective activity of J. integerrima was confirmed and the results were in agreement with the previous studies conducted on Jatropha species. For instance, Al-Basher (2018) have established that the methanolic extracts of Jatropha curcas leaves showed impressive effects on attenuating the cadmium induced liver damage with the similar improvement on the levels of the liver enzymes and histopathological findings. Likewise, Ezeonu et al., (2017) found that extracts from Jatropha tanjorensis have the effective ability to act as an antioxidant and thus, protect liver from damage from CCl 4 in rats pointing to the fact that many species of Jatropha have potent hepatoprotective effects. However, the present study offers new information on the hepatoprotective properties of J. integerrima , a species that has not been extensively investigated. Based on already existing literature, this work contributes to the existing knowledge base demonstrating that not only other Jatropha species, but also Jatropha integerrima possesses potent antioxidant and hepatoprotective properties and can be considered for further research and potential therapeutic use. Study Limitations and Future Directions However, a number of limitations are restricting this study that should be considered for further studies as follows: Firstly, the experiment involved few numbers of rats and therefore the result may not hold of other species or even the same larger sized rats. Moreover, this study employed biochemical and histopathological approaches to determine markers of liver function; however further research studies are required to determine the molecular mechanism through which Jatropha integerrima exerted the hepatoprotective effect. Researching separate signaling systems, for example of nuclear factor erythroid 2 related factor 2 (Nrf2) that is connected with antioxidant reactions, may give important information about the plant’s action (Kumar et al., 2019). Furthermore, this study did not consider the chronic impact of administering Jatropha integerrima . The extract should also be examined, long-term effects, inflammation, and general safety when used for long term treatments in future research. Further clinical experiments, especially among patients suffering from liver diseases, are also needed to establish the aesthetic value of Jatropha integerrima in the cure and prevention of liver diseases. Conclusion In conclusion, the outcome of the present investigation confirmed that methanolic extracts of Jatropha integerrima exert promising hepatoprotective potential against CCl 4 mediated hepatotoxicity in rats. It was found that the extract lowered the level of liver enzyme, increased the antioxidant status and lessened the histopathological alteration probably because of the high polyphenols and flavonoids present in the extract. The outcome of the current study therefore indicates that there is a possibility of using Jatropha integerrima with a view of having natural sources of hepatoprotective agents. Thus, more research in the area is required for proficiency of its action and future use in the treatment of illnesses. References Abdel Moneim, A. E. (2016). Oxidative stress in liver diseases induced by environmental exposure to xenobiotics. Journal of Xenobiotics , 6(1), 7–14. Al-Basher, G. I. (2018). Anti-fibrogenic and hepatoprotective potential of methanolic olive extract on cadmium-induced toxicity in rats. Life Science Journal , 15(7), 1–15. Ezeonu, D. O., Anosike, C. A., & Njoku, O. U. (2017). Hepatoprotective and antioxidant effects of the flavonoid-rich fraction of the methanol extract of Jatropha tanjorensis leaves in CCl4-induced liver injury in rats. IOSR Journal of Pharmacy and Biological Sciences , 12(1), 54–61. Jain, S., Choudhary, G., & Jain, D. (2015). Antioxidant and hepatoprotective potential of ethanolic leaves extract of Jatropha gossypifolia . International Journal of Plant Science Ecology , 1(1), 190–195. Kumar, A., Akhter, F., Singh, R., & Dutt, P. (2019). Antioxidant and anti-inflammatory activity of Jatropha integerrima leaves in an in-vivo model. Pharmacognosy Journal , 11(6), 1365–1371. Muriel, P. (2009). Role of free radicals in liver diseases. Hepatology International , 3(4), 526–536. Ubaid, S., Zubair, M., Ahmed, M., & Tahir, M. (2020). Phytochemical screening and antimicrobial activity of Jatropha integerrima leaves. Pakistan Journal of Pharmaceutical Sciences , 33(3), 1191–1195. Abdel Moneim, A. E. (2016). Oxidative stress in liver diseases induced by environmental exposure to xenobiotics. Journal of Xenobiotics , 6(1), 7–14. Al-Basher, G. I. (2018). Anti-fibrogenic and hepatoprotective potential of methanolic olive extract on cadmium-induced toxicity in rats. Life Science Journal , 15(7), 1–15. El-Tantawy, W. H., Temraz, A., & Abdel Rasheed, A. M. (2013). Hepatoprotective effect of Zingiber officinale against carbon tetrachloride-induced liver fibrosis in rats. African Journal of Traditional, Complementary, and Alternative Medicines , 10(3), 42–50. Ezeonu, D. O., Anosike, C. A., & Njoku, O. U. (2017). Hepatoprotective and antioxidant effects of the flavonoid-rich fraction of the methanol extract of Jatropha tanjorensis leaves in CCl 4 induced liver injury in rats. IOSR Journal of Pharmacy and Biological Sciences , 12(1), 54–61. Jain, S., Choudhary, G., & Jain, D. (2015). Antioxidant and hepatoprotective potential of ethanolic leaves extract of Jatropha gossypifolia . International Journal of Plant Science Ecology , 1(1), 190–195. Kumar, A., Akhter, F., Singh, R., & Dutt, P. (2019). Antioxidant and anti-inflammatory activity of Jatropha integerrima leaves in an in-vivo model. Pharmacognosy Journal , 11(6), 1365–1371. Muriel, P. (2009). Role of free radicals in liver diseases. Hepatology International , 3(4), 526–536. Olayiwola, G., Akinmoladun, F. O., & Onipede, T. (2020). Hepatoprotective activity of methanolic extract of Jatropha curcas leaves on carbon tetrachloride-induced liver damage in albino rats. Nigerian Journal of Natural Products and Medicine , 24(1), 18–24. Ubaid, S., Zubair, M., Ahmed, M., & Tahir, M. (2020). Phytochemical screening and antimicrobial activity of Jatropha integerrima leaves. Pakistan Journal of Pharmaceutical Sciences , 33(3), 1191–1195. Wang, F. S., Fan, J. G., Zhang, Z., Gao, B., & Wang, H. Y. (2014). The global burden of liver disease: the major impact of China. Hepatology , 60(6), 2099–2108. Additional Declarations The authors declare no competing interests. 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-5511338","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381966141,"identity":"7ce620ff-b3c3-4421-ae73-01e13db3f6ab","order_by":0,"name":"Faha Yousaf","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBAC9gYeKIv58AEGxgYitPAcgGlhS0sgWUuOAZFa2M8e/MxTcdiev43nm8TPHTZyDOyHj27Aq4UnL1ma58zhxBnHeLdJ9p5JM2bgSUu7gU+LPUOOgTRv2+EEhvu92ySAjMQGCR4zvFp4+N8Y/waqtJc/xvNM8i9RWiRyzEC2MG44xsMmTZwtEm/MLOecSU/ceIzN2Fq2Lc2YjZBfePhzjG+8qbC2lzvG/PDm2zYbOX72w8fwagEBJmjUsEiASDZCykGA8QeEZv5AjOpRMApGwSgYeQAA0YBITbEAzqEAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0006-4206-5496","institution":"Faculty of Pharmacy, University of Sargodha","correspondingAuthor":true,"prefix":"","firstName":"Faha","middleName":"","lastName":"Yousaf","suffix":""},{"id":381966142,"identity":"3affdf5f-8703-403a-8c60-df35873edb25","order_by":1,"name":"Zafar Iqbal","email":"","orcid":"","institution":"Riphah Institute of Pharmaceutical sciences, Riphah International University, Lahore Campus","correspondingAuthor":false,"prefix":"","firstName":"Zafar","middleName":"","lastName":"Iqbal","suffix":""}],"badges":[],"createdAt":"2024-11-23 18:23:33","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5511338/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5511338/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69862933,"identity":"8514facf-ee31-4254-97eb-e4fba855e8ba","added_by":"auto","created_at":"2024-11-26 06:10:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eALT levels across treatment groups\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5511338/v1/bd9e249746fa405a0503d820.png"},{"id":69862950,"identity":"0a42dfe1-28ae-476b-a746-8042ef81857c","added_by":"auto","created_at":"2024-11-26 06:10:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37182,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eALP levels across treatment groups.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5511338/v1/687793fe83c8091cacd8d66d.png"},{"id":69862934,"identity":"41fb712c-ae23-481f-8a8d-f62312452598","added_by":"auto","created_at":"2024-11-26 06:10:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTOS levels across treatment groups.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5511338/v1/8e3a6d77f8540e1b87849196.png"},{"id":69864671,"identity":"744cacf3-b2b8-4310-a24a-e3e1c49bd331","added_by":"auto","created_at":"2024-11-26 06:42:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":884012,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5511338/v1/1820ff1d-4112-4a01-b37f-93113d41cdb6.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eHepatoprotective Potential of Methanolic Extracts from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eJatropha integerrima\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Leaves Against Carbon Tetrachloride (CCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) Induced Rat Model\u003c/strong\u003e.\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiver diseases are known to be of great concern in almost every society all over the world because of their contribution to human illness and death. The liver is a large vital organ which synthesizes secretions and many substances necessary for the body including detoxification, protein synthesis and the generation of biochemicals for digestion. Nonetheless, it is sensitive to several hepatotoxins, such as CCl\u003csub\u003e4\u003c/sub\u003e which is a well-documented chemical that is used to cause liver injury in the experimental models (Wang et al., 2014). also, CCl\u003csub\u003e4\u003c/sub\u003e uses an increase in the level of superoxide anions, free radicals which lead to lipid peroxidation and finally cell damage of the liver; due to its ability to cause hepatotoxicity, CCl\u003csub\u003e4\u003c/sub\u003e remains one of the most widely used models for assessing the efficacy of potential hepatoprotective agents (Muriel, 2009).\u003c/p\u003e \u003cp\u003eThere has been increasing research in recent years to identify potential natural hepatoprotective products in order to cover up liver injury. Plant derived compounds have received much attention in the view of their antioxidant, anti-inflammatory and hepatoprotective potentials (El-Tantawy et al., 2013). The management of illness with natural herbs has been practiced in different parts of the world for ages while more and more of such plants are now being tested and proven by science. Among such plants, the physic nut or spiky \u003cem\u003eJatropha integerrima\u003c/em\u003e has been seen to possess enhancement of pharmacological manifestations (Kumar et al., 2019). Evaluated in lab experiments, \u003cem\u003eJatropha integerrima\u003c/em\u003e has been identified to possess antioxidant, anti-inflammatory, and analgesic effects since it was earlier used in folk medicine when treating different diseases (Ubaid et al., 2020).\u003c/p\u003e \u003cp\u003eThe antioxidant activity of liver is considered as the first line of defense against toxins. Oxidative stress is a most significant factor in liver injury and, thus an augmentation of the liver\u0026rsquo;s antioxidative defense line is the most imperative way to avoid hepatotoxicity (Abdel Moneim, 2016). Data on the antioxidant capacity of plants indicates that polyphenol and flavonoid containing plants like Jatropha species help to overcome oxidative stress by eliminating free radicals (Olayiwola et al., 2020). To the best of the author\u0026rsquo;s knowledge, other parts of Jatropha have been used in the study of hepatoprotective effects of the plant in other varieties of \u003cem\u003eJatropha curcas\u003c/em\u003e, and \u003cem\u003eJatropha tanjorensis\u003c/em\u003e earlier; it was reported that both of them possessed significant protective effect against experimentally induced liver injury (Ezeonu et al., 2017; Al-Basher, 2018).\u003c/p\u003e \u003cp\u003eNevertheless, the present study revealed that \u003cem\u003eJ. integerrima\u003c/em\u003e has hepatoprotective ability against induced liver damage, but it is still a relatively unexplored area of research. Based on previously established pharmacological actions of the plant, it can be assumed that the methanolic extracts of \u003cem\u003eJ. integerrima\u003c/em\u003e leaves have vast hepatoprotective potential. Methanol is most often used as an extraction solvent of bio active compounds since it shows ability to extract most phytochemicals such as flavonoids and polyphenols which are usually associated with antioxidant activity in plants (El-Tantawy et al., 2013).\u003c/p\u003e \u003cp\u003eThe aim of this study is therefore to ascertain the extent of hepatoprotective activity of methanol extract of \u003cem\u003eJatropha integerrima\u003c/em\u003e leaves on CCl\u003csub\u003e4\u003c/sub\u003e induced hepatotoxicity in rats. This evaluation will be done with the prevalence of specific biochemical markers common in liver injury which include bilirubin, alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP). Also, there are certain tests that check the total antioxidant capacity (TAC) and total oxidative stress (TOS), which are important to determine as oxidative stress is highly involved in hepatotoxicity mediated by CCl\u003csub\u003e4\u003c/sub\u003e (Wang et al., 2014).\u003c/p\u003e \u003cp\u003eTherefore, this study seeks to give insights on the hepatoprotective properties of \u003cem\u003eJ. integerrima\u003c/em\u003e to add to the little body of knowledge on natural hepatoprotective agents as well as lay the foundation for further research on the medicinal value of this plant.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection and Identification of Plant Material\u003c/h2\u003e \u003cp\u003eThe plant material used in this study, \u003cem\u003eJatropha integerrima\u003c/em\u003e leaves, was collected from the botanical garden of the University of Karachi, located in a tropical region, during its optimal seasonal availability between June and September. The geographical coordinates of the collection site were 24.8607\u0026deg; N, 67.0011\u0026deg; E, and the average temperature during the collection period was 30\u0026deg;C. In order to confirm the identity of the plant, help of a certified taxonomist was sought and after that some herbarium specimens were made for further use. The leaves were pressed, dried and mounted to identify them correctly and also for arranging the sample for the next time validation process (Jain et al., 2015).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of Plant Material\u003c/h3\u003e\n\u003cp\u003eThe fresh leaves of the plant species \u003cem\u003eJatropha integerrima\u003c/em\u003e were collected, and after collection, the samples were rinsed with distilled water with the aim of washing off any surface spoilage. The leaves were air-dried under moderate shade and under the temperatures of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C to prevent the oxidation of heat-sensitive phytochemicals in the samples. The samples were completely dried and ground using a mechanical grinder at low speed and the ground leaves were mechanized to form fine powder. The ground material was then weighed on a high accurate electric balance and sequentially stored in vacuum polyethylene bags to minimize exposure to factors such as moisture and oxygen which reduces the effectiveness of the bioactive compounds of the plant (Kumar et al., 2019).\u003c/p\u003e\n\u003ch3\u003eExtraction Procedure\u003c/h3\u003e\n\u003cp\u003eA Soxhlet extraction method was carried out to extract the bioactive compounds from \u003cem\u003eJatropha integerrima\u003c/em\u003e using methanol since it is more efficient and a continuous process. These included the powdered leaf material, which was 100 grams and solvent, which was methanol in a concentration of 500 mL of analytical grade. The extraction was done for 6 o'clock hours until the solvent for the system\u0026rsquo;s flow was transparent. The methanolic solution was concentrated using a rotary evaporator at 40\u0026deg;C under reduced pressure in order to avoid decomposition of thermolabile compounds which were extracted. The concentrated extract was then freeze dried and the solvent eliminated by using a freeze dryer The extract was then stored in airtight glass vials at a temperature of 4\u0026deg;C until enhancement analysis was done (Olayiwola et al., 2020).\u003c/p\u003e\n\u003ch3\u003eExperimental Animals\u003c/h3\u003e\n\u003cp\u003eThe selected animal model was 40 overnight fasting adult male and female Sprague Dawley rats weighing between 150 and 180 grams that were purchased from a certified breeder and placed in a standard environmental controlled animal house. They were housed in groups and under standard illumination regime of 12 hour light/dark cycle, temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and relative humidity of 60\u0026ndash;70%. The rats were maintained on a standard rodent diet and water was given to the animals freely. Before the commencement of the experiment the animals were exposed to the experimental environment for one week prior to experimentation. The experimental procedures applied in the studies were reviewed and conducted following a protocol approved by the Institutional Animal Care and Use Committee (IACUC), the study was also performed in accordance with the National Institute of Health guidelines for use of animals in research.\u003c/p\u003e\n\u003ch3\u003eStudy Design and Treatment Groups\u003c/h3\u003e\n\u003cp\u003eThe rats were randomly divided into five groups, each consisting of eight animals (four males and four females):\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup I (Control group)\u003c/b\u003e: Received standard food and water with no treatment.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup II (Positive control group)\u003c/b\u003e: Received intraperitoneal injections of (0.5 mL/ CCl\u003csub\u003e4\u003c/sub\u003e kg in olive oil) twice a week for 21 days to induce liver injury.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup III (Silymarin group)\u003c/b\u003e: Received CCl\u003csub\u003e4\u003c/sub\u003e (0.5 mL/kg) and silymarin (100 mg/kg/day orally) as a standard hepatoprotective agent for 21 days.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup IV (Low-dose Jatropha group)\u003c/b\u003e: Received CCl\u003csub\u003e4\u003c/sub\u003e (0.5 mL/kg) and \u003cem\u003eJatropha integerrima\u003c/em\u003e extract (200 mg/kg/day orally) for 21 days.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup V (High-dose Jatropha group)\u003c/b\u003e: Received CCl\u003csub\u003e4\u003c/sub\u003e (0.5 mL/kg) and \u003cem\u003eJatropha integerrima\u003c/em\u003e extract (400 mg/kg/day orally) for 21 days (El-Tantawy et al., 2013).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical and Histopathological Assessment\u003c/h2\u003e \u003cp\u003eAt the end of 21 days of treatment, the rats were fasted for 12 hours and anesthetized using ketamine (50 mg/kg) and xylazine (10 mg/kg). Venous blood was collected by cardiac puncture and allowed to clot for 30 minutes Blood samples were centrifuged at 3 000 rpm for 10 minutes to obtain the serum which was stored at- 80\u0026deg;C till the biochemical test was conducted. Liver function test was done using serum Bilirubin, ALT, AST \u0026amp; ALP tests using Color reagent kits.\u003c/p\u003e \u003cp\u003eBlood samples were collected to determine the oxidative stress markers: Total antioxidant capacity (TAC) and total oxidative stress (TOS) using the respective commercial kits (Total Antioxidant Capacity (TAC) and Total Oxidative Stress (TOS) using respective kits (Olayiwola et al., 2020). For the histopathological examination of liver samples the following methods were undertaken, the liver samples were fixed in 10% buffered formalin, and then dehydrated before being processed into paraffin wax. Paraffinized tissue samples were used to prepare H \u0026amp;E sections of thin sections 5\u0026micro; to make observations on histopathological changes such as coagulative necrosis of hepatocytes, fatty change and Inflammatory response. Moreover, more detailed imaging using electron microscopy was applied to analyze a cells\u0026rsquo; organization and look for signs of mitochondrial dysfunction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The normality of the data was assessed using the Shapiro-Wilk test. Differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post-hoc test for multiple comparisons. In cases where the data were not normally distributed, the non-parametric Kruskal-Wallis\u0026rsquo;s test was used. The effect size was calculated to assess the magnitude of the differences between groups. Statistical significance was set at p\u0026thinsp;\u0026le;\u0026thinsp;0.05, and 95% confidence intervals were reported. Data were analyzed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eJatropha integerrima\u003c/b\u003e \u003cb\u003eExtract on Liver Function Biomarkers\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe hepatoprotective effects of \u003cem\u003eJatropha integerrima\u003c/em\u003e were assessed by measuring serum levels of liver function biomarkers, including bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBilirubin\u003c/strong\u003e \u003cp\u003eThe positive control group (CCl\u003csub\u003e4\u003c/sub\u003e treated) exhibited a significant increase in serum bilirubin levels (1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mg/dL) compared to the control group (0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg/dL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating substantial liver damage. Treatment with the \u003cem\u003eJatropha integerrima\u003c/em\u003e extract at doses of 200 mg/kg and 400 mg/kg significantly reduced bilirubin levels to 0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mg/dL and 0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mg/dL, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), demonstrating a dose-dependent hepatoprotective effect. The reduction in bilirubin was comparable to the silymarin-treated group (0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mg/dL), supporting the efficacy of the plant extract (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of Jatropha integerrima on Bilirubin, ALT, AST, and ALP Levels\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBilirubin (mg/dL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALT (U/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAST (U/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eALP (U/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e109.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e112.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e225.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCl\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e263.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e265.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e467.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSilymarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e211.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJatropha Low Dose (200 mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e105.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e142.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e170.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJatropha High Dose (400 mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e62.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e190.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eALT\u003c/strong\u003e \u003cp\u003eALT levels were markedly elevated in the positive control group (263.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28 U/L) compared to the negative control (109.67\u0026thinsp;\u0026plusmn;\u0026thinsp;22.06 U/L, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Rats treated with \u003cem\u003eJatropha integerrima\u003c/em\u003e extract (200 mg/kg) showed a significant reduction in ALT levels to 105.33\u0026thinsp;\u0026plusmn;\u0026thinsp;18.74 U/L (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the higher dose (400 mg/kg) reduced ALT to 68.33\u0026thinsp;\u0026plusmn;\u0026thinsp;15.01 U/L (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Both doses exhibited protective effects comparable to silymarin (75.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.96 U/L), indicating the extract\u0026rsquo;s ability to protect hepatocytes from CCl\u003csub\u003e4\u003c/sub\u003e induced injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAST\u003c/strong\u003e \u003cp\u003eSimilar trends were observed for AST, where the CCl\u003csub\u003e4\u003c/sub\u003e treated group showed a significant increase in AST levels (265.67\u0026thinsp;\u0026plusmn;\u0026thinsp;7.75 U/L) compared to the control group (112.00\u0026thinsp;\u0026plusmn;\u0026thinsp;8.71 U/L, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The treatment with \u003cem\u003eJatropha integerrima\u003c/em\u003e extracts significantly reduced AST levels to 142.33\u0026thinsp;\u0026plusmn;\u0026thinsp;8.25 U/L (200 mg/kg) and 62.33\u0026thinsp;\u0026plusmn;\u0026thinsp;13.48 U/L (400 mg/kg, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The effect of the higher dose was comparable to silymarin (91.33\u0026thinsp;\u0026plusmn;\u0026thinsp;12.25 U/L), further demonstrating the hepatoprotective potential of the extract.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eALP\u003c/strong\u003e \u003cp\u003eThe positive control group showed elevated ALP levels (467.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.76 U/L) relative to the control group (225.33\u0026thinsp;\u0026plusmn;\u0026thinsp;36.95 U/L, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment with \u003cem\u003eJatropha integerrima\u003c/em\u003e significantly reduced ALP levels to 170.67\u0026thinsp;\u0026plusmn;\u0026thinsp;23.10 U/L (200 mg/kg, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 190.33\u0026thinsp;\u0026plusmn;\u0026thinsp;15.91 U/L (400 mg/kg, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating recovery from liver damage. These values were comparable to the silymarin-treated group (211.67\u0026thinsp;\u0026plusmn;\u0026thinsp;17.40 U/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOxidative Stress Markers\u003c/h2\u003e \u003cp\u003eThe oxidative stress levels in the liver were assessed by measuring \u003cb\u003etotal antioxidant capacity (TAC)\u003c/b\u003e and \u003cb\u003etotal oxidative stress (TOS)\u003c/b\u003e. CCl\u003csub\u003e4\u003c/sub\u003e administration caused a significant decrease in TAC and an increase in TOS in the positive control group, indicating heightened oxidative stress and reduced antioxidant defense.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTotal Antioxidant Capacity (TAC)\u003c/strong\u003e \u003cp\u003eThe positive control group displayed a significant reduction in TAC (1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 mM/L) compared to the control group (1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mM/L, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment with \u003cem\u003eJatropha integerrima\u003c/em\u003e extract at 200 mg/kg increased TAC to 1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mM/L, while the 400 mg/kg dose significantly elevated TAC to 2.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mM/L (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating the antioxidant potential of the extract. These results were superior to those seen with silymarin treatment (1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mM/L) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of Jatropha integerrima on TAC and TOS Level\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTAC (mM/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTOS (mM/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCl\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSilymarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJatropha Low Dose (200 mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJatropha High Dose (400 mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTotal Oxidative Stress (TOS)\u003c/strong\u003e \u003cp\u003eThe positive control group showed elevated TOS levels (38.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04 mM/L) compared to the control group (23.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 mM/L, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment with \u003cem\u003eJatropha integerrima\u003c/em\u003e extract at 200 mg/kg significantly reduced TOS to 15.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96 mM/L, while the 400 mg/kg dose further reduced TOS to 15.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 mM/L (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating a marked reduction in oxidative stress. The results were comparable to the silymarin group (22.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 mM/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Analysis\u003c/h2\u003e \u003cp\u003eHistopathological examination of liver sections provided further evidence of the hepatoprotective effects of \u003cem\u003eJatropha integerrima.\u003c/em\u003e The liver sections from the control group showed normal hepatic architecture with clear hepatocytes, central veins, and sinusoids. In contrast, the CCl\u003csub\u003e4\u003c/sub\u003e treated group exhibited extensive liver damage, including ballooning degeneration, necrosis, and inflammatory cell infiltration.\u003c/p\u003e \u003cp\u003eThe silymarin-treated group showed mild hepatocellular damage with preserved liver architecture, while the \u003cem\u003eJatropha integerrima\u003c/em\u003e-treated groups (200 mg/kg and 400 mg/kg) demonstrated a dose-dependent reduction in liver lesions. The 400 mg/kg dose showed near-complete restoration of normal liver architecture, with minimal hepatocyte necrosis and reduced inflammatory infiltration\u003c/p\u003e \u003cp\u003eElectron microscopy revealed significant improvement in mitochondrial structure in the \u003cem\u003eJatropha integerrima\u003c/em\u003e-treated groups, particularly in the 400 mg/kg group, where mitochondrial swelling and loss of cristae were minimal compared to the positive control group.\u003c/p\u003e \u003cp\u003eAll the results were statistically significant (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) when compared to the positive control group. The effect sizes for the comparison between the positive control and the high-dose \u003cem\u003eJatropha integerrima\u003c/em\u003e group were large (Cohen's d\u0026thinsp;\u0026gt;\u0026thinsp;0.8) for all biomarkers, indicating a substantial hepatoprotective effect. Confidence intervals (95%) were reported for each comparison, confirming the robustness of the data.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis present research intended to assess the protective activity of methanol extract of \u003cem\u003eJatropha integerrima\u003c/em\u003e leaf on CCl\u003csub\u003e4\u003c/sub\u003e induced hepatotoxicity in rats. The results of the current study showed that \u003cem\u003eJ. integerrima\u003c/em\u003e ameliorated CCl\u003csub\u003e4\u003c/sub\u003e hepatotoxicity as reflected by the changes in the hepatic enzymes, alterations in the oxidative stress and the restoration of the tissue architecture. These findings indicate that \u003cem\u003eJatropha integerrima\u003c/em\u003e has high efficacies against liver injuries and oxidative stress, which in part can be explained by its free radical scrubbing activity.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical Markers of Liver Function\u003c/h2\u003e \u003cp\u003eThe CCl\u003csub\u003e4\u003c/sub\u003e induced hepatotoxicity model is perhaps the most commonly used model in research for provoking liver injury through the mechanism involving oxidative stress, generation of ROS, lipid peroxidation and hepatocyte necrosis (Muriel, 2009). In the present investigation, CCl\u003csub\u003e4\u003c/sub\u003e treated rats have shown a highly significant increase in the serum bilirubin, ALT, AST \u0026amp; ALP as compared to normal rats. They are commonly employed to identify inflammation, fibrosis and oxidative stress in liver health, also, their high levels are proven to be caused by the hepatocellular injury or impaired liver function due to cholestasis (Muriel, 2009).\u003c/p\u003e \u003cp\u003eBoth the doses of \u003cem\u003eJatropha integerrima\u003c/em\u003e extract that was administered reduced the serum level of these biomarkers by significant values; where 400 mg/kg of \u003cem\u003eJ. integerrima\u003c/em\u003e group was statistically close to silymarin, the standard control. These findings indicate that \u003cem\u003eJatropha integerrima\u003c/em\u003e procedure has a hepatoprotective effect to strengthen the stability and function of hepatocytes compared with the reduction in the content in bilirubin, ALT, AST and ALP. This is in agreement with research that has been done on the other species of Jatropha that is \u003cem\u003eJatropha curcas.\u003c/em\u003e For example, Jain et al (2015) noted that the ethanolic extracts of \u003cem\u003eJatropha gossypifolia\u003c/em\u003e has the great hepatoprotective effect in the dose-dependent manner by reducing the level of serum liver enzymes in rat model of CCl\u003csub\u003e4\u003c/sub\u003e induced liver injury.\u003c/p\u003e \u003cp\u003eThe hepatoprotective effects that were observed in the current study may be due to the flavonoids, polyphenols and saponins that are found in \u003cem\u003eJatropha integerrima\u003c/em\u003e. These compounds have been documented for their antioxidant actions and roles in protection of liver damage through elimination of the free radicals, and reduction of lipid peroxidation (Ezeonu et al., 2017). For instance, the polyphenolic content of \u003cem\u003eJatropha integerrima\u003c/em\u003e may act as an important factor for cell membrane stabilization and against the oxidative injury to hepatocytes as postulated in the other experimental studies of natural hepatoprotective (Abdel Moneim, 2016).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOxidative Stress and Antioxidant Activity\u003c/h2\u003e \u003cp\u003eIn addition, oxidative stress is the key factor that has been reported to be involved in the pathogenesis of CCl\u003csub\u003e4\u003c/sub\u003e induced hepatotoxicity, since CCl\u003csub\u003e4\u003c/sub\u003e metabolites produce free radicals which enhance peroxidation of lipids and inflict cellular damage. Chlorinated carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e) treatment enhanced the total oxidative stress (TOS) and reduced total antioxidant capacity (TAC) in the liver implying that there are increased oxidative damage and impaired antioxidant defense in the liver.\u003c/p\u003e \u003cp\u003e \u003cem\u003eJatropha integerrima\u003c/em\u003e extract has a considerable potential to boost the liver antioxidant capability since the treatment improved both TAC as well as reduced TOS in a dose-dependent manner. Similar findings have been reported in the other parts of Jatropha, in other species of this plant. Methanolic extract of \u003cem\u003eJatropha tanjorensis\u003c/em\u003e leaf have marked antioxidant potential to augment SOD and GPx level with reduced OS markers in CCl\u003csub\u003e4\u003c/sub\u003e intoxicated rats, according to (Ezeonu et al. 2017). Likewise, the antioxidant and hepatoprotective potentials of \u003cem\u003eJatropha curcas\u003c/em\u003e have also been documented in other studies which adds to the fact that Jatropha species are useful in reducing oxidative stress and liver injury (Al-Basher, 2018).\u003c/p\u003e \u003cp\u003eThe presence of flavonoids and polyphenolic compounds in \u003cem\u003eJatropha integerrima\u003c/em\u003e could be responsible for the antioxidant activity, as these compounds counteract free radicals and stimulate antioxidant enzyme function. The rising TAC protein data revealed in this study implies that \u003cem\u003eJ. Integerrima\u003c/em\u003e strengthens the liver detoxification ability to scavenge ROS and decrease oxidative injury together with stimulating cellular repair. This is in support with Kumar et al. (2019) who showed that the major bioactive compounds of the \u003cem\u003eJatropha integerrima\u003c/em\u003e leaves were polyphenols and these had vigorous radical scavenging activity \u003cem\u003ein vitro.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Findings\u003c/h2\u003e \u003cp\u003eThe biochemical and oxidative stress results were supported and reinforced by the histopathological analysis of the liver tissues. Tissues of the liver from the CCl\u003csub\u003e4\u003c/sub\u003e treated group exhibited; extensive hepatocellular necrosis, inflammation and sinusoidal distension which when viewed were interpreted as significant liver damage. Nevertheless, after the \u003cem\u003eJatropha integerrima\u003c/em\u003e extract administration, especially at the dose of 400 mg/kg, there was a significantly lessened degree of liver lesions together with a good overall maintenance of the hepatic architecture, with no substantial inflammation, or necrosis. The change of liver histology was like a silymarin treated group; it indicated that \u003cem\u003eJatropha integerrima\u003c/em\u003e possesses the ability to prevent hepatocytes from damage due to CCl\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThese protective effects are supported by other studies on Jatropha species that have been published. Jain et al. (2015) revealed similar tissue morphological changes in the rats which were treated by \u003cem\u003eJatropha gossypifolia\u003c/em\u003e extracts, it was further concluded that hepatic tissues were protected against CCl\u003csub\u003e4\u003c/sub\u003e induced necrosis and inflammation. Similarly, Al-Basher (2018) proved that extracts from \u003cem\u003eJatropha curcas\u003c/em\u003e possess appreciable histopathological lesions in cadmium-intoxicated rats as possible evidence of hepatoprotective effects of Jatropha species.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMechanism of Action\u003c/h2\u003e \u003cp\u003eThe hepatoprotective action of \u003cem\u003eJatropha integerrima\u003c/em\u003e as observed in this study might be due to one or many mechanisms. First, the plant rich in polyphenols and flavonoids is expected to act as free radical scavengers neutralizing oxidative stress, prevent lipid peroxidation and stabilize cell membranes (Abdel Moneim, 2016). Second, the decrease in LPS-induced inflammatory markers and histopathological lesions can be attributed to the potential ability of \u003cem\u003eJatropha integerrima\u003c/em\u003e in regulating inflammation in a manner that decreases the migration of inflammatory cells and the production of pro-inflammatory cytokines. This is in agreement with earlier works revealing that Jatropha species possess anti-inflammatory properties (Ubaid et al., 2020).\u003c/p\u003e \u003cp\u003eAdditionally, the capacity to recover the mitochondrial structure by \u003cem\u003eJatropha integerrima\u003c/em\u003e, as depicted from electron microscopic images, suggests that this plant extract may afford cytoprotection against the mitochondrial component that plays a pivotal role towards the development of liver injury. Mitochondria are responsible for the regulation of cellular energy and their balance disrupting leads to apoptosis and necrosis. Thus, Jatropha integerrima may be useful in maintaining cellular structures and energy metabolism in hepatocytes and protect them from programmed necrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eComparison with Other Studies\u003c/h2\u003e \u003cp\u003eIn this study, hepatoprotective activity of \u003cem\u003eJ. integerrima\u003c/em\u003e was confirmed and the results were in agreement with the previous studies conducted on Jatropha species. For instance, Al-Basher (2018) have established that the methanolic extracts of \u003cem\u003eJatropha curcas\u003c/em\u003e leaves showed impressive effects on attenuating the cadmium induced liver damage with the similar improvement on the levels of the liver enzymes and histopathological findings. Likewise, Ezeonu et al., (2017) found that extracts from \u003cem\u003eJatropha tanjorensis\u003c/em\u003e have the effective ability to act as an antioxidant and thus, protect liver from damage from CCl\u003csub\u003e4\u003c/sub\u003e in rats pointing to the fact that many species of Jatropha have potent hepatoprotective effects.\u003c/p\u003e \u003cp\u003eHowever, the present study offers new information on the hepatoprotective properties of \u003cem\u003eJ. integerrima\u003c/em\u003e, a species that has not been extensively investigated. Based on already existing literature, this work contributes to the existing knowledge base demonstrating that not only other Jatropha species, but also \u003cem\u003eJatropha integerrima\u003c/em\u003e possesses potent antioxidant and hepatoprotective properties and can be considered for further research and potential therapeutic use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStudy Limitations and Future Directions\u003c/h2\u003e \u003cp\u003eHowever, a number of limitations are restricting this study that should be considered for further studies as follows: Firstly, the experiment involved few numbers of rats and therefore the result may not hold of other species or even the same larger sized rats. Moreover, this study employed biochemical and histopathological approaches to determine markers of liver function; however further research studies are required to determine the molecular mechanism through which \u003cem\u003eJatropha integerrima\u003c/em\u003e exerted the hepatoprotective effect. Researching separate signaling systems, for example of nuclear factor erythroid 2 related factor 2 (Nrf2) that is connected with antioxidant reactions, may give important information about the plant\u0026rsquo;s action (Kumar et al., 2019).\u003c/p\u003e \u003cp\u003eFurthermore, this study did not consider the chronic impact of administering \u003cem\u003eJatropha integerrima\u003c/em\u003e. The extract should also be examined, long-term effects, inflammation, and general safety when used for long term treatments in future research. Further clinical experiments, especially among patients suffering from liver diseases, are also needed to establish the aesthetic value \u003cem\u003eof Jatropha integerrima\u003c/em\u003e in the cure and prevention of liver diseases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the outcome of the present investigation confirmed that methanolic extracts of \u003cem\u003eJatropha integerrima\u003c/em\u003e exert promising hepatoprotective potential against CCl\u003csub\u003e4\u003c/sub\u003e mediated hepatotoxicity in rats. It was found that the extract lowered the level of liver enzyme, increased the antioxidant status and lessened the histopathological alteration probably because of the high polyphenols and flavonoids present in the extract. The outcome of the current study therefore indicates that there is a possibility of using \u003cem\u003eJatropha integerrima\u003c/em\u003e with a view of having natural sources of hepatoprotective agents. Thus, more research in the area is required for proficiency of its action and future use in the treatment of illnesses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel Moneim, A. E. (2016). Oxidative stress in liver diseases induced by environmental exposure to xenobiotics. \u003cb\u003eJournal of Xenobiotics\u003c/b\u003e, 6(1), 7\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Basher, G. I. (2018). Anti-fibrogenic and hepatoprotective potential of methanolic olive extract on cadmium-induced toxicity in rats. \u003cb\u003eLife Science Journal\u003c/b\u003e, 15(7), 1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEzeonu, D. O., Anosike, C. A., \u0026amp; Njoku, O. U. (2017). Hepatoprotective and antioxidant effects of the flavonoid-rich fraction of the methanol extract of \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eJatropha tanjorensis\u003c/span\u003e leaves in CCl4-induced liver injury in rats. \u003cb\u003eIOSR Journal of Pharmacy and Biological Sciences\u003c/b\u003e, 12(1), 54\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain, S., Choudhary, G., \u0026amp; Jain, D. (2015). Antioxidant and hepatoprotective potential of ethanolic leaves extract of \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eJatropha gossypifolia\u003c/span\u003e. \u003cb\u003eInternational Journal of Plant Science Ecology\u003c/b\u003e, 1(1), 190\u0026ndash;195.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, A., Akhter, F., Singh, R., \u0026amp; Dutt, P. (2019). Antioxidant and anti-inflammatory activity of \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eJatropha integerrima\u003c/span\u003e leaves in an in-vivo model. \u003cb\u003ePharmacognosy Journal\u003c/b\u003e, 11(6), 1365\u0026ndash;1371.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuriel, P. (2009). Role of free radicals in liver diseases. \u003cb\u003eHepatology International\u003c/b\u003e, 3(4), 526\u0026ndash;536.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUbaid, S., Zubair, M., Ahmed, M., \u0026amp; Tahir, M. (2020). Phytochemical screening and antimicrobial activity of \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eJatropha integerrima\u003c/span\u003e leaves. \u003cb\u003ePakistan Journal of Pharmaceutical Sciences\u003c/b\u003e, 33(3), 1191\u0026ndash;1195.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdel Moneim, A. E. (2016). Oxidative stress in liver diseases induced by environmental exposure to xenobiotics. \u003cb\u003eJournal of Xenobiotics\u003c/b\u003e, 6(1), 7\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Basher, G. I. (2018). Anti-fibrogenic and hepatoprotective potential of methanolic olive extract on cadmium-induced toxicity in rats. \u003cb\u003eLife Science Journal\u003c/b\u003e, 15(7), 1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Tantawy, W. H., Temraz, A., \u0026amp; Abdel Rasheed, A. M. (2013). Hepatoprotective effect of Zingiber officinale against carbon tetrachloride-induced liver fibrosis in rats. \u003cb\u003eAfrican Journal of Traditional, Complementary, and Alternative Medicines\u003c/b\u003e, 10(3), 42\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEzeonu, D. O., Anosike, C. A., \u0026amp; Njoku, O. U. (2017). Hepatoprotective and antioxidant effects of the flavonoid-rich fraction of the methanol extract of \u003cb\u003eJatropha tanjorensis\u003c/b\u003e leaves in CCl\u003csub\u003e4\u003c/sub\u003e induced liver injury in rats. \u003cb\u003eIOSR Journal of Pharmacy and Biological Sciences\u003c/b\u003e, 12(1), 54\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain, S., Choudhary, G., \u0026amp; Jain, D. (2015). Antioxidant and hepatoprotective potential of ethanolic leaves extract of \u003cb\u003eJatropha gossypifolia\u003c/b\u003e. \u003cb\u003eInternational Journal of Plant Science Ecology\u003c/b\u003e, 1(1), 190\u0026ndash;195.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, A., Akhter, F., Singh, R., \u0026amp; Dutt, P. (2019). Antioxidant and anti-inflammatory activity of \u003cb\u003eJatropha integerrima\u003c/b\u003e leaves in an in-vivo model. \u003cb\u003ePharmacognosy Journal\u003c/b\u003e, 11(6), 1365\u0026ndash;1371.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuriel, P. (2009). Role of free radicals in liver diseases. \u003cb\u003eHepatology International\u003c/b\u003e, 3(4), 526\u0026ndash;536.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlayiwola, G., Akinmoladun, F. O., \u0026amp; Onipede, T. (2020). Hepatoprotective activity of methanolic extract of \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eJatropha curcas\u003c/span\u003e leaves on carbon tetrachloride-induced liver damage in albino rats. \u003cb\u003eNigerian Journal of Natural Products and Medicine\u003c/b\u003e, 24(1), 18\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUbaid, S., Zubair, M., Ahmed, M., \u0026amp; Tahir, M. (2020). Phytochemical screening and antimicrobial activity of \u003cb\u003eJatropha integerrima\u003c/b\u003e leaves. \u003cb\u003ePakistan Journal of Pharmaceutical Sciences\u003c/b\u003e, 33(3), 1191\u0026ndash;1195.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, F. S., Fan, J. G., Zhang, Z., Gao, B., \u0026amp; Wang, H. Y. (2014). The global burden of liver disease: the major impact of China. \u003cb\u003eHepatology\u003c/b\u003e, 60(6), 2099\u0026ndash;2108.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Lahore","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":"Jatropha integerrima, hepatoprotective, carbon tetrachloride, liver damage, oxidative stress, antioxidants, natural therapy","lastPublishedDoi":"10.21203/rs.3.rs-5511338/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5511338/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eIntroduction:\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLiver diseases are a significant public health issue globally, often caused by exposure to hepatotoxins like carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e), which induces oxidative stress and liver injury. Natural compounds, particularly plant-based products, have gained attention for their hepatoprotective properties. \u003cem\u003eJatropha integerrima\u003c/em\u003e, a plant known for its antioxidant and anti-inflammatory effects, has not been extensively studied for its hepatoprotective effects against CCl\u003csub\u003e4\u003c/sub\u003e induced liver damage.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethodology:\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMethanolic extracts of \u003cem\u003eJatropha integerrima\u003c/em\u003e leaves were prepared using a Soxhlet extraction method. Forty Sprague Dawley rats were divided into five groups: a control group, a CCl\u003csub\u003e4\u003c/sub\u003e-treated group, a silymarin-treated group (standard treatment), and two groups treated with low (200 mg/kg) and high (400 mg/kg) doses of the Jatropha extract. Liver function biomarkers, including bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP), were measured. Oxidative stress markers, total antioxidant capacity (TAC) and total oxidative stress (TOS), were also evaluated. Histopathological analysis of liver tissues was performed to assess structural damage.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults:\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCCl\u003csub\u003e4\u003c/sub\u003e-induced liver damage resulted in significant increases in serum liver enzymes and oxidative stress markers. Treatment with \u003cem\u003eJatropha integerrima\u003c/em\u003e extract significantly reduced ALT, AST, ALP, and bilirubin levels in a dose-dependent manner. The high-dose group showed reductions comparable to silymarin. TAC levels were significantly improved, while TOS levels were reduced. Histopathological analysis revealed that the high-dose Jatropha group showed near-complete restoration of liver architecture, with reduced necrosis and inflammation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDiscussion:\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe hepatoprotective effects of \u003cem\u003eJatropha integerrima\u003c/em\u003e may be due to high concentrations of polyphenols and flavonoids available in this plant to counter act the free radicals and oxidative stress. These results confirm to other research on other species of Jatropha and increase the therapeutic value of \u003cem\u003eJatropha integerrima\u003c/em\u003e as a natural means to treating hepatotoxicity ailments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion:\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMethanolic extracts of \u003cem\u003eJatropha integerrima\u003c/em\u003e provide substantial hepatic protection against CCl\u003csub\u003e4\u003c/sub\u003e induced liver injury in rats through marked improvement in liver function, reduction of oxidative stress and better maintenance of hepatic morphology. These findings indicated that \u003cem\u003eJatropha integerrima\u003c/em\u003e might provide a promising alternative for treating any kind of liver diseases and this study has to pave the way for further research in order to discover the full potential and efficacy of \u003cem\u003eJatropha integerrima\u003c/em\u003e in treating diseases of the liver.\u003c/p\u003e","manuscriptTitle":"Hepatoprotective Potential of Methanolic Extracts from Jatropha integerrima Leaves Against Carbon Tetrachloride (CCl4) Induced Rat Model.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 06:10:45","doi":"10.21203/rs.3.rs-5511338/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":"cd3e2cd0-6e99-4d12-b688-98eb4c8d7243","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40678545,"name":"Biochemical Research Methods"}],"tags":[],"updatedAt":"2024-11-26T06:10:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-26 06:10:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5511338","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5511338","identity":"rs-5511338","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}