Effects Of Gongronema Latifolium (Benth.) Leaf Extract on Cardiac Glutathione and Dopamine Antioxidant Systems

Effects Of Gongronema Latifolium (Benth.) 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Leaf Extract on Cardiac Glutathione and Dopamine Antioxidant Systems Daniel Kehinde Omosebi, Ojo Olubukola Benedicta, Olowookere, Boyede Dele This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6758129/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 This study investigates the effects of Gongronema latifolium methanolic leaf extract (GLL) on antioxidant enzyme activities and dopamine levels in cardiac tissue. Female Wistar rats (150 ± 30g) were divided into four groups, one control group and three treatment groups (treated with GLL at doses of 250, 500, and 1000 mg/kg for 28 days). The animals were sacrificed after 28 days and the hearts were excised for biochemical analysis (reduced glutathione (GSH) level, glutathione peroxidase (GPx) activity, glutathione S-transferase (GST) activity, superoxide dismutase (SOD) activity, and dopamine level). Results showed dose-dependent modulation of these parameters. GSH and dopamine levels significantly increased at higher doses, enhancing antioxidative defences and potentially supporting cardiovascular function. GPx activity showed sustained elevation across doses, while SOD activity showed a pronounced dose-independent increase, highlighting robust antioxidant effects. GST activity, however, peaked at moderate doses (250 mg/kg), suggesting a biphasic response. These findings show the potential of GLL as a cardioprotective agent. This study contributes to the understanding of its role in mitigating oxidative stress and improving cardiovascular health. Higher doses could overwhelm detoxification pathways, showcasing the need for well-optimized dosages. Future research should explore the molecular pathways underlying these effects and assess GLL’s therapeutic potential against cardiovascular disorders. Applied Biochemistry Gongronema latifolium Cardiovascular health Oxidative stress Dopaminergic systems Antioxidant Systems Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The use of medicinal plants in developing nations is a major fabric of both traditions and culture, with these traditions being intergenerational (Nwangwu et al. , 2009). Medicinal plants are composed of diverse kinds of compounds with medicinal properties, making them important candidates for the development of new antibacterial and antioxidant medications (Sibanda et al. , 2007). G. latifolium has been especially singled out for its rich phytochemical compositions which confer on it its antioxidant, anti-inflammatory, and cardio-protective properties (Ezejiofor et al., 2020 ). G. latifolium is an edible rainforest plant of West African Origin (Nelson, 1965). It is indigenous to both the South-Eastern as well as South-Western parts of Nigeria (Owu et al., 2012 ). It is widely dispersed in the African forest and family farms as wild, semi-wild and cultivated (Okafor, 1980). It can be propagated by seed or stem (Softwood, semi-hardwood and hardwood) cuttings (Agbo and Obi, 2006). Known locally as 'Utazi' or 'Arokeke,' it serves as a remedy for ailments like diabetes, hypertension, and malaria, among others. It is highly valued for its medicinal and nutritional contributions, often used as both a spice and a vegetable in Western African cuisine and has been historically employed to regulate weight in lactating women and enhance female fertility. Earlier studies have highlighted its potential hypoglycemic and antihyperglycemic properties (Ugochukwu and Babady, 2002 ), further placing emphasis on its medicinal importance. G. latifolium extracts have shown radioprotective effects, reducing oxidative stress in irradiated rats, and suggesting potential prophylactic applications (Okeke et al., 2022 ). Knowledge can be inferred from existing literature about the biochemical mechanism through which it promotes cardiac antioxidant systems. A key mechanism is through the modulation of oxidative stress and inflammatory biomarkers. Studies have illustrated that phytochemical constituent in the plant (flavonoids, saponins, and alkaloids) are useful in restoring antioxidant enzyme activity in cardiac tissues under pathological conditions. In models of myocardial injury and diabetic cardiomyopathy, administration of G. latifolium extracts significantly increased the activity of several important antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), while reducing malondialdehyde (MDA), a marker of lipid peroxidation (Okesola et al., 2020 ; Imo, 2017 ). These effects are likely mediated by the plant’s ability to rummage reactive oxygen species (ROS), chelate transition metals, and upregulate endogenous antioxidant systems. G. latifolium has also been shown to suppress inflammation-related cardiac injury by reducing levels of tumor necrosis factor-alpha (TNF-α) and C-reactive protein, (Okon et al., 2022 ). These findings suggest that the cardioprotective antioxidant mechanism of G. latifolium is based on its ability to temper redox balance and inflammatory pathways. The main aim of this study is to provide preliminary evidence supporting the use of extracts of G. latifolium as potential cardio-protective agents and lays a foundation for future molecular studies. This study, therefore, outlines the effects of G. latifolium on key antioxidant systems, such as SOD, GPx, and GST, suggesting that G. latifolium enhances the heart's antioxidant defence mechanisms. The study also evaluated the potential of G. latifolium to modulate cardiac dopamine levels, linking it to oxidative stress response and cardiovascular function. While the antioxidant benefits of G. latifolium are well-supported, the literature does not extensively address the potential side effects of overconsumption. This gap suggests a need for further research to explore any adverse effects associated with high intake levels, ensuring safe consumption practices. MATERIALS AND METHODS Chemicals and Reagents Thiobarbituric acid, trichloroacetic acid, reduced glutathione, 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB/Ellman’s reagent), epinephrine, ferric cyanide, Folin-Ciocâlteu’s reagent, and 1,2-dichloro-4-nitrobenzene (CDNB), Reagents were sourced from British Drug House and Sigma-Aldrich. Methanol was supplied by Scharlau (Spain), and all other chemicals were of analytical grade. Plant Collection and Extract Preparation Fresh G. latifolium leaves were bought from a local market in Ife, air-dried, pulverized, and sequentially macerated in diethyl ether and 80% methanol. The methanolic extract was obtained by evaporation, dried, and stored in a desiccator until use. Animal Handling and Experimental Design Female Wistar rats (150 ± 30g) were used. The animals were kept under standard environmental conditions in the animal facility of the Department of Chemical Sciences, Kings University, Odeomu, Nigeria. They were fed with standard rat chow (Vital Feeds, Lagos, Nigeria) and water, ad libitum . Animal handling and use also conformed to the standard guidelines of the Committee on Care and Use of Experimental Animal Resources published by the National Institutes of Health, in 1985. Age-matched animals were divided into four groups (n = 5 per group) and were treated as follows: one control group received vehicle (1% DMSO and distilled water), and three treatment groups received G. latifolium at doses of 250, 500, and 1000 mg/kg respectively. Treatments were administered orally for 28 days, after which animals were sacrificed, and their hearts excised for biochemical analyses. Tissue Processing for Biochemical Estimations Heart tissues (10% w/v) were homogenized in 0.1M phosphate-buffered saline (PBS, pH 7.4), using a Teflon homogenizer and the mixture was centrifuged at 10,000 x g at 4°C for 15 minutes. The supernatants obtained were used for biochemical estimations. Assay for Reduced Glutathione (GSH) Level GSH concentration in tissue homogenates was estimated as previously reported (Beutler et al. , 1963; Sunil et al. , 2011). Tissue homogenates (0.2 ml) were added to a mixture having 1.8 ml of distilled water and 3 ml of 4% Sulfosalicylic acid (SSA) and centrifuged at 1500 x g for 5 min. The supernatant (1 ml) was mixed with 4 ml of 0.1 M phosphate buffer (pH 7.4) and then reacted with 0.5 ml of 5,5-dithio-bis-(2-nitrobenzoic acid (DTNB). The yellow chromophore produced was read at 412 nm against a reagent blank. The glutathione concentration in the homogenate was estimated by extrapolation from a standard glutathione curve. Assay for Determination of Glutathione peroxidase (GPx) activity Glutathione peroxidase activity was determined according to the method of Haque et al. , (2003). The reaction mixture contained 500 µl of 0.1M phosphate buffer pH 7.4 100µl of 10mM Sodium azide, 200 µl of 4mM GSH, 100 µl of 2.5mM H 2 O 2 were added to 500 µl of the heart homogenate sample, after which 600 µl of distilled water were added and mixed thoroughly. The reaction mixture was incubated at 37°C for 3 mins after which 0.5 ml 10% trichloroacetic acid (TCA) was added and thereafter centrifuged at 3000 rpm for 5 mins. To 1 ml of each of the supernatants, 2 ml of K 2 HPO 4 and 1 ml of 0.04% DTNB were added, and the absorbance was read at 412 nm against a blank. Assay for Glutathione Transferase Activity Glutathione transferase (GST) activity was determined according to the method of Habig et al. (1974). The reaction medium consisted of 30 µl reduced glutathione (0.1 M), 150 µl CDNB (20 mM), 2.79 ml phosphate buffer (0.1 M, pH 6.5) and 30 µl supernatant in a cuvette inside the spectrophotometer. The blank cuvette, on the other hand, did not have supernatant but instead 2.82 ml of buffer. The reaction was allowed to run for 3 min and the absorbance was taken every 60 s each time against the blank at 340 nm. The temperature was kept at approximately 31°C throughout the experiment. GST activity was calculated using the molar extinction coefficient of CDNB ( 9.6 mm − 1 cm − 1 ) Assay for Superoxide Dismutase Activity Superoxide dismutase activity (SOD) was evaluated according to the method of Kakkar and others (Kakkar et al. , 1984). Tissue homogenates (1 ml) were mixed with 2.5 ml of 0.05 M carbonate buffer (pH 10.2) and equilibrated in a spectrophotometer before the addition of 0.3 ml of freshly prepared 0.3 mmol epinephrine to start the reaction. The absorbance was read at 480 nm every 30 s for 150 s. The blank solution contained PBS instead of homogenates. The change in absorbance per minute of the test sample was divided by the change in absorbance per minute of the blank to obtain percentage inhibition. The amount of protein required for 50% inhibition of the oxidation of adrenaline to adrenochrome was estimated as one unit of SOD activity in the tissue. Evaluation of Dopamine Levels The level of dopamine was measured by the method of Guo et al. , (2009). Homogenate (0.1 ml) was incubated with a mixture having potassium ferricyanide (5 mM, 0.1 ml), FeCl 3 (5 mM, 0.1 ml) and NaH 2 PO 4 (pH 4.0, 2.5 ml) in room temperature for 35 min. The absorbance was taken at 735 nm against a reagent blank and dopamine level was estimated from the dopamine hydrochloride standard curve. Determination of Total Protein Content Total protein content was estimated by the method of Lowry and others (1951). Tissue homogenate (0.2 ml) was mixed with freshly prepared Reagent C (1 ml) which is made up of Reagent A (2% Sodium Carbonate and 0.1N Sodium hydroxide) and Reagent B (0.5% Copper Sulphate and 1% Potassium tartrate) and allowed to stand for 10 min at room temperature. Then 0.1 ml of Reagent D which is a mixture of Folin Ciocalteu reagent and distilled water (1:2) was added and the absorbance was read at 750 nm after 30 min. The total protein content was estimated from a bovine serum albumin standard curve. Statistical Analysis All data derived from the animal experiment analysis was expressed as group mean ± standard deviation. The experimental results were analysed using the one-way analysis of variance (ANOVA) followed by Tukey multiple comparison tests. In all the tests, P < 0.05 was taken as statistical significance. The statistical significance software used to analyse the data was GraphPad Prism 6.01 (GraphPad Software Inc., CA, USA). RESULTS Effect of Gongronema latifolium Methanolic Leaf Extract on Cardiac Reduced Glutathione Levels The effect of G. latifolium leaf methanolic extract (GLL) on reduced glutathione (GSH) levels in the hearts of rats is shown in Fig. 1 . There was a significant decrease in the GSH level in the heart of rats administered 500 (P = 0.0011) and 1000 (P < 0.0001) mg/kg GLL in a dose-dependent manner compared to the control. The cardiac GSH level in rats treated with 250 mg/kg GLL were not significantly (P = 0.1020) different from the control. Effects of Gongronema Latifolium Methanolic Leaf Extract on Cardiac Glutathione Peroxidase Activity Figure 2 shows the effect of GLL on glutathione peroxidase (GPx) activity in the heart of rats. The activity of GPx decreased significantly (P < 0.0001) in the hearts of rats administered 500 and 1000 mg/kg GLL compared to the control. The GPx activity in the heart of rats administered 250 mg/kg GLL was not significantly (P = 0.0813) different from control. Effect of Gongronema Latifolium Methanolic Leaf Extract on Cardiac Glutathione S-Transferase Activity The effect of GLL on glutathione S-transferase (GST) activity in the hearts of rats is shown in Fig. 3 . The GST activity in the hearts of rats administered 250 mg/kg GLL increased significantly (P = 0.0343) compared to control. Cardiac GST activity of rats administered 500 mg/kg (P = 0.0548) and 1000 mg/kg (P = 0.9388) GLL were not significantly different from control. The results also show a biphasic response in glutathione S-transferase (GST) activity, which peaked at moderate doses (250 mg/kg), highlighting the importance of dosage optimization for effective detoxification. Effect of Gongronema latifolium Methanolic Leaf Extract on Cardiac Superoxide Dismutase Activity Figure 4 shows the effect of GLL on superoxide dismutase activity in the hearts of rats. The activity of superoxide dismutase (SOD) the heart of rats administered 500 mg/kg decreased significantly (P < 0.0001) but increased significantly (P < 0.0001) in rats administered 1000 mg/kg GLL compared to control. The SOD activity in the heart of rats administered 250 mg/kg GLL was not significantly (P = 0.7054) different from control. Superoxide dismutase (SOD) activity showed a pronounced, dose-independent alteration. Effects of Gongronema latifolium Methanolic Leaf Extract on Cardiac Dopamine Levels The effect of GLL on dopamine levels in the hearts of rats is shown in Fig. 5 . Dopamine levels decreased significantly (P = 0.0044) in the hearts of rats administered 250 mg/kg GLL, did not change significantly (P = 0.9977) in rat treated with 500 mg/kg but increased significantly (P = 0.0063) in 1000 mg/kg GLL compared to control. The results indicate that GLL modulates key antioxidant enzymes and dopamine levels, enhancing cardiac defences against oxidative stress. DISCUSSION Gongronema latifolium methanolic leaf extract is widely recognized for its antioxidant properties, which are generally considered beneficial. The plant's extracts have been previously reported to show significant antioxidant activities, including the ability to reduce lipid peroxidation and scavenge free radicals (Okoko et al. , 2021). These effects are attributed to the presence of various phytochemicals and essential oils (Chinwe and Usunomena, 2017; Eze, 2018 ). G. latifolium extracts have also shown radioprotective effects, reducing oxidative stress in irradiated rats, and suggesting potential prophylactic applications (Okeke et al., 2022 ). The plant's abundance of phytochemicals, including phenolic compounds, alkaloids, saponins, and flavonoids, has been reported to be responsible for its health advantages (Olugbodi et al., 2019 ). Though the antioxidant benefits are well-documented, caution about overconsumption is not extensively discussed in existing literature. In this study, the effects of Methanolic Leaf extract of G. latifolium on Cardiac glutathione and dopaminergic antioxidant systems are shown to be dosage dependent. This supports earlier reports that methanol extracts of G. latifolium potentiated a concentration-dependent increase in antioxidant activity, comparable to standard drugs used for such purposes (Chidi et al., 2017 ). This shows that though traditional herbal administrations of the leaves of G. latifolium have been without caution towards the side effects of overconsumption, the effects of G. latifolium extracts on antioxidant systems are overall generally positive. Reduced glutathione (GSH) concentrations are decreased in a dosage-dependent manner. This indicates that the extracts of G. latifolium may act by directly increasing the mechanisms of synthesis of antioxidant enzymes and molecules. Similarly, glutathione peroxidase (GPx) activity decreased at higher doses of G. latifolium , showing a potential improving impact on the heart's ability to detoxify peroxides. Lower dosages of G. latifolium extracts seem to improve GST activity but at higher dosages, activity is less pronounced showing a biphasic response, with moderate doses enhancing detoxification pathways and higher doses overwhelming the system. G. latifolium extracts dose-independent alteration on cardiac SOD activity. This result supports earlier studies on the function of SOD in attenuating oxidative damage in cardiovascular tissues (Griendling & FitzGerald, 2003 ). In addition, higher doses of extracts of G. latifolium resulted in significant increases in cardiac dopamine (DA) levels, showing a potential adaptive response to oxidative stress with elevated dopamine potentially helping to mitigate damage and support cardiac function. G. latifolium extract has been previously reported to increase the activities of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione transferase (GST) in numerous studies. This enhancement in enzyme activity helps in reducing oxidative stress markers in cardiac tissues (Okesola et al., 2020 ; Okon et al., 2022 ). The medication of G. latifolium extract has been shown to regulate neurotransmitter levels (including dopamine), in diabetic rats, which is crucial for supporting cardiac function and reducing oxidative stress (Ojo et al. , 2020). The observed modulation of GSH, GPx, GST, and SOD activities, accompanied by changes in cardiac dopamine levels, suggests that G. latifolium extracts can improve the heart's antioxidant defences and protect against oxidative stress-induced damage. These results are a foundation of proof of G. latifolium as a potential cardio-protective agent, though further studies are needed to clarify molecular mechanisms and find the best therapeutic doses. The findings thus provide insights into the cardio-protective mechanisms of GLL. The plant's ability to influence and modulate key antioxidant systems and its impact on cardiac dopamine levels, expose its multi-pronged cardioprotective mechanisms. This study has offered significant insights into the effects of G. latifolium on cardiac antioxidant systems and its potential medico-therapeutic implications for cardiovascular health. While the methanolic leaf extract of G. latifolium shows promise in modulating cardiac glutathione and dopamine antioxidant systems, it is important to put into consideration the potential for variability in individual responses to different dosages. For example, 250 mg/kg of GLL did not significantly alter cardiac GSH level, GPx and SOD activity; 500 and 1000 mg/kg of GLL did not significantly change cardiac GST activity; and 500 mg/kg GLL did not significantly alter cardiac dopamine levels in rats compared to control in rats. Further research is needed to prove standardized dosing regimens and to explore the long-term effects of the extract on cardiac and dopaminergic health. Future studies should focus on finding and characterizing the specific molecular pathways by which G. latifolium affects cardiac antioxidant systems. This could involve further research into the plant’s impact on gene expression with oxidative stress and antioxidant defence mechanisms. Adequate research into the dosage response relationship of G. latifolium to decide the best dosage for therapeutic effects is also necessary. Studies should include a wider range of dosages and possibly different forms of extracts to ascertain the most effective and safe concentrations for clinical use. Declarations All procedures involving animal handling and care were conducted in accordance with international guidelines for the use and care of laboratory animals. Ethical approval for the study was granted by the Health Research Ethics Committee of the Institute of Public Health, Obafemi Awolowo University, Ile-Ife, Nigeria. Funding Details: This research did not receive any specific grant from funding agencies. Declaration of Interest: The authors report there is no conflict of interest to declare. Data Availability: Enquiries about data availability should be directed to the first author and corresponding author. References Akande IS (2019) Therapeutic potentials of Gongronema latifolium (Benth.) in diseases management: A review. J Med Plants Res 13(3):73–83 Akpan HD, Ekpo AJ (2015) Protective role of diets containing Gongronema latifolium leaves on streptozotocin- induced oxidative stress and liver damage. J Appl Pharmaceut Sci 5(3):85–90 Ballatori N et al (2009) Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem 390:191–214 Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL (2009) Glutathione dysregulation and the etiology and progression of human diseases. 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Int J Trend Sci Res Dev, Volume-1(Issue-4). https://doi.org/10.31142/ijtsrd87 Okesola MA, Ojo OA, Onikanni SA, Ajiboye BO, Oyinloye BE, Agboinghale PE, Kappo AP (2020) Ameliorative effect of Gongronema latifolium leaf extract on alloxan-induced diabetic cardiomyopathy in Wistar rats model. Comp Clin Pathol 29(4):865–872. https://doi.org/10.1007/s00580-020-03134-8 Okoko T, Silas-Olu DI (2021) In-vitro Anti-lipid Peroxidation and Other Antioxidant Potentials of Gongronema latifolium Leaf Extract. Asian J Res Biochem 35–41. https://doi.org/10.9734/ajrb/2021/v8i130172 Additional Declarations The authors declare potential competing interests as follows: I hereby declare that authors have no interests, affiliations, or associations that might be perceived to influence the results and/or discussion reported in this preprint submission. 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GLL: \u003cem\u003eGongronema Latifolium\u003c/em\u003e Methanolic Leaf Extract.\u003c/p\u003e","description":"","filename":"image1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6758129/v1/bf272ed9eebdf1ce6311910d.jpg"},{"id":83974649,"identity":"656769ce-6409-45bf-b03b-1fa2d6fd124c","added_by":"auto","created_at":"2025-06-05 08:45:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46449,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eGongronema Latifolium\u003c/em\u003e Methanolic Leaf Extract on Cardiac Glutathione Peroxidase Activity; Results are expressed as mean ± SD (n = 5) and analysed using a one-way ANOVA and Tukey’s multiple comparisons test. GLL: \u003cem\u003eGongronema Latifolium\u003c/em\u003e Methanolic Leaf Extract.\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6758129/v1/c7b19a40f87ce6e7e34b7ec3.jpg"},{"id":83974647,"identity":"bdc026e1-09c0-4c6b-80dc-d9de82374549","added_by":"auto","created_at":"2025-06-05 08:45:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49861,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eGongronema Latifolium\u003c/em\u003e Methanolic Leaf Extract on Cardiac Glutathione S-Transferase Activity;\u003cstrong\u003e \u003c/strong\u003eResults are expressed as mean ± SD (n = 5) and analysed using a one-way ANOVA and Tukey’s multiple comparisons test. GLL: \u003cem\u003eGongronema Latifolium\u003c/em\u003e Methanolic Leaf Extract.\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6758129/v1/b208796279afd95f59987bf3.jpg"},{"id":83975901,"identity":"ed03cae8-f2c6-4812-a331-aa9add7eead9","added_by":"auto","created_at":"2025-06-05 09:01:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39646,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eGongronema latifolium\u003c/em\u003e Methanolic Leaf Extract on Cardiac Superoxide Dismutase Activity; Results are expressed as mean ± SD (n = 5) and analysed using a one-way ANOVA and Tukey’s multiple comparisons test. GLL: \u003cem\u003eGongronema Latifolium\u003c/em\u003e Methanolic Leaf Extract.\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6758129/v1/4d7c70cda4366118b9f3cb01.jpg"},{"id":83974909,"identity":"7668a442-f77d-4c89-9589-81b8e986fb5f","added_by":"auto","created_at":"2025-06-05 08:53:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37381,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eGongronema Latifolium\u003c/em\u003e Methanolic Leaf Extract on Cardiac Dopamine Levels; Results are expressed as mean ± SD (n = 5) and analysed using a one-way ANOVA and Tukey's multiple comparisons tests. GLL: \u003cem\u003eGongronema Latifolium\u003c/em\u003e Methanolic Leaf Extract.\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6758129/v1/9abbcb39fd1e40bf2357c11d.jpg"},{"id":83975902,"identity":"ce5db044-78c0-46c8-82fb-2511fe48e497","added_by":"auto","created_at":"2025-06-05 09:01:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":966178,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6758129/v1/8a38ff66-cc71-416b-8402-7812e494789f.pdf"}],"financialInterests":"The authors declare potential competing interests as follows: I hereby declare that authors have no interests, affiliations, or associations that might be perceived to influence the results and/or discussion reported in this preprint submission.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEffects Of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGongronema Latifolium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (Benth.) Leaf Extract on Cardiac Glutathione and Dopamine Antioxidant Systems\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe use of medicinal plants in developing nations is a major fabric of both traditions and culture, with these traditions being intergenerational (Nwangwu \u003cem\u003eet al.\u003c/em\u003e, 2009). Medicinal plants are composed of diverse kinds of compounds with medicinal properties, making them important candidates for the development of new antibacterial and antioxidant medications (Sibanda \u003cem\u003eet al.\u003c/em\u003e, 2007). \u003cem\u003eG. latifolium\u003c/em\u003e has been especially singled out for its rich phytochemical compositions which confer on it its antioxidant, anti-inflammatory, and cardio-protective properties (Ezejiofor et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eG. latifolium\u003c/em\u003e is an edible rainforest plant of West African Origin (Nelson, 1965). It is indigenous to both the South-Eastern as well as South-Western parts of Nigeria (Owu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). It is widely dispersed in the African forest and family farms as wild, semi-wild and cultivated (Okafor, 1980). It can be propagated by seed or stem (Softwood, semi-hardwood and hardwood) cuttings (Agbo and Obi, 2006). Known locally as 'Utazi' or 'Arokeke,' it serves as a remedy for ailments like diabetes, hypertension, and malaria, among others. It is highly valued for its medicinal and nutritional contributions, often used as both a spice and a vegetable in Western African cuisine and has been historically employed to regulate weight in lactating women and enhance female fertility. Earlier studies have highlighted its potential hypoglycemic and antihyperglycemic properties (Ugochukwu and Babady, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), further placing emphasis on its medicinal importance. \u003cem\u003eG. latifolium\u003c/em\u003e extracts have shown radioprotective effects, reducing oxidative stress in irradiated rats, and suggesting potential prophylactic applications (Okeke et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eKnowledge can be inferred from existing literature about the biochemical mechanism through which it promotes cardiac antioxidant systems. A key mechanism is through the modulation of oxidative stress and inflammatory biomarkers. Studies have illustrated that phytochemical constituent in the plant (flavonoids, saponins, and alkaloids) are useful in restoring antioxidant enzyme activity in cardiac tissues under pathological conditions. In models of myocardial injury and diabetic cardiomyopathy, administration of \u003cem\u003eG. latifolium\u003c/em\u003e extracts significantly increased the activity of several important antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), while reducing malondialdehyde (MDA), a marker of lipid peroxidation (Okesola et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Imo, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These effects are likely mediated by the plant\u0026rsquo;s ability to rummage reactive oxygen species (ROS), chelate transition metals, and upregulate endogenous antioxidant systems. \u003cem\u003eG. latifolium\u003c/em\u003e has also been shown to suppress inflammation-related cardiac injury by reducing levels of tumor necrosis factor-alpha (TNF-α) and C-reactive protein, (Okon et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These findings suggest that the cardioprotective antioxidant mechanism of \u003cem\u003eG. latifolium\u003c/em\u003e is based on its ability to temper redox balance and inflammatory pathways.\u003c/p\u003e \u003cp\u003eThe main aim of this study is to provide preliminary evidence supporting the use of extracts of \u003cem\u003eG. latifolium\u003c/em\u003e as potential cardio-protective agents and lays a foundation for future molecular studies. This study, therefore, outlines the effects of \u003cem\u003eG. latifolium\u003c/em\u003e on key antioxidant systems, such as SOD, GPx, and GST, suggesting that \u003cem\u003eG. latifolium\u003c/em\u003e enhances the heart's antioxidant defence mechanisms. The study also evaluated the potential of \u003cem\u003eG. latifolium\u003c/em\u003e to modulate cardiac dopamine levels, linking it to oxidative stress response and cardiovascular function. While the antioxidant benefits of \u003cem\u003eG. latifolium\u003c/em\u003e are well-supported, the literature does not extensively address the potential side effects of overconsumption. This gap suggests a need for further research to explore any adverse effects associated with high intake levels, ensuring safe consumption practices.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and Reagents\u003c/h2\u003e \u003cp\u003eThiobarbituric acid, trichloroacetic acid, reduced glutathione, 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB/Ellman\u0026rsquo;s reagent), epinephrine, ferric cyanide, Folin-Cioc\u0026acirc;lteu\u0026rsquo;s reagent, and 1,2-dichloro-4-nitrobenzene (CDNB), Reagents were sourced from British Drug House and Sigma-Aldrich. Methanol was supplied by Scharlau (Spain), and all other chemicals were of analytical grade.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlant Collection and Extract Preparation\u003c/h3\u003e\n\u003cp\u003eFresh \u003cem\u003eG. latifolium\u003c/em\u003e leaves were bought from a local market in Ife, air-dried, pulverized, and sequentially macerated in diethyl ether and 80% methanol. The methanolic extract was obtained by evaporation, dried, and stored in a desiccator until use.\u003c/p\u003e\n\u003ch3\u003eAnimal Handling and Experimental Design\u003c/h3\u003e\n\u003cp\u003eFemale Wistar rats (150\u0026thinsp;\u0026plusmn;\u0026thinsp;30g) were used. The animals were kept under standard environmental conditions in the animal facility of the Department of Chemical Sciences, Kings University, Odeomu, Nigeria. They were fed with standard rat chow (Vital Feeds, Lagos, Nigeria) and water, \u003cem\u003ead libitum\u003c/em\u003e. Animal handling and use also conformed to the standard guidelines of the Committee on Care and Use of Experimental Animal Resources published by the National Institutes of Health, in 1985. Age-matched animals were divided into four groups (n\u0026thinsp;=\u0026thinsp;5 per group) and were treated as follows: one control group received vehicle (1% DMSO and distilled water), and three treatment groups received \u003cem\u003eG. latifolium\u003c/em\u003e at doses of 250, 500, and 1000 mg/kg respectively. Treatments were administered orally for 28 days, after which animals were sacrificed, and their hearts excised for biochemical analyses.\u003c/p\u003e\n\u003ch3\u003eTissue Processing for Biochemical Estimations\u003c/h3\u003e\n\u003cp\u003eHeart tissues (10% w/v) were homogenized in 0.1M phosphate-buffered saline (PBS, pH 7.4), using a Teflon homogenizer and the mixture was centrifuged at 10,000 x g at 4\u0026deg;C for 15 minutes. The supernatants obtained were used for biochemical estimations.\u003c/p\u003e\n\u003ch3\u003eAssay for Reduced Glutathione (GSH) Level\u003c/h3\u003e\n\u003cp\u003eGSH concentration in tissue homogenates was estimated as previously reported (Beutler \u003cem\u003eet al.\u003c/em\u003e, 1963; Sunil \u003cem\u003eet al.\u003c/em\u003e, 2011). Tissue homogenates (0.2 ml) were added to a mixture having 1.8 ml of distilled water and 3 ml of 4% Sulfosalicylic acid (SSA) and centrifuged at 1500 x g for 5 min. The supernatant (1 ml) was mixed with 4 ml of 0.1 M phosphate buffer (pH 7.4) and then reacted with 0.5 ml of 5,5-dithio-bis-(2-nitrobenzoic acid (DTNB). The yellow chromophore produced was read at 412 nm against a reagent blank. The glutathione concentration in the homogenate was estimated by extrapolation from a standard glutathione curve.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssay for Determination of Glutathione peroxidase (GPx) activity\u003c/h2\u003e \u003cp\u003eGlutathione peroxidase activity was determined according to the method of Haque \u003cem\u003eet al.\u003c/em\u003e, (2003). The reaction mixture contained 500 \u0026micro;l of 0.1M phosphate buffer pH 7.4 100\u0026micro;l of 10mM Sodium azide, 200 \u0026micro;l of 4mM GSH, 100 \u0026micro;l of 2.5mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were added to 500 \u0026micro;l of the heart homogenate sample, after which 600 \u0026micro;l of distilled water were added and mixed thoroughly. The reaction mixture was incubated at 37\u0026deg;C for 3 mins after which 0.5 ml 10% trichloroacetic acid (TCA) was added and thereafter centrifuged at 3000 rpm for 5 mins. To 1 ml of each of the supernatants, 2 ml of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and 1 ml of 0.04% DTNB were added, and the absorbance was read at 412 nm against a blank.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAssay for Glutathione Transferase Activity\u003c/h3\u003e\n\u003cp\u003eGlutathione transferase (GST) activity was determined according to the method of Habig \u003cem\u003eet al.\u003c/em\u003e (1974). The reaction medium consisted of 30 \u0026micro;l reduced glutathione (0.1 M), 150 \u0026micro;l CDNB (20 mM), 2.79 ml phosphate buffer (0.1 M, pH 6.5) and 30 \u0026micro;l supernatant in a cuvette inside the spectrophotometer. The blank cuvette, on the other hand, did not have supernatant but instead 2.82 ml of buffer. The reaction was allowed to run for 3 min and the absorbance was taken every 60 s each time against the blank at 340 nm. The temperature was kept at approximately 31\u0026deg;C throughout the experiment. GST activity was calculated using the molar extinction coefficient of CDNB \u003cb\u003e(\u003c/b\u003e9.6 mm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n\u003ch3\u003eAssay for Superoxide Dismutase Activity\u003c/h3\u003e\n\u003cp\u003eSuperoxide dismutase activity (SOD) was evaluated according to the method of Kakkar and others (Kakkar \u003cem\u003eet al.\u003c/em\u003e, 1984). Tissue homogenates (1 ml) were mixed with 2.5 ml of 0.05 M carbonate buffer (pH 10.2) and equilibrated in a spectrophotometer before the addition of 0.3 ml of freshly prepared 0.3 mmol epinephrine to start the reaction. The absorbance was read at 480 nm every 30 s for 150 s. The blank solution contained PBS instead of homogenates. The change in absorbance per minute of the test sample was divided by the change in absorbance per minute of the blank to obtain percentage inhibition. The amount of protein required for 50% inhibition of the oxidation of adrenaline to adrenochrome was estimated as one unit of SOD activity in the tissue.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Dopamine Levels\u003c/h2\u003e \u003cp\u003eThe level of dopamine was measured by the method of Guo \u003cem\u003eet al.\u003c/em\u003e, (2009). Homogenate (0.1 ml) was incubated with a mixture having potassium ferricyanide (5 mM, 0.1 ml), FeCl\u003csub\u003e3\u003c/sub\u003e (5 mM, 0.1 ml) and NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (pH 4.0, 2.5 ml) in room temperature for 35 min. The absorbance was taken at 735 nm against a reagent blank and dopamine level was estimated from the dopamine hydrochloride standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Total Protein Content\u003c/h2\u003e \u003cp\u003eTotal protein content was estimated by the method of Lowry and others (1951). Tissue homogenate (0.2 ml) was mixed with freshly prepared Reagent C (1 ml) which is made up of Reagent A (2% Sodium Carbonate and 0.1N Sodium hydroxide) and Reagent B (0.5% Copper Sulphate and 1% Potassium tartrate) and allowed to stand for 10 min at room temperature. Then 0.1 ml of Reagent D which is a mixture of Folin Ciocalteu reagent and distilled water (1:2) was added and the absorbance was read at 750 nm after 30 min. The total protein content was estimated from a bovine serum albumin standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data derived from the animal experiment analysis was expressed as group mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The experimental results were analysed using the one-way analysis of variance (ANOVA) followed by Tukey multiple comparison tests. In all the tests, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was taken as statistical significance. The statistical significance software used to analyse the data was GraphPad Prism 6.01 (GraphPad Software Inc., CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eGongronema latifolium\u003c/b\u003e \u003cb\u003eMethanolic Leaf Extract on Cardiac Reduced Glutathione Levels\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effect of \u003cem\u003eG. latifolium\u003c/em\u003e leaf methanolic extract (GLL) on reduced glutathione (GSH) levels in the hearts of rats is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. There was a significant decrease in the GSH level in the heart of rats administered 500 (P\u0026thinsp;=\u0026thinsp;0.0011) and 1000 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) mg/kg GLL in a dose-dependent manner compared to the control. The cardiac GSH level in rats treated with 250 mg/kg GLL were not significantly (P\u0026thinsp;=\u0026thinsp;0.1020) different from the control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of\u003c/b\u003e \u003cb\u003eGongronema Latifolium\u003c/b\u003e \u003cb\u003eMethanolic Leaf Extract on Cardiac Glutathione Peroxidase Activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the effect of GLL on glutathione peroxidase (GPx) activity in the heart of rats. The activity of GPx decreased significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the hearts of rats administered 500 and 1000 mg/kg GLL compared to the control. The GPx activity in the heart of rats administered 250 mg/kg GLL was not significantly (P\u0026thinsp;=\u0026thinsp;0.0813) different from control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eGongronema Latifolium\u003c/b\u003e \u003cb\u003eMethanolic Leaf Extract on Cardiac Glutathione S-Transferase Activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effect of GLL on glutathione S-transferase (GST) activity in the hearts of rats is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The GST activity in the hearts of rats administered 250 mg/kg GLL increased significantly (P\u0026thinsp;=\u0026thinsp;0.0343) compared to control. Cardiac GST activity of rats administered 500 mg/kg (P\u0026thinsp;=\u0026thinsp;0.0548) and 1000 mg/kg (P\u0026thinsp;=\u0026thinsp;0.9388) GLL were not significantly different from control. The results also show a biphasic response in glutathione S-transferase (GST) activity, which peaked at moderate doses (250 mg/kg), highlighting the importance of dosage optimization for effective detoxification.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eGongronema latifolium\u003c/b\u003e \u003cb\u003eMethanolic Leaf Extract on Cardiac Superoxide Dismutase Activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the effect of GLL on superoxide dismutase activity in the hearts of rats. The activity of superoxide dismutase (SOD) the heart of rats administered 500 mg/kg decreased significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) but increased significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in rats administered 1000 mg/kg GLL compared to control. The SOD activity in the heart of rats administered 250 mg/kg GLL was not significantly (P\u0026thinsp;=\u0026thinsp;0.7054) different from control. Superoxide dismutase (SOD) activity showed a pronounced, dose-independent alteration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of\u003c/b\u003e \u003cb\u003eGongronema latifolium\u003c/b\u003e \u003cb\u003eMethanolic Leaf Extract on Cardiac Dopamine Levels\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effect of GLL on dopamine levels in the hearts of rats is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Dopamine levels decreased significantly (P\u0026thinsp;=\u0026thinsp;0.0044) in the hearts of rats administered 250 mg/kg GLL, did not change significantly (P\u0026thinsp;=\u0026thinsp;0.9977) in rat treated with 500 mg/kg but increased significantly (P\u0026thinsp;=\u0026thinsp;0.0063) in 1000 mg/kg GLL compared to control.\u003c/p\u003e \u003cp\u003eThe results indicate that GLL modulates key antioxidant enzymes and dopamine levels, enhancing cardiac defences against oxidative stress.\u003c/p\u003e "},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cem\u003eGongronema latifolium\u003c/em\u003e methanolic leaf extract is widely recognized for its antioxidant properties, which are generally considered beneficial. The plant's extracts have been previously reported to show significant antioxidant activities, including the ability to reduce lipid peroxidation and scavenge free radicals (Okoko \u003cem\u003eet al.\u003c/em\u003e, 2021). These effects are attributed to the presence of various phytochemicals and essential oils (Chinwe and Usunomena, 2017; Eze, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eG. latifolium\u003c/em\u003e extracts have also shown radioprotective effects, reducing oxidative stress in irradiated rats, and suggesting potential prophylactic applications (Okeke et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The plant's abundance of phytochemicals, including phenolic compounds, alkaloids, saponins, and flavonoids, has been reported to be responsible for its health advantages (Olugbodi et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Though the antioxidant benefits are well-documented, caution about overconsumption is not extensively discussed in existing literature. In this study, the effects of Methanolic Leaf extract of \u003cem\u003eG. latifolium\u003c/em\u003e on Cardiac glutathione and dopaminergic antioxidant systems are shown to be dosage dependent. This supports earlier reports that methanol extracts of \u003cem\u003eG. latifolium\u003c/em\u003e potentiated a concentration-dependent increase in antioxidant activity, comparable to standard drugs used for such purposes (Chidi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This shows that though traditional herbal administrations of the leaves of \u003cem\u003eG. latifolium\u003c/em\u003e have been without caution towards the side effects of overconsumption, the effects of \u003cem\u003eG. latifolium\u003c/em\u003e extracts on antioxidant systems are overall generally positive.\u003c/p\u003e \u003cp\u003eReduced glutathione (GSH) concentrations are decreased in a dosage-dependent manner. This indicates that the extracts of \u003cem\u003eG. latifolium\u003c/em\u003e may act by directly increasing the mechanisms of synthesis of antioxidant enzymes and molecules. Similarly, glutathione peroxidase (GPx) activity decreased at higher doses of \u003cem\u003eG. latifolium\u003c/em\u003e, showing a potential improving impact on the heart's ability to detoxify peroxides. Lower dosages of \u003cem\u003eG. latifolium\u003c/em\u003e extracts seem to improve GST activity but at higher dosages, activity is less pronounced showing a biphasic response, with moderate doses enhancing detoxification pathways and higher doses overwhelming the system. \u003cem\u003eG. latifolium\u003c/em\u003e extracts dose-independent alteration on cardiac SOD activity. This result supports earlier studies on the function of SOD in attenuating oxidative damage in cardiovascular tissues (Griendling \u0026amp; FitzGerald, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In addition, higher doses of extracts of \u003cem\u003eG. latifolium\u003c/em\u003e resulted in significant increases in cardiac dopamine (DA) levels, showing a potential adaptive response to oxidative stress with elevated dopamine potentially helping to mitigate damage and support cardiac function. \u003cem\u003eG. latifolium\u003c/em\u003e extract has been previously reported to increase the activities of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione transferase (GST) in numerous studies. This enhancement in enzyme activity helps in reducing oxidative stress markers in cardiac tissues (Okesola et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Okon et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The medication of \u003cem\u003eG. latifolium\u003c/em\u003e extract has been shown to regulate neurotransmitter levels (including dopamine), in diabetic rats, which is crucial for supporting cardiac function and reducing oxidative stress (Ojo \u003cem\u003eet al.\u003c/em\u003e, 2020). The observed modulation of GSH, GPx, GST, and SOD activities, accompanied by changes in cardiac dopamine levels, suggests that \u003cem\u003eG. latifolium\u003c/em\u003e extracts can improve the heart's antioxidant defences and protect against oxidative stress-induced damage.\u003c/p\u003e \u003cp\u003eThese results are a foundation of proof of \u003cem\u003eG. latifolium\u003c/em\u003e as a potential cardio-protective agent, though further studies are needed to clarify molecular mechanisms and find the best therapeutic doses. The findings thus provide insights into the cardio-protective mechanisms of GLL. The plant's ability to influence and modulate key antioxidant systems and its impact on cardiac dopamine levels, expose its multi-pronged cardioprotective mechanisms. This study has offered significant insights into the effects of \u003cem\u003eG. latifolium\u003c/em\u003e on cardiac antioxidant systems and its potential medico-therapeutic implications for cardiovascular health. While the methanolic leaf extract of \u003cem\u003eG. latifolium\u003c/em\u003e shows promise in modulating cardiac glutathione and dopamine antioxidant systems, it is important to put into consideration the potential for variability in individual responses to different dosages. For example, 250 mg/kg of GLL did not significantly alter cardiac GSH level, GPx and SOD activity; 500 and 1000 mg/kg of GLL did not significantly change cardiac GST activity; and 500 mg/kg GLL did not significantly alter cardiac dopamine levels in rats compared to control in rats.\u003c/p\u003e \u003cp\u003eFurther research is needed to prove standardized dosing regimens and to explore the long-term effects of the extract on cardiac and dopaminergic health. Future studies should focus on finding and characterizing the specific molecular pathways by which \u003cem\u003eG. latifolium\u003c/em\u003e affects cardiac antioxidant systems. This could involve further research into the plant\u0026rsquo;s impact on gene expression with oxidative stress and antioxidant defence mechanisms. Adequate research into the dosage response relationship of \u003cem\u003eG. latifolium\u003c/em\u003e to decide the best dosage for therapeutic effects is also necessary. Studies should include a wider range of dosages and possibly different forms of extracts to ascertain the most effective and safe concentrations for clinical use.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll procedures involving animal handling and care were conducted in accordance with international guidelines for the use and care of laboratory animals. Ethical approval for the study was granted by the Health Research Ethics Committee of the Institute of Public Health, Obafemi Awolowo University, Ile-Ife, Nigeria.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding Details:\u0026nbsp;\u003c/strong\u003eThis research did not receive any specific grant from funding agencies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest:\u003c/strong\u003e The authors report there is no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eEnquiries about data availability should be directed to the first author and corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkande IS (2019) Therapeutic potentials of Gongronema latifolium (Benth.) in diseases management: A review. 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Int Invention J Med Med Sci 2(6):88\u0026ndash;95\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUsunomena U, Chinwe IV \u0026amp; Faculty of Science, Edo University, Iyamho, Edo State, Nigeria. (2017). Analysis of phytochemicals, minerals and in vitro antioxidant activities of Gongronema latifolium leaves. Int J Trend Sci Res Dev, Volume-1(Issue-4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.31142/ijtsrd87\u003c/span\u003e\u003cspan address=\"10.31142/ijtsrd87\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkesola MA, Ojo OA, Onikanni SA, Ajiboye BO, Oyinloye BE, Agboinghale PE, Kappo AP (2020) Ameliorative effect of Gongronema latifolium leaf extract on alloxan-induced diabetic cardiomyopathy in Wistar rats model. Comp Clin Pathol 29(4):865\u0026ndash;872. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00580-020-03134-8\u003c/span\u003e\u003cspan address=\"10.1007/s00580-020-03134-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkoko T, Silas-Olu DI (2021) In-vitro Anti-lipid Peroxidation and Other Antioxidant Potentials of Gongronema latifolium Leaf Extract. Asian J Res Biochem 35\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.9734/ajrb/2021/v8i130172\u003c/span\u003e\u003cspan address=\"10.9734/ajrb/2021/v8i130172\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"Kings University Odeomu","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"Gongronema latifolium, Cardiovascular health, Oxidative stress, Dopaminergic systems, Antioxidant Systems","lastPublishedDoi":"10.21203/rs.3.rs-6758129/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6758129/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the effects of \u003cem\u003eGongronema latifolium\u003c/em\u003e methanolic leaf extract (GLL) on antioxidant enzyme activities and dopamine levels in cardiac tissue. Female Wistar rats (150\u0026thinsp;\u0026plusmn;\u0026thinsp;30g) were divided into four groups, one control group and three treatment groups (treated with GLL at doses of 250, 500, and 1000 mg/kg for 28 days). The animals were sacrificed after 28 days and the hearts were excised for biochemical analysis (reduced glutathione (GSH) level, glutathione peroxidase (GPx) activity, glutathione S-transferase (GST) activity, superoxide dismutase (SOD) activity, and dopamine level). Results showed dose-dependent modulation of these parameters. GSH and dopamine levels significantly increased at higher doses, enhancing antioxidative defences and potentially supporting cardiovascular function. GPx activity showed sustained elevation across doses, while SOD activity showed a pronounced dose-independent increase, highlighting robust antioxidant effects. GST activity, however, peaked at moderate doses (250 mg/kg), suggesting a biphasic response. These findings show the potential of GLL as a cardioprotective agent. This study contributes to the understanding of its role in mitigating oxidative stress and improving cardiovascular health. Higher doses could overwhelm detoxification pathways, showcasing the need for well-optimized dosages. Future research should explore the molecular pathways underlying these effects and assess GLL\u0026rsquo;s therapeutic potential against cardiovascular disorders.\u003c/p\u003e","manuscriptTitle":"Effects Of Gongronema Latifolium (Benth.) Leaf Extract on Cardiac Glutathione and Dopamine Antioxidant Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-05 08:45:05","doi":"10.21203/rs.3.rs-6758129/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":"e616dcf6-596e-4c4b-9d47-73c6accf635c","owner":[],"postedDate":"June 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49174582,"name":"Applied Biochemistry"}],"tags":[],"updatedAt":"2025-06-05T08:45:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-05 08:45:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6758129","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6758129","identity":"rs-6758129","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}