Antioxidant Effect of Ethanol Extract of C. sativus on Streptozotocin-Induced Diabetic Rat Liver

Antioxidant Effect of Ethanol Extract of C. sativus on Streptozotocin-Induced Diabetic Rat Liver

^1*Abu, O.D., ²Ojo, I., ³Enagbonma, B.J. and ⁴Akerele, O.R.

¹Department of Biochemistry, Faculty of Life Sciences, University of Benin, Benin City, Nigeria.

²Thermo Fisher Scientific Ltd., Carlsbad California, USA.

³Department of Environmental Management and Toxicology, Faculty of Life Sciences, University of Benin, Benin City, Nigeria.

⁴Department of Biochemistry, Faculty of Basic Medical Sciences, College of Medical Sciences, Edo State University, Uzairue, Edo State, Nigeria.

*Corresponding Author

DOI: https://doi.org/10.51584/IJRIAS.2023.81218

Received: 11 December 2023; Revised: 14 December 2023; Accepted: 19 December 2023; Published: 15 January 2024

ABSTRACT

The present study investigated the antioxidant effect of ethanol extract of Cucumis sativus on streptozotocin (STZ)-induced diabetic rat liver. Adult male Wistar albino rats (n = 25, mean weight = 215 ± 15 g) were randomly assigned to five groups of 5 rats each: normal control, diabetic control, metformin, 200 mg/kg body weight (bwt) extract and 300 mg/kg bwt extract groups. Diabetes mellitus was induced in the rats via intraperitoneal injection of 50 mg/kg bwt STZ. The diabetic rats were then treated for 21 days with either metformin (50 mg/kg bwt) or the extract at doses of 200 and 300 mg/kg bwt, respectively. Activities of antioxidant enzymes such as catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) as well as antioxidant molecules like retinoic acid, ascorbic acid and α-tocopherol were evaluated in liver homogenate. The results showed that induction of diabetes mellitus with STZ significantly increased the fasting blood glucose (FBG) concentrations of the rats (p < 0.05). However, treatment of the diabetic rats with the extract markedly reduced the FBG concentration and body weights of rats (p < 0.05). Treatment of diabetic Wistar rats with ethanol extract of C. sativus significantly increased activities of the antioxidant enzymes and molecules as well as concentrations of nitric oxide (NO), but it markedly reduced the concentrations of hepatic total protein (TP) and malondialdehyde (MDA) (p < 0.05). These results suggest that ethanol extract of the medicinal plant can enhance antioxidant defense in the liver of STZ-induced diabetic rats.

Keywords:  Antioxidants, Hepatocytes, Lipid peroxidation, Oxidative stress, Reactive oxygen species.

INTRODUCTION

Oxidative stress is defined as an imbalance in the oxidant-to-antioxidant ratio, causing the generation of free radicals [1]. The liver is the main detoxification organ of the body and plays an important role in controlling normal glucose homeostasis [2]. The production of oxidants such as reactive oxygen species (ROS)-like superoxide anions, hydrogen peroxide and hydroxyl radicals by activated Kupffer cells has been identified as central to hepatic injuries [3]. Kupffer cells, also known as hepatic macrophages, are one type of non-parenchymal cell that help maintain the integrity of hepatocytes. However, these phagocytic cells are also susceptible to the effects of oxidative stress produced by the surrounding cells and its own immune reactions [4, 5].

Excessive ROS production results in several deleterious events, including an irreversible oxidative modification of lipids, proteins and carbohydrates [6, 7]. In addition, it will induce apoptosis in hepatocytes and the release of inflammatory cytokines, thereby increasing the expression of adhesion molecules and the infiltration of leukocytes. A combination of all of these processes causes massive tissue destruction in the liver [8, 9]. However, the liver is equipped with potent antioxidants such as SOD, catalase and the glutathione enzyme family, including glutathione-S-transferases (GSTs) and glutathione peroxidases (GPXs)—so as not only to neutralize free radicals but also to protect liver cells from oxidative damage [7, 10]. Studies have demonstrated that a decrease in SOD and catalase activities within a hyperglycaemic state leads to an increase in ROS, which eventually contributes to oxidation-induced liver damage [10, 11].

The liver also plays a pivotal role in the homeostasis of the glutathione enzyme family. Different cell components such as the endoplasmic reticulum (ER), mitochondria and nucleus consist of separate pools of GSH. Among these, mitochondrial GSH is more essential than cytoplasmic GSH in maintaining cell viability [10]. Acting as the first line of defense as an endogenous antioxidant, the oxidation process takes place in the thiol group of GSH [10]. Reduced levels of GSH in diabetic rat livers have been associated with depletion of decreased GST, GPx and GR activity and with the accumulation of oxidative stress products, such as advanced glycation end-products (AGEs), protein oxidation products (POPs) and lipid peroxidation (LPO) [12 – 14]. However, measuring certain redox couples in cells such as oxidized nicotinamide adenine dinucleotide (NAD)/reduced NAD and oxidized NAD phosphate (NADP)/reduced NADP—is difficult; as a result, the GSH-to-glutathione disulphide ratio in the liver is considered representative of a redox state [10]. This study investigated the antioxidant effect of ethanol extract of C. sativus on diabetic rat liver.

MATERIALS AND METHODS

Chemicals

All chemicals and reagents used in this study were of analytical grade and they were products of Sigma-Aldrich Ltd. (USA).

Plant Extraction  

Freshly harvested Cucumis sativus fruits were purchased from a major fruit/vegetable market in Benin City, Nigeria and identified by Dr. Henry Akinnibosun of Plant Biology and Biotechnology Department, University of Benin. They were thereafter washed, and air-dried for about 4 weeks at the Department of Biochemistry. The dry plant was ground with a mechanical blender.  The pulverized sample was cold macerated in absolute ethanol for three days (72 h) in a bell jar and filtered using Whatmann filter paper No. 42 (125 mm). The ethanol extract was thereafter concentrated using rotary evaporator and freeze-dried using a lyophilizer [15, 16].

Experimental Animals

Mature male Wistar albino rats (n = 25) weighing 200 to 230 g (mean weight = 215 ± 15 g) were bought from the Department of Anatomy, University of Benin and housed in wooden cages. They were acclimatized for two weeks before commencement of the study, and had free access to feed and water.

Experimental Design 

The rats were randomly assigned to five groups (5 rats/group): normal control, diabetic control, metformin, 200 mg/kg bwt extract and 300 mg/kg bwt extract groups. Diabetes mellitus was induced in the rats via intraperitoneal injection of 50 mg/kg bwt STZ. The diabetic rats were then treated with either metformin (50 mg/kg bwt) or the extract at doses of 200 and 300 mg/kg bwt, respectively, for 21 days.

Tissue Sample Collection and Preparation

At the end of day 21 of treatment, the rats were euthanized under mild chloroform anaesthesia after an overnight fast. Their liver were excised, and used to prepare 20 % tissue homogenate. The homogenate was centrifuged at 2000 rpm for 10 min to obtain supernatant which was used for biochemical analysis.

Biochemical Analyses

The activities of catalase, SOD and GPx were determined [17 – 19]. Hepatic levels of TP, MDA, GSH, NO as well as vitamins A, E and C were also measured [20 – 26]. The activity of GR was determined using a previously described method [27].

Statistical Analysis

Data are presented as mean ± SEM (n = 5). Statistical analysis was performed using SPSS version 21. Statistical differences between means were compared using Duncan multiple range test. Statistical significance was assumed at p < 0.05.

RESULTS

Effect of Ethanol Extract of C. sativus on Weight and Blood Glucose of Rats

As shown in Tables 1 and 2, induction of diabetes mellitus using STZ significantly increased the blood glucose concentrations of the rats (p < 0.05). However, treatment of the diabetic rats with the extract markedly reduced their FBG concentration and body weights (p < 0.05).

Table 1: Effect of Ethanol Extract of C. sativus on Weight and Blood Glucose Parameters

Data are weight and FBG parameters and are expressed as mean ± SEM (n = 5).

Table 2: Comparison of the Weights of Rat Liver Among the Groups

Data are relative liver weights and are expressed as mean ± SEM (n = 5).

Values with superscript “a” are significantly different from the diabetic control group.

Oxidative Status in Diabetic Rats Treated with the Medicinal Plant Extract

Treatment of diabetic Wistar rats with ethanol extract of Cucumis sativus significantly increased the activities of the antioxidant enzymes and molecules as well as concentrations of NO, but it markedly reduced the concentrations of hepatic TP and MDA (p < 0.05). These results are shown in Tables 3 to 6.

Table 3: Effect of Ethanol Extract of C. sativus on Oxidative Status in Rat Liver

Data are markers of oxidative stress and are expressed as mean ± SEM (n = 5).

Values with superscript “a” are significantly different from the diabetic control group.

Table 4: Effect of Ethanol Extract of C. sativus on Hepatic TP and Glutathione Level

Data are hepatic TP and GSH levels and are expressed as mean ± SEM (n = 5).

Values with superscript “a” are significantly different from the diabetic control group.

Table 5: Effect of Ethanol Extract of C. sativus on Hepatic Glutathione Peroxidase Activity and NO Level

Data are hepatic glutathione peroxidase activity and NO level, and are expressed as mean ± SEM (n = 5).

Values with superscript “a” are significantly different from the diabetic control group.

Table 6: Effect of Ethanol Extract of C. sativus on Hepatic Concentrations of Antioxidant Molecules

Data are hepatic levels of antioxidant molecules, and are expressed as mean ± SEM (n = 5).

Values with superscript “a” are significantly different from the diabetic control group.

DISCUSSION

The World Health Organization (WHO) describes diabetes mellitus as a chronic, progressive disease characterized by elevated levels of blood glucose which causes complications in many parts of the body and increases the overall risk of dying prematurely [28]. This chronic non-communicable disease of the endocrine system arises from multiple etiologies in the secretion of insulin [29, 30]. Diabetes mellitus describes a group of metabolic disorders characterized by a state of chronic hyperglycemia due to defects in insulin secretion, insulin action or both, which currently affects about 463 million people worldwide [31, 32]. Over time, elevated blood glucose, a common effect of uncontrolled diabetes mellitus, may lead to serious damage to the heart, blood vessels, eyes, kidneys and nerves [28]. This metabolic disorder is often described as “a silent killer” since it may be asymptomatic at onset. Therefore, the disease usually goes undiagnosed until major complications arise. The disease is associated with reduced life expectancy, significant morbidity as well as diminished quality of life [33 – 35]. It has been reported that 10 % of global health expenditure is spent on diabetes [36, 37]. There is profound reason to suggest that this figure might increase in the coming years giving the myriad of complications that result from diabetes mellitus. Unfortunately, four out of five people with diabetes are living in low and middle-income countries, where healthcare budgeting is not even prioritized [38]. The lack of prioritization of healthcare in these countries consequently creates a yawning gap in efforts aimed at possibly managing and tackling the disease scourge. Diabetes mellitus has ceased to be a disease of affluence and has become a disease of globalization. Currently, two out of three people with diabetes are living in urban areas of the world [38]. Therefore, it is a considered opinion that the enormous increase in prevalence of cases of diabetes mellitus may not be unrelated to factors such as increasing urbanization, increasing prevalence of overweight and obesity, lack of physical activity as well as changes in socio-demographic characteristics of the population [39, 40].

Many of the drugs currently used for the treatment of diabetes mellitus produce adverse effects: sulfonylureas stimulate pancreatic islet cells to secrete insulin, while metformin slows down hepatic glucose production [41]. All these therapies have limited effectiveness, thereby necessitating the search for novel plant-based compounds that can effectively reduce blood glucose. According to World Health Organization, about 80 % of the world’s population rely essentially on plants for primary health care [42]. There is growing interest in the exploitation of plants for medicinal purposes, especially in Africa [43 – 53]. The antidiabetic effect of plant-derived compounds is due to their capacity to alter carbohydrate digestion/absorption, stimulate beta cell function, mimic insulin action, and mop up ROS [54- 57]. The aim of this study was to investigate the antioxidant effect of ethanol extract of C. sativus on STZ-induced diabetic rat liver. The antioxidants measured were catalase, SOD, GPx, GSH, ascorbic acid, retinoic acid and α-tocopherol. The concentrations of nitric oxide, TP and MDA were also determined.

Oxidative stress has mediatory role in the pathogenesis of diabetes mellitus and its related complications via promotion of free radicals production and impairment of antioxidant defense systems. The results of this study showed that induction of diabetes mellitus with STZ significantly increased the blood glucose concentrations of the rats. However, treatment of the diabetic rats with the extract markedly reduced the FBG concentration and body weights of rats. Similarly, treatment of the diabetic Wistar rats with the medicinal plant extract significantly increased the activities of the antioxidant enzymes and molecules as well as concentrations of NO, but it markedly reduced the concentrations of hepatic TP and MDA. The effect of the extract was comparable to that of metformin. The evaluation of plasma TP alone may not tell the actual picture of the metabolic state of an individual, since the concentration of the various proteins are not affected by each other. An elevated level of TP may be due to dehydration or infection. Plasma concentration may decrease due to impaired synthesis that can result from malnutrition, malabsorption, over-hydration and some forms of liver diseases [58]. The results of this study are consistent with those of earlier studies [59 – 61]. It is likely the medicinal plant extract contains important phytochemicals that can potentiate inherent antioxidant defense mechanism in rats [62 – 72].

CONCLUSION

Ethanol extract of C. sativus has protective effect in diabetes complications and can be considered a suitable drug candidate for reducing oxidative stress typically observed in diabetes mellitus.

REFERENCES   

1. Palsamy, P., Sivakumar, S. and Subramanian, S. (2010). Resveratrol attenuates hyperglycemia-mediated oxidative stress, proinflammatory cytokines and protects hepatocytes ultrastructure in streptozotocin-nicotinamide-induced experimental diabetic rats. Chem Biol Interact. 186: 200–210.
2. Leclercq, I.A., Da Silva-Morais, A., Schroyen, B., Van-Hul, N. and Geerts, A. (2007). Insulin resistance in hepatocytes and sinusoidal liver cells: Mechanisms and consequences. J Hepatol. 47: 142–156.
3. Wei, Y., Chen, P., de Bruyn, M.D., Zhang, W., Bremer, E. and Helfrich, W. (2010). Carbon monoxide-releasing molecule-2 (CORM-2) attenuates acute hepatic ischemia reperfusion injury in rats. BMC Gastroenterol. 10: 42.
4. Reid, A.E. (2006). Non-alcoholic fatty liver disease. In: Feldman, M., Friedman, L.S., Brandt, L.J., Eds. Sleisenger and Fordtran’s Gastrointestinal and Liver Disease: Pathophysiology/diagnosis/management, 8th ed. St. Louis, Missouri, USA: Saunders. Pp. 1772–99.
5. Crawford, J.M. and Iacobuzio-Donahue, C. (2009). Liver and biliary tract. In: Kumar V, Abbas AK, Fausto N, Aster JC, Eds. Robbins and Cotran Pathologic Basis of Disease. 8th ed. Philadelphia, Pennsylvania, USA: Saunders. Pp. 833–890.
6. Leung, T.M. and Nieto, N. (2013). CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease. J Hepatol. 58: 395–398.
7. Parveen, K., Khan, M.R., Mujeeb, M. and Siddigui, W.A. (2010). Protective effects of Pycnogenol on hyperglycemia-induced oxidative damage in the liver of type 2 diabetic rats. Chem Biol Interact. 186: 219–227.
8. Welt, K., Weiss, J., Martin, R., Dettmer, D., Hermsdorf, T. and Asayama, K. (2004). Ultrastructural, immunohistochemical and biochemical investigations of the rat liver exposed to experimental diabetes and acute hypoxia with and without application of Ginkgo extract. Exp Toxicol Pathol. 55: 331–345.
9. Wei, Y., Chen, P., de Bruyn, M.D., Zhang, W., Bremer, E. and Helfrich, W. (2010). Carbon monoxide-releasing molecule-2 (CORM-2) attenuates acute hepatic ischemia reperfusion injury in rats. BMC Gastroenterol. 10: 42.
10. Han, D., Hanawa, N., Saberi, B., Kaplowitz, N. (2006). Mechanisms of liver injury: III – Role of glutathione redox status in liver injury. Am J Physiol Gastrointest Liver Physiol. 291: G1–7.
11. Manna, P., Das, J., Ghosh, J. and Sil, P.C. (2010). Contribution of type 1 diabetes to rat liver dysfunction and cellular damage via activation of NOS, PARP, IkappaBalpha/NF-kappaB, MAPKs, and mitochondria-dependent pathways: Prophylactic role of arjunolic acid. Free Radic Biol Med. 48: 1465–1484.
12. Ahmed, N. (2004). Advanced glycation endproducts: Role in pathology of diabetic complications. Diabetes Res Clin Pract. 67: 3–21.
13. Horiuchi, S. (2002). The liver is the main site for metabolism of circulating advanced glycation end products. J Hepatol. 36: 123–125.
14. Yagmur, E., Tacke, F., Weiss, C., Lahme, B., Manns, M.P. and Keifer, P. (2006). Elevation of Nepsilon-(carboxymethyl)lysine-modified advanced glycation end products in chronic liver disease is an indicator of liver cirrhosis. Clin Biochem. 39: 39–45.
15. Abu, O.D., Imafidon, K.E., Obayuwana, H.O. and Okuofu, E.D. (2017). Phytochemical, proximate, and metal content analysis of citrullus lanatus (watermelon) seeds. FUDMA Journal of Sciences, 2 (2): 153 – 156.
16. Abu, O.D. and Onoagbe, I.O. (2019a). Biochemical effect of aqueous extract of Dialium Guineense stem bark on oxidative status of normal Wistar rats. International Journal of Clinical Biology and Biochemistry. 1 (2): 15 – 18.
17. Cohen, G., Dembie, C.D. and Marcus, J. (1970). Measurement of catalase activity in tissue extracts. Analytic Biochemistry. 34: 30 – 38.
18. Misra, H.R. and Fridovich, I. (1972). The role of superoxide anions in the auto oxidation of epinephrine and a single assay for superoxide dismutase. J Biol. Chem. 247: 3170 – 3175.
19. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Hafeman, D.G. and Hockstra, W.G. (1973). Selenium biochemical role as a component of glutathione peroxidase. Science. 179: 588 – 590
20. Henry, R.J., Sobel, C. and Beckman, S. (1957). Determination of serum protein by the Biuret reaction. Anal. Chem. 92 (149): 1 – 5.
21. Ellman, G.L. (1959). Tissue sulphydryl groups. Archive of Biochemistry and Biophysics. 82 (1): 70 – 77.
22. Guttridge, J.M.C. and Wilkins, C. (1982). Cancer dependent hydroxyl radical damage to ascorbic acid. Formation of thiobarbituric acid reactive product. FEBS Lett. 137: 327 – 340.
23. Marcocci L, Packer L, Droy Lefaix MT, Sekaki A, et al. (1994) Antioxidant action of Ginkgo biloba extract EGb 761. Methods in Enzymology 234: 462-475.
24. Neeld, J.B. and Pearson, W.N. (1963) Macro-and Micro-Methods for the Determination of Serum Vitamin a Using Trifluoroacetic Acid. Journal of Nutrition. 79: 454 – 462.
25. Omaye, S.T. Turabull, J.D. and Sanberlich, H.E. (1979). “Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids”. Methods Enzymology. 62: 1 – 11.
26. Desai, I.D. (1984). Vitamin E methods for animal tissues. Methods Enzymol. 105: 138 – 143.
27. Abu, O.D. and Ikponmwosa-Eweka, O. (2022a). Evaluation of the Potential of Total saponins and Tannins of Dialium guineense Stem Bark in the Amelioration of Carbon Tetrachloride-Induced Renal Oxidative Stress. SAU Science-Tech. Journal. 7 (1): 42 – 50.
28. World Health Organization (WHO). (2016). Global Report on Diabetes. Part 1: Global burden of diabetes. World Health Organization: Geneva.
29. Todkar, S.S. (2016). Diabetes mellitus the ‘Silent Killer’ of mankind: An overview on the eve of upcoming World Health Day! Journal of Medical and Allied Sciences. 6 (1): 39 – 44.
30. Omodanisi, E.I., Aboua, Y.G. and Oguntibeju, O.O. (2017). Assessment of the anti-hyperglycaemic, anti-inflammatory and antioxidant activities of the methanol extract of Moringa oleifera in diabetes-induced nephrotoxic male wistar rats. Molecules. 22 (4): E439.
31. Ozougwu, J.C., Obimba, K.C., Belonwu, C.D. and Unakalamba, C.B. (2013). The pathogenesis and pathophysiology of type 1 and type 2 diabetes mellitus. Journal of Physiology and Pathophysiology. 4 (4): 46 – 57.
32. International Diabetes Federation (IDF). (2019). Diabetes Atlas (9th edition). International Diabetes Federation: Brussels, Belgium.
33. Abbott, A.D., Brand, F.N. and Kannel, W.B. (1990). Epidemiology of some Peripheral Arterial Findings in Diabetic Men and Women- Experiences from the Framingham Study. Am J Med. 88: 376 – 381.
34. Barceló, A. and Rajpathak, S. (2001). Incidence and prevalence of diabetes mellitus in the Americas. Rev Panam Salud Publica. 10 (5): 300 – 308.
35. Funke, I. and Melzig, M.F. (2006). “Traditionally used plants in diabetes therapy-phytotherapeutics as inhibitors of α-amylase activity”. Revista Brasileira De Farmacognosia. 16: 1 – 5.
36. Cho, N.H., Shaw, J.E., Karuranga, S., Huang, Y., da Rocha Fernandes, J.D., Ohlrogge, A.W. and Malanda, B. (2018). IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Research and Clinical Practice. 138: 271 – 281.
37. International Diabetes Federation (IDF). (2000). Diabetes Atlas (1st edition). International Diabetes Federation: Brussels, Belgium.
38. International Diabetes Federation (IDF). (2017). Diabetes Atlas (8th edition). International Diabetes Federation: Brussels, Belgium.
39. Waugh, N.R., Shyangdan, D., Taylor-Phillips, S., Suri, G. and Hall, B. (2013). Screening for type 2 diabetes: a short report for the National Screening Committee. Health Technology Assessment. 17: 1 – 90.
40. Saleem, S.M., Khan, S.M.S. and Jan, S.S. (2017). Finnish Diabetic Risk Score: A Tool for Predicting Risk of Undiagnosed Type 2 Diabetes Mellitus. Annals of Medical and Health Science Research. 7: 295 – 298.
41. Moller, D.E. (2001). “New drug targets for type 2 diabetes and the metabolic syndrome”. Nature. 414 (6865): 821 – 827.
42. Abu, O. D., Imafidon, K. E. and Iribhogbe M. E. (2015). Biochemical effect of aqueous leaf extract of Icacina trichanta Oliv. on urea, creatinine and kidney oxidative status in CCl₄-induced Wistar rats. Nigerian Journal of Life Sciences. 5 (1): 85 – 89.
43. Abu, O.D., Onoagbe, I.O., and Obahiagbon, O. (2020a). Alpha amylase and alpha glucosidase inhibitory activities of extracts of Dialium guineense stem bark. International Journal of Clinical Biology and Biochemistry. 2 (1): 7 – 10.
44. Abu, O.D., Onoagbe, I.O., and Osemwenoyenmwen, O. (2020b). Alpha amylase and alpha glucosidase inhibitory activities of isolated total saponins and tannins of Dialium guineense stem bark. Journal of Cellular and Molecular Biology Research. 1 (2): 1 – 3.
45. Abu, O.D., Onoagbe, I.O. and Obahiagbon, O. (2020c). In Vitro Antioxidant Activities of Isolated Total Saponins and Tannins of Dialium Guineense Stem Bark. IAR Journal of Medical Sciences. 1 (4): 193 – 199.
46. Abu, O.D., Onoagbe, I.O. and Obahiagbon, O. (2020d). Vitamin contents of extracts of Dialium guineense stem bark. Biomed J Sci and Tech Res. 30 (2): 23263 – 23267.
47. Abu O.D., Onoagbe I.O. and Obahiagbon O. (2020e). Analyses of metal and amino acid compositions of aqueous and ethanol stem bark extracts of Dialium guineense. Journal of Biogeneric Science and Research. 6 (4): 1 – 3.
48. Abu, O.D., Onoagbe, I.O. and Obahiagbon, O. (2020f). Phenolic contents of extracts of Dialium guineense stem bark. American Journal of Sciences and Engineering Research. 3 (4): 92 – 96.
49. Abu, O.D. and Onoagbe, I.O. (2021). Acute toxicity of aqueous and ethanol extracts of Dialium guineense stem bark. Journal of Bioinnovation. 10 (2): 427 – 432.
50. Abu, O.D., Adeogun, E.F. and Ebhohon S.O. (2019b). Oral LD₅₀of total saponins and tannins isolated from Dialium guineense stem bark. European Journal of Experimental Biology. 9 (2): 11 – 13.
51. Abu, O.D., Onoagbe, I.O., and Ojo, I. (2021). Determination of effective dose for ethanol extract of Dialium guineense stem bark. Journal of Medical Research and Case Reports. 3 (2): 1 – 4.
52. Abu, O.D. and Ikponmwosa-Eweka, O. (2022b). Potential of Extracts of Dialium   guineense Stem Bark in the Mitigation of Carbon Tetrachloride-induced Renal Oxidative Stress. BIU Journal of Basic and Applied Sciences. 7 (1): 62 – 69.
53. Abu, O.D. and Ikponmwosa-Eweka, O. (2022j). Potential of Total Saponins and Tannins Isolated from the stem bark of Dialium guineense in the Amelioration of Kidney Dysfunction Caused by CCl₄. Journal of Basic and Applied Medical Sciences. 2 (1): 1 – 6.
54. Tiwari, A.K. and Rao, J.M. (2002). Diabetes mellitus and Multiple Therapeutic Approaches of Phytochemicals- Present Status and Future Prospects. Current Sci. 83: 30 – 38.
55. Abu, O.D., Imafidon K.E. and Obayuwana, O. (2020). Ethanol leaf extract of Anacardium occidentale ameliorates alloxan-induced changes on blood glucose level and lipid profile of Wistar rats. IAR Journal of Medical Sciences. 1 (5): 257 – 262.
56. Abu, O.D., Imafidon K.E. and Obayuwana, O.(2020). Effect of aqueous extract of Anacardium occidentale leaves on blood glucose level and lipid profile of diabetic rats. Global Scientific Journal. 8 (10): 977 – 987.
57. Obayuwana, , Imafidon, K.E. and Abu, O.D. (2020). Phytochemical and proximate composition of leaves of Anacardium occidentale. IAR Journal of Agriculture Research and Life Sciences. 1 (5): 139 – 142.
58. Tietz, N.W., Finley, P.R. and Pruden, E.L. (1990) Clinical Guide to Laboratory Tests. 2^nd Edition, W.B. Saunders, Philadelphia. Pp. 304 – 306.
59. Chandrasekar, B., Mukherjee, B. and Mukherjee, S.K. (1989). Blood lowering potentiality of selected Cucurbitaceae plants of Indian origin. Indian J. Med Res. 90: 300 – 305.
60. Minaiyan, M., Zolfaghari, B., Kamal, A. (2011). Effect of hydroalcoholic and butanolic extract of Cucurmis sativus seeds on blood glucose level of normal and streptozotocin-induced diabetic rats. Iran J Basic Med Sci. 14: 436 – 442.
61. Atta, A.H., Saad, S.A., Atta, S.A., Mouneir, M., Nasr, S.M., Desouky, H.M. and Shaker, H.M. (2020). Cucumis sativus and Cucurbitamaxima extract attenuate diabetes-induced hepatic and pancreatic injury in a rat model. J. Physiol Pharmacol. 71 (4).
62. Abu, O.D., Avenbuan, S.E. and Osarhenomase, E.G. (2023). Renal Oxidative Status in Diabetic Wistar Rats Administered Ethanol Extract of Cucumis sativus Whole Fruit. Int. J. of Clinical Studies and Medical Case Reports. 30(1): 1-4
63. Abu O.D., Awhin E.P. and Ozedu M.E. (2023). Evaluation of Cardiovascular Disease Risk Factors in Diabetic Rats Administered Ethanol Extract of Cucumis sativus Fruit. African Journal of Health, Safety and Environment. 4(1): 108 – 117.
64. Abu O.D., Awhin E.P. and Iyare H.E. (2023). Assessment of Renal Function in Diabetic Wistar Rats Treated with Ethanol Extract of Cucumis sativus. African Journal of Health, Safety and Environment. 4(1): 101-107.
65. Abu O.D., Awhin E.P. and Ifekwe, J.C. (2023). Liver Function Status of Diabetic Wistar Rats Treated with Ethanol Extract of Cucumis sativus Fruit.  Biomedical Journal of Scientific and Technical Research. 51 (2): 42440 – 42445.
66. Abu, O.D., Ojo, I. and Ezike, T.V. (2023). Methanol Fraction of Ethanol Extract of Dialium guineense Stem Bark Mitigates STZ-Induced Oxidative Stress in Rat Biomedical Journal of Scientific and Technical Research. 51 (2): 42594 – 42600.
67. Abu, O.D., Osime, E.C. and Ngedaa, O.S. (2023). Cardiac Oxidative Status in Diabetic Wistar Rats Exposed to Ethanol Extract of Cucumis sativus Fruit. J. Diagnostics and Case Reports. 4 (2): 1 – 5.
68. Abu, O.D., Ojo, I. and Awhin, E.P. (2023). Protective Property of Ethanol Extract of C. sativus on STZ-Induced Diabetic Rat Pancreas. Biomedical Journal of Scientific and Technical Research. 52(2): 43613-43618.
69. Abu, O.D., Awhin, E.P. and Iyare, H.E. (2023). Investigation of Renal Function in Diabetic Rats Treated with Methanol Fraction of Ethanol Extract of Dialium guineense (MEDG) Stem Bark. Journal of Urology and Nephrology Studies, 4 (4): 513 – 518.
70. Abu, O.D., Obaze, G.E., Egili, S. and Idehen, I.O. (2023). Ethanol Extract of C. sativus Modulates the Activity of Glucose 6-phosphatase/ Aminotransferases and Levels of Lipids in Tissues of STZ-Induced Diabetic Rats. Biomedical Journal of Scientific and Technical Research. 53(4): 44989-44994.
71. Abu O.D., Ohikhuare F. and Ezike T.V. (2023). in vitro Antioxidant Activity of Aqueous and Ethanol Extracts of Cucumis sativus. Journal of Clinical Epidemiology and Public Health. 01 (03): 1 – 6.
72. Abu, O.D., Awhin, E.P. and Ohikhuare, F. (2023). Effect of Methanol Fraction of Ethanol Extract of Dialium guineense Stem Bark on Cardiovascular Disease Risk Factors in Diabetic Rats. Journal of Biology and Medicine, 4 (1): 128.