Ameliorative Effect of Ethanolic Extract of Cucumis Sativus (Cucumber) Pulp on Alloxan Induced Kidney Toxicity in Male Adult Wistar Rats

Nephropathy is a common complication of diabetes, managed with both orthodox and herbal remedies. Cucumis sativus is a creeping plant belonging to the Cucurbitaceae family. Its edible fruits possess diverse pharmacological activities. This study investigated the effect of ethanolic extract of cucumis sativus pulp (EECSP) on alloxan induced renotoxicity in adult male Wistar rats. Thirty adult male Wistar rats weighing between 170-190g were purchased and acclimatized for 2 weeks and afterwards grouped into 6 groups (A-F) of 5 rats each. Group A (Control) received distilled water and rat feed ad libitum . Diabetes was induced in Groups B, C, D, and E with consequent renotoxicity using a single intraperitoneal injection of 150mg/kg of Alloxan after an overnight fast. Group C, D and E were afterwards treated with 100, 250 and 500mg/kg of ethanolic extract of cucumis sativus pulp respectively. Group F received 500mg/kg of EECSP. Extract administration was oral and once daily for 28days. At the end of the experiment, blood samples were collected via ocular puncture and sera separated for kidney function test. The animals were sacrificed and their kidneys collected, and prepared for light microscopy. Results showed that treatment with EECSP caused a dose dependent restoration of kidney architecture and renal functions when compared with untreated alloxan group that showed severe distortions in renal architecture and higher than normal serum urea and creatinine levels. Ethanolic extract of cucumis sativus pulp thus caused a dose dependent ameliorative effect on alloxan induced diabetic nephropathy in adult male Wistar rats.

Treatment of diabetes induced nephrotoxicity has over the years been achieved not only via generic drugs but also by plant/herbal concoctions. These concoctions are derived from plants rich in phytochemicals that possess vital cell, and tissue soothing properties. Such plants and herbs are not only cheap, but readily accessible. One of such plants is Cucumis sativus (Cucumber).
Cucumis sativus (Cucumber) is a widely distributed plant. It belongs to the Cucurbitaceae family, a creeping vine that roots in the ground and grows up trellises or other supporting frames, wrapping around supports with thin, spiraling tendrils (Huang et al., 2009;Trease et al., 2002). C. sativus bears edible fruit that possess diverse pharmacological activities such as antibacterial, antifungal, antidiabetic, cytotoxic, antacid, carminative, hepatoprotective, and wound healing activities, providing a significant role in the prevention and treatment of diseases (Jony et al., 2013). This is as a result of its nutritional (Vitamin B1, ascorbic acid, proteolytic enzyme, rutin, oxidase, succinic and maleic dehydrogenases) and phytochemical constituents (steroids, flavonoids, and tanins) (Kumar et al., 2010).
Limited literature exists on the effects of Cucumis sativus (Cucumber) fruit on alloxan induced diabetic kidney toxicity thus the need to carry out this research to investigated the effect of ethanolic extract of cucumis sativus pulp (EECSP) on alloxan induced diabetic renotoxicity in adult male Wistar rats.

Procurement of Alloxan and Induction of Experimental Diabetes
Alloxan in the form of Alloxan Monohydrate (Sigma-Aldrich, St. Louis, MO, USA) was purchased from Cyleon-C Pharmacy at Nnewi, Anambra State, Nigeria. Alloxan (2, 4, 5, 6-tetraoxypyrimidine; 2, 4, 5, 6-pyrimidinetetrone) is a pyrimidine derivative, and a toxic glucose analogue that selectively destroys insulin-producing beta cells of the pancreas of experimental animal (Onyeka et al., 2013). This causes symptoms similar to type 1 diabetes in humans with consequent renotoxicity. 150mg of 0.5grams of alloxan monohydrate (Sigma chemicals, USA) was dissolved in 5ml of normal saline to form a stock solution, and 150mg/kg was withdrawn and injected intraperitoneally to the experimental animals were applicable after 48 hour fast (allowed access to only water) to cause the development of diabetes. Diabetes was confirmed in alloxan-treated rats with a fasting blood sugar concentration 126mg/dl.

Procurement of and Preparation of Cucumis Sativus (Cucumber) Fruit Extract.
Cucumis Sativus fruits were purchased from the Nkwo market in Nnewi. They were afterwards washed in running tap water to remove dirt, and subsequently cut into pieces. These cut pieces were then shed dried and milled into coarse powder using Local grinder. 50g of the cucumis sativus pulp powder was macerated in 95% ethanol for 48hours, which was then filtered using porcelain cloth and further filtered using Whatman No 1 filter paper. The filtrate was concentrated using a rotatory evaporator and was further dried using a laboratory oven at 50°C into a semi solid gel-like form. Part of the extract was used for phytochemical analysis while the remainder was preserved in a refrigerator at -4 0 C for further usage.

Phytochemical Screening
Phytochemical screening was carried out using a small portion of the ethanolic extract of Cucumis sativus pulp to determine the presence of tannins, flavonoids, alkaloids, saponins and steroids using modified methods of Harborne, (1998), Tiwari et al., (2011), and Ebirien-Agana et al., (2016 Campus, Nnamdi Azikiwe University, Anambra State. Animals were kept in standard plastic rat cages at a room temperature of 27±2°C, and exposed to atleast a period of 12hours light and dark cycles. These animals were fed with rat chow (Growers Vital top feed, Jos, Nigeria) and water ad libitum for a period of two weeks prior to the onset of substrate administration. This procedure was to allow for proper acclimatization.

Experimental Design and Protocol
After acclimatization, these adult male Albino wistar rats were grouped into 6 groups (A-F) of 5 rats each. The administration lasted for duration of 28 days. Group A served as Control group, and received only distilled water and rat chow ad libitum throughout the duration of the experiment. Diabetes and consequent renotoxicity was induced in Groups B, C, D, and E using a single intraperitoneal injection of 150mg/kg of Alloxan after an overnight fast. Group C, D and E were afterwards treated with 100, 250 and 500mg/kg of ethanolic extract of cucumis sativus pulp respectively 48 hours after confirmation of diabetes. While Group F received 500mg/kg of ethanolic extract of Cucumis Sativus. All extract were orally administered and lasted for duration of 28 days.
2.6 Termination of Experiment, Serum Blood collection, and Organ collection At the end of the experiment which lasted for 30 days, the experimental animals were carefully injected with ketamine hydrochloride 24hours after the last administration of extract so as to anaesthetize the animals. Blood samples were collected from the rats before sacrificing via ocular puncture for Kidney Function Test. These blood samples were put in plain sterilized glass tubes devoid of anticoagulant. The samples were centrifuged with a laboratory ultracentrifuge machine (New life model) and serum collected and stored in the refrigerator. Afterwards, the animals were sacrificed via cervical dislocation, dissected through the anterior abdominal region and their kidneys harvested, washed in normal saline, weighed and then transferred into specimen bottles containing freshly prepared 10% formal saline for preservation prior to histological processing. The remains of the experimental animals were properly disposed.

Kidney Function Test
Estimation of creatinine was carried out according to the method described by Owen et al., (1954), while estimation of urea was carried out using the method described by Marsh et al., (1965).

Tissue Processing and Microscopy
The harvested kidneys were fixed in 10% formal saline in specimen bottles with light fitting lids for three days to prevent autolysis; improve their staining qualities and aid optical differentiation of cell and their components. Afterwards, the fixed kidney tissues were dehydrated to remove water using ascending grades of alcohol ranging from 50% to absolute Alcohol for 30 minutes each. Thereafter, the dehydrated tissues were cleared by removing the alcohol via immersion through three changes of xylene for 30 minutes each. These cleared tissues were then impregnated and infiltrated to remove the xylene by passing it through three changes of molten paraffin wax in a hot oven temperature of 60 o C for 30 minutes. The infiltrated tissues were embedded in molten paraffin wax using an embedding mould and allowed to cool and solidify. The paraffin blocks obtained afterwards were trimmed so as to expose the surface of the tissue by adjusting the microtome knife to 15µm. Thereafter, and sections were cut with rotary microtome and the ribbons placed on 20% alcohol, and picked on a large slide (5cm X 7.5cm slide) to remove minor folds and creases from sections. These ribbons were placed on a water bath preheated to about 45 o C in order to float out the tissues. The tissues were then collected with clean slides, allowed to dry, and well labelled with the aid of a diamond pencil. These slides were afterwards dried on a hot plate at 5 o C for the tissue to adhere to the slide. The tissues were dewaxed in xylene for 10 minutes and then rinsed in descending order of alcohol ranging from absolute 95%, 90%, 70% and 50% for two seconds each. The tissues were then washed in two changes of water and differentiated in 1% acid alcohol. Afterwards, the tissues were stained using Hematoxylin and washed in tap water until the tissues colour changed to blue. The tissues were counterstained in Eosin for 5 minutes and cleared in xylene prior to mounting in DPX, and dried for photomicrography and histopathological interpretation.

Statistical Analysis
Data obtained from this study were analyzed using Statistical Package for Social Sciences (SPSS Version 23). The results were expressed as mean ± Standard Error of Mean (S.E.M.). Data for Kidney Function Test (Urea &Creatinine) was analyzed using One-way ANOVA, followed by Post hoc LSD. While body weights were analyzed using Student dependent T-test. Values were considered significant at P<0.05.

Physical and Behavioural Observations
At the beginning of the experiment, all animals were apparently healthy and agile. During the two weeks of acclimatization, their stool was dark brown and they adapted well to their environment. However, during the first week of alloxan injection, most of the rats showed signs of weakness, reduction in food and water intake and polyuria. In the group that received alloxan and were left untreated, feeding habits improved slightly, although they got weaker as the experiment progressed. In the groups that received alloxan and were later treated with graded doses of ethanolic extract of Cucumis Sativus, feeding habits and urine outputs returned to normal as the experiment progressed. In group 5 (that received only 500mg/kg of the ethanolic leaf extract of Cucumis Sativus), they were apparently healthy and agile throughout the duration of the experiment.  Result from the tables 1 and 2 above shows that there was a significant increase in the body weight in group A when comparing the Initial weight to the Final weight. In group B, there was a significant decrease in the body weight when comparing the initial weight to the final weight. In group C, there was a significant increase in the body weight when comparing the Initial weight to the Final weight. In group D, there was an insignificant increase in the body weight when comparing the initial weight to the final weight. In group E, there was an insignificant increase in the body weight when comparing the initial weight to the final weight. In group F, there was a significant increase in the body weight when comparing the Initial weight to the Final weight.

Effects on Body weight
The result showed that the body weight difference increase in group B and F, while a decrease in group C and C, D and E when compared with group A, but showed a significance in group B, C and F while D and E was not significant. Result from table 3 above showed that there was a significant increase in the serum Urea concentration in group B, C, D, E and F when compared to group A (Control). Group B that received a single intraperitoneal injection of 150mg/kg of alloxan monohydrate only, had the highest value of serum urea levels. Result from table 4 above showed that there was a significant increase in the serum creatinine concentration in group B, and C when compared to group A (Control). Group B that received a single intraperitoneal injection of 150mg/kg of alloxan monohydrate only, had the highest value of serum creatinine levels.

Discussion
Plant derived remedies for various ailments have gained popularity especially in the rural areas of most developing countries. These plants contain phytochemicals that have been documented to be medicinal, promoting good health, aiding healing process, while at the same time causing cellular regeneration (Onyeka et al., 2013;Ofoego et al., 2017;. Findings from this study showed that a single intraperitoneal injection of 150mg/kg of alloxan to male wistar rats weighing between 170-190g and left untreated, caused alterations in feeding habits which slightly improved as the experiment progressed, although the experimental animals got weaker as the experiment progressed with signs and symptoms characteristic of diabetes type 1. This alterations in feeding habits caused a significant decrease in the body weight (P<0.05) in the untreated diabetic group. This findings support the findings of Akbarzadeh et al., (2007), Muhammad et al., (2010), Ewenighi et al., (2015, and  who all reported loss of weights in diabetic experimental animals. Diabetes is also usually accompanied with increased biochemical activities such as glycogenolysis, lipolysis, gluconeogenesis causing muscles wasting and loss of tissue protein (Ewenighi et al., 2015). However, in diabetic groups treated with ethanolic extract of Cucumis Sativus pulp, their feeding habits were improved, with resultant weight gains in a dose dependent manner although these gains were lower than that of control (P<0.05).
Kidneys maintain body homeostasis by optimizing the composition of body fluid through the removal of metabolic wastes such as urea and creatinine. Blood urea and creatinine levels, are routinely used as markers of renal functions, and are sensitive markers of renal damage (Olubunmi and Adeyemi, 2015). An increase in urea and creatinine concentration in all groups that received intraperitoneal administration of alloxan was observed in the current research when compared to control group indicating diabetic nephropathy, one of the most common complications due to diabetes (Donnelly et al., 2000;Lepedda et al., 2016). The increased levels of urea and creatinine observed could be as a result of impaired function of the kidney following damage caused by alloxan. Alloxan and its end product of reduction, dialuric acid, have been found to be involved in a redox cycle causing the production of large amounts of superoxide radicals. These radicals are converted to hydrogen peroxide by undergoing dismutation, causing alkylation of DNA. This leads to liberation of large toxic amounts of nitric oxide that inhibits aconitase activity causing DNA damage (Szkudelski, 2001), which could have led to the severe damage and necrosis of kidney tissue as observed in the micrograph of the untreated diabetic rat in this study. These findings agree with those of Hohenegger and Rudas, (1971), Abdulazeez, (2014), and Sharma et al., (2014). It has been documented, that serum levels of creatinine and urea increases when more than 50% of the renal mass is lost (Braun and Lefebvre, 2008). This is further confirmed by the kidney micrograph of untreated alloxan induced diabetic rats seen in this current research, which showed extensive necrosis.
However, it was evidenced that in the current research, administration of ethanolic extract of cucumis sativus  Vol.9, No.4, 2019 20 pulp to alloxan induced diabetic rats caused a marked reduction in the serum levels of urea and creatinine in a dose dependent manner. Flavonoids, saponins, alkaloid, and steroids found in the ethanolic extract of cucumis sativus pulp used in this research has been documented to improve general diabetic conditions through various pathways such as improving glycolytic and glycogenic processes with simultaneous decline in glycogenolysis and gluconeogenesis (Andrade-Cetto et al., 2003). This could be behind the improvements in urea and creatinine levels observed after cucumis sativus administration to the diabetic rats, as well as improved feeding habits and renal histology seen in their respective micrographs. These improvements were however dose dependent. This pattern of activity could be as a result of these dietary flavonoids from cucumber synergizing with antioxidants for optimal cellular functions. They easily cross the cell membrane and cope with the intracellular ROS formation (Heidari et al., 2012). Studies have also isolated isovitexin and isoorientin, two C-glycosylflavones from cucumis sativus pulp. Both C-glycosylflavones have been reported to exhibit protective and antioxidant effects on animal tissues (Krauze- Baranowska and Cisowski, 2001).
The histopathological findings corroborated that Alloxan caused kidney damage with severe necrosis of tubular cell, coagulative necrosis of glomeruli, severe fatty change and tubular necrosis in untreated Alloxan group. This could without doubt, be as a result of the generation of reactive oxygen species (ROS) that caused damage to the kidney architecture by interfering with various biochemical activities thus impairing the function of the kidney (Donnelly et al., 2000;Lepedda et al., 2016). There was however a dose dependent restoration of the architecture of alloxan induced renal histology with treatment with graded doses of ethanolic extract of cucumis sativus pulp. 100mg/kg of ethanolic extract of cucumis sativus pulp showed mild healing and restoration, 250mg/kg of ethanolic extract of cucumis sativus pulp showed moderate healing of restoration of renal tissue architecture while treatment with 500mg/kg of ethanolic extract of cucumis sativus pulp showed a superlative renal histology restoration, bring it near normal renal architecture. The presence of phytonutrients such as flavonoids and tannins found in cucumis sativus has the ability to cause regeneration of damaged tissues (Tapas et al., 2008).

Conclusion
This present study has shown that ethanolic extract of Cucumis sativus (Cucumber) pulp was able to ameliorate kidney damage resulting from alloxan induced diabetic nephropathy. This activity was however dose dependent.