Nutritional content of selected species of tropical eggplant fruit (Solanum spp) diet Attenuates hepatic inflammation in high‐fat fed male Wistar rats induced with streptozotocin

Abstract Tropical Solanum species contains a high level of phenolic acids and flavonoids, which were found to inhibit some key enzymes associated with the incidence of type 2 diabetes in in vitro and in vivo models based on earlier studies. This study was further designed to compare the nutritional properties, glycemic index, and hypolipidemic and the antioxidant effects of three species of tropical eggplant fruit (Solanum kumba, Solanum aethiopicum, and Solanum gilo) diet on streptozotocin (STZ)‐induced nephrotoxicity in male Wistar rats. The animal model was subjected to high‐fat diet prior to interperitional administration of streptozotocin (35 mg/kg wt); thereafter, the rats were given supplemented eggplant fruit diet, which lasted for 14 days. Determination of lipid content [triglycerides (TG), low‐density lipoproteins (LDLs), high‐density lipoproteins (HDLs), and total cholesterol (TC)], was assessed, while the liver biomarker enzymes alanine aminotransferase (ALT) alkaline phosphatase (ALP) and aspartate aminotransferase (AST), also endogenous enzymes such as superoxide dismutase (SOD) and catalase (CAT), were determined. Histopathological assessment of inflammation was carried out on kidney while the blood urea nitrogen (BUN), uric acid, and creatinine level on the kidney function were determined. The results showed that the groups with supplemented eggplant diet had significant (p < 0.05) reduction in lipid profile, decreased leakages of the liver, and kidney function enzymes while there was restoration of depleted endogenous antioxidant enzymes. The inflammatory cells and fat deposit from the histopathological view were reduced. However, S. kumba had the best nutritional output.

to proteins or lipids which have caused elevated blood glucose, and these have led to increased level of generation of reactive oxygen. Increased oxidative stress invariably increased the production of reactive oxygen species that is known as free radicals.
These radicals are involved in the oxidation of protein in cellular structures, also in the oxidation of lipids which have led to cell injury associated with the pathology of vascular disease resulting in poor health and death in type II diabetes (Ghoul, 2012). Different reports have emphasized on natural products such as medicinal plants or plant fruits (Anosike, Abonyi, & Ubaka, 2011) and vegetables, based on folklore medicine to treat or manage diabetes mellitus (DM) (Nwanna et al., 2014). The use of natural products is pharmacologically active, not costly with little or minimal reactions as compared to other synthetic hypoglycemic agents. Fruits and vegetables have served a therapeutic role in different metabolic syndrome such as obesity, diabetes (Suhaila, 2014).
Eggplant commonly called garden egg or scarlet eggplants in West Africa are part of folklore remedy, which is used to curb elevated blood sugar and increased weight gain in Nigeria. According There is a need to scientifically explore these commonly found eggplant indigenous to sub-Saharan Africa (Anosike et al., 2011;Nagaoka, Watanabe, Sakata, & Yoshihara, 2001). Nwanna et al. (2014) and Nwanna, Ibukun, and Oboh (2016) reported on the in vitro and the in vivo activities of these eggplant species in the management of type 2 diabetes. However, this present study further determines the effect of this eggplant diet supplementation on STZ-induced nephrotoxicity in male Wistar rats and also assesses their hypolipidemia and antioxidant properties as well as the nutritional and glycemic index. The reagents and chemicals were purchased from Sigma-Aldrich chemical company, the USA. In addition, some kits were ordered from Randox Laboratories.

| Determination of proximate composition of eggplant species
Proximate analysis was carried out on the eggplant samples according to the method of Association of Official Analytical Chemists (AOAC) (2005) in order to determine the percentage of composition of protein, fat, fiber, ash, and the total carbohydrate.

| Starch and sugar determination
The method of Onitilo, Sanni, Daniel, Maziya-Dixon, and Dixon (2007) was used to determine the sugar and starch content of the sample. Hot ethanol 80% was used to digest 0.02 g of the sample, which was centrifuged at 2,000 g for 10 min. The supernatant was aspirated and used to determine the free sugar, while the residue was used to determine the start content according to the protocol.
Glucose was used as the standard.

| Determination of glycemic index
According to the method of Brounds et al. (2005), 25 mg of the eggplant powdered sample was weighed into a beaker, which was mixed with 5 ml of the stomach solution potassium chloride buffer at 1.5 pH and allowed in a shaker bath for 60 min at 40°C. It was then diluted with phosphate buffer pH 6.9 before the addition of 2.5 ml α-amylase solution and allowed to stay at 37°C for 10 min.
Thereafter, 200 μl of the digested sample was poured into test tube at 30-min interval (0, 30, 60, 90, 120, 150, and 180) min. The aliquots were boiled for 15 min before addition of 500 μl sodium acetate pH 4.75 followed by 5 μl of the α-glucosidase solution and then incubated for 45 min at 60°C. Two hundred microliters of DNSA solution was added and incubated for 5 min at 100°C; this was followed by an addition of 2 ml distilled water; and the mixture undergoes centrifugation at 3,000 g for 5 min. The supernatant was aspirated and read 540 nm. The sum of area under the curve for each sample was divided by the sum of area under the curve for standard glucose and multiplied by 100. The value obtained is the glycemic index.

| Amylose content determination
According to the method of Goñi, Garcia-Alonso, and Saura-Calixto (1997) and Brounds et al. (2005), 1 ml of 95% ethanol and 9 ml of 1 M NaOH were added to 0.1 g of eggplant plant sample and standard (amylose). The mixture was heated for 10 min, 1 ml was taken and made up to 10 ml with distilled water, and 0.5 ml was taken out from the diluted sample while 0.1 ml of acetic acid and 0.2 ml of iodine solution were added. It was eventually made up to 10 ml using distilled water and was allowed to stay at room temperature for 10 min., after which vortexes and absorbance were taken at 620 nm. The results were calculated using the following formula: [Amylose of sample (%) = (Absorbance standard) % − Absorbance sample %)/Absorbance standard].

| Amylopectin content determination
This was done using the method of Goñi et al. (1997). The percentage of amylose value of the amylose content determined was subtracted from 100%.

| Experimental design
The methods of Oboh and Ogunruku (2010), Srinivasan, Viswanad, Asrat, Kaul, and Ramara (2005), and Nwanna et al. (2016) were used for the formulation of the food and the experimental design. Induction of rats to make them diabetic and the grouping were done according to the earlier studies reported by Nwanna et al. (2016). Various biochemical parameters were analyzed, the kidney was cut into small slices, and the method of Maynard and Downes (2014) was used to preserve it and later use for histopathological structure viewing.
Total cholesterol concentration was determined according to the method used by Adefegha and Oboh (2012). Triglyceride concentration was determined according to the method used by Oboh, Bello, and Ademosun (2014). High-density lipoprotein cholesterol was determined using the method used by Adefegha et al. (2017). Low-density lipoprotein cholesterol was determined using the method used by Oboh et al. (2014). Determination of malondialdehyde produced was done using the method of Ohkawa, Ohishi, and Yagi (1979). Plasma superoxide dismutase (SOD) activity was determined according to the method of Rietman and Frankel (1957). Catalase activity was determined using the method of Misra and Fridovich (1972). The methods of Beers and Sizer (1952) were used to determine the plasma transaminases such as alkaline phosphatase, ALP, aspartate aminotransferase, AST, and alanine aminotransferase, ALT. Protein content in all the tissues used was determined using the method of Lowry, Rosebrough, Farr, and Randall (1951).

| Statistical analysis
The results of replicate readings were presented as means of standard deviation ± SD. Tukey's test and one-way analysis of variance were used with GraphPad Prism version 7 software. A significant difference was taken to be p < 0.05 (Zar (1984)).

| RE SULTS
Tropical eggplant proximate composition as shown in Table 1 revealed that the eggplant species had moderate content of carbohydrate (61.78%-70.06%), high fiber (7.46%-9.97%), low fat (11.05-14.675) low protein (5.04%-5.75%), and low ash (5.68%-8.44%) contents. Table 2 depicts the amylopectin between 30.22 and 3.64 g/100 g, while amylose ranges between 4.96 and 9.93 g/100 g. PG had significantly highest amylopectin values than PGW and PW. Table 3 shows the total sugar content ranges between 8.19 and 12.24 g/100 g), while the starch content ranges between 31.07 and 37 g per 100 g. The glycemic index is presented in Table 4, and it ranges between 30.16% and 38.65%. All the eggplant had low glycemic index below 50% (Table 5).  Also, Figure 3a-c shows the effect of the STZ induction and treatment diet liver function such as ALT, AST, and ALP enzymatic activities was elevated but was reduced as a result of the eggplant treatment; these followed the same trend of earlier reports. Figure 4a-c shows the histopathology results carried out on the kidney. The uric acid level, creatinine, and the blood urea nitrogen were elevated for the induced group with STZ without treatment, but the eggplant treatment diet was able to ameliorate the effect of the STZ and reduced the leakages significantly (p < 0.05). The endogenous antioxidant assess in this experiment catalase (CAT) and SOD was compromised; however, treatment with eggplant supplemented diet was able to manage as well as reduce the effect of the free radicals generated as a result of the STZ induction.
Although metformin-treated group (control drug) was able to ameliorate the effect better than the eggplant treatment group for SOD, eggplant diet elevated CAT especially S. kumba than S. gilo and S. aethiopicum as seen in Figure 5a,b. Plate 1a-f reveals the histopathology for kidney at 400 magnification from each of the study groups. Plate 1a is the control group, which shows mild congestion of the vessels which is focal, there is fatty infiltration of the cortex, the glomeruli appear normal, and there is no evidence of effacement of the glomerular basement membrane. Plate 1b is the induced group with STZ, which shows area of interstitial infiltration by inflammation as well as congestion of vessels, and there is also more fatty infiltration of the cortex. Plate 1c is the metformin-treated group, which shows congestion of vessels as well as less fatty infiltration of cortex with mild inflammation, the glomeruli appear normal, and there is no evidence of effacement of the    (2016) on Mangifera indica kernel, which could be due to the polyphenol constituents as reported earlier (Nwanna, Ibukun, & Oboh, 2013). Reports from Jenkins et al. (1981) showed that glucose responses are products from carbohydrate foods consumed, the types of food, and the extent to which it was processed.

| D ISCUSS I ON
The slower the degradation of carbohydrate-containing foods, the lower the level of glucose in the blood, which is relative to the value of glycemic index; thus, the food source or that particular diet can be termed to have low GI. Therefore, GI is the ability to rank carbohydrate-containing foods associated with the glucose level in the blood after consumption on a scale of 100% with 100 representing pure glucose. The benefits of assessing the glycemic index of foodstuffs serve as glucose control, control of insulin demands, and low concentration of lipids in the blood, which are invariably linked to the control and/or prevention of diseases such as cardiovascular disease, diabetes, and hypertension. Previous studies on carbohydrates, "isomaltulose," by Ines (2010) (2001) and Jeya et al. (2007) categorized foods within the content of low glycemic index (<55), medium glycemic index (56-69), and high glycemic index (>70).
This tropical eggplant falls below 55, a low GI diet, which is encouraged for good health. Hyperlipidemia is a known contributing risk factor, which has led to high accumulation and deposits of fats in the vessels (Mengesha, 2006), and also predisposed the Malondialdehyde produced in the diabetic group without treatment was more as observed in the kidney and liver tissues when compared to a normal healthy group and the diabetic groups treated with metformin and eggplant supplementation. This is because the antioxidant defense mechanism has been compromised but supplemented eggplant diet/metformin was able to manage or treat the diabetic subjects, which invariably reduced the radical species generation during auto-oxidation of glucose which could be due to the phenolic acids and flavonoids in the eggplant (Nwanna et al., 2016).
In disease investigation and diagnosis, ability to measure enzyme activity in tissue is paramount (Malomo, 2000). This measurement or the level of activity of this enzyme could give information concerning the organ or tissue wherever there is damage. The activities of ALP, AST, and ALT in the liver tissue are important in amino acid metabolism, which provide by-products or intermediates for another pathway like gluconeogenesis (Malomo, 2000 supplementation that was used for the histopathology view because 40% inclusion was better in their output in this study. The incidence of diabetic nephropathy showed elevated kidney markers like creatinine, uric acid, and blood urea nitrogen in the animal model due to the inability of the kidney to filter out these waste products of metabolism efficiently; the eggplant supplemented diet was able to reduce these products to restore the function of the kidney to normal as evidenced in the kidney enzyme activities. This report agrees with the study of Kalaivanan and Pugalendi (2011). During diabetes, there is overload and excess production of different by-products during the process of different metabolism whether anabolism or catabolism such as P L AT E 1 The effect of eggplant diet on the kidney. Histopathology view at *400. (a) Control rat revealed mild congestion of vessels, there is fatty infiltration of cortex, the glomeruli appear normal, and there is no evidence of effacement of the glomerular basement membrane. (b) Diabetic rat without treatment revealed focal area of interstitial fatty infiltration with inflammation (Black arrow) and congestion of vessels. (c) Diabetic rat given treatment with metformin revealed mild interstitial infiltration with inflammation (black arrow) and congestion of vessels with fatty infiltration of cortex. (d) Diabetic rat given treatment with 40% S. gilo revealed mild congestion with fatty infiltration of cortex, the glomeruli appear normal, and there is no evidence of effacement of the glomerular basement membrane. (e) Diabetic rat given treatment with 40% S. aethiopicum revealed mild congestion of vessels with less fatty infiltration of cortex, the glomeruli appear normal, and there is no evidence of effacement of the glomerular basement membrane. (f) Diabetic rat given treatment with 40% S. kumba revealed mild congestion of vessels, with less fatty infiltration of cortex, the glomeruli appear normal, and there is no evidence of effacement of the glomerular basement membrane (a) (b) (c) (d) (e) (f) glycogenolysis gluconeogenesis after glycolysis (Latner, 1985) but a better therapeutic source such as functional foodstuffs with a low glycemic index could manage these effects.
In the same vein, the inclusion of the tropical eggplant in the diet could restore and reverse the diabetic state of the diabetic model due to the presence of polyphenol and flavonoid constitutes as reported earlier by Nwanna et al. (2014Nwanna et al. ( , 2016 from the high-performance liquid chromatogram, which depicts the individual phenolic acids and flavonoids. It was reported that the concentration of these constituents was more in S. kumba, which could have contributed to the observed activities.

| CON CLUS ION
Our findings suggest that tropical eggplant diet could have a holistic effect on various pathology as a result of its abundant polyphenol constituents and its low glycemic index, which could serve as therapeutic functional food that can be used to manage/control diabetes and diabetes-related complications such as hepatic inflammation.

ACK N OWLED G M ENT
The Author acknowledges the Staff Development Support of the Federal University of Technology, Akure, Nigeria.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

E TH I C A L A PPROVA L
Approval was obtained from the relevant School's ethics committee responsible for the use of laboratory animals. The handling and use of the experimental animals was as approved by the Animal