Effects of aqueous leaf extract of Vernonia amygdalina and seed extract of Fenugreek on Body weight in diabetic rats

When compared to a diabetic control rat, treatment with Vernonia and Vernonia combined with Fenugreek improved body weight reduction (P<0.01) as shown in Figure 1.

Effect of aqueous leaf extract of *Vernonia amygdalina* and seed extract of Fenugreek on body weight in diabetic rats. N=2-3 and data are expressed as means ± SE. *p<0.05 vs. control, #p<0.05 vs. Diabetic.

Effects of aqueous leaf extract of Vernonia amygdalina and seed extract of Fenugreek on plasma total blood glucose level in diabetic rats

Treatment with alloxan (140 mg/kg) induced hyperglycemia in Groups B, C, D, E, and F. This hyperglycemia was reduced by treating them with Vernonia, Fenugreek, Vernonia with Fenugreek and Amyral in C, D, E and F groups, respectively where Vernonia and Vernonia with Fenugreek treatment in Group-C and Group-E had better result than Amyral treated groups. On the other hand, a significant difference was observed (p<0.05). The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on blood glucose level in alloxan-induced diabetic Rats are presented in Figure 2.

Impacts of aqueous leaf extract of *Vernonia amygdalina* and seed extract of Fenugreek on plasma total blood glucose level in diabetic rats. N=2-3 and data are expressed as means ± SE. *p<0.05 vs. control, #p<0.05 vs. Diabetic.

Effects of aqueous leaf extract of Vernonia amygdalina and seed extract of Fenugreek on plasma total cholesterol in diabetic rats

The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on plasma total cholesterol (TC) are shown in Figure 3. It was found that the total cholesterol level was raised (67.32±5.6) significantly (P<0.01) in diabetic rats (Group-B) in comparison to the control group (A) (122.75±9.98). There were no significant changes in total cholesterol values after treating them with Vernonia, Fenugreek, Vernonia with Fenugreek, and Amyral. But Vernonia and Vernonia with Fenugreek treatment prevent the further increase in the cholesterol level of alloxan-induced diabetic animals.

Effects of aqueous leaf extract of *Vernonia amygdalina* and seed extract of Fenugreek on plasma total cholesterol in diabetic rats. N=2-3 and data are expressed as means ± SE. *p<0.05 vs. control, #p<0.05 vs. Diabetic.

Effects of aqueous leaf extract of Vernonia amygdalina and seed extract of Fenugreek on plasma triglycerides in diabetic rats

The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on plasma triglyceride (mg/dL) values are expressed in Figure 4. It is clear that, in alloxan-induced diabetes, plasma triglyceride values were increased in the diabetic group (194.12±14.02) significantly (P<0.01) compared to the control (A) group (87.875±9.47). After treating them with Vernonia, Fenugreek, and Vernonia with Fenugreek, but the Vernonia and Vernonia with Fenugreek indicated that the triglyceride values were decreased compared to Amaryl treated group (96.9.00±10.23), which was not statistically significant.

Effects of aqueous leaf extract of *Vernonia amygdalina* and seed extract of Fenugreek on plasma triglycerides in diabetic rats. N=2-3 and data are expressed as means ± SE. *p<0.05 vs. control, #p<0.05 vs. Diabetic.

Effects of aqueous leaf extract of Vernonia amygdalina and seed extract of Fenugreek on plasma HDL and LDL in diabetic rats

The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on HDL (mg/dL) values are expressed in Figure 5A. After 7 weeks of treatment, it is clear that, in alloxan-induced diabetes, HDL values decreased in the diabetic control (B) group (32.26±6.9) significantly (P<0.01) compared to the control (A) group (67.25±3.39). After treating them with Vernonia, Fenugreek, and Vernonia with Fenugreek, it was found that the HDL values increased compared to the Amaryl-treated group (70±1.23), which was not statistically significant.

The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on LDL (mg/dL) values are expressed in Figure 5B. After 7 weeks of treatment, it is clear that, in alloxan-induced diabetes, LDL values increased in diabetic control (B) group (69.01) significantly (P<0.01) compared to the normal control (A) group (113.75). After treating them with Vernonia, Fenugreek, and Vernonia with Fenugreek where Vernonia and Vernonia with Fenugreek, it was found that the LDL values were decreased compared to the Amaryl-treated group (105.9), which was not statistically significant.

Effects of aqueous leaf extract of *Vernonia amygdalina* and seed extract of Fenugreek on plasma A) HDL and B) LDL in diabetic rats. N=2-3 and data are expressed as means ± SE. *p<0.05 vs. control, #p<0.05 vs. Diabetic.

Effects of aqueous leaf extract of Vernonia amygdalina and seed extract of Fenugreek on blood urea nitrogen and creatinine in diabetic rats

The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on blood urea nitrogen (BUN) (mg/dL) values are expressed in Figure 6A. After 7 weeks of treatment, it is clear that, in alloxan-induced diabetes, BUN values increased in the diabetic control (B) group (66.445) significantly (P<0.01) compared to the normal control (A) group (24.955). After treating them with Vernonia, Fenugreek, and Vernonia with Fenugreek, it was found that the BUN values were decreased compared to the Amaryl-treated group (23.85), which was not statistically significant.

The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on Creatinine (mg/dL) values are expressed in Figure 6B. After 7 weeks of treatment, it is clear that, in alloxan-induced diabetes, Creatinine values increased in the diabetic control (B) group (1.27) significantly (P<0.01) compared to the normal control (A) group (0.645). After treating them with Vernonia, Fenugreek, and Vernonia with Fenugreek, it was found that the Creatinine values were decreased compared to the Amaryl-treated group (0.68), which was not statistically significant.

Effects of aqueous leaf extract of *Vernonia amygdalina* and seed extract of Fenugreek on A) blood urea nitrogen and B) creatinine in diabetic rats. N=2-3 and data are expressed as means ± SE. *p<0.05 vs. control, #p<0.05 vs. Diabetic.

Effects of aqueous leaf extract of Vernonia amygdalina and seed extract of Fenugreek on ALT, AST, and ALP in diabetic rats

The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on Alanine aminotransferase (ALT) (U/L) values are expressed in Figure 7A. After 7 weeks of treatment, it is clear that, in alloxan-induced diabetes, creatinine values increased in the diabetic control (B) group (54.345) significantly (P<0.01) compared to the normal control (A) group (31.21). After treating them with Vernonia, Fenugreek, and Vernonia with Fenugreek, it was found that the Creatinine values were decreased compared to the Amaryl-treated group (31.865), which was not statistically significant.

The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on Aspartate aminotransferase (AST) (U/L) values are expressed in Figure 7B. After 7 weeks of treatment, it is clear that, in alloxan-induced diabetes Creatinine values increased in the diabetic control (B) group (172.46) significantly (P<0.01) compared to the normal control (A) group (95.225). After treating them with Vernonia, Fenugreek, and Vernonia with Fenugreek, it was found that the Creatinine values were decreased compared to the Amaryl-treated group (99.595), which was not statistically significant.

The effects of aqueous leaf extract of Vernonia and seed extract of Fenugreek on Alkaline Phosphotransferase (ALP) (U/L) values are expressed in Figure 7C. After 7 weeks of treatment, it is clear that, in alloxan-induced diabetes, creatinine values increased in diabetic control (B) group (117.9) significantly (P<0.01) compared to the normal control (A) group (36.945). After treating them with Vernonia, Fenugreek, and Vernonia with Fenugreek, it was found that the Alkaline Phosphotransferase (ALP) values were decreased compared to the Amaryl-treated group (39.15), which was not statistically significant.

Effects of aqueous leaf extract of *Vernonia amygdalina* and seed extract of Fenugreek on A) ALT, B) AST, and C) ALP in diabetic rats. N=2-3 and data are expressed as means ± SE. *p<0.05 vs. control, #p<0.05 vs. Diabetic.

Effects of aqueous leaf extract of Vernonia amygdalina and seed extract of Fenugreek on histopathological parameters in diabetic rats

Following completion of the experimental protocol, the pancreas, liver, and kidneys were harvested for histopathological evaluation. Electron microscope examination of pancreatic tissue from the control group (Group A) revealed a preserved structural organization of the islets of Langerhans, surrounded by intact and healthy exocrine pancreatic tissue (data not shown). Histological analysis of renal sections from the normal control rats (Figure 8) demonstrated well-maintained kidney architecture, characterized by intact tubular structure with wide, clear lumina and well-defined, undamaged glomeruli and Bowman’s capsules, accompanied by clearly visible cell nuclei.

In contrast, pronounced histopathological abnormalities were observed in the kidneys of diabetic rats (Figure 8). These alterations included marked structural damage to both glomerular and tubular components, manifested by narrowing of Bowman’s capsule, distortion of glomerular morphology, and evidence of glomerular necrosis. Treatment with the test extracts resulted in partial to substantial restoration of renal histoarchitecture, as illustrated in Figure 8. Notably, the most prominent histological recovery was observed in animals receiving the highest extract dose (500 mg/kg) as well as in those treated with metformin. In these groups, reductions in Bowman’s space and normalization of glomerular dimensions were evident, along with improved integrity of the tubular epithelium. Quantitative assessment further confirmed a significant decrease in Bowman’s space diameter following administration of 500 mg/kg aqueous extract and 200 mg/kg metformin (Figure 8). Importantly, treatment with the aqueous extract at 500 mg/kg produced a greater reduction in Bowman’s space compared with metformin, indicating a superior reno-protective effect.

Figure 9 displays normal hepatic features, which are histologically described as a large number of hepatocytes with circular euchromatic nucleoli and sinusoidal lining. Figure 9 displays cellular abnormalities with areas of necrosis, vascular blockage, and cellular degradation in the liver tissues of the diabetic rats compared to those of the healthy group. Comparing the liver tissue of diabetic rats treated with extracts at a dose of 250 mg/kg to that of the normal and diabetic control groups (Figure 9), the liver tissue of the diabetic rats treated with extracts at a dose of 500 mg/kg displayed moderate areas of nuclei, vascular congestion, and cellular restoration. In contrast, the liver tissue of the diabetic rats treated with extracts at a dose of 250 mg/kg showed noticeable cellular degeneration and vascular congestion along with a small area of cellular repair. Along with some mild degenerative alterations, the metformin group displayed certain typical healthy traits similar to those of the normal control rats (Figure 9). The group with diabetes displayed the highest degree. This impact was lessened in treatments using 250 and 500 mg/kg aqueous extracts. The figures had dropped to 50% and 70%, respectively. Treatment with metformin at a dose of 200 mg/kg reduces this liver damage by 30%.

Effects of aqueous leaf extract of *Vernonia amygdalina* and seed extract of Fenugreek on kidney histology in diabetic rats. This figure provides a visual representation of the kidney histopathological findings, where black arrows show the glomerulus, yellow arrows show the proximal tubules, and green arrows show Bowman's space.

Effects of aqueous leaf extract of *Vernonia amygdalina* and seed extract of Fenugreek on liver histology in diabetic rats. This figure provides a visual representation of the kidney histopathological findings, where black arrows show hepatocytes, yellow arrows show vacuolization, and green arrows show the sinusoidal layer.