Table 2 presents experimental runs, blocks, graded values of independent variables (X1 to X3) and dependent variables (Y1 to Y7).
Table 2
Independent and dependent variables
Runs
|
Block
|
X1
|
X2
|
X3
|
Y1
|
Y2
|
Y3
|
Y4
|
Y5
|
Y6
|
Y7
|
4
|
1st
|
100
|
100
|
0
|
98
|
220
|
7.45
|
300
|
21.71
|
300
|
0.501
|
6
|
1st
|
100
|
0
|
50
|
245
|
107
|
6.23
|
170
|
12.45
|
298
|
0.605
|
11
|
1st
|
50
|
50
|
25
|
83
|
111
|
6.01
|
75
|
21.09
|
300
|
0.506
|
12
|
1st
|
50
|
50
|
25
|
79
|
116
|
6.5
|
71
|
20.11
|
298
|
0.506
|
9
|
1st
|
50
|
50
|
25
|
91
|
120
|
6.91
|
89
|
19.9
|
287
|
0.576
|
3
|
1st
|
0
|
100
|
0
|
213
|
90
|
4.99
|
201
|
12.13
|
180
|
0.561
|
5
|
1st
|
0
|
0
|
50
|
213
|
80
|
4.12
|
200
|
12.89
|
178
|
0.506
|
1
|
1st
|
0
|
0
|
0
|
220
|
79
|
7.79
|
287
|
11.89
|
173
|
0.581
|
10
|
1st
|
50
|
50
|
25
|
89
|
115
|
5.11
|
102
|
19.98
|
289
|
0.419
|
8
|
1st
|
100
|
100
|
50
|
69
|
226
|
7.9
|
301
|
22.11
|
321
|
0.509
|
2
|
1st
|
100
|
0
|
0
|
220
|
90
|
6.01
|
298
|
12.9
|
162
|
0.605
|
7
|
1st
|
0
|
100
|
50
|
221
|
89
|
4.76
|
276
|
11.94
|
168
|
0.657
|
19
|
2nd
|
50
|
50
|
25
|
82
|
122
|
4.23
|
100
|
20.21
|
300
|
0.512
|
18
|
2nd
|
50
|
50
|
68
|
80
|
123
|
5.33
|
98
|
20.21
|
298
|
0.634
|
16
|
2nd
|
50
|
134
|
25
|
93
|
200
|
6.9
|
254
|
19.21
|
162
|
0.566
|
15
|
2nd
|
50
|
34
|
25
|
223
|
92
|
5.79
|
289
|
11.98
|
165
|
0.56
|
17
|
2nd
|
50
|
50
|
17
|
84
|
100
|
5
|
170
|
12.16
|
170
|
0.569
|
13
|
2nd
|
34
|
50
|
25
|
213
|
98
|
5.41
|
288
|
11.23
|
166
|
0.5
|
14
|
2nd
|
134
|
50
|
25
|
100
|
229
|
7.5
|
297
|
22.12
|
298
|
0.51
|
20
|
2nd
|
50
|
50
|
25
|
77
|
115
|
5.64
|
167
|
16.32
|
176
|
0.65
|
X = Independent variables: X 1 = % concentration of ethanolic extract of M. oleifera, X2 = Exposure (in days), X3 = Sex of animal model (male = 1, female = 2). (Yn) = Responses: Y1 = Blood glucose (mmol/l), Y2 = Body weight (g), Y3 = Red blood cell concentration (%), Y4 = Glutathione (GSH), Y5 = Malonaldehyde (MDA) Umol/g, Y6 = Superoxide dismutase (mmol/ml), Y7 = Weight of kidney (g).
3.1. Blood glucose level
Figure 1 presents variation of blood glucose level in the diabetic rat at subnormal and normal treatment. The blood glucose levels decreased in the diabetic rats (≥ 250 mg/dL) to sub-normal (120 mg/dL), and normal (65–100 mg/dL) values with an increase in the concentration of the extract and period of exposure of the rats to the extracts. Analysis of variance on the data revealed that the model of blood glucose was significant (p < 0.05), predictable R2 = 0.9962, with a mean value of 139.65 mg/mL (Fig. 1). Equ. 3 shows strong contribution of the variables to the model parameter. The observation in the work agrees with the trend reported by Ewis and Abel-Rahman (1995) on ‘effect of metformin on glutathione and magnesium in normal and STZ-induced diabetic rats. The slight differences could be attributed to nutritional status, and age of the rats.
Blood glucose level is an important biomarker in the diagnosis, treatment and management of type 2 diabetes (T2D). In this study, blood glucose level of STZ-induced diabetic rats on treatment decreased significantly (p<0.05) from ≥250 mg/dL to normal level of 70-90 mg/dl. The concentration and exposure-time to the extract affected the blood glucose level. Diabetes (T2D) involves insulin resistance, insulin deficiency, and glucose overload, which emanate from the malfunctioning of the pancreas. Any substance which can restore these factors is antidiabetic (Peter et al., 2019). Untreated diabetic rats had higher blood glucose level ≥250 mg/dl, exhibited low body weight, high liver and kidney weight. Omotoso et al. (2018) had observed that moringa product can restore an injured pancreas. Hypoglycemic potency of moringa leaf has been reported by Awodele et al. (2012). Adedapo et al. (2009) reported the restoration of biological enzymes to normal levels by aqueous extract of Moringa leaf.
3.2. Body weight
Figure 2 presents the relationship between body weight of diabetic rat and dose of the extract as well as exposure time. The Figure shows that the weight of diabetic rats increased with doses and exposure time of rats to the extract. Body weight of the diabetic rats were lower than that of normal rats. The trend is elucidated by Fig. 2, equ. 7
According to Figure 2, rats gained weight as treatment progressed according to the dose administered and time of exposure to the extract. Analysis of variance on the body weight data revealed that the model was significant (p< 0.05), R2 = 0.8895, and mean body weight of 241.95 g was observed (Fig. 2 and Eq. 5). Stunting and death were observed in rats which did not receive the treatment. Rats treated with low dose of the extract at any exposure time exhibited low recovery from the effect of STZ. The result of the work was like the one reported by Adedapo et al. (2009) on safety of Moringa products. Body weight of rats increased significantly (p<0.05) as treatment progressed at rates proportional to days of exposure and concentration of the extract. Untreated animals continued to lose weight possibly due to increased lipolysis, glycogenolysis, gluconeogenesis (Ali et al., 2017). M. oleifera leaf extract restored the diabetic animals to a healthy state. This finding is similar to reports by Re et al. (1999) and Williams et al. (2015). Garima et al. (2011) and Tahmasse et al. (2013) attributed the activity of Moringa to the content of polyphenolic compounds many of which are antioxidants.
3.3 Red blood cell count
Response of red blood count of STZ-induced diabetic rats to treatment with extract leaf of M. oleifera is presented in Figure 3. The result revealed that red blood cell count increased from the diabetic state (≤ 11.25 to 15.88 mmol/L) to normal value as the dose of the extract and time increased (Fig. 3, Equ. 8). Analysis of variance on the data showed that the model appeared significant (p<0.05) with the coefficient of variation, R2 = 0.5663. The mean value of the parameter was observed to be 5.98 mg/mL. The observation in the work agrees with that of Veerasamy et al. (2008) who reported a rapid increase in red blood cell count and packed cell volume in diabetic rat treated with extract of M. oleifera.
Red blood cell count =
6.46 - 011X1-0.0091X2-0.030X3 +0.006X1X2-0.00048X1X3 8
Low haemoglobin concentration is associated with low postprandial peptide concentration and low β-cell responsiveness (Khawaja et al. 2010). Untreated animals and those that received low concentrations of the plant extract showed weakness and stunting. Since diabetic progression is a function of β-cell dysfunction as result of damage to pancreatic islet cells, the increase levels in the red blood cell could be from blood forming nutrients in M. oleifera leaf.
3.4 Glutathione
Effect of concentration of glutathione in the blood serum samples of the STZ-induced diabetic rats is presented in Figure 4. The values ranged from 75 mg/mL in diabetic rats to 300 mg/mL in normal rats and rats fully treated with the extract, respectively. The result revealed that all the diabetic rats at the beginning of the experiment exhibited glutathione deficiency of approximately 70-98 mg/ml. The level improved with increase in the dose, and period of exposure of the rats to the extract. Mathematical model of the glutathione was significant (p<0.05), regression analysis, showed a linearity coefficient of R2 = 0.8880, the mean of the parameter was 166.25 mmol/L and standard deviation of 30.61 (Fig. 4; eq. 9) showed steady increase of the value of the parameter at a dose- and exposure time-response trend. The period of storage of the leaf did not show any effect on the parameter (Fig. 4, Equ. 9). The observation agreed with the observation of Adeeyo et al. (2013) who reported on the effect of M. oleifera on the antioxidant status of diabetic rats.
Glutathione = 285.58-2.59X1-3.10X2– 2.216X3+0.029X1X2
+0.027X12+0.024X2X2 9
GSH plays an important synergistic role in defense against oxidative stress in mammalian models (Barsha et al., 2021). The synthesis of GSH starts with the formation of α-glutamyl-cystine-cysteine, the reaction is catalyzed by α-glutamyl-cystine-synthase. The value for glutathione varied between 106-220 Umol/g and was significantly (p<0.05) different among the animal models. The range of values are at variance with that reported by Barsha et al. (2021) on ‘beneficial effect of the methanolic leaf extract of Allium hookeri on stimulating glutathione biosynthesis and preventing impaired glucose metabolism in type 2 diabetes’. The level of reduced glutathione is lower in diabetics because of increased oxidative stress (Kalkan and Suher, 2013). Depletion of glutathione could arise from its utilization in diabetes. The high glutathione levels in animals fed with moringa extracts could be attributed to the high content of nutrients required for blood formation in the moringa extract (Barsha et al., 2021; Verma et al., 2021).
3.6 Malondialdehyde (MDA)
Dose of Moringa leaf extract and exposure of diabetic rats to the extract significantly affected (p .05) the level of MDA in STZ-induced diabetic rats (Figure 5). According to the Figure, MDA level was reduced significantly (p<0.05) by treatment of diabetic rats with moringa leaf extract (0.50). The model of MDA was significant (p<0.05), the coefficient of linearity was R2=0.8930, and the mean value was 16.63 mg/ml. The model also showed negligible differences in the values (standard deviation of 0.0780. Fig. 5 and Equ. 10 show trends of the variation with changing value and contribution of each independent variable to the parameter. The observation in the work is like the report of Adeeyo et al. (2013) that SOD and MDA were increasing in STZ-induced diabetic Wistar rat as treatment with extract of leaf of Moringa continued. Also, Tahmasse et al. (2013) reported that grape and black rice anthocyanins could effectively reduce oxidation stress in vitro and in vivo due to increased levels of antioxidant
MDA =11.58+0.060X1+ 0.0627X2+0.002x3+0.00095X1X2
– 0.00051X12-0.00067X22+0.00023X32 10
Lipid peroxidation has a high positive association with hyperglycemia. Hyperglycaemic subjects experience low insulin levels because beta-cells of the pancreas are not functional. Under diabetic conditions, normal metabolism is diverted the use of fatty acid and acetyl-CoA (Verma et al., 2021). Oleic acid could enhance the release of insulin and help normalize insulin secretion thereby preventing peroxidation. The high MDA levels of diabetic animals was reduced by treatment with M. oleifera extract. The results suggest that the treatment with ethanol extract of M. oleifera leaf led to reduction in lipid peroxidation. Verma et al. (2021) and Fahey et al. (2018) reported similar results. Malonaldehyde is one of the final oxidation products of poly unsaturated fatty acids and its overproduction could cause brain damage. Adeeyo et al. (2013) and Dorcely et al. (2017) respectively, also reported healing of target organs of tested animal models. The low-level MDA in the blood of treated animal models indicate that Moringa products can protect against tissue damage.
3.7 Superoxide dismutase (SOD)
Serum concentration of SOD of STZ-induced type 2 diabetic Albino rats which were treated with ethanolic extract of leaf of M. oleifera increased from 170 to 300 mmol/mL in diabetic to normal (fully treated rats) respectively. Mathematical model of the parameter was significant (p<0.05), R2=0.682, Adj. R2=0.5918). The analysis of variance showed a mean value of 234.0 mg/mL, the increment of SOD concentration exhibited steady increment towards normal value of ≤300 mmol/ml as treatment with the plant extract progressed. Fig. 6 and Eq. 11 are pictorial and mathematical responses respectively of the interaction between the parameter and the extract.
The equation further showed the magnitude of influence of the independent variables on the SOD level. The observation agreed with the values which were reported by Adeeyo et al. (2013) on ‘increased SOD levels in STZ-Nicotinic-induced diabetic Albino rats to normal within a week of treatment with Moringa extract’. Ali et al. (2017) on ‘the effect of Egyptian M. oleifera Lam. on blood haematology, serum biochemical parameters and lipid profile with special reference to kidney function in Albino rats’ reported a stable value of SOD of about 220 mmol/ml.
SOD =138.864+0.88X1+1.563X2+1.069X3 -0.13X22 11
Superoxide dismutase (SOD) of the STZ-induced type 2 diabetes rats treated with 0 to 80% ethanolic extract of shade-dried leaf of M. oleifera for 40 days varied significantly (p<0.05). Reduction in the levels of SOD enzymes in the animals treated with low doses of the extract were not significant (p>0.05). Untreated animals did not exhibit good health throughout the period of exposure to the test substance. SOD boosts the natural immune system by enhancing the activity of the antioxidant enzymes against the formation of free radical in mammalian tissues. Tissue integrity of antioxidants affects the development of diabetic complications as was reported by Dachana et al. (2010). The enzymatic antioxidant, SOD, is one of the primary enzymes that directly eliminates reactive oxygen species (ROS), (Semenova et al., 2022). It is an important defense enzyme and a scavenger of O2 from H2O2, the intervention diminishes the toxic effects due to free radical injury from secondary reaction (Abdel-Monsef et al., 2023). Besides, SOD prevents diabetes mellitus due to non- enzymatic glycosylation and oxidation. In the present study, the depletion of SOD in the diabetic rats could be attributed to inactivation caused by STZ-generated ROS and the SOD level was eventually improved by the bioactive components in the plant extract (Rani et al., 2018; Islam et al., 2021).
3.8 Weight of kidney
Influence of the moringa leaf extract on the weight of kidney of diabetic and fully treated, non-diabetic rats is presented in Figure 7. The Figure shows that the diabetic organ was 0.553g and 0.501 g for the treated rat. ANOVA on the data revealed that the model was not significant (p>0.05), R2=22.03%. The observation is like the reports of Zafar and Naqvi (2010) who reported on effects of STZ-induced diabetes on the relative weights of kidney, liver and pancreas in Albino Rats. Differences in finding could be attributed to the breed and strain of the animal models, exposure time of the animal models could be too short to achieve total restoration.
3.9The effects of major bioactive compounds in the plant product on the biomarkers of type 2 diabetes.
Overall, bioactive compounds in plant products are responsible for many of their health benefits (Vennila et al., 2017) and the extraction solvents used affect their potencies (Ojimelukwe et al., 2019). Ethanol soluble bioactive compounds in Moringa oleifera positively enhance metabolism of Wistar rats normalizing the negative effects of streptozotocin induced diabetes. Ethanolic extract of Moringa oleifera leaves have been found to contain a lot of phytochemicals (lutein, beta-carotene, phytyl fatty acid ester, beta-sitosterol (90 μg/mL), phenols (9 μg/mL), flavonoids (27μg/mL) etc) and nutrients. Flavonoids, phenols, alkaloids and saponins are found in appreciable amounts (Soraya et al. 2022). Soraya et al. (2022) identified 17 chemical components in the ethanol extract of Moringa oleifera leaves with quinic acid, glycerol, and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) as the most abundant compounds. Flavonoids help to regulate glucose metabolism, improve the activities of hepatic enzymes as well as the lipid profile (Al-Ishaq et al., 2019; Singh et al., 2022; Kusmuyati et al., 2022). Saponins may inhibit the hydrolysis of sugars by inhibiting amylases and glucosidases that break down starch into sugars thereby slowing down the rate of loading glucose into the blood stream (El-Barky et al., 2017).
3.10 Histopathology of Kidney and Liver of Experimental animals
The kidney cortex of diabetic Wistar rats had abnormal glomerulus (GM) and renal tubules (RT), it also showed some degenerating glomeruli (arrow) and tubule with distorted epithelium (open arrowhead).
The kidney of the diabetic Wistar rats after treatment with 80% ethanolic extract of M. oleifera leaves had normal parenchymal architecture, renal tubules (Rt), and glomeruli (GM), and the pathological lesions observed in diabetic rats were no longer there.
Sections of the kidney of untreated diabetic Wistar rat and after full treatment with 80% ethanolic extract of M. oleifera administered for 30 days.
The liver of untreated diabetic Wistar rats showed dilated central vein (CV) arrays of hepatocytes (black arrowhead), and several inflammatory infiltrates (blue arrowhead), hepatocyte disarray, as well as inflammation. After exposure to 80% ethanolic M. oleifera extract for 30 days, the liver architecture became normal, with arrays of hepatocytes (arrowhead) and normal sinusoidal spaces (open head arrow). No pathological lesions were seen.
Figure Liver of diabetic Wistar rats before and after exposure to 80% M. oleifera ethanolic leaf extract for 30 days.
Observations, made on the kidney and liver of STZ-induced diabetic Wistar rats after 30 days of exposure to M. oleifera ethanol extract indicated regeneration of the cells and the loss of pathological lesions that were observed in the diabetic control group. This implies that treatment with ethanolic extract of M. oleifera ameliorates type 2 diabetes in streptozotocin induced diabetic Wistar rats. Optimization data depicted 28 days of exposure, but histological experiments were carried out after 30 days of exposing the diabetic animals to M. oleifera ethanolic extract.