The methanolic leaf extract of
Diabetes mellitus is a disorder of carbohydrate, protein and fat metabolism and can represent absolute insulin deficiency, impaired release of insulin by the pancreatic
There are two predominant forms of diabetes mellitus, type 1 and type 2. Type 1 diabetes mellitus is characterised by absolute insulin deficiency that results from an autoimmune destruction of pancreatic islet cells; therefore, it is referred to as insulin-dependent diabetes mellitus (Achenbach et al.
Medicinal plants have formed the basis of health care throughout the world since the earliest days of humanity and have remained relevant in both developing and the developed nations of the world for various chemotherapeutic purposes (Johnson et al.
Despite the introduction of hypoglycaemic agents from natural and synthetic sources, diabetes and its secondary complications continue to be a major medical problem. In the search for alternative herbal treatment for diabetes, this study was conducted to evaluate the antidiabetic and antioxidant properties of the methanol leaf extract of
Fresh leaves of
A total of 70 male albino rats (150 g–220 g) of the Wistar strain were obtained and kept in the experimental animal house of the Faculty of Veterinary Medicine, University of Ibadan, throughout the period of this experiment. They were housed in rat cages and were fed the standard rat diet. They were given access to clean water at all times and allowed to acclimatise for a period of 2 weeks. After acclimatisation, fasting blood glucose (pre-induction) was measured before induction of diabetes.
Diabetes was induced in these rats by a single intraperitoneal (I.P.) injection of freshly prepared solution of alloxan monohydrate (100 mg/kg). Forty-eight hours after induction, fasting blood glucose level was assessed using ACCUCHEK active blood glucometer and a total of 40 animals with blood glucose >200 mg/dL were selected for the antidiabetic study.
A total of 50 rats were randomly allotted to five groups of 10 animals each. Group A animals were not diabetic and received vehicle + normal saline and served as control, Group B animals were diabetic rats and did not receive any treatment, Group C comprised diabetic rats that received glibenclamide at 4 mg/kg and Groups D and E received the MLVA at 200 mg/kg and 400 mg/kg, respectively. All treatments were done daily via the oral route and lasted for 28 days. Blood glucose level and weight of rats were measured weekly and blood was collected for haematology and serum for biochemical assays on days 14 and 28 post-treatment.
Fasting blood glucose was determined at intervals of 7 days during the 28-day experimental period using a glucometer (ACCUCHEK). Body weight of animals was also determined at intervals of 7 days using a weighing balance.
Blood was collected for haematological evaluation on days 14 and 28 post-treatment. From each rat, 5 mL of fresh whole blood was collected through the retro-orbital venous plexus. Of the 5 mL of blood, 2 mL was used for haematological analysis. Haematological analysis was done for determining packed cell volume (PCV), haemoglobin concentration (Hb), red blood cell count (RBC), white blood cell (WBC) count, white blood cell differential count and platelets count. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined using appropriate calculations. The remaining 3 mL of blood was also collected into sterile tubes and left for about 30 min to clot. The clotted blood was thereafter centrifuged at 4000 rpm for 10 min. Serum was harvested into sample bottles and stored at –20°C until the time of analysis. The animals were not anaesthetised.
After collection of blood from each of the animals, all rats were sacrificed by cervical dislocation after the 28th day of the treatment and a portion of the intestine was harvested on ice, rinsed and homogenised in aqueous potassium buffer (0.1 M, pH 7.4) and the homogenate was centrifuged at 10 000 rpm (4°C) for 10 min to obtain the post-mitochondrial fraction (PMF).
A portion of each blood sample was collected (on days 14 and 28 post-treatment) into plain bottles and thereafter centrifuged to obtain serum which was used to estimate total cholesterol, triglycerides (TGs) and high-density lipoproteins (HDLs), using commercial kits (Randox Laboratories, Ltd. UK) and following standard procedures as outlined by the producer.
Small pieces of pancreas were collected in 10% formalin for proper fixation. These tissues were processed and embedded in paraffin wax. Sections of 5–6 µm in thickness were made and stained with haematoxylin and eosin for histopathological examination (Drury, Wallington & Cancerson
Results are expressed as mean ± SD. Statistical analysis was performed by one-way analysis of variance (ANOVA), using GraphPad Prism version 6. The level of statistical significance was considered as
All experimental procedures were in conformity with the University of Ibadan Ethics Committee on Research in Animals as well as internationally accepted principles for laboratory animal upkeep and use.
Alloxan monohydrate induces hyperglycaemia in rats. The MLVA caused a significant (
Effect of
Groups | Day 0 | Day 7 | Day 14 | Day 21 | Day 28 |
---|---|---|---|---|---|
A | 122.3 ± 7.7 |
103.7 ± 8.6 |
113.6 ± 13.4 |
105.4 ± 6.19 |
124.0 ± 5.2 |
B | 489.0 ± 189.7 |
427.5 ± 38.9 |
481.0 ± 53.4 |
420.0 ± 59.4 |
483.5 ± 70.0 |
C | 600.0 ± 0 |
406.0 ± 68.2 |
419.33 ± 85.47 |
427.00 ± 27.58 |
442.00 ± 196.99 |
D | 333.4 ± 114.5 |
118.2 ± 42.9 |
163.6 ± 45.2 |
163.6 ± 74.5 |
108.4 ± 37.7 |
E | 465.7 ± 84.6 |
162.3 ± 52.3 |
188.4 ± 89.4 |
181.2 ± 84.4 |
176.0 ± 15.3 |
Values are mean ± SD,
Effects of
Groups | Day 0 | Day 7 | Day 14 | Day 21 | Day 28 |
---|---|---|---|---|---|
A | 173.1 ± 28.9 | 197.1 ± 29.2 |
212.4 ± 30.1 |
222.3 ± 45.7 |
233.6 ± 18.3 |
B | 145.3 ± 7.4 | 159.0 ± 3.1 | 168.4 ± 4.7 |
160.2 ± 13.8 |
156.2 ± 13.8 |
C | 160.0 ± 13.2 | 140.0 ± 15.7 |
131.4 ± 20.7 |
138.4 ± 24.1 |
127.2 ± 19.3 |
D | 186.0 ± 25.5 | 170.0 ± 12.7 | 166.8 ± 18.4 |
203.7 ± 12.6 |
200.4 ± 25.8 |
E | 171.9 ± 25.5 | 183.0 ± 33.7 |
177.7 ± 41.3 | 180.6 ± 38.7 | 218.6 ± 25.9 |
Values are mean ± SD,
The induction of diabetes caused statistically significant (
Effects of
Parameters | Groups | Basal | 14 days | 28 days |
---|---|---|---|---|
PCV (%) | A | 47.00 ± 1.00 | 50.60 ± 3.97 | 44.20 ± 1.92 |
B | 43.40 ± 0.54 | 44.00 ± 2.00 |
43.50 ± 2.65 |
|
C | 44.20 ± 1.92 | 40.00 ± 1.00 |
46.75 ± 2.50 | |
D | 43.40 ± 1.67 | 43.00 ± 1.41 |
47.40 ± 1.34 |
|
E | 47.00 ± 1.40 | 43.5 ± 0.58 |
48.20 ± 2.86 |
|
HB (g/dL) | A | 15.54 ± 0.89 | 17.53 ± 0.42 | 16.22 ± 0.95 |
B | 14.23 ± 0.41 | 14.70 ± 0.08 |
14.00 ± 0.70 |
|
C | 14.64 ± 0.77 | 13.37 ± 0.51 |
15.80 ± 1.10 |
|
D | 14.83 ± 1.04 | 14.48 ± 0.40 |
15.88 ± 0.38 |
|
E | 15.73 ± 1.62 | 14.70 ± 0.08 |
16.24 ± 0.85 |
|
RBC (×106/µL) | A | 7.74 ± 0.32 | 8.83 ± 0.04 | 7.89 ± 0.72 |
B | 7.34 ± 0.34 | 7.47 ± 0.48 |
7.16 ± 0.30 | |
C | 7.37 ± 0.38 | 6.87 ± 0.06 |
7.40 ± 0.15 | |
D | 7.44 ± 0.51 | 7.49 ± 0.20 |
7.89 ± 0.41 | |
E | 7.54 ± 0.47 | 7.65 ± 0.06 |
7.45 ± 0.29 |
Values are mean ± SD,
PCV, packed cell volume; HB, haemoglobin concentration; RBC, red blood cell count; MLVA, methanol leaf extract of
Concerning haematometric indices, the following observations were noted: significant increase in the MCHC in rats treated with MLVA when compared with the diabetic control and glibenclamide-treated groups (
Effects of
Parameters | Groups | Basal | 14 days | 28 days |
---|---|---|---|---|
MCV (fl) | A | 59.24 ± 1.61 | 63.50 ± 3.58 | 60.04 ± 0.99 |
B | 58.31 ± 2.72 | 57.15 ± 2.36 | 58.05 ± 0.21 | |
C | 60.08 ± 3.88 | 64.38 ± 2.25 | 58.45 ± 1.25 | |
D | 60.34 ± 3.23 | 61.04 ± 3.47 | 58.13 ± 3.64 | |
E | 61.13 ± 3.72 | 63.95 ± 3.70 | 56.67 ± 0.75 | |
MCH (pg) | A | 20.10 ± 0.99 | 20.06 ± 0.91 | 19.84 ± 0.51 |
B | 19.42 ± 0.76 | 19.75 ± 0.20 | 19.20 ± 0.53 | |
C | 19.88 ± 0.46 | 20.46 ± 1.27 | 19.41 ± 0.70 | |
D | 19.97 ± 0.85 | 20.25 ± 0.77 | 19.33 ± 0.22 | |
E | 20.85 ± 1.48 | 21.55 ± 1.85 | 19.23 ± 0.15 | |
MCHC (g/dL) | A | 33.73 ± 2.17 | 33.31 ± 0.63 | 33.55 ± 0.41 |
B | 32.82 ± 0.89 | 33.09 ± 1.67 | 32.56 ± 0.16 |
|
C | 33.41 ± 1.98 | 32.49 ± 2.78 | 33.41 ± 0.51 |
|
D | 32.48 ± 0.95 | 33.02 ± 0.92 | 34.71 ± 0.41 |
|
E | 32.71 ± 2.64 | 32.97 ± 0.82 | 34.00 ± 0.53 |
Values are mean ± SD,
MCV, Mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MLVA, methanol leaf extract of
Platelet count of MLVA-treated diabetic rats was significantly (
Effects of
Groups | Basal | 14 days | 28 days |
---|---|---|---|
A | 12.3 ± 2.5 | 12.93 ± 0.99 | 13.58 ± 0.97 |
B | 12.4 ± 1.6 | 20.05 ± 1.49 |
24.15 ± 9.Z55 |
C | 11.2 ± 1.5 | 17.43 ± 2.95 |
18.00 ± 1.56 |
D | 12.2 ± 3.1 | 18.75 ± 0.97 |
14.64 ± 0.85 |
E | 10.8 ± 1.9 | 13.40 ± 0.62 |
14.94 ± 1.07 |
Values are mean ± SD,
MLVA, methanol leaf extract of
Effects of
Groups 4 | Basal | 14 days | 28 days |
---|---|---|---|
A | 5.3 ± 1.8 | 4.81 ± 0.36 | 8.58 ± 1.09 |
B | 4.9 ± 1.9 | 5.90 ± 0.56 |
10.92 ± 1.56 |
C | 3.9 ± 1.1 | 5.39 ± 0.51 | 10.63 ± 0.51 |
D | 5.2 ± 2.1 | 4.67 ± 0.51 |
10.48 ± 0.40 |
E | 4.8 ± 2.3 | 5.13 ± 0.63 | 9.95 ± 0.79 |
Values are mean ± SD,
MLVA, methanol leaf extract of
Effects of
Parameters | Groups | Basal | 14 days | 28 days |
---|---|---|---|---|
Lymphocyte (103/µL) | A | 66.6 ± 6.2 | 79.00 ± 1.00 | 53.60 ± 1.94 |
B | 68.9 ± 5.6 | 71.75 ± 5.44 |
60.67 ± 6.66 |
|
C | 70.1 ± 3.8 | 60.33 ± 7.09 |
53.25 ± 5.68 |
|
D | 66.1 ± 3.9 | 71.17 ± 3.81 | 58.33 ± 1.15 |
|
E | 68.7 ± 5.0 | 62.00 ± 3.16 |
62.69 ± 0.58 |
|
Neutrophil (103/µL) | A | 32.50 ± 0.58 | 13.67 ± 2.51 | 26.75 ± 2.22 |
B | 30.00 ± 3.81 | 29.67 ± 3.05 |
45.00 ± 3.61 |
|
C | 27.17 ± 3.06 | 24.00 ± 5.66 |
31.00 ± 5.20 |
|
D | 30.00 ± 3.16 | 22.00 ± 1.63 |
27.00 ± 3.56 |
|
E | 27.14 ± 4.63 | 34.00 ± 1.83 |
28.25 ± 3.40 |
|
Monocyte (103/µL) | A | 1.86 ± 0.38 | 2.75 ± 0.50 | 2.67 ± 0.50 |
B | 2.20 ± 0.45 | 2.50 ± 0.58 |
2.67 ± 0.58 | |
C | 1.17 ± 0.41 | 3.50 ± 0.58 | 2.50 ± 0.55 | |
D | 1.20 ± 0.45 | 3.75 ± 0.50 |
2.83 ± 0.41 | |
E | 1.60 ± 0.55 | 3.40 ± 0.55 | 2.38 ± 0.52 | |
Eosinophil (103/µL) | A | 2.75 ± 0.50 | 2.67 ± 0.58 | 2.63 ± 0.52 |
B | 2.50 ± 0.71 | 2.75 ± 0.50 |
1.25 ± 0.50 |
|
C | 2.60 ± 0.55 | 1.67 ± 0.58 |
1.40 ± 0.55 |
|
D | 2.80 ± 0.45 | 2.60 ± 0.55 | 2.57 ± 0.53 |
|
E | 2.14 ± 1.07 | 2.25 ± 0.50 | 2.33 ± 0.52 |
Values are mean ± SD,
MLVA, methanol leaf extract of
Eosinophil count was significantly reduced in the untreated diabetic rats when compared with the normal control and the MLVA-treated groups. There were no significant changes in the monocyte counts (
From this study, treatment with
Effect of
Parameter | Groups | Pre-induction | 14 days post-induction | 28 days post-induction |
---|---|---|---|---|
Triglyceride (mg/dL) | A | 49.25 ± 4.92 | 41.67 ± 6.11 | 43.33 ± 3.51 |
B | 49.67 ± 2.89 | 41.50 ± 3.51 | 65.67 ± 4.04 |
|
C | 49.00 ± 2.83 | 35.00 ± 5.19 | 44.00 ± 4.24 |
|
D | 48.00 ± 4.83 | 34.25 ± 3.86 | 37.33 ± 4.73 |
|
E | 49.33 ± 4.93 | 39.40 ± 3.13 | 43.33 ± 3.21 |
|
Cholesterol (mg/dL) | A | 45.75 ± 5.32 | 44.33 ± 3.21 | 60.67 ± 4.04 |
B | 45.67 ± 7.64 | 47.67 ± 2.08 |
72.50 ± 3.53 |
|
C | 45.00 ± 4.36 | 45.50 ± 3.54 | 69.00 ± 4.24 |
|
D | 51.50 ± 9.19 | 41.75 ± 2.06 |
55.00 ± 1.41 |
|
E | 46.67 ± 4.51 | 44.33 ± 2.08 | 59.50 ± 0.71 |
|
High-density lipoproteins (mg/dL) | A | 19.67 ± 2.31 | 33.67 ± 1.53 | 20.67 ± 1.15 |
B | 18.00 ± 2.58 | 32.00 ± 1.73 |
17.50 ± 4.43 |
|
C | 18.00 ± 1.15 | 34.33 ± 2.31 |
21.50 ± 2.12 |
|
D | 17.75 ± 5.25 | 29.67 ± 2.31 |
20.50 ± 0.71 |
|
E | 16.00 ± 2.65 | 32.75 ± 1.50 |
21.00 ± 1.00 |
|
Low-density lipoproteins (mg/dL) | A | 20.83 ± 3.59 | 21.60 ± 1.41 | 21.06 ± 0.76 |
B | 21.64 ± 2.20 | 26.87 ± 3.93 |
21.60 ± 1.06 | |
C | 21.25 ± 1.80 | 23.40 ± 0.92 | 15.90 ± 5.80 | |
D | 20.48 ± 1.27 | 21.70 ± 1.43 |
20.20 ± 0.35 | |
E | 20.92 ± 2.96 | 24.75 ± 1.84 | 20.53 ± 0.46 |
Values are mean ± SD,
MLVA, methanol leaf extract of
Histopathological analysis of the pancreas was carried out in this study; diabetic control shows islets in varying sizes, few and far between, but the treated group show mild pathologies (
Photomicrograph of the pancreas of alloxan-induced diabetic rats (×400). (a) Normal control shows normal exocrine acini (blue arrows); the intralobular and interlobular ducts are essentially normal and contain pancreatic secretion (slender arrows). (b) Diabetic control shows islets in varying sizes, few and far between (slender arrows). (c) Glibenclamide-treated group shows islets in varying sizes, few and far between (blue arrows). (d) 200 mg/kg MLVA and (e) 400 mg/kg MLVA islets show hyalinisation in the centre while the remnant islet cells are limited to the periphery; these are few and far between (blue arrows).
The results obtained from this study revealed that induction of diabetes with alloxan monohydrate led to a significant (
Effect of methanol leaf extract of
A significant (
Effect of methanol leaf extract of
A significant (
Effect of methanol leaf extract of
Diabetes mellitus, a global burden for a developing economy, is characterised by hyperglycaemia and results in disturbances in carbohydrate, fat and protein metabolism, which arise because of defects in insulin secretion or insulin action. It is presently a very prevalent disease, especially in Africa (Aguocha et al.
In this study, Groups B, C, D and E induced with diabetes using alloxan developed hyperglycaemia. Alloxan is a diabetogenic agent with two distinct effects interfering with the physiological function of the pancreatic beta cells by selectively inhibiting glucose-induced insulin secretion through specific inhibition of glucokinase, the glucose sensor of the beta cell, and it also causes a state of insulin-dependent diabetes through its ability to induce ROS formation, resulting in the selective necrosis of beta cells (Lenzen
In this study, blood glucose level of diabetic rats treated with 200 mg/kg and 400 mg/kg doses of the MLVA was reduced to that comparable with those of the normal control (
A wide range of haematology laboratory values change significantly in patients with diabetes. Anaemia has been reported as a complication of diabetes mellitus (Kothari & Bokariya
Platelets of diabetic patients are characterised by dysregulation of several signalling pathways and have been suggested to be hyperreactive, showing increased adhesion, activation and aggregation (Randriamboavonjy & Fleming
Peripheral blood leukocytes are composed of polymorphonuclear cells, including monocytes as well as lymphocytes. Polymorphonuclear and mononuclear leukocytes can be activated by advanced glycation end products (Pertynska-Marczewska et al.
The pathogenesis of diabetes mellitus is associated with disturbances in carbohydrate, fat and protein metabolism. Under normal circumstances, insulin activates the enzyme lipoprotein lipase, which hydrolyses TGs. However, in diabetic state, lipoprotein lipase is not activated because of insulin deficiency, resulting in hyper triglyceridaemia (Cianflone, Paglialunga & Roy
Oxidative stress has recently been recognised as a significant player in the pathogenesis of gastrointestinal complications of diabetes (Kashyap & Farrugia
The results of this study showed that there is oxidative damage in the intestine of diabetic rats evidenced by the increase in H2O2 levels and MDA content of diabetic control rats when compared with the normal control. Furthermore, significant decrease in reduced glutathione, protein thiol (PT) and non-protein thiol (NPT) levels in diabetic control rats when compared with the normal control was observed. Also, the activities of the antioxidant enzyme systems (SOD, GPx and GST) were significantly (
Treatment with MLVA resulted in a decrease in the levels of H2O2 and MDA compared with the diabetic control rats. A number of earlier investigators have shown that the antioxidant effect of plant products is mainly because of radicals scavenging activity of phenolic compounds such as flavonoids, polyphenols, tannins and phenolic terpenes (Hu et al.
Oxidative stress acts on signal transduction and, via NF-κB, affects gene expression of antioxidant enzymes, thereby reducing the expression of antioxidant enzymes. Also, hyperglycaemia can simply inactivate existing enzymes by glycating these proteins (Wiernsperger
This study concludes that
This study was supported with a grant (TETFUND/DESS/NRF/UIIBADAN/STI/VOL. 1/B2.20.11) received from the National Research Foundation of the Tertiary Education Trust Fund (TETFUND), Nigeria.
The authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article.
A.A.A. was the project leader and was responsible for experimental and project design. A.T.A., A.A.O., T.O.O. and A.D.A. performed most of the experiments. A.E.A. made conceptual contributions and plant identification. All authors read and approved the final draft of the manuscript.