Reagents and chemicals
Methanol, concentrated H2SO4, acetic anhydride, ammonia solution, hydrochloric acid, citric acid, sodium citrate, streptozotocin, tris, EDTA, ATP, trichloroacetic acid, sodium chloride, sodium hydroxide, sodium hypochlorite, mono-potassium phosphate, di-potassium phosphate, ethanol, sodium carbonate, sodium acetate, acetic acid, tri-chloro acetic acid, ammonium molybdate, amino naphtol sulfonic acid, sodium bisulfate, sodium sulfite, ascorbic acid, dimethyl sulfoxide, sucrose, glucose, maltose, 3,5-dintrosalicylic acid, potassium chloride, magnessium chloride, calcium chloride, dodium bicarbonate, HumulinR and metformin were obtained from Sigma Chemical Company, St. Lious, Mo, U.S.A., and British Drug House (BDH) chemical Ltd., Poole, England. The diagnostic kits were obtained from Randox Laboratories Ltd., Crumlin, Co. Antrim, UK. All reagents and chemicals used were of analytical grade.
Collection and preparation of Tetracarpidium conophorum seed
Tetracarpidium conophorum seeds were purchased from a local market in Osogbo, Osun State, Nigeria. The plant was identified and authenticated by Mr. G.A. Ademoriyo at Ife Herbarium, Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria, where specimen copy was deposited. The herbarium identification number was 17,713. The shells were removed, and the seeds shade dried for 4 weeks. The T. conophorum seeds were pulverized and weighed into the sample container.
Extraction of Tetracarpidium conophorum seed
Dried powder of T. conophorum seed (500 g) was subjected to cold maceration with frequent agitation in 5 L of 100% methanol for 72 h at room temperature (Ajilore and Adesokan 2018; Asare and Oseni 2012; Santos et al. 2018). The filtrate was concentrated using standard procedure. Methanolic extract of T. conophorum was stored in the fridge until used.
GC–MS analysis of Tetracarpidium conophorum seed extract
GC–MS was utilized to identify compounds in the methanol extract of the plant seed according to the method described by Santos et al. (2018).
Extraction and determination of the α-amylase activity
α-Amylase was extracted from sorghum grains according to the method described by Adewale et al. (2006) and the activity was determined spectrophotometrically at 540 nm according to the dinitro salicylic acid (DNSA) procedure of Bernfeld (1955). One unit of enzyme activity is defined as the amount of the enzyme that produces 1 μmol maltose /min under the assay conditions. Activity was calculated using enzyme activity extinction coefficient of 0.354 cm2/mM.
Estimation of α-amylase inhibitory activity of Tetracarpidium conophorum seed
α-amylase inhibitory activity of T. conophorum seed extract was estimated using DNSA method as follows:
Procedure
|
Test (µl)
|
Control (µl)
|
Blank (µl)
|
---|
Extract (1.25–10.00 mg/ml)
|
250
|
–
|
–
|
1% starch solution
|
500
|
500
|
–
|
1% NaCl
|
250
|
250
|
250
|
Phosphate buffer (0.02 M, pH 7)
Pre-incubate for 5 min at 37 °C
|
250
|
250
|
250
|
α-Amylase solution
Incubate again for 15 min at 37 °C
|
200
|
250
|
–
|
2 M NaOH
Boil for 1 min
|
200
|
200
|
–
|
Add DNSA solution
|
500
|
500
|
–
|
The assay mixtures were incubated again for 2 min and cooled. The OD was read @ 540 nm against blank.
Calculation:
Concentrations of extracts resulting in 50% inhibition of enzyme activity (IC50) were determined.
Mode of α-amylase inhibition
The mode of inhibition of α-amylase by T. conophorum seed extract was determined according to the method described by Ali et al. (2006). The amount of reducing sugar released was determined spectrophotometrically at 540 nm against blank. Concentration of maltose released from starch solution was calculated from the absorbance using maltose standard curve and converted to reaction velocities. The type of inhibition in the presence and absence of the extract on α-amylase activity was determined by analysis of the Michaelis–Menten kinetics plot.
α-Glucosidase inhibitory assay
The effect of the plant extract on α-glucosidase activity was determined according to the method described by Dahlqvist (1964). The amount of glucose liberated was measured by RANDOX commercial glucose kit.
Concentrations of extracts resulting in 50% inhibition of enzyme activity (IC50) were determined.
Mode of α-glucosidase inhibition
The mode of inhibition of α-glucosidase by T. conophorum seed extract was determined according to the method described by Ali et al. (2006). The amount of glucose liberated in the presence and absence of extract was measured by RANDOX commercial glucose kit. The amount of reducing sugars released was converted to reaction velocity. The type of inhibition in the presence and absence of the extract on α-glucosidase activity was determined by analysis of the Michaelis–Menten kinetics plot.
Preparation and extraction of glucokinase eenzyme
An albino rat was fasted for 24 h after which the basal blood glucose level was determined by glucose oxidase method. The study was conducted according to the institutional guidelines and conforms to national guidelines for animal usage in research. The rat was subjected to monitored glucose tolerance test (as shown in the table below) following administration of 0.3 g/kg, 50% dextrose intra-peritoneal glucose load given over one minute.
Duration (Min)
|
Blood sugar (mmol/L)
|
---|
0
|
2.89
|
5
|
6.67
|
10
|
11.11
|
15
|
25.00
|
20
|
33.33
|
25
|
35.33
|
30
|
33.06
|
The rat was sacrificed after 30 min and liver immediately harvested and homogenized (tissue: buffer = 1: 10) in glucokinase buffer containing 150 mM KCl, 50 mM Tris–HCl (pH 7.6), 4 mM EDTA, 4 mM Dithiothreitol and 7.5 mM MgCl2 (Zhang et al. 2009). The homogenate was centrifuged at 3,000 rpm for 10 min at 4 °C following overnight lysis at 4 °C. The supernatant collected was used as enzyme extract immediately.
Assay for glucokinase enzyme activity
The effects of T. conophorum seed extract (TECOSE) on glucokinase activity was measured by estimating the amounts of glucose consumed and glucose-6-phosphate produced during phosphorylation of glucose in glucokinase assay mixture as follows:
Procedure
|
Negative control (µl)
|
Positive control (µl)
|
TECOSE (µl)
|
---|
TECOSE (5–10 mg/ml)
|
–
|
–
|
100
|
Glucokinase extract
|
–
|
100
|
100
|
Glucose (100 mM and 50 mM)
|
100
|
100
|
100
|
4 mM ATP
|
–
|
100
|
100
|
7.5 mM MgCl2
|
–
|
100
|
100
|
The assay mixture was incubated for 10 min at 30 °C. Glucokinase activity was calculated as mU/mg protein in the presence and absence of TECOSE as the difference between 100 and 0.5 mM glucose. Glucose concentration was determined using RANDOX commercial glucose kit while the amount of glucose-6-phosphate was measured using modified Fiske and Subbarow (1925) method by addition of ascorbic acid into the assay mixture to stabilize the phosphate ester. Protein in the liver extract was measured using RANDOX total protein kit.
Determination of glucose uptake in muscle and diaphragm
Tissue glucose uptake was determined according to the method described by Chattopadhyay (1992). Five groups, with each group containing five test tubes (n = 5) for each of the tissue, were considered as follows:
-
Group 1: Perfusion solution only (negative control).
-
Group 2: Perfusion solution + tissue (muscle or diaphragm).
-
Group 3: Perfusion solution + tissue (muscle or diaphragm) + extract.
-
Group 4: Perfusion solution + tissue (muscle or diaphragm) + metformin.
-
Group 5: Perfusion solution + tissue (muscle or diaphragm) + insulin.
Glucose concentration was determined using RANDOX commercial glucose kit.
Statistical analysis
Data obtained were analyzed using One Way Analysis of Variance (SPSS version 20.0). Levene statistic was used for tests of homogeneity of variance. Tukey’s test was used for multiple comparisons and homogenous subsets. A p-value of less than 0.05 was considered statistically significant.