Collection of plant material and preparation of the crude methanol extract of N. cadamba
Fresh flowers of N. cadamba were collected during the month of August 2015 from the district of Sirajganj, Bangladesh. The flower sample were authenticated by the Bangladesh National Herbarium, Mirpur, and sample specimen were submitted for future referencing purpose. The collected flowers were then sun dried for several days followed by heating in the oven for a 24 h. period but at low temperature (< 40 °C). The dried fruits were then grounded into coarse powder using high capacity grinding machine in the Phytochemical Research Laboratory, State University of Bangladesh. 600 g of the powdered material were measured, equally divided into two parts (300 g each) and later transferred into 2 amber colored reagent bottles both containing 2.0 L of methanol and chloroform. The containers with their contents were sealed by bottle cap and kept for a period of 15 days accompanied by occasional shaking and stirring. The mixtures were then filtered several times using fresh cotton plug and finally with Whatman No.1 filter paper. The volume of the filtrates were then allowed to evaporate at ambient temperature until approximately 70% solvents were evaporated. The leftover slugs were used as the crude methanol and chloroform extracts respectively .
Folin-Ciocalteu reagent, vincristine sulfate and alloxan were the products of Sigma chemical company, USA. BHT was manufactured by Merck, Germany. Gallic acid was purchased from Wako pure chemicals Ltd., Japan and metformin from Chadwell health essex, England. All the materials were ensured to be of analytical grade.
Nine weeks old male White-Evans rat (100–120 g) were purchased from Jahangirnagar University (JU), Dhaka, Bangladesh and kept in animal cages under standard environmental conditions (22–25 °C, humidity 60–70%, 12 h light: 12 h dark cycle). During the in vivo bioassay (14 days), all the rats were fed with standard laboratory diet (Purina rat chow) obtained from the JU and water ad libitum. All procedures were performed according to the institutional guidelines for animal experimentation of Department of Pharmacy, State University of Bangladesh. The animals were randomly assigned into group I, II, III, IV, V and VI; 5 rats in each group following the respective treatment protocols for the determination of blood glucose level. Two groups (I and II) were treated as the normal and the diabetic control respectively. Groups III and IV received the crude extract at two different doses. Group V served as the positive control and Group VI received the combined dose of Metformin and NCFE.
Collection of blood and analytical procedure
Blood samples were collected from the tail vein of the rats followed by measurement using the glucose monitoring system purchased from Tyson Bioresearch, Based Industrial Park, Chu-Nan, Taiwan.
Determination of total phenolic and flavonoid content
Total phenolic content of various fractions of NCFE were determined employing the method described by Singleton et al., 1965, involving the Folin-Ciocalteu reagent as the oxidizing agent, gallic acid as the standard and the results expressed as the milligram of gallic acid equivalent (GAE) per gram of plant extract . The total flavonoid content was determined using the well-known aluminum chloride colorimetric method and the results expressed as the milligram of quercetin equivalent (QE) per gram of plant extract .
Determination of antioxidant activity
The widely used method of Brand-William et. al., 1995 were followed in order to assess the antioxidant activity of the soluble fractions . The antioxidant potential was evaluated by the ability of the fractions to neutralize the radicals of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and compared with the standard Tetra-butyl-1-hydroxytoluene (BHT). The IC50 value, concentration of reagent required to neutralize 50% of the oxidant molecule, was calculated using the formula:
Percent inhibition = (1 – A1/A0) × 100%.
Here, A0 = absorbance of the blank (methanol).
A1 = absorbance of the sample being analyzed.
In vitro antidiabetic activity
The α-amylase inhibitors are a class of antidiabetic drugs that block the hydrolysis of carbohydrates, decreasing its absorption from the gastrointestinal tract and aiding in controlling the blood glucose level . The in vitroα-amylase inhibitory test were performed following the method of Bhandari et al., 2003, using acarbose (ABS) as the reference standard .
In vivo antidiabetic activity
The in vivo antidiabetic potential of the plant extracts were analyzed exclusively as well as in combination with metformin, after inducing diabetes in White-Evans rat by alloxan (120 mg/kg.bd.wt.) as mentioned in the works of Pari et al., 2002 . NCFE at doses 250 and 500 mg/kg.bd.wt. and metformin at 850 mg/kg.bd.wt. were given orally to group III, IV and V respectively for consecutive 14 days. Same procedures were followed for group VI receiving the combined dose of NCFE (250 mg/kg) and metformin (425 mg/kg). Blood glucose level were monitored using glucose monitoring system purchased from Tyson Bioresearch, Based Industrial Park, Chu-Nan, Taiwan.
All the results were recorded in triplicates and results expressed as the mean ± standard error mean (SEM). In order to determine statistical significance between groups, Dunnett’s test and One way ANOVA were performed using SPSS version 25.0 and p < 0.05 was considered to be statistically significant.