Plant material
Plants belonging to the species B. diffusa (Fig. 1) were collected from in and around Coimbatore. The entire plant was authenticated by Botanical Survey of India (BSI), TNAU, Coimbatore (BSI/SRC/5/23/2013-14/Tech/1041).
Extraction and synthesis of AgNPs
Ethanolic extract of whole plant of B. diffusa was prepared using the Soxhlet equipment. The extract was then subjected with AgNPs and analyzed for in vitro antidiabetic activity (Akhtar et al. 2016).
In vitro antidiabetic activities
The α-amylase inhibition assay
From 1 mg/ml stock solution, different concentrations of plant extracts were prepared in phosphate buffer (10, 20, 40, 60, 80 and 100 µg/ml) of B. diffusa extract and acarbose were combined with 500µl of α-amylase (0.5 mg/ml) and incubated for 10 minutes at room temperature. Following that, 500µl of 1% starch solution was added and incubated for 10 minutes. The reaction mixture was then treated with 1 ml of dinitro salicylic acid DNS, a colouring reagent, and heated for 15 minutes in a boiling water bath before adding 10 ml of distilled water. A blank was prepared by replacing the enzyme with buffer for each concentration of the sample set to quantify the absorbance of the coloured extracts. The absorbance was determined at 540nm. With the help of the formula, the inhibition percentage was computed (Ishwarya et al. 2022).
% inhibition = Abs Control – Abs Sample/ Abs Control × 100
The alpha-glucosidase Assay
The alpha glucosidase inhibitory activity was determined using the (Palanuvej et al. 2009) technique. Yeast alpha glucosidase was dissolved at 0.1U/ml in 100mM phosphate buffer, pH 7.0, including 2000 mg/l bovine serum albumin and 200 mg/ml sodium azide as an enzyme source. As a substrate, para-nitro phenyl-alpha-D-glucopyranoside was utilized. B. diffusa (5%) plant extract was weighed, and serial dilutions of 62.5, 31.25, 15.6, 7.8, 3.9 and 1.95 mg/ml dimethyl sulfoxide and distilled water was prepared equal volume. For 5 minutes, ten microliters of plant extract dilutions were incubated with a 50microliter enzyme source. Following the incubation, 50 microliters of substrate were added and incubated for 5 minutes at room temperature. A microtitre reader was used to measure the absorbance at 405 nm.
% inhibition = Abs Control – Abs Sample/ Abs Control × 100
Non-Enzymatic Glycosylation of Haemoglobin Method
In phosphate buffer 0.01 M, pH 7.4, solutions of glucose (2%), haemoglobin (0.6%) and gentamycin (0.02%) were made. 1.0 ml of each of the above solutions was combined with 1.0 ml of the B. diffusa extract of varying concentrations (10, 20, 40, 60, 80 and 100 µg/ml). The reaction mixture was incubated for 72 hours at room temperature in the dark, and after that it was determined by colorimetry at 520 nm that much the haemoglobin had been glycosylated. Metformin was used as a standard drug by which further the percentage inhibition was determined using the formula, (Gupta et al. 2012).
% inhibition = Abs Sample – Abs Control/ Abs Sample × 100
Inhibition in vitro protein glycation
Fructose (1000 mM, in 200 mM, phosphate buffer pH 7.4) (4.0 ml) with 5.0 ml of BSA (20 mg/ml, in 200 mM phosphate buffer, pH 7.4) was incubated for 24 hours, either in the absence or presence of varied 1.0 ml of final concentrations of plant extract B. diffusa (250, 500, 750, and 1000 µg). Following the incubation time, the fluorescence intensities of the reaction mixtures were assessed throughout an emission wavelength range of 370–650 nm at an excitation wavelength of 360 nm. The amount of fluorescent advanced glycation end-products (AGEs) produced was correlated with the fluorescence intensity. At final concentrations of 1.25, 0.75, and 0.25 mg/ml, the common anti-glycation drug Pioglitazone served as a positive control. The percentage inhibition of fluorescent AGE formation was calculated using the following equation, (Avwioroko et al. 2022)
% inhibition = (FC- FB) – (FS -FSB)/ (FC- FB) × 100
Where FC is the fluorescence intensity of the control, FCB is the fluorescence intensity of the control blank, FS is the fluorescence intensity of the sample and FSB is the fluorescence intensity of the sample blank.
Glucose uptake by yeast cell
A 10% (v/v) suspension of commercial baker's yeast was made in distilled water after the yeast was repeatedly centrifuged (3,000 × g; 5 min) until the supernatant fluids were clear. 1ml of the glucose solution (5,10 and 25mM) was added to various concentrations of plant extract B. diffusa (20, 40, 60, 80 and 100 µg/ml) and incubated for 10 min at 37°C. After adding 100µl of yeast suspension and vortexing the mixture, the reaction was allowed to continue for 60 min at 37°C. The tubes were centrifuged (2,500 × g, 5 min) after 60 min to determine the amount of glucose in the supernatant. Metronidazole was taken as a standard drug. The percentage increase in glucose uptake by yeast cells was calculated using the following formula (Ramanathan et al. 2025).
% inhibition = Abs Sample – Abs Control/ Abs Sample × 100
Glucose diffusion assay
To measure the in vitro glucose diffusion and absorption, a cellulose ester dialysis tube (CEDT) was filled with 2 mL of 0.15 M NaCl and 0.22 mM glucose in either the presence (treated) or absence (control) of plant extract (50µg/ml). CEDT was transferred into a 50 ml centrifuge tube containing a 45 ml solution of 0.15 M NaCl that had been firmly sealed on both ends and maintained at room temperature in an orbital shaker. Every 60 minutes, the concentration of glucose in the external solution was measured to track the diffusion of glucose (Ansari et al. 2022).