Collection of plant material
The Argyreia pierreana and Matelea denticulata used in this study were collected and authenticated by Dr. Madhava Chetty, Department of Botany, S.V. University, Tirupathi, India (A voucher test number of 1364 and 1596).
Preparation of the extracts
The leaves were collected, cleaned, and shade dried for 1 week. The aqueous extract was prepared by maceration (50 g/500 mL) at room temperature for 72 h. The ethanolic (90%) extract was prepared using the Soxhlet mechanical assembly at 60–75 °C for 48 h. The ethanolic extricate was sifted, dried under reduced temperature at 40 °C in a hot air oven and stored below 20 °C until further use [24, 25].
Characterization of prepared extract
Determination of preliminary phytochemicals
The bioactive components of the extracts (aqueous and ethanolic extracts of both plants) were identified using standard qualitative phytochemical tests [26].
Estimation of total phenol content
0.2 mL (from 10 μg/mL stock) of test substance and standard were blended with Folin-Ciacalteau reagent (1 mL). Then, sodium carbonate (0.8 mL of 10% w/w) was added and incubated (Biovision, India) for 60 min at 27 °C. A total of 100 μL of the above reaction mixture was transferred to a microplate (Gilson, USA) and absorbance was measured at 750 nm using an ELISA plate reader (Biotech, USA). The total phenol content of the test sample was expressed as gallic acid (monohydrate) in mg/gm of the extract using the calibration curve of gallic acid (20 μg/mL to 200 μg/mL) [27].
Estimation of total flavonoid content
The test substance (0.2 mL of 10 μg/mL stock) and standard were blended with demineralized water (1.8 mL). Then, 0.5 mL of this solution was mixed with 95% ethanol (1.5 mL), 1 M potassium acetate (0.1 mL), 0.1 mL of aluminium chloride hexahydrate (AlCl3), and deionized water (2.8 ml) and incubated (Biovision, India) for 40 min at 27 °C. The above solution (100 μL) was placed into a microplate (Tarsons, India) and the absorbance was measured at 415 nm using an ELISA plate reader (Biotech, USA). The total flavonoid content of the test sample was compared with quercetin as standard (mg/gram) of the extract using the calibration curve (20 to 200 μg/mL) [27].
Scavenging activity by DPPH assay
The assay was carried out in a 96-well microtitre plate (Tarsons, India). Briefly, the methanolic solution of DPPH (200 μL) was added to wells previously containing 10 μL of aqueous and ethanolic extracts separately. The wells containing extracts (10 μL) and methanol (200 μL) were considered as test blank. The wells containing methanolic DPPH (200 μL) and DMSO (10 μL), and wells containing plain methanol (200 μL) and DMSO (10 μL) were considered as control and control blank, respectively. The plate was then incubated for 30 min at 37 °C and then the absorbance was measured at 490 nm using an ELISA plate reader (Biotech, USA) [28,29,30].
$$ \%\mathsf{of}\ \mathsf{scavenging}\ \mathsf{activity}=\left[\left(\mathsf{Optical}\ \mathsf{density}\ \mathsf{of}\ \mathsf{control}-\mathsf{Optical}\ \mathsf{density}\ \mathsf{of}\ \mathsf{sample}\right)/\mathsf{OD}\ \mathsf{control}\right]\ \mathsf{x}\ \mathsf{100} $$
Hydroxyl (OH•) radical scavenging activity
In Eppendorf tubes (10 mL capacity), EDTA (0.1 mL of 1 mM), FeCl3 (0.01 mL of 10 mM), H2O2 (0.1 mL of 30%), deoxyribose (0.36 mL of 10 mM), test or standard substance (1 mL) of various concentrations, phosphate buffer saline (0.33 mL, pH 7.4), and ascorbic acid solution (0.1 mL of 0.1 mM) were taken and incubated (Biovision, India) at 37 °C for 1 h. Distilled water was used as a test blank and control blank instead of a reagent mixture. Post incubation, 0.5 mL of the response blend containing OH• radical is pipetted out and TCA and TBA reagents (0.5 mL) was added to all tubes except control blank. The vials were kept in a boiling water bath for 20 min and cooled to room temperature and the supernatant (0.2 mL) was transferred to the microtitre plate. The absorbance was measured at 532 nm using an ELISA plate reader (Biotech, USA) [30]. The % of OH• radical scavenging activity was determined, as was the DPPH assay.
Superoxide radical scavenging activity
Briefly, freshly prepared alkaline DMSO (1 mL of 5 mM NaOH in DMSO), test and standard substances (0.3 mL prepared in distilled DMSO) of various concentrations, and Nitro Blue Tetrazolium (NBT, 0.1 mL of 1 mg/mL stock) were mixed to get a final volume of 1.4 mL. 0.1 mL of distilled water was used in place of NBT for test blank and control blank. 0.1 mL of the blend was then transferred to the microtitre plate and the absorbance was measured at 560 nm using an ELISA microplate reader (Biotech, USA) [30]. The % of superoxide radical scavenging activity was determined, as was the DPPH assay.
Reducing power assay
Briefly, 2 mL of phosphate buffer (0.2 M, pH 6.6) and potassium ferric cyanide (2.5 mL of 1% stock) were added to test or standard samples (0.5 mL). The distilled water, instead of potassium ferric cyanide, was added to test blank and control blank. Then, the reaction mixture was heated at 50 °C for 30 min. The resulting mixture was cooled to room temperature, mixed with 2.5 mL (10%) of trichloroacetic acid, and then centrifuged for 10 min at 3000 rpm. The supernatant (5 mL) was then mixed with condensed water (5 mL) and ferric chloride (1 mL of 0.1% stock) and incubated at room temperature for 10 min. The blend (0.1 mL) was then placed into a microtitre plate and the absorbance was measured at 700 nm using an ELISA plate reader (Biotech, USA). The obtained findings were presented in terms of ascorbic acid equivalent/g of extract. The increase in reducing power is indicated by an increase in absorbance [31].
Total antioxidant capacity
Briefly, the test solution (0.1 mL, prepared in DMSO) containing a reducing species was mixed with a reagent solution (1 mL, a mixture of 0.6 M sulphuric acid, 4 mM ammonium molybdate and 28 mM sodium phosphate) and heated at 95 °C for 90 min. Then, the samples were cooled to room temperature and 0.1 mL was transferred to the microtitre plate. The absorbance was measured at 695 nm using an ELISA plate reader (Biotech, USA). The obtained values were expressed as mM equivalent of ascorbic acid [30].
In vitro antidiabetic activity by glucose uptake method
The effect of extracts on glucose uptake was examined using differentiated rat skeletal muscle cells (L-6 cells). The cell cultures (70–80% confluence) were allowed, for 4–6 days, to differentiate in Dulbecco’s modified eagle growth medium (DMEM) comprising 2% FBS. Then, the differentiated cells were serum-starved overnight, washed once with HEPES buffered Krebs Ringer Phosphate solution (KRP buffer), and incubated at 37 °C for 30 min in KRP buffer containing 0.1% BSA. The cells were further incubated with test and standard drugs (non-toxic concentrations) and negative controls for 30 min at 37 °C. The d-glucose solution (1 M, 20 μL) was added at the same time to all wells before incubation. Post incubation, the supernatant solutions were aspired from wells and the cells were washed three times using the KRP buffer solution (ice-cold). Aliquots of cell lysates (prepared in 0.1 M NaOH solution) were analysed for cell-associated glucose using a glucose assay kit (ERBA) [32, 33].
In vitro gene expression study on GLUT-4 and PPAR-gamma
The effect of test substances on GLUT-4 and PPARγ gene expressions of L-6 cells was determined with respect to untreated cells [34, 35].
RNA isolation and cDNA synthesis
Post-treatment with the test substances, the L-6 cells were lysed using Tri-extract reagent and the prepared cell lysates were treated chloroform to isolate the RNA. The upper layer (amongst three distinct layers observed), in a fresh tube, was mixed with isopropanol (an equal volume) and incubated at − 20 °C for 10 min. The samples were then centrifuged and the pellet was resuspended in an appropriate volume of ethanol. Ethanol was then evaporated, the pellet was air-dried, and the appropriate volume of TAE buffer was added. The isolated total RNA was further used for cDNA synthesis.
The cDNA was synthesized by priming with oligo dT primers followed by reverse transcriptase enzyme treatment according to the manufacturer’s protocol (Thermo scientific). The cDNA thus synthesized was subjected to PCR (polymerase chain reaction) for the amplification of collagen, elastin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, internal control gene).
RT-PCR procedure
The mRNA expression levels of GLUT-4 and PPARγ were determined using semi-quantitative RT-PCR (reverse transcriptase-polymerase chain reaction). GLUT-4 and PPARγ cDNAs (in 50 μL of the reaction mixture) were amplified using specifically designed primers (Eurofins, India), and GAPDH (Housekeeping gene) was co-amplified with each reaction. The amplification conditions and primers used in the present study are in accordance with the earlier research paper [36].
Statistical analysis
The obtained findings were presented as mean ± standard deviation (SD). The findings were analysed by GraphPad Prism (8.01) using nonlinear regression for in vitro antioxidant activity and ANOVA (analysis of variance) for glucose uptake activity. The differences were considered statistically significant when p < 0.05.