In vitro free radical scavenging and antidiabetic activity of aqueous and ethanolic leaf extracts: a comparative evaluation of Argyreia pierreana and Matelea denticulata

Abstract

Background

Oxidation is believed to play a vital role in the pathogenesis of diabetes mellitus by lipid peroxidation; DNA and protein damage leads to the development of vascular complications like coronary heart disease, stroke, neuropathy, retinopathy, and nephropathy. The herbal preparations are complementary and alternative medicines to allopathic drugs which are believed to cause adverse events. Therefore, the current study was aimed to identify the novel plants, which belong to the genera Argyreia (Argyreia pierreana (AP)) and Matelea (Matelea denticulata (MD)), and assess the aqueous and ethanolic leaf extracts for in vitro antioxidant and antidiabetic potential by DPPH, OH^•, superoxide, and glucose uptake and gene expression (GLUT-4 and PPARγ) studies using the L-6 cell line respectively.

Results

The preliminary scrutiny revealed the presence of polyphenols, flavonoids, terpenoids, steroids, tannins, alkaloids, and glycosides. The total phenolic and flavonoid contents of ethanolic extracts were found higher than those of aqueous extracts. The ethanolic extracts exhibited the superior antioxidant capacity when compared with aqueous extracts. However, the ethanolic extract of MD was shown superlative glucose uptake activity (72.54%) over control (0.037%) and GLUT-4 and PPARγ gene expressions (1.17 and 1.20) in term of folds respectively over cell control (1.00).

Conclusion

The ethanolic leaf extracts of both plants showed significant in vitro antioxidant and antidiabetic activities compare to aqueous extracts. The Matelea denticulata ethanolic leaf extract exhibited superior activity. This superior activity might be due to their higher phenolic and flavonoid content. However, further approaches are needed to define these activities.

Background

Traditional herbal medicines have shaped the basis of human health care, and further research will improve global health [1, 2]. Presently, about 80% of the world population (according to WHO) uses herbal drugs for some aspects of primary health care. Globally, the use of medicinal plants predates antibiotics and other contemporary drugs [3, 4]. In addition, many culinary herbs and spices were tested for their biological activities in Alzheimer’s disease management and other chronic diseases [5, 6].

The natural antioxidant defence mechanism, in all human and other aerobic organisms, prevents the oxidative damage. Since the natural antioxidant defence mechanism is inadequate on its own, the nutritional consumption of antioxidants is suggested [7, 8]. Currently, synthetic antioxidants are replaced by natural antioxidants as the former are reported to have carcinogenic properties. Plants are the primary source of natural antioxidant molecules capable of eliminating or neutralizing the harmful reactive oxygen species (ROS) [9]. The natural antioxidants are also free-radical scavengers, reduction agents, pro-oxidant metal complexes, singlet oxygen quenchers, etc. They can also safeguard the human body from free radicals and delay the progression of many chronic illnesses (such as cancer, heart disease, and stroke) and boost the plasma’s antioxidant ability and prevent lipid oxidative rancidity in foods [10, 11].

The incidence of diabetes mellitus (DM) is very high all over the world. Natural antidiabetic drugs are now becoming increasingly popular due to severe financial burdens and side effects connected with allopathic therapy strategies [12,13,14]. The herbal preparations are complementary and alternative medicine (CAM) and the search for the discovery of novel compounds derived from natural sources like herbs or plant is growing mainly due to acquired resistance, side effects, and adverse events (AE) of allopathic medication [15,16,17,18]. In search of new medicinal plants that pose concurrent antioxidant and antidiabetic activity, we have screened the leaf extracts of two novel plants belonging to the genera Argyreia (Argyreia pierreana) and Matelea (Matelea denticulata).

Argyreia pierreana (AP), a new flowering plant, which belongs to the family Convolvulaceae, genus Argyreia, is found throughout India and China. In India, it is commonly found in Assam, West Bengal, Bihar, Orissa, and South India. There are several supporting pharmacological activities that have been proven for various species or genus of Argyreia belongs to family Convolvulaceae [19,20,21].

Matelea denticulata (MD) belongs to family Apocynaceae, genus Matelea. Matelea is a genus of flowering plants and contains about 200 species, which are commonly known as milkvine. It is found in Bell, Burnet, Palo Pinto, and Parker counties; Brown, Comanche, Eastland, and Johnson counties; and India [22, 23].

Previous studies of Argyreia and Matelea genus plant extracts indicated the presence of flavonoids, phenols, tannins, and saponins which are the supporting compounds with various pharmacological activities. These plants are novel belonging to the same genus which might have the new kind of phytochemicals for the concurrent diseases. Hence, we selected these plants with the aim and objective to find the supportive phytochemicals for in vitro antioxidant and antidiabetic activity and comparison of their potency to determine the superlative plant extract for discovering the potential phytoformulation. Further, our study extended to develop the plant-derived chemicals for concomitant metabolic disorders by applying suitable bioavailability-enhancing techniques.

Methods

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.

Results

Preliminary phytochemical screening

The prepared AEAP, EEAP, AEMD, and EEMD were screened for the presence of various phytochemicals such as polyphenols, flavonoids, terpenoids, steroids, saponins, tannins, alkaloids, and glycosides. The steroids were found absent in aqueous extracts of both plants (AEAP and AEMD) whereas the glycosides were found absent in ethanolic extracts of both plants (EEAP and EEMD) (Table 1). These results clearly revealed the solubility pattern of the above-screened phytoconstituents.

Table 1 The phytochemical profile of the prepared plant leaf extracts

Total phenol and flavonoid content of the extracts

The total phenol and flavonoid concentrations of the plant extracts are presented in Table 2. All plant extracts (AEAP, EEAP, AEMD, and EEMD) showed a considerably elevated amount of phenolic compounds over flavonoids. Further, the ethanolic extracts showed a substantially increased concentration of phenolic compounds than aqueous extracts (AEAP and AEMD). In addition, the results revealed no significant difference in total flavonoid compounds amongst aqueous and alcoholic extracts.

Table 2 Total phenolic and flavonoid content of the prepared plant leaf extracts

Free radical scavenging activity

The prepared extracts were screened for their antiradical activity against various free radicals (DPPH, OH^•, and superoxide radicals). The scavenging activity of all prepared extracts, against all free radicals tested, is found to be concentration-dependent (Fig. 1a–c). The IC₅₀ values obtained with the tested extracts are presented in Table 3.

Fig. 1

Free radical scavenging activity of extracts: a Scavenging activity by DPPH assay. b Hydroxyl radical scavenging activity. c Superoxide radical scavenging activity. The values are presented as mean ± SD, (n = 3)

Table 3 IC₅₀ Free radical scavenging activity of prepared plant leaf extracts

In the DPPH assay, both ethanolic extracts (EEAP and EEMD) showed significantly higher scavenging activity over aqueous extracts (AEAP and AEMD) at a tested amount of 1000 μg/mL (Fig. 1a and Additional file 1: Table S1). Amongst the ethanolic extracts, the EEMD displayed significantly higher scavenging activity than EEAP indicating superior anti-oxidant activity of the Matelea denticulata plant leaf. The IC₅₀ value for EEMD is found to be 827.3 μg/mL over other extracts (more than 1000 μg/mL). However, the ascorbic acid (test standard) displayed significantly higher antioxidant activity (IC₅₀ 88.4 μg/mL) over all extracts tested.

In the OH^• radical scavenging assay, all the extracts inhibited the OH^• free radicals. We observed a dose-dependent quenching of hydroxyl free radicals for all extracts. The scavenging activity of ethanolic extracts (EEAP and EEMD), as compared with their aqueous extracts, are found similar to standard ascorbic acid at the tested amount of 1000 μg/mL (Fig. 1b and Additional file 1: Table S2). Both EEAP and EEMD showed scavenging activity that is comparable with standard ascorbic acid against hydroxyl free radicals as compared with DPPH free radicals. Further, EEAP showed scavenging activity comparable with EEMD against hydroxyl free radicals than DPPH free radical.

Similarly, the superoxide free radical assay revealed the scavenging activity of EEAP, EEMD, and AEMD which is comparable with standard ascorbic acid whereas AEAP displayed significantly less scavenging activity over other tested extracts and standard (Fig. 1c and Additional file 1: Table S3). Surprisingly, the aqueous extract of Matelea denticulata plant leaf also displayed scavenging activity comparable with ethanolic extracts and standard ascorbic acid against superoxide free radicals than DPPH and OH^• free radicals. Overall, the EEMD displayed scavenging activity that is significantly higher than other extracts tested and comparable with standard ascorbic acid.

Reducing power activity

The reducing power activity of the tested extracts and standard ascorbic acid is presented in Fig. 2. All extracts displayed concentration-dependent reducing activity. The increase in the extract amount results in increased reducing activity. All extracts showed significantly high reducing activity over standard ascorbic acid at a tested amount of 1000 μg/mL. However, the reducing activity of ascorbic acid was found higher than all extracts at lower concentrations (up to 250 μg/mL). Amongst extracts tested, the EEMD showed high reducing activity over other extracts tested.

Fig. 2

Reducing power activity of the extracts. The values are presented as mean ± SD, (n = 3)

Total antioxidant capacity

The total antioxidant activity of all tested extracts is presented in Fig. 3. The ethanolic extracts of AP and MD displayed the superior antioxidant capacity (107.80 ± 0.08 and 214.10 ± 10.03) when compared with aqueous extracts AP and MD (34.74 ± 1.04 and 52.83 ± 1.98) mg equivalent. ascorbic acid/g extract respectively. Amongst both ethanolic plant extracts, the EEMD showed significantly higher antioxidant capacity. These findings clearly revealed the superior antioxidant activity of the plant Matelea denticulata (leaf) as compared with Argyreia pierreana (leaf).

Fig. 3

Total antioxidant capacity of the extracts. The values are presented as mean ± SD, (n = 3)

In vitro antidiabetic activity by glucose uptake method

In vitro, glucose uptake by L-6 cells was measured to determine the effect of extracts on cellular glucose uptake behaviour. Further, the antidiabetic activity of the extracts was compared with standard marketed antidiabetic drugs (Insulin and Metformin) (Table 4) and (Fig. 4). Insulin (1 IU/mL) and Metformin (100 μg/mL) treatments caused significantly increased glucose uptake (about 120% and 92% respectively) over the untreated control group. The treatment of L-6 cells with ethanolic extracts caused significantly higher glucose uptake as compared with aqueous extracts. Amongst ethanolic extracts, the EEMD caused about 2.26-fold higher glucose uptake as compared with EEAP. When compared with the standard drug (Insulin, 1 IU/mL) the EEMD (200 μg/mL) caused significantly less (about 47%) glucose uptake. Further, the glucose uptake caused by EEMD was found about 22% less than the standard metformin (100 μg/mL). Although EEMD caused significantly less L-6 glucose uptake as compared with standard drugs at the tested amount of 200 μg/mL the EEMD could cause L-6 glucose uptake comparable with standard drugs at higher concentrations. However, further studies are needed to validate this fact.

Table 4 In vitro glucose uptake activity by L-6 myotubes in the presence of plant leaf extracts

Fig. 4

Glucose uptake activity of extracts on the L-6 cell line. Values presented are mean ± SD, (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 over control

In vitro gene expression study on GLUT-4 and PPAR-gamma

The effect of EEAP and EEMD treatments GLUT-4 and PPARγ mRNA expressions in L-6 cells is determined using a semi-quantitative RT-PCR technique (Fig. 5). The scanning densitometric analysis was performed to determine the GLUT-4 and PPARγ mRNA expressions of the untreated and Pioglitazone, EEAP and EEMD treated L-6 cells. The results revealed the elevated level of GLUT-4 expression in treated cells as compared with control cells. The Pioglitazone (standard drug), EEMD and EEAP treatments resulted in about 1.22, 1.17 and 1 fold increased GLUT-4 transcription levels when compared with untreated cells (Fig. 6a and Additional file 1: Table S4).

Fig. 5

Gene expression study: a Effect of extracts on GLUT-4 and b PPARγ transcripts in L6 myotubes determined by RT-PCR technique

Fig. 6

Densitometric analysis of gene transcripts: a The relative level of GLUT-4 and b PPARγ gene expression is normalized to GAPDH. Values shown depict arbitrary units

Additionally, the PPARγ expression of untreated and pioglitazone-, EEAP-, and EEMD-treated L-6 cells was determined. Surprisingly, the EEMD treatment (200 μg/mL) showed about a 1.2-fold increase in PPARγ expression when compared with untreated cells (Fig. 6b and Additional file 1: Table S4). Besides, the PPARγ expression of EEMD treated cells was found comparable with standard pioglitazone treated cells. Similarly, the EEAP treatment showed elevated (about 1.07 fold) PPARγ expression when compared with untreated cells. The L-6 cells treatment with further increased concentration of EEMD and EEAP may result in further increased GLUT-4 and PPARγ expressions. To ascertain these facts, however, further studies are needed. The above findings indicate the correlation between increased cellular glucose uptake and elevated GLUT4 and PPARγ gene expression.

Discussion

In recent years, the hunt for antioxidant phytoconstituents has been significantly increased due to their potential therapeutic application in diverse chronic and infectious diseases. In search of new plants, in the current research, we have screened the in vitro antioxidant and antidiabetic activities of aqueous and ethanolic leaf extracts of two plants which belong to the genera Argyreia (AP) and Matelea (MD).

The free radicals (associated with one or more unpaired electrons) are highly unstable and attain stability by extracting electrons of other molecules. Further, the free radicals, due to their highly reactive nature, damage the transient chemical species. The increased risk of many chronic diseases in humans was associated with the elevated free radical level as a result of a failure in endogenous antioxidant defence mechanism or exposure to environmental oxidants or damage to cell structures [37]. Therefore, we can avoid chronic disease progression and risks associated with them by supplementing the patients with proven antioxidants or by increasing the antioxidant defence.

The role of medicinal plants in decreasing free radical–caused tissue injury reveals their antioxidant activity [10]. The current research, therefore, explores the antioxidant activity of leaves aqueous and ethanolic extracts of plants AP and MD. As free radicals are different chemical entities, the extracts were investigated against many free radicals in order to prove their antioxidant activity via various mechanisms (such as free radical scavenging, reducing activity, potential complexing of pro-oxidant metals, and quenching of singlet oxygen) [38, 39].

The antioxidant characteristics of the extracts are first assessed on the basis of their capacity to trap free radical DPPH. By their capacity to donate hydrogen, the antioxidants decrease and decolorate the DPPH. The experimental findings revealed that the ethanolic extracts, as compared with aqueous extracts, have significantly high DPPH free radical scavenging activity. The leaves of the plant MD showed superior DPPH free radical scavenging activity than the leaves of the plant AP.

The OH^• radical formation in the liver or other iron-rich tissues through Fenton reaction contributes to the lipid peroxidation initiation. Besides, the OH^• radicals cause DNA mutagenesis and various proteins inactivation (owing to their high reactivity) [29, 40] are found involved in the initiation of inflammation and cancer and are extremely harmful to the tissues [41]. Therefore, the OH^• radical scavenging activity can be considered as one of the best indicators of a compound’s antioxidant potential. In the current study, the obtained results revealed that the EEAP and EEMD have significantly high OH^• radical scavenging activity which is found similar to ascorbic acid.

Superoxide radical is very harmful to cellular components (as a precursor of more reactive species) [30] and can result in in vivo H₂O₂ formation through a dismutation reaction. The formed H₂O₂ (not very reactive itself) further produces OH^• radicals in the cells which eventually causes cell damage. Therefore, the removal of H₂O₂ from the cell system is imperative for the antioxidant defence system. The obtained experimental data revealed the comparable antioxidant activity of EEAP, EEMD, and AEMD with standard ascorbic acid. The AEAP displayed significantly less antioxidant activity over other tested substances and standards. Overall, the EEMD displayed significantly high free radical scavenging activity than other extracts tested, and is comparable with ascorbic acid.

The alkaloids, tannins, glycosides, flavonoids, and polyphenols are found in the extracts. Many researchers have shown that most of these compounds (polyphenols and flavonoids) have antioxidant properties [42]. Further, the polyphenols (ortho-hydroxyl phenolic compounds like quercetin, gallic acid, caffeic acid, and catechin) prevents the H₂O₂ caused by mammalian cell damage [43, 44]. In the current research, the observed superior free radical scavenging activities of ethanolic leaf extracts of plants AP and MD could be correlated to their high flavonoid and phenolic compounds. Moreover, the antiradical activity relies on the availability and capacity of these extracts to provide hydrogen or electron atom [10]. In the present research, the findings acquired indicate the free radical stabilizing ability of extracts by giving them electron or hydrogen.

Extracts are composed of a number of scavenging compounds which may act synergetically to enhance the antiradical activity in a variety of oxidative stress and in diseases like diabetes [44]. The difference in antiradical activity observed amongst the extracts in the present study, it may be due to the presence of different level of several bioactive compounds (such as phenols and flavonoids) which have the ability to donate hydrogen atoms to stabilize the free radicals [43, 44].

The reducing power of compounds would serve as an indicator of their antioxidant potential. Thus, in the current research, we assessed the reducing power of the extracts by measuring their capacity to reduce Fe³⁺ to Fe²⁺ by donating electron [45]. All extracts showed the highest reduction capacity (optical density) as compared with ascorbic acid at a tested amount of 1000 μg/mL. This capacity of the extracts to reduce Fe³⁺ could be ascribed to the presence of reduction agents, such as polyphenols, and the number and/or the position of the hydroxyl groups on polyphenols [45].

In addition, in the current research, the total antioxidant activity of the extracts is assessed and compared with the standard ascorbic acid. The obtained results revealed the superior antioxidant activity of the ethanolic extracts as compared with aqueous extracts. Further, the EEMD displayed a significantly higher antioxidant activity than EEAP. Many researchers have reported a strong relationship between the antioxidant activity of many plant species and their total phenol content [46, 47]. These results further validate the potential antioxidant nature of polyphenols. Thus, in the present study, the obtained higher antioxidant activity of the extracts could be correlated to their high amount of polyphenols which functions as reduction agents, hydrogen donors, free radical scavenger, singlet oxygen quenchers, and metal chelators [48]. However, further comprehensive studies are required to isolate the antioxidant components of these extracts and determine their in vivo biological activities.

The skeletal muscle is the main part of the human body responsible for postprandial glucose use and its function is essential for maintaining normal levels of blood glucose [49]. The non-insulin-dependent diabetes mellitus (NIDDM) is characterized by a defect in insulin-stimulated skeletal muscle glucose uptake [50]. In this context, in the current research, the effect of extracts on skeletal muscle cell (L-6) glucose utilization is determined in vitro. All aqueous and ethanolic extracts have significantly enhanced glucose uptake when compared with the control group. The glucose utilization by L-6 cells is found significantly high in the presence of EEMD than all other extracts. Further, the EEMD effect on glucose utilization is found almost comparable with standard metformin. This increased glucose utilization in the presence of extracts could be correlated to their effect on a number of L-6 cell glucose transporters or other receptors that are involved in the glucose transport across the cell membrane.

In the current research, an additional effort is taken to define the mechanism behind enhanced glucose utilization by L-6 cells in the presence of extracts. The GLUT-4 is the main transporter of glucose present in insulin-responsive tissues (skeletal muscle and adipose tissue) [51], and the abnormal glucose transport in insulin-resistant type 2 diabetes is linked with poor GLUT-4 translocation (from the intracellular membrane storage site to the plasma membrane) and/or defective insulin signaling cascade. The significant increase in GLUT-4, P13 kinase, and mRNA expressions, in the presence of insulin, in euglycemic and hyperinsulinemic clamp further validates the function of GLUT-4 and P13 kinase in the insulin-mediated transfer of glucose. Along with insulin, the PPARγ agonists (such as rosiglitazone and pioglitazone) have also been reported to facilitate the transport of glucose in type 2 diabetes by acting as insulin sensitizers [52].

In the present study, the effect of extracts on L-6 cell GLUT-4 and PPARγ expressions is determined to validate their role in glucose uptake. The obtained results revealed the elevated expressions of GLUT-4 and PPARγ in L-6 cells treated with all extracts as compared with untreated L-6 cells. The EEMD treatment caused increased L-6 cellular GLUT-4 and PPARγ expressions as compared with other extracts. Thus, its effect on increased glucose uptake by L-6 cells is might be due to its ability to elevate GLUT-4 and PPARγ expressions. In addition, these results are consistent with the literature reports which revealed the increased glucose uptake as a result of enhanced GLUT-4 and PPARγ levels in L-6 cells [35, 51, 52]. The obtained results in the current research are in accord to support the glucose uptake-enhancing property of EEMD. Based on gene expression study results, we can conclude that the EEMD would trigger glucose transport through the up-regulation of GLUT-4 and PPARγ expressions. However, additional research is required to reveal the other mechanisms or molecules which resulted in increased glucose transport in the presence of EEMD.

The promising in vitro antioxidant and antidiabetic activity of these plant extracts might be due to the presence of supportive phytochemicals such as polyphenols, flavonoids, terpenoids, and tannins. Additionally, the individual component identification and biological activities are underway.

Conclusion

The preliminary phytochemical analysis indicated that, the newly identified plant’s leaf extracts contain supportive phytochemicals such as flavonoids, polyphenols, steroids, and terpenoids. The study results revealed that the ethanolic extracts of both plants’ leaf extracts showed significant in vitro antioxidant and antidiabetic activity when compared with aqueous extracts. The ethanolic leaf extract of Matelea denticulata exhibited superior in vitro free radical scavenging and antidiabetic activity amongst the other extracts. This superior activity might be due to their high phenolic and flavonoid content than the leaves of Argyreia pierreana. Besides, other parts of this plant or whole plant would show similar or superior antioxidant and antidiabetic activities than the leaf. However, further research is needed to determine the study-specific phytochemicals with the mechanisms behind these activities.