3.1. Elemental analysis
Results presented in Table 1 showed the levels of ETEs (Iron, Zinc and Calcium) concentration in GD extract-FMB and GD extract, respectively. From the result, it is ascertained that ETEs were successfully replenished into a conventional extract, i.e., GD extract-FMB.
Table 1
Comparative content of ETEs analyzed by ICP-OES (n = 3)
(ETEs) | GD extract-FMB (Units in ppm) | GD extract (Units in ppm) |
Iron | 93 ± 4.01 | 12.00 ± 0.61 |
Zinc | 36.89 ± 3.22 | 18.69 ± 0.97 |
Calcium | 3100.00 ± 43.65 | 139.66 ± 2.04 |
Iron plays a direct and causal involvement in the aetiology of diabetes, and two significant mediators of this role are insulin resistance and beta-cell failure. Iron is used by the majority of tissues involved in fuel homeostasis to control metabolism, with the adipocyte being crucial in iron sensing [16]. Previous studies reported that concurrent administration of ETE including iron reduces oxidative damage and increases antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase [17].
Zinc plays a significant role in antioxidant defence in type 2 diabetic patients by functioning as a cofactor of the superoxide dismutase enzyme, regulating glutathione metabolism and metallothionein expression, competing with iron and copper in the cell membrane, and inhibiting the nicotinamide adenine dinucleotide phosphate-oxidase enzyme. Zinc reduces persistent hyperglycemia, which reduces oxidative stress in these people. It fact it promote insulin receptor phosphorylation by enhancing glucose transport into cells [18, 19]. A previous study reported that the Zn-coupling extract of Catharanthus roseus showed better α-amylase inhibitory activity as compared with a standard drug (Acarbose) [20].
Calcium is essential for insulin-mediated intracellular activities in tissues that respond to insulin, such as skeletal muscle and adipose tissue. However, effective insulin-mediated activities only need a small number of calcium ions for cellular performance [21]. Calcium decreases reactive oxygen species and boosted antioxidant enzyme activity in the cells. [22]. Additionally, a previous study found that patients with type 2 diabetes have abnormalities in skeletal muscle, adipocytes, and liver that are associated with impaired cellular calcium homeostasis. Calcium also aids in reducing de novo lipogenesis by increasing insulin activity and reducing fat accumulation [23].
3.2. Estimation of total phenolic and flavonoid content
The calibration curves of gallic acid (r2 = 0.9954) and quercetin (r2 = 0.9929) were used to calculate total phenolic and flavonoid content. The total phenolic and flavonoid content per gram of gallic acid and quercetin was found to be 16.34 ± 0.46 mg/g and 2.18 ± 0.74 mg equivalents, respectively. Our findings showed that G. sylvestre is high in phenolics and flavonoids.
Phytoconstituents especially polyphenols which are abundant in plants may have a beneficial effect on hyperglycemia by inhibiting the activity of carbohydrate-digesting enzymes (α-amylase and α-glucosidase), stimulating insulin secretion from pancreatic beta-cells, and decreasing blood glucose levels [24]. Moreover, the cited studies demonstrated that phytoconstituents protected beta-cells and their integrity by modulating hyperglycemia and oxidative stress [25]. The identification of gallic acid and quercetin in the present work strongly supported the pharmacological significance of G. sylvestre for its antioxidant and antidiabetic potential.
3.3. Free radical scavenging assay
DPPH free radicals scavenging ability of extract was calculated as percentage inhibition. In the present study, DPPH screening of GD extract-FMB and GD extract had clearly shown the dose-dependent antioxidant activity at the different concentrations tested (33–500 µg/mL). At maximum tested concentration, the inhibition potential of GD extract-FMB, GD extract and quercetin was 84.29 ± 1.06, 79.21 ± 1.13 and 95.29 ± 0.48%, respectively. The obtained results revealed antioxidant inhibition by the GD extract-FMB is more effective and similar to the reference compound as compared to the GD extract. The overall data was presented in Table 2.
Concentration (µg/mL) | DPPH activity |
Quercetin | GD extract-FMB | GD extract |
33 | 33.87 ± 0.16 | 18.44 ± 0.30 | 13.67 ± 0.34 |
66 | 56.43 ± 0.37 | 31.57 ± 0.54 | 26.87 ± 0.68 |
120 | 69.08 ± 0.72 | 45.87 ± 0.48 | 39.01 ± 0.19 |
250 | 81.34 ± 0.28 | 69.54 ± 0.96 | 56.07 ± 0.66 |
500 | 95.29 ± 0.48 | 84.29 ± 1.06 | 79.21 ± 1.13 |
IC50 | 57.00 ± 0.62 | 129.20 ± 0.98 | 176.60 ± 0.51 |
The DPPH evaluation is a simple and reliable procedure to assess the antioxidant properties of herbal products. G. sylvestre contains a lot of phytoconstituents, which slow down the oxidation of organic matter by adding a hydrogen atom to the chain-carrying ROO* radicals [26]. Through this mechanism, phytoconstituents inhibit the formation of free radicals and play a pivotal role in reactive oxygen species metabolism in the biological system.
3.4. In vitro α-amylase and α-glucosidase inhibition activity
FMB extract and conventional extract of G. sylvestre were tested against the carbohydrates digesting enzyme α-amylase and α-glucosidase and their inhibitory potential was found in a concentration-dependent manner, implying its potential role in diabetes management (33–500 µg/mL). In the case of α-amylase, the IC50 value of GD extract-FMB and GD extract was found 118.23 ± 0.42 and 153.45 ± 0.52µg/mL, respectively whereas in the case of α-glucosidase IC50 value was found to 109.52 ± 0.34 and 138.72 ± 0.48µg/mL, respectively. The overall data were presented in Table 3.
Concentration (µg/mL) | Acarbose | α-amylase | α-glucosidase |
GD extract-FMB | GD extract | GD extract-FMB | GD extract |
33 | 21.16 ± 0.14 | 16.26 ± 0.56 | 12.56 ± 0.67 | 17.22 ± 0.64 | 14.62 ± 0.47 |
66 | 35.56 ± 0.47 | 28.43 ± 0.67 | 25.56 ± 0.86 | 34.38 ± 0.93 | 29.88 ± 0.38 |
120 | 48.29 ± 0.36 | 52.69 ± 0.48 | 41.42 ± 0.48 | 51.69 ± 0.46 | 46.48 ± 0.48 |
250 | 76.36 ± 0.70 | 71.80 ± 0.62 | 65.36 ± 0.94 | 75.73 ± 0.49 | 63.78 ± 0.39 |
500 | 97.21 ± 0.92 | 91.82 ± 1.02 | 83.28 ± 0.47 | 90.78 ± 0.38 | 87.54 ± 0.84 |
IC50 | 105.20 ± 0.88 | 118.23 ± 0.42 | 153.45 ± 0.52 | 109.52 ± 0.34 | 138.72 ± 0.48 |
One treatment strategy for diabetes management is to hinder/block the absorption of glucose in the gastrointestinal tract and also maintain glucose metabolism in the liver by inhibiting the carbohydrate-metabolizing enzymes α-amylase and α-glucosidase [27, 28]. Likewise, in the current study, α-amylase and α-glucosidase inhibitory activity of GD extract-FMB and GD extract is most likely to be due to number of phytoconstituents including gymnemic acid, gymnemagenin, caffeic acid, gallic acid, quercetin, kaempferol, etc. In the case of both the carbohydrate-digesting enzymes such as amylase and glucosidase, it was found that GD extract-FMB showed a more pronounced effect as compared to standardized extract i.e. GD extract. Furthermore, there has been substantial scientific studies evidence suggesting that inhibition of α-amylase and α-glucosidase by phytoconstituents and ETEs makes it possible to ameliorate hyperglycemia to combat the occurrence of diabetes [29].
3.5. Gene disease association network and PPI network with essential trace elements (ETEs)
In this study, a target-disease interaction network was created using NetworkAnalyst to better understand the relationship between target proteins and disease association (Fig. 2). A total of 44 proteins were screened to describe the pathways involving diabetes and its associated disease. In addition, we have also created an interaction network between proteins associated with diabetes and ETEs (iron, zinc, calcium) using Cytoscape to better understand the relationship between target proteins and ETEs (Fig. 3). These target proteins were found to be primarily involved in diabetes mellitus, insulin resistance, impaired glucose intolerance, hyperglycemia, hyperinsulinemia, and obesity. According to network analysis, multiple target proteins and ETEs exist in one pathway, and the same target protein and ETEs exist in multiple pathways. In essence, the interaction between one target protein and multiple pathways with ETEs is more significant than the interaction between multiple target proteins and a single pathway with no ETEs. Because we all know that ETEs play a cofactor at the site of receptors that may enhance the bioactivity of ligands (bioactive phytoconstituents). These findings imply that the potent pharmacological components of GD extract-FMB may act on various proteins and signalling pathways to alleviate the levels of insulin, glucose, inflammation, and oxidative stress associated with diabetes and other related diseases [30, 31].
Moreover, biopotential genes (IRS1, TNF-α, IL6, MAPK3, DPP4, LEPR, PPARA and PIK3CG etc.) in the same PPI network were selected in the study to understand the interaction of ETEs of G. sylvestre (Fig. 3). The network created in Fig. 2 provided an understanding of the complex network relationships between proteins and disease while Fig. 3 provided the network interaction between proteins and ETEs. The above networks provide multiple target strategies to treat or manage diabetes and associated disease and disorders.
3.6. Molecular docking analysis
For examining the relationship between a protein and its ligand or inhibitor, AutoDock is a widely utilised platform. In order to investigate the interactions between gymnemic acid an active phytoconstituents of G. sylvestre and the proteins (α-amylase and α-glucosidase), molecular docking was carried out. The types of chemical bonds that were produced and the binding locations of amino acid residues provided evidence of the interaction between ligand and protein (Table 4). The molecular docking results are presented in Fig. 4. The results of docking studies revealed that gymnemic acid had strong binding with both the enzymes with the lowest energy at 6.88 kcal/mol and 7.74 kcal/mol, respectively that calculated from the docking experiment, and this result agreed with our in vitro results presented above [7].
Table 4
The results of molecular docking of gymnemic acid with the enzymes α-amylase and α-glucosidase.
Compounds | Protein | Binding energy | H-bonds Interactions |
Gymnemic acid | α-amylase | -6.88 kcal/mol | Lys200 (A); Glu240 (A) |
α-glucosidase | -7.74 kcal/mol | Lys91 (A); Try307 (A), Glu242(B), Glu48(B), Arg46(B), |
Furthermore, the strong interaction of gymnemic acid with α-amylase and α-glucosidase suggested that G. sylvestre reduced carbohydrate digestion and absorption. It is also reported that these compounds controlled insulin levels in the pancreatic beta-cells by enhancing the 5′ adenosine monophosphate-activated protein kinase pathway [32, 33]. Furthermore, previous findings also suggested that gymnemic acid may control oxo-inflammations, insulin resistance, and glucose metabolism, which are beneficial for the management of diabetes.
Overall, previous and present evidence reveals that consumption of ETEs and bioactive phytoconstituents exert better pharmacological activity and their collective intake is more beneficial as compared to the solitary application of either phytoconstituents or ETEs. Moreover, the in vitro studies, network pharmacology and in silico molecular studies revealed that FMB extracts are more beneficial and effective as compared to conventional extracts.