1 Introduction

The statistics of Type 2 diabetes mellitus (T2DM) is prevailing as a main non-communicable disease wondering ∼366 million of world population. One of the major studies led by diabetic researchers, by 2030, it is estimated as a high rate reaching 439–552 million (Whiting et al., 2011). Risk associated with the T2DM is metabolic syndromes and micro–macro vascular complications increased the risk of cardiovascular indications (Tahrani et al., 2011). The majority of patients required different types of medications to balance their blood-glucose levels worldwide. The catastrophe of use of metformin, sulphonylureas and/or gliptins is often followed by the introduction of insulins. Though productive, this strategy had several limitations, which eventually lead to therapeutic inertia and compromise the targets for glycemic control. Hence an urge of safe and efficient modality for its management is urgently needed (Lankatillake et al., 2019; Dias et al., 2012).

Plants like Pterocarpus marsupium Roxb. and Andrographis paniculata Nees are few such candidates who have high possibilities in administering diabetes. Pterocarpus marsupium Roxb. extensively used to treat Type 2 diabetes mellitus for thousands of years (Jung et al., 2006). Similarly, Andrographis paniculata Nees. has also been used as an anti-diabetic plants with other activities such as anti-analgesic, antioxidants, and hepatoprotector (Jayakumar et al., 2013). These plants biosynthesized a major class of phytoactive compounds, pterostilbene (1) and andrographolide (2) respeticvely, have been known as anti-diabetic (Titi and Sabtanti, 2015; Perera, 2016; Fig. 1). More recently, Chakraborty et al. (2010) and Islam (2017) has studied Andrographis paniculata Nees. and Pterocarpus marsupium Roxb. impact against diabetes and evaluated their interactions with GLUT4, PPARα and PPARγ.

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Fig. 1

Structures of pterostilbene (1) and andrographolide (2).

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The development of a new agent of plant-based origin that assures safety with moderate but definite efficacy is the need of the time. Several clinical and pre-clinical studies on the traditional medicinal plants have shown their antidiabetic potential. Various attempts are being made to prepare formulations with optimized contents of phytoactive compounds of the plant by other (Lankatillake et al., 2019). The combined activity of these phytoactive materials, in a defined reference window, against multiple selected targets can be pooled together as a complementary agent with specific actions for the management of T2DM.

2 Material and methods

3 Results

In this study, we have observed that there is significant decrease in fasting blood glucose (FBG) level the fasting blood glucose (FBG) in Ptyrone^TM treated diabetic group (184 ± 44.54 mg/dl) in comparison with a diabetic group (344.3 ± 56.62 mg/dl) (Table 1) with an increase in PPARγ expression in Ptyrone^TM treated diabetic group (2.25 ± 1) in comparison with the diabetic control group (1 ± 0.01) (Table 1). The study of SGLT2 and PPARα expression was shown a reduction in SGLT 2 (0.90 ± 0.02) but does not affect PPARα expression in Ptyrone^TM treated diabetic group (0.97 ± 0.13) in comparison with control diabetic group (1 ± 0.0.01), evocative the PPARγ activity (Table 2). Interestingly, for the first-time metformin has also shown statistically significant SGLT2 inhibitory activity in this study. Similarly, the GSK3β and GLUT4 expression level, (0.9 ± 0.1) and (1.1 ± 0.15) in Ptyrone^TM treated diabetic group as compared to the GSK3β and GLUT4 expression level (1 ± 0.01) and (1 ± 0.01) in the diabetic control group (Table 2). The Ptyrone^TM treated diabetic group (Table 3) did not exhibit significant DPP4 inhibition (39.01 ± 4.54) in comparison with the control diabetic group (0 ± 0).

3.1

3.1 Molecular interactions

In this study, we predicted homology models for the four protein targets namely GPR40, GLUT4, SGLT2, and AMPKγ. Ramachandran plot (RC Plot) was used to study the torsion angles of the models built. Ramachandran plot for all the four homology models is shown in Fig. 2. Nanosecond dynamic simulations were executed using GROMACS 2018.1 version. Before simulations, all the homology models were energy minimized and potential energy was checked (for stable structure the average potential energy should be in the scale of −1*10⁵ kcal/mol) shown in Table S4. As per the RC Plots, potential energy, and the RMSD, these homology models are reasonably good for further in silico studies. For molecular docking studies, ligands are directed to the active site as predicted. The active site prediction for our target proteins is described in Table S5. The images showing the actives site are given in Fig. 3. The ligands pterostilbene and andrographolide were docked with 3D structures of protein targets using AutodockTools (Table S6). Binding modes for pterostilbene and andrographolide with all the protein targets are shown in Fig. 3. Molecular docking studies and predicted activity with minimum energy interactions concerning their active sites are shown in Table S7 for pterostilbene and andrographolide.

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Fig. 2

RC Plots of GPR40(A), GLUT4(B), SGLT2(C) & AMPKγ(D).

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Fig. 3

Images showing binding mode of Pterostilbene with (a) GSK3β, (b) PPARγ, (c), DPPIV (d) GPR40, (e) SGLT2, (f) GLUT4, (g) AMP; binding mode of Andrographolide (h) GSK3β, (i) PPARα, (j) PPARγ, (k) DPPIV , (l) GPR40, (m) SGLT2, (n) GLUT4 & (o) AMPK.

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4 Discussion

Type 2 diabetes mellitus (DM), is a metabolic disorder wherein the currently used therapeutic modalities for the management of Type 2 DM have limitations and side effects. Hence, there is a need for the development of natural multi-targeted agents for the comprehensive management of diabetes. In the current article, for the first time, we have used hydroalcoholic extracts of Andrographis paniculata Nees. and Pterocarpus marsupium Roxb. plants combined in the certain proportion for the diabetes management. The diabetic rat was induced by a low-dose STZ to assess the outcome of Ptyrone^TM (hydroalcoholic extracts of Pterocarpus marsupium Roxb. and Andrographis paniculata Nees.) and evaluating the FBG and mRNA expression levels of PPARα, PPARγ, GLUT4, SGLT2, GSK-3β and DPPIV to check the inhibition of Type 2 diabetes in diabetic rats as compared to metformin. Afterwards, we have confirmed the anti-diabetic nature of pterostilbene and andrographolide and their binding interctions with GSK3β, PPARγ, DPPIV, GPR40, SGLT2, GLUT4 and AMPK through in silico studies (Fig. 3).

PPARs are known for nuclear hormone receptors that acts as transcription factors which regulates the genetic expression of both lipid metabolism as well as insulin resistance (Lamichane et al., 2018). Notably, PPARγ plays an imperative role in adipocyte differentiation, glucolipid metabolism and insulin resistance (Jia et al., 2015), which is why PPARγ regulates the usual physiological processes of cells. Also, PPARγ is linked with obesity, fat differentiation and insulin resistance as PPARγ is a type of ligand-activated nuclear transcription factor (Wasik et al., 2017). Metformin treatment group has shown prominent PPARγ upregulation but also a significant downregulation in PPARα mRNA expression. This could also be one of the possible reason for the cardiovascular side effects of metformin. Similar results were also observed with PPARγ agonist glitazones such as pioglitazones and rosiglitazones. One of the major concern with PPAR is their downregulated expression in diabetic patient is by the disease itself. If a drug has an antagonist activity or downregulates the expression of PPARα, it may lead to water retention and CHF (Schernthaner et al., 2013). However, with the use of Ptyrone^TM formulation, it has been observed that no significant decrease in PPARα was heeded with a slight upregulation in PPARγ expression while comparing the diabetic control group. This further establishes the safety of the Ptyrone^TM formulation. The beneficial effects of increased production of enzymes via PPARγ activiation is increased glucose tolerance and improved insulin sensitivity followed by, improved energy expenditure in white adipose tissue (Kaupang and Hansen, 2020). So, the need of the hour is that of selective PPARγ agonist and with no effect on PPARα wherein our formulation suffices this issue.

GLUT4 is a transmembrane transport protein which boosts glucose transport to insulin-sensitive tissues for intracellular exploitation (Irudayaraj et al., 2016) which is not only found in skeletal muscle and adipocytes, but also in the brain and heart (Wang et al., 2020). Our research reveals that in T2DM rats, the expression level of GLUT4 were increased by Ptyrone^TM. Its upregulation can increase the translocation into the cell membranes which thereby increases the glucose uptake in the cell which will be helpful in achieving the glucose homeostasis (Wan et a., 2020). It was also monitored that the same formulation also downregulated the SGLT2 expression, suggesting the inhibitory affect of SGLT2 activity. Ferrannini et al. (2013) reported that the renal SGLT2 threshold levels for glucose reabsorption is paradoxically augmented in hyperglycemia in an experiment on diabetic rodents and humans. Also, its inhibitors exhibit glucose-dependent as well as -independent renoprotective effects (Kawanami et al., 2017). Since the inhibitors of SGLT2 shows pleiotropic effects, hence, they may exert beneficial effects on peripheral nerves, thereby addressing diabetic peripheral neuropathy.

The role of a serine/threonine kinase based glycogen synthase kinase-3 (GSK-3) is significant in the regulation of glycogen metabolism together with other numerous cellular activities. Along with this, obese people suffering from type 2 diabetic are also related to defective capability of insulin to stimulate the dumping of glucose and glycogen synthase due to overexpression and over-activity of GSK-3 in skeletal muscles. A supplementary imperative accomplishment of these serine/threonine kinase inhibitors in the framework of obese-associated diabetes (type 2) caused the decrease of hepatic glucose level may be due to the downregulation mechanism of genes related to gluconeogenesis (Henriksen and Dokken, 2006). It is also advocated that during the treatment of CNS neuropathy of diabetes mellitus, GSK-3 might be a significant target since it has shown a vital role in the pathogenesis (Qu et al., 2014). GSK-3β expression in our study was downregulated in comparison to the diabetic control group. Its inhibition will further improve the insulin production and glucose metabolism in human skeletal muscle (Nikoulina et al., 2020) which will further improve the diabetes patient health.

Dipeptidyl peptidase IV (DPPIV) is generally a scattered multifunctional transmembrane glycoprotein in organs and tissues of humans that selectively dissects dipeptides after proline or alanine residues (Yang et al., 2007; Longenecker et al., 2006). It is acclaimed for its deactivation of incretin hormones engaged in glucose homeostasis, specifically, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) (Green et al., 2006), which stops the secretion of glucagon and activates insulin secretion. DPPIV inhibitors are involved in lowering glucose levels and hence they stall the active time of GLP-1 and GIP as their elementary pharmacological action. In this study, after STZ treatment, the Ptyrone^TM group showed DPPIV inhibition.

Molecular docking performed for the ligands (pterostilbene and andrographolide) with protein targets (GSK-3β, PPARα, PPARγ, DPPIV, GPR40, SGLT2, GLUT4 and AMPK) showing good mode of binding or interactions (in terms of hydrogen bond formation) with each ligand with protein. Interacting residues, binding energy for all the protein–ligand and their activity is shown in Tables S6 and S7.

5 Conclusion

The authors for the first time developed a formulation (Ptyrone^TM) of two major hydroalcoholic extracts based phytoactives each from both plants for its action against type 2 diabetes mellitus. The in vivo studies have shown that this formulation significantly reduced fasting blood glucose, along with an increase in PPARγ and GLUT4 expressions with a reduction in SGLT2 and GSK-3β expressions. The formulation did not even exhibit significant DPPIV inhibition in comparison with the diabetic control group. These findings are further confirmed with in silico studies. The docking studies were excuted to better understand the interaction between the ligands-pterostilbene, andrographolide along with eight protein targets. Both the ligands formed hydrogen bonds with all the target proteins except pterostilbene which showed no interaction with PPARα. Thus, it can be concluded that Ptyrone^TM formulation with the phytoactives from two plants in specific proportions is a potential antidiabetic candidate with its multi-targeted effects. This is for the first time such a plant-based product derived from natural origin has emerged with so many multiple targets in diabetes.