Exploring the potential of pheophorbide A, a chlorophyll-derived compound in modulating GLUT for maintaining glucose homeostasis.

Introduction: Pheophorbide A, a chlorophyll-breakdown product, is primarily investigated for its anti-oxidant and anti-inflammatory activity. Recent reports on pheophorbide A have shown its potential in lowering blood glucose levels, thus leading to the exploration of its use in diabetes management. Literature has also shown its effect on enhanced insulin secretion, whereas its mechanism on glucose stimulated insulin secretion (GSIS) in pancreatic ß cells remains unexplored. Methods: In-silico and in-vitro investigations were used to explore the effect of pheophorbide A on class I glucose transporters (GLUTs). In-silico studies include - Molecular docking studies and stability assessment using GROMACS. In-vitro studies include - MTT assay, Glucose uptake assay, Live-cell imaging and tracking of GLUTs in presence of Pheophorbide A compared to control. Results: Molecular docking studies revealed better binding affinity of pheophorbide A with GLUT4 (-11.2 Kcal/mol) and GLUT1 (-10.7 Kcal/mol) when compared with metformin (-5.0 Kcal/mol and -4.9 Kcal/mol, respectively). Glucose levels are largely regulated by GLUTs where GLUT1 is one of the transporters that is ubiquitously present in human ß cells. Thus, we confirmed the stability of the complex, that is, pheophorbide A-GLUT1 using GROMACS for 100 ns. We further assessed its effect on a pancreatic ß cell line (INS-1) for its viability using an MTT assay. Pheophorbide A (0.1-1 µM) showed a dose-dependent response on cell viability and was comparable to standard metformin. To assess how pheophorbide A mechanistically acts on GLUT1 in pancreatic ß cell, we transfected INS-1 cells with GLUT1-enhanced green fluorescent protein and checked how the treatment of pheophorbide A (0.50 µM) modulates GLUT1 trafficking using live-cell imaging. We observed a significant increase in GLUT1 density when treated with pheophorbide A (0.442 ± 0.01 µm-2) at 20 mM glucose concentration when compared to GLUT1 control (0.234 ± 0.01 µm-2) and metformin (0.296 ± 0.02 µm-2). The average speed and distance travelled by GLUT1 puncta were observed to decrease when treated with pheophorbide A. The present study also demonstrated the potential of pheophorbide A to enhance glucose uptake in ß cells. Conclusion: The current study's findings were validated by in-silico and cellular analyses, suggesting that pheophorbide A may regulate GLUT1 and might be regarded as a potential lead for boosting the GSIS pathway, thus maintaining glucose homeostasis.

Exploring antidiabetic potential of a polyherbal formulation Madhurakshak Activ: An in vitro and in silico study.

Madhurakshak Activ (MA), a commercial polyherbal antidiabetic preparation is known to manage diabetes mellitus (DM) by reducing blood glucose levels. However, lacks systematic mechanistic evaluation for their molecular and cellular mode of actions. In the present study, hydro-alcoholic and aqueous extract of MA were evaluated for their effects on glucose adsorption, diffusion, amylolysis kinetics and transport across the yeast cells using in vitro techniques. Bioactive compounds identified from MA by LC-MS/MS were assessed for their binding potential against DPP-IV and PPARγ via an in silico approach. Our results revealed that the adsorption of glucose increased dose dependently (5 mM -100 mM). Both extracts exhibited linear glucose uptake into the yeast cells (5 mM - 25 mM), whereas glucose diffusion was directly proportional to time (30-180 min). Pharmacokinetic analysis revealed drug-like properties and low toxicity levels for all the selected compounds. Among the tested compounds, 6-hydroxyluteolin (-8.9 against DPP-IV and PPARγ) and glycyrrhetaldehyde (DPP-IV -9.7 and PPARγ -8.5) have exhibited higher binding affinity compared to the positive control. Therefore, the above compounds were further considered for molecular dynamics simulation which showed stability of the docked complexes. Hence, studied mode of actions might produce a concerted role of MA in increasing the rate of glucose absorption and uptake followed by the in silico studies which suggest that the compounds identified from MA may inhibit DPP-IV and PPARγ phosphorylation.

Multimode Assessment of Commercial Polyherbal Formulation: an In Vitro and In Silico Approach.

Antidiabetic polyherbal formulations (APH) are used in management of diabetes mellitus (DM). High glucose levels in DM are related to oxidative stress leading to its associated complications. Therefore, assessing antioxidant activity of various APH might unveil an antioxidant-rich formulation for management of DM and its associated complications. Subsequently selecting an antioxidant assessment method is a challenging aspect, considering various in vitro assays working with diverse mechanism of action. Therefore, present study aims to validate the sensitivity/capacity of different antioxidant assay, thereby assessing the antioxidant potential of 9-APH. Obtained results revealed the ABTS·+ values were higher compared to DPPH+ assay. I-9-HAE (DPPH+: IC50 53.31 µg/ml), NK-HAE (ABTS·+: IC50 2.71 µg/ml), and MN-HAE (FRAP and TAC) exhibited highest antioxidant capacity. A significant correlation was obtained between TPC-DPPH+ (r2: 0.8187****). Furthermore, three APH with better antiradical potential was chosen for various in vitro and in silico method, for validating scientific antidiabetic propensities. Among the tested extracts, I-9-HAE (α-amylase inhibition: IC50 831.84 µg/ml) and MN-HAE (α-glucosidase inhibition: IC50 558.64 µg/ml and antiglycation: IC50 883.74 µg/ml) have showed highest antihyperglycemic and antiglycation properties. Finally, the secondary-metabolites of selected APH were screened through literature search, Lipinski rule, ADMET, and ProTox-II. Subsequently, in molecular docking for the selected 9 secondary metabolites, highest binding affinity was observed in apigenin-7-glucuronide for DPPiv (- 9.6), GLP-1 (- 8.8), NADPH (- 8.7), and HSA (- 9.4). Thus, obtained result proposes synergistic interaction with high antioxidant potential of the selected 3-APH and can be considered an alternative for management of DM, where multiple secondary metabolites exert holistic biological effects. Furthermore, our study also provides data on sensitivity/capacity of different in vitro antioxidant assays.

Comparative study of six antidiabetic polyherbal formulation for its multimodal approaches in diabetes management.

Commercial antidiabetic polyherbal formulations (APH) are available with claimed hypoglycemic activities; yet they lack systematic scientific studies leading to their limited global acceptance. In the present study, six selected APH from the Indian market were evaluated for their phytochemical contents, anti-hyperglycemic, anti-hyperlipidemic, antioxidant activities and further identifying the major antidiabetic bioactive compound of "MA" by HPLC-ESI-MS/MS. Our results revealed highest TPC (136.97 ± 0.6 µg GAE/mg) and TFC (128.85 ± 0.74 µg QE/mg) in APH-DB and APH-SN, respectively. APH-MA has exhibited highest α-amylase 72.5% (IC50-579.65 µg/ml), α-glucosidase 88.02% (IC50-261.03 µg/ml) and moderate lipase inhibition 57.7% (IC50 159.57 µg/ml). A variable free radical scavenging activity was observed by all the tested APH. Further significant linear positive correlations were observed between TPC-Lipase (r 2-0.985****), TFC-α-amylase (r 2-0.868**) and DPPH-α-amylase inhibition (r 2-0.8098*). HPLC-ESI-MS/MS of MA showed the presence of anti-hyperglycemic compounds, Pheophorbide a and Pyropheophorbide a, as the major peaks. Among the tested extracts, MA exhibited better activities while BG, MH, SN, DB, and DT have showed comparable/mild anti-hyperglycemic, anti-hyperlipidemic and antioxidant potential. Hence the tested APH may be considered effective for DM management which can further be assessed for their other targets of inhibition.

Health risk assessment of Macro, Trace-elements and heavy metal in various Indian Antidiabetic Polyherbal formulations.

Diabetes mellitus, a global pandemic, can be holistically managed with the use of polyherbal formulations which is an accessible form of treatment in developing countries due to fewer side effects, economical and easily available. Commercial polyherbal formulation lacks systemic based scientific study, thus it is suspected to be associated with many contaminations and related toxicities, one of which is considered to be elemental health hazards. Therefore, the present study is designed to assess six selected antidiabetic polyherbal formulations from the Indian market for their element contents, quality, and health risk assessment. Concentrations of 35 essential and non-essential trace-elements were quantified by Handheld X-ray spectrophotometer and health risk assessment was calculated by estimated daily intake (EDI) and Total hazard quotient (THQ). Elements were found to be in a vast range of concentration in the tested APH. Among the 35 elements analyzed, Ca (23100±0.033ppm) and K (14800±0.021 ppm) in "MH" and Zn (15600±0.025 ppm) in "DB" were found to be the highest. The lowest concentrations of Rb and Nb (3±2 ppm) were observed in the formulation "MH" and "SN" respectively. THQ of all the elements was calculated to be less than unity except for Rb in the formulation "MA". Rb is rarely associated with toxicities as it is rapidly excreted in sweat and urine. V, Co, Ni, Cu, As, Se, Y, Ag, Sn, Sb, Ba, W, and Hg were absent in all the APH. Therefore the present study indicated the presence of essential elements some of which are important for the management of diabetes and hence can be considered safe for use.