Diabetes is one of the most common and thus important diseases in the world, hence it requires special attention with more emphasis on translational research. In the past few decades, several treatment regimes have been practiced; unfortunately, their associated adverse effects are not yet satisfactorily resolved (WHO, 2021, fact sheet; Nugraha, R. V et al., 2020). Studies have revealed that the phytochemicals present in polyherbal formulations can be a constitutive source (Dinda, B., & Dinda, M. (2022; Mukherjee P. K. et al., 2021) and their amalgamation into the main treatment regime can be the solution we have been seeking. Researchers have found that owing to their complex composition ayurvedic/polyherbal formulations tend to interact with multiple targets, and resonate in a dynamic way providing an enhanced effect.
Proteomics is a crucial tool for understanding the disease/physiological condition and associated pathways, and in mapping the mechanism of action of respective drugs/compounds. Here, we have prepared a polyherbal formulation by mixing 6 plant powdered materials namely Rubia cordifolia, Gymnema sylvestre, Terminalia Arjuna, Pterocarpus Marsupium, Holarrhena antidysenterica, and Tinospora cordifolia in equal proportions. There are no reports of toxicity for the individual components of formulation in the current study (Balkrishna, A et al., 2022; Eskandarzadeh et al., 2023; Verma, P et a., 2022; Rekha, B. C et al., 2016; Raji, R. O et al., 2021; Patil, S. G et al., 2016; Anantharaman et. al., 2016). Due to synergism, poly-herbalism offers benefits not deliverable by a single herbal drug. It enables better therapeutic effects with lower doses, reducing the risk of adverse side effects of individual plants, if any (Kotmire, S et al., 2024). Also, presence of multiple active compounds together can provide a potentiating effect that may not be achievable by any single compound. Thus, a polyherbal formulation can provide a complete therapy against a disease condition (Karole, S et al., 2019).
In the present study, the formulation at the dose level of 2000 mg/kg body weight, did not cause any mortality or toxicity thus confirming that after combination the formulation is nontoxic. This indicates that the maximum tolerated dose of the polyherbal formulation was more than 2000 mg/kg and thus, no observed adverse effect level (NOAEL) of the formulation should be above 2000 mg/kg with approximate LD50 more than 2500 mg/kg. Thus, according to the "Globally Harmonized System (GHS) for classification of chemicals which cause acute toxicity, OECD series on testing and assessment, Number 33; Harmonized Integrated Classification System for Human and Environmental Hazards of Chemical Substances and Mixtures (ENV/JM/MONO (2001) 6)", the Polyherbal formulation can be classified as GHS Category 5 or Unclassified for the obligatory labeling requirement for oral toxicity.
Although there can be side effects when taken alongside other medicine prescribed in diabetic condition. As some of the individual components have been known to enhance the immune response, lower the blood glucose levels, expedite liver metabolism, and have anticoagulant activity (Parasuraman, S et al., 2014). When co-consumed, patients with autoimmune response can experience severe reaction, it can lead to hypoglycemic conditions, and it may also all together alter the pharmacokinetics of drugs. In the current study the combination effects of modern medicine and the formulation has not been studied.
For the formation of polyherbal formulations, drug antagonism is a critical factor
to be considered. In the preparation of polyherbal, it is crucial to note that some herbs are incompatible (viruddha) and should not be taken in combination. Such discordance can be due to quantitative incompatibility, energetic incompatibility or functional incompatibility (Parasuraman, S et al., 2014). In our formulation we have not observed any adverse effect during and after treatment in animal model.
The formulation was found to be most effective at 500 mg/kg concentration in regulating body weight and maintaining blood glucose levels in the STZ induced diabetic rat model. It was able to revert the declined bodyweight and elevated blood glucose levels to normal (Figs. 1 and 2). Suggesting that the formulation has an positive effect on glucose uptake and metabolism (Kang et. al, 2009; Xu et al., 2019; Liu et al., 2021, Sharma et. al., 2021; Li Y et. al., 2019; Kim et. al., 2017; Biswas et. al, 2011; Mohanty et. al., 2019; Pari et. al., 2018; Mishra et. al., 2013; Ali et. al., 2011; Sharma et. al., 2021; Jamadagni et al., 2017; Chandrasekaran et al., 2009; Joshi et al., 2004; Kumari et al., 2021; Ogawa et al., 2004). Here we aimed to provide a better understanding of the molecular mechanisms involved in the treatment of diabetes using a polyherbal formulation, and the importance of natural resources for improving the quality of life.
Hallmark of diabetes mellitus is hindered glucose uptake and metabolism due incapacitated or damaged pancreatic β-cells. There are reports suggesting the pancreatic β-cells repair/regenerative potential displayed by the individual component plants on the STZ-induced rat model (Shanmugasundaram et al., 1990; Bolkent et al., 2000; Daisy et al., 2009; Mishra et. al., 2013; Rajalakshmi and Anita., 2016). Here, we have tested the synergistic effect of the polyherbal formulation on the STZ-induced rat model for the same. By studying the protein-protein interactions, we can link cellular pathways and their intricate cross-connectivity. In the Healthy control group, the PPIs show overall growth, progression, maintenance, and homeostasis. As per the condition in the disease control group, the PPIs displays an ongoing active repair mechanism resultant of streptozotocin treatment. Also the interaction depicting disturbed calcium homeostasis which can be correlated with diabetic cardiomyopathy were observed (Pereira, L., et al, 2014). For the Glibenclamide standard group, PPIs reveal microtubule assembly promotion, metabolism of lipoproteins, and membrane trafficking in the trans-Golgi network (TGN). In the 250 mg/kg drug treatment group, the PPIs related to cell growth, cell cycle regulation, developmental processes and cell survival that unveil the active cell proliferation, differentiation, and growth in response to treatment of formulation. In the 500 mg/kg treatment group, PPIs have shed light on significant ongoing cellular processes that hint towards a repair mechanism being activated in response to damage incurred to pancreatic β-cells. As we were able to identify insulin receptor protein which is an integral component of glucose metabolism, and a key protein, murinoglobulin-1’s functional interactions are responsible for maintenance of pancreatic well being (Umans, L., et al., 1999).
The DAVID-KEGG pathway analysis of identified proteins uncovered the possible pathways that were triggered in respective study groups. Namely the HIF-1 signaling pathway in the healthy control group, Insulin secretion and circadian entrainment pathways in the disease control group, ECM-receptor interaction pathway in the 250 mg/kg group, and circadian entrainment pathway in the 500 mg/kg group.
In the healthy control group, three proteins from our study namely Hexokinase-2, Angiopoietin-2, and Protein kinase C gamma type were found to be associated with the HIF-1 signaling pathway. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that functions as a master regulator of oxygen homeostasis in all metazoan species. HIF-1 controls oxygen delivery, by regulating angiogenesis and vascular remodeling, and oxygen utilization, by regulating glucose metabolism and redox homeostasis (Cerychova and Pavlinkova., 2018; Krock et al., 2011; Semenza., 2014). Bosch-Marce et. al., (2007) have demonstrated that the therapeutic enhancement of HIF activity can overcome age and diabetes, as ectopic expression of HIF-1α can partially rescue limb perfusion in old mice.
In the disease control category, three proteins from our study were found to be involved with Insulin secretion and circadian entrainment pathways; these were Inositol 1,4,5-trisphosphate receptor type 3, Voltage-dependent T-type calcium channel subunit alpha-1H and Protein piccolo. The Inositol triphosphate pathway mobilizes the calcium ions from organelles and consequently increases the secretion of insulin (Onaolapo et al., 2018). Mechanisms for Ca2+ entry include voltage-operated channels (VOC), receptor-operated channels (ROC), and store-operated channels (SOC) as well as two families of intracellular Ca2+ release channels, the ryanodine receptor (RyR) and InsP3R (Hagar and Ehrlich, 2000; Yang et al., 2013). In 2002, Fujimoto et. al. demonstrated the importance of the cAMP-GEFIIּ Rim2ּ Piccolo complex in cAMP-induced insulin secretion. Thus indicating the effort of the body to regulate the imbalanced insulin levels due to damaged pancreas (islet of Langerhans).
In the 250 mg/kg formulation treatment category, three proteins were found to be engaged with ECM-receptor interaction; the Collagen alpha-1(XI) chain, Collagen alpha-1(I) chain, and Collagen alpha-2(I) chain. During the onset and progression of Diabetes, the ECM protein expression and functioning are impaired, leading to structural alteration of the ECM network as well as normal tissues and cell behavior including cell-cell interactions (Bansode and Gacche., 2019; Shirolkar et al., 2022). Subsequently, these changes lead to diabetes-induced organ-dependent diseases such as nephropathy, retinopathy, and diabetic cardiomyopathy. Spiro and Crowley (1993) have also demonstrated that Alloxan-induced diabetes in rats displayed a significant increase in collagen type VI when compared to collagen types I and IV and other ECM proteins. A high glucose environment and other changes in diabetes lead to alterations in ECM, such as decreased collagen deposition and abnormal collagen metabolism mitigate wound healing (Huang and Kyriakides., 2020). Thus, indicating the therapeutic effect of formulation in mitigating the diabetic condition via up regulation of ECM network.
In the 500 mg/kg treatment category, three proteins were found to be involved in the circadian entrainment pathway; those were Ryanodine receptor 2, Glutamate receptor ionotropic NMDA 2B, and Voltage-dependent T-type calcium channel subunit alpha-1G. The CLOCK and BMAL1 proteins are known to activate the transcription of genes in the pancreas, which are responsible for insulin synthesis/transport and glucose-stimulated secretion (Stenvers et al., 2019). Marcheva et. al., (2010) have demonstrated using both CLOCK and BMAL1 mutants that disruption of the biological clock components leads to hypoinsulinemia and diabetes. Uehara et. al., (2004) have suggested that the mGluR4-mediated signaling and GABAA receptor-mediated cascade; both individually act through the L-glutamatergic system that may act as autocrine transmitter and inhibit glucagon, in the islet of Langerhans. This displays the fringe benefit of the polyherbal formulation at 500 mg/kg in diabetes condition, as it maneuvers towards normalization of the metabolism. As discussed herewith we have demonstrated that diabetes-specific proteins and pathways are associated with progressions and recovery using a polyherbal formulation holistically.