Diabetes mellitus (DM) is one of the most prevalent non-communicable disorders globally, with its incidence rising steadily across all age groups. The global diabetic population is projected to exceed 700 million by 2045, posing significant healthcare and economic challenges (IDF, 2025). Diabetes mellitus (DM) is one of the most prevalent non-communicable disorders worldwide, with extensive research conducted across medical history (Swain et al., 2022). Across medical history, Diabetes Mellitus (DM) is the field that has maximum research. Diabetes was reported more than 2000 years ago, the first documented account of diabetes can be found in the Ebers Papyrus, an assemblage of Egyptian medical manuscripts composed in 552 BC (Hagedorn et al., 1936; Khan and Siddiqui, 2025). The term “Diabetes” was later coined by Aretaeus of Cappadocia between 129 and 199 AD, derived from the Greek word ‘Siphon.’ Historical texts from India and China also reference diabetes and its traditional treatments (Evans et al., 2011).
       
Diabetes mellitus (DM) is characterized by impaired metabolism and chronic hyperglycemia resulting from either inadequate insulin secretion or a combination of insulin resistance and insufficient insulin production. Clinical manifestations include polyphagia, polydipsia, polyuria, weight loss and vision disturbances. If left untreated, diabetes can lead to severe complications, including diabetic ketoacidosis and coma (Ma et al., 2025).
       
As of 2019, approximately 463 million people worldwide were living with diabetes, with projections suggesting this number will rise to 700 million by 2045. DM is primarily classified into two types: Type 1 DM, which is insulin-dependent due to the complete lack of insulin secretion by pancreatic β-cells and Type 2 DM, which is characterized by insulin resistance and partial insulin deficiency, accounting for over 90% of cases.
       
Animal models have been instrumental in diabetes research, sharing notable similarities with human diabetes. Dogs experience Type 1 diabetes, while cats exhibit Type 2 diabetes, making them valuable in pharmacological studies. Recent research indicates a rising incidence of DM in pets, influenced by factors such as obesity and genetics. Experimental models like diabetic rodents induced by alloxan or streptozotocin have provided significant insights into diabetes mechanisms.
       
Therapeutic strategies for diabetes include insulin therapy and oral hypoglycemic agents, alternative therapies-particularly those based on medicinal plants-have gained prominence due to their potential efficacy and reduced side effects. Ethnobotanical research has identified numerous plants with antidiabetic properties, emphasizing the relevance of traditional medicine as a complementary therapeutic approach (Firdous et al., 2025).
       
Medicinal and aromatic plants serve as significant alternatives in traditional and complementary medicine, offering therapeutic and protective benefits in various diseases. These benefits are attributed to their antioxidant, antidiabetic and antimicrobial properties, derived from valuable phytochemicals. In particular, numerous medicinal aromatic plants and fruits have demonstrated protective effects against diabetes induced by streptozotocin (Manikandan et al., 2018; Song et al., 2019; Wang et al., 2020).
       
While numerous antidiabetic plants are available, this study selects four well-researched, locally accessible medicinal plants-Trigonella foenum-graecum (Fenugreek), Momordica charantia (Bitter melon), Syzygium cumini (Jamun) and Catharanthus roseus-for formulation into a polyherbal combination aimed at maximizing antidiabetic efficacy. It is hypothesized that the synergistic interaction, such synergy is hypothesized to enhance metabolic indices more effectively than individual extracts, as supported by recent findings on multi-herb formulations (Jain et al., 2025). Traditional systems of medicine often favour polyherbal formulations over single-herb treatments due to enhanced therapeutic efficacy and reduced side effects. Despite existing studies on polyherbal formulations, a considerable research gap persists. This study aims to evaluate the antidiabetic and antioxidant potential of the selected plants in streptozotocin-induced diabetic rat model, providing further insight into their therapeutic benefits.

The present investigation was conducted in the Department of Veterinary Pharmacology and Toxicology, Bihar Veterinary College, Bihar Animal Sciences University, Patna. The study aimed to assess the antidiabetic and antioxidant potential of a polyherbal formulation comprising Trigonella foenum-graecum (Fenugreek), Momordica charantia (Bitter melon), Syzygium cumini (Jamun) and Catharanthus roseus in streptozotocin-induced diabetic Wistar rats.
 
Experimental animals
 
This study involved 54 male albino Wistar rats, aged 8-10 weeks, with an average weight of 200-250 g. Prior to initiation, ethical approval was obtained from the Institute Animal Ethical Committee (Decision date and number: 31.10.2022-2022/30). All experimental procedures adhered to ethical guidelines, ensuring animal welfare and compliance with regulatory standards.
 
Experimental design and diabetes induction
 
The study was conducted over a 30-day period, with 54 male albino Wistar rats (aged 8-10 weeks, weighing 200-250 g) randomly allocated into nine distinct experimental groups (six rats per group) to evaluate the antidiabetic effects of a polyherbal formulation (PHF) in a streptozotocin-induced diabetic model. Randomization was employed to minimize bias and ensure statistically reliable results.
       
Diabetes was induced in experimental rats via a single intraperitoneal injection of streptozotocin (STZ) at a dose of 60 mg/kg body weight. Following confirmation of diabetes, diabetic rats were divided into different treatment groups (group IV o IX). The detailed experimental design has been presented as Table 1.


 
Preparation and composition of polyherbal formulations
 
Two polyherbal formulations were developed using four medicinal plants with well-documented antidiabetic properties: Trigonella foenum-graecum (Fenugreek), Momordica charantia (Bitter melon), Syzygium cumini (Jamun) and Catharanthus roseus (Sadabahar). The plant materials were shade-dried, powdered and blended in specific ratios to create two distinct combinations, as detailed in Table 2.


       
The powdered mixtures were soaked in a 50:50 hydromethanol solvent using cold extraction method to extract bioactive constituents (Kumar et al., 2023). The resulting extracts were filtered, concentrated and stored under refrigeration until use.
       
PHF-I emphasized Trigonella foenum-graecum and Syzygium cumini, leveraging their potent hypoglycemic and insulinotropic effects. This formulation was designed to enhance glucose metabolism and pancreatic function.
       
PHF-II prioritized Momordica charantia and Catharanthus roseus, both known for their insulin-mimetic and β-cell regenerative properties. This combination aimed to exploit their complementary mechanisms for improved glycemic control.
       
The formulation strategy was based on the hypothesis that synergistic interactions among these botanicals would yield superior therapeutic outcomes compared to individual plant extracts. By evaluating both combinations in a standardized diabetic rat model, the study sought to identify the most effective blend for managing hyperglycemia and oxidative stress.

Collection of serum samples
 
Blood samples were obtained from the tail vein of rats at predetermined intervals-days 0, 10, 20 and 30. Following collection, the samples were allowed to clot in non-heparinized test tubes at room temperature for 3-4 hours. The resulting serum, separated from the retracted clot, was carefully collected and subjected to centrifugation at 4^oC for 20 minutes at 4000 rpm. The supernatant serum fraction was then isolated and stored at -20^oC for subsequent in vivo parameter analysis (Kumar et al., 2025).
 
Measurement of In-vivo parameters for assessment of antidiabetic effect
 
Blood insulin estimation
 
Plasma samples obtained from collected blood were analyzed for insulin levels using an enzyme-linked immunosorbent assay (ELISA). The procedure involved incubating plasma with an insulin antibody-coated microplate, followed by the introduction of a secondary enzyme-conjugated antibody. Subsequently, a substrate was added, triggering a colorimetric reaction. The optical density of the resultant colour change was measured and insulin concentrations were quantified by referencing a standard calibration curve (Chevenne et al., 1999).
 
Blood glucose estimation
 
Blood glucose levels were assessed by obtaining a sample via tail vein puncture, followed by immediate analysis using a glucometer. The device measured glucose concentration based on a biochemical reaction within the test strip, providing rapid and accurate readings. (Dr. Morepen Laboratories Ltd., Gurgaon, India).
 
Glycosylated haemoglobin estimation
 
Glycosylated haemoglobin (GHb) was estimated using a commercial GHb determination kit, following the manu-facturer’s instructions. Hemolysate preparation to 0.5  ml of lysing reagent in labelled test and control tubes, 0.1 ml of reconstituted control or well-mixed sample was added. The mixture was gently agitated until complete lysis occurred. GHb Separation Ion-exchange resin tubes (labelled test/control) were uncapped and 0.1 ml of the prepared hemolysate was added to each. A resin separator was inserted such that the rubber sleeve rested ~1 cm above the resin surface. Tubes were vortexed for 5 minutes, allowing resin to settle, after which the separator was pushed down to compact the resin. The supernatant was collected and its absorbance was read at 415 nm against distilled water (Spectrum Diagnostics, 2022).
 
Total haemoglobin estimation
 
In test and control tubes, 0.02 ml of hemolysate was diluted with 5 ml of distilled water. The mixture was thoroughly mixed and absorbance was measured against distilled water as blank (Dacie and Lewis, 2023).
 
Calculations
 
Results for the unknowns and controls are calculated as follows.

Statistical analysis
 
Statistical analyses and high-quality graphical represent-ations were performed using GraphPad Prism software (version 10.3.1, trial version). The interaction between groups and time for repeated measurements (0 and 30 days) was evaluated using analysis of variance (ANOVA). A significance threshold of p<0.05 was applied to determine statistical significance in the results.

Diabetes mellitus is one of the leading causes of mortality and disability worldwide (Sun et al., 2022). In this study, the effects of hydromethanolic extracts of two polyherbal formulations (PHF I and PHF II) on blood insulin levels were evaluated in streptozotocin (STZ)-induced diabetic Wistar rats over a 30-day period. STZ induced diabetes by selectively destroying insulin-producing beta cells in the pancreas, leading to a significant reduction in insulin levels in the diabetic control group. These findings are consistent with the observations of Sayeed et al., (2015), who reported similar insulin level declines following STZ administration.
 
Effect of polyherbal formulations on blood insulin levels
 
Table 3 presents the effects of hydromethanolic extracts of two polyherbal formulations, PHF-I and PHF-II, on blood insulin levels in streptozotocin (STZ)-induced diabetic Wistar rats over a 30-day period. As expected, the diabetic control group exhibited significantly reduced insulin levels due to STZ-mediated pancreatic beta-cell destruction.


       
Administration of PHF-I and PHF-II resulted in notable improvements in insulin levels, particularly at the higher dosage of 400 mg/kg. By day 30, insulin concentrations in rats treated with 400 mg/kg of PHF-I (11.57±1.04 mU/L) were comparable to those of the standard drug control (11.9±1.13 mU/L), while PHF-II at 400 mg/kg yielded insulin levels of 9.8±0.91 mU/L. Lower dosages (200 mg/kg) also showed improvements, albeit less pronounced, suggesting a dose-dependent response. These findings align with previous research, such as (Mishra et al., 2010), who reported similar insulin-enhancing effects following administration of hydroethanolic extract (1:1) of Jatropha curcas leaves at doses of 250 mg/kg and 500 mg/kg in alloxan-induced diabetic rats over 21 days. Additionally, observations by (Olayiwola et al., 2004) further support the efficacy of herbal interventions in diabetes management.
       
These findings are consistent with earlier studies, such as Mishra et al. (2010), who reported insulin-enhancing effects of hydroethanolic extracts of Jatropha curcas leaves in alloxan-induced diabetic rats. Similarly, Olayiwola et al. (2004) highlighted the efficacy of herbal interventions in improving insulin levels and glycemic control. Arulmozhi et al. (2010) further demonstrated that polyherbal formulations could restore insulin secretion and enhance metabolic regulation in diabetic models.
       
Recent investigations continue to support the therapeutic potential of multi-herb combinations. Bhaskarrao et al., (2022) showed that polyherbal formulations significantly elevated insulin levels in STZ-induced diabetic rats, while Akhtar et al., (2023) reported that Cichorium intybus extract improved insulin secretion and glycemic control. Additionally, Gupta and Kori (2023) observed that novel polyherbal blends enhanced insulin activity and pancreatic function in diabetic Wistar rats. These outcomes collectively reinforce the hypothesis that synergistic interactions among bioactive phytochemicals in PHF-I and PHF-II contribute to their insulinotropic effect.
 
Effect of polyherbal formulations on blood glucose levels
 
The present study evaluated the antihyperglycemic effects of hydromethanolic extracts of two polyherbal formulations (PHF-I and PHF-II) in streptozotocin (STZ)-induced diabetic Wistar rats over a 30-day period. Both formulations demonstrated a dose-dependent reduction in blood glucose levels, with higher doses (400 mg/kg) exhibiting more pronounced effects. By day 30, PHF-I at 400 mg/kg significantly decreased blood glucose levels from 353.01 mg/dL to 222.54 mg/dL, while PHF-II at the same dosage reduced glucose levels from 384.34 mg/dL to 270.81 mg/dL. These findings suggest substantial antihyperglycemic properties, with PHF-I showing slightly greater efficacy than PHF-II.
       
Table 4 presents the effects of PHF-I and PHF-II on blood glucose levels (mg/dL) over the 30-day study period. As expected, the diabetic control group exhibited persistent hyperglycemia. Both PHF-I and PHF-II displayed dose-dependent glucose-lowering effects, with the higher dosage (400 mg/kg) yielding significant reductions. While both formulations improved glucose regulation, PHF-I demonstrated greater antihyperglycemic potency compared to PHF-II in STZ-induced diabetic rats.


       
These results highlight the potential of hydromethanolic extracts of PHF-I and PHF-II as effective interventions for improving glycaemic control in diabetes. Their efficacy appears to be dose-dependent, with higher doses providing more substantial reductions in blood glucose levels.
 
Effect of polyherbal formulations on glycosylated haemoglobin (HbA1c) levels
 
Table 5 summarizes the impact of PHF-I and PHF-II on glycosylated haemoglobin (HbA1c) levels in various rat groups, measured at four intervals: day 0 (baseline), day 10, day 20 and day 30. The control group maintained stable HbA1c levels throughout the study, demonstrating normal glycaemic regulation. In contrast, the diabetic control group exhibited a significant increase in HbA1c, progressing from 88.45 mg/dL on day 0 to 111.33 mg/dL on day 30, indicating declining glycaemic control due to diabetes.


       
Treatment with PHF-I and PHF-II resulted in comparatively stable HbA1c levels, suggesting their potential role in maintaining blood sugar balance. Despite gradual increases at doses of 200 mg/kg and 400 mg/kg, the formulations kept HbA1c levels lower than those observed in the diabetic control group, demonstrating their antihyperglycemic effects. Higher doses (400 mg/kg) showed greater efficacy, reinforcing a dose-dependent response. Similarly, the standard drug control exhibited a gradual increase in HbA1c levels; however, the values remained consistently lower than those of the diabetic control group, confirming its effectiveness in managing glycaemic regulation.
       
The glucose-lowering effects of both formulations are comparable to those observed with standard antidiabetic drugs, as demonstrated by the standard drug control group, which achieved a blood glucose level of 190.25±8.60 mg/dL by day 30. This dose-dependent response is consistent with findings reported by (Petchi et al., 2014), who documented similar antihyperglycemic effects of polyherbal formulations in diabetic models.
       
The observed reduction in blood glucose levels can be attributed to the synergistic action of various bioactive compounds present in the polyherbal formulations. Our findings align with those of (Pari and Saravanan, 2004), who highlighted the potential of combined herbal extracts in enhancing antidiabetic effects through multiple mechanisms, including improved insulin sensitivity, increased glucose uptake and modulation of carbohydrate metabolism.
       
The study examined the effect of hydromethanolic extracts of two polyherbal formulations (PHF I and PHF II) on glycosylated haemoglobin (HbA1c) levels in streptozotocin (STZ)-induced diabetic Wistar rats over 30 days. The HbA1c-lowering effects observed in the PHF I and PHF II treatment groups reflect their potential to confer sustained glycaemic control in diabetic rats. By day 30, both formulations effectively attenuated the STZ-induced rise in glycosylated haemoglobin, demonstrating values that approached those of the standard drug-treated group. These findings indicate a dose-dependent modulation of chronic hyperglycaemia, analogous to the glucose-lowering patterns observed. This trend aligns with the results of Singh and Ilango (2024), who reported a similar reduction in HbA1c with antidiabetic polyherbal interventions. The improved glycation profile may be attributed to the integrated action of phytoconstituents that enhance insulin activity, limit oxidative stress and suppress glucose-mediated protein glycation pathways=mechanisms echoed in the observations of Kumar and Bhowmik (2010).
       
The results of this study indicate that administration of polyherbal formulations to diabetic rats led to a significant reduction in glucose levels. This effect is likely mediated by the phytochemicals contained within the formulations, which exhibit antidiabetic properties and contribute to improved glycaemic control.
 
Histopathological analysis
 
Histopathological examination of the liver, kidney and pancreas sections from the diabetic control group revealed significant pathological alterations associated with diabetes-induced organ damage (Fig 1-3).






       
Liver sections exhibited pronounced fatty changes accompanied by hepatocellular necrosis, indicating severe hepatic injury and metabolic disruption. Additionally, congestion and hyperplasia of Kupffer cells were observed, alongside substantial infiltration of inflammatory cells, highlighting the extensive inflammatory response and hepatic stress induced by diabetes (Fig 1).
       
In kidney sections, focal necrosis was evident in the tubular epithelial lining, reflecting extensive renal damage. These degenerative changes are characteristic of diabetic nephropathy and indicate impaired renal function (Fig 2).
       
Pancreatic analysis revealed considerable degeneration and necrosis of the Islets of Langerhans, underscoring the disruption of insulin production and the profound impact of diabetes on pancreatic health. Microscopic features of pancreatic islet degeneration are depicted in Fig 3.
       
Treatment with the polyherbal formulations (Groups V, VI, VII and VIII) and the Glibenclamide-treated group (Group IX) demonstrated notable improvements in tissue morphology. In these treatment groups, partial regeneration of damaged hepatocytes was observed, accompanied by a reduction in hepatic inflammatory changes. This suggests a therapeutic effect of PHF-I and PHF-II, particularly at the higher dosage of 400 mg/kg, which exhibited a dose-dependent regenerative impact. Additionally, treatment resulted in mitigated pathological alterations in the kidneys and pancreas. Improvements included a decrease in focal necrosis within the tubular epithelial lining and partial restoration of the normal architecture of the Islets of Langerhans, highlighting a potential protective effect of the formulations on pancreatic integrity.
       
The partial regeneration of damaged hepatocytes and the reduction in inflammatory changes observed in groups treated with PHF-I, PHF-II and Glibenclamide suggest a promising therapeutic potential of these interventions. These findings indicate that both the polyherbal formulations and Glibenclamide not only mitigate diabetes-induced tissue damage but also facilitate repair and regeneration.
       
The higher dosage of 400 mg/kg exhibited particularly significant effects, reinforcing a dose-dependent response. Similar regenerative and protective effects of bioactive compounds in diabetic models have been reported by various workers (Abdollahi et al., 2011; Honjo et al., 1986).

The administration of polyherbal formulations containing Trigonella foenum-graecum, Momordica charantia, Syzygium cumini and Catharanthus roseus demonstrates significant antidiabetic effects in streptozotocin-induced diabetic rats. The formulations effectively improved blood insulin and glucose levels, with the higher dosage of 400 mg/kg showing particularly pronounced results comparable to the standard antidiabetic drug, Glibenclamide. Histopatho- logical analysis further validated the study’s findings, showing that polyherbal formulations treatments reduced tissue damage and encouraged regeneration of damaged cells in the liver, kidney and pancreas. The observed reduction in fatty changes, necrosis and inflammation underscores the therapeutic potential of these polyherbal formulations in managing diabetes and its complications.

This manuscript is a part of the Ph.D. thesis of the first author. The authors wish to acknowledge their gratitude to the DRI-cum-Dean, PGS, BASU, Patna and Dean, Bihar Veterinary College, Patna for providing financial support and necessary facilities to carry out this research work.
 
Informed consent
 
All animal procedures for experiments were approved by the Institute Animal Ethics Committee of Bihar Veterinary College, Bihar Animal Sciences University, Patna duly approved by CCSEA, New Delhi.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.