Evaluation of antidiabetic potential of Plumbago indica Roots: insights from in vitro, in vivo and in silico studies

Evaluation of antidiabetic potential of Plumbago indica Roots: insights from in vitro, in vivo and in silico studies Abdulkadir Abdu Mohmmednor Ibrahim1, Brahmjot Singh1, Hossamaldeen Bakrey1, Hasandeep Singh2, Atamjit Singh1, Balbir Singh1, Sarabjit Kaur1 1 Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, 143005, Punjab, India 2 Department of Pharmaceutical Sciences, Amritsar Group of Colleges, Amritsar, 143005, Punjab, India Corresponding author: Sarabjit Kaur (sarabjit.pha[email protected]) Received 14 July 2025♦ Accepted 24 September 2025♦ Published 19 December 2025 Citation: Ibrahim AAM, Singh B, Bakrey H, Singh H, Singh A, Singh B, Kaur S (2025) Evaluation of antidiabetic potential of Plumbago indica Roots: insights from in vitro, in vivo and in silico studies. Pharmacia 72: 1–17. https://doi.org/10.3897/pharmacia.72.e165113 Abstract Diabetes mellitus (DM) is a chronic metabolic disorder characterized by elevated blood glucose levels due to insufficient insulin production or decreased insulin sensitivity. Despite advances in antidiabetic pharmacotherapy, the limitations and adverse effects of current treatments highlight the need for natural products as alternative therapies. The present investigation was carried out to evaluate the anti-diabetic activity of the methanol extract (PIME) and ethyl acetate fraction (PIEAF) of roots of Plumbago indica using in vitro, in vivo and in silico models. The in vitro study was done by α-glucosidase and αamylase inhibition assays. The effect of PIME and PIEAF on serum parameters and histopathological changes was assessed in streptozotocin/nicotinamide-induced type 2 diabetes mellitus in rats. The in vitro studies showed that PIEAF and PIME inhibited α-glucosidase and αamylase enzymes in a dose dependent manner. In vivo studies indicated that administration of PIME and PIEAF led to the reduction in serum glucose, cholesterol, triglycerides, hepatic enzyme levels and significantly improved renal function and oxidative stress markers. Taking Plumbagin as the marker compound in P. indica, the HPLC analysis of PIME was carried out and it showed that the plumbagin content was 0.26%. In silico studies revealed that plumbagin inhibited α-glucosidase activity through optimal orientation and strong molecular affinity. P. indica roots showed strong promising potential as a treatment for diabetes, probably due to its antioxidant properties and higher content of plumbagin than other species of Plumbago. Keywords Plumbago indica, Antidiabetic, Streptozotocinnicotinamide, Plumbagin, Molecular docking Introduction Diabetes mellitus is a metabolic disorder causing elevated blood sugar levels due to reduced insulin production and sensitivity (Mukhtar et al. 2020). Type 2 diabetes mellitus (T2DM) is the most predominant type, accounting for around 90% of diabetes cases worldwide (Galicia-Garcia et al. 2020). T2DM is strongly linked to obesity rates, particularly among individuals aged 45 and older (Hillier and Pedula 2001). However, there is an alarming increase in T2DM cases among children and teenagers due to the rising prevalence of obesity and sedentary lifestyles (Amschler 2002). Diabetes mellitus has been considered as a silent killer disease since its evoluCopyright Ibrahim AAM et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pharmacia 72: 1–17 DOI 10.3897/pharmacia.72.e165113 Research Article Ibrahim AAM et al.: Hypoglycaemic potential of P. indica Roots2 tion (Joseph and Vadasseril 2023). Pancreatic β-cells are responsible for glucose homeostasis and the cells can be degenerated in diabetic patients with a loss of the ability to control the high glucose level, resulting in diabetic complications (Khin et al. 2023). A decrease in secretion of insulin from the pancreas, an increase in blood glucose level and unutilized blood glucose by tissues leads to symptoms of diabetes, such as polyphagia, polyuria and polydipsia (Adu-Sarkodie 2017). Recent research has shown that the prevalence of diabetes is increasing rapidly in many developing and developed countries (Khan et al. 2019). Currently, DM is controlled either by administration of exogenous insulin, hypoglycemic agents or by insulin secretagogues (Satin et al. 2021). However, these approaches are associated with various side effects and toxicity. Some hypoglycemic synthetic agents are even known to produce life-threatening hypoglycemia (Mishra et al. 2021; Tasnim et al. 2024). The drawback of current medications arises from the lack of efficacy, high risk and high cost of the treatment. Due to adverse effects of various synthetic antidiabetic agents, there is an increasing interest in the prediction of antidiabetic potential of herbal plants. Therefore, the plantbased therapy for diabetes using traditional medicines is growing widely which are in high demand for safe, economical and readily available treatments (Ansari et al. 2023; Mishra et al. 2024). P. indica Linn. is a medicinal plant belonging to Plumbaginaceae family, used in Thai traditional medicine for gastric stimulation, flatulence, hemorrhoids and improving appetite. Moreover, in eastern Africa and India, it is used as gastric stimulant, abortifacient and oral contraceptive (Ibrahim et al. 2018). The roots of this plant serve as a major source of plumbagin, a naturally occurring yellow pigment with a wide range of pharmacological properties (Lenora et al. 2012). There is substantial evidence indicating that Plumbagin have a significant role in the treatment of inflammation, breast cancer, brain tumors, lung cancer, hepatocellular carcinoma, cardiovascular diseases and other conditions (Sharma et al. 2024). As the principal and bioactive component present in the roots of this plant is plumbagin, therefore its initial extraction and subsequent testing by HPLC was done in this study to verify the presence of the intended bioactive compound. P. indica is rich in naphthoquinones, flavonoids and terpenoids which have shown their potential in diabetes management (Priyanjani et al. 2021). It has been reported that this plant possesses antioxidant activity and reduces chronic inflammation which are linked to diabetes and its complications (Ittiyavirah et al. 2012; Eldhose et al. 2013). Moreover, other species of genus Plumbago eg. P. zeylanica and P. auriculata have been reported to exhibit antidiabetic activity (Zia et al. 2024). As there is no scientific report available on hypoglycemic activity of P. indica, therefore, this study was carried out to evaluate the antidiabetic potential of this plant. Materials and methods Drugs and chemicals Streptozotocin (STZ) at a purity of 98% and nicotinamide at a purity of 99.5% were procured from Sisco Research Laboratories Pvt. Ltd India. Nitro blue tetrazolium (NBT), reduced glutathione (GSH) and 5, 5’-dithio-bis [2-nitrobenzoic acid] (DTNB) were obtained from Loba Chemie, Mumbai, India. All remaining reagents utilized in the experiment were of analytical grade. Plant material Plumbago indica roots were procured from Jagdeesh Herbals, Kerala and its authentication was done by Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar and voucher specimen no. (3979) has been kept at the Department Herbarium. Preparation of plant extracts and their active fractions The plant material, after cleaning and authentication, was grounded into a coarse powder using a mechanical grinder. The dried plant material (1000 g) was first defatted with hexene and successively extracted with chloroform and methanol in a Soxhlet apparatus for 48 hours. Further fractions were obtained from the most potent extract using ethyl acetate and n-butanol. Each extract and fractions were concentrated using a rotary evaporator after filtration and their weight was measured to calculate the percentage yield from the air-dried plant material. The concentrated extracts/fractions were stored in a refrigerator to prevent microbial contamination (Rajput et al. 2023). Preliminary phytochemical screening Preliminary testing for the presence of secondary metabolites was performed for the most bioactive extract using standard protocols for various classes of phytoconstituents such as alkaloids, terpenoids, flavonoids, glycosides and tannins (Afzal et al. 2022). In Vitro studies Antioxidant assay 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) free radical scavenging assay To assess the hydrogen-donating capacity of natural compounds or plant extracts, DPPH assay was used. The process of converting the DPPH radical to a stable molecule allows for the measurement of the capacity to scavenge free radicals. The IC50 value for the DPPH scavenging activity was calculated. Different concentrations of extracts and Pharmacia 72: 1–17 3 fractions (25, 50, 100, 200 and 400 μg/ml) were prepared in methanol, added to test tubes and mixed with methanolic DPPH solution. After incubation, absorbance was measured at 517 nm against a blank with lower absorbance indicating higher scavenging capacity (Rajput et al. 2023). The equation for calculating scavenging activity is as follows: Inhibition potential = (Ac – As) / Ac × 100, where Ac is the absorbance of control and As is the absorbance of sample. Ferric reducing antioxidant power assay (FRAP) The FRAP assay was conducted using various concentrations of extracts and fractions ranging from 25–400 μg/ml. This assay assessed the ability of the extracts/fractions to convert potassium ferricyanide (Fe3+) into potassium ferrocyanide (Fe2+). This Fe2+ then reacts with ferric chloride, forming a ferricferrous complex measurable via spectrophotometry. Each concentration (1ml) was mixed with 2.5 ml of 1% potassium ferricyanide and 2.5 ml of phosphate buffer (200 mM, pH 6.6) and the mixture was incubated at 50 °C for 20 minutes. Eventually, 2.5ml of 10% TCA (trichloroacetic acid) was added and centrifuged at 3000 rpm for 10 minutes. Later, 2.5 ml of supernatant was mixed with 2.5ml of double distilled water and 0.5 ml of Ferric chloride (0.1%) was added and the absorbance was measured at 765 nm (Koffi et al. 2022). Ascorbic acid was used as the standard drug. Inhibition potential was calculated using the following formula: Ferricyanide reduction Potential = Absorbance of ascorbic acidAbsorbance of sample/ Absorbance of ascorbic acid ×100. Antidiabetic assay Alpha-glucosidase inhibition assay For the α-glucosidase inhibition assay, a microtiter 96well plate was utilized. In this assay, wells of the plate were filled with a solution containing 50 μl of phosphate buffer (pH 6.8), 10 μl of α-glucosidase enzyme solution (1U/ml) and the extracts and fractions (25, 50, 100, 150, 200 and 250 μg/ml) were added and pre-incubated for 5 minutes at 37 °C (Wang and Zhao 2019). After the pre-incubation period, 20 μl of a 2 mmol/l solution of p-nitrophenol-α-Dglucopyranoside (pNPG) substrate from HiMedia was added. The mixture was then incubated at 37 °C for 30 minutes. Afterwards, 50 μl of sodium carbonate solution (100 mmol/l) was added to stop the reaction. Acarbose served as the reference compound. The conversion of pNPG into yellow-colored p-nitrophenol was determined by measuring the absorbance at 405nm using a spectrophotometer. The assay was conducted in triplicate along with appropriate blanks. Alpha-amylase inhibition assay The α-amylase enzyme inhibition assay was conducted using a microliter 96-well plate. A solution comprising 20% methanol, 25 μL of phosphate buffer (pH 6.9) and porcine αamylase, was dispensed into each well of the plate, after which extracts and fractions (25–250 μg/ml) were added. The reaction mixture was incubated at 25 °C for 10 minutes. The enzymatic reaction was halted by adding 50 μL of 96 mM 3, 5-dinitrosalicylic acid (DNS) color reagent. The IC50 values of the samples were determined and reported as the mean ± standard deviation of three independent experiments (Wang and Zhao 2019). List of abbreviations PIME: Plumbago indica Methanol Extract; PICE: Plumbago indica Chloroform Extract; IEAF: Plumbago indica Ethyl Acetate Fraction; PIBF: Plumbago indica n-Butanol Fraction; STZ: Streptozotocin; NBT: Nitro blue tetrazolium; DTNB: Dithio-bis 2-Nitrobenzoic acid; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FRAP: Ferric reducing antioxidant power assay; DNS: Dinitro salicylic acid; IAEC: Institutional Animal Ethics Committee; CCSEA: Committee for Control and Supervision of Experiments on Animals; NA: Nicotinamide; CMC: Carboxymethylcellulose; SGOT: Serum glutamic oxaloacetic transaminase; SGPT: Serum glutamate pyruvate transaminase; ALP: Alkaline phosphate; HDL: High-Density lipoproteins; TBARS: Thiobarbituric acid reactive substances; SAG: Superoxide anion generation; GSH: Glutathione. In vivo study Animals Wistar rats (200-250 gram) were procured from Guru Nanak Dev University, Amritsar, Punjab, India. They were housed in the animal house with free access of water and laboratory pellet diet. The rats were exposed to normal cycle of light and dark. The animals were allowed to acclimatize to the environment for 7 days before the commencement of experiments. The experimental protocol no. (226/ CPCSEA/2023/22) of Guru Nanak Dev University, Amritsar was duly approved by the Institutional Animal Ethics Committee (IAEC) and animals were taken care of as per the guidelines of Committee for Control and Supervision of Experiments on Animals (CCSEA). Acute toxicity study The acute toxicity test for the most potent extract i.e methanol extract of P. indica (PIME) was conducted in accordance with the Organisation for Economic Co-operation and Development (OECD) 423 guidelines (OECD 2001). Before administering the dose, the animals were fasted overnight, with access to water only. The following morn- Ibrahim AAM et al.: Hypoglycaemic potential of P. indica Roots4 ing, methanol extract was given orally at a dose of 2000 mg/ kg. After dosing, the animals were allowed to resume normal access to food and water. Observations for signs of toxicity and behavioral changes were made every 24 hours over the course of 14 days to assess any potential mortality. Induction of diabetes mellitus Diabetes was induced in the rats through intraperitoneal injection of streptozotocin (STZ) and nicotinamide (NA) at doses of 50 and 110 mg/kg body weight respectively (Sadasivam and Manickam 1996). STZ was dissolved in freshly prepared citrate buffer at pH 4.5 and administered to overnight fasted rats, 15 minutes following intraperitoneal administration of nicotinamide at a dose of 110 mg/ kg body weight. To prevent acute hypoglycemic conditions resulting from the release of a high concentration of insulin during beta islet destruction, rats were immediately provided with 10% dextrose in their drinking water for 24 hours post-STZ injection. Subsequently, rats were allowed access to regular food and drinking water. After one-week, fasting blood glucose levels were measured using a digital glucometer (BG-03 Gluco One Glucometer, Dr. Morepen) and animals showing blood glucose levels exceeding 200 mg/dl were selected for further experimentation. The experimental animals received daily treatment with methanol extract of P. indica (PIME) at the doses of 100, 200 and 400mg/kg and ethyl acetate fraction of roots of P. indica (PIEAF) at the doses of 10, 20 and 40 mg/kg for 21 days, with 5mg/kg Gliclazide serving as the standard drug. After that blood samples were collected from fasted and anesthetized rats via retro-orbital sinus vein puncture and serum was obtained through centrifugation. Subsequently, all rats were euthanized and their kidneys and pancreas were harvested for analysis of biochemical, hematological and histopathological parameters. Experimental design Animals were divided into 9 groups (with n=6 in each group). Animals were treated with streptozotocin (STZ) and nicotinamide (NA) to induce diabetes. Diabetic rats were treated with Methanol extract of P. indica and its ethyl acetate fraction (PIME and PIEAF) in different doses orally which were prepared in 0.5% carboxymethylcellulose (CMC). STZ was dissolved in freshly prepared citrate buffer solution (pH=4.5) and gliclazide was used as standard drug. The experimental groups for animal studies were as follows: • Normal control (n=6): rats received 0.5 ml of 0.5% CMC daily for 3 weeks. • Diabetic control (n=6): rats received STZ (50 mg/kg) and NA (110 mg/kg) once and after the induction of diabetes, vehicle was administered daily for 3 weeks. • Standard drug (n=6): diabetic rats of this group received gliclazide (5 mg/kg/day) for 3 weeks. Diabetic rats were administered daily with PIME suspended in CMC for 3 weeks at doses of 100, 200 and 400 mg/ kg and PIEAF at doses of 10, 20 and 40 mg/kg. After completion of the treatment period, rats were placed in an individual metabolic cage for 24h and kept fasting for 12h. The rats were anesthetized with sodium pentobarbital and blood was collected using retro-orbital puncture and then they were sacrificed. Both the kidneys and pancreas were harvested immediately and rinsed with KCl buffer solution (pH7.4). Serum was isolated from the collected blood by centrifugation at 5000×g using REMI C24 PLUS centrifuge for biochemical estimations. The harvested kidney and small part of pancreas were preserved in 10% formalin solution for histopathological investigations. Rest part of pancreas was homogenized (10% w/v) in potassium chloride solution with the help of a Teflon homogenizer, then centrifuged at 5000×g for 20 minutes to isolate the supernatant for estimation of oxidative stress biomarkers (Singh et al. 2020). Evaluation of serum glucose, liver enzymes and lipid profiles Glucose, serum glutamic oxaloacetic transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphate (ALP), cholesterol, triglycerides, low density lipoproteins (LDL) and high-density lipoproteins (HDL) in serum were assessed using commercially available kits (Erba Diagnostics India) (Singh et al. 2022). Renal functions parameters The estimation of creatinine, urea and uric acid in serum was carried out using commercially available estimation kits (Erba Diagnostics India). Estimation of oxidative stress parameters in pancreatic tissue Oxidative stress markers were assessed in tissue homogenates of pancreas from each experimental group. The level of lipid peroxidation, a crucial indicator of oxidative damage, was quantified by measuring thiobarbituric acid reactive substances (TBARS). Superoxide anion generation (SAG) in the tissues was evaluated by assessing the reduction in nitro blue tetrazolium. Additionally, the concentration of reduced glutathione (GSH) in the tissue was determined spectrophotometrically (Ahmed et al. 2015). These analyses provided insights into the oxidative stress status and antioxidant capacity of the pancreatic tissues in each experimental group. Hematoxylin and Eosin Staining Preserved kidneys and pancreas were initially submerged in a 10% formalin solution before undergoing a series of preparatory steps. These included dehydration through successive ethanol concentration, followed by immersion in xylene and subsequent fixation in paraffin wax. The resulting tissue sections, with a thickness of four micrometers, were then mounted onto glass slides using commercial Bakers mounting fluid. To ensure proper adhesion, the slides were gently heated to melt the paraffin wax and then rinsed with xylene. Subsequent washing steps in- Pharmacia 72: 1–17 5 volved washing with absolute alcohol and hydration with water. Finally, the sections were stained with hematoxylin and eosin (Rowley et al. 2019). The prepared slides were examined under a microscope for any gross histopathological changes. High performance liquid chromatography (HPLC) of PIME The methanol extract was analyzed on Agilent technology HPLC system, provided with a photodiode array (PDA) detector using C-18 (150 mm × 4.6 mm, i.d. 5 µm) column. The mobile phase consisting of methanol and water in the proportion of 3:1 v/v was used. The column was equilibrated with mobile phase, pumped at the flow rate of 0.8ml/min and the injection volume was 5 mL. For calibration curve, plumbagin was prepared as standard at different concentrations 1–20 µg/ml in ethanol as solvent. Both the standard and extract were performed in triplicate and the detection was monitored at the wavelength of 265 nm. Identification and quantification of plumbagin were performed based on retention time and peak area was analyzed using EZChrome Elite chromatography data system software (CDS) (Tiwari et al. 2021). Molecular docking studies Previously developed and validated homology model of α-glucosidase on isomaltase (Saccharomyces cerevisiae) template (PDB: 3A4A) was utilized to analyze plumbagin interactions within the most favorable binding site in α-glucosidase (Singh et al. 2024). Structure of plumbagin was drawn on ChemDraw Ultra (2013) and its energy was minimized by using MM2 (molecular mechanics) force field in Chem3D Ultra software. The prepared structure of plumbagin was protonated in aqueous solution using ligand preparation module in LeadIt and docked into defined binding site using FlexX docking module in LeadIt (Singh et al. 2022). All FlexX solutions yielded were scored using Consensus scoring function (CScore) and ranked accordingly. The top best pose with the highest score was selected for investigation of interactions, HYDE assessment and calculation of free energy of binding (ΔG). 2D and 3D enzyme-hybrid interactions were visualized using Discovery Studio Visualizer (Singh et al. 2023). Briefly, the prepared homology model on the isomaltase (Saccharomyces cerevisiae) template using SWISS-MODEL pipeline was verified by overlapping with the template crystallographic structure and visualizing binding site residues (including His111, Asp214, Glu276, His348, and Asp349) of the co-crystalized ligand alpha-D-glucopyranose. Binding site residues were devoid of any deviations. The model was further validated using PROCHECK, which showed 89.5% residues in most favorable regions in the generated Ramachandran plot (Suppl. material 1). The accuracy of the docking protocol was established by docking the co-crystalized parent template protein ligand isomaltose in the binding site, which generated ligand confirmation with a root mean square deviation (RMSD) value of 0.5614, indicating the reliability of the docking protocol. Statistical analysis All the results were calculated as mean ± S.E.M. Statistical evaluation was performed using Graph Pad Prism software version 9.5.1 by using one-way ANOVA. P < 0.05 was considered as significant. Results Preliminary phytochemical screening The extracts prepared from the roots of P. indica were subjected to preliminary phytochemical screening in order to check the presence of particular class of components present. Methanol extract showed the presence of alkaloids, flavonoids, tannins and cardiac glycosides. In vitro antioxidant studies The antioxidant ability of methanol, chloroform extracts and the separated fractions ethyl acetate fraction (PIEAF) and n-butanol fraction (PIBF) of active methanol extract of P. indica roots were assessed using in vitro assays such as DPPH and ferricyanide ion reduction assay. DPPH method The inhibitory potential of the extracts and fractions from P. indica root was evaluated in DPPH assay for its radical scavenging activity was compared to ascorbic acid as standard. Ethyl acetate fraction of P. indica (PIEAF) showed the highest antioxidant potential with an IC50 value of 121.86 µg/mL, followed by the methanol extract of P. indica (PIME) with an IC50 of 148.25 µg/mL compared to ascorbic acid (IC50 53.10 μg/mL) as shown in Fig. 1. The results revealed that free radical scavenging activity of the PIME and PIEAF increased with an increase in the concentration. Ferric-reducing antioxidant power assay The ferric reducing antioxidant power (FRAP) assay was carried out at different concentrations of all extracts and fractions of P. indica. PIEAF and PIME with IC50 value of 122.75 µg/mL and 134.56 µg/mL respectively, exhibited the highest reducing power, while PICE (IC50 value 302.24 µg/mL) and PIBF (IC50 241.01 µg/mL) showed relatively weaker activities as shown in Fig. 2. From these observations, it was concluded that among all the extracts and fractions, methanol extract and its fraction showed maximum antioxidant activity in DPPH and FRAP assays. Pharmacological reports have shown that many plants with antioxidant activity may help manage diabetes by mitigating oxidative stress, a key factor in the disease’s progression, therefore further invitro antidia- Ibrahim AAM et al.: Hypoglycaemic potential of P. indica Roots6 Figure 2. Percentage inhibition of hydroxyl radical scavenging activity of extacts and fractions of P.indica: A. PIME (Methanol extract of P. indica); B. PICE (Chloroform extract of P. indica); C. PIEAF(ethyl acetate fraction of P. indica); D. PIBF(butanol fraction of P. indica) at different concentrations (25–400 μg/ml), compared with ascorbic acid as antioxidant standard. Figure 1. Percentage inhibition of DPPH free radical activity of extracts and fractions of P.indica: A. PIME (Methanol extract of P. indica); B. PICE (Chloroform extract of P. indica); C. PIEAF(ethyl acetate fraction of P. indica); D. PIBF (butanol fraction of P. indica) at different concentrations (25–400 μg/ml), compared with standard antioxidant ascorbic acid. Pharmacia 72: 1–17 7 betic assays were done on extracts and fractions prepared from this plant to test its antihyperglycemic effect. In vitro anti-diabetic studies Alpha-glucosidase inhibition assay P. indica extracts and fractions were tested for α-glucosidase inhibitory activity at doses ranging from 25 to 250 µg/ mL. The standard drug used was acarbose (IC50 = 70.92 µg/mL). The results showed that the PIEAF exhibited the highest inhibitory effect (IC50=99.89 µg/mL), followed by the PIME (IC50=109.82 µg/mL). The butanol fraction (PIBF) and chloroform extract (PICE) showed minimum inhibition with IC50 = 170.34 µg/mL and IC50 = 214.71 µg/ mL) as represented in Fig. 3. PIEAF and PIME showed better αglucosidase enzyme inhibition than PICE and PIBF. Alpha-amylase inhibition assay Alpha-amylase inhibitory activity of P. indica extracts and fractions was done at doses ranging from 25 to 250 µg/mL, using acarbose as the standard reference (IC50 = 75.29 µg/mL). The PIEAF showed the highest inhibition (IC50 = 106.09 µg/mL), followed by the PIME (IC50 = 112.85 µg/mL). The PICE and PIBF exhibited moderate inhibitory activity (IC50 = 197.59 µg/mL and 213.56 µg/ mL) as shown in Fig. 4. The results from the above assays indicated that the methanol extract of roots of this plant and its ethyl acetate fraction possess notable hypoglycemic activity whereas its chloroform extract and butanol fraction was found to be less effective. Based on these aforementioned results, the bioactive extract and its fraction namely PIME and PIEAF were further subjected to in vivo testing using streptozotocin (STZ) and nicotinamide (NA) model of Type 2 diabetes in rats. Acute toxicity studies In acute toxicity studies, rats showed no signs of toxicity in the methanol extract of P. indica at a dose of 2000 mg/ kg within the first 24 hours. Based on the OECD guidelines 423, a dose equal to one-tenth (200mg/kg) of this safe amount was chosen for experimental use, while lower (half, 100mg/kg) and higher (double, 400mg/kg) doses were used to evaluate any dosedependent effects. In-vivo evaluation of antidiabetic activity The antidiabetic activity of methanol extract and its ethyl acetate fraction was investigated using STZ/NA induced diabetes in rats and various renal, hepatic and oxidative stress parameters were assessed in rats. Serum glucose, liver enzymes and lipid profiles The effects of PIME and PIEAF treatment on various biochemical parameters was assessed in STZ/NA induced diabetic rats. The diabetic control group had significantly higher levels of glucose, triglycerides, cholesterol, LDL, SGOT, Figure 3. α-Glucosidase enzyme inhibition potential of extracts and fractions of P.indica: A. PIME (Methanol extract of P. indica); B. PICE (Chloroform extract of P. indica); C. PIEAF (ethyl acetate fraction of P. indica); D. PIBF (butanol fraction of P. indica) at different concentrations (25–250 μg/ml), compared with acarbose as standard. Ibrahim AAM et al.: Hypoglycaemic potential of P. indica Roots8 Figure 4. αAmylase enzyme inhibition potential of extacts and fractions of P. indica: A. PIME; B. PICE; C. PIEAF; D. PIBF at different concentrations (25–250 µg/ml), compared with acarbose as standard. SGPT and ALP, while also having lower HDL levels, compared to the normal control group. The treatment with PIME (100, 200 and 400 mg/kg) and PIEAF (10, 20 and 40 mg/kg) resulted in a significant reduction in glucose, triglycerides, cholesterol, LDL, SGOT, SGPT and ALP levels, in addition to a significant improvement in HDL levels, when compared to the diabetic control group as shown in Fig. 5A–H. Among the doses evaluated, the higher dose of PIEAF (40 mg/kg) had the maximum effect on returning biochemical parameters to normal levels, suggesting its significant antihyperglycemic activity. Renal functions and oxidative stress parameters The effects of PIME and PIEAF on renal function and oxidative stress indicators in STZ/NAinduced diabetic rats was observed in this study. The diabetic control group had considerably higher levels of creatinine, urea, uric acid, TBARS (Thio barbituric Acid Reactive Substances) and SAG (Superoxide Anion Generation) as well as a significant decrease in GSH(Glutathione) as compared to the normal control group. When compared to the diabetic control, PIME (200 and 400 mg/kg) and PIEAF (20 and 40 mg/kg) significantly reduced the high levels of creatinine, urea, uric acid, TBARS and SAG while raising GSH levels in a dose-dependent manner Fig. 6A–G. Among the groups, the higher dose of PIEAF (40 mg/kg) exhibited significant improvement, demonstrating nephroprotective and antioxidant properties in diabetic rats. Histopathological evaluation of tissues Histopathological study of pancreatic tissue on STZ/ NA-induced diabetic rats showed that the effects of PIME and PIEAF on islet cell morphology were dose-dependent. The control group exhibited pancreatic tissue with normally situated islets of Langerhans (Fig. 7A). The diabetic control group showed extensive degeneration and distortion of islet cells, indicating serious pancreatic damage (Fig. 7B). The standard Gliclazide 5mg/kg drug treatment showed protection of islet cells (Fig. 7C). The groups treated with methanol extract of P. indica at 100 and 200mg/kg showed mild protective effects, but significant improvement was observed at the dose of 400 mg/kg, restoring islet structure (Fig. 7D–F). PIEAF-treated groups at lower dose showed minimal protection, but at the dose of 40 mg/kg significant improvement in islet cell structure was observed Fig. 7G–I. These findings suggest that PIME and PIEAF may have dose-dependent protective effects in islets of Langerhans of pancreatic tissue which has been degenerated and distorted by diabetes and the maximum effect was observed at 40mg/kg dose of ethyl acetate fraction of P.indica. After assessing the effect of different groups on pancreatic tissue, significant morphological differences were also observed in renal tissue of various groups. The normal control group had well-preserved glomeruli and intact tubules, indicating normal renal anatomy (Fig. 8A). The diabetic control group showed severe kidney injury, while the standard drug-treated group had typical glomerular Pharmacia 72: 1–17 9 Figure 5. Effect of PIME and PIEAF on A. Glucose; B. Triglyceride; C. Cholesterol; D. HDL; E. LDL; F. SGOT; G. SGPT; H. ALP of diabetic rats. Data are expressed as mean ± SEM [ n = 6 in each group], ‘a’ shows the mean significant difference when compared to the normal control group (P < 0.05). ‘b’ shows the mean significant difference when compared to the diabetic control group (P < 0.05). Figure 6. Effect of PIME and PIEAF on A. Creatinine; B. Urea; C. Uric acid; D. Bilirubin; E. TBARS (Thio barbituric acid reactive substances); F. SAG (Superoxide anion Generation); G. GSH (glutathione) in diabetic rats. Data are expressed as mean ± SEM [n = 6 in each group], ‘a’ shows the mean significant difference when compared to the normal control group (P < 0.05). ‘b’ shows the mean significant difference when compared to the diabetic control group (P < 0.05). Ibrahim AAM et al.: Hypoglycaemic potential of P. indica Roots16 Solanum elaeagnifolium Cav. (Solanaceae): In vitro and in silico investigations. 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Pharmacological Research – Modern Chinese Medicine 10: 1–12. https://doi.org/10.1016/j.prmcm.2023.100351 Supplementary material 1 Supplementary figure Authors: Abdulkadir Abdu Mohmmednor Ibrahim, Brahmjot Singh, Hossamaldeen Bakrey, Hasandeep Singh, Atamjit Singh, Balbir Singh, Sarabjit Kaur Data type: png Explanation note: Ramachandran Plot. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/ odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/pharmacia.72.e165113.suppl1