Pharmacological Evaluation of Antidiabetic Activity of Novel Herbal Combination in Experimental Model

Diabetes mellitus is one of the most prevalent endocrine disorders worldwide and is characterized by chronic hyperglycemia resulting from impaired insulin secretion, insulin resistance, or both. According to the International Diabetes Federation, the incidence of diabetes continues to rise globally, posing a significant public health challenge. Long-term uncontrolled diabetes can lead to severe complications such as cardiovascular diseases, nephropathy, neuropathy, retinopathy, and impaired wound healing. [1] Current antidiabetic therapies, including insulin and oral hypoglycemic agents, effectively control blood glucose levels but may cause adverse effects such as hypoglycemia, weight gain, gastrointestinal disturbances, and hepatic toxicity. Therefore, there is increasing interest in identifying natural products with antidiabetic potential that can provide effective glycemic control with fewer side effects. [2] Medicinal plants have been used traditionally for the treatment of diabetes for centuries. Various phytoconstituents, including flavonoids, alkaloids, glycosides, tannins, terpenoids, and phenolic compounds, have demonstrated significant antihyperglycemic activity through multiple mechanisms such as stimulation of insulin secretion, enhancement of glucose uptake, inhibition of carbohydrate digestion, and antioxidant activity. [3] A novel herbal combination consisting of selected medicinal plants with complementary mechanisms of action may provide synergistic therapeutic benefits. The present investigation was designed to evaluate the pharmacological antidiabetic activity of a novel herbal combination using an experimentally induced diabetic animal model and compare its efficacy with a standard antidiabetic drug. [5]

Table 1: Comparative Pharmacognostic and Pharmacological Profile of Selected Plants Used in the Novel Herbal Antidiabetic Formulation.

Figure 1. Schematic Representation of the Antidiabetic Activity of the Novel Herbal Combination.

MATERIALS

The stem of Tinospora cordifolia (Guduchi), seeds of Linum usitatissimum (Flax), and bulbs of Allium sativum (Garlic) were used for the preparation of the novel herbal formulation. Streptozotocin (STZ) was used for the induction of diabetes, while Metformin hydrochloride was used as the standard antidiabetic drug. Ethanol (70%), distilled water, citrate buffer (pH 4.5), and commercially available biochemical diagnostic kits were employed for extraction and biochemical estimations. Healthy Wistar albino rats (150–250 g) of either sex were used as experimental animals. The study also utilized standard laboratory equipment including a Soxhlet extraction apparatus, rotary vacuum evaporator, electronic balance, glucometer, centrifuge, UV–Visible spectrophotometer, and microscope for experimental procedures and analysis.

METHODOLOGY

Collection and Authentication of Plant Materials

The flax seeds (Linum usitatissimum), fresh garlic bulbs (Allium sativum), and stems of Guduchi (Tinospora cordifolia) were procured from the local market of Chikhali, District Buldana, Maharashtra, India. The plant materials were authenticated by Dr. Pramod R. Padole, Professor of Botany, Shivaji Science and Arts College, Chikhali, Buldana. Voucher specimens (ACP/HF-489, ACP/HF-490, and ACP/HF-491) were deposited in the Herbarium Department for future reference.The authenticated plant materials were cleaned, shade-dried at room temperature under controlled humidity conditions until free from moisture, and then pulverized using a mechanical grinder. The powdered materials were passed through sieve No. 40 and stored in airtight containers for further pharmacognostic, phytochemical, and pharmacological investigations.

Morphological and Microscopic Evaluation

The crude drugs were evaluated for organoleptic characteristics including colour, odour, taste, shape, and size. Microscopic examination of flax seeds, Guduchi stem, and garlic bulb was carried out using suitable staining reagents and observed under a compound microscope to confirm their diagnostic characteristics. [6]

Powder Microscopy

The powdered plant materials were passed through sieve No. 60. A small quantity of powder was mounted on a glass slide with suitable reagents such as water, glycerin, iodine solution, or phloroglucinol-HCl. The slides were observed under low (10×) and high (40×) magnifications for identification of characteristic cellular structures. [7]

Fluorescence Analysis

One gram of powdered drug was treated separately with different reagents including distilled water, 1N sodium hydroxide, 1N hydrochloric acid, nitric acid, sulfuric acid, chloroform, ethanol, and ammonia solution. The resulting colour changes were observed under daylight and ultraviolet light (254 nm and 366 nm) for authentication and detection of adulteration. [8]

Extraction of Plant Materials

The powdered flax seeds were defatted with petroleum ether and extracted with 80% ethanol using a Soxhlet apparatus. The extract was concentrated using a rotary evaporator. The powdered stems of Tinospora cordifolia were defatted with petroleum ether and extracted using hydroalcoholic solvent (ethanol, 80:20) by ultrasound-assisted extraction. The extract was filtered, concentrated under reduced pressure, and stored at 4°C.Fresh garlic cloves were peeled, crushed, and allowed to stand for 10–15 minutes to facilitate allicin formation. Extraction was carried out using ethanol (60:40) under ultrasound-assisted conditions. The extract was concentrated under reduced pressure below 35°C and stored at 4°C until use. [9]

Micromeritic Evaluation

The powdered extracts were evaluated for mean particle size, angle of repose, bulk density, tapped density, compressibility index, and Hausner ratio using standard procedures to determine flow properties and packing characteristics. [10]

Heavy Metal Analysis

Quantitative estimation of heavy metals including arsenic, cadmium, mercury, and lead was carried out according to WHO guidelines to ensure the safety and quality of Table [11]

Experimental Animals

Healthy male Wistar albino rats (130–180 g, 2–2.5 months old) were used for the study. Animals were housed in polypropylene cages under standard laboratory conditions (25 ± 2°C, 12 h light/dark cycle) with free access to standard pellet diet and water. The animals were acclimatized for seven days before the experiment. The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) in accordance with CPCSEA guidelines. [12]

Experimental Design

Thirty rats were divided into five groups (n = 6):

Table 3. Formulation of polyherbal formulation

Induction of Diabetes

Diabetes was induced by intraperitoneal administration of Streptozotocin (STZ) (60 mg/kg) dissolved in citrate buffer (pH 4.5), following nicotinamide (110 mg/kg, i.p.). After one week, rats with fasting blood glucose levels above 200 mg/dL were considered diabetic and included in the study. [14]

Oral Glucose Tolerance Test (OGTT)

Following overnight fasting, animals received glucose (2 g/kg, p.o.) 30 min after administration of saline, standard drug, or test formulation. Blood glucose levels were measured at 0, 0.5, 1, 2, 3, and 4 h using a glucometer. [15]

Hypoglycemic Activity

Based on OGTT results, diabetic rats were treated orally with Metformin or the polyherbal formulation once daily for 28 days. Fasting blood glucose levels and body weight were recorded periodically throughout the study. [16]

Evaluation Parameters [17]

• Fasting Blood Glucose (FBG)
• Oral Glucose Tolerance Test (OGTT)
• Body Weight
• Lipid Profile
• Serum Insulin
• Liver and Kidney Function Tests
• Oxidative Stress Markers
• Histopathological Examination of Pancreas and Liver

Histopathological Study

At the end of the study, pancreas and liver tissues were collected and fixed in 10% formalin. The tissues were processed, embedded in paraffin wax, sectioned at 5 µm thickness, stained with Hematoxylin and Eosin (H&E), and examined under a light microscope for histopathological changes [18]

Statistical Analysis

Data were expressed as Mean ± SEM and analyzed using One-Way ANOVA followed by Dunnett’s multiple comparison test. Values of p < 0.05 were considered statistically significant. [18]

RESULTS

Fluorescence Analysis

Fluorescence analysis of Tinospora cordifolia, Allium sativum, and Linum usitatissimum powders was carried out using various chemical reagents under daylight and UV light (366 nm). The observed fluorescence characteristics were found to be consistent with standard pharmacognostic parameters and showed no evidence of adulteration. The results confirmed the identity and purity of the selected crude drugs.

Table 4. Fluorescence Analysis of Crude Drugs

Extraction Yield

Hydroalcoholic extracts of Linum usitatissimum, Tinospora cordifolia, and Allium sativum were prepared using suitable extraction techniques. The percentage yield of extracts was calculated based on the weight of dried extract obtained from 100 g of crude drug.

Table 5 Percentage Yield of Plant Extracts

Values are expressed as Mean ± SD (n = 3).

Among the selected plants, Tinospora cordifolia exhibited the highest extractive yield (23.54 ± 2.81%), followed by Linum usitatissimum (18.32 ± 3.18%), whereas Allium sativum showed the lowest yield (13.27 ± 1.37%). The extraction methods employed provided stable and concentrated extracts suitable for further phytochemical and pharmacological evaluation.

Physical Properties of Extracts

The prepared extracts of Linum usitatissimum, Tinospora cordifolia, and Allium sativum were evaluated for their physical characteristics, including colour, pH, and texture. The extracts exhibited characteristic colours ranging from reddish-brown to yellowish-green and showed a soft sticky texture. The pH of aqueous extracts ranged between 6.2 and 6.8, indicating their suitability for further pharmaceutical applications.

Table 6: Physical Properties of Plant Extracts

Micromeritic Properties of Extracts

Micromeritic evaluation revealed that all extracts possessed acceptable flow and compressibility characteristics. The angle of repose values were below 30°, indicating good flow properties, while Hausner’s ratio and compressibility index suggested fair to good powder flow behavior.

Table 7 Micromeritic Properties of Extracts

Values represent mean of triplicate determinations.

Physicochemical Evaluation

Physicochemical parameters such as loss on drying, ash values, and extractive values were determined to assess the quality and purity of the extracts. The results indicated acceptable moisture content and ash values, while higher water-soluble and alcohol-soluble extractive values suggested the presence of significant amounts of polar phytoconstituents.

Table 8: Physicochemical Evaluation of Plant Extracts (% w/w)

The obtained values confirmed the quality, purity, and suitability of the extracts for further phytochemical and pharmacological investigations.

Quantitative Estimation of Heavy Metals

Heavy metal analysis was performed according to WHO guidelines to evaluate the safety of the selected plant materials. The concentrations of lead, mercury, arsenic, iron, and cadmium were found to be within permissible limits, indicating that the extracts were safe for pharmacological studies.

Table 9: Heavy Metal Content of Plant Extracts (mg/kg)

The results demonstrated that all heavy metal concentrations were below the permissible limits prescribed by WHO, confirming the safety and quality of the plant materials used in the study.

Formulation and Evaluation of Novel Polyherbal Tablets

The polyherbal tablets were prepared using extracts of Linum usitatissimum, Tinospora cordifolia, and Allium sativumalong with spray-dried lactose, sodium starch glycolate, magnesium stearate, talc, and aerosil. Due to the sticky nature of the extracts, no additional binder was required. The prepared formulations (F1 and F2) were evaluated for organoleptic and physical parameters. Both formulations complied with official pharmacopoeial limits for hardness, friability, thickness, and disintegration time.

Table 10 Evaluation of Polyherbal Tablet Formulations

Antidiabetic Activity of Polyherbal Formulation

The antidiabetic activity of the polyherbal formulation was evaluated in streptozotocin (STZ)-induced diabetic Wistar rats following OECD-423 guidelines. Blood glucose levels, food intake, water intake, and body weight were monitored throughout the 28-day study period.

Table 11 Parameters Monitored During Study

Oral Glucose Tolerance Test and Hypoglycemic Activity

The polyherbal formulation demonstrated dose-dependent glucose-lowering activity in glucose-loaded and STZ-induced diabetic rats. Significant reductions in blood glucose levels were observed after continuous administration for 28 days. The effect was comparable to the standard antidiabetic drug, Glibenclamide (5 mg/kg).

Table 12 Effect of Polyherbal Formulation on Blood Glucose Levels (mg/dL)

The diabetic control group showed a continuous increase in blood glucose levels, whereas treatment with the polyherbal formulation significantly reduced glucose levels from Day 14 onwards. The formulation exhibited antidiabetic activity comparable to Glibenclamide. This effect may be attributed to the presence of phenolic and flavonoid compounds, which improve insulin sensitivity, reduce oxidative stress, and enhance glucose metabolism. The results suggest that the developed polyherbal formulation possesses promising antidiabetic potential.

Table 13 Comparative Antidiabetic Activity of Glibenclamide and Formulation

Figure 4 Comparative Antidiabetic Activity of Glibenclamide and Formulation

Blood Glucose Levels

The fasting and non-fasting blood glucose levels were significantly increased in the diabetic control group throughout the study. Treatment with Glibenclamide and the polyherbal formulation significantly reduced blood glucose levels compared to diabetic control animals. Although Glibenclamide produced a greater reduction, the test formulation showed considerable antihyperglycemic activity and effectively controlled both fasting and postprandial blood glucose levels. These findings indicate the potential antidiabetic efficacy of the developed polyherbal formulate

Table 14 Blood Glucose Levels

Values expressed as Mean ± SEM (n = 6).

The diabetic control group showed continuously elevated fasting and non-fasting blood glucose levels throughout the study. Treatment with Glibenclamide and the polyherbal formulation significantly reduced blood glucose levels (p < 0.05). Although Glibenclamide produced greater antihyperglycemic activity, the formulation also showed effective control of both fasting and postprandial hyperglycemia, indicating promising antidiabetic potential.

Histopathological Study

Histopathological examination of the pancreas and liver revealed significant tissue damage in diabetic control rats. Pancreatic sections showed a marked reduction in the number of islets of Langerhans and β-cells, along with fibrosis and inflammatory cell infiltration. Liver sections exhibited fatty changes, sinusoidal dilation, congestion, and necrosis. Treatment with the polyherbal formulation resulted in considerable recovery of pancreatic and hepatic architecture, with improved β-cell population, reduced inflammation, and near-normal liver histology. The findings were comparable to those observed with Glibenclamide treatment, indicating the protective and antidiabetic potential of the polyherbal formulation.