Development and Comparative Evaluation of Polyherbal Formulations Comprising Herbal Extracts for Diabetes Management

Diabetes mellitus (DM) is one of the most prevalent metabolic disorders globally, characterized by chronic hyperglycaemia due to defects in insulin secretion, insulin action, or both (American Diabetes Association, 2022). The International Diabetes Federation (IDF, 2021) reports that approximately 537 million adults worldwide live with diabetes, a number projected to rise significantly by 2045. Current therapeutic approaches primarily involve synthetic drugs such as sulfonylureas, biguanides, and insulin, but their long-term use is associated with limitations including hypoglycaemia, gastrointestinal discomfort, weight gain, and decreased efficacy over time (Alqahtani et al., 2022). Hence, there is growing interest in herbal and polyherbal formulations that can provide safe, effective, and affordable alternatives.

Polyherbalism, rooted in Ayurvedic and traditional medicine practices, emphasizes the synergistic therapeutic potential of combining multiple plant extracts (Patwardhan et al., 2015). Several medicinal plants have been extensively studied for their antidiabetic properties. Trigonella foenum-graecum (Fenugreek) seeds are rich in soluble fibers and bioactive compounds such as trigonelline and 4-hydroxyisoleucine, which enhance insulin sensitivity and reduce postprandial glucose (Basch et al., 2003). Azadirachta indica (Neem) leaves exhibit hypoglycemic activity through modulation of glucose uptake and pancreatic β-cell protection (Chattopadhyay, 1999). Cinnamomum verum (Cinnamon) bark improves glucose metabolism by enhancing insulin receptor signaling and glycogen synthesis (Anderson et al., 2004). Curcuma longa (Turmeric) rhizomes contain curcumin, known for its potent antioxidant and anti-inflammatory properties, which contribute to amelioration of insulin resistance and oxidative stress in diabetes (Aggarwal & Harikumar, 2009). Piper nigrum (Black pepper) contains piperine, which not only exhibits antidiabetic effects but also enhances the bioavailability of curcumin and other phytoconstituents (Atal et al., 1985).

Considering their complementary mechanisms of action, the rational combination of these herbal extracts in a polyherbal formulation may provide a holistic approach to diabetes management. The present study focuses on the development and comparative evaluation of such polyherbal formulations, assessing their physicochemical properties, phytochemical profiles, and in vitro antidiabetic potential.

MATERIALS AND METHODS

Materials

The following materials were used in the formulation:

• Active pharmaceutical ingredient (API): Metformin (Sun Pharmaceutical Industries Ltd, India).
• Excipients: Microcrystalline cellulose, Sodium starch glycolate, Magnesium stearate (S.D. Lab Chem, Mumbai, India).
• Herbal extracts:
  • Trigonella foenum-graecum (Fenugreek seeds) extract
  • Azadirachta indica (Neem leaves) extract
  • Cinnamomum verum (Cinnamon bark) extract
  • Curcuma longa (Turmeric rhizomes) extract
  • Piper nigrum (Black pepper seeds) extract
    (All procured from IndiaMART, India).

Methods

1. Preformulation Studies

a. Organoleptic characterization: The appearance, colour, odour, and solubility of the herbal extracts were observed and recorded.

2. Evaluation of Pre-compression Parameters of Powder Blend

The polyherbal powder blend was evaluated for flow properties including angle of repose, bulk density, tapped density, Hausner’s ratio, and Carr’s compressibility index.

a. Angle of Repose: The fixed funnel method was used. Powder was allowed to freely flow through a funnel, forming a heap. The radius (r) and height (h) of the pile were measured, and the angle of repose (θ) was calculated using Equation (1):

θ=tan?−1(h/r)(1)\theta = \tan^{-1}(h/r) \tag{1}

Where:

• θ = Angle of repose
• h = Height of pile
• r = Radius of pile base

Measurements were performed in triplicate.

b. Bulk Density and Tapped Density: A 100 ml graduated cylinder was filled with 10 g of powder, and the initial volume was noted (bulk volume). The cylinder was tapped at 2-second intervals from a height of 2.5 cm until the volume remained constant (tapped volume). Bulk density (LBD) and tapped density (TBD) were calculated using Equations (2) and (3):

LBD=Weight of powder Bulk volume (2) LBD = \frac{Weight\ of\ powder}{Bulk\ volume} \tag{2}

TBD=Weight of powder Tapped volume (3) TBD = \frac{Weight\ of\ powder}{Tapped\ volume}

c. Carr’s Compressibility Index (CI): The CI was calculated using Equation (4):

CI=TBD−LBDTBD×100(4) CI = \frac{TBD - LBD}{TBD} \times 100

Lower CI values indicate better flowability of the powder blend.

d. Hausner’s Ratio (HR): Hausner’s ratio was calculated using Equation (5):

HR=TBDLBD(5)HR = \frac{TBD}{LBD}

Values of HR < 1.25 indicate good flow properties, while values > 1.25 indicate poor flow.

Formulation of Tablet

Tabel 1:Herbal Drug dose

Table 2 Formulation table of polyherbal tablet

Direct Compression Method

Tablet compression involves converting powder or granulated material into a uniform solid dosage form using a tablet press machine. In the present study, the dried herbal extracts were blended with pharmaceutical excipients such as binders, disintegrants, diluents, and lubricants to ensure adequate flow properties and compressibility. The final blend was subjected to direct compression using either a single-punch or rotary press machine.

The powder mixture was fed into the die cavity and compressed between the upper and lower punches under controlled pressure to yield tablets of uniform size and shape. Compression pressure was carefully adjusted to achieve adequate hardness and avoid defects such as lamination or capping. The prepared tablets were subjected to post-compression evaluation to ensure their physicochemical quality and consistency.

EVALUATION OF POST-COMPRESSION PARAMETERS

1. Thickness- The thickness of randomly selected tablets was measured using a digital Vernier calliper.

2. Hardness- The crushing strength of tablets was determined using a Monsanto hardness tester. Ten tablets were randomly selected from each batch, and hardness was expressed in kg/cm².

3. Friability- Friability was determined using a Roche Friabilator, Pre-weighed 20 tablets were rotated at 25 rpm for 4 minutes (100 revolutions). Tablets were dedusted and reweighed. Percentage friability was calculated using Equation:

% Friability = Winitial−WfinalWinitial×100

%Friability = \ frac{W_{initial} -W_{final}}{W_{initial}} \times 100 \tag

where W initialW_{initial} = initial tablet weight, and W finalW_{final} = final tablet weight.

4. Weight Variation- Twenty tablets were randomly selected and weighed individually. The average weight was calculated and compared with individual tablet weights. Percentage deviation from the mean was determined as per pharmacopeial standards.

5. Wetting Time- A tissue paper was placed in a 6.5 cm diameter Petri dish containing 6 ml of distilled water. A tablet was placed on the tissue paper surface, and the time required for water to completely wet the tablet surface was recorded. The experiment was performed in triplicate.

EVALUATION OF TABLETS

In Vitro Dissolution Study

Dissolution testing was carried out in USP Apparatus II (paddle method) containing 900 ml of pH 1.2 buffer (citric acid–sodium phosphate) at 37 ± 0.5 °C with a paddle speed of 50 rpm. At intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours, 5 ml samples were withdrawn, filtered through a 0.45 µm Millipore filter, diluted appropriately, and analyzed at 233 nm using a UV spectrophotometer. Cumulative percentage drug release was calculated and dissolution profiles were plotted.

ANIMAL STUDY

Experimental Design

To assess the antidiabetic potential of Polyherbal Formulation (PHF) in vivo studies were conducted on Wistar rats.

• Species: Wistar albino rats
• Age: 8–10 weeks
• Weight: 180–220 g
• Group size: 10 rats per group

Grouping of Animals

• Group I (Normal Control): No induction, no treatment
• Group II (Diabetic Control): STZ-induced, no treatment
• Group III (PHF-treated): PHF (100 mg/kg, oral)
• Group IV (APHF-treated): APHF (50 mg/kg, oral)
• Group V (Standard Control): Metformin (90 mg/kg, oral)

Induction of Diabetes

Diabetes was induced by intraperitoneal injection of Streptozotocin (STZ) at a dose of 50 mg/kg. Blood glucose levels were measured 72 hours post-injection, and animals with glucose levels >250 mg/dL were considered diabetic.

Parameters Evaluated

• Blood glucose levels: Monitored weekly
• Biochemical markers:
  • Lipid profile (Total cholesterol, Triglycerides, HDL, LDL)
  • Liver function (ALT, AST, ALP)
  • Kidney function (Creatinine, BUN)
• Oxidative stress markers: Superoxide dismutase (SOD), Catalase, Malondialdehyde (MDA)
• Histopathological examination:
  Liver and pancreatic tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned (3–5 µm), and stained with hematoxylin-eosin (H&E). Slides were examined under a light microscope (Motic, USA) to assess histopathological changes.

RESULTS – PREFORMULATION STUDY

Organoleptic Characterization of Plant Extracts

The selected plant extracts were evaluated for colour, odour, and taste. The findings (Table 3) correspond well with the reported characteristics of the respective botanicals, thereby confirming their identity and suitability for formulation.

Table 1: Organoleptic Characteristics of Plant Extracts

Moisture content was determined by loss on drying. As presented in Table 4, values ranged from 7.6% to 26.4%. Among the extracts, Tinospora cordifolia exhibited the highest moisture content (26.4%), while Azadirachta indica had the lowest (7.6%). All extracts were adequately dried for further formulation work.

Table 4: Loss on Drying of Plant Extracts

The micromeritic parameters of powder blends, including bulk and tapped density, Hausner’s ratio, Carr’s index, and angle of repose, were evaluated. Most blends demonstrated good flow characteristics (Hausner’s ratio <1.25, Carr’s Index <15%, angle of repose 25–35°). However, sample no. 6 showed poor flowability (Carr’s Index 27.59%, angle of repose ~45°), indicating the possible requirement of flow enhancers.

Differential Scanning Calorimetry (DSC)

DSC thermograms confirmed the thermal behavior of formulations:

• Polyherbal formulation (F2): Broad endothermic peak at 138.76 °C, attributed to excipients, without evidence of incompatibility.

Evaluation of Tablet Properties

The prepared formulations (F1–F4) were assessed for physical and quality control parameters. As shown in Table 5, all batches met pharmacopeial standards. Hardness values (5.1–5.5 kg/cm²) indicated sufficient mechanical strength, while friability values (<0.54%) confirmed durability. Drug content uniformity (89.89–98.87%) was within the acceptable range (±5% of the labelled claim).

Table 5: Evaluation of Prepared Tablet Formulations

The thermal behavior of the polyherbal formulation (PHF) was evaluated using Differential Scanning Calorimetry (DSC), and the thermogram is presented in Figure 1. The DSC curve revealed a prominent endothermic peak at approximately 187.72 °C, with an onset temperature of 132.22 °C and an offset at 210.83 °C. The measured peak height was –100.75 mW, corresponding to a heat flow change of approximately –154.65 J/g.

The observed endothermic transition can be attributed to the melting and/or decomposition of phytoconstituents present in the PHF. The broadness of the peak indicates the presence of multiple bioactive components with overlapping thermal events, which is expected in polyherbal formulations due to the coexistence of alkaloids, flavonoids, phenolics, and terpenoids.

The absence of sharp, multiple melting peaks suggests that the formulation is in a partially amorphous state, which may enhance its solubility and bioavailability compared to crystalline phytochemicals. The broad endothermic transition also reflects the thermal stability of the formulation up to ~130 °C, indicating that the PHF remains intact under moderate processing or storage conditions.

When compared to reported DSC thermograms of individual plant extracts, the endothermic peak of the PHF appears shifted and broadened. This shift is suggestive of molecular interactions among the bioactive constituents, which may result in synergistic stabilization and altered thermodynamic behavior. Such interactions are particularly relevant for herbal formulations, as they often contribute to improved pharmacological activity.

The DSC profile therefore confirms that the PHF is thermally stable up to ~130 °C, exhibits an amorphous nature, and possesses strong intermolecular interactions between its phytoconstituents, supporting its suitability for pharmaceutical applications such as oral dosage forms.

The preformulation studies confirmed that both pure Metformin and the selected herbal extracts met pharmacopeial quality standards. Organoleptic characterization and LOD values indicated stability and suitability for formulation. Micrometric properties demonstrated generally good flowability, although one sample required optimization.

Spectroscopic (UV, FTIR) and thermal (DSC) analyses confirmed the absence of significant incompatibilities between drug and excipients. Tablet evaluation further established the robustness, uniformity, and reproducibility of the prepared formulations.

Among the batches, F2 exhibited superior results, with the highest drug content (98.87%), optimal hardness, and acceptable friability. These findings support the potential of the developed polyherbal and allopolyherbal formulations as effective antidiabetic agents.

Fig1 : DSC of Polyherbal formulation F2

Blood Glucose Levels

The changes in fasting blood glucose levels across experimental groups were recorded weekly over a period of 4 weeks (Table 6).

Table 6: Effect of PHF and Metformin on Blood Glucose Levels (mg/dL)

In diabetic control rats, blood glucose progressively increased from 280 ± 10 mg/dL to 300 ± 12 mg/dL by week 4. In contrast, PHF treatment significantly reduced glucose levels from 275 ± 12 mg/dL to 130 ± 11 mg/dL by week 4, representing ~52% reduction. Metformin treatment produced a stronger effect, reducing glucose levels from 276 ± 9 mg/dL to 95 ± 6 mg/dL (~65% reduction).

The normal control group maintained stable glucose levels (90–96 mg/dL) throughout the experiment.

Observation: Both PHF and Metformin significantly reduced blood glucose levels compared to the diabetic control. However, Metformin exerted a slightly more pronounced hypoglycemic effect compared to PHF.

Biochemical Parameters

The effect of PHF and Metformin on lipid profile and hepatic–renal biomarkers is shown in Table 7.

Table 7: Effect of PHF and Metformin on Biochemical Parameters

Treatment with PHF significantly improved these biochemical markers. Total cholesterol decreased to 165 ± 10 mg/dL, triglycerides to 140 ± 8 mg/dL, and LDL to 80 ± 5 mg/dL, while HDL increased to 40 ± 3 mg/dL. Similarly, hepatic marker ALT decreased from 90 ± 5 IU/L (diabetic control) to 65 ± 4 IU/L (PHF), and creatinine reduced from 1.6 ± 0.1 mg/dL to 1.2 ± 0.1 mg/dL, indicating hepatoprotective and nephroprotective effects.

Metformin-treated animals showed near-normal values for all parameters, demonstrating its superior efficacy.

Observation: PHF treatment improved lipid metabolism and protected liver and kidney function in diabetic rats, though effects were less pronounced compared to Metformin.

Oxidative Stress Markers

The antioxidant status of experimental groups was evaluated using SOD, Catalase, and MDA assays (Table 8).

Table 8: Effect of PHF and Metformin on Oxidative Stress Markers