Formulation and Evaluation of Chewable Tablets of Catharanthus Roseus Leaf

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia resulting from defects in insulin secretion, insulin action, or both [1]. It has emerged as one of the most significant global health challenges, affecting millions of individuals worldwide [2]. The long-term complications associated with diabetes, including neuropathy, nephropathy, retinopathy, and cardiovascular diseases, considerably reduce the quality of life and increase mortality [3]. Although several synthetic antidiabetic agents are available, their use is often associated with limitations such as adverse effects, high cost, and poor patient compliance, particularly in long-term therapy [4]. These drawbacks have prompted increasing interest in the exploration of safer and more effective alternatives, especially those derived from natural sources [5]. In recent years, herbal medicines have gained considerable attention due to their therapeutic efficacy, minimal side effects, and better patient acceptance [6]. Plants have historically served as a rich source of bioactive compounds with diverse pharmacological properties [7]. Among these, Catharanthus roseus (commonly known as Sadabahar or Madagascar periwinkle) is a well-known medicinal plant belonging to the family Apocynaceae [8]. It is widely distributed in tropical and subtropical regions and has been traditionally used in various systems of medicine for the management of diabetes and other ailments [9]. The pharmacological potential of Catharanthus roseus is attributed to its rich phytochemical profile, particularly the presence of indole alkaloids such as vincristine, vinblastine, vindoline, and catharanthine [10]. While vincristine and vinblastine are extensively used in cancer chemotherapy, several studies have also demonstrated the antidiabetic potential of leaf extracts of the plant [11]. These extracts are reported to reduce blood glucose levels through multiple mechanisms, including enhancement of insulin secretion, improvement of glucose utilization, and inhibition of carbohydrate-digesting enzymes [12]. In addition, the antioxidant properties of the plant help in reducing oxidative stress, which plays a crucial role in the progression of diabetes and its complications [13]. Despite its significant therapeutic potential, the clinical application of Catharanthus roseus is often limited by issues related to dosage form, palatability, and patient adherence [14]. Conventional dosage forms such as decoctions or tablets may not be suitable for all patient populations, particularly pediatric and geriatric groups [15]. In this context, chewable tablets represent an attractive alternative dosage form, offering advantages such as ease of administration without water, improved patient compliance, enhanced palatability, and faster onset of action [16]. Furthermore, chewable tablets can be formulated to mask the bitter taste of herbal extracts, thereby improving acceptability [17].

Figure 1: Catharanthus roseus

In addition to its antidiabetic properties, Catharanthus roseus also possesses well-established anticancer activity due to the presence of vinca alkaloids, which interfere with microtubule formation and inhibit cell division [18]. This dual therapeutic potential makes the plant a promising candidate for the development of multifunctional herbal formulations [19]. Therefore, the present study aims to formulate and evaluate chewable tablets containing Catharanthus roseus leaf extract with a primary focus on antidiabetic activity, while also exploring its potential anticancer properties [20]. The study involves extraction of bioactive constituents, formulation of chewable tablets using suitable excipients, and evaluation of physicochemical as well as pharmacological parameters to assess the efficacy and quality of the developed formulation.

MATERIALS AND METHODS

MATERIALS

Fresh leaves of Catharanthus roseus were collected from the local region of Nashik, Maharashtra, India, and authenticated by a qualified botanist. The leaves were washed, shade-dried, and powdered for further use. The following excipients were used for the formulation of chewable tablets: mannitol (diluent and sweetening agent), microcrystalline cellulose (MCC PH-102) as a binder and filler, sodium saccharin as an artificial sweetener, talc as a glidant and magnesium stearate as a lubricant. For in vitro pharmacological evaluation, MCF-7 human breast cancer cell lines (ATCC) were used for anticancer studies. RPMI-1640 medium, fetal bovine serum (FBS) and MTT reagent were used for cell culture and cytotoxicity assays. For in vitro antidiabetic studies, α-amylase and α-glucosidase enzymes along with their respective substrates were used. Acarbose was used as a standard reference drug. All chemicals and reagents used in the study were of analytical grade.

METHODS

Collection and Authentication of Plant Material

Fresh leaves of Catharanthus roseus were collected from the Nashik region, Maharashtra, India and authenticated by a botanist.

Preparation of Leaf Extract

The collected leaves were washed thoroughly and shade-dried for 7–10 days. The dried leaves were powdered using a mechanical grinder. The powdered material was subjected to Soxhlet extraction using ethanol as a solvent for 6–8 hours. The extract was filtered and concentrated under reduced pressure using a rotary evaporator [21]. The concentrated extract was dried and stored in a desiccator until further use.

Pre-compression Evaluation of Powder Blends

Prior to compression, the prepared powder blends were evaluated for various micromeritic properties to assess flowability and packing characteristics. This included bulk density, tapped density, angle of repose, Carr’s index and Hausner’s ratio.

Bulk Density: A pre-weighed quantity of the powder blend was carefully transferred into a 100 mL graduated measuring cylinder without compacting. The initial volume occupied by the powder was recorded as the bulk volume [22]. Bulk density was calculated using the following formula:

Bulk density = Mass of the powder / Bulk volume

Tapped Density: The same sample was then subjected to 100 mechanical tappings using a tapped density apparatus. The volume after tapping was recorded as the tapped volume [22]. Tapped density was calculated using the formula:

Tapped density (g/cm³) = Weight of the powder / Tapped volume

Angle of Repose: The angle of repose was determined using the fixed funnel method. The funnel was positioned vertically and the powder blend was allowed to flow through it onto a flat surface, forming a conical pile. The height (h) of the cone and the radius (r) of its base were measured. The angle of repose (θ) was then calculated using the formula:

tan θ = h​ /r

Carr’s Index: Carr’s compressibility index was calculated to evaluate the flowability and compressibility of the blend using the formula [22,23]:

% Carr’s index = Tapped density - Bulk density / Tapped density Х 100

Hausner’s Ratio: Hausner’s ratio was calculated to further assess powder flow characteristics using the following equation [22,23]:

Hausner’s ratio = Tapped density / Bulk density Х 100

Formulation of Chewable Tablets

Chewable tablets were prepared by the direct compression method. The extract and excipients were passed through sieve no. 60, mixed uniformly, and lubricated with talc and magnesium stearate before compression.

Table 1: Formulation Composition of Chewable Tablets

Post-formulation Evaluation

Thickness and Diameter

Three tablets from each formulation batch were randomly selected, and their thickness and diameter were measured using a digital vernier caliper. The values were recorded individually, and the average measurements were calculated to ensure uniformity among the tablets [23].

Hardness

The hardness of the tablets was determined using a Monsanto type hardness tester. Three tablets from each batch were tested, and the force required to break each tablet was recorded. The average hardness was then calculated and expressed in kg/cm².

Percentage Friability

Friability testing was performed using a Roche friabilator. Twenty pre-weighed tablets were placed in the apparatus and rotated at 25 rpm for 4 minutes. After the test, the tablets were dedusted and reweighed. The percentage friability was calculated using the following formula:

% Friability = (Initial Weight−Final / Weight Initial Weight) × 100

Weight Variation

The weight variation test was conducted in accordance with Indian Pharmacopoeia (IP) 2007 guidelines. Twenty tablets were randomly selected from each batch and individually weighed using a digital balance. The average weight was calculated, and individual weights were compared to ensure that no more than two tablets deviated by more than ±5% from the average weight [23,24].

Drug Content Uniformity

Drug content uniformity was determined to ensure uniform distribution of the active constituent in the formulated chewable tablets of Catharanthus roseus. Twenty tablets from each formulation batch were accurately weighed and finely powdered. An amount of powder equivalent to 100 mg of C. roseus extract was transferred into a 100 mL volumetric flask and dissolved in a suitable solvent (e.g., methanol or ethanol). The solution was sonicated for 15–20 minutes to ensure complete extraction of the active constituents and then filtered through Whatman filter paper. The filtrate was appropriately diluted with the same solvent, and absorbance was measured using a UV–visible spectrophotometer at the predetermined λmax of the extract. A calibration curve of the extract was used to determine the drug content [24,25]. The drug content was calculated using the following formula:

% Drug Content=Actual amount of drug present / Theoretical amount of drug ×100

In Vitro Dissolution Study

The in vitro dissolution study of chewable tablets of Catharanthus roseus was performed using the USP Type II (paddle) dissolution apparatus. The dissolution medium consisted of 900 mL phosphate buffer (pH 6.8), maintained at a temperature of 37 ± 0.5°C, and stirred at a constant speed of 50 rpm. One tablet from each formulation batch was placed in the dissolution medium. At predetermined time intervals (5, 10, 15, 20, 30, 45, and 60 minutes), 5 mL samples were withdrawn and filtered. An equal volume of fresh dissolution medium was replaced to maintain sink conditions. The collected samples were analyzed using a UV–visible spectrophotometer at the λmax of the extract. The cumulative percentage drug release was calculated using a previously prepared calibration curve. The dissolution profile of each formulation was plotted as percentage cumulative drug release versus time and the optimized formulation was selected based on maximum drug release and acceptable tablet properties [24,25].

In Vitro Antidiabetic Activity

α-Amylase Inhibition Assay

The assay was carried out using the DNSA method. The reaction mixture containing α-amylase enzyme and sample solution was incubated with starch substrate. The reaction was terminated using DNSA reagent and absorbance was measured at 540 nm [26]. % Inhibition was calculated using:

% Inhibition=Ac−As / Ac × 100

Where,

Ac = Absorbance of the control (enzyme + substrate without sample)

As​ = Absorbance of the test sample (enzyme + substrate + extract/formulation)

α-Glucosidase Inhibition Assay

The assay was performed using p-nitrophenyl-α-D-glucopyranoside as substrate. After incubation with enzyme and sample, absorbance was measured at 405 nm. IC₅₀ values were calculated from the inhibition data [26,27].

In Vitro Anticancer Activity

MCF-7 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and incubated at 37°C in a humidified atmosphere containing 5% CO₂.

MTT Assay

Cells were seeded in 96-well plates and treated with different concentrations of the extract/tablet formulation. After incubation, MTT reagent was added and incubated for 3–4 hours. The formed formazan crystals were dissolved using DMSO and absorbance was measured at 570 nm. IC₅₀ values were calculated to determine cytotoxic activity [28].

% Cell Viability=As / Ac × 100

Where,

Ac = Absorbance of the control (enzyme + substrate without sample)

As​ = Absorbance of the test sample (enzyme + substrate + extract/formulation).

Stability Study

Stability studies were carried out on the optimized formulation (F3) under accelerated conditions of 40°C ± 2°C and 75% ± 5% relative humidity (RH) for a period of three month. Individual tablets were packed in butter paper, sealed in aluminum foil and stored in a stability chamber. After the specified period, the samples were evaluated for physical appearance, drug content and in vitro drug release [23,25].

Statistical Analysis

All experiments were performed in triplicate, and results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA, and p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Pre-compression Parameters

The powder blends of all six formulations exhibited satisfactory flow properties, indicating their suitability for direct compression. The angle of repose values ranged from 28.5° to 26.2°, suggesting good flowability. Bulk density and tapped density values showed minimal variation, while Carr’s index (14.2–16.0%) and Hausner ratio (1.16–1.19) confirmed acceptable compressibility.

Table 2: Results of Pre-compression Parameters

Post-compression Evaluation