Formulation And Evaluation of An Anti-Inflammatory and Anti-Microbial Gel Using Clitoria Ternatea Linn

Oral ulcers are common lesions characterized by the loss of the epithelial lining of the oral mucosa, leading to exposure of the underlying connective tissue. These lesions are often associated with pain, inflammation, redness, a burning sensation, and difficulty in eating and speaking, thereby significantly affecting the patient’s quality of life. Oral ulcers may arise due to various factors, including trauma, microbial infections, nutritional deficiencies, immune disorders, stress, or the use of certain medications. Conventional treatment approaches primarily involve the use of topical corticosteroids, antiseptics, analgesics, and antimicrobial agents; however, prolonged use of these medications may result in adverse effects such as mucosal irritation, opportunistic infections, and the development of drug resistance.¹ ²

Herbal medicines have gained considerable attention as safer and effective alternatives due to the presence of diverse bioactive phytochemicals possessing anti-inflammatory, antimicrobial, antioxidant, and wound-healing properties. Natural products are widely accepted because of their lower incidence of side effects, improved patient compliance, and significant therapeutic potential in managing oral inflammatory conditions.³

Clitoria ternatea Linn. (Family: Fabaceae), commonly known as butterfly pea, is a well-known medicinal plant extensively utilized in traditional Ayurvedic medicine. The flowers are rich in anthocyanins, flavonoids, phenolic compounds, tannins, and other phytoconstituents that exhibit antioxidant, anti-inflammatory, antimicrobial, analgesic, and wound-healing activities. These pharmacological properties make Clitoria ternatea a promising candidate for the development of herbal formulations for the treatment of oral ulcers and other inflammatory conditions of the oral cavity.???

Topical drug delivery systems are preferred for the management of oral ulcers as they enable direct delivery of the therapeutic agent to the affected site, resulting in rapid onset of action, prolonged local drug retention, reduced systemic absorption, and enhanced patient compliance. Among the various topical dosage forms, gels are widely favored due to their excellent spreadability, non-greasy nature, ease of application, bio-adhesive properties, and ability to maintain prolonged contact with the oral mucosa.???

Therefore, the present study aimed to formulate and evaluate an anti-inflammatory and antimicrobial herbal gel using Clitoria ternatea flower extract for the effective management of oral ulcers.

MATERIALS AND METHODS

1. 1.   Plant Material :

The flowers of Clitoria ternatea Linn. (Family: Fabaceae), commonly known as Butterfly Pea or Aparajita, were used in the present study. Fresh, healthy, and mature flowers were collected from the botanical garden of Shivajirao S. Jondhale College of Pharmacy during the flowering season. The plant material was authenticated by Dr. Harshad M. Pandit, Director, Botany Research Laboratory, Azadnagar Road, Andheri (West), Mumbai–400058, Maharashtra, India.?

Biological Source

• Scientific name: Clitoria ternatea Linn.
• Family: Fabaceae
• Common names: Butterfly Pea, Blue Pea, Aparajita, Gokarna
• Plant part used: Dried flowers?

Figure 1 Clitoria ternatea Linn.

3.2 CHEMICALS AND REAGENT

Carbopol 940 (Loba Chemie Pvt. Ltd., Mumbai, India), Hydroxypropyl Methylcellulose (HPMC K15M) (HiMedia Laboratories Pvt. Ltd., Mumbai, India), Glycerin (Merck Life Science Pvt. Ltd., Mumbai, India), Propylene Glycol (Loba Chemie Pvt. Ltd., Mumbai, India), Triethanolamine (Merck Life Science Pvt. Ltd., Mumbai, India), Potassium Sorbate (Loba Chemie Pvt. Ltd., Mumbai, India), Citric Acid (SD Fine-Chem Ltd., Mumbai, India), Methanol (Analytical grade, Merck Life Science Pvt. Ltd., Mumbai, India), and Distilled Water were used throughout the study. All chemicals and reagents used were of analytical reagent (AR) grade.⁹

3.3Preparation of Plant Material 

The collected flowers were washed thoroughly with distilled water to remove dust and other impurities and shade-dried under well-ventilated conditions at room temperature. After complete drying, the flowers were coarsely powdered and stored in an airtight container until further use.^8,10

3.4 Extraction of Plant Material 

The powdered flowers of Clitoria ternatea Linn. were extracted by the maceration method using a hydroalcoholic solvent system. The powdered plant material was soaked in the solvent for an appropriate period with intermittent stirring to facilitate the extraction of phytoconstituents. The extract was filtered using Whatman No. 1 filter paper and concentrated under reduced temperature. The concentrated extract was stored in an airtight container at 4 °C until further use for phytochemical screening and gel formulation.^10,11

3.5 Preliminary Phytochemical Testing

The hydroalcoholic extract of Clitoria ternatea Linn. flowers were subjected to preliminary phytochemical screening using standard qualitative methods to detect the presence of major phytoconstituents. The screening was performed to identify bioactive compounds that may contribute to the anti-inflammatory and antimicrobial activities of the formulated herbal gel.^8,9

The extract was qualitatively evaluated for the presence of anthocyanins, flavonoids, phenolic compounds, tannins, alkaloids, glycosides, saponins, and steroids using established pharmacognostic procedures. ?,?,¹¹. Anthocyanins were identified by the pH differential method in acidic and alkaline media, while flavonoids were confirmed by the Shinoda test and lead acetate test.¹¹. Additional tests, including the ammonia test, sodium bisulfite test, ferric chloride test, and alkaline reagent test, were performed to detect the respective phytochemical constituents.??¹¹. The results were interpreted based on characteristic colour changes or precipitate formation and recorded accordingly. ?

3.6. Procedure

The herbal gel containing the hydroalcoholic extract of Clitoria ternatea Linn. was prepared using Carbopol 940 and Hydroxypropyl Methylcellulose (HPMC) as gelling agents. Initially, the required quantity of purified water was taken in a beaker, and the gelling agent was gradually dispersed with continuous stirring to prevent lump formation. The dispersion was allowed to hydrate for 30–60 minutes to obtain a uniform gel base.??

The hydroalcoholic extract of Clitoria ternatea was mixed separately with glycerin and propylene glycol under continuous stirring until a homogeneous solution was obtained. The prepared extract phase was then added slowly to the hydrated gel base while stirring continuously at 500–800 rpm to ensure uniform distribution of the extract throughout the formulation.??

Triethanolamine was added dropwise to neutralize the Carbopol dispersion and develop the desired gel consistency. Potassium sorbate was incorporated as a preservative, and the pH of the formulation was adjusted to 5.5–6.5 using citric acid or a suitable buffer solution. Finally, the volume was adjusted with purified water wherever required, and the gel was stirred for an additional 20–25 minutes to achieve complete homogenization. The prepared formulation was transferred into clean, sterile containers and stored in a cool, dry place until further evaluation.??

3.7 Evaluation of Formulated Gel:

3.7.1.   Organoleptic Evaluation: The formulated herbal gel was evaluated for its organoleptic characteristics, including colour, odour, appearance, consistency, and homogeneity. The evaluation was carried out by visual inspection to assess the physical appearance and overall acceptability of the formulation. The observations were recorded for each formulation batch. ⁷

3.7.2.   pH Determination: The pH of the formulated herbal gel was determined using pH paper to evaluate its suitability for topical application. The pH was measured at room temperature, and values within the range of 5.0–6.5 were considered acceptable for application to the oral mucosa. ⁶

3.7.3. In Vitro Anti-microbial Activity (Escherichia Coli no-8739 Staphylococcus aureus ATCC no-6538[56-58]:  The antimicrobial activity of the test formulation was evaluated by determining its ability to inhibit the growth of selected microorganisms, which serves as an in-vitro marker for antimicrobial potential. The study was carried out against Escherichia coli (ATCC no8739) and Staphylococcus aureus (ATCC no-6538) using the agar well diffusion method. The formulation was tested at different concentrations (5 mg/mL and 10 mg/mL), and the activity was assessed by measuring the zone of inhibition. The results indicated a concentration-dependent increase in antimicrobial activity, confirming the formulation possesses mild antibacterial potential.^12-14

The test microorganisms used for the anti-microbial activity are shown in Figure 2

Figure 2 a) Escherichia Coli ATCC no-8739 b) Staphylococcus aureus ATCC no-6538

5. In vitro Anti-inflammatory activity by Protein Denaturation Method The in vitro anti-inflammatory activity of the formulated herbal gel was evaluated using the protein denaturation method. Diclofenac sodium was used as the standard drug. The absorbance of the reaction mixtures was measured using a UV–Visible spectrophotometer, and the percentage inhibition of protein denaturation was calculated to determine the anti-inflammatory potential of the formulation. ^15-16

The control, standard diclofenac sodium, and the sample formulation used in the protein denaturation assay are shown in figure 3.

Figure 3 (a)Control, (b) Std Diclofenac sodium, (c) Sample Anti-inflammatory activity

4.Result:

4.1. Preliminary studies:

The preliminary phytochemical screening of the hydroalcoholic extract of Clitoria ternatea flowers revealed the presence of anthocyanins and flavonoid, as summarized in Table 1

Table 1 Preliminary studies

A) Oragnoleptic properties of flower:

B) Physicochemical Studies

4.2 Evaluation of Poly-herbal gel:

A) Organoleptic properties:

The organoleptic characteristics of the formulated herbal gel are presented in Table 2.

Table 2 Organoleptic properties:

Table 3 Organoleptic properties observation

b) pH determination:

The pH values obtained for the different formulation batches are presented in Table 4.

Table 4 pH determination

c) In-vitro anti-microbial activity(Escherichia Coli ATCC no8739 Staphylococcus aureus ATCC no-6538)

The in vitro antimicrobial activity of the herbal gel formulated using Clitoria ternatea was evaluated by the agar diffusion method and compared with the standard drug Streptomycin, a widely used antibiotic. The standard drug exhibited significant antimicrobial activity in a concentration-dependent manner, showing a progressive increase in the zone of inhibition with increasing concentration. The herbal gel also demonstrated noticeable antimicrobial activity, indicating the presence of bioactive phytoconstituents responsible for its antimicrobial effect. The results confirmed the effectiveness of the formulation and the validity of the experimental model.

Result: The     gel       shows  mild    antibacterial    activity,           which  increases            with concentration but remains significantly lower than the standard antibiotic

1.The gel exhibited very low activity at 5 mg/mL with inhibition zones of 1 mm for both organisms.

2.At a higher concentration of 10 mg/mL, the activity improved to 4 mm against E. coli and 6 mm against S. aureus, indicating slightly better effectiveness against Gram-positive bacteria.

d) In vitro Anti-inflammatory activity by Protein Denaturation Method

The percentage inhibition of protein denaturation observed for the standard diclofenac sodium and the sample formulation at different concentrations is presented in Table 5.

Table 5 In vitro Anti-inflammatory activity by Protein Denaturation Method