INTRODUCTION:

The risk to human health posed by fungal infections is substantial, especially for those with compromised immune systems. The growing problem of antifungal resistance—combined with a shortage of effective treatments—underscores a critical need to find and develop novel, potent antifungal agents.¹ Fungal pathogens demonstrate a sophisticated ability to manipulate the host immune system, either by suppressing or activating it to ensure their survival. This manipulation contributes to the significant health risks posed by fungi.

For instance, infections from Candida and Aspergillus can cause life-threatening conditions in immunodeficient patients. Beyond systemic disease, fungal spores are also known to provoke allergic reactions, asthma, and various respiratory problems. Fungi also cause substantial discomfort and pain through common skin infections like ringworm and athlete's foot. The impact of fungi extends to the global food supply, where pathogens like powdery mildew, rust, and leaf spot cause extensive agricultural diseases. Furthermore, species such as Aspergillus and Fusarium produce mycotoxins that pose a considerable threat by contaminating food supplies.² Severe pneumonia is often linked to Cryptococcus. Antifungal medications, including ketoconazole, miconazole, and econazole, are among the active treatments available. Tridax procumbens L is recognized for its chemical constituents, including flavonoids, alkaloids, tannins, carotenoids, and fumaric acid. These compounds exhibit beneficial properties such as anti-inflammatory, antimicrobial, antioxidant, anticancer, and anti-diabetic effects. This plant is particularly noted for its potent antibacterial activity, attributed to the phenolic compounds it contains³.

MATERIALS AND METHODS:

Materials required:

The study utilized an ethanolic extract of Tridax procumbens Linn. as the primary active antifungal agent, with the plant material specifically collected from the medicinal garden at the Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad. This active extract was incorporated into a cream base designed for topical application. The formulation relied on a combination of solvents (absolute ethanol, water, propylene glycol), thickeners and emulsifiers (bees wax, lanette wax, cetyl alcohol, stearyl alcohol, stearic acid), the occlusive emollient white soft paraffin, and preservatives (methyl paraben and propylparaben) to ensure stability and microbial safety. The inclusion of triethanolamine helped neutralize the stearic acid, creating an in situ emulsifying system essential for the cream's stable structure.

Instrument required:

Electronic weighing balance, Soxhlet extractor, Brookfield viscometer, laminar air flow, BOD incubator, autoclave, pH meter, hot air oven, magnetic stirrer, water bath.

Apparatus required:

Round bottom flask, condenser petri dishes, beakers, glass rod, and spatula.

Method of Extraction:

The preparation of the active antifungal component began with the meticulous processing of 100 grams of fresh Tridax procumbens Linn. leaves, which were collected and botanically identified. After being washed, the leaves were shade-dried for 24–48 hours and subsequently ground into a coarse powder. This powder was then subjected to Soxhlet extraction for approximately 6 hours using 500 mL of absolute ethanol as the solvent, ensuring exhaustive extraction of the plant's phytochemicals. Finally, the resulting ethanolic extract was collected, and the solvent was gently evaporated on a water bath at 50°C. The concentrated extract was further dried in a desiccator to guarantee complete removal of residual solvent before being incorporated into the cream formulation.

Table 1: Procedure and observation of Standard phytochemical tests

S. No	Phyto-constituent	Standard test	Method	Observation
1	Alkaloids	Dragendroff’s test	1ml of ethanolic extract + fecl₃reagent.	Blue to green colour
2	Tannins	Ferric chloride test	1ml of ethanolic extract + lead acetate reagent	Yellow colour
3	Flavonoids	Lead acetate test	1ml of ethanolic extract + dragendroff’s reagent	Orange to red precipitate

Phytochemical Screening:

Standard phytochemical test was carried out for qualitative analysis of phytochemicals including, flavonoids, alkaloids, tannins, were tested by standard test and confirmed⁴.

Method of Preparation of Cream:

The Oil Phase (Internal Phase): Accurately weighed lipophilic (oil-soluble) ingredients—including Stearic acid, stearyl alcohol, cetyl alcohol, bees wax, and lanette wax—were combined in a primary beaker. This mixture was heated on a water bath at 70°C with continuous magnetic stirring until all components were uniformly melted and homogenized. These ingredients primarily function as emulsifiers, thickeners, and emollients. The Water Phase (External Phase): Simultaneously, the hydrophilic (water-soluble) ingredients—propylene glycol, triethanolamine, and purified water—were combined in a separate beaker. This phase was also heated to a matching temperature of 70°C on the same water bath. Triethanolamine acts as a neutralizing agent for stearic acid, forming an in situ emulsifier (triethanolamine stearate) when the phases are combined.

The molten oil phase was then slowly poured into the water phase under continuous, vigorous stirring to facilitate the formation of a stable O/W emulsion. Once the primary emulsion formed, the mixture was allowed to cool. At an optimal temperature (typically below 40°C to prevent degradation), the optimum quantity of the herbal Tridax procumbens extract (the active pharmaceutical ingredient) was incorporated into the base with continuous mixing using a mechanical stirrer to ensure uniform dispersion. Finally, the preservatives (Methylparaben and Propylparaben) and perfume were added after the formulation had completely cooled, yielding the final topical cream product.

Table 2: Formulation table of Tridax procumbens Linn cream.

OIL PHASE	F1 quantity %w/v	F2 quantity %w/v	F3 quantity %w/v
White bees wax	0	0.2	0.4
Lanette wax	0.4	0	0
Stearyl alcohol	1	1	1
Liquid paraffin	0.6	0.8	0.6
Stearic acid	0.2	0.2	0.2
Cetyl alcohol	1	1	1
WATER PHASE	---	---	---
Ethanolic extract	1	1	1
Methylparaben	0.004	0.004	0.004
Triethanolamine	0.4	0.4	0.4
Propylene glycol	1.1	1.1	1.1
Propylparaben	0.01	0.01	0.01
Water	20	20	20

Evaluation of Topical Formulations:

· Organoleptic evaluation:

Visual inspection is performed for their physical appearance, colour, texture, phase separation, homogeneity, texture and homogeneity were tested by placing cream in between thumb and index finger and pressing it⁵.

· Determination of viscosity:

Viscosity of different formulations was determined using a Brookfield viscometer using spindle S-04 at 20rpm⁵.

· Stability test:

Stability of the formulations were tested by placing the formulations in environmental stability chamber at 25-27°C for 14 days and creaming or coalescence is inspected.^5,6.

· Determination of pH:

Dissolve 1gram from different formulations in 10ml of distilled water and homogenised using magnetic stirrer pH was determined using digital pH metre.ph of formulation was noted and the average results were shown⁵.

· Spreadability:

The spread ability was determined by applying 1g of each formulation on a glass plate over which another plate was placed. The sample was pressed between two plates. A weight of 100g was allowed to rest on plate for 5mins. The increased in diameter due to spreading of cream was noted⁵.

Anti-Fungal Assay:

Test organism: Standard fungal strain of Aspergillus niger (grown culture).

Antifungal Assay of Tridax procumbens Linn Extract:

The antifungal assay of ethanolic extract of Tridax procumbens Linn was evaluated using modified agar well diffusion assay. Sabouraud’s dextrose agar (30ml) was poured in sterile petri dishes (n=3) and allowed to solidify. Two wells (10mm diameter) were created in 2 petri dishes containing media with a cork borer, and plates were inoculated with standardized inoculum of Aspergillus niger using a cotton swab. The ethanolic extract of Tridax procumbens Linn (1mg) were added to the wells made in two petri dishes and another petri dish was kept as control. These petri dishes were labelled and were incubated at 25°C for 7days after 1hour room stabilization period. The diameter of the zone of inhibition was subsequently measured to assess antifungal activity^6,7.

Anti-Fungal Assay of Cream:

The antifungal efficacy of topical agents was evaluated using modified agar well diffusion assay. Sabouraud’s dextrose agar (30ml) was poured in sterile petri dishes (n=4) and allowed to solidify. Three wells (10mm diameter) were created in 3 petri dishes containing media with a cork borer, and plates were inoculated with standardized inoculum of Aspergillus niger using a cotton swab. The topical formulations (1mg) were added to the wells made in three petri dishes and another petri dish was kept as control. These petri dishes were labelled and were incubated at 25°C for 7days after 1hour room stabilization period. The diameter of the zone of inhibition was subsequently measured to assess antifungal activity⁷.

RESULTS AND DISCUSSIONS:

Phytochemical analysis: Extract of tridax was used to perform standard phytochemical test and the results are given below in table

Table 3: Results of phytochemical analysis.

Sr. No	Evaluation parameters	Formulation 1	Formulation 2	Formulation 3
1	Colour	Light green	Olivacues green	Pisum green
2	Physical state	Semi-solid	Semi-solid	Semi-solid
3	Homogeneity	Homogenous	Homogenous	Homogenous
4	Texture	Uniform	Non uniform	Uniform
5	Fluidity	Viscous	Viscous	Viscous
6	Appearance	Soft	Soft	Soft
7	Stability	Stable	Stable	Stable
8	pH	8.39	8.37	8.32
9	Spreadability (cm)	5.6	5.1	7.5
10	Viscosity (cp)	348.3	295.3	318.2
11	Loss on drying	41%	31%	20%

Figure 1: Standard chemical tests for tridax procumbens.

EVALUATION OF PHYSICAL PARAMETERS OF PREPARED FORMULATIONS:

The physical characteristics of three formulations are presented in the table below. The results of creaming, coalescence test indicated that the formulations exhibit excellent stability and aesthetic appearance and uniformity.

Table 4: Evaluation of physical parameters of prepared formulations.

S. No	Phytoconstituent	Standard test	Results of plant extract
1	Tannins	Ferric chloride test	+
2	Flavonoids	Lead acetate test	+
3	Alkaloids	Dragendroff’s test	+

ANTI-FUNGAL ACTIVITY STUDY OF Tridax procumbens Linn EXTRACT:

The antifungal assay of 100ppm and 200ppm extract was performed andthe results of zone of inhibition are as follows:

Table 5: Antifungal activity of herbal extracts (Zone of inhibition)

Strain used	Zone of inhibition (in diameter mm)
Aspergillus niger	Control	100ppm Extract	200ppmExtract
Aspergillus niger	0	20.5	19.2

Figure 2: Zone of inhibition of extract against Aspergillus niger

ANTI-FUNGAL ACTIVITY STUDY:

The antifungal assay of 3 formulations was performed and the results of zone of inhibition are as follows:

Table 6: Antifungal activity of herbal creams (Zone of inhibition)

Strain used	Zone of inhibition (in diameter mm)
Aspergillus niger	Control	F1	F2	F3
Aspergillus niger	0	9	9.5	10.5

Figure 3: Zone of inhibition of different formulations.

SUMMARY:

The work aims to formulate and evaluate an antifungal herbal cream of Tridax procumbens Linn extract. The various herbal creams are prepared with different oil phase ingredients and are characterized with various parameters such as pH, appearance, phase separation, viscosity, spread ability, and antimicrobial activity.

This study demonstrates the antifungal activity efficacy of Tridax procumbens Linn leaf extract base cream against Aspergillus niger. The results show that cream of Tridax procumbens Linn exhibits superior antifungal activity. The extract’s complex chemical composition likely contributes to its broad-spectrum antifungal properties, making it a promising natural alternative for treating skin infections.

· All the creams were observed to be green and had strong odour with smooth texture.

· pH of the creams was within the range of 8.32-8.39

· The viscosity of the herbal creams was found to be in the range of 295-398 cps.

· No phase separation was observed in the 3 herbal creams.

· The spread ability of all the herbal creams was found to be in the range of 5.1 -7.9 g.cm/s.

· All the developed herbal creams showed good antifungal activity. Amongst all the herbal creams, the F3 formulation showed highest zone of inhibition against fungi as compared to F1 and F2 formulations. The zone of inhibition was higher i.e. 10.5 ± 00.1mm in case of Aspergillus niger.

· The texture of F3 was uniform and the pH was 8.2 with a viscosity of 318.2 cps.

· The Spreadability of F3 was 7.5

· All the herbal creams were found to be stable at the end of storage period and can be safely used on skin. Among them F3 formulation showed better results.

Hence, it can be concluded that herbal cream of Tridax procumbens Linn showed promising formulation for future application.

CONCLUSION:

This study successfully demonstrated the phytoformulation of a stable oil-in-water O/W topical cream containing the ethanolic leaf extract of Tridax procumbens Linn. Furthermore, the investigation into its antifungal efficacy confirmed that the Tridax procumbens Linn. cream formulation exhibits superior antifungal activity against the challenging fungal pathogen Aspergillus niger in an in vitro setting. This potent activity is likely attributable to the extract’s complex chemical composition and the resulting broad-spectrum antifungal properties previously established in the literature. Collectively, these results strongly support the use of Tridax procumbens as a promising natural alternative for the development of effective and innovative topical treatments aimed at managing various fungal skin infections. Further in vivo clinical evaluation and detailed phytochemical characterization are warranted to fully validate its therapeutic potential.

ACKNOWLEDGEMENTS:

We are grateful to generous support for this work by Gokaraju Rangaraju College of Pharmacy, Hyderabad.

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Received on 11.03.2025 Revised on 18.07.2025

Accepted on 29.10.2025 Published on 15.04.2026

Available online from April 18, 2026

Asian J. Pharm. Res. 2026; 16(2):123-127.

DOI: 10.52711/2231-5691.2026.00018

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.