Formulation And Evaluation Of Polyherbal Antifungal Topical Gel

Superficial fungal infections of the skin, hair and nails are among the most frequently encountered dermatological complaints worldwide, with dermatophytes, yeasts and moulds accounting for the majority of cases. Factors such as a hot and humid climate, occlusive clothing, excessive perspiration, poor hygiene, immunosuppression and the indiscriminate use of antibiotics have all contributed to a steady rise in the prevalence of these infections. Synthetic antifungal agents, although effective, are increasingly limited by the emergence of resistant fungal strains, recurrent relapses, and adverse effects ranging from local irritation to hepatotoxicity on prolonged systemic use. This has prompted a search for safer, cost-effective and sustainable alternatives, with medicinal plants offering an attractive resource because of their multi-component, multi-target mode of action and generally favourable safety profile.

Herbal drugs owe their therapeutic versatility to a wide array of secondary metabolites-flavonoids, tannins, alkaloids, terpenoids and phenolic acids-that individually and synergistically interfere with fungal cell membrane integrity, ergosterol biosynthesis and other essential metabolic pathways. Polyherbal formulations, in which two or more plant extracts are combined, are often reported to produce an additive or synergistic therapeutic response that exceeds the activity of either component alone, while simultaneously broadening the spectrum of action.

Leucas aspera (Willd.) Link, a member of the family Lamiaceae and commonly known as Thumbai, is a widely distributed wayside herb of peninsular India traditionally applied to wounds, skin eruptions and insect bites. Its aerial parts are reported to contain flavonoids, phenolics, tannins, saponins, terpenoids and triterpenoids such as oleanolic and ursolic acid, constituents that underlie its documented antimicrobial, anti-inflammatory, antioxidant and wound-healing properties. Psidium guajava L. (Myrtaceae), the common guava, is similarly valued in folk medicine; its leaves are rich in flavonoids such as quercetin and guaijaverin, tannins and polyphenolic acids, and have been repeatedly shown to possess broad-spectrum antimicrobial, antidiarrhoeal and antioxidant activity.

Topical gels are a preferred dosage form for the management of localized cutaneous infections because they are non-greasy, easily spreadable, cosmetically acceptable, and deliver the active constituents directly to the site of infection while minimizing systemic exposure. Against this background, the present study was undertaken to develop a polyherbal antifungal gel combining ethanolic extracts of L. aspera and P. guajava, and to evaluate its physicochemical characteristics and in-vitro antifungal efficacy against Aspergillus niger and Aspergillus flavus, with the broader aim of validating a safe and effective plant-based topical option for superficial fungal infections.

AIM AND OBJECTIVES

The study aimed to formulate and evaluate a polyherbal antifungal topical gel using extracts of Leucas aspera and Psidium guajava, with the specific objectives of

 (i) preparing the two plant extracts by a suitable extraction method (ii) incorporating them into an appropriate gel base, and (iii) evaluating the formulated gel for physicochemical parameters-appearance, homogeneity, pH, viscosity, spreadability and extrudability-and for in-vitro antifungal activity.

II. MATERIALS AND METHODS

Collection and Authentication of Plant Material

Fresh aerial parts of Leucas aspera and fresh leaves of Psidium guajava were collected locally and identified and authenticated by the Botanical Survey of India, Southern Regional Centre, Coimbatore (authentication reference nos. BSI/SRC/5/23/2025-26/Tech./1092 for L. aspera and BSI/SRC/5/23/2025-26/Tech./1094 for P. guajava). Voucher specimens were preserved in the departmental herbarium. The collected material was washed under running tap water followed by distilled water to remove adhering soil and debris, shade-dried at ambient temperature away from direct sunlight to protect thermolabile constituents, and then mechanically ground to a coarse powder that was passed through sieve no. 60 and stored in airtight, amber-coloured containers until further use.

Preparation of Extracts

The powdered plant materials were extracted separately by the cold maceration technique using 70% v/v ethanol as solvent. Each powder was soaked in the solvent for 48-72 hours at room temperature with intermittent shaking, after which the macerate was filtered successively through muslin cloth and Whatman filter paper. The filtrates were concentrated under reduced pressure on a rotary evaporator at a controlled temperature, and the resulting semi-solid extracts were dried, weighed and stored separately in airtight labelled containers. Percentage yield was calculated as the ratio of the weight of dried extract to the weight of powdered plant material taken, expressed as a percentage.

Preliminary Phytochemical Screening

Both extracts were subjected to standard qualitative tests to detect the major phytoconstituent classes: alkaloids (Dragendorff's test), flavonoids (alkaline reagent test), tannins (ferric chloride test), saponins (foam test), terpenoids (Salkowski test), phenolic compounds (ferric chloride test), anthraquinone glycosides (Borntrager's test), steroids (Liebermann-Burchard test), lipids (Sudan III test), amino acids (ninhydrin test) and proteins (biuret test), following conventional phytochemical procedures.

Formulation of the Polyherbal Gel

Carbopol 940 was selected as the gelling agent, and four formulations (F1-F4) were prepared by varying its concentration while keeping the quantity of extract and other excipients constant, as summarised in Table 1.

Ingredient	F1	F2	F3	F4
Leucas aspera extract	1 g	1 g	1 g	1 g
Psidium guajava extract	1 g	1 g	1 g	1 g
Carbopol 940	0.5 g	0.75 g	1 g	1.25 g
Propylene glycol	5 mL	7.5 mL	10 mL	12.5 mL
Methyl paraben	0.1 g	0.1 g	0.1 g	0.1 g
Triethanolamine	0.5 mL	0.5 mL	0.5 mL	0.5 mL
Glycerin	5 mL	5 mL	5 mL	5 mL
Ethanol	2 mL	2 mL	2 mL	2 mL
Distilled water	q.s.	q.s.	q.s.	q.s.

Table 1: Composition of polyherbal antifungal gel formulations (F1-F4)

Fig. 1: Leucas aspera (aerial parts with flower)

Fig. 2: Psidium guajava (leaves and fruit)

Fig. 3: Prepared polyherbal gel formulations F1-F4 (left to right)

Plant Extract	Solvent (Ethanol:Water)	Powder Taken (g)	Extract Obtained (g)	% Yield
Leucas aspera	308:132 mL	44	13.25	30.11%
Psidium guajava	350:150 mL	50	14.11	28.22%

Table 2: Extractive yield of Leucas aspera and Psidium guajava (70% ethanol, maceration)

Phytoconstituent	Leucas aspera		Psidium guajava
Alkaloids	+		+
Flavonoids	+		+
Phenols	+		+
Tannins	+		+
Saponins	+		+
Terpenoids	+		+
Glycosides	+		+
Amino acids	+		+
Proteins	-		-
Steroids	-		-
Lipids	-		-
Fig. 4: Phytochemical screening of L. aspera extract		Fig. 5: Phytochemical screening of P. guajava extract

Table 3: Preliminary phytochemical screening of the two extracts (+ present, - absent)

Parameter	Result (Formulation F4)
Colour	Pale green
Odour	Pleasant, characteristic
Appearance	Smooth and elegant
Clarity	Clear
Homogeneity	Good
Lumps	Absent
Air bubbles	Absent
Spreadability	7.2 g·cm/sec
Extrudability	Good
Viscosity	5000 cP
Washability	Easily washable
Grittiness	Absent
pH	5.8

Table 4: Physicochemical evaluation of the optimized formulation (F4)

Test Organism	10 µL Gel	20 µL Gel		Fluconazole Disc (25 µg)	DMSO
Aspergillus niger	11 mm	13 mm		10 mm	Nil
Aspergillus flavus	14 mm	16 mm		13 mm	Nil
Fig. 6: Agar-well diffusion assay - Aspergillus niger			Fig. 7: Agar-well diffusion assay - Aspergillus flavus

Table 5: Zone of inhibition (mm) of the optimized gel (F4) against Aspergillus niger and Aspergillus flavus

Fig. 8: Zone of inhibition of the optimized gel (F4) against A. niger and A. flavus

Cold maceration in 70% ethanol furnished extractive yields of 30.11% w/w for L. aspera and 28.22% w/w for P. guajava (Table 2), indicating that the hydro-ethanolic solvent system was efficient at solubilising the polar-to-moderately-polar metabolites typically implicated in antifungal action.

Phytochemical screening (Table 3) confirmed the presence of alkaloids, flavonoids, phenolic compounds, tannins, saponins, terpenoids, glycosides and amino acids in both extracts, while proteins, steroids and lipids were not detected. The consistent presence of flavonoids, tannins and terpenoids in both plants is noteworthy, since these classes are widely reported to compromise fungal cell membrane integrity and interfere with ergosterol biosynthesis, and their co-occurrence in the two extracts provides a plausible chemical basis for an additive or synergistic antifungal effect in the combined formulation.

All four gel bases (Table 1) were physically acceptable, but they differed in consistency as the Carbopol 940 concentration was increased from 0.5% to 1.25% w/v. Formulation F4 gave the best overall combination of viscosity, spreadability and extrudability and was accordingly selected as the optimized batch. On detailed evaluation (Table 4), F4 was a pale green, smooth, clear gel free of lumps, air bubbles and grittiness, with good homogeneity and easy washability. Its spreadability (7.2 g·cm/s) and extrudability indicate suitability for convenient topical application, while a viscosity of 5000 cP is consistent with a semi-solid preparation that will remain at the site of application without excessive run-off. The measured pH of 5.8 lies close to the normal physiological pH of human skin (approximately 4.5-6.0), suggesting a low likelihood of irritation on repeated topical use.

In the agar-well diffusion assay (Table 5), formulation F4 produced clear, concentration-dependent zones of inhibition against both test organisms. At the lower test volume (10 µL) the zones were 11 mm against A. niger and 14 mm against A. flavus, increasing to 13 mm and 16 mm respectively at 20 µL. These values compare favourably with the 25 µg fluconazole standard, which produced zones of 10 mm and 13 mm against the same organisms, while the DMSO control produced no zone of inhibition, confirming that the observed activity originated from the herbal constituents of the gel rather than the vehicle. A. flavus was consistently more susceptible than A. niger across both test volumes, and the increase in inhibition with increasing gel volume points to a concentration-dependent antifungal effect, plausibly arising from the combined action of the flavonoid, tannin and terpenoid constituents contributed by both plant extracts.

Taken together, these results indicate that combining L. aspera and P. guajava extracts in a Carbopol-based gel yields a stable, cosmetically acceptable and skin-compatible topical preparation with antifungal efficacy approaching that of a standard synthetic agent, while offering the additional safety and acceptability generally associated with herbal formulations.

CONCLUSION

A polyherbal antifungal topical gel combining Leucas aspera and Psidium guajava extracts was successfully formulated and optimized using Carbopol 940 as the gelling agent. The optimized formulation (F4) exhibited satisfactory physicochemical characteristics-including a skin-compatible pH of 5.8, appropriate viscosity, and good spreadability, extrudability and homogeneity-along with a rich phytochemical profile encompassing flavonoids, tannins, terpenoids and phenolic compounds known to contribute to antifungal action. The formulation displayed significant, concentration-dependent antifungal activity against Aspergillus niger and Aspergillus flavus, comparable to standard fluconazole, supporting a synergistic contribution from the two plant extracts. These findings substantiate the traditional antimicrobial use of both plants and identify the developed gel as a promising, safe and effective natural alternative for the topical management of superficial fungal infections; further work on long-term stability, dermal irritation and clinical efficacy is warranted before therapeutic application.

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