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Formulation, evaluation, and optimization of polyherbal gel for effective antifungal action
*Corresponding author: Dhiraj Kumar, Department of Pharmacy, Sunder Deep Group of Institutions, Sunder Deep Pharmacy College, Ghaziabad, Uttar Pradesh, India. pharmamylife2@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Kumar D, Jain N, Singh S, et al. Formulation, evaluation, and optimization of polyherbal gel for effective antifungal action. Am J Pharmacother Pharm Sci 2025:015
Abstract
Objectives:
To develop and evaluate a polyherbal gel as a potential alternative to synthetic antifungal agents, addressing the increasing prevalence of fungal infections and microbial resistance.
Materials and Methods:
Polyherbal gels were formulated using extracts of Tora, Fenugreek, and Neem seeds incorporated into a Carbopol 940 base. Various batches containing 1%–4% of each seed extract were prepared. The formulations were evaluated for physicochemical properties (pH, viscosity), spreadability, extrudability, in vitro antifungal activity against fungal strains, and stability.
Results:
Formulations exhibited pH values between 5.5–6.5, suitable for topical application, and viscosities ranging from 3455.98–4614.96 cP. Spreadability ranged from 5.4–6.84 cm, indicating good application properties. All formulations required 3.5–4.5 kg of force for extrusion. Antifungal activity increased with extract concentration, reaching a plateau at 4% (polyherbal gel 4 - PHG4), which demonstrated statistically significant results comparable to the standard antifungal drug.
Conclusion:
The optimized polyherbal gel (PHG4) demonstrated effective antifungal activity, favorable physicochemical properties, and potential as a safe herbal alternative to synthetic antifungal topical preparations, with no risk of microbial resistance.
Keywords
Antifungals
Extracts
Fenugreek
Neem
Polyherbal gels
INTRODUCTION
Topical antifungal preparations are used to treat burns, wounds, bacterial infections, and superficial fungal infections. They include creams, lotions, gels, and ointments. Topical preparations are thought to be advantageous for treating localized skin infections, mainly fungal, given the drug concentration and level of solubility.[1] People have used plants with therapeutic characteristics as traditional medicine for millennia. Various medicinal plants’ leaves, stems, and roots have been used as a source of extracts to treat a variety of diseases.[2] The primary source of medicines for both human and animal ailments has always been plants. For the preservation and well-being of health and other bodily functions, herbal therapies may traditionally be provided holistically.[3] A treatment of choice for particular medical problems with a better clinical outcome may be offered by current herbal formulations based on scientifically demonstrated efficacy.[2] For the treatment of skin-related conditions, including wounds and ringworms, as well as for use as an antimicrobial agent and for cosmetic purposes, the active ingredients responsible for such medicinal properties are extracted and manufactured as creams, soaps, and ointments.[4] Growing interest has been shown in using plants and plant-based products to treat illnesses or enhance the health of both humans and animals.[5] Scientists and pharmaceutical companies throughout the world have expressed a keen interest in the topic of phytochemistry because plants are one possible source of new molecules.[6] Conventional herbal preparations are still used today as an alternative to Western medications. Basic research reveals phytochemical data that necessitate more investigation into pharmaceutical formulations, pharmaceutical assays for medication validation, and pharmaceutical standardization. More than 95% of traditional medicine, which is utilized as the main form of healthcare worldwide[7], comes from medicinal plants. There are thought to be over 250,000 different species of medicinal plants on earth. The majority of the medications that are frequently utilized in therapeutic settings come from tropical plants.[7,8] Botany, pharmacology, ethnology, and anthropology are all used in the current efforts to develop drugs from therapeutic plants. The prevalence of fungal infections has recently increased, necessitating immediate intervention. Fungal infections are rarely identified in the early stages, which increases the severity of illnesses and complicates treatment. Fungal pathogens employ a range of tactics to get around the host immune system and worsen diseases. Different superficial and systemic infections are treated with antifungal drugs chosen from a variety of possibilities. Antifungal therapy, however, offers challenges due to well-documented evidence of microbial resistance to the majority of antifungal drugs.[9] This makes it challenging to treat these diseases. Numerous physiological adaptations, genetic mutations, and selection of these alterations in the presence of antifungal medications all contribute to the development of resistance in fungi. The availability of a limited antifungal arsenal, the emergence of resistance, and biofilm-conferred resistance are driving the need for the development of novel drugs and alternative treatment modalities for better treatment outcomes against mycoses.[10] Numerous fungal diseases, their etiologic agents, pathophysiology, antifungal treatment kinds, and resistance mechanisms, including host immune responses and evasion strategies, were all clearly explained in this graphical analysis. To combat fungal infections, we have utilized recent advancements in new antifungal medicines and various alternative strategies.[11]
Since the beginning of time, people have employed natural herbal treatments to treat disease and advance health. Natural phytoconstituent-based formulations have gained universal acceptance as therapeutic agents for the treatment of inflammatory disorders, infections, arthritis, hyperglycemia, depression, anxiety, human immunodeficiency virus, and other diseases.[12] Due to their historical roots, financial sustainability, and patient compliance, the development of polyherbal formulations has received increased attention. There are numerous topical, biological, and systemic drugs that can be used to treat a fungal infection. They do, however, have undesirable side effects, such as dry skin and etching. While certain drugs can help with sickness symptoms, they can also have unfavorable side effects. Producing a medicine that is both extremely effective and has few side effects is crucial in the interim.[13] A plant is considered medicinal if it contains elements that can be utilized to create semi-synthetic medicines or that have therapeutic advantages. The phytochemicals that are present in plants but are not nutrients act as the plants’ defense mechanisms against microbial illnesses. Neem (Azadirachta indica) is a member of the Meliaceae family.[14] It attributes its significance as a health-promoting agent to its high antioxidant content. Through its antifungal activity, it has long been used in Chinese, Ayurvedic, and Unani medicines, notably in the Indian Subcontinent, to treat and prevent a wide range of ailments. Numerous studies have shown that fenugreek, a different plant seed, possesses antibacterial, antifungal, and antidandruff activities in its seed extracts, whereas fenugreek leaf extracts have only sometimes proven antibacterial and antifungal action. Numerous studies have shown that Cassia tora seeds might also be antifungal.[15] The fact that no single herb could perform or provide activity similar to that of synthetic drugs was what led to the development of polyherbal gel. Since the majority of the fungal infections are treated with topical treatment, a topical preparation was chosen for the current experiment. A gel formulation is the finest topical preparation for an antifungal medication since it can keep the application site wet, minimizing irritation. Among other essential functions, gels are utilized to provide the optimal cutaneous and percutaneous medication dispersion. The formulation and assessment of the polyherbal gel was the primary goal of this investigation. The physicochemical attributes and in vitro anti-microbial testing of the produced formulations, and antifungal efficacy were evaluated.[16]
MATERIALS AND METHODS
Materials
Neem, Fenugreek, and tora seeds were purchased from authentic sources. All other chemicals were purchased from standard suppliers that are of analytical grade.
Methods
Extraction
The seeds were collected and finely crushed. 500 g of the powdered, crushed seeds were extracted using the Soxhlet method using ethanol as the solvent. Until the solvent was confirmed to be clear, the treatment was continued. The extract was dried to a powdery form using a desiccator. The same procedure was used to handle all types of seeds.[17]
Evaluation of extract
Characteristics of the extract were evaluated for its physical state, color, and odor.
Phytochemical investigation of the extract
The presence of several phytoconstituents, including flavonoids, proteins, amino acids, phenols, and organic acids, was qualitatively screened for in the alcoholic extract of all seeds.[18]
Formulation
Formulation of gel base and optimization
The gelling agent Carbopol 940 was dispersed in a range of concentrations in an acceptable amount of water. Propylene glycol, which is used as a plasticizer or humectant, is also included. The preservatives, methylparaben and propylparaben, were moved and combined. To achieve a pH of neutral, triethanolamine was added, and distilled water was used to raise the gel’s final weight to 50 g. To get rid of any air bubbles and keep the mixture at room temperature, the aforementioned mixture was stirred for 2 h at a speed of 500 rpm.[19]
Formulation of polyherbal gel
The polyherbal gel was made by varying the amounts of ethanolic seed extract that were added to the optimized Carbopol gel in accordance with the suggested recipe. After 2 h of agitation at 500 rpm, the entire mixture was left undisturbed at room temperature. The individual seed extract gel was created using the exact same process.
Physical Characterization of the Formulated polyherbal gel
The formulation was subjected to physical characterization, such as color, appearance, pH, viscosity, and spreadability.
Physical appearance
After being placed in the container, the prepared gel was examined for its organoleptic properties, viscosity, and homogeneity, as well as for the appearance and presence of any aggregates.
Determination of pH
The gel was mixed with about 1 g of deionized water per 100 mL. The pH of each unique formulation was measured using a digital pH meter (Model MK-VI, Kolkata, India). In order to get results in triplicate, readings were taken 3 times.
Determination of viscosity
The viscosity of the designed gel was measured using a rotational viscometer (Brookfield viscometer RVT, spindle No. 64) of the cup-and-bob variety.
Homogeneity
The consistency and homogeneity of the gel were assessed by pressing a tiny amount between the thumb and index finger, noting if the gel was homogeneous or not and whether any coarse particles had detached or appeared on the fingers.
Spreadability
The spreadability of the gel was computed to determine how much of an area it covers when applied to skin. 100 g of the gel was lightly layered on top of two slides with a 6 cm boundary in between them. Gels 2023, 9, 163-12 of 14 were then used to attach the slide to an unchanged platform such that only the upper slide could be released by the weight that was linked to it. A 20 g mass was used to secure the upper slide. The upper slide’s time to progress a certain distance before colliding with another object was timed. Three copies were produced.
Analysis of gel extrudability
Extrudability: The effort necessary to expel the gel formulations from the tubes was used to calculate their extrudability. Plastic syringes (10 mL) were filled with gel formulation and stacked to a chosen height (3 cm). Then, using the TA_XT@ Texture Analyzer (Stable Micro Systems, UK) in compression mode with the analytical probe’s pre-test speed set to 3 mm/s, the formulation was evacuated from the syringes.[20] Using the TA-XT2, a calibrated texture analyzer, the strength of the created gel was evaluated. The device was first calibrated to measure force and distance at room temperature. The 45° cap was partly filled with 1 g of the gel and set on the platform of the analyzer. To disseminate and detect the dynamics of spreading and retracting forces as they traveled vertically toward the bottom of the cap before withdrawing to their original location, a similar 45° cone was utilized as a probe. The assembly of the cone and cap was coaxially oriented. The cone probe descended during the test at a speed of 3 mm/s until it was 1 mm away from the bottom of the cap. The probe then moved upward at a rate of 10 mm/s (i.e., in retraction mode) right after that. The gel strength is represented by the sum of the work done to spread 1 g of the gel between the cone-cap surfaces and the sum of the work needed to retract from the spread gel. The TA-XT2 texture analyzer (UK)’s HDP/FE forward extrusion cell was used to test extrudability. The compression force is the amount of pressure necessary to push a piston disc through a regular-sized exit in the sample container’s base to extrude a product. A piston disc was connected to a load cell (5000 g) using a probe adaptor, and the sample was placed in a centralizing insert poured into a sturdy platform. The sample was squeezed by the plunger, which also caused forward flow through the disc’s annulus. The compression force sensed by the load cell represents the extrudability.[21]
Antimicrobial Activity by cup plate method
The test organism (C. albicans) was then put at the correct dilution in the sterile Petri dishes after the Mueller–Hinton agar medium had been added. On each plate, four cylinders, or cups, were created in the media by using a sterile borer. Consistently pouring the solution into the cup, which was then kept at 37°C for 24 h, was the procedure. Three iterations of the study were conducted, and the results are shown as mean inhibition in diameter (mm).[22]
Determination of antifungal activity
The standard Candida albicans (ATCC-18804) and another fungal strain were used to test the extract’s anti-fungal effectiveness against infectious agents.[23]
Standardization of inoculum
The inoculum was standardized using the serial dilution agar-plate method by aseptically transferring 1 mL of the bacterial suspension tube into sterile water of a predetermined volume, where the culture was diluted 10 times from 10-1 to 10-10. The diluted suspensions were gently rotated as they were added to a Mueller–Hinton agar medium, cultured for 24 h at 37°C, and then counted using a colony counter. By multiplying the visible cells on each plate by the dilution factor, the sample’s colony count was determined.
RESULTS
Seeds extract
Tora, Fenugreek, and neem seeds were extracted, and powder from the extracts was gathered for future usage. The extraction process used a methanol-based solvent. For each variety of seed, 10 g of the extract was stored in a dry, cool environment.
Physical evaluation of seed extract
Physical evaluation of seed extract was done separately in terms of color, physical state, and % yield [Table 1].
| S. No. | Items | Fenugreek seeds | Tora seeds | Neem seeds |
|---|---|---|---|---|
| 1. | Color | Pale yellow | Light to dark brown | Brownish |
| 2. | Physical state | Solid | Semi-solid to solid | Solid |
| 3. | Yield value | 10.22% | 9.89% | 11.28% |
| 4. | Stickiness | None sticky | Little bit sticky | None sticky |
Phytochemical investigation of the extract
Table 2 concludes that alkaloids are present in almost all seed extracts.
| S. No. | Items | Qualitative chemical analysis of methanolic extract of seeds | ||
|---|---|---|---|---|
| Fenugreek seeds | Tora seeds | Neem seeds | ||
| 1. | Glycoside | + | ++ | + |
| 2. | alkaloid | ++ | +++ | +++ |
| 3. | Saponins | + | - | - |
| 4. | Flavonoids | + | ++ | + |
| 5. | Tannin | - | + | ++ |
+++: Highly present, ++: Moderately present, +: Present, -: Absent
Formulation of gel base and optimization
As a gelling agent, Carbopol 940 was employed. 0.5%, 1%, 1.5%, 2.0%, 2.5%, and 3% Carbopol 940 were used to optimize the percentage of gelling agent that has accurate consistency in accordance with the pharmacopoeia standard, along with necessary preservatives and other pH-balancing agents [Table 3]. Table 3 lists the ingredients in the gel base.
| S. No. | Ingredients | Percentage |
|---|---|---|
| 1. | Carbopol 940 | 2% |
| 2. | Propyl paraben | 0.01% w/w |
| 3. | Methanol | 10% |
| 4. | Triethanolamine | q.s |
| 5. | Water | Up to 100 g |
Because Carbopol 940 is made of carbohydrate and is a good source for the growth of microorganisms, propyl paraben is utilized as a preservative. Triethanolamine was added to keep the gel’s pH stable because a change in pH could irritate and swell the skin.
Formulation of polyherbal gel
Different formulations [Table 4] of poly herbal gel were created using varied concentrations of methanolic extract from each seed. For each batch, the other ingredients’ composition is the same.
| S No. | Formulation code | % composition of seeds extract | |
|---|---|---|---|
| 1. | PHG1 | 0.5% w/w of each seeds extract added in the optimized gel base | All other ingredients remain same as mentioned in optimized gel base. |
| 2. | PHG2 | 1% w/w of each seeds extract added in optimized gel base | |
| 3. | PHG3 | 1.5% w/w of each seeds extract added in optimized gel base | |
| 4. | PHG4 | 2% w/w of each seeds extract added in optimized gel base | |
| 5. | PHG5 | 2.5% w/w of each seeds extract added in optimized gel base | |
| 6. | PHG6 | 3% w/w of each seeds extract added in optimized gel base | |
| 7. | PHG7 | 3.5% w/w of each seeds extract added in optimized gel base | |
| 8. | PHG8 | 4% w/w of each seeds extract added in optimized gel base | |
PHG: Prepared polyherbal gel.
Formulations with varying percentages of content were created, and they were then further assessed based on research on their antifungal efficacy. The minimum percentage composition has antifungal action on par with that of common antifungal medications.
Physical characterization of polyherbal gels
All of the formulations’ pH and viscosity were confirmed to be within the acceptable range for a topical gel. The pH ranges from 5.5 to 6.5, making it perfect for topical application. A fluid’s viscosity, which measures its thickness, ranges from 300 to 5000 cP, and is used to describe fluids used in gel formation. The viscosity was likewise within the ideal range for the semi-solid gel preparation of 3455.98–4614.96 cP [Table 5].
| S. No. | Formulation code | Color | Physical appearance | pH | Viscosity (cP) |
|---|---|---|---|---|---|
| 1. | PHG1 | Faint brown | Semi solid | 5.55±0.43 | 3455.98±380.17 |
| 2. | PHG2 | Faint brown | 5.43±0.56 | 3467.91±340.89 | |
| 3. | PHG3 | Faint brown | 5.81±0.61 | 3605.88±390.12 | |
| 4. | PHG4 | Brown | 5.98±0.33 | 3985.95±467.18 | |
| 5. | PHG5 | Brown | 6.26±0.69 | 4405.17±489.95 | |
| 6. | PHG6 | Brown | 6.09±0.12 | 4505.09±440.88 | |
| 7. | PHG7 | Brown | 6.12±0.23 | 4514.97±333.09 | |
| 8. | PHG8 | Brown | 6.49±43 | 4614.96±352.66 |
PHG: Prepared polyherbal gel.
Homogeneity
The consistency and homogeneity of the gel were assessed by pressing a small amount of gel between the thumb and index finger, noting whether the gel was homogeneous or not and whether any coarse particles had detached or appeared on the fingers. It was discovered that none of the gel formulations had any coarse particles.
Spreadability
The capacity of the gel to spread across the skin’s surface is referred to as the spreadability of gel preparations. The surface area that the gel can reach increases with the scatter diameter. When applied to the skin, a gel with good spreadability is evenly distributed; this spreadability is measured in the range of 5–7 cm. Figure 1 shows the spreadability test results for the various gel preparations, which show a value between 5.4 and 6.84 cm, indicating that the gel has an excellent spreadability.
- Spreadability of prepared polyherbal gel (PHG).
Analysis of gel extrudability
Extrudability when creating semi-solid formulations, viscosity is crucial because the finished goods must be both fluid enough to allow extrusion from the container and viscous enough to delay phase separation. When too much viscosity is used, the formulation’s fluidity may suffer, producing a non-fluidic gel rather than a syringeable formulation. The effort necessary to extrude the gel formulations from the tubes was used to calculate their extrudability. The findings show that all polyherbal gels need between 3.5 kg and 4.5 kg of force to push through the outlet, as shown in Figure 2. As a result, the extrudability of all the gel formulations is perfect.
- Extrudability study of formulated gel.
Determination of antifungal activity
To assess the antimicrobial activity of plant or microbial extracts, the agar well-diffusion method is frequently utilized. The agar plate surface is inoculated using a process similar to the disk-diffusion approach in which a volume of the microbial inoculum is dispersed across the entire agar surface. The antimicrobial agent or extract solution is then added to the well in the prescribed amount at the specified concentration after a hole measuring 6–8 mm in diameter is aseptically punched. The test microorganism is then placed on an appropriate agar plate, and the incubation process is continued. The antibiotic ingredient spreads across the agar media and stops the tested microbial strain from growing. The zone of inhibition expands as the polyherbal extract concentration rises, although the effects peak at 4% concentration or the PHG4 coded formulation. In addition to the ceiling effect, it is also extremely similar to a regular drug. Results that were statistically significant were obtained when compared to the standard. The most efficient antifungal gel, then, is PHG4 [Figure 3].
- Evaluation of antifungal activity by agar well-diffusion method.
Statistical analysis
One-way analysis was used for the statistical analysis. Statistics were judged to be significant at P < 0.05.
DISCUSSION
Fungal infections are becoming increasingly common, and the rise of multidrug-resistant strains has created an urgent demand for treatments that are both effective and less likely to induce resistance. This study was designed to meet that need by developing and assessing a polyherbal gel formulated with extracts of Neem (Azadirachta indica), Fenugreek (Trigonella foenum-graecum), and Tora (Cassia tora), using Carbopol 940 as the gelling agent.
The results of the physicochemical evaluations demonstrated that the prepared gels were well-suited for topical use. All batches exhibited a pH between 5.5 and 6.5, which closely matches the natural pH of human skin and helps to minimize irritation. The viscosity values (3455.98– 4614.96 cP) indicated that the gels had a stable, semi-solid consistency, which is essential for ease of application and therapeutic performance. These characteristics play a vital role in ensuring patient comfort and improving adherence to treatment.
Spreadability and extrudability are critical for the practical use of topical formulations. The prepared gels showed spreadability in the optimal range (5.4–6.84 cm), ensuring even application over the affected area. Extrudability tests confirmed that the gels could be dispensed with moderate force (3.5–4.5 kg), indicating convenience in handling without compromising product integrity. These findings align with established benchmarks for high-quality topical preparations.
Antifungal efficacy was evaluated against Candida albicans and other pathogenic strains using the agar well-diffusion method. Increasing the concentration of herbal extracts improved antifungal activity, with the formulation containing 4% of each extract (PHG4) demonstrating the largest zone of inhibition. This activity was statistically comparable to that of the standard antifungal agent (P<0.05). The enhanced effectiveness of PHG4 is likely due to the synergistic action of its phytoconstituents: Neem’s azadirachtin, Fenugreek’s bioactive saponins and flavonoids, and Tora’s anthraquinones, all of which are known for their antimicrobial properties.
The absence of visible particulate matter and the overall stability of the formulation further support the quality of the developed gel. Importantly, using a combination of plant-based extracts may help reduce the likelihood of microbial resistance, a significant limitation of many synthetic antifungal medications.
Despite these promising results, certain limitations remain. The present investigation was limited to in vitro assessments, and additional studies involving in vivo testing, skin compatibility evaluations, and detailed mechanism-of-action analyses are necessary to confirm clinical efficacy. Conducting stability tests under accelerated conditions would also help determine the product’s shelf life.
In summary, the optimized polyherbal gel (PHG4) demonstrated excellent physical attributes and strong antifungal potential. Its natural composition and favorable performance profile suggest that it could be developed as a safe, effective, and patient-friendly alternative to existing antifungal therapies.
CONCLUSION
Based on the aforementioned information, our formulation PHG4 was chosen as the best polyherbal gel among all formulations. The antifungal activity of the chosen formulation was remarkably similar to that of the prescription medicine. Even though we are aware that synthetic antifungal medications may develop resistance, our polyherbal gel may not result in any resistance against any microorganisms. Further research is required to investigate the mechanistic basis of antifungal action.
Acknowledgment:
The authors are thankful to the institution for providing all facilities to complete this work.
Ethical approval:
Institutional review board approval is not required.
Declaration of patient consent:
Patient’s consent not required, as there are no patients in this study.
Conflicts of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript, and no images were manipulated using AI.
Financial support and sponsorship: Nil.
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