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INTRODUCTION
Fungi are parasitic eukaryotic microorganisms which possess a cell wall and lack the ability to photosynthesize. Medically, fungi represent an important group of microbial pathogens, responsible for several potentially fatal diseases in humans. They commonly infect the dermis and deeper layers of the skin and most fungal infections can cause itching, blisters, swelling, and severe scaling on the dermis, epidermis or stratum corneum. It has been reported that Candida species colonize the skin and mucous membranes without causing any damage under normal conditions. However, factors like increased moisture, heat and weakened local or systemic immune defenses can create an ideal environment for their proliferation and pathogenicity (Nurdin et al., 2021). These infections may remain superficial, by affecting the epidermis or mucous membranes, or become systemic by entering the bloodstream and spreading to internal organs.
Collection and authentication of plants material
Senna auriculata (Common name: Senna; Family: Fabaceae) and Lawsonia inermis (Common name: Henna; Family: Lythraceae) are commonly used in the folk medicine in Tamil Nadu (Jangid and Begum, 2022). The plant materials were collected from local areas of Chennai during the optimal time of rainy and early winter season and evaluated for antimycotic activity (Al-Snafi, 2019). The collected plant materials were authenticated at the Department of Botany, Madras Christian College, Chennai.
Cleaning and drying of plant material
The aerial parts of the plants were cleaned and washed with tap water to remove dirt and contaminants. The leaves were then separated and dried under shade until they were completely moisture-free (Ben et al., 2011). The dried leaves were finely powdered using a blender and stored in a clean airtight container for further analysis.
Despite the individual antifungal efficacy of Lawsonia inermis and Senna auriculata being studied, there has been no direct comparative study of their methanolic leaf extracts employing the same extraction techniques, phytochemical analysis, FTIR characterization, and antifungal testing against Candida albicans. Thus, the current research aimed to undertake a comparative assessment of the phytochemical constituents and antifungal properties of these two traditionally employed medicinal plants under standardized experimental conditions to identify the comparatively more promising extract for future antifungal drug formulation.
MATERIALS AND METHODS
Extraction of Plant Material
The extraction process was done by Maceration with agitation method (Abubakar AR & Haque M (2020); World Health Organization, 2011). 30 g of leaf powder sample of Henna and Senna were taken in two different conical flasks, and 150 mL of methanol (analytical grade) was added in both the conical flasks and kept for 72 hr at 25ºC temperature. The entire mixture of both the samples was filtered and concentrated to dryness by evaporating on a vacuum evaporator. The extracted crude sample was kept in airtight glass container and stored at 4ºC until use. Percentage yield of extracts was calculated for the extracts (Table 1) using the following formula:
| Plant material | Solvent system | Weight of Plant powder (g) | Weight of Dried extract (g) | Percentage yield (%) |
|---|---|---|---|---|
| Lawsonia inermis | Methanol | 30 | 5.209 | 17.36 |
| Senna auriculata | Methanol | 30 | 3.043 | 10.14 |
Preliminary Phytochemical analysis
The preliminary phytochemical screening of the methanolic extracts of Senna auriculata and Lawsonia inermis was carried out using standard qualitative methods to identify the presence of major phytoconstituents, including flavonoids, tannins, saponins, alkaloids, glycosides, carbohydrates, and proteins (Evans, 2009; Harborne, 1998).
Infrared (IR) Spectroscopy Analysis of PLANT Extract
The dried crude extracts were mixed with KBr (Potassium Bromide) and pressed into pellets for Fourier-Transform Infrared (FTIR) spectroscopy analysis. The FTIR spectra were recorded in the 4000-400 cm⁻¹ range, and the absorption peaks corresponding to various functional groups were analyzed and interpreted (Arockia et al.,, 2015; Bhalodia, 2012).
Evaluation of Antifungal Activity
Preparation of control plate
The control plates were prepared for each extract. Candida albicans (ATCC strain) was used as the test organism. The inoculum was standardized to 0.5 McFarland turbidity. The targeted microorganisms were cultured in Potato dextrose broth and incubated for 24 hr. The petri dishes containing Potato Dextrose Agar (PDA) medium were cultured with diluted fungal strain.
Preparation of test concentrations of the extracts
A stock solution of 10 mg/mL was prepared by taking 100 mg) of each extract in 10 mL of sterile distilled water. From this solution, appropriate dilutions were made to obtain concentrations of 1000 µg/mL, 2000 µg/mL and 4000 µg/mL.
Evaluation of anti-fungal activity by Disc diffusion method
The antifungal activity was evaluated by disc diffusion method (Kavita, 2024). Sterile Whatman No.1 filter paper discs (6 mm) were impregnated with 20 µL of each extract solution to obtain final extract loadings of 20 µg/disc, 40 µg/disc and 80 µg/disc (corresponding to stock solutions of 1000 µg/mL, 2000 µg/mL and 4000 µg/mL). The impregnated discs were air-dried under aseptic conditions before being placed on inoculated Potato Dextrose Agar plates. Commercial fluconazole discs (25 µg/disc) served as the positive control, and sterile distilled water was the negative control. The inoculated plates were incubated at 28ºC for 24 hr and the zone of inhibition was measured and expressed in millimetres (Akwongo et al.,, 2024).
Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)
The MIC of methanolic extracts of Senna auriculata and Lawsonia inermis against Candida albicans was determined by the broth microdilution method as per the guidelines of Clinical and Laboratory Standards Institute (CLSI) (M27-A3) with slight modifications. Each extract was serially diluted in sterile Potato Dextrose Broth to give a range of concentrations. Each well was inoculated with one standardized suspension of fungus equivalent to 0.5 McFarland standard and incubated at 35ºC for 24-48 hr. The MIC was defined as the lowest concentration of the extract at which no visible growth of the fungus was observed. Samples from wells that did not show visible growth were plated onto Potato Dextrose Agar plates for determination of the Minimum Fungicidal Concentration (MFC). Plates were incubated for 24-48 hr. The minimum concentration that showed no visible growth of fungal colonies was recorded as MFC indicating complete fungicidal activity.
Statistical Analysis
Statistical analysis was performed using IBM SPSS statistics Version 23.0. Results are expressed as mean±standard deviation of three independent experiments. Comparisons among groups were performed using one-way Analysis of Variance (ANOVA) followed by Tukey's multiple comparison test. A p-value<0.05 was considered statistically significant.
RESULTS
As presented in Table 2, the percentage yield of methanolic extracts of Lawsonia inermis produced a higher extraction yield (17.36%) than Senna auriculata (10.14%), indicating a greater recovery of methanol-soluble constituents under identical extraction conditions. The preliminary phytochemical screening depicted in Table 3 revealed that the phytochemical profiles of the two plants were largely comparable. The methanolic extracts of both plants contained alkaloids, carbohydrates, cardiac glycosides, flavonoids, proteins, resins, saponins and tannins, whereas starch, steroids and triterpenoids were absent. The FTIR analysis (Tables 4 and 5; Figures 1 and 2) confirmed the presence of several functional groups including hydroxyl (O-H), carbonyl (C=O), aromatic (C=C), amine (C-N), carboxylic acid (C-O), alkane (C-H), and ester functional groups in both extracts. The variation in their chemical composition was indicated by the minor differences in peak positions between the two medicinal plants.
| Sl. No. | Phytoconstituents | Methanolic extract | |
|---|---|---|---|
| Senna auriculata | Lawsonia inermis | ||
| 1 | Alkaloids | (+) | (+) |
| 2 | Carbohydrate | (+) | (+) |
| 3 | Cardiac Glycoside | (+) | (+) |
| 4 | Flavonoids | (+) | (+) |
| 5 | Proteins | (+) | (+) |
| 6 | Resins | (+) | (+) |
| 7 | Saponins | (+) | (+) |
| 8 | Starch | (-) | (-) |
| 9 | Steroids | (-) | (-) |
| 10 | Tannins | (+) | (+) |
| 11 | Triterpenoids | (-) | (-) |
| Observed peak (cm⁻¹) | Functional group | Interpretation |
|---|---|---|
| 3307.13 | O-H stretching | Phenols/alcohols |
| 2924.12 | C-H stretching | Alkanes |
| 2853.05 | C-H stretching | Alkyl groups |
| 1721.81 | C=O stretching | Carbonyl compounds |
| 1613.22 | C=O stretching | Ketones |
| 1505.28, 1453.36 | C=C stretching | Aromatic rings |
| 1355.12 | C-H bending | Methyl groups |
| 1233.79 | C-N stretching | Amines |
| 1203.8 | C-O stretching | Carboxylic acids |
| 1155.21, 1109.33 | C-O stretching | Ethers/esters |
| 1009.29 | C-O-R stretching | Esters |
| 892.64 | P-O-C stretching | Phosphate groups |
| 834.35 | C-H bending | Aromatic compounds |
| Observed peak (cm⁻¹) | Functional group | Interpretation |
|---|---|---|
| 3309.68 | O-H stretching | Phenols/alcohols |
| 2925.11 | C-H stretching | Alkanes |
| 2855.14 | C-H stretching | Alkyl groups |
| 1720.53 | C=O stretching | Carbonyl compounds |
| 1632.00, 1603.57 | C=C/C=O stretching | Aromatic/ketones |
| 1509.62 | C=C stretching | Aromatic rings |
| 1398.65 | O-H bending | Phenolic compounds |
| 1342.54 | O-H bending | Hydroxyl groups |
| 1258.98 | C-N stretching | Amines |
| 1202.38 | C-O stretching | Carboxylic acids |
| 1014.01 | C-O-R stretching | Esters |
| 889.77 | P-O-C stretching | Phosphates |
| 819.46 | C-H bending | Aromatic compounds |
| Treatment | Concentration (µg/mL) | Zone of inhibition (mm, Mean±SD, n=3) | Statistical significance |
|---|---|---|---|
| Negative control | - | 0.0±0.0 | - |
| Senna auriculata | 1000 | 0.0±0.0 | ns (p>0.05) |
| 2000 | 9.4±0.6 | p<0.05 | |
| 4000 | 12.2±0.5 | p<0.01 | |
| Lawsonia inermis | 1000 | 0.0±0.0 | ns (p>0.05) |
| 2000 | 9.0±0.4 | p<0.05 | |
| 4000 | 13.0±0.6 | p<0.01 | |
| Positive control (Fluconazole) | 25 µg/disc | 20.4±0.2 | - |
The antifungal susceptibility test showed that C. albicans was susceptible to Senna auriculata and Lawsonia inermis by having a clear zone of inhibition. Figures 3 and 4 show the growth inhibitory effect of standard drug and Senna auriculata and Lawsonia inermis at 1000 µg/mL, 2000 µg/mL and 4000 µg/mL. Table 6 explains the antifungal activity of the methanolic extracts against Candida albicans. No inhibition was observed with either extract at 1000 µg/mL. At 2000 µg/mL, both Senna auriculata and Lawsonia inermis produced comparable inhibition zones of approximately 9 mm. At 4000 µg/mL, the inhibition zone increased to 12.2±0.5 mm for Senna auriculata and 13.0±0.6 mm for Lawsonia inermis, demonstrating concentration-dependent antifungal activity. Fluconazole (25 µg/disc) produced the largest inhibition zone (20.4±0.2 mm). Statistical analysis showed significant inhibition at 2000 µg/mL (p<0.05) and highly significant inhibition at 4000 µg/mL (p<0.01) compared with the negative control.
| Treatment | MIC (mg/mL) | MFC (mg/mL) | MFC/MIC ratio | Interpretation |
|---|---|---|---|---|
| /Senna auriculata | 2 | 4 | 2 | Fungicidal |
| Lawsonia inermis | 1 | 2 | 2 | Fungicidal |
| Fluconazole | 0.016 | 0.032 | 2 | Fungicidal |
The MIC and MFC values of the extracts are presented in Table 7. Lawsonia inermis exhibited lower MIC and MFC values than Senna auriculata, indicating comparatively greater antifungal potency.
| Extract | Concentration (µg/mL) | Zone of inhibition (mm) |
|---|---|---|
| Senna auriculata | 1000 | - |
| 2000 | 9±0.57 | |
| 4000 | 12±0.86 | |
| Lawsonia inermis | 1000 | - |
| 2000 | 9±0.68 | |
| 4000 | 13±0.94 |
DISCUSSION
The present study evaluated the antifungal activity of two commonly used medicinal plants, Senna auriculata and Lawsonia inermis against Candida albicans through preliminary phytochemical analysis, Fourier-Transform Infrared (FTIR) spectral studies and antifungal evaluation. The phytochemical analysis revealed that both methanolic extracts contained various phytoconstituents like alkaloids, carbohydrates, glycosides, flavonoids, proteins, resins, saponins, and tannins attributing to different biological activities.
Methanolic extracts of Senna auriculata and Lawsonia inermis showed concentration-dependent antifungal activity against Candida albicans by disc diffusion assay. The observed antifungal activity may be due to the presence of these phytoconstituents such as flavonoids, tannins and phenolic compounds which are known to inhibit fungal enzymes, disrupt cell membrane integrity and interfere with fungal growth and metabolism (Clinical and Laboratory Standards Institute., 2008).
Lawsonia inermis showed a marginally greater zone of inhibition at the highest concentration tested, but the difference between the two extracts was relatively small and suggestive of similar antifungal efficacy under the experimental conditions employed. The FTIR analysis also confirmed the presence of functional groups associated with phenolic compounds, aromatic rings, alcohols, carbonyl compounds and amines which have been reported to contribute to antimicrobial activity (Doss, 2009).
In contrast to earlier research that examined these medicinal plants individually, the current study offers a direct comparative evaluation of the methanolic extracts of Senna auriculata and Lawsonia inermis sourced from the same geographical area and subjected to analogous extraction and laboratory conditions (Manoj et al.,, 2024). The phytochemical analysis, FTIR characterization, and biological assessment under controlled experimental conditions provide valuable initial insights into their relative antifungal efficacy and help identify the more promising extract for future phytopharmaceutical development. These findings also establish a scientific foundation for the isolation and characterization of bioactive antifungal agents.
Consistent with the previous reports, the present study demonstrated the antifungal potential of Lawsonia inermis, particularly against Candida species and dermatophytes, which has led to the development of several herbal formulations, including nail lacquers for the management of onychomycosis caused by Trichophyton rubrum and Trichophyton interdigitale. Likewise, studies have also reported that Senna auriculata possesses antimicrobial activity attributable to its rich phytochemical composition (Bello, 2020 and Balasankar et al., 2013). The comparable antifungal activity observed in the present study further supports its potential as a medicinal plant with antifungal properties. Future studies should evaluate these extracts against multiple clinically relevant fungal species, including other Candida species and filamentous fungi, to determine their broader antifungal spectrum.
The MIC and MFC values further confirmed the disc diffusion results. Lawsonia inermis showed lower MIC and MFC values than Senna auriculata, indicating relatively higher antifungal activity. The MFC/MIC ratio of 2 for both extracts indicates that the extracts are fungicidal under the experimental conditions.
In the present study, Senna auriculata demonstrated antifungal activity comparable to that of Lawsonia inermis, suggesting that both plants may serve as promising, cost-effective sources of natural antifungal agents. However, this study has some limitations such as evaluation against a single fungal species and lack of in vivo validation. MIC and MFC values were determined but further studies such as isolation of active phytoconstituents, elucidation of antifungal mechanisms, toxicity evaluation and in vivo efficacy studies are required before clinical application.
CONCLUSION
The study concludes that Lawsonia inermis and Senna auriculata both have strong antifungal activity against Candida albicans. Both plants showed similar antifungal potential, however Lawsonia inermis exhibited a higher activity at higher concentrations. Together, the phytochemical, FTIR, disc diffusion and MIC/MFC results support their potential as sources of bioactive compounds for the development of new antifungal agents. Further studies involving isolation of active compounds, elucidation of antifungal mechanisms and evaluation of toxicity and in vivo evaluation are recommended before clinical application.