The antimicrobial activity of Centella asiatica, Wedelia trilobata, and Eucalyptus citriodora oil (EC oil) was evaluated using the agar well diffusion assay (Magaldi et al., 2004) with minor modifications. Briefly, crude extracts of C. asiatica and W. trilobata were dissolved in DMSO to obtain final concentrations of 20, 40, 60, and 80 mg mL⁻¹, while EC oil was prepared at concentrations ranging from 3 to 14% (v/v). A 100 μL aliquot of freshly prepared broth cultures of Malassezia furfur and Staphylococcus epidermidis was spread uniformly on the surface of Dixon’s agar (Himedia, India) and nutrient agar (Himedia, India) plates, respectively. Thereafter, 6 mm-diameter wells (as per the experimental design) were aseptically created with the help of a sterile cork borer. Different concentrations (abovementioned) of the selected herbal extracts and oil (100 μL) were then aseptically added to the wells, and the plates were kept in a laminar flow hood until the plant extracts were completely diffused into the wells. Plates inoculated with M. furfur were incubated at 30 ± 2 °C for 5–7 days to allow visible lawn formation, whereas those inoculated with S. epidermidis were incubated at 28 ± 2 °C for 24 h. The diameter (in mm) of inhibition zones around the well was measured to evaluate the antimicrobial efficacy of herbal leads. All assays were conducted in triplicate, and mean inhibition values were recorded.

The extracts of C. asiatica, W. trilobata, and EC oil were blended in specified ratios (based on primary screening) to prepare formulation 1 (3% C. asiatica, 4% W. trilobata, 8% EC oil), formulation 2 (5% C. asiatica, 3% W. trilobata,10% EC oil), and formulation 3 (5% C. asiatica, 4% W. trilobata,12% EC oil).

The antifungal efficacy of the formulated oil, comprising a blend of the three herbal components, was similarly assessed using the agar well diffusion assay. In addition, different strategic experiments were executed to further examine the efficacy of the different combinations of the formulated oil.

Dandruff-causing microbes, such as M. furfur and S. epidermidis, were separately cultured in Sabouraud Dextrose Broth (SDB) and Nutrient Broth (NB), followed by incubation at 30 ± 2 °C (for M. furfur) and 28 ± 2 °C (for S. epidermidis) under their respective growth conditions. Spores/cells of microbial growth were harvested through centrifugation at 12000 rpm for 10 min, washed twice in saline (0.8%NaCl), and re-suspended to achieve the standardized inoculum densities of 2×10⁵ spores/cells ml⁻¹ (M. furfur) and 1×10⁸ CFU ml⁻¹ (S. epidermidis), respectively.

The treatments were as follows: formulations 1, 2, and 3 (F1, F2, and F3; different permutation combinations of the selected herbal leads), along with two anti-fungal standards (Standard 1: 50 mg ketoconazole; Standard 2; 50 mg fluconazole) and two commercially available herbal anti-dandruff market products (MP1 and MP2) containing plant-derived components such as rosemary, tea tree oil, neem, and other herbal extracts, which are commonly claimed to exhibit anti-dandruff activity, and a control without treatment.

Different parameters of the selected herbal ingredients in the formulated oil were evaluated as per guidelines (International, 2005; WHO, 2007; Medicine and Homoeopathy, 2008). The obtained data are tabulated in Supplementary Tables S1, S2.

A rapid, double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) was applied to confirm the antimicrobial activity of the developed formulation through the determination of cell viability in cell suspensions (Zhu et al., 2023). Treated cells of dandruff-causing organisms were harvested from different treatments and incubated with a mixture of FDA (2 mg/mL) and PI (1 mg/mL). The stained cells were washed aseptically using PBS (pH 7.0). The viability of live and dead cells after the supplementation of the formulated product was visualized using a confocal laser scanning microscope and compared with respective controls and standards (Carl Zeiss LSM 510 META) using FDA (green) and PI (red) filters with excitation and emission at 480/40 nm and 630/30 nm, respectively. Furthermore, the fluorescence intensities of FDA and PI were individually extracted from replicate micrographs using the ZEISS ZEN software integrated with the confocal microscope and quantitatively analyzed to assess live and dead cell populations.

The antagonistic effects of the most effective final formulation against M. furfur cells were visualized using scanning electron microscopy (SEM), following the procedure outlined by Mishra et al. (2018). Treated M. furfur cells (supplemented with formulated herbal oil) were harvested by centrifugation at 10000 rpm for 5 min. Subsequently, the treated cells were fixed with 2.5% (v/v) glutaraldehyde containing 0.1 M sodium cacodylate buffer, followed by the treatment of OsO₄ (1%w/v) for 40 min. Thereafter, dehydration of the treated cells was achieved using increasing concentrations of ethanol (30–100%). The cells were dried using a critical point dryer and coated with gold–palladium using a sputter coater (Q150T ES, Quorum, East Sussex, UK). The treated cells were then visualized using a scanning electron microscope (eSEM-FEI Quanta 250). All experimental procedures were performed in triplicate.

The inhibitory effect of EC oil (at different concentrations) was investigated against dandruff-causing fungal (M. furfur) and bacterial (S. epidermidis) inhabitants using the agar well diffusion assay. The findings revealed that EC oil showed very promising antimicrobial activity against these dandruff-causing pathogens, and the effect gradually increased with concentration (Figures 1A,B). Notably, the highest inhibition zone (37.3 ± 1.8 mm) was observed at 12% concentration and showed a 1.32- and 1.39-fold increase compared to standard 1 (STD1; ketoconazole 50 mg) and standard 2 (STD2; fluconazole 50 mg), respectively (Supplementary Table S5.1). Similarly, a 1.27- and 1.35-fold increase in antimicrobial activity was observed at 10% concentration of EC oil (36 ± 2.08 mm inhibition zone) compared to STD1 and STD2 against M. furfur. In contrast, 8% EC oil displayed the least inhibitory effect against M. furfur. Noticeably, no statistically significant difference was observed in the inhibitory effect of 12 and 10% concentrations of EC oil against M. furfur (Figure 1A; Supplementary Table S5.1). Whereas, 10% concentration of EC oil was depicted maximum inhibitory activity against S. epidermidis (38.7 ± 0.9mm), estimated 2.8 and 2.76-fold increment (Figure 1B; Supplementary Table S5.2). Moreover, 8% EC oil also exhibited a considerable inhibition zone (24 ± 0.9 mm) against S. epidermidis, which was 1.76- and 1.71-fold higher than the respective standards (Supplementary Table S5.2). The antimicrobial activity of C. asiatica and W. trilobata was not as pronounced (Supplementary Table S4). However, these herbs are traditionally known for their various other properties such as wound healing and maintaining the luster and texture of hair strands (Bylka et al., 2013; Arribas-López et al., 2022).

Based on the physiological and physicochemical characteristics, along with the IC₅₀ value of the selected herbs against dandruff-causing pathogens, different permutation combinations (n = 3) were prepared to identify a novel and effective herbal formulation. The permutation combinations of synergistic herbal formulation comprised the pharmaceutically acceptable concentrations of Centella asiatica (3.0–6.0 w/v), Wedelia trilobata (2.0–5.0 w/v), and EC oil (8–12% v/v).

The formulated combinations were tested against major dandruff-causing microbes using the agar well diffusion assay to evaluate their synergistic antimicrobial activity compared to standards and market products (MP1 and MP2). The observations indicated that the formulated combinations exhibited higher inhibitory activity against M. furfur (Table 1) and S. epidermidis (Table 2) compared to the individual herbal ingredients.

As evidenced by the agar well diffusion assay, the developed formulation diminished the growth and proliferation of dandruff-causing microbes. The results showed the lowest yeast cell viability at 10 and 12% concentrations of EC oil. Moreover, a 1.8- and 1.9-fold reduction in the average cell count of M. furfur was recorded in formulation 2 (10% EC oil) and 3 (12% EC oil), respectively, compared to their respective controls (Table 3). Furthermore, the reduction in M. furfur growth observed with formulations 2 and 3 was significantly higher than that with STD1 (1.4- and 1.5-fold), STD2 (1.3- and 1.4-fold), MP1 (1.73- and 1.84-fold), and MP2 (1.68- and 1.80-fold), respectively. Among all developed permutations and combinations, formulation 1 (8% EC oil) exhibited the least inhibitory activity against M. furfur.

Similarly, the reduced average CFU count of S. epidermidis following treatment with formulations 1, 2, and 3 further demonstrates their inhibitory effect. The results, showing the least average CFU count of S. epidermidis in the formulated hair oil treatment, suggest that formulations 2 and 3 were the most effective combination for inhibiting the growth of S. epidermidis (2- and 2.5-fold) compared to the control and recorded 1.76- and 1.97-fold greater reductions than STD2 (Table 3).

The intensity of FDA fluorescence was comparatively low when the cells were treated with the formulated oil. Moreover, the lowest fluorophore uptake (3- and 2.8-fold compared to the control) in the M. furfur cells was detected after treatment with formulations 3 and 2. These reductions were greater than those observed with MP1 (2.46- and 2.28-fold), MP2 (1.92- and 1.78-fold), STD1 (1.67- and 1.54-fold), and STD2 (1.21- and 1.13-fold). Formulation 1 resulted in higher FDA fluorescence (1.3 and 1.2-fold) in the M. furfur cells compared to formulations 3 and 2, although it still showed a 2.26-fold reduction compared to the respective control (Figure 2A).

Treatment with the formulated herbal anti-dandruff hair oil (in different permutation combinations) was most effective against S. epidermidis and recorded 3.59-, 3.78-, and 4.72-fold reductions in the intensity of FDA compared to their respective control (Figure 2B). Among these, treatment with formulations 3 and 2 resulted in the most significant reduction in fluorophore uptake by S. epidermidis cells, compared to MP1 (2.77- and 2.22-fold), MP2 (2.57- and 2.06-fold), STD1 (1.47- and 1.18-fold), and STD2 (2.8- and 2.2-fold), respectively. Although formulation 1 exhibited a 2.1-, 1.95-, 1.1-, and 2.1-fold greater reduction in FDA intensity compared to MP1, MP2, STD1, and STD2, respectively, this reduction was still lower than that observed in other permutations and combinations of the formulated herbal anti-dandruff hair oil (Figure 2B).

C. asiatica, W. trilobata, and EC oil were subjected to physicochemical analysis to ascertain the identity, purity, and strength of the raw material used in the study (Supplementary Tables S1, S2). Data suggest that the quality of the raw material complied with the standards of the Ayurvedic Pharmacopoeia of India.

HPLC profiling confirmed the presence of bioactive markers, namely MS (3.69 μg/mg), AS (1.87 μg/mg), and MA (1.084 μg/mg), with retention times (R_t) of 4.121 min, 5.017 min, and 7.667 min, respectively, in the C. asiatica extract, while AA could not be detected due to its very low content (Supplementary Figure S1). Similarly, WDL (32.6 μg/mg) was detected at 6.273 min in the W. trilobata extract (Supplementary Figure S1). The EC oil was found to be rich in isopulegol (42.26%), followed by citronellol (8.54%), p-Methane-3-one (4.79%), and citronella formate (3.64%). I-Hepta triacontanol was found in the least concentration, i.e., 0.87% (Supplementary Figure S2; Supplementary Table S3).

Molecular docking analysis of EC oil revealed the binding of all phytomolecules at the inhibition site of LDM with different affinities (Supplementary Table S6). The binding at the inhibition site was confirmed by visualizing the overlapping interactions between the co-crystallized ligand and the phytomolecules, which showed hydrophobic contacts with His601, Thr318, Phe236, Leu380, Gly314, Ile139, and Gly310, along with hydrogen bonding to Tyr140 (Figure 3). Among the quantified phytomolecules of EC oil, β-caryophyllene showed the highest docking score (∆G = −8.9 Kcal/Mol), followed by eucalyptol (∆G = −6.8 Kcal/Mol) and isopulegol (∆G = −6.5), compared to respective standards—ketoconazole (∆G = −10.3) and fluconazole (∆G = −8.2).

As per the docking results, β-caryophyllene, eucalyptol, and isopulegol were selected for MD simulation to examine the dynamic behavioral effects of their binding on the target protein. The stability of the protein and the protein–ligand complex during the 5 ns simulation was monitored using the time evolution of the root mean square deviation (RMSD), as shown in Figure 4. The RMSD of all complexes and the unbound protein remained >0.20 nm, indicating the stability of the systems. In the β-caryophyllene–bound complex (A), the final RMSD was slightly lower than that of the protein, suggesting increased rigidity of the protein upon ligand binding. However, in both the eucalyptol (B) and isopulegol complexes (C), no significant deviation in RMSD was observed compared to the unbound protein, indicating no significant alteration in protein stability due to ligand binding. The root mean square fluctuation (RMSF) of the residues was compared to assess the mobility of the residues in the protein and protein–ligand complex systems, as influenced by ligand binding. The RMSF of all the complexes exhibited no significant fluctuation compared to the unbound protein, indicating that ligand binding does not disrupt or restrict the motion of residues. RMSF values in all systems ranged below 0.25 nm throughout the binding pocket (except for the terminals of the protein), suggesting the stability of the active site after ligand binding.

The findings of molecular docking were validated via live/dead cell imaging by confocal microscopy. The localization of live/dead cells under different treatments clearly depicted that formulations 3 and 2 (containing 12 and 10% EC oil) were the most effective combinations compared to market products, standards, and the respective control. Detailed analysis of confocal micrographs showed the highest number of live cells in the untreated microbial population (Figure 5A), whereas treatments with MP1, MP2, STD1, and STD2 resulted in a reduction of live cells. The fewest live cells were observed in the formulated oil treatments (in various permutation combinations), as evidenced by the strongest PI fluorescence under the same conditions. Moreover, the findings of confocal microscopy were corroborated by the average intensity ratios of PI (Figure 5B) and FDA (Figure 5C) fluorescence.

It is noteworthy that formulations 2 and 3 did not show significant variation in terms of antimicrobial activity against dandruff-causing microbes, which is consistent with the findings of previous experiments. Therefore, formulation 2 (containing 10% EC oil) was selected as the best-suited anti-dandruff hair oil.

Scanning electron microscopy (SEM) was performed to evaluate the effect of the final formulation of the anti-dandruff herbal hair oil against M. furfur. The SEM micrographs clearly showed punctured M. furfur cells with rough, disintegrated, and degraded surfaces after exposure to the final formulation (Figure 6). In contrast, untreated control M. furfur cells appeared smooth and intact (Figure 6).