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. 2022 Nov 24:13:1067697.
doi: 10.3389/fphar.2022.1067697. eCollection 2022.

Appraisal of selected ethnomedicinal plants as alternative therapies against onychomycosis: Evaluation of synergy and time-kill kinetics

Syeda Aroosa Mohsin  1 , Shazia Shaukat  2 , Marya Nawaz  1 , Tofeeq Ur-Rehman  1 , Nadeem Irshad  1 , Muhammad Majid  3 , Syed Shams Ul Hassan  4   5 , Simona Bungau  6 , Humaira Fatima  1
Affiliations

Appraisal of selected ethnomedicinal plants as alternative therapies against onychomycosis: Evaluation of synergy and time-kill kinetics

Syeda Aroosa Mohsin et al. Front Pharmacol. .

Abstract

Introduction: This study aims at the biological profiling of Allium sativum, Zingiber officinale, Nigella sativa, Curcuma longa, Mentha piperita, Withania somnifera, Azadirachta indica, and Lawsonia inermis as alternatives against onychomycosis to combat the treatment challenges. Methods: An extract library of aqueous (DW), ethyl acetate (EA), and methanol (M) extracts was subjected to phytochemical and antioxidant colorimetric assays to gauge the ameliorating role of extracts against oxidative stress. RP-HPLC quantified therapeutically significant polyphenols. Antifungal potential (disc diffusion and broth dilution) against filamentous (dermatophytes and non-dermatophytes) and non-filamentous fungi (yeasts; Candida albicans), synergistic interactions (checkerboard method) with terbinafine and amphotericin-B against resistant clinical isolates of dermatophytes (Trichophyton rubrum and Trichophyton tonsurans) and non-dermatophytes (Aspergillus spp., Fusarium dimerum, and Rhizopus arrhizus), time-kill kinetics, and protein estimation (Bradford method) were performed to evaluate the potential of extracts against onychomycosis. Results: The highest total phenolic and flavonoid content along with noteworthy antioxidant capacity, reducing power, and a substantial radical scavenging activity was recorded for the extracts of Z. officinale. Significant polyphenolics quantified by RP-HPLC included rutin (35.71 ± 0.23 µg/mgE), gallic acid (50.17 ± 0.22 µg/mgE), catechin (93.04 ± 0.43 µg/mgE), syringic acid (55.63 ± 0.35 µg/mgE), emodin (246.32 ± 0.44 µg/mgE), luteolin (78.43 ± 0.18 µg/mgE), myricetin (29.44 ± 0.13 µg/mgE), and quercetin (97.45 ± 0.22 µg/mgE). Extracts presented prominent antifungal activity against dermatophytes and non-dermatophytes (MIC-31.25 μg/ml). The checkerboard method showed synergism with 4- and 8-fold reductions in the MICs of A. sativum, Z. officinale, M. piperita, L. inermis, and C. longa extracts and doses of amphotericin-B (Amp-B) and terbinafine (against non-dermatophytes and dermatophytes, respectively). Furthermore, the synergistic therapy showed a time-dependent decrease in fungal growth even after 9 and 12 h of treatment. The inhibition of fungal proteins was also observed to be higher with the treatment of synergistic combinations than with the extracts alone, along with the cell membrane damage caused by terbinafine and amp-B, thus making the resistant fungi incapable of subsisting. Conclusion: The extracts of A. sativum, Z. officinale, M. piperita, L. inermis, and C. longa have proven to be promising alternatives to combat oxidative stress, resistance, and other treatment challenges of onychomycosis.

Keywords: HPLC; antifungal; onychomycosis; resistance; synergistic studies.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Free radical scavenging activity of test extracts. Values (mean ± SD) are the average of the triplicate analysis of each plant extract (n value of 1 × 3). The columns with different superscript (a-g) letters show significantly (p < 0.05) different means. FRSA, free radical scavenging activity; A. S, Allium sativum; Z. O, Zingiber officinale; M. P, Mentha piperita; N. S, Nigella sativa; L. I, Lawsonia inermis; C. L, Curcuma longa; W. S, Withania somnifera; EA, ethyl acetate extract; DW, distilled water extract; M, methanolic extract.
FIGURE 2
FIGURE 2
Graphical display of total antioxidant capacity and total reducing power of selected extracts. Values (mean ± SD) are the average of the triplicate analysis of each plant extract (n value of 1 × 3). The columns with different superscript (a–f) letters show significantly (p < 0.05) different means. A. S, Allium sativum; Z. O, Zingiber officinale; M. P, Mentha piperita; N. S, Nigella sativa; L. I, Lawsonia inermis; C. L, Curcuma longa; W. S, Withania somnifera; EA, ethyl acetate extract; DW, distilled water extract; M, methanolic extract.
FIGURE 3
FIGURE 3
Graphical presentation of total phenolic (TPC) and total flavonoid content (TFC). Values (mean ± SD) are average of triplicate analysis of each plant extract (n value of 1 × 3). The columns with different superscript (a–e) letters show significantly (p < 0.05) different means. A. S, Allium sativum; Z. O, Zingiber officinale; M. P, Mentha piperita; N. S, Nigella sativa; L. I, Lawsonia inermis; C. L, Curcuma longa; W. S, Withania somnifera; EA, ethyl acetate extract; DW, distilled water extract; M, methanolic extract.
FIGURE 4
FIGURE 4
HPLC chromatogram of L. inermis M extract.
FIGURE 5
FIGURE 5
HPLC chromatogram of M. piperita M extract.
FIGURE 6
FIGURE 6
HPLC chromatogram of A. indica EA extract.
FIGURE 7
FIGURE 7
Time-kill curves of selected extracts against dermatophytes. M, methanolic extract; DW, distilled water extract.
FIGURE 8
FIGURE 8
Time-kill curves of selected extracts against dermatophytes. M, methanolic extract; DW, distilled water extract.
FIGURE 9
FIGURE 9
Time-kill curves of selected extracts against non-dermatophytes. EA, ethyl acetate extract; M, methanolic extract; DW, distilled water extract.
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