Abstract

Fungal infections are increasingly recognized as an emerging threat to public health. They are treated by antifungal drugs; however fungal resistance continues to increase and complicate patient management, despite the introduction of new antifungal drugs. Antifungal activities of crude extracts of seven plant species were evaluated against ten species of fungi causing cutaneous mycoses. The antifungal susceptibility testing of five commercial antifungal drugs against the tested fungi were also investigated. The plant extract showing the strongest activity was submitted to two-fold dilution broth method to determine the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). All tested fungal species, except C. albicans, were susceptible to nystatin with inhibition zone diameters in the range of 20−28 mm. All fungal species were found to be resistant for fluconazole. The aqueous extract of A. nilotica showed a greater antifungal activity against all tested fungi, with inhibition zone diameters in the range of 13−24 mm. The lowest MIC value of A. nilotica was 0.62 mg/ml against all tested fungi, except M. canis, E. floccosum, and C. albicans, which showed MIC values of 1.25 mg/ml. Three plant species, namely Cissus rotundifolia, Psiadia arabica, and Pulicaria jaubertii were inactive against all tested fungi. In conclusion, A. nilotica had the most potent antifungal activity against the tested fungi. Therefore, A. nilotica might be potentially valuable as a source of natural antifungal agents.

1. Introduction

Infectious diseases represent a major health problem and considered as one of the main causes of morbidity and mortality in the world ¹. Fungal infections (mycoses) are increasingly recognized as an emerging threat to public health ². Cutaneous mycoses are a group of fungal infections, which can affect immunocompromised and immunocompetent individuals, resulting in a wide spectrum of clinical manifestations that vary from mild to severe ^{3, 4}. The prevalence rate of cutaneous mycoses has been reported to be 20-25% of the world's population, and vary with respect to occupational groups, gender, age group, etc. ^{4, 5}. The treatment of these diseases is expensive and costs approximately 500 million dollars annually in the USA ⁶. Therefore, these infections are considered as a major burden for patients worldwide. This burden is the greatest in resource-poor countries and the tropical regions ⁷.

Clinically, there are two types of cutaneous mycoses: dermatophytoses (tineas or ringworms) and cutaneous candidiasis. Dermatophytoses affect individuals worldwide, but their incidence is higher in tropical countries, because of high temperatures and humidity. They are classified according to the site of infection. Tinea capitis (infection of the scalp) is more common in children, while other tineas are more common in postpubertal individuals. Dermatophytoses are mainly caused by filamentous fungi called dermatophytes, namely species of the genera Trichophyton, Microsporum, and Epidermophyton that can invade and feed on keratinized tissues, such as skin, hairs, and nails ^{5, 7}. Cutaneous candidiasis is also a common skin disease that affect all ages and caused by several species of the genus Candida, but Candida albicans is the most common cause. Cutaneous candidiasis may be a primary infection or a secondary infection to other skin diseases, such as atopic dermatitis, psoriasis, or existing diaper dermatitis ⁸. The most frequent clinical forms of cutaneous candidiasis include intertrigo, cheilitis, diaper dermatitis, and interdigital candidiasis ⁹. In general, candidiasis is the earliest infection to manifest in immunocompromised patients, and both primary drug and azole-resistance have been reported ^{10, 11, 12}.

Generally, human pathogenic fungi are controlled by synthetic antifungal drugs; however, a few antifungal drugs are available and licensed for use in human being treatment ¹³. The treatment of fungal infections has lagged behind bacterial chemotherapy and fewer antifungal than antibacterial drugs are available ¹⁴. The use of antifungal drugs is limited due to their unwanted side effects, particularly systemic antifungal drugs, and is increasingly restricted by the ability of pathogenic fungi to develop resistance against most of the antifungal drugs ^{15, 16}. Antifungal resistance continues to grow and evolve and complicate patient management, despite the introduction of new antifungal agents ¹⁷. The use of amphotericin B, considered as a good standard antifungal, is limited because of its nephrotoxicity and infusion-related problems ¹⁸.

Medicinal plants are still major sources of new active molecules for a wide variety of human diseases for thousands of years all over the world. The antifungal activity of plant extracts against dermatophytes and Candida spp. has been previously reported ^{19, 20, 21, 22, 23, 24}. In developing countries, particularly in Yemen, a large number of people still rely on folk medicine to treat microbial infections. The objective of this study is to evaluate the antifungal activity of ethanolic and aqueous crude extracts of seven selected plants against some species of dermatophytes and C. albicans, causing cutaneous mycoses. The choice of plants is based on traditional usage and information provided by elders.

2. Materials and Methods

Seven fresh plant species were collected from Ibb governorate, southwest of Yemen, in October-November 2020. The voucher samples were authenticated by Assistant Prof. Dr. Esam Aqlan, Department of Biology, Faculty of Science, Ibb University, Yemen. The plants with their common names and families are listed in Table 1. Once plants were harvested, they were cleaned, shade dried at room temperature, and cut into small pieces before grinding in a manual mill. The powdered plant material was stored in an airtight container prior to extraction.

According to Harborne ²⁵ with some modifications, the powdered plant sample (20 g) was macerated with 200 ml 80% ethanol at room temperature for 48 h accompanied by constant, continuous shaking. The mixture was filtered through Whatman filter paper (No. 1), and then dried with an evaporator. The crude extract was weighed and preserved at −20°C until use.

Based on the method reported by Kumari et al. ²⁶ with some modifications, the powdered plant material (20 g) was extracted sequentially with 100 ml of hot deionized water. The mixture obtained was filtered to obtain the extract, which dried and preserved as the same method mentioned above.

The tested fungi, namely Trichophyton rubrum AUMC 5469, T. mentagrophytes (AUMC 5504), T. violaceum (AUMC 5459), Microsporum canis (AUMC 5490), Epidermophyton floccosum (AUMC 5496), Candida albicans (AUMC 1299), C. parapsilosis (AUMC 8068), C. tropicalis (AUMC 4917), C. glabrata (AUMC 8175), and C. krusei (AUMC 3958) were obtained from Assiut University Mycological Centre (AUMC), Assiut, Egypt. The fungi were maintained on Sabouraud Dextrose Agar (SDA, Oxoid) slants at 4°C, and sub-cultured monthly on the same medium at 28°C until the assays were carried out.

Laboratory antifungal susceptibility testing of five commercial antifungals against the tested fungi were investigated using disc diffusion technique following the guidelines of CLSI ²⁷ for dermatophytes and CLSI ²⁸ for yeasts, with some minor modifications. The fungal inocula were prepared and adjusted spectrophotometrically to obtain ~10⁴ spore/ml for dermatophytes and ~10³ cell/ml for Candida spp. The inocula were evenly spread onto the surface of SDA with a sterile cotton swab. Commercially available discs preloaded with amphotericin B, fluconazole, itraconazole, miconazole, and nystatin were dispensed onto the surface of agar plates. All plates were incubated at 28°C for up to three weeks for dermatophytes and two days for Candida spp. Following incubation, the resultant inhibition zones were measured in millimeters.

Evaluation of antifungal activities of plant extracts was accomplished by using the agar well-diffusion method on SDA ²⁹. 2 g of plant extract was dissolved in 5 ml of the solvent used for the extraction to give 400 mg/ml as stock. The fungal inocula were prepared as mentioned above. The prepared inocula were spread on SDA. Thereafter, four holes of 6 mm in diameter were made on the media plate using a sterile cork borer and agar discs were removed. Using automatic microliter pipette, holes were aseptically filled with 100 μl of 100 mg/ml of plant extract and allowed to diffuse at room temperature for 2 h. The antifungal drug amphotericin B was used as a positive control. All plates were incubated at 28°C for up to three weeks for dermatophytes and for two days for Candida spp ³⁰. Antifungal activity was assessed by the appearance of inhibition zone around the hole, without fungal growth.

The plant extracts, which showed the best results in the preliminary assays, were chosen for further evaluation of the minimal inhibitory concentration (MIC) using two-fold dilution broth method ³¹. An aliquot of 1 ml of plant extract was added to test tubes containing 1 ml of sterile Sabouraud Dextrose Broth (SDB) to prepare 62.5 to 2000 μg/ml. The tubes were then inoculated with 50 μl of tested dermatophytes (~10⁴ spore/ml) and Candida spp. (~10³ cell/ml), and incubated at 28°C for up to three weeks for dermatophytes and two days for Candida spp. Un-inoculated tubes, containing SDB, ethanol, and fungal inoculum, and those containing SDB with no extract, and fungal inoculum were used as positive and negative controls, respectively. The MIC values were determined as the antifungal concentrations, where there were no growths, by comparison with the control (fungi grown without extract).

The MFC was determined by sub-culturing the test dilution (used in MIC) onto a fresh medium of SDA and incubated further at 28°C for up to three weeks for dermatophytes and two days for Candida spp. The lowest concentration of plant extract that completely killed the fungus was recorded as MFC.

3. Results

In this study, all tested dermatophytes and Candida spp. were subjected to antifungal susceptibility testing using disc diffusion technique for amphotericin B, fluconazole, itraconazole, miconazole, and nystatin. The results presented in Table 2 revealed that all tested fungi, except C. albicans, were susceptible to nystatin with inhibition zone diameters in the range of 20−28 mm, and to some extent to miconazole and amphotericin B. On the other hand, all fungi were found to be resistant for fluconazole and to some extent to itraconazole. Only T. mentagrophytes and M. canis were sensitive to itraconazole.

Ethanolic and aqueous extracts of selected seven plants were screened in vitro for their antifungal activity against ten fungal species of dermatophytes and Candida spp. and the results were presented in Table 3. A noteworthy observation was the antifungal activity of only hot aqueous extract of A. nilotica among several plant extracts tested against all tested fungi, with inhibition zone diameters in the range of 13−24 mm. In addition, C. tropicalis and C. parapsilosis showed susceptibility for ethanolic extract of Buxus dioica, with inhibition zone diameters of 13 and 15 mm, respectively. In addition, ethanolic extract of Psidium guajava showed activity against four yeast species, namely C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis, with inhibition zone diameters of 13, 16, 14, and 20 mm, respectively. Also, only C. albicans and C. tropicalis showed susceptibility for only ethanolic extract of Salvadora persica. Both ethanolic and aqueous extracts of Cissus rotundifolia, Psiadia arabica, and Pulicaria jaubertii did not show antifungal activity against all tested fungi.

The antifungal activities of A. nilotica in terms of MIC and MFC were carried out and the results were presented in Table 4. The MIC values of the aqueous extracts of A. nilotica were found to be 0.62 mg/ml against T. mentagrophytes, T. rubrum, T. violaceum, C. tropicalis, C. glabrata, C. krusei, and C. parapsilosis, while 1.25 mg/ml was the MIC value against M. canis, E. floccosum, and C. albicans. Considering that in this study only crude extracts were applied, extracts with MIC values of 1.25 mg/ml or below against any of the tested fungi, were considered highly active. The MFC of A. nilotica was within two-twofold dilution of the MIC against all tested fungi.

4. Discussion

To eradicate dermatophytes and other fungal pathogens, the in vitro antifungal susceptibility test is very important ³². Antifungal susceptibility testing for fungi against antifungal drugs is a dynamic field in medical mycology to choose antifungal drugs with respect to their antifungal profiles for treating the mycoses, and also when used as a reference in searching of new antifungal agents from natural sources, such as medicinal plants.

In the antifungal susceptibility test, the results revealed that all tested fungi, except C. albicans, were susceptible to nystatin and to some extent to miconazole and amphotericin B. On the other hand, all fungi were found to be resistant for fluconazole and to some extent to itraconazole. Farrag et al. ³³ also found that nystatin was the most effective antifungal drug against a panel of filamentous fungi isolated from farm animals. There are many studies indicating that dermatophytes ^{13, 34, 35} and Candida spp. ^{11, 12, 36} showed resistance against fluconazole. Saranya et al. ¹¹ screened antifungal susceptibility for 26 clinical isolates of C. albicans against six antifungal drugs, including nystatin. The isolates showed complete resistance towards itraconazole (100%), clotrimazole (100%), nystatin (92.31%), fluconazole (80.77%), and ketoconazole (65.38%), whereas they showed highly sensitivity only to amphotericin B (88.46%). The early 1990s was the start of a drastic increase in resistance among fungal clinical isolates ¹⁶. The resistance of fungi against antifungal agents may be primary (intrinsic) or secondary (acquired). Primary resistance was found among certain fungi without prior exposure to the antifungal drug, while secondary resistance was developed among previously susceptible strains after exposure to the antifungal drug and was usually dependent on altered gene expression ³⁷. For example, the resistance developed by Candida spp. against fluconazole may be primary as in the C. krusei ³⁸ or secondary as in the C. albicans ³⁹. There are four major mechanisms to counteract the antifungal (fungicidal/fungistatic) effects of azoles, all of which rely on altered gene expression. These mechanisms include: (i) decreased azole concentration, (ii) target site alteration, (iii) up-regulation of target enzyme, and (iv) development of bypass pathways ³⁷.

Extracts from plant species have long been used for treatment of various diseases, including skin diseases, and natural products may tend to have less harmful side effects than corresponding synthetic drugs ⁴⁰. In this study, hot aqueous extract of A. nilotica showed an interesting antifungal activity against all tested fungi, but yeasts were more susceptible than dermatophytes. To the best of our knowledge, this is the first time that the antifungal activity of A. nilotica is reported against dermatophytes. This plant species is one of the most popular plant remedies in Yemen, and it is used in traditional medicine for skin infections and other diseases. The activity of this plant could be attributed to the presence of some bioactive components in the extract. Aqueous extract of A. nilotica bark was reported to contain tannins, alkaloids, saponins, phenol, steroids, and glycosides ^{41, 42}. The plant also contains a chemical compound called methyl gallate ⁴³, which showed antimicrobial activity against bacteria ^{44, 45} and fungi ⁴⁶. Tannins are water-soluble polyphenols that are present in nearly all plants. They have been known to display different biological activities including antimicrobial activity ^{47, 48}. Tannins can across the cell membrane, because they can precipitate proteins. They may also be able to complex with metabolic ions, leading to the decrease of essential ions available to the metabolism of microorganisms ⁴⁹. Plant-derived alkaloids are important bioactive substances, and their antimicrobial activity have been reported ^{50, 51}. The mechanism of action of aromatic planar quaternary alkaloids was attributed to their ability to intercalate with microbial DNA, resulting in impaired cell division and cell death ⁵².

Antifungal activity of P. guajava shown in the present study was consistent with the finding of Morais-Braga et al. ⁵³, where hydro-ethanolic extract of this plant revealed inhibition growth of C. albicans and C. tropicalis. Buxus dioica (black henna), also called Katam, is a plant that grows in Yemen. The antimicrobial activity of Yemeni B. dioica was not studied. This is the first report indicating the antifungal activity of B. dioica. The leaves of this plant have an ash grey dye and traditionally mixed with Henna powder (Lawsonia inermis) to create a light brunette hair color. According to the results reported in this study, B. dioica may help significantly in the treatment of fungal infections caused by C. albicans and C. parapsilosis.

Previous reports demonstrated that many medicinal plants had antifungal activities against dermatophytes and Candida spp. Screening of the antifungal activity of 56 plant species from Tanzania against yeasts was carried out by Hamza et al. ⁵⁴. They found that the strong antifungal activity was exhibited by extracts of ten plants including A. nilotica and A. robusta. The most susceptible yeasts were C. neoformans, followed by C. krusei, C. tropicalis, and C. parapsilosis, but the least susceptible were C. albicans and C. glabrata. Antifungal activity of other plant extracts against some dermatophytes and Candida spp. was also reported by other researchers. Souza et al. ⁵⁵ showed that Hyptis ovalifolia extract inhibited 25 out of the 30 dermatophytes at a concentration of 250 μg/ml, and the strains of T. rubrum were the most susceptible. In another study conducted in Mexico, Webster et al. ⁵⁶ demonstrated the activity of fourteen plants for their antifungal activity against the dermatophytes (T. mentagrophytes, T. rubrum, T. tonsurans, Microsporum canis, and E. floccosum) and the yeasts, including C. albicans, C. krusei, C. parapsilosis, C. tropicalis, C. glabrata, and C. lusitaniae. Three plants, namely Fragaria virginiana, Epilobium angustifolium, and Potentilla simplex showed strong antifungal potential overall. Alnus viridis, Betula alleghaniensis, and Solidago gigantea also demonstrated a significant degree of activity against many of the yeast isolates.

In the present study, the activity of A. nilotica against dermatophytes, particularly T. rubrum has particular interest, because this fungus is the most common species of dermatophytes, and represents about 80 to 93% of all chronic and recurrent infections produced by dermatophytes ⁵⁷. In addition, this fungus also shows resistance to the antifungal drugs used for the treatment of dermatomycoses ⁵⁸. The results of this study suggested the possibility of using the extract of A. nilotica as starting point for the finding of new antifungal agents that selectively inhibit the most common etiological agent in cutaneous mycoses. The MFC of A. nilotica was within two-twofold dilution of the MIC against all tested fungi, suggesting that the extract of this plant was fungistatic at lower concentration, but fungicidal at higher concentration. Although the mechanism of action was not elucidated, the complexation with ergosterol in the cell membrane of the fungal cell may be occurred.

5. Conclusion

The development of resistance in common pathogenic fungi against available antifungal drugs demonstrates the urgent importance of identifying novel natural antifungal agents. The present study showed that the aqueous extract of A. nilotica had the most potent antifungal activity against the tested fungal pathogens, suggesting the presence of active compounds in A. nilotica stem that could be a potential source of natural antifungal agents, and this may justify the traditional use of this plant in the treatment of skin diseases in Yemen. This will give support to the ethno-pharmacological use of this plant and makes it interesting for further studies.

Acknowledgements

The authors would like to thank Aljazeera University, Ibb, Yemen for partial financial support for this study. The authors would also like to thank Dr. Esam Aqlan, Department of Biology, Faculty of Science, Ibb University, Yemen for helping in plants identification. Furthermore, we would like to thank the Assiut University Mycological Centre for the significant reduction in the price of the fungal strains to be tested.

References