BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
URL: http://ijmm.ir/article-1-1426-en.html
2- Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran
3- Student Research Committee, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4- Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran , ensiehlotfali@yahoo.com
5- Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran
Fungal infections are among the most prevalent infections affecting approximately a quarter of the world’s population, which is observing an increasing trend since the number of immunocompromised patients is rising due to the increased number of patients receiving immunosuppressive drugs in such conditions as cancers and HIV infection (1, 2). Dermatophytosis, also known as tinea or ringworm, is a well-known superficial fungal disease, mostly caused by Trichophyton species such as Trichophyton menta-grophytes and Trichophyton rubrum (3). Trichophyton mentagrophytes are divided into two distinct strains, namely anthropophilic and zoophilic (3).
Various synthetic components with different structural properties are available adopted to treat dermatophytosis. In this regard, today, terbinafine )TRB( and azoles are used as the main treatments of dermatophytosis (4). However, the emergence of TRB and azole-resistant Trichophyton strains showed that these strains should be included in searching for new inhibitory agents to treat drug-resistant organisms (5-7).
Plants are considered the main source of secondary metabolites showing degrees of antifungal activity such as flavonoids, phenols, saponins, and terpenoids (8).
Prosopis farcta (Banks & Sol.) J.F.Macbr. from the Leguminosae family, commonly known as mesquite, is a native plant of Asia, Eastern Europe, and Northern Africa (mostly distributed from India to Iran). Similar to other species of genus Prosopis, this plant is mostly less than 1 m (usually 40 cm) tall and well adapted to drought (Figure 1) (9, 10).
Figure 1. Prosopis farcta grown in Yazd province, Iran.
P. farcta has been mainly used as a traditional medical means in the south of Iran, and nowadays, it has attracted attention for ethnobotany and medical purposes, such as antimicrobial activity, anti-tumor, and antioxidant properties (11, 12). To the best of our knowledge, few studies have been performed inves-tigating the biological activities of P. farcta root. The previous studies merely determined the antibacterial, anti-diabetic, and nitrogen fixative activity of root parts (12, 13) and aerial parts (9, 14) of P. farcta.
In a report obtained from the researchers, Afghans living in Dehnow region of Yazd province used the root of this plant as an anti-parasitic and topical antifungal. It is called “Jenjengok” in the Yazdi dialect. The purpose of this study is to assess the antifungal activity of P. farcta extract on T. mentagrophytes strain in vitro and in vivo for the first time.
2.1 Plant Materials and Extraction Procedure
The roots of P. farcta were collected from Dehnow area (Yazd Province, Iran) in July 2018. The plants were identified by Dr. V. Mozaffarian at Iran Agriculture and Natural Resources Research and Education Center, Tehran, Iran. The root parts were cleaned, dried, and grounded to a fine powder. Subsequently, 100 g of each powdered part was immersed in 300 mL methanol: water (4:1 or 80%) (Chem-Lab, Belgium) and held in a shaking incubator (GFL, Germany) at room temperature for 24 h (maceration method). This process was performed three times more, followed by the filtration (Whatman, UK) and evaporation of solvents using a rotary evaporator (Heidolph, Germany). The concentrated extracts were dried in a dry-oven (Dena, Iran) at 40°C to remove the remained solvents, and the yield of extracts was calculated. For further tests, the resulting extracts were kept in sterile containers at 4°C. To prepare the topical solution of 1%, the above-mentioned amount was solved in water. Then it was subjected to the ultra-sound waves for ten minutes. It is worth mentioning that the solution is made under GLP conditions in a reliable laboratory.
2.2. Fungi Isolates and in vitro Anti-dermatophytic Assay of P. farcta
The five archived clinical isolates of terbinafine resistant T. mentagrophytes strains were used in this study. T. mentagrophytes reference strain (PTCC 5054) was obtained from the Iranian Research Organization for Science and Technology.
The strains were cultured on potato dextrose agar (PDA; Merck, Germany). Inoculum suspensions were prepared by covering fresh cultures of T. menta-grophytes with saline solution and tween 80, according to the Clinical and Laboratory Standards Institute guidelines (CLSI, M38-A2) (15). The suspensions were added into a tube containing 2 mL of saline solution, and densities were adjusted at 530 nm. The final density of inoculum was 1×103 to 3 × 103 CFU/mL. The inocula were diluted 1:50 in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, USA).
The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were evaluated with methanol plant extract and ampho-tericin B (AMB; as a positive control).
The first, 100 μL of RPMI 1640 medium was added to a 96-well microplate, and 100 µL extract suspension (64 mg of concentrated extracts in 1 mL distilled water) was added to the first well. The contents were mixed, and 100 µL was transferred to a second well. This serial dilution was repeated through to the ninth well. An aliquot (100 µL) was discarded from the ninth well.
The serial dilution of plant extract was prepared with concentrations ranging from 0.00625-32 μg/mL. Then 100 µL of inoculum was added to wells 1-10, and the contents were mixed. The microplate was incubated at 30°C for 72 h.
50 μL was inoculated onto PDA from the solution without growth in the MIC test regarding MFC. Moreover, the lowest concentration without growth was recorded as the MFC value. The tests were performed in duplicate.
2.3. In vivo Anti-dermatophytic Activity of P. farcta
A suspension of T. mentagrophytes (PTCC 5054) was prepared by washing the surface of the fresh culture tube (on PDA media after 7-10 days at 25°C) with sterile distilled water and tween 80 adjusted to 1×106 conidia/mL (16).
Male Swiss albino mice (Mus musculus) of approximately 5-7 weeks old and 30-40 g weight were used for the present investigation. Mice were immunosuppressed by subcutaneous injection of 500 mg of estradiol valerate 4 days before the infection (17). The hair of the back of each mouse (2 cm2) was shaved, and the skin was slightly scraped by a single-use scalpel. Afterward, 50 μL of the suspension of T. mentagrophytes was inoculated to the surface within the shaved zone and was gently rubbed with the flat part of a sterile blade (17).
The animals were assigned to three groups, including a test group (n=3), a positive control group (n=3) receiving treatment with TRB (the reference antifungal drug), and a negative control group taking only distilled water without infection (n=3). After 21 days of primary infection, when the dermatophytosis appeared, methanol extract of P. farcta root was used as a solution (1%) in the test group. The animals were fed with autoclaved water and food in clean cages. The skin lesions were scored on a scale ranging from 0-3 (no visible lesions to significant crusting and erythema) based on the results of a previous study (18). The effect of the P. farcta solution against T. mentagrophytes strains was examined by shaving the hair and scraping the skin of infected mice (Figure 2).
Figure 2. A: Preparing the animal for the test. B: Dressing the contaminated area with a solution containing the extract of P. farcta.
2.6. Ethical Statement
The ethics approval was obtained from the animal ethics committee of Tehran University of Medical Science, Tehran, Iran (IR.TUMS.VCR.REC.1398.196).
2.7. Statistical Analysis
The values of MIC and MFC were calculated in SPSS 19.0 for windows (SPSS Inc., Chicago, IL, USA) and the differences between the groups were determined using ANOVA. A P-value of ≤ 0.05 was considered significant.
3.1. Yield of Extraction
The extraction yield of P. farcta was obtained as 11.8%.
3.2. In vitro Anti-dermatophytic Activity
The hydro-alcoholic extract revealed a growth inhibition effect against tested isolates (P=0.042). The MIC and MFC of the methanol extract are tabulated in Table 1.
Table 1. Minimum inhibitory concentration and minimum fungicidal concentration of the plant extracts
| Methanol extract | amphotericin B | |||
| Test isolates | MIC* (µg/mL) | MFC* (µg/mL) | MIC (µg/mL) | MFC (µg/mL) |
| T. mentagrophytes (PTCC 5054) | 0.125 | 0. 25 | 0.031 | 0.062 |
| 1 | 0.125 | 0.25 | 0. 25 | 0.25 |
| 2 | 0.031 | 0.062 | 0.062 | 0.125 |
| 3 | 0.031 | 0.062 | 0.125 | 0.25 |
| 4 | 0.062 | 0.125 | 0.125 | 0.25 |
| 5 | 0.031 | 0.031 | 0.125 | 0.25 |
*MIC: Minimum Inhibitory Concentration, MFC: Minimum Fungicidal Concentration, Clinical isolates of Trichophyton mentagrophytes: 1, 2, 3, 4, 5
3.3. In vivo Anti-dermatophytic Activity
It was revealed that P. farcta solution (1%) was a perfect cure compared with terbinafine (P<0.05). The complete cure using the terbinafine occurred on day 21, while using P. farcta solution (1%) reduced this period to 10 days. The hair culture exhibited 100% recovery within 6-15 days; however, the positive control group was grown in the hair culture.
One of the major concerns in modern medicine is the organisms developing resistance to synthetic drugs (7, 19). This issue made conventional medicine a point of interest to search for any evidence allowing it to be applicable for drug-resistant organisms (20). Over time, the application of herbal medicine in treating superficial fungal lesions has received attention among different parts worldwide (21, 22). Secondary metabolites produced by herbal medical species are the main mediators to achieve the desired effect of herbal medicine on the subject (23). The antimicrobial activity of P. farcta, is related to active metabolites in all parts of P. farcta like; flavonoids, saponins, phenols, alkaloids, tannins, resins, and glycosides.
It was reported that the root of P. farcta contained high concentrations of flavonoids, saponins, and phenols and moderate concentrations of other active compounds noted previously (12).
For anti-parasite activity, the fresh root extract was used as a herbal remedy in Iran (24).
The source of variable degree of resistance or sensitivity of fungi against plant extract may be due to the combinations and nature of compounds present in the plant extract (25). On the other hand, the inherent tolerance of the fungi species can play a role in this pattern. The main phytochemicals in P.farcta are alkaloids, tannins, and glycosides. These phytoche-micals have antimicrobial activity (12).
Recent reports have indicated that terbinafine-resistant T. mentagrophytes harbors a mutation in the squalene epoxidase (SQLE) gene, which aroused concern for the spread rate of this resistance throughout the world (26). In 2020, this mutation-mediated resistance was reported in Iran and India and made this organism a target for herbal medicine research (27, 28).
According to the review of previous studies, anti-organism topics were mainly investigated on aerial parts of P. farcta (9, 29). In those with the root parts examined, only the antibacterial properties under-went evaluation (30). To the best of our knowledge, the present study was the first to investigate both in vitro and in vivo antifungal activity of the root part of P. farcta. According to Saad et al., ethyl-acetate extract of aerial parts of P. farcta demonstrated significant antifungal activity against Candida albicans with a 7.3 mm inhibition zone in the filter paper disc method (29). Based on another study, a MIC value of 32 μg/mL against C. albicans was reported for P. farcta ethanol fruit extract. In the mentioned research, the MIC value was estimated at 256 μg/mL for Aspergillus niger (9). The only study investigating the in vitro antifungal activity against dermatophytes was performed by Maoz and Neeman. Accordingly, a MIC of 10 % was reported for aqueous extract of upper parts of P. farcta against Microsporum canis and Trichophyton rubrum (31).
A comparative review of previous studies concerning the antifungal activity of herbs against T. mentagrophytes revealed the potentiality of P. farcta against this fungus species. According to Balakunar et al., the Ocimum sanctum showed antifungal activity against T. mentagrophytes with a MIC of 125±25 μg/mL (32). Essential oils of Thymus serpillum (MIC=0.1 %, area percentage), Origanum vulgare (MIC=0.5 %), and Rosmarinus officinalis (MIC=5 %) were demonstrated to have in vivo anti-derma-tophytic activity on T. mentagrophytes (33). In a study, the antifungal activity of ethyl-acetate extracts of 36 herbs grown in Japan was evaluated against T. mentagrophytes based on an agar diffusion test. The results of the mentioned research showed the best inhibitory diameter for Monarda fistulosa seeds (48 mm), Lavandula x intermedia flowers (36 mm), and Salvia officinalis flowers and leaves (35 mm) (34). Inula helenium and Curcuma longa were demonstrated to have antifungal activity against T. mentagrophytes with an inhibition zone of 23 mm and 14 mm, respectively (35).
Herein, the hydro-alcoholic root extract of P. farcta showed excellent anti-dermatophytic properties compared with AMB in terbinafine resistant clinical isolates. The results of this study confirmed the ethnobotanical uses of this plant in fungal skin infections. This finding can be considered a promising antifungal agent. Furthermore, to reveal the effective compounds in the methanol extract, it is essential to screen the anti-dermatophytic assay.
Thanks to Ms. Maasoumeh Anousheh for collecting the herb.
No financial support exit.
E.S.S and E.L designed the project. E.S.S prepared the extracts and solution. E.L performed an antifungal susceptibility assay. M.F performed the animal assay. K.R and A.Kh wrote the draft of the manuscript. E.S.S, M.F, and E.L revised the manuscript. All of the authors approved the final version of the manuscript.
Conflicts of Interest
The authors declared no conflict of interest.
Received: 2021/08/11 | Accepted: 2021/12/29 | ePublished: 2022/02/10
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
Copyright Policy
Iranian Journal of Medical Microbiology by Farname is licensed under CC BY-NC 4.0
