Abstract
Background:
The ethanolic extract of Myrtus communis Linn. leaves was assayed in vitro as a growth inhibitor against opportunistic fungi such as Candida and Aspergillus species. Myrtus communis Linn. (Family, Myrtaceae) is an aromatic evergreen shrub or small tree. It is native to the Mediterranean region.Objectives:
This study aimed to assess antifungal activity (in vitro) of the ethanolic extracts of Myrtus communis leaves as a growth inhibitor against 24 clinical isolates of Candida, including C. albicans, C. glabrata, and C. tropicalis also three species of Aspergillus, including A. niger, A. flavus, and A. terreus.Materials and Methods:
The ethanolic extract of myrtle leaves was prepared by maceration method and minimal inhibitory concentration (MIC) of Myrtus communis leaves extract was determined by agar-well diffusion technique. Amphotericin B and clotrimazole were used as the positive control in this assay.Results:
The minimal inhibitory concentration (MICs) values of Myrtus communis leaves extract ranged 0.625-5.0 µg/µL and 5-40 µg/µL against tested Candida spp. and Aspergillus spp., respectively.Conclusions:
Results revealed that the ethanolic extract of Myrtus communis leaves have antifungal potency against both pathogenic tested fungi, and it can be used as a natural antifungal agent.Keywords
1. Background
According to the World Health Organization (WHO) report, more than 80% of the world's population use traditional medicine for treatment (1). Herbal medicines have been used in Asia for a long time(1). Evaluation of medicinal plants for antimicrobial activity is important for finding new therapeutic compounds. Myrtus communis Linn. (Family, Myrtaceae) is an aromatic evergreen shrub or small tree, with 1.8-2.4 m in height. This plant has small foliage and deep fissured bark. It is native to Northern Africa, Southern Europe, and Western Asia and widespread in the Mediterranean region (2). Myrtus is a Greek name for myrtle, and communis means a plant, which grows in groups (3).
This study was carried out to find a replacement agent for amphotericin B to be used in treatment of mycotic infections. Amphotericin B has been used for the treatment of fungal infections since the 1950s. It has side effects such as hepatotoxicity and nephrotoxicity and often accompanied by azoles (4). Aspergillus species are filamentous and commonly isolated from environment. Some Aspergillus species such as A. flavus cause food contamination (5) and the source of opportunistic infections in human such as aspergillosis(6). Also, Candida albicans is responsible for opportunistic infections in oral cavity, digestive tract and vaginal cavity (7). Candida species are yeast and the most common cause of fungal opportunistic infections.
Myrtus communis (myrtle) has been recognized in Iran and has several therapeutic properties such as antioxidant (8, 9), antimicrobial (10, 11), anti-hyperglycemic (12), and analgesic effects (13). The antimicrobial activity of myrtle oil was known against Escherichia coli, Staphylococcus aureus and Candida albicans by Yadegarinia et al. (8). Also, antibacterial property of this plant was reported by Degtyareva et al. (14). Gunduz et al. reported that myrtle oil possess antibacterial action against Salmonella typhimorium (15), Lactobacillus spp., Yersinia enterocolitica (16, 17) and Helicobacter pylori (18). Weak antifungal activity of myrtle oil against Fusariumsolani, Rhizoctonia solani, and Colletotrichum linelemuthianum was reported (10). Previous studies have revealed the chemical composition of myrtle oil from leaves (8, 19-22).
2. Objectives
The present investigation aimed to assess the antifungal efficacy of the ethanolic extract of Myrtus communis.
3. Materials and Methods
3.1. Plant Material
Leaves of Myrtus communis L. were obtained from Agricultural and Natural Resources Center, Ahvaz, Iran. They were dried in shade, powdered in miller, and stored in an airtight bottle for future work.
3.2. Plant Extraction
Ten grams of plant powder were soaked in 100 mL of 80% ethanol and kept on a rotary shaker for 3 × 24 hours at room temperature. Then crude ethanol extract was filtered using Whatman filter paper No.1, and the supernatant of extract was evaporated to dryness at room temperature. Dried extract was stored in sterile bottles and kept at -20°C for assay.
3.3. Fungal Isolates and Fungal Suspensions Preparation
Twenty-one Candida isolates, including 9 C. albicans, 6 C. glabrata, and 6 C. tropicalis from oral cavity and also one environmental isolate from each of A. niger, A. flavus and A. terreus were tested in this study. The fungi from stock were subcultured on Sabauraud dextrose agar (SDA; Merck, Germany). A loopful of spore from the stock cultures was added to the test tubes of Sabauraud dextrose broth (SDB) and grown overnight on a rotary shaker at room temperature. Then broth cultures were centrifuged at 12000 rpm for 10 minutes, and then the supernatant was discarded. The fungal cells were pelleted by centrifugation washed with phosphate-buffered saline (pH 7.5) three times and fungal suspensions adjusted to 108 colony-forming units (CFU/mL) to obtain the turbidity of 0.5 McFarland by the same buffer (23)
3.4. Positive and Negative Controls
The commercial antifungal drugs such as clotrimazole and amphotericin B (4 µg/µL) were used as positive control and absolute dimethyl sulfoxide (DMSO) was used as the negative control.
3.5. Determination of minimum inhibitory concentration (MIC)
Aliquots of 1000 mg dried extract were dissolved in 5 mL absolute of DMSO for obtaining concentration of 200 µg/µL as stock solution. Serial dilutions of plant extracts were prepared in a concentration range of 0.156-20 µg/µL according to the previous study (24). The ethanolic extracts of Myrtus communis leaves were assayed for antifungal activity using an agar well diffusion method (25). One hundred microliters inoculum (108 CFU/mL) of each tested Candida and Apergillus species was uniformly spread by sterile bent glass rod onto SDA medium. The plates were allowed to dry for 5-10 minutes and punched 5 well (7 mm diameter) onto SDA agar medium by using a sterile borer. Subsequently, 100 µL of different concentrations of the ethanolic extracts of Myrtus communis leaves were poured into the wells of agar plates. The minimum inhibition concentration (MIC) value was defined as the lowest extract concentration that inhibits the growth at 35°C for 24 hours.
4. Results
Tables 1 and 2 present data for the MIC assay of the tested plant. The strongest activity was shown against C. glabrata with the MIC values of 0.625-5.0 µg/µL and the range of 15-30 mm inhibition zones while the highest concentration showed a weak inhibitory effect against Aspergillus spp. with the MIC values of 5.0-40 µg/µL. In this research, clotrimazole and amphotericin B were used as positive controls. MIC value of clotrimazole was 0.25 µg/µL and its inhibition zone against C. albicans was 18-25 mm. Also, MIC value of amphotericin B was 0.125 µg/µL, and its inhibition zone against Aspergillus niger was 17-22 mm as shown in Tables 1 and 2.
The MIC values in (µg/µL) of the Ethanolic Extract of Myrtus Communis Leaf Against candida spp. and Aspergillus spp. Isolatesa,b,c
| A. terrus (No.1) | A. flavus (No.1) | A. niger (No.1) | C. tropicalis (No.6) | C. glabrata (No.6) | C. albicans (No.9) | |
|---|---|---|---|---|---|---|
| MIC values, µg/µL | 20-40 | 20-40 | 5.0-40 | 1.25-5.0 | 0.625-5.0 | 2.5-20 |
| Positive controls, µg/µL | ||||||
| Clotrimazole | - | - | - | - | - | 0.25 |
| Amphotericin B | - | - | 0.125 | - | - |
The inhibition zone (mm) of Myrtus communis leaf extract against Candida spp. and Aspergillus spp. Isolatesa
| A. terrus (No.1) | A. flavus (No.1) | A. niger (No.1) | C. tropicalis (No.6) | C. glabrata (No.6) | C. albicans (No.9) | |
|---|---|---|---|---|---|---|
| MIC values, mm | 13-21 | 0-20 | 0-11 | 10-25 | 15-30 | 7-23 |
| Positive controls, mm | ||||||
| Clotrimazole | - | - | - | - | - | 18-25 |
| Amphotericin B | - | - | 17-22 | - | - |
5. Discussion
In the present study, the antifungal efficacy of Myrtus communis was assessed against clinical isolates of Candida spp. and Aspergillus spp. Among the tested fungi, the strongest activity belonged to C. glabrata with MIC values of 0.625-5.0 µg/µL .Similar results were reported by other researchers (26). For example, Mahboubi and Ghazian Bidgoli reported that the essential oil of Myrtus communis had significant activity against C. albicans (P < 0.01) (26). In the present study, MIC value of clotrimazole was 0.25 µg/µL for C. albicans. Also, the MIC value of amphotericin B was 0.125 µg/µL against A. niger. Similar finding reported by Aali et al. that methanolic extract of Myrtus communis extract was greater than that of clotrimazole (P < 0.001) (27). Martinetz et al. reported the leaf extract of Myrtus communis was effective on Fusarium spp. while, it was ineffective on Penicillium (28). While, in another study, Ouelhadj et al. reported the oil of Myrtus communis was most active against Penicillium spp. and Aspergillus niger (59.5 mm and 46 mm), respectively with the lowest MIC value for both pathogens (125 μL/mL) (29).
Also, previous studies revealed that the essential oil of Myrtus communis L. was tested in vitro against phytopathogenic fungi such as Rhizoctonia solani, Fusariumsolani and Colletotrichum lindemuthianum. The essential oil of M. communis inhibited the growth of fungi by 60% in the concentration of 1600 ppm and microscopic observation revealed that the essential oil of M. communis caused morphological alterations of hyphae of all fungi (10). Antifungal activity of the methanolic extract of Myrtus communis leaves against dematiaceous such as Alternaria spp. was reported by Sacchetti et al. (30). Ameziane et al. revealed antifungal activity of the leaf extract of Myrtus communis against Geotrichum candidum (31).Furthermore, previous studies revealed the essential oil of Myrtus communis L. was tested in vitro activity against phytopathogenic fungi such as Rhizoctonia solani, Fusariumsolani and Colletotrichum lindemuthianum.
Shahidi Bonjar et al. reported that the essential oil of Myrtus communis inhibited the growth of bacteria such as Staphylococcus aureus; Escherichia coli; Klebsiella sp. and fungi such as Candida albicans but, was not effective against Saccharomyces cerevisiae (32). The anti-Candida activity of the leaf extract of Myrtus communis was confirmed (33). Miller et al. reported anti-fungal activity of this plant against Cladosporrium spp. (34). The inhibition efficacy of the leaf extract of Myrtus communis in the concentrations of 1%, 10% and 15% against Salmonella typhimorium, Staphylococcus aureus, Candida rugosa and Aspergillusniger (35). Other studies showed that this plant has the most efficacy on the mosquito Culex pipiens (36) and the plant was effective at pH 6.8 against Trichomonas vaginalis but, it was ineffective at pH 6.
In conclusion, the MIC values showed that the tested plant extract besides its antimicrobial effects was quite active against fungi at tested concentrations. Generally, finding alternative drugs derived from natural agents (herbal sources) is important in the future, because they are safer and cheaper than commercial drugs with respect to their antifungal and antibacterial therapy.
Acknowledgements
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