Chemical composition and antifungal effect of hydroalcoholic extract of Allium tripedale (Tvautv.) against Candida species

Background and Purpose: Treatment of life-threatening fungal infections caused by Candida species has become a major problem. Candida spp. are the most important causative agents of candidiasis. Allium tripedale is a medicinal plant that has been traditionally used to treat infections. In the present study, we aimed to determine the chemical compounds and antimicrobial activity of hydroalcoholic extract of A. tripedale against different species of Candida. Materials and Methods: Phytochemical analysis was performed to identify the possible bioactive components of this extract by using gas chromatography and mass spectroscopy (GC-MS). The hydroalcoholic extract of A. tripedale were collected. Different concentrations of A. tripedale (50, 25, 12.5, and 6.25 mg/ml) were used to evaluate its antifungal activity against Candida species (C. albicans, C. parapsilosis, and C. krusei) using disk diffusion assay. Results: The GC-MS analysis revealed the presence of 40 different phytoconstituents with peak area; the major compounds were tetracosane, hexadecanoic acid, 1-eicosanol, 1,2-dihydro-pyrido[3,2,1-kl]phenothiazin-3-one, 2-hexadecen-1-ol, and 3,7,11,15-tetramethyl. Hydroalcoholic extract showed strong antimicrobial activity (inhibition zone ⩾ 20 mm), moderate antimicrobial activity (inhibition zone < 12-20 mm), and no inhibition (zone < 12 mm). In addition, the hydroalcoholic extract exhibited the highest antimicrobial properties against C. albicans strains. Conclusion: A. tripedale extract had a considerable inhibitory effect against various Candida species, but its highest inhibitory effect was against Candid albicans. Further investigations are required to detect the performance of this plant in the treatment of Candida infection.


Introduction
lants are a great source of useful phytochemicals, which have inhibitory effects against some microorganisms in vitro and are effective in the treatment of various conditions [1]. Generally, 1-10% of plants (out of approximately 250,000-500,000 species) on earth are used by humans [2]. In recent years, there has been a growing global interest in the use of medicinal plants for disease prevention and treatment, especially in Iran [3]. Limited success in the treatment of human diseases, undesirable side effects of chemical drugs, and growing emergence of drug resistance, particularly to antibiotics, have led to increased use of medicinal plants [4].
Medicinal plants are a widespread source of biologically active compounds including alkaloids, tannins, flavonoids, and phenolic compounds. Accordingly, they are of marked significance to the health of individuals and communities and are widely used for disease treatment [2].
A. tripedale belonging to the Liliaceae family, is a wild Allium species native to the Caucasus (North + South), Iraq, Turkey, and Iran. This plant has long and strong stems (50-90 cm in length) and some what unpleasant taste [5,6]. A. tripedale has been extensively used by locals as a spicy vegetable and for the treatment of infections. Given the presence of saponins in the structure of this plant, it is expected to have inhibitory effect against pathogenic fungi [7].
Since the early 1990s, the increase in the number of infections caused by pathogenic and opportunistic fungi has been introduced as the leading cause of P Curr Med Mycol, 2017, 3(1): 6-12 7 mortality among hospitalized patients [8]. In other words, a large number of people are suffering from fungal infections, and these infections are posing a great threat to mankind [9]. In addition, the increased use of antifungal agents has led to the development of resistance to the available drugs. Candida albicans as an opportunistic pathogen plays an important role in the infection and is the most common cause of cutaneous, oral, and systemic diseases in immunodeficiency patients [10]. Although Candida albicans is still the major species isolated from clinical samples in the majority of individuals, it is well known that some other non-albicans Candida spp. such as Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis infections are significantly widespread. Candidiasis associated with this kind of non-albicans Candida spp. pose a clinical challenge because they are resistant to common antifungal agents such as fluconazole and amphotericin B [11,12].
Regarding the increase in the use of antifungal agents and resistance to some types of Candida spp. and the undesirable side effects of chemical drugs, it is essential to explore new sources of treatment, particulary among herbal plants [8,13]. To the best of our knowledge, no has yet explored the antifungal activity of A. tripedale against Candida isolates. The purpose of this study was to evaluate chemical composition and antimicrobial activity of hydroalcoholic extract of A. tripedale against different Candida spp.

Plant collection
Tripedale was collected from the highlands of Shahrekord in southeast of Iran (Isfahan Province). The collected samples were identified in Ahvaz Agricultural and Natural Research Centre (Herbarium No. A151640100AP). Extraction and laboratory examinations were carried out in Ahvaz University of Medical Sciences, Ahvaz, Iran. The aerial parts of the plants were aired indoors at room temperature and then finely powdered using an electric grinder (Busch, MKM6003, Slovenia). It took two days to extract 20 g of plant materials by soxhlet with 120 ml ethanol 80%. The extract was filtered using Whatman qualitative filter paper, Grade 1. The extract was preserved in sterilized airtight bottles at 4ºC, and then to prepare the dried extracts, the solution was placed in a bain-marie at 40ºC for 24 h prior to use [14].

Gas chromatography and mass spectroscopy (GC-MS) analysis
GC-MS analysis of ethanolic extract of the whole A. tripedale was performed on GC 7890A equipped with MS 5975C detector and HP-5ms capillary column (30 × 0.25 m, 0.25 µm; Agilent Co., USA). The initial column temperature was set at 60ºC, then increased from 60ºC to 190ºC (heating rate: 5ºC per minute), from 190ºC to 270ºC for 30 min, and finally kept at 270ºC for approximately 5 min; the total analysis time was about 34 min.

Compound identification
Interpretation of GC-MS was performed via the National Institute Standard and Technology (NIST) database. The spectra of the unknown components were compared with those of the known ones registered in the NIST library. The name, molecular weight, and structure of the components of the test materials were determined.

Preparation of organisms
Standard strains of C. albicans (ATCC 3153), C. parapsilosis (ATCC 2195), and C. krusei (ATCC 573) were obtained from the Department of Mycology, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran. The strains were cultured on Sabouraud Dextrose Agar (SDA) (Merck, Germany) medium. Fungal suspension was prepared with concentration adjusted to 1.5˟10 6 CFU/ml in sterile distilled water as described by Forbes et al. [15].

Well diffusion assay
Agar well diffusion method is extensively used to evaluate the antimicrobial activity of plants or microbial extracts. To determine the effective concentration, inhibition zones of hydroalcoholic extract of A. tripedale were examined against C. albicans (ATCC 3153), C. parapsilosis (ATCC 2195), and C. krusei (ATCC 573) strains by using well assay technique. The agar plate surface was inoculated overnight by spreading inoculum of Candida spp. over the entire SDA surface. A hole 6 to 8 mm in diameter was punched with a sterile tip. Then, the extract was added to the pits in the agar medium and incubated under suitable conditions at 27°C for 24 h [16]. The diameter of the inhibitory zone was measured, and the corresponding effective concentration was chosen for subsequent experiments [17].

Disk diffusion method
The fungal broth culture aliquots were added to SDA. Sterile paper disks (Merck, Germany) were impregnated with 50 μl of extract solution and placed on the culture plates. The plates were incubated at 37°C for 24 h. Antifungal activity was evaluated by measuring the inhibition zone diameter [18]. Fluconazole was used as positive control [19], whereas paper disks loaded with solvents (ethanol and distilled water) were used as negative controls.

Statistical analysis
Statistical analysis was performed using SPSS, version 10.0. The inhibition diameters of the test substances were expressed as mean and standard deviation. Group comparisons were performed using One-way analysis of variance (ANOVA) followed by Waller-Duncan Post Hoc test. P-value less than 0. was considered statistically significant.

The phytochemical analysis
The phytocomponents present in the hydroalcoholic extract of A. tripedale were identified by GC-MS analysis; GC-MS running time is 34 min. The active compounds in the hydroalcoholic extract of the plant, their retention time (RT), molecular formula, and molecular weight are provided in Table 1, and GC-MS chromatograms are presented in Figure 1.
The gas chromatogram is used to help identify a mixture of compounds by separating compounds according to each compound's retention time. The heights of the peaks indicate the relative concentrations of the components present in the plant. GC-MS analysis revealed the presence of 40 compounds by dichloromethane solvent; the major compounds included tetracosane, hexadecanoic acid, 1-eicosanol, 1,2-dihydro-pyrido[3,2,1-kl]phenothiazin-3-one, 2hexadecen-1-ol, and 3,7,11,15-tetramethyl.

Disk and well diffusion assay
Preliminary screening of the antifungal activity of hydroalcoholic extracts of A. tripedale was performed against Candida spp. using the disk and well diffusion assay. The results showed variation in the antifungal properties of hydroalcoholic extract of A. tripedale ( Table 2). The extract showed strong activity (inhibition zone ⩾ 20 mm), moderate activity (inhibition zone < 12-20 mm), and no inhibition (zone < 12 mm). Fluconazole, a known antifungal antibiotic, as a positive   control significantly inhibited the growth of Candida spp. (Figure 2). Based on the available evidence, the major effective antifungal activity by the hydroalcoholic extract was achieved against C. albicans ( Figure 3). The hydroalcoholic extract of A. tripedale inhibited the growth of C. parapsilosis by well diffusion assay and C. krusei by disk diffusion assay in a dose-dependent manner (Figures 4, 5).

Discussion
Evidence suggests that fungal infections annually affect more than a billion people, and this rate is ever increasing. Candida spp. can be systemic or infect different parts of the body such as skin, nails, respiratory tract, urogenital system, and alimentary canal [20]. Although several species of Candida are potentially pathogenic in humans, Candida albicans is the most important cause of severe candidiasis [21]. Trizoles initially appear to be highly effective against fungal infections, but nowadays, increased resistance is being reported and azole resistant has instigated extensive research to evaluate the effect of antifungal agents from different sources, especially medicinal plants [19]. The most famous antifungal medicinal plants belong to Liliaceae family, where more reports are found on antifungal activity of Allium genus [22]. The antifungal properties of the Allium genus were mentioned in some studies. Shams-Ghahfarokhi et al. (2007) reported that aqueous extracts of Allium cepa and Allium sativum had antifungal activity against Malassezia furfur, Candida spp. and several strains of various dermatophyte species in a dose-dependent manner with the maximum of 100% at defined concentrations [23]. Another study by Amin and Kapadnis proved the antifungal activity of Allium ascalonicum against 23 fungal strains [24].
In this study, we examined the antifungal effect of hydroalcoholic extract of A. tripedale against different strains of Candida by disk and well diffusion assay. Our results revealed that the hydroalcoholic extract (50 mg/ml) had the greatest effect on C. albicans. However, it also had inhibitory effect against C. parapsilosis and C. krusei.
Based on the analysis conducted on the hydroalcoholic extract components using GC-MS method, 40 compounds were identified in this plant that had different properties. We found that tetracosane and other higher alkenes had antioxidant, antitumor, and antifungal properties, particularly against fungal spores and germination [25]. Tetradecanoic acid and eicosane had antioxidant and antimicrobial activities [26]. Hexadecanoic acid is known to have antioxidant and hypocholesterolemic properties and is a constituent of nematicides, pesticides, lubricants, antiandrogens, flavoring agents, hemolytics 5-alpha reductase inhibitors, Curr Med Mycol, 2017, 3(1): 6-12 11 antifeedants, and insect-repellents [27]. Benzoic acid derivatives possess antibacterial and antifungal properties. Phenazopyridine hydrochloride is a topical analgesic that relieves the irritative symptoms associated with urinary tract infection through acting on the mucosal lining of the urinary tract. This agent is compatible with antibiotics and relieves pain before the antibiotic begins to control the infection. Propionic acid is an important chemical commonly used as a raw material in different industries [28]. Propionic acid, the biopreservative produced by Propionibacterium spp., is capable of inhibiting the growth of molds, bacteria, and dairy-spoilage yeasts such as Zygosaccharomyces bailii and Candida spp. [29]. Phenolic compounds, esters, alkanes, aldehydes, alkenes, and ketones are the major volatile compounds, which have anti-inflammatory, antiarthritic, antidiabetic, antiulcer, hypolipidemic, antiatherosclerotic, anti-HIV, and cytotoxic activities [30]. Based on the results of the present study, hydroalcoholic extract of A. tripedale had a significant inhibitory effect against the growth of various strains of Candida. In sum, it seems that A. tripedale is a major source of anti-fungal compounds, which can be applied for the treatment of infectious diseases.

Conclusion
This is the first report on the GC-MS analysis of A. tripedale. It can be concluded that A. tripedale contains various important bioactive compounds. Therefore, it is recommended as a plant of phytochemical and pharmaceutical importance. Further studies are required to isolate the active ingredients of the extract and elucidate its mechanism of action in various diseases.