Original Article in Vitro Evaluation of Antifungal Activity of Three Traditionally Used Medicinal Plants; Umbilicus Intermedius

International Journal of Medical Laboratory 2019;6(4):266-274. Original Article In Vitro Evaluation of Antifungal Activity of Three Traditionally Used Medicinal Plants; Umbilicus Intermedius Boiss, Cuminum Cyminum and Zingiber Officinale Extracts .Ph.D ٭Ameneh Takesh1 M.Sc., Mahnaz Fatahinia2 Ali Zarei Mahmoudabadi2 Ph.D. 1Department of Medical Mycology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 2Infectious and Tropical Diseases Research Center, Health Research Institute and Department of Medical Mycology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. A B S T R A C T Article history Background and Aims: The study aimed to determine the effectiveness of Received 12 Mar 2019 three medicinal plant extracts on fungi with three methods and to compare Accepted 2 Oct 2019 methods. Available online 10 Dec 2019 Material and methods: This study examined the antifungal properties of Key words cumin (Cuminum cyminum L), ginger (Zingiber officinale Roscoe) and Nafe Antifungal Venus (Umbilicus intermedius boiss) extracts against fungi including, Cumin Aspergillus spp., Penicillium spp., Mucor spp., Stemphylium spp., Drechslera Ginger Medicinal plants spp., Alternaria spp., Cladosporium spp., and Aureobasidium pullulans. Umbilicus intermedius boiss Furthermore, 17 candida isolates including, C. albicans, C. glabrata and C. dubliniensis were tested. In the present study two methods of disc diffusion method, agar wells diffusion method were used for assay. Then, the mixing with culture medium method was used for assessment of the antifungal activity of extracts against Alternaria sp.(as black mold), A. terreus (as hyaline mold) and C. albicans (as yeast) to compare methods as well. Results: No fungi were susceptible to extracts in disc diffusion method and agar wells diffusion method. But, this study showed that in mixing with culture medium method, cumin extract has valuable anti-fungal property and Umbilicus intermedius boiss has the inhibitory properties against the black fungi. Furthermore, it is found that mixing with culture medium method is more efficient than disc and agar well diffusion methods. Alternaria sp. and C. albicans were susceptible and resistant to all extracts. Conclusions: it is found that mixing with culture medium method is more efficient than disc and agar well diffusion methods and inhibitory potency of the extracts varies according to the type of extraction and their concentration. *Corresponding Author: Infectious and Tropical Diseases Research Center, Health Research Institute and Department of Medical Mycology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. Tel: +989163102952, Email: [email protected] A. Takesh et al. Introduction The use of medicinal plants for treating fungal urinary tract infections and kidney stone and bacterial diseases has a long history. disposal [5-7]. Today, the use of herbal medicines has Cumin (Cuminum cyminum L.) is an aromatic increased due to microorganisms’ resistance flowering plant in the family Apiaceae or to chemical drugs and the side effects of Umbelliferae [8, 9]. The important and these drugs. Most of the drugs have a effective components of cumin include: chemical origin, but about one-third of all myrtenal, trans-carveol, O-cymene, cuminique pharmaceutical preparations have plant origin alcohol and many other materials [9-11]. or extractions which are modified. For Several medicinal properties are attributed to example, approximately 25% of drugs in the cumin, including healing the common cold, United States are made of medicinal herbs[1, jaundice, diarrhea, indigestion and bloating. 2]. One of these herbs is Nafe Venus plant [3] . Moreover, therapeutic properties against fungi, Nafe Venus (Umbilicus intermedius boiss, bacteria and viruses for cumin have been Genus: Umbilicus) is a hydrous perennial reported [8, 12]. floral plant, which includes approximately 11 Ginger consists of thick squamate root of the to 30 species. It belongs to the stonecrop monocotyledonous plant Zingiber officinale, family Crassulaceae (including approximately belonging to the family Zingiberaceae [13]. 1400 species and 34 or 35 genera). Nafe Various studies have shown that ginger has Venus is 20-35 cm high with simple green many therapeutic properties including leaves including alternate, scutate, convex, lob antidiabetic, anti-arthritic, anti-microbial ate-crenate, stem leaves linear, wedge-shaped activities against various bacteria, fungi, and and serrate. It produces white, yellow flower nematodes, anti-inflammatory and anti- clusters from March to June. The plant flowers thrombotic. Ginger (Zingiber officinale) is are pendent in compact spike, urceolate- known throughout history as a medicinal tubular, congregations [4]. Although plants [14-18] . Umbilicus intermedius has worldwide Saprophytic fungi include some of the most distribution, it is found more frequently in common indoor and outdoor molds which southern Africa, deserts of Israel, Lebanon can cause different infectious diseases in and in mountains of Jordan and Saudi Arabia immunocompromised patients [19]. and in some regions of Iran such as Ilam [5]. Furthermore, some fungi are resistant to The leaves of Nafe Venus is used freshly or routinely used antifungals and some patients dried mixed with yogurt for treating burned do not tolerate chemical antifungals. For skin. Moreover, the decoction of the leaves this reason, the present study aimed to was used to treat skin infections, carbuncles, investigate the antifungal activity of methanol- chloroform, alcoholic and aqueous extract of International Journal of Medical Laboratory 2019;6(4):266-274. 267 EVALUATION OF ANTIFUNGAL ACTIVITY OF THREE MEDICINAL PLANTS Umbilicus intermedius boiss and alcoholic Finally, the extract was prepared as extract of ginger and cumin against fungi explained above [21]. such as Aspergillus, Penicillium and Mucor In a dark bottle, 25 ml of 80% methanol was species. In addition, some dematiaceous fungi mixed with 25 ml of chloroform; it was (Stemphylium sp., drechslera sp., Alternaria then,added to 7.87 grams of powdered plant. sp., Cladosporium sp., Aureobasidium Later, it was shaken in a refrigerator incubator pullulans sp.) and Candida species (C. in 10°C at 100 rpm for 72 hours. Finally, albicans, C. glabrata and C. dubliniensis) supernatant was filtrated twice by using a filter were used to compare methods. paper and extract was prepared as mentioned Materials and Methods above [22] . Preliminary preparation of fungal samples Plant Preparation Twenty nine fungal strains were collected in The Umbilicus plant (herbarium code: 296) the laboratories of mycology department of was collected from the mountains of Ilam and Ahvaz Jundishapur University of Medical Cumin (herbarium code: A170106OFP) and Sciences. The strains were confirmed by using Ginger (herbarium code: A173211ORP) standard methods [23-25]. All strains were plants were purchased from groceries in cultured in Sabouraud dextrose agar (SDA) Ahvaz and Yazd, respectively. Plants were (Bio life, Italian) and incubated at 35°C for completely washed with cold water and then 24 hours for yeasts and filamentous fungi at dried in a dark place. Then, they were 25°C for 3-7 days. completely powdered by a household mill and Preparation of standard fungal suspension stored at a dark and dried place at the room The fungi, mostly collected in autumn temperature until use. and spring, were re-cultured on SDA. Preparation of alcoholic, aqueous and A suspension of the yeast strains were metanol chloroform extracts adjusted according to CLSI M44-A using a Ten grams of plant powder were added to spectrophotometer in a concentration of 1- 250 ml of 80% ethanol in a dark vessel and 5×106 (a 0.5 McFarland standard). In addition, completely mixed on a shaker at 25-30°C for the suspensions of filamentous fungi were 72 hours at 150 rpm. The mixture was prepared spectrophotometrically to optical filtered twice using a filter paper (Whatman densities ranging from 0.09 to 0.11 in No-1) and filtrate was placed in an oven at accordance with CLSI M51-A [26]. 40°C until the elimination of the alcohol, Disc diffusion method: One hundred μL of Then it was stored at 4°C until use [6, 20]. fungal suspensions were spread on SDA Ten grams of plant powder were added in 50 medium and kept at room temperature for 10- ml of boiling distilled water, while shaking 15 minutes. After the microbial suspensions once every few minutes for 72 hours. were completely absorbed, blank discs (diameter of 6.4 mm, PADTAN TEB Co., 268 International Journal of Medical Laboratory 2019;6(4): 266-274. A. Takesh et al. Iran) impregnated with 25 μL different concentrations (200, 100 and 50 mg/ml) of the extracts, were placed on the plate's surface. IP = Percentage of growth inhibition Yeasts were then incubated at 35°C for 24 C = Mean diameter of fungus colony in control hours and saprophytic fungi at room T = Mean diameter of fungus colony in the temperature for 3-7 days [6]. Finally, diameter tested concentration of extracts inhibition of zone by extracts around each disc Results was measured. The inhibitory efficacy of extracts was Agar well diffusion method evaluated by disc diffusion, agar wells and According to disc diffusion method, medium mixing the extracts with culture medium was prepared and inoculated with fungi. The methods against several molds and yeasts wells were made using a 6.0 mm diameter pathogens. In this study,

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