The effect of Methanolic Extract of Anthemis gayana on Candida Spp.

Transcription

1 International Journal of Agriculture and Crop Sciences. Available online at IJACS/213/51/ ISSN X 213 IJACS Journal The effect of Methanolic Extract of Anthemis gayana on Candida Spp. L. Amjad *1, Z. Rezvani 2, M. Madani 3 1. Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran. 2. Young Researchers and Elite Club, Science and Research Branch, Islamic Azad University Fars Iran. 3. Department of Microbiology, Falavarjan Branch, Islamic Azad University Isfahan Iran. * Corresponding author: amjad@iaufala.ac.ir, amjad.leila@gmail.com ABSTRACT: The genus Anthemis L. belongs to the family Asteraceae. Anthemis gayana is an annual endemic plant of this genus. The aim of this study was to appraise of effect of the flower and leaf methanol extract of the Anthemis gayana on Candida spp. In this study leaves and flowers powder were extracted by Soxhlet extraction for 8 hours. Antimicrobial effect of methanolic extract were assessed by using microdilution MIC determination, disk diffusion assay and agar well diffusion assay and dates were analyzed using ANOVA test in the p <.5. The methanolic extract of Anthemis gayana flowers and leaves were not active against CBS 2175 and ATCC 6261, ATCC Flowers and leaves methanolic extract had more effect against. Thus, there are significant changes of antifungal effects between effective fungi and control sample (P<.5). The results showed that the methanolic extract of Anthemis gayana flowers and leaves were able to inhibit numerous of the fungi used in this study with different degree of inhibition. Keywords: Anthemis gayana, antifungal, Candida, extract. INTRODUCTION Candida are one of the most pathopgens. The most common surface mycotic infections of the skin, nails or hairs are known to be caused by dermatophytes and yeasts (Vollekova et al., 23). Medicinal plants may demonstrate a valuable, original source of recent antifungal drugs and have been shown to contain diverse biochemical and pharmacological actions. Their antifungal activity has been showed against some Candida species (Khan et al., 23; Vollekova et al., 23). The genus Anthemis is the second largest genus in the Compositae (Asteraceae) family, tribe Anthemideae, consists of 39 annual and perennial species distributed all over of Iran. These plants choose dry, open sites on hillsides and grow particularly on calcic substrates in nature (Uzel et al., 24). The species of the Anthemis genus are widely used in the pharmaceutics, cosmetics and food craft. The flowers of the genus have welldocumented use as disinfectant and healing herbs, the main components being natural flavonoids and essential oils (Uzel et al., 24). Some Anthemis spp. Essential oils contain antiaging activity (Savoglou et al., 26). While Anthemis cotula essential oil has been proved to possess antioxidant confidants and essential oil from Anthemis nobilis flowers is generally used for pharmaceuticals, food additives, as well as an main source in aromatic and cosmetic industries (Savoglou et al., 26). Although the Anthemideae is one of the chemically best studied tribes of Asteraceae, only the extract of leaves and flowers of Anthemis gayana Boiss. have been studied. Anthemis gayana is an annual endemic plant of Asteraceae family (Sonboli et al., 25). This plant grows in the Isfahan west region from Iran. Essential oil flowers and leaves were first investigated by Sonboli et al, 25 and more than 45 compounds of the flowers oil representing 94% of total oil were identified, germacrened (14.7%), caryophyllene (9%), spathulenol (7.3%) and bicyclogermacrene (6.1%) were the original compounds. In the oil of the leaves of Anthemis gayana 34 components, accounting for 92.4% of total oil were distinguished with germacrened (3.2%), geranyl isovalevate (7.4%), bicyclogermacrene (6.7%) and caryophyllene (5.5%) as the major compounds (Sonboli et al., 25). The information about the in vitro antifungal activity features of the Anthemis gayana has not been reported earlier. In

2 Intl J Agri Crop Sci. Vol., 5 (1), , 21 this paper were showed the antifungal activity of the methanolic extracts from the flowers and leaves of Anthemis gayana for the first time. MATERIALS AND METHODS Plant material and preparation of extracts Anthemis gayana Boiss. was collected from around area of Isfahan city (Iran) in the June of 212. The voucher specimen was deposited at the herbarium of Research Institute of Isfahan Forests and Rangelands. The plant materials were dried under shade and ground into fine powder using electric blender, then, 2g of the flowers and leaves powder were extracted with 11 ml methanol (CH 3 OH) by Soxhlet extraction for 8 hours. The residue was dried over night and then evaporated by using a rotary evaporator. The dried extracts were stored at 4 C until used. The extracts were dissolved in dimethylsulfoxide (DMSO) (AlJanabi et al., 211; Bajpai et al., 28). Assessment of antifungal activity Seven strains of fungi, NCPF 3153, ATCC 6261, ATCC 1677, CBS 94, CBS 2175, CBS 25 and CBS 573, were used. For the study of antifungal activity, total count of isolated strains were standardized to equivalent a.5 Macfarland nephelometer standard ( cfu/ml) by sabouraud dextrose broth and then diluted as then diluted as 1:1. Antifungal activity was studied by the agar well diffusion method. Sabouraud dextrose agar was used as the fungal medium. The effect of extract antimicrobial were assessed in the concentration include 15, 3, 6, 12 using agar well diffusion method and also by determination minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) using microdilution test method Kachuei et al., 212). The extracts were diluted in dimethylsulphoxide at the concentrations of 15, 3, 6, 12 sabouraud dextrose agar (SDA). Petridishes (8cm diameter) contining 2 ml of SDA were used for antifungal activity assay, performed on solid media by the well diffusion metod sterile. Wells were prepared in the seeded agar petridishes. The study compound was introduced in the well (6mm). The plate were incubated at 37 c for 24 48h. Growth inhibitation of each fungal strains was counted as the percentage of prevent of radial grow relative to the control, fluconazol. A standard antifungal agent, fluconazol served as a positive control and dimethysulfoxide was used as a negative control (Oliveira et al., 26). Statistical analysis The experiment was performed three times to minimize the error and the mean values are presented. Data were analyzed using ANOVA test in the P<.5. RESULTS AND DISCUSSION Results were analyzed and fungi growth inhibition were confirmed after inoculation in abouraud dextrose agar. The extracts showed some degree of inhibition of fungi growth at different concentrations. The antifungal activity of the methanolic extract of the Anthemis gayana flowers and leaves were determined against 7 fungi strains which is showed in Table 1, 3. The MIC and MFC of these extracts are currently being obtained in Table 2, 4. The methanolic extracts of the flower and leaf exhibited antifungal activity at against 7 fungi. In the present study, leaf methanolinc extracts in concentration 12 mg/ml have more inhibitory activity against fungi. Thus, there are significant changes of antifungal effects between effective fungi and control sample (P<.5). The inhibitory effect of the extracts were compared with standard antifungal, fluconazole. The increasing public and economic implications caused by fungi means there is a stable fighting to produce safer food crops and to develop new antifungal agents (Bajpai et al., 28). Plant extracts and essential oils are potential sources of novel antimicrobial compounds against bacteria and fungi pathogens. plants have severe ability to synthesize phenolic products. These products are secondary metabolites and serve as plant defense mechanisms by microorganisms Amjad et al., 211). Their activity is due to their ability to complex with extracellular and soluble proteins (Amjad et al., 212). Thus, previous reports have demonstrated that: saponins, tannins, alkaloids, terpenes, carotenoids and flavonoides possessed antifungal activity (Amjad et al., 212). Antimicrobial flavonoids have multiple cellular targets, Instead of

3 Intl J Agri Crop Sci. Vol., 5 (1), , 21 one specific site of action. Alternatively, these common structural features may simply be necessary for flavonoids to gain presence to or uptake into the microbial cell. It may be that flavonoids are not killing microbial cells but singly inducing the formation of microbial aggregates and thereby reducing the number of CFUs in viable counts (Kachuei et al., 212). However, there are different antimicrobial mechanisms of action of various flavonoids: 1) Inhibition of nucleic acid synthesis; 2) Inhibition of cytoplasmic membrane function; 3) Inhibition of energy metabolism (Amjad et al., 212; Uzel et al., 24). Kosalec et al. (25) determined the differences in MIC values. They showed that the differences in MIC values can be explained by different concentrations of antifungal components in the extracts, but also in the application of methods for determining MIC values. MIC values are also influenced by a number of variables, such as the composition of the culture medium, inoculums density, etc. (Clark et al., 9). Current antifungal drugs against fungal pathogens are demonstrated by azole and polyene antimycotics.their antifungal action depends on the presence of ergostrol, an important fungal membrane part. While the azole agents inhibit the intracellular biosynthesis of ergostrol, polyene antimycotics act by direct binding to ergostrol in the cell membrane (Vollekova et al., 23). Therefore, it has also been reported that the antifungal activity of secondary metabolites against fungi, results from their ability to inhibit sterol and chitin synthase, key enzymes in the patways of both ergostrol and chitin biosynthesis (Vollekova et al., 23). The finding from the present study indicates that the Anthemis gayana evaluated antifungal properties. The plants of Asteraseae family have a large amount of triterpenoid compounds similar to saponin and studies have shown that the triterpenoids have activity against yeast species (Amjad et al., 211; Fawzy et al., 213). This explains the use of this plant in folk medicine for the treatment of various diseases whose symptoms might involve fungal infections. Future phytochemical research is needed to identify the active principles responsible for the antifungal effects of this medicinal plant. In vitro antifungal activity of the methanolic extract of Anthemis gayana flowers and leaves on fungi was tested. The methanolic extract of Anthemis gayana flowers and leaves showed antifungal activity against Candida albicans,, and. Major investigations should be performed on a wider range of fungi in order to determine which component of the extract shows the most potent antifungal activity. The antifungal activity could also be a result of the synergism of its components. ACKNOWLEDGEMENT This work was supported by Islamic Azad University, Falavarjan Branch. The authors also thank Dr. Monajjemi and Dr. Ranjbar from the Department of Biology, Islamic Azad University, Falavarjan Branch for their aid. Table 1. The antifungal activity of methanolic extract of Anthemis gayana flowers on Candida spp. (X±SD) Control() Control(+) Concentration (6261) 25 33±.97 28± ± ± ±.92 25± ±.21 2±.6 (25) 29± ± ± ± ± ± ± ±.17 23

4 Intl J Agri Crop Sci. Vol., 5 (1), , 21 Table 2. Determination of MIC and MFC of flowers methanolic extract for Candida spp. MFC MIC (6261) (25) Table 3. The antifungal activity of methanolic extract of Anthemis gayana leaves on Candida spp. (X±SD) Control () Control (+) Concentration (6261) 25 32±.2 29±.5 26±.65 24± ± ± ± ±.16 (25) 32± ± ± ± ±.22 26±.2 22±.5 2±.1 23 Table 4. Determination of MIC and MFC of leaves methanolic extract for Candida spp. MFC MIC

5 Intl J Agri Crop Sci. Vol., 5 (1), , 21 (6261) (25) REFERENCES ALJanabi AAS Potential activity of the purine compounds caffeine and aminophylline on bacteria. J Glob Infect Dis 3: Amjad L, Mohammadisichani M, Mohammadikamalabadi M Potential activity of the Achillea wilhelmsii leaves on bacteria. Int J Biosci Biochem Bioinform 1: Amjad L, Mousavidehmourdi K, Saghazadeh M Antifungal potential of Achillea wilhelmsii flowers methanolic extract on different strains of. Int J Biol Med Res 3(3): Bajpai VK, Shukla S, Kang SC. 28. Chemical composition and antifungal activity of essential oil and various extract of Silene armeria L. Bioresour Techno 99: Clark AM, Jurgens TM, Hufford HD. 9. Antimicrobial activity of juglone. Phytother Res 4: Fawzy GA, ElGamal AA, AlAti HY Antimicrobial and cytotoxic potentials of Buddleja polystachya extracts. Bangladesh J Pharmacol 8: Hammer KA, Carson CF, Riley TV. 99. Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol 86(6): Kachuei R, Emami M, Naemi B, Diba K Isolation of keratinophilic fungi from soil in Isfahan province, Iran. J Mycol Med 22: 813. Khan MAU, Ashfaq MK, Zuberi HS, Mahmood MS, Gilani AH. 23. The In vivo antifungal activity of the aqueous extract from Nigella sativa seeds. Phytother Res 17: Kosalec I, Pepeljnjak S, Kutrak D. 25. Antigungal activity of fluid extract and essential oil from anise fruits (Pimpinella anisum L., Apiaceae). Acta Pharmaceut 55(4): Oliveira ACP, Shinobu CS, Longhini R, Franco SL, Svidzinski TIE. 26. Antifungal activity of propolis extract against yeasts isolated from Onychomycosis lesions. Mem Inst Oswaldo Cruz 11(5): Saroglou V, Dorizas N, Kypriotakis Z, Skaltasa HD. 26. Analysis of the essential oil composition of eight Anthemis species from Greece. J Chromatogr A 114: Sonboli A, Kanani MR, Salehi P. 25. Volatile constituents of the leaves and flowers of Anthemis gayana from Iran. Dig 1 th Sem Med Nat Prod Chem Iran 74. Tim Cushnie TP, Lamb AJ. 25. Antimicrobial activity of flavonoids. Int J Antimicrob Ag 26: Uzel A, Guvensen A, Cetin E. 24. Chemical composition and antimicrobial activity of the essential oils of Anthemis xylopoda O. Schwarz from Turkey. J Ethnopharmacol 95: Vollekova A, Kostalova D, Kettmann V, Toth J. 23. Antifungal activity of Mahonia aquifolium extract and its major protoberb erine alkaloids. Phytother Res 17: