ARTICLE INFO

Article Type

Original Research

Authors

Sarkhani Moghaddam   F (1)
Fakoor   MH (2)
Sabokbar   A (*)
Ebrahimzadeh   M (1)






(*) Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran
(1) Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran
(2) 2 Department of Microbiology, Hidaj Branch, Islamic Azad University, Hidaj, Iran

Correspondence

Address: Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran
Phone: +98 (26) 32773730
Fax: +98 (26) 32773730
azar.sabokbar2017@gmail.com

Article History

Received:  May  18, 2018
Accepted:  May 23, 2018
ePublished:  July 23, 2018

BRIEF TEXT


Aspergillus species are a group of filamentous fungi that are generally grown in soil, corrosive vegetables, and seeds as saprophytic fungi. Aspergillus can be harmful to humans at times [1]. … [2-6]. Green tea polyphenols reduce the formation of free radicals in the body and protect the body against diseases such as atherosclerosis, cardiovascular disease, and cancers [7].

Studies conducted by Chrruiyot et al. in 2015 showed that green tea root extract possesses antifungal properties against Candida species, including Candida tropicalis ATCC 750, Candida Famata, Candida Lusitaniae, and Dermatophytes species, including Trichophyton mentagrophytes. Candida albicans and Cryptococcus neoformans reacted to green tea root extract [8]. In another study, the results of Jigisha et al.'s research in 2015 showed that methanolic and acetonic extracts of green tea have antibacterial properties. Also, in the combination of methanol extract of green tea with ampicillin on Staphylococcus aureus and Pseudomonas aeruginosa, synergistic effects were observed; in a way that the diameter of the inhibition zone against Staphylococcus aureus and Pseudomonas aeruginosa increased from 36±0.07 to 39 ±1.414 mm and 26±0.07 to 29±0.07, respectively, after the combination of the drug and the extract [9]. In 2016, Madhura et al. proved the antifungal effects of green tea extract on Candida species by t-test compared to the control group [10].

The aim of this study was to evaluate the antifungal activity of aqueous and methanolic extracts of green tea in Lahijan province on the 4 strains of Aspergillus including Aspergillus flavus ATCC 39, Aspergillus fumigatus ATCC 278, Aspergillus trous ATCC 274, and Aspergillus niger ATCC 9142 as well as the synergistic effects of these two extracts in combination with itraconazole and voriconazole.

This research is an experimental study.

The population of the present study included 4 strains of Aspergillus including Aspergillus flavus ATCC 39, Aspergillus fumigatus ATCC 278, Aspergillus trous ATCC 274, and Aspergillus niger ATCC 9142.

4 strains of Aspergillus including Aspergillus flavus ATCC 39, Aspergillus fumigatus ATCC 278, Aspergillus trous ATCC 274, and Aspergillus niger ATCC 9142 were prepared from the Department of Mycology, Mazandaran University of Medical Sciences and Faculty of Basic Sciences, Shahed University, and then were transmitted to the Laboratory of Microbiology at Azad University of Karj to carry out the study.

Each fungal spore suspension was prepared according to 38-A2 CLSI protocol. After culturing the fungus in a slender graft of sabouraud dextrose agar for 2 weeks, the spores were extracted and shaken with sterile water and Tween® 80 (E.Merck). In order to prepare the drug stock, the concentration of 0.0032 g of itraconazole and voriconazole with 5 mL of pure DMSO was combined as a drug solvent and also the elimination of possible contaminants. The initial concentration of drugs was 1 μg/ml. The compound was placed at room temperature for 30 minutes until the drugs were completely dissolved in DMSO. 100 μl of the solution was poured into a sterile test tube and the 900 μl RPMI1640 medium containing sterilized MOPS was added. The concentration of drugs used in the first well of microplate Elisa was diluted 0.1 and equal to 64 μg/ml. The rest of the wells were considered serial dilutions of 32, 16, 8, 4, 2, 1, and 0.5 μg/ml. Dilution was performed by RPMI1640 medium containing sterilized MOPS [11]. In order to prepare the aqueous and methanolic extracts of green tea leaves, the dried leaves of this plant, which were the product of the year in 2015 and the produced by Lahijan tea production, were extracted by maceration method [12]. To investigate the antifungal effects of the aqueous and methanolic extracts of green tea, the calculations were performed in such a way that the concentration of each of the extracts was 128 μg/ml in the first well and the subsequent dilutions in the microplate wells from left to right were 0.5, 1, 2, 4, 8, 16, 32, 64 μg/ml in the second well until the end. Then, 0.25 g of each of the extracts was weighed, and the steps in the preparation of drug stock for itraconazole and voriconazole were the same. Finally, the concentration of extracts in the first well was 128 μg/ml [11]. In order to prepare the stock for the combination of drugs and extracts together, all stages of preparation of stock of aqueous and methanolic extracts were performed, except that half of the weight of drugs or powder of the extracts was tested (0.25/2=0.125 g) [11]. Investigations were conducted to determine the minimum inhibitory concentration (MIC) of drugs, extracts, and their combinations to evaluate the synergistic effects of drugs and extracts in accordance with 38-A2 CLSI Protocol by inter-microplate dilution of 96-well Elisa. The minimum fungicidal concentration (MFC) and MIC of drugs and their extracts and their compounds were determined by observation after 5 times repetition. Then, using SPSS 20 software, Duncan multiple test was performed.

Itraconazole has effective and desirable results compared with voriconazole as a result of synergy with aqueous and methanolic extracts of green tea. The synergistic effects of voriconazole with the extracts, except Aspergillus fumigatus, were worthless. The aqueous and methanolic extracts of green tea with the name of the product range of Lahijan alone did not have an antifungal effect on Aspergillus strains. The aqueous and methanolic extracts of Lahijan green tea did not have an antifungal effect on Aspergillus strains. The results of the MIC indicated that there were synergistic effects in compounds such as the combination of aqueous extract of green tea and itraconazole on the Aspergillus fumigatus ATCC 278 with a MIC of 4 μg/ml (Table 1). As shown in Tables 1 and 2, methanolic extracts of green tea together with voriconazole have no MFC and MIC value, while the combination of aqueous extract of green tea is valuable for the treatment of Aspergillus fumigatus infection.

… [13-19]. Haramain et al. carried out some studies to investigate the antimicrobial effect of methanolic and aqueous extracts of Chinese green tea via release from agar on a gram-positive bacterium such as Staphylococcus aureus and 5 gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, and Salmonella typhi as well as 2 fungi, including Aspergillus niger and Candida albicans. The results of this study demonstrated that methanolic extracts of green tea leaf showed antifungal effects (24-28 mm) against all bacteria in the test [20]. In 2017, Naeini et al. conducted a study to investigate the antimicrobial effect of aqueous extract of green tea on various species of Candida, including Candida albicans, Candida glabrata, Candida Krusei, and Candida tropicalis, using the punch-hole method. In this study, aqueous extracts of green tea on the candidate species was ineffective (with diameter of the inhibition zone). [21]. Park et al. examined the antifungal effects of Epigallocatechin gallate in green tea on dermatophyte species, including Trichophyton rubrum ... [22] and Trichophyton Mentagrophytes. In this study, both of these fungi were sensitive to Epigallocatechin gallate, with a range of MIC 50=2-4 μg/ml and MIC 50=4-8 μg/ml, respectively [23]. Abraham et al. investigated the synergistic effect of methanolic extract of green tea with chloramphenicol by disc plate and examined the diameter of the inhibition zone. The result showed that it was 16 mm for Escherichia coli, 24 mm for Enterococcus faecalis, 12 mm for Salmonella typhi, 6 mm for Staphylococcus aureus, and 10 mm for Pseudomonas aeruginosa, and Aspergillus niger, Candida albicans, Fusarium, and Vibrio cholera were found to be ineffective [24].

It is suggested to investigate the antimicrobial and antifungal properties of the effective compounds of Iranian tea, especially on those compounds that have the highest percentage in green tea.

None declared by the authors.

The results indicate that there are significant synergistic effects between the compounds of the drugs and the extracts.

The research is the dissertation of Mrs. Farnoosh Sarkhani Moghaddam. Therefore, we thank the Islamic Azad University of Karaj, which provided the conditions for conducting the researches.

None declared by the authors.

None declared by the authors.

All financial resources belong to dissertation of Mrs. Farnoosh Sarkhani Moghaddam, which is provided in accordance with the directives of Islamic Azad University of Karaj.

TABLES and CHARTS

Show attach file


CITIATION LINKS

[1]Mousavi B, Hedayati MT, Hedayati N, Ilkit M, Syedmousavi S. Aspergillus species in indoor environments and their possible occupational and public health hazards. Curr Med Mycol, 2016;2(1):36-42.
[2]Lamoth F. Aspergillus fumigatus-Related Species in Clinical Practice. Front Microbiol. 2016;7(683):1-8.
[3]Van Der Linden JW, Arendrup MC, Warris A, Lagrou K, Pelloux H, Hauser PM, et al. Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus. Emerg Infect Dis. 2015;21(6):1041–4.
[4]Lazzarini C, Esposto MC, Prigitano A, Cogliati M, De Lorenzis G, Tortorano AM. Azole Resistance in Aspergillus fumigatus clinical isolates from an Italian culture collection. Antimicrob Agents Chemother. 2016;60(1):682-5.
[5]Zia udden N, Shahid M. Determination of bioactive properties of different temperature Camellia sinensis (Green tea). Am J Food Nutr. 2017;5(1):10-8.
[6]Farhad Mollashahi N, Bokaeian M, Farhad Mollashahi L, Afrougheh A. Antifungal efficacy of green tea extract against Candida albicans biofilm on tooth substrate. J Dent. 2015;12(8):592-8.
[7]Sigei EC, Muturi M, Bii C. Antifungal activities of Camellia sinensis crude extract, mixture with milk, on selected pathogenic and mycotoxic fungi. J Med Plants Res. 2015;9(42):1070-80.
[8]Sigei EC, Muturi M, Bii C. Antifungal activities of Camellia sinensis crude extract on selected pathogenic and mycotoxic fungi. J Bacteriol Mycol. 2015;2(2):1015.
[9]Anand J, Gautam P, Rai N. Comparative study of antibacterial and anti-proliferative potential of grean tea from different geographical locations in India. Asian J Pharm Clin Res. 2015;8(1):253-8.
[10]Madhura MG, Shweta RD, Weerendra Kumar B, Savithri D, Gajalakshmi S, Soumya BV. Antifungal effect of green tea extracts on oral Candida species: An in vitro study. J Adv Clin Res Ins. 2016;3(1):1-4.
[11]Kate G, Davey, Ann D. Holmes, Elizabeth M. Johnson, Adrien Szekely, David W. Warnock. Comparative evaluation of fungitest and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans. J Clin Microbiol. 1998;36(4):926-30.
[12]Naeini A, Jalayer Naderi N, Shokri H. Analysis and in vitro anti-Candida antifungal activity of Cuminum cyminum and Salvadora persica herbs extracts against pathogenic Candida strains. J Med Mycol. 2014;24(1):13-8.
[13]Fakoor MH, Rasooli I. Pathogen control by antioxidative characteristics of Cuminum cyminum and Rosmarinus officinalis essential oil. Acta Hortic. 2008;786:125-36.
[14]Mohammadpour H, Moghimipour E, Rasooli I, Fakoor MH, Alipoor Astaneh Sh, Shehni Moosaie S, Jalili Z. Chemical composition and antifungal activity of Cuminum cyminum L. essential oil from Alborz mountain against Aspergillus species. Jundishapur J Nat Pharm Prod. 2012;7(2):50-5.
[15]Rasooli I, Fakoor MH, Yadegarinia D, Gachkar L, Allameh A, Rezaei MB. Antimycotoxigenic characteristics of Rosmarinus officinalis and Trachyspermum copticum L. essential oils. Int J Food Microbiol. 2008;122(1-2):135-9.
[16]Rasooli I, Fakoor MH, Allameh AA, Rezaee MB, Owlia P. Phytoprevention of aflatoxin production. J Med Plants. 2009;8(5):97-104.
[17]Mousavi B, Hedayati MT, Hedayati N, Ilkit M, Syedmousavi S. Aspergillus species in indoor environments and their possible occupational and public health hazards. Curr Med Mycol. 2016;2(1):36-42.
[18]Kemoi EK, Nyerere A, Gross U, Bader O, Gonoi T, Bill C. Diversity of azoles resistant Aspergillus species isolated from experience and naïve soils in Nairobi County and Naivasha sub-county Kenya. Eur Sci J. 2017;13(36):301-11.
[19]Reygaert CW. An Update on the health benefits of green tea. Beverages J. 2017;3(6):1-14.
[20]Haramain, S E, Almaghboul, A Z, Ahmed, A O. Antimicrobial activity and phytochemical screening of Chinese green tea (Camellia sinensis L). ARPN J Sci Technol. 2015 May; 5(5):246-52.
[21]Naeini AR, Shayegh S, Shokri H. In vitro antifungal effect of herbal mixture (Nigella sativa, Foeniculum vulgare and Camellia sinensis) against Candida species isolated from denture wearers. J Herbmed Pharmacol. 2017;6(2):74-9.
[22]Almada A. Leveraging the science behind tea. J Funct Foods. 2005;3:34-5.
[23]Park BJ, Taguchi H, Kamei K, Matsuzawa T, Hyon SH, Park JC. In vitro antifungal activity of epigallocatechin 3-O-gallate against clinical isolates of dermatophytes. Yonsei Med J. 2011;52:535-8.
[24]Archana S, Abraham J. Comparative analysis of antimicrobial activity of leaf extracts from fresh green tea, commercial green tea and black tea on pathogens. J Appl Pharm Sci. 2001;8(1):149-52.