Efficacy of Some Medicinal Plants Extracts for Potential Antifungal Activity
Background: Peganum harmala, Glycyrrhiza glabra, Mentha spicata and Rosmarinus officinalis are often used in traditional medicine.
Objectives: The current study aimed to screen the antifungal activity of ethanolic extracts of P. harmala, G. glabra, M. spicata and R. officinalis leaves collected from Sistan region, Iran against Fusarium oxysporum.
Methods: Minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of herbal extracts were determined.
Results: Of the total herbal extracts, four showed fungistatic activities and two showed fungicidal activities. MIC values, ranged 6.25 - 50 ppm and MFC values, ranged 12.5 - 100 ppm. The ethanolic extract of G. glabra had the lowest MIC (6.25 ppm); while the ethanolic extract of R. officinalis had the highest MIC (50 ppm). Minimum bactericidal concentration (MBC) of the alcoholic extract of G. glabra showed the highest efficiency against F. oxysporum at the low value of 12.5 ppm.
Conclusions: The results of the study showed the antifungal activity of medicinal plants against F. oxysporum. Especially, the bioactive compounds of G. glabra were effective to inhibit the growth of F. oxysporum.
Keywords: Antimicrobial Effect; Ethanol Extract; Medicinal Plants; Fusarium oxysporum
The Fusarium oxysporum that is one of the most important Fusarium species does not show sexual reproduction. Crown and root rot caused by these fungi are reported (1). The species were isolated from several European countries, Asia, Africa and North America (2). In Iran, for the first time in 1964, the causal agent was isolated from melon in Mashhad (3). Although the fungi is an option, due to practical difficulties including non-target effects, high cost and environmental and health threatening risks, it is not at least always preferred (4). The growing popularity of organic production requires the development and adoption of other methods to control fungi diseases.
Iran has a long history of traditional medicine with recent academic facilities. At present, there is little evidence on the antimicrobial properties of herbal medicines under review against F. oxysporum.
The current study aimed to investigate the potential antifungal activity of aqueous extracts of herbal medicines collected from Sistan region, Iran, against F. oxysporum to verify possible inhibitory effects.
Different herbal tissues of various species used in traditional medicine (Table 1) were collected from Sistan region, Iran, in 2016. After collecting the plants, they were dried at 25°C. To prepare the extracts, 10 g dry powder of the plants was placed in a liter flask containing 100 mL of 96% ethanol. Extracts of Peganum harmala, Glycyrrhiza glabra, Mentha spicata and Rosmarinus officinalis were prepared using a rotary device (5). Spores of F. oxysporum were collected from corn meal agar cultures after seven days (6). Sporangial suspension concentration was prepared using a cell counting chamber adjusted to 1 × 106 spores mL-1 (7). A microplate method was utilized to determine MIC values of the herbal extracts (8). The extracts were diluted ranging from 1/1 to 1/64 of crude extract. In each well, 100 microliters of each dilution of the extract with 10 microliter of the spore suspension (1 × 106 mL-1 spores) were mixed. Microplates were incubated for 72 hours at 28°C. The MIC was measured by microplate reader at 595 nm and was determined by comparing the growth in the control wells. The MIC was defined as the lowest concentration of the extract that inhibits the growth of more than 90% at 72 hours as compared to that of the control.
3.1. Plant Material
3.2. Preparation of Plant Extracts
3.3. Fungal Strain
3.4. Minimal Inhibitory Concentration and Minimum Fungicidal Concentration
Different herbal tissues of various species used in traditional medicine (Table 1) were collected from Sistan region, Iran, in 2016.
After collecting the plants, they were dried at 25°C. To prepare the extracts, 10 g dry powder of the plants was placed in a liter flask containing 100 mL of 96% ethanol. Extracts of Peganum harmala, Glycyrrhiza glabra, Mentha spicata and Rosmarinus officinalis were prepared using a rotary device (5).
Spores of F. oxysporum were collected from corn meal agar cultures after seven days (6). Sporangial suspension concentration was prepared using a cell counting chamber adjusted to 1 × 106 spores mL-1 (7).
A microplate method was utilized to determine MIC values of the herbal extracts (8). The extracts were diluted ranging from 1/1 to 1/64 of crude extract. In each well, 100 microliters of each dilution of the extract with 10 microliter of the spore suspension (1 × 106 mL-1 spores) were mixed. Microplates were incubated for 72 hours at 28°C. The MIC was measured by microplate reader at 595 nm and was determined by comparing the growth in the control wells. The MIC was defined as the lowest concentration of the extract that inhibits the growth of more than 90% at 72 hours as compared to that of the control.
The effect of different concentrations of herbal extracts on F. oxysporum is summarized in Table 2. Thirteen ethanol extract dilutions (ranging from 1/1 to 1/32) inhibited the growth of F. oxysporum more than 90%. The ethanol extract of G. glabra performed in 1/32 dilution showed growth inhibition at low concentrations.
Antifungal Activity of Various Dilutions of Ethanolic Herbal Extractsa
MIC and MFC were performed for ethanol extract of each of the two species. Table 3 shows MIC and MFC of active herbal extracts. Of the total extracts, four showed inhibitory effects on fungi and two showed no activity.
MIC values ranged 6.25 - 50 ppm and MFC values ranged 12.5 - 100 ppm. The ethanol extract of G. glabra showed the lowest MIC (6.25 ppm), while the ethanol extract of R. officinalis had the highest MIC (50 ppm). MBC of the alcoholic extract of G. glabra had the highest inhibitory effects on F. oxysporum at 12.5 ppm (Table 3).
Minimal Inhibitory Concentration and Minimum Fungicidal Concentration of Ethanolic Herbal Extracts Against Fusarium oxysporuma
The antifungal effects of G. glabra extract, and its active constituent glabridin, on antibiotic-resistant Candida albicans species were studied in different studies (9). The current research showed antifungal effects of G. glabra extracts on C. albicans species. The inhibitory effects of glycyrrhetinic acid derived from G. glabra on Candida spp. were evaluated in different studies and the results showed the efficiency of the combination (10). The researches indicated that G. glabra can inhibit the growth of Aspergillus parasiticus and prevent the production of aflatoxin (11). Another research reported that the methanol extract of G. glabra had high fungicidal effects on Chaetomium funicola and Arthrinium sacchari (12).
In addition, Glycyrrhiza species include glycyrrhizin, which prevents protein synthesis in bacteria (13). A study indicated the antipseudomonal activity of G. glabra and one of its compounds (14). The inhibitory effect of G. glabra on Helicobacter pylori was studied in-vitro and indicated that it can have effects similar to those of metronidazole (15). Another research also indicated the inhibitory effects of G. glabra extract on H. pylori (16). Some Glycyrrhiza species, e g, G. glabra, inhibit the growth of some Gram-negative bacteria such as Shigella spp. and Salmonella spp. (17). Glycyrrhizin also showed anti-viral and anti-tuberculosis properties (18, 19).
The results of the current research showed antifungal activities of herbal medicines against F. oxysporum. In particular, G. glabra extract presented bioactive compounds effective to inhibit the growth of F. oxysporum.
The authors wish to acknowledge their gratitude to Mr. Hasan Ahmadi from the University of Zabol for providing the research facilities.
- 1. Saremi H. Fusarium biology, ecology and taxonomy. Mashhad,Iran: Jahade Daneshghahi Press; 2005.
- 2. Ficcadenti N, Sestili S, Annibali S, Campanelli G, Belisario A, Maccaroni M, et al. Resistance to Fusarium oxysporum f. sp. melonis race 1,2 in muskmelon lines Nad-1 and Nad-2. Plant Dis. 2002;86:897-900. [DOI]
- 3. Banihashemi Z. The existence of Fusarium wilt of melon in Iran. Proc First Nat Cong. Plant Med. 1968;47-8.
- 4. Harris CA, Renfrew MJ, Woolridge MW. Assessing the risks of pesticide residues to consumers: recent and future developments. Food Addit Contam. 2001;18(12):1124-9. [DOI] [PubMed]
- 5. Jahani S, Saeidi S, Javadian F, Akbarizadeh Z, Sobhanizade A. Investigating the Antibacterial Effects of Plant Extracts on Pseudomonas aeruginosa and Escherichia coli. Int J Infect. 2016;3(2):34081. [DOI]
- 6. Broekaert WF, Terras FRG, Cammue BPA, Vanderleyden J. An automated quantitative assay for fungal growth inhibition. FEMS Microbiol Lett. 1990;69:55-60. [DOI]
- 7. Abril M, Curry KJ, Smith BJ, Wedge DE. Improved microassays used to test natural productbased and conventional fungicides on plant pathogenic fungi. Plant Dis. 2008;92:106-12. [DOI]
- 8. Eloff JN. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med. 1998;64(8):711-3. [DOI] [PubMed]
- 9. Fatima A, Gupta VK, Luqman S, Negi AS, Kumar JK, Shanker K, et al. Antifungal activity of Glycyrrhiza glabra extracts and its active constituent glabridin. Phytother Res. 2009;23(8):1190-3. [DOI] [PubMed]
- 10. Pellati D, Fiore C, Armanini D, Rassu M, Bertoloni G. In vitro effects of glycyrrhetinic acid on the growth of clinical isolates of Candida albicans. Phytother Res. 2009;23(4):572-4. [DOI] [PubMed]
- 11. Mohseni R, Nasrollahi A, Norbakhsh F, Rezaie S, Hosseinjani H. A Survey of the Effect of Licorice Plant Extract on aflR Gene Expression and Aflatoxin Production in Aspergillus Parasiticus via Real-time PCR. Modares J Med Sci. 2012;15(3):63-77.
- 12. Hojo H, Sato J. Antifungal Activity of Licorice (Glycyrrhiza glabra) and Potential Applications in Beverage Foods. J Food Ingredients. 2002;203:45-9.
- 13. Kim HK, Park Y, Kim HN, Choi BH, Jeong HG, Lee DG, et al. Antimicrobial mechanism of b-glycyrrhetinic acid isolated from licorice, Glycyrrhiza glabra. Biotechnol Lett. 2002;24:1899-902.
- 14. Chakotiya AS, Tanwar A, Narula A, Sharma RK. Effects of Glycyrrhiza glabra on membrane permeability and inhibition of efflux activity and biofilm formation in Pseudomonas aeruginosa and its in vitro time-kill activity.Alternative to antibiotics against Pseudomonas aeruginosa. Microb Pathog. 2016;98:98-105.
- 15. Krausse R, Bielenberg J, Blaschek W, Ullmann U. In vitro anti-Helicobacter pylori activity of Extractum liquiritiae, glycyrrhizin and its metabolites. J Antimicrob Chemother. 2004;54(1):243-6. [DOI] [PubMed]
- 16. Nariman F, Eftekhar F, Habibi Z, Falsafi T. Anti-Helicobacter pylori activities of six Iranian plants. Helicobacter. 2004;9(2):146-51. [DOI] [PubMed]
- 17. Shirazi MH, Ranjbar R, Eshraghi S, Sadeghi G, Jonaidi N, Bazzaz N, et al. An evaluation of antibacterial activity of Glycyrrhiza glabra extract on the growth of Salmonella, Shigella and ETEC E. coli. J Biol Sci. 2007;7:827-9. [DOI]
- 18. Fiore C, Eisenhut M, Krausse R, Ragazzi E, Pellati D, Armanini D, et al. Antiviral effects of Glycyrrhiza species. Phytother Res. 2008;22(2):141-8. [DOI] [PubMed]
- 19. Gupta VK, Fatima A, Faridi U, Negi AS, Shanker K, Kumar JK, et al. Antimicrobial potential of Glycyrrhiza glabra roots. J Ethnopharmacol. 2008;116(2):377-80. [DOI] [PubMed]