{"id":218,"date":"2015-01-20T08:15:55","date_gmt":"2015-01-20T08:15:55","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=218"},"modified":"2017-01-04T07:06:09","modified_gmt":"2017-01-04T07:06:09","slug":"antifungal-activity-of-the-essential-oil-of-cinamomum-zeynalicum-on-candida-albicans-aspergillus-niger-and-aspergillus-flavus","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol1no1\/antifungal-activity-of-the-essential-oil-of-cinamomum-zeynalicum-on-candida-albicans-aspergillus-niger-and-aspergillus-flavus\/","title":{"rendered":"Antifungal Activity of the Essential Oil of Cinamomum Zeynalicum on Candida Albicans, Aspergillus Niger and Aspergillus Flavus."},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Fungal infections have been increasing in recent years due to a growing number of high-risk patients, particularly immunocompromised hosts. Candida<\/em> is the third- or fourth-most-common isolate in nosocomial bloodstream infections in the USA. In addition, The mortality rate due to invasive aspergillosis increased by 357 % between 1980 and 1997 in the USA .In spite of the introduction of new antifungal drugs, they are limited in number. The increase of fungal resistance to classical drugs, the treatment costs, and the fact that most available antifungal drugs have only fungistatic activity, justify the search for new strategies (Eugenia pinto, 2006).<\/p>\n

Aromatic plants have been widely used in folk medicine. It isknown that most of their properties are due to their volatileoils. Essential oils from many plants are known to possess antifungalactivity, but only limited informationexists about activity toward human fungal pathogens. They havebeen empirically used as antimicrobial agents, but the mechanismsof action are still unknown.<\/p>\n

Some essential oils show an important antifungal activity against yeasts, dermatophyte fungi and Aspergillus<\/em> strains, which could predict therapeutic benefits, mainly for diseases with mucosal, cutaneous and respiratory tract involvement. (Pina-Vaz et al.<\/em>, 2004; Salgueiro et al.<\/em>, 2003, 2004), \u00a0<\/sup><\/p>\n

The objective of our present research was to evaluate the antifungal activity and investigate the mechanism of action of Cinamomum zeynalicum <\/em>\u00a0<\/strong>essential oil from Iran.<\/p>\n

Method<\/strong><\/p>\n

Essential oil analysis<\/strong><\/p>\n

Essential oil from the bark parts of the plant was obtained by hydro distillation . Cinnamon sticks were finely chopped before being boiled, and analysed by GC and GC-MS. Gas chromatography (GC) and GC\u2013mass spectrometry (MS) analysis of essential oil were performed.<\/p>\n

Plant material and chemicals<\/strong><\/p>\n

Bark parts of the plants were collected from Tokestan, 11th<\/sup> Km. Gorgan-Mashhad Road,Gorgan, Iran.<\/p>\n

Essential oil analysis<\/strong><\/p>\n

Essential oil was isolated by water distillation for 3\u00a0h from air-dried material, using a Clevenger-type apparatus, according to the procedure described in the European Pharmacopoeia (Council of Europe, 1997).<\/p>\n

Gas chromatography (GC\u2013FID): Gas chromatography analysis was performed on a Hewlett-Packard Model 5890 Series II gas chromatograph equipped with flame ionization detector and capillary column HP-101 (Methyl silicone fluid), 25\u00a0m\u00a0\u00d7\u00a00\u00b72\u00a0mm i.d., coating thickness 0\u00b72\u00a0\u03bc<\/em>m. Chromatographic conditions were as follows: helium as carrier gas at 1\u00b70\u00a0ml\u00a0min\u22121<\/sup>; injector and detector temperatures, 250\u00b0C and 300\u00b0C. Oven temperature was isothermal at 70\u00b0C for 2\u00a0min, then increased to 200\u00b0C, at a rate of 3\u00b0C min\u22121<\/sup> and held isothermal for 15\u00a0min. Volume injected 1\u00a0\u03bc<\/em>l. Split ratio 1\u00a0:\u00a050.<\/p>\n

Gas chromatography\u2013mass spectrometry: Essential oil was also analysed by Hewlett Packard GC\u2013MS (model 5890 series II) with mass selective detector (model 5971A). Two columns of different polarity were used: an HP-101 column (Methyl silicone fluid, Hewlett Packard; 25\u00a0m\u00a0\u00d7\u00a00\u00b72\u00a0mm i.d., film thickness 0\u00b72\u00a0\u03bc<\/em>m) and an HP-20M column (Carbowax 20M, Hewlett Packard; 50\u00a0m\u00a0\u00d7\u00a00\u00b72\u00a0mm i.d., film thickness 0\u00b72\u00a0\u03bc<\/em>m). Oven temperature was programmed as follows: isothermal at 70\u00b0C for 4\u00a0min, then increased to 180\u00b0C, at a rate of 4\u00b0C min\u22121<\/sup> and subsequently held isothermal for 15\u00a0min (for HP-20M column); isothermal at 70\u00b0C for 2\u00a0min, then increased to 200\u00b0C, at a rate of 3\u00b0C min\u22121<\/sup> and held isothermal for 15\u00a0min (for HP-101 column). Carrier gas was helium, flow rate: 1\u00a0ml\u00a0min\u22121<\/sup>; injector temperature: 250\u00b0C; volume injected: 1\u00a0\u03bc<\/em>l; split ratio: 1\u00a0:\u00a050. MS conditions: ionization voltage: 70\u00a0eV; ion source temperature: 280\u00b0C; mass range: 30\u2013300 mass units.<\/p>\n

Qualitative and quantitative determination: The individual peaks were identified by comparison of their retention indices to those of authentic samples, as well as by comparing their mass spectra with the Wiley 6\u00b70 library (Wiley, New York, NY, USA) and NIST98 (National Institute of Standards and Technology, Gaithersburg, MD, USA) mass spectral database and literature (Adams 1995).<\/p>\n

The percentage composition of the samples was computed from the GC peak areas by using the normalization method (without correction factors). Quantitative results are mean of data derived from duplicate GC-FID analyses.<\/p>\n

Isolation and detection of Fungi<\/h4>\n

The antifungal activity of Cinamomum zeynalicum <\/em>\u00a0essential oil was evaluated against Candid albicans<\/em> ATCC 10231 , Aspergillus niger<\/em> ATCC 9642 , and Aspergillus flavus<\/em> ATCC 9643.The fungal isolates were identified by standardmicrobiology methods and stored in Sabouraud dextrose brothwith glycerol at \u201370 \u00b0C.<\/p>\n

Fungi were plated on Sabouraud 2% (w\/v) glucose agar (SGA),\u00a0 and incubated at 25\u00a0\u00b1\u00a02\u00b0C for the 5\u20137\u00a0days.<\/p>\n

For the antifungal activity testing, Cinamomum zeynalicum <\/em>\u00a0<\/strong>essential oil was dissolved in 96% (v\/v) ethanol and then diluted with 30% (v\/v) ethanol in distilled water with 0\u00b71% (w\/v) Tween 80. Final concentrations of the essential oil was 2% (w\/v) .<\/p>\n

Antifungal activity testing by dilution method: Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined by using the serial broth dilution method described by Pepeljnjak et\u00a0al.<\/em> (2003). Serial twofold dilutions in DMSO, ranging from 0.02 to 20\u00a0\u00b5l\u00a0ml\u20131<\/sup>, were tested for essential oil. In addition, the reference antifungal compounds, fluconazole (Pfizer) for Candida albicans<\/em> or amphotericin B (Sigma) for Aspergillus<\/em>, were used as standard antifungal drugs. Twofold serial dilutions ranging from 0.25 to 128\u00a0\u00b5g\u00a0ml\u20131<\/sup> for fluconazole and 0.016 to 16\u00a0\u00b5g\u00a0ml\u20131<\/sup> for amphotericin B were used. Quality control determinations of the MICs of fluconazole and amphotericin B were performed by testing C. parapsilosis<\/em> ATCC 90018 and C. krusei<\/em> ATCC 6258. The results obtained were withinthe recommended limits.<\/p>\n

MIC is defined as the lowest concentration of extract or essential oil that allows no more than 20% growth of the fungus, visualized as a reduced number of colonies after removing the loop with approx. 10\u00a0\u03bc<\/em>l of each dilution, and then inoculated on SGA and incubated at 25\u00a0\u00b1\u00a02\u00b0C for 7\u00a0days. MFC is defined as the lowest concentration of essential oil that completely inhibited the growth of fungi. These experiments performed in duplicate were repeated independentlythree times and yielded essentially the same results. A rangeof values is presented where different results were obtained.Two growth controls, RPMI medium and RPMI with 2.0 % (v\/v) DMSO,were included for each strain.<\/p>\n

Statistics<\/h4>\n

The data obtained as MIC and MFC of \u00a0essential oil, expressed in \u03bc<\/em>g\u00a0ml\u22121<\/sup>, were statistically analysed by using the Wilcoxon matched pairs test. The level of P<\/em>\u00a0<\u00a00\u00b705 was considered statistically significant.<\/p>\n

Results and discussion<\/strong><\/p>\n

The oil was obtained from air-dried plant material in a yield of 1.8 % (v\/w).<\/p>\n

Evaluation of MIC showed that the oil was active against all the tested strains (Table\u00a01). Cinamomum zeynalicum <\/em>essential oil exhibited significant antifungal activity. MIC value 0.125\u00a0\u00b5l\u00a0ml\u20131<\/sup> against Aspergillus<\/em> strains and Candida<\/em>. It is difficult to attribute the activity of a complex mixture to particular constituents. The importance of the phenolic hydroxyl groups for the antimicrobial activity of the monoterpenoids has previously been reported (Adam et al.<\/em>, 1998; Aligiannis et al.<\/em>, 2001; Dorman & Deans, 2000; Nostro et al.<\/em>, 2004; Sivropoulou et al.<\/em>, 1996).<\/p>\n

Table 1:\u00a0Antimicrobial activity (MIC) and (MFC) of the essential oil of the Cinamomum zeynalicum <\/em>\u00a0for Candida albicans<\/em>, Aspergillus niger<\/em> and Aspergillus flavus.<\/em><\/strong><\/p>\n

Results were obtained from three independent experiments performed in duplicate.<\/p>\n\n\n\n\n\n
Candida albicans<\/em>,<\/strong><\/td>\nAspergillums niger<\/em><\/strong><\/td>\nAspergillus flavus<\/em><\/strong><\/td>\nCinamomum zeynalicum <\/em>\u00a0effects<\/strong><\/td>\n<\/tr>\n
0.125<\/td>\n0.125<\/td>\n0.125<\/td>\nMIC in \u03bc<\/em>g\u00a0ml\u22121<\/sup><\/td>\n<\/tr>\n
0.25<\/td>\n0.25<\/td>\n0.25<\/td>\nMFC in \u03bc<\/em>g\u00a0ml\u22121<\/sup><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

In conclusion, the findings of the present study indicate that Cinamomum zeynalicum <\/em>\u00a0<\/strong>essential oil has potential as a topical antifungal agent against fungi that are pathogenic to humans. This essential oil is a broad-spectrum agent that inhibites Aspergillus<\/em> and Candida<\/em> species,<\/p>\n

Given the results described above, particularly the possiblemechanisms of action, which might induce side-effects in humans,these antifungals require further investigation.<\/p>\n

The results presented should stimulate studies on toxicity,improved formulations and the determination of optimal concentrationsfor clinical applications, as well as comparative studies alongsidecurrently used drugs of the therapeutic efficacy of essentialoils to control infections.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Adam, K., Sivropoulou, A., Kokkini, S., Lanaras, T. & Arsenakis, M. (1998). Antifungal activities of Origanum vulgare<\/em> subsp. hirtum<\/em>, Mentha spicata<\/em>, Lavandula angustifolia<\/em>, and Salvia fruticosa<\/em> essential oils against human pathogenic fungi. J Agric Food Chem<\/em> 46, 1739\u20131745.[CrossRef]<\/li>\n
  2. Adams, R. P. (1995). Identification of Essential Oil Components by Gas Chromatography\/Mass Spectroscopy<\/em>. Carol Stream, IL: Allured Publishing Corporation.<\/li>\n
  3. Aligiannis, N., Kalpoutzakis, E., Mitaku, S. & Chinou, I. B. (2001). Composition and antimicrobial activity of the essential oils of two Origanum<\/em> species. J Agric Food Chem<\/em> 38, 4168\u20134170.[CrossRef].<\/li>\n
  4. Council of Europe (1997). Methods of Pharmacognosy. In European Pharmacopoeia<\/em>, 3rd edn, pp.\u00a0121\u2013122. Strasbourg: European Department for the Quality of Medicines.<\/li>\n
  5. Dorman, H. J. & Deans, S. G. (2000). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol<\/em> 88, 308\u2013316.[CrossRef][Medline]<\/li>\n
  6. Nostro, A., Blanco, A. R., Cannatelli, M. A., Enea, V., Flamini, G., Morelli, I., Roccaro, A. S. & Alonzo, V. (2004). Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol. FEMS Microbiol Lett<\/em> 230, 191\u2013195.[CrossRef][Medline]<\/li>\n
  7. Pepeljnjak, S., Kosalec, I., Kalodera, Z. and Ku\u0161trak, D. (2003) Natural antimycotics from Croatian plants. In Plant-Derived Antimycotics ed. Rai, M. and Mares, D. pp. 49\u201381. Binghampton<\/em>: The Haworth Press.<\/li>\n
  8. Pina-Vaz, C., Rodrigues, A. G., Pinto, E., Costa-de-Oliveira, S., Tavares, C., Salgueiro, L. R., Cavaleiro, C., Gon\u00e7alves, M. J. & Martinez-de-Oliveira, J. (2004). Antifungal activity of Thymus<\/em> oils and their major compounds. J Eur Acad Dermatol<\/em> 18, 73\u201378.<\/li>\n
  9. Pinto, E., Pina-Vaz, C ., Salgueiro, L. R., Gon\u00e7alves, M. J., Costa-de-Oliveira, S., Cavaleiro, C., Palmeira, A., Rodrigues , A. & Martinez-de-Oliveira, J. (2006). Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species.J Med Microbiol 55,1367-1373.<\/li>\n
  10. Salgueiro, L. R., Cavaleiro, C., Pinto, E. & 7 other authors (2003). Chemical composition and antifungal activity of the essential oil of Origanum virens<\/em> on Candida<\/em> species. Planta Med<\/em> 69, 871\u2013874.[CrossRef][Medline]<\/li>\n
  11. Salgueiro, L. R., Pinto, E., Gon\u00e7alves, M. J. & 7 other authors (2004). Chemical composition and antifungal activity of the essential oil of Thymbra capitata<\/em>. Planta Med<\/em> 70, 572\u2013575.[CrossRef][Medline]<\/li>\n
  12. Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S., Lanaras, T. & Arsenakis, M. (1996). Antimicrobial and cytotoxic activities of Origanum<\/em> essential oils. J Agric Food Chem<\/em> 44, 1202\u20131205.[CrossRef]<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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