Journal of Food Protection. Vol. 58. No. 10, Pages lJ47-1149 Copyright". International Association of Milk. FOOd and Environmental Sanitarians

A Research Note

Antifungal Activity of the Essential Oil of syriacum L.

RASHA K. DAOUK, SHAWKY M. DAGHER* and ELSA J. SATTOUT

Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon; and Faculty of Agriculture, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/58/10/1147/1665570/0362-028x-58_10_1147.pdf by guest on 26 September 2021 Universite St. Esprit, Kaslik, Lebanon

(MS# 94-292: Received 13 December 1994/Accepted 29 March 1995)

ABSTRACT major constituent, while the other is rich in its stereoisomer, carvacrol (11). The volatile oil of the Lebanese Za' atar (Origanum syriacum L.) was characterized for its thymol and carvacrol content using gas- Za'atar has a very strong but pleasant aroma imparted to liquid chromatography. These two compounds constituted the major it by its high content of essential oils. Many such oils are components of the oil and were present in equal proponions of 30% known for their antifungal activity. A recent study on the in the volatile oil extracted from the leaves and shoot tips of the antifungal activity of the essential oils of five tropical Origanum plant during the preflowering stage. The percentage of species, Apium leptophylum, Bidens formosa, Blumea carvacrol in the essential oil increased to 62% after flowering and decurrens, Conyza summasterensis and Tagetes minuta, maturation, while the concentration of thymol decreased to 14%. against the black mold Aspergillus niger reported that con- Origanum oil extracted from collected during midseason was centrations as low as 1 Ill/ml growth medium of anyone of evaluated for its antifungal activity against Aspergillus niger, these oils showed marked antifungal activity (8). A similar Fusarium oxysporum, and Penicillium species. The oil exhibited study was conducted on A. parasiticus using the essential oils strong inhibitory action against the three fungi tested. The minimum of several plants; it was shown that cumin and thyme oils inhibitory concentration (MIC) of the oil was found to be 0.1 Ill/ml produced a highly significant decrease in both mycelial growth of yeast extract sucrose broth for the fungi tested. and aflatoxin production when used at a concentration of 0.2 mg/ml and completely inhibited growth at higher levels (9). Key words: Essential oil, Origanum syriacum, antifungal activity, Another study performed to evaluate the antifungal activity of Za'atar mmjoram oil (from Origanum majorana L.) on several As- pergillus species demonstrated differing degrees of inhibition The aromatic plant Origanumsyriacum L., also known as of the various molds, with A. niger showing a higher degree of Majorana syriaca L. Rafin (17, 18) belongs to the Labiatae susceptibility in its growth compared toA, flavus, A. ochraceus family and is one ofthe type of herbs. In the Middle or A. parasitic us (7). The oil of marjoram was also studied East where it grows abundantly it has the common name of along with the phenols thymol and carvacrol against Trichophyton concentricum, T. mentagraphytes and Za' atar and is usually collected for human consumption. The T. rub rum. In this investigation the agar overlay technique leaves and fiowers are dried, ground, and mixed with pow- was used; the results obtained showed that all three organisms dered seed coats of (Rhus coriaria L.) which serves as were strongly inhibited by the oil and by either one of the two an acidulant and imparts a reddish brown color to the mixture. phenolic compounds used (21). Studies done on Aspergillus This mixture is a very popular breakfast food that is consumed parasiticus using thymol alone showed that a concentration by all sectors of the population in Lebanon, particularly in the as low as 10 Ilg/ml produced a transitory inhibition of growth form of a baked pie after adding some olive oil to it. It is also in this mold (2). used in cooking as a spice or condiment. A comparative study of the antifungal activity of essen- Several investigations and chemical analyses were car- tial oils extracted from thyme, rosemary, eucalyptus, and ried out on the essential oils of O. syriacum and other related three chemotypes of mug wort was carried out by a group of plants of the Labiatae family to determine their constituents investigators against 39 mold strains. These workers seeded (10,11,12,20). The composition of these oils was found to agar plates with the test organisms and incubated them in an vary with geographical origins, climatic conditions, stage of upside-down position. The essential oil of thyme was found to plant maturity, and degree of freshness of the plant material be the most effective. It caused total inhibition of most of the analyzed (13,19,20). The essential oil of O. syriacum exists molds when 5 III were placed on a filter paper in the middle of in two phenolic chemotypes, one containing thymol as the the petri dish covers (1).

JOURNAL OF FOOD PROTECTION, VOL. 58, OCTOBER 1995 1148 DAOUK, DAGHER AND SATTOUT

Essential oils from other plants such as the African Assessment of inhibition of fungal growth wormwood (Artemisia afra Jacq.) (14), the lavender tree The fungal spores were washed from the surface of agar plates (Heteropyxis natalensis) (15) and sweet gale (Myrica gale) with sterile 0.75% saline containing 0.1 % Tween 80 (vol/vol) (23). (3) have been demonstrated to have inhibitory effects against The spore suspension was filtered through sterile cheesecloth and a broad spectrum of fungal species. Yeasts were also shown to adjusted with sterile saline to a concentration of approximately 1.0 x be inhibited by the oils of garlic, allspice, cinnamon, clove, 105 spores per ml (7) with the aid of a hemocytometer. The inocula onion, oregano, and thyme (4). were stored at 4°C for further use. Dilutions of the inocula were The purpose of this study was to estimate the quantities of cultured on solid PDA to verify the absence of contamination and carvacrol and thymol found in the Lebanese Za'atar and to to check the validity of the inoculum (16). A O.5-ml portion of the investigate the antifungal activities of the volatile oil of this spore suspension was added to a 250-ml Erlenmeyer flask containing plant. The minimum inhibitory concentration (MIC) of the oil 25 ml of YES broth. Four such flasks were prepared for each oil was also determined for each of the organisms used in this dilution per organism. The essential oils extracted from plant samples study. collected during the month of July were added to the flasks at a concentration ranging from 0.01 to 5 Ill/ml of medium. The flasks

MATERIALS AND METHODS which received no essential oil ofZa' atar were used as controls. All Downloaded from http://meridian.allenpress.com/jfp/article-pdf/58/10/1147/1665570/0362-028x-58_10_1147.pdf by guest on 26 September 2021 flasks were incubated at 30 ± 1°C in the dark for 6 days on a shaking Distillation and characterization of the oil ofZa'atar incubator (New Brunswick Scientific Co., Inc., Edison, NJ, USA) Four samples of leaves and flowers of the Za' atar plant were with a shaking frequency of 100 cycles per minute (23). After collected from one location on the hills of Lebanon during the months incubation, the mycelia produced were collected by filtration under of April, July, August, and October 1993. The samples were dried in vacuum through Whatman no. I filter paper (7 cm in diameter). The the shade, and the stems and petioles were removed. Distilled water collected mycelia were washed three times with sterile distilled was added to the dried leaves and flowers at a ratio of 6: 1 (wt/wt) and water and dried overnight in an oven at 105°C (9). the mixture was distilled by simple distillation using a I-I round- The level of inhibition relative to the control was calculated bottomed flask. The distillate was collected in a glass cylinder and using the following formula: % Inhibition = [(C - T)/C] x 100, where the essential oil layer that floated on the surface of the distillate was C = mean weight of mycelia from control flasks and T = mean weight collected and stored at 4°C until used. of mycelia from test flasks (6). The components of the essential oil were separated by gas- The values reported for percentage inhibition were the means of liquid chromatography on a Perkin Elmer Autosystem gas chromato- four determinations. The minimum inhibitory concentration (MIC) graph. The stainless steel column, 6 ft. with 1I8-inch diameter, was of the oil was considered to be the lowest concentration of the test oil packed with carbowax 20 M (10%) coated on chromosorb W (1001 that prevented visible growth and gave 100% inhibition on a mycelial 120). The injection port temperature was maintained at 240°C and weight basis (22). the detector was kept at 250°C. The column temperature was programmed from 75° to 178°C in two stages according to the RESULTS AND DISCUSSION following program: Tl' 75°C; T2' 125°C, T3' 178°C; and initial time, 5 min; intermediate time, 0 min; and final time, 28 min. Rate lmponance of Za' atar and the phenolic compounds in its I was 6°C Imin and rate 2 was 8°C Imin. Nitrogen was used as the essential oil carrier gas and was maintained at a head pressure of 30 Ib/in2 and a In this study it was found that the essential oil content of flow rate of 30 ml/min. The attenuation used was 32 and the range the edible part of the Za'atar plant (Origanum syriacum L.) was 20. The volume of the sample injected into the chromatograph was about 3% (wt/vol) and that the major components of this was 2 III of 1% thyme oil containing 0.2% anisaldehyde which oil were carvacrol and thymol. These ingredients were present served as the internal standard. The solvent used was hexane and at equal levels in the plant during the preflowering stage in samples were assayed in triplicate. The values for the percentage area early spring. Each of these compounds constituted about 30% ratios for the major components of the oil to that of the internal (wt/wt) of the volatile oil. As the plant matured in late spring standard were within the linear portions of the standard curves and early summer the percentage of carvacrol in its oil obtained for these components. increased until it reached 62 % towards the end of the growing season, while the level of thymol decreased to 4% during Fungal strains August and then increased to 14% during October. The The fungi used in this study were Aspergillus niger, Penicillium origanum oil sample that was used for the assessment of spp. and Fusarium oxysporum. The first two test organisms are fungal inhibition was found to contain 59% carvacrol and considered to be food spoilage organisms while Fusarium oxysporum 10% thymol. is a phytopathogenic fungus. These organisms were obtained from the Department of Plant Patholo gy, Faculty of Agricultural and Food The antifungal effectiveness of the essential oil Sciences, at the American University of Beirut. The volatile oil of Lebanese Za'atar showed inhibitory effects on the three fungi tested when present in concentra- Media used tions as low as 0.01 ~l/ml of broth (Table 1). At an oil The three fungi were maintained on potato dextrose agar (PDA) concentration of 0.05 ~Uml of broth, the most susceptible and the cultures were stored at room temperature and subcultured organism was Penicillium sp., which showed 93% inhibition. once a month (7). The medium used for the susceptibility test was The other organisms tested, namely Aspergillus niger and yeast extract sucrose broth (YES) prepared by adding 20% sucrose Fusarium oxysporum, showed more than 50% inhibition at to 2% yeast extract (5). Both PDA and YES media were obtained the same concentration of the volatile oil. A concentration of from Difco Laboratories Inc. (Detroit, MI). 0.1 )..lUmlbroth completely inhibited the mycelial growth of all

JOURNAL OF FOOD PROTECTION, VOL. 58, OCTOBER 1995 ANTIFUNGAL ACTIVITY OF ORIGANUM OIL 1149 organisms tested; this was taken to represent the MIC value. 4. Conner, D. E., and L. R. Beuchat. 1984. Effects of essential oils from This low value obtained for MIC indicates a strikingly potent plants on growth offood spoilage yeasts. J. Food Sci. 49:429-434. 5. Davis, N. D., U. L. Diener, and D. W. Edridge. 1966. Production of effect of the Za' atar oil against the growth of molds. Aflatoxins B and G1 by Aspergillusflavus in semi-synthetic mediums. An earlier study on thymol rich thyme-oil reported a Appl. Microbiol. 14:368-380. minimum inhibitory concentration of 0.4 mg/ml of the oil 6. Deans, S. G. 1991. Evaluation of antimicrobial activity of essential against Aspergillus parasiticus (9). Also, A. niger was re- (volatile) oils; p. 309-320. In H. G. Liskens and J. F. Jackson (ed.), ported to show a 99% inhibition with the essential oil of Essential oils and waxes, vol. 12. Springer-Verlag, Berlin. 7. Deans, S. G. and K. P. Svoboda. 1990. The Antimicrobial Properties of Heteropyxis natalensis (15) and 89% inhibition with marjo- Majorarn (Origanum ITUljoramL.) volatile oils. Flav. Fragr. J. 5:187 -190. ram oil at a concentration of 10 Ill/ml (7). Another study 8. Deans, S. G., K. P. Svoboda, M. Gundidza, and E. Brechany. 1992. Essential showed that a concentration of 10 Ill/ml of the volatile oil oil profiles of several temperate and tropical aromatic plants: their antimicro- from A rtemisia afra Jacq. was needed to cause 92% inhibition bial and antioxidant activities. Acta Hort. 306:229-232. in the growth of Fusarium culmorum (14). The more potent 9. Farag, R, Z. Daw, and S. Abo-Raya. 1989. Influence of some spice essential oils on Aspergillus parasiticus growth and production of effect observed in our oil samples may have been due to its afIatoxins in a synthetic medium. J. 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