Essential Oil Constituents of Leaves and Fruits of Myrtus communis L. from Iran

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1 ISSN X Essential Oil Constituents of Leaves and Fruits of Myrtus communis L. from Iran Maryam Pezhmanmehr 1, Dara Dastan 2, Samad Nejad Ebrahimi 2 and Javad Hadian 2 * 1 Department of Horticultural Sciences, Faculty of Agriculture, University of Tehran, Karaj , Iran 2 Medicinal Plants and Drug Research Institute, Shahid Beheshti University, G.C. Evin, Tehran , Iran Abstract: Myrtus communis L. is an evergreen aromatic plant growing wild in Iran. Leaf samples were collected from two origins and in two different growth stages. Myrtle fruits were collected at full ripening stage. The oils of all samples were obtained by hydrodistillation using a Clevenger-type apparatus and their constituents were analyzed by GC and GC-MS. The major oil components of leaves of two origins at flowering stage were α-pinene ( %), 1,8-cineole ( %), limonene ( %), linalool ( %) and α-terpinyl acetate ( %). The leaf oil compositions at fruit ripening stage was highly similar to those of flowering stage as 1,8-cineole (24.0 %), α-pinene (22.1 %), limonene (17.6 %), linalool (11.4 %), linalyl acetate (4.5 %), α-terpinyl acetate (2.2 %), and geranyl acetate (1.2 %) were main components. Major constituents of fruit oil were α-pinene (28.6 %), 1,8-cineole (26.7 %), limonene (18.0 %), α-terpinyl acetate (5.4 %), linalyl acetate (3.4 %) and linalool (2.3 %). Key words: Myrtus communis, essential oil, leaves, fruits. Introduction: Myrtus communis L. (Myrtaceae) is a typical evergreen shrub of the Mediterranean, which grows wild in Africa, Europe and western parts of Asia 1. Myrtle is cultivated elsewhere as a garden plant and in some countries like Tunesia, Spain, France and Italy for oil production 2. Myrtle is wildly distributed in sub-tropical areas in north and south of Iran. The lanceolate leaves of Myrtle are dark glossy green and strongly scented. The flowers are fragrant and white or pinkish. The fruit is a round, reddish blue to violet berry 1. The ancient Persians regarded Myrtle as a holy plant and in pharaonic Egypt women *Corresponding author (Javad Hadian) E- mail: < javadhadian@gmail.com >

2 wore its blossoms together with Punica granatum on festive occasions 2. Dioscorides prescribed extract of the leaves and berries for lung and bladder infections 2. In Europe, the leaves and flowers were the major ingredients of a skin lotion known as angel s water. Herbal drugs of M. communis are used in folk medicine because of its astringent and balsamic properties 2, 3. The ripe berries have high vitamin contents. The decoction of leaves and fruits are useful for sore washing also for enemas and against respiratory diseases 4. The essential oil obtained from Myrtle has been widely investigated 5, 6, 7. Its composition is quite variable depend on the origin. The main constituents of myrtle essential oil are normally α-pinene, 1,8-cineole and myrtenyl acetate 5. Myrtle berry oil is not produced commercially but is used in bitters and certain liqueurs. Berries contain essential oils and fatty acids 2. Major component of oil are monoterpenes such as 1,8-cineole, geranyl acetate and linalool and no myrtenyl acetate while predominant fatty acids are linoleic, palmitic and oleic acids 2, 6, 8. The oil and extracts of M. communis possess several antimicrobial, antioxidant and other biological activities 10, 11, 12. The essential oil and different extracts of M. communis are used in Iran as major ingredient of several drugs such as Aftoplex, Myrtenol, Rectol, Belfarol, Myrtoplex, etc. In present work, essential oil constituents of leaves (at flowering and fruit ripening stages) and fruits of M. communis from Iran are reported. Material and methods Plant material: The aerial parts of M. communis were collected during flowering stage in July from two locations in Booshehr and Lorestan provinces of Iran. Beside leaves and fruit were collected at fruit ripening stage from Lorestan province. All plant materials were dried in shade at room temperature. Voucher specimen has been deposited in the Medicinal Plants and Drugs Research Institute Herbarium (MPRIH), Shahid Beheshti University, Tehran, Iran. Oil Isolation: The oil was obtained by hydrodistillation of air-dried leaves and fruits using a Clevenger-type apparatus for 3 h. The oil was dried over anhydrous sodium sulfate and stored in tightly closed dark vials at +4 C until the analysis. GC analysis: GC analysis was performed by using a Thermoquest-Finnigan gas chromatograph with a flame ionization detector (FID). The analysis was carried out using a fused silica capillary DB-5 column (60 m 0.25 mm i.d.; film thickness 0.25 μm). The operating conditions were as follows: injector and detector temperatures were 250 C and 300 C, respectively. Nitrogen was used as the carrier gas at a flow rate of 1 ml/min; oven temperature was programmed from 60 C-250 C at the rate of 5 C/min, and finally held isothermally for 10 min. Split ratio was 1/50. GC-MS analysis: GC-MS analysis was performed by using a Thermoquest-Finnigan gas chromatograph equipped with above mentioned column and coupled to a TRACE mass quadrapole analyzer. Helium was used as the carrier gas with ionization voltage of 70 ev. Ion source and interface temperatures were 200 C and 250 C, respectively. Mass

3 range was from m/z Gas chromatographic conditions were as given for GC. The constituents of the oil were identified by calculation of their retention indices under temperature programmed conditions for n-alkanes (C6-C24) and the oil on a DB-5 column under the same chromatographic conditions. Identification of individual compounds was made by comparison of their mass spectra with those of the internal reference mass spectra library or with authentic compounds and confirmed by comparison of their retention indices with authentic compounds or with those reported in the literature 13. For quantification purpose, relative area percentages obtained by FID were used without the use of correction factors. Results: The oil yield of leaf sample collected at flowering stage from Booshehr province was 1.8 %. The oil yield of samples collected from Lorestan province obtained 2.61 % for leaves at flowering stage, 1.3 % for leaves at fruit ripening stage and 0.17 % for fruits. The oils had a whitish yellow color with distinct sharp odor. Qualitative and quantitative analytical results are shown in Table 1 along with the retention indices of the identified compounds, where all constituents are listed in order of their elution from the DB-5 column. Thirty three constituents were identified among all analyzed oils. The essential oil profiles of two samples collected at flowering stage from Booshehr province and Lorestan were relatively different in quality and quantity. Major oil components of two origins at flowering stage were α-pinene ( %), 1,8-cineole ( %), limonene ( %), linalool ( %) and α-terpinyl acetate ( %). Oil components of leaves sample collected from Lorestan at fruit ripening stage was similar to those of identified at flowering stage. Concentrations of major oil components were 1,8-cineole (24.0 %), α-pinene (22.1 %), limonene (17.6 %), linalool (11.4 %), α- linalyl acetate (4.5 %), terpinyl acetate (2.2 %), and geranyl acetate (1.2 %). The oil composition of fruits was similar to those of leaves. Major constituents of fruit oil were α- pinene (28.6 %), 1,8-cineole (26.7 %), limonene (18.0 %), α-terpinyl acetate (5.4 %), linalyl acetate (3.4 %) and linalool (2.3 %). Discussion: The oil content of Myrtle obtained in this study was higher than those of previously reported. Oil yield of aerial parts of M. communis from Italy and Turkey has been reported to be 0.33 % and 0.38 % 14. Akgul and Bayrak 15 reported that the essential oil content of air-dried leaves of plants growing wild in Antalya, Balikesir and Sinop yielded 0.9, 0.6 and 0.6 %, respectively. Wannes et al 16 reported that oil yield of Myrtle fruit varied during maturation from % to 0.01 % and showed a remarkable increase at 60 days after flowering to reach a maximum of 0.11 %. Up to now, the essential oils obtained from M. commonis from different parts of the world have been widely investigated. The essential oil composition of M. communis from two different geographical origins from Italy has been reported 17. The main identified compounds were α-pinene ( %), 1,8-cineole ( %), limonene ( %) and trans-myrtanol acetate ( %). linalool (11.7 %) was present in higher concentration in one of samples and α-terpineol (3.6 %) in another one. Gardeli et al 18 reported that during flowering stage essential oil of Myrtle contain high oxygenated

4 monoterpene fraction which is responsible for strong antioxidant activity of the oil. Laurentis, et al. 19 studied seasonal variation of chemical composition in M. communis essential oil. Monoterpene hydrocarbons were the main compounds of Myrtle essential oil (37.1 %), with α-pinene (30.6 %) as the dominant constituent. Oxygenated monoterpenes were presented in less amount (32.1 %), with eucalyptol as the main compound (20.6 %), followed by sesquiterpenes (6.6 %), with caryophyllene (4.7 %) as the main constituent. Monoterpene hydrocarbons reached maximum values in February and minimum values in September. Oxygenated monoterpenes showed maximum and minimum values in June and October, respectively. Sesquiterpenes showed maximum values in July. Mentioned data suggested a high variability of chemical composition during the collection period, with the maximum values generally observed in spring. The composition of the essential oils from aerial parts of M. communis from Italy and Turkey has been reported 14. The Italian oil was characterized by α-pinene (15.7 %) and 1,8-cineole (16.5 %). Linalool (36.5 %) and linalyl acetate (16.3 %) were the most abundant components in the Turkish oil. The Turkish oil showed more antibacterial activity compared to the Italian oil. AnLian and Lei 3 reported myrtenyl acetate (35.3 %), linalool (18.5 %), 1,8-cineole (15.9 %), α-pinene (10.6 %) and myrtenol (4.5 %) as major components of dry leaves during fructification. Messaoud, et al. 20 studied variability of the essential oil composition in natural populations of M. communis in Tunisia. The major constituents were α-pinene (19.2 %) and 1,8-cineole (16.0 %). The other important constituents were linalool (7.7 %), α-terpineol (7.5 %) and limonene (5.8 %). The percentage of all components varied within and among populations according to their bioclimatic appurtenance. α-pinene, camphene and 1,8-cineole were the main compounds to differentiate the populations according to the bioclimate. Akgul and Bayrak 15 studied oil composition of Myrtle leaves from different origins of Turky. The major components of Myrtle leaves growing wild in Antalya were α- pinene (40.0 %), limonene (26.3 %) and linalool (13.1 %), of the Balikesir sample were linalool (32.2 %), limonene (22.0 %) and linalyl acetate (14.4 %) and of the Sinop sample were α-pinene (28.8 %), linalool (20.1 %) and limonene (10.3 %). Myrtenol (7.7 %) and myrtenyl acetate (6.5 %) were detected in the Sinop oil. Rasooli, et al. 11 reported α-pinene (29.4 %), limonene (21.2 %), 1,8-cineole (18.8 %), linalool (10.6 %), linalyl acetate (4.6 %) and α-terpineole (3.1 %) as the major components of Myrtle oil from Iran. The high monoterpenes hydrocarbons such as α-pinene and limonene seem to contribute to the strong antimicrobial activity of M. communis 11. Myrtle fruit essential oil has been reported by Wannes et al. 16. Forty-seven volatile compounds were identified of which 1,8-cineole ( %), geranyl acetate ( %), linalool ( %) and α-pinene ( %) were the main monoterpene compounds. The most representative components of the essential oil of the fruits from two different geographical origin of Italy were α-pinene ( %), 1,8-cineole ( %), limonene ( %) and trans-myrtanol acetate ( %) 21. Myrtle oils can be separated into two groups according to the amounts of myrtenyl acetate. In several papers 22, 23, 24 myrtenol, myrtenal and myrtenyl acetate have been reported in high concentrations among the constituents of the essential oil of leaves and fruits of M. communis.

5 In present study, no myrtenol and myrtenyl acetate was identified in the oil of leaves at flowering stage. Myrtenyl acetate was present in the oil of leaves collected from Booshehr province in a concentration of 1.1 %. Oil composition was relatively different across different origins, may be due to different geographic conditions of studied populations. Monoterpene hydrocarbons constitute 43.3 % of the oil of Lorestan sample and 9.4 % of the Booshehr sample. Oxygenated monoterpenes represented in a concentration of 46.7 % and 67.6 % of oil of Lorestan and Booshehr provinces, respectively. Regarding wide distribution of M. communis in north and south of Iran, the study of oil of populations growing in different ecological conditions, is recommended. Acknowledgments: We are grateful to Shahid Beheshti University Research Council for financial support of this work. References 1. Rechinger, K.H. (1982). Flora Iranica. Akademische Druck and Verlagsanstalt, Graz, Austria. 2. Weiss, E.A. (1997). Essential Oil Crops. CAB International, 600 pp. 3. Zargari, A. (1989). Medicinal Plants. Tehran University, Vol. 4: 969 pp. 4. Marchini, G., and Maccioni, S. (1998). Liguria in parole povere. La bassa Val di Magra. Genova: Sagep. 5. Lawrence, B.M. (1996). Progress in essential oils. Myrtle oil. Perfumer and Flavorist, 21: Chalchat, J., Garry, R.P. and Michet, A. (1998). Essential Oils of myrtle (Myrtus communis L.) of the mediterranean littoral. J. Essent. Oil Res., 10: Bradesi, T.P., Casanova, J., Costa, J. and Bernardini, A.F. (1997). Chemical composition of myrtle leaf oil from Corsica (France). J. Essent. Oil Res., 9: Cakir, A. (2004). Essential oil and fatty acid composition of the fruits of Hippophae rhamnoides L. (Sea Buckthorn) and Myrtus communis L. from Turkey. Biochem. Sys. and Ecol., 32(9): Weyerstahl, P., Marschall, H. and Rustaiyan, A. (1994). Constituents of the essential oil of Myrtus communis L. from Iran. Flav. and Frag. J., 9: Elfellah, M.S., Akhter, M.H. and Khan, M.T. (1984). Anti-hyperglycaemic effect of an extract of Myrtus communis in streptozotocin-induced diabetes in mice. J. Ethnopharmacol., 11: Rasooli, I., Moosavi, M.L., Rezaee, M.B., Jaimand, K. (2002) Susceptibility of microorganisms to Myrtus communis L. essential oil and its chemical composition. J. Agri. Scie. and Tech. 4(3/4): Romani, A., Coinu, R., Carta, S., Pinelli, P., Galardi, C., Vincieri, F.F. and Franconi, F. (2004). Evaluation of antioxidant effect of different extracts of Myrtus communis L. Free Radic Res., 38: Adams, R.P. (1995). Identification of essential oil components by gas chromatography/mass spectroscopy. Carol Stream: Allured.

6 14. Senatore, F., Formisano, C., Napolitano, F., Rigano, D., Ozcan, M. (2006). Chemical composition and antibacterial activity of essential oil of Myrtus communis L. growing wild in Italy and Turkey. J. Essent. Oil Bearing Plants, 9(2): Akgul, A., Bayrak, A. (1989). The essential oil content and composition of Myrtle (Myrtus communis L.) leaves. Doga, Turk Tarim ve Ormancilik Dergisi, 13(2): Wannes, W.A,. Mhamdi, B., Marzouk, B. (2009). Variations in essential oil and fatty acid composition during Myrtus communis var. italica fruit maturation. Food Chemistry, 112(3): Flaminia, G., Cionia, P. L., Morellia, I., Maccionib, S. and Baldini, R. (2004). Phytochemical typologies in some populations of Myrtus communis L. on Caprione Promontory (East Liguria, Italy). Food Chem., 85: Gardeli, C., Vassiliki, P., Athanasios, M., Kibouris, T., Komaitis, M. (2008). Essential oil composition of Pistacia lentiscus L. and Myrtus communis L.: Evaluation of antioxidant capacity of methanolic extracts. Food Chem., 107(3): AnLian, Z., Lei, Y. (2006). Composition analysis of essential oil of Myrtus communis L. in different vegetal periods and in dry and fresh leaves. Journal of Shanghai Jiaotong University Agricultural Science, 24(4): Messaoud, C., Zaouali, Y., Ben Salah, A., Khoudja, M.L. and Boussaid, M. (2005). Myrtus communis in Tunisia: variability of the essential oil composition in natural populations. Flavour Fragr. J., 20: Laurentis, N. de., Rosato, A., Morlacchi, F., Armenise, D., Leone, L., Milillo, M. A. (2006). Seasonal variation of chemical composition in Myrtus communis essential oil. Rivista Italiana EPPOS, 42: Asllani, U. (2000). Chemical composition of albanian myrtle oil (Myrtus communis L.). J. of Essent. Oil Res., 12: Boelens, M.H. and Jimenez, R. (1992). The chemical composition of Spanish myrtle oils. J. of Essent. Oil Res., 4: Ozek, T., Demirci, B. and Baser, K.H.C. (2000). Chemical composition of Turkish myrtle oil. J. of Essent. Oil Res., 12:

7 Table 1. Constituents of the essential oil composition of Myrtus communis L. No. Component RI Lorestan Booshehr Leaves fruits flowering ripening flowering 1 E-2-Hexenal Isobutyl isobutyrate α-thujene α-pinene β-pinene Myrcene α-phellandrene Δ 3 -Carene α-terpinene p-cymene Limonene ,8-Cineole trans-β-ocimene Methyl acetophenone Terpinolene Linalool Terpineol α-terpineol p-arill arisol Linalyl acetate Methyl citronella Methyl citronellate Myrtenyl acetate α-terpinyl acetate Neryl acetate Geranyl acetate Methyl eugenol trans-caryophyllene α-humulene Cinerolon Flavesone Caryophyllene oxide Humulene oxide Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others Total Oil-content (%)