Chemical variability of peel and leaf essential oils of sour orange

Transcription

1 FLAVOUR AND FRAGRANCE JOURNAL Flavour Fragr. J. 2001; 16: Chemical variability of peel and leaf essential oils of sour orange Marie-Laure Lota, 1 Dominique de Rocca Serra, 1 Camille Jacquemond, 2 Félix Tomi and Joseph Casanova 1 1 Université de Corse, Equipe Chimie et Biomasse, URA CNRS 2053, Route des Sanguinaires, Ajaccio, France 2 SRA INRA-CIRAD, San Ghjuliano, France Received 31 March 2000 Revised 31 July 2000 Accepted 11 August 2000 ABSTRACT: Peel and leaf oils of 30 sour orange cultivars, belonging to four different species (C. aurantium L., C. myrtifolia Raf., C. natsudaidai Hay. and C. neo-aurantium Tan.) were obtained from fruits and leaves collected on sour orange trees submitted to the same pedoclimatic and horticultural conditions. Their chemical composition was investigated by capillary GC, GC MS and 13 C-NMR. Two chemotypes, limonene and limonene/ˇ-pinene, were distinguished for peel oils, while four chemotypes, linalool/linalyl, sabinene/(e)-ˇ-ocimene, limonene/ˇpinene and ˇ-pinene/linalool, were observed for leaf oils. Copyright 2001 John Wiley & Sons, Ltd. KEY WORDS: Citrus; sour orange; peel and leaf oils; essential oil composition; GC; GC MS; 13 C-NMR Introduction Bigarade (family Rutaceae, genus Citrus), commonly named sour orange, is cultivated in Central and South America (Haiti and Paraguay) and in the Mediterranean countries. According to Tanaka, 1 sour oranges are classified into four botanical species. Citrus aurantium L., which has several varieties, is the best known. C. myrtifolia Raf., C. natsudaidai Hay. and C. neo-aurantium Tan. are characterized by a small number of varieties. The sour orange tree is very resistant to cold, to excess of water and to several diseases. For these main qualities, sour orange tree was the most popular rootstock before the appearance of the Citrus Tristeza Virus (CTV). Although the bigarade fruit looks like the sweet orange, it is differentiated by the acidity of the pulp and the bitterness of the rind. If the sour orange is not consumed fresh, the fruit is used for the preparation of marmalades. The peel is used for the preparation of liquors and, in pharmacy, for the aromatization of several drugs. 2 The essential oil of bigarade (Petitgrain) is mainly produced by France, Italy, Spain and Paraguay. It possesses a pleasant and characteristic fragrance and is widely used in perfumery for the sweet and fresh note that it gives to eau de Colognes and lotions. The oil is also used for *Correspondence to: F. Tomi, Université de Corse, Equipe Chimie et Biomasse, URA CNRS 2053, Route des Sanguinaires, Ajaccio, France. tomi@vignola.univ-corse.fr Contract/grant sponsor: Délégue Régional à la Recherche et à la Technologie pour la Corse; Contract/grant number: Contract/grant sponsor: Collectivité Territorial de Corse. the fabrication of soaps because of its good resistance for the alkaline medium. The production of petitgrain oils from Paraguay (used for domestic detergents) and Mediterranean countries (reserved for luxury articles) is estimated at 260 tonnes year. 3 Conversely, the production of peel oils reached no more than 25 to 30 tonnes year. The chemical composition of peel essential oils of sour orange (C. aurantium), described in the literature, 3 13 was dominated by limonene (71 97%): (a) alone for the samples of the kabusu, sudachi, iyokan, natsudaidai, daidai cultivars from Japan, 6 amara cultivar from Spain and Italy, 8 cyathifera cultivar from Japan, 9 bittersweet, bouquet de fleurs, chinotto, daidai, granito, kinkoji, paraguay, séville, gadadehi, konejime, nanshô daidai, bigaraldin, kharna and sour cultivars from California 13 or unspecified cultivars from Italy, China, Brazil, Cuba or from unspecified origin, 3 7,10 12 (b) associated with myrcene (24%) for one sample of the sumikan cultivar from China. 6 Studies concerning the chemical composition of leaf or petitgrain oils of sour orange have been reviewed. 14,15 Several chemical compositions have been described, nevertheless, the linalool/linalyl chemotype (12 66%/13 62%) was the most frequently reported for commercial samples (unspecified cultivar) as well as for the oils extracted in the laboratory from the following cultivars: bittersweet, bouquet de fleurs, chinotto, granito, kinkoji, myrtifolia, paraguay, salicifolia, séville, daidai and sour from California, 13 apepu from Paraguay 14 or unspecified cultivars from Japan, China, North America, Paraguay, Egypt, Tunisia, Copyright 2001 John Wiley & Sons, Ltd.

2 90 M.-L. LOTA ET AL. Morocco, Spain, France or unspecified origin. 3,14 17 Seven other compositions were more infrequantly described: (a) ˇ-pinene (17 30%) associated with nerol (10 11%) and/or linalool (19%) for two oils of the konejime and nanshô daidai cultivars; 13 (b) sabinene (34%) followed by ˇ-ocimene (12%) and linalool (11%) for one sample of the bigaraldin cultivar; 13 (c) limonene alone (15 44%) or associated with citronellol C geranial (17%), nerol (18%), ˇ-ocimene (16%) and neral (13%) for two samples, kharna and gadadehi cultivars; 13 (d) -phellandrene (30%) followed by decanal (10%) and limonene (9%) for one oil of the kinkoji cultivar; 13 (e) ˇ-ocimene (47%) for one sample of the poorman cultivar; 13 (f) myrcene (43%); and (g) linalool/terpinen- 4-ol (66%/21%) for samples of unspecified cultivars from China and Mauritius. 15,18 It has to be pointed out that the -terpinene/p-cymene composition (31%/18%) was described for a solvent extract from a Japanese natsudaidai cultivar (C. natsudaidai). 19 The aim of our work was to study the chemical variability of 30 cultivars of the sour orange family that were grown in the Station de Recherches Agronomiques (SRA) of the INRA-CIRAD in San Ghjulianu (Corsica, France). We will compare the chemical compositions of peel and leaf oils. The composition of only three of the cultivars analysed in this work has been previously reported in the literature. 13 The samples were analysed by GC, GC MS and/or 13 C-NMR. This latter technique was carried out following a methodology first reported by Formácek and Kubecska, 20 developed in our laboratory 21 and well suited for chemical polymorphism studies Experimental Plant Material Clonal propagated trees, grafted on Troyer citrange rootstock, were 12 years old and grown in the germplasm collection orchard of the SRA of INRA-CIRAD, located at San Ghjulianu (latitude N, longitude E; Mediterranean climate, average rainfall 840 mm per year and average temperature 15.2 C; soil derived from alluvial deposits and classified as fersiallitic, ph range ). The Citrus varieties collection of INRA- CIRAD in Corsica is one of the FAO-recognized Citrus collections in the world. In this arboretum, each tree has a computerized identification number. Trees are in good vigour, disease-free and without visible insect infection. For each cultivar of sour orange, about 500 g of leaves from the least autumn leaf flush and at least 30 ripe fruits were collected from many parts of the same tree, early in the morning and in dry weather during the period December 1997 April The following cultivars were investigated: ž C. aurantium L.: Apepu (No. 1), Brazilian (No. 2), Doux amer (No. 3), Menton (No. 4), Alibert de Corse (No. 5), Commune de Tuléar (No. 6), Sae Algérie (No. 7), Bouquetier de Nice à fruits plats (No. 8), A fleurs ferrando (No. 9), Granito (No. 10), Bouquetier de Nice (No. 11), Doux (No. 12), Sans épine (No. 14), Luisi (No. 15), Santucci (No. 16), De Floride (No. 17), Algerian (No. 18), Alibert Hybride 12 (No. 19), Corsigliese (No. 20), Tunisian (No. 21), Bouquet de Fleurs (No. 22), Maroc (No. 23), Petit Pierre (No. 24), Australian (No. 26), Gou Tou (No. 28), Espagne (No. 29). ž C. myrtifolia Raf.: Chinois à grandes feuilles (No. 25) and Chinensis (No. 30). ž C. natsudaidai Hay.: Natsudaidai (No. 13). ž C. neo-aurantium Tan.: Tosu (No. 27). Peel and Leaf Essential Oils The peel of fresh fruits was cold-pressed and then the essential oil was separated from the crude-extract by centrifugation (10 min at rpm). Fresh leaves were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. Yields ranged between 0.3% and 0.5%. GC, GC MS and 13 C-NMR Analyses The GC analysis was carried out using a Perkin-Elmer 8500 apparatus equipped with FID and fused capillary columns (50 m ð 0.22 mm, film thickness 0.25 µm) BP-20 (polyethylene glycol) and BP-1 (dimethyl siloxane). The oven temperature was programmed ( C at 2 C/min); injector temperature, 250 C; detector temperature, 260 C; carrier gas, helium (20 psi). GC MS analysis was performed on a Perkin-Elmer quadrupole MS apparatus (Model 910) coupled with the above gas chromatograph (BP- 1 column). The MS operating parameters were: ionization voltage, 70 ev; ion source temperature, 220 C; scan mass range, u. 13 C-NMR spectra were recorded on a Bruker AC 200 Fourier Transform spectrometer operating at MHz, in deuterated chloroform, with all shifts referred to internal tetramethylsilane (TMS). The spectra were recorded with the following parameters: pulse width (PW), 5.0 µs (flip angle 45 ); spectral width, Hz (250 ppm); CPD mode decoupling. The number of accumulated scans was for each sample (200 mg of the oil in 2 ml CDCl 3 ). An exponential multiplication of the free induction decay with the line broadening of 1.0 Hz was applied before Fourier transformation.

3 PEEL AND LEAF ESSENTIAL OILS OF SOUR ORANGE 91 Identification of Components Identification of the individual components of samples was based on: (a) comparison of their GC retention indices (RI) on apolar and polar columns, determined relative to the retention time of a series of n-alkanes with linear interpolation, with those of authentic compounds; (b) computer matching with mass spectral libraries (NIST, WILEY) and comparison with spectra of authentic samples or literature data; (c) comparison of the resonances in the 13 C-NMR spectrum of the mixture with those of the reference spectra compiled in our spectral library, with the help of laboratory-produced software. 21 All peel and leaf oils were investigated by GC. All leaf oils and 13 peel oils were analysed by 13 C-NMR, while three peel oils and four leaf oils were analysed by GC MS. Samples submitted to GC MS and 13 C-NMR analysis were selected on the basis of their chromatographic profile in such a manner that all of the 47 components were identified by mass spectrometry/retention indices and the major components ½0.5% by 13 C-NMR spectroscopy. Results and Discussion It was possible to realise a real comparative study of the composition of peel and leaf oils of 30 cultivars of sour orange, since all trees are grown in the same pedoclimatic and cultural conditions. Similarly, extraction conditions were identical for all samples (see Experimental). Therefore, the influences of environmental and technical parameters on the chemical composition of essential oils were considered negligible. Peel Oils The 39 components identified accounted for % of the total amount of the oil (Table 1). Peel oils consisted almost exclusively of hydrocarbons, with limonene as major component. Almost all samples (29 of 30) belong to the same group, while the last sample (No. 30) presents an atypical composition. Behind the main cluster, 26 of 29 samples exhibited a very high content of limonene ( %). The other compounds were inevitably present at low contents. Among them, ˇ-pinene (tr 2.4%), myrcene ( %), linalool (tr 1.5%), linalyl (0 3.9%), -pinene (0 0.6%) were identified in almost all samples. Among these 26 samples, two subgroups can be distinguished relatively to the content of limonene ( % and % respectively). The last three samples of the cluster (Nos 27, 28 and 29) exhibited a slightly lower content of limonene ( %). The two first samples (tosu cultivar of C. neo-aurantium; gou tou cultivar of C. aurantium) were also characterized by appreciable percentages of -terpinene and ˇ-pinene (approximately 4 5%) and a third sample (Espagne cultivar of C. aurantium) exhibited higher contents of linalool and linalyl (approximately 5%), (E)-nerolidol (3.2%) and germacrene-d (2.1%). The atypical composition of the chinensis cultivar (No. 30, C. myrtifolia) exhibited a significantly lower content of limonene (48.8% vs %) and appreciable amounts of ˇ-pinene (19.3% vs. tr 5.2%), -terpinene (7.8%), p-cymene (6.5%), sabinene (3.2%), geranial (1.4%), terpinen-4-ol (1.2%), geranyl (1.1%) and neral (1.0%). The 26 cultivars from C. aurantium, the monovarietal C. natsudaidai and C. neoaurantium sp., as well as one sample of C. myrtifolia sp., belonged to the main group. The chinensis cultivar (No. 30), which was discriminated by an atypical limonene/ˇ-pinene chemotype, belongs to the C. myrtifolia species. The composition of the 29 samples of the main cluster is very close to that reported in the literature for 24 other cultivars. Conversely, to our knowledge the limonene/ˇ-pinene composition has never been reported in the literature for the bigarade group. Leaf Oils The chemical composition of the 30 oils is reported in Table 2. The total of the 47 components identified accounted for % of the oil. Oxygenated compounds were identified at appreciable contents in most of the oils: linalool ( %), linalyl (0 36.8%), -terpineol ( %), terpinen-4-ol (0 7.0%), geraniol (0 6.7%), nerol (tr 2.3%), geranyl and neryl ( % and %, respectively). Note that the percentages of sabinene ( %), limonene ( %), ˇ-pinene ( %) and (E)-ˇ-ocimene ( %) were sometimes important. In Table 2, two clusters (I, II) have been distinguished: (I) 26 samples (Nos 1 25 and 29), belonging to the same group, exhibited a very homogeneous composition, dominated by linalyl ( %) and linalool ( %), associated with -terpineol ( %), geraniol ( %), geranyl ( %), neryl ( %) and nerol ( %); (II) the four samples (Nos and 30) of the second group, were distinguished from those of the first group by the absence of linalyl, the low contents of - terpineol ( %), geraniol (0 0.9%), geranyl ( %), neryl ( %). Nevertheless, this group was characterized by a great variability. Indeed, two samples (Nos 27 and 28) exhibited the ˇ- pinene/linalool chemotype (32.8/23.1% and 36.7/22.6%). The oil No. 30 was dominated by limonene (44.0%),

4 92 M.-L. LOTA ET AL. Table 1. Chemical composition of peel essential oils of sour orange Constituents BP-20 BP Thujene Ł Pinene Ł Camphene tr tr tr tr tr tr tr tr tr tr 0.1 ˇ-Pinene tr Sabinene Myrcene p-cymene tr tr tr tr tr tr tr Terpinolene tr tr tr tr tr 0.1 tr Octanal tr tr tr 0.1 tr tr tr Nonanal tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr Phellandrene -Terpinene Limonene ˇ Phellandrene (Z)-ˇ tr tr tr tr tr tr tr 0.1 Ocimene -Terpinene tr 0.1 tr tr 0.4 tr tr tr tr tr tr tr tr (E)-ˇ Ocimene cis-linalool tr 0.4 oxide THF trans tr 0.1 Limonene-1,2 oxide Octyl tr tr tr 0.1 tr tr 0.1 tr Decanal

5 PEEL AND LEAF ESSENTIAL OILS OF SOUR ORANGE 93 Table 1. Continued Constituents BP-20 BP Linalool tr Linalyl trans- - Bergamotene (E) tr tr tr Caryophyllene Terpinen-4-ol tr 1.2 -Humulene tr tr tr tr tr tr tr tr tr tr Neral tr tr tr 0.1 tr 0.1 tr 0.1 tr tr tr tr 0.1 tr tr tr 0.1 tr 1.0 -Terpinyl tr tr -Terpineol tr tr tr tr tr Germacrene tr D ˇ-Bisabolene (Z) tr tr Bisabolene Neryl tr tr tr tr tr 0.1 tr tr tr tr 0.2 tr Geranial tr tr tr tr 1.4 Geranyl Nerol tr tr tr tr 0.1 Geraniol tr tr tr tr tr tr (E)-Nerolidol Total tr < 0.05%. The correspondence between numbers and samples is reported in Experimental. Order of elution and percentages of components is given in the BP-20 column, except for compounds with an asterisk (percentages given in BP-1 column). All the components were identified by GC RI on polar and apolar columns. All the compounds of samples Nos 17, 28 and 30 were also identified by GC MS. The major components (in bold) of samples Nos 4, 7, 9, 11, 15, 17, were identified by 13 C-NMR.

6 94 M.-L. LOTA ET AL. Table 2. Chemical composition of leaf oils of sour orange I II Constituents BP-20 BP Thujene Ł tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr 0.5 -Pinene Ł Camphene tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr ˇ-Pinene Sabinene Carene tr Myrcene Phellandrene tr tr Terpinene tr tr 0.1 tr tr tr tr tr tr tr tr tr 0.1 tr tr tr tr tr tr tr tr tr 0.1 tr ˇ-Phellandrene Ł { 0.1 { 0.1 { 0.1 { 0.1 { 0.1 { 0.1 { 0.1 { 0.1 { { { { { { { { { { { { { { { tr tr tr Limonene { 0.7 { { ,8-Cineole Ł (Z)-ˇ-ocimene Terpinene tr tr tr 0.1 tr tr tr 0.1 tr 0.1 tr 0.1 tr tr (E)-ˇ-Ocimene p-cymene tr tr tr Terpinolene Octanal tr tr 6-Methylhept-5- en-2-one allo-ocimene tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr Nonanal tr tr tr 0.3 cis-linalool tr tr tr 0.1 tr 0.1 tr tr tr tr 0.1 tr 0.1 tr 0.1 tr tr tr oxide THF trans-sabinene tr tr tr hydrate trans-linalool tr 0.1 tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr 0.1 tr tr oxide THF Citronellal tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr tr Decanal Linalool Linalyl

7 PEEL AND LEAF ESSENTIAL OILS OF SOUR ORANGE 95 Table 2. Continued I II Constituents BP-20 BP cis-p-menth-2- en-1-ol tr Terpinen-4-ol Undecanal Citronellyl tr tr 0.1 tr -Humulene tr tr tr tr tr tr tr tr Neral tr tr tr tr tr tr tr 0.1 tr tr tr tr tr tr tr tr tr tr -Terpinyl (E)- Caryophyllene -Terpineol (Z) Bisabolene Neryl Geranial tr Piperitone Geranyl Citronellol Nerol tr Geraniol (E)-Nerolidol Spathulenol Total tr < 0.05%. The correspondence between numbers and samples is reported in Experimental. Order of elution and percentages of components is given in the BP-20 column, except for compounds with an asterisk (percentages given in the BP-1 column). All the components were identified by GC RI on polar and apolar columns. All the compounds of samples Nos 17, 26, 28 and 30 were also identified by GC MS. The major components (in bold) of all samples were identified by 13 C-NMR.

8 96 M.-L. LOTA ET AL. ˇ-pinene (16.6%) and citral (14.7%). At the end, sample No. 26 was differentiated by a high content of sabinene (52.6%) associated with (E)-ˇ-ocimene (15.1%) and terpinen-4-ol (7.0%). Globally, the leaf oils of sour orange showed a great homogeneity in their composition. Indeed, 24 of 26 samples that belonged to C. aurantium exhibited the linalool/linalyl chemotype (widely described in the literature). 3,14 17 Nevertheless, the two last cultivars, Nos 26 and 28 (australian and gou tou cultivars of C. aurantium) were distinguished, respectively, by the atypical sabinene/(e)-ˇ-ocimene and ˇ-pinene/linalool chemotypes, also described in the literature. 13 To our knowledge, the limonene/ˇ-pinene chemotype, observed for the chinensis sample (No. 30), has never been reported in sour orange oils. In our sampling, the sour orange group exhibited a weak chemical variability and was characterized by limonene as major component (peel oils) and by linalool/linalyl chemotype (leaf oils). Nevertheless, some cultivars were differentiated. Indeed, the Espagne cultivar (No. 29, C. aurantium) was discriminated by the peel oil and the australian cultivar (No. 26, C. aurantium) was differentiated by the leaf oil. The tosu and gou tou cultivars (Nos 27 and 28), which belonged to two different species (C. neoaurantium and C. aurantium, respectively) showed the same chemical characteristics and were discriminated by the composition of peel oils as well as that of leaf oils. Concerning C. myrtifolia, the oil of the chinois à grande feuilles cultivar (sample No. 25) exhibited the same chemotype observed in most of the sour orange oils, whereas the composition of the chinensis sample (No. 30) was atypical. Finally, the oil from the monovarietal C. natsudaidai (No. 13) exhibited a composition close to that of the 24 cultivars from C. aurantium. Acknowledgements The authors are indebted to the Délégué Régional à la Recherche et à la Technologie pour la Corse for financial support (Convention No ), to the Collectivité Territoriale de Corse for a research grant (MLL) and to INRA-CIRAD of Corsica for welcome and availability of plant material. References 1. Citrus of the World, SRA INRA-CIRAD: San Giuliano, France, Bruneton J. Pharmacognosie, Phytochimie Plantes Médicinales, Technique et Documentation-Lavoisier: 000, 1993; Huet R. Fruits 1991; 46: Mondello L, Dugo P, Bartle KD. Flavour Fragr. J. 1995; 10: Chouchi D, Barth D, Reverchon E, Della Porta G. J. Agric. Food Chem. 1996; 44: Lawrence BM. Bitter orange oil. In Essential Oils Allured: Carol Stream, IL, 1995; (a) 19, 25 ( ); (b) 55 57, ( ); (c) ( ); (d) , ( ). 7. Shaw PE. J. Agric. Food Chem. 1979; 27: Boelens MH, Jimenez R. J. Essent. Oil Res. 1989; 1: Njoroge SM, Ukeda H, Kusunose H, Sawamura M. Flavour Fragr. J. 1994; 9: Dugo G, Verzera A, Stagno d Alcontres I, Cotroneo A, Ficarra R. Flavour Fragr. J. 1993; 8: Lawrence BM. Progress in essential oils: bitter orange oil. Perfum. Flavor. 1994; 19: Dugo G. Perfum. Flavor. 1994; 19: Ortiz JM, Kumamoto J, Scora RW. Int. Flavour Food Addit. 1978; 5: Lawrence BM. Bitter orange leaf (petitgrain) oil. In Essential Oils Allured: Carol Stream, IL, 1995; (a) 19 ( ); (b) ( ). 15. Mondello L, Dugo G, Dugo P, Bartle KD. J. Essent. Oil Res. 1996; 8: Mondello L, Dugo P, Dugo G, Bartle KD. J. Chromatogr. Sci. 1996; 37: Kamiyama S, Amaha M. Bull. Brew. Sci. 1972; 18: Gurib-Fakim A, Demarne F. J. Essent. Oil Res. 1995; 7: Kamiyama S. J. Agric. Biol. Chem. 1967; 31: Formácek V, Kubeczka KH. Essential Oils Analysis by Capillary Gas Chromatography and Carbon-13 NMR Spectroscopy. Wiley: Chichester, Tomi F, Bradesi P, Bighelli A, Casanova J. J. Magn. Reson. Anal. 1995; 1: Salgueiro L, Vila R, Tomi F, Tomas X, Cañigueral S, Casanova J, Proença da Cunha A, Adzet T. Phytochemistry 1997; 45: Lota M-L, de Rocca Serra D, Tomi F, Bessiere J-M, Casanova J. Flavour Fragr. J. 1999; 14: Lota M-L, de Rocca Serra D, Tomi F, Casanova J. Biochem. Syst. Ecol. 2000; 26: Castola V, Bighelli A, Casanova J. Biochem. Syst. Ecol. 2000; 26: Jennings W, Shibamoto T. Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography. Academic Press. New York, McLafferty FW, Stauffer DB. The Wiley/NBS Registry of Mass Spectral Data. Wiley: San Diego, A, Joulain D, König WA. The Atlas of Spectral Data Of Sesquiterpene Hydrocarbons, E.B.-Verlag: Hamburg, 1998.