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Culture conditions and salt effects on essential oil composition of sweet marjoram ( Origanum majorana ) from Tunisia

Culture conditions and salt effects on essential oil composition of sweet marjoram ( Origanum... Acta Pharm. 62 (2012) 251­261 DOI: 10.2478/v10007-012-0019-9 Short communication OLFA BAÂTOUR1* IMEN TARCHOUNE1* HELA MAHMOUDI1 NAWEL NASSRI1 WISSAL ABIDI1 RYM KADDOUR1 GHAITH HAMDAOUI2 MOUHIBA BEN NASRI-AYACHI1 MOHTAR LACHAÂL1 BRAHIM MARZOUK2 1 Unité de Physiologie et Biochimie de la Tolérance au Sel des Plantes Département de Biologie, Faculté des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisie 2 Laboratoire des Substances Bioactives Centre de Biotechnologie de Borj-Cedria BP 901, 2050 Hammam-lif, Tunisie O. majorana shoots were investigated for their essential oil (EO) composition. Two experiments were carried out; the first on hydroponic medium in a culture chamber and the second on inert sand in a greenhouse for 20 days. Plants were cultivated for 17 days in hydroponic medium supplemented with NaCl 100 mmol L­1. The results showed that the O. majorana hydroponic medium offered higher essential oil yield than that from the greenhouse. The latter increased significantly in yield (by 50 %) under saline constraint while it did not change in the culture chamber. Under greenhouse conditions and in the absence of salt treatment, the major constituents were terpinen-4-ol and trans-sabinene hydrate. However, in the culture chamber, the major volatile components were cis-sabinene hydrate and terpinen-4-ol. In the presence of NaCl, new compounds appeared, such as eicosane, spathulenol, eugenol, and phenol. In addition, in the greenhouse, with or without salt, a very important change of trans-sabinene hydrate concentration in EO occurred, whereas in the culture chamber change appeared in cis-sabinene hydrate content. Keywords: Origanum majorana, greenhouse, culture chamber, salt, yield, terpinen-4-ol Accepted May 3, 2012 Sweet marjoram (Origanum majorana L., syn. Majorana hortensis Moench) is a herbaceous and perennial plant native to Cyprus and the Eastern Mediterranean (1). It is an appreciated herb species and its essential oil is also used in perfumery because of its spicy herbaceous notes (2). In addition, essential oil (EO) of O. majorana is utilized in the manufacture of fungicides, and various pharmaceutical and industrial products (2). Two chemotypes characterize O. majorana essential oil: terpinen-4-ol/sabinene hydrate chemotype (3), and thymol (or carvacrol) chemotype (4). In the first chemotype, * Correspondence; e-mail: baatourolfanaiimi@gmail.com; These authors have equally participated in the preparation of the manuscript. O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. the two major constituents are responsible for the characteristic flavor and fragrance of marjoram oil (5). In medicinal and aromatic plants, the biosynthesis of secondary metabolites such as essential oils and their constituents is strongly influenced by environmental factors (6). One of the most important environmental constraints that affect almost 50 % of irrigated areas is salinity (7). This constraint generally modifies essential oil biosynthesis and its secretion (8). The essential oil yield of some species can change with age, growth cycle, climatic conditions, soil type and cropping pattern (9). Culturing conditions can affect the quality of essential oil (10). Moreover, the age of the plant has a significant effect on its essential oil composition (11). However, there is no clear information on the possible influence of culture conditions on essential oil yield and composition in O. majorana. In this paper, we report on combined effects of NaCl treatment and culturing conditions of Tunisian marjoram aerial parts. EXPERIMENTAL Plant material Marjoram (Origanum majorana L., Lamiaceae) plants were collected at the 6-leaf stage from a nursery located in Soliman in northeastern Tunisia (latitude 36° 41' 47 N; longitude 10° 29' 30 E; altitude 1500 m). These 11 days old plants were divided into two lots. The first was used for a hydroponic culture in eight-strength Hoagland's solution (12). Plants were placed in a culture chamber under 16 h light/8 h dark conditions at 22/18 °C, photosynthetically active radion at 150 mmol m­2 s­1. The second lot of plants was transferred to plastic pots (1 plant per pot) filled with inert sand. Plants were irrigated with the same nutrient medium and placed in a greenhouse at 35 °C (day) and 26 °C (night). After 20 days of acclimatization, individual plants were grown without salt (control) or with NaCl (100 mmol L­1) for 17 days prior to harvest. Essential oil isolation Essential oil was extracted by classical hydrodistillation of fresh shoots (50 g) during 90 min according to Msaada et al. (13). The distillate was submitted to a liquid-liquid extraction in diethyl ether and the organic phase was concentrated at 35 °C using a Vigreux column. In order to quantify EO and its constituents, 6-methyl-5-hepten-2-one was used as an internal standard. Essential oil obtained was stored at ­20 °C prior to analysis. Each extraction was made in triplicate. GC-FID Gas chromatography analysis was carried out on a Hewlett-Packard 6890 gas chromatograph (Agilent Technologies, USA) equipped with a flame ionization detector (FID) and an electronic pressure control (EPC) injector. A polar column HP Innowax (PEG) and an apolar HP-5 column (30 m ´ 0.25 mm, 0.25 mm film thickness, Agilent Technologies) O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. were used. The nitrogen flow rate was 1.6 mL min­1 and the split ratio was 60:1. EO analysis was performed using the following temperature program: oven 35 °C for 10 min, from 35 to 205 °C at a rate of 3 °C min­1, and isotherm at 225 °C for 10 min. Injector and detector temperature, were maintained at 250 and 300 °C, respectively. Retention indices were calculated using a homologous series of n-alkanes C6­C20. GC-MS GC-MS analysis was performed on a gas chromatograph HP 5890 (II) interfaced with a HP 5972 mass spectrometer with electron impact ionization (70 eV). A HP-5MS capillary column (apolar, 30 m ´ 0.25 mm, 0.25-mm film thickness, Agilent Technologies) was used. Column temperature was programmed to rise from 50 to 240 °C at a rate of 5 °C min­1. The carrier gas was helium with a flow rate of 1.2 mL min­1; split ratio was 60:1. Scan time and mass range were 1 s and 40­300 m/z, respectively. Statistical analysis All extractions and analyses were conducted in triplicate. Data were expressed as means ± SD. RESULTS AND DISCUSSION Essential oil yield Plants growing under different culture conditions did not produce equal yields of EO and gave rise to different contents and compositions in plant shoots (Table I). Thus, in the culture chamber, the essential oil yield based on dry mass, was 0.8 % in the control, and in the presence of NaCl (100 mmol L­1). In the greenhouse, EO decreased by a factor of two in the presence of salt. In our previous work (14) we showed that Canadian O. majorana can tolerate a moderate NaCl concentration (50 mmol L­1) without modification in EO yield and composition. However, at a high NaCl concentration (100 mmol L­1), significantly modified essential oil yield and quality occured. Similar findings were reported for Salvia hispanica (8). In contrast, salt addition enhanced EO yield in Oenothera biennis (8). In the case of Table I. Impact of salinity on essential oil yield of marjoram shoots after 2 weeks of treatment Essential oil yield (%) NaCla (100 mmol L­1) Greenhouse Culture chamber a b NaCl (100 mmol L­1)a 0.2 ± 0.03b 0.8 ± 0.02b 0.4 ± 0.1 0.8 ± 0.04 Mean ± SD, n = 3. Significantly different after salt addition at p < 0.05. O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. Mathiola tricuspidata, salinity did not exert any effect, on EO yield (8). Diversity of the findings showed that the effect of salt on essential oil yield depends on the salt concentration, culture medium and tolerance of the species. Culture chamber conditions: essential oil composition of O. majorana shoots Gas chromatography analysis of essential oils from the culture chamber showed 44 compounds accounting for 99.86 % of the total EO (Table II), as represented in Figs. 1 c,d. In control plant shoots, the main compound was cis-sabinene hydrate (43.66 %) followed by terpinen-4-ol (23.21 %), trans-sabinene hydrate (8.17 %) and a-humulene (7.30 %). Salt addition induced a decrease estimated at 4.40 % for cis-sabinene hydrate, 5.66 % terpinen-4-ol, 4.83 % trans-sabinene hydrate and 2.89 % a-humulene as compared to the control. In that case, EO was of cis-sabinene hydrate/terpinen-4-ol chemotype, which is responsible for the characteristic flavor and fragrance of marjoram. Our results are in accord with those of Bronchio et al. (3). Our previous data (14) showed that the essential oil of Canadian Origanum cultivated under the same conditions as the Tunisian one was found to be rich in trans-sabinene hydrate (47.67 %), and terpinen-4-ol (20.82 %). Salinity had a significant effect (p < 0.05) on the composition and content of the major EO. In the culture camber cis-sabinene hydrate and terpinen-4-ol proportions were decreased, while their concentrations increased. In fact, NaCl (100 mmol L­1) increased cis-sabinene hydrate and terpinen-4-ol contents significantly (125 times to reach 20.60 mg g­1 and 6 times to reach 0.556 mg g­1) respectively. Despite these changes, O. majorana retained the same chemotype after salt addition. In the culture chamber, an important bioconversion of EO composition of O. majorana shoots from trans-sabinene hydrate into cis-sabinene hydrate was detected (Table II). These two isomers are biosynthesized by the same enzyme, sabinene hydrate synthase (1). In fact, these culture conditions activate this enzyme, favouring the biosynthesis of cis-sabinene hydrate. Indeed, this is an intensive and spic component of marjoram, whereas the trans-sabinene hydrate does not have any typical properties of marjoram (1). Essential oil composition of shoots was characterized by the prevalence of terpenic alcohols in a proportion of 86.20 %, standing for cis-sabinene hydrate, trans-sabinene hydrate, and terpinen-4-ol, and 8.48 % for monoterpene hydrocarbons (Table II). In the same context, Baatour et al. (14) indicated that the most represented class of EO components of O. majorana native to Canada was that of oxygenated monoterpenes, followed by monoterpene hydrocarbons and esters in control plants. A decrease in these different compound proportions was observed after salt addition, except for monoterpene hydrocarbons and phenols. Greenhouse conditions: Essential oil composition O. majorana shoots Thirty-two compounds were found to represent 99.55 % of EO (Table III), as shown in Figs. 1 a,b. Independent of the applied treatment, terpinen-4-ol was detected as the major compound (31.77 %), followed by trans-sabinene hydrate (24.18 %) and a-ter- O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. FID1 A, (LSBA\PA000000.D) pA 225 200 175 150 125 100 75 50 25 0 1 2 3 4 5 6 10 7 8 9 11 20 30 15 16 18 21 19 20 24 23 25 26 40 27 28 50 min SI 12 13 14 17 22 10 FID1 A, (LSBA\PA000100.D) pA 120 100 80 60 40 20 0 10 20 2 213 10 12 IS SI 28 32 60 min FID1 A, (LSBA\02010802.D) pA 90 80 70 60 50 40 30 20 0 23 1 4 6 5 10 8 7 9 10 12 14 11 13 15 20 16 30 SI 19 20 22 24 26 21 25 27 17 18 23 32 60 min FID1 A, (LSBA\03010801.D) pA 90 80 70 60 50 40 30 20 0 4 2 13 5 14 11 SI IS 15 23 17 22 15' 27 13' 24 10 8 11 79 8'12 9 8 13 10 17 15 11' 16 13'' 25 12' 39 28 36 27' 25 18 18' 33 27'' 35 41 2921 27 23 24 37 38 40 30 26 27 19 32 40 32' 47 48 33 min Fig. 1. Chromatogram of essential oil compounds from Origanum majorana. a) 100 mmol L­1 NaCl in the greenhouse after 17 days of treatment; b) 100 mmol L­1 NaCl in the greenhouse, after 17 days of treatment; c) 100 mmol L­1 NaCl, in the culture chamber, after 17 days of treatment; d) 75 mmol L­1 NaCl, in the culture chamber, after 17 days of treatment. SI: internal standard (6-methyl 5 heptene 2 one). 1: Tricyclene, 2: a-pinene; 3: a-tujene; 4: sabinene; 5: b-myrcene; 6: a-phellandrene; 7: a-terpinene; 8: limonene; 8': 1,8-cineole; 9: g-terpinene; 10: p-cymene; 11: terpinolene; 11': octanol; 12: cis-3-hexanol; 12': hexanol; 13: trans-sabinene hydrate; 13': linalyl acetate; 13'': bornyl acetate; 14: linalool, 15: cis-sabinene hydrate; 15': cis-p-menth-en-1-ol; 16: b-elemene, 17: terpinen-4-ol, 18: b-caryophyllene, 18': a-humulene; 19: aromadendrene; 20: bicyclogermacrene; 21: a-terpenyl acetate; 22: a-terpineol; 23: borneol; 24: neryl acetate, 25: geranyl acetate, 26: nerol, 27: myrthenol; 27': geraniol; 27'': cis-caveol; 28: nonadecane; 29: eiocasane; 30: spathuneol; 31: eugenol; 32: phenol; 32': thymol; 33: carvacrol. 255 O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. Table II. Essential oil composition of Origanum majorana in the culture chamber by GC-MS NaCl 0 100 100 0 (mmol L­1) (mmol L­1) (mmol L­1) (mmol L­1) Compound Tricyclene a-Pinene a-Thujene Sabinene b-myrcene a-Phellandrene a-Terpinene Limonene 1.8-Cineole g-Terpinene p-Cymene Terpinolene Octanal Hexanol cis-3-Hexanol trans-Sabinene hydrate Linalool cis-Sabinene hydrate cis-p-Menthen-1-ol Linalyl acetate Bornyl acetate b-Elemene Terpinene-4-ol B-Caryophyllene Aromadendrene a-Humulene Bicyclogermacrene a-Terpenyl acetate Myrtenyl acetate a-Terpineol Borneol Neryl acetate Geranyl acetate Nerol tR (min) 5.32 5.54 5.66 6.11 8.74 9.7 13.04 13.43 14.56 14.99 16.12 17.33 19.39 25.12 26.95 29.74 29.89 32.06 32.34 32.49 32.58 33.33 34.11 35.19 36.07 36.85 37.06 37.43 37.43 40.08 39.60 40.08 40.35 41.68 KIa 927 931 939 976 991 1006 1016 1030 1033 1062 1026 1088 ­ ­ ­ 1053 1098 1082 1129 1257 1295 1391 1176 1419 1443 1454 1344 1494 1335 1189 1165 1385 1383 1228 KIb 1014 1035 1032 1132 1174 1176 1188 1203 1213 1266 1280 1290 ­ ­ 1385 1474 1553 1556 1562 1556 1597 1601 1611 1612 1628 1687 1705 1755 1701 1713 1702 1733 1765 1797 (%)c 0.142 0.07 0.339 0.094 ND 0.562 1.292 0.517 0.582 0.062 0.050 0.069 0.474 0.210 ND 8.178 ND 43.667 4.892 0.993 2.272 0.329 23.209 0.450 ND 7.300 0.199 ND ND 4.619 0.203 0.133 ND 0.170 (%)c 0.193 0.106e 0.106e 0.134e 0.016 0.787e 1.730e 0.742e 0.782e 0.030e 0.054 0.107e 0.725e 0.020e 0.010 7.783e 0.043 41.745e 3.544e 1.472e 1.974e 0.837e 21.894e 0.507e 0.0169 7.089e 0.227e 0.021 0.021 4.995e 0.241e 0.193e 0.007 0.161e (mg g­1)c,d (mg g­1)c,d 0.045 0.532 0.265 0.352 ND 2.106 4.839 1.937 2.179 0.235 0.187 0.261 1.777 0.789 ND 0.030 0.289 0.163 0.018 3.720 8.507 1.234 0.086 1.688 ND 1.301 0.746 ND ND 0.017 0.763 0.499 ND 0.638 0.409e 0.011e 0.021e 0.290e 0.138 1.041e 0.046e 0.091e 0.022e 0.843e 0.334e 0.042e 0.144e 0.592e 0.010 1.600e 0.084e 20.595e 1.808e 8.845e 8.845e 0.289e 0.556e 58.240e 3.834 1.160e 0.285e 2.049 2.049 9.820e 41.500e 1.740e 0.811 0.468e O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. Table II. continued Myrtenol Geraniol cis-Caveol Nonadecane Eicosane Spathulenol Phenol Eugenol Thymol Carvacrol Classes Monoterpene hydrocarbons Terpenic alcohols Sesquiterpene hydrocarbons Terpenic esters Aliphatic hydrocarbons Phenols 8.483 86.207 0.714 3.265 0.003 0.025 1194 1255 1247 1581 1996 ­ ­ 1401 1295 1302 1804 1857 1804 1900 2000 2144 ­ 2030 ­ ­ 0.027 ND 0.449 ND 0.036 ND ND ND ND 0.025 0.024e 0.025 0.462e 0.011 0.034e 0.012 0.056 0.013 0.041 0.045e 11.126e 81.671e 0.156e 3.306e 0.004e 0.156e 0.104 ND 1.683 ND 0.138 ND ND ND ND 0.096 3.253e 0.021 0.392e 0.010 0.065e 0.008 0.093 5.186 0.063 0.023e 4.744e 48.012e 0.162e 0.048e 0.076e 0.193e tR ­ retention time, KI ­ Kovat's index on: a apolar column HP-5MS, n-alkane), ND ­ not detected. c Mean of 3 replicates. d Dry mass basis. e Significantly different after salt addition at p < 0.05. polar column HP-Innowax (relative to pineol (7.30 %). In the presence of NaCl (100 mmol L­1), a small increase was observed in terpinen-4-ol percentage, while percentages of trans-sabinene hydrate and a-terpineol were not affected. In the absence or presence of salt, O. majorana EO was of trans-sabinene hydrate/ terpinen-4-ol chemotype. These results are in agreement with those of Hamrouni et al. (9), who reported that the major constituents were terpinen-4-ol, cis-sabinene hydrate, and trans-sabinene hydrate. Later investigations showed that some of the above main constituents were found in numerous EO samples of O. majorana. Novak et al. (1) mentioned that the main compounds of marjoram EO were the epimeric monoterpene alcohols, trans-sabinene hydrate, cis-sabinene hydrate, and cis-sabinene hydrate acetate. In fact, according to Vera and Chane-Ming (2), the main components were terpinen-4-ol and cis-sabinene hydrate in EO of O. majorana originating from Island. Our results showed that NaCl modified the essential oil composition. In fact, new compounds appeared: eicosane, spathulenol, eugenol, and phenol (Table III). However, myrcene, present in the control, disappeared under saline conditions. Indeed, a very important modification of EO composition was bioconversion from cis-sabinene hydrate into trans-sabinene hydrate. Culture conditions had an important effect on the transformation of these two isomers: from cis- to trans-sabinene hydrate under greenhouse conditions and from trans- to cis-sabinene hydrate under culture cham257 O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. ber conditions. These results suggest that culture conditions activated sabinene hydrate synthase in favor of cis-sabinene hydrate or trans-sabinene hydrate. Under saline conditions, contents of all compounds evolved in the same way (Table III). The composition of EO of O. majorana shoots is primarily made up of terpenic alcohols (78.27 %), followed by monoterpene hydrocarbons in the control (18.74 %). Salinity decreased the content of each class, except for aliphatic hydrocarbons and terpenic alcohols (Table III). The quality of the EO produced and its composition in Origanum majorana depends on culturing conditions (15). Our findings are in accord with those of Avry and Gallouin (9) and Kotan et al. (16). Essential oil of O. majorana plants cultivated in a greenhouse had lower level of flavor than EO of plants cultivated in the culture chamber due to the great difference in the cis-sabinene hydrate production. Therefore marjoram cultivated Table III. Essential oil composition of Origanum majorana in the greenhouse by GC-MS. NaCl (mmol L­1) 0 Compound Tricyclene a-Pinene a-Thujene Sabinene b-Myrcene a-Phellandrene a-Terpinene Limonene g-Terpinene p-Cymene Terpinolene cis-3-Hexanol trans-Sabinene hydrate Linalool cis-Sabinene hydrate b-Elemene Terpinen-4-ol b-Caryophyllene Aromadendrene Bicyclogermacrene a-Terpenyl acetate a-Terpineol tR (min) 3.28 3.55 3.69 5.74 6.45 9.84 11.39 11.49 11.61 14.25 16.62 26.62 29.74 29.89 30.283 31.729 31.91 32.47 33.00 34.86 35.40 35.78 RIa ­ 931 939 976 991 1006 1016 1030 1033 1062 1026 1088 1053 1098 1082 1053 1098 1082 1129 1257 1295 1391 RIb ­ 1035 1032 1132 1174 1176 1188 1203 1213 1266 1280 1290 1474 1553 1556 1474 1553 1556 1562 1556 1597 1601 (%)c 0.252 0.316 0.506 0.181 3.807 4.145 0.710 0.162 1.448 5.883 1.183 7.428 24.181 3.994 2.235 0.197 31.77 0.210 0.864 0.335 0.320 7.300 100 (%)c ND 0.121e 0.430e 2.067e ND 3.602e 0.724e 2.205e 1.903e 3.215e 1.081e 10.650e 24.541e 3.724e 2.492e 0.225e 29.050e 0.311e 0.545e 0.223e 0.238e 7.089e 0 100 (mg g­1)c,d (mg g­1)c,d 0.045 0.056 0.090 0.032 0.678 0.738 0.126 0.028 0.258 1.048 0.210 1.323 4.309 0.711 0.398 0.035 5.662 0.037 0.154 0.059 0.057 1.301 ND 0.019e 0.070e 0.338e ND 0.589e 0.118e 0.361e 0.311e 0.526e 0.176e 1.743e 4.017 0.609e 0.408e 0.036 4.756e 0.051e 0.089e 0.036e 0.039e 1.160e O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. Table III. continued Borneol Neryl acetate Geranyl acetate Nerol Myrtenol Nonadecane Eicosane Spathulenol Eugenol Phenol Classes Monoterpene hydrocarbons Terpenic alcohols Sesquiterpene hydrocarbons Terpenic esters Aliphatic hydrocarbons Phenols 18.749 78.270 1.410 1.320 0.123 0 15.577e 79.365e 1.081e 1.005e 0.377e 0.115 3.313 1.395 0.251 0.235 0.021 0 2.550e 1.195e 0.177e 0.110e 0.059e 0.044 37.19 37.49 38.13 39.49 41.27 43.61 52.95 54.162 55.482 61.071 1176 1385 1419 1228 1191 1454 1344 1494 1335 ­ 1611 1733 1612 1797 1804 1687 1705 1755 1701 ­ 0.885 0.495 0.145 0.112 0.361 0.123 ND ND ND ND 0.786e 0.289e 0.147 ND ND 0.147 0.229 0.329 0.142 0.115 0.157 0.088 0.025 0.020 0.064 0.025 ND ND ND ND 0.128e 0.047e 0.024 0.037e 0.053e 0.024 0.039 0.023 0.018 0.020 ND ND ND tR ­ Retention time. KI ­ Kovat's index on: a apolar column HP-5MS, b polar column HP-Innowax (relative to n-alkane). ND ­ not detected. c Mean of 3 replicates. d Drug mass basis. e Significantly, different after salt addition at p < 0.05. in the greenhouse was of higher quality than that cultivated in the culture chamber because of its high terpinen-4-ol concentration (Tables II, III). The EO is believed to have had medicinal properties because of the presence of biologically active compounds such as terpinen-4-ol (5). In fact, terpinen-4-ol, a-terpineol, and linalool exhibited high antibacterial activity. O. majorana EO is known for its strong antimicrobial activity and could therefore be used in food applications. CONCLUSIONS Our data indicate high variations in quantitative and qualitative composition of O. majorana EOs produced under two conditions: greenhouse and culture chamber. The study showed that EO yield and composition depend on the culturing and climatic conditions. We observed an important modification in EO composition of O. majorana shoots by converting trans-sabinene hydrate into cis-sabinene hydrate in the culture chamber and cis- to trans-sabinene hydrate in the greenhouse. The EO of the latter was considered to be of higher quality than that cultivated in the culture chamber, because of its higher concentration of oxygenated compounds, particularly bioactive compounds such as terpinen-4-ol. O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Pharmaceutica de Gruyter

Culture conditions and salt effects on essential oil composition of sweet marjoram ( Origanum majorana ) from Tunisia

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Abstract

Acta Pharm. 62 (2012) 251­261 DOI: 10.2478/v10007-012-0019-9 Short communication OLFA BAÂTOUR1* IMEN TARCHOUNE1* HELA MAHMOUDI1 NAWEL NASSRI1 WISSAL ABIDI1 RYM KADDOUR1 GHAITH HAMDAOUI2 MOUHIBA BEN NASRI-AYACHI1 MOHTAR LACHAÂL1 BRAHIM MARZOUK2 1 Unité de Physiologie et Biochimie de la Tolérance au Sel des Plantes Département de Biologie, Faculté des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisie 2 Laboratoire des Substances Bioactives Centre de Biotechnologie de Borj-Cedria BP 901, 2050 Hammam-lif, Tunisie O. majorana shoots were investigated for their essential oil (EO) composition. Two experiments were carried out; the first on hydroponic medium in a culture chamber and the second on inert sand in a greenhouse for 20 days. Plants were cultivated for 17 days in hydroponic medium supplemented with NaCl 100 mmol L­1. The results showed that the O. majorana hydroponic medium offered higher essential oil yield than that from the greenhouse. The latter increased significantly in yield (by 50 %) under saline constraint while it did not change in the culture chamber. Under greenhouse conditions and in the absence of salt treatment, the major constituents were terpinen-4-ol and trans-sabinene hydrate. However, in the culture chamber, the major volatile components were cis-sabinene hydrate and terpinen-4-ol. In the presence of NaCl, new compounds appeared, such as eicosane, spathulenol, eugenol, and phenol. In addition, in the greenhouse, with or without salt, a very important change of trans-sabinene hydrate concentration in EO occurred, whereas in the culture chamber change appeared in cis-sabinene hydrate content. Keywords: Origanum majorana, greenhouse, culture chamber, salt, yield, terpinen-4-ol Accepted May 3, 2012 Sweet marjoram (Origanum majorana L., syn. Majorana hortensis Moench) is a herbaceous and perennial plant native to Cyprus and the Eastern Mediterranean (1). It is an appreciated herb species and its essential oil is also used in perfumery because of its spicy herbaceous notes (2). In addition, essential oil (EO) of O. majorana is utilized in the manufacture of fungicides, and various pharmaceutical and industrial products (2). Two chemotypes characterize O. majorana essential oil: terpinen-4-ol/sabinene hydrate chemotype (3), and thymol (or carvacrol) chemotype (4). In the first chemotype, * Correspondence; e-mail: baatourolfanaiimi@gmail.com; These authors have equally participated in the preparation of the manuscript. O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. the two major constituents are responsible for the characteristic flavor and fragrance of marjoram oil (5). In medicinal and aromatic plants, the biosynthesis of secondary metabolites such as essential oils and their constituents is strongly influenced by environmental factors (6). One of the most important environmental constraints that affect almost 50 % of irrigated areas is salinity (7). This constraint generally modifies essential oil biosynthesis and its secretion (8). The essential oil yield of some species can change with age, growth cycle, climatic conditions, soil type and cropping pattern (9). Culturing conditions can affect the quality of essential oil (10). Moreover, the age of the plant has a significant effect on its essential oil composition (11). However, there is no clear information on the possible influence of culture conditions on essential oil yield and composition in O. majorana. In this paper, we report on combined effects of NaCl treatment and culturing conditions of Tunisian marjoram aerial parts. EXPERIMENTAL Plant material Marjoram (Origanum majorana L., Lamiaceae) plants were collected at the 6-leaf stage from a nursery located in Soliman in northeastern Tunisia (latitude 36° 41' 47 N; longitude 10° 29' 30 E; altitude 1500 m). These 11 days old plants were divided into two lots. The first was used for a hydroponic culture in eight-strength Hoagland's solution (12). Plants were placed in a culture chamber under 16 h light/8 h dark conditions at 22/18 °C, photosynthetically active radion at 150 mmol m­2 s­1. The second lot of plants was transferred to plastic pots (1 plant per pot) filled with inert sand. Plants were irrigated with the same nutrient medium and placed in a greenhouse at 35 °C (day) and 26 °C (night). After 20 days of acclimatization, individual plants were grown without salt (control) or with NaCl (100 mmol L­1) for 17 days prior to harvest. Essential oil isolation Essential oil was extracted by classical hydrodistillation of fresh shoots (50 g) during 90 min according to Msaada et al. (13). The distillate was submitted to a liquid-liquid extraction in diethyl ether and the organic phase was concentrated at 35 °C using a Vigreux column. In order to quantify EO and its constituents, 6-methyl-5-hepten-2-one was used as an internal standard. Essential oil obtained was stored at ­20 °C prior to analysis. Each extraction was made in triplicate. GC-FID Gas chromatography analysis was carried out on a Hewlett-Packard 6890 gas chromatograph (Agilent Technologies, USA) equipped with a flame ionization detector (FID) and an electronic pressure control (EPC) injector. A polar column HP Innowax (PEG) and an apolar HP-5 column (30 m ´ 0.25 mm, 0.25 mm film thickness, Agilent Technologies) O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. were used. The nitrogen flow rate was 1.6 mL min­1 and the split ratio was 60:1. EO analysis was performed using the following temperature program: oven 35 °C for 10 min, from 35 to 205 °C at a rate of 3 °C min­1, and isotherm at 225 °C for 10 min. Injector and detector temperature, were maintained at 250 and 300 °C, respectively. Retention indices were calculated using a homologous series of n-alkanes C6­C20. GC-MS GC-MS analysis was performed on a gas chromatograph HP 5890 (II) interfaced with a HP 5972 mass spectrometer with electron impact ionization (70 eV). A HP-5MS capillary column (apolar, 30 m ´ 0.25 mm, 0.25-mm film thickness, Agilent Technologies) was used. Column temperature was programmed to rise from 50 to 240 °C at a rate of 5 °C min­1. The carrier gas was helium with a flow rate of 1.2 mL min­1; split ratio was 60:1. Scan time and mass range were 1 s and 40­300 m/z, respectively. Statistical analysis All extractions and analyses were conducted in triplicate. Data were expressed as means ± SD. RESULTS AND DISCUSSION Essential oil yield Plants growing under different culture conditions did not produce equal yields of EO and gave rise to different contents and compositions in plant shoots (Table I). Thus, in the culture chamber, the essential oil yield based on dry mass, was 0.8 % in the control, and in the presence of NaCl (100 mmol L­1). In the greenhouse, EO decreased by a factor of two in the presence of salt. In our previous work (14) we showed that Canadian O. majorana can tolerate a moderate NaCl concentration (50 mmol L­1) without modification in EO yield and composition. However, at a high NaCl concentration (100 mmol L­1), significantly modified essential oil yield and quality occured. Similar findings were reported for Salvia hispanica (8). In contrast, salt addition enhanced EO yield in Oenothera biennis (8). In the case of Table I. Impact of salinity on essential oil yield of marjoram shoots after 2 weeks of treatment Essential oil yield (%) NaCla (100 mmol L­1) Greenhouse Culture chamber a b NaCl (100 mmol L­1)a 0.2 ± 0.03b 0.8 ± 0.02b 0.4 ± 0.1 0.8 ± 0.04 Mean ± SD, n = 3. Significantly different after salt addition at p < 0.05. O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. Mathiola tricuspidata, salinity did not exert any effect, on EO yield (8). Diversity of the findings showed that the effect of salt on essential oil yield depends on the salt concentration, culture medium and tolerance of the species. Culture chamber conditions: essential oil composition of O. majorana shoots Gas chromatography analysis of essential oils from the culture chamber showed 44 compounds accounting for 99.86 % of the total EO (Table II), as represented in Figs. 1 c,d. In control plant shoots, the main compound was cis-sabinene hydrate (43.66 %) followed by terpinen-4-ol (23.21 %), trans-sabinene hydrate (8.17 %) and a-humulene (7.30 %). Salt addition induced a decrease estimated at 4.40 % for cis-sabinene hydrate, 5.66 % terpinen-4-ol, 4.83 % trans-sabinene hydrate and 2.89 % a-humulene as compared to the control. In that case, EO was of cis-sabinene hydrate/terpinen-4-ol chemotype, which is responsible for the characteristic flavor and fragrance of marjoram. Our results are in accord with those of Bronchio et al. (3). Our previous data (14) showed that the essential oil of Canadian Origanum cultivated under the same conditions as the Tunisian one was found to be rich in trans-sabinene hydrate (47.67 %), and terpinen-4-ol (20.82 %). Salinity had a significant effect (p < 0.05) on the composition and content of the major EO. In the culture camber cis-sabinene hydrate and terpinen-4-ol proportions were decreased, while their concentrations increased. In fact, NaCl (100 mmol L­1) increased cis-sabinene hydrate and terpinen-4-ol contents significantly (125 times to reach 20.60 mg g­1 and 6 times to reach 0.556 mg g­1) respectively. Despite these changes, O. majorana retained the same chemotype after salt addition. In the culture chamber, an important bioconversion of EO composition of O. majorana shoots from trans-sabinene hydrate into cis-sabinene hydrate was detected (Table II). These two isomers are biosynthesized by the same enzyme, sabinene hydrate synthase (1). In fact, these culture conditions activate this enzyme, favouring the biosynthesis of cis-sabinene hydrate. Indeed, this is an intensive and spic component of marjoram, whereas the trans-sabinene hydrate does not have any typical properties of marjoram (1). Essential oil composition of shoots was characterized by the prevalence of terpenic alcohols in a proportion of 86.20 %, standing for cis-sabinene hydrate, trans-sabinene hydrate, and terpinen-4-ol, and 8.48 % for monoterpene hydrocarbons (Table II). In the same context, Baatour et al. (14) indicated that the most represented class of EO components of O. majorana native to Canada was that of oxygenated monoterpenes, followed by monoterpene hydrocarbons and esters in control plants. A decrease in these different compound proportions was observed after salt addition, except for monoterpene hydrocarbons and phenols. Greenhouse conditions: Essential oil composition O. majorana shoots Thirty-two compounds were found to represent 99.55 % of EO (Table III), as shown in Figs. 1 a,b. Independent of the applied treatment, terpinen-4-ol was detected as the major compound (31.77 %), followed by trans-sabinene hydrate (24.18 %) and a-ter- O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. FID1 A, (LSBA\PA000000.D) pA 225 200 175 150 125 100 75 50 25 0 1 2 3 4 5 6 10 7 8 9 11 20 30 15 16 18 21 19 20 24 23 25 26 40 27 28 50 min SI 12 13 14 17 22 10 FID1 A, (LSBA\PA000100.D) pA 120 100 80 60 40 20 0 10 20 2 213 10 12 IS SI 28 32 60 min FID1 A, (LSBA\02010802.D) pA 90 80 70 60 50 40 30 20 0 23 1 4 6 5 10 8 7 9 10 12 14 11 13 15 20 16 30 SI 19 20 22 24 26 21 25 27 17 18 23 32 60 min FID1 A, (LSBA\03010801.D) pA 90 80 70 60 50 40 30 20 0 4 2 13 5 14 11 SI IS 15 23 17 22 15' 27 13' 24 10 8 11 79 8'12 9 8 13 10 17 15 11' 16 13'' 25 12' 39 28 36 27' 25 18 18' 33 27'' 35 41 2921 27 23 24 37 38 40 30 26 27 19 32 40 32' 47 48 33 min Fig. 1. Chromatogram of essential oil compounds from Origanum majorana. a) 100 mmol L­1 NaCl in the greenhouse after 17 days of treatment; b) 100 mmol L­1 NaCl in the greenhouse, after 17 days of treatment; c) 100 mmol L­1 NaCl, in the culture chamber, after 17 days of treatment; d) 75 mmol L­1 NaCl, in the culture chamber, after 17 days of treatment. SI: internal standard (6-methyl 5 heptene 2 one). 1: Tricyclene, 2: a-pinene; 3: a-tujene; 4: sabinene; 5: b-myrcene; 6: a-phellandrene; 7: a-terpinene; 8: limonene; 8': 1,8-cineole; 9: g-terpinene; 10: p-cymene; 11: terpinolene; 11': octanol; 12: cis-3-hexanol; 12': hexanol; 13: trans-sabinene hydrate; 13': linalyl acetate; 13'': bornyl acetate; 14: linalool, 15: cis-sabinene hydrate; 15': cis-p-menth-en-1-ol; 16: b-elemene, 17: terpinen-4-ol, 18: b-caryophyllene, 18': a-humulene; 19: aromadendrene; 20: bicyclogermacrene; 21: a-terpenyl acetate; 22: a-terpineol; 23: borneol; 24: neryl acetate, 25: geranyl acetate, 26: nerol, 27: myrthenol; 27': geraniol; 27'': cis-caveol; 28: nonadecane; 29: eiocasane; 30: spathuneol; 31: eugenol; 32: phenol; 32': thymol; 33: carvacrol. 255 O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. Table II. Essential oil composition of Origanum majorana in the culture chamber by GC-MS NaCl 0 100 100 0 (mmol L­1) (mmol L­1) (mmol L­1) (mmol L­1) Compound Tricyclene a-Pinene a-Thujene Sabinene b-myrcene a-Phellandrene a-Terpinene Limonene 1.8-Cineole g-Terpinene p-Cymene Terpinolene Octanal Hexanol cis-3-Hexanol trans-Sabinene hydrate Linalool cis-Sabinene hydrate cis-p-Menthen-1-ol Linalyl acetate Bornyl acetate b-Elemene Terpinene-4-ol B-Caryophyllene Aromadendrene a-Humulene Bicyclogermacrene a-Terpenyl acetate Myrtenyl acetate a-Terpineol Borneol Neryl acetate Geranyl acetate Nerol tR (min) 5.32 5.54 5.66 6.11 8.74 9.7 13.04 13.43 14.56 14.99 16.12 17.33 19.39 25.12 26.95 29.74 29.89 32.06 32.34 32.49 32.58 33.33 34.11 35.19 36.07 36.85 37.06 37.43 37.43 40.08 39.60 40.08 40.35 41.68 KIa 927 931 939 976 991 1006 1016 1030 1033 1062 1026 1088 ­ ­ ­ 1053 1098 1082 1129 1257 1295 1391 1176 1419 1443 1454 1344 1494 1335 1189 1165 1385 1383 1228 KIb 1014 1035 1032 1132 1174 1176 1188 1203 1213 1266 1280 1290 ­ ­ 1385 1474 1553 1556 1562 1556 1597 1601 1611 1612 1628 1687 1705 1755 1701 1713 1702 1733 1765 1797 (%)c 0.142 0.07 0.339 0.094 ND 0.562 1.292 0.517 0.582 0.062 0.050 0.069 0.474 0.210 ND 8.178 ND 43.667 4.892 0.993 2.272 0.329 23.209 0.450 ND 7.300 0.199 ND ND 4.619 0.203 0.133 ND 0.170 (%)c 0.193 0.106e 0.106e 0.134e 0.016 0.787e 1.730e 0.742e 0.782e 0.030e 0.054 0.107e 0.725e 0.020e 0.010 7.783e 0.043 41.745e 3.544e 1.472e 1.974e 0.837e 21.894e 0.507e 0.0169 7.089e 0.227e 0.021 0.021 4.995e 0.241e 0.193e 0.007 0.161e (mg g­1)c,d (mg g­1)c,d 0.045 0.532 0.265 0.352 ND 2.106 4.839 1.937 2.179 0.235 0.187 0.261 1.777 0.789 ND 0.030 0.289 0.163 0.018 3.720 8.507 1.234 0.086 1.688 ND 1.301 0.746 ND ND 0.017 0.763 0.499 ND 0.638 0.409e 0.011e 0.021e 0.290e 0.138 1.041e 0.046e 0.091e 0.022e 0.843e 0.334e 0.042e 0.144e 0.592e 0.010 1.600e 0.084e 20.595e 1.808e 8.845e 8.845e 0.289e 0.556e 58.240e 3.834 1.160e 0.285e 2.049 2.049 9.820e 41.500e 1.740e 0.811 0.468e O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. Table II. continued Myrtenol Geraniol cis-Caveol Nonadecane Eicosane Spathulenol Phenol Eugenol Thymol Carvacrol Classes Monoterpene hydrocarbons Terpenic alcohols Sesquiterpene hydrocarbons Terpenic esters Aliphatic hydrocarbons Phenols 8.483 86.207 0.714 3.265 0.003 0.025 1194 1255 1247 1581 1996 ­ ­ 1401 1295 1302 1804 1857 1804 1900 2000 2144 ­ 2030 ­ ­ 0.027 ND 0.449 ND 0.036 ND ND ND ND 0.025 0.024e 0.025 0.462e 0.011 0.034e 0.012 0.056 0.013 0.041 0.045e 11.126e 81.671e 0.156e 3.306e 0.004e 0.156e 0.104 ND 1.683 ND 0.138 ND ND ND ND 0.096 3.253e 0.021 0.392e 0.010 0.065e 0.008 0.093 5.186 0.063 0.023e 4.744e 48.012e 0.162e 0.048e 0.076e 0.193e tR ­ retention time, KI ­ Kovat's index on: a apolar column HP-5MS, n-alkane), ND ­ not detected. c Mean of 3 replicates. d Dry mass basis. e Significantly different after salt addition at p < 0.05. polar column HP-Innowax (relative to pineol (7.30 %). In the presence of NaCl (100 mmol L­1), a small increase was observed in terpinen-4-ol percentage, while percentages of trans-sabinene hydrate and a-terpineol were not affected. In the absence or presence of salt, O. majorana EO was of trans-sabinene hydrate/ terpinen-4-ol chemotype. These results are in agreement with those of Hamrouni et al. (9), who reported that the major constituents were terpinen-4-ol, cis-sabinene hydrate, and trans-sabinene hydrate. Later investigations showed that some of the above main constituents were found in numerous EO samples of O. majorana. Novak et al. (1) mentioned that the main compounds of marjoram EO were the epimeric monoterpene alcohols, trans-sabinene hydrate, cis-sabinene hydrate, and cis-sabinene hydrate acetate. In fact, according to Vera and Chane-Ming (2), the main components were terpinen-4-ol and cis-sabinene hydrate in EO of O. majorana originating from Island. Our results showed that NaCl modified the essential oil composition. In fact, new compounds appeared: eicosane, spathulenol, eugenol, and phenol (Table III). However, myrcene, present in the control, disappeared under saline conditions. Indeed, a very important modification of EO composition was bioconversion from cis-sabinene hydrate into trans-sabinene hydrate. Culture conditions had an important effect on the transformation of these two isomers: from cis- to trans-sabinene hydrate under greenhouse conditions and from trans- to cis-sabinene hydrate under culture cham257 O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. ber conditions. These results suggest that culture conditions activated sabinene hydrate synthase in favor of cis-sabinene hydrate or trans-sabinene hydrate. Under saline conditions, contents of all compounds evolved in the same way (Table III). The composition of EO of O. majorana shoots is primarily made up of terpenic alcohols (78.27 %), followed by monoterpene hydrocarbons in the control (18.74 %). Salinity decreased the content of each class, except for aliphatic hydrocarbons and terpenic alcohols (Table III). The quality of the EO produced and its composition in Origanum majorana depends on culturing conditions (15). Our findings are in accord with those of Avry and Gallouin (9) and Kotan et al. (16). Essential oil of O. majorana plants cultivated in a greenhouse had lower level of flavor than EO of plants cultivated in the culture chamber due to the great difference in the cis-sabinene hydrate production. Therefore marjoram cultivated Table III. Essential oil composition of Origanum majorana in the greenhouse by GC-MS. NaCl (mmol L­1) 0 Compound Tricyclene a-Pinene a-Thujene Sabinene b-Myrcene a-Phellandrene a-Terpinene Limonene g-Terpinene p-Cymene Terpinolene cis-3-Hexanol trans-Sabinene hydrate Linalool cis-Sabinene hydrate b-Elemene Terpinen-4-ol b-Caryophyllene Aromadendrene Bicyclogermacrene a-Terpenyl acetate a-Terpineol tR (min) 3.28 3.55 3.69 5.74 6.45 9.84 11.39 11.49 11.61 14.25 16.62 26.62 29.74 29.89 30.283 31.729 31.91 32.47 33.00 34.86 35.40 35.78 RIa ­ 931 939 976 991 1006 1016 1030 1033 1062 1026 1088 1053 1098 1082 1053 1098 1082 1129 1257 1295 1391 RIb ­ 1035 1032 1132 1174 1176 1188 1203 1213 1266 1280 1290 1474 1553 1556 1474 1553 1556 1562 1556 1597 1601 (%)c 0.252 0.316 0.506 0.181 3.807 4.145 0.710 0.162 1.448 5.883 1.183 7.428 24.181 3.994 2.235 0.197 31.77 0.210 0.864 0.335 0.320 7.300 100 (%)c ND 0.121e 0.430e 2.067e ND 3.602e 0.724e 2.205e 1.903e 3.215e 1.081e 10.650e 24.541e 3.724e 2.492e 0.225e 29.050e 0.311e 0.545e 0.223e 0.238e 7.089e 0 100 (mg g­1)c,d (mg g­1)c,d 0.045 0.056 0.090 0.032 0.678 0.738 0.126 0.028 0.258 1.048 0.210 1.323 4.309 0.711 0.398 0.035 5.662 0.037 0.154 0.059 0.057 1.301 ND 0.019e 0.070e 0.338e ND 0.589e 0.118e 0.361e 0.311e 0.526e 0.176e 1.743e 4.017 0.609e 0.408e 0.036 4.756e 0.051e 0.089e 0.036e 0.039e 1.160e O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261. Table III. continued Borneol Neryl acetate Geranyl acetate Nerol Myrtenol Nonadecane Eicosane Spathulenol Eugenol Phenol Classes Monoterpene hydrocarbons Terpenic alcohols Sesquiterpene hydrocarbons Terpenic esters Aliphatic hydrocarbons Phenols 18.749 78.270 1.410 1.320 0.123 0 15.577e 79.365e 1.081e 1.005e 0.377e 0.115 3.313 1.395 0.251 0.235 0.021 0 2.550e 1.195e 0.177e 0.110e 0.059e 0.044 37.19 37.49 38.13 39.49 41.27 43.61 52.95 54.162 55.482 61.071 1176 1385 1419 1228 1191 1454 1344 1494 1335 ­ 1611 1733 1612 1797 1804 1687 1705 1755 1701 ­ 0.885 0.495 0.145 0.112 0.361 0.123 ND ND ND ND 0.786e 0.289e 0.147 ND ND 0.147 0.229 0.329 0.142 0.115 0.157 0.088 0.025 0.020 0.064 0.025 ND ND ND ND 0.128e 0.047e 0.024 0.037e 0.053e 0.024 0.039 0.023 0.018 0.020 ND ND ND tR ­ Retention time. KI ­ Kovat's index on: a apolar column HP-5MS, b polar column HP-Innowax (relative to n-alkane). ND ­ not detected. c Mean of 3 replicates. d Drug mass basis. e Significantly, different after salt addition at p < 0.05. in the greenhouse was of higher quality than that cultivated in the culture chamber because of its high terpinen-4-ol concentration (Tables II, III). The EO is believed to have had medicinal properties because of the presence of biologically active compounds such as terpinen-4-ol (5). In fact, terpinen-4-ol, a-terpineol, and linalool exhibited high antibacterial activity. O. majorana EO is known for its strong antimicrobial activity and could therefore be used in food applications. CONCLUSIONS Our data indicate high variations in quantitative and qualitative composition of O. majorana EOs produced under two conditions: greenhouse and culture chamber. The study showed that EO yield and composition depend on the culturing and climatic conditions. We observed an important modification in EO composition of O. majorana shoots by converting trans-sabinene hydrate into cis-sabinene hydrate in the culture chamber and cis- to trans-sabinene hydrate in the greenhouse. The EO of the latter was considered to be of higher quality than that cultivated in the culture chamber, because of its higher concentration of oxygenated compounds, particularly bioactive compounds such as terpinen-4-ol. O. Baâtour et al.: Culture conditions and salt effects on essential oil composition of sweet marjoram (Origanum majorana) from Tunisia, Acta Pharm. 62 (2012) 251­261.

Journal

Acta Pharmaceutica – de Gruyter

Published: Jun 1, 2012

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