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Essential-Oil Composition of Daucus carota ssp. major (Pastinocello Carrot) and Nine Different Commercial Varieties of Daucus carota ssp. sativus Fruits by Guido Flamini* a ) b ), Elena Cosimi a ), Pier Luigi Cioni a ), Ilaria Molfetta c ), and Alessandra Braca a ) b ) a ) Dipartimento di Farmacia, Universita` di Pisa, via Bonanno 33, I-56126 Pisa (phone: þ 39-050-2219686; fax: þ 39-050-2219660; e-mail: [email protected].) b ) Centro Interdipartimentale die Ricerca Nutraceutica e Alimentazione per la Salute, Universit di Pisa, Via del Borghetto 80, I-56124 Pisa c ) Dipartimento di Scienze Agrarie, Alimentari ed Agro-Ambientali, via del Borghetto 80, I-56124 Pisa
The chemical composition of the essential oils obtained by hydrodistillation from the pastinocello carrot, Daucus carota ssp. major (Vis.) Arcang. (flowers and achenes), and from nine different commercial varieties of D. carota L. ssp. sativus (achenes) was investigated by GC/MS analyses. Selective breeding over centuries of a naturally occurring subspecies of the wild carrot, D. carota L. ssp. sativus, has produced the common garden vegetable with reduced bitterness, increased sweetness, and minimized woody core. On the other hand, the cultivation of the pastinocello carrot has been abandoned, even if, recently, there has been renewed interest in the development of this species, which risks genetic erosion. The cultivated carrot (D. carota ssp. sativus) and the pastinocello carrot (D. carota ssp. major) were classified as different subspecies of the same species. This close relationship between the two subspecies urged us to compare the chemical composition of their essential oils, to evaluate the differences. The main essential-oil constituents isolated from the pastinocello fruits were geranyl acetate (34.2%), apinene (12.9%), geraniol (6.9%), myrcene (4.7%), epi-a-bisabolol (4.5%), sabinene (3.3%), and limonene (3.0%). The fruit essential oils of the nine commercial varieties of D. carota ssp. sativus were very different from that of pastinocello, as also confirmed by multivariate statistical analyses.
Introduction. – Cultivated carrot, Daucus carota ssp. sativus [1], was obtained in the Middle East by hybridization between strains of D. carota ssp. carota and D. carota ssp. maximus, and its cultivation in the Middle Age was spread to Western Europe. Through the improved agronomic selection, currently, carrot has an edible, plump, and sweet root [2]. D. carota ssp. major, known as pastinocello, seems to be an ancient variety, perhaps dating back to the Roman times, selected from the wild carrot. This variety would have preserved the characteristics of the first breeding, which are very different from those obtained with the latest improvements, as well in color, shape, and organoleptic requirements. Pastinocello carrots were traded together with other important products, such as olive oil, and were used by farmers for special celebrations. Not only the root, but also the aerial parts, leaves, and stems are eaten. The leaves, called erbuccio, tender and tasty, are eaten fresh in salads or boiled; in boiling water they are curative for kidney disease; the leaves are also used for preparing omelets or diuretic infusions. Pastinocello is one of the earliest forms of domestication of wild 2014 Verlag Helvetica Chimica Acta AG, Zrich
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carrots and has a yellow-brown root, a sweet and distinctive nutty taste, and a mellower and softer texture than other carrots [3]. The D. carota L. group is characterized by a wide polymorphism and comprises species such as D. carota sensu stricto and its subspecies D. carota L. ssp. carota, D. carota ssp. maritimus (Lam.) Batt., D. carota ssp. major (Vis.) Arcang., D. carota ssp. maximus (Desf.) Ball, and D. carota ssp. sativus (Hoffm.) Arcang. [1]. D. carota ssp. major is widespread in uncultivated and grass lands [1] and along the river banks, from sea level up to high altitude [3]. Its features are considered intermediate between D. carota ssp. carota and D. carota ssp. maximus. Similarly, it is believed that D. carota ssp. sativus, with its enlarged root that is usually orange colored, resulted from D. carota ssp. carota D. carota ssp. maximus crossing [1]. The pastinocello carrot, identified as D. carota ssp. major (Vis.) Arcang. is generally more or less hispid with usually ascending stems; the leaves are green and shiny; the stem, 30 cm to 2 m long, is striated and branched. The white flowered umbels are 6 – 10 cm wide and appear during spring. According to the different areas in which the plants grow, D. carota L. essential oil belongs to distinct chemotypes such as a carotol ( 77.5%), geranyl acetate ( 81.2%), and sabinene ( 60.4%) chemotype and those containing ca. equal percentages of these compounds. In some habitats, carrot species are able to accumulate in the fruit essential oil considerable amounts of a-pinene ( 55.5%), geraniol ( 50.0%), b-bisabolene ( 35.0%), b-caryophyllene ( 29.0%), g-bisabolene (87.0%, China), and (E)asarone (40.3%, Japan) [4]. Previous studies on the composition of the essential oil of the flowers from wild carrot (D. carota L. ssp. carota) grown in Poland showed that the main constituents were sabinene and geranyl acetate, while carotane-type sesquiterpenes were absent [5] [6]. Some authors reported an allelopathic activity for the aqueous extract of carrot seeds [7] and for their essential oil [8]. The low sensitivity of carrot seeds to fungal infections seems to be due to carotol, a fungitoxic compound active against Alternaria sp. strains [9] and Fusarium oxysporum [10]. The fruit essential oil showed antibacterial activity [4] against Bacillus subtilis [11], Staphylococcus aureus [12], and Campylobacter jejuni [13]. Thanks to the herbicidal, fungicidal, and insecticidal activities of its constituents, this essential oil may be useful for the preparation of biopesticides [14]. The essential oil of the fruits also showed a hypotensive and sedative action and has a moderate anticonvulsant protective effect against strychnine and metrazol poisoning [15]. Results and Discussion. – Note on the Plant-Material Selection. The close relationship between D. carota ssp. sativus and D. carota ssp. major (pastinocello) urged us to compare the chemical composition of their essential oils. Seeds (fruits) of nine commercial varieties of cultivated carrot and pastinocello fruits and dried flowers were selected. In Table 1, the names and the manufacturers of the selected samples are listed. Essential-Oil Yield. The average yield of fruit essential oil of the D. carota ssp. sativus varieties ranged between 0.5 and 0.8% (w/w), whereas the D. carota ssp. major (pastinocello) samples showed oil yields of 0.5 and 0.8% for the fruits and dried flowers, respectively. Although the first three samples (Samples 1 – 3; Table 1) belonged to the same variety (Nantese 2), the seeds of the different brands showed significant differences in their essential-oil yields.
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 1. Analyzed Carrot Samples: Fruits (seeds) of Nine Commercial Varieties of Daucus carota ssp. sativus (cultivated carrot; Samples 1 – 9) and Flowers and Fruits of D. carota ssp. major (pastinocello; Samples 10 and 11, resp.) Sample Sample abbreviation Variety 1 2 3
Nant_mez_lun Nant_prec Nant_Chiog
4 5
Nant_ibr_Asc Nant_biol
6 7 8 9 10 11
Berlicum2 Ortolana Rubrovit Flakke`e2 Pastin_flow Pastin_fruit
Mezza lunga Nantese 2 Nantese 2 precoce Nantese migliorata 2 or Nantese Chioggia 2 Nantese ibrida Ascania F1 Nantese 5 (biological)
Origin/Company Quagliano sementi ( Pisa, Italy) Landen ( The Netherlands) SAIS (Cesena, Italy)
Compagnia delle Sementi (Cesena, Italy) Bio-coop, Cooperativa agricola Cesenate (Cesena, Italy) Berlicum 2 Gargini sementi ( Lucca, Italy) Ortolana SAIS selezione conservatrice (Cesena, Italy) Rubrovitamina SAIS selezione conservatrice (Cesena, Italy) Flakke`e 2 Blumen (Piacenza, Italy) Pastinocello dried flowers Regional germplasm bank ( Tuscany, Italy) Pastinocello fruits Regional germplasm bank ( Tuscany, Italy)
Essential-Oil Composition. Altogether, 212 compounds were identified in the investigated oil samples, accounting from 92.7 to 99.8% of the whole essential-oil compositions (Table 2). The main constituents of the essential oil isolated from the fruits of D. carota ssp. major (pastinocello; Sample 11) were geranyl acetate (123 1)), 34.2%), a-pinene (7, 12.9%), geraniol (94, 6.9%), myrcene (14, 4.7%), epi-a-bisabolol (208, 4.5%), sabinene (11, 3.3%), and limonene (24, 3.0%). The essential oils of the nine commercial varieties of D. carota ssp. sativus were very different from that of pastinocello fruits. The content of geranyl acetate was considerably lower in the oils of the D. carota ssp. sativus varieties, with the only exception being variety Rubrovitamina (Sample 8), which contained a similar percentage than the pastinocello-fruit oil (23%). The same was observed for geraniol, which was completely absent in some varieties, while it appeared in small amounts in others. The closest geraniol oil content with respect to pastinocello fruits was found again for Rubrovitamina (4.3%); a comparison of the relative amounts of the major constituents highlighted many similarities between the pastinocello-fruit oil and the oil of the Rubrovitamina variety of cultivated carrot. Most of the oils of the commercial varieties contained carotol (184), with particularly high contents observed for varieties Flakke`e 2 (Sample 9; 49.8%) and Mezza Lunga Nantese 2 (Sample 1; 37.2%). On the contrary, carotol was not detected at all in the pastinocello-fruit oil. The a-pinene (7) oil contents varied between 2.5% in variety Flakke`e 2 and 21.7% in variety Nantese 2 precoce (Sample 2), while the pastinocellofruit oil showed intermediate values (12.9%). In the essential oil of the pastinocello flowers, the major compounds were a-pinene (7, 24.4%), sabinene (11, 13.3%), geranyl acetate (123, 13.0%), epi-a-cadinol (198, 8.5%), myrcene (14, 4.8%), and b-oplopenone (189, 4.3%). A comparison of the flower oil with the fruit essential oil revealed higher and more significant contents of apinene and sabinene and lower amounts of geranyl acetate and geraniol. Considering 1)
Italic numbers in parentheses refer to the entries in Table 2.
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Table 2. Chemical Composition of the Essential Oils Isolated from the Fruits (seeds) of Nine Commercial Varieties of Daucus carota ssp. sativus ( Samples 1 – 9) and the Flowers and Fruits of D. carota ssp. major ( Samples 10 and 11, resp.) Entry Compound name and class
LRI a ) Content [%] b ) 1 c)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Hexanal Hexan-1-ol Heptanal Heptan-2-one a-Thujene Tricyclene a-Pinene Camphene Thuja-2,4(10)-diene Benzaldehyde Sabinene b-Pinene 6-Methylhept-5-en-2-one Myrcene 6-Methylhept-5-en-2-ol Octan-3-one Yomogi alcohol ( E )-Hex-3-enyl acetate b-Phellandrene Octan-1-al a-Phellandrene a-Terpinene p-Cymene Limonene 1,8-Cineole ( Z )-b-Ocimene Benzene acetaldehyde ( E )-b-Ocimene Bergamal g-Terpinene Artemisia ketone cis-Sabinene hydrate cis-Linalool oxide Artemisia alcohol p-Mentha-2,4(8)-diene Terpinolene Dehydrolinalool p-Cymenene trans-Linalool oxide 6,7-Epoxymyrcene Camphen-6-one a-Pinene oxide Linalool Nonanal Camph-6-enol p-Mentha-1,3,8-triene
802 833 871 892 902 908 910 925 931 937 950 954 963 966 969 984 998 1002 1003 1007 1008 1021 1029 1034 1037 1043 1045 1054 1057 1062 1064 1073 1080 1087 1088 1090 1091 1092 1095 1096 1097 1099 1103 1106 1112 1113
2
3
– – – – – tr tr tr tr – – tr 0.2 0.2 0.7 tr – tr 8.5 21.7 4.1 0.8 1.7 0.5 tr tr tr tr tr tr 10.7 17.8 28.8 2.3 3.0 1.1 tr – – 5.8 10.5 1.7 – – – – – tr – – – – – – – – – tr – tr – tr – tr tr 0.2 5.0 0.3 0.7 2.6 5.1 1.9 tr tr tr tr 0.2 – – – – tr tr – – – – 1.0 0.2 0.5 – – – 0.3 0.1 1.6 – – tr – tr – 0.2 – – – 0.2 0.2 0.2 – 0.2 – – – – – – tr tr 0.2 – – – – – 0.7 1.3 1.8 2.8 – tr tr 1.2 – – – – –
4
5
6
7
1.0 – – – – – – – – tr tr tr – – – – tr 0.2 0.4 tr – tr – 0.1 3.4 12.6 13.7 15.0 0.3 1.4 1.5 2.8 tr tr 0.5 1.0 tr tr tr tr 6.3 10.2 20.7 0.6 0.3 3.2 1.5 3.8 tr 0.2 tr tr 0.5 5.4 3.4 1.0 – – – – – – – tr – – – – – – – tr – – – tr – tr – – – – – tr tr – 0.3 tr 1.2 7.2 3.2 0.3 1.0 6.0 3.2 2.3 tr – tr – – – – tr – – tr – – – tr – – tr – – tr 0.4 0.7 tr – – – – 0.7 0.4 0.7 0.2 – tr tr 0.2 – – – – – – – – – 0.5 – – 0.5 0.4 – 1.3 – – – tr – – – tr tr tr – tr tr 0.2 – 0.6 3.0 – – 1.4 1.7 6.7 2.4 1.0 – – – – – 3.0 – – – – – –
8
9
– – tr – tr tr 6.3 1.1 0.2 tr 0.8 0.8 0.1 1.6 tr – – – – – – – 1.7 1.3 tr tr – tr tr tr – tr 0.3 – – – 0.7 – 0.8 tr 0.3 – 3.2 – 3.2 –
– – – – – – – – 0.1 – – – tr 0.3 0.2 – – – 2.5 24.4 12.9 0.3 2.2 1.1 tr 0.1 0.8 tr – – 7.9 13.3 3.3 0.3 1.3 1.8 tr – tr 1.4 4.8 4.7 – – – tr – – – – tr – – – – – – tr – – – 0.2 – tr 0.4 0.3 1.7 0.4 1.5 1.6 2.9 3.0 tr – – tr 0.2 0.4 – – tr tr 0.3 0.5 – – – 0.4 0.6 0.4 – – tr 0.3 0.2 tr 0.2 – tr – – – – – – 0.2 0.3 0.6 – – – – – – – – – tr tr tr – – – 1.4 – – 1.5 0.7 2.2 – – – – – – – – tr
10
11
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 2 (cont.) Entry Compound name and class
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92
b-Thujone exo-Fenchol trans-p-Mentha-2,8-dien-1-ol Dehydrosabinaketone cis-p-Menth-2-en-1-ol a-Campholenal cis-Limonene oxide cis-p-Mentha-2,8-dien-1-ol trans-Sabinol cis-Sabinol trans-Pinocarveol trans-Verbenol trans-p-Menth-2-en-1-ol cis-Verbenol Sabinaketone b-Pinene oxide trans-Pinocamphone ( E )-Non-2-enal Pinocarvone p-Mentha-1,5-dien-8-ol Borneol Umbellulone d-Terpineol cis-Pinocamphone Terpinen-4-ol Cryptone a-Thujenal p-Cymen-8-ol trans-p-Mentha-1,(7)8-dien1-ol a-Terpineol Myrtenol Myrtenal p-Cymen-9-ol trans-Piperitol Verbenone trans-Carveol ( Z )-Ocimenone Nerol cis-p-Mentha-1,(7)8-dien1-ol cis-Carveol Pulegone Neral Cumin aldehyde Carvone Piperitone Chavicol
LRI a ) Content [%] b ) 1 c)
2
3
1114 1117 1123 1127 1128 1131 1138 1141 1142 1143 1144 1145 1146 1150 1159 1162 1163 1165 1167 1168 1170 1171 1172 1175 1181 1186 1187 1188 1189
tr – – – – 0.9 – – – – 1.5 2.8 – tr 0.6 tr – tr 0.6 – 0.2 – 0.3 tr 0.2 – 0.3 – –
– – – – tr 0.3 tr – – – 0.4 0.7 – – – 0.2 – – 0.3 – tr – – – 0.6 – tr – –
1193 1195 1196 1205 1211 1213 1223 1229 1230 1232
0.1 – 1.2 – – 0.4 0.2 – – –
1234 1238 1240 1246 1249 1258 1259
– – – 0.2 0.2 – –
4
5
6
– 0.2 – – – 0.3 tr – 0.3 – 1.7 1.6 0.3 tr tr tr – – – – 3.0 3.7 2.8 10.2 – 0.7 – – 1.7 0.7 0.7 0.5 – – – – 0.8 1.0 1.1 – tr – tr – – 1.2 – – 1.4 0.9 tr – 0.9 0.6 – – – –
0.2 – 0.2 – – 1.6 tr tr 0.6 – 2.6 tr – 5.9 0.7 0.4 – – 1.1 – 0.6 – – – 0.4 – 0.5 – –
0.2 – 0.2 – tr tr tr – – –
tr 0.8 1.4 – tr 0.2 0.2 – – –
tr – 2.6 – – 1.4 1.1 – – –
– – – – tr tr –
– – – 0.8 0.2 – –
– – – 0.4 0.9 – 0.2
7
8
9
10
11
– tr 0.2 – tr – – – – – – 0.2 0.2 0.3 – 1.7 1.8 1.8 tr – tr tr – tr tr 1.6 – – tr – 1.8 6.1 2.7 0.1 17.1 13.4 1.4 – – 7.7 – 1.1 1.1 tr tr – – – – 0.4 0.4 – – – 0.8 2.8 1.1 0.5 0.8 – – – 0.5 tr – – – – 0.5 – tr tr 0.9 0.2 0.2 – – – 0.6 – – – 0.3 0.5 – tr tr
0.1 – 0.1 – – 1.0 tr tr – – 1.8 4.2 – tr 0.6 0.4 – tr 0.5 – 0.2 – – – 0.3 tr 0.4 – –
– – – – tr 0.3 tr tr – – 0.2 0.9 – 0.2 – – – – 0.3 0.1 – – – – 0.6 – tr – –
– tr tr – – 0.6 – tr – – 0.9 2.4 – 0.2 – 0.2 – 0.3 0.5 – 0.2 – – – 0.2 – – 0.1 –
0.2 – 1.9 – tr 0.6 0.5 – 0.3 –
0.1 0.4 1.5 – – 1.5 0.7 – – –
0.2 – 3.7 tr tr 3.9 2.0 – – tr
0.1 – 2.3 – – 3.0 1.4 – 0.3 –
0.1 0.4 1.2 – 0.1 0.5 0.5 tr – –
tr – 0.4 – – 0.1 0.1 – tr –
0.2 1.0 – – – 0.3 0.3 – – –
– – tr 0.2 0.8 – –
tr – – 0.2 0.3 – –
tr – – tr 1.1 – –
– tr – tr 1.0 – –
tr – – 0.3 0.3 tr –
– – – – tr – –
0.1 – tr tr 0.1 – –
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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Table 2 (cont.) Entry Compound name and class
93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140
Carvenone Geraniol Linalyl acetate trans-Myrtanol cis-Carvone oxide ( E )-Cinnamaldehyde p-Menth-1-en-7-al Geranial trans-Carvone oxide cis-Verbenyl acetate a-Terpinen-7-al Isobornyl acetate p-Cymen-7-ol trans-Sabinyl acetate Thymol Tridecane trans-Pinocarvyl acetate Carvacrol Undecanal Dihydrocarvyl acetate Myrtenyl acetate Piperitenone a-Terpinyl acetate Eugenol Neryl acetate Cyclosativene a-Copaene Daucene b-Patchoulene trans-Myrtenyl acetate Geranyl acetate Isolongifolene b-Cubebene 7-Episesquithujene b-Elemene Isoitalicene Italicene Methyl eugenol Longifolene a-Gurjunene a-Santalene cis-a-Bergamotene b-Caryophyllene g-Elemene trans-a-Bergamotene ( Z )-b-Farnesene Aromadendrene epi-b-Santalene
LRI a ) Content [%] b )
1260 1261 1262 1263 1269 1276 1277 1278 1281 1283 1287 1288 1290 1291 1295 1300 1302 1303 1308 1311 1330 1347 1355 1358 1370 1373 1378 1382 1381 1383 1388 1387 1392 1393 1394 1402 1408 1409 1411 1412 1418 1419 1421 1433 1439 1443 1447 1450
1 c)
2
3
4
5
6
7
– – – – – – tr – – tr – 0.7 – – – – – – – – tr – tr – tr – – 0.3 – – 0.8 – – tr – – – tr – – – 0.2 0.7 – 0.4 tr 0.2 –
– – – – – – tr – – – – 1.6 – – – – – – – – – – 0.2 – tr – tr 0.3 – – 0.2 – – tr – – tr – tr – – 0.5 1.6 tr 0.8 – 0.3 tr
tr – – – – – 0.2 – – tr 0.3 0.4 – tr – – – tr tr – tr – 0.6 – tr – – 0.2 – – 0.3 0.4 – – tr – tr – – – – 0.2 0.4 – 0.3 – 0.2 –
– – 0.3 tr – tr 0.3 – – – – 1.6 – – – – – – – – – – 0.8 – – – – tr tr – 3.8 – tr – – – – – – – – – 0.3 – 0.3 – – –
– 2.7 – – – – tr 0.2 – – – 0.9 – – – – – – – – – – tr – tr – – tr – – 6.1 – – tr – – tr – – – – 0.3 1.3 – 0.4 – 0.2 –
– – tr – – – 0.1 – – – tr 0.6 tr – – – – tr – – – – 0.2 – tr tr tr 0.3 – – – – – – tr tr – – – – – tr 0.4 – 0.3 – – –
– – 0.2 4.3 – – tr – tr – – – tr – – 0.4 tr – – – – – 1.0 0.9 – – – – tr – – – tr – – – – – tr – tr tr tr – 0.1 tr – – tr tr – – – – – tr – – – – 4.8 23.0 – – – – tr – – – – – – tr – – – – – – tr – tr tr 0.2 0.2 – – tr tr – – tr tr – –
8
9
10
– – tr – – – 0.2 – – – 0.2 0.8 – – 0.1
– – 0.2 6.9 – – – – – – – – – – – 0.2 – – – – – – 0.7 – – – – – – – tr – – – – – – – – – tr tr – – tr 0.2 – tr 0.8 0.1 tr 0.1 0.1 tr – – – – – tr 13.0 34.2 – – – – – – – – – – – – – – – – 0.1 – – – – tr 1.3 0.4 – – tr – – – – – – –
– tr – – tr – 0.4 – tr – tr 0.6 – – 0.2 – – – tr – tr – – – – 0.2 1.0 – 0.6 – – 0.4
11
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 2 (cont.) Entry Compound name and class
LRI a ) Content [%] b ) 1 c)
141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188
b-Himachalene a-Humulene a-Patchoulene ( E )-b-Farnesene g-Decalactone cis-Muurola-4(14),5-diene b-Acoradiene trans-Cadina-1(6),4-diene 9-epi-(E )-Caryophyllene g-Muurolene Germacrene D g-Himachalene b-Selinene Bicyclogermacrene a-Muurolene a-Selinene ( E )-Methyl isoeugenol Pentadecane g-Patchoulene ( Z )-a-Bisabolene b-Bisabolene d-Amorphene 10-Epiitalicene ether ( Z )-g-Bisabolene Myristicin d-Cadinene b-Sesquiphellandrene ( E )-a-Bisabolene a-Cadinene Selina-3,7(11)-diene cis-Sesquisabinene hydrate cis-Cadinene ether Elemol Germacrene B Italicene epoxide trans-Cadinene ether Silphiperfol-5-en-3-ol A Dimethyl ionone trans-Nerolidol Longicamphenilone Caryophyllenyl alcohol Spathulenol Caryophyllene oxide Carotol Guaiol trans-Sesquisabinene hydrate Cedrol Sesquithuriferol
1451 1458 1459 1461 1463 1465 1471 1472 1475 1478 1480 1483 1486 1494 1497 1498 1499 1500 1502 1505 1510 1512 1516 1517 1519 1525 1526 1531 1538 1547 1547 1554 1555 1557 1558 1559 1560 1565 1567 1568 1572 1578 1583 1594 1595 1598 1599 1605
2
3
4
– – – – tr 0.1 – – – – – – 1.5 2.5 0.9 0.7 – – – – – tr – – – – – – 0.1 tr tr tr – – – – – – – – tr 0.1 – – – – – – 0.3 0.4 tr – – 0.1 tr – – – – – tr – – – 0.4 – – – – – – 0.5 – – – tr – tr – – 0.5 1.3 0.8 1.1 – – – – – – – – – – – – 0.4 – – 1.3 – – – – 0.1 0.2 – – – – – – – – – – – – – – 0.2 – – 0.2 – – – 0.1 0.1 – – – tr tr tr – – – – – – – 0.1 – – – – – – – – – – – – – – – – – – – – – 0.1 tr 0.1 – 2.5 0.8 2.7 4.4 37.2 22.0 20.3 24.2 – – – – – – – – – 0.2 – – tr – – –
5
6
– – tr – – – 1.4 0.5 tr – – tr – – – – – – – tr tr – – – 0.2 0.3 – – – – – – – – – – – – – – 0.2 0.4 – – – – – – – – – – tr tr – – – – – – tr – – – tr – tr – – – – – – – – – – – – – – – 0.2 tr 2.8 1.0 4.7 17.5 – – – – – – – –
7
8
9
– tr tr 0.3 – – – tr – – – – tr tr – – – – – – 0.1 – – – tr – – – – – – – – tr 0.1 – 0.4 – – – – tr 2.6 8.6 – tr – –
– 0.2 tr 0.1 – – 0.2 1.3 0.2 – – – – – – 0.2 tr – – – – – – – 0.1 0.9 tr 0.2 – – 0.2 0.2 – – – – – – – – 0.2 1.3 – – – – tr – 0.1 tr – – tr tr – tr – – – – – 0.3 – – 0.2 – tr tr – – – – – – tr 0.1 – 0.2 tr – – 0.1 tr 0.3 3.0 3.4 6.4 49.8 – – – – – – – –
10
11
– 0.5 – 0.6 – – 1.1 – – – 0.5 – 0.3 0.2 – – – – – – 0.8 – 2.0 – – 0.1 – – – 0.4 – – – – – – – – – – – 0.4 0.4 0.2 tr – – –
– 0.2 – 0.4 – tr – – – – 0.2 2.0 0.1 – tr – – – – – 0.2 tr – – – 0.2 – – tr – – – – – – – – – – – – 0.7 0.7 – – – – –
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Table 2 (cont.) Entry Compound name and class
189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212
b-Oplopenone Humulene epoxide II Bisabol-11-ol Benzophenone 1-epi-Cubenol b-Cedren-9-one Caryophylla-4(14),8(15)-dien5-ol Epoxyalloaromadendrene Daucol epi-a-Cadinol Agarospirol b-Eudesmol a-Cadinol Bulnesol 14-Hydroxy-9-epi-( E)caryophyllene b-Bisabolol ( Z )-a-Santalol Elemol acetate a-Bisabolol epi-a-Bisabolol Apiol Junicedranol Juniper camphor Aristolone
LRI a ) Content [%] b ) 1 c)
2
3
4
5
6
7
8
9
10
11
1606 1609 1626 1628 1631 1633 1637
– tr – – – – –
– tr – – 0.1 – –
– 0.3 – 3.4 – – –
– 0.5 – 7.8 – – –
– tr – – – – –
– tr – – – – –
– tr – 0.3 – – tr
– 0.3 – 2.7 – – tr
– 0.4 0.1 – – 0.3 –
4.3 – – – – – –
– 1.2 – – – – –
1639 1641 1643 1648 1649 1656 1667 1670
tr 1.7 – – – – 0.3 –
– 0.1 tr – – tr tr –
– 2.0 – – – 0.2 – –
– 3.3 – – – – – –
– 0.3 – – – tr – –
– 0.7 – – – – – –
0.2 5.0 – – – – tr 0.2
– 1.6 – – tr 0.1 tr 0.4
0.1 2.2 – – – – – –
– – 8.5 – tr 0.1 – –
– – 0.2 tr – – – –
1674 1675 1681 1683 1686 1687 1693 1696 1751
0.6 – – 0.4 0.2 – – tr –
0.2 – – 0.1 – – – tr –
0.2 – – tr – – – 0.6 –
– – – tr – – – tr –
0.3 – – tr – – – 0.5 –
– – – tr – – – tr –
– 0.2 – – – 1.2 – tr –
– 0.1 0.2 0.1 – – – 0.1 0.5
– – – – – – – – –
– – – – 0.3 – – – –
– – – 0.2 4.5 – 0.1 tr –
Total identified [%]
99.6 99.5 99.8 99.7 99.5 96.0 99.7 99.6 99.7 92.7 94.3
Monoterpene hydrocarbons Oxygenated monoterpenes Total monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Total sesquiterpenes Non-terpene derivatives
37.0 14.3 51.3 4.3 43.2 47.5 0.8
60.7 6.7 67.4 8.3 23.7 32.0 tr
40.3 26.3 66.6 3.4 26.2 29.7 3.4
13.0 40.9 53.9 2.5 32.5 35.0 10.8
47.0 40.2 87.2 3.9 8.7 12.7 0.2
49.1 25.5 74.6 2.2 19.2 21.4 tr
26.9 53.2 80.1 0.6 17.4 18.0 1.6
13.7 67.7 81.4 1.2 13.0 14.2 3.1
16.1 18.2 34.3 7.1 57.3 64.4 0.1
51.7 18.8 70.5 6.0 16.2 22.2 tr
31.5 51.1 82.6 3.7 7.6 11.3 0.4
a ) LRI: Linear retention indices determined on a DB-5 column. b ) Content given as percentage of the total oil composition; –, not detected; tr, trace. c ) For the variety and origin of Samples 1 – 11, cf. Table 1.
the chemical compound classes, the main differences between the oils of pastinocello flowers and fruits were observed for monoterpenes: the content of monoterpene hydrocarbons was 51.7% in the flowers and 31.5% in the fruits, while the oxygenated monoterpenes reached 18.8% in the flowers and 51.1% in the fruits, respectively. Multivariate Statistical Analyses. To evidence a possible correlation between the compositions of the essential oils, the oil contents were submitted to multivariate statistical analysis, in particular to hierarchical cluster analysis (HCA) and principal
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component analysis (PCA). HCA is a method in which the samples are considered as lying in an n-dimensional space and distances between samples are calculated joining the objects with an agglomerative procedure [16] [17]. PCA is a well-known method where the principal components (PCs) are calculated in order to keep most of the information present in the original data set in the least possible number of new variables, usually two or three. The PCs can be plotted for visual inspection of the data, to point out patterns hidden in the data set [18]. The HCA (Fig. 1) allowed to separate the essential oils into two main groups: one formed by the oils of the pastinocello fruits and the fruits of the cultivated-carrot varieties Ortolana, Rubrovitamina, and Nantese ibrida Ascania F1 (Group I) and the other formed by the oils of the remaining cultivated-carrot varieties and the pastinocello flowers (Group II). Within each group, two subgroups could further be evidenced: for Group I, a subgroup formed by the sole Nantese ibrida Ascania F1 variety and a subgroup constituted of the three other Group I oil samples and for Group II, a subgroup composed of the Mezza Lunga Nantese 2 and Flakke`e 2 varieties and a subgroup comprising the other Group II oil samples. The PCA (Fig. 2) substantially confirmed this classification. The essential oil of the pastinocello fruits grouped together with those of varieties Ortolana and Rubrovitamina in the lower left quadrant of the PCA plot. This placement was due to the high loadings of oxygenated monoterpenes and monoterpene hydrocarbons on the negative PC1 and PC2 axes. The main compounds responsible for these loadings were geraniol (94) and geranyl acetate (123) and a-pinene (7) and limonene (24), respectively. The placement in the same quadrant of the oils of the Ortolana and Rubrovitamina varieties was more influenced by oxygenated monoterpenes, particularly for the latter, because of the high loadings of trans-sabinol (55), transpinocarveol (57), and trans-verbenol (58) along the negative PC1 axis and of geraniol (94) and geranyl acetate (123) along the negative PC2 axis. For the oils of both
Fig. 1. Dendrogram obtained by hierarchical cluster analysis (Wards method with squared Euclidian distances as a measure of similarity) of the essential oils of the fruits of nine commercial varieties of Daucus carota ssp. sativus and of the flowers and fruits of D. carota ssp. major. For Sample abbreviations and details, cf. Table 1.
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Fig. 2. Principal component analysis of the essential oils of the fruits of nine commercial varieties of Daucus carota ssp. sativus and of the flowers and fruits of D. carota ssp. major. For Sample abbreviations and details, cf. Table 1.
varieties, the contents of monoterpene hydrocarbons played a minor role. The composition of the oil of the Nantese ibrida Ascania F1 variety was less close to those of the other three oil samples of the same HCA group (Group I), because, even if oxygenated monoterpenes (trans-pinocarveol (57), myrtenal (78), and isobornyl acetate (104)) had a high loading on the negative PC1 axis, the contribution of nonterpene derivatives (hexanal (1), myristicin (165), and benzophenone (192)) on the negative PC1 and on the positive PC2 axes and of oxygenated sesquiterpenes (caryophyllene oxide (183), carotol (184), and daucol (197)) on the positive PC1 axis cannot be neglected. Even if the plant material was different, the oil obtained from pastinocello flowers grouped with those obtained from the fruits of some cultivatedcarrot varieties, in particular with that of variety Berlicum 2, because of the high loadings of monoterpene hydrocarbons (a-pinene (7), myrcene (14), and limonene (24)), sesquiterpene hydrocarbons (b-caryophyllene (135) and b-acoradiene (147)), and oxygenated sesquiterpenes (epi-a-cadinol (198)) on the positive PC1 axis. The other subgroup of HCA Group II (Mezza Lunga Nantese 2 and Flakke`e 2) was placed in the upper right quadrant of the PCA plot, because of the high loadings of oxygenated sesquiterpenes (carotol) and sesquiterpene hydrocarbons ((E)-b-farnesene (144) and b-bisabolene (161)) on the two positive axes. The loading of the monoterpene
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
hydrocarbons sabinene (11) and p-cymene (23) on the positive PC1 and negative PC2 axes also substantially contributed to this placement. Conclusions. – The fruit and flower essential oil of D. carota ssp. major (pastinocello) was characterized by the comparison of the chemical composition of the volatile compounds with that of nine cultivars of D. carota ssp. sativus grown for commercial purposes. The cultivation of pastinocello has been abandoned, and the small area currently allocated to this species confirms the danger of its genetic erosion. Considering that the genetic erosion in a century has consumed 75% of the biodiversity, the special quality of this carrot may be directed to the reintroduction of the species in extensive cultivation and in the food chain. Experimental Part Plant Material. Seeds (fruits) of nine commercial varieties of D. carota L. ssp. sativus (cultivated carrot) and seeds and flowers of D. carota ssp. major (Vis.) Arcang. (pastinocello) were selected (Table 1). D. carota ssp. major was not of commercial origin, but was obtained from a regional bank of germplasm (Universita` di Pisa, Dipartimento di Scienze Agrarie, Alimentari e Agro-Alimentari, Sezione della Banca Regionale del Germoplasma). Essential-Oil Isolation. The dried plant material (50 g each) was separately hydrodistilled in a Clevenger-type apparatus for 2 h. The essential oil was collected, dried (anh. Na2SO4 ), and stored at 48 until analysis. GC/MS Analysis. The GC/EI-MS analyses were performed with a Varian CP-3800 apparatus equipped with a DB-5 cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm) and a Varian Saturn 2000 ion-trap mass detector. The oven temp. was programmed rising from 60 to 2408 at 38/min; injector temp., 2208; transfer-line temp., 2408; carrier gas, He (1 ml/min); split ratio, 1 : 80; injection volume, 0.2 ml (10% hexane soln.); split ratio, 1 : 30; scan time, 1 s; mass range, 35 – 400 amu. The identification of the constituents was based on the comparison of i) their retention times (tR ) with those of pure authentic samples and ii) their linear retention indices (LRIs), determined rel. to the tR of a series of n-alkanes, and mass spectra with those listed in the commercial libraries NIST 98 and ADAMS and in a home-made mass-spectral library, built up from pure substances and components of known oils, and MS literature data [19 – 25]. Furthermore, the molecular weights of all the identified compounds were confirmed by GC/CI-MS using MeOH as the chemical ionizing gas. Statistical Analysis. The statistical analyses were carried out with the JMP software package (SAS Institute, Cary, NC, USA). The hierarchical cluster analysis (HCA) was performed using Wards method with squared Euclidian distances as a measure of similarity. The contents of the essential-oil compounds were used as input data and no treatment was performed before PCA processing. REFERENCES [1] S. Pignatti, Flora dItalia, Edagricole, Bologna, 1982. [2] F. Bardeau, Curarsi con i Fiori, Mondadori, Milano, 1977. [3] Regione Toscana, http://www.regione.toscana.it/regione/multimedia/RT/documents/12224203 64820_psr2.pdf (accessed in June 2013). [4] D. Mockute, O. Nivinskiene, J. Essent. Oil Res. 2004, 16, 277. [5] M. Staniszewska, J. Kula, M. Wieczorkiewicz, J. Essent. Oil Res. 2005, 17, 579. [6] M. Staniszewska, J. Kula, J. Essent. Oil Res. 2001, 13, 439. [7] I. Jasicka-Misiak, P. P. Wieczorek, P. Kafarski, Phytochemistry 2005, 66, 1485. [8] I. Jasicka-Misiak, J. Lipok, Biotechnologia 2000, 3, 100. [9] I. Jasicka-Misiak, J. Lipok, E. M. Nowakowska, P. P. Wieczorek, P. Mlynarz, P. Kafarski, Z. Naturforsch., C: Biosci. 2004, 59, 791.
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[10] J. Lipok, A. Czernicka, I. Jasicka-Misiak, P. P. Wieczorek, P. Kafarski, Second European Allelopathy Symposium, Pulawy, Poland, June 3 – 5, 2004. [11] S. B. Glisic, D. R. Misic, M. D. Stamenic, I. T. Zizovic, R. M. Asanin, D. U. Skala, Food Chem. 2007, 105, 346. [12] X. Imamu, A. Yili, H. A. Aisa, V. V. Maksimov, O. N. Veshkurova, S. I. Salikhov, Chem. Nat. Compd. 2007, 43, 495. [13] P. G. Rossi, L. Bao, A. Luciani, J. Panighi, J. M. Desjobert, J. Costa, J. Casanova, J. M. Bolla, L. Berti, J. Agric. Food Chem. 2007, 55, 7332. [14] P. P. Wieczorek, J. Lipok, I. Jasicka-Misiak, Chemik 2006, 59, 55. [15] H. E. A. Saad, S. H. El Sharkawy, A. F. Halim, Pharm. Acta Helv. 1995, 70, 79. [16] J. Davis, Statistics and Data Analysis in Geology, Wiley, New York, 1986. [17] B. S. Everitt, Cluster Analysis, Heineman, London, 1980. [18] E. Marengo, C. Baiocchi, M. C. Gennaro, P. L. Bertolo, S. Lanteri, W. Garrone, Chemom. Intell. Lab. Syst. 1991, 11, 75. [19] E. Stenhagen, S. Abrahamsson, F. W. McLafferty, Registry of Mass-Spectral Data, John Wiley & Sons, New York, 1974. [20] Y. Massada, Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry, John Wiley & Sons, New York, 1976. [21] W. Jennings, T. Shibamoto, Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Chromatography, Academic Press, New York, 1980. [22] A. A. Swigar, R. M. Silverstein, Monoterpenes, Aldrich Chemical Company, Milwaukee, 1981. [23] N. W. Davies, J. Chromatogr., A 1990, 503, 1. [24] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, Allured Publishing Corporation, Carol Stream, IL, 1995. [25] R. P. Adams, T. A. Zanoni, A. Lara, A. F. Barrero, L. G. Cool, J. Essent. Oil Res. 1997, 9, 303. Received December 13, 2013
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Essential-Oil Composition of Daucus carota ssp. major (Pastinocello Carrot) and Nine Different Commercial Varieties of Daucus carota ssp. sativus Fruits by Guido Flamini* a ) b ), Elena Cosimi a ), Pier Luigi Cioni a ), Ilaria Molfetta c ), and Alessandra Braca a ) b ) a ) Dipartimento di Farmacia, Universita` di Pisa, via Bonanno 33, I-56126 Pisa (phone: þ 39-050-2219686; fax: þ 39-050-2219660; e-mail: [email protected].) b ) Centro Interdipartimentale die Ricerca Nutraceutica e Alimentazione per la Salute, Universit di Pisa, Via del Borghetto 80, I-56124 Pisa c ) Dipartimento di Scienze Agrarie, Alimentari ed Agro-Ambientali, via del Borghetto 80, I-56124 Pisa
The chemical composition of the essential oils obtained by hydrodistillation from the pastinocello carrot, Daucus carota ssp. major (Vis.) Arcang. (flowers and achenes), and from nine different commercial varieties of D. carota L. ssp. sativus (achenes) was investigated by GC/MS analyses. Selective breeding over centuries of a naturally occurring subspecies of the wild carrot, D. carota L. ssp. sativus, has produced the common garden vegetable with reduced bitterness, increased sweetness, and minimized woody core. On the other hand, the cultivation of the pastinocello carrot has been abandoned, even if, recently, there has been renewed interest in the development of this species, which risks genetic erosion. The cultivated carrot (D. carota ssp. sativus) and the pastinocello carrot (D. carota ssp. major) were classified as different subspecies of the same species. This close relationship between the two subspecies urged us to compare the chemical composition of their essential oils, to evaluate the differences. The main essential-oil constituents isolated from the pastinocello fruits were geranyl acetate (34.2%), apinene (12.9%), geraniol (6.9%), myrcene (4.7%), epi-a-bisabolol (4.5%), sabinene (3.3%), and limonene (3.0%). The fruit essential oils of the nine commercial varieties of D. carota ssp. sativus were very different from that of pastinocello, as also confirmed by multivariate statistical analyses.
Introduction. – Cultivated carrot, Daucus carota ssp. sativus [1], was obtained in the Middle East by hybridization between strains of D. carota ssp. carota and D. carota ssp. maximus, and its cultivation in the Middle Age was spread to Western Europe. Through the improved agronomic selection, currently, carrot has an edible, plump, and sweet root [2]. D. carota ssp. major, known as pastinocello, seems to be an ancient variety, perhaps dating back to the Roman times, selected from the wild carrot. This variety would have preserved the characteristics of the first breeding, which are very different from those obtained with the latest improvements, as well in color, shape, and organoleptic requirements. Pastinocello carrots were traded together with other important products, such as olive oil, and were used by farmers for special celebrations. Not only the root, but also the aerial parts, leaves, and stems are eaten. The leaves, called erbuccio, tender and tasty, are eaten fresh in salads or boiled; in boiling water they are curative for kidney disease; the leaves are also used for preparing omelets or diuretic infusions. Pastinocello is one of the earliest forms of domestication of wild 2014 Verlag Helvetica Chimica Acta AG, Zrich
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carrots and has a yellow-brown root, a sweet and distinctive nutty taste, and a mellower and softer texture than other carrots [3]. The D. carota L. group is characterized by a wide polymorphism and comprises species such as D. carota sensu stricto and its subspecies D. carota L. ssp. carota, D. carota ssp. maritimus (Lam.) Batt., D. carota ssp. major (Vis.) Arcang., D. carota ssp. maximus (Desf.) Ball, and D. carota ssp. sativus (Hoffm.) Arcang. [1]. D. carota ssp. major is widespread in uncultivated and grass lands [1] and along the river banks, from sea level up to high altitude [3]. Its features are considered intermediate between D. carota ssp. carota and D. carota ssp. maximus. Similarly, it is believed that D. carota ssp. sativus, with its enlarged root that is usually orange colored, resulted from D. carota ssp. carota D. carota ssp. maximus crossing [1]. The pastinocello carrot, identified as D. carota ssp. major (Vis.) Arcang. is generally more or less hispid with usually ascending stems; the leaves are green and shiny; the stem, 30 cm to 2 m long, is striated and branched. The white flowered umbels are 6 – 10 cm wide and appear during spring. According to the different areas in which the plants grow, D. carota L. essential oil belongs to distinct chemotypes such as a carotol ( 77.5%), geranyl acetate ( 81.2%), and sabinene ( 60.4%) chemotype and those containing ca. equal percentages of these compounds. In some habitats, carrot species are able to accumulate in the fruit essential oil considerable amounts of a-pinene ( 55.5%), geraniol ( 50.0%), b-bisabolene ( 35.0%), b-caryophyllene ( 29.0%), g-bisabolene (87.0%, China), and (E)asarone (40.3%, Japan) [4]. Previous studies on the composition of the essential oil of the flowers from wild carrot (D. carota L. ssp. carota) grown in Poland showed that the main constituents were sabinene and geranyl acetate, while carotane-type sesquiterpenes were absent [5] [6]. Some authors reported an allelopathic activity for the aqueous extract of carrot seeds [7] and for their essential oil [8]. The low sensitivity of carrot seeds to fungal infections seems to be due to carotol, a fungitoxic compound active against Alternaria sp. strains [9] and Fusarium oxysporum [10]. The fruit essential oil showed antibacterial activity [4] against Bacillus subtilis [11], Staphylococcus aureus [12], and Campylobacter jejuni [13]. Thanks to the herbicidal, fungicidal, and insecticidal activities of its constituents, this essential oil may be useful for the preparation of biopesticides [14]. The essential oil of the fruits also showed a hypotensive and sedative action and has a moderate anticonvulsant protective effect against strychnine and metrazol poisoning [15]. Results and Discussion. – Note on the Plant-Material Selection. The close relationship between D. carota ssp. sativus and D. carota ssp. major (pastinocello) urged us to compare the chemical composition of their essential oils. Seeds (fruits) of nine commercial varieties of cultivated carrot and pastinocello fruits and dried flowers were selected. In Table 1, the names and the manufacturers of the selected samples are listed. Essential-Oil Yield. The average yield of fruit essential oil of the D. carota ssp. sativus varieties ranged between 0.5 and 0.8% (w/w), whereas the D. carota ssp. major (pastinocello) samples showed oil yields of 0.5 and 0.8% for the fruits and dried flowers, respectively. Although the first three samples (Samples 1 – 3; Table 1) belonged to the same variety (Nantese 2), the seeds of the different brands showed significant differences in their essential-oil yields.
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Table 1. Analyzed Carrot Samples: Fruits (seeds) of Nine Commercial Varieties of Daucus carota ssp. sativus (cultivated carrot; Samples 1 – 9) and Flowers and Fruits of D. carota ssp. major (pastinocello; Samples 10 and 11, resp.) Sample Sample abbreviation Variety 1 2 3
Nant_mez_lun Nant_prec Nant_Chiog
4 5
Nant_ibr_Asc Nant_biol
6 7 8 9 10 11
Berlicum2 Ortolana Rubrovit Flakke`e2 Pastin_flow Pastin_fruit
Mezza lunga Nantese 2 Nantese 2 precoce Nantese migliorata 2 or Nantese Chioggia 2 Nantese ibrida Ascania F1 Nantese 5 (biological)
Origin/Company Quagliano sementi ( Pisa, Italy) Landen ( The Netherlands) SAIS (Cesena, Italy)
Compagnia delle Sementi (Cesena, Italy) Bio-coop, Cooperativa agricola Cesenate (Cesena, Italy) Berlicum 2 Gargini sementi ( Lucca, Italy) Ortolana SAIS selezione conservatrice (Cesena, Italy) Rubrovitamina SAIS selezione conservatrice (Cesena, Italy) Flakke`e 2 Blumen (Piacenza, Italy) Pastinocello dried flowers Regional germplasm bank ( Tuscany, Italy) Pastinocello fruits Regional germplasm bank ( Tuscany, Italy)
Essential-Oil Composition. Altogether, 212 compounds were identified in the investigated oil samples, accounting from 92.7 to 99.8% of the whole essential-oil compositions (Table 2). The main constituents of the essential oil isolated from the fruits of D. carota ssp. major (pastinocello; Sample 11) were geranyl acetate (123 1)), 34.2%), a-pinene (7, 12.9%), geraniol (94, 6.9%), myrcene (14, 4.7%), epi-a-bisabolol (208, 4.5%), sabinene (11, 3.3%), and limonene (24, 3.0%). The essential oils of the nine commercial varieties of D. carota ssp. sativus were very different from that of pastinocello fruits. The content of geranyl acetate was considerably lower in the oils of the D. carota ssp. sativus varieties, with the only exception being variety Rubrovitamina (Sample 8), which contained a similar percentage than the pastinocello-fruit oil (23%). The same was observed for geraniol, which was completely absent in some varieties, while it appeared in small amounts in others. The closest geraniol oil content with respect to pastinocello fruits was found again for Rubrovitamina (4.3%); a comparison of the relative amounts of the major constituents highlighted many similarities between the pastinocello-fruit oil and the oil of the Rubrovitamina variety of cultivated carrot. Most of the oils of the commercial varieties contained carotol (184), with particularly high contents observed for varieties Flakke`e 2 (Sample 9; 49.8%) and Mezza Lunga Nantese 2 (Sample 1; 37.2%). On the contrary, carotol was not detected at all in the pastinocello-fruit oil. The a-pinene (7) oil contents varied between 2.5% in variety Flakke`e 2 and 21.7% in variety Nantese 2 precoce (Sample 2), while the pastinocellofruit oil showed intermediate values (12.9%). In the essential oil of the pastinocello flowers, the major compounds were a-pinene (7, 24.4%), sabinene (11, 13.3%), geranyl acetate (123, 13.0%), epi-a-cadinol (198, 8.5%), myrcene (14, 4.8%), and b-oplopenone (189, 4.3%). A comparison of the flower oil with the fruit essential oil revealed higher and more significant contents of apinene and sabinene and lower amounts of geranyl acetate and geraniol. Considering 1)
Italic numbers in parentheses refer to the entries in Table 2.
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Table 2. Chemical Composition of the Essential Oils Isolated from the Fruits (seeds) of Nine Commercial Varieties of Daucus carota ssp. sativus ( Samples 1 – 9) and the Flowers and Fruits of D. carota ssp. major ( Samples 10 and 11, resp.) Entry Compound name and class
LRI a ) Content [%] b ) 1 c)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Hexanal Hexan-1-ol Heptanal Heptan-2-one a-Thujene Tricyclene a-Pinene Camphene Thuja-2,4(10)-diene Benzaldehyde Sabinene b-Pinene 6-Methylhept-5-en-2-one Myrcene 6-Methylhept-5-en-2-ol Octan-3-one Yomogi alcohol ( E )-Hex-3-enyl acetate b-Phellandrene Octan-1-al a-Phellandrene a-Terpinene p-Cymene Limonene 1,8-Cineole ( Z )-b-Ocimene Benzene acetaldehyde ( E )-b-Ocimene Bergamal g-Terpinene Artemisia ketone cis-Sabinene hydrate cis-Linalool oxide Artemisia alcohol p-Mentha-2,4(8)-diene Terpinolene Dehydrolinalool p-Cymenene trans-Linalool oxide 6,7-Epoxymyrcene Camphen-6-one a-Pinene oxide Linalool Nonanal Camph-6-enol p-Mentha-1,3,8-triene
802 833 871 892 902 908 910 925 931 937 950 954 963 966 969 984 998 1002 1003 1007 1008 1021 1029 1034 1037 1043 1045 1054 1057 1062 1064 1073 1080 1087 1088 1090 1091 1092 1095 1096 1097 1099 1103 1106 1112 1113
2
3
– – – – – tr tr tr tr – – tr 0.2 0.2 0.7 tr – tr 8.5 21.7 4.1 0.8 1.7 0.5 tr tr tr tr tr tr 10.7 17.8 28.8 2.3 3.0 1.1 tr – – 5.8 10.5 1.7 – – – – – tr – – – – – – – – – tr – tr – tr – tr tr 0.2 5.0 0.3 0.7 2.6 5.1 1.9 tr tr tr tr 0.2 – – – – tr tr – – – – 1.0 0.2 0.5 – – – 0.3 0.1 1.6 – – tr – tr – 0.2 – – – 0.2 0.2 0.2 – 0.2 – – – – – – tr tr 0.2 – – – – – 0.7 1.3 1.8 2.8 – tr tr 1.2 – – – – –
4
5
6
7
1.0 – – – – – – – – tr tr tr – – – – tr 0.2 0.4 tr – tr – 0.1 3.4 12.6 13.7 15.0 0.3 1.4 1.5 2.8 tr tr 0.5 1.0 tr tr tr tr 6.3 10.2 20.7 0.6 0.3 3.2 1.5 3.8 tr 0.2 tr tr 0.5 5.4 3.4 1.0 – – – – – – – tr – – – – – – – tr – – – tr – tr – – – – – tr tr – 0.3 tr 1.2 7.2 3.2 0.3 1.0 6.0 3.2 2.3 tr – tr – – – – tr – – tr – – – tr – – tr – – tr 0.4 0.7 tr – – – – 0.7 0.4 0.7 0.2 – tr tr 0.2 – – – – – – – – – 0.5 – – 0.5 0.4 – 1.3 – – – tr – – – tr tr tr – tr tr 0.2 – 0.6 3.0 – – 1.4 1.7 6.7 2.4 1.0 – – – – – 3.0 – – – – – –
8
9
– – tr – tr tr 6.3 1.1 0.2 tr 0.8 0.8 0.1 1.6 tr – – – – – – – 1.7 1.3 tr tr – tr tr tr – tr 0.3 – – – 0.7 – 0.8 tr 0.3 – 3.2 – 3.2 –
– – – – – – – – 0.1 – – – tr 0.3 0.2 – – – 2.5 24.4 12.9 0.3 2.2 1.1 tr 0.1 0.8 tr – – 7.9 13.3 3.3 0.3 1.3 1.8 tr – tr 1.4 4.8 4.7 – – – tr – – – – tr – – – – – – tr – – – 0.2 – tr 0.4 0.3 1.7 0.4 1.5 1.6 2.9 3.0 tr – – tr 0.2 0.4 – – tr tr 0.3 0.5 – – – 0.4 0.6 0.4 – – tr 0.3 0.2 tr 0.2 – tr – – – – – – 0.2 0.3 0.6 – – – – – – – – – tr tr tr – – – 1.4 – – 1.5 0.7 2.2 – – – – – – – – tr
10
11
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 2 (cont.) Entry Compound name and class
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92
b-Thujone exo-Fenchol trans-p-Mentha-2,8-dien-1-ol Dehydrosabinaketone cis-p-Menth-2-en-1-ol a-Campholenal cis-Limonene oxide cis-p-Mentha-2,8-dien-1-ol trans-Sabinol cis-Sabinol trans-Pinocarveol trans-Verbenol trans-p-Menth-2-en-1-ol cis-Verbenol Sabinaketone b-Pinene oxide trans-Pinocamphone ( E )-Non-2-enal Pinocarvone p-Mentha-1,5-dien-8-ol Borneol Umbellulone d-Terpineol cis-Pinocamphone Terpinen-4-ol Cryptone a-Thujenal p-Cymen-8-ol trans-p-Mentha-1,(7)8-dien1-ol a-Terpineol Myrtenol Myrtenal p-Cymen-9-ol trans-Piperitol Verbenone trans-Carveol ( Z )-Ocimenone Nerol cis-p-Mentha-1,(7)8-dien1-ol cis-Carveol Pulegone Neral Cumin aldehyde Carvone Piperitone Chavicol
LRI a ) Content [%] b ) 1 c)
2
3
1114 1117 1123 1127 1128 1131 1138 1141 1142 1143 1144 1145 1146 1150 1159 1162 1163 1165 1167 1168 1170 1171 1172 1175 1181 1186 1187 1188 1189
tr – – – – 0.9 – – – – 1.5 2.8 – tr 0.6 tr – tr 0.6 – 0.2 – 0.3 tr 0.2 – 0.3 – –
– – – – tr 0.3 tr – – – 0.4 0.7 – – – 0.2 – – 0.3 – tr – – – 0.6 – tr – –
1193 1195 1196 1205 1211 1213 1223 1229 1230 1232
0.1 – 1.2 – – 0.4 0.2 – – –
1234 1238 1240 1246 1249 1258 1259
– – – 0.2 0.2 – –
4
5
6
– 0.2 – – – 0.3 tr – 0.3 – 1.7 1.6 0.3 tr tr tr – – – – 3.0 3.7 2.8 10.2 – 0.7 – – 1.7 0.7 0.7 0.5 – – – – 0.8 1.0 1.1 – tr – tr – – 1.2 – – 1.4 0.9 tr – 0.9 0.6 – – – –
0.2 – 0.2 – – 1.6 tr tr 0.6 – 2.6 tr – 5.9 0.7 0.4 – – 1.1 – 0.6 – – – 0.4 – 0.5 – –
0.2 – 0.2 – tr tr tr – – –
tr 0.8 1.4 – tr 0.2 0.2 – – –
tr – 2.6 – – 1.4 1.1 – – –
– – – – tr tr –
– – – 0.8 0.2 – –
– – – 0.4 0.9 – 0.2
7
8
9
10
11
– tr 0.2 – tr – – – – – – 0.2 0.2 0.3 – 1.7 1.8 1.8 tr – tr tr – tr tr 1.6 – – tr – 1.8 6.1 2.7 0.1 17.1 13.4 1.4 – – 7.7 – 1.1 1.1 tr tr – – – – 0.4 0.4 – – – 0.8 2.8 1.1 0.5 0.8 – – – 0.5 tr – – – – 0.5 – tr tr 0.9 0.2 0.2 – – – 0.6 – – – 0.3 0.5 – tr tr
0.1 – 0.1 – – 1.0 tr tr – – 1.8 4.2 – tr 0.6 0.4 – tr 0.5 – 0.2 – – – 0.3 tr 0.4 – –
– – – – tr 0.3 tr tr – – 0.2 0.9 – 0.2 – – – – 0.3 0.1 – – – – 0.6 – tr – –
– tr tr – – 0.6 – tr – – 0.9 2.4 – 0.2 – 0.2 – 0.3 0.5 – 0.2 – – – 0.2 – – 0.1 –
0.2 – 1.9 – tr 0.6 0.5 – 0.3 –
0.1 0.4 1.5 – – 1.5 0.7 – – –
0.2 – 3.7 tr tr 3.9 2.0 – – tr
0.1 – 2.3 – – 3.0 1.4 – 0.3 –
0.1 0.4 1.2 – 0.1 0.5 0.5 tr – –
tr – 0.4 – – 0.1 0.1 – tr –
0.2 1.0 – – – 0.3 0.3 – – –
– – tr 0.2 0.8 – –
tr – – 0.2 0.3 – –
tr – – tr 1.1 – –
– tr – tr 1.0 – –
tr – – 0.3 0.3 tr –
– – – – tr – –
0.1 – tr tr 0.1 – –
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Table 2 (cont.) Entry Compound name and class
93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140
Carvenone Geraniol Linalyl acetate trans-Myrtanol cis-Carvone oxide ( E )-Cinnamaldehyde p-Menth-1-en-7-al Geranial trans-Carvone oxide cis-Verbenyl acetate a-Terpinen-7-al Isobornyl acetate p-Cymen-7-ol trans-Sabinyl acetate Thymol Tridecane trans-Pinocarvyl acetate Carvacrol Undecanal Dihydrocarvyl acetate Myrtenyl acetate Piperitenone a-Terpinyl acetate Eugenol Neryl acetate Cyclosativene a-Copaene Daucene b-Patchoulene trans-Myrtenyl acetate Geranyl acetate Isolongifolene b-Cubebene 7-Episesquithujene b-Elemene Isoitalicene Italicene Methyl eugenol Longifolene a-Gurjunene a-Santalene cis-a-Bergamotene b-Caryophyllene g-Elemene trans-a-Bergamotene ( Z )-b-Farnesene Aromadendrene epi-b-Santalene
LRI a ) Content [%] b )
1260 1261 1262 1263 1269 1276 1277 1278 1281 1283 1287 1288 1290 1291 1295 1300 1302 1303 1308 1311 1330 1347 1355 1358 1370 1373 1378 1382 1381 1383 1388 1387 1392 1393 1394 1402 1408 1409 1411 1412 1418 1419 1421 1433 1439 1443 1447 1450
1 c)
2
3
4
5
6
7
– – – – – – tr – – tr – 0.7 – – – – – – – – tr – tr – tr – – 0.3 – – 0.8 – – tr – – – tr – – – 0.2 0.7 – 0.4 tr 0.2 –
– – – – – – tr – – – – 1.6 – – – – – – – – – – 0.2 – tr – tr 0.3 – – 0.2 – – tr – – tr – tr – – 0.5 1.6 tr 0.8 – 0.3 tr
tr – – – – – 0.2 – – tr 0.3 0.4 – tr – – – tr tr – tr – 0.6 – tr – – 0.2 – – 0.3 0.4 – – tr – tr – – – – 0.2 0.4 – 0.3 – 0.2 –
– – 0.3 tr – tr 0.3 – – – – 1.6 – – – – – – – – – – 0.8 – – – – tr tr – 3.8 – tr – – – – – – – – – 0.3 – 0.3 – – –
– 2.7 – – – – tr 0.2 – – – 0.9 – – – – – – – – – – tr – tr – – tr – – 6.1 – – tr – – tr – – – – 0.3 1.3 – 0.4 – 0.2 –
– – tr – – – 0.1 – – – tr 0.6 tr – – – – tr – – – – 0.2 – tr tr tr 0.3 – – – – – – tr tr – – – – – tr 0.4 – 0.3 – – –
– – 0.2 4.3 – – tr – tr – – – tr – – 0.4 tr – – – – – 1.0 0.9 – – – – tr – – – tr – – – – – tr – tr tr tr – 0.1 tr – – tr tr – – – – – tr – – – – 4.8 23.0 – – – – tr – – – – – – tr – – – – – – tr – tr tr 0.2 0.2 – – tr tr – – tr tr – –
8
9
10
– – tr – – – 0.2 – – – 0.2 0.8 – – 0.1
– – 0.2 6.9 – – – – – – – – – – – 0.2 – – – – – – 0.7 – – – – – – – tr – – – – – – – – – tr tr – – tr 0.2 – tr 0.8 0.1 tr 0.1 0.1 tr – – – – – tr 13.0 34.2 – – – – – – – – – – – – – – – – 0.1 – – – – tr 1.3 0.4 – – tr – – – – – – –
– tr – – tr – 0.4 – tr – tr 0.6 – – 0.2 – – – tr – tr – – – – 0.2 1.0 – 0.6 – – 0.4
11
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 2 (cont.) Entry Compound name and class
LRI a ) Content [%] b ) 1 c)
141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188
b-Himachalene a-Humulene a-Patchoulene ( E )-b-Farnesene g-Decalactone cis-Muurola-4(14),5-diene b-Acoradiene trans-Cadina-1(6),4-diene 9-epi-(E )-Caryophyllene g-Muurolene Germacrene D g-Himachalene b-Selinene Bicyclogermacrene a-Muurolene a-Selinene ( E )-Methyl isoeugenol Pentadecane g-Patchoulene ( Z )-a-Bisabolene b-Bisabolene d-Amorphene 10-Epiitalicene ether ( Z )-g-Bisabolene Myristicin d-Cadinene b-Sesquiphellandrene ( E )-a-Bisabolene a-Cadinene Selina-3,7(11)-diene cis-Sesquisabinene hydrate cis-Cadinene ether Elemol Germacrene B Italicene epoxide trans-Cadinene ether Silphiperfol-5-en-3-ol A Dimethyl ionone trans-Nerolidol Longicamphenilone Caryophyllenyl alcohol Spathulenol Caryophyllene oxide Carotol Guaiol trans-Sesquisabinene hydrate Cedrol Sesquithuriferol
1451 1458 1459 1461 1463 1465 1471 1472 1475 1478 1480 1483 1486 1494 1497 1498 1499 1500 1502 1505 1510 1512 1516 1517 1519 1525 1526 1531 1538 1547 1547 1554 1555 1557 1558 1559 1560 1565 1567 1568 1572 1578 1583 1594 1595 1598 1599 1605
2
3
4
– – – – tr 0.1 – – – – – – 1.5 2.5 0.9 0.7 – – – – – tr – – – – – – 0.1 tr tr tr – – – – – – – – tr 0.1 – – – – – – 0.3 0.4 tr – – 0.1 tr – – – – – tr – – – 0.4 – – – – – – 0.5 – – – tr – tr – – 0.5 1.3 0.8 1.1 – – – – – – – – – – – – 0.4 – – 1.3 – – – – 0.1 0.2 – – – – – – – – – – – – – – 0.2 – – 0.2 – – – 0.1 0.1 – – – tr tr tr – – – – – – – 0.1 – – – – – – – – – – – – – – – – – – – – – 0.1 tr 0.1 – 2.5 0.8 2.7 4.4 37.2 22.0 20.3 24.2 – – – – – – – – – 0.2 – – tr – – –
5
6
– – tr – – – 1.4 0.5 tr – – tr – – – – – – – tr tr – – – 0.2 0.3 – – – – – – – – – – – – – – 0.2 0.4 – – – – – – – – – – tr tr – – – – – – tr – – – tr – tr – – – – – – – – – – – – – – – 0.2 tr 2.8 1.0 4.7 17.5 – – – – – – – –
7
8
9
– tr tr 0.3 – – – tr – – – – tr tr – – – – – – 0.1 – – – tr – – – – – – – – tr 0.1 – 0.4 – – – – tr 2.6 8.6 – tr – –
– 0.2 tr 0.1 – – 0.2 1.3 0.2 – – – – – – 0.2 tr – – – – – – – 0.1 0.9 tr 0.2 – – 0.2 0.2 – – – – – – – – 0.2 1.3 – – – – tr – 0.1 tr – – tr tr – tr – – – – – 0.3 – – 0.2 – tr tr – – – – – – tr 0.1 – 0.2 tr – – 0.1 tr 0.3 3.0 3.4 6.4 49.8 – – – – – – – –
10
11
– 0.5 – 0.6 – – 1.1 – – – 0.5 – 0.3 0.2 – – – – – – 0.8 – 2.0 – – 0.1 – – – 0.4 – – – – – – – – – – – 0.4 0.4 0.2 tr – – –
– 0.2 – 0.4 – tr – – – – 0.2 2.0 0.1 – tr – – – – – 0.2 tr – – – 0.2 – – tr – – – – – – – – – – – – 0.7 0.7 – – – – –
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Table 2 (cont.) Entry Compound name and class
189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212
b-Oplopenone Humulene epoxide II Bisabol-11-ol Benzophenone 1-epi-Cubenol b-Cedren-9-one Caryophylla-4(14),8(15)-dien5-ol Epoxyalloaromadendrene Daucol epi-a-Cadinol Agarospirol b-Eudesmol a-Cadinol Bulnesol 14-Hydroxy-9-epi-( E)caryophyllene b-Bisabolol ( Z )-a-Santalol Elemol acetate a-Bisabolol epi-a-Bisabolol Apiol Junicedranol Juniper camphor Aristolone
LRI a ) Content [%] b ) 1 c)
2
3
4
5
6
7
8
9
10
11
1606 1609 1626 1628 1631 1633 1637
– tr – – – – –
– tr – – 0.1 – –
– 0.3 – 3.4 – – –
– 0.5 – 7.8 – – –
– tr – – – – –
– tr – – – – –
– tr – 0.3 – – tr
– 0.3 – 2.7 – – tr
– 0.4 0.1 – – 0.3 –
4.3 – – – – – –
– 1.2 – – – – –
1639 1641 1643 1648 1649 1656 1667 1670
tr 1.7 – – – – 0.3 –
– 0.1 tr – – tr tr –
– 2.0 – – – 0.2 – –
– 3.3 – – – – – –
– 0.3 – – – tr – –
– 0.7 – – – – – –
0.2 5.0 – – – – tr 0.2
– 1.6 – – tr 0.1 tr 0.4
0.1 2.2 – – – – – –
– – 8.5 – tr 0.1 – –
– – 0.2 tr – – – –
1674 1675 1681 1683 1686 1687 1693 1696 1751
0.6 – – 0.4 0.2 – – tr –
0.2 – – 0.1 – – – tr –
0.2 – – tr – – – 0.6 –
– – – tr – – – tr –
0.3 – – tr – – – 0.5 –
– – – tr – – – tr –
– 0.2 – – – 1.2 – tr –
– 0.1 0.2 0.1 – – – 0.1 0.5
– – – – – – – – –
– – – – 0.3 – – – –
– – – 0.2 4.5 – 0.1 tr –
Total identified [%]
99.6 99.5 99.8 99.7 99.5 96.0 99.7 99.6 99.7 92.7 94.3
Monoterpene hydrocarbons Oxygenated monoterpenes Total monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Total sesquiterpenes Non-terpene derivatives
37.0 14.3 51.3 4.3 43.2 47.5 0.8
60.7 6.7 67.4 8.3 23.7 32.0 tr
40.3 26.3 66.6 3.4 26.2 29.7 3.4
13.0 40.9 53.9 2.5 32.5 35.0 10.8
47.0 40.2 87.2 3.9 8.7 12.7 0.2
49.1 25.5 74.6 2.2 19.2 21.4 tr
26.9 53.2 80.1 0.6 17.4 18.0 1.6
13.7 67.7 81.4 1.2 13.0 14.2 3.1
16.1 18.2 34.3 7.1 57.3 64.4 0.1
51.7 18.8 70.5 6.0 16.2 22.2 tr
31.5 51.1 82.6 3.7 7.6 11.3 0.4
a ) LRI: Linear retention indices determined on a DB-5 column. b ) Content given as percentage of the total oil composition; –, not detected; tr, trace. c ) For the variety and origin of Samples 1 – 11, cf. Table 1.
the chemical compound classes, the main differences between the oils of pastinocello flowers and fruits were observed for monoterpenes: the content of monoterpene hydrocarbons was 51.7% in the flowers and 31.5% in the fruits, while the oxygenated monoterpenes reached 18.8% in the flowers and 51.1% in the fruits, respectively. Multivariate Statistical Analyses. To evidence a possible correlation between the compositions of the essential oils, the oil contents were submitted to multivariate statistical analysis, in particular to hierarchical cluster analysis (HCA) and principal
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
component analysis (PCA). HCA is a method in which the samples are considered as lying in an n-dimensional space and distances between samples are calculated joining the objects with an agglomerative procedure [16] [17]. PCA is a well-known method where the principal components (PCs) are calculated in order to keep most of the information present in the original data set in the least possible number of new variables, usually two or three. The PCs can be plotted for visual inspection of the data, to point out patterns hidden in the data set [18]. The HCA (Fig. 1) allowed to separate the essential oils into two main groups: one formed by the oils of the pastinocello fruits and the fruits of the cultivated-carrot varieties Ortolana, Rubrovitamina, and Nantese ibrida Ascania F1 (Group I) and the other formed by the oils of the remaining cultivated-carrot varieties and the pastinocello flowers (Group II). Within each group, two subgroups could further be evidenced: for Group I, a subgroup formed by the sole Nantese ibrida Ascania F1 variety and a subgroup constituted of the three other Group I oil samples and for Group II, a subgroup composed of the Mezza Lunga Nantese 2 and Flakke`e 2 varieties and a subgroup comprising the other Group II oil samples. The PCA (Fig. 2) substantially confirmed this classification. The essential oil of the pastinocello fruits grouped together with those of varieties Ortolana and Rubrovitamina in the lower left quadrant of the PCA plot. This placement was due to the high loadings of oxygenated monoterpenes and monoterpene hydrocarbons on the negative PC1 and PC2 axes. The main compounds responsible for these loadings were geraniol (94) and geranyl acetate (123) and a-pinene (7) and limonene (24), respectively. The placement in the same quadrant of the oils of the Ortolana and Rubrovitamina varieties was more influenced by oxygenated monoterpenes, particularly for the latter, because of the high loadings of trans-sabinol (55), transpinocarveol (57), and trans-verbenol (58) along the negative PC1 axis and of geraniol (94) and geranyl acetate (123) along the negative PC2 axis. For the oils of both
Fig. 1. Dendrogram obtained by hierarchical cluster analysis (Wards method with squared Euclidian distances as a measure of similarity) of the essential oils of the fruits of nine commercial varieties of Daucus carota ssp. sativus and of the flowers and fruits of D. carota ssp. major. For Sample abbreviations and details, cf. Table 1.
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Fig. 2. Principal component analysis of the essential oils of the fruits of nine commercial varieties of Daucus carota ssp. sativus and of the flowers and fruits of D. carota ssp. major. For Sample abbreviations and details, cf. Table 1.
varieties, the contents of monoterpene hydrocarbons played a minor role. The composition of the oil of the Nantese ibrida Ascania F1 variety was less close to those of the other three oil samples of the same HCA group (Group I), because, even if oxygenated monoterpenes (trans-pinocarveol (57), myrtenal (78), and isobornyl acetate (104)) had a high loading on the negative PC1 axis, the contribution of nonterpene derivatives (hexanal (1), myristicin (165), and benzophenone (192)) on the negative PC1 and on the positive PC2 axes and of oxygenated sesquiterpenes (caryophyllene oxide (183), carotol (184), and daucol (197)) on the positive PC1 axis cannot be neglected. Even if the plant material was different, the oil obtained from pastinocello flowers grouped with those obtained from the fruits of some cultivatedcarrot varieties, in particular with that of variety Berlicum 2, because of the high loadings of monoterpene hydrocarbons (a-pinene (7), myrcene (14), and limonene (24)), sesquiterpene hydrocarbons (b-caryophyllene (135) and b-acoradiene (147)), and oxygenated sesquiterpenes (epi-a-cadinol (198)) on the positive PC1 axis. The other subgroup of HCA Group II (Mezza Lunga Nantese 2 and Flakke`e 2) was placed in the upper right quadrant of the PCA plot, because of the high loadings of oxygenated sesquiterpenes (carotol) and sesquiterpene hydrocarbons ((E)-b-farnesene (144) and b-bisabolene (161)) on the two positive axes. The loading of the monoterpene
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
hydrocarbons sabinene (11) and p-cymene (23) on the positive PC1 and negative PC2 axes also substantially contributed to this placement. Conclusions. – The fruit and flower essential oil of D. carota ssp. major (pastinocello) was characterized by the comparison of the chemical composition of the volatile compounds with that of nine cultivars of D. carota ssp. sativus grown for commercial purposes. The cultivation of pastinocello has been abandoned, and the small area currently allocated to this species confirms the danger of its genetic erosion. Considering that the genetic erosion in a century has consumed 75% of the biodiversity, the special quality of this carrot may be directed to the reintroduction of the species in extensive cultivation and in the food chain. Experimental Part Plant Material. Seeds (fruits) of nine commercial varieties of D. carota L. ssp. sativus (cultivated carrot) and seeds and flowers of D. carota ssp. major (Vis.) Arcang. (pastinocello) were selected (Table 1). D. carota ssp. major was not of commercial origin, but was obtained from a regional bank of germplasm (Universita` di Pisa, Dipartimento di Scienze Agrarie, Alimentari e Agro-Alimentari, Sezione della Banca Regionale del Germoplasma). Essential-Oil Isolation. The dried plant material (50 g each) was separately hydrodistilled in a Clevenger-type apparatus for 2 h. The essential oil was collected, dried (anh. Na2SO4 ), and stored at 48 until analysis. GC/MS Analysis. The GC/EI-MS analyses were performed with a Varian CP-3800 apparatus equipped with a DB-5 cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm) and a Varian Saturn 2000 ion-trap mass detector. The oven temp. was programmed rising from 60 to 2408 at 38/min; injector temp., 2208; transfer-line temp., 2408; carrier gas, He (1 ml/min); split ratio, 1 : 80; injection volume, 0.2 ml (10% hexane soln.); split ratio, 1 : 30; scan time, 1 s; mass range, 35 – 400 amu. The identification of the constituents was based on the comparison of i) their retention times (tR ) with those of pure authentic samples and ii) their linear retention indices (LRIs), determined rel. to the tR of a series of n-alkanes, and mass spectra with those listed in the commercial libraries NIST 98 and ADAMS and in a home-made mass-spectral library, built up from pure substances and components of known oils, and MS literature data [19 – 25]. Furthermore, the molecular weights of all the identified compounds were confirmed by GC/CI-MS using MeOH as the chemical ionizing gas. Statistical Analysis. The statistical analyses were carried out with the JMP software package (SAS Institute, Cary, NC, USA). The hierarchical cluster analysis (HCA) was performed using Wards method with squared Euclidian distances as a measure of similarity. The contents of the essential-oil compounds were used as input data and no treatment was performed before PCA processing. REFERENCES [1] S. Pignatti, Flora dItalia, Edagricole, Bologna, 1982. [2] F. Bardeau, Curarsi con i Fiori, Mondadori, Milano, 1977. [3] Regione Toscana, http://www.regione.toscana.it/regione/multimedia/RT/documents/12224203 64820_psr2.pdf (accessed in June 2013). [4] D. Mockute, O. Nivinskiene, J. Essent. Oil Res. 2004, 16, 277. [5] M. Staniszewska, J. Kula, M. Wieczorkiewicz, J. Essent. Oil Res. 2005, 17, 579. [6] M. Staniszewska, J. Kula, J. Essent. Oil Res. 2001, 13, 439. [7] I. Jasicka-Misiak, P. P. Wieczorek, P. Kafarski, Phytochemistry 2005, 66, 1485. [8] I. Jasicka-Misiak, J. Lipok, Biotechnologia 2000, 3, 100. [9] I. Jasicka-Misiak, J. Lipok, E. M. Nowakowska, P. P. Wieczorek, P. Mlynarz, P. Kafarski, Z. Naturforsch., C: Biosci. 2004, 59, 791.
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[10] J. Lipok, A. Czernicka, I. Jasicka-Misiak, P. P. Wieczorek, P. Kafarski, Second European Allelopathy Symposium, Pulawy, Poland, June 3 – 5, 2004. [11] S. B. Glisic, D. R. Misic, M. D. Stamenic, I. T. Zizovic, R. M. Asanin, D. U. Skala, Food Chem. 2007, 105, 346. [12] X. Imamu, A. Yili, H. A. Aisa, V. V. Maksimov, O. N. Veshkurova, S. I. Salikhov, Chem. Nat. Compd. 2007, 43, 495. [13] P. G. Rossi, L. Bao, A. Luciani, J. Panighi, J. M. Desjobert, J. Costa, J. Casanova, J. M. Bolla, L. Berti, J. Agric. Food Chem. 2007, 55, 7332. [14] P. P. Wieczorek, J. Lipok, I. Jasicka-Misiak, Chemik 2006, 59, 55. [15] H. E. A. Saad, S. H. El Sharkawy, A. F. Halim, Pharm. Acta Helv. 1995, 70, 79. [16] J. Davis, Statistics and Data Analysis in Geology, Wiley, New York, 1986. [17] B. S. Everitt, Cluster Analysis, Heineman, London, 1980. [18] E. Marengo, C. Baiocchi, M. C. Gennaro, P. L. Bertolo, S. Lanteri, W. Garrone, Chemom. Intell. Lab. Syst. 1991, 11, 75. [19] E. Stenhagen, S. Abrahamsson, F. W. McLafferty, Registry of Mass-Spectral Data, John Wiley & Sons, New York, 1974. [20] Y. Massada, Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry, John Wiley & Sons, New York, 1976. [21] W. Jennings, T. Shibamoto, Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Chromatography, Academic Press, New York, 1980. [22] A. A. Swigar, R. M. Silverstein, Monoterpenes, Aldrich Chemical Company, Milwaukee, 1981. [23] N. W. Davies, J. Chromatogr., A 1990, 503, 1. [24] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, Allured Publishing Corporation, Carol Stream, IL, 1995. [25] R. P. Adams, T. A. Zanoni, A. Lara, A. F. Barrero, L. G. Cool, J. Essent. Oil Res. 1997, 9, 303. Received December 13, 2013
