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Chemical Composition of the Tuber Essential Oil from Helianthus tuberosus L. (Asteraceae) by Niko S. Radulovic´* and Miljana R. ord–evic´ Department of Chemistry, Faculty of Science and Mathematics, University of Nisˇ, Visˇegradska 33, 18000 Nisˇ, Serbia (phone: þ 381-628049210; fax: þ 381-18533014; e-mail: [email protected])
Helianthus tuberosus L. (Jerusalem artichoke) is cultivated in Europe and other parts of the world as a food crop and ornamental plant. The volatile oils of the aerial parts of H. tuberosus were investigated more than 30 years ago, but no study could be found to date on the constituents of the tuber essential oil. Herein, the first characterization by GC-FID, GC/MS, and 13C-NMR analyses of a hydrodistilled essential oil of Jerusalem artichoke tubers was reported. Fresh plant material collected in Serbia (Sample A) and a commercial sample (Sample B) yielded only small amounts of oil (0.0014 and 0.0021% (w/w), resp.). In total, 195 constituents were identified, representing 88.2 and 93.6% of the oil compositions for Samples A and B, respectively. The main constituents identified were b-bisabolene (1; 22.9 – 30.5%), undecanal (0 – 12.7%), a-pinene (7.6 – 0.8%), kauran-16-ol (2; 6.9 – 9.8%), 2-pentylfuran (0.0 – 5.7%), and (E)-tetradec-2-enal (0.0 – 4.9%). Several rare compounds characteristic for Helianthus ssp. were also detected: helianthol A (6; 2.1 – 1.9%), dihydroeuparin (10; 0.0 – 2.3%), euparin (9; 0.0 – 0.4%), desmethoxyencecalin (7; traces – 0.2%), desmethylencecalin (8; 0.0 – 0.4%), and an isomer of desmethylencecalin (0.0%-traces). The essential oils isolated from the tuber and the aerial parts share the common major component 1.
Introduction. – The genus Helianthus L. comprises about 50 annual and perennial sunflower species (tribe Heliantheae) [1]. Jerusalem artichoke (Helianthus tuberosus L., syn. H. serotinus Tausch) and sunflower (H. annuus L.) are the only cultivated crops of this genus. H. tuberosus is a perennial plant species native to North America, widespread in all parts of the world including Serbia, where it can be frequently found as a weed species of pastures and fallow land or an ornamental garden plant [2]. The tubers of H. tuberosus have a high content of the polysaccharide inulin, which makes them interesting for the production of sweeteners, low-caloric food additives, and bioethanol/liquors [3]. Its tubers are often used in the human diet, referred to as the food of poor, and the folk medicine for the treatment of diabetes [4] and rheumatism, for aiding digestion and preventing constipation, or as a diuretic [5]. Above-ground parts and tubers are used as cattle feed as well [2]. A number of biological activities as antimicrobial [6] and cytotoxic [5] properties have already been reported for the extracts of this species. Previous phytochemical investigations of this species resulted in the identification of nonvolatile coumarin [7] and polyacetylene [8] derivatives and a number of sesquiand diterpenoids [6] [9], along with the constituents of essential oils of aerial and underground parts of the plant [10]. Some 30 years ago, MacLeod et al. [10a] first published an article on the volatile flavor components of the extract of the hypogeal 2014 Verlag Helvetica Chimica Acta AG, Zrich
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parts. They reported the successful identification of only 16 constituents and a number of others that remained unidentified. A year later, two Japanese scientists isolated a new bisabolane sesquiterpenol, helianthol A, from the essential oil of the aerial parts of H. tuberosus [9c]. The only other piece of information on the composition of H. tuberosus oil was that its major constituent was b-bisabolene, as previously found for the tubers by MacLeod et al. [10a]. Only very recently, Bach et al. [10b] studied the volatiles of the underground parts (tubers) of different varieties of this species by dynamic headspace sampling and assessed their impact on the sensory quality of Jerusalem artichoke. In total, 27 compounds were successfully identified by these authors. Several additional components, among which five sesquiterpenoids, were detected, but their exact chemical structure was not identified. These 27 compounds, together with the compounds from the other two previous studies [9c] [10a], make 40 volatile compounds in total reported for this plant species. All of the mentioned reports [10] indicate that many more volatile compounds are still left unidentified. The information mentioned above and the fact that the hydrodistilled tuber essential oil has never been studied before prompted us to perform the first detailed characterization (using GC-FID, GC/MS, and 13C-NMR analyses) of the chemical composition of the hydrodistilled essential oil of two samples of fresh H. tuberosus tubers from Serbia. Results and Discussion. – The hydrodistilled essential oils from fresh tubers of Jerusalem artichoke, collected in southern Serbia (Sample A) or purchased as a commercial sample intended for human consumption (Sample B), were obtained in very low yields (0.0014 and 0.0021% (w/w), resp.). Their characterization by GC-FID, GC/MS, and 13C-NMR analyses allowed the identification of 195 compounds in total, 106 compounds, representing 88.2% of the oil composition, for Sample A and 141 compounds, corresponding to 93.6% of the oil composition, for Sample B. More than 170 of these compounds were reported here for the first time for H. tuberosus. The results of the analyses are compiled in the Table. A typical total-ion chromatogram (TIC) of the analyzed oils (Sample A) is given in Fig. 1. The oils were rich in terpenoids, which constituted more than 50% of the oil composition. More specifically, sesquiterpenoids represented the major compound class, with total contents of 27.8 and 37% for Samples A and B, respectively, followed by oxygenated diterpenes (10.8 and 25.4%, resp.) and monoterpene hydrocarbons (8.1 and 0.8%, resp.). Thus, the two samples differed mainly in the content of monoterpene hydrocarbons, with a-pinene constituting the majority of this fraction in both samples. The main constituents of Sample A were b-bisabolene (1; 22.9%; Fig. 2), undecanal (12.7%), a-pinene (7.6%), kauran-16-ol (2; 6.9%; Fig. 2), 2-pentylfuran (5.7%), and (E)-2-tetradecenal (4.9%). Compound 1 was also the major component of Sample B (30.5%), which also contained a considerable amount of 2 (9.8%). However, the contents of other dominant oil components of Sample B differed from those of Sample A: (Z,Z)-9,12octadecadienoic acid (15.4 vs. 0.0%), hexadecanoic acid (10.5 vs. 0.5%), communic acid (3; 9.2 vs. 0.0%), phyllocladanol (4; 3.3 vs. 1.8%), and kaur-16-en-18-oic acid (5; 2.7 vs. 0.0%). A closer inspection of the Table revealed that the differences go further and that only 50 identified compounds were in common to both oils. Several compounds with a very limited natural occurrence were identified during the course of the analyses (Fig. 1), and these components are characteristic for Helianthus ssp., i.e., the
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Table. Chemical Composition of the Tuber Essential Oil Isolated from a Wild-Growing Sample ( A) and a Commercial Sample ( B) of Helianthus tuberosus L. Compound a )
RIDB-5MS b ) RIDB-1 c ) Content [%] d ) A
3-Methylbutan-1-ol Pyridine g ) Pentan-1-ol ( Z )-Pent-2-en-1-ol 3-Methylbut-2-en-1-ol 3-Methylbut-2-enal Hexanal g ) h ) Octane Furfural g ) ( E )-Hex-2-enal ( Z )-Hex-3-en-1-ol ( Z )-Hex-2-en-1-ol Hexan-1-ol g ) h ) ( Z )-Hept-4-enal Heptan-2-one Heptanal Methional Nonane Tricyclene a-Thujene a-Pinene h ) Camphene h ) ( Z )-Hept-3-en-1-ol Benzaldehyde Thuja-2,4(10)-diene Heptan-1-ol Phenol Oct-1-en-3-ol Sabinene h ) b-Pinene h ) ( E )-Oct-4-enal 2-Pentylfuran g ) h ) 2,3-Didehydro-1,8-cineole 2-Acetylthiazole Phenylacetaldehyde p-Cymene b-Phellandrene Limonene h ) ( Z )-b-Ocimene ( E )-Oct-2-enal Acetophenone ( Z )-Oct-5-en-1-ol g-Terpinene Octan-1-ol Non-8-en-2-one 2-Methyldecane a-Pinene oxide ( Z )-Non-6-enal
747 762 763 765 778 801
718 719
0.1 tr tr tr tr tr
800 828 846 850 859 863 889 902 903
848 861 876
tr tr tr
900 922 931 932 947 956 958 960
971 976 986
1041
1026 1030 1052 1065
1092
921 928 941
944 951 960 961 963 966 977 987 981 1007 1010 1018 1019 1025 1030 1042 1047 1053 1056 1058 1066 1070
B tr tr
tr 0.1 tr tr tr 1.6
tr tr tr tr
tr tr 7.6 tr tr tr tr
0.1 tr 5.7
0.4 tr tr tr
tr
Class e ) Identification f )
tr 0.8 tr
tr tr tr tr tr 0.1 tr tr tr tr tr tr tr tr tr tr tr 0.1 tr tr tr 0.2
O O O O O O O O O O O O O O O O O O O MT MT MT O O MT O O O MT MT O O MT* O O O MT MT MT O O O MT O O O MT* O
MS, RI, CoI, NMR MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI, CoI, NMR MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI, NMR MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI
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Table (cont.) Compound a )
RIDB-5MS b ) RIDB-1 c ) Content [%] d ) A
p-Cymenene Terpinolene Nonanal h ) Linalool h ) 2-Phenylethanol Camph-6-enone a-Campholenal Camphor trans-Pinocarveol cis-Verbenol trans-Verbenol Camphene hydrate ( E )-Non-2-enal trans-Pinocamphone Pinocarvone p-Mentha-1,5-dien-8-ol cis-Pinocamphone p-Methylacetophenone p-Mentha-1,8-dien-4-ol Cymen-9-ol Terpinene-4-ol cis-Pinocarveol p-Cymen-8-ol a-Terpineol Verbenone Myrtenol Myrtenal a-Campholenol Decanal Nopol Verbenone Dodec-1-ene trans-Carveol trans-Car-3-en-2-ol Cuminal Carvone Carvotanacetone Geraniol Decan-1-ol cis-Carvone oxide ( E )-Anethole Bornyl acetate (2E,4Z )-Deca-2,4-dienal Indole 2-Octylfuran Undecan-2-one Thymol Carvacrol
1100 1101 1112 1125 1139 1140 1143 1155 1161 1162 1165
1177 1183 1190 1196 1197 1201
1071 1077 1081 1083 1084 1091 1102 1117 1120 1123 1126 1129 1133 1134 1135 1144 1147 1151 1156 1158 1159 1163 1170 1174 1175 1176
tr 0.2 0.4 0.5 tr tr 1.7 0.2 tr 0.1 0.2
tr tr tr tr tr tr
1183 1184 1210 1217 1234 1243
1263
tr tr tr tr tr tr 0.4 tr 0.1 0.1 0.4 tr tr tr 0.1 0.2 tr tr tr tr 0.1 tr tr 0.1 0.1 tr tr tr
0.2 0.1 tr
tr tr tr 0.1 tr tr tr
3.1 1241 1259 1266
1290 1293 1294 1295
tr tr tr tr tr tr tr
1270 1299
B
0.7 1187 1196 1198 1209 1212 1217 1234
tr tr
Class e ) Identification f )
MT MT O MT* O MT* MT* MT* MT* MT* MT* MT* O MT* MT* MT* MT* O MT* MT* MT* MT* MT* MT* MT* MT* MT* MT* O MT* MT* O MT* MT* MT* MT* MT* MT* O MT* O MT* O O O O MT* MT*
MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI, NMR MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI
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Table (cont.) Compound a )
RIDB-5MS b ) RIDB-1 c ) Content [%] d ) A
trans-Pinocarvyl acetate p-Vinylguaiacol Tridecane h ) ( E,E )-Deca-2,4-dienal Myrtenyl acetate Undecanal 7aH-Silphiperfol-5-ene (11) Eugenol ( E )-Undec-2-enal 7bH-Silphiperfol-5-ene (12) Silphiperfola-5,7(14)-diene (13) a-Copaene h ) Modheph-2-ene (14) a-Isocomene Tetradecene h ) Dodecanal cis-a-Bergamotene Geranylacetone ( E )-b-Farnesene g ) Sesquisabinene B Dodecan-1-ol g-Curcumene ( Z,E )-a-Farnesene a-Muurolene b-Bisabolene (1) g ) h ) i ) Tridecanal Myristicin d-Cadinene a-Calacorene g-Undecalactone Elemicin cis-Sesquisabinene hydrate ( E )-Tridec-2-enal ( E )-Nerolidol Dodecanoic acid Tridecan-1-ol trans-Sesquisabinene hydrate Tetradecanal 6-Methoxyelemicin Helianthol isomer 1 j ) k ) Helianthol A (6) i ) k ) Desmethoxyencecalin (7) k ) cis-Methyl dihydrojasmonate ( E )-Tetradec-2-enal Intermedeol Neointermedeol g-Dodecalactone b-Atlantone
1277 1282 1300 1313
1287 1302
1323 1356 1357
1376
1340 1349 1371 1375 1381 1387
tr 1.7
tr
0.1 tr 0.1 tr 0.1 tr tr tr
tr 1398 1427
1454
tr tr 0.6
1443 1458 1468 1482 1501
1512 1525
1549 1555
0.5 tr 0.2 tr tr 22.9 tr 1.0 0.7
30.5
0.1 0.2
0.1 0.2 1537
1564
1593 1595 1606 1619 1650
tr tr
12.7 1321 1327
1408
1499 1507 1509 1517 1523
B tr tr
tr 0.4
tr tr
1546 1547 1559 1571 1590 1587 1601 1610 1614
1658 1661
0.2 0.7 1.1 2.1 0.2
tr tr tr tr 0.1 1.0 1.9 tr tr
4.9 tr 1636
1667
0.1 0.1
1645
Class e ) Identification f )
tr
MT* O O O MT* O ST O O ST ST ST ST ST O O ST O ST ST O ST ST ST ST O O ST ST O O ST* O ST* O O ST* O O ST* ST* O O O ST* ST* O ST*
MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, MS, RI, MS, RI, CoI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI, CoI, NMR MS, RI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS MS, NMR MS MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI
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Table (cont.) Compound a )
RIDB-5MS b ) RIDB-1 c ) Content [%] d ) A
Tetradecan-1-ol Apiole Helianthol isomer 2 j ) k ) Pentadecanal Cryptomerione Acetyl-hydroxy-2,2dimethylchromene isomer j ) k ) Tetradecanoic acid Eupatoriochromene (8) k ) b-Bisabolenal ( Z )-Lanceol Pentadecan-1-ol g-Bicyclohomofarnesal Hexadecanal Cyclopentadecanolide Dihydroeuparin (10) k ) Pentadecanoic acid Euparin (9) k ) Nonadecane g ) Heptadecanal Methyl hexadecanoate Isopimara-8,15-diene Pimara-8,15-diene Pimara-8(14),15-diene Hexadecanoic acid ( Z )-Biformene 13-Epimanool oxide Kaur-16-ene Manool 13-Epimanool Heptadecanoic acid Methyl linoleate Henicosane Nonadecanal ( Z,Z )-Octadeca-9,12-dienoic acid Octadecanoic acid Kaur-16-en-18-al Trachyloban-19-al Kauran-16-ol (2) 3-Methylheneicosane Phyllocladanol (4) Communic acid (3) Pimara-7,15-dien-3-one Tricosane Docosanal Kaur-16-en-18-oic acid (5) Heptacosane Squalene
1659 1677 1686 1703 1726 1736
1666 1692 1697
1744 1762 1766 1774
1755 1758 1768
1808
tr 2.3
1845 1889 1900 1911
0.4 0.2 tr tr
1907 1922 1928 1934 1961 1970 1998 2023 2035 2036 2045 2070 2100
2133
0.5 0.5 0.2
2199 2185 2211 2234
2260
15.4 tr 0.6 1.3 6.9 1.8
9.8 tr 3.3 9.2
tr 2300
2396
2833
tr 0.1 0.5 tr 10.5 0.8 0.4 tr tr tr tr tr 0.1
0.2 2127 2150
2235
0.4 tr tr
tr
1857
2196 2209 2222
1.7 0.2 0.1
0.1 0.4 tr 0.2
1805
1955 1987 2016
B tr
0.1 0.7 0.2 tr tr
tr tr
2390 2700 2810
tr
Class e ) Identification f )
2.7 tr 0.1
O O ST* O ST* O
MS, RI, CoI MS, RI, CoI MS MS, RI, CoI MS, RI MS
O O ST* ST* O ST* O O O O O O O O DT DT DT O DT DT* DT DT* DT* O O O O O O DT* DT* DT* O DT* DT* DT* O O DT* O TT
MS, RI, CoI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI
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Table (cont.) Compound a )
Nonacosane Hentriacontane Tritriacontane Pentatriacontane Total identified [%] Monoterpene hydrocarbons ( MT) Oxygenated monoterpenes ( MT*) Diterpene hydrocarbons ( DT ) Oxygenated diterpenes ( DT*) Sesquiterpene hydrocarbons ( ST ) Oxygenated sesquiterpenes ( ST*) Triterpene hydrocarbons ( TT ) Others (O ) Number of components
RIDB-5MS b ) RIDB-1 c ) Content [%] d )
2900
2900 3100 3300 3500
A
B
tr
tr tr 0.1 tr
88.2
93.6
8.1 3.7 0.5 10.8 24.2 3.6 37.3 106
Class e ) Identification f )
O O O O
MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI
0.8 1.7 1.4 25.4 31.8 5.2 0.1 27.2 141
a
) Compounds are listed in the order of their elution on the DB-5MS and/or DB-1 columns. b ) c ) RIDB-5MS and RIDB-1 : Retention indices determined relative to a homologous series of n-alkanes (C7 – C36 ) on the DB-5MS or DB-1 columns, respectively. d ) Values are means of three individual analyses; tr, trace amounts ( < 0.05%). e ) Identification method: RI, retention indices matching with literature data [11]; MS, mass spectra matching with those listed in the Wiley 6, NIST11, MassFinder 2.3, and a homemade mass spectral library; CoI, coinjection with pure reference compound; NMR, characterization by 13 C-NMR spectroscopy. g ) Constituent previously reported for this species [10a]. h ) Constituent previously reported for this species [10b]. i ) Constituent previously reported for this species [9c]. j ) Correct isomer not determined. k ) EI-MS of helianthol A (6): 220 (11, M þ ), 205 (2), 202 (11), 187 (11), 159 (36), 121 (33), 119 (76), 93 (86), 91 (66), 85 (28), 79 (100), 59 (35); desmethoxyencecalin (7): 202 (10, M þ ), 188 (12), 187 (100), 159 (1), 145 (3), 144 (16), 115 (7), 43 (9); eupatoriochromene (8): 218 (13, M þ ), 204 (13), 203 (100), 185 (17), 175 (1), 160 (3), 145 (2), 128 (4), 94 (9), 91 (8), 77 (8), 69 (5), 43 (11); dihydroeuparin (10): 218 (100, M þ ), 204 (13), 203 (95), 185 (20), 175 (59), 160 (30), 157 (9), 129 (14), 105 (16), 94 (14), 77 (19), 69 (21), 43 (58); euparin (9): 216 (75, M þ ), 201 (100), 173 (20), 124 (8), 115 (16), 98 (47), 81 (37), 67 (58), 55 (54), 43 (50), 41 (64); helianthol isomer 1: 220 (5, M þ ), 202 (17), 187 (25), 174 (11), 159 (47), 134 (37), 119 (75), 105 (76), 93 (100), 91 (93), 85 (24), 79 (89), 69 (80), 67 (60), 55 (51); helianthol isomer 2: 220 (2, M þ ), 202 (7), 187 (11), 159 (33), 134 (66), 119 (64), 105 (54), 93 (77), 91 (66), 85 (29), 79 (100), 67 (67), 59 (45); acetyl-hydroxy-2,2-dimethylchromene isomer: 218 (22, M þ ), 204 (18), 203 (100), 91 (18), 77 (30), 69 (19), 55 (33), 44 (60).
sesquiterpene alcohol helianthol A (6), the chromanes desmethoxyencecalin (7) and desmethylencecalin (8; syn. eupatoriochromene), and the benzofurans euparin (9) and dihydroeuparin (10) [12]. Helianthol A is closely biosynthetically linked to the major oil constituent 1. Indeed, allylic oxidation of one of the side-chain C¼C bonds of 1, followed by an allylic rearrangement of the intermediary secondary alcohol into the final tertiary one results in compound 6. Two additional compounds that seem to be isomers of compound 6 were detected, since both showed almost identical mass spectra to 6. One of these components might be helianthol B, originally proposed to be a constituent of H. tuberosus [13]. Interestingly, the majority of the identified sesquiterpenoids had a bisabolane-type skeleton with summed-up contents of 27.0
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Fig. 1. Total ion chromatogram (TIC) of the tuber essential oil of Helianthus tuberosus Sample A. 1, bBisabolene; 2, kauran-16-ol; 6, helianthol A; 7, desmethoxyencecalin; 8, desmethylencecalin; 9, euparin; 10, dihydroeuparin.
and 36.7% vs. 27.8 and 37.0% of total sesquiterpenes for Samples A and B, respectively (cf. Table). Moreover, based on the characteristic MS fragmentation pattern similar to compound 8, it was hypothesized that Sample A contains an acetyl-hydroxy-2,2dimethylchromene isomer. Another fact worth noting is the existence of a number of triquinane sesquiterpenes, viz., 7aH-silphiperfol-5-ene (11), 7bH-silphiperfol-5-ene (12), silphiperfola5,7(14)-diene (13), and modheph-2-ene (14; Fig. 2), reported for the first time in this genus. This might have been expected, since triquinanes are characteristic for Asteraceae. Indeed, they have previously been reported for more than 60 genera of this family, with a few rare exceptions outside the Asteraceae [14]. Conclusions. – The first detailed characterization by GC-FID, GC/MS, and C-NMR analyses of hydrodistilled essential oils of Jerusalem artichoke tubers (collected in southern Serbia or purchased as a commercial sample) was presented here. The analyses allowed the identification of 195 compounds in total with bbisabolene as the major component of both oil samples. More than 170 of these compounds were reported for the first time for H. tuberosus. Additionally, chemotaxonomically important compounds characteristic for Helianthus ssp. were identified, i.e., helianthol A, desmethoxyencecalin, desmethylencecalin, euparin, and dihydroeuparin. 13
This work was supported by the Ministry of Education, Science, and Technological Development of Serbia (Project No. 172061).
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Fig. 2. Chemical structures of the noteworthy compounds identified in the tuber essential oils of Helianthus tuberosus. 1, b-Bisabolene; 2, kauran-16-ol; 3, communic acid; 4, phyllocladanol; 5, kaur-16en-18-oic acid; 6, helianthol A; 7, desmethoxyencecalin; 8, desmethylencecalin; 9, euparin; 10, dihydroeuparin; 11, 7aH-silphiperfol-5-ene; 12, 7bH-silphiperfol-5-ene; 13, silphiperfola-5,7(14)-diene; 14, modheph-2-ene. Experimental Part Plant Material. Underground parts (tubers) of Sample A were collected in October 2012 from natural Helianthus tuberosus L. populations growing near the city of Leskovac, southern Serbia. The plant material constituting Sample A represented the second year tubers growing in a loose loam in full sun on a deserted field next to the banks of the river Juzˇna Morava. Sample B was purchased from a local producer (vicinity of the city of Nisˇ ), who planted the crop early in spring of 2012 in alkaline, well-worked soil with an average moisture content of 30%. After a growing season of 125 d at 18 – 278, the tubers were collected from the fields. Voucher specimens have been deposited with the Herbarium Collection of the Faculty of Science and Mathematics, University of Nisˇ, under the accession number MDJ05102012. Chemicals. For the determination of retention indices (RIs), two n-alkane mixtures (Sigma-Aldrich, USA) ranging from heptane to eicosane (C7 – C20) and from heneicosane to hexatriacontane (C21 – C36 ) were used. All solvents (HPLC grade) were purchased from Sigma-Aldrich. Authentic chemical samples were obtained from Sigma-Aldrich and Fluka in the highest available purity. Extraction of Essential Oils. Fresh, finely chopped tubers (1000 g) of H. tuberosus were subjected to hydrodistillation with ca. 2.5 l of dist. H2O for 3 h, using an original Clevenger-type apparatus [15]. The
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obtained oils were separated by extraction with Et2O and dried (anh. MgSO4 ). The solvent was evaporated under a gentle stream of N2 at r.t., to exclude any loss of the essential oils, and the samples immediately analyzed. For the determination of the oil yields, after the bulk of Et2O was removed under a stream of N2 , the residue was exposed to vacuum at r.t. for a short period of time, to eliminate the solvent completely. The pure oils were then measured on an analytical balance, and multiple gravimetric measurements were taken during 24 h to ensure that all of the solvent had evaporated. GC-FID and GC/MS Analyses. The chemical composition of the oils was determined by GC-FID and GC/MS analyses (three repetitions for each sample). The GC/MS analyses were performed with a Hewlett-Packard 6890N gas chromatograph equipped with two fused-silica cap. columns (30 m 0.25 mm, film thickness 0.25 mm, Agilent Technologies, USA), a DB-5MS (5% phenylmethylsiloxane) and a DB1 (100% dimethylpolysiloxane), and coupled with a 5975B mass selective detector (MSD) from the same company. The injector and interface were operated at 250 and 3008, resp. The oven temp. was raised from 70 to 2908 at 58/min and then isothermally held at 2258 for 10 min; carrier gas, He (1.0 ml/ min). The samples, 1 ml of the oil solns. in Et2O (1 : 100), were injected in a pulsed split mode (flow of 1.5 ml/min for the first 0.5 min and then 1.0 ml/min throughout the remainder of the analysis; split ratio, 40 : 1), which enabled sufficient separation (narrower peaks due to a pulsed injection mode despite starting the run at 708) and positive identification of numerous components with RIs lower than 900. The MSD was operated at the ionization energy of 70 eV, over the mass range 35 – 500 amu, with a scan time of 0.34 s. The identification of partially overlapping peaks was aided by the software NIST AMDIS (automated mass-spectral deconvolution and identification system) version 2.4, National Institute of Standards and Technology (NIST, USA). The GC-FID analyses were carried out under the same experimental conditions and using the same columns as described for the GC/MS analyses. The percentage composition (relative content in %) was computed from the GC-FID peak areas without the use of correction factors. Identification of Constituents. The identification of the essential-oil constituents was based on the comparison of their linear retention indices (RIs), determined rel. to the retention times (tR ) of a series of n-alkanes (C7 – C36 ) on the DB-5MS and DB1 columns, to those reported in the literature [11] and their mass spectra to those of authentic standards as well as to those listed in the Wiley 6, NIST11, and MassFinder 2.3 mass spectral libraries. Moreover, a homemade mass spectral library built with the spectra of pure substances and components of known essential oils was used, and, finally, whenever possible, the identification was achieved by coinjection with an authentic sample (cf. Table). 13 C-NMR Analysis. The 13C-NMR spectra were recorded with a Bruker AVANCE III 400 spectrometer operating at 100 MHz and equipped with a 5-mm DUALPLUS probe, in (D6 )benzene (ca. 20 mg of oil in 0.7 ml of (D6 )benzene), with all shifts expressed in ppm and referred to internal Me4Si, with the following parameters: pulse width, 0.6 ms (flip angle, 458); acquisition time, 1.705 s; relaxation delay D1, 0.294 s (total recycling time, 1.999 s); for 64 K data table with a spectral width of 18863.2 Hz; globally optimized alternating-phase rectangular pulses; digital resolution, 0.469 Hz/pt; scans accumulated, 5000. An exponential multiplication of the free induction decay with the line broadening of 1.0 Hz was applied before Fourier transformation.
REFERENCES [1] E. E. Schilling, Flora of North America, Oxford University Press, New York, Oxford, 2006. [2] V. Sikora, J. Berenji, D. Latkovic, Helia 2010, 33, 37. [3] W. J. Mullin, H. W. Modler, E. R. Farnworth, A. Payne, Food Chem. 1994, 51, 263; R. Jaiswal, S. Deshpande, N. Kuhnert, Phytochem. Anal. 2011, 22, 432. [4] J. L. Kim, C. R. Bae, Y. S. Cha, J. Korean Soc. Food Sci. Nutr. 2010, 39, 31; N. Kaur, A. K. Gupta, J. Biosci. 2002, 27, 703. [5] L. Pan, M. R. Sinden, A. H. Kennedy, H. Chai, L. E. Watson, T. L. Graham, D. Kinghorn, Phytochem. Lett. 2009, 2, 15. [6] S. J. Kays, S. F. Nottingham, Biology and Chemistry of Jerusalem Artichoke Helianthus tuberosus L., CRC Press, Taylor & Francis Group, Boca Raton, 2008.
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[7] F. Tesio, L. A. Weston, A. Ferrero, Sci. Hortic. 2011, 129, 361; F. Cabello-Hurtado, F. Durst, J. V. Jorin, D. Werck-Reichhart, Phytochemistry 1998, 49, 1029. [8] H. Matsuura, T. Yoshihara, A. Ichihara, Y. Kikuta, Y. Koda, Biosci., Biotechnol., Biochem. 1993, 57, 1253; H. Matsuura, T. Yoshihara, A. Ichihara, Biosci., Biotechnol., Biochem. 1993, 57, 1492. [9] a) O. Spring, Phytochemistry 1991, 30, 519; b) H. Baba, Y. Yaoita, M. Kikuchi, J. Tohoku Pharm. Univ. 2005, 52, 21; c) M. Miyazawa, H. Kameoka, Phytochemistry 1983, 22, 1040; d) H. Morimoto, H. Oshio, J. Nat. Prod. 1981, 44, 748; e) H. Morimoto, Y. Sanno, H. Oshio, Tetrahedron 1966, 22, 3173; f) X. Yuan, M. Cheng, M. Gao, R. Zhuo, L. Zhang, H. Xiao, Phytochem. Lett. 2013, 6, 21; g) F. Bohlmann, J. Jakupovic, R. M. King, H. Robinson, Phytochemistry 1980, 19, 863. [10] a) A. J. MacLeod, N. M. Pieris, N. Gonzales de Troconis, Phytochemistry 1982, 21, 1647; b) V. Bach, U. Kidmose, G. K. Bjørn, M. Edelenbos, Food Chem. 2012, 133, 82. [11] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, Allured Publishing Corporation, Carol Stream, IL, 2007. [12] F. Bohlmann, M. Grenz, Chem. Ber. 1970, 103, 90. [13] H. Kameoka, M. Miyazawa, M. Mega, Perfumer & Flavorist 1977, 2, 80. [14] a) N. S. Radulovic´, P. D. Blagojevic´, B. K. Zlatkovic´, R. M. Palic´, S. Afr. J. Chem. 2009, 62, 30; b) N. S. Radulovic´, M. S. Denic´, Chem. Biodiversity 2013, 10, 658. [15] N. S. Radulovic´, M. Z. Mladenovic´, N. ord–evic´, Chem. Biodiversity 2012, 9, 1320. Received August 7, 2013
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Chemical Composition of the Tuber Essential Oil from Helianthus tuberosus L. (Asteraceae) by Niko S. Radulovic´* and Miljana R. ord–evic´ Department of Chemistry, Faculty of Science and Mathematics, University of Nisˇ, Visˇegradska 33, 18000 Nisˇ, Serbia (phone: þ 381-628049210; fax: þ 381-18533014; e-mail: [email protected])
Helianthus tuberosus L. (Jerusalem artichoke) is cultivated in Europe and other parts of the world as a food crop and ornamental plant. The volatile oils of the aerial parts of H. tuberosus were investigated more than 30 years ago, but no study could be found to date on the constituents of the tuber essential oil. Herein, the first characterization by GC-FID, GC/MS, and 13C-NMR analyses of a hydrodistilled essential oil of Jerusalem artichoke tubers was reported. Fresh plant material collected in Serbia (Sample A) and a commercial sample (Sample B) yielded only small amounts of oil (0.0014 and 0.0021% (w/w), resp.). In total, 195 constituents were identified, representing 88.2 and 93.6% of the oil compositions for Samples A and B, respectively. The main constituents identified were b-bisabolene (1; 22.9 – 30.5%), undecanal (0 – 12.7%), a-pinene (7.6 – 0.8%), kauran-16-ol (2; 6.9 – 9.8%), 2-pentylfuran (0.0 – 5.7%), and (E)-tetradec-2-enal (0.0 – 4.9%). Several rare compounds characteristic for Helianthus ssp. were also detected: helianthol A (6; 2.1 – 1.9%), dihydroeuparin (10; 0.0 – 2.3%), euparin (9; 0.0 – 0.4%), desmethoxyencecalin (7; traces – 0.2%), desmethylencecalin (8; 0.0 – 0.4%), and an isomer of desmethylencecalin (0.0%-traces). The essential oils isolated from the tuber and the aerial parts share the common major component 1.
Introduction. – The genus Helianthus L. comprises about 50 annual and perennial sunflower species (tribe Heliantheae) [1]. Jerusalem artichoke (Helianthus tuberosus L., syn. H. serotinus Tausch) and sunflower (H. annuus L.) are the only cultivated crops of this genus. H. tuberosus is a perennial plant species native to North America, widespread in all parts of the world including Serbia, where it can be frequently found as a weed species of pastures and fallow land or an ornamental garden plant [2]. The tubers of H. tuberosus have a high content of the polysaccharide inulin, which makes them interesting for the production of sweeteners, low-caloric food additives, and bioethanol/liquors [3]. Its tubers are often used in the human diet, referred to as the food of poor, and the folk medicine for the treatment of diabetes [4] and rheumatism, for aiding digestion and preventing constipation, or as a diuretic [5]. Above-ground parts and tubers are used as cattle feed as well [2]. A number of biological activities as antimicrobial [6] and cytotoxic [5] properties have already been reported for the extracts of this species. Previous phytochemical investigations of this species resulted in the identification of nonvolatile coumarin [7] and polyacetylene [8] derivatives and a number of sesquiand diterpenoids [6] [9], along with the constituents of essential oils of aerial and underground parts of the plant [10]. Some 30 years ago, MacLeod et al. [10a] first published an article on the volatile flavor components of the extract of the hypogeal 2014 Verlag Helvetica Chimica Acta AG, Zrich
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parts. They reported the successful identification of only 16 constituents and a number of others that remained unidentified. A year later, two Japanese scientists isolated a new bisabolane sesquiterpenol, helianthol A, from the essential oil of the aerial parts of H. tuberosus [9c]. The only other piece of information on the composition of H. tuberosus oil was that its major constituent was b-bisabolene, as previously found for the tubers by MacLeod et al. [10a]. Only very recently, Bach et al. [10b] studied the volatiles of the underground parts (tubers) of different varieties of this species by dynamic headspace sampling and assessed their impact on the sensory quality of Jerusalem artichoke. In total, 27 compounds were successfully identified by these authors. Several additional components, among which five sesquiterpenoids, were detected, but their exact chemical structure was not identified. These 27 compounds, together with the compounds from the other two previous studies [9c] [10a], make 40 volatile compounds in total reported for this plant species. All of the mentioned reports [10] indicate that many more volatile compounds are still left unidentified. The information mentioned above and the fact that the hydrodistilled tuber essential oil has never been studied before prompted us to perform the first detailed characterization (using GC-FID, GC/MS, and 13C-NMR analyses) of the chemical composition of the hydrodistilled essential oil of two samples of fresh H. tuberosus tubers from Serbia. Results and Discussion. – The hydrodistilled essential oils from fresh tubers of Jerusalem artichoke, collected in southern Serbia (Sample A) or purchased as a commercial sample intended for human consumption (Sample B), were obtained in very low yields (0.0014 and 0.0021% (w/w), resp.). Their characterization by GC-FID, GC/MS, and 13C-NMR analyses allowed the identification of 195 compounds in total, 106 compounds, representing 88.2% of the oil composition, for Sample A and 141 compounds, corresponding to 93.6% of the oil composition, for Sample B. More than 170 of these compounds were reported here for the first time for H. tuberosus. The results of the analyses are compiled in the Table. A typical total-ion chromatogram (TIC) of the analyzed oils (Sample A) is given in Fig. 1. The oils were rich in terpenoids, which constituted more than 50% of the oil composition. More specifically, sesquiterpenoids represented the major compound class, with total contents of 27.8 and 37% for Samples A and B, respectively, followed by oxygenated diterpenes (10.8 and 25.4%, resp.) and monoterpene hydrocarbons (8.1 and 0.8%, resp.). Thus, the two samples differed mainly in the content of monoterpene hydrocarbons, with a-pinene constituting the majority of this fraction in both samples. The main constituents of Sample A were b-bisabolene (1; 22.9%; Fig. 2), undecanal (12.7%), a-pinene (7.6%), kauran-16-ol (2; 6.9%; Fig. 2), 2-pentylfuran (5.7%), and (E)-2-tetradecenal (4.9%). Compound 1 was also the major component of Sample B (30.5%), which also contained a considerable amount of 2 (9.8%). However, the contents of other dominant oil components of Sample B differed from those of Sample A: (Z,Z)-9,12octadecadienoic acid (15.4 vs. 0.0%), hexadecanoic acid (10.5 vs. 0.5%), communic acid (3; 9.2 vs. 0.0%), phyllocladanol (4; 3.3 vs. 1.8%), and kaur-16-en-18-oic acid (5; 2.7 vs. 0.0%). A closer inspection of the Table revealed that the differences go further and that only 50 identified compounds were in common to both oils. Several compounds with a very limited natural occurrence were identified during the course of the analyses (Fig. 1), and these components are characteristic for Helianthus ssp., i.e., the
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Table. Chemical Composition of the Tuber Essential Oil Isolated from a Wild-Growing Sample ( A) and a Commercial Sample ( B) of Helianthus tuberosus L. Compound a )
RIDB-5MS b ) RIDB-1 c ) Content [%] d ) A
3-Methylbutan-1-ol Pyridine g ) Pentan-1-ol ( Z )-Pent-2-en-1-ol 3-Methylbut-2-en-1-ol 3-Methylbut-2-enal Hexanal g ) h ) Octane Furfural g ) ( E )-Hex-2-enal ( Z )-Hex-3-en-1-ol ( Z )-Hex-2-en-1-ol Hexan-1-ol g ) h ) ( Z )-Hept-4-enal Heptan-2-one Heptanal Methional Nonane Tricyclene a-Thujene a-Pinene h ) Camphene h ) ( Z )-Hept-3-en-1-ol Benzaldehyde Thuja-2,4(10)-diene Heptan-1-ol Phenol Oct-1-en-3-ol Sabinene h ) b-Pinene h ) ( E )-Oct-4-enal 2-Pentylfuran g ) h ) 2,3-Didehydro-1,8-cineole 2-Acetylthiazole Phenylacetaldehyde p-Cymene b-Phellandrene Limonene h ) ( Z )-b-Ocimene ( E )-Oct-2-enal Acetophenone ( Z )-Oct-5-en-1-ol g-Terpinene Octan-1-ol Non-8-en-2-one 2-Methyldecane a-Pinene oxide ( Z )-Non-6-enal
747 762 763 765 778 801
718 719
0.1 tr tr tr tr tr
800 828 846 850 859 863 889 902 903
848 861 876
tr tr tr
900 922 931 932 947 956 958 960
971 976 986
1041
1026 1030 1052 1065
1092
921 928 941
944 951 960 961 963 966 977 987 981 1007 1010 1018 1019 1025 1030 1042 1047 1053 1056 1058 1066 1070
B tr tr
tr 0.1 tr tr tr 1.6
tr tr tr tr
tr tr 7.6 tr tr tr tr
0.1 tr 5.7
0.4 tr tr tr
tr
Class e ) Identification f )
tr 0.8 tr
tr tr tr tr tr 0.1 tr tr tr tr tr tr tr tr tr tr tr 0.1 tr tr tr 0.2
O O O O O O O O O O O O O O O O O O O MT MT MT O O MT O O O MT MT O O MT* O O O MT MT MT O O O MT O O O MT* O
MS, RI, CoI, NMR MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI, CoI, NMR MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI, NMR MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table (cont.) Compound a )
RIDB-5MS b ) RIDB-1 c ) Content [%] d ) A
p-Cymenene Terpinolene Nonanal h ) Linalool h ) 2-Phenylethanol Camph-6-enone a-Campholenal Camphor trans-Pinocarveol cis-Verbenol trans-Verbenol Camphene hydrate ( E )-Non-2-enal trans-Pinocamphone Pinocarvone p-Mentha-1,5-dien-8-ol cis-Pinocamphone p-Methylacetophenone p-Mentha-1,8-dien-4-ol Cymen-9-ol Terpinene-4-ol cis-Pinocarveol p-Cymen-8-ol a-Terpineol Verbenone Myrtenol Myrtenal a-Campholenol Decanal Nopol Verbenone Dodec-1-ene trans-Carveol trans-Car-3-en-2-ol Cuminal Carvone Carvotanacetone Geraniol Decan-1-ol cis-Carvone oxide ( E )-Anethole Bornyl acetate (2E,4Z )-Deca-2,4-dienal Indole 2-Octylfuran Undecan-2-one Thymol Carvacrol
1100 1101 1112 1125 1139 1140 1143 1155 1161 1162 1165
1177 1183 1190 1196 1197 1201
1071 1077 1081 1083 1084 1091 1102 1117 1120 1123 1126 1129 1133 1134 1135 1144 1147 1151 1156 1158 1159 1163 1170 1174 1175 1176
tr 0.2 0.4 0.5 tr tr 1.7 0.2 tr 0.1 0.2
tr tr tr tr tr tr
1183 1184 1210 1217 1234 1243
1263
tr tr tr tr tr tr 0.4 tr 0.1 0.1 0.4 tr tr tr 0.1 0.2 tr tr tr tr 0.1 tr tr 0.1 0.1 tr tr tr
0.2 0.1 tr
tr tr tr 0.1 tr tr tr
3.1 1241 1259 1266
1290 1293 1294 1295
tr tr tr tr tr tr tr
1270 1299
B
0.7 1187 1196 1198 1209 1212 1217 1234
tr tr
Class e ) Identification f )
MT MT O MT* O MT* MT* MT* MT* MT* MT* MT* O MT* MT* MT* MT* O MT* MT* MT* MT* MT* MT* MT* MT* MT* MT* O MT* MT* O MT* MT* MT* MT* MT* MT* O MT* O MT* O O O O MT* MT*
MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI, NMR MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI
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431
Table (cont.) Compound a )
RIDB-5MS b ) RIDB-1 c ) Content [%] d ) A
trans-Pinocarvyl acetate p-Vinylguaiacol Tridecane h ) ( E,E )-Deca-2,4-dienal Myrtenyl acetate Undecanal 7aH-Silphiperfol-5-ene (11) Eugenol ( E )-Undec-2-enal 7bH-Silphiperfol-5-ene (12) Silphiperfola-5,7(14)-diene (13) a-Copaene h ) Modheph-2-ene (14) a-Isocomene Tetradecene h ) Dodecanal cis-a-Bergamotene Geranylacetone ( E )-b-Farnesene g ) Sesquisabinene B Dodecan-1-ol g-Curcumene ( Z,E )-a-Farnesene a-Muurolene b-Bisabolene (1) g ) h ) i ) Tridecanal Myristicin d-Cadinene a-Calacorene g-Undecalactone Elemicin cis-Sesquisabinene hydrate ( E )-Tridec-2-enal ( E )-Nerolidol Dodecanoic acid Tridecan-1-ol trans-Sesquisabinene hydrate Tetradecanal 6-Methoxyelemicin Helianthol isomer 1 j ) k ) Helianthol A (6) i ) k ) Desmethoxyencecalin (7) k ) cis-Methyl dihydrojasmonate ( E )-Tetradec-2-enal Intermedeol Neointermedeol g-Dodecalactone b-Atlantone
1277 1282 1300 1313
1287 1302
1323 1356 1357
1376
1340 1349 1371 1375 1381 1387
tr 1.7
tr
0.1 tr 0.1 tr 0.1 tr tr tr
tr 1398 1427
1454
tr tr 0.6
1443 1458 1468 1482 1501
1512 1525
1549 1555
0.5 tr 0.2 tr tr 22.9 tr 1.0 0.7
30.5
0.1 0.2
0.1 0.2 1537
1564
1593 1595 1606 1619 1650
tr tr
12.7 1321 1327
1408
1499 1507 1509 1517 1523
B tr tr
tr 0.4
tr tr
1546 1547 1559 1571 1590 1587 1601 1610 1614
1658 1661
0.2 0.7 1.1 2.1 0.2
tr tr tr tr 0.1 1.0 1.9 tr tr
4.9 tr 1636
1667
0.1 0.1
1645
Class e ) Identification f )
tr
MT* O O O MT* O ST O O ST ST ST ST ST O O ST O ST ST O ST ST ST ST O O ST ST O O ST* O ST* O O ST* O O ST* ST* O O O ST* ST* O ST*
MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, MS, RI, MS, RI, CoI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI, CoI, NMR MS, RI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS MS, NMR MS MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI
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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table (cont.) Compound a )
RIDB-5MS b ) RIDB-1 c ) Content [%] d ) A
Tetradecan-1-ol Apiole Helianthol isomer 2 j ) k ) Pentadecanal Cryptomerione Acetyl-hydroxy-2,2dimethylchromene isomer j ) k ) Tetradecanoic acid Eupatoriochromene (8) k ) b-Bisabolenal ( Z )-Lanceol Pentadecan-1-ol g-Bicyclohomofarnesal Hexadecanal Cyclopentadecanolide Dihydroeuparin (10) k ) Pentadecanoic acid Euparin (9) k ) Nonadecane g ) Heptadecanal Methyl hexadecanoate Isopimara-8,15-diene Pimara-8,15-diene Pimara-8(14),15-diene Hexadecanoic acid ( Z )-Biformene 13-Epimanool oxide Kaur-16-ene Manool 13-Epimanool Heptadecanoic acid Methyl linoleate Henicosane Nonadecanal ( Z,Z )-Octadeca-9,12-dienoic acid Octadecanoic acid Kaur-16-en-18-al Trachyloban-19-al Kauran-16-ol (2) 3-Methylheneicosane Phyllocladanol (4) Communic acid (3) Pimara-7,15-dien-3-one Tricosane Docosanal Kaur-16-en-18-oic acid (5) Heptacosane Squalene
1659 1677 1686 1703 1726 1736
1666 1692 1697
1744 1762 1766 1774
1755 1758 1768
1808
tr 2.3
1845 1889 1900 1911
0.4 0.2 tr tr
1907 1922 1928 1934 1961 1970 1998 2023 2035 2036 2045 2070 2100
2133
0.5 0.5 0.2
2199 2185 2211 2234
2260
15.4 tr 0.6 1.3 6.9 1.8
9.8 tr 3.3 9.2
tr 2300
2396
2833
tr 0.1 0.5 tr 10.5 0.8 0.4 tr tr tr tr tr 0.1
0.2 2127 2150
2235
0.4 tr tr
tr
1857
2196 2209 2222
1.7 0.2 0.1
0.1 0.4 tr 0.2
1805
1955 1987 2016
B tr
0.1 0.7 0.2 tr tr
tr tr
2390 2700 2810
tr
Class e ) Identification f )
2.7 tr 0.1
O O ST* O ST* O
MS, RI, CoI MS, RI, CoI MS MS, RI, CoI MS, RI MS
O O ST* ST* O ST* O O O O O O O O DT DT DT O DT DT* DT DT* DT* O O O O O O DT* DT* DT* O DT* DT* DT* O O DT* O TT
MS, RI, CoI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI, CoI MS, RI, CoI MS, RI MS, RI, CoI MS, RI, CoI
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433
Table (cont.) Compound a )
Nonacosane Hentriacontane Tritriacontane Pentatriacontane Total identified [%] Monoterpene hydrocarbons ( MT) Oxygenated monoterpenes ( MT*) Diterpene hydrocarbons ( DT ) Oxygenated diterpenes ( DT*) Sesquiterpene hydrocarbons ( ST ) Oxygenated sesquiterpenes ( ST*) Triterpene hydrocarbons ( TT ) Others (O ) Number of components
RIDB-5MS b ) RIDB-1 c ) Content [%] d )
2900
2900 3100 3300 3500
A
B
tr
tr tr 0.1 tr
88.2
93.6
8.1 3.7 0.5 10.8 24.2 3.6 37.3 106
Class e ) Identification f )
O O O O
MS, RI, CoI MS, RI, CoI MS, RI, CoI MS, RI, CoI
0.8 1.7 1.4 25.4 31.8 5.2 0.1 27.2 141
a
) Compounds are listed in the order of their elution on the DB-5MS and/or DB-1 columns. b ) c ) RIDB-5MS and RIDB-1 : Retention indices determined relative to a homologous series of n-alkanes (C7 – C36 ) on the DB-5MS or DB-1 columns, respectively. d ) Values are means of three individual analyses; tr, trace amounts ( < 0.05%). e ) Identification method: RI, retention indices matching with literature data [11]; MS, mass spectra matching with those listed in the Wiley 6, NIST11, MassFinder 2.3, and a homemade mass spectral library; CoI, coinjection with pure reference compound; NMR, characterization by 13 C-NMR spectroscopy. g ) Constituent previously reported for this species [10a]. h ) Constituent previously reported for this species [10b]. i ) Constituent previously reported for this species [9c]. j ) Correct isomer not determined. k ) EI-MS of helianthol A (6): 220 (11, M þ ), 205 (2), 202 (11), 187 (11), 159 (36), 121 (33), 119 (76), 93 (86), 91 (66), 85 (28), 79 (100), 59 (35); desmethoxyencecalin (7): 202 (10, M þ ), 188 (12), 187 (100), 159 (1), 145 (3), 144 (16), 115 (7), 43 (9); eupatoriochromene (8): 218 (13, M þ ), 204 (13), 203 (100), 185 (17), 175 (1), 160 (3), 145 (2), 128 (4), 94 (9), 91 (8), 77 (8), 69 (5), 43 (11); dihydroeuparin (10): 218 (100, M þ ), 204 (13), 203 (95), 185 (20), 175 (59), 160 (30), 157 (9), 129 (14), 105 (16), 94 (14), 77 (19), 69 (21), 43 (58); euparin (9): 216 (75, M þ ), 201 (100), 173 (20), 124 (8), 115 (16), 98 (47), 81 (37), 67 (58), 55 (54), 43 (50), 41 (64); helianthol isomer 1: 220 (5, M þ ), 202 (17), 187 (25), 174 (11), 159 (47), 134 (37), 119 (75), 105 (76), 93 (100), 91 (93), 85 (24), 79 (89), 69 (80), 67 (60), 55 (51); helianthol isomer 2: 220 (2, M þ ), 202 (7), 187 (11), 159 (33), 134 (66), 119 (64), 105 (54), 93 (77), 91 (66), 85 (29), 79 (100), 67 (67), 59 (45); acetyl-hydroxy-2,2-dimethylchromene isomer: 218 (22, M þ ), 204 (18), 203 (100), 91 (18), 77 (30), 69 (19), 55 (33), 44 (60).
sesquiterpene alcohol helianthol A (6), the chromanes desmethoxyencecalin (7) and desmethylencecalin (8; syn. eupatoriochromene), and the benzofurans euparin (9) and dihydroeuparin (10) [12]. Helianthol A is closely biosynthetically linked to the major oil constituent 1. Indeed, allylic oxidation of one of the side-chain C¼C bonds of 1, followed by an allylic rearrangement of the intermediary secondary alcohol into the final tertiary one results in compound 6. Two additional compounds that seem to be isomers of compound 6 were detected, since both showed almost identical mass spectra to 6. One of these components might be helianthol B, originally proposed to be a constituent of H. tuberosus [13]. Interestingly, the majority of the identified sesquiterpenoids had a bisabolane-type skeleton with summed-up contents of 27.0
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Fig. 1. Total ion chromatogram (TIC) of the tuber essential oil of Helianthus tuberosus Sample A. 1, bBisabolene; 2, kauran-16-ol; 6, helianthol A; 7, desmethoxyencecalin; 8, desmethylencecalin; 9, euparin; 10, dihydroeuparin.
and 36.7% vs. 27.8 and 37.0% of total sesquiterpenes for Samples A and B, respectively (cf. Table). Moreover, based on the characteristic MS fragmentation pattern similar to compound 8, it was hypothesized that Sample A contains an acetyl-hydroxy-2,2dimethylchromene isomer. Another fact worth noting is the existence of a number of triquinane sesquiterpenes, viz., 7aH-silphiperfol-5-ene (11), 7bH-silphiperfol-5-ene (12), silphiperfola5,7(14)-diene (13), and modheph-2-ene (14; Fig. 2), reported for the first time in this genus. This might have been expected, since triquinanes are characteristic for Asteraceae. Indeed, they have previously been reported for more than 60 genera of this family, with a few rare exceptions outside the Asteraceae [14]. Conclusions. – The first detailed characterization by GC-FID, GC/MS, and C-NMR analyses of hydrodistilled essential oils of Jerusalem artichoke tubers (collected in southern Serbia or purchased as a commercial sample) was presented here. The analyses allowed the identification of 195 compounds in total with bbisabolene as the major component of both oil samples. More than 170 of these compounds were reported for the first time for H. tuberosus. Additionally, chemotaxonomically important compounds characteristic for Helianthus ssp. were identified, i.e., helianthol A, desmethoxyencecalin, desmethylencecalin, euparin, and dihydroeuparin. 13
This work was supported by the Ministry of Education, Science, and Technological Development of Serbia (Project No. 172061).
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Fig. 2. Chemical structures of the noteworthy compounds identified in the tuber essential oils of Helianthus tuberosus. 1, b-Bisabolene; 2, kauran-16-ol; 3, communic acid; 4, phyllocladanol; 5, kaur-16en-18-oic acid; 6, helianthol A; 7, desmethoxyencecalin; 8, desmethylencecalin; 9, euparin; 10, dihydroeuparin; 11, 7aH-silphiperfol-5-ene; 12, 7bH-silphiperfol-5-ene; 13, silphiperfola-5,7(14)-diene; 14, modheph-2-ene. Experimental Part Plant Material. Underground parts (tubers) of Sample A were collected in October 2012 from natural Helianthus tuberosus L. populations growing near the city of Leskovac, southern Serbia. The plant material constituting Sample A represented the second year tubers growing in a loose loam in full sun on a deserted field next to the banks of the river Juzˇna Morava. Sample B was purchased from a local producer (vicinity of the city of Nisˇ ), who planted the crop early in spring of 2012 in alkaline, well-worked soil with an average moisture content of 30%. After a growing season of 125 d at 18 – 278, the tubers were collected from the fields. Voucher specimens have been deposited with the Herbarium Collection of the Faculty of Science and Mathematics, University of Nisˇ, under the accession number MDJ05102012. Chemicals. For the determination of retention indices (RIs), two n-alkane mixtures (Sigma-Aldrich, USA) ranging from heptane to eicosane (C7 – C20) and from heneicosane to hexatriacontane (C21 – C36 ) were used. All solvents (HPLC grade) were purchased from Sigma-Aldrich. Authentic chemical samples were obtained from Sigma-Aldrich and Fluka in the highest available purity. Extraction of Essential Oils. Fresh, finely chopped tubers (1000 g) of H. tuberosus were subjected to hydrodistillation with ca. 2.5 l of dist. H2O for 3 h, using an original Clevenger-type apparatus [15]. The
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obtained oils were separated by extraction with Et2O and dried (anh. MgSO4 ). The solvent was evaporated under a gentle stream of N2 at r.t., to exclude any loss of the essential oils, and the samples immediately analyzed. For the determination of the oil yields, after the bulk of Et2O was removed under a stream of N2 , the residue was exposed to vacuum at r.t. for a short period of time, to eliminate the solvent completely. The pure oils were then measured on an analytical balance, and multiple gravimetric measurements were taken during 24 h to ensure that all of the solvent had evaporated. GC-FID and GC/MS Analyses. The chemical composition of the oils was determined by GC-FID and GC/MS analyses (three repetitions for each sample). The GC/MS analyses were performed with a Hewlett-Packard 6890N gas chromatograph equipped with two fused-silica cap. columns (30 m 0.25 mm, film thickness 0.25 mm, Agilent Technologies, USA), a DB-5MS (5% phenylmethylsiloxane) and a DB1 (100% dimethylpolysiloxane), and coupled with a 5975B mass selective detector (MSD) from the same company. The injector and interface were operated at 250 and 3008, resp. The oven temp. was raised from 70 to 2908 at 58/min and then isothermally held at 2258 for 10 min; carrier gas, He (1.0 ml/ min). The samples, 1 ml of the oil solns. in Et2O (1 : 100), were injected in a pulsed split mode (flow of 1.5 ml/min for the first 0.5 min and then 1.0 ml/min throughout the remainder of the analysis; split ratio, 40 : 1), which enabled sufficient separation (narrower peaks due to a pulsed injection mode despite starting the run at 708) and positive identification of numerous components with RIs lower than 900. The MSD was operated at the ionization energy of 70 eV, over the mass range 35 – 500 amu, with a scan time of 0.34 s. The identification of partially overlapping peaks was aided by the software NIST AMDIS (automated mass-spectral deconvolution and identification system) version 2.4, National Institute of Standards and Technology (NIST, USA). The GC-FID analyses were carried out under the same experimental conditions and using the same columns as described for the GC/MS analyses. The percentage composition (relative content in %) was computed from the GC-FID peak areas without the use of correction factors. Identification of Constituents. The identification of the essential-oil constituents was based on the comparison of their linear retention indices (RIs), determined rel. to the retention times (tR ) of a series of n-alkanes (C7 – C36 ) on the DB-5MS and DB1 columns, to those reported in the literature [11] and their mass spectra to those of authentic standards as well as to those listed in the Wiley 6, NIST11, and MassFinder 2.3 mass spectral libraries. Moreover, a homemade mass spectral library built with the spectra of pure substances and components of known essential oils was used, and, finally, whenever possible, the identification was achieved by coinjection with an authentic sample (cf. Table). 13 C-NMR Analysis. The 13C-NMR spectra were recorded with a Bruker AVANCE III 400 spectrometer operating at 100 MHz and equipped with a 5-mm DUALPLUS probe, in (D6 )benzene (ca. 20 mg of oil in 0.7 ml of (D6 )benzene), with all shifts expressed in ppm and referred to internal Me4Si, with the following parameters: pulse width, 0.6 ms (flip angle, 458); acquisition time, 1.705 s; relaxation delay D1, 0.294 s (total recycling time, 1.999 s); for 64 K data table with a spectral width of 18863.2 Hz; globally optimized alternating-phase rectangular pulses; digital resolution, 0.469 Hz/pt; scans accumulated, 5000. An exponential multiplication of the free induction decay with the line broadening of 1.0 Hz was applied before Fourier transformation.
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