J. Agric. Food Chem. 2000, 48, 3463−3469
3463
Volatile Chemicals Identified in Extracts from Leaves of Japanese Mugwort (Artemisia princeps Pamp.) Katumi Umano,† Yukio Hagi,† Kazuaki Nakahara,† Akihiro Shoji,† and Takayuki Shibamoto* Department of Environmental Toxicology, University of California, Davis, California 95616
Extracts from leaves of Japanese mugwort (Artemisia princeps Pamp.) were obtained using two methods: steam distillation under reduced pressure followed by dichloromethane extraction (DRP) and simultaneous purging and extraction (SPSE). A total of 192 volatile chemicals were identified in the extracts obtained by both methods using gas chromatography (GC) and gas chromatographymass spectrometry (GC-MS). They included 47 monoterpenoids (oxygenated monoterpenes), 26 aromatic compounds, 19 aliphatic esters, 18 aliphatic alcohols, 17 monoterpenes (hydrocarbon monoterpenes), 17 sesquiterpenes (hydrocarbon sesquiterpenes), 13 sesquiterpenoids (oxygenated sesquiterpenes), 12 aliphatic aldehydes, 8 aliphatic hydrocarbons, 7 aliphatic ketones, and 9 miscellaneous compounds. The major volatile constituents of the extract by DRP were borneol (10.27 ppm), R-thujone (3.49 ppm), artemisia alcohol (2.17 ppm), verbenone (1.85 ppm), yomogi alcohol (1.50 ppm), and germacren-4-ol (1.43 ppm). The major volatile constituents of the extract by SPSE were 1,8-cineole (8.12 ppm), artemisia acetate (4.22 ppm), R-thujone (3.20 ppm), β-caryophyllene (2.39 ppm), bornyl acetate (2.05 ppm), borneol (1.80 ppm), and trans-β-farnesene (1.78 ppm). Keywords: Volatile chemicals; gas chromatography; Japanese mugwort; yomogi INTRODUCTION
Japanese mugwort (yomogi; Artemisia princeps Pamp.) is a plant native to China, Japan, and Korea. It generally grows in mountainous areas. The plants including stem (1 cm), branches, and leavessgrows to ∼60-120 cm. Young leaves of yomogi have a characteristic greenish aroma. In the spring in Japan, yomogi leaves are mixed with rice cakes to give them a fresh aroma and a greenish color. The green juice of yomogi leaves is also used in traditional Japanese folk medicine to treat skin injuries. In Korea, the juice of yomogi leaves is used to treat gastrointestinal disorders (Kim et al., 1994). The extracts from yomogi leaves have received attention from researchers in Japan and Korea since the mid-1960s. Before appropriate gas chromatography-mass spectrometry (GC-MS) was developed, 22 constituents of extracts from yomogi leaves were identified using fractional distillation and packed column GC (Tsubaki et al., 1966). Later, nearly 100 components of yomogi extracts were identified using an advanced GC-MS with a capillary column (Fujita and Nogami, 1994, 1995). In the present study, volatile chemicals in extracts obtained from young yomogi leaves were identified by GC and GC-MS. EXPERIMENTAL PROCEDURES Materials. Japanese mugwort leaves or yomogi leaves (A. princeps Pamp.) were bought from a local market. Authentic compounds were purchased from Aldrich Chemical Co. (Milwaukee, WI), Tokyo Kasei Organic Chemicals (Tokyo, Japan), * Author to whom correspondence should be addressed [telephone (530) 752-4543; fax (530) 752-3394; e-mail
[email protected]]. † Present address: Takata Koryo Co., Ltd., 22-2, 7-Chome, Tsukaguchi-Honmachi, Amagasaki, Hyogo-Pref., 661 Japan.
Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and Fluka Chemical Co. (Ronkonkoma, NY) or obtained from Takata Koryo Co., Ltd. (Osaka, Japan) as a gift. Yomogi alcohol, artemisia alcohol, and caryophylla-2(12),6(13)-dien5-one were synthesized according to the methods of Thomas and Pawlak (1971), Wilson and Guazzaroni (1989), and Kaiser and Lamparsky (1983), respectively. Isolation of Volatile Chemicals by Steam Distillation under Reduced Pressure (DRP). Yomogi leaves (250 g) were washed with cold water three times and once with hot water and then cut into small pieces (1 cm) using a knife. The pieces of yomogi leaves were homogenized in a blender with 1800 mL of distilled water and 200 mL of saturated sodium chloride solution. The homogenized sample was placed in a 3 L round-bottom flask. The solution was steam distilled at 32 °C for 30 min under reduced pressure (32 mmHg). The distillate (900 mL) was extracted with 130 mL of dichloromethane using a liquid-liquid continuous extractor for 6 h. After the extract was dried over anhydrous sodium sulfate, the solvent was removed by distillation with a Vigreux column. The distillation was stopped when the volume of extract was reduced to 1 mL, and then the solvent was further reduced under a purified nitrogen stream until the extract weight was 100 mg. The sample was stored at 5 °C until analysis. Headspace Isolation by Simultaneous Purging and Solvent Extraction (SPSE). Yomogi leaves (350 g) were washed with cold water three times and once with hot water and then cut into small pieces (1 cm) using a knife. The pieces of yomogi leaves were homogenized in a blender with 2000 mL of distilled water and 200 mL of saturated sodium chloride solution. The homogenized sample was placed in a 5 L roundbottom flask. The flask was connected to an SPSE apparatus previously prepared (Umano and Shibamoto, 1987, 1988). The flask was kept at 40 °C with a water bath. Headspace volatile chemicals were purged into 200 mL of distilled water, which was extracted with 30 mL of dichloromethane simultaneously and continuously for 2 h. After the extract was dried over anhydrous sodium sulfate, the solvent was removed by distillation with a Vigreux column. Distillation was stopped when the volume of extract was reduced to 1 mL. The excess solvent was further reduced under a purified nitrogen stream until
10.1021/jf0001738 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/08/2000
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Table 1. Aroma Chemicals Identified in Yomogi Leaf Extracts concentration (ppm) compound monoterpenes R-pinene R-thujene camphene β-pinene sabinene R-phellandrene myrcene R-terpinene D-limonene β-phellandrene cis-β-ocimene γ-terpinene trans-β-ocimene 1-(1-methylethyl)-4-methylbenzene (p-cymene) terpinolene 1-(1-methylethyl)-2-methylbenzene (o-cymene) 1-(1-methylethenyl)-4-methylbenzene (dehydro-p-cymene) monoterpenoids 2,3-dehydro-1,8-cineol 1,8-cineol 5,5-trimethyl-2,6-heptadien-4-one (artemisia ketone) 2,5,5-trimethylhepta-3,6-dien-2-ol (yomogi alcohol) R-thujone 2,5,5-trimethylhepta-2,6-dien-4-yl acetate (artemisia acetate) β-thujone trans-sabinene hydrate chrysanthenone 2,5,5-trimethylhepta-2,6-dien-4-ol (artemisia alcohol) camphor thujyl acetate cis-sabinene hydrate pinocarvone p-menth-2-en-1-olg bornyl acetate 4-terpineol lavandulyl acetate myrtenal bornyl isobutanoate bornyl propanoate pinocarveol thujyl alcohol (first) thujyl alcohol (second) trans-verbenol R-terpinyl acetate R-terpineol borneol verbenone R-citral L-carvone L-carvenyl acetate piperitol chrysanthenol citronellol L-perillaldehyde myrtenol p-mentha-1(7),8-dien-2-ol isopiperitenone trans-carveol p-cymen-8-ol geraniol trans-myrtanol cis-myrtanol cis-carveol piperityl acetate L-perillyl alcohol sesquiterpenes R-copaene R-bourbonene β-cubebene trans-R-bergamotene β-caryophyllene aromadendrene R-humulene trans-β-ndarnesene
Ib
DRPc
SPSEd
9 10 15 18 19 24 25 27 32 33 37 39 41 46 50 55 83
1021 1024 1064 1107 1119 1161 1161 1176 1197 1206 1230 1243 1249 1266 1279 1298 1432
nde nd 0.28 nd trf 0.11 nd 0.24 0.09 0.13 tr 0.26 nd tr tr tr tr
1.10 0.34 1.88 0.66 0.47 nd 1.06 0.15 1.29 tr 0.47 0.21 0.22 tr tr nd nd
31 34 64 78 80 81 84 88 93 94 95 99 100 102 103 104 107 108 111 113 114 115 117 121 122 123 124 125 126 130 132 133 134 136 138 140 141 142 144 145 146 147 148 149 150 154 161
1190 1208 1346 1404 1417 1417 1436 1467 1501 1509 1510 1542 1550 1561 1563 1578 1601 1606 1621 1638 1641 1654 1664 1679 1679 1694 1697 1701 1701 1726 1729 1731 1741 1751 1769 1777 1793 1797 1832 1833 1847 1850 1856 1861 1866 1900 2004
tr 0.13 nd 1.50 3.49 tr 0.20 0.40 0.16 2.17 1.06 tr 0.45 tr 0.20 0.43 0.34 0.07 tr 0.18 0.16 0.12 0.09 1.37 nd nd nd 10.27 1.85 0.04 0.05 nd 0.36 0.04 0.13 0.13 0.07 0.03 0.01 0.28 0.02 0.10 tr 0.03 0.34 0.02 0.31
nd 8.12 tr 0.96 3.20 4.22 0.22 0.25 0.45 0.67 0.14 tr tr tr 0.02 2.05 0.07 0.15 0.01 0.35 0.34 0.02 nd nd 0.06 nd 0.01 1.79 nd nd nd tr nd 0.03 nd nd nd nd nd nd nd nd nd nd nd nd nd
91 96 98 105 106 109 116 118
1487 1513 1524 1583 1591 1610 1664 1668
nd nd nd nd 0.07 nd nd 0.09
0.05 tr tr tr 2.39 tr 0.77 1.78
GC
peaka
Volatile Chemicals Identified in Japanese Mugwort
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Table 1 (Continued) concentration (ppm) compound sesquiterpenes β-guaieneg γ-muurolene germacrene D zingiberene R-muurolene cis,trans-R-farnesene trans,trans-R-farnesene δ-cadinene ar-curcumene sesquiterpenoids caryophyllene oxide R-humulene oxide (first) elemyl acetate R-humulene oxide (second) caryophylla-2(12),6(13)-dien-5-one trans-nerolidol germacren-4-olg globulol caryophylla-2(12),6-dien-5-one δ-cadinolg R-bisabolol R-cadinolg caryophylla-2(12),6(13)-dien-5-ol aliphatic aldehydes 2-methylpropanal 2-methyl-2-propenal (E)-2-butenal 4-methylpentanal hexanal heptanal (E)-2-hexenal octanal (E)-2-heptenal nonanal (E)-2-octenal (E,Z)-2,4-heptadienal aliphatic esters ethyl butanoate 2-methylpropyl 2-methylpropanoate 3-methylbutyl propanoate butyl 2-methylbutanoate hexyl acetate 3-methylbutyl 2-methylbutanoate 2-methylbutyl 2-methylbutanoate 1-hepten-3-yl acetate 2-methylbutyl 3-methylbutanoate (Z)-3-hexenyl acetate pentyl 2-methylbutanoate 1-octen-3-yl acetate 4-methylpentyl 2-methylbutanoate (Z)-3-hexenyl 2-methylpropanoate (Z)-3-hexenyl 2-methylbutanoate 1-nonen-3-yl acetate ethyl hexadecanoate ethyl oleate ethyl linoleate aliphatic hydrocarbons 1-octene tricyclene docosane tricosane tetracosane pentacosane hexacosane heptacosane aliphatic ketones acetone 3-buten-2-one butane-2,3-dione octan-3-one 6-methyl-5-hepten-2-one 2-cyclopentenone 2-hydroxy-2-methyl-4-pentanone
Ib
DRPc
SPSEd
119 120 127 128 129 131 135 137 139
1671 1679 1705 1718 1722 1726 1747 1755 1770
nd nd nd nd 0.15 0.04 nd 1.03 nd
tr 0.04 1.23 0.21 tr 0.71 0.21 0.21 0.02
159 162 164 165 166 167 168 169 170 174 177 178 180
1977 2010 2026 2033 2037 2041 2050 2073 2079 2169 2214 2232 2294
0.30 0.02 0.01 0.12 0.20 0.27 1.43 nd 0.07 0.64 0.06 1.36 0.41
0.01 nd nd tr 0.01 tr nd tr nd 0.06 nd nd tr
2 5 13 14 16 29 35 53 57 76 82 85
818 893 1037 1038 1080 1183 1215 1286 1321 1391 1426 1460
0.02 nd tr nd tr nd 0.05 tr nd tr tr tr
0.99 0.05 nd tr 0.04 0.01 0.14 0.02 tr 0.02 nd nd
11 17 30 38 47 48 51 52 54 56 62 71 72 73 89 90 179 186 188
1033 1092 1188 1232 1271 1276 1279 1284 1295 1316 1327 1379 1381 1385 1472 1477 2255 2476 2521
tra nd nd nd nd nd nd nd nd 0.04 nd 0.01 nd nd nd nd 0.08 0.03 0.02
nd tr tr tr tr tr 0.06 tr 0.02 0.27 0.01 0.48 tr 0.02 tr 0.01 nd nd nd
4 8 175 181 184 187 190 192
892 1009 2200 2300 2400 2500 2600 2700
nd nd nd nd tr tr tr 0.03
0.01 0.06 tr tr tr tr tr tr
3 6 7 43 63 66 68
846 943 972 1252 1335 1349 1359
0.03 tr nd nd tr tr 0.01
nd nd nd tr trace nd nd
GC
peaka
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Table 1 (Continued) concentration (ppm) compound aliphatic alcohols butanol 1-penten-3-ol 2-methyl-1-buten-3-ol 3-methyl-3-buten-1-ol pentanol 2-methyl-(E)-2-butenol hexanol (E)-3-hexenol (Z)-3-hexenol octan-3-ol (E)-2-hexenol 1-octen-3-ol 2-ethylhexanol 1-nonen-3-ol (E)-2-octenol dodecanol hexadecanol octadecanol aromatic compounds toluene 1,4-dimethylbenzene 1,3-dimethylbenzene 1,2-dimethylbenzene 3-ethyl-1-methylbenzene 2-ethyl-1-methylbenzene 1,2,4-trimethylbenzene 5-ethyl-1,3-dimethylbenzene 1,2,3-trimethylbenzene 2-methyl-1-vinylbenzene 4-ethyl-1,3-dimethylbenzene 4-ethyl-1,2-dimethylbenzene 3-ethyl-1,2-dimethylbenzene benzaldehyde phenylacetaldehyde phenylethyl acetate benzyl alcohol phenylethyl 2-methylpropanoate phenylethyl propanoate phenylethyl alcohol phenylethyl 2-methylbutanoate phenylethyl 3-methylbutanoate methyl anisate cinnamyl alcohol eugenol 6-methyl-3-isopropylphenol miscellaneous dimethyl sulfide 2-methylpyrazine 3-(methylthio)propanal 2,6-di-tert-butyl-4-methylphenol (BHT) 12-hydroxy-cis-9-dodecenoic acid lactoneg diethyl phthalate δ-dodecalactone dibutyl phthalate d
Ib
DRPc
SPSEd
22 23 26 40 42 59 67 70 74 77 79 86 92 101 110 158 183 189
1143 1158 1169 1248 1250 1322 1355 1365 1386 1397 1408 1453 1492 1554 1615 1969 2377 2581
tr tr tr 0.01 tr 0.05 tr tr 0.96 0.03 0.01 0.62 0.02 0.06 tr nd tr 0.02
nd nd tr nd nd nd nd nd 0.03 nd nd 0.25 0.02 0.02 nd tr nd nd
12 20 21 28 36 44 49 58 60 61 65 69 75 97 112 143 151 152 153 155 157 160 171 172 173 176
1037 1131 1137 1180 1222 1257 1276 1321 1327 1327 1348 1362 1390 1514 1633 1811 1874 1877 1880 1908 1968 1986 2085 2098 2162 2210
tr nd nd nd nd nd nd nd nd tr nd nd 0.01 tr 0.24 0.10 0.05 0.13 0.08 0.15 0.11 0.07 0.03 0.08 0.88 0.03
0.02 tr tr tr tr tr tr tr tr nd tr tr nd nd nd tr nd 0.02 nd nd tr 0.02 nd nd nd nd
1 45 85 156 163 182 185 191
748 1264 1449 1909 2018 2361 2416 2680
nd tr 0.03 nd 0.04 nd 0.03 0.27
tr nd nd tr nd tr nd 0.11
GC
peaka
a Refer to Figures 1 and 2. b Kovats index on DB-Wax column. c Volatiles obtained by steam distillation under reduced pressure. Volatiles obtained by simultaneous purging and solvent extraction. e Not detected. f Concentration 1 ppm in extracts obtained by DRP. Sesquiterpenes (hydrocarbon sesquiterpenes) were also recovered by SPSE (18.97% of the total GC peak area) with greater efficiency than by DRP (3.48% of the total GC peak area). Extracts obtained from yomogi leaves consisted of many terpenoids (oxygenated terpenes). These extracts also contained many oxygenated aliphatic compounds including 12 aldehydes, 19 esters, 7 ketones, and 18 alcohols. Some of these compounds play an important role in the aroma of this extract. For example, aliphatic aldehydes, such as (E)-2-hexenal, (E)-2-heptenal, and (E)-2-octenal, possess a strong green note (Arctander, 1969). It follows that the characteristic strong green aroma of this extract may be due to the presence of these oxygenated aliphatic compounds in addition to the presence of terpenoids. A total of 26 aromatic compounds were identified in the present study: 16 by DPC and 15 by SPSE. p- and o-cymenes are aromatic compounds, but they are listed as monoterpenes in Table 1. Alkyl aromatic hydrocarbons such as toluene, dimethylbenzenes, and methyl ethylbenzenes are not generally present in extracts isolated from plants. Therefore, these may be formed from monoterpenes via disproportionation reaction during experiments. Two phthalate compounds found in the
extracts by DRP are probably contaminants from unknown sources. LITERATURE CITED Arctander, S. Perfume and Flavor Chemical; Arctander: Montclair, NJ, 1969. Fujita, S.; Nogami, K. Constituents of the essential oil of Artemisia princeps. In Proceedings of the 38th Symposium on the Chemistry of Terpenes, Essential Oils, and Aromatics (TEAC38); 1994; pp 1-3. Fujita, S.; Nogami, K. Constituents of the essential oil of Artemisia princeps Pampan. In Proceedings of the 39th Symposium on the Chemistry of Terpenes, Essential Oils, and Aromatics (TEAC39); 1995; pp 267-269. Grandi, R.; Messerotti, W.; Pagnoni, U. M. Monoterpene variation in two Achillea ageratum chemotypes. Phytochemistry 1976, 15, 1770-1771. Hayashi, S.; Yano, K.; Matsuura, T. A new monoterpene alcohol from Artemisia feddei Lev. et Van. Tetrahedron Lett. 1968, 6241-6243. Kaiser, R.; Lamparsky, D. New carbonyl compounds in the high-boiling fraction of lavender oil. Helv. Chim. Acta 1983, 66, 1843-1849. Kalemba, D. Constituents of the essential oil of Artemisia asiatica Nakai. Flav. Fragr. 1999, 14, 173-176. Kim, Y. S.; Lee, J. H.; Kim, M. N.; Lee, W. G.; Kim, J. O. Volatile flavour compounds from raw mugwort leaves and parched mugwort tea. Korean Soc. Food Nutr. 1994, 23, 261-264; Chem. Abstr. 1994, 121, 29965g. Kovats, E. Gas chromatographic characterization of organic substances in the retention index system. Adv. Chromatogr. 1965, 1, 229-247. Thomas, A. F.; Pawlak, W. The synthesis and reactions of yomogi alcohol. Helv. Chim. Acta 1971, 54, 1822-1845.
Volatile Chemicals Identified in Japanese Mugwort Tsubaki, N.; Nishimura, K.; Hirose, Y. The volatile constituents of some plants of the Compositae family. Bull. Chem. Soc. Jpn. 1966, 39, 312-315. Umano, K.; Shibamoto, T. Analysis of headspace volatiles from overheated beef fat. J. Agric. Food Chem. 1987, 35, 14-18. Umano, K.; Shibamoto, T. A new method of headspace sampling: grapefruit volatiles. In Flavors and Fragrances: A World Perspective, Proceedings of the 10th International Congress of Essential Oils, Fragrances and Flavors, Washington, DC; 1988; pp 981-998.
J. Agric. Food Chem., Vol. 48, No. 8, 2000 3469 Willhalm, B.; Thomas, A. F. Synthesis and structure of yomogi alcohol, an alcohol related to artemisia alcohol. Chem. Commun. 1969, 1380. Wilson, S. R.; Guazzaroni, M. E. Synthesis of homoallylic alcohol in aqueous media. J. Org. Chem. 1989, 54, 30873091. Received for review February 9, 2000. Accepted May 8, 2000. JF0001738
