Abstract
Pinus species are important in traditional medicine throughout their ranges, and pine essential oils are of interest in aromatherapy and as topical treatments. In this work, the leaf (needle) essential oils of Pinus ponderosa var. ponderosa and Pinus contorta subsp. contorta from Oregon and Pinus flexilis growing in Idaho, have been obtained by hydrodistillation and analyzed by gas chromatographic techniques. The leaf essential oil of P. ponderosa was dominated by β-pinene (21.5–55.3%), methyl chavicol (8.5–41.5%), α-pinene (3.6–9.6%), δ-3-carene (3.6–6.2%), and α-terpineol (1.4–5.3%). The major components of P. contorta essential oil were β-phellandrene (23.8%), terpinen-4-ol (11.0%). The essential oil of P. flexilis was dominated by α-pinene (37.1%), β-pinene (21.9%), bornyl acetate (12.8%), and camphene (8.5%). Chiral gas chromatography revealed the enantiomeric ratios of α-pinene and limonene to be variable, but (−)-β-pinene predominated in Pinus essential oils.
Keywords: ponderosa pine, shore pine, limber pine, monoterpenoids, enantiomers, chiral GC-MS
1. Introduction
Numerous members of the genus Pinus (Pinaceae) are used in traditional medicine in their native ranges [1] and several essential oils derived from the genus are commercially important for use in aromatherapy and topical therapy applications, such as Scots pine (Pinus sylvestris L.), black pine (Pinus nigra J.F. Arnold), jack pine (Pinus bansksiana Lamb.), and white pine (Pinus strobus L.) [2]. In this work, the leaf essential oils of Pinus ponderosa Douglas ex C. Lawson var. ponderosa, Pinus contorta Douglas ex Loudon subsp. contorta, and Pinus flexilis E. James have been investigated for their chemical compositions and terpenoid enantiomeric distributions. In the case where essential oils are used therapeutically (e.g., aromatherapy) the different compositions and enantiomers may have very different biological activities. For commercial essential oils, the chemical compositions and enantiomeric distribution can be valuable for assessing the quality and consistency of the essential oil as well as a potential screen for adulteration or contamination.
Pinus ponderosa, the ponderosa pine (Figure 1), is the most widespread species of pine in western North America and ranges from British Columbia, south through the Cascade Range, the Sierra Nevada range of California, the Rocky Mountains and into the southwestern mountains of Utah, Arizona, and New Mexico. World Flora Online currently lists 11 subtaxa for the species [3], but the taxonomy is not resolved [4]. However, two varieties of the species are generally recognized: Pinus ponderosa var. ponderosa, the Pacific ponderosa pine, which ranges from southern British Columbia, south through the mountains of Washington, Oregon, and California, and Pinus ponderosa var. scopulorum Engelm., the Rocky Mountain ponderosa pine, found in eastern Montana, western North and South Dakota and Nebraska, Wyoming, Nebraska, northern and central Colorado and Utah [5]. Flathead Native Americans used the boughs of P. ponderosa in sweat lodges to treat muscular pains, while the Navajo people took a decoction of the needles for coughs and fever [6].
Figure 1.
Pinus ponderosa var. ponderosa from central Oregon. (A) Leaves (needles) and cone. (B) bark.
The native range of P. contorta is western North America, where there are three recognized subspecies: P. contorta subsp. latifolia (Engelm.) Critchf., the Rocky Mountain lodgepole pine, is found in the Rocky Mountains from the Yukon, south through Colorado; P. contorta subsp. murrayana (Balf.) Engelm., the Sierra lodgepole pine, found along the Cascade Range from Washington, through Oregon, and into northern California, and the Sierra Nevada Range in California; and P. contorta subsp. contorta, the shore pine (Figure 2), which ranges along the Pacific coast from southern Alaska, south to northwestern California [7,8]. The Haisla and Hanaksiala Native Americans used smoldering twigs of P. contorta subsp. contorta to alleviate pain and swelling of arthritic or injured joints [6].
Figure 2.
Pinus contorta subsp. contorta from the central Oregon coast. (A) Leaves (needles) and cone. (B) bark.
Pinus flexilis (Figure 3) naturally ranges in the Rocky Mountains of western North America, from southwest Alberta and southeast British Columbia, south through Colorado and New Mexico. It is also found in the mountains of Utah, Idaho, Nevada, and California [9]. The Navajo people used P. flexilis as cough medicine and to reduce fever [6]. As part of our investigation into the essential oil compositions of Pinus species [10,11], we have examined the compositions of the leaf essential oils of P. ponderosa var. ponderosa from La Pine, Oregon, P. contorta subsp. contorta from Ona Beach, Oregon, and Pinus flexilis from Boise, Idaho. As far as we are aware, this is the first report on the leaf oil composition of P. flexilis and the first report on the enantiomeric distributions of terpenoids in these Pinus species.
Figure 3.
Pinus flexilis from southwestern Idaho. (A) Leaves (needles) and cone. (B) bark.
2. Results and Discussion
2.1. Chemical Composition of Pinus ponderosa var. ponderosa
Hydrodistillation of three samples of fresh leaves of P. ponderosa var. ponderosa gave colorless essential oils in 0.321%, 0.399%, and 0.463% (w/w) yield, which are comparable to those obtained in previous studies (0.1–0.6%) [12,13,14]. The essential oil compositions are presented in Table 1. A total of 118 compounds were identified in the essential oils accounting for >99% of the composition. The major components in the essential oils were β-pinene (21.5–55.3%), methyl chavicol (8.5–41.5%), α-pinene (3.6–9.6%), δ-3-carene (3.6–6.2%), and α-terpineol (1.4–5.3%).
Table 1.
Chemical composition of Pinus ponderosa var. ponderosa leaf essential oil.
| RIcalc | RIdb | Compound | % Composition | ||
|---|---|---|---|---|---|
| Tree #1 | Tree #2 | Tree #3 | |||
| 919 | 919 | Hashishene | tr | --- | --- |
| 922 | 923 | Tricyclene | tr | tr | tr |
| 925 | 926 | α-Thujene | tr | tr | tr |
| 932 | 932 | α-Pinene | 3.6 | 5.7 | 9.6 |
| 946 | 948 | α-Fenchene | tr | tr | tr |
| 948 | 950 | Camphene | 0.1 | 0.2 | 0.3 |
| 970 | 970 | 3,7,7-Trimethyl-1,3,5-cycloheptatriene | tr | tr | tr |
| 971 | 971 | Sabinene | 0.1 | 0.1 | 0.1 |
| 978 | 978 | β-Pinene | 21.5 | 35.3 | 55.3 |
| 988 | 989 | Myrcene | 1.7 | 1.3 | 1.7 |
| 999 | 1000 | δ-2-Carene | tr | --- | --- |
| 1006 | 1006 | α-Phellandrene | tr | tr | tr |
| 1009 | 1008 | δ-3-Carene | 3.6 | 5.5 | 6.2 |
| 1015 | 1015 | 1,4-Cineole | tr | tr | tr |
| 1016 | 1017 | α-Terpinene | tr | 0.1 | 0.1 |
| 1019 | 1022 | m-Cymene | tr | tr | tr |
| 1024 | 1025 | p-Cymene | 0.1 | 0.1 | 0.1 |
| 1028 | 1030 | Limonene | 0.8 | 1.1 | 1.3 |
| 1030 | 1031 | β-Phellandrene | 0.9 | 1.3 | 1.7 |
| 1034 | 1034 | (Z)-β-Ocimene | 0.7 | 0.7 | tr |
| 1045 | 1045 | (E)-β-Ocimene | 0.1 | tr | tr |
| 1057 | 1057 | γ-Terpinene | 0.1 | 0.1 | 0.1 |
| 1070 | 1069 | cis-Linalool oxide (furanoid) | tr | tr | tr |
| 1080 | 1082 | p-Mentha-2,4(8)-diene | tr | tr | tr |
| 1084 | 1086 | Terpinolene | 0.4 | 0.7 | 0.8 |
| 1086 | 1086 | trans-Linalool oxide (furanoid) | 0.1 | --- | 0.1 |
| 1089 | 1091 | p-Cymenene | tr | --- | tr |
| 1090 | 1090 | 2-Nonanone | --- | 0.1 | tr |
| 1099 | 1101 | Linalool | 2.1 | 0.4 | 0.3 |
| 1104 | 1104 | Nonanal | 0.1 | tr | tr |
| 1118 | 1119 | endo-Fenchol | 0.1 | tr | tr |
| 1124 | 1124 | cis-p-Menth-2-en-1-ol | tr | tr | tr |
| 1126 | 1126 | α-Campholenal | tr | tr | tr |
| 1127 | 1127 | allo-Ocimene | --- | tr | --- |
| 1137 | 1137 | Nopinone | 0.2 | tr | 0.1 |
| 1140 | 1140 | trans-Pinocarveol | 0.4 | 0.1 | tr |
| 1142 | 1142 | trans-p-Menth-2-en-1-ol | --- | 0.1 | --- |
| 1145 | 1145 | Camphor | --- | tr | --- |
| 1154 | 1156 | Camphene hydrate | 0.1 | 0.1 | 0.1 |
| 1155 | 1155 | Hexyl isobutyrate | --- | --- | tr |
| 1160 | 1160 | trans-Pinocamphone | 0.2 | 0.2 | 0.3 |
| 1161 | 1164 | Pinocarvone | 0.3 | 0.1 | 0.1 |
| 1170 | 1170 | (2E)-Nonen-1-ol | 0.1 | tr | 0.1 |
| 1171 | 1171 | p-Mentha-1,5-dien-8-ol | 0.1 | tr | tr |
| 1175 | 1176 | cis-Pinocamphone | 0.2 | 0.2 | 0.2 |
| 1180 | 1180 | Terpinen-4-ol | 0.4 | 0.3 | 0.2 |
| 1187 | 1186 | p-Cymen-8-ol | 0.3 | 0.1 | --- |
| 1196 | 1195 | α-Terpineol | 5.3 | 1.4 | 3.0 |
| 1199 | 1197 | Methyl chavicol (= Estragole) | 41.5 | 27.4 | 8.5 |
| 1206 | 1206 | Decanal | --- | 0.1 | 0.1 |
| 1208 | 1208 | Verbenone | tr | tr | --- |
| 1228 | 1229 | Thymol methyl ether | --- | --- | tr |
| 1252 | 1253 | (Z)-Anethole | --- | tr | --- |
| 1253 | 1254 | Piperitone | --- | tr | --- |
| 1278 | 1276 | (2E)-Decen-1-ol | --- | 0.1 | --- |
| 1283 | 1282 | Bornyl acetate | 0.2 | 0.1 | 0.1 |
| 1285 | 1285 | (E)-Anethole | 2.3 | 1.6 | 0.1 |
| 1292 | 1293 | 2-Undecanone | --- | 0.1 | --- |
| 1313 | 1314 | Carvenolide | 0.1 | --- | --- |
| 1322 | 1322 | Myrtenyl acetate | 0.1 | 0.1 | tr |
| 1345 | 1346 | α-Terpinyl acetate | 0.3 | 0.3 | 0.2 |
| 1372 | 1370 | (2E)-Undecen-1-ol | 0.7 | 0.3 | 0.3 |
| 1375 | 1375 | α-Copaene | 0.1 | 0.2 | 0.2 |
| 1383 | 1382 | β-Bourbonene | --- | tr | --- |
| 1387 | 1387 | β-Cubebene | tr | 0.1 | tr |
| 1389 | 1390 | trans-β-Elemene | 0.1 | --- | --- |
| 1389 | 1389 | (5Z)-Decen-1-yl acetate | --- | 0.5 | 0.4 |
| 1399 | 1403 | Methyl eugenol | 0.1 | tr | --- |
| 1409 | 1410 | Dodecanal | 0.1 | 0.1 | 0.1 |
| 1419 | 1417 | (E)-β-Caryophyllene | 0.5 | 0.5 | 0.1 |
| 1429 | 1430 | β-Copaene | tr | tr | tr |
| 1432 | 1432 | trans-α-Bergamotene | 0.5 | 0.1 | 0.1 |
| 1438 | 1438 | Aromadendrene | 0.3 | tr | 0.2 |
| 1442 | 1442 | Guaia-6,9-diene | --- | --- | tr |
| 1447 | 1447 | Geranyl acetone | --- | tr | --- |
| 1448 | 1448 | cis-Muurola-3,5-diene | --- | tr | tr |
| 1452 | 1452 | (E)-β-Farnesene | 0.1 | tr | tr |
| 1455 | 1454 | α-Humulene | 0.1 | 0.1 | tr |
| 1459 | 1457 | allo-Aromadendrene | --- | --- | tr |
| 1461 | 1463 | cis-Muurola-4(14),5-diene | 0.1 | 0.1 | tr |
| 1467 | 1469 | Ethyl (E)-cinnamate | 0.2 | --- | 0.1 |
| 1469 | 1470 | (2E)-Undecenyl acetate | 0.1 | 0.3 | tr |
| 1471 | 1472 | trans-Cadina-1(6),4-diene | tr | 0.1 | 0.1 |
| 1474 | 1475 | γ-Muurolene | 0.2 | 0.4 | 0.2 |
| 1480 | 1480 | Germacrene D | 0.4 | 0.9 | 0.3 |
| 1488 | 1489 | β-Selinene | 0.4 | 0.1 | 0.2 |
| 1491 | 1492 | trans-Muurola-4(14),5-diene | 0.1 | 0.1 | 0.1 |
| 1495 | 1495 | 2-Tridecanone | --- | 0.3 | --- |
| 1496 | 1497 | Bicyclogermacrene | 0.8 | --- | 0.5 |
| 1498 | 1497 | α-Muurolene | 0.3 | 0.5 | 0.3 |
| 1512 | 1512 | γ-Cadinene | 0.9 | 1.5 | 1.0 |
| 1518 | 1518 | δ-Cadinene | 1.6 | 2.8 | 1.9 |
| 1519 | 1519 | trans-Calamenene | tr | tr | 0.1 |
| 1522 | 1521 | Zonarene | tr | tr | 0.1 |
| 1532 | 1533 | trans-Cadina-1,4-diene | tr | 0.1 | 0.1 |
| 1536 | 1538 | α-Cadinene | 0.1 | 0.1 | 0.1 |
| 1540 | 1541 | α-Calacorene | tr | tr | tr |
| 1561 | 1561 | (E)-Nerolidol | --- | 1.0 | --- |
| 1561 | 1560 | Dodecanoic acid | 0.5 | 0.2 | 0.3 |
| 1574 | 1574 | Germacrene D-4α-ol | --- | 0.6 | --- |
| 1577 | 1576 | Spathulenol | 1.0 | --- | 0.6 |
| 1581 | 1582 | Caryophyllene oxide | 0.2 | 0.1 | tr |
| 1586 | 1590 | Globulol | 0.1 | 0.1 | 0.1 |
| 1593 | 1598 | Ethyl dodecanoate | 0.1 | --- | --- |
| 1625 | 1624 | Muurola-4,10(14)-dien-1β-ol | tr | 0.1 | tr |
| 1627 | 1628 | 1-epi-Cubenol | tr | 0.1 | 0.1 |
| 1642 | 1643 | τ-Cadinol | 0.3 | 0.5 | 0.3 |
| 1644 | 1644 | τ-Muurolol | 0.3 | 0.6 | 0.5 |
| 1655 | 1655 | α-Cadinol | 0.5 | 0.7 | 0.5 |
| 1664 | 1664 | Brevifolin (= Xanthoxylin) | 0.1 | --- | --- |
| 1675 | 1670 | (6Z)-Pentadecen-2-one | 0.1 | 0.2 | --- |
| 1765 | 1769 | Benzyl benzoate | --- | 0.1 | tr |
| 1794 | 1796 | (9Z)-Hexadecenal | --- | 0.1 | tr |
| 1816 | 1817 | Hexadecanal | tr | 0.1 | 0.1 |
| 1866 | 1869 | Benzyl salicylate | --- | 0.1 | --- |
| 1991 | 1989 | Manoyl oxide | 0.2 | 0.1 | 0.1 |
| 1995 | 1997 | 9β-Isopimara7,15-diene | --- | 0.1 | 0.1 |
| 2290 | 2297 | Methyl isopimarate | 0.1 | 0.1 | tr |
| Monoterpene hydrocarbons | 33.6 | 52.3 | 77.3 | ||
| Oxygenated monoterpenoids | 10.5 | 3.4 | 4.5 | ||
| Sesquiterpene hydrocarbons | 6.7 | 7.7 | 5.5 | ||
| Oxygenated sesquiterpenoids | 2.4 | 3.7 | 2.0 | ||
| Diterpenoids | 0.3 | 0.3 | 0.1 | ||
| Benzenoid aromatics | 44.2 | 29.2 | 8.7 | ||
| Others | 1.7 | 2.5 | 1.4 | ||
| Total identified | 99.5 | 99.1 | 99.5 | ||
There have been several investigations into the essential oil composition of P. ponderosa from different geographical locations, including California (USA) [13,14], British Columbia (Canada) [19], Washington (USA) [20], Poland [21], and Arizona (USA) [22]. Although there is much variation in the concentrations, the major components of P. ponderosa leaf essential oils reported in the literature have been α-pinene (10.2–69.3%), β-pinene (2.1–66.0%), myrcene (1.4–7.4%), δ-3-carene (up to 41.8%), α-terpineol (up to 7.5%) and methyl chavicol (1.8–20.4%). Thus, the essential oil compositions of Oregon P. ponderosa, subsp. ponderosa in this work are qualitatively similar to previous reports for P. ponderosa, and the wide chemical variations are likely due to geographical locations and/or genetic differences.
2.2. Chemical Composition of Pinus contorta Subsp. contorta
The fresh leaves of P. contorta subsp. contorta were hydrodistilled to give a colorless essential oil in 0.674% (w/w) yield. A previous report by Adams and co-workers indicated an essential oil yield of only 0.1% [23]. The essential oil composition is summarized in Table 2. A total of 55 compounds were identified accounting for 98.2% of the essential oil composition. The dominant components in the essential oil were the monoterpenoids β-phellandrene (23.8%), terpinen-4-ol (11.0%), thymol (6.6%), and chavicol (5.3%). Adams and co-workers have reported the leaf essential oils of P. contorta subsp. contorta, P. contorta subsp. latifolia, and P. contorta subsp. murrayana [23]. There are some notable differences between the leaf essential oil composition of the Oregon sample (this work) and those from coastal Washington [23]. The β-phellandrene concentration was lower than the Washington samples (39.2–61.5%), but γ-terpinene and terpinen-4-ol concentrations were higher than the Washington samples (0.6–1.7% and 0.3%, respectively), and neither chavicol nor thymol were detected in the Washington samples.
Table 2.
Chemical composition of Pinus contorta subsp. contorta leaf essential oil.
| RIcalc | RIdb | Compound | % Composition |
|---|---|---|---|
| 782 | 782 | Prenol | 1.1 |
| 801 | 801 | Hexanal | 0.6 |
| 848 | 849 | (2E)-Hexenal | 0.5 |
| 851 | 853 | (3Z)-Hexenol | 0.3 |
| 923 | 923 | Tricyclene | 0.1 |
| 925 | 927 | α-Thujene | 0.2 |
| 933 | 932 | α-Pinene | 1.2 |
| 949 | 950 | Camphene | 0.2 |
| 959 | 959 | Benzaldehyde | 2.0 |
| 972 | 971 | Sabinene | 0.2 |
| 977 | 978 | β-Pinene | 0.5 |
| 989 | 989 | Myrcene | 1.0 |
| 989 | 990 | Dehydro-1,8-cineole | 0.1 |
| 1007 | 1006 | α-Phellandrene | 0.6 |
| 1009 | 1008 | δ-3-Carene | 0.2 |
| 1014 | 1015 | 1,4-Cineole | 3.7 |
| 1017 | 1017 | α-Terpinene | 3.6 |
| 1024 | 1024 | p-Cymene | 1.5 |
| 1029 | 1030 | Limonene | 2.0 |
| 1030 | 1031 | β-Phellandrene | 23.8 |
| 1035 | 1034 | (Z)-β-Ocimene | 1.1 |
| 1057 | 1057 | γ-Terpinene | 6.8 |
| 1070 | 1069 | cis-Linalool oxide (furanoid) | 0.2 |
| 1085 | 1086 | Terpinolene | 2.2 |
| 1086 | 1086 | trans-Linalool oxide (furanoid) | 0.4 |
| 1089 | 1091 | p-Cymenene | 0.3 |
| 1100 | 1099 | Linalool | 0.1 |
| 1124 | 1124 | cis-p-Menth-2-en-1-ol | 1.8 |
| 1135 | 1136 | Terpin-3-en-1-ol | 2.3 |
| 1142 | 1142 | trans-p-Menth-2-en-1-ol | 1.2 |
| 1146 | 1145 | Camphor | 0.6 |
| 1177 | 1179 | 2-Isopropenyl-5-methyl-4-hexenal | 0.6 |
| 1180 | 1180 | Terpinen-4-ol | 11.0 |
| 1187 | 1186 | p-Cymen-8-ol | 1.7 |
| 1187 | 1188 | trans-β-Ocimenol | 0.3 |
| 1195 | 1195 | α-Terpineol | 2.4 |
| 1196 | 1197 | Estragole (= Methyl chavicol) | 0.4 |
| 1199 | 1200 | γ-Terpineol | 0.9 |
| 1237 | 1237 | Pulegone | 0.4 |
| 1249 | 1250 | Chavicol | 5.3 |
| 1277 | 1277 | Phellandral | 0.3 |
| 1286 | 1285 | (E)-Anethole | 0.3 |
| 1289 | 1289 | Thymol | 6.6 |
| 1353 | 1356 | Eugenol | 0.3 |
| 1444 | 1442 | Guaia-6,9-diene | 0.8 |
| 1483 | 1480 | Germacrene D | 0.2 |
| 1564 | 1560 | Dodecanoic acid | 1.7 |
| 1573 | 1571 | (3Z)-Hexenyl benzoate | 1.6 |
| 1579 | 1576 | Spathulenol | 0.5 |
| 1627 | 1627 | Benzophenone | 0.2 |
| 1766 | 1769 | Benzyl benzoate | 0.5 |
| 1868 | 1869 | Benzyl salicylate | 0.5 |
| 1960 | 1958 | Palmitic acid | 0.6 |
| 2012 | 2016 | Juvabione | 0.6 |
| 2052 | 2053 | Manool | 0.4 |
| Monoterpene hydrocarbons | 45.3 | ||
| Oxygenated monoterpenoids | 34.2 | ||
| Sesquiterpene hydrocarbons | 0.9 | ||
| Oxygenated sesquiterpenoids | 0.5 | ||
| Diterpenoids | 0.4 | ||
| Benzenoid aromatics | 11.5 | ||
| Others | 5.5 | ||
| Total identified | 98.2 |
β-Phellandrene also dominated the essential oils of P. contorta subsp. latifolia from Alberta, Canada (34.3% β-phellandrene) [24] and P. contorta subsp. murrayana (37.2% β-phellandrene) [11]. In contrast, however, the concentration of terpinen-4-ol was relatively minor in both P. contorta subsp. latifolia (0.5%) and P. contorta subsp. murrayana (1.9%). Thymol was a minor component (0.3%) in P. contorta subsp. murrayana, and not observed in P. contorta subsp. latifolia. Chavicol was not observed either the latifolia or murrayana subspecies. Conversely, β-pinene was an abundant constituent of P. contorta subsp. latifolia (30.5%) and P. contorta subsp. murrayana (17.0%) as was α-terpineol (4.3% and 11.6%, respectively).
2.3. Chemical Composition of Pinus flexilis
Hydrodistillation of the fresh leaves (needles) of P. flexilis gave a colorless essential oil in 0.273% (w/w) yield. There have been no previous reports on P. flexilis essential oil yields. However, essential oils from Pinus species have been obtained in yields ranging from 0.08% (P. rigida) to 2.33% (P. pumila) [14]. The essential oil composition is presented in Table 3. A total of 102 compounds were identified in the leaf essential oil of P. flexilis, accounting for 99.7% of the composition. The major components in the essential oil were α-pinene (37.1%), β-pinene (21.9%), bornyl acetate (12.8%), and camphene (8.5%).
Table 3.
Chemical composition of Pinus flexilis leaf essential oil.
| RIcalc | RIdb | Compound | % Composition |
|---|---|---|---|
| 801 | 801 | Hexanal | 0.2 |
| 848 | 849 | (2E)-Hexenal | 0.7 |
| 850 | 853 | (3Z)-Hexenol | 0.2 |
| 863 | 867 | 1-Hexanol | 0.1 |
| 880 | 880 | Santene | 0.1 |
| 900 | 900 | Nonane | tr |
| 923 | 923 | Tricyclene | 0.7 |
| 925 | 925 | α-Thujene | tr |
| 933 | 933 | α-Pinene | 37.1 |
| 951 | 953 | Camphene | 8.5 |
| 953 | 953 | Thuja-2,4(10)-diene | tr |
| 972 | 972 | Sabinene | 0.3 |
| 979 | 978 | β-Pinene | 21.9 |
| 989 | 989 | Myrcene | 1.5 |
| 1007 | 1007 | α-Phellandrene | 0.1 |
| 1017 | 1017 | α-Terpinene | 0.1 |
| 1024 | 1024 | p-Cymene | 0.1 |
| 1030 | 1030 | Limonene | 3.3 |
| 1031 | 1031 | β-Phellandrene | 2.2 |
| 1034 | 1034 | (Z)-β-Ocimene | 0.1 |
| 1045 | 1045 | (E)-β-Ocimene | tr |
| 1057 | 1057 | γ-Terpinene | 0.2 |
| 1085 | 1086 | Terpinolene | 1.0 |
| 1088 | 1090 | Fenchone | 0.1 |
| 1089 | 1093 | p-Cymenene | tr |
| 1096 | 1099 | 6-Camphenone | 0.1 |
| 1100 | 1100 | Undecane | 0.4 |
| 1104 | 1104 | Nonanal | tr |
| 1119 | 1120 | endo-Fenchol | tr |
| 1124 | 1124 | cis-p-Menth-2-en-1-ol | tr |
| 1126 | 1126 | α-Campholenal | 0.2 |
| 1138 | 1139 | Nopinone | tr |
| 1140 | 1141 | trans-Pinocarveol | 0.2 |
| 1142 | 1142 | trans-p-Menth-2-en-1-ol | tr |
| 1145 | 1145 | trans-Verbenol | 0.1 |
| 1147 | 1145 | Camphor | 0.1 |
| 1150 | 1150 | α-Phellandren-8-ol | 0.1 |
| 1155 | 1156 | Camphene hydrate | 0.1 |
| 1160 | 1160 | trans-Pinocamphone | tr |
| 1162 | 1164 | Pinocarvone | tr |
| 1171 | 1171 | p-Mentha-1,5-dien-8-ol | 0.3 |
| 1171 | 1173 | Borneol | 0.2 |
| 1180 | 1180 | Terpinen-4-ol | 0.2 |
| 1186 | 1186 | p-Cymen-8-ol | 0.1 |
| 1195 | 1195 | α-Terpineol | 1.5 |
| 1206 | 1205 | Verbenone | tr |
| 1228 | 1229 | Thymyl methyl ether | 0.2 |
| 1286 | 1287 | Bornyl acetate | 12.8 |
| 1291 | 1293 | 2-Undecanone | 0.3 |
| 1294 | 1294 | trans-Pinocarvyl acetate | tr |
| 1300 | 1300 | Tridecane | tr |
| 1357 | 1357 | 2-Methylundecanal | 0.1 |
| 1376 | 1375 | α-Copaene | 0.1 |
| 1409 | 1410 | Dodecanal | 0.1 |
| 1410 | 1408 | Acora-3,7(14)-diene | tr |
| 1420 | 1417 | (E)-β-Caryophyllene | 0.2 |
| 1430 | 1430 | β-Copaene | tr |
| 1452 | 1152 | (E)-β-Farnesene | 0.2 |
| 1455 | 1154 | α-Humulene | tr |
| 1475 | 1175 | γ-Muurolene | tr |
| 1481 | 1480 | Germacrene D | 0.2 |
| 1494 | 1494 | 2-Tridecanone | 0.3 |
| 1498 | 1497 | α-Muurolene | 0.2 |
| 1507 | 1508 | β-Bisabolene | 0.6 |
| 1512 | 1512 | γ-Cadinene | 0.1 |
| 1518 | 1518 | δ-Cadinene | 0.3 |
| 1548 | 1549 | α-Elemol | tr |
| 1560 | 1560 | (E)-Nerolidol | tr |
| 1576 | 1576 | Spathulenol | 0.1 |
| 1627 | 1628 | 1-epi-Cubenol | tr |
| 1641 | 1640 | τ-Cadinol | 0.1 |
| 1643 | 1644 | τ-Muurolol | 0.1 |
| 1647 | 1651 | α-Muurolol (= δ-Cadinol) | tr |
| 1655 | 1655 | α-Cadinol | 0.2 |
| 1664 | 1665 | Intermedeol | tr |
| 1668 | 1667 | (6Z)-Pentadecen-2-one | tr |
| 1684 | 1683 | epi-α-Bisabolol | tr |
| 1687 | 1688 | α-Bisabolol | 0.7 |
| 1696 | 1697 | 2-Pentadecanone | 0.1 |
| 1707 | 1706 | (2E,6Z)-Farnesal | tr |
| 1717 | 1714 | (2E,6Z)-Farnesol | 0.1 |
| 1734 | 1737 | (2E,6E)-Farnesal | tr |
| 1782 | 1779 | Dodecyl butyrate | tr |
| 1815 | 1817 | Hexadecanal | tr |
| 1830 | 1832 | Farnesyl acetate | tr |
| 1964 | 1968 | Sandaracopimara-8(14),15-diene | 0.1 |
| 1993 | 1994 | Manoyl oxide | 0.3 |
| 1997 | 2000 | 9β-Isopimara-7,15-diene | 0.1 |
| 2013 | 2007 | 18-Norabieta-8,11,13-triene | 0.1 |
| 2085 | 2086 | Abietadiene | tr |
| 2145 | 2147 | Abienol | tr |
| 2182 | 2180 | Sandaracopimarinal | 0.1 |
| 2222 | 2231 | Isopimarinal | 0.2 |
| 2230 | 2236 | Palustrinal | 0.2 |
| 2234 | --- | Levopimarinal a | tr |
| 2241 | 2238 | Methyl pimarate | tr |
| 2262 | 2267 | Dehydroabietal | tr |
| 2292 | 2297 | Methyl isopimarate | tr |
| 2296 | 2302 | Methyl levopimarate | tr |
| 2307 | 2312 | Abietal | tr |
| 2330 | 2341 | Methyl dehydroabietate | tr |
| 2365 | 2366 | Neoabietic acid | tr |
| Monoterpene hydrocarbons | 77.3 | ||
| Oxygenated monoterpenoids | 16.0 | ||
| Sesquiterpene hydrocarbons | 1.9 | ||
| Oxygenated sesquiterpenoids | 1.2 | ||
| Diterpenoids | 0.9 | ||
| Fatty acid derivatives | 2.3 | ||
| Total identified | 99.7 |
RIcalc = Retention index calculated with respect to a homologous series of n-alkanes on a ZB-5ms column. RIdb = Reference retention index obtained from the databases [15,16,17,18]. tr = trace (<0.05%). a Identification tentative; the MS is a good match (93% similarity match), but there is no reference RI available.
2.4. Enantiomeric Distribution of Terpenoids
The enantiomeric distributions of several terpenoid essential oil components have been determined by chiral gas chromatography-mass spectrometry. The enantiomeric distributions of terpenoid components of P. ponderosa var. ponderosa, P. contorta subsp. contorta, and P. flexilis essential oils are summarized in Table 4.
Table 4.
Enantiomeric distribution of terpenoids of Pinus ponderosa var. ponderosa, Pinus contorta subsp. contorta, and Pinus flexilis leaf essential oils.
| Terpenoid Compound | Enantiomeric Distribution, (+):(−) | ||||
|---|---|---|---|---|---|
| P. ponderosa | P. contorta | P. flexilis | |||
| Tree #1 | Tree #2 | Tree #3 | |||
| α-Pinene | 53.3:46.7 | 20.3:79.7 | 6.2:93.8 | 27.5:72.5 | 4.8:95.2 |
| Camphene | 47.9:52.1 | 10.6:89.4 | 8.2:91.8 | --- | 1.8:98.2 |
| Sabinene | --- | --- | --- | --- | 100:0 |
| β-Pinene | 1.9:98.1 | 1.7:98.3 | 1.7:98.3 | 0:100 | 3.2:96.8 |
| α-Phellandrene | --- | --- | --- | 8.4:91.6 | 17.2:82.8 |
| δ-3-Carene | 72.1:27.9 | 0.7:99.3 | 1.0:99.0 | --- | --- |
| Limonene | 38.7:61.3 | 41.1:58.9 | 41.2:58.8 | 13.2:86.8 | 33.0:67.0 |
| β-Phellandrene | 2.3:97.7 | 0.9:99.1 | 1.3:98.7 | 0.6:99.4 | 3.5:96.5 |
| Fenchone | --- | --- | --- | --- | 100:0 |
| Linalool | 7.6:92.4 | 9.3:90.7 | 9.7:90.3 | --- | --- |
| Camphor | --- | --- | --- | 0:100 | --- |
| Borneol | --- | --- | --- | --- | 0:100 |
| Terpinen-4-ol | 37.2:62.8 | 30.7:69.3 | 39.3:60.7 | 53.0:47.0 | 43.5:56.5 |
| α-Terpineol | 2.6:97.4 | 3.6:96.4 | 2.8:97.2 | 35.5:64.5 | 8.8:91.2 |
| Pulegone | --- | --- | --- | 100:0 | --- |
| Bornyl acetate | 0:100 | 0:100 | 0:100 | --- | 0:100 |
| α-Terpinyl acetate | 0:100 | 0:100 | 0:100 | --- | --- |
| (E)-β-Caryophyllene | 0:100 | 0:100 | 0:100 | --- | 0:100 |
| Germacrene D | 0:100 | 0:100 | 0:100 | --- | 0:100 |
| β-Bisabolene | --- | --- | --- | --- | 100:0 |
| δ-Cadinene | 0:100 | 0:100 | 0:100 | --- | 0:100 |
| (E)-Nerolidol | --- | 0.6:99.4 | --- | --- | --- |
--- = not detected.
In P. ponderosa var. ponderosa essential oil, the (−)-enantiomer was the dominant stereoisomer in all monoterpenoids assessed. In the case of limonene and terpinen-4-ol, the (−)-enantiomer was only is slight excess over the (+)-enantiomer, however. In the case of P. contorta subsp. contorta, the (−)-enantiomer was dominant in α-pinene, β-pinene, α-phellandrene, limonene, β-phellandrene, borneol, and α-terpineol, which is comparable to the distribution found in P. contorta subsp. murrayana [11] as well as P. ponderosa var. ponderosa (above). Interestingly, the enantiomeric distribution for terpinen-4-ol was (+)53.0:(−)47.0 in P. c. subsp. contorta, but reversed in P. c. subsp. murrayana, (+)39.9:(−)60.1. In P. flexilis, the (−)-enantiomers dominated in α-pinene, camphene, β-pinene, α-phellandrene, β-phellandrene, and α-terpineol, while the (+)-enantiomers were exclusively observed for sabinene, fenchone, and β-bisabolene. As observed in P. ponderosa var. ponderosa, the (−)-enantiomers were slightly higher than the (+)-enantiomers for limonene and for terpinen-4-ol in P. flexilis.
The enantiomeric distributions for α-pinene, β-pinene, and limonene have been assessed for several Pinus species, which are listed in Table 5 for comparison. A perusal of Table 5 reveals that the enantiomeric distribution of α-pinene and limonene in Pinus species is variable both between species and within species. Allenspach and co-workers found that (+)-α-pinene generally predominated in primary essential oils of P. sylvestris and P. cembra, but that P. mugo and P. nigra were generally dominated by (−)-α-pinene [25]. The enantiomeric distribution in β-pinene in Pinus species, however, is consistently dominated by (−)-β-pinene.
Table 5.
Enantiomeric distribution, (+):(–), of monoterpene hydrocarbons in Pinus species leaf essential oils.
| Pinus Species | Geographical Source | α-Pinene | β-Pinene | Limonene | Ref. |
|---|---|---|---|---|---|
| Pinus banksiana Lamb | Eastern Canada | 74.5:25.5 | 3.0:97.0 | 8.4:91.6 | APRC |
| Eastern Canada | 74.4:25.6 | 3.0:97.0 | 8.4:91.6 | ||
| Pinus cembra L. | Italy | 64.4:35.6 | 0.8:99.2 | 0:100 | [26] |
| Pinus contorta subsp. murrayana (Balf.) Engelm. | Oregon, USA | 20.1:79.7 | 2.2:97.8 | 0:100 | [11] |
| Pinus contorta Douglas ex Loudon subsp. contorta | Oregon, USA | 27.5:72.5 | 0:100 | 13.2:86.8 | This work |
| Pinus flexilis E. James | Idaho, USA | 4.8:95.2 | 3.2:96.8 | 33.0:67.0 | This work |
| Pinus halepensis Mill. | Portugal | 59.1:40.9 | 4.7:95.3 | --- | [27] |
| Pinus mugo Turra (syn. P. montana Mill.) | Austria | 49.2:50.8 | 0.9:99.1 | 28.1:71.9 | [26] |
| Italy | 63.3:36.7 | 1.4:98.6 | 13.4:86.6 | ||
| Korea | 43.8:56.2 | 19.1:80.9 | 62.7:37.3 | ||
| Pinus nigra J.F. Arnold | Austria | 16.9:83.1 | 6.7:93.3 | 23.8:76.2 | [26] |
| Albania | 3.9:96.1 | 18.0:82.0 | 23.6:76.4 | APRC | |
| Pinus peuce Griseb. | Germany | 26.8:73.2 | 3.7:96.3 | 29.2:70.8 | [28] |
| Germany | 31.0:69.0 | 3.3:96.7 | 20.2:79.8 | ||
| Pinus pinaster Aiton | Italy | 71.3:28.7 | 2.6:97.4 | 17.8:82.2 | [26] |
| Portugal | 30.3:69.7 | 0.6:99.4 | 31.0:69.0 | [27] | |
| Pinus pinea L. | Portugal | 48.3:51.7 | 0:100 | 0.4:99.6 | [27] |
| Pinus ponderosa Douglas ex C. Lawson var. ponderosa | Oregon, USA | 53.3:46.7 | 1.9:98.1 | 38.7:61.3 | This work |
| Oregon, USA | 20.3:79.7 | 1.7:98.3 | 41.1:58.9 | ||
| Oregon, USA | 6.2:93.8 | 1.7:98.3 | 41.2:58.8 | ||
| Pinus resinosa Aiton | Eastern Canada | 61.2:38.8 | 2.9:97.1 | 44.0:56.0 | APRC |
| Eastern Canada | 63.0:37.0 | 2.5:97.5 | 38.8:61.2 | ||
| Pinus strobus L. | Eastern Canada | 39.8:60.2 | 2.2:97.8 | 16.5:83.5 | APRC |
| Eastern Canada | 40.2:59.8 | 2.4:97.6 | 16.5:83.5 | ||
| Pinus sylvestris L. | Poland | 76.2:23.8 | 1.8:98.2 | 98.1:1.9 | [26] |
| Austria | 23.2:76.8 | 3.5:96.5 | 25.9:74.1 | ||
| Italy | 13.5:86.5 | 3.6:96.4 | 29.3:70.7 | ||
| Korea | 33.4:66.6 | 4.9:95.1 | 66.7:33.3 | ||
| Portugal | 27.2:72.8 | 0.9:99.1 | --- | [27] | |
| Eastern Canada | 67.1:32.9 | 2.3:97.7 | 21.8:78.2 | APRC | |
| Eastern Canada | 67.3:32.7 | 2.4:97.6 | 21.7:78.3 | ||
| Pinus uncinata subsp. uliginosa (G.E.Neumann ex Wimm.) Businský | Poland | 65.6:34.4 | 11.7:88.3 | 63.6:36.4 | [29] |
| Pinus uncinata Ramond ex DC. | Poland | 58.4:41.6 | 9.1:90.9 | 11.7:88.3 | [29] |
APRC = Data from the commercial essential oil samples from the collection of the Aromatic Plant Research Center (Lehi, Utah, USA). --- = not detected.
The dominant enantiomer for α-pinene and β-pinene in P. flexilis were the (−)-enantiomers. α-Pinene enantiomeric distributions are generally variable in Pinus species, but (−)-β-pinene generally predominates in the genus (see above). The enantiomeric distribution of limonene also seems to be variable in Pinus species (see above), but (−)-limonene was the major enantiomer in P. flexilis. (−)-Camphene was the dominant enantiomer in P. flexilis, which was also found to be the case for Pinus uncinata subsp. uliginosa (G.E.Neumann ex Wimm.) Businský, Pinus uncinata Ramond ex DC., Pinus peuce Griseb., Pinus mugo Turra, Pinus nigra J.F. Arnold, Pinus pinaster Aiton, and Pinus cembra L. [26]. Interestingly, the enantiomeric distribution for camphene in Pinus sylvestris L. is variable depending on geographical source; (−)-camphene dominated in P. sylvestris from Poland and from Korea, whereas (+)-camphene dominated the essential oils from Austria and Italy [26]. (−)-Borneol and (−)-bornyl acetate were the exclusive enantiomers in P. flexilis essential oil, which was also observed in P. contorta subsp. latifolia (Engelm.) Critchf. [11].
3. Materials and Methods
3.1. Plant Material
Fresh plant material of P. ponderosa was collected from three individual mature trees growing near La Pine, Oregon (#1, 43°46′28″ N, 121°32′33″ W, elev. 1288 m; #2, 43°46′24″ N, 121°32′30″ W, elev. 1283 m; #3, 43°45′51″ N, 121°31′47″ W, elev. 1294 m), on 18 May 2021. Pinus contorta subsp. contorta was collected from a mature tree near Ona Beach, Oregon (44°31′16″ N, 124°4′13″ W, 3.0 m elevation) on 6 July 2021. The trees were identified in the field by E. Ankney using the field guide by Turner and Kuhlmann [30] and confirmed by comparison with samples from the C.V. Starr Virtual Herbarium, New York Botanical Garden (http://sweetgum.nybg.org/science/vh/, accessed on 14 January 2022). Leaves of P. flexilis were collected from a mature tree growing on the grounds of the Idaho Botanical Garden (43°36′04″ N, 116°09′35″ W, 862 m elevation) on 29 July 2021. The tree was identified by Daniel Murphy, Collections Curator of the Idaho Botanical Garden. Voucher specimens have been deposited in the University of Alabama in Huntsville herbarium. The fresh leaves (needles) of each tree sample were hydrodistilled for 3 h using a Likens-Nickerson apparatus to give colorless essential oils (Table 6). The essential oils were stored under refrigeration (−20 °C) until analysis. Commercial Pinus essential oil samples from the collection from the Aromatic Plant Research Center (APRC) were analyzed as received.
Table 6.
Collection and hydrodistillation details of Pinus species.
| Tree Sample | Voucher Number | Mass Leaves | Mass Essential Oil |
|---|---|---|---|
| Pinus ponderosa var. ponderosa #1 | EA-50553 | 33.25 g | 106.6 mg |
| Pinus ponderosa var. ponderosa #2 | 33.39 g | 133.2 mg | |
| Pinus ponderosa var. ponderosa #3 | 67.72 g | 313.8 mg | |
| Pinus contorta subsp. contorta | EA-50554 | 15.82 g | 106.7 mg |
| Pinus flexilis | KS-58231 | 115.62 | 315.7 mg |
3.2. Gas Chromatography–Mass Spectrometry
Gas chromatographic–mass spectral (GC-MS) analysis of the Pinus essential oils was carried as previously described [31]: Shimadzu GCMS-QP2010 Ultra, ZB-5ms fused silica capillary column (60 m length, 0.25 mm diameter, 0.25 μm film thickness), He carrier gas, 2.0 mL/min flow rate, injection and ion source temperatures 260 °C; GC oven program 50 °C to 260 °C at 2.0 °C/min; 0.1 μL of a 5% (w/v) sample of essential oil in CH2Cl2 injected, split mode, 24.5:1 split ratio. Retention index (RI) values were calculated using a linear equation by Van den Dool and Kratz [32]. Identification of the essential oil components was carried out by comparison of MS fragmentation and comparison of retention indices (RI) with those available in the databases [15,16,17,18]. Representative gas chromatograms of the Pinus species are shown in supplementary Figure S1.
3.3. Gas Chromatography–Flame Ionization Detection
The GC-FID analysis was carried out as previously described [33]: Shimadzu GC 2010 equipped with flame ionization detector, a split/splitless injector, and Shimadzu autosampler AOC-20i; ZB-5 capillary column (60 m × 0.25 mm i.d.; film thickness 0.25 μm); He carrier gas, 1.0 mL/min flow rate; GC oven program as above for GC-MS; injector and detector temperatures maintained at 260 °C; 0.1 μL of a 5% (w/v) solution in CH2Cl2 injected, split mode, 31:1 split ratio. The percent compositions of the essential oil components were determined from peak areas and standardized using external standards of representative compounds from each compound class (α-pinene, β-pinene, camphene, limonene, menthol, borneol, (E)-β-caryophyllene, eugenol, and methyl chavicol).
3.4. Chiral Gas Chromatography–Mass Spectrometry
Chiral GC-MS of the leaf essential oils was carried out, as reported previously [34]: Shimadzu GCMS-QP2010S, electron impact (EI) mode, electron energy = 70 eV; scan range = 40–400 amu, scan rate = 3.0 scans/s; Restek B-Dex 325 chiral capillary GC column (30 m length × 0.25 mm inside diameter × 0.25 μm film thickness). Oven temperature program: starting temperature = 50 °C, temperature increased 1.5 °C/min to 120 °C, then 2 °C/min to 200 °C, and kept at 200 °C for an additional 5 min; carrier gas was helium, flow rate = 1.8 mL/min. For each essential oil sample, a 3% w/v solution in CH2Cl2 was prepared, and 0.1 μL was injected using a split ratio of 1:45. The enantiomers of the monoterpenoids were identified by comparison of retention times with authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA). The enantiomer percentages were determined from peak areas.
4. Conclusions
The leaf essential oil compositions of P. ponderosa var. ponderosa and P. contorta subsp. contorta from Oregon, USA, have been determined. The enantiomeric distributions of these two Pinus species are reported for the first time. The chemical composition as well as the enantiomeric distribution for P. flexilis from Idaho, USA, are reported for the first time. Both α-pinene and limonene show considerable variation in enantiomeric distribution between and within Pinus species, but (−)-β-pinene is consistently the more dominant enantiomer. This work adds to our knowledge of the essential oil compositions of the genus Pinus. Additional studies on chemical compositions as well as enantiomeric distributions of members of the Pinaceae are underway in our laboratories.
Acknowledgments
We are grateful to Daniel Murphy, Collections Curator of the Idaho Botanical Garden for collection and identification of Pinus flexilis. This work was carried out as part of the activities of the Aromatic Plant Research Center (APRC, https://aromaticplant.org/).
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27175658/s1, Figure S1: Gas chromatograms of Pinus ponderosa var. ponderosa (A), Pinus contorta subsp. contorta (B), and Pinus flexilis (C).
Author Contributions
Conceptualization, W.N.S.; methodology, P.S. and W.N.S.; software, P.S.; validation, P.S. and W.N.S.; formal analysis, P.S. and W.N.S.; investigation, E.A., K.S., P.S. and W.N.S.; data curation, W.N.S.; writing—original draft preparation, W.N.S.; writing—review and editing, E.A., K.S., P.S. and W.N.S. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
All data are available in the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
Sample Availability
Essential oil samples are no longer available.
Funding Statement
This research received no external funding.
Footnotes
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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