Phytologia (April2012) 94(1) 133 CHEMOSYTEMATICS OF DOUGLAS FIR (PSEUDOTSUGA MENZIESII): EFFECTS OF LEAF DRYING ON ESSENTIAL OIL COMPOSITION Robert P. Adams Biology Department, BaylorUniversity, Box 97388, Waco, TX 76798, USA email [email protected] ABSTRACT A comparison of the essential oil from fresh and air-dried leaves ofDouglas fir {Pseudotsuga menziesi'x) revealed relatively minor changes in the oil composition (on a percent total oil basis), except for terpinen-4-ol, which declined from 12.2% to 7.8 and 8.6% when dried at 2rC and 42°C (Table 1). Citronellyl and geranyl acetates appear to increase with drying and storage for the first week, then are stable. It I seems that careful air drying of Douglas fir leaves can result in the conservation ofthe terpenoid profile in the composition. This appears to be a solution to the problem ofthe transport offresh materials across international borders. Phytologia 94(1):133-138 (April2, 2012). KEY WORDS: Pseudotsuga menziesii, Douglas fir, oils from dried I leaves, chemosystematics. j Recently, Adams (2010, 2011) reported the effects of air- drying leaves on the essential oil composition ofJuniperus pinchotii Sudw. and J. virginiana L. He found that gently air-drying (21-42°C) had generally small effects on the oil compositions in these Juniperus species, similar to the results reported for J. thurifera (Achak et al., 2008, 2009). Our lab is currently involved in a study of Douglas fir {Pseudotsuga menziesii) from throughout its range. The transport of fresh materials from Mexico to our lab has presented considerable difficulties with government customs agents. However, herbarium vouchers are generally (in the author's experience) permitted without 134 Phytologia (April2012) 94(1) too much difficulty. Part ofthe ease ofimporting herbarium specimens is because specimens are frozen to kill insects, then air dried. In order to facilitate this project, a small study was undertaken to evaluate the effects ofleaf-drying onthe essential oils from leaves ofDouglas fir. The purpose ofthis study was to determine ifthe changes in oil composition upon air-drying the leaves ofDouglas fir would preclude theiruse in chemosystematics. MATERIALS AND METHODS Plant material - Pseudotsuga menziesii, Adams 12918, Olympic National Forest, PortAngeles, WA, 48°02' 48.1" N, 123°25' 04.08" W, 525 m. Voucher specimen is deposited in the Herbarium, Baylor University (BAYLU). Fresh (200 g.) and air dried (100 g) leaves were steam distilled for 2 h using a circulatory Clevenger-type apparatus (Adams, 1991). The oil samples were concentrated (diethyl ether trap removed) with nitrogen and the samples stored at -20° C until analyzed. The extracted leaves were oven-dried (48h, 100° C) for the determination of oil yields. The oils were analyzed on a HP5971 MSD mass spectrometer, scan time 1/ sec, directly coupled to a HP 5890 gas chromatograph, using a J & W DB-5, 0.26 mm x 30 m, 0.25 micron coating thickness, fused silica capillary column (see Adams, 2007 for operating details). Identifications were made by library searches ofour volatile oil library (Adams, 2007), using the HP Chemstation library search routines, coupled with retention time data of authentic reference compounds. Quantitation was by FID on anHP 5890 gas chromatograph using aJ & W mm DB-5, 0.26 x 30 m, 0.25 micron coating thickness, fused silica capillary column usingthe HP Chemstation software. RESULTS AND DISCUSSION Table 1 shows the composition ofthe leafoil ofPseudotsuga menziesii, and a comparison ofcomponents, over the 5 month storage period. The compounds are remarkably stable during the drying and Phytologia (April2012) 94(1) 135 storage tests. The major components (a-pinene, sabinene, P-pinene, y- terpinene, terpinolene) show small changes, except for terpinen-4-ol, which declined from 12.2% to 7.8 and 8.6% when dried at 21°C and 42°C, then increased after one and 2 months (Table 1). Citronellyl and geranyl acetates appear to increase with drying and storage for the first week, then they are stable. Comparing the storage tests oils, with var. menziesii (var. menz. Table 1) and var. glauca (var. glauc, Table 1) shows that oils from both fresh and dried leaves ofcoastal Douglas fir (var. menziesii, Olympic Natl. Forest) are clearly in the published ranges of von Rudloff(1973). Three kinds ofvariation seem apparent (Fig. 1): a-pinene, P- pinene and sabinene; y-terpinene and terpinolene; and terpinen-4-ol. The decline of terpinen-4-ol in leaves air-dried (24 h, at 21°C and 42°C), and similar decline in the 5 month sample (Table 1), is not understood and deserves additional study. Douglas Fir: Effects ofdrying on leafessential oil 30 p-pinene ^.-''>. ^ 25 s 20 terpino)ene ^^-^ — ^ " sabinene ; tefpinen-4-ot 10 'V. a-pinene 7.^-*-"^'*-'- Y-terpiriene fresh 1 day 1 day 7day 1 mo 2mo 5mo 42X 2VC 2rc 2rc 2rc 2rc Ivs. Figure 1. Variation in the major terpenoids of Douglas fir with leaf drying. 136 Phytologia (April2012) 94(1) In conclusion, it appears that careful air-drying ofDouglas fir leaves can result in the conservation of the terpenoid profile in the composition. This appears to be a solution to the problem oftransport offreshmaterials across international borders. ACKNOWLEDGEMENTS Thanks to Art Tucker and Billie Turner for reviews. Thanks to Tonya Yanke for lab assistance. This research was supported in part withfunds fromBaylorUniversity. LITERATURE CITED Achak,N., A. Romane, M. Alifriqui andR. P. Adams. 2008. Effect of the leafdryingand geographic sources onthe essential oil composition ofJuniperus thurifera L. var. africana Maire fromthe Tensift-Al Haouz, Marrakech region. J. Essential Oil Res. 20: 200- 204. Achak,N., A. Romane, M. Alifriqui and R. P. Adams. 2009. Chemical studies ofthe leafessential oils ofthree species ofJuniperus from TensiftAl Haouz-Marrakech Region (Morocco). J. Essential Oil Res. 21:337-341. Adams, R. P. 1991. Cedarwood oil - analysis andproperties. In Modem Methods ofPlantAnalysis: Oils and Waxes. Edits., H. F. Linskins andJ. F. Jackson, pp. 159 - 173, Springier-Verlag, Berlin, Germany. Adams, R. P. 2007. Identification ofessential oils by gas chromatography/ mass spectrometry, 4th edition. Allured Publ., Carol Stream, IL, USA. Adams, R. P. 2010. Chemosystematics ofJuniperus: Effects ofleaf dryingon essential oil composition. Phytologia 92(2): 186-198. Adams, R. P. 2011. Chemosystematics Juniperus: Effects ofleaf drying on essential oil composition: II. Phytologia 93(1): 51-62. Von Rudloff, E. 1973. Geographical variation inthe terpene composition ofthe leafoil ofDouglas fir. PureApplied Chem. 34: 401-410. J J f Phytologia (April2012) 94(1) 137 Table 1. Comparison ofleafoil compositions forPseudotsuga menziesiivar. menziesii(Olympic National Forest, WA): fresh leaves, airdried 42°C then distilled, airdried at 21°C, then stored at 21°C for 1 day, 1 month, 2 months and 5 months, var. menz. = coastal type (var. menziesii), von Rudloff(1973), var.glauc=Rocky Mtn. type (var. glaiica), von Rudloff(1973). fresh 21°C 42°C 21°C 21°C 21°C var. var. KT compound Ivs. Z'tn z4n 1 mo 2mo 5 mo menz. glauc santene - J-j 921 tricyclene t t t t t t 2^.A4-4A 924 a-thujene 0.4 A0.6 A0.17 AU.C6 A0.C6 A0.O6 932 a-pinene 0.2 7.6 6. 4A.6O 4A.6O C6.4A T/-IICd 11C5-O2A0 94o camphene rU\.A4 AU.A4 AU.A4 AU.31 AU.J1 Au.A4 AU-UA.02 2OUA-1J(U\ 997o49 spa-bpiinneennee "21t5/CI.A6o 2166..30 O215C3.A.94 2O111111..oT22 O21111..132 215J..2J O2T2-C5-1131<(0\ UAjC.-Il11-A0UA..< 988 myrcene 1.3 1.5 1.5 11.o2 11.o2 11.13 1002 a-phellandrene 0.2 A0."2S A0.31 A0.5C A0.4A A0.T3 1008 5-3-carene 0.5 0.7 0.8 0.5 0.4 0.3 i 1012 1,4-cineole t t t t t t 1014 a-terpinene 3.4 2.1 3.3 4.3 4.2 3.5 2-5 0-0.3 1020 p-cymene 0.1 0.1 0.2 0.3 0.2 0.4 1024 limonene 0.8 0.8 1.0 1.0 1.0 0.9 0.5-1. 5-10 1025 P-phellandrene 1.7 1.8 2.0 2.0 2.0 1.9 1054 y-terpinene 5.8 4.5 5.6 7.4 7.2 5.6 3-8 0.1-1 1065 cis-sabinenehydrate 0.4 0.3 0.4 0.4 0.4 0.4 1086 terpinolene 16.8 15.8 17.3 18.5 18.0 15.6 5-20 0.5-3 1098 trans-sabinenehydrate 0.3 0.2 0.4 0.2 0.2 0.3 1098 linalool 0.2 0.1 0.1 0.3 0.3 0.2 1118 endo-fenchol t t t t t t 111c cis-p-menth-2-en-1-ol AU.1/ AU.JC 0.6 0.6 AU.1/ AU.O 1130 1-terpineol t 0.1 0.1 0.1 0.2 0.1 1136 trans-p-menth-2-en- 0.5 0.4 0.4 0.6 0.7 0.5" l-ol 1145 camphenehydrate t t 0.1 1148 citronellal 0.6 0.5 0.3 0.7 0.8 0.5 1165 bomeol t t t t t t 1174 terpinen-4-ol 12.2 7.8 8.6 10.2 11.4 8.2^ 5-15 0.5-3 1186 a-terpineol 1.9 0.9 1.0 1.1 1.3 0.3 1-3 0.2-1 1195 cis-piperitol t t t 0.2 0.1 t 1207 trans-piperitol 0.2 0.1 0.2 0.2 0.2 0.2 1223 citronellol 0.5 0.4 0.3 0.8 0.5 0.8 1-5 0.1-1 "1287 bomylacetate 0.1 0.1 0.1 0.2 0.2 0.1 0-0.3 20-30 1350 citronellylacetate 1.9 1.9 2.9 2.5 2.6 2.6 2-4 0.1-2 1379 geranylacetate 1.9 1.7 2.3 2.4 2.7 2.1 1-3 0.1-1 138 Phytologia (April2012) 94(1) fresh 21°C 42°C 21°C 2rc 2rc var. var. KI compound Ivs. 24h 24h 1 mo 2mo 5mo menz. glauc 1452 a-humulene 0.1 O.I 0.1 0.3 0.3 0.2 — 1483 a-amorphcne t t t t t 1483 germacreneD t t t t t 1638 epi-a-cadinol t t t t t 1638 epi-a-muurolol t t t t t 1652 a-cadinol 0.1 0.1 t 0.1 0.1 0.2 2300 tricosane(C23) 0.1 0.1 0.1 t 0.1