Phytochemistry 71 (2010) 1545–1557 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Irregular sesquiterpenoids from Ligusticum grayi roots Laurence G. Cool a,*, Karl E. Vermillion b, Gary R. Takeoka a, Rosalind Y. Wong a a United States Department of Agriculture, Agricultural Research Service, 800 Buchanan St., Albany, CA 94710, USA b United States Department of Agriculture, Agricultural Research Service, 1815 N. University St., Peoria, IL 61604, USA article info abstract Article history: Root oil of Ligusticum grayi (Apiaceae) contains numerous irregular sesquiterpenoids. In addition to the Received 26 March 2010 known acyclic sesquilavandulol and a sesquilavandulyl aldehyde, two thapsanes, one epithapsane, and 14 Received in revised form 26 May 2010 sesquiterpenoids representing eight hitherto unknown carbon skeletons were found. These skeletons are: Available online 6 July 2010 prethapsane, i.e. 1,1,2,3a,7,7-hexamethyloctahydro-1H-indene; isothapsane, i.e. 1,2,3a,6,7,7a-hexamethyl- octahydro-1H-indene; ligustigrane, i.e. 1,1,2,7,7,7a-hexamethyloctahydro-1H-indene; isoligustigrane, i.e. Keywords: 1,1,2,6,7,7a-hexamethyloctahydro-1H-indene; preisothapsane, i.e. 1,1,2,3a,6,7-hexamethyloctahydro-1H- Ligusticum grayi indene; isoprethapsane, i.e. 1,1,2,4,7,7-hexamethyloctahydro-1H-indene; allothapsane, i.e. 1,1,2,3a,7,7a- Apiaceae hexamethyloctahydro-1H-indene; and oshalagrane, i.e. 1,1,2,4,6,6-hexamethylspiro[4.4]nonane. Gray’s lovage Root The bicyclic sesquiterpenoids are presumably biosynthesized by head-to-head coupling of geranyl Sesquiterpenoids diphosphate and dimethylallyl diphosphate, followed by a cyclization sequence leading to a hydroindane Thapsane skeleton with six one-carbon substituents. Subsequent rearrangements—primarily methyl migrations— Epithapsane account for the remarkable variety of structures represented in L. grayi root oil. Prethapsane Ó 2010 Elsevier Ltd. All rights reserved. Isothapsane Preisothapsane Ligustigrane Isoligustigrane Allothapsane Isoprethapsane Oshalagrane 1. Introduction to the usual ‘‘head-to-tail” 10–4 coupling of GPP with DMAPP or GPP that generates FPP or GGPP. Van Klink et al. (2005) have pre- The plant family Apiaceae has been a rich source of novel natu- sented evidence supporting such a biosynthetic sequence for the ral products. Of particular interest are several terpenoids whose anisotomenes. structures are incompatible with biosynthetic mechanisms involv- ing the usual acyclic substrate molecules farnesyl diphosphate (FPP, leading to most sesquiterpenoids) and geranylgeranyl diphosphate (GGPP, leading to most diterpenoids). Thus, Thapsia villosa L. produces sesquiterpenoids based on the irregular thap- sane carbon skeleton A (Pascual Theresa et al., 1985, 1986a,b) while Anisotome lyallii Hook. f. generates anisotomane diterpenoids (skeleton B; van Klink et al., 1999; Zidorn et al., 2002). Their abso- lute stereochemistry has been proven to be as indicated (Srikrishna and Anebouselvy, 2002, 2003; van Klink et al., 2004). Biosynthesis Ligusticum grayi J.M. Coult. & Rose (oshala, Gray’s lovage or licorice- of these terpenoids apparently involves 10–2 or ‘‘head-to-head” root; Apiaceae) grows in the Sierra Nevada, Klamath, and Cascade coupling of geranyl diphosphate (GPP) with dimethylallyl Mountains of California, Nevada, Oregon and Washington, and in diphosphate (DMAPP) or a second GPP molecule. This is in contrast similar habitats in Idaho, Montana and westernmost Utah. The roots of this species and the more easterly L. porteri (‘‘osha”) were valued by Native Americans for medicinal purposes. Though L. porteri and some Asian and European Ligusticum species have been chem- * Corresponding author. Present address: 2408 McKinley Ave., Berkeley, CA 94703, USA. Tel.: +1 510 549 3972. ically analyzed, composition of L. grayi root essential oil, which E-mail address: [email protected] (L.G. Cool). has a strong, distinctive fragrance, has not been reported. Our 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.06.003 1546 L.G. Cool et al. / Phytochemistry 71 (2010) 1545–1557 analysis of root oil from a Sierran L. grayi population by gas The non-terpenoid hydrocarbon viridene was originally found chromatography–mass spectrometry (GC–MS) found an abundance in roots of two other members of Apiaceae, Meum athamanticum of the non-terpenoid hydrocarbon viridene, as well as numer- Jacq. and L. mutellina (L.) Crantz (Kubeczka et al., 1980, 1982). ous monoterpenes and sesquiterpenoids. Most of the latter The authors noted the striking ‘‘green” odour of viridene, and we appeared to be alcohols, but of these only the irregular acyclic likewise found it to be a major contributor to the fragrance of L. sesquilavandulol could be identified from existing GC–MS data grayi root oil. Kubeczka et al. (1980, 1982) depicted the structure collections. The composition of the root oil, and in particular, of viridene as 1-[(2E)-pent-2-en-1-yl]cyclohexa-1,3-diene, but we the identity of 18 new sesquiterpenoids, is the subject of this consider this to be questionable since no NMR spectroscopic data report. was given, nor did they discuss how they deduced the location or geometry of the double bonds. Our re-examination of the structure of viridene will be published separately. 2. Results and discussion The minor known sesquiterpenes a- and b-barbatene and iso- bazzanene, originally identified from liverworts, have also been re- The composition of the Ligusticum grayi root oil, as determined ported from another member of the Apiaceae, Meum athamanticum by GC–MS and GC-FID, is given in Table 1. Unidentified compounds (König et al., 1996), where they were found to be enantiomeric to are designated by abbreviations consisting of a species and organ the corresponding sesquiterpenes from liverworts. Sufficient b- identifier (LiGrR = L. grayi root), followed by apparent molecular barbatene was isolated from L. grayi root oil to measure its optical mass and a suffix letter (e.g. 222a). rotation (ca. +30°), proving that its absolute stereochemistry is the same as that from M. athamanticum. The acyclic irregular sesquiterpenoid sesquilavandulol, i.e. (4E)-5,9-dimethyl-2-(1-methylethenyl)deca-4,8-dien-1-ol, had 1D Table 1 NMR spectroscopic data identical to that previously reported Composition of Ligusticum grayi root oil from population 2 (38.45459°N, (Faure et al., 2000). The optical rotation of the L. grayi material 119.75941°W). is +7°, but rotation data on material of known absolute stereo- Compound Percent Identification chemistry has not been reported. The depicted chirality is pro- a-Thujene 0.1 GC–MS posed because the dextrorotatory monoterpenoid homologue a-Pinene 0.2 GC–MS (+)-lavandulol has S absolute stereochemistry (Piva, 1995). Also, Sabinene 2.4 GC–MS Thapsia villosa produces thapsanes with 2S absolute stereochemis- b-Pinene 0.4 GC–MS try (Srikrishna and Anebouselvy, 2002, 2003), and it is likely that Myrcene 0.3 GC–MS a-Phellandrene 3.7 GC–MS the new thapsanes and related structures in L. grayi described be- a-Terpinene 0.1 GC–MS low share this stereochemistry. Since thapsane biosynthesis p-Cymene 6.3 GC–MS doubtless derives from a chiral sesquilavandulyl precursor (van b-Phellandrene 1.7 GC–MS Klink et al., 2000), 2S stereochemistry for sesquilavandulol in L. Limonene 0.2 GC–MS c-Terpinene 9.2 GC–MS grayi is inferred. LiGrR132a, -134a 1.4 Compound 1, a minor component, is an achiral aldehyde Terpinolene 1.2 GC–MS with the sesquilavandulane skeleton. The 1H NMR spectrum (Ta- Viridene 13.9 GC–MS ble 2) showed an aldehydic proton (d 10.02), five vinyl methyl Methyl carvacrol 0.4 GC–MS signals between d 1.49 and 1.72, two vinyl methines (d 5.13 a-Barbatene 0.1 GC–MS Isobazzanene 0.1 GC–MS and 5.16), and three allylic methylene signals (d 2.04, 2.13 (+)-Isothapsadiene 4 4.2 NMR, polarimetry and bis-allylic 3.10, two protons each); a total of 24 protons (+)-Thapsadiene 16 0.7 NMR, polarimetry was indicated by these data. The 13C spectrum (Table 4) con- (+)-b-Barbatene 0.3 GC–MS, polarimetry sisted of 15 signals, seven of which were in the olefin region. (+)-b-Acoradiene 0.1 GC–MS, polarimetry Myristicin 0.6 GC–MS One at d 189.3 was clearly the aldehydic carbon, and two others Elemicin 0.4 GC–MS at d 124.8 and 122.6 represented vinyl methines, as confirmed Dihydroagarofuran 0.2 GC–MS by DEPT experiments. Four quaternary vinyl carbons were evi- Dill apiole 0.5 GC–MS dent (singlets between d 131 and 153). Taken together, the 1D (+)-Sesquilavandulol 4.9 GC–MS, NMR, polarimetry NMR spectroscopic data indicated a molecular formula of LiGrR222a 0.2 LiGrR222b 0.1 C15H24O, which was confirmed by HRMS (Section 4.3.1). Assign- 1 13 (+)-a-Isothapsenol 6 4.5 NMR, polarimetry ment of protons to carbons was by H– C HSQC, and the LiGrR222d 0.1 1H–13C HMBC and 1H–1H NOESY spectra completely determined LiGrR222e 0.2 the structure (key correlations in Table 5). A NOESY correlation (+)-a-Preisothapsenol 11 1.5 NMR, polarimetry from H-3 to Me-12 established the E geometry of the central (À)-a-Thapsenol 17 0.4 NMR, polarimetry (À)-a-Prethapsenol 2 15.5 NMR, polarimetry double bond. Sesquilavandulal 1 0.3 NMR Compounds 2–17 proved to be irregular hydroindane sesqui- (+)-b-Isothapsenol 5 0.5 NMR, polarimetry terpenoids that had substitution patterns reminiscent of the thaps- (+)-b-Prethapsenol 3 3.3 NMR, polarimetry anes and anisotomane diterpenoids. Their atom numbering in the LiGrR222j 0.1 (+)-Isoprethapsenol 14 0.2 NMR, polarimetry tables and text follows that given to the thapsanes by Srikrishna LiGrR222l 0.1 et al. (2000), except in Sections 4.3.2–4.3.19 where IUPAC names (+)-Allothapsenol 13 0.4 NMR, polarimetry and numbering are given.