J. Mass Spectrom. Soc. Jpn. Vol. 58, No. 6, 2010 Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii Shuichi Y6B6BDID,1῎ Robert E. CDM,2 and Bernd R. T. S>BDC:>I3, 4 1 Department of Environmental Engineering for Symbiosis, Faculty of Engineering, Soka University, Hachioji, Tokyo, JAPAN 2 24 Fancis Street, Blackburn, Victoria, AUSTRALIA 3 Department of Chemistry, Oregon State University, Corvallis, OR 97331, U.S.A. 4 COGER, King Saud University, 11451 Riyadh, SAUDI ARABIA The total extract of resin from Callitris preissii was analyzed by gas chromatography/mass spectrometry and 34 lignans, underivatized and as trimethylsilyl ether derivatives, were identified by interpretation of the mass spectra. The lignans in the resin include 13 new lignans, i.e., four secoisolariciresinol derivatives, four lariciresinol derivatives, 3῏,4῏-methylenedioxy-7῏-hydroxylariciresinol, 3,4-methylenedioxypinoresinol isomer, and two matairesinol derivatives. Furthermore, 17 lignans of the resin are characterized by methylenedioxy substitution on either or both guaiacylpropyl units or the syringylpropyl unit: i.e., five secoisolariciresinol derivatives, three lariciresinol derivatives, 3῏,4῏-methylenedioxy-7῏-hydroxylariciresinol, three pinoresinol deriv- atives, and five matairesinol derivatives, have methylenedioxy structures. Eleven lignans, including two secoisolariciresinol derivatives, four lariciresinol derivatives, two pinoresinol derivatives, and three matairesinol derivatives, contain a syringyl moiety. (Received June 28, 2010; Accepted August 12, 2010) of their mass spectra. Here we report many other and 1. Introduction novel lignans in the resin of C. preissii by GC/MS Lignans are synthesized via the shikimate pathway analysis of total underivatized and silylated extracts. and their carbon skeletons are derived from oxidative We present their mass spectra with interpretations of coupling of two phenyl propane units.1), 2) The lignans the fragmentation patterns of the underivatized com- are widely distributed in the plant kingdom and pounds and of their trimethylsilyl (TMS) derivatives. known for about one thousand species.3) They are of 2. Experimental utility in development of new drugs, e.g., cytotoxins of enzymes, and cancer chemotherapy,2), 3) as well as bio- 2.1 Sample marker tracers for plants in the environment.4), 5) Mass The resin was collected as hardened extrusions on spectrometric data for such biomarker tracers are cru- the bark of a C. preissii (Cypress pine) tree in the Royal cial to advances in environmental sciences. The Botanic Gardens Melbourne, Australia. Pinales, i.e., Araucariaceae, Cupressaceae, Pinaceae, 2.2 Extraction and derivatization of extracts Podocarpaceae, and Taxaceae, are the major source The resin from C. preissii was crushed and sonicated plants of lignans. Many lignans have been character- three times with dichloromethane : methanol (1 : 1; v/ ized especially in the Cupressaceae (9 genera, 29 spe- v) for 15 min. The total extracts were combined and cies) and Pinaceae (6 genera, 31 species).2), 3), 6), 7) Howev- filtered through a glass fiber filter (Whatman GF/A). er, although Callitris preissii (Cypress pine) belongs to The filtrate was first concentrated with a rotary evap- the Cupressaceae, the chemical constituent of Callitris orator and then with a stream filtered nitrogen. An preissii (Cupressaceae) has not been investigated in aliquot (50 mL) of the total extract was converted detail. This is the first survey of the lignan composi- to trimethylsilyl (TMS) derivatives by reaction with tion in resin of C. preissii based on the lignan mass N,O-bis-(trimethylsilyl)trifluoroacetamide and pyridine spectra. for 3 hrs at 80῍. The excess reagent was removed by Although absolute structures of unknown lignans blow-down using a dry nitrogen stream and the tri- cannot easily be determined by NMR analysis in mix- methylsilylated extract dissolved in n-hexane for GC/ tures, gas chromatography/mass spectrometry (GC/ MS. MS) analysis of such mixtures is very useful to deter- 2.3 Gas chromatography/mass spectrometry mine compositions and possible structures of various GC/MS analyses of the underivatized and deriva- lignans by interpretation of the fragmentation patterns tized extracts were carried out using an Agilent model 6890 GC coupled to an Agilent model 5973 quadrupole ῎ Correspondence to: Shuichi Y6B6BDID, Department of MSD. A fused silica capillary column (Agilent DB-5MS, Environmental Engineering for Symbiosis, Faculty of 30 mῌ0.25 mm i.d., film thickness 0.25 mm) was used Engineering, Soka University, 1῍236 Tangi-cho, Hachioji, with helium as the carrier gas. The oven temperature Tokyo 192῍8577, JAPAN, e-mail: [email protected] was programmed as follows: temperature hold at 65῍ ῌ195ῌ S. Yamamoto, R. E. Cox, and B. R. T. Simoneit Fig. 1. GC/MS traces (TIC) of the total extract (a) underivatized, and (b) TMS-derivatives of the extract of Callitris preissii resin. Peak annotation, see Table 1. ῌ196ῌ Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii for 2 min, increase from 65 to 300ῌ at a rate of 6ῌ penoids are not significant components in the resin, minῌ1, and with a final isothermal hold at 300ῌ for 20 because they comprise only 12.7 (4.2῍), whereas the min. The samples were injected in the splitless mode lignans, consisting of thirty-four compounds comprise with the injector temperature at 300ῌ. The mass 268 (88.1῍), are the major resin components. The spectrometer was operated in the electron ionization lignans include the dibenzylbutanediol type, i.e., mode at 70 eV ionization energy and scanned from m/z secoisolariciresinol (1, Table 1) and the derivatives 3,3῎, 50 to 650. GC/MS data were acquired and processed 4,4῎-dimethylenedioxysecoisolariciresinol-9-methyl with the Agilent Chemstation software. ether-9῎-acetate (2), secoisolariciresinol-9-methyl ether- Identifications of compounds were based on compar- 9῎-acetate (3), 3,3῎,4,4῎-dimethylenedioxysecoisolaricire- isons with standards, literature mass spectra, Wiley sinol (4), 3,4-methylenedioxysecoisolariciresinol 275 library data, and interpretation of mass spectro- (5), secoisolariciresinol-9῎-methylether (6), 3,4-meth- metric fragmentation patterns. The relative abun- ylenedioxy-5῎-methoxysecoisolariciresinol (7), and dance of each compound was calculated by using its 3,4-methylenedioxy-5῎-hydroxysecoisolariciresinol (8). peak area in the total ion current (TIC) chromatogram Their total relative abundance is 22.6, which is not of TMS derivatives (Fig. 1b), and the relative abun- a dominant portion of the extract (Table 1). The dance detected in only the underivatized extract TIC tetrahydrofuran type lignans are comprised of the fol- (Fig. 1a) was normalized by the peak area of pinore- lowing classes (Table 1): shonanin (9); lariciresinol sinol which is the maximum compound in both derivatives: lariciresinol (10), its isomer (11), 3῎,4῎-meth- analyses. When two compounds overlapped, each area ylenedioxylariciresinol (12), 3῎,4῎-methylenedioxy- was allocated proportionally by the area of each base 5-hydroxylariciresinol (13), 3῎,4῎-methylenedioxy-5- peak. methoxylariciresinol (14), 5-methoxylariciresinol (15), and 5-methoxylariciresinol isomer (16); and 7῎-hy- 3. Results and Discussion droxylariciresinol derivatives: 7῎-hydroxylariciresinol The GC/MS (TIC) traces of the underivatized and (17), 7῎-hydroxylareciresinol isomer (18), and 3῎,4῎- TMS derivatized total extract from the C. preissii resin methylenedioxy-7῎-hydroxylariciresinol (19). The tetra- are shown in Fig. 1. The lignans identified and their hydrofuran type lignan including lariciresinol is a relative abundances versus the pinoresinol TMS deriv- major component for the TIC area in the resin, amount- ative on the basis of TIC area are listed in Table 1. The ing to 101.1 (33.2῍). The seven pinoresinol derivatives numbers in Table 1 refer to the chemical structures of (Table 1) are furofuran types and comprise: pinoresinol the lignans, which are shown in the plots of the mass (20), epi-pinoresinol (its isomer) (21), sesamin (22), 3,4- spectra (Figs. 2῍6), and the classification of the lignans methylenedioxypinoresinol (23), 3,4-methylenedioxy- based on chemical structure is according to Castro et epi-pinoresinol (an isomer) (24), 5-hydroxypinoresinol al.2) (25), and 5-methoxypinoresinol (26). The furofuran 3.1 Resin composition class including pinoresinol is also a dominant compo- The underivatized analysis of the resin extract in- nent for the TIC area in the resin, comprising a total of cludes twenty lignans, three sesquiterpenoids and four 129.2 (42.4῍). Matairesinol derivatives, which are diterpenoids (Fig. 1a and Table 1). On the other hand, lignanolides, consist of: matairesinol (27), hinokinin twenty-five lignans were identified in the analysis of (28), 3,4-methylenedioxymatairesinol (29), 3῎,4῎-meth- the TMS derivatives of the resin extract besides ylenedioxymatairesinol (30), 3῎,4῎-methylenedioxy-5- vanillic acid (35) and four diterpenoids (Fig. 1b methoxymatairesinol-4-methyl ether (31), 3῎,4῎-meth- and Table 1). The following eight lignans: 3,3῎,4,4῎- ylenedioxy-5-methoxymatairesinol (32), and 5-hydroxy- dimethylenedioxysecoisolariciresinol-9-methyl ether- matairesinol (thujaplicatin) (33). The class is not a 9῎-acetate (2), secoisolariciresinol-9-methyl ether-9῎- major component, with total of 14.2 (4.7῍). Isolaricire- acetate (3), shonanin (9), sesamin (22), 3,4-methyl- sinol (34), which is the cyclolignan type, is a trace