Phytochemistry 71 (2010) 1714–1728 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Lignans in flowering aerial parts of Linum species – Chemodiversity in the light of systematics and phylogeny Thomas J. Schmidt a,*, Shiva Hemmati b, Michael Klaes a, Belma Konuklugil c, Abdolali Mohagheghzadeh d, Iliana Ionkova e, Elisabeth Fuss f, A. Wilhelm Alfermann b a Institut für Pharmazeutische Biologie und Phytochemie IPBP, Westfälische Wilhelms-Universität Münster, Hittorfstraße 56, D-48149 Münster, Germany Institut für Entwicklungs- und Molekularbiologie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, D-40225 Düsseldorf, Germany Faculty of Pharmacy, University of Ankara, 06100 Tandogan, Ankara, Turkey d Pharmaceutical Sciences Research Center and Department of Pharmacognosy, Shiraz University of Medical Sciences and Health Services, Shiraz, IR, Iran e Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria f Interfakultäres Institut für Biochemie, Eberhard Karls Universität Tübingen, Hoppe-Seyler-Str. 4, D-72076 Tübingen, Germany b c a r t i c l e i n f o Article history: Received 22 March 2010 Received in revised form 22 June 2010 Available online 23 July 2010 Keywords: Linum Linaceae Lignan HPLC–MS Chemosystematics Evolution a b s t r a c t The aerial parts of 54 accessions representing 41 Linum species and four species of related genera were analysed for lignans by means of HPLC-ESI/MS–MS-UV/DAD. In total, 64 different lignans of the aryltetralin-, arylnaphthalene-, aryldihydronaphthalene-, dibenzylbutyrolactone-, and furofuran type were identified. According to their lignan profile, the Linum species can be divided in two groups accumulating as major lignan types either cyclolignans of the aryltetralin-series on one hand, or aryldihydronaphthalenes/ arylnaphthalenes, on the other. Five of the investigated Linum species did not contain any detectable amounts of these lignans under the chosen analytical conditions. Furthermore, none of the lignans identified in Linum species was detectable in representatives of three related genera, namely, Reinwardtia (Linaceae, Linoideae), Hugonia and Indorouchera (Linaceae, Hugonioideae). The two species groups differing in the types of the dominating cyclolignans comprise representatives of the major taxonomic sections. Representatives of sections Syllinum, Cathartolinum and Linopsis accumulate mainly aryltetralins while those of sections Linum and Dasylinum were found to contain mainly aryldihydronaphthalenes/-naphthalenes. These phytochemical data correlate very well with a recent study on the molecular phylogeny of Linum/Linaceae, where a subdivision of Linum into two major clades comprising representatives of the two mentioned groups was found. Thus, the distribution of lignans apparently reflecting phylogenetic interrelations at the infrageneric level, a plausible scenario for the evolution of lignan biosynthesis in the genus Linum can now be presented. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Lignans are a large class of dimeric phenylpropanoids widely distributed in higher plants (Umezawa, 2003a,b). Although based on few simple building blocks, their structures show considerable diversity paralleled by a wide spectrum of biological activities (Ayres and Loike, 1990; Apers et al., 2003; Lee and Xiao, 2003; Saleem et al., 2005). Approximately 3000 different structures of lignans and associated natural products such as neo-, sesqui-, and flavonolignans are listed in the Dictionary of Natural Products. Lignans have attracted considerable attention because of numerous pharmacological activities. Some of them have been developed into clinically approved therapeutics and others are considered lead structures to new drugs (e.g., Apers et al., 2003; Lee and Xiao, 2003; Saleem et al., 2005). * Corresponding author. Tel.: +49 251 83 33378; fax: +49 251 83 38341. E-mail address: thomschm@uni-muenster.de (T.J. Schmidt). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.06.015 The genus Linum (Linaceae) consists of approximately 200 species world wide. It is divided on grounds of classical taxonomy in five (Rogers, 1982) or six sections (Diederichsen and Richards, 2003). The molecular phylogeny of Linum has recently been studied extensively (McDill et al., 2009). Due to the presence of the anticancer agent podophyllotoxin in Linum album (Weiss et al., 1975), there was an increasing interest in investigation of Linum species for lignans. Furthermore, it has been reported that some Linum species accumulate arylnaphthalene lignans such as justicidin B which were found in in vitro cultures of Linum austriacum (Mohagheghzadeh et al., 2002), Linum lewisii, Linum altaicum (Konuklugil et al., 2007), Linum narbonense, Linum leonii (Vasilev et al., 2004) and Linum glaucum (Mohagheghzadeh et al., 2009). Arylnaphthalene glycosides were identified in Linum perenne cultivar ‘‘Himmelszelt” (Hemmati et al., 2007a). Besides arylnaphthalenes, we recently reported on the presence of 70 -aryl-7,8-dihydronaphthalenes in L. perenne. This type of lignans was previously unknown to occur in the genus Linum where it T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 1715 Table 1 Distribution of lignans in the samples under study. Numerical values express approximate peak areas in ion chromatograms of the characteristic base peak for each lignan. Taxa are arranged with respect to their sections within the genus Linum (A: Syllinum; B: Cathartolinum; C: Linopsis; D: Dasylinum; E: Linum) and phylogenetic branches were drawn in accordance with McDill et al. (2009). 1716 Table 1 (continued) T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 1717 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 Table 2 Investigated plant material. Sections: A: Syllinum; B: Cathartolinum; C: Linastrum/Linopsis; D: Dasylinum; E: Linum/Eulinum. Sample number Taxon Sect. Sample origin 1 L. flavum L. Aa,b,g Bot. Garden, Düsseldorf a,b,h h 2 3 4 5 6 Wild location, Bulgaria L. flavum L. ssp. sparsiflorum (Stoj.) Petrova L. elegans Spruner ex Boiss. A Aa,b,h Wild location, Bulgariah L. scabrinerve Davis (=L. flavum ssp. scabrinerve Davis) L. tauricum Willd. (=L. tauricum Willd. ssp. tauricum Petrova) (1) Aa,b Wild location, Turkey A b,h Wild location, Bulgaria h Ab,h Wild location, Bulgariah Ab,h Wild location, Bulgariah b,h h 9 L. tauricum Willd. (=L. tauricum Willd. ssp. tauricum Petrova) (2) L. bulgaricum Podp. (=L. tauricum Willd. ssp. bulgaricum (Podp.) Petrova) L. linearifolium (Lindem.) Jáv. (=L. tauricum Willd. ssp. linearifolium (Lindem.) Petrova) L. dolomiticum Borbás 10 L. campanulatum L. Aa,b,g 11 L. arboreum L. Aa,b,g Greenhouse; seeds from Jelittok Greenhouse; seeds from IPKi: acc. no. LIN 1760/ 98 Wild location, Turkey 12 L. capitatum Kit. ex Schult. var. laxiflorum (Stoj.) Petrova Aa,b,h Wild location, Bulgariah 13 Ab,h Wild location, Bulgariah 14 L. serbicum Podp. (=L. tauricum Willd. ssp. serbicum (Podp.) Petrova) L. nodiflorum L. A 15 L. album Kotschy. ex Boiss. Aa,g Wild location, Iran 16 L. persicum Kotschy. ex Boiss. Aa Wild location, Iran 7 8 A Ab a,b,g c,d,e Wild location, Bulgaria Wild location, Iran 17 L. mucronatum Bertol. A 18 Ac,e Wild location, Turkey 19 L. mucronatum Bertol. ssp. armenum (Bordz.) Davis L. boissieri Aschers. et Sind. Aa Wild location, Turkey 20 L. pamphylicum Boiss. et Held Ad Wild location, Turkey d Wild location, Turkey 21 L. triflorum Davis A 22 L. trigynum L. (1) Cb,g 23 L. trigynum L. (2) Cb,g Wild location, Iran Greenhouse; seeds from IPKi: acc. no. LIN 1554/ 95 Wild location, Turkey a,b,g Wild location, Iran 24 L. corymbulosum Reichenb. (=L. strictum L. ssp. corymbulosum (Reichenb.) Rouy) C 25 L. tenuifolium L. (1) Wild location, Iran 26 L. tenuifolium L. (2) 27 L. suffruticosum L. 28 L. catharticum L. (1) Ea Cb,g Ea Cb,g Ea Cb,g Ba,b,g 29 L. catharticum L. (2) Ba,b,g Wild location, Gotland, Sweden Wild locations Schliersee area (Bavaria), Germany Wild location Ischgl (Tir.), Austria Wild location, Iran a,b,g 30 31 32 33 L. catharticum L. (3), (4), (5) B L. catharticum L. (6) Ba,b,g 34 L. austriacum L. Ea,b,g 35 L. lewisii Pursh (=L. perenne subsp. lewisii) E a,g Bot. Garden, Düsseldorf Bot. Garden, Düsseldorf Wild location, Iran Greenhouse; seeds from Coll. date, persons Voucher number Type of voucher material Voucher location 06/2003 TJS 1969 AP 1968 AP 05/1989 BK Summer 2004 NV 1969 AP 1969 AP 1969 AP TS_Lflav_01 Specimen IPBP, Münster SOM 130 523 Specimen SOM 126 580 Specimen AEF (19566) Specimen FAF 0001 Specimen SOM 126 657 Specimen SOM 126 808 Specimen SOM 130 107 Specimen Bulgarian Academy of Science, Inst. of Botany, Sofia Bulgarian Academy of Science, Inst. of Botany Ankara Univ., Dept. of Pharmacy Dept. of Pharmacognosy, Fac. of Pharmacy, Med. Univ. Sofia Bulgarian Academy of Science, Inst. of Botany, Sofia Bulgarian Academy of Science, Inst. of Botany, Sofia Bulgarian Academy of Science, Inst. of Botany, Sofia TS_Ldol_01 Freeze dried plant mat. Freeze dried plant mat. IPBP, Münster AEF (22946) Specimen FAF 0002 Specimen SOM 126 692 Specimen SUMS-FPH No. 230 SUMS-FPH No. 233 SUMS-FPH No. 229 SUMS-FPH No. 237 AEF (3918) Specimen AEF (19559) Specimen AEF (19561) Specimen AEF (23138) Specimen TS_Ltrig_01 Freeze dried plant mat. Ankara Univ., Dept. of Pharmacy Dept. of Pharmacognosy, Fac. of Pharmacy, Med. Univ. Sofia Bulgarian Academy of Science, Inst. of Botany, Sofia Shiraz Univ. Med. Sci., Fac. Pharm. Shiraz Univ. Med. Sci., Fac. Pharm. Shiraz Univ. Med. Sci., Fac. Pharm. Shiraz Univ. Med. Sci., Fac. Pharm. Ankara Univ., Dept. of Pharmacy Ankara Univ., Dept. of Pharmacy Ankara Univ., Dept. of Pharmacy Ankara Univ., Dept. of Pharmacy IPBP, Münster AEF (25798) Specimen TS_Lcory_01 (HHU-PZK No.1) SUMS-FPH No. 240 TS_Lten_01 Specimen Specimen Shiraz Univ. Med. Sci., Fac. Pharm. IPBP, Münster TS_Lsuf_01 Specimen IPBP, Münster SUMS-FPH No. 234 TS_Lcat_01 Specimen Shiraz Univ. Med. Sci., Fac. Pharm. IPBP, Münster TS_Lcat_02a TS_Lcat_02b TS_Lcat_02c TS_Lcat_03 Specimen Specimen Specimen Specimen IPBP, Münster SUMS-FPH No. 239 TS_Llew_01 Specimen Shiraz Univ. Med. Sci., Fac. Pharm. IPBP, Münster 2006 EF 2006 EF 07/2001 BK Summer 2004 NV 1968 AP 04/2001 AM 05/2003 AM 05/2003 AM 04/2003 AM 06/1973 BK 05/1989 BK 05/1988 BK 06/2003 BK 2006 EF 06/2001 BK 06/1999 AM 08/2001 AM 06/2003 06/2003 TJS 08/2001 AM 07/2004 SE 07/2007 TJS 08/2008 TJS 08/2001 AM 2005 TS_Lcamp_01 Specimen Specimen Specimen Specimen Specimen Specimen Freeze dried plant IPBP, Münster Ankara Univ., Dept. of Pharmacy IPBP, Münster IPBP, Münster (continued on next page) 1718 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 Table 2 (continued) Voucher number Type of voucher material TS_LP_01 mat. Specimen IPBP, Münster TS_LP_02 Specimen IPBP, Münster TS_LPDia_01 crushed aer. parts IPBP, Münster TS_LPHz_01 crushed aer. parts IPBP, Münster TS_Lalp_01 Freeze dried plant mat. IPBP, Münster 2006 EF TS_Lleo_01 Freeze dried plant mat. IPBP, Münster 07/2001 BK 07/2001 BK 2006 EF AEF (22846) Specimen AEF (22951) Specimen TS_Lalt_01 Freeze dried plant mat. Ankara Univ., Dept. of Pharmacy Ankara Univ., Dept. of Pharmacy IPBP, Münster SUMS-FPH No. 330 SUMS-FPH No. 241 TS_LU_01 Specimen SUMS-FPH No. 231 TS_L_mon_01 Specimen Sample number Taxon Sect. Sample origin Coll. date, persons 36 L. perenne L. (1) Ea,b,g ARSj: acc. no. 263511 Bot. Garden, Düsseldorf 37 L. perenne L. (2) Ea,b,g Bot. Garden, Düsseldorf 38 L. perenne L. cv. ‘‘Diamant” E Bot. Garden, Düsseldorf 39 L. perenne cv. ‘‘Himmelszelt” E Bot. Garden, Düsseldorf 40 L. alpinum L. = L. perenne L. ssp. alpinum (Jack.) Ockendon Ea,b 41 L. leonii F.W. Schultz Eb,g 42 L. meletonis Hand.-Mazz. Ea Greenhouse; seeds from IPKi: acc. no. LIN 1905/ 00 Greenhouse; seeds from IPKi: acc. no. LIN 1672/ 92 Wild location, Turkey TJS 06/2003 TJS 06/2003 TJS 09/2004 EF 09/2004 EF 2006 EF 43 L. olympicum Boiss. Ea Wild location, Turkey 44 L. altaicum Ledeb. Ef 45 L. glaucum Boiss. et Noe Ec Greenhouse; seeds from IPKi: acc. no. LIN 1632/ 84 Wild location, Iran 46 L. bungei Boiss. (=L. nervosum Waldst. et Kit. var. bungei (Boiss.) Sharifnia) L. usitatissimum L. Ea Wild location, Iran Ea,b,g Bot. Garden, Düsseldorf 47 b,g 48 L. bienne Mill. = L. angustifolium Huds. E 49 L. monogynum Forst. f. Eg 50 L. decumbens Desf. Aa,b,g 51 L. grandiflorum Desf. Ea,g 52 L. hirsutum L. 53 L. viscosum L. 54 L. stelleroides Planch. Ea Db,g Ea Db,g Ea,g Non-Linum Linaceae 55 Reinwardtia indica Dum. Wild location, Iran Greenhouse; plant from DPl Greenhouse; seeds from IPKi: acc. no. LIN 1754/ 94 Cultivated Univ. Shiraz, Iran Greenhouse; seeds from ARSj: acc. no. 502406-08 Bot. Garden, Düsseldorf Greenhouse; seeds from IPKi: acc. no. LIN 1655/ 84 08/2001 AM 05/2001 AM 06/2003 TJS 05/2003 AM 2006 EF 2006 EF 05/2001 AM 2005 EF 06/2003 TJS 2006 EF TS_Ldec_01 TS_Lvisc_01 Specimen IPBP, Münster TS_Lstel_01 Freeze dried plant mat. IPBP, Münster Crushed aerial parts, powdered material Whole leaves, twigs IPBP, Münster TS_Htom_01 Whole leaves, twigs IPBP, Münster TS_Indgr_01 Whole leaves IPBP, Münster Summer 2006 TB Summer 2006 TB Summer 2004 GI TS_Hser_01 57 H. tomentosa Cav. Wild location, Mauritius 58 Indorouchera griffithiana H. Hallier Wild location, Indonesia IPBP Münster Shiraz Univ. Med. Sci., Fac. Pharm. IPBP, Münster TS_Rind_01 Wild location, Mauritius Freeze dried plant mat. Freeze dried plant mat. Shiraz Univ. Med. Sci., Fac. Pharm. IPBP Münster Specimen 2006 AWA H. serrata Lam. Specimen Shiraz Univ. Med. Sci., Fac. Pharm. Shiraz Univ. Med. Sci., Fac. Pharm. IPBP, Münster SUMS-FPH No. 238 TS_Lhirs_01 Greenhouse, Univ. Tübingen/Düsseldorf 56 Specimen Voucher location IPBP, Münster Collecting persons: TJS: Schmidt, T.J.; EF: Fuss, E.; AM: Mohagheghzadeh, A., Mehregan, I., Soltani, M., Sharifnia, F. (Shiraz); BK: Konuklugil, B., Bahadır, Ö. (Ankara); NV: Vasilev, N.; AP: Petrova, A. (Sofia); SE: Etges, S. (Botanical Garden, Univ. Düsseldorf); AWA: Alfermann, A.W.; TB: Bahorun, T. (University of Mauritius); GI: Indrayanto, G. (Airlangga State University, Dharmawangsa Dalam, Surabaya, Indonesia). a Engler and Harms (1931). b Ockendon and Walters (1968). c Sharifnia and Assadi (2001). d Davis (1967). e Rechinger (1974). f Shishkin (1949). g McDill et al. (2009). h Vasilev et al. (2008). i IPK: Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, D-06466 Gatersleben, Germany. j ARS: US Agricultural Research Service, Iowa State University, Ames, Iowa, USA. k Jelitto Staudensamen GmbH, D-29685 Schwarmstedt, Germany. l Desirable Plants (S.&J. Sutton), Pentamar, Crosspark, Totnes, Devon TQ9 5BQ, UK. 1719 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 OCH3 OR 6 O 7 1 7’ 4 O O 9 O O 9’ H O 1’ 4’ OCH3 H3CO R'' a R H CH3CO C2H5CO C3H7CO C4H9CO C4H9CO C5H11CO β-glucose H CH3CO c hexose H CH3CO c hexose H3CO R’ CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 H H H CH3 CH3 CH3 R’’ OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 H H H a 4a b 4b b 4d b 4g b 4h b 5a b 5b b 5h c 5h’ R H CH3CO C3H7CO C5H11CO glucose H CH3CO c hexose H O OCH3 OR' OR' OR' 1a a 1b b 1c b 1d b 1e b 1f a 1g a 1h a 2a b 2b b 2h b 3a b 3b b 3h H O 5’ 3’ O O O 8’ H H3CO H H 8 O OR OR H R’ CH3 CH3 CH3 CH3 CH3 H H H hexose a 6a b 6h a 7a b 7h R H glucose H glucose R’ CH3 CH3 H H Fig. 1. Structures of aryltetralin lignans detected in Linum species. aIdentical with authentic reference compound. bStereochemistry assigned in analogy with parent compound or known constituent of Linum species. cStructure assigned tentatively; stereochemistry of sugar moiety unknown. has, up to present, only been found in this species (Schmidt et al., 2007). Most Linum taxa reported to contain lignans of the arylnaphthalene type are members of section Linum and it thus appears that arylnaphthalenes are typical for this section. Linum usitatissimum and Linum bienne are somewhat untypical representatives of section Linum that were found to contain dibenzylbutyrolactone- and furofuranolignans, respectively, while none of the aforementioned cyclolignans were hitherto detected in aerial parts of these species (Schmidt et al., 2006, 2008; Mohagheghzadeh et al., 2009). Recently we reported that the aerial parts and roots of several taxa of section Syllinum contain a wide variety of aryltetralin lignans which appear widespread in this section (Vasilev et al., 2008; earlier reports summarized by Westcott and Muir (2003)). Our studies on the diversity of lignans in the genus Linum have now been extended to further species from all the major sections, as well as to some representatives of related genera. This communication presents the chemical data obtained for 54 accessions representing 41 different species. We demonstrate a strong correlation of these chemical data with the systematics and molecular phylogeny of Linum/Linaceae (McDill et al., 2009). Implications with respect to evolutionary events leading to the observed diversity in lignan chemistry within the genus are discussed. 2. Results 2.1. Lignan types and their detection in Linum species Dichloromethane extracts of flowering aerial parts of 41 different Linum species (54 separate accessions, see Tables 1 and 2) were analysed by HPLC-ESI/MS–MS, complemented by HPLC-UV/DAD (Schmidt et al., 2006, 2008; Vasilev et al., 2008). Sixty-four different lignans were detected, whose structures are presented in Fig. 1, 3 and 5. Their HPLC retention data are reported in Table 3. The occurrence of the lignans in each sample is reported in Table 1, where the analysed taxa are ordered according to their assignment to the five sections used by Rogers (1982) and their phylogenetic relations according to the recently published molecular data (McDill et al., 2009). The first group (henceforth termed ‘‘group A”) comprises taxa of sections Syllinum, Linopsis and Cathartolinum. Most representatives of these groups accumulate aryltetralin (AT) lignans as major lignans (structures in Fig. 1). The second group (‘‘group B”), comprising taxa of sections Linum and Dasylinum, contain arylnaphthalene (AN) and aryldihydronaphthalene (ADN) lignans as predominant metabolites (structures in Fig. 3). Some taxa of both groups contain dibenzylbutyrolactone (DBBL) lignans (structures in Fig. 5) in varying amounts along with the mentioned AT and AN/ADN lignans. DBBL lignans are considered biogenetic precursors of cyclolignans of both mentioned types (Suzuki and Umezawa, 2007). Two species so far, namely, L. usitatissimum and its closest relative, L. bienne, were found to contain DBBL and furofuran (FF) type compounds, respectively, as the major lignans (structures in Fig. 3) while cyclolignans were not detected (Schmidt et al., 2006, 2008). FF-type lignans are known to be biogenetic precursors of DBBL and represent the biogenetically most simple type of lignans synthesized via the monolignol pathway (Umezawa, 2003a,b; Suzuki and Umezawa, 2007). Intermediates between furofurans and dibenzylbutyrolactones, such as the dibenzylbutane secoisolariciresinol are accumulated as diglucosides in the seeds of L. usitatissimum (Ford et al., 2001) but were not found during the present study in any of the investigated samples of aerial parts of flowering Linum plants. Six Linum species (Linum viscosum, Linum decumbens, L. glaucum, Linum grandiflorum, Linum monogynum and Linum suffruticosum) did not contain detectable amounts of lignans under the analytical conditions chosen. Furthermore, samples of related Linaceae taxa, namely, Reinwardtia indica (Linaceae, subfamily Linoideae), 1720 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 Fig. 2. HPLC-ESI/MS–MS analysis of L. flavum (sample 1 in Table 1). Depicted are the total ion chromatogram (TIC, obtained in positive ESI mode) as well as various extracted ion chromatograms characteristic for the lignans under study. Hugonia serrata, Hugonia tomentosa and Indorouchera icosandra (Linaceae, subfamily Hugonioideae) were not found to contain any of the lignans detected in the genus Linum. 2.2. Group A: Linum species accumulating mainly aryltetralin lignans Applying the analytical methods previously described (Schmidt et al., 2006; Vasilev et al., 2008), lignans of the AT-type (1a–7f) were now detected in further taxa occurring in Iran, Turkey and Western Europe. The resulting profiles are reported in Table 1. Among the lignans previously reported (Vasilev et al., 2008), an ester of 6-methoxypodophyllotoxin with a hexanoic acid, 1g, was described on grounds of its ESI-mass spectrum alone. The full structure has very recently been elucidated after isolation as 6methoxypodophyllotoxin-7-O-n-hexanoate (Klaes et al., 2010). As a result of the present study, this very lipophilic ester has been detected in the aerial parts of 12 further accessions of different species. It also occurs as a major constituent in the seeds of Linum 1721 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 L. glaucum (Mohagheghzadeh et al., 2009). Compound 8 was also detected in aerial parts and roots of Linum linearifolium, a species very rich in AT-type lignans (Vasilev et al., 2008). Besides AN (8, 9 and 12), we isolated the ADN lignans 13–16 from flowering aerial parts of L. perenne (Schmidt et al., 2007). The diphyllin diglycosides 11i and 11j were moreover reported as constituents in cell cultures of L. perenne cv. ‘‘Himmelszelt” (Hemmati et al., 2007a). Diphyllin (11a) and its glycosides could now also be detected along with 8, 9 and 12 in the flowering aerial parts of this cultivar. However, 11a and its derivatives were present neither in the other investigated representatives of L. perenne nor in any of the other samples containing arylnaphthalenes. On the other hand, ADN lignans (13–16), abundant in the other L. perenne samples and further taxa of this group, were not found in cv. ‘‘Himmelszelt”. Significant amounts of AN- (8–10, 12) accompanied by ADN lignans (13–16) were identified in another cultivar of L. perenne, cv. ‘‘Diamant” as well as in L. austriacum, L. lewisii, L. leonii, Linum alpinum, Linum meletonis, Linum hirsutum and Linum stelleroides. As a representative example, HPLC-ESI/MS–MS analysis of L. perenne (sample 37 in Table 1) is shown in Fig. 4. It appears noteworthy that despite of the justicidin B production by in vitro cultures of L. glaucum (Mohagheghzadeh et al., 2009), neither this compound nor any other lignan could be found in differentiated plants of the same progeny. Two hitherto unreported aryldihydronaphthalenes, 130 and 160 , were detected in several samples and characterised by their mass- and UV spectra (see Supplementary information) as well as retention behaviour (Table 3) as isomers of 7,8-dihydroisojusticidin B (13) and of linoxepin (16), respectively. flavum var. compactum (Klaes et al., 2010) and several other Linum species (Klaes et al., unpublished). Along with this product, two further esters of 6-methoxypodophyllotoxin 1a were detected in five different accessions of Linum catharticum and characterised by their mass-spectral and retention behaviour as isomeric pentanoates (1e and 1f). In addition, two hitherto unreported esters of podophyllotoxin 4a, namely, the butanoate 4d and the hexanoate 4g, were detected in Linum scabrinerve. Full structure elucidation of these isomers must await their isolation. Furthermore, the acetate of 3a (50 -demethoxy-6-methoxypodophyllotoxin), 3b, not hitherto reported as a natural product, was detected in Linum nodiflorum and L. scabrinerve. Aryltetralins with 6,7-dioxygenation (6-methoxypodophyllotoxin-type, 1a–1h, 2a–2h, 3a–3h) represent the most predominant lignans within this group of Linum species. Such compounds occur in all samples that contain significant amounts of AT lignans. 6Methoxypodophyllotoxin 1a and its glucoside 1h usually represent the major constituents. As an example for this group of species, the HPLC-ESI/MS–MS analysis of L. flavum (sample 1 in Table 1) is shown in Fig. 2. 2.3. Group B: Linum species accumulating mainly arylnaphthalene and aryldihydronaphthalene lignans In recent years, arylnaphthalene lignans such as justicidin B (8) were reported from cell cultures of L. austriacum (Mohagheghzadeh et al., 2002) L. austriacum ssp. euxinum, L. lewisii and L. altaicum (Konuklugil et al., 2007), Linum campanulatum (Vasilev and Ionkova, 2005), L. narbonense, L. leonii (Vasilev et al., 2004) and R'' R O O H3CO O R' O O O O O O O 8 9 10 11a 11i 11j R OCH3 H OH OCH3 OCH3 OCH3 R’ H OCH3 H H H H R’’ H H H OH O-apiose-xylose O-hexose-pentose H H H H O 5 H3CO 12 O H3CO O O 3 H3CO O O O O O O O 4 O O 3’ 13 13’: Isomer of 13, most probably 4,5dimethoxylated. 14 O O O O 5’ 4’ O O O O 15 Fig. 3. Structures of arylnaphthalene and aryldihydronaphthalene lignans detected in Linum species. 16 16’: Isomer of 16, possibly 4’,5’-methylenedioxy analog. 1722 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 Fig. 4. HPLC-ESI/MS–MS analysis of L. perenne (sample 37 in Table 1). Depicted are the total ion chromatogram (TIC, obtained in positive ESI mode) as well as various extracted ion chromatograms characteristic for the lignans under study. 2.4. Overlap between groups A and B Trace amounts of AN and ADN-type compounds were detected in several samples containing AT derivatives as dominant lignans (Linum bungei, one of two accessions of Linum tenuifolium, Linum trigynum, Linum corymbulosum, one of five accessions of L. catharticum and L. linearifolium). In L. linearifolium, known to contain a small amount of 8 (Vasilev et al., 2008) along with AT lignans, a low amount of the ADN 13 was now detected as well. On the other hand, AT lignans (1a, 1h and some others) were also detectable in very small quantities in samples characterised by AN/ADN structures as major constituents, namely, L. perenne (2 of 4 accessions), L. austriacum and L. meletonis (see Table 1). The investigated sample of Linum olympicum (a member of section Linum and thus a close relative of L. perenne (Engler and Harms, 1931)) was found to contain traces of 1a while AN/ADN compounds, otherwise abundant in the L. perenne group, were not detectable. 1723 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 R R R OCH3 OCH3 O O OH A B H3CO OCH3 OCH3 OCH3 C D H H ar1 R O O ar1 ar2 O 8 H H 7 ar2 17 18 19 20 21 22 23 24 25 26 H ar1 A B C A B B A C B C O ar2 A A A C B C D C D D 27a 27b 28a 28b 29a 29b 30a 30b 31a 31b ar1 O ar2 ar1 B B B B B B C C C C ar2 B B C C D D C C D D 7,8 conf. ∆ E Z E Z E Z E Z E Z 32 33 34 35 36 O ar1 B B C B C ar2 B C C D D Fig. 5. Structures of dibenzylbutyrolactone- and furofuran type lignans detected in Linum species. 2.5. Species accumulating mainly dibenzylbutyrolactone- and furofuranolignans Schmidt et al. (2006, 2008) reported on lignans in the aerial parts of L. usitatissimum and its closest relative, L. bienne. L. usitatissimum was found to contain ten lignan compounds of the DBBL type and eight 7,8-dehydro-DBBL derivatives. All of them are biosynthetically closely related; hence, several parallel pathways may form a biosynthetic network yielding the same compounds (Schmidt et al., 2008). L. bienne contained exclusively furofuranolignans (33–37) (Schmidt et al., 2006). As a result of the present study, these compounds were detectable exclusively in L. bienne and not in any of the other samples investigated. 2.6. Occurrence of dibenzylbutyrolactone-lignans as minor constituents Several DBBL lignans found in L. usitatissimum were also detected in minor amounts in some other Linum species (see Table 1). Among these, hinokinin 21 and bursehernin 22 were the most widespread, the former being detectable somewhat more frequently in species containing AN/ADN lignans, the latter more often in samples containing mainly AT lignans. Of the 7,8-dehydro-DBBL derivatives occurring in L. usitatissimum (28a–31b), only one E/Z-isomeric pair, 28a/28b, could be detected in another species, L. meletonis, where it occurred together with another pair of such isomers, 27a/27b. These compounds were also found in L. lewisii. They had previously been isolated from L. corymbulosum in vitro cultures (Mohagheghzadeh et al., 2006), but were not detectable in the present accession of differentiated plant material of this species. 3. Discussion 3.1. Biosynthetic pathways to different lignan types in Linum According to literature (Suzuki and Umezawa, 2007), the biosynthetic pathway to cyclolignans of the AT, ADN and AN types (see Scheme 1) proceeds from FF-type lignans such as pinoresinol via secoisolariciresinol to DBBL lignans such as matairesinol (17). Further cyclisation is presumed to lead to the AT skeleton, and, by stepwise aromatization, to ADN and, ultimately, to AN-type lignans. A number of enzymes involved in these biosynthetic steps are known. Lewis and coworkers isolated a ‘‘dirigent protein” from Forsythia which mediates the stereospecific coupling of two molecules of coniferyl alcohol by a laccase to chiral 8R,80 R-pinoresinol (Davin et al., 1997). Dirigent proteins could be isolated from other plant species as well, e.g., from Arabidopsis (Pickel et al., 2010) and are possibly also involved in lignan biosynthesis in Linum (Davin et al., 2008). From pinoresinol, the dibenzylbutyrolactone lignan matairesinol 17 is formed by pinoresinol–lariciresinol reductase (PLR) and by secoisolariciresinol dehydrogenase (SDH) (Davin and Lewis, 2003). The ring closure between C-7 and C-60 of matairesinol or similar DBBL lignans catalysed by yet unknown enzymes leads to cyclolignans, finally resulting in formation of podophyllotoxin/6methoxypodophyllotoxin derivatives (in L. album) or justicidin B 8 (L. perenne cv. ‘‘Himmelszelt”), respectively. Enzymes involved in hydroxylation of justicidin B to diphyllin 11a and of deoxypodophyllotoxin to b-peltatin and methylation to peltatin A methylether could be identified (Molog et al., 2001; Kranz and Petersen, 2003; Hemmati et al., 2007b). Together with previous investigations, our data presented here show that representatives of all major lignan types occurring along this pathway indeed accumulate in Linum species. None of the investigated extracts obtained from flowering green aerial parts, however, was found to contain secoisolariciresinol derivatives. This is in contrast with existing reports about Linum seeds, which are known to accumulate such compounds in glycosylated form as major lignans (Ford et al., 2001; further literature cited in Westcott and Muir (2003)). It is worth mentioning at this point, that recent studies of our group have shown that the seeds of a variety of Linum species belonging to group A also accumulate AT lignans (Klaes et al., 2010; Klaes et al., unpublished) while such of group B contain AN and ADN lignans. Interesting correlations were found between the occurrence of such less polar cyclolignans and the 1724 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 Table 3 HPLC retention data of lignans detected in Linum species in the methanol- and acetonitrile–water gradients described in the experimental section. All data are expressed as relative retention times in relation to 6-methoxypodophyllotoxin (1a). Compound rRt (MeOH) rRt (ACN) 1a 1b 1c 1d 1e 1f 1g 1h 2a 2b 2h 3a 3b 3h 4a 4b 4d 4g 4h 5a 5b 5h 5h0 6a 6h 7a 7h 8 9 10 11a 11i 11j 12 13 130 14 15 16 160 17 18 19 20 21 22 23 24 25 26 27a 27b 28a 28b 29a 29b 30a 30b 31a 31b 32 33 34 35 36 1.000 1.154 1.276 1.387 1.465 1.481 1.531 0.860 0.844 1.044 0.662 0.966 1.161 0.794 0.882 1.079 1.368 1.447 0.750 0.714 0.922 0.618 0.581 0.899 0.721 0.741 0.573 1.195 1.143 1.016 1.147 1.044 0.908 1.326 1.090 n.d. 1.133 1.249 1.044 0.986 0.685 0.934 0.837 0.839 1.223 1.104 0.894 0.991 1.085 0.963 1.272 1.363 1.168 1.303 1.161 1.279 1.068 1.173 1.065 1.178 1.328 1.207 1.081 1.199 1.079 1.000 1.443 1.672 1.880 2.050 2.073 2.315 0.451 0.683 1.058 0.268 0.963 1.302 0.440 0.753 1.295 n.d n.d 0.392 0.563 0.949 0.226 0.169 0.846 0.318 0.531 0.177 1.440 1.531 n.d. 1.106 0.729 0.488 1.638 1.506 1.307 1.569 1.512 1.218 1.165 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. presence of secoisolariciresinol diglucoside (Klaes et al., unpublished). A full account of these studies will be reported separately. Quite conspicuously, the AT lignans found in Linum all show a methylene dioxy substitution at the tetrahydronaphthalene ring system whereas all AN/ADN-type compounds reported have a methylene dioxy group at the pendant benzene ring (compare structures 1a–7h with 8–16). In the AT-type compounds, this latter ring is exclusively decorated with methoxy or hydroxy groups, whereas in the AN/ADN-group, methoxy substitution occurs only at the naphthalene/dihydronaphthalene core. This fact provokes the hypothesis that the AT compounds found in Linum are not direct biosynthetic precursors of the AN-lignans as might be conceived (Suzuki and Umezawa, 2007). We would rather propose that the two types of cyclolignans observed in Linum represent distinct end points of separate branches of lignan biosynthesis. Interestingly, the types of aromatic substitution observed in the DBBL lignans of L. usitatissimum (17–26, 28a–31b) and in the biogenetically even simpler FF lignans of L. bienne (32–36) are closely related and comprise compounds showing the ‘‘decoration pattern” found in both types of cyclolignans. The same or very similar enzymes responsible for the aromatic substitution pattern observed in the AT and AN/ADN lignans hence appear to be fully active also in these two cases forming cyclolignans only at a negligibly low level. 3.2. Evolution of the biosynthetic pathways for lignans in Linum Table 1 clearly shows that the distribution of the two mentioned classes of cyclolignans, especially their occurrence as dominant end points of lignan biosynthesis, correlates strongly with the taxonomic segregation of Linum sections. Representatives of sections Syllinum, Cathartolinum and, to a lesser extent, also of section Linopsis, accumulate AT lignans while among the species belonging to sections Linum and Dasylinum AN/ADN-type lignans are dominant. A recent study on the molecular phylogeny of Linum (McDill et al., 2009), has shown that these two groups (yellow- and blue-flowering clades, respectively) were indeed the first to be separated during evolution of the genus, forming two major clades, presumably about 60 million years ago (mya). Since both types of cyclolignans are occasionally observed along with each other in both clades, it may be hypothesized that the genes for both branches of cyclolignan biosynthesis are present throughout the genus and should hence also have been present in the last common ancestor. This assumption, however, could not be confirmed by our studies. Representatives of three closely related genera, namely, Reinwardtia, Hugonia and Indorouchera were not found to contain any of the lignans present in Linum. It must be mentioned however, that one single report exists on low amounts of podophyllotoxin-type AT lignans and pinoresinol in H. tomentosa (Konuklugil, 1996). Although we found neither leaves nor twigs of this species to contain such lignans, it is possible that the biosynthetic machinery to form them is present but not always expressed at a detectable level. A strong dependence of lignan accumulation on developmental stage and plant organ studied has recently been found in L. usitatissimum (Hemmati et al., 2010). The finding that, e.g., L. glaucum when cultured in vitro produces justicidin B (Mohagheghzadeh et al., 2009) while no lignans were detectable in the aerial parts collected at a natural habitat (present study) points in the same direction. Thus, lignans might indeed be formed by the mentioned non-Linum species under certain conditions and it is also conceivable that lignan biosynthesis was expressed, possibly at low levels, in the last common ancestors of Linum and the mentioned Linoid taxa. Further investigations on Hugonia and other non-Linum Linoideae are necessary in order to clarify the ancestral state of lignan pattern in Linum. It appears clear, however, from the results presented here, that the evolutionary events leading to accumulation of high amounts of AT- or AN/ADN lignans as major metabolites in the two groups of species occurred after the segregation of the genus into two separate lines (yellow- and blue-flowering clades, according to McDill et al. (2009)). The common ancestor the yellow-flowering group 1725 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 H3CO OH 2x Laccase (EC 1.10.3.2) + Dirigent protein (Davin et al., 1997) HO Coniferyl alcohol OH OH H CH3O O OCH3 OH H H CH3O O HO 8R,8'R-(+)-pinoresinol HO H Pinoresinol-lariciresinolreductase (PLR; EC 1.1.1.-; e.g. Hemmati et al., 2010)* (-)-8R,8'R-secoisolariciresinol9,9'-diglucoside*, constituent in seeds of L. perenne and related species (Westcott and Muir, 2003; Klaes et al., unpublished). OCH3 OH 8R,8'R-(−)-secoisolariciresinol* Secoisolariciresinoldehydrogenase (SDH; EC 1.1.-; e.g. Youn et al., 2005) H Furofuranolignans as isolated from L. bienne (Schmidt et al., 2006; compounds 32-36, see Fig. 5) CH3O Further dibenzylbutyrolactone lignans as isolated from L. usitatissimum (Schmidt et al., 2006, 2008; compounds 18-31b, see Fig. 5). O HO H O Enzymes unknown OCH3 OH 8R,8'R-(−)-Matairesinol (17) Arylnaphthalene and aryldihydronaphthalene lactones (compounds 8 - 16', see Fig. 3). Aryltetralin lactones (compounds 1a-7h, see Fig. 1) Scheme 1. Biogenetic relationships of the lignans found in aerial flowering parts of Linum species. *Recently, Hemmati and coworkers (2010) demonstrated that in L. usitatissimum two PLR-enzymes of opposite enantiospecificity (PLR-Lu1 and PLR-Lu2, respectively) are expressed differentially. The former utilizes 8S,80 S-, the latter 8R,80 Rpinoresinol. Thus, the seeds of this species accumulate 8S, 80 S-(+)-secoisolariciresinol diglucoside, formed from 8S,80 S-( )-pinoresinol as major constituent while all lignans found in the aerial green parts possess the 8R,80 R-configuration. (See above-mentioned references for further information.) most probably specialised on the formation of AT lignans in high amounts, so that these lignans became dominant throughout the clade. It is worth mentioning at this point that AT lignans such as podophyllotoxin 4a are potent antimitotic agents (e.g., Ayres and Loike, 1990; Apers et al., 2003; Lee and Xiao, 2003; Saleem et al., 2005) and thus of ecological value for the plants as a chemical defence mechanism. The question whether specialisation on formation of these toxic agents occurred immediately after separation from the other major group (blue-flowering clade) and before the next segregations, when the ancestors of American and South African Linopsis, and of Radiola linoides were separated (see McDill et al., 2009), must be answered in further studies, since none of these plants could be obtained so far in sufficient amounts for analysis. In any case, the specialisation on AT lignans certainly occurred before the emergence of section Cathartolinum (i.e., the ancestor of present day L. catharticum about 50 mya). L. catharticum produces AT lignans in very high amounts, with a qualitative and quantitative pattern very similar to the analysed representatives of the Syllinum group. Quite noteworthy, the lignan pattern found in L. catharticum was found essentially unchanged in five different populations from three geographically very remote origins (Gotland, Bavarian/Austrian Alps, Iran). The collection sites in Sweden and Iran cover almost the maximum distance within the range of distribution of L. catharticum (McDill et al., 2009), so that lignan formation in L. catharticum has obviously remained stable over a very long period of time. Finding almost the same lignans in representatives of Syllinum, such as L. album, L. flavum, and many others, it appears safe to conclude that production of AT lignans must have represented the ancestral state in the Syllinum/Linopsis/Cathartolinum clade. Compounds of the AN/ADN type are also likely to represent components of the plants’ chemical defence system. Justicidin B, found as major constituent in most of the blue clade Linum species, possesses, e.g., antifungal, antiprotozoal, cytotoxic and toxic activity to crustacean arthropods (Hui et al., 1986; Gertsch et al., 2003). The common ancestors of the blue-flowering Linum clade should in a similar way have specialised on the production of such lignans as stated above for AT in the yellow-flowering group. This specialisation is likely to have occurred before the next dichotomy within the blue clade, when the ancestors of L. stelleroides and those of section Dasylinum were separated from those of section Linum, about 45 mya. The presence of AN and ADN lignans in L. stelleroides and in L. hirsutum indicates that accumulation of these lignans represented the ancestral state in the blue clade. Within section Linum, the last common ancestor of the L. usitatissimum/ L. bienne group and the L. perenne group most probably had retained that trait. After segregation of these two groups (estimated about 35 mya), it was further retained up to present time by the latter, while it was lost within the former, whose extant 1726 T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 representatives do not form significant amounts of cyclolignans. Although we could detect no lignans in L. glaucum, a member of the L. usitatissimum group, recent results on cell and plantlet cultures of this species have shown the accumulation of justicidin B (Mohagheghzadeh et al., 2009), so that the ability to form such lignans under certain conditions may have been retained at least in parts of the group. The lignan pattern observed in L. usitatissimum, which only produces DBBL lignans (however, in considerable amounts), might be explained by the loss of a cyclase enzyme converting DBBL lignans to cyclolignans. More recently, after the separation of L. usitatissimum from L. bienne, the latter must even have abandoned the accumulation of DBBL compounds at significant levels since it contains only FF-type compounds in high concentration. This change may possibly be explained by a decrease in pinoresinol–lariciresinol reductase (PLR, Hemmati et al., 2010) activity. The capability to synthesize trace amounts of AT lignans apparently was not completely lost in L. bienne, as well as in some representatives of the L. perenne group (here along with large amounts of AN/ADN lignans), which supports our hypothesis that there are two partly independent pathways in Linum leading to cyclolignans. This scenario is also supported by the occurrence of small amounts of naphthalenoid lignans in some of the representatives of the yellow-flowering clade. L. bienne (=Linum angustifolium) is commonly considered ancestral to cultivated flax, L. usitatissimum (McDill et al., 2009). In the proposed scenario of successive loss of biosynthetic steps in the pathway to more complex lignans, however, L. usitatissimum would appear less advanced with respect to this trait of secondary metabolism than L. bienne. On this background and taking into account the hypothesis, that L. usitatissimum descended by a single domestication event from L. bienne (Allaby et al., 2005; Fu and Allaby, 2010) it will be of high interest to analyse the lignans in a larger number of samples of both species, best complemented by investigations on PLR expression, in order to shed light on this difference between the two closely related species whose separation presumably occurred only a few thousand years ago. 4. Conclusions In conclusion, as the major result of the present investigations, a picture of lignan accumulation in Linum has emerged which is much more detailed than could be obtained on grounds of previous phytochemical studies. Strong parallels of these patterns with the molecular phylogeny of Linum (McDill et al., 2009) were observed so that a plausible scenario of the progress of evolution of lignan patterns in the genus Linum could be deduced. The results of this study indicate that the capability to form complex cyclolignans of both, the aryltetralin- and arylnaphthalene types was probably inherited by Linum from a common ancestor and that a specialisation on the predominant accumulation of either type occurred early in the phylogeny of the two major clades of Linum species. It will be of high interest to continue this part of our investigations by extending the analyses to the hitherto unstudied South American and African representatives of Linum and to further related genera, which may then yield even more detailed aspects on the evolution of lignan biosynthesis in the Linaceae and their relatives within Malpighiales. 5. Experimental 5.1. General experimental procedures HPLC-ESI/MS–MS analysis was performed with a Finnigan LCQ Deca XP mass spectrometer (Thermo Finnigan, Dreieich, Germany) coupled to an Agilent (Agilent, Waldbronn, Germany) 1100 series HPLC system as reported previously (Schmidt et al., 2006, 2008; Vasilev et al., 2008). Briefly, separations were achieved with a Knauer (Berlin, Germany) Eurosphere RP C18 column (250  2 mm i.d., 5 lm) using acetonitrile:water and/or methanol:water (the latter containing 0.1% formic acid in each case) for elution in a gradient from 3:7 to 7:3 in 30 min, followed by isocratic elution with 7:3 between 30 and 40 min, a further increase from 7:3 to 100:0 between 40 and 55 min, and finally isocratic elution with acetonitrile from 55 to 65 min. The flow rate was 0.4 ml/min throughout. The following ESI-MS traces were recorded: (1) positive ions from m/z 100 to 1000, (2) wideband MS/MS of the most intense ion from (1), (3) negative ions from m/z 100 to 1000, and (4) wideband MS/MS of the most intense ion from (3). The four different modes were cycled through every second. For the MS/MS spectra, the normalized collision energy was set at 35% according to the manufacturer’s specifications. The capillary temperature was set at 300 °C, and the source voltage was 5 kV. Simultaneously, UV absorbance was monitored between 210 and 400 nm with the DAD and chromatogram traces extracted at k = 220, 254 and 280 nm. Mass spectrometric identification of most lignans reported here was described previously (Schmidt et al., 2006, 2008; Vasilev et al., 2008; Klaes et al., 2010). The numerical data reported in Table 1 are to serve as estimates for comparison of the relative amounts of each particular lignan between the various samples and do not express exact quantitative measures. Data represent peak area units (counts  s  10 9) obtained by integration of single ion chromatograms of the base ions characteristic for each compound (Schmidt et al., 2006, 2008; Vasilev et al., 2008). For the two major lignans, 6-methoxypodophyllotoxin 1a and justicidin B 8, 27 and 16 area units, respectively, corresponded to a concentration of 0.1 mg/ml under the analytical conditions chosen. 5.2. Plant material The origin of the investigated plant material, their collection dates and voucher locations are reported in Table 2. In short, aerial parts of each accession were typically collected at the flowering stage (usually with flowers and capsules), in few instances (only in case of cultivated plants of specified origin), plants had to be harvested without flowers. The material was dried at max. 40 °C and stored at ambient temperature until extraction. In case of the Bulgarian accessions, voucher material was used (Vasilev et al., 2008). 5.3. Sample preparation Dried and powdered plant material was extracted exhaustively in a Soxhlet apparatus with dichloromethane. In cases where only very small amounts of sample material were available, extraction was performed by repeated maceration with dichloromethane (3  50 ml) in Erlenmeyer flasks under reflux and stirring. The extract was evaporated under reduced pressure and an aliquot of the residue was reconstituted in methanol to a concentration corresponding to 1 g dried plant material/ml. The non-soluble part after sonication was removed by centrifugation at 2500 g for 10 min. The supernatant was used directly for HPLC analysis. A volume of 10 ll was generally injected. T.J. Schmidt et al. / Phytochemistry 71 (2010) 1714–1728 5.4. Analytical data 5.4.1. Relative retention times (rRt) Relative retention times (rRt) of all compounds with respect to 6-methoxypodophyllotoxin (1a) are reported in Table 3 for MeOH and acetonitrile gradients, respectively. 5.4.2. ESI-MS data of lignans not previously reported as constituents of Linum species 1e: ESI-MS (positive ion mode): m/z (%) 551 [M+Na]+ (97); 546 [M+NH4]+ (12); 449 [M+Na C4H9COOH]+ (13); 427 + [M+H C5H10O2] (100); 343 [A+H]+ (8); 259 [B+H C4H9COOH]+ (5); MS/MS of m/z 551: 551 (30); 449 [M+Na C4H9COOH]+ (100); MS/MS of m/z 427: 427 (46); 409 [427 H2O]+ (28); 399 [427 CO]+ (9); 385 [427 O@C@CH2]+ (25); 381 [427 H2O CO]+ (67); 343 [A+H]+ (100); 312 (4); 275 [10] (10); 259 [B+H C4H9COOH]+ (5). 1f: ESI-MS (positive ion mode): m/z (%) 551 [M+Na]+ (87); 546 [M+NH4]+ (9); 449 [M+Na C4H9COOH]+ (10); 427 + [M+H C4H9COOH] (100); 343 [A+H]+ (12); 259 [B+H C5H10O2]+ (6); MS/MS of m/z 551: 551 (2); 449 [M+Na C4H9COOH]+ (100); MS/MS of m/z 427: 427 (36); 409 [427 H2O]+ (37); 399 [427 CO]+ (8); 385 [427 O@C@CH2]+ (29); 381 [427 H2O CO]+ (59); 343 [A+H]+ (100); 312 (12); 275 (6); 259 [B+H C4H9COOH]+ (3). 3b: ESI-MS (positive ion mode); m/z (%): 479 [M+Na]+ (87); 419 [M+Na CH3COOH]+ (35); 397 [M+H CH3COOH]+ (100); 313 [A+H]+ (18); 259 [B+H CH3COOH]+ (20); 215 [259 CO2]+ (15); MS–MS of m/z 397: 397 (32); 379 [397 H2O]+ (36); 351 [397 CO H2O]+ (64); 313 [A+H]+ (100), 259 [B+H CH3COOH]+ (14); 245 (9); 215 [259 CO2] (4); 195 (3); 165 (5). 4d: ESI-MS (positive ion mode); m/z (%): 507 [M+Na]+ (30); 397 [M+Na C3H7COOH]+ (100); 313 [A+H]+ (11); 229 [B+H C3H7COOH]+ (6); MS–MS of m/z 397: 397 (50); 379 [397 H2O]+ (22); 351 [397 CO H2O]+ (67); 313 [A+H]+ (100); 229 [B+H C3H7COOH]+ (10); 195 (3). 4g: ESI-MS (positive ion mode); m/z (%): 535 [M+Na]+ (46); 397 (100); 313 [A+H]+ (8); 229 [M+Na C5H11COOH]+ [B+H C6H11COOH]+ (8); MS–MS of m/z 397: 397 (67); 379 [397 H2O]+ (27); 351 [397 CO H2O]+ (77); 313 [A+H]+ (100); 229 [B+H C5H11COOH]+ (7); 195 (7). 5h0 (isomer of compound 5h (Vasilev et al., 2008)): ESI-MS (positive ion mode); m/z (%): 585 [M+Na]+ (90); 580 [M+NH4]+ (64); 563 [M+H]+ (14); 463 (26); 401 [M+H C6H10O5]+ (12); 383 [M+H C6H12O6]+ (100); 299 [A+H]+ (8); 267 [A+H CH3OH]+ (12); 229 [B+H C6H12O6]+ (16); MS–MS of m/z 383: 383 (58); 365 [383 H2O]+ (27); 337 [383 2H2O]+ (52); 299 [A+H]+ (100); 267 [A+H CH3OH]+ (24); 229 [B+H C6H12O6]+ (8); 181 (5). 130 (isomer of compound 13 (Schmidt et al., 2007)): ESI-MS (positive ion mode); m/z (%): 755 [2M+Na]+ (16); 367 [M+H]+ (100); MS–MS of 367: 367 (100); 319 (14); 291 (7); 177 (26). 160 (isomer of linoxepin 16 (Schmidt et al., 2007)): ESI-MS (positive ion mode); m/z (%): 751 [2M+Na]+ (25); 387 [M+Na]+ (11); 365 [M+H]+ (100); MS–MS of 365: 365 (100); 335 (25); 289 (18); 259 (18); 231 (5). For a definition of A and B fragments and further details on the fragmentation pathways see Schmidt et al. (2006) and Vasilev et al. (2008). Mass and UV spectra of all compounds are available as Supplementary information. Acknowledgements Donation of seed material of several Linum accessions by US Agricultural Research Service (ARS) and by Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Gatersleben, Germany, 1727 is most gratefully acknowledged. Furthermore, the authors cordially thank Profs. Theeshan Bahorun (Mauritius), Shuming Li (Marburg), and G. Indrayanto (Indonesia) for providing plant material of the investigated non-Linum taxa. We are indebted to Drs. R. Edrada-Ebel, R. Ebel, and J. Sendker (Düsseldorf) for their invaluable help in performing HPLC–MSanalyses. The help of M. Repplinger (Mainz) in the identification of some accessions as well as valuable discussions are gratefully acknowledged. 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