J Wood Sci (2002) 48:440-445 The Japan Wood Research Society 2002 Keigo Mikame Norikazu Sakakibara Toshiaki Umezawa Mikio Shimada Lignans of Linum flavum var. compactum Received: July 11, 2001 / Accepted: November 14, 2001 Abstract A new dibenzylbutyrolactone lignan 7,6'- plants and its biotechnological production) Several dihydroxybursehernin, together with six known lignans woody plants (e.g., Juniperus sabina, Thujopsis dolabrata, (pinoresinol, lariciresinol, secoisolariciresinol, a-peltatin, Callitrus drummondii, Hernandia ovigera) produce podo- fi-peltatin, 5-methoxypodophyllotoxin) were isolated from phyllotoxin, its congeners, or both. 1 Hence, production the methanol extracts of Linum flavum vat. compactum. of the antitumor lignan by woody plants is a challenging The enantiomeric analysis of pinoresinol and lariciresinol subject in the field of wood chemistry and biochemistry. As isolated from the species, which are upstream lignans in the the first step to accessing biotechnological production it lignan biosynthetic pathway, indicated that they are not is necessary to understand the biosynthetic mechanisms, optically pure, which is in accordance with our recent but much remains unknown about the biosynthesis of this findings on lignans occurring in other plant species. lignan. 4 In addition to these woody plants and Podophyllum Key words Lignan 7,6'-Dihydroxybursehernin Linum spp., some herbaceous plants 1 such as Anthriscus sylvestris5 flavum vat. compactum Linaceae Biosynthesis and Linum spp. (especially those belonging to the section Syllinum including Linum flavum and Linum album) 6-1~ have been known to produce podophyllotoxin and related lignans. In addition, suspension, root, and hairy root cul- Introduction tures of Linum species producing significant amounts of 5-methoxypodophyllotoxin (Fig. 1) and related lignans An aryltetralin lignan, podophyllotoxin (Fig. 1), has been have been established, s'11-14 Thus, Linum plants are attrac- isolated from herbaceous perennial Podophyllum species tive as plant systems for elucidating the biosynthetic mecha- and has long been reputed to have antitumor activity.~ 4 The nisms of podophyllotoxin congeners, and the detailed lignan is exploited commercially as a source of a semisyn- knowledge of the mechanisms can be applied to biotechno- thetic anticancer drug, etoposide, which is being applied logical production of the lignans in woody plants as well successfully as cancer chemotherapy. 1-3 Because of the as Linurn spp. As for A. sylvestris, this species exhibits limited supply of Podophyllum emodi, much attention has good growth behavior and contains large amounts of been focused on the availability of the lignan in various desoxypodophyllotoxin (deoxypodophyllotoxin, anthricin) and yatein (Fig. 1), which were reported to be precursors of podophyllotoxin. 15'16 Thus, the application of this species to the studies of podophyllotoxin biosynthesis is also of interest; we have reported the lignan analysis in A. sylvestris and formation of lignans by an enzyme preparation of the K. Mikame ~ N. Sakakibara - T. Umezawa ([]) . M. Shimada plant] Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Recently, enzymatic reactions of upstream steps in the Japan Tel. +81-774-38-3625; Fax +81-774-38-3682 lignan biosynthetic pathway in L. flavum have been re- e-mail: [email protected] ported: conversion of pinoresinol to 7'-hydroxymatairesinol (Fig. 1) via lariciresinol, secoisolariciresinol, and mataire- Present address sinol. 17 However, the enantiomeric compositions of the 1Faculty of Bioresources, Mie University, Mie 514-8507, Japan upstream lignans and other possible precursors of aryltetralin lignans occurring in this Linum species remains Parts of this report were presented at the 42nd Lignin Symposium, Sapporo, October 1997; and the 43rd Lignin Symposium, Fuchu, unknown. Herein we report isolation of a new lignan, 7,6'- October 1998 dihydroxybursehernin, and six known lignans (Fig. 1) from 441 Fig. l. Structures of lignans A ~.~oH ,.~x./OH It OH CH30 OH OH isolated from Linum flavum var. o , H c.3o.~- A-~-oa ~o ~o compactum (A) and related lignans (B) (y-o. 222 o :o OCH3 OCH3 OH OCH 3 OCH 3 OR (+)-Pinoreslnol (+)-Lariciresinol (-)-Secoiso- 7,6'-Dihydroxyburse- 5-Methoxypodo- R-H: ~x-Peltati. lariciresinol hernin (TR*, 8S ~, 8'R*) phyllotoxin R-CH3: ffPeltatin B _OH o : o .3ooR @oO Mataireszrtol ..,~1t ~3 j~ J~ ~1 Epipodorhizol R=oH: n "] CH30 OCH 3 Podophyllo- Clt30""~'OCH 3 RI=OH , R2=H: 7'-Hydroxy-. "~'OCH3 CH30" y "OCtt 3 OCH3 toxin OC}I3 Podorhizol matairesinol OH OCH3 Yatein L. flavum var. compactum and the stereochemical charac- Extraction and isolation terization of the upstream lignans. Freeze-dried aerial parts (6.3g) of L. flavum var. compactum were pulverized and extracted six times with Experimental hot MeOH (total 260ml). To the suspension of the MeOH extracts (1.3g) in 100ml of 0.1M NaOAc buffer (pH 5.0) Plant material were added 500 units of fi-glucosidase (Sigma G-0395, 5.0U/rag, from almond). Following incubation at 37~ for Seeds of Linum flavum var. compactum (Harris Seeds, 24h, the reaction mixture was extracted with CH2C12. An Rochester, NY, USA) were sowed in a gardening soil and aliquot (381mg) of the CH2C12 extracts (454mg) thus ob- grown in an incubator at 23~ under a day (ca. 200001ux)/ tained was submitted to repeated column chromatography night regime (14/10h) for 3 years. (solvents: appropriate mixtures of MeOH-CH2C12), TLC (solvents: appropriate mixtures of MeOH-CH2CI2, EtOAc- n-hexane, or benzene-EtOAc), and reversed-phase HPLC General experimental procedures (solvent system A, B, or C) to afford 7,6'-dihydroxy- bursehernin (8.0mg), a-peltatin (13.0mg), /3-peltatin One-dimensional and two-dimensional nuclear magnetic (13.8mg), pinoresinol (5.1mg), lariciresinol (2.3mg), and resonance (NMR) spectra were obtained with a JNM- secoisolariciresinol (2.2mg). LA400MK FT-NMR system (JEOL). Chemical shift and Freeze-dried roots (7.0g) of the plant were pulverized coupling constants (J) were expressed in d and Hz, respec- and extracted with hot MeOH as above. The MeOH ex- tively. Low- and high-resolution electron impact-mass spec- tracts (2.0 g) thus obtained were treated with/3-glucosidase trometry (EI-MS), gas chromatography-mass spectrometry and extracted with CH2C12 as above. An aliquot (ca. 500mg) (GC-MS), and high-performance liquid chromatography of the CH2C12 extracts (954mg) was subjected to repeated (HPLC) were conducted as previously described. 18 24 The TLC to afford 5-methoxypodophyllotoxin (15.0mg). reversed-phase HPLC column used was a Waters Symme- 7,6'-Dihydroxybursehernin: ~H- and 13C-NMR, try C18 (150 • 3.9 mm), which was eluted with the following 1H-~H correlated spectrometry (COSY), and IH-detected three solvent systems: (1) Solvent system A was for linear heteronuclear multiple-bond quantum correlation gradient elution at lml/min by CH3CN-H20 (25:75) at t = (HMBC) are summarized in Table 1. MS m/z (%): 402 0-15 min and then to 50:50 at t = 20rain, the latter compo- (2.1), 400 (3.1), 384 (19.6), 233 (15.6), 166 (13.4): 165 (11.0), sition being held for an additional 5 min. (2) Solvent system 151 (100), 139 (10.0). High-resolution MS m/z (M+), calcu- B was for linear gradient elution at lml/min by CH3CN- lated for C2~H22Os: 402.1315, found: 402.1287. H20 (20:80) at t = 0-12 rain and then to 50:50 at t = 18rain, 5-Methoxypodophyllotoxin, ct-peltatin, and fi-pettatin: the latter being held for an additional 5min. (3) Solvent ~H- and 13C-NMR (CDC13) data coincided with data system C was for linear gradient elution at l ml/min by in the literature. 7'~'zs Pinoresinol, Iariciresinol, and CHgCN-H20 (20:80) at t = 0-10min and then to 50:50 at secoisolariciresinol: tH-NMR (CDC13) data were t = 16min, the latter being held for an additional 5min. superimposable on those of chemically synthesized authen- The elution conditions for chiral HPLC were the same tic samples, respectively. as reported previously: pinoresinol, ~s lariciresinol, 22 and secoisolariciresinol.22 Infrared (IR) spectra were obtained by a JASCO IR-810 infrared spectrophotometer with solu- Enantiomeric compositions of lignans tion cells. Silica gel column chromatography and silica gel thin-layer chromatography (TLC) employed Kieselgel 60 The enantiomeric composition of secoisolariciresinol iso- (Merck, 70-230 mesh) and Kieselgel 60 F254 (Merck, 20 • lated from L. flavum var. compactum was determined by 20cm, 0.50 and 0.25ram), respectively. All chemicals used chiral HPLC analysis, as previously described. ~* Enantio- were reagent grade unless otherwise stated. meric compositions of pinoresinol and lariciresinol isolated 442 Table 1. NMR data for 7,6'-dihydroxybursehernin isolated from L. flavum var. cornpacturn in CDC13 Carbon no. ~3Ca IH~ H-H COSY b HMBCc 1 132.5 H-5, 7 2 109.6 6.95 (1H, m) H-6, 7 3 149.3~ / H-2, 5 OCH~ 4 149.4~ J 5 110.9 6.85 (1H, d, J = 8.1) H-6 6 119.1 6.95 (1H, m) H-5 H-2, 7 7 74.4 4.92 (1H, d, J 7.6) H-8 H-2, 6, 8 8 51.1 2.69 (1H, dd, J - 7.6, 9.5) H-7, 8' H-9, 7' 9 178.8 H-7, 8, 9' 1' 115.2 H-5', 7', 7' 2' 109.7 6.25 (1H, s) H-7', 7' 3' 141.5d H-2', 5', --CH?O-- 4' 147.0d H-2', 5', --CH20-- 5' 98.5 6.30 (1H, s) 6' 148.1 H-2', 5', 7' 7' 31.9 2.14 (1H, dd, J = 4.9, 14.2) H-7', 8' H-8, 2', 8', 9' 2.37 (1H, dd, J = 8.5, 14.2) H-7', 8' 8' 38.4 2.59 (1H, m) H-7', 7', 8, 9', 9' H-7, 8, 7', 7', 9' 9' 71.6 3.95 (1H, t, J = 8.9) U-8', 9' H-7' 4.13 (1H, t, J - 8.7) U-8', 9' H-7' MeO 56.0 3.87 (3H, s) 56.0 3.88 (3H, s) --OCH~O-- 101.2 5.86 (2H, s) NMR, nuclear magnetic resonance; COSY, correlated spectroscopy; HMBC, ~H-detected heteronuclear multiple-bond quantum correlation aChemical shifts are d values; coupling constants (aT in parentheses) are given in Hz bCorrelation between H-2 and H-6 was not observed clearly ~ correlated with carbon resonances d.~May be interchanged Fig.