Flavonoids from Pseudotsuga Menziesii

Flavonoids from Pseudotsuga menziesii Mirosława Krauze-Baranowskaa,*, Paweł Sowińskib, Anna Kawiakc,d, and Barbara Sparzakd a Department of Pharmacognosy, Medical University of Gdańsk, Gen. J. Hallera 107 str., 80-416 Gdańsk, Poland. Fax: +48583493160. E-mail: [email protected] b NMR Laboratory, Faculty of Chemistry, Technical University, Majakowskiego 11/12 str., Gdańsk, Poland c Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Kładki 24 str., 80-822 Gdańsk, Poland d Laboratory of Human Physiology, Faculty of Health Sciences with Subfaculty of Nursing, Medical University of Gdańsk, Tuwima 15 str., 80-210 Gdańsk, Poland * Author for correspondence and reprint requests Z. Naturforsch. 68 c, 87 – 96 (2013); received February 14, 2012/February 1, 2013 Four O-acylated fl avonol glycosides, new in the plant kingdom, were isolated from the needles of Pseudotsuga menziesii. Their structures were established by 1D and 2D NMR and MS data as: daglesioside I [kaempferol 3-O-[2’’,5’’-O-(4’’’,4IV-dihydroxy)-β- truxinoyl]-α-L-arabinofuranoside] (1), daglesioside II [kaempferol 3-O-[2’’,5’’-O-(4’’’- hydroxy)-β-truxinoyl]-α-L-arabinofuranoside] (2), daglesioside III [kaempferol 3-O-[2’’,5’’- di-O-(E)-p-coumaroyl]-α-L-arabinofuranoside] (3), and daglesioside IV [kaempferol 3-O-[3’’,6’’-di-O-(E)-cinnamoyl]-β-D-glucopyranoside] (4). In addition, the known fl avonoids (E)-tiliroside, (E)-ditiliroside, astragalin (kaempferol 3-O-β-D-glucopyranoside), isorhamnetin, kaempferol, and quercetin were identifi ed. The cytotoxic activity of compounds 1 and 3 was evaluated towards the HL-60, HeLa, and MDA-MB468 cell lines. Key words: Pseudotsuga menziesii, O-Acylated Flavonol Glycosides, β-Truxinic Acid Deriva- tives

Introduction the wood industry, as a source of medicinal and industrial compounds (Foo et al., 1992; Foo and Various species of the genus Pseudotsuga Karchesy, 1989a, b). (Pinaceae), including P. menziesii (Douglas fi r), Chromatographic analyses (TLC, HPLC) of are used in traditional medicine as antirheumatic, the chemical composition of P. menziesii leaves analgesic, diaphoretic, and diuretic agents (John- have also been performed (Krauze-Baranowska son, 1999). Previous phytochemical investigations et al., 2002, 2004). of the leaves, bark, and heartwood of P. menziesii In the present study, the isolation and structure focused on natural compounds belonging to the elucidation of four new O-acylated fl avonol gly- groups of fl avonoids (Dellus et al., 1997; Foo et al., cosides, namely daglesiosides I – IV (compounds 1992; Foo and Karchesy, 1989a; Niemann, 1988; 1 – 4), along with known compounds: acylated Stafford et al., 1989, 1986; Zou and Cates, 1995, fl avonol O-glycosides (tiliroside, ditiliroside), fl a- 1997), phenolic acids (Zou and Cates, 1997; Kuit- vonol O-glycoside (kaempferol 3-O-β-D-glu co- ers and Sarink, 1986), catechins (Stafford et al., pyranoside), and fl avonol aglycones (isorhamne- 1986; Foo and Karchesy, 1989b), fl avonolignans tin, kaempferol, quercetin) from the needles of P. (Foo and Karchesy, 1989b), lignans (Dellus et al., menziesii, growing in Poland, is described. 1997), and essential oils (Buchbauer et al., 1994; Montes et al., 1993; Tanaka et al, 1993). Exten- Experimental sive research on the chemical composition of P. menziesii was mainly undertaken in the USA or General Canada. These studies were aimed at using vari- Thin-layer chromatography (TLC) was per- ous parts of the plant, which are by-products in formed on glass plates covered with cellulose

(20 cm x 20 cm) (Merck, Darmstadt, Germany) amide column (50 g, 23 cm x 3 cm) eluted with and polyamide (20 cm x 20 cm) (Merck) using the MeOH/H2O (70:30) and MeOH. Fractions 9 – 18 following mobile phases: BuOH/H2O/CH3COOH were separated by chromatography over a Se- (4:1:5, v/v/v) (I), CHCl3/MeCOEt/MeOH (4:2:3) phadex G-10 column, eluted with MeOH, to give (II) (Krauze-Baranowska et al., 2002). Total hy- kaempferol 3-O-β-D-glucopyranoside (3 mg). drolysis was done by heating 1 mg of compounds Fractions 24 – 29 and 32 – 36 were subjected to 1 – 4 with 1 M HCl (100 °C, 30 min). Sugar analysis column chromatography over a polyamide col- was carried out on aluminium sheets precoated umn eluted with BuOH/MeOH/H2O (3:1:1) and with silica gel 60 F254 (0.2 mm thick; Merck) us- a Sephadex G-10 column eluted with MeOH to ing the mobile phase AcCN/H2O (15:85) (III). afford kaempferol 3-O-[6’’-O-(E)-p-coumaroyl]- The chromatograms were visualized by spray- β-D-glucopyranoside (tiliroside) (6 mg) and iso- ing with aniline phthalate, followed by heating rhamnetin (10 mg). Fractions 37 – 41 and 46 – 51 at 105 °C. For alkaline hydrolysis, 1 mg of com- were subjected to column chromatography over a pounds 1 – 4 was suspended in 4% aqueous KOH Sephadex G-10 column eluted with MeOH/H2O (4 h, room temperature). Analysis of phenolic ac- (70:30) to afford kaempferol (8 mg) and querce- ids was performed on aluminium sheets, precoat- tin (8 mg). Fractions 67 – 92, 93 – 99, and 100 – 107 ed with cellulose DC (20 cm x 20 cm) (Merck) were separately subjected to column chromato- with HCOONa/85% HCOOH/H2O (10:1:200) graphy over a Sephadex LH-20 column eluted (IV); the following spray reagents were used for with MeOH to afford kaempferol 3-O-[3’’,6’’- visualization of the spots: NH3, 1% (w/v) FeCl3, di-O-(E)-p-coumaroyl]-β-D-glucopyranoside 0.5% diazotized sulfanilic acid solution, and 10% (ditiliroside) (30 mg) and two further fractions: Na2CO3. subfractions 12 – 19 and 16 – 28. The subfractions NMR spectra were recorded on a Bruker MSL 12 – 19 were separated over a polyamide col- 300 instrument (Coventry, UK) at 300 MHz (for 1 13 umn eluted with CHCl3/MeOH/Me2CO (4:2:1) H) and 75.5 MHz (for C) in DMSO-d6 using to afford compound 3 (10 mg). The subfractions tetramethylsilane (TMS) as an internal standard. 16 – 28 were rechromatographed over a Sephad- ESI mass spectra were recorded on a Finnigan ex LH-20 column to afford compound 1 (10 mg). MAT (+) TSQ 700 spectrometer (Bremen, Ger- The concentrated chloroform extract was subject- many) and FAB-MS on a Trio-3 VG instrument ed to column chromatography over a polyamide (Masslab, Manchester, UK). column (60 g, 30 cm x 2.5 cm) eluted with CHCl3 (300 mL) and CHCl /MeOEt (4:3) (200 mL). Fla- Plant material 3 vonoids were eluted with MeOH (7 x 300 mL). The needles of Pseudotsuga menziesii (Mirb.) The methanol fractions were concentrated and Franco (Pinaceae) were collected from the Me- subjected to column chromatography over a dicinal Plants Garden of the Medical University Sephadex LH-20 column (25 g, 50 cm x 1.5 cm) of Gdańsk (Gdańsk, Poland) in August 1997; a eluted with MeOH. From the obtained fractions voucher specimen (No. 99-001) has been depos- 6 – 12 compound 2 (10 mg) was separated using ited in the herbarium of the Medicinal Plants preparative TLC on silica gel RP-18 with MeOH/ Garden. H2O/HCOOH (75:25:6) and rechromatography over a Sephadex LH-20 column (5 g, 8 cm x 1 cm) Extraction and isolation with MeOH. From the second obtained fractions Dried and pulverized leaves of P. menziesii 14 – 16, compound 4 (15 mg) was separated em- (3 kg) were extracted initially with petroleum ploying column chromatography over polyamide ether in a Soxhlet apparatus. Next, the puriﬁ ed with CHCl3/MeOEt/MeOH (4:0.5:3). material was extracted with chloroform, in a Sox- Daglesioside I [kaempferol 3-O-[2’’,5’’-O-(4’’’,4IV- hlet apparatus, and after drying, with methanol dihydroxy)-β-truxinoyl]-α-L-arabinofuranoside or (3 x 8 L) at 60 °C. The methanol extract was con- 3-O-(2’’,5’’-O-[7’’’-(4’’’-hydroxy)-phenyl,7IV-(4IV- IV IV centrated, suspended in H2O/MeOH (1:1), and hy droxy)-phenyl-(7’’’α,7 α,8’’’β,8 β)]-cyclo buta- IV successively extracted with EtOAc (6 x 300 mL). ne- 9’’’,9 -dioyl)-α-L-arabinofuranoside-3,5,7,4’- The extracts obtained were combined and con- tetrahydroxyﬂ avone, 1]: White powder. – TLC centrated, and next fractionated over a poly- (polyamide): Rf (II) = 0.40. – UV: λmax (MeOH) = M. Krauze-Baranowska et al. · Flavonoids from Pseudotsuga menziesii 89

267, 295sh, 348; (+AlCl3) 276, 316sh, 349, 399; NMR (DMSO-d6): see Table I. – Acid hydrolysis

(+AlCl3/HCl) 277, 315sh, 349, 403; (+CH3ONa) gave the aglycone kaempferol, arabinose, and a

274, 335sh, 403; (+CH3COONa) 274, 317sh, 376, compound with Rf (IV) = 0.08. Alkaline hydroly-

403sh; (+CH3COONa/H3BO3) 267, 295sh, 353 nm. sis gave the secondary glycoside [Rf (I) = 0.82] 2+ – Positive FAB-MS: m/z = 712 [M+2H] (25), 425 and a compound with Rf (IV) = 0.08. 2+ 1 [M+2H–287] (100). – H NMR (DMSO-d6): δ = Daglesioside III [kaempferol 3-O-[2’’,5’’-di- 6.21 (1H, d, J = 2.1 Hz, H-6), 6.42 (2H, d, J = 8.8 Hz, O-(E)-p-coumaroyl]-α-L-arabinofuranoside or IV IV H-3 ,5 ), 6.45 (2H, d, J = 8.3 Hz, H-3’’’,5’’’), 6.48 3-O-[2’’,5’’-di-O-(E)-p-coumaroyl]-α-L-arabino- (1H, d, J = 2.1 Hz, H-8), 6.77 (2H, d, J = 8.8 Hz, furanoside-3,5,7,4’-tetrahydroxyﬂ avone, 3]: Yel- H-2IV,6IV), 6.81 (2H, d, J = 8.3 Hz, H-2’’’,6’’’), 6.89 lowish powder. – TLC (polyamide): Rf (II) = 0.72. (2H, d, J = 8.8 Hz, H-3’,5’), 8.16 (2H, d, J = 8.8 Hz, – UV: λmax (MeOH) = 269, 317, 335sh; (+AlCl3) H-2’,6’), 5.72 (1H, s, H-1’’-O-arabinose), 5.96 (1H, 277, 319, 336sh, 403sh; (+AlCl3/HCl) 279sh, 318, d, J = 4.4 Hz, OH-3’’), 5.07 (1H, s, H-2’’), 4.60 336sh, 403sh; (+CH3ONa) 274, 314sh, 375, 408sh; (1H, d, J = 2.2 Hz, H-5’’a), 4.32 (1H, d, J = 4.4 Hz, (+CH3COONa) 276, 312, 403sh; (+CH3COONa/

H-3’’), 4.12 (1H, brs, H-4’’), 4.10 (1H, d, J = 2.2 Hz, H3BO3) 269, 313, 335sh nm. – Positive FAB-MS: IV H-5’’e), 3.75 (1H, dd, J = 6.4/10.4 Hz, H-8 ), 4.19 m/z = 712 [M+2H]2+ (30), 425 [M+2H–287]2+ 1 (1H, dd, J = 7.8/10.8 Hz, H-7’’’), 4.23 (1H, dd, J = (100). – H NMR (DMSO-d6): δ = 6.16 (1H, d, 7.8/10.2 Hz, H-8’’’), 3.98 (1H, dd, J = 6.4/10.4 Hz, J = 1.5 Hz, H-6), 6.28 (1H, d, J = 15.6 Hz, H-8IV), H-7IV ), 9.18 (2H, s, OH-4’’’,4IV), 10.24 (1H, s, OH- 6.42 (1H, d, J = 1.5 Hz, H-8), 6.43 (1H, d, J = 4’), 10.91 (1H, s, OH-7), 12.41 (1H, s, OH-5). – 13C 15.6 Hz, H-8’’’), 6.76 (2H, d, J = 8.3 Hz, H-3IV,5IV), NMR (DMSO-d6): see Table I. – Acid hydrolysis 6.78 (2H, d, J = 8.3 Hz, H-3’’’,5’’’), 6.88 (2H, d, J = IV gave the aglycone kaempferol [Rf (I) = 0.87], ara- 8.8 Hz, H-3’,5’), 7.47 (1H, d, J = 15.8 Hz, H-7 ), IV IV binose [Rf (III) = 0.28], and a compound with Rf 7.52 (2H, d, J = 8.3 Hz, H-2 ,6 ), 7.53 (2H, d, J = (IV) = 0.11. Alkaline hydrolysis gave the second- 8.3 Hz, H-2’’’,6’’’), 7.63 (1H, d, J = 15.6 Hz, H-7’’’), ary glycoside [Rf (I) = 0.82] and a compound with 7.96 (2H, d, J = 8.8 Hz, H-2’,6’), 5.74 (1H, s, H-1’’- Rf (IV) = 0.11. O-arabinose), 5.85 (1H, brs, OH-3’’), 5.36 (1H, dd, Daglesioside II [kaempferol 3-O-[2’’,5’’-O- J = 1.0/3.4 Hz, H-2’’), 4.21 (1H, dd, J = 2.9/9.6 Hz, (4’’’-hydroxy)-β-truxinoyl]-α-L-arabinofuranoside H-5’’a), 4.05 (1H, t, H-5’’e), 4.00 (1H, m, H-3’’), or 3-O-(2’’,5’’-O-[7’’’-(4’’’-hydroxy)-phenyl-7IV- 3.87 (1H, td, J = 2.7/13.0 Hz, H-4’’), 10.15 (1H, phenyl-(7’’’α,7IVα,8’’’β,8IVβ)]-cyclobutane-9’’’,9IV- brs, C-OH-4’,4’’’,4IV), 12.45 (1H, s, C-OH-5). – 13C di oyl)-α-L-arabinofuranoside-3,5,7’,4’-tetrahydrox- NMR (DMSO-d6): see Table I. – Acid hydrolysis yﬂ avone, 2]: White powder. – TLC (polyamide): gave the aglycone kaempferol, arabinose, and p-

Rf (II) = 0.51. – UV: λmax (MeOH) = 267, 293sh, coumaric acid [Rf (IV) = 0.39]. Alkaline hydroly-

354; (+AlCl3) 276, 316sh, 347, 403; (+AlCl3/HCl) sis gave the secondary glycoside [Rf (I) = 0.82]

276sh, 315sh, 353, 403; (+CH3ONa) 274, 336sh, and p-coumaric acid.

400; (+ CH3COONa) 275, 319sh, 395, 403sh; Daglesioside IV [kaempferol 3-O-[3’’,6’’-di-O- (+CH3COONa/H3BO3) 269, 294sh, 352 nm. – Pos- (E)-cinnamoyl]-β-D-glucopyranoside or 3-O-[3’’, 2+ itive FAB-MS: m/z = 696 [M+2H] (100). – Posi- 6’’-di-O-(E)-cinnamoyl]-β-D-glucopyranoside- tive ESI-MS: m/z = 695 [M+H]+ (100). – 1H NMR 3,5,7,4’-tetrahydroxyﬂ avone, 4]: Yellowish pow-

(DMSO-d6): δ = 6.20 (1H, s, H-6), 6.39 (2H, d, der. – TLC (polyamide): Rf (II) = 0.62. – UV:

J = 8.3 Hz, H-3’’’,5’’’), 6.42 (1H, s, H-8), 6.79 (2H, λmax (MeOH) = 264, 303sh, 316, 335sh; (+AlCl3) d, J = 8.3 Hz, H-2’’’,6’’’), 6.82 (2H, d, J = 8.8 Hz, 273, 304sh, 318sh, 351, 403sh; (+AlCl3/HCl) 271, IV IV H-3’,5’), 6.98 (1H, d, J = 7.3 Hz, H-3 ,5 ), 7.02 303sh, 316sh, 350, 403sh; (+CH3ONa) 272, 305sh, IV IV IV (1H, m, H-4 ), 7.08 (2H, d, J = 7.3 Hz, H-2 ,6 ), 380, 400sh; (+CH3COONa) 273, 304sh, 315,

8.20 (2H, d, J = 8.8 Hz, H-2’,6’), 5.72 (1H, s, H- 352sh, 400sh; (+CH3COONa/H3BO3) 264, 302sh, 1’’-O-arabinose), 5.96 (1H, d, J = 4.9 Hz, OH-3’’’), 315, 345sh nm. – Positive FAB-MS: m/z = 710 5.07 (1H, s, H-2’’), 4.60 (1H, d, J = 2.7 Hz, H-5’’a), [M+2H]2+ (100), 732 [M+2H+Na]2+ (20). – 1H

4.30 (1H, m, H-3’’), 4.26 (2H, m, H-7’’’, 8’’’), 4.12 NMR (DMSO-d6): δ = 6.12 (1H, d, J = 1.5 Hz, (3H, m, H-4’’, H-5’’e, H-7IV), 3.88 (1H, dd, J = H-6), 6.35 (1H, d, J = 1.5 Hz, H-8), 6.40 (1H, d, 6.4/10.4 Hz, H-8IV), 9.10 (1H, s, C-OH-4’’’), 10.30 J = 16.0 Hz, H-8’’’), 6.67 (1H, d, J = 16.0 Hz, H- (1H, s, C-OH-4’), 12.41 (1H, s, C-OH-5). – 13C 8IV), 6.88 (2H, d, J = 8.8 Hz, H-3’,5’), 7.30 (5H, 90 M. Krauze-Baranowska et al. · Flavonoids from Pseudotsuga menziesii m, H-3’’’,5’’’, 3IV,5IV, 4’’’), 7.40 (1H, d, J = 16.0 Hz, Cytotoxicity assay H-7’’’), 7.55 (2H, d, J = 8.1 Hz, H-2’’’,6’’’), 7.65 The viability of the cell lines was determined IV IV (1H, m, H-4 ), 7.70 (1H, d, J = 16.0 Hz, H-7 ), using the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5- IV IV 7.72 (2H, d, J = 8.1 Hz, H-2 ,6 ), 7.98 (2H, d, J = diphenyltetrazolium bromide] assay. Cells were 8.8 Hz, H-2’,6’), 5.78 (1H, d, J = 4.8 Hz, OH-2’’), seeded in 96-well microtitre plates (104 cells/well) 5.56 (1H, d, J = 7.8 Hz, H-1’’-O-glucose), 5.47 and treated for 24 h with the test compounds. MTT (1H, d, J = 6.8 Hz, OH-4’’), 5.00 (1H, t, H-3’’), (0.5 mg/mL) was added and the mixture incubat- 4.26 (1H, d, J = 112 Hz, H-6’’a), 4.09 (1H, dd, ed for 3 h at 37 °C followed by lysis of cells with J = 6.0/11.2 Hz, H-6’’e), 3.59 (1H, m, H-5’’), 3.45 dimethyl sulfoxide (DMSO). The optical density (2H, m, H-2’’,4’’), 9.98 (1H, s, C-OH-4’), 10.70 of the formed formazan solution was measured at 13 (1H, s, C-OH-7), 12.35 (1H, s, C-OH-5). – C 550 nm with a plate reader (Victor, 1420 multila- NMR (DMSO-d6): δ = 177.4 (C-4), 166.4, 166.3 bel counter; Perkin Elmer, Turku, Finland). (C-9’’’,9IV), 164.9 (C-7), 161.8 (C-5), 160.7 (C-4’), IV 157.2 (C-2), 157.0 (C-9), 145.2, 145.1 (C-7’’’, 7 ), Results and Discussion 134.8, 134.4 (C-1’’’,1IV), 133.6 (C-3), 131.5 (C- 2’,6’), 130.3, 129.6 (C-4’’’,4IV), 129.5 (C-2IV,6IV), Separation of the compounds in the chloro- 129.0 (C-2’’’,6’’’; 2IV,6IV), 128.6, 128.5 (C-3’’’,5’’’, form and methanol extracts from the leaves of 3IV,5IV), 121.3 (C-1’), 119.2, 118.1 (C-8’’’,8IV), 115.8 P. menziesii by column chromatography on poly- (C-3’,5’), 104.5 (C-10), 101.5 (C-1’’), 99.5 (C-6), amide, Sephadex LH-20, and G-10 columns and 94.4 (C-8), 77.8 (C-3’’), 74.6 (C-5’’), 72.7 (C-2’’), preparative TLC led to the isolation of a number 68.6 (C-4’’), 63.5 (C-6’’). – Acid hydrolysis gave of fl avonoids up to date unknown in this species: the aglycone kaempferol, glucose, and cinnaminic kaempferol 3-O-β-D-glucopyranoside (astraga- acid [Rf (IV) = 0.50]. Alkaline hydrolysis gave the lin), kaempferol 3-O-[6’’-O-(E)-p-coumaroyl]- 3-O-glucoside of kaempferol [Rf (I) = 0.70] and β-D-glucopyranoside (tiliroside), isorhamnetin, cinnamic acid. kaempferol, quercetin, kaempferol 3-O-[3’’,6’’- di-O-(E)-p-coumaroyl]-β-D-glucopyranoside (di- Cytotoxic activity tiliroside), daglesioside I (1), daglesioside II (2), daglesioside III (3), and daglesioside IV (4), to- Chemicals gether with the previously described quercetin All cell culture material was purchased from 3-O-galactoside and chlorogenic acid (Zou and Gibco-Invitrogen (Paisley, UK). All other chemi- Cates, 1995, 1997). The aglycones and kaempfer- cals were purchased from Sigma-Aldrich (St. ol 3-O-glucoside were identifi ed on the basis of Louis, MO, USA). co-chromatography with standards and UV and mass spectra (Markham, 1982). Cell culture The identifi cation of two known O-acylated The cell lines were cultured on the following fl avonol glycosides, kaempferol 3-O-[6’’-O -(E)- media: the HL-60 (human promyelocytic leuke- p-coumaroyl]-β-D-glucopyranoside and kaemp- mia) cell line on the RPMI 1640 medium sup- ferol 3-O-[3’’,6’’-di-O-(E)-p-coumaroyl]-β-D- plemented with 10% fetal bovine serum, 2 mM glucopyranoside, was carried out by spectral glutamine, 100 units/mL penicillin, and 100 μg/mL analysis, 1H, 13C NMR, NOE, COSY, HMBC, streptomycin; the HeLa (human cervical adeno- HSQC experiments, FAB-MS, and also by the carcinoma) cell line on minimal Eagle’s medium comparison of 1H and 13C NMR spectra with (MEM) supplemented with 10% fetal bovine se- those published (Markham, 1982; Budzianowski rum, 2 mM glutamine, 100 units/mL penicillin, and and Skrzypczak, 1995; Liu et al., 1999). (E)-Tili- 100 μg/mL streptomycin; human skin fi broblasts roside is a known constituent of some species of and the MDA-MB468 (human breast cancer) the genus Pinus, i.e. P. concorta and P. banksiana cell line on Dulbecco’s modifi ed Eagle’s medium (Harborne and Baxter, 1999), however, (E)-ditili- (DMEM) supplemented with 10% fetal bovine roside has only been recognized in Picea obovata serum, 2 mM glutamine, 100 units/mL penicillin, (Liu et al., 1999) and Picea silvestris (Harborne and 100 μg/mL streptomycin. All cultures were and Baxter, 1999). maintained in a humidifi ed atmosphere with 5% Alkaline hydrolysis of compound 3 yielded p-

CO2 at 37 °C. coumaric acid, arabinose, and a ﬂ avonoid glyco- M. Krauze-Baranowska et al. · Flavonoids from Pseudotsuga menziesii 91

side with Rf values higher than those of kaemp- ton (Markham and Geiger, 1994). Moreover, the ferol 3-O-glucoside in the used TLC systems. The 1H NMR resonances of two coupled doublets at

UV spectrum of 3 against chromogenic reagents δH 6.16 and 6.42 ppm with a small coupling con- revealed the presence of free OH groups in the stant (J = 1.5 Hz) were characteristic for two me- positions C-7, C-4’, and C-5 (Markham, 1982). ta-coupled H-6 and H-8 protons of the A-ring of The positive FAB mass spectrum of 3 gave an kaempferol. The furanose form of the sugar arab- [M+2H]2+ peak at m/z 712 consistent with the inose in compound 3 was deduced from the NOE, molecular mass of kaempferol arabinoside di- DEPT-HMBC, DQF-COSY (Fig. 1), ROESY, and coumaroyl ester (710 Da). The structure of com- HMBC spectra Table I. The following basic corre- pound 3 was elucidated on the basis of 1D-1H, lations were observed in the HMBC spectrum: H- 13 C and 2D NMR, HSQC, HMBC, DQF-COSY 1’’/C-4’’ at δH 5.74 ppm/δC 82.8 ppm (Fig. 1) and in spectra (Fig. 1) and ROESY experiments. In the the ROESY spectrum: H-1’’/H-3’’, H-2’,6’/H-4’’, 1H NMR spectrum of 3, three pairs of aromatic H-2’,6’/H-2’’, H-3’,5’/H-4’’, H-3’,5’/H-2’’. protons of the system AA’BB’ appeared, which were assigned to two moieties of p-coumaric acid from the data of DQF-COSY, ROESY, Table I. 13C NMR spectroscopic data of daglesiosides HSQC – respectively the doublets at δH 6.76 and I – III isolated from P. menziesii (the δ values were de- 7.52 ppm (H-3IV,5IV, H-2IV,6IV, J = 8.3 Hz,) as well termined on the basis of HSQC, HMBC, 1H-1H COSY measurements). as at δH 6.78 and 7.53 ppm (H-3’’’,5’’’, H-2’’’,6’’’, J = 8.3 Hz) (Markham, 1982; Liu et al, 1999; C Daglesio- Daglesio- Daglesio- Markham and Geiger, 1994) – whereas the one side III side I side II pair – the doublets at δH 6.88 and 7.96 ppm (H- Kaempferol 3’,5’, H-2’,6’, J = 8.8 Hz) – was assigned to a para- 2 157.0 157.1 156.9 substituted phenyl group of the ﬂ avonoid skele- 3 132.8 134.3 134.3 4 177.2 178.0 177.4 5 161.1 160.9 161.8 6 98.8 99.5 99.5 7 165.5 165.1 165.3 8 93.8 94.5 94.5 9 156.4 156.9 157.1 10 104.4 104.6 104.5 1’ 120.3 121.0 121.0 2’ 130.3 131.7 131.7 3’ 115.8 115.2 115.2 4’ 160.1 161.0 160.9 5’ 115.8 115.2 115.2 6’ 130.3 131.0 131.1 Arabinofuranose 1’’ 105.4 106.4 106.7 2’’ 83.3 89.0 89.0 3’’ 75.5 73.1 73.1 4’’ 82.8 82.4 82.4 5’’ 62.6 63.8 63.8 Acyl group 1’’’/1IV 124.9/124.8 129.4/129.4 129.2/139.4 2’’’/2 IV 130.7/130.6 129.5/129.7 129.5/128.4 3’’’/3 IV 116.4/116.4 115.3/116.1 116.1/128.7 4’’’/4 IV 160.5/160.4 156.1/156.0 156.0/126.7 5’’’/5 IV 116.5/116.4 115.3/116,1 116.1/128.7 6’’’/6 IV 130.7/130.6 129.5/129.7 129.5/128.4 7’’’/7 IV 146.0/145.1 43.6/43.3 43.9/43.5 Fig. 1. Selected HMBC (heteronuclear multiple bond IV 1 1 8’’’/8 113.0/113.5 42.6/45.7 42.8/45.1 correlation) and H- H COSY (correlation spectros- IV copy) correlations of compound 3. 9’’’/9 165.9/166.1 172.8/172.1 172.7/172.0 92 M. Krauze-Baranowska et al. · Flavonoids from Pseudotsuga menziesii

The coupling constant of the anomeric pro- at m/z 696 [M+2H]2+, respectively, for 1 and 2. Dou- ton was determined as JH-1’’-H-2’’ = 1 Hz from the bly protonated molecules were also present in O- data of the DQF-COSY spectrum (the correla- acylated fl avonoids of Stenochlaena palustris (Liu tion signal between H-1’’ and H-2’’), confi rming et al., 1999). The difference of 16 Da in the molecu- the α-confi guration of the sugar in 3 (Markham lar masses of both compounds presumably indicat- and Geiger, 1994). The linkage of the coumaroyl ed the lack of one OH group in 2. The values of  substituents to 2’’-OH and to 5”-OH of arabi- signals, displayed in the aromatic and sugar range nose were established by the signifi cant downfi eld of the 1H and 13C NMR spectra of 1 and 2, are shifts of H-2’’ by ca.  = 6.9 ppm and H-5’’ by similar to the δ values of daglesioside III, suggest- ca.  = 2.8 ppm, from normally expected values ing that these compounds may be the more highly (Markham, 1982; Markham and Geiger, 1994). esterifi ed kaempferol 3-O-α-arabinofuranosides, The HMBC spectrum of 3, in which one of the es- with the acyl substituents in positions C-2’’ and ter carbonyl carbon atoms at δC 165.9 ppm (C-9’’’) C-5’’ of the sugar residues (Table I). 1 was correlated to H-2’’ (δH 5.36 ppm) and the sec- In the H NMR spectrum of 1, four pairs of IV ond at δC 166.1 ppm (C-9 ) to H-5’’ (δH 4.21 ppm, ortho-coupled doublets at δH 6.42 ppm (J = 4.05 ppm), further substantiated these fi ndings. 8.8 Hz, H-3IV,5IV), 6.77 ppm (J = 8.8 Hz, H-2IV,6IV), The HMBC experiment also supported the glyco- 6.45 ppm (J = 8.3 Hz, H-3’’’,5’’’), and 6.81 ppm sidation at 3-OH of kaempferol – correlation be- (J = 8.3 Hz, H-2’’’,6’’’) indicated the presence of tween H-1’’ and C-3 (δH 5.74 ppm/δC 132.8 ppm) two para-substituted phenyl groups. Moreover, (Fig. 1). The confi guration of protons H-7’’’/H-8’’’ the 1H-1H COSY (Fig. 2) and HSQC, NOESY ex- and H-7IV/H-8IV in both p-coumaric acid moieties periments revealed the presence of a four-mem- was elucidated as trans, regarding the value of the bered carbon ring in the structure of 1, linked to coupling constant J = 17.0 Hz (Liu et al., 1999; the phenyl substituents via carbonyl groups of Abdel-Shafeek et al., 2000; Lin et al., 1999). Thus, esterifi ed kaempferol 3-O-arabinoside; the signals the structure of compound 3 was determined as kaempferol 3-O-[2’’,5’’-di-O-(E)-p-coumaroyl]-α- L-arabinofuranoside, and named daglesioside III. In nature, among the acylated fl avonol arabinosides, quercetin arabinopyranosides occur more frequently than kaempferol arabinosides (Har- borne and Baxter, 1999), for example calabrico- side B from Putoria calabrica (Rubiaceae) (Calis et al., 2001). In the group of kaempferol arabinopyranosides, two compounds, i.e. 3-O-(2’’-O- p-coumaroyl)-arabinoside from Picea koranensis and Larix decidua (Pinaceae) and 3-O-(2’’-O- galloyl)-arabinoside from Eucalyptus rostrata (Myrtaceae), have been described (Harborne and Baxter, 1999). To date, kaempferol 3-O-arabinofuranoside has been isolated from some species of the families Aspleniaceae, Juglandaceae, Po- lygonaceae, Rosaceae, and Tiliaceae (Harborne and Baxter, 1999). The maxima of the band I absorption in the UV spectra of compounds 1 and 2 were batho- chromically shifted by ca. 20 nm, in contrast to the other isolated O-acylated glycosides tiliroside, ditiliroside, and 3. The UV analysis suggested that both compounds are derivatives of kaempferol 3-glycosides (Markham, 1982). Fig. 2. Selected HMBC (heteronuclear multiple bond In the FAB mass spectra, doubly protonated correlation) and 1H-1H COSY (correlation spectros- molecules were observed at m/z 712 [M+2H]2+ and copy) correlations of compound 1. M. Krauze-Baranowska et al. · Flavonoids from Pseudotsuga menziesii 93 of the ester carbonyl carbon atom were, respectively, at δC 172.8 ppm (C-9’’’) and 172.1 ppm (C- 9IV). In the HSQC spectrum, the observed signals of four protons of methine groups at δH 3.75 ppm (1H, dd, J = 6.4/10.4 Hz, H-8IV), 4.19 ppm (1H, dd, J = 7.8/10.8 Hz, H-7’’’), 4.23 ppm (1H, dd, J = 7.8/10.2 Hz, H-8’’’), and 3.98 ppm (1H, dd, J = 6.4/10.4 Hz, H-7IV) were assigned, respec- IV tively, to carbon signals at δC 45.7 ppm (C-8 ), 43.6 ppm (C-7’’’), 43.3 ppm (C-7IV), and 42.6 ppm (C-8’’’). In the COSY spectrum, the following correlations: H-7’’’/H-8’’’, H-7IV/H-8IV and H- 7IV/H-7’’’, H-8IV/H-8’’’ were present as evidence of a cyclobutane ring. In addition, the correlation between H-7’’’/H-2’’’,6’’’, H-7IV/H-2IV,6IV and C- 9’’’/H-2’’, C-9IV/H-5’’a/e in the HMBC spectrum of 1 showed the presence of phenyl groups in positions C-7’’’ and C-7IV, and on the other hand, the sites of acylation of the OH groups at C-2’’ and C-5’’ arabinofuranose (Fig. 2). Moreover, the other correlations from the HMBC experiment, Fig. 3. Selected HMBC (heteronuclear multiple bond i.e. between H-8’’’/C-1’’’ and H-8IV/C-1IV, led to correlation) and 1H-1H COSY (correlation spectros- the conclusion that compound 1 is a di-(4’’’,4IV- copy) correlations of compound 2. dihydroxy)-phenyl-cyclobutanedicarboxylic acid derivative of kaempferol 3-O-arabinoside. The confi guration of the substituents in the cyclobu- plants. To date, only two diglucosylfl avonoids con- tane ring of 1 was established from the NOESY taining truxinic acid moieties have been identi- spectrum, in which signifi cant NOE correlations fi ed in the plant kingdom, namely stachysetin and between protons H-7’’’ and H-7’ as well as H-8’’’ monochaetin from Stachys aegyptiaca (El-Ansari and H-8’ were observed against signifi cantly et al., 1995) and Monochaetum multifl orum (Isaza weaker correlations between protons H-7 (7’) et al., 2001), respectively. and H-8 (8’). From these data, the acid moiety Daglesioside IV (4) was isolated as a yellow was deduced to be b-truxinic acid with cis-confi g- amorphous powder. The UV spectrum of 4 pos- uration of the proton pairs H-7,7’ and H-8,8’ and sessed an intense absorption band in the range trans-confi guration of the proton pairs H-7,8 and 310 – 320 nm, which indicated the acylation of the H-7’, 8’ in the cyclobutane ring (Sudo et al., 2000). fl avonoid moiety by cinnamic acid derivatives Accordingly, the structure of compound 1 was (Markham, 1982). In the positive FAB mass spec- established as kaempferol 3-O-[2’’,5’’-O-(4’’’,4IV- trum of 4, the doubly protonated molecule at m/z 2+ dihydroxy)-β-truxinoyl]-α-L-arabinofuranoside or 710 [M+2H] confi rmed that this compound is a daglesioside I. kaempferol glycoside dicinnamoyl ester. In the The structure of compound 2 was elucidated on HSQC spectrum of 4, the signal of an anomeric the basis of the same spectroscopic methods, in- proton at δH 5.56 ppm was correlated with the sig- cluding 2D NMR-HSQC, HMBC, COSY (Fig. 3), nal of an anomeric carbon atom at δC 101.5 ppm, and NOESY experiments. The data collected were and the values of the chemical shifts of the other almost identical with those of 1 (Table I) and in- carbon atoms in the range 77 – 63 ppm were char- dicated that the difference between the two com- acteristic of glucose with β-confi guration (JH-1’’,2’’ = pounds was the lack of an OH group at C-4IV in 7.8 Hz) substituted in the positions 3’’ and 6’’ with 2. Thus, the structure of 2 was assigned as kaemp- two moieties of cinnamic acid (Liu et al., 1999; ferol 3-O-[2’’,5’’-O-(4’’’-hydroxy)-β-truxinoyl]-α- Atta-ur-Rahman et al., 1998), as indicated by the L-arabinofuranoside or daglesioside II. HMBC spectrum which showed correlations be- IV Daglesiosides I and II are the fi rst fl avonol tween H-3’’/C-9 at δH 5.00 ppm/δC 166.3 ppm and monoglycoside β-truxinic esters isolated from H-6’’a/e/C-9’’’ at δH 4.26/4.09 ppm/δC 166.4 ppm, 94 M. Krauze-Baranowska et al. · Flavonoids from Pseudotsuga menziesii respectively (Fig. 4). On the other hand, the pres- and Rosazza, 1998). Therefore, the structure of 4 ence of an O-glycosidic bond at C-3 of kaemp- was elucidated as kaempferol 3-O-[3’’,6’’-di-O- ferol was confi rmed by the correlation signal be- (E)-cinnamoyl]-β-D-glucopyranoside or daglesio- tween C-3 and H-1’’ at δC 133.6 ppm/δH 5.56 ppm. side IV, a new compound in the plant kingdom. It In the 1H NMR spectrum of 4, trans-confi guration is worth noticing that kaempferol 3-O-[6’’-O-(Z)- of the cinnamic acid moieties was evidenced by cinnamoyl]-glucoside has been previously identi- four proton signals at δH 6.40 (H-8’’’), 6.67 (H- fi ed only in Solanum eleagnifolium (Solanaceae) 8IV), 7.40 (H-7’’’), and 7.70 ppm (H-7IV) with a (Harborne and Baxter, 1999). coupling constant of J = 16.0 Hz (Markham, 1982; The cytotoxic activity of two of the newly Markham and Geiger, 1994; Abdel-Shafeek et al., isolated compounds, besides of tiliroside and 2000; Hosny and Rosazza, 1998). The values of the kaempferol 3-O-glucoside, was examined against chemical shifts of the proton pairs H-8’’’,H-7’’’ the following human tumour cell lines: HL-60, and H-8IV,H-7IV were assigned on the basis of HeLa, and MDA-MB468. Human skin fi broblasts the correlation displayed in COSY and HSQC were used as a non-neoplastic control (Table II). spectra. Furthermore, the HMBC spectrum of 4 The cytotoxic activities of tiliroside and kaemp- showed the long-range correlations between H- ferol 3-O-glucoside were signifi cantly lower than IV IV IV 2 ,6 /C-4 at δH 7.72 ppm/δC 130.3 ppm and H- those of daglesiosides I and III, and for all exam- 2’’’,6’’’/C-4’’’ at δH 7.55 ppm/δC 129.6 ppm, respec- ined cell lines their IC50 values were equal to or tively, confi rming the structure of cinnamic acid above 50 μg/mL. Daglesioside I and daglesioside (Atta-ur-Rahman et al., 1998; Hosny and Rosazza, III exerted the highest cytotoxic activity towards

1998) (Fig. 4). The other assignments of d values the HL-60 cell line with IC50 values of 8.5 μg/mL for protons and carbon atoms of cinnamic acid, and 6 μg/mL, respectively. Compared to the HL- from the HSQC and HMBC spectra, were in 60 cell line, daglesioside III displayed a two-fold good agreement with the literature data (Hosny lower activity towards the MDA-MB468 cell line,

Fig. 4. Selected HMBC (heteronuclear multiple bond correlation) correlations of compound 4.

Table II. Cytotoxic activity (expressed as IC50 values  SD in μg/mL) of daglesiosides I and III against human tumour cell lines. Compound/extract Cell line HL-60 HeLa MDA-MB468 Fibroblasts Daglesioside I 8.5  0.5 32  1.6 25  1 37  1.8 Daglesioside III 6  0.3 17  1 13  0.5 27  1 Tiliroside 50  3.0 >50 >50 - Kaempferol 3-O-glucoside >50 >50 >50 - Etoposide 0.45  0.022 >50 >50 - Mitoxantrone 0.06  0.004 0.4  0.012 0.2  0.008 -

Experiments were carried out in triplicate. M. Krauze-Baranowska et al. · Flavonoids from Pseudotsuga menziesii 95 and a three-fold lower activity towards the HeLa difference in their chemical structures, due to the cell line, with the lowest activity observed towards occurrence of a cyclobutane ring in daglesioside I fi broblasts. Daglesioside I was less active than as a result of the dimerization of two p-coumaric daglesioside III towards the remaining cell lines, acid moieties in daglesioside III. again with the lowest activity observed towards fi broblasts. Despite the lower activity of dagle- Conclusions sioside I, its activity towards the HL-60 cell line was comparable to that of daglesioside III, but The leaves of Pseudotsuga menziesii are a much lower than that of daglesioside III towards source of fl avonoids with diverse structures, from the other examined cell lines, hence it exhibited fl avonol aglycones through fl avonol monoglyco- greater selectivity towards the HL-60 cell line. sides to fl avonol monoglycoside esters, including The observed differences between the activities derivatives of truxinic acids, which very rarely oc- of the two compounds may be explained by the cur in the plant kingdom.

Abdel-Shafeek K. A., El-Menssiry M. M., Shahat A. A., Hosny M. and Rosazza J. P. N. (1998), Gmelinosides Apers S., Peters L., and El-Nasr M. (2000), A new A – L, twelve acylated iridoid glycosides from Gmeli- acylated fl avonol triglycoside from Carrichtera anna arborea. Phytochemistry 47, 845 – 850. nua. J. Nat. Prod. 63, 845 – 847. Isaza J. H., Ito H., and Yoshida T. (2001), A fl avonol- Atta-ur-Rahman K. F., Nighat F., Shabbir M., Hemalal lignan ester and accompanying acylated glucosides K. D., and Tillerkerante L. M. (1998), Dimeric tro- from Monochaetum multifl orum. Phytochemistry 58, pane alkaloids from Erythroxylum moonii. Phyto- 321 – 327. chemistry 48, 377 – 383. Johnson T. (1999), Ethnobotany Desk Reference. CRC Buchbauer G., Jirovetz L., Wasichy M., and Kikiforov Press, New York, USA. A. (1994), Composition of Douglas-fi r essential oil Krauze-Baranowska M., Malinowska I., and and extracts. J. Agric. Food Chem. 42, 2852 – 2854. Skwierawska J. (2002), TLC of fl avonol truxinic es- Budzianowski J. and Skrzypczak L. (1995), Phenylpro- ters from Pseudotsuga menziesii. J. Planar Chroma- panoid esters from Lamium album fl owers. Phyto- togr. 15, 437 – 441. chemistry 38, 997 – 1001. Krauze-Baranowska M., Bączek T., Głód D., Kaliszan Calis I., Heilmann J., Tasdemir D., Linden A., Ireland R., and Wollenweber E. (2004), HPLC separation of C. M., and Sticher O. (2001), Flavonoid, iridoid, and O-acylated fl avonoids and bifl avones from some spe- lignan glycosides from Putoria calabrica. J. Nat. Prod. cies of Gymnospermae. Chromatographia 60, 9 – 15. 64, 961 – 964. Kuiters A. T. and Sarink H. M. (1986), Leaching of phe- Dellus V., Milla I. N., Scalbert A., Menard C., Michon V., nolic compounds from leaf and needle litter of sev- and Herve du Penhoat C. L. M. (1997), Douglas-fi r polyphenols and heartwood formation. Phytochem- eral deciduous and coniferous trees. Soil Biol. Bio- istry 45, 1573 – 1578. chem. 18, 475 – 480. El-Ansari M. A., Nawwar M. A., and Saleh N. A. M. Lin X. C., ElSohly H. N., Nimrod A. C., and Clark A. M. (1995), Stachysetin, an apigenin-7-glucoside-p,p’- (1999), Antifungal jujubogenin saponins from Colub- dihydroxy-truxinate from Stachys aegyptiaca. Phyto- rina retusa. J. Nat. Prod. 62, 674 – 677. chemistry 40, 1543 – 1548. Liu H., Orjala J., Sticher O., and Rali T. (1999), Acylat- Foo L. Y. and Karchesy J. (1989a), Pseudotsuganol from ed fl avonol glycosides from leaves of Stenochlaena bark of Pseudotsuga menziesii. J. Chem. Soc. Chem. palustris. J. Nat. Prod. 62, 70 – 75. Commun., 217 – 219. Markham K. R. (1982), Techniques of Flavonoids Iden- Foo L. Y. and Karchesy J. (1989b), Polyphenolic glyco- tifi cation. Academic Press, London, UK, pp. 36 – 49 sides from Douglas fi r inner bark. Phytochemistry 28, and pp. 72 – 93. 1237 – 1240. Markham K. R. and Geiger H. (1994), 1H nuclear mag- Foo L. Y., Helm R., and Karchesy J. (1992), [5’,5’]-Bis- netic spectroscopy of fl avonoids and their glycosides dihydroquercetin: a B-ring linked bifl avonoid in hexadeuterodimethylsulfoxide. In: The Flavonoids. from Pseudotsuga menziesii. Phytochemistry 31, Advances in Research since 1986 (Harborne J. B., 1444 – 1445. ed.). Chapman and Hall, London, UK, pp. 44 – 497. Harborne J. B. and Baxter H. (1999), The Handbook of Montes M. A., Valenzuela L., Wilkomirsky T., and Bello Natural Flavonoids. Wiley, Chichester, UK. H. (1993), Essential oil from Pseudotsuga menziesii. 96 M. Krauze-Baranowska et al. · Flavonoids from Pseudotsuga menziesii

Composition and antimicrobial activity. An. Real 4,4’-dimethoxy-β-truxinic acid catalpol diester from Acad. Nal. Farm. 59, 467 – 476. the leaves of Premma subcandens MERR. Chem. Niemann G. J. (1988), Distribution and evolution of the Pharm. Bull. 48, 542 – 546. fl avonoids in gymnosperms. In: The Flavonoids (Har- Tanaka R., Takaoka Y., and Yamaguchi H. (1993), borne J. B., ed.). Chapman and Hall, London, UK, 3β-Methoxycycloartan-27-oic acid and other constit- p. 475. uents from the leaves of Pseudotsuga japonica. J. Nat. Stafford H. A., Kreitlow K. S., and Lester H. H. (1986), Prod. 56, 1753 – 1757. Flavonoids in leaves of Ginkgo biloba, Pseudotsuga Zou J. and Cates R. G. (1995), Foliage constituents of menziesii and Ribes sanguineum. Plant Physiol. 82, Douglas fi r (Pseudotsuga menziesii Mirb.) Franco 1132 – 1138. (Pinaceae): Their seasonal variation and potential Stafford H. A., Smith E. C., and Weider R. M. (1989), role in Douglas fi r resistance and silviculture man- The development of proanthocyanidins (condensed agement. J. Chem. Ecol. 21, 387 – 402. tannins) and other phenolics in bark of Pseudotsuga Zou J. and Cates R. G. (1997), Effects of terpenes menziesii. Can. J. Bot. 67, 1111 – 1118. and phenolic and fl avonoid glycosides from Doug- Sudo H., Ide T., Otsuka H., Hirata E., Takushi A., las fi r on western spruce budworm larval growth, Shinzato T., and Takeda Y. (2000), Megastigmane, pupal weight, and adult weight. J. Chem. Ecol. 23, benzyl and phenethyl alkohol glycosides and 2313 – 2327.