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Contribution of flavonoids and catechol to the reduction of ICAM-1 expression in endothelial cells by a standardised Willow bark extract.

ARTICLE INFO

Article history:

Cordially dedicated to Prof. Dr. Otto Sticher (Zurich) on the occasion of his 75th birthday.

Keywords:

Salix purpurea Salicaceae Willow bark extract ICAM-1 Catechol Flavonoids

ABSTRACT

Introduction: A quantified aqueous Willow bark extract (STW 33-1) was tested concerning its inhibitory activity on TNF-a induced ICAM-1 expression in human microvascular endothelial cells (HMEC-1) and further fractionated to isolate the active compounds.

Results: At 50 [micro]g/ml the extract, which had been prepared from Salix purpurea L, decreased ICAM-1 expression to 40% compared to control cells without showing cytotoxic effects. Further liquid-liquid partition revealed an ethyl acetate phase with potent reduction of ICAM-1 expression to 40% at 8 [micro]g/ml. This fraction was comprehensively characterised by the isolation of flavanone aglyca and their corresponding glycosides, chalcone glycosides, salicin derivatives, cyclohexane-1,2-diol glycosides, catechol and trans-p-coumaric acid. All compounds were investigated for their activity on TNF-eft induced ICAM-1 expression. The flavonoid and chalcone glycosides were not active up to 50 p,M, whereas catechol and eriodictyol at the same concentration showed a significant reduction of ICAM-1 expression to 50% of control. Interestingly, other isolated flavanone aglyca like taxifolin, dihydrokaempferol and naringenin showed only weak or moderate inhibitory activity. Eriodictyol was a minor compound in the extract, whereas the catechol content in the extract (without excipients) reached 2.3%, determined by HPLC. One of the isolated cyclohexan-1,2-diol glucosides, 6'-O-4-hydroxybenzoyl-grandidentin, is a new natural compound.

Conclusion: As catechol is quantitatively important in Willow bark extracts it can be concluded from the in vitro data that not only flavonoids and salicin derivatives, but also catechol can probably contribute to the anti-inflammatory activity of Willow bark extracts.

Introduction

Willow bark extracts (Salicis cortex, Salix spec., Salicaceae) are used since ancient times for the treatment of fever, pain and inflammation (Hairier and Everts 1998). Salicylic acid, the in vivo metabolite of salicylic alcohol derivatives, was postulated to be the main active principle of these extracts. Due to the low achievable salicylic acid plasma concentration, which is equivalent to only ~100 mg of acetylsalicylic acid after oral application of 240 mg Willow bark extract (Schmid et al. 2001), recent work concentrated on the additional contribution of polyphenolic compounds on the anti-inflammatory and analgesic effect. Standardised extracts rich in polyphenols showed significant clinical effects (for review Vlachojannis et al. 2009) and extracts as well as fractions containing polyphenols also showed inhibitory activity in vitro on several molecular targets like transcription factors, pro-inflammatory cytokines (Bonaterra et al. 2010), cyclooxygenases and radical production (Khayyal et al. 2005). Consequently, the overall effect of Willow bark extracts can be most likely explained by different effects of various phenolic compounds on distinct targets (Nahrstedt et al. 2007). Wagner and Ulrich-Merzenich (2009) drafted a multi-component/multi-target concept with regard to the importance of multi-target synergy effects for combinations of plant constituents and demonstrated its importance not only for Willow bark extracts, but also for several other phytophar-maceuticals. As further compounds and targets may contribute to the anti-inflammatory effect of Willow bark, a quantified aqueous extract (STW 33-I; Steigerwald) was investigated in a microvascular endothelial cell line (HMEC-1) on its ability to reduce the expression of the adhesion molecule ICAM-1. Adhesion molecules like ICAM-1, VCAM and the selectins are known to be involved in inflammatory processes at endothelial cells (Gearing and Newman 1993). Endothelial cells communicate substantially via adhesion molecules with their counter-receptors on leukocytes to generate intracellular signals followed by inflammation processes under active participation of the endothelium (Cook-Mills and Deem 2005). Furthermore, several in vivo and in vitro models revealed the particular importance of endothelial cells and their expression of adhesion molecules in atherosclerosis and the inflammatory processes initiated therewith (Galkina and Ley 2007). For example, VCAM-1 and ICAM-1 dependent recruitment of immune cells in regions of plaque neovascularization is likely also an important step during the development of atherosclerosis. Therefore, these molecules are rational targets for in vitro testing of compounds showing anti-inflammatory effects in vivo. As the ethyl acetate fraction of STW 33-1 was most active all phenolic compounds (Fig. 1) significantly present in this fraction were isolated and tested as single compounds. To exclude false positive results caused by cytotoxicity, a MTT assay under the same assay conditions was always done in parallel. As the phytochemical characterisation of the polyphenolic pattern of the Salix species S. purpurea L, S. daphnoides Via. and S. fragilis L. is rudimentary (all particularly mentioned in the Pharmacopeia Europeia (PhEur) for Willow bark) and the extract studied had been prepared from Salix purpurea L, the present study is also a first step towards a systematic phytochemical characterisation of pharmaceutically used Willow bark species and resulting extracts.

Materials and methods

Extract

Dry extract from Willow bark (Phan. 6.1, Salix purpurea L., Salicaceae; STW 33-I Steigerwald) obtained from water extraction; drug-to-extract ratio (DEV nativ) 16-23:1, overall salicin content 23-26% (m/m), excipients: silica, magnesium stearate.

Isolation

10 g extract was separated from the excipients by vacuum filtration and suspended in water (250 m1). The resulting suspension was partitioned against n-hexane (2 x 100 ml) to extract the lipophilic compounds. The hexane phase was dried under vacuum to yield only 50 mg residue which was excluded from further tests due to the low amount. The water phase was subsequently partitioned against ethyl acetate (EA, 3 x 100 ml) and n-butanol (BUT, 3 x 100 ml) to yield 1.3 and 2.5 g extract after drying over [Na.sub.2]S[O.sub.4] and evaporation of solvent. The volume of the water phase was reduced to 100 ml on a rotary evaporator and 500 ml EtOH was added to yield the EtOH-soluble part and a precipitate only soluble in pure water ([H.sub.2]0-phase) after stirring. After centrifugation and drying 3.0 g EtOH and 0.5 g H2O phase were obtained (partition process modified according to Nahrstedt et al. 2007). The procedure was repeated five times. An aliquot of the ethyl acetate phase (6.3 g) was divided in two portions and fractionated by CC on Sephadex LH-20 (length 67 cm, [phi] 4.0 cm) eluting with 75% EtOH (1 ml/min) to yield fractions EA1-EA6. Fractions were combined according to similarity in TLC using Naturstoff A (5% (m/v) in Me0H) and anisaldehyde reagent (0.5% anisaldehyde, 84.5% Me0H, 10% glacial acetic acid, 5% sulphuric acid (98%), each v/v) as spray reagent for detection and resulted in 1.74g EA1 (~0-800 ml), 0.15 g EA2 (~800-850 ml), 2.29 g EA3 (~850-1000 ml), 0.70 g EA4 (~1000-1100 ml), 0.47 g EA5 (~1100-1300 ml) and 0.50g EA6 (~1300-1800 ml). Further isolation protocol of compounds 1-20 is described in Chart 1.

The identity of all compounds was confirmed by comparison of spectral data ([1.sup]H NMR, [13.sup]C NMR, ESI-MS, melting points, UV-spectrum, and optical rotation) with literature data of isogran-didentatin A (1, Si et al. 2009), trichocarposide (3, Fernandes et al. 2009), populoside B (4, Zhang et al. 2006a), dihydrokaempferol-7-0-[beta]-D-glucoside (5, Foo and Karchesy 1989), (2R,2S)-naringenin-7-0-[beta]-D-glucoside (6, Zapesochnaya et al. 2002), catechol (7, Smith and Proulx 1976), (2R)-naringenin-5-0-[beta]-D-glucoside (8, Ibrahim et al. 2007, Tyukavkina et al. 1989, Zapesochnaya et al. 2002), (2S)-naringenin-5-0-[beta]-D-glucoside (9, Ibrahim et al. 2007; Tyukavkina et al. 1989; Zapesochnaya et al. 2002), (2R,2S)-eriodictyol-7-0-[beta]-D-glucoside (10, Pan et al. 2008), trans-p-coumaric acid (11, Kort et al. 1996), isosalipurposide (12, Zapesochnaya et al. 2002), 6"-trans-p-coumaroyl-(2R)-naringenin-5-0-[beta]-D-glucoside (13, Vinokurov and Skrigan 1969), 6"-trans-p-coumaroyl-(2S)-naringenin-5-0-[beta]-D-glucoside (14, Vinokurov and Skrigan 1969), taxifolin (16, Markham and Ternai 1976; Han et al. 2007), dihydrokaempferol (17, Foo and Karchesy 1989; Han et al. 2007), eriodictyol (18, Pan et al. 2008; Zhang et al. 2006b), naringenin (15, Markham and Ternai 1976; Zhang et al. 2006b), 6"-trans-p-coumaroylisosalipurposide (19, Zapesochnaya et al. 2002) and phelligrin A (20, Mo et al. 2003). Furthermore, 2D NMR mea-surements ([1.sup]H,[1.sup]H COSY, [1.sup]H,[13.sup]C HSQC, [1.sup]H,[13.sup]C HMBC and [1.sup]H,[1.sup]H ROESY/[1.sup]H,[1.sup]H NOESY) for confirmation of the literature signal assignment were done. In case of R or S assignment also CD spectra were measured. For determination of the D- or L-configuration of a sugar a method based on capillary electrophoresis (CE) was applied (Noe and Freissmuth 1995).

Instrumentation

NMR spectra were measured on a Bruker Avance 600 ([1.sup]H 600 MHz, [13.sup]C 150 MHz at 298.01<) and referenced against undeuterated solvents. LRMS spectra were recorded on a Finnigan MAT SSQ 710A (EI-MS: 70 eV), ThermoQuest Finnigan TSQ 7000 (ESI-MS: 3 kV, capillary: 250 [degrees]C). HRESI-MS was determined on a UHD accurate mass Q-TOF (Agilent Technologies). UV spectra were measured on a Cary 50 Scan UV Visible spectrophotometer (Varian) in Me0H (Uvasol[R] ). Optical rotation was recorded on a Unipol L1000 (Schmidt +Haensch) in Me0H (Uvasol ), cell length: 5 cm. CD spectra were measured on a J-815 spectropolarimeter (Jasco), c=1.5 mg/mL in Me0H at room temperature, quartz cuvette of 0.1 cm path length. HPLC (semi-preparative): Varian ProStar, autosampler 410, 2 pumps 210, detector 335, columns: Varian Pursuit XRs, C18, 5[micro]m, 250 mm x 10 mm, Varian Polaris C8-A, 5[micro]m, 250 mm x 10 mm, Knauer Eurosphere-100, C18, 7[micro]m, 16mm x 25 mm. HPLC (analytical): Elite LaChrom VWR Hitachi, autosampler L-2200, pump L-2130, column oven L-2350, DAD L-2455, column: Purospher[R] STAR RP-18e, 250 mm x 4.6 mm. Flash chromatography system: Armen Spot Liquid Chromatography Flash, columns: SPV D40 RP 18, 25-40 p.m, 90 g (Merck) and SVF D26 Si60, 15-40 [micro]m, 30g (Merck). Absolute configurations of the sugars were measured on a Bio-Rad Biofocus 3000 CE system (70/75 cm, 50 [micro]m quartz capillary) according to Noe and Freissmuth 1995 (modified). Shortly, the glucoside was hydrolyzed (4 M aqueous TFA, 120 [degrees]C, 60 min) and afterwards evaporated to remove the acid until dryness. The hydrolysis product and D-and L-glucose reference substances (Sigma-Aldrich, Roth) were derivatised with 0.1 M S-(-)-1-phenylethylamine respectively and reduced with 0.46 M aqueous sodium cyanoborohydride solution. The resulting diastereomers were subjected to capillary electrophoresis (buffer: 50 mM [Na.sub.2][B.sub.4][O.sub.7] in 23% aqueous MeCN, pH 10.3, injection: 3 psi s, voltage: 30 kV, detection: 200nm, temperature: 27 [degrees]C). Comparison of migration times confirmed the presence of D-glucose.

61-0-4-Hydroxybenzoylgrandidentin (2)

White amorphous powder; UV (MeOH) [[lambda].sub.max] 257 nm; [[alpha].sub.D.sup.22] +41[degrees] (c= 0.0005); [1.sup]H NMR (600MHz, 298K, C[D.sub.3]OD): 1.11-1.19 (1H, m, H-Sb), 1.22-1.30 (1H, m, H-4b), 1.43-1.51 (2H, m, H-3b, H-6b), 1.52-1.61 (21-1, m, H-4a, H-5a), 1.71-1.78 (1H, m, H-3a), 1.85-1.92 (1H, m, H-6a), 3.24-3.28 (1H, m, H-2'), 3.33-3.38 (1H, m, H-4'), 3.57 (1H, ddd, J=2.1, 7.0, 9.2, H-5'), 3.68-3.72 (1H, m, H-1), 3.73-3.78 (1H, m, H-2), 3.42-3.76 (1H, m, H-3'), 4.40 (1H, dd, J=6.9, 11.8, H-6'b), 4.41 (1H, d, J=7.9, H-1'), 4.57 (1H, dd, j=2.1, 11.8, H-6'a), 6.81 (2H, d, J=8.6, H-3", H-5"), 7.88 (2H, d, J=8.6, H-2", H-6"); [13.sup]C NMR (150MHz, 298K, C[D.sub.3]OD): 22.6 (C-4), 23.1 (C-5), 30.0 (C-6), 31.3 (C-3), 64.9 (C-6'), 70.3 (C-2), 72.1 (C-4'), 75.4 (C-2', C-5'), 77.8 (C-3'), 81.6 (C-1), 104.4 (C-1'), 116.1 (C-3", C-5"), 122.2 (C-1"), 132.9 (C-2", C-6"), 163.6 (C-4"), 168.0 (C-7"). LRESI-MS (pos.) m/z 399 [M+[H.sup.+]]; HRESI-MS (pos.) m/z 397.1497 [M-H1, calculated for [C.sub.19][H.sub.25][O.sub.9] 397.1504.

6"-trans-p-Coumaroyl-(2S)-naringenin-5-0-[beta]-D-glucoside (14)

White amorphous powder (MeOH) [[lambda].sub.max]: 228, 287, 311 nm; [[alpha].sub.D.sup.20] = -69[degrees] (c=0.001). [1.sup]H NMR (600MHz, 298 K, C[D.sub.3]OD): 2.68 (1H, dd, J=3.0, 17.3, H-3b), 2.98 (1H, dd, J=13.1, 17.3, H-3a), 3.48 (1H, dd, J=8.7, 9.3, H-4"), 3.51 (1H, dd, J=8.6, 8.6, H-3"), 3.59 (1H, dd, J=7.8, 8.6, H-2"), 3.74 (1H, dd, J=2.2, 6.7, H-5"), 4.32 (11-1, dd, J=6.6, 11.9, H-6"b), 4.59 (1H, dd, J=2.3, 11.9, H-6"a), 4.83 (1H, d, J=7.7, H-1"), 5.29 (1H, dd, J=2.9, 13.2, H-2), 6.11 (1H, d, J=2.2, H-8), 6.37 (1H, d, J=16.1, H-8"9, 6.44 (1H, d, J=2.2, H-6), 6.79 (2H, d, J=8.6, H-3', H-5'), 6.80 (2H, d, J=8.6, H-31", H-5'"), 7.28 (2H, d, J=8.6, H-2', H-6'), 7.46 (2H, d, J=8.6, H-2"', H-6"9, 7.63 (1H, d, J=16.1, H-7'"); [13.sup]C NMR (150MHz, 298 K, C[D.sub.3]OD): 46.3 (C-3), 645 (C-6"), 71.7 (C-4"), 74.7 (C-2"), 75.9 (C-5"), 77.3 (C-3"), 80.2 (C-2), 99.4 (C-8), 100.5 (C-6), 104.3 (C-1"), 107.0 (C-10), 114.9 (C-8'", 116.3 (C-5', C-3'), 116.8 (C-3'", C-5'"), 127.2 (C-1'" ), 129.0 (C-2', C-6'), 131.0 (C-1'), 131.3 (C-2'", C-6'"), 146.9 (C-7'", 159.0 (C-4'), 161.3 (C-4'"), 162.1 (C-5), 166.5 (C-9), 167.3 (C-7), 169.1 (C-9'"), 193.0 (C-4). LRESI-MS (pos.) m/z 581 [M+[H.sup.+]].

EAI (1.5 g): flash chromatography (column SPV D40 RP 18, 25-40 [micro]m, 90 g, precolumn Lichroprep RP-18 25-40 [micro]m (10 a); mobile phase A: [H.sub.2]O, B: Me0H; 0-60 min 70/30 [right arrow] 40/60, 60-65 min 40/60 [right arrow] 0/100: 20 ml/min flow) [right arrow] 10 subtractions EA1.1-10. EA1.5 (160 mg, [R.sub.t]~37-42 min, polarity: Me0H ~48-51%): semi-preparative HPLC on RP-8 (5 pm; isocratic 0-15 min Me0H/[H.sub.2]0 45/55; 4 ml/min) [right arrow] fraction at R, ~10.7 min [right arrow] HPLC (same conditions) [right arrow] 17 mg of 1 and 3 mg of 2.

EA2 (140 mg): semi-preparative HPLC (RP-18 7 [micro]m; 0-16 min Me0H/[H.sub.2]0 55/45 [right arrow] 66/34, flow 10 ml/min) [right arrow] trichocarposide (3, 15 mg, [R.sub.t] ~8.7 min) and populoside B (4, 17 mg. [R.sub.t] ~10.5 min).

EA3 (1.5 g): flash chromatography (column SPV D40 RP 18, 25-40 pm, 90 g, precolumn Lichroprep RP-18 25-40 [micro]m (10 g); mobile phase A: [H.sub.2]O + 0.05% trifluoracetic acid (TEA), B: Me0H + 0.05% TEA; 0-90 min 70/30 [right arrow] 40/60, 90-95 min 40/60 [right arrow] 0/100, 15 ml/min flow) [right arrow] subtractions EA3.1-EA3.5. EA3.1 (110 mg. [R.sub.t]~0-20 min, polarity: 30-37% Me0H): RP-18 HPLC (semi-preparative, 5 [micro]m; 0-15 min Me0H/[H.sub.2]O 35/65 [right arrow] 42/58; flow 3 ml/min) [right arrow] dihydrokaempferol-7-0-[beta]-D-glucoside (5. 13 mg, R, -10 min). EA3.3 (390 mg, [R.sub.t] ~40-52 min, polarity: 43-47% Me0H) was stored over night in the fridge [right arrow] precipitate [right arrow] washing with cooled Me0H [right arrow] 37 mg of (2R/S)-naringenin-7-0-[beta]-D-glucoside (6).

EA4 (550 mg) [right arrow] CC on silica gel (63-200 [micro]m, length 30 cm.[phi] 0 3.0 cm) Et0Ac/Me0H (100/0 [right arrow 0/100) as mobile phase [right arrow] subtractions EA4.1-EA4.5. E4.1 (100% EtOAc) was catechol (7, 24 mg). EA4.2 (40 mg, 95/5 [right arrow] 50/50) and EA4.4 (70 mg, 30/70 [right arrow] 20/80) -> HPLC (RP-18, semi-preparative 7 [micro]m; EA4.2: 0-25 min MeOH/[H.sub.2]0 40/60 [right arrow] 60/40, 25-30 min 60/40 isocratic; EA4.4: 0-15 min MeOH/[H.sub.2]0 32/68 [right arrow] 35/65, 15-30 min 35/65, flow 10 ml/min each) [right arrow] (2R)-naringenin-5-0-[beta]-D-glucoside (8, 3 mg, [R.sub.t] ~20 min), (2S)-naringenin-5-O-[beta]-D-glucoside (9, 10 mg, [R.sub.t] ~22 min) and a fraction containing (2R,25)-eriodictvol-7-0-[beta]-D-glucoside (10, 14 mg, [R.sub.t] ~18 min), Impure 10 [right arrow] HPLC (same conditions) [right arrow] pure 10 (8 mg). From EA4.2 (11 mg): impure 11 [right arrow] HPLC (RP-8, semi-preparative 5 [micro]m; 0-11 min isocratic Me0H/[H.sub.2]O 30/70, 11-12 min 30/70 [right arrow] 40/60, 12-16 min 40/60 isocratic, 16-20 min 40/60 90/10, isocratic 20-24 min 90/10; flow 4 [right arrow] trans-p-coumaric acid (11, 3 mg, [R.sub.t] ~22 min).

EA5 (420 mg) [right arrow] flash chromatography (column SPV D40 RP 18, 25-40 [micro]m, 90 g, precolumn Lichroprep RP-18 25-40 [micro]m (10 g)); mobile phase A: [H.sub.2]O + 0.05% TFA, B: Me0H + 0.05% TFA; 0-90 min 60/40 [right arrow] 40/60, 90-95 min 40/60 [right arrow] 0/100; 15 ml/min flow) [right arrow] subtractions EA5. EA5.4 (170 mg, [R.sb.t] ~3.5-50 min, polarity: 48-51% Me0H) [right arrow] flash chromatography on silica gel (15-40 [micro]m SVF D26 Si60 30 g; 0-15 min Et0Ac/Me0H 100/0, 15-20 min 100/0 [right arrow] 95/5, 20-35 min 95/5, 35-40 min 95/5 [right arrow] 80/20, 40-60 min 80/20. flow 7 ml/min). Impure isosalipurposide (12, 90 mg, [R.sub.t] ~41-54 min polarity: EtOAc/MeOH 80/20) [right arrow] semi-preparative HPLC (RP-18, 7 [micro]m, 0-10 min Me0H/[H.sub.2]O 50/50 [right arrow] 60/40, 10-13 min 60/40 [right arrow] 90/10 flow 10 ml/min) [right arrow] 20 mg of 12 ([R.sub.t] ~8 min). EA5.6 (80 mg, [R.sub.t] ~80-87 min, polarity: 58-59% Me0H) [right arrow] semi-preparative HPLC (RP-18, 7 [micro]m, Me0H/[H.sub.2]0 0-7 min 50/50 [right arrow] 58/42, 7-20 min 58/42 [right arrow] 63/37; flow 10 ml/min) [right arrow] enriched fraction (70 mg, [R.sub.t] 13-15 min) of 6"-trans-p-coumaroy1-(2R)-naringenin-5-0-[beta]-D-glucoside (13). This fraction [right arrow] HPLC (RP-18, semi-preparative column 5 [micro]m; Me0H/[H.sub.2]0 0-15 min 70/30 [right arrow] 78/22; 3 ml/min) [right arrow] 13 (26 mg, [R.sub.t] ~7 min). EA5.7 (80 mg, [R.sub.t] ~88-95 min polarity: 60-100% WOW a fraction containing 6"-trans-p-coumaroyl-(25)-naringenin-5-0-[beta]-D-glucoside (14) precipitated after cooling over night. The precipitate (50 mg) was washed with cool Me0H [right arrow] semi-preparative HPLC (RP-18 7 pm; 0-14 min Me0H/[H.sub.2]0 60/40 [right arrow] 67/33; flow 10 ml/min) [right arrow] 14 (38 mg).

EA6 (460 mg) [right arrow] flash chromatography (column SPV D40 RP 18, 25-40 [micro]m, 90 g, precolumn Lichroprep RP-18 25-40 [micro]m (10 g); mobile phase A: [H.sub.2]0, B: Me0H; 0-10 min 80/20 [right arrow] 60/40, 10-80 min 60/40 [right arrow] 30/70; 80-85 min 30/70, 85-90 min 30/70 [right arrow] 0/100, 90-95 min 0/100, 20 ml/min flow) 9 suhfractions EA6.l - EA6.9. EA6.2 (50 mg, [R.sub.t] ~22-29 min polarity: 45-48 % Me0H) [right arrow] semi-preparative HPLC (RP-18, 5 [micro]m; 0-18 min Me0H/[H.sub.2]0 45/55 [right arrow] 48/52, 18-20 min 48/52 [right arrow] 95/5; flow 3 ml/min) [right arrow] impure taxifolin (16, 11 mg, [R.sub.t] 13 min). Impure 16 [right arrow] HPLC (RP-18, 5 [micro]m, 0-15 min MeOH\[H.sub.2]0 32/68 [right arrow] 38/62; flow 3.5 ml/min) [right arrow] pure 16 (4 mg, [R.sub.t] 10.5 min). EA6.3 (40 mg, [R.sub.t] ~29-35 min, polarity: 48-51% Me0H) [right arrow] semi-preparative HPLC (RP-18, 5 [micro]m; 0-15 min Me0H/[H.sub.2]0 55/45 [right arrow] 65/35, flow 3 ml/min) [right arrow] dihydrokaempferol (17. 15 mg, [R.sub.t] ~11 min). EA6.4 (50 mg, [R.sub.t] ~36-48 min, polarity: 51-56% Me0H) [right arrow] semi-preparative HPLC (RP-18, 5 pm; 0-21 min Me0H/[H.sub.2]0 55/45 [right arrow] 64/36: flow 3 ml/min) [right arrow] (2R,2S)-eriodictyol (18, 21 mg). EA6.5 (120 mg, R, -48-58 min, 56-61% MeOH) consisted of naringenin (15). EA6.7 (30 mg, [R.sub.t] ~70-75 min, 66-68% Me0H) [right arrow] HPLC (RP-18. 5 [micro]m; 0-5 min Me0H/[H.sub.2]0 68/32 [right arrow] 70/30, 5-20 min 70/30 isocratic; flow 3 ml/min) [right arrow] 6"-trans-p-coumaroylisosalipurposide (19, 11 mg, [R.sub.t] ~12 min). EA6.9 (20 mg, [R.sub.t] ~82-89 min, 70-94% Me0H) was phclligrin A (20, 4 mg).

Chart 1. Detailed isolation protocol of compounds 1-20.

Determination of catechol

The content of catechol (7) was determined by HPLC. STVV 33-1 (recipients removed) was dissolved in HPLC grade water and sonicated for 15 min. 75 [micro]l were injected and analyzed by analytical HPLC, n = 3. Conditions: A: H2O + 0.1% formic acid, B: MeCN: [H.sub.2]O 95/5; 0-10 min 90/10 [right arrow] 87/13, 20-30 min 87/13 isocratic; 0.75 ml/min flow, oven temperature 30 [degrees]C. A calibration curve of catechol was established (12 point, n = 3, range 0.05-125[micro]g/ml) by height of the catechol peaks (y = 26,591x + 9823.2, [R.sup.2]=0.9982). The content of catechol in the extract was determined to be 2.3 [+ or -] 0.1%.

Cell culture

Confluent grown human microvascular endothelial cells (HMEC-1, Ades et al. 1992) were pretreated either with test compounds, parthenolide (Calbiochem, purity [greater than or equal to]97%, 5 or 10 [micro]M, positive control) or medium (ECGM, endothelial cell growth medium (Provitro)+ 10% FCS + antibiotics + supplements (both Provitro)) as negative control in 24-well plates. 30 min later, 10 ng/m1 TNF-[alpha] (Sigma-Aldrich) were added to stimulate the ICAM-1 expression. After 24 h of incubation (New Brunswick Scientific, 37 [degrees]C, 5% [CO.sub.2]), cells were washed with PBS, removed from the plate with trypsin/EDTA and fixed with formalin. After incubating with FITC-labelled mouse antibody against ICAM-1 (Biozol) for 20 min, the fluorescence intensity was measured by FACS analysis (Becton Dickinson Facscalibur[TM] ). ICAM-1 expression of cells treated with TNF-[alpha] only was set as 100%.

The MTT viability assay was performed according to Mosman 1983 (modified). Briefly, confluent grown HMEC-1 cells were incubated with fractions and test compounds (concentration according to the ICAM-1 assay) in 96-well plates in medium (pure medium for negative control; n = 6 for each value). After 24 h the fractions/substances and the supernatants were removed and 10 [micro]l MTT (5 mg/ml) in 90 [micro]l medium was added for 3 h. This solution was exchanged for 10% sodium dodecyl sulphate and 24 h later the absorbance was measured with a SpectraFluor plus plate reader (Tecan, Crailsheim, Germany) at 560 nm.

Statistical analysis

Each experiment was performed 3 times in duplicates for ICAM-1 assay and 3 times in sextuplicates for viability assay. Results are expressed as mean [+ or -] SD. Statistical significance between control and test compounds was determined by one-way ANOVA (p < 0.05) followed by Dunetts Post hoc test.

Results

An aqueous extract of Willow bark was separated from its excipients (magnesium stearate and silicium dioxide) and in vitro tested at 10 and 50 [micro]g/ml in HMEC-1 for reduction of TNF-[alpha] induced ICAM-1 expression. The extract showed a potent and dose-dependent reduction of ICAM-1 expression to 70% and 40% of control (Fig. 2A) without exhibiting cytotoxic effects (Fig. 5A) Following a liquid-liquid separation strategy ("Material and methods") of the extract four fractions were obtained for further pharmacological testing. The ethyl acetate (EA), n-butanol (BUT), ethanol (EtOH) and [H.sub.2]O soluble phase were tested in different concentrations resembling their yield from the extract. The EA phase showed the strongest activity and decreased the ICAM-1 expression to ~40% of control at 8 pig/m1 and was chosen for further fractionation. The BUT (15 [micro]g/m1) and EtOH (19 [micro]g/ml) phase showed moderate effects with a decrease of ICAM-1 expression to 75 and 82% of control, respectively, whereas the [H.sub.2]O-soluble phase was not active at 3 [micro]g/m1 (Fig. 2A). None of the tested phases showed cytotoxic effects at the used concentration (Fig. 5A).

The EA phase was worked up with column chromatography on Sephadex LH-20 to obtain 6 fractions showing a significant activity in fraction EA4 (Fig. 2B). All fractions were worked up to yield the flavanone glycosides (2R)-naringenin-5-0-[beta]-D-glucoside (8), (2S)-naringenin-5-O-[beta]-D-glucoside (9), (2R,2S)-naringenin-7-0-[beta]-D-glucoside (6), 6"-trans-p-coumaroy1-(2R)-naringenin-5-0-[beta]-D-glucoside (13), 6"-trans-p-coumaroy1-(2S)-naringenin-5-0-[beta]-D-glucoside (14), (2R,2S)-eriodictyol-7-0-[beta]-D-glucoside (10), the flavanonol glycoside dihydrokaempferol-7-O-[beta]-D-glucoside (5), the chalcone glycosides isosalipurposide (12), 6"-trans-p-coumaroylisosalipurposide (19), the flavanone or flavanonol aglycones naringenin (15), taxifolin (16), dihydrokaempferol (17), eriodictyol (18) and phelligrin A (20), the salicyl alcohol derivatives trichocarposide (3) and populoside B (4) as well as catechol (7) and trans-p-coumaric acid (11, Fig. 1) by subsequent open column chromatography, flash chromatography and HPLC. Additionally, compounds 1 and 2, representing rare coumaroyl- and benzoyl-glycosides with cyclohexanol substitution were isolated of which compound 2 is a new one. Isolated compounds were tested at 20 and 50 [micro]M for their ability to reduce ICAM-1 expression in HMEC-1. While none of the glucosides were active (data not shown), probably due to their high hydrophilicity, only the aglyca catechol (7) and the flavanone eriodictyol (18, Figs. 3 and 4) exhibited significant activity. Catechol was further tested in different concentrations showing a dose dependent decrease of ICAM-1 expression (Fig. 4) without any signs of cytotoxicity in the MTT assay (Fig. 5B). Interestingly, neither the isolated flavanonol aglyca (introduction of an additional hydroxyl group at C-3) nor naringenin (15) showed comparable activity to 18. Whereas 18 is only a minor compound, the amount of 7 in the extract is noteworthy and reached 2.3%. Despite of several phytochemical investigations regarding the genera Salix and Populus (both Salicaceae) also a hitherto unknown esterified cyclohexandiol-[beta]-O-glucoside (2) was isolated. The [1.sup]H NMR spectrum substantiated the presence of a [beta]-configurated glucose residue (Domisse et al. 1986; Si et al. 2009). Furthermore, the downfield region showed two doublets at [delta] = 6.81 and 7.88 (J = 8.6, 2H, each) indicating together with a quaternary carbon signal at [delta] = 168.0, which was coupled to the phenyl protons in HMBC, the presence of a 4-hydroxybenzoyl moiety. The resonance signals of the protons at C-6 of glucose ([delta] =4.40, 4.57) were shifted significantly downfield in comparison with unsubstituted glucose pointing to C-6' as position for esterification. This was confirmed by a HMBC experiment showing [3.sup]J correlations between the ester carbonyl and H-6'. The 1,2-dihydroxycyclohexane unit was deduced from 4 pairs of broad geminal proton signals resonating between [delta] =1.11-1.92 as well as from two additional downfield shifted and coupling proton signals at [delta] =3.68-3.78. This unit is linked at position 1 to the glucose at C-1' what was substantiated in the NOESY spectrum as well as by the corresponding [3.sup]J C,H correlations in the HMBC. Extensive [13.sup]C and 2D NMR measurements corroborated the presence of a 1,2-dihydroxycyclohexyl-6'-0-4-hydroxy-benzyl-[beta]-D-glucopyranoside.

In order to investigate the stereochemistry of the 1,2-hydroxycyclohexane-ring, the acetal was cleaved by acid hydrolysis (TFA 1 M, 50% Me0H in a Wheaton V-Vial, 120 [degrees]C, 15 min) and subsequent TLC of the reaction products on silica gel (mobile phase: hexane/ethyl acetate/tetrahydrofuran/methanol/formic acid 12/10/3/3). Cis and trans 1,2-hydroxycyclohexane (Sigma) were co-chromatographed as reference substances. After derivatisation with anisaldehyde reagent and heating at 120 [degrees] C, cis-1,2-hydroxycyclohexane was identified by its purple colour and [R.sub.f] value. Thus, structure of 2 was unambiguously elucidated as the new cis-1,2-dihydroxycyclohexyl-6'-O-4-hydroxybenzyl-[beta]-D-glucopyranoside and named 6'-O-4 hydroxy-benzoylgrandidentin.

For compounds 13 and 14, isolated the first time by Vinokurov and Skrigan (1969), the NMR data were not given in the literature and thus are presented for compound 14 in the experimental part. Compound 13 showed identical mass and UV absorption, but optical rotation was different [[alpha].sub.D.sup.20] = 34. [1.sup]H NMR signals of 13 were identical to those of 14 ( [+ or -]0.05 ppm) except for H-6: [delta] = 6.36, d, J= 2.2 Hz, H-1": [delta] = 4.90, d, J= 7.5 Hz, H-3a: [delta] = 3.04, dd, J= 12.9, 16.4 Hz and H-3b: [delta] =2.61, dd, J= 3.0 and 16.4 Hz. Signals in the [13.sup]C NMR spectrum were also identical ([delta][+ or -] 0.3 ppm), except for C-5 ([delta] = 161.4 ppm), C-10 ([delta] = 106.4 ppm) and C-1" ([delta] = 103.3 ppm). Stereochemical assignment (8/9 and 13114) was carried out by CD spectroscopy and comparison with the literature (Tyukavkina et al. 1989).

Discussion

As the tested Willow bark extract showed significant activity on the TNF-[alpha] induced inhibition of the adhesion molecule ICAM-1 in HMEC-1 cells, a liquid/liquid partition protocol was applied to identify the compounds responsible for the observed activity. The EA-phase, containing a variety of phenols from different structural classes, exhibited the most pronounced activity which resulted at least mostly from the presence of the compound catechol (7). Among the other isolated phenols only the flavanone aglycone eriodictyol (18) showed significant in vitro activity. The activity of 18 is interesting from the structural point of view as neither 15 nor the corresponding flavanonols 16 and 17 were active. These points to a relatively specific mechanism besides the always postulated effect caused by the 3,4-dihydroxyphenyl moiety of flavonoids. Unfortunately, this part of investigation was not expendable to flavones and flavonols, as luteolin, apigenin and quercetin are active in the ICAM-1 assay, but showed partially unspecific cytotoxic effects in concentrations above 20 [micro]M (data not shown). In literature, specific effects of 18 in comparison to luteolin or 15 are relatively scarce and reported for example in the field of TNF-[alpha] mediated cytotoxicity (Haptemariam 1997), whereas a stronger activity of luteolin in comparison to 18 is more common (e.g. van Zanden et al. 2004; Takano-lshikawa et al. 2003; Sasaki et al. 2003) and often attributed to the additional 2,3-double bond. The importance of the latter structural feature is also addressed in the field of attenuation of VCAM-1, ICAM-1 and E-selectin, in other endothelial cells, but interestingly without testing eriodictyol (Lotito and Frei 2006). Nevertheless, Takano-Ishikawa et al. (2003) reported in accordance with our results on a significant inhibition of E-selectin (evoked by TNF-[alpha] addition) in HUVECs by eriodictyol (18). As luteolin and 18 were both reported to be able to reduce Toll like receptor mediated inflammatory processes in vitro (Lee et al. 2009), an in vitro reduction of adhesion factor expression by 18 is likely. As it is only a trace compound in the extract a substantial contribution to the anti-inflammatory in vivo effects seems to be questionable. In contrast, 7 is present in higher amounts (>2%) likely resulting from the degradation of the 1-hydroxy-6-oxo-2-cyclohexene-1-carboxylic acid moiety of salicin and catechin derivatives. Catechol (7) is not only able to reduce the ICAM-1 expression in HMEC-1 cells, but is also reported as a potent inhibitor of the transcription factor NF-kB (Ma et al. 2003) resulting in the inhibition of LPS induced NO-production (Zheng et al. 2008). Ruuhola et al. 2003 observed the facile degradation of 1-hydroxy-6-oxo-2-cyclohexene-1-carboxylic acid moiety of salicortin to 7 in plant material under different pH conditions, what is confirmed here by the presence of free 7 in the aqueous extract. Furthermore, Knuth et al. (2011) were able to show that salicortin can produce 7 also under cell culture conditions. As this process is also mentionable in vivo for all compounds exhibiting a cyclohexenon carboxylic acid moiety, like the salicyl alcohol derivatives salicortin, 2'-acetylsalicortin and tremulacin (Poblecka-Olech et al. 2007) or the catechin derivative catechin-3-O-(1-hydroxy-6-oxo-2-cyclohexene-1-carboxylic acid)-ester (Jurgenliemk et al. 2007) the amount of 7 would further increase in vivo. Thus, the release, presence and metabolism of 7 in vivo and its possible contribution on the anti-inflammatory effects of Willow bark preparations is an interesting topic of further research currently in progress. As catechol is a simple phenol also occurring in food like coffee, malt, bread crust or cocoa powder (Lang et al. 2008) the data are of general interest and complement the results published by Zheng et al. 2008 on possible beneficial effects of this compound.

Noticeable from the phytochemical point of view is, besides the isolation of the new compound 6'-0-4-hydroxybenzoylgrandidentin (2), the presence of flavanone (13, 14) and chalcone glycosides (19) substituted at 6-OH of glucose with a coumaroyl moiety. It has to be evaluated by comprehensive phytochemical analyses whether this is characteristic for S. purpurea in comparison to other Salix species. Moreover, the isolation of 20 is noticeable as this compound has only been isolated from Phellinus igniarius (L ex Fr.) Quel. an edible and pharmaceutically used mushroom of the Hymenochaetaceae (Agaricomycetidae) family (Mo et al. 2003). As there is no further report on the isolation and production of flavonoids associated to orthohydroxybenzyl moiety (typical for salicyl alcohol derivatives), and P. igniarius is often growing on Salix spec., the question arises whether P. igniarius uses the services of the secondary metabolite spectrum of its respective host for its own secondary metabolite profile.

Acknowledgements

Special thanks are due to Steigerwald Arzneimittelwerk GmbH for financial support, to Dr. U. Kroll and Dr. E.-U. Heinrich from this company for providing the extract, to Dr. E. Ades, F.J. Candal of CDC (USA) and to Dr. T. Lawley of Emory University (USA) for providing the HMEC-1. We thank J. Kiermaier and W. Sollner (Central Analytic of NWF IV, University of Regensburg) for measuring the mass spectra. We are indebted to Dr. T. Burgemeister and F. Kastner (Central Analytic of NWF IV, University of Regensburg) for recording the NMR spectra. S. Knuth (Department of Pharmaceutical Biology, University of Regensburg) is acknowledged for measuring the ICAM-1 assays of phelligrin A.

0944-711 3/$ - see front matter [C] 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j,phyrned.2011.08.065

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* Corresponding author. Tel.: +49 09419434759; fax: +49 09419434990. E-mail address: Joerg.Heilmann@chemie.uni-regensburg.de (J. Heilmann).

A. Freischmidt (a), G. Jurgenliemk (a), B. Kraus (a), S.N. Okpanyi (b), J. Muller (b), 0. Kelber (b), D. Weiser (b), J. Heilmann (a), *

(a.) Lehrstuhl fur Pharmazeutische Biologie, Universitat Regensburg, 93040 Regensburg, Germany

(b.) Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany
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Author:Freischmidt, A.; Jurgenliemk, G.; Kraus, B.; Okpanyi, S.N.; Muller, J.; Kelber, O.; Weiser, D.; Heil
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Mar 1, 2012
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