NPC
Natural Product Communications
Poly[3-(3,4-dihydroxyphenyl)glyceric Acid] from Anchusa italica Roots
2010 Vol. 5 No. 7 1091 - 1095
Vakhtang Barbakadzea,∗, Lali Gogilashvilia, Lela Amiranashvilia, Maia Merlania, Karen Mulkijanyana, Manana Churadzea, Antonio Salgadob and Bezhan Chankvetadzec a
I.Kutateladze Institute of Pharmacochemistry, 36 P. Sarajishvili str., 0159 Tbilisi, Georgia
b
Departamento de Química Médica, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain
c
Department of Physical and Analytical Chemistry and Molecular Recognition and Separation Science Laboratory, School of Exact and Natural Sciences, Tbilisi State University, 3 Chavchavadze Ave., 0179 Tbilisi, Georgia
[email protected]
Received: March 10th, 2010; Accepted: April 26th, 2010
Elucidation of the main structural unit of a water-soluble, high-molecular weight preparation from the crude polysaccharides of Anchusa italica Retz. roots has been carried out. According to 13C NMR, 1H NMR and 2D heteronuclear 1H/13C HSQC spectral data, the main structural element of the high-molecular, water-soluble preparation was a regularly substituted polyoxyethylene chain, namely poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene]. Most carboxylic groups of this caffeic acid-derived polymer of A. italica are methylated. Keywords: Anchusa italica, Boraginaceae, structure elucidation, caffeic acid-derived polymer, chemotaxonomic marker, poly[3-(3,4-dihydroxyphenyl)glyceric acid], poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene].
As reported in our previous studies, the main chemical constituent of the high molecular fractions from water extracts of Symphytum asperum, S. caucasicum and S. officinale (Boraginaceae) is either a caffeic acid-derived polymer, namely poly[3-(3,4-dihydroxyphenyl)glyceric acid] or poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl) ethylene] [1,2]. This polymer possesses various types of biological activities. For instance, it shows anticomplementary, antioxidant and anti-inflammatory properties [3-5], it can modulate the apoptosis of Bchronic lymphocytic leukemia cells and the cell cycle progression [6,7], and it completely abrogates the adhesion of murine B16 melanoma cells to tumoractivated hepatic sinusoidal endothelium (HSE) [8]. Within our ongoing search for biologically active caffeic acid-derived polymers in plant species belonging to different genera of the Boraginaceae family, we have now carried out the isolation and structure elucidation of a water-soluble, high-molecular preparation from the crude polysaccharides of Anchusa italica roots. Crude polysaccharides from the roots of A. italica (CP-AI) were extracted, as previously described [9]. A
Table 1: Total sugars and monosaccharide composition of CP-AI and HM-AI of A.italica. Preparation CP-AI HM-AI
Total Monosaccharides, % sugars, Uronic Fru Rha Xyl Ara Glc Gal % acid 64.0 18.25 15.0 1.8 0.6 10.3 6.25 11.8 27.3 13.5 1.4 0.4 5.8 6.2
Fru, fructose; Rha, rhamnose; Xyl, xylose; Ara, arabinose; Glc,glucose; Gal, galactose.
high-molecular (>500 kDa) fraction (HM-AI) was isolated from the crude polysaccharides by means of ultrafiltration through membrane filters with a cut-off value of 500 kDa, as previously reported [1,2]. The sugar composition of CP-AI and HM-AI is presented in Table 1. According to monosaccharide composition, we suggested that the main polysaccharide component of A. italica roots, as opposed to S. asperum and S. caucasicum [10], was acidic arabinogalactan, and the minor component, glucofructan. Fractionation procedures led to the elimination of most ballast polysaccharides.
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Table 2: Signal assignments in the 13C and 1H NMR spectra of poly[3(3,4-dihydroxyphenyl)glyceric acid] from A. italica roots (δ, ppm)
1' COOR O
HC 1
2
CH
С atom no.
1'' 6''
2''
5''
3'' 4''
Barbakadze et al.
1' OH
OH
Figure 1: The repeating unit of poly[3-(3,4-dihydroxyphenyl)glyceric acid] of A. italica roots. R=H, CH3.
The HM-AI contained neither fructose nor glucose. Thus, the ultrafiltration of crude polysaccharides allowed the complete removal of glucofructan and to obtain a preparation, which still contained some amount of acidic arabinogalactan. The UV and IR spectra of HM-AI were similar to those of previously isolated polyethers from S. asperum, S. caucasicum and S. officinale [1,2]. The IR spectrum of HM-AI showed all characteristic bands corresponding to the hydroxyl groups attached to the aromatic ring, as well as the carboxyl and ether groups. The IR spectrum contained bands at 1605 and 1404 cm-1 for the carboxyl group and 1736 cm-1 for its ester form [11]. HM-AI was further characterized by using different NMR spectroscopy techniques. The 1H NMR, 13C NMR, and 2D heteronuclear 1H/13C HSQC spectra of HM-AI showed a complete set of resonances characteristic of poly[3-(3,4-dihydroxyphenyl)glyceric acid], which was earlier found in water-soluble high molecular fractions of S. asperum, S. caucasicum and S. officinale [1,2]. The 13C NMR spectrum showed nine resonances that were characteristic of a 3-(3,4dihydroxyphenyl)glyceric acid residue (Figure 1). Two signals with chemical shifts at δ 78.84 and 80.96 ppm were thought to belong to oxygen-bound protonated aliphatic carbon atoms. Another six signals were assigned to aromatic carbon atoms (protonated carbon atoms at δ 118.02, 119.20 and 122.98 and quaternary carbon atoms at δ 132.19, 144.46, and 145.25). Then, two non-sharp signals (δ 172.84 and 175.56) were thought to be due to two carboxyl groups. A resonance in the 13C NMR spectrum at δ 54.86, which correlated with the 1H resonance at δ 3.85, suggested the presence of methoxy groups in carboxylic acid methyl esters. Thus, the signals at δ 175.56 and δ 172.84 were assigned to carboxylic acid groups and methyl ester carbonyl functions, respectively [11-15]. About 70 % of the carboxyl groups of the polymer chain were methylated (MeO: 13C, δ 54.86; 1H, δ 3.85). The extent of methyl esterification was calculated by comparing the integral intensity of the methyl ester signal (δ 3.85, 0.5 H) to that of the aliphatic proton signal at
1 2 1'' 2'' 3'' 4'' 5'' 6''
13
C chemical shift, δC, ppm 175.56 (COOH) 172.84 (COOCH3) 54.86 (OCH3) 78.84 80.96 132.19 118.02 145.25 144.46 119.20 122.98
1
H chemical shift, δH, ppm 3.85 (OCH3) 5.24 4.71 7.16 7.07 7.06
H1 (δ 5.24, 0.7 H) in a 1H NMR experiment that included a WATERGATE water suppression routine. The presence of methoxy groups at C3'' and/or C4'' in the aromatic ring is excluded as there are no downfield shifts of the signals of C3'' (δ 145.25) and/or C4'' (δ 144.46), which would be expected from methylation of any of these hydroxyl groups [14,15]. Moreover, the 2D DOSY experiment gave similar diffusion coefficients for the methylated and non-methylated signals. Both sets of signals fell in the same horizontal. This would imply a similar (same order of magnitude) molecular weight for methylated and non-methylated polymers. This was further evidenced by graphic representations of the intensity decay of the 1H signals of aromatic H-2″ and H-1 at δ 7.16 and 5.24 (Figures 2a and 2b, respectively), and that of the methoxy group at δ 3.85 (Figure 2c). These three 1H signals essentially showed the same curve shape, whereas the resonance due to residual water at δ 4.35 (Figure 2d) followed a different decay pattern (faster diffusion). Thus, we conclude that the NMR signals of both methylated and non-methylated carboxylic groups originate from the same poly[3-(3,4-dihydroxyphenyl) glyceric acid] polymer. The total assignment of the complete set of resonances characteristic for poly[3-(3,4-dihydroxyphenyl)glyceric acid] in the 13C NMR and 1H NMR spectra, based on correlations between protons and carbon atoms by means of the 2D 1H/13C HSQC spectrum, were carried out in accordance with the previous paper [1] and are listed in Table 2. Apart from the above-mentioned signals, which were characteristic for a 3-(3,4-dihydroxyphenyl)glyceric acid residue, the 13C NMR, 1H NMR and 2D 1H/13C HSQC spectra of HM-AI showed the following additional resonances: δ 70.33, 72.52, 101.57, 102.21 (13C NMR); δ 3.78, 4.02 (1H NMR). 2D Correlations were visible between the following proton and carbon atom pairs: δ 3.78/70.33, 4.02/72.52. These additional signals might have appeared in the spectra of HM-AI
Poly[3-(3,4-dihydroxyphenyl)glyceric acid] from Anchusa italica
Natural Product Communications Vol. 5 (7) 2010 1093
a
b
c
d
Figure 2: Intensity decay of H signals at δ 7.16 (Ar H-2″) (a), δ 5.24 (H-1) (b), δ 3.85 (OMe) (c) and δ 4.35 (residual water) (d). X-axis, s/cm2; Y-axis, relative intensity (dimensionless). 1
due to the presence of residual polysaccharide impurities, namely acidic arabinogalactan and/or rhamnogalacturonan [16-18]. Thus, according to the aforementioned NMR data, the main structural element of a high-molecular, watersoluble preparation isolated from the roots of A. italica is a regularly substituted polyoxyethylene, namely poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene]. As in the corresponding preparations of S. asperum, S. caucasicum and S. officinale [1,2], the compound identified in this research is a representative of natural polyethers with a 3-(3,4-dihydroxyphenyl)glyceric acid residue as the repeating unit (Figure 1). However, in contrast with the Symphytum polymer, most of the carboxylic groups of this caffeic acid-derived polymer of A. italica are methylated. The repeating unit of this polymer contains two asymmetric carbon atoms. The establishment of the absolute configuration of these chiral atoms, further purification of HM-AI from residual polysaccharides and/or determination of structural significance of these residual carbohydrates will be the subject of further research. Also, future study should clarify the physiological function of this polyether in plants. There is no information on the biosynthesis of such a polymer in plants but, from the chemical viewpoint, this process might be conceived as the epoxidation of the double bond in caffeic acid, followed by the polymerization of the resulting epoxide [1]. The detection of poly[oxy-1carboxy-2-(3,4-dihydroxyphenyl)ethylene] in the genus Anchusa shows that its biosynthesis is not an unique
feature of the genus Symphytum. The detection of this compound in other genera of Boraginaceae would be interesting from the chemotaxonomic point of view. This unusual caffeic acid-derived polymer could be a chemotaxonomic marker for genera of Boraginaceae. The biosynthetic pathway responsible for this compound might also be unique for Boraginaceae. Besides, the presence of poly[3-(3,4dihydroxyphenyl)glyceric acid] in different Boraginaceae genera expands the source of raw materials of this biologically active polymer. Experimental General methods: UV spectra were recorded on a DU 520 UV/Vis spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA). IR spectra in KBr disc were obtained on an Avatar 370 FT-IR spectrophotometer (Thermo Nicolet Corporation, Madison, WI, USA). GC was performed on a HP 5890A chromatograph (Palo Alto, CA, USA), equipped with a flame ionization detector (FID), a HP 3393A integrator, and fitted with a WCOT HP-1MS fused silica capillary column (30 m x 0.25 mm i.d., 0.25 µm film thickness) (Agilent Technologies Inc., Santa Clara, CA, USA). Chromatographic conditions: sample volume – 1 µL; injection mode - splitless; injector and detector temperatures - 285oC and 300oC, respectively; initial temperature - 160°C, 1 min; final temperature - 290°C, 1 min; temperature ramp rate 7°/min; carrier gas - N2. 1 H, 1H/13C HSQCED and 2D DOSY NMR spectra of 1% solutions in D2O were recorded on a Bruker AVANCE 700 NMR spectrometer (operating 1H and
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C NMR frequencies at 700.015 and 176.05 MHz, respectively), fitted with a 5 mm inverse QXI 700 MHz S4 probe head, XYZ-gradient unit and temperature controller. All spectra were recorded at 80ºC, and with acetone-d6 as the internal standard (δC 31.42 ppm, δH, 2.225 ppm vs. Me4Si). Monodimensional, fully protondecoupled 13C NMR spectra were recorded at 80ºC on a Bruker AVANCE II 300 NMR spectrometer (operating 1 H and 13C NMR frequencies at 300.150 and 75.472 MHz, respectively), fitted with an inverse 5 mm QNP 300 MHz S1 probe head, Z-gradient unit and temperature controller. In every case, the proton 90º hard pulse was optimized after the sample was equilibrated at 80ºC. For the 1H and the phase sensitive 1 H/13C HSQCED experiments, a standard Bruker pulse sequence was employed. For the 2D DOSY experiments, a Bruker pulse sequence that incorporated stimulated echo and longitudinal eddy currents and bipolar gradients for diffusion was used for both optimization of diffusion time and gradient pulse duration (1D) and then diffusion measurement (2D) [19]. For the monodimensional 13C spectra, two different pulse sequences were tried. One was a conventional Bruker experiment that included power gated decoupling and 30º flip angle. The second was a UDEFT monodimensional pulse sequence that used a flip back pulse at the end of the acquisition to decrease the relaxation delay (d1) and NOE [20]. Plant material: Fresh roots (1.31 kg) of A.italica were harvested in the neighborhood of Tbilisi (Georgia) in June 2009 and identified by Dr M. Churadze. A voucher specimen # 224 of A.italica was deposited in the Herbarium of the Institute of Pharmacochemistry, Tbilisi, Georgia. Extraction and isolation: Roots were cut into small pieces, air-dried (yield 183 g, 14% based on fresh biomass), and ground in a mill. Lipids, pigments and low molecular weight compounds (monosaccharides, phenolics, etc) were removed using Soxhlet extractions with chloroform, methanol and acetone. Hot water extraction of 11 g of pretreated roots followed by dialysis [9] afforded crude water-soluble polysaccharides (0.6 g, 5.4 %, based on dry biomass).
Barbakadze et al.
Further fractionation in a stirred ultrafiltration cell, model 8200 (Millipore Corporation, Billerica, MA, USA) on a Biomax-500 ultrafiltration disc (500 000 NMWL), as reported in [21], yielded 0.36 g watersoluble, high-molecular (>500 kDa) preparation HM-AI (3.3% based on dry biomass). Carbohydrate analysis of A. italica CP-AI and HMAI: Total sugars, fructose, uronic acid and glucose were determined by photocolorimetry, as described [22-25]. Monosaccharide composition was analyzed after hydrolysis of samples (5-10 mg) with 2M CF3COOH at 121°C for 2 h. The acid was removed by multiple evaporations to dryness of methanol solutions. The monosaccharides were identified by means of either paper chromatography (PC) or TLC with monosaccharide standards for reference. Descending PC was run on Filtrak FN-1 paper in a 6: 4: 3 n-butanol-pyridine-water system. TLC was performed on 0.25 mm pre-coated silica gel plates (Merck 60, GF254; Merck, Darmstadt, Germany) treated with nbutanol-acetic acid-water (3:1:1). The sugars were visualized by spraying the paper and plates with aniline hydrogen phthalate and heating at 105OC for 10 min. The neutral sugars were converted into alditol acetates, identified by comparison of GC retention times with authentic standards, and quantified using peak area ratios of investigated compounds and an internal standard - meso-inositol acetate, taking account of the detector response factors [26]. HM-AI UV (H2O) λmax: 213, 236 (sh), 282 (sh), 286 nm. IR (KBr) υ: 3425, 2924, 1736, 1605, 1512, 1443, 1404, 1160, 1126, 1080, 1018, 872, 818 cm-1. 1 H NMR (300 or 700 MHz, D2O, δ, ppm) and 13C NMR (75 MHz, D2O, δ, ppm): Table 2. Acknowledgments – Dr Antonio Salgado thanks the Laboratori de Ressonancia de Barcelona (LRB) for a training fellowship.
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