Jan 30, 2012 - E. purpurea roots were supplied by Hans Peter. Abrahamsen, (Mercurialis, Sorø, Sealand, Denmark). They were stored dry at a temperature ...
Journal of Medicinal Plants Research Vol. 6(4), pp. 580-592, 30 January, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1085 ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Difference in structure and complement fixing activity of pectic polysaccharides from different plant parts of Echinacea purpurea (L.) Moench. Hilde Barsett1*, Torun Helene Aslaksen1, Lea Dalby-Brown1,3 and Terje E. Michaelsen1,2 1
School of Pharmacy, University of Oslo, P. O. Box 1068 Blindern, N-0316 Oslo, Norway. 2 Norwegian Institute of Public Health, P. O. Box 4404 Nydalen, N-0403 Oslo, Norway. 3 Department of Medicinal Chemistry, the Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 København Ø, Denmark. Accepted 13 October, 2011
Water soluble pectic polysaccharide fractions were isolated from different parts of Echinacea purpurea in order to elucidate any structural and biological differences. Water extracts of stems, leaves, flower buds, open flowerheads, post fertile flowers and roots were purified by ion exchange chromatography and by gelfiltration. The polysaccharides consisted of a high amount of galacturonic acid, but also rhamnose, arabinose and galactose with glycosidic linkages compatible with rhamnogalacturonan type I (RG-I), arabinogalactan I (AG-I) and arabinogalactan II (AG-II) structures. Precipitation with Yarivreagent supports the presence of AG-II. The polysaccharides isolated from the different plant part showed largely the same structure, but with some differences mainly in the arabinan component. The complement fixing activity varied considerable between the plant parts. The structural elucidation indicated that the glycosidic linkages to arabinose are important for the complement fixating activity, and thus a diminished activity after weak hydrolysis of the pectic fractions was observed. Key words: Complement fixing activity, Echinacea, glycosidic linkages, pectic polysaccharides, Yariv-reagent.
INTRODUCTION Preparations of Echinacea species are amongst the most widely used herbal medicines. The beneficial effects are assumed at least partly to be due to their reported immunostimulatory properties. Echinacea purpurea (L.) Moench. is the best studied of the nine species of the genus Echinacea (Asteraceae). Two other of the species, Echinacea angustifolia (DC.) Hell. and Echinacea pallida (Nutt.) Nutt. are also used medicinally (Foster, 1984). Echinacea spp. has a long history of medicinal use for a wide variety of conditions, mainly microbial infections (Bauer, 1998). The current medicinal use of Echinacea spp. is probably due to its effects on the immune system, particularly in the treatment and prevention of common cold, influenza and other upper respiratory tract infection (Barrett, 2003). The most consistent results identified by
*Corresponding author. E-mail: hilde.barsett@farmasi.uio.no. Tel: +47 22856573. Fax: +47 22854402.
the majority of studies so far indicate that Echinacea has nonspecific immunomodulatory properties through the triggering of the innate immune system (Zhai et al., 2007). The compounds assumed to be responsible for the activities include caffeic acid derivatives, alkamides, polyacetylenes, polysaccharides and glycoproteins. Polysaccharides from E. purpurea have previously been investigated for their immunostimulatory properties (Stimpel et al., 1984; Wagner et al., 1984). Two polysaccharides (PS I and PS II) were isolated from aqueous extracts of aerial parts, and structural analysis showed PS I to be a 4-O-methyl-glucuronoarabinoxylan with average MW of 35 kDa, PS II was an acidic arabinorhamnogalactan of MW 45 kDa (Proksch and Wagner, 1987). A xyloglucan, MW 79.5 kDa was polysaccharide isolated from leaves and stems of E. purpurea, while a pectin-like polysaccharide was isolated from the expressed juice (Stuppner, 1985). The biological activity of these polysaccharides has been reviewed by Bauer (1999), Emmendörffer et al. (1999) and Hall
Barsett et al.
(2003). Thude and Classen (2005) have isolated a highly branched arabinan from E. pallida, and in addition biological active arabinogalactan-proteins have been isolated from all three species (Bauer, 1998; Classen et al., 2006; Thude et al., 2006). Structure of polymeric fructans from E. purpurea roots has also been determined (Wack and Blaschek, 2006). Several of the medicinal plants used in traditional medicine contain polysaccharides showing biological effects related to different parts of the immune system (Yamada and Kiyohara, 1999, 2007; Yamada et al., 2009). Polysaccharides with biological activity often contain uronic acids, as in pectins. Among the structural moiety of pectins, the ramified region often contains the active sites for the complement fixing activity, mitogenic and enhancement of immune complex clearance (Paulsen and Barsett, 2005). There is no single identified compound in Echinacea preparations that can be associated with the medicinal effects. To date more than 216 different phytochemicals have been identified in the literature based on E. purpurea (Zheng et al., 2006). The content of phytochemicals has shown to vary between the different plant parts, and also differ as a result of cultivating systems, nutrients, water and physical environment. Zheng (2006) showed that caffeic acid derivatives also vary from one plant part to another. Liu et al. (2007) showed that E. purpurea reached its highest content of caffeic acid derivatives in the middle stage of full blossoming. In the present study, pectic polysaccharide fractions were isolated by the same procedure from roots, stems, leaves, flower buds, open flower heads (early stage of full blossoming) and post fertile flowers (just before withered state) of E. purpurea and compared with respect to carbohydrate composition, structural patterns and complement fixating activity. MATERIALS AND METHODS Plant material The different plant parts of E. purpurea; stems, leaves, flower buds, open flower heads (early stage of full blossoming) and post fertile flowers ( just before withering) were supplied by Steinar Dragland (Norwegian Institute of Plant Research, 2350 Nes, Norway). The plant material was stored at -20°C, prior to freeze-drying and pulverization. E. purpurea roots were supplied by Hans Peter Abrahamsen, (Mercurialis, Sorø, Sealand, Denmark). They were stored dry at a temperature below 5°C for 4 days during transport to Oslo, Norway, where they were stored in ethanol overnight, then freeze-dried until stable weight, pulverized to a fine powder by a mechanical grinder and stored in closed vessels below -18°C until extraction.
Extraction The extraction and fractionation procedure is outlined in Figure 1. All extractions were performed under reflux and with thorough
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stirring, using plant material/solvent ratios of 1:20 (w/v). The extracts were filtered through Whatman GF/A glass fiber filter. Low molecular weight compounds and lipophilic constituents such as phenolics and alkamides, were removed from the aerial plant material of E. purpurea by extraction with boiling 80% aqueous ethanol four times for 1 h each. Low molecular weight and lipophilic constituents were removed from the roots by extraction with boiling 96% ethanol, followed by extraction with boiling 50% aqueous ethanol four times for 1 h each. The residual plant material of the stems, leaves, flower buds, open flower heads and post fertile flowers were further extracted with 50°C H2O, pH 5, two times for 2 h. The combined water extracts for each plant part were concentrated by rotary evaporation under diminished pressure and 40°C, dialyzed against distilled water in a Spectra/Por Membrane dialysis tube (Spectrum) with molecular weight cut off of 3.5 kDa, then freeze dried and called “water extract - E. purpurea-stems” (WE-EP-S), “-leaves” (WE-EPL),” - flower buds” (WE-EP-FB), “ - open flower heads” (WE-EP-OF) and “ - post fertile flowers” (WE-EP-PFF), respectively. The residual root material after removal of lipophilic and low molecular weight compounds was extracted three times with 100°C water for 1 h, to obtain the ethanol insoluble compounds such as polysaccharides (Dalby-Brown et al., 2005). The combined H2Oextracts were concentrated by rotary evaporation under diminished pressure and 40°C, dialyzed at cut-off 3500 Da, freeze-dried and called “water extract – E. purpurea - root” (WE-EP-R).
Anion exchange chromatography The water extracts were fractionated by anion exchange chromatography on a DEAE Sepharose fast flow column (Ø: 5 cm, L: 14.5 cm, GE Health) with chloride as counter ion. Extracts were filtered through Acro® 50A 5 μm filter (Gelman Sciences) and thereafter through Millex®-HA 0.45 μm filter (Millipore) prior to application on the column. The column was eluted with app. 600 mL of water resulting in one neutral fraction (WE-EP -“plant part”N”). The column was then eluted with a linear gradient, 0 to 1 M NaCl, in 2 L, at 2 ml/min, 11 ml fractions were collected. The carbohydrate profile was monitored using the phenolsulphuric acid method (Dubois et al., 1956). The absorption at 490 nm was plotted as a function of elution volume. The elution profiles from the different extract were similar and gave one broad main peak with a small peak in front and a small peak in just after the main peak. The root extract gave three acidic polysaccharide fractions (WE-EP-R-A1, -A2, -A3) of which WE-EP-R-A2 was the main peak (Dalby-Brown et al., 2005). For the other extracts, the main peak after ion exchange chromatography was divided into two fractions; WE-EP-”plant part”-A1, -A2a, -A2b and –A3 (Figure 2). The relevant fractions were pooled. All these fractions were dialyzed (cut-off =3500 Da) to remove NaCl, reduced in vacuo and freeze-dried.
Gel filtration on Sephacryl S-400 The polysaccharides fractions, WE-EP-FB-A2a, WE-EP-OF-A2a, WE-EP-PFF-A2a from ion exchange chromatography (20 mg) was dissolved in distilled water (10 ml), filtered through Millex®-HA 0.45 μm filter (Millipore) and applied onto a Sephacryl S-400 column (Ø: 1.5 cm, L: 54 cm). The samples were eluted with 10 mM NaCl at 0.5 ml/min, and 2 ml fractions were collected. The carbohydrate profile was determined as described above. The absorption at 490 nm was plotted as a function of elution volume, and the relevant fractions pooled. Dextran standards of 475, 233, 98.4, and 16.4 kDa were used for calibration of the column. Two sub fractions, A2a-I (2000 to 500 kDa) and -A2a-II (500 to 100 kDa) were identified for each fraction based on their elution profile, dialyzed
Scheme 1mmmmmmmmmmmmmmmmmmmmmmmmmmmmmm J. Med. Plants Res.
Plant material Echinacea purpurea Extractions with 80% ethanol
Residue Extraction with water 50oC
Water extract (WE-EP-) DEAE-Sepharose fast flow ion exchange chromatography
-N
-A1
-A2a
-A3
-A2b Sephacryl 400
Weak acid hydrolysis
-A2a-HYD
-A2a-I
-A2a-II
2000-500KDa
500-100KDa
Figure 1. Procedure for extraction and fractionation of different aerial plant parts of Echinacea purpurea.
1.4
-A2a
-A1
1.2
-A2b
-A3 NaCl gradient (M)
1
A (490nm) (M NaCl)
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0.8 0.6 0.4 0.2 0 0
50
100
150
200
250
Fractions Figure 2. A typical polysaccharide elution profile of water extracts of Echinacea purpurea fractionated by anion exchange chromatography on a DEAE Sepharose fast flow column.
Barsett et al.
and freeze-dried.
Weak acid hydrolysis The polysaccharides fractions WE-EP-FB-A2a, WE-EP-OF-A2a, WE-EP-PFF-A2a from ion exchange chromatography (5 mg) were hydrolyzed with 0.05 M oxalic acid (2 ml) at 100°C for 2 h (Cartier et al., 1987). The acid was neutralized with sodium carbonate, and the mixture dialysed (Mw cut off 3,500 Da) against distilled water to eliminate salt and carbohydrate monomers. The acid resistant parts, WE-EP-FB-A2a-HYD, WE-EP-OF-A2a-HYD and WE-EPPFF-A2a-HYD were freeze-dried.
Carbohydrate composition The samples (1 mg) were subjected to methanolysis with 4 M HCl in anhydrous MeOH for 24 h at 80°C (Chambers and Clamp, 1971; Barsett and Smestad, 1991). Mannitol was used as an internal standard. After methanolysis the reagents were removed under a stream of N2 and the methyl-glycosides dried in vacuum over P2O5 prior to conversion into the corresponding TMS-derivates. The samples were subjected to capillary gas chromatography (DB-5, Carlo Erba 6000 Vegas Series 2) as described by Barsett and Smestad (1991).
Quantitative determination of phenolic content The total phenolic compounds were determined according to the Folin-Ciocalteu assay (Swain and Hillis, 1959), and modified as described by Rombouts et al. (1986). The absorbance was measured at 750 nm in a 4049 Novaspec spectrophotometer (LKB Biochrom using ferulic acid as standard. The total phenolic content was determined as ferulic acid equivalents (FA/sample) • 100%.
Linkages analysis of the polysaccharides Prior to methylation of the polymers, the uronic acids were reduced to their corresponding neutral sugar as described by Sims and Bacic (1995). The reduced polymers were methylated using the method of Ciucanu and Kerek (1984), modified by McConville et al. (1990). The fully methylated polysaccharides were hydrolysed with TFA, the monomers reduced with 1 M NaBD4 in 2 M NH4OH and the resulting partly methylated alditols were acetylated to partially methylated alditol acetates, PMAA, by adding 200 µl 1methylimidazole and 2 ml acetic acid anhydride. The PMAA were dissolved in 100 µl MeOH and analyzed by GC-MS on Fisons GC 8065 using split injection and a Fisons fused silica column (30 m x 0.2 mm i.d.) with a film thickness of 0.20 mm coupled with a Hewlett-Packard Mass Selective Detector 5970. The compounds present were identified by retention time and interpretation of the mass spectra. The relative amounts of each linkage type were estimated from the total amount of each monosaccharide obtained from the methanolysis analysis and the relative amount of each PMAA present. The Yariv- β-glucosyl test The presence of arabinogalactan type II (AGII) structures was detected by the single radial diffusion in an agarose gel containing the β-glucosyl Yariv reagent as described by Van and Clarke (1985). A positive reaction was identified by a reddish circle (halo) around the well into which the samples were applied. Gum Arabic was used as a positive control.
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Complement fixating assay The complement fixing test is based on inhibition of haemolysis of antibody sensitized sheep red blood cells, SRBC, by human sera as described by Michaelsen et al. (Method A) (2000). PMII, a pectin fraction from the leaves of Plantago major, was used as a positive control (Samuelsen et al., 1996). Inhibition of lysis induced by the test samples were calculated by the formula (Acontrol – Atest)/ Acontrol) x 100 % From these data a dose-response curve was constructed and the concentration of test sample giving 50% inhibition of lysis (IC50) was calculated. A low IC50 value means a high complement fixing activity. This biological test system can have some day to day variation and thus the ration IC50 PMII / IC50 sample was calculated. A high ratio means high complement fixing activity.
RESULTS AND DISCUSSION Isolation of polysaccharide fractions Pulverized plant material of stems (S), leaves (L), flower buds (FB), open flower heads (early stage of full blossoming) (OP) and post fertile flowers (just before withering) (PFF) of E. purpurea (EP), was pre-extracted using boiling 80% aqueous ethanol in order to remove low molecular weight and lipophilic constituents such as low molecular weight carbohydrates, polyphenols and alkamides before extraction with water at 50°C. The root material of E. purpurea was similarly pre-extracted with boiling 96% ethanol followed by boiling 50% aqueous ethanol in order to remove low molecular weight and lipophilic constituents prior to the extraction with boiling water. All the water extracts (WE) were then further fractionated according to Figure 1. The so-called flowers of Asteraceae are really clusters of flowers called inflorescences (flower heads). The individual flowers are called florets, and they are usually tiny and numerous, arranged in a compact inflorescence (flower head) so that they resemble a single flower (Stern, 1994).
Monosaccharide composition, content of phenols, detection of AG II and the complement fixinging activity of the water extracts The monosaccharide composition of the water extracts from the different plant part of E. purpurea is presented in Table 1. A rather high amount of arabinose, galactose and galacturonic acid is seen in all the fractions, indicating pectic type polysaccharides. The monosaccharide composition of the water extracts of flower buds (WE-EP-FB), open flowers (WE-EP-OF) and post fertile flowers (WE-EP-PFF) are similar, and these three fractions have the highest content of galactose and galacturonic acid compared to the water extracts from the other plant parts. The water extract of the stems (WE-EPS) contain 46% of glucose, which is higher than the
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Table 1. Monosaccharide composition and content of phenolic substances of different water extracts of Echinacea purpurea.
Monosaccharide composition Ara Rha Xyl Man Gal Glc GlcA GalA Total phenol content (%, w/w) a
a
WE-EP-S 17.7 2.7 2.9 1.9 9.5 46.0 2.8 16.5 5.2
WE-EP-L 35.0 3.7 5.7 2.6 8.1 16.7 28.2 42.2
WE-EP-FB 27.0 3.1 2.0 2.7 14.6 11.1 39.3 29.0
WE-EP-OF 20.0 3.8 2.5 3.2 15.2 9.7 45.6 19.8
WE-EP-PFF 24.0 4.1 3.0 2.6 15.6 10.5 40.2 22.6
WE-EP-R 49.0 5.9 1.4 6.2 1.9 35.6 2.6
% of total carbohydrate content.
IC50 PMII / IC50 Sample
IC50 PMII / IC50 Sample
3,5 3.5 33
2.5 2,5
22
1.5 1,5
11
0.5 0,5
00
Figure 3. The complement fixing activity of the water extracts, assayed as IC 50 values relative to IC50 of a polysaccharide standard PMII from Plantago major (IC 50 PMII / IC50 test sample).
glucose content from the other plant parts. This could be due to a high portion of starch. The total content of phenols is also shown in Table 1. The stems and roots had lowest content of phenolic compounds than the other plant parts, while the leaves had the highest. The Yariv test gave positive response for all extracts tested. The extracts from flower buds, open flowers and post fertile flowers gave stronger response than those from stems and leaves. This indicates the presence of arabinogalactan type II structures in the polysaccharides from all crude water extracts. The complement fixing test is based on inhibition of hemolysis of antibody sensitized sheep erythrocytes. Inhibition of lysis of the fractions were assayed as IC 50 values relative to IC50 of a polysaccharide standard PMII from Plantago major (IC50 PMII / IC50 sample), which are less influenced by day to day variations in the biological
test system (Michaelsen et al., 2000). The relative IC50 values of the extracts are given in Figure 3. The water fraction from the post fertile flowers (WE-EP-PFF) was the fraction with the highest activity, higher than all the other water extracts and approximately 3 times stronger than the standard polysaccharide (PMII). It is interesting to note that even though the monosaccharide composition of the water extracts of flower buds, open flowers and post fertile flowers are very similar, the activity of these extracts differs.
Characterization of the acidic fractions The crude water extracts of leaves, flower buds, open flower heads, post fertile flowers and roots were subjected to further fractionation by anion exchange
Barsett et al.
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Table 2. Monosaccharide composition of the acidic fractions after ion exchange chromatography.
Monosaccharide a,b Composition Ara Rha Xyl Man Gal Glc GalA a
WE-EP-L -A1 -A2a -A2b
WE-EP-FB -A1 -A2a -A2b
20.1 1.7 16.1 3.1 27.1 17.8 14.1
23.4 3.9 12.8 3.1 36.2 7.2 13.4
7.6 3.5 24.7 2.8 8.8 3.2 49.4
8.2 5.6 8.4 7.3 3.1 67.4
14.6 4.5 1.0 15.4 3.0 61.5
7.8 4.7 4.9 9.1 2.7 70.8
-A1
WE-EP-FB -A2a -A2b
-A3
20.4 1.9 10.5 2.4 28.7 5.4 30.7
12.7 3.4 1.2 12.3 1.3 69.1
12.5 6.0 13.3 3.2 65.0
11.4 5.5 10.6 2.9 69.6
-A1
WE-EP-PFF -A2a -A2b
21.1 2.6 12.2 3.3 27.1 8.2 25.5
10.7 3.6 1.2 11.9 3.0 69.6
10.9 8.4 10.9 3.7 66.1
-A3
WE-EP-R -A1 -A2
-A3
15.6 8.7 1.2 18.7 4.8 51.1
38.9 12.0 6.0 15.3 1.0 26.6
50.8 9.0 2.0 15.6 1.4 21.2
34.7 7.0 7.3 49.8
% of total carbohydrate content. b Amounts below 1.0% are not included.
chromatography. The elution profiles from the different extract were similar and showed one broad main peak with a small peak in connection in front of the main peak, and a small peak after the main peak. These shoulder peaks contained too little material for characterization analyses. For the water extract of the leaves (WE-EP-L), the flower buds (WE-EP-FB), the open flowers (WEEP-OF) and the post fertile flowers (WE-EP-PFF) the resulting main peak was divided into two fractions (-A2a, and -A2b) and further characterized (Figure 2). The main peak from the root extract (WE-EP-R) was kept as one fraction (A2) (Dalby-Brown et al., 2005). The obtained neutral fractions devoid of complement fixing activity did not precipitate with the Yariv-reagent and were not further studied. Monosaccharide composition of the obtained acidic fractions is presented in Table 2. The results of the linkage analysis of the same fractions are shown in Table 3, and the complement fixing activity assayed as relative IC50 values (IC50 PM II / IC50 test sample), are given in Figure 4. All the front shoulder (-A1) peaks, had lower amount of galacturonic acid and
rhamnose, and higher amount of arabinose and galactose than the main fractions (-A2). All - A1 fractions also had a higher content of xylose and glucose compared to the other fractions. The monosaccharide composition of the - A2 fractions after anion exchange chromatography, showed all a relatively high amount of galacturonic acid and the neutral monomers rhamnose, arabinose and galactose which lead to the assumption that these fractions contain pectic polymers. The galacturonic acid is mainly 1-4 linked. Rhamnose is terminally, 1 - 2 and 1 - 3 linked and some branched units mainly as 1-2,4, but also as 1-3,4 linked units. The arabinose is mostly terminally and 1-5 linked with some branch points having 1-3,5 linkages. Galactose is terminally and 1-4 linked, but also 1-3 linked, and 1-3,6 linked units as branch points. Root fraction (WE-EP-RA2) contained only terminally, 1-3 linked and 1-3,6 linked galactose, and had higher amount of arabinose than the other plant parts. Besides being terminally and 1-5 linked, arabinose is here highly branched in positions 1-3,5. Pectins were earlier thought tomainly consist of galacturonic
acid, but it has now long been recognized that pectins are a very complex group of polysaccharides and they are divided into two main types: Rhamnogalacturonan I (RG-I) and Rhamnogalacturonan II (RG-II). This is reviewed by Albersheims group (McNeil et al., 1982; Ishii et al., 1989) and also by Waldron and Faulds (2007) and Paulsen and Barsett (2005). RG-I is the main part of the total amount of pectins present and consist of a polymer that have a core of alternating α-1,4-linked D-galacturonic acid and α1,2-L-rhamnose units. The rhamnose units are frequently found as branch points, primarily on position 4, carrying galactan and arabinan side chains of varying structures. The arabinogalactans attached to the rhamnose units are frequently found to be of the arabinogalactan type I (AG-I) and/ or arabinogalactan type II (AG-II) (precipitates with the Yariv-reagent). AG-I is composed of a β-1,4 linked galactan backbone with side chains of arabinans basically linked through position 3 of the galactose units. AG-II has as its main core a galactan that can have either 3 or 6 linkages in the main chain and is
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Table 3. Linkage composition of the acidic polysaccharide fractions from different plant parts of Echinaca purpurea.
Tf Ara 1→2f Ara 1→3f Ara 1→5f Ara 1→2,5f Ara 1→3,5f Ara
WE-EP-L -A2a -A2b 4.9 5.3 0.1 0.2 0.1 0.2 1.7 1.7 0.4 0.8 0.4
Tp Xyl 1→3p Xyl 1→4p Xyl
20.2 3.4 1.1
Tp Rha 1→2p Rha 1→3p Rha 1→2,3p Rha 1→2,4p Rha 1→3,4p Rha
1.8 0.7 0.3 0.3 0.3 0.1
Type of linkage
-A1 14.0 0.4 0.4 6.9 1.0 0.7
WE-EP-FB -A2a 8.0 0.1 0.1 4.3 2.1
WE-EP-OF -A2a -A2b 6.7 6.4 0.1 0.2 0.1 0.2 4.0 3.1 1.8 1.5
-A2b 4.4 0.2 0.2 2.1 0.9
-A1 12.6 0.3 0.3 5.6 0.8 0.8
6.7 1.3 0.4
12.8
1.0
-
1.4 9.1
1.2
-
3.6 0.7 0.7 0.2 0.4
3.6 0.3 -
2.1 1.2 0.4 0.8 -
1.9 1.6 0.7 0.5 -
1.2 0.7 -
0.8 0.4 1.7 0.4 0.1
2.0 1.8 0.6 0.7 0.4
WE-EP-PFF -A1 -A2a -A2b 11.8 5.2 4.7 0.4 0.2 0.2 0.4 0.2 0.2 5.6 3.4 3.6 1.4 1.5 1.7 2.2
-A1 13.6 0.2 0.2 9.3 15.6
-
2.0 10.2
0.5 0.1 0.6
-
6.0 -
-
2.0 -
2.6 1.9 0.3 0.9 0.3
2.3 0.1 0.2 -
1.7 1.1 0.2 0.6 -
2.5 4.6 0.6 0.3 0.1 0.3
0.2 5.4 6.4 -
1.0 4.2 0.2 1.6 -
0.5 5.6 2.9 -
-A4 6.4 0.2 0.2 3.9 1.8
Tp Man 1→ 3p Man 1→6p Man
WE-EP-R -A2 -A3 10.0 23.8 0.2 0.5 0.2 0.5 9.5 1.8 14.8 24.2
0.5 2.3 0.5
Tp Glc 1→4p Glc 1→6p Glc
0.5 1.7 1.0
0.4 1.2 1.5
0.7 0.9 5.6
0.2 0.7 2.1
0.4 2.3 -
0.3 0.2 4.9
0.2 0.2 0.9
0.4 1.6 0.9
0.4 1.4 1.4
0.8 1.1 6.3
0.2 0.9 1.9
0.4 1.0 2.3
0.6 0.4
-
0.1 0.7 0.6
Tp Gal 1→3p Gal 1→4p Gal 1→6p Gal 1→3,4p Gal 1→3,6p Gal 1→4,6p Gal 1→3,4,6p Gal
0.8 0.3 6.5 0.4 0.6 0.1 0.1
0.5 0.4 4.6 0.4 1.2 0.1 0.1
3.5 6.0 3.8 22.5 0.4 -
0.7 0.4 12.9 0.2 1.2 -
0.4 0.3 8.2 0.1 0.1 -
1.2 2.5 12.2 12.8 -
0.3 0.2 10.8 0.1 0.8 0.1 -
0.4 0.4 9.0 0.2 0.6 -
0.6 0.3 11.1 0.2 1.1 -
4.4 3.0 6.2 0.2 0.2 12.8 0.2 0.1
0.9 0.4 8.7 0.1 1.5 0.2 0.1
1.0 0.4 8.3 1.0 0.1 0.1
4.1 0.8 9.7 0.7 -
5.3 1.2 0.8 -
8.0 1.8 3.9 1.9 -
Barsett et al.
Table 3. Contd.
2.7 44.1 7.8 12.8
1.2 12.2 -
2→1f Fru Tp 4-O-Me-GlcA
1.6 58.6 0.2 1.1
2.2 67.9 0.2 0.5
1.1 66.9 0.3 0.8
1.9 66.3 0.3 1.1
1.8 61.9 0.2 1.1
3.3 21.9 0.3 -
Trace
3.3 65.2 0.4 0.7
5.3 58.3 0.8 1.7
Trace
23.6 0.6 2.4
2.0 46.0 0.7 1.1
3.3 16.6 1.3
Trace Trace
Trace
Trace Trace
PMII
WE-EP-R-A3
WE-EP-R-A2
WE-EP-R-A1
WE-EP-PFF-A3
WE-EP-PFF-A2b
WE-EP-PFF-A2a
WE-EP-PFF-A1
WE-EP-OF-A3
WE-EP-OF-A2b
WE-EP-OF-A2a
WE-EP-OF-A1
WE-EP-FB-A2a
WE-EP-FB-A1
WE-EP-L-A2b
4.5 4,5 44 3.5 3,5 33 2.5 2,5 22 1.5 1,5 11 0.5 0,5 00
WE-EP-L-A1
IC50 PMII / IC50 Sample
Trace Trace
4.2 26.5 -
WE-EP-FB-A2b
2.6 40.1 2.7 4.0
WE-EP-L-A2a
Tp GalA 1→4p GalA 1→2,4p GalA 1→3,4p GalA
Figure 4. The complement fixing activity of acidic fractions after anion exchange chromatography, assayed as IC50 values relative to IC50 of a polysaccharide standard PMII from Plantago major (IC50 PMII / IC50 test sample).
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highly branched with the 1,3,6-linked galactose units at the branching points. The arabinan may be linear or branched, and primarily linked through positions 3 or 5. Linkages through C-2 are also observed, but generally they are less frequent than the 3-linkage. It is generally accepted that the core linkage of the arabinans are the 5linkages and the branches occur at C-3 or C-2. All the presented fractions isolated from ion exchange chromatography, gave to a different extent, precipitation with the Yariv-reagent. Concomitant with the linkage analyses showing the presence of 1-3,6 linked galactose these results confirmed that all fractions contain AG-II, and with high amount of galacturonic acid this support the assumption that the fractions contain pectic polysaccharides. High amount of 1-4 linked galactose (Table 3) support the presence of AG-I side chains as well. The amount present of galacturonic acid is normally increasing when eluted with increasing gradient concentration. The fractionation of the water extract of the roots is an exception of this as the last fraction has lower percentage of galacturonic acid than the main peak eluated at a lower concentration. This could be due to a higher amount of methyl esterification of the galacturonic acid in the main fraction. Presence of methyl esterified galacturonic acid in pectins is well established (Voragen et al., 2000). The complement fixing test (Figure 4) showed that most of the fractions isolated after anion exchange chromatography had lower activity than their parent extracts, apart from the – A3 fractions from post fertile flowers and from roots that both gave much higher activity than any of the other fractions. The – A3 fraction from the root extract had the highest activity. From the linkage analysis, this polysaccharide fraction showed higher amount of terminally linked and branched arabinose and higher amount of terminally linked and branched galactose. The monosaccharide composition together with the elution range from the anion exchange column gives the assumption that this polysaccharide has a lower degree of methyl esterified galacturonic acid than the other polymers.
Characterization of high molecular weight fractions The fractions from the first part of the main peak (- 2Aa) after anion exchange chromatography of the extracts of flower buds, open flower heads and post fertile flowers, were further fractionated on a Sephacryl S - 400 gelfiltration column. The column was calibrated with dextrans ranging from 2000 to 16.4 kDa. Dextrans have other physical properties than pectins, and cannot be used for exact molecular weight determination of pectins, but the calibration of the column with these standards can give a certain lead towards the molecular weight of the fractions. The isolated fractions eluted in the range of 2000 to 500 and 500 to 100 kDa, were named – A2a - I
and – A2a – II, respectively. Monosaccharide composition of the obtained fractions after gel filtration was determined and presented in Table 4. The results of the linkage analysis of the same fractions are shown in Table 5, and the complement fixing activity assayed as relative IC50 values as shown in the foregoing (Figure 5).Comparison of the fractions with respect to the molecular weight showed that the fractions with the highest molecular weight of the two from the same original extracts, showed the highest complement fixing activity. The activity rises with stage of blossoming, and the highest molecular weight fraction from post fertile flowers gave the highest immunomodulating activity. The monosaccharide composition and linkage analysis of the two molecular weight fractions originating from the flower buds (WE-EP-FB-A2a-I and WE-EP-FB-A2a-II ) showed that the two fractions were very similar, only a small amount of terminally linked mannose was found in addition in the highest molecular weight fraction. The two different molecular weight fractions originating from the water extract of open flower heads (WE-EP-OF-), showed also similarities. Both gave high content of galacturonic acid, mainly 1-4 linked, and similar amount of terminally, 1-3 linked, 1-4linked and 1-3,6 linked galactose. The high molecular weight (-A2a-I) fraction contained a small amount of 1-6 linked galactose in addition. The high molecular weight fraction showed much higher content of arabinose, given as higher amount of terminally linked, 1-5 linked and 1-3,5 linked monomers. The same differences were seen between the two molecular weight fractions (-A2a-I and –A2a-II) originating from the water extract of post fertile flowers (WE-EP-PFF-). This could lead to the conclusion that the structural elements of arabinose are important for complement fixating activity.
Characterization of hydrolysed fractions The fractions WE-EP-FB-A2a, WE-EP-OF-A2a and WEEP-PFF-A2a after ion exchange chromatography were also subjected to weak acid hydrolysis. Monosaccharide compositions of the high molecular weight material after hydrolysis are presented in Table 6, and results of the linkage analysis shown in Table 7. The most striking differences between the original –A2a fractions and the hydrolyzed fractions (-A2a-HYD) were the total disappearance of arabinose after hydrolysis. A part of the rhamnose and a smaller part of the galactose were also lost afterhydrolysis. The linkage analysis showed a higher structure element after hydrolysis, the 1-4 linked relative amount of terminally linked galactose which could be due to arabinose bound to galactose in the original fractions. Also, a somewhat lower amount of 1-3,6 linked galactose could be due to branching with arabinose in 3 position or 6 position of the galactose. The relatively higher amount of terminally linked galacturonic acid after the weak
Barsett et al.
Table 4. Monosaccharide composition of acidic fractions after gelfiltration on Sephacryl S400.
Monosaccharide a,b composition Ara Rha Xyl Man Gal Glc GalA a
WE-EP-FB -A2a-I 2000-500 -A2a-II 500-100 KDa KDa 11.9 11.0 2.7 3.3 1.9 1.3 1.4 14.1 16.8 4.8 4.7 63.2 62.9
WE-EP-OF -A2a-I 2000-500 -A2a-II 500-100 KDa KDa 13.5 4.2 3.0 1.4 1.2 1.6 14.6 15.2 2.5 1.0 63.6 78.2
WE-EP-PFF -A2a-I 2000-A2a-II 500-100 500KDa KDa 18.0 9.2 4.8 3.0 3.4 1.0 2.5 22.6 12.2 5.3 1.9 43.4 72.7
% of total carbohydrate content. b Amounts below 1.0% are not included.
Table 5. Linkage composition of the acidic polysaccharide fractions after gelfiltration on Sephacryl S400.
Type of linkage Tf Ara 1→2f Ara 1→3f Ara 1→5f Ara 1→3,5f Ara
WE-EP-FB -A2a-I -A2a-II 6.7 5.9 0.2 0.2 3.3 3.4 1.9 1.3
WE-EP-OF -A2a-I -A2a-II 6.4 2.2 0.3 0.1 0.3 0.1 4.0 1.2 2.5 0.6
WE-EP-PFF -A2a-I -A2a-II 9.5 5.0 0.4 0.1 0.4 0.1 4.7 2.7 3.0 1.3
Tp Xyl 1→4p Xyl Tp Rha 1→2p Rha 1→3p Rha 1→2,4p Rha
1.9 1.9 0.8
0.2 1.1 2.0 0.7 0.6
1.2 1.8 0.5 0.7
0.3 0.4 0.3 0.4
0.4 3.0 2.9 0.7 1.2
0.3 0.7 1.9 0.5 0.3 0.3
Tp Man
1.4
-
1.6
-
2.5
-
Tp Glc 1→4p Glc 1→6p Glc
0.5 2.4 1.9
0.5 2.2 2.0
0.2 1.7 0.6
0.2 0.4 0.4
0.4 3.0 1.9
0.2 0.6 1.1
Tp Gal
1.9
1.5
0.8
1.6
1.5
1.0
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Table 5. Contd.
1→3p Gal 1→4p Gal 1→6p Gal 1→3,4p Gal 1→3,6p Gal
0.5 8.1 0.2 3.4
0.6 10.1 1.0 3.6
0.3 11.3 0.6 1.6
0.3 11.8 0.1 1.4
0.6 17.2 0.5 2.8
0.2 9.7 0.1 1.2
Tp GalA 1→4p GalA 1→2,4p GalA 1→3,4p GalA
3.4 59.8 -
4.6 58.0 0.3 -
3.0 60.6 -
1.2 75.5 0.4 1.1
2.4 40.2 0.1 0.7
3.8 68.4 0.4 0.1
Tp 4-O-Me-GlcA
trace
trace
trace
trace
trace
trace
44
IC50 PMII / IC50 Sample
590
3,5 3.5 33
2,5 2.5 22
1,5 1.5 11
0,5 0.5 00
Figure 5. The complement fixing activity of high molecular weight subfractions (2000kDa500kDa and 500kDa-100kDa) after gelfiltration of acidic polysaccharides on a Sephacryl S-400 column, assayed as IC50 values relative to IC50 of a polysaccharide standard PMII from Plantago major (IC50 PMII / IC50 test sample).
Barsett et al.
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Table 6. Monosaccharide composition of acidic fractions after weak acid hydrolysis.
Monosaccharide composition Rha Gal Glc 4-O-Me-GlcA GalA a
a,b
WE-EP-FB -A2a-HYD 1.6 10.6 3.1 1.0 83.7
WE-EP-OF -A2a-HYD 1.3 8.9 2.8 87.0
WE-EP-PFF -A2a-HYD 1.6 8.5 2.4 87.5
% of total carbohydrate content. b Amounts below 1.0% are not included.
Table 7. Linkage composition of the acidic fractions from after weak acid hydrolysis.
WE-EP-FB -A2a-HYD 0.4 0.7 0.3 0.2
WE-EP-OF -A2a-HYD 0.8 0.3 0.2
WE-EP-PFF -A2a-HYD 0.7 0.5 0.4
Tp Glc 1→4p Glc 1→6p Glc
0.6 1.3 1.2
0.4 0.9 1.5
0.4 0.9 1.1
Tp Gal 1→3p Gal 1→4p Gal 1→3,4p Gal 1→3,6p Gal
0.9 0.2 8.7 0.3 0.5
1.1 0.1 7.2 0.1 0.4
1.4 0.1 6.4 0.2 0.4
Tp GalA 1→4p GalA 1→3,4p GalA
8.3 74.8 0.6
12.0 74.5 0.5
9.3 78.0 0.2
Tp 4-O-Me-GlcA
1.0
trace
trace
Type of linkage Tp Rha 1→2p Rha 1→3p Rha 1→2,4p Rha
hydrolysis can be difficult to explain when referring to what is known about pectic polymers. Smidsrød et al. (1966) showed that poly- and oligouronides showed a particularly high rate of hydrolysis at low acid concentrations due to a high proton concentration near the negatively charged polysaccharide chain in solution. This will result in increased galacturonic acid as nonreducing end. The main galacturonic acid, show that this is the main chain of the polymer. The hydrolysis of the –A2a fractions gave lower complement fixating activity in all the flower part fractions. In conclusion, pectic polysaccharide fractions from roots and post fertile flowers seemed to have higher complement fixing activity than similar fractions from the other plant parts. High molecular weight polysaccharide fractions showed higher content of 1-5 linked and 1-3,5-linkedarabinose and also
higher complement fixing activity than the lower molecular weight fractions originating from the same water extracts. Weak acid hydrolysis removed all arabinose units in the flower part polysaccharide fractions, which resulted in lower complement fixing activity. This lead to the assumption that the structural elements of arabinose are important for the biological activity.
ACKNOWLEDGEMENT We thank Steinar Dragland, Norwegian Institute of Plant Research, 2350 Nes Norway, for supplying the plant material.
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REFERENCES Barrett B (2003). Medicinal properties of Echinacea: a critical review. Phytomedicine, 10: 66-86. Barsett H, Smestad PB (1991). Separation, isolation and characterization of acidic polysaccharides from the inner bark of Ulmus glabra Huds. Carbohydr. Polym., 17: 137-144. Bauer R (1998). Echinacea: biological effects and active principles. In Lawson LD,Bauer R, (Eds.) Phytomedicines in Europe. Chemistry and biological activity. ACS symposium series, American Chemical Society: Washington DC., 691: 140-157. Bauer R (1999). Chemistry, analysis and immunological investigations of Echinacea phytopharmaceuticals. In Wagner H, (Ed.) Immunomodulatory agents from plants., Birkhäuser Verlag: Basel, pp. 41-88. Cartier N, Chambat G, Joseleau JP (1987). An arabinogalactan from the culture medium of Rubus fruticosus cells in suspension. Carbohydr. Res., 168: 275-283. Chambers RE, Clamp JR (1971). An Assessment of methanolysis and other factors used in the analysis of carbohydrate-containing materials. Biochem. J., 125: 1009-1018. Ciucanu I, Kerek F (1984). A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131: 209-217. Classen B, Thude S, Blaschek W, Wack M, Bodinet C (2006). Immunomodulatory effects of arabinogalactan-proteins from Baptisia and Echinacea. Phytomedicine, 13: 688-694. Dalby-Brown L, Barsett H, Landbo AK, Meyer AS, Mølgaard P (2005). Synergistic antioxidative effects of alkamides, caffeic acid derivatives, and polysaccharide fractions from Echinacea purpurea on in vitro oxidation of human low-density lipoproteins. J. Agric. Food Chem., 53: 9413-9423. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956). Colorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356. Emmendörffer A, Wagner H, Lohmann-Matthes ML (1999). Immunologically active polysaccharides from Echinacea purpurea plant and cell culture. In Wagner H, (Ed.) Immunomodulatory agents from plants., Birkhäuser Verlag: Basel, pp. 89-104. Foster S (1984). Echinacea exalted! The Botany, Culture, History and Medicinal Uses of the Purple coneflowers., Ozark Beneficial Plant Project, Brixey, MO. Hall C 3rd (2003). Echinacea as a functional food ingredient. Adv. Food Nutr. Res., 47: 113-173. Ishii T, Thomas J, Darvill A, Albersheim P (1989). Structure of plant cell walls : XXVI. The walls of suspension-cultured sycamore cells contain a family of rhamnogalacturonan-I-like pectic polysaccharides. Plant Physiol., 89: 421-428. Liu YC, Zeng JG, Chen B, Yao SZ (2007). Investigation of phenolic constituents in Echinacea purpurea grown in China. Planta Med., 73: 1600-1605. McConville MJ, Homans SW, Thomas-Oates JE, Dell A, Bacic A (1990). Structures of the glycoinositolphospholipids from Leishmania major. A family of novel galactofuranose-containing glycolipids. J. Biol. Chem., 265: 7385-7394. McNeil M, Darvill AG, Albersheim P (1982). Structure of plant cell walls : XII. Identification of seven differently linked glycosyl residues attached to O-4 of the 2,4-linked L-rhamnosyl residues of rhamnogalacturonan I. Plant Physiol., 70: 1586-1591. Michaelsen TE, Gilje A, Samuelsen AB, Hogasen K, Paulsen BS (2000). Interaction between human complement and a pectin type polysaccharide fraction, PMII, from the leaves of Plantago major L. Scand. J. Immunol., 52: 483-490. Paulsen BS, Barsett H (2005). Bioactive pectic polysaccharides. Adv. Polym. Sci., 186: 69-101. Proksch A, Wagner H (1987). Structural analysis of a 4-O-methylglucuronoarabinoxylan with immunostimulating activity from Echinacea purpurea. Phytochemistry, 26: 1989-1993. Rombouts FM, Thibault JF (1986). Enzymatic and chemical degradation and the fine-structure of pectins from sugar-beet pulp. Carbohydr. Res., 154: 189-203.
Samuelsen AB, Paulsen BS, Wold JK, Otsuka H, Kiyohara H, Yamada H, Knutsen SH (1996). Characterization of a biologically active pectin from Plantago major L. Carbohyd. Polym., 30: 37-44. Sims IM, Bacic A (1995). Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia. Phytochemistry, 38: 1397-1405. Smidsrød O, Haug A, Larsen B (1966). The influence of pH on rate of hydrolysis of acidic polysaccharides. Acta Chem. Scand., 20: 10261034. Stern KR (1994). Introductory plant biology. Dubuque, Wm. C. Brown Publishers. Stimpel M, Proksch A, Wagner H, Lohmann-Matthes ML (1984). Macrophage activation and induction of macrophage cytotoxicity by purified polysaccharide fractions from the plant Echinacea purpurea. Infect. Immun., 46: 845-849. Stuppner H (1985). Chemische und Immunologische Untersuchung von Polysacchariden zu der Gewebekultur von Echinacea purpurea (L.) Moench. PhD Ludwig-Maximilians Univ., Germany. Swain T, Hillis WE (1959). Phenolic constituents of Prunus domestica. I. Quantitative analysis of phenolic constituents. J. Sci. Food Agric., 10: 63-68. Thude S, Classen B (2005). High molecular weight constituents from roots of Echinacea pallida: an arabinogalactan-protein and an arabinan. Phytochemistry, 66: 1026-1032. Thude S, Classen B, Blaschek W, Barz D, Thude H (2006). Binding studies of an arabinogalactan-protein from Echinacea purpurea to leucocytes. Phytomedicine, 13: 425-427. van Holst GJ, Clarke AE (1985). Quantification of arabinogalactanprotein in plant extracts by single radial gel diffusion. Anal. Biochem., 148: 446-450. Voragen AGJ, Daas PJH, Schols HA (2000). Enzymes as tools for structural studies of pectins. In Paulsen BS, (Ed.) Bioactive carbohydrate polymers. Proceedings of the phytochemical society of Europe, Kluwer Academic Publishers: Dordrecht, 44: 129-145. Wack M, Blaschek W (2006). Determination of the structure and degree of polymerisation of fructans from Echinacea purpurea roots. Carbohydr. Res., 341: 1147-1153. Wagner H, Proksch A, Riessmaurer I, Vollmar A, Odenthal S, Stuppner H, Jurcic K, Leturdu M, Heur YH (1984). Immunostimulating polysaccharides (heteroglycanes) of higher plants. ArzneimittelForsch, 34: 659-661. Waldron KW, Faulds CB (2007). Cell Wall Polysaccharides: Composition and Structure. In Kamerling JP, Boons GJ, Lee YC, Suzuki A, Taniguchi N,Voragen AGJ, (Eds.) Comprehensive Glycoscience, Elsevier: Oxford, 1: 181-201. Yamada H, Kiyohara H (1999). Complement-activating polysaccharides from medicinal herbs. In Wagner H, (Ed.) Immunomodulatory Agents from Plants, Birkhäuser Verlag: Basel., pp. 161-202. Yamada H, Kiyohara H (2007). Immunomodulating Activity of Plant Polysaccharide Structures. In Kamerling JP, (Ed.) Comprehensive Glycoscience - From Chemistry to Systems Biology, 1st ed.; Elsevier: Oxford, 4: 663-694. Yamada H, Kiyohara H, Matsumoto T (2009). Recent studies on structures and intestinal immunity modulating activities of pectins and pectic polysaccharides from medicinal herbs. In Schols HA, Visser RGF,Voragen AGJ, (Eds.) Pectins and Pectinases, Wageningen Academic Publishers: Wageningen, pp. 293-304. Zhai Z, Liu Y, Wu L, Senchina DS, Wurtele ES, Murphy PA, Kohut ML, Cunnick JE (2007). Enhancement of innate and adaptive immune functions by multiple Echinacea species. J. Med. Food, 10: 423-434. Zheng Y, Dixon M, Saxena PK (2006). Growing environment and nutrient availability affect the content of some phenolic compounds in Echinacea purpurea and Echinacea angustifolia. Planta Med., 72: 1407-1414.
