Differentiation Between the Complement Modulating Effects of an Arabinogalactan-Protein from Echinacea purpurea and Heparin

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Test compounds
• Results
• Activity in the haemolytic complement assay for the
  classical pathway
• Activity in the haemolytic complement assay for the
  alternative pathway
• Haemolytic activity of serum in the complement assay for the alternative pathway
• Preincuabtion time-dependent activity in the haemolytic complement assay for the alternative pathway
• Discussion
• References

Abstract

Due to the important physiological role of the complement system, complement modulation, either inhibition or stimulation, is an interesting target for drug development. Several plant polysaccharides are known to exhibit complement modulating activities. Sometimes these effects are described as complement inhibition, although the basic mechanism is a stimulation of the complement activation. This misinterpretation is due to the observed reduced haemolysis in the widely used haemolytic complement assay, which does not allow to differentiate between complement activators and inhibitors, when it is performed in the classical manner. The aim of the presented study was to demonstrate that by simple modifications of the classical procedure this assay becomes an efficient tool to distinguish between real complement inhibitors and complement activating compounds without performing expensive, molecular mechanistic investigations. As practical examples heparin with proven complement inhibiting activity and AGP, a new arabinogalacatan-protein type II isolated from pressed juice of the aerial parts of Echinacea purpurea, as a potential complement activating compound were included in the study. By means of varying the preincubation time of the test compound with complement, AGP was clearly identified as a stimulator of both the classical and alternative pathway of complement activation. These findings correspond to the results of molecular mechanistic investigations. Selective removal of the arabinose side chains of AGP resulted in considerably reduced activity. Therefore, the three-dimensional structure of the polysaccharide, i. e., a backbone branched by side chains, is supposed to be important for the interactions with the complement system. The complement activating effects of AGP may contribute to the well-established immunostimulating effects of the pressed juice from Echinacea purpurea.

Abbreviations

AGP:arabinogalactan-protein

AGP-hydr.:hydrolysed arabinogalactan-protein

AP-CA:haemolytic complement assay for the alternative
pathway

CP-CA:haemolytic complement assay for the classical
pathway

EGTA-VB:veronal buffered saline containing EGTA and Mg²⁺

HPS:human pooled serum

RT:room temperature

LPS:lipopolysaccharide

RaE:rabbit erythrocytes

RT:room temperature

ShE(A):(sensitised) sheep erythrocytes

VB:veronal buffered saline containing Ca²⁺ and Mg²⁺

Introduction

The complement system is part of the innate immune system and comprises a family of at least 20 plasma and membrane proteins that react in a regulated cascade [1]. Its main physiological functions are non-specific host defence and mediation of inflammation, bridging innate and adaptive immunity, and disposing of immune complexes and the products of inflammatory injury. Depending on the particular clinical situation, either inhibition or activation of the complement system may be worthwhile. As an example, complement inhibition will reduce tissue damage resulting from organ transplantation, ischaemia-reperfusion injury, cancer, glomerulonephritis and the use of extracorporeal circuits [2]. Complement activation plays an important role in host defense against many microbial pathogens [3] and in terms of a non-specific immunostimulation it may support wound-healing, but also antitumor therapy.

Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components [3]. The classical pathway is usually activated by antibody bound to a foreign particle such as viruses and Gram-negative bacteria, whereas the alternative pathway is antibody-independently triggered by certain activating surfaces, e. g., from microoganisms and foreign cells [1]. Polysaccharides as components of bacterial lipopolysaccharides (LPS) and capsules play an important role in complement activation by activating either alternative or classical or both pathways [4], [5], [6]. However, besides a stimulating effect, they may also be responsible for complement resistance, e. g., by reducing the binding of the complement component C3 [7]. Moreover, sulfated polysaccharides like heparin, dextran sulfate or fucoidan are known to inhibit both classical and alternative complement activation [8]. Therefore, polysaccharides represent interesting candidates for a therapeutic modulation of the complement system.

During the last years, several polysaccharides isolated from plants used in phytotherapy and folk medicine have been tested for complement modulating properties. Especially pectic polysaccharides and acidic, branched heteroglycans, e. g., glucuronoarabinoxylan, turned out to stimulate complement activation via both the classical and alternative pathway in most cases [9], [10], [11], [12], [13]. But sometimes the found effects are carelessly described as anti-complementary activities [12], [13]) and occasionally complement inhibiting and activating effects are even mixed up [14]. This may result from the widely applied haemolytic complement assay, also known as complement fixation test, which is originally used in clinical diagnostics. In this simple screening assay performed in the classical manner, both complement activating and inhibiting effects lead to reduced haemolysis and do not allow one to differentiate between these two modes of action.

The aim of the present study was to demonstrate that it is possible to clearly distinguish complement activating and inhibiting compounds by using the haemolytic complement assay. Besides heparin as a complement inhibitor, an arabinogalactan-protein type II (AGP) was included in the study. This AGP was isolated from pressed juice of the aerial parts of Echinacea purpurea L. Moench and recently structurally characterized [15]. Extracts and pressed juices from Echinacea purpurea are widely used as non-specific immunostimulants, namely orally for prevention and treatment of the common cold as well as treatment of chronic infections of the upper respiratory and lower urinary tract, topically for wound healing and parenterally for immunostimulation. The activity is mainly directed towards the non-specific cellular immune system. Besides glycoproteins, caffeic acid derivatives (cichoric acid) and alkamides, polysaccharides are discussed as pharmacologically active constituents [16]. Therefore, it was of a special interest to investigate AGP for any influences on the complement system, which could contribute to the proven immunomodulatory cinical effects of pressed juice from this herb.

Materials and Methods

Test compounds

Arabinogalactan-protein (AGP): The isolation and characterization of AGP was recently described in detail by Classen et al. [15]. Briefly, AGP was isolated from pressed juice of the aerial parts of Echinacea purpurea L. Moench, which was a kind gift from Madaus AG (Köln, Germany). After removal of free proteins and components with MW < 50 kd, an arabinogalactan-protein was obtained by precipitation with an aqueous solution of 1 mg/ml β-glucosyl Yariv reagent (1,3,5-tris-[4-β-D-glucopyranosyl-oxyphenylazo]-2,4,6-trihydroxybenzene) [17]. Preparative gel permeation chromatography on Sepharose CL-4B resulted in AGP which was eluted as a narrow peak with a mean MW of about 1200 kD. By means of the methods of carbohydrate structure elucidation, AGP was identified as an arabino-3,6-galactan, classified by Aspinall [18] as type II arabinogalactan. Besides a low protein content (7 %), it is mainly composed of a highly branched core polysaccharide of 3-, 6-, and 3,6-linked Galp residues with terminal Araf, GlcAp and terminal units of Araf-(1→5)-Araf-(1→.

To exclude any contamination with LPS, AGP was treated with 0.1 N NaOH for 17 h at 30 °C. To verify the absence of LPS, AGP was tested in the limulus amoebocyte lysate (LAL) assay (Pyroquant® 50, Pyroquant Diagnostic GmbH, Mörfelden-Waldorf, Germany).

Hydrolyzed arabinogalactan-protein (AGP-hydr.): Partial acid hydrolysis of AGP with 12.5 mM oxalic acid resulted in loss of about 75 % of Araf residues at the periphery of the molecule, whereas the major part of the galactose content of the starting material was recovered in the core-polysaccharide [15]. The linkages between the protein and polysaccharide moiety were stable under these hydrolytic conditions. This AGP-hydr. showed no more reactivity toward the β-glucosyl Yariv antigen.

Heparin: Unfractionated heparin of porcine mucosal origin (147 USP-U/mg) was bought from Sigma (Deisenhofen, Germany). As a lipopolysaccharide LPS from E. coli (Serotype 0111:B4) (Sigma, Deisenhofen, Germany) was used.

Haemolytic complement assays: The effects of the test compounds on the classical and alternative pathway complement activation were determined by haemolytic complement assays. Instead of a macro or semi-macro scale, a microtitre plate technique was used, which was first described by Klerx et al. [19].

Buffers: For the CP-CA, veronal buffered saline (pH 7.3 ± 0.1) (0.145 M sodium chloride, 1.82 mM barbital sodium, 3.12 mM barbital) supplemented with 0.25 mM calcium chloride and 0.83 mM magnesium chloride (VB) was purchased from Institut Virion-Serion (Würzburg, Germany). For the AP-CA, veronal buffered saline (0.142 M sodium chloride, 4.95 barbital sodium) was supplemented with 5.0 mM magnesium chloride and 8.0 mM ethylene glycol-bis-(β-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA) and adjusted to pH 7.2 with 0.1 N sodium hydroxide (EGTA-VB).

Human pooled serum: Human pooled serum (HPS) was used as source of complement. From at least 8 healthy volunteers, venous blood was drawn into S-Monovettes® Z (Sarstedt, Nümbrecht, Germany) for serum preparation. After coagulation of the blood at RT, the serum was separated by centrifugation (2500 g, 20 min, 15 °C), pooled and stored at -70 °C until use.

Erythrocytes: Sheep blood (Institut Virion-Serion, Würzburg, Germany) and rabbit blood (Charles River, Kißlegg, Germany) diluted in citrate-buffered glucose (Alsever's solution) served as sources of sheep erythrocytes (ShE) for the CP-CA and rabbit erythrocytes (RaE) for the AP-CA, respectively.

After centrifugation (1000 g, 10 min, 20 °C) and removal of the supernatant, the erythrocytes were washed three times with 0.9 % sodium chloride and once with VB (ShE) and EGTA-VB (RaE), respectively. Subsequently, ShE were resuspended in VB (2.5 × 10⁸ cells/ml) and sensitised by incubation with an equal volume of diluted (1 : 2500 with VB) haemolytic amboceptor, i. e., rabbit anti-ShE antibodies (Institut Virion-Serion, Würzburg, Germany). The sensitised ShEA (1.25 × 10⁸ cells/ml) were directly used for the CP-CA. RaE were resuspended in EGTA-VB (2.5 × 10⁸ RaE/ml).

Haemolytic complement assays for the classical and alternative pathway (CP-CA and AP-CA): The tests were performed in V-well microtitre plates (Nunc, Wiesbaden, Germany). The test compounds were dissolved in VB for the CP-CA and EGTA-VB for the AP-CA, respectively and stepwise diluted resulting in sample concentrations ranging from 0.25 μg/ml to 1000 μg/ml. HPS was freshly thawed-off at RT. A 1 : 80 dilution (1.25 %) in VB was used for the CP-CA, a 1 : 2 (50 %) dilution in EGTA-VB for the AP-CA. These HPS concentrations induced 50 % of the total haemolysis of the respective erythrocytes. For the assays with preincubation (CP-CA-30, AP-CA-30), 75 μl of the sample dilution were preincubated with 25 μl of the HPS dilution in the wells of the microtitre plates for 30 min at 37 °C. Subsequently, 50 μl of the ShEA and RaE suspensions, respectively, were added. For the assays without preincubation (CP-CA-0, AP-CA-0), first 50 μl erythrocytes suspensions were pipetted into the wells containing 75 μl sample solutions immediately followed by addition of 25 μl of the corresponding HPS dilution. Next, the microtitre plates were generally incubated for 45 min at 37 °C. To determine the degree of haemolysis, the microtitre plates were centrifuged (1000 g, 15 min, 4 °C) and 100 μl aliquots of the supernatants were mixed with 100 μl of water in 96-well flat-bottom microtitre plates (Greiner, Frickenhausen, Germany). The absorbance at 405 nm was measured using a microplate reader MRX Relevation (Dynex Technologies, Frankfurt, Germany).

In addition to the AP-CA-0 and AP-CA-30, a kinetic assay was carried out by varying the periods of preincubation of sample solution and HSP dilution, i. e., 0, 15, 30, 45, 60, and 90 min.

The following controls were included in each microtitre plate:

1. Total haemolysis by incubating the erythrocytes with 100 μl water instead of 75 μl sample solution and 25 μl HSP dilution.

2. 100 % haemolysis, i. e., 100 % complement activity, by using 25 μl buffer instead of sample solution. The corresponding absorbance at 405 nm in the CP-CA-0 and CP-CA-30 was 0.570 ± 0.015, that in the AP-CA-0 was 1.595 ± 0.020.

3. 0 % haemolysis, i. e. 0 % complement activity, by using 25 μl heat-inactivated HPS (by incubation for 30 min at 56 °C). The corresponding absorbance at 405 nm in the CP-CAs was 0.033 ± 0.003, that in the AP-CAs was 0.136 ± 0.010.

The results are presented as mean ± SD of triplicate determinations. All the tests were repeated on another day. The concentrations indicated in the text and the figures represent final concentrations in the mixtures containing test compounds, complement and erythrocytes. The IC₅₀ [μg/ml] corresponds to the concentration inhibiting the haemolysis by 50 %.

Results

Activity in the haemolytic complement assay for the
classical pathway

According to the usual strategy to test natural compounds for interactions with the complement system, AGP was first examined in the CP-CA-30, i. e., the complement fixation assay. As shown in Fig. [1] A, AGP concentration-dependently inhibited the haemolysis. Its IC₅₀ of 37 μg/ml was higher than that of heparin (IC₅₀ = 23 μg/ml), however at higher concentrations it was as active as heparin. In contrast to heparin, AGP concentrations lower than 2.5 μg/ml induced a slight, but reproducible activation of haemolysis. This effect was also observed with AGP-hydr. But AGP-hydr. was much less active than AGP, it only reduced haemolysis at concentrations higher than 20 μg/ml and its IC₅₀ of 454 μg/ml was about 12 times higher.

There are two different mechanisms leading to a reduced haemolysis in the CP-CA-30: On the one hand, a compound may inhibit the complement activation triggered by the sensitised erythrocytes as it is known for heparin [8]. On the other hand, a compound may induce complement activation. Due to the short half-live of activated complement factors, after the 30 min incubation less complement is left to be activated by the sensitised erythrocytes. Based on this fact, the CP-CA-0, which is performed without preincubation, should allow us to distinguish between these two mechanisms of apparent haemolysis inhibition. In fact, whereas the concentration-haemolysis curve of heparin in this assay (Fig. [1] B) was similar to that in the CP-CA-30, AGP and AGP-hydr. were much less active. Only AGP concentrations higher than 25 μg/ml inhibited haemolysis (IC₅₀ = 119 μg/ml), whereas low concentrations caused an about 10 % activation of haemolysis. Apart from the highest concentration (inhibition of 23.5 ± 1.7 % at 500 μg/ml), AGP-hydr. neither inhibited nor activated the haemolysis.

Activity in the haemolytic complement assay for the
alternative pathway

In the AP-CA-30, the three test compounds were considerably less active than in the CP-CA-30 (Fig. [2] A). In the presence of AGP-hydr., no significant changes in haemolysis were noticed. In contrast to the CP-CA-30, AGP was as active as heparin regarding the IC₅₀ values (124 μg/ml for AGP, 131 μg/ml for heparin). At low concentrations, AGP was even superior to heparin, which up to 25 μg/ml showed a slightly, but significantly increased haemolysis. Concentrations higher than 125 μg/ml AGP caused some turbidity due to a limited solubility in the buffer system of AP-CA.

When the AP-CA was performed without preincubation (AP-CA-0) (Fig. [2] B), AGP completely lost its activity. In contrast to the CP-CA-0, also the effect of heparin was weaker as obvious by its 3.3 times higher IC₅₀ of 433 μg/ml.

Haemolytic activity of serum in the complement assay for the alternative pathway

Since the absorbance corresponding to 100 % haemolysis was generally 10 - 14 % lower in the AP-CA-30 than in the AP-CA-0, the haemolytic activity of the HPS dilution in dependence on the preincubation time was investigated. As shown in Fig. [3], the extent of haemolysis linearly (R² = 0.9928) decreased with increasing incubation time. After 90 min preincubation of the HPS dilution at 37 °C, its haemolytic activity amounted to only 61.0 ± 2.2 % of the original one. Probably due to the lower HPS concentration, the time-dependent haemolytic activity decrease in the CP-CA was at most about 15 %.

Preincuabtion time-dependent activity in the haemolytic complement assay for the alternative pathway

These findings and the activity differences of both AGP and heparin in the AP-CA-0 and AP-CA-30 caused us to examine the effects of these compounds on the haemolysis in dependence on the preincubation time similar to Beukelman et al. [20].

With increasing preincubation time, AGP turned from an inactive compound at t = 0 min, i. e., without preincubation, to a more and more potent inhibitor of haemolysis (Fig. [4] A). The IC₅₀ exponentionally (exponentional function second order, R² = 0.9986) decreased from 304 μg/ml at t = 15 min to 13.2 μg/ml at t = 90 min (Fig. [5] A, black columns). At times ≥ 30min, AGP was considerably more active than heparin. This increase in activity becomes even more pronounced by calculating the IC₅₀ on the basis of the original haemolytic activity of the HPS dilution at t = 0 min (Fig. [5] A, white columns). After a preincubation time of 90 min, already 0.7 μg/ml AGP were sufficient to inhibit the original haemolytic activity by 50 %.

The preincubation time-dependent shifts in the concentration-haemolysis of heparin were completely different from those of AGP (Fig. [4]B). A strong activity increase was noticed between t = 0 min and t = 15 min. Higher incubation times resulted in an increasing activation of haemolysis at low concentrations as it was already observed in the AP-CA-30. Regarding the inhibiting parts of the curves, a considerable activity increase occurred between t = 0 min and t = 15 min similar to AGP. Longer incubation times led in turn to an apparent consecutive drop in activity as demonstrated by the IC₅₀ values (Fig. [5] B, black columns). However, the IC₅₀ values calculated on the basis of the original haemolytic activity of the HSP dilution did not significantly differ at t > 15 min (Fig. [5] B, white columns).

Discussion

AGP, an arabinogalactan-protein type II isolated from the pressed juice of Echinacea purpurea, was tested for any interactions with the complement-induced haemolysis. Its apparent concentration-dependent inhibitory activity in the CP-CA-30 and the AP-CA-30 suggests an interference with both pathways of complement activation as it has been described for other arabinogalactan-proteins type II [21], [22]. In the AP-CAs, higher concentrations of both AGP and heparin were required to achieve efficient inhibition. However, the interactions with the alternative complement pathway may not be really weaker, because the serum concentration used for the AP-CAs was 20 times higher than in the CP-CAs.

Removal of the arabinofuranoside residues by weak acid hydrolysis (AGP-hydr.) resulted in considerably lower activity in the CP-CA-30 and a complete loss of activity in the AP-CA-30. As the calculated MW of AGP-hydr. is only reduced by about 30 % and its hydrodynamic volume is nearly identical with that of AGP (1200 kd), the lower MW can be excluded as reason for that. However, the assumption that intact arabinofuranosyl residues are essential for the activity [22] is questionable, since Yamada [10] found that a pectin containing a variety of neutral galactosyl chains attached to the rhamnogalacturonan core exhibited only complement activating activity in the presence of these galactosyl chains. Therefore, rather the three-dimensional structure of exposed, flexible side chains than a specific type of monosaccharide may be important for the activity.

At first sight, AGP inhibits haemolysis like heparin. The highly complex regulatory effects of heparin on the complement system have been investigated for many decades, but there are still open questions [8]. Regardless of some specific interactions with complement factors, the overall activity of heparin is considered as an inhibition of complement activation. But this is not at all the case for AGP. Molecular mechanistic studies [23] revealed that AGP binds to the complement segment C1q similar to established activators of the classical pathway like immune complexes and LPS. Further, it strongly enhances the C3-consumption, a key component of both the classical and alternative pathway of complement activation. Finally, AGP induces an increase of the membrane-attack complex C5b-9, which is the final product of complement activation and responsible for cell lysis. By these mechanisms, AGP has been proven to represent a potent complement activating compound, which may contribute to the in vivo immunostimulating effects of Echinacea purpurea products. However, in vitro in the CP-CA-30 and AP-CA-30, these effects simulate an inhibition of the complement-induced haemolysis. Responsible for this misleading result is the fast elimination of activated complement factors [1]: the incubation of AGP with HPS leads to a consumption of complement so that less intact complement is left for the haemolysis, when the erythrocytes are added later on.

The presented study demonstrates that by simple modifications the classical haemolytic complement assays still allow us to differentiate between real complement inhibitors and activators. A first indication of the complement activating properties of AGP is already given by the slightly increased haemolysis in the CP-CA in the presence of low AGP concentrations, whose action is too weak to ultimately lead to a complement consumption. Performing the CP-CA without preincubation (CP-CA-0), AGP cannot activate and thus consume complement in advance but only during the coincubation with the ShEA. As a consequence, the activation-inhibition curve is shifted to higher concentrations. That means that only concentrations > 12 μg/ml are able to activate the complement in addition to the ShEA to an extent resulting in its decay during the 45 min incubation. Contrary to this, a real complement activation inhibitor like heparin retains its activity.

In the AP-CAs, a 20-fold higher HPS concentration was used. This explains the generally lower activity of the test compounds and the complete inactivity of AGP in the AP-CA-0. In contrast to the CP-CAs, not only AGP, but also heparin differs in its activity in the AP-CA-0 and the AP-CA-30. Still by further variations of the incubation times, striking differences between the influences of AGP and heparin became obvious. The underlying mechanism of the non-specific complement activation via the alternative pathway is completely different from the specific classical pathway activation [1]. In biological fluids, there is a constant slight basic activation being present as C3b, which may be the product of classical pathway activation or may derive from spontaneous hydrolysis of C3 [24]. This C3b is sufficient to trigger the amplifying loop under certain conditions such as activating surfaces of microorganisms and foreign cells. Under the conditions of preparation of HPS, this basic activation reaches a relatively high level [25]. After careful defrosting of the frozen HSP at RT, this complement activation proceeds during incubation at 37 °C with the consequence of a time-dependent consumption of the haemolytic activity (Fig. [3]). Coincubation with complement inhibitors like heparin partly prevents this further activation. The longer the incubation time the more pronounced becomes the difference between the haemolytic activity of the HPS with and without such an inhibitor (Fig. [4] B). But this effect can only be noticed at concentrations being too low to efficiently inhibit the burst activation induced by the RaE. The inhibiting parts of the curves and the corresponding IC₅₀ values suggest a decreasing potency of heparin with increasing incubation time (Fig. [4] B and Fig. [5] B, black columns). As heparin, however, conserves the original complement activity, the correct IC₅₀ values have all to be calculated on the basis of the 100 % haemolysis at t = 0 min. The resulting similar IC₅₀ values prove that the inhibition of the burst complement activation by addition of the RaE occurs independent of the preincuabtion time. (Since the mechanisms of heparin and other sulfated polysacchrides are not subject of the presented paper, further details, e. g., the activity increase between t = 0 min and t = 15 min, and the experimental proofs will be published elsewhere.) Coincubation with complement activators like AGP results in completely different effects. They stimulate the ongoing activation and thus even potentiate complement consumption. The longer they are incubated with the complement the less is left to induce haemolysis, when the RaE are added as evident by the time-dependent increase in the haemolysis inhibiting potency of AGP (Fig. [4] A and Fig. [5] A).

In conclusion, completely different modes of action, i. e., complement activation and inhibition, may result in similar haemolysis-concentration curves in the haemolytic complement assay performed in the classical way (CP-CA-30, AP-CA-30). But simple modifications turn them into efficient tools to distinguish between real complement inhibitors and complement activating compounds without performing expensive, molecular mechanistic investigations. For this, the CP-CA and the AP-CA have to be performed just with as well as without preincubation of the test compound with the HPS. An additional laborious kinetic study as additionally presented here and described earlier [22] is not necessary to get a clear discrimination. The differences between the resulting concentration-haemolysis curves and IC₅₀ values allow to identify the type of interaction with the complement system. As a practical example, AGP, a new arabinogalactan-protein type II isolated from pressed juice of the aerial parts of Echinacea purpurea [15], was identified as a stimulator of both the classical and alternative pathway of complement activation in this way. These results agree with sophisticated, but expensive and time-consuming methods such as ELISA, immunonephelometry assays or electrophoretic methods for molecular mechanistic investigations on the interactions of AGP with the complement system [23]. By enhancing the endogenous basic complement activation, AGP may support efficient activation of the non-specific immune system as, e. g., required for host defence against microbial pathogens. Whereas there are warranted doubts on absorption of AGP after oral administration of this Echinacea purpurea preparation, it may still substantially contribute to the well-established immunostimulating effects of this herbal medicinal drug, when it is topically or parenterally applied.

References

• 1 Morgan B P. Physiology and pathophysiology of complement: progress and trends.  Critical Reviews in Clinical Laboratory Sciences. 1995;  32 265-98
  PubMedSearch in Google Scholar
• 2 Marsh J E, Pratt J R, Sacks S H. Targeting the complement system.  Current Opinion in Nephrology and Hypertension. 1999;  8 557-62
  CrossrefPubMedSearch in Google Scholar
• 3 Figueroa J E, Densen P. Infectious diseases associated with complement deficiencies.  Clinical Microbiology Reviews. 1991;  4 359-95
  PubMedSearch in Google Scholar
• 4 Saxen H, Reima I, Makela P H. Alternative complement pathway activation by Salmonella O polysaccharide as a virulence determinant in the mouse.  Microbial Pathogenesis. 1987;  2 15-28
  CrossrefPubMedSearch in Google Scholar
• 5 Levy N J, Kasper D L. Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway.  Journal of Immunology. 1986;  136 4157-62
  PubMedSearch in Google Scholar
• 6 Schifferle R E, Wilson M E, Levine M J, Genco R J. Activation of serum complement by polysaccharide-containing antigens of Porphyromonas gingivalis .  Journal of Periodeontal Research. 1993;  28 248-54
  PubMedSearch in Google Scholar
• 7 Alvarez D, Merino S, Tomas J M, Benedi V J, Alberti S. Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates.  Infection and Immunity. 2000;  68 953-5
  CrossrefPubMedSearch in Google Scholar
• 8 Edens R E, Linhardt R J, Weiler J M. Heparin is not just an anticoagulant anymore: six and one-half decades of studies on the ability of heparin to regulate complement activity. In: Cruse JM, Lewis RE Jr, editors Complement Profiles. Basel; Karger 1993: 96-120
  Search in Google Scholar
• 9 Yamada H, Nagai T, Cyong J C, Otsuka Y, Tomoda M, Shimizu N, Gonda R. Relationship between chemical structure and activating potencies of complement by an acidic polysaccharide, Plantago-mucilage A, from the seed of Plantago asiatica .  Carbohydrate Research. 1986;  156 137-45
  CrossrefPubMedSearch in Google Scholar
• 10 Yamada H. Pectic polysaccharides from Chinese herbs: structure and biological activity.  Carbohydrate Polymers. 1994;  25 269-76
  CrossrefPubMedSearch in Google Scholar
• 11 Michaelsen T E, Gilje A, Samuelsen A B, Hogasen K, Paulsen B S. Interaction between human complement and a pectin type polysaccharide fraction, PMII, from the leaves of Plantago major L.  Scandavian Journal of Immunology. 2000;  52 483-90
  PubMedSearch in Google Scholar
• 12 Shin K S, Kwon K S, Yang H C. Screening and characteristics of anti-complementary polysaccharides from Chinese medicinal herbs.  Han’guk Nonghwa Hakhoechi. 1992;  35 42-50
  PubMedSearch in Google Scholar
• 13 Samuelsen A B, Lund I. Djahromi JM, Paulsen BS, Wold JK, Knutsen SH. Structural features and anti-complementary activity of some heteroxylan polysaccharide fractions from the seeds of Plantago major L.  Carbohydrate Polymers. 1999;  38 133-43
  CrossrefPubMedSearch in Google Scholar
• 14 Zvyagintseva T N, Shevchenko N M, Nazarova I V, Scobun A S, Luk’yanov P A, Elyakova L A. Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds.  Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 2000;  126C 209-15
  PubMedSearch in Google Scholar
• 15 Classen B, Witthohn K, Blaschek W. Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the b-glucosyl Yariv reagent.  Carbohydrate Research. 2000;  327 497-504
  CrossrefPubMedSearch in Google Scholar
• 16 Bauer R. Echinacea drugs-effects and active ingredients.  Zeitschrift für Ärztliche Fortbildung. 1996;  90 111-5
  PubMedSearch in Google Scholar
• 17 Yariv J, Rapport M M, Graf L. The interaction of glycosides and saccharides with antibody to the corresponding phenylazo glycosides.  Biochemical Journal. 1962;  85 383-8
  PubMedSearch in Google Scholar
• 18 Aspinall G O. Carbohydrate Polymers of Plant Cell Walls. In: Loewus F, editor Biogenesis of Plant Cell Wall Polysaccharides. New York; Academic Press 1973: 95
  Search in Google Scholar
• 19 Klerx J P, Beukelman C J, Van Dijk H, Willers J M. Microassay for colorimetric estimation of complement activity in guinea pig, human and mouse serum.  Journal of Immunological Methods. 1983;  63 215-20
  CrossrefPubMedSearch in Google Scholar
• 20 Beukelman C J, Rademaker P M, van Dijk H, Aerts P C, Berrens L, Willers J M. House dust allergen activates the classical complement pathway in mouse serum.  Immunological Letters. 1986;  13 159-64
  PubMedSearch in Google Scholar
• 21 Yamada H, Kiyohara H, Cyong J C, Otsuka Y. Studies on polysaccharides from Angelica acutiloba-IV. Characterization of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa.  Molecular Immunology. 1985;  22 295-304
  CrossrefPubMedSearch in Google Scholar
• 22 Diallo D, Paulsen B S, Liljeback T HA. Michaelsen TE. Polysaccharides from the roots of Entada africana Guill. et Perr., Mimosaceae, with complement fixing activity.  Journal of Ethnopharmacology. 2001;  74 159-71
  CrossrefPubMedSearch in Google Scholar
• 23 Odenthal K P, Schwarz T, Witthohn K, Loos M. Bioassay-guided identification of immunomodulating constituents in Echinacin®. International Congress and 48th Annual Meeting of the Society for Medicinal Plant Research Zürich; 3. - 7.9.2000: P4B/16
  Search in Google Scholar
• 24 Lachmann P J, Hughes-Jones N C. Initiation of complement activation.  Springer Seminars in Immunopathology. 1984;  7 143-62
  CrossrefPubMedSearch in Google Scholar
• 25 Kirschfink M. The clinical laboratory: Testing the complement system. In: Rother K, Till GO, Hänsch GM, editors The complement system. Berlin; Springer 1998: 522-4
  Search in Google Scholar

Priv-Doz. Dr. Susanne Alban

Institute of Pharmacy

University of Regensburg

Universitätsstr. 31

93040 Regensburg

Germany

Phone: +49-941-943 4792

Fax: +49-941-943 4762

Email: Susanne.Alban@chemie.uni-regensburg.de

References

• 1 Morgan B P. Physiology and pathophysiology of complement: progress and trends.  Critical Reviews in Clinical Laboratory Sciences. 1995;  32 265-98
  PubMedSearch in Google Scholar
• 2 Marsh J E, Pratt J R, Sacks S H. Targeting the complement system.  Current Opinion in Nephrology and Hypertension. 1999;  8 557-62
  CrossrefPubMedSearch in Google Scholar
• 3 Figueroa J E, Densen P. Infectious diseases associated with complement deficiencies.  Clinical Microbiology Reviews. 1991;  4 359-95
  PubMedSearch in Google Scholar
• 4 Saxen H, Reima I, Makela P H. Alternative complement pathway activation by Salmonella O polysaccharide as a virulence determinant in the mouse.  Microbial Pathogenesis. 1987;  2 15-28
  CrossrefPubMedSearch in Google Scholar
• 5 Levy N J, Kasper D L. Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway.  Journal of Immunology. 1986;  136 4157-62
  PubMedSearch in Google Scholar
• 6 Schifferle R E, Wilson M E, Levine M J, Genco R J. Activation of serum complement by polysaccharide-containing antigens of Porphyromonas gingivalis .  Journal of Periodeontal Research. 1993;  28 248-54
  PubMedSearch in Google Scholar
• 7 Alvarez D, Merino S, Tomas J M, Benedi V J, Alberti S. Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates.  Infection and Immunity. 2000;  68 953-5
  CrossrefPubMedSearch in Google Scholar
• 8 Edens R E, Linhardt R J, Weiler J M. Heparin is not just an anticoagulant anymore: six and one-half decades of studies on the ability of heparin to regulate complement activity. In: Cruse JM, Lewis RE Jr, editors Complement Profiles. Basel; Karger 1993: 96-120
  Search in Google Scholar
• 9 Yamada H, Nagai T, Cyong J C, Otsuka Y, Tomoda M, Shimizu N, Gonda R. Relationship between chemical structure and activating potencies of complement by an acidic polysaccharide, Plantago-mucilage A, from the seed of Plantago asiatica .  Carbohydrate Research. 1986;  156 137-45
  CrossrefPubMedSearch in Google Scholar
• 10 Yamada H. Pectic polysaccharides from Chinese herbs: structure and biological activity.  Carbohydrate Polymers. 1994;  25 269-76
  CrossrefPubMedSearch in Google Scholar
• 11 Michaelsen T E, Gilje A, Samuelsen A B, Hogasen K, Paulsen B S. Interaction between human complement and a pectin type polysaccharide fraction, PMII, from the leaves of Plantago major L.  Scandavian Journal of Immunology. 2000;  52 483-90
  PubMedSearch in Google Scholar
• 12 Shin K S, Kwon K S, Yang H C. Screening and characteristics of anti-complementary polysaccharides from Chinese medicinal herbs.  Han’guk Nonghwa Hakhoechi. 1992;  35 42-50
  PubMedSearch in Google Scholar
• 13 Samuelsen A B, Lund I. Djahromi JM, Paulsen BS, Wold JK, Knutsen SH. Structural features and anti-complementary activity of some heteroxylan polysaccharide fractions from the seeds of Plantago major L.  Carbohydrate Polymers. 1999;  38 133-43
  CrossrefPubMedSearch in Google Scholar
• 14 Zvyagintseva T N, Shevchenko N M, Nazarova I V, Scobun A S, Luk’yanov P A, Elyakova L A. Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds.  Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 2000;  126C 209-15
  PubMedSearch in Google Scholar
• 15 Classen B, Witthohn K, Blaschek W. Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the b-glucosyl Yariv reagent.  Carbohydrate Research. 2000;  327 497-504
  CrossrefPubMedSearch in Google Scholar
• 16 Bauer R. Echinacea drugs-effects and active ingredients.  Zeitschrift für Ärztliche Fortbildung. 1996;  90 111-5
  PubMedSearch in Google Scholar
• 17 Yariv J, Rapport M M, Graf L. The interaction of glycosides and saccharides with antibody to the corresponding phenylazo glycosides.  Biochemical Journal. 1962;  85 383-8
  PubMedSearch in Google Scholar
• 18 Aspinall G O. Carbohydrate Polymers of Plant Cell Walls. In: Loewus F, editor Biogenesis of Plant Cell Wall Polysaccharides. New York; Academic Press 1973: 95
  Search in Google Scholar
• 19 Klerx J P, Beukelman C J, Van Dijk H, Willers J M. Microassay for colorimetric estimation of complement activity in guinea pig, human and mouse serum.  Journal of Immunological Methods. 1983;  63 215-20
  CrossrefPubMedSearch in Google Scholar
• 20 Beukelman C J, Rademaker P M, van Dijk H, Aerts P C, Berrens L, Willers J M. House dust allergen activates the classical complement pathway in mouse serum.  Immunological Letters. 1986;  13 159-64
  PubMedSearch in Google Scholar
• 21 Yamada H, Kiyohara H, Cyong J C, Otsuka Y. Studies on polysaccharides from Angelica acutiloba-IV. Characterization of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa.  Molecular Immunology. 1985;  22 295-304
  CrossrefPubMedSearch in Google Scholar
• 22 Diallo D, Paulsen B S, Liljeback T HA. Michaelsen TE. Polysaccharides from the roots of Entada africana Guill. et Perr., Mimosaceae, with complement fixing activity.  Journal of Ethnopharmacology. 2001;  74 159-71
  CrossrefPubMedSearch in Google Scholar
• 23 Odenthal K P, Schwarz T, Witthohn K, Loos M. Bioassay-guided identification of immunomodulating constituents in Echinacin®. International Congress and 48th Annual Meeting of the Society for Medicinal Plant Research Zürich; 3. - 7.9.2000: P4B/16
  Search in Google Scholar
• 24 Lachmann P J, Hughes-Jones N C. Initiation of complement activation.  Springer Seminars in Immunopathology. 1984;  7 143-62
  CrossrefPubMedSearch in Google Scholar
• 25 Kirschfink M. The clinical laboratory: Testing the complement system. In: Rother K, Till GO, Hänsch GM, editors The complement system. Berlin; Springer 1998: 522-4
  Search in Google Scholar

Priv-Doz. Dr. Susanne Alban

Institute of Pharmacy

University of Regensburg

Universitätsstr. 31

93040 Regensburg

Germany

Phone: +49-941-943 4792

Fax: +49-941-943 4762

Email: Susanne.Alban@chemie.uni-regensburg.de