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The presence of natural human antibodies reactive against pharmacologically active pectic polysaccharides from herbal medicines.

Abstract

Direct ELISA was performed using normal human sera and human colostrum, to analyse the presence of antibodies which react with pharmacologically active pectic polysaccharides isolated from plants used in traditional Japanese herbal (Kampo) medicine. All sera and colostrum were shown to contain IgM, IgG, IgA and secretory IgA class antibodies which react with the active pectic polysaccharides to different degrees. The reacting IgG antibody in normal human serum recognized the ramified regions (rhamnogalacturonan core with carbohydrate side-chains) of the pharmacologically active pectic polysaccharides as the active sites for complement-activating activity. Correlation analysis indicated that a significant and positive correlation was observed between reactivity with the reacting antibody of IgG class and the degree of complement-activating activity of the active polysaccharides.

The reacting IgG class antibody, which was purified from normal human serum by affinity chromatography on bupleuran 2IIc (a pharmacologically active pectic polysaccharide from the roots of Bupleurum falcatum)-immobilized Sepharose, showed cross-reactivity not only with some other pharmacologically active pectic polysaccharides from other medicinal herbs but also with autoantigens such as single-strand DNA, myosin and tublin from mammals.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Natural antibodies; Human; Pectic polysaccharides; Herbal medicines; Complement-activating activity

Introduction

Herbal medicines comprise numerous types of ingredients, of low and high molecular weight. Macro-molecular polysaccharides have been described as essential ingredients for the expression of pharmacological activities in some herbal medicines, including the traditional Japanese herbal (Kampo) medicines Juzen-taiho-to (TJ-48, Shi-Quan-Da-Bu-Tang; Yamada, 1999) and Sho-saiko-to (TJ-9, Xiao-Chai-Hu-Tang; Yamaoka et al., 1995) as well as Echinacea purpurea (Barrett, 2003). Clarification of the modes of action of the bioactive polysaccharides in herbal medicines is required in order to understand their roles in herbal medicines and further application.

Numerous complement-activating polysaccharides have been isolated from various kinds of medicinal plants and fungi, including Kampo medicines, and some of these polysaccharides have also shown several other immunomodulating activities (Yamada, 1999; Yamada and Kiyohara, 1999). Complement activation appears to be intrinsically associated with several immunomodulation phenomena, including immunopotentiation and anti-inflammatory effects (Law and Reid, 1995). Therefore, the complement-activating polysaccharides in herbal medicines are assumed to contribute to the expression of their immunopharmacological activities.

The complement-activating polysaccharides found in medicinal plants are grouped into pectic polysaccharides (pectins, pectic arabinogalactans, and pectic herteroglycans), arabinogalactans, arabinans and other types such as mucilage, and it is suggested that they activate the complement system mainly through the classical pathway (Yamada and Kiyohara, 1999). Activation of the complement system through the classical pathway is initiated by the formation of immune complexes (Law and Reid, 1995). When IgG-depleted normal human serum was used to measure the complement-activating activity of pectic polysaccharides (AAFIIb-2 and IIb-3) from leaves of Artemisia princeps PAMP, the activity was significantly reduced (Yamada et al., 1991). This observation raises the hypothesis that normal human serum contains the antibody against complement-activating polysaccharides from the herbal medicines. Because immunoglobulin receptors are expressed in some immune cells (Flesch and Neppert, 2000), this antibody may play an important role in the expression of immunopharmacological activity via the complement-activating pectic polysaccharides in herbal medicines.

This report describes naturally occurring antibodies that react with pharmacologically active pectic polysaccharides from the component herbs prescribed in Kampo medicine.

Materials and Methods

Materials

Sera and antibodies

Normal human sera were obtained from healthy volunteers, and all the sera used for detection and purification of polysaccharide-reacting natural antibody were incubated at 56 [degrees]C for 30 min in order to inactivate the complement system and stored at -80 [degrees]C until use. The sera used for measurement of the complement-activating activity of the samples were stored at -80 [degrees]C without any treatment until use. Secretory IgA from human colostrum was purchased from Sigma.

Medicinal herbs

The roots of Angelica acutiloba Kitagawa were obtained from Tochimoto-Tenkaido Co. Ltd. (Osaka), and roots of Bupleurum falcatum L., roots of Glycyrrhiza uralensis Fisch et DC and leaves of Artemisia princeps PAMP were purchased from Uchida-Wakan-Yaku Co. Ltd. (Tokyo).

Polysaccharides and antigens

Pharmacologically active polysaccharides from the following single herbs were prepared as described previously (Yamada and Kiyohara, 1999): roots of A. acutiloba Kitagawa (AR-2IIa, AR-2IIb and AR-2IId: pectins, AGIIb-1: pectic arabinogalactan), roots of B. falcatum L. (bupleuran 2IIb and bupleuran 2IIc: pectins), roots of G. uralensis Fisch et DC. (GR-2IIb: pectin), and leaves of A. princeps PAMP (AAF-IIb-2: pectic heteroglycan). Various carbohydrate digestions of the ramified region (rhamnogalacturonan possessing carbohydrate side-chains rich in neutral sugars) of bupleuran 2IIc were performed with endo-[beta]-D-(1 [right arrow] 4)-galactanase, endo-[alpha]-L-(1 [right arrow] 5)-arabinanase, exo-[beta]-D-(1 [right arrow] 3)-galactanase and rhamnogalacturonase A following the procedure of Sakurai et al. (1998). Myosin from rabbit muscle, single-strand DNA from calf thymus, and tublin from bovine brain were purchased from Sigma.

Detection of polysaccharide-reacting antibodies

Solutions of polysaccharides and other antigens in phosphate-buffered saline (PBS, 100 [micro]l/well, 125-1000 ng/well) were added to 96-well microtiter plates (Sumitomo, MS-3596 F/H), and incubated at 37 [degrees]C overnight. Unbound antigens on the plate were removed by washing with PBS containing 0.05% Tween 20 (PBST, 250 [micro]l) four times. The plate was further incubated with 1% skim milk (SM) in PBST (SM-PBST)(250 [micro]l/well) at 37 [degrees]C for 1 h, and washed with PBST four times. Dilutions of normal human sera (1:200) or purified polysaccharide-reacting IgG with SM-PBST were added to the wells (100 [micro]l/well), and incubated at 37 [degrees]C for 1 h. After the plate was washed with PBST five times, alkaline phosphatase-labelled anti-human IgA, IgM or IgG (Kappel, 100 [micro]l), which was diluted with SM-PBST (1:1000), was added to the wells and incubated at 37 [degrees]C for 1 h. The plate was washed with PBST five times, and each well was incubated with 150 [micro]l of chromogenic substrate solution (1 mg of p-nitrophenylphosphate disodium salt in 1 ml of 10% diethanolamine buffer, pH 9.8) at room temperature. The absorbance at 405 nm was measured during incubation using a Microplate reader (Model 450, Bio-Rad).

Measurement of complement-activating activity

Complement-activating activity was measured as anti-complementary activity by the method described previously (Kiyohara et al., 1986). All the active pectic polysaccharides used in this study have been suggested to show anti-complementary activity via activation of the complement system, mainly through the classical pathway (Yamada and Kiyohara, 1999).

Purification of bupleuran 2IIc-reacting natural IgG antibody from normal human sera

Protein G-Sepharose chromatography

Inactivated normal human sera were loaded on prepacked columns of Protein G-Sepharose (Pharmacia Biotech) according to the manufacturer's instructions. After the unabsorbed fraction was removed by washing the column with PBS, the absorbed IgG fraction was eluted using 0.1 M glycine-HCl buffer (pH 2.7). The IgG fraction was dialyzed with PBS and concentrated by ultrafiltration using a YM-3 membrane (Amicon, USA).

Affinity chromatography on bupleuran 2IIc-immobilized Sepharose 4B

Bupleuran 2IIc was immobilized to EAH-Sepharose (Pharmacia Biotech) with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride according to the procedure of Miskiel and Pazur (1991). The IgG fraction from 2 ml of normal human serum was loaded onto a column (2 ml) of bupleuran 2IIc-immobilized Sepharose 4B equilibrated with PBS, and an unabsorbed fraction was eluted with PBS. The absorbed fraction was eluted with 1 M ammonium thiocyanate (pH 4.9) into tubes containing 1 M glycine-NaOH buffer (pH 7.5), and proteins were monitored at 280 nm. The natural IgG obtained, which recognizes bupleuran 2IIc, was dialyzed and concentrated as above.

Results

Natural antibodies active against pharmacologically active pectic polysaccharides from component herbs prescribed in Kampo medicines

The presence of pharmacologically active pectic polysaccharide-reacting immunoglobulins in normal human serum was analyzed by the direct ELISA method. Seven pharmacologically active pectic polysaccharides purified from component herbs frequently prescribed in Kampo medicine such as G. uralensis (GR-2IIb), A. acutiloba (AR-2IIa, AR-2IIb, AR-2IIc AR-2IId, and AGIIb-1), B. falcatum (bupleuran 2IIb and bupleuran 2IIc) and A. princeps (AAF-IIb-2) were each coated on ELISA plates. After the diluted serum was reacted with the coated polysaccharides, the bound antibodies of IgM, IgG and IgA classes were detected by alkaline phosphatase-labelled anti-immunoglobulin antibodies. Fig. 1 clearly shows that normal human serum contained antibodies of the IgM, IgG and IgA classes, which could react with all the active pectic polysaccharides tested, although the degrees of reactivity with the antibodies were different among the pectic polysaccharides. When sera from 6 normal adults were analyzed for the presence of the IgG class antibody, reacting with 6 kinds of the above pharmacologically active pectic polysaccharides, all were shown to contain various contents of the reacting antibody of IgG class (data not shown). However, IgE class antibody was not detected in noticeable amounts as the pectic polysaccharide reacting antibody, in the direct ELISA (data not shown).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Secretory IgA is the dominant immunoglobulin in the digestive system. If polysaccharide-reacting IgA is present in the digestive system, it is possible that the reacting IgA may interact with the pharmacologically active polysaccharides of herbal medicines in the intestinal fluid. In order to examine whether humans also possess polysaccharide-reacting secretory IgA antibody in the mucosal site, direct ELISA was performed using a secretory IgA preparation from human colostrum. IgA-secreting lymphocytes in the mammary gland are known to derive from gut-associated lymphoid tissue (GALT), and it is reported that recognition patterns of secretory IgA for antigens in colostrum and intestinal fluid are similar (Roux et al., 1977; McGhee et al., 1999). The IgA preparation from normal human colostrum reacted significantly with some pharmacologically active pectic polysaccharides, bupleuran 2IIb, AAFIIb-2 and AR 2IId from B. falcatum, A. princeps and A. acutiloba, respectively (Fig. 2).

The pharmacologically active pectic polysaccharides used in the present study express their complement-activating activities via their ramified regions (rhamno-galacturonan with complex side-chains rich in neutral sugars) as one of their structural units (Yamada and Kiyohara, 1999). In order to elucidate the contribution of the pectic polysaccharide-reacting antibody to expression of the complement-activating activity of the polysaccharides, we tested the reactivity of the ramified regions of the active pectic polysaccharides with the antibody. All the ramified regions of the pharmacologically active pectins (AR-2IIb, bupleuran 2IIc and GR-2IIb) tested showed stronger reactivity with the IgG class antibody in serum than the original active pectins (data not shown). The correlation between the reactivity of the polysaccharides with the reacting IgG antibody and the degree of complement-activating activity of the polysaccharides were also analyzed. As shown in Fig. 3, a statistically significant and positive correlation ([gamma] = 0.847, n = 7, p < 0.05) was observed among 7 types of the complement-activating pectic polysaccharides. These results suggest that the polysaccharide-reacting antibody contributes to expression of the complement-activating activity of the polysaccharides.

[FIGURE 3 OMITTED]

Property of the IgG class antibody reacting with a pectic polysaccharide (bupleuran 2IIc) from B. falcatum

In order to characterize pectic polysaccharide-reacting antibodies, bupleuran 2IIc-reacting IgG was purified from normal human serum. Crude IgG fractions, prepared from 3 different normal human sera by Protein G-Sepharose affinity chromatography, were each further fractionated by bupleuran 2IIc-immobilized-Sepharose, and the bound fraction was obtained as bupleuran 2IIc-reacting IgG (contents in total IgG; 0.1-0.26%, data not shown). Sakurai et al. (1998) have reported that some carbohydrate side-chains and rhamnogalacturonan in the ramified region of bupleuran 2IIc can be trimmed by various structure-specific carbohydrases. When the ramified region of bupleuran 2IIc was digested with endo-[beta]-D-(1 [right arrow] 4)-galactanase, endo-[alpha]-L-(1 [right arrow] 5)-arabinanase or exo-[beta]-D-(1 [right arrow] 3)-galactanase, each resulting digestion product decreased about 16, 23 and 38% of the reactivity of the ramified region with bupleuran 2IIc-reacting IgG, respectively, and the combined digestion with endo-[alpha]-L-(1 [right arrow] 5)-arabinanase and rhamnogalacturonase A reduced the reactivity by about 53% (data not shown).

These results indicate that bupleuran 2IIc-reacting IgG preparation contains polyclonal IgG antibody, which recognizes the various carbohydrate chains in the ramified region of bupleuran 2IIc. Bupleuran 2IIc-reacting IgG could react significantly with some other pharmacologically active polysaccharides such as bupleuran 2IIb, bupleuran 2IIc, AGIIb-1 and AAFIIb-2 (Fig. 4). Bupleuran 2IIb, which was not an antigen for affinity chromatography, showed stronger reactivity with the purified IgG than bupleuran 2IIc. This result suggests that these polysaccharides contain the carbohydrate epitope(s) for bupleuran 2IIc-reacting IgG in different amounts.

It is well known that various anti-carbohydrate natural antibodies such as anti-[alpha]-Gal antibody exist in normal human sera (Galili et al., 1999), and that many naturally occurring antibodies including [alpha]-Gal antibody show polyreactivity against autoantigens (Satapathy et al., 1999). Polyreactivity of the purified bupleuran 2IIc-reacting IgG was also tested for single-strand DNA, myosin and tubulin, and the IgG showed weak but significant cross-reactivities against these autoantigens (Fig. 5).

[FIGURE 4 OMITTED]

Discussion

Detectable antibodies, which react with the body's own molecules, or foreign molecules without known immunization, are termed natural antibodies (Coutinho et al., 1995). The present study strongly suggests the presence of antibodies which react with carbohydrate chains of pharmacologically active pectic polysaccharides from the component herbs prescribed in Kampo medicine, in sera and mucosal sites such as the digestive system of normal humans, and that the polysaccharide-reacting antibodies are natural antibodies. It has recently been reported that natural antibodies active against 1,3/1,6-[beta]-D-glucans from fungi are present in sera from humans and mice (Harada et al., 2003; Nasuzawa et al., 2003). The bupleuran 2IIc-reacting IgG purified in the present study did not show cross-reactivity with schizophylan, 6-branched-[beta]-D-1,3-glucan (Kiyohara, unpublished results). It is therefore suggested that the pectic polysaccharide-reacting antibodies are present as the other natural antibody in human serum. Although the bupleuran 2IIc-reacting IgG cross-reacted with some pectic polysaccharides from the other medicinal herbs, the present study also suggests that human serum contains many natural antibodies, which interact with various kinds of other polysaccharide antigens obtained from the other medicinal herbs.

[FIGURE 5 OMITTED]

Natural antibodies recognizing carbohydrate chains have been found asanti-[alpha]-Gal- and anti-blood-type antigens (Galili et al., 1999). Anti-[alpha]-Gal natural antibodies can be detected in human and old-world monkeys, but not in other mammals such as rodents (Galili et al., 1999). Because pectic polysaccharide-reacting antibodies could be detected even in the sera of mice, and the purified bupleuran 2IIc-reacting IgG did not show any cross-reactivity with neoglycoconjugates bearing oligosaccharide epitopes for anti-[alpha]-Gal- and anti-blood-type antibodies (Kiyohara, unpublished results), pectic polysaccharide-reacting natural antibodies are probably not related to the known anti-[alpha]-Gal- and anti-blood-type antibodies. Polyreactive characteristics of natural antibodies against a variety of autoantigens have been proposed as an explanation for many functional aspects of this natural antibody (Satapathy and Ravindran, 1999). The present study also indicates that the pectic polysaccharide-reacting antibody has a polyreactive feature for some autoantigens from mammals. Ishikawa et al. (1998) have reported that some anti-carbohydrate monoclonal antibodies cross-reacted with peptides, and that the three-dimensional features of some oligosaccharides are similar to those of peptide moieties. These reports suggest the possibility that pectic polysaccharide-reacting natural antibodies may be produced originally to act against autoantigens of human and rodents, and that these autoantibodies may cross-react with oligosaccharide moieties in pharmacologically active pectic polysaccharides from the medicinal herbs.

Sakurai et al. (1996) have reported that bupleuran 2IIc was detected in the liver of mice after oral administration, and this observation postulated that bupleuran 2IIc can be absorbed from the digestive system into the blood stream. Because some immunoglobulin receptors are expressed in B cells, macrophages, dendritic cells, NK cells and mast cells (Flesch and Neppert, 2000), the pectic polysaccharide-reacting natural antibodies are assumed to contribute to the expression of immunopharmacological activities of the pectic polysaccharides through the immunoglobulin receptors after absorption. The present study also indicated that pectic polysaccharide-reacting natural and secretory IgA antibody exists in mucosal sites such as the human intestine. The IgA receptor has been found to be located on M cells of Peyer's patches in the human intestine, and the receptor contributes to the uptake of antigen-IgA immune complexes into Peyer's patches (Mantis et al., 2002). Sakurai et al. (1996) have also reported that bupleuran 2IIc is accumulated in Peyer's patches by oral administration to mice. Meanwhile, dimeric IgA has been assumed to contribute to activate the alternative complement pathway to produce some biologically active complement fragments, and complement components such as C3 and C4 are known to be produced by the epithelial cells of the intestinal tract (Michael et al., 1999; Andoh, 1993).

It is also speculated that the formation of immune complexes of pectic polysaccharide-reacting natural IgA antibody with the active pectic polysaccharides in the intestinal fluid may not only participate in incorporation of the active pectic polysaccharides into Peyer's patches but also activate complement components in the fluid to result in certain modulations of the intestinal immune system. Nevertheless, herbal medicines cause inflammatory reactions such as acute hepatitis as their side-effects in isolated cases. The possibility still remains that the polysaccharide-reacting natural antibody participates in the hyperreaction, although the natural antibody of IgE class was not detected in normal human sera in the present study.

Clarification of the roles and biological functions of the pectic polysaccharide-reacting natural antibodies awaits further study.

Acknowledgements

A part of the present work was supported by the 21st Century COE Program, Ministry of Education, Culture, Sports, Science and Technology of Japan. A part of this work was also supported by a fund from Tsumura & Co. Ltd., Japan. We would like to thank Ms. Y. Higuchi, M. Itoh and E. Ikeda for their technical assistance.

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H. Kiyohara (a,b), T. Matsumoto (a,b), T. Nagai (a,b), S.-J. Kim (b), H. Yamada (a,b,*)

(a) Kitasato Institute for Life Sciences, Kitasato University, Tokyo 108-8641, Japan

(b) Oriental Medicine Research Center, The Kitasato Institute, Tokyo 108-8642, Japan

Received 27 January 2005; accepted 24 March 2005

*Corresponding author. Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Tel.: +81 3 5791 6364; fax: +81 3 3445 1351.

E-mail address: yamada@lisci.kitasato-u.ac.jp (H. Yamada).
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Author:Kiyohara, H.; Matsumoto, T.; Nagai, T.; Kim, S.-J.; Yamada, H.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Jul 1, 2006
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