Chemical composition and immunomodulatory activity of a pectic polysaccharide from the ground thistle Cirsium esculentum Siev.

Food Chemistry 126 (2011) 870–877

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Chemical composition and immunomodulatory activity of a pectic polysaccharide from the ground thistle Cirsium esculentum Siev. Daria S. Khramova a,⇑, Victoria V. Golovchenko a, Alexandre S. Shashkov b, Dorjgoo Otgonbayar c, Aria Chimidsogzol c, Yury S. Ovodov a a b c

Institute of Physiology, Komi Science Centre, The Urals Branch of the Russian Academy of Sciences, 50, Pervomaiskaya str., Syktyvkar 167982, Russia N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47, Leninsky Prospect, Moscow 119991, Russia Institute of Chemistry and Chemical Technology, The Mongolian Academy of Sciences, Ulaanbaatar-51, Mongolia

a r t i c l e

i n f o

Article history: Received 28 May 2010 Received in revised form 9 October 2010 Accepted 10 November 2010 Available online 19 November 2010 Keywords: Cirsium esculentum Siev. Pectin Structural features Enzymatic digestion by 1,4-a-Dpolygalacturonase NMR spectroscopy of polysaccharides Oral tolerance abrogation Anti-allergic activity

a b s t r a c t The pectic polysaccharide cirsiuman CE was extracted from the stems of the ground thistle Cirsium esculentum Siev. using 0.7% aqueous ammonium oxalate, yielding 10.4% of the air-dried material. The backbone of cirsiuman was proved to consist of 1,4-a-D-galactopyranosyluronan blocks interconnected by 1,2-linked L-rhamnose residues. The side chains were attached to the 4-position of the L-rhamnopyranose residues. The side chains include 1,4-b-galactopyranan, which contains 3,4- and 4,6-substituted D-galactopyranose residues as branched points, and 1,5-a-arabinofuranan bearing 3,5-substituted a-L-arabinofuranose residues as the branching points. Oral administration of cirsiuman was found to prevent induction of oral tolerance to ovalbumin (OVA), increased the levels of serum IgG2a, and downregulated serum IgE responses, therefore it was tested for anti-allergic properties. The score of systemic anaphylactic reaction and the levels of both serum IgE and IL-4 were found to decrease; however, IFN-c response was upregulated by cirsiuman feeding. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Oral tolerance has long been recognised as a physiological mechanism of immune unresponsiveness to dietary and bacterial microflora antigens (Smith, Eaton, Finlayson, & Garside, 2000). Development of oral tolerance represents an undesirable side effect in oral immunisation strategies because soluble antigens require the application of adjuvants. It has been hypothesised that the food components may affect immunogenic potential of ingested proteins (Wijk et al., 2005). Therefore, an elucidation of dietary immunopotentiator that are able to evoke an immune response on co-administrated protein antigens is of great interest. Pectins are considered to be the most complicated polysaccharides in relation to their structural features. They have irregular block sugar chains and contain various macromolecular segments of the linear and ramified regions. The linear region consists of a-1,4-D-galacturonan chain as the backbone of all pectins. The ramified regions contain various heteroglycanogalacturonan segments, such as rhamnogalacturonan I, rhamnogalacturonan II, xylogalacturonan, and apiogalacturonan (O’Neill, Albersheim, & Darvill, 1990; Perez, Rodriguez-Carvajal, & Doco, 2003). Pectins ⇑ Corresponding author. Tel./fax: +7 8212 241001. E-mail address: [email protected] (D.S. Khramova). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.11.062

are present in the diet and are widely used in food industry as gelling agents. Several recent papers have described the immune stimulating activity of pectic polysaccharides at oral administration. Oral administration of a polysaccharide fraction from dried extract (TJ-48) of Japanese herbal (Kampo) to mice showed the intestinal immune system modulating activity (Kiyohara, Matsumoto, & Yamada, 2002). An acidic polysaccharide fraction prepared from a constituent herb of Juzen-taiho-to has been shown to have a protective activity against Candida-infected mice (Inagaki et al., 2001). An adjuvant effect has been noted for apiogalacturonan from duckweed Lemna minor L. (Popov et al., 2006). But all of these pectins were isolated from various medicinal plants, which are not present in the diet. The data indicated that the immune stimulation of dietary pectins or pectic polysaccharides isolated from food plants is rare. Dietary pectin stimulated IgA secretion by lymphocytes in mesenteric lymph nodes (Lim, Lee, Park, & Choue, 2003). Citrus pectin was found to affect cytokine production by human peripheral blood cells (Salman, Bergman, Djaldetti, Orlin, & Bessler, 2008), and abrogated oral tolerance in mice (Khramova et al., 2009). Pectic polysaccharide from the fruit pulp of Spondias cytherea stimulated peritoneal macrophages (Iacomini et al., 2005). The stems and roots of the ground thistle Cirsium esculentum Siev. (distributed in Central Asia) are widely present in the Mongolian diet, and the easy cultivation of C. esculentum Siev.,

D.S. Khramova et al. / Food Chemistry 126 (2011) 870–877

compensates for the lack of fruit and vegetables in the dry climatic zone of steppes. In this paper, we report the isolation and identification of immune modulating pectic polysaccharides from the stems of the ground thistle C. esculentum Siev. named cirsiuman. 2. Materials and methods 2.1. Raw plant material and the isolation of cirsiuman Cirsiuman CE was isolated from fresh stems of ground thistle C. esculentum Siev. and purified as described earlier in details (Ovodova, Vaskovsky, & Ovodov, 1968). 2.2. General analytical methods The glycuronic acid content was determined by the reaction with 3,5-dimethylphenol in the presence of concentrated sulphuric acid. A calibration plot was constructed for D-galacturonic acid, and photocolorimetry was carried out at the following two wavelengths: 400 and 450 nm (Usov, Bilan, & Klochkova, 1995). Protein concentration was calculated using Lowry’s procedure (Lowry, Roserbourgh, Farr, & Randall, 1951) with a standard curve using bovine serum albumin (BSA). The amount of methoxyl groups was determined by a previously described method (Wood & Siddiqui, 1971), and from the calibration plot constructed for methanol, photocolorimetry was carried out at 412 nm. Spectra were measured on an Ultrospec 3000 spectrophotometer (UK). The specific optical rotations were determined on a Palatronic MHZ polarimeter (Germany). The solutions were concentrated in a rotary evaporator under reduced pressure at 40–45 °C, centrifuged at 5000–6000 rpm for 10–20 min, and lyophilised. Samples were lyophilised from the frozen state using the VirTis lyophiliser (USA) with a constant vacuum of <10mTorr at 65 °C. NMR spectral data were recorded on a DRX-500 spectrometer (Bruker, Germany) for 3–5% solutions of polysaccharides in D2O at 303 K (acetone as internal standard; d H 2.225 ppm, d C 31.45 ppm). The two-dimensional spectra were run using the standard Bruker procedures. The molecular weight of the pectic fractions (3 mg/ml) was determined by high-performance liquid chromatography (HPLC). The chromatographic system for analysis included a LC-20AD pump (Shimadzu, Japan), degasser DGU-20A3 (Shimadzu, Japan), a CTO-10AS thermostat (Shimadzu, Japan), a RID-10A refractometer (Shimadzu, Japan) as a detector, and a column of Shodex OH-pak SB-804 HQ (7.6 mm  30 cm) with a precolumn Shodex GS-2G 7B (7.6 mm  5 cm) (Shimadzu, Japan) . HPLC was performed at 40 °C, at a flow rate of 0.3 ml/min. Detection was obtained by refractive index detection (RID 10A, Shimadzu, Japan). The column was conditioned with 0.15 M sodium chloride, containing 0.02% NaN3 as a preservative, and elution was carried out with the same solution. Deionised water supplied by a Simplicity 185 Milllipore water purification system (France) was used to prepare eluents and sample. Pullulans from Fluka, Germany (1.3, 6, 12, 22, 50, 110, 200, 400, 800 kDa) were used as standards. Weight average molecular weight (Mw), number average molecular weight (Mn) and polydispersity factor (Mw/Mn) were calculated by the LCsolutionGPC program (LCsolution, version 1.24 SP1). The samples and standards were injected in duplicate. 2.3. The complete acidic hydrolysis and analysis of monosaccharide composition Two molar trifluoroacetic acid (TFA) (1 ml) containing myo-inositol (0.5 mg/ml) was added to a weighed portion (3–5 mg) of

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polysaccharide fractions. The mixture was incubated for 5 h at 100 °C. Excess acid was removed by repeated evaporation of the hydrolysate to dryness with methanol. The mixture of monosaccharides was transformed to alditol acetates and identified by gas–liquid chromatography (GLC) on a Hewlett–Packard 4890A chromatograph (United States) equipped with a flame ionisation detector and an HP 3395A integrator using a capillary column RTX-1 (30 m  0.25 mm). The carrier gas was helium. GLC of alditol acetates was carried out under temperature programming from 175 °C (1 min) to 250 °C (2 min) at a rate of 3 °C/min. The content of monosaccharides as a percent of the total preparation mass was calculated from the area of peaks using the coefficients of detector response (York, Darvill, McNeil, Stevenson, & Albersheim, 1985). Myo-inositol was used as the internal standard. 2.4. Ion-exchange chromatography of cirsiuman on DEAE-cellulose Cirsiuman CE (70.4 mg) was dissolved in 0.01 M NaCl (3 ml) and was fractionated on a column with DEAE-cellulose OH – form (40 cm  2.5 cm). Fractions were eluted consecutively with 0.01; 0.1; 0.2; 0.3, 0.4 and 0.5 M NaCl at a rate of 48 ml/h. Fractions were collected and analysed by Smith’s procedure (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Fractions corresponding to separate peaks on an elution curve were combined, concentrated, dialysed and lyophilised. Four combined polysaccharide fractions were obtained as follows: CE-1 (eluted with 0.01 M NaCl, yielding 1.6 mg), CE-2 (eluted with 0.1 M NaCl, yielding 1.5 mg), CE-3 (eluted with 0.2 M NaCl, yielding 12.6 mg), and CE-4 (eluted with 0.3 M NaCl, yielding 34.0 mg). Fractions were subjected to a complete acid hydrolysis with 2 M TFA to yield similar monosaccharides. 2.5. The partial acid hydrolysis of cirsiuman Cirsiuman CE (109 mg) was heated with 0.5 M TFA (5 ml) at 100 °C for 3 h. TFA was removed by evaporation and the reaction mixture was poured into ethanol. The precipitate obtained was dissolved in water and the solute was dialysed and lyophilised to produce galacturonan CEH (34 mg). 2.6. The enzymatic digestion of cirsiuman Cirsiuman CE (681.3 mg) was dissolved in water (100 ml), then the aqueous solution (1 ml) of exo- and endo-1,4-a-D-polygalacturonase (15 mg, activity 500U/mg, EC 3.2.1.15.; Fluka, Germany) was added, and the mixture was incubated at 37 °C for 4 h. The digestion was controlled according to Nelson (1944) in order to estimate the reducing sugar quantities. The 1,4-a-D-polygalacturonase was inactivated by boiling at 100 °C and removed by centrifugation. The supernatant was concentrated and precipitated with three volumes of 96% ethanol. The precipitate was separated by centrifugation and washed with methanol until there was an absence of free galacturonic acid. The residual material was dissolved in water and lyophilised to yield the polysaccharide fragment CEF (476.1 mg). 2.7. The saponification and enzymatic hydrolysis of cirsiuman Cirsiuman CE (512 mg) was dissolved in distiled water (75 ml). Two molar sodium hydroxide (25 ml) was added to a solution of cirsiuman, and the mixture was vigorously stirred at 20 °C for 2 h. The excess of alkali was neutralised by acetic acid up to pH 4.5. The resulting precipitate was separated by centrifugation and 1,4-a-D-polygalacturonase (15 mg, activity 500U/mg, EC 3.2.1.15.; Fluka, Germany) was added to the solution. This mixture was incubated at 37 °C under increasing amounts of reducing sugars following the procedure of Nelson and Somogyi (Nelson, 1944).

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Polygalacturonase was deactivated by boiling at 100 °C, and the precipitate was removed by centrifugation. The solution was concentrated and the polysaccharides were precipitated with fourfold volumes of 96% ethanol. The precipitate was separated by centrifugation, dissolved in distiled water and lyophilised to yield the polysaccharide fragment CESF (173.8 mg).

and cytokine concentration, mice were bled by cardio puncture after the systemic anaphylaxis measurement. Blood was centrifuged for 5 min at 400g, and serum was collected and stored frozen at 20 °C until assayed.

2.8. Ion-exchange chromatography of cirsiuman fragment CESF on DEAE-cellulose

Fifteen days after subcutaneous sensitisation, the mice were challenged intragastrically with 50 mg of OVA dissolved in 0.2 ml of PBS. Systemic anaphylaxis-like symptoms appeared within 30 min. The severity of symptoms was evaluated using a scoring system that was previously described (Li et al., 2003).

Cirsiuman CESF (70.0 mg) was dissolved in 0.01 M NaCl (2 ml) and was fractionated on a column with DEAE-cellulose OH – form (40 cm  2.5 cm). Fractions were eluted consecutively with 0.01; 0.1; 0.2; 0.3, 0.4 and 0.5 M NaCl at a rate of 48 ml/h. Fractions were collected in accordance with Smith’s procedure (Dubois et al., 1956). Fractions corresponding to separate peaks on an elution curve were combined, concentrated, dialysed and lyophilised. Two combined polysaccharide fractions were obtained as follows: CESF-1 (eluted with 0.1 M NaCl, yielding 33.6 mg) and CESF-2 (eluted with 0.2 M NaCl, yielding 21.7 mg). 2.9. Induction and assessment of oral tolerance and pectins administration The structure of this study and animal experimental procedures were approved by the ethical committee of the Komi Science Center of the Russian Academy of Sciences on animal care and use. The female A/HeJ mice (20–25 g) were fed a cereal-based diet that consisted of 12.7% protein, 5.6% fat and 54.1% carbohydrate, with a total fibre content of 3.7%. The diet was supplemented with a vitamin–mineral premix according to the recommendation of the American Institute of Nutrition (AIN-93 M diet) (Reeves, Nielsen, & Fahey, 1993). In experiment 1, mice were immunised with 100 mg of ovalbumin (OVA, Appli Chem GmbH, Germany) in 50 ml of complete Freund’s adjuvant (CFA; MP Biomedicals) subcutaneously at the base of the tail. To induce oral tolerance, seven days before subcutaneous immunisation, mice were intragastrically given 20 mg of OVA dissolved in 0.2 ml of phosphate buffered solution (PBS) (tolerant group, n = 21) or PBS only (control group, n = 7) (Kweon, Fujihashi, Wakatsuki, & Koga, 1999). Cirsiuman CE or apple pectin (AP; MP Biomedicals) (1 mg/day) were administered to tolerant mice intragastrically via a polyethylene tube continuously for 14 days: 7 days before and 7 days after the OVA gavage (CE-treated, n = 7 and APtreated, n = 7, groups). Untreated mice (n = 7) received PBS. To assess oral tolerance, delayed type hypersensitivity (DTH) and serum antibody responses were measured in the control, tolerant untreated, tolerant CE-treated and tolerant AP-treated mice. Mice were injected with 25 lg of heat aggregated OVA in 25 ll of PBS in the right footpad and PBS was injected in the left footpad. The volume of the injected paws was measured by a water displacement plethysmograph (Ugo Basil, Comerio, Italy) 24 h after injection, and data were recorded as a difference between the left and right foot pad (Williamson, Bilsborough, & Viney, 2002). 2.10. Oral treatment and sensitisation In experiment 2, mice were sensitised according to a previously established protocol (Hino et al., 2005). Briefly, on the first day of the experiment, mice were primed by subcutaneous injection of 1 mg of OVA in complete CFA. Mice were orally immunised three times with five-day intervals of 1 mg of OVA only (group OVA, n = 8) or OVA mixed with CE (group OVA + CE, n = 8) dissolved in PBS. Control animals received orally only PBS (control group, n = 8). Mice received the first oral ingestion simultaneously with subcutaneous sensitisation. To determine the antibody response

2.11. Oral challenge and assessment of anaphylaxis-like symptoms

2.12. Detection of antibody and cytokine responses Antibody titers to OVA were assessed by ELISA as previously described (Kato, Fujihashi, Kato, Yuki, & McGhee, 2001). Flat-bottom polystyrene plates (Costar, NY) were coated overnight at 4 °C with 5 lg of OVA diluted in 0.1 ml of 0.05 M carbonate buffer (pH 9.6) per well, washed with saline containing 0.05% (v/v) Tween-20, blocked with 1% BSA in PBS, washed again, and then the mouse serum samples were added to the wells at serial dilutions starting with 1:10. After incubation for 2 h at 25 °C, HRP-labelled goat anti-mouse IgG antibodies (1/2000; MP Biomedicals) were added to the wells and incubated for 2 h at 25 °C. The colour reaction was developed by the addition of H2O2 and ortho-phenylene-diamine (Sigma, Germany). The reaction was interrupted after 15 min by the addition of 0.1 ml of 2.5 M HCl and the absorbance (OD) was read at 492 nm (Power Wave 200, BioTek Instruments, USA). Endpoint antibody titers were expressed as the last dilution showing OD 0.1 units above the negative control values at 492 nm. Sandwich ELISA was used to measure total IgE. A monoclonal affinity purified anti-mouse IgE (clone 23G3) (eBiosciences) was used as the capture antibody for IgE, with a biotin-conjugated anti-mouse IgE (clone 23G3) (eBiosciences) as the detection antibody. Indirect ELISA was used to measure serum OVA-specific IgE. To generate a standard curve, the upper two rows of each plate were coated with the purified goat anti-mouse IgE (BD PharMingen, USA). HRP-labelled streptavidin (BD PharMingen, USA) was used for detection. A biotin-conjugated monoclonal antibody specific to IgG1 (clone LO-MG1-2) or IgG2a (clone LO-MG2a-9) (Acris Antibodies GmbH, Germany) was used for the analysis of IgG1 and IgG2a antibody subclasses (Bashir, Andersen, Fuss, Shi, & Nagler-Anderson, 2002). The serum cytokine levels were assayed by sandwich ELISA for IFN-c and IL-4 using a monoclonal affinity purified anti-mouse IL-4 (clone 1D11) (Pierce Biotechnology, Inc., CIA) antibody and antimouse IFN-c (clone XMG1.2) (Pierce Biotechnology, Inc., CIA), according to the manufacture’s protocol. The monoclonal affinity purified anti-mouse IL-4 and the anti-mouse IFN-c antibodies were used as the capture antibodies, and biotin-conjugated IL-4 and IFNc antibodies were used as the detection antibodies, respectively. HRP-labelled streptavidin (BD PharMingen) was used for detection. Samples were run in duplicate and the results were related to standard preparations of IL-4 (Pierce Biotechnology, Inc., CIA) and IFN-c (Cytolab, USA). The sensitivity of the ELISA assays was 60 pg/ml for IL-4 and 15 pg/ml for IFN-c. 2.13. Detection of serum immunoreactive OVA Serum was collected 30 min after OVA challenge, and immunoreactive OVA was measured by indirect competitive ELISA. Ninetysix well microplates (Costar, USA) were coated for 18 h at 4 °C with 100 ll/well of a 25 lg/ml solution of OVA in carbonate buffer (pH 9.6). The plates were washed four times with PBS containing 0.05% (w/v) Tween-20, blocked with 1% BSA in PBS for 60 min. Samples

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containing 250 ll of each test serum or a standard (0–15 lg/ml OVA in saline) were pre-incubated with 250 ll an HRP-labelled anti-OVA rabbit polyclonal antibody (Acris Antibodies GmbH, Germany) diluted in PBS-Tween-20 for 60 min at room temperature. After preincubation, samples were added to the wells (100 ll/well) and incubated for 60 min at 37 °C. After incubation and subsequent washing, the colour reaction was developed with ortho-phenylenediamine (Sigma, Germany). The OD (k = 492 nm) of the samples were compared to those obtained from the OVA standards included for each plate (Fujihashi et al., 2001). The data shown are expressed as mean ± standard deviation. The significance of differences was calculated using the Student’s t-test. P-values <0.05 were regarded as significant. 3. Results and discussion 3.1. Extraction of cirsiuman and analysis of its homogeneity Cirsiuman CE was isolated from the fresh stems of ground thistle C. esculentum Siev. as previously described (Ovodova et al., 1968). The plant material was preliminarily treated with an aqueous formalin to bind pigments and remove contaminants. Protopectin was destroyed by dilute hydrochloric acid added at pH 4.0 during rigorous stirring and heating to 50 °C. Cirsiuman was extracted with 0.7% (w/v) aqueous ammonium oxalate followed by precipitation with 96% ethanol. The precipitate was separated by centrifugation and dissolved in water followed by dialysis and lyophilisation. Crude cirsiuman was obtained with a 10.4% yield from the air-dried plant material. A complete acidic hydrolysis of cirsiuman CE with 2 M aqueous TFA at 100 °C for 5 h led to the following monosaccharides in the hydrolysate: galacturonic acid, rhamnose, arabinose, galactose, xylose, mannose, and glucose. These sugars are the constituents of the sugar chains of the macromolecule. In addition, cirsiuman CE was found to contain protein (Table 1). Cirsiuman CE was subjected to ion-exchange chromatography on a column with DEAE-cellulose to afford two main fractions: CE-3 eluted with 0.2 M NaCl (18% yield) and CE-4 eluted with 0.3 M NaCl (48% yield) (Table 1). 3.2. Partial acidic hydrolysis of cirsiuman The cirsiuman CE obtained was subjected to partial acid hydrolysis (1 M TFA, 100 °C, 5 h) in order to furnish the crude galacturonan CEH; the further purification led to a virtually pure galacturonan, which contained 99% galacturonic acid. Rhamnose

was the single neutral monosaccharide and was detected at trace levels, less than 1% (Table 1). These data reveal that galacturonan appeared to be the backbone of cirsiuman. The high positive spe cific rotation of galacturonan CEH, ½a20 589 þ 241 (c 0.1; aqueous ammonia), suggested an a-configuration of the glycoside linkages in the backbone of galacturonan. Galacturonan CEH was determined to contain 1.4% methoxyl groups. 3.3. Enzymatic hydrolysis of cirsiuman An insignificant cleavage of the carbohydrate chain of cirsiuman CE was observed during treatment of cirsiuman with 1,4-a-D-polygalacturonase, which possesses both endo- and exo- activities. Digestion of CE with enzyme yielded some amounts of free galacturonic acid and oligogalacturonides. Free galacturonic acid was detected by paper chromatography in the supernatant after digestion. The residual material resistant to 1,4-a-D-polygalacturonase action and designated as CEF (yield ca. 70% of the parent cirsiuman CE) was found to contain a considerable amount of methoxyl groups, and ca. 30% of the galacturonic acid residues in CEF are present as methyl esters (Table 1). A substantial content of methoxyl groups appeared to contribute to the stability of CEF during the digestion. The sugar chain of CEF was shown to consist of residues from galacturonic acid, arabinose, galactose, and rhamnose as main constituents and residues from xylose, glucose, mannose as minor constituents (Table 1). 3.4. Saponification and enzymatic hydrolysis of cirsiuman, and NMR spectroscopy of fragment CESF Saponification followed by digestion with 1,4-a-D-polygalacturonase of cirsiuman CE (containing ca. 20 % of methyl-esterified D-galacturonic acid residues) yielded the digested fragment CESF (yield 33.9% of the parent cirsiuman CE). The sugar composition of the polysaccharide fraction of CESF is shown in Table 1. A reduction of the galacturonic acid content (up to 63%) after saponification followed by digestion indicates that the methyl-esterified D-galacturonic acid residues are supposed to be localised in the linear branches of cirsiuman. Fragment CESF was subjected to ion-exchange chromatography (DEAE-cellulose) to produce the following two fractions: CESF-1 eluted with 0.1 M NaCl (yield 48%) and CESF-2 eluted with 0.2 M NaCl (yield 31%) (Table 1). The polysaccharide fractions obtained were analysed using NMR spectroscopy and revealed to have similar structure. The 13C NMR spectra of CESF-1 and CESF-2 are shown in Fig. 1.

Table 1 Chemical characteristics of cirsiuman and its fragments. Pectin

Yield, %

Proteind

OMed

DEe

GalAd

Rhad

Arad

Xyld

Mand

Glcd

Gald

CE CE-1 CE-2 CE-3 CE-4 CEH CEF CESF CESF-1 CESF-2

10.4a 2.3b 2.1b 17.9c 48.3b 31.2b 69.9b 33.9b 48.0c 31.0c

5.6 9.8 0.0 0.0 3.7 0.0 0.0 0.0 0.0 0.0

2.7 n.d. n.d. 1.9 2.1 1.4 3.7 0.2 0.6 0.0

20.2 n.d. n.d. 13.9 13.8 8.6 31.1 2.0 18.1 0.0

81.0 44.0 72.0 83.0 92.0 99.0 72.0 63.0 20.0 45.0

2.7 3.7 2.9 5.1 2.5 0.8 4.5 2.7 10.0 9.8

2.5 15.2 13.5 12.5 2.8 0.0 11.7 17.1 51.5 34.8

9.5 0.5 0.4 0.0 0.7 0.0 0.3 0.0 0.0 0.8

0.1 0.2 0.7 0.0 0.5 0.0 0.2 0.1 0.0 0.2

0.6 0.5 2.5 0.4 1.0 0.0 0.7 1.0 3.4 0.3

2.2 12.7 11.1 4.5 1.6 0.0 3.9 9.3 15.1 7.8

n.d. – not determined. a Yield of the plant raw material. b Yield of the parent cirsiuman CE. c Yield of the CESF fraction. d Data are calculated as weight %. e Data are given in molar %.

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Fig. 1. The 13C NMR spectra of polysaccharide fragments CESF-1 (a) and CESF-2 (b).

As determined by the 13C NMR spectra, the sugar chain contains regions composed of the 1,4-a-linked D-galacturonic acid residues containing the non-methyl-esterified carboxyl groups in cirsiuman fragments (Fig. 1, Table 2). This statement was confirmed by an analytical estimation (Wood & Siddiqui, 1971) of low levels of methoxyl groups in CESF-1 and CESF-2 (0 and 0.6% respectively). In addition, data from the heteronuclear 1H/13CHSQC and homonuclear TOCSY and COSY spectroscopy demonstrated that the residues of L-rhamnopyranose are present in the backbone by 1,2-linkages (Table 2). The combined analysis of the correlation spectra (COSY, TOCSY, HSQC) revealed that the side sugar chains consisted of the a-Larabinofuranose residues substituted in the 5- and 3,5- positions, as well as the terminal residues of a-L-arabinofuranose (Table 2). The signals in the heteronuclear 1H/13C HSQC spectra of the polysaccharide fragments demonstrated the presence of terminal b-D-galactopyranose, b-1,4-linked D-galactopyranose and b-Dgalactopyranose substituted in the 3,4-and 4,6-positions (Table 2). 3.5. The effect of pectin on oral tolerance Oral tolerance, resulting in a nonreactive immune response to ingested proteins, produces a hurdle for mucosal vaccine development, therefore protein antigens require a potent adjuvant for maximisation of immune responses. In our laboratory, we develop pectic polysaccharides as dietary immunomodulators. The present study postulates a possibility to use cirsiuman as a dietary immunopotentiator evoking an immune response on co-administrated protein antigen. The data obtained demonstrate that cirsiuman administered orally with OVA inhibits oral tolerance induction to the same protein. Mice fed OVA and treated with cirsiuman were shown to develop a DTH response with an anti-OVA IgG level

greater than that of untreated tolerant animals (Fig. 2A, B). The DTH response and the levels of serum antigen-specific IgG are commonly used for oral tolerance assessment (Friedman & Weiner, 1994). Cirsiuman administrated with OVA decreased the IgE response; the serum level of IgE was lower when compared to mice fed only OVA (Table 3). Treatment with cirsiuman changed the pattern of IgG subclasses. The induction of oral tolerance was found to decrease the levels of both IgG1 and IgG2a antibodies (Table 4). Cirsiuman was demonstrated to increase the levels of IgG2a threefold and failed to affect the IgG1 response (Table 4). This pattern of response was found in every individual mouse tested. Apple pectin was used as the reference compound and failed to have an affect on oral tolerance. An enhanced DTH reaction in mice in response to OVA as a thymus-dependent antigen, the recovery of anti-OVA IgG2a serum levels induced by cirsiuman, and the downregulation of IgE response together, indicate the stimulatory effect of cirsiuman on Th1 lymphocytes and accessory cell types, which are required for the expression of the DTH reaction. DTH requires the specific recognition of a given antigen by memory Th1 cells, which subsequently proliferate and release cytokines. In turn, cytokines increase vascular permeability, induce vasodilatation and the accumulation of inflammatory cells (neutrophils, macrophages) (Descotes, Choquet-Kastylevsky, van Ganse, & Vial, 2000). The activation of the Th1 immune deviation by pectic polysaccharides has been recently noted. Dietary pectin-derived acidic oligosaccharides enhanced systemic Th1-dependent immune responses in a murine vaccination model (Vos et al., 2007). In contrast, apiogalacturonanic pectin of duckweed Lemna minor L. named lemnan appeared to elicit adjuvant activity via induction of both Th1 and Th2-type responses (Popov et al., 2006). The dose of pectin used was approximately equivalent to the estimated pectin intake in humans (4 g/day). This dose is much more than the effective doses of other

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D.S. Khramova et al. / Food Chemistry 126 (2011) 870–877 Table 2 Chemical shifts of signals in Residue

?4)-a-D-GalpA-(1?4 ?4)-a-D-GalpA-(1?2 ?2)-a-L-Rhap-(1? ?2)-a-L-Rhap-(1? 4) " b-D-Galp-(1? ?4)-b-D-Galp-(1?4 ?4)-b-D-Galp-(1?6 ?4)-b-D-Galp-(1? 6) " ?4)-b-D-Galp-(1?6 3) "

a-L-Araf-(1?5 a-L-Araf-(1?3 ?5)-a-L-Araf-(1?5 ?5)-a-L-Araf-(1?5 3) "

13

C and 1H NMR spectra of cirsiuman fragments CESF-1 and CESF-2. 13

C NMR chemical shifts (dC acetone 31.45) and 1H (italic, dH TSP 0.0)

C-1 H-1

C-2 H-2

C-3 H-3

C-4 H-4

C-5 H-5,5’

C-6 H-6,6’

100.7; 100.8 5.09, 5.07 99.0 5.02

70.2 4.02 69.3 3.87

70.0 4.14 71.3 4.12

79.8 4.44 79.0 4.42

72.6 4.85 72.3 4.80

175.5

100.2 5.24 100.2 5.24

77.9 4.11 78.1 4.12

70.1 3.88 70.0 4.09

73.4 3.42 81.8 3.74

69.6 3.75 69.2 3.81

18.0 1.24 18.2 1.31

105.9 4.64 104.9 4.62 104.9 4.48 104.9 4.62

73.3 3.67 73.2 3.52 72.3 3.53 73.2 3.52

74.1 3.66 74.2 3.66 73.8 3.76 74.2 3.66

70.2 3.92 79.2 4.16 79.2 4.16 78.0 4.14

76.7 3.68 76.1 3.71 76.1 3.71 75.1 3.92

62.6 3.82, 62.6 3.82, 62.6 3.82, 70.9 4.04,

104.7 4.53

71.3 3.65

77.2 3.74

76.6 4.17

74.9 3.94

70.9 4.04, 3.94

109.2 5.08 108.5 5.15 108.5 5.15 108.9 5.11

82.3 4.13 82.7 4.13 82.7 4.13 80.8 4.28

78.3 4.00 78.3 4.00 78.1 4.03 83.9 4.09

85.4; 85.7 4.12; 4.07 85.5 4.03 83.9 4.21 82.9 4.30

62.7 3.83; 62.7 3.83; 68.4 3.88; 68.2 3.94;

175.5

3.82 3.82 3.82 3.94

3.72 3.72 3.79 3.82

Table 3 Serum IgE responses induced by subcutaneous immunisation with OVA/CFA of mice fed OVA and treated with CE and APa. Group

Total IgE, lg/ml

Control Untreated tolerant CE-treated tolerant AP-treated tolerant

930 ± 159 590 ± 48* 480 ± 32  630 ± 150

AP, apple pectin; CFA, complete Freund’s adjuvant; CE, cirsiuman; IgE, immunoglobulin E; OVA, ovalbumin. a Data are presented as mean ± SD (n = 7). * P < 0.05 versus control.   P < 0.05 versus untreated tolerant.

Table 4 Serum anti-OVA IgG subclass responses induced by subcutaneous immunisation with OVA/CFA of mice fed OVA and treated with CE and APa.

Fig. 2. The effect of cirsiuman (CE) or apple pectin (AP) on the induction of oral tolerance. The delayed type hypersensitivity reaction (A) and the anti-ovalbumin immunoglobulin G production in tolerant untreated (white columns), tolerant CEtreated (grey columns), tolerant AP-treated (striped columns) and control (black columns) mice. Data are present as mean ± SD, n = 7, ⁄P < 0.05 versus controls;   P < 0.05 versus tolerant.

Group

IgG1, reciprocal log2

IgG2a, reciprocal log2

Control Untreated tolerant CE-treated tolerant AP-treated tolerant

13.0 ± 2.1 7.3 ± 2.0* 9.4 ± 1.8 5.6 ± 2.4

10.8 ± 2.0 4.4 ± 1.5* 13.1 ± 0.8  5.0 ± 2.4

AP, apple pectin; CFA, complete Freund’s adjuvant; CE, cirsiuman; IgG, immunoglobulin G; OVA, ovalbumin. a Data are presented as mean ± SD (n = 7). * P < 0.05 versus control.   P < 0.05 versus untreated tolerant.

known compounds influencing oral tolerance. Cholera enterotoxin abrogated oral tolerance at a single oral dose of 10 lg (Bagley, Abdelwahab, Tuskan, & Lewis, 2003). The oral tolerance abrogation

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by citrus pectin has been recently revealed in our laboratory (Khramova et al., 2009), but citrus pectin administered orally with OVA dramatically enhanced IgE production, therefore it provides proallergic effect and can not be advised as a dietary immunopotentiator.

Table 5 Serum anti-OVA IgE and cytokine responses induced by oral immunisation with OVA and CEa.

3.6. An inhibitory effect of cirsiuman on anaphylaxis

CE, cirsiuman; IFN-c, interferon-c; IgE, immunoglobulin E; IL-4, interleukin 4; OVA, ovalbumin. a Data are presented as mean ± SD (n = 8). * P < 0.05 versus OVA fed mice.

Wijk et al. (2005) showed that the food matrix may affect the immune responses to individual peanut allergens. The present study indicated that cirsiuman supplemented together with OVA and activated the Th1 immunity appeared to inhibit an allergenspecific immune response to the protein. The anaphylactic reaction was evaluated and the severity of anaphylactic symptoms were scored 30 min following an intragastrically OVA challenge. One hundred percent of control sensitised mice exhibited severe anaphylactic symptoms, and OVA feeding failed to alter anaphylactic symptoms development; however, when OVA was administrated with cirsiuman, the symptom scores were twofold lower when compared to OVA treated mice (Fig. 3). The anaphylaxis inhibition by cirsiuman was accompanied by a decrease in the antigenspecific IgE response: the serum level of anti-OVA-IgE was lower when compared to mice fed only OVA (Table 5). The data indicate that oral administration of cirsiuman leads to a decrease in IL-4 concentration with a simultaneous increase in IFN-c levels (Table 5). The production of IgE antibodies is dependent upon the help from IL-4 (Hsieh, Heimberger, Gold, O’Garra, & Murphy, 1992). The IFN-c produced by Th1 lymphocytes inhibits IgE production and switches the deviation of IgG subclasses from IgG2a prevalence to IgG1 (Comoy, Capron, & Thyphronitis, 1997). Evidently the IFN-c levels induced by cirsiuman and the downregulation of IL-4 response led to the suppression of IgE production, and therefore inhibited the allergic reaction at the sensitising stage. In addition, IFN-c is known to abrogate mast cell degranulation (Nieuwenhuizen, Herbert, Lopata, & Brombacher, 2007), and to date, cirsiuman is suggested to possess an anti-allergic effect at an effector stage. Activation of IFN-c production is known to depend on the stimulation of IL-12 secretion by antigen-presenting cells (APCs) (Trinchieri & Gerosa, 1996). Several studies have revealed that the anti-allergic effect of different substances is caused by IL-12 dependent inhibition of IgE synthesis via the stimulation of IFN-c production. Continuous oral treatment of mice with hot water extract of Chloella vulgas prior to sensitisation decreased the Th2-dependent allergy due to the enhanced IFN-c and IL-12 production (Hasegawa et al., 1999). The Lactobacillus casei strain Shirota suppressed serum IgE and IgG1 responses and systemic

Immunisation

Anti-OVA IgE, ng/ml

IL-4, ng/ml

IFN-c, ng/ml

OVA OVA + CE

50 ± 15 28 ± 9*

73 ± 18 45 ± 15*

690 ± 140 1142 ± 180*

allergic reaction in a food allergy model, and the switching of Th2 responses on the Th1 type reaction was mediated by IL-12 induction (Shida et al., 2002). The decreasing of IgE-response is confirmed by Lee et al. (2004) showed that the administration of Asian pear pectin-sol in presensitised mice suppressed IgE production and an allergic asthma symptoms severity. Our previous results indicate that oral administration of OVA and lemnan, apiogalacturonanic pectin of duckweed Lemna minor L., failed to elevate levels of either total and antigen-specific IgE responses (Popov et al., 2006). 3.7. The effect of cirsiuman on antigenic OVA concentration in the blood The first property of cirsiuman which we considered might be important for its immunomodulatory activity was an effect on uptake of OVA from the intestine. To test this idea, the level of antigenic OVA was measured in the serum 30 min after challenge. Consistent with previous reports (Fujihashi et al., 2001) we detected some amount of antigenic OVA in the serum by labelling with anti-OVA antibodies 30 min after feeding (548 ± 168 ng/ml, n = 8). Serum OVA levels were the same in mice fed with OVA mixed with cirsiuman (500 ± 105 ng/ml) as compared to OVA fed mice (548 ± 168 ng/ml, n = 8, P > 0.05). The inability to alter OVA penetration via the intestinal wall 30 min after feeding has also been observed recently for the citrus pectin (Khramova et al., 2009); at the same time both citrus pectin (Khramova et al., 2009) and apiogalacturonan from the duckweed Lemna minor L. (Popov et al., 2006a) enhanced OVA penetration in the blood through 3 and 1 h after feeding, respectively. Food components were tested to evaluate protein penetration from the intestine. When the major soybean allergen, Gly m Bd 30 K, was co-administered with dietary fats, absorption was enhanced via fat carrier– mediated transport (Weangsripanaval, Moriyama, Kageura, Ogawa, & Kawada, 2005). Palmitoyl coupling OVA was demonstrated to enhance protein absorption to the blood (Oliveira et al., 2007). 4. Conclusions

Fig. 3. The inhibition of anaphylaxis by oral cirsiuman (CE) treatment. Sensitised (subcutaneous OVA in CFA) mice were orally administered with PBS (control), 1 mg OVA (OVA), or 1 mg OVA plus 1 mg CE (OVA + CE). n = 8, ⁄P < 0.05 versus OVA fed mice.

In conclusion, cirsiuman CE, a pectic polysaccharide isolated from the stems of C. esculentum Siev., was found to have a branched structural pattern bearing a backbone containing a linear 1,4-a-Dgalacturonan. Linear 1,2-a-L-rhamno-1,4-a-D-galacturonan appeared to be present in the core of the ramified regions. The side chains attached to the 4-position of the L-rhamnopyranose residues of rhamnogalacturonan appeared to contain 1,4-b-Dgalactopyranan, which consists of 3,4- and 4,6-substituted Dgalactopyranose residues as branched points. In addition, ramified regions contain side chains with branched 1,5-a-arabinofuranan bearing 3,5-substituted a-L-arabinofuranose residues as branching points. Cirsiuman administered orally was found to abrogate oral tolerance and evoke a predominant Th1 immune response to coadministered protein. Therefore, it appears to be a useful immunopotentiator or adjuvant for oral vaccines. The Th1 response

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