Structure of pectic polysaccharides isolated from onion Allium cepa L. using a simulated gastric medium and their effect on intestinal absorption

Food Chemistry 134 (2012) 1813–1822

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Structure of pectic polysaccharides isolated from onion Allium cepa L. using a simulated gastric medium and their effect on intestinal absorption Victoria V. Golovchenko a,⇑, Daria S. Khramova a, Raisa G. Ovodova a, Alexandre S. Shashkov b, Yury S. Ovodov a a b

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

a r t i c l e

i n f o

Article history: Received 7 December 2011 Received in revised form 12 March 2012 Accepted 21 March 2012 Available online 30 March 2012 Keywords: Onion Allium cepa L. Pectin structural features Gastric medium NMR spectroscopy of polysaccharides Gastrointestinal protein absorption Serum ovalbumin level

a b s t r a c t The polysaccharide fraction extracted with simulated gastric juice from onion bulbs contained a mixture of galactan with short-length sugar chains, pectic polysaccharides and evident content of proteinaceous material. Galacturonan and rhamnogalacturonan were the main constituents of the linear regions of the sugar chains of the pectic polysaccharides. The ramified regions included rhamnogalacturonan-I. NMR data revealed that the side chains of the ramified region contained mainly 1,4-linked b-D-galactopyranose residues and lesser content of 1,3-linked b-D-galactopyranose and 1,5-linked a-L-arabinofuranose residues. Furthermore, the proteinaceous material was determined to be partly linked to the sugar chains. The polysaccharide fraction was found to decrease absorption of ovalbumin (OVA) to the blood from the gut lumen. The serum OVA level was threefold lower in mice fed with OVA mixed with the onion pectins compared with the control group, which was administered OVA alone. Protein removal failed to abolish the inhibitory effect of the onion polysaccharides, confirming that the polysaccharide chains are the active component of onion gastric juice extract. Ó 2012 Published by Elsevier Ltd.

1. Introduction Food allergies are an increasingly prevalent problem in westernised countries (Nowak-Wegrzyn & Sampson, 2011). Food allergens are mostly proteins that invoke IgE-mediated immune reactions (Aalberse, 1997). Allergies to eggs are especially common in children, affecting an estimated 1.3% of children and 0.2% of adults in the United States (Shek, Soderstrom, Ahlstedt, Beyer, & Sampson, 2004). Ovalbumin (OVA), the major protein in egg white (58% w/w), is considered to be a dominant allergen (Mine & Yang, 2007). The epithelium of the gastrointestinal tract is an important area of contact between the organism and its external environment (Fujita, Baba, Shimamoto, Sakuma, & Fujimoto, 2007) and prevents food antigen absorption to the blood. The impairment of the mucosal barrier function increases the penetration of immunoreactive proteins into circulation and consequently amplifies the immune response to food (Tlaskalova-Hogenova et al., 2002). Therefore, elucidating the nutrient factors that are present in the diet and that affect protein absorption through the mucosal barrier is of great interest. Food components were shown to improve protein absorption from the intestine. For example, the absorption of the major ⇑ Corresponding author. Tel./fax: +7 8212 241001. E-mail address: [email protected] (V.V. Golovchenko). 0308-8146/$ - see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodchem.2012.03.087

soybean allergen, Gly m Bd 30 K, was enhanced by co-administering it with corn oil (Weangsripanaval, Moriyama, Kageura, Ogawa, & Kawada, 2005). Palmitoyl coupling of OVA was used to improve protein transport to the blood from the gut lumen (Oliveira et al., 2007). Food-grade surfactants (sucrose monoester fatty acids) were found to increase the paracellular uptake of ovomucoid through the Caco-2 cell monolayers (Mine & Zhang, 2003). The inhibition of protein absorption by food components is rare. However, the peptide NPWDQ (Asn-Pro-Trp-Asp-Gln, aa 107-111 of alpha(2)-casein), which is isolated from enzyme-modified cheese, inhibited both the paracellular and transcellular transport of OVA through the Caco-2 cell monolayers (Isobe, Suzuki, Oda, & Tanabe, 2008). Food based pectic polysaccharides were noted to modulate immune activity. Dietary pectin stimulated IgA- and decreased IgE 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). Apiuman, pectin from the stalks of the celery Apium graveolens var. Dulce, and pectin from the common cranberry were shown to possess anti-inflammatory activity after oral administration in mice (Ovodova et al., 2009; Popov, Markov, et al., 2006). Pectic polysaccharide from the fruit pulp of Spondias cytherea stimulated peritoneal macrophages (Iacomini

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et al., 2005). A complement fixing activity was revealed for pectin from cabbage (Samuelsen et al., 2007; Westereng, Yousif, Michaelsen, Knutsen, & Samuelsen, 2006). Pectins with different origin in vivo possessed anti-allergic activity. Asian pear pectinsol administration in presensitised mice suppressed IgE production and allergic asthmatic reaction (Lee et al., 2004). Pectic polysaccharide fraction from the Japanese soy sauce Shoyu orally administered had a significant suppressive effect on passive cutaneous anaphylaxis induced in the ears of mice and inhibited the release of histamine from rat basophilic leukaemia (RBL-2H3) cells (Kobayashi et al., 2004). The score of systemic anaphylactic reaction and the levels of both serum IgE and IL-4 were decreased by cirsiuman, a pectin from the stems of Cirsium esculentum Siev. (Khramova et al., 2011). In the human diet, pectins occur as a part of dietary fibre in fruit and vegetables, as gelling, stabilising and emulsifier agents in jams and jellies, and more recently in low-calorie foods (Thakur, Slugh, & Handa, 1997). Enhancement of OVA absorption by pectins was recently observed in our laboratory. Citrus pectin (Khramova et al., 2009) and apiogalacturonan from the duckweed Lemna minor L. (Popov, Golovchenko, et al., 2006) increased OVA penetration to the blood after feeding. The pectins were isolated by treating plant material with extraction methods, which differ significantly from the conditions of the gastro-intestinal medium. The effects of pectins on food allergen absorption through the mucosal barrier are assumed to depend on the isolation of the pectins from plant material during digestion. Onion bulbs are widely used in human nutrition across the world. The structure of the cell wall polysaccharides from onion bulbs has been studied in detail (Ishii, 1982; Mankarios, Hall, Jarvis, Threlfall, & Friend, 1980; Redgwell & Selvendran, 1986). The pectic polysaccharides, xyloglucan and a small amount of hemicellulose–pectic complexes were isolated from onion bulbs using classical methods of extraction (Ha, Evans, Jarvis, Apperley, & Kenwright, 1996; Mankarios et al., 1980; Redgwell & Selvendran, 1986). The pectin content of the onion bulbs was determined to be ca. 0.5 g/100 g fresh weight (O‘Donoghue et al., 2004). The high content of galactose residues has been reported to be a structural feature of onion pectin (Ha et al., 1996; O‘Donoghue et al., 2004; Redgwell & Selvendran, 1986). The objective of the present investigation was to isolate and characterise pectin from onion bulbs by extracting with simulated gastric juice and to elucidate its effect on OVA absorption to the blood from the gut lumen.

2. Materials and methods 2.1. Isolation of polysaccharides from Allium cepa L. onion bulbs The plant material (3 kg) was chopped into small pieces and then homogenised in 1 L of distilled water using a domestic blender for 5 min at medium speed. The obtained mixture was added to a solution of simulated gastric juice (19 L, pH 1.5). The artificial gastric juice was used to mimic the conditions of the stomach as previously described (Corcoran, Stanton, Fitzgerald, & Ross, 2007) with slight modifications. Simulated gastric juice was formulated from HCl (1.34 g/L), NaCl (2.16 g/L), KH2PO4 (0.63 g/L), CaCl2 (0.12 g/L), KCl (0.39 g/L), and pepsin (0.53 g/L). Extraction was performed at 37 °C, 4 h. The extract was filtered and centrifuged with a flow centrifuge at 10,000 rpm (Avanti J-25I; Beckman Coulter, Fullerton, CA) for 1 h at 4 °C. The supernatant was collected and passed through an ultrafiltration cell with membrane pores of 300 kDa (Vladisart, Vladimir, Russia). The ultrafiltration lasted until the removal of all chemicals used during isolation. The residual

materials with molecular mass above 300 kDa were collected and lyophilised to yield polysaccharide fraction AC (9.7 g). The plant material (0.67 kg) was chopped into small pieces and homogenised in 1 L of distilled water using a domestic blender for 5 min at medium speed, mixture volume was increased up to 6 L by distilled water. Polysaccharides were extracted at 70 °C for 4 h. Water extract was separated from plant material by filtration. Residual plant material was treated with aqueous hydrochloric acid (pH 4.0, 3 h at 50 °C) in order to protopectin destroying. The onion pectic polysaccharides were extracted with 0.7% aqueous ammonium oxalate (6 L) for 5 h at 70 °C. Both extracts were centrifuged at 10,000 rpm for 20 min, concentrated in vacuo at 40 °C and precipitated by three volumes of 96% ethanol. The crude polysaccharides were separated by centrifugation, dissolved in distilled water, dialysed for 2 days against distilled water at 10 °C and lyophilised. Thus, polysaccharide fractions AC-W (0.077 g) and AC-O (1.541 g) from onion were obtained by water and ammonium oxalate extraction, respectively. 2.2. General analytical methods The monosaccharide composition was determined after hydrolysis of the polysaccharides with 2 M aqueous trifluoroacetic acid (TFA), as previously described (Khramova et al., 2011). The glycuronic acid content was determined by reaction with 3,5-dimethylphenol in the presence of concentrated sulphuric acid. A calibration plot was constructed for D-galacturonic acid (DGalUA), and photocolorimetry was carried out at 400 and 450 nm (Usov, Bilan, & Klochkova, 1995). The proteinaceous material concentration was calculated using Lowry’s procedure (Lowry, Roserbourgh, Farr, & Randall, 1951) with a standard curve using bovine serum albumin (BSA). Spectra were measured on an Ultrospec 3000 spectrophotometer (Pharmacia Biotech, Amersham, UK). The specific optical rotations were determined with a Palatronic MHZ polarimeter (Schmidt + Haensch, Berlin, Germany). The solutions were concentrated with a Laborota 4002 rotary evaporator (Heidolph, Schwabach, Germany) under reduced pressure at 40–45 °C. The samples were centrifuged with a 6 K 15 centrifuge (Sigma, Osterode am Harz, Germany) at 5000–11,000g for 10–20 min and then lyophilised from the frozen state using a VirTis lyophiliser (SP Industries, Warminster, PA) with a constant vacuum of <10 mTorr at 65 °C. The samples were periodically removed and weighed to assure a constant mass after 6 h, and they were dried further if the sample mass changed by more than 5% during the last 2 h of lyophilisation. NMR spectra were recorded using a Bruker DPX-500 spectrometer (Bruker BioSpin, Rheinstetten, Germany) for 3–5% solutions of polysaccharides in D2O at 30 °C (the internal standard – acetone, dy 2.225 ppm, dC 31.45 ppm). The number of scans was 20,000. Two-dimensional spectra were run using standard Bruker procedures. The molecular weight of the polysaccharide samples (3 mg/mL) was determined by high-performance liquid chromatography (HPLC). The chromatographic system for the analysis included an LC-20AD pump (Shimadzu, Kyoto, Japan), a DGU-20A3 degasser (Shimadzu,), a CTO-10AS thermostat (Shimadzu), an RID-10A refractometer (Shimadzu) as a detector, a Shodex OH-pak SB-804 HQ column (7.6 mm  30 cm) and a Shodex GS-2G 7B precolumn (7.6 mm  5 cm) (Shimadzu). The HPLC experiments were performed at 40 °C with a flow rate of 0.3 mL/min. 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 the Simplicity 185 Milllipore water purification system (France) was used to prepare the eluents and samples. Pullulans (Fluka, Buchs, Germany) (1.3, 6, 12, 22, 50,

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110, 200, 400 and 800 kDa) were used as standards. The weightaverage molecular weight (Mw), the number average molecular weight (Mn) and the polydispersity index (Mw/Mn) were calculated by the LCsolution GPC program (LCsolution, Version 1.24 SP1). The samples and standards were injected in duplicate. 2.3. Separation of polysaccharide fraction AC by anion-exchange chromatography

tion with three volumes of 96% ethanol, at 5 °C for 2 h, and centrifugation. The obtained pellets were washed twice with a cold mixture of ethanol and diethyl ether (1:1), solubilised in distilled water and dialysed extensively against water using dialysis tubing with a cellulose membrane (Sigma Aldrich, Mw 1.2–2000 Da). The residual material was centrifuged and lyophilised to yield polysaccharide fraction ACP (148.5 mg). 2.6. OVA and pectin administration

Polysaccharide fraction AC (224.4 mg) dissolved in 0.01 M NaCl (5 mL) was fractionated with a DEAE-cellulose (OH form) column (2.5  40 cm). The column was eluted with a stepwise gradient of different concentrations of NaCl solutions (0.01, 0.1, 0.2, 0.3 and 1.0 M NaCl) at a flow rate of 0.8 mL/min. The obtained fractions were combined based on the total sugar content quantified by the phenol–sulphuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The relevant fractions were collected, concentrated using a rotary evaporator at 40 °C, dialysed against distilled water and lyophilised. The obtained polysaccharide fractions were as follows: AC-1 (eluted with 0.01 M NaCl, yield 63.2 mg), AC-2 (eluted with 0.1 M NaCl, yield 29.1 mg), AC-3 (eluted with 0.2 M NaCl, yield 80.8 mg), AC-4 (eluted with 0.3 M NaCl, yield 3.9 mg) and AC-5 (eluted with 1.0 M NaCl, yield 5.3 mg). The sugar compositions of the polysaccharide fractions obtained are in Table 1.

This study and the animal experimental procedures were approved by the Ethical Committee of the Komi Science Centre 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, which consisted of 12.7% protein, 5.6% fat and 54.1% carbohydrates, 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) (Reese et al., 2005). The mice were fasted for a night before administration of OVA (AppliChem) and pectin but had free access to drinking water. The mice were intragastrically given 20 mg of free OVA or 20 mg of OVA mixed with 1 mg of the polysaccharide fractions (AC, AC-1, AC-3 or ACP) or citrus pectin (MP Biomedicals, Solon, OH) used as the reference compound dissolved in 0.2 mL of phosphate buffered solution (PBS).

2.4. Deproteinisation of polysaccharide fraction AC by Sevag’s method

2.7. Detection of serum immunoreactive OVA

Polysaccharide fraction AC was deproteinised three times by Sevag’s method (Sevag, Lackman, & Smolens, 1938). Fraction AC (100 mg) was dissolved in water (100 mL). Chloroform (25 mL) and n-butanol (10 mL) were added to the solution. The mixture was shaken for 30 min and then centrifuged, resulting in separation into two layers. The lower layer contained a stable chloroform-protein gel. The upper layer was separated from the lower one by decantation. This procedure was repeated twice. The solution was dialysed against distilled water, centrifuged, concentrated and lyophilised to yield polysaccharide fraction ACS (75.4 mg).

Serum was collected 1, 2 and 3 h after the challenge of OVA mixed with 1 mg of the polysaccharide fractions AC and 3 h after feeding with the polysaccharide fractions AC-1, AC-3 or ACP or citrus pectin, and immunoreactive OVA was measured by an indirect competitive enzyme-linked immunosorbent assay (ELISA). Ninetysix well CostarÒ microplates (Corning, New York, NY) 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 and then blocked with 1% BSA in PBS for 60 min. Samples containing 250 lL of each test serum or a standard (0–15 lg/mL OVA in saline) were pre-incubated with 250 lL of an HRP-labelled anti-OVA rabbit polyclonal antibody (Acris Antibodies GmbH, Herford, Germany) diluted in PBS-Tween-20 for 60 min at room temperature. After pre-incubation, 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 o-phenylenediamine (Sigma– Aldrich, Germany). The optical density (OD) (k = 492 nm) of the samples was compared to that obtained from OVA standards included in each plate (Fujihashi et al., 2001). Results were expressed as mean ± standard deviation. Statistical significance was determined using the two-tailed non-parametric

2.5. Deproteinisation of polysaccharide fraction AC by trichloroacetic acid Treatment with trichloroacetic acid (TCA) was used to deproteinate polysaccharide fraction AC. Polysaccharide fraction AC (200 mg) was dissolved in distilled water (60 mL). The obtained aqueous solution was subjected to acid treatment by addition of 100% (w/v) TCA to a final concentration of 15%. The solution was incubated in a cold box at 20 °C for 15 min and then centrifuged at 11,000g for 20 min at 5 °C. The supernatant was concentrated (to 30 mL), and then the polysaccharides were separated by precipita-

Table 1 Chemical characteristics of the polysaccharide fraction from onion.

AC AC-1 AC-2 AC-3 ACP ACS AC-W AC-O a b c

Yield (%)

Proteinc

GalAc

Rhac

Arac

Xylc

Glcc

Galc

Mw (kDa)

Mn (kDa)

Mw/Mn

0.31a 28.2b 13.0b 36.0 b 74.3b 75.4 b 0.01a 0.23a

23.0 6.6 5.6 6.6 1.1 8.8 25.0 9.1

42.0 29.0 40.0 71.0 45.0 49.7 27.2 68.4

2.1 3.5 4.0 1.7 2.7 1.6 0.7 0.6

2.8 2.9 5.3 1.8 3.8 3.6 8.6 1.8

0.6 1.1 0.6 0.8 1.7 0.6 1.5 0.3

0.7 0.6 5.6 0.4 0.5 0.1 2.4 1.0

28.3 53.3 41.0 17.2 41.9 35.8 33.8 18.2

559 718 317 113 443 331 777 495

41 60 59 26 38 22 544 142

13.6 12.0 5.4 4.3 11.7 15.0 1.4 3.5

Yield of the plant raw material. Yield of the parent polysaccharide fraction AC. Data were calculated as weight %.

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Mann–Whitney test. P-values were calculated using Statistica 6.0 (StatSoft Inc., Tulsa, OK). P-values of 0.05 or less were considered significant.

3. Results and discussion 3.1. Isolation of polysaccharides from Allium cepa L. onion with simulated gastric juice and analysis of homogeneity Pectic polysaccharides, xyloglucan and small amounts of hemicellulose–pectic complexes have been extracted from cell walls by oxalate–citrate buffer, cyclohexane-trans-1,2-diamine tetraacetate, polygalacturonase and dilute alkali. In addition, pectic polysaccharides, xyloglucan (Ishii, 1982; Mankarios et al., 1980; Redgwell & Selvendran, 1986). The monosaccharide composition and structure of onion pectins depend on extraction procedure (Ishii, 1982; Mankarios et al., 1980; Redgwell & Selvendran, 1986) and batches of onions (O‘Donoghue et al., 2004); the authors showed prevalence of GalUA and galactose residues in pectic macromolecules. Ishii (1982) demonstrated high content of arabinan in ramified regions of pectins. 1,4-linked galactans were determined to be the major component of side chains of polysaccharides extracted from cell walls by oxalate–citrate buffer at pH 4 (Mankarios et al., 1980). In the present work, we reveal the composition and structural features of pectic polysaccharides, which were extracted from onion bulbs under conditions closely mimicking the normal fasting human stomach. The plant material was treated with simulated gastric juice (pH 1.5) at 37 °C for 4 h. The pH level suitable for simulation of the fasting gastric condition is revealed to be between pH 1.5 and pH 2 (Dressman, Amidon, Reppas, & Shah, 1998); therefore, we extracted onion pectins with simulated gastric juice at pH 1.5. The extraction time of 4 h was selected to cover residence time for gastric exposure as most solids are emptied within 4 h (Kong & Singh, 2011). Polysaccharides were fractionated with a 300 kDa membrane, and the fraction with a molecular mass over 300 kDa was collected and lyophilised. The lyophilisate was obtained with a 0.31% yield from the raw plant material. Complete acid hydrolysis of fraction AC performed by treatment with 2 M aqueous TFA allowed for the detection of the following monosaccharides in the hydrolysate, which were identified by gas chromatography after being transformed to alditol acetates: rhamnose, arabinose, galactose, xylose and glucose. The total uronic acid content was determined colorimetrically. Consistent with previous reports (Ishii, 1982; Mankarios et al., 1980; Redgwell & Selvendran, 1986), the monosaccharides GalUA and galactose were identified as the main constituents of the AC sugar chains (Table 1). In addition, fraction AC was found to contain a considerable quantity of proteinaceous material (Table 1). The separation of fraction AC by anion-exchange chromatography using a DEAE-cellulose column yielded pectic polysaccharides differing in the content of the main components: galactose and GalUA (Table 1). Fraction AC was subjected to anion-exchange chromatography with a DEAE-cellulose column to afford three main polysaccharides: AC-1 eluted with 0.01 M NaCl (28.2% yield), AC-2 eluted with 0.1 M NaCl (13.0% yield) and AC-3 eluted with 0.2 M NaCl (36.0% yield) (Table 1). The fraction eluted with 1.0 M NaCl did not contain any carbohydrate components. Analysis of the sugar composition of the polysaccharide fractions revealed GalUA and galactose as the main monosaccharides. Small contents of rhamnose, arabinose, xylose and glucose were also detected in these fractions. The contents of galactose and GalUA residues differed in the fractions. Polysaccharide fraction AC-1 (½a20 D + 137.6° (c 0.1; H2O)) was characterised by the minimum content of GalUA residues (29%) and the maximum content of D-galactose (53.3%), a high polydispersity in-

dex (12) and high Mw (718 kDa), as indicated by HPLC. Fraction AC-3 (½a20 D + 171.5° (c 0.1; H2O)) contained the pectic polysaccharides and the maximum content of D-GalUA (71%). The Mw of AC-3 was approximately 113 kDa, and the polydispersity index was 4. All fractions obtained by anion-exchange chromatography contained proteinaceous material (up to 6.6%). In the control extraction water-soluble and cell wall onion polysaccharides were isolated by water and 0.7% aqueous ammonium oxalate, yielding fraction AC-W (13.0% yield) and fraction AC-O (13.0% yield), respectively. Monosaccharide composition of the fraction AC-W and AC-O was similar to that of the fraction AC-1 and AC-3, respectively (Table 1). These results indicated the coextraction of water soluble and cell wall polysaccharides from onion bulbs in gastric condition. In contrast to the fraction AC-W, the fraction AC-1 is characterised by the decreasing content of arabinose and increasing content of galactose. Consistent with previous reports (Ishii, 1982) the ramified regions of onion pectins extracted under neutral conditions included significant content of arabinose residues. The decrease of arabinose content in the polysaccharides extracted by simulated gastric medium compared to the polysaccharides extracted by water could be due to hydrolysis of acidic labile arabinofuranose residues as demonstrated previously (Voragen, Pilnik, Thibault, Axelos, & Renard, 1995; Westereng, Michaelsen, Samuelsen, & Knutsen, 2008). In addition, the fractions AC-1 and AC-3 were characterised by lower Mw, and higher polydispersity index than the fractions AC-W and AC-O (Table 1). This result confirmed destruction of sugar chains by simulated gastric medium during the extraction procedure. 3.2. Deproteinisation of polysaccharide fraction AC Fraction AC was extracted from onion with simulated gastric conditions and contained a considerable content of proteinaceous material (up to 23%). Several methods, including anionexchange chromatography with a DEAE-cellulose column, Sevag’s method and TCA treatment, were used for proteinaceous material removal. Fraction ACS was generated by treatment with Sevag’s method and was determined to include 8.8% proteinaceous material. All fractions obtained by anion-exchange chromatography (AC-1, AC-2 and AC-3) contained approximately 7% proteinaceous material. Treatment of fraction AC by 15% (w/v) TCA led to a reduction in the proteinaceous material content (to 1%). The molecular mass of the polysaccharide fractions generated with Sevag’s method and TCA treatment was lower than that of fraction AC, as indicated by HPLC (Table 1); this appeared to be caused by proteinaceous material removal. TCA treatment led to a significant reduction (up to 1.1%) of proteinaceous material content. However, in contract to the fraction ACP, the fraction ACS was characterised by higher content of proteinaceous material and lower Mw (Table 1), which indicated partial co-precipitation of carbohydrates with proteinaceous material during Sevag’s treatment. The failure of proteinaceous material removal from the polysaccharides by Sevag’s method and anion-exchange chromatography indicated the presence of linkage between the proteins and sugar chains in the fraction AC. Proteins and pectic polysaccharides were co-extracted from various sources using different extraction conditions, due to the different matrix polysaccharide and protein content in plant cell walls (Carpita & Gibeaut, 1993). Polysaccharides and proteins extracted from the cell walls, were observed directly with the electron microscope after rotary shadow casting (Stafstrom & Staehelin, 1986). Data of atomic force microscopy demonstrated the extraction of protein–pectin complexes consisting of pectin molecules attached to the pectin chain with protein (Kirby, MacDougal, & Morris, 2008). According to Strahm, Amado, and Neukom (1981), a-4-hydroxyprolinegalac-

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toside linkage was established to connect the arabinogalactan side chains to the peptide of water-extractable arabinogalactan (Strahm et al., 1981). 3.3. Interpretation of NMR data The resonances in the 1H and 13C NMR spectra of polysaccharides fractions AC, AC-1 and AC-3 were assigned by means of two-dimensional COSY, TOCSY, ROESY, HMBC and HSQC spectra. The chemical shifts are given in Tables 2–4. The anomeric region of 13C NMR (Fig. 1a) and 1H/13C HSQC (Fig. 2b) spectra of the polysaccharide fraction AC identified the signals of terminal (d 104.9 and 105.5 ppm) and 1,4-linked b-D-galactopyranose (d 105.6 ppm), 1,5-linked a-L-arabinofuranose (d 108.8 ppm), 1,4-linked a-D-galactopyranosyluronic acid (d 100.6 ppm) residues and reducing terminal residues of 4-substituted b-D-galactopyranose (1,4-b-D-GalOH) (d 97.5 ppm). In addition, the signals of low intensity at d 100.0 and 93.4 ppm belonged to the anomeric carbons of 1,2-linked and 2,4-substituted a-L-rhamnose residues and reducing terminal residues of 4-substituted a-D-galactopyranose (1,4-a-D-GalOH), respectively (Fig. 2b). The occurrence of 1,2-linked and 2,4-substituted a-L-rhamnose residues in fraction AC was confirmed by C/H cross-peaks in HSQC spectrum at d 17.8/1.25 ppm and d 18.0/1.31 ppm belonging to the methyl group of 1,2-linked and 2,4-substituted a-L-rhamnose residues, respectively (Fig. 2a). HMBC spectrum revealed an

Table 2 Chemical shifts of the signals in the 1H and

interaction between the methyl proton (d 1.25 ppm) and C-4 (d 73.1 ppm) and C-5 (d 70.2 ppm) of 1,2-linked a-L-rhamnose residues. The other signals of H/C correlation failed to be determined. COSY spectra identified cross-peaks between methyl proton (d 1.25 ppm) and H-5 (d 3.74 ppm) of 1,2-linked a-L-rhamnose residues and between methyl proton (d 1.31 ppm) and H-5 (d 3.82 ppm) of 2,4-substituted a-L-rhamnose residues. In TOCSY spectrum the chemical shifts of non-anomeric protons of 1,2linked a-L-rhamnose residues (H-5 – d 3.74; H-3 – d 3.88 ppm) were observed to correlate with methyl proton (d 1.25 ppm). In addition, positions of H-4 (d 3.69 ppm), H-5 (d 3.82 ppm) and H-2 (d 4.10 ppm) of 2,4-substituted a-L-rhamnose residues were determined in TOCSY spectrum. The signal of low intensity at d 175.2 ppm (Fig. 1a) downfield were attributed to the C-6 of the nonmethyl-esterified 1,4-linked a-D-galactopyranosyluronic acid residues (Catoire, Goldberg, Pierron, Morvan, & Herve du Penhoat, 1998). The C-6 of the methyl-esterified GalUA residues resonated at d 172.4 ppm (Odonmazˇig, Badga, Ebringerová, & Alföldi, 1992), in addition to the carbon of methoxyl group resonating at d 54.1 ppm. The data of heteronuclear 1H/13C HSQC (Fig. 2c) and homonuclear TOCSY, COSY and ROESY spectroscopy of fraction AC demonstrated 1,4-linked a-D-GalUA residues attached to the 2-position of a-rhamnopyranose residues. Thus, rhamnopyranose residues were involved in the backbone of onion pectic macromolecule by 1,2-linkages as has been shown for sugar beet pectins (Renard, Lahaye, Mutter, Voragen, & Thibault, 1998). Consistent

13

C NMR spectra of the polysaccharide fraction AC. 13

Residue

C NMR chemical shifts (dC acetone 31.45) and 1H (italic, dH 2.225)

C-1 H-1

C-2 H-2

C-3 H-3

C-4 H-4

C-5 H-5; H-50

C-6 H-6; H-60

?4)-a-D-GalpA-(1 ? (GA)

100.6 5.11; 5.06

69.5 3.77

69.9 4.02

79.4 4.44

73.2 4.82

175.2

?4)-a-D-GalpA-(1 ? 2)-a-L-Rhap-(1 ? (GA0 )

100.0 5.24

69.5 3.77

71.5 4.12

78.8 4.38

72.2 4.85

175.2

?5)-a-L-Araf-(1 ? (Ara)

108.8 5.09

82.0 4.13

78.0 4.01

83.7 4.21

68.2 3.80; 3.89

?4)-b-D-Galp-(1 ? (G)

105.6 4.64

73.1 3.68

74.5

78.9 4.18

75.8 3.72

62.0 3.70; 3.90

?4)-b-D-GalpOH(G0 )

97.5 4.61

72.5 3.59

74.5 3.77

78.9 4.18

75.8 3.72

62.0 3.70; 3.90

b-D-Galp-(1 ? 4)-b-D-Galp(Gt)

105.5 4.60

73.5 3.57

74.0 3.65

69.9 3.91

76.4 3.68

62.0 3.70; 3.90

b-D-Galp-(1 ? 6)-b-D-Galp(Gt0 )

104.9 4.45

71.9 3.53

74.0 3.65

69.9 3.95

76.4 3.68

62.0 3.70; 3.90

Table 3 Chemical shifts of the signals in the 1H and Residue

13

C NMR spectra of the polysaccharide fraction AC 1. Chemical shifts

13

C (dC acetone 31.45) and 1H (italic, dH 2.225)

C1 H1

C2 H2

C3 H3

C4 H4

C5 H5; H50

C6 H6; H60

?5)-a-L-Araf-(1 ? (Ara)

108.9 5.09

82.1 4.13

78.0 4.01

83.7 4.21

68.2 3.81; 3.89

?4)-b-D-Galp-(1 ? (G)

105.9 4.64

73.1 3.68

74.6 3.78

78.9 4.18

75.8 3.72

62.0 3.70; 3.90

?4)-b-D-GalpOH(G0 )

97.8 4.60

72.5 3.60

74.6 3.77

78.9 4.18

75.8 3.72

62.0 3.70; 3.90

b-D-Galp-(1 ? 4)-b-D-Galp(Gt)

105.8 4.60

73.5 3.57

74.0 3.66

70.0 3.91

76.4 3.68

62.0 3.70; 3.90

b-D-Galp-(1 ? 6)-b-D-Galp(Gt0 )

104.9 4.45

72.0 3.53

74.0 3.66

70.0 3.95

76.4 3.68

62.0 3.70; 3.90

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Table 4 Chemical shifts of the signals in the 1H and Residue

a

13

C NMR spectra of the polysaccharide fraction AC 3. Chemical shifts

13

C (dC acetone 31.45) and 1H (italic, dH 2.225)

C-1 H-1

C-2 H-2

C-3 H-3

C-4 H-4

C-5 H-5; H50

C-6 H-6; H60

?4)-a-D-GalpA-(1 ? (GA)

100.5 5.09; 5.05

69.7 3.75

70.3 3.98

79.5 4.42

72.6 4.78

176.1

?2)-a-L-Rhap-(1 ? (Rha)

100.0 5.25

77.8 4.11

70.0 3.85

73.4 3.40

71.2 3.76

18.0a 1.24

b-D-Galp-(1 ? 4)-b-D-Galp(Gt)

105.8 4.63

73.4 3.62

74.2 3.66

70.3 3.89

76.6 3.68

62.2 3.7; 3.81

b-D-Galp-(1 ? 6)-b-D-Galp(Gt0 )

105.0 4.44

72.2 3.54

74.2 3.66

70.3 3.89

76.6 3.68

62.2 3.79; 3.81

?4)-b-D-Galp-(1 ? 4(G)

105.6 4.63

73.3 3.68

74.6 3.77

78.9 4.17

75.7 3.72

62.0 3.7; 3.81

?3)-b-D-Galp-(1 ? (G00 )

104.3 4.51

72.6 3.77

81.5 3.73

69.8 4.14

76.6 3.68

62.0 3.80; 3.85

a-L-Araf-(1 ? (Ara)

110.7 5.24

82.8 4.21

78.1 3.96

85.4 4.13

62.8 3.82; 3.71

?5)-a-L-Araf-(1 ? (Arat)

108.7 5.09

82.1 4.13

78.0 4.00

83.6 4.21

68.2 3.8; 3.80

Corresponding chemical shifts of the 2,4-substituted Rhap at dC 18.2 and dH 1.30.

Fig. 1. The

13

C NMR spectra of the polysaccharide fraction AC (a), AC-1 (b) and AC-3 (c).

with previous reports (Cardoso, Ferreira, Mafra, Silva, & Coimbra, 2007; Polle, Ovodova, Chizhov, Shashkov, & Ovodov, 2002; Wang, Duan, & Fang, 2004) the assignments of signals in 13C (Fig. 1a) and cross-peaks in 1H/13C HSQC (Fig. 2b and c) spectra of fraction AC indicated the occurrence of arabinofuranose residues. The downfield position of the resonance of C-5 (d 68.2 ppm) indicated a substitution of arabinofuranose residues in 5-position. The other

signals of 1,5-linked arabinofuranose residues were determined from heteronuclear 1H/13C HSQC (Fig. 2c) and homonuclear TOCSY, COSY spectra (Table 2). The 13C (Fig. 1a) and 1H/13C HSQC (Fig. 2b and c) spectral data of fraction AC revealed the sugar chains contained regions of 1,4-linked b-D-galactopyranose residues. The assignments of 1,4-linked b-D-galactopyranose residues in the current study were confirmed by comparing them with the litera-

V.V. Golovchenko et al. / Food Chemistry 134 (2012) 1813–1822

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Fig. 2. The 1H/13C HSQC spectrum of the polysaccharide fraction AC.

Fig. 3. The 1H/13C HSQC spectrum of the polysaccharide fraction AC-1.

ture value (Keenan, Belton, Matthew, & Howson, 1985; Pressy & Himmelsbach, 1984). 1H and 13C NMR (Fig. 1a) spectra and two-dimensional COSY, TOCSY, and HSQC spectra (Fig. 2b and c) of fraction AC indicated the occurrence of two different states of terminal b-D-galactopyranose residues (Gt and Gt0 in Table 2) attached to 4- and 6-positions of b-D-galactopyranose residues.

These residues were differed by assignments of C-1, C-2 and H-4 (Fig. 2b and c and Table 2). 1H/13C HSQC spectra (Fig. 2b and c) indicated the presence of intensive signals of 1,4-b-D-GalOH and low signals of 1,4-a-D-GalOH. Downfield position of the crosspeaks of C-4/H-4 (d 78.9/4.18 ppm and d 79.5/4.23 ppm) in the 1 H/13C HSQC spectrum (Fig. 2b and c) indicated the substitution

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Fig. 4. The 1H/13C HSQC spectrum of the polysaccharide fraction AC-3.

at the 4-position of galactopyranose residues. The other signals of 1,4-b-D-GalOH were detected from heteronuclear 1H/13C HSQC (Fig. 2c) and homonuclear TOCSY and COSY spectra (Table 2). H/C correlation of 4-substituted a-D-galactopyranose was detected only for C-1/H-1 at d 93.4/5.26 ppm and C-4/H-4 at d 79.5/ 4.22 ppm (Fig. 2b and c). COSY spectrum revealed an interaction between H-1 (d 5.26 ppm) and H-2 (d 3.90 ppm). In the TOCSY spectrum several cross-peaks indicated the correlation of H-1 (d 5.26 ppm) with protons at d 3.89, 3.95 and 4.23 ppm of 1,4-a-DGalOH. The presence of reducing terminal units of 4-substituted galactopyranose indicated the presence of short-length sugar chains of galactan in fraction AC. 13 C (Fig. 1b) and HSQC (Fig. 3) spectra of fraction AC-1 were similar to those of fraction AC, but differed in the intensity of the signals of 1,4-b-D-GalOH, 1,4-a-D-GalOH and 1,5- linked a-L-arabinofuranose residues. The spectra contained intensive resonances of terminal and 1,4-b-linked D-galactopyranose residues. In contrast to fraction AC, NMR spectra of fraction AC-1 revealed the absence of signals of 1,4-a-D-galacturonan (Figs. 1b and 3b and c). The assignments of proton and carbon chemical shifts obtained from homonuclear COSY, TOCSY and heteronuclear HMQC, HMBC spectra are shown in Table 3. 13 C NMR (Fig. 1c) and HSQC spectra (Fig. 4) of fraction AC-3 revealed the intensive signals belonged to 1,4-linked a-D-GalUA and 1,4-linked b-galactopyranose residues. In addition, the sugar chains of fraction AC-3 contained regions of 1,3-linked b-D-galactopyranose residues (G00 ) confirmed by downfield position of the resonance of C-3 (d 81.5 ppm), which were not determined in fraction AC and AC-1. The other signals of 1,3-linked b-D-galactopyranose residues were determined from heteronuclear 1H/13C HSQC (Fig. 4b and c) and homonuclear TOCSY, COSY spectra and their assignments are shown in Table 4. NMR spectra of fraction AC-3 included signals of a-L-arabinofuranose residues with two different states (Fig. 4b and c). Two cross-peaks belonging to terminal and 1,5-linked a-L-arabinofuranose residues were detected in anomeric region of HSQC spectrum at 108.7/5.09 ppm and 110.7/5.24 ppm (Fig. 4b). Intensity of signals of 1,5-linked a-L-arabinofuranose residues was lower than that of signals of terminal a-L-arabinofura-

nose residues. The cross-peak of low intensity at d 100.0/ 5.25 ppm belonging to the anomeric carbons of 1,2-linked and 2,4-substituted a-L-rhamnose residues was detected in the 1H/13C HSQC spectrum (Fig. 4b). The occurrence of 1,2-linked and 2,4substituted a-L-rhamnose residues in the fraction AC-3 was confirmed by cross-peaks at d 17.8/1.25 ppm and d 18.0/1.31 ppm belonging to methyl group of 1,2-linked and 2,4-substituted a-Lrhamnose residues, respectively (Fig. 4a). The cross-peaks on the dotted line in TOCSY spectrum of fraction AC-3 are the correlations between the methyl proton (d 1.24 ppm) and the other protons H-4 (d 3.40 ppm), H-5 (d 3.76 ppm), H-3 (d 3.85 ppm) and H-2 (d 4.11 ppm). The correlation of protons and carbons of 1,2-linked a1 13 L-rhamnose residues was assigned in heteronuclear H/ C HSQC spectrum (Fig. 4c). The chemical shifts of signals in NMR spectra of polysaccharide fraction AC-3 are given in Table 4. All fractions AC, AC-1, AC-3 included signals of 1,2-linked and 2,4-substituted a-L-rhamnose residues (Figs. 2–4), that allowed us to suggest the presence of rhamnogalacturonan-I in ramified regions with rhamnopyranose residues involved in the backbone pectic macromolecule of onion by 1,2-linkages partly substituted at the 4-position of side chains, as has been previously shown for the majority of pectins (Ridley, O’Neill, & Mohnen, 2001; Yapo, 2011). The side chains of onion pectin extracted with simulated gastric juice included single neutral glycosyl residues and polymeric sugar chains of 1,4-linked b-D-galactopyranose (mainly) and 1,3-linked b-D-galactopyranose, 1,5-linked a-L-arabinofuranose (scarcely) residues. This study demonstrated the fraction AC, which was extracted under conditions closely mimicking the normal fasting human stomach, was found to contain a mixture of galactan with shortlength sugar chains, pectic polysaccharides and significant content of proteinaceous material partially linked with sugar chains. Galacturonan and rhamnogalacturonan were confirmed to be the main constituents of the linear regions of the sugar chains in pectic polysaccharides. The ramified regions included rhamnogalacturonan-I with side chains containing mainly 1,4-linked b-D-galactopyranose residues and lesser content of 1,3-linked b-D-galactopyranose and 1,5-linked a-L-arabinofuranose residues (Tables 2–4, Figs. 1–4). In

V.V. Golovchenko et al. / Food Chemistry 134 (2012) 1813–1822

addition, occurrence of galactan with short-length sugar chains in the onion polysaccharide fraction suggested the presence of galactan in onion bulbs and/or partial acidic hydrolysis of the side chains of onion pectin during the extraction procedure. 3.4. Effect of the polysaccharides from onion on OVA absorption to the blood Consistent with previous reports (Fujihashi et al., 2001), we detected some immunogenic OVA in the serum by labelling with anti-OVA antibodies 1, 2 and 3 h after feeding (3.3 ± 0.6 lg/mL; 1.2 ± 0.2 lg/mL; 1.3 ± 0.1 lg/mL, respectively, n = 24). The serum OVA level was threefold lower in mice fed with OVA mixed with the AC pectin (0.4 ± 0.01 lg/mL; 0.4 ± 0.05 lg/mL; 0.5 ± 0.1 lg/ mL, respectively, n = 24, p < 0.05) compared with the control group, which was administered OVA alone. Pectic polysaccharides from onion revealed a similar level of activity to citrus pectin used as the reference compound. The serum OVA level was threefold higher 3 h after administration with OVA mixed with citrus pectin used as the reference compound, as compared with the control group (3.4 ± 0.7 lg/mL for citrus pectin vs. 1.3 ± 0.1 lg/mL in controls, n = 24, p < 0.05). A number of studies have determined that pectins decrease glucose absorption due to their viscosity and interaction with other molecules (Fuse, Bamba, & Hosoda, 1989; Kim, 2005). We previously reported the ability of citrus pectin (Khramova et al., 2009) and apiogalacturonan from the duckweed Lemna minor L. (Popov, Golovchenko, et al., 2006) to increase the absorption of OVA 3 h and 1 h after feeding, respectively. By contrast, a pectic polysaccharide, which was isolated from the stems of C. esculentum Siev. (Khramova et al., 2011), did not affect the absorption of OVA. Thus, the structural features of the pectic polysaccharides are likely to determine their effects. The inhibitory effect was exhibited by the fractions AC, AC-1 and ACP, which contained a mixture of galactan with short-length sugar chains and pectic polysaccharides and differed in galactose, D-GalUA and protein content. The OVA absorption was similar to the controls for the fraction AC-3 (Fig. 5), which contained the pectic polysaccharides and the maximum content of D-GalUA. The higher activity level observed for the fraction ACP, which was characterised by the minimum content of proteinaceous material, indicates that proteinaceous material removal failed to abolish the inhibitory effect of the onion polysaccharides, confirming that the polysaccharide chains are the active component of onion gastric juice extract. It cannot be excluded that in addition to the side chain architecture, the Mw of polymers

1821

could have an influence on activity. The complement-fixing activity of pectic polysaccharides from cabbage was noted to be determined by Mw of the polysaccharide fractions (Westereng et al., 2008). HPLC revealed that the Mw of fraction AC-3 was lower than Mw of active AC, AC-1 and ACP fractions. In addition, HPLC results indicated that citrus pectin used as control sample had a high Mw (407 kDa, data not shown). These results allow us to speculate the necessity of the high Mw of polysaccharides for affecting OVA absorption. However, we did not identify the specific size of the polysaccharide macromolecule required for the activity in the present work. The low Mw galactan structures were not isolated and the activity for those fragments was not detected. The failure to enhance the complement-fixing activity of pectic polysaccharides from cabbage with low Mw was noted in a recent study (Westereng et al., 2008), although the different content of galactose in the polysaccharide fraction from cabbage should not be overlooked. The complement-fixing activity was found to correlate to large neutral side chains with high content of galactose and low content of D-GalUA of pectins from cabbage (kale and red kale) but not to molecular weight distribution of the polymers (Samuelsen et al., 2007). Further studies are essential in order to determine the molecular mechanism(s) underlying the inhibition of the intestinal absorption of OVA by the pectic polysaccharides and the structure–activity relations of onion pectins. 4. Conclusion This work demonstrated that a mixture of galactan with shortlength sugar chains, pectic polysaccharides and evident content of proteinaceous material was extracted with simulated gastric juice from onion bulbs. Galacturonan and rhamnogalacturonan were the main constituents of the linear regions of the sugar chains of the pectic polysaccharides. The ramified region contained mainly 1,4linked b-D-galactopyranose residues and a lesser content of 1,3linked b-D-galactopyranose and 1,5-linked a-L-arabinofuranose residues. The polysaccharides from onion bulbs were found to decrease absorption of protein to the blood from the gut lumen. The pathophysiology of food allergies is accompanied with an increased transport of intact food proteins across the gut epithelial barrier; therefore, the inhibition of immunogenic protein absorption suggests a potential health benefit of onions for preventing food allergies. On the other hand the inhibition of the protein antigen uptake by onion polysaccharides extracted during digestion might be interesting in oral immunisation strategies, due to its hypothetical ability in reducing the effectiveness of the oral adjuvants, which transport the antigen via the intestinal barrier. Acknowledgement The work was supported by Russian Fund for Basic Research (Grants 09-04-90202, 11-04-12110-ophi-m-2011). References

Fig. 5. The effect of the AC fractions on OVA absorption to the blood. The data are presented as the mean ± SD, n = 8, ⁄p < 0.05 vs. OVA.

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