Polysaccharides from the Styrian oil-pumpkin with antioxidant and complement-fixing activity

Industrial Crops and Products 41 (2013) 127–133

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Polysaccharides from the Styrian oil-pumpkin with antioxidant and complement-fixing activity Zuzana Koˇst’álová a,∗ , Zdenka Hromádková a , Anna Ebringerová a , Martin Polovka b , Terje Einar Michaelsen c , Berit Smestad Paulsen c a

Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovak Republic Department of Chemistry and Food Analysis, Food Research Institute, Priemyselná 4, P.O. Box 25, SK-824 75 Bratislava, Slovak Republic c Department of Pharmacognosy, School of Pharmacy, University of Oslo, Oslo, Norway b

a r t i c l e

i n f o

Article history: Received 2 March 2012 Received in revised form 11 April 2012 Accepted 14 April 2012 Keywords: Oil-pumpkin Pectin Structure Antioxidant Immunomodulatory activity

a b s t r a c t Pectic polysaccharides have attracted great attention due to their health-promoting potential. From the Styrian oil-pumpkin biomass representing an alternative pectin source, two series of acidic polysaccharide fractions were isolated using in succession hot water, EDTA and dilute HCl. Chemical and spectroscopic (FTIR, NMR) analyses of the fractions revealed the predominance of partially methylesterified and acetylated pectins containing homogalacturonan and ramified rhamnogalacturonan elements, and a minority of phenolic compounds, protein and hemicelluloses. The pectins exhibited moderate antioxidant activities tested by colorimetric DPPH and FRAP assays and EPR method. The activities correlated with the total phenolic content. In the complement-fixing test, most of the pectin fractions exhibited potent effects comparable to the positive control – a pectin from Plantago major. The oil-pumpkin pectins represent potential antioxidant and immunoenhancing additives applicable in food and nutraceuticals. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Pectic polysaccharides from fruits and vegetables have attracted great attention of both food producers and consumers due to their physical properties, their health-promoting and diseasepreventing potential and, as such, became employed also in cosmetic and pharmaceutical products (Willats et al., 2006). Pectin in all higher plants represents a structurally diverse group of acidic polymers, which are composed of several structural elements known as homogalacturonan, substituted galacturonans (xylogalacturonan and apiogalacturonan) and rhamnogalacturonans (RG I and RG II) with attached neutral carbohydrate side chains. The pectic polysaccharides are strongly bound in the cell walls of parenchymtous tissues, the most abundant in fruits and vegetables (Voragen et al., 2009). During the last decade, crop-derived products as renewable raw materials have potential benefits recognized also in case of pectins. Accordingly, the number of papers dealing with the isolation of pectin and antioxidant phytochemicals from the by-products formed during processing of fruits and vegetables steadily increased.

∗ Corresponding author. Tel.: +421 2 59410284; fax: +421 2 59410222. E-mail address: [email protected] (Z. Koˇst’álová). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.04.029

Since ancient time, many varieties of pumpkins (genus Cucurbita; family Cucurbitaceae) have been cultivated throughout the world for use as vegetable, fruit and folk medicine. The flesh and peel of the fruit represent rich sources of pectin-type dietary fiber and antioxidants (Caili et al., 2006). The many beneficial physiological effects, immunological activity and other pharmacological activities of various pumpkin extracts have been published (Caili et al., 2007; Yadav et al., 2010; Adams et al., 2011), and antioxidant activity was reported by Nara et al. (2009) for a water-soluble polysaccharide from the fruit of pumpkin (Cucurbita maxima Duchesne). The oil-pumpkin (Cucurbita pepo L.) and its hull-less seed mutant – Styrian oil-pumpkin (C. pepo L. var. styriaca) became economically important plants for oil production in Austria and adjacent countries. After removal of the seeds, the fruit residues of these pumpkin species are by-products which have been mainly left in the field and less used for feeding. This biomass, comprising about 90% of the fresh fruit, represents a potential source of pectin and became the object of our research. The proximate composition of both oil-pumpkin de-seeded fruit biomasses was determined (Koˇst’álová et al., 2009) and the extractability of their non-cellulosic polysaccharides by conventional and ultrasoundassisted extraction procedures reported (Koˇst’álová et al., 2010). In a further paper some of the isolated pectic polysaccharides were shown to exhibit significant antitussive effects (Nosál’ová et al., 2011).

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The purpose of presented study was to isolate pectic polysaccharides from the seeded fruit biomass of the Styrian oil-pumpkin by sequential extraction permitting some separation of this complex of polysaccharides and to characterize the obtained polysaccharide fractions by chemical composition and main structural features. The bioactive properties of the pectic polysaccharides were evaluated for antioxidant and immunomodulating activities using various in vitro tests. 2. Materials and methods 2.1. Raw material and chemicals The Styrian oil-pumpkin (C. pepo L. var. styriaca) was grown ˇ at fields belonging to the scholar enterprise (Kolínany, Slovakia) and harvested in September 2005. After removal of the seeds, the biomass was grated yielding an aqueous suspension. One part of the suspension was dewatered by solvent exchange using ethanol (biomass EOP) and the second part was after filtration dried on air (biomass DOP). More details were described in a previous paper (Koˇst’álová et al., 2009). Finally, both samples were ground into small particles (0.6–0.8 mm) and used as starting materials. Gallic acid, d-galacturonic acid and 2,4,6-tri(2-pyridyl)s-triazine (TPTZ) were obtained from Fluka (Germany). The Folin–Ciocalteu’s phenol reagent was from Merck GmbH (Darmstadt, Germany). The stable free radical 1,1-diphenyl-2-picryl-hydrazyl 6-hydroxy-2,5,7,8(• DPPH), tetramethylchroman-2-carboxylic acid (Trolox) and bovin serum albumin (≥96%) were purchased from Sigma–Aldrich (Germany). Coomassie Brilliant Blue G-250 was from Serva, 2,2 -azinobis(3-ethylbenthiazoline-6-sulfonic acid) salt (ABTS•+ ) from Polysciences, Inc. (Warrington, PA) and ethylenediaminetetraacetic acid (EDTA) from Lachema Brno (Czech Republic). All other used chemicals were of analytical grade. 2.2. Sequential extraction The biomasses EOP and DOP were exhaustively extracted with ethanol–chloroform (1:4, v/v) to remove extractives and then subjected to a sequential extraction procedure more detailed described earlier (Nosál’ová et al., 2011). As extracting agents were used: water (60 ◦ C for 2 h) in step 1, 0.05 M EDTA in 0.33 M KH2 PO4 (pH 4.8, 25 ◦ C for 2 h) in step 2, and dilute HCl (pH 2.5, 60 ◦ C for 30 min) in steps 3 and 4. The solubilized material from each step was separated by filtration and the filtrate exhaustively dialyzed (Serva, MWCO 3,5 kDa). Lyophilization of the retentates yielded polysaccharide fractions EP1–EP4 from EOP and DP1–DP4 from DOP. 2.3. Ion exchange chromatography The EDTA-extracted fraction (EP2) and acid-extracted fraction (DP4) were fractionated on DEAE Sephadex A25 (Cl− form) into five subfractions by step-wise elution with water and (0.1–1 M) sodium chloride solutions as previously described (Nosál’ová et al., 2011; Koˇst’álová et al., 2011). In experiments, the major subfractions (EP2/C, DP4/C) were eluted with 0.3 M NaCl, dialyzed (Serva, MWCO 1.0 kDa) and freeze-dried. 2.4. General methods The neutral sugar composition of the isolated polysaccharides was determined after hydrolysis with 2 M trifluoroacetic acid (reflux, 2 h) by gas chromatography (Hewlett-Packard instrument, Model HP 5890) in the form of the alditol trifluoracetates (Hromádková and Ebringerová, 1995). The uronic acid content (UA) was determined by the 3-hydroxydiphenyl assay (Blumenkrantz

and Asboe-Hansen, 1973) using galacturonic acid as standard. The identification of uronic acids was performed by paper chromatography (p.c.) as described in a previous paper (Koˇst’álová et al., 2009). Quantitative determination of total phenolics (TP) was performed by the Folin–Ciocalteu’s assay (Yu et al., 2002) using gallic acid as standard. UV-spectra were recorded on a Shimadzu UV-1800 device in the 340–280 nm region using gallic acid as standard. The content of protein was determined by a modified Bradford method (Sedmak and Grossberg, 1977) with bovine serum albumin as standard. All yields were calculated on moisture-free basis and represent mean values of at least two extractions. FTIR spectra (in KBr pellets) were measured using the NicoletMagna 750 spectrophotometer operating at 4 cm−1 resolution. 1 H and 13 C NMR spectra were recorded at 40 or 60 ◦ C on a Varian 400 MR spectrometer operating at 400 MHz. The chemical shifts were referenced to the 1 H/13 C signals of the methoxyl group of methylesterified pectin (3.81/53.7 ppm). The samples were measured in D2 O (99.99 atom%) in a 5-mm inverse probe. The spectra were adjusted using the software MestReNova 7.0. 2.5. Statistics Measurements of the content of phenolic components and uronic acids were performed (at least) in triplicate. The values were averaged and reported along with the standard deviation (mean ± SD). 2.6. Antioxidant activity assays 2.6.1. DPPH radical-scavenging activity The capability of the polysaccharide fractions to scavenge the free stable radical (• DPPH) was measured by the DPPH method (Rao and Muralikrishna, 2006). Briefly, 1 mL of the sample (2 mg/mL H2 O, pH 7) was added to 1 mL of the freshly prepared • DPPH solution in methanol (0.08 mg/mL). After the incubation period (1 h) at room temperature in dark, the absorbance at  = 517 nm was read against a blank (sample solution/methanol, 1:1) in the Spectronic 20 Genesis spectrophotometer. The decline in the radical concentration indicated the radical scavenging activity of the sample. Gallic acid (0.7 mg/mL) was used as a reference corresponding to 100% activity. The radical scavenging activity (RSA) was expressed as the percentage disappearance of • DPPH in relation to that of gallic acid (100%) according to the equation: RSA (%) =

A0 − Atest × 100 A0 − Aref

where A0 is the initial absorbance of • DPPH solution in water, Atest is the absorbance of the tested sample in the • DPPH solution, and Aref is the absorbance of gallic acid in the • DPPH solution, all measured after 1 h. The analyses were performed in duplicate mode. 2.6.2. Ferric reducing antioxidant power (FRAP) The reduction of ferric tripyridyltriazine complex (Fe3+ -TPTZ) to ferrous tripyridyltriazine (Fe2+ -TPTZ) by a reductant at low pH was measured according to a slight modification of the FRAP method (Rao and Muralikrishna, 2006). Briefly, 0.2 mL of the sample (6 mg/mL) was mixed with 1.8 mL of the freshly prepared FRAP reagent consisting of 25 volumes of 300 mmol/L acetate buffer (pH 3.6), 2.5 volumes of 20 mmol/L FeCl3 and 2.5 volumes of 10 mmol/L TPTZ in 40 mmol/L HCl warmed up to 37 ◦ C. After the incubation period (up to 4 min) at room temperature, the absorbance at  = 595 nm was read against water. Ferrous sulphate solutions (100–1000 ␮M) were used for calibration. The FRAP value was calculated by constructing a linear regression and expressed as ␮mol Fe2+ /g sample. All experiments were performed in duplicate mode.

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2.6.3. EPR spectroscopy The antioxidant activity of the individual polysaccharide samples dissolved in water was followed by means of portable X-band EPR spectrometer e-scan (Bruker BioSpin, GmbH, Karlsruhe, Germany) with accessory. The decay of concentrations of the oxidants (semi-stable free radicals ABTS•+ and • DPPH) after the addition of the respective polysaccharide sample was monitored. The ABTS•+ water solution was prepared according to Re et al. (1999) and its concentration was determined via the measurement of UV absorbance at 735 nm using the value of molar extinction coefficient, 735 nm = 14.8 L/(mol cm). The initial concentration of • DPPH was determined from the UV absorbance at 515 nm using the value of molar extinction coefficient, 515 nm = 11.6 L/(mol cm). Briefly, 1 mL of the aqueous sample solution was placed into one syringe and 1 mL of the solution of • DPPH in ethanol (initial concentration of • DPPH in system, c0(• DPPH) = 0.1 mmol/L) or of the solution of ABTS•+ in water (initial concentration of ABTS•+ in the system c0(ABTS•+ ) = 72 ␮mol/L) was placed in the second one. Both syringes were attached to a small mixing chamber connected directly to the quartz EPR flat cell (internal volume, 300 ␮L) fixed in the proper position in the cavity of the EPR spectrometer. After simultaneous injection of both, oxidant and test sample solutions, the respective radical concentration was monitored in time domain for 12 min. A set of 15 EPR spectra was recorded; whereby, each spectrum represents an average of 30 individual scans. The experiments were performed in triplicate mode and the relative standard deviation among the individual measurements was less than 5%. The response and settings of EPR spectrometer was checked by means of Strong pitch standards (Bruker) daily before starting the experiments. Evaluation of the recorded spectra was performed as previously described by Polovka et al. (2010), and the results were expressed as Trolox Equivalent Antioxidant Capacity (TEAC). 2.7. Complement-fixing assay The complement-fixing assay was performed as previously described (Michaelsen et al., 2000) using PMII, a bioactive pectin fraction from the medicinal herb Plantago major L. (Samuelsen et al., 1996) as positive control. The method is based on the inhibition of hemolysis of antibody-sensitized sheep erythrocytes by human sera. A dose–response curve was constructed to calculate the concentration of the tested sample able to give 50% inhibition of lysis (ICH50 ). Low ICH50 means high complement fixing activity. Samples were run in quadruplicates. 3. Results and discussion 3.1. Polysaccharide composition and structural features Because the pumpkin biomasses dried by both methods (EOP and DOP) contained about 7% extractives (lipids, carotenoids and other phenolic compounds), these were removed by refluxing with a mixture of chloroform and ethanol (Koˇst’álová et al., 2009). The extractive-free biomasses were subjected to four sequential extraction steps using as solvents hot water, aqueous solution of EDTA and twice dilute HCl (Fig. 1). Chemical analyses data of the polysaccharides fractions isolated from EOP (EP1–EP4) and DOP (DP1–DP4) were summarized in Table 1. The content of uronic acids in the fractions of the E-series and D-series ranging at 41–52% and 40–63%, respectively, and the prevalence of galacturonic acid (identified by p.c.) indicated the pectic polysaccharides to be major components. Comparing the hot-water extracted fractions EP1 and DP1 from both biomasses, differences were observed mainly in the proportions of neutral sugars (rhamnose, arabinose, and galactose) known to form neutral branches ramifying rhamnogalacturonan

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Fig. 1. Sequential extraction scheme of DOP and EOP.

(RG I) domains (Voragen et al., 2009). These fractions showed also the highest content of phenolic compounds and protein. The presence of xylose, glucose and mannose indicated contamination with hemicelluloses. All pumpkin polysaccharides showed considerable molecular polydispersity measured by HPSEC. The apparent molar masses ranged between 2.5 and 900 kDa (Nosál’ová et al., 2011; Koˇst’álová et al., 2011). The FTIR spectra of all polysaccharide fractions tested were illustrated in Fig. 2. The absorption bands at 1145, 1104 and 1022 cm−1 in the finger-print region (1200–800 cm−1 ) are typical of pectin polymers, whereby, the bands (f) at 1077 and 1050 cm−1 varying in intensity corresponded to vibrations of neutral arabinoseand galactose-based glycans (Kaˇcuráková et al., 2000). The last bands showed, in accord with the sugar analysis, the highest intensity in the spectra of DP1 and EP1. The pectins were present in the partly methylesterified form showing stretching vibration of the ester carbonyl at ∼1745 cm−1 , whereas, the strong bands at 1640–1610 cm−1 corresponded to asymmetric carbonyl stretching of carboxylate groups, overlapped by the water absorption band. Comparison of the intensity of both vibration bands revealed that fractions isolated by the chelating agent (EP2 and DP2) consisted mainly of non-esterified pectin (pectate) what was more pronounced in the spectrum of the purified fraction EP2/C. All other fractions contained pectinates differing in the degree of methyl-esterification. Proteins were identified by the weak band at ∼1546 cm−1 (amide II) in EP1 and DP1 and as shoulders in other fractions. The usually stronger vibration band of amide I (∼1655 cm−1 ) is overlapped by vibrations of the carboxyl groups and vibration bands of phenolic compounds occurring in the region 1700–1500 cm−1 could not be distinguished. NMR spectroscopy was used to verify the presence of pectic polysaccharides assumed from the chemical analyses and FTIR spectroscopy data. As the spectral patterns of the corresponding hot water-, EDTA- and acid-extracted fractions from both EOP and DOP biomasses showed large similarities, for illustration, the 13 C NMR and 1 H NMR spectra of selected fractions were depicted in Fig. 3.

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Table 1 Analytical data of acidic polysaccharide fractions isolated from ethanol-dried (E-series) and air-dried (D-series) pumpkin biomasses. Fraction

Yield (%)a

TP (%)b

Protein (%)b

UA (%)b

EP1 EP2 EP2/C EP3 EP4 DP1 DP2 DP3 DP4 DP4/C

2.9 2.5 38.7c 2.1 0.8 3.7 2.4 1.0 1.1 32.8c

0.9 ± 0.3 0.6 ± 0.3 0.2d 0.4 ± 0.1 0 2.2 ± 0.5 0.9 ± 0.3 0.8 ± 0.3 0.6 ± 0.1 0.2d

2.7 0.9 0 0.8 0 3.2 2.2 2.2 0.9 0

41.2 48.8 75.3 51.6 42.5 39.5 54.3 47.7 63.1 62.3

± ± ± ± ± ± ± ± ± ±

Neutral sugar composition (mol%)

3.4 2.7 2.1 2.2 0.5 2.1 0.6 2.0 3.0 2.3

Rha

Fuc

Ara

Xyl

4.0 10.1 26.3 17.3 11.7 2.8 2.6 2.5 20.3 16.2

1.6 14.7 15.8 6.4 2.4 10.4 4.0 7.0 8.0 2.8

16.9 21.7 21.1 29.6 41.2 36.8 15.4 18.4 23.3 40.4

2.7 6.6 8.9 3.3 2.3 10.9 1.7 3.3 4.7 13.5

Man 4.9 9.3 0 6.0 6.0 3.2 10.1 5.1 6.9 0

Glc

Gal

51.5 25.4 14.2 20.6 16.4 13.6 55.2 48.2 17.8 5.7

18.4 12.2 13.7 16.8 20.0 22.3 11.0 15.5 19.0 21.4

TP, total phenolic content expressed as gallic acid equivalents; UA, content of uronic acids (as galacturonic acid equivalents); (±) standard deviation. a Calculated on oven-dried EOP or DOP. b Calculated on moisture-free basis of individual fractions. c % related to starting EP2 and DP4. d Determined by UV absorption at 280 nm (calibration with gallic acid).

The subfraction EP2/C (Fig. 3A) displayed a simple spectrum with six major signals at ı 175.5, 99.9, 78.9, 72.0, 69.7 and 69.1 assigned to C-6, C-1, C-4, C-5, C-3 and C-2 of 4-linked ˛-GalpA residues. The chemical shifts of protons H-1, H-2, H-3 and H5 appeared as dominant signals at ı 5.00, 3.69, 3.90 and 4.68. The data coincided with those reported for 1,4-␣-d-galacturonans (Catoire et al., 1998). In contrast, the 13 C and 1 H NMR spectra of the crude EDTA-extracted, water and acid-extracted fractions (shown for DP1, EP4 and DP4 in Fig. 3A and for EP1, DP2, DP3 and EP4 in Fig. 3B) were much more complex and contained signals of both the non-esterified (G unit) and methylesterified (E unit) ˛-GalpA residues. E units showed signals at ı 53.7 (OCH3 ), 171.4 (C-6), 100.9 (C-1), 79.4 (C-4) and 71.3 (C-5), whereby, the couples of signals at ı ∼ 68.9–69.6 corresponding to C-2 and C-3 were not distinguishable for the G and E units. These data accorded with assignments published for non-methylesterified and methylesterified citrus pectins (Catoire et al., 1998; Tamaki et al., 2004). As shown in Fig. 3B, next to the methoxyl proton (ı 3.81), several signals appeared in the anomeric region of all fractions. The H-1 and H-5 of G units resonated at ı ∼ 5.11 and 4.68, respectively. The H-1 of E units gave split signals at ı 4.95–4.90 and H-5 at ı 5.05 what accorded with published data (Renard and Jarvis, 1999). The shifting of G and E protons was proven to be dependent on the

various positions of both units in diads and triads or larger groupings in the pectin chains. The presence of rhamnose was evidenced by signals of the C-6 methyl group at ı ∼ 17.6, not always well distinguishable from the noise. However, the corresponding methyl protons showed signals at ı 1.18 and 1.27 in all fractions. They were assigned to 2- and 2,4-linked ˛-Rhap residues belonging to the RG I domains (Habibi et al., 2004; Catoire et al., 1998). Weak signals at ı 21.2–20.8 indicated the presence of acetyl groups which were well distinguished in the 1 H NMR spectra of all fractions and were absent only in EP2/C. The split signals at ı ∼ 2.16 and 2.09 were attributed to the methyl protons of acetyl groups located at positions 2 and/or 3 of ˛-GalpA residues (Renard and Jarvis, 1999). The signals at ı 108.5–108.3, 84.8 and 81.7 were assigned to C1, C-4 and C-2 of terminal and 5-linked ˛-Araf residues and those at ı 106.1–105.1, 77.6 and 4.20 to C-1, C-4 and H-4 of 4-linked ˇGalp residues (Habibi et al., 2004). They were the most prominent in both water-extracted pectin fractions. The signals at ∼96 and 93 ppm in DP1 were attributable to C-1 of ˇ- and ˛-reducing end sugars of oligomeric pectin fragments (Catoire et al., 1998), what accorded with the high proportion of low molecular weight components in this fraction (Nosál’ová et al., 2011). The assignments of all presented signals were established by more detailed investigation of the structural features of subfraction DP4/C using 1D

Fig. 2. FTIR spectra of polysaccharide fractions isolated from the (A) ethanol-dried and (B) air-dried pumpkin biomasses, and of purified subfractions EP2/C and DP4/C. The letters denote absorption bands of carboxyl groups: (a) ␯C O of COOH, COOCH3 and acetyl groups, (b) ␯as C O and (c) ␯s C O of COO− ; (d) amide I, (e) amide II; FPR, finger-print region; (f) vibrations of arabinan and arabinogalactans.

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Fig. 3. 13 C NMR spectra (A) and partial 1 H NMR spectra (B) in D2 O of pectin fractions. (1) DP1, (2) EP2/C, (3) EP4, (4) DP4, (5) EP1, (6) DP2 and (7) DP3. A: ˛-Araf, U: both forms of 4-linked GalpA and the ␣,␤ reducing end units, G: ˛-GalpA, E: ˛-GalpA(6Me), R: ˛-Rhap, Ga: ␤-Galp, OCH3 : methoxyl group of E units, and AcCH3 : methyl of acetyl group. The numbers denote the carbon or proton of the sugars.

and 2D heteronuclear NMR spectroscopy analyses (Koˇst’álová et al., 2011). The results confirmed that all isolated polysaccharide fractions comprised pectin polymers to various extent methylesterified, slightly acetylated and containing minor amounts of ramified RG I domains. The presence of phenolic compounds was evidenced by a complex of signals in the region ı 6.3–7.5 which were detectable only in the water-extracted fractions and in accord with the highest TP content of these samples (Table 1). 3.2. Antioxidant activity The pectin fractions isolated from both pumpkin biomasses were tested for antioxidant activity involving two colorimetric methods and EPR spectroscopy. The antioxidant capacity of respective fractions was expressed by their ability to scavenge/eliminate the stable free radical • DPPH and the cation-radical ABTS•+ as well as in terms of the ferric ion reducing antioxidant power (FRAP). The results of spectrophotometric experiments are given in Table 2. As seen, fractions EP1 and DP1 exhibited the highest free radical scavenging/antioxidant activities in both assays. The maximum DPPH radical scavenging activity (RSA) of the samples (at 1.2 mg/mL) reached 83% and 55% of the activity of gallic acid, used as standard, what indicated moderate antioxidant capacity. Similar RSA activities (68–90% at 1 mg/mL) were reported for water-soluble acidic polysaccharides isolated from Acanthopanax senticosus leaves (Chen et al., 2011) and also for a commercial pectin (RSA ∼ 58% at 1 mg/mL) (Tomida et al., 2010). The FRAP values of the pectin fractions from the E-series (2.6–40 ␮mol Fe2+ /g) and Dseries (5.5–79 ␮mol Fe2+ /g) were markedly lower (10–20 times) than that of the Acanthopanax polysaccharides showing maximum FRAP values between 550 and 790 ␮mol Fe2+ /g at much lower concentration (0.2 mg/mL) which are indicative of a pronounced reductive power. The antioxidant activity of the individual pectin fractions was also proved by means of EPR spectroscopy which allowed to compare quantitatively and qualitatively the antioxidant capacity of

the fractions. As is clearly depicted in Fig. 4, when no antioxidant was present in the sample (reference sample) the signal of both • DPPH and ABTS•+ radicals remained practically stable revealing only small fluctuations caused by the concentration level and the experimental conditions (scan rate) used. However, as a result of adding the tested pectin fraction, the EPR spectra intensity gradually decreased indicating the elimination/termination of either • DPPH or ABTS•+ radicals from the experimental system by the antioxidant capacity of the added sample. From the quantitative point of view, the lower EPR signal the stronger the antioxidant. Using this approach it is evident that sample DP1 revealed to be a more effective ABTS•+ and • DPPH radical-scavenger than the DP4 one. All these results were in good agreement with the results of the colorimetric analyses (Table 2). As mentioned in Section 2, quantification of antioxidant action in case of EPR experiments was performed via the calculation of effective TEAC values (Staˇsko et al., 2006; Polovka et al., 2010). Results obtained (data not

Table 2 Antioxidant activities of pectic polysaccharides isolated from ethanol- and air-dried pumpkin biomasses measured by the DPPH and FRAP assays. Fraction EtOH-dried biomass EP1 EP2 EP2/C EP3 EP4 Air-dried biomass DP1 DP2 DP3 DP4 DP4/C

DPPH (%)a

FRAP c(Fe2+ ) ␮mol/g b

55.0 28.5 33.0 23.2 8.7

± ± ± ± ±

3.6 2.1 3.2 1.3 0.6

40.2 ± 5.1 3.8 ± 0.5 nd 3.6 ± 0.6 2.6 ± 0.5

83.0 15.1 11.6 17.4 11.1

± ± ± ± ±

3.0 1.7 0.1 0.9 0.2

78.7 ± 6.9 5.8 ± 0.5 5.5 ± 0.8 14.7 ± 3.1 nd

a Radical scavenging activity (RSA) related to 100% activity of gallic acid, measured at sample concentration 1.2 mg/mL. b Antioxidant activity expressed as ␮mol FeSO4 equivalents per 1 g sample.

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Fig. 5. Complement-fixing activity of the pumpkin pectic polysaccharides. The activity of samples was referred to ICH50 of the positive control (PMII).

Fig. 4. Time evolution of EPR spectra of • DPPH and ABTS• + radicals recorded in the presence of (a) pure ethanol and deionized water, respectively, as reference samples, and of polysaccharide solutions in water (b) DP1 (c = 0.08 g/100 mL) and (c) DP4 (c = 0.08 g/100 mL). Spectra were recorded at 25 ◦ C. Concentration of radicals: c0(•DPPH) = 1.0 × 10−4 M and c0(ABTS• + ) = 7.2 × 10−5 M.

presented) are coherent with the above described trends. The values of TEACABTS•+ varied from 60 to 100 ␮mol/g and those of TEAC• DPPH from 25 to 50 ␮mol/g sample. In Table 2, a same trend of antioxidant activities was observed in the E- and D-series, i.e. a decrease of the antioxidant capacity with decreasing content of phenolic compounds. Both phenolic substances and protein might induce or stimulate the antioxidant activity. It has to be stressed that in case of EOP, a part of these compounds were lost during the ethanol-drying process. Moreover, some extractives were removed from both pumpkin biomasses before starting the extraction process and the isolated pectin fractions were purified from low molecular weight components by dialysis. Therefore, the present phenolic compounds must be strongly physically and/or covalently associated with polysaccharide components. By comparing fractions EP1, EP2 and EP2/C, the last mentioned one comprising a pectate polymer (75% UA) with minor amount of phenolics and no protein showed still a weak antioxidant activity in the DPPH test. Of course, phenolic compounds covalently bound to polysaccharides were reported to enhance the antioxidant activity such in case of phenolic acid–polysaccharide complexes (Yuan et al., 2005; Rao and Muralikrishna, 2006) and protein-bound polysaccharides (Chen et al., 2008). 3.3. Immunomodulatory activity The immunomodulatory activity of selected pectin fractions from both pumpkin biomasses was tested by the in vitro complement-fixing assay using the highly methylesterified pectic polysaccharide (PMII) from P. major L. as positive control (Michaelsen et al., 2000; Samuelsen et al., 1996). The

complement-activating potency expressed as ICH50 represents the concentration of the tested sample able to give 50% inhibition of lysis of sensitized red blood cells from sheep. However, the ICH50 values of PMII in various experiments ranged between 35 and 60 ␮g/mL. In order to facilitate the comparison of the immunomodulatory effects of the tested pectin samples with corresponding data already published for other polysaccharides, the ICH50 values of the pectin samples were related to the value obtained with the control (PMII). The yielding relative values Rel-ICH50 were calculated as: ICH50 of sample/ICH50 of PMII. The lower the Rel-ICH50 values the higher the complement-fixing activity. As illustrated in Fig. 5, all pectin fractions tested showed complement-fixing activity with Rel-ICH50 values ranging from 0.4 to 4.3. Large differences were observed with the hot waterextracted EP1 and DP1 fractions. EP1 showed about 40% higher activity than the positive control and a 2.5 times higher activity than DP1. The complement-fixing activities of the pumpkin pectic polysaccharides with Rel-ICH50 0.6–1.3 indicated that they exhibited a higher immunomodulatory potency than the pectic polysaccharides isolated from white cabbage with Rel-ICH50 values of 4.5–21.5 (Westereng et al., 2006) or those from roots of Cochlospermum tinctorium with Rel-ICH50 values of 1.3–12.7 (Nergard et al., 2005). The highest activities seen appear to be of the same order as of polysaccharides isolated from various other medicinal plants from Mali that are used for woundhealing, e.g. Combretum glutinosum (Glæserud et al., 2011), Biophytum petersianum (Inngjerdingen et al., 2006), and Opilia celtidifolia (Gronhaug et al., 2010); all having different structural features of pectic nature. The differences observed are likely less related to the presence and content of non-carbohydrate components, because the subfraction DP4/C free of protein and phenolics exhibited the highest immunological activity, whereas, the pure subfraction EP2/C showed the weakest activity. Evidently, of importance are the different structural features of the pumpkin pectin fractions. Fraction EP2/C comprised polygalacturonan chains with a very low proportion of ramified RG I regions, whereas, the most active DP4/C was built of partially methylesterified homogalacturonan chains with branched RG I regions (Koˇst’álová et al., 2011). The results were in accord with previous studies on immunoactive pectins isolated from various herbal plants (Yamada and Kiyohara, 1999; Paulsen and Barsett, 2005; Glæserud et al., 2011; Inngjerdingen et al., 2005) where the authors suggested the complement-fixing potency to be mainly affected by the ramified RG regions. 4. Conclusions This study has shown that the corresponding pectic polysaccharide fractions isolated from the differently dried oil pumpkin

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biomasses showed large similarity in FTIR and NMR spectral patterns. All fractions contained partially methylesterified polygalacturonans with minor amounts of rhamnogalacturonan RG I regions ramified with side chains rich in ˛-Araf and 4-linked ˇGalp residues, and were slightly acetylated. However, the pectin fractions differed in the proportion of these structural features. Whereas, the hot-water extracted fractions represented a mixture of polysaccharides with pectic arbinogalactans prevailing, the EDTA-extracted ones consisted predominantly of non-acetylated and weakly methylesterified homogalacturonans (typical of pectates). Both acid-extracted pectins comprised pectinates differing in the extent of methylesterification and acetylation and presence of ramified RG I. Phenolic compounds and protein occurred mainly in both water-extracted fractions. The pectic polysaccharides displayed moderate antioxidant and radical scavenging activities which correlated with the content of phenolic compounds; the most active were both water-extracted pectic polysaccharides. The evaluation of the immunomodulatory activity using the in vitro complement-fixing assay revealed significant activities for most of the pectin fractions which were comparable and even higher than the herbal pectin from P. major L. used as positive control. The results implied that the presented results are of importance from the scientific as well as technological points of view. Depending on the extraction conditions, natural bioactive pectins with potential antioxidant and immuno-enhancing activities might be produced from the Styrian oil-pumpkin seeded fruits for use as additives in functional foods and as nutraceuticals. Acknowledgements Authors acknowledge for financial support the Slovak Grant Agency VEGA, grant No. 2/0062/09 and the COST action FP0901. This contribution is the result of the project implementation: “Centre of excellence for white-green biotechnology, ITMS: 26220120054” and the “Centre of Excellence for Contaminants and Microorganisms in Food, ITMS: 26240120024” supported by the Research and Development Operational Programme funded by the ERDF. References Adams, G.G., Imrand, S., Wang, S., Mohammad, A., Kok, S., Gray, D.A., Channell, G.A., Morris, G.A., Harding, S.E., 2011. The hypoglycaemic effect of pumpkins as antidiabetic and functional medicines. Food Research International 44, 862–867. Blumenkrantz, N., Asboe-Hansen, G., 1973. New method for quantitative determination of uronic acids. Analytical Biochemistry 54, 484–489. Caili, F., Huan, S., Quanhong, L., 2006. A review on pharmacological activities and utilization technologies of pumpkin. Plant Foods for Human Nutrition 61, 73–80. Caili, F., Haijun, T., Tongyi, C., Yi, L., Quanhong, L., 2007. Some properties of an acidic protein-bound polysaccharide from the fruit of pumpkin. Food Chemistry 100, 944–947. Catoire, L., Goldberg, R., Pierron, M., Morvan, C., Hervé du Penhoat, C., 1998. An efficient procedure for studying pectin structure which combines limited depolymerization and 13 C NMR. European Biophysics Journal 27, 127–136. Chen, H.X., Zhang, M., Qu, Z.S., Xie, B., 2008. Antioxidant activities of different fractions of polysaccharide conjugates from green tea (Camellia sinensis). Food Chemistry 106, 559–563. Chen, R., Liu, Z., Zhao, J., Chen, R., Meng, F., Zhang, M., Ge, W., 2011. Antioxidant and immunobiological activity of water-soluble polysaccharide fractions purified from Acanthopanax senticosus. Food Chemistry 127, 434–440. Glæserud, S., Grønhaug, T.E., Michaelsen, T.E., Inngjerdingen, M., Barsett, H., Diallo, D., Paulsen, B.S., 2011. Immunomodulating polysaccharides from the leaves of the Malian medicinal tree Combretum glutinosum: structural differences between small and large leaves can substantiate the preference for small leaves by the healers. Research Journal of Medicinal Plant 5, 2781–2790. Gronhaug, T.E., Ghildyal, P., Barsett, H., Michaelsen, T.E., Morris, G., Diallo, D., Inngjerdingen, M., Paulsen, B.S., 2010. Bioactive arabinogalactans from the leaves of Opilia celtidifolia Endl. ex Walp. (Opiliaceae). Glycobiology 20, 1654–1664. Habibi, Y., Heyraud, A., Mahrouz, M., Vignon, M.R., 2004. Structural features of pectic polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits. Carbohydrate Research 339, 1119–1127.

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