Chemical characterization of fructooligosaccharides, inulin and structurally diverse polysaccharides from chamomile tea

Chamomile is one of most known species of medicinal plants. It has valuable pharmacological properties that produce positive effects in many therapeutical uses. Some of these properties are attributed to the presence of secondary metabolites but is already known that primary metabolites can also produce positive effects.

Carbohydrate Polymers 214 (2019) 269–275 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Chemical characterization of fructooligosaccharides, inulin and structurally diverse polysaccharides from chamomile tea Pedro Felipe P Chaves, Marcello Iacomini, Lucimara M.C Cordeiro T ⁎ Department of Biochemistry and Molecular Biology, Federal University of Paraná, CP 19.046, CEP 81.531-980, Curitiba, PR, Brazil A R T I C LE I N FO A B S T R A C T Keywords: Chamomile tea Inulin Fructooligosaccharides Homogalacturonan Arabinogalactan Chamomile is one of most known species of medicinal plants It has valuable pharmacological properties that produce positive effects in many therapeutical uses Some of these properties are attributed to the presence of secondary metabolites but is already known that primary metabolites can also produce positive effects In this study we elucidate the fine chemical structure of polysaccharides present in the infusion of chamomile flower chapters After ethanolic precipitation, polysaccharides were obtained from the tea (fraction MRW, 3.2% yield), purified and characterized as an inulin type fructan, a highly methyl esterified and acetylated homogalacturonan (DE = 87% and DA = 19%), and a type II arabinogalactan From ethanolic supernatant (20.2% yield), fructooligosaccharides (FOS) ranging from GF2 (m/z 543) to GF10 (m/z 1839) were detected Inulin and FOS are well-established prebiotics, as well as the pectic polysaccharides Thus, chamomile could be a source of structurally diverse dietary fibers with potential prebiotic, gastrointestinal and immunological functions Introduction Medicinal plants have a fundamental role in the world health, they can be used as sources of direct therapeutic agents, can serve as a raw material for the elaboration of semi-synthetic pharmaceuticals or the discovery of new compounds (Akerele, 1993) Hence, every year more species have their chemical components described, their therapeutic effectiveness are proven and also the discovery of new therapeutic uses occurs (Halberstein, 2005) Numberless species are explored for their pharmacological effects, among them are the chamomile Chamomilla recutita [L.] Rauschert, commonly called German chamomile, is one of most known medicinal species and is included in the pharmacopoeia of almost all countries (Franke & Schilcher, 2005) It is consumed in infusion or decoction form from its floral chapters, to obtain the positive effects as improver of digestion, to facilitate the elimination of gases, to stimulate the appetite, to relief anxiety, to treat colic, wounds or diseases of the skin, as healing agent and mainly as an anti-inflammatory medicine (Lorenzi & de A.Matos, 2008; Sousa, Matos, Matos, Machado, & Craveiro, 1991) Moreover, the chamomile oil is extensively used in perfumery, cosmetics, aromatherapy and in pharmaceutical and food industries Thus, there is a great demand for chamomile in the market and it is the fifth top selling herb in the world (Singh, Khanam, Misra, & Srivastava, 2011) ⁎ The pharmacological properties exhibit by medicinal plants are usually attributed to the presence of specific secondary metabolites, however it is already known that some primary metabolites, such as polysaccharides, can work together to produce these properties and also can exhibit strong biological effects per se (Halberstein, 2005; Liu, Willför, & Xu, 2015) Polysaccharides can also have prebiotic effect (Roberfroid, 2007a) Their capacity of escaping the digestion in the upper gastrointestinal tract and become available for fermentation by microbiota is already known and can be linked to their structural characteristics, such as monosaccharide composition, glycosidic bond configuration, amount and size of branches and molar mass (Roberfroid, 2007a; CantuJungles, Cipriani, Iacomini, Hamaker, & Cordeiro, 2017, 2007b) Thus, in the present study we described the purification process of polysaccharides obtained from chamomile infusion, its structural characterization and with the results we suggested a new therapeutical use to the species, as a source of prebiotic polysaccharides Material and methods 2.1 Plant material Dried floral C recutita chapters were kindly provided by Chamel® Produtos Naturais Industry The plant material was stored in a sealed Corresponding author E-mail address: lucimaramcc@ufpr.br (L.M.C Cordeiro) https://doi.org/10.1016/j.carbpol.2019.03.050 Received February 2019; Received in revised form 11 March 2019; Accepted 14 March 2019 Available online 16 March 2019 0144-8617/ © 2019 Elsevier Ltd All rights reserved Carbohydrate Polymers 214 (2019) 269–275 P.F.P Chaves, et al Fig Scheme of extraction and purification of polysaccharides from infusion of Chamomilla recutita floral chapters 2.3 Determination of monosaccharide composition plastic container at room temperature until use In addition, a voucher specimen of industry’s crop was collected (Campo Largo - PR, Brazil, 25º24.58’’S 49º27.64’’W, in 2013 September) to confirm the botanical identity and deposited in the Museu Botânico Municipal de Curitiba, under registration number 382674 All fractions (except MRW-30E) were hydrolyzed in 500 μL M TFA at 100 °C for h MRW-30E was hydrolyzed with 500 μL 0.2 M TFA at 80 °C for 30 The TFA was evaporated and the samples were converted to alditol acetates by NaBH4 reduction at 100 °C for 10 followed by acetylation with Ac2O-pyridine (1:1, v/v, mL) at 100 °C for 30 The resulting alditol acetates were then extracted with CHCl3 and analyzed by GC-MS using a Varian 3800 gas chromatograph coupled to a Varian Ion-Trap 2000R mass spectrometer (Varian, Palo Alto, CA) The column was DB-225 MS (30 m 0.25 mm i.d.; Agilent Santa Clara, CA) programmed from 50 to 220 °C at 40 °C/min, with helium as carrier gas, at a flow rate of mL/min The inlet temperature was 250 °C, and the MS transfer line was set at 250 °C MS acquisition parameters included scanning from m/z 50–550 in electron ionization mode (EI) at 70 eV Components were identified by their retention times and EI spectra Fructose upon reduction and acetylation gives glucitol and mannitol acetates on GC–MS analysis The amounts of both derivatives have been summed up to give the amount of fructose present in the sample Uronic acid contents were determined using the modified m-hydroxybiphenyl method (Filisetti-Cozzi & Carpita, 1991) 2.2 Extraction of polysaccharides The floral chapters were reserved in a beaker and boiling distilled water was added (40 g/L), the beaker was closed and let rest for about 30 The extract (tea) was filtered, concentrated under reduced pressure and the polysaccharides precipitated with 95% ethanol (3 vol.) The polysaccharides were recovered by filtration, dialyzed in semipermeable membrane (Cellulose Spectrumlabs 6–8 kDa cut-off) and freeze-dried (MRW fraction) (Fig 1) These procedures were repeated several times to enable the extraction of 628 g of floral chapters MRW was further fractionated by ultrafiltration on 100 kDa cutoff membrane (Fig 1), giving MRW-100R (retained on the membrane) and MRW-100E (eluted) This latter was ultrafiltrated on 30 kDa membrane The retained fraction (MRW-30R) was treated with Fehling solution (Jones & Stoodley, 1965), and the resulting insoluble Cu2+ complex isolated by centrifugation Both (Fehling supernatant and precipitated fractions, SF and PF, respectively) were neutralized with acetic acid, dialyzed, and deionized with H+ form cation-exchange resin SF was then treated with endo-inulinase enzyme (316 U/mg, Megazyme) in acetic acid/sodium acetate buffer (pH 4.6) for 16 h at 45 °C and then dialyzed (Cellulose Spectrumlabs 6–8 kDa cut-off), giving SF-EN fraction Finally, it was submitted to anion exchange chromatography on DEAE Sepharose Fast Flow (GE Healthcare) and eluted with water, to give fraction SF-EN-AG All the fractionation steps are summarized in Fig Yields of polysaccharide fractions were expressed as percent based on the weight of dried floral chapters that were submitted to extraction (628 g) 2.4 Determination of homogeneity and relative molecular weight The homogeneity and relative molecular weight (Mw) of water-soluble polysaccharides were evaluated by high performance steric exclusion chromatography (HPSEC), with a Waters 2410 differential refractometer as equipment for detection A series of four columns, with exclusion sizes of × 106 Da (Ultrahydrogel 2000, Waters), × 105 Da (Ultrahydrogel 500, Waters), × 104 Da (Ultrahydrogel 250, Waters) and × 103 Da (Ultrahydrogel 120, Waters) was used The eluent was 0.1 M aq NaNO2 containing 200 ppm aq NaN3 at 0.6 mL/min The sample, previously filtered through a membrane (0.22 μm, Millipore), was injected (250 μl loop) at a concentration of mg/mL To obtain the 270 Carbohydrate Polymers 214 (2019) 269–275 P.F.P Chaves, et al relative Mw, standard dextrans (487 kDa, 266 kDa, 124 kDa, 72.2 kDa, 40.2 kDa, 17.2 kDa and 9.4 kDa, from Sigma) were employed to obtain the calibration curve The relative Mw of the sample was calculated according to the calibration curve Table Monosaccharide composition of fractions obtained from chamomile (C recutita) tea Fraction 2.5 Methylation analysis MRW MRW-100R MRW-30E MRW-30R SF-EN SF-EN-AG Fraction SF-EN-AG was carboxyl reduced by the carbodiimide method, using NaBH4 as the reducing agent, giving products with the eCOOH groups of its uronic acid residues reduced to eCH2OH (Taylor & Conrad, 1972) The carboxyl reduced sample was O-methylated according to Ciucanu and Kerek (1984) method, using powdered NaOH in DMSO-MeI The per-O-methylated polysaccharide was then submitted to methanolysis in 3% HCl–MeOH (80 °C, h) followed by hydrolysis with H2SO4 (0.5 M, 12 h) and neutralization with BaCO3 The material was then submitted to reduction and acetylation as described above for sugar composition, except that the reduction was performed using NaBD4 The products (partially O-methylated alditol acetates) were examined by capillary GC–MS A capillary column (30 m × 0.25 mm i.d.) of DB-225, held at 50 °C during injection for and then programmed at 40 °C/min to 210 °C and held at this temperature for 31 min, was used for separation The partially O-methylated alditol acetates were identified by their typical electron impact breakdown profiles and retention times (Sassaki, Gorin, Souza, Czelusniak, & Iacomini, 2005) Neutral sugarsa Uronic acidb Rha Ara Xyl Gal Fruc 3.0 1.1 – 2.6 3.0 – 24.6 4.0 4.7 18.5 40.6 37.4 8.6 1.9 – 6.9 18.4 – 12.0 2.2 – 11.6 24.0 58.0 12.6 – 95.3d 16.3 tr – 39.2 91.0 nde 44.1 14.0 4.6 a % of peak area relative to total peak area, determined by GC–MS Determined using the m-hydroxybiphenyl method (Filisetti-Cozzi & Carpita, 1991) c The amounts of glucitol and mannitol acetates on GC–MS analysis have been summed up to give the amount of fructose present in the sample d Hydrolysis with 0.2 M TFA at 80 °C followed by GC–MS analysis e Not determined b 2.6 Nuclear magnetic resonance spectroscopy The 1H, 13C and heteronuclear single quantum coherence (HSQCDEPT 135) spectra were obtained from samples dissolved in D2O, at 70 °C using a 400 MHz Bruker model DRX Avance III spectrometer, operating at 9.5 T, observing 1H at 400.13 MHz and 13C at 100.61 MHz, equipped with a 5-mm multinuclear inverse detection probe with zgradient The chemical shifts are expressed in ppm relative to CH3 signal from internal reference acetone (δ 30.2/2.22) All pulse programs were supplied by Bruker 2.7 Electrospray ionization mass spectroscopy analysis A syringe pump was used at a flow rate of μL/min to infuse fraction MRW-ET (at 200 μg/mL) directly into the mass spectrometer The positive high-resolution mass spectroscopy analysis was carried out with electrospray ionization (ESI) at atmospheric pressure ionization (API) in an LTQ-OrbiTrap-XL (Thermo-Scientific), using N2 for sample desolvation with sheath gas at a flow rate of UA and auxiliary gas at UA with a source temperature of 300 °C The ionization was performed following the operational parameters: electrospray voltage at kV, capillary voltage 25 V, tube lens offset 125 V The spectra were processed and analysed with Thermo Xcalibur 1.0.0.42 software Fig HSQC correlation map of MRW fraction in D2O at 70 °C, the chemical shifts are expressed as δ ppm Ara = arabinose, GalA = galacturonic acid, GalA’= methyl esterified galacturonic acid, Fru = fructose δ 81.1/3.86 (C5-H5) and δ 62.0/3.76 and 3.83 (C6-H6) (CorrêaFerreira, Noleto, & Oliveira Petkowicz, 2014; de Oliveira et al., 2011; Perrone et al., 2002; Popov et al., 2011; Vriesmann & de Oliveira Petkowicz, 2009) Small amounts of an arabinogalactan may also be present by the observed anomeric signals of β-D-Galp units at δ 102.9/ 4.47 and that of α-L-Araf at δ 107.6/5.07 and δ 109.0/5.25 (do Nascimento, Iacomini, & Cordeiro, 2017; de Oliveira, Nascimento, Iacomini, Cor deiro, & Cipriani, 2017) These two main polysaccharide types were also observed in homogeneity analysis, where a heterogeneous profile with two evident peaks (I and II) (Fig 3) were present To isolate them, the fraction was submitted to ultrafiltration using a 100 kDa cutoff membrane The process was highly efficient, once peak II was concentrated in the eluted fraction (MRW-100E, 1.4% yield), while peak I remained retained on the membrane (MRW-100R, 1.0% yield) This latter contained the pectic homogalacturonan It had mainly uronic acid (Table 1) on sugar analysis, identified as galacturonic acid by GC–MS of carboxyl-reduced sample, and a relative Mw of 500 kDa 13C NMR spectrum (Fig 4A) showed typical signals of the methyl esterified HG (as cited above) The Results and discussion The process of extraction by infusion of C recutita floral chapters produced a crude polysaccharide fraction named MRW with 3.2% yield from the dry weight and an ethanolic supernatant (MRW-ET, 20% yield) The sugar composition, which showed uronic acids, arabinose, galactose, xylose, rhamnose and fructose (Table 1) together with HSQC correlation map analysis of MRW (Fig 2) allowed a preliminary identification of two main polysaccharide types present in chamomile tea: (1) a methyl esterified homogalacturonan (HG) could be detected due to the signals at δ 100.0/4.97 (C1-H1 from methyl esterified GalpA), δ 99.3/5.18 (C1-H1 from GalpA), δ 68.0/3.75 (C2), δ 68.3/3.98 (C3), δ 78.6/4.46 (C4), δ 70.5/5.05 (C5 from methyl esterified GalpA) and δ 52.8/3.82 (eCOOCH3); and (2) a fructan of inulin-type, due to the signals at δ 61.0/3.73 (C1-H1), δ 103.2 (C2, visible only in the 13C spectrum, data not shown), δ 77.2/4.23 (C3-H3), δ 74.6/4.09 (C4-H4), 271 Carbohydrate Polymers 214 (2019) 269–275 P.F.P Chaves, et al Mw < 9.4 kDa) also showed a third small peak in HPSEC analysis (with relative Mw of 60 kDa) (Fig 3) and thus was submitted to a new ultrafiltration procedure using a 30 kDa cutoff membrane Peak II was eluted in the membrane (MRW-30E fraction) and had fructose on sugar analysis as the major constituent (Table 1) 13C NMR analysis (Fig 4B) indicated the presence of the inulin-type fructan, with six typical signals of →1)-β-D-Fruf-(2→ at δ 61.2 (C1), δ 103.2 (C2), δ 77.5 (C3), δ 74.8 (C4), δ 81.3 (C5) and δ 62.3 (C6) (Corrêa-Ferreira et al., 2014; de Oliveira et al., 2011) Looking for the presence of fructooligosaccharides (FOS) in chamomile tea, we analyzed fraction MRW-ET, which was obtained in high yield (20%), using the LTQ Orbitrap-XL Hybrid Ion Trap-Orbitrap Mass Spectrometer The MS spectra (Fig 5) showed besides sucrose, FOS ranging from GF2 (m/z 543) to GF10 (m/z 1839) Thus, the results showed that chamomile tea contains as main polysaccharides a highly methyl esterified and acetylated homogalacturonan and inulin, besides high amounts of fructooligosaccharides A previous study about C recutita polysaccharides pointed out the existence of a polysaccharide containing (1→4)-linked α-D-GalpA residues (Yakovlev & Gorin, 1977), but the structural characterization of the polymer has not been performed by the authors Later, Füller and Franz (1993) observed the presence of a fructan of the inulin type in their C recutita extracts, but the presence of FOS in chamomile tea has not been reported in the literature yet Fructans are commonly found in species from the Asteraceae family, to which C recutita belongs These can be found as reserve polymers in the tuberous roots of Jerusalem artichoke (Helianthus tuberosus) (Saengthongpinit & Sajjaanantakul, 2005), chicory (Cichorium intybus) (Toneli, Park, Ramalho, Murr, & Fabbro, 2008) and yacon (Smallanthus sonchifolius) (Paredes et al., 2018) In the aerial parts, fructans have already been found in artemisia Fig HPSEC elution profile of fractions MRW, MRW-100E and MRW-100R Refractive index detector Elution volume of dextran standards of molecular weight 487 kDa, 266 kDa, 124 kDa, 72.2 kDa, 40.2 kDa, 17.2 kDa and 9.4 kDa (left to right) were employed to construct the calibration curve degree of methyl esterification was determined by 1H NMR following the method of Grasdalen, Einar Bakøy, and Larsen, (1988) giving a value of 87%, characterizing the chamomile pectin as a HM pectin (Silva et al., 2006) Due to the presence of acetyl signals at δ 20.3 in the 13 C NMR spectrum, the degree of acetylation was also determined by 1H NMR following the method of An et al (2011) and spectrophotometrically by Hestrin (1949) methodology, giving a value of 19% Fraction MRW-100E containing the peak II of MRW (with relative Fig 13 C NMR spectra of fractions MRW-100R (A), MRW-100E (B) and SF-EN (C) in D2O at 70 °C, the chemical shifts are expressed as δ ppm 272 Carbohydrate Polymers 214 (2019) 269–275 P.F.P Chaves, et al Fig MS spectra (+ve mode) of MRW-ET fraction obtained in LTQ Orbitrap-XL Hybrid Ion Trap-Orbitrap Mass Spectrometer 103.4 (anomeric carbon of β-D-Galp) and at δ 107.6 and δ 109.0 (anomeric carbons of α-L-Araf units), probably from an arabinogalactan (Nascimento et al., 2017; Oliveira et al., 2017) Finally, fraction SF-EN was further purified by anion exchange chromatography in DEAE Sepharose Fast Flow The column was eluted with water, giving a fraction (SF-EN-AG) composed mainly of galactose and arabinose (Table 1) Methylation analysis of carboxyl reduced sample (Table 2) confirmed the presence of an arabinogalactan The main methylated derivative was 2,4-Me2-Gal-ol acetate, from 3,6-di-O-substituted Galp units Other Gal derivatives were 2,3,4,6-Me4-Gal-ol, 2,3,4-Me3-Gal-ol, 2,4,6-Me3Gal-ol and 4-Me-Gal-ol acetates, from terminal, 6-O-, 3-O- and 2,3,6-triO-substituted Galp units, respectively Arabinose was present as terminal, 5-O- and 3,5-di-O-substituted Araf units Terminal Glcp units were also observed, from GlcpA units Its HSQC-DEPT correlation map (Fig 6) showed anomeric cross peaks at δ 109.0/5.24 and δ 107.3/5.07 from terminal and →5)-α-L-Araf-(1→ units, at δ 103.8/4.69, δ 103.2/ 4.46 and δ 103.0/4.51 from terminal, →3)-β-D-Galp-(1→/→3,6)-β-DGalp-(1→ and →6)-β-D-Galp-(1→, respectively Inverted DEPT signals were at δ 69.2/3.92–4.04 from 6-O-linked β-D-Galp units and at δ 66.6/ 3.80–3.87 from 5-O-linked α-L-Araf units Other inverted signals were at δ 61.2/3.80, δ 61.1/3.73 and δ 60.9/3.77 from unsubstituted C-6/H- (Artemisia vulgaris) (Corrêa-Ferreira et al., 2014), stevia (Stevia rebaudiana) (de de Oliveira et al., 2011) and another Matricaria species (M maritima (Cérantola et al., 2004) They were also extracted from the monocotyledon agave plant (Agave tequilana var azul) (Praznik, Löppert, Cruz Rubio, Zangger, & Huber, 2013) It is well stablished in the literature that inulin is a versatile substance with numerous health benefits Inulin and FOS are the most studied and well-established prebiotics They escape digestion in the upper gastrointestinal tract and reach the large intestine virtually intact, where they modulate the composition and activities of the gut microbiota (Roberfroid, 2007a) Moreover, it has been demonstrated that pectic polymers from different sources can also be prebiotics, being extensively fermented in the colon and are able to modulated the gut microbiota (Cantu-Jungles et al., 2017; Gulfi, Arrigoni, & Amadò, 2005; Jonathan et al., 2012; Licht et al., 2010; Min et al., 2015; Titgemeyer, Bourquin, Fahey, & Garleb, 1991) It is worth noting that inulin, FOS and pectins can also specifically affect several other gastrointestinal functions (for example, mucosal functions, endocrine activities and mineral absorption) as well as systemic functions (especially glucose and lipid homeostasis and immune functions) (Lunn & Buttriss, 2007; Popov & Ovodov, 2013; Roberfroid, 2007a; Vogt et al., 2015) To a comprehensive identification of chamomile polysaccharides, the low-yield fraction MRW-30R which corresponded to the peak III (Fig 3) was also chemically characterized It had a very complex monosaccharide composition, composed of rhamnose, arabinose, xylose, fructose, galactose and uronic acid (Table 1) Galacturonic acid and fructose came from HG and inulin, that were still present in this fraction (observed in its 13C NMR spectrum, data not shown) To further purification and characterization of other polysaccharides, MRW-30R was treated with Fehling reagent once homogalacturonans interact with copper and precipitate Thus, due to alkaline pH of Fehling reagent, deesterified and deacetylated HG remained in PF fraction, as could be observed in its 13C NMR spectrum (Suppl Fig 1) Fraction SF was also treated with endo-inulinase, due to the presence of some amounts of contaminating inulin On sugar analysis, fraction SF-EN presented rhamnose, arabinose, galactose, xylose and uronic acids (Table 1) Its 13 C NMR spectrum (Fig 4C) showed signals at δ 101.1 and δ 101.7 assigned to anomeric β-D-Xylp units, and at δ 97.6 (C1) and 59.4 (eOCH3) assign to 4-O-Me-α-D-GlcpA units, probably from an acid xylan (Dinand & Vignon, 2001; Vignon & Gey, 1998), and signals at δ Table Linkage types based on analysis of partially O-methyl alditol acetates obtained from methylated type II arabinogalactan (fraction SF-EN-AG) from chamomile (C recutita) tea Partially O-methylalditol acetate SF-EN-AGa Linkage typeb 2,3,5-Me3-Arafc 2,3,4,6-Me4-Glcp 2,3,4,6-Me4-Galp 2,3-Me2-Araf 2-Me-Araf 2,4,6-Me3-Galp 2,3,4-Me3-Galp 2,4-Me2-Galp 4-Me-Galp 11.5 7.2 14.9 8.8 1.1 2.3 4.1 45.2 4.9 Araf-(1→ Glcp-(1→ Galp-(1→ →5)-Araf-(1→ →3,5)-Araf-(1→ →3)-Galp-(1→ →6)-Galp-(1→ →3,6)-Galp-(1→ →2,3,6)-Galp-(1→ a Fraction was carboxyl reduced by Taylor and Conrad (1972) method % of peak area of O-methyl alditol acetates relative to total area, determined by GC–MS b Based on derived O-methyl alditol acetates c 2,3,5-Me3-Ara = 2,3,5-tri-O-Methylarabinitolacetate, etc 273 Carbohydrate Polymers 214 (2019) 269–275 P.F.P Chaves, et al Appendix A Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.03.050 References Akerele, O (1993) Summary of WHO guidelines for the assessment of herbal medicines Pharmacology & Pharmacy, 28, 13 An, T N., Thien, D T., Dong, N T., Dung, P., Le, Du, N., Van (2011) Isolation and characteristics of polysaccharide from Amorphophallus corrugatus in Vietnam Carbohydrate Polymers, 84(1), 64–68 https://doi.org/10.1016/j.carbpol.2010.10 074 Brecker, L., Wicklein, D., Moll, H., Fuchs, E C., Becker, W M., & Petersen, A (2005) Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.) Carbohydrate Research, 340(4), 657–663 https://doi.org/10.1016/j.carres.2005.01.006 Cantu-Jungles, T M., Cipriani, T R., Iacomini, M., Hamaker, B R., & Cordeiro, L M C (2017) A pectic polysaccharide from peach palm fruits (Bactris gasipaes) and its fermentation profile by the human gut microbiota in vitro Bioactive Carbohydrates and Dietary Fibre, 9(August 2016), 1–6 https://doi.org/10.1016/j.bcdf.2016.11.005 Capek, P., Matulová, M., Navarini, L., & Suggi-Liverani, F (2010) Structural features of an arabinogalactan-protein isolated from instant coffee powder of Coffea arabica beans Carbohydrate Polymers, 80(1), 180–185 https://doi.org/10.1016/j.carbpol 2009.11.016 Cérantola, S., Kervarec, N., Pichon, R., Magné, C., Bessieres, M.-A., & Deslandes, E (2004) NMR characterisation of inulin-type fructooligosaccharides as the major water-soluble carbohydrates from Matricaria maritima (L.) Carbohydrate Research, 339(14), 2445–2449 https://doi.org/10.1016/j.carres.2004.07.020 Ciucanu, I., & Kerek, F (1984) A simple and rapid method for the permethylation of carbohydrates Carbohydrate Research, 131(2), 209–217 https://doi.org/10.1016/ 0008-6215(84)85242-8 Corrêa-Ferreira, M L., Noleto, G R., & Oliveira Petkowicz, C L (2014) Artemisia absinthium and Artemisia vulgaris: A comparative study of infusion polysaccharides Carbohydrate Polymers, 102(1), 738–745 https://doi.org/10.1016/j.carbpol.2013.10 096 de Oliveira, A J B., Gonỗalves, R A C., Chierrito, T P C., Dos Santos, M M., De Souza, L M., Gorin, P A J., Iacomini, M (2011) Structure and degree of polymerisation of fructooligosacchari des present in roots and leaves of Stevia rebaudiana (Bert.) Bertoni Food Chemistry, 129(2), 305–311 https://doi.org/10.1016/j.foodchem 2011.04.057 Dinand, E., & Vignon, M R (2001) Isolation and NMR characterisation of a (4-O-methylD-glucurono)-D-xylan from sugar beet pulp Carbohydrate Research, 330(2), 285–288 https://doi.org/10.1016/S0008-6215(00)00273-1 Dong, Q., & Fang, J N (2001) Structural elucidation of a new arabinogalactan from the leaves of Nerium indicum Carbohydrate Research, 332(1), 109–114 https://doi.org/ 10.1016/S0008-6215(01)00073-8 Filisetti-Cozzi, T.m.c.c., & Carpita, N.c (1991) Measurement of uranic acids without from neutral sugars Analytical Biochemistry, 162, 157–162 https://doi.org/10.1016/00032697(91)90372-Z Franke, R., & Schilcher, H (2005) Chamomile industrial profiles Boca Raton: CRC Press Taylor & Francis Group Füller, V E., & Franz, G (1993) Neues von den kamillenpolysacchariden Deutsche Apotheker Zeitung, 133, 4224–4227 Goellner, E M., Utermoehlen, J., Kramer, R., & Classen, B (2011) Structure of arabinogalactan from Larix laricina and its reactivity with antibodies directed against typeII-arabinogalactans Carbohydrate Polymers, 86(4), 1739–1744 https://doi.org/10 1016/j.carbpol.2011.07.006 Grasdalen, H., Einar Bakøy, O., & Larsen, B (1988) Determination of the degree of esterification and the distribution of methylated and free carboxyl groups in pectins by 1H-n.m.r spectroscopy Carbohydrate Research, 184, 183–191 https://doi.org/10 1016/0008-6215(88)80016-8 Gulfi, M., Arrigoni, E., & Amadò, R (2005) Influence of structure on in vitro fermentability of commercial pectins and partially hydrolysed pectin preparations Carbohydrate Polymers, 59(2), 247–255 https://doi.org/10.1016/j.carbpol.2004.09 018 Halberstein, R A (2005) Medicinal plants: Historical and cross-cultural usage patterns Annals of Epidemiology, 15(9), 686–699 https://doi.org/10.1016/j.annepidem.2005 02.004 Hestrin, S (1949) The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application The Journal of Biological Chemistry, 180(1), 249–261 Jonathan, M C., Van Den Borne, J J G C., Van Wiechen, P., Souza Da Silva, C., Schols, H A., & Gruppen, H (2012) In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans Food Chemistry, 133(3), 889–897 https://doi.org/ 10.1016/j.foodchem.2012.01.110 Jones, J K N., & Stoodley, R J (1965) Fractionation using copper complexes Methods in Carbohydrate Chemistry, 5, 36–38 Liang, F., Hu, C., He, Z., & Pan, Y (2014) An arabinogalactan from flowers of Chrysanthemum morifolium: Structural and bioactivity studies Carbohydrate Research, 387(1), 37–41 https://doi.org/10.1016/j.carres.2013.09.002 Licht, T R., Hansen, M., Bergström, A., Poulsen, M., Krath, B N., Markowski, J., Wilcks, A (2010) Effects of apples and specific apple components on the cecal environment of conventional rats: Role of apple pectin BMC Microbiology, 10(13), Fig HSQC-DEPT correlation map of SF-EN-AG fraction in D2O at 50 °C, the chemical shifts are expressed as δ ppm Inverted signals in DEPT experiment are shown in blue color (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article) or C-5/H-5 from α-L-Araf-(1→, β-D-Galp-(1→ and →3)-β-D-Galp-(1→ units The assignments are in agreement with published literature data and methylation analysis described above (2015, Brecker et al., 2005; Capek, Matulová, Navarini, & Suggi-Liverani, 2010; Dong & Fang, 2001; Goellner, Utermoehlen, Kramer, & Classen, 2011; Liang, Hu, He, & Pan, 2014; Wang, Shi, Bao, Li, & Wang, 2015) and shows the presence of a type II arabinogalactan in SF-EN-AG fraction In their preliminary characterization of C recutita polysaccharides, Füller and Franz (1993) also suggested the presence of a rhamnogalacturonan with type II arabinogalactan and a glucuronoxylan in the aqueous chamomile extracts However, the fine chemical structure of these polysaccharides had not been determined Matricaria chamomilla belongs to a major group of cultivated medicinal plants, often referred to as the “star among medicinal species” More than 120 chemical constituents have been identified in chamomile flower as secondary metabolites, which gives to chamomile its multitherapeutic, cosmetic, and nutritional values, that have been established through years of traditional and scientific use and research (Singh et al., 2011) The presence of inulin, FOS, highly methyl esterified homogalacturonan, type II arabinogalactan and acid xylan in chamomile tea shows that not only can the secondary metabolites be the responsible molecules by the health benefits of chamomile consumption and adds to chamomile a new property, as a source of structurally diverse dietary fibers with potential prebiotic, gastrointestinal and immunological functions Acknowledgements This research was supported by CAPES (Process 1264763), Fundaỗóo Araucỏria and by Universal Project (Process 404717/2016-0) provided by CNPq foundation (Brazil) The authors are grateful to Chamel® Produtos Naturais Industry who kindly provided the dried floral C recutita chapters, to the NMR Center of UFPR for recording the NMR spectra and to Dr Lauro M de Souza for the mass spectroscopy analysis 274 Carbohydrate Polymers 214 (2019) 269–275 P.F.P Chaves, et al 1016/j.postharvbio.2005.03.004 Sassaki, G L., Gorin, P A J., Souza, L M., Czelusniak, P A., & Iacomini, M (2005) Rapid synthesis of partially O-methylated alditol acetate standards for GC-MS: Some relative activities of hydroxyl groups of methyl glycopyranosides on Purdie methylation Carbohydrate Research, 340(4), 731–739 https://doi.org/10.1016/j.carres 2005.01.020 da Silva, J A L., & Rao, M A (2006) Pectins: Structure, functionality, and uses In A M Stephen, G O Phillips, & P A Williams (Eds.) Food polysaccharides and their applications (pp 353–412) (2nd ed.) Boca Raton: CRC Press Singh, O., Khanam, Z., Misra, N., & Srivastava, M (2011) Chamomile (Matricaria chamomilla L.): An overview Pharmacognosy Reviews, 5(9), 82–95 https://doi.org/10 4103/0973-7847.79103 Sousa, M P., Matos, M E O., Matos, F J A., Machado, M I L., & Craveiro, A A (1991) Constituintes químicos ativos de plantas medicinais brasileiras Fortaleza: EUFC Laboratório de Produtos Naturais Taylor, R L., & Conrad, H E (1972) Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl group Biochemistry, 11(8), 1383–1388 https://doi org/10.1021/bi00758a009 Titgemeyer, E C., Bourquin, L D., Fahey, G C., & Garleb, K A (1991) Fermentability of various fiber sources by human fecal bacteria in vitro The American Journal of Clinical Nutrition, 53(6), 1418–1424 https://doi.org/10.1093/ajcn/53.6.1418 Toneli, J T C L., Park, K J., Ramalho, J R P., Murr, F E X., & Fabbro, I M D (2008) Rheological characterization of chicory root (Cichorium intybus L.) inulin solution Brazilian Journal of Chemical Engineering, 25(3), 461–471 https://doi.org/10.1590/ S0104-66322008000300004 Vignon, M R., & Gey, C (1998) Isolation, 1H and 13C NMR studies of (4-O-methyl-dglucurono)-d-xylans from luffa fruit fibres, jute bast fibres and mucilage of quince tree seeds Carbohydrate Research, 307(1–2), 107–111 https://doi.org/10.1016/ S0008-6215(98)00002-0 Vogt, L., Meyer, D., Pullens, G., Faas, M., Smelt, M., Venema, K., De Vos, P (2015) Immunological properties of inulin-type fructans Critical Reviews in Food Science and Nutrition, 55(3), 414–436 https://doi.org/10.1080/10408398.2012.656772 Vriesmann, L C., & de Oliveira Petkowicz, C L (2009) Polysaccharides from the pulp of cupuassu (Theobroma grandiflorum): Structural characterization of a pectic fraction Carbohydrate Polymers, 77(1), 72–79 https://doi.org/10.1016/j.carbpol.2008.12 007 Wang, H., Shi, S., Bao, B., Li, X., & Wang, S (2015) Structure characterization of an arabinogalactan from green tea and its anti-diabetic effect Carbohydrate Polymers, 124, 98–108 https://doi.org/10.1016/j.carbpol.2015.01.070 Wang, P., Zhang, L., Yao, J., Shi, Y., Li, P., & Ding, K (2015) An arabinogalactan from flowers of Panax notoginseng inhibits angiogenesis by BMP2/Smad/Id1 signaling Carbohydrate Polymers, 121, 328–335 https://doi.org/10.1016/j.carbpol.2014.11 073 Yakovlev, A I., & Gorin, A C (1977) Structure of the petic acid of Matricaria chamomilla Khimiya Prirodnykh Soedinenlii, 2, 186–189 https://doi.org/10.1186/1471-2180-10-13 Liu, J., Willför, S., & Xu, C (2015) A review of bioactive plant polysaccharides: Biological activities, functionalization, and biomedical applications Bioactive Carbohydrates and Dietary Fibre, 5(1), 31–61 https://doi.org/10.1016/j.bcdf.2014.12.001 Lorenzi, H., & de A.Matos, F J (2008) Plantas Medicinais Do Brasil Nativas E Exóticas (2nd ed.) Nova Odessa São Paulo: Instituto Plantarum Lunn, J., & Buttriss, J L (2007) Carbohydrates and dietary fibre Nutrition Bulletin, 32(1), 21–64 https://doi.org/10.1111/j.1467-3010.2007.00616.x Min, B., Kyung Koo, O., Park, S H., Jarvis, N., Ricke, S C., Crandall, P G., Lee, S.-O (2015) Fermentation patterns of various pectin sources by human fecal microbiota Food and Nutrition Sciences, 06(12), 1103–1114 https://doi.org/10.4236/fns.2015 612115 Nascimento, G E., Iacomini, M., & Cordeiro, L M C (2017) New findings on green sweet pepper (Capsicum annum) pectins: Rhamnogalacturonan and type I and II arabinogalactans Carbohydrate Polymers, 171, 292–299 https://doi.org/10.1016/j carbpol.2017.05.029 de Oliveira, A F., Nascimento, G E., Iacomini, M., Cor deiro, L M C., Cipriani, T R (2017) Chemical structure and anti-inflammatory effect of polysacchari des obtained from infusion of Sedum dendroideum leaves International Journal of Biological Macromolecules, 105, 940–946 https://doi.org/10.1016/j.ijbiomac.2017.07.122 Paredes, L L R., Smiderle, F R., Santana-Filho, A P., Kimura, A., Iacomini, M., & Sassaki, G L (2018) Yacon fructans (Smallanthus sonchifolius) extraction, characterization and activation of macrophages to phagocyte yeast cells International Journal of Biological Macromolecules, 108, 1074–1081 https://doi.org/10.1016/j.ijbiomac 2017.11.034 Perrone, P., Hewage, C M., Thomson, A R., Bailey, K., Sadler, I H., & Fry, S C (2002) Patterns of methyl and O-acetyl esterification in spinach pectins: New complexity Phytochemistry, 60(1), 67–77 https://doi.org/10.1016/S0031-9422(02)00039-0 Popov, S V., & Ovodov, Y S (2013) Polypotency of the immunomodulatory effect of pectins Biochemistry Biokhimiia, 78(7), 823–835 https://doi.org/10.1134/ s0006297913070134 Popov, S V., Ovodova, R G., Golovchenko, V V., Popova, G Y., Viatyasev, F V., Shashkov, A S., Ovodov, Y S (2011) Chemical composition and anti-inflammatory activity of a pectic polysaccharide isolated from sweet pepper using a simulated gastric medium Food Chemistry, 124(1), 309–315 https://doi.org/10 1016/j.foodchem.2010.06.038 Praznik, W., Löppert, R., Cruz Rubio, J M., Zangger, K., & Huber, A (2013) Structure of fructo-oligosaccharides from leaves and stem of Agave tequilana Weber, var azul Carbohydrate Research, 381, 64–73 https://doi.org/10.1016/j.carres.2013.08.025 Roberfroid, M B (2007a) Inulin-type fructans: Functional food ingredients The Journal of Nutrition, 137(11), 2493S–2502S https://doi.org/10.1093/jn/137.11.2493S Roberfroid, M B (2007b) Prebiotics: The concept revisited The Journal of Nutrition, 137(3), 830S–837S https://doi.org/10.1093/jn/137.3.830S Saengthongpinit, W., & Sajjaanantakul, T (2005) Influence of harvest time and storage temperature on characteristics of inulin from Jerusalem artichoke (Helianthus tuberosus L.) tubers Postharvest Biology and Technology, 37(1), 93–100 https://doi.org/10 275 ... 269–275 P.F.P Chaves, et al Fig Scheme of extraction and purification of polysaccharides from infusion of Chamomilla recutita floral chapters 2.3 Determination of monosaccharide composition plastic... ranging from GF2 (m/z 543) to GF10 (m/z 1839) Thus, the results showed that chamomile tea contains as main polysaccharides a highly methyl esterified and acetylated homogalacturonan and inulin, ... fructose came from HG and inulin, that were still present in this fraction (observed in its 13C NMR spectrum, data not shown) To further purification and characterization of other polysaccharides,