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.

Contents lists available atScienceDirect Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol

Chemical characterization of fructooligosaccharides, inulin and structurally

diverse polysaccharides from chamomile tea

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 L E I N F O

Keywords:

Chamomile tea

Inulin

Fructooligosaccharides

Homogalacturonan

Arabinogalactan

A B S T R A C T 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 thefine 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), fruc-tooligosaccharides (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 struc-turally diverse dietaryfibers with potential prebiotic, gastrointestinal and immunological functions

1 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 itsfloral chapters, to obtain the positive effects as improver

of digestion, to facilitate the elimination of gases, to stimulate the

ap-petite, 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,

cos-metics, aromatherapy and in pharmaceutical and food industries Thus,

there is a great demand for chamomile in the market and it is thefifth

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; Cantu-Jungles, Cipriani, Iacomini, Hamaker, & Cordeiro, 2017,2007b) Thus,

in the present study we described the purification process of poly-saccharides obtained from chamomile infusion, its structural char-acterization and with the results we suggested a new therapeutical use

to the species, as a source of prebiotic polysaccharides

2 Material and methods 2.1 Plant material Driedfloral C recutita chapters were kindly provided by Chamel® Produtos Naturais Industry The plant material was stored in a sealed

https://doi.org/10.1016/j.carbpol.2019.03.050

Received 7 February 2019; Received in revised form 11 March 2019; Accepted 14 March 2019

⁎Corresponding author

E-mail address:lucimaramcc@ufpr.br(L.M.C Cordeiro)

Available online 16 March 2019

0144-8617/ © 2019 Elsevier Ltd All rights reserved

T

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

2.2 Extraction of polysaccharides

Thefloral 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 min The extract (tea) was filtered, concentrated under reduced

pressure and the polysaccharides precipitated with 95% ethanol

(3 vol.) The polysaccharides were recovered byfiltration, dialyzed in

semipermeable membrane (Cellulose Spectrumlabs 6–8 kDa cut-off)

and freeze-dried (MRW fraction) (Fig 1) These procedures were

re-peated several times to enable the extraction of 628 g offloral 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

frac-tion 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 1 Yields of polysaccharide fractions were expressed as percent

based on the weight of dried floral chapters that were submitted to

extraction (628 g)

2.3 Determination of monosaccharide composition All fractions (except MRW-30E) were hydrolyzed in 500μL 2 M TFA

at 100 °C for 8 h MRW-30E was hydrolyzed with 500μL 0.2 M TFA at

80 °C for 30 min The TFA was evaporated and the samples were con-verted to alditol acetates by NaBH4 reduction at 100 °C for 10 min followed by acetylation with Ac2O-pyridine (1:1, v/v, 1 mL) at 100 °C for 30 min The resulting alditol acetates were then extracted with CHCl3and 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 aflow rate of 1 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 pre-sent in the sample

Uronic acid contents were determined using the modified m-hy-droxybiphenyl method (Filisetti-Cozzi & Carpita, 1991)

2.4 Determination of homogeneity and relative molecular weight The homogeneity and relative molecular weight (Mw) of water-so-luble polysaccharides were evaluated by high performance steric ex-clusion chromatography (HPSEC), with a Waters 2410 differential re-fractometer as equipment for detection A series of four columns, with exclusion sizes of 7 × 106Da (Ultrahydrogel 2000, Waters), 4 × 105Da (Ultrahydrogel 500, Waters), 8 × 104Da (Ultrahydrogel 250, Waters) and 5 × 103Da (Ultrahydrogel 120, Waters) was used The eluent was 0.1 M aq NaNO2 containing 200 ppm aq NaN3at 0.6 mL/min The sample, previouslyfiltered through a membrane (0.22 μm, Millipore), was injected (250μl loop) at a concentration of 1 mg/mL To obtain the

Fig 1 Scheme of extraction and purification of polysaccharides from infusion of Chamomilla recutita floral chapters

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

2.5 Methylation analysis

Fraction SF-EN-AG was carboxyl reduced by the carbodiimide

method, using NaBH4as 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

ac-cording toCiucanu 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, 2 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 1 min and then

pro-grammed 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)

2.6 Nuclear magnetic resonance spectroscopy

The1H,13C and heteronuclear single quantum coherence

(HSQC-DEPT 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, observing1H at 400.13 MHz and13C at 100.61 MHz,

equipped with a 5-mm multinuclear inverse detection probe with

z-gradient 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 5 μL/min to infuse

fraction MRW-ET (at 200μg/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 N2for sample

desolvation with sheath gas at aflow rate of 8 UA and auxiliary gas at 2

UA with a source temperature of 300 °C The ionization was performed

following the operational parameters: electrospray voltage at 4 kV,

capillary voltage 25 V, tube lens offset 125 V The spectra were

pro-cessed and analysed with Thermo Xcalibur 1.0.0.42 software

3 Results and discussion

The process of extraction by infusion of C recutitafloral 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

iden-tification of two main polysaccharide types present in chamomile tea:

(1) a methyl esterified homogalacturonan (HG) could be detected due

to the signals atδ 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 the13C

spectrum, data not shown),δ 77.2/4.23 (C3-H3), δ 74.6/4.09 (C4-H4),

δ 81.1/3.86 (C5-H5) and δ 62.0/3.76 and 3.83 (C6-H6) ( Corrêa-Ferreira, 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 ofβ-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, do Nascimento, Iacomini, Cor deiro, & Cipriani, 2017)

These two main polysaccharide types were also observed in homo-geneity analysis, where a heterogeneous profile with two evident peaks (I and II) (Fig 3) were present To isolate them, the fraction was sub-mitted to ultrafiltration using a 100 kDa cutoff membrane The process was highly efficient, once peak II was concentrated in the eluted frac-tion (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 ana-lysis, 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

Table 1 Monosaccharide composition of fractions obtained from chamomile (C recutita) tea

a % of peak area relative to total peak area, determined by GC–MS

b

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

Fig 2 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

degree of methyl esterification was determined by1H 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 atδ 20.3 in the

13C NMR spectrum, the degree of acetylation was also determined by1H

NMR following the method of An et al (2011) and

spectro-photometrically byHestrin (1949)methodology, giving a value of 19%

Fraction MRW-100E containing the peak II of MRW (with relative

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 ul-trafiltration 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 fructooligosacchar-ides (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 be-sides 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 homo-galacturonan and inulin, besides high amounts of fructooligosacchar-ides A previous study about C recutita polysaccharides pointed out the existence of a polysaccharide containing (1→4)-linked α-D-GalpA re-sidues (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 3 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

Fig 4.13C NMR spectra of fractions MRW-100R (A), MRW-100E (B) and SF-EN (C) in D2O at 70 °C, the chemical shifts are expressed asδ ppm

(Artemisia vulgaris) (Corrêa-Ferreira et al., 2014), stevia (Stevia

re-baudiana) (dede 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

sub-stance 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

in-tact, 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,

xy-lose, 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 its13C 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 its13C 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

13C NMR spectrum (Fig 4C) showed signals atδ 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 atδ

103.4 (anomeric carbon of β-D-Galp) and at δ 107.6 and δ 109.0 (anomeric carbons ofα-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 Se-pharose 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-Me3 -Gal-ol and 4-Me Gal-ol acetates, from terminal, 6-O-, 3-O- and 2,3,6-tri-O-substituted Galp units, respectively Arabinose was present as term-inal, 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 atδ 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 andδ 103.0/4.51 from terminal, →3)-β-D-Galp-(1→/→3,6)-β-D-Galp-(1→ and →6)-β-D-Galp-(1→, respectively Inverted DEPT signals were atδ 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

atδ 61.2/3.80, δ 61.1/3.73 and δ 60.9/3.77 from unsubstituted

C-6/H-Fig 5 MS spectra (+ve mode) of MRW-ET fraction obtained in LTQ Orbitrap-XL Hybrid Ion Trap-Orbitrap Mass Spectrometer

Table 2 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-AG a Linkage type b

a Fraction was carboxyl reduced byTaylor 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

6 or C-5/H-5 fromα-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

ex-tracts However, the fine chemical structure of these polysaccharides

had not been determined

Matricaria chamomilla belongs to a major group of cultivated

med-icinal plants, often referred to as the“star among medicinal species”

More than 120 chemical constituents have been identified in

chamo-mile flower as secondary metabolites, which gives to chamomile its

multitherapeutic, cosmetic, and nutritional values, that have been

es-tablished through years of traditional and scientific use and research

(Singh et al., 2011) The presence of inulin, FOS, highly methyl

ester-ified 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

con-sumption and adds to chamomile a new property, as a source of

structurally diverse dietary fibers with potential prebiotic,

gastro-intestinal and immunological functions

Acknowledgements

This research was supported by CAPES (Process 1264763),

Fundação Araucária 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

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