Arabinoxylan from Mucilage of Tomatoes (Solanum lycopersicum L

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Arabinoxylan from mucilage of tomatoes (Solanum lycopersicum L.): structure and antinociceptive effect in mice models Geórgia Erdmann do Nascimento, Cristiane H. Baggio, Maria Fernanda de Paula Werner, Marcello Iacomini, and Lucimara M.C. Cordeiro J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05134 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on January 30, 2016

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Journal of Agricultural and Food Chemistry

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Arabinoxylan from mucilage of tomatoes (Solanum lycopersicum L.):

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structure and antinociceptive effect in mice models

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Georgia Erdmann do Nascimento*, Cristiane H. Baggio#, Maria Fernanda de Paula

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Werner#, Marcello Iacomini*, Lucimara M. C. Cordeiro*ξ

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*

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19.046, CEP 81.531-980, Curitiba, PR, Brazil.

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, CP

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#

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Curitiba, PR, Brazil.

Departamento de Farmacologia, Universidade Federal do Paraná, CEP 81.531-980,

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ξ

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address: [email protected]

Corresponding author. Phone: +55 (41) 33611655; Fax: +55 (41) 32662042; E-mail

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ACS Paragon Plus Environment


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ABSTRACT

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Tomato is a known functional food due to its content of bioactive compounds. Herein,

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polysaccharides were extracted from mucilage of tomatoes and a purified fraction

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(PTOK) was analyzed by sugar composition, methylation and NMR spectroscopy

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analysis. The results showed the presence of an arabinoxylan, having (1→4)-linked β-D-

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Xylp units in the main chain, which carried low proportion of branching (~5.6%), at O-

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2 and O-3 position, with side-chains constituted by single Araf or Xylp units.

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Intraperitoneal administration of the arabinoxylan in mice significantly reduced the

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number of abdominal constrictions induced by 0.6% acetic acid and the inflammatory

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phase of nociception induced by 2.5% formalin, indicating that it had an antinociceptive

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effect on inflammatory pain models, amplifying the biological roll displayed by

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arabinoxylans of diet. Furthermore, this study reports the presence of an arabinoxylan in

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a dicotyledon plant and also it is the first study about polysaccharides from mucilage of

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tomatoes.

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Keywords: Tomatoes mucilage, Solanum lycopersicum L., arabinoxylan,

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antinociceptive activity.

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INTRODUCTION

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Tomato (Solanum lycopersicum) is one of the most important crops present in

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the human diet. Tomatoes are good sources of potassium, folate, vitamins C, E and A

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and flavonoids (rutin and naringerin). Among its bioactive compounds stands out the

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carotenoid lycopene, that supply over 85% of the total lycopene consumed in the diet

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and it is often assumed to be responsible for the positive health effects of tomato. The

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strong antioxidant effect of the lycopene is correlated with prevention of prostate cancer

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and chronic degenerative and cardiovascular diseases in epidemiological studies where

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tomatoes consumption is increased 1, 2.

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Furthermore, tomatoes are important source of dietary fiber with about 1g/100 g

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of fresh weight 3 and about 18g/100g of dry weight 4. The concept of dietary fibre is

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complex and can be summarized as nondigestible carbohydrates that pass into large

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bowel, where they can be partially or completely fermented by microbiota 5, 6. Dietary

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fibers act in all processes of the gut as the digestion, absorption, gastrointestinal motility

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and its control, gastrointestinal immunity and prebiotic effects, which as result may

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have impact on cardiovascular/systemic health, immune function, weight management,

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colonic health and on the levels of blood total cholesterol and postprandial glucose 7. In

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addition, several other biological properties for plant polysaccharides, both in vitro and

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in vivo experiments, also have been attracted attention, such as: antioxidant 8, 9,

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immunomodulatory 10-12, anticomplement 13, 14, gastroprotective 15-17 and anti-

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inflammatory 18, 19. These properties are correlated with structural parameters such as

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monosaccharide composition, type and configuration of the glycosidic linkage, size and

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frequency of branched points and molecular mass of the polysaccharides 20. The

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polysaccharide structural features display wide ranges in abundance and occurrence



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according to taxonomy and vegetal tissue 21. For instance, xylans are hemicellulosic

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polysaccharides constituted by a β-(1→4) linked D-xylopyranosyl polymer as the

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backbone. In Poaceae family (monocotyledon), which include grasses and cereals (e.g.

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wheat, rye, corn, barley, oat, rice, and sorghum), neutral heteroxylans represent the

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main xylan component in the endosperm, where the backbone is heavily substituted

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with L-arabinofuranosyl (Araf) residues through α-(1→2) and/or α-(1→3) linkages and

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are known as arabinoxylans. Araf substituents can be further esterified with p-coumaric

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or ferulic acids esters at O-3 position. On the other hand, in dicotyledon plants, like

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tomato, the main substituents of xylan backbone are α-glucuronic acid and/or 4-O-

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methyl-glucuronic acid and are termed as glucuronoxylans. Araf could be also present

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in these acidic xylans in small amounts 22, 23. Considering the importance of plant foods

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as a source of polysaccharides and that their biological activities are directly related to

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their structure, in this work, we extracted the hemicellulosic polysaccharides from

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mucilage of ripe tomato fruits. An arabinoxylan was characterized and its

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antinociceptive activity was evaluated using models of pain in mice.

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MATERIALS AND METHODS

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Plant material Ripe fruits of tomatoes (Solanum lycopersicum L.), variety Santa Clara, were purchased at municipal market of Curitiba, State of Paraná, Brazil.

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Extraction and purification of polysaccharides


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Seeds surrounded by mucilage were manually separated from pulp (pericarp) of tomato fruits (1.6 kg). The mucilage was separated from the seeds with the aid of a

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sieve. The mucilage obtained (226 g) was freeze-dried and defatted with chloroform-

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methanol (1:1) by sequential extraction through Soxhlet apparatus, in order to remove

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lipids, pigments and other hydrophobic material. The polysaccharides were firstly

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extracted from the residue with water at 100 ºC under reflux for 2 h (× 4, l L each).

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The aqueous extracts were obtained by centrifugation (12,000 × g, 20 min at

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10°C), joined and concentrated below 60 C under reduced pressure. The

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polysaccharides were precipitated with EtOH (3 vol.). The resulting precipitate was

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dialyzed and freeze-dried, to give fraction TOW (2.6%). In order to obtain the

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hemicellulosic polysaccharides, the remaining residue of aqueous extraction was then

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submitted to extraction with aq. 10% KOH at 100°C under reflux for 2h (×4, 1 L each).

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The alkaline extracts were neutralized with acetic acid, dialyzed for 48h with tap water

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using 12–14 kDa cut-off membrane, concentrated under reduced pressure and freeze-

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dried, originating fraction TOK (6.6%).

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A freeze-thaw treatment was applied in fraction TOK, to give cold-water soluble

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(STOK, 5.0% yield), and insoluble (PTOK, 1.6% yield) fractions (Fig. 1). In this

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procedure, the sample was dissolved in water, frozen and then thawed at room

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temperature and the fractions recovered by centrifugation. The freeze-thaw treatment

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was repeated until no more precipitate appeared.

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The yields of polysaccharide fractions were expressed as % based on the weight

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of dried and defatted tomato mucilage that was submitted to extraction (7.6 g), while the

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moisture and non-polar compounds were expressed as % based on the weight of wet

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tomato mucilage (226 g).



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Sugar composition Neutral monosaccharide components of the polysaccharides fractions (2 mg) and

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their ratio were determined by hydrolysis with 2 M TFA (1 mL) for 8 h at 100°C. The

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hydrolyzates were converted to alditol acetates by successive NaBH4 reduction,

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followed by acetylation with Ac2O-pyridine (1:1, v/v, 1mL) at 100 °C for 30min. The

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resulting alditol acetates were analyzed by GC−MS using a Varian gas chromatograph

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and mass spectrometer, model Saturn 2000R, with He as carrier gas. A capillary column

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(30 m x 0.25 mm i.d.) of DB-225, held at 50 ºC during injection for 1 min, then

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programmed at 40 ºC/min to 220 ºC and held at this constant temperature for 19.75 min.

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was used for the quantitative analysis. The alditol acetates were identified by their

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typical electron impact breakdown profiles and retention times.

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Uronic acid contents were determined spectrofotometrically using the mhydroxybiphenyl method 24.

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Methylation analysis of polysaccharide The purified polysaccharide PTOK (4 mg) was O-methylated according to the

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method of Ciucanu & Kerek 25, using powdered NaOH in Me2SO-MeI. The per-O-

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methylated derivatives were then submitted to methanolysis in 3% HCl–MeOH (80 ºC,

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2 h) followed by hydrolysis with H2SO4 (0.5 M, 12 h, at 100 ºC) and neutralization with

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BaCO3. The resulting mixture of partially O-methylated monosaccharides was

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successively reduced with NaBD4 and acetylated with Ac2O-pyridine. The products

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(partially O-methylated alditol acetates) were analyzed by GC-MS. A capillary column

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(30 m × 0.25 mm i.d.) of DB-225, held at 50 ºC during injection for 1 min, then


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programmed at 40 ºC/min to 210 ºC and held at this temperature for 31 min was used

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for separation. The partially O-methylated alditol acetates were identified by their

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typical electron impact breakdown profiles and retention times 26.

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Nuclear magnetic resonance (NMR) spectroscopy 13

C 1{H}NMR and HSQC spectra were obtained with a Bruker AVANCE III

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400 NMR, operating at 9.5 T, observing 13C at 100.61 MHz and 1H at 400.13 MHz.

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Analyses were performed using a 5 mm multinuclear inverse detection probe with z-

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gradient, at 70 °C. The sample was acquired in Me2SO-d6 with chemical shifts

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expressed as δ ppm, using the resonances of CH3 (δ 39.7) and 1H (δ 2.60) groups of

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Me2SO-d6, as internal references. All pulse programs were supplied by Bruker.

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Animals Experiments were conducted using female Swiss mice (25–35 g), provided by

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the Federal University of Paraná colony. Animals were kept under standard laboratory

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conditions (12 h light/dark cycles, temperature 22 ± 2 ºC) with food and water provided

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ad libitum. Animals were acclimatized to the laboratory for at least 12 h before testing

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and were used only once for experiments. The mice were placed individually into glass

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cylinders over 30 min before the beginning of the experiment. All the experiments were

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performed after approval of the respective protocols by the Committee of Animal

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Experimentation of Federal University of Paraná (CEUA/BIO - UFPR; approval

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number 832). The study was conducted in accordance with the “Principles of

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Laboratory Animal Care” (NIH Publication 85-23, revised 1985) and with the ethical

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guidelines for investigations of experimental pain in conscious animals. The number of



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animals and intensity of noxious stimuli used were the minimum necessary to

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demonstrate consistent effects of the drug treatments.

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Abdominal constriction induced by acetic acid

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The animals were pretreated with vehicle (saline, 10 mL/kg) or PTOK fraction

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(0.1, 1 and 10 mg/kg) by intraperitoneal (i.p.) route, 30 min before the i.p. injection of

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0.6% aqueous acetic acid (0.45 mL/mouse, made up in saline). The numbers of

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abdominal constrictions were cumulatively counted over a period of 20 min 27.

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Nociception induced by formalin Animals were intraperitoneally treated with vehicle (saline, 10 mL/kg, i.p.) or

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PTOK fraction (1, 3 and 10 mg/kg). After 30 min, mice received 20 µl of a 2.5%

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formalin solution (0.92% formaldehyde, in saline) via an intraplantar injection in the

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ventral surface of the right hind paw. Animals were observed from 0 to 5 min (early

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phase) and 15 to 30 min (late phase) and the time that they spent licking the injected

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paw was considered as indicative of nociception 27.

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Statistical analysis

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Data were expressed as means ± standard error of mean (S.E.M.) with 5-6

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animals per group. Comparisons between experimental and control groups were

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performed by one-way analysis of variance (ANOVA) followed by Newman Keul’s

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test. P values less than 0.05 were considered as indicative of significance.

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RESULTS AND DISCUSSION

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Isolation and structural characterization of the arabinoxylan

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The mucilage from ripe tomatoes was freeze-dried and then defatted with

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chloroform-methanol (1:1) in Sohxlet apparatus, yielding a moisture and nonpolar

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compounds content of 95% and 0.6%, respectively. The defatted residue was then

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submitted to successive extraction with water and 10% aq. KOH, both at 100ºC, and the

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extracted polysaccharides (fraction TOW and TOK, respectively) recovered by EtOH

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precipitation and dialysis, respectively (Fig. 1).

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The fraction TOK, on monosaccharide analysis, revealed the presence of

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rhamnose (4.5%), arabinose (23.4%), xylose (53.1%), mannose (12.1%), galactose

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(2.4%) and glucose (4.6%). This fraction was submitted to freeze-thaw treatment,

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giving rise to supernatant STOK and precipitate PTOK. Monosaccharide analysis of

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STOK showed arabinose (10.9%), xylose (12.1%), mannose (35.6%), galactose

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(14.0%), glucose (24.0%) and uronic acids (3.4%). Otherwise, PTOK revealed only

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arabinose (4.0%) and xylose (96.0%), indicating the presence of an arabinoxylan in this

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fraction.

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Methylation analysis of PTOK is reported in Table 1 and it is in agreement with

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monosaccharide composition. The main observed derivative was 2,3-Me2-Xyl-ol

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acetate, which correspond to (1→4)-linked Xylp residues, indicating a (1 → 4)-linked

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xylan as main chain. A low proportion of branching (~5.6%) could be observed, either

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at O-2 or O-3, due to the presence of 3-Me-Xyl-ol and 2-Me-Xyl-ol, respectively. The

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Araf units were present only as terminal units, together with small amounts of Xylp

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units.



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NMR spectral data (13C and HSQC) of the arabinoxylan were in good agreement

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with the results of the linkage analysis. Thus, its 13C NMR spectrum is dominated by

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five signals of the (1→4)-linked β-D-Xylp units of the main chain, with resonances at δ

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101.7 (C-1), δ 75.5 (C-4), δ 74.0 (C-3), δ 72.6 (C-2), δ 63.2 (C-5) (Fig. 2A). Their

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coupled hydrogens were seen in the HSQC experiment, at δ 4.40, δ 3.65, δ 3.40, δ 3.19

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and δ 4.01/3.33 respectively (Fig. 2B). Signals of low intensity are also present at δ

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107.2/5.43 (C-1/H-1), 85.9/4.10 (C-4/H-4), δ 80.5/3.95 (C-2/H-2), δ 77.8/3.76 (C-3/H-

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3) and δ 61.8/3.60, 3.53 (C-5/H-5, H-5´) and could be assigned to terminal Araf units

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(Fig. 2A and B). These assignments are in agreement with published literature data 28, 29.

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Concerning the polysaccharides from of ripe tomatoes fruits, pectins and the

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hemicellulosic polymers xyloglucans and galactoglucomanns are undoubtedly the main

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polysaccharides present in the pulp 30-32. Although quantitatively in small amounts,

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xylans have also been reported, but as acid xylans (glucuroxylans) and mainly

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complexed with pectins or glucomannans 31-34. Regarding chemical characterization of

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arabinoxylans in dicotyledons plants, there are only reports in literature about

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arabinoxylans present in leaves and bark of tropical Litsea species 35-37.

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Thus, this study reports the presence of an arabinoxylan in another dicotyledon

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plant and also it is the first study that describes the chemical structure of a

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polysaccharide extracted from mucilage of tomatoes.

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Antinociceptive activity of PTOK Our results showed that intraperitoneal administration of fraction PTOK at 1 and

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10 mg/kg produced dose related inhibition of the abdominal constrictions induced by

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acetic acid, a screening model for assessment of antinociceptive and anti-inflammatory


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properties of new agents because of its simplicity and sensitivity (Fig. 3). In this model

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PTOK displayed a mean ID50 of 2.60 mg/kg (95% confidence limits 0.90–7.48), and at

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10 mg/kg inhibited the number of writhes by 84 ± 8%. To confirm the effect of PTOK

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on inflammatory pain, we performed the formalin test. This model is constituted by two

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distinct phases: the neurogenic pain (early phase), which results from the direct

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irritating effect on nociceptors; and the inflammatory pain (late phase), mediated by a

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combination of peripheral input and spinal cord sensitization 38. However, the inhibition

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of the inflammatory phase of formalin-induced nociceptive response occurred only

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when PTOK was administered at 10 mg/kg (inhibition of 90 ± 5%) (Fig. 4A and B).

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These findings suggested that the arabinoxylan present in the tomatoes mucilage

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promotes antinociceptive effects through anti-inflammatory mechanisms. Both models

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were also used for Nascimento 39 to evaluate the antinociceptive activity of an

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arabinoglucuronoxylan from tamarillo pulp. This complex acid heteroxylan showed a

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mean ID50 of 0.47 mg/kg (95% confidence limits 0.18–1.19), and at 10 mg/kg inhibited

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the number of abdominal writhes induced by acetic acid by 78 ± 6%. In the formalin

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test, this heteroxylan inhibited the inflammatory phase and displayed a mean ID50 of

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0.09 mg/kg (0.03–0.23) and a maximal inhibition of 80 ± 6% at 1 mg/kg. Structurally,

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this heteroxylan fraction contained glucuronic acid and a higher degree of branching

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(~20%) compared to neutral PTOK fraction (~5.6%) evaluated in this work, indicating

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that differences in the fine structure of side chains of xylan contribute to biological

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response. It is important to note that differences in the structures of xylans, like a higher

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content of arabinose or acid monosaccharides were also related to more potent

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biological activities, such as those observed for anti-ulcer 40, immunological 28, anti-

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complementary 41 and antitussive 42 activities.



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Concerning arabinoxylans, different biological activities have already been

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attributed to this biomolecule. Dietary fiber effects are well documented for

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arabinoxylans, particular from Poaceae family 20, 43. Their poly- and oligosaccharides

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have gained interest as prebiotics 44, 45. Effects on innate and acquired immune response

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in mice 46, anti-inflammatory 47, antioxidant 48 and anti-complementary activities 49

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were also attributed to arabinoxylans as well as antitumor activity probably due

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immunostimulatory properties 50. Finally, the arabinoxylan extracted from green leaves

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of a dicotyledon Litsea glutinosa was also biologically active, once showed splenocyte,

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thymocyte, and macrophage activations 35.

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Therefore, fraction PTOK isolated from mucilage of tomatoes was composed of

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an arabinoxylan, which is an unusual polysaccharide for dicotyledons. Furthermore, this

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fraction significantly reduced the number of abdominal writhes induced by 0.6% acetic

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acid, and inhibited the inflammatory phase of formalin induced nociceptive response.

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These results amplify the roll of biological activities displayed by arabinoxylans, as

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molecules with analgesic effects, probably through an anti-inflammatory mechanism.

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However, more extensive study is necessary to identify the specific mechanism(s)

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behind this inhibition of pain.

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Funding Sources The authors would like to thank the Brazilian funding agencies CNPq foundation

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and PRONEX-Carboidratos for provided financial support for this research (Projeto

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Universal - Process 477971/2012-1).

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Abbreviations used


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2,3,5-Me3-Ara-ol, 2,3,5-tri-O-methylarabinitolacetate; Ac2O, acetic anhydride; Araf,

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arabinofuranosyl; BaCO3, barium carbonate; EtOH, ethanol; HCl, hydrochloric acid;

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H2SO4, sulfuric acid; HSQC, Heteronuclear Single-Quantum Coherence; KOH,

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potassium hydroxide; NaBD4, sodium borodeuteride; NaBH4; sodium borohydride;

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MeI, methyl iodide; MeOH, methanol; Me2SO, dimethyl sulfoxide; Me2SO-d6,

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deuterated dimethyl sulfoxide; NMR, nuclear magnetic resonance; NaOH; sodium

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hydroxide; PPM, parts per million; TFA, trifluoroacetic acid; Xylp, xylopyranosyl.

297 298 299

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502

Figure 1. Scheme of extraction and purification of arabinoxylan from mucilage of ripe

503

tomatoes. The yields were expressed as % based on the weight of dried and defatted

504

mucilage of tomatoes that was submitted to extraction (7.6 g).

505 506

Figure 2. 13C-NMR spectrum (A) and 2D 1H–13C HSQC correlation map showing some

507

carbon and hydrogen assignments (B) of fraction PTOK obtained from mucilage of ripe

508

tomatoes. Sample was dissolved in Me2SO-d6 and data collected at probe temperature of

509

70ºC.

510 511

Figure 3. Effect of intraperitoneal administration of PTOK on abdominal constriction

512

induced by 0.6% acetic acid in mice. The animals were treated with vehicle (VEH, 10

513

mL/kg, i.p.) or PTOK (0.1, 1 or 10 mg/kg, i.p.). Data are expressed as means ± S.E.M.

514

(n = 6–8) and statistical comparison was performed using one-way ANOVA followed

515

by post hoc Newman Keul’s test. Differences from vehicle group (*p < 0.05).

516 517

Figure 4. Effect of intraperitoneal administration of PTOK on neurogenic phase (A)

518

and inflammatory phase (B) of nociception induced by 2.5% formalin in mice. The

519

animals were treated with vehicle (VEH, 10 mL/kg, i.p.) or PTOK (1, 3 or 10 mg/kg,

520

i.p.). Data are expressed as means ± S.E.M. (n = 6–8) and statistical comparison was

521

performed using one-way ANOVA followed by post hoc Newman Keul’s test.

522

Differences from vehicle group (*p < 0.05).

523 524


Page 23 of 28


23

Table 1 Linkage types based on analysis of partially O-methyl alditol acetates obtained from methylated arabinoxylan from tomatoes mucilage (Solanum lycopersicum).

PTOKb

Linkage typec

2,3,5-Me3-Araa

3.5

Araf-(1→

2,3,4-Me3-Xyl

1.8

Xylp-(1→

2,3-Me2-Xyl

89.3

→4)-Xylp-(1→

2-Me-Xyl/3-Me-Xyl d

5.3

→3,4)-Xylp-(1→/→2,4)-Xylp-(1→

Partially O-methylalated aditol acetate

a

2,3,5-Me3-Ara = 2,3,5-tri-O-Methylarabinitolacetate, etc.

b

% of peak area of O-methylalditol acetates relative to total area, determined by GC-MS.

c

Based on derived O-methylalditol acetates.

d

The ratio of 2-Me-xylitol acetate and 3-Me-xylitol estimated by their specific fragmentation patterns in

GC-MS was 2.7: 1.



Page 24 of 28

24

Mucilage of ripe tomatoes Freeze-dried and defatted

Aq. extraction, 100°C

Residue

Fraction TOW (2.6%)

10% KOH extraction, 100°C

Fraction TOK (6.6%)

Residue

Freeze-thaw

Supernatant STOK (5%)

Precipitate PTOK (1.6%)

Arabinoxylan

Figure 1


Page 25 of 28


25

A

61.8/3.60

61.8/3.52

B 63.2/4.01

63.2/3.33

72.6/3.19 74.0/3.40 75.5/3.65

80.5/3.95

77.8/3.76 85.9/4.10 101.7/4.40 107.2/5.43

Figure 2



Page 26 of 28

26

Figure 3


Page 27 of 28


27

Figure 4



Page 28 of 28

28

O

O O

OH

O O

OH

O O

O O

OH

O OH O

OH

OH

HO OH

O

OH

OH

OH

OH O

O

O

OH

HO

OH HO

HO OH

Arabinoxylan

Inhibition of pain

TOC Graphic


Arabinoxylan from Mucilage of Tomatoes (Solanum lycopersicum L

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