Structural features of polysaccharides from edible jambo (Syzygium jambos) fruits and antitumor activity of extracted pectins

Accepted Manuscript Structural features of polysaccharides from edible jambo (Syzygium jambos) fruits and antitumor activity of extracted pectins

Camila Silva Tamiello, Eliana Rezende Adami, Natalia Mulinari Turin de Oliveira, Alexandra Acco, Marcello Iacomini, Lucimara M.C. Cordeiro PII: DOI: Reference:

S0141-8130(18)30894-8 doi:10.1016/j.ijbiomac.2018.06.164 BIOMAC 10004

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

24 February 2018 29 May 2018 27 June 2018

Please cite this article as: Camila Silva Tamiello, Eliana Rezende Adami, Natalia Mulinari Turin de Oliveira, Alexandra Acco, Marcello Iacomini, Lucimara M.C. Cordeiro , Structural features of polysaccharides from edible jambo (Syzygium jambos) fruits and antitumor activity of extracted pectins. Biomac (2018), doi:10.1016/ j.ijbiomac.2018.06.164

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ACCEPTED MANUSCRIPT 1

Structural features of polysaccharides from edible jambo (Syzygium jambos) fruits and antitumor activity of extracted pectins

Camila Silva Tamielloa, Eliana Rezende Adamib, Natalia Mulinari Turin de Oliveirab,

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

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Alexandra Accob, Marcello Iacominia, Lucimara M. C. Cordeiroa*

b

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

Departamento de Farmacologia, Universidade Federal do Paraná, CP 19.046, CEP 81.531-

*

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980, Curitiba, PR, Brazil.

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

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address: [email protected]

ACCEPTED MANUSCRIPT 2 ABSTRACT

Jambo is a tropical fruit cultivated in Southeast Asia and tropical regions of America and Africa. After extraction, its polysaccharides were structurally characterized. Water soluble polysaccharides (WSP) were composed of GalA:Ara:Gal:Glc:Rha in

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51:22:16:5:6 molar ratio, indicating the presence of pectic polysaccharides. Methylation

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analysis and NMR spectroscopy indicated the presence of homogalacturonan (HG), type

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II arabinogalactan and type I rhamnogalacturonan (RG-I). The HG/RG-I ratio was 88%, indicating greater amounts of smooth than hairy pectic regions. Hemicellosic

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polysaccharides were extracted from the residue and fractionated by freeze-thaw procedure in two fractions (ASP-S and ASP-I). ASP-S was composed of

and

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Glc:Gal:Xyl:Ara:Man:Fuc:UA in a 45:16:20:3:8:5:2 molar ratio. Methylation analysis 13

C NMR spectroscopy indicated xyloglucan, xylan and mannan. Their relative

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proportion estimated on sugar linkage was 89%, 6% and 3%, respectively. ASP-I was

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composed mainly of xylose (99.5%) and its

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C NMR indicated a linear (1→4)-β-D-

xylan. The biological activity of WSP was tested in Ehrlich tumor-bearing mice. After

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21 days of oral treatment, the doses of 150 and 250 mg/kg WSP reduced expressively the tumor growth, similarly to the positive control methotrexate, and improved the body

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weight of tumor-bearing mice. Further studies are necessary to investigate the mechanisms of the WSP antitumor effect.

Keywords: Jambo; Syzygium jambos; polysaccharides; pectins; hemicelluloses; Ehrlich tumor.

ACCEPTED MANUSCRIPT 3 1 Introduction The structure of plant polysaccharides differs according to their monosaccharide composition (mainly glucose, galactose, fructose, mannose, arabinose, xylose, rhamnose, fucose, uronic acids), linkage types, main and branched chains. There is great evidence that the polysaccharide structures affects physiological responses providing

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many health benefits by modulating directly the gut microbiota (Jones, 2014), by

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exerting immunological (Ferreira, Passos, Madureira, Vilanova, & Coimbra, 2015;

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Simas-Tosin et al., 2012), antitumor (Zhao et al., 2013), anti-inflammatory (Popov et al., 2014), antioxidant (Yang et al., 2006) and hypoglycemic (Luo, Cai, Yan, Sun, &

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Corke, 2004) effects, among others.

Syzygium jambos (L.) Alston is a tropical plant from Myrtaceae family whose

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fruits are edible and known as rose apple or jambo. It was first described in Southeast Asia and propagated to tropical regions of American and African continents. The seeds

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are small or nonexistent, the peel is thin and usually consumed with the pulp and it is

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finding during the entire year in many Brazilian States. Due to the high production by the tree of jambo and the lack of knowledge of technological processing procedures,

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many fruits are wasted in the harvest (Cardoso, 2008; Clerici & Carvalho-Silva, 2011). The prior knowledge of the chemical structures of polysaccharides consumed in

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human diet may contribute for the knowledge of health benefits related to fruit consumption and this is of great interest. Despite well-known and largely consumed in many tropical countries, so far there are no reports in the literature about S. jambos nutritional composition, however, it was observed for S. malaccense (red jambo) that the main macronutrient is carbohydrate. Recently, we have observed the immunomodulating properties of a type II arabinogalactan extracted from S. jambos on THP-1 macrophages (Tamiello, do Nascimento, Iacomini & Cordeiro, 2018). It

ACCEPTED MANUSCRIPT 4 increased TNF-α, IL-1β and IL-10 macrophage secretion in a concentration-dependent manner as well as attenuated the inflammatory response induced by LPS at the highest concentration (100 μg/mL) tested. Here, we further report the in vivo antitumor effects of pectic polysaccharides in mice bearing solid Ehrlich tumor as well describe structural

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features of the extracted pectins and hemicelluloses from edible jambo fruits.

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2 Materials and methods

2.1 Plant material

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Ripe fruits of jambo (S. jambos (L.) Alston) were purchased in the supply center

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(Ceasa) in Paraná, Brazil.

2.2 Extraction of pectic polysaccharides

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Fruits with peel (2.2 kg) were cut, freeze-dried and milled. Dried fruit powder

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(281 g) was defatted with chloroform-methanol (1:1) in a Soxhlet apparatus. Pectins, present as water soluble polysaccharides (WSP), were extracted as described in

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Tamiello et al. (2018) and shown schematically in Fig. 1.

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2.3 Extraction of hemicellulosic polysaccharides The remaining residue after the WSP extraction contained polysaccharides such as cellulose and hemicelluloses. To solubilize some of the hemicelluloses, the residue was treated with aq. 10% NaOH under reflux at 100 ºC for 2 h (x 4, 1 L each) in the presence of NaBH4. Then, it was centrifuged to give rise the cellulosic residue and the alkali soluble polysaccharides (ASP fraction) (Fig. 1). Both were neutralized with acetic acid and dialyzed against tap water (Cellulose Spectrumlabs 6-8 kDa cut-off). The

ACCEPTED MANUSCRIPT 5 cellulosic residue was freeze-dried while ASP fraction was submitted to freeze-thaw treatment. In this procedure, the sample was frozen and then thawed at room temperature and the fractions recovered by centrifugation. The freeze-thaw treatment was repeated until no more precipitate appeared, giving rise to cold-water soluble (ASPS), and insoluble (ASP-I) fractions (Fig. 1).

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The yields were expressed as percentages based on the weight of dried fruit

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subjected to extraction (281 g), while the moisture and non-polar compounds were

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expressed as percentages based on the weight of wet fruit (2.2 kg).

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2.4 Sugar composition

Neutral sugars were determined after hydrolysis of the polysaccharides with 2 M

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trifluoroacetic acid (TFA) for 8 h at 100 C, followed by conversion to alditol acetates by reduction with NaBH4 and acetylation with acetic anhydride-pyridine (1:1, v/v, at

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100 C for 30min). These 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 (25 m x 0.25 mm i.d.) of VF-5, programmed at 10 ºC/min to 280 ºC and held at this

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constant temperature for 35 min was used for the quantitative analysis. The alditol acetates were identified by their typical electron impact breakdown profiles and

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retention times compared with standards. Uronic acid contents were determined using the modified m-hydroxybiphenyl colorimetric method and galacturonic acid as standard (Filisetti-Cozzi & Carpita, 1991). The identity of uronic acid was identified by GCMS analysis (as cited above) of the carboxyl-reduced sample. This was done employing the method of Taylor and Conrad (1972).

ACCEPTED MANUSCRIPT 6 2.5 Sugar linkage analysis Sugar linkage analysis was performed by the methylation of the polysaccharides. Due to the presence of uronic acids, fractions were submitted to carboxyl-reduction with carbodiimide, according to Taylor and Conrad (1972). The carboxyl-reduced samples were O-methylated using powdered NaOH in DMSO-MeI following the method of

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Ciucanu and Kerek (1984). The products were submitted to methanolysis in 3% HCl–

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MeOH (80 ºC, 2 h) followed by hydrolysis with H2SO4 (0.5 M, 14 h) and neutralization

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with BaCO3. After that the material was submitted to reduction and acetylation as described above for sugar composition, except that the reduction was performed using

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NaBD4. The products (partially O-methylated alditol acetates) were examined by capillary GC–MS and identified by their typical retention times and electron impact

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spectra (Sassaki, Gorin, Souza, Czelusniak, & Iacomini, 2005).

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2.6 Nuclear magnetic resonance (NMR) spectroscopy C NMR and DEPT-135 spectra were acquired at 70 C on a Bruker AVANCE

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III 400 NMR spectrometer, operating at 9.5 T, observing 13C at 100.61 MHz, equipped

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with a 5-mm multinuclear inverse detection probe with z-gradient. The water-soluble samples were acquired in D2O and the water-insoluble ones in DMSO-d6. Chemical

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shifts were expressed as  ppm relative to CH3 signal from acetone at  30.2 or DMSOd6 at  39.7 as internal references. The degree of methyl esterification (DE) and acetylation (DA) were determined by 1H NMR spectroscopy. Briefly, the fractions were deuterium-exchanged three times by freeze-drying with D2O solutions, finally dissolved in D2O, and transferred into 5mm NMR tube. The 1H NMR spectra were acquired at 70 °C, with 256 scans, at pD 5.0, on a Bruker AVANCE III 400 NMR spectrometer, operating at 9.5 T, observing 1H at

ACCEPTED MANUSCRIPT 7 400.13 MHz. Chemical shifts were expressed as δ ppm, using the resonances of acetone at δ 2.22 as internal reference. The values of DM and DA were calculated by hydrogen integration according to Grasdalen, Bakøy and Larsen (1988) and Nguyen, Do, Nguyen, Pham, & Nguyen (2011), respectively. All pulse programs were supplied by Bruker.

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2.7 Determination of homogeneity and molecular weight of polysaccharides

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The homogeneity and molecular weight of water-soluble polysaccharides were

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determined by gel permeation chromatography (GPC). The procedure was carried out as previously reported by Leivas, Iacomini and Cordeiro (2015). Briefly, four columns

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were used in series (7 x 106 Da, 4 x 105 Da, 8 x 104 Da and 5 x 103 Da, Ultrahydrogel, Waters) in the gel permeation chromatography (Waters equipment) and a Waters 2410

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refractometer was used as detection equipment. The eluent was 0.1 M aq. NaNO2 containing 200 ppm aq. NaN3 at 0.6 mL/min. The samples, previously filtered through a

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membrane (0.22 µm, Millipore), were injected at a concentration of 1 mg/mL. To obtain

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the molecular weight, standard dextrans (487kDa, 266kDa, 124kDa, 72.2kDa, 40.2kDa,

2.8 Animals

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17.2kDa and 9.4kDa, from Sigma) were employed to obtain the calibration curve.

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Female Swiss mice weighing between 25-30 g, obtained from the vivarium of Federal University of Paraná, were kept under standard laboratory conditions. The animals were housed in collective plastic cages (maximum of 7 mice per cage) with pine shavings bedding and free access to water and food (Nuvilab®, Brazil), under a 12 h light/dark cycle and at controlled temperature (22 ± 2 ºC). All experiments were conducted in agreement with the “Guide for the Care and Use of Laboratory Animals” (8th edition, National Research Council, 2011) and approved by the Committee of

ACCEPTED MANUSCRIPT 8 Animal Experimentation of the Federal University of Paraná (CEUA/BIO − UFPR; approval number 984).

2.9 In vivo antitumor activity of WSP The biological activity of pectic polysaccharides present in WSP was tested in

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female Swiss mice bearing the Ehrlich solid tumor. Ehrlich cells were maintained in

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ascitic form by intraperitoneal passages (2 × 106 cells) in mice (~25-30 g), until cells

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reach ≥ 95% viability, evaluated by Tripan blue exclusion method. For solid model implantation, Ehrlich cells were inoculated subcutaneously into the right pelvic member

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(2 × 106 cells/mice), and then the animals were separated in groups: vehicle (distilled water; n = 14), WSP 100 mg/kg (n = 7), WSP 150 mg/kg (n = 7), WSP 250 mg/kg (n =

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7), and the positive control Methotrexate (MTX) 2.5 mg/kg (n = 7). Animals were treated daily by gavage with vehicle and WSP, or each 3 days intraperitoneally with

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MTX, for 21 days after the tumor cells inoculation. The body weight was daily

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measured. The tumor volume (V) was assessed from day 7 until the day 21 and calculated according to Mizuno et al. (1999) as: V(cm3) = 4/3•a2•(b/2), where a is the

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smallest tumor diameter and b is the largest tumor diameter (in centimeters). The inhibitory effect on the tumor volume in last day of treatment was calculated using the

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following formula: Tumor suppression (%) = (1 - T/C), where T is the average tumor volume in the treated groups, and C is the average tumor volume in the vehicle group.

2.10. Statistical Analysis The in vivo data are presented as mean  standard error of the mean (SEM). Tumor growth and body weight curves were analyzed by two-way ANOVA followed of Bonferroni's multiple comparisons test. The area under the curve (AUC) of tumor

ACCEPTED MANUSCRIPT 9 growth was individually calculated, grouped by treatments, and then compared using one-way ANOVA followed by Bonferroni´s multiple comparisons test, considering p<0.05 statistically significant.

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3. Results and discussion

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To extract the polysaccharides, ripe S. jambos fruits were cut into small pieces,

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freeze-dried (yielding approximately 87% of moisture), milled and defatted with chloroform-methanol (1:1). Pectic polysaccharides (WSP) were extracted from the

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defatted and dried powder employing boiling water and precipitated from the aq. extract with EtOH (3 vol.), dialyzed and freeze-dried, yielding 12 g of material (4.3% dry

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weight) (Fig. 1).

On monosaccharide composition analysis (Table 1), the WSP showed mainly

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Ara and Gal as neutral sugars and 50.6% of uronic acids, indicating the presence of

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pectic polysaccharides. The identity of the uronic acids was determined by sugar analysis of the carboxyl-reduced sample, showing the presence of GalA due to the

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increased amounts of Gal compared with native WSP. On gel permeation chromatography (Fig. 2A), two peaks were observable in WSP, with relative Mw of 355

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kDa and 110 kDa.

The glycosidic linkage determination of the polysaccharides present in WSP was performed by methylation analysis (Table 2). It showed methylated derivatives that indicated the presence of hairy regions of pectins, such as arabinan, arabinogalactans (AG) and type I rhamnogalacturonan (RG-I) as well as of smooth regions formed by homogalacturonan (HG). That arising from arabinan moiety were 2,3,5-Me3-Ara-olacetate, 2,3-Me2-Ara-ol-acetate and 2-Me-Ara-ol-acetate, indicating a (1→5)-linked

ACCEPTED MANUSCRIPT 10 arabinan branched at O-3 by Araf units. The main methylated derivative of Gal was 2,3,6-Me3-Gal-ol-acetate that revealed a (1→4)-linkage in the backbone, probably derived from (1→4)-linked GalpA units from homogalacturonan. Typical methylated derivatives arriving from type II arabinogalactan (AG-II) have also been observed: 2,3,4-Me3-Gal-ol-acetate,

2,4,6-Me3-Gal-ol-acetate

and

2,4-Me2-Gal-ol-acetate

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corresponding to 6-O-, 3-O- and 3,6-di-O-substituted Galp units. Araf, Arap, Glcp

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(probably from carboxyl-reduced GlcpA) and Galp were found as non-reducing

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terminal units. Besides, 3,4,6-Me3-Gal-ol-acetate, 2,3-Me2-Gal-ol-acetate and 2-MeGal-ol-acetate derivative, which were derived from 2-O-, 4,6-O- and 3,4,6-tri-O-

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substituted Galp units were also present. These derivatives have already been reported for AG-II isolated from various sources (Carlotto et al., 2016; Corrêa-Ferreira, Noleto,

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& Oliveira Petkowicz, 2014; Lim, Yu, Kim, & Chung, 2016; Ponder & Richards, 1997; Zhang, Kiyohara, Sakurai, & Yamada, 1996) as well as for AG-I (Cantu-Jungles et al.,

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2014; Do Nascimento et al., 2015; Iacomini et al., 2005; Leivas et al., 2015; Oechslin,

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Lutz, & Amado, 2003; Yashoda, Prabha, & Tharanathan, 2005). The presence of some AG-I in jambo WSP cannot be ruled out.

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Concerning RG-I, the observed methylated derivatives of rhamnose units were 3,4-Me2-Rha-ol-acetate and 3-Me-Rha-ol-acetate corresponding to 2-O- and 2,4-di-O-

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substituted Rhap units.

In order to estimate the molar proportion of homogalacturonan relative to the type I rhamnogalacturonan chain, the ratio HG/RG-I (%) was estimated using the relation proposed by Koffi, Yapo and Besson (2013): HG/RG-I(%) = 100 x

[GalA(%) – Rha(%)] [2Rha(%)+Ara(%)+Gal(%)]

ACCEPTED MANUSCRIPT 11 The obtained ratio was 88%, indicating a high homogalacturonan amount in the jambo pectic polysaccharides. This is in agreement with our previous results (Tamiello et al., 2018), where the homogalacturonan was precipitated with Fehling’s solution from WSP in high yield. The 13C NMR spectrum (Fig. 3A) of WSP is in agreement with the methylation

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data and monosaccharide analysis. It showed intense signals of the HG domain. That at

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δ 100.0 and δ 70.5 corresponded to C-1 and C-5 of methyl-esterified α-D-GalpA units, respectively, while the signals at δ 99.3 and δ 71.6 corresponded to C-1 and C-5 of α-D-

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GalpA unesterified units. The remaining assignments of D-GalpA ring carbons were at δ

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78.7 (O-substituted C-4), δ 68.0 (C-3 and C-2, overlapped). Moreover, the signals at δ 170.9 and δ 175.1 were attributed to carboxyl groups (C-6) of esterified and unesterified

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α-D-GalpA units, respectively. Signals of methyl and acetyl groups linked to α-D-GalpA units appeared at δ 52.8 and δ 20.5, respectively. Due to the presence of esterified α-D-

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GalpA units, the degree of methyl esterification and degree of acetylation were

The

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determined by 1H NMR spectroscopy, giving values of 83% and 6%, respectively. C NMR anomeric signals of arabinan moiety and of α-L-Araf units from

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AG side chains appeared at δ 107.6, δ 108.4, δ 109.1 and δ 109.4, while that of -DGalp units of the galactan chain were observed at δ 103.5. The

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C-DEPT NMR

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spectrum showed inverted signals at δ 68.8 and δ 61.5 which corresponded to Osubstituted and free C-6 of -D-Galp units, respectively, and at δ 67.0 and δ 63.3 from O-substituted and free C-5 of α-L-Araf units, respectively. Finally, the C-6 of Rhap signals from RG-I could be seen at δ 16.5. The assignments are in agreement with published literature data and were summarized in Table 4. (Capek, Matulová, Navarini, & Suggi-Liverani, 2010; Shakhmatov, Toukach, Kuznetsov, & Makarova, 2016).

ACCEPTED MANUSCRIPT 12 Regarding the hemicellulosic polysaccharides, they were present in the residue after WSP extraction. To solubilize them, the residue was treated with alkaline solution and centrifuged to give rise the cellulosic residue (8 g yield, 2.8% dry weight) and alkali soluble polysaccharides (ASP, 10 g yield, 3.6% dry weight) (Fig.1). We then applied the freeze-thaw treatment on ASP, where some polysaccharides can be fractionated by

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their solubility. In this procedure, the sample was frozen and then thawed at room

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temperature and the fractions recovered by centrifugation, giving rise to cold-water

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soluble (ASP-S, 5.7 g yield, 2% dry weight), and insoluble (ASP-I, 3.4 g yield, 1.2% dry weight) fractions (Fig. 1). This treatment was highly efficient, once a purified xylan

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was present in ASP-I. It showed 99.5% of xylose on monosaccharide analysis and its 13

C NMR spectrum (Fig. 3B, Table 4) had the five Xyl signals at δ 101.9 (C-1), δ 72.8

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(C-2), δ 74.2 (C-3), δ 75.8 (O-substituted C-4) and δ 63.4 (C-5), indicating the presence of a linear (14)-β-D-linked xylan. In fruits, this type of hemicellulosic polysaccharide

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has been reported only for pericarps of Argania spinosa (Habibi, Mahrouz & Vignon,

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2005), buriti (Cordeiro, De Almeida, & Iacomini, 2015) and açaí (Cantu-Jungles et al., 2017).

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The polysaccharides present in ASP-S fraction were mainly composed of Glc, Gal and Xyl. Few amounts of uronic acids, arabinose, mannose and fucose were also

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detected (Table 1). On gel permeation chromatography (Fig. 2B), two peaks were observable in this fraction, with relative Mw of 40 kDa and 5 kDa. The glycosidic linkage determination of the polysaccharides present in ASP-S was also performed by methylation analysis (Table 3). It showed methylated derivatives that indicated the presence of a xyloglucan, xylan and mannan. The xyloglucan backbone was represented by the 2,3,6-Me3-Glc-ol-acetate and 2,3-Me2-Glc-ol-acetate derivatives. Using the nomenclature of a single-letter code (Fry et al., 1993; Tuomivaara, Yaoi, O’Neill, &

ACCEPTED MANUSCRIPT 13 York, 2015), the side chains X (Xylp→), L (Galp-(1→2)-Xylp→), F (Fucp-(1→2)Galp-(1→2)-Xylp→) and S (Araf-(1→2)-Xylp→) may be present in jambo xyloglucan, due to the presence of 2,3,4-Me3-Xyl-ol-acetate, 2,3,5-Me3-Ara-ol-acetate, 2,3,4-Me3Fuc-ol-acetate, 2,3,4,6-Me4-Gal-ol-acetate, 3,4-Me2-Xyl-ol-acetate, 3,4,6-Me3-Gal-olacetate derivatives. It was also observed in methylation analysis 4-O- and 2,4-di-O-

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substituted Xylp units, and despite its already reported occurrence in xyloglucans of

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some sources (Busato et al., 2005; Tuomivaara et al., 2015), we believe that they may arise from a β-D-xylan, due to the downfield anomeric carbon of (1→4)-linked β-Xylp 13

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units seen at δ 101.6 in the

C NMR spectrum (Fig. 3C). Moreover, terminal GlcpA

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units may constitute the xylan side chains, due to the presence of 2,3,4,6-Me4-Glc-olacetate derivative. The derivatives that indicate the presence of small amounts of a

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mannan were 2,3,4,6-Me4-Man-ol-acetate and 2,3,6-Me3-Man-ol-acetate. The relative proportion of these polysaccharides in ASP-S were estimated based on the % of sugar

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linkage (Table 3) attributed to that polysaccharide, according to Pettolino, Walsh,

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Fincher and Bacic (2012). Xyloglucan content was estimated to be ~89%, while xylan and mannan were ~6% and 3%, respectively. Accordingly, ASP-S

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C NMR spectrum

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showed characteristic signals of these polymers (Fig. 3C, Table 4). Anomeric signals of the xyloglucan sugar units were at δ 102.2 (4- and 4,6-linked-β-D-Glcp), δ 98.9

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(terminal and 2-linked-α-D-Xylp), δ 104.3 (terminal β-D-Galp), δ 103.2 (2-linked-β-DGalp), and δ 99.4 (terminal α-L-Fucp). Signals of α-L-Araf units were not visible, probably due to their small amounts. Additionally, the signal of the C-6 of α-L-Fucp units appeared at δ 15.8 and an inverted downfield signal at δ 66.4 from substituted C-6 of β-D-Glcp units was observable in DEPT experiment. Anomeric signal of β-D-xylan was at δ 101.6 (4-β-D-Xylp), while that of β-D-mannan was at δ 99.9 (4-β-D-Manp). The

ACCEPTED MANUSCRIPT 14 assignments are in agreement with published literature data (Busato et al., 2005; Cordeiro et al., 2015; Guo et al., 2012). Finally, it is very well stablished that complex carbohydrates from plant cell walls consumption is associated with many health benefits, including beneficial effects on gut microbiota, mineral absorption, gut barrier function, fat metabolism and

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cholesterol levels, glycaemic and insulin responses, bile acid excretion, and reduced

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incidence of colon carcinoma (Tungland & Meyer, 2002). Moreover, some studies have demonstrated that polysaccharides are able to cross the small intestinal epithelium and

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can be found in blood and Peyer’s patches, where they can interact directly with

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immune cells (Vos, M’Rabet, Stahl, Boehm, & Garssen, 2007) and also modulate systemic immune responses (Ferreira et al., 2015). There is growing evidence that the

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observed biological responses can strongly be affected by the polysaccharide structural features (Ferreira et al., 2015; Popov & Ovodov, 2013; Vos et al., 2007). Thus, we

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evaluated the antitumor activity of WSP in an in vivo model of Ehrlich solid tumor.

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Both doses of 150 and 250 mg/kg WSP reduced significantly the tumor volume, similarly to the MTX (Fig. 4B, C). In the last day of treatment, the tumor suppression

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was in order of 66%, 72% and 80% for WSP 150, WSP 250 and MTX 2.5 mg/kg, respectively, compared with the vehicle tumor. Additionally, both 150 and 250 mg/kg

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dose WSP improved the body weight of mice compared with vehicle group (Fig. 4A). Interestingly, the group that gained less weight was that treated with MTX, an antitumor drug used in the treatment of several kinds of solid tumors. Despite studies already reported the antitumor activity of structurally different polysaccharides from several sources, such as mushrooms, algae, roots and leaves of medicinal plants (Zong, Cao & Wang, 2012), the observations of the in vivo antitumor effect of native pectic polysaccharides from edible fruits is still in the beginning. Saima

ACCEPTED MANUSCRIPT 15 et al. (2000) showed that a pectin from pulp of Feronia limonia fruits had significant in vivo Ehrlich ascites carcinoma cell growth inhibition in mice while a high-methoxyl homogalacturonan pectin, from the Hippophae rhamnoides berry could significantly inhibit the Lewis lung carcinoma growth in tumor-bearing mice and the induced antitumor effects might be mediated through immunological activities (Wang et al.,

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2015). Growth inhibition of Sarcoma 180 tumor transplanted in mice was promoted by

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a pectin from passion fruit (Passiflora edulis flavicarpa) (Silva et al., 2012). In vivo

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tumor growth inhibition was also presented by polysaccharides from cactus pear fruit (Liang, Liu & Cao, 2008) , longan (Dimocarpus longan) pulp (Zhong, Wang, He & He,

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2010) and fruits of Schisandra chinensis (Zhao et al., 2013). We believe that this is the first report of the antitumor activity of pectins isolated from edible jambo fruits. The

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action mechanism of WSP upon tumor cells and the toxicological effects upon nontumor cells still needs investigation, since this information may be helpful to clarify its

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health benefits. However, the induced antitumor effect might be mediated at least in part

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through immunological activities, once as observed before type II arabinogalactans present in WSP fraction could interact with macrophages and modulate cytokine (TNF-

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α, IL-1β and IL-10) secretion (Tamiello et al., 2018). Hirazumi and Furusawa (1999) also found a polysaccharide from fruit juice of Morinda citrifolia (noni) with antitumor

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activity in the Lewis lung (LLC) peritoneal carcinomatosis model. The polysaccharide did not exert significant cytotoxic effects on LLC cells in vitro, but indirectly it exerted significant cytotoxic effects against tumor cells by eliciting the tumoricidal activity of peritoneal exudate cells (mainly macrophages). Noni polysaccharides effectively enhanced their production of NO, TNF-α, IL-1β and IL-12 p70, which were considered important mediators of tumor cytostasis and/or cytotoxicity.

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Acknowledgements

This research was supported by Projeto Universal (Process 404717/2016-0) provided by CNPq foundation (Brazil) and CAPES. MI, AA and LMCC received

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fellowship from CNPq (Bolsa Produtividade em Pesquisa). The authors are grateful to

SC

RI

NMR Center of UFPR for recording the NMR spectra.

NU

References

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ACCEPTED MANUSCRIPT 24 Table 1 Monosaccharide composition of fractions obtained from the fruit of jambo (S. jambos) Monosaccharide composition (%)a Fractions Fuc

Ara

Xyl

Man

Gal

Glc

Uronic acidb

WSP

6.0

trc

22.2

trc

trc

16.5

4.7

50.6

WSP-CRd

5.0

trc

12.0

trc

trc

76.0

7.0

-

ASP-S

-

5.0

3.0

20.5

8.3

16.0

45.0

2.2

ASP-I

-

-

-

99.5

trc

-

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Rha

-

-

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

b

Determined spectrophotometrically using the m-hydroxybiphenyl method (Filisetti-Cozzi & Carpita,

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a

Trace amounts.

d

Sample was carboxyl-reduced by the carbodiimide method (Taylor & Conrad, 1972), prior to

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monosaccharide composition analysis.

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c

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1991).

ACCEPTED MANUSCRIPT 25 Table 2 Linkage types based on analysis of partially O-methyl alditol acetates from WSP.

WSP-CRb

Linkage typec

2,3,5-Me3-Araa

10.9

Araf-(1→

2,3,4-Me3-Ara

6.7

2,5-Me2-Ara

2.2

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Partially O-methylalditol

2,3-Me2-Ara

3.3

2-Me-Ara

1.4

2,3,4,6-Me4-Glc

3.3

a

acetate

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→3)-Araf-(1→

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→5)-Araf-(1→

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→3,5)-Araf-(1→

8.6

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2,3,4,6-Me4-Gal

Glcp-(1 Galp-(1

21.4

→4)-Galp-(1

6.4

→6)-Galp-(1

4.3

→3)-Galp-(1

3,4,6-Me3-Gal

3.0

→2)-Galp-(1

2,3-Me2-Gal

12.9

→4,6)-Galp-(1

2,4-Me2-Gal

4.2

→3,6)-Galp-(1

2-Me-Gal

5.2

→3,4,6)-Galp-(1

3,4-Me2-Rha

3.3

→2)-Rhap-(1

3-Me-Rha

3.0

→2,4)-Rhap-(1

2,3,6-Me3-Gal

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2,4,6-Me3-Gal

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2,3,4-Me3-Gal

a

Arap-(1→

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. Sample was carboxyl-reduced by the carbodiimide method (Taylor & Conrad, 1972), prior to methylation analysis. c Based on derived O-methylalditol acetates.

ACCEPTED MANUSCRIPT 26 Table 3 Linkage types based on analysis of partially O-methyl alditol acetates from ASP-S fraction. Partially O-methylalditol ASP-Sb

Linkage typec

2,3,5-Me3-Araa

2.6

Araf-(1→

2,3,4-Me3-Fuc

3.6

2,3,4-Me3-Xyl

6.6

2,3- / 3,4-Me2-Xyl

15.0d

3-Me-Xyl

0.8

2,3,4,6-Me4-Gal

5.3

3,4,6-Me3-Gal

10.2

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2,3,4,6-Me4-Man 2,3,6-Me3-Man

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→4)-Xylp-(1→ / →2)-Xylp-(1→

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→2,4)-Xylp-(1 Galp-(1→ →2)-Galp-(1→ Glcp-(1→

18.5

→4)-Glcp-(1→

1.0

→6)-Glcp-(1→

31.7

→4,6)-Glcp-(1→

0.5

Manp-(1→

3.2

→4)-Manp-(1→

2,3,4-Me3-Ara = 2,3,4-tri-O-Methylarabinitolacetate, etc. b % of peak area of O-methylalditol acetates

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a

Xylp-(1→

1.1

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2,3,6-Me3-Glc

2,3-Me2-Glc

Fucp-(1→

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2,3,4,6-Me4-Glc

2,3,4-Me3-Glc

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acetate

relative to total area, determined by GC-MS. Sample was carboxyl-reduced by the carbodiimide method (Taylor & Conrad, 1972), prior to methylation analysis. c Based on derived O-methylalditol acetates. d The ratio of 2,3-/3,4-Me2-Xyl was estimated by their fragmentation patterns in GC-MS was 1:2.75

ACCEPTED MANUSCRIPT 27 Table 4 13

C-Resonances observed in the polysaccharide fractions obtained from jambo (S.

jambos) fruits. Linkage

Resonance (δ)

Carbon

C-1

100.0

units

C-2 and C-3, overlapped

68.0

C-4 (O-substituted)

78.7

C-5

70.5

170.9

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C-6 Unesterified (1→4)-α-D-GalpA C-1

99.3

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C-2 and C-3, overlapped

68.0

C-4 (O-substituted)

78.7

C-5

71.6

C-6

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units

175.1

O−CH3

52.8

Acetyl

O=C−CH3

20.5

α-L-Araf units

C-1

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Methyl

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α-L-Rhap

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-D-Galp units

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Esterified (1→4)-α-D-GalpA

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WSP fraction (pectic polysaccharides)

107.6, 108.4, 109.1 and 109.4

C-5 (free)

63.3

C-5 (O-substituted)

67.0

C-1

103.5

C-6 (free)

61.5

C-6 (O-substituted)

68.8

C-6

16.5

C-1

101.9

C-2

72.8

C-3

74.2

C-4 (O-substituted)

75.8

C-5

63.4

ASP-I fraction (xylan) (14)-β-D-Xylp units

ASP-S fraction (xyloglucan+xylan+mannan) (1→4)- and (1→4,6)-β-D-Glcp

C-1

102.2

ACCEPTED MANUSCRIPT 28 66.4

terminal and (1→2)-α-D-Xylp

C-1

98.9

terminal β-D-Galp

C-1

104.3

(1→2)-β-D-Galp

C-1

103.2

terminal α-L-Fucp

C-1

99.4

C-6

15.8

(14)-β-D-Xylp units

C-1

101.6

(14)-β-D-Manp units

C-1

99.9

AC

CE

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D

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SC

RI

PT

C-6 (O-substituted)

ACCEPTED MANUSCRIPT 29 Fig. 1. Scheme of extraction and fractionation of pectic (WSP fraction) and hemicellulosic (ASP fraction) polysaccharides from jambo (S. jambos) fruits. Legend: dw= dry weight.

Fig. 2. GPC elution profile of (A) pectic polysaccharides present in WSP fraction and

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(B) hemicellulosic polysaccharides present in ASP-S fraction from jambo fruits.

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Refractive index detector. Elution volume of dextran standards of molecular weight

Fig. 3.

13

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employed to construct the calibration curve.

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487kDa, 266kDa, 124kDa, 72.2kDa, 40.2kDa, 17.2kDa and 9.4kDa (left to right) were

C NMR spectra of (A) pectic polysaccharides (WSP), in D2O; (B) ASP-I

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fraction, in DMSO-d6 and (C) ASP-S fraction, in DMSO-d6 obtained from jambo fruits. Inverted signals in DEPT-135 experiment are market with asterisk. Experiments were

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performed at 70 °C.

Fig. 4. Body weight (A), tumor volume (B) and area under the curve (AUC) of tumor

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growth (C). Ehrlich tumor-bearing mice were treated with vehicle (Veh), WSP fraction (100, 150 or 250 mg/kg) or methotrexate (2.5 mg/kg) during 21 days. The data are

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presented as mean  SEM, and were compared by two-way ANOVA (A, B) or one-way ANOVA (C) followed of Bonferroni´s multiple comparisons test. Symbols: *p<0.05, **p<0.01, ***p<0.001 compared with Veh group.

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Syzygium jambos (L.) Alston fruit Freeze-dried and defatted Aq. extraction, 100 °C

Water-soluble polysaccharides WSP (12 g, 4.3% dw)

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ASP-I Insoluble (3.4 g, 1.2% dw)

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CE

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ASP-S Soluble (5.7 g, 2% dw)

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Freeze-thawing

Cellulosic Residue (8 g, 2.8 % dw)

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Alkali soluble polysaccharides ASP (10 g, 3.6% dw)

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NaOH 10%

Figure 1

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Residue

ACCEPTED MANUSCRIPT

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SC

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31

Figure 2

ACCEPTED MANUSCRIPT 32

A *

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*

*

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*

* *

AC

C

CE

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D

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B

*

Figure 3

* *

ACCEPTED MANUSCRIPT

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PT

33

Figure 4