The Tea and Polysaccharide Fractions of Ximenia americana Stem Barks Inhibits Inflammatory Parameters Induced by Zymosan in Mice

Ximenia americana or “ameixa-do-sertão” is a plant used in the folk medicine of Northeast-Brazil in inflammatory and painful processes. The literature has shown anti-inflammatory and antinociceptive activities for the aqueous or hydroalcoholic extracts and polysaccharide fractions of X. americana stem barks. This study investigated the anti-inflammatory effect of polysaccharide fractions and tea prepared from X. americana stem barks in mice models of paw edema and peritonitis induced by zymosan. Tea (50,100, 150 mg/kg; p.o.) and polysaccharide fractions (FI, FII: 1 mg/kg; i.v.), containing carbohydrate (44.8, 25.0, 44.4%) and polyphenols (28.3, 0.78, 1.50%), respectively, were administered 1 h before zymosan (500 g; s.c. or i.p). FT-IR analysis of the tea showed bands indicating C=O and C-O groups vibration, typical of phenolic compounds. Zymosan-induced paw edema (AUC: 355.0 ± 38.1) was inhibited by the tea by 38% (100 mg/kg) and 36% (150 mg/kg). FI and FII reduced the edema by 47% (933.8 ± 158.1 AUC) and 30% (1263 ± 124.8 AUC), respectively. Zymosan i.p. induced hypernociception (4.7 ± 0.3 g) and increased the peritoneal leukocyte migration (10979 ± 1475 cells/l). FII increased by 66% the nociceptive threshold (13.87 ±1.59 g) and reduced by 63% leukocyte migration (3993 ± 439.1 cells/l). FII decreased plasma protein by 64% (92.6 ± 4.5 vs. zymosan: 119.6 ± 5.4 g/ml) and MDA by 63% (57.5 ± 3.5 vs. zymosan: 88.9 ± 6.3 g/ml). The tea, containing polysaccharides and polyphenols, and polysaccharide fractions FI/FII of X. americana barks possess antiinflammatory effect in mice models of acute inflammation induced by zymosan.


Introduction
Natural compounds isolated from medicinal plants have been widely investigated due to its anti-inflammatory and analgesic properties, allowing the validation of the popular use and directing several clinical researches. These compounds can be obtained from various plant parts, such as stem barks, leaves, fruits, seeds, roots, and are popularly used as infusion, maceration, decoction, etc (Balunas & Kinghorn, 2005).
Among plant compounds, polysaccharides are highly bioavailable and have low or no toxicity (Ovodov, 1998). Regarding its biological activities in the inflammatory process, the polysaccharide fractions obtained from pods of Caesalpinia ferrea (jucá ) and seeds of Azadirachta indica (neem) inhibited leukocyte migration and acute phase inflammatory mediators in rats (Pereira et al., 2012a;2012b). In nociceptive processes, Thladiantha dubia fruit polysaccharides reduced the abdominal contortions elicited by acetic acid (Wang et al., 2011).
Ximenia americana L. (Olacaceae), popularly known as ameixa-do-sertã o, is widely distributed in Northeast Brazil (Le et al., 2012), being its barks prepared as decoction ("tea") to treat inflammatory and painful conditions. Experimental studies demonstrated the antiinflammatory effect for the hydroethanolic bark extracts in acute and chronic inflammation models (da Silva et al., 2018). The aqueous extract of X. americana roots reduced paw edema, neutrophil migration and vascular permeability induced by carrageenan in mice (Olabissi et al., 2011). The antinociceptive activity of the aqueous extract obtained from stem barks was also demonstrated in the inflammatory phase of the formalin test (Soro et al., 2009). In addition, total polysaccharide and fractions isolated from X. americana presented inhibitory effect in inflammatory models (Silva-Leite et al., 2017. Moreover, the inhibitory effect on neutrophil migration was demonstrated in the gastritis model for the tea of X. americana, prepared following the same protocol used in the present study (da Silva-Pantoja et al., 2018).
This study aimed to evaluate the anti-inflammatory activity of the tea and polysaccharides obtained from X. americana stem barks in the models of paw edema and peritonitis induced by zymosan in mice.

Experimental Design
The anti-inflammatory and anti-hypernociceptive activities of the tea preparation and polysaccharide fractions of X. americana stem barks were evaluated after stimulation of mice paws or peritonium with the flogistic agent zymosan for analysis of the following parameters: edema, protein leakage, leukocyte infiltrate, oxidative stress markers (malondialdehyde) and hyperalgesia. Control animals received sterile saline by subcutaneous (s.c.) route in the paws or intraperitoneal (i.p.).

Plant
Barks of X. americana were collected in Custódio county (Quixadá -CE) and the plant exsiccate was deposited at FECLESC/Quixadá and Herbarium Prisco Bezerra -UFC (nº 46794). In accordance to the Brazilian Federal Law No. 13123/2015, the assessment activity was registered in the Brazilian System for the Management of the Genetic Heritage and the Associated Traditional Knowledge (SISGEN -A8B2C79).

Tea Preparation, Extraction and Isolation of Polysaccharides of X. americana
The tea was obtained by decoction of X. americana stem barks in distilled water (100º C/5 min) and lyophilization before used in the experimental protocols (da Silva-Pantoja et al., 2018).

Fractioning, Biochemical and Structural Caracterization of Tea and Polysaccharides of X. americana
TPL-Xa was dissolved in distilled water (1:2, w/v) and applied to chromatographic column (resin: DEAE-cellulose), equilibrated and washed with distilled water, and eluted with NaCl (0.1 and 0.25M). Polysaccharide fractions were monitored for the carbohydrate content by the phenol-sulfuric acid method (Silva-Leite et al., 2017;Dubois et al., 1956).
The tea was analyzed by infrared spectroscopy (FT-IR). For this, samples (2 mg) were mixed with powdered potassium bromide and pressed into a disc in the medium infrared region at 500-4000 cm -1 , with resolution of 3 cm −1 (Perkin Elmer Spectrum 100, USA).

Animals
Swiss mice (25-35 g) were maintained with free Access to food and water and controlled environmental conditions (12/12 h light/dark cycle, temperature of 25 °C). The experimental protocols were approved by the Animal Care and Use Committee of the State University of Ceara (UECE n°: 4153018/2018).

Paw Edema Model
Paw edema was induced by zymosan (500 g; s.c.) and measured by hydroplethysmometry at Journal of Biology and Life Science ISSN 2157-6076 2020 zero time and selected time intervals thereafter (0.5, 1 -5 h). Control animals received saline (0.1 mL/10 g body mass). Results were expressed as the variation in paw volume (l) or area under curve (AUC).
For evaluation of mechanical visceral hypernociception, mice were placed in clear acrylic box 15 min before the test for adaptation to the environment. The nociceptive threshold of the animals was tested by application of a gradual pressure (g) using polypropylene tip, coupled to a hand-held force transducer (Electronic Von Frey Anesthesiometer; Insight). Hypernociception was also evaluated at baseline (time zero) (Le Bars et al., 2001).

Statistical Analysis
Results were presented as mean ± SEM. Analysis of variance (ANOVA) followed by Bonferroni's test were applied. P values< 0.05 were considered significant.
FTIR spectra of X. americana tea revealed absorption peaks around 3375 cm -1 and 2925 cm -1 corresponding to stretching of O-H and C-H, respectively (Sun et al., 2009). Peaks at 1442 cm -1 , 1525 cm -1 and 1612 cm -1 were also detected, indicating presence of uronic acid (group COOH) . Absortion regions between 1500 and 700 cm −1 correspond to carbohydrate range (Karnik et al., 2016), in which peak at 1113 cm −1 , characterizes glycosidic bonds (Xu et al., 2016). The peak observed at 1442 cm -1 was attributed to the elongation of the C=O group of aromatic rings, typical of phenolic compounds and the peak at 1296 cm -1 to C-O in the flavonoids found in the tea (Bagheri et al., 2013) (Figure 1).

Figure 1. X. americana tea analysis by infrared spectroscopy (FTIR)
The spectral region examined was from 500 to 4000 cm −1 , with resolution of 3 cm −1 .

FII Inhibits Inflammatory Parameters and Oxidative Stress Makers in the Peritonitis Induced
Zymosan i.p. (500 (g) elicited hypernociception in mice, observed by the behavioral response of abdomen withdrawal in response to the pressure applied, reducing the nociceptive threshold compared to the control (zymosan: 4.7 ± 0.3 g vs. saline: 14.7 ± 0.9 g). FII increased by 66% the animal's nociceptive threshold, restoring the values close to the controls (FII: 13.87 ±1.59) ( Figure 3A).

Discussion
The present study demonstrates the obtention and partial chemical characterization of deproteinized polysaccharide fractions and tea from X. americana stem barks, along with its anti-inflammatory activity in the mice models of paw edema and peritonitis induced by zymosan. ISSN 2157-6076 2020 The tea presented high content of total carbohydrates and polyphenols with low protein content. Previous studies had already demonstrated this biochemical pattern in teas and polysaccharide fractions obtained from plants (Zhu et al., 2019, Yang et al., 2018, Chen et al., 2018.

Journal of Biology and Life Science
TPL-Xa and FII fraction presented carbohydrate content similar to the tea, but different uronic acid content, being FII superior in the ratio uronicacid/hexoses, and substantial lower levels of polyphenols compared to the tea. It is well reported in the literature that polysaccharide fractions of several plants present similar biochemical composition compared to the decoction extracts (teas) from which they were extracted (Chen et al., 2016).
The tea FTIR spectra demonstrates peaks related to carbohydrate range, uronic acid (group COOH), phenolic compounds and flavonoids (Sun et al., 2009;Karnik et al., 2016;Li et al., 2014;Bagheri et al., 2013). Our data reinforce the suggestion that despite of different extraction methods the total carbohydrates content does not significantly change. These results corroborate the absorption ranges found for the non-deproteinated FII fraction extracted from X. americana barks, already published by our research group (Silva-Leite et al., 2017).
The animal's treatment p.o. with the tea and i.v. with the polysaccharide fractions of X. americana inhibited the zymosan-induced paw edema. Zymosan, a polysaccharide of yeast cell wall, induces an inflammatory response involving dose and time-dependent edema, accompanied by severe leukocyte infiltrate (Suo et al., 2014). Studies had shown the antiedematogenic effect of plant polysaccharide extracts, such as those from Caesalpinea ferrea, Azadirachta indica (Pereira et al., 2012a;2012b) and X. americana (roots aquous etanol extract) (Olabissi et al., 2011) in carrageenan-induced edema, which is also accompanied by protein exudate and intense leukocyte infiltrate (DiRosa et al., 1971).
Zymosan, administered i.p. induces acute inflammation characterized by severe leukocyte infiltrate, increase in vascular permeability, hypernociception and inflammatory mediators (Leite et al., 2015). The animal's treatment with FII reduced neutrophil migration and vascular permeability, seen by the decrease in protein leakage, and also increased the abdominal nociceptive threshold, showing significant action on inflammatory pain. In previous studies, similar results had been reported for other plant polysaccharides (Pereira et al., 2012a;2012b;Wang et al., 2011;Olabissi et al., 2011;Silva-Leite et al., 2017).
Moreover, our results showed that the polysaccharide fraction FII was able to reduce MDA levels in the peritoneal fluid of animals stimulated with zymosan. Malondialdehyde is one of the main product of lipid peroxidation, therefore an important oxidative stress determinant in clinical situations, such as inflammation (Giera et al., 2012). Accordingly, antioxidant and free-radical scavenging activities had been described for plant polysaccharides (Chen et al., 2004;Liu et al., 2011;Mengome et al., 2014;Lin et al., 2017).
The chemical analysis showed high carbohydrate content in the tea and polysaccharide fractions composition, especially FII, which reinforce the supposition that both the anti-inflammatory effect of tea and fractions, as well as the antioxidant effect of FII may be accounted for the polysaccharides. In addition, the tea also presents considerable content of polyphenols, substance well described for its anti-inflammatory and antioxidant properties (Xu et al., 2017).
In conclusion, the polysaccharide fractions and the tea, containing polysaccharides and polyphenols, obtained from X. americana stem barks possess anti-inflammatory effect in the mice models of paw edema and peritonitis induced by zymosan via reduction of edema, protein exudate, neutrophil migration and inflammatory pain. These effects may validate the popular use of X. americana as anti-inflammatory.