Exopolysaccharides from Klebsiella oxytoca : anti-inflammatory activity

Exopolysaccharides (EPS) produced by Klebsiella oxytoca are of environmental, pharmaceutical, and medicinal interest. However, studies about the anti-inflammatory activity of EPS produced by this microorganism still remain limited. The aim of this study was to produce, characterize, and evaluate the anti-inflammatory activity of EPS from K . oxytoca in a pleurisy model. Colorimetric analysis revealed that precipitated crude exopolysaccharides (KEPSC) and deproteinated exopolysaccharides (KEPS) present high levels of total carbohydrates (65.57% and 62.82%, respectively). Analyses of uronic acid (7.90% in KEPSC and 6.21% in KEPS) and pyruvic acid (3.01% in KEPSC and 1.68% in KEPS) confirm that the EPS are acidic. Gas chromatography-mass spectrometry analyses demonstrated that the EPS consisted of rhamnose (29.83%), glucose (11.21%), galactose (52.45%), and mannose (6.50%). The treatment of an experimental pleurisy model in rats through subcutaneous administration of 50, 100, 200, and 400 mg/kg of KEPS decreased both the volume of inflammatory exudate and the number of leukocytes recruited to the pleural cavity. The present data showed that EPS production by K . oxytoca using the method described is easy to perform and results in a good yield. In addition, we show that KEPS exhibit anti-inflammatory activity when administered subcutaneously in rats.

rhizosphere (Hirota et al., 1978). Strains of K. oxytoca are also known to produce EPS of environmental and pharmaceutical interest (Baldi et al., 2001;Sugihara et al., 2000). Furthermore, according to Sugihara et al. (2000;, the AZ9 polysaccharide produced by K. oxytoca has been shown to exhibit important immunosuppressive activity in experimental models of chronic inflammatory and allergic diseases.
However, to the best of our knowledge, no studies have described the anti-inflammatory activity of EPS produced by K. oxytoca. Thus, the aim of this study was to produce EPS from K. oxytoca and to evaluate the antiinflammatory activity in a pleurisy model in rats.

Strain and medium
The microorganism K. oxytoca was isolated from the rhizosphere of Aspidosperma polyneuron and identified according to biochemical tests (Celloto et al., 2012). The strain was also phylogenetically identified by partial 16S rDNA sequence analysis and the GenBank data homology research search result was 99% according to the methodology described by Nogueira et al. (2004) and Procópio et al. (2009).
The K. oxytoca strain (1.5 × 10 8 CFU/mL standard bacterial suspension) was cultured in liquid medium (10 mL/L inoculum) as described by Sugihara et al. (2001). Cultures were grown in Sugihara medium (SM) (5.0 g/L K 2 HPO 4 , 0.5 g/L MgSO 4 .7H 2 O, 2.0 g/L polypeptone, and 20 g/L glucose) in 100 mL Erlenmeyer flasks at 28 °C with shaking at 200 rpm for 48 h. The bacterial culture was then transferred aseptically to 2000 mL Erlenmeyer flasks containing 900 mL of SM, incubated at 28 °C for 120 h then maintained at 4 °C prior to EPS isolation.

Isolation and purification of K. oxytoca EPS
Bacterial cells were separated by centrifugation at 12,000 × g for 20 min at 5 °C. The supernatant was collected and concentrated to 100 mL in a rotary evaporator at 50 °C. Following this, the concentrated solution containing EPS was precipitated from the clear supernatant by adding three volumes of cold acetone and maintained at 4 °C for 48 h. The crude exopolysaccharides from K. oxytoca (KEPSC) were obtained by filtration, lyophilized and weighed to calculate the yield (Kazy et al., 2002;Sugihara et al., 2000;Sugihara et al., 2001;Sugihara et al., 2002). Once isolated, the KEPSC solution was deproteinized with trichloracetic acid (20%, w/v) at a ratio of 1:1, maintained at 4 °C for 2 h, and then centrifuged (12,000 × g for 20 min at 5 °C). Deproteination and precipitation were performed twice to remove remnant proteins. The polysaccharides were dissolved in bidistilled water and dialyzed (cellulose membrane, MWCO: 12,000, Sigma-Aldrich, St. Louis, MO, USA) at 4 °C for 4 days. Finally, the deproteinated exopolysaccharides (KEPS) were lyophilized, weighed to calculate the yield and stored at 4 °C until further use (Ruas-Adiedo, Creyes-Gavilan, 2005;Marcial et al., 2013).

Colorimetric analyses
The total sugar content was determined using the phenol sulfuric acid method (Dubois et al., 1956); determination of reducing sugars was performed according to the spectrophotometric method of p-hydroxybenzoic acid hydrazide (Lever, 1972); uronic acid was assayed using the carbazol sulfuric acid method (Chaplin, Kennedy, 1994); pyruvic acid was analyzed using the colorimetric method of 2,4-dinitropheny-hydrazine reagent (Sloneker, Orentas, 1962); and total protein was quantified using the Hartree method (Hartree, 1972).

Analyses of the monosaccharide composition of KEPSC
Neutral monosaccharide components of the KEPSC (1 mg) were determined by gas chromatography-mass spectrometry. Their ratios were determined by hydrolysis with 1 mL of 2 M trifluoroacetic acid for 8 h at 100 °C, followed by conversion to alditol acetates by successive NaBH 4 (1 mg) reduction and acetylation with 1 mL acetic anhydride-pyridine (1:1, v/v) at room temperature for 14 h. The resulting alditol acetates were extracted with chloroform. The alditol acetate analysis was carried out using a Varian model 3300 gas chromatograph linked to Helium was used as the carrier gas in the quantitative analysis. The alditol acetates were identified by their retention times and typical electron impact breakdown profiles compared with standards. The results were given as weight percentages, which take into account the coefficients from the detector response (York et al., 1985).

1
H and 13 C nuclear magnetic resonance spectroscopy (NMR) 1 H and 13 C NMR spectroscopy was performed using a Varian spectrometer, Mercury Plus model, operating at 300.06 MHz for 1 H and 75.45 MHz for 13 C. The 1 H and 13 C NMR spectra of the samples of polysaccharides were obtained using deuterated water as a solvent. The chemical shifts (δ) were expressed in ppm and compared with data obtained from the literature.

Evaluation of the anti-inflammatory activity of EPS from K. oxytoca on carrageenan-induced pleurisy
The experimental protocol was approved by the Ethics Committee for Animal Experimentation of the State University of Maringá (012/2013). Groups of Wistar rats (n = 6-7 per group), weighing 200-220 g, were kept under controlled temperature (22 ± 2 °C) and a light/dark cycle of 12 h with water and food ad libitum. The animals were anesthetized using a solution of 10% ketamine and 2% xylazine at a dose of 40 mg/ kg and pleurisy was induced by injecting 0.25 mL of a carrageenan suspension (Cg; 200 μg) into the intra-pleural cavity (Vinegar, Truax, Selph, 1976;Goodman et al., 1993). In this experiment, the crude EPS obtained from the Sugihara culture (KEPSC) and KEPS were dissolved in water and saline, respectively. Groups of rats fasted for 12 h were treated orally (gavage) at a single dose of 400 mg/kg KEPSC and KEPS, or subcutaneously at a dose of 50, 100, 200, or 400 mg/kg KEPS, 1 h before the induction of pleurisy. Another group of rats were treated with indomethacin (Indo) at a dose of 5 mg/kg (positive control), or water or saline (negative controls), by the same routes of administration.

Statistical analyses
The results of the anti-inflammatory and chemical assays were expressed as the mean ± standard error of the mean. The data obtained in the anti-inflammatory and chemical assay were evaluated with GraphPad Prism 5.0 and Origin 7.0 ® software, respectively, and analyzed using analysis of variance followed by Tukey's test. A value of p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION
EPS produced by K. oxytoca was isolated from the Sugihara culture medium at yields of 1.18 ± 0.20 and 0.96 ± 0.10 g/L (dry weight/volume) for KEPSC and KEPS, respectively. Table I shows the contents of total carbohydrate, reducing sugars, uronic acid, pyruvic acid, and protein.
The low concentration of reducing sugars and the high content of total carbohydrate present in the KEPS indicated that the carbohydrates are present in the structural form of polysaccharides. Analyses of uronic acid and pyruvic acid confirmed that the EPS extracted from K. oxytoca are acidic. It is likely that these differences in composition are responsible for the different rheological behavior of the solutions, which provide strong gels (Lamb et al., 2004;Junhua et al., 2013). Gas chromatography revealed that the monosaccharide composition of the crude EPS consisted of rhamnose (Rha; 29.83%), glucose (Glc; 11.21%), galactose (Gal; 52.45%), and mannose (Man; 6.50%); thus, galactose and rhamnose are the major components. These data are in agreement with those found by Dlamini et al. (2007) and Feng, Li, Chen (2009), who used different media cultures for the production of EPS from K. oxytoca.
The 1 H NMR spectrum of KEPS contained seven signals in the anomeric region between 5.25 and 4.50 ppm (labeled A-G in order of decreasing chemical shift, Figure 1 A, B); these were assigned by monosaccharide composition through comparison with the structure and spectroscopic data of exopolysaccharide isolated from K. oxytoca BAS-10 described by Leone et al. (2007). The signals of anomeric hydrogens δ 5.23, 5.16, 5.14, and 5.04 were suggested to be α-Rha di-substituted (2 → 1, A and/or B), α-Rha di-substituted (3 → 1, C), and α-Rha tri-substituted (3 → 1,4, D), respectively. The signals at δ 4.79 and 4.69 were suggested to be β-GlcA terminal (E) and 4-β-GlcA (F), respectively. The signal at δ 4.55 was suggested to be 3-β-Gal (G). Moreover, between 1.11 and 1.31 ppm, the signals of four methyl groups were observed and each was suggested to be the 6-deoxy position of a Rha unit.
Colorimetric determination of glucuronic acid present in KEPS was 6.20% and its presence was confirmed by the carboxyl group signal at 172.95 ppm. The presence of C-6 (CH 3 ) of rhamnose units was evidenced by signals at δ 16.83 and 16.69. The signal at 61.18 ppm (C-6) represents galactose present in the biopolymer.
This partial chemical characterization of EPS showed that their structures could be related to the EPS isolated from K. oxytoca BAS-10 by Leone et al. (2007).

Anti-inflammatory activity of EPS from K. oxytoca
Cg-induced pleurisy in animals is an effective model that has often been used to investigate the pathophysiology of acute inflammatory response and to evaluate the anti-inflammatory activity of numerous compounds. The intrapleural injection of Cg in rats induces an acute inflammatory response characterized by a significant increase in the volume of pleural inflammatory exudate and the cells that migrate into the cavity when compared to normal animals (without injection of Cg). Polymorphonuclear leukocytes are the dominant cell type, which are recruited to the site up to 12 h after injection, after which they are replaced by mononuclear leukocytes (Amdekar et al., 2012;Adebayo et al., 2012).
Oral administration of 400 mg/kg EPS (KEPSC, KEPS) prior to Cg-induced pleurisy did not significantly change the volume of inflammatory exudate or the number of recruited leukocytes into the pleural cavity ( Figure 2; Table III). Possible explanations for this include: 1) when administered orally, EPS (KEPSC, KEPS) isolated from K. oxytoca may be degraded in the acidic environment of the stomach, or 2) that the high molecular weight polysaccharides may not be absorbed in the systemic circulation via oral administration (Wang et al., 2017).   Pleurisy was induced by intrapleural carrageenan injection (Cg -200 µg/cavity) in rats (n = 6 -7) 1 h following oral administration of polysaccharides at 400 mg/kg or saline (Cg). Indomethacin (Indo; 5 mg/kg) was administered subcutaneously as an antiinflammatory reference drug (positive control). N = normal animals that received no pleural injection of carrageenan; TL = total leukocytes; MN = mononuclear leukocytes; PMN = polymorphonuclear leukocytes. Each point represents the mean ± standard error of the mean of the number of leukocytes recruited into the pleural cavity 4 h following injection of carrageenan. a p < 0.05 compared with normal rats (ANOVA, Tukey's test).
FIGURE 3 -KEPS effect on the volume of pleural inflammatory exudate. The pleurisy was induced by intrapleural carrageenan injection (Cg -200 µg/cavity) in rats (n = 6-7), 1 h following subcutaneous administration of the EPS at 50, 100, 200, 400 mg/ kg or saline (Cg). Indomethacin (Indo, 5 mg/kg) was subcutaneously administered as an anti-inflammatory reference drug (positive control). N = normal animals that received no pleural injection of carrageenan. Each point represents the mean ± standard error of the mean of the volume of exudate 4 h after injection of carrageenan. a p < 0.05 when compared with normal rats; b p < 0.05 when compared with group of rats injected with carrageenan and untreated (Cg) (ANOVA, Tukey's test). Literature data report that K. oxytoca EPS isolated from the soil, and produced under the same cultivation conditions as that employed in this work, have an average molecular weight of approximately 200 kDa (Sugihara et al., 2000;Sugihara et al., 2001) and KEPSC and KEPS are probably high molecular weight macromolecules.
The treatment of rats, by subcutaneous administration, with KEPS significantly reduced both the volume of the pleural inflammatory exudate (edema) and the number of recruited leukocytes into the pleural cavity (polymorphonuclear and mononuclear) ( Figure  3, Table IV). The inhibitory effect on edema was similar for all four doses tested ( Figure 3). However, KEPS treatment at doses of 50, 100, 200 and 400 mg/ kg reduced the number of recruited leukocytes by 68%, 84%, 83% and 90%, respectively. No significant difference was found between the 100 and 200 mg/kg doses of KEPS (Table IV).
Although cell migration is important for the body's defense in inflammatory processes, heightened mobilization to the site of injury can damage tissue depending on the activity of metalloproteinases and the generation of reactive oxygen/nitrogen species (Paula-Neto et al., 2011). Thus, a decrease in the number of recruited leukocytes into the pleural cavity following treatment with KEPS indicates potential clinical benefits of this compound by reducing the extent of the injury.
Corroborating our data, previous studies utilizing other experimental models (in vitro and in vivo) have shown that exopolysaccharides isolated from a variety of microorganisms (bacteria and fungus) have antiinflammatory activity (Du et al., 2016;Du et al., 2017;Gangalla et al., 2018). These studies have also shown that the anti-inflammatory response can be associated with an inhibitory action on enzymes and mediators involved in the inflammatory response (COX-2, 5-LOX, NO, cytokines) (Zha et al., 2015).
In both experimental assays, indomethacin treatment (oral and subcutaneous administration), which was used as an anti-inflammatory drug reference, caused a reduction in the exudate volume but did not change the total number of recruited leukocytes. This can be explained by the fact that indomethacin was given at a low dose (5 mg/kg). Previous reports have shown that the effect of indomethacin on leukocyte migration depends on the administered dose and the experimental model used (Higgs et al., 1980;Jain, Parmar, 2011).
It is important to highlight that in the present study, in the evaluation of anti-inflammatory activity administered subcutaneously, only KEPS was used, as it is a more purified compound than KEPSC, and therefore less likely to interfere with the inflammatory response and confound interpretation of the results (Hamuro et al., 2017).
Overall, our data showed that the production of EPS by K. oxytoca is viable using the method described in this study, easy to perform, and results in a good yield. In addition, we showed that EPS exhibit important antiinflammatory activity when administered subcutaneously in rats, and that the choice of drug administration route is a crucial factor in the experimental model used (Hamuro et al., 2017).

CONCLUSION
Overall, the deproteinated exopolysaccharides from K. oxytoca exhibited anti-inflammatory activity. Subcutaneous administration of KEPS decreased both the volume of inflammatory exudate and the number of leukocytes recruited to the pleural cavity in an experimental rat pleurisy model.
It could be suggested that EPS from K. oxytoca may be a candidate for the development of new therapeutic The pleurisy was induced by intrapleural carrageenan injection (Cg; 200 µg/cavity) in rats (n = 6-7) 1 h after the respective treatments with polysaccharide concentrations of 50, 100, 200, or 400, mg/kg or saline (Cg). Indomethacin (Indo; 5 mg/kg) was administered subcutaneously as an anti-inflammatory reference drug (positive control). N = normal animals that received no pleural injection of carrageenan; TL = total leukocytes; MN = mononuclear leukocytes; PMN = polymorphonuclear leukocytes. Each point represents the mean ± standard error of the mean of the number of leukocytes recruited into the pleural cavity 4 h following injection of carrageenan. a p < 0.05 compared with normal rats; b p < 0.05 compared with rats injected with carrageenan and untreated (Cg); c p < 0.05 compared with rats injected with carrageenan and treated with KEPS 50 mg/kg; d p < 0.05 compared with rats injected with carrageenan and treated with KEPS 100 and 200 mg/kg (ANOVA, Tukey's test).
anti-inflammatory agents, nevertheless, further studies are warranted to isolate and to identify their mechanisms in modulating the inflammatory process.