Polysaccharide extract of Mimosa tenuiflora stem barks stimulates acute inflammatory response via nitric oxide

Mimosa tenuiflora (Mimosaceae) or “jurema-preta” is well distributed in the northeast Brazil, being popularly used to treat skin lesions, burns and inflammation. The healing effect of the alcoholic extract prepared with its barks corroborates the popular use. This study aimed to evaluate the inflammatory response of polysaccharides extracted from M. tenuiflora barks (EP-Mt) by methanol/NaOH and ethanol precipitation. Inflammatory activity was assessed in rat models of acute inflammation (paw edema and peritonitis), by the following parameters: edema, vascular permeability, leukocyte migration, myeloperoxidase activity and pharmacological modulation of nitric oxide and prostaglandins. EP-Mt presented 3.8% yield, 41% carbohydrate and 0.34% protein. EP-Mt (0.01, 0.1, 1.0 mg kg) injected by subcutaneous route elicited paw edema that lasted from 30-420 min, with maximal effect at 1 mg kg (40x vs. saline), and was inhibited by L-NAME (52%) and dexamethasone (26%). EP-Mt (1 mg kg, via intraperitoneal) stimulated leukocytes migration (2.2x), mainly neutrophils (6.5x) and MPO activity (96%). The leukocyte migration elicited by EP-Mt was inhibited by dexamethasone (39%) and L-NAME (38%). EP-Mt containing high carbohydrate content induces acute inflammation via nitric oxide, which open perspectives of application in pathological conditions of immunosuppression.


Plant material
Barks of M. tenuiflora were collected directly in nature, in the District of Daniel de Queiroz-Quixadá, Ceará, Brazil.The plant was identified by the biologist Regis C. Paulino and its exsiccate (n° 46085) was deposited at the Botanical Laboratory (Universidade Estadual do Ceará) and Herbarium Prisco Bezerra (Universidade Federal do Ceará).

Animals
Wistar rats (150-250 g) were maintained under 12/12 h light/dark cycle, at 25°C, with free access of food and water.Experimental protocols were in compliance with the Guide for the Care and Use of Laboratory Animals of the US Department of Health and Human Services (NIH n° 85-23, 1985) and approved (n° 10130208-8/40) by the Animal Care and Use Committee of the Universidade Estadual do Ceará, Brazil.

Inflammation models
Paw edema was induced by subcutaneous (s.c.) intraplantar injection of EP-Mt (0.01, 0.1 and 1 mg kg -1 ) as inflammatory stimulus.Control animals received sterile saline (0.9% NaCl).Dexamethasone (5 mg kg -1 ) or L-NAME (25 mg kg -1 ) was injected by intravenous route (i.v.) 30 min prior to EP-Mt.The edema was measured by plethysmometry (Panlab LE-7500) before edema induction (zero time), at 30 min, and from 60 to 420 min thereafter, and expressed in μL or area under the curve-AUC (arbitrary units) (Landucci et al., 1995).For evaluation of plasma leakage, animals received Evans blue (25 mg kg -1 ) 30 min before edema induction by EP-Mt.The paws were excised, incubated with 1 mL formamide (37°C, 72 hours) and the increase in vascular permeability assessed by spectrophotometry (A 600 nm).Data was expressed in μg Evans blue/g paw wet weight using linear regression based on Evans blue standard curve (Wilhelm, 1982).
The activity of mieloperoxidase (MPO) was measured in peritoneal fluid collected 180 min after stimulation.The samples were homogenized, mixed with o-dianisidine dihydrochloride (0.167 mg mL -1 in 50 mM phosphate buffer), containing H 2 O 2 (0.005%) and measured at A 460 nm.Results were expressed as units of MPO (UMPO mL -1 ).One unit of MPO was defined as the conversion of 1 μmol of hydrogen peroxide to water in 1 min at 22°C (Bradley, Chritensen, & Rothstein, 1982).

Statistical analysis
Results were expressed as mean ± S. E. M. (n=6) and analyzed by One-way/Two-way ANOVA and Bonferroni test (Prism 5.0, GraphPad Software Inc., California, USA).Differences were considered for p≤0.05.
Although in the present study the analysis of phenolic compounds had not been assessed, in other study performed with M. tenuiflora barks, the presence of these compounds (minor residual amounts) was observed in the total polysaccharide extract.It is important to note that the authors could correlate the presence of phenolic compounds with the toxicity observed in cultured skin fibroblasts.Such effect was abolished after removal of the phenolic compound, which was associated to the polysaccharide (Zippel et al., 2009).In addition, previous phytochemical studies demonstrated presence of phenolic compounds (Jiag et al., 1991;Rivera-Arce et al., 2007), saponin, alkaloids, glucose, xylose, rhamnose, arabinose, lupeol, phytosterols, lipids, calcium oxalate crystals and starch (Jiag et al., 1991) in M. tenuiflora barks.
The vascular permeability evaluated by Evans blue leakage at 30 min in the paw edema model was not altered, indicating that EP-Mt edematogenic effect is not of osmotic nature, different from that elicited by the polysaccharide dextran (Lo, Almeida, & Beaven, 1982).This data was confirmed by the lack of alteration in vascular permeability, seen by the protein dosage by the Bradford method evaluated at 180 min after peritonitis induction (Table 1).In acute inflammatory processes, macrophages, mast cells and lymphocytes have a modulator role in neutrophil migration releasing chemotactic factors, such as leukotrienes (LTB 4 ) and cytokines: tumor necrosis factor-alpha (TNF-α) and interleukin 1 (IL-1) (Ranki, Sylvester, Smith, Yoshimura, & Leonard, 1990).The inhibitory effect of dexamethasone on EP-Mt chemotactic effect could be explained by the blockage of leukotrienes and/or cytokines release.Besides, the anti-inflammatory mechanism of dexamethasone includes the EP-Mt (mg kg -1 ) Leukocyte x 10 3 mL -1 Leukocyte x 10 3 mL -1 MPO (U mL -1 ) inhibition of the expression of various inflammatory genes, such as that of the inducible nitric oxide synthase (iNOS), leading to decreased production of NO (Korhonen, Lahti, Hämäläinen, Kankaanranta, & Moilanen, 2002;Secco et al., 2006;Moncada, Palmer, & Higgs, 1991).This data reinforces the inhibitory effect of L-NAME on the neutrophil migration stimulated by EP-Mt.Prostaglandins are mediators that also play a complex role in primary aspects of acute inflammatory response, such as pain, neutrophil migration, swelling and plasma exudation (Menezes et al., 2008).Our data demonstrated that the pre-treatment of animals with indomethacin did not alter the leukocyte migration, suggesting that prostaglandins are not crucial for the occurrence of leukocyte migration elicited by EP-Mt.In addition, it is possible to suggest that EP-Mt stimulates neutrophil chemotaxis, rather than vascular permeability.Furthermore, although not detected at the time-points evaluated in the peritonitis model, the possible EP-Mt effect in permeability could occur prior to its neutrophil chemotactic effect, which partially explains the inhibitory effect of L-NAME.
The effect of polysaccharides of higher plants in leukocytes, via macrophage activation, is extensively described in literature (Park et al., 2001;Sugawara, Lee, & Wong, 1984;Schepetkin, Faulkner, Nelson-Overton, Wiley, & Quinn, 2005;Schepetkin & Quinn, 2006).It has been shown that these compounds might stimulate macrophage phagocytic and cytotoxic activities against tumoral cells and micro-organisms, production of reactive oxygen species (ROS), and secretion of cytokines and chemokines.The polysaccharide of Juniperus scopolorum showed potent immunostimulant effect, via increase of NO production and release of proinflammatory cytokines by macrophages (IL-1, IL-6, TNF-α) (Schepetkin et al, 2005).In addition, the polysaccharide obtained from Dendobrium officinal raised macrophage levels of TNF-α, IL-1β (He et al., 2016) and NO production (Xia et al., 2012).Based on these, we believe that EP-Mt might stimulate leukocytes to release chemotactic substances, being considered a promisor candidate to be used in immunostimulant therapies.

Conclusion
The results reported in the present study clearly demonstrate that the polysaccharides extracted from M. tenuiflora stem barks, possessing high content of carbohydrate, elicit acute inflammation with the involvement of nitric oxide.It is worth mentioning the necessity to investigate molecules from natural sources presenting immunomodulatory effect, aiming the development of novel therapeutic agents for being used in pathological conditions of the immune system.

Table 1 .
EP-Mt does not alter vascular permeability.