BR102013005019A2 - pharmaceutical compositions containing procyanidins from rizophora mangle bark fractions - Google Patents

Abstract

Procyanidins-containing pharmaceutical compositions of rhizophora mangle fractions The present invention describes two pharmaceutical compositions derived from two rhizophora mangle fractions alone. Additionally, new molecular fractions with potential for treatment of gastrointestinal inflammatory diseases (dll) as well as the possible systemic immunological interactions involved in their administration are also described. The drug compositions are primarily indicated for treatment of nonspecific ulcerative rectolitis and Crohn's disease, both characterized by chronic and uncontrolled inflammatory process in the intestine mediated by immune cells. Thus, the drug compositions described in this invention allow for improvement in colonic mucosal damage, antioxidant (gsh) and enzyme (gsh-px, catalase) contents, as well as modulation of inflammatory markers (mpo, tbars, cox-1). and cox-2) and cytokines (tnf-α, yl-6, yl-12 and yl-12).

Description

FIELD OF THE INVENTION The present invention is a Certificate of Addition to patent application BR 10 2012 028815 0 and describes two drug compositions derived from two fractions of Rizophora mangle, in isolation. Additionally, new molecular fractions with potential for treatment of gastrointestinal inflammatory diseases (D11) as well as the possible systemic immunological interactions involved in their administration are also described.

The drug compositions are primarily indicated for treatment of nonspecific ulcerative rectolitis and Crohn's disease, both characterized by chronic and uncontrolled inflammatory process in the intestine mediated by immune system cells.

BACKGROUND OF THE INVENTION

Inflammatory bowel diseases (Dll) include Crohn's disease and nonspecific ulcerative rectolitis, both characterized by chronic and uncontrolled inflammatory process in the intestine mediated by immune cells.

The main features of these diseases are a robust inflammatory response of unknown etiology associated with damage to the intestinal mucosa and increased epithelial permeability, invasion of commensal bacteria into the subepithelial space or lamina propria, and massive neutrophil recruitment [1]. Ulcerative colitis mainly affects the lining of the colon and rectum, while Crohn's disease usually affects the entire bowel wall and potentially can extend to any part of the gastrointestinal tract [2]. Intestinal homeostasis requires a microbiota-controlled innate immune response, which is recognized by toll-like receptors (TLRs) and intracellular nucleotide oligomerization domain (NOD) receptors on epithelial and immune cells. This recognition process contributes to tolerance, but when the process is deregulated, inflammation continues.

In Crohn's disease, innate immunity-related abnormalities are linked to receptor gene variations, that is, those responsible for normally mediating microbial recognition [3]. Several proinflammatory mediators such as cyclooxygenases (COX-1 and COX-2), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-12 (IL-12) , in the presence of activated inflammatory cells, such as neutrophils, dendritic cells, macrophages, and excessive production of reactive oxygen species (ROS), may implicate in the pathogenesis of this disease [4]. Many polyphenolic compounds have been studied for their activity. anti-inflammatory agents, while the main effect of these compounds on conditions of inflammation is due to inhibition of proinflammatory markers [5, 6]. In this context, medicinal plants that accumulate polyphenolic compounds play an important role in the investigation of new therapeutic agents.

Rhizophora mangle is a mangrove plant rich in phenolic compounds, especially condensed and hydrolysable tannins; Its bark is known by traditional Brazilian medicine and Garibian countries [7}. Thus - as indicated in patent application BR 10 2012 028815 0, R. mangle has antiulcer [11] and antioxidant [8] potential and the present invention, in turn, describes novel fractionally derived molecules described in the previous patent application and its application in inflammatory bowel diseases (Dll) acting on the immune system, mainly pediatricians of inflammation as described below; The production of proinflammatory cytokines such as interleukin-6, interleukin-12, and tumor necrosis factor (TNF-α) is increased in patients with inflammatory bowel disease. Abnormalities in adaptive immunity that differentiate ulcerative colitis from Crohn's disease are defined by CD4 + mucosal T cells, which were initially divided into two strains: Th1 and Th2 cells. Crohn's disease is a Th 1 type disease [3]. The characteristic inflammatory response begins with an infiltration of neutrophils and macrophages, which then release chemokines and cytokines. Neutrophil infiltration into the inflamed mucosa is one of the most prominent histological features observed in Dll. This is because activated neutrophils produce reactive oxygen and nitrogen species in the intestinal mucosa inducing oxidative stress, which plays an important role in the pathogenesis of Dll [28].

Inflammatory mediators, including reactive oxygen species (ROS) and cytokines, contribute to the inflammatory cascade that modulates the immune response in Dll, while TNF-α may activate oxidative stress genes responsible for prolonging inflammation [29]. Several studies have shown that ERQs play an important role in the pathogenesis of Dll, as their overproduction by mucosal cells induces an immediate immune inflammatory response that directly or indirectly enhances damage to intestinal epithelial cells, subsequently impairing integrity. mucosa and leading to severe bowel injury [30, 31]. Oxidative stress, and its consequent lipid peroxidation, can lead to free radical chain reactions, affect the integrity of the intestinal mucosal barrier and activate inflammatory mediators. Additionally, it has been shown that catalase (CAT) present in the colon, GSH-Px activity and GSH levels are reduced, while lipid peroxidation (TBARS levels) is increased in experimental and clinical studies [32, 33]. CAT, GSH-Px and GSH present themselves as primary defenses and therefore could reduce oxidative stress and activation of inflammatory mediators. R. mangle, therefore, acting as a free radical scavenger acts contrary to the function of ROS and can directly eliminate hydroxyl and reactive peroxicity species [31].

Cytokines are key mediators in interactions between active immune cells and nonimmune cells, including epithelial and mesenchymal cells. TNF-α, in turn, is an important mediator of inflammation in the intestine, synthesized by various cell types including intestinal epithelial cells, but predominantly by monocytic lineage cells and T lymphocytes. TNF-α induces the expression of several genes, which It has multiple biological effects, such as increased leukocyte recruitment, modulation of nitric oxide production, induction of proinflammatory eitokine secretion, and proliferation and differentiation of immune cells [3]. Thus, TNF-α and cytokines are found to be one of the therapeutic targets for chronic inflammatory disorders, such as Dll [37]. Overproduction of IL-12, a macrophage-derived cytokine, shifts the immune response in the Th 1 direction and this response is characterized by increased production of TNF-α and IL-6, resulting in a self-sustaining activation cycle [38]. ].

Anti-IL-12 or interferon-γ-specific antibodies antagonize spontaneous Dll development in IL-10 deficient mice, but only IL-12 neutralization improved the disease [4]. IL-12 blockade inhibits the generation of the Th1 response and aborts the production of chain eitokines. IL-6, in turn, is a pleiotropic cytokine that plays a crucial role in inflammation, immune regulation, hematopoiesis, and oncogenesis. There is increasing evidence that IL-6 signaling pathway plays a key role in the uncontrolled inflammatory process of Dll [39]. These proinflammatory eitokines act on local cell populations promoting intracellular death (superoxides, peroxides). ), improving the recruitment of other inflammatory cells, increasing secretion of chemotactic cytokines (chemokines), and promoting the destruction of local tissue [40].

Another possible way to promote bowel tolerance; falls on mucosal dendritic cells, because the expansion of these cells, including those present in the intestinal tissues, promote the ability to induce oral tolerance. Additionally, mucosal dendritic cells residing on the lamina propria or Peyer's plaques of the intestine indicate preferentially producing IL-10, rather than IL-12 [41]. IL-10 is a cytokine secreted by various cell types and has pleiotropic effects on T cells, B cells, myeloid cells, and a variety of cells. IL-10 has suppressive anti-inflammatory activity in T cells, macrophages and dendritic cells in humans, as well as in animal models of inflammatory diseases. However, although IL-10 effectively treats colitis in rat models and suppresses inflammatory cytokine production in vivo in intestinal cells of IBD patients [3], in those diseases in which there is absolute or relative IL-10 deficiency, persistent activation of the immune system still exists [42].

Accordingly, the present invention describes two drug compositions containing two Rizophora mangle fractions alone and are capable of promoting systemic immune interactions to ameliorate tissue injured by inflammatory bowel diseases, particularly nonspecific ulcerative colitis and Crohn's disease. Additionally, new molecules are also proposed, derived from these potential fractions whose immunological interaction potentials are also described herein.

BRIEF DESCRIPTION OF THE INVENTION The present invention is Certificate of Addition to patent application BR 10 2012 028815 0, and describes two drug compositions, each containing a different fraction of Rhizopbora mangle bark with application in nonspecific ulcerative rectoitis. Crohn's disease, two common gastrointestinal inflammatory diseases (Dll) in the population.

Both fractions are obtained from the extract of R. mangle bark as described in item 1 of the Detailed Description of the Invention of patent application BR 10 2012 028815 0, without any modification. The fractions are classified as butanolic (BuOH) or ethyl acetate (EtOAc), depending on the process stage at which they were obtained. From the above two fractions, the present invention describes two drug compositions, each containing only one of the fractions alone, which were used in experimental procedures to advance their fractionation, enabling the proposition of new molecules that actively act in the treatment of gastrointestinal injuries, as well as how these molecules act in relation to their interaction with the immune system.

Through the induction of colitis in animals and treatment with the compositions containing BuOH butanolic fraction and EtOAc ethyl acetate fraction alone, it was possible to analyze the damage that the TNBS stimulus caused and the recovery capacity of the injured tissue after treatment. Ex vivo experiments were performed to measure the antioxidant activity attributed to both compounds, including glutathione levels (GSH), glutathione peroxidase activity (GSH-Px), catalase activity (CAT), myeloperoxidase (MPO) and estimation. Peroxidation Test (TBARS).

In addition, fraction mass spectra and multistage fragmentation patterns (MS2, MS3) were verified.

Full-scari mass spectra investigation revealed the presence of various substances, which are represented by the precursor ions of the deprotonated molecules [M-H] 'and the other fingerprints. Based on the information obtained, the present invention presents two possibilities for drug compositions capable of reducing the extent of gastrointestinal lesions, especially those caused by nonspecific ulcerative rectolitis and Crohn's disease, although only the BuOH fraction has caused significant changes. adhesion, extent of lesions, and disease activity index. The present invention indicates that BuOH and EtOAc fractions maintain GSH levels in physiological standards, as well as GSH-Px and catalase activity, and cause GSH-Px activity to be increased in both treatments.

Only the BuOH fraction exerts effects on MPO activity and lipid peroxidation in colon tissue, causing decreased MPO activity and TBARS levels.

Both fractions were able to modulate cytokine levels in the inflamed colon, and the EtOAc fraction caused a reduction in pro-inflammatory cytokine levels TNF-α, IL6 and IL-12, and increased levels of IL-10, a cytokine. anti-inflammatory. The BuOH fraction, in turn, reduced the levels of TNF-α and IL12.

Drug compounds made from both fractions alone (BuOH and EtOAc) were not able to decrease COX-2 expression. However, COX-1 expression was found at physiological levels in all EtOAc groups, while the compound containing the BuOH fraction showed increased expression. The expression of COX-2, but not COX-1, is stimulated following TNBS-induced colitis [34]. Thus, during the development of colitis, both COX isoforms could together exert an anti-inflammatory effect.

Comparisons between the non-colitic group regarding CAT, GSH-Px activities and GSH levels decreased significantly in the colonic tissues of the untreated colitic group (control). Treatment with drug compositions containing the BuOH or EtOAc fractions for 21 days resulted in a marked increase in CAT and GSH-Px activities in colon tissues. However, only treatment with the BuOH fraction was able to reduce TBARS levels. In addition, these treatments resulted in increased colon GSH levels, indicating that both drug compositions containing the BuOH fraction and EtOAc fraction inhibited TNBS-induced oxidative stress.

Thus, the two drug compositions described in the present invention, derived from fractions of R. mangle for application in nonspecific ulcerative colitis and Crohn's disease, allow improvement in colon mucosal injury, improvement of antioxidant (GSH) and enzyme contents. (GSH-Px, catalase), as well as in the modulation of inflammatory markers (MPO, TBARS, COX-1 and COX-2) and cytokines (TNF-α, IL-6, IL-12 and IL-12).

The finger prints of the acetonatwater extract (7: 3, v / v) of R. mangle and the BuOH and EtOAc fractions obtained by FIA-ESI-ITMS / MS indicated the presence of tannins from catechin polymerization. Suggested fragmentations for the tones of some identified molecules reveal the presence of proanthocyanidins and O-glucosylated derivatives.

Thus, the drug compound containing the BuOH fraction described herein causes decreased MPO activity and may be related to the lower damage found in animals treated with this fraction, although this finding is related to the evaluation of other ERR mediators. Thus, the present invention discloses that drug compounds containing the BuOH fraction exert their anti-inflammatory effect by elevating both COX-1 and COX-2 expression, whereas the compound containing the EtOAc fraction exhibits similar levels of expression of these. enzymes in the group with untreated colitis.

Additionally, both the BuOH-containing and EtOAc-containing compounds provided downregulation of TNF-α levels, indicating that these compounds may act upon the activation of this cytokine. Additionally, both drug compounds (BuOH and EtOAc) also reduced IL-12 levels, which are linked to TNF-α suppression, indicating by what mechanism the two treatments can improve the inflammation scenario. Evaluation of IL-6, proinflammatory cytokine, revealed that the drug compound containing the EtOAc fraction decreased its level, however, the one containing the BuOH fraction showed no effect on its levels.

In relation to IL-10, the drug compound containing the EtOAc fraction promotes elevation of its level, while the drug composition containing the BuOH fraction showed no effect on the IL-10 level.

Thus, the present invention indicates that the drug composition containing the EtOAc moiety acts on anti-inflammatory IL-10 activation and down-regulation of IL-12, thereby resulting in decreased levels of TNF-α and IL-6.

Similarly, the BuOH fraction-containing drug compound has anti-inflammatory action by regulating cyclooxygenases as well as activity, levels of ROS mediators, in particular those related to lipid peroxidation (TBARS) and MPO which They are also linked to neutrophil infiltration, which promotes recovery of injured tissue.

BRIEF DESCRIPTION OF THE FIGURES

In FIGURE 1, the full-scan first-order mass spectrum of: A) acetone: water (7: 3, v / v); B) aqueous fraction; C) butanolic fraction; D) ethyl acetate fraction of R. mangle barks. FIGURE 2 shows structures of the monomer (m / z 289) and the [epi] catechin dimer (m / z 577). The second-order mass spectrum of the precursor ion of m / z 885, obtained in negative mode with 20% collision energy, can be seen in FIGURE 3. FIGURE 4 shows the proposed structure for the precursor ion of m / z 885. z 1173. ___ FIGURE 5 shows the proposed fragmentation of a possible trimeric proanthocyanidin identified in the extract of R. mangle barks. The main fragmentation mechanisms involved are: RDA (Retro-Diels-Alder), and QM (Quinone Metid). The second-order mass spectrum of the precursor ion m / z 597, obtained in negative mode with 20% collision energy, can be seen in FIGURE 6.

DETAILED DESCRIPTION OF THE INVENTION The present invention is Certificate of Addition to Patent Application BR 10 2012 028815 0, and describes two pharmaceutical compositions, each containing a different fraction of Rhizophora mangle bark with application in nonspecific ulcerative rectoitis and Crohn's disease, two common gastrointestinal inflammatory diseases (Dll) in the population.

Both fractions are obtained from the extract of R. mangle bark as described in item 1 of the Detailed Description of the Invention of patent application BR 10 2012 028815 0, without any modification. The fractions are classified as butanolic (BuOH) or ethyl acetate (EtOAc), depending on the process stage at which they were obtained. From the above two fractions, the present invention describes two drug compositions, each containing only one of the fractions alone, which were used in experimental procedures to advance their fractionation, enabling the proposition of new molecules that actively act in the treatment of gastrointestinal injuries, as well as how these molecules act in relation to their interaction with the immune system.

Experimental procedures, including the fractionation of molecules of interest with pharmacological potential and the systemic immunological action caused by the proposed molecules are described below. 1. Experimental colitis Male Unib-WH rats were randomly assigned to four groups (n = 8), two of which (the non-colitic and control groups) received no treatment. The other groups received orally, daily for 3 weeks, 0.5 mg BuOH fraction, 1.5 mg / kg EtOAc fraction (suspended in 10 ml / kg saline). Both non-colitic and control groups (with untreated colitis) received 10ml / kg of saline daily.

Two weeks after initiation of treatment, the mice were fasted overnight and was induced to colitis in the control and treatment groups by the method originally described by Morris et al. [12] They were anesthetized with halothane and received 10 mg of TNBS dissolved in 0.25 ml of 50% (v / v) ethanol through an 8 cm Teflon cannula inserted through the anus. Rats in the non-colitic group were administered 0.25 mL of saline phosphate buffer through the colon instead of TNBS.

Some parameters such as behavior, body weight and stool consistency were analyzed daily throughout the tests. All rats were sacrificed with a halothane overdose 1 week ... after colitis induction, and the colon was removed aseptically, placed on cold ice plate and sectioned longitudinally. 2. Colon damage assessment 10 centimeter distal were removed from the colon of each animal and then washed in physiological saline to remove fecal debris and then weighed. Points were awarded for macroscopic regions of inflammation based on their characteristics, as follows: score 0-10: 0 (no damage), 1 (focal hyperemia), 2 (ulceration without hyperemia or thickening of the bowel wall), 3 (ulceration with local inflammation), 4 (> 2 ulceration and inflammation sites), 5 (major inflammation sites 1 centimeter along the organ), 6-10 (major inflammation sites 2cm along the organ) . Stool consistency (score 0-1) was assessed according to the criteria of Bailon et al. [13] and Vicario et al. [14] Excerpts of colonic inflammation were collected, further divided into longitudinal segments and frozen in liquid nitrogen to measure additional biochemical parameters. 3. Determination of ex vivo antioxidant activity (a) Glutathione (GSH) levels - GSH levels of animal colon tissue were determined by Ellman reaction using 5.5 dithiobis (2-nitrobenzoic) acid in methanol (DTNB - 0, 01 mM) as described by Anderson [15]. The intensity of the yellow color was read at 412nm. b) Glutathione oeroxidase activity (GSH-Px) - GSH-Px activity was quantified following the decrease in absorbance at 365 nm induced by 0.25 mM H202 in the presence of reduced glutathione (10 mM), NADPH , (4 mM), and 1U enzymatic activity of GSH-Px [16]. c) Catalase (CAT) - Catalase activity was verified according to the method described by Aebi [17]. Samples were homogenized (1: 200) in phosphate buffer (50 mM, pH 7.0) and triton X- 100 (9: 1). They were then centrifuged (7000G, 4 ° C, 15 minutes), and in 200 µl of homogenate 100 µl of H2 O (30 mM) was added and the absorbance was measured at 240 nm for 2 minutes. Catalase activity was expressed in U / mg protein, where U is enzyme activity, defined as the amount of enzyme required to degrade 1pmol H202 / s / mg protein. d) Myeloperoxidase (MPO) activity - MPO activity in the colon mucosa was measured by the method proposed by Krawisz et al. [18] to assess neutrophil accumulation. The tissue was triturated with hexadecyltrimethylammonium bromide buffer (HTAB) on ice and homogenized three times. The homogenate was subjected to an ultrasonic bath for 10 seconds, followed by a triple freezing and thawing process, which facilitates the rupture of cell structures and, consequently, the enzyme release. Aliquots of homogenate were then mixed with 50 mM phosphate buffer, pH 6.8, containing 0.005% H2 O2 and 1.25 mg / mL o-dianisidine hydrochloride and then measured at 460 nm. e) Estimated lipid peroxidation (TBARS) - Colon homogenate was diluted in 0.15 M KCI (1:10 ratio). Then, to 0.5 ml of this homogenate, 0.2 ml of (8.1%) dodecyl sulfate, 1.5 µl were added, then the samples were treated with Laemmili buffer (0.5M PB, pH 6, 8, glycerol, 10% sodium dodecyl sulfate (SDS), 0.1% bromophenol, β-mercaptoethanol) in a 1: 1 ratio. Equal amounts of sample protein (100 µg) were separated by 10% acrylamide gel electrophoresis and polyacrylamide dodecyl sulfate.

In the next step, proteins were electrophoretically transferred to a nitrocellulose membrane and incubated with the primary specific antibodies: COX-1- (160109, Cayman Chemical, USA) and COX-2 (160126, Cayman Chemical, USA) at 1: 500. dilution Each membrane was washed three times for 10 minutes and incubated with goat anti-rabbit HRP (Invitrogen 656,120) (for COX-1 and COX-2, diluted 1: 5000). To prove equal charge, the blots were analyzed with the ponceau dye protein standard [21]. Immunodetection was performed using enhanced chemiluminescence through light detection kit (SuperSignal West Femto Chemiluminescent Substrate, Pierce, IL, USA). Densitometric data were performed with G-BOX, Syngene and normalization was followed by ponceau using GeneSys software.

6. FIA-ESI-IT-MS

Mass spectra were obtained on a Thermo Analytical LCQ Fleet mass spectrometer equipped with a direct sample insertion device via continuous flow injection analysis (FIA). The sample was analyzed in electrospray ionization mode (ESI) and multistage fragmentation (MS2, MS3) performed on an ion-trap (IT) interface. The negative mode was chosen for generation and analysis of first-order mass spectra (MS) as well as for the other multistage experiments (MSn) under the following conditions: Capillary voltage -4 V, spray voltage -5 kV, capillary temperature 280 ° C, carrier gas (N2) flow 60 (arbitrary units). The acquisition range was m / z 50-1200, with two or more scan events performed simultaneously on the LCQ mass spectrometer. The first event was a full-scan mass spectrum to acquire the ion data in the established m / z range. The other events were MSn experiments performed from the first scan data for preselected precursor ions, with collision energy between 25 and 30% of the total energy of the instrument. Xcalibur version 1.0 software (Thermo Scientific®) was used during the acquisition and processing of spectrometric data.

Full-scan investigation of the mass spectra (Figure 4) revealed the presence of various substances, which are represented by the precursor ions of the deprotonated molecules [M-H] 'for the extract and fractions of R. mangle. All fingerprints in figure 4 (A-D) had less intense ions of m / z 885,723 and the other ESI-MS fingerprints had characteristic ions at m / z 597,471 (highest intensity). In a first class of secondary metabolites, the [epi] catechin precursor ion formed by the interflavonoid bond between m / z 577 (dimer) catechin units, [MH] '(Figure 5), was easily found in the full mass spectrum. -scan of figure 1 A.

MS / MS fragmentation of the precursor ion of m / z 1173 (Figure 1A) yielded the base peak of m / z 885; This ion was generated by the elimination of a [epi] catechin [M-288-H] 'unit (Figure 3) from the [epi] catechin unit of this trimer (Figure 6). Third-order fragmentation (MS3) of the product ion in m / z 733 refers to the loss of a gaoyl unit (Figure 6) [M-288-152-H]. Another consistent proposal was used to explain the presence of the product ion of m / z 587, generated by the fragmentation mechanism, methoxy quinone (QM), which occurred in the upper unit D ring with loss of glucosylated 1,3,5-trihydroxybenzene residue and following, deletion of the RDA (Retro-Diels-Alder) fragment in the central unit's C-ring [M-288-152-H] \ In the fragmentation mechanism involving QM splitting, the location of sugar in the upper unit of the molecule The deprotonated protein was postulated based on the interflavonoid bond divar between the upper unit C-ring and the central unit D-ring (Figure 7). The evidence that the sugar is in the upper trimeric unit is provided by the m / z ion. 885. This ion is the result of the elimination of the lower catechin unit (rings G1), with the consequent formation of a fragment whose upper unit is a diglucosylated catechin (rings A, B, C) and the lower unit contains a system. to QM (rings D, E, F). Such a fragment cannot be formed if the sugar is in the lower trimer unit.

Proanthocyanidins consisting solely of equivalent units of [epijate catechins and having an interflavonoid bond are called B-type procyanidins. Thus, the precursor ions of m / z 577, m / z 885, m / z 1173, are indicative of B-type procyanidins [9]. However, as with all MS techniques, no differentiation between stereoisomers was performed, as well as no information on the position and stereochemistry of interflavonoid bonding.

Another catechin derivative containing two hexoses was identified at m / z 597. The MS2 spectrum showed the loss of 146. a.m.a. (arbitrary mass unit) probably due to the loss of a terminal raminose, providing the product ion at m / z 451 (figure 6), structure proposed in figure 7. From the above information, the present invention presents two possibilities of compositions drugs capable of reducing the extent of gastrointestinal lesions, especially those caused by nonspecific ulcerative rectolitis and Crohn's disease, although only the BuOH fraction caused significant changes in adhesion, extent of lesions, and disease activity index.

BuOH and EtOAc fractions maintain GSH levels in physiological standards, as well as GSH-Px and catalase activity, as well as increasing GSH-Px activity in both treatments.

Only the BuOH fraction exerts effects on MPO activity and lipid peroxidation in colon tissue, causing decreased MPO activity and TBARS levels.

Both fractions were able to modulate cytokine levels in the inflamed colon, and the EtOAc fraction caused a reduction in pro-inflammatory cytokine levels TNF-α, IL6 and IL-12, and increased levels of IL-10, a cytokine. anti-inflammatory. The BuOH fraction, in turn, reduced the levels of TNF-α and IL12.

Drug compounds made from both fractions alone (BuOH and EtOAc) were unable to decrease COX-2 expression (Figure 8). However, COX-1 expression was found at physiological levels in all EtOAc groups, while the compound containing the BuOH fraction showed increased expression (Figure 8). The expression of COX-2, but not COX-1, is stimulated following TNBS-induced colitis [34], although Hyun et al. [35] demonstrated that in rats with PAR2 deficiency, COX-1 and COX-2 have their expression increased after induction of TNBS colitis, and this has been associated with a reduction in inflammation. Thus, during the development of colitis, both COX isoforms could together exert an anti-inflammatory effect. Reuter et al. [36] indicate that COX-1 and COX-2 play an active role in the inflammatory modulation process in various experimental models of colitis, since the administration of selective COX-1 or COX-2 inhibitors in TNBS induced colitis increased the severity of colitis and impaired the healing process.

Comparisons between the non-colitic group regarding CAT, GSH-Px activities, and GSH levels decreased significantly in the colon tissues of the untreated colitic group (control). Treatment with drug compositions containing the BuOH or EtOAc fractions for 21 days resulted in a marked increase in CAT and GSH-Px activities (Table 1) in colon tissues. However, only treatment with the BuOH fraction was able to reduce TBARS levels (Table 2). In addition, these treatments resulted in increased colon GSH levels (Table 2), indicating that both drug compositions containing the BuOH fraction and EtOAc fraction inhibited TNBS-induced oxidative stress.

Table 1: Effect of BuOH and EtOAc treatments on GSH levels and activity of GSH-Px and CAT enzymes in colic mucosa of rats submitted to chronic treatment TNBS-induced colitis model.

Data expressed as mean ± standard deviation, ANOVA followed by Dunnett's posterior test, * p <0.05, ** p <0.01 and *** p <0.001.

Therefore, the present invention indicates that in the drug compounds there should be 0.5 mg / kg BuOH fraction and 1.5 mg / kg EtOAc fraction. The present invention describes two drug compositions derived from fractions of phi. mangle, for application in nonspecific ulcerative colitis and Crohn's disease, which allow for improvement in colonic mucosal damage (Table 3), improvement in antioxidant (GSH) and enzyme (GSH-Px, catalase) contents, as well as in modulation of inflammatory markers (MPO, TBARS, COX-1 and COX-2) and cytokines (TNF-α, IL-6, IL-12 and IL-12). TABLE 3: Effect of BuOH and EtOAc fraction on parameters observed in colon of rats submitted to TNBS colitis in rats.

The results were expressed as mean ± standard deviation (dp) or standard error (ep) and subjected to one-way analysis of variance (ANOVA) followed by Dunnett and / or Tuckey posterior test with a significance level of * p < 0.05, ** p <0.01 and *** p <0.001.

Finger prints of acetone: water (7: 3, v / v) extract from R mangle and from the BuOH and EtOAc fractions obtained by FIA-ESI-ITMS / MS indicated the presence of tannins from the catechin polymerization. Suggested fragmentations for the ions of some identified molecules reveal the presence of proanthocyanidins and O-glucosylated derivatives. The present invention describes that the drug compound containing the BuOH fraction causes decreased MPO activity (Table 4) which may be related to the minor damage found in animals treated with this fraction, although this finding is related to the evaluation. from other ERO mediators, as shown below. TABLE 4: Effect of BuOH and EtOAc fraction on MPO activity and TBARS levels in TNBS colitis in rats.

Results were expressed as mean ± standard deviation (dp) or standard error (ep) and subjected to one-way analysis of variance (ANOVA) followed by Dunnett and / or Tuckey a posteriori test with a significance level of * p < 0.05, ** p <0.01 and *** p <0.001.

Thus, the present invention discloses that drug compounds containing the BuOH fraction exert their anti-inflammatory effect by elevating both COX-1 and COX-2 expression, whereas the a. EtOAc fraction shows similar levels of expression of these enzymes in the untreated colitis group (Figure 8).

Additionally, both the BuOH-containing and EtOAc-containing compounds provided downregulation of TNF-α levels (Table 5), indicating that these compounds may act upon the activation of this cytokine. Additionally, both drug compounds (BuOH and EtOAc) also reduced IL-12 levels (Table 5), which are linked to TNF-α suppression, which indicates by which mechanism the two treatments can improve the inflammation scenario.

Table 5 Effect of BuOH and EtOAc treatments on levels of TNF-α, IL-12, IL-1 β and IL-6 in colic mucosa of mice submitted to acute model of SSD-induced colitis.

Data expressed as mean ± standard error, ANOVA followed by Dunnett's a posteriori test, * p <0.05, ** p <0.01 and *** p <0.001. Evaluation of IL-6, proinflammatory cytokine, revealed that the drug compound containing the EtOAc fraction decreased its level, however, the one containing the BuOH fraction showed no effect on its levels.

In turn, in relation to IL-10 (Table 5), the drug compound containing the EtOAc fraction promotes elevation of its level, while the drug composition containing the BuOH fraction showed no effect on the IL-10 level.

Thus, the present invention indicates that the drug composition containing the EtOAc moiety acts on anti-inflammatory IL-10 activation and down-regulation of IL-12, thereby resulting in decreased levels of TNF-α and IL-6. . __ ..

Similarly, the BuOH fraction-containing drug compound has anti-inflammatory action by regulating cyclooxygenases as well as activity, levels of ROS mediators, in particular those related to lipid peroxidation (TBARS) and MPO which They are also linked to neutrophil infiltration, which promotes recovery of injured tissue.

EXAMPLE

TNBS-INDUCED COUTE WITH CHRONIC TREATMENT IN MICE - HISTOLOGICAL EVALUATION OF LESID TISSUE AND ITS RECOVERY

This experiment focused on the beneficial effects of the BuOH fraction and EtOAc fraction of Rhizophora mangle bark in chronic TNBS-induced colitis in rats. The TNBS-induced colitis model is used to study chronic and acute colon damage and ROS-associated phenomena [10]. It has been shown in an old study [43] that 30% ethanol only acts by breaking the barrier to facilitate TNBS entry into the mucosa, but it does not intensify colon damage. TNBS can be metabolized, enzymatically or non-enzymatically by the hydrogen peroxide (H202) -coverbar, which suggests that the induced colitis in this model may be partially mediated by ROS generated by the oxidative metabolism of TNBS [44], Thus, considering that the TNBS model of colitis induces a transmural lesion with pathological features that resemble Crohn's human disease. In this context, and in order to evaluate the importance of the therapeutic use of procyanidins from the peel of. Rhizophora mangle, the colitis-induced TNBS protocol in rats was used. Oral administration of the BuOH fraction and EtOAc fraction for 21 days attenuates the macroscopic colon injury caused by TNBS at 0.5 mg and 1.5 mg, respectively, and demonstrated efficacy in the acute model of TNBS-induced colitis. Thus, they were also tested at these doses in chronic colitis. Figure 9 shows the evolution of animal weight over the experimental protocol; After the fasting and colitis induction period, the animals show great weight loss, and this process is reversed with the return of feeding. In non-coyitic animals, the gain is noticeably higher than in those receiving experimental treatments, ie, the coyitic groups show difficulty in regaining weight due to the inflammatory process installed in the colon; however, weight gain was observed in BuOH and EtOAc groups in relation to animals in the colitic group (untreated).

Tables 6 and 7 below show the results of tissue damage and enzymatic activity, respectively, obtained in the TNBS-induced acute colitis model in rats whose macroscopic parameters were evaluated during and after the experimental protocol. Table 6 Macroscopic evaluation of damage caused by intrarectal administration of TNBS in an experimental model of acute colitis.

Data expressed as mean ± standard error, ANOVA followed by Dunnett's posfer / or / test, * p <0.05, ** p <0.01 and *** p <0.001.

Table 7: Effect of BuOH and EtOAc treatments on GSH levels and GSH-Px and MPQ enzyme activity in colic mucosa of rats submitted to acute model of TNBS-induced colitis.

Data expressed as mean ± standard error, ANOVA followed by Dunnett's a posteriori test, * p <0.05, ** p <0.01 and *** p <0.001.

The data thus expressed show that there was a significant reduction in colonic mucosal damage by TNBS only in the groups treated with BuOH (0.5) and EtOAc (1.5 mg) fractions. Although the 3.0 mg EtOAc dose significantly reduced the lesion score, it did not occur with the other parameters evaluated.

An observation of Fig. 10 shows that BuOH (Fig. 22C) reduced the inflammatory infiltrate as well as maintained part of the mucosa intact or poorly compared to the colitic group (Fig. 22B, untreated). In the very organized mucosa of the EtOAc-treated animals, only the extension of the lesion was statistically significant (Table 8).

Table 8 Assessment of damage caused by intrarectal administration of TNBS, rats treated for 21 days with BuOH 0.5 mg.Kg'1 and EtOAc 1.5mg.Kg'1.

Data expressed as mean ± standard deviation, ANOVA followed by Dunnett's a posteriori test, * p <0.05, ** p <0.01 and *** p <0.001. Histological evaluation shows representative of normal colon mucosa (Figure 10 (a)), compared to colitic animals: untreated (Figure 10 (b)), BuOH fraction (Figure 10 (c)), and EtOAc fraction (Figure 10 (d)). Despite a non-quantitative analysis, it is noteworthy that in the BuOH fraction the mucosa presents itself without marked reduction of the inflammatory infiltrate.

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Claims (23)

1. Compounds characterized by the following structural formulas Compounds according to claim 1, characterized in that they are intended to treat gastrointestinal ulcers and injuries. Compounds according to claim 2, characterized in that they are preferably for treating nonspecific recto-ulcerative colitis. Compounds according to claim 2, characterized in that they are preferably for treating Crohn's disease. Medicament characterized in that it contains one or more of the compounds described in claim 1. Medicament according to Claim 5, characterized in that it is for treating inflammatory bowel diseases. Medicament according to Claim 5, characterized in that it is preferably for treating recto-ulcerative colitis. Medicament according to claim 5, characterized in that it is preferably for treating Disease and Crohn. Pharmaceutical composition characterized in that it contains Rhizophora mangle bark fraction associated with one or more pharmaceutically active excipients. Pharmaceutical composition according to Claim 8, characterized in that the fraction was obtained using butanol. Pharmaceutical composition according to Claim 9, characterized in that it contains a concentration ranging from 0.5 to 1.5 mg / kg of the butanolic fraction. Pharmaceutical composition according to Claim 10, characterized in that it preferably contains 0.5 mg / kg of the butanolic fraction. Pharmaceutical composition according to Claim 8, characterized in that the fraction was obtained using ethyl acetate. Pharmaceutical composition according to Claim 12, characterized in that it contains a concentration ranging from 1.0 to 3.0 mg / kg of the ethyl acetate fraction. Pharmaceutical composition according to Claims 8 to 13, characterized in that they maintain the concentration of glutathione at a normal level. Pharmaceutical composition according to Claims 8 to 13, characterized in that they maintain the concentration of glutathione peroxidase at a normal level. - Pharmaceutical composition according to Claims 8 to 13, characterized in that they maintain the catalase concentration at a normal level. Pharmaceutical composition according to Claims 8 to 11, characterized in that it reduces the activity of the enzyme myeloperoxidase. Pharmaceutical composition according to Claims 8 to 11, characterized in that it reduces lipid peroxidation. Pharmaceutical composition according to claims 8 to 13, characterized in that it reduces the level of expression of interleukin 6. Pharmaceutical composition according to Claims 8 to 13, characterized in that it reduces the expression of TNF-α. Pharmaceutical composition according to Claims 8 to 13, characterized in that it reduces the expression of interleukin. Pharmaceutical composition according to Claims 8 to 13, characterized in that it reduces the expression of interferon-γ. Pharmaceutical composition according to Claims 8, 12 and 13, characterized in that it increases the expression of interleukin.