BR102012028815A2 - Pharmaceutical compositions containing fraction obtained from rhizophora mangle extract and their use - Google Patents

Abstract

Pharmaceutical compositions containing fraction obtained from rhizophora mangle extract and its use. The present invention describes a form of fractionation of r extract. Mangle through which it is possible to efficiently concentrate a significant amount of complex tannins. The fraction obtained by the proposed method has a surprising effect on the treatment and gastrointestinal ulcers and injuries, when compared to the medication that is currently used for this purpose, as it uses a dose up to 60 times lower than this and with the same efficiency. Thus, drug or pharmaceutical compositions involving the obtained butanolic fraction are described in the present invention, as well as the proposition of the compounds contained in that fraction.

Description

Field of the Invention The present invention describes a form of fractionation of Rhizophora extract. mangle through which it is possible to efficiently concentrate a significant amount of complex tannins. Thus, the fraction obtained by the proposed method has a surprising effect on the treatment of gastrointestinal ulcers and injuries, when compared with the drug that is currently used for this purpose, because it uses a dose up to 60 times lower than this and with the same efficiency.

Therefore, the present invention describes the use of the butanol fraction obtained in pharmaceutical or pharmaceutical compositions with acceptable excipients.

Background of the Invention Protection of the gastric mucosa The gastric mucosa maintains its structural and functional integrity despite being constantly exposed to harmful factors such as HCI and pepsin, which are capable of digesting tissue. Under normal conditions, mucosal integrity is maintained by defense mechanisms, which include pre-epithelial factors, an epithelial barrier (surface of juxtaposed epithelial cells secreting mucus, generating bicarbonate, peptides, prostaglandins, and heat shock proteins), renewal progenitor cell-perfected cells (regulated by growth factors and PGE2), continuous blood flow through mucosal microvessels, endothelial barrier, sensory innervation, generation of PGs and nitric oxide (NO) and antioxidant mechanisms (Laine et al. /., 2008). Prostaglandins Prostaglandins are involved in a number of physiological processes in the stomach, including acid secretion, mucus production and blood flow in the gastric mucosa (Robert & Ruwart, 1982). It has been established that suppression of PG synthesis in the stomach by inhibition of cyclooxygenases (1 and 2) is the key component of the gastrointestinal tract ulceration mechanism. DAINES induce gastric mucosal ulceration by reducing PG synthesis via cyclooxygenase inhibition. The consequences of this reduction are evidenced by the decrease in mucus and bicarbonate secretion and local blood flow (Cryer, 2000). In addition, the direct contact of NSAIDs with the gastric mucosa attacks the phospholipids present in both mucus and gastric epithelium. which severely interferes with the hydrophobicity of the mucus layer, thereby causing a back diffusion of hydrogen ions (Bjorkman, 1996, and Berstad et al, 2002). The biological effect of PGs, in this case PGE2, is mediated via membrane-specific receptors, called EP (EP1, EP2, EP3 and EP4), which are coupled to membrane G-proteins, linked to different intracellular signal transduction pathways. (Sugimoto et al., 2000; Dey et al., 2006). PGE2 ligands to EP1 receptors result in intracellular release of inositol triphosphate (IP3) and diacylglycerol (DAG); EP2 and EP4 ligands activate the adenylate cyclase-cAMP system; Finally, EPS binders inhibit this system (Pawlick, 2002; Dey et al., 2006). PG exerts protection by increasing both mucus and bicarbonate secretion and epithelial cell resistance against cytotoxin damage and maintaining mucosal blood flow (Hawkey & Rampton, 1985). Moreover, the demonstration that PGE2 inhibits acid secretion also contributes to this mediator being considered an important factor in the protection of the gastric mucosa. Nitric oxide (NO) Nitric oxide plays an important role in modulating gastric mucosal defense, such as: regulator of mucus secretion (Brown et al., 1993), vasodilator, producing increased local blood flow (Wallace, 2000), inhibitor neutrophil migration (Banick et al., 1997) and assist in the healing process of gastric ulcer (Jadeski & Laia, 1999). In the latter, the action of NO produces increased collagen deposition by fibroblasts in addition to stimulating angiogenesis (Wallace, 2000). NO exerts a broad spectrum of in vivo biological activities dependent in part on cyclic guanosine-3 ', 5'-monophosphate (cGMP) produced after stimulation of soluble guanylate cyclase (Moncada, et al., 1991). In smooth muscle cells this stimulation results in relaxation. NO can also act directly on calcium-dependent potassium channels, leading to endothelium-dependent hyperpolarization, resulting in vasodilation (Bolotina et al., 1994).

The three NO-producing enzymes are NO synthases: nNOS (neuronal), eNOS (endothelial), and induced (or inflammatory) iNOS, which have been characterized in the gastrointestinal tract. INOS produces large amounts of NO causing damage and therefore specific inhibition of this enzyme is beneficial. Constitutive NOS keeps the mucosal barrier intact (Kubes & McCafferty, 2000).

Appropriate stimuli, such as inflammatory responses (eg, the presence of gastric ulcer), increase iNOS activity (Kristjansson et al., 2005). NO blockade increases oxidative stress by activating mast cells, which are cells found in large quantities in the gastrointestinal tract, which release mediators such as histamine and platelet activating factor (PAF), causing increased epithelial permeability (Kanwar et al, 1994). .

Mucus and bicarbonate The first line of defense against acid is the mucus barrier (Phillipson et al., 2002). The gastric epithelium is covered by a continuous layer of mucus adhered to the mucosal surface. This mucus, together with bicarbonate secreted by the epithelium, serves as a barrier against self-digestion caused by acid and pepsin (Allen & Flemstrom, 2005). The mucus is viscous, elastic and adherent in the form of a clear gel composed of 95% water and 5% glycoprotein, which covers the surface of the gastrointestinal mucosa (Reppeto & Lleusly, 2002). Gastric mucus also has antioxidant activity due to glycoproteins, and potent sugars sequester reactive oxygen species (ROS) (Mojzis etal., 2000). Gastric mucus secretion is controlled by several factors in different ways. PGs, NO and secretin stimulate mucus secretion (Tani et al., 1997); In receptors that mediate mucus secretion, many physiologically active substances, including neurotransmitters, autacoids, and hormones serve as secretagogue factors (Johnson & Alpers, 1994).

Blood Flow One of the roles of blood flow is to supply the mucosa with oxygen, nutrients and hormones, and to participate in the regulation of acid output, mucus production, bicarbonate secretion and removal of tissue products, including ion back diffusion. hydrogen thus blood flow contributes substantially to the physiological maintenance of mucosal integrity. Its reduction is involved in the formation of gastric mucosal lesions caused by stress, ethanol and DAINES (Kawano & Tsuji, 2000). Microcirculation is modulated by the nervous system and mediators such as NO, bradykinin and PGs. Diffusion of acid or toxins in the mucosa results in critical elevation of afferent sensory neuron-mediated blood flow, limiting damage and facilitating repair. Blood dilutes and / or neutralizes acid / toxin and prevents the accumulation of high mucosal concentrations (Wallace & Ma, 2001).

Sulfhydryl Clusters (GSH) The gastroprotective role of endogenous sulfhydryl clusters (sulfhydryl compounds) present in gastric mucus and various enzymes of the xanthine oxidase system has already been demonstrated in various ulcer induction models (ethanol, DAINES, and stress). in which depletion of these compounds occurs (Hernandez-Munoz et al., 2000 and Bayir et al., 2006). Pretreatment with blockers of sulfhydryl groups such as N-ethylmaleimide (NEM) has been shown to significantly potentiate gastric ulcer induction (Hiraishi et al., 1994), while significant increases in these groups promote gastroprotection (Sener-Muratoglu et al., 2001).

In the inflammatory process, ROS are generated and initiate a chain reaction that culminates in lipid peroxidation and cell death (Tariq et al., 2006). Sulfhydryl compounds bind to free radicals formed during this process or produced upon exposure to harmful agents, thereby protecting the gastric mucosa (Avilla et al, 1996).

These agents are also important in the production and maintenance of gastric mucus, since their glycoprotein subunits are joined together by disulfide bridges which, once reduced, make the mucus hydrosoluble (Salim, 1992 and Avilla et al., 1996).

Reconstitution and Renewal of Gastric Epithelium The stomach has several ways to protect itself when continuously exposed to high acid concentrations; One of the most important structures is the gastric epithelium. It is often renewed, with “old” cells shifting toward the lumen. The human gastric epithelium is completely renewed every 2-4 days. The ability to allow old cells to be replaced by younger cells without significant breakage of the barrier is attributed to a process of cell extension, ie neighboring cells gradually “squeeze” aged cells at the base (Wallace & Granger, 1996). ). The term reconstitution refers to the process of epithelial repair of the mucosa, which involves rapid migration of healing cells to injured sites at the base of the unprotected membrane. Gastric cells are attached to the epithelial cell basement membrane and this site is very sensitive to acid-induced damage (Paimela et al, 1995). Mucosal healing in gastric ulcer requires reconstitution of the epithelial glandular structure (reepithelization), restoration of the lamina propria including a microvascular network in the mucosa, nerves, and connective tissue cells (Milani & Calabro, 2001). Ulcer healing is accompanied by increased gastric blood flow in the ulcer area, plasma gastrin, and pro-inflammatory cytokines such as TNF-α and 1L-1 β. Hypergastrinemia that occurs during the period prior to ulcer healing can be attributed to suppression of gastric acidity and expression of growth factors (Brzozowski et al., 2001). The gastric mucosa, located on the ulcer margin, forms a “healing zone”, glands in this region begin to dilate, and the lining cells of these glands undergo differentiation. Activation of epidermal growth factor (EGF) and cell proliferation begins three days after ulcer implantation and is essential for healing. In general, growth factors promote proliferation and migration of epithelial cells to the ulcer crater, leading to re-epithelialization of this crater and maturation of the glands. At the base of the ulcer tissue granulation occurs with continuous remodeling. Angiogenesis (microvessel formation) facilitates this remodeling by releasing oxygen and nutrients. Inflammatory cells are replaced by fibroblasts and microvessels in the final healing phase (Chan & Sung, 2001). Angiogenesis is therefore important for the repair of both acute and chronic damage during the healing of gastroduodenal ulcers (Malara et al., 2005).

Free radicals and antioxidant defense The history of free radicals in biology and medicine actually began when in McCord and Fridovich (1969) they discovered superoxide dismutase (SOD), an enzyme capable of destroying the superoxide anion radical by univalent oxygen reduction. From then on, there was an exponential growth of scientific articles that showed the excessive production of “reactive oxygen species” (ROS) or “free radicals” implicated in the pathogenesis of various diseases in humans. ROS may be formed by the activation of inflammatory cells during xenobiotic metabolism or from xanthine oxidase during ischemia-reperfusion injury (Blake et al., 1987; Cross et al., 1987; Grisham & Granger, 1988; Braganza, 1989).

Free radicals are involved in the pathophysiology of various gastrointestinal (ulcers and inflammations) and liver diseases (Arthur et al., 1988; Braganza, 1989; Rozga, 1989; Verspaget et al., 1991; Babbs, 1992; Harris et al., 1992 Stark & Szurszewski 1992; Van der Vliet & Bast 1992; Simmonds 1995; Yoshida 1995). Arachidonic acid, macrophage, and neutrophil metabolism generate ROS that may contribute to damage to the gastric mucosa (Rosen et al., 1995). ROS hijackers are used to protect the gastric mucosa from oxidative damage and accelerate healing of ulcers. The imbalance between the formation and removal of free radicals in the body, due to the decrease in endogenous antioxidants or the increase in the generation of oxidizing species, generates a prooxidant state that favors the occurrence of oxidative lesions in macromolecules and cell structures, which may even result. in cell death (Gutteridge, 1993; Halliwell & Gutteridge, 2005).

According to Augusto (2006) the body's antioxidant defenses may be enzymatic and non-enzymatic endogenous, and may also come from the diet.

Enzymes consist of superoxide dismutases, catalases, GSH peroxities, GSH reductases, peroxyredoxins, thioredoxins, repair enzymes, which synthesize GSH and replenish NADPH, while non-enzymatic ones are GSH, uric acid and albumin.

Among those coming from the diet are found: ascorbic acid, α-tocopher! (vitamin E), β-carotene, polyphenols, flavonoids, etc. Under normal conditions the concentration of these species within cells is extremely low because there are antioxidant enzymes that remove them or prevent their formation. These radicals tend to be eliminated from the body by the enzymes superoxide dismutase, catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) (McCord & Fridovich, 1969; Halliwel! & Gutteridge, 2005).

Endogenous antioxidants and dietary derivatives are indispensable for maintaining health in the face of free radical attack; Dietary derivatives include a wide variety of antioxidants, many of which are represented by potent polyphenols. These compounds can exert their beneficial effects on the gastrointestinal tract, as this is possibly the main site of pro and antioxidant actions. All pathological conditions found in the gastrointestinal tract involve reactive species and oxidative damage during their progression and even before their origin (Halliwell et al, 2000; Halliwell & Gutteridge, 2005). Polyphenols, Plant Tannins Tannins are a class of compounds, water-soluble phenolic substances with molecular weight up to 3000 Dalton, which have the ability to form water-insoluble complexes with alkaloids, gelatin and other proteins.

Tannins are particularly important as taste components and are responsible for the astringency of many fruits or peels in vegetable food products. The complexation between tannins and proteins is the basis for their properties as insect, fungal and bacterial control factors as well as for their pharmacological activities (Santos & Mello, 2004). The biological role of tannins in plants has been investigated; They are believed to be involved in the chemical defense of plants against herbivory and pathogenic microorganisms. In defense against herbivory the decrease in palatability due to astringent taste, difficulties in digesting by complexing tannins with digestive enzymes and / or plant proteins and, finally, formation of toxic products in the digestive tract from tannin hydrolysis would be the modes of action proposed as an evolutionary strategy of species with tannins as secondary metabolites (Santos & Mello, 2004).

It is believed that the pharmacological activities of tannins are, at least in parle, due to three common general characteristics, to a greater or lesser extent to those condensed and hydrolysable: 1) Compiexation with metal ions (iron, manganese, vanadium, copper, aluminum). , calcium, among others); 2) antioxidant and free radical scavenger activity; 3) ability to complex with macromolecules such as proteins and polysaccharides.

It has been suggested that the possible modes of action of tannins in treating diseases are closely linked to these three properties. Tannins help in the healing process of wounds, burns and inflammation by forming a protective layer (tannin-protein and / or polysaccharide complex) on the damaged skin or mucosa, which favors the healing process below (restructuring). epithelium and vessel formation).

Similar process probably occurs in cases of gastric ulcer, where a tannin-protein layer would be responsible for protecting the lesion and ensuring better healing (Haslam, 1996).

Tannins with different chemical structures occur all over the planet in medicinal and food plants; have several biological and pharmacological activities, which are often specific to certain structures and significant to human health (Okuda, 2005). Tannins are therefore worthy of further investigation not only in phytochemistry but also in pharmacology.

Rhizophora mancile and its importance as a source of tannins Species of the genus Rhizophora, are known for the medicinal properties of their tannins. For example, R. apiculata has free radical scavenging and antioxidant activity (Vijayavel et al., 2006; Rahim et al., 2008; Loo et al., 2007; Loo et al., 2008), and antiviral activity (Premanathan et al. al., 1999); R. stylosa also has antioxidant activity (Li et al, 2007; Takara et al., 2008). R. mangle is popularly known as “Mangue-Vermelho”, “Candapuva”, “Guaraparaíba”, “Mangue Garobeira”, “Espeto Mangrove”, “Pendão Mangue”, “Mangue Preto”, “Mangue Sapateiro”, “ Paxiubarana ”and“ Mangue True ”, mainly in northeastern Brazil; is known worldwide as "Mangle Rojo", "Mangle Colorado" and "Red Mangrove". It is found throughout the coasts of Brazil, the Antilles, Mexico, Africa and the Pacific Islands; It is economically important as a supplier of tannins (which comprise 15 to 36% of the dry mass) extracted from bark and the plant is one of the most important astringent for industrial use (Pio-Corrêa, 1984; Chapman, 1976).

According to Pio-Corrêa (1984), the R. mangle bark containing tannin is used as an infusion against diarrhea, dysentery and hemorrhage. In several countries of America (Panama, Costa Rica, Jamaica, Cuba) the species is used as firewood, and has its decoction mixed with flaxseed oil to wax the floor of housing, including palaces and large hotels.

Roig (1974) reports that R. mangle has long been known in folk medicine from different Caribbean countries. Its bark has been used as astringent, antiseptic, hemostatic and has antifungal and antiulcerogenic properties.

Some recent work, especially from Cuba, reporting several medicinal properties of R. mangle, such as antioxidant (Sánchez et al, 2006 and Berenguer et al., 2006), antibacterial (Melchor et al., 2001), antiulcerogenic (Perera et al ., 2001 and Berenguer et al., 2006), healing (Fernandez st., 2001 and Armas et al., 2005), anti-inflammatory (Marrero et al., 2006) and anti-diabetic (Alarcon-Aguilara et al. , 1998).

In view of the foregoing, therefore, the present invention describes a form of fractionation of R. mangle extract through which it is possible to efficiently concentrate a significant amount of tannins of interest.

Thus, the fraction obtained by the proposed method has a surprising effect on the treatment of gastrointestinal ulcers and injuries, when compared with the drug that is currently used for this purpose, because it uses a dose up to 60 times lower than this and with the same efficiency.

BRIEF DESCRIPTION OF THE INVENTION The present patent application refers to compounds - and compositions - obtained by a process, also subject of the present patent application, in order to have surprising effect in the treatment of gastrointestinal ulcers and injuries.

The compounds are those shown in figure 32.

The compositions are obtained by combining one or more compounds described in Figure 32 together with one or more pharmaceutically active excipients. The obtaining process is basically comprised of: A) Maceration of Rhizophora mangle bark in water and aceion solution; B) Filtration of the extract obtained in (A) C) Dissolution of the extract obtained in (B) in water; D) Liquid-liquid partition of the solution obtained in (C); E) Dissolution of the extract obtained in (D) in ethyl acetate; F) Partition of the fraction obtained in (E) by 3 times; G) Partition of the aqueous phase obtained in (F) with n-butanol 3 times. These compounds had a surprising effect in relation to the state of the art and required 60 times less than the main similar found to obtain the same pharmacological effect. .

Brief description of the figures In Figure 1 it is possible to observe the representative flowchart of the extraction methods (A) and fractionation (B) of the R. mangle barks. It is possible to verify, in Figure 2 the thin layer chromatography, (A) anisaldehyde development / H2SC> 4; (B) developing with NP / PEG; (C) disclosure with FeC! 3 1%. EC: crude extract; AcF: ethyl acetate fraction; AqF: aqueous fraction; BuF: butanolic fraction; GA: gallic acid; Ru: rutin; Ca: catechin; Ep: epicaequin. . Figure 3 shows HPLC-DAD chromatograms of injections of (A) acetone extract: water (7: 3); (B) aqueous fraction; (C) ethyl acetate fraction; (D) butanolic fraction.

In Figure 4 we observe the negative mode mass spectrum of the acetone: water extract (7: 3) of Rhizophora mangle barks.

Figure 5 shows the negative mode mass spectrum of the aqueous fraction of the acetone: water extract (7: 3) of Rhizophora mangle barks. Figure S shows the negative mode mass spectrum of the ethyl acetate fraction of the acetone: water extract (7: 3) of Rhizophora mangle barks.

Figure 7 shows the negative mode mass spectrum of the butanolic fraction of the acetone: water extract (7: 3) of Rhizophora mangle shells. Figure 8 shows the reducing potential of the crude extract (CE) and R. mangle fractions on the DPPH radical. Gastric protection promoted by the R. mangle aqueous fraction (fr-Aq) in the rat-induced ethanol ulcer model can be seen in Figure 9. Data expressed as mean ± standard deviation. ANOVA, with Dunnett's posterior test, ** P <0.01, *** P <0.001 compared to Saline control. Figure 10 shows the gastric protection promoted by the R. mangle ethyl acetate (fr-EtOAc) fraction in the ethanol-induced ulcer model in rats. Data expressed as mean ± standard deviation. ANOVA, with Dunnett's posterior test, * P <0.05, ** P <0.01 and *** P <0.001 compared to the Saline control.

Figure 11 shows the gastric protection promoted by the R. mangle butanolic fraction (fr-BuOH) in the ethanol-induced ulcer model in rats. Data expressed as mean ± standard deviation. ANOVA, with Dunnett's posterior test, * P <0.05, ** P <0.01 and *** P <0.001 compared to the Saline control.

Figure 12 shows the gastric protection promoted by the butanolic fraction (0 5 mg.Kg'1) of R. mangle in the rat ischemia / reperfusion-induced ulcer model. Data expressed as mean ± standard deviation. ANOVA, with Dunnett's a posteriori test, *** P <0.001 compared to Salina contract.

Figure 13 shows the effect of oral administration of fr-BuOH (0.5 mg, Kg'1) on the ethanol-induced ulcer model in N-ethyl maleimide (NEM) pretreated rats. Data expressed as mean ± standard deviation (n = 7), ANOVA followed by a posterior Tuckey test, *** P <0.001 compared to the Saline group. The effect of oral administration of fr-BuOH (0.5 mg.Kg) on the ethanol-induced ulcer model in L-NAME pretreated rats is shown in Figure 14. Data expressed as mean ± standard deviation (n = 7 ), ANOVA followed by a posterior Tuckey test, * P <0.05, *** P <0.001 compared to the Saline group. Figure 15 shows the effect of oral administration of fr-BuOH (0.5 mg.Kg "1) on the production of mucus adhered by the gastric mucosa of pyloric ligated rats. Data expressed as mean ± standard deviation (n = 7), ANOVA followed by Dunnett's posterior test, ** P <0.01 compared to the Saline group.

In Figure 16 we see the effect of oral administration of fr-BuOH (0.5 mg.Kg'1) on prostaglandin E2 (PGE2) production in rat gastric mucosa. Data expressed as mean ± standard error (n = 6), ANOVA followed by Dunnett's a posteriori test, ** P <0.01, *** P> 0.001 compared to the Indomethacin group.

Figure 17 shows the effect of oral administration of fr-BuOH (0.5 mg.Kg'1) during (A) evolution, (B) 4 ° (C) 82 and (D) 14 days on healing of acetic acid-induced injury in rats. Data expressed as mean ± standard deviation (n = 4), ANOVA followed by Dunnett's a posteriori test, ** P <0.01 compared to the Saline group. Figure 18 shows the evaluation of fr-BuOH toxicity (0.5 mg.Kg'1) in the acetic acid-induced cancer model in rats. Effect of fr-BuOH administered for 14 consecutive days on animal weight. Data expressed as mean ± standard deviation (n = 12). ANOVA followed by Dunnett a posteriori test. P> 0.05 compared to the Saline group. Figure 19 shows in turn the evaluation of fr-BuOH toxicity (0.5 mg.Kg'1) in the rat acetic acid-induced ulcer model. Effect of fr-BuOH administered for 14 consecutive days on animal organ weight. Data expressed as mean ± standard deviation (n = 4). ANOVA followed by Dunnett's a posteriori test, ** P <0.01 compared to the Saline group. The effect of oral administration of fr-BuOH (0.5 mg.Kg'1) for 4 days on cycloxygenase 1 (COX-1) expression during acetic acid-induced wound healing in rats is seen in Figure 20. Data expressed as mean ± standard error (n = 4), ANOVA followed by Dunnett's posterior test, P> 0.05 compared to the Saline group.

In Figure 21 we see the effect of oral administration of fr-BuOH (0.5 mg.Kg'1) for 8 days on cycloxygenase 1 (COX-1) expression during acetic acid-induced wound healing in rats. Data expressed as mean ± standard error (n = 4), ANOVA followed by Dunnett's a posteriori test, P> 0.05 compared to the Saline group. Figure 22.shows the effect of oral administration of fr-BuOH (0.5 mg.Kg'1) for 14 days on cycloxygenase 1 (COX-1) expression during acetic acid-induced wound healing in rats. Data expressed as mean ± standard error (n = 4), ANOVA followed by Dunnett's posterior test, P> 0.05 compared to the Saline group. Figure 23 shows the effect of oral administration of fr-BuOH for 4 days on cycloxygenase 2 (COX-2) expression during acetic acid-induced injury wound healing in rats. Data expressed as mean ± standard error (n = 4), ANOVA followed by Dunnett's posterior test, P> 0.05 compared to the Saline group.

Figure 24 shows the effect of 8-day oral administration of fr-BuOH (0.5 mg.Kg'1) on cycloxygenase 2 (COX-2) expression during acetic acid-induced wound healing in rats . Data expressed in aver; a. ± standard error (n = 4), ANOVA followed by Dunnett's posterior test, P> 0.05. Compared to the Saline group. Figure 25 shows the effect of oral administration of fr-BuOH (0.5 mg.Kg'1) for 14 days on cycloxygenase 2 (COX-2) expression during acetic acid-induced wound healing in rats. Data expressed as mean ± standard error (n = 4), ANOVA followed by Dunnett's posterior test, * P <0.05, ** P <0.01 compared to the Saline group.

Figure 26 shows the effect of oral administration of fr-BuOH (0.5 mg.Kg'1) for 14 days on epidermal growth factor expression! (EGF) during healing of acetic acid-induced injury in rats. Data expressed as mean ± standard error (n = 4), ANOVA followed by Dunnett's posterior test, * P <0.05, ** P <0.01 compared to the Saline group. Figure 27.shows photomicrographs of rat stomachs with acetic acid-induced ulcer following treatment with fr-BuOH (0.5 mg.Kg "1). Histological sections stained with hematoxylin and eosin (HE) for analysis of mucosal morphological organization where the different experimental groups are observed, gl - glands; m - mucosa; mm - mucosal muscle; SM - sub-mucosa Figure 28 shows photomicrographs of rat stomachs with acetic acid-induced ulcer after treatment with fr-BuOH ( 0.5 mg.Kg'1) Histological sections stained with periodic Schiff acid (PAS) glycoprotein marker for visualization of gastric mucus produced in the different experimental groups Arrows indicate polysaccharide compounds.

Figure 29 shows photomicrographs of stomachs of rats with acetic acid-induced ulcer following treatment with fr-BuOH (0.5 mg.Kg'1). Histological sections submitted to immunolocation of Heat Shock Protein (HSP) 70 revealed with 3'3 diaminobenzidine (DAB), which promotes the brown color as a positive reaction response. The participation of HSP-70 in the healing process in the different experimental groups was verified. Arrows indicate immunolocation. Figure 30 shows photomicrographs of rat stomachs with acetic acid-induced ulcer following treatment with fr-BuOH (0.5 mg.Kg · 1). Histological sections submitted to immunolocation of Proliferation cell nuclear antigen (PCNA) revealed with ABC 3'3 diaminobenzindin complex (DAB) and peroxidase, which promotes brown staining as a positive reaction response. The process of cell division during the healing of the lesions in the different experimental groups was verified. Arrows indicate immunolocation.

In Figure 31 we see photomicrographs of rat stomachs with acetic acid-induced ulcer after treatment with fr-BuOH (0.5 mg.Kg '1). Histological sections submitted to immunolocation of cyclooxygenase 2 (COX-2) revealed with ABC 3'3 diaminobenzindine (DAB), which promotes brown staining as a positive reaction response. The participation of the enzyme COX-2 in the healing process of the lesions in the different experimental groups was verified. Arrows indicate immunolocation.

Figure 32 shows the structures of the polyphenolic compounds proposed from the profile (ESI! V <S) of the acetone: water (7: 3) extract obtained from the R. mangle shells. (;) catechin or epicatechin, condensed tannin monomer; (2) and (3) (epi) heteroside catechin; (4), (5), (6) and (7) elagitanine derivatives, hydrolyzable tannin; (8) and (9) galotanine derivatives, hydrolyzable tannin; (10) and (11) complex tannin derivatives.

Detailed Description of the Invention The present invention discloses pharmaceutical compositions involving fractions obtained from Rhizophora mangle whose main use is in the treatment of digestive system injuries. More specifically, there are described drug compositions involving fractions obtained through different solvents, as well as the proposition of the compounds contained in these fractions.

The fractions obtained from R. mangle bark extract have their chemical, pharmacological and biological response characteristics described here, as well as the proposition of the compounds contained in these fractions. The preparation of extracts and fractions is from R. Mangle bark. It was dried for seven days at 40 ° C (213 g) and ground to a powder (3 mm). The powder in acetone: water (7: 3) was added, obtained an extract prepared by maceration in 31.4% yield (66.9 g). The R. mangle bark extract (CE) was submitted to liquid-liquid partition with increasing polarity solvents with semi-purified fractions. This methodology provides adequate cleaning of polar extracts. About 20g of the extract was partitioned between 150 mL of water and 50 mL of ethyl acetate via a 3-fold separatory funnel. Then the aqueous phase was partitioned with 50 mL n-butanol (3 times). All fractions were concentrated in vacuo to give dry fractions: aqueous fraction (fr-Aq; 6.34 g, 31.7%), ethyl acetate fraction (fr-AcOEt; 4.82 g, 24.10 %), and the butanol fraction (Bu OH, 7.73 g, 38.65%). 1) Preparation of the extract and fractions The preparation of the extract proposed herein describes the preferred way of obtaining the fractions of interest, and should not be limited to the present invention. It is emphasized that it is important, after fractionation of the extract, to obtain a butanolic fraction. The preparation of the extract began by drying the R. mangle barks in an oven at 40 ° C for seven days and then milling them. Extraction is performed using acetone: water (7: 3) by the maceration method, which lasted seven days and yielded 31.4%. The acetone: water extract of R. mangle was then subjected to liquid-liquid partition with solvents of increasing polarity, thus obtaining semi-purified fractions. This methodology provides an adequate clean up of polar extracts (Calixto & Yunes, 2001).

Approximately 20 g of the extract was dissolved in 150 ml of water in a separatory funnel. 50 mL of ethyl acetate was added to the separatory funnel and partitioned 3 times. Then, the aqueous phase was also partitioned with 50 ml of n-butanol (3 times), as can be seen in figure 1. After this step, three fractions were obtained, the Ethyl Acetate Fraction (fr-EtOAc ), Butanolic Fraction (fr-BuOH) and Aqueous Fraction (Aq-F). All of them will be considered in the present description so as to show the efficiency of the Butanolic Fraction over the others.

In order to determine the components of the fractions of interest, as well as to obtain efficiency indications in the obtained method presented here, thin layer chromatography (CCD), high performance liquid chromatography (HPLC) and ionization mass spectrometry tests were performed. eletrospray (ES! MS) as described below. Descriptions of the embodiments of the tests are detailed in Examples 2, 3 and 4, respectively. 2) Thin Layer Chromatography (CCD) Through the three revelations used, the CCD showed important information about the compounds present in the tested samples through the colorings acquired by the compounds after reaction with the respective developers, besides visualizing the efficiency in the proposed fractionation process. in the present invention. It can be seen from Figure 2 (A) that the revelation of the chromatographic plate with anideide / H2SO4 allowed to observe the presence of catechin in the extract samples and fractions. The revelation with NP / PEG (natural products / polyethylene glycol), in turn, represented by Figure 2 (B), also points to this latter observation. Figure 2 (C), where the chromatographic plate revealed with FeCI3 1% is observed, draws attention to the possible presence of complex tannins, observation made by the grayish tint, probably a result of the mixture of complex (greenish) tannin with hydrolysable tannin. (bluish). 3) High performance liquid chromatography (HPLC) The HPLC-DAD analyzes were performed to verify the fractionation process and the separation observed in the CCD, so the method was not employed to identify substances, since no standards were injected for this purpose. The chromatographic profile obtained for each sample indicates that each of the fractions has its own chemical characteristics, evidencing the quality and success of the extractive procedures proposed here, as can be seen in figure 3. 4) Electrosprav ionization mass spectrometry (ESIMS) constituents of the fractions of interest were identified by electrospray ionization mass spectrophotometry tests. The constituents were identified by comparing molecular weights with literature data.

As in the initial chemical analysis performed by CCD and HPLC analysis, the mass spectra obtained by electrospray ionization of the compounds (Figures 4, 5, 6 and 7) corroborated the efficiency of the fractionation process proposed here for acetone extract: water (7: 3) from R. mangie barks.

These methods of analysis, one common and qualitative (CCD) and the other modern and with higher analytical resolution (HPLC and ESIMS), allowed to raise data set that provided necessary results for the proposition of structural alternatives within the class with greater efficiency. The analysis of these results indicated the chromatographic (figure 6) and mass fingerprint profiles (figures 8 to 11) obtained for extract and organic fractions indicated that the fractionation method described in this process is the most efficient, but not the only one. to obtain the fraction with the necessary quality and purity. The CCD, HPLC and ESIMS tests confirm the good execution of the fractionation. Spectra analysis was performed by molecular weight comparison and bibliographic search in databases such as SciFinder, PubMed, and ISI of Knowledge, while the proposed molecules composition (shown below) was performed using ACD / LabsChemSkeích 12.0 free version.

The proposed molecules based on the analysis of their molecular weight (m / z) constitute the class under study, being found representatives of the three classes of tannins known today, condensed, hydrolysable and as'exex3. Additionally, it has been observed that some heteroside condensed and hydrolysable tarin monomers as well as the isolated monomer, in this case catechin or epicatechin, are present in the obtained fractions.

The structures of the proposed polyphenolic compounds from the profile (ESIMS) of the acetone: water extract (7: 3) obtained from the R. mangle barks, (1) catechin or epicatechin, condensed tannin monomer; (2) and (3) (epi) heteroside catechin; (4), (5), (6) and (7) derivatives of elagitanines, hydrolysable tannin; (8) and (9) galotanine derivatives, hydrolyzable tannin; (10) and (11) complex tannin derivatives.

Table 1 - Description of the proposed molecules with their real mass (m / z) and the mass found in the ESIMS (m / z - H) From the extract profile and fractions, two in vitro antioxidant activity experiments, DPPH and Folin-Ciocalteu, the Folin-Ciocalteu Determination and DPPH radical reduction tests described below were carried out, and their execution is detailed in example 8, items A and B.

5) Determination of In Vitro Antioxidant Activity by Folin-Ciocalteu Test and DPPH Radical Reduction

Table 1 shows the results for these two models and the concentration of polyphenols in both extract and fractions is high.

The data obtained are in agreement with those found by Chapman (1976) in the R. mangle shells. The EC50 values for the antioxidant activity (DPPH) of both the crude extract and the fractions are low. The crude extract presented a lower value than the quercetin control. Figure 8 illustrates the DPPH reduction curve for the different extract concentrations and fractions. There is therefore an indication that extract and fractions were efficient in sequestering free radicals in vitro and present polyphenol concentration between 20 and 27%.

Table 2 - Reducing potential (DPPH) and total polyphenols (Folin-Ciocalteu) content of the R. mangle extract and fractions.

Then, to confirm the antioxidant potential of the fractions, which had already been indicated in the in vitro tests, in vivo tests were performed. 6) Determination of in vivo antioxidant activity 6A) Ethanol-induced gastric ulcer Ethane gastric lesions may be associated with the generation of ROS and these lesions produce imbalance between oxidative and antioxidant cellular processes (Repetto & Llesuy, 2002). Administration of ethanol may further result in changes in membrane permeability with reduced mucosal resistance and disturbances in H + secretion (Vasquez-Ramirez et al., 2006).

Ulcers appear due to the necrotizing action of ethanol on the gastric mucosa, with less participation of acid secretion (Lewis & Hanson, 1991).

Classically, in the search for antiulcerogenic compounds, the ethanol-induced experimental ulcer model in rats represents the first step of this research line, as it provides basic information about this activity, besides being useful to determine the treatment doses. In this model it was verified the antiulcerogenic activity of the aqueous (fr-Aq), ethyl acetate (fr-EtOAc) and butanolic (fr-BuOH) fractions in variable doses from 0.5 to 100.0 mg.Kg'1 for both. the latter (Figures 10 and 11) and 6.5 to 100.0 mg.Kg'1 for the first (Figure 9).

The effects of ethanol on the gastric mucosa can be classified as: 1) caustic damage with high concentrations of ethanol (above 20%), which allows lesions to penetrate deep into the vascular plexus causing stasis, rupture of the blood vessel wall, favoring hemorrhage. and mucosal necrosis; 2) damage by retrofusion of H + ions with low concentrations of ethanol (8 to 20%), which may lead to epithelial cell exfoliation and, in the presence of gastric juice, the rupture of the protective barrier of the gastric mucosa; 3) increased mucosal resistance, a paradoxical effect, with concentrations ranging from 5 to 20%, which may trigger adaptive cytoprotection. Moderately irritated sparingly used stimulate prostaglandin synthesis, which in turn favors mucosal regenerative processes (Slkiric et al, 1999). The ulcer induction method by ethanol administration, as well as the procedures for verifying the most active fractions, are presented in detail in Example 7A.

Figures 9, 10 and 11 demonstrate antiulcerogenic activity of organic fractions, the most effective dose to prevent the generation of gastric lesions by ethanol administration and evaluate the activity of antioxidant enzymes from the collected tissues. After more active fractions were defined, the lowest effective doses of each were selected from the dose-response curves obtained for fr-Aq, fr-EtOAc and fr-BuOH in the ethanol-induced ulcer model. Fr-Aq has anti-glycerogenic activity at doses of 12.5 and 100.0 mg.Kg'1, while fr-EtOAc demonstrates this activity at all doses tested (from 0.5 to 100.0 mg.Kg'1 ), with 1.5 mg.Kg'1 being the best, since among the most significant data there was no difference; As in the previous fraction, fr-BuOH was effective in all doses tested, and 0.5 mg.Kg1 was more interesting because it was the lowest dose and had the same significance among the most significant.

The results showed that, although the fr-EtOAc curve plot was more compatible with that of a dose-response curve, fr-BuOH proved to be much more effective from the point of view of statistical significance, and extremely high doses were used. low. Previous work has shown that tannins, especially condensates, have very low dose pharmacological activity (Iwasaki et al, 2004).

Thus, it is possible to evidence that the fractionation of R. mangie extract as described in the present invention, in order to obtain the butanoic fraction, allows us to concentrate sufficiently condensed tannins so that the fraction has surprising effect in the treatment of ulcerative lesions, as evidenced. the tests presented in this document. C) Antioxidant activity of sulfidic clusters, lipid peroxidation, and enzymes 6B) Ischemia-reperfusion-induced gastric ulcer In the ischemia-reperfusion-induced gastric lesion model, lesions occur without the use of chemical agents (Cabeza et al, 2001). Ischemia weakens the gastric mucosa barrier by increasing acid diffusion, which causes damage to the mucosa (Kawai etal., 1994).

During ischemia, there is a reduction in blood flow in the organ leading to a sequence of chemical reactions that result in dysfunction, cell necrosis and the appearance of toxic metabolites contributing to cell death (De Groot, 2005). With reperfusion, ROSs are generated, especially in the xanthine oxidase system and neutrophil activation, causing lipid peroxidation in the tissue; Thus, the combination of ROS with acid secretion promotes damage to the gastric mucosa (Wada et al., 1996).

Gastric lesions occurring in the reperfusion phase are considered more severe than those occurring during ischemia, as there is participation of ROS (including biomolecules such as 02 ', OH' and H202 that attack membrane lipids, nucleic acids, enzymes and receptors). , causing changes in structure, cell activity and protein transport, as well as changes, for example, in the influx of calcium into cells (Schoenberg & Berger, 1993; Cerqueira et al., 2005). The ischemia-reperfusion model was used to determine the antioxidant activity promoted by fr-BuOH without the participation of a chemical agent in lesion induction.

According to the results presented in the gastric ulcer tests, pretreatment with lansoprazole (30 mg.Kg "1) and fr-BuOH (0.5 mg.kg'1) was able to reduce the appearance of lesions induced by ischemia-reperfusion Gastric mucosa scraping was collected for the evaluation of antioxidant enzyme activity, as detailed in example 7B.

In this model, fr-BuOH was also effective in significantly reducing lesion formation (Figure 12), pointing to the protection given to R. mangie tannins and thus confirming the antioxidant activity in vivo.

Additionally, as performed in the ethanol model, antioxidant mechanisms of action were evaluated for compound levels and antioxidant enzyme activity.

Table 7 shows GSH levels and SOD activity. In this model, it was determined that administration of fr-BuOH maintained the GSH level high, and increased SOD activity, which was also elevated by lansoprazole. Table 8 shows that fr-BuOH promoted increased activity of GPx and GR enzymes, while table 9 shows fr-BuOH reduced LPO and MPO activity.

Table 3 - Table 7. GSH levels and SOD enzymatic activity of rats submitted to ischemia / reperfusion induced ulcer.

Table 4 - Enzyme activity of GPx and GR of rats submitted to Ischemia / reperfusion induced ulcer.

Table 5 - Enzymatic activity of OLP and MPO of rats submitted to ischemia / reperfusion induced ulcer. The set of data obtained for ex vivo antioxidant activity indicates that fr-BuOH has antioxidant action and among its mechanisms we can highlight the maintenance and increase of GSH levels, SOD, GPx and GR activity, reduction of OLP and MPO activity. Thus the anti-ulcer activity of ir-BuOH can be attributed in part to this antioxidant action. ROS are continuously produced during normal physiological events and are removed by the antioxidant defense mechanism. Under pathological conditions, ROS results in lipid peroxidation and ovidative damage. The imbalance between ROS and antioxidant defense leads to oxidative modification in the cell membrane or intracellular molecules (El-Habi et al, 2000). The first antioxidant enzyme in the gastric mucosa is SOD, which catalyzes the less harmful 02 'dismutation in H2O2. The second step of H202 metabolism depends on GPx activity. The reduction of H2 O in water by GPx is accompanied by the conversion of the reduced form gluatic (GSH) to the oxidized form (GSSG), which is then converted to GSH by the GR (Kwiecién et al., 2002).

Cell membranes are constant targets of ROS because they are rich in enzymatic complexes belonging to the O 2 -reducing chains. In them, the large amount of polyunsaturated fatty acids (AGP) favors the formation of ROS generating sites, causing lipoperoxidation due to the easy access of ROS to fatty acid unsaturation (Buege & Aust, 1978; McCord, 2000; Andreoli, 2000). This degenerative process, involving formation, propagation of lipid radicals peroxyl 3 alkoxy! (ROO, RO), O2 uptake, and double-binding rearrangement of AGP alters membrane integrity and flow (Buege and Aust, 1989) Gastric mucosa integrity depends on factors such as maintenance of mycocircution, mucus secretion, and enzyme activity. antioxidants such as SOD and GPx; Such factors also imply gastroprotection against exogenous and endogenous irritants in the gastric mucosa (Kwiecién st al, 2004). Thus, substances capable of guaranteeing mucosal integrity are potentially useful in the treatment and / or prevention of gastric lesions, which shows once again that the butanolic fraction of R. mangle extract concentrates compounds in order to cause a surprising effect due to the its ability to maintain the GSH level high. 7) Mechanism of antiulcerogenic activity 7A) Involvement of sulfhydric clusters in Q-protection It is observed from Figure 13 that fr-BuOH is not able to attenuate ethanol-induced injury in MEN pre-treated rats (a GSH blocker), indicating that its antiulcerogenic activity is dependent on sulfhydryl groups; 7B) NO involvement in oestroprotection Pretreatment of rats with L-NAME showed, as Figure 14 illustrates, that the anti-ulcer activity of fr-BuOH is lost when c and NO synthesis is blocked, indicating that NO participates in gastroprotection. promoted by fr-BuOH.

Table 6 - Effect of oral administration of fr-BuOH (0.5 mg.Kg'1) on rats submitted to puoro ligation, evaluation of secreted volume and its pH. The table 3 brings the data obtained in the pyloric ligation model, an antisecretive action of fr-BuOH was verified where the pH was significantly increased while there was a reduction in the volume of gastric secretion. 7C) Production of mucus adhered to the gastric mucosa In this model it was evaluated the production of mucus adhered to the gastric mucosa in drains submitted to pyloric ligation. Figure 15 shows that fr-BuOH was able to increase it by more than 100. %. 7D1 PGE2 production in the gastric mucosa Figure 15 presents the results obtained for prostaglandin production in the gastric mucosa. Pretreatment with fr-BuOH was able to stimulate the synthesis of PGE2 in the presence of indomethacin, a cyclooxygenase inhibitor. 8) Evaluation of gastric ulcer healing in a chronic model Figure 17 shows that fr-BuOH accelerates the healing process of a gastric ulcer in a chronic model. Since the beginning of the treatments, healing promoted by fr-BuOH occurred faster. 8A) Sub-chronic toxicity Monitoring of animal weight during chronic treatment for 14 consecutive days is shown in Figure 18. It is noted that the groups receiving lansoprazole and fr-BuOH showed lower weight gain from day 8 However, this difference was not significant.

Continuing the evaluation of fr-BuOH toxicity after 14 consecutive days of treatment, the relationship between organ weight and animal weight was analyzed, as shown in Figure 19. A significant difference was found in this relationship for the liver of animals treated with ir-BuOH; This evaluation suggests possible hepatotoxicity of this treatment, however increased liver mass is more associated with toxicity than penca.

8B1 Expression of COX-1, CQX-2 and EGF

Figures 20 to 26 show the graphs corresponding to the densitometric irtensities obtained in the membrane revelations used to analyze the expression of COX-1, COX-2 and EGF, using β-actin protein as internal control. The densitometry was calculated for each membrane, and for the analyzes the densitometric values of the proteins of interest were divided by the β-actin density found on the same membrane.

Figures 19, 20 and 21 demonstrate COX-1 expression after 4, 8 and 14 days of treatment respectively. No differences were verified as expected.

Then, in figures 23 and 24, the expression of COX-2 was verified, after 4 and 8 days of treatment respectively, no difference was found in the expression of the same on those days. However in figure 29, where the expression of COX-2 was verified after 14 days of treatment, a solid increase was found in the groups treated with lansoprazoi and fr-BuOh. There was an increase in the expression of epidermal growth factor after 14 days. treatment with lansoprazole and fr-BuOH, as shown in Figure 2. The increase in EGF expression is critical for faster ulcer healing in this model. 80 Histoanalytical analysis Figures 27 to 31 below show the results obtained in the histological (figures 27 and 28) and immunohistochemical (figures 29, 30 and 1) studies of the stomachs of animals submitted to the chronic ulcer model and treated for 14 consecutive days. . Figure 27 shows the best structural arrangement at the edge (especially of the glands) of the lesion in the treatments with lansoprazole (30 mg.Kg "1) and ir-BuOH (0.5 mg.Kg'1), the same is observed in Figure 28, however, the presence of mucus in the gastric glands is highlighted.

Figures 29, 30 and 31 show the immunolocalization of HSP-70, PCNA and COX-2 respectively; their presence was greater in the lansoprazole and fr-BuOH group, being more evident in the latter; Thus, the participation of HSP-70, PCNA and COX-2 was observed in the healing process, especially in groups where this process was more efficient and faster.

After observing all the tests and characterizations performed with the fractions obtained from R. mangle extract, it is possible to highlight the advantageous action of the butanolic fraction in the treatment of gastrointestinal injuries, including the ulcer. The obtained butanolic fraction concentrates complex tannins in surprising quantity, which allows the effect of its use to promote similar and even better results than the medicine currently used to treat these gastric injuries in considerably lower dose, being up to 60 times. lower The form of action of the butanol fraction is mainly as an antioxidant and epithelium-recovering agent by promoting the maintenance and / or elevation of GSH levels, which in turn favored the activity of SOD, GPx and GR, while reducing the values. of LPO and MPO. It also accelerates the healing process and also increases the production of mucus adhering to the gastric mucosa, all factors that contribute to the protection against gastric injuries.

Accordingly, the present invention describes the use of the butanol fraction in pharmaceutical or pharmaceutical compositions with acceptable excipients, as demonstrated above.

EXAMPLES

Example 1 - Species Collection The collection of biological material was performed in the continental area of the Santos / SP estuary system, taking advantage of a vegetation suppression authorized by IBAMA (authorization number: 116/2006) and performed by EMBRAPQRT under the care of from the vegetable taxonomist Msc. Paulo de Sa: ss Penieado Sampaio, also responsible for species identification.

Example 2 - Thin Layer Chromatography (CCD) In comparative CCD analysis, ready-made silica ge: 60 (Merck) plates of various sizes were used. The fractions obtained on the partitions were analyzed by CCD using as mobile phase CHCl3: MeOH: PrOH: H2O (5: 6: 1: 4 v / v / v / v, organic phase) and as standards: rutin, gallic acid, catechin epicatechin, lupeol, reserpine, friedelin. The following were used as developer: light LJV (254 and 366 nm), anisaldehyde / H2SO4, NP / PEG and 1% FeCh in methane! (Wagner et al, 1984).

Example 3 - High Performance Liquid Chromatography (HPLC) The oronatographic profiles of the R. mangle extract and fractions were obtained using HFL.C-UV / DAD system (Shimadzu) and ACE 5 C18 column (250 x 4.6 mm, id 5 um ). The mobile se was formed by acetonitrile (pump A) and H2 O with 1% acetic acid pH 2.88 (pump B), gradient [0 to 15min - 15% A; 15 to 22 min - 15 to 20% A; 22 to 25 min - 20% A; 25 to 30 min - 20 to 40% A; 30 to 35 min - 40% A; 35 - 50 min - 40 to 100% A; 50 to 55 min -100 to 15% A; 55 to 60 min - 15% A]. The injected sample volume was 20 pl with a concentration of 1 mg / ml and the effluent was monitored at 280 nm.

Example 4 - Electrospray Ionization Mass Spectrometry (ESIMS) Extracts were diluted in a solution containing 50% (v / v) chromatographic grade methanol (Tedia, Fairfield, OH, USA), 50% (v / v) water deionized acid and 0.5% ammonium hydroxide (Merck, Darmstadt, Germany). The fingerprints or ESIMS profile of the negative mode extracts were acquired and accumulated over 60s, and the spectra were scanned at m / z 100 to 1000 using a Micromass-Waters Q-TOF mass spectrometer. (Waters, Manchester, England). Capillary and cone voltages were set at -3000 V and -40 V, respectively, at a temperature of 100 ° C. The ESI-MS was made by direct injection, with typical 10 | 1.min -1 using a pump-coupled micro-syringe (Harvard Apparatus, MA, U.S.A.). Example 5 - Animals All animals come from the UNICAMP Bioterism Center (CEMIB). Male Wistar rats (150 to 250 g) were used for gastric lesion experiments and determination of antiulcerogenic and antioxidant mechanisms of action.

The animals were acclimated to laboratory conditions for at least seven (7) days prior to experimental handling, with temperature (23 ± 2 ° C) and 12-hour controlled light-dark cycles fed Nuvital ration (Nuvüab) and water ad übitum. The animals were randomly assigned to the different experimental groups and fasted (with ad libiturn water) for at least 12 hours, depending on the type of experiment.

All experimental protocols were approved by the Animal Experimentation Committee - CEEA / IB / UNICAMP. It is important to remember that the possibility offered by the model animates! It only affects the quality of the research and increases its applicability from this data. However, it is important to emphasize that in vivo experiments suffer interference from any kind of novelty or irritation to the anima! experimental, all manipulation, sounds, smells, all this means in research where the substrate is an animal model. In addition to all the natural stress to which the animals are subjected, there is the whole question that involves a certain experimental protocol. In this study, fasting is fundamental for investigations to be carried out. However, this fact can increase the stress of the animals. and awakens in some of them attitudes that may influence the results obtained. In fact, the greatest influence can be seen in reducing the linearity of acute model results, for example, and this does not occur with in vitro experiments where all conditions and variables are determined and controlled by the researcher. Example β - Used Drugs The drugs used to determine antiulcerogenic and antioxidant activity and their mechanisms of action were: Lansoprazole (MEDLEY, Campinas, Brazil), Indomethacin, Νω-nitro-L-arginine methyl ester (L-NAME), N -ethyl-maleimide (NEM), Quercetin, Gallic Acid (SIGMA Chemical Co, St. Louis, USA), PA acetic acid and PA sodium chloride ((CHEivICO, Campinas, Brazil). All drugs were prepared immediately for use. .

Example 7 - Experimental delinea In antiulcerogenic activity tests, the drugs were always administered orally at a dose volume of 10 mL.Kg'1. A control (negative) group was used which received an equivalent volume of a 0.5 'saline vehicle in which fractions and / or the standard drug were dissolved.

Rc a.r rats kept in special cage without cutter and subjected to Wi m at least 12 h, depending on the experimental protocol. In all injury-inducing models, stomachs were opened in the cs. greater curvature, pressed on glass plates (except chronic ulcer model) and photographed. The lesion area was calculated using the AVSoft BioView 4 program. A) Ethane-induced gastric ulcer After 24 hours of fasting, groups of rats were orally treated with the chronic fractions at varying doses (0.5; 1.5 ; 3.0; 6.25; 12.5; 25.0; 50.0 and 100.0 mg.Kg'1) with Lansoprazole (30 mg.Kg'1) and saline (10 mL.Kg) "1), except Sham group (manipulated animals) one hour before is induction of gastric injury. Gastric injury was induced by oral administration of 1 ml of absolute ethane. Animals were sacrificed 1 hour after administration. of the injurious agent, and the stomachs removed for counting ulcerative lesions (Morim.oío et al., 1991) B) Saemia-reperfusion-induced ulcer The rats were fasted for 24 hours with free access to water. Butanolic fraction - fr-BuOH (0.5 mg.Kg "1), Lansoprazole (30 mg.Kg'1) and Saline (10 mL.Kg'1) were performed orally except Sham group (a manipulated animals), 30 min before anesthetizing the animals. Under anesthesia, the animals underwent a surgical procedure to clamp the celiac artery.

After half an hour of ischemia the clamp was removed and then the rats were subjected to one hour of reperfusion and then sacrificed (Ueda, et al., 1883). Example 8 - In vitro antioxidant activity assays A ) Folin-Ciocalteu The extract and fractions were analyzed for their scL total phenolic content using the Folin-Ciocalteu colorimetric method (Piccinelli et al., 200; Wu eia /., 2004). For this, extract and fractions were solubilized in ethanol, and dilutions were prepared with concentrations between 20 to 200 ppm. For the reference substance (gallic acid) an analytical curve was drawn at the concentrations of 3,125, 6.25, 12.5, 25, 50, 100 and 2Z0 ppm. The absorbance of the samples and standard sample was measured by an elisa reader (λ = 730 nm) and the results were expressed as mg of gallic acid equivalents (AGeq) per gram of extract or fraction on dry basis (mg AGeq / g). . As negative control was used the diluent of samples and as positive control, galic acid (200 ppm). The experiments were performed in triplicate.

B) Evaluation of DPPH Radical Reduction DPPH (2,2-diphenyl-1-picrylhydrazyl) radica is stable, purple in color, but when reduced it becomes yellow in color. Through this assay the ability of the test samples and standard sample to produce the DPPH radical was evaluated. For this, the extract and fractions (2, S mg) were dissolved in ethanol (1 mL), obtaining a stock solution. Several dilutions were prepared in ethanol to obtain final assay concentrations of 3.125 to 200 ppm (extract and fractions). For each sample (10 µl) 100 µl were added. μΙ_ ethanol, 100 μΙ_ phosphate buffer (100 mM) and 50 μΙ_ DPPH solution (250 μΜ). After 30min the absorbance was measured in an elisa reader (λ = 517 nm) and the percentage of radical reduction was calculated by the equation:% reduction = 100 - [(Abs. Sample - Abs. Negative control) / Abs. DPPH control - Abs. negative control) x 100] (1-Huang et al, 2005; Prior et al, 2005; Cuendet et al., 1997). The sample diluent was used as the final control and the positive control was the quercetin flavonoid.

All men and boys were performed in triplicate.

Example 9 - In vivo Detoxifying Activity Assays For the determination of antioxidant mechanisms, the stomachs obtained had their glandular portions scraped thoroughly with glass slides and homogenized in 1 mL of 0.1 M phosphate buffer (pH --- 7 *). and frozen at -70 ° C until biochemical tests are performed.

Enzyme activities, or levels of antioxidant compounds, were determined spectrophotometrically in ELISA readers. Protein concentration was determined according to the method described by Bradforc (1976) using bovine serum albumin to establish the standard curve. A) Levels of sulfhydric salts (GSH) The samples were centrifuged (12000 rpm at 4 ° C for 15 minutes) and the supernatant diluted (1:10) in sodium phosphate buffer (0.1 M, pH = 7.4). Then, the absorbance of 100 µl of the sample was read, plus 100 µl of Tris solution (1.0 mM) and EDTA (0.02 mM) at 412 nm (A1). After this reading 20 µl of 5.5 dithiobis (2-nitrobenzoic acid) diluted in methanol (DTNB - 0.01 mM) was added and then read again (-2) at 412 nm after 15 min. reaction, for determination tíe gr parn-; non-protein sulfhydryl compounds (GSH). Concentration of sulfhydryl (thio!) Parentheses is given by (A1-A2) x 1.57 (Faure & Lafond, 19S5; B) QluíatTna reductase (GR) activity To determine GR activity, the enzymatic reaction was prepared with 100 μί_ of the sample (after centrifugation and dilution of the supernatant in thalamus phosphate pH = 7.4 at 1:10 ratio) and 150 μΙ_ of the cocktail formed by EDTA (0.20 mM), oxidized glutathione (1 mM) and NADPH (0.1 mM). The absorbance was read at 365 nm between 1 and 10 min (Carlberg & Mannervik, 1985). C) Glutatic activity on peroxidase (GPx) The samples were centrifuged at 12000 rpm at 4 ° C for 15 min and the supernatant diluted in 0.1 M phosphate buffer (pH = 7.4) in 1:10 ratio. To a 100 µl aliquot of the scrape, 150 µl of reduced glutanoru (10 mM), NADPH (4 mM) and glutathione reductase (1U) and 20 A in H2Q2 (25 mM) solution were added according to Yoshikawa method. et al., (199 Osorbance was determined at 365 nm, between 1 and 10 min.

Dl Supermutase (SQD) activity In this assay the method described by Winterbourn et al. (1975), where samples were centrifuged and diluted in phosphate buffer (0.1 M, pH = 7.4) at 1:20 ratio. To 100 μΙ of the homogenate was added 150 pu of the hypoxanthine solution (0.1 mM), xanthine oxidase (0.07 U) and r. trobluetetrazolium (NBT - 0.6 mM).

The absoibanias were read at 560 nm. Results were expressed as U / rng and protein. E) Myeloosroxiaase Activity (MPO) MPO activity in the gastric mucosa was measured according to the method proposed by Krawisz et al., (1984), as minor modifications in Fa ■ ns-S: lva et a! 2007). Aliquots of the supernatant were used (6.7 µL) as 0.05 M phosphate buffer, pH 6.8 containing 0.0005% Acirogen percxid and 1.25 mg / mL o-dianisidine dihydrochloride, following reading at 460 ° C. nm for 10 minutes at 1 minute intervals. F) Determination of Lipid Peroxidation Index (LPQ) The homogenate of the glandular portion of the stomach was diluted in 0.15 M KCI (1 * 0 ratio). SBguids, 3 0.5 ml dà © stomomomoGnato 0.2 ml dodecyl sulfate (8.1%) were added; 1.5 mL acetic acid (20%, pH 3.5 adjusted solution with pH); 1.5 mL thiobarbituric acid (0.8% w / v) and 0.3 mL distilled water. All samples were left in a water bath with thermostat adjusted to 95-C for 1 hour. After this time, the samples were cooled and added with 1 mL distilled water and 5 mL n-buanol + pyridine mixture (15: 1, v / v), vortexed for 1 min and centrifuged at 1400 G for 10 min. minutes The absorbance of the organic layer was determined at 532 nm. Tetraethoxypropanc (t-pp): diluted in ethanol was used as standard. Results were expressed as pie; of thiobarbituric acid reacting substances (TBARS color mg protein (pmol TBARS / mg protein) (Ohkawa et al., 1973 '.

Example 10 - Meear. rics of antiulcerogenic activity A) Involvement of ç. .. sufydrylic comments on gastroprotection 24-hour fasting male VVistar rats were divided into groups according to the treatments (Salina + Salina, Salina + NOR, Lansoprazol + Salina, Lansoprazol - NOR, fr-BuOH + Salina and fr-BuOH + NOR). Controls received iniraperitoneal ejection of saline and the other animals from NEM (10 mg.Kg'1), a blocker of the suphydryl group. After 30 minutes the groups received orally the treatments Saline (10 mL.Kg'1), Lansoprazole (30 mg.Kg'1) and fr-BuOH (0.5 mg.Kg'1). After one hour (1h), the animals received orally 1mL of ethanol. One hour (1h) after treatment with ethanol the animals were sacrificed and stomachs removed for counting ions (Matsuda, 1999). B) Involvement of fats, Cats, and fats protection 24-hour fasting male VV ctar rats were divided into groups according to treatments (Saline + Saline, Saline + L-NAME, Lansoprazol Saline, Lansoprazol + L-NAME, fr-BuOH + Saline and fr-BuOH + L-NAME). Controls received intraperitoneal injection of saline and the other animals from L-NAME (70 mg.Kg'1), a suphydryl group blocker. After 30 minutes the groups received orally the treatments Saline (10 mL.Kg'1), Lansoprazole (30 mg.Kg'1) and fr-BuOH (0.5 mg.Kc ').

After one hour, the animals received orally 1 ml of ethanol. One hour after ethanol treatment the animals were sacrificed and their stomachs removed to count the lesions (Matsuda, 1999). C) Assessment of acid secretion by the Shav method After 24 hours of fasting, animals under anesthesia (ketamine 30 mg.Kg'1 and xylazlna 0.3 rng Kg1) underwent a longitudinal incision just below the apophysis x'3. de for pylorus location and tying. Saline (C mL.Kg "1), Lansoprazole (30 mg.Kg'1) and fr-BuOH (0.5 mg.Kg'1) treatments were administered intraduodenally shortly after pyloric ligation, and in segiuria, sutured incisions Four hours after the surgical procedure, more air was sacrificed, incisions reopened, and stomachs removed Stomach contents were collected to determine the volume and cH of gastric secretion (Shay et al, 1945). D) Production of mucus adhering to gastric mucosa Gastric wall mucc was determined in rats submitted to pyloric ligation.The different experimental groups of rats received the treatments Saline (10 ml.Kg'1), Lansoprazol (30 mg.Kg "1). and fr-BuOH (0.5 mg.Kg-1) via the route, one hour later these anesthetized animals were subjected to pyloric thinning according to the method described above.

After the animals were sacrificed, the stomachs were removed and opened in the direction of great curvature, the glandular segments of the stomach were removed and weighed.

Each segment was immediately transferred to a tube containing 10 mL 0.1% Aian blue (in 0.05 M sodium acetate buffered 0.16 M sucrose solution, pH 5). After soaking for 2 h in this solution, the excess stomach was removed by two successive washes with 10 mL of 0.25 M sucrose solution, first for 15 min and then for 45 min. Aician Blue complexed with gastric wall mucus was extracted with 10 ml 0.5 M MgCl 2 with intermittent stirring for 1 min at 30 min intervals for 2 h. A 4 ml aliquot of Aician Blue's extract was vigorously stirred with an equal volume of cyclic ether; The resulting emulsion was centrifuged at 3600 rpm for 20 min at the absorbance of the aqueous layer determined at 580 nm. The amount of Aiciane extracted per gram of glandular tissue was then calculated, and expressed as dye concentration in pg per ml of solution per gram and glandular fraction (Corne et al, 1974). E) Production of PGE2 rs. Gastric gastrosis The experimental groups were treated v. with Saline (10 mLKg'1) r iY-BuOH (0.5 rng.Kg'1), except sham (manipulated animals), Thirty-maturity after treatment, 30 rng.Kg'1 indom was administered to the animals. sreeina sc, and 30 minutes later the animals were sacrificed, the wounds removed and opened in the direction of greatest curvature. The mucosa was scraped with the aid of two glass slides, weighed and suspended in 1 ml sodium phosphate buffer (10 mM; pH 7.4). The tissue was homogenized with the aid of a Polytron®, then not at 37 ° C for 20 min. PGE2 in the buffer was determined by a radioimmunoassay (R&D System KGE004) and all data on PGE2 synthesis capacity were expressed in PG / mg gland tissue weight / (Curtis, et al, 1995 F) Evaluation of the scarring gastric ulcer action in a chronic model Fc.arn rats anesthetized with ketamine (30 rng.Kg'1) and xylazine (0.3 rng.Kg'1) for stomach exposure through an incision approximately 1 c 'performed below the xiphoid apophysis, and was then deposited, with au cs, a micro pipette, in an area demarcated with a plastic tube, 50 μ., of 40% acetic acid in the serous layer of the junction. bottom of the stomach with the antrum of the stomach.The next day one animal from each group experimented to determine the initial area of the lesion.Two days after the rash, treatments (Salina, Lansoprazol and fr-BuGA) were started, per via ora !, which persisted for 14 consecutive days (Okabe & Ameçase, 2005).

After 4, 8, and 4 days the animals were sacrificed and their stomachs removed open in the direction of greatest curvature and the stomachs removed for determination of the area of bonding, and immunohistochemistry and western blot assays. F.1) Subcritical Toxicity The subchronic toxicity test is an estimate of the toxic properties of the test substance at its therapeutic dose (Lima et al., 2003). The weight in the above described model was recorded during this time: 15 days, the animals were sacrificed and the organs (heart. Acaac, kidneys, lungs, testicles) removed for weighing. Ages were expressed by arcosene organ weight / animal weight.

F-2) Expression of CQX-J1, COX-2 and EGP The stomach scrape was centrifuged at 12000 rpm at 49 ° C for 15 minutes. Protein concentration was quantified by the Bradford method (1976). Determinated protein sweats (50 pg) were applied on polyacrylic gel (SDS-PAGE) and electrophoresed with buffer solution (Trism case 25 mM, glycine 1.92 mM, 1% SDS). The SDS-PAGE was initially subjected to 40V until the passage of the demarcated line through the phase ie protein stacking, and 120V to the thin! from ge! resolving. Then, the proteins separated in the SDS- "AGE were transferred to a nitrocelluiosis membrane in electrotransfer mode, with the membranes soaked in transfer buffer (25 mM base Trism, 192 mM glycine, rnet; maintained at a constant voltage of 90V for 2 hours Nitrocelluiosis mmbs containing the transferred proteins were bloated in a biochemical solution (10mM NaCM50, 10mM Tween 20, 0% milk). 5% skimmed powder) for one hour to decrease nonspecific protein protein, then the membranes will be washed with basic buffer (150 mM NaCI, Trism base 1C mH, Tween 20 at 0.05%) at 10 minute intervals The member is incubated at 4 ° C overnight using specific antibody "for COX-1 and COX-2 (Cayman), and EGF (Santa Cruz). The next morning, the nitrocellulose membrane was washed in basic buffer for 40 minutes and then incubated at room temperature for 1h with secondary antibody. Scientific Goat Anti-Rabbit IgG (H + L) 31460 for COX-1 and COX-? and Zvmed San Francisco, CA, USA ZyMax Rabbit Anti-Goat IgG (H + L) HR: "2 ocrvj3ate 81-1620 for EGF. To detect the immunoreactive bands. blots were exposed to chemiluminescence solution ΈΓΓ pius-LumiGo chemiluninescense substrate, Kirkegard and Ps. " .Η. rsuoro, MD. USA) for 1 minute, followed by exposure to a single for chemiluminescence detection (HyperfilmTM ECLTM - AmsrSham Pharmacia Biotech, Buckinghamshire, England).

Census data of the film samples were captured for qi, optical densitometric donation using AVSoft Bioview 4 software. F.3) Histological analysis After ε fixation of the material in ALFAC (formalin, 80% alcohol, acetic acid) by 24 hours, the pieces were dehydrated and included in parapiasl.

Posteriorly. the paraplast blocks were cut (7 pm thick) in micro orb. in a serious way. Samples were subjected to colo 1 and gO r hematoxylin-eosin (HE) (Behmer et al., 1976) for general observation of structure and cellularity, and periodic acid of Schifà ° AS (Vacca, 1985) for analysis. of mucopolysaccharide substances air. light microscopy. F.4) Immunohistochemistry: · For immunohistochemical rheumatism, a slide was used representative of each ray of the animals submitted to acetic acid-induced ulcer, which was deparaffinized, rehydrated, and destined for immunohistochemistry. with development method for peroxidase. The mespecific reaction blockade was performed with skim milk solution after rsc-, sniggenic waxing as shown in the table below, and samples were incubated with specific antibodies in overnight blockage solution. Postencmeots samples were washed in phosphate buffer (0.01 mol / L, pH 7.4) and incubated in secondary antibody (ABC Vector Kit) and stained with Avndma-Siotin associated with 3-3'diaminobenzine tetrahydrochioride · DA3 Sigma), and further analyzed by Leica nomicroscope. D * v · coupled with Leica QWir Stanoard image capture software, UK.

Example 11 - Rings * .V! Ca All smnacological results were expressed as mean ± standard deviation, or standard error, and subjected to one-way analysis of variance (ANOVA) and Dunnett's and / or Tuckey's s-test. with a significance level of * P <C.Ò5. "° <0.01 and *** P <0.001.

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Claims (13)

1. Compounds characterized by having the structural formulas Compounds according to Claim 1, characterized in that they are for treating gastrointestinal ulcers and injuries. Medicament characterized in that it contains one or more of the compounds described in claim 1. 4. Pharmaceutical composition characterized in that it contains Rhizophora mangle fraction associated with one or more pharmaceutically active excipients. Pharmaceutical composition according to Claim 5, characterized in that the fraction was obtained using butanol. Pharmaceutical composition according to Claim 5, characterized in that it stimulates prostaglandin E2 synthesis in the presence of indomethacin. Pharmaceutical composition according to Claim 5, characterized in that it maintains or raises the level of sulphide groups. Pharmaceutical composition according to Claim 5, characterized in that it maintains or raises the level of the superoxide dismutase enzyme. Pharmaceutical composition according to Claim 5, characterized in that it maintains or raises the level of the enzyme glutathione peroxidase. Pharmaceutical composition according to Claim 5, characterized in that it maintains or raises the level of the enzyme glutathione reductase. Pharmaceutical composition according to Claim 5, characterized in that it reduces lipid peroxidation. Pharmaceutical composition according to Claim 5, characterized in that it reduces the activity of the enzyme myelperoxidase. Process for obtaining the compounds described in claim 1, characterized in that the butanolic fraction is preferably obtained by the following steps: a. Rhizophora mangle bark maceration in water and acetone solution; B. Filtration of extract obtained from (Â) c. Dissolution of the extract obtained in (B) in water; d. Liquid-liquid partition of the solution obtained at (C); and. Dissolution of the extract obtained in (D) in ethyl acetate; f. Partition of the fraction obtained in (E) by 3 times; g. Partition of the aqueous phase obtained in (F) with n-butanol 3 times