BRPI0306167B1 - PROCEDURE FOR OBTAINING SYLVESTRIS CASEARIA EXTRACTS AND ACTIVE FRACTIONS AND THEIR USES - Google Patents

Abstract

"process of obtaining extracts from casearia sylvestris, processes for obtaining active fractions, extracts, active fractions, use of extracts and active fractions, composition, dosage unit, method to prevent, treat, combat or suspend gastrointestinal disorders, medicine and principle". active". The present invention relates to an antiulcerogenic drug, more particularly to a drug comprising an active fraction of the sylvestris casearia plant as an effective component. In particular the invention deals with a drug useful in the treatment of disorders of the gastrointestinal tract, especially in the treatment of gastroduodenal ulcer. It has cytoprotective action, acts on both mild and moderate and severe ulcers and, being selective, has no abortifacient action.

Description

(54) Title: PROCESS OF OBTAINING EXTRACTS AND ACTIVE FRACTIONS FROM SYLVESTRIS HOUSEHOLD AND ITS USES (51) Int.CI .: A61K 31/78; A61P 1/04 (73) Holder (s): SÃO PAULO STATE RESEARCH FOUNDATION - FAPESP. UNIVERSITY OF SAO PAULO. PAULISTA STATE UNIVERSITY JÚLIO DE MESQUITA FILHO - UNESP (72) Inventor (s): JAYME ANTONIO ABOIN SERTIÉ; RICARDO GOMIDE WOISKY; ALBERTO JOSÉ CAVALHEIRO; VANDERLAN DA SILVA BOLZANI; ANDRÉ GONZAGA DOS SANTOS; ARISTEU GOMES TININIS

1/28 “PROCESS OF OBTAINING EXTRACTS AND ACTIVE FRACTIONS FROM SYLVESTRIS CASEERY AND ITS USES”

Field of the Invention [1] The present invention relates to an antiulcerogenic drug, more particularly, to a drug comprising an active fraction of the Casearia sylvestris plant as an effective component. In particular, the invention deals with a drug useful in the treatment of disorders of the gastrointestinal tract, especially in the treatment of gastroduodenal ulcers, since it has a cytoprotective action, acts on both mild, moderate and severe ulcers and, being selective, does not have an abortive action.

Background of the Invention [2] Disorders of the gastrointestinal tract, especially ulcerative pathologies are considered to be one of the characteristic conditions of modern life. Less than a decade ago, these pathologies predominated in the male population, economically active and living in large centers (executive disease). Nowadays, such disorders no longer respect sex, socio-economic conditions and much less age, a fact that is easily justified, given that 90% of gastroduodenal ulcers result mainly from stress and eating habits.

[3] As a result of the intense urbanization that has been occurring in the last decades, the level of stress increased considerably, and it was found that the growth of this pathology was proportional to the growth in the consumption of antacid and anti-ulcer medications. According to data from Pharmaceutical Marketing Brazil (IMS) in the year 2000, the world pharmaceutical market had an amount of 380 billion dollars, with an estimated annual growth of around 10%. Among the most consumed medicines that account for 31% of the global market, the group of antiulcer drugs, for the tenth consecutive year, tops the list of the most

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2/28 sold, with omeprazole standing out, which accounts for 35% of sales in this specialty. According to data from the National Health Surveillance Agency (ANVISA), the Brazilian drug market, with annual sales of around 13 billion dollars, is the fifth largest market in the world.

[4] Antacids are used by doctors in the treatment of hyperchlorhydria, defined as the excessive presence of hydrochloric acid in gastric juice, as well as by lay people as a form of self-medication aimed at a large number of symptoms. Gastric antacids are a group of drugs whose use is extremely abusive and are used in any gastric rash or disorder. The incidence of placebo effects in individuals with minor gastrointestinal disorders, further induces the layperson to the inappropriate use of antacids.

[5] Antiulcer drugs commonly used worldwide include H2 receptor antagonists (histamine receptor), M3 receptor antagonists (muscarinic receptor), Na ⁺ / H ⁺ pump inhibitors and peptic antiulcer agents, the latter comprising cytoprotective drugs.

[6] The presence of histamine in the stomach induces an increase in the synthesis of hydrochloric acid (HCl) from the degradation of carbonic acid (H2CO3), releasing an H ⁺ proton that reacts with the chloride anion (Cl ^- ) giving rise to the acid hydrochloric, which when in high concentrations, in addition to causing pain, as a result of spasm caused in the gastric smooth muscle, leads to an increase in the formation of pepsin; this enzyme in the absence of cytoprotective mechanisms is responsible for the destruction of the mucosa and the appearance of gastric ulcers.

[7] Histamine H ₂ receptor antagonists, for example, cimetidine and ranetidine, are drugs that have a similar structure to that of

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3/28 histamine and competitively bind to the H ₂ receptor in the gastric mucosa preventing the binding of histamine or displacing it from its receptive site, resulting in an elevation in the pH of the medium.

[8] M (muscarinic) receptors are receptors whose activation produces an effect similar to those produced by extracts from the mushroom Amanita muscaria, hence the name received. Once activated, they produce effects such as increased secretion of salivary and sweat glands, contraction of smooth muscles in the digestive and urinary tracts, among others, that is, an increase in the secretion of hydrochloric acid in the stomach. These effects can be blocked by antimuscarinic drugs such as atropine, which is widely used in the treatment of gastrointestinal disorders, especially as an antispasmodic. Muscarinic receptors are named depending on their location in the body and their specific antagonists. Specifically, M ₁ (neural) receptors are present mainly in the central nervous system, ganglia and in gastric enterochromaffin cells, M ₃ (glandular), are in exocrine glands, smooth muscles and vascular endothelium and parietal cells of the stomach.

[9] Drugs that inhibit the action of the proton pump, also called a proton pump (Na ⁺ / H ⁺ ), among which omeprazole stands out, also raise the pH of gastric juice too much, in addition to requiring, in its production , packaging in acid-resistant capsules, which makes the final product more expensive.

[10] Thus, most drugs used to treat gastrointestinal disorders result in an increase in the pH of gastric juice.

[11] As is already known to the person skilled in the art, protein digestion begins in the stomach, where pepsin breaks down proteins into proteases, peptones and polypeptides. However, this important peptic enzyme requires an acidic medium for it to function. Pepsin is more active

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4/28 at pH between 1.6 and 3.2, being completely inactive at pH close to 5.0, that is, the digestive enzyme activity of pepsin decreases with the increase of gastric pH (GUYTON, AC, Textbook of medical Physiology , WB Saunders company, Philadelphia, pp.816-826, 1981. AND GANONG, WF, Review of Medical Physiology, Lange Medical Publications, Los Altos, California, pp.384393, 1983.).

[12] According to BASILE, AC, et. al., “Pharmacological assay of Casearia sylvestris. I: Preventive anti-ulcer activity and toxicity of the leaf crude extract ”. J. Ethnopharmacol. 30: 185-197, 1990; SERTIÉ, JAA, et. al., “Preventive anti-ulcer activity of the rhizome extract of Zingiber officinale. Phytotherapy 63: 55-59, 1992; and BACCHI, EM & SERTIÉ, JAA, “Anti-ulcer action and toxicity of Styrax camporum and Caesalpina ferrea”. Planta Medica 60: 117-120, 1994, H ₂ receptor blockers (cimetidine, ranetidine) and M ₃ (pyranzepine) are drugs that have the property of raising gastric pH (more emphatic elevation with M ₃ blockers), a factor this which, in addition to altering protein digestion, can alter the sterility of the environment, facilitating the installation of opportunistic infections, combined with the fact that they do not present cytoprotective activity.

[13] It should be noted that, in 1987, Hornick raised the hypothesis that the peptic ulcer was the result of an infection caused by Helicobacter pylori (HORNICK, RB, “Peptic ulcer desease: a bacterial infection?” N. Engl. J Med. 316: 1518-1523. 1987.). However, until now, technicians on the subject have not reached a conclusion as to whether this bacterium would be the cause of ulcers or whether it would have its growth facilitated by the change in the pH of gastric juice, which is motivated by the use of anti-ulcer agents with local or systemic action , thus contributing to the maintenance of the injured area, especially in chronic ulcer patients.

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5/28 [14] Another fact to highlight is the prolonged use of some of these drugs, especially the H ₂ receptor blockers, which can lead to tachyphylaxis, that is, the patient does not respond to the usual doses, requiring continuous elevation of the dose to obtain the desired effect.

[15] In addition, in the last 15 years there has been a huge advance in the study of pathophysiology, pathology and pharmacology of the gastric mucosa, and the knowledge about the mechanisms involved in the formation and secretion of acid and pepsin has greatly expanded. The discovery of H ₂ receptors and their pharmacological inhibitors allowed the use of modern therapies to treat gastroduodenal ulcers. However, even these potent drugs are unable to prevent acute damage to the gastric mucosa produced by necrotizing agents.

[16] On the other hand, another area of research, called "cytoprotection", has been studied aiming at the development of new ways to increase the defense and protection of the gastric mucosa. This new area of research comprises, as one of its main themes, the study of the effects caused by prostaglandins in the body.

[17] Prostaglandin is the generic name for several substances, called autacoids, found in numerous tissues, such as, for example, in the prostate, in the seminal fluid, in the muscles, in the brain, among others. These substances have remarkable and very diverse biological properties, interfering with mechanisms that lead to hypotension, dilation of the bronchi, stimulation of intestinal peristalsis and uterine contractions (during childbirth), abortion and contraception. Its therapeutic indications are not yet sufficiently clarified, but prostaglandins have been shown to be efficient in increasing the defense and protection of the gastric mucosa against acute injuries produced by a variety of ulcerogenic and necrotizing agents through different mechanisms of inhibition of acid secretion.

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Synthetic analogs of prostaglandin are already used in the treatment of gastric ulcers.

[18] The concept of cytoprotection has increased knowledge about the mechanisms involved in the protection of gastric mucosa, encompassing not only mucus and bicarbonate secretion but also aspects related to cell proliferation and renewal and microvascular blood flow. In addition to prostaglandins, other agents such as essential fatty acids, sucralfate, antacids containing aluminum and extracts from known Chinese plants (suflacone) have proved to be important protective agents of gastric mucosa against acute injuries. Some of these agents act by increasing the release of prostaglandins and / or inhibiting their deactivation, while others probably act by mechanisms unrelated to prostaglandins.

[19] The main advantage of cytoprotective drugs, such as misoprostol, is that they interfere very little in the parameters of gastric secretion, especially in pH, acting basically by physiological mechanisms. These drugs have as main disadvantages, however, the undesirable abortion action, which requires control in their commercialization, the fact that they do not act on ulcers of mild and moderate intensity, and their cost is quite high.

[20] In view of the facts described above, it is necessary to search for new drugs effective in the treatment of disorders of the gastrointestinal tract, mainly in the treatment of ulcers, more particularly still in the treatment of gastroduodenal ulcers, which act at all levels of ulceration (mild, moderate and severe), and which have lesser side effects.

[21] Casearia sylvestris (popularly known as guaçatonga) is a plant in the family Flacourtiaceae originally from tropical America that can

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7/28 be found from Mexico to Argentina. It is known to technicians in the subject for exerting a high healing power and for presenting significant antiulcerogenic action.

[22] Previous studies were carried out more than a decade ago by one of the inventors using extracts obtained from the plant Casearia sylvestris. These studies have shown advantageous properties, in relation to classic drugs, of said extracts when applied to the treatment of stress-induced ulcers. (Sertié, et. Al. “Pharmacological assay of Casearia sylvestris. I” Preventive antiulcer activity and toxicity of the leaf extract. Journal of ethnopharmacology, 30: 185-97, 1990; and Sertié, et. Al. “Antiulcer activity of the crude extract from the leaves of Casearia sylvestris. ”Pharmaceutical Biology, 38 (2): 112-9, 2000).

[23] The present invention relates to new drugs with cytoprotective action useful in the treatment of disorders of the gastrointestinal tract, particularly ulcers, and more particularly gastroduodenal ulcers, which have advantageous properties when compared to existing drugs for the same purpose.

Brief Description of the Invention [24] A first object of the present invention are potentized extracts and active fractions, from leaves of the Casearia sylvestris plant, useful in the treatment of gastrointestinal disorders, particularly of antiulcerogenic action, more particularly for the treatment of gastroduodenal ulcers.

[25] By potentiated action, as used in this text, it is understood that the extract or fraction is capable of achieving the desired effect even if applied in lower doses than those used with extracts known in the art.

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8/28 [26] A second object of the present invention are drugs of potentiated action, useful in the treatment of gastrointestinal disorders, particularly drugs of antiulcerogenic action, more particularly for the treatment of gastroduodenal ulcers comprising the active fraction or extract, obtained from leaves of the Casearia sylvestris plant.

[27] Another object of the present invention are pharmaceutical compositions that comprise a pharmaceutically acceptable carrier and a therapeutically effective amount of the Casearia sylvestris potentiated extract and / or active fraction.

[28] Additionally, the object of the present invention is a method of treating gastrointestinal disorders, particularly ulcers, which comprises administering to a body in need of such treatment a therapeutically effective amount of the said potentiated extract or active fraction. In a preferred embodiment, the dosage used in this treatment is reduced.

[29] Yet another object of the present invention is the use of said extracts or potentiated active fraction or their compositions for the manufacture of a medicament for the treatment of gastrointestinal disorders, particularly ulcers, more particularly still gastroduodenal ulcers.

Brief Description of the Figure [30] Figures 1 and 2 illustrate the flowcharts of the general procedures for obtaining the active fractions of Casearia sylvestris, from ethanolic extracts.

[31] Figure 3 illustrates a graph comparing the reduction in the injury rate when Extracts I and III are used. Effect of oral administration of Extracts I and III at doses of 57.5, mg / kg, compared to control (1% Agar) in gastric lesions induced by stress and immersion in water.

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9/28 [32] Figure 4 illustrates a graph showing the Chromatogram of the active fraction A - Characterization of the active fraction A, by High Performance Liquid Chromatography, of Casearia sylvestris, consisting predominantly of casearins (A unidentified Casearinas).

[33] Figure 5 illustrates a graph showing the Chromatogram of the active fraction B - Characterization of the active fraction, by High Performance Liquid Chromatography, of Casearia sylvestris, consisting predominantly of casearins (A unidentified Casearinas).

[34] Figure 6 illustrates the typical UV spectrum of casearins. The typical UV spectrum of the main components of fractions A and B has only one band, with maximum absorption at 235 nm, (ε 13500), attributed to the C ₁₂ terminal diene. and C ₁₄ , characteristic of casearins.

Detailed Description of the Invention [35] Firstly, the objective was to obtain a purified extract, derived from potentiated extract of the plant Casearia sylvestris, to extract the active fraction of the plant that is responsible for its desirable pharmacological effects.

Process of obtaining potentiated extract from Casearia sylvestris [36] The methodology applied for industrial extraction of the active fraction of Casearia sylvestris, with the objective of establishing a viable method of operation was carried out according to a process that comprises at least the steps of:

- A. Extraction with solvent;

- B. Phase separation; and

- C. Elimination of the solvent;

[37] In particular achievements, some details are noted, and some other steps can be inserted, for example as shown below.

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1) Preparation of Raw Material: the leaves of Casearia sylvestris are collected and then prepared according to means known to the person skilled in the art, for example, dried in greenhouses with air circulation at a temperature of approximately 30 to approximately 40 ^o C.

2) Weighing and grinding plant material: Casearia sylvestris leaves are weighed and ground according to known techniques, for example, in a disintegrator-type electric mill or electric knife mill.

3) Extraction: the dried and ground leaves of Casearia sylvestris are extracted with solvents that have different polarities, for example, hexane, dichloromethane, chloroform, ethyl acetate, methanol and preferably ethanol, for approximately 10 minutes, particularly approximately 20 minutes. According to one of the preferred embodiments of the present invention, said extraction is carried out for approximately 2 hours. The extraction can occur, for example, by decoction, percolation, maceration or sonication, both cold, hot and / or with agitation, preferably by sonication in an ultrasound bath or by mechanical agitation.

[38] According to preferred embodiments, an Extract I of the present invention is obtained by performing the extraction for approximately 20 minutes of sonication, and an Extract II of the present invention is obtained by performing the extraction for approximately 2 hours under mechanical agitation.

4) Phase separation: after extraction, the material undergoes a separation step, such as filtration. This treatment is preferably repeated, more particularly twice.

5) Elimination of the solvent: it is carried out by distillation under reduced pressure (about 170 mbar), for example in a rotary evaporator, at a temperature of approximately 40 ^o C thus resulting in a potentiated extract of the present invention, being preferably an ethanolic extract resulting in potentiated extracts I and II, according to the

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11/28 extraction method used.

Procedure 1

Process of obtaining the most purified potentiated extract of

Casearia sylvestris [39] From the potentialized Extract, particularly Extract I, a more purified extract can be obtained, through a process that comprises at least the steps of:

- D. Partition of the extract; and

- E. Isolation of the most purified phase;

6) Partition: the Potentiated Extract, preferably dry ethanolic, is partitioned, preferably between dichloromethane (DCM) and methanol (MeOH), by any method of separation familiar to the man of the art, such as decantation or liquid-liquid extraction. The lower phase, preferably dichloromethane, is separated. This treatment is preferably repeated, more particularly twice.

7) Isolation of the most purified phase: the lower fraction obtained is separated and the solvent is eliminated, preferably by means of distillation, thus resulting in a more purified potentiated extract that is preferably dry (Extract I.1).

Process of Obtaining the Active Fraction [40] After obtaining the most purified extract, Extract I.1, at least the following steps are taken to obtain the potentiated active fraction of the Casearia sylvestris plant:

- F. Extraction; and

- G. Separation of phases for isolation of the active fraction.

[41] In particular achievements, some details are noted, and some other steps can be inserted, for example, as shown below:

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8) Extraction: when the extract is more purified in solid state, then the solid phase extraction (EFS) occurs, which is preferably carried out on a glass column filled with adsorbent, such as silica. Three fractions are obtained. After sample application, fraction 1 is eluted with dichloromethane, fraction 2 with (dichloromethane / methanol, preferably in the 9: 1 ratio), and fraction 3 with methanol.

9) Separation of phases for isolation of the active fraction: it occurs preferentially by means of chromatography, more particularly still column chromatography (CC) of the fraction 2 obtained. The elution of fraction 2 is done in gradient mode, in stages, using hexane / ethyl acetate in different proportions, ethyl acetate and methanol. In a preferred embodiment of the present invention, equal parts of hexane / ethyl acetate in the proportions 80:20, 60:40 and 40:60, plus ethyl acetate and methanol are used in the elution. 100 subfractions are collected, with the potentiated active fraction (active fraction A) being supplied by the first 50 subfractions, more particularly subfractions 32 to 42.

Characterization of the active fraction A [42] The characterization of the active fraction of Casearia sylvestris, consisting predominantly of casearins, was made from the chromatographic profile obtained in HPLC (Figure 4), according to the following conditions: Sample preparation:

[43] An aliquot of fraction 2 was diluted in MeOH 95% (1mg / mL). 1 ml of this solution was subjected to EFS-C18 (1.5 X 0.9 cm d.i.), eluted with an additional 1 ml of 95% MeOH. The 2 ml obtained were filtered and analyzed. Chromatographic conditions:

Column: Supelcosil LC-18, 5 pm, 250 x 4.6 mm, with pre-column; Elution:

MeOH / Water 75:25; Flow: 0.5 mL / min; Detection: UV at 235 nm. Injected volume: 10 pL.

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Identification of the components of the active fraction:

[44] The UV spectrum of the main components of the sample has only one band, with maximum absorption at 235 nm, (ε 13500), attributed to the terminal diene in C12 and C14, characteristic of casearins (Figure

6).

Procedure 2

Process of obtaining the most purified potentiated extract of

Casearia sylvestris [45] From the potentiated extract, Extract II, a more purified extract can be obtained, through a process that comprises at least the steps of:

- D. Partition of the extract; and

- E. Isolation of the most purified phase

6) Partition: the Potentiated Extract, preferably dry ethanolic is partitioned, preferably between hexane (Hex) and 60:40 ethanol / water (60% EtOH) by any method of separation familiar to the man of the art, such as by liquid-extraction liquid or decantation. The upper phase, preferably hexane, is separated. This treatment is preferably repeated, more particularly twice.

7) Isolation of the most purified phase: the upper fraction obtained is separated and the solvent is eliminated, preferably by means of distillation, thus resulting in a more purified potentiated extract, which is preferably dry (Extract II.1).

Process of Obtaining the Active Fraction [46] After obtaining the most purified extract, Extract II.1, at least the following step is used to obtain the potentiated active fraction of the Casearia sylvestris plant:

- F. Extraction and separation of phases for isolation of the active fraction

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14/28 [47] In particular achievements, some details are observed, and some other steps can be inserted, for example, as shown below.

8) Extraction: when the extract is more purified in solid state, then the solid phase extraction (EFS) occurs, which is preferably carried out on a glass column filled with adsorbent, such as, for example, silica / active carbon. After sample application, elution is performed with hexane, hexane / ethyl acetate in the proportions 75:25, 65:35, 50:50, 40:60, 20:80, plus ethyl acetate and methanol. 26 subfractions are collected, with the potentiated active fraction (active-B fraction) being provided by the initial subfractions, particularly subfraction 9.

Characterization of the active fraction B [48] The characterization of the active fraction of Casearia sylvestris, consisting predominantly of casearins, was made from the chromatographic profile obtained in HPLC (Figure 5), according to the following conditions: Sample preparation:

[49] An aliquot of fraction 2 was diluted in 98% MeOH (1mg / mL). 1 ml of this solution was subjected to EFS-C18 (1.5 X 0.9 cm d.i.), eluted with an additional 5 ml of 98% MeOH. The 6 ml obtained were filtered and analyzed. Chromatographic conditions:

Column: Supelcosil LC-18, 5 pm, 250 x 4.6 mm, with pre-column; Elution: MeOH / Water 70:30; Flow: 1.0 mL / min; Detection: UV at 235 nm; Injected volume: 10 pL.

Identification of the components of the active fraction:

[50] The UV spectrum of the main components of the sample has only one band, with maximum absorption at 235 nm, (ε ^ 13500), attributed to the terminal diene (C ₁₂ and C _14- linked to the molecule), characteristic of casearins ( Figure 6).

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15/28 [51] The potentiated extracts or active fraction, obtained in accordance with that described in the present invention, have an activity superior to that of the state of the art in the treatment of gastrointestinal disorders such as disorders of the oropharynx, thrush, herpes and bad breath , disorders whose treatment is favored by the healing action, in particular antiulcerogenic activity, surprisingly due to the reduced amount of active material necessary to obtain the desired effect, not achieved by extracts known in the art. After a decade of studies, the inventors managed to develop an improved active material, whose action results in a full recovery of the organism in faster treatment, an effect attributed to the fact that this extract or active fraction of the natural product has an effective action in the treatment of ulcerative processes of all levels (mild, moderate and severe), do not interfere with the pH of gastric juice, have a cytoprotective action, are selective and inexpensive.

[52] By selectivity, as applied in the present application, it is understood the fact that it does not present an abortive action, unlike what was observed with cytoprotective drugs known until today.

Dosage and Formulation [53] Compositions comprising at least an active fraction or extract, obtained according to the described process, also constitute an object of the present invention.

[54] The active fractions or potentiated extracts and compositions of the present invention can be administered in oral dosage forms, such as capsules, lozenges, pills, powders, granules, elixirs, suspensions, syrups and emulsions (each including formulations of scheduled or delayed release). They can also be administered intravenously (mixture or infusion), subcutaneously or intramuscularly. Can be administered alone, but also associated with a vehicle

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16/28 pharmacist selected based on route of administration and standard pharmaceutical practice.

[55] Compositions comprising at least a fraction or extract according to the present invention may contain still other active ingredients depending on the desired effect.

[56] For oral administration in tablet or capsule form, for example, the active drug component can be combined with a pharmaceutically acceptable inert carrier, such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate , calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid forms, the oral drug components can be combined with any inert, pharmaceutically acceptable carrier, such as ethanol, glycerol, water and the like. In addition, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

[57] The extracts and active fractions according to the present invention can also be administered in the form of liposome delivery systems or coupled to soluble polymers as drug delivery vehicles.

[58] Liquid dosage forms for oral administration may contain dyes and flavorings to increase patient acceptance. In

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17/28 general form, water, an appropriate oil, saline, aqueous dextrose (glucose), related sugar solutions and glycols, such as propylene glycol or polyethylene glycols, phosphate buffer, are suitable vehicles in liquid dosage forms.

[59] Another object of the present invention is a method of preventing, combating or suspending disorders of the gastrointestinal tract, particularly ulcerative progress, comprising administering a substantially effective dose of a potent active fraction or extract, or composition, described herein. According to an embodiment of the present invention, a substantially effective dose corresponds to about 10 to about 300 mg of extract per kilogram of the patient, preferably between about 10 to 100 mg / kg, and more particularly about 30 to 50 mg / kg. kg; a substantially effective dose further corresponds to about 0.1 to about 50 mg of the active fraction per kilo of the patient, preferably between about 1 to 30 mg / kg, and more particularly about 3 to 10 mg / kg.

[60] The extracts or compositions according to the present invention can be administered in a single daily dose, or the total daily dosage can be administered in doses divided into two, three, four times or more per day. For certain treatments, however, application on alternate days is appropriate, cyclically or not.

[61] Dosage forms of compositions suitable for administration contain about 0.01 to 500 mg, more particularly about 1 to 100 mg of active ingredient per unit. In these pharmaceutical compositions, the extract or active fraction will normally be present in an amount of about 0.01 to 99% by weight, particularly about 1 to 70% and more particularly about 10 to 40% by weight, based on total weight of the composition, which may further comprise at least one pharmaceutically acceptable carrier.

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Examples [62] Embodiments of the present invention are illustrated in the examples that follow. However, it should be understood that these examples, while indicating particular embodiments of the present invention, are given by way of illustration only, in no way limiting the scope of the invention as defined in the claims.

Example 1

Obtaining Potentialized Dry Dichloromethane Extract [63] Leaves of the Casearia sylvestris plant were collected and then dried in air circulation greenhouses until reaching approximately 40 ° C. The dried leaves were then ground in a knife mill (Marconi MA 680) and then weighed. A total weight of 1 kg of dry ground leaves was obtained.

[64] The total of dried and ground leaves was extracted with 96 ° GL ethanol (1: 3 w / v) by sonication at room temperature (about 37 ° C) for 20 minutes.

[65] After extraction, the resulting material was then filtered through cotton. This procedure was repeated three times and the filtrates were combined, resulting in 9L of liquid ethanolic extract.

[66] The liquid ethanolic extract was then distilled on a rotary evaporator. The internal pressure of the evaporator was adjusted to about 170 mbar in order to allow the distillation of the solvent to occur at a temperature of approximately 40 ^° C. This process resulted in a total weight of 65 g of a dry ethanolic extract.

[67] The dry ethanolic extract was partitioned between 650 ml of dichloromethane (DCM) and 1000 ml of 70% methanol (MeOH) (MeOH / Water 70:30). The lower phase (dichloromethane), after decanting, was separated. This procedure was repeated three times and the dichloromethane phases were

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19/28 combined, providing a volume of about 1800 mL.

[68] The liquid dichloromethane phase was then distilled on a rotary evaporator. The internal pressure of the evaporator was adjusted to around 700 mbar in order to allow the distillation of the solvent to occur at a temperature of approximately 40 ° C. This process resulted in a total weight of 32.5 g of a potentized dry dichloromethane extract.

Example 2

Obtaining Potentialized Dry Hexanic Extract [69] Leaves of the Casearia sylvestris plant were collected and then dried in air circulation ovens until reaching approximately 40 ° C. The dried leaves were then ground using the DEFACAS apparatus (Marconi MA 680) and then weighed. A total weight of 1 kg of dry ground leaves was obtained.

[70] The total of dried and ground leaves was extracted with 96 ° GL (1: 3 w / v) ethanol for 2 hours under mechanical stirring at room temperature (about 37 ° C) [71] After extraction, the material resultant was then filtered through cotton. This procedure was repeated three times and the filtrates were combined, resulting in 9L of liquid ethanolic extract.

[72] The liquid ethanolic extract was then distilled on a rotary evaporator. The internal pressure of the evaporator was adjusted to about 170 mbar in order to allow the distillation of the solvent to occur at a temperature of approximately 40 ° C. This process resulted in a total weight of 114 g of a dry ethanolic extract.

[73] The dry ethanolic extract was partitioned between 735 ml of hexane (HEX) and 2250 ml of 60% EtOH (60:40 v / v EtOH / Water). The lower phase (hexane), after decanting, was separated. This procedure was repeated three times and the hexane phases were combined.

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20/28 [74] The liquid hexane phase was then distilled on a rotary evaporator. The internal pressure of the evaporator was adjusted to around 700 mbar in order to allow the distillation of the solvent to occur at a temperature of approximately 40 ° C. This process resulted in a total weight of 73.5 g of an enhanced hexane dry extract.

Example 3

Obtaining potentiated dry hexanic extract [75] The extracts were obtained from the dried and ground leaves, using three consecutive extractions (3 X 3.0 L of 96 ° GL ethanol for each 1000 g of dry and ground leaves) under mechanical stirring for 2 hours. At each stage, the ethanolic solution was filtered through cotton, obtaining 9 liters of ethanolic extract for every 1000 g of dry leaves. The extracts were concentrated in a rotary evaporator.

[76] The extract obtained was subjected to liquid-liquid extraction (hexane: ethanol 60% in a 1: 3 ratio). The ratio used was 50 g of extract to 500 ml of hexane. The upper phase was collected and the hydroalcolic phase extracted twice more.

[77] After the liquid-liquid extraction process, 1.5 L of the hexane phase and 1.5 L of the hydroalcolic phase were obtained for each 50 g of extract. The solvent from both phases was eliminated in a rotary evaporator. Each kilo of dry leaf provides about 70 g of dry hexane extract.

Example 4

Obtaining the Potentialized Active Fraction (procedure 1) [78] The extraction in solid phase (EFS) of 32.5 g of the potentiated dry dichloromethane extract, obtained in Example 1, was promoted. The extraction was carried out in a glass column containing the bottom a sintered glass plate and an adaptation for connection to the vacuum line. Silica gel 60 - particles of approximately 40-63 pm were used as adsorbent. At

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21/28 dimensions of the chromatographic bed were 10 cm high X 9.5 cm in internal diameter. By said extraction three fractions (1, 2 and 3) were obtained. After sample application, fraction 1 was eluted with approximately 2250 ml of DCM (dichloromethane), fraction 2 with approximately 1000 ml of DCM / methanol (MeOH) 9: 1 and fraction 3 with approximately 2000 ml of methanol.

[79] The aforementioned extraction resulted in a total weight of 16.42 g of fraction 2. Shortly thereafter, chromatography was performed on a Michel-Miller® glass column (30 cm height X 4 cm internal diameter), packaged with Silica gel 60 - 40-63 pm particles. The elution of fraction 2 was performed in gradient mode, in stages, using 900 ml of Hexane / ethyl acetate 80:20, 900 ml of Hexane / ethyl acetate 60:40, 900 ml of Hexane / ethyl acetate 40:60 , 900 ml of ethyl acetate and 900 ml of methanol. 100 subfractions of 50 ml each were collected. Subfractions 32 to 42 combined gave the potentiated active fraction (active fraction A).

Example 5

Obtaining the Potentialized Active Fraction (procedure 2) [80] Extraction in solid phase (EFS) of 73.5 g of the potentized dry hexane extract, obtained in Example 2, was promoted. The extraction was carried out in a glass column containing the bottom a cotton filter and an adaptation for connection to the vacuum line. Silica gel 60 was used as an adsorbent - particles of approximately 40-63 pm in the same mass proportions, with powdered active carbon. The dimensions of the chromatographic bed were 12 cm high X 14 cm in internal diameter. By said extraction, 26 fractions were obtained. After sample application, the 26 fractions (1000 ml each) were eluted with the solvents: hexane, ethyl acetate and methanol in mixtures with increasing polarity (2000 mL - HEX; 3000 mL - HEX / AcOEt (75:25) ; 3000mL - HEX / AcOEt (65:35); 3000 mL

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22/28

HEX / AcOEt (50:50); 3000 mL - HEX / AcOEt (40:60); 3000 mL HEX / AcOEt (20:80); 3000 mL - AcOEt; 6000 mL - MeOH). The active fraction (F9) was eluted with 50:50 v / v HEX / AcOEt.

[81] The extraction mentioned above resulted in a total weight of 6.5 g of fraction 9 (active fraction B).

Example 6

Obtaining the Potentialized Active Fraction (procedure 2) [82] Fractionation of the hexane extract, obtained as described in Example 3, was carried out by solid phase extraction (EFS), using a 14 cm high glass column by 12 cm in height. di, filled with a mixture of active carbon / silica gel 60, 40-63 qm, in the proportion 1: 1, applying 105.4 g of hexane extract of CS 101, and then eluted with gradient in stages: 3, 0 L hex, 3.0 L hex / AcOEt (75:25), 3.0 L hex / AcOEt (65:35), 3.0 L hex / AcOEt (50; 50), 3.0 L hex / AcOEt (40:60), 3.0 L hex / AcOet (20:80), 3.0 L AcOEt, 5.0 L MeOH. 26 fractions of = 1.0 L. were collected.

Fraction 9 provided the potentiated active fraction (active fraction B). Comparative Test 1 [83] Comparative tests were carried out between the potentiated extracts obtained according to the present invention (Extract I - dry leaf / ethanol / ultrasound and Extract II - dry leaf / ethanol / mechanical stirring), the two extracts of the Casearia plant sylvestris already known in the art (Extract III (dry leaves / hydroalcoholic - percolation) and Extract IV (fresh leaves / hydroalcoholic - maceration) and drugs commonly used in the treatment of ulcers (cimetidine, atropine and misoprostol).

[84] The present comparative test proved that the extracts have advantageous properties when compared to existing drugs:

Such advantages include:

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23/28 (a) High safety margin (LD50 32 times greater than the DE50), according to the method of Thompson and Weil, (Biometrics, 8: 51-4, 1952) with LD50 representing the dose that kills 50% of the animals tested and DE50 represents the dose that shows 50% effectiveness in the animals tested.

(b) Extracts I, III and IV do not modify the pH of gastric juice, according to the method of Sertié et al, (Pharmaceutical Biology, (38) 2: 112-9, 2000) differently from that observed with H2 receptor blocking drugs, for example cimetidine, and with M3 receptor blocking drugs, for example pyranzepine;

(c) Extracts I, III and IV act on stress induction models, according to the method of Basile et al, (Journal of Ethnopharmacology, 30: 185-97, 1990), on mild, moderate and severe ulcers, similar to drugs H2 blockers, whereas cytoprotective drugs, for example misoprostol, only act on more severe ulcers; and (d) In chronic ulcers, induced by acetic acid, according to the method of Albelda et al, (Rev. Esp. Enferm. Apar. Dig., 41: 771, 1973) Extracts III and IV similar to the already existing cytoprotective drugs , present a faster healing rate than that observed with H2 blocking drugs.

[85] The method consists of mating virgin rats, treated orally with the extracts until the day of delivery, with virgin rats. Then, the number of rats that become pregnant is evaluated; number of pups per rat; presence of malformation; size, weight and length of offspring. It should be noted that initial toxicological studies for Extracts I, II, III and IV did not show an abortive action or induce bone malformation in the offspring of treated females during the gestation phase.

[86] Extracts III and IV show cytoprotective activity, reducing the area of gastric lesion by approximately 87%. Extracts I and II, obtained according to the present invention, reduced by up to 100% the number

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24/28 of lesions when administered orally in the same doses, which represents a surprising result.

[87] In view of these extremely relevant results achieved with Extract I, it was fractionated by liquid-liquid partition obtaining the extract CH ₂ Cl ₂ (Extract I.1), showing that the latter is the main responsible for the action cytoprotective. Chromatographic fractionation of the CH2Cl2 extract led to the active fraction A, which, like Extract I and CH ₂ Cl ₂ , reduced the number of ethanol-induced ulcer lesions by 100%, revealing that this fraction was responsible for the cytoprotective activity, being observed, still, a maintenance of the reduction of the lesion area at its maximum level even with the reduction of about 40% to 50% of the administered dose.

[88] Extract II was fractionated by liquid-liquid partition, obtaining the hexanic extract (Extract II.1), showing that the latter is the main responsible for the cytoprotective action. The chromatographic fractionation of the hexane extract led to the active fraction B, which reduced, similarly to Extract II and the hexane extract, by 100% the number of ethanol-induced ulcer lesions, revealing that this fraction is responsible for the cytoprotective activity.

Comparative Test 2 [89] A comparative test was carried out between Extracts I (the present invention) and III (known in the art). This test is illustrated by Figure 3.

[90] As can be seen in this experimental model, according to the model recommended by Basile et al, (Journal of Ethnopharmacology, 30: 185-97, 1990), Extract I, administered orally at a dose of 57.5 mg / kg, reduced the gastric lesion index by approximately 57% when compared to the control group, while this reduction in Extract III administered by the same route, in the same dose, was approximately 29%, showing the greater activity of the extract obtained by the sonication process

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25/28 of the present invention.

[91] In addition, gastric secretion parameters between Extract I and the control group were evaluated. Extract I, when administered orally at 57.5 mg / kg, did not change either the volume of secretion or the pH of gastric juice when compared to the control group.

[92] Table 1 below shows the effect of oral administration of Extract I on gastric secretion parameters.

Table 1

Samples Dose / kg Gastric secretion Volume (mL) pH Control (1% Agar) 4.0 mL 1.92 ± 0.60 2.64 ± 0.24 Extract I 57.5 mg 2.10 ± 0.26 2.88 ± 0.32

Each value represents the mean ± E.P.M. of 5 rats

Comparative Tests 3- 5 [93] Comparative studies were carried out between the active fractions of the present invention, misoprostol, cimetidine and Extracts I, II, III and IV, aiming to evaluate their cytoprotective activity.

Comparative Test 3 [94] Table 2 below shows the result of oral administration in an ethanol-induced ulcer, according to the model recommended by Robert, A. (Adv. Prostaglandin Thromboxane Res., 8: 1533-41, 1980) where it was observed that the extracts prepared by the process of the present invention (Extracts I and II) reduced the lesion area by 100%, proving to be more efficient than misoprostol, a classic cytoprotective drug

Table 2

Treatment Dose / kg ^r Q Injury area (mm ² ) Reduction(%) Control (1% Agar) 4.0 mL 3,840.60 ± 196.24 Extract I 57.5 mg Zero 100%

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Treatment Dose / kg ^r Q Injury area (mm ² ) Reduction(%) Extract II 57.5 mg Zero 100% Extract III 57.5 mg 657.07 ± 40.64 83 Extract IV 57.5 mg 657.09 ± 50.94 82 Misoprostol 0.5 mg 219.47 ± 12.36 ^a , ^b 94.3 Cimetidine 32.0 mg 3,790.00 ± 89.63 1.3

Each value represents the mean ± S.E.M of 8 animals aSignificantly different from the control (Tukey, p <0.05) bSignificantly different from Extracts I, II and III and cimetidine (Tukey, p <0.05)

Comparative Test 4 [95] Table 3 below shows the comparative study between Extracts I, dichloromethane, and active fraction A; Extracts II, hexanic and active fraction B. The administration also took place orally in gastric lesions induced by ethanol, according to the model recommended by Robert, A. (Adv. Prostaglandin Thromboxane Res., 8: 1533-41, 1980).

Table 3

Treatment Number of injuries

Control (4.0 mL / kg agar) Treatment Extract I (57.5 mg / kg) 12.7 ± 1.1 * Zero Dichloromethane extract (I.1) 20.3 ± 1.78 ** Zero (17.97mg / kg) Active fraction A (3.5 mg / kg) 9.4 ± 1.5 ** Zero Extract II (57.5 mg / kg) 18.8 ± 1.7 * Zero Hexanic extract (II.1) 15.6 ± 1.8 ** Zero (34.5 mg / kg) Active fraction B 18.6 ± 2.9 ** Zero

(2.267 mg / kg) * mean ± E.P.M. of 8 rats ** mean ± E.P.M. of 10 rats.

Comparative Test 5

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27/28 [96] Table 4 shows the evaluation of the cytoprotective activity of active fraction A of the present invention administered in different doses orally, according to the model recommended by Robert, A. (Adv. Prostaglandin Thromboxane Res., 8: 1533- 41, 1980) showing the intense cytoprotective activity of the active fraction of Casearia sylvestris, emphasizing that such activity proves to be dose dependent, which proves to be its systemic and not local action, characterizing cytoprotective activity.

[97] The main aspect of the present test lies in the fact that only small doses of the active fraction are needed to achieve a 100% reduction in the area of injury without causing apparent side effects.

Table 4

Treatment Dose / kg Reduction of the injury area (%) Control (1% Agar) 4.0 mL 1.6 mg 60.4 Active fraction A 2.1 mg 83.1 2.7 mg 97.4 3.5 mg 100

Each value represents mean ± E.P.M. of 8 rats.

Comparative Test 6 [98] An evaluation of the uterine musculature response of rats “in vitro” was carried out, according to the experimental model recommended by Acevedo and Contreras, (Comp Biochem Physiol C. 1987; 87 (2): 425-8), where the active fraction A administered in volume of 100 pL, at a dose of 420 pg / 100 pL of DMSO inhibited the uterine contraction induced by 3x10 ^-6 M carbacol and partially at the concentration 10 ^-5 M.

[99] Active fraction A, administered in a volume of 100 pL, at a dose of 420 pg / 100 pL of DMSO inhibited uterine contraction caused by prostaglandin PGF _2a 10 ^-6 M. At three times greater doses, active fraction A

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28/28 caused an additional increase in inhibition compared to that observed with the lower dose.

Comments [100] The antiulcerogenic drugs developed by the present invention act on all levels of ulceration (mild, moderate and severe), do not interfere with the pH of gastric juice (keeping it within normal physiological levels), have a cytoprotective action, do not contract the uterine musculature (being, therefore, devoid of abortive activity), and have low cost.

[101] In addition, the antiulcerogenic drugs of the present invention result in maintaining the reduction of the lesion area at its maximum level, even with a 40 to 50% decrease in the administered dose.

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1/3

Claims (16)

Claims 1. PROCESS OF OBTAINING EXTRACT AND ACTIVE FRACTIONS from Casearia sylvestris, characterized by understanding the steps of: - A. Extraction from the dried and ground leaves of the plant by solvent that has different polarities, selected from the group consisting of hexane, dichloromethane, chloroform, ethyl acetate, methanol and ethanol through the method chosen between decoction, percolation, maceration, sonication or mechanical agitation, both cold, hot or with agitation; - B. Separation of the phases obtained in step A by filtration carried out particularly 3 times; - C. Obtaining ethanolic extract by eliminating the solvent used in step A; - D. Partition by decantation or liquid-liquid extraction of the extract obtained in step C using dichloromethane and methanol or hexane and ethanol / water; - E. Distillation isolation of the phase obtained in step D to obtain the dry extract; - F. Extraction in solid phase (EFS) to obtain the fraction contained in the extract, preferably carried out in a glass column, followed by elution of three fractions, the first fraction being preferably with dichloromethane, the second with dichloromethane / methanol; and the third with methanol. - G. Isolation of the desired fraction contained in the extract obtained in phase F using hexane / ethyl acetate in different proportions, ethyl acetate and methanol. 2. PROCESS, according to claim 1, characterized by the fact that the solvent used is ethanol. 3. PROCESS, according to claim 1, characterized by the fact that step A is performed by sonication. Petition 870180024263, of March 26, 2018, p. 36/39 2/3 4. PROCESS, in wake up with the claim 3, featured because step A last 10 minutes, particularly 20 minutes. 5. PROCESS, in wake up with the claim 1, characterized by the fact that step A is performed by mechanical stirring. 6. PROCESS, according to claim 5, characterized by the fact that step A lasts 2 hours. 7. PROCESS, according to claim 1, characterized by the fact that step D is performed particularly 3 times. 8. PROCESS, according to claim 1, characterized in that in step F it is used for elution of dichloromethane / methanol in the proportion 9: 1. 9. PROCESS, according to claim 8, characterized by the fact that when the elution takes place by dichloromethane / methanol, step G will be carried out from the second fraction obtained in step F. 10. PROCESS, according to claim 9, characterized by the fact that when the elution takes place by dichloromethane / methanol, the elution of step G is carried out by column chromatography. 11. PROCESS, according to claim 1, characterized in that in step G five equal parts of hexane / ethyl acetate 80:20, 60:40, 40:60, ethyl acetate and methanol are used. 12. PROCESS, according to claims 1 and 11, characterized by the fact that 100 samples are collected, with the potentiated fraction being supplied by the first 50 samples, particularly by samples 32 to 42. 13. PROCESS, according to claim 1, characterized by the fact that in step G the elution is carried out with hexane, Petition 870180024263, of March 26, 2018, p. 37/39 3/3 hexane / ethyl acetate 75:25, 65:35, 50:50, 40:60 and 20:80, ethyl acetate and methanol. 14. PROCESS, according to claims 1 and 13, characterized by the fact that 26 samples are collected, with the potentiated fraction being supplied by the initial samples, particularly sample 9. 15. USE OF EXTRACTS obtained according to the process defined in claims 1 to 12, characterized by the fact that it is in the manufacture of a medication to treat gastrointestinal disorders. 16. USE, according to claim 15, characterized by the fact that the gastrointestinal disorder is an ulcerative process. 17. USE OF THE ACTIVE FRACTION obtained according to the process defined in claims 1 to 14, characterized by the fact that it is in the manufacture of a medication to treat gastrointestinal disorders. 18. USE, according to claim 17, characterized by the fact that the gastrointestinal disorder is an ulcerative process. Petition 870180024263, of March 26, 2018, p. 38/39 1/6