WO2005099726A1

WO2005099726A1 - ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L.

Info

Publication number: WO2005099726A1
Application number: PCT/BR2004/000138
Authority: WO
Inventors: Maria Tereza Grombone Guaratini; Célia Akiko HIRUMA LIMA; Vera Nisaka Solferini
Original assignee: Universidade Estadual De Campinas - Unicamp
Priority date: 2004-04-15
Filing date: 2004-07-29
Publication date: 2005-10-27
Also published as: BRPI0401592A

Abstract

The present invention is in general related to phytotherapeutic compounds, mainly those that belong to the class of terpenes and acetylenes, present in a plant extract of the family Asteraceae, or specifically of the species Bidens alba, whose occurrence in Brazil was confirmed by phytochemical and genetic studies. Compounds present in the dichloromethanic extract of this plant show anti-ulcerogenic activity against gastric lesions induced by HCl/ethanol and pyroxican models. The extract was able to inhibit formation of gastric ulcers by acting simultaneously preventing the generation, or the necrosis action of mediators on the gastric microvascularization and the endogenous production of prostaglandin.

Description

ANTI-ULCEROGENIC ACTIVITIES OF SESQUITER-PENS AND POLYACETY ENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. FIELD OF THE INVENTION The present invention is related to phytotherapeutic compounds, a plant extract, a phytotherapeutic composition, use of the pharmacological composition, preparation process of a medicament, a medicament, and one process of preparing the extract of a new plant species of the family Asteraceae, which presents active substances with pharmacological functions. More specifically, the present invention is related to phytotherapeutic compounds found in an extract of a plant of the genus Bidens, or its synthetic equivalents that present anti-ulcerogenic activities. More specifically, the present invention is related to a dichloromethanic extract of a new species of the genus Bidens in the Brazilian territory, with preventive and healing activity for duodenal ulcer. BACKGROUND OF THE INVENTION Although in many cases the etiologic agent of the ulcer is unknown, it is accepted that ulcerative processes occur as results of a balance between aggressive endogenous factors such as acid, pepsin and the maintenance of the integrity of the gastric mucosa, and environmental factors like the use of non-steroidal anti- inflammatory drugs (NSAID), the presence in the gastro-intestinal mucosa of Helicobacter pylori, smoking habit, environmental stress, and population feeding habits (Lanza, F. L. 1984. American Journal of Medicine 11: 19-24). For centuries, therapeutic solutions besides diets were always prepared to neutralize the gastric content of chloride acid by using antacids, or surgery. Evolution of the treatment for general dyspepsia was satisfactory until the late 80' s, wb-en antacids, anticholinergic agents, antagonists of the histamine H₂ receptor, cytoprotectors and inhibitors of the protonic bomb, and antimicrobials used individually or in association, were alternated as therapeutic solutions for the peptic ulcer disease. All these drugs produce side effects (Wallace, J.L. Granger, D.N., 1996. FASEB Journal, 10: 731-740). Traditionally, duodenal ulcer treatment aims to: relieve pain, accelerate ulcer cicatrization, and prevent formation of new ulcers and their complications. Currently, drugs used for treatment comprise: Antimicrobials For eradication or control of Helicobacter pylori. Amoxicilline, tetracycline, clarithromicine, metronidazol (Friedman, L. S. ; Peterson, W. L. 1998. Harrison 's principles of internal medicine - 14^th Edition CD-ROM). Antacids They are currently used to relive pain caused by ulcer. Most preparations are composed of a mixture of aluminum and magnesium hydroxides that neutralize HC1. Complications of this treatment include: constipation, phosphate depletion that may result in weakness and anorexia in patients with a low phosphate diet. Magnesium may stimulate gastrine secretion and gastric secretion (Friedman, L. S.; Peterson, W. L. 1998. Harrison 's principles of internal medicine - 14^th Edition CD-ROM). Antagonists of the H₂ receptor They are powerful inhibitors of acid secretion. Many side effects of their use have been observed. Increase in the number hepatic enzymes, increase in prolactin levels, inhibition of liver enzyme systems, and tendency to gynecomastia are some of the several side effects of these drugs, mainly cimetidine (Friedman, L. S.; Peterson, W. L. 1998. Harrison 's principles of internal medicine - 14^th Edition CD-ROM). Anticholinergic agents Used in the past, but seldom or not used nowadays (Friedman, L. S.; Peterson, W. L. 1998. Harrison 's principles of internal medicine - 14^th Edition CD- ROM). Prostaglandins A wide variety of prostaglandins, especially those from series E (PGE 1 and PGE 2), have been efficient for ulcer treatment. They reduce acid secretion and act also by increasing defenses of the mucosa: 1. they stimulate mucous secretion; 2. stimulate secretion of gastric and duodenal bicarbonate; 3. maintain or increase blood flow to the mucosa; 4. maintain mucous barriers of the stomach, avoiding ionic hydrogen difϊusion; 5. stimulate cell renewal of the gastric mucosa. They have many side effects: diarrhea, uterine contraction, abortion, and fetal malformation (Friedman,

L. S.; Peterson, W. L. 1998. Harrison 's principles of internal medicine - 14 ^& Edition

CD-ROM; .M.; Wolfe, M. M.; Lichtenstein, D.R.; Singh, G. 1999. The New England

Journal of Medicine 340 (24): 1888-1899A.; Goldberg, A. B.; Greenberg, M. B.; Darney, P. D. 2001. The New England Journal of Medicine 344 (1): 38-47). Inhibitors of the proton bomb The last step in the secretion of the hydrogen ion by parietal cells is carried out by a proton bomb [H+, K+ - ATPase] that exchanges hydrogen for potassium. Inhibition of this bomb by omeprazol and lanzoprazol causes decreases in gastric acid secretion (US 2001/0006649). Its side effects include: skin hypersensitivity reactions; muscular-skeletal reactions like muscular weakness and myalgia; hematological reactions like plaketopenia; presence of gastric cysts with extended treatment (Friedman, L. S.; Peterson, W. L. 1998. Harrison 's principles of internal medicine - 14^tilEdition CD-ROM). Because there is no drug that produces 100% remission of gastroduodenal ulcers (Friedman, L. S.; Peterson, W. L. 1998. Harrison 's principles of internal medicine - 14^th Edition CD-ROM), and because there are thousands of cases in Brazil involving this kind of illness, what constitutes a huge health problem, to study substances with potential anti-ulcerogenic activity is not only important, but vital. It is necessary to seek anti-ulcerogenic agents that are more effective, less toxic and cheaper. The possibility of finding such solutions in a plant that is widely distributed, herbaceous, and easily bred may satisfy such needs. It is known that many therapeutic agents that include plant parts are used to inhibit gastric secretion or to increase defensive mechanisms of the mucosa, increasing mucus production, and stabilizing the epithelial surface of cells or interfering with prostaglandin synthesis (Di Stasi, L. C, Santos, E. M. C, Moreira dos Santos, C,

Hiruma, C. A., Plantas Medicinais da Amazonia, Editora UNESP, Sao Paulo, 1989, pp.127-128). The family Asteraceae includes about 25,000 species, and is one of the world's largest. Chemical substances like polyacetylene, sesquiterpenoids, diterpenoids, triterpenoids, flavonoids, cumarines, benzofurans and benzopirans are particularly abundant in some dicotyledonous families, and are frequently found in the Asteraceae.

(Hertz, W. 1996. Compositae: systematics. Whitstablle Litho Printers Ltd, United Kingdom, pp 229-251). Currently, there are records of chemical compounds of 5,000 species and about 7,000 components that were isolated and identified (Zdero, C. and

Bohlmann, F. 1990. Plant Systematic and Evolution 111: 1-14). Some of the several plant species of the Asteraceae have phytotherapeutic compounds that have been already studied, also with verified pharmacological activity both in vitro and in vivo. For instance, Lychnophora ericoides, the Brazilian arnica, whose analgesic and anti-inflammatory properties were verified by Dr. Noberto Peporine Lopes' staff (2001). Another genus of this family, Bidens L., is subcosmopolitan and occurs in North and South Americas; it comprises, according to Sherff, E. E. (1937) (Field Museum of Natural History 16:16-4841937) 233 species distributed over temperate and tropical regions of the world, with about 284 varieties and 148 forms. Bidens alba (L.) DC. (Asteraceae, Heliantheae) is originally from the eastern region of Mexico, occurring throughout Florida and the Caribbean, and also in the coast of the Mexican Gulf in heights over 2,000 m, under influence of the tropical wet climate. The occurrence of this species, exclusively in the costal region of Brazil, was initially reported by M.D. Moraes (A familia Asteraceae na planicie litoranea de Picinguaba, Municipio de Ubatuba - Sao Paulo. Master Thesis, Universidade Estadual de Campinas, Campinas, 1998) and M. Magenta (As subtribos Ambrosiinae, Galinsoginae e Coreopsidinae (Heliantheae - Asteraceae) no Estado de Sao Paulo, Sao Paulo. 1998, 133p. Dissertaςao de Mestrado, Universidade de Sao Paulo, 1999). Afterwards, the identification and verification of the occurrence of Bidens alba in Brazil, and of a new species, was carried out based on molecular and phytochemical data (M.T. Grombone-Guaratini; K. L. SilvaV. J. Semir, V.N. Solferini & J.R. Trigo 2004 Sesquiterpenes and polyacetylene profile of Bidens pilosa complex - Asteraceae: Heliantheae - from Southeast of Brazil. Biochemical and Systematics Ecology). Bidens alba together with B. pilosa and B. subalternans form a species complex in the southeastern region of Brazil (Grombone-Guaratini, M. T., Mansanares, M.E., Semir, J.; Solferini, V. N. (in press). Chromosomal studies of three species of Bidens (L.) (Asteraceae). Genetics and Molecular Biology). Phytochemical and pharmacological studies carried out in other species of the genus Bidetτs, e.g. B. pilosa, have shown that different extracts present antimicrobial, anti-helminthic, protozoocide, and anti-ulcerogenic activities (NTDounga, M., Balansard, G, Babadjamian, A., Timon, D.P., Gasquet, M. 1983. Plantes Medicinales et Phytotherapie 17: 64-75; Bondarenko, A.S., Petrenko, G.T., Aizenman, B.E., Evsenko, O.N., 1985. Mib-obiology Zh (Kiev) 47: 81-83; Geissberger, P., Sequin, N. 1991. Acta Tropica 48: 251-261; Alvarez, A., Pomar, F., Sevilla, M.A., Montero, M.J. 1999. Journal of Eihnopharmacology 61: 333-340). Studies carried out in plants of the genus Bidens have tried to explain scientifically why they are used in traditional medical practices in Africa and Amazonia. Geissberger, P., Sequin, N., 1991 (Acta Tropica 48: 251-261) using aerial parts of Bidens pilosa found the substance phenylheptatriyne, to which they attribute strong anti-microbial action against gram-positive bacteria, and weak activity against gram-negative bacteria. According to these authors, the action of this substance would be mainly against bacterial infections in the gastrointestinal tract, and not in the H₂ bomb nor in the production of prostaglandins. M. G. L. Brandao, A. U. Krettli, L. S. R Soares, C. G C. Νery & H. C. Marinuzzi (1997) suggested that phenylacetylene compounds found in leaves and roots of Bidens alba are responsible for the anti-malarial activity observed in vitro and in vivo. Brandao, M. G. L.; Krettli, A. U; Soares, L. S. R; Νery, C. G. C;

Marinu-zzi, H. C. (1997. Journal of Ethnopharmacology 57: 131-138) demonstrated that the ethanolic extract of Bidens alba decreased the volume of gastric juice, gastric secretion, and pepsin secretion in rats that had their pylorus bonded. According to these authors, the extract inhibited effectively ethanol-induced hemorrhagic lesions. This study concluded that flavonoids, possibly present in the ethanolic extract of Bidens pilosa, have a cytoprotector effect associated to the anti-secretory gastric activity. Tan, P. N.; Dimo, T.; Dongo (2000. Journal of Ethnopharmacology 73: 415-421) demonstrated that the dichloromethanic extract of Bidens pilosa is an excellent protector of the gastric mucosa. Besides, according to these authors, the extract presents cytoprotector activity against irritants, probably mediated by the protective action of endogenous prostaglandins. These authors attribute such activities to the presence of flavonoids in leaves of Bidens pilosa. Otherwise, several patents were given to herbicide and soil decontaminant activities of Bidens alba, see for instance United States Patent 6,492,301; 6,280,500; 6,302,942; 6,713,434. However, there is no patent record for the anti- ulcerogenic activity of compounds extracted in a dichloromethanic extract of Bidens alba. Therefore, it would be desirable to obtain new phytotherapeutic compounds that have pharmacological activity able to protect the gastric mucosa, acting at least in two physiological mechanisms (such as inhibition of the H₂ bomb and stimulation of prostaglandin production), preventing and treating gastroduodenal ulcers. BRIEF DESCRIPTION OF THE INVENTION The present invention describes the preparation process and the composition of a dichloromethanic extract of substances found exclusively in Bidens alba, to which we describe inedited mechanisms of physiological action of polyacetylenes (mainly phenylheptatriyne) and sesquiterpenes. Phytotherapeutic substances found in this extract present anti-ulcerogenic properties, acting at the same time in the inhibition of the formation of gastric ulcers induced by the HCl/ethanol and pyroxican models, at 85.4% and 72.6%, respectively. That is, the extract was able to inhibit the formation of gastric ulcers by simultaneously preventing the generation or the necrotic action of mediators on the gastric microvascularization, and in the production of endogenous prostaglandin. A first modality of this invention is related to phytotherapeutic compounds of the terpene and acetylene class. A second modality of this invention is related to a dichloromethanic extract of aerial parts of Bidens alba. A third modality of this invention is related to the pharmacological composition derived from this dichloromethanic extract of aerial parts of Bidens alba added to an pharmacologically acceptable vehicle. A fourth modality of this invention is related to the use of this composition for the treatment of ulcers. A fifth modality of this invention consists of a preparation process of medicament. A sixth modality of this invention is related to the preparation process of this Bidens alba extract. BRIEF DESCRIPTION OF FIGURES Figure 1 presents a plot of ulcer indexes in mm (mean ± standard deviation). Gastric lesion induced by HCL/ethanol (1), protector effect of lanzoprazol (2) and extract of Bidens alba leaves in CH₂C1₂ in doses of 135 mg/kg (3), 250 mg/kg (4) and 500 mg/kg (5). Figure 2 presents an ulcer index in mm (mean ± standard deviation). Gastric lesion induced by pyroxican (1), protector effect of cimetidine (2), and extract of Bidens alba leaves in CH₂C1₂ in doses of 135 mg/kg (3), 250 mg/kg (4) and 500 mg/kg (5) _. Figure 3 presents an effect of vehicle administration (A), of CH₂C1₂- 135mg/Kg (B), CH2C12-250mg Kg (C) and CH2C12-500mg/Kg (D) in gastric lesion

induced by HCl/ethanol in the gastric mucosa of mice (— »). Figure 4 shows and effect of vehicle administration (E), of CH₂C1₂- 135mg Kg (F), CH2C12-250mg/Kg (G) and CH2C12-500mg/Kg (H) in gastric lesion induced by pyroxican in the gastric mucosa of mice (-»).

DETAILED DESCRIPTION OF THE INVENTION Bidens alba (voucher UEC 128.915 e 128.916) is a plant of short life cycle (2-3 years), whose habit varies from a herb to a small tree (0.4-1.2m); straight to dorsally decumbent stalk, quadrangular, green to olive, straight or decumbent; with radial capitula; radius flowers with white and reflex ligules, 6-16 mm, discus flowers yellow tubular, ca. 5.5 mm, and cipsels 6-10 mm, angular, costade, hispid, predominantly with two arists (Grombone-Guaratini, M.T.; Solferini, V. N.; Semir, J. (in press). Reproductive Biology In Three Species Of Bidens L. (Asteraceae). Scientia Agricola). In order to verify the activity of the dichloromethanic extract of Bidens alba we used male albino Swiss mice (25-35 g), descendant from the "Bioterio Central" of the Universidade Estadual Paulista (UNESP) - Botucatu campus. Animals were fed

certified ration Nuvilab CR-a^® (Nuvital), and given free access to water under standardized conditions of 12h light and 12h dark, moisture (60 ± 1.0%) and temperature (21 ± 1.0%). Animals remained unfed, because drugs and extract were

administered orally or intra-peritoneally, using a 12% Tween 80^®(10 ml/kg) solution as vehicle. Animals were kept in cages with elevated floor to prevent coprophagy. Protocols were approved by the "Instituiςao de Cuidados com Animais" [Animal Care Institution] and the "Comite de Utiliza?ao" [Use Comitee] of the UNESP, following recommendations of the Canadian Council on Animal Care.⁸ EXAMPLES: Preparation of the plant extract Example 1: Fresh leaves of Bidens alba were collected in Cubatao - SP. Fresh material (ca. 200 g) was kept for 24h in environmental temperature with 2 L of distillate dichloromethane (CH2CI2). Afterwards, this material was vacuum filtered, washed with distillate water, to form two phases and to remove the excess of water. As a drying agent 200 g of anhydrous sodium sulfate were added to the solution (Na₂SO₄). Posteriorly, the volume was reduced in a rotoevaporator without heating and under reduced pressure, removing predominantly all solvent. The concentrate resulted in 2.3 g of a dark solid substance. Example 2: Fresh leaves of Bidens alba were collected in Cubatao - SP. Fresh material (ca. 200 g) was kept for 24h in environmental temperature with 2 L of distillate dichloromethane (CH₂CI₂). Posteriorly, this material was vacuum filtered, washed with distillate water, to form two phases and to remove the excess of water. As a drying agent 200 g of magnesium sulfate were added to the solution (MgSO4). Afterwards, the volume was reduced in a rotoevaporator without heating and under reduced pressure, removing predominantly all solvent. The concentrate resulted in 2.3 g of a dark solid substance. Production process of the pharmacological composition Example 3: the process to produce a pharmacological composition of Bidens alba consists of adding to the extract a pharmacologically acceptable and/or not toxic vehicle, more specifically, a buffered solution, with pH between 5 and 11. Preparation process of the composition for test in an animal subject Example 4: The concentrate obtained from the extraction process described above was made soluble with an emulsificant that induces ulcer, like 10

mL/Kg of Tween 80^®(Synth, Brazil) at 12%. Besides the Bidens alba extract, we also uses the following anti-

ulcerogenic drugs: lanzoprazol (Hexal - Brasil) and cimetidine (Tagamet^® SmithKline,

Brazil); and ulcerogenic drugs: Tween 80^® (Synth, Brazil) and pyroxican (Sigma Chemical Co., U.S.A.). Chemicals used for preparation of other substances were all of analytical quality. Tests of the pharmacological properties of the Bidens alba extract Example 5: ulcer induced by HCl/ethanol in mice treated with lanzoprazol The anti-ulcerogenic activity of the dichloromethanic extract of Bidens alba was conducted as described by Mizui and Doteuchi (1983). Mice were divided into five groups of 10 animals that remained unfed for 24 h. Before beginning the experiment, mice were weighted and numbered. Posteriorly, each of the groups received

orally the following treatments: 12% Tween 80^® (10 ml kg), lanzoprazol (30 mg/kg),

dichloromethanic extract of Bidens alba (135, 250 and 500 mg/kg). After 50 min, all groups were treated orally with 0.2 mL of 0.3 M HCl/60% ethanol solution for the induction of gastric ulcer. Animals were killed 1 h after the administration of

HCl/ethanol, and stomachs were removed and washed with water. The extension of lesions was measured and the index of lesion was expressed and the sum of all lesions as described by I. Szelenyi and K. Thiemer (Arch. Toxicol. 41, 99-105 (1978). Example 6: Induction of gastric ulcer by pyroxican in mice treated with cimetidine The experiment was carried out following the modified Rainsford method. Mice were divided into five groups of 10 animals that remained unfed for 36 h.

Before beginning the experiment mice were weighted and numbered. Posteriorly each

of the groups received orally the following treatments: 12% Tween 80^® (10 ml/kg), cimetidine (100 mg/kg), dichloromethanic extract (135, 250 and 500 mg/kg). After 30 min gastric lesions were induced by pyroxican (30mg/kg) administered hypodermically. Animals were killed 4 h after treatment with the ulcerogenic substance. Stomachs were removed and gastric damage determined as described above. Results and Discussion The dichloromethanic extract of Bidens alba was a powerful protector of the gastric mucosa against lesion produced by the HCl/methanol method. Besides, this extract showed to be effective in the control of lesion caused by the pyroxican treatment. When the dichloromethanic extract of Bidens alba was administered orally in mice before gastric lesion were induced by HCl/ethanol, an dose-dependent anti-ulcerogenic activity was observed (Table 1, Figure 1).

Treatment Dose N Ulcer Index (mm) Inhibition

(p.o.) (mg K *) (%)

Control - 8 30.88 ± 14.16 * -

Lanzoprazol 30 8 5.25 ± 4.74 * 83

CH₂C1₂ 135 8 14.87 ± 11.41* 43.7 250 8 9.75 ± 5.15 * 68.4 500 8 4.5 ± 6.14 * 85.4

Control - 7 20.43 ± 8.14 ** -

Cimetidine 100 8 12.63 ± 4.95 ** 38

CH₂C1₂ 135 5 5.6 ± 3.29 ** 72.6 250 5 14.8 ± 4.87 27.6 500 4 13.25 ± 5.32 35.14 TABLE 1. Effect of lanzoprazol, cimetidine and different doses of the leaf extract of

Bidens alba in CH₂Cl₂on gastric lesions induced by HCL/ethanol in mice. Results are presented as mean

± standard deviation. CH₂C1₂ and lanzoprazol: ANO VA *F _{c > 35)} = 11.66 (P < 0.01) for the ulcer index and

CH₂C1₂ and cimetidine ANOVA **F_{t , 24J} = 4.993 (P < 0.05) for the ulcer index. The oral administration of the HCl/ethanol solution in the control group clearly produced lesions (Figure 3). The lesion index for the control group with gastric ulcer induced by HCl/ethanol was 30.88 ± 14.16 mm. Anti-ulcerogenic drugs lanzoprazol and dichloromethanic extract of Bidens alba (135, 250 and 500 mg/kg) significantly inhibited ulcer formation in 83, 43.7, 68.4 and 85.4 %, respectively

The effect of the dichloromethanic extract of Bidens alba, administered hypodermically (135mg/Kg) before gastric lesion induced by pyroxican, was also efficient (72.6%; p<0,05) in the reduction of lesion (Table 1, Figure 2). In the control group, the oral administration of pyroxican clearly produced lesion (Figure 4). Nevertheless the best inhibitory effect on the ulcerative effect was observed in the model HCl/ethanol (85.-4 %) followed by the pyroxican model (72.6 %). HCl/ethanol is highly corrosive in the gastric mucosa. HCl/ethanol induces the dissolution of the mucous layer of the stomach, increasing the flow of Na⁺ and K⁺ inside the lumen, pepsine secretion and the loss of ions (Hiruma-Lima C. A.; Toma, W.; Gracioso J. S.; Almeida, A. B. A.; Batista, L. M.; Magri, L.; Paula A.C. B.; Soares F. R; Nunes, D. S.; Souza Brito, A. R. M. 2002. Biological Pham Bull 25 (4): 452-456). This pathogenic action system involves an aggressive superficial cellular necrosis, and the liberation of mediators like histamine and leucotriene C₄. These mediators act on the microvascularization, triggering a series of events that results in the destruction of the tissue of the mucosa and submucosa (Oates, P. J., Hakkinen, J.P. 1985. Gastroenterology 77: 433-443). Results obtained by the present study point out that the dichloromethanic extract of Bidens alba presents phytochemical components, which prevent the generation or the necrotic action of mediators on the gastric microvascularization. Pyroxican is a prostaglandin inhibitor that suppresses the gastro uodenal secretion of bicarbonate, breaking the mucosa barrier, decreasing the endogenous biosynthesis of prostaglandin and the flow of gastric mucus (Midler, T.A. American

Journal of Physiology Gastrointestinal Liver Physiology) 1982. 8) 245, G6O1-G623;

Midler, T.A. American Journal of Physiology (Gastrointestinal Liver Physiology) 1982.

8) 245, G601-G623). Otherwise, large-scale prostaglandin synthesis is associated to cytoprotection of the gastric mucosa (Robert, A. 1981. Physiology of the gastrointestinal tract. R-aven Press, New York, pp.1407-1434). The anti-ulcerogenic effect presented by the dichloromethanic extract of Bidens alba points out to the presence of phytochemical compounds in this extract that act on the endogenous production of prostaglandin. The present study found that the polyacetylene (phenylheptatriyne) and

sesquiterpenes, like ε-caryophillene and β-gurgenene extracted by the dichloromethane

of Bidens alba present anti-ulcerogenic properties, acting simultaneously in the inhibition of formation of gastric ulcers induced by HCl/ethanol and pyroxican models in 85.4%o and 12.6%, respectively. That is, the extract was able to inhibit formation of gastric ulcers by acting simultaneously preventing the generation or the necrosis action of mediators on the gastric microvascularization, and acting on the endogenous production of prostaglandin. Advantages of the extract of the present invention could be related to the discovery of phytotherapeutic compounds and/or the discovery of new action mechanisms of these phytotherapeutic medicaments that might be more powerful, present less side effects and serve as an alternative to conventional chemicals found in the market, and also reduce the cost of prevention and treatment of gastroduodenal ulcers. The description of the present invention was presented aiming only to illustrate and describe. Besides, the description does not intend to limit the invention to the form here used. Consequently, variations and modifications compatible with the knowledge presented, and the ability or knowledge of the relevant technique, are inside the scope of the present invention. Above described modalities aim to better explain known ways to practice the invention, and to allow that technicians in the area use the invention in such, or other, modalities and with several modifications necessary for specific appliances, or uses of the present invention. It is our intention that the present invention comprises all its modalities and variations, within the scope described in the report and in attached claims.

Claims

CLAIMS 1 ANTI-ULCEROGENIC ACTIVITIES OF

SESQXJITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC

EXTRACT OF Bidens alba L. , Phytotherapeutic compounds, characterized by deriving from an extract of Bidens alba, having pharmacological activity, or being synthetically produced.

2. ANTI-ULCEROGENIC ACTIVITIES OF

SESQXJITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC

EXTRACT OF Bidens alba L. , Phytotherapeutic compounds of claim 1, characterized by deriving from a dichloromethanic extract of Bidens alba, having anti-ulcerogenic activity" and belonging to the class of acetylenes and terpenes.

3 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Phytotherapeutic compounds of claim 2, derived from a dichloromethanic extract of Bidens alba, having anti-ulcerogenic activity, and belonging to the class of polyacetylenes and sesquiterpenes.

4 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Phytotherapeutic compounds of claims 1 to 3, comprising a phenylheptathriyene and, at least, ε-caryophillene e β-gurjenene.

5. ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Plant extract derived from Bidens alba, and having pharmacological activity. 6 ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Plant extract of claim 5, wherein phytotherapeutic compounds of claims 1 to 4 are present. 7 ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Plant extract of claim 5, wherein compounds of claims 1 to 4 are present, and which is extracted by using an apolar solvent. 8 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC

EXTRACT OF Bidens alba L. , Plant extract of claim 7, comprising preferentially the solvent dichloromethane. 9 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Plant extract of claim 5, characterized as an extract of aerial parts of Bidens alba. 10 ANTI-ULCEROGENIC ACTIVITIES OF SESQXJITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. Plant extract of claim 9, characterized specifically as an extract of fresh leaves of Bidens alba. 11 ANTI-ULCEROGENIC ACTIVITIES OF SESQXJITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Plant extract of claim 5, having pharmacological activity for treatment or prevention of ulcers. 12 ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Plant extract of claim 11, characterized by being preferentially destined to prevention and treatment of gastroduodenal ulcers. 13 ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Pharmacological composition, comprising phytotherapeutic compounds of claims 5 to 12, adding an effective amount of, at least, one pharmacologically acceptable vehicle. 14 ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Pharmacological composition of claim 13, wherein the pharmacologically acceptable vehicle is preferentially a buffered solution, with pH between 5 an 11. 15 ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Use of the composition of claims 13 and 14, characterized by the use for ulcer treatment. 16 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC

EXTRACT OF Bidens alba L. , Use of the composition of claims 13 and 14, characterized by use for treatment of gastroduodenal ulcers. 17 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Preparation process of a medicament comprising the pharmacological composition of claims 13 and 14, comprising the stage of mixing phytotherapeutic compounds of claims 1 to 4, and/or the extract of claims 5 to 12, wherein a pharmacologically acceptable vehicle and/or proper pharmacological excipients are present. 18 ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Medicament, characterized by being administered as a capsule, dragees, solutions, suspension, dust, granules or pills. 19 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC

EXTRACT OF Bidens alba L. , Preparation process of the plant extract, characterized as an extract of Bidens alba and consisting of the following stages: (a) Collection of aerial parts of the plant; (b) Infusion with an apolar solvent for enough time at environmental temperature; (c) Filtration; (d) Washing with distillate water; (e) Addition of an enough amount of a drying agent; (f) Decrease of the volume in a rotoevaporator, without heating and under reduced pressure. 20 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Preparation process of the plant extract of claim 19, characterized by stage (a) consisting of the collection of fresh leaves of the plant. 21 ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Preparation process of the plant extract of claims 19 and 20, comprising preferentially the apolar solvent dichloromethane (CH₂C1₂). 22 ANTI-ULCEROGENIC ACTIVITIES OF

SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC EXTRACT OF Bidens alba L. , Preparation process of the plant extract of claims 19 to

21, wherein the infusion time is preferentially 24 h. 23 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC

EXTRACT OF Bidens alba L. , Preparation process of the plant extract of claims 19 to

22, comprising the drying agent of stage (e), which is selected between anhydrous sodium sulfate (Na₂SO₄) and magnesium sulfate (MgSO₄). 24 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC

EXTRACT OF Bidens alba L. Preparation process of the plant extract of claim 23, in which the drying agent of stage (e) is present in a concentration of 10% to 80% of fresh weight of Bidens alba leaves. 25 ANTI-ULCEROGENIC ACTIVITIES OF SESQUITERPENS AND POLYACETYLENES OF A DICHLOROMETHANIC

EXTRACT OF Bidens alba L. , Preparation process of the plant extract of claim 24, wherein the drying agent of stage (e) is preferentially in a concentration of 30% to 60% of fresh weight of Bi dens alba leaves.