BRPI0804525A2 - process of obtaining a standardized extract, obtained extract, pharmaceutical composition, method of treatment and use of said extract - Google Patents

Abstract

The present invention relates to a process for obtaining a standardized extract from at least a part of a plant of the genus Aleurites with antinociceptive, anti-inflammatory and antipyretic properties from a plant of the genus Aleurites. Additionally, the present invention provides a pharmaceutical composition which comprises as active ingredient a pharmaceutically effective amount of a standardized extract of at least a part of a plant of the genus Aleurites. The present invention relates to a method of treating and using said extract, alone or in a pharmaceutical composition, for the prevention, control or treatment of painful, inflammatory and febrile disorders.

Description

Patent Descriptive Report for: "PROCESS FOR OBTAINING A STANDARDIZED EXTRACT, OBTAINED EXTRACT, PHARMACEUTICAL COMPOSITION, METHOD OF TREATMENT AND USE OF DITOEXTRATE".

FIELD OF INVENTION

The present invention relates to a process for obtaining a standardized extract with antinocioactive, antiinflammatory and antipyretic properties from a plant of the genus Aleurites. The invention too

0 includes a pharmaceutical composition comprising said standardized extract, methods of treating, preventing or controlling painful, febrile and inflammatory disorders using said standardized extract.

BACKGROUND OF THE INVENTION

Pain is the most common symptomatic manifestation in humans. It is a complex condition, often with undefined etiology. While acute pain usually results from trauma, visceral dyskinesias, acute inflammatory processes and infections, chronic pain usually

0 is related to musculoskeletal, neuropathic, oncological disorders and chronic inflammatory processes.

Several mechanisms are related to the acute donor occurrence: trauma of musculoskeletal structures and soft tissue, dysfunctional abnormalities, acute inflammatory processes, abdominal, thoracic or oropharyngeal visceral dyskinesias can generate physical, mechanical, thermal and / or chemical stimuli that activate bone fibers. peripheral nervous system. These carry nociceptive information to the spinal cord and subsequently to the hypothalamus and cortical areas involved in the processing of sensitivity, giving rise to pain.

The most common chronic manifestations of pain are associated with chronic inflammatory processes, usually observed in osteoarthritis, tendonitis, bursitis, spondylitis and other degenerative processes, usually autoimmune. More than the pain itself, in this process, the phenomenon of the mechanical thermal hyperalgesia related to the decrease of the nociceptors threshold is highlighted, which generates hypersensitivity to movement, mechanical deformation and heat. Hyperalgesia is caused by the action of numerous inflammatory mediators released in the affected regions, especially bradykinin, acetylcholine, histamine and cytokines such as interleukins and tumor necrosis factor. The resulting hypersensitivity justifies the fact that the inflamed tissue is painless at rest and becomes painful when stimulated.

Proper treatment and control of pain and inflammation is often done through pharmacotherapy, in which various known pharmacological classes of the technical state are used for anti-inflammatory analgesic purposes, the vast majority of which are obtained by chemical synthesis.

The most commonly used analgesic and anti-inflammatory drugs are those of the non-steroidal anti-inflammatory drugs (NSAIDs) class, such as salicylates, acetic acid derivatives, indolic derivatives, enolic acid derivatives, among others.

However, the main problem related to the anti-inflammatory methods specified above is due to their side effects, especially during prolonged administration. Such effects include gastritis, ulcerations, dyspepsia, nausea, vomiting, allergies, renal failure, irreversible nephropathy, deeye syndrome, metabolic acidosis, hypertensive crises and risk cardiovascular diseases.

In the case of non-selective NSAIDs, non-specific inhibitors of the enzyme cyclooxygenase (COX) inhibit both constitutive aCOX-1, responsible for organ and stomach homogeneous processes, and increased inducible aCOX-2 in the body during agenesis of inflammation. COX-2 is responsible for the production of proinflammatory prostanoid mediators, such as some prostaglandins and leukotrienes. Inhibition of this enzyme (COX-2) is important for reducing the inflammatory process. However, inhibition of COX-1 generates the adverse effects described above, especially gastrointestinal ones, as it is an important regulator of hydrochloric acid production as well as the production of bicarbonate and the mucoprotector lining the stomach, protecting it against the acid and enzymes present in the gastric juice.

NSAID use has been associated with serious complications. Such problems sometimes prevent adherence to the pharmacological treatment itself, sometimes make the treatment of painful and / or chronic inflammatory processes unfeasible.

In light of the foregoing, it is understandable that the development of safer therapies or, in other words, better risk-benefit for donor treatment and inflammation is necessary, especially considering the risks associated with commonly used drugs.

In this context, herbal medicine is a relevant alternative to the predominantly synthetic therapeutic arsenal.

The importance and chemical potentiality of medicinal plants can be identified with data from scientific research where approximately 50% of the medicines used in clinical practice worldwide originate from natural products and derivatives (Gurib-Fakim, A., 2006, Mol. Aspects med ., 27, 1-93).

Frequently, the pharmacological effect of a herbal medicine is not due to the action of a single compound, but to the joint activity of the various active substances contained / associated therein. Generally, the active substance alone presents different activity, or with lower power than that presented by the phytocomplex.

Unlike a synthetic drug in a herbal medicine, the active substances are almost always in low concentrations, which generally translates into a lower incidence of adverse effects.

Despite the limitations encountered during the procurement, purification and identification of natural products, advancement technology has made it possible to obtain them with improvements and power when compared to synthetic products (Koehn, F.E. and Carter, G. T., 2005, Nature, 4, 206-220).

A common problem in the use and production of herbal medicines relates to the concentration-decay variation of the active ingredient (s), usually secondary metabolites. These metabolites represent an interface between plants and the environment and their synthesis is often affected by environmental conditions (seasonality, circadian rhythm, plant development stage, plant age, temperature, water availability, ultraviolet radiation (UV), soil nutrients, altitude. , atmospheric composition and damage to vegetable tissues). These factors, as well as others, may affect the final content of secondary plant metabolites, such as collection conditions, stabilization, storage, and industrial processing, and thus may have a major influence on the quality of herbal preparations. Therefore, in addition to quality control, involving modern analytical techniques and standardization of intermediate products, the source and quality of raw material play a central role in obtaining products that are consistent in their composition and reproducible therapeutic properties (Gobbo-Neto, L. and Lopes, NP, 2007, Quim. Nova, 30, 374-381).

The Euphorbiaceae family consists of herbaceous plants, shrubs or trees that usually produce a latexwood, are species originating from Tropical Asia and Pacific Islands, found in Brazil in tropical areas, widely cultivated on the coast, and can also be found in temperate zones. This family contains approximately 7,000 species with 317 genera (Webter, G.L., 1994, Annals of the Missouri. Botan. Garden, 81, 1, 3-32). Previous investigations conducted with this family have found the presence mainly of lipids, terpenoids, alkaloids and hydrocarbons (Hui, W.H. and Hoi, C.T., 1968, Aust. J. Chem., 21, 1675-7). Studies reveal the importance of this family in food, medicine, as well as in industry (Gneco, S. et al., 1996, Bull. Soc. Chil. Quim., 41, 229-233; Villalobos, MJP and Castellanos, E. C, 1992 , Gras Y accepted, 43, 1, 39-44).

Among the various genera belonging to the family Euphorbiaceae, they can be cited as the most important due to their diversity, application and economic interest: Euphorbia, Croton, Phyllanthus, Jatropa, Sapium, Ricinus, Aleurites (Webter, GL, 1984, Annals of the Missouri. Botan. Garden, 81, 1, 3-32). The genus Aleuritessubdivides into A. trisperma, A. cordata, A. montana, A.fordii, A. montance, A. rockinghamensis, and A.moluccana (Villalobos, MJP and Castellanos, E. C, 1992, Grasas y Accepted, 43, 1, 39-44; Misra, DR and Khastgir, HN, 1970, Tetrahedron, 26, 12, 3017-3021; Cruz, GLD Dictionary of Useful Plants of Brazil 4 Ed Rio de Janeiro: Bertrand, 1964; Pio Correia, M. Dictionary of Useful Plants of Brazil and Cultivated Exotic Plants IBDF, Brasilia - DF (1984) V: 294-295; Fozdar, BI et al 1989, Phytochem., 28, 9, 2459-2461; Agarwal , R. et al., 1995, Fett Wissench, Technologie-Fat Science Technol., 97, 526-527 (Forster, PI, 1996, Muelleria, 9, 5-13).

Moluccan Aleurites L. Willd., Whose synonyms are JR & G. Forst Triloba Aleurites, Croton moluccanus L., Jatropa moluccana is an exotic tree, native to Indonesia and widely introduced in Brazil from the state of Sao Paulo to Rio Grande do Sul. especially abundant in the state of Santa Catarina. It is popularly known in the country as "Iguape Walnut", "India Nut", "India Walnut", "Bangkok Nut", or simply "Walnut" (Duke, JA, Handbook of medicinalherbs. USA) : CRC Press, 1991). In the popular context, the use of Moluccan Aleurites, as with any medicinal plant, is very wide, being used for the empirical control and treatment of the following diseases or symptoms: fever, inflammation, asthma, conjunctivitis, hepatitis, headaches, ulcers, diarrhea, gonorrhea and also used as antitumor, stimulant-relaxant and sweat and anti-rheumatic (Pio Correia, M. Dictionary of Useful Plants of Brazil and Cultivated Exotic. IBDF, Brasília - DF, 1984, 294-295; Villalobos, MJP and Castellanos, E. C, 1992, Grasas Y Accepted, 43.1, 39-44; Forster, PI, 1996, Muelleria, 9, 5-13; Duke, James A. Handbook of medicinal herbs. : CRC Press, 1991; Stuppy, W. et al., 1999, Blumea, 44, 1, 73-98; Locher, CP et al. al., 1995, J. Ethnopharmacol., 49, 23; Hope, BE et al., 1993, Hawaii Med., 56, 6, 160-166). However, very few studies that can systematically justify this broad therapeutic potential are described in the literature. .

Previous studies by a Belgian research group have shown that plant extracts collected in Hawaii exert antiviral actions, especially against the HIV virus (Locher, CP et al., 1996, Phytomedicine, 2, 259), as well as antibacterial effect. Staphylococus aureus and Pseudomonas aeruginosa (Locher, CP et al., 1995, J. Ethnopharmacol., 49, 23).

Other preliminary studies conducted by the inventors and collaborators also demonstrated that non-standard hydroalcoholic extracts of moluccan Aleurites as well as hexanic fractions showed analgesic potential in acetic acid-induced pain model in mice. In addition, the analysis of the isolated actives led to the identification of n-hentriacontane, alpha-amyrin, beta-amyrin, alpha-amirinone, beta-amirinone, stigmasterol, beta-sitosterol, campesterol, acetylaleuritolic acid, swertisine and 2 '' - O -ramnosyl-swertisine, and some of these substances significantly inhibited acetic acid-induced abdominal writhing in mice (Meyre-Silva, C. et al., 1998, Phytomedic., 5,2, 109-113; Meyre-Silva, C. et al. ., 1999, Plant Med., 65,3, 293-294).

In 1999, the inventors and collaborators also demonstrated that swertisine, a flavonoid isolated from the moluccan Aleuritis leaves, has no analgesic effect on the acetic acid-induced abdominal writhing model in mice. However, its derivative, 2 '' - 0-ramnosyl-swertisine, had about 16 times more potent analgesia than aspirin in this model, thus suggesting that the ramnosil group is important for the analgesic action of these compounds (Meyre-Silva, C. et al. ., 1999, Plant Med., 65, 3, 293-294).

Thus, studies previously conducted by the inventors and collaborators were able to identify a potential use of the species to develop a new herbal medicine.

Studies and research presented by the inventors demonstrate new antinociceptive, antiinflammatory and antipyretic activities related to A. moluccana extracts as evidenced by the data and tests described in this report.

The inventors have also identified a set of surprising procedures, practices and conditions compared to the state of the art as described below which contribute to the use of Aleuritesmoluccana as a pharmaceutical with assurances that its specification, safety and efficacy with respect to analgesic properties, anti-inflammatory drugs are reproducible and consistent.

In view of the above, a process of obtaining a standardized extract from moluccan Aleurites, a process of isolation of its marker, a process of preparation of a herbal medicine composition, analgesic, anti-inflammatory properties and

antipyretics, a method of treatment and its use as an alternative therapy to NSAIDs in view of their therapeutic potential and possibly better profile of safety and tolerability.

The present invention relates to a process of obtaining a standardized extract with antinociceptive, anti-inflammatory and antipyretic properties from a plant of the genus Aleurites, preferably Aleurites moluccana L. Willd, using at least a part of the plant, preferably the leaves, being said standardized extract distinguished by the marker 2 '' - O-ramnosil-swertisina.

The invention also relates to the pharmacological characterization of the standardized extracts cited above for their antinociceptive, antiinflammatory, antipyretic, antiedematogenic, antihypernociceptive activities, and assessment of their acute toxicity.

Still further, the invention provides a pharmaceutical composition which comprises as active ingredient a pharmaceutically effective amount of a standardized extract of moluccan leurites containing at least one active ingredient selected from the group consisting of alpha-amyrin, beta-amirin, alpha-amirinone, beta-amirinone, swertisine, saidextrate. being standardized to its 2 '' - 0-ramnosil-swertisine marker. Preferably, the pharmaceutical composition of the invention comprising the standardized extract of moluccan Aleurites contains a content of 2 '' - 0-ramnosyl swertisine in the range of 0.05 to 15%.

Finally, as a further aspect, the present invention comprises a method of treating and using this extract, alone or in a pharmaceutical composition, for the prevention, control or treatment of painful, inflammatory and febrile disorders.

BRIEF DESCRIPTION OF DRAWINGS

The following figures are part of this report and are included herein to illustrate certain aspects of the invention. The object of the present invention may be better understood with reference to one or more of these figures, in combination with the detailed description of the preferred embodiment set forth herein.

Figure 1 shows the chromatographic profile of the results obtained with the soft extract and dry extract of Moluccan leurites, in (A) using the AcOEt: Acetone: H20 25: 8: 2 mixture and (B) using as eluent the hexane mixture. : AcOEt 85:15.

Figure 2 shows the results obtained from High Performance Liquid Chromatography (HPLC) analysis of moluccan Aleurite extract, in (A) with the dry extract and (B) with the soft extract.

Figure 3 shows the results obtained from the 2 '-0-ramnosyl-swertisine marker analysis by Carbon 13 Nuclear Magnetic Resonance (C13 NMR) (300 MHz) in methanol deuterated (MeOD), being in (A) in the region of 65 to 0 ppm (B) in the region of 86 to 54 ppm (300 MHz) and in (C) the region of 205 to 85 ppm (300 MHz). Figure 4 shows the results obtained from the 2 "-0- marker analysis. Hydrogen Nuclear Magnetic Resonance Ramnosyl-Swertisine (H1 NMR) (300 MHz) in deuterated methanol (MeOD), being (A) in the region of 13 to 0 ppm (300MHz); in (B) in the region of 8.5 to 5.5 ppm (300 MHz) and in (C) in the region of 4.5 to 0.5 ppm (300 MHz).

Figure 5 shows the HPLC chromatogram of the CCDP purified standard 2 '' -O-ramnosyl-swertisine (AMSR170707) at 145 µg / ml, being at (A) at 338 nm and (B) in the deultraviolet region at 213, 8, 270.3 and 336.8 nm.

Figure 6 shows the results obtained with the 2 '-0-ramnosyl-swertisine marker when evaluated by Infrared Spectroscopy with KBr pellet.

Figure 7 shows the results obtained with the moluccan Aleurites extracts, in (A) with dry extract and (B) with soft extract and in (C) the control group.

Figure 8 shows the results obtained with the extraction of moluccan Aleurites, being (A) Phase I, (B) Phase II and (C) Edema (control group).

Figure 9 shows the results obtained with the moluccan Aleurites extratomole, being (A) Phase I, (B) Phase II and (C) Edema (control group).

Figure 10 represents the results obtained with the moluccan Aleurites extracts, being in (A) with dry extract and (B) with soft extract.

Figure 11 shows the effect of orally administered dry extract of Alucite moluccana (125 to 500 mg / kg) and dexamethasone on the paw edema model, being induced in (A) by carrageenan, in (B) porbradicinin and in (C) by histamine.

Figure 12 shows the results obtained with the oral extract of moluccan Aleurites (125 to 500 mg / kg) and indomethacin (10 mg / kg) administered orally in the carrageenan-induced pleurisy model in mice used in (A), total leukocytes; in (B), with exudation; in (C) neutrophils; and in (D) mononuclear cells.

Figure 13 shows the results obtained with the oral extract of moluccan Aleurites (125 to 500 mg / kg) and indomethacin (10 mg / kg) administered orally in the substance P-induced pleurisy model in mice, being used in (A ) total leukocytes; in (B) exudation, in (C) neutrophils and (D) mononuclear cells.

Figure 14 shows the results obtained with oral extract of moluccan Aleurites (125 to 500 mg / kg) and indomethacin (10 mg / kg) administered orally in the bradykinin-induced pleurisy model in mice used in (A) leukocytes. totals; in (B) exudation; in (C) neutrophils; in (D) mononuclear cells.

Figure 15 shows the results obtained with oral extract of moluccan Aleurites (125 to 500 mg / kg) and indomethacin (10 mg / kg) administered orally in the histamine-induced pleurisy model in mice used in (A) leukocytes. totals; in (B) exudation; in (C) neutrophils and in (D) mononuclear cells.

Figure 16 shows the results obtained with the extract of moluccan Aleurites (125 to 500 mg / kg) and indomethacin (10 mg / kg) administered orally in the croton oil-induced ear edema model.

Figure 17 shows the results obtained with the oral extract of moluccan Aleurites (5 to 10 mg / kg) and Dipyrone (10 mg / kg) administered orally in the LPS-induced hyperthermia model.

Figure 18 shows the results obtained with the oral extract of moluccan Aleurites (5 to 10 mg / kg) and indomethacin (10 mg / kg) to verify the appearance of lesion in the gastric mucosa.

Figure 19 shows the antihypernociceptive effect of dry Mucurcan Aleurites extract (125 to 500 mg / kg, v.o.) on the injection-induced mechanical hypernociception model. carrageenan (300 µg / paw).

Figure 20 shows the antihypernociceptive effect of dry Mucurcan Aleurites extract (125 to 500 mg / kg, v.o.) on the mechanical injection-induced hypernociception model. of PGE 2 (0.1 nmol / paw).

Figure 21 shows the effect of preventive treatment with dry extract of moluccan Aleurites (125 to 500 mg / kg, v.) On mechanical injection-induced hypernociception i.pl. CFA (20 µl / paw). Figure 22 shows the effect of curative treatment as a dry extract of moluccan Aleurites (125 to 500 mg / kg, v.o.) on injection-induced mechanical hypernociception i.pl. CFA (20 µl / paw) on the paw, on (A) ipsilateral and (B) contralateral to injection.

Figure 23 shows the results obtained with the dichloromethane fraction of moluccan Aleurites evaluated by Gas Chromatography (GC).

Figure 24 shows the effect of treatment with dry Mucurcan Aleurites extract (125 to 500 mg / kg, v.o.) on CPNC-induced mechanical hypernociception.

DETAILED DESCRIPTION OF THE INVENTION

In order to facilitate the understanding of the present invention, the invention will be further explained by identifying its particularities and preferred embodiments. However, the present disclosure should not be construed as limiting the embodiments of the invention.

PROCEDURES FOR OBTAINING STANDARDIZED EXTRACTS

A process of obtaining a standardized extract is particularly useful in the development of a phytotherapeutic drug because of its quality, its phytochemical constitution by the use of markers, its physicochemical characteristics, its pharmacological properties, and, consequently, its therapeutic action in a reproducible manner. , the process of obtaining a standardized extract from at least a part of the dog-like Aleurites plant with antinociceptive, anti-inflammatory and antipyretic properties according to the present invention comprises the following steps:

collection, drying and subdivision of plant material;

pre-extraction;

extraction with solvent extraction;

extract filtration;

extract concentration;

pausteurization;

drying; and

qualitative and quantitative analysis (standardization) of the extract.

The process steps according to the present invention are sufficient to ensure an adequate concentration of at least one of the following compounds: alpha-amyrin, beta-amirin, alpha-amirinone, beta-amirinone, swertisine and 2 '' - O -ramnosyl-swertisine. In particular, the process steps are capable of providing the standardized extract with a total flavonoid concentration of 0.1 to 15% of their constitution. More preferably, the process steps are capable of providing a 2 'O-ramnosyl-swertisine concentration of 0.05 to 15% of its composition.

In accordance with the present invention, the collection, drying and subdivision step is carried out with plant material obtained from a part of a plant of the genus Aleurites, which includes: A. trisperma, A. cordata, A. montana, A. fordii, A. montance, A. rockinghamensis and A.moluccana. Preferably, part of the Aleurites moluccana L. Willd species, and more specifically its leaves, is used as a vegetable material.

The plant material can be obtained, with advantages, from healthy individuals of the species A. moluccana, whether they are adults 10 years old, young (<10 years), intact or in the bud stage, and can be collected in any month of the year, in any period. seasonal, any time or period of the day. However, the collection of plant material is especially useful when carried out in winter, given the best phytochemical profile of the species during this period.

In accordance with the present invention, the plant material may be used fresh, dried, whole, scratched, minced, ground, ground, pulverized or micronized. More specifically, it is useful to use dry and ground particulate vegetable material with dimensions <50 mm.

The process of obtaining standardized extract according to the present invention requires a pre-extraction step corresponding to the washing of the suitable solvent plant material, preferably ethanol. The pre-extraction step is especially advantageous for removing contaminants or impurities adsorbed on plant material, particularly heavy metals (eg lead and mercury) adsorbed on the leaf surface, which can contaminate the final standardized extract if not eliminated. In a preferred embodiment of the pre-extraction step, the plant material is washed with sufficient 96 ° GL ethanol under constant agitation at room temperature for 15 to 30 minutes and the solvent is exhausted and removed after this time.

The extraction solvent extraction process step according to the present invention comprises an extraction procedure such as, but not limited to maceration, percolation, decoction, infusion, leaching, distillation, turbolysis (or turbo extraction), supercritical extraction or the like. employing a suitable extractor solvent.

Examples of extracting solvents include, but are not limited to water, methanol, ethanol, propanol, isopropanol, propylene glycol, acid solution, ethyl acetate, dichloromethane, chloroform, hexane, glycerin, acetone, petroleum ether, supercritical fluid, a combination of the same and similar.

Preferably, according to the present invention, the extraction process consists of maceration and the solvent extractant in a water: ethanol hydroalcoholic solution, wherein the water content is not higher than the alcohol content, for example, using the reasons 1: 9, 2: 8, 3: 7, 4: 6 and 1: 1. More preferably, the water: alcohol ratio is 3: 7. Extraction may occur under the presence or absence of light and / or oxygen, with or without agitation, for approximately <10 days.

The extracting solvent: plant material ratio employed in the extraction procedure may vary from 1: 1 to 20: 1. More specifically, the extracting solvent: vegetable material ratio is 10: 1.

In accordance with the present invention, the process of obtaining the extracts requires a filtration procedure to eliminate the residue of the plant material. Filtration procedures include, but are not limited to, deep or surface filtration using different types of filter materials.

The step of the extract concentration process may be carried out using a procedure selected from the techniques of reduced pressure evaporation, heat convention evaporation, heating, a combination thereof and the like.

More preferably, the extract concentration procedure relates to the reduced pressure evaporation technique, employing a heating temperature of approximately <70 ° C and a vacuum of 200 to 400 mmHg, which is capable of providing a concentration of approximately 20% to 50%. of solids in the extract, and sufficient for the total elimination of alcohol.

After extract concentration it is especially useful to process a soft extract pasteurization step to extinguish any microbial contamination. The pasteurization step is particularly applicable when carried out at a temperature of about 95 ° C for at least 3 minutes.

Plant extracts are generally known to contain pigments, such as chlorophyll, and insoluble materials that can provide undesirable visual appearance to the extracts. Thus, according to a preferred aspect of the present invention, a step of eliminating these elements or depigmentation may optionally be employed after the extract concentration step in the process of obtaining a standardized extract. Insoluble pigments and materials may be partially or totally removed from the extracts, for example by treatment with activated charcoal or adsorption resin, membrane ultrafiltration and the like.

More particularly, the use of ultrafiltration on pore size membranes between 5 and 100 kDa driven under gravity or pressure is useful.

Membrane ultrafiltration may also be useful for removing oxidases from extracts, especially those with high molecular weight, such as laccase. Elimination of oxidases may be especially desirable in stabilizing standardized extracts. Other processes are known in the art for performing an extract stabilization step. For example, an extract may be stabilized by the addition of a sufficient amount of one or more stabilizing agents or antioxidants such as, but not limited to: butylhydroxytoluene, butylhydroxyanisole, ascorbic acid and its derivatives, octyldodecanol, cysteine, glutathione, and the like. Thus, optionally, an extract stabilization step relating to the addition of a stabilizing agent at a concentration of 0.01 to 5%, preferably 0.2 to 1% combase in the extract may be included in the process. As a preferred aspect, a stabilizing agent may be added during or after the extraction procedure.

According to an alternative embodiment of the present invention, to obtain a standardized dry extract, an extract drying step can be performed after the concentration step.

The drying procedure according to the present invention may be selected from a group consisting of deliophilization spray-drying, reduced pressure evaporation, heat-convention drying, or a combination thereof. More preferably, the extract drying procedure relates to the spray-drying technique.

In the spray-drying procedure, drying aids may be advantageously employed. Examples of drying aids include, but are not limited to: colloidal silicon dioxide, mandiocamodified starch, tricalcium phosphate, maltodextrin, cyclodextrins, microcrystalline cellulose , lactose, a combination thereof and the like. Addition of drying adjuvants in an amount of 10 to 40% relative to the total solids content of the final product is especially useful.

According to an optional aspect, an additional microbial decontamination step may be performed after the drying step. Gamma radiation, ultraviolet radiation and the like techniques are particularly useful in this case.

The process of obtaining a standardized extract, according to the invention, requires a qualitative and quantitative analysis step through duly validated analytical methodologies regarding the parameters of delineability, accuracy, precision, specificity and robustness.

In accordance with the invention, particularly useful analytical methodologies for equitative qualitative characterization include, but are not limited to, thin layer chromatography (CCD), paper chromatography, gas chromatography (GC), column chromatography (CC), high performance liquid chromatography (HPLC). ), mass spectrometry (MS), thermo-analytical techniques and the like.

A standardized extract according to the present invention may be particularly analyzed by CCD, presenting a peculiar chromatographic profile as shown in Figure 1 (A and B) and by HPLC as shown in Figure 2 (A and B). Figure 1 (A) shows the chromatographic profile results obtained with the soft extract and dry extract of moluccan Aleurites, when evaluated in a chromatographic plate, developed using a 25: 8: 2 AcOEt: Acetone: H20 mixture as revealed with ferric chloride solution. 3%. The first spot refers to the soft extract (EtOH: H2 O 9: 1), the second to the dry extract, the third to the swertisine standard and the fourth to 2-O-ramnosyl-swertisine. Figure 1 (B) shows the profile chromatographic analysis of the results obtained with the soft extract and the dry extract of Aleurites moluccana, when evaluated by placachromatography, using as the eluent the mixture of hexane: AcOEt 85:15 and developed with hot anisaldehydesulfuric solution.The first spot refers to the extratomole (EtOH: H20 9: 1), the second to the dry extract, the third standard of alpha and beta-amirin, the fourth alpha-beta-amirinone standard and the fifth standard of the mixture of stigmasterol and beta-sitosterol Figure 2 (A) shows the results obtained. with the dry extract of Aleuritesmoluccana when evaluated by High Performance Liquid Chromatography (HPLC) The extract was initially dissolved in methanol (20% of final volume), sonicating for 5 min, followed by the addition of acidified water to pH 3.57 (20% of final volume). The volume was then made up with a 50:50 methanol / acid water mixture at a concentration of 1 to 2 mg / ml. After filtration of the sample into a 0.22 µm membrane filter, 20 µl of the solution was injected using a reverse phase analytical column, where Cia groups are chemically bonded to silica, having a length of 10 to 25 cm and an internal diameter of 4 to 6 mm. The detector used was a PDA (diode array) type monitoring the chromatographic run at 254 and 338 nm. Separation was performed using a mobile phase linear gradient, initially composed of methanol: acidified water pH 3.57: acetonitrile (20:15:65) in a flow rate of 0.5 to 1 ml / min, up to 10: 15: 75, after 20 min, with pressure range from 2,000 to 3,000 psi.

Figure 2 (B) shows the results obtained with the moluccan Aleurites extratomole when evaluated by High Performance Liquid Chromatography (HPLC) of the same as in Figure 2 (A).

A standardized dry extract or soft extract according to the present invention is particularly characterized in that it has a 2 '' - O-ramnosyl swertisine marker content of 0.05 to 15% of its composition.

A fractionation step of a standardized extract according to the present invention may be especially useful for concentration of substances potentially related to the pharmacological activities of the species. Numerous fractionation processes are known in the art. More particularly, it is useful to use sparingly with different chromatographic supports (eg, silica, Sephadex®, alumina, cellulose and the like) and suitable eluents or liquid-liquid partition using hexane, dichloromethane, chloroform, ethyl acetate, butanol. and the like. More preferably, hexane, dichloromethane and chloroform may be employed to obtain a concentrated fraction relative to the supporting substances of an extract, such as beta-amyrin, alpha-amyrin; and ethyl acetate and butanol to obtain a concentrated fraction relative to the polar substances of the extract, such as wertisine and 2 '' - O-ramnosyl swertisine.

Preferred embodiments of the present invention for obtaining and analyzing a standardized extract are detailed in Examples I, II and III.

MARKER INSULATION, PURIFICATION AND CHARACTERIZATION

The election, isolation, purification and characterization of an extract constituent / marker become particularly necessary for the procedures of extract quantification and standardization.

According to the present invention, 2 '' - O-ramnosyl-swertisine flavonoid has been determined as the most suitable marker for a standardized extratation process. Such election is justifiable because it is a major compound in the plant, because it is more discriminatory of the species, because it is related to the pharmacological properties of the extract and the fact that it is a flavonoid serum, which may be indicative of the stability of the extract, during technological processing or storage (Sonaglio , D. et al., Technology Development and Production of Phytotherapics In: SIMões et al. (Org.) Pharmacognosy: From Plant to Drug PortoAlegre: University Publisher, 1999, 221-258).

These quantification and standardization procedures for extracts occur according to the following steps:

I. Isolation,

II. Purification, and

III. Marker characterization.

Isolation of the 2 '' - O-ramnosyl-swertisine marker (step I) can be accomplished by techniques which include, but are not limited to, liquid-liquid partition, preparative CCD, column chromatography, centrifugal chromatography, preparative HPLC ( CLAEP) or semi-preparative, a combination of the same and similar.

More preferably, the isolation of the 2 '' -O-ramnosyl-swertisine marker can be accomplished by sequentially employing liquid-liquid partition, open column chromatography and preparative CCD techniques, using silica in the latter two. 60 as a stationary phase and chloroform-methanol mixture as eluent or preparative or semi-preparative HPLC using preparative or semi-preparative C18 type reverse phase columns with a particle size of 10 µm using a linear gradient of methanol: acetonitrile: acidified water monitored at 254 and 338. nm.

Purification of the 2 '' - 0-ramnosyl-swertisine marker (step II) can be performed by techniques that include, but are not limited to, preparative CCD and preparative or semi-preparative HPLC.

More preferably, marker purification is carried out by preparative CCD using stationary phase silica 60 and chloroform: methanol mixture as eluent. Alternatively, the preparative or semipreparative HPLC technique using preparative or semipreparative Cie type reverse phase phase columns of approximately 10 µm particle size, using a linear gradient of methanol: acetonitrile: acidified water, monitored approximately 254 to 338 nm.

According to the present invention, by using the procedures described above, a purity of approximately> 90% should be achieved.

In accordance with the present invention, the quantitative and qualitative analysis of the isolated and purified 2-O-ramnosyl-swertisine marker, i.e. its characterization (step III) can be performed by techniques which include, but are not limited to, nuclear magnetic resonance (NMR) of hydrogen and carbon-13, infrared (IR) spectrophotometry, ultraviolet (ÜV) spectrophotometry, HPLC, CCD, thermal analysis.

The isolated 2 '-O-ramnosyl swertisine marker is particularly characterized by NMR and HPLC.

Figure 3 shows the results obtained with the marker when evaluated by carbon 13 Nuclear Magnetic Resonance (C13 NMR) (300 MHz) in deuterated methanol (MeOD), being (A) in the region of 65 to 0 ppm (300 MHz); (B) in the region from 86 to 54 ppm (300 MHz) and (C) in the region from 205 to 85 ppm (300 MHz). Figure 4 shows the results obtained with the 2 "-0-ramnosyl-swertisine marker when evaluated by Hydrogen Nuclear Magnetic Resonance (H1 NMR) (300MHz) in deuterated methanol (MeOD), being (A) in the region of 13a 0 ppm (300 (B) in the region of 8.5 to 5.5 ppm (300 MHz) and (C) in the region of 4.5 to 0.5 ppm (300 MHz) Characterization of the marker by HPLC is shown in Figure 5

Figure 5 (A) shows the HPLC chromatographic profile of the CCDP purified 2 '' - O-ramnosyl-swertisine standard (AMSR170707) at 145 µg / ml, at 338 nm, which eluted in about 17 min. demonstrating successful marker purification as indicated by the presence of a single peak on the chromatogram. Figure 5 (B) shows the results, specifically the absorption profile of the typical flavonoid peak, obtained with the 2 "-0-ramnosyl-swertisine marker with maximal absorption of 213.8, 270.3 and 336.8 nm when evaluated. by scanning the ultraviolet spectrum on the PDA (Photo DiodeArray) detector of the Liquid Chromatograph, as in Figure 2. (A) Figure 6 shows the typical absorption spectrum of the flavonoid infrared.

A preferred embodiment of the present invention for label isolation, purification and analysis is detailed in Example IV.

PHARMACOLOGICAL CHARACTERIZATION OF STANDARDIZED EXTRACTS

Standardized extracts, according to the present invention, can be characterized as to their pharmacological activity in animal models of pain by nociception, hypernociception, neuropathic pain, inflammation and fever.

Several animal models can be applied for the pharmacological characterization of an extract with antinociceptive, antiinflammatory and antipyretic properties, among which are: 1. Test of abdominal writhing induced by acetic acid; 2. Randal-Sellito test; 3.Mechanical hypernociception models (of inflammatory and / or neuropathic origin); 4. formalin test; 5. hot plate test; 6. neuropathic pain tests; 7. Rat and / or mouse paw-ear edema assay induced by different phlogistic agents; 8. Pleurisy model, induced by different phlogistic agents; 9. Freund's adjuvant induced arthritis models; 10. LPS-induced hyperthermia models; 11. Evaluation of undesirable effects; and the like.

Standardized extracts according to the present invention, when characterized for their pharmacological activity, show antinociceptive, antiinflammatory and antipyretic properties, as detailed below.

Antinociceptive effect of extracts of AZ.euri.tes moluccana through the model of abdominal writhing induced by acetic acid.

Oral administration of the dry and soft extracts is capable of significantly and dose-dependent inhibition of the number of i.p. of acetic acid in mice, and ID50s of 62.65 (24.99-157.64) mg / kg, 99.84 (85.30-116.85) mg / kg, respectively. Figure 7 shows the results obtained with the extracts of moluccan laurites, in (A) (dry extract) and (B) (soft extract), administered orally, in the model of abdominal acetic acid induced contortions. Cadacoluna represents the average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differed significantly from the control group (see Figure 7 (C)), ** P <0.01. The inhibition values (IM) obtained are set at 68.64 ± 1.22%; 55.12 ± 2.13% and 64.0 ± 1.65%, 63.5%, and 96.9% for the dry and soft extracts, respectively, as shown in Table 1 below, compared to the drugs used. at the clinic.

Table 1 - Comparative data of DI50 and MI in the model of abdominal writhing induced by acetic acid: orally.

<table> table see original document page 33 </column> </row> <table>

Antinociceptive effect of moluccananauritis extracts through the formalin-induced pain model.

Oral administration of the dry extract at doses of 50 to 500 mg / kg orally is capable of significantly and dose-dependent inhibition of both the neurogenic phase and the inflammatory phase of nociception induced by the i.pl. injection. formalin, with inhibition of69 ± 2.34% and 15.84 ± 1.98% for the first and second formalin phase, respectively, and DI50% of 271.83 (248.84 -296.94) mg / kg for the first phase of the test. Figure 8 shows the results of this experiment, being (A) Phase I, (B) Phase II and (C) Edema. Each column represents an average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differs significantly from the control group (C), * P <0.05 and ** P <0.01, so the same extract was unable to reduce formalin-induced edema depata (Figure 8 (C)) .

Administration of the soft extract of Aleuritesmoluccana at doses of 50 to 500 mg / kg orally is capable of significantly and dose-dependent inhibition of both the neurogenic and inflammatory phase of nociception induced by i.pl. injection. deformalin, with inhibition of 46.5 ± 3.41% and 79, 08 ± 2.54% for the first and second formalin phases, respectively, and ID50% greater than 500 mg / kg for the second phase of the test. Figure 9 shows the results of this experiment in the formalin-induced pain model, being (A) Phase I, (B) Phase II and (C) Edema.

Each column represents an average of 8 to 10 animals and vertical bars indicate the E.P.M. The result differed significantly from the control group (C), * P <0.05 and ** P <0.01. The same extract is able to reduce formalin-induced paw edema by 34% inhibition (Figure 9 C). The values of MI and ID50 compared to the drugs used in the clinic are shown in Table 2 (Phase I) and Table 3 (Phase II) below.

Table 2 - Comparative data of DI50 and IM - formalin-induced donor model - Phase I: oral.

<table> table see original document page 34 </column> </row> <table> <table> table see original document page 35 </column> </row> <table>

Table 3 - Comparative data of DI50 and MI - formalin-induced pain model - Phase II: oral route.

<table> table see original document page 35 </column> </row> <table>

Table 4 below shows the values of DI50 and model - formalin - induced pain in the evaluation of edema compared to drugs used in clinical trials.

Table 4 - Comparative data of DI50 and IM - formalin-induced donor model - edema: oral route.

<table> table see original document page 35 </column> </row> <table>

Antinociceptive effect of zaoluccana Aleurites extracts through the hot plate model.

Figure 10 represents the results obtained with the dry (A) and Mole (B) extracts of orally administered moluccan Aleurites in the hot plate model. Each point represents the average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differed significantly from the control group (C), ** P <0.01. None of the extracts, when administered orally, is capable of significantly altering the animals' latency in the hot plate test when compared to morphine, the drug of choice for positive control, as shown in Figure 10.

Antiinflammatory effect of the dried extract of Aleuritesmoluccana through the phlogistic agent-induced paw edema model.

As can be seen from Figure 11 (A and B), oral administration of the dried extract of moluccan Aleurites (125 to 500 mg / kg) is able to significantly reduce carrageenan-induced paw edema and bradykinin. However, the same extract is able to significantly inhibit histamine-induced paw edema in only one point (30 min) (Figure 11 C).

Antiinflammatory effect of the dried extract of Aleuritesmoluccana through the model of pleurisy induced by intrapleural injection of phlogistic agents

Figure 12 shows the results obtained with the extraction of moluccan Aleurites (125 to 500 mg / kg) and indomethacin (10 mg / kg), administered orally, in the carrageenan-induced pleurisy model in mice. Each point represents the average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differed significantly from the control group (C), * P <0.05 and ** P <0.01. # represents the difference between the group control and the saline group. In (A) total leukocytes are used; in (B) with exudation; in (C) comneutrophils; and in (D) mononuclear cells. As can be seen in Figure 12, pretreatment with oral extract of moluccan Aleurites at doses of 125-500 mg / kg is able to inhibit exudation as well as amicable inflammatory cell counts, represented by total leukocyte counts. , eneutrophic mononuclear cells, in the model of pelacarragenin-induced pleurisy in mice. However, it is capable of interfering only with plasma extravasation and mononuclear cell naming in the P-induced pleurisy model (see Figure 13 A to D). Figure 13 shows the results obtained with the dried extract of Moluccan Aleurites (125 to 500 mg / kg) and Indomethacin (10 mg / kg) orally administered in the P-induced pleurisy model in mice. Each score represents an average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differs significantly compared to the control group (C), * P <0.05 and ** P <0.01. #represents the difference between the control group and the salt solution group. The experiment was performed on leukocyte totals (Figure 13 (A)); by exudation (Figure 13 (B)); comneutrophils (Figure 13 (C)); and with mononuclear cells (Figure 13 (D)).

Figure 14 shows the results obtained with the extract of moluccan Aleurites (125 to 500 mg / kg) and indomethacin (10 mg / kg) administered orally in the bradykinin-induced pleurisy model in mice. Each point represents the average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differed significantly from the control group (C), * P <0.05 and ** P <0.01. # represents the difference between the group control and the saline group. The experiment was performed on (A) total leukocytes; in (B) exudation; in (C) neutrophils; and in (D) mononuclear cells. As shown in Figure 14, oral pretreatment with A. moluccana extract at doses of 125 to 500mg / kg is able to inhibit exudation as well as the migration of inflammatory cells, represented by leukocyte count. mononuclear cells and neutrophils, bradykinin-induced pleurisy model in mice. However, it is capable of interfering only with plasma extravasation (dose-dependent) and mononuclear cell migration using the Histamine-induced pleurisy model (see Figure 15B-D). Figure 15 shows the results obtained with dry moluccan Aleurites extract (125 to 500 mg / kg) and orally administered Indomethacin (10 mg / kg), as the histamine-induced pleurisy model in mice.

Each dot represents the average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differed significantly from the control group (C), * P <0.05 and ** P <0.01. # represents the difference between the group control and the saline group. The experiment was performed on total leukocytes (Figure 15 (A)); with exudation (Figure 15 (B)); in neutrophils (Figure 15 (C)); and in mononuclear cells (Figure 15 (D)).

Antiedematogenic effect of the dried extract of Aleuritesmoluccana through the inflammation model the ear edema induced by Croton oil.

Figure 16 shows the results obtained with the extract of moluccan Aleurites (125 to 500 mg / kg) and indomethacin (10 mg / kg) administered orally in the croton oil-induced ear edema model. Each score represents an average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differs significantly compared to the control group (black bar), * P <0.05 and ** P <0.01. Figure 16 shows that oral administration of dry oral extract is effective in reducing croton oil-induced ear edema with 43.75 ± 2.47%. Antipyretic effect of moluccananauria extracts through the hyperthermia model induced by LPS in rats.

Figure 17 shows the results obtained with the oral extract of moluccan Aleurites (5 to 10 mg / kg) and Dipyrone (10 mg / kg) administered orally in the LPS-induced hyperthermia model. Each dot represents the average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differs significantly from the control group, * P <0.05. # means the difference between the control group and the saline group. Figure 17 shows the fact that oral administration of dry extract causes a statistically significant reduction in febrile response of animals compared to controls (dipyrone, vehicle) and does not produce changes in the gastric mucosa, as usual in antipyretic agents.

Effect of moluccan Aleurites extracts on gastric mucosal induction in rats.

Figure 18 shows the results obtained with the oral extract of moluccan Aleurites (5 to 10 mg / kg) and indomethacin (10 mg / kg) to verify the appearance of lesion in the gastric mucosa. Each score represents an average of 8 to 10 animals and the vertical bars indicate the E.P.M. The result differs significantly compared to the control group, * P <0.05. # means the difference between the control group and the saline solution group. Figure 18 shows the fact that oral administration of soft extract does not produce mucosagastric changes, so common in antipyretic agents.

Effect of dry extract of moluccan Aleurites on mechanical hypernociception induced by carrageenan intraplantar injection.

Figure 19 shows the antihypernociceptive effect of dry Mucurcan Aleurites extract (125 to 500 mg / kg, v.o.) on the injection-induced mechanical hypernociception model. carrageenan (300 µg / paw). Each dot represents an average of 6 to 8 animals and the vertical bars indicate the E.P.M. The result differs significantly from the control group, * P <0.05; ** P <0.01 and *** <0.001. As can be seen in Figure 19, oral treatment with A. moluccana dry extract, when administered at doses of 125, 250 and 500 mg / kg, is capable of significantly and dose-dependent reduction of carrageenan-induced mechanical hypernociception with ID50% of 433 (295 to 407) mg / kg. This inhibition is observed for up to 6h after carrageenan injection. The observed inhibitions in relation to the control response are set at 33 ± 5% and 51 ± 2% for the 250 and 500 mg / kg doses, respectively.

Effect of dry extract of moluccan Aleurites on mechanical injection-induced prostaglandin E2 (PGE2) mechanical hypernociception Figure 20 shows the antihypernociceptive effect of dry extract of Moluccan Aleurites (125 to 500 mg / kg, vo) on induced mechanical hypernociception model by injectioni.pl. of PGE 2 (0.1 nmol / paw). Each dot represents an average of 6 to 8 animals and the vertical bars indicate the E.P.M. The result differs significantly from the control group, * P <0.05; ** P <0.01 and *** <0.001. Figure 20 shows the fact that oral treatment with A. moluccana dry extract (125, 250 and 500 mg / kg) is able to significantly and dose-dependently reduce the mechanical injection-induced hypernociceptive response. PGE2 (for up to 2 h after PGE2 injection), with an ID50% of 201 (91 to 445) mg / kg. Inhibitions of 8 ± 6%, 11 ± 8% and 63 ± 7% were obtained for the doses of 125, 250 and 500 mg / kg, respectively.

Effect of dry extract of Moluccan Aleurites on mechanical hypernociception induced by Freund's complete adjuvant intraplantar injection (CFA).

Figure 21 shows the effect of preventive treatment with dry extract of moluccan Aleurites (125 to 500 mg / kg, v.) On mechanical injection-induced hypernociception i.pl. of CFA (20 µl / paw). Each dot represents an average of 6 to 8 animals and the vertical bars indicate the E.P.M. The result differs significantly from the control group, * P <0.05; ** P <0.01 and *** <0.001. Figure 21 shows the fact that preventive oral administration with the dry extract of A. moluccana (125, 250 and 500 mg / kg) is able to significantly reduce the mechanical injection-induced hypernociception. deCFA, for up to 6 h after induction of hypernociception, with ID50% of 275 (239 to 316) mg / kg and inhibitions of 29 ± 6% and 53 ± 6% at 250 and 500 mg / kg, respectively.

To evaluate the effect of A.moluccana dry extract on mechanical hypernociception, the animals were first submitted to i.pl. of CFA, and after 24 h were treated with the extract at doses of 125, 250 and 500 mg / kg 2 times daily for 5 consecutive days.

Figure 22 shows the effect of curative treatment as dry extract of moluccan Aleurites (125 to 500 mg / kg, v.o.) On mechanical injection-induced hypernociception i.pl. CFA (20 ul / paw) in the ipsilateral and contralateral injection paw (Figure 16 (A)) (Figure 16 (B)). Each point represents the average of 6 to 8 animals and the vertical bars indicate the E.P.M. The result differed significantly from the control group (C), * P <0.05; ** P <0.01 and *** <0.001. As shown in Figure 22, the administration of A. moluccana dry extract is capable of reversing the mechanical hypernociception condition previously installed in the ipsilateral injection foot, in a dose-dependent manner, with an ID50% of 272 (130 to 568) mg / kg. The inhibitions were 8 ± 6%, 11 + 8 I and 63 ± 7 at the doses of 125, 250 and 500 mg / kg, respectively (Figure 22 A). In a similar manner, the same treatment is able to prevent the emergence of mechanical hypernociception in the patacontralateral to the injected paw, with inhibition of 56 ± 8% of 500 mg / kg nadose (Figure 22 B).

A preferred embodiment of the pharmacological characterization assays of the standardized extracts of Moluccan leuritis obtained according to the above described procedures for their antinociceptive, antiinflammatory and antihypernociceptive activity can be found in detail in Examples V, VI and VII.

Toxicological evaluation of soft extract of Aleuritesmoluccana.

Preliminary mouse studies have shown that oral administration of the 5000 mg / kg A. moluccana nadose soft extract (n = 10, 5 males and 5 females) did not cause any death within 3 to 24 hours. In addition, no signs of irritability, tachycardia, tremors, and piloerection were evidenced, as detailed in Tables 5 and 6.

Table 5 - Evaluation of behavioral parameters in male mice treated with soft extract (5000 mg / kg) of Moluccan Aleurites using the Hippocratic test. <table> table see original document page 45 </column> </row> <table>

Table 6 - Evaluation of behavioral parameters in female mice treated with soft extract (5000 mg / kg) of Moluccan Aleurites using the hippocratic test.

<table> table see original document page 46 </column> </row> <table>

A preferred embodiment of the pharmacological characterization assays for the standardized extracts of Moluccan leurites obtained according to the above described procedures for toxicity can be found in detail in Example VIII.

PHARMACEUTICAL COMPOSITIONS

A moluccan aieurite extract obtained and characterized as described above may be administered alone, but will generally be administered in combination with one or more pharmaceutical vehicles or adjuvants, selected based on the route of administration and galenic practice.

Pharmaceutical compositions with antinociceptive, anti-inflammatory and antipyretic properties, intended for use in mammals, containing as an active ingredient one of the effective of at least one standardized extract obtained and characterized according to the processes described above, in combination with one or more pharmaceutical vehicles or adjuvants, are also aspects of present invention.

More particularly, it is an object of the present invention to provide a pharmaceutical composition comprising at least one moluccan Aleurite extract which comprises alpha-amyrin, beta-amirin, alpha-amirinone, beta-amirinone, stigmasterol, beta-sitosterol, campesterol, swertisine and standard compounds. with respect to its 2 '' -O-ramnosyl-swertisine marker at 0.05 to 15% content.

The pharmaceutical composition according to the present invention may contain a standardized extract as defined above in an amount representing 5 to 90% of the total weight of the composition, preferably an amount representing 20 to 80% of the total weight of the composition.

The pharmaceutical composition according to the present invention may contain a standardized extract as defined above at a concentration of 0.01 to 2000 mg per dosage unit, preferably a concentration of 50 to 1000 mg per dosage unit.

Standard extracts and compositions of the present invention may be administered orally by oral pharmaceutical forms such as lozenges, capsules (hard or soft), pills, powders, granules, simple tablets, coated tablets, chewable tablets, effervescent tablets, sublingual tablets, tablets. controlled release, dragees, globules, elixirs, suspensions, syrups and emulsions, each of which includes immediate, controlled, prolonged or delayed release formulations.

Compositions containing the extract of the invention may also be administered topically by pharmaceutical forms such as ointments, ointments, cream, emulsions, gels, solutions, pastes, aerosols, transdermal systems and others known in the art.

Intravenous (bolus or infusion), subcutaneous or intramuscular administration is also possible using pharmaceutical forms known in the art.

Administration may also be by intravaginal and rectal administration using as pharmaceutical forms: suppositories, ova, ointments, creams and others known in the art.

According to the present invention, the solid pharmaceutical composition for oral administration should preferably be in capsule or tablet form containing at least one standardized extract of Aleuritesmoluccan which may be combined with one or more pharmaceutically acceptable inert carriers such as lubricant, lubricants , non-sticking agents, slipping agents, binding agents, disintegrating agents, as well as preservatives, colorants, sweeteners, coating agents, plasticizers, opacifiers and the like as required. The amounts of these pharmaceutical vehicles or adjuvants may be equal to those conventionally used in the art.

Examples of diluents include, but are not limited to starch, lactose monohydrate, spray-dried lactose, microcrystalline cellulose, dicalcium phosphate, mannitol, cellulose powder and the like.

Examples of lubricants and glidants include, but are not limited to: stearic acid, silicon oxide, magnesium stearate, calcium stearate, talc, sodium stearyl fumarate, glyceryl stearate, polyethylene glycol and the like.

Examples of binders include, but are not limited to, pregelatinized starch, hydroxypropylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, gelatin, natural sugars such as glucose, syrups, natural and synthetic gums, acacia, sodium alginate polyethylene, , waxes and the like.

Examples of disintegrants include, but are not limited to cross-linked sodium carboxymethylcellulose, low-substitution sodium carboxymethylcellulose, sodium croscarraelose, sodium starch glycolate, cross-linked polyvinylpyrrolidone and the like.

Examples of glidants include, but are not limited to: colloidal silicon dioxide, hydrophobic colloidal desilicon dioxide, and the like.

Examples of preservatives include, but are not limited to ascorbic acid, peroxybenzoic acid esters, chlorobutanol, benzyl alcohol, alcohololfenethyl, dehydroacetic acid and the like.

Preferred examples of dyes include, but are not limited to, water-insoluble lacquer pigments, natural pigments such as p-carotene and chlorophyll, iron oxide, yellow iron sesquioxide, red iron sesquioxide, black iron oxide, and the like.

Examples of sweeteners include, but are not limited to: dextrose, mannitol, sorbitol, sucrose, glycerin, sodium saccharin, dipotassium glycyrrizate, aspartame, stevia and the like.

Examples of film coating polymers include, but are not limited to, hydroxypropyl methylcellulose (e.g. Opadry®), hydroxypropylcellulose, cellulose acetophthalate and methacrylic acid derivatives and methacrylates (e.g. Eudragit®) and the like.

Examples of plasticizers and opacifiers include, but are not limited to, glycerine fatty acid esters, triethyl citrate, propylene glycol, polyethylene glycol, titanium dioxide and the like.

Preferred embodiments of the present invention for obtaining a tablet and capsule are detailed in Example IX and Example X respectively.

Pharmaceutical compositions for oral administration in accordance with the present invention also include solutions, syrups, elixirs and suspensions, which may contain, but are not limited to solubilizers, solvents, co-solvents, dissolving aids, wetting agents, suspending agents, preservatives, stabilizers, alkalizers, acidifiers, viscosity modifiers, sweeteners and flavorings. In general, water, ethyl alcohol, appropriate oils, saline, aqueous dextrose (glucose), sucrose, glycerine, related sugar solutions and glycols such as propylene glycol or polyethylene glycols, phosphate buffer, cyclodextrins, agar, bentonite, carbomer, methylcellulose,

hydroxypropyl methylcellulose are suitable carriers in liquid pharmaceutical forms.

Preferred embodiments of the present invention for obtaining a suspension or syrup are detailed in Example XI and Example XII, respectively.

Pharmaceutical compositions according to the present invention may be pharmaceutical forms which provide modified, controlled, sustained, extended, prolonged, programmed, slow, delayed, enteric, colon-specific or site-specific release of the active ingredient.

Examples of pharmaceutical forms for these purposes include, but are not limited to, osmotic pump systems, matrix systems, reservoir systems, ion-ionic systems, enteric coated forms, pH dependent coating forms, transdermal systems, pulsatile devices, implants, liposomal systems, nanostructured systems, similar microstructured systems.

Pharmaceutical compositions according to the present invention may be obtained by industrial pharmaceutical processes and unit operations known to the art such as, but not limited to mixing, milling, sieving, grinding, micronizing, lubricating, grading, wetting, drying, dry granulation, wet granulation, compression, encapsulation, coating, dissolution and homogenization, or a combination thereof.

According to the present invention, a pharmaceutical composition for topical administration, preferably but not limited to emulsions, creams or ointments, may contain one or more pharmaceutically acceptable inert carriers or adjuvants. Such adjuvants may be phasase constituents, oil phase constituents, emollients, humectants, emulsifiers, surfactants, preservatives, stabilizers, antioxidants, chelating agents, cerasauto-emulsifiers, ointment bases, dyes, and the like, as needed. The amounts of these pharmaceutical vehicles or adjuvants may be equal to those conventionally used in the art.

Examples of oil phase constituents, hardening agents, emulsifiers and self-emulsifying waxes include, but are not limited to stearic acid, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, cetostearyl alcohol 20oe (20 moles of ethylene oxide), glyceryl monostearate, monostearate depropylene glycol, sorbitan monostearate, ceramic crystalline, anionic self-emulsifying wax (eg, Lanete), nonionic self-emulsifying wax (eg Cosmowax®), nonionic self-emulsifying wax (eg Polawax®), nonemonic emulsion-self-emulsifying wax ®) and the like.

Examples of emollients include, but are not limited to, isopropyl myristate, mineral oil, myristyl alcohol, cetyl palmitate, octyl palmitate, myristyl myristate, glyceryl stearate, polyoxidoethylene stearate, hydrogenated coconut glycerides, lanolin, lanolin alcohols, oleate decyl, other esters and fatty alcohols and the like.

Examples of aqueous phase constituents, wetting agents, wetting agents and polymers include, but are not limited to carbomer (e.g. Carbopol®), hydroxyethylcellulose (e.g. Natrozol® and Cellosize®), polyacrylic acid (e.g. Pemulem®), propylene glycol, sorbitol, glycerin, dimethicone, and similar urea.

Examples of surfactants include, but are not limited to tween 20, tween 80, triethanolamine, polysorbate 80, oxtoxynol, sorbitan monopalmitate, sodium lauryl sulfate and the like.

Examples of ointment bases include, but are not limited to, lanolin, petroleum jelly, hydrophilic petrol, yellow ointment, white ointment, hydrophilic ointment, depolyethylene glycol ointment and the like.

Examples of preservatives, stabilizers, antioxidants and chelating agents include, but are not limited to, methylparaben (e.g., Nipagin®), propylparaben (eg, Nipazol®), butylparaben, thimeroal, benzalkonium chloride, benzethonium chloride, phenoxyethanol + parabens (e.g. Phenonip®), imidazolidinylurea (e.g. Germall®), imidazolidinylurea + iodopropinyl butyl carbamate (GermallPlus®), edetic acid, disodium EDTA, ascorbic acid, sodium bisulfite, and the like.

In order to complement the therapeutic activity presented herein, the pharmaceutical compositions comprising at least one extract according to the present invention may further combine other pharmacologically active compounds such as synthetic, semi-synthetic substances, biological molecules (eg proteins), vitamins and other derivatives of plant origin, depending on the desired effect.

TREATMENT METHOD

Another aspect of the present invention relates to a method for preventing, controlling or treating clinical conditions of pain, inflammation and fever, some of the above, comprising administering to mammals a pharmaceutically effective dose of a standardized Moluccan Aleurite extract, or pharmaceutical composition. containing the same for an appropriate period.

The method according to the present invention relates to the clinical effects of an effective dose of a standardized extract of Moluccan Aleurites on diseases and symptoms in which antinociceptive, anti-inflammatory and antipyretic substances are particularly useful.

The method according to the present invention is particularly useful in controlling pain symptoms of nociceptive origin under acute, subacute or chronic conditions.

According to one embodiment of the present invention, the dose should correspond to about 0.01 to 100 mg of patient's extract per kilogram, preferably about 0.1 to 50 mg / kg, and more particularly about 0.5 to 20 mg / kg. kg

Extracts or pharmaceutical compositions according to the present invention may be advantageously administered in a single daily dose, or alternatively the total daily dosage may be divided to be administered two, three or more times daily.

According to one embodiment of the present invention a dose of the extract or pharmaceutical composition may advantageously be administered for a period of from 1 day to several months.

For certain treatments, however, it is appropriate to apply on alternate days, cyclically or otherwise.

The dosage regimen for a standardized extract or pharmaceutical composition according to the present invention naturally varies according to known factors, such as the route of administration, the species, age, sex, health, medical conditions and weight of the patient, the nature and extent of symptoms. , the type of concurrent treatment, the severity and degree of the disease, the frequency of treatment, the patient's renal and hepatic function and the desired effect. A doctor, dentist or veterinarian may determine and prescribe the effective amount of medication necessary to prevent, control or treat a medical condition, inflammation or fever.

USES

One aspect of the invention relates to the use of the standardized extract for the preparation of a medicament for treating clinical conditions of pain, inflammation and stroke.

The invention also relates to the use of the standardized extract for the preparation of a medicament for controlling painful symptoms of nociceptive origin in acute, subacute or chronic conditions.

The invention further relates to the use of the pharmaceutical composition containing the standardized extract for the preparation of a medicament for the treatment of clinical conditions of pain, inflammation and fever.

Finally, this invention also relates to the use of the pharmaceutical composition containing the standardized extract for the preparation of a medicament for the control of painful nociceptive symptoms in acute, subacute or chronic conditions.

The following are some more detailed examples of preferred embodiments of the present invention. The proposed procedures and data given are representative examples of the applications of the present invention and should not be construed as indicative of limiting use of the product.

EXAMPLE I

Obtaining the standardized soft extract from the leaves of Moluccana leurltes

A. moluccana leaves are washed, dried, crushed and sorted by sieving with ground leaves less than 20 mm in size. The plant material is then subjected to extraction with 7: 3 hydroalcoholic ethanol / water solution. The volume used for extraction corresponded to 10 parts of extracting liquid to 1 part of plant material. Extraction was done at room temperature by the maceration method without the use of stirring. After 5 days, the solution was percolated and filtered on filter paper. The filtrate was concentrated at a maximum temperature of 70 ° C in specific evaporators (Bernhauer) under vacuum (400 mmHg) to a soft extract without alcoholic strength and a total solids content of 20% to 40%.

Obtaining the dry extract of the leaves of Aleurltes moluccana

The soft extract was obtained as described above. As a result, a variable amount of 10 to 40% drying aid is added to the extract relative to the desolate content of the final product. The mixture was dehydrated in a spray dryer using centrifugal rotating disc, 45 Hz pump feed speed, generating 6.5 liters concentrate / hour feed, inlet temperature from 160 to 180 ° C and outlet temperature from 90 to 110 ° C.

EXAMPLE II

Qualitative analysis of standardized Aleuritesmoluccana extracts

Qualitative analysis of extracts by CCD

Thin layer chromatography (CCD) was employed in the analysis of the constituents of the Aleuritesmoluccan extracts according to the present invention using alpha-amyrin and beta-amyrin, alpha-amirinone ebeta-amirinone, stigmasterol, beta-sitosterol, swertisinae 2 standards. '' -O-ramnosil-swertisine, isolated from the plant itself.

In the identification of polar compounds, with emphasis on naswertisine and 2 '' - O-ramnosil-swertisine, 1 g of the ethyl acetate fraction obtained from the 9: 1 EtOH: H20 extract of A. moluccana leaves was solubilized with methanol. Swertisine and 2 '' - O-ramnosyl-swertisine standards were weighed (1 mg each) and solubilized with 0.1 ml methanol. To the silica gel 60 plate (length 8 cm and width 5.5 cm) was applied 10 µl of cavernation and after drying, the plate was eluted with AcOEt: Acetone: H2 O (25: 8: 2). The elution was repeated three times. The plate was analyzed in a UV chamber and then revealed with 3% ferric chloride solution in EtOH, calculating the retention factor (Rf) of the compounds (Brazilian Pharmacopoeia, part II. 4. Ed. São Paulo: Atheneu, 1996, 200p) .

Comparison of the results showed the presence of these compounds in the dry and soft extracts, as shown in Figure 1 (A).

In the identification of nonpolar compounds, with emphasis on alpha-, beta-amyrin, alpha-, beta-amirinone, stigmasterol, beta-sitosterol, 1 g of the dichloromethane fraction obtained from the 9: 1 EtOH: H20 extract of the A. leaf was weighed. moluccan, solubilizing with dichloromethane. Alpha / beta-amyrin, alpha / beta-amirinone, stigmasterol, beta-sitosterol standards were weighed (1 mg each) and solubilized with 0.1 ml dichloromethane. To the desilic gel 60 plate was applied 10 µl of each solution and after drying the plate was eluted with Hexane: AcOEt (7: 3). The plaque was analyzed in a UV chamber and later revealed with sulfuric anisaldehyde solution and heated in a hot plate at 60 ° C, calculating the Rf of the compounds (Brazilian Pharmacopoeia, part II. 4. Ed. São Paulo: Atheneu (1996) 200p ).

Comparison of the results showed the presence of these compounds in the extracts, as shown in Figure 1 (B). Qualitative analysis of the extracts by Gas Chromatography (GC)

Gas chromatographic analysis was performed on a gas chromatograph equipped with a flame-ionization detector (CG-FID), with a DB1 (Agilent) column with 30m x 0.25u internal diameter.

Test solution: Exactly 10 mg of the dichloromethane fraction obtained from EtOH: H20 (9: 1) extract was weighed and solubilized with 0.3 ml HPLC grade dichloromethane. 1 µl of the dichloromethane fraction was injected with the aid of a microsyringe.

Reference solution: 1 æL of e (3-amyrin (commercial reference standards - Sigma) and 5 mg / ml concentration were injected with the aid of a microsyringe).

Chromatographic conditions: column temperature at 200 ° C for 2 min, rising at a rate of 10 ° C / min to 290 ° C remaining at that temperature for 40 minutes. Temperature injector 260 ° C and detector 290 ° C. 30 ml / min split with 10 ml / min septum purge. Hydrogen as carrier gas with flow rate of 1 ml / min.

Figure 23 shows the results obtained with the moluccan Aleurites dichloromethane fraction when evaluated by Gas Chromatography (GC) in a chromatograph equipped with flame ionization detector (CG-FID), DB1 (Agilent) column with 30 mx 0.25 mm internal diameter. The test solution (dichloromethane fraction of the EtOH: H2 O extract = 9: 1) was solubilized with 0.3 ml HPLC grade dichloromethane. 1 µl of the dichloromethane fraction was injected with the aid of a microsyringe. The reference solution (p-amyrin, standard Sigma) was injected at a concentration of 5 mg / ml with the aid of a microsyringe. The chromatographic conditions were: column temperature at 200 ° C for 2 min, subindone at a rate of 10 ° C / min to 290 ° C while remaining at this temperature for 40 minutes; injector temperature 260 ° C and detector 290 ° C; 30 ml / min split with 10 ml / min purge deseptum; Hydrogen as a carrier gas with a flow rate of 1 ml / min. As shown in Figure 23, the presence of the beta-amyrin standard (5.0mg / ml) at the retention time of 19.2 ± 0.2 min and alpha-amirine at the retention time of 20, respectively. 23 ± 0.2 on the chromatogram dry and soft extracts.

EXAMPLE III

Quantitative analysis and standardization of extracts by HPLC

HPLC (High Performance Liquid Chromatography) analysis was performed by initially dissolving moluccan leurite extracts in methanol (20% volumefinal), sonicating for 5 min, followed by the addition of acidified water pH 3.57 (20% final volume). Then the volume was made up with a 50:50 methanol / acidified mixture at a concentration of 1 to 2 mg / ml. After filtering the sample onto a 0.22 µm membrane filter, 20 µl of the solution was injected using a reverse phase analytical column where C18 groups are chemically bonded to silica, having a length of 10 to 25 cm and an internal diameter of 4 to 6 mm. . The detector used was a PDA (diode array) type monitoring the chromatographic run at 254 and 338 nm. Separation was performed using a linear gradient of the mobile phase, initially composed of methanol: acidified water pH3.57: acetonitrile (20:15:65) in a flow rate of 0.5 to 1ml / min, as follows:

<table> table see original document page 63 </column> </row> <table>

The pressure ranged from 2,000 to 3,000 psi.

Figure 5 (A) shows the CCDP purified 2 '' - O-ramnosyl-swertisine HPLC chromatogram (AMSR170707) at 145 µg / ml at 338 nm, which eluted at about 17 min, demonstrating success in pathway purification as indicated by the presence of a single nocromatogram peak.

Figure 5 (B) shows the absorption profile of the flavonoid-typical 2 '' -O-ramnosyl-swertisine pattern. Figure 2 (A) shows the chromatogram of the 2 mg / ml moluccan Aleurites extract showing the resolution dope of marker 2 '' - O-ramnosyl-swertisine (about 17min) from the next peak (swertisine).

In addition to the coincidence in retention time, the UV-absorption profile of the peak eluting at about 17 min in the dry extract chromatogram (Figure 5 B) is identical to the 2 '' - O-ramnosil-swertisine standard.

Figure 2 (B) shows the chromatogram of the 1.72 mg / ml moluccan Aleurites molede extract, showing a profile very similar to that of the dry extract (Figure 23 A), with the presumed peak for 2 '' - O-ramnosil-swertisine. (about 17 min) from the next peak (swertisine).

Thus, it is possible to confirm and quantify the presence of 2 '' - O-ramnosil-swertisine in the extracts, observing a variation of 0.05 to 15% of this marker in the extracts.

The quantification method of 2 '' - O-ramnosil-swertisinans extracts has been validated according to official standards (Brazil. National Health Surveillance Agency. RE 899, May 29, 2003).

EXAMPLE IV

Isolation, purification and analysis process of the marker 2 '' - O-ramnosyl-swertisine from Aleuritesmoluccana extract by preparative CCD. Since the selected marker is not a commercial product, it was isolated and purified from the ethyl acetate fraction of the hydroalcoholic extract of A.moluccana.

From the soft extract, described above, it was made a liquid-liquid partition, using initially dichloromethane, and later ethyl acetate. From the concentrated acetyl fraction to dryness on a rotary evaporator at 50 ° C, open column chromatography was performed with silica gel 60 support starting from 5 to 20 g of the concentrated ethyl acetate fraction with which a pellet was prepared. by mixing with silica gel 60. As the mobile phase, chloroform was initially used with gradual polarity increase, using increasing methanol ratios (from 9: 1 to 5: 5 CHCl 3: methanol, ending with 100% methanol). Isolation of 2 '' - 0-ramnosyl-swertisine was monitored by CCD as described below (CCD Profile of Aleuritesmoluccana extracts).

From the semipurified fractions obtained according to the above methodology, the final purification was performed using Preparative Thin Layer Chromatography (CCDP). The CCDP was performed with a 60F254 silica gel plate (thickness 1 mm, Merck), in which a solution of the semipurified fraction dissolved in methanol (10mg in 0.5 to 1.0 ml) was applied and eluted with the chloroform: methanol mixture 7. : 3. After elution and drying of the plate at room temperature, UV analysis was performed and the 2 '' - O-ramnosyl-swertisine stain (identified by staining a portion of the plate with 3% ferric chloride) was removed and solubilized with methanol, followed by of paper filtration to obtain the marker. The filtrate containing the isolated label was subjected to drying at room temperature.

After obtaining, this standard was analyzed through analytical CCD (according to the methodology described in example II above), HPLC (according to the methodology described in example III), KBr pellet infrared spectrum, H1 NMR and C13 NMR, in order to prove its accuracy. purity, establishing the lot number of the standard in its own form, attaching the result of the analyzes performed, in order to allow the traceability of the process of obtaining the standard.

The C13 NMR, H1 and IR NMR spectra of the marker (lotAMSR170707), mentioned above, are found respectively in Figures 3 (A, B, C), 4 (A, B, C) and 6.

The HPLC elution profile and the UV absorption profile (lot AMSR170707) are found respectively in Figures 5 A and B.

EXAMPLE V

Assays for the determination of antinociceptive activity of A. moluccana extract. in acute nociception models.Assessment of the antinociceptive effect of moluccan leuritis extracts through the acetic acid induced abdominal writhing model.

The experimental model of acetic acid-induced abdominal writhing allows us to evaluate the antinociceptive activity of various substances that act on both central and peripheral levels. The nociceptive response is induced by intraperitoneal injection of acetic acid (0.6%) diluted in saline (0.9%). Abdominal contortions consist of contraction of the abdominal muscles together with the extension of one hind leg. The antinociception index is evaluated by reducing the number of abdominal writhes when compared to the group receiving animals (negative control) (Collier, H. D. J. et al., 1968, Br. J. Pharmacol., 32, 295-310).

For each analysis, 8-10 male animals (Swiss Webster mice) weighing 25 to 30 g were used. The dose was calculated individually for each animal, according to its weight, the amount in mg / kg (mg / 1,000 g) of the substance to be administered. The injected volume of the solutions follows the 0.1 ml / 10 g animal weight rule.

After treatment with the compounds and injection of acetic acid the animals were individually observed, placed under glass funnels and the number of abdominal contortions quantified cumulatively over a period of 20 minutes. Results were quantified through the arithmetic mean of the number of writhes observed in the animals (all animals in a specific treatment) followed by the SEM (standard errors of the mean).

Evaluation of the antinociceptive effect of moluccan leuritis extracts through the formalin induced pain model.

This model is more specific than the acetic acid-induced abdominal contortion test. It allows the evaluation of two distinct types of pain: pain of neurogenic origin (direct stimulation of nociceptive neurons) and pain of inflammatory origin, representing the tonic response to pain, accompanied by an inflammatory response related to the release of inflammation mediators. The nociceptive response was induced by using 20 μL formalin, which consists of a 0.92% (2.5% intra-plant) formaldehyde solution injected into the dorsal region of the right hind paw and vehicle on the back paw. Soon after formalin injection, the animals were placed individually under glass funnels, surrounded by mirrors to facilitate visualization of the emitted behavior. Immediately the observation of the burning reaction began, and the first 5 minutes were initially timed after formalin injection (corresponding to the dorneurogenic period). After an interval of 10 minutes, the licking time was restarted for a further 15 minutes, which corresponds to the inflammatory pain. The total test time was 30 '.

At the end of the observation time, the animals were sacrificed by cervical dislocation and the hind legs were cut in the tibiotarsal region and weighed on an analytical balance to quantify formalin-induced edema. The weight difference (in mg) between right (injected with formalin) and left (injected with saline) was considered as an index of edema. (Dubuisson, D. et al., 1977, Pain., 4, 161-174; Hunskaar, S. and Hole, K., 1987, Pain, 28, 343-355). For each analysis, 8-10 male animals (Swiss Webster mice) weighing 25 to 30 g were used.

The dose was calculated individually for each animal according to its weight, the amount in mg / kg (mg / 1000 g) of the substance to be administered. The injected volume of the resolutions follows the rule of 0.1 ml / 10 g of animal weight.

Animals were observed after intra-implant formalin injection for 30 min. The reaction time in seconds (time the animal remains licking the injected paw) was then quantified in each phase: phase I (neurogenic pain - 5 min observation) and phase II (inflammatory pain - 15 min observation).

Evaluation of the antinociceptive effect of moluccan leuritis extracts through the hot plate model. The hot plate model is generally used to check analgesic drugs whose mechanism of action may involve the opioid pathway. Animals are placed on a warm plate (UGO BASILE, model L1912-06), set at a temperature of 56 ° C (+1) and the time in seconds that each animal takes to lick, bite or raise its paws is considered indicative of pain. The maximum time allowed for animals to remain on the plate is 30 seconds to prevent tissue damage caused by heating. For this model, the animals were pre-selected 24 hours before the test for pain check without introducing any treatments. Animals whose threshold was below 10 s (observed in the pre-test) were submitted to the test. A positive control of the test was also performed with animals submitted to subcutaneous morphine (5 mg / kg) treatment (Souza, MM et al. Methods for the evaluation of biological reactivity of natural and synthetic products. InCechinel Filho, V., Bresolin, TMB Pharmaceutical Sciences: Contribution to the development of new drugs and medicines, 2003, 108-166).

For each analysis, 8-10 male animals (Swiss Webster mice) weighing 25 to 30 g were used. Results were quantified by the arithmetic mean reaction time in seconds (time the animals took to have thermal sensitivity that was expressed by the licking of the hind legs as well as the escape attempt through the rearing position) after their placement on the hot plate, followed by the EPMs (standard errors of the mean).

Statistical analysis:

For the models described above, parametric statistical tests were performed. Results were submitted to ANOVA analysis of variance followed by appropriate post hoc tests. For pain and inflammation models the results were presented as mean ± standard error of the mean (SEM), except for the DI50 (dose that reduces the response to 50% compared to the control group), which was presented as the geometric mean accompanied by their respective limited confidence in 95% level. Statistical analyzes of the results were performed by means of the variance analysis followed by the multiple comparison test using the Dunnett method, when appropriate. Values of p <0.05 or less were considered as indicative of significance. The ID50 was estimated from individual experiments in the Graphpad Instat statistical program.

EXAMPLE VI

Tests for the determination of the antiinflammatory activity of A. moluccana extract.

Evaluation of the antiinflammatory effect of the dry extract of Auclides moluccana through the phlogistic agent induced paw edema model.

The pharmacological model of paw edema is not considered as a model of inflammation per se, but as a model of anti-hematogenic activity, since it only evaluates one of the parameters of the inflammatory process that is the formation of edema. Experiments are generally carried out using carrageenan as a phlogistic agent. During "inflammation" induced by this agent, several process steps can be evaluated. From 0 to 2 hours after its application, the histamine, serotonin and substance P mediators are released. At the peak of the maximum inflammation response (from the fourth hour of the experiment) prostaglandins, nitric oxide and various stachykinins are released.

In this way, it is verified in which phase of the inflammatory process a compound acts, and, later, it is possible to induce the edema precisely with such mediator. Thus, it is characterized not only the antiedematogenic effect of the compound studied, but also its mechanism. which mediators of the inflammatory process the compounds are supposedly inhibiting.

Carrageenan-induced paw edema - During the trials, the animals received a phlegistic intraocular injection, carrageenan, at the concentration of 300ug / paw diluted in saline solution in the right paw (0.025ml / paw), and physiological solution in the left paw (0.025ml / paw). ). The various concentrations of the dry extract as well as negative control (physiological solution) were administered orally (0.3 ml / 100 g animal weight) 1 hour before carrageenan injection. The positive control (dexamethasone 0.5 mg / kg) was administered subcutaneously 4 hours before. Paw edema was measuredletletometrically (plethysmometer Ugobasile, Italy) at time intervals of 0.5, 1, 2, 3 and 4 hours after injection of the phlogistic agent. The variation in paw volume expressed in ml and the difference between right and left paws volume is taken as the edema index (Souza, M.M. et al. (2003)).

Bradykinin-Induced Paw Edema - During the trials, the animals received intraplantar injection of the phlogistic agent bradykinin at a concentration of 300ug / paw diluted in saline in the right paw (0.025ml / paw) and physiological solution in the left paw (0.025ml / paw). ). The various concentrations of the dry extract as well as the negative control (physiological solution) were orally administered (0.3 ml / 100 g animal weight) 1 hour before bradykinin injection. The positive control (dexamethasone 0.5 mg / kg) was administered subcutaneously 4 hours before. Paw edema was measuredletletometrically (plethysmometer UGOBASILE, Italy) at time intervals of 0.5, 1, 2 hours after phlogistic agent injection. The variation in paw volume is expressed in ml and the difference between right and left paw volume is taken as an edema index (Souza, M. M. et.al. (2003)).

Histamine-Induced Paw Edema - During the trials, animals received a phlogistic agent intraplantar injection, histamine, at a concentration of 0.1% / patinated in saline solution in the right paw (0.025 ml / paw), and physiological solution in the left paw (0.025 ml / paw). Various concentrations of the dry extract as well as negative control (physiological solution) were administered orally (0.3 ml / 100 g animal weight) 1 hour before carrageenan injection. Positive control (dexamethasone 0.5 mg / kg) was administered subcutaneously 4 hours before. Paw edema was measured plethysmetrically (UGOBASILE plethysmometer, Italy) at time intervals of 0.5, 1, 2 hours after injection of the phytological agent. Paw volume malfunction is expressed in ml and the difference between right and left paw volume is taken as an edema index (Souza, M. M. et. Al. (2003)).

For each of the above analyzes, 8-10 male animals (RatsWistar) weighing 200 to 300 g were used.

Results were quantified by the arithmetic mean obtained from the paw edema of each treatment group, followed by the EPMs (mean standard errors), along the measurement times during each experiment: carrageenan (6 measurements in 4 hours of experiments), bradykinin and histamine (3 measurements within 2 hours of experimentation).

Evaluation of the antiinflammatory effect of the dry extract of Moluccan aurias through the model of pleurisy induced by intrapleural injection of phlogistic agents

Among the animal models available for the evaluation of antiinflammatory activity of substances, the depleurisy model is considered one of the most complete, since from the pleural cavity wash it is possible to analyze and quantify the cellular and humoral components of the inflammation, without resorting to detailed extraction and quantification procedures ( Saleh TSF, Calixto, JBMediros, YS, 1997, Eur J. Pharmacol., 331, 43-52). Apleurisy can be induced in animals by phlogogenic agents such as carrageenan, bradykinin, Substance P, Prostaglandin E2 etc. and, as occurs in the paw edema model, the first induction of the process is with carragenin. The animals were treated with different concentrations (125, 250, 500 mg / kg) of the dry extract of A.moluccana, and vehicle (saline) by oral route. They were then injected (0.2 ml) with an intravenous Evans blue solution (25 mg / kg). One hour after the injection of the treatments the administration of the phlogistic agent (carrageenin 1.0%, Substance P (20 nmol), and bradykinin (10 nmol), histamine (100 µg / well) was administered. treated with the extract and phlogistic agent, there is a negative group control (pretreated with saline that receives saline i.pL) and another control group (pretreated with saline and receiving phlogistic agent i.pL) .

After 4 hours of treatment with the phlogistic agent, the animals were sacrificed and the abdominal region was opened with diaphragm rupture. The pleural cavity was washed with heparinized saline solution (1.0 ml) (20 µl / ml). Then the pleural wash was collected and an aliquot of it (10 µl) was added to a Turk solution (2% acetic acid) at a ratio of 1:20.

Of this material, 10 µl were transferred to the Neubauer chamber for total cell counting procedure.

The remainder of the pleural wash was centrifuged (10,000 rpm X10 min). The supernatant was removed and used for ELISA reading (620 nm) to measure plasma extravasation (edema) after inflammatory stimulus. The pellet was resuspended with 100 µl egg albumin (3%) and 10 µl was added to a slide covered with perforated filter paper. After drying, staining was performed by the May-Grunwald-Giemsa method for differential cell counting by microscopy (Aum 100 times).

Evaluation of the antiinflammatory effect of extracts of Moluccan laurites through the croton oil-induced ear edema model.

The ear edema model is specific and widely used to research the activity of topical anti-inflammatory agents. The irritating agent used is croton oil and edema is measured by the difference in ear thickness of the animal before and after treatment (Montello, MSAG, Effect of Low Power Laser Therapy (HeNe and AsGa) on Ear Croton Oil Dermatitis). São José dosCampos, 2002. 82p. Dissertation Master's Degree in Biomedical Engineering - Research and Development InstituteUniversity of Paraiba Valley).

A "basal" measurement of the ear thickness was made and soon after, the control substance or extracts were applied to the inner surface of the right ear. After 30 min the croton oil was applied to the outer face of the same ear. A new ear measurement was then made 4 hours after the oil treatment.

For each analysis, 8-10 male animals (Swiss Webster mice) weighing 25 to 30 g were used. The results were quantified by the arithmetic mean obtained from the ear of each treatment group over the measurement times during the experiment (2 measurements in 6 hours of experiments), followed by the SEM (mean standard errors). Evaluation of the antipyretic effect of the Aleuritesmoluccana extracts through the LPS-induced hyperthermia model in rats.

Antipyretic property is also observed with NSAIDs, and evaluation of such pharmacological property is also important in evaluations with analgesic and anti-inflammatory substances. The genesis of processofebril involves mediators such as prostaglandins, interleukins, among others, whose action is related to the processes of analgesia and inflammation, and drugs that interfere with the activity of these mediators exhibit the three pharmacological properties. In order to justify the antipyretic effect of A.moluccana soft extract, the LPS-induced hyperthermia model was used. Groups of animals were treated with plant extratomole in two doses (5.0 and 10 mg / kg), with positive (sodium dipyrone, 10 mg / kg) and / or negative (vehicle) control via the intraperitoneal route. After 60 minutes the basal temperatures of all groups were read. After this time, the groups (treated and control) received intraperitoneal injection of LPS (10 µg / kg, diluted in physiological solution). Temperature checks were performed with a veterinary thermometer, which was carefully inserted into each animal's rectal canal, remaining for about 1 minute on site. Checks were made after 40 minutes of LPS injection at 40-minute intervals for a period of 7 hours and 20 consecutive minutes, ie 40, 80, 120, 160, 200, 240, 280,320, 400, and 1,440 minutes. Results were expressed as the difference between final and basal temperatures as described by Roth and De Souza (Roth, J. De-Souza, G.E.P., 2001, Brazilian Journal of Medicinal and Biological Research 34,301-314).

Male Wistar rats (250 to 300 g) were pretreated with A. moluccana (5 to 10 mg / kg), and / or vehicle and dipyrone (10 mg / kg) and submitted after 1 hour to the LPS fever induction model. (10 ng / kg). Temperature variations were observed at 40-minute intervals over an 8-hour period. It was also verified if the plant would produce alterations in the gastric mucosa of the animals, which received the same treatment above, besides Indomethacin (30 mg / Kg).

Statistical analysis:

Statistical analysis of the data was performed by one-way analysis of variance (ANOVA) followed by Dunnett's test. P values <0.05 were considered as indicative of significance. The ID50 (dose that reduces the response by 50% compared to the control group) are presented with the geometric means accompanied by their respective confidence limits, at a level of 95% and were estimated from individual experiments using the linear regression method using GraphPad® program. The calculation of the ID50 is used to compare the potency between the test substance and the traditional drugs. Inhibition percentages were quoted as the mean ± standard error of the mean difference (percentage) for each individual experiment relative to the corresponding control group.

EXAMPLE VII

Assays for determination of antihypernociceptive activity of A. moluccana extract in persistent donor models.

Evaluation of the effect of the dry extract of moluccan Aleurites on mechanical hypernociception induced by intra-implant carrageenan injection.

Induction of inflammatory hypernociception in mice was performed by i.pl. 50 µl of carrageenan (300 µg / site) on the plantar surface of the rear patellar. This dose is capable of producing edema, hypernociception, and a significant increase in patellar size, but animals continue to show normal behavior (De Campos et al., 1996, Eur JPharmacol., 316, 2-3, 277-86; Bortolanza et al. , 2002, Eur. J. Pharmacol., 453, 2-3, 203-8; Quintão et al., 2005, Anesth Analg., 101, 6, 1763-9; Quintão et al., 2006, Neuropharmacol. , 5, 614-620). Initially, the animals were pretreated with the dry oral A. moluccana extract (v.; 125-500 mg / kg) 1h before induction of hypernociception. Then the animals received an i.pl injection. carrageenan (300 µg / paw), and were evaluated for mechanical hypernociception by von Frey doping, as described below and at different times (1, 3, 4, 6, 24 and 48 h). The control group was treated with the vehicle (10 ml / kg) used for extract addition (0.9% saline).

Evaluation of the effect of the dry extract of moluccan Aleurites on mechanical hypernociception induced by intra-implant injection of prostaqlandin E2 (PGE2).

Initially, the animals were pretreated with dry A. moluccana extract orally (v.; 125-500mg / kg), 1h before induction of hypernociception. After treatment time, the animals received injection i.pl. of PGE 2 (0.1 nmol / paw) (Kassuya et al., 2007, Br J Pharmacol., 150 (6), 727-737). The animals were then evaluated for mechanical hypernociception by von Frey 0.6 g, as described below and at different times (0.5, 1, 2, 4, 6 and 24 h). The control group was treated with the vehicle (10 ml / kg) used for extract addition (0.9% saline).

Evaluation of the preventive and curative effect of the Moluccan Aleurites secoda extract on the mechanical hypernociception induced by the intraplantar injection of Freund's complete adjuvant (CFA).

To induce persistent inflammatory response, animals received 20 nl i.pl injection of Freund's complete adjuvant (CFA; 1 mg / ml heat-inactivated mycobacteriumtuberculosis bacillus; each milliliter (ml) should contain 0.85 ml paraffin oil + 0.15 ml manida monooleate) on the plantar surface of the right hind leg (Cao et al., 1998, Nature, 392, 390-394; Ferreiraet al., 2001, Neuropharmacol., 41, 8, 1006-1012). Mechanical and thermal hypernociception were evaluated by the von Frey filament as described below in item e.To evaluate the preventive effect on mechanical hypernociception, the animals were previously orally treated with dry extract of A. moluccana (125-500 mg / kg) or vein ( 10 ml / kg). After 1 h of treatment, the animals received an i.pl. CFA, and mechanical hypernociception were evaluated at different time intervals (1, 2, 4, 6, 8, 12, 24 and 48 h).

In order to verify the effect of curative treatment, the animals received the i.pl. after 24 h CFA were treated orally with A. moluccana dry extract (125-500 mg / kg) or vehicle (10 ml / kg), 2 times daily (12 in 12 h) for 5 consecutive days, and The mechanical hypernociception of the lateral and contralateral patas to the injection of the CFA was evaluated 6 h after the first administration of the day, as previously described. In order to investigate the extent of the antinociceptive effect of A. moluccana in CFA experiments, treatment was discontinued 5 days after the first administration and then restarted after 2 days to investigate the possible development of tolerance. Evaluation of the preventive and curative effect of Aucurana moluccana extract on mechanical hypernociception induced by partial sciatic nerve constriction (NSCLC).

The procedure used was similar to the one described by Seltzer et al. (1990) and modified for mice by Mamberg and Basbaum (1998). Mice were anesthetized with 7% chloral hydrate (8 ml / kg; i.p.). Sciatic nerve constriction was performed by tying 1/3 to 1/2 of the dorsal portion of the 8-0 silk-threaded sciatic nerve (Ethicon®). One group of animals had exposed sciatic nerve, however, no mooring was performed (false-operated group). On the 4th postoperative day, the animals were evaluated for mechanical hypernociception by von Frey monofilament, as described in item e, and treated orally with dry A. moluccana (125-500 mg / kg) or vehicle (10ml / kg) extract. ) Twice a day (12 every 12 h) for 5 consecutive days, and mechanical hypernociception was evaluated 6 h after the first administration of the day.

Mechanical threshold analysis through the VonFrey filament. To evaluate mechanical hypernociception, animals subjected to the carcinogen, CFA or CPNC induced hypernociception models were placed individually in individual transparent acrylic compartments (9X7X 11 cm) located on a raised wire platform to allow access to the ventral surface of the patellas. The animals were acclimatized for at least 30min before the behavioral tests. The frequency of withdrawal was obtained through 10 applications (duration 1 s each) of the von Frey 0.6 g filament (VFH, Stoelting, Chicago, USA). The stimuli were performed on the plantar surface of the right hind paw of the animal (Quintão et al. 2005, Anesth Analg., 101, 6, 1763-9; Quintão et al., 2006, Neuropharmacol., 50, 5, 614-620). In order to determine the basal mechanical threshold (B), all groups of animals underwent prior evaluation and again re-evaluated at different times after carrageenan or CFA injections, or after CPNC.

Statistical analysis:

Statistical analysis of the data was performed by two-way analysis of variance (ANOVA) followed by Bonferroni test. P values <0.05 were considered as indicative of significance.

The ID50 (dose that reduces the response by 50% in relation to the control group) are presented with the geometry averages followed by their respective confidence limits, at a level of 95% and were estimated from individual experiments using the linear regression method through the program. GraphPad®. The DI50 calculus is used to compare the potency between the test substance and the traditional drugs. Inhibition percentages were cited as the mean ± standard error of the mean difference (in percentage) between the areas under the curves obtained for each individual experiment relative to the corresponding control group.

EXAMPLE VIII

Pharmacological evaluation of A.moluccana dry extract in mice by neuropathic pain assay.

For the pharmacological tests, female Swiss mice (n = 8 to 10 animals), female weighing 25 to 30 g, kept under controlled light temperature, with feed and water ad libitum (water at will) were used, except during For the performance of each experiment, 8 to 10 animals were used in each treatment group and identified, after weighing, with overhead projector under sequential numbers.

Induction by partial sciatic nerve constriction (NSCLC)

The procedure used was similar to the one described by Seltzer et al. (1990) and modified for mice by Mamberg and Basbaum (1998). Mice were anesthetized with 7% chloral hydrate (8 ml / kg; i.p.). Sciatic nerve constriction was performed by tying 1/3 to 1/2 of the dorsal portion of the 8-0 silk-threaded sciatic nerve (Ethicon®). One group of animals had exposed sciatic nerve, however, no mooring was performed (false-operated group). On the 4th postoperative day, the animals were evaluated for mechanical hypernociception by von Frey monofilament, as described in item e, and treated orally with dry A. moluccana (125-500 mg / kg) or vehicle (10ml / kg) extract. ) Twice a day (12 every 12 h) for 5 consecutive days, and mechanical hypernociception was evaluated 6 h after the first administration of the day.

Mechanical threshold analysis by Von Frey filament

To assess mechanical hypernociception, the animals subjected to the CPNC-induced hypernociception models were placed individually in individual transparent 9 cm x 7 cm (11 x 7 x 11 cm) compartments located on a raised wire platform to allow access to the ventral surface of the hind paws. acclimatized for at least 30 min before behavioral testing. The melted response frequency was obtained through 10 applications (duration 1s each) of the von Frey 0.6 g filament (VFH, Stoelting, Chicago, USA). The stimuli were performed on the plantar surface of the animal's right hind paw (Quintão et al., 2005, Anesth Analg., 101, 6, 1763-9; Quintão et al., 2006, Neuropharmacol., 50, 5, 614-620) . Aiming to determine basal mechanical olimiar (B), all groups of animals were submitted to previous evaluation and re-evaluated at different times after CPNC.

Statistical analysis of the data was performed by two-way analysis of variance (ANOVA) followed by Bonferroni test. P values <0.05 were considered as indicative of significance.

Effect of A. moluccana extract on mechanic hypernociception induced by partial sciatic nerve constrictionOral treatment with A. moluccana dry extract (industrial batch), when administered at a dose of 125 to 500 mg / kg, was able to significantly reduce Depending on the dose, the mechanical hypernociception induced by the CNCP. This inhibition was significant for up to 8 days, where treatment was discontinued, as shown in Figure 24.

Figure 24 shows the effect of treatment with dry moluccan Aleurites extract (125 to 500 mg / kg, v.o.) on CPNC-induced mechanical hypernociception. Each point represents the average of 6 to 8 animals and the vertical bars indicate the E.P.M. It differs significantly from the control group (C), * P <0.05; ** P <0.01 and *** <0.001.

EXAMPLE IX

Acute Toxicity Assay for Aleuritesmoluccana ExtractThis study refers to a preliminary and preliminary assessment of the toxic properties of a test substance, providing information on the risks to health resulting from short-term exposure via the chosen pathway. Acute toxicity is also the basis for establishing a dose regimen for research on subchronic, chronic and repeated dose toxicity, and provides initial information on the mode of toxic action of the test substance.

In the experiment, 10 male and 10 female mice were used, which were divided into 4 groups: a) vehicle-treated male group (saline), b) male group treated with A. moluccana soft extract (5000 mg / kg); c) group of females treated with vehicles and group of females treated with the extract (5000 mg / kg). After 6 h fasting, the animals received the treatments by gavage, and immediately transferred to individual box for observation through the Hippocratic test as described by Brito (1994) (Brito, A. S. Manual of in vivo Toxicological Tests. Campinas: UNICAMP, 199, 41-121). The same were observed individually and systematically up to 24 h after treatment at intervals of (0 to 10 min, 30 min, 60 min, 180 min and 1,440 min). During the first 4 hours of observation the water supply was restricted. Behaviors and / or manifestations were observed such as: piloerection, salivation, cage movement, tremors, tachycardia, irritability, urination, defecation, ptosis, seizure and the number of deaths. Special attention has been paid to symptoms such as tremors, convulsions, salivation, diarrhea, irritability, lethargy and coma. During the experiment, deaths as well as the time of occurrence should be recorded and whenever possible to investigate the cause. At the end of the tests the animals were weighed and later sacrificed. Results were quantified on average in terms of onset of behavioral response to be observed at the specified time of observation. For example, if of the 5 animals in the experiment, only 3 had defecation, which was recorded in table 3/5. The highest dose used was 5000 mg and as no deaths were found, the LD50 (value statistically derived from the administration of a single dose that can kill 50% of animals in one experiment) could not be calculated. Test substance with DL50 greater than 5 g is very unlikely to exhibit toxic effects.

Statistical analysis of the data was performed by means of analysis of variance followed by the T-test. Values of p <0.05 were considered as indicative of significance.

EXAMPLE X

An example of a pharmaceutical composition in coated decompressed form can be obtained by the following formulation 1: <table> table see original document page 90 </column> </row> <table>

Preparation Method: For core preparation, the components are individually weighed and screened. Then components A, B, C and D are mixed in a "v" type mixer for 20 min. Then components F and G are added and mixed for a further 5 min. As a result, the mixture is subjected to direct compression for forming of the compressed cores. The compressed cores are then coated by spraying (spraying) a solution containing components H, I, J, K and L forming the coating film.

EXAMPLE XI

An example of a pharmaceutical composition in capsule form can be obtained by the following formulation 2:

<table> table see original document page 91 </column> </row> <table>

Method of Preparation: For preparation of capsules components A, B, C and D are weighed, mixed and sieved and then subjected to wet granulation in suitable equipment, gradually adding a solution containing the binder E. Afterwards, the formed granules are immediately submitted to oven drying at 30 to 40 ° C until reaching a humidity of approximately 2%. As a result, the dried granules are graded and mixed with the sliding agent F for 5 minutes. The graded dried granules are then encapsulated.

EXAMPLE XII

An example of a pharmaceutical composition in the suspension form can be obtained by the following formulation 3: <table> table see original document page 92 </column> </row> <table>

Preparation Method: For preparation of a suspension components A and F are weighed and mixed in a homogenizer tank. Afterwards, part of K and the other components are gradually added to the mixture gradually and under constant agitation. Then the remaining K is added to the desired final volume. Mechanical stirring proceeds until complete homogenization of the mixture.

EXAMPLE XIII

An example of a pharmaceutical composition in the form of syrup can be obtained by the following formulation 4: All publications mentioned in the above specification, and references cited therein. publications are incorporated herein by reference. Various modifications and variations of the processes, extracts, compositions, methods and uses described in the present invention will be apparent to those skilled in the art without departing from the scope of the present invention.

Claims (41)

1. Process for obtaining a standardized extract, characterized in that it employs at least a part of a plant of the genus Aleurites, and comprises the steps of: (i) collecting, drying and subdividing plant material; (ii) pre-extraction; (iii) extraction with solvent extraction; (iv) extract filtration; (v) extract concentration; (v) pasteurization; (vi) drying; and (vii) qualitative equantitative analysis (standardization) of the extract, being said standardized extract distinguished by the marker 2 '' - O-ramnosil-swertisine. Process according to Claim 1, characterized in that said plant is selected from the group consisting of A. trisperma, A. cordata, A. montana, A. fordii, A. montance, A. rockinghamensis and A.moluccana. Process according to Claim 1 or 2, characterized in that said plant of the genus Aleuritesser Aleurites moluccana L.Wild. Process according to Claim 3, characterized in that it comprises at least the leaves of L. molycana Aleurites L.Wild as a vegetable material. Process according to Claim 1, characterized in that the collection stage of the vegetable material takes place during the dry season. Process according to Claim 1, characterized in that the pre-extraction step is carried out with stirring in an alcoholic medium at ambient temperature to remove contaminants from the vegetable material. Process according to Claim 1, characterized in that in the extraction solvent extraction step, the extraction solvent is selected from a group consisting of, but not limited to, water, methanol, ethanol, propanol, isopropanol, propylene glycol, acidic solution, ethyl acetate, dichloromethane, chloroform, hexane, glycerine, acetone, petroleum ether, supercritical fluid, a combination thereof and the like. Process according to Claim 7, characterized in that said extracting solvent is a water: ethanol hydroalcoholic solution in which the water content is not greater than the alcohol content. Process according to Claim 7, characterized in that said extracting solvent is a water: ethanol hydroalcoholic solution in a selected ratio between 2: 8, 3: 7, 4: 6 and 1: 1. Process according to Claim 7, characterized in that said extracting solvent is a water: ethanol hydroalcoholic solution in the ratio of 3: 7. Process according to Claim 1, characterized in that, in the solvent extraction extraction step, the solvent-vegetable-extracting solvent ratio ranges from approximately 1: 1 to 20: 1. Process according to Claim 1, characterized in that said solvent-extractor: plant material ratio is 10: 1. Process according to Claim 1, characterized in that the pasteurisation step is carried out at a temperature of about 95 ° C for approximately 3 minutes. Process according to claim 1, characterized in that the extract concentration step provides a concentration of approximately 20% to 50% solids in the extract and is capable of completely eliminating alcohol. Process according to Claim 1, characterized in that the drying step is carried out under appropriate conditions to maintain the quantitative and qualitative characteristics of the extract. Process according to Claim 15, characterized in that the drying step is carried out by the spray-drying technique in the presence of a drying aid. Process according to Claim 16, characterized in that the drying aid is selected from a group consisting of silicon dioxide, modified cassava starch, tricalcium phosphate, maltodextrin, cyclodextrins, microcrystalline cellulose, lactose or a combination thereof. , added in a proportion of approximately 10 to 40% based on the total desolate content of the extract. Process according to Claim 1, characterized in that it optionally comprises an extract depigmentation step, using the selected procedure from a group consisting of activated charcoal treatment, resorption resin treatment or membrane ultrafiltration. Process according to Claim 1, characterized in that it optionally comprises an extract stabilization step in the presence of a stabilizing agent, said agent being selected from a group consisting of thiol derivatives, ascorbic acid and derivatives thereof, cysteine, glutathione , a combination of similar ones. Standardized extract, characterized in that it is obtained according to claims 1 to 19. Extract according to claim 20, characterized in that it contains at least one ingredient selected from the group consisting of alpha-amyrin, beta-amyrin, alpha-amirinone, beta-amirinone, swertisine, said standardized extract with respect to your marker-2 '' -O-ramnosil-swertisine. Extract according to claim 21, characterized in that said standardized extract of moluccanleurites contains a content of 2 '' - 0'-ramnosyl-swertisine in the range of 0.05 to 15%. 23. Standardized extract from at least a part of a plant of the genus Aleurites, characterized in that it is distinguished by at least one of the selected aspects of the group consisting of: (i) high performance liquid chromatography specific chromatography of soft extract and dry extract of said plant (ii) C13 NMR spectra of the 2 '' - 0-ramnosyl swertisine marker measured at 300 MHz in deuterated methanol, (iii) H13 NMR spectra of the 2 '' - 0-ramnosyl swertisine marker measured at 300 MHz MHz in deuterated methanol, (iv) high performance liquid chromatography chromatography of the standard 2 '' - O-ramnosyl-swertisine purified by preparative thin layer chromatography and (v) 2 '' - O-ramnosyl-swertisine marker infrared spectrometer. Extract according to Claim 23, characterized in that said high-performance liquid chromatography and dry extract-specific high performance liquid chromatograms are shown but not limited as in Figure 2. Extract according to claim 23, characterized in that said C 13 NMR spectra are represented, but not limited to, as in Figure 3. Extract according to claim 23, characterized in that said H 13 NMR spectra are represented, but not limited to, as in Figure 4. Extract according to Claim 23, characterized in that said high performance liquid chromatography chromatograms of the 2 '' - O-ramnosyl-swertisine standard are shown but not limited as in Figure 5. Extract according to claim 23, characterized in that said 2-O-ramnosyl-swertisine marker infra-red spectrum is shown but not limited as in Figure 6. Extract according to Claims 20 to 28, characterized in that it has analgesic, antiinflammatory and anti-fever properties in mammals. Pharmaceutical composition, characterized in that it comprises: (i) a pharmacologically effective amount of a standardized extract as defined in claims 20 to 29 and (ii) at least one pharmaceutically acceptable carrier. Pharmaceutical composition according to claim 30, characterized in that said standardized extract is present in an amount representing from 5 to 90% of the total weight of the composition. Pharmaceutical composition according to claim 31, characterized in that said standardized extract is present in an amount representing from 20 to 80% of the total weight of the composition. 33. Pharmaceutical composition according to claim 30, characterized in that it additionally contains at least one pharmacologically active substance selected from the group consisting of synthetic, semi-synthetic, biological molecules, vitamins and other substances derived from plant origin. Pharmaceutical composition according to claim 30, characterized in that they are appropriate pharmaceutical forms for oral, topical, intravenous, subcutaneous, intramuscular, intravaginal and / or rectal administration. 35. Pharmaceutical composition according to claim 34, characterized in that they are selected from tablets, hard or soft capsules, pills, powders, granules, single tablets, coated tablets, chewable tablets, effervescent tablets, sublingual tablets, controlled deliberation tablets, dragees. , globules, elixirs, suspensions, syrups and emulsions, each including immediate, controlled, prolonged or delayed release formulations, ointments, ointments, creams, emulsions, gels, solutions, pastes, aerosols, subcutaneous or intramuscular bolus or infusion , suppositories, ova, ointments, and similar creams. A method of treatment, wherein a mammalian animal is administered an effective amount of standardized extract as defined in claims 20 to 29 to prevent, control or treat conditions of pain, inflammation and / or fever. Method of treatment, characterized in that at least one effective amount of the pharmaceutical composition according to any one of claims 30 to 35 is administered to a mammalian animal to prevent, control or otherwise control conditions of pain, inflammation and fever. Method of treatment according to claims 36 and 37, characterized in that the dose to be administered provides from 0.1 to 50 mg of said standardized extract per kilogram of the patient. Method of treatment, characterized in that an isolated and purified effective amount of 2 '' - O-ramnosyl swertisine is administered to a mammalian animal from the extract according to claims 20 to 29. Use of the standardized extract as defined in claims 20 to 29, characterized in that it is for the preparation of a medicament for the prevention, control and treatment of clinical conditions of pain, inflammation and effusion. 41. Use of the pharmaceutical composition as defined in claims 30 to 35, characterized in that it is for the preparation of a medicament for the prevention, control and treatment of clinical conditions of pain, chronic inflammation.