BRPI1002115A2 - biomarker isolation and purification process; process of obtaining a standardized extract with antinociceptive and antiinflammatory properties, topical pharmaceutical compositions, preparation process thereof - Google Patents

Abstract

BIOMARKER ISOLATION AND PURIFICATION PROCESS, PROCESS OF OBTAINING A STANDARDIZED EXTRACT WITH ANTINOCICEPTIVE AND ANTI-INFLAMMATORY PROPERTIES, TOPICAL PHARMACEUTICAL COMPOSITIONS, PROCESSES PREPARATION. The present invention relates to a process of obtaining a standardized extract with antinociceptive and anti-inflammatory properties, as from a plant of the genus Sphagneticola trilobata, previously known as Acmella brasilìensis and Wedelia paludosa, using at least part of the plant, preferably the aerial parts. The present invention also relates to a process of isolation and purification of the standardized extract biomarker. In addition, providing a pharmaceutical formulation based on said extract in a sufficient and effective amount, being useful in the treatment of clinical manifestations such as pain and inflammation in mammals, as well as a method of preparing it.

Description

Patent Invention Descriptive Report

"BIOMARKER INSULATION AND PURIFICATION PROCESS, PROCEDURE FOR OBTAINING A STANDARDIZED EXTRACT WITH ANTI - CINCEPTIVE AND ANTI - INFLAMMATORY PROPERTIES, PHARMACEUTICAL COMPOSITIONS OF THE SAME PREPARATION.

Field of the invention

The present invention relates to a process of obtaining a standardized extract with antinociceptive and anti-inflammatory properties from a plant identified as Sphagneticola trilobata, also called Acmella brasiliensis and / or Wedelia paludosa, using at least one part of the plant, preferably the aerial parts. The present invention also relates to a process of isolating and purifying the standardized extract biomarker. Additionally, providing a topical pharmaceutical formulation based on said extract in a safe and effective amount, being useful in the treatment of clinical manifestations such as pain and inflammation in mammals, as well as a method of preparation thereof.

Background and Background of the Invention

For a better understanding of the present invention, a detailed explanation of the state of the art and the problem surrounding it is necessary to aid in better understanding.

Chronic pain is a health problem of frightening proportions. Data provided by the World Health Organization (WHO) about chronic pain (which lasts for more than three months) indicates an annual loss of approximately 550 million working days worldwide. In the United States, pain was considered the disease of the decade. According to Holden and Pizzi (2003), 48 million Americans suffer from chronic pain and spend approximately $ 100 billion on treatments, losing about 4 billion working days annually at a cost of $ 65 billion in productivity losses (Hollen, JE Pizzi JA, 2003, Advanced Drug DeIiveryReviews, v. 55, pp. 935-948,).

The most commonly used analgesic and anti-inflammatory drugs today are those in the non-steroidal anti-inflammatory (NSAID) class. Unfortunately, when it comes to drugs for the treatment of painful and inflammatory processes, despite the extensive arsenal available on the market, most of them have numerous side and / or adverse effects that prevent their use in chronic treatments (Calixto et al., 2000, Phytother, Res. V. 14, no. 6, pp. 401-418; Calixto et al., 2001, Expert Opinion, Emerging Drugs. V. 6, pp. 261-279; Calixto et al. Br. J. Pharmacol, v. 143, pp. 803-818). Such effects include gastritis, ulcerations, dyspepsia, nausea, vomiting, allergies (eg, skin urticaria and erythema), renal failure (reversible with cessation of medication), irreversible nephropathy (eg associated with continuous paracetamol medication), Reye's syndrome (a serious condition rarely caused by aspirin in children), metabolic acidosis (eg aspirin overdose), hypertensive crises and risk of cardiovascular disease (eg selective COX-2 inhibitors).

Moreover, individuals living with chronic pain are four times more likely to develop depression and anxiety because the available treatments do not lead to effective pain healing, only relieving symptoms and not addressing the real cause of pain. More than 70% of cancer patients suffer from pain caused by the development of the disease itself or its treatment (Brennan et al. 2007, Anest Analg. V. 75, p. 199-207).

Currently available therapies for the treatment of chronic pain include, besides the (1) non-steroidal anti-inflammatory drugs (NSAIDs) already mentioned, (2) opioid drugs and (3) a group of drugs, with actions various pharmacological agents, known as adjuvant, comprising antidepressants, anticonvulsants, local anesthetics, α2-adrenergic receptor agonists, and gabergic agonists, among others. However, as already mentioned above, these therapies also have their use restricted by the emergence of serious side effects.

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

In this context, phytotherapy presents itself as a relevant alternative to the predominantly synthetic therapeutic arsenal, constituting a form of medicine that has been growing visibly over the years. It is believed that the main factor contributing considerably to the growth in question is the evolution of scientific studies, particularly chemical and pharmacological studies, which increasingly prove the efficacy of medicinal plants, especially those used in folk medicine for medical purposes. Therapeutics (Cechinel Filho, V .; Yunes, RA 1998, Quim. Nova, v. 21, no. 1, pp. 99-105, 1998; Malheiros, Bittencourt, Niero and Cechinel Filho, in: Drugs and Medicines, an Approach Multidisciplinary, Chapter 2, pp. 17-44, 2009).

Herbal medicines are products obtained using exclusively raw materials of plant origin, such as oils and extracts. They are characterized by knowledge of the effectiveness and risks of their use, as well as the reproducibility and consistency of their quality. Its active ingredients are chemically characterized substances whose pharmacological action is known and responsible, in whole or in part, for the therapeutic effects of herbal medicine (Brazil, ANVISA - National Health Surveillance Agency. Resolution RDC No. 48, of 16 March 2004).

The potential of plants as a source of research in the search for new drugs with new modes of action, high selectivity and pharmacological activity is still little explored, as about 250,000 to 350,000 plant species in the world, only 15%. have been studied phytochemically and 6% biologically (Di Stasi, LC, 2005, Plant Genetic Resources, 3, 2, 199-205; Gurib-Fakim, A., 2006, Mol. aspects med., 27, 1-93; Molina -Salinas et al., 2006, Archives Of Medical Research, 37, 45-49).

W. paiudosa is used by popular therapy in infectious, inflammatory, painful, respiratory (expectorant) processes, among others (Corrêa, M.. M. Dictionary of Brazilian Useful Plants and Cultivated Exotic Plants. Rio de Janeiro: National Press, v 5, pp. 137.1984). In the form of tinctures, it is indicated for bruising, bruising, injury, neuralgia, anemia, and general inflammation (Michalak, E. Sister Eva Michalak's Phytotherapic Notes. Florianópolis: EPAGRI, 1997. 94p.).

However, very few studies that can systematically justify this broad therapeutic potential are described in the literature. Studies by the inventors and collaborators with S. trílobata have shown a higher concentration of compounds of terpenic, steroid and phenolic nature (flavonoids) and absence of alkaloids. The different parts of the plant (root, stem, leaf and flower) have distinct chromatographic profiles, indicating a different quantitative chemical constitution among them, but with some similarity with some compounds. Cauterenoic diterpene is found in all parts of the plant, but in higher concentrations in the root, and considering the seasonal variation, it is produced in higher concentration in the root and stem during autumn (Bresciani, LFV; Yunes, RA; Bürger, C. de Oliveira, LE; Bóf, KL; Cechinel Filho, 2004, V. Zeitschrift Fur Naturforschung, v. 59, No. 3-4, pp. 229-232,). Already the flavonoid Iuteolin is concentrated only in leaves and flowers.

Two cauranic acids (3α-tigloyloxycar-16-en-19-oic acid and 3α-cinnamoyloxycar-16-en-19-oic acid), stigmasterol, a mixture of 3b-0-bD-glycopyranosylitosterol and 3b were also identified in the flowers. -0-bD-glycopyranosylstigmasterol and a mixture of three 3b-O-acyloleanic esters (Carvalho, JA; Carvalho, MG; Ferreira, DT; Faria, TJ; Braz-Filho, R. Quim. Nova, v. 24, n. 1, pp. 24-26, 2001), and more recently, a new eudesmanolide lactone, paludolactone (Cechinel Filho, V .; Block1 LC; Yunes, RA; Monache, FD, 2004,, / ai. Prod. Res., V. 18, no. 5, pp. 447-451). The strong analgesic effect of the crude S. trilobata extract was also evidenced, being much more potent than some clinically used drugs, such as acetylsalicylic acid, acetaminophen, dipyrone and sodium diclofenac. In addition, the extract inhibited carrageenan-induced rat paw edema, dextran and bradykinin, showing anti-inflammatory potential (Block, LC; Scheidt, C.; Quintão, NLM; Santos, ARS; Cechinel Filho, V. Pharmazie, v. 53, No. 10, pp. 716-718,1998). Both hydroalcoholic extract and compounds (kaurenoic acid, iuteoline and paludolactone) obtained from the plant showed significant antinociceptive effect in various models of chemical nociception, such as acetic acid, formalin and capsaicin in mice. The root extract was about 2 to 4 times more potent than the extract obtained from the stems and leaves, that is, the active ingredients responsible for the effect of this plant are in higher concentration in the roots, or even due to the presence of other different compounds (Scheidt, C. Study of the antinociceptive and antiinflammatory action of hydroalcoholic extract and compounds isolated from Wedelia paludosa DC (Compositae). 1998. 53f. Vale do Itajai, Itajai, 1998; Calixto, JB et al., 2000, Phytotherapy Research, v. 14, pp. 401-418).

Witek and Bretzke (1999) reported that extracts obtained from W. paludosa had significant antihyperalgesic effect when analyzed in intraplantar injection-induced hyperalgesia of carrageenan, bradykin and capsaicin, being more effective than the clinically used drug (diclofenac) (Witek LM , Bretzke PE 1999. Antihyperalgesic activity of Wedelia paludosa and Moluccan Aleurites in rats Itajaí, 50p Course Conclusion Paper (Undergraduate) - Pharmacy Course, University of Vale do Itajaí)

The hexane and dichloromethane fractions obtained from S. trilobata were more active in the pain model analyzed than the ethyl acetate and butanol fractions, suggesting that the more nonpolar compounds are partly responsible for the analgesic potential of the plant. Isolated pure compounds (kaurenoic acid and luteoline) are also responsible for the analgesic activity presented by the hexane and dichloromethane fractions, respectively, being more potent than aspirin, acetoaminophen, indomethacin and dipyrone. With the exception of flowers, all other parts of the plant (roots, stems and leaves) dose-dependently inhibited abdominal writhing induced by acetic acid in mice, and methanolic root extract was more effective (Block, LC. active principles of Wedelia paludosa DC, (Compositae) 1997. 63f Course Completion Paper (Undergraduate) -Pharmacy Course, University of Vale do Itajaí, Itajaí, 1997; Schlemper, SRM; Cordeiro, F .; Cechinel Filho, V, Block, LC Reach (Research), year V, no. 2, pp. 14-18,1998).

In addition, the hydroalcoholic extract inhibited carrageenan, dextran and bradykinin-induced rat paw edema, suggesting anti-inflammatory potential (Block, LC; Scheidt, C.; Santos, ARS; Yunes, RA; Souza, MM; Monache, FD; Cechinel Filho, V., 1998, Ethnopharmacoi.,: 61, pp. 85-89). Results from Bretzke and Witek (1999) demonstrated that extracts obtained from S. triobata as well as diclofenac sodium had significant antihyperalgesic effect when analyzed for intra-plantar injection-induced hyperalgesia of carrageenan, substance P, bradykinin and capsaicin. However the extracts used were more effective than the clinically used drug (diclofenac - 30 mg / kg, i.p.).

Luteolin was about five times more effective than aspirin and paracetamol used as a positive control, corroborating popular uses in the treatment of painful and inflammatory processes. The extracts and compounds analyzed were effective in significantly reducing the migration of total leukocytes as well as only neutrophils in the pleural cavity, without interfering with the extravasation of Evans blue (exudation) caused by the intrapleural injection of carrageenan. Probably, the extracts and compounds studied are inhibiting factors involved in the migration of cells to the inflammatory site, without interfering with exudation (Niero, R. Obtaining new molecules with analgesic and anti-inflammatory activity from Brazilian medicinal plants. 2000. Thesis (Doctorate) - Postgraduate Course in Chemistry, Federal University of Santa Catarina, Florianópolis, 2000). W. paludosa has also been shown to have antibacterial properties in hexane and dichloromethane extracts (nonpolar compounds), mainly against pyogenic cocci. Pure compounds isolated from semi-purified hexane and dichloromethane fractions, caurenoic acid and lactone eudesmanolide, were effective against Gram-positive bacteria (Block, LC; Scheidt, C.; Santos, ARS; Yunes, RA; Souza Monache, FD; Cechinel Filho, V., 1998, J. Ethnopharmacol., V. 61, pp. 85-89).

In addition to all these pharmacological activities, W. paludosa flowers were effective against dermatophyte fungi, validating the popular use of one part of the plant that is used in folk medicine to treat skin diseases, but did not exert activity against systemic mycoses (Sartori, MRK; Pretto, JB; Cruz, AB; Bresciani, LFV; Yunes, RA; Sortino, M .; Zacchino, SA; Cechinel Filho, V., 2003, Pharmazie, v. 58, no. 8, pp. 567-569). On the other hand, this plant also presented trypanosomicidal activity (Trypanosoma cruzi protozoan) attributed to the existing diterpenes in the plant (Batista, R.; Chian, E.; Oliveira, AB, 1999, Planta Med., V. 65, p. 283- 284, 1999; Vieira, HS; Takahashi, JA; Bonaventura, MAD, 2001, Fitoterapla, v. 72, pp. 854-856).

The acute and subacute toxicological results demonstrate that the LD50 selected for the W. paludosa hydroalcoholic extract is greater than 4000 mg / kg, having low toxicity when administered orally in mice proving that the extract is practically devoid of toxicity in toxicity studies. Acute and subacute (Fischer, DR; Cordenunzzi, DA Preclinical toxicological evaluation of Wedelia paludosa hydroalcoholic extract in mice. 2000. 48 f. Monograph (Undergraduate) - Health Sciences Center, University of Vale do Itajaí, Itajaí, 2000 ; Burger, C.; Ficher, DR; Cordenunzzi, DA; Batschauer, AP; Cechinel Filho, V .; Soares, AR, 2005, J. Pharm. Pharm. Sci., V. 19, no. 8 (2), 3870-3873).

Cavalcanti et al (2006) determined the in vitro genotoxicity of kaurenoic acid using Chinese mouse (V79) pulmonary fibroblast cells. The duration of the test was 3 hours and methyl methanesulfonate (MMS) was used as positive control. At concentrations of 2.5, 5 and 10 µg / m L no damage to cellular DNA occurred. However, at high concentrations (30 and 60 µg / mL) of kaurenoic acid there was a significant increase in cell damage.

In an assay to evaluate cytotoxicity, Saccharomyces cerevisiae yeasts were cultivated at different concentrations of W. paludosa extract and acetylsalicylic acid was used as a control. Apparently the cytotoxic action of the extract plays an acute action, and thereafter there is a reduction of its toxic effects (Miguel, CO Bosco, B.. B. Analysis of the cytotoxicity of Acmeia brasiiiensis using Saccharomyces cerevisiae cells. 2005. 40 f. Monograph ( Graduation) - Health Sciences Center, University of Vale do Itajaí, Itajaí, 2005).

It was developed and validated a method for HPLC quantification of kaurenoic acid in W. paludosa extracts (Zanela, AH et al., Development and validation of method of determination of kaurenoic acid by clae in Wedelia paludosa dc compositae and aerial parts extract). topical herbal remedies In: V Scientific Initiation Seminar, 2006, Itajaí Annals of the V Scientific Initiation Seminar, Itajaí: Univali Publisher, 2006. v. 1. p. 1-1) and obtaining a soft extract standardization of this plant (Baccarin.et al. Standardization of hydroalcoholic extracts hi Acmela brasiiiensis (Wedelia paludosa) (Asteraceae). In: 5th International Congress of Pharmaceutical Sciences, 2005, Ribeirão Preto. RBCF - Brazilian Journal of Pharmaceutical Sciences. 2005. v. 41. pp. 469-469) using the turbo-extraction method.

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 and anti-inflammatory activities related to S. trilobata extracts as evidenced by the data and tests described in this report.

The inventors have also identified procedures and methods not yet reported in the literature, as described below, that contribute to the use of S. trilobata recognized as a safe and effective safety / efficacy drug for analgesic and anti-inflammatory properties to be reproducible and consistent.

Some patents can be found that use the same plant by treating another condition or using different processes from those presented.

An example is CN 1631106 (A) - which provides a method of reproducing S. trilobata which comprises the following five steps: (1) disinfecting the implant with 65-75% ethanol and 0.1% mercury chloride, (2) obtaining sterile shoots through MS employing 15- to 30-day culture medium, (3) generating incentive agglomeration buds, (4) inducing root to generate, (5) transplanting regenerated stresses . It can be verified from this short summary of the order, which the object of the requests.

Another example is patent application PI0701125-3, PRODUCT PROCESSING - PLANT EXTRACT BASIS Sphagneticola trílobata, which provides a process for obtaining medicament for use as an anesthetic, anti-inflammatory and healing agent, which can also be used in insect burns and the elimination of skin blemishes, characterized in that the process for obtaining drug based on the extract of the Sphagneticola tribolata plant is started by collecting leaves and flowers of the plant, cleaning and selecting for infusion in a volume of alcohol in the Proportion of: for each liter of alcohol a volume of leaves and flowers. This ratio (alcohol + leaves / flowers) is placed in a container, which is hermetically sealed, and then infused at room temperature for twenty four (24) hours. After this period the infusion is filtered to obtain an ideal solution where the extract is maintained with all its pharmacological properties in solution with alcohol to be used in drops and in the form of dermatological cream in association with its own elements. Said patent application (PI0701125-3) discloses a popular artisan process which does not reach the complexity of the process presented in this application. In view of the above, a process of obtaining a standardized extract from S. trilobata, a process for isolating its biomarker, a process for preparing a phytotherapeutic pharmaceutical composition with analgesic and anti-inflammatory properties, is of particular interest. a method of treatment and its use as an alternative therapy to topical NSAIDs in view of the potential therapeutic potential devoid of side effects.

Object of the Invention

The main object of the present invention is to provide a process for obtaining a standardized extract with antinociceptive and antiinflammatory properties from a plant of the genus Spagneticola. Preferably, the standardized extract is obtained from a part of the S. trilobata plant, more preferably from the aerial parts of S. trilobata.

It is also an object of this invention to provide a process of isolation, purification and characterization of the extract biomarker and is primarily responsible for the pharmacological effect, more particularly Caurenoic Acid.

Another object of this invention is to provide the pharmacological characterization of the standardized extract cited above for its antinociceptive and antiinflammatory, antiedematogenic, antihypernociceptive activities, as well as the evaluation of its acute toxicity and cytotoxicity.

Furthermore, the invention provides pharmaceutical compositions comprising as active ingredient a pharmaceutically effective amount of a standardized extract with respect to one or more biomarkers, preferably kaurenoic acid, as well as a method of preparing the same.

Brief Description of the Figures

The figures shown demonstrate the present patent, by way of non-limiting example, providing a better understanding thereof.

Figure 1 - Shows the HPLC chromatographic profile of the isolated kaurenoic acid standard (lot AC001 / 06) at 210 nm.- isocratic system Figure 2 - Shows the absorption spectrum of the isolated kaurenoic acid standard (lot AC001 / 06) in the infrared region.

Figure 3 - Shows the 1H NMR spectrum of the isolated kaurenoic acid standard (lot AC001 / 06).

Figure 4 - Shows the 13 C NMR spectrum of the isolated kaurenoic acid standard (lot AC001 / 06).

Figure 5 - Shows the CCD Profile of the soft extracts of S. trilobata collected in spring 2006. P: Acid. kaurenoic (biomarker; 1: soft extract (batch WP311106a, b, c); 2: soft extract (batch WP311106d, e, f); 3: soft extract (batch WP311106 shade a, b, c); 4: soft extract ( lot WP311106 shadow d, e, f)

Figure 6- Shows the HPLC chromatographic profile of standardized S. trilobata soft extract (batch WP010307) at 210 nm - isocratic system.

Figure 7- Shows the HPLC chromatographic profile of the scaled-up dry S. trilobata extract (lot 3453) at 210 nm - isocratic system.

Figure 8 - Shows the HPLC chromatographic profile of the scaled up dry S. trilobata extract (batch 3403) by HPLC at 210 nm. - gradient system

Figure 9 - Shows the dry extracts of S. trilobata lots: 1: ES001 / 08 (20% aerosi); 2: ES002 / 08 (25% aerosil) and 3: ES003 / 08 (30% aerosil) - laboratory scale

Figure 10 - Effect of topical cream treatment containing 3 standardized dry extracts of S. trilobata (lots ES001 / 08, ES002 / 08 and ES003 / 08), isolated kaurenoic acid standard (lot AC00108), soft extract (lot SE001 / 08) and dexamethasone in croton oil-induced ear edema (2.5%) in mice. η = 6, ** ρ <0.01 and *** ρ <0.001. ANOVA in one way followed by Dunnett's post hoc test.

Figure 11 - Effect of topical treatment with cream containing standardized scaling up dry extract of S. trilobata (lot 3453) at 1, 2, 3 and 5% and 0.5% dexamethasone in oil-induced ear edema of croton (2.5%) in mice. η = 6, ** ρ <0.01 and *** ρ <0.001. ANOVA in one way followed by Dunnett's post hoc test.

Detailed Description of the Invention

The present invention, as will be apparent from the presented figures, is a process of obtaining a standardized extract with antinociceptive and anti-inflammatory properties. Process for isolating and purifying the biomarker, pharmaceutical compositions, processes for preparing them, which can be better understood from the presentation of the Wedelia genus plant knowledge and the processes used to achieve the objects of this invention.

The plant W. paludosa, also known as Acmella brasiliensis Spreng and Sphagneticola trilobata (1996) (Hind, N. Electronic publication), is popularly referenced by the synonyms: Vedelia, Margaret, Gold-drop, Marigold, Picão -Beach, Pseudo-Arnica. It is a native plant in Brazil, widely found in various regions of the country, including the state of Santa Catarina. Occurs spontaneously along the coast, formed a uniform green mattress (15-30 thick), showcasing showy yellow flowers almost all year and producing excellent ornamental effect. Evergreen plant reproduced by seed; adapted to wetlands; tolerates a certain degree of shading, blooming with intensity if well lit (Kismann, K. G .; Groth, D. Weeding and harmful plants. Rio de Janeiro: BASF Brasileira, 1992. p.383-385).

The Asteraceae family consists of prostrate, root-knotted plants with sparse reddish-brown stems; opposite leaves, short-petiolate, membranous, hairy on both sides, most pronounced on the dorsal, narrowed toward the base above the middle, provided with two small lateral lobes and a larger, jagged terminal, 6 cm long and 3 cm long. width; semicylindrical petiole, ciliated, 4 mm long; solitary, long-pedunculated, axillary chapters; hairy peduncle, 10 cm long; foliaceous involucral bracts, in 2 series, hairy on the back, 8-12 mm long; conical receptacle, fleshy, paleaceous; yellow flowers, the female marginal, about 13, such as ligature corolla, trilobated at the apex, 8 mm long and hermaphrodite, with tubular corolla; feminine flower stiletto with glabrous, fleshy, obtuse, semi-cylindrical branches; black anthers, of sacred base; turgid achene, triplet, glabrous, narrow at base, with 1 mm long scythe papus (Corrêa, Ρ. M. Dictionary of Brazilian Useful Plants and Cultivated Exotic Plants. Rio de Janeiro: National Press, v. 5, p. 137,1984; Kismann, KG; Groth1 D. Weeding and noxious plants (Rio de Janeiro: Brazilian BASF, 1992. p.383-385).

The leaves are concentrated in the final part of the stems and branches, disappearing in the older parts that are inside the plants and that do not receive adequate illumination; underground system composed of superficial roots; simple leaves, subsessible, occurring opposite. The limbus is membranous, subastated in shape, with regularly attenuated base, two lateral lobes, acute apex and irregularly dented margins; prominent ribs on the dorsal surface, the central rib being single at the attenuated part of the base; dark green color and bright surface on the ventral face and lighter on the dorsal surface; tiny hyaline hairs on the dorsal surface especially on the ribs; The leaves are also slightly rough on both sides. The chapters are isolated, with thin and straight peduncle, reaching 10 cm in length from a leaf armpit; flowering occurs almost every year, with greater intensity in the summer months; flowers with showy chapters, 2-3cm in diameter, intense yellow in color, with dark points (anthers) protruding from the central florets; a shell made up of two series of five lanceolate filarias, half the length of the ligules, welded at the base, green and with fine hyaline hair on the outer surface; in the periphery there are about 15 female lobules, with developed lobules, reaching 8mm in length, spaciform and apex with 2-3 small entrances, intense yellow in color; in the central area a large number of hermaphroditic florets, with a pale yellow lanceolate membranous bract, accompanying the tubular corolla, opening into 5 wolves having tiny hairs, yellow as the corolla wolves; androecium with linear anthers, arranged in the shape of a tube, dark brown in color; the anthers are projected out of the corolla; he wiggled with stilettos projecting a bifid, yellow stigma out of the corolla; conical receptacle, fleshy, paleaceous; aquoneous from obconoidal to ovoid, long acuminate obtuse base; truncated apexes, narrower than the body of the achene; depressed elliptical epigenous disc at the base of the thick whitish stylus; absent papilla; seedling with cylindrical, smooth and glabrous hypocotyl; suborbicular cotyledonary leaves, hairy epicotylus; true oval leaves, with a round base and short acute apex, jagged margins, well-marked veins, smooth and glabrous surfaces, green (Kismann, KG; Groth, D. Weeding and harmful plants. Rio de Janeiro: BASF Brasileira, 1992. p. 383-385).

BIOMARKER INSULATION, PURIFICATION AND CHARACTERIZATION

The election, isolation, purification and characterization of one or more constituents / biomarkers of an extract become particularly necessary for the quantification and standardization procedures of the extracts.

According to the present invention, preferably kaurenoic acid (Figures 1 to 4) has been determined as the most appropriate biomarker for a process of obtaining a standardized extract. Such selection is justifiable because it is a major compound in the plant and is related to the pharmacological properties of the extract (Figure 10). In addition to this compound, other substances present in the extract may be used as biomarkers as observed in HPLC analysis in gradient system (Figure 8). Possibly these compounds are choreopsin, luteolin, paludolactone or caffeic acid already isolated from this plant (Bresciani, LFV; Yunes, RA; Bürger, C.; De Oliveira, LE; Bóf, KL; Cechinel Filho, 2004, V. Zeitschrift Fur Naturforschung, v. 59, no. 3-4, pp. 229-232; Cechinel Filh Cechinel Filho, V .; Block, LC; Yunes, RA; Monache, FD, 2004, Nat. Prod. Res., V. 18 No. 5, ¶ 447-451, V .; Block, LC; Yunes, RA; Monache, FD, 2004, Nat. Prod. Res., v. 18, No. 5, pp. 447-451), among others not identified.

These procedures for quantification and standardization of extracts occur according to the following steps: I. Isolation,

II. Purification, and

III. Biomarker characterization.

Isolation of the kaurenoic acid biomarker (step I) can be accomplished by techniques that include, but are not limited to, liquid-liquid partition, preparative CCD (CCDP), column chromatography, centrifugal chromatography, preparative HPLC (CLAEP) or semi-preparative , a combination of the same and similar.

The isolation of the caurenoic acid biomarker can be accomplished by sequentially employing liquid-liquid partition and open column chromatography techniques, using different stationary phases (silica, sephadex-LH20, chitin, etc.), preferably silica. and solvent mixtures as eluent (hexane, dichloromethane, acetone, ethyl acetate), preferably Hexane: Ethyl Acetate (100: 0 to 0: 100), and preferably in the ratio 85:15; or flash column chromatography, or preparative or semi-preparative HPLC using normal or reverse phase, preparative or semi-preparative columns, using different mobile solvents as mixtures of acetonitrile, methanol and acidified ultrapure water (pH 3.5) as mobile phase. preferably 40:45:15, monitored at 210 nm.

Purification of the caurenoic acid biomarker (step II) can be accomplished by techniques including dissolution and recrystallization with solvents such as hexane, methanol, ethyl acetate, among others, with or without heating. More preferably, the biomarker purification is performed by washing and recrystallization with hexane.

According to the present invention, by using the procedures described above, an equivalent biomarker purity content of> 90% should be achieved.

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

Isolated and purified kaurenoic acid biomarker is particularly characterized by FTIR (Figure 2), NMR, having an H1 (Figure 3) and C13 (Figure 4) NMR spectrum, as well as HPLC (Figure 1), with absorption profile characteristic, with maximum at 210 nm, characteristic Rf in CCD analysis (Figure 5) and characteristic melting range 176-180 ° C (Boeck, P .; Sá, MM; Souza, BS; Cercená, R .; Escalante, AM ; Zachino, SA; Cechinel Filho, V .; Yunes1 R. Α., 2005, J. Braz. Chem. Soc., V. 16, η. 6B, p. 1360-1366).

Caurenoic acid (CA) was isolated from S. trílobata roots and received lot number AC001 / 06, submitted to the above analyzes and complying with the specifications (Boeck, P .; Sá, MM; Souza, BS; Cercená , R., Escalante, AM; Zachino, SA; Cechinel Filho, V.; Yunes, RA, 2005, J. Braz. Chem. Soc., V. 16, η. 6B, pp. 1360-1366).

As shown in Figure 2 the spectrum of the isolated compound in the IR region showed the main peaks related to the chemical groupings of the molecule (main bands: 3300-2500, 1710-1680 and 1320-1210 cm "1) (Boeck, P .; Sá , MM; Souza, BS; Cercená, R.; Escalante, AM; Zachino, SA; Cechinel Filho, V.; Yunes, R. Α., 2005, J. Braz. Chem. Soc., V. 16, η. 6B, pp. 1360-1366).

The 1 H NMR spectrum of the AC (Figure 3) presents as main information: two singles at 0.9 and 1.20 ppm for the two methyls and two singles at 4.74 and 4.80 ppm for the olefinic hydrogens. Signals between 0.6 and 2.8 ppm refer to methylenes and methines. The data are in agreement with those obtained with the reference AC (lot 001/06) and those described in the literature (Boeck, P.; Sá, MM; Souza, BS; Cercená, R.; Escalante, AM; Zachino, SA; Cechinel Filho, V., Yunes, RA, 2005, J. Braz. Chem. Soc., V. 16, η. 6B, pp. 1360-1366).

Figure 4 shows the 13 C NMR spectrum of AC. Signals are observed at 184.2 ppm for carbonyl and at 155.8 and 102.9 ppm for olefinic carbons. The other signals between 60 and 15 ppm refer to methylene, methyl and methane carbons. The obtained spectrum is in agreement with the data obtained with reference AC (lot 001/06) and those described in the literature (BOECK et al., 2005). The selected biomarker was used for the development of a quantitative method, by HPLC1 for the standardization of the adapted extract (Zanela et al., Development and validation of a method for the determination of clae caurenoic acid in Wedelia paludosa dc compositae and topical herbal remedies In: V Scientific Initiation Seminar, 2006, Itajaí Annals of the V Scientific Initiation Seminar, Itajaí: Univali Publisher, 2006. v. 1. p. 1-1).

Purity was determined by the principal peak area relative to the sum of the HPLC CA standard chromatogram areas and the CA alone had a purity of 95 ± 1% (Figure 1). The HPLC analysis method can be performed using normal mode or reverse phase, preferably using a 10-25 cm long and 3-4.6 mm diameter C18 column using various HPLC grade solvents in the mobile phase such as as acetonitrile (ACN), methanol (MeOH), ultrapure water, tetrahydrofuran, among others, in gradient or isocratic mode. In isocratic mode preferably with MeOH: ACN :: water pH 2.6 at a ratio of 35:45:17 in a flow rate of 0.3-2.0 mL / min, preferably at 1.0 mL / min at 20-60 ° C, preferably at 35 ° C, with peak detection of 200-300 nm, preferably 210 nm. In gradient mode, preferably according to Table 1:

Table 1. Scheme of gradient used in HPLC analysis of dry extract of S. trilobata

<table> table see original document page 18 </column> </row> <table> <table> table see original document page 19 </column> </row> <table>

PROCEDURE FOR OBTAINING THE STANDARDIZED EXTRACT OF S. trilobata

A standardized extract comprises a soft or dry extract, standardized to one of its major biomarkers according to the following steps: (a) collection, drying and subdivision of plant material; b) extraction with solvent extraction; c) extract filtration; d) extract concentration (soft extract) and drying (dry extract); e) qualitative and quantitative analysis (standardization) of the extract.

After harvesting the plant, preferentially select the aerial parts of S. trilobata, drying the newly collected plant in a circulating air oven, or outdoors at room temperature, or using other drying methods.

The process steps according to the present invention are sufficient to ensure an adequate concentration of kaurenoic acid. In particular, the process steps are capable of providing the standardized extract with a total flavonoid concentration of 0.2 to 2% of its constitution. More preferably, the process steps are capable of providing a caurenoic acid concentration of from 0.05 to 15% of its constitution, preferably from 5-8%.

In the stage of collection, drying and subdivision of plant material, the plant material according to the present invention comprises a part of a plant of the genus Sphagneticota, more specifically, a part of the species S. trilobata is used as plant material. and most preferably the aerial parts including stems and leaves.

Plant material can be obtained with advantage from healthy individuals of the species S. tritobata, whether these are adults (£ 1 year), young (<3-12 years), intact or in the regrowth stage.

The plant material according to the present invention may be used fresh, dried, whole, scratched, minced, ground, ground, powdered or micronized. More specifically, it is useful to employ dry and crushed particulate material with dimensions <50 mm.

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, soxlet, using a suitable extracting 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, glycerine, acetone, petroleum ether, supercritical fluid, a combination. of the same and similar.

Preferably, according to the present invention, the extraction process consists of dynamic maceration and the extracting solvent in a water: ethanol hydroalcoholic solution, with the water content not greater than the alcohol content, preferably 60% ethanol A Extraction may occur under the presence or absence of light and / or oxygen under agitation for a period of 2-12 h, preferably 8 h.

The solvent-to-plant material ratio employed in the extraction procedure may range from 1: 1 to 20: 1. More specifically, the extractant solvent: plant 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, employing different types of filter materials. 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.

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

More preferably, the extract concentration procedure refers to the technique of evaporation under reduced pressure, using a heating temperature of approximately <70 ° C and a vacuum of 200 to 400 mmHg, which is capable of providing a concentration of approximately 90% solids in the extract, and sufficient for the total elimination of alcohol.

A useful procedure for stabilizing an extract relates to microbial decontamination. Gamma radiation, ultraviolet radiation, pasteurization and the like are particularly useful in this case.

In the step of obtaining the dry extract the addition of drying aids such as colloidal silicon dioxide, cellulose derivatives, starch and derivatives, sugars such as maltodextrose, lactose, among others can be used. Particularly useful is the use of colloidal silicon dioxide in proportions ranging from 0.05-90%, preferably 20-30%. Mixing can be performed using different equipment, such as tanks with different types of agitation, with or without addition of solvents such as ethanol, water, glycerine, propylene glycol, acetone, among others, preferably ethanol. As for the drying method, methods such as spray dryer, circulating air oven, vacuum oven, lyophilization, among others can be employed. Drying can take place at temperatures of 20-200 ° C, preferably up to 130-170 ° C in the spray dryer method, 30-70 ° C in circulating and vacuum greenhouses.

Preferred embodiments of the present invention for obtaining a standardized dry extract are detailed in Example I.

A fractionation step of a standardized extract according to the present invention may be especially useful for concentration of the substance (s) potentially related to the pharmacological activity of the species. More particularly, it is useful to use open-column or flash-type chromatography with different chromatographic supports (eg, silica, Sephadex®, alumina, cellulose and the like) and suitable eluents or liquid-liquid partition. hexane, dichloromethane, chloroform, ethyl acetate, butanol and the like. More preferably, hexane and ethyl acetate may be employed to obtain a concentrated fraction relative to the support / polar character of an extract, such as kaurenoic acid.

The process of obtaining a standardized extract, according to the invention, requires a qualitative and quantitative analysis step through duly validated analytical methodologies according to official standards (BRASIL, 2003).

According to the invention, analytical methodologies useful for qualitative and quantitative characterization include, but are not limited to, thin layer chromatography (CCD), paper chromatography, gas chromatography (GC), column chromatography (CC), liquid chromatography of high efficiency (HPLC), mass spectrometry (MS), thermo-analytical techniques and the like.

A standardized extract according to the present invention is presented as a fine, greenish yellow powder (Figure 9) which can be particularly analyzed by CCD having a characteristic chromatographic profile (Figure 5). The CCD can be performed using GF254 silica plate, preferably using the 80:20 to 95: 5 hexane: ethyl acetate, sulfuric anisaldehyde staining mixture as eluents. HPLC is also an important analytical tool and can be performed by dissolving the sample in a suitable solvent such as acetonitrile, methanol or water, preferably 50:50 methanol: water followed by sonication for 1-30 min, and membrane filtration, applying the same method used in the analysis of the caurenoic acid pattern previously described. Depending on the system, isocratic or gradient, the standardized S. trilobata extract has a characteristic chromatographic profile at 210 nm, as shown in the figures (isocratic-Figure 7 and gradient-Figure 8).

The following is a more detailed example of the preferred process of the present invention for obtaining the soft and dry extract. The proposed procedures and data presented are representative examples of the applications of the present invention and should not be construed as indicative of the limited use of the product.

EXAMPLE I

Process for obtaining soft and dry extract from S. trilobata aerial parts

The volume used for extraction corresponded to 10 parts of extracting liquid to 1 part of plant material. The extraction was done at room temperature, by the dynamic maceration method, for 8 h. To obtain the soft extract, the aerial parts are dried, crushed and classified by passing through sieves, using a vegetable drug of smaller size. 50 mm, after being extracted with hydroalcoholic ethanol / water solution in the ratio of 60:40. The solution was filtered on filter paper. The filtrate was concentrated at a maximum temperature of 70 ° C in a specific evaporator (Bernhauer) under vacuum (400 mmHg) to a soft extract without alcoholic strength and a total solids content of 20% to 40%. In order to obtain the dry extract, a variable amount of 20-30% of drying aid in relation to the solids content of the final product is added to the extract. The mixture was spray-dried dehydrated using a centrifugal rotating disc, the pump feeding speed of 45 Hz1 generating 6.5 liters of concentrate / hour feed, inlet temperature of 150 to 170 ° C and outlet temperature of 80-100 ° C.

The extract was dried according to the parameters described above. The dried extracts were analyzed for physical characteristics (color, density, flow), moisture content and hygroscopicity, resulting in an extract with characteristic CCD chromatographic profile (Figure 5), HPLC (Figures 7 and 8) and pharmacological activity. as shown in figure 11.

PHARMACOLOGICAL CHARACTERIZATION OF STANDARDIZED EXTRACTS -

Standardized extracts according to the present invention can be characterized for pharmacological activity in experimental models nociception pain, hypernociception, neuropathic pain and inflammation.

Several animal models can be applied for the pharmacological characterization of an extract with antinociceptive and antiinflammatory properties, including: 1. Thermal and / or mechanical hypernociception models of inflammatory origin (induced by carrageenan, CFA, PGE2, LPS injection). epinephrine, etc.), neuropathic (partial sciatic nerve constriction, brachial plexus avulsion, diabetic neuropathy) and / or cancer (melanoma or sarcoma); 2. Rat and / or mouse ear and paw edema assay induced by different phlogistic agents; 3. Pleurisy model, induced by different phlogistic agents; 4. LPS-induced hyperthermia models; 5. Evaluation of undesirable effects; and similar (evaluation of locomotor activity, motor performance, body temperature, gastric ulcer and interference in the basal thresholds of thermal and mechanical sensitivity, etc.).

Standardized extracts according to the present invention, when characterized for their pharmacological activity, show antinociceptive and antiinflammatory properties, as detailed below. For pharmacological analysis the dried extracts and the concentrated extract were incorporated in nonionic creams (Polawax wax) at a concentration of 3%. The correction factor was calculated in order to eliminate the influence of the moisture content of the extracts as well as the proportion of adjuvant in the dry extracts. Positive controls were creams containing 0.1% kaurenoic acid and 0.5% dexamethasone cream.

The effect of topical treatment on croton oil-induced ear edema (2.5%) in mice was analyzed.

Dry extracts (ES001 / 08, ES002 / 08 and ES003 / 08) showed similar activity in edema inhibition, as the effect of dry extracts did not differ significantly from concentrated extract (Figure 13). The results show that even dry extracts having a lower biomarker content, pharmacological activity was not compromised.

From the results obtained with the study of development of the dry extract of S. trilobata in laboratory scale, it is proposed to use Aerosil® at a concentration of 20% as a drying aid of the concentrated extract of the plant.

The dry extract obtained with this formulation presented good technological characteristics, higher biomarker content and similar pharmacological effect to the other dry extracts with higher content of this excipient.

Pilot scale extracts showed maintenance of pharmacological activity (Figure 11).

PHARMACEUTICAL COMPOSITIONS AND PREPARATION PROCESSES

A standardized soft or dry S. trilobata extract obtained and characterized as described above with topical antinociceptic and / or antiinflammatory action may be administered alone, but preferably incorporated in a liquid, semi-solid, solid or adhesive pharmaceutical form obtained in combination with a or more pharmaceutical vehicles or adjuvants and technological transformation process, resulting in an effective, safe and quality herbal medicine. Liquid pharmaceutical composition for topical use according to the present invention may contain a standardized extract as defined above, in an amount representing 0.05 to 90% of the total weight of the composition, preferably an amount representing 0, 5 to 20% of the total weight of the composition in solution or suspension form and may be administered in the form of drops, spray or aerosol, among others known in the state of the art.

According to the present invention, a semi-solid composition for topical use, preferably, but not limited to ointments, ointments, creams, lotions, gels, pastes and the like known in the art, may contain one or more pharmaceutical excipients. Such excipients may include carrier, humectants, surfactants, suspending agents, preservatives, antioxidants, chelators, colorants and essence.

Such adjuvants may be water phase constituents, oil phase constituents, emollients, humectants, emulsifiers, surfactants, preservatives, stabilizers, antioxidants, chelating agents, self-emulsifying waxes, ointment bases, dyes, essences and the like as required. The amounts of these pharmaceutical carriers or adjuvants may be equal to those conventionally used in the prior art. Examples of oil phase constituents, consistency 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, propylene glycol monostearate, sorbitan monostearate, microcrystalline wax, anionic self-emulsifying wax (e.g. Lanette®), nonionic self-emulsifying wax (e.g. Polawax®) 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. , decyl oleate, other esters and fatty alcohols and the like. Examples of aqueous phase constituents, consistency agents, wetting agents and polymers include, but are not limited to carbomer (e.g., Carbopol®), polyacrylic acid (eg, Pemulem®), propylene glycol, sorbitol, glycerin, silicone and derivatives thereof. and the like. Examples of surfactants include, but are not limited to, sorbitan monopalmitate polysorbates (tween and span), sodium lauryl sulfate and the like.

Examples of ointment bases include, but are not limited to lanolin, vaseline, hydrophilic petrolatum, yellow ointment, white ointment, hydrophilic ointment, polyethylene glycol ointment and the like.

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

An example of a cream pharmaceutical composition can be obtained by the formulation described in Table 2.

Table 2. Example of solution containing standardized S. trilobata extract for topical use.

<table> table see original document page 27 </column> </row> <table>

According to the present invention, a liquid composition for topical use, preferably but not limited to solutions and suspensions may contain one or more pharmaceutical excipients. Such excipients may include carrier, humectants, surfactants, suspending agents, preservatives, antioxidants, chelators, colorants and essence. Exemplary vehicles include, but are not limited to water, ethanol, propylene glycol, liquid petroleum jelly, vegetable oils, among others. Examples of humectants include, but are not limited to, propylene glycol, polyethylene glycol, glycerine, isopropyl myristate, mineral oil, myristyl alcohol, cetyl palmitate, octyl palmitate, glyceryl stearate, polyoxidoethylene stearate, hydrogenated coconut glycerides. , lanolin, lanolin alcohols, decyl oleate, other esters and fatty alcohols and the like, among others. Examples of surfactants include, but are not limited to polysorbates such as tweens and spans, glyceryl monostearate, sodium lauryl sulfate and the like, among others. Examples of suspending agents include, but are not limited to cellulose derivatives, starches and derivatives, gums, among others. Examples of preservatives, antioxidants and chelators include, but are not limited to methylparaben, propylparaben, butylparaben, benzalkonium chloride, benzethonium chloride, phenoxyethanol, phenoxyethanol + parabens (e.g. Phenonip®), imidazolidinylurea (e.g. Germall®), imidazolidinylurea + iodopropynyl butylcarbamate (germall plus®), edetic acid, disodium EDTA, ascorbic acid, sodium bisulfite, sodium metabisulfite and the like.

An example of a pharmaceutical composition in solution or suspension form may be obtained by the formulation described in Table 3 or 4, respectively. Table 3. Example of solution containing standardized S. trilobata extract for topical use.

<table> table see original document page 28 </column> </row> <table> Table 4. Example of suspension containing standardized S. trilobata extract for topical use.

<table> table see original document page 29 </column> </row> <table>

According to the present invention, a solid composition for topical use, preferably but not limited to powder, may contain one or more pharmaceutical excipients. Such excipients may include diluents such as talc, starch, cellulose derivatives, zinc oxide among others.

Pharmaceutical compositions according to the present invention may be obtained by industrial pharmaceutical processes and unit operations known in the art such as, but not limited to mixing, milling, levigating, micronizing, lubricating, wetting, dissolving, emulsifying and homogenizing.

For preparation of a cream (Table 2) the components are fractionated; Items B, C, E and F are heated to 80 ° C in a homogenizing tank under heating. Then 50% of the required volume of water (H) containing item G is maintained under stirring and heating for emulsification; The remaining volume of water is added under stirring. Separately, in a homogenizer tank, extract (A) with item D and incorporate it into the base emulsion and mix.

For preparation of a solution (Table 3) the components are fractionated and, in a stirring tank, the extract (A) is dissolved in B and C, together with D. Following this, the mixture is gradually and gradually stirred under constant stirring. purified water to the desired final volume and filter. For preparation of a suspension (Table 4) the components are fractionated and, in homogenization tank, the extract (A) is mixed with B and C. Then, a portion of G e is added to the mixture gradually and under constant agitation. the other components. Then the remainder of G is added to the desired final volume. Mechanical stirring proceeds until complete homogenization of the mixture.

Claims (37)

1. BIOMARKER INSULATION, PURIFICATION AND CHARACTERIZATION PROCESS "characterized by understanding the use of kaurenoic acid as the most appropriate biomarker. BIOMARKER INSULATION, PURIFICATION AND CHARACTERIZATION PROCESS according to claim 1, characterized in that it comprises the following steps: I. Isolation, II. Purification, and III. Marker characterization. 3. "BIOMARCATOR INSULATION, PURIFICATION AND CHARACTERIZATION PROCESS" characterized by the fact that the isolation of the caurenoic acid biomarker (step II) can be performed by sequentially using liquid-liquid partition and open column chromatography techniques. 4. "BIOMARCATOR INSULATION, PURIFICATION AND CHARACTERIZATION PROCESS" according to claim 3, characterized in that it comprises different stationary phases (silica, sephadex-LH20. Chitin, etc.), preferably silica and solvent mixtures as eluent ( hexane, dichloromethane, acetone, ethyl acetate), preferably from Hexane: Ethyl Acetate (100: 0 to 0: 100), and preferably 85:15; or flash column chromatography, or preparative or semi-preparative HPLC using normal or reverse phase, preparative or semi-preparative columns. 5. "BIOMARCATOR INSULATION, PURIFICATION AND CHARACTERIZATION PROCESS" characterized in that the purification of the caurenoic acid biomarker (step II) can be performed by techniques that include dissolution and recrystallization with solvents such as hexane, methanol, ethyl acetate, among others. with or without heating. 6. "BIOMARKER INSULATION, PURIFICATION AND CHARACTERIZATION PROCESS" according to claim 5, characterized in that it preferably comprises the purification of the biomarker by washing and recrystallization with hexane. 7. "BIOMARKER INSULATION, PURIFICATION AND CHARACTERIZATION PROCESS" according to claim 1 (step III), characterized in that it comprises HPLC analysis using as stationary phase, reverse phase analytical column, using the isocratic mode, with eluent consisting of MeOH: ACN :: water pH 2.6 at a ratio of 35:45:17, in a flow rate of 0.3-2.0 mL / min, preferably at 1.0 mL / min, at a temperature of 20-30 ° C. 60 ° C, preferably 35 ° C, with peak detection of 200-300 nm, preferably 210 nm or, in gradient mode, further solvents such as tetrahydrofuran, propyl alcohol, among others (Table 1). 8. "BIOMARKER INSULATION, PURIFICATION AND CHARACTERIZATION PROCESS" characterized in that its characterization (step III) can be performed by techniques that also include, but are not limited to, hydrogen and carbon-13 nuclear magnetic resonance (NMR). , infrared (IR) spectrophotometry, ultraviolet (UV) spectrophotometry, HPLC, CCD, thermal analysis. 9. "BIOMARKER INSULATION, PURIFICATION AND CHARACTERIZATION PROCESS" characterized by achieving a biomarker purity equivalent to approximately> 90%. 10. "PROCESS FOR OBTAINING THE STANDARDIZED EXTRACT OF S. trilobata WITH ANTINOCICEPTIVE AND ANTIINFLAMMATORY PROPERTIES" characterized by comprising a standardized soft or dry extract in relation to one of its main biomarkers. 11. "PROCESS FOR OBTAINING THE STANDARDIZED EXTRACT OF S. trilobata WITH ANTINOCICEPTIVE AND ANTI-INFLAMMATORY PROPERTIES" characterized by the following steps: a) collection, drying and subdivision of plant material; b) extraction with solvent extraction; c) extract filtration; d) extract concentration (soft extract) and drying (dry extract); e) qualitative and quantitative analysis (standardization) of the extract. PROCEDURE FOR OBTAINING THE STANDARDIZED EXTRACT OF S. trilobata WITH ANTINOCICEPTIVE PROPERTIES AND "IN FLAM ATORY" according to claim 11, characterized in that it comprises an extract stabilization step. 13. "PROCEDURE FOR OBTAINING THE STANDARDIZED EXTRACT FROM THE S. trilobata PLANT WITH ANTINOCICEPTIVE AND ANTI-INFLAMMATORY PROPERTIES" according to claim 11 (step c), is characterized in that the procedure is selected from a group consisting of, but not limited to filtration depth or surface filtration, using different types of filtering materials to eliminate plant material residues such as filter paper, polymeric membranes and the like. Process for obtaining the standardized S. trilobata extract with antinociceptive and anti-inflammatory properties "according to claim 11, characterized in that it comprises a fractionation step of a standardized extract. 15. "PROCESS FOR OBTAINING THE STANDARDIZED EXTRACT OF THE S. trilobata PLANT WITH ANTINOCICEPTIVE AND ANTI-INFLAMMATORY PROPERTIES" characterized by the fact that the standardized extract comprises in its constitution a concentration of 0.2 to 2% of total flavonoids. 16. "PROCEDURE FOR OBTAINING THE STANDARDIZED EXTRACT OF THE S. trilobata PLANT WITH ANTINOCICEPTIVE AND ANTIINFLAMMATORY PROPERTIES" according to claim 1, characterized in that it comprises a concentration of caurenoic acid of 0.05 to 15% of its constitution, preferably, between 5-8% 17. "PROCEDURE FOR OBTAINING THE STANDARDIZED EXTRACT FROM THE S. trílobata PLANT WITH ANTINOCICEPTIVE AND ANTI-INFLAMMATORY PROPERTIES" characterized in that it comprises preferably the steps of; a) crushing; b) sorting through sieving, provided with a plant drug preferably less than 50 mm in size; c) Extraction preferably by dynamic maceration at room temperature for 8 h .; d) Filtration by filter paper e) Concentration of the extract; f) Spray drying. 18. "PROCEDURE FOR OBTAINING THE STANDARDIZED EXTRACT FROM THE S. trílobata PLANT WITH ANTINOCICEPTIVE AND ANTI-INFLAMMATORY PROPERTIES" according to claim 17 (step c), characterized in that it comprises preferably 60% V / V hydroalcoholic solution. 19. "PROCESS FOR OBTAINING THE STANDARDIZED EXTRACT FROM THE S. trílobata PLANT WITH ANTINOCICEPTIVE AND ANTIINFLAMMATORY PROPERTIES" characterized by the fact that the extraction is done at room temperature, preferably by the dynamic maceration method for 8 h. "PROCEDURE FOR OBTAINING THE STANDARDIZED EXTRACT OF THE S. TRILOBATA PLANT WITH ANTINOCICEPTIVE AND ANTI-INFLAMMATORY PROPERTIES" according to claim 17 (step e), comprising the use of a group consisting of, but not limited to evaporation under reduced pressure. evaporation by heat convention and the like, preferably at a pressure <£% - reduced to a maximum temperature of 70 ° C under vacuum (400 mmHg) until a soft extract without alcoholic strength and a total solids content of 20% to 40% 21. PROCESS FOR OBTAINING THE STANDARDIZED EXTRACT OF THE S. trilobata PLANT WITH ANTIINOCICEPTIVE AND ANTIINFLAMMATORY PROPERTIES "according to claim 17 (step f), characterized in that methods such as spray dryer, circulating air oven, vacuum drying, freeze drying, etc. Drying can take place at temperatures of 20-200 ° C, preferably up to 130-170 ° C in the spray dryer method, 30-70 ° C in circulating and vacuum greenhouses . 22. "PROCEDURE FOR OBTAINING THE STANDARDIZED EXTRACT OF THE S. trilobata PLANT WITH ANTINOCICEPTIVE AND ANTI-INFLAMMATORY PROPERTIES" according to claim 17 (step f), characterized in that it consists in obtaining the dry extract from the spray drying parts of the plant S. trilobata the following steps; a) preferably adding from 20% to 30% of drying aid to the solids content of the soft extract end product; b) dehydrate in a spray-dryer preferably using centrifugal rotary disc, the pump feeding speed of 45 Hz, generating a feed of 6.5 liters of concentrate / hour, inlet temperature from 150 to 170 ° C and outlet temperature from 80-1000 ° C. 23. "PROCEDURE FOR OBTAINING THE STANDARDIZED EXTRACT FROM THE S. trilobata PLANT WITH ANTINOCICEPTIVE AND ANTIINFLAMMATORY PROPERTIES" according to claim 21, preferably comprising the use of colloidal silicon dioxide at a concentration of 20% as a drying aid for the concentrated extract. of the plant. 24. "STANDARDIZED EXTRACT OF S. trilobata PLANT WITH ANTINOCICEPTIVE AND ANTIINFLAMMATORY PROPERTIES" characterized by the fact that the standardized extract of S. trilobata has a characteristic chromatographic profile at 210 nm. (isocratic - Figure 7 and gradient - Figure 8). 25. "PHARMACOLOGICAL CHARACTERIZATION OF STANDARDIZED EXTRACTS OF THE S. trilobata PLANT" characterized by the fact that it comprises antinociceptive and antiinflammatory properties. 26. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" characterized in that it comprises topical antinociceptic and / or antiinflammatory action which may be administered alone but preferably incorporated in a liquid, semi-solid, solid or adhesive pharmaceutical form obtained in combination with one or more vehicles or pharmaceutical adjuvants. 27. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" characterized in that it comprises the use of the standardized extract in an amount representing 0.05 to 90% of the total weight of the composition, preferably an amount representing 0.5 to 20%. % of the total weight of the composition in solution or suspension form and may be administered in drops, spray or aerosol form, among others known in the prior art. 28. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" characterized in that it comprises as bases for ointments, not limited to: lanolin, vaseline, hydrophilic petrolatum, yellow ointment, white ointment, hydrophilic ointment, polyethylene glycol ointment and the like. 29. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" characterized in that it comprises in its preparation of aqueous phase constituents, consistency agents, wetting agents and polymers, not limited to carbomer (e.g. Carbopol®), polyacrylic acid (eg , Pemulem®), propylene glycol, sorbitol, glycerin, silicone and its derivatives and the like. 30. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" characterized in that it comprises surfactants which include, but are not limited to, sorbitan monopalmitate polysorbates (tween and span), sodium lauryl sulfate and the like. 31. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME", comprising in its preparation preservatives, stabilizers, antioxidants and chelating agents, including, but not limited to, methylparaben, propylparaben, butylparaben, benzalkonium chloride, benzethonium chloride , phenoxyethanol, phenoxyethanol + parabens (eg Phenonip®), imidazolidinylurea (eg Germall®), imidazolidinylurea + iodopropinyl butylcarbamate (germall plus®), edetic acid, EDTA disodium, ascorbic acid, sodium bisulfite, and sodium metabisulphite similar. 32. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" characterized in that it comprises a semi-solid composition containing standardized S. trilobata extract for topical use - Table 2 - Cream-like pharmaceutical composition: a) Standardized S. trilobata extract 3% b) Nonionic wax: 15% c) Acetylated Ianoline Alcohol: 3% d) Propylene Glycol: 5% e) Methylparaben: 0.1% f) Propylbaraben: 0.1% g) EDTA: 0.1% h) Purified Water qsp: 100% 33. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" characterized in that it comprises a liquid composition containing standardized S. trilobata extract for topical use - Table 3 - pharmaceutical composition in solution form: a) Standardized S. trilobata extract: 10% b ) Ethanol: 10% c) Propylene Glycol: 5% d) Methylparaben: 0.1% e) Purified water qsp: 100% 34. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" characterized in that it comprises liquid composition containing standardized extract of S. trilobata for topical use - Table 4 - pharmaceutical composition in suspension form: a) Standardized extract of S. trilobata: 30% b ) Tween 40: 3% c) Propylene glycol: 5% d) Hydroxyethylcellulose: 0.8% e) Methylparaben: 0.1% f) Propylbaraben: 0.1% g) Purified water qsp: 100% 35. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" according to claim 32, characterized in that it comprises in the preparation of the cream the following fractional components; a) Items B, C, E and F are heated to 80 ° C in a homogenizer tank under heating. b) Then 50% of the required volume of water (H) containing item G1 kept under stirring and heating for emulsification is added; c) The remaining volume of water is added under stirring. d) Separately, in a homogenizer tank, extract (A) with item D and incorporate it into the base emulsion and mix. 36. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" according to claim 33, comprising in preparing the solution or suspension the following fractionated components; a) In a mixing tank, the extract (A) and item D are dissolved in B and C. b) Subsequently, the mixture is gradually and under stirring added to the desired final volume. 37. "PHARMACEUTICAL COMPOSITION AND PREPARATION PROCESS FOR THE SAME" according to claim 34, comprising in preparing the solution or suspension the following fractionated components and; a) In homogenization tank, extract (A) is irrigated in B and C. b) In auxiliary tank, EeF items are dissolved in part of G1 under heating and stirring. Item D is dispersed in this solution under stirring and heating. c) The dispersion of items E, F and D in G is gradually added to the mixture containing extract (A) and the remainder of item G is added to the desired final volume.