BRPI1000802A2 - process of obtaining copula spheres microparticles langsdorffii (aerial parts) and isolation of the active principles; microparticles and compounds thus obtained, having antilithiasis (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic activity, their formulations, products and uses - Google Patents

Abstract

PROCESS OF OBTAINING MATERIAL MICROPARTICLES FROM COPAìFERA LANGSDORFFII (AIR PARTS) AND ISOLATION OF THE ACTIVE PRINCIPLES; MICROPARTICLES AND COMPOUNDS SO OBTAINED, WITH ANTILITIASIS ACTIVITY (KIDNEY CALCULATION), ANALGESICS, ANTI-SPASMÓDICA, ANTI-INFLAMMATORY, DIURETICS AND ANTI-SEPTIC, ITS FORMULATIONS, PRODUCTS AND USES. The present invention relates to a process for obtaining matrix microparticles of Copaifera Iangsdorffii (aerial parts) and the isolation of the active compounds and / or markers of the aerial parts of that plant, as well as the biological activities found for such matrix microparticles and the isolated compounds, namely: antilitiásica activity (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic, as well as the use of these in the treatment of renal lithiasis (renal calculus), arthritis, tartar, muscle pain and spasmodic in general (abdominal, renal cramps, etc.). It also refers to the other diseases related to the activities previously mentioned, in addition to the formulations that can be originated with the matrix microparticles of Copaifera Iangsdorffii; and / or its isolated compounds, such as medicines, personal hygiene products, cosmetics, food, veterinary and dental products, etc.

Description

Process for obtaining Iangsdorffii (aerial parts) matrix microparticles and isolation of the active ingredients; microparticles and compounds thus obtained, having antilithiasis (renal calculus), analgesic, antispasmodic, anti-inflammatory, diuretic and antiseptic activity, their formulations, products and uses.

The present invention relates to a process of obtaining Copaifera Iangsdorffii matrix (aerial parts) microparticles and the isolation of the active compounds and / or markers of the aerial parts of said plant, as well as the biological activities found for such matrix microparticles and isolated compounds, namely: antilithiasis (renal stone), analgesic, antispasmodic, anti-inflammatory, diuretic and antiseptic activity, as well as their use in the treatment of renal lithiasis (renal stone), arthritis, tartar, muscle pain and spasmodic in general (abdominal cramps, renal, etc.). Other diseases related to the previously mentioned activities are also mentioned, besides the formulations that may originate with the matrix microparticles of Copaifera Iangsdorffii; and / or their isolated compounds, such as medicines, toiletries, cosmetics, food, veterinary and dental products, etc.

BACKGROUND OF THE INVENTION

Today, the world herbal market represents a significant portion of the drug industry, moving $ 21.7 billion a year globally. The main movement of the world herbal market is concentrated in Europe, 50% in Germany. Then appear Asia and Japan. However, no other market grows as large as the US market (CALIXTO, 2000). In Brazil, there is no updated official data, but it is estimated that this market is around US $ 160 million per year (CARVALHO et al., 2008).

The largest plant biodiversity in the world, estimated at about 200,000 plant species, is located in Brazil and, according to some authors, at least half may have some useful therapeutic property for the population. However, the data reveal that only 15 to 17% of the species were studied for their medicinal potential, justifying the search for further development in this area (GUERRA & NODARI, 2001). This huge genetic heritage currently has invaluable economic value, with great potential for the development of new drugs.

In the case of renal lithiasis, more than 70 species with antilithiasis properties are described. The plant species Phyllanthus niruri, popularly known as rock breakers, stands out for the treatment of urolithiasis (UNANDER et al., 1991; CALIXTO et al, 1998, BOIM and SCHOR, 2003).

Phyllanthus niruri is a plant used in folk medicine for the treatment of urolithiasis. P. niruri has previously been shown to have no toxicity, increased stone elimination in lithiasic patients, reduced bladder stone growth in an experimental rat model, and inhibited adhesion and internalization of calcium oxalate (CaOx) crystals in MOCK cells. . To add evidence of the protective role of P. niruri, the effect of its aqueous extract on in vitro CaOx crystallization (0.25 mg / mL urine) was evaluated. The presence of P. niruri did not inhibit CaOx precipitation and even a larger number of crystals were observed, but smaller. Moreover, in the presence of P. niruri there was a predominance of dihydrate crystals, which is less adherent. When crystals were observed 24 hours after crystallization, large aggregation was observed in the absence of P. niruri, and in their presence aggregation was inhibited. The results show that P. niruri interferes with the growth and aggregation of crystals in human urine suggesting a potential role of this plant in preventing the development of urinary stone (BARROS, 2002).

In addition to the stone-breaker plant (Phyllanthus niruri), presented above, it is common to use Uva-ursi (Arctostaphylos uva-ursi), a traditional herbal medicine recognized by the National Health Surveillance Agency (ANVISA) with the biological activity approved as an anti- urinary septic. Thus, it is indicated for patients with cystitis and often also for those with renal lithiasis, however, being only a compound for relief and asepsis of the urinary tract, with no antilithiasis effect.

The wide variety of species popularly indicated for treatment of lithiasis can be attributed to the high prevalence of the disease. It is quite curious to note that despite the large number of species (including products marketed for these purposes), there are few serious scientific studies that claim to establish how true these properties are. An example of this is the absence in Brazil of patents related to active ingredients and / or medicines capable of effectively exercising such action.

Currently, INPI's patent application database contains only four processes related to kidney calculations, as follows:

- One (i), of American origin, addresses the use of "rare earth" compounds, basically based on lanthanum, with properties of chelating oxalate in the gastrointestinal tract, and therefore being preventive of the formation of kidney stones. for preventing oxalate absorption (PI 0209072-4 - Application of rare earth compounds for the prevention of kidney stones)]

- another (ii) of Chinese origin deals with the use of hydrochloric acid (HCI) solution for the treatment of hypertension and general calculations (PI0608022-7 A2 - Process for the manufacture of a medicament for the manufacture of a medicament for treating hypertension, antihypertensive formulation and process for manufacturing a drug to treat calculus)]

- a third (iii), of Brazilian origin, entitled "Process for obtaining and using extracts, fractions and substances isolated from Copaifera species in the treatment of urinary iathiasis in humans and animals" (PI0401631-9 A2), deposited on 22 / 04/2004;

- and (iv) a grant to an application of Japanese origin entitled "Amino-ethyl-phenoxy acetic acid derivatives and pain-relieving drugs and promoting the removal of stones in urinary lithiasis" (PI9811483- 2 B1), with deposit made on July 15, 1998 and the concession in January 2010.

It should be noted that there is no application for a technological product with inventive activity concerning the use of derivatives of plant origin, as is advantageously our case, since the application for Copaifera species according to PI0401631-9 A2, described above, is not liable to grant as it is a discovery as well established in the Industrial Property Law. Since as knowledge, teas, plant drugs, extracts are not endowed with inventive activity, but are subject to scientific discoveries, and these can be used as a matter for technological development and generation of inventions that, if well characterized and carefully evaluated, can bring benefits to society. In addition, the claims referred to in item (i) and (ii), detailed above, are requests directed to oxalate chelating action and therefore only of a preventive character, completely different from the object of the present invention, which is advantageously a technological product, with antilithiasis, anti-inflammatory, analgesic, antispasmodic, antiseptic and diuretic activities, having medicinal and curative character.

The last application (iv) above is an innovative molecule obtained by chemical synthesis and its medicaments, which is not comparable to the invention object of the present application. The current order involves the transformation of a discovery into an innovative and revolutionary technology product for the pharmaceutical industry, that is, it transforms what the present team discovered with the Copaifera plant into an unprecedented pharmaceutical ingredient and can be used in pharmaceutical and cosmetic formulations. and veterinary. Because the treatment of nephrolithiasis is expensive and in many cases invasive and with many side effects, it is important to seek alternatives to these conventional methods using herbal medicine. In fact, in many countries medicinal herbs are used that appear to cure urolithiasis patients. However, the effectiveness and mechanism by which these plants function have not been demonstrated by scientific and objective methods. Thus, it is recommended that new drugs be researched from medicinal plants for the treatment and prevention of kidney stone formation (ATMANI, 2003; MACIEL et al, 2002) and that such scientific discoveries be transformed into technological development, such as the present one. such innovative products are effectively applied in the treatment of diseases and generate benefits for the community and the country.

Among the plants for medicinal and traditional use is Copaifera sp, a native Brazilian plant popularly used since the 16th century. The literature provides several studies of the biological activities of oil-resin of several genera of Copaifera sp (CARVALHO, 2004; VEIGA JR, 2005; PAIVA et al., 1998; 2003; 2004; BASILE et al., 1988; COSTA-LOTUFO et al., 2002), as its healing action, antiseptic and anti-inflammatory airway (CARVALHO, 2004). However, studies on the leaves of Copaifera sp are scarce in the literature except for the study of their constituents in ecological interactions (LANGENHEIM et al., 1981; 1986a and 1986b).

- Copaifera langsdorffii

The species Copaifera langsdorffi belongs to the botanical family Leguminasae, subfamily Caesalpinioideae and can be shrubs or leafy trees from 15 to 25 m. This species is found in the north, northeast, southeast, midwest and part of southern Brazil, as well as in Venezuela, southern Bolivia, northeast Argentina, and northern Paraguay. Other species of the genus Copaifera are distributed throughout South and Central America, with citations occurring in western Africa (CARVALHO, 2004; VEIGA JÚNIOR, 2005; VEIGA JÚNIOR and PINTO, 2002).

In Brazil alone, there are more than twenty species of the genus Copaifera used with the same therapeutic actions. Some species have their own popular denomination in Portuguese and, eventually, in other languages. In particular, C. Iangsdorffi is called red copaíba, red oil or copaíba floodplain in Amazonas, copaibeira de Minas, cupiúva or podoi in Piauí and Ceará and potter or cabimo in Venezuela (CARVALHO, 2004). In general, the Copaifera genus has infinite denominations that can be found in the literature (CARVALHO, 2004; VEIGA JÚNIOR, 2005; VEIGA JÚNIOR and PINTO, 2002). The literature shows that the oil-resin of C. Iangsdorffi species has as main chemical constituents the kaurane tetracyclic diterpenes such as kaurenoic and kauranoic acids (FERRARI et al, 1971), clerodanes as hardwickiic acid (OHSAKI et al., 1994 ) and labdanic diterpenes such as copalic acid (CASCON and GILBERT, 2000; MACIEL et al., 2002) among others, as well as sesquiterpenes, predominantly β-caryophyllene, β-bisabolene, a-bergamotene and β-selinene (CARVALHO, 2004; VEIGA JR, 2005; VEIGA JR and PINTO, 2002).

The chemical composition of Copaifera Iangsdorffi leaves presents 3% of the amino acid, N-methyl-frans-4-hydroxy-L proline and sesquiterpenes such as σ-cubeben, σ-copaene, cyperene, β-copaene, caryophyllene, β humulene, muurolene, β-selinene, δ-cadinene and y-cadinene (LANGENHEIM et al, 1986a; MACEDO and LANGENHEIM, 1989a; MACEDO and LANGENHEIM, 1989b).

Among the most studied pharmacological activities for Copaifera sp (oil-resin) are: anti-inflammatory, analgesic, antibacterial, antitumor, antioxidant, healing and gastroprotective, anthelmintic, leishmanicide, trypanomicide, and muscle relaxant (CARVALHO, 2004; VEIGA JÚNIOR) , 2005; VEIGA JÚNIOR and PINTO, 2002).

The scientific literature does not contemplate any data regarding the study of the leaves of Copaifera langsdorffii, residing here the main discovery used as material for the present invention. Thus, the present proposal involved the study of processes for obtaining matrix microparticles containing extracts of Copaifera langsdorffii leaves, phytochemical processes for the isolation and identification of their chemical compounds, the study of their efficacy and safety, aiming to obtain pharmaceutical supplies, medicines, products for medical, veterinary, dental, among others.

Studies conducted by our group have shown that standardized C. langsdorffii extract and / or its isolated substances, obtained from Copaifera aerial parts, by the processes described herein, have antilithiasis, anti-inflammatory, analgesic, antispasmodic, antiseptic and diuretic properties. , unprecedented discovery that underlies the technological development carried out. Justifying the studies that originated the present invention, that is, the process of obtaining matrix microparticles containing extracts of Copaifera langsdorffii, a very advantageous technological system in relation to the use of lyophilized extracts, which provides greater protection and stability to the compounds identified in the extract. system homogeneity, modified release, reduced particle size and morphology that provide better pharmacotechnical development, endowed with excellent biological activity for the purposes intended in this application. It is noteworthy that the lyophilized extracts can undergo degradation processes, decomposition of the compounds of interest, besides offering larger particles, with great particle size variation, deformed and difficult to handle pharmacotechnically. Furthermore, it is noteworthy that the chemical composition of Copaifera sp aerial parts is completely different from that observed for copaíba oil, which is obtained from the plant stem. The chemical composition of the aerial parts consists mainly of phenolic compounds mostly soluble in water. Copaiba oil, on the other hand, consists mainly of compounds belonging to the terpene class and mostly soluble in oil. Therefore, this invention should not be compared to what has already been described for copaiba oil, although it is not indicated for the treatment of lithiasis.

Thus, several studies have been performed as shown in the examples below to demonstrate the claims of the present application, such as: (a) description of the process of obtaining standardized extracts that will be processed and transformed into microparticles; (b) the process of obtaining the matrix microparticles of the aerial parts of Copaifera langsdorffii; (c) the microparticles themselves; (d) compound isolation process, characterization and identification of such substances; (e) preclinical and clinical safety and efficacy (preliminary). An antilithiasis activity study was carried out comparing the activity of the matrix microparticles of Copaifera Iangsdorffii (according to EXAMPLE 6) by the same group that evaluated the biological activity of Phyllanthus niruri tea, showing that the results obtained with the technological product Matrix Microparticles of C Langsdorffii, were far superior to the previous ones, demonstrating the relevance of the present application for industrial protection.

Description of the Invention

The present invention relates to a process for obtaining Copaifera langsdorffii matrix microparticles; microparticles themselves and the isolation of the majority compounds of the Copaifera leaves, as well as the biological activities of the obtained matrix microparticles and the isolated compounds of Copaifera, namely: antilithiasis (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretics and antiseptics, as well as their uses, especially in their use in the treatment of renal lithiasis (renal calculi), arthritis, tartar, muscle and spasmodic pain in general (abdominal, renal colic, etc.), inflammation and infections of the urinary tract or other diseases, as well as other diseases related to the previously mentioned activities, besides the present invention contemplate the microparticles themselves, their isolated compounds and the formulations that can be originated with them and / or their isolated compounds, such as: medicines, toiletries, cosmetics, food, veterinary and odo technologies, etc.

Description of the Figures

For a better understanding of the present invention, the following examples characterize it in detail, as well as the figures which may clarify the purposes, applications and claims of the present application. Thus, follow the captions of the figures:

Figure 1: Photographs showing the matrix microparticles obtained in the process described in the present invention, compared to the photographs of Copaifera aerial parts lyophilisate, showing the differences and advantages of obtaining the microparticles.

Figure 2: Schematic procedure for obtaining Copaifera Iangsdorffii Matrix Microparticles;

Figure 3: Fractionation procedure of Standardized Copaifera Extract for compound isolation;

Figure 4: Chromatographic profiles showing the separation of the compounds: (i) quercethrin-3-O-ramnoside (first major) and (ii) canferol-3-O-ramnoside (second major) with methodology described in the isolation example;

Figure 5: Quercethrin-3-O-ramnoside mass spectrum with MS - MS 447 - characterizing compound identification;

Figure 6: Mass spectrum of canferol-3-O-ramnoside compound - characterizing compound identification;

Figure 7: Chemical structures of the quercetrin and kaempferol 3-O-a-L-rhamnopyranoside flavonols isolated from the standardized extract of C. langsdorffii aerial parts;

Figure 8: Chromatographic profile of standardized C. langsdorffii extract (alcohol: water - 7: 3) at 257 nm. 1 - quercethrin-3-O-ramnoside and 2 - canferol-3-O-ramnoside; Figure 9: Chromatographic profiles of samples at 257 nm. A - FCFRP-USP Campus; B - Campus FFCLRP-USP; C - Marilia Region - SP. 2 - quercethrin-3-O-ramnoside, 3 - canferol-3-O-ramnoside and internal standard (pi): benzophenone; Figure 10: Chromatographic profiles of the standardized extracts obtained, (A) by extraction with 7: 3 hydroalcoholic solution; (B) by extraction with 4: 6 hydroalcoholic solution by aqueous extraction; (C) by extraction with water at room temperature and (D) by extraction with boiling water. Figure 11: Kidney and satellite calculations taken from animals with renal lithiasis from each group studied.

EXAMPLES:

EXAMPLE 1 - Obtaining Copaifera langsdorffii Microparticles;

Stage 01 - Obtaining Extracts: The plant material for obtaining the extracts was collected and properly identified, whose exsiccates are deposited in the herbarium of the Department of Biology of the Faculty of Philosophy, Sciences and Letters of Ribeirão Peto / University of São Paulo (FFCLRP / USP) under code SPFR 10120. After collection of plant material (branches and mainly leaves), it was dried and stabilized in a circulating hot air oven at 40 ° C and then subjected to knife milling. previously sterilized. The powder obtained after milling of the plant material was macerated in hydroalcoholic solution in the ratio of 6: 4 or 7: 3 followed by percolation three times, every 72 hours. The extract may also be obtained by infusing the ground vegetable drug with boiling water. The extracts are advantageously PURIFIED by a process consisting of sequential passage in a fixed bed separation system composed of triple layer stationary phase composed of diatomaceous earth, activated magnesium silicate and active charcoal in order to remove pigmented and waxy compounds. which hinder the desired bioactive activity for the present invention. The plant drug, by exhaustive extraction in Soxhlet apparatus, has yield of 20 to 40% of soluble solids, for information. Obtaining Matrix Microparticles: The extracts obtained, as described above, purified, should be added with drying aid, film former or film with suitable pharmacotechnical properties to the subsequent formulation of the drug, such as colloidal silicon dioxide, modified sugars and starches, cellulosic polymers, acrylates, bioadhesive or nonadhesive biopolymers, natural gums and waxes, as well as arabic gum, xanthan gum, chitosan, and others, including mixtures or combinations thereof in any proportions, relative amounts, drug / adjuvant, ranging from one to ten up to ten to one. It is followed by a spray drying process involving the following conditions: temperature 10-450 ° C, air flow 0.01 to 1000 cubic meters per minute and sample flow rate of 0.1 to 1000 liters per hour. The obtained powder characterized by matrix matrix of Copaifera Iangsdorffii presents the following characteristics: particle size between 1 and 200 micrometres, massive spherical morphology, and the presence of the markers and / or active ingredients quercethrin and canferol 3-O-raminoside at concentrations of 2 10% and 0.5 to 10%, respectively. The obtained microparticles may be diluted with various solvents such as hydroalcoholic solutions, water added with preservatives or buffering systems, polyols (sorbitol, propylene glycol, etc.), as well as with excipients or carriers for specific application formulations such as tableting. , capsules, etc.

EXAMPLE 2 - Methodology for Characterization of Standardized Cooaifera Iangsdorffii Extract (used to obtain microparticles)

1 mL of the standardized extract (containing 50 μg benzophenone (Pi) / ml) was collected for filtration through the MiIIex-LCR-PTFE analytical filter (0.45 μηη χ 13 mm) and transferred to an appropriate injector vial. automatic (1 mL).

Next, a 10 pL aliquot of each sample was analyzed by high performance liquid chromatography. A Shimadzu high performance liquid phase chromatograph equipped with SCL-10Avp controller, equipped with three LC-10AD pumps, SPD - MIOAvp diode array detector, automatic injector (SIL-10ADvp) and computerized controller system with Shimadzu CIass-VP version 5.02 software. Two monolithic columns (Onyx ™ 100 X 4.6 mm - C18 Phenomenex) were also serially interconnected and protected by a column of the same material. As mobile phase was used water in phase A and acetonitrile (MeCN) in phase C under flow rate of 1 mL / min. and a multilinear gradient developed as follows: starting at 5-6% phase C in 1 min., 6-8% C (1-2 min.), 8-10% C (2-5 min. .), 10 - 15% C (5 - 12 min.), Maintaining 15% of phase C up to 22 minutes, rising from 15-25% C (22 - 27 min.), Remaining 25%. Phase C up to 35 minutes, increasing from 25 - 40% C (35 - 39 min.), maintaining 40% Phase C up to 42 minutes, rising from 40-100% C (42 - 47 min.), Remaining 100% phase C for 1 minute, plus another 13 min. to return to initial conditions and rebalance the spine. All HPLC chromatographic profiles were obtained at 257 nm absorption. The method was validated in accordance with current standards.

EXAMPLE 3 - Process of Isolation of Compounds from

Copaifera Iangsdorffii

3.1. Fractionation of crude extract by liquid-liquid partition

The standardized extract of Copaifera Iangsdorffii leaves was partitioned with organic solvents in order to isolate their constituents. To this end, 100 g of this extract was solubilized in 1000 mL of methanol-water (9: 1 v / v) and then transferred to the separatory funnel and partitioned 3 times with 400 mL of hexane. In this resulting fraction, called hexane, anhydrous sodium sulfate was added to remove water and then it was filtered into a round bottom flask, where it was concentrated under reduced pressure at 40 ° C with the aid of rotary evaporator. Once concentrated, the hexane fraction was transferred to a previously weighed flask and the gravimetric difference was calculated.

The remaining hydromethane solution was rotary evaporated to remove methanol. Then, to the remaining aqueous solution was added 600 mL of distilled water and the same partition procedure as for the hexane fraction was performed, but using the dichloromethane and ethyl acetate solvents in sequence. The extract fractionation process is summarized in Figure 3.

3.2. By column chromatography with standard silica gel

After pilot work, in which the ethyl acetate fraction was fractionated by CLV, it was observed that it could be fractionated using normal phase silica, but increasing to the stationary phase to improve selectivity. For this, about 8 g of this fraction was solubilized in 50 mL of ethyl acetate and, subsequently, this sample was applied to the top of the normal phase silica, which was packed in a ratio of 1 g: 100 g in a column. 6.5 cm internal diameter glass by 100 cm high. In this procedure the compacted stationary phase measured 75 cm in height. Through this, 40 fractions with a volume of 1 L each were collected, the first six using 100% ethyl acetate, followed by the following gradient: AcOEt: MeOH 9: 1 (fractions 7 to 9); 8: 2 (10 to 15); 7: 3 (16 to 22); 5: 5 (23 to 29). Fraction 30 was eluted with 100% methanol, which was kept until fraction 40 was obtained, completing the procedure. 3.3 Using Sephadex® LH 20-filled column chromatography

In order to isolate the chemical constituents of interest with better purity, to serve as chromatographic standards in the validation of the method and to perform biological assays, it was decided to perform a new fractionation modifying chromatographic procedure, in this case using Sephadex® LH 20 as a stationary phase. For this purpose, a 5 g aliquot of the ethyl acetate fraction was solubilized in 50 mL of methanol P. A. with the aid of a sonicator. Soon after, this sample was filtered through analytical filter (0.45 μm). A 1.0 mL aliquot of the supernatant was placed on the surface of a previously weighed watch glass. This, in turn, was dried for 1 hour in a circulating air oven at 50 ° C. After this procedure, making the gravimetric difference, the relative soluble solids content was calculated, which was 91 mg / mL. Approximately 43 mL of this supernatant was used to perform the chromatographic procedure. Therefore, about 4 g of the sample was applied to the top of the Sephadex® LH 20, which was packed in a 1 g: 100 g ratio on a glass column measuring 3.5 cm internal diameter by 120 cm high. .

Chromatographic separations of compounds having Sephadex® LH 20 as stationary phase are influenced by the mechanism of molecular exclusion. Therefore, the mobile phases used in this technique are usually isocratic. Thus, in this fractionation 159 fractions were collected, 155 with 25 mL volume and 4 with 500 mL volume using methanol as mobile phase.

All subfractions obtained by this methodology were analyzed by CCDC using as a mobile phase a mixture of butanol, acetic acid and water in the ratio of 5: 1: 4 v / v / v. These subfractions were pooled based on the similarity profile, which was observed after the chromatographic plates were developed. UV light at 254 and 366 nm length and 5% sulfuric acid in methanol were used as developers. These meetings resulted in 16 fractions, which were later monitored by HPLC.

3.4 Purification of samples obtained by column chromatography

Through CCDC it was possible to select promising fractions obtained from the methodologies described above. Thus these fractions were purified by HPLC in the preparative mode, aiming to obtain the compounds of interest in adequate amounts for the development of the analytical method. For this purpose, a Shimadzu-brand chromatograph consisting of two Shimadzu LC-6A pumps, Shimadzu SPD-6AV UV / VIS spectrophotometric detector, SCL-IOAvp controller system, connected to a computer with Shimadzu CIass-VP version 5.02 software and SIL- 10ADvp. Semi-preparative reverse phase chromatographic column CLC-ODS (M) - Shimadzu, 10 mm x 250 mm, particle diameter 5μm and C18 reverse phase precolumn manufactured by the same company. As mobile phase was used water in phase A and methanol in phase B, under flow rate of 6 mL / min. and a gradient starting at 20-60% phase B (0-15 min.), maintaining 60% phase B up to 25 minutes, rising from 60-100% B (25-35 min. ), remaining 100% phase B for up to 37 minutes, plus another 8 min. to return to initial conditions and rebalance the spine.

EXAMPLE 4 - Iangsdorffii Copaifera Isolated Compounds

The study of the phytochemistry of Copaifera Iangsdorffi Standardized Extract (obtained from the aerial parts) showed that its major constituents, approximately 60%, are polar, differing from the chemical composition of the resin oil. The chemical structures of isolated and purified metabolites were elucidated using spectroscopic methods such as 1H and 13C1 IR Nuclear Magnetic Resonance spectroscopy and mass spectrometry, as well as two-dimensional spectrometric techniques such as HMBC, HMQC and NOESY. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on Brucker-Advance DRX400 spectrometers operating at 400 MHz (1 H NMR) and 100 MHz (13 C NMR) using deuterated Aldrich solvents. In addition, other methods can be used to assist in identification such as melting point determination and [a] D measurement. Figures 4, 5, 6 and 7 show the characterization of the structures (i) quercethrin-3-O-ramnoside and (ii) canferol-3-β-ramnoside.

Table 01: 1 H and 13 C NMR data obtained for quercethrin.

<table> table see original document page 13 </column> </row> <table>

* Signals may be switched. Markham et al., 1978.8 Kosuge et al., 1985.

Table 02: HeC NMR data obtained for canferol 3-O-α-L-ramnopyranoside.

<table> table see original document page 13 </column> </row> <table> <table> table see original document page 14 </column> </row> <table>

Nugroho et al „2009. 1H and C13 = DMSO-d6

EXAMPLE 5 - Standardization of Copaifera langsdorffii- Extract to be used to obtain matrix microparticles

The standardization of the extracts that will be passed through the specific process to obtain the matrix microparticles involves the identification and quantification of at least the compounds: (i) quercethrin-3-O-ramnoside and (ii) canferol-3-O-ramnoside (figure 7), which should be in the range 0.25% to 2.0% corresponding to the plant drug or 120 to 460 μg / mL in the extracts. The pharmacological effects of the present invention consist of: antilithiasis (renal calculus), analgesic, antispasmodic, anti-inflammatory, diuretic and antiseptic activity, as well as their use in the treatment of renal lithiasis (renal calculus), arthritis, tartar, muscle and spasmodic pain in general (abdominal cramps, kidney, etc.) and other diseases related to the activities previously mentioned. Formulations which may be derived from Copaifera langsdorffii matrix microparticles and / or their isolated compounds, such as medicines, toiletries and cosmetics, food, veterinary and dental products, etc. should consider the concentration of the isolated compounds in the daily dosage to be defined for each product. Thus, the Copaifera matrix microparticles should be inserted in the respective formulations taking into consideration their standardization and the desired final concentrations so that the final objectives are met, and the effective dosages of the final product are in the range of 5 to 20g of the product. plant material and their counterparts in standardized extracts and markers as detailed below for antilithiasis, antiseptic activities; anti-inflammatory; antispasmodic; analgesic, etc.

EXAMPLE 6 - Antilithiasis Activity and Safety in a Preclinical Model

Considering the need for a drug for the treatment of nephrolithiasis and the great potential of C. langsdorffii, Copaifera langsdorffii matrix microparticles were evaluated in an experimental rat lithiasis model. In this model, lithiasis was induced in the animal by introducing a calcium oxalate tablet into its bladder, which serves as a niche for bladder stone growth. The protocol lasted 48 days, and in the last 18 days the animals were treated with C. langsdorffii matrix microcapsules at a dose of 20 pg / g body mass / day (concentration referring to dry mass of plant material), diluted. in 1 mL of water by gavage. The control group underwent the same protocol, except for extract administration in the last 18 days (BARROS et al, 2006). At the end of the experiment, no significant biochemical changes were observed between the control and treated groups, that is, the administration of C. Iangsdorffii matrix microparticles did not change urinary volume, urinary excretion and serum sodium, potassium and creatinine concentration. However, in the group of treated animals, there was a reduction in total mass (0.286 ± 0.042 vs. 0.164 ± 0.066) and in the total number of stones formed (25.25 ± 6.96 vs. 7.40 ± 3.67 *, P <0.05). In addition, comparing the hardness of the calculations quantitatively, it was observed that the pressure required to disintegrate the calculations of the control group animals is at least twice as high as that required to disintegrate the calculations of the treated group (6.90 ± 3; 45 vs. 3.00 ± 1.51). Thus, it was found that the treatment not only reduces the number and total mass of formed stones, but also modifies their morphology, reinforcing the therapeutic action of C. Iangsdorffii matrix microparticles in the treatment of renal lithiasis.

<table> table see original document page 15 </column> </row> <table> <table> table see original document page 16 </column> </row> <table>

EXAMPLE 7 - Demonstration of the Safety of Copaifera Iansdorffii Matrix Microparticles in Cytotoxicity and Mutagenic Potential Models

C. langsdorffii matrix microparticles were evaluated for mutagenic and / or antimutagenic potential by micronucleus testing (preclinical safety assessment test) in peripheral blood of Swiss mice. The results showed no cytotoxicity of C. langsdorffi matrix microparticles. In addition, an antimutagenic effect was observed when the animals were simultaneously treated with different doses of C. langsdorffi microparticles (10, 20, 40 and 80 mg / kg - concentration corresponding to the dry mass of plant material) and doxorubicin (15 mg / kg ).

The C. Iangsdorffii matrix microparticles were also evaluated for mutagenic and / or antimutagenic potential in V79 cells (hamster lung fibroblasts) also by the micronucleus test. The results showed that C. langsdorffi microparticles did not show mutagenic activity at concentrations of 30, 60 and 120 pg / mL (concentration corresponding to the dry mass of plant material) and had a chemopreventive effect when combined with methyl methanesulfonate (400 μΜ ).

EXAMPLE 8 - Demonstration of antioxidant activity of Copaifera Iansdorffii Extracts "in vitro". The antioxidant activity was evaluated using the Brand-Williams (1995) method, which uses the free radical 2,2-diphenyl-1-picrylhydrazine (DPPH). Copaifera langsdorffii extracts, obtained according to example 1, ie, aqueous extracts (1), (2) 4: 6 hydroalcoholic solution (ethanol: water) and (3) 7: 3 hydroalcoholic solution (ethanol: water), were diluted. in ethanol to a concentration of 50 pg / mL. In test tubes, 1 ml of sodium acetate / 0.1 M acetic acid buffer, pH 5.5; 1 mL of 125 M DPPH solution and 50 μί sample. After 30 minutes of rest, the samples were measured by spectrophotometer at 517 nm. The 50% inhibition concentration (IC50) of the DPPH radical was used to compare the antioxidant activity between the samples. The results showed that the IC50 found for the standardized aqueous extract of Copaifera langsdorffii (1) was 0.8518 Mg / mL, while for the extract (2) it was IC50 = 0.5281 pg / mL and for (3). IC 50 = 0.4716 pg / mL. When the antioxidant activity of the same extracts was quantified through the total flavonoids of the extracts, we obtained: (i) IC50 = O1IOO Mg / mL for the standardized extract (aqueous); (ii) IC50 = 0.0955 pg / mL (40% hydroalcoholic solution) and (iii) IC50 = O11534 pg / mL for the extract obtained with 70% hydroalcoholic solution, demonstrating the antioxidant power of Copaifera langsdorffii, through the proposed methodology. , material used in the present invention.

EXAMPLE 9 - Demonstration of Preclinical Safety of Copaifera langsdorffii Matrix Microparticles - Preclinical Research

Toxicological tests were carried out for the acute and sub-chronic phases of the Copaifera matrix microparticles using Wistar and Rabbits rats. The results obtained for both protocols showed that the treatment of animals at doses three times higher than the recommended doses for humans, did not cause any change in biochemical, hematological and histopathological parameters in the studied animals. It is noteworthy that it was not possible to obtain the 50% lethal dose, which demonstrates the safety of the therapeutic index (report attached to the CEP / FAMEMA protocol under No. 128/05).

EXAMPLE 10 - Demonstration of Clinical Safety of Copaifera Iangsdorffi Matrix Microparticles - phase I clinical research

A Phase I clinical study was performed to evaluate the safety of use of Copaifera Iangsdorffii Microparticles in 28 healthy volunteers. For this purpose, laboratory tests were performed on renal function (creatinine, urea and urine I), liver (TGO, TGP, gamma-GT and prothrombin time) and cardiac (pressure and frequency) before and after product ingestion. The volunteers had an average age of 24.1 years (SD = 2.6 years), being 9 (32.1%) females and 19 (67.9%) males. The results found for the physical examinations of the volunteers demonstrated the absence of alterations after the administration of the microparticles. The average results were: 111.4 mmHg (SD = 12.4) χ 74.4 mmHg (SD = 8.3) for blood pressure, 72.2 bat./min (SD = 10.9) for heart rate, 18.0 (SD = 0.4) for RF, 36.2 (SD = 0.4) for body temperature , 70.1 kg (SD = 12.6) for body weight and 170.0 cm (SD = 10.2) for height. Laboratory test results for changes in renal and hepatic function showed normal results, both before and after ingestion of Copaifera microparticles, for all parameters. Blood and urine I results were normal before and after administration of Copaifera Microparticles for all parameters. The results obtained with healthy volunteers after routine medical procedures (history taking, physical examination and complementary laboratory tests for renal and hepatic functions - blood and urine) showed that the ingestion of Copaifera microparticles, equivalent to a daily intake of 10g of the material. vegetable (divided into three shots) in short-term treatment was devoid of toxic effects for the volunteers who participated in the study.

EXAMPLE 11 - Demonstration of Clinical Effectiveness of Copaifera Ianpsdorffi Microparticles - Phase II Clinical Research

In a phase II clinical study, regarding efficacy, preliminary data demonstrated the great potential for therapeutic use of the product, since of eight patients with urolithiasis, 3 (37.5%) demonstrated complete elimination of the stone, 3 (37.5) demonstrated a decrease in diameter and alteration of the stone coloration and in 2 (25%) patients, the treatment was not satisfactory. Considering that the clinical study protocol addressed three distinct treatments, namely: (i) 1 dose + 1 dose, where patients ingested a dose equivalent to 10 g of plant material (divided into three shots / day) for three days, underwent reevaluation. doctor and again received the equivalent of 10g of plant material (divided into three shots / day) to be administered for three days; (ii) 2 doses + 1 dose, 10 g of plant material (divided into 3 shots / day) being administered for a period of six days and after a medical evaluation the administration of 10 g / day (divided into three shots / day) by another three days, and finally (iii) the administration of 10g (divided into three shots / day) for six days, followed by a medical evaluation, product administration for a further three days, a new evaluation and finally the treatment by Three more days. Thus, the data obtained by only eight patients can already infer satisfactory results, since 75% had satisfactory results, being 37.5% with complete elimination of the calculus, results that are difficult to obtain in conventional treatments and that point to the success of the product. .

Brief Description of the Invention

The invention is characterized by the obtaining and use of Matrix Microparticles of Copaifera langsdorffii, and isolated substances (aerial parts) of Copaifera, mainly Copaifera langsdorffii Der., But also of other species such as C. beyrichii Hayne .; C. bijuga Hayne .; C. bracteata Bth .; C. confertiflora Bth .; C. coriaceae M .; C. duckei Dwyer .; C. elliptical M .; C. glabra Vog .; C. guianensis Def .; C. jacquini Def .; C. jacquimana Def .; C. jussieuii Hayne .; C. laxa Hayne .; C. luetzelburgii Harms .; etc.

Thus, the present invention relates to a process of obtaining Copaifera langsdorffii Matrix Microparticles and the isolation of their compounds, as well as the biological activities of the microparticles and the isolated compounds of the plant, namely: antilithiasis activity (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic agents, as well as their uses, especially in their use in the treatment of renal lithiasis (renal calculus), arthritis, tartar, muscle and spasmodic pain in general (abdominal cramps, etc.), urinary tract or other inflammations and infections, as well as other diseases related to the aforementioned activities, in addition to the present invention contemplate the matrix microparticles themselves, their isolated compounds and the formulations that may originate with them and / or their isolated compounds, such as: medicines, toiletries and cosmetics, there veterinary and dental products, etc.

It is noteworthy that, as already presented in the prior art, there is currently no medicine obtained from the medicinal plant, endowed with inventive activity, that is, that uses scientific discoveries to obtain a technological product that is efficient in disintegrating. the structure of the calculation. The procedures used are the hydration of the patient, aiming at the elimination of calculus and palliative treatments, and the plants studied did not culminate in obtaining drugs or efficient products for the treatment of lithiasis.

It is also worth mentioning that the process of obtaining the matrix microcapsules of Copaifera langsdorffii provides obtaining technological advantageous system in relation to the lyophilisates of the conventionally obtained extracts, not only regarding the biological activity of the product but also in relation to the industrial technological process. The microparticles advantageously offer homogeneous, spherical, solid, more chemically stable particles in relation to lyophilized extracts, microencapsulated systems capable of offering modified release, and easier to obtain pharmaceutical preparations such as tablets, capsules, solutions, etc. Lyophilisates, in turn, are heterogeneous systems, subjected to oxidation and degradation processes of bioactive chemical compounds previously extracted from the plant drug, which do not have fluidity, flowability and are difficult to handle pharmacotechnically. In addition, the extracts used as material for obtaining the microcapsules require purification step, which consists of the passage of the extract in a sequential fixed bed separation system composed of triple layer stationary phase consisting of diatomaceous earth, activated magnesium silicate and charcoal. active for removal of pigmented materials and waxy compounds that make it difficult to obtain the desired biological effect. Thus, the present invention is socially and economically relevant, endowed with inventive activity and perfectly applicable industrially, justifying the present application for industrial property.

Detailed Description of the Invention

Aerial parts, leaves and branches of Copaifera species are submitted to the drying process in hot and circulating air oven at 40 ° C and then subjected to knife milling until fine powder is obtained. The powder thus obtained is then subjected to extraction processes by cold or hot maceration in water or 4: 6 or 7: 3 hydroalcoholic solution (ethanol: water). The extracts are filtered on filtering canvas to separate the vegetable pie. After filtration, the extract is sequentially purified in a triple layer stationary phase fixed bed separation system consisting of diatomaceous earth, activated magnesium silicate and active charcoal. Specifically, the process of the present invention comprises the steps of:

(a) drying aerial parts, leaves and branches of Copaifera species in a circulating hot air oven at 40 ° C;

b) grinding the dry material in a knife mill to fine powder;

c) extraction by cold or hot maceration in water or 4: 6 or 7: 3 hydroalcoholic solution (ethane-water);

d) filtration of the extracts in filtration canvas to separate the vegetable pie;

e) a fundamental purification step consisting of sequential passage in a fixed bed separation system composed of a triple layer stationary phase consisting of diatomaceous earth, activated magnesium silicate and active carbon.

g) the standardized extracts obtained by (i) extraction in water (hot or cold), maceration followed by percolation in hydroalcoholic solutions in the proportions (ii) 4: 6 or (iii) 7: 3 (ethanol: water) are transformed in matrix microcapsules, by the addition of a drying aid, film-forming or film-forming agent with suitable pharmacotechnical properties to the later formulation of the medicament, such as colloidal silicon dioxide, modified sugars and starches, cellulosic polymers, acrylates, bioadhesive or non-gum biopolymers, natural gums and waxes, such as gum arabic, xanthan gum, chitosan and the like, including mixtures or combinations thereof in any proportions, relative amounts, drug / adjuvant, ranging from one to ten to ten to one. It is followed by a spray drying process involving the following conditions: temperature 10-450 ° C, air flow 0.01 to 1000 cubic meters per minute, sample flow rate from 0.1 to 1000 liters per hour. The microparticles are characterized by the chemical profiles shown in Figure 10, and also by having the major compounds quercethrin-3-O-ramnoside (first major) and (ii) canferol-3-β-ramnoside (second major) in their respective ranges. concentrations: (i) 30 - 100 mg / g and 10 - 60 mg / g; (ii) 20-200 mg / g and 10-150 mg / g and (iii) 10-90 mg / g and 5-50 mg / g. g) Process of obtaining the majority isolated compounds of Copaifera, which involves (Figure 3): fractionation of standardized extracts obtained from aerial parts of Copaifera species through filtration processes in columns packed with silica gel-based stationary phases or obtaining the fractions rich in the active principles by liquid-liquid partition processes from the hydroalcoholic extract of Copaifera species branches, using hexane, dichloromethane and ethyl acetate in sequence; fractionation of the extract in ethyl acetate using a silica gel based stationary packed column; purification of the major component-rich fraction obtained by fractionation of the ethyl acetate extract using high performance liquid chromatography (HPLC) 1 providing the major compounds quercethrin-3-O-ramnoside (first major) and canferol-3 -Nos- ramnoside (second major) (figure 7) according to chromatogram obtained in HPLC figure 4, as well as other minority phenolic compounds. Pointing out that the extracts can be fractionated by filtration processes on stationary phase packed columns such as: silica gel, activated magnesium silicate, reverse phase silica (C-8, C-18), Sephadex LH20, etc. Fractions may also be obtained from partitioning processes using organic solvents as shown in Figure 3. Organic solvents are removed in vacuum concentrators and the aqueous phases are lyophilized. (h) Copaifera matrix microparticles may be used as such or conveyed in suitable preparations (medicines, toiletries and cosmetics, veterinary, dental or other products), in treatments of three to 28 days for each adult patient for which three daily doses of microparticles, corresponding to 5 to 20 g of dry plant material each, equivalent to the dose of 1.0 to 5.40 g of C. Iangsdorffii (aqueous) microparticles, 1.0 to 6, are used. 0 g of microparticles (obtained with 40% alcohol) and 1.20 to 7.0 g of microparticles (obtained with 70% alcohol), with the respective markers guercethrin-3-O-ramnoside (first majority) and canferol-3 O-ramnoside (second majority). For animal treatment, 1.50 to 80 mg / kg body weight / day of microparticles should be employed. i) Matrix Microparticles and / or their isolated compounds, whether or not contained in solvents or excipients or carriers of drug formulations, personal care and cosmetic products, veterinary, dental or other products for the treatment of renal lithiasis (renal calculus), arthritis , tartar, muscle and spasmodic pain in general (abdominal cramps, kidney, etc.), inflammation and infections of the urinary tract or others, as well as other diseases related to the activities previously mentioned, due to the biological activities of the extract and its isolated compounds which are: antilithiasis (renal calculus), analgesic, antispasmodic, antiinflammatory, diuretic and antiseptic activity.

j) There are no medicines on the market, whether herbal or allopathic, that are effective in disintegrating kidney stones. Thus, the products of the present invention will promote the development of an unprecedented product, as the existing ones act as diuretics, anti-inflammatory drugs, analgesics and, at most, as prophylactic.

Claims (8)

1. Process of obtaining Copaifera Iangsdorffii matrix microparticles (aerial parts) and isolation of Copaifera active ingredients and / or markers; microparticles and compounds thus obtained, with antilithiasis (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic activity, their formulations, products and uses, characterized by the following steps: a) drying of the aerial parts, leaves and branches of Copaifera species in hot air circulating at 40 ° C; b) grinding the dry material in a knife mill to fine powder; c) extraction by cold or hot maceration in water or 4: 6 or 7: 3 hydroalcoholic solution (ethane-water); d) filtration of the extracts in filtration canvas to separate the vegetable pie; e) purification of the extracts sequentially in a fixed bed separation system composed of a triple layer stationary phase consisting of diatomaceous earth, activated magnesium silicate and active carbon; (f) Obtaining the Matrix Microparticles by drying the extracts added, preferably, but not exclusively, from drying aid, film-forming agent or film having suitable pharmacotechnical properties for the subsequent formulation of the medicinal product, such as colloidal silicon dioxide, modified sugars and starches, cellulosic polymers, acrylates, bioadhesive or nonadhesive biopolymers, natural gums and waxes, such as gum arabic, xanthan gum, chitosan, and the like, including mixtures or combinations thereof in any proportions, in relative amounts, drug / adjuvant, ranging from one to ten to Ten to one. It is followed by a spray drying process involving the following conditions: temperature 10-450 ° C, air flow 0.01 to 1000 cubic meters per minute, sample flow rate from 0.1 to 1000 liters per hour; Process according to Claim 1, characterized in that the fractions are obtained by partitioning processes using organic solvents, which are eliminated in vacuum concentrators and the aqueous phases are lyophilized; Process according to Claim 1, characterized in that the Copaifera species is preferably Copaifera Iangsdorffii Def .; Process according to Claim 1, characterized in that the Copaifera species are C. beyrichii Hayne .; C. bijuga Hayne .; C. bracteata Bth .; C. confertiflora Bth .; C. coriaceae M .; C. duckei Dwyer .; C. elliptical M .; C. glabra Vog .; C. guianensis Def .; C. jacquini Def .; C. jacquimana Def .; C. jussieuii Hayne .; C. langsdorffii Def .; C. Iaxa Hayne .; C. Iuetzelburgii Harms .; C. martii Hayne .; C. nitida Hayne .; C. oblongifolia M .; C. officinalis L .; C. publiflora Bth .; C. reticulata Ducke .; C. rigid Bth .; C. rondonii Hoehne .; C. sellowii Hayne; C. trapezifolia Hayne, among others; Matrix microparticles obtained by claim -1, the extracts being obtained to obtain the microparticles obtained by (i) extraction in water (hot or cold), maceration followed by percolation in hydroalcoholic solutions in the proportions (ii) 4: 6 or (iii ) 7: 3 (ethane-water), with subsequent addition of drying and microencapsulation adjuvants, followed by spray drying, characterized by chemical profiles containing the markers quercethrin-3-O-ramnoside and canferol-3-O-ramnoside Matrix microparticles obtained by claim -1, being the extracts for obtaining the microparticles obtained (i) extraction in water (hot or cold), maceration followed by percolation in hydroalcoholic solutions in the proportions (ii) 4: 6 or (iii ) 7: 3 (ethanol: water), with subsequent addition of drying and microencapsulation adjuvants, followed by spray drying, characterized by having the major compounds quercethrin-3-O-ramnoside (first major) and (ii) canferol-3 -O-ramnoside (second largest) in the respective concentration ranges: (i) 30-100 mg / g and 10-60 mg / g; (ii) 20-200 mg / g and 10 -150 mg / g and (iii) -10-90 mg / g and 5-50 mg / g. The use or uses of the matrix microparticles, objects of claim 1, 3, 4, 5 and 6, used as such or conveyed in suitable preparations (medicaments, toiletries and cosmetics, veterinary, dental or other products) , for applications in the treatment of renal lithiasis (renal calculi), arthritis, tartar, muscle and spasmodic pain in general (abdominal, kidney, etc.), urinary tract or other inflammation and infections, as well as other activity-related diseases previously mentioned, due to the biological activities of microparticles and C. langsdorffii isolated compounds, namely: antilithiasis (renal calculus), analgesic, antispasmodic, anti-inflammatory, diuretic and antiseptic activity; 8. Isolation process and obtaining the major compounds of quercethrin-3-O-ramnoside and canferol-3-O-ramnoside, characterized by the steps: fractionation of standardized extracts obtained from aerial parts of Copaifera species through filtration processes. in columns packed with silica gel-based stationary phases or obtaining the fractions rich in the active principles by liquid-liquid partition processes from the hydroalcoholic extract of the Çopaifera species branches, using hexane, dichloromethane and ethyl acetate in sequence; fractionation of the extract in ethyl acetate using a silica gel based stationary packed column; purification of the fraction rich in major components, obtained by fractionation of the extract in ethyl acetate, using high performance liquid chromatography (HPLC), providing the major compounds quercethrin-3-O-ramnoside (first major) and canferol-3 -O-ramnoside (second major) (figure 7) according to HPLC chromatogram, figure 4, as well as other minority phenolic compounds. Pointing out that the extracts can be fractionated by filtration processes on stationary phase packed columns such as: silica gel, activated magnesium silicate, reverse phase silica (C-8, C-18), Sephadex LH20, etc. The fractions obtained, rich in the active ingredients, may be used in doses corresponding to their equivalent contents in 10 g of the dried plant material. Fractions may also be obtained from partitioning processes using organic solvents as shown in Figure -3. Organic solvents are removed in vacuum concentrators and aqueous phases are lyophilized; (I) quercethrin-3-O-ramnoside (first major) and (ii) canferol-3-O-ramnoside (second major) compounds, as claimed in claim 7, for applications in the treatment of renal lithiasis (renal calculus); arthritis, tartar, muscle and spasmodic pain in general (abdominal cramps, kidney, etc.), inflammation and infections of the urinary tract or other, as well as other diseases related to the activities mentioned above, due to the biological activities of the extract and its compounds. isolated, namely: antilithiasis (renal calculus), analgesic, antispasmodic, antiinflammatory, diuretic and antiseptic activity, directly or in various pharmaceutical compositions, such as solutions, tablets, capsules, or others.