BRPI1000802B1 - process of obtaining langsdorffii copaiferous matrix microparticles (aerial parts) and langsdorffii copaiferous matrix microparticles - Google Patents

Abstract

Process of obtaining matrix microparticles of copaiferous langsdoffíi (aerial parts) and isolation of the active ingredients; microparticles and compounds thus obtained, with antilitiásica (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic activity, their formulations, products and uses. The present invention relates to a process of obtaining matrix microparticles of Copaifera Iangsdorffii (aerial parts) and of 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, antispasmodic, anti-inflammatory, diuretic and antiseptic, as well as the use of these in the treatments 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

[001] The present invention refers to a process of obtaining matrix microparticles of Copaifera langsdorffii (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 microparticles matrix and isolated compounds, which are: antilitiásica (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic activity, as well as their use in the treatment of kidney stones (kidney stones), arthritis, tartar , muscle and spasmodic pain 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 originate with the matrix microparticles of Copaifera langsdorffii ', and / or its isolated compounds, such as medicines, personal hygiene products, cosmetics, food, veterinary products and dental, etc.

BACKGROUND OF THE INVENTION

[002] Currently, the world herbal medicine market represents a significant share in the medicines sector, with a global turnover of US $ 21.7 billion per year. The main movement of the world herbal medicine market is concentrated in Europe, with 50% in Germany. Then, Asia and Japan appear. However, no other market grows as much as the North American market (CALIXTO, 2000). In Brazil, there are no updated official data, but it is estimated that this market is around 160 million dollars per year (CARVALHO et al., 2008).

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

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

[005] Phyllanthus niruri is a plant used in folk medicine to treat urolithiasis. It was previously demonstrated that P. niruri does not present toxicity, increased the elimination of stones in lithiasic patients, reduced the growth of bladder stones in an experimental model in rats and inhibited the 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 CaOx crystallization in vitro (0.25 mg / mL of urine) was evaluated. The presence of P. nirurinão inhibited CaOx precipitation and even a greater number of crystals was observed, although of a smaller size. In addition, in the presence of P. niruri there was a predominance of crystals in the dihydrated form, which is less adherent. When the crystals were observed 24 hours after crystallization, there was a great aggregation in the absence of P. niruri, and in its presence the aggregation was inhibited. The results obtained demonstrate 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 calculus (BARROS, 2002).

[006] In addition to the stone-breaking 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 approved biological activity as a urine antiseptic. Thus, it is indicated for patients who have cystitis and often also for those who have renal lithiasis, however, being only a compound for relief and asepsis of the urinary tract, with no anti-lithiasis effect.

[007] The wide variety of species popularly indicated for the 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 intend to establish how true these properties are. An example of this is the absence in Brazil of patents related to active ingredients and / or drugs capable of effectively exercising such action.

[008] Currently, in the INPI patent application bank there are only four processes related to kidney stones, being: - one (i), of American origin, addresses the use of compounds obtained from "rare earth", basically the base lanthanum, with chelation properties of oxalate in the gastrointestinal tract, and for that reason they are preventive of the formation of kidney stones by preventing the absorption of oxalate (PI 0209072-4 - Application of rare earth compounds for the prevention of kidney stone) ', - another (ii), of Chinese origin, addresses the use of hydrochloric acid solution (HCI) for the treatment of hypertension and calculations in general (PI0608022-7 A2 - Process for the manufacture of a drug for the manufacture of a drug to treat hypertension, anti-hypertension formulation and process to manufacture a medicine to treat calculus); - a third (iii), of Brazilian origin, entitled “Process of obtaining and using extracts, fractions and substances isolated from Copaifera species in the treatment of urinary lithiasis in humans and animals” (PI0401631-9 A2), deposited in 22 / 04/2004; - and finally, (iv) a concession to an order of Japanese origin, entitled “Derivatives of amino-ethyl-phenoxy-acetic acid and drugs for the remission of pain and promoting the removal of stones in urinary lithiasis” (PI9811483- 2 B1), with deposit made on 07/15/1998 and the concession in January 2010.

[009] It is noticed that there is no request for a technological product, endowed with inventive step regarding the use of derivatives of plant origin, as advantageously is our case, since the request referring to Copaifera species, according to PI0401631- 9 A2, described above, is not eligible for concession, since it is a discovery, as well established in the Industrial Property Law. Since as of knowledge, teas, plant drugs, extracts are not endowed with inventive step, but are subject to scientific discoveries, and these can be used as material for technological development and generation of inventions that, if well characterized and carefully evaluated, they can generate benefits to society. In addition, the orders related to that described in item (i) and (ii), detailed above, are orders aimed at chelating action of oxalate and, therefore, only of preventive character, completely different from the objective of the present invention, which advantageously is a technological product, with antilitiásica, anti-inflammatory, analgesic, antispasmodic, antiseptic and diuretic activities, having a medicinal and curative character.

[0010] The last request (iv) mentioned above is about an innovative molecule obtained by chemical synthesis and its medications, which is not comparable to the invention object of the present petition. The current order, on the other hand, involves the transformation of a discovery into an innovative and revolutionary technological product for the pharmaceutical industry, that is, it transforms what the present team discovered with the Copaifera plant into an unprecedented pharmaceutical input, which can be used in pharmaceutical, cosmetic and veterinary

[0011] Because the treatment of nephrolithiasis is expensive and in many cases invasive and with many side effects, it is considered important to search for alternatives to these conventional methods using herbal medicine. In fact, in many countries medicinal herbs are used that appear to cure patients with urolithiasis. However, the effectiveness and the mechanism by which these plants work 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 the formation of kidney stones (ATMANI, 2003; MACIEL et al, 2002) and that such scientific discoveries be transformed into technological development, as the present invention, so that such innovative products are applied effectively in the treatment of diseases and generate benefits for the community and the country.

[0012] Among the plants for medicinal and traditional use is Copaifera sp, a native Brazilian plant that has been widely used since the 16th century. The literature provides several studies of biological activities of oil-resin from different genera of Copaifera sp (CARVALHO, 2004; VEIGA JÚNIOR, 2005; PAIVA et al., 1998; 2003; 2004; BASILE et a /., 1988; COSTA-LOTUFO et al., 2002), as its healing, antiseptic and anti-inflammatory action on the respiratory tract (CARVALHO, 2004). However, studies on Copaifera sp leaves are scarce in the literature except for the study of their constituents in ecological interactions (LANGENHEIM et al., 1981; 1986a and 1986b).

Copaifera langsdorffii

[0013] The species Copaifera langsdorffi belongs to the botanical family Leguminasae, sub-family Caesalpinioideae and may be bushes or leafy trees from 15 to 25 m. This species is found in the north, northeast, southeast, central-west and part of the southern region of Brazil, as well as in Venezuela, southern Bolivia, northeast Argentina, and northern Paraguay. Other species of the Copaifera genus are distributed throughout South and Central America, with citations occurring in the western part of Africa (CARVALHO, 2004; VEIGA JÚNIOR, 2005; VEIGA JÚNIOR and PINTO, 2002).

[0014] 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 both in Portuguese and, eventually, in other languages. In particular, C. langsdorffi is called red copaiba, red oil or copaiba da várzea 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 names that can be found in the literature (CARVALHO, 2004; VEIGA JÚNIOR, 2005; VEIGA JÚNIOR and PINTO, 2002).

[0015] In the literature it is said that the oil-resin of the species of C. langsdorffi has as main chemical constituents tetracyclic diterpenes of the kauran series as kaurenoic and kauranoic acids (FERRARI et al., 1971), clerodanes as hardwickiic acid (OHSAKI et al., 1994) and labdanic diterpenes such as copalic acid (GASCON and GILBERT, 2000; MACIEL et al., 2002) among others, as well as sesquiterpenes, predominantly / 3-karyophylene, β-bisabolene, orbergamotene and / 3- selineno (CARVALHO, 2004; VEIGA JÚNIOR, 2005; VEIGA JÚNIOR and PINTO, 2002).

[0016] In the chemical composition of Copaifera langsdorffi leaves, 3% of the amino acid, N-methyl-trans-4-hydroxy-L proline and sesquiterpenes such as cr-cubebene, cr-copaene, cyperene, / 3-copaene are presented , karyophylene, / 3-humulene, muurolene, / 3-selinene, õ-cadinene and y-cadinene (LANGENHEIM et al., 1986a; MACEDO and LANGENHEIM, 1989a; MACEDO and LANGENHEIM, 1989b).

[0017] Among the most studied pharmacological activities for Copaifera sp (oil-resin) are: anti-inflammatory, analgesic, antibacterial, anti-tumor, antioxidant, healing and gastroprotective, anthelmintic, leishmanicide, trypanomycin, and muscle relaxant (CARVALHO, 2004 ; VEIGA JÚNIOR, 2005; VEIGA JÚNIOR and PINTO, 2002).

[0018] The scientific literature does not include any data referring to the study of Copaifera langsdorffii leaves, here residing the main discovery used as material for the present invention. Thus, based on this, the present proposal involved the study of processes for obtaining matrix microparticles containing extracts from the leaves of Copaifera langsdorffii, phytochemical processes for the isolation and identification of their chemical compounds, the study of their effectiveness and safety, aiming at obtaining pharmaceutical supplies, medicines, products for medical, veterinary, dental use, among others.

[0019] Studies carried out by our group have shown that the standardized extract of C. langsdorffii and / or its isolated substances, obtained from the aerial parts of Copaifera, by the processes described here, have antilitiásica, anti-inflammatory, analgesic, antispasmodic, anti-spasmodic properties. septic and diuretic, an 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 that contain 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, homogeneity of the system, 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, in addition to offering larger particles, with great granulometric variation, deformed and difficult to handle pharmacotechnically. Still, it is noteworthy that the chemical composition of the aerial parts of Copaifera sp is completely different from that observed for copaiba oil, which is obtained from the stem of the plant. 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 with what has already been described for copaiba oil, although it is not indicated for the treatment of lithiasis.

[0020] Thus, several studies have been carried out as demonstrated in the examples presented below to demonstrate the claims of the present application, such as: (a) description of the process of obtaining the standardized extracts that will be processed and transformed into microparticles; (b) the process of obtaining the matrix microparticles of the aerial parts of Copaifera langsdorffif, (c) of the microparticles themselves; (d) the process of isolating compounds, characterizing and identifying such substances; (e) pre-clinical and (preliminary) safety and efficacy. In addition, an antilitiásica activity study was carried out comparing the activity of the matrix microparticles of Copaifera langsdorffii (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 from C langsdorffii, were much higher than the previous ones, demonstrating the relevance of the present application for industrial protection.

Description of the Invention

[0021] The present invention relates to a process of obtaining matrix microparticles of Copaifera langsdorffif, microparticles themselves and the isolation of the major compounds from Copaifera leaves, as well as the biological activities of the obtained matrix microparticles and the isolated compounds of Copaifera , namely: antilitiásica (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic activity, as well as their uses, especially in the use of these in the treatment of renal lithiasis (renal calculus), arthritis, tartar , muscle and spasmodic pains in general (abdominal, renal cramps, etc.), inflammation and infections of the urinary tract or others, as well as other diseases related to the previously mentioned activities, in addition to the present invention to include the microparticles themselves, their isolated compounds and the formulations that can be originated with the same and / or their isolated compounds, such as: medicines, personal care products, cosmetics, food, veterinary and dental products, etc.

Description of the Figures

[0022] For a better understanding of the present invention, the examples presented below characterize it in detail, as well as the figures that can clarify the objectives, applications and claims of the present application. Thus, the captions of the figures follow: - Figure 1: Photographs showing the matrix microparticles obtained in the process described in the present invention, in comparison with the photographs of the lyophilisate of the extract of the aerial parts of Copaifera, showing the differences and advantages of obtaining the microparticles . - Figure 2: Schematic procedure for obtaining the Matrix Microparticles of Copaifera langsdorffif, - Figure 3: Fractionation procedure of the Standardized Copaifera Extract for isolation of compounds; - Figure 4: Chromatographic profiles showing the separation of the compounds: (i) quercetrin-3-O-ramnoside (first major) and (ii) canferol-3-O-ramnoside (second major) with methodology described in the example regarding isolation; - Figure 5: Mass spectrum of quercetrin-3-O-ramnoside with EM - EM 447 - featuring the identification of the compound; - Figure 6: Mass spectrum of the compound canferol-3-O-ramnoside - characterizing the identification of the compound; - Figure 7: Chemical structures of flavonols quercetrin and kaempferol 3-O-a-L-ramnopyrananoside isolated from the standardized extract of aerial parts of C. langsdorffii; - Figure 8: Chromatographic profile of the standardized extract of C. langsdorffii (alcohol-water - 7: 3), at 257 nm. 1 - quercetrin-3-O-ramnoside and 2 - canferol-3-O-ramnoside; - Figure 9: Chromatographic profiles of the samples at 257 nm. A - Campus FCFRP USP; B - Campus FFCLRP-USP; C - Marília Region - SP. 2 - quercetrin-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: Renal stones and satellites taken from animals with renal lithiasis in each studied group.

EXAMPLES: EXAMPLE 1 - Obtaining the Microparticles of Copaifera langsdorffii ',

[0023] Step 01 - Obtaining the 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 the code SPFR 10120. After collecting the plant material (branches and mainly leaves), it was dried and stabilized in a hot air circulating oven at 40 ° C and then subjected to grinding in a knife mill, previously sterilized. The powder obtained, after grinding the plant material, was subjected to maceration in a hydroalcoholic solution in the proportion of 6: 4 or 7: 3 followed by percolation three times, every 72 hours. The extract can also be obtained by infusing the ground vegetable drug with boiling water. The extracts are advantageously PURIFIED by a process consisting of sequential passage through a fixed bed separation system composed of a stationary phase in a triple layer consisting of diatomaceous earth, activated magnesium silicate and active carbon, in order to remove pigmented and waxy compounds that hinder the desired bioactive activity for the present invention. The vegetable drug, by exhaustive extraction in a Soxhlet apparatus, presents a yield of 20 to 40% of soluble solids, for information purposes. Obtaining the Matrix Microparticles: The extracts obtained, as described above, purified, must be added with drying adjuvant, film or film former with pharmacotechnical properties suitable for the later formulation of the drug, such as colloidal silicon dioxide, sugars and modified starches, cellulosic polymers, acrylates, bioadhesive biopolymers or not, natural gums and waxes, as well as arabic gum, xanthan gum, chitosan, and others, including mixtures or combinations in any proportions, in relative quantities, drug / adjuvant, varying 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 from 0.1 to 1000 liters per hour. The powder obtained characterized by matrix microparticles of Copaifera langsdorffii has the following characteristics: particle size between 1 and 200 micrometers, massive spherical morphology, and the presence of markers and / or active ingredients quercetrin and 3-0-raminoside in concentrations of 2 to 10% and 0.5 to 10%, respectively. The microparticles obtained can 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 vehicles of specific formulations for different applications, such as obtaining tablets. , capsules, etc.

EXAMPLE 2 - Methodology for the Characterization of the Standardized Extract of Copaifera langsdorffii (used in obtaining the microparticles)

[0024] 1 mL of the standardized extract (containing 50 μg benzophenone (Pi) / mL) was collected for filtration through a Millex-LCR-PTFE analytical filter (0.45 μm x 13 mm), which was transferred to a flask suitable for automatic injector (1 mL). Continuing, a 10 μL aliquot of each sample was analyzed by high performance liquid chromatography. For this, a Shimadzu high-efficiency liquid phase chromatograph was used, equipped with a SCL-10Avp controller, equipped with three LC-10AD pumps, a SPD - M10Avp diode array detector, automatic injector (SIL-10ADvp) and computerized controller system with Shimadzu Class-VP software version 5.02. Two monolithic columns (Onyx ™ 100 X 4.6 mm - Cie Phenomenex) were also used interconnected in series, protected by a column of the same material. The mobile phase used water in phase A and acetonitrile (MeCN) in phase C, at a flow rate of 1 mL / min. and a multilinear gradient developed as follows: starting from 5 - 6% of phase C in 1 min., 6 - 8% of C (1 - 2 min.), 8 - 10% of 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% of phase C up to 35 minutes, increasing from 25 - 40% of C (35 - 39 min.), maintaining 40% of phase C up to 42 minutes, rising from 40 - 100% of C (42 - 47 min.), Remaining 100% phase C for 1 minute, in addition to another 13 min. to return to initial conditions and rebalance the column. All HPLC chromatographic profiles were obtained at 257 nm absorption. The method has been validated in accordance with current regulations.

EXAMPLE 3 - Isolation Process of Copaifera langsdorffii Compounds 3.1. Fractionation of crude extract by liquid-liquid partition

[0025] The standardized extract of the leaves of Copaifera langsdorffii was subjected to partition with organic solvents in order to isolate its constituents. For this purpose, 100 g of this extract were solubilized in 1000 mL of methanol-water (9: 1 v / v), and then it was transferred to a separatory funnel and partitioned 3 times with 400 mL of hexane. In this resulting fraction, called hexane, anhydrous sodium sulfate was added to eliminate water, and then it was filtered into a round bottom flask, in which it was concentrated under reduced pressure at 40 ° C with the aid of a rotary evaporator. After concentrating, the hexane fraction was transferred to a previously weighed flask and the gravimetric difference was calculated.

[0026] The remaining hydromethanolic solution was rotated in order to eliminate methanol. Then, 600 ml of distilled water were added to the remaining aqueous solution and the same partition procedure described for the hexane fraction was carried out, but using the solvents dichloromethane and ethyl acetate in sequence. The extract fractionation process is summarized in Figure 3.

3.2. By column chromatography filled with common silica gel

[0027] After a 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 on the way to the stationary phase to improve selectivity . For this, about 8 g of this fraction were solubilized in 50 ml of ethyl acetate and, subsequently, this sample was applied on top of the normal phase silica, which was packed in the proportion of 1 g: 100 g in a column of 6.5 cm internal diameter glass per 100 cm height. 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 and, immediately afterwards, obeying the following gradient: AcOELMeOH 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 maintained until fraction 40 was obtained, completing the procedure.

3.3. Using column chromatography filled with Sephadex® LH 20

[0028] In order to isolate the chemical constituents of interest with a better degree of purity, to serve as chromatographic standards in the validation of the method and to perform biological tests, it was decided to perform a new fractionation modifying the chromatographic procedure, in this case with the use of Sephadex® LH 20 as a stationary phase. For this purpose, a 5 g aliquot of the fraction in ethyl acetate was solubilized in 50 mL of methanol P. A. with the aid of a sonicator. Soon after, this sample was filtered through an 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 subjected to drying 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 were applied on top of Sephadex® LH 20, which was packaged, in the proportion of 1 g: 100 g, in a glass column measuring 3.5 cm in internal diameter by 120 cm in height .

[0029] Chromatographic separations of compounds, having Sephadex® LH 20 as the stationary phase, are influenced by the molecular exclusion mechanism. Therefore, the mobile phases used in this technique are usually isocratic. Thus, in this fractionation 159 fractions were collected, 155 with a volume of 25 ml_ and 4 with a volume of 500 ml_ using methanol as the mobile phase.

[0030] All sub-fractions obtained through this methodology were analyzed by CCDC using a mixture of butanol, acetic acid and water in the proportion of 5: 1: 4 v / v / v as the mobile phase. These sub-fractions were assembled based on the similarity profile, which was observed after the development of the chromatographic plates. UV light in the lengths of 254 and 366 nm and 5% sulfuric acid in methanol were used as developers. These meetings resulted in 16 fractions, which were subsequently monitored by CLAE.

3.4. Purification of samples obtained by column chromatography

[0031] Through CCDC it was possible to select the promising fractions obtained from the methodologies described above. Thus, these fractions were purified by HPLC in the preparative way, aiming to obtain the compounds of interest in quantities suitable for the development of the analytical method. For this purpose, we used a Shimadzu chromatograph composed of two Shimadzu LC-6A pumps, a Shimadzu SPD-6AV UV / VIS spectrophotometric detector, a SCL-10AVP controller system, connected to a computer with Shimadzu Class-VP software version 5.02 and SIL- 10ADvp. A semi-prepared reverse phase chromatographic column CLC-ODS (M) - Shimadzu, 10 mm x 250 mm, particle diameter 5 μm and Cie reverse phase pre-column manufactured by the same company were used. The mobile phase used water in phase A and methanol in phase B, at a flow rate of 6 mL / min. and a gradient starting from 20 - 60% of phase B (0 - 15 min.), maintaining 60% of phase B up to 25 minutes, rising from 60 - 100% of B (25 - 35 min. ), remaining 100% phase B up to 37 minutes, in addition to another 8 min. to return to initial conditions and rebalance the column.

EXAMPLE 4 - Isolated Compounds of Copaifera langsdorffii

Tabela 02 dados de RMN He C obtidos para o canferol 3 o l ramnopiranosideo

[0032] The study of the phytochemistry of the Standardized Extract of Copaifera langsdorffi (obtained from the aerial parts) demonstrated that its major constituents, approximately 60%, are polar, differing from the chemical composition of the oil-resin. The chemical structures of isolated and purified metabolites were elucidated using spectroscopic methods such as 1H and 13C Nuclear Magnetic Resonance spectroscopy, IV, UV and mass spectrometry, in addition to two-dimensional spectrometric techniques, such as HMBC, HMQC and NOESY. The 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on Brucker-Advance DRX400 spectrometers, operating at 400 MHz (1H NMR) and 100 MHz (13C NMR), using Aldrich deuterated solvents. In addition, other methods can be used to assist in identification, such as determining the melting point and measuring [a] D. Figures 4, 5, 6 and 7 show the characterization of the structures (i) quercetrin-3-O-ramnoside and (ii) canferol-3-O-ramnoside. Table 01: 1H and 13C NMR data obtained for quercetrin.

Table 02 He C NMR data obtained for canferol 3 ol ramnopyranoside

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

[0033] The standardization of the extracts that will be passed through the specific process to obtain matrix microparticles involves the identification and quantification of at least the compounds: (i) quercetrin-3-O-ramnoside and (ii) canferol-3-O- ramnoside (figure 7), and they must be included in the ranges of 0.25% to 2.0% corresponding to the vegetable drug or from 120 to 460 μg / mL in the extracts. The pharmacological effects object of the present invention consist of: antilitiásica (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic activity, as well as the use of these in the treatment of renal lithiasis (renal calculus), arthritis, tartar, muscle and spasmodic pain in general (abdominal, renal cramps, etc.) and, also, other diseases related to the activities previously mentioned. The formulations that can be originated with the matrix microparticles of Copaifera langsdorffii and / or its isolated compounds, such as medicines, personal hygiene 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 matrix microparticles of Copaifera should be inserted in the respective formulations taking into account 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 plant material and its correspondents in standardized extracts and markers as detailed below, for antilitiásica, antiseptic activities; anti-inflammatory; antispasmodic; painkiller, etc.

EXAMPLE 6 - Antilithiasis activity and safety in a preclinical model

Tabela 04: Parâmetros bioquímicos urinários dos animais.

Tabela 5: Parâmetros bioquímicos séricos dos animais.

Tabela 6: Parâmetros de função renal dos animais. 

Tabela 7: Dados referentes aos cálculos retirados dos animais.

[0034] Considering the need for a drug for the treatment of nephrolithiasis and the great potential of C. langsdorffii, the matrix microparticles of Copaifera langsdorffii were evaluated in an experimental model of lithiasis in rats. In this model, lithiasis was induced in the animal through the introduction of a calcium oxalate tablet in its bladder, which serves as a niche for the growth of the bladder stone. The protocol lasted 48 days, and in the last 18 days the animals were treated with matrix microcapsules of C. langsdorffii, at a dose of 20 μg / g body weight / day (concentration referring to the dry mass of plant material), diluted in 1 mL of water, per gavage. The control group was submitted to the same protocol, except for extract administration in the last 18 days (BARROS et al., 2006). At the end of the experiment, no important biochemical changes were observed between the control and treated groups, that is, the administration of the matrix microparticles of C. langsdorffii did not change the urinary volume, urinary excretion and the serum concentration of sodium, potassium and creatinine. However, in the group of treated animals, there was a reduction in the total mass (0.286 ± 0.042 vs. 0.164 ± 0.066) and in the total number of calculations 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 animals in the control group is at least twice as high as that required to disintegrate the calculations in the treated group (6.90 ± 3, 45 vs. 3.00 ± 1.51). Thus, it was found that the treatment not only acts to reduce the number and total mass of stones formed, but also changes their morphology, reinforcing the therapeutic action of the matrix microparticles of C. langsdorffii in the treatment of renal lithiasis. Table 03: General parameters of the animals.

Table 04: Urinary biochemical parameters of animals.

Table 5: Serum biochemical parameters of animals.

Table 6: Renal function parameters of animals.

Table 7: Data related to calculations taken from the animals.

EXAMPLE 7 - Demonstration of the safety of Copaifera lansdorffii Matrix Microparticles in models of cytotoxicity and mutagenic potential

[0035] The matrix microparticles of C. langsdorffii were evaluated for mutagenic and / or antimutagenic potential by means of the micronucleus test (test for the assessment of preclinical safety) in peripheral blood of Swiss mice. The results showed no cytotoxicity of the matrix microparticles C. langsdorffi. In addition, an antimutagenic effect was observed when the animals were treated simultaneously 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 ).

[0036] The matrix microparticles of C. langsdorffii were also evaluated for mutagenic and or antimutagenic potential in V79 cells (hamster lung fibroblasts) also by the micronucleus test. The results found demonstrated that the C. langsdorffi microparticles did not show mutagenic activity at concentrations of 30, 60 and 120 μg / mL (concentration corresponding to the dry mass of plant material) and, they showed a chemopreventive effect when combined with methyl methanesulfonate (400 μM ).

EXAMPLE 8 - Demonstration of the antioxidant activity of Copaifera lansdorffii Extracts "in vitro".

[0037] Brand-Williams method (1995) was used to evaluate the antioxidant activity, which uses the free radical 2,2-diphenyl-1-picrilhidrazil (DPPH). The extracts of Copaifera langsdorffii, obtained according to example 1, that is, extracts (1) aqueous, (2) hydroalcoholic solution 4: 6 (ethanol-water) and (3) hydroalcoholic solution 7: 3 (ethanol-water), were diluted in ethanol until concentration 50 μg / mL. In test tubes, 1 ml of 0.1 M sodium acetate / acetic acid buffer, pH 5.5 was added; 1 mL of DPPH 125 M solution and 50 μL of the sample. After 30 minutes of rest, the samples were measured on a 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 0 standardized aqueous extract of Copaifera langsdorffii (1) was 0.8518 μg / mL, while for extract (2) it was IC5O = O, 5281μg / mL and for 0 (3) it was IC5o = 0.4716 μg / mL. When the antioxidant activity of the same extracts was quantified through the total flavonoids of the extracts, it was obtained: (i) ICso = 0.100 μg / mL for the standardized (aqueous) extract; (ii) IC5o = O, O955 μg / mL (40% hydroalcoholic solution) and (iii) IC5o = O, 1534 μg / mL for the extract obtained with 70% hydroalcoholic solution, demonstrating the antioxidant power of Copaifera langsdorffii, through proposed methodology, material used in the present invention.

EXAMPLE 9 - Demonstration of the Pre-Clinical Safety of the Matrix Microparticles of Copaifera langsdorffii-pre-clinical research

[0038] Toxicological tests were carried out for the acute and sub-chronic phases of the matrix microparticles of Copaifera, using Wistar rats and rabbits. The results obtained for both protocols showed that the treatment of animals in doses three times higher than the doses recommended for humans, did not cause any change in the biochemical, hematological and histopathological parameters in the animals studied. 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 langsdorffii Matrix Microparticles - Phase I Clinical Research

[0039] A Phase I clinical study was conducted to assess the safety of use of Copaifera langsdorffii Microparticles in 28 healthy volunteers. Therefore, laboratory tests of renal function (creatinine, urea and urine I), liver (TGO, TGP, gamma-GT and prothrombin time) and cardiac (pressure and frequency) tests were performed before and after ingestion of the product. The volunteers had an average age of 24.1 years (SD = 2.6 years), with 9 (32.1%) being female and 19 (67.9%) being male. The results found for the physical examinations of the volunteers demonstrated the absence of alterations after the administration of the microparticles, being the average results: 111.4 mmHg (SD = 12.4) x 74.4 mmHg (SD = 8.3) for the blood pressure, 72.2 beats / 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. The results of laboratory tests to verify the occurrence of changes in kidney and liver function showed normal results, both before and after ingestion of Copaifera microparticles, for all parameters. The results found in blood and urine I tests were normal before and after the administration of Copaifera Microparticles, for all parameters. The results obtained with healthy volunteers, after routine medical procedures (anamnesis, physical examination and complementary laboratory tests for kidney and liver function - blood and urine) showed that the intake of Copaifera microparticles, equivalent to a daily intake of 10g of the material vegetable (divided into three shots) under short treatment, was devoid of toxic effects for the volunteers who participated in the study.

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

[0040] In a Phase II Clinical study, related to efficacy, preliminary data demonstrated the great potential for therapeutic use of the product, as of eight patients affected by urolithiasis, 3 (37.5%) demonstrated complete elimination of the stone, 3 (37 , 5) demonstrated a decrease in diameter and color change in the stone and in 2 (25%) patients, the treatment was not satisfactory. Considering that the clinical study protocol covered three different treatments, namely: (i) 1 dose + 1 dose, where patients ingested a dose equivalent to 10 g of plant material (divided into three doses / day) for three days, underwent reassessment medical and received again the equivalent of 10g of plant material (divided into three doses / day) to be administered for three days; (ii) 2 doses + 1 dose, with 10 g of plant material (divided into 3 doses / day) for a period of six days and after a medical evaluation the administration of 10 g / day (divided into three doses / day) per another three days, and finally, (iii) the administration of 10g (divided into three doses / day) for six days, followed by a medical evaluation, administration of the product for three more days, a new evaluation and finally, the treatment by three more days. Thus, the data obtained by only eight patients, can already infer a satisfactory result, since 75% had a satisfactory result, 37.5% with complete elimination of the stone, results that are very difficult to obtain in conventional treatments and that point to the success of the product. .

Brief Description of the Invention

[0041] The invention is characterized by obtaining and using Matrix Microparticles of Copaifera langsdorffii, and of the isolated substances (aerial parts) of Copaifera, mainly Copaifera langsdorffii Desf., But also from other species such as C. beyrichii Hayne .; C. bijugaHayne .; C. bracteata Bth .; C. confertiflora Bth .; C. coriaceae M .; C. duckei Dwyer .; C. elliptica M .; C. glabraVog .; C. guianensis Desf .; C.jacquini Desf .; C. jacquimana Desf .; C.jussieuii Hayne .; C. laxaHayne .; C. luetzelburgiiHarms .; etc.

[0042] Thus, the present invention relates to a process of obtaining Matrix Microparticles from Copaifera langsdorffii and the isolation of their compounds, as well as the biological activities of the microparticles and the isolated compounds of the plant, namely: antilitiásica activity (calculation analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic, as well as their uses, especially in their use in the treatment of kidney stones (kidney stones), arthritis, tartar, muscle and spasmodic pain in general ( abdominal, renal colic, etc.), inflammation and infections of the urinary tract or other, as well as other diseases related to the activities previously mentioned, in addition to the present invention to include the matrix microparticles themselves, their isolated compounds and the formulations that can be originated with them and / or their isolated compounds, such as: medicines, personal hygiene and cosmetics, food, veterinary products and dental, etc.

[0043] It is worth mentioning that, as already presented in the state of the art (previous), there is currently no medicine obtained from a medicinal plant, endowed with an inventive step, that is, that uses scientific discoveries to obtain a technological product, efficient in disintegrating the structure of the calculation. The procedures used are the patient's hydration, aiming at the elimination of calculus and palliative treatments, in addition to the studied plants having not culminated in obtaining efficient medicines or products for the treatment of lithiasis.

[0044] It should also be noted that the process of obtaining the matrix microcapsules of Copaifera langsdorffii provides obtaining an advantageous technological system in relation to the freeze-dried extracts conventionally obtained, not only with regard to the biological activity of the product but also in relation to the technological process industrial. Microparticles advantageously offer homogeneous, spherical, massive particles, more chemically stable in relation to lyophilized extracts, microencapsulated systems capable of offering modified release, in addition to greater ease in obtaining pharmacotechnical preparations, such as tablets, capsules, solutions, etc. Lyophilizates, in turn, are heterogeneous systems, subject to oxidation and degradation processes of bioactive chemical compounds previously extracted from the plant drug, which do not show fluidity, flowability and are difficult to handle pharmacotechnically. In addition, the extracts used as material for obtaining the microcapsules require a purification step, which consists of passing the extract through a sequential separation system through a fixed bed composed of a stationary phase in a triple layer consisting of diatomaceous earth, activated magnesium silicate and coal. active to remove 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 an inventive step and perfectly applicable industrially, justifying the present request for industrial property.

Detailed description of the invention

[0045] Aerial parts, leaves and branches, of Copaifera species are submitted to the drying process in a hot air circulating oven at 40 ° C and, then, they are submitted to grinding in a knife mill until obtaining fine powder. 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 a filter canvas to separate the vegetable cake. After filtration, the extract is purified sequentially in a fixed bed separation system composed of a stationary phase in a triple layer consisting of diatomaceous earth, activated magnesium silicate and active carbon.

[0046] Specifically, the process of the present invention comprises the steps of: a) drying the aerial parts, leaves and branches of Copaifera species in a hot air circulating oven at 40 ° C; b) grinding the dry material in a knife mill until obtaining fine powder; c) extraction by cold or hot maceration, in water or 4: 6 or 7: 3 hydroalcoholic solution (ethanol); d) filtration of extracts on a filtration canvas to separate the vegetable cake; e) fundamental, purification step that consists of sequential passage in a fixed bed separation system composed of a stationary phase in a triple layer consisting of diatomaceous earth, activated magnesium silicate and active carbon. f) the standardized extracts obtained through (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), are transformed into microcapsules matrix, by the addition of a drying aid, film or film former with suitable pharmacotechnical properties for the subsequent formulation of the drug, such as colloidal silicon dioxide, modified sugars and starches, cellulosic polymers, acrylates, bioadhesive biopolymers or not, natural gums and waxes , such as gum arabic, xanthan gum, chitosan and others, including mixtures or combinations in any proportions, in relative quantities, drug / adjuvant, varying 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.01 to 1000 cubic meters per minute, sample flow from 0.1 to 1000 liters per hour. The microparticles are characterized by the chemical profiles shown in figure 10 and, also, by presenting the major compounds quercetrin-3-O-ramnoside (first major) and (ii) canferol-3-O-ramnoside (second major) in the respective ranges of 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 major isolated compounds of Copaifera, which involves (Figure 3): fractionation of the standardized extracts obtained from aerial parts of Copaifera species through filtration processes in columns packed with stationary phases based on silica gel or obtaining the fractions rich in active ingredients by liquid-liquid partition processes from the hydroalcoholic extract of the branches of Copaifera species, using hexane, dichloromethane and ethyl acetate, in sequence; fractionation of the extract in ethyl acetate, using a column package with stationary phases based on silica gel; purification of the fraction rich in major components, obtained by fractionating the extract in ethyl acetate, using high performance liquid chromatography (HPLC), providing the major compounds quercetrin-3-O-ramnoside (first major) and canferol-3 -O- rhodnoside (second major) (figure 7) according to the chromatogram obtained in HPLC, figure 4, as well as other minority phenolic compounds. Emphasizing that the extracts can be fractionated by filtration processes in columns packed with stationary phases, such as: silica gel, activated magnesium silicate, reverse phase silica (C-8, C-18), Sephadex LH20, etc. Fractions can also be obtained from partitioning processes using organic solvents, as shown in Figure 3. Organic solvents are eliminated in vacuum concentrators and the aqueous phases are lyophilized. h) Copaifera matrix microparticles can be used as such or conveyed in appropriate preparations (medicines, personal hygiene and cosmetics, veterinary, dental or other products), in treatments of three to 28 days for each adult patient, for which are used three daily doses of microparticles, corresponding to 5 to 20 g of dry plant material each, equivalent to a dose of 1.0 to 5.40 g of microparticles of C. langsdorffii (aqueous), 1.0 to 6, 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 guercetrina-3-O-rnosnoside (first major) and canferol-3- O-ramnosid (second major). For the treatment of animals, 1.50 to 80 mo / kg of body weight / day of microparticles should be used. i) Matrix microparticles and / or their isolated compounds, properly said or conveyed in solvents or excipients or vehicles for formulating medications, personal hygiene and cosmetic products, veterinary, dental or other products for the treatment of kidney stones (kidney stones), arthritis , tartar, muscle and spasmodic pain in general (abdominal, renal cramps, etc.), inflammation and urinary tract infections or others, as well as other diseases related to the activities previously mentioned, depending on the biological activities of the extract and its isolated compounds , which are: antilitiásica activity (renal calculus), analgesic, anti-spasmodic, anti-inflammatory, diuretic and antiseptic. j) There are no drugs on the market, whether herbal or allopathic, that are effective in disintegrating kidney stones. Thus, the products of the present invention will foster the development of an unprecedented product, since the currently existing ones act as diuretics, anti-inflammatories, analgesics and, at most, as prophylactics.

Claims (8)

1. Process of obtaining matrix microparticles of Copaifera langsdorffii (aerial parts), with antilithiasis activity (renal calculus), characterized by the fact that it comprises the following steps: a) drying of aerial parts, leaves and branches of Copaifera species in a greenhouse. hot and circulating air at 40 ° C; b) grinding the dry material in a knife mill until obtaining fine powder; c) extraction by cold or hot maceration, in water or 4: 6 or 7: 3 hydroalcoholic solution (ethanol: water); d) filtration of extracts on a filtration canvas to separate the vegetable cake; e) purification of extracts, sequentially, in a fixed bed separation system composed of a stationary phase in a triple layer consisting of diatomaceous earth, activated magnesium silicate and active carbon; f) obtaining Matrix Microparticles by drying the extracts added with, drying adjuvant, film or film former with appropriate pharmacotechnical properties for the subsequent formulation of the drug, such as colloidal silicon dioxide, sugars and modified starches, cellulosic polymers, acrylates, biopolymers bioadhesive or not, natural gums and waxes, such as arabic gum, xanthan gum, chitosan, including mixtures or combinations, in relative quantities, drug / adjuvant, varying from one to ten to ten to one; it is followed by a spray drying process, involving the following conditions: temperature 10-450oC, air flow 0.01 to 1000 cubic meters per minute, sample flow from 0.1 to 1000 liters per hour; for the end to obtain matrix microparticles with the following characteristics: particle size between 1 and 200 micrometers, massive spherical morphology, and the presence of markers and / or active principles quercetrin and canferol-3-O-rnosnoside in concentrations from 2 to 10% and 0.5 to 10%, respectively. 2. Process, according to claim 1, characterized by the fact that the fractions are obtained by partitioning processes using organic solvents, which are eliminated in vacuum concentrators and the aqueous phases are lyophilized. 3. Process, according to claim 1, characterized by the fact that Copaifera species are C. beyrichii Hayne .; C. Hayne bijuga .; C. bracteata Bth .; C. confertiflora Bth .; C. coriaceae M .; C. duckei Dwyer .; C. elliptica M .; C. glabra Vog .; C. guianensis Desf .; C. jacquini Desf .; C. jacquimana Desf .; C. jussieuii Hayne .; C. langsdorffii Desf .; C. laxa Hayne .; C. luetzelburgii Harms .; C. martii Hayne .; C. nitida Hayne .; C. oblongifolia M .; C. officinalis L .; C. publiflora Bth .; C. Ducke reticulata .; C. rigida Bth .; C. rondonii Hoehne .; C. sellowii Hayne and C. trapezifolia Hayne. 4. Process, according to claim 3, characterized by the fact that the species of Copaifera is, Copaifera langsdorffii Desf. 5. Process, according to claim 1, characterized by the fact that the extracts for obtaining the microparticles are 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), with subsequent addition of drying and microencapsulation adjuvants, followed by spray drying, and presenting chemical profiles containing the markers quercetrin-3-O-ramnoside and canferol-3-O -ramnosid. 6. Process, according to claim 5, characterized by the fact that the microparticles have the major compounds quercetrin-3-O-ramnoside (first major) and (ii) canferol-3-O-ramnoside (second major) in the respective ranges of 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. 7. Copaifera langsdorffii matrix microparticles obtained according to the process as defined in any one of claims 1 to 6, characterized in that they are diluted with polyols (sorbitol, propylene glycol), drying aids: colloidal silicon dioxide, sugars and modified starches , cellulosic polymers, acrylates, bioadhesive biopolymers or not, natural gums and waxes, as well as arabic gum, xanthan gum, chitosan, including mixtures or combinations in relative quantities, drug / adjuvant, varying from one to ten to ten to one. 8. Matrix microparticles of Copaifera langsdorffii obtained according to claim 7, characterized in that the matrix microparticles of Copaifera langsdorffii have the following characteristics: particle size between 1 and 200 micrometers, massive spherical morphology, and the presence of markers and / or principles assets quercetrin and canferol 3-Oraminoside in concentrations of 2 to 10% and 0.5 to 10%, respectively.

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