BRPI0905584B1 - ANTIMALARIC PHARMACEUTICAL COMPOSITION CONTAINING EXTRACT AND / OR STANDARD FRACTION OF ASPIDOSPERMA PARVIFOLIUM AND / OR ULEIN SHELL AND ITS DERIVATIVES AND USE - Google Patents

Abstract

standardized extract and fraction of aspidosperma parvifolium husks and their pharmaceutical composition the present invention describes the process for obtaining standardized extract and fraction of aspidosperma parvifolium, popularly called "pau-pereira, guatambu or peroba", in addition to the isolation and purification of the ulein. the invention further comprises obtaining pharmaceutical compositions containing the extract, derived fraction and / or ulein, as well as its use for the treatment of malaria. the extract, fraction and ulein presented were tested and showed antiplasmodic activity, particularly against plasmodium falciparum.

Description

FIELD OF THE INVENTION

The present invention describes the process for obtaining standardized extract and fraction of Aspidosperma parvifolium, popularly called “pau-pereira, guatambu or peroba”, in addition to the isolation and purification of ulein. The invention further comprises obtaining pharmaceutical compositions containing the extract, standardized fraction and / or ulein and its pharmaceutically acceptable derivatives and excipients, as well as its use for the treatment of human malaria.

The extract, fraction and ulein presented were tested and showed antiplasmodic activity, particularly against Plasmodium falciparum.

TECHNICAL STATUS

Malaria chemotherapy started in the 17th century. The Jesuits who came to South America observed that the Peruvian Indians used plants of the genus Cinchonaspp (family Rubiaceae), popularly known as quinas, for the treatment of febrile illnesses. Phytochemical studies of Cinchonaspp, carried out in France, by Pelletier and Caventou, in the early 19th century, of quinine, which showed antimalarial activity. Quinine was a model for the synthesis of antimalarials such as mefloquine and chloroquine.

Chloroquine has been used for a long time in the treatment of malaria, but recently there has been the introduction of new drugs such as artemisinin and atovacone.

Artemisinin is an active substance in Artemisia annua, used for thousands of years in China, having been isolated in 1972. Semi-synthetic derivatives such as artemeter, arteeter and sodium artesunate are also in clinical use (ROSENTHAL, PJ; GOLDSMITH, RS Antiprozoários. Basic & Clinical Pharmacology (Editor: Katzung, BGC Guanabara- Koogan, 8th ed, chapter: 53, p. 769-783, 2003.)

Atovacone is a synthetic naphthoquinone, an analogue of lapachol, which is common in species of Tabebuia, a genus to which Ipe trees belong, trees that occur in South America (MIRAGLIA, MCM Chemical study of Tabebuia serratifolia (Vahl.) Nichols (Bignoniaceae ) and synthesis of pyranonephtoquinones, furanonephtoquinones and anthraquinones (Thesis (PhD in Chemistry) - Federal University of Minas Gerais, 1991).

In this context, plants have made an important contribution to malaria chemotherapy, such as quinine and artemisinin.

The antiplasmodic activity of alkaloids has been widely reported in the literature, and in the period from 1990 to 2000, more than a hundred active substances of this class have been described, some more potent than chloroquine, according to a recent review (SAXENA, S. et al. Antimalarial agents from plant sources. Current Sci, v. 85, pp. 1314-1329, 2003).

These alkaloids have often been isolated from plants traditionally used for the treatment of malaria in different regions of the world. A literature survey indicated that 1,200 plant species are employed on three continents for the treatment of malaria (WILLCOX, M.L .; BODEKER, G. Traditional herbal medicines for malaria. BMJ, v. 329, p. 1156 -1159, 2004).

The genus Aspidosperma belongs to the family Apocynaceae which includes about 200 genera and 2000 species. Species of this genus occur in tropical and subtropical regions, from Mexico to Argentina, being found in different habitats, such as in the savannahs of the central south region of Brazil, inundated areas on the banks of rivers in the Amazon, Paraguay, Argentina, Mexico and regions in elevation from Peru and Bolivia (WOODSON, RS Studies in the Apocynaceae VIII. An interim revision of the genus Aspidosperma Mart & Zucc. Ann. Missouri Botanical Garden, v. 38, p. 119,1951).

From species of the genus Aspidospermajá, about 250 indole alkaloids were isolated (JÁCOME, RLRP Chemical study of Zeyheria montana M. and Aspidosperma parvifoliumA.DC. Quantification by CLAE of naphthoquinones isolated from Z. montana. UFMG / ICEx / DQ thesis. Belo Horizonte , 1998).

Regarding the traditional use and pharmacological activity of species of this genus, very little is found in the literature. An ethnopharmacological survey carried out in the municipality of Igarapé Mirim (PA, Brazil) revealed that the local population uses Aspidosperm shells, including A. auriculatum, for the treatment of malaria and fever (BARBOSA, WLR; TAVARES, JCC; SOARES, DC Alkaloids of Aspidosperma auriculatum Standi (Rev. Bras. Farmacogn, v. 13, p. 06-08, 2003).

In general, in the Amazon, some species of Aspidosperm are used by indigenous and caboclo populations, for various medicinal purposes, such as the treatment of inflammation of the uterus and ovaries, diabetes, cancer, as contraceptives and treatment of malaria (MILLIKEN, W. Plants for malaria for fever: medicinal species in Latin America-a bibliography survey. Kew: the Royal Botanic Gardens. Monograph. The Royal Botanic Gardens, 1997). The most used species are A. nitidum, A. marcgravianum, A. carapanauba and A. desmantun (BRANDÃO, M. et al. Survey of medicinal plants used as antimalarials in the Amazon. Journal of Ethnopharmacology, v. 36, p. 175-182, 1992).

The A. quebracho-bianco species is used as a febrifuge for the treatment of asthma, bronchitis, emphysema and fever (BOWN, D. Encyclopedia of herbs & their uses. Dorling, Kindersley, London, 1995). A. excelsum is indicated for stomach problems and indigestion (CHEVALIER, A. The encyclopedia of medicinal plants. Dorling Kindersley. London, 1996).

A. pynfolium and A. megalocarpononze alkaloids with aspidosperman skeleton were isolated and their antiplasmodic activity (sensitive and chloroquine resistant strains) and cytotoxicity were evaluated. Of these alkaloids, seven are tetracyclic, with free ethyl group, and showed IC50 between 3.2 and 15.4 μM, after 72 hours of exposure. However, four pentacyclic alkaloids showed reduced antiplasmodic activity, that is, IC50 between 22.6 and 52.6 μM. The alkaloid and chloroquine association was also evaluated, having observed synergism in the following associations: chloroquine - N-formyl-aspidospermidine and chloroquine-aspidospermine. Regarding cytotoxicity (NiH 3T3 cells), alkaloids with high antiplasmodic activity showed less cytotoxicity (MITAINE-OFFER, AC et al. Antiplasmodial activity of Aspidosperma indole alkaloids. Phytomedicine, v. 9, n. 2, p. 142-145 , 2002).

Aspidosperma parvifolium is popularly known as guatambú, peroba or pau-pereira. The development of the plant in the field is fast, reaching 3.5-4.0 m at 2 years (LORENZI, H. Brazilian Trees: Manual for Identification and Cultivation of Native Brazilian Tree Plants. Nova Odessa, SP: Editora Plantarum, 1992).

From the ethanolic extract of A. parvifolium husks, N-methyltetrahydroelipticin alkaloids (BURNELL, RH; CASA, DD Alkaloids of Aspidosperma vargasii A.DC. Can J Chem., V. 45, p. 89, 1967) were isolated, ulein, epiulein, aparicin, demethylulein, in addition to a triterpene, lupeol, and a steroid, stigmasterol (JÁCOME, RLR et al. Chemical study and chromatographic profile of the barks of Aspidosperma parvifoliumA. DC. (“Pau- Pereira”). Química Nova , v. 27, n. 6, p. 897-900, 2004). It is worth mentioning that, in this study, however, the extract's antimalarial activity was not evaluated.

Ulein has already been obtained from other species of Aspidosperma, such as A. olivaceum and A. tomentosum (GILBERT, B. et al. Alkaloids studies L-The alkaloids of twelve Aspidosperma species Tetrahedron, v. 21, p. 1141-1161, 1965 ).

The ethanolic extract of A. parvifoliu peels evaluated for lethality in Artemia salina and callus inhibition, induced by Agrobacterium tumefaciens, in potato discs, showed a high antitumor potential, that is, the 50% lethal concentration (CL50) was 103, 8 g / mL for Artemia salina and, in this concentration, 94.4% of the callus formation in potato discs was inhibited. This extract also presented anti-Trypanosoma cruzi activity (DOLABELA, MF Master's Dissertation: Screening for anti-tumor and anti-Trypanossoma cruzi activity of plant extracts, natural products and synthetic substances. Belo Horizonte: Dept of Physiology and Pharmacology, ICB, UFMG, 1997). The toxicity observed in Artemia salina, is probably related to the presence of indole alkaloids.

A mixture of indole alkaloids, predominantly containing ulein (53%) reduced acid secretion in the stomach of rodents, and this reduction may be related to the blocking of the H +, K + -ATPase pump (BAGGIO, CH et al. Gastroprotective mechanisms of indole alkaloids from Himatanthus lancifolius, Planta Med, v. 71, n. 8, p. 733-8, 2005).

Another indole alkaloid, present in A. parvifolium, aparicin, was tested for antifungal activity, but no growth inhibition of the microorganism was observed (HERNANDEZ, NMR; PEREZ, CD Fungal activity of various alkaloids isolated from Catharanthus roseus G. Don Rev Cubana Med Trop, v. 29, n. 3, pp. 147-52, 1977).

Currently, some patent documents related to the present invention are available.

Patent application BE814772 describes obtaining new therapeutic alkaloids by extracting the barks of Aspidosperma spegazzinii. The shell was extracted with benzene or chloroform and the resulting extract is extracted with acidic solution. The acid solution obtained is treated with an alkaline solution to precipitate the total alkaloids, which are separated by organic solvent extraction and later by chromatography on alumina.

The patent FR2865652 reports the use of plant extracts or active ingredients isolated from these, in the preparation of pharmaceutical, cosmetic and nutraceutical compositions, such as lipolytic agents and anti-cellulite. The extracts are selected from plants of the genus: Aspidosperma; Catharanthus; Eschscholzia; Bocconia; Vismia; Orbignya; Terminalia; Entada; Hypericum; Ginkgo; Viburnum; Papavere Butea.

The US3225053 patent reports the isolation of quebranchidine and derivatives from the extract of Aspidosperma quebracho-blanco and its use as psycho-sedatives and β-adrenergic blockers.

PROBLEMS OF THE STATE OF THE TECHNIQUE

The drugs available for the treatment of malaria do not always show satisfactory results. Chloroquine, due to its low toxicity and being economically viable, was widely used in the treatment of malaria for many years, however, due to the increasing resistance of parasites, new drugs had to be introduced, such as artemisinin and atovacone.

However, atovacone, as well as artemisinin and derivatives are of high cost, which makes their use in poor endemic regions unfeasible, in addition to the possibility of developing parasite resistance to these drugs, which has already been observed for artemisinin. There is, therefore, a need for new antimalarial agents that are effective against chloroquine-resistant strains of P. falciparum, promoting healing within a reasonable time to ensure adherence to treatment, that are safe and inexpensive (FIDOCK, DA et al. Antimalarial drug discovery: efficacy models for compound screening (Nat Rev Drug Discov, v. 3, n. 6, p. 509-20, 2004).

TECHNOLOGY ADVANTAGES

Although pharmaceutical compositions are available on the market for the treatment of malaria, none of these refers to pharmaceutical compositions that contain A. parvifolium bark extract, a standardized fraction rich in alkaloids and / or ulein.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Acid-base extraction from the ethanolic extract of A. parvifolium husks

Figure 2: Chromatogram of the EEPPVA fraction

Figure 3: Ulein chromatogram

Figure 4: EEPPVA + ulein chromatogram

Figure 5: Spectrum in the IV of APT 13-16 (8) (ulein)

Figure 6: Mass spectrum by electronic impact of APT 13-16 (8) (ulein)

DETAILED TECHNOLOGY DESCRIPTION

The present invention describes the process for obtaining standardized extract and fraction of Aspidosperma parvifolium, popularly called “pau-pereira, guatambu or peroba”, in addition to the isolation and purification of ulein. The invention further comprises obtaining pharmaceutical compositions containing the extract, derived fraction and / or ulein combined with pharmaceutically acceptable excipients, as well as their use for the treatment of malaria.

Examples of excipients include water, saline, phosphate buffered solutions, Ringer's solution, dextrose solution, Hank's solution, biocompatible saline solutions containing or not polyethylene glycol. Non-aqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglyceride can also be used. Compositions can be prepared with such an excipient or mixture.

Excipients may also contain minor amounts of additives such as substances that increase the isotonicity and chemical stability of substances or buffers. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard compositions can be liquid or solid. Thus, in a solid formulation, the excipient can include dextrose, preservatives, to which water or sterile saline can be added before administration. In addition to binders, disintegrants, thinners, lubricants, surfactants.

These compositions can be administered via intramuscular, intravenous, subcutaneous, oral, inhalation or as devices that can be implanted or injected, being preferably administered orally.

The present invention can be better understood through the following, non-limiting examples of technology:

Example 1: Obtaining standardized extract and fractions of Aspidosperma parvifolium husks

The powdered shells of A. parvifolium were subjected to extraction by exhaustive percolation, with ethanol at 96 ° GL. Then, the ethanolic extract was concentrated in a rotary evaporator, at 50 ° C, under reduced pressure, until residue, being kept in a desiccator until constant weight.

The ethanolic extract of A. parvifolium husks (EEPPV) (10.0 g) was solubilized in a reduced volume of ethanol (<10 ml_) and 1N aqueous HCI (250 ml) followed by extraction with dichloromethane (3 x 250 ml) ) or diethyl ether, or dichloromethane or ethyl acetate, obtaining a layer of organic solvent (EEPPVN) and an acidic aqueous layer (EEPPVAAC). To this was added NH4OH until pH 9 and extracted with dichloromethane (3 x 250 mL), obtaining an alkaline aqueous layer (EEPPVAAL) and an organic layer (alkaloid fraction) (EEPPVA). The flowchart for these extractions is shown in Figure 1.

The fraction rich in alkaloids (EEPPVA) presented epiulein, demethylulein and aparicin as constituents, according to the chromatogram recorded by reverse phase high performance liquid chromatography. Ulein is the major component as indicated by the percentage of peak area 69% (Figure 2) and standardized because it represents 45 to 60% w / w of the fraction rich in alkaloids.

Example 2: Isolation and identification of ulein

The ethanolic extract of A. parvifolium (EEPPV) was submitted to acid-base extraction and the alkaloidic fraction (EEPPVA) was obtained. From this fraction, after successive chromatography on silica gel and Sephadex LH-20 columns, the fractions, APT 13-16 (7) and APT 13-16 (8) were obtained, which presented a single stain on CCDS (developers: anisaldehyde- sulfuric acid and Dragendorff's reagent). The fractions obtained were compared with a sample of ulein isolated by Jácome (1998), showing much similarity. (JÁCOME, R.L.R.P., 1998).

The HPLC-FR chromatograms coupled to a UV detector confirmed that it was ulein, with peaks at TR 25.820 min for the reference ulein (Figure 3) and 25. 556 min for the ulein present in the EEPPVA (Figure 2). The identification of the peak corresponding to the ulein in the EEPPVA chromatogram was confirmed by co-injection with the reference ulein (Figure 4) and by the UV spectra recorded online that have a maximum absorption at 304.2 nm.

The spectrum in the APT 13-16 (8) IV (Figure 5) showed absorptions corresponding to axial deformation of CH bonds (3176 cm'1), NH stretch (3040 cm'1), stretching of the C = C bond in an aromatic system (1535 and 1613 cm'1) and deformation of CH bonds in ortho-disubstituted benzene ring, which is the most intense in the spectrum (730 cm'1). This spectrum showed much similarity to that of the ulein (JÁCOME, R.L.R.P., 1998), as expected.

The mass spectrum of APT 13-16 (8) showed the expected molecular ion, in rryk 266, corresponding to the ulein (Figure 6).

Example 3: Evaluation of antiplasmodic activity

The antiplasmodic activity of the ethanolic extract of A. parvifolium husks (EEPPV), alacaloidic fraction (EEPPVA) and ulein [APT 13-16 (8)] was tested by the traditional method (RIECKMAN, KH et al. Drug sensitity of Plasmodium falciparum. An in vitro microtechnique, Lancet, v. I, p. 22-23, 1978; CARVALHO, LH Dissertation thesis: Experimental chemotherapy with crude plant extracts and chemically defined compounds. UFMG, 1990).

The evaluation using the traditional method showed the following results: in clone W2 (resistant to chloroquine), the ethanolic extract of A. parvifolium (EEPPV) showed IC50 32.75 + 1.06 μg / mL. The fraction of alkaloids (EEPPVA) and ulein were shown to be active (IC50 <10 μg / mL). In clone 3d7 (sensitive to mefloquine), the ethanolic extract (EEPPV) showed moderate antiplasmodic activity, whereas the alkaloidic fraction (EEPPVA) and ulein were active (IC50 <10 μg / mL) (Table 1).

Table 1: Data on antiplasmodic activity, in vitro, by the traditional method, of the ethanolic extract of A. parvifolium husks (EEPPV), fraction of alkaloids (EEPPVA) and ulein

Example 4: Evaluation of cytotoxicity in vitro

The assessment of in vitro vitrotoxicity was performed using the MTT colorimetric assay (TWENTYMAN, PR; LUSCOMBE, MA Study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. British J. Cancer, v. 56, p 279-285, 1987). EEPPV was not very toxic to VERO cells (CCso> 500 μg / mL), while the fraction of alkaloids (EEPPVA) and ulein showed moderate cytotoxicity (100 <CC5o <500 μg / mL). When the selectivity index (IS = CC50 / CI50) was determined using the IC50 obtained for clone W2 (Table 1), it was observed that the purer the sample, the higher its selectivity index, that is, the ulein presented higher IS than the extract (EEPPV) and the fraction (EEPPVA). However, when using the results obtained with clone 3d7 (Table 1), the changes in the SI are discrete and less favorable (Table 2). Table 2: Cytotoxicity data (CC50) and selectivity index (IS) of ethanolic extract from Aspidosperma parvifolium husks (EEPPV), fraction of alkaloids (EEPPVA) and ulein

Example 5: In vivo toxicity assessment

The toxic effects of alcoholic extract (EEPPV) and the fraction rich in alkaloids (EEPPVA) were evaluated in mice after acute administration (observational screening, assessment of motor coordination, motor activity and acute toxicity) and in rats after repeated administration of 28 days ( behavioral assessment, hematology, serum biochemistry and anatomopathology). In observational screening, where the extracts were administered orally at doses of 10, 100 and 1000 mg / kg, there was greater toxicity for the EEPPVA fraction.

Toxicological studies carried out with the EEPPV extract and the fraction

EEPPVA indicate a certain degree of toxicity of these extracts when in high doses, but good tolerability in low doses, that is, up to 250 mg / kg (acute) or 150 mg / kg (chronic) in the case of EEPPV; or 75 and 50 mg / kg (acute and chronic, respectively) of EEPPVA.

Claims (1)

1. PROCESS OF OBTAINING ANTIMALARIC FRACTION FROM ASPIDOSPERMA PARVIFOLIUM SHELLS, characterized by obtaining a fraction rich in alkaloids, containing a range of 45 to 60% w / w of ulein, in addition to the constituents epiulein, demethylulein and aparicina, through following steps: a. Solubilization of the ethanolic extract of A. parvifolium husks in ethanol and 1N HCL solution; B. Extraction with dichloromethane or diethyl ether, or dichloromethane or ethyl acetate, obtaining a dichloromethane layer and an acidic aqueous layer; ç. Addition of NH4OH to pH 9 to the acidic aqueous layer; d. Extraction with dichloromethane, obtaining an alkaline aqueous layer and an organic layer (alkaloid fraction); and. Recovery of the alkaloid fraction, which has high concentrations of ulein (45 to 60% w / w), in addition to epiulein, demethylulein and aparicin.

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