BRPI0905584A2 - standardized extract and fraction of barks of aspidosperma parvifolium and / or uleìna and their pharmaceutical composition - Google Patents

Abstract

STANDARDIZED EXTRACT AND FRACTION OF ASPIDOSPERMA PAR VIFOLIUM SHELLS AND THEIR PHARMACEUTICAL COMPOSITION The present invention describes the process for obtaining standardized extract and fraction of Aspidosperma paà'vifolium, popularly called "pau-pereira, guatambu or peroba", in addition to the isolation and peroba ", ulein purification. The invention also 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

"STANDARDIZED EXTRACT AND FRACTION OF SHELLS OF ASPIDOSPERMA PARVIFOLIUM AND / OR ULEIN AND ITS PHARMACEUTICAL COMPOSITION"

FIELD OF INVENTION

The present invention describes the process for obtaining standardized extract and extract of Aspidosperma parvifolium, popularly termed "pereira, guatambu or peroba", in addition to isolation and purification of ulein. The invention further comprises obtaining pharmaceutical compositions containing the extract. , derived fraction and / or ulein and derivatives thereof, as well as their use for the treatment of malaria.

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

TECHNICAL STATE

Malaria chemotherapy began in the 17th century. The Jesuits coming to South America observed that the Indians of Peru used plants of the genus Cinchona spp (family Rubiaceae), popularly known as kinas, for the treatment of febrile diseases. Phytochemical studies of Cinchona spp, carried out in Europe, led to isolation in France by Pelletier and Caventou in the early nineteenth 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, more recently there have been new drugs such as artemisinin and atovacone.

Artemisinin is an active substance of Artomisia annua, of usomilenar in China, having been isolated in 1972. Semi-synthetic derivatives such as artemeter, arteeter and sodium artesunate are also in clinical use (ROSENTHAL, PJ and GOLDSMITH, RS. Editor: Katzung, BGC Guanabara-Koogan, 8th ed, chapter: 53, p.769-783,2003.) Atovacone is a synthetic naphthoquinone, an analog of Iapachol1 that is frequent in Tabebuia species, a genus to which they belong. ipês, trees that occur in South America (MIRAGLIA, MCM Chemical study of Tabebuia serratifolia (Vahl.) Nichols (Bignoniaceae) and synthesis of pyranonaphthoquinones, furanonaptoquinones and anthraquinones. Thesis (Doctorate in Chemistry) - Federai University of Minas Gerais, 1991).

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

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

Often these alkaloids have been isolated from plants traditionally used for malaria treatment in different regions of the world. A survey of the literature indicated that 1200 plant species are used on three continents for the treatment of malaria (WILLCOX, M.L. And BODEKER, G. Traditional herbal medicines for malaria. BMJ, Nov 2004; 329: 1156-1159).

The genus Aspidosperma belongs to the Apocynaceae family which includes about 200 genera and 2000 species. Species of this genus occur in tropical and subtropical regions, from Mexico to Argentina, with different habitats found, such as in the cerrados of central southern Brazil, flooded areas on the banks of the Amazon, Paraguay, Argentina, and Peru's rising regions. and Bolivia (WOODSON, RS Studies in the Apocynaceae VIII. An Interim Review of the Genus Aspidosperma Mart & Zucc.Ann. Missouri Botanical Garden. v.38, p.119, 1951).

From species of the genus Aspidosperma, approximately 250 indole alkaloids have already been isolated (JÁCOME, RLRP Zeyheria montanaM. And Aspidosperma parvifolium A.DC. Quantification by HPLC denaftoquinones isolated from Z montana. UFMG / ICEx / DQ thesis Belo Horizonte, 1998 , 157p).

Regarding the traditional use and pharmacological activity of species of this genus, very little is reported in the literature. A pharmacological survey conducted in the municipality of Igarapé Mirim (PA, Brazil) revealed that the local population uses Aspidosperm husks, including A.auriculatum, for the treatment of malaria and fever. (BARBOSA, W.L.R.; TAVARES, J.C.C. and SOARES, D.C. Alkaloids of Aspidosperma auriculatumStandI. Rev. Bras. Farmacogn. 13 (supl), p.06-08, 2003).

In general, in the Amazon, some species of Aspidosperma are used by indigenous and cabocla populations for various medicinal purposes, such as the treatment of uterus and ovarian inflammation, diabetes, cancer, contraception and treatment of malaria. (MILLIKEN, W. Plants for malaria forfever: medicinal species in Latin America-a bibliography survey. Kew: the Royal Botanic Gardens. Monograph. The Royal Botanic Gardens, 27, 1997). The most commonly used species are A. riitidum, A. marcgravianum, A. carapanauba eA. (BRANDÃO, M., GRANDI, T., ROCK, E., SAWYER, D., KRETTLI, A. Survey of medicinal plants used as antimalarials in the Amazon.Journal of Ethnopharmacology, v.36, p. 175- 182, 1992).

The species A. quebracho-blanco is used as a fever for the treatment of asthma, bronchitis, emphysema and fever. (BOWN, D. Encyclopedia of Herbs & Their Uses. Dorling1 Kindersley, London, 1995). A. excelsum is indicated for stomach problems and indigestion (CHEVALIER, A. The encyclopedia of medicinal plants. Dorling Kindersley. London, 1996).

Eleven alkaloids with aspidospermanskeleton were isolated from A. pyrifolium and A. megalocarpon and their antiplasmodic activity (cepassensitive and chloroquine resistant) and cytotoxicity were evaluated. Of these alkaloids, seven are tetracyclic, presenting free ethyl group, and showed CUo between 3.2 and 15.4μ / W, after 72h of exposure. However, four pentacyclic alkaloids showed reduced antiplasmodic activity, that is, IC50 between 22.6 and 52.6 μΜ. The association between alkaloid and chloroquine was also evaluated, and the following associations were observed: chloroquine - N-formyl-aspidospermidina and chloroquine-aspidospermine. Regarding cytotoxicity (NiH 3T3 cells), alkaloids with high antiplasmodic activity showed lower cytotoxicity. (MITAINE-OFFER, AC; SAUVAIN, M .; VALE NTI Ν, A.; CALLAPA, J .; MALLIE, M. and ZECHES-HANROT, M .. Antiplasmodial activity of Asidosperma indole alkaloids. Phytomedicine, v.9 (2) , p.142-145, 2002).

Aspidosperma parvifoiium is popularly known as guatambú, peroba or pereira. In botanical terms, this species is characterized by having glabrous, membranous leaves, silver-colored at the bottom, 5-10 cm long. The tree is 10-15 m high, with the troncode 40-60 cm in diameter. It produces annually large quantities of seeds that are widely spread by the wind. It blooms in late August with the emergence of new foliage. Plant development in the field is rapid and can reach 3.5-4.0 m at 2 years (LORENZI, H.Brazilian Trees: Manual of Identification and Cultivation of Brazilian Native Trees. Nova Odessa, SP: Publisher Plantarum, 1992) .

From the ethanolic extract of A. parvifoiium husks were isolated N-methylthihydrohydroelipticin alkaloids (BURNELL, RH and CASA, DD Alkaloids of Aspidosperma vargasii A.DC. Can J Chem. aparicin, demethylulein, in addition to a triterpene, lupeol, and a steroid, stigmasterol (JÁCOME, RLR; OLIVEIRA, AB; RASLAN, DS and WAGNER, H. Chemical study and chromatographic profile of Aspidospermaparvifoiium A. DC shells. ("Pau -Pereira "). New Chemistry, v.27 (6), p.897-900, 2004)

Ulein has already been obtained from other species of Aspidosperma, such as A.olivaceum and A. tomentosum. (GILBERT, B .; DUARTE, AP; NAKAWAY, Y.; JOULE, JA; FLORES, SE; BRISSOLE, JA; CAMPELO, J.; CARRAZZONI, EP; OWELLEN, RJ; BROSEY, EC; BROWN Jr .; KS and DJERASSI, C. Alkaloids studies (The alkaloids of twelve Aspidosperma species (Tetrahedron, v. 21, p.1141-1161, 1965)).

The ethanolic extract of A. parvifoiium peels evaluated for its salinity inhibition and callus inhibition, induced by Agrobacteriumtumefaciens, in potato discs, showed a high antitumor potential, ie, the lethal concentration 50% (LC50) was 103.8 g. / ml for salineemia and, at this concentration, 94.4% inhibition of callus formation on potato discs was inhibited. This extract also presented trypanosoma cruzi activity. (DOLABELA, M.F. In vitro screening for tumor and anti-Trypanosoma cruzi activity of plant extracts, natural products and synthetic substances. Belo Horizonte: Department of Physiology and Pharmacology, ICB, UFMG, 1997. 13Gp. Master's Dissertation). The toxicity observed in salineemia is probably related to the presence of indole alkaloids.

A mixture of indole alkaloids, predominantly containing aulein (53%), reduced acid secretion in the rodent's stomach, and this reduction may be related to H +, K + -ATPase pump block. (BAGGIO, CH; MARTINI OTOFUJI, G .; SOUZA, WM; MORAES SANTOS, CA; TORRES, LM; RIECK, L .; ANDRADE MARQUES, MC and MESIA-VELA, S. Gastroprotective mechanisms of indole alkaloids from Himatanthuslancifolius. Plant Med, v.71 (8), p.733-8 , 2005).

Another indolic alkaloid, present in A. parvifolium, aparicina, was submitted to the antifungal activity assay, but no inhibition of microorganism growth was observed. (HERNANDEZ, N.M.R. and PEREZ, C.D. Fungai activity of various alkaloids isolated from Catharanthus roseus G.Don. Rev Cuban Med Trop, v.29 (3), pp. 147-52, 1977).

We currently have some patents related to this invention available:

Patent application BE814772-A describes the obtainment of novel therapeutic alkaloids by extracting Aspidospermaspegazzinii peels. The shell was extracted with benzene or chloroform, the extractant is washed with acidic solution. The wash solution is treated with alkaline solution for precipitation of total alkaloids, which are subsequently separated by alumina chromatography.

FR2865652 discloses the use of plant extracts or active ingredients isolated from them in the preparation of pharmaceutical, cosmetic and nutraceutical compositions as lipolytic and anticellulite agents. The extracts are selected from plants of the genus: Aspidosperma; Catharanthus; Eschscholzia; Bocconia; Vismia; Orbignya; Terminalia; Entada; Hypericum; Ginkgo; Viburnum; Papaver and Butea.

US3225053 discloses the isolation of derivatized quebranchidine from the Aspidosperma extract of whitewash and its use as psychoeducatives and β-adrenergic blockers.

TECHNICAL STATE PROBLEMS

Drugs available for malaria treatment do not always yield satisfactory results. Because chloroquine is low in toxicity and economically viable, it has been 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 aartemisinin and atovacone.

However, atovacone, as well as artemisinin and its derivatives are of high cost, which makes its use in poor endemic regions unfeasible, besides the possibility of development of resistance of the parasites 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 P.falciparum strains, promoting a timely cure to ensure safe and cost-effective adherence to treatment (FIDOCK, DA; ROSENTHAL, PJ; CROFT, SL ; BRUN, S. and NWAKA, S. Antimalarialdrug discovery: efficacy models for compound screening Nat Rev Drug Discov, Jun 2004; 3 (6): 509-20).

TECHNOLOGY ADVANTAGES

Although pharmaceutical compositions for the treatment of malaria exist on the market, as well as their processes for obtaining them, none of these proposes a process of obtaining and pharmaceutical compositions of standardized extract of A. parvifolium houses, a fraction rich in eulein alkaloids with antiplasmodic activity. BRIEF DESCRIPTION OF THE FIGURES

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

Figure 2: Chromatogram of EEPPVA fraction

Figure 3: Ulein chromatogram

Figure 4: EEPPVA + ulein chromatogram

Figure 5: APT 13-16 IR Spectrum (8) (ulein)

Figure 6: APT 13-16 (8) electronic impact mass spectrum (ulein)

DETAILED DESCRIPTION OF TECHNOLOGY

The present invention describes the process for obtaining standardized extract and extract of Aspidosperma parvifolium, popularly termed "pereira, guatambu or peroba", in addition to isolation and purification of ulein. The invention further comprises obtaining pharmaceutical compositions containing the extract. , derived fraction and / or ulein, as well as its use for malaria treatment.

The composition of the present invention is characterized by the use of the extract, fraction or ulein combined with pharmaceutically acceptable excipients. Examples of excipients include water, saline, phosphate buffered solutions, Ringer's solution, dextrose solution, Hank's solution, saline solutions. biocompatible materials whether or not containing polyethylene glycol. Non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglyceride may also be used. Compositions may be prepared with such an excipient or mixture thereof.

Excipients may also contain smaller amounts of additives as substances that increase the isotonicity and chemical stability of the substances or buffers. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris1 buffer while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. The standard compositions may be liquid or solid. Thus, in a solid formulation, the excipient may include dextrose, preservatives, to which water or sterile saline solution may be added prior to administration. In addition to binders, disintegrants, diluents, lubricants, surfactants.

Such compositions may be administered intramuscularly, intravenously, subcutaneously, orally, by inhalation or as devices that may be implanted or injected, preferably administered orally.

The present invention may be better understood by the following non-limiting examples of technology:

Example 1: Obtaining the standardized extract and fraction of Asparsperma parvifolium barks

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

Ethanolic extract of A. parvifolium bark (EEPPV) (10.0 g) was solubilized in reduced ethanol (<10ml) and 1N aqueous HCL (250ml) followed by extraction with dichloromethane (3 χ 250 ml) or diethyl ether, or dichloromethane or ethyl acetate to give a dichloromethane layer (EEPPVN) and an acidic aqueous layer (EEPPVAAC). NH 4 OH was added to pH 9 and extracted with dichloromethane (3 x 250 ml) to give an alkaline aqueous layer (EEPPVAAL) and an organic layer (alkaloid fraction) (EEPPVA). The flowchart of these extractions is shown in Figure 1. Under controlled conditions, the alkaloid-rich fraction (EEPPVA) was standardized to contain 45 to 60% w / w ulein and also presented as epiulein, demethylulein and aparicin constituents according to the chromatography-standardized fingerprint. reverse phase high efficiency liquid (Figure 2). Example 2: Isolation and identification of ulein

The ethanolic extract of A. parvifolium (EEPPV) was submitted to acid-base extraction and the alkaloid fraction (EEPPVA) 1 of this fraction was obtained. After successive chromatography on Sephadex LH-20 column and silica gel, the fractions APT 13- 16 (7) and APT 13-16 (8) which presented a single CCDS stain (developers: anisaldehyde-sulfuric acid and Dragendorff's reagent). The fractions obtained were compared with the ulein sample isolated by Jácome (1998), showing very similarity. (JÁCOME, RLRP Chemical study of Zeyheria montana M. and Aspidospermaparvifolium A.DC. HPLC quantification of isolated Zmontana naphthoquinones. UFMG / ICEx thesis Belo Horizonte, 1998, 157p.)

HPLC-RF-coupled chromatograms confirmed that this was ulein, with peaks with RT 25.820 min for reference ulein (Figure 3) and 25.556 min for ulein present in the EEPPVA (Figure 2). The identification of the peak corresponding to the EEPPVA nocromatogram ulein was confirmed by co-injection with reference ulein (Figure 4) and by online UV spectra that show a maximum absorption at 304.2 nm.

APT 13-16 IR spectrum (8) (Figure 5) showed absorptions corresponding to axial deformation of CH bonds (3176 cm-1), N-H stretch (3040 cm-1), C = C bond stretch in aromatic system (1535 and 1613 cm "1) and deformation of CH bonds in an ortho-disubstituted benzene ring, which is the most intense in the spectrum (730 cm" 1). This spectra showed much similarity to that of ulein (JÁCOME, RLRP Zeyheria montana M. and Aspidosperma parvifolium A.DC.Quantification by HPLC of Z. montana isolated naphthoquinones. Thesis UFMG / ICEx / DQ. Belo Horizonte, 1998, 157p. ) as expected.

The APT mass spectrum 13-16 (8) showed the expected molecular ion in rrVz. 266, corresponding to ulein (Figure 6). Example 3: Evaluation of antiplasmodic activity

The antiplasmodic activity of the ethanolic extract of A.parvifolium husks (EEPPV), alacaloid fraction (EEPPVA) and ulein [APT 13-16 (8)] was tested by the traditional method (RIECKMAN, KH; SAX1 LJ; CAMPBELL, GH; MRENA, JF Drug sensitity of Plasmodium falciparum An in vitromicrotechnique Lancet, vi, p.22-23, 1978. OAK, LH Experimental chemotherapy with crude plant extracts and chemically defined compounds).

The evaluation by the traditional method presented the following results: in clone W2 (chloroquine resistant), the ethanolic extract of A.parvifolium (EEPPV) presented IC50 32.75 + 1.06 pg ^ nl. The alkaloid fraction (EEPPVA) and ulein were active (Cl50 <10 pg ^ nl). In clone3d7 (sensitive to mefloquine), the ethanolic extract (EEPPV) presented moderate antiplasmodic activity, whereas the alkaloid fraction (EEPPVA) and the foramative ulein (IC50 <10 pg ^ fnl) (Table 1)

Table 1: Traditional in vitro antiplasmodic activity of A. parvifolium bark extract (EEPPV), alkaloid fraction (EEPPVA) and ulein

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Example 4: In vitro cytotoxicity assessment

In vitro cytotoxicity was assessed by MTT assay (TWENTYMAN, PR, LUSCOMBE, Μ. A study of somevariables in a tetrazolium dye (MTT) based assay for cell growth andchemosensitivity. British J. Cancer, v. 56, p 279-285, 1987.) EEPPV was found to be less toxic to VERO cells (CC50> 500 pg ^ nl), while alkaloid fraction (EEPPVA) and ulein showed moderate cytotoxicity (100 <CC50 <500 pg ^ fnl ). When the selectivity index (IS = CC50 / IC50) was determined using the IC50 obtained for cloneW2 (Table 1), it was observed that the purer the sample, the higher its selectivity index, that is, the ulein presented more IS higher than the extract (EEPPV) and the fraction (EEPPVA). However, when using the results obtained with clone 3d7 (Table 1), changes in SS are slight and favorable (Table 2).

Table 2: Cytotoxicity (CC50) and selectivity index (IS) of Aspidosperma parvifolium bark extract (EEPPV), dealkaloid fraction (EEPPVA) and ulein.

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Example 5: In vivo toxicity assessment

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

Toxicological studies carried out with the EEPPV extract and the EEPPVA fraction indicate a certain degree of toxicity of these extracts at high doses, but good tolerability at low doses, ie up to 250 mg / kg (acute) or 150 mg / kg (chronic) in the case. EEPPV; or 75 and 50 mg / kg (acute screen, respectively) of the EEPPVA.

Claims (7)

1. STANDARDIZED EXTRACT AND FRACTION OF SPASTERSPERMA PARVIFOUUM SHELLS, characterized in that it comprises standard alkaloid-rich fraction containing 45 to 60% w / w deulein, in addition to the constituents epiulein, demethylulein and aparicin; STANDARDIZED EXTRACT AND FRACTION OF DEPASIDOSPERMA PARVIFOUUM SHELLS according to claim 1, characterized in that it comprises standardization based on the optimization of extraction and fractionation processes, as well as on the determined antiplasmodic activity for the main chemical marker of the fraction: ulein; STANDARDIZED EXTRACT AND FRACTION OF DEVASPIDOSPERMA PARVIFOUUM SHELLS according to claims 1 and 2, characterized in that said standard alkaloid-enriched fraction and the characteristic HPLC profile are similar to those selected in Fig. 3; 4. PHARMACEUTICAL COMPOSITION OF STANDARDIZED EXTRACT AND FRACTION OF ASPIDOSPERMA PARVIFOUUM EL OUULEINE SHELVES AND THEIR DERIVATIVES, characterized in that it comprises a standard fraction rich in alkaloids containing a range of 45 to 60% m / m deulein, in addition to the epiulein and demethylaminoin constituents pharmaceutically and pharmacologically acceptable excipient; PHARMACEUTICAL COMPOSITION OF STANDARDIZED EXTRACT AND FRACTION OF ASPIDOSPERMA PARVIFOUUM AND / OUULEINE SHELVES AND THEIR DERIVATIVES according to claim 4, characterized in that it has antiplasmodic activity; PHARMACEUTICAL COMPOSITION OF STANDARDIZED EXTRACT AND FRACTION OF ASPIDOSPERMA PARVIFOUUM AND / OUULEINE SHELVES AND THEIR DERIVATIVES according to claims 4 and 5, characterized in that it is administered by oral, intramuscular, intravenous, intraperitoneal, subcutaneous, or as a device that may transdermally implanted or injected; being preferably administered orally; PHARMACEUTICAL COMPOSITION OF STANDARDIZED EXTRACT AND FRACTION OF ASPIDOSPERMA PARVIFOLIUM AND / OUULEINE SHELVES AND THEIR DERIVATIVES according to claims 4 to 7, characterized in that they are in the preparation of medicaments for treating malaria;