Preliminary study of the extract of the barks of Licania macrophylla Benth : phytochemicals and toxicological aspects

The objective of this research was to evaluate the phytochemical, physicochemical and toxicity profile of ethanol crude extract of L. macrophyllain relation to Artemiasalina L. The Phytochemical analysis of the ethanol crude extract of the barks and stem and physicochemical calculation were performed by methods found in the Brazilian Pharmacopoeia. Phytochemical analysis detected saponins, organic acids, reducing sugars, tannins, anthraquinones, and depsidedepsidone. In relation to the physicochemical parameters, it showed pH = 4.64, due to the occurrence of saponins, organic acids and tannins; lipid = 0.55% indicates that the material presents few lipids of low molecular weight; humidity = 12.09% ± 0.12, relates to a small amount of water, indispensable for non occurrence of development of microorganisms or enzymatic degradation. Waste by incineration is within the pharmacognostic standards of 8.30% ± 0.54. The crude extract is practically nontoxic with LC = 1253ìg/mL, since the mortality rate and LC should be higher than 1000ìg/mL 50 50 to be considered nontoxic. Phytochemical analysis have confirmed, in part, the use of species for phytotherapic purposes, however, the type of treatment and packaging can influence the determination of secondary metabolites. The adopted physicochemical parameters have shown that the species is free of decomposersagents.


Introduction
stimulated the popular and institutional interest in Medicinal plants were used for many years as the first strengthening the use of medicinal plants in its several preventive measure in treatment and cure of diseases pharmaceutical ways, defined by herbal medicine, (CARVALHO et al., 2010).From popular and ethnicities without the use of isolated compounds.In Brazil, during the use there were known and discovered several species with 80's, begins the interest of inserting herbal medicines in pharmacological potential, thus, the efficacy of the use of the Unified Health System (SUS -in Portuguese), however, the plant contribute to the relevance and realization of only in mid-2006 it was approved by the National Policy the associated therapeutic properties (HOCAYEN et al., on Traditional Medicine and Herbal Medicines by Decree 2012).Herbal medicine (Phytotherapy) has emerged as a 5833, 22nd June 2006(BRASIL, 2006 apud KUNZ, medical option well accepted and accessible to people of 2007).the world, and in the case of Brazil it is pleasant to the In the Chrysobalanaceae family there are 18 genera needs of hundreds of local municipalities in primary and presents 531 species, commonly distributed in health care (MELO et al., 2007).
tropical and subtropicas areas (HEMSING; ROMERO, The functional activity of a natural product has 2010).The stem bark of the species was separated for drying 30m height and upper limbs in its top emerging in the at room temperature and powdered in a cutting mill at the rainforest.The flower, by difference, is compared, Bioprospecting and Atomic Absorption Lab -UNIFAP.After although almost every genus is characterized by drying and pulverizing, the vegetal material was placed in fundamental uniformity of inflorescence and floral a round-bottomed flask with ethanol 96ºGL in proportion structure (ZUQUE et al., 2004).Its leaves are 1:2 (w/v), comprising an extraction period of 4 days until characterized by a simple and alternate shape, exhaustion.Subsequently, the extract was concentrated on pentamerous flowers, free petals, superior ovary and a rotary evaporator under reduced pressure and placed ovaries and erect ovules.In its composition there is in an erlenmeyer in a desiccator.composition of flavonoids, triterpenes, diterpenes, steroids and tannins (YAKANDAWALA et al., 2001;LOCATELLI, 2009).

Physicochemical analyzes In African continent, traditional population widely uses
The adopted physicochemical parameters in this study some species of Chrysobalanaceae family for treatment were: pH, Waste by Incineration (Ash), Lipids and of epilepsy, malaria, dysentery and toothache.The Licania Humidity.They were carried out according to standards macrophylla Benth is popularly known as "anauera" or of the Adolfo Lutz Institute (2008) and Brazilian "anuera", and in Brazil, specifically in the Amazon region, Pharmacopoeia (2010).the species is used in the prophylaxis of parasitic amoebic and dysentery disorders (MEDEIROS et al., 2012).
Phytochemical study and sample preparation for Secondary metabolites comprise a rich and diverse toxicological testing class of compounds intended to provide functional A phytochemical screening of the obtained extract was activities or properties of a given plant.However, the conducted with the use of specific revealing reagents, quality of plant material does not guarantee itself the according to the methodology proposed by the Brazilian safety and efficacy of the final product, since the Pharmacopoeia (2010)and Macêdo (2005) for organic photochemical analysis is guided of qualitative tests for acids, reducing sugars, saponins, polysaccharides, phenols the detection of metabolites classes.Then, the and tannins, flavonoids, alkaloids, purines, steroids and p h y s i c o c h e m i c a l a n a l y s i s a s s o c i a t e d w i t h triterpenes, and depsides and depsidone, anthraquinones pharmacological ensures, partly, the effectiveness through and catechins.

pre-clinical and clinical tests of the recommended effects
The cytotoxicity assay of Artemia salina was based on (SIMÕES et al., 2010).
the technique of Araujo et al., (2010) and Lôboet al., In the methanol extract of the stem barks and flowers of (2010) with some modifications.Initially, it was prepared L. macrophylla Benth, were isolated and determined 250 mL of synthetic sea salt solution (35.5 g/L) for structures of flavanol (-)-4'-O-methyl-epi-gallocatechinincubation of 25mg of eggs of A. salina, which were 3'-O-á-L-rhamnoside, licanol, along with nine compounds exposed to artificial light for 24h for larvae hatching known and identified as (-)-4'-O-methyl-epi (metanauplius), then, the metanauplius were separated gallocatechin, pheophytin A, 132-hydroxy-(132-S)and placed in the dark environment for a period of 24h.pheophytin A, pheophytin B, sitosterol, stigmasterol, The mother solution was prepared containing 62.5 mg of sitosterol-â-O-glucoside, alcohol betulinic and oleanolic the crude extract from the stem barks, added 28 mL of acid (MEDEIROS et al., 2012).synthetic sea salt solution and 2 mL of dimethylsulfoxide The toxicological analysis of Artemia salina L. is a (DMSO) to facilitate the solubilization.preliminary bioassay that allows to estimate the Subsequently, at the end of the dark period they were evaluation of the toxicity of the extract, through the lethal selected and divided into 7 groups with 10 subjects in each concentration (LC ) of the fraction or active components 50 test tube, and in each group it was added an aliquot with facing to a marine organism.It is an essential test to draw al from mother solution (3125, 2500, 1250, 625, determine the biological activity potential of the 250, 25 to 2.5 ìL) in which the volume was completed to 5 compound or extract (NASCIMENTO et al., 2008).This mL with the synthetic sea salt solution, and the final work aimed to evaluate the phytochemical and solutions had concentrations varying from 1250, 1000, physicohemical profile of the plant species and determine 500, 250, 100, 10 to 1 µg/mL, thereby, the groups were the toxicity of the ethanol crude extract of L. macrophylla designated according to their respective concentration Benth in relation to larvae of Artemia salina L. and all tests were performed in triplicate.

Collection and identification of vegetal material
The preliminary phytochemical analysis of the ethanolic The vegetal material was collected in the Fazendinha extract of the stem barks of L. macrophylla Benth revealed Environmental Protection Area, in Macapá.The species the presence of secondary metabolites: saponins, organic was identified by Prof. Dr. Wellgliane Pararício, at the acids, reducing sugars, tannins, anthraquinones, depsides Herbarium of the Federal University of Amapá-UNIFAP.
and depsidones.The results can be observed in Table 1.
The determined values in physicochemical analysis significant with (p=0.0011),thereby, increases the of stem bark of L. macrophylla Benth are shown in mortality rate due to reduction in the determination of

Discussion
daily activities (CANIATO et al., 2004).The saponins present anthelmintic, antiviral and anti- The tannins presented activity in the treatment of inflammatory activities, the anthraquinones stand out as diarrhea, rheumatism, burns, and renal and urinary allelopathic, laxative, antibacterial and antifungal system problems (SIMÕES et al., 2010).The presence of properties, the presence of these secondary metabolites tannins in the plants is associated with the biological role corroborates, in part, with the population use of the of chemical defense against microorganism, and plant.
vertebrate or invertebrate herbivores attacks.It's Saponines, in general, presents amphiphilic character believed that their activities are due to, by part, the and capability of complex to form steroids, proteins and ability to form complexes with other molecules including membrane phospholipids, behaviors that determines the macromolecules, as proteins and polysaccharides number of biological activities for this compound, as (OLIVEIRA; RAMOS; ALMEIDA, 2013).described in the literature, such as hemolytic, ichtyotoxic, Depsides and depsidones have been recognized for molluscicide, anthelmintic and spermicidal (SIMÕES et presenting antioxidant, antiviral, antitumor, analgesical al., 2010;OLIVEIRA;RAMOS;ALMEIDA, 2013).The and antipyretic activities.This class of secondary mechanism of action purposed for some saponines is in metabolic gives a bitter taste in various species of plants the capability of break the plasma membrane of in that they are present, a property related also to microorganisms, resulting in extravasation of cellular organic acids (MENDES et al., 2011).content and, finally, death.Another action of saponines is Anthraquinones and some isolated substances, like their use in human diet for reduction of serum levels, aloine, are used as laxative, and usually have toxic because this compound interacts with cholesterol in their properties, for which is attributed the chemical defense complexation and oxidation (KAISER et. al., 2010).In function, related to alleopathic activity, that is, that way, it reinforces the use of this species by production and excretion of substances to environment traditional communities and points for a more detailed capable of inhibit the growing of other germinative study of the isolated and purified active principles.
species, act against termites and raise the value of Organic acids presents antifungal activity, as commercial woods that have such secondary metabolic described in literature (SIMÕES, 2010), are largely used (SIMÕES, 2010). in food industry as additives, can act since as There is absence of studies records for the species antimicrobial agents until antioxidants (FIORUCCI; aphrodisiac activity, because, according to Medeiros et SOARES; CAVALHEIRO, 2002;OLIVEIRA;RAMOS;al., (2012), there are reports of use of stem bark by ALMEIDA, 2013).
indigenous communities as a sexual stimulant.

Plants have the ability of cumulate organic acids in
In study of Gomes et al. (2006) on the chemical their vacuoles; this can be evidenced in citric fruit juices, composition of leaves of species L. macrophyllaBenth due to the presence citric acid.These acids are not from the Combu Island -Pa region, specifically of restricted to the fruits and can appear also in many ethanol crude extrate, detected the positive presence for plants leaves.Organic acids have bacteriostatic power flavonoids saponnins, phenols, steroids and triterpenoids.and bactericidal gram-negative, in-vitro, provided acid In comparison to results found, it's observed equivalence molecules find themselves ionized (IHEJIRIKA, 2011; in results.However, the presence of flavonoids in the KUMAR et al., 2011) Technological Research Institute, in Porto Grande, of sugars in which are reduced, these compounds are demonstrated activity against bacteria multiresistant to readily excreted from the body without help from drugs, assigning to (-)-4'-O-methyl-epi-gallocatechin-3'carriers, decreasing cellular activity without causing O-á-L-rhamnoside, a flavonoid never before described stress or premature cellular aging (SILVA et al., 2003;in scientific literature. QUADROS et al., 2010).
The pH 4.64 value of L. macrophylla Benth, Reducing sugars are characterized by the end of characterizes the presence of potentially acidic carbon chain,bycarbon atoms not spatially prevented to substances.The presence of secondary metabolites as react, that is, carbons that are not evolved in glycoside saponins, organic acids and tannins tend to lower the pH bonds, for instance, fructose and galactose.In the other of the species, since the presence of a carboxyl group on hand, saccharose is an antioxidant but not a reducing aglycone or the sugar chain increases the release of ions sugar.Utilization of fruits of species are considerable + H the reaction medium (SIMÕES et al., 2010).source of nutrients and energy for the maintenance of

Table 2 .
The crude extract, according to the toxicity the LC value.Graphic 1 expresses the relation 50 test, is nontoxic, because the LC calculated was between mortality and extract concentrations for

Table 1 .
Preliminary phytochemical analysis of ethanol crude extract of stem barks of L. macrophylla Benth.

Table 2 .
Physicochemical characterization of the stem barks of L. macrophylla Benth.Toxicity test of the extract of L. macrophylla retated to A. salina L. in different concentrations.Ramos Preliminary study of the extract of the barks of Licania macrophylla Benth et al. | . These compounds were detected in branches was not observed, because the non detection ethanolic leaves' brute extract, what justifies, in part, the may be associated to the low concentration of this use of the vegetal species for these activities.