Phytochemical investigation and antioxidant activity of extracts of Lecythis pisonis Camb

Phytochemical investigation of the ethanol extract of leaves, twigs and fruit shell of Lecythis pisonis Camb. revealed the presence of squalene, e -amyrin, lupeol, 3-friedelinol, ursolic and oleanolic acids, (E)-phytol, sitosterol, stigmasterol, campesterol, quercetin-3-O-rutinoside and kaempferol-3-Orustinoside. Structural elucidation was achieved using ultraviolet (UV), nuclear magnetic resonance (NMR), and mass spectrometry. This is the first report of the occurrence of flavonoids, together with squalene, lupeol and campesterol in L. pisonis. The EtOH extract of the leaves showed high antioxidant activity, which can be associated in part with the high level of phenols and flavonoids.


INTRODUCTION
Plants of the Lecythidaceae family are representatives of many neotropical forests in the Americas, Africa and Asia.This family has about 25 genera and 400 species in three subfamilies: Foetidioideae, Planchonioideae and Lecythidoideae (Mori, 2001).The genus Lecythis is little studied from the chemical point of view.There are reports in the literature on the chemical speciation of selenium (Se) in nuts of Lecythis minor (Dernovics et al., 2007), chemical analysis of the essential oils from flowers of Lecythis usitata (Andrade et al., 2000) and leaves of Lecythis persistens and Lecythis poiteau (Courtois et al., 2009).The species Lecythis pisonis Camb popularly known as sapucaia, is distributed in Brazil, within the state of Piaui and from Pernambuco to São Paulo as well as in the Amazon region (Corrêa, 1978).Traditionally, the infusion prepared from the bark of the tree is astringent and used in the treatment of diarrhea, while the leaves are used as diuretic and tonicardiac in tea or infusion or in baths to relieve itching (pruritus) and the fruits are used in the treatment of diarrhea and syphilis (Braga et al. 2007;Denadai, 2006).The phytochemical study of the leaves of this species led to the isolation of pentacyclic triterpenoids, phytol, sitosterol and stigmasterol (Oliveira et al., 2012).The ethanol extract and the mixture of ursolic and oleanolic acids obtained from the leaves demonstrated antipruritic and cytotoxic activities (Silva et al., 2012;Oliveira et al., 2012).Other studies also showed that ethanol extract, eterea fraction and mixture of ursolic and oleanolic acids from leaves of L. pisonis exhibited antinociceptive activity in models of acute pain in mice (Brandão et al., 2013).
As part of our studies aimed at exploring the chemical constituents and pharmacological potential of plant species of the cerrado and transition area in the state of Piauí, this present work describes the isolation and identification of chemical constituents from the ethanol extract of leaves, twigs and fruit shell of L. pisonis as well as the evaluation of the antioxidant potential and determination of content phenols and flavonoids total.

General experimental procedures
1 H and 13 C NMR spectra were recorded on a Varian INOVA and Bruker Avance III spectrometer at 500/400 and 125/100 MHz, respectively, using CDCl3 or DMSO-d6 as solvents.The samples were analysed by gas chromatography-mass spectrometry (GC-MS) on an Agilent Technologies 7890A GC system coupled to a 5975 VLMSD mass spectrometer equipped with a 7683B series injector device and DB-5 column (J&W, 30 m × 250 mm × 0.25 mm), with injector temperature at 250°C and temperature at the interface of 310°C.The injected volume was 1.0 ml (5 mg ml -1 ) in the split mode (10:1).Helium was used as carrier gas, at a velocity of 1 ml min -1 .Column temperature was initially maintained at 200°C for 4 min, followed by a heating gradient of 6°C min -1 until reaching 290°C.This temperature was maintained for 15 min after which a new heating gradient of 2°C min -1 was applied until reaching 305°C.This temperature was maintained for 5 min.The obtained mass spectra were compared to library data Nist 0.5.The absorption measurements were determined by an UV-Vis spectrophotometer PerkinElmer, Lambda 25.The chromatographic plates were prepared using Fluka silica gel G and the revealed were made by spraying the plates with a solution of Ce(SO4)2.The atmospheric pressure chromatographic columns were prepared with silica gel 60 (70 to 230 mesh) from Acros Organics or Sephadex LH-20 (Aldrich).For low-pressure chromatography column Büchi Switzerland with silica gel (40-60 µm; 12 × 150 mm) coupled to a Büchi B-688 pump was used.The semipreparative high performance liquid chromatography (HPLC) was performed on a Shimadizu LC-6AD system equipped with a Phenomenex column (Luna C18, 5 µm, 150 × 21.2 mm).

Plant
The leaves and twigs L. pisonis Camb.were collected in July, 2008 and fruit shell in July, 2010, on the Campus Ministro Petronio Portela of the Universidade Federal do Piauí -UFPI, in the city of Teresina, Piauí State, (South latitude = 05° 02' 53.2",West longitude = 42° 47' 16.8", at the level of 68 m).A voucher specimen was identified and deposited in the Herbarium Graziela Barroso at the UFPI, under accession number TEPB 26488.

Determination of antioxidant activity and dosage of total phenolics and flavonoids
The antioxidant activity was evaluated by 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical consumption, and the quantification of phenolic compounds determined by Folin-Ciocalteu method and expressed in gallic acid equivalent (GAE) according to Sousa et al. (2007) and Costa et al. (2010).The total flavonoid content was determined by molecular absorption spectrometry, following the methodology described by Sobrinho et al. (2010).Stock solutions of EtOH extracts and fractions (1000 µg ml -1 ) were prepared and then an aliquot of 300 µl of these solutions was transferred to 10 ml flasks to which 0.24 ml of acetic acid, 4 ml of a methanolic solution of pyridine at 20% and 1 ml of a methanolic solution of aluminum chloride (50 mg ml -1 ) were added, completing the volume with distilled water.Control was prepared in parallel.

Compounds Hexane fractions Leaves
a Only identified by GC-MS.After 30 min, the absorbance of samples was measured at 420 nm using glass cuvets.The total flavonoid content (TFC) was determined by interpolating the absorbance of the samples against a calibration curve constructed with standard rutin at concentrations of 3, 6.5, 10, 13.5, 17 and 21 mg l -1 , obtained from a stock solution of 1000 mg l -1 in MeOH:H2O (7:3).To each flask containing 10 ml of these solutions, 0.24 ml of acetic acid, 4 ml of a methanolic solution of pyridine at 20% and 1 ml of methanol solution of aluminum chloride (50 mg ml -1 ) were added completing with distilled water.
After 30 min at room temperature, lecture was performed using a spectrophotometer at 420 nm.Values are expressed as milligrams of equivalent rutin per gram of dried plant material (mg of ER g -1 of DPM).The straight line equation is: A = 0.0262C -0.0072,where A is the absorbance, C is concentration and linear correlation coefficient of r 2 = 0.999.All analyzes were performed in triplicate (n = 3).

RESULTS
The phytochemical study of hexane fractions obtained after partition of the EtOH extract of the leaves, twigs and fruit shell of L. pisonis resulted in the isolation and identification by GC-MS, 1 H and 13 C NMR of seven triterpenoids (1 to 7), three steroids (8 to 10) and a diterpenoid (11) listed in Table 1 and Figure 1.The triterpenoid lupeol (3), α-and β-amyrin (4 and 5) and steroids sitosterol (8) and stigmasterol (9) were identified in all samples.Squalene (1) and 3β-friedelinol (2) were identified only in leaves, while campesterol (10) was identified only in the twigs.The ursolic (6) and oleanolic (7) acids were identified in the leaves and twigs, but were not obtained from fruit shells.Fractionation of the EtOAc fraction, resulting from the partition of EtOH extract of the leaves of L. pisonis resulted in the isolation of the flavonoids quercetin-3-O-rutinoside (12) and kaempferol-3-O-rutinoside (13) (Figure 1).The structural identification of these compounds was based on analysis of UV spectra, NMR (1D and 2D).
The antioxidant activity (AA%) of the EtOH extracts of leaves, twigs and fruit shells of L. pisonis and positive control (rutin) in concentrations ranging from 25 to 250 μg ml -1 , are shown in Figure 2. The extracts and control showed concentration dependent antioxidant activity.Table 2 presents the results of the evaluation of EtOH extracts of leaves, twigs and fruit shells on the content of flavonoids and phenolics and total antioxidant activity expressed by EC 50 .The EtOH extract of the fruit shell   showed no significant antioxidant activity.The total phenols and flavonoid contents, determined in relation to the dry plant material, lying in the range 0.88 to 56.78 mg of GAE g -1 and of 0.73 to 30.04 mg of ER g -1 , respectively, with the highest concentrations of these constituents being obtained from the EtOH extract of the leaves (Table 2).Table 3 shows the levels of total phenols and flavonoids of partition fractions of EtOH extracts of leaves and twigs, which proved to be richer in these constituents.Significant differences between fractions were observed (p < 0.05).The highest total phenols and flavonoids contents were recorded for the aqueous fraction of the leaves; however it was not possible to determine the flavonoid in hexane fractions of leaves and twigs due to the absence or low content of these constituents.

DISCUSSION
The structural identification of substances 1 to 11 was performed by GC-MS analysis, 1 H and 13 C NMR and comparison with literature data (Olea and Roque, 1990;Rahman and Ahmad, 1992;Mahato and Kundu, 1994;Junges et al., 2000;Salazar et al., 2000;De-Ekankul et al., 2003).The lupeol (3, leaves) and campesterol (10, twigs) were identified solely on analysis by GC-MS due to the low concentration of these substances in samples.Squalene, lupeol and campesterol are reported for the first time in L. pisonis.
The EtOH extracts of leaves and twigs, in concentrations of 200 and 250 μg ml -1 showed antioxidant activity comparable to percentage of the positive control (rutin), while the EtOH extract of the fruit shell was the least active in all concentrations tested.Antioxidant activity was also evaluated by EC 50 , thus the EtOH extract of the leaves (EC 50 = 49.04 ± 1.65 μg ml -1 ) was comparable to the positive control rutin (EC 50 = 47.08 ± 4.65 μg ml -1 ).According to Rufino et al. (2010), the content of phenolic compounds, expressed as gallic acid equivalents per gram of dried plant material may be classified into low (< 10 mg GAE g -1 ), medium (10 to 50 mg GAE g -1 ) and high (> 50 mg GAE g -1 ).In this study, the content of phenolic compounds was high for the EtOH extract of leaves, medium for the twigs and low for the fruit shell.A positive correlation between the EC 50 and the content of phenols and flavonoids of EtOH extracts of leaves, twigs and fruit shells was observed.This behavior is generally expected, considering that the phenolic compounds and in particular flavonoids are free radicals scavengers and consequently exhibit antioxidant properties.
The phenolic contents for the fractional partition EtOH extract of the leaves ranged from 0.06 to 42.81 mg GAE g -1 of DPM while flavonoids contents ranged from 2.37 to 10.59 mg ER g -1 of DPM.Although the total phenols of EtOAc fraction of leaves was lower than in the EtOAc fraction of the twigs, the content of flavonoids was higher, being explained partly by the presence of flavonoids quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside, that were detected in the EtOAc fraction of leaves but not from twigs, according to TLC analysis.

Table 3 .
Total phenols (TP) and total flavonoid content (TFC) in fractions of a partition of ethanol extracts of L. pisonis.