Myricitrin as the main constituent of two species of Myrcia

Article history: Received on: 04/12/2013 Revised on: 22/01/2014 Accepted on: 17/02/2014 Available online: 27/02/2014 Myricitrin was isolated as the major constituent from Myrcia splendens (2 g %) and Myrcia palustris (0.5g %) of their fractions soluble in ethyl acetate. The presence of this compound was confirmed by chromatographic analyses using HPLC-DAD. The antioxidant activity was measured using reducing power in relation to the reduction of iron (III) to iron (II) ions and DPPH radical scavenging assay. The ethyl acetate and butanol fractions showed the highest antioxidant activity for both species. In relation to the ability of the sample to scavenge DPPH radicals the EC50 values were 8.44 and 9.35 μg/ mL for M. splendens and 17.83 and 15.34 μg/ mL for M. palustris for the ethyl acetate and butanol fractions, respectively. The antioxidant activity may be related to the myricitrin in these extracts that showed an EC50 = 13.93 μg/mL.


INTRODUCTION
The family Myrtaceae is one of the most characteristic in Brazil, presenting significant potential economic benefits.The species of this large family are used as medicinal plants, ornamental plants, fruit trees and to obtain wood and paper.The family belongs to the order Myrtiflorae (Myrtales) and comprises around 150 genera with an estimated 5500 species predominantly distributed in tropical America, South Australia and Southeast Asia (Cronquist, 1981;Barroso, 1991;Wilson, 2011).Myrcia is one of the largest genera of American Myrtaceae, with more than 300 species distributed from Mexico to southern Brazil.Seventeen of these species are found in southern Brazil (Marchiori, 1997).Myrcia spp.are popularly known as "insulin plants", notably M. multiflora (pedra-ume-caá), which is used as a hypoglycemic agent in the form of an infusion or decoction (Brito & Lanetti, 1996;Batista et al., 2011).M. splendens (Sw.)DC., known in Brazil as "guamirim" or "folha-miúda", has been widely observed from Mexico to southern Brazil.There are several botanical names for this species, including M. acutata DC., M. splendens DC., . .

M. communis
Berg & M..fallax (Rich.)DC. (Oliveira-Filho, 2006;Morais & Lombardi, 2006).Myrcia palustris DC., known in Brazil as "pitangueira-do-mato", "murta-do-brejo" or "baga-desabiá", is a native species found mainly in the south and southeast of Brazil.This species is commonly reported in the literature with the names M. banisteriifolia (DC.), M. garopabensis Cambess., Gomidesia palustris (DC.)Kausel, G. banisteriifolia (DC.)O.Berg, G. gardneriana O.Berg, G. garopabensis (Cambess.)D. Legrand (Sobral et al., 2010).The plants of the Myrcia genus contain triterpenes and steroids (Gottlieb et al., 1972) as well as flavanones, acetophenones and flavonoid glycosides (Yoshikawa et al., 1998;Ferreira et al., 2006) and these compounds show different activities, notably inhibition of aldose reductase and alpha-glucosidase (Matsuda et al., 2002a,b).Studies on essential oils conducted on these species have demonstrated a high content of cyclic sesquiterpenes and varying amounts of monoterpene and aliphatic compounds (Zoghbi et al., 2003;Cole et al., 2008;de Cerqueira et al., 2009;Nakamura et al., 2010).The aim of this study was to test the crude hydroalcoholic extract and n-hexane, ethyl acetate, nbutanol and insoluble fractions of M. splendens and M. palustris in order to investigate their antioxidant activity using DPPH free radical scavenging and reducing potential assays.Also, the major compounds in the most active fractions were identified by HPLC-DAD.

Chemicals and reagents
All reagents and solvents were analytical or HPLC grade (J.T. Baker, USA).The water used in all experiments was purified in-house using a Milli-Q system (Millipore, Florianópolis, SC, Brazil) with a conductivity of 0.60 μS/cm.Rutin (Sigma, USA) of the highest grade (purity > 98.0%) and myricitrin (as isolated compound) were used as the external standards.

Plant Material
Plant

Preparation of Vegetal Material
The plant samples were dried and macerated with 96% ethanol for seven days.After filtration, the extracts were concentrated to dryness in order to obtain the crude extracts.The crude extracts were dissolved in 30% EtOH and left in a refrigerator for one day and an insoluble fraction was precipitated.The remaining filtrates were sequentially extracted with solvents of increasing polarity, yielding n-hexane, ethyl acetate, butanol and aqueous fractions.The crude extracts and fractions were subjected to antioxidant assays.

Determination of total phenolics
The total phenolic content of the samples was determined using the Folin-Ciocalteu reagent.In this method, the test solution was composed of 0.5 mL of sample (crude extracts and fractions, diluted in methanol at a concentration of 1.0 mg.mL -1 ), 5.0 mL of distilled water and 0.5 mL of the Folin-Ciocalteu reagent.After a period of 3 min, 1.0 mL of saturated sodium carbonate solution was added.These mixtures were shaken and allowed to stand for 1 h.The absorbance was measured at 725 nm against a blank (solution without sample).The mean of three readings was used and expressed as gallic acid (GA) equivalents (mg gallic acid/g dry extract).

Determination of flavonoid content
The flavonoid content was determined spectrophotometrically according to Lamaison and Carnat (Quettier-Deleu et al., 2000) with modifications.Briefly, 0.5 mL of 2% aluminum chloride (AlCl 3 ) in ethanol was mixed with the same volume of sample (1.0 mg.mL -1 ).Absorption readings at 415 nm were taken after 1 h against a blank (ethanol).The mean of three readings was used and expressed as quercetin equivalent (QE); (mg quercetin/g of dry extract).

Determination of reducing power
In this assay, 100 µL of sample (1.0 mg.mL -1 ) was added to 8.5 mL of deionized water followed by 1.0 mL of 0.1 M FeCl 3 in 0.1 M HCl and after 3 min 1.0 mL of 0.008 M potassium ferricyanide was added.After 15 min the absorbance was measured at 720 nm.A solution prepared as described above but without the addition of FeCl 3 solution was used as the blank.The analysis was performed in triplicate and the results were expressed as ascorbic acid (AA) equivalents (mg ascorbic acid/g dry extract).

DPPH radical scavenging assay
The free radical scavenging activity of the samples was measured using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay.DPPH is a stable free radical that accepts an electron or hydrogen atom to form a stable molecule.A change of color from purple to yellow indicates a decline in the absorbance of DPPH radicals at 517 nm caused by the reaction between the antioxidants present in sample and the free radical.For this assay 1 mL of sample (5-200 µg/mL -1 ) was added to 2 mL of a solution of DPPH in methanol (0.004%).The mixture was shaken vigorously and allowed to stand for 30 min at room temperature.In order to subtract the absorbance promoted by the staining of the sample, a solution of ethanol (2.0 mL) and the sample (1.0 mL) was used as the blank (Abs blank ).A solution of DPPH (2.0 mL) and ethanol (1.0 mL) was used as the control (Abs control ) which is regarded as 100% of DPPH.The absorbance of the resulting solution (Abs sample ) was measured at 517 nm and converted into percentage of antioxidant activity (AA) using the following formula: AA% = 100-{[(Abs sample -Abs blank )×100]/Abs control }.Ascorbic acid, quercetin and myricitrin were used as standards.The radical scavenging activity was expressed in terms of the amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (EC 50 ).The EC 50 value for each sample was determined graphically by plotting the percentage disappearance of DPPH as a function of the sample concentration.

Statistical analysis
The results were subjected to analysis of variance (ANOVA) and the Fisher's test for multiple comparisons, considering statistical significance as p<0.05.

HPLC analysis
In the HPLC analysis, a Shimadzu HPLC system consisting of a SCL-10AV system controller, LC-10AT pump, CTO-10AS column oven and SPD-10AV DAD was used.The column was a Varian C18 column (250 mm × 4.6 mm; 5µm) and the analysis was carried out at 25 °C.The mobile phase was methanol (A) and water (B); both acidified with 1.0 % formic acid (FA).The optimized gradient profile began at time t = 0 min with the solvents A (35%) and B (65%), changing after 10 min to eluent B (55%), 25 min to B (45%), 30 min to B (40%) and 32 min to B (0%).The flow-rate was 1.0 mL/min, injection volume was 10 µL and the UV-Vis diode array detector (DAD) reading was acquired at 354 nm.Data acquisition was performed using the Shimadzu Class VP Software.The samples (ethyl acetate and butanol fractions) were injected without pre-treatment at a concentration of 200 µg/mL -1 in MeOH/H 2 O (2:8).
The same compound was obtained from the ethyl acetate fraction of M. palustris after the process of liquid-liquid partitioning in the form of a precipitate.Was obtained 166.0 mg of the compound from 30.3 g of the ethyl acetate fraction which corresponds to approximately 0.5%.

RESULTS AND DISCUSSION
The crude hydroalcoholic extract and the fractions of the liquid-liquid partition (insoluble fraction, n-hexane, ethyl acetate, butanol and aqueous fractions) were subjected to four different assays: determination of the content of total phenolics and flavonoids, reducing power and DPPH radical scavenging.According to Table 1, the concentrations of flavonoids and phenolic compounds were observed in the ethyl acetate and butanol fractions of M. splendens and M. palustris.Likewise, in relation to the antioxidant assays, for the reducing power and DPPH radical scavenging the best results were also obtained for the ethyl acetate and butanol fractions.The EC 50 values for the ethyl acetate and butanol fractions of M. splendens were very similar to those of the positive controls.These results are consistent with those obtained in studies on the chemotaxonomy of the Myrcia genera, highlighting a large number of flavonoid glycosides (Gottlieb et al., 1972, Matsuda et al., 2002a, Ferreira et al., 2006;Imatomi et al., 2013) and tannins (Yoshikawa et al., 1998) mainly in the more polar .fractions obtained from liquid-liquid partition.The results for the determination of phenolic and flavonoid contents and the antioxidant assays indicated that the flavonoids in the more polar fractions of the two plant species, that is, the ethyl acetate and butanol fractions, merit further investigation.Therefore, a methodology for the analysis of flavonoids by HPLC was developed for the ethyl acetate and butanol fractions of M. splendens and M. palustris.
The chromatographic profile of the fractions can be seen in Fig. 1.The ethyl acetate fraction of M. splendens (Fig. 1-a) showed five major peaks at 10.8 min (11.3 %), 11.8 min (6.4 %), 12.8 min (35.7 %), 14.3 min (6.9 %) and 14.4 min (7.2%), which together represent almost 80 % of the total area.The chromatogram showed a more intense peak at 12.8 min and its UV spectrum, obtained in the HPLC analysis, showed maximum absorption bands at 265 nm and 356 nm.This result suggests the presence of a flavonoid containing, as the aglycone core, a flavonol which has two characteristic absorption bands: band II (250-280 nm) and band I (330-360 nm for substituted flavonol 3-OH or 350-385 nm for unsubstituted flavonol 3-OH) (Markham, 1982).The butanol fraction provided a different profile (Fig. 1-b), with 10 peaks and two major retention times of 11.1 min (35.4 %) and 13.0 min (24.6 %), which together represent 60% of the total area.Furthermore, this fraction contained a low concentration (24.5%) of flavonoids in relation to the total phenolics.In contrast to the other fractions, the flavonoids in the ethyl acetate fraction of M. splendens represented 86.8 % of the total phenolics and in the ethyl acetate and butanol fractions of M. palustris they represent 97.1% and 74.0% of the total phenolics, respectively (Table 1).The chromatographic profiles for Myrcia palustris (Fig. 1-c and 1d) were similar to that for the ethyl acetate fraction of M. splendens.The ethyl acetate and butanol fractions of M. palustris showed an intense peak with the same retention time of 12.8 min, corresponding to 77.8 % and 71.7 %, respectively.
The HPLC analysis was performed with two standards of flavonol heterosides, myricitrin and rutin, using the analysis conditions applied for the fractions, in order to determine the major compounds observed in the fractions of the two species.The major compound in myricitrin showed the same retention time as that of the fractions (12.8 min), as seen in Fig. 2. The peak at 14.3 min relates to rutin and a small signal at 17.7 min is an impurity, since the myricitrin standard was an isolated compound and not a commercial standard.
The myricitrin standard was therefore used for the coinjection analysis in order to confirm the presence of this flavonoid in the samples.The chromatograms with and without co-injection were superimposed to check for similarity (Fig. 3).A substantial increase in the peak at 12.8 min for the flavonoid myricitrin was observed in the co-injection chromatograms.
According to the chromatograms, it can be inferred that the flavonoid myricitrin is present in all fractions with the exception of the butanol fraction of Myrcia splendens (Fig. 3).The chromatographic profiles were almost the same for the other three fractions with an increase in the area of the compound at 12.8 min.The profile of the co-injection chromatogram for the butanol fraction of M. splendens (Fig. 3-b) did not indicate the presence of the flavonoid myricitrin and in Fig. 1-b it can be seen that the most intense peak is at a retention time of 11.1 min and not at 12.8 min as in the case of other fractions.
The liquid-liquid partitioning of a crude hydroalcoholic extract of species leaves resulted in the isolation of myricitrin (1) from the ethyl acetate fraction of M. splendens and M. palustris.Physical data, spectroscopic data ( 1 H and 13 C NMR, IR), and comparison with the data from the literature were utilized to identify the compound 1 (Agrawal, 1989).
Myricitrin has been isolated from other species of the Myrcia genera and shown to exert multiple pharmacological activities.The aqueous partition of the methanolic extract of M. uniflora and its isolated myricitrin were able to inhibit thyroid peroxidase activity in vitro (Ferreira et al., 2006).The methanolic extract and ethyl acetate-soluble portion obtained from leaves of Myrcia multiflora demonstrated the inhibition of aldose reductase and α-glucosidase.One of the components isolated was myricitrin which also showed potent inhibitory activity (Matsuda et al., 2002b).

CONCLUSIONS
This study demonstrated the antioxidant activity of the more polar fractions of the two Myrcia species obtained in the liquid-liquid partitioning.The antioxidant activity may be related to the flavonoid composition of the extracts, most fractions being mainly composed of myricitrin.

Table . 1
: Antioxidant activity of crude extract and fractions of M. splendens and M. palustris.All means, except those marked by ( a , b , c ), showed significant differences in the Fisher test (p<0.05).