Biological Activities of Lignoids from Amazon Myristicaceae Species : Virola michelii

*e-mail: myoshida@iq.usp.br # This paper is dedicated to Prof. Otto R. Gottlieb, who made an important phytochemical contribution to plant species from the Amazon rain forest, describing the occurrence of neolignans in Lauraceae and Myristicaceae species (Gottlieb, O. R.; Yoshida, M.; Lignans, in Natural Products of Woody Plants: Chemicals Extraneous to the Lignocellulosic Cell Wall, J.W. Rowe, ed., Springer-Verlag, Berlin, 1989, ch. 7.3, pp. 439-511). Biological Activities of Lignoids from Amazon Myristicaceae Species: Virola michelii, V. mollissima, V. pavonis and Iryanthera juruensis #


Introduction
Myristicaceae is a botanical family composed of 18 genera and about 500 species with pantropical distribution.The species are normally trees, exceptionally shrubs, frequently found in lowland forests.In Brazil, this family is represented by the genera Compsoneura, Iryanthera, Osteophloeum, Otoba (syn.Dialyanthera) and Virola, which are concentrated in the Amazon region, where the myristicaceous species are popularly known as "ucuúba". 1,2he species belonging to this family became the target of intense study when Schultes reported that some indigenous tribes of the Amazon used the barks and resins of Virola spp.for the preparation of hallucinogenic snuffs and arrow poisons for hunting. 1 The phytochemical investigations on myristicaceous species have shown the accumulation of a variety of lignoids in their tissues, which have relevant pharmacological activities that are well documented in the literature. 3,4While there are several studies about the chemical composition of Amazon Myristicaceae species, the biological activities of these species could be better studied.This paper aims to contribute to the chemosystematics of Myristicaceae species and to assay some biological activities in vitro, by reporting a phytochemical re-investigation of the fruits from Iryanthera juruensis and Virola pavonis and of the leaves from Virola michelii, as well as a new study of the fruits of Virola mollissima as well as the results of biological assays of isolated lignoids.Plant material About 1.0 kg of ripe fruits from I. juruensis Warburg were collected on the campus of the Universidade Federal do Amazonas (March 2003), in Manaus-AM, while the fruits of V. mollissima (Poepp.ex A. DC.) Warburg and V. pavonis (A.DC.) A.C. Smith were obtained from the Adolpho Ducke Reserve, Manaus-AM, in November 2003, and March 2004, respectively.The botanical materials collected were identified by José Ferreira Ramos.Virola michelii Heckel leaves were collected on Campus I of the Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus-AM, in December 2004.Vouchers were deposited at the INPA herbarium under the numbers: 213356 (I.j.), 179760 (V.mo.), 214917 (V.p.) and 215688 (V.mi.).

Extraction and isolation
The ripe fruits (I.juruensis, V. mollissima and V. pavonis) were manually separated into pericarps and seeds and the latter into arils, seed coats and almonds, which were dried, milled and macerated individually with hexane followed by EtOH, during a total period of fourteen days.After the extractions, the solutions were concentrated under reduced pressure yielding their hexane and EtOH extracts.The crude hexane extracts were dissolved in 9:1 MeOH:water, and submitted to partition with hexane, yielding the hexane and hydromethanolic fractions.Similarly, the crude EtOH extracts were partitioned with hexane, CHCl 3 and EtOAc, generating four additional fractions.
The ground leaves of V. michelii (730.0 g) were extracted for six hours in a Soxhlet apparatus, first with hexane and then with EtOH.After that, the solutions were concentrated under reduced pressure yielding 28.4 g of hexane extract and 89.5 g of EtOH extract.Then, the crude EtOH extract was dissolved in MeOH:water 9:1, and partitioned with hexane and EtOAc yielding three residues.After TLC analysis, the EtOAc residue (29.2 g) was submitted to column chromatography using silica gel and CHCl 3 :MeOH in mixtures of increasing polarity, collecting 13 fractions.Fractions 1-5, after fractionation on preparative TLC using for elution the systems: hexane:EtOAc (98:2), hexane:Et 2 O (8:2) and CHCl 3 : Me 2 CO (9:1), allowed the isolation of eudesmin (18, 27.3 mg) 18 and phylligenol (19, 4.8 mg). 19

Antioxidant assay
The assay was performed with DPPH (Sigma-Aldrich) in MeOH solution and the 50% effective dose (ED 50 ) was determined following the procedure described by Chang et al. 20

In vitro antileishmanial assay
Promastigote forms of Leishmania amazonensis (WHO/BR/00/LT0016), L. braziliensis (MHOM/BR/75/ M2903) and L. chagasi (MHOM/BR/72/strain 46) were maintained in M199 liquid media supplemented with 10% fetal calf serum 21 and 2% human urine 22 at 26 o C.About 1×10 7 promastigote cells were separately incubated in 24well plates at 26 o C for 72 h in the presence of increasing concentrations of samples.The number of cells in each culture was estimated by counting in a Neubauer chamber.

Results and Discussion
The use of adsorption chromatographic methods allowed the isolation and identification of two lignoids from the fruits of Iryanthera juruensis, three from Virola mollissima, twelve from V. pavonis and two from V. michelii leaves.The identification of isolated compounds was based especially on interpretations of their 1 H and 13 C NMR spectra and compared with reported data, since these are known compounds.
The NMR data of 5 and 19 allowed the unambiguous assignment of chemical shifts.The structure of secolignoid 5 was suggested 9 to be produced by a retro Friedel-Crafts reaction from aryltetralone neolignan.In this work, the extensive studies using NMR techniques such as gCOSY, gHSQC and gHMBC allowed the correct assignment agreeing with those previously described (see Table 1).Through a 1D nOe (Figure 2) experiment with 5 it is possible to observe the effect of the oxy-methylene hydrogens H-1a and H-1b with the methyl hydrogens CH 3 -3 and of H-4 with CH 3 -2.Figure 2 represents a unique possible conformation of 5, according to the observed nOe, confirming the stereochemistry previously proposed.
The literature does not register the chemical shifts of the aromatic hydrogens phylligenol 19, because aromatic hydrogens in the 1 H NMR spectrum of guaiacyl and veratryl rings present continuous absorption.An experiment was performed at 40 o C, using Gaussian transformation on the FID in order to result in better resolution of aromatic hydrogens, where it was possible to observe the aromatic hydrogens (Figure 3).Between d 6.83-6.94can be observed hydrogens at d 6.94 (d, J 1.5 Hz, H-2), at d 6.91 (d, J 2.0 Hz, H-2'), at d 6.89 (d, J 8.0 Hz, H-5'), centered at d 6.86 (H-6 and H-5, close to an A 2 system), d 6.85 (d, J 8.0 Hz, H-5), d 6.85 (dd, J 8.0 and 2.0 Hz, H-6').
The allocation of the hydrogens from ring furofuran of 16 was confirmed by analysis of its gCOSY spectrum, in which observed correlations are consistent with a spin system of H-7:H-8:H-9a:H-9b, H-7':H-8':H-9'a:H-9'b, besides the correlation between the hydrogens H-8:H-8'.The analysis of this system was based by comparison of chemical shifts of hydrogens H-7 and H-7' with models available in the literature.
In our previous work, we communicated that compound 4 showed promising results in the bioautographic test and in a solid medium quantitative activities against xylophagus Vol. 20, No. 6, 2009 fungi Pycnoporus sanguineus, Trametes villosa and Lenzites work were determined.This experiment revealed the potential of V. mollissima in the development of antifungal products against wood decaying fungi. 24n antioxidant assay by the radical scavenger DPPH was performed on V. michelii extract, their fractions and phylligenol.The 50% effective dose (ED 50 ) of 16.0 mg mL -1 was found for the crude EtOH extract and for its aqueous EtOH fraction.The EtOAc fraction obtained after partition of crude extract showed a ED 50 of 20.4 mg mL -1 .After purification of the EtOAc fraction by preparative TLC, the major fraction lost the antioxidant activity, with ED 50 = 278.0mg mL -1 .The phylligenol isolated from this fraction presented ED 50 = 108.0mg mL -1 .The ED 50 determined for quercetin was 5.3 mg mL -1 .
The lignoids 1 and 2 obtained from I. juruensis arils presented higher antileishmanial activity among the assayed lignoids against the promastigote forms of Leishmania amazonensis when compared with promastigote forms of L. braziliensis and L. chagasi, as shown in the Table 2.The 50% inhibition concentration, IC 50 , was 27.0 and 45.0 mg mL -1 of 1 and 2, observed for L. amazonensis, suggests further studies for potential use of these compounds.

General 1 H
and 13 C NMR spectra were recorded on spectrometers from Bruker AC-200 and Varian INOVA 200, 4.7 T, operating at 200 MHz and 50 MHz, respectively, Varian INOVA 300, 7.4 T, operating at 300 MHz and 75 MHz, respectively, and Varian INOVA 500, 11.7 T, operating at 500 MHz and 125 MHz, respectively.The samples were dissolved in CDCl 3 or CD 3 CN, purchased from Aldrich or CIL.