Chemical Defenses of the Endemic Brazilian Gorgonian Lophogorgia violacea Pallas (Octocorallia, Gorgonacea)

O mecanismo de defesa contra predadores no octocoral brasileiro Loph gorgia violacea foi investigado através de ensaios de preferência alimentar em campo frente a predadores. Os ensaios de palatabilidade com o extrato bruto da gorgônia demonstraram que seus metabólitos secundários possuem atividade deterrente significativa frente a peixes generalistas. O fracionamento do extrato bruto, guiado pelos ensaios em campo, revelou que uma mistura complexa de furanocembranolídeos é a responsável pela defesa química de L. violacea. O diterpeno lophotoxina ( 1) foi a principal substância isolada, seguida por outras duas similares, deoxilophotoxina ( 2) e 13-acetoxi-11 β,12βepoxipukalídeo ( 3), além de dois furanocembranolídeos inéditos na literatura, 7-acetoxi-8hidroxilophotoxina ( 4) e 3-metoxi-8-hidroxilophotoxina ( 5). A lophotoxina ( 1), uma importante neurotoxina, originalmente isolada de L. rigida do Pacífico, demonstrou ser a mais potente dentre as substâncias fagoinibidoras presentes no extrato bruto. Por outro lado, os outros quatro furanocembranolídeos isolados também parecem contribuir, de maneira aditiva, para a atividade total observada. Estes resultados corroboram estudos prévios, confirmando que furanocembranolídeos são uma classe de substâncias de defesa frente ao consumo por peixes em ambientes tropicais e temperados.

The chemical defenses of the Brazilian gorgonian octocoral Lophogorgia violacea Pallas have been investigated using feeding preference experiments performed in situ with an ecologically relevant, natural assemblage of predatory fishes.Feeding experiments incorporating the crude extract of the animal into palatable foods showed that the organic constituents of L. violacea provide a powerful chemical deterrence toward consumption by generalist fish carnivores.Bioassayguided fractionation of the extract and evaluation of the fractions and purified compounds obtained in the same in situ assay revealed that a complex mixture of furanocembranolides was responsible for the overall feeding deterrence observed.The most potent feeding deterrent identified was lophotoxin (1), followed by two previously reported and structurally related compounds, deoxylophotoxin (2), and 13-acetoxy-11β,12β-epoxypukalide (3), as well as two new furanocembranolides: 7-acetoxy-8-hydroxylophotoxin (4) and 3-methoxy-8-hydroxylophotoxin (5).Lophotoxin (1), a neurotoxin originally isolated from the Pacific gorgonian L. rigida, was the most potent feeding deterrent among the furanocembranolides isolated.However, the four other related furanocembranolides (2-5) appear to contribute, in an additive manner, to the overall deterrent effects observed.These results add to earlier studies providing further evidence that compounds of this structural class provide effective chemical defenses against fish predators in both temperate and tropical environments.

Introduction
Gorgonian octocorals (Gorgonaceae, Octocorallia) are a unique group of marine cnidarians, which thrive in tropical to warm temperate oceans, being most abundant and diverse in shallow Caribbean waters.Diverse from the stony corals, they do not have an external calcium carbonate skeleton, but calcitic sclerites embedded in their coenenchyme that are thought to serve as structural support for the colony 1,2 .Despite their relative abundance in coral reefs, habitats characterized by high levels of predation and nutrient scarcity, octocorals seem to be free of predation with the exception of some specialist consumers (e.g.polychaetes, mollusks, and butterflyfishes) 1 .Explanations for the low predation rates on gorgonians may include both physical and chemical defenses but only very recently have relevant laboratory or field experiments been employed to test these hypotheses 3 .
Sclerites from Pseudopterogorgia acerosa have been demonstrated to deter predation in field assays 2 .This study indicated the important role that structural elements can play in the defense against some potential consumers in gorgonians corals, or why some gorgonian specialists fishes (e.g.chaetodontids) consume only the polyps and not the coenenchyme 2 .
The extensive Brazilian coastline is rich in marine organisms, but assessments of ecological roles of their natural products have been rare and restricted to only a few studies [14][15][16][17][18][19][20][21] .Recently, investigation of the chemical defenses in two Brazilian gorgonians revealed that different compounds, like the Caribbean species studied, are responsible for their chemical defense.Although several compounds have been isolated from Phyllogorgia dilatata 21 only the diterpene 11β,12β-epoxypukalide showed deterrent activity in field assays 14 .Likewise, investigation of the Brazilian gorgonian Heterogorgia uatumani demonstrated that defenses were derived from the additive effects of an eunicellane diterpenoid and a sesquiterpene lactone, heterogorgiolide, isolated from the bioactive crude extract 15 .
As part of our continuing interest in the natural products chemistry and chemical ecology of Brazilian marine invertebrates [14][15][16][21][22][23][24][25] , we have investigated the chemistry and feeding deterrence properties of Lophogorgia violacea Pallas, an endemic species occurring in the southeast regions of Brazil 26 . In ths report, we describe the isolation and identification of the secondary metabolites (Figure 1) responsible for the observed deterrence.

Palatability assay
Food strips were prepared using the established methodology 7,8,14,16 , which involved homogenizing 2.5 g carrageenan (Sigma C-1013 type 1), 20 cm 3 commercial tunafish puree (packed in oil) in 60 cm 3 of water.In separate experiments, crude gorgonian extract and fractions were volumetrically reconstituted in a matrix of the carrageenanbased food at the same concentration as they occurred in the gorgonian fresh tissues (final volume of 60 cm 3 ).Compound 1 was assayed in two different concentrations (0.9 and 1.5 mg cm -3 ).For each experiment, 20 treated and 20 control strips (1.0 x 0.6 x 5.0 cm each) were arranged in pairs and attached to 20 ropes.The ropes were anchored slightly above the bottom near to the same site the L. violacea colonies were collected.Within 3 hr the ropes were retrieved and the amount of each strip eaten was measured.During the experiments, several common tropical fishes, well known to occur in the studied area, using SCUBA at 10-12 meters depth at Arraial do Cabo, Rio de Janeiro state, Brazil on March, 1994.The specimens were frozen immediately after collection.

Results and Discussion
Isolation and structure elucidation of diterpenes Specimens of the gorgonian L. violacea collected at Arraial do Cabo, RJ, were immediately frozen and freeze dried prior the extraction with dichloromethane and methanol.The combined extracts were fractionated by vacuum flash chromatography on silica gel to yield five fractions.Proton-NMR analysis revelead that fractions B and C were rich in furanocembranolides by the characteristic deshielded signals between δ H 9.0 to 10.0 and 4.0 to 7.0.Subsequent purification of fraction B by normal phase HPLC, using 1:1 isooctane:ethyl acetate, afforded compounds 1, 2 and 3. Purification of fraction C by normal phase HPLC using the same solvent system also yielded compounds 1 and 2, along with the diterpene 4. Compound 5 was obtained by HPLC purification of fraction D (normal phase, 4:6 hexane:ethyl acetate).
The structures and relative stereochemistries of the new diterpenes 4 and 5 were established by 1D and 2D NMR experiments, including 1 H-1 H-COSY, 1 H-1 H-NOESY, HMQC and HMBC.The 1 H and 13 C NMR chemical shifts assignments are shown in Table 1 for the cembranolides 4 and 5, which are described here for the first time.
These results led us to conclude that a mixture of diverse furanocembranolide diterpenes, with lophotoxin (1) as the major metabolite, is responsible for the potent feeding deterrent effects observed for the L. violacea crude extract.Indeed, it appears common that the overall effects of deterrence are derived not by a single compound, but by complex mixtures of metabolites, whose additive effects provide amazingly effective defenses against predators 15 .
Our results, combined with previous studies [11][12][13][14] , and coupled with the wide distribution of the furanocembranolides, suggest that this class of diterpenoids is responsible for the defenses of diverse octocorals from both tropical and temperate marine communities.