A multidisciplinary approach to the study of the fluminense vegetation *

The fluminense vegetation, more specifically the flora from the Jurubatiba restinga has been investigated by a multidisciplinary team of botanists, chemist, radiobiologist, insect physiologists and geneticist. Vouchers of 564 specimens have been collected, identified, organized in an herbarium, and a database is being build up containing, in addition to classical botanical data, chemical data and information on the potential economic use either for landscape gardening, alternative foods or as medicinal plants. Phytochemical studies of the Guttiferae,Clusia hilariana, yielded oleanolic acid and nemorosone. Their biological activities against the haematophagous insect Rhodnius prolixus vector of Chagas disease have been investigated. Finally, it has been observed that aquatic plants possessed high levels of the natural radionuclide polonium-210, which seems to be originated mainly from soil rather than from atmospheric supply.

Atlantic tropical rain forest and the typical restinga vegetation, object of the present study.
Restingas were formed, along the Brazilian coast, during the Holocene period, as a result of consecutive transgressions and regressions of the sea.They are characterized by large sandy plains of sedimentary origin that are rippled by rows of dunes isolating lagoons, lakes, ponds, bogs and marshes.Such a diversity of physical conditions gives rise to a great diversity of habitats that are colonized by a great variety of vegetal communities.Restingas are thus complex ecosystems in very delicate equilibrium that possess a typical flora, well adapted to the edaphic conditions (Araújo and Lacerda 1987).A number of investigations have appeared dealing mainly with geomorphologic, limnological, botan-ical and ecological aspects of Brazilian restingas, most of which situated in the state of Rio de Janeiro (Lacerda et al. 1982(Lacerda et al. , 1984)).The restinga of Jurubatiba, however, has been almost overlooked although it possesses a rich and well preserved vegetation including some endemic species and some species in extinction.This restinga is located 250 km Northeast of Rio de Janeiro City (22 • to 22 • 23'S and 41 • 15' to 41 • 45'W), extending from Macaé to Carapebus and Quissamã.The climate of the Jurubatiba area vary from warm and rainy in the summer to dry in the winter, the mean temperature oscillates from 22 to 24 • , and the yearly precipitations are comprised between 1,000 and 1,350mm.
The vegetation of the Jurubatiba restinga and the structure of the vegetal communities have been described only recently (Araújo et al. 1998(Araújo et al. , 2000)).Although little anthropic impact has been observed so far, there is a need for a general study of the Jurubatiba restinga, since the littoral area is being rapidly occupied by holiday houses and the border area, distant from the sea, is being threatened by an extensive sugar cane culture.All its area is now protected by law since April 29, 1998.
The following work is part of a broad multidisciplinary study of the Fluminense vegetation aiming the identification of plants with potential economic use (e.g. for landscape gardening, alternative foods or as medicinal plants) and that can eventually be useful to the local population.

MATERIAL AND METHODS
Botanical work -Ten collections of botanical material have been organized since May 1995.Until now, 564 plant samples have been collected.Photographic documentation of all plants has been done in situ.Collected material has been herborized following conventional techniques, and identified by us (GLR, PCAF, MGS and JGS).Incomplete botanical material or highly uncommon samples were identified by specialists from the Botanical Garden and from the National Museum of Rio de Janeiro.Ethnobotanical and ethnopharmacological data were obtained from the local population and from an herbalist well acquainted with the vegetation of the Jurubatiba restinga.
A data-base is being build up containing all necessary information, such as voucher number, botanical family, scientific name, local names, synonymies, short description, habitat, name of the collector, date and local of collection, number of replicates, geographic distribution, local uses, and any other pertinent information such as chemical data, literature data on pharmacological uses, etc.
Phytochemical work -Fruits and flowers of the Guttiferae (=Clusiaceae) Clusia hilariana Schlecht were collected every year at the end of September/beginning of October, since 1995 (voucher numbers: 106, 369, 518 and 568).Fruits and flowers were air-dried in the shadow, at room temperature.Ground fruits (199g), male (418g) and female (74g) flowers were separately extracted exhaustively by room temperature percolation with hexane.Filtration and evaporation of the extraction solvent under vacuum furnished a viscous reddish-brown gum (fruits: 29.8g, 15%; male flowers: 49.4g, 12% and female flowers: 4.3g, 5.8%).TLC examination in several solvent systems indicated that the male and female flowers extracts were almost identical except for one compound, hilarione A, much less abundant or even absent in the female flowers and whose structure will be reported elsewhere.The fruits extract was more complex and contained, in addition to the metabolites reported here, a series of triterpenes that were not investigated.Each extract was purified in the same way.A type purification is as follows.
All the samples were dried at constant weight at 80-100 • C, ground in a mortar, weighted (1.0-2.0gdry mass) and mineralized at 100 • C with a mixture of concentrated HNO 3 and HClO 4 (12:1 ml/g), for 10-15h.The wet mineral residues were treated with HCl 12N (1-2ml), to produce chlorides and then allowed to dry.Spontaneous electrodeposition of 210 Po on stainless steel discs was carried out, in the usual way (Gouvea et al. 1987), by treatment of the mineral residue with 100ml HCl 0.5N and 250mg L-ascorbic acid at 80 • C, under continuous stirring for 2.5h.The disks were then washed with distilled water, dried and counted by total alpha scintillometry using a ZnS(Ag) crystal (Halden and Harley 1960) coupled to a photomultiplier and a pulse counter.Calculations of the 210 Po concentrations (in mBq.g -1 ) took into consideration radiochemical yields (98%), radiometric efficiency (31.4%) and the equivalent of conversion d.p.h./mBq.

RESULTS AND DISCUSSION
The flora of the Jurubatiba restinga is constituted by several vegetal communities: restinga forest, marsh forest, Clusia habitats, Ericaceae habitats, Palmae habitats, periodically inundated areas, permanently inundated areas, lakes and beach environments, inter alii (Araújo et al. 1998).Plants distribution in these communities has been the object of recent studies, and 618 vascular species have been identified.They belong to 381 genera and 120 families (Araújo et al. 2000).Among these species, several are used not only as foods or as medicinal plants, but also for a number of other applications that will be briefly commented.

Botanical, Ethnobotanical and Ethnopharmacological Results
Ten collections of botanical material have been organized in the Jurubatiba restinga between May 1995 and April 2000.Collected plants were herborized, identified taxonomically and an herbarium is being organized.Some species are endemic and some are almost extinguished, for example Couepia schottii Fritshi, Jacquinia brasiliensis Mez, Molinedia glabra (Sprengel) Perkins and Pavonia alnifolia St. Hill.Production of plantlets and cuttings is underway, aiming preservation and propagation of these species.
So far, 564 specimens have been collected.One hundred and eighteen species belonging to 98 genera and 48 families have been identified as ''useful species'', either because they are effectively used by local population or because they are potentially useful species.The complete list of all these species is beyond the scope of this paper and will be published elsewhere (Reis et al. 2001).The most economically important families are: Myrtaceae (with 10 species), Clusiaceae (6) and Rubiaceae ( 6), but the Leguminosae (5), Asteraceae (5) and Bromeliaceae (5) are also of great interest.Considering the number of species reported for these families in the Jurubatiba restinga (Araújo et al. 2000), it can be deduced that 47% of the Myrtaceae found in Jurubatiba are used or useful, 26% of the Bromeliaceae, 21% of the Rubiaceae, 16% of the Asteraceae, but only 11% of the Leguminosae.
A database is being built up containing, in addition to classical botanical data, chemical data and informations on the potential economic uses.Thus, among the 118 useful species: 26% are medicinal plants, 23% are edible (some being very appreciated), 20% are timber-trees, 16% are ornamental and can be used for landscape gardening, 5% furnish fibers used to make ropes and whips, and that have potential use in the textile industry, 3% are ingredients for religious practices and 7% have miscellaneous uses (flavors, aromatics, pigments, etc.).
The edible species belong to three major families: Myrtaceae (9 species), Arecaceae (4) and Cactaceae (3), the principal species being identified as: Ethnobotanical and ethnopharmacological information was furnished by local population and by Jorge Inácio Barcelos, an herbalist habitant of the Jurubatiba restinga where he was born.The medicinal plants belong to 17 families, with Myrtaceae and Asteraceae (4 species each), and Rubiaceae and Verbenaceae (3 species each) as the most important ones.Plants are mainly used as anti-diarrheic (10 species), to heal wounds (4), against fever (4), as anesthetic (3) and also for a number of other minor uses.For a more complete information about these popular uses, one should refer to the work of Reis and coworkers (Reis et al. 2001).One should note that the diarrhea problem is a typical problem of tropical countries, where poor population usually uses bad quality water to drink to cook their food.

Phytochemical Results
Besides the studies of Kaplan and co-workers on leaf waxes, tannins and cyanogenic glycosides, little is known about secondary metabolites from the restinga vegetation in Brazil (e.g.Kaplan et al. 1983Kaplan et al. , 1979Kaplan et al. -2000 and references) and references).Almost nothing has been experimentally established on the biological activities associated to plants from Brazilian restingas, although, as discussed above, several species are used in popular medicine.This is, however, not the case of the Guttiferae (Clusiaceae) a very abundant family in the Jurubatiba restinga, whose principal genus, Clusia, is found associated to one of the most characteristic habitats (Araújo et al. 1998).
The hexane extracts of the fruits, male and female flowers of Clusia hilariana were submitted to solvent partition followed by several column chromatographies.This procedure led to the isolation of an unidentified mixture of sesquiterpenes, of one triterpene, oleanolic acid, and of the benzophenone nemorosone.
Oleanolic acid (1) was isolated in large amounts from fruits and in smaller quantities from the resin of both male and female flowers.It was identified as its methyl ester derivative (3) by direct comparison of its 1 H and 13 C NMR spectral data with those of an authentic sample.This triterpene had already been described from Clusia rosea (whole plant) (Mathur 1972).As far as we are aware, this is the first report of the presence of this triterpene in the floral resin of a Clusia species.Oleanolic acid possesses a number of interesting biological activities that have been reviewed recently (Liu 1995).Remarkably, when tested in vitro against HIV-1 reverse transcriptase, oleanolic acid showed moderate activity, inhibiting the enzyme in 15% (Pereira et al. 1998).Nemorosone was isolated as a tautomeric mixture in relative amount 2:3.In what follows, we will refer to these tautomers as nemorosonesA (2a) and B (2b) respectively.Nemorosones are the major benzophenones in the resins of both male and female flowers.They are also present in the hexane extract from the fruits.The structure of nemorosones came from comparison of their 1 H and 13 C-NMR spectra with data on nemorosone A methyl ether (5) (Oliveira et al. 1996).Nemorosone A (2a) has already been isolated, as the methyl ether 5, from several species of Clusia (Oliveira et al. 1996(Oliveira et al. , 1999)), but the NMR data of the natural underived metabolite 2a remained unpublished, and its tautomer (2b) had never been described before.Interestingly, methylation (CH 2 N 2 , Et 2 O, r.t.) of nemorosones afforded only methyl ether derivative 5. On the contrary, acetylation (Ac 2 O and Pyridine, 65 • C, 4h) of the tautomeric mixture yielded exclusively the monoacetate corresponding to the other tautomer (4).Nemorosone, as the mixture of tautomers, showed moderate activity against HIV-1 reverse transcriptase, inhibiting the enzyme in 30% at a concentration of 100µM.Its acetylated derivative (4) was less active (Pereira et al. 1998).

Biological Activities of Clusia Crude Extracts, and of Compounds (1) and (2)
Besides the ethnopharmacological activities cited above, the other plants are being submitted to a systematic screening, in order to detect toxicity, phagorepellent or anti-ecdysis properties against insect vectors of tropical diseases.
Thus, the hexane, methylene chloride and MeOH crude extracts of male flowers of C. hilariana have been assayed against the haematophagous insect, Rhodnius prolixus, one of the vectors of Chagas disease, at the concentrations of 1, 10 and 100µg/ml blood.No feeding inhibition could be observed, except with the MeOH extract at the higher dose, that reduced the feeding of the insects in 98.7%.Similarly, the crude extracts did not induce delay of the ecdysis, except for the insects partially fed with blood containing 100µg/ml of the MeOH extract.
A preliminary study of the biological activities, on R. prolixus, of oleanolic acid and of the mixture of nemorosone A and B has also been carried out.Figures 3 and 4 show the toxicity and the antiecdysis effect of oleanolic acid (1) fed at 1, 10 and 100µg/ml blood.Dose-dependent toxicity and potent anti-molting effect were observed for oleanolic acid.These unexpected activities are now the object of further studies.Figures 5 and 6 show the activities of nemorosones A and B (2a+2b) fed at 1 and 10µg/ml blood.It appears that the nemorosones are almost non toxic to R. prolixus, but a dose-dependent anti-molting activity could be observed that needs further investigation.

Radioecological Studies
Several habitats of the Jurubatiba restinga are constantly or seasonally under water.The existence of lagoons, lakes, ponds and marches stimulated us to carry out a radioecological study on aquatic plants in order to estimate their capacity to bioaccumulate polonium-210 ( 210 Po), one of the most important contributors to the active deposit of interest in natural radioactive contamination of the environment (Santos et al. 1970), and that most contributes to the internal radiation dose to man (Parfenov 1974).
The occurrence of 210 Po in the terrestrial and marine fauna and flora has been studied for almost three decades (Parfenov 1974, Folson andBeasley 1972).It is known that terrestrial plants are able to accumulate 210 Po from the soil by their roots, but translocation of this radionuclide to the other parts of the vegetal is usually insignificant (Tso and Fisenne 1968).Indeed, the major contamination of plants occurs through deposition onto the leaves, particularly the hairy ones, by dry rather than by wet deposition.On the contrary, in the marine environment 210 Po is directly accumulated from water or from particulate material and is frequently concentrated along food chains (Stegnar and Kobal 1982).Little attention, however, has been paid to freshwater plants and vegetation from Brazilian restingas had never been examined (Lacerda et al. 1982, INIS Fig. 3 -Toxicity of oleanolic acid (1).Concentrations in µg/ml.Fig. 4 -Anti-molting activity of (1).Concentrations in µg/ml.data base .
Six plants, one green alga and five aquatic plants, have been collected from the ''Blau-Blau'' marsh, and analyzed for their 210 Po content.The results that have been published recently (Kelecom et al. 1999) are reproduced and complemented in Table I.The alga showed elevated concentrations of 210 Po, similar to those observed for marine algae.All the other plants showed the lowest concentration of 210 Po in the stems and the highest in the roots.Intermediate values were observed in the leaves.The unexpected high concentrations of 210 Po in the roots must be due to the elevated level of this radionuclide  Concentrations in µg/ml. in associated soils.For a deeper discussion about the significance of these results, see Kelecom et al. (1999).

CONCLUSIONS
The multidisciplinary study described here resulted in the obtainment of a rich collection of plants from the Jurubatiba restinga.An herbarium and an associated database are being built up.This has furnished leads for the rational choice of plants for phytochemical research.Hence, the abundant Gut- tiferae, C. hilariana has been investigated.So far, two secondary metabolites have been isolated and identified: the triterpene oleanolic acid and the benzophenone nemorosone (in tautomeric equilibrium).These substances showed interesting physiological effects on the insect R. prolixus.On the other hand, radioecological studies of six aquatic plants showed unexpected high capacity to concentrate polonium-210.We are now planning to extend our work to the study of the interactions between insects and plants.Thus, the almost extinguished butterfly, Parides ascanius Cramer 1775, specific of the marshy restingas of the State of Rio de Janeiro, maintains a specific relationship with the plant Aristolochia macroura, since the butterfly larvae only develop on cited plant.P. ascanius is a brightly colored butterfly (aposematic colors) that seems to possess a chemical protection from dietary origin.The study of this insectplant interaction is now under way in our laboratories.

ACKNOWLEDGMENTS
AK, CBMN and MSG thank the Scientific Research Council (CNPq) for research fellowships.We are indebted to our university for travel facilities to Carapebus.We are grateful to Prof. M. Cecília B.V. de Souza (Department of Organic Chemistry, UFF) for recording the NMR spectra of nemorosones.A number of students were trained in laboratory tech-niques and research methodologies.The following students participated of parts of this study: A.A. Ribeiro, Alba Valéria dos Santos, Ana Maria Martins Lucas, Artur Maciel, Elaine Coelho Majdalani, F.D. Martins, José Mauro Lima da Silva, Marco Antonio Rocha, P.G.Vargas and Vanuse Rangel Tavares.Their help is acknowledged.Finally, we are grateful to Jorge Inácio Barcelos for the invaluable information he furnished and for his constant help during the collecting trips.An.Acad. Bras. Cienc., (2002) 74 (1) Palavras-chave: taxonomia, uso econômico, ácido oleanólico, nemorosona, polonium-210, atividades biológicas.