The susceptibility tests applied in the assays that investigate the potential of plant derivatives to be used as source of medicine against mycobacteria are often performed by using the broth microdilution method in microtiter plates. A quantitative interferometric method has been used to investigate the susceptibility of slowly growing mycobacteria. In the present article, this interferometric method is applied to rapidly growing mycobacteria. Samples of Mycobacterium smegmatis were exposed to methanol extract of fruits from Stifftia chrysantha and in this case, the interferometric method also complemented the conventional method by adding quantitative information.

From the methanol extract of the aerial parts of Peperomia blanda (Piperaceae), two chromenes were isolated and characterized mainly through application of 2D-NMR spectroscopy. The structures were 2S-(4-methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7-dimethyl-2H-chromene and 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7-dimethyl-2H-chromene named as blandachromenes I and II, respectively.

The triterpenoid pool from the dichloromethane fraction obtained from the methanolic extract of roots of Cecropia lyratiloba Miquel was submitted to CCC using a gradient elution consisting of Hex/EtOAc/MeOH/H2O-1/2/X/1 (X = 0.5 (A); 0.75 (B); 1.0 (C); 1.5 (D); 2.0 (E)) in five steps. The lower aqueous phase was used as mobile phase, 2 mL/min at 850 rpm. This procedure led to the isolation of tormentic acid and a mixture of tormentic and euscaphic acids. In order to improve the triterpene separation the fractions Fr 31-49 were submitted to a new CCC run using a fine adjustment of the methanol concentration in the gradient elution system. This separation procedure led to the isolation of euscaphic acid, 3-acetyl tormentic acid and a mixture of tormentic and isoarjunolic acids.

The project encompasses plants from the following families: Palmae, Lamiaceae, Acanthaceae, Leguminosae and Gesneriaceae. Regarding the pharmacology, several models have been used like antinociceptive, antiinflammatory, antioxidant, molluscicidal, anti-diabetes, anti-microbial and nitric oxide production inhibition. Results showed that utilizing ethnopharmacological information is a very important way to search for new bioactive molecules. It is noteworthy to mention the activity of Acai fruit extracts in the inhibition of nitric oxide production. It was also possible to identify flavonoids responsible for the antidiabetic activity in plants belonging to the family Leguminosae. Acanthaceae extracts showed important antinociceptive and anti-inflammatory activities, as they are very rich in steroids and triterpenes. The same could be said about plants belonging to Lamiaceae that gave several examples of this kind of pharmacological property due to its steroid and triterpenoid compounds. One specie of Lamiaceae also produced a great amount of dihydroxylated triterpenoids with great molluscicidal potencial. Palmae species, rich in fatty acids and steroids led to enriched extracts responsible for the anti-BPH activities. Plants belonging to Gesneriaceae were antioxidant due to their flavonoid content. Polar extracts and isolated molecules, isolated from many species were able to donate hydrogen radical to DPPH. INTRODUCTION Brazil is one of the greatest centers of plant biodiversity. This research project was started to identify this diversity from chemical and pharmacological point of view. The scientific approach could be the only way to avoid losing this biological value. Ethnopharmacology has been used for a long time as a tool for the study of medicinal plants (Holmstedt, 1991; Holmstedt and Bruhn, 1983). Thus, the chemistry of natural products isolated from medicinal plants is now facilitated with the previous knowledge from popular information of use of plant. By this way, we have studied Proc. WOCMAP III, Vol. 1: Bioprospecting & Ethnopharmacology Eds. J. Bernath, E. Nemeth, L.E. Craker and Z.E.Gardner Acta Hort 675, ISHS 2005 90 several plants belonging to different families trying to validate their chemical constituents as well as their uses by Brazilian people. MATERIAL AND METHODS Table 1 lists the plants/parts/families used in this study. All of these plants were previously reported to treat some disease or group of diseases. However, the plants were collected in different periods of time and an herbarium sample of each plant was deposited in the Universitary Botanical Center. All collected plants were submitted to a successive extraction procedure using ethanol as solvent. Each ethanolic extract was dried under reduced pressure and after that, suspended in water. A liquid-liquid extraction procedure was done in order to obtain extracts of different polarities: hexane, dichloromethane, ethyl acetate and n-butanol. These extracts of different polarities were treated separately according to their physicalchemical properties. Crude extracts, isolated compounds and semi purified extracts were tested in pharmacological models. Several pharmacological models were performed in order to validate the information given by people traditionally using the plant. In this sense, techniques of antioxidant measurement (as discoloration of a DPPH solution), techniques for the evaluation of anti-inflammatory activity (as induce paw edema with several inductors), techniques for the evaluation of the antinociceptive potential (as the count of writhings after intraperitoneal injection of acetic acid), techniques for the evaluation of hypoglicemiant activity (as the peroxidation of glucose) and toxicological tests were performed. Tea made of species listed in Table 2 in 10% w/v was prepared and administered to mice. The glucose concentration of each mouse was measured at this time in order to achieve the basal blood glucose concentration (time 0h). After four hours of tea ingestion each group of mice that received each plant tea had their blood glucose measured again (time 4h). Statistical analyses were done. Student’s T test was used for a comparison between two means and a one-way analysis of variance (ANOVA) was used for comparison of more than two means complemented by the Tukey’s test (Runyon and Haber, 1984). A difference was considered statistically when p<0.05. RESULTS AND DISCUSSION Plants belonging to the family Lamiaceae were studied after the collection of popular information about their uses (Table 1). People from the Northeast Region in Brazil use plants of this family to produce syrup to treat cough and also in inflammatory processes, primarily skin inflammation. Several constituents were isolated from Raphiodon echinus, Marsypianthes chamaedrys, Hyptis tetracephala, Hyptis heterodon and Hyptis fascidulata. There is no doubt that the major constituents produced from these plants were triterpenes and steroids. Concerning the pharmacological properties it could be proven that some antinociceptice and anti-inflammatory activities probably due to the higher content of triterpene and steroids (sitosterol and stigmasterol) (Jie, 1995). It was also very interesting that the molluscicidal activity found for the hexanic extract of M. chamaedrys and also the bactericidal and antimicotic activity found for some extracts belonging to species of the genus Hyptis and the species R. echinus and M. chamaedrys. It is also noteworthy to mention the antioxidant activity found for the more polar extracts belonging to plants of the Lamiaceae family and also for rosmarinic acid isolated from M. chamaedrys and Hyptis species and for methylpiperitol isolated from Hyptis fasciculata. Regarding Acanthaceae family, we have studied several extracts from the leaves and stems of B. palisatii and the flowers of P. lutea (Table 1). Among the extracts of B. palisatii it is noteworthy to mention the activity found for hexanic and dichloromethane extracts both for leaves and stems regarding inflammatory processes and also antinociception and specially for the stems (dichloromethane extract) it was shown the inhibition of nitric oxide production in a dose-dependant way achieving the maximum of

Several plants from the Brazilian Tropical Forest are used in folk medicine for treatment of hypertension and asthma. In this study, we investigated the effects of hexanic extracts of leaves (HLE) and stems (HSE) from Piper truncatum on the contractility of cardiac, vascular and tracheal smooth muscles. Twitches of cardiac muscles obtained with electrical stimulation were recorded before and after exposure to increasing concentrations of hexanic extracts. HLE and HSE respectively reduced significantly the amplitude of twitches to 57.05 +/- 11.63 and 61.58 +/- 5.70% of control in the presence of 100 mug mL(-1). Contractile response to a single concentration of adrenaline (epinephrine) was measured before and after exposure of rat aorta rings to HLE and HSE. Both extracts inhibited aorta contraction in a concentration-dependent manner. The concentration of 50% inhibitory effect (IC50) was 32.3 +/- 13.8 and 47.0 +/- 23.8 mug mL(-1) for HLE and HSE, respectively, in aorta with intact endothelium. HLE and HSE also reduced the acetylcholine-precontracted trachea in a concentration-dependent manner with maximal effect observed at 250 and 350 mug mL(-1), respectively. Vasodilatation and trachea relaxation induced by HLE and HSE could explain the use of Piper extracts to reduce blood pressure and bronchospasm.

This article describes the evaluation of immunomodulatory activity of Mollugo verticillata L. (Molluginaceae), a weed plant common in warm and/or wet regions of the American continent. Nitric oxide (NO) release was evaluated in mice peritoneal cell cultures treated in vivo using the ethanolic extract of M. verticillata with and without BCG. The plant extract showed immunostimulatory activity when peritoneal cells were stimulated in vitro with BCG antigen only. However, mice peritoneal cells treated with M. verticillata plus BCG showed a drastic reduction in NO production when they received the additional stimulus in vitro with BCG. Ethanolic extracts of M. verticillata could directly increase NO release by peritoneal cells, but suppress the immune response of these cells when treated with BCG antigen and Mycobacterium tuberculosis whole antigen (TB). Preliminary phytochemical tests allowed the detection of quercetin and triterpenoid glycosides in the ethanolic extract of M. verticillata, and those compounds are probably responsible for the effect of this plant material on the immune system.

Plants are known as important source in the search for new anti-cancer agents. Cytotoxicity-guided fractionation of leaves and fruits from Licania tomentosa Bench and leaves from Chrysobalanus icaco L. resulted in the isolation of betulinic, oleanolic and pomolic acids. These triterpenoids inhibited the growth and induced apoptosis of K562, an erythroleukemia cell line. Most importantly, they also inhibited the proliferation of Lucena 1, a vincristine-resistant derivative of K562 that displays several multidrug resistance (MDR) characteristics. Taken together, our findings emphasize the anti-tumor activity of these triterpenes on leukemia cell lines and call attention to their potential as anti MDR agents.

EVOLUTIONARY TENDENCY OF COUMARIN-BEARING FAMILIES IN ANGIOSPERMAE. Coumarins are special metabolites well distributed in the Angiospermae, either in Monocotyledoneae or Dicutyledoneae. Simple coumarins, the most widespread type, is found in all coumarin-producing families, such as: Apiaceae, Rutaceae, Asteraceae, Fabaceae, Oleaceae, Moraceae e Thymelaeaceae. The other types, linear- and angular furanocoumarins, linear- and angular pyranocoumarins, lignocoumarins, his- and triscoumarins, are of more restricted circumscription. Among the families with occurrence numbers (NO) < 100, the more advanced ones are specialized in the production of only one or two coumarin types, while the primitive families are very well diversified in types. Calculations of percentual numbers of occurrence (%NO) show relevant meaning of coumarin-types in the taxonomic positioning of the producing taxa.

The flavone-flavolol ratio (fo/fl) for Dicotyledoneae is studied through graphic comparisons between indices indicative of morphological tendencies: the herbaceousness and Sporne indices. These comparisons show a tendency toward the reduction of flavone biosynthesis in arboreous and shrubby taxa. Ligneous families (HI ≤50.0) and morphologically more primitive families (SI ≤55) possess fo/fl <1.00. However, the relationship between evolutionary status of families with fo/fl values is not simply linear in Dicotyledoneae, because more advanced families are also characterized by a wider range of variation in fo/fl values.

The volatile composition of ten Piperaceae species, comprising three genera (Ottonia, Piper and Peperomia) has been studied. The species were collected in a typical fragment of the Brazilian Atlantic forest, constantly being damaged by human action. The essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus and their analyses were performed by GC and GC/MS. Identification of the substances was made by comparison of mass spectra and retention indices with literature records. A great number of non-oxygenated monoterpenes and sesquiterpenes were identified. Most of these sesquiterpenes are derived from E,E-farnesyl-PP, suggesting a preference of these species to synthesize metabolites from this pathway. Arylpropanoids, although being common compounds produced by Piperaceae species, were not found in these oils.

Dorstenin, 5-[3-(4,5-dihydro-5,5-dimethyl-4-oxo-2-furanyl)-butoxy]-7H-furo[3, 2-g] benzopyran-7-one, is a psoralen analog recently isolated from Dorstenia species (Moraceae). In order to characterize its biological activity, its photosensitizing and mutational properties were measured in wild-type E. coli and S. cerevisiae and also in strains carrying mutations which affect DNA repair. Compared to the high activities of psoralen and bergapten, dorstenin showed lower genotoxic effect.

The essential oil from Piper solmsianum leaves and its major compound (sarisan) were tested to verify their influences upon mice behaviour. The essential oil was obtained by hydrodistillation in a modified Clevenger extractor and analysed by GC/ MS. This analysis revealed in the oil the presence of monoterpenes, sesquiterpenes and of arylpropanoids. The compound sarisan, a myristicin analogue, was isolated from the oil to perform the pharmacological tests. Emulsions of the oil and of sarisan (5.0 and 10.0% v/v) were used in the tests. Pentobarbital (30 mg/ kg s.c.) or diazepam (2.5 mg/ kg s.c.) were tested as standard drugs to verify depressant or anxiolytic effects, respectively. Both essential oil and sarisan showed to have exciting and depressant effects in the tested animals.

From the leaves of Renealmia chrysotrycha (Zingiberaceae), we isolated a sesquiterpene alcohol for which spectral data suggested the structure of a 10-aromadendranol. A meticulous NMR investigation, based mainly on vicinal proton-proton coupling constants and NOE interactions, and confirmed by a molecular mechanics calculation, established its relative stereochemistry as that of ledol. This finding resolves several recent literature ambiguities. Three known compounds (aromadendrene, cis-calamenene and palustrol) were also isolated.

The dichloromethane fraction obtained from the methanolic extract of leaves of P. lhotzkyanum afforded a C-glucosylflavone (kaplanin) together with sakuranetin, methyl 4-methoxydihydroferulate and a mixture of methyl ferulate and dihydroferulate derivatives. Spectrometric methods were used to elucidate the structures of these compounds.

Polygodial, isopolygodial, mukaadial and polygonone were isolated for the first time from Capsicodendron dinisii. Thus, with respect to drimane-type sesquiterpenoids of the Canellaceae, the close affinity of the South American genus Capsicodendron with the African genus Warburgia rather than the Madagascarian genus Cinnamosma is established.

The volatile composition of four species of the genus Peperomia collected near to Rio de Janeiro, Brazil, has been studied. These essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus. The analysis was performed by GC/MS and the identification of the substances was made by comparison of their mass spectra with literature records and retention indices. According to this methodology it has been possible to identify in the studied essential oils monoterpenes, sesquiterpenes and arylpropanoids.

ABSTRACT 2′,6′-Dihydroxy-4′-methoxychalcone (DMC) was purified from the dichloromethane extract of Piper aduncum inflorescences. DMC showed significant activity in vitro against promastigotes and intracellular amastigotes of Leishmania amazonensis, with 50% effective doses of 0.5 and 24 μg/ml, respectively. Its inhibitory effect on amastigotes is apparently a direct effect on the parasites and is not due to activation of the nitrogen oxidative metabolism of macrophages, since the production of nitric oxide by both unstimulated and recombinant gamma interferon-stimulated macrophages was decreased rather than increased with DMC. The phagocytic activity of macrophages was functioning normally even with DMC concentrations as high as 80 μg/ml, as seen by electron microscopy and by the uptake of fluorescein isothiocyanate-labeled beads. Ultrastructural studies also showed that in the presence of DMC the mitochondria of promastigotes were enlarged and disorganized. Despite destruction of intracellular amastigotes, no disarrangement of macrophage organelles were observed, even at 80 μg of DMC/ml. These observations suggest that DMC is selectively toxic to the parasites. Its simple structure may well enable it to serve as a new lead compound for the synthesis of novel antileishmanial drugs.

The inhibition of intracellular Leishmania amazonensis growth by 2', 6'-dihydroxy-4'-methoxychalcone (DMC) isolated from Piper aduncum was further enhanced after encapsulation of DMC in polymeric nanoparticles. Encapsulated DMC also showed increased antileishmanial activity in infected BALB/c mice, as evidenced by significantly smaller lesions and fewer parasites in the lesions.

The hexane extract from leaves of P. lhotzkyanum yielded lhotzchromene (2-methyl-2-[4′-methyl-3′-pentenyl]-2H-1-benzopyran-6-carboxylic acid) together with the known (E) and (Z) isomers of 4-hydroxy-3-(3′,7′-dimethyl-1′-oxo)-2′,6′-octadienylbenzoic acid and a mixture of six hydroxylated sesquiterpenes along with phytol. Spectroscopic methods were used to identify these compounds.

Marsypianthes chamaedrys is a common herb that occurs in the North and Northeast regions of Brazil. Phytochemical studies showed this plant to be rich in triterpenoids. The hexane extract of M. chamaedrys yielded, after chromatographic separations, two mixtures: the first containing monohydroxylated-compounds (alpha-amyrin, beta-amyrin, lupeol and germanicol) and the second, dihydroxylated compounds (chamaedrydiol, castanopsol, 2alpha-hydroxylupeol and epigermanidiol). Molluscicidal tests were performed for the crude hexane extract and for mixtures I and II. Mixture II showed high activity.