Essential Oils of Aromatic Plant Species from the Atlantic Rainforest Exhibit Extensive Chemical Diversity and Antimicrobial Activity.

Microbial resistance, caused by the overuse or inadequate application of antibiotics, is a worldwide crisis, increasing the risk of treatment failure and healthcare costs. Plant essential oils (EOs) consist of hydrophobic metabolites with antimicrobial activity. The antimicrobial potential of the chemical diversity of plants from the Atlantic Rainforest remains scarcely characterized. In the current work, we determined the metabolite profile of the EOs from aromatic plants from nine locations and accessed their antimicrobial and biocidal activity by agar diffusion assays, minimum inhibitory concentration, time-kill and cell-component leakage assays. The pharmacokinetic properties of the EO compounds were investigated by in silico tools. More than a hundred metabolites were identified, mainly consisting of sesqui and monoterpenes. Individual plants and botanical families exhibited extensive chemical variations in their EO composition. Probabilistic models demonstrated that qualitative and quantitative differences contribute to chemical diversity, depending on the botanical family. The EOs exhibited antimicrobial biocidal activity against pathogenic bacteria, fungi and multiple predicted pharmacological targets. Our results demonstrate the antimicrobial potential of EOs from rainforest plants, indicate novel macromolecular targets, and contribute to highlighting the chemical diversity of native species.

Piper tectoniifolium Kunth: A New Natural Source of the Bioactive Neolignan (-)-Grandisin.

The Piper species are a recognized botanical source of a broad structural diversity of lignans and its derivatives. For the first time, Piper tectoniifolium Kunth is presented as a promising natural source of the bioactive (-)-grandisin. Phytochemical analyses of extracts from its leaves, branches and inflorescences showed the presence of the target compound in large amounts, with leaf extracts found to contain up to 52.78% in its composition. A new HPLC-DAD-UV method was developed and validated to be selective for the identification of (-)-grandisin being sensitive, linear, precise, exact, robust and with a recovery above 90%. The absolute configuration of the molecule was determined by X-ray diffraction. Despite the identification of several enantiomers in plant extracts, the major isolated substance was characterized to be the (-)-grandisin enantiomer. In vascular reactivity tests, it was shown that the grandisin purified from botanical extracts presented an endothelium-dependent vasorelaxant effect with an IC50 of 9.8 ± 1.22 µM and around 80% relaxation at 30 µM. These results suggest that P. tectoniifolium has the potential to serve as a renewable source of grandisin on a large scale and the potential to serve as template for development of new drugs for vascular diseases with emphasis on disorders related to endothelial disfunction.

Using online databases to produce comprehensive accounts of the vascular plants from the Brazilian protected areas: The Parque Nacional do Itatiaia as a case study.

BACKGROUND: Brazil is one of the most biodiverse countries in the world, with about 37,000 species of land plants. Part of this biodiversity is within protected areas. The development of online databases in the last years greatly improved the available biodiversity data. However, the existing databases do not provide information about the protected areas in which individual plant species occur. The lack of such information is a crucial gap for conservation actions. This study aimed to show how the information captured from online databases, cleaned by a protocol and verified by taxonomists allowed us to obtain a comprehensive list of the vascular plant species from the "Parque Nacional do Itatiaia", the first national park founded in Brazil. All existing records in the online database JABOT (15,100 vouchers) were downloaded, resulting in 11,783 vouchers identified at the species level. Overall, we documented 2,316 species belonging to 176 families and 837 genera of vascular plants in the "Parque Nacional do Itatiaia". Considering the whole vascular flora, 2,238 species are native and 78 are non-native. NEW INFORMATION: The "Parque Nacional do Itatiaia" houses 13% of the angiosperm and 37% of the fern species known from the Brazilian Atlantic Forest. Amongst these species, 82 have been cited as threatened, following IUCN categories (CR, EN or VU), seven are data deficient (DD) and 15 have been classified as a conservation priority, because they are only known from a single specimen collected before 1969.

Local Anesthetic Activity from Extracts, Fractions and Pure Compounds from the Roots of Ottonia anisum Spreng. (Piperaceae).

Piperaceae species can be found worldwide in tropical and subtropical areas and many of them have been used for centuries in traditional folk medicine and in culinary. In Brazil, species of Piperaceae are commonly used in some communities as local anesthetic and analgesic. Countrified communities have known some species of the genus Ottonia as "anestesia" and it is a common habit of chewing leaves and roots of Ottonia species to relief toothache. The purpose of this study is to report our findings on new molecules entities obtained from the roots of Ottonia anisum Spreng, in which local anesthetic activity (sensory blockage) is demonstrated for the first time in vivo guinea pig model. Phytochemical investigation led to the isolation of three amides (pipercallosidine, piperine and valeramide) and in an enriched mixture of seven amides (valeramide, 4,5-dihydropiperlonguminine, N-isobutil-6-piperonil-2-hexenamide, piperovatine, dihydropipercallosidine, pipercallosidine and pipercallpsine). Our findings demonstrated the anesthetic potential for the methanolic extract from roots, its n-hexane partition and amides from O. anisum and it is in agreement with ethnobotanical survey.

Bioactivity-guided isolation of laevicarpin, an antitrypanosomal and anticryptococcal lactam from Piper laevicarpu (Piperaceae).

Crude CH2Cl2 extract from leaves of Piper laevicarpu (Piperaceae) displayed antitrypanosomal activity against trypomastigote forms of Trypanosoma cruzi (Y strain) and antimicrobial potential against Cryptococcus gattii (strain-type WM 178). Bioactivity-guided fractionation of crude extract afforded one new natural bioactive lactam derivative, named laevicarpin. The structure of isolated compound, which displayed a very rare ring system, was elucidated based on NMR, IR and MS spectral analysis. Using MTT assay, the trypomastigotes of T. cruzi demonstrated susceptibility to laevicarpin displaying IC50 value of 14.7µg/mL (49.6µM), about 10-fold more potent than the standard drug benznidazole. The mammalian cytotoxicity of laevicarpin was verified against murine fibroblasts (NCTC cells) and demonstrated a CC50 value of 100.3µg/mL (337.7µM-SI=7). When tested against Cryptococcus gattii, laevicarpin showed an IC50 value of 2.3µg/mL (7.9µM) and a MIC value of 7.4µg/mL (25µM). Based in the obtained results, laevicarpin could be used as a scaffold for future drug design studies against the Chagas disease and anti-cryptococosis agents.

Antifungal and cytotoxic 2-acylcyclohexane-1,3-diones from Peperomia alata and P. trineura.

Bioactivity-guided fractionation of the separate CH2Cl2 extracts from the aerial parts of Peperomia alata and P. trineura yielded seven polyketides: alatanone A [3-hydroxy-2-(5'-phenylpent-4'E-enoyl)cyclohex-2-en-1-one, 1a] and alatanone B [3-hydroxy-2-(3'-phenyl-6'-methylenedioxypropanoyl)cyclohex-2-en-1-one, 2a] from P. alata and trineurone A [3-hydroxy-2-(11'-phenylundec-10'E-enoyl)cyclohex-2-en-1-one, 1b], trineurone B [3-hydroxy-2-(15'-phenyl-18'-methylenedioxypentadecanoyl)cyclohex-2-en-1-one, 2b], trineurone C [3-hydroxy-2-(17'-phenyl-20'-methylenedioxyheptadecanoyl)cyclohex-2-en-1-one, 2c], trineurone D [3-hydroxy-2-(hexadec-10'Z-enoyl)cyclohex-2-en-1-one, 3a], and trineurone E [(6R)-(+)-3,6-dihydroxy-2-(hexadec-10'Z-enoyl)cyclohex-2-en-1-one, 3b] from P. trineura. The isolated compounds were evaluated for antifungal activity against Cladosporium cladosporioides and C. sphaeospermum and for cytotoxicity against the K562 and Nalm-6 leukemia cell lines.

Cytotoxic non-aromatic B-ring flavanones from Piper carniconnectivum C. DC.

The EtOAc extract from the leaves of Piper carniconnectivum C. DC. was subjected to chromatographic separation to afford two non-aromatic B-ring flavanone compounds: 5-hydroxy-2-(1'-hydroxy-4'-oxo-cyclohex-2'-en-1'-yl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (1) and 5-hydroxy-2-(1',2'-dihydroxy-4'-oxo-cyclohexyl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (2). The absolute configuration of (+)-1 was unambiguously determined as 2S,1'R by electronic circular dichroism (ECD) spectroscopy and comparison to simulated spectra that were calculated using time-dependent density functional theory (TDDFT). This methodology allowed the assignment of the absolute configuration of (+)-2 also as 2S,1'R, except for the stereogenic center at C-2', which was assigned as R because of the evidence drawn from high resolution NMR experiments. The cytotoxic activity of both compounds and 3 (hydrogenated B-ring derivative of 1) was evaluated on twelve human leukemia cell lines, and the IC50 values (<10 µM) indicated the activity of 1 against seven cell lines.

Chemical study and larvicidal activity against Aedes aegypti of essential oil of Piper aduncum L. (Piperaceae).

Piper aduncum L. is used in folk medicine to treat respiratory and inflammatory diseases. The aim of this study was to analyze the essential oil from leaves of P. aduncum collected in the Brazilian Cerrado, North of Minas Gerais, as well as to evaluate the larvicidal activity of this oil and of its major constituent. The essential oil was analyzed by gas chromatography coupled to flame ionization detector and gas chromatography coupled to mass spectrometry that allowed characterizing 23 compounds (monoterpenes: 90.4%; sesquiterpenes: 7.0%). The major component was 1,8-cineole (53.9%). This oil showed to be very different from those obtained from the same species. Larvae of A. aegypti were exposed to different concentrations of the essential oil and 1,8-cineole. The mortality rate of 100% was obtained after 24 h of treatment with the oil at concentrations of 500 and 1,000 ppm. After 48 h of treatment, the mortality rate was 80% and 50% for concentrations of 250 and 100 ppm, respectively. The LC50 obtained after 24h was estimated in 289.9 ppm and after 48 h was 134.1 ppm. The major compound 1,8-cineole showed no larvicidal activity.

Growth study and essential oil analysis of Piper aduncum from two sites of Cerrado biome of Minas Gerais State, Brazil

Piper aduncum L., Piperaceae, stands out due to its biological activities, however, it is still found in the wild and little is known about its agronomic point of view. The aim of this study was to evaluate the growth and to analyze the chemical composition of essential oils from leaves of P. aduncum collected in two different sites of Cerrado as well as in cultivated plants. The cultivation was installed out in a greenhouse using cuttings of adult specimens. Essential oils were obtained from fresh leaves. Plants from the two studied locations showed erect growth habit and behavior of linear growth. The essential oils composition of P. aduncum from Bocaiuva did not differ between wild and cultivated plants, as the major substance identified as 1,8-cineole. The plants from Montes Claros site showed a distinct concentration for the two samples, being the major substance characterized as transocimene (13.4%) for wild and 1,8-cineole (31.3%) for cultivated plants. Samples from both locations showed a similar essential oil composition in cultivars. Our results showed that P. aduncum cultivation is feasible and the variation in chemical composition of the two sites may indicate an environmental influence, since chemical and isoenzyme analysis did not show great differences.

Larvicidal activities and chemical composition of essential oils from Piper klotzschianum (Kunth) C. DC. (Piperaceae).

BACKGROUND: Volatile oils from fresh roots, stems, leaves and seeds of Piper klotzschianum (Piperaceae) were obtained by hydrodistillation and analysed by GC-FID and GC-MS. In total, 25 components, representing more than 95% of the examined oils, were identified. The essential oils were evaluated against Artemia salina Leach nauplii and fourth-instar Aedes aegypti larvae. RESULTS: The major chemical constituents that were identified from various parts of this plant were 1-butyl-3,4-methylenedioxybenzene and 2,4,5-trimethoxy-1-propenylbenzene in the root, 1-butyl-3,4-methylenedioxybenzene in the stems and leaves and 1-butyl-3,4-methylenedioxybenzene, limonene and α-phellandrene in the seeds. The biological activities of these essential oils generally exhibited high toxicity against A. salina, with LC50 values that ranged from 7.06 to 15.43 µg mL(-1), and significant larvicidal activity against fourth-instar A. aegypti larvae was observed in the essential oils from the seeds (LC50 of 13.27 µg mL(-1)) and roots (LC50 of 10.0 µg mL(-1)) of the plant. CONCLUSION: The present study indicates that both essential oil of P. klotzsdhianum and the isolate 1-butyl-3,4-methylenedioxybenzene are potential resources for A. aegypti larva control. This is the first report of the biological activities of the oil and isolated compound.

Leishmanicidal activity of an alkenylphenol from Piper malacophyllum is related to plasma membrane disruption.

Leishmaniasis and Chagas disease are parasitic protozoan infections that affect the poorest population in the world, causing high mortality and morbidity. As a result of highly toxic and long-duration treatments, novel, safe and more efficacious drugs are essential. In this work, the methanol (MeOH) extract from the leaves of Piper malacophyllum (Piperaceae) was fractioned to afford one alkenylphenol, which was characterized as 4-[(3'E)-decenyl]phenol (gibbilimbol B) by spectroscopic methods. Anti-protozoan in vitro assays demonstrated for the first time that Leishmania (L.) infantum chagasi was susceptible to gibbilimbol B, with an in vitro EC(50) of 23 µg/mL against axenic promastigotes and an EC(50) of 22 µg/mL against intracellular amastigotes. Gibbilimbol B was also tested for anti-trypanosomal activity (Trypanosoma cruzi) and showed an EC(50) value of 17 µg/mL against trypomastigotes. To evaluate the cytotoxic parameters, this alkenylphenol was tested in vitro against NCTC cells, showing a CC(50) of 59 µg/mL and absent hemolytic activity at the highest concentration of 75 µg/mL. Using the fluorescent probe SYTOX Green suggested that the alkenylphenol disrupted the Leishmania plasma membrane upon initial incubation. Further drug design studies aiming at derivatives could be a promising tool for the development of new therapeutic agents for leishmaniasis and Chagas disease.

Antioxidant capacity and larvicidal and antifungal activities of essential oils and extracts from Piper krukoffii.

The leaves and twigs of Piper krukoffii, collected in the Carajás National Forest, north Brazil, yielded essential oils (2.0% and 0.8%), the main constituents of which were myristicin (40.3% and 26.7%), apiole (25.4% and 34.1%) and elemicin (2.8% and 3.0%). The antioxidant activities of the oils, methanol extract and its sub-fractions were evaluated. The DPPH EC50 values varied from the ethyl acetate sub-fraction (73.4 +/- 3.7 microg/mL) to the methanol extract (24.9 +/- 0.8 microg/mL), and the ABTS TEAC values ranged in the same order from 265.7 to 349.2 microMol TE/g. These results indicated a significant antioxidant activity for the plant. The lignans (-)-kusunokin, yatein, (-)-hinokin and cubebin were identified in the methanol extract. The hydro-methanolic sub-fraction showed a high value for total phenol content (106.5 +/- 0.7 mg GAE/g), as well as 1H NMR signals for sugar moieties. Crude extracts and sub-fractions were also able to inhibit beta-carotene bleaching, varying from 22.4 to 47.1%. The oils from the leaves and twigs showed strong larvicidal (21.4 and 3.6 microg/mL) and fungicide (0.5 and 0.1 microg/mL) activities.

Aristolactams from roots of Ottonia anisum (Piperaceae).

The Piperaceae species are known worldwide for its medicinal properties and its chemical compounds. In Brazil, many species of this family are distributed mainly in Amazon Region and in the Atlantic Forest. The genus Ottonia is known as source of amides, flavonoids, arilpropanoids and terpenes with record biological activities. Six aristolactams, including, aristolactam BII, piperolactam C, goniothalactam, stigmalactam, aristolactam AII and aristolactam BIII were isolated from roots of this species. GC-MS, 1H NMR and NOESY techniques were used to characterize these compounds. This is the first report about the occurrence of aristolactams in the Ottonia anisum Sprengel.

Essential oil composition, antioxidant capacity and antifungal activity of Piper divaricatum.

The major components of the essential oil of Piper divaricatum from Marajó Island, PA, Brazil, were methyleugenol (63.8%) and eugenol (23.6%), which were identified by GC and GC-MS. Essential oils of Piper species from the Brazilian Amazon are rich in either mono- and sesquiterpenes or phenylpropanoids, as seen in this study. The oil was able to scavenging the DPPH radical, displaying an inhibition varying from 19% to 74%, with an EC50 value of 16.2 +/- 1.9 microg mL(-1). In the bleaching of beta-carotene, the inhibition of the oil oxidation was 60.0 +/- 2.7%. The oil gave minimum inhibitory concentrations for the fungi Cladosporium cladosporioides and C. sphareospermum of 0.5 microg and 5.0 microg, respectively. The oil also showed significant brine shrimp larvicidal activity (LC50, 38.8 +/- 0.8 microg mL(-1)).

Chemistry and biological activity of essential oils from Piper claussenianum (Piperaceae).

Analyses of essential oils obtained from fresh and dried leaves and inflorescences of Piper clausenianum were performed using GC-FID, GC-MS and NMR techniques. Forty compounds were detected for these four oils with the total of identified constituents ranging from 88.7% for the dried inflorescences to 97.7% for the dried leaves. Sesquiterpenes were the main constituents in the volatile fraction from leaves with a high percentage of (E)-nerolidol (up to 83%). However, monoterpenes were identified in greater amount in the inflorescences, with linalool percentages from 50% up. The essential oils from fresh leaves and inflorescences were submitted to anti-parasitic activity against a strain of Leishmania amazonensis. Both samples showed biological activity, but the essential oil from P. claussenianum fresh leaves, which was rich in (E)-nerolidol, showed effective growth inhibition of L. amazonensis due to the high percentage of this metabolite in the mixture.

C-glycosyl flavones from Peperomia blanda.

The methanol extract from aerial parts of the Peperomia blanda (Piperaceae) yielded two C-glycosyl-flavones. Their structures were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR, chemical transformation and comparison with the related known compounds. The structure of the new flavonoids were established as 4'-methoxy-vitexin 7-O-beta-D-xylopyranoside (1) (7-O-beta-D-xylopyranosyl-8-C-beta-D-glucopyranosyl-4'-methoxy-apigenin) and vicenin-2 (2). The antioxidant activity of both compounds was investigated using the DPPH assay. Both compounds showed only modest activity, with IC50 values of 357.2 microM for 1, and 90.5 microM for 2.

Caracterização de derivado arilbutanoídico em folhas e raízes de Ottonia anisum Sprengel / Characterization of an arylbutanoic derivative from leaves and roots of Ottonia anisum Sprengel

The family Piperaceae encompasses 5 genera, Piper, Peperomia, Pothomorphe, Ottonia and Sarcorhachis, and approximately 2,000 species widely distributed in the tropical and subtropical regions of the world. Most of the Ottonia species are native to Brazil and they are used in folk medicine as diuretic, salivating, and anaesthetic. Chemical studies of non-polar fractions from leaves and roots of Ottonia anisum Sprengel lead to the isolation of an arylbutanoic metabolite, the 1-butyl-3,4-methylenedioxybezene.

A família Piperaceae é formada por 5 gêneros, Piper, Peperomia, Pothomorphe, Ottonia e Sarcorhachis, com cerca de 2000 espécies de ampla distribuição nas regiões tropicais e subtropicais do mundo. Das 23 espécies descritas para o gênero Ottonia, a maioria é nativa do Brasil. Essas plantas são popularmente utilizadas no país como diuréticas, salivantes e anestésicas. O estudo químico dos extratos apolares de folhas e de raízes de Ottonia anisum Sprengel levou aoisolamento de um componente majoritário identifi cado como o derivado arilbutanoídico: 1-butil-3,4-metilenodioxibeneno.

Variability in essential-oil composition of Piper marginatum sensu lato.

This paper contains data on the chemical composition of the essential oils of 22 leaf samples of Piper marginatum Jacq. collected in different areas and ecosystems of the brazilian Amazon, as well as an overview of the available literature. The species presents a large synonymy based on their different leaf characteristics and distinct scents where some of them smell like anise or very close compounds. By GC, GC/MS, and cluster analysis, we identified seven chemotypes for the leaf oils. The main components found in chemotype I were safrole (1) and 3,4-(methylenedioxy)propiophenone (2). The chemotype II was dominated by 3,4-(methylenedioxy)propiophenone (2) and p-mentha-1(7),8-diene (10). The major compounds identified in chemotype III were 3,4-(methylenedioxy)propiophenone (2), myristicin (3), (E)-beta-ocimene (11), and gamma-terpinene (12). In the chemotype IV, the principal constituents were beta-caryophyllene (13), alpha-copaene (14), and 3,4-(methylenedioxy)propiophenone (2). The chemotype V was dominated by (E)-isoosmorhizole (6), (E)-anethole (8), and isoosmorhizole (7). The main compounds found in the chemotype VI were 2-methoxy-4,5-(methylenedioxy)propiophenone (4), methoxy-4,5-(methylenedioxy)propiophenone isomer 5, and (E)-isoosmorhizole (6). The major constituents in chemotype VII were beta-caryophyllene (13), bicyclogermacrene (15), and (E)-asarone (9).

Insect feeding preferences on Piperaceae species observed in São Paulo city, Brazil

Piperaceae species have been placed among the basal angiosperm and are adapted to a variety of habitats including moist forests, secondary vegetation and dry high lands. The major anatomical/morphology features are of small trees, vines, and shrubs for Piper species, while the epiphytic and succulent characteristics are predominant forms among Peperomia species. Their secondary chemistry can be mostly represented by amides, phenylpropanoids/lignoids, and chromenes in addition to a phletoria of biosynthetically mixed-origin secondary compounds. Although several amides and lignans are known as insecticides, several phytophagous insects, among which some considered pests of economic importance, have been observed feeding vigorously on Piperaceae species. Herein we describe the feeding preferences of fourteen phytophagous species of Coleoptera, Lepidoptera and Hemiptera over approximately fifty Piperaceae species observed in São Paulo, SP, Brazil, in a long-term basis.

As espécies de Piperaceae têm sido posicionadas entre as angiospermas basais e são frequentemente encontradas em habitats diversificados que incluem matas ciliares, vegetação secundária e campos rupestres. As espécies de Piper possuem hábitos de plantas herbáceas, arboretos e trepadeiras enquanto que, no caso de Peperomia, é freqüente o hábito de epífitas e suculentas. As classes de metabólitos secundários que caracterizam espécies de Piperaceae são amidas, fenilpropanóides/lignóides e cromenos, além de diversos outros de origem biossintética mista de menor representatividade. Apesar de muitos desses possuírem atividades inseticidas, diversos insetos fitófagos, alguns considerados pragas de importância econômica, foram observados alimentando-se de espécies de Piperaceae. Neste trabalho são relatadas as preferências alimentares de quatorze espécies fitófagas de Coleoptera, Lepidoptera e Hemiptera sobre aproximadamente cinqüenta espécies de Piperaceae observadas em São Paulo, SP, Brasil, durante um período de quatro anos.

Antifungal amides from Piper scutifolium and Piper hoffmanseggianum.

Chromatographic fractionation of a dichloromethane extract from the leaves of Piper scutifolium yielded two new isobutyl amides, scutifoliamide A ( 1) and scutifoliamide B ( 2), together with the known compounds piperolactam C ( 3), piperovatine ( 4), piperlonguminine ( 5), corcovadine ( 6), isopiperlonguminine ( 7), and isocorcovadine ( 8). From the dichloromethane extract from the leaves of P. hoffmannseggianum two new isobutyl amides, hoffmannseggiamide A ( 9) and hoffmannseggiamide B ( 10), were obtained together with the known compounds isopiperlonguminine ( 7) and isocorcovadine ( 8), sitosterol, and stigmasterol. The structures of the new compounds were established on the basis of spectroscopic data analysis. The inhibitory activity of compounds 1-10 against the growth of the fungi Cladosporium sphaerospermum and C. cladosporioides was determined by bioautography.