Mass spectrometry as a tool for the dereplication of saponins from Ampelozizyphus amazonicus Ducke bark and wood.

INTRODUCTION: Mass spectrometry in natural products research has been used as a first step to identify possible chemical structures and to guide subsequent efforts to isolate novel compounds. Preparations of Ampelozizyphus amazonicus Ducke (AA) are known for their high content of saponins, especially dammarane-type triterpenoid aglycones. In the Amazon, where it is known as "saracura-mirá", roots and bark are widely used for the treatment and prevention of malaria, while the wood is discarded. The extract prepared from the wood is also saponin-rich, but its exact chemical composition has not been described. OBJECTIVE: This study provides information on the chemical profiling and tentative structural identification of the major compounds (saponins) present in aqueous and ethanol extracts of bark and wood of AA by mass spectrometry. METHODS: The strategy used to identify compounds present in all samples was ultra-high-performance liquid chromatography with an ultraviolet detector coupled to tandem mass spectrometry (UHPLC-UV-MS/MS) for the analysis of fragmentation patterns through product ion scan using MZmine 2 software. Also, direct sample injection and electrospray ionisation combined with high-resolution mass spectrometry (DI-ESI-HRMS) measurements were performed. RESULTS: The extracts showed chemical similarity, and 95 saponins were tentatively identified in AA wood and bark, including 73 which are described for the first time as tentative structures for this plant species. CONCLUSION: This research describes a useful method for the fast and simultaneous tentative identification of major saponins in AA, contributing to the study of the chemical properties of this genus and family. Furthermore, it demonstrates the importance of the qualitative dereplication process, allowing a straightforward way to propose the tentative identification of compounds.

Isolation of quinoline alkaloids from three Choisya species by high-speed countercurrent chromatography and the determination of their antioxidant capacity

Abstract Choisya ternata Kunth, C. ternata var. sundance Kunth and the hybrid Choisya 'Aztec-Pearl' are three related species belonging to the Rutaceae family. Ethanol extracts were prepared from the leaves of these three species and evaluated in relation to their antioxidant activity using in vitro and ex vivo models. The ethanol extracts belonging to the three species produced a very high antioxidant profile as evidenced by the DPPH radical scavenging activity, the determination of total phenolics and flavonoid equivalent. The generation of reactive species of oxygen in leukocytes stimulated with LPS was dramatically reduced when the three ethanol extracts were used. The alkaloids anhydroevoxine and choisyine were isolated from the ethanol extract of C. ternata using HEMWat (4:6:5:5) as the solvent system by means of high-speed countercurrent chromatography. This was the first time quinoline alkaloids were isolated from this species using HSCCC. These compounds were also assayed for their capacity to inhibit the generation of ROS in leukocytes stimulated by LPS and the results also suggested that they are reactive oxygenase inhibitors.

Diterpenes and a new benzaldehyde from the mangrove plant Rhizophora mangle

ABSTRACT This work describes the isolation, by high-speed counter-current chromatography, of the diterpenes manool, jhanol and steviol and the benzaldehyde p-oxy-2-ethylhexyl benzaldehyde from the stilt roots hexane extract of the mangrove plant Rhizophora mangle L., Rhizophoraceae. For this, a non-aqueous biphasic solvent system composed of hexane–acetonitrile–methanol 1:1:0.5 (v/v/v) was applied. As far as we know, only steviol was previously isolated in Rhizophoraceae and this is the first time that p-oxy-2-ethylhexyl benzaldehyde is reported.

Alternating isocratic and step gradient elution high-speed counter-current chromatography for the isolation of minor phenolics from Ormocarpum kirkii bark.

A total of 14 compounds were isolated from the ethanol bark extract of O. kirkii S. Moore (Fabaceae) by alternating isocratic and step gradient elution high-speed counter-current chromatography (HSCCC) methods, using several solvent systems with reference to the polarity of compounds being purified. The extract was successively fractionated with generic solvent systems including n-hexane-ethanol-water (4:2:2) and ethyl acetate-water (1:1). Resulting fractions were further purified using the following preparative gradient elution consisting of ethyl acetate-n-butanol-water (X:Y:10), (X:Y=9:1 (I); 8:2 (II); 7:3 (III); 6:4 (IV); 5:5 (V); 4:6 (VI) 3:7 (VII) and n-hexane- ethyl acetate-methanol-water (1:X:1:1), X=1, 2, 2.5, 3 solvent systems. Two flavone glycosides, apigenin-6-C-ß-d-glucopyranosyl-4'-O-[ß-d-glucopyranosyl-(1→5)]-ß-d-apiofuranoside (1) and apigenin-6-C-ß-d-glucopyranosyl-4'-O-ß-d-apiofuranoside (2), and one biflavanone diglycoside 7,7″-di-O-ß-d-glucosylliquiritigeninyl-(I-3,II-3)-naringenin (4) were isolated as new compounds along with other 11 known ones. The structures of the isolated compounds were identified by HPLC-UV, ESI-MS, 1D and 2D NMR and comparison with literature data. Thus, over common traditional chromatographic methods, the present study shows that HSCCC is a useful and fast method for natural product research with no losses and lower solvent use.

In vitro and in vivo evaluation of efficacy and safety of photoprotective formulations containing antioxidant extracts

ABSTRACT Chronic exposure to solar radiation could contribute to premature skin aging and skin cancer. Skin presents its own antioxidant defense, however when defenses are out of balance, reactive oxygen species could damage biological structures. In the present work, an oil-in-water photoprotective emulsion was developed and Bauhinia microstachya var. massambabensis Vaz, Fabaceae, extracts at 1% (obtained by extraction with different solvents) were added to this emulsion. In vitro and in vivo efficacy and safety of the formulations were evaluated. Spectrophotometric methods and in vivo Colipa test were performed to evaluated efficacy of the formulations, through sun protection factor (SPF) determination and UVA protection factor assessment. To the in vitro safety assessment HET-CAM, CAM-TBS and Red Blood Cell tests were performed. Results showed that both extracts contributed to a higher in vivo photoprotection (SPF 18) when compared to the formulation without extract (SPF 13), this result could be attributed to the antioxidant activity of the plant extracts that act by capturing reactive oxygen species. Concerning safety, all formulations were considered non-irritant according to in vitro tests. Formulations containing extracts could be considered efficient and safe for cosmetic use since they presented higher sun protection factor and passed the toxicity tests.

Ethnopharmacological evaluation of medicinal plants used against malaria by quilombola communities from Oriximiná, Brazil.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is the most important parasitic disease in the world, including in the Amazon region, due to its high incidence. In addition, malaria is difficult to control because of the geographical characteristics of the endemic Amazon region. The quilombola communities of Oriximina, located in remote rainforest areas, have extensive experience with medicinal plants due to their close contact with and dependence on local biodiversity as a therapeutic resource. To search for active bioproducts against malaria, based on in vitro tests using blood culture-derived parasites and plants selected by an ethno-directed approach in traditional quilombola communities of Oriximiná, in the Amazon region of Brazil. MATERIALS AND METHODS: Ethnobotanical data were collected from 35 informants in the quilombola communities of Oriximiná, Brazil, by a free-listing method for the survey of species locally indicated to be effective against malaria and related symptoms. Data were analyzed by salience index (S) and major use agreement. The activity of extracts from 11 plants, selected based on their Salience values (four plants with S>1; seven plants with S<0.1), was measured in vitro in cultures of W2 clone Plasmodium falciparum parasites resistant to chloroquine. RESULTS: Thirty-five ethnospecies comprising 40 different plants belonging to 23 botanical families and 37 genera were listed as antimalarials by the ethno-directed approach. Among these, 11 species selected based on their S values were assayed against P. falciparum. The most active plant extracts, with an IC50 as low as 1.6µg/mL, were obtained from Aspidosperma rigidum (Apocynaceae), Bertholletia excelsa (Lecythidaceae) and Simaba cedron (Simaroubaceae), all of which displayed an S value>1. CONCLUSION: A strong correlation between the consensus of the informants from quilombola communities living in a malaria endemic area and the salience index indicating antiplasmodial activity was observed, where the ethnospecies mostly cited to be effective against malaria produced the most active plant extracts in vitro. It was also evident from the data that these groups approached the treatment of malaria with an holistic view, making use of purgative, depurative, emetic and adaptogen plants.

Isolation of achyrobichalcone from Achyrocline satureioides by high- speed countercurrent chromatography.

Achyrobichalcone is a biflavonoid recently found in Achyrocline satureioides. This substance has unprecedented chemical structure and occurrence, but resembles other bioactive bichalcones, which have important pharmacological properties, such as anticancer activity. The major challenge for evaluation of the physicochemical and biological properties of this new molecule is the isolation step, which affects the purity and yield of the isolated product. Thus, the objective of this work was to develop a semi-preparative method for achyrobichalcone isolation from Achyrocline satureioides by high-speed countercurrent chromatography. The high-speed countercurrent chromatography separation was achieved in two steps. In the first step, an enriched fraction of achyrobichalcone from the freeze-dried extract was obtained, using the solvent system hexane-ethyl acetate-methanol-water 0.8:1:0.8:1, v/v. The purification of achyrobichalcone from the enriched fractions was achieved by further high-speed countercurrent chromatography fractionation with hexane-ethyl acetate-methanol-water 0.9:0.9:0.8:1, v/v. The final isolated product was obtained using preparative thin layer chromatography and crystallization procedure. A yellow semi-crystalline solid with purity close to 90% was obtained as the final product. The mass recovery of achyrobichalcone isolation was near 67%. The structural identification from spectroscopic and chromatographic techniques confirmed the achyrobichalcone structure. This is the first report of achyrobichalcone isolation on a semi-preparative scale by high-speed countercurrent chromatography. This method afforded achyrobichalcone in good yield and purity for further biopharmaceutical studies.

Ethnopharmacological studies of Lippia origanoides

Lippia origanoides Kunth. Verbenaceae, is of great importance in the Brazilian traditional medicine. Because of it, this work had the purpose to contribute to the ethnopharmacological knowledge of L. origanoides through an ethnobotanical survey conducted within quilombola (maroon) communities of Oriximiná, Pará, Brazil. Among 254 plants cited in the survey, L. origanoides stood out among the ten most versatile species. The agreed main uses were to treat menstrual cramps, stomachache, and baby and postpartum colic. This could indicate a consensus of the informants to possible antispasmodic, anti-inflammatory and analgesic activities of L. origanoides.Therefore, anti-inflammatory and analgesic activities of L. origanoides extract (aerial parts) were assessed through thermal (hot plate) and chemical (formalin and acetic acid) models of nociception. A dose-dependent reduction in acetic acid-induced writhing was observed after treating mice with L. origanoides extract. The same extract also inhibited significantly formalin-induced licking response and proved to have a central antinociceptive effect, in the hot plate test. This work demonstrates that L. origanoides is used specially by quilombola women from Oriximiná for disorders of the genitourinary system and that biological activities of this species could contribute to these uses. Furthermore, it was also observed antispasmodic, analgesic and antimicrobial uses of other species of the genus Lippia (Goniostachyum section), rich in thymol and carvacrol.

Application of pH-zone-refining countercurrent chromatography for the separation of indole alkaloids from Aspidosperma rigidum Rusby.

Species of Aspidosperma (Apocynaceae) are characterized by the occurrence of indole alkaloids, but few recent reports on Aspidosperma rigidum Rusby chemical constituents were found. The present work shows the application of pH-zone refining countercurrent chromatography on the separation of alkaloids from the barks of A. rigidum. In this study, the dichloromethane extract was fractionated with the solvent system composed of methyl-tert-butyl ether and water with different concentrations of the retainer triethylamine in the organic stationary phase and formic or hydrochloric acids as eluters in the aqueous mobile phase, in order to evaluate the most suitable condition. In each experiment, from circa 200mg of the dichloromethane extract of A. rigidum, three major alkaloids were isolated and identified as 3α-aricine (circa 17mg), isoreserpiline (ca. 22mg) and 3ß-reserpiline (ca. 40mg), with relative purity of 79%, 89% and 82% respectively, in a one-step separation of 2h. Two of them - 3α-aricine and isoreserpiline - were isolated and identified for the first time in this species.

Ethnopharmacological versus random plant selection methods for the evaluation of the antimycobacterial activity

The municipality of Oriximiná, Brazil, has 33 quilombola communities in remote areas, endowed with wide experience in the use of medicinal plants. An ethnobotanical survey was carried out in five of these communities. A free-listing method directed for the survey of species locally indicated against Tuberculosis and lung problems was also applied. Data were analyzed by quantitative techniques: saliency index and major use agreement. Thirty four informants related 254 ethnospecies. Among these, 43 were surveyed for possible antimycobacterial activity. As a result of those informations, ten species obtained from the ethnodirected approach (ETHNO) and eighteen species obtained from the random approach (RANDOM) were assayed against Mycobacterium tuberculosis by the microdilution method, using resazurin as an indicator of cell viability. The best results for antimycobacterial activity were obtained of some plants selected by the ethnopharmacological approach (50 percent ETHNO x 16,7 percent RANDOM). These results can be even more significant if we consider that the therapeutic success obtained among the quilombola practice is complex, being the use of some plants acting as fortifying agents, depurative, vomitory, purgative and bitter remedy, especially to infectious diseases, of great importance to the communities in the curing or recovering of health as a whole.

Isolation of chavibetol from essential oil of Pimenta pseudocaryophyllus leaf by high-speed counter-current chromatography.

Counter-current chromatography (CCC) was used to isolate chavibetol from the essential oil of leaves of Pimenta pseudocaryophyllus (Gomes) Landrum. Chavibetol was obtained in high purity (98%) and mass recovery (94.4%). Methyleugenol was also isolated. The CCC biphasic solvent system used was composed of hexane:n-butanol:methanol:water (12:4:4:3, v/v/v/v).

Anatomia e análise de óleo essencial das folhas de Hennecartia omphalandra J Poisson (Monimiaceae) / Anatomy and essential oil analysis of the leaves from Hennecartia omphalandra J Poisson (Monimiaceae)

Descreve-se a morfo-anatomia e a análise de óleo essencial das folhas de Hennecartia omphalandra J. Poisson (Monimiaceae), uma planta tóxica. A ocorrência de tricomas unicelulares, estômatos paracíticos; braquiesclereídes em grupos no pecíolo e na lâmina foliar, além de esclereídes vermiformes e ramiformes; sistema vascular em arco, com extremidades voltadas para o centro no pecíolo e na forma de arco atenuado na lâmina foliar, sendo o padrão de venação uma variação do tipo craspedódromo-semicraspedódromo, além de óleo essencial armazenado em idioblastos oleíferos não especializados no mesofilo, foram considerados caracteres importantes que podem auxiliar na sua morfo-diagnose. A análise do óleo essencial das folhas revelou a presença de monoterpenos, sesquiterpenos e álcoois sesquiterpênicos, sendo este o grupo mais significativo. Os componentes majoritários identificados foram β-felandreno (24,0 por cento), seguido por germacreno D (10,5 por cento), sabineno (9,3 por cento), biciclogermacreno (8,4 por cento) e β-elemeno (5,3 por cento).

This paper describes the morphological and anatomical aspects and the essential oil analysis of the leaves from Hennecartia omphalandra J. Poisson (Monimiaceae), a toxic plant. The presence of trichomes formed by a single cell, paracitic stomata, groups of braquisclereids in the petiole and leaf blade; vermiform and ramiform sclereids, vascular system in arc softly invaginated at ends in the petiole, and in a flat arc in the middle vein, being the venation pattern a variation of craspedodromous-semicraspedodromous type, and essential oil stored in oil idioblasts in the mesophyll were the most important characters that can help the morphological diagnosis. The analysis of essential oil of the leaves showed the presence of monoterpenes, as well as sesquiterpenes hydrocarbons and alcohols, being the latter the most significant group. The major identified compounds were β-phelandrene (24.0 percent), germacrene D (10.5 percent), sabinene (9.3 percent), bicyclogermacrene (8.4 percent) and β-elemene (5.3 percent).

Considerações anatômicas e análise de óleo essencial do hipanto e do fruto de Hennecartia omphalandra J. Poisson (Monimiaceae) / Anatomical considerations and essential oil analysis of the hypanthium and the fruit of Hennecartia omphalandra J. Poisson (Monimiaceae)

Descreve-se o estudo morfo-anatômico e a análise de óleo essencial do fruto de Hennecartia omphalandra J. Poisson (Monimiaceae), uma planta tóxica. A ocorrência de feixes recurrentes e de um anel descontínuo de braquiesclereides no hipanto; assim como a presença de células oleíferas no mesocarpo, de "idioesclereídes" formando o endocarpo e de células com espessamento parietal em rede, constituindo a testa da semente, foram considerados caracteres importantes que podem auxiliar na sua morfo-diagnose. A análise do óleo essencial dos frutos revelou a presença de monoterpenos, sesquiterpenos e álcoois sesquiterpênicos, sendo este o grupo mais significativo. Os componentes majoritários identificados foram o biciclogermacreno (19,6 por cento) e o β-felandreno (8,7 por cento), além do germacreno D (5,3 por cento), α-bulneseno (5,1 por cento) e valerianol (4,9 por cento).

The morphological and anatomical aspects of flower and fruit and the essential oil analysis of fruits of Hennecartia omphalandra J. Poisson (Monimiaceae), a toxic plant, are described in this paper. The presence of vascular recurrent bundles and of a ring of brachysclereids in the hypanthium; the oil cells in the mesocarp, the "idiosclereids" that constitutes the endocarp and cells with reticulated thick walls, shaping the seed coat were described as characters that may help in its diagnosis. The analysis of the essential oil of fruits showed the presence of monoterpenes, as well as sesquiterpenes hydrocarbons and alcohols, being the latter the most significant group. The major identified compounds were bicyclogermacrene (19.6 percent); β-felandrene (8.7 percent); germacrene D (5.3 percent); α - bulnesene (5.1 percent) and valerianol (4.9 percent).

Applications of counter-current chromatography in organic synthesis purification of heterocyclic derivatives of lapachol.

This work describes the application of counter-current chromatography (CCC) as a useful, fast and economic alternative for the isolation and purification of heterocyclic derivatives from lapachol and beta-lapachone, two naturally occurring compounds from Tabebuia species, and nor-beta-lapachone, a synthetic congener of lapachol. The discussed data comprise four examples of purification of synthetic reactions with different solvent systems - the mixture of the oxazole and the imidazole from beta-lapachone; the quinoxaline from nor-beta-lapachone; and the purification of the N-oxides from the quinoxaline and the phenazine from nor-beta-lapachone from their respective not fully reacted substrates by means of aqueous reversed- and normal-phase elution modes and non-aqueous solvent systems. Traditional purification of these reaction products by silica gel column chromatography demanded a large amount of solvent and time and, in some cases, serious degradation of the products occurred, leading to low yield of the reaction. High-speed counter-current chromatography (HSCCC) was used as an alternative to optimize the process and raise the yield of the reactions.

Ethnopharmacological study of two Lippia species from Oriximiná, Brazil.

Lippia alba (Miller) N.E. Brown is an aromatic herb that occurs in practically all regions of Brazil and has a large importance in Brazilian folk medicine. This species is characterized by a variability in morphology and in the chemical composition of the essential oil. The present work focused on the ethnopharmacological investigation of Lippia alba, locally known as "erva-cidreira" and Lippia alba f. intermedia, known as "carmelitana". In addition, the chemical composition and antimicrobial activity of their essential oils was investigated in order to correlate to their traditional uses. The ethnopharmacological study showed a good agreement of the major use (MUA) of Lippia alba (MUA=92.0%) and to a lesser extent, for Lippia alba f. intermedia (MUA=66.7%), as sedatives. The analyses of the essential oils allowed the identification of Lippia alba as a myrcene-citral chemotype (15% and 37.1%, respectively) and Lippia alba f. intermedia as a citral chemotype (22.1%). The essential oils of both species were active against all microorganisms assayed (bacteria and fungi) by the drop test, with inhibition halos ranging from 1.1 to 5.0 cm, probably due to the high content of oxygenated monoterpenes (51.0% and 40.1%, respectively), specially represented by aldehydes and alcohols. The chemical and pharmacological data of Lippia alba are in agreement with the ethnobotanical survey.

Screening of Central and South American plant extracts for antimycobacterial activity by the Alamar Blue test / Triagem de extratos de plantas das Américas Central e do Sul para atividade antimicobacteriana pelo teste do Alamar Blue

Forty eight ethanolic crude extracts and fractions (hexane, dichloromethane, ethyl acetate and n-butanol) from ten Brazilian plants (Leguminosae, Monimiaceae and Verbenaceae), 1 from Costa Rica (Verbenaceae) and 1 from Argentina (Verbenaceae) were screened for anti-mycobacterium activity against Mycobacterium tuberculosis (ATCC-27294H37Rv), by the Alamar Blue test, at a fixed concentration of 100 µg/mL. Out of the forty eight, seven were active at this concentration, corresponding to Lantana trifolia (hexane and dichloromethane extracts from leaves), Vitex cooperi (methanol:water, 1:1 extract from barks), Lippia lacunosa (hexane and dichloromethane extracts from leaves) and Lippia rotundifolia (hexane and dichloromethane extracts from leaves), all from the Verbenaceae family.

Quarenta e oito extratos brutos etanólicos e frações (em hexano, diclorometano, acetato de etila e n-butanol) de dez plantas brasileiras pertencentes às famílias Leguminosae, Monimiaceae e Verbenaceae; uma da Costa Rica (Verbenaceae) e uma da Argentina (Verbenaceae) foram ensaiados para verificação da atividade anti-micobacteriana contra Mycobacterium tuberculosis (ATCC-27294H37Rv), pelo teste do Alamar Blue, a uma concentração fixa de 100 µg/mL. Dentre os quarenta e oito extratos e frações estudados, sete mostraram-se ativos na concentração ensaiada - frações em hexano e diclorometano de folhas de Lantana trifolia, extrato em metanol:água, 1:1 de cascas de Vitex cooperi, frações em hexano e diclorometano de folhas de Lippia lacunosa e de Lippia rotundifolia, sendoque todas essas plantas pertencem à família Verbenaceae.

Quick preparative separation of natural naphthopyranones with antioxidant activity by high-speed counter-current chromatography.

The natural naphthopyranones paepalantine [structure: see text], paepalantine-9-O-beta-D-glucopyranoside [structure: see text] and paepalantine-9-O-beta-D-allopyranosyl-(1-->6)-O-beta-D-glucopyranoside [structure: see text] were separated in a preparative scale from the ethanolic extract of the capitula of Paepalanthus bromelioides by high-speed counter-current chromatography (HSCCC). The solvent system used was composed of water-ethanol-ethyl acetate-hexane (10:4:10:4, v/v/v/v). This technique led to the separation of the three different naphthopyranone glycosides in pure form in approximately 7 hours. Paepalantine showed a good antioxidant activity when assayed by the DPPH radical spectrophotometric assay.