Danilo Ribeiro de Oliveira

Screening of Central and South American plant extracts for antimycobacterial activity by the Alamar Blue test

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.

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

The municipality of Oriximina, 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% ETHNO x 16,7% 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.

Analysis of the chemical composition of the essential oils extracted from Lippia lacunosa Mart. & Schauer and Lippia rotundifolia Cham. (Verbenaceae) by gas chromatography and gas chromatography-mass spectrometry

Lippia lacunosa and L. rotundifolia (Verbenaceae) are two Brazilian species of complex taxonomic delimitation. The composition of the essential oils from leaves and flowers of these plants was investigated by gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC-MS) analysis. The major components of the essential oils of flowers and leaves of L. lacunosa were: myrcene (14.7% and 11.9%), myrcenone (45.2% and 64.2%), Z-ocimenone (5.7% and 5.2%), and E-ocimenone (14.7% and 4.1%), respectively; whereas in L. rotundifolia (flowers and leaves) were a-pinene (8.7% and 1.8%), myrcene (5.1% and 3.6%), limonene (26.0% and 7.9 %), cis-pinocamphone (4.5% and 3.1%) and myrtenal (22.3% and 16.7%), respectively. The essential oils from L. lacunosa exhibited a strong and pleasant mango aroma, which was related to the presence of myrcene and myrcenone. The marked differences in the chemical composition of their essential oils may represent a powerful tool for the botanical classification.

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.

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 3β-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.

Immunobiologic and Antiinflammatory Properties of a Bark Extract from Ampelozizyphus amazonicus Ducke

Ampelozizyphus amazonicus is used in the treatment and prevention of malaria. The effect of an aqueous extract from this plant (SART) on the immune response was investigated by measuring immunoglobulin production induced by immunization with the antigen TNP-Ficoll in Plasmodium chabaudi-infected mice. SART treatment increased antigen-specific IgM and IgG levels in TNP-Ficoll-immunized mice. The B cell response during malarial infection was also modified by SART. There was an increase in total serum IgM and IgG and a decrease in the percentage of splenic plasma cells (CD138+ cells) in P. chabaudi-infected, SART-treated animals. SART (1, 3 or 10 mg/kg, p.o.) and the reference drug dexamethasone (5 mg/kg) were also tested in carrageenan-induced leukocyte migration to the subcutaneous air pouch (SAP). All SART doses significantly reduced leukocyte migration into the SAP. The protein concentration resulting from extravasation into the peritoneum was also significantly reduced. Our data indicate that SART possesses immunomodulatory properties, inducing an in vivo modification of the B lymphocyte response and anti-inflammatory properties, which are partly due to a reduction in cell migration and are most likely due to an inhibition of the production of inflammatory mediators. Preliminary HPLC-ESI-MS/MS analysis of SART shows a complex saponin profile with deprotonated molecule [M-H]− ions in the range of m/z 800–1000.

Essential oils of Protium spp. samples from Amazonian popular markets: chemical composition, physicochemical parameters and antimicrobial activity

Species belonging to the Burseraceae family, especially of the genus Protium, are well known for producing fragrant oleoresins known in Brazil as breus. In this study, the physicochemical properties and antimicrobial activities of essential oils obtained from commercials samples of breu from the Brazilian Amazon were evaluated. Essential oils were extracted by hydrodistillation using a modified Clevenger apparatus and analyzed by gas chromatography/mass spectrometry (GC/MS) and GC/flame ionization detector (GC/FID). p-Cymene was the major constituent in all of the analyzed breu samples, with concentrations ranging from 21.9% to 51.9%, except for the white breu sample from Adolfo Lisboa Popular Market (ALPM), which contained α-pinene (22.7%). Other common monoterpenes were β-phellandrene, α-phellandrene, β-pinene, trans-dihydro-α-terpineol, α-terpineol and α-terpinene. The refractive indices ranged from 1.4769 to 1.4849 and the optical rotation ranged from −14.15° to +29.96°. The antimicrobial activity was low for all of the samples. The essential oil of black breu from ALPM was the only sample exhibiting antimycobacterial activity.

Essential oil from Philodendron fragrantissimum, an aromatic Araceae from Amazonia, Brazil

In the Amazon region of Brazil, the roots of different species of Philodendron are used in the traditional medicine. Philodendron fragrantissimum (Hook.) G. Don, locally known as ‘cipó-tracuá’, has very aromatic roots and is used for baths and fumigation. Here, we describe the chemical composition of the essential oil from the roots of P. fragrantissimum obtained by hydrodistillation in a modified Clevenger-type apparatus for 4 hours. The oil was analyzed by gas chromatography (GC) and GC/mass spectrometry (GC/MS). Thirty-five substances were detected and the major components were β-caryophyllene (29.9%), limonene (15.8%), β-selinene (11.5%), α-selinene (15.7%), α-pinene (3.4%), α-copaene (3.2%), β-pinene (2.5%) and δ-cadinene (2.9%). To the best of our knowledge, this is the first report on the chemical composition of the essential oil from P. fragrantissimum. The high content of β-caryophyllene was not observed in the other oils studied so far and might be considered a tool in taxonomy studies.

Counter-current chromatography with off-line detection by ultra high performance liquid chromatography/high resolution mass spectrometry in the study of the phenolic profile of Lippia origanoides

Lippia origanoides (Verbenaceae) is an important Brazilian medicinal plant, also used for culinary purposes. Most chemical studies with this plant have been focused on its volatile composition. In this work, we combined High-Speed Counter-current Chromatography (HSCCC) and High Performance Liquid Chromatography coupled to Ultra Violet detection and High Resolution Mass Spectrometry (HPLC-UV-HRMSn) methodologies to access the non-volatile chemical composition of L. origanoides. The crude ethanol extract of L. origanoides (LOEF) was first analyzed by HPLC-UV-HRMSn and allowed the identification of 7 major compounds. Among them, eriodictyol, naringenin and pinocembrin, were determined and are phytochemical markers of this plant. However, owing to the complexity of this plant matrix, LOEF was fractionated by HSCCC (hexane-ethanol-water, 4:3:1) as a tool for preparative pre-purification, affording a flavonoid-rich fraction. A column screening with the chromatographic stationary phases ZIC-HILIC, monolithic and particulate RP18 was performed. The best column separation was achieved with a Purospher STAR RP18e, which was used for HPLC-DAD-HRMSn studies. By this approach 12 compounds were further identified in addition to the major ones identified in the raw extract. Two of them, 6,8-di-C-hexosyl-luteolin and 6,8-di-C-glucosyl-apigenin, are being reported for the first time in the family Verbenaceae. This work shows the integration of HSCCC as a preparative tool for the fractionation and purification of natural products from a complex plant extract with other analytical techniques, with the purpose of showing each techniques potential.

Spray-dried extract from the Amazonian adaptogenic plant Ampelozizyphus amazonicus Ducke (Saracura-mirá): Chemical composition and immunomodulatory properties

Ampelozizyphus amazonicus Ducke is a medicinal plant used in the Amazon region to prepare a drink with tonic, immunomodulatory and adaptogenic properties. Due to the growing interest in dietary supplements with these properties and, to provide a new functional ingredient, barks from A. amazonicus were extracted. The extract was spray dried without drying adjuvants, resulting in a powder (SARF), which was characterized by its physico-chemical properties and proximate, mineral and saponin contents. The SARF saponins were characterized by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry (HPLC-HRMSn) analysis. The SARF particles tended to have a spherical shape and a unimodal size distribution. The particles also had good rehydration characteristics and high saponin content (33%). The effect of SARF on antibody production was investigated, and we found that SARF increased the basal levels of anti-ovalbumin, anti-LPS and anti-dextran IgM antibodies, and the anti-dextran IgG antibodies in unimmunized mice. No increase in antibody titers was observed after SARF treatment in immunized mice. These results suggest that SARF could be an interesting new functional ingredient for food applications or pharmaceutical products.