Hector Henrique Ferreira Koolen

Comprehensive characterization of lipids from Amazonian vegetable oils by mass spectrometry techniques

An integrative approach in mass spectrometry (MS) comprising gas chromatography coupled to MS (GC-MS), ultra-efficiency liquid chromatography coupled to MS (UPLC-MS) and easy ambient sonic-spray ionization MS (EASI-MS) is proposed for the comprehensive characterization of Amazonian oils. Coconut, andiroba and castor seed oils, which are vastly sold in markets of the Amazonian region of Brazil, were selected as a representative test set. These oils were found to contain several lipids such as triacylglycerides (TAGs), fatty acids (FAs), phytosterols and limonoids. In the analyzed samples 30 different TAGs, 11 FAs, 6 phytosterols and 7 limonoids were identified. The antioxidant capacity (AOC) of the oils, as measured by their oxygen radical absorbance capacity (ORAC), was also used to evaluate their potential biological properties as well as their possible consumption as food. Edible virgin coconut oil was the most active (0.720±0.001 Trolox eq./mmol), whereas considerable lower activity was observed for andiroba and castor seed oils. The antimicrobial activities of the oils were also recorded against a panel of pathogenic bacteria and fungi in which andiroba oil was the only one that was active, solely against Enterococcus aeruginosa.

An antimicrobial alkaloid and other metabolites produced by Penicillium sp. An endophytic fungus isolated from Mauritia flexuosa L. f.

The alkaloid glandicoline B (1) and six other compounds: ergosterol (2), brassicasterol (3), ergosterol peroxide (4), cerevisterol (5), mannitol (6) and 1-O-α-D-glucopyranoside (7) were isolated from Penicillium sp. strain PBR.2.2.2, a fungus from Mauritia flexuosa roots. The structures of the isolated metabolites were established by spectral analysis. MeOH extract of the fungal mycelium at 500 µg mL-1 exhibited antimicrobial activity against Staphylococcus aureus and the compound 1 at 100 µg mL-1 was active against S. aureus, Micrococcus luteus and Escherichia coli. The relationship between the bioactive properties of the fungus PBR.2.2.2 and those achieved for glandicoline B, as well the potential of this substance as bactericide is discussed.

Triterpenes and flavonoids from the roots of Mauritia flexuosa

Mauritia flexuosa L. f., Arecaceae, is an endemic species of South America. This species was studied with the intent to isolate the constituents of its roots. After the fractionation of the n-hexane and methanolic extracts from the roots of M. flexuosa, six triterpenes were obtained: friedelin, taraxerone, lupenyl acetate, lupenone, betulin and betulinic acid, along with three flavonoids: rutin, quercitrin and quercetin. All the compounds were identified by analysis of NMR and MS data and comparison with the literature. All those compounds are been reported for the first time in Mauritia, and the chemosystematic significance of the flavonoids isolated in this genus is discussed.

DESREPLICAÇÃO DE ALCALOIDES APORFÍNICOS E OXOAPORFÍNICOS DE Unonopsis guatterioides POR ESI‑IT‑MS

The dereplication of aporphine and oxoaporphine alkaloids by direct infusion in ESI-IT-MSn system was applied for alkaloidal fractions of the Unonopsis guatterioides (Annonaceae). Its main advantage over other dereplication methods is the ability to quickly identify substances in complex mixtures without the use of coupled techniques and expensive databases. By only the fragmentation keys and comparison with literature data the aporphine alkaloids anonaine, asimilobine and nornuciferine were identified. The nornuciferine is being reported for the first time in Unonopsis. The oxoaporphine alkaloids liriodenine and lisycamine were identified in the alkaloidal fractions by comparison with the fragmentations of authentic samples.

An antimicrobial diketopiperazine alkaloid and co-metabolites from an endophytic strain of Gliocladium isolated from Strychnos cf. toxifera

From an endophytic strain of Gliocladium sp. isolated from the Amazonian plant Strychnos cf. toxifera, we obtained the diketopiperazine alkaloid cyclo-(glycyl-L-tyrosyl)-4,4-dimethylallyl ether (1), the steroids ergosterol (2), ergosterol peroxide (3), cerevisterol (4) and the citric acid (5). The AcOEt extract of the fermented broth by Gliocladium sp. showed potent activity against the cancer cell lines MDA-MB435 (human breast cancer cells), HCT-8 (human colorectal cancer cells) and SF-295 (human glioblastoma cancer cells). Compound 1 exhibited a strong antimicrobial activity against Micrococcus luteus at a concentration of 43.4 µM.

Aproveitamento do Óleo das Amêndoas de Tucumã do Amazonas na Produção de Biodiesel

RESUMO A falta de disponibilidade de energia eletrica e um dos principais motivos pelo baixo Indice de Desenvolvimento Humano das comunidades isoladas localizadas na Amazonia. O biodiesel produzido a partir de oleos vegetais extraidos de especies oleaginosas nativas de forma sustentada e uma das melhores alternativas energeticas para a regiao. O tucuma do amazonas, Astrocaryum aculeatum, e uma especie de palmeira que produz um fruto muito apreciado na regiao, a partir do qual se obtem uma amendoa com alto teor de oleo. Nesse estudo, foi avaliada a producao de biodiesel etilico, a partir de diferentes lotes de oleos de tucuma do amazonas, com indices de acidez baixos e elevados, pela transesterificacao por catalise basica e acida homogeneas, respectivamente. Na catalise acida, foram testados HCl e H 2 SO 4 como catalisadores nas concentracoes de 0,0625 a 1,000 M, empregando etanol hidratado na proporcao molar de 1:6 e a reacao conduzida a 90 oC por 24 h. Na catalise basica, foram testados NaOH e KOH, nas proporcoes de 0,5 a 2,0 %, empregando etanol anidro na proporcao molar de 1:12 e a reacao conduzida a 80 oC por 2 h. O biodiesel obtido em cada experimento foi analisado por metodos fisicos (massa especifica) e cromatograficos (CLAE em fase reversa). Analises cromatograficas indicaram que as melhores conversoes foram alcancadas por amostras de biodiesel com massas especificas inferiores a 0,87 g.cm -1 . As amostras de biodiesel obtidas com melhor qualidade foram obtidas utilizando-se os catalisadores acidos a 1,0 M com rendimentos superiores a 90%. No caso da catalise basica, obteve-se biodiesel de boa qualidade empregando-se o catalisador NaOH a 2,0%, porem com rendimento inferior a 60 %. Contudo, em ambos os casos, foi possivel identificar um excelente potencial de producao de biocombustivel, a partir do oleo das amendoas de tucuma. PALAVRAS-CHAVE: Sustentabilidade, Arecaceae, Energia eletrica, Amazonia. The use of tucuma of amazonas kernel oil in the biodiesel production

Direct infusion ESI‐IT‐MSn alkaloid profile and isolation of tetrahydroharman and other alkaloids from Bocageopsis pleiosperma maas (Annonaceae)

INTRODUCTION The Annonaceae family is known as a promising abundant source of secondary metabolites, especially annonaceous acetogenins, terpenoids and isoquinoline-derived alkaloids. Although widely investigated from the phytochemical viewpoint, this family still presents some largely unexplored genera, e.g. the Bocageopsis. OBJECTIVE To investigate the alkaloid content of Bocageopsis pleiosperma Maas using direct infusion electrospray ionisation ion trap tandem mass spectrometry (ESI-IT-MS(n)) analysis. METHODOLOGY Dichloromethane extracts of aerial parts were subjected to acid-base partitioning to yield the alkaloidal fractions. These fractions were analysed by direct infusion into a (+)ESI-IT-MS(n) system. The alkaloidal fraction from the leaves was also obtained on a large scale and subjected to chromatographic separation. RESULTS The tentative MS(n) -based identification of alkaloids in leaves, twigs and trunk bark showed that aporphine alkaloids were restricted to the leaves and twigs, tetrahydroprotoberberine alkaloids were only found in the twigs and trunk bark while benzylisoquinoline alkaloids were found in the leaves, twigs and trunk bark. Chromatographic separation of the leaf alkaloidal fraction yielded the aporphine alkaloids nornuciferine, asimilobine and isoboldine, the β-carboline alkaloid tetrahydroharman and some mixtures containing benzylisoquinoline and aporphine alkaloids, all described for the first time in the Bocageopsis genus. Furthermore, tetrahydroharman has not previously been reported in the Magnoliales order. CONCLUSION Direct infusion ESI-IT-MS(n) analysis of alkaloids allowed fast recognition of alkaloidal classes previously reported in the Annonaceae family, aiding the chromatographic step and allowing a selective isolation of compounds previously not identified in the Bocageopsis genus.

Talaroxanthone, a novel xanthone dimer from the endophytic fungus Talaromyces sp. associated with Duguetia stelechantha (Diels) R. E. Fries

DgCr22.1b, an endophytic isolate of Talaromyces sp., was collected in the Amazonian Rainforest from the medicinal plant Duguetia stelechantha. From the fractionation of the methanolic mycelial extract, a new xanthone dimer talaroxanthone was isolated. The structure of this new compound was elucidated based on spectroscopic analyses including 2D nuclear magnetic resonance (NMR) experiments.

Chemotaxonomy of the Amazonian Unonopsis Species Based on Leaf Alkaloid Fingerprint Direct Infusion ESI-MS and Chemometric Analysis

Unonopsis (Annonaceae) is a neotropical genus constituted by nearly fifty species, with fifteen described in Brazil. In the state of Amazonas seven species are found, including U. guatterioides, which displays problems from the botanical viewpoint. Previous studies showed this genus as a promising source of aporphinoid alkaloids. In order to investigate the potential of the leaf alkaloid fingerprint for chemotaxonomic approaches, twelve Unonopsis specimens, representing five species commonly found in the state of Amazonas were subjected to acid-base partitioning to yield the respective alkaloidal fractions. These fractions were analysed by direct infusion electrospray ionization multiple stage mass spectrometry (ESI-MSn). The obtained data were treated through chemometric tools [principal components analysis (PCA) and hierarchical cluster analysis (HCA)]. Multivariate analysis pointed to aporphine, proaporphine and tetrahydroprotoberberine alkaloids as the responsible compounds for segregation of the investigated species, being these alkaloids tentatively identificated by multiple stage mass spectrometry. The alkaloid fingerprint along with multivariate analysis provided a simple and effective approach to differentiateUnonopsis species commonly found in the state of Amazonas.

Chemical constituents of Penicillium chrysogenum, an endophytic fungus from Strychnos toxifera

Strychnos toxifera is a poisonous plant used by Amazon indians in the manufacture of curare poison [1]. Its toxicity is clue to indole alkaloids, which are the major constituents of this poison [2]. Endophytes are considered outstanding and underexplored sources of novel chemically diverse and bioactive compounds. These microorganisms can be detected at a particular moment within the tissues of apparently healthy plant hosts, and they have been found in all plant species examined to date [3]. As they occupy unique biological niches, the complex web of interactions with other endophytes and with the host might give rise to new chemical diversity and bioactive compounds [4]. In the course of screening for biologically active agents from Amazonian endophytic fungi [5], Penicillium chrysogenum, an endophytic fungus from the trunk of S. toxifera, was chosen for further phytochemical investigation. The MeOH extract of the mycelial mass from P. chrysogenum was subjected to chromatographic separation to afford four steroids [6] [ergosterol (1), brassicasterol (2), ergosterol peroxide (3), and cerevisterol (4)], two aromatic acids [7] [cinnamic acid (5) and p-hydroxybenzoic acid (6)], and three mycotoxins [8] [kojic acid (7), penicillic acid (8), and patulin (9)]. These compounds were obtained and characterized by a comparison of their physical and spectral data (UV, IR, NMR and MS), with values in the literature. S. toxifera was collected from Manaus, Brazil in July, 2008. The strain of P. chrysogenum was obtained, purified, and identified from the healthy tissues of the trunk using a previous methodology [9]. After cultivation in liquid media [5], the mycelial mass (1.1 kg) was extracted with MeOH (3 500 mL) at room temperature, and a MeOH extract (30.4 g) was obtained upon concentration under reduced pressure. The MeOH extract was chromatographed over silica gel (600 g, 70–230 mesh) using n-hexane containing increasing amounts of EtOAc to obtain nine fractions. Fraction 1 (321.7 mg) was purified on a silica gel column with n-hexane–CHCl3 with increasing gradient, yielding 1 (22 mg), 2 (7 mg), 3 (12 mg), and 4 (9 mg). Fraction 5 (507 mg) was subjected to silica gel chromatography with CHCl3–MeOH with increasing gradient, yielding 5 (4 mg), 6 (11 mg), 7 (89 mg), 8 (9 mg), and 9 (5 mg).