Benzylated Dihydroflavones and Isoquinoline-Derived Alkaloids from the Bark of Diclinanona calycina (Annonaceae) and Their Cytotoxicities.

Diclinanona calycina R. E. Fries popularly known as "envira", is a species of the Annonaceae family endemic to Brazil. In our ongoing search for bioactive compounds from Annonaceae Amazon plants, the bark of D. calycina was investigated by classical chromatography techniques that yielded thirteen compounds (alkaloids and flavonoids) described for the first time in D. calycina as well as in the genus Diclinanona. The structure of these isolated compounds were established by extensive analysis using 1D/2D-NMR spectroscopy in combination with MS. The isolated alkaloids were identified as belonging to the subclasses: simple isoquinoline, thalifoline (1); aporphine, anonaine (2); oxoaporphine, liriodenine (3); benzyltetrahydroisoquinolines, (S)-(+)-reticuline (4); dehydro-oxonorreticuline (3,4-dihydro-7-hydroxy-6-methoxy-1-isoquinolinyl)(3-hydroxy-4-methoxyphenyl)-methanone) (5); (+)-1S,2R-reticuline Nß-oxide (6); and (+)-1S,2S-reticuline Nα-oxide (7); tetrahydroprotoberberine, coreximine (8); and pavine, bisnorargemonine (9). While the flavonoids belong to the benzylated dihydroflavones, isochamanetin (10), dichamanetin (11), and a mixture of uvarinol (12) and isouvarinol (13). Compound 5 is described for the first time in the literature as a natural product. The cytotoxic activity of the main isolated compounds was evaluated against cancer and non-cancerous cell lines. Among the tested compounds, the most promising results were found for the benzylated dihydroflavones dichamanetin (10), and the mixture of uvarinol (12) and isouvarinol (13), which presented moderate cytotoxic activity against the tested cancer cell lines (<20.0 µg·mL-1) and low cytotoxicity against the non-cancerous cell line MRC-5 (>25.0 µg·mL-1). Dichamanetin (11) showed cytotoxic activity against HL-60 and HCT116 with IC50 values of 15.78 µg·mL-1 (33.70 µmol·L-1) and 18.99 µg·mL-1 (40.56 µmol·L-1), respectively while the mixture of uvarinol (12) and isouvarinol (13) demonstrated cytotoxic activity against HL-60, with an IC50 value of 9.74 µg·mL-1, and HCT116, with an IC50 value of 17.31 µg·mL-1. These cytotoxic activities can be attributed to the presence of one or more hydroxybenzyl groups present in these molecules as well as the position in which these groups are linked. The cytotoxic activities of reticuline, anonaine and liriodenine have been previously established, with liriodenine being the most potent compound.

BcXyl, a ß-xylosidase Isolated from Brunfelsia Calycina Flowers with Anthocyanin-ß-glycosidase Activity.

Brunfelsia calycina flowers lose anthocyanins rapidly and are therefore well suited for the study of anthocyanin degradation mechanisms, which are unclear in planta. Here, we isolated an anthocyanin-ß-glycosidase from B. calycina petals. The MS/MS (Mass Spectrometry) peptide sequencing showed that the enzyme (72 kDa) was a ß-xylosidase (BcXyl). The enzyme showed high activity to p-Nitrophenyl-ß-d-galactopyranoside (pNPGa) and p-Nitrophenyl-ß-d-xylopyranoside (pNPX), while no activity to p-Nitrophenyl-ß-d-glucopyranoside (pNPG) or p-Nitrophenyl-ß-D-mannopyranoside (pNPM) was seen. The optimum temperature of BcXyl was 40 °C and the optimum pH was 5.0. The enzyme was strongly inhibited by 1 mM D-gluconate and Ag⁺. HPLC (High Performance Liquid Chromatography) analysis showed that BcXyl catalyzed the degradation of an anthocyanin component of B. calycina, and the release of xylose and galactose due to hydrolysis of glycosidic bonds by BcXyl was detected by GC (Gas Chromatography) /MS. A full-length mRNA sequence (2358 bp) of BcXyl (NCBI No. MK411219) was obtained and the deduced protein sequence shared conserved domains with two anthocyanin-ß-glycosidases (Bgln and BadGluc, characterized in fungi). BcXyl, Bgln and BadGluc belong to AB subfamily of Glycoside hydrolase family 3. Similar to BcPrx01, an anthocyanin-degradation-related Peroxidase (POD), BcXyl was dramatically activated at the stage at which the rapid anthocyanin degradation occurred. Taken together, we suggest that BcXyl may be the first anthocyanin-ß-glycosidase identified in higher plants.

In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers.

In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated.

Phenolic compounds and extracts from Crotalaria calycina Schrank potentially alleviate pain and inflammation through inhibition of cyclooxygenase-2: An in vivo and molecular dynamics studies.

Crotalaria calycina Schrank is a local Bangladeshi plant well-accepted by the tribal population for its medicinal properties. The primary approach of our study was to uncover the analgesic and anti-inflammatory potential of methanol extract of C. calycina stem in mice model with in silico molecular docking and molecular dynamics simulation approach. Phenolic compounds were identified and quantified from the extract through high-performance liquid chromatography-diode array detector (HPLC-DAD) analysis. Writhing assay through injection of acetic acid, licking assay through formalin injection, and finally, hot plate assay was employed to observe the analgesic activity. The carrageenan-induced paw edema model was employed to determine the anti-inflammatory potential of the extract. In silico molecular docking and molecular dynamics were also run to validate the in vivo study results. Eight polyphenolic compounds from the extract were identified and quantified via HPLC-DAD analysis, and (-) epicatechin was most abundantly distributed (87.15 ± 0.24 mg/100 g dry extract). In vivo study revealed that 400 mg/kg dose significantly inhibited (P < 0.01) the writhing response in the writhing assay and demonstrated the highest percent of inhibition of licking (70.67%) in the late part of the licking test. The same extract dose produced the highest (74.71%) percent of maximal effect (% MPE) in the hot plate assay. It demonstrated the highest percent of edema inhibition (68.00%) in the fourth hour of the paw edema assay. Molecular docking and molecular dynamics simulation of (-) epicatechin, caffeic acid, and kaempferol with cyclooxygenase-2 revealed that they have similar interactions to the standard inhibitor celecoxib. These valuable bioactive compounds may induce significant analgesic and anti-inflammatory properties in MECCS. Therefore, based on the findings of this study, it can be concluded that C. calycina stem can be a prospect in the medicinal field due to its remarkable analgesic and anti-inflammatory effect.

Species of the common discomycete genus Bisporella reassigned to at least four genera.

Bisporella as typically conceived is a genus of noticeable, bright yellow inoperculate discomycetes. This interpretation of the genus, however, is at odds with Bisporella pallescens, the current name of the type species of the genus; furthermore, the genus has been interpreted as including the unusual species Bisporella resinicola. By comparing morphological and molecular traits of species traditionally included in Bisporella, we show that the genus is polyphyletic, with many "typical" members of the genus belonging instead in Calycina in Pezizellaceae. Bisporella pallescens is conclusively linked with its asexual morph, Bispora antennata, and the genus Bisporella is abandoned as a later synonym of the monotypic genus Bispora (previously applied only to asexual fungi) and placed as sister to Hymenoscyphus in Helotiaceae. Bisporella resinicola is shown to represent an independent monotypic genus, Eustilbum, which so far is placed incertae sedis in Helotiales. Finally, "Bisporella" subpallida, like Bispora, belongs to Helotiaceae but is instead related to "Phaeohelotium" epiphyllum.

Exploration of genetic, morphological and essential oil variation reveals tools for the authentication and breeding of Salvia pomifera subsp. calycina (Sm.) Hayek.

Salvia pomifera subsp. calycina (Sm.) Hayek (Lamiaceae), is an Eastern Mediterranean element, which is used in traditional medicine and cuisine in the same manner as S. fruticosa Mill. and S. officinalis L.. The essential oil (EO) and the extracts of S. pomifera possess bioactive compounds with anti-proliferative, anticholinesterase, antioxidant, antiviral and antifungal properties. In this study, the chemical (EO), genetic (DNA microsatellites, SSRs) and morphological diversity of forty-nine individuals of Salvia pomifera subsp. calycina, originating from five natural populations of the Peloponnese (Greece) were determined, in order to explore the potential for successful breeding and to reveal tools and biomarkers for identification and authentication. Chemical and genetic analyses revealed high levels of variation both within and among populations, while morphological analysis mainly within populations. Essential oil yield ranged from 1.79 to 5.79 ml 100 g-1 dry wt, among individuals while ß-thujone ranged from 6.04 to 64.75%. Consistency was found in the EO yield and composition of specific individuals, when sampled during the same period, for three consecutive years, while the analysis during spring and summer months showed differentiation that still retained individual's discrimination. Genetic analysis using SSRs showed that the observed population heterozygosity (Ho) ranged from 0.48 to 0.67, while high number of private alleles were revealed in all populations. Considerable genetic differentiation was observed among the three Salvia taxa (S. pomifera subsp. calycina, S. fruticosa, S. officinalis) (Fst values ranged from 0.27 to 0.48) and lower among S. pomifera subsp. calycina populations (Fst values ranged from 0.06 to 0.13). The great variation that was revealed in all measured traits, in combination with the demonstrated, genetically based, consistency of their EO yield and composition, advocates to a successful breeding, whereas SSR genotyping presents a strong identification and authentication tool.

Eugenia calycina Cambess extracts and their fractions: Their antimicrobial activity and the identification of major polar compounds using electrospray ionization FT-ICR mass spectrometry.

Eugenia calycina, which is described as "red pitanga or pitanga cherry of cerrado," is widely distributed in the Cerrado area of Brazil. Its leaf and bark extracts are used in folk medicine for many applications. In this study, the compositions of the major polar compounds of the bark and leaf extracts and their fractions were obtained from a liquid-liquid extraction using hexane, dichloromethane, ethyl acetate, and water. They were then evaluated using electrospray ionization negative FT-ICR mass spectrometry (ESI(-) FT-ICR MS), which revealed a large number of oxygen-containing compounds, such as flavonoids, terpenes, tanins, steroids, and fat acids. The biological activity of these extracts towards several bacterial and fungal strains was then evaluated. The highest activity was found using aqueous fractions, in which the ESI(-) FT-ICR MS analysis revealed compounds with a high content of oxygen (e.g., glycosed flavonoids, tannins, and polyphenolic compounds) against Cryptococcus sp. D (minimum inhibitory concentration [MIC]=15.62µg/mL). Strong activity was also found using the hexanic fractions-in which the ESI(-) FT-ICR MS analysis revealed that the compounds contained a decreased amount of oxygen (e.g., fat acids and steroids)-towards Cryptococcus gatti L48, Cryptococcus neoformans L3 (MIC=31.2µg/mL), and Cryptococcus sp. D (MIC=62.5µg/mL). Therefore, antimicrobial assays using the bark/leaf extracts of E. calycina present prospects for the research of active substances that may be used for the treatment of cryptococcosis, a disease that is common in immunosuppressed patients.

Inhibition of human platelet aggregation in vitro by standardized extract of Wendtia calycina

Wendtia calycina (Griseb.) Griseb., Vivianiaceae, is a Paraguayan herbaceous plant commonly known as burrito. Our previous study indicated that burrito leaves are a very good source of phenylpropanoid glycosides, principally verbascoside. From W. calycina leaves, a standardized, water-soluble extract rich in phenylpropanoid glycosides (WSE) has been developed on an industrial scale to be used as a food supplement, cosmetic, phytomedicine, and ingredient of different formulations. In this study, we investigated the effect of the WSE on human platelet aggregation in vitro induced by adenosine diphosphate (ADP), epinephrine (EPN), collagen (COL) or arachidonic acid (AA). WSE, concentration-dependently, inhibited ADP and EP-induced human platelet aggregation (IC50 were 0.82±0.15 mg/mL and 0.41±0.02 mg/mL, respectively). It did not inhibit collagen-induced platelet aggregation, thus suggesting a selectivity for the ADP-induced platelet activation pathways.

In vitro screening of Amazonian plants for hemolytic activity and inhibition of platelet aggregation in human blood

In the present study, different aerial parts from twelve Amazonian plant species found in the National Institute for Amazon Research's (INPA's) Adolpho Ducke Forest Reserve (in Manaus, Amazonas, Brazil) were collected. Separate portions of dried, ground plant materials were extracted with water (by infusion), methanol and chloroform (by continuous liquid-solid extraction) and solvents were removed first by rotary evaporation, and finally by freeze-drying which yielded a total of seventy-one freeze-dried extracts for evaluation. These extracts were evaluated initially at concentrations of 500 and 100 µg/mL for in vitro hemolytic activity and in vitro inhibition of platelet aggregation in human blood, respectively. Sixteen extracts (23 % of all extracts tested, 42 % of all plant species), representing the following plants: Chaunochiton kappleri (Olacaceae), Diclinanona calycina (Annonaceae), Paypayrola grandiflora (Violaceae), Pleurisanthes parviflora (Icacinaceae), Sarcaulus brasiliensis (Sapotaceae), exhibited significant inhibitory activity towards human platelet aggregation. A group of extracts with antiplatelet aggregation activity having no in vitro hemolytic activity has therefore been identified. Three extracts (4 %), all derived from Elaeoluma nuda (Sapotaceae), exhibited hemolytic activity. None of the plant species in this study has known use in traditional medicine. So, these data serve as a baseline or minimum of antiplatelet and hemolytic activities (and potential usefulness) of non-medicinal plants from the Amazon forest. Finally, in general, these are the first data on hemolytic and inhibitory activity on platelet aggregation for the genera which these plant species represent.

No presente estudo, partes aéreas obtidas de doze (12) espécies vegetais da Amazônia encontradas na Reserva Florestal Adolpho Ducke (localizada na cidade de Manaus, Estado do Amazonas, Brasil) do Instituto Nacional de Pesquisas da Amazônia foram coletadas, secadas e moídas. Porções dos materiais vegetais em pó foram extraídas com água (por infusão), metanol e clorofórmio (por extração líquido-sólido contínua) e os solventes foram removidos por evaporação rotatória e finalmente liofilização, forneceram um total de setenta e one (71) extratos liofilizados. Esses extratos foram avaliados inicialmente para atividade hemolítica in vitro e atividade inibitória da agregação plaquetária em sangue humano in vitro em concentrações de 500 e 100 µg/mL, respectivamente. Dezesseis (16) extratos (23 % dos extratos testados, 42 % das espécies vegetais) representando as seguintes plantas, apresentaram inibição significativa frente a agregação de plaquetas humanas in vitro: Chaunochiton kappleri (Olacaceae), Diclinanona calycina (Annonaceae), Paypayrola grandiflora (Violaceae), Pleurisanthes parviflora (Icacinaceae), Sarcaulus brasiliensis (Sapotaceae). Como principal resultado, um grupo de extratos apresentando atividade inibitória da agregação plaquetária e em que não há atividade hemolítica in vitro foi identificado. Três (3) extratos (4 % do total de extratos testados), todos obtidos a partir de Elaeoluma nuda (Sapotaceae), apresentaram atividade hemolítica. Nenhuma das espécies vegetais nesse estudo tem uso medicinal conhecido. Assim, esses dados servem de linha base ou mínimas das atividades antiplaquetária e hemolítica (e utilidade potencial) de plantas da floresta Amazônica. Finalmente, em geral, esses dados são os primeiros disponíveis sobre ação hemolítica e inibição da agregação plaquetária dos gêneros representados por essas espécies de plantas.

Screening of Amazonian plants from the Adolpho Ducke forest reserve, Manaus, state of Amazonas, Brazil, for antimicrobial activity

Tropical forests are species-rich reserves for the discovery and development of antimicrobial drugs. The aim of this work is to investigate the in vitro antimicrobial potential of Amazon plants found within the National Institute on Amazon Research's Adolpho Ducke forest reserve, located in Manaus, state of Amazonas, Brazil. 75 methanol, chloroform and water extracts representing 12 plant species were tested for antimicrobial activity towards strains of Mycobacterium smegmatis, Escherichia coli, Streptococcus sanguis, Streptococcus oralis, Staphylococcus aureus and Candida albicans using the gel-diffusion method. Active extracts were further evaluated to establish minimum inhibitory concentrations (MIC) and antimicrobial profiles using bioautography on normal-phase thin-layer chromatography plates. Diclinanona calycina presented extracts with good antimicrobial activity and S. oralis and M. smegmatis were the most sensitive bacteria. D. calycina and Lacmellea gracilis presented extracts with the lowest MIC (48.8 µg/ml). D. calycina methanol and chloroform leaf extracts presented the best overall antimicrobial activity. All test organisms were sensitive to D. calycina branch chloroform extract in the bioautography assay. This is the first evaluation of the biological activity of these plant species and significant in vitro antimicrobial activity was detected in extracts and components from two species, D. calycina and L. gracilis.