Erythroxylum pungens Tropane Alkaloids: GC-MS Analysis and the Bioactive Potential of 3-(2-methylbutyryloxy)tropan-6,7-diol in Zebrafish (Danio rerio).

Tropane alkaloids are specialized plant metabolites mostly found in the Erythroxylaceae and Solanaceae families. Although tropane alkaloids have a high degree of structural similarity because of the tropane ring, their pharmacological actions are quite distinct. Brazil is one of the main hotspots of Erythroxylum spp. diversity with 123 species (almost 66% of the species catalogued in tropical America). Erythroxylum pungens occurs in the Caatinga, a promising biome that provides bioactive compounds, including tropane alkaloids. As part of our efforts to investigate this species, 15 alkaloids in specimens harvested under different environmental conditions are presented herein. The occurrence of 3-(2-methylbutyryloxy)tropan-6,7-diol in the stem bark of plants growing in their natural habitat, greenhouse controlled conditions, and after a period of water restriction, suggests that it is a potential chemical marker for the species. This alkaloid was evaluated for several parameters in zebrafish (Danio rerio) as a model organism. Regarding toxicity, teratogenic effects were observed at 19.5 µM and the lethal dose for embryos was 18.4 µM. No mortality was observed in adults, but a behavioral screen showed psychostimulatory action at 116.7 µM. Overall, the alkaloid was able to cause zebrafish behavioral changes, prompting further investigation of its potential as a new molecule in the treatment of depression-like symptoms. In silico, targets involved in antidepressant pathways were identified by docking.

Trichomonas vaginalis NTPDase inhibited by lycorine modulates the parasite-neutrophil interaction.

Lycorine is an Amaryllidaceae alkaloid that presents anti-Trichomonas vaginalis activity. T. vaginalis causes trichomoniasis, the most common non-viral sexually transmitted infection. The modulation of T. vaginalis purinergic signaling through the ectonucleotidases, nucleoside triphosphate diphosphohydrolase (NTPDase), and ecto-5'-nucleotidase represents new targets for combating the parasite. With this knowledge, the aim of this study was to investigate whether NTPDase and ecto-5'-nucleotidase inhibition by lycorine could lead to extracellular ATP accumulation. Moreover, the lycorine effect on the reactive oxygen species (ROS) production by neutrophils and parasites was evaluated as well as the alkaloid toxicity. The metabolism of purines was assessed by HPLC. ROS production was measured by flow cytometry. Cytotoxicity against epithelial vaginal cells and fibroblasts was tested, as well as the hemolytic effect of lycorine and its in vivo toxicity in Galleria mellonella larvae. Our findings showed that lycorine caused ATP accumulation due to NTPDase inhibition. The alkaloid did not affect the ROS production by T. vaginalis; however, it increased ROS levels in neutrophils incubated with lycorine-treated trophozoites. Lycorine was cytotoxic against vaginal epithelial cells and fibroblasts; conversely, it was not hemolytic neither exhibited toxicity against the in vivo model of G. mellonella larvae. Overall, besides having anti-T. vaginalis activity, lycorine modulates ectonucleotidases and stimulates neutrophils to secrete ROS. This mechanism of action exerted by the alkaloid could enhance the susceptibility of T. vaginalis to host immune cell, contributing to protozoan clearance.

Isoflavone production in hairy root cultures and plantlets of Trifolium pratense.

OBJECTIVES: The aim of this study was to develop a Trifolium pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments. RESULTS: We obtained 13 independent HR lines, producing approximately three times more isoflavonoids than seedlings (3.3 mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140 mg/L, allowed the maximum isoflavonoid production in plantlets (11.9 mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9 mg/g dry weight) was obtained using sucrose 60 g/L, for 3.5 days. CONCLUSION: This work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.

Non-target molecular network and putative genes of flavonoid biosynthesis in Erythrina velutina Willd., a Brazilian semiarid native woody plant.

Erythrina velutina is a Brazilian native tree of the Caatinga (a unique semiarid biome). It is widely used in traditional medicine showing anti-inflammatory and central nervous system modulating activities. The species is a rich source of specialized metabolites, mostly alkaloids and flavonoids. To date, genomic information, biosynthesis, and regulation of flavonoids remain unknown in this woody plant. As part of a larger ongoing research goal to better understand specialized metabolism in plants inhabiting the harsh conditions of the Caatinga, the present study focused on this important class of bioactive phenolics. Leaves and seeds of plants growing in their natural habitat had their metabolic and proteomic profiles analyzed and integrated with transcriptome data. As a result, 96 metabolites (including 43 flavonoids) were annotated. Transcripts of the flavonoid pathway totaled 27, of which EvCHI, EvCHR, EvCHS, EvCYP75A and EvCYP75B1 were identified as putative main targets for modulating the accumulation of these metabolites. The highest correspondence of mRNA vs. protein was observed in the differentially expressed transcripts. In addition, 394 candidate transcripts encoding for transcription factors distributed among the bHLH, ERF, and MYB families were annotated. Based on interaction network analyses, several putative genes of the flavonoid pathway and transcription factors were related, particularly TFs of the MYB family. Expression patterns of transcripts involved in flavonoid biosynthesis and those involved in responses to biotic and abiotic stresses were discussed in detail. Overall, these findings provide a base for the understanding of molecular and metabolic responses in this medicinally important species. Moreover, the identification of key regulatory targets for future studies aiming at bioactive metabolite production will be facilitated.

Screening and bioguided fractionation of Amaryllidaceae species with anti-Trichomonas vaginalis activity.

The Amaryllidaceae family is known by its ornamental and medicinal value and has attracted considerable attention due to the content of alkaloids of its species, which showed interesting biological properties. The present study evaluated the anti-Trichomonas vaginalis activity of eighteen extracts (12.5 to 0.19 mg/mL) and six isolated alkaloids (125 to 1.9 µg/mL) from Amaryllidaceae species. The alkaloids diminished the trophozoites viability (from 15 to 40 %). The extracts from Hippeastrum breviflorum demonstrated the highest anti-T. vaginalis activity (viability was 60 % reduced), and a bioguided study was conducted. Six fractions with antiprotozoal activity had lycorine and lycosinine as major components suggesting a synergistic effect, taking into account the higher anti-T. vaginalis activity of extracts when compared to isolated alkaloids. Our results point out the antiprotozoal potential of the Amaryllidaceae species against T. vaginalis. This parasite causes trichomonosis, the most prevalent nonviral sexually transmitted diseases (STD) worldwide and a public health problem that requires new therapeutic alternatives as well bioactive natural products.

Erythrina velutina Willd. alkaloids: Piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves.

Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. Erythrina species are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids (BIAs), which can act on several pathology-related biological targets. Objectives: In this sense, in an unprecedented approach used with a non-model Fabaceae species grown in its unique arid natural habitat, a combined transcriptome and metabolome analyses (seeds and leaves) is presented. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a NextSeq 500 platform. Regarding metabolite profiling, the High-resolution Liquid Chromatography was coupled to DAD and a micrOTOF-QII mass spectrometer by using electrospray ionization (ESI) and Time of Flight (TOF) analyzer. The tandem MS/MS data were processed and analyzed through Molecular Networking approach. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids, several of them unique. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: Overall, these results could contribute by indicating potential biotechnological targets for modulation of erythrina alkaloids biosynthesis as well as improve molecular databases with omic data from a non-model medicinal plant, and reveal an interesting chemical diversity of Erythrina BIA harvested in Caatinga.

Hydroxypropyl-ß-cyclodextrin-containing hydrogel enhances skin formononetin permeation/retention.

OBJECTIVES: This study was aimed to investigate the in vitro permeation potential of hydrogel formulations containing the isoflavones formononetin and biochanin A and cyclodextrins in different combinations. METHODS: The permeation assay was performed using porcine skin discs on Franz diffusion cells model. The isoflavone contents of the formulations were quantified in the different layers of the skin using a validated HPLC-PDA method. KEY FINDINGS: The isoflavones individually incorporated into the formulations showed high permeation potential, especially formononetin, after the incorporation of hydroxypropyl-ß-cyclodextrin that enhanced its permeation in the epidermis and dermis. Biochanin A showed 2.7 times of permeation capacity in the epidermis and dermis mainly after incorporation of cyclodextrins in the formulations. Formononetin showed reduction in its permeation when incorporated in the formulations together to biochanin A, showing the absence of synergism. CONCLUSIONS: Our results indicated a noticeable skin permeation promoting effect of HPßCD in formononetin formulation. Furthermore, formononetin and biochanin A can permeate the skin being mostly retained in the epidermis and dermis, revealing its potential use in cosmetic preparations intended to prevent skin aging.

Forebrain glutamate uptake and behavioral parameters are altered in adult zebrafish after the induction of Status Epilepticus by kainic acid.

The development of new antiepileptic drugs is a high-risk/high-cost research field, which is made even riskier if the behavioral epileptic seizure profile is the unique approach on which the development is based. In order to increase the effectiveness of the screening conducted in the zebrafish model of status epilepticus (SE), the evaluation of neurochemical markers of SE would be of great relevance. Epilepsy is associated with changes in the glutamatergic system, and glutamate uptake is one of the critical parameters of this process. Therefore, we evaluated the levels of glutamate uptake in the zebrafish brain and analyzed its correlation with the progression of behavioral changes in zebrafish at different times after the administration of kainic acid (5 mg/kg). The results showed that the zebrafish suffered with lethargy while swimming for up to 72 h after SE, had reduced levels of GFAP cells 12 h after SE, reduced levels of S100B up to 72 h after SE, and reduced levels of glutamate uptake in the forebrain between 3 h and 12 h after SE. The forebrain region of adult zebrafish after SE present similar changes to the neurochemical limbic alterations that are seen in rodent models of SE. This study demonstrated that there is a time window in which to use the KA zebrafish model of SE to explore some of the known neurochemical alterations that have been observed in rodent models of epilepsy and epileptic human patients.

Root exudation of oxalic acid in Lotus corniculatus in response to aluminum toxicity

The objective of this research was to identify the existence of root exudation of organic acid in Lotus corniculatus germplasms subjected to toxic aluminum (Al) levels and investigate the effect of this mechanism on the Al content in the root tissue and in morphological parameters of plant development. Two experiments were performed in nutrient solution to evaluate the Al accumulation and exudation of organic acids, using cultivars INIA Draco and São Gabriel and genotypes UFRGS and UF-T2. The plants were cultivated in Al-free solution, which was applied on the 45th day in half of the pots of each genotype. Root exudation was highly correlated with the reduced accumulation of Al in the root tissue (r2 = 0.75 at 72 h). Genotype UF-T2, selected for Al tolerance, extruded 80% more oxalic acid in the presence of Al compared with the other germplasms, indicating that this mechanism is involved with Al tolerance in L. corniculatus. This experiment showed strong evidence that L. corniculatus exhibits Type I tolerance, in which anionic channels are rapidly activated by Al exposure. Exudation of oxalic acid is likely a crucial mechanism that allows the maintenance of L. corniculatus growth when exposed to toxic Al conditions, and this characteristic should be used to identify tolerant genotypes in the future.(AU)

Anxiolytic-, antidepressant- and anticonvulsant-like effects of the alkaloid montanine isolated from Hippeastrum vittatum.

Compounds isolated from different members of the Amaryllidaceae family are becoming relevant options for the treatment of neurological disorders and neurodegenerative diseases. In particular, species of the Hippeastrum genus are important source of alkaloids with a wide profile of putative therapeutical applications. Here, we report on the behavioral and pharmaco-toxicological characterization of montanine, an isoquinoline alkaloid isolated from Hippeastrum vittatum, an ornamental plant found throughout the world. In mice, montanine showed a LD(50) of 64.7 mg/kg and 67.6 mg/kg for male and female, respectively. When given i.p., montanine dose-dependently decreased sodium pentobarbital-induced sleep, protected against pentylenetetrazole-provoked convulsions, increased the number of entries and the time spent in the open arms of an elevated plus maze and augmented the time spent struggling during a forced swimming test. When given immediately after inhibitory avoidance training, montanine did not affect avoidance memory retention in rats. Our results suggest that montanine, as other alkaloids isolated from Amaryllidaceae species, has psychopharmacological activities including anxiolytic, antidepressive and anticonvulsive effects.

Toxicity of Glandularia selloi (Spreng.) Tronc. leave extract by MTT and neutral red assays: influence of the test medium procedure.

Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of plant extracts with potential phytotherapeutic properties. This study compared three methods to prepare culture media for the exposure of Vero cells to plant extracts. Leaves of Glandularia selloi (Spreng.) Tronc. were used to prepare culture medium with aqueous extract, extract in culture medium and methanol extract. Toxicity was assessed using the MTT and neutral red (NR) assays. In general, alterations in the cellular functions were found in all extracts and assays. Cytotoxic effect occurred at lower doses in aqueous extract and the range of effect of the methanol extract was small. The procedure of preparing the test medium has an effect on the outcome of the assay. Cytotoxicity of plant extract can be assessed by MTT and NR assays. Aqueous extract added to the culture medium presented the best profile to assess cytotoxicity.

Trifolium pratense: Friable calli, cell culture protocol and isoflavones content in wild plants, in vitro and cell cultures analyzed by UPLC

Abstract Trifolium pratense L., Fabaceae, is a rich source of isoflavones and has become the focus of several studies related to its phytoestrogenic activity. The aim of this study was to establish germination and cell cultures protocol for T. pratense and quantify isoflavones content in cell cultures, in vitro cultured and wild plants harvested in two different seasons. Murashige Skoog medium supplemented with naphthalene acetic acid and kinetin was able to produce the highest formation of friable calli. Calli cultures were analyzed qualitatively after 60 days of culture, and in vitro plants after 30, 45 and 60 days of cultivation. The chemical analysis was performed by ultra performance liquid chromatography, using the linearity curves of daidzein, genistein, formononetin and biochanin A as standards. The concentrations of isoflavones detected in wild plants were different in the two harvest periods and contrasted in content when compared to the in vitro plants. Cell cultures exhibited diverse profiles and concentration of isoflavones, none of which presented the isoflavonoid biochanin A. Pectinase was used to promote reduction of clumps and ended up altering the characteristics of secondary metabolites production in some cultures. Formononetin showed higher concentration in wild red clover samples (15.407 mg g-1), and in the in vitro grown plants the highest concentration was daidzein (17.591 mg g-1) at 60 days. The methods used for this research were effective, and the red clover plants of the analyzed variety can be cultivated in vitro aiming the commercial productivity by having contents greater than or equal to the wild plants in the periods studied, even without the use of elicitors during the cultivation.

New lycosinine derivative from Hippeastrum breviflorum

AbstractA new lycosinine derivative, 9-O-demethyllycosinine B, was isolated from the native Brazilian Hippeastrum breviflorumHerb., Amaryllidaceae, along with the well-known alkaloids lycosinine B and lycorine. The structure of the new compound was established by physical and spectroscopic methods. 9-O-demethyllycosinine B is the third lycosinine variant identified in the Amaryllidaceae family.