Browsing by Author "Garcia, Maria Rita"
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- Chemical characterization and in vitro studies on the impact of the 'Dream Herb' Calea zacatechichiSchltdl. upon neuronal cellsPublication . Garcia, Maria Rita; Mineiro, Tiago; Andrade, Paula B.; Ferreres, Federico; Gil-Izquierdo, Ángel; Seabra, Vítor; Silva, Diana; Gomes, Nelson G. M.An increasing number of psychoactive plants, particularly those with long history of use on ritualistic practices, are now being progressively used in recreational context, many of which remaining unregulated. Calea zacatechichiis traditionally used in divination rituals, due to its oneirogenic properties. Yet, considering the psychoactive effects of this ‘Dream Herb’and easiness of purchase, the potential for recreational use is high, with scarce information concerning its toxicity. We aimed to characterize C. zacatechichiaqueous extracts, mimicking those typically consumed, to identify bioactives that underlie the psychoactive or toxic effects, and evaluate their impact upon neuronal function, neurotransmission and radical stress. Chemical characterization was attained by HPLC-DAD-ESI(Ion Trap)/MSnand HPLC-DAD. Impact upon SH-SY5Y and BV-2 cell viability was assessed by the MTT assay and LDH release (up to 1000 μg/mL). Impact on neuromodulation and neuroinflammation was evaluated through acetylcholinesterase and 5-lipoxygenase inhibition, while antiradical properties were approached upon nitric oxide (•NO) and superoxide (O2•-). Statistical comparisons among groups performed by one-way ANOVA followed by Dunnett post hoc test. Qualitative analyses enabled the identification of 28 compounds, the majority being hydroxycinnamic acid derivates, namely 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, followed by flavonoid derivates, particularly quercetin-3-O-rutinoside. Cytotoxic effects were verified at concentrations above 125 μg/mL with LDH leakage starting at 250 μg/mL. Acetylcholinesterase inhibition was recorded at 1000 μg/mL, and a concentration-dependent inhibition of 5-lipoxygenase was found (IC50= 71.12 μg/mL). Concentration-dependent scavenging effects upon •NO and O2•-were verified at concentrations higher than 62.5 μg/mL. Even though apparent antiradical and anti-inflammatory properties were attained with C. zacatechichi, thepronounced cytotoxic effects upon neuronal cells cannot be overlooked, requiring further investigation to elucidate the underlying mechanisms that might be involved, given the possible deleterious consequences thisplant can induce among its consumers.
- Mexican calea (Calea zacatechichi Schltdl.) interferes with cholinergic and dopaminergic pathways and causes neuroglial toxicityPublication . Garcia, Maria Rita; Gil-Izquierdo, Ángel; Ferreres, Federico; Andrade, Paula B.; Mineiro, Tiago; Seabra, Vítor; Videira, Romeu A.; Silva, Diana Dias da; Gomes, Nelson G.M.; Dias da Silva, Diana CristinaThe use of “Mexican calea” (Calea zacatechichi Schltdl.) in ritualistic ceremonies, due to its dream-inducing effects, was until recently limited to indigenous communities in Mexico. However, the plant has recently gained popularity in Western societies being commonly used in recreational settings. Despite the traditional and recreational uses, mechanisms underlying its reported oneirogenic effects remain unknown, with no data available on its neurotoxic profile. The scarcity of toxicological data and the unknown role of major neurotransmitter systems in the dream-inducing properties of the plant prompted us to investigate which neurotransmitters might be affected upon its consumption, as well as the potential cytotoxic effects on neurons and microglial cells. Furthermore, we aimed to explore a relationship between the recorded effects and specific constituents. Effects on cholinergic and monoaminergic pathways were investigated using enzymatic assays, with the latter also being conducted in neuronal SH-SY5Y cells along with the impact on glutamate-induced excitotoxicity. Investigation of the neurotoxic profile was approached in neuronal SH-SY5Y and microglial BV-2 cells, evaluating effects on metabolic performance and membrane integrity using MTT and LDH leakage assays, respectively. Potential interference with oxidative stress was monitored by assessing free radical's levels, as well as 5-lipoxygenase mediated lipid peroxidation. Phenolic constituents were identified through HPLC-DAD-ESI(Ion Trap)MSn analysis. Based on the significant inhibition upon acetylcholinesterase (p < 0.05) and tyrosinase (IC50 = 60.87 ± 7.3 μg/mL; p < 0.05), the aqueous extract obtained from the aerial parts of C. zacatechichi interferes with the cholinergic and dopaminergic systems, but has no impact against monoamine oxidase A. Additionally, a notable cytotoxic effect was observed in SH-SY5Y and BV-2 cells at concentrations as low as 125 and 500 μg/mL (p < 0.05), respectively, LDH leakage suggesting apoptosis may occur at these concentrations, with necroptosis observed at higher ones. Despite the neurocytotoxic profile, these effects appear to be independent of radical stress, as the C. zacatechichi extract scavenged nitric oxide and superoxide radicals at concentrations as low as 62.5 μg/mL, significantly inhibiting also 5-lipoxygenase (IC50 = 72.60 ± 7.3 μg/mL; p < 0.05). Qualitative and quantitative analysis using HPLC-DAD-ESI(Ion Trap)MSn enabled the identification of 28 constituents, with 24 of them being previously unreported in this species. These include a series of dicaffeoylquinic, caffeoylpentoside, and feruloylquinic acids, along with 8 flavonols not previously known to occur in the species, mainly 3-O-monoglycosylated derivatives of quercetin, kaempferol, and isorhamnetin. Our findings regarding the neuroglial toxicity elicited by C. zacatechichi emphasize the necessity for a thorough elucidation of the plant's toxicity profile. Additionally, evidence is provided that the aerial parts of the plant inhibit both acetylcholinesterase and tyrosinase, potentially linking its psychopharmacological effects to the cholinergic and dopaminergic systems, with an apparent contribution from specific phenolic constituents previously unknown to occur in the species. Collectively, our results lay the groundwork for a regulatory framework on the consumption of C. zacatechichi in recreational settings and contribute to elucidating previous contradictory findings regarding the mechanisms underlying the dream-inducing effects of the plant.
- Preliminary chemical profile and in vitro pharmacological evaluation of the hallucinogenic plant Diplopterys cabreranaPublication . Garcia, Maria Rita; Dutka, Mykhaylo; Guimarães, Sofia; Andrade, Paula B.; Seabra, Vítor; Diana Dias da Silva; Gomes, Nelson G. M.; Dias da Silva, Diana CristinaFor the last few years, Ayahuasca ceremonies have been gaining popularity in recreational settings in Europe and North America [1]. Similar to Psychotria viridis, Diplopterys cabreranais also suggested to contain the psychoactive compound N,N-dimethyltryptamine, and is therefore used in Aya-huasca rituals for its ability to induce hallucinations, euphoria and entheogenic effects [1-3]. However, while information on the toxic profile of D. cabreranaremains very limited, its acquisition is easily ac-complished by consumers. We aimed to characterize the aqueous extracts of D. cabreranaleaves, mimicking those typically consumed, to identify bioactives that underlie the psychoactive or toxic effects, and evaluate their impact on neuronal function, neurotransmission and radical stress. Chemical characterization was attained by HPLC-DAD. Impact upon neuronal viability was assessed by the MTT assay (up to 1000 μg/mL) in SH-SY5Y neuroblastoma cells. Impact on neuromodulation and neuroinflammation was evaluated through acetylcholinesterase and 5-lipoxygenase inhibition, while an-tiradical properties were assessed by evaluating nitric oxide (•NO) and xanthine oxidase (XO) activity. Inhibition of the α-glucosidase enzyme was also evaluated. Statistical comparisons among groups per-formed by one-way ANOVA followed by Dunnett post hoc test. Preliminary characterization results revealed the presence of several catechin derivates, alongside two apigenin derivates and one tryp-tamine derivate. Cytotoxicity was not verified up to the highest concentration tested. Acetylcholinesterase inhibition was recorded starting at 250 μg/mL, and a concentration-dependent inhibition of 5-lipoxygen-ase was found (IC50=79.77 μg/mL). Concentration-dependent scavenging effects upon •NO and XO inhi-bition were verified at concentrations higher than 1.953 μg/mL and 31.25 μg/mL, respectively. At last, inhibition of α-glucosidase occurred with concentration-dependency and an IC50of 4.78 μg/mL. Conclu-sions:Although antiradical, anti-inflammatory and antidiabetic properties were verified, with no in vitrocytotoxicity being detected, further research is needed to elucidate the underlying mechanisms that might be involved in our preliminary results.