RESUMO

Enzyme-substrate interactions play a fundamental role in elucidating synthesis pathways and synthetic biology, as they allow for the understanding of important aspects of a reaction. Establishing the interaction experimentally is a slow and costly process, which is why this problem has been addressed using computational methods such as molecular dynamics, molecular docking, and Monte Carlo simulations. Nevertheless, this type of method tends to be computationally slow when dealing with a large search space. Therefore, in recent years, methods based on artificial intelligence, such as support vector machines, neural networks, or decision trees, have been implemented, significantly reducing the computing time and covering vast search spaces. These methods significantly reduce the computation time and cover broad search spaces, rapidly reducing the number of interacting candidates, as they allow repetitive processes to be automated and patterns to be extracted, are adaptable, and have the capacity to handle large amounts of data. This article analyzes these artificial intelligence-based approaches, presenting their common structure, advantages, disadvantages, limitations, challenges, and future perspectives.

RESUMO

Skin cancer has high rates of mortality and therapeutic failure. In this study, to develop a multi-agent strategy for skin cancer management, the selective cytotoxicity of several alkaloid fractions and pure alkaloids isolated from Amaryllidaceae species was evaluated in melanoma cells. In addition, UVB-stimulated keratinocytes (HaCaT) were exposed to seven alkaloid fractions characterized by GC-MS, and the production of intracellular reactive oxygen species (ROS) and IL-6, were measured to evaluate their photoprotection effects. The Eucharis caucana (bulb) alkaloid fraction (20 µg/ml) had a clear effect on the viability of melanoma cells, reducing it by 45.7% without affecting healthy keratinocytes. This alkaloid fraction and tazettine (both at 2.5 µg/ml) suppressed UVB-induced ROS production by 31.6% and 29.4%, respectively. The highest anti-inflammatory potential was shown by the Zephyranthes carinata (bulb) alkaloid fraction (10 µg/ml), which reduced IL-6 production by 90.8%. According to the chemometric analysis, lycoramine and tazettine had a photoprotective effect on the UVB-exposed HaCaT cells, attenuating the production of ROS and IL-6. These results suggest that Amaryllidaceae alkaloids have photoprotective and therapeutic potential in skin cancer management, especially at low concentrations.

Assuntos

Alcaloides , Alcaloides de Amaryllidaceae , Melanoma , Neoplasias Cutâneas , Humanos , Alcaloides de Amaryllidaceae/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Interleucina-6 , Alcaloides/farmacologia , Queratinócitos , Neoplasias Cutâneas/tratamento farmacológico , Melanoma/tratamento farmacológico

RESUMO

Cancer is a major cause of death and an impediment to increasing life expectancy worldwide. With the aim of finding new molecules for chemotherapeutic treatment of epidemiological relevance, ten alkaloid fractions from Amaryllidaceae species were tested against six cancer cell lines (AGS, BT-549, HEC-1B, MCF-7, MDA-MB 231, and PC3) with HaCat as a control cell line. Some species determined as critically endangered with minimal availability were propagated using in vitro plant tissue culture techniques. Molecular docking studies were carried out to illustrate binding orientations of the 30 Amaryllidaceae alkaloids identified in the active site of some molecular targets involved with anti-cancer activity for potential anti-cancer drugs. In gastric cancer cell line AGS, the best results (lower cell viability percentages) were obtained for Crinum jagus (48.06 ± 3.35%) and Eucharis bonplandii (45.79 ± 3.05%) at 30 µg/mL. The research focused on evaluating the identified alkaloids on the Bcl-2 protein family (Mcl-1 and Bcl-xL) and HK2, where the in vitro, in silico and statistical results suggest that powelline and buphanidrine alkaloids could present cytotoxic activity. Finally, combining experimental and theoretical assays allowed us to identify and characterize potentially useful alkaloids for cancer treatment.

Assuntos

Alcaloides , Alcaloides de Amaryllidaceae , Amaryllidaceae , Antineoplásicos , Neoplasias , Extratos Vegetais/farmacologia , Amaryllidaceae/química , Simulação de Acoplamento Molecular , Alcaloides/química , Alcaloides de Amaryllidaceae/farmacologia

RESUMO

Zephyranthes carinata Herb., a specie of the Amaryllidoideae subfamily, has been reported to have inhibitory activity against acetylcholinesterase. However, scientific evidence related to their bioactive alkaloids has been lacking. Thus, this study describes the isolation of the alkaloids of this plant, and their inhibition of the enzymes acetylcholinesterase (eeAChE) and butyrylcholinesterase (eqBuChE), being galanthine the main component. Additionally, haemanthamine, hamayne, lycoramine, lycorine, tazettine, trisphaeridine and vittatine/crinine were also isolated. The results showed that galanthine has significant activity at low micromolar concentrations for eeAChE (IC50 = 1.96 µg/mL). The in-silico study allowed to establish at a molecular level the high affinity and the way galanthine interacts with the active site of the TcAChE enzyme, information that corroborates the result of the experimental IC50. However, according to molecular dynamics (MD) analysis, it is also suggested that galanthine presents a different inhibition mode that the one observed for galanthamine, by presenting interaction with peripheral anionic binding site of the enzyme, which prevents the entrance and exit of molecules from the active site. Thus, in vitro screening assays plus rapid computer development play an essential role in the search for new cholinesterase inhibitors by identifying unknown bio-interactions between bioactive compounds and biological targets.

Assuntos

Alcaloides , Amaryllidaceae , Acetilcolinesterase/metabolismo , Alcaloides/farmacologia , Amaryllidaceae/química , Amaryllidaceae/metabolismo , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Simulação de Acoplamento Molecular

RESUMO

The expression of HIGD2A is dependent on oxygen levels, glucose concentration, and cell cycle progression. This gene encodes for protein HIG2A, found in mitochondria and the nucleus, promoting cell survival in hypoxic conditions. The genomic location of HIGD2A is in chromosome 5q35.2, where several chromosomal abnormalities are related to numerous cancers. The analysis of high definition expression profiles of HIGD2A suggests a role for HIG2A in cancer biology. Accordingly, the research objective was to perform a molecular biosystem analysis of HIGD2A aiming to discover HIG2A implications in cancer biology. For this purpose, public databases such as SWISS-MODEL protein structure homology-modelling server, Catalogue of Somatic Mutations in Cancer (COSMIC), Gene Expression Omnibus (GEO), MethHC: a database of DNA methylation and gene expression in human cancer, and microRNA-target interactions database (miRTarBase) were accessed. We also evaluated, by using Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), the expression of Higd2a gene in healthy bone marrow-liver-spleen tissues of mice after quercetin (50 mg/kg) treatment. Thus, among the structural features of HIG2A protein that may participate in HIG2A translocation to the nucleus are an importin -dependent nuclear localization signal (NLS), a motif of DNA binding residues and a probable SUMOylating residue. HIGD2A gene is not implicated in cancer via mutation. In addition, DNA methylation and mRNA expression of HIGD2A gene present significant alterations in several cancers; HIGD2A gene showed significant higher expression in Diffuse Large B-cell Lymphoma (DLBCL). Hypoxic tissues characterize the "bone marrow-liver-spleen" DLBCL type. The relative quantification, by using RT-qPCR, showed that Higd2a expression is higher in bone marrow than in the liver or spleen. In addition, it was observed that quercetin modulated the expression of Higd2a gene in mice. As an assembly factor of mitochondrial respirasomes, HIG2A might be unexpectedly involved in the change of cellular energetics happening in cancer. As a result, it is worth continuing to explore the role of HIGD2A in cancer biology.

Assuntos

Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Quercetina/administração & dosagem , Biologia de Sistemas/métodos , Animais , Medula Óssea/metabolismo , Linhagem Celular Tumoral , Simulação por Computador , Metilação de DNA , Bases de Dados Genéticas , Humanos , Fígado/metabolismo , Masculino , Camundongos , Mutação , Proteínas de Neoplasias/química , Transplante de Neoplasias , Neoplasias/metabolismo , Transporte Proteico , Quercetina/farmacologia , Baço/metabolismo , Distribuição Tecidual

RESUMO

In Alzheimer's disease (AD), amyloid beta (Aß) plaques initiates a cascade of pathological events where the overactivation of N-methyl-d-aspartate receptors (NMDA) by excess glutamate (Glu) triggers oxidative processes that lead to the activation of microglial cells, inflammation, and finally neuronal death. Amaryllidaceae alkaloids exert neuroprotective activities against different neurotoxin-induced injuries in vitro, and although their biological potential is well demonstrated, their neuroprotective activity has not been reported in an in vivo model of AD. The aim of our study was to determine the in vitro and in vivo neuroprotective potential of standardized alkaloidal fractions of Zephyranthes carinata. In this work, the neuroprotective effect of two alkaloidal fractions extracted from Z. carinata (bulbs and leaves) was analyzed in an in vitro excitotoxicity model in order to select the most promising one for subsequent evaluation in a triple transgenic mouse model of AD (3xTg-AD). We found that Z. carinata bulbs protect neurons against a Glu-mediated toxic stimulus in vitro, as evidenced by the decrease in apoptotic nuclei, the reduction in the lipid peroxidation product malondialdehyde and the conservation of dendritic structures. The effects of intraperitoneal administration of Z. carinata bulbs (10 mg/kg) every 12 h for 1 month on 3xTg-AD (18 months old) showed improved learning and spatial memory. Histopathologically, the alkaloidal fraction-treated 3xTg-AD mice exhibited a significant reduction in tauopathy and astrogliosis, as well as a significant decrease in the proinflammatory marker COX-2 and an increase in pAkt. The results suggest that Z. carinata bulbs provide neuroprotective effects both in vitro and in 3xTg-AD mice by intervening in the inflammatory processes, regulating the aggregation of pair helical filaments (PHFs) and activating survival pathways.

Assuntos

Alcaloides/uso terapêutico , Doença de Alzheimer/prevenção & controle , Amaryllidaceae , Aprendizagem em Labirinto/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Alcaloides/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Aprendizagem em Labirinto/fisiologia , Memória/efeitos dos fármacos , Memória/fisiologia , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Neuroproteção/fisiologia , Distribuição Aleatória , Ratos , Ratos Wistar , Resultado do Tratamento

RESUMO

Amaryllidaceae plants are the commercial source of galanthamine, an alkaloid approved for the clinical treatment of Alzheimer's disease. The chemistry and bioactivity of Chilean representatives of Rhodophiala genus from the family of Amaryllidaceae have not been widely studied so far. Ten collections of five different Chilean Rhodophiala were analyzed in vitro for activity against enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) as well as for their alkaloid composition by GC-MS. To obtain an insight into the potential AChE and BuChE inhibitory activity of the alkaloids identified in the most active samples, docking experiments were carried out. Although galanthamine was found neither in aerial parts nor in bulbs of R. splendens, these plant materials were the most active inhibitors of AChE (IC50: 5.78 and 3.62 µg/mL, respectively) and BuChE (IC50: 16.26 and 14.37 µg/mL, respectively). Some 37 known alkaloids and 40 still unidentified compounds were detected in the samples, suggesting high potential in the Chilean Amaryllidaceae plants as sources of both novel bioactive agents and new alkaloids.

Assuntos

Alcaloides/farmacologia , Amaryllidaceae/química , Inibidores da Colinesterase/farmacologia , Simulação de Acoplamento Molecular , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Alcaloides/química , Biomassa , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Termodinâmica

RESUMO

AIMS: Determine a relationship between the neuroprotective activity and the antioxidant capacity of the Amaryllidaceae alkaloids in a model of Glu excitotoxicity in rat cortical neurons. MATERIALS AND METHODS: Was evaluated several alkaloidal fractions isolated from Amaryllidaceae species, a family known to contain neuroprotective alkaloids, in a model of Glu excitotoxicity in rat cortical neurons. In addition, several mechanisms of antioxidant activity were used, and a theoretical study of the antioxidants was performed. KEY FINDINGS: The results of this study suggest that a possible neuroprotective mode of action of the alkaloidal fractions of Eucharis bonplandii (Kunth) Traub bulbs, Eucharis caucana Meerow bulbs, and Clivia miniata Regel leaves, is through their antioxidant activity and ability to stabilize free radicals generated from an excitotoxic process mediated by Glu. The chemical structure characterization and antioxidant activity of the fractions suggest that the phenol and enol groups in the structures of the alkaloids are critical for the stabilization of ROS and RNS. Additionally, the pair of free electrons on the N is spatially close to a hydroxyl group, which benefits the cleavage of this group and, consequently, the stabilization of the generated O. SIGNIFICANCE: The versatility of the structures of the studied Amaryllidaceae alkaloids suggests that they have potential as neuroprotective agents against an oxidative stimulus.

Assuntos

Alcaloides de Amaryllidaceae/farmacologia , Antioxidantes/farmacologia , Embrião de Mamíferos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos/citologia , Estrutura Molecular , Neurônios/patologia , Oxirredução , Ratos , Ratos Wistar

RESUMO

Phospholipid alterations in the brain are associated with progressive neurodegeneration and cognitive impairment after acute and chronic injuries. Various types of treatments have been evaluated for their abilities to block the progression of the impairment, but effective treatments targeting long-term post-stroke alterations are not available. In this study, we analyzed changes in the central and peripheral phospholipid profiles in ischemic rats and determined whether a protective monoterpene, Linalool, could modify them. We used an in vitro model of glutamate (125 µM) excitotoxicity and an in vivo global ischemia model in Wistar rats. Linalool (0.1 µM) protected neurons and astrocytes by reducing LDH release and restoring ATP levels. Linalool was administered orally at a dose of 25 mg/kg every 24 h for a month, behavioral tests were performed, and a lipidomic analysis was conducted using mass spectrometry. Animals treated with Linalool displayed faster neurological recovery than untreated ischemic animals, accompanied by better motor and cognitive performances. These results were confirmed by the significant reduction in astrogliosis, microgliosis and COX-2 marker, involving a decrease of 24:0 free fatty acid in the hippocampus. The altered profiles of phospholipids composed of mono and polyunsaturated fatty acids (PC 36:1; 42:1 (24:0/18:1)/LPC 22:6)/LPE 22:6) in the ischemic hippocampus and the upregulation of PI 36:2 and other LCFA (long chain fatty acids) in the serum of ischemic rats were prevented by the monoterpene. Based on these data, alterations in the central and peripheral phospholipid profiles after long-term was attenuated by oral Linalool, promoting a phospholipid homeostasis, related to the recovery of brain function.

Assuntos

Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Monoterpenos/farmacologia , Doenças Neurodegenerativas/metabolismo , Fármacos Neuroprotetores/farmacologia , Fosfolipídeos/metabolismo , Monoterpenos Acíclicos , Animais , Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Ácido Glutâmico/toxicidade , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Distribuição Aleatória , Ratos Wistar , Recuperação de Função Fisiológica/efeitos dos fármacos

RESUMO

INTRODUCTION: Enzymatic inhibition of acetylcholinesterase (AChE) is an essential therapeutic target for the treatment of Alzheimer's disease (AD) and AChE inhibitors are the first-line drugs for it treatment. However, butyrylcholinesterase (BChE), contributes critically to cholinergic dysfunction associated with AD. Thus, the development of novel therapeutics may involve the inhibition of both cholinesterase enzymes. OBJECTIVE: To evaluate, in an integrated bioguided study, cholinesterases alkaloidal inhibitors of Amaryllidaceae species. METHODOLOGY: The proposed method combines high-performance thin-layer chromatography (HPTLC) with data analysis by densitometry, enzymatic bioautography with different AChEs and BChEs, the detection of bioactive molecules through gas chromatography mass spectrometry (GC-MS) analysis of spots of interest, and theoretical in silico studies. RESULTS: To evaluate the bioguided method, the AChE and BChE inhibitory activities of seven Amaryllidaceae plant extracts were evaluated. The alkaloid extracts of Eucharis bonplandii exhibited a high level of inhibitory activity (IC50  = 0.72 ± 0.05 µg/mL) against human recombinant AChE (hAChE). Regarding human serum BChE (hBChE), the bulb and leaf extracts of Crinum jagus had the highest activity (IC50  = 8.51 ± 0.56 µg/mL and 11.04 ± 1.21 µg/mL, respectively). In the HPTLC spots with high inhibitory activity, several alkaloids were detected using GC-MS, and some of these alkaloids were identified. Galanthamine, galanthamine N-oxide and powelline should be the most prominent inhibitors of substrate accommodation in the active site of the Torpedo californica AChE (TcAChE), hAChE and hBChE enzymes. CONCLUSIONS: These results are evidence of the chemical relevance of the Colombian's Amaryllidaceae species for the inhibition of cholinesterases and as potent sources for the palliative treatment of AD. Copyright © 2017 John Wiley & Sons, Ltd.

Assuntos

Acetilcolinesterase/efeitos dos fármacos , Alcaloides/isolamento & purificação , Amaryllidaceae/química , Butirilcolinesterase/efeitos dos fármacos , Inibidores da Colinesterase/isolamento & purificação , Alcaloides/farmacologia , Animais , Inibidores da Colinesterase/farmacologia , Cromatografia em Camada Fina/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Cavalos , Humanos , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Folhas de Planta/química , Raízes de Plantas/química , Proteínas Recombinantes/efeitos dos fármacos , Torpedo

RESUMO

AIMS: Amaryllidaceae alkaloids exhibit a wide range of physiological effects, of which the acetylcholinesterase (AChE) inhibitory activity is the most relevant. However, scientific evidence related to their neuroprotective effectiveness against glutamate-induced toxicity has been lacking. Thus, the purpose of this study was to conduct a comparative study of the neuroprotective activity and the AChE inhibitory activity of species of Amaryllidaceae. MAIN METHODS: The neuroprotective activity against glutamate-induced toxicity was measured in rat cortical neurons and the Ellman method was employed for the quantification of acetylcholinesterase inhibitory activity of alkaloidal extracts of five species of Amaryllidaceae (Crinum jagus, Crinum bulbispermum, Hippeastrum barbatum, Hippeastrum puniceum and Zephyranthes carinata). The alkaloid Amaryllidaceae patterns based on GC/MS analyses were also investigated. KEY FINDINGS: The results showed that the alkaloidal extract from C. jagus presented a high neuroprotective activity in both pre- and post-treatments against a glutamate excitotoxic stimulus. Furthermore, the alkaloid extracts from C. jagus and Z. carinata revealed an inhibitory activity of AChE from the electric eel with IC50 values of 18.28±0.29 and 17.96±1.22µg/mL, respectively. In addition, 46 alkaloids were detected by GC/MS, and 20 of them were identified based on their mass spectra and retention index. The results suggest that the neuroprotective effects might be associated with lycorine and crinine-type alkaloids, whereas the acetylcholinesterase enzyme inhibitory activity could be related to galanthamine and lycorine-type alkaloids, although not based on synergistic processes. SIGNIFICANCE: In summary, Amaryllidaceae species are sources of alkaloids with potential use for Alzheimer's disease.

Assuntos

Acetilcolinesterase/metabolismo , Córtex Cerebral/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Liliaceae/química , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Alcaloides/farmacologia , Animais , Western Blotting , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/enzimologia , Imunofluorescência , Cromatografia Gasosa-Espectrometria de Massas , L-Lactato Desidrogenase/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Ratos , Ratos Wistar

RESUMO

Acetylcholinesterase (AChE) enzymatic inhibition is an important target for the management of Alzheimer disease (AD) and AChE inhibitors are the mainstay drugs for its treatment. In order to discover new sources of potent AChE inhibitors, a combined strategy is presented based on AChE-inhibitory activity and chemical profiles by GC/MS, together with in silico studies. The combined strategy was applied on alkaloid extracts of five Amaryllidaceae species that grow in Colombia. Fifty-seven alkaloids were detected using GC/MS, and 21 of them were identified by comparing their mass-spectral fragmentation patterns with standard reference spectra in commercial and private library databases. The alkaloid extracts of Zephyranthes carinata exhibited a high level of inhibitory activity (IC50 = 5.97 ± 0.24 µg/mL). Molecular modeling, which was performed using the structures of some of the alkaloids present in this extract and the three-dimensional crystal structures of AChE derived from Torpedo californica, disclosed their binding configuration in the active site of this AChE. The results suggested that the alkaloids 3-epimacronine and lycoramine might be of interest for AChE inhibition. Although the galanthamine group is known for its potential utility in treating AD, the tazettine-type alkaloids should be evaluated to find more selective compounds of potential benefit for AD.

Assuntos

Alcaloides/análise , Inibidores da Colinesterase/farmacologia , Cromatografia Gasosa-Espectrometria de Massas/métodos , Liliaceae/metabolismo , Extratos Vegetais/farmacologia , Alcaloides/química , Alcaloides/farmacologia , Doença de Alzheimer/tratamento farmacológico , Humanos , Modelos Moleculares

RESUMO

Taraxacum officinale F. H. Wigg, Asteraceae, is frequently misidentified or substituted with Hypochaeris radicata L., Asteraceae (false dandelion). To increase our knowledge of T. officinale and differentiate it from H. radicata, we investigated the two species using a combination of taxonomy, microscopy, and chromatographic studies via fingerprint profiles. Micromorphological characteristics were studied using scanning electron microscopy, while optic light microscopy was used for histochemical observations. Fingerprint profiles were constructed using HPTLC. T. officinale was found to have a morphologically distinct type of pluricellular trichomes that can be used to differentiate the two species, as these structures were not identified in H. radicata samples. Furthermore, two types of laticiferous vessels may also be distinctive characteristics of T. officinale at species level. In addition, the HPTLC data derived from methanolic extracts of H. radicata and T. officinale roots showed clearly different chemical profiles. Thus this study establishes the authenticity of T. officinale, and the observed parameters could help minimize drug substitutions in herbal medicines.