RESUMO
The indole alkaloid gramine, 3-(dimethylaminomethyl)indole, is a defensive specialized metabolite found in some barley cultivars. In its biosynthetic process, the tryptophan (Trp) side chain is shortened by two carbon atoms to produce 3-(aminomethyl)indole (AMI), which is then methylated by N-methyltransferase (HvNMT) to produce gramine. Although side chain shortening is one of the crucial scaffold formation steps of alkaloids originating from aromatic amino acids, the gene and enzyme involved in the Trp-AMI conversion reactions are unknown. In this study, through RNA-seq analysis, 35 transcripts were shown to correlate with gramine production; among them, an uncharacterized cytochrome P450 (CYP) gene, CYP76M57, and HvNMT were identified as candidate genes for gramine production. Transgenic Arabidopsis thaliana and rice overexpressing CYP and HvNMT accumulate AMI, N-methyl-AMI, and gramine. CYP76M57, heterologously expressed in Pichia pastoris, was able to act on Trp to produce AMI. Furthermore, the amino group nitrogen of Trp was retained during the CYP76M57-catalyzed reaction, indicating that the C2 shortening of Trp proceeds with an unprecedented biosynthetic process, the removal of the carboxyl group and Cα and the rearrangement of the nitrogen atom to Cß. In some gramine-non-accumulating barley cultivars, arginine 104 in CYP76M57 is replaced by threonine, which abolished the catalytic activity of CYP76M57 to convert Trp into AMI. These results uncovered the missing committed enzyme of gramine biosynthesis in barley and contribute to the elucidation of the potential functions of CYPs in plants and undiscovered specialized pathways.
Assuntos
Sistema Enzimático do Citocromo P-450 , Hordeum , Alcaloides Indólicos , Proteínas de Plantas , Triptofano , Hordeum/genética , Hordeum/enzimologia , Hordeum/metabolismo , Triptofano/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alcaloides Indólicos/metabolismo , Plantas Geneticamente Modificadas , Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Oryza/genética , Oryza/enzimologia , Oryza/metabolismo , Regulação da Expressão Gênica de Plantas , Metiltransferases/genética , Metiltransferases/metabolismoRESUMO
Alkaloids are natural compounds useful as scaffolds for discovering new bioactive molecules. This study utilized alkaloid gramine to synthesize two groups of C3-substituted indole derivatives, which were either functionalized at N1 or not. The compounds were characterized by spectroscopic methods. The protective effects of the new compounds against in vitro oxidative hemolysis induced by standard oxidant 2,2'-azobis(2-amidinopropane dihydro chloride (AAPH) on human erythrocytes as a cell model were investigated. Additionally, the compounds were screened for antimicrobial activity. The results indicated that most of the indole derivatives devoid of the N1 substitution exhibited strong cytoprotective properties. The docking studies supported the affinities of selected indole-based ligands as potential antioxidants. Furthermore, the derivatives obtained exhibited potent fungicidal properties. The structures of the eight derivatives possessing indole moiety bridged to the imidazole-, benzimidazole-, thiazole-, benzothiazole-, and 5-methylbenzothiazoline-2-thiones were determined by X-ray diffraction. The C=S bond lengths in the thioamide fragment pointed to the involvement of zwitterionic structures of varying contribution. The predominance of zwitterionic mesomers may explain the lack of cytoprotective properties, while steric effects, which limit multiple the hydrogen-bond acceptor properties of a thione sulfur, seem to be responsible for the high hemolytic activity.
Assuntos
Eritrócitos , Hemólise , Indóis , Humanos , Hemólise/efeitos dos fármacos , Indóis/química , Indóis/farmacologia , Eritrócitos/efeitos dos fármacos , Simulação de Acoplamento Molecular , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Relação Estrutura-Atividade , Antioxidantes/farmacologia , Antioxidantes/química , Testes de Sensibilidade Microbiana , Citoproteção/efeitos dos fármacos , AmidinasRESUMO
The natural alkaloid gramine has attracted significant attention in both academic and industrial circles because of its potential and diverse biological activities, including antiviral, antibacterial, antifungal, anti-inflammatory and antitumor activities; application in therapy for Alzheimer's disease; serotonin-receptor-related activity; insecticidal activity; and application as an algicide. In this review, we focus on the research advances that have been made for gramine-based molecules since their discovery, providing key information on their extraction and separation, chemical synthesis and diverse biological activities. Data regarding their mechanisms of action are also presented. This comprehensive and critical review will serve as a guide for developing more drug candidates based on gramine skeletons.
Assuntos
Alcaloides , Alcaloides Indólicos , Alcaloides Indólicos/farmacologia , Alcaloides/farmacologia , Alcaloides/químicaRESUMO
In the search for new bioactive compounds, a methodology based on combining two molecules with biological properties into a new hybrid molecule was used to design and synthesize of a series of ten indole derivatives bearing imidazole, benzothiazole-2-thione, or benzoxazole-2-thione moieties at the C-3 position. The compounds were spectroscopically characterized and tested for their antioxidant, antibacterial, and fungicidal activities. The crystal structures were determined for five of them. Comparison of the closely related structures containing either benzothiazole-2-thione or benzoxazole-2-thione clearly shows that the replacement of -S- and -O- ring atoms modify molecular conformation in the crystal, changes intermolecular interactions, and has a severe impact on biological activity. The results indicate that indole-imidazole derivatives with alkyl substituent exhibit an excellent cytoprotective effect against AAPH-induced oxidative hemolysis and act as effective ferrous ion chelating agents. The indole-imidazole compound with chlorine atoms inhibited the growth of fungal strains: Coriolus versicolor (Cv), Poria placenta (Pp), Coniophora puteana (Cp), and Gloeophyllum trabeum (Gt). The indole-imidazole derivatives showed the highest antibacterial activity, for which the largest growth-inhibition zones were noted in M. luteus and P. fluorescens cultures. The obtained results may be helpful in the development of selective indole derivatives as effective antioxidants and/or antimicrobial agents.
Assuntos
Antioxidantes , Tionas , Antioxidantes/farmacologia , Tionas/química , Benzoxazóis/química , Imidazóis/farmacologia , Antibacterianos/química , Benzotiazóis/química , Antifúngicos/farmacologia , Indóis/farmacologia , Estrutura MolecularRESUMO
In recent years, a large number of studies have reported that neuroinflammation aggravates the occurrence of secondary injury after spinal cord injury. Gramine (GM), a natural indole alkaloid, possesses various pharmacological properties; however, the anti-inflammation property remains unclear. In our study, Gramine was investigated in vitro and in vivo to explore the neuroprotection effects. In vitro experiment, our results suggest that Gramine treatment can inhibit release of pro-inflammatory mediators. Moreover, Gramine prevented apoptosis of PC12 cells which was caused by activated HAPI microglia, and the inflammatory secretion ability of microglia was inhibited by Gramine through NF-κB pathway. The in vivo experiment is that 80 mg/kg Gramine was injected orthotopically to rats after spinal cord injury (SCI). Behavioural and histological analyses demonstrated that Gramine treatment may alleviate microglia activation and then boost recovery of motor function after SCI. Overall, our research has demonstrated that Gramine exerts suppressed microglia activation and promotes motor functional recovery after SCI through NF-κB pathway, which may put forward the prospect of clinical treatment of inflammation-related central nervous diseases.
Assuntos
Alcaloides Indólicos/farmacologia , Inflamação/tratamento farmacológico , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Linhagem Celular , Feminino , Inflamação/imunologia , Inflamação/metabolismo , Microglia/metabolismo , Microglia/patologia , NF-kappa B/genética , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Gramine can be intelligently and efficiently supplied with N, N-dimethylamino group and then reacted with the corresponding sulfonyl chlorides to synthesize N, N-dimethylarylsulfonamides. We herein designed and controlled synthesis of N, N-dimethylarylsulfonamide derivatives, and first reported the results of the nematicidal activity of 15 title compounds 3a-o against Meloidogyne incongnita in vitro, respectively. Among all of the title derivatives, compounds 3a, 3c, 3k, and 3o exhibited potent nematicidal activity with median lethal concentration (LC50) values ranging from 0.22 to 0.26 mg/L. Most noteworthy, N, N-dimethyl-4-methoxyphenylsulfonamide (3c) and N, N-dimethyl-8-quinolinesulfonamide (3o) showed the best promising and pronounced nematicidal activity, with LC50 values of 0.2381 and 0.2259 mg/L, respectively.
Assuntos
Antinematódeos , Tylenchoidea , Animais , Antinematódeos/farmacologia , Estrutura MolecularRESUMO
Following the premises of the multitarget-directed ligands approach for the drug R&D against neurodegenerative diseases, where Alzheimer's disease (AD) outstands, we have synthesized and evaluated analogues of the gramine derivative ITH12657 (1-benzyl-5-methyl-3-(piperidin-1-ylmethyl-1H-indole, 2), which had shown important neuroprotective properties, such as blocking effect of voltage-gated Ca2+ channels (VGCC), and prevention of phosphoprotein phosphatase 2A (PP2A) inhibition. The new analogues present different substitutions at the pending phenyl ring, what slightly modified their pharmacological characteristics. The VGCC blockade was enhanced in derivatives possessing nitro groups, while the pro-PP2A feature was ameliorated by the presence of fluorine. Chlorine atoms supplied good activities over the two biological targets aimed; nevertheless that substitution provoked loss of viability at 100-fold higher concentrations (10⯵M), what discards them for a deeper pharmacological study. Overall, the para-fluorine derivative of ITH12657 was the most promising candidate for further preclinical assays.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Bloqueadores dos Canais de Cálcio/farmacologia , Alcaloides Indólicos/farmacologia , Indóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Piperidinas/farmacologia , Proteína Fosfatase 2/metabolismo , Alcaloides/síntese química , Alcaloides/química , Alcaloides/farmacologia , Alcaloides/toxicidade , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/toxicidade , Linhagem Celular Tumoral , Humanos , Alcaloides Indólicos/síntese química , Alcaloides Indólicos/química , Indóis/síntese química , Indóis/química , Indóis/toxicidade , Simulação de Acoplamento Molecular , Estrutura Molecular , Neurônios/metabolismo , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/toxicidade , Ácido Okadáico/farmacologia , Piperidinas/síntese química , Piperidinas/química , Piperidinas/toxicidade , Proteína Fosfatase 2/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Gramine is a natural indole alkaloid that has been isolated from different raw plants occurring mainly in Avena sativa, etc. The study was aimed to investigate the possible in vitro antioxidant, in vitro mutagenic, in vitro antimutagenic, and in vivo genotoxic activity of gramine using ferric reducing ability of plasma (FRAP) assay, Metal chelating, Ames bacterial reverse mutation test, and the mouse bone marrow micronucleus assay as well as chromosomal aberration. Four concentrations of gramine viz. 250, 500, 1000, and 2000 µg/mL were evaluated for its antioxidant activity in FRAP Assay and Metal Chelating Test. Four concentrations of gramine (1250 µg/plate, 2500 µg/plate, 5000 µg/plate, and 10 000 µg/plate) were employed in Salmonella typhimurium strains to study the mutagenicity in the presence and absence of standard mutagens, 2-aminofluorene (2-AF), sodium azide (SA), and 2-nitrofluorene (2-NF). Three doses, i.e. 0.1, 0.2, and 0.3 × the LD50 of gramine (i.e. 50 mg/kg, 100 mg/kg, and 150 mg/kg) were administered orally to either sex of Swiss albino mice for 48 h to study the genotoxic activity in micronucleus assay as well as chromosomal aberration. Gramine showed potent antioxidant activity in both the assay. Gramine at the given dose lacks mutagenicity as well as found to possess antimutagenic efficacy. Interestingly, S9 enzymes increase the antimutagenic activity in a dose-dependent manner. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs), as well as no significant difference in the percentage of chromosomal aberrations was observed between the gramine groups and the negative groups but percentage of polychromatic erythrocytes (PCEs) is found to be higher in all the gramine groups. These results indicate significant antioxidant, non-mutagenic as well as non-genotoxic activity of gramine in vitro and in vivo in the given doses.
Assuntos
Alcaloides/farmacologia , Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Avena , Grão Comestível , Testes de Mutagenicidade , Alcaloides/química , Alcaloides/isolamento & purificação , Alcaloides/toxicidade , Animais , Antimutagênicos/química , Antimutagênicos/isolamento & purificação , Antimutagênicos/toxicidade , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/toxicidade , Avena/química , Avena/toxicidade , Relação Dose-Resposta a Droga , Grão Comestível/química , Grão Comestível/toxicidade , Feminino , Ferricianetos/química , Alcaloides Indólicos , Quelantes de Ferro/farmacologia , Masculino , Camundongos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Testes para Micronúcleos , Mutação , Oxirredução , Ratos Wistar , Medição de Risco , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genéticaRESUMO
The Poaceae is a large taxonomic group consisting of approximately 12,000 species and is classified into 12 subfamilies. Gramine and benzoxazinones (Bxs), which are biosynthesized from the tryptophan pathway, are well-known defensive secondary metabolites in the Poaceae. We analyzed the presence or absence of garamine and Bxs in 64 species in the Poaceae by LC-MS/MS. We found that Hordeum brachyantherum and Hakonechloa macra accumulated gramine, but the presence of gramine was limited to small groups of species. We also detected Bxs in four species in the Pooideae and six species in the Panicoideae. In particular, four species in the Paniceae tribe in Panicoideae accumulaed Bxs, indicating that this tribe is a center of the Bx distribution. Bxs were absent in the subfamilies other than Pooideae and Panicoideae. These findings provide an overview of biased distribution of gramine and Bxs in Poaceae species.
Assuntos
Alcaloides/metabolismo , Benzoxazinas/metabolismo , Poaceae/metabolismo , Triptofano/metabolismo , Alcaloides/análise , Benzoxazinas/análise , Cromatografia Líquida/métodos , Alcaloides Indólicos , Redes e Vias Metabólicas , Metabolismo Secundário , Espectrometria de Massas em Tandem/métodos , ortoaminobenzoatos/análise , ortoaminobenzoatos/metabolismoRESUMO
Twenty-four gramine derivatives were synthesized and evaluated on MT1 and 5-HT1A receptors in vitro. Among them, seven derivatives (7, 8, 16, 19, 20, 21, and 24) exhibited higher agonisting activities on MT1 or 5-HT1A receptors. Compared with gramine, derivatives 7, 8, 16, 19, 20, 21, and 24 displayed 1.6-3.5-fold increase in agonistic rates on 5-HT1A receptor. Particularly, derivatives 7, 19, and 21 exhibited significant agonistic activities on MT1 and 5-HT1A receptors with EC50 values of 0.51, 0.39, 0.50 mΜ and 0.28, 0.46, 0.23 mΜ, respectively. The preliminary structure-activity relationships of gramine derivatives were summarized for further investigation on MT1 and 5-HT1A receptors as new potential agonists.
Assuntos
Alcaloides/síntese química , Alcaloides/farmacologia , Agonistas do Receptor 5-HT1 de Serotonina/síntese química , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Alcaloides/química , Animais , Alcaloides Indólicos , Estrutura Molecular , Agonistas do Receptor 5-HT1 de Serotonina/química , Relação Estrutura-AtividadeRESUMO
Clavicipitic acid is an ergot alkaloid, which was isolated from Claviceps strain and Claviceps fusiformis. Its unique tricyclic azepinoindole skeleton has attracted synthetic chemists, and various strategies have been developed for its total synthesis. These strategies can be generally categorized into two types based on the synthetic intermediates, namely, 4-substituted gramine derivatives and 4-substituted tryptophan derivatives. This Minireview summarizes the reported total syntheses from the point of these two key intermediates.
Assuntos
Alcaloides de Claviceps/química , Alcaloides de Claviceps/síntese química , Triptofano/análogos & derivados , Catálise , Estrutura Molecular , Estereoisomerismo , Triptofano/químicaRESUMO
Gramine (GRM), which occurs in Gramineae plants, has been developed to be a biological insecticide. Exposure to GRM was reported to induce elevations of serum ALT and AST in rats, but the mechanisms of the observed hepatotoxicity have not been elucidated. The present study aimed to identify reactive metabolites that potentially participate in the toxicity. In rat liver microsomal incubations fortified with glutathione or N-acetylcysteine, one oxidative metabolite (M1), one glutathione conjugate (M2), and one N-acetylcysteine conjugate (M3) were detected after exposure to GRM. The corresponding conjugates were detected in the bile and urine of rats after GRM administration. CYP3A was the main enzyme mediating the metabolic activation of GRM. The detected GSH and NAC conjugates suggest that GRM was metabolized to a quinone imine intermediate. Both GRM and M1 showed significant toxicity to rat primary hepatocytes.
Assuntos
Ativação Metabólica , Citocromo P-450 CYP3A , Hepatócitos , Ratos Sprague-Dawley , Animais , Ratos , Masculino , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/genética , Microssomos Hepáticos/metabolismo , Glutationa/metabolismo , Inseticidas/toxicidade , Inseticidas/metabolismo , Alcaloides/metabolismoRESUMO
BACKGROUND: Sepsis and its associated heart failure are among the leading causes of death. Gramine, a natural indole alkaloid, can be extracted from a wide variety of raw plants, and it exhibits therapeutic potential in pathological cardiac hypertrophy. However, the effect of gramine on inflammatory cardiomyopathy, particularly sepsis-induced myocardial injury, remains an unexplored area. PURPOSE: To determine the role of gramine in sepsis-induced myocardial dysfunction and explore its underlying mechanism. STUDY DESIGN AND METHODS: In mice, sepsis was established by intraperitoneally injecting lipopolysaccharide (LPS, 10 mg/kg). Subsequently, the effects of gramine administration (50 or 100 mg/kg) on LPS-triggered cardiac dysfunction in mice were investigated. For in vitro studies, isolated primary cardiomyocytes were used to assess the effect of gramine (25 or 50 µM) on LPS-induced apoptosis and inflammation. Additionally, molecular docking, co-immunoprecipitation and ubiquitination analyzes were conducted to explore the underlying mechanisms. RESULTS: Gramine visibly ameliorated sepsis-induced cardiac dysfunction, inflammatory response, and mortality in vivo. Moreover, it significantly alleviated LPS-induced apoptotic and inflammatory responses in vitro. Furthermore, target prediction for gramine using the SuperPred website indicated that the nuclear factor NF-κB p105 subunit was one of the molecules ranked in priority order with a high model accuracy and a high probability score. Molecular docking studies demonstrated that gramine effectively docked to the death domain of NF-κB p105. Mechanistic studies revealed that gramine suppressed the processing of NF-κB p105 to p50 by inhibiting NF-κB p105 ubiquitination. Additionally, the protective effect of gramine on cardiac injury was almost abolished by overexpressing NF-κB p105. CONCLUSION: Gramine is a promising bioactive small molecule for treating sepsis-induced myocardial dysfunction, which acts by docking to NF-κB p105 and inhibiting NF-κB p105 ubiquitination, thus preventing its processing to NF-κB p50. Therefore, gramine holds potential as a clinical drug for treating myocardial depression during sepsis.
Assuntos
Cardiomiopatias , Cardiopatias , Sepse , Animais , Camundongos , NF-kappa B/metabolismo , Lipopolissacarídeos , Simulação de Acoplamento Molecular , Alcaloides Indólicos , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/etiologia , Ubiquitinação , Sepse/complicações , Sepse/tratamento farmacológico , Sepse/metabolismoRESUMO
BACKGROUND: The presence of plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 and its related variants has been associated with heightened resistance to tigecycline, thus diminishing its effectiveness. In this study, we explored the potential of gramine, a naturally occurring indole alkaloid, as an innovative adjuvant to enhance the treatment of infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters. METHODS: The synergistic potential of gramine in combination with antibiotics against both planktonic and drug-tolerant multidrug-resistant Enterobacterales was evaluated using the checkerboard microbroth dilution technique and time-killing curve analyses. Afterwards, the proton motive force (PMF) of cell membrane, the function of efflux pump and the activity of antioxidant system were determined by fluorescence assay and RT-PCR. The intracellular accumulation of tigecycline was evaluated by HPLC-MS/MS. The respiration rate, bacterial ATP level and the NAD+/NADH ratio were investigated to reveal the metabolism state. Finally, the safety of gramine was assessed through hemolytic activity and cytotoxicity assays. Two animal infection models were used to evaluate the in vivo synergistic effect. RESULTS: Gramine significantly potentiated tigecycline and ciprofloxacin activity against tmexCD1-toprJ1 and its variants-positive pathogens. Importantly, the synergistic activity was also observed against bacteria in special physiological states such as biofilms and persister cells. The mechanism study showed that gramine possesses the capability to augment tigecycline accumulation within cells by disrupting the proton motive force (PMF) and inhibiting the efflux pump functionality. In addition, the bacterial respiration rate, intracellular ATP level and tricarboxylic acid cycle (TCA) were promoted under the treatment of gramine. Notably, gramine effectively restored tigecycline activity in multiple animal infection models infected by tmexCD1-toprJ1 positive K. pneumoniae (RGF105-1). CONCLUSION: This study provides the first evidence of gramine's therapeutic potential as a novel tigecycline adjuvant for treating infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.
Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Animais , Tigeciclina/metabolismo , Tigeciclina/farmacologia , Tigeciclina/uso terapêutico , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Minociclina/farmacologia , Minociclina/metabolismo , Minociclina/uso terapêutico , Espectrometria de Massas em Tandem , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Farmacorresistência Bacteriana , Antibacterianos/farmacologia , Alcaloides Indólicos/farmacologia , Trifosfato de Adenosina/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Oxidative stress caused due to the perturbations in the oxidant-antioxidant system can damage molecules and cause cellular alteration leading to the pathogenesis of multiple diseases. This study was designed and performed to investigate the antioxidant and anti-inflammatory effects of an alkaloid, gramine on H2O2-induced oxidative stress on HEK 293 cells. Cell viability and morphometric analysis of cells treated with H2O2 and gramine were studied. Oxidative stress and inflammatory and antioxidant enzymes such as ROS, LPO, NO, SOD, GSH, and CAT were analyzed. Furthermore, mRNA expression of SOD, CAT, and COX-2 was also evaluated. H2O2 at concentration > 0.3 mM and gramine at concentration > 80 µg/mL affect the proliferation. Viability and morphometric analysis showed that gramine has protective effects. Treating cells with gramine suppressed oxidative stress and inflammatory enzymes, whereas antioxidant enzymes were enhanced. SOD and CAT mRNA levels were overexpressed and COX-2 mRNA levels were decreased in the treated groups. Gramine possesses effective antioxidant potential and can regulate oxidative stress and damages associated with it.
RESUMO
Barley (Hordeum vulgare) is one of the most widely cultivated crops for feedstock and beer production, whereas lupins (Lupinus spp.) are grown as fodder and their seeds are a source of protein. Both species produce the allelopathic alkaloids gramine and hordenine. These plant-specialized metabolites may be of economic interest for crop protection, depending on their tissue distribution. However, in high concentrations they pose a health risk to humans and animals that feed on them. This study was carried out to develop and validate a new method for monitoring these alkaloids and their related metabolites using fluorescence detection. Separation was performed on an HSS T3 column using slightly acidified water-acetonitrile eluents. Calibration plots expressed linearity over the range 0.09-100 pmol/µL for gramine. The accuracy and precision ranged from 97.8 to 123.4%, <7% RSD. The method was successfully applied in a study of the natural range of abundance of gramine, hordenine and their related metabolites, AMI, tryptophan and tyramine, in 22 barley accessions and 10 lupin species. This method provides accurate and highly sensitive chromatographic separation and detection of tryptophan- and tyrosine-derived allelochemicals and is an accessible alternative to LC-MS techniques for routine screening.
RESUMO
Plants accumulate various secondary metabolites, and the biosynthetic reactions responsible for their scaffold construction are the key steps that characterize their structural categories. Gramine, an indole alkaloid, is a defensive secondary metabolite biosynthesized in barley (Hordeum vulgare) from tryptophan (Trp) via aminomethylindole (AMI). While the two sequential N-methylation steps following the formation of AMI have already been characterized both genetically and enzymatically, the step preceding AMI formation, which includes the Trp side chain-shortening, has not yet been revealed. To gain further insight into these biosynthetic reactions, barley seedlings were fed Trp labeled with stable isotopes (13C and 15N) at various positions, and the isotope incorporation into gramine was analyzed by liquid chromatography/mass spectrometry. Significant increases in the abundance of isotopic gramine were detected in experimental sets in which Trp was labeled at either the indole ring, the ß-carbon, or the amino group, whereas the isotopolog composition was not affected by α-carbon-labeled Trp. Although absorbed Trp presumably undergoes transamination in plants, this reaction did not seem to be related to gramine productivity. The data indicated that AMI directly inherited the amino group from Trp, while the α-carbon was removed, suggesting that the Trp-AMI conversion includes a novel intramolecular rearrangement reaction. The results of this study provide novel insights into scaffold formation in plant secondary-metabolite synthesis.
RESUMO
A series of indole-1,4-disubstituted-1,2,3-triazole conjugates were synthesised by click chemistry. The haemolytic properties and cytoprotective activity of all the newly synthesised indole-triazole conjugates were tested in vitro. In addition, molecular docking was performed in silico for the selected conjugates to determine their antibacterial and antifungal properties. The results indicate that indole-triazole derivatives effectively protect human erythrocytes against free radical-induced haemolysis in a structure-dependent manner and that bis-indole-bis-triazole derivatives with alkyl linkers are excellent cytoprotective agents against oxidative haemolysis. The tested series of indole-1,4-disubstituted-1,2,3-triazole conjugates may have an affinity for the active sites of specific protein domains (PDB IDs: 2Q85 and 5V5Z) according to molecular docking studies.
RESUMO
BACKGROUND: Gramine, also named 3-(N,N-dimethylaminomethyl) indole, is a indole alkaloid. It is mainly extracted from various natural raw plants. Despite being the simplest 3-aminomethylindole, Gramine has broad pharmaceutical and therapeutic effects, such as vasodilatation, antioxidation, mitochondrial bioenergetics-related effects, and angiogenesis via modulation of TGFß signaling. However, there is little information available about Gramine's role in heart disease, especially pathological cardiac hypertrophy. PURPOSE: To investigate Gramine's effect on pathological cardiac hypertrophy and clarify the mechanisms behind its action. METHODS: In the in vitro experiment, Gramine (25 µM or 50 µM) was used to investigate its role in Angiotensin II-induced primary neonatal rat cardiomyocytes (NRCMs) hypertrophy. In the in vivo experiment, Gramine (50 mg/kg or 100 mg/kg) was administrated to investigate its role in transverse aortic constriction (TAC) surgery mice. Additionally, we explored the mechanisms underlying these roles through Western blot, Real-time PCR, genome-wide transcriptomic analysis, chromatin immunoprecipitation and molecular docking studies. RESULTS: The in vitro data demonstrated that Gramine treatment obviously improved primary cardiomyocyte hypertrophy induced by Angiotensin II, but had few effects on the activation of fibroblasts. The in vivo experiments indicated that Gramine significantly mitigated TAC-induced myocardial hypertrophy, interstitial fibrosis and cardiac dysfunction. Mechanistically, RNA sequencing and further bioinformatics analysis demonstrated that transforming growth factor ß (TGFß)-related signaling pathway was enriched significantly and preferentially in Gramine-treated mice as opposed to vehicle-treated mice during pathological cardiac hypertrophy. Moreover, this cardio-protection of Gramine was found to mainly involved in TGFß receptor 1 (TGFBR1)- TGFß activated kinase 1 (TAK1)-p38 MAPK signal cascade. Further exploration showed that Gramine restrained the up-regulation of TGFBR1 by binding to Runt-related transcription factor 1 (Runx1), thereby alleviating pathological cardiac hypertrophy. CONCLUSION: Our findings provided a substantial body of evidence that Gramine possessed a potential druggability in pathological cardiac hypertrophy via suppressing the TGFBR1-TAK1-p38 MAPK signaling axis through interaction with transcription factor Runx1.
Assuntos
Angiotensina II , Subunidade alfa 2 de Fator de Ligação ao Core , Ratos , Camundongos , Animais , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Angiotensina II/farmacologia , Simulação de Acoplamento Molecular , Cardiomegalia/metabolismo , Miócitos Cardíacos , Transdução de Sinais , Alcaloides Indólicos/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
The new series of triazole-bearing gramine derivatives were synthesized through a CuAAC procedure. The structures of all newly obtained compounds were confirmed by spectroscopic analysis and DFT methods. The obtained derivatives were screened for their protective potency against oxidative haemolysis induced by free radicals generated from 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). Our work demonstrates that derivatives with propyl or octyl linker and phthalimide group associated with indole-triazole moiety, which have a folded structure, effectively protect human erythrocytes against oxidative stress-induced haemolysis.