RESUMO
The global population is aging, and intervention strategies for anti-aging and the prevention of aging-related diseases have become a topic actively explored today. Nicotinamide adenine dinucleotide (NAD+) is an important molecule in the metabolic process, and its content in tissues and cells decreases with age. The supplementation of nicotinamide mononucleotide (NMN), an important intermediate and precursor of NAD+, has increased NAD+ levels, and its safety has been demonstrated in rodents and human studies. However, the high content of NMN in natural plants has not been fully explored as herbal medicines for drug development. Here, we identified that the leaf of Cinnamomum verum J. Presl (C. verum) was the highest NMN content among the Plant Extract Library (PEL) with food experience, using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To validate this result, the extraction and quantitative analysis of bark, leaf, root, and stem of fresh C. verum was conducted. The results revealed that the bark had the highest NMN content in C. verum (0.471 mg/100 g). Our study shed light on the prospects of developing natural plants in the context of NMN as drugs for anti-aging and prevention of aging-related diseases. The future should focus on the development and application of C. verum pharmaceutical formulations.
Assuntos
NAD , Mononucleotídeo de Nicotinamida , Humanos , NAD/metabolismo , Cinnamomum zeylanicum , Cromatografia Líquida , Casca de Planta/metabolismo , Espectrometria de Massas em Tandem , Extratos Vegetais/farmacologia , Preparações FarmacêuticasRESUMO
Effective drugs that can cure cognitive impairments remain elusive. Because synaptic dysfunction has been correlated with cognitive impairments, drug development to target synaptic dysfunction is important. Recently, natural compounds and crude drugs have emerged as potential therapeutic agents for cognitive disorders. However, their effects on synaptic function remain unclear, because of lack of evaluation system with high reproducibility. We have recently developed highly reproducible in vitro high-content imaging analysis system for evaluation of synaptic function using drebrin as a marker for synaptic states. Therefore, we aimed to examine the direct effects of well-known natural compounds and crude drugs on synaptic states using this system. Rat hippocampal neurons were treated using natural compounds (nobiletin, diosgenin and tenuifolin) and crude drugs (Uncaria Hook [UH], Bezoar Bovis [BB], Coptis Rhizome [CR], Phellodendron Bark [PB] and Polygala Root [PR]). Immunocytochemical analysis was performed, and dendrite lengths and drebrin cluster densities were automatically quantified. We found that diosgenin, tenuifolin, CR, PB and PR decreased drebrin cluster densities, and the effects of PB and PR were partially dependent on N-methyl-D-aspartic acid-type glutamate receptors (NMDARs). Nobiletin and UH did not show any effects, whereas low-dose BB treatment increased drebrin cluster densities. Our results showed that diosgenin, tenuifolin, BB, CR, PB and PR appeared to directly change synaptic states. Particularly, the NMDAR dependency of PB and PR appears to affect synaptic plasticity.
Assuntos
Preparações Farmacêuticas , Receptores de N-Metil-D-Aspartato , Animais , Ratos , Hipocampo/metabolismo , Neuropeptídeos , Receptores de N-Metil-D-Aspartato/metabolismo , Reprodutibilidade dos Testes , Sinapses/metabolismoRESUMO
(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC50 = 2.1 µM, and 20.2 µM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Embrião não Mamífero/patologia , Neoplasias da Próstata/patologia , Saponinas/toxicidade , Styrax/química , Peixe-Zebra/embriologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Embrião não Mamífero/efeitos dos fármacos , Masculino , Saponinas/química , Ovinos , Testes de Toxicidade AgudaRESUMO
Endotoxin is an unintentional contaminant that has numerous activities and can affect various biological experiments using cells. In this study, we measured the endotoxin activity of samples from a plant extract library (PEL) and determined their degrees of contamination. Endotoxin was detected in approx. 48% (n = 139) and approx. 4% (n = 5) of field-collected and crude drug samples, respectively, and in concentrations >5.0 EU/mL in some samples. The concentrations of endotoxin that affect cells in vitro vary depending on the target cell type. Although the degree of contamination varied in the present study, it was considered to have little effect on the cell experiments. More than 150 PEL samples had problems with reaction courses or recovery rates of Limulus amoebocyte lysate (LAL) tests. In the LAL tests, using three plant extracts [Sanguisorba officinalis L. (Rosaceae), Oenothera biennis L. (Onagraceae), and Lythrum salicaria L. (Lythraceae)], the polyphenolic compounds in the plant extracts affected LAL test and their effects differed depending on the plant species. When the 16 single polyphenol compounds were added to the LAL tests, the compounds with caffeoyl and pyrogallol moieties were found to affect the LAL reaction course and recovery rate. Furthermore, none of the compounds had any effects at concentrations of 1 µM. Because the plant extracts contained analogs of various polyphenolic compounds, they were presumed to actually act synergistically. Our findings demonstrated that attention must be paid to the recovery rate and reaction process of LAL tests with samples containing polyphenolic compounds.
Assuntos
Contaminação de Medicamentos/prevenção & controle , Endotoxinas/análise , Teste do Limulus/normas , Extratos Vegetais/química , Animais , Lythrum/química , Oenothera biennis/química , Extratos Vegetais/normas , Polifenóis/química , Sanguisorba/químicaRESUMO
Epithelial-mesenchymal transition (EMT) has recently been associated with cancer invasion, metastasis, and resistance. In our previous study, we discovered nanaomycin K, a natural growth inhibitor for EMT-induced Madin Darby canine kidney (MDCK) cells, from the cultured broth of actinomycetes. However, the screening method was undeveloped, because the activity of nanaomycin K was discovered accidentally. In this study, we established a screening method by analyzing the characteristics of nanaomycin K in MDCK cells. Nanaomycin K showed the characteristic growth inhibitory activity on MDCK cells cultured under four conditions: medium containing dimethyl sulfoxide, SB431542, TGF-ß, and a mixture of SB431542 and TGF-ß. The activity was stronger in TGF-ß-treated cells than in DMSO-treated cells. In the mixture of SB431542 and TGF-ß-treated cells, the activity of nanaomycin K was suppressed. The anti-cancer agents, mitomycin C, cisplatin, and staurosporine, lacked the characteristics as that of nanaomycin K for these four treatment conditions. Since these four conditions distinguish between the effects of nanaomycin K and other anti-cancer agents in EMT-induced cells, the screening method was established. Among the 13,427 plant extracts tested, Piper betle leaf extract displayed growth inhibitory activity against EMT-induced cells. Through the purification of the extract via bio-guided fractionation, hydroxychavicol was isolated as an active compound. The cytotoxic activity of hydroxychavicol was stronger in EMT-induced MDCK cells than in control cells. However, its cytotoxic activity was suppressed in EMT-inhibited cells. Furthermore, hydroxychavicol exhibited same activity against SAS cells (human squamous cell carcinoma of the tongue). Thus, we have successfully established a screening method for growth inhibitors of EMT-induced cells and have discovered an inhibitor from plant-based sources.
Assuntos
Transição Epitelial-Mesenquimal , Piper betle , Fator de Crescimento Transformador beta , Animais , Cães , Humanos , Benzamidas/farmacologia , Benzamidas/química , Proliferação de Células/efeitos dos fármacos , Dioxóis/farmacologia , Dioxóis/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Eugenol/farmacologia , Eugenol/análogos & derivados , Inibidores do Crescimento/farmacologia , Inibidores do Crescimento/química , Inibidores do Crescimento/isolamento & purificação , Células Madin Darby de Rim Canino , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Fator de Crescimento Transformador beta/metabolismo , Piper betle/químicaRESUMO
Chronic kidney disease (CKD) induces cardiac inflammation and fibrosis and reduces survival. We previously demonstrated that G protein-coupled receptor 68 (GPR68) promotes cardiac inflammation and fibrosis in mice with 5/6 nephrectomy (5/6Nx) and patients with CKD. However, no method of GPR68 inhibition has been found that has potential for therapeutic application. Here, we report that Cephalotaxus harringtonia var. nana extract and homoharringtonine ameliorate cardiac inflammation and fibrosis under CKD by suppressing GPR68 function. Reagents that inhibit the function of GPR68 were explored by high-throughput screening using a medicinal plant extract library (8,008 species), and we identified an extract from Cephalotaxus harringtonia var. nana as a GPR68 inhibitor that suppresses inflammatory cytokine production in a GPR68 expression-dependent manner. Consumption of the extract inhibited inflammatory cytokine expression and cardiac fibrosis and improved the decreased survival attributable to 5/6Nx. Additionally, homoharringtonine, a cephalotaxane compound characteristic of C. harringtonia, inhibited inflammatory cytokine production. Homoharringtonine administration in drinking water alleviated cardiac fibrosis and improved heart failure and survival in 5/6Nx mice. A previously unknown effect of C. harringtonia extract and homoharringtonine was revealed in which GPR68-dependent inflammation and cardiac dysfunction were suppressed. Utilizing these compounds could represent a new strategy for treating GPR68-associated diseases, including CKD.
Assuntos
Mepesuccinato de Omacetaxina , Extratos Vegetais , Receptores Acoplados a Proteínas G , Insuficiência Renal Crônica , Animais , Camundongos , Citocinas/metabolismo , Fibrose , Cardiopatias/tratamento farmacológico , Cardiopatias/etiologia , Mepesuccinato de Omacetaxina/farmacologia , Mepesuccinato de Omacetaxina/uso terapêutico , Camundongos Endogâmicos C57BL , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/complicaçõesRESUMO
To explore useful natural compounds from indigenous medicinal plants, the cytotoxic properties from a methanolic extract of Boehmeria sieboldiana leaves against human cancer cell lines were isolated in the present study. After purification of the extract, seco-dehydroantofine B (1) together with two known phenanthroindolizine alkaloids, seco-dehydroantofine A (2) and septicine (3), were isolated. The structure of seco-dehydroantofine B was elucidated by performing comprehensive one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The cytotoxicity of these compounds against five human tumor cell lines was evaluated. Compound 3 exhibited anti-tumor activity at IC50 values of 50.0, 66.9, 50.0, and 153.7 µM against MKN1, SAS, HL-60, and THP-1 cells, respectively.
Assuntos
Alcaloides , Antineoplásicos Fitogênicos , Boehmeria , Neoplasias , Alcaloides/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Humanos , Estrutura Molecular , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Folhas de Planta/químicaRESUMO
Adjuvants are important vaccine components, composed of a variety of chemical and biological materials that enhance the vaccine antigen-specific immune responses by stimulating the innate immune cells in both direct and indirect manners to produce a variety cytokines, chemokines, and growth factors. It has been developed by empirical methods for decades and considered difficult to choose a single screening method for an ideal vaccine adjuvant, due to their diverse biochemical characteristics, complex mechanisms of, and species specificity for their adjuvanticity. We therefore established a robust adjuvant screening strategy by combining multiparametric analysis of adjuvanticity in vivo and immunological profiles in vitro (such as cytokines, chemokines, and growth factor secretion) of various library compounds derived from hot-water extracts of herbal medicines, together with their diverse distribution of nano-sized physical particle properties with a machine learning algorithm. By combining multiparametric analysis with a machine learning algorithm such as rCCA, sparse-PLS, and DIABLO, we identified that human G-CSF and mouse RANTES, produced upon adjuvant stimulation in vitro, are the most robust biological parameters that can predict the adjuvanticity of various library compounds. Notably, we revealed a certain nano-sized particle population that functioned as an independent negative parameter to adjuvanticity. Finally, we proved that the two-step strategy pairing the negative and positive parameters significantly improved the efficacy of screening and a screening strategy applying principal component analysis using the identified parameters. These novel parameters we identified for adjuvant screening by machine learning with multiple biological and physical parameters may provide new insights into the future development of effective and safe adjuvants for human use.
Assuntos
Adjuvantes de Vacinas , Vacinas , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Adjuvantes Farmacêuticos , Animais , Citocinas , Medicina Herbária , Aprendizado de Máquina , CamundongosRESUMO
One of the major challenges in development of antienterovirus (EV) drugs is in the safety of the drug. Here, we attempted to identify anti-EV compounds from an edible plant extract library and found potent antienterovirus D68 (EV-D68) activity in avocado (Persea americana). The purified identity is determined as 2R,4R-(12Z,15Z)-heneicosa-12,15-diene-1,2,4-triol, named avoenin. Avoenin shows an EC50 of 2.0 µM for EV-D68 (Fermon) infection with CC50 of >150 µM in RD cells by targeting the uncoating step of EV-D68 infection. Resistant mutations of EV-D68 (VP3-V24I, S173P, and S180G) to avoenin confer cross-resistance to pleconaril, an uncoating inhibitor of EV-D68. The inhibitory effect of avoenin is substantially specific to EV-D68 among the EVs. This work reveals avoenin as the identity of anti-EV-D68 activity in avocado and offers insights into development of a novel and effective strategy to overcome EV-D68 infection and its related respiratory diseases.