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1.
PLoS One ; 15(11): e0243066, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33253287

RESUMO

Candidiasis causes high morbidity and mortality among immunocompromised patients. Antifungal drug resistance and cytotoxicity highlight the need of effective antifungal therapeutics. In this study, we found that kalopanaxsaponin A (KPA), a triterpenoid saponin natural product, could inhibit the proliferation of various Candida species, and exerted a fungicidal effect against C. albicans. To further explore its antifungal action mode, spectrofluorophotometer, fluorescence microscopy and transmission electron microscopy were performed, showing that KPA treatment induced the accumulation of intracellular reactive oxygen species (ROS), resulting in mitochondrial dysfunction. Meanwhile, KPA treatment also broke down the membrane barrier of C. albicans causing the leakage of intracellular trehalose, the entrance of extracellular impermeable substance and the decrease of ergosterol content. Both ROS accumulation and membrane destruction contributed to the death of C. albicans cells. Our work preliminarily elucidated the potential mechanisms of KPA against C. albicans on a cellular level, and might provide a potential option for the treatment of clinical candidiasis.


Assuntos
Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Antifúngicos/farmacologia , Candida albicans/patogenicidade , Candidíase/microbiologia , Candidíase/patologia , Membrana Celular/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Ergosterol/farmacologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/genética , Ácido Oleanólico/farmacologia , Espécies Reativas de Oxigênio/metabolismo
2.
Mol Biol (Mosk) ; 54(5): 813-825, 2020.
Artigo em Russo | MEDLINE | ID: mdl-33009791

RESUMO

Conyzasaponins produced by the traditional Chinese herb Conyza blinii are oleanane-type saponins with a wide range of biological activities. Here, we identified a gene, designated CbCYP716A261, encoding a ß-amyrin 28-hydroxylase in conyzasaponins biosynthesis. Ten full putative CYP sequences were isolated from Conyza blinii transcript tags. The CbCYP716A261 gene product was selected as the putative ß-amyrin 28-hydroxylase by phylogenetic analysis and transcriptional activity analysis of methyl jasmonate-treated Conyza blinii. To identify the enzymatic activity, we performed enzymatic activity experiments in vitro and in vivo. The HPLC results revealed that CbCYP716A261 catalyzes the hydroxylation of ß-amyrin at the C-28 position to yield oleanolic acid. Our findings provide new information about the conyzasaponin biosynthesis pathway and widen the list of isolated ß-amyrin 28-hydroxylases.


Assuntos
Conyza/enzimologia , Oxigenases de Função Mista/metabolismo , Saponinas/biossíntese , Conyza/genética , Oxigenases de Função Mista/genética , Ácido Oleanólico/análogos & derivados , Filogenia
3.
PLoS One ; 15(10): e0241053, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33104740

RESUMO

The relationship between methylglyoxal (MGO) and D-lactate during saikosaponin C (SSC) treatment of mice with accelerated nephrotoxic serum (NTS) nephritis was investigated. NTS nephritis was induced by administration of anti-basement membrane antibodies to C57BL/6 mice and three dosages of SSC were administered for 14 days. Proteinuria, blood urea nitrogen, serum creatinine, renal histology, urinary MGO and d-lactate changes were examined. Compared to the NTS control group, the middle dosage (10 mg/kg/day) of SSC significantly alleviated the development of nephritis based on urine protein measurements (34.40 ± 6.85 vs. 17.33 ± 4.79 mg/day, p<0.05). Pathological observation of the glomerular basement membrane (GBM) revealed monocyte infiltration, hypertrophy, and crescents were alleviated, and injury scoring also showed improved efficacy for the middle dose of SSC during nephritis (7.92 ± 1.37 vs. 3.50 ± 1.14, p<0.05). Moreover, the significant decreases in urinary levels of MGO (24.71 ± 3.46 vs. 16.72 ± 2.36 µg/mg, p<0.05) and D-lactate (0.31 ± 0.04 vs. 0.23 ± 0.02 µmol/mg, p<0.05) were consistent with the biochemical and pathological examinations. This study demonstrates that MGO and D-lactate may reflect the extent of damage and the efficacy of SSC in NTS nephritis; further studies are required to enable clinical application.


Assuntos
Glomerulonefrite/tratamento farmacológico , Ácido Láctico/urina , Ácido Oleanólico/análogos & derivados , Aldeído Pirúvico/urina , Saponinas , Animais , Camundongos , Camundongos Endogâmicos C57BL , Ácido Oleanólico/administração & dosagem , Ácido Oleanólico/uso terapêutico , Saponinas/administração & dosagem , Saponinas/uso terapêutico
4.
Sci Rep ; 10(1): 15543, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968148

RESUMO

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3's has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ácido Oleanólico/análogos & derivados , Fatores de Transcrição/metabolismo , Azepinas/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Humanos , Ácido Oleanólico/metabolismo , Ácido Oleanólico/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Triazóis/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
5.
Yakugaku Zasshi ; 140(8): 1063-1069, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741864

RESUMO

Quantitative NMR (qNMR) has been developed as an absolute quantitation method to determine the purity or content of organic compounds including marker compounds in crude drugs. The "qNMR test" has been introduced into the crude-drug section of the Japanese Pharmacopoeia (JP) for determining the purity of reagents used for the assay in the JP. In Supplement II to the JP 17th edition published in June 2019, fifteen compounds adopted qNMR test were listed as the reagents for the assay. To establish the "qNMR test" in the crude drug section of the JP, there were several problems to be solved. Previously, we reported that the handling impurity signals from reference substances and targeted marker compounds, chemical shifts of reference substances, and peak unity of signals of targeted marker compounds are important factors to conduct qNMR measurements with intended accuracy. In this study, we investigated that the hygroscopicity of reagents could cause the changes in the compounds' purity depending on increasing their water content. Twenty-one standard products used for the crude-drug test in JP were examined by water sorption-desorption analysis, and ginsenosides and saikosaponins were found to be hygroscopic. To prepare a sample solution of saikosaponin b2 for qNMR analysis, samples need to be maintained for 18 h at 25°C and 76% relative humidity; further, samples need to be weighed at the same humidity for the qNMR analysis.


Assuntos
Contaminação de Medicamentos/prevenção & controle , Higroscópicos/química , Higroscópicos/normas , Indicadores e Reagentes/normas , Espectroscopia de Ressonância Magnética/métodos , Farmacopeias como Assunto/normas , Ginsenosídeos/química , Ginsenosídeos/normas , Umidade , Japão , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/química , Ácido Oleanólico/normas , Psicoterapia Breve , Saponinas/química , Saponinas/normas , Temperatura , Água/análise
6.
J Med Chem ; 63(17): 9965-9976, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32787104

RESUMO

Synthetic triterpenoids including CDDO, its methyl ester (CDDO-Me, bardoxolone methyl), and its imidazolide (CDDO-Im) enhance Nrf2-mediated antioxidant and anti-inflammatory activity in many diseases by reacting with thiols on the adaptor protein, Keap1. Unlike monofunctional CDDO-Me, the bifunctional analog, CDDO-Im, has a second reactive site (imidazolide) and can covalently bind to amino acids other than cysteine on target proteins such as glutathione S-transferase pi (GSTP), serum albumin, or Keap1. Here we show for the first time that bifunctional CDDO-Im (in contrast to CDDO-Me), as low as 50 nM, can covalently transacylate arginine and serine residues in GSTP and cross-link them to adjacent cysteine residues. Moreover, we show that CDDO-Im binds covalently to Keap1 by forming permanent Michael adducts with eight different cysteines, and acyl adducts with lysine and several tyrosine residues. Modeling studies suggest that the Tyr 85 adduct stabilizes the Keap1-Cul3 complex, thereby enhancing the potency of CDDO-Im.


Assuntos
Imidazóis/química , Proteína 1 Associada a ECH Semelhante a Kelch/química , Ácido Oleanólico/análogos & derivados , Sequência de Aminoácidos , Proteínas Culina/química , Proteínas Culina/metabolismo , Glutationa S-Transferase pi/química , Glutationa S-Transferase pi/metabolismo , Humanos , Imidazóis/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Simulação de Acoplamento Molecular , Ácido Oleanólico/química , Ácido Oleanólico/metabolismo , Multimerização Proteica/efeitos dos fármacos , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo
7.
Sci Rep ; 10(1): 10534, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601436

RESUMO

The cytotoxic mechanism of the saponin QS-21 and its aglycone quillaic acid (QA) was studied on human gastric cancer cells (SNU1 and KATO III). Both compounds showed in vitro cytotoxic activity with IC50 values: 7.1 µM (QS-21) and 13.6 µM (QA) on SNU1 cells; 7.4 µM (QS-21) and 67 µM (QA) on KATO III cells. QS-21 and QA induce apoptosis on SNU1 and KATO III, as demonstrated by TUNEL, Annexin-V and Caspase Assays. Additionally, we performed in silico docking studies simulating the binding of both triterpenic compounds to key proteins involved in apoptotic pathways. The binding energies (∆Gbin) thus calculated, suggest that the pro-apoptotic protein Bid might be a plausible target involved in the apoptotic effect of both triterpenic compounds. Although QA shows some antiproliferative effects on SNU1 cells cultured in vitro, our results suggest that QS-21 is a more powerful antitumor agent, which merits further investigation regarding their properties as potential therapeutic agents for gastric cancer.


Assuntos
Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Quillaja , Saponinas/química , Neoplasias Gástricas/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Ácido Oleanólico/uso terapêutico , Saponinas/farmacologia , Saponinas/uso terapêutico
8.
Phytochemistry ; 177: 112434, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32544729

RESUMO

Drought stress affects vegetative and reproductive growth processes and synthesis of secondary metabolites in plants. We assessed relevant indicators of vegetative and reproductive growth in Bupleurum chinense DC. during drought stress. Samples were collected on days 4, 8, 12, 20, and 24 of a drought treatment according to drought stress severity in order to elucidate potential effects on synthesis of flavonoids in leaves and saikosaponins in roots of B. chinense. The results showed that B. chinense can adapt to drought stress mainly by increasing concentrations of osmoregulatory substances (soluble protein and proline) and increasing activity of protective enzymes (superoxide dismutase and catalase), as observed on days 12 and 20 of the treatment. Secondary metabolite concentrations in B. chinense roots and leaves showed significant differences-drought stress increased saikosaponin concentrations in roots by 9.85% and 6.41% during vegetative and reproductive growth, respectively, on day 20, and saikosaponin concentrations in roots were higher during vegetative growth than during reproductive growth. In leaves, large amounts of antioxidants were consumed owing to drought stress, which decreased leaf rutin concentrations by 38.79% and 30.11% during vegetative and reproductive growth, respectively, as observed on day 20; overall, leaf rutin concentrations were lower during vegetative growth than during reproductive growth. Changes in soil water content are known to affect synthesis of secondary metabolites in medicinal plants by altering gene transcription, and affected genes may synergistically respond to soil water changes and alter concentrations of flavonoid in leaves and of saikosaponin in roots. The gene F3H down-regulates flavonoid production in leaves. Squalene epoxidase and ß-amyrin synthase genes may be key genes regulating saikosaponin accumulation, and changes in their expression corresponded to accumulation of saikosaponins. Our results provide insights in B. chinense adaptation to drought stress through physiological changes and regulation of secondary metabolite production in different plant tissues.


Assuntos
Bupleurum , Secas , Flavonoides , Ácido Oleanólico/análogos & derivados , Folhas de Planta , Raízes de Plantas , Saponinas
9.
J Agric Food Chem ; 68(26): 6977-6986, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502339

RESUMO

Six new pentacyclic triterpenoid saponins, centelloside F (1), centelloside G (2), 11-oxo-asiaticoside B (3), 11-oxo-madecassoside (4), 11(ß)-methoxy asiaticoside B (5), and 11(ß)-methoxy madecassoside (6), along with seven known ones, asiaticoside (7), asiaticoside B (8), madecassoside (9), centellasaponin A (10), isoasiaticoside (11), scheffoleoside A (12), and centelloside E (13), were separated from the 80% MeOH extract of the whole plant of Centella asiatica, which has been used as a medicinal plant and is now commercially available as a diatery supplement in many countries. Compounds 1 and 2, 3 and 4, and 5 and 6 are three pairs of isomers with oleanane- or ursane-type triterpenes as aglycones. The chemical structures of the new triterpene saponins were fully characterized by extensive analysis of their nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry data. The protective effects of compounds 1-13 on PC12 cells induced by 6-OHDA were screened, and compound 3 displayed the best neuroprotective effect, with 91.75% cell viability at the concentration of 100 µM. Moreover, compound 3 also attenuated cell apoptosis and increased the mRNA expression of antioxidant enzymes, including superoxide dismutase and catalase. Additionally, compound 3 activated the phosphatidylinositol 3-kinase/Akt pathway, including PDK1, Akt, and GSK-3ß. These findings suggested that triterpene saponins from C. asiatica were worthy of further biological research to develop new neuroprotective agents.


Assuntos
Centella/química , Fármacos Neuroprotetores/farmacologia , Ácido Oleanólico/análogos & derivados , Extratos Vegetais/química , Triterpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Células PC12 , Extratos Vegetais/farmacologia , Ratos , Superóxido Dismutase/metabolismo , Triterpenos/química
10.
Arthritis Rheumatol ; 72(10): 1707-1720, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32500632

RESUMO

OBJECTIVE: Pristane-induced lupus is associated with nonresolving inflammation and deficiency of proresolving macrophages. Proresolving nonclassic macrophages (NCMs) are less responsive to type I interferon (IFN) than classic macrophages (CMs; which are proinflammatory), reflecting their relative expression levels of the type I IFN receptor (IFNAR). This study was undertaken to investigate the regulation of IFNAR expression in macrophages. METHODS: We carried out gene expression profiling of purified CMs and NCMs from mice treated with pristane (which develop lupus) or mineral oil (non-lupus controls). Macrophage differentiation and IFNAR expression were examined in mice treated with NF-E2-related factor 2 (Nrf2) activators and inhibitors and in Nrf2-deficient mice. Nrf2 activity was also assessed in blood cells from patients with systemic lupus erythematosus (SLE). Significant differences were determined by Student's t-test. RESULTS: RNA sequencing revealed increased expression of genes regulated by the transcription factor Nrf2 in NCMs from mineral oil-treated versus pristane-treated mice and in NCMs versus CMs. The Nrf2 activator CDDO-imidazole (CDDO-Im) decreased CMs (P < 0.0001) and promoted the development of proresolving NCMs (P = 0.06), whereas the Nrf2 inhibitor brusatol increased CMs (P < 0.05) and decreased NCMs (P < 0.001). CDDO-Im decreased Ifnar1 (P < 0.001) and IFN-stimulated gene (ISG) expression in macrophages and alleviated oxidative stress (P < 0.05), whereas brusatol had the opposite effect (P < 0.01). Moreover, Ifnar1 and ISG expression levels were higher in Nrf2-knockout mice than controls (P < 0.05). As seen in mice with lupus, SLE patients showed evidence of low Nrf2 activity. CONCLUSION: Our findings indicate that Nrf2 activation favors the resolution of chronic inflammation in lupus. Since autoantibody production and lupus nephritis depend on IFNAR signaling, the ability of Nrf2 activators to repolarize macrophages and reduce the INF signature suggests that these agents may warrant consideration for treating lupus.


Assuntos
Polaridade Celular/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/metabolismo , Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Receptores de Interferon/metabolismo , Animais , Expressão Gênica , Imidazóis/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Quassinas/farmacologia
11.
Biochim Biophys Acta Biomembr ; 1862(10): 183383, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522531

RESUMO

The paper considers the effects of plant triterpenoid betulin and its derivative betulonic acid on rat liver mitochondria and liposomes. It was found that betulonic acid and, to a lesser extent, betulin, activate mitochondrial respiration in states 2 and 4 and inhibit ADP- and DNP-stimulated (uncoupled) respiration. The effect of betulonic acid resulted in a significant decrease of the respiratory control and ADP/O ratios and decrease in Δψ. The effects of both compounds were most pronounced in the case of succinate-fueled mitochondrial respiration. This may include both the possible protonophore effect of betulonic acid and the inhibition of respiratory chain complexes by both compounds. Both agents enhanced H2O2 production in succinate-fueled mitochondria, while betulonic acid exerted an antioxidant effect with NAD-dependent substrates. Betulin was found to induce mitochondrial aggregation, but had no effect on membrane permeability. A similar pattern was found on liposomes. As revealed by the laurdan generalized polarization (GP) technique, betulin increased laurdan GP in lecithin liposomes, indicating a decrease in membrane fluidity. Measurements of GP as a function of fluorescence excitation wavelength gave an ascending line for high concentrations of betulin, which can be interpreted as phase heterogeneity of the lipid/betulin system. High concentrations of betulin (> 60 mol%) was also demonstrated to cause permeabilization of lecithin liposomes. Betulonic acid was much less effective in inducing the aggregation of mitochondria and liposomes and had no effect on membrane permeability. The possible mechanisms of betulin and betulonic acid effect on rat liver mitochondria and liposomes are discussed.


Assuntos
Lipossomos , Mitocôndrias Hepáticas/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Triterpenos/farmacologia , Animais , Transporte de Elétrons , Metabolismo Energético/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Fosforilação Oxidativa , Ratos
12.
BMC Complement Med Ther ; 20(1): 167, 2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493316

RESUMO

BACKGROUND: Previous studies indicate that soyasaponins may reduce inflammation via modulating toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling. However, its underlying mechanisms are still not fully understood. METHODS: Lipopolysaccharide (LPS)-challenged inflamed male ICR mice were intervened by intragastrical administration with 10 and 20 µmol/kg·BW of soyasaponin A1, A2 or I for 8 weeks. The serum inflammatory markers were determined by commercial kits and the expression of molecules in TLR4/MyD88 signaling pathway in liver by real-time PCR and western blotting. The recruitments of TLR4 and MyD88 into lipid rafts of live tissue lysates were detected by sucrose gradient ultracentrifugation and western blotting. LPS-stimulated RAW264.7 macrophages were treated with 10, 20 and 40 µmol/L of soyasaponin A1, A2 or I for 2 h. MyD88-overexpressed HEK293T cells were treated with 20 and 40 µmol/L of soyasaponins (A1, A2 or I) or 20 µmol/L of ST2825 (a MyD88 inhibitor) for 6 h. The expression of molecules in TLR4/MyD88 signaling pathway were determined by western blotting. Data were analyzed by using one way analysis of variance or t-test by SPSS 20.0 statistical software. RESULTS: Soyasaponins A1, A2 or I significantly reduced the levels of tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and nitric oxide (NO) in serum (p < 0.05), and decreased the mRNA levels of TNFα, IL-6, IL-1ß, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) (p < 0.05), the protein levels of myeloid differentiation protein 2 (MD-2), TLR4, MyD88, toll-interleukin1 receptor domain containing adaptor protein (TIRAP), phosphorylated interleukin-1 receptor-associated kinase 4 (p-IRAK-4), phosphorylated interleukin-1 receptor-associated kinase 1 (p-IRAK-1) and TNF receptor associated factor 6 (TRAF6) (p < 0.05), and the recruitments of TLR4 and MyD88 into lipid rafts in liver (p < 0.05). In LPS-stimulated macrophages, soyasaponins A2 or I significantly decreased MyD88 (p < 0.05), soyasaponins A1, A2 or I reduced p-IRAK-4 and p-IRAK-1 (p < 0.05), and soyasaponin I decreased TRAF6 (p < 0.05). In MyD88-overexpressed HEK293T cells, soyasaponins (A1, A2 or I) and ST2825 significantly decreased MyD88 and TRAF6 (p < 0.05). CONCLUSION: Soyasaponins can reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts. This study provides novel understanding about the anti-inflammatory mechanism of soyasaponins.


Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Fator 88 de Diferenciação Mieloide/metabolismo , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Receptor 4 Toll-Like/metabolismo , Animais , Modelos Animais de Doenças , Regulação para Baixo , Células HEK293 , Humanos , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ácido Oleanólico/farmacologia
13.
PLoS One ; 15(5): e0231980, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357188

RESUMO

Triterpenoids are high-value plant metabolites with numerous applications in medicine, agriculture, food, and home and personal care products. However, plants produce triterpenoids in low abundance, and their complex structures make their chemical synthesis prohibitively expensive and often impossible. As such, the yeast Saccharomyces cerevisiae has been explored as an alternative means of production. An important triterpenoid is oleanolic acid because it is the precursor to many bioactive triterpenoids of commercial interest, such as QS-21 which is being evaluated as a vaccine adjuvant in clinical trials against HIV and malaria. Oleanolic acid is derived from 2,3-oxidosqualene (natively produced by yeast) via a cyclisation and a multi-step oxidation reaction, catalysed by a ß-amyrin synthase and a cytochrome P450 of the CYP716A subfamily, respectively. Although many homologues have been characterised, previous studies have used arbitrarily chosen ß-amyrin synthases and CYP716As to produce oleanolic acid and its derivatives in yeast. This study presents the first comprehensive comparison of ß-amyrin synthase and CYP716A enzyme activities in yeast. Strains expressing different homologues are compared for production, revealing 6.3- and 4.5-fold differences in ß-amyrin and oleanolic acid productivities and varying CYP716A product profiles, which are important to consider when engineering strains for the production of bioactive oleanolic acid derivatives.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Transferases Intramoleculares/metabolismo , Ácido Oleanólico/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/análise , Plasmídeos/genética , Plasmídeos/metabolismo , Alinhamento de Sequência
14.
J Agric Food Chem ; 68(21): 5910-5916, 2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32351112

RESUMO

Erythrodiol, a typical pentacyclic triterpenic diol in olive oil and its byproduct, olive pomace, frequently appears in food additives for the prevention of cardiovascular diseases because of its antioxidation, anti-inflammatory, and antitumor activities. To develop new derivatives of erythrodiol (1), preparative biotransformations were investigated through Streptomyces griseus ATCC 13273, Penicilium griseofulvum CICC 40293, and Bacillus subtilis ATCC 6633, and ten new (1a-1j) and one known metabolites were isolated. Their structures were elucidated by high resolution electrospray ionization mass spectrometry (HR-ESI-MS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy. Furthermore, relative to 1, most metabolites exhibited lower toxicity and more potent inhibitory activities against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In particular, the glycosylated metabolite 1k showed a dramatically increased inhibitory effect with an IC50 value of 2.40 µM, which is even lower than that of quercetin. Thus, biotransformation of erythrodiol is a viable strategy for discovering new triterpenes as food supplements with anti-inflammatory properties.


Assuntos
Anti-Inflamatórios/metabolismo , Ácido Oleanólico/análogos & derivados , Penicillium/metabolismo , Streptomyces griseus/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Biotransformação , Suplementos Nutricionais/análise , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , Óxido Nítrico/imunologia , Ácido Oleanólico/química , Ácido Oleanólico/metabolismo , Ácido Oleanólico/farmacologia , Células RAW 264.7 , Espectrometria de Massas por Ionização por Electrospray
16.
Sci Rep ; 10(1): 6323, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286347

RESUMO

We screened some phytochemicals for cytotoxic activity to human cancer cells and identified Soyasapogenol-A (Snol-A) as a potent candidate anti-cancer compound. Interestingly, Soyasapogenin-I (Snin-I) was ineffective. Viability assays endorsed toxicity of Snol-A to a wide variety of cancer cells. Of note, wild type p53 deficient cancer cells (SKOV-3 and Saos-2) also showed potent growth inhibitory effect. Molecular analyses demonstrated that it targets CARF yielding transcriptional upregulation of p21WAF1 (an inhibitor of cyclin-dependent kinases) and downregulation of its effector proteins, CDK2, CDK-4, Cyclin A and Cyclin D1. Targeting of CARF by Snol-A also caused (i) downregulation of pATR-Chk1 signaling leading to caspase-mediated apoptosis and (ii) inactivation of ß-catenin/Vimentin/hnRNPK-mediated EMT signaling resulting in decrease in migration and invasion of cancer cells. In in vivo assays, Snol-A caused suppression of tumor growth in subcutaneous xenograft model and inhibited lung metastasis in tail vein injection model. Taken together, we demonstrate that Snol-A is a natural inhibitor of CARF and may be recruited as a potent anti-tumor and anti-metastasis compound for treatment of p53-deficient aggressive malignancies.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Ácido Oleanólico/análogos & derivados , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteína Supressora de Tumor p53/deficiência , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Invasividade Neoplásica/prevenção & controle , Ácido Oleanólico/farmacologia , Ácido Oleanólico/uso terapêutico , Proteínas de Ligação a RNA/metabolismo , Saponinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Sci Rep ; 10(1): 6560, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32300202

RESUMO

The tumor microenvironment (TME) is an essential contributor to the development and progression of malignancy. Within the TME, tumor associated macrophages (TAMs) mediate angiogenesis, metastasis, and immunosuppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ T cells. In previous work, we demonstrated that the synthetic triterpenoid CDDO-methyl ester (CDDO-Me) converts breast TAMs from a tumor-promoting to a tumor-inhibiting activation state in vitro. We show now that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of splenic CD4+ T cells were reduced, while the proportion of CD8+ T cells was significantly increased in both tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of CD4+ Foxp3+ regulatory T cells within tumors. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast TME and unleashes host adaptive anti-tumor immunity.


Assuntos
Neoplasias Mamárias Animais/patologia , Ácido Oleanólico/análogos & derivados , Receptores Estrogênicos/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Neoplasias Mamárias Animais/imunologia , Camundongos Endogâmicos C57BL , Ácido Oleanólico/farmacologia , Baço/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Microambiente Tumoral/imunologia
18.
Mini Rev Med Chem ; 20(3): 252-257, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32134368

RESUMO

BACKGROUND: Based on the biological significance of hederagenin-type saponins found in our previous investigation, a series of new hederagenin derivatives were designed and synthesized. METHODS: Their in vitro antiproliferative activities were evaluated against the HepG2 liver cancer cell line and normal cell line L929 by MTT assay. RESULTS: The preliminary bioassay results demonstrated that all the tested compounds 1-7 showed potent anti-hepatoma activities, and some compounds exhibited better effects than 5-fluorouracil against human hepatocellular carcinoma HepG2 cell line. Furthermore, compound 5 showed a significant antihepatoma activity against HepG2 cells with an IC50 value of 1.88 µM. Besides, all of the tested compounds showed a low cytotoxic effect against the normal cell line L929. CONCLUSION: All the compounds 1-7 displayed superior selectivity against human hepatocellular carcinoma HepG2 cell line, and the results suggest that the structural modifications of C ring on the hederagenin backbone are vital for modulating anti-hepatoma activities.


Assuntos
Antineoplásicos/síntese química , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Fígado/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Fígado/patologia , Estrutura Molecular , Ácido Oleanólico/síntese química , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Relação Estrutura-Atividade
19.
Cancer Res ; 80(9): 1819-1832, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32127355

RESUMO

RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFß/SMAD signaling. SMAD3 and its adaptors, such as ß2SP, are important mediators of TGFß signaling and regulate gene expression to suppress stem cell-like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/ß2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3 +/-), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFß1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients.Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFß in liver carcinogenesis.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação para Baixo , Deleção de Genes , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Fosforilação , RNA Interferente Pequeno , Proteínas Smad/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/deficiência , Proteína Smad3/genética , Espectrina/genética , Espectrina/metabolismo , Células-Tronco/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Regulação para Cima , Sequenciamento Completo do Exoma
20.
Molecules ; 25(5)2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32120971

RESUMO

Saponins are an important group found in Chenopodium quinoa. They represent an obstacle for the use of quinoa as food for humans and animal feeds because of their bitter taste and toxic effects, which necessitates their elimination. Several saponins elimination methods have been examined to leach the saponins from the quinoa seeds; the wet technique remains the most used at both laboratory and industrial levels. Dry methods (heat treatment, extrusion, roasting, or mechanical abrasion) and genetic methods have also been evaluated. The extraction of quinoa saponins can be carried out by several methods; conventional technologies such as maceration and Soxhlet are the most utilized methods. However, recent research has focused on technologies to improve the efficiency of extraction. At least 40 saponin structures from quinoa have been isolated in the past 30 years, the derived molecular entities essentially being phytolaccagenic, oleanolic and serjanic acids, hederagenin, 3ß,23,30 trihydroxy olean-12-en-28-oic acid, 3ß-hydroxy-27-oxo-olean-12en-28-oic acid, and 3ß,23,30 trihydroxy olean-12-en-28-oic acid. These metabolites exhibit a wide range of biological activities, such as molluscicidal, antifungal, anti-inflammatory, hemolytic, and cytotoxic properties.


Assuntos
Chenopodium quinoa/química , Saponinas/química , Saponinas/isolamento & purificação , Sementes/química , Extração em Fase Sólida/métodos , Anti-Inflamatórios/análise , Chenopodium quinoa/genética , Cromatografia Líquida de Alta Pressão , Temperatura Alta , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/análise , Extratos Vegetais/farmacologia , Saponinas/análise , Saponinas/genética , Sementes/genética , Sonicação/métodos , Água
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