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1.
Food Chem ; 307: 125558, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31644977

RESUMO

In this work, steam explosion (SE) was exploited as a green and facile process to deconstruct liquorice's structure and deglycosylate glycyrrhizic acid (GL) to improve conversion and diffusion efficacy of GL and its hydrolyzed products. Results showed SE induced auto-hydrolysis of GL into glycyrrhetic acid 3-O-mono-ß-D-glucuronide (GAMG) and glycyrrhetinic acid (GA), by which 30.71% of GL conversion, 5.24% and 21.47% of GAMG and GA formation were obtained. GL hydrolytic pathways were revealed by reaction kinetics and thermodynamics, which possessed complex consecutive and parallel reactions with endothermic, non-spontaneous and entropy-decreasing features. SE referred to cause cleavage of the ß-1,3 glycosidic bond in GL which was hydrolyzed to GA as a main product and GAMG and glucuronic acids as minor products. Diffusion of hydrolyzed products was accelerated by raising the diffusion coefficient and shortening the equilibrium time by over 90%. This work provides a sustainable and efficient route for product conversion and function enhancement of bioactive components.


Assuntos
Glycyrrhiza/química , Ácido Glicirrízico/metabolismo , Vapor , Ácido Glicirretínico/metabolismo , Temperatura Alta , Hidrólise , Cinética , Termodinâmica
2.
J Agric Food Chem ; 67(42): 11694-11702, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31558015

RESUMO

Sucrose synthase (SUS) plays an important role in carbohydrate metabolism in plants. The SUS genes in licorice remain unknown. To reveal the sucrose metabolic pathway in licorice, all the 12 putative SUS genes of Glycyrrhiza uralensis were systematically identified by genome mining, and two novel SUSs (GuSUS1 and GuSUS2) were isolated and characterized for the first time. Furthermore, we found that the flexible N-terminus was responsible for the low stability of plant SUSs, and deletion of redundant N-terminus improved the stability of GuSUS1 and GuSUS2. The half-life of both GuSUS1 and GuSUS2 mutants was increased by 2-fold. Finally, the GuSUS1 mutant was coupled with UGT73C11 for the glycosylation of glycyrrhetinic acid (GA) with uridine 5'-diphosphate disodium salt hydrate (UDP) in situ recycling, and GA conversion was increased by 7-fold. Our study not only identified the SUS genes in licorice but also provided a stable SUS mutant for the construction of an efficient UDP-recycling system for GA glycosylation.


Assuntos
Glucosiltransferases/metabolismo , Glycyrrhiza uralensis/enzimologia , Proteínas de Plantas/metabolismo , Difosfato de Uridina/metabolismo , Biocatálise , Glucosiltransferases/química , Glucosiltransferases/genética , Glicosilação , Ácido Glicirretínico/metabolismo , Glycyrrhiza uralensis/química , Glycyrrhiza uralensis/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Difosfato de Uridina/química
3.
J Sep Sci ; 42(15): 2534-2549, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31144455

RESUMO

A simple and sensitive liquid chromatography with tandem mass spectrometry method was developed for simultaneous quantification of paeoniflorin, albiflorin, oxypaeoniflorin, liquiritin, liquiritigenin, glycyrrhetinic acid, and glycyrrhizin in rat plasma after oral administration of Shaoyao-Gancao decoction, which is traditionally used in the treatment of polycystic ovary syndrome. The plasma samples were pretreated with methanol as precipitant. The method exhibited good linearity (correlation coefficient (R2 ) > 0.99) with lower quantification limits of 0.595-4.69 ng/mL for all analytes. Intra- and interbatch precision, accuracy, recovery, and stability of the method were all within accepted criteria. The results showed that the pharmacokinetic behaviors of the seven compounds were altered in the pathological status of polycystic ovary syndrome. Furthermore, a total of 36 metabolites were structurally identified based on their accurate masses and fragment ions. The major metabolic pathway involves phase I metabolic reactions (such as hydroxylation), phase II metabolic reactions (such as sulfation and glucuronidation conjugation) as well as the combined multiple-step metabolism. This study is the first report on the pharmacokinetic and metabolic information of Shaoyao-Gancao decoction in both normal and model rats, which would provide scientific evidences for the bioactive chemical basis of herbal medicines and also promote the clinical application of Shaoyao-Gancao decoction for treating polycystic ovary syndrome.


Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Síndrome do Ovário Policístico/tratamento farmacológico , Administração Oral , Animais , Hidrocarbonetos Aromáticos com Pontes/sangue , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/metabolismo , Medicamentos de Ervas Chinesas/farmacocinética , Feminino , Flavanonas/sangue , Flavanonas/metabolismo , Flavanonas/farmacocinética , Glucosídeos/sangue , Glucosídeos/metabolismo , Glucosídeos/farmacocinética , Ácido Glicirretínico/sangue , Ácido Glicirretínico/metabolismo , Ácido Glicirretínico/farmacocinética , Ácido Glicirrízico/sangue , Ácido Glicirrízico/metabolismo , Ácido Glicirrízico/farmacocinética , Monoterpenos/sangue , Monoterpenos/metabolismo , Monoterpenos/farmacocinética , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem
4.
Microb Cell Fact ; 18(1): 95, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138208

RESUMO

BACKGROUND: Glycyrrhetinic acid (GA) is the most important ingredient in licorice due to its outstanding anti-inflammatory activity and wide application in the medicine and cosmetics industries. Contemporary industrial production of GA by acid hydrolysis of glycyrrhizin which was extracted from Glycyrrhiza plants, is not environment-friendly and devastates farmland since the Glycyrrhiza rhizomes grow up to 10 m underground. RESULTS: In this study, GA was produced through metabolically engineering Saccharomyces cerevisiae by introducing the entire heterogeneous biosynthetic pathway of GA. Codon optimized CYP88D6 and CYP72A154, combined with ß-AS (ß-amyrin synthase encoding gene) and the NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana were introduced into S. cerevisiae. The resulting strain (Y1) produced 2.5 mg/L of ß-amyrin and 14 µg/L of GA. The cytochrome b5 from G. uralensis (GuCYB5) was identified and the introduction of this novel GuCYB5 increased the efficiency of GA production by eightfold. The joint utilization of the GuCYB5 gene along with 10 known MVA pathway genes from S. cerevisiae were overexpressed in a stable chromosome integration to achieve higher GA production. Using the combined strategy, GA concentration improved by 40-fold during batch fermentation. The production was further improved to 8.78 mg/L in fed-batch fermentation, which was increased by a factor of nearly 630. CONCLUSIONS: This study first investigated the influence of carbon flux in the upstream module and the introduction of a newly identified GuCYB5 on GA production. The newly identified GuCYB5 was highly effective in improving GA production. An integrated strategy including enzyme discovery, pathway optimization, and fusion protein construction was provided in improving GA production, achieving a 630 fold increase in GA production. The metabolically engineered yeast cell factories provide an alternative approach to glycyrrhetinic acid production, replacing the traditional method of plant extraction.


Assuntos
Vias Biossintéticas/genética , Ácido Glicirretínico/metabolismo , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados , Saccharomyces cerevisiae , Citocromos b5/genética , Fermentação , Transferases Intramoleculares/genética , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
5.
Planta Med ; 85(1): 56-61, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30086557

RESUMO

In this study, the biocatalysis of 18ß-glycyrrhetinic acid by two strains of filamentous fungi, namely Rhizopus arrhizus AS 3.2893 and Circinella muscae AS 3.2695, was investigated. Scaled-up biotransformation reactions yielded 14 metabolites. Their structures were established based on extensive nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry data analyses, and seven of them are new compounds. The two fungal strains exhibited distinct biocatalytic features. R. arrhizus could catalyze hydroxylation and carbonylation reactions, whereas C. muscae preferred to catalyze hydroxylation and glycosidation reactions. These highly specific reactions are difficult to achieve by chemical synthesis, particularly under mild conditions. Furthermore, we found that most of the metabolites exhibited pronounced inhibitory activities on lipopolysaccharides-induced nitric oxide production in RAW264.7 cells. These biotransformed derivatives of 18ß-glycyrrhetinic acid could be potential anti-inflammatory agents.


Assuntos
Anti-Inflamatórios/farmacologia , Ácido Glicirretínico/análogos & derivados , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Biotransformação , Catálise , Ácido Glicirretínico/química , Ácido Glicirretínico/metabolismo , Ácido Glicirretínico/farmacologia , Hidroxilação , Camundongos , Mucorales/metabolismo , Células RAW 264.7 , Rhizopus/metabolismo
6.
Biomed Chromatogr ; 33(3): e4449, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30513133

RESUMO

Xuanmai Ganjie Granules (XMGJ), a widely used Chinese herbal formula in the clinic, is used for treatment of sore throats and coughs. Despite the chemical constituents having been clarifying by our previous studies, both of the metabolism and pharmacokinetic studies of XMGJ are unclear. This study aimed to explore the disposition process of XMGJ in vivo. A sensitive and selective ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method was developed to analyze the absorbed components and metabolites in rat plasma and urine after oral administration of XMGJ. A total of 42 absorbed components, including 16 prototype compounds and 26 metabolites, were identified or tentatively characterized in rat plasma and urine after oral administration of XMGJ. Moreover, the pharmacokinetic studies of five compounds of XMGJ were investigated using ultra-high liquid chromatography with tandem mass spectrometry method. The results indicated that liquiritin, harpagoside, glycyrrhetic acid, liquiritigenin, formononetin and their metabolites might be the major components involved in the pharmacokinetic and metabolism process of XMGJ. This research showed a comprehensive investigation of XMGJ in vivo, which could provide a meaningful basis for further material basis and pharmacological as well as toxicological research.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas , Espectrometria de Massas em Tandem/métodos , Administração Oral , Animais , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/metabolismo , Medicamentos de Ervas Chinesas/farmacocinética , Flavonoides/sangue , Flavonoides/metabolismo , Flavonoides/farmacocinética , Flavonoides/urina , Glicosídeos/sangue , Glicosídeos/metabolismo , Glicosídeos/farmacocinética , Glicosídeos/urina , Ácido Glicirretínico/sangue , Ácido Glicirretínico/metabolismo , Ácido Glicirretínico/farmacocinética , Ácido Glicirretínico/urina , Limite de Detecção , Modelos Lineares , Metaboloma , Piranos/sangue , Piranos/metabolismo , Piranos/farmacocinética , Piranos/urina , Ratos , Reprodutibilidade dos Testes
7.
J Agric Food Chem ; 66(43): 11380-11389, 2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30296070

RESUMO

In this study, computation-aided design on the basis of structural analysis was employed to rationally identify a highly dynamic C-terminal region that regulates the stability, expression level, and activity of a GH2 fungal glucuronidase from Aspergillus oryzae Li-3 (PGUS). Then, four mutants with a precisely truncated C-terminal region in different lengths were constructed; among them, mutant D591-604 with a 3.8-fold increase in half-life at 65 °C and a 6.8 kJ/mol increase in Gibbs free energy showed obviously improved kinetic and thermodynamic stability in comparison to PGUS. Mutants D590-604 and D591-604 both showed approximately 2.4-fold increases in the catalytic efficiency kcat/ Km and 1.8-fold increases in the expression level. Additionally, the expression level of PGUS was doubled through a C-terminal region swap with bacterial GUS from E. coli (EGUS). Finally, the robust PGUS mutants D590-604 and D591-604 were applied in the preparation of glycyrrhetinic acid with 4.0- and 4.4-fold increases in concentration through glycyrrhizin hydrolysis by a fed-batch process.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Glucuronidase/metabolismo , Engenharia de Proteínas , Biotransformação , Biologia Computacional , Proteínas Fúngicas/genética , Glucuronidase/genética , Ácido Glicirretínico/metabolismo , Ácido Glicirrízico/metabolismo , Microbiologia Industrial , Simulação de Dinâmica Molecular , Proteínas Mutantes/metabolismo , Estrutura Terciária de Proteína
8.
Appl Microbiol Biotechnol ; 102(21): 9193-9205, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30109395

RESUMO

Glycyrrhizin (GL), the principal sweet-tasting bioactive ingredient of licorice (root of Glycyrrhiza glabra), shows poor oral absorption and gut microbial transformation of GL to glycyrrhetinic acid (GA) plays a major role for its multiple pharmacological effects. Co-administration of GL-hydrolyzing bacteria appears to be a feasible strategy to enhance GA exposure. This study reported a gut bacterial strain Staphylococcus pasteuri 3I10 which exhibited moderate p-nitrophenyl-ß-D-glucuronide (PNPG)-hydrolyzing activity but low GL deglucuronidation activity in its crude lysate. The gus gene encoding S. pasteuri 3I10 ß-glucuronidase was successfully cloned and overexpressed in Escherichia coli BL21(DE3). The purified ß-glucuronidase (SpasGUS) was 71 kDa and showed optimal pH and temperature at 6.0 and 50 °C, respectively. Comparing to E. coli ß-glucuronidase (EcoGUS), SpasGUS displayed lower velocity and affinity to PNPG hydrolysis (Vmax 16.1 ± 0.9 vs 140.0 ± 4.1 µmolmin-1 mg-1; Km 469.4 ± 73.4 vs 268.0 ± 25.8 µM), but could selectively convert GL to GA at much higher efficiency (Vmax 0.41 ± 0.011 vs 0.005 ± 0.002 µmolmin-1 mg-1; Km 116.9 ± 15.4 vs 53.4 ± 34.8 µM). Molecular docking studies suggested SpasGUS formed hydrogen bond interactions with the glucuronic acids at Asn414, Glu415 and Leu450, and Val159, Tyr475, Ala368, and Phe367 provided a hydrophobic environment for enhanced activity. Two special substrate interaction loops near the binding pocket of SpasGUS (loop 1 ß-glucuronidase) may account for the selective and efficient bioconversion of GL to GA, predicting that loop 1 ß-glucuronidases show high possibility in processing GL than mini-loop 1 and loop 2 ß-glucuronidases. These findings support potential applications of SpasGUS in cleaving GL to facilitate GA production in vivo or in pharmaceutical industry.


Assuntos
Glucuronidase/metabolismo , Ácido Glicirrízico/metabolismo , Staphylococcus/metabolismo , Escherichia coli/metabolismo , Microbioma Gastrointestinal/fisiologia , Glucuronatos/metabolismo , Glucuronídeos/metabolismo , Ácido Glicirretínico/metabolismo , Hidrólise , Intestinos/microbiologia , Simulação de Acoplamento Molecular/métodos
9.
Appl Environ Microbiol ; 84(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30054355

RESUMO

Glycyrrhetinic acid 3-O-mono-ß-d-glucuronide (GAMG), which possesses a higher sweetness and stronger pharmacological activity than those of glycyrrhizin (GL), can be obtained by removal of the distal glucuronic acid (GlcA) from GL. In this study, we isolated a ß-glucuronidase (TpGUS79A) from the filamentous fungus Talaromyces pinophilus Li-93 that can specifically and precisely convert GL to GAMG without the formation of the by-product glycyrrhetinic acid (GA) from the further hydrolysis of GAMG. First, TpGUS79A was purified and identified through matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF-TOF MS) and deglycosylation, indicating that TpGUS79A is a highly N-glycosylated monomeric protein with a molecular mass of around 85 kDa, including around 25 kDa of glycan moiety. The gene for TpGUS79A was then cloned and verified by heterologous expression in Pichia pastoris TpGUS79A belonged to glycoside hydrolase family 79 (GH79) but shared low amino acid sequence identity (<35%) with the available GH79 GUS enzymes. TpGUS79A had strict specificity toward the glycan moiety but poor specificity toward the aglycone moiety. Interestingly, TpGUS79A recognized and hydrolyzed the distal glucuronic bond of GL but could not cleave the glucuronic bond in GAMG. TpGUS79A showed a much higher catalytic efficiency on GL (kcat/Km of 11.14 mM-1 s-1) than on the artificial substrate pNP ß-glucopyranosiduronic acid (kcat/Km of 0.01 mM-1 s-1), which is different from the case for most GUSs. Homology modeling, substrate docking, and sequence alignment were employed to identify the key residues for substrate recognition. Finally, a fed-batch fermentation in a 150-liter fermentor was established to prepare GAMG through GL hydrolysis by T. pinophilus Li-93. Therefore, TpGUS79A is potentially a powerful biocatalyst for environmentally friendly and cost-effective production of GAMG.IMPORTANCE Compared to chemical methods, the biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid 3-O-mono-ß-d-glucuronide (GAMG), which has a higher sweetness and stronger pharmacological activity than those of GL, via catalysis by ß-glucuronidase is an environmentally friendly approach due to the mild reaction conditions and the high yield of GAMG. However, currently available GUSs show low substrate specificity toward GL and further hydrolyze GAMG to glycyrrhetinic acid (GA) as a by-product, increasing the difficulty of subsequent separation and purification. In the present study, we succeeded in isolating a novel ß-glucuronidase (named TpGUS79A) from Talaromyces pinophilus Li-93 that specifically hydrolyzes GL to GAMG without the formation of GA. TpGUS79A also shows higher activity on GL than those of the previously characterized GUSs. Moreover, the gene for TpGUS79A was cloned and its function verified by heterologous expression in P. pastoris Therefore, TpGUS79A can serve as a powerful biocatalyst for the cost-effective production of GAMG through GL transformation.


Assuntos
Proteínas Fúngicas/química , Glucuronidase/química , Glucuronidase/metabolismo , Glucuronídeos/metabolismo , Ácido Glicirretínico/metabolismo , Ácido Glicirrízico/metabolismo , Talaromyces/enzimologia , Biotransformação , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucuronidase/genética , Glucuronídeos/química , Ácido Glicirretínico/química , Ácido Glicirrízico/química , Hidrólise , Cinética , Estrutura Molecular , Especificidade por Substrato , Talaromyces/química , Talaromyces/genética , Talaromyces/metabolismo
10.
Arch Pharm Res ; 41(4): 409-418, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29532412

RESUMO

Hizikia fusiformis (Harvey) Okamura is a brown seaweed widely used in Korea and Japan, and it contains different therapeutically active constituents. In the present study, we investigated the activities of glycyrrhizin isolated from H. fusiformis, including its metabolites, 18α- and 18ß-glycyrrhetinic acid against Alzheimer's disease (AD) via acetyl and butyrylcholinesterase and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibition. Among these three compounds, 18ß-glycyrrhetinic acid (IC50 = 8.93 ± 0.69 µM) demonstrated two fold potent activity against BACE1 compared to the positive control, quercetin (IC50 = 20.18 ± 0.79 µM). Additionally, glycyrrhizin with an IC50 value of 20.12 ± 1.87 µM showed similarity to quercetin, while 18α-glycyrrhetinic acid showed moderate activity (IC50 = 104.35 ± 2.84 µM). A kinetic study revealed that glycyrrhizin and 18ß-glycyrrhetinic acid were non-competitive and competitive inhibitiors of BACE1, demonstrated via K i values of 16.92 and 10.91 µM, respectively. Molecular docking simulation studies evidently revealed strong binding energy of these compounds for BACE1, indicating their high affinity and capacity for tighter binding to the active site of the enzyme. These data suggest that glycyrrhizin isolated from the edible seaweed, H. fusiformis and its metabolite, 18ß-glycyrrhetinic acid demonstrated selective inhibitory activity against BACE1 to alleviate AD.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Simulação por Computador , Ácido Glicirretínico/análogos & derivados , Ácido Glicirrízico/farmacologia , Simulação de Acoplamento Molecular/métodos , Alga Marinha , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Electrophorus , Ácido Glicirretínico/isolamento & purificação , Ácido Glicirretínico/metabolismo , Ácido Glicirretínico/farmacologia , Ácido Glicirrízico/isolamento & purificação , Ácido Glicirrízico/metabolismo , Cavalos , Humanos
11.
Bioorg Chem ; 78: 210-219, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29602045

RESUMO

We present an efficient approach to the synthesis of a series of glycyrrhetinic acid derivatives. Six derivatives, five of them new compounds, were obtained through chemoenzymatic reactions in very good to excellent yield. In order to find the optimal reaction conditions, the influence of various parameters such as enzyme source, nucleophile:substrate ratio, enzyme:substrate ratio, solvent and temperature was studied. The excellent results obtained by lipase catalysis made the procedure very efficient considering their advantages such as mild reaction conditions and low environmental impact. Moreover, in order to explain the reactivity of glycyrrhetinic acid and the acetylated derivative to different nucleophiles in the enzymatic reactions, molecular docking studies were carried out. In addition, one of the synthesized compounds exhibited remarkable antiviral activity against TK + and TK- strains of Herpes simplex virus type 1 (HSV-1), sensitive and resistant to acyclovir (ACV) treatment.


Assuntos
Antivirais/farmacologia , Ácido Glicirretínico/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Lipase/metabolismo , Simulação de Acoplamento Molecular , Aciclovir/farmacologia , Antivirais/química , Antivirais/metabolismo , Biocatálise , Candida/enzimologia , Carica/enzimologia , Relação Dose-Resposta a Droga , Eurotiales/enzimologia , Ácido Glicirretínico/química , Ácido Glicirretínico/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Rhizomucor/enzimologia , Rhizopus/enzimologia , Relação Estrutura-Atividade
12.
Metab Eng ; 45: 43-50, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29196123

RESUMO

Glycyrrhetinic acid (GA) and its precursor, 11-oxo-ß-amyrin, are typical triterpenoids found in the roots of licorice, a traditional Chinese medicinal herb that exhibits diverse functions and physiological effects. In this study, we developed a novel and highly efficient pathway for the synthesis of GA and 11-oxo-ß-amyrin in Saccharomyces cerevisiae by introducing efficient cytochrome P450s (CYP450s: Uni25647 and CYP72A63) and pairing their reduction systems from legume plants through transcriptome and genome-wide screening and identification. By increasing the copy number of Uni25647 and pairing cytochrome P450 reductases (CPRs) from various plant sources, the titers of 11-oxo-ß-amyrin and GA were increased to 108.1 ± 4.6mg/L and 18.9 ± 2.0mg/L, which were nearly 1422-fold and 946.5-fold higher, respectively, compared with previously reported data. To the best of our knowledge, these are the highest titers reported for GA and 11-oxo-ß-amyrin from S. cerevisiae, indicating an encouraging and promising approach for obtaining increased GA and its related triterpenoids without destroying the licorice plant or the soil ecosystem.


Assuntos
Sistema Enzimático do Citocromo P-450 , Fabaceae/genética , Ácido Glicirretínico/metabolismo , Ácido Oleanólico/análogos & derivados , Proteínas de Plantas , Saccharomyces cerevisiae , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Fabaceae/enzimologia , Ácido Oleanólico/biossíntese , Ácido Oleanólico/genética , Oxirredução , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
13.
J Biol Chem ; 293(2): 433-443, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29146597

RESUMO

Glycoside hydrolases (GHs) have attracted special attention in research aimed at modifying natural products by partial removal of sugar moieties to manipulate their solubility and efficacy. However, these modifications are challenging to control because the low substrate specificity of most GHs often generates undesired by-products. We previously identified a GH2-type fungal ß-glucuronidase from Aspergillus oryzae (PGUS) exhibiting promiscuous substrate specificity in hydrolysis of triterpenoid saponins. Here, we present the PGUS structure, representing the first structure of a fungal ß-glucuronidase, and that of an inactive PGUS mutant in complex with the native substrate glycyrrhetic acid 3-O-mono-ß-glucuronide (GAMG). PGUS displayed a homotetramer structure with each monomer comprising three distinct domains: a sugar-binding, an immunoglobulin-like ß-sandwich, and a TIM barrel domain. Two catalytic residues, Glu414 and Glu505, acted as acid/base and nucleophile, respectively. Structural and mutational analyses indicated that the GAMG glycan moiety is recognized by polar interactions with nine residues (Asp162, His332, Asp414, Tyr469, Tyr473, Asp505, Arg563, Asn567, and Lys569) and that the aglycone moiety is recognized by aromatic stacking and by a π interaction with the four aromatic residues Tyr469, Phe470, Trp472, and Tyr473 Finally, structure-guided mutagenesis to precisely manipulate PGUS substrate specificity in the biotransformation of glycyrrhizin into GAMG revealed that two amino acids, Ala365 and Arg563, are critical for substrate specificity. Moreover, we obtained several mutants with dramatically improved GAMG yield (>95%). Structural analysis suggested that modulating the interaction of ß-glucuronidase simultaneously toward glycan and aglycone moieties is critical for tuning its substrate specificity toward triterpenoid saponins.


Assuntos
Glucuronidase/metabolismo , Glicosídeo Hidrolases/metabolismo , Saponinas/metabolismo , Triterpenos/metabolismo , Glucuronidase/genética , Glicosídeo Hidrolases/genética , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/metabolismo , Engenharia de Proteínas , Especificidade por Substrato
14.
Life Sci ; 188: 186-191, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28768154

RESUMO

Glycyrrhetinic acid (GA) is a natural active component from licorice, which is broadly used in traditional Chinese medicine. Lots of glycyrrhetinic acid receptors (GA-R) are proved to locate on the surface of liver cells. Many reports about the hepatocellular carcinoma (HCC) treatment were dependent on GA modified carriers. However, the reality of GA-R in HCC cells was not clear. In this paper, 18ß-glycyrrhetinic acid (18ß-GA) was labeled with fluorescence (FITC) by chemical synthesis. Together with the binding effect of fluorescence labeled glycyrrhetinic acid (FITC-GA), the competitive action of 18ß-GA with GA-R was investigated in HCC cells. The results showed that in HepG2 cells, 18ß-GA and FITC-GA presented similar cytotoxicity. The specific binding saturation of GA showed the dissociation constant (Kd) was 7.457±2.122pmol/L and the maximum binding counts (Bmax) was 2.385±0.175pmol/2.5×106 cells, respectively. FITC-GA bound to cytomembrane specifically and 18ß-GA competed to bind the sites significantly in HepG2 cells. Therefore, there is binding effect between fluorescence labeled GA and GA-R. The GA-R on HCC cells is confirmed as expected, which provides a useful reference of active target modified by GA and a novel approach for receptors and ligands study.


Assuntos
Carcinoma Hepatocelular/metabolismo , Ácido Glicirretínico/análogos & derivados , Ligantes , Apoptose/efeitos dos fármacos , Ligação Competitiva , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacologia , Ácido Glicirretínico/química , Ácido Glicirretínico/metabolismo , Ácido Glicirretínico/farmacologia , Humanos
15.
Drug Metab Pharmacokinet ; 32(4): 218-223, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28754329

RESUMO

Glycyrrhetinic acid (GA) is an active metabolite of glycyrrhizin, which is a main constituent in licorice (Glycyrrhiza glabra). While GA exhibits a wide variety of pharmacological activities in the body, it is converted to a toxic metabolite GA 3-O-glucuronide by hepatic UDP-glucuronosyltransferases (UGTs). To avoid the development of the toxic metabolite-induced pseudohyperaldosteronism (pseudoaldosteronism), there is a limitation in maximum daily dosage of licorice and in combined usage of other glycyrrhizin-containing natural medicine. In this study, we investigated the inhibitory effects of various polyphenols and triterpenoids on the UGT-mediated GA 3-O-glucuronidation. In human liver microsomes, UGT-mediated GA glucuronidation was significantly inhibited by protopanaxadiol with an IC50 value of 59.2 µM. Isoliquiritigenin, rosmarinic acid, alisol B, alisol acetate, and catechin moderately inhibited the GA glucuronidation with IC50 values of 96.4 µM, 125 µM, 160 µM, 163 µM, and 164 µM. Other tested 19 polyphenols and triterpenoids, including liquiritigenin, did not inhibit UGT-mediated GA glucuronidation in human liver microsomes. Our data indicate that relatively higher dosage of licorice can be used without a risk of developing pseudohyperaldosteronism in combination of natural medicine containing protopanaxadiol such as Panax ginseng. Furthermore, supplemental protopanaxadiol and isoliquiritigenin might be useful in preventing licorice-inducing pseudoaldosteronism.


Assuntos
Inibidores Enzimáticos/farmacologia , Glucuronídeos/metabolismo , Glucuronosiltransferase/antagonistas & inibidores , Ácido Glicirretínico/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Polifenóis/farmacologia , Triterpenos/farmacologia , Relação Dose-Resposta a Droga , Glucuronosiltransferase/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade
16.
Int J Biol Macromol ; 102: 92-103, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28377238

RESUMO

In this research, the interactions of glycyrrhizin (GL) and glycyrrhetinic acid (GA) with bovine serum albumin (BSA) have been investigated by the novel method of spectrophotometric- gradient flow injection titration technique. The hard-modeling multivariate approach to binding was used for calculation of binding constants and estimation of concentration-spectral profiles of equilibrium species. The stoichiometric ratio of binding was estimated using eigenvalue analysis. Results showed that GL and GA bind BSA with overall binding constants of KGL-BSA=3.85 (±0.09)×104Lmol-1, KGA-BSA=3.08 (±0.08)×104Lmol-1. Ligand-BSA complexes were further analyzed by combined docking and molecular dynamics (MD) simulations. Docking simulations were performed to obtain a first guess on the binding structure of the GL/GA-BSA complex, and subsequently analyzed by 20 ns MD simulations in order to evaluate interactions of GL/GA with BSA in detail. Results of MD simulations indicated that GL-BSA complex forms mainly on the basis of hydrogen bonds, while, GA-BSA complex forms on the basis of hydrophobic interactions. Also, water molecules can bridge between the ligand and protein by hydrogen bonds, which are stable during the entire simulation and play an important role in stabilization of the GL/GA-BSA complexes.


Assuntos
Ácido Glicirretínico/metabolismo , Ácido Glicirrízico/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Análise de Injeção de Fluxo , Ligação Proteica , Conformação Proteica , Soroalbumina Bovina/química , Espectrofotometria
17.
New Phytol ; 212(1): 123-35, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27252088

RESUMO

Glycyrrhizin is an important bioactive compound that is used clinically to treat chronic hepatitis and is also used as a sweetener world-wide. However, the key UDP-dependent glucuronosyltransferases (UGATs) involved in the biosynthesis of glycyrrhizin remain unknown. To discover unknown UGATs, we fully annotated potential UGATs from Glycyrrhiza uralensis using deep transcriptome sequencing. The catalytic functions of candidate UGATs were determined by an in vitro enzyme assay. Systematically screening 434 potential UGATs, we unexpectedly found one unique GuUGAT that was able to catalyse the glucuronosylation of glycyrrhetinic acid to directly yield glycyrrhizin via continuous two-step glucuronosylation. Expression analysis further confirmed the key role of GuUGAT in the biosynthesis of glycyrrhizin. Site-directed mutagenesis revealed that Gln-352 may be important for the initial step of glucuronosylation, and His-22, Trp-370, Glu-375 and Gln-392 may be important residues for the second step of glucuronosylation. Notably, the ability of GuUGAT to catalyse a continuous two-step glucuronosylation reaction was determined to be unprecedented among known glycosyltransferases of bioactive plant natural products. Our findings increase the understanding of traditional glycosyltransferases and pave the way for the complete biosynthesis of glycyrrhizin.


Assuntos
Biocatálise , Glucuronosiltransferase/metabolismo , Ácido Glicirretínico/metabolismo , Ácido Glicirrízico/metabolismo , Glicosilação , Ácido Glicirretínico/química , Glycyrrhiza/enzimologia , Glycyrrhiza/genética , Ácido Glicirrízico/química , Sequenciamento de Nucleotídeos em Larga Escala , Simulação de Acoplamento Molecular , Anotação de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Raízes de Plantas/genética , Transcriptoma/genética , Difosfato de Uridina/metabolismo
18.
Exp Anim ; 65(4): 353-361, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27245118

RESUMO

We examined the effect of Yokukansankachimpihange (YKSCH), a form of Yokukansan containing parts of two herbaceous plants, Citrus Unshiu Peel (Chimpi) and Pinellia Tuber (Hange), on aggressive behavior of mice housed individually. Mice were fed a zinc-deficient diet for 2 weeks. In a resident-intruder test, the cumulative duration of aggressive behavior was decreased in zinc-deficient mice administrated drinking water containing YKSCH (approximately 300 mg/kg body weight/day) for 2 weeks. We tested mice for geissoschizine methyl ether (GM), which is contained in Uncaria Hook, and 18ß-glycyrrhetinic acid (GA), a major metabolite of glycyrrhizin contained in Glycyrrhiza, which were contained in YKS and YKSCH. In hippocampal slices from zinc-deficient rats, excess exocytosis at mossy fiber boutons induced with 60 mM KCl was attenuated in the presence of GA (100-500 µM) or GM (100 µM). The intracellular Ca2+ level, which showed an increase induced by 60 mM KCl, was also attenuated in the presence of GA (100-500 µM) or GM (100 µM). These results suggest that GA and GM ameliorate excess glutamate release from mossy fiber boutons by suppressing the increase in intracellular Ca2+ signaling. These ameliorative actions may contribute to decreasing the aggressiveness of mice individually housed under zinc deficiency, potentially by suppressing excess glutamatergic neuron activity in the hippocampus.


Assuntos
Agressão/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Exocitose/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Zinco/deficiência , Animais , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/metabolismo , Hipocampo/química , Alcaloides Indólicos/metabolismo , Masculino , Camundongos , Ratos , Ratos Wistar
19.
Antioxid Redox Signal ; 25(16): 855-869, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-26886723

RESUMO

AIMS: Proteasomes are constituents of the cellular proteolytic networks that maintain protein homeostasis through regulated proteolysis of normal and abnormal (in any way) proteins. Genetically mediated proteasome activation in multicellular organisms has been shown to promote longevity and to exert protein antiaggregation activity. In this study, we investigate whether compound-mediated proteasome activation is feasible in a multicellular organism and we dissect the effects of such approach in aging and Alzheimer's disease (AD) progression. RESULTS: Feeding of wild-type Caenorhabditis elegans with 18α-glycyrrhetinic acid (18α-GA; a previously shown proteasome activator in cell culture) results in enhanced levels of proteasome activities that lead to a skinhead-1- and proteasome activation-dependent life span extension. The elevated proteasome function confers lower paralysis rates in various AD nematode models accompanied by decreased Aß deposits, thus ultimately decelerating the progression of AD phenotype. More importantly, similar positive results are also delivered when human and murine cells of nervous origin are subjected to 18α-GA treatment. INNOVATION: This is the first report of the use of 18α-GA, a diet-derived compound as prolongevity and antiaggregation factor in the context of a multicellular organism. CONCLUSION: Our results suggest that proteasome activation with downstream positive outcomes on aging and AD, an aggregation-related disease, is feasible in a nongenetic manipulation manner in a multicellular organism. Moreover, they unveil the need for identification of antiaging and antiamyloidogenic compounds among the nutrients found in our normal diet. Antioxid. Redox Signal. 25, 855-869.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Ácido Glicirretínico/análogos & derivados , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Morte Celular , Células Cultivadas , Progressão da Doença , Ácido Glicirretínico/metabolismo , Ácido Glicirretínico/farmacologia , Longevidade , Neurônios/efeitos dos fármacos , Oxirredução , Fenótipo , Agregação Patológica de Proteínas/metabolismo
20.
J Enzyme Inhib Med Chem ; 31(6): 1457-63, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26899462

RESUMO

A series of hybrids, which are composed of glycyrrhetic acid (GA) and slowly hydrogen sulfide-releasing donor ADT-OH, were designed and synthesized to develop anticancer and anti-inflammatory agents. Most of the compounds, whose inhibitory rates were comparable to or higher than those of GA and aspirin, respectively, significantly inhibited xylene-induced ear edema in mice. Especially, compound V4 exhibited the most potent inhibitory rate of 60.7%. Furthermore, preliminary structure-activity relationship studies demonstrated that 3-substituted GA derivatives had stronger anti-inflammatory activities than the corresponding 3-unsubstituted GA derivatives. In addition, anti-proliferative activities of compounds V1-9 were evaluated in three different human cancer cell lines. Compound V4 showed the most high potency against all three tumor cell lines with IC50 values ranging from 10.01 µM in Hep G2 cells to 17.8 µM in MDA-MB-231 cells, which were superior to positive GA.


Assuntos
Ácido Glicirretínico/metabolismo , Sulfeto de Hidrogênio/metabolismo , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Ácido Glicirretínico/análogos & derivados , Humanos , Camundongos , Análise Espectral/métodos
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