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1.
Phytochemistry ; 171: 112236, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31923723

RESUMO

Glycyrrhiza glabra (licorice) is a medicinal plant with valuable specialised metabolites such as triterpene sweetener glycyrrhizin. Salinity stress is the main environmental stress limiting plant growth and development. The effects of six levels of NaCl (0, 100, 200, 400, 600, and 800 mM) on growth, osmolyte content, oxidative stress markers, antioxidant enzyme activities, K+/Na+ ratio, glycyrrhizin content, and gene expression of glycyrrhizin biosynthesis (bAS, CYP88D6, and CYP72A154) were investigated in licorice rhizomes of two populations. The results showed that the salt stress progressively reduced the growth parameters and increased the proline concentrations in the rhizomes. K+/Na+ ratio showed a significant decrease under salinity as compared to the controls. Salt stress resulted in oxidative stress on the rhizomes, as indicated by increased lipid peroxidation and hydrogen peroxide concentrations and elevated the activities of antioxidant enzymes (i.e., ascorbate peroxidase and superoxide dismutase). The glycyrrhizin content increased only under 100 and 200 mM NaCl treatments. The same trend was observed in the expression of bAS, CYP88D6, and CYP72A154 genes in Fars population. Fars population was found to have more glycyrrhizin content than Khorasan population. But, growth, glycyrrhizin content, and biosynthesis genes of glycyrrhizin showed more reduction in Khorasan population as compared to those of Fars population. The results indicate that the application of 100 mM NaCl up-regulated the expression of key genes involved in the biosynthesis of triterpenoid saponins and directly enhanced the production of glycyrrhizin. Accordingly, G. glabra can be introduced as a halophyte plant.


Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glycyrrhiza/efeitos dos fármacos , Ácido Glicirrízico/metabolismo , Compostos Fitoquímicos/biossíntese , Cloreto de Sódio/farmacologia , Química Física , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica de Plantas/genética , Glycyrrhiza/química , Glycyrrhiza/genética , Ácido Glicirrízico/química , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Compostos Fitoquímicos/química , Salinidade , Cloreto de Sódio/química
2.
J Agric Food Chem ; 68(5): 1480-1493, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31899641

RESUMO

Licorice (Glycyrrhiza uralensis Fisch) possesses a substantial share of the global markets for its unique sweet flavor and diverse pharmacological compounds. Cultivated licorice is widely distributed in northwest regions of China, covered with land with a broad range of salinities. A preliminary study indicated that suitable salt stress significantly increased the content of bioactive constituents in licorice. However, the molecular mechanisms underlying the influence of salinity on the accumulation of these constituents remain unclear, which hinders quality breeding of cultivated licorice. In our study, flavonoid-related structural genes were obtained, and most of them, such as phenylalanine ammonia-lyases, cinnamate 4-hydroxylases, 4-coumarate: CoA ligases, chalcone synthases, chalcone-flavanone isomerase, and flavonol synthase, showed high levels after salt treatment. In the biosynthesis of glycyrrhizin, three key enzymes (bAS, CYP88D6, and CYP72A154) were identified as differentially expressed proteins and remarkably upregulated in the salt-stressed group. Combining these results with the contents of 14 bioactive constituents, we also found that the expression patterns of those structural proteins were logically consistent with changes in bioactive constituent profiles. Thus, we believe that suitable salt stress increased the accumulation of bioactive constituents in licorice by upregulating proteins involved in the related biosynthesis pathways. This work provided valuable proteomic information for unraveling the molecular mechanism of flavonoid and glycyrrhizin metabolism and offered fundamental resources for quality breeding in licorice.


Assuntos
Glycyrrhiza uralensis/química , Extratos Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Cloreto de Sódio/metabolismo , Flavonoides/metabolismo , Glycyrrhiza uralensis/genética , Glycyrrhiza uralensis/metabolismo , Ácido Glicirrízico/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raízes de Plantas/química , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Proteômica , Estresse Salino
3.
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
4.
Environ Sci Pollut Res Int ; 26(34): 34924-34930, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31659708

RESUMO

Atrazine (ATR) is a common herbicide used worldwide. It is a potent endocrine disruptor that causes hormonal imbalance. We investigated the modulatory role predisposed by glycyrrhizic acid (GA) against the hazardous effects caused by the ATR in the rabbit spleen. Sixty rabbits were assigned into 4 groups. The first group is the negative control; the ATR group received 1/10 of the oral LD 50 ATR; the GA group received 50 mg/kg body weight daily intraproteinally; and group 4 received both ATR and GA concurrently. ATR and GA administrations were done for 60 days. ATR-induced humoral immunotoxicity was illustrated by decreased serum total protein, albumin, and globulin levels and rabbit hemorrhagic disease virus antibody titer, 4 weeks after vaccination. Moreover, upregulation of spleen Fas and caspase-III genes was recorded in ATR-exposed rabbits. Clear splenocyte apoptosis was observed in the immunohistochemical examination by the caspase-III technique. GA diminished the ATR-induced splenocyte apoptosis through downregulation of Fas and caspase-III expressions. In conclusion, our findings bounced a new perspective into the mechanism by which ATR induces immunotoxicity and assumed the potential modulatory role of GA.


Assuntos
Atrazina/toxicidade , Ácido Glicirrízico/metabolismo , Herbicidas/toxicidade , Baço/fisiologia , Animais , Apoptose/efeitos dos fármacos , Atrazina/metabolismo , Caspases/metabolismo , Herbicidas/metabolismo , Baço/metabolismo
5.
Molecules ; 24(20)2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614687

RESUMO

The demand for licorice and its natural product derivatives in domestic and foreign market is considerably huge. The core production areas of licorice are covered with salinity and drought land in northwestern China. Studies have shown that suitable environmental stress can promote the accumulation of glycyrrhizin and liquiritin to improve its quality as medicinal materials. However, there are few reports on other bioactive constituents of licorice, not to mention their dynamic accumulation under stressed conditions. To explore the quality formation of licorice from the perspective of salt influence, a reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-MS/MS) was established for simultaneous determination of sixteen bioactive constituents, including triterpenoids, flavonoids, chalcones and their glycosides. Physiological experiments were performed to investigate salt tolerance of licorice under different salinity treatments. The expressions of crucial genes (bAS and CHS), key enzymes of triterpenoid and flavonoid synthesis, were also tested by qRT-PCR. Our study found that 50 mM NaCl treatment (low stress) was the most favorable to promote the accumulation of bioactive constituents in the long term, without harming the plants. Flavonoid accumulation of non-stressed and low-stressed groups became different in the initial synthesis stage, and glycosyltransferases may have great influence on their downstream synthesis. Furthermore, bAS and CHS also showed higher levels in low-stressed licorice at harvest time. This work provides valuable information on dynamic variations in multiple bioactive constituents in licorice treated by salt and insight into its quality formation under stressed conditions.


Assuntos
Medicamentos de Ervas Chinesas/química , Flavonoides/química , Glycyrrhiza/química , Extratos Vegetais/química , Chalconas/química , Chalconas/metabolismo , Cromatografia Líquida , Medicamentos de Ervas Chinesas/metabolismo , Flavanonas/química , Flavanonas/metabolismo , Flavonoides/metabolismo , Glucosídeos/química , Glucosídeos/metabolismo , Ácido Glicirrízico/química , Ácido Glicirrízico/metabolismo , Humanos , Extratos Vegetais/metabolismo , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Folhas de Planta/metabolismo , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Estresse Salino , Espectrometria de Massas em Tandem , Triterpenos/química , Triterpenos/metabolismo
6.
Molecules ; 24(9)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086079

RESUMO

As calcium signal sensors, calcium-dependent protein kinases (CPKs) play vital roles in stimulating the production of secondary metabolites to participate in plant development and response to environmental stress. However, investigations of the Glycyrrhiza uralensis CPK family genes and their multiple functions are rarely reported. In this study, a total of 23 GuCPK genes in G. uralensis were identified, and their phylogenetic relationships, evolutionary characteristics, gene structure, motif distribution, and promoter cis-acting elements were analyzed. Ten GuCPKs showed root-specific preferential expressions, and GuCPKs indicated different expression patterns under treatments of CaCl2 and NaCl. In addition, under 2.5 mM of CaCl2 and 30 mM of NaCl treatments, the diverse, induced expression of GuCPKs and significant accumulations of glycyrrhizic acid and flavonoids suggested the possible important function of GuCPKs in regulating the production of glycyrrhizic acid and flavonoids. Our results provide a genome-wide characterization of CPK family genes in G. uralensis, and serve as a foundation for understanding the potential function and regulatory mechanism of GuCPKs in promoting the biosynthesis of glycyrrhizic acid and flavonoids under salt stress.


Assuntos
Flavonoides/metabolismo , Glycyrrhiza uralensis/efeitos dos fármacos , Glycyrrhiza uralensis/metabolismo , Ácido Glicirrízico/metabolismo , Proteínas Quinases/metabolismo , Cloreto de Cálcio/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glycyrrhiza uralensis/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas Quinases/genética , Estresse Salino , Cloreto de Sódio/farmacologia
7.
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
8.
Appl Microbiol Biotechnol ; 103(12): 4813-4823, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31055652

RESUMO

In this study, we aimed to shift the optimal pH of acidic ß-glucuronidase from Aspergillus oryzae Li-3 (PGUS) to the neutral region by site-directed mutagenesis, thus allowing high efficient biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid (GA) under higher pH where the solubility of GL could be greatly enhanced. Based on PGUS structure analysis, five critical aspartic acid and glutamic acid residues were replaced with arginine on the surface to generate a variant 5Rs with optimal pH shifting from 4.5 to 6.5. The catalytic efficiency (kcat /Km) value of 5Rs at pH 6.5 was 10.7-fold higher than that of PGUS wild-type at pH 6.5, even 1.4-fold higher than that of wild-type at pH 4.5. Molecular dynamics simulation was performed to explore the molecular mechanism for the shifted pH profile and enhanced pH stability of 5Rs.


Assuntos
Aspergillus oryzae/metabolismo , Glucuronidase/metabolismo , Ácido Glicirrízico/metabolismo , Mutagênese Sítio-Dirigida , Ácido Aspártico/metabolismo , Biotransformação , Catálise , Glucuronidase/genética , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Simulação de Dinâmica Molecular
9.
Protoplasma ; 256(3): 827-837, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30623261

RESUMO

Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoid and phenolic families. The aim of this study is to evaluate the effect of salinity stress on the expression of key genes involved in the biosynthetic pathway of triterpenoids such as glycyrrhizin, betulinic acid, soyasaponins, and phytosterols in licorice root, as well as providing a phonemic platform to characterize antioxidant properties, glycyrrhizin, and total phenolic content. This study also includes measuring the gene expression level and glycyrrhizin content in leaves and roots of control plants. The studied genes included squalene synthase (SQS1 and SQS2), ß-amyrin synthase (bAS), lupeol synthase (LUS), cycloartenol synthase (CAS), ß-amyrin 11-oxidase (CYP88D6), and ß-amyrin 24-hydroxylase (CYP93E6). Our results revealed that all of the mentioned genes were upregulated following the stress condition with different transcription rates. The highest increase (12-fold) was observed for the expression of the LUS gene, which is related to the betulinic acid pathway. Also, the highest content of glycyrrhizin was observed at 72 h post-treatment, which was consistent with the upregulated transcription levels of the glycyrrhizin pathway genes especially SQS1 and CYP88D6 at the same time. Correlation and stepwise regression analysis proved the key role of SQS1 gene in the biosynthetic pathway of glycyrrhizin. Antioxidant activity and phenolic content also were increased following stress condition. A comparison between the expression levels of SQS1 and other genes involved in the production of glycyrrhizin, phytosterols, and soyasaponins revealed a similar transcription trend, which shows the gene expression in the roots was significantly higher than the leaves. In contrast, SQS2 and LUS genes displayed a higher expression in leaf tissues. The genes related to betulinic acid biosynthetic pathway exhibited an expression rate different from other triterpenoid pathway genes, which could be observed in the leaves and roots of control plants and the roots of salt-treated plants. Furthermore, results showed that these two SQS genes have different expression rates due to different plant tissues (roots and leaves) and stress conditions. Importantly, in contrast to previous reports, we detected the glycyrrhizin in leaf tissues. This result may indicate the presence of a different genetic background in native Iranian licorice germplasm.


Assuntos
Regulação da Expressão Gênica de Plantas , Glycyrrhiza/genética , Glycyrrhiza/metabolismo , Compostos Fitoquímicos/metabolismo , Estresse Salino/genética , Triterpenos/metabolismo , Antioxidantes/metabolismo , Ácido Glicirrízico/metabolismo , Fenóis/análise , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Regressão
10.
Int J Pharm ; 559: 271-279, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30690130

RESUMO

Glycyrrhizic acid is the main active component of Licorice root which has been known in traditional Chinese and Japanese medicine since ancient times. In these cultures glycyrrhizic acid (GA) is one of the most frequently used drugs. However, only in 21-st century a novel unusual property of the GA to enhance the activity of other drugs has been discovered. The review describes briefly the experimental evidences of wide spectrum of own biological activities of glycyrrhizic acid as well as discusses the possible mechanisms of the ability of GA to enhance the activity of other drugs. We have shown that due to its amphiphilic nature GA is able to form self-associates in aqueous and non-aqueous media, as well as water soluble complexes with a wide range of lipophilic drugs. The main purpose of our review is to focus reader's attention on physicochemical studies of the molecular mechanisms of GA activity as a drug delivery system (DDS). In our opinion, the most intriguing feature of glycyrrhizic acid which might be the key factor in its therapeutic activity is the ability of GA to incorporate into the lipid bilayer and to increase the membrane fluidity and permeability. The ability of biomolecules and their aggregates to change the properties of cell membranes is of great significance, from both fundamental and practical points of view.


Assuntos
Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Ácido Glicirrízico/química , Ácido Glicirrízico/metabolismo , Animais , Membrana Celular/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Bicamadas Lipídicas/metabolismo , Permeabilidade/efeitos dos fármacos
11.
Biomed Chromatogr ; 33(3): e4421, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30362136

RESUMO

Diarrhoeal diseases alter the composition of intestinal flora, thereby affecting the efficacy of herbal medicinal formulations. Gegen Qinlian decoction (GQD), a Chinese traditional herbal formulation, is widely used to treat infectious diarrhoea. However, little is known about the microbial disposition of GQD in the diarrhoeal state. In this study, the comparative metabolism of components of GQD by diarrhoeal and normal intestinal flora was investigated in vitro. UPLC-MS/MS was performed for simultaneous analysis of eight ingredients of GQD in bacterial solution. The type, activities, and sources of microbial enzymes were also investigated. Microbial metabolism of daidzin, genistin and liquiritin (metabolized by ß-glucosidase); baicalin, wogonoside and glycyrrhizin (metabolized by ß-glucuronidase); and berberine and coptisine (metabolized via nitroreductase) was faster in the diarrhoeal group than in the normal group. Moreover, the activities of these enzymes in the diarrhoeal group were higher than those in the normal group. This difference might be associated with the increase in Escherichia spp. Thus, a change in the metabolism of components by diarrhoeal intestinal flora is associated with a preponderance of Escherichia spp., which might improve the efficacy of GQD. These findings have implications for understanding the action mechanism of GQD for diarrhoea treatment in terms of the microbial milieu.


Assuntos
Bactérias/metabolismo , Diarreia/microbiologia , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/metabolismo , Microbioma Gastrointestinal/fisiologia , Animais , Bactérias/enzimologia , Berberina/análise , Berberina/metabolismo , Medicamentos de Ervas Chinesas/química , Flavonoides/análise , Flavonoides/metabolismo , Ácido Glicirrízico/análise , Ácido Glicirrízico/metabolismo , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos
12.
Acta Pharmacol Sin ; 40(2): 288-296, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29773886

RESUMO

The influence of broad-spectrum antibiotics on the pharmacokinetics and biotransformation of major constituents of Shaoyao-Gancao decoction (SGD) in rats was investigated. The pharmacokinetic behaviors of paeoniflorin (PF), albiflorin (AF), liquiritin (LT), isoliquiritin (ILT), liquiritin apioside (LA), isoliquiritin apioside (ILA), and glycyrrhizic acid (GL), seven major constituents of SGD, as well as glycyrrhetinic acid (GA), a major metabolite of GL, were analyzed. A 1-week pretreatment with broad-spectrum antibiotics (ampicillin, metronidazole, neomycin, 1 g L-1; and vancomycin, 0.5 g L-1) via drinking water reduced plasma exposure of the major constituents. The AUC0-24 h of PF and LT was significantly decreased by 28.7% and 33.8% (P < 0.05 and P < 0.005), respectively. Although the differences were not statistically significant, the AUC0-24 h of AF, ILT, LA, ILA, and GL was decreased by 31.4%, 50.9%, 16.9%, 44.1%, and 37.0%, respectively, compared with the control group. In addition, the plasma GA exposure in the antibiotic-pretreated group was significantly lower (P < 0.005) than the control group. The in vitro stability of the major constituents of SGD in the rat intestinal contents with or without broad-spectrum antibiotics was also investigated. The major constituents were comparatively stable in the rat duodenum contents, and the biotransformation of GL mainly occurred in the rat colon contents. In summary, broad-spectrum antibiotics suppressed the absorption of the major constituents of SGD and significantly inhibited the biotransformation of GL to GA by suppressing the colon microbiota. The results indicated a potential clinical drug-drug interaction (DDI) when SGD was administered with broad-spectrum antibiotics.


Assuntos
Antibacterianos/farmacologia , Medicamentos de Ervas Chinesas/farmacocinética , Interações Ervas-Drogas , Administração Oral , Animais , Medicamentos de Ervas Chinesas/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Ácido Glicirrízico/metabolismo , Ácido Glicirrízico/farmacocinética , Absorção Intestinal/efeitos dos fármacos , Masculino , Ratos Sprague-Dawley
13.
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
14.
J Phys Chem B ; 122(43): 9938-9946, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30299964

RESUMO

It is known that glycyrrhizic acid (GA) promotes the enhancement of the activity of several medicines. This is attributed to the fact that GA increases the membrane permeability of small drug molecules. There is an opinion that GA facilitates the formation of additional large voids in the membrane, which enhance the passive diffusion of molecules across the membrane. In this work, we investigate how GA influences the intermolecular voids using the molecular dynamics simulation. We calculate the interstitial spheres (empty spheres inscribed between molecules) in model DPPC and DOPC bilayers, both pure and with the addition of cholesterol. It was observed that the addition of GA does not lead to the formation of new large interstitial spheres; i.e., new large voids do not appear. The distribution of empty volume inside the bilayers is also studied. We calculated the profiles of the empty volume fraction both from the middle plane of the bilayer and from its outer surface (from the lipid-water interface). This analysis has shown that the addition of GA does not cause the increase of the empty volume in the bilayer; moreover, there is a slight decrease in the bilayers with cholesterol. Thus, we have not found a confirmation of the simplest hypothesis that individual GA molecules induce pores in the membrane.


Assuntos
Ácido Glicirrízico/química , Bicamadas Lipídicas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Ácido Glicirrízico/metabolismo , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química
15.
DNA Cell Biol ; 37(12): 941-946, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30325653

RESUMO

Autoimmune encephalomyelitis is a chronic autoimmune disease caused by immune-mediated sterile inflammatory response and demyelination in the central nervous system (CNS). High-mobility group box protein 1 (HMGB1) is a ubiquitous nuclear protein, which can be released from damaged cells and induce proinflammatory responses in autoimmune encephalomyelitis. Glycyrrhizin (GL), a major constituent of licorice root, can inhibit the proinflammatory bioactivities of HMGB1. In this article, we bring some insight into the effects of GL on CNS inflammatory diseases and discuss the therapeutic potential of GL in autoimmune encephalomyelitis in the future.


Assuntos
Encefalomielite/tratamento farmacológico , Ácido Glicirrízico/metabolismo , Ácido Glicirrízico/farmacologia , Animais , Doenças Autoimunes/tratamento farmacológico , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Proteína HMGB1/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos
16.
Phytochemistry ; 156: 124-134, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30278303

RESUMO

Glycyrrhiza glabra L. (licorice) is a medicinal species rich in the specialised plant metabolite glycyrrhizin. It has been previously proposed that drought, which is increasing in importance due to the climatic change and scarcity of water resources, can promote the synthesis of glycyrrhizin. The effects of slight, moderate and intense drought (70, 35 and 23% of the regular irrigation, respectively) on growth parameters, osmolyte content, oxidative stress markers, antioxidant enzymes, glycyrrhizin biosynthesis genes and root glycyrrhizin concentration and contents, have been assessed in five Iranian licorice genotypes grown in the field. Drought decreased progressively biomass and leaf relative water contents, and increased progressively osmolyte (proline, glycine-betaine and soluble sugars) concentrations in leaves and roots. Drought caused oxidative stress in leaves, as indicated by lipid peroxidation and hydrogen peroxide concentrations, and increased the activities of antioxidant enzymes in leaf extracts (catalase, peroxidase, superoxide dismutase and pholyphenoloxidase). Drought promoted the synthesis of glycyrrhizin, as indicated by the increases in the expression of the glycyrrhizin biosynthesis pathway genes SQS1, SQS2, bAS, CYP88D6, CYP72A154 and UGT73, and increased the root concentrations of glycyrrhizin with drought in some genotypes. However, the large decreases in root biomass caused by drought led to general decreases in the amount of glycyrrhizin per plant with moderate and intense drought, whereas the slight drought treatment led to significant decreases in glycyrrhizin content in only one genotype. Under intense drought two of the genotypes were still capable to maintain half of the control glycyrrhizin yield, whereas in the other three genotypes glycyrrhizin yield was 22-33% of the control values. Results indicate that under intense drought, with only 23% of the normal water dose being applied, an appropriate choice of genotype can still lead to acceptable glycyrrhizin yields.


Assuntos
Antioxidantes/metabolismo , Secas , Glycyrrhiza/metabolismo , Ácido Glicirrízico/metabolismo , Estresse Fisiológico , Betaína/metabolismo , Glycyrrhiza/química , Glycyrrhiza/crescimento & desenvolvimento , Ácido Glicirrízico/química , Concentração Osmolar , Prolina/metabolismo , Açúcares/metabolismo
17.
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
18.
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
19.
Yakugaku Zasshi ; 138(6): 743-750, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-29863039

RESUMO

 The scientific evaluation of crude drugs and kampo medicines (KMs) was demonstrated using the eastern blotting method with monoclonal antibodies (MAbs) against bioactive natural compounds. Scutellariae radix is one of the most important crude drugs used in KMs. Part of its pharmaceutical properties is due to the flavone glycoside baicalin (BI). A quantitative analysis method based on eastern blotting was developed for BI using an anti-BI MAb. A rapid, simple, sensitive, specific analytical system was subsequently established for BI with the eastern blotting technique using dot-blot and chemiluminescent methods. This system was useful as a high-throughput analytical method for the determination of BI in KMs as well as HPLC and enzyme-linked immunosorbent assay systems. Furthermore, an eastern blotting method was applied to the biological metabolic study of glycyrrhizic acid (GL), the major active constituent of licorice, for investigation of metabolites of GL such as 3-monoglucuronyl-glycyrrhetinic acid (3MGA) because licorice causes pseudoaldosteronism as a side effect. This approach may make it possible to determine the pathogenic agents of licorice-induced pseudoaldosteronism.


Assuntos
Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/metabolismo , Immunoblotting/métodos , Medicina Kampo , Animais , Anticorpos Monoclonais , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Glycyrrhiza/química , Ácido Glicirrízico/efeitos adversos , Ácido Glicirrízico/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Síndrome de Liddle/induzido quimicamente , Síndrome de Liddle/prevenção & controle , Scutellaria baicalensis/química
20.
Mol Biotechnol ; 60(6): 412-419, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29687371

RESUMO

Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces various triterpene saponins such as glycyrrhizin. Beta-amyrin 11-oxidase (CYP88D6) plays a key role in engineering pathway of glycyrrhizin production and converts an intermediated beta-amyrin compound to glycyrrhizin. In this study, pBI121GUS-9:CYP88D6 construct was transferred to G. glabra using Agrobacterium rhizogene ATCC 15834. The quantitation of transgene was measured in putative transgenic hairy roots using qRT-PCR. The amount of glycyrrhizin production was measured by HPLC in transgenic hairy root lines. Gene expression analysis demonstrated that CYP88D6 was over-expressed only in one of transgenic hairy root lines and was reduced in two others. Beta-amyrin 24-hydroxylase (CYP93E6) was significantly expressed in one of the control hairy root lines. The amount of glycyrrhizin metabolite in over-expressed line was more than or similar to that of control hairy root lines. According to the obtained results, it would be recommended that multi-genes of glycyrrhizin biosynthetic pathway be transferred simultaneously to the hairy root in order to increase glycyrrhizin content.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Glycyrrhiza/enzimologia , Ácido Glicirrízico/metabolismo , Engenharia Metabólica , Agrobacterium/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/metabolismo , Técnicas de Transferência de Genes , Glycyrrhiza/genética , Glycyrrhiza/metabolismo , Ácido Glicirrízico/análise , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo
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