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1.
Comput Biol Chem ; 84: 107163, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31767507

RESUMO

The present study was to illustrate the agonistic property of arjungenin and arjunic acid towards farnesoid X receptor protein (FXR).The pharmacokinetic properties like molecular interactions, absorption, distribution, metabolism, elimination and toxicity (ADMET) of the ligands were checked through in-silico studies. Protein-ligand docking was carried out using autodock software. Molecular docking analysis confirmed strong binding energy and interaction of arjungenin and arjunic acid with the target protein and the ADMET profiles identified for both compounds were promising.Further in vitro studies were performed in 3T3-L1 adipocyte to verify the agonistic property of arjungenin and arjunic acid. Oil red O staining was done to check differentiation induction. Adiponectin, leptin, triglycerides and total cholesterol levels were quantified. The mRNA expression of FXR, Cyp7a1, PPAR-γ and SREBP-1c were quantified using fluorescent real-time PCR. Cytotoxicity assay was confirmed that up to 150 µM concentration there is no significant cell death on treatment with arjunic acid and arjungenin. Treatment with arjungenin and arjunic acid confirms increased differentiation of the cells with significant (P < 0.05) increase in adiponectin (118.07% and 132.92%) and leptin (133.52% and 149.74%) protein levels compared to the negative control group. After treatment with arjungenin and arjunic acid in 3T3-L1 preadipocytes the mRNA expression of FXR, PPAR-γ and SREBP-1c were significantly (P < 0.01) increased and cyp7a1 was significantly (P < 0.01) decreased when compared with the negative control group. Overall, our results suggest that arjungenin and arjunic acid acts as an FXR agonist and may be useful for rational therapeutic strategies as a novel drug to treat cholesterol mediated metabolic syndrome and insulin resistance.


Assuntos
Receptores Citoplasmáticos e Nucleares/agonistas , Triterpenos/farmacologia , Células 3T3-L1 , Adiponectina/metabolismo , Animais , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Expressão Gênica/efeitos dos fármacos , Leptina/metabolismo , Camundongos , Simulação de Acoplamento Molecular , PPAR gama/genética , PPAR gama/metabolismo , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triterpenos/metabolismo , Triterpenos/farmacocinética
2.
Phytochemistry ; 170: 112225, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31855780

RESUMO

Sixteen previously undescribed lanostane-type triterpenoids (1-16), together with fourteen known compounds, were isolated from cultivated fruiting bodies of the basidiomycete Ganoderma casuarinicola, a recently described species. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. Two of these compounds, 9 and 10, showed antimalarial activity with IC50 values of 9.7 and 9.2 µg/ml, respectively.


Assuntos
Antimaláricos/farmacologia , Antituberculosos/farmacologia , Ganoderma/química , Lanosterol/farmacologia , Malária/tratamento farmacológico , Compostos Fitoquímicos/farmacologia , Triterpenos/farmacologia , Animais , Antimaláricos/química , Antimaláricos/metabolismo , Antituberculosos/química , Antituberculosos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Carpóforos/química , Carpóforos/metabolismo , Ganoderma/metabolismo , Lanosterol/análogos & derivados , Lanosterol/química , Lanosterol/metabolismo , Testes de Sensibilidade Microbiana , Conformação Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Compostos Fitoquímicos/química , Compostos Fitoquímicos/metabolismo , Triterpenos/química , Triterpenos/metabolismo , Células Vero , Madeira/química , Madeira/metabolismo
3.
Zhongguo Zhong Yao Za Zhi ; 44(20): 4476-4480, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31872635

RESUMO

The 70% ethanol extract of the whole plant of Souliea vaginata was purified by multi-chromatographic methods including macroporous resin,silica gel,Sephadex LH-20,and C18-reversed-phase column chromatography. A new spirocyclic cycloartane triterpenoid was isolated and identified as( 16 R*,20 R*,23 S*,24 R*,25 S*)-16,23: 23,26-diepoxy-15α,24,25-trihydroxy-9,19-cycloart-3ß-O-ß-D-xylopyranoside( 1),and named as soulieoside S. Its planar structure and relative configuration were determined by spectroscopic techniques including 2 D NMR and HRESI-MS. As one of the main components of S. vaginata,compound 1 was evaluated for its anti-inflammatory activity by a lipopolysaccharide( LPS)-stimulated NO production model in RAW264. 7 macrophages,but it didn't show NO production inhibitory effect.


Assuntos
Actaea/metabolismo , Triterpenos/metabolismo , Actaea/química , Glicosídeos , Lipopolissacarídeos , Estrutura Molecular , Triterpenos/análise
4.
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
5.
Zhongguo Zhong Yao Za Zhi ; 44(16): 3562-3568, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602923

RESUMO

The mass spectrometry-based metabolomics method was used to systematically investigate the formation of celastrol metabolites,and the effect of celastrol on endogenous metabolites. The mice plasma,urine and feces samples were collected after oral administration of celastrol. Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry( UPLC-QTOF-MS) was applied to analyze the exogenous metabolites of celastrol and its altered endogenous metabolites. Mass defect filtering was adopted to screen for the exogenous metabolites of celastrol. Multivariate statistical analysis was used to identify the endogenous metabolites affected by celastrol. Celastrol and its eight metabolites were detected in urine and feces of mice,and 5 metabolites of them were reported for the first time. The hydroxylated metabolites were observed in the metabolism of both human liver microsomes and mouse liver microsomes. Further recombinant enzyme experiments revealed CYP3 A4 was the major metabolic enzyme involved in the formation of hydroxylated metabolites. Urinary metabolomics revealed that celastrol can affect the excretion of intestinal bacteria-related endogenous metabolites,including hippuric acid,phenylacetylglycine,5-hydroxyindoleacetic acid,urocanic acid,cinnamoylglycine,phenylproplonylglycine and xanthurenic acid. These results are helpful to elucidate the metabolism and disposition of celastrol in vivo,and its mechanism of action.


Assuntos
Metabolômica , Triterpenos/farmacocinética , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Camundongos , Microssomos Hepáticos/metabolismo , Triterpenos/metabolismo
6.
Plant Physiol Biochem ; 144: 73-84, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31561200

RESUMO

Cleome arabica is a medicinal plant contains diverse bioactive compounds and terpenoids are the major components. However, the isolation and purification of the active triterpenes from this plant involve long and complicated procedures. The present work investigates the triterpenes profiles of different tissues, besides that, describes the isolation, heterologous expression and functional characterization of C. arabica gene coding for triterpenes synthases. The phytochemical investigation through GC-MS revealed significant accumulation of pentacyclic triterpenes in leaves and siliques at mature stage compared to the stems and roots of C. arabica. Among the pentacyclic triterpenes, the lupeol reached the highest level of 320 µg/g DW in leaves at maturity stage compared to the other tissues. The biosynthesis of a pentacyclic triterpene was investigated through isolation and cloning of a full-length oxidosqualene cyclase cDNA (CaOSC) from mature leaves of C. arabica. The bioinformatic analyses revealed that CaOSC was highly homologous with the characterized lupeol synthases and shared 79.3% identity to camelliol C synthase from A. thaliana. Heterologous expression of CaOSC gene in Saccharomyces cerevisiae synthesized lupeol as a single product. The lupeol biosynthesis was exponentially increased after induction through the fermentation process reaching the maximum of 2.33 µg/ml for 240 h. Furthermore, organ-specific expression of lupeol gene was exactly matched the accumulation pattern in different tissues of C. arabica during phenological cycle. Thus, the identified CaOSC will be useful in enhancing triterpene yield for industrial purposes.


Assuntos
Cleome/enzimologia , Cleome/metabolismo , Transferases Intramoleculares/metabolismo , Triterpenos/metabolismo , Triterpenos Pentacíclicos/metabolismo
7.
Mol Immunol ; 114: 571-577, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31525576

RESUMO

Asthma is a chronic inflammatory disorder of airway affecting people from childhood to old age, and is characterized by airway epithelial dysfunction. Cucurbitacin E (CuE), a tetracyclic triterpene isolated from Cucurbitaceae plants, has been recently proved to exert anti-inflammation and immunology regulation activities. Nevertheless, its roles in asthma remains poorly defined. In the current study, CuE had little cytotoxicity on cell viability of human bronchial epithelial cell line BEAS-2B. Moreover, lipopolysaccharide (LPS) exposure inhibited cell viability and induced cell apoptosis, which was reversed following CuE pretreatment. Additionally, CuE administration suppressed LPS-induced inflammatory cytokine production, including TNF-α, IL-6, and IL-8. Simultaneously, supplementation with CuE decreased the transcripts and releases of mucin 5AC (MUC5AC) in LPS-treated BEAS-2B cells. Intriguingly, CuE inhibited LPS-evoked activation of the high-mobility group box1 (HMGB1)-TLR4-NF-κB signaling by reducing the expression of HMGB1, TLR4 and p-p65 NF-κB. Notably, restoring this pathway by elevating HMGB1 expression largely offset the protective function of CuE against LPS-triggered cell injury, inflammatory response and MUC5AC expression. Consequently, these findings highlight that CuE can ameliorate human bronchial epithelial cell insult and inflammation under LPS-simulated asthmatic conditions by blocking the HMGB1-TLR4-NF-κB signaling, thereby supporting its usefulness as a promising therapeutic agent against asthma.


Assuntos
Brônquios/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Inflamação/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Triterpenos/farmacologia , Apoptose/efeitos dos fármacos , Asma/tratamento farmacológico , Asma/metabolismo , Brônquios/metabolismo , Linhagem Celular , Células Cultivadas , Células Epiteliais/metabolismo , Proteína HMGB1/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Mucina-5AC/metabolismo , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Triterpenos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
8.
Molecules ; 24(19)2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31554155

RESUMO

Ganoderic acid A (GAA) is a bioactive triterpenoid isolated from the medicinal fungus Ganoderma lucidum. Our previous study showed that the Bacillus subtilis ATCC (American type culture collection) 6633 strain could biotransform GAA into compound (1), GAA-15-O-ß-glucoside, and compound (2). Even though we identified two glycosyltransferases (GT) to catalyze the synthesis of GAA-15-O-ß-glucoside, the chemical structure of compound (2) and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the same B. subtilis strain, for the biotransformation of GAA into compound (2) through acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a scaled-up production, compound (2) was successfully isolated using preparative high-performance liquid chromatography and identified to be a new triterpenoid glucoside (GAA-26-O-ß-glucoside) by mass and nuclear magnetic resonance spectroscopy. The results of kinetic experiments showed that the turnover number (kcat) of BsGT110 toward GAA at pH 6 (kcat = 11.2 min-1) was 3-fold higher than that at pH 7 (kcat = 3.8 min-1), indicating that the glycosylation activity of BsGT110 toward GAA was more active at acidic pH 6. In short, we determined that BsGT110 is a unique GT that plays a role in the glycosylation of triterpenoid at the C-26 position under acidic conditions, but loses most of this activity under alkaline ones, suggesting that acidic solutions may enhance the catalytic activity of this and similar types of GTs toward triterpenoids.


Assuntos
Bacillus subtilis/enzimologia , Glucosídeos/biossíntese , Glicosiltransferases/metabolismo , Ácidos Heptanoicos/metabolismo , Lanosterol/análogos & derivados , Proteínas Recombinantes , Triterpenos/metabolismo , Sequência de Aminoácidos , Biotransformação , Catálise , Cromatografia Líquida de Alta Pressão , Glucosídeos/química , Glicosilação , Ácidos Heptanoicos/química , Cinética , Lanosterol/química , Lanosterol/metabolismo , Triterpenos/química
9.
Med Sci Monit ; 25: 6846-6854, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31545303

RESUMO

BACKGROUND Worldwide, gastric cancer is one of the most common malignant tumors. Ursolic acid is a plant metabolite and pentacyclic triterpenoid used in traditional Chinese medicine. This study aimed to investigate the effects of ursolic acid the growth and apoptosis of SGC7901 and BGC823 human gastric cancer cells in vitro. MATERIAL AND METHODS SGC7901 and BGC823 human gastric cancer cells and normal GES-1 gastric epithelial cells were cultured with increasing doses of ursolic acid at 50, 60, and 100 µM. Cell viability and proliferation were assessed using an MTT assay. Flow cytometry was used to assess cell apoptosis. Western blot was used to measure procaspase-8, procaspase-9, procaspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) expression. The expression of receptor interaction protein 3 (RIP3) was examined by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Morphological changes in the gastric cancer cells were determined using Hoechst 33342 staining following ursolic acid treatment. RESULTS Ursolic acid inhibited the viability of SGC7901 and BGC823 cells but not GES-1 cells. Ursolic acid treatment significantly induced apoptosis in SGC7901 and BGC823 cells when compared with GES-1 cells (P<0.05), and significantly increased the activation of caspase-3, caspase-8, caspase-9, poly ADPribose polymerase (PARP), and the production of reactive oxygen species (ROS). Treatment of SGC7901 and BGC823 cells with ursolic acid for 72 h did not induce necroptosis. CONCLUSIONS Ursolic acid inhibited the proliferation of SGC7901 and BGC823 human gastric cancer cells in vitro through a caspase-dependent apoptotic pathway.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Triterpenos/farmacologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , China , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Triterpenos/metabolismo
10.
Med Sci Monit ; 25: 7158-7168, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31545785

RESUMO

BACKGROUND We previously reported that astragaloside IV (As-IV) can alleviate myocardial damage induced by lipopolysaccharide (LPS). However, the anti-inflammatory effects of As-IV following LPS stimulation in mice and H9C2 cardiomyocytes remain unclear. The present study was designed to explore the mechanism of action of As-IV. MATERIAL AND METHODS In vivo, C57BL/6J mice were randomly divided into 5 groups: the control group, the LPS group (10 mg/kg), and 3 LPS groups receiving different doses of As-IV (20, 40, and 80 mg/kg). The protective effect of As-IV on LPS-stimulated H9C2 cardiomyocytes was evaluated in vitro. Cardiac function was detected by echocardiography, and H&E staining was used to evaluate morphologic changes. Cardiomyocyte viability was detected by MTT assay. ELISA was used to detect free fatty acid (FFA), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor alpha (TNF-alpha) levels in mouse serum and in cell supernatant. Adenosine triphosphate (ATP) and adenosine monophosphate (AMP) contents in myocardial tissues and cells were detected by high-performance liquid chromatography. ATP5D and TLR4/NF-kappaB/PPARalpha signaling pathway proteins (TLR4, NF-kappaB, p65, and PPARalpha) were detected by Western blotting. RESULTS As-IV significantly improved cardiac function, myocardial cell viability, and pathological changes and reduced FFA, IL-1ß, IL-6, and TNF-alpha levels. The ATP/AMP ratio in the cardiac tissues of mice and in H9C2 cardiomyocytes was increased compared to that in the LPS group. In addition, As-IV enhanced ATP synthase and PPARalpha protein expression. In H9C2 cardiomyocytes, the p65-specific inhibitor BAY11-7082 exerted similar effects as As-IV. CONCLUSIONS As-IV alleviates LPS-induced myocardial damage by modulating TLR4/NF-kappaB/PPARalpha signaling-mediated energy biosynthesis.


Assuntos
Coração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , China , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , PPAR alfa/metabolismo , Saponinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Triterpenos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
11.
Chin J Nat Med ; 17(8): 575-584, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31472894

RESUMO

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.


Assuntos
Diterpenos/metabolismo , Regulação da Expressão Gênica de Plantas , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Fenantrenos/metabolismo , Proteínas de Plantas/metabolismo , Tripterygium/metabolismo , Acetilcoenzima A/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Domínio Catalítico , Compostos de Epóxi/metabolismo , Hidroximetilglutaril-CoA Sintase/química , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Interferência de RNA , Terpenos/metabolismo , Tripterygium/enzimologia , Tripterygium/genética , Triterpenos/metabolismo
12.
Int J Mol Sci ; 20(18)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487924

RESUMO

Chaga (Inonotus obliquus) is a medicinal fungus used in traditional medicine of Native American and North Eurasian cultures. Several studies have demonstrated the medicinal properties of chaga's bioactive molecules. For example, several terpenoids (e.g., betulin, betulinic acid and inotodiol) isolated from I. obliquus cells have proven effectiveness in treating different types of tumor cells. However, the molecular mechanisms and regulation underlying the biosynthesis of chaga terpenoids remain unknown. In this study, we report on the optimization of growing conditions for cultured I. obliquus in presence of different betulin sources (e.g., betulin or white birch bark). It was found that better results were obtained for a liquid culture pH 6.2 at 28 °C. In addition, a de novo assembly and characterization of I. obliquus transcriptome in these growth conditions using Illumina technology was performed. A total of 219,288,500 clean reads were generated, allowing for the identification of 20,072 transcripts of I. obliquus including transcripts involved in terpenoid biosynthesis. The differential expression of these genes was confirmed by quantitative-PCR. This study provides new insights on the molecular mechanisms and regulation of I. obliquus terpenoid production. It also contributes useful molecular resources for gene prediction or the development of biotechnologies for the alternative production of terpenoids.


Assuntos
Basidiomycota/genética , Genes Fúngicos , Transcriptoma , Triterpenos/metabolismo , Basidiomycota/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica
13.
Int J Mol Sci ; 20(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394870

RESUMO

Platycodin D (PD), a major saponin (platycoside) in Platycodi radix (balloon flower root), has higher pharmacological activity than the other major platycosides; however, its content in the plant root is only approximately 10% (w/w) and the productivities of PD by several enzymes are still too low for industrial applications. To rapidly increase the total PD content, the ß-glucosidase from Caldicellulosiruptor bescii was used for the deglucosylation of the PD precursors platycoside E (PE) and platycodin D3 (PD3) in the root extract into PD. Under the optimized reaction conditions, the enzyme completely converted the PD precursors into PD with the highest productivity reported so far, increasing the total PD content to 48% (w/w). In the biotransformation process, the platycosides in Platycodi radix were hydrolyzed by four pathways: deapiosylated (deapi)-PE → deapi-PD3 → deapi-PD, PE → PD3 → PD, polygalacin D3 → polygalacin D, and 3″-O-acetyl polygalacin D3 → 3″-O-acetyl polygalacin D.


Assuntos
Biotransformação , Firmicutes/metabolismo , Raízes de Plantas/metabolismo , Platycodon/metabolismo , Saponinas/metabolismo , Triterpenos/metabolismo , beta-Glucosidase/metabolismo , Hidrólise , Redes e Vias Metabólicas , Estrutura Molecular , Saponinas/química , Especificidade por Substrato , Triterpenos/química , beta-Glucosidase/química
14.
Plant Cell Physiol ; 60(11): 2496-2509, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31418782

RESUMO

Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted ß-amyrin into 24-hydroxy-ß-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.


Assuntos
Lotus/metabolismo , Triterpenos/metabolismo , Regulação da Expressão Gênica de Plantas , Ácido Oleanólico/metabolismo , Proteínas de Plantas/metabolismo
15.
Molecules ; 24(16)2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31405141

RESUMO

The aim of the study was the evaluation of the efficiency of selected abiotic elicitors, i.e., silver and cadmium ions, ultrasound, and UV-C irradiation, in the stimulation of triterpenoid biosynthesis, accumulation, and saponin secretion in Calendula officinalis hairy root cultures. Apart from the possible enhancement of triterpenoid production, the relationship between primary and secondary metabolism (represented respectively by sterols and pentacyclic triterpenes), modifications of the sterol compositional profile, and fluctuations in the total triterpenoid content were monitored in the performed experiments. The main phenomenon observed as a response to heavy metal treatment was the stimulation (up to 12-fold) of the secretion of saponins, accompanied by significant changes in sterol composition. Ultrasound stimulated the secretion of saponins (up to 11-fold); however, it exerted diverse influences on the triterpenoid content in hairy root tissue (stimulating or decreasing) depending on the duration of the exposure to the elicitor. UV-C radiation caused a slight increase in the content of both sterols and saponins in hairy root tissue, and stimulated saponin secretion up to 8.5-fold. The expected symptoms of the competition between the biosynthetic pathways of sterols and pentacyclic triterpenoids were less evident in reactions to abiotic stressors than those reported previously for biotic elicitors.


Assuntos
Calendula/metabolismo , Raízes de Plantas/metabolismo , Saponinas/metabolismo , Triterpenos/metabolismo , Raios Ultravioleta
16.
Prep Biochem Biotechnol ; 49(10): 1010-1019, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31385749

RESUMO

Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem's inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150 mg l-1 on the 8th day. The highest content of BA was 1.3 mg g-1 DW after treatment with 1 mg l-1CCC on the 4th and 6th days and 200 mg l-1CTS NF on the 10th day. The highest B content (0.94 mg g-1DW) was obtained in the treated hairy root by 2 mg l-1 CCC after 4 and 6 days.


Assuntos
Betula/metabolismo , Raízes de Plantas/metabolismo , Triterpenos/metabolismo
17.
Int J Mol Sci ; 20(15)2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31362345

RESUMO

The dried sclerotia of Wolfiporia cocos (Schwein.) Ryvarden & Gilb., a traditional Chinese medicine, has triterpenoid as its main active component. Breeding high-yield triterpenoid in W. cocos is an important research topic at present. We screened out two monosporal strains from the same W. cocos 5.78, high-yielding DZAC-Wp-H-29 (H) and low-yielding DZAC-Wp-L-123 (L), and cultured mycelia for 17 days, 34 days, and 51 days, respectively. Transcriptome analysis results showed that triterpenoid synthesis is closely related to gene expression in triterpenoid synthesis pathways (hydroxymethyl glutaryl-CoA reductase (HMGCR), farnesyl diphosphate synthase (FDPS), 4-hydroxybenzoate polyprenyltransferase (COQ2), C-8 sterol isomerase (ERG2), sterol O-acyltransferase (ACAT), tyrosine aminotransferase (TAT), torulene dioxygenase (CAO2), and sterol-4alpha-carboxylate 3-dehydrogenase (erg26)), and is limited by the expression of enzyme M20 combined with domain protein peptide (Pm20d2), aryl-alcohol dehydrogenase (norA), ISWI chromatin-remodeling complex ATPase ISW2, GroES-like protein (adh), cytochrome P450 (ftmP450-1), and unknown proteins unigene0001029 and unigene0011374. In addition, maintaining high triterpenoid accumulation in W. cocos may require a stable membrane structure, so the accumulation ability may be related to the high synthesis ability of sterols. The low accumulation of triterpenoid in W. cocos may be due to the products of key enzymes increasing flow to other pathways.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Triterpenos/química , Wolfiporia/química , Wolfiporia/genética , Vias Biossintéticas , Biologia Computacional/métodos , Medicamentos de Ervas Chinesas/química , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Triterpenos/metabolismo , Wolfiporia/metabolismo
18.
Appl Microbiol Biotechnol ; 103(17): 7029-7039, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31309269

RESUMO

Betulinic acid (BA) and its derivatives possess potent pharmacological activity against cancer and HIV. As with many phytochemicals, access to BA is limited by the requirement for laborious extraction from plant biomass where it is found in low amounts. This might be alleviated by metabolically engineering production of BA into an industrially relevant microbe such as Saccharomyces cerevisiae (yeast), which requires complete elucidation of the corresponding biosynthetic pathway. However, while cytochrome P450 enzymes (CYPs) that can oxidize lupeol into BA have been previously identified from the CYP716A subfamily, these generally do not seem to be specific to such biosynthesis and, in any case, have not been shown to enable high-yielding metabolic engineering. Here RoCYP01 (CYP716A155) was identified from the BA-producing plant Rosmarinus officinalis (rosemary) and demonstrated to effectively convert lupeol into BA, with strong correlation of its expression and BA accumulation. This was further utilized to construct a yeast strain that yields > 1 g/L of BA, providing a viable route for biotechnological production of this valuable triterpenoid.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Rosmarinus/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Triterpenos/metabolismo , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/genética , Bases de Dados Genéticas , Expressão Gênica , Triterpenos Pentacíclicos/metabolismo , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rosmarinus/genética , Rosmarinus/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato
19.
J Pharm Biomed Anal ; 175: 112762, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31336286

RESUMO

As a triterpene saponin, pedunculoside is one of the most abundant, representative and active components in plants of genus Ilex (Aquifoliaceae). Pedunculoside has been used to treat myocardial ischemia, ameliorate hyperlipidemia and prevent liver injury. In this paper, a systemic in vitro liver microsomes / S9 and intestinal bacteria incubation, and in vivo animal experiment were performed, using LC-Q-TOF/MS analysis and a three-step data processing protocol. As a result, Bifidobacterium adolescentis and Bifidobacterium breve were identified to potentially metabolize pedunculoside among the intestinal bacteria tested. A total of 11 metabolites were found and tentatively identified, with 6 in both microsomal and bacterial incubation systems, and 9 after rats orally administered with pedunculoside. The metabolites detected involving both phase I and phase II metabolism, mainly through deglycosylation (hydrolyzation), dehydrogenation, hydroxylation and conjugation, and some of them underwent more than one-step metabolic reactions. Most of the metabolites have not been reported before. In vitro, liver microsome and intestinal bacteria prefer to metabolize pedunculoside in totally different ways; while in vivo, intestinal tract is the most important site for the metabolism and excretion of pedunculoside, where both intestinal bacteria and the host metabolic enzymes participate in its metabolism and disposition. The importance of intestinal bacteria should be highlighted. This study would contribute to a better understanding of pedunculoside metabolism, which can provide scientific evidence for its pharmacodynamic mechanism research and prove its clinical application.


Assuntos
Biotransformação/fisiologia , Glucose/análogos & derivados , Metaboloma/fisiologia , Triterpenos/química , Triterpenos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/metabolismo , Fezes/química , Glucose/química , Glucose/metabolismo , Hidroxilação/fisiologia , Ilex/metabolismo , Intestinos/fisiologia , Masculino , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Saponinas/química , Saponinas/metabolismo , Espectrometria de Massas em Tandem/métodos
20.
Plant Cell Physiol ; 60(11): 2510-2522, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31350564

RESUMO

The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids.


Assuntos
Indolquinonas/metabolismo , Maytenus/metabolismo , Triterpenos/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oxirredução , Folhas de Planta/metabolismo , Saccharomyces cerevisiae/metabolismo , Tabaco/metabolismo
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