RESUMO
Epithelial-mesenchymal transition (EMT) has recently been associated with cancer invasion, metastasis, and resistance. In our previous study, we discovered nanaomycin K, a natural growth inhibitor for EMT-induced Madin Darby canine kidney (MDCK) cells, from the cultured broth of actinomycetes. However, the screening method was undeveloped, because the activity of nanaomycin K was discovered accidentally. In this study, we established a screening method by analyzing the characteristics of nanaomycin K in MDCK cells. Nanaomycin K showed the characteristic growth inhibitory activity on MDCK cells cultured under four conditions: medium containing dimethyl sulfoxide, SB431542, TGF-ß, and a mixture of SB431542 and TGF-ß. The activity was stronger in TGF-ß-treated cells than in DMSO-treated cells. In the mixture of SB431542 and TGF-ß-treated cells, the activity of nanaomycin K was suppressed. The anti-cancer agents, mitomycin C, cisplatin, and staurosporine, lacked the characteristics as that of nanaomycin K for these four treatment conditions. Since these four conditions distinguish between the effects of nanaomycin K and other anti-cancer agents in EMT-induced cells, the screening method was established. Among the 13,427 plant extracts tested, Piper betle leaf extract displayed growth inhibitory activity against EMT-induced cells. Through the purification of the extract via bio-guided fractionation, hydroxychavicol was isolated as an active compound. The cytotoxic activity of hydroxychavicol was stronger in EMT-induced MDCK cells than in control cells. However, its cytotoxic activity was suppressed in EMT-inhibited cells. Furthermore, hydroxychavicol exhibited same activity against SAS cells (human squamous cell carcinoma of the tongue). Thus, we have successfully established a screening method for growth inhibitors of EMT-induced cells and have discovered an inhibitor from plant-based sources.
Assuntos
Transição Epitelial-Mesenquimal , Piper betle , Fator de Crescimento Transformador beta , Animais , Cães , Humanos , Benzamidas/farmacologia , Benzamidas/química , Proliferação de Células/efeitos dos fármacos , Dioxóis/farmacologia , Dioxóis/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Eugenol/farmacologia , Eugenol/análogos & derivados , Inibidores do Crescimento/farmacologia , Inibidores do Crescimento/química , Inibidores do Crescimento/isolamento & purificação , Células Madin Darby de Rim Canino , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Fator de Crescimento Transformador beta/metabolismo , Piper betle/químicaRESUMO
Chronic kidney disease (CKD) induces cardiac inflammation and fibrosis and reduces survival. We previously demonstrated that G protein-coupled receptor 68 (GPR68) promotes cardiac inflammation and fibrosis in mice with 5/6 nephrectomy (5/6Nx) and patients with CKD. However, no method of GPR68 inhibition has been found that has potential for therapeutic application. Here, we report that Cephalotaxus harringtonia var. nana extract and homoharringtonine ameliorate cardiac inflammation and fibrosis under CKD by suppressing GPR68 function. Reagents that inhibit the function of GPR68 were explored by high-throughput screening using a medicinal plant extract library (8,008 species), and we identified an extract from Cephalotaxus harringtonia var. nana as a GPR68 inhibitor that suppresses inflammatory cytokine production in a GPR68 expression-dependent manner. Consumption of the extract inhibited inflammatory cytokine expression and cardiac fibrosis and improved the decreased survival attributable to 5/6Nx. Additionally, homoharringtonine, a cephalotaxane compound characteristic of C. harringtonia, inhibited inflammatory cytokine production. Homoharringtonine administration in drinking water alleviated cardiac fibrosis and improved heart failure and survival in 5/6Nx mice. A previously unknown effect of C. harringtonia extract and homoharringtonine was revealed in which GPR68-dependent inflammation and cardiac dysfunction were suppressed. Utilizing these compounds could represent a new strategy for treating GPR68-associated diseases, including CKD.
Assuntos
Mepesuccinato de Omacetaxina , Extratos Vegetais , Receptores Acoplados a Proteínas G , Insuficiência Renal Crônica , Animais , Camundongos , Citocinas/metabolismo , Fibrose , Cardiopatias/tratamento farmacológico , Cardiopatias/etiologia , Mepesuccinato de Omacetaxina/farmacologia , Mepesuccinato de Omacetaxina/uso terapêutico , Camundongos Endogâmicos C57BL , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/complicaçõesRESUMO
This study used two types of analyses and statistical calculations on powdered samples of Polygala root (PR) and Senega root (SR): (1) determination of saponin content by an independently developed quantitative analysis of tenuifolin content using a flow reactor, and (2) near-infrared spectroscopy (NIR) using crude drug powders as direct samples for metabolic profiling. Furthermore, a prediction model for tenuifolin content was developed and validated using multivariate analysis based on the results of (1) and (2). The goal of this study was to develop a rapid analytical method utilizing the saponin content and explore the possibility of quality control through a wide-area survey of crude drugs using NIR spectroscopy. Consequently, various parameters and appropriate wavelengths were examined in the regression analysis, and a model with a reasonable contribution rate and prediction accuracy was successfully developed. In this case, the wavenumber contributing to the model was consistent with that of tenuifolin, confirming that this model was based on saponin content. In this series of analyses, we have succeeded in developing a model that can quickly estimate saponin content without post-processing and have demonstrated a brief way to perform quality control of crude drugs in the clinical field and on the market.
Assuntos
Saponinas , Espectroscopia de Luz Próxima ao Infravermelho , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Controle de Qualidade , Análise dos Mínimos QuadradosRESUMO
Peony root is an important herbal drug used as an antispasmodic analgesic. To evaluate peony roots with different botanical origins, producing areas, and post-harvest processing, 1H NMR-based metabolomics analysis was employed. Five types of monoterpenoids, including albiflorin (4), paeoniflorin (6), and sulfonated paeoniflorin (25), and six other compounds, including 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (18), benzoic acid (21), gallic acid (22), and sucrose (26) were detected in the extracts of peony root samples. Among them, compounds 4, 6, 18, and total monoterpenoids including 21 were quantified by quantitative 1H NMR (qHNMR). Compound 25 was detected in 1H NMR spectra of sulfur-fumigated white peony root (WPR) extracts indicating that 1H NMR was a fast and effective method for identifying sulfur-fumigated WPR. The content of 26, the main factor affecting extract yield, increased significantly in peony root after low-temperature storage for one month, whereas that in WPR did not increase due to the boiling treatment after harvesting. We investigated the impact of preprocessing methods to such analysis for NMR data from commercial samples, resulting that the data matrix transformed from qHNMR spectra and normalized to internal standard were optimum for multivariate analysis. The multivariate analysis demonstrated that among commercial samples derived from P. lactiflora, peony root samples in Japanese market (PR) had high contents of 18 and 22, and red peony root (RPR) samples had high content of monoterpenoids represented by 6; and among RPR samples, those derived from P. veitchii showed higher contents of 18 and 22 than those from P. lactiflora. The 1H NMR-based metabolomics method coupled with qHNMR was useful for evaluation of peony root and would be applicable for other crude drugs.
Assuntos
Paeonia , Extratos Vegetais , Espectroscopia de Ressonância Magnética , Extratos Vegetais/análise , Monoterpenos/análise , Paeonia/química , Enxofre/análise , Metabolômica , Análise Multivariada , Raízes de Plantas/químicaRESUMO
Apricot and Peach Kernels are commercial crude drugs used in many formulas of traditional Japanese medicine, Kampo. Although their applications are quite different, it is difficult to distinguish them using conventional methods such as HPLC. The study aimed at near-infrared (NIR) metabolic profiling to discriminate Apricot and Peach Kernels (Armeniacae Semen and Persicae Semen) collected from Japanese markets. A fast, simple, non-destructive, and robust NIR measurement of kernel surface with no sample pre-treatment was achieved in situ. Principal component analysis and orthogonal partial least squares discriminant analysis (OPLS-DA) models showed discrimination between the two crude drugs with good fitting and prediction values. These results indicate that NIR metabolic profiling is useful for discriminating Apricot and Peach Kernels based on their chemical constituents using a simple and non-destructive procedure.
Assuntos
Prunus armeniaca , Prunus persica , Metabolômica , Análise de Componente Principal , Cromatografia Líquida de Alta PressãoRESUMO
The global population is aging, and intervention strategies for anti-aging and the prevention of aging-related diseases have become a topic actively explored today. Nicotinamide adenine dinucleotide (NAD+) is an important molecule in the metabolic process, and its content in tissues and cells decreases with age. The supplementation of nicotinamide mononucleotide (NMN), an important intermediate and precursor of NAD+, has increased NAD+ levels, and its safety has been demonstrated in rodents and human studies. However, the high content of NMN in natural plants has not been fully explored as herbal medicines for drug development. Here, we identified that the leaf of Cinnamomum verum J. Presl (C. verum) was the highest NMN content among the Plant Extract Library (PEL) with food experience, using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To validate this result, the extraction and quantitative analysis of bark, leaf, root, and stem of fresh C. verum was conducted. The results revealed that the bark had the highest NMN content in C. verum (0.471 mg/100 g). Our study shed light on the prospects of developing natural plants in the context of NMN as drugs for anti-aging and prevention of aging-related diseases. The future should focus on the development and application of C. verum pharmaceutical formulations.
Assuntos
NAD , Mononucleotídeo de Nicotinamida , Humanos , NAD/metabolismo , Cinnamomum zeylanicum , Cromatografia Líquida , Casca de Planta/metabolismo , Espectrometria de Massas em Tandem , Extratos Vegetais/farmacologia , Preparações FarmacêuticasRESUMO
To explore useful natural compounds from indigenous medicinal plants, the cytotoxic properties from a methanolic extract of Boehmeria sieboldiana leaves against human cancer cell lines were isolated in the present study. After purification of the extract, seco-dehydroantofine B (1) together with two known phenanthroindolizine alkaloids, seco-dehydroantofine A (2) and septicine (3), were isolated. The structure of seco-dehydroantofine B was elucidated by performing comprehensive one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The cytotoxicity of these compounds against five human tumor cell lines was evaluated. Compound 3 exhibited anti-tumor activity at IC50 values of 50.0, 66.9, 50.0, and 153.7 µM against MKN1, SAS, HL-60, and THP-1 cells, respectively.
Assuntos
Alcaloides , Antineoplásicos Fitogênicos , Boehmeria , Neoplasias , Alcaloides/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Humanos , Estrutura Molecular , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Folhas de Planta/químicaRESUMO
A discriminant LC/MS quantitative analysis of ephedrine (EP) and pseudoephedrine (PEP) in Ephedrae herba was performed. Aerial parts of three Ephedra species were separated into internodes and extracted using Finger Masher with minimum loss. The contents of EP and PEP were measured by LC/MS/MS using the multiple reaction monitoring (MRM) method. Their contents in old-year branches were lower than those current-year branches and tended to be higher in the middle part than in the tip of each branch. The content ratio of EP and PEP was reversed in some branches depending on their extent of growth. In E. sinica, the contents were low at the first internode closest to the central main stem at each branch. The contents drastically increased from the second internode and were highest at the third internode. There was a strong correlation between the internode distance and alkaloid (EP + PEP) contents. The distribution of alkaloids in one internode was examined and the results showed that the part closest to the node had the lowest contents.
Assuntos
Ephedra/química , Efedrina/análise , Pseudoefedrina/análise , Alcaloides/análise , Cromatografia Líquida , Medicamentos de Ervas Chinesas/análise , Ephedra/classificação , Caules de Planta/química , Espectrometria de Massas em TandemRESUMO
To evaluate the safety and efficacy of Glycyrrhiza uralensis root extracts produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems, we investigated anti-allergic action in mice using IgE-mediated immediate hypersensitivity. Hot water extracts obtained from the roots of Glycyrrhiza uralensis cultivated using two systems were orally administered at a dose of 100 mg/kg as glycyrrhizin (GL) and compared with the commercial crude drug, Glycyrrhizae Radix. Both the artificial hydroponic and artificial hydroponic-field hybrid cultivated root extracts showed anti-allergic effects on IgE-mediated immediate hypersensitivity in mice, as did the commercial crude drugs. These results highlight the potential for artificially cultivated roots of Glycyrrhiza uralensis to be used as an alternative medicinal source.
Assuntos
Glycyrrhiza uralensis/química , Medicina Kampo/métodos , Extratos Vegetais/química , Raízes de Plantas/química , Água/química , Animais , Feminino , Temperatura Alta , CamundongosRESUMO
Plants produce various prenylated phenolic metabolites, including flavonoids, phloroglucinols, and coumarins, many of which have multiple prenyl moieties and display various biological activities. Prenylated phenylpropanes, such as artepillin C (3,5-diprenyl-p-coumaric acid), exhibit a broad range of pharmaceutical effects. To date, however, no prenyltransferases (PTs) involved in the biosynthesis of phenylpropanes and no plant enzymes that introduce multiple prenyl residues to native substrates with different regio-specificities have been identified. This study describes the isolation from Artemisia capillaris of a phenylpropane-specific PT gene, AcPT1, belonging to UbiA superfamily. This gene encodes a membrane-bound enzyme, which accepts p-coumaric acid as its specific substrate and transfers two prenyl residues stepwise to yield artepillin C. These findings provide novel insights into the molecular evolution of this gene family, contributing to the chemical diversification of plant specialized metabolites. These results also enabled the design of a yeast platform for the synthetic biology of artepillin C.
Assuntos
Artemisia/enzimologia , Dimetilaliltranstransferase/isolamento & purificação , Fenilpropionatos/metabolismo , Proteínas de Plantas/isolamento & purificação , Artemisia/genética , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Genes de Plantas , Fenilpropionatos/química , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prenilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Biologia Sintética/métodosRESUMO
Numerous Foods with Function Claims that contain the extract of Pueraria flower (kudzu) isoflavones (PFI) are available in the Japanese market. These are labelled with function claims of reducing visceral fat. However, these foods have not undergone proper safety assessment such as the evaluation of their oestrogenic activity and effects on drug-metabolising enzymes (cytochrome P-450: CYP) in the liver. This study evaluated the estrogenic effect and the hepatic CYP activity and mRNA expression in normal female mice as a safety assessment of PFI (Experiment 1). In addition, the bone mineral density and visceral fat weight in ovariectomised mice (OVX) compared to soy isoflavones (SI) was evaluated to assess the efficacy of PFI (Experiment 2). OVX control fed a control diet, OVX fed a PFI diet (the recommended human intake of PFI), OVX fed a PFI20 diet (20- times the recommended PFI), OVX fed an SI diet (the recommended human intake of SI), and OVX fed an SI20 diet (20 -times the recommended intake of SI) for 28 days in Experiment 2. Body, liver, and visceral fat weights were not affected by the PFI, PFI20, SI, or SI20 diets. The hepatic CYP1A and CYP3A activities were elevated by the SI20 treatment. Ovariectomy-induced bone loss was inhibited by the SI20 treatment, but not by the PFI20 treatment. These results suggest that (1) PFI intake in human doses had no oestrogenic properties and did not affect CYP activity in the liver; (2) there was no evidence that PFI affects the amount of visceral fat in OVX mice.
Assuntos
Flores/química , Isoflavonas/química , Isoflavonas/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Pueraria/química , Gordura Abdominal/efeitos dos fármacos , Gordura Abdominal/metabolismo , Animais , Biomarcadores , Densidade Óssea/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Modelos Animais , Osteogênese/efeitos dos fármacos , OvariectomiaRESUMO
The pharmacological properties of Eleutherococcus senticosus leaf have not been clarified although it is taken as a food item. In this study, the effects of water extract of Eleutherococcus senticosus leaves on memory function were investigated in normal mice. Oral administration of the extract for 17 days significantly enhanced object recognition memory. Compounds absorbed in blood and the brain after oral administration of the leaf extract were detected by LC-MS/MS analyses. Primarily detected compounds in plasma and the cerebral cortex were ciwujianoside C3, eleutheroside M, ciwujianoside B, and ciwujianoside A1. Pure compounds except for ciwujianoside A1 were administered orally for 17 days to normal mice. Ciwujianoside C3, eleutheroside M, and ciwujianoside B significantly enhanced object recognition memory. These results demonstrated that oral administration of the leaf extract of E. senticosus enhances memory function, and that active ingredients in the extract, such as ciwujianoside C3, eleutheroside M, and ciwujianoside B, were able to penetrate and work in the brain. Those three compounds as well as the leaf extract had dendrite extension activity against primary cultured cortical neurons. The effect might relate to memory enhancement.
Assuntos
Encéfalo/efeitos dos fármacos , Eleutherococcus/química , Memória/efeitos dos fármacos , Extratos Vegetais/farmacologia , Folhas de Planta/química , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Córtex Cerebral/química , Córtex Cerebral/embriologia , Córtex Cerebral/ultraestrutura , Dendritos/efeitos dos fármacos , Dendritos/fisiologia , Glicosídeos/análise , Glicosídeos/farmacocinética , Glicosídeos/farmacologia , Masculino , Camundongos , Extratos Vegetais/análise , Extratos Vegetais/farmacocinética , Saponinas/análise , Saponinas/farmacocinética , Saponinas/farmacologiaRESUMO
The article Impact of different postharvest processing methods on the chemical compositions of peony root.
RESUMO
In order to develop new domestic production of Rhei Rhizoma (RR) from Rheum specimens cultivated in the Sugadaira Medicinal Plant Cultivation Test Field (SMPCF), the ITS sequences of 12 SMPCF specimens and Chinese Rheum specimens of four species, as well as RR samples produced in North Korea, China and Japan, were determined by subcloning and their sequences were compared. As the ITS sequences of 10 SMPCF specimens showed significant intra-individual polymorphism, identification of pseudogenes was conducted by detecting the three motifs of the 5.8S sequence and the stability of the 5.8S secondary structure. Approximately 46% of sequences obtained from the SMPCF specimens were putative pseudogenes. The maximum likelihood tree based on ITS sequences showed three main groups-the outer group and inner clusters I and II; clones from 10 SMPCF specimens including putative pseudogenes belonged to the outer group. Cluster I was composed of two clades, one including clones from R. officinale specimens and R. palmatum-derived samples with matK genotype Rp9, and another including clones from R. coreanum-derived samples. Cluster II consisted of three clades, one including clones from R. palmatum specimens with genotype Rp5, another including clones mainly from R. tanguticum specimens with genotype Rt4, and the third including clones from R. palmatum or R. tanguticum specimens with various matK genotypes. Clones from SMPCF specimen RC5 showed a close relationship with those from R. tanguticum specimens with matK genotype Rt4, whereas those from specimen RC9 related to R. coreanum-derived samples. As a result, specimens RC5 and RC9 were considered as candidates for the development of domestic RR.
Assuntos
DNA de Plantas/genética , DNA Espaçador Ribossômico/genética , Polimorfismo Genético/genética , Rheum/genética , Sequência de Bases , China , Clonagem Molecular , Genótipo , Japão , Medicina Tradicional Chinesa , Plantas Medicinais/genética , Rheum/química , Análise de Sequência de DNARESUMO
To evaluate the safety and efficacy of Glycyrrhiza uralensis root produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems, we investigated the pharmacokinetics of a major metabolite of glycyrrhizin (GL), glycyrrhetinic acid (GA). Hot water extracts obtained from the roots of the artificial hydroponic-field hybrid cultivated Glycyrrhiza uralensis were orally administered at a dose of 100 mg/kg as GL in mice and, compared with a commercial crude drug, Glycyrrhizae Radix. The temporal changes in serum GA concentration was found to depend on the GL concentration of the hot-water extracts. When hot-water extracts containing relatively high GL were administered, bimodal peaks appeared. In contrast, a broad single peak was detected when a hot-water extract containing relatively low GL content was administered. These tendencies in the serum GA concentration time course were observed for all samples, regardless of their derivation. Moreover, we compared the pharmacokinetic parameters and found that the Cmax and AUC0-48 values after oral administration of the extracts from Glycyrrhiza uralensis roots produced by the artificial cultivation system are within the range of variation for the commercial crude drugs. These results suggest the possibility that roots of Glycyrrhiza uralensis cultivated by the artificial hydroponic-field hybrid cultivation system can be used in addition to currently available commercial crude drugs produced from wild plant resources.
Assuntos
Ácido Glicirretínico/química , Glycyrrhiza uralensis/química , Hidroponia/métodos , Medicina Kampo/métodos , Extratos Vegetais/química , Raízes de Plantas/química , Animais , Feminino , Camundongos , Raízes de Plantas/metabolismoRESUMO
As a part of the investigation of the safety and efficacy of the cultivated Coptis japonica rhizome extracts using an artificial hydroponic cultivation system, the mutagenetic and anti-allergic activities were evaluated. Some extracts of commercial crude drugs of Coptis sp. were also evaluated for the comparison. None of the extracts showed a significant mutagenicity in Salmonella typhimurium TA102 by the Ames tests, but all the extracts showed in S. typhimurium TA98. The extracts of the hydroponically cultivated rhizomes showed anti-allergic activities against contact hypersensitivity as well as those of commercial crude drugs of Coptis sp. These results suggested the potential of the hydroponically cultivated rhizomes as one of the alternative sources for the medicinal usage.
Assuntos
Antialérgicos/farmacologia , Berberina/análise , Coptis/química , Coptis/imunologia , Mutagênicos/química , Extratos Vegetais/farmacologia , Antialérgicos/química , Berberina/química , Dermatite de Contato , Hidroponia , Extratos Vegetais/química , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genéticaRESUMO
The three Forsythia species, F. suspensa, F. viridissima and F. koreana, have been used as herbal medicines in China, Japan and Korea for centuries and they are known to be rich sources of numerous pharmaceutical metabolites, forsythin, forsythoside A, arctigenin, rutin and other phenolic compounds. In this study, de novo transcriptome sequencing and assembly was performed on these species. Using leaf and flower tissues of F. suspensa, F. viridissima and F. koreana, 1.28-2.45-Gbp sequences of Illumina based pair-end reads were obtained and assembled into 81,913, 88,491 and 69,458 unigenes, respectively. Classification of the annotated unigenes in gene ontology terms and KEGG pathways was used to compare the transcriptome of three Forsythia species. The expression analysis of orthologous genes across all three species showed the expression in leaf tissues being highly correlated. The candidate genes presumably involved in the biosynthetic pathway of lignans and phenylethanoid glycosides were screened as co-expressed genes. They express highly in the leaves of F. viridissima and F. koreana. Furthermore, the three unigenes annotated as acyltransferase were predicted to be associated with the biosynthesis of acteoside and forsythoside A from the expression pattern and phylogenetic analysis. This study is the first report on comparative transcriptome analyses of medicinally important Forsythia genus and will serve as an important resource to facilitate further studies on biosynthesis and regulation of therapeutic compounds in Forsythia species.
Assuntos
Forsythia/química , Folhas de Planta/química , Plantas Medicinais/química , Metabolismo Secundário/genética , Transcriptoma/genética , Plantas Medicinais/genéticaRESUMO
The impact of key processing steps such as boiling, peeling, drying and storing on chemical compositions and morphologic features of the produced peony root was investigated in detail by applying 15 processing methods to fresh roots of Paeonia lactiflora and then monitoring contents of eight main components, as well as internal root color. The results showed that low temperature (4 °C) storage of fresh roots for approximately 1 month after harvest resulted in slightly increased and stable content of paeoniflorin, which might be due to suppression of enzymatic degradation. This storage also prevented roots from discoloring, facilitating production of favorable bright color roots. Boiling process triggered decomposition of polygalloylglucoses, thereby leading to a significant increase in contents of pentagalloylglucose and gallic acid. Peeling process resulted in a decrease of albiflorin and catechin contents. As a result, an optimized and practicable processing method ensuring high contents of the main active components in the produced root was developed.
Assuntos
Paeonia/química , Raízes de Plantas/químicaRESUMO
Aconitum carmichaelii is an important medicinal herb used widely in China, Japan, India, Korea, and other Asian countries. While extensive research on the characterization of metabolic extracts of A. carmichaelii has shown accumulation of numerous bioactive metabolites including aconitine and aconitine-type diterpene alkaloids, its biosynthetic pathway remains largely unknown. Biosynthesis of these secondary metabolites is tightly controlled and mostly occurs in a tissue-specific manner; therefore, transcriptome analysis across multiple tissues is an attractive method to identify the molecular components involved for further functional characterization. In order to understand the biosynthesis of secondary metabolites, Illumina-based deep transcriptome profiling and analysis was performed for four tissues (flower, bud, leaf, and root) of A. carmichaelii, resulting in 5.5 Gbps clean RNA-seq reads assembled into 128,183 unigenes. Unigenes annotated as possible rate-determining steps of an aconitine-type biosynthetic pathway were highly expressed in the root, in accordance with previous reports describing the root as the accumulation site for these metabolites. We also identified 21 unigenes annotated as cytochrome P450s and highly expressed in roots, which represent candidate unigenes involved in the diversification of secondary metabolites. Comparative transcriptome analysis of A. carmichaelii with A. heterophyllum identified 20,232 orthogroups, representing 30,633 unigenes of A. carmichaelii, gene ontology enrichment analysis of which revealed essential biological process together with a secondary metabolic process to be highly enriched. Unigenes identified in this study are strong candidates for aconitine-type diterpene alkaloid biosynthesis, and will serve as useful resources for further validation studies.
Assuntos
Aconitum/genética , Alcaloides/biossíntese , Diterpenos/metabolismo , Proteínas de Plantas/genética , Metabolismo Secundário/genética , Transcriptoma , Aconitina/química , Aconitina/isolamento & purificação , Aconitina/metabolismo , Aconitum/classificação , Aconitum/metabolismo , Alcaloides/química , Alcaloides/isolamento & purificação , Diterpenos/química , Diterpenos/isolamento & purificação , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas MedicinaisRESUMO
The roots of Platycodon grandiflorus are widely used as a crude drug. The active components include a variety of triterpenoid saponins. Recent studies have revealed that Cyt P450 monooxygenases (P450s) function as triterpene oxidases in triterpenoid saponin biosynthesis in many plant species. However, there have been no reports regarding triterpene oxidases in P. grandiflorus. In this study, we performed transcriptome analysis of three different P. grandiflorus tissues (roots, leaves and petals) using RNA sequencing (RNA-Seq) technology. We cloned six P450 genes that were highly expressed in roots, and classified them as belonging to the CYP716A, CYP716D and CYP72A subfamilies. We heterologously expressed these P450s in an engineered yeast strain that produces ß-amyrin, one of the most common triterpenes in plants. Two of the CYP716A subfamily P450s catalyzed oxidation reactions of the ß-amyrin skeleton. One of these P450s, CYP716A140v2, catalyzed a three-step oxidation reaction at C-28 on ß-amyrin to produce oleanolic acid, a reaction performed by CYP716A subfamily P450s in a variety of plant species. The other P450, CYP716A141, catalyzed the hydroxylation of ß-amyrin at C-16ß. This reaction is unique among triterpene oxidases isolated to date. These results enhance our knowledge of functional variation among CYP716A subfamily enzymes involved in triterpenoid biosynthesis, and provide novel molecular tools for use in synthetic biology to produce triterpenoid saponins with pre-defined structures.