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1.
ACS Infect Dis ; 2020 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-32567833

RESUMO

One of the major challenges in development of anti-enterovirus (EV) drug is in the safety of the drug. Here, we attempt to identity anti-EV compound from an edible plant extract library, and identify potent anti-enterovirus 68 (EV-D68) activity in avocado (Persea americana). The purified identity is determined as 2R,4R-(12Z,15Z)-heneicosa-12,15-diene-1,2,4-triol, and named avoenin. Avoenin shows EC50 of 2.0 µM for EV-D68(Fermon) infection with CC50 of > 150 µM in RD cells, by targeting uncoating step of EV-D68 infection. Resistant mutations of EV-D68 (VP3-V24I, S173P, and S180G) to avoenin confer cross resistance to pleconaril, an uncoating inhibitor of EV-D68. The inhibitory effect of avoenin is substantially specific to EV-D68 among the EVs. This work reveals avoenin as the identity of anti-EV-D68 activity in avocado, and offers insights into development of novel and effective strategy to overcome EV-D68 infection and its related respiratory diseases.

2.
J Nat Med ; 74(3): 550-560, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32333306

RESUMO

Previously, we established a 1H NMR metabolomics method using reversed-phase solid-phase extraction column (RP-SPEC), and succeeded in distinguishing wild from cultivated samples of Saposhnikoviae radix (SR), and between SR and its substitute, Peucedanum ledebourielloides root (PR). Herein, we performed LC-HR/MS metabolomics using fractions obtained via RP-SPEC to identify characteristic components of SR and PR. One and three characteristic components were respectively found for SR and PR; these components were isolated with their m/z values and retention times as a guide. The characteristic component of SR was identified as 4'-O-ß-D-glucosyl-5-O-methylvisamminol (1), an indicator component used to identify SR in the Japanese Pharmacopoeia. In contrast, the characteristic components of PR were identified as xanthalin (2), 4'-O-ß-D-apiosyl (1 → 6)-ß-D-glucosyl-5-O-methylvisamminol (3), and 3'-O-ß-D-apiosyl (1 → 6)-ß-D-glucosylhamaudol (4) based on spectroscopic data such as 1D- and 2D-NMR, MS, and specific optical rotation. Among them, 4 is a novel compound. For the correlation between the NMR metabolomics results in the present and our previous report, only 1 and 2 were found to correlate with the chemical shifts, and the other compounds had no correlation. As the chemical shifts for compounds 1, 3, and 4 were similar to each other, especially for the aglycone moiety, they could not be distinguished because of the sensitivity and resolution of 1H NMR. Accordingly, combining NMR and LC/MS metabolomics with their different advantages is considered useful for metabolomics of natural products. The series of methods used in our reports could aid in quality evaluations of natural products and surveying of marker components.

3.
J Nat Med ; 74(1): 135-141, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31407152

RESUMO

LC-MS analyses of saponin fractions of Achyranthes roots in the Japanese market revealed that there were three patterns for the saponin fraction of their water extracts, i.e., the saponins with a sugar moiety at position 28 [achyranthosides B (3), C (4) and D (5)] were the major constituents, the saponins without sugar moiety at position 28 [betavulgarosides II (10) and IV (11)] were the major constituents, and mixtures of these saponins. In a decoction prepared from the sample which contained 10 and 11 as the major saponins, their amounts were largely decreased compared with those of the water extract. As large amounts of these saponins were found in the precipitates formed by heating of the water extract, these saponins were seemed to precipitate out under heating. When hot water was used for the extraction, 3, 4 and 5 were detected even from the samples whose water extract did not contain these saponins. This was attributed to inhibition of endogenous esterase which hydrolyzes the ester linkage at position 28. When saponins were extracted with reagent grade 1-butanol, in addition to the decrease of the amounts of highly polar saponins, oxidative decarboxylation of 3 and 10 occurred resulting in formation of achyranthoside E (6) and spinacoside D (12), respectively. As these changes were not observed with HPLC grade 1-butanol, which contain not more than 5 ppm of peroxide impurities, the change was attributable to the peroxide impurities contained in the reagent grade 1-butanol.


Assuntos
Achyranthes/química , Ácido Oleanólico/análogos & derivados , Extratos Vegetais/química , Raízes de Plantas/química , Saponinas/química , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Japão , Ácido Oleanólico/química , Espectrometria de Massas em Tandem/métodos
4.
J Nat Med ; 74(2): 463-466, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31808065

RESUMO

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.

5.
J Nat Med ; 74(1): 170-188, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31578667

RESUMO

Saposhnikoviae Radix (SR) is a commonly used crude drug that is obtained from the root and rhizome of Saposhnikovia divaricata which is distributed throughout China, Korea, Mongolia, and Russia. To evaluate the quality of Mongolian S. divaricata, metabolomic profiling of 43 plant specimens from different regions of Mongolia, as well as 8 SR samples and 2 plant specimens from China, were conducted by liquid chromatography-ion-trap-time-of-flight-mass spectrometer (LC-IT-TOF-MS). LC-MS profiles of the specimens showed uniformity and 30 compounds were tentatively identified, including 13 chromones and 17 coumarins. Among them, 16 compounds were isolated and unambiguously verified by comparing them with the spectroscopic data of standard compounds. Orthogonal partial least squares-discriminant analysis (OPLS-DA) based on LC-MS data from 7 Mongolian specimens and 8 Chinese SR samples as well as 2 plant specimens revealed that these 2 groups were clearly distinguishable and that Mongolian specimens were characterized by an abundance of prim-O-glucosylcimifugin (1). Moreover, the OPLS-DA of the Mongolian specimens showed that they can be discriminated by their growing regions based on the content of 8 chromones. The total content of dihydrofurochromones 1-3 was relatively higher in the specimens from Khalkhgol in the far eastern part of Mongolia, while contents of 10, 11, 15, and 16 were higher in those from Holonbuir in the eastern part. Based on this research, the roots of S. divaricata from Mongolia have potential as a new resource of SR in Kampo medicine.


Assuntos
Apiaceae/química , Cromonas/análise , Cromonas/química , Cumarínicos/química , Monossacarídeos/química , Xantenos/química , China , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Medicamentos de Ervas Chinesas/química , Medicina Kampo , Mongólia , Raízes de Plantas/química , Rizoma/química , Espectrometria de Massas em Tandem/métodos
6.
J Nat Med ; 74(1): 65-75, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31342251

RESUMO

1H NMR-based metabolomics has been applied in research on food, herbal medicine, and natural products. Although excellent results were reported, samples were directly extracted with a deuterated solvent (e.g., methanol-d4 or D2O) in most reports. As primary metabolites account for most of the results, data for secondary metabolites are partially reflected. Consequently, secondary metabolites tend to be excluded from factor loading analysis, serving as a significant unfavorable feature of 1H NMR-based metabolomics when investigating biologically active or functional components in natural products and health foods. Reversed-phase solid-phase extraction column (RP-SPEC) was applied for sample preparation in 1H NMR-based metabolomics to overcome this feature. The methanol extract from Saposhnikoviae radix (SR), an important crude drug, was fractionated with RP-SPEC into 5% methanol-eluting fractions, and the remaining fraction was collected. Each fraction was subjected to 1H NMR-based metabolomics and compared to results from conventional 1H NMR-based metabolomics. Based on principal component analysis (PCA) and partial least squares projections to latent structures discriminant analysis (PLS-DA), the 5% methanol fraction and conventional method reflected the amount of saccharides such as sucrose on the PC1/PLS1 axes, and wild and cultivated samples were discriminated along those axes. The remaining fraction clearly distinguished SR from Peucedanum ledebourielloides root. The compounds responsible for this discrimination were deemed falcarindiol derivatives and other unidentified secondary metabolites from the s-plot on PLS-DA. The secondary metabolites from original plants were, therefore, presumed to be concentrated in the remaining fraction by RP-SPEC treatment and strongly reflected the species differences. The developed series is considered effective to perform quality evaluation of crude drugs and natural products.


Assuntos
Apiaceae/química , Misturas Complexas/química , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Raízes de Plantas/química , Extração em Fase Sólida/métodos , Espectroscopia de Prótons por Ressonância Magnética
7.
Commun Biol ; 2: 384, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31646187

RESUMO

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/métodos
8.
Int J Mol Sci ; 20(12)2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31212773

RESUMO

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 , Ovariectomia
9.
J Nat Med ; 73(3): 661-666, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31028662

RESUMO

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/metabolismo
10.
J Nat Med ; 73(3): 608-613, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30847756

RESUMO

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/genética
11.
J Nat Med ; 73(2): 369-380, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30547286

RESUMO

Gleditsia sinensis is widely used as a medicinal plant in Asia, especially in China. Triterpenes, alkaloids, and sterols were isolated from Gleditsia species. Among them, triterpenoid saponins are very important metabolites owing to their various pharmacological activities. However, the triterpenoid saponin biosynthesis pathway has not been well characterized. In the present study, we performed de novo transcriptome assembly for 14.3 Gbps of clean reads sequenced from nine tissues of G. sinensis. The results showed that 81,511 unique transcripts (unitranscripts) (47,855 unigenes) were constructed, of which 31,717 unigenes were annotated with Gene Ontology and EC numbers by Blast2GO against the NCBI-nr protein database. We also analyzed the metabolite contents in the same nine tissues by LS-MS/MS, and saponins including gleditsioside I were found in fruit at higher levels. Many of the genes with tissue-specific expression in fruit are involved in the flavonoid biosynthesis pathway, and many of those have UDP-glucosyltransferase (UGT) activity. We constructed a saponin biosynthesis pathway and identified two key enzyme families in the triterpenoid saponin biosynthesis pathway, cytochrome P450 and UDP-glucosyltransferase, that are encoded by 37 unigenes and 77 unigenes, respectively. CYP72A, CYP716A, and CYP88D, which are known as key enzymes for saponin biosynthesis, were also identified among the P450s. Our results provide insight into the secondary metabolite biosynthesis and serve as important resources for future research and cultivation of G. sinensis.


Assuntos
Gleditsia/genética , Saponinas/biossíntese , Transcriptoma , Triterpenos/metabolismo , China , Perfilação da Expressão Gênica , Ontologia Genética , Gleditsia/química , Gleditsia/metabolismo , Metaboloma , Plantas Medicinais/química , Plantas Medicinais/genética , Saponinas/análise , Saponinas/genética , Análise de Sequência de RNA , Espectrometria de Massas em Tandem
12.
Chem Pharm Bull (Tokyo) ; 66(12): 1174-1180, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30504632

RESUMO

Polygala Root (the root of Polygala tenuifolia WILLDENOW; Japanese name "Onji"), a well-known crude drug, traditionally used as an expectorant and sedative, has been attracting increased interest in recent years owing to its newly found pharmacological effect related to neuroprotection. However, there is no specific method for identifying and estimating the quality of this crude drug in the Japanese Pharmacopoeia, 17th edition. Therefore, in order to develop a TLC-based simple and convenient identification method using characteristic chemical marker(s) for the drug and its extract products, UV-sensitive constituents of Polygala Root were first investigated. A total of 23 aromatic compounds were isolated and characterized. Two new compounds, namely, polygalaonjisides A (1) and B (2), were characterized as syringic acid 4-O-(2'-O-ß-D-apiosyl)-ß-D-glucoside and 2-O-(ß-D-glucosyl)-3'-O-benzoylsucrose, respectively. Based on these phytochemical results, a TLC method focusing on three marker spots with Rf value of approximately 0.4-0.5 due to tenuifolisides A and B and 3,6'-di-O-sinapoylsucrose was proposed as a simple and convenient test to identify Polygala Root or its single-extract products on the market. The data presented in this paper could be useful in stipulating a confirmation test to identify Polygala Root.


Assuntos
Hidrocarbonetos Aromáticos/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Raízes de Plantas/química , Polygala/química , Raios Ultravioleta , Biomarcadores/análise , Cromatografia em Camada Delgada , Hidrocarbonetos Aromáticos/química , Estrutura Molecular , Extratos Vegetais/química , Controle de Qualidade
13.
J Nat Med ; 72(4): 867-881, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29736697

RESUMO

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/genética
14.
J Nat Med ; 72(3): 774-778, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29464510

RESUMO

The root of Angelica acutiloba Kitagawa is an important crude drug in Kampo medicines (traditional Japanese medicine). Chemical evaluation of crude drugs is crucial to ensuring the safety and efficacy of herbal medicine; however, there is currently no chemical standard for the A. acutiloba crude drug in Japanese pharmacopoeia. (E)-ferulic acid (FA) is an important active ingredient of Angelica spp., including A. sinensis (Oliv.) Diels, and has been suggested as a marker for quality evaluation of those crude drugs. However, it has been controversial whether FA is a reliable marker constituent of A. acutiloba. To achieve effective extraction of FA from A. acutiloba, we compared three different extraction methods: alkaline hydrolysis, ethanol extraction, and hexane extraction. FA levels in these extracts were assessed using high performance liquid chromatography (HPLC), and alkaline hydrolysis was found to be the most effective. Furthermore, in the hydrolysate, FA was distinctly identified by thin layer chromatography (TLC) analysis. These results provide useful information for the quality control of the A. acutiloba crude drug.


Assuntos
Angelica/química , Ácidos Cumáricos/química , Raízes de Plantas/química , Plantas Medicinais/química , Controle de Qualidade
15.
J Nat Med ; 72(1): 267-273, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29149424

RESUMO

Saposhnikoviae radix (SR) is described in the Japanese Pharmacopoeia as a crude drug derived from the root of Saposhnikovia divaricata Schischkin (Umbelliferae). According to Flora of China, the root of Peucedanum ledebourielloides K. F. Fu is used as a regional substitute for SR. Therefore, we surveyed the botanical origin of the drug used in China, especially Shaanxi and the surrounding regions, through nucleotide sequence analysis of the internal transcribed spacer region of rDNA. As a result, several samples from Shaanxi () and Shanxi () provinces were identified as Peucedanum ledebourielloides. To prevent this substitute from being distributed as genuine SR, we developed a thin-layer chromatography analysis condition to enable a specific compound of this species to be easily detected. The specific compound was identified as xanthalin, based on 1D- and 2D-NMR and high-resolution mass spectrometry data. The established TLC conditions were as follows-extraction solvent, n-hexane; applied volume, 5 µL; chromatographic support, silica gel; developing solvent, n-hexane:ethyl acetate:acetic acid (20:10:1); developing length, 7 cm; detection, UV (365 nm); R f value, 0.4 (blue fluorescence; xanthalin).


Assuntos
Medicamentos de Ervas Chinesas/química , Raízes de Plantas/química , China
16.
Molecules ; 22(12)2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29206203

RESUMO

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 Medicinais
17.
Skin Pharmacol Physiol ; 30(4): 205-215, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28697505

RESUMO

Cosmetic industries have an interest in exploring and developing materials that have the potential to regulate melanin synthesis in human skin. Although melanin protects the skin from ultraviolet irradiation, excess melanin can be undesirable, particularly on the face where spots or freckles are associated with an appearance of aging. In this study, we found that ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11α-OH KA) in Pteris dispar Kunze strongly inhibited melanin synthesis by suppressing tyrosinase gene expression. The melanogenic transcription factor microphthalmia-associated transcription factor (MITF) is required for this suppression. However, 11α-OH KA did not modulate the expression level or activity of MITF. Structure-activity relationship analyses suggested that the 11α-OH, 15-oxo, and 16-en moieties of 11α-OH KA are essential for the suppression of melanin synthesis. On the other hand, the 19-COOH moiety is important for preventing cellular toxicity associated with 11α-OH KA and its related compounds. These results suggest that 11α-OH KA is an attractive target for potential use in the production of cosmetic items.


Assuntos
Diterpenos de Caurano/farmacologia , Melaninas/biossíntese , Preparações Clareadoras de Pele/farmacologia , Pele/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Monofenol Mono-Oxigenase/genética , Extratos Vegetais , Folhas de Planta , Pteris , Pele/metabolismo , Relação Estrutura-Atividade
19.
J Integr Med ; 15(3): 182-185, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28494848

RESUMO

There had been no standardized rules for citing ethical Kampo products used in clinical trials in journal articles. Although the name of a Kampo manufacturer was described in 77.9% of research articles, the name and ratios of crude drug components of Kampo formulas were not described in 77.5% of these papers. Considering the importance of proper characterization of interventions in the Consolidated Standards of Reporting Trials (CONSORT) checklist, we hereby propose the use of the Standards of Reporting Kampo Products (STORK) website, http://mpdb.nibiohn.go.jp/stork, as a reference for Kampo products. This will provide an official source on the internet for verified information on individual Kampo formulations for citation purposes in clinical research articles.


Assuntos
Acesso à Informação , Pesquisa Biomédica , Lista de Checagem , Medicamentos de Ervas Chinesas , Internet , Medicina Kampo , Relatório de Pesquisa/normas , Humanos , Japão , Padrões de Referência
20.
Plant Cell Physiol ; 58(5): 874-884, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28371833

RESUMO

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.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Plantas/metabolismo , Platycodon/metabolismo , Saponinas/metabolismo , Triterpenos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Plantas Medicinais/enzimologia , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Platycodon/enzimologia , Platycodon/genética
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