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Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe2+ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.
Subject(s)
Arabidopsis/metabolism , Rhododendron/metabolism , Amino Acid Sequence , Anthocyanins/metabolism , Phylogeny , Flavonoids/metabolism , Cloning, Molecular , Gene Expression Regulation, Plant , Plant Proteins/metabolismABSTRACT
This study was to investigate the chemical constituents from the aerial parts of Thymus przewalskii. The chemical consti-tuents were separated and purified by column chromatography on silica gel, ODS, Sephadex LH-20 and semi-prepared HPLC, and their structures were determined by physicochemical properties and spectroscopic data. Four flavanones were isolated from the ethanol extract of the aerial parts of T. przewalskii, and identified as(2S)-5,6-dihydroxy-7,8,4'-trimethoxyflavanone(1), 5,4'-dihydroxy-6,7-dimethoxyflavanone(2),(2S)-5,4'-dihydroxy-7,8-dimethoxyflavanone(3), sakuranetin(4), respectively. Compound 1 was a new compound and its configuration was determined by CD spectrum, compound 3 was natural product which was isolated for the first time and their configurations were determined by CD spectra. Compound 2 was isolated from the genus Thymus for the first time and compound 4 was isolated from T. przewalskii for the first time. Furthermore, cytotoxicity test was assayed for the four flavanones. They exhibited weak cytotoxicity against human lung cancer cells(A549), with the IC_(50) from 74.5 to 135.6 μmol·L~(-1).
Subject(s)
Humans , Chromatography, High Pressure Liquid , FlavanonesABSTRACT
(2S)-taxifolin is an important flavonoid that has anti-inflammatory and anti-oxidation effects. It is widely used in pharmaceutical and nutraceutical industries. Flavone 3-hydroxylase (F3H) can catalyze the synthesis of (2S)-taxifolin and other 3-hydroxylated flavonoids from (2S)-eriodictyol. Due to the low catalytic efficiency of F3H, the titer of many 3-hydroxyflavones, such as taxifolin, synthesized by microbial method is relatively low. In this study, a SmF3H was identified from the transcriptome of Silybum marianum (L.) Gaertn. The results of fermentation showed that SmF3H can catalyze the flavone 3-hydroxylation reaction, and its catalytic efficiency was significantly higher than that of commonly used SlF3H from Solanum lycopersicum. Six promoters with different transcription strength were selected to optimize the synthesis pathway from the flavonoid precursor (2S)-naringenin to (2S)-taxifolin. The results showed that the highest titer of (2S)-taxifolin (695.90 mg/L in shake flask) could be obtained when the P(GAL7) promoter was used to control the expression of SmF3H. The titer of (2S)-taxifolin was further improved to 3.54 g/L in a 5-L fermenter, which is the highest titer according to current available literatures.
Subject(s)
Antioxidants , Flavonoids , Milk Thistle , Quercetin/analogs & derivativesABSTRACT
Objective To obtain the key enzyme gene involved in flavone C-glycosides biosynthesis pathway, a flavanone 2-hydroxylase (F2H) gene was cloned from Microcos paniculata, and its bioinformatics analysis and gene expression pattern were also performed. Methods The specific primers were designed according to Unigene in F2H annotated in the transcriptome data of M. paniculata. The open reading frame (ORF) of MpF2H gene was amplified by PCR. Then the PCR product was purified and ligated to pET30a, and finally a prokaryotic expression vector pET30a-MpF2H was constructed. The bioinformation of F2H gene cDNA sequences was analyzed by some online tools. Using RT-qPCR with suitable primers, the quantitative expression analysis of MpF2H gene in different tissues, namely, buds, leaves, twigs, flowers and fruits was carried out. Results The length of MpF2H gene ORF was 1 557 bp (GenBank accession number KY652921), which encoded a protein with 518 amino acid residues, relative molecular weight of 54 500, theory isoelectric point of 5.49. In which was no transmembrane domain. It was hypothesized that this protein located in chloroplast. MpF2H gene was expressed in different tissues, with the highest expression in leaves and the lowest expression in twigs and flowers. Conclusion The expression of MpF2H gene varied widely in different tissues. The MpF2H gene was cloned from M. paniculata based on pET30a-MpF2H expression vector. This study will provide the fundamental information for the further preparation and functional research of MpF2H protein in flavone C-glycosides biosynthesis pathway.
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Objective In order to study the key genes involved in flavonoid biosynthesis pathway, the flavanone-3-hydroxylase (F3H) gene was isolated from Lepidium apetalum, which is named as LaF3H. Meanwhile, the sequence analysis, prokaryotic expression, and purification were also performed. Methods Specific primers were designed according to LaF3H gene sequences in the transcriptome data of L. apetalum, and the cDNA sequence of LaF3H gene was isolated from L. apetalum. By construction the prokaryotic expression vector pET-32a-LaF3H, the recombinant LaF3H protein was expressed in Escherichia coli BL21 (DE3) cells under IPTG induction. Results The open reading frame (ORF) of LaF3H was 1 080 bp, which encoded a protein of 359 amino acid residues, with a predicted molecular mass of 40 320. Sequence analysis showed that LaF3H contains five conserved motifs of F3H protein. The phylogenetic analysis indicated that LaF3H protein showed the highest homology with F3H protein from cruciferous plants (such as AtF3H from Arabidopsis thaliana). The prokaryotic expression vector pET-32a-LaF3H was constructed and the recombinant LaF3H protein was successfully expressed in E. coli BL21 (DE3) cells. Furthermore, the recombinant LaF3H protein was purified through Ni2+ affinity chromatography. Conclusion The LaF3H gene was isolated from L. apetalum and the recombinant LaF3H protein was obtained. The results of this study provided the foundation for the further preparation of LaF3H antibody and detection of LaF3H enzyme activity, and were helpful for functional characterization of LaF3H gene involved in flavonoid biosynthesis pathway of L. apetalum.
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Objective: To clone flavanone 3-hydroxylase (F3H) gene from Lithocarpus polystachyus, and to understand its gene characteristics and initially investigate its expression level in different organs. Methods: The total RNA and genomic DNA from blade of L. polystachyus were extracted. Based on the result of RNA-seq, a pair of specific primers were designed. cDNA and DNA sequences of F3H gene from L. polystachyus were amplified by PCR, then bioinformation analysis was performed after sequencing. The expression level of F3H gene in different organs of L. polystachyus was detected by qRT-PCR. Results: The full length of cDNA of F3H gene was 1 340 bp containing a 1 092 bp open reading frame that encoded 393 amino acids, and F3H was located in the cytoplasm. The result of qRT-PCR showed that F3H gene expressed in different organs of L. polystachyus, and the expression levels of F3H gene were significantly different in different organs (P < 0.05). Conclusion: The F3H gene of L. polystachyus was cloned and its bioinformation was analyzed for the first time, proving that the expression level of F3H gene in different organs of L. polystachyus was different. This finding lays a foundation for the studies on secondary metabolism of flavonoids in L. polystachyus.
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Two flavanone glucosides were isolated from the 80% ethanol extract of Glycyrrhiza uralensis using various chromatographic techniques, including macroporous adsorbent resin, RP-C18, Sephadex LH-20, MCI and preparative HPLC. On the basis of chemical properties and spectra data, these compounds were identified as (2S)-liquiritigenin-4'-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranoside (1), (2R)-liquiritigenin-4'-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranoside (2), respectively. Compounds 1 and 2 are new compounds, and their aglycones are enantiomers.
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Research progress of flavanone compounds synthesis was reviewed ,various types of synthesis methods of fla‐vanone compounds were summarized ,and methodological support for flavanone compounds in new drug research and develop‐ment was provided .Research progress in synthesis of flavanone compounds includes chalcone cyclization ,Friedel‐Crafts reac‐tion ,Knoevenagel condensation ,Hoesch single acetoxylation ,and asymmetric synthesis of flavanone compounds .Solvent‐free synthesis of flavanone compounds and other green chemistry methods were also introduced .
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In this work, a bivalent RNA interference (RNAi) plant-transformation vector was constructed to silence both the flavanone 3-hydroxylase (F3H) gene and the flavone synthase II (GmFNSII) gene in soybean (Glycine max). Two further unit RNAi vectors were constructed for each of these two genes. RNAi-mediated suppression of these genes effectively regulated flavone and isoflavone production in hairy roots that arose from soybean cotyledons transformed with Agrobacterium rhizogenes ATCC15834. Notably, the bivalent RNAi vector had a significantly higher effect for increasing isoflavone production compared with the two unit RNAi vectors. The study highlighted molecular methods that could be used to enhance isoflavone production in soybean and demonstrated the challenges associated with such metabolic engineering for the production of plant natural products.
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Objective: To study the phenolic constituents of Draconis Resina. Methods: The compounds were isolated and purified by silica gel and sephadex LH-20 column chromatography, and their structures were identified by physiochemical properties and MS and NMR spectroscopic data. Results: Fourteen compounds were isolated from the ethyl acetate extract of Draconis Resina and identified as 4'-hydroxy-1', 4″-dimethoxychalcane (1), 7-hydroxy-5, 4'-dimethoxy-2-arylbenzofuran (2), pterostilbene (3), 3, 4'-dihydroxy-5- methoxystilbene (4), 5, 7, 4'-trihydroxyflavanone (5), pinocembrin (6), 7-hydroxyflavanone (7), liquiritigenin (8), methyl 4- hydroxybenzoate (9), methyl 4-hydroxy-3-methoxybenzoate (10), ethyl 4-hydroxy-3-methoxybenzoate (11), 2, 4-dihydroxy acetophenone (12), 3, 4-dihydroxyallybenzene (13), and β-sitosterol (14). Conclusion: Compound 1 is a new natural product. Compound 2 is firstly obtained from the plants of Liliaceae. Compounds 5 and 9-12 are firstly isolated from the plants of Dracaena Vand. ex L., and compounds 6 and 7 are isolated from Dracaena cochinchinensis for the first time.
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<p><b>OBJECTIVE</b>To investigate the butanol fraction of the water/acetone extract and isolate of the new flavonoids from Launeae arboescens.</p><p><b>METHODS</b>The compounds were isolated by liquid chromatographic methods and their structures were identified by using spectroscopic analysis.</p><p><b>RESULTS</b>THE ISOLATED COMPOUNDS WERE IDENTIFIED AS: 7-O-[α-rhamnopyranosyl 4',5,6-Trihydroxy flavone 1,4',5'-Di-Methoxy 7-(5″-Me Hexan)1-oyl flavanone 2, 3″-isopropyl pyrano [1″:7,4″:6] 3',4',5',5-Tetrahydroxy flavanone 3,5,4',5'-Tri-Hydroxy 7-(3″-Me butan) -yl flavanone 4, 5,7-Dihydroxy-2',4',5' -trimethoxy-isoflavanone 5,5,6,7,4'-tetrahydroxy flavonol 6,7-O-[α-rhamnopyranosyl-(1->6)-β-glucopyranosyl]- 4',5,7-tri-hydroxy-flavanone 7,7-O-[α-rhamnopyranosyl-(1->6)-β-glucopyranosyl] 3',5-Dihydroxy 4'-Methoxy flavanone 8.</p><p><b>CONCLUSIONS</b>The presence of different types of bioactive flavonoids in Launeae arboescens extract can explain the large ethnopharmacological uses and the potential activity of this medicinal plant.</p>
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Objective: To investigate the butanol fraction of the water/acetone extract and isolate of the new flavonoids from Launeae arboescens. Methods:The compounds were isolated by liquid chromatographic methods and their structures were identified by using spectroscopic analysis. Results:The isolated compounds were identified as: 7-O-[α-rhamnopyranosyl 4',5,6-Trihydroxy flavone 1,4’,5’-Di-Methoxy 7-(5’’-Me Hexan)1-oyl flavanone 2, 3”-isopropyl pyrano [1”:7,4”:6] 3’,4’,5’,5-Tetrahydroxy flavanone 3,5,4’,5’-Tri-Hydroxy 7-(3’’-Me butan) -yl flavanone 4, 5,7-Dihydroxy-2',4',5' –trimethoxy-isoflavanone 5,5,6,7,4'-tetrahydroxy flavonol 6,7-O-[α-rhamnopyranosyl-(1->6)-β-glucopyranosyl]- 4',5,7-tri-hydroxy-flavanone 7,7-O-[α-rhamnopyranosyl-(1->6)-β-glucopyranosyl] 3',5-Dihydroxy 4’-Methoxy flavanone 8. Conclusions:The presence of different types of bioactive flavonoids in Launeae arboescens extract can explain the large ethnopharmacological uses and the potential activity of this medicinal plant.
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Chromatographic procedures from the aerial parts of Abies nobilis and stem barks of Prosopis africana led to the isolation of two (antimicrobial and cytotoxic) compounds. Their structures were established as 7, 3’, 4’- trihydroxy-3-methoxyflavanone and dehydroabietic acid on the basis of spectroscopic techniques.
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OBJECTIVE: To investigate the chemical constituents of the leaves of Engelhardtia roxburghiana. METHODS: After the plant materials were extracted and fractionated with solvents, column chromatography was used to isolate and purify the compounds. The structures were elucidated on the basis of 1H-NMR, 13C-NMR, MS analysis and comparison with the literature data. RESULTS: Thirteen compounds were identified as following; taxilfolin (1), 5,7,4'-trihydroxy flavanone(2), kaempferol (3), dihydrokaempferol (4), quercetin(5), isoengeletin(6), engeletin (7), astilbin(8), isoastilbin(9), kaempferol-3-O-α-L-rhamnoside(10), eucryphin (11), 5-hydroxy-3,7,3',4'-tetramethoxyflavone(12), quercetin-3-O-α-L-rhamnoside (13). CONCLUSION: Compounds 2, 4, 9, 11 and 12 were identified from the title plant for the first time.
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AIM: To develop a rapid HPLC method for quality control of traditional Chinese medicinal ingredients consisted of citrus flavonoids, naringin, hesperidin, neohesperidin, sinensetin and nobiletin. METHODS:Gradient elution with non-salt mobile phase ( methanol and water only) HPLC method on a Kromasil column ( 100-5C18-250A, 4.6 mm ×250 mm, 5 μm, C18 reverse phase) with peaks identification through DAD full UV wavelength scan. UV 284 nm and 332 nm profiles were observed. RESULTS: Satisfactory resolution, linearity, 95%～ 102% of recovery and 1.88 ～ 2.93% of repeatability were obtained for those five citrus flavonoids. Content of 6 Citrus aurantium L. based TCM ingredients were analyzed and identified. CONCLUSION: Rapid HPLC test method on citrus flavonoids was developed and can be in LC-MS identification.
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The aim of this study was to look for the chemical constituents from the rhizomes of Dryopteris sublaeta. The fresh plant was extracted twice with boiling water, the extract was concentrated to small volume under reduced pressure at 50 ℃. The concentrated material was partitioned with ether, ethyl acetate and n-butanol. The fraction of ethyl acetate was repeatedly chromatographied over silica gel and Sephadex LH-20 columns. Structures of pure compounds were established on the basis of their physiochemical and spectral data. Nine compounds were obtained and identified as sublaetentin A (1), sublaetentin B (2), sublaetentin C (3), sublaetentin D (4), matteuorienate A (5), matteuorienate C (6), arbutin (7), 3-methoxy-4-hydroxyphenyl-1-O-β-D-glucopyranoside (8) and 3,4-dimethoxyphenyl-1-O-β-D-glucopyranoside (9). Compounds 1-4 are new compounds, the others were isolated from this plant for the first time.
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Aim To study the chemical constituents of Dryopteris sublaeta Ching et Hsu. Methods Fresh plant of Dryopteris sublaeta Ching et Hsu was extracted twice with boiling water, the extract was concentrated to small volume under reduced pressure at 50 ℃. The concentrated material was partitioned with ether, ethyl acetate, and n-butanol. The fraction of ether extract was chromatographed over silica gel column. The compounds were identified on the basis of their physiochemical and spectral data. Results Four compounds were obtained and identified as 2 (S)-5,7, 3'-trihydroxy-6,8-dimethy1-5'-methoxyflavanone ( 1 ), matteucinol ( 2 ), desmethoxymatteucinol ( 3 ) and 5,7,2'-trihydroxy-6,8-dimethy1-flavanone (4). Conclusion Compound 1 is a new one, the others were isolated from Dryopteris for the first time.
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Objective To study the chemical constituents in the leaves of Mallotus apelta. Methods Constituents isolation and purification were carried out on silica gel and polyamide column. Their structures were identified by physicochemical properties and spectral analysis. Results Five compounds were isolated and elucidated as taraxerol (Ⅰ), ?-sitosterol (Ⅱ), 5, 7-dihydroxy-6-prenyl-4′-methoxy-flavanone (Ⅲ), apigenin (Ⅳ), and apigenin-7-O[WTBZ]-?-D-glucoside (Ⅴ). Conclusion Compound Ⅲ is a new compound named as mallotusin. Compounds Ⅰ and Ⅲ-Ⅴ are isolated from the leaves of M. apelta for the first time.