Identification and expression analysis of cellulose synthase family genes in Aquilaria sinensis / 药学学报

Cellulose synthase (CesA), one of the key enzymes in the biosynthesis of cellulose in plants, plays an important role in plant growth and plant resistance. In this study, a total of 21 AsCesA genes from Aquilaria sinensis were systematically identified and the physico-chemical characteristics were analyzed based on genome database and bioinformatical methods. The phylogenetic tree was constructed and the gene location on chromosome, cis-acting elements in the 2 000 basepairs upstream regulatory regions and conservative motifs were analyzed. The AsCesA proteins were mainly located on the plasma membrane. The number of amino acids of the proteins ranged from 390 to 1 261. The isoelectric point distributed from 5.67 to 8.86. All of the 21 AsCesA proteins possessed the transmembrane domains, the number of which was from 6 to 8. The genes were classified into 3 groups according to the phylogenetic relationship. Obvious differences were observed in motif composition in genes from different groups. However, motif2, motif6, motif7 and motif10 were observed in all of AsCesA proteins. Analysis of cis-acting elements indicated that AsCesA genes family has cis-acting elements related to plant hormones, abiotic stresses, and biological processes. Seven AsCesA genes with differential expression were selected according to the calli transcriptome data induced by NaCl at different times and their expression levels under different abiotic stresses were analyzed by quantitative real-time PCR. The results indicated that salt, low temperature, drought, and heavy metal stresses could affect the expression level of AsCesA genes, and the abundance of AsCesA1, AsCesA3 and AsCesA20 showed a significant change, implying their potential important roles to the abiotic stresses. The accumulation pattern of cellulose content under different abiotic stresses was similar to the expression trend of AsCesA genes. Our results provide valuable insights into the role of cellulose synthase in A.sinensis in plant defense.

LC-MS analysis of 2-(2-phenylethyl) chromones in sodium chloride-treated suspension cells of Aquilaria sinensis / 中国中药杂志

Qualitative and quantitative analysis of 2-(2-phenylethyl) chromones in sodium chloride(NaCl)-treated suspension cells of Aquilaria sinensis was conducted by UPLC-Q-Exactive-MS and UPLC-QQQ-MS/MS. Both analyses were performed on a Waters T3 column(2.1 mm×50 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as mobile phases at gradient elution. MS data were collected by electrospray ionization in positive ion mode. Forty-seven phenylethylchromones was identified from NaCl-treated suspension cell samples of A. sinensis using UPLC-Q-Exactive-MS, including 22 flindersia-type 2-(2-phenylethyl) chromones and their glycosides, 10 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and 15 mono-epoxy or diepoxy-5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones. Additionally, 25 phenylethylchromones were quantitated by UPLC-QQQ-MS/MS. Overall, the rapid and efficient qualitative and quantitative analysis of phenylethylchromones in NaCl-treated suspension cells of A. sinensis by two LC-MS techniques, provides an important reference for the yield of phenylethylchromones in Aquilariae Lignum Resinatum using in vitro culture and other biotechnologies.

Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products / 中国中药杂志

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.

Cloning, expression analysis and enzyme activity verification of dihydroflavonol 4-reductase from Cistanche tubulosa (Schenk) Wight flower / 药学学报

Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.

Cloning, expression and functional identification of cytochrome P450 reductase gene in Aquilaria sinensis / 药学学报

Cytochrome P450 reductase (CPR) is essential for the electron transport chain of cytochrome P450s, playing an indispensable role in electron transfer in vivo. In this study, one cDNA encoding cytochrome P450 reductase (Ascpr1) was identified from the callus of Aquilaria sinensis. Ascpr1 contains an open reading frame of 2 124 bp. The deduced protein is composed of 707 amino acids, with a predicted molecular weight of 78.82 kD. Phylogenetic analysis revealed that AsCPR1 is a type Ⅱ CPR protein closely related to the CPR from Theobroma cacao. Transmembrane prediction using TMHMM 2.0 indicated that the amino acids 52-71 of AsCPR1 comprise a transmembrane region. After truncating of 67 amino acid residues from N-terminal, the truncated AsCPR1 was successfully expressed in E. coli Transetta (DE3). Further purification of the recombinant AsCPR1 by affinity chromatography and determination of the enzymatic activity allowed the reducing ability of AsCPR1 to cytochrome C in vitro. The results pave the way for further study on the synthesis of defensive chemicals involved in P450s and the functions of CPR in self-defense of A. sinensis.

Cloning and expression analysis of chalcone isomerase from Aquilaria sinensis / 药学学报

Chalcone isomerases (CHIs) play an essential role in the biosynthesis of flavonoids important in plant self-defense. Based on the transcriptome data of Aquilaria sinensis Calli, a full-length cDNA sequence of CHI1 (termed as AsCHI1) was cloned by reverse transcription PCR. AsCHI1 contains a complete open frame (ORF) of 654 bp. The deduced protein is composed of 217 amino acids, with a predicted molecular weight of 23.11 kDa. The sequence alignment and phylogenetic analysis revealed that AsCHI1 has conserved most of the active site residues in type I CHIs, indicating a close relationship with the CHI from Gossypium hirsutum. The recombinant AsCHI1 protein was obtained by heterologous expression of AsCHI1 in E. coli BL21(DE3). The purified AsCHI1 protein exhibited CHI activity by catalyzing the production of naringenin from naringenin chalcone. Remarkably, AsCHI1 expression in A. sinensis Calli treated with various abiotic stresses including salt, mannitol, cold, and heavy metals could be markedly increased, and plant hormones such as abscisic acid (ABA), gibberellin (GA3), and salicylic acid (SA) could also increase the expression of AsCHI1, suggesting that AsCHI1 might play an important role in plant self-defense. The results expand our understanding of the biosynthesis of flavonoids in A. sinensis and give further insight into the defensive responses of A. sinensis to abiotic and biotic stresses.

Isolation and identification of endophytic fungi from Huperzia serrata and their metabolites' inhibitory activities against acetylcholinesterase and anti-inflammatory activities / 中国中药杂志

A total of 27 endophytic fungal strains were isolated from Huperzia serrata,which were richly distributed in the stems and leaves while less distributed in roots. The 27 strains were identified by Internal Transcribed Spacer( ITS) r DNA molecular method and one of the strains belongs to Basidiomycota phylum,and other 26 stains belong to 26 species,9 general,6 families,5 orders,3 classes of Ascomycota Phylum. The dominant strains were Colletotrichum genus,belonging to Glomerellaceae family,Glomerellales order,Sordariomycetes class,Ascomycota Phylum,with the percentage of 48. 15%. The inhibitory activities of the crude extracts of 27 endophytic fungal strains against acetylcholinesterase( ACh E) and nitric oxide( NO) production were evaluated by Ellman's method and Griess method,respectively. Crude extracts of four fungi exhibited inhibitory activities against ACh E with an IC50 value of 42. 5-62. 4 mg·L~(-1),and some fungi's crude extracts were found to inhibit nitric oxide( NO) production in lipopolysaccharide( LPS)-activated RAW264. 7 macrophage cells with an IC50 value of 2. 2-51. 3 mg·L~(-1),which indicated that these fungi had potential anti-inflammatory activities.The chemical composition of the Et OAc extract of endophytic fungus HS21 was also analyzed by LCMS-IT-TOF. Seventeen compounds including six polyketides,four diphenyl ether derivatives and seven meroterpenoids were putatively identified.

Expression analysis of allene oxide synthase gene from Aquilaria sinensis / 药学学报

Jasmonic acid (JA) is an important signal molecule involved in plant resistance, and allene oxide synthase (AOS) is a key enzyme in the biosynthesis of jasmonates. In this study, a full-length cDNA of AsAOS1 gene was cloned from Aquilaria sinensis. Meanwhile, the sequence analysis, prokaryotic expression, purification, tissue-specific expression analysis and expression analysis under different abiotic stresses and hormone treatments were performed. The open reading frame (ORF) of AsAOS1 gene was 1 575 bp, encoding a protein of 524 amino acid residues, with a predicted molecular mass of 58.70 kDa. AsAOS1 protein possessed the conserved sequences of cytochrome P450 (CYP450). The phylogenetic analysis indicated that AsAOS1 protein had the highest level of homology with AOS protein of Citrus sinensis. The recombinant AsAOS1 protein was successfully expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-AsAOS1 and the recombinant AsAOS1 was purified by Ni2+ affinity chromatography. Expression analysis results in different tissues showed that AsAOS1 was primarily observed in stems, and then roots, followed by leaves. AsAOS1 transcript level was significantly induced after 12 h treatment of NaCl, cold temperature and CdCl2. Furthermore, AsAOS1 expression level was enhanced upon methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. However, mannitol and gibberellin (GA3) treatments had little influence on the expression level of AsAOS1. These results provides valuable insights into the role of JA in the mechanism of agarwood formation and plant resistance.

Cloning and expression analysis of transcription factor AsMYB1 and AsMYB2 from Aquilaria sinensis / 中国中药杂志

The MYB gene family comprises one of the richest groups of transcription factors in plants. The full length of two MYB genes were isolated through heterologous screening of Aquilaria sinensis calli transcriptome data, and the reverse transcription PCR was performed to obstain the corrected MYB clones, named AsMYB1, AsMYB2. The MYB transmembrane domain and phylogenetic analysis were predicted by different software to analyze the bioinformatics of MYB proteins. The transcript level of AsMYB1, AsMYB2 was performed by real-time quantitative RT-PCR in different tissues and in responds to abiotic stresses including salt, cold, metal and drought stress, and hormone treatments including abscisic acid (ABA), salicylic acid (SA), gibberellins (GA3) and methyl jasmonate (MeJA) treatment. The AsMYB1 cDNA sequence had an ORF of 1 063 nucleotides, encoding a protein of 353 amino acids. The largest AsMYB2 ORF was 1 081 nucleotides, and its predicted translation products consisted of 359 amino acids. Two MYB genes had a tissues-specific pattern in A. sinensis. Moreover, the expression level of AsMYB1 and AsMYB2 was regulated by different abiotic stresses and hormone treatments, suggesting the transcription factors AsMYB1 and AsMYB2 play an important role in plant defense and hormone signal transduction in A. sinensis.

Triterpenoids from the roots of Rubus parvifolius / 中国天然药物

Two new oleanane-type triterpenoids, parvifolactone A (1) and rubuside P (2), together with 11 known triterpenoids, fupenzic acid (3), 18,19-seco,2α,3α-dihydroxyl-19-oxo-urs-11,13(18)-dien-28-oic acid (4), euscaphic acid (5), maslinic acid (6), 1β- hydroxyeuscaphic acid (7), 2α,3α,19α,23-tetrahydroxyolean-12-en-28-oic acid (8), 2α,3β,19α,23-tetrahydroxyurs-12-en-28-oic acid (9), glucosyl pinfaensate (10), rubuside J (11), 2α,3α,19α,23-tetrahydroxyurs-12-en-24,28-dioic acid (12), and 2α,3β,19α- trihydroxyurs-12-en-23,28-dioic acid (13), were isolated from the roots of Rubus parvifolius.

Research progress of plant BAHD acyltransferase family / 中国中药杂志

Acylation conducted by acyltransferase is a ubiquitous process in structure modification of secondary metabolites. It plays an important role in the structural diversity of natural products and contributes significantly to their improved stabilities, increased solubilities, and enhanced bioavailabilities. BAHD acyltransferase family is a typical kind of acyltransferase original from plants, which involved in the biosynthesis of various bioactive acylated natural products. In order to provide references for future investigations of BAHD acyltransferase family, research progresses on basic properties, three-dimensional structures, catalytic mechanisms, enzymatic functional identifications and phylogenetic analyses of BAHD family from plants is summarized in this paper.

Simultaneous determination of 14 organic acids in Shenfu injection by hydrophilic interaction chromatography-tandem mass spectrometry / 中国中药杂志

Organic acids are widely distributed in plants and related products, and participate in a wide range of metabolic pathways (e.g. tricarboxylic acid cycle), showing diverse pharmacological activities. As a widely used Chinese patent medicine, its adverse reactions are often reported. Therefore, we should further clarify the chemical components of Shenfu injection, and prepare strict quality standards to ensure the safety and effectiveness of its clinical use. Shenfu injection is prepared from red ginseng (steamed roots of Panax ginseng) and black prepared lateral roots of Aconitum carmichaelii (Heishunpian) by using modern extraction process, and organic acids are regarded as one of its main components. In current study, a hydrophilic interaction chromatography (HILIC) coupled with mass spectrometric method (HILIC-LC-MS) was developed and validated for the simultaneous determination of 14 organic acids, including cinnamic acid, ferulic acid, 4-hydroxylbenzoic acid, L-(+)-lactic acid, adipic acid, fumaric acid, caffeic acid, succinic acid, maleic acid, malonic acid, D-malic acid, (-)-shikimic acid, D-tartaric acid, and quinic acid in Shenfu injection. Satisfactory retention and separation were achieved for all organic acids on HILIC chromatographic column. Except cinnamic acid (231 μg•L⁻¹), lactic acid (113 μg•L⁻¹) and malonic acid (32.5 μg•L⁻¹), the limit of quantitation for the remaining 11 compounds were less than 10 μg•L⁻¹. D-Malic acid, malonic acid, quinic acid, L-(+)-lactic acid, and cinnamic acid were observed to have higher contents in Shenfu injection (>1.89 mg•L⁻¹), whereas caffeic acid and adipic acid were undetectable in all batches. Above all, the developed method is suitable for the simultaneous determination of organic acids in Shenfu and some other traditional Chinese medicine injections.

Cloning and expression analysis of glucose-6-phosphate dehydrogenase 1 (G6PDH1) gene from Chimonanthus praecox / 中国中药杂志

Glucose-6-phosphate dehydrogenase is main regulatory enzyme for pentose phosphate pathway. To amplify the core sequence of G6PDH gene from Chimonanthus praecox, the primers were synthesized, based on the conserved nucleotide sequence of other reported plant G6PDH genes. The specific primers were designed according to the major fragment. The full length cDNA of the G6PDH1 gene was isolated by the 3' and 5' rapid amplification of cDNA ends approach. Transcript levels of G6PDH1 isoform was measured by real-time quantitative RT-PCR in different tissues and in responds to cold treatment. The G6PDH1 subcellular localization, transmembrane domain, three-dimensional structure, and phylogenetic analysis were predicted by different software to analysis the bioinformatics of G6PDH1 protein. The G6PDH1 cDNA sequence was 2 011 bp in length and consisted of 1 551 bp Open Reading Frame (ORF) , encoding a protein of 516 amino acids. Expression analysis results in different tissues showed that G6PDH1 was primarily observed in flowers and roots, as opposed to the leaves and stems. Cold treatment experiments indicated that cold treatment caused a rapid increase in G6PDH1 expression in flowers within 12 h. The full-length cDNA of G6PDH1 and its expression analysis will play an important role for further study on cold stress responses in Ch. praecox.

Studies on chemical constituents from root of Clematis hexapetala / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents from the roots and rhizomes of Clematis hexapetala.</p><p><b>METHOD</b>The compounds were separated by means of solvent extraction, repeated chromatography with silica gel and HPLC. The structures were determined by spectral analysis.</p><p><b>RESULT</b>Nine compounds were isolated as friedelin (1) , anemonin (2) , beta-sitosterol (3) , palmitic acid (4) , vanillic acid (5) , isolariciresinol (6) , 5-hydroxumethyl-5H-furan-2-one (7) , n-nonane (8) , daucosterol (9).</p><p><b>CONCLUSION</b>All the compounds were isolated from the plant for the first time.</p>

The isolation and identification of a new lignanoside from Selaginella tamariscina (Beauv.) Spring / 药学学报

<p><b>AIM</b>To study the chemical constituents of Selaginella tamariscina (Beauv.) Spring.</p><p><b>METHODS</b>The compounds were isolated and purified by macroporous adsorption resin, Sephadex LH-20 and silica gel column chromatography and identified on the basis of their physicochemical and spectral data.</p><p><b>RESULTS</b>Four compounds were obtained from the n-BuOH fraction of 70% acetone extracts. Their structures were elucidated as (7S, 8R)-7, 8-dihydro-7-(4-hydroxy-3,5-dimethoxyphenyl)-8-hydroxymethyl-[1'-( 7'-hydroxyethyl)-5' methoxyl] benzofuran-4-O-beta-D-glucopyranoside (tamariscinoside C, I), D-mannitol (II), tyrosine (II), shikimic acid (IV).</p><p><b>CONCLUSION</b>Compound I is a new compound, compounds II and III were obtained from the genius for the first time, compound IV was yielded from the plant for the first time.</p>

Isolation and structural identification of chemical constituents from Selaginella tamariscina (Beauv.) Spring / 药学学报

<p><b>AIM</b>To study the chemical constituents of the water-extracts of Selaginella tamariscina (Beauv.) Spring.</p><p><b>METHODS</b>Various chromatographic techniques were used to separate and purify the constituents. Their physico-chemical properties and spectral data were used to elucidate the structures.</p><p><b>RESULTS</b>Nine compounds were isolated and identified as (2R,3S)-dihydro-2- (3',5'-dimethoxy-4'-hydroxyphenyl)-7-methoxy-5-acetyl-benzofuran (1), 3-hydroxy-phenpropionic acid-(2'-methoxy-4'-carboxy-phenol) ester (tamariscina ester A, 2), sygringaresinol (3), 1-(4'-hydroxyl-3'-methoxyphenyl)glycerol (4), ferulic acid (5), caffeic acid (6), vanillic acid (7), syringic acid (8), and umbelliferone (9).</p><p><b>CONCLUSION</b>Compound 1 and 2 are new compounds, and the others were isolated from Selaginella for the first time.</p>

Study on the chemical constituents of Selaginella tamariscina (Beauv.) Spring / 药学学报

<p><b>AIM</b>To study the chemical constituents of Selaginella tamariscina (Beauv.) Spring.</p><p><b>METHODS</b>Various chromatographic techniques were used to separate and purify the chemical constituents. Their physico-chemical properties and spectral data were used to elucidate the structures.</p><p><b>RESULTS</b>Four compounds were isolated from the n-BuOH fraction of the water-extracts. Their structures were identified as 1-hydroxy-2-[2-hydroxy-3-methoxy-5-(1-hydroxyethyl)-phenyl]-3-(4-hydroxy-3,5-dimethoxy)-propane-1-O-beta-D-glucopyranoside (tamariscinoside B, I), adenosine (II), guanosine (III), arbutin (IV).</p><p><b>CONCLUSION</b>Tamariscinoside B (I) is a new compound, while the others were isolated from Selaginella for the first time.</p>