ABSTRACT
Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical 'seahorse' shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41 ℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.
Subject(s)
Phenylalanine Ammonia-Lyase/metabolism , Podophyllotoxin , Phylogeny , Cloning, MolecularABSTRACT
Objective: To study the chemical constituents from Sinopodophyllum hexandrum and their antitumor activities. Methods: The constituents were separated by chromatography of silica gel, ODS, Sephadex LH20 and pre-TLC. Their structures were elucidated by spectroscopic means. The in vitro cytotoxic activities of the isolated compounds were studied by MTT method. Results: Nine compounds were isolated and identified as 8,2'-diprenylquercetin 3-methyl ether-4'-O-β-D-glucoside (1), 8,2'-diprenyl quercetin-3- methylether (2), 5,7,4'-trihydroxy-3'-(3-methylbut-2-enyl)-3-methoxy flavone (3), 8-prenylkaempferol (4), sophoflavescenol (5), podoverine A (6), sinoflavonoid K (7), diosmetin (8) and acacetin (9). Conclusion: Compound 1 is a new compound named sinoflavonoid glycosides A, and compounds 5-9 are isolated from S. hexandrum for the first time. Compounds 1-5 show cytotoxicities against HeLa cells with IC50 of 42.6, 46.9, 26.9, 16.1 and 31.2 μmol/L, respectively.
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Objective To establish the preparative isolation technique of grahislactone A from the fruits of Sinopodophyllum hexandrum, and to investigate the action mechanism for apoptosis induction of graphislactone A in MCF-7/ADR cell line (human breast adencarcinoma cell line). Methods The fruits of S. hexandrum were extracted under ultrasound by EtOAc. Graphislactone A was enriched by silica gel column chromatography, and then purified by recrystallization. The cytotoxic activities of graphislactone A were tested against MCF-7 and MCF-7/ADR cell lines by MTT. The apoptosis of MCF-7/ADR cell line was evaluated by flow cytometry. RT-PCR was used to detect the expression of apoptosis-related genes in MCF-7/ADR cell line. Expression of P-gp in MCF-7/ADR cell line was investigated by western blotting. Results Graphislactone A was isolated from the fruits of S. hexandrum, which showed the potent cytotoxic activities against MCF-7 and MCF-7/ADR cell lines, with IC50 values of 2.38 and 9.35 μmol/L, respectively. With the increased dose of graphislactone A, the expression levels of p53, bax, caspase-3 genes were up-regulated, while bcl-2 and P-gp down-regulated in MCF-7/ADR cell line. Conclusion Graphislactone A as phenolic compoundswas firstly found in higher plants. The preparative isolation has the advantages of high-efficiency, high sample recovery, and high-purity compared with etoposide. Graphislactone A showed strong cytotoxic activities and apoptosis induction action in MCF-7 and MCF-7/ADR cell line.
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Objective To explore a new identification method for medicinal materials of Dysosma, and analyze the second internal transcribed spacer (ITS2) barcode sequences of Diphylleia sinensis and Dysosma versipellis, Sinopodophyllum hexandrum, Dysosma difformis and Dysosma pleiantha in five kinds of podophyllum. Methods The ITS2 of ribosomal DNA of medicinal materials of podophyllum was amplified and sequenced by bi-directional sequencing of PCR products. Sequence assembly and consensus sequence generation were performed by using CodonCode Aligner. Phylogenetic study was performed using software MEGA 5.1 in accordance with Kimura-2-parameter (K2P) model. Genetic distances were calculated and analyzed and the phylogenetic tree was constructed by using the neighbor-joining (NJ) method. Results There were significant differences among five kinds of Dysosma. Their maximum intraspecific genetic distance (K2P distance) was far lower than their minimum interspecific genetic distance with the other species. In the cluster dendrogram, all species showed monophyletic. Conclusion ITS2 sequence as DNA barcoding technique can be used to identify Chinese herbal materials of Dysosma.
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Objective: To explore the resource diversity of endophytic fungi from the endangered Sinopodophyllum hexandrum and its antimicrobial activity. Methods: The endophytic fungi were isolated by tissue block method. Five pathogenic fungi and four bacteria were used as indicators to test the antimicrobial activity by agar plate antagonistic action and improved agar gel diffusion methods. Results: The results indicated that 49 endophytic fungi strains were isolated from the roots, stems, and leaves of S. hexandrum, the most was from the roots (22), then from the stems (18), but the least was from the leaves (19); The isolated strains attributed to nine genera, three families, and two orders, and Fusidium sp. was the dominant genera based on morphological characters; For the isolated strains, 22 of them expressed the inhibition to one or more pathogenic fungi, amounting to 44.8% of the total isolates, and eight strains were found to have significant inhibition against two or more pathogenic fungi, making up 16.3% of the total isolated strains, and three isolated endophytic fungal strains showed the evident inhibition against four or more indicating pathogenic fungi, and the two isolated strains showed the obvious antibacterial activity against the two indicating bacteria. Conclusion: The endophytic fungi isolated from S. hexandrum have the diversity and evident inhibition against exterior fungi, active strains belong to the fungi of Aspergillus sp., Hormodendrum sp., and Fusidium sp., respectively, and its research and development are of ecological and economic significances.
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Objective To study the genetic diversity of Sinopodophyllum hexandrum so as to provide basic molecular genetic evidence for protecting and exploiting the resource of S.hexandrum.Methods Six populations of S.hexandrum were analyzed by DALP molecular markers.To make up the genetic diversity correlation data by PopGene 1.31 software and cluster by UPGMA method,and establish the dendrogram.The tree was subsequently visualized with Treeview software.Results Five primer groups were screened and a total of 150 DNA fragments were amplified,among which 104 were polymorphic,i.e.the percentage of polymorphic bands(PPB) was 69.33%,the average number of DNA polymorphic band amplified by each primer group was 20.8 and the average PPB was 14.22%.In the six populations,the total observed number of alleles(Na) was 1.693 3 and the average Na was 1.142 2;The total effective number of alleles(Ne) was 1.417 1 and the average Ne was 1.083 8;The total Nei′s gene diversity(H) was 0.244 3 and the average H was 0.048 8;The total Shannon′s information index(I) was 0.364 3 and the average I was 0.073 0;The total gene diversity(Ht) was 0.244 3 while the gene diversity within a population(Hs) was 0.048 7.The coefficient of gene differentiation(Gst) was 0.800 5 between populations,namely 80.05% genetic variation occurring between populations and the rest 19.95% within population,and estimated gene flow(Nm) was 0.124 6.Conclusion The result indicats that S.hexandrum has much larger genetic differentiation among populations and gene flow has been blocked.This might be a result of species breeding system and population habitat.