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Xanthocillin is a unique natural product with an isonitrile group and shows remarkable antibacterial activity. In this study, the genome of an endophytic fungus Penicillium chrysogenum MT-40 isolated from Huperzia serrata was sequenced, and the gene clusters with the potential to synthesize xanthocillin analogues were mined by local BLAST and various bioinformatics analysis tools. As a result, a biosynthetic gene cluster (named for) responsible for the biosynthesis of xanthocillin analogues was identified by further heterologous expression of the key genes in Aspergillus oryzae NSAR1. Specifically, the ForB catalyzes the synthesis of 2-formamido-3-(4-hydroxyphenyl) acrylic acid, and the ForG catalyzes the dimerization of 2-formamido-3-(4-hydroxyphenyl) acrylic acid to produce the xanthocillin analogue N, N'-(1, 4-bis (4-hydroxyphenyl) buta-1, 3-diene-2, 3-diyl) diformamide. The results reported here provide a reference for further discovery of xanthocillin analogues from fungi.
Subject(s)
Penicillium chrysogenum/genetics , Huperzia/microbiology , Acrylates , Multigene FamilyABSTRACT
Objective: To investigate the chemical metabolites from Cercospora lagenariae MT-45, an endophytic fungus isolated from Huperzia serrata (Thunb.) Trev. Methods: The compounds were isolated and purified by using silica gel column chromatography and semi-preparative liquid chromatography. The structures were established using physicochemical properties and MS and NMR. The anti-inflammatory activities of all the isolates were also preliminarily investigated by using in vitro model. Results: Nine polyketide derivatives including cercolagenlic acid A (1), alternariol (2), alternariol 9-methyl ether (3), (+)-nigrosporaol A (4), alternarienonic acid B (5), 2-methyl-5-carboxymethyl-7-hydroxychromone (6), 2,5-dimethyl-7-hydroxychromone (7), 1-deoxy- rubralactone (8), and (-)-alternarlactam (9) were isolated from C. lagenariae MT-45 fermented on brown rice solids. Conclusion: Compound 1 is a new compound, and compound 6 can exhibit a certain inhibition on the nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells with an IC50 value of (57.5 ± 1.2) μmol/L.
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Objective: To analyze the differences in protein of thallus of Huperzia serrata (in vitro cultures, strains A, B, and C, respectively) with different Hup A content, and to explore related enzymes that may be synthesized with Hup A accumulation. Methods: Quantitative proteomics was performed in vitro cultures of Huperzia serrata with different Hup A content using quantitative proteomics tandem mass tag (TMT) techniques, followed by differential protein analysis: GO, KEGG and other biological information analysis. Results: Strain B hadthe lowest Hup A content and strain C had the highest Hup A content, which was twice than that of B. There were 78 differential proteins between the strain B and C. Analysis of differential protein GO enrichment showed that MF accounted for 28.75%; Analysis of differential protein expression showed that three strains shared two differential proteins (P93541, Q8RXU4) in the alkaloid metabolic pathway starting from amino acids. P93541 protein was down-regulated in the low-yield strain B and up-regulated in the relatively high-yield strains A and C. The Q8RXU4 protein was up-regulated in the low-yield strain B and down-regulated in the relatively high-yield strains A and C. Conclusion: This study found that the difference in Hup A content was positively correlated with the protein expression. Two enzymes P93541 and Q8RXU4 that may be related to Hup A accumulation were analyzed, providing a basis for bioinformatics analysis of Hup A biosynthesis.
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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.
Subject(s)
Animals , Mice , Acetylcholinesterase , Anti-Inflammatory Agents , Pharmacology , Ascomycota , Chemistry , Classification , Cholinesterase Inhibitors , Metabolism , Endophytes , Classification , Huperzia , MicrobiologyABSTRACT
Objective To investigate the chemical constituents from Penicillium chrysogenum MT-12, an endophytic fungus isolated from Huperzia serrata. Methods The compounds were isolated and purified by using various column chromatographies including silica gel, Sephadex LH-20, and semi-preparative HPLC. The structures were established using extensive spectroscopic techniques such as IR, MS, and NMR. The anti-inflammatory and AChE inhibitory activities of all the isolates were also preliminarily investigated by using in vitro models. Results Eight diphenyl ether derivatives including penicichrysogenillide A (1), penicichrysogenillide B (2), talaromyone A (3), isopenicillide (4), penicillide (5), hydroxypenicillide (6), purpactin A (7), and penicichrysogenillide C (8) were isolated from the solid fermentation cultures of P. chrysogenum MT-12. Conclusion Compounds 1 and 2 are new compounds, and this is the first report for the isolation of compound 3 from Penicillium species and compounds 4-8 from P. chrysogenum. Compounds 1 and 2 exhibited inhibitory activities anginst the nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 value of (72.6 ± 2.3) μmol/L and (41.2 ± 1.4) μmol/L, respectively. In contrast, all the compounds didn't exhibit inhibitory activities on AChE at the concentration of 100 μmol/L.
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Objective To prepare an immobilized acetylcholinesterase (AChE) microreactor and establish a rapid screening method for Chinese materia medica (CMM) acetylcholinesterase inhibitors (AChEIs). Methods A novel immobilized AchE microreactor was prepared by crosslinking with glutaraldehyde, using aminated magnetic microspheres as carrier. The characterizations were conducted by physicochemical properties and chromatographic performance. The immobilized AChE reactor was used to screen AChEIs from the Huperzia Serratum extracts. Results In the enzyme reaction system, the optimum substrate concentration was 50 μmol/L, and the incubation time was 5 min, respectively. IR characterization, specificity verification, enzyme kinetics, and stability study results all demonstrated the effectiveness of the enzyme reactor. The CMM AChEI, huperzine A, was obtained from the screening of H. Serratum extracts. Conclusion A high throughput screening method for AChEIs is established in this paper, which will be further applied and popularized.
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In this study, we established a rapid and efficient HPLC method to determine the accumulation of Huperzine A and Huperzine B in the fermentation broth of endophytic fungus Colletotrichum gloesporioides from Huperzia serrate. The chloroform extracts of fermentation broth were dissolved in methanol and filtered before injection for HPLC analysis. The analysis was performed on an Agilent Eclipse plus-C18 column (250 mm×4.6 mm, 5 μm) by isocratic elution. The mobile phase was 0.015 mol/L ammonium acetate-methanol (70:30, V/V), the flow rate was 1 mL/min and the detection wavelength was set at 308 nm. Huperzine A and Huperzine B could be well separated within 25 min. Good linearity of Huperzine A was found in the range of 1.50-48.00 μg/mL (r=0.999 5), and that of huperzine B was in 0.25-7.50 μg/mL (r=0.999 7). The average recoveries of Huperzine A and Huperzine B were 106.83% and 108.06%, respectively (RSD=3.34%, 3.60%). The results demonstrate that this method can detect the content of huperzine A and huperzine B in fermentation broth simply, rapidly, accurately and in good reproducibility. Under the optimized conditions, the accumulated content of huperzine A and huperzine B were measured from the sixth to the fifteenth day. Huperzine A and Huperzine B reached the highest (12.417 0 μg/mL and 4.660 3 μg/mL, respectively) at the fourteenth and eighth days. The analysis methodology could contribute to the future study of huperzine A and huperzine B biosynthesis in C. gloeosporioides, consequently facilitate the development of new drug resources.
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Objective: To investigate the non-alkaloids in Huperzia serrata. Methods: The constituents were mainly isolated and purified by means of silica gel, RP18, and Sephadex LH-20 column chromatographies. The structures were identified based on analyses of the spectral data of NMR and MS. Results: Totally 11 constituents were obtained from dichloromethane fraction of the plant and they were 3β-hydroxyserrat-14-en-21β-yl-p-dihydrocoumarate (1), seratenediol-3,21-diacetate (2), seratenediol-3-acetate (3), 21-epi-serratenediol-3-acetate (4), 3α,21β,24-trihydroxyserrat-14-en (5), 21-epi-serratenediol (6), serratenediol (7), 3α,21β-dihydroxy- serrat-14-en-16-one (8), 3β,21β,24-trihydroxyserrat-14-en (9), 3α,21β,24-trihydroxyserrat-14-en-16-one (10), and 1-dibenzofuranol (11). Conclusion: Compounds 1-10 are serratene-type triterpenoids and compound 11 is a dibenzofuranol. Compounds 1 is a new compound named as serratcoumarate and compounds 2 and 11 are isolated from the plant for the first time.
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Objective: To investigate the influence factors and dynamic varitation rule for the accumulation of huperzine A (Hup A) from thallus of Huperzia serrata, which could produce Hup A. Methods: The optimal concentration of plant growth substances for the growth of thallus of H. serrata were screened using orthogonal design method. The content of target extract from thallus of H. serrata were tested using HPLC. The kinetic curve of thallus growth was described using Logistic growth model. Results: The thallus of H. serrata could produce the most Hup A (72.45 μg/L) when cultured in 1/4 MS solid medium complemented with 12 h/d white light and the relative growth rate could reach 3 174.5%. According to the growth curve of thallus of H. serrata like "S" shape, the best successive generation date of callus culture of H. serrata was 50 d. The concentration of Hup A increased obviously after 60 d when the thallus reached the stable period and the maximal value of Hup A was 7.453 μg/g at 85 d, which meant that the rapid growth of thallus was priority to the accumulation of Hup A. Conclusion: The relational model between the accumulation of Hup A from thallus and its growth is non-growth conjugation. In addition, the growth law of thallus is consistent with Logistic growth model and the maximum specific growth rate (μmax) was 0.071 d-1.
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Objective: Due to the low reproductive capacity and scarce resources of Huperzia serrata, and in vitro culture of H. serrata has not been really successful, we wish to make a good progress in rapid propagation in this study. Methods: Living shoot tips were used as the materials, after a variety of surface sterilization, malachite green and AAS were used to eliminate the endophytic fungi of the seedling. Then, the aseptic seedlings were transplanted to the medium with 1/2 MS + 25 g/L sucrose to be cultured. After the new dichotomous branching occurred, and the branches grew to 4-5 cm high, we cut it and subcultured for the new dichotomous branching. Results: After four times of subculture of dichotomous branching, the aseptic seedlings got the different number of proliferating lateral buds, which came from the middle or top of the stem. Comparing the structure of lateral buds with the gemma, we found it was significantly different from the gemma. The number of lateral buds from different individuals was different, the most lateral buds could get to 14 in one seedling, some lateral bud generating point can produce two or more lateral buds. After removing the apical dominance of stem tip, the lateral buds in the middle of the stem grew faster. The surviving rate of different height lateral buds was different when they separated from the stock plant. Lateral buds which beyond 5 mm, the surviving rate could get 90%, while cultured in medium of 1/2 MS + 30 g/L sucrose, after 120 d, it grew into seedling about 2-3 cm high, lateral buds less 5 mm had high mortality with the same cultured medium. Conclusion: Though in vitro culture, H. serrata can break the reproductive way by which the leaf axil could generate spore only but no lateral bud naturally, and can produce the multiple lateral bud proliferation. The lateral buds can easily produce adventitious roots by stripping, which could provide an important view for rapid reproduction of H. serrata.
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Objective: To obtain and analyze the full-length L-lysine decarboxylase (LDC) gene sequence of Huperzia serrata var. longipetiolata and predictively analyze its protein structure on the basis of cloning the coding region of LDC gene from four species of Huperziaceae. The species are Huperzia serrata var. longipetiolata, Phlegariurus minchegensis, Phlegariurus austrosinicus, and Phlegariurus petiolatus. Methods: The LDC coding region sequences were cloned by RT-PCR strategy with the template of total RNA extracted from the leaves. Then the sequences were analyzed by means of BLAST and MEGA 5.0. The full-length of LDC gene sequence of H. serrata var. longipetiolata was obtained by RACE technology. And then the secondary structure and three-dimensional structure of LDC protein were predictively analyzed. Results: The coding region sequences were highly similar to the lysine decarboxylase in the database. And the encoding protein of H. serrata var. longipetiolata was highly similar to the amino acid sequences of H. serrata in NCBI, and with high homology to Selaginella moellendorffii. The full-length LDC gene sequence of H. serrata var. longipetiolata contained 1 266 bp open reading frame and encodes a predicted protein of 403 amino acids. The GenBank accession number for this gene is KF040056. Conclusion: The LDC genes of the four species of Huperziaceae are cloned in this study. The full-length LDC gene sequence of H. serrata var. longipetiolata is obtained and analyzed, and its protein structure is predictively analyzed. The result will provide a foundation for exploring the mechanism of huperzine A biosynthesis in the plants of Huperziaceae.
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OBJECTIVE: To study the chemical constituents in the whole plant of Huperzia leishangensis. METHODS: Compounds were isolated and purified by Prep-TLC, silica gel chromatography, Sephadex LH-20 and preparative high performance liquid chromatography, and their structures were identified on the basis of comprehensive analysis of their physical and chemical properties, NMR data and literatures. RESULTS: Ten compounds were isolated and elucidated as (15R)-12,16-epoxy-11,14-dihydroxy-8, 11, 13-abietatrien-7-one (I), 21-epi-serratenediol-3, 21-acetate (II), serratenediol-3-acetate (III), 21-epi-serratenediol-3-acetate (IV), 21α-hydroxy-serrat-14-en-3β-ol(V), 21β-hydroxy-serrat-14-en-3β-ol(VI), huperzine A (VII), sucrose (VIII), β-sitosterol (IX) and β-daucosterol(X). CONCLUSION: Compound I-X were for the first time isolated from H. leishangensis.
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Objective: Huperzia serrata, whose growth is limited by high temperature, is a rare medicinal plant with the treatment function for Alzheimer's disease (AD). To research the effect of high temperature on the structure and function of cell membrane and chloroplast, and to provide the evidence for production practices. Methods: H. serrata was processed at 25, 30, 35, and 40°C, respectively, then the content changes of malondial dehyde (MDA) and conductivity rate, and the content changes of total chlorophyll, chlorophyll a, chlorophyll b, and chlorophyll a/b values were measured. The changes of the chloroplast ultra microstructure were observed under the transmission electron microscope (TEM). Results: The changes of MDA and conductivity rate in the process at 35 and 40°C were significantly higher than those of the control group; After processed at 40°C for 4 d, the total chlorophyll was decreased significantly, and became the lowest on the day 6, just was 58% compared to the control group; the change trends to the contents of chlorophyll a, chlorophyll b, and total chlorophyll were similar; TEM observation revealed that after processed at 35°C for 4 d, the chloroplast structure appeared deformation, and after processed at 40°C for 4 d, the chloroplast structure subjected obvious destruction capsule fuzzy, fracture in different degrees, thylakoid in disorder, matrix lamellar irregular, and so on. Conclusion: According to the changes of physiological index, ultramicroscopic structure, and external morphology of chloroplast, the suitable temperature for H. serrata is 25-30°C, 40°C is the limited temperature, causing death after 4 d stress, and 35°C has obvious impact on the growth, long-time stress in 35°C could also cause plant deaths.
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The transcript encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) was discovered from the transcriptome data of Huperzia serrata. The transcript contained an open reading frame with length of 1,440 bp and coded 479 amino acids. The full length of HsDXR1 had been cloned using RT-PCR method. Ac-cording the bioinformatic analysis, the molecular weight of HsDXR1 protein was 51.4961 kDa and the pI was 6.44. No signal peptide and transmembrane site was discovered in HsDXR1, and the protein was most likely to be located in chloroplast. HsDXR1 had the same domain similar to the DXR protein of Arabidopsis and Oryza sativa. The expression level of HsDXR1 was most abundantly in H. serrata stem, followed by root and leaf. This study cloned and analyzed HsDXR1 gene from H. serrata for the first time. The result will provide a foundation for exploring the mechanism of terpene biosynthesis in H. serrata plants.
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Sporangia ontogeny and sporogenesis of the lycopodium Huperzia brevifolia (Lycopodiaceae) from the high mountains of Colombia. Huperzia brevifolia is one of the dominant species of the genus Huperzia living in paramos and superparamos from the Colombian Andes. A detailed study of the sporangiums ontogeny and sporogenesis was carried out using specimens collected at 4200m above sea level, in Parque Natural Nacional El Cocuy, Colombia. Small pieces of caulinar axis bearing sporangia were fixed, dehydrated, paraffin embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made, stained with aqueous Toluidine Blue (TBO). The sporangia develops basipetally, a condition that allows observation of all the developmental stages taking place throughout the caulinar axis of adult plants. Each sporangium originates from a group of epidermal cells, axilar to the microphylls. These cells undergo active mitosis, and produce new external and internal cellular groups. The sporangium wall and the tapetum originate from the external group of cells, while the internal cellular group leads to the sporogenous tissue. Meiosis occur in the sporocytes and produce simultaneous types tetrads, each one giving rise four trilete spores, with foveolate ornamentation. During the sporangium ripening, the outermost layer of the wall develops anticlinally, and inner periclinal thickenings and the innermost one perform as a secretory tapetum, which persists until the spores are completely mature. All other cellular layers colapse. Rev. Biol. Trop. 57 (4): 1141-1152. Epub 2009 December 01.
Se describe la ontogenia y la esporogénesis en H. brevifolia, en material recolectado en el Parque Nacional Natural El Cocuy (Boyacá-Colombia) a 4200m de altitud. Los esporangios se desarrollan de forma basípeta sobre el eje caulinar: los iniciales y juveniles se localizan en el ápice y los adultos a maduros, en la base. El desarrollo se inicia a partir de un grupo de células epidérmicas localizadas en las axilas que forman los microfilos con el eje caulinar. Estas células se dividen activamente por mitosis formando una masa celular externa y otra interna. La primera da origen a la pared del esporangio, de varios estratos celulares; de éstos, el estrato externo desarrolla engrosamientos en las paredes anticlinales y en la periclinal interna. El estrato celular interno se diferencia para formar el tapete secretor. Los demás estratos celulares de la pared se degradan durante la maduración del esporangio. La masa celular interna da origen al tejido esporógeno que forma los esporocitos, que experimentan la meiosis I hasta la etapa de díada. La meiosis II concluye con la formación de tétradas, constituidas por esporas en disposición tetraédrica. Las esporas son foveoladas con abertura trilete y son liberadas del esporangio a través de la dehiscencia.
Subject(s)
Huperzia/physiology , Spores/growth & development , Colombia , Huperzia/cytologyABSTRACT
Objective To obtain the endophytic fungi which produce Huperzine A from four species in Hupziaceae and improve the culture conditions of screening endophytic fungi. Methods Plant materials were cultivated in culture medium after sterilization and their endophytic fungi were isolated. Hupzine A from metabolic was determined by HPLC, and the strains were identified by microscopic features. Results Thirty-two endophytic fungi were isolated from Huperzia serrata (Thunb. ex Murray) Trev, H. serrata (Thunb. ex Murray) Trev var. longipetiolata (Spring) H. M. Chang, H. appressa (Desv.) Love et D. Love, Phlegmariurus cryptomerianus (Maxim.) Ching ex L. B. Zhang et H. S. Kung. Two of these strains were isolated from P. cryptomerianus, HA15 (Blastomyces sp.) and HA23 (Botrytis sp.), which produced Huperzine A. Conclusions Endophytic fungi producing Hupzine A has been successfully isolated from P. cryptomerianus.
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Aim To establish a HPLC method for determination of the content of Huperzine A in Huperzia serrata and compare it in different parts of the plants. Method DiamonsilTm C_ 18 column(200 mm?4.6 mm,5 ?m)was used; the mobile phase was methanol ∶0.08 mol?L-1ammonium acetate buffer(40 ∶60,pH=6.0),the detection wavelength was 308 nm, with flow rate at 1.0 ml?min-1;the column temperature was room temperature.Results There is a good linear relationship within the range of 5～100 mg?L-1(r=0.9997).The average recovery was 99.10%,with RSD 2.44%(n=5).Regarding the content of Huperzine A in different parts of Huperzia serrata, it was found that the quantity of Huperzine A in leaf was higher than that in stem and it was the lowest in root. Conclusion The method is simple, accurate and reliable etition. Huperzine A can be accumulated in the aboveground part of the plant.
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Object To compare the hupzine A (Hup A) in Huperzia serrata (Thunb.) Trev. obtained by different extracting methods and investigate the amount of alkaloids and the content of Hup A from different parts of the plants and from different places. Methods Using HPLC for the determination of Hup A. Results The content of Hup A in the stem and leaf is richer than that in the root. The content of Hup A from Guizhou, Guangdong and Anhui Provinces is 0.018%, 0.021% and 0.020% repectively; The difference of extract method of Hup A is no prominence. Conclusion The content of Hup A in the ground is richer than that of underground, and there are some difference in the content of Hup A obtained from different places.
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In this study,nearly 200 endophytic bacteria were isolated from different part of Huperzia serrata, over 60 bacterium with clear antifungal activity were selected from those cultures.Among them,strain H-6 exhibited the highest antifungal activity which was strongly inhibits the growth of many plant pathogenic fungi such as Sclerotinia scleroliorum,Fusarium graminearumt,Sclerotinia libertiana,Phytophthora capsici Leonia and Sesame fusarium wilt.According to the characteristics of morphology,physiology and biochem- istry tests and the comparison of 16S rDNA sequence,the strain H-6 was similar to the Burkholderia.So strain H-6 was identified as Burkholderia sp.H-6.The results also showed that Burkholderia sp.H-6 was markedly different from Burkholderia cepacia that was applied widely in agriculture as antagonistic bacteria. The medium and culture conditions of the strain all were optimized by single factor and orthogonal experiments.In the investigation of the culture condition,growth was carried out in a basal medium(potato juice)and gradually supplemented with the various ingredients to be investigated.The major ingredients be- ing investigated included carbon sources and nitrogen sources.The optimal antifungal activity production condition is growth in a medium(potato juice with 2.5%mannitol and 0.1%NaNO3),initial pH 4.0 at 28℃.