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.

Active anti-acetylcholinesterase component of secondary metabolites produced by the endophytic fungi of Huperzia serrata

Background An endophytic fungus lives within a healthy plant during certain stages of, or throughout, its life cycle. Endophytic fungi do not always cause plant disease, and they include fungi that yield different effects, including mutual benefit, and neutral and pathogenic effects. Endophytic fungi promote plant growth, improve the host plant's resistance to biotic and abiotic stresses, and can produce the same or similar biologically active substances as the host. Thus, endophytic fungal products have important implications in drug development. Result Among the numerous endophytic fungi, we identified two strains, L10Q37 and LQ2F02, that have anti-acetylcholinesterase activity, but the active compound was not huperzine A. The aim of this study was to investigate the anti-acetylcholinesterase activity of secondary metabolites isolated from the endophytic fungi of Huperzia serrata. Microbial cultivation and fermentation were used to obtain secondary metabolites. Active components were then extracted from the secondary metabolites, and their activities were tracked. Two compounds that were isolated from endophytic fungi of H. serrata were identified and had acetylcholine inhibitory activities. In conclusion, endophytic fungal strains were found in H. serrata that had the same anti-acetylcholinesterase activity. Conclusion We isolated 4 compounds from the endophytic fungus L10Q37, among them S1 and S3 are new compounds. 6 compounds were isolated from LQ2F02, all 6 compounds are new compounds. After tested anti acetylcholinesterase activity, S5 has the best activity. Other compounds' anti acetylcholinesterase activity was not better compared with huperzine A.

Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata / 生物工程学报

Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures.

A new Lycopodium alkaloid from Phlegmariurus fargesii / 中国天然药物

AIM@#To investigate the chemical constituents from the whole plants of Phlegmariurus fargesii.@*METHOD@#Compounds were isolated by repeated silica gel column chromatography. Their structures were elucidated by spectroscopic methods and chemical correlation. The acetylcholinesterase (AChE) inhibitory activity of the isolated compounds was evaluated.@*RESULTS@#A new Lycopodium alkaloid, lycopodine N-oxide (1), along with lycopodine (2), 8,15-dehydrolycopodine (3), 6α-hydroxylycopodine (4), deacetyllycoclavine (5), N-methylhuperzine B (6), lycodine (7), and phlegmarine (8), was isolated.@*CONCLUSION@#Compound 1 is a new Lycopodium alkaloid, and compound 3 was obtained from nature for the first time. Other alkaloids are isolated from this plant for the first time.

Screening of endophytic fungi from Huperzia serrata for acetylcholinesterase inhibitory activity and its taxonomic identification / 中国中药杂志

<p><b>OBJECTIVE</b>To screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata.</p><p><b>METHOD</b>Endophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics.</p><p><b>RESULT</b>Fungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium.</p><p><b>CONCLUSION</b>Endophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.</p>

Cloning and bioinformatics analysis of cycloartenol synthase (HcCAS1) gene in Huperzia carinata / 中国中药杂志

<p><b>OBJECTIVE</b>To clone and sequence the open reading frame of cycloartenol synthase (CAS) from Huperzia carinata.</p><p><b>METHOD</b>After searching the transcriptome dataset of H. carinata, one unique sequence containing oxide squalene cyclases domain was discovered. The primers were designed according to the cDNA sequence of CAS from the dataset. And then, the open reading frame of CAS was cloned by RT-PCR strategy with the template of mixed RNA extracted from root, stem and leaf of H. carinata. The bioinformatic analysis of this gene and its corresponding protein was performed.</p><p><b>RESULT</b>One unique sequence of CAS, named as HcCAS1 (GenBank accession number JN790125) , was cloned from H. carinata. The open reading frame of HcCAS1 consists of 2 474 bp, encoding one polypeptide with 757 amino acids.</p><p><b>CONCLUSION</b>This study cloned and analyzed CAS from H. carinata for the first time. The result will provide a foundation for exploring the mechanism of sterol biosynthesis in Huperziaceae plants.</p>

Cloning and analysis of squalene synthase (HsSQS1) gene in Huperzia serrata / 药学学报

Squalene synthase (SQS) is a key enzyme in plant terpenoid biosynthetic pathway. This study focused on cloning and analysis of Huperzia serrata SQS (HsSQS1) gene. After searching the transcriptome dataset of H serrata, one unique sequence encoding SQS was discovered. The primers were designed according to the transcript sequence of HsSQS1 from the H. serrata transcriptome dataset. The open reading frame of HsSQS1 was cloned using RT-PCR strategy. The bioinformatic analysis of this gene and its corresponding protein were performed. The cDNA (named as HsSQS1) contains a 1263 bp open reading frame and encodes a predicted protein of 420 amino acids. The GenBank accession number for this gene is JQ004938. HsSQS1 contains two transmembrane regions, without signal peptide. The conserved domain of squalene synthase was presented in HsSQS1. HsSQS1 was more abundant in H. serrata root than in leaf and stem. This study cloned and analyzed squalene synthase gene from H. serrata for the first time. The result will provide a foundation for exploring the mechanism ofterpenoid biosynthesis in H. serrata plants.

Isolation of endophytic fungi from Huperzia serrata and their acetylcholinesterase inhibitory activity / 中国中药杂志

A total of 127 strains of endophytic fungi were isolated from roots, branches and leaves of Huperzia serrata. These strains were identified into 19 genera based on morphological characters and ribosomal DNA (rDNA) sequence analysis, there into Penicillium, Aspergillus and Podospora were dominant populations in H. serrata. From analysis results we found some endophytic fungi showed a certain degree of tissue preference. The isolation rate and colonization rate of stems were both larger than those of leaf and roots. After testing the acetylcholinesterase (AChE) inhibitory activity of these endophytic fungi, a total of 39 endophytic fungi belonging to 15 genera showed AChE inhibition. Eleven endophytic fungi showed potent AChE inhibition, 7 of which were isolated from leaf. The research not only provided theoretical basis for developing and utilizing the resources of endophytic fungi in H. serrata but also showed a new path for searching medicines resource which has AChE inhibitory activity.

Cloning, expression and functional identification of a type III polyketide synthase gene from Huperzia serrata / 药学学报

A cDNA encoding novel type III polyketide synthase (PKS) was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata (Thunb.) Trev. by RT-PCR using degenerated primers based on the conserved sequences of known CHSs, and named as H. serrata PKS2. The terminal sequences of cDNA were obtained by the 3'- and 5'-RACE method. The full-length cDNA of H. serrata PKS2 contained a 1212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids. The deduced amino acid sequence of H. serrata PKS2 showed 50%-66% identities to those of other chalcone synthase super family enzymes of plant origin. The recombinant H. serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 1, 3-dihydroxy-N-methylacridone. Interestingly, H. serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).

Analysis of dynamic change of huperzine A content in Huperzia serrata / 中国中药杂志

A precise and selective reversed phase high-performance liquid chromatographic method (HPLC) was used to quantify the levels of huperzine A in samples of three Huperzia serrata populations with a total of 73 individuals located in Zhejiang, Guangxi, Chongqing, respectively, as well as in one-to-one samples of these 73 individuals introduced in same site after one year. Huperzine A content variation both among and within populations, and the dynamic change of this alkaloid occurring in same population after one year introduction, were analyzed using SPSS 13.0 software (Coefficient of variation, One-way ANOVA analysis, Paired-samples T tests). The results indicated that huperzine A content varied significantly by geographical locations, especially change with longitude, i. e., the order of the huperzine A content was CQ population > GX population > ZJ population. The coefficients of variation (CV) were as follows: 0.36 (CQ), 0.44 (GX) and 0.40 (ZJ). This indicated that there was plentiful diversity concerned with huperzine A content among individuals within population. Moreover, this high diversity was still maintained after one year introduction. ANOVA analysis showed that there was significant difference among populations in huperzine A content. Finally, the significant change of huperzine A content was not observed in all three populations after one year introduction. The results presented in this study could provide evidence that the huperzine A content variation of H. serrata is the results of an interaction between genes and the environment, by comparison, is mainly controlled by genetic factor.

Ontogenia del esporangio y esporogénesis del licopodio Huperzia brevifolia (Lycopodiaceae) de las altas montañas de Colombia

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 sporangium’s 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.

Research advances of Huperzia serrata (Thunb.) Trev / 中国中药杂志

Qiancengta, a traditional Chinese medicine produced from the whole plant of the club moss Huperzia serrata, has been used for a long history in China for treatment of a number of ailments, including contusions, strains, swellings, schizophrenia, myasthenia gravis and noworganophosphate poisoning. It has become known worldwide as a medicinal plant since Chinese scientists discovered huperzine A from it in the 1980s, which is reversible, potent and selective acetylcholine esterase (AChE) inhibitors by in vitro and in vivo pharmacological, and produce definite effects in the treatment of Alzheimer's disease. Now, Qiancengta is popular in all over the word for his famous pharmacological actions. For further exploitation this valuable resource under protection of nature environmental, its biological and ecological features, pharmaceutical active ingredients, artificial propagation and in vitro tissue culture, were reviewed, and the sustainable use of Qiancengta natural resource through plant biotechnology was put on the agenda.

Two diterpenoids from herbs of Huperzia serrata / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the constituents from Huperzia serrata.</p><p><b>METHOD</b>Two diterpenoid compounds were isolated by column chromatography with silica gel and Sephadex LH-20. The structures were elucidated by spectral analysis and X-ray crystallographic analysis.</p><p><b>RESULT</b>Their structures were determined as (15R)-12, 16-epoxy-11, 14-dihydroxy-8, 11, 13-abietatrien-7-one (1) and 3beta-hydroxysandaracopimaric acid (2).</p><p><b>CONCLUSION</b>Compound 1 was a new natural abietane-type diterpenoid, compound 2 was isolated from this plant for the first time.</p>

Effect of environmental factors on growth of Huperzia serrata / 中国中药杂志

<p><b>OBJECTIVE</b>To establish and optimize the cultivation systems for wild Huperzia serrata, through assessing the effects of environmental factors on its growth.</p><p><b>METHOD</b>H. serrata was planted under natural field condition, the growth status of H. serrata under different light intensities, temperatures, relative humidity (R. H.), fertilizer levels, and so on were investigated and analyzed.</p><p><b>RESULT</b>H. serrata grew nicely under following environmental conditions: temperature ranging from 18.9-26.3 degrees C, R. H. ranging from 81%-90% and 1330-3000 lx light intensity. A cultivation system, in which H. serrata was planted in the bamboo forest and did not occupy the arable land, was established through the study.</p><p><b>CONCLUSION</b>Concluded that light intensity and R. H are the two factors which greatly confine the growth of H. serrata. As long as the levels of the above two factors meets the demands, H. serrata could be cultivated.</p>

Effects of microbes on rooting and on several physiological and biochemical indexes of cuttings of Huperzia serrata / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects of soil microbes of habitats, endophytic fungi and root surface fungi of Huperzia serrata on rooting rate of the cuttings and on several physiological and biochemical indexes.</p><p><b>METHOD</b>The cuttings of H. serrata were planted on soil I of primary habitat, the soil II had been inoculated endophytic fungi and root surface fungi, the soil III had been auto-claved, rooting rate was examined, and soluble protein and soluble sugar contents, activities of PPO and POD, flavonoids and huperzine A contents were determined.</p><p><b>RESULT</b>Compared with the H. serrata that were planted on soil III, soil I and soil II were found to increase the rooting rate by 10% and 16%, soil II increased the soluble protein contents of stem of H. serrata (P < 0.05), Soil I increased the soluble sugar contents of leaves and stem of H. serrata (P < 0.05), soil I and Soil II increased the flavonoids contents of H. serrata (P < 0.05).</p><p><b>CONCLUSION</b>Soil microbes from primary habitat, endophytic fungi and root surface fungi promote rooting, they also increase plant metabolism level of H. serrata.</p>

Phylogeny relationship and molecular identification of ten Huperzia species (Huperziaceae) based on matK gene sequences / 中国中药杂志

<p><b>OBJECTIVE</b>To study the phylogeny relationship and molecular identification of 10 species from Huperzia (Huperziaceae) based on matK gene sequences data.</p><p><b>METHOD</b>Total DNA of nine species from Huperzia was extracted; matK gene sequence was amplified by PCR. PCR product was directly sequenced after purification.</p><p><b>RESULT</b>The chloroplast matK gene nucleotide sequences from 9 species of Huperzia species were sequenced. The matK gene nucleotide sequences length was 1 589 bp. Analysis with Huperzia lucidula matK gene nucleotide sequences (download from GenBank) and taking Lycopodiella cernua as outgroup, Maximum Parsimony, Neighbor-Joining analyses and genetic distances were conducted using MEGA 3.1 software. 35 variable sites and 35 parsimony informative sites have been found. Pairwise genetic distances among 10 species of Huperzia was 1.59% - 0.25%.</p><p><b>CONCLUSION</b>The results were consistent with the taxonomy in morphological of Huperzia. But H. longipetiolata and H. serrata were resolved into in different clade. There are 19 different sites of matK gene sequences between H. longipetiolata and H. serrata, the genetic distances is 0.121%. It is suggested H. longipetiolata should be as an independent species.</p>

Mechanism of effects of soil microbes on cuttings rooting of Huperzia serrata / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects of soil microbes of habitats on the cuttings rooting of Huperzia serrata and its mechanism.</p><p><b>METHOD</b>The cuttings of H. serrata were planted on the primary habitat soil and on the soil autoclaved, rooting rates were examined respectively. Changes of contents of phenolic compounds (catechin, quercetin, chlorogenic acid, ferulic acid) and indoleacetic acid (IAA) , and changes of activities of polyphenoloxidase (PPO) and peroxydase (POD) during the cuttings rooting were determined.</p><p><b>RESULT</b>Soil microbes of primary habitat could increase the rooting rate and the number of roots, could increase contents of catechin, chlorogenic acid, ferulic acid and IAA during rooting, and could affect activities of PPO and POD.</p><p><b>CONCLUSION</b>During the rooting higher contents of catechin, chlorogenic, ferulic acid and IAA caused by soil microbes of primary habitat are important factor for rooting rate.</p>

Structural identification of huperzinine C / 药学学报

<p><b>AIM</b>To study the alkaloid constituents of Huperzia serrata (Thunb.) Trev..</p><p><b>METHODS</b>Chromatographic methods were used for the isolation and purification. Structure was elucidated on the basis of chemical analysis and spectroscopic data.</p><p><b>RESULTS</b>An alkaloid constituent was isolated from H. serrata (Thunb.) Trev..</p><p><b>CONCLUSION</b>The compound was found to be a novel lycodine type alkaloid with tricyclic structure named huperzinine C.</p>

A novel phlegmariurine type alkaloid from Huperzia serrata (Thunb.) Trev / 药学学报

<p><b>AIM</b>To study the alkaloid constituents of Huperzia serrata (Thunb.) Trev..</p><p><b>METHODS</b>Chromatographic methods were used for the isolation and purification. Structure was elucidated on the basis of chemical analysis and spectroscopic data.</p><p><b>RESULTS</b>An alkaloid constituent was isolated from H. serrata (Thumb.) Trev..</p><p><b>CONCLUSION</b>The compound was found to be a novel phlegmariurine type alkaloid, named 8 beta-hydroxy phlegmariurine B.</p>