The Huperzia selago Shoot Tip Transcriptome Sheds New Light on the Evolution of Leaves.

Lycopodiophyta-consisting of three orders, Lycopodiales, Isoetales and Selaginellales, with different types of shoot apical meristems (SAMs)-form the earliest branch among the extant vascular plants. They represent a sister group to all other vascular plants, from which they differ in that their leaves are microphylls-that is, leaves with a single, unbranched vein, emerging from the protostele without a leaf gap-not megaphylls. All leaves represent determinate organs originating on the flanks of indeterminate SAMs. Thus, leaf formation requires the suppression of indeterminacy, that is, of KNOX transcription factors. In seed plants, this is mediated by different groups of transcription factors including ARP and YABBY.We generated a shoot tip transcriptome of Huperzia selago (Lycopodiales) to examine the genes involved in leaf formation. Our H. selago transcriptome does not contain any ARP homolog, although transcriptomes of Selaginella spp. do. Surprisingly, we discovered a YABBY homolog, although these transcription factors were assumed to have evolved only in seed plants.The existence of a YABBY homolog in H. selago suggests that YABBY evolved already in the common ancestor of the vascular plants, and subsequently was lost in some lineages like Selaginellales, whereas ARP may have been lost in Lycopodiales. The presence of YABBY in the common ancestor of vascular plants would also support the hypothesis that this common ancestor had a simplex SAM. Furthermore, a comparison of the expression patterns of ARP in shoot tips of Selaginella kraussiana (Harrison CJ, etal. 2005. Independent recruitment of a conserved developmental mechanism during leaf evolution. Nature 434(7032):509-514.) and YABBY in shoot tips of H. selago implies that the development of microphylls, unlike megaphylls, does not seem to depend on the combined activities of ARP and YABBY. Altogether, our data show that Lycopodiophyta are a diverse group; so, in order to understand the role of Lycopodiophyta in evolution, representatives of Lycopodiales, Selaginellales, as well as of Isoetales, have to be examined.

Phytochemical and ethnomedicinal study of Huperzia species used in the traditional medicine of Saraguros in Southern Ecuador; AChE and MAO inhibitory activity.

ETHNOBOTANICAL AND ETHNOMEDICINAL RELEVANCE: This study concerns seven Huperzia species (Lycopodiaceae), namely H. brevifolia, H. columnaris, H. compacta, H. crassa, H. espinosana, H. tetragona, H. weberbaueri, which are considered sacred plants by the Saraguro community, living in the Southern Andes of Ecuador; these plants are widely used in traditional medicine and ritual ceremonies. MATERIAL AND METHODS: The plants were selected on the basis of written interviews with 10 visionary healers (yachak) (2 women, 8 men), indicated as the most credible by the Saraguro Healers Council. The Informant Consensus Factor (Fic) was determined. The first phytochemical study of the plants was performed by standard procedures, while the AChE and MAO-A inhibition by fractions enriched in high MW alkaloids, was measured in vitro. AIMS OF THE STUDY: i) to investigate the uses of some Huperzia plants in healing and magical-religious practices of Saraguros; ii) to identify the main components of plant hydromethanolic extracts; iiì) to test the effects of alkaloidal fractions on the activity of two enzymes linked to mental health. RESULTS: All the interviewed Saraguro yachak showed a high consensus about the uses of the seven Huperzia plants as purgatives and against supernatural diseases, such as the "espanto" (startle). In admixtures with other plants, some species also induce a state of trance or hallucinations in participants in magical-religious rituals. GC-MS of the volatile alkaloid fractions allowed the identification of some lycodine-type and lycopodine-type alkaloids (1-5) in H. compacta, H. columnaris, and H. tetragona. The flavones selgin) (6) and tricin (7) were isolated from H. brevifolia and H. espinosana. Tricin (7) was also detected in the other five species. The rare serratene triterpenes serratenediol (8) serratenediol-3-O-acetate (9), 21-episerratenediol (10), and 21-episerratenediol-3-O-acetate (11) were isolated from H. crassa. In addition, the presence of an unprecedented group of high molecular weight alkaloids has been determined. Alkaloid fractions of H. brevifolia, H. compacta, H. espinosana, and H. tetragona significantly inhibited AChE and MAO-A activities in vitro. CONCLUSIONS: The first phytochemical and ethnopharmacological study of seven Huperzia plants, widely used by Saraguro healers, led to the identification of several alkaloids and triterpenoids with different remarkable biological activities. In addition, alkaloid fractions exhibited a significant AChE and MAO-A inhibitory activity. These results may support the use of these plants in brews prepared for inducing psychoactive effects in participants in magical-religious ceremonies. This study confirms the rich traditional medical knowledge of Saraguro healers which must be documented and preserved for future generations.

Production of huperzine A and other Lycopodium alkaloids in Huperzia species grown under controlled conditions and in vitro.

A UPLC-MS method was developed for quantifying huperzine A (HupA), an anti-Alzheimer's disease (AD) drug candidate from the traditional Chinese medicine Qian Ceng Ta (Huperzia serrata), in samples of 11 Huperzia genus plants. The highest content of HupA was found in Huperzia pinifolia. The accumulation of various Lycopodium alkaloids was monitored in these tissues using high resolution Q-IMS-TOFMS analysis. Tissue culture of various Huperzia species has been achieved and production of HupA has been confirmed in the callus of H. pinifolia. Furthermore, it was established that the major alkaloid produced by the naturally grown plant and the callus of H. pinifolia changed dramatically from HupA to nankakurine B.

Modification and validation of a high-performance liquid chromatography method for quantification of Huperzine A in Huperzia crispata.

The present study describes a rapid and sensitive HPLC method for the quantification of huperzine A (HupA) in Huperzia crispata (Huperziaceae). The sample extraction and preparation involved a simple, time-saving, single-solvent extraction, with each sample being analyzed within 12 min. The mobile phase was ammonium acetate (0.1 M, pH 6.0)--methanol (64 + 36, v/v) at a flow rate of 1.0 mL/min. Detection was at 308 nm. The calibration curve was linear from 0.049 to 7.84 microg (R2 = 0.9997), with intraday and interday precision RSD of less than 2%. The extraction recovery rate was over 98.49%. Quantification of HupA was performed using this modified method, and the content of HupA was 1.86 times higher in the whole plant of H. crispata (218.17 +/- 1.55 microg/g) than in that of H. serrata (117.03 +/- 2.97 microg/g). In the whole plant of H. crispata, HupA mainly accumulated in the actively growing shoot tips, the apical bud, and the 10 youngest leaves, reaching 455.23 +/- 2.97 microg/g. The content of HupA in the samples from sunshine-sheltered sites was 3.45 times higher than in that from sunshine-abundant sites. The satisfactory results indicate that this modified method can be applied in the quality control of large-scale Huperziaceae plant extracts and that changes should be made in the cultivation of H. crispata so as to maximize the production of HupA.

Comparison of 454-ESTs from Huperzia serrata and Phlegmariurus carinatus reveals putative genes involved in lycopodium alkaloid biosynthesis and developmental regulation.

BACKGROUND: Plants of the Huperziaceae family, which comprise the two genera Huperzia and Phlegmariurus, produce various types of lycopodium alkaloids that are used to treat a number of human ailments, such as contusions, swellings and strains. Huperzine A, which belongs to the lycodine type of lycopodium alkaloids, has been used as an anti-Alzheimer's disease drug candidate. Despite their medical importance, little genomic or transcriptomic data are available for the members of this family. We used massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform to generate a substantial EST dataset for Huperzia serrata (H. serrata) and Phlegmariurus carinatus (P. carinatus) as representative members of the Huperzia and Phlegmariurus genera, respectively. H. serrata and P. carinatus are important plants for research on the biosynthesis of lycopodium alkaloids. We focused on gene discovery in the areas of bioactive compound biosynthesis and transcriptional regulation as well as genetic marker detection in these species. RESULTS: For H. serrata, 36,763 unique putative transcripts were generated from 140,930 reads totaling over 57,028,559 base pairs; for P. carinatus, 31,812 unique putative transcripts were generated from 79,920 reads totaling over 30,498,684 base pairs. Using BLASTX searches of public databases, 16,274 (44.3%) unique putative transcripts from H. serrata and 14,070 (44.2%) from P. carinatus were assigned to at least one protein. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology annotations revealed that the functions of the unique putative transcripts from these two species cover a similarly broad set of molecular functions, biological processes and biochemical pathways.In particular, a total of 20 H. serrata candidate cytochrome P450 genes, which are more abundant in leaves than in roots and might be involved in lycopodium alkaloid biosynthesis, were found based on the comparison of H. serrata and P. carinatus 454-ESTs and real-time PCR analysis. Four unique putative CYP450 transcripts (Hs01891, Hs04010, Hs13557 and Hs00093) which are the most likely to be involved in the biosynthesis of lycopodium alkaloids were selected based on a phylogenetic analysis. Approximately 115 H. serrata and 98 P. carinatus unique putative transcripts associated with the biosynthesis of triterpenoids, alkaloids and flavones/flavonoids were located in the 454-EST datasets. Transcripts related to phytohormone biosynthesis and signal transduction as well as transcription factors were also obtained. In addition, we discovered 2,729 and 1,573 potential SSR-motif microsatellite loci in the H. serrata and P. carinatus 454-ESTs, respectively. CONCLUSIONS: The 454-EST resource allowed for the first large-scale acquisition of ESTs from H. serrata and P. carinatus, which are representative members of the Huperziaceae family. We discovered many genes likely to be involved in the biosynthesis of bioactive compounds and transcriptional regulation as well as a large number of potential microsatellite markers. These results constitute an essential resource for understanding the molecular basis of developmental regulation and secondary metabolite biosynthesis (especially that of lycopodium alkaloids) in the Huperziaceae, and they provide an overview of the genetic diversity of this family.

[Effect of environmental factors on growth of Huperzia serrata].

OBJECTIVE: To establish and optimize the cultivation systems for wild Huperzia serrata, through assessing the effects of environmental factors on its growth. METHOD: 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. RESULT: 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. CONCLUSION: 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.

[Effects of microbes on rooting and on several physiological and biochemical indexes of cuttings of Huperzia serrata].

OBJECTIVE: 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. METHOD: 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. RESULT: 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). CONCLUSION: Soil microbes from primary habitat, endophytic fungi and root surface fungi promote rooting, they also increase plant metabolism level of H. serrata.

In vitro production of huperzine A, a promising drug candidate for Alzheimer's disease.

Alzheimer's disease (AD) is growing in impact on human health. With no known cure, AD is one of the most expensive diseases in the world to treat. Huperzine A (HupA), a anti-AD drug candidate from the traditional Chinese medicine Qian Ceng Ta (Huperzia serrata), has been shown to be a powerful and selective inhibitor of acetylcholinesterase and has attracted widespread attention because of its unique pharmacological activities and low toxicity. As a result, HupA is becoming an important lead compound for drugs to treat AD. HupA is obtained naturally from very limited and slowly growing natural resources, members of the Huperziaceae. Unfortunately, the content of HupA is very low in the raw plant material. This has led to strong interest in developing sources of HupA. We have developed a method to propagate in vitro tissues of Phlegmariurus squarrosus, a member of the Huperziaceae, that produce high levels of HupA. The in vitro propagated tissues produce even higher levels of HupA than the natural plant, and may represent an excellent source for HupA.

[Mechanism of effects of soil microbes on cuttings rooting of Huperzia serrata].

OBJECTIVE: To study the effects of soil microbes of habitats on the cuttings rooting of Huperzia serrata and its mechanism. METHOD: 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. RESULT: 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. CONCLUSION: 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.