An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns.

PREMISE: Apomixis in ferns is relatively common and obligatory. Sterile hybrids may restore fertility via apomixis at a cost of long-term genetic stagnation. In this study, we outlined apomixis as a possible temporary phase leading to sexuality and analyzed factors relating to transitioning to and away from apomixis, such as unreduced and reduced spore formation in apomict and apo-sex hybrid ferns. METHODS: We analyzed the genome size of 15 fern species or hybrids ("taxa") via flow cytometry. The number of reduced and unreduced gametophytes was established as a proxy for viable spore formation of either type. We also calculated the spore abortion ratio (sign of reduced spores) in several taxa, including the apo-sex hybrid Dryopteris × critica and its 16 apomictically formed offspring. RESULTS: Four of 15 sampled taxa yielded offspring variable in genome size. Specifically, each variable taxon formed one viable reduced plant among 12-451 sampled gametophytes per taxon. Thus, haploid spore formation in the studied apomicts was very rare but possible. Spore abortion analyses indicated gradually decreasing abortion (haploid spore formation) over time. In Dryopteris × critica, abortion decreased from 93.8% to mean 89.5% in one generation. CONCLUSIONS: Our results support apomixis as a transitionary phase toward sexuality. Newly formed apomicts hybridize with sexual relatives and continue to form haploid spores early on. Thus, they may get the genomic content necessary for regular meiosis and restore sexuality. If the missing relative goes extinct, the lineage gets locked into apomixis as may be the case with the Dryopteris affinis complex.

Antibacterial activity and antibacterial mechanism of flavaspidic acid BB against Staphylococcus haemelyticus.

BACKGROUND: Staphylococcus haemolyticus (S. haemolyticus) is the main etiological factor in skin and soft tissue infections (SSTI). S. haemolyticus infections are an important concern worldwide, especially with the associated biofilms and drug resistance. Herein, we investigated the inhibitory effect of Flavaspidic acid BB obtained from plant extractions on clinical S. haemolyticus strains and their biofilms. Moreover, we predicted its ability to bind to the protein-binding site by molecular simulation. Since the combination of Hsp70 and RNase P synthase after molecular simulation with flavaspidic acid BB is relatively stable, enzyme-linked immunosorbent assay (ELISA) was used to investigate Hsp70 and RNase P synthase to verify the potential antimicrobial targets of flavaspidic acid BB. RESULTS: The minimum inhibitory concentrations (MIC) of flavaspidic acid BB on 16 clinical strains of S. haemolyticus was 5 ~ 480 µg/mL, and BB had a slightly higher inhibitory effect on the biofilm than MUP. The inhibitory effect of flavaspidic acid BB on biofilm formation was better with an increase in the concentration of BB. Molecular simulation verified its ability to bind to the protein-binding site. The combination of ELISA kits showed that flavaspidic acid BB promoted the activity of Hsp70 and inhibited the activity of RNase P, revealing that flavaspidic acid BB could effectively inhibit the utilization and re-synthesis of protein and tRNA synthesis, thus inhibiting bacterial growth and biofilm formation to a certain extent. CONCLUSIONS: This study could potentially provide a new prospect for the development of flavaspidic acid BB as an antibacterial agent for resistant strains.

Anthelmintic activity of European fern extracts against Haemonchus contortus.

Most drugs used in the treatment of helminthiasis in humans and animals have lost their efficacy due to the development of drug-resistance in helminths. Moreover, since anthelmintics, like many pharmaceuticals, are now recognized as hazardous contaminants of the environment, returning to medicinal plants and their products represents an environmentally friendly way to treat helminthiasis. The goal of the present study was to test the anthelminthic activity of methanol extracts of eight selected European ferns from the genera Dryopteris, Athyrium and Blechnum against the nematode Haemonchus contortus, a widespread parasite of small ruminants. Eggs and adults of H. contortus drug-susceptible strain ISE and drug-resistant strain WR were isolated from experimentally infected sheep. The efficacy of fern extracts was assayed using egg hatch test and adults viability test based on ATP-level measurement. Among the ferns tested, only Dryopteris aemula extract (0.2 mg/mL) inhibited eggs hatching by 25% in comparison to control. Athyrium distentifolium, Dryopteris aemula and Dryopteris cambrensis were effective against H. contortus adults. In concentration 0.1 mg/mL, A. distentifolium, D. aemula, D. cambrensis significantly decreased the viability of females from ISE and WR strains to 36.2%, 51.9%, 32.9% and to 35.3%, 27.0%, 23.3%, respectively in comparison to untreated controls. None of the extracts exhibited toxicity in precise cut slices from ovine liver. Polyphenol's analysis identified quercetin, kaempferol, luteolin, 3-hydroxybenzoic acid, caffeic acid, coumaric acid and protocatechuic acid as the major components of these anthelmintically active ferns.

Dryoptkirbioside, A New Fructofuranoside Glycerol, and Other Constituents from Dryopteris kirbi Hook et Grav Rhizomes.

A new fructofuranoside glycerol, dryoptkirbioside (1), along with thirteen known compounds (2-14), was isolated from the MeOH extract of Dryopteris kirbi rhizomes by silica gel column chromatography, Sephadex LH-20 column chromatography, and semipreparative HPLC. The structure of the new compound was determined by analyses of its spectroscopic data including nuclear magnetic resonance (NMR), and high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) and chemical conversions. The hexane-soluble portion and the EAFA fraction showed strong activities against lung (A549), breast (MCF-7), and cervical (HeLa) human cancer cell lines (IC50 values ranging from 4.0 to 8.8 µg/mL). Aspidinol P (5) and aspidinol B (6) exhibited moderate to low cytotoxicity on the three cell lines (IC50 values ranging from 20.4 to 58.7 µM). The MeOH extract and hexane-soluble portion had excellent activities against Staphylococcus aureus and Bacillus subtilis (MICs 11.7 and 23.4 µg/mL), whereas the AcOEt- and BuOH-soluble portions were significantly active on S. aureus (MICs 46.9 and 93.8 µg/mL). The main fractions EAFB , EAFC and nBFB displayed excellent activity against S. aureus (MICs 11.7 and 23.4 µg/mL). Aspidinol B (6) had significant activity, while aspidinol P (5) was moderately active against S. aureus and B. subtilis (MICs 42.0 and 89.5 µM).

Proteome and Interactome Linked to Metabolism, Genetic Information Processing, and Abiotic Stress in Gametophytes of Two Woodferns.

Ferns and lycophytes have received scant molecular attention in comparison to angiosperms. The advent of high-throughput technologies allowed an advance towards a greater knowledge of their elusive genomes. In this work, proteomic analyses of heart-shaped gametophytes of two ferns were performed: the apomictic Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. In total, a set of 218 proteins shared by these two gametophytes were analyzed using the STRING database, and their proteome associated with metabolism, genetic information processing, and responses to abiotic stress is discussed. Specifically, we report proteins involved in the metabolism of carbohydrates, lipids, and nucleotides, the biosynthesis of amino acids and secondary compounds, energy, oxide-reduction, transcription, translation, protein folding, sorting and degradation, and responses to abiotic stresses. The interactome of this set of proteins represents a total network composed of 218 nodes and 1792 interactions, obtained mostly from databases and text mining. The interactions among the identified proteins of the ferns D. affinis and D. oreades, together with the description of their biological functions, might contribute to a better understanding of the function and development of ferns as well as fill knowledge gaps in plant evolution.

Two New Compounds from the Endophytic Fungi of Dryopteris crassirhizoma and Their Antimicrobial Activities.

Two endophytic fungi Trichoderma afroharzianum (HP-3) and Alternaria alstroemeriae (HP-7) were isolated and purified from the fresh root of Dryopteris crassirhizoma. Chemical investigation of the two fungi resulted in the isolation of two new phenols 2,4-dihydroxy-3-farnesyl-5-methoxy benzoic acid (1) and 2-hydroxyphenethyl 2-phenylacetate (2), together with 22 known compounds. Their structures were elucidated by NMR, UV, IR, HRESIMS, and comparison to the literature data. Compounds 15 and 16 showed significant antibacterial activity against Micrococcus lysodeikticus with MIC value of 6.25 µg/mL, while 8 and 14 displayed moderate inhibitory activities against several plant pathogenic fungi and clinically important bacterial strains. This is the first study to report the isolation, identification, and antimicrobial properties of metabolites from endophytic fungi of D. crassirhizoma. Our findings may provide lead compounds for the development of new antibacterial agents.

A potential role of salicylic acid in the evolutionary behavior of Trichoderma as a plant pathogen: from Marchantia polymorpha to Arabidopsis thaliana.

MAIN CONCLUSION: Recognition of the interaction of Trichoderma during the evolution of land plants plays a potential key role in the development of the salicylic acid defense pathway and the establishment of a mutualistic relationship. Marchantia polymorpha is a common liverwort considered in recent years as a model plant for evolutionary studies on plant-microorganism interactions. Despite the lack of research, remarkable results have been reported regarding the understanding of metabolic and evolutionary processes of beneficial and/or harmful interactions, owing to a better understanding of the origin and evolution of different plant defense pathways. In this study, we have carried out work on the direct and indirect interactions (exudates and volatiles) of M. polymorpha with different species of the fungal genus Trichoderma. These interactions showed different outcomes, including resistance or even growth promotion and disease. We have analyzed the level of tissue colonization and defense-related gene expression. Furthermore, we have used the pteridophyte Dryopteris affinis and the angiosperm Arabidopsis thaliana, as subsequent steps in plant evolution, together with the plant pathogen Rhizoctonia solani as a control of plant pathogenicity. Trichoderma virens, T. brevicompactum and T. hamatum are pathogens of M. polymorpha, while exudates of T. asperellum are harmful to the plant. The analysis of the expression of several defense genes in M. polymorpha and A. thaliana showed that there is a correlation of the transcriptional activation of SA-related genes with resistance or susceptibility of M. polymorpha to Trichoderma. Moreover, exogenous SA provides resistance to the virulent Trichoderma species. This beneficial fungus may have had an evolutionary period of interaction with plants in which it behaved as a plant pathogen until plants developed a defense system to limit its colonization through a defense response mediated by SA.

Apomictic fern fathers: an experimental approach to the reproductive characteristics of sexual, apomict, and hybrid fern gametophytes.

PREMISE: Apomixis and hybridization are two essential and complementary factors in the evolution of plants, including ferns. Hybridization combines characteristics from different species, while apomixis conserves features within a lineage. When combined, these two processes result in apo-sex hybrids. The conditions leading to the formation of these hybrids are poorly understood in ferns. METHODS: We cultivated spores from 66 fern samples (43 apomicts, 7 apo-sex hybrids, and 16 sexuals), and measured their development in vitro over 16 weeks. We evaluated germination, lateral meristem formation rates, sexual expression, and production of sporophytes and then compared ontogenetic patterns among the three groups. RESULTS: The three examined groups formed antheridia (male gametangia) but differed in overall gametophyte development. Sexual species created archegonia (female, 86% of viable samples), but no sporophytes. Apomicts rarely created nonfunctional archegonia (8%) but usually produced apogamous sporophytes (75%). Surprisingly, apomictic and sexual species showed similar development speed. The sexually reproducing parents of viable studied hybrids formed about twice as many meristic gametophytes as the apomictic parents (39% vs. 20%, respectively). CONCLUSIONS: We present the most thorough comparison of gametangial development of sexual and apomictic ferns, to date. Despite expectations, apomictic reproduction might not lead to earlier sporophyte formation. Apomicts produce functional sperm and thus can contribute this type of gamete to their hybrids. The development patterns found in the parents of hybrids indicate a possible increase of hybridization rates by antheridiogens. The apo-sex hybrids always inherit the apomictic reproductive strategy and are thus capable of self-perpetuation.

Structure elucidation, biogenesis, and bioactivities of acylphloroglucinol-derived meroterpenoid enantiomers from Dryopteris crassirhizoma.

Twenty-four racemic acylphloroglucinol meroterpenoids including eighteen unusual stuctures (3 âˆ¼ 10, 13, 14, and 17 âˆ¼ 24), and a major component filixic acid ABA (25), were isolated from Dryopteris crassirhizoma. Structurally, the dimeric acylphloroglucinol derivatives possess unprecedented skeletons of mixed acylphloroglucinol and sesquiterpene biosynthetic origin. The stereochemistries of six reported meroterpenoids with undefined chiral centers were reassigned. Two intriguing methods by analyzing a) the regularity of chemical shift variation of protons and carbons around the stereogenic centers, and b) pyridine-induced deshielding effect of hydroxy groups, to discriminate relative configurations of flexible long-chain alcohol with chiral centers separated by three or seven covalent bonds, were successfully applied. A non-enzymatic biosynthesis of 1 âˆ¼ 24 was assumed based on a rare single-crystal cluster formed with two diastereomeric enantiomer pairs (±1/±2) and chiral HPLC analyses. Meroterpenoids 13 and 14 showed obvious inhibitory effects on NO production in LPS-induced RAW264.7, and suppressed the expression of iNOS, COX-2, IL-1ß, and IL-18. Their anti-inflammatory activity was closely related to the inhibition of the formation and function of inflammasomes. Additionally, the known 25 showed antiviral efficacy against the influenza viruse A/Puerto Rico/8/1934 (H1N1).

Two new cadinane-type sesquiterpenoid glycosides from Dryopteris fragrans with anti-inflammatory activities.

Two new cadinane-type sesquiterpenoid glycosides, dryopteristerpeneA (1) and dryopteristerpeneB (2), were isolated from the aqueous extract of Dryopterisfragrans. Their structures were determined by spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 exhibited inhibition on nitric oxide production in lipopolysaccharide induced RAW 264.7 macrophages with their IC50 values of 60.5 and 59.8 µM, respectively.

The Shared Proteome of the Apomictic Fern Dryopteris affinis ssp. affinis and Its Sexual Relative Dryopteris oreades.

Ferns are a diverse evolutionary lineage, sister to the seed plants, which is of great ecological importance and has a high biotechnological potential. Fern gametophytes represent one of the simplest autotrophic, multicellular plant forms and show several experimental advantages, including a simple and space-efficient in vitro culture system. However, the molecular basis of fern growth and development has hardly been studied. Here, we report on a proteomic study that identified 417 proteins shared by gametophytes of the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. Most proteins are predicted to localize to the cytoplasm, the chloroplast, or the nucleus, and are linked to enzymatic, binding, and structural activities. A subset of 145 proteins are involved in growth, reproduction, phytohormone signaling and biosynthesis, and gene expression, including homologs of SHEPHERD (SHD), HEAT SHOCK PROTEIN 90-5 (CR88), TRP4, BOBBER 1 (BOB1), FLAVONE 3'-O-METHYLTRANSFERASE 1 (OMT1), ZEAXANTHIN EPOXIDASE (ABA1), GLUTAMATE DESCARBOXYLASE 1 (GAD), and dsRNA-BINDING DOMAIN-LIKE SUPERFAMILY PROTEIN (HLY1). Nearly 25% of the annotated proteins are associated with responses to biotic and abiotic stimuli. As for biotic stress, the proteins PROTEIN SGT1 HOMOLOG B (SGT1B), SUPPRESSOR OF SA INSENSITIVE2 (SSI2), PHOSPHOLIPASE D ALPHA 1 (PLDALPHA1), SERINE/THREONINE-PROTEIN KINASE SRK2E (OST1), ACYL CARRIER PROTEIN 4 (ACP4), and NONHOST RESISTANCE TO P. S. PHASEOLICOLA1 (GLPK) are worth mentioning. Regarding abiotic stimuli, we found proteins associated with oxidative stress: SUPEROXIDE DISMUTASE[CU-ZN] 1 (CSD1), and GLUTATHIONE S-TRANSFERASE U19 (GSTU19), light intensity SERINE HYDROXYMETHYLTRANSFERASE 1 (SHM1) and UBIQUITIN-CONJUGATING ENZYME E2 35 (UBC35), salt and heavy metal stress included MITOCHONDRIAL PHOSPHATE CARRIER PROTEIN 3 (PHT3;1), as well as drought and thermotolerance: LEA7, DEAD-BOX ATP-DEPENDENT RNA HELICASE 38 (LOS4), and abundant heat-shock proteins and other chaperones. In addition, we identified interactomes using the STRING platform, revealing protein-protein associations obtained from co-expression, co-occurrence, text mining, homology, databases, and experimental datasets. By focusing on ferns, this proteomic study increases our knowledge on plant development and evolution, and may inspire future applications in crop species.

Comparison of Constituents and Antioxidant Activity of Above-Ground and Underground Parts of Dryopteris crassirhizoma Nakai Based on HS-SPME-GC-MS and UPLC/Q-TOF-MS.

Dryopteris crassirhizoma Nakai is a Chinese traditional medicinal fern plant for heat-clearing and detoxifying, promoting blood circulation and dissipating blood stasis. Previous researches showed that many factors could influence the components of medicinal plants, and the plant part is one of the main factors. So far, only the underground part of D. crassirhizoma, called "Mianma Guanzhong", has been widely sold in the market. However, the above-ground part was usually at low utilization, resulting in a waste of medicinal resources. In order to further develop and utilize the medicinal resources of D. crassirhizoma, the constituents, total flavonoid contents and antioxidant activity of the above-ground and underground parts of D. crassirhizoma were tentatively analyzed and compared based on HS-SPME-GC-MS and UPLC/Q-TOF-MS. The results showed that (1) the volatile components were mainly focused in the above-ground part of D. crassirhizoma, including 3-carene, isoledene, ionene, 4-amino-1-naphthol and furfural. (2) Nonvolatile components of the underground part of D. crassirhizoma contained phenolic acid, flavonoids, phloroglucinol and less fatty acid. (3) The common compounds of the above-ground and underground parts of D. crassirhizoma were phenolic acid and flavaspidic acid AB. (4) Antioxidant activity of the underground part was stronger than that of the above-ground part of D. crassirhizoma. In conclusion, both the above-ground and underground parts of D. crassirhizoma are important medicinal resources worthy of further development.

[A new phloroglucinol from Dryopteris fragrans and its antibacterial activity in vitro].

A new phloroglucinol was isolated from 50% ethanol extract of Dryopteris fragrans by silica gel column chromatography, Sephadex LH-20 gel column chromatography, thin-layer chromatography(TLC), and preparative liquid column chromatography. On the basis of MS, ~1H-NMR, ~(13)C-NMR, and reference materials, compound 1 was identified as 2,5-cyclohexadien-1-one, 2-{[2,6-dihydroxy-4-methoxy-3-methyl-5-(1-isobutyl)phenyl]methyl}-3,5-dihydroxy-4,4-dimethyl-6-(1-oxobutyl)(1), and named disaspidin BB. Compound 1 was evaluated for its antibacterial activity. The experimental results showed that compared with the commonly used topical antibiotics erythromycin or mupirocin, disaspidin BB exhibited significant antibacterial activities against Staphylococcus epidermidis(SEP), S. haemolyticus(SHA), and methicillin-resistant S. aureus(MRSA)(P<0.05). Additionally, disaspidin BB was sensitive to ceftazidime-resistant SEP1-SEP4, SHA5-SHA7, MRSA8, and MRSA9. The MIC values of disaspidin BB against SEP and SHA were 1.67-2.71 µg·mL~(-1) and 10.00-33.33 µg·mL~(-1) respectively. Disaspidin BB has good antibacterial activities and deserves development as a new anti-infective drug for external use.

Anti-diabetogenic and in vivo antioxidant activity of ethanol extract of Dryopteris dilatata in alloxan-induced male Wistar rats.

AIMS: Oxido-inflammatory stress has been implicated as the main targets in alleviating diabetic complications induced by hyperglycaemia. Dryopteris dilatata: a bioactive plant serves great medicinal benefits in ethnopharmacology to ameliorate pathological conditions. This study investigated the protective effects of ethanol extract of Dryopteris dilatata (EEDD) in alloxan-induced diabetic rats through mechanism involving inhibition of oxidative stress and liver and kidney inflammatory markers. METHODOLOGY: Male Wistar rats were made diabetic via alloxan monohydrate (100 mg/kg) administration intraperitoneally. Diabetic rats were post-treated with EEDD (800 mg/kg) and Metformin (50 mg/kg) orally for two weeks. Fasting blood sugar (FBS), body and organ weight change, markers of oxidative stress, liver and kidney inflammation were evaluated. RESULTS: Our results revealed that EEDD significantly reduced alloxan-induced hyperglycaemia in the diabetic rats after 5, 10 and 15 days of treatment. Markers of oxidative injury were also significantly ameliorated in the pancreas, liver and kidney of the diabetic rats following treatment with EEDD. However, liver and kidney injury markers were significantly attenuated with marked decreased organ weight in the diabetic rats after treatment with EEDD. CONCLUSION: Here in, we found that Dryopteris dilatata could be used as nutraceuticals in the prevention and treatment of diabetes and its related complications through positively modulating oxidative stress and liver and kidney inflammatory markers.

[A new phloroglucinol compound from Dryopteris fragrans].

Two phloroglucinol compounds(1-2) were isolated and purified from 95% ethanol extract of Dryopteris fragrans through various column chromatographies on silica gel, Sephadex LH-20, medium pressure column chromatography, and preparative HPLC. Their structures were elucidated as 2',4',6'-trihydroxy-5'-methyl acetate-3'-methyl-1'-butyrophenone(1) and aspidinol B(2) based on their chemical and physicochemical methods and spectroscopic data. Compound 1 is a new phloroglucinol compound named "dryofraginol".

Transcriptomic Analysis and Specific Expression of Transcription Factor Genes in the Root and Sporophyll of Dryopteris fragrans (L.) Schott.

Background: Dryopteris fragrans, which is densely covered with glandular trichomes, is considered to be one of the ferns with the most medicinal potential. The transcriptomes from selected tissues of D. fragrans were collected and analyzed for functional and comparative genomic studies. The aim of this study was to determine the transcriptomic characteristics of wild D. fragrans sporangium in tissues from the SR (root), SL (sporophyll), and TRL (sporophyll with glandular trichomes removed). Results: Cluster analysis identified genes that were highly expressed in an organ-specific manner according to read mapping, feature counting, and normalization. The functional map identified gene clusters that can uniquely describe the function of each tissue. We identified a group of three tissue-specific transcription factors targeting the SL, SR, and TRL. In addition, highly expressed transcription factors (TFs) were found in each tissue-specific gene cluster, where ERF and bHLH transcription factors were the two types showing the most distinct expression patterns between the three different tissues. The specific expression of transcription factor genes varied between the different types of tissues. The numbers of transcription factors specifically expressed in the roots and sporophylls were 60 and 30, respectively, while only seven were found for the sporophylls with glandular trichomes removed. The expression of genes known to be associated with the development of glandular trichomes in flowering plants, including MIXTA, ATML1, and MYB106, were also validated and are discussed. In particular, a unigene encoding MIXTA was identified and exhibited the highest expression level in SL in D. fragrans. Conclusions: This study is the first report of global transcriptomic analysis in different tissues of D. fragrans, and the first to discuss these findings in the context of the development of homologous glandular trichomes. These results set the stage for further research on the development, stress resistance, and secondary metabolism of D. fragrans glandular trichomes.

Phloroglucinol Derivatives from Dryopteris crassirhizoma as Potent Xanthine Oxidase Inhibitors.

Dryopteris crassirhizoma rhizomes are used as a traditional medicine in Asia. The EtOAc extract of these roots has shown potent xanthine oxidase (XO) inhibitory activity. However, the main phloroglucinols in D. crassirhizoma rhizomes have not been analyzed. Thus, we investigated the major constituents responsible for this effect. Bioassay-guided purification isolated four compounds: flavaspidic acid AP (1), flavaspidic acid AB (2), flavaspidic acid PB (3), and flavaspidic acid BB (4). Among these, 1 showed the most potent inhibitory activity with a half-maximal inhibitory concentration (IC50) value of 6.3 µM, similar to that of allopurinol (IC50 = 5.7 µM) and better than that of oxypurinol (IC50 = 43.1 µM), which are XO inhibitors. A comparative activity screen indicated that the acetyl group at C3 and C3' is crucial for XO inhibition. For example, 1 showed nearly 4-fold higher efficacy than 4 (IC50 = 20.9 µM). Representative inhibitors (1-4) in the rhizomes of D. crassirhizoma showed reversible and noncompetitive inhibition toward XO. Furthermore, the potent inhibitors were shown to be present in high quantities in the rhizomes by a UPLC-QTOF-MS analysis. Therefore, the rhizomes of D. crassirhizoma could be used to develop nutraceuticals and medicines for the treatment of gout.

A plastid phylogeny of the fern genus Arachniodes (Dryopteridaceae).

Arachniodes (Dryopteridaceae) is one of the most confusing and controversial fern genera in terms of its circumscription, nomenclature, and taxonomy. Estimates of species number range from 40 to 200. Previous molecular works included only 2-17 accessions representing 2-12 species of Arachniodes and allied genera, leaving most of the Asian species remain unsampled and the infragneric relationships unclear. In this study DNA sequences of seven plastid markers of 343 accessions representing ca. 68 species of Arachniodes (275 accessions), and 64 outgroup accessions from subfam. Dryopteridoideae and subfam. Polybotryoideae were used to infer a phylogeny with maximum likelihood, Bayesian inference, and maximum parsimony approaches. Our major results include: (1) Two species currently assigned in Arachniodes (A. macrostegia and A. ochropteroides are resolved outside of the core Arachniodes making the currently defined Arachniodes polyphyletic, confirming earlier findings; (2) Lithostegia, Leptorumohra, and Phanerophlebiopsis are indeed synonyms of Arachniodes; (3) Leptorumohra is confirmed to be monophyletic, but Phanerophlebiopsis is polyphyletic; (4) The New World species of Arachniodes are confirmed to be not monophyletic with A. denticulata being nested within the Old World species, suggesting that this species is dispersed from the Old World; (5) Arachniodes s.s is resolved into 12 major clades, some of which are further divisable into recognizable subclades and groups, with A. mutica from Japan being resolved as the sister to the rest of the genus; (6) A number of systematic implications of the phylogeny have been suggested; and (7) the genus is estimated to contain ca. 83 species.

Cloning and functional analysis of squalene synthase gene from Dryopteris fragrans (L.) Schott.

Dryopteris fragrans (L.) Schott is a traditional herbal medicine containing medicinal sterols and triterpenoids. Squalene synthase (SQS) is the first crucial enzyme in the biosynthesis pathway of sterols and triterpenoids. The full-length cDNA named DfSQS1 was isolated by RACE. It was predicted that DfSQS1 contained an open reading frame (ORF) of 1239 bp coding 412 amino acid residues with molecular weight of 46.6 kDa. It had 18 potential phosphorylation sites, 1 potential N-glycosylation site and 2 transmembrane domains. In neighbor-joining (NJ) phylogenetic tree, DfSQS1 was away from branch of gymnosperms and angiosperms. One hydrophobic domain at the C-terminal of DfSQS1 was deleted to express soluble recombinant enzyme. The truncated DfSQS1 (tDfSQS1) was expressed in Escherichia coli BL21 (DE3). Then, tDfSQS1 was obtained and incubated with farnesyl diphosphate (FPP) to identify its enzymatic activity. The result demontrated that squalene, the product of enzyme catalyzed reaction, was detected by HPLC. Quantitative real-time PCR (qRT-PCR) analysis revealed that the transcription level of DfSQS1 in D. fragrans was the highest in roots, followed by leaves and rhizomes. This work is the first report on cloning, characteration and expression of SQS from D. fragrans. It will be helpful to understand the regulatory role of SQS on the biosynthesis of triterpenoids in the fern.

Potent in Vitro α-Glucosidase Inhibition of Secondary Metabolites Derived from Dryopteris cycadina.

α-glucosidase is responsible for the hydrolysis of complex carbohydrates into simple absorbable glucose and causes postprandial hyperglycemia. α-glucosidase inhibition is thus the ideal target to prevent postprandial hyperglycemia. The present study was therefore designed to analyze the effects of various compounds isolated from Dryopteris cycadina against α-glucosidase including ß-Sitosterol 1, ß-Sitosterol3-O-ß-d-glucopyranoside 2, 3, 5, 7-trihydroxy-2-(p-tolyl) chorman-4-one 3, Quercetin-3-0-ß-d-glucopyranoside (3/→0-3///)- ß-d- Quercetin -3-0- ß â»d-galactopyranoside 4 and 5, 7, 4/-Trihydroxyflavon-3-glucopyranoid 5. The in vitro spectrophotometric method was used for the analysis of test compounds against possible inhibition. Similarly, molecular docking studies were performed using the MOE software. These compounds showed concentration-dependent inhibition on α-glucosidase, and compounds 1 (IC50: 143 ± 0.47 µM), 3 (IC50:133 ± 6.90 µM) and 5 (IC50: 146 ± 1.93 µM) were more potent than the standard drug, acarbose (IC50: 290 ± 0.54 µM). Computational studies of these compounds strongly supported the in vitro studies and showed strong binding receptor sensitivity. In short, the secondary metabolites isolated from D. cycadina demonstrated potent α-glucosidase inhibition that were supported by molecular docking with a high docking score.