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Dryopteris sect. Diclisodon is a small section of ferns with about 12 species mainly distributed in East Asia. Here, we carried out morphological and phylogenetic analyses of this section. A new species from southwest China, D. gaoligongensis, is described and illustrated. Dryopteris gaoligongensis resembles D. indonesiana and D. sparsa, but differs by having a creeping rhizome and large 4-pinnate fronds. We also show that D. glabrior Ching & Z.Y. Liu is a distinct species; however, because it is a later homonym of D. glabrior Copeland, it should be renamed D. renchangiana. We conclude that a species previously known as D. nitidula, also an illegitimate homonym, should be recognized with a new name, D. sinonepalensis. We resolve the phylogenetic position of D. yoroii as sister to other sampled species of D. sect. Diclisodon. Our phylogenetic analyses confirm the distinctiveness of D. gaoligongensis, D. renchangiana, and D. sinonepalensis. A key to species of D. sect. Diclisodon in China is provided.

By various chromatographic methods, 30 phloroglucinols (1−30) were isolated from a methanol extract of Dryopteris crassirhizoma, including two new dimeric phloroglucinols (13 and 25). The structures of the isolates were confirmed by HR−MS, 1D, and 2D NMR as well as by comparison with the literature. The protein tyrosine phosphatase 1B (PTP1B) effects of the isolated compounds (1–30) were evaluated using sodium orthovanadate and ursolic acid as a positive control. Among them, trimeric phloroglucinols 26–28 significantly exhibited the PTP1B inhibitory effects with the IC50 values of 1.19 ± 0.13, 1.00 ± 0.04, 1.23 ± 0.05 μM, respectively. In addition, the kinetic analysis revealed compounds 26–28 acted as competitive inhibitors against PTP1B enzyme with Ki values of 0.63, 0.61, 1.57 μM, respectively. Molecular docking simulations were performed to demonstrate that these active compounds can bind with the catalytic sites of PTP1B with negative binding energies and the results are in accordance with that of the kinetic studies. In vitro and in silico results suggest that D. crassirhizoma rhizomes together with compounds 26–28 are potential candidates for treating type 2 diabetes.

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction.

The stems of Dryopteris crassirhizoma, one of the main components of Lianhua-Qingwen Formula (LQF) was traditionally used for heat-clearing and detoxifying. Dryocrassin ABBA is a key antiviral component in the herbal medicine while the compound is hard to get in large amounts with the features of homologous compounds, polyphenol groups, and low contents. Therefore, the present work aims to seek influenza H7N9 virus inhibitors from natural source by synthesis of dryocrassin ABBA and its analogues. As a result, total synthesis of the compound was achieved in nine steps with an over-all yield of 4.6%. Neuraminidases (NAs) inhibitory activities of the synthesized product and its analogues were evaluated afterward. Comparing with the positive control, OSV (9.6 μM), it was very exciting that dryocrassin ABBA and its analogues (b5 and e2) showed better NAs inhibitory activity against Anhui H7N9 with IC50 values of 3.6 μM, 2.5 μM and 1.6 μM. For the highly resistant Shanghai N9, these compounds can also show medium inhibitory activities. Docking results indicated the direct interaction of synthesized 3 hits with the key K294 by hydrogen bonds, but no direct interaction of OSV with the key K294 was observed in Shanghai N9. This study suggested that dryocrassin ABBA and its analogues especially AB, which consisted of polyphenol groups may have beneficial effects on treating avian influenza H7N9 virus.

The District Charsadda has a wide diversification with nature beauty and greenery. The area has rich biodiversity and natural resources. The District is geographically organized into two primary parts: Hashtnagar and Do Aaba. Research was carried out during 2017–18, in blooming season to collect different taxa from the area. The study depicted 253 taxa floristically with 189 genera and 71 families in which 4 families were pteridiophytes, 3 were gymnosperms and 64 were angiospermic families included 11 Monocot and 53 Dicot. The number of taxa per family varies from 1 to 56. Poaceae was the leading family in term of number of genera and species with 36 genera (19.04%) and 56 species (22.13%) followed by Asteraceae with 15 genera (7.936%) and 17 species (6.719%). The important groups in terms of species percentage were Dicots with a share of (53 species) followed by Monocots (11 species), Pteridophytes (5 species) and Gymnosperms (4 species). Herbaceous layer consists of 173 species (68.37%) followed by trees 46 species (18.18%), 25 species (9.881%) were shrub, 8 species (3.162%) were climbers and only 1 species (0.395%) was parasitic in the area. Biological spectrum depicted that Therophytes (126 species, 49.80%) and Microphyll (78 species, 30.83%) were the leading life form and leaf size classes. Ethnobotanical study proved that majority of species 164 were used as fodder/forage, 27 species were fruits, 28 species were vegetables, 28 species ornamental, 47 species fuel, 66 species were medicinals, 21 species used as timber, 8 species for thatching, 29 species for insect attractant and only 4 species used as spices (condiments) while some species have no local use in research area like Dryopteris stewartii, Equisetum arvense L., Marsilea L., Eichhornia crassipes (Mart.) Solms, and Potamogeton nodosus Poir. This is the first ever record of the area, no prior work exists in this regard. This work might help for future intensive and extensive researches.

Ethnopharmacological relevance Dryopteris crassirhizoma (DC) is used as a traditional herbal remedy to treat various diseases, the tapeworm infection, common cold, and cancer in Korea, Japan, and China. DC also has the antioxidant anti-inflammatory and antibacterial activities. However, the anti-allergic inflammatory effect of DC and some of its mechanisms in allergic rhinitis model are unknown well. Aim of this study The purpose of this study is to investigate the anti-allergic inflammatory effect of DC on the allergic rhinitis model, mast cell activation and histamine release. Materials and methods Allergic rhinitis was induced in BALB/c mice by sensitization and challenge with ovalbumin (OVA). Different concentration of DC and dexamethasone was administrated by oral gavage on 1 h before the OVA challenge. Mice of the control group were treated with saline only. Then mice were evaluated for the presence of nasal mucosa inflammation, the production of allergen-specific cytokine response and the histology of nasal mucosa. Results DC significantly ameliorated the nasal symptoms and the inflammation of nasal mucosa. DC also reduced the infiltration of eosinophils and mast cells in these tissues and the release of histamine in blood. Meanwhile, DC evidently inhibited the overproduction of Th2 cytokines and increased the Th1 and Treg cytokines in nasal lavage fluid by OVA. DC also reduced the levels of OVA-specific IgE, IgG1 and IgG2a in blood. Conclusions This study suggests that DC has a significant anti-allergic inflammatory effect in the nasal cavity. DC may have the therapeutic effect of allergic rhinitis.

Ethnopharmacological relevance Dryopteris fragrans (L.) Schott is a deciduous perennial herb, which has been used traditionally for treatment of ringworm infections and others skin diseases in the north of China. Aim of the study To characterize the chemical composition, evaluate the antifungal activity and explore possible mechanisms about action of ethanol extracts of D. fragrans. Materials and methods The chemical components in the ethanol extracts of D. fragrans were detected by high-performance liquid chromatography coupled with electrospray ionization and quadruple time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS). The minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of the ethanol extracts of D. fragrans were determined by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method against 62 isolates of dermatophytes. The kinetics of fungal kill, synergy testing by checkerboard dilution and quantitation of sterol by ultra-performance liquid chromatography (UPLC) on Trichophyton rubrum and Trichophyton mentagrophytes were also investigated. Results Fourteen derivatives of phloroglucinol were identified in the ethanol extracts of D. fragrans. The MIC of the ethanol extracts of D. fragrans ranged from 0.059 to 3.780 mg/mL while MFC ranged from 0.118 to 3.780 mg/mL. The ethanol extracts of D. fragrans exerted fungicidal activity after 12 h of incubation against Trichophyton rubrum while it required 36 h of incubation against Trichophyton mentagrophytes at concentrations of 8 × MIC. In synergy testing, the interaction between miconazole (MCZ) and terbinafine (TBF) with the ethanol extracts of D. fragrans proved to be indifferent by testing fractional inhibitory concentration (FIC) values. Sterol in samples of fungal cells treated with the ethanol extracts of D. fragrans was significantly reduced. Conclusions The ethanol extracts of D. fragrans had antifungal and fungicidal activity against dermatophytes and was likely a strain-dependent fungicidal agent. Interaction between drugs was indifferent on tested isolates. The inhibition of ergosterol biosynthesis was one of the antifungal mechanisms of the ethanol extracts of D. fragrans. These results showed that the ethanol extracts of D. fragrans could be explored for promising antifungal drugs. Dozens of phloroglucinol derivatives may contribute to high antifungal activity of the ethanol extracts of D. fragrans.

Twenty-four racemic acylphloroglucinol meroterpenoids including eighteen unusual stuctures (3 ∼ 10, 13, 14, and 17 ∼ 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 ∼ 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-1β, 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).

Enzootic bovine haematuria (EBH) in cattle occurs in upland areas of the world. In India, the disease is present in isolated pockets in the Himalayas and in the Nilgiri Hills. The variation in the disease incidence has been attributed to different environmental conditions and animal rearing practices followed in the different regions. The aim of the study was to conduct field surveys in parts of EBH endemic regions of Himachal Pradesh, a north-western Himalayan state of India. Out of the total 103 plant samples collected, a total of 95 samples were identified as ferns. The major ferns identified included, Onychium japonicum (Thunb.) Kunze, Polystichum piceopaleaceum Tagawa, Dryopteris juxtaposita Christ, Pseudocyclosorus canus (Baker) Holttum and J.W. Grimes, Onychium contiguum C. Hope, Dryopteris nigropaleacea (Fraser-Jenk.), Pteridium aquilinum (L.) Kuhn, Diplazium esculentum (Retz.) Sw., Allantodia maxima (D. Don) Ching, Woodwardia unigemmata (Makino) Nakai, Pteris cretica L., Pteris vittata L., Asplenium trichomanes L., Thelypteris phegopteris (L.) Sloss. ex Rydb, Adiantum venustum D. Don and Paraceterach vestita (Hook.) R.M. Tryon. The concentration of ptaquiloside (PTA) and pterosin B (PtB) in some of the ferns collected from Kullu and Chamba regions ranged from 0 to 358.6 ± 70.5 μg/g and 0 to 652.4 ± 50.0 μg/g, respectively. In addition, field cases of the disease in cattle were also studied in the EBH endemic districts. A total of sixteen cattle urine samples and one urinary bladder of EBH affected cattle were collected. On physical, chemical (benzidine test) and microscopic examination of urine sediment, all the sixteen field samples were found to be positive for erythrocytes and the cases were diagnosed as macrohaematuria. The clinico-pathological studies on the field cases and the presence of PTA and PtB in the ferns indicated that EBH is a prevalent disease and there is an association between chronic fern ingestion and EBH in cattle. On the basis of gross pathology, histopathology and immunohistochemistry (p53 and H-ras nuclear expression in the urothelial cells) of the urinary bladder tissue, the field case was diagnosed as transitional cell adenocarcinoma with chronic lymphocytic cystitis.

The seasonal dynamics of the total flavonoid contents in various parts of Dryopteris erythrosora, a traditional Chinese medicinal fern, and their antioxidant activity were investigated. The total flavonoids content in various parts of D. erythrosora showed an obvious seasonal dynamic change. The total flavonoid contents in stems (from 4.3% to 12.5%) were much higher than that in leaves with an average content of 2.01%. In spring, the total flavonoid contents in stems were relatively low, but increased rapidly from summer to winter. However, the seasonal dynamics of total flavonoid contents in leaves showed different model. The total flavonoid contents in the stems showed a negative correlation with that in the leaves from January to July. The correlation coefficient of about -0.7 was obtained. The antioxidant activity of the extracts also altered in proportion to the change of total flavonoid contents. In general, the extracts from stems always showed highest antioxidant potentials and it was suggested that the stems can be used as crude medicine.

Keloid is an overgrowth of scar tissue that develops around a wound. The mechanisms of keloid formation and development still remain unknown, and no effective treatment is available. Searching for active natural resources may develop better prevention and treatment approaches for keloids. Aspidin PB is a natural resource with lower toxicity. We explored its effect on the regulation of TGF-β1-induced expression of type I collagen, CTGF, and α-SMA in keloid fibroblasts (KFs). Western blotting was used to detect the expression levels of type I collagen, CTGF, α-SMA, PI-3K/Akt and Smad-dependent and Smad-independent signaling pathway. The effect of aspidin PB on cell viability in human keloid fibroblasts was measured by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). The percentage of the apoptotic cells was studied by flow cytometry. Based on our results, we revealed that aspidin PB inhibited the production of type I collagen, CTGF, and α-SMA in TGF-β1-induced KFs by blocking PI-3K/Akt signaling pathway. The TGF-β1-mediated phosphorylated levels of Smad2/3 were inhibited by aspidin PB pretreatment. Conclusively, our study suggests that aspidin PB has an inhibitory effect on fibrogenesis in TGF-β1-induced KFs. Our findings imply that aspidin PB has a therapeutic potential to intervene and prevent keloids and other fibrotic diseases.

Ethnopharmacological relevance Dryopteris fragrans (L.) Schott (D. fragrans), a deciduous perennial herb, has been traditionally used for treatment of various skin diseases in Heilongjiang province of China for many years. Phloroglucinol derivatives extracted from D. fragrans were the most effective fraction against dermatophytes. Isoflavaspidic acid PB is a typically phloroglucinol derivative which extracted from D. fragrans and has been reported to exert anti-fungal activities against several dermatophytes. Aim of the study. This study aimed to evaluate anti-fungal and anti-biofilm activity of isoflavaspidic acid PB on planktonic and biofilm growth of dermatophytes and explore possible mechanisms of anti-biofilm. Materials and methods Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of isoflavaspidic acid PB against 25 isolates of dermatophytes were determined by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method. The effects of isoflavaspidic acid PB on dermatophytes biofilm formation and pre-formed biofilm were assessed by 2.3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl (phenylamino)]-2H-tetrazolium hydroxide (XTT) assay. Morphology of mature biofilm were observed by Scanning Electron Microscope (SEM). Biomass, exopolysaccharide and ergosterol content of mature biofilm were analyzed by gravimetric analysis, anthranone sulfuric acid method and Ultra Performance Liquid Chromatography (UPLC) assay respectively. Result The MIC and MFC ranges of isoflavaspidic acid PB against 25 isolates of dermatophytes were 20–80 μg/mL and 40–80 μg/mL respectively. Isoflavaspidic acid PB (2 MIC) inhibited not only Trichophyton biofilm formation (54.8% ∼ 81.2%) but also the metabolic activity of mature biofilm (20.7% ∼ 44.2%). The result of SEM showed that isoflavaspidic acid PB (8 MIC) could destroy the morphology of hyphae seriously. Comparing with control group, biomass, exopolysaccharide and ergosterol content of the mature biofilm under isoflavaspidic acid PB (8 MIC) were significantly decreased (P Conclusion Isoflavaspidic acid PB had anti-fungal and fungicidal activities against dermatophytes. Isoflavaspidic acid PB could inhibit the biofilm of Trichophyton. The mechanism might be related to the decline of the biofilm biomass, exopolysaccharide and ergosterol content. These results showed that isoflavaspidic acid PB could be explored for promising anti-biofilm drugs.

The Dryopteris varia species complex belongs to D. subg. Erythrovariae sect. Variae and includes eight closely related, controversial species. In Korea, six species belong to the complex; D. varia s. str., Dryopteris p acifica , Dryopteris sacrosancta , Dryopteris bissetiana , Dryopteris saxifraga , and Dryopteris saxifragivaria . The morphology of these species is highly variable due to the frequent occurrence of agamospory, hybridization, and/or polyploidization. We analyzed sequences of the nDNA pgi C and cpDNA rbc L, trn L– trn F IGS and atp F– atp H IGS regions from all six of these species to elucidate their relationships and origins, and to investigate the parentage of the putative hybrid taxon, D. saxifragivaria . Fourteen nDNA pgi C haplotypes and five cpDNA haplotypes were obtained from accessions of the D. varia complex in Korea. In particular, all accessions examined appear to have multiple pgi C haplotypes. Maximum parsimony and Bayesian inference analyses of the nDNA and cpDNA sequence data show that the agamosporous triploids D. sacrosancta and D. pacifica are of allopolyploid origin involving interspecific hybridization. The results also indicate that agamosporous triploid individuals of D. saxifragivaria in Korea were derived from hybridization between sexual diploid individuals of D. saxifraga (maternal) and agamosporous diploid individuals of D. bissetiana (paternal). In addition, our results strongly suggest that the agamosporous triploid D. varia s. str. is probably of autopolyploid origin, and cytoplasmic gene flow has occurred from D. sacrosancta to Dryopteris chinensis of D. subg. Dryopteris .

Biogeochemical samples were collected from the Norra Karr Alkaline Complex, a rare earth element (REE) and zirconium enriched deposit in Southern Sweden, to determine which sample medium is the most effective grassroots exploration tool for delineating concealed REE mineralization. The fern species Dryopteris filix-mas and Athyrium filix-femina were found to be widespread in the study area and surrounding countryside, and particularly efficient at concentrating high levels of REEs in their leaf tissue. There was distinct elemental fractionation in all three fern species. Each species showed enrichment in the LREEs (light rare earth elements), especially A. filix-femina. D. filix-mas showed the most enrichment in HREEs (heavy rare earth elements); Pteridium aquilinum had lower levels of REEs than the other two species. The best contrast was observed in D. filix-mas samples from areas over mineralization compared to samples taken over barren Vaxjo granites, which suggests that D. filix-mas is the preferred biogeochemical sample medium for REE exploration in this environment.

Ethnopharmacological relevance Dryopteris crassirhizoma has been traditionally used for the treatment of tapeworm infestation, the common cold and cancer in Korea, China and Japan. Despite various pharmacological properties of Dryopteris crassirhizoma , there is no available information about the safety of Dryopteris crassirhizoma . Aim of this study To ensure more information about the safety of Dryopteris crassirhizoma , we performed the acute oral toxicity and genotoxicity tests of Dryopteris crassirhizoma . Materials and methods The acute oral toxicity test of Dryopteris crassirhizoma was performed in rats. Genotoxicity of Dryopteris crassirhizoma was evaluated by bacterial reverse mutation, chromosomal aberration and bone marrow micronucleus tests. Results In acute toxicity test, Dryopteris crassirhizoma exhibited no mortality, body weight and behavioral changes and adverse effects in male and female rats. Dryopteris crassirhizoma did not significantly increase the number of the bacterial revertant and chromosomal aberration in both in vitro assays. Moreover, the Dryopteris crassirhizoma -related increases of micronucleated polychromatic erythrocytes (MNPCE) in mouse bone marrow were not observed. Conclusion Therefore, Dryopteris crassirhizoma is non-genotoxic in a three standard battery of tests and the oral LD 50 of Dryopteris crassirhizoma is >2000 mg/kg.

The profiles and bioactivities of flavonoids extracted from Dryopteris erythrosora were investigated. The total flavonoid content in full plant of D. Erythrosora is about 14.33%. The main flavonoids in D. Erythrosora were identified as gliricidin 7-O-hexoside, apigenin7-O-glucoside, quercetin 7-O-rutinoside, quercetin 7-O-galactoside, keampferol 7-O-gentiobioside, keampferol-3-O-rutinoside, myricetin 3-O-rhamnoside and quercitrin by means of HPLC-DAD-ESI-MS. Flavonoids (0.36mg/ml) extract from D. erythrosora showed similar 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH()), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS()), superoxide anion scavenging potential and ferric reducing antioxidant potential (FRAP) with that of rutin (0.80mg/ml). However, the antioxidant power by FRAP assay of 0.36mg/ml flavonoids extract from D. erythrosora was much weaker than that of 0.80mg/ml rutin. Moreover, the flavonoids extract from D. erythrosora showed obvious cytotoxic effects on A549 cells. The antioxidant activities of flavonoids extracts from 69 ferns showed a significant reciprocal proportion to the total flavonoids contents. The flavonoids extract from D. erythrosora exhibited a dose-dependent inhibition against acetylcholinesterase. Moreover, the anticancer activity slightly increased with improving antioxidant potential of fern flavonoids. Fern flavonoids are excellent function foods.

Dryofragin is a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott. In this study, the anticancer activity of dryofragin on human breast cancer MCF-7 cells was investigated. Dryofragin inhibited the growth of MCF-7 cells in a time and concentration-dependent manner. The cell viability was measured using MTT assay. After treatment with dryofragin for 72, 48 and 24 h, the IC₅₀ values were 27.26, 37.51 and 76.10 μM, respectively. Further analyses of DNA fragmentation and Annexin V-PI double-labeling indicated an induction of apoptosis. Dryofragin-treatment MCF-7 cells had a significantly accumulation of reactive oxygen species (ROS), as well as an increased percentage of cells with mitochondrial membrane potential (MMP) disruption. These phenomena were blocked by pretreatment for 2 h of MCF-7 cells with the antioxidant compound N-acetyl-L-cysteine (NAC, 5 mM). These results speak for the involvement of a ROS-mediated mitochondria-dependent pathway in dryofragin-induced apoptosis. Western blot results showed that dryofragin inhibited Bcl-2 and induced Bax expression which led to an activation of caspases-9 and -3 in the cytosol, and further cleavage of poly ADP-ribose polymerase (PARP) in the nucleus, then induced cell apoptosis. In conclusion, the present study provides evidence that dryofragin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway.

Solvent-free microwave extraction (SFME) of the essential oil from Dryopteris fragrans and its antioxidant activity were investigated. A central composite design combined with response surface methodology was applied to study the influences of extraction time, irradiation power and humidity (proportion of water pretreatment). A maximal extraction yield of 0.33% was achieved under optimal conditions of extraction time 34 min, irradiation power 520 W and humidity 51%. Sixteen compounds, representing 89.65% of the oil, were identified, of which the major ones, (1R,4S,11R)-4,6,6,11-tetramethyltricyclo[5.4.0.0(4,8)]undecan-1-ol (30.49%), 1R,4S,7S,11R-2,2,4,8-tetramethyltricyclo[5.3.1.0(4,11)]undec-8-ene (22.91%) and, 1,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethyl-1-naphthalenemethanol (15.11%), accounted for 68.51% of the oil. The antioxidant activity of the essential oil was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene/linoleic acid, and reducing power assay, the IC50 values were 0.19, 0.09 and 0.18 mg/mL, respectively. All these results suggest that SFME represents an excellent alternative protocol for production of essential oils from plant materials.

Arsenic accumulation and speciation in different plant species growing in two polluted sites were examined, in addition to arsenic transfer from soils to plants. The studied areas were two abandoned mercury mines in Northern Spain, La Soterrana and Los Rueldos. Plants accumulated extremely high amounts of arsenic in their tissues. Accumulation rates depend on both the grade of pollution of the site and on the plant species itself. Total arsenic concentrations varied depending on the part of the plant, with roots accumulating the most arsenic in all the studied plants (up to 1400 mg·kg − 1 ). A suitable extraction method for arsenic speciation in the studied samples was developed and optimized using 0.3 M orthophosphoric acid as the extracting agent in a graphite block digestion system at 90 °C for 1 h. Extraction efficiencies were above 80% and speciation was not altered, since recoveries of the spiked arsenic species (As(III), As(V), DMA (dimethylarsonic acid) and MMA (monomethylarsonic acid)) were between 94 and 102%. Speciation studies were performed on the parts of each plant species by means of HPLC-ICP-MS with the results indicating no significant differences in the speciation between parts of the plants. As(V) was always the predominant species, although a non-negligible amount of As(III) was found for all samples. Little amounts of MMA and DMA occurred in certain samples. Arsenic uptake by plants depends on the plant available arsenic fraction in soils, here considered as the sum of water-soluble and phosphate-extractable fractions. Although soils from La Soterrana were less polluted, arsenic bioavailability was much higher than in Los Rueldos. Soil-to-plant transfer factors, based on total and available arsenic in soils, were evaluated for the studied plants. Dryopteris filix-mas and Calluna vulgaris (L.) Hull , from La Soterrana and Los Rueldos respectively, were the plants with the highest abilities to extract arsenic from their corresponding soils.

Aim of the study The inhibitory effect of Dryopteris crassirhizoma on the proliferation of human metastatic prostate PC3-MM2 cells and the mechanism of action were examined to identify its anti-cancer properties. The effect of the extract on cell cycle progression and its combined cytotoxic effect with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on PC3-MM2 cells were also investigated. Materials and methods The anti-proliferative effects of Dryopteris crassirhizoma were examined by culturing PC3-MM2 cells in the presence or absence of various concentrations of Dryopteris crassirhizoma extract, and the inhibitory effects on cell proliferation were determined by Cell Counting Kit (CCK)-8 analysis. The quantities of apoptosis-inducing proteins were measured by western blotting analysis. Cell cycle progression was analyzed by PI staining using flow cytometry. Results Dryopteris crassirhizoma (50 and 100 μg/ml) inhibited markedly the proliferation of PC-3 and PC3-MM2 cells without cytotoxicity to normal (spleen) cells from BALB/C mice. Dryopteris crassirhizoma (100 μg/ml) effectively induced apoptosis through the activation of caspase-3, -8, -9, bid, and PARP in PC3-MM2 cells. The cells exposed to Dryopteris crassirhizoma increased significantly the accumulation of the DNA contents in the G0/G1 phase and sub-G1 phase in contrast to the control. The combined cytotoxic effects of Dryopteris crassirhizoma and TRAIL induced the increased activity of 29% in contrast to the sum of the inhibitory effects of each agent alone. Conclusions Dryopteris crassirhizoma has anti-cancer properties by inducing cell cycle arrest and apoptosis through the extrinsic and intrinsic pathway in PC3-MM2 cells. The extract also showed a combined effect with TRAIL on the inhibition of proliferation in the cells. These findings suggest that possibly its extract could be used for treating androgen-independent prostate cancer with minimal side effects.

One of the main concerns of current environmental toxicology is the low number of taxa used for standard bioassays. Ferns, with more than 10 000 living species, are the second largest group of vascular plants and are important components of numerous plant communities. Fern spores and gametophytes have long been recognized as useful models for plant research since they constitute a naturally miniaturised and economic higher plant model. Mitochondria are the main energy source in eukaryotic cells and any toxic damage will affect the whole organism. The reduction of tetrazolium salts to water-insoluble coloured formazan salts by the respiratory chain has been used for more than 50 years as a measure of cell mitochondrial activity and viability in eukaryotic organisms. Here, the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by mitochondria is adapted and optimized to measure fern spore or gametophyte viability. Procedures selected as optimum in the model species Dryopteris guanchica are as follows: bleach sterilization, incubation without shaking at 20 °C in the dark for 1–4 h with 0.05–1.5% TTC in Dyer medium supplemented with 0.001–0.005% Tween 20 at pH 8. We conclude that this method constitutes a promising low cost bioassay for higher plant toxicity during development.

Sesquiterpene glucoside was isolated from the aqueous extract of the aerial parts of Dryopteris fragrans (L.) schot. Three new sesquiterpene glucoside xianglinmaojuesides A–C ( 1 – 3 ), 3β,11-dihydroxy-drim-8 (12)-en-11-O-β- d -glucopyranoside ( 1 ), 3β,11-dihydroxy-drim-8(12)-en-3-O-β- d -glucopyranoside ( 2 ), and 11,14-dihydroxy-drim-8(12)-en-11-O-β- d -glucopyranoside ( 3 ) were finally obtained by reversed-phase HPLC and their structures were elucidated by HRESIMS and 1D, 2D NMR analyses.

Ferns produce a variety of cyclic triterpene hydrocarbons in large amount. Squalene cyclases (SCs) are responsible enzymes for formation of cyclic triterpene hydrocarbon skeletons. Although more than ten bacterial SCs have been cloned and four of them characterized for their enzymatic products, the only example of a fern SC is ACH, from Adiantum capillus-veneris, which produces hydroxyhopane. To obtain a deeper understanding of the molecular evolution of SCs and the origin of the structural diversity of fern triterpenes, further cloning and characterization of SCs have been pursued. In this study, a SC cDNA, DCD, was cloned from Dryopteris crassirhizoma by homology-based RT-PCR. DCD contains a 2058-bp open reading frame that encodes a 685 amino acid polypeptide exhibiting 66% identity to the previously identified fern SC, ACH, and 35-40% identity to bacterial SCs. Heterologous expression of DCD in yeast established it to be a dammaradiene synthase affording dammara-18(28),21-diene, a tetracyclic triterpene hydrocarbon. Although neither this compound nor any derived metabolites have been previously reported from D. crassirhizoma, re-investigation of the leaflets demonstrated the presence of dammara-18(28),21-diene. DCD represents the first SC that produces a tetracyclic triterpene hydrocarbon.

Methanol extract and its fractions (hexane, EtOAc, n-BuOH, and H2O) of Dryopteris crassirhizoma were investigated for antibacterial activity against methicillin-resistant Staphylococcus aureus. The hexane fraction showed a good antibacterial activity against all tested strains.

Summary Mention of fern populations at railway stations are extremely rare in the literature. Some older publications go so far as to expressly state that ferns do not occur at railway stations. Nevertheless, an inventory of fern species carried out at 63 railway stations in Central Europe has shown that this statement is no longer valid: ferns were found at more than 90% of the railway stations examined. An average of two fern species were found at each railway station, with up to six species detected at some locations. The most frequently encountered species were Dryopteris filix-mas , Asplenium ruta-muraria and A. trichomanes . The species found at railway stations included species of the Red List for Germany or at least for the individual German states. A repeat study performed in the Rhine-Main region shows that the number of fern species at railway stations has distinctly increased in recent years. The factors which are possibly responsible for this increase include the decreasing use of herbicides to combat weeds, the increasing age of the walls and platforms, and the changed emission situation.

Summary Lanthanum was demonstrated to be beneficial to the growth of Dryopteris erythrosora , a fern species which accumulates rare earth elements (REEs) under natural conditions. The enhancement of its growth by La was much greater than that by Ca. The uptake rates of REEs were evaluated to be higher than that of Zn and almost equal to those of Sr and Co. The region in the mesophyll where REEs were concentrated was not the same as that of Ca. In mesophyll cells, REEs were observed mainly in chloroplasts. These observations suggest that REEs do not behave as a ‹super Ca› as was suggested for some plants, but that they are transferred to chloroplasts by special mechanisms, possibly for fulfilling yet unknown functions or being stored as a mechanism of detoxification.

Spectroscopic properties, amino acid sequence, electron transfer kinetics, and crystal structures of the oxidized (at 1.7 A resolution) and reduced form (at 1.8 A resolution) of a novel plastocyanin from the fern Dryopteris crassirhizoma are presented. Kinetic studies show that the reduced form ofDryopteris plastocyanin remains redox-active at low pH, under conditions where the oxidation of the reduced form of other plastocyanins is inhibited by the protonation of a solvent-exposed active site residue, His87 (equivalent to His90in Dryopteris plastocyanin). The x-ray crystal structure analysis of Dryopteris plastocyanin reveals π-π stacking between Phe12 and His90, suggesting that the active site is uniquely protected against inactivation. Like higher plant plastocyanins, Dryopteris plastocyanin has an acidic patch, but this patch is located closer to the solvent-exposed active site His residue, and the total number of acidic residues is smaller. In the reactions of Dryopteris plastocyanin with inorganic redox reagents, the acidic patch (the “remote” site) and the hydrophobic patch surrounding His90 (the “adjacent” site) are equally efficient for electron transfer. These results indicate the significance of the lack of protonation at the active site of Dryopteris plastocyanin, the equivalence of the two electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer system of the first vascular plant fern, including its molecular evolutionary aspects. This is the first report on the characterization of plastocyanin and the first three-dimensional protein structure from fern plant.

The sparse resinous leaf exudates of Dryopteris villarii and D. arguta contain acylphloroglucinols that have previously only been found in the rhizomes of Dryopteris species. The phloroglucinol derivatives were characterized as representatives of the para-aspidin, desaspidin and albaspidin series by 2-D NMR spectroscopy and atmospheric pressure chemical ionization mass spectrometry (APCI-MS-MS). Mass fragmentation proved to be of diagnostic value to identify the acyl side chains and also allowed distinction between desaspidins and isomeric albaspidin analogues. The frond exudate of Currania robertiana was devoid of acylphloroglucinols, but was found to contain flavonoid aglycones. The acylphloroglucinols and flavonoid aglycones are presumably produced by external glands on the leaves, the presence and ultrastructure of which was examined by scanning electron microscopy.

The leaves of Leucosidea sericea (Rosaceae) are used medicinally by some indigenous South African people as a vermifuge and astringent. No information on the biological activity and phytochemistry of the plant could be found in the literature. Bioassay-guided fractionation was used to identify the active compound. Partial purification of the petroleum ether extract of the leaves and flowers was followed by fractionation using chromatographic methods. This process yielded the Phloroglucinol derivatives, aspidinol and desaspidinol, which were previously reported to be present in a Dryopteris species. Aspidinol was further isolated and structurally elucidated. This is the first report of the presence of these compounds in Leucosidea sericea. The disk diffusion method used in our study, indicated that the plant has antimicrobial activity against Staphylococcus aureus , Bacillus subtilis and Candida albicans .

The low-fluence-rate response of light-oriented chloroplast movement in Dryopteris sparse protonemata was induced by continuous irradiation with either red (R) or blue light (B). Effects of concomitant irradiation with far-red light (FR) indicated that the photoreceptor of R was phytochrome and that of B, a specific B photoreceptor. Action dichroism of light suggested that the absorption axes of the R-absorbing form of phytochrome and the B photoreceptor were parallel to the cell surface, and that of the FR-absorbing form of phytochrome (Pfr), perpendicular. When irradiation was terminated, the B-induced chloroplast arrangement began to decay immediately. In contrast, the R-induced arrangement survived for approximately 3 h, but when R irradiation was terminated with an FR pulse, the decay began after 1 h. When R irradiation was terminated before the response reached the maximum, it stayed at the attained level for 3 h, but did not increase further. These results suggest that the R induction of chloroplast movement required not only the presence of a Pfr gradient, but also the continued asorption of R, the former is only effective in determining the direction of the movement and in holding the chloroplasts at the area of higher Pfr concentration.

The phloroglucinol composition in two neotropical American ferns Dryopteris fuscoatra and D. hawaiiensis has been investigated. Dryopteris fusco-atra contains flavaspidic acids and filixic acids, which are a characteristic of the section Fibrillosae . The other member of the same section, D. hawaiiensis , lacks these compounds and instead contains much para-aspidin and the aromatic compounds margaspidin and phloraspidinol. Para-aspidin AB and margaspidin AB have been isolated for the first time from Dryopteris .

Summary In contrast to what is known about growth of gametophytes of the ferns Adiantum capillus-veneris and Dryopteris filix-mas in red light, the results presented demonstrate that a certain percentage of gametophytes of both species can undergo a transition to biplanar growth under red irradiation whether this is given continuously or interrupted by a dark period of several weeks.Unlike blue light-grown prothallia, two-dimensional development in red light only occurs after the formation of a fairly long filamentous part.

The effects of exposing plants of Dryopteris filix-mas (L.) Schott, Phyllitis scolopendrium (L.) Newman and Polypodium vulgare L. to 60 nl litre −1 (122 μg m −3 ) NO 2 for 37 weeks were investigated in a closed chamber fumigation system. There was no effect of NO 2 on the numbers of fronds produced for any species at any time during the exposure period. However, at the end of the study, there was a lower dry weight yield of green shoots of D. filix-mas and P. scolopendrium and a higher yield of green shoots of P. vulgare for plants in the NO 2 treatment as compared to control plants. These differences in shoot dry weights were not accompanied by an effect of NO 2 exposure on total plant dry weights.

A new phenolic glycoside, 3,5-dimethyl-6-hydroxy-2-methoxy-4-O-D-glucopyranosyl-oxy-acetophenone (1), was isolated from the aerial parts of Dryopteris fragrans. The structure was elucidated on the basis of spectroscopic methods.

Summary Dryopteris filix-mas (L.) S chott is known as a summer-green fern in Europe, though plants in the juvenile phase sometimes retain green leaves until the following spring. To determine the stage at which the fern shifts from evergreen to summer-green phenology, the number of midrib branches of the leaf (NV, number of venation) was used to quantify successive development of the sporophyte. Sterile leaves were quantitatively expressed as having 0 to 70-NV and fertile leaves with sori had 57 to 82-NV. The juvenile sporophyte was successively quantified below 55- NV. A clear phenological shift from the evergreen-like to the summergreen stage was quantitatively determined from the 55 to 60-NV stage, at which point the sporophyte changes from the juvenile to the adult life phase.

Summary Spores of Dryopteris filix-mas have an absolute light requirement for germination, which is mediated by phytochrome. The effect of a single red pulse 24 h after sowing is potentiated by a light pretreatment, given before the spores acquire competence to P fr . This pretreatment (the inducing effect of which is abolished by a far-red pulse) can consist of a single pulse as well; P fr has to be present for several hours, and is most effective towards the end of the pretreatment period. The effect does not require the maximum possible P fr level, and even a level of 10–20% P fr /P tot can significantly enhance the result of the subsequent inducing pulse. Generally, the pretreatment effect is greatest, if the inducing pulse is given with a non-saturating fluence or with a wavelength establishing an intermediate P fr level. It is assumed, and will be tested in future experiments, that P fr resulting from both pretreatment and inducing pulses act upon the same system, and that their products add to each other to surpass a threshold allowing germination.

Surface sample studies on the composition of pollen assemblages in the subalpine meadows and the adjacent Fagus scrub/forest in the crest region of the Vosges mountains indicate that: 1. (1) The proportions between the regional pollen types in the forest are different from those in the meadows. 2. (2) Forest- and meadow-type vegetation can be recognised on the basis of the local pollen assemblages. 3. (3) Among the pollen and spores produced by the Fagus scrub/forest only those of Fagus and Dryopteris are transported into the meadows upslope, giving rise to distinctly higher values than the regional ones.

The phloroglucinol composition of species and synthesised hybrids of the Dryopteris carthusiana complex has been examined. Hybrids appear to show the influence of either, or both, of the parental species. The significance of these results to the evolution of species within the complex is discussed.

Each diastereomer of N-(−)-camphanoyl-(3-methylthio)-1-aminopropane chirally deuteriated at C-1 has been synthesized and compared to deuteriated samples derived from L-methionine via enzymic decarboxylation. Here we report that decarboxylatlon occurs in a retentive mode.

The separation of a series of naturally occuring Dryopteris, Hagenia and Mallotus compounds and several artefacts by reversed-phase high-performance liquid chromatography is reported. A particularly good separation of bicyclic Dryopteris phloroglucinols including side-shain homologues was achieved. Moreover, the easily occuring decomposition of polycyclic phloroglucinols during chromatography could be totally avoided using slightly acidic conditions.

The phloroglucinol composition in two tropical American ferns, Dryoperis parallelogramma and D. patula has been investigated. Dryopteris parallelogramma , a member of the D. filix-mas complex, contains filixic acids, which are a characteristic of the group. Dryopteris patula contains aspidin, as do its relatives in the D. dilatata complex, and albaspidin- AA .

The red pigments in ferns were identified for the first time as 3-desoxyanthocyanins. Apigeninidin and luteolinidin were found, mainly as the 5-glucosides but also in several other combined forms, in the juvenile fronds of eight species. They also occurred in the red sori of Dryopteris erythrosma. The earlier report of common anthocyanins in Dryopteris could not be confirmed, but pelargonidin and cyanidin derivatives were definitely identified in Davallia divaricata. The distribution of 3-desoxyanthocyanins in the Gesneriaceae, the family from which they were first isolated, was also studied. They were found in 18 of 21 species in the sub-family Gesnerioideae but were absent from 25 species in the sub-family Cyrtandroideae. These results support Burtt's recent re-classification of the two sub-families on the basis of presence or absence of anisocotyly and geographical distribution but do not fit Fritsch's division of the family based on the position of the ovary. A new 3-desoxyanthocyanidin, columnidin, was isolated from Columnea x banksii and provisionally identified as 5,7,8,3′,4′-pentahydroxyflavylium. While common anthocyanins (e.g. malvidin 5-glucoside-3-rutinoside and pelargonidin-3-rutinoside) also occur in the Gesneriaceae, flavonols are absent. The flavones apigenin, luteolin and diosmetin were identified in nine species. It is suggested that 3-desoxyanthocyanin production is basically a primitive plant character but that these pigments are synthesized in the highly advanced Gesneriaceae in response to natural selection for a bright orange-red flower colour.

The gametophyte differentiation of a fern Dryopteris erythrosora has been investigated in relation to the behavior of ribonucleic acid (RNA). It has been found that two-dimensional growth was specifically inhibited with 8-azaguanine. In parallel to the inhibition, the analog was found to incorporate into RNA, when tested with C 14 -8-azaguanine. The study of changes in the RNA content during the gametophyte development has demonstrated no simple correlation of over-all concentration of RNA or of the rate of increase of RNA to the mode of growth. Conversion of the two-dimensional growth to the one-dimensional growth by the action of 8-azaguanine is followed by a cessation of increase in the RNA content or by degradation of RNA. Finally, it has been shown that RNA from the prothallium in the two-dimensional growth possesses a nucleotide composition distinct from that in the one-dimensional growth. RNA found in the prothallium in the one-dimensional growth induced artificially by 8-azaguanine has been shown to have practically the same nucleotide composition as that of the primary one-dimensional growth. From these observations, it has been tentatively inferred that a special RNA fraction which is sensitive to the action of 8-azaguanine is closely related to the two-dimensional growth.

The effect of desaspidin, ortho-desaspidin, flavaspidic acid, nor-flavaspidic acid and desaspidinol on respiration and oxidative phosphorylation in rat liver mitochondria was studied. All these substances uncouple oxidative phosphorylation. Desaspidin and orthodesaspidin show maximum activity in the concentration 2–4 × 10 −6 M, the other substances in concentrations of 1.6–6.4 × 10 −5 M. Respiration is inhibited more or less distinctly by these concentrations, especially when glutamate or succinate are used as substrate. When pyruvate and malate are employed as the substrate, this effect is less pronounced. In a medium free of terminal phosphate acceptor respiration is stimulated by desaspidin and by flavaspidic acid to values corresponding to those obtained with phosphate acceptor or with 2:4-dinitrophenol. The connection between chemical structure and uncoupling effect as well as the possible connection between the former and the biological effects of the substances tested are discussed briefly.

Summary Changes in isoenzyme patterns, induced by active phytochrome were studied in germinating fern spores of Pteridium aquilinum and Dryopteris filixmas. The functional analysis of soluble proteins by a sensitive electrophoretic method supplemented with enzyme specific staining showed characteristic phytochrome effect in NADP-IDH of D. filix-mas and G6PD of both species. This effect was virtually prevented by inhibitors of protein synthesis.

Investigation of Dryopteris marginalis has resulted in the isolation of a new phloroglucinol derivative, m.p. 178–180°, designated margaspidin. Characterization studies have indicated that it is a two-ring compound consisting of butyryl-3-methylphloroglucinol (II) and aspidinol (V) linked by a methylene bridge (I). The structure (I) has been confirmed by synthesis. Six other phloroglucinol derivatives have been identified and four of them isolated in pure form. These compounds are albaspidin, desaspidin, flavaspidic acid, para -aspidin, phloraspin, and phloraspidinol, of which the first three have been chromatographically detected by previous workers. Margaspidin has been found to be the major compound in D. marginalis extracts, the content being about ten times higher than that of the other phloroglucinol derivatives present in this Dryopteris species.