Anti-inflammatory activities of several diterpenoids isolated from Hemionitis albofusca.

Hemionitis albofusca (Baker) Christenh is a plant that grows in various regions of China. Although it is not recognized as a traditional medicine, it is often mistakenly labelled and used as Aleuritopteris argentea (S. G. Gmél.) Fée to alleviate menstruation-related issues. Recently, several diterpenoids such as ent-16-oxo-17-norkauran-19-oic acid (Compound A), 14-oxy-7ß,20-dihydroxycyath-12,18-diene (Compound B), ent-8(14),15-pimaradiene-2ß,19-diol (Compound C), ent-kaurane-16-ene-2ß,18α-diol (Compound D), ent-kaurane-2ß,16α,18α-triol (Compound E), and onychiol B have been extracted from H. albofusca. In this study, we investigated the anti-inflammatory activity of these diterpenes. We confirmed that compounds A ~ D suppressed the amount of cellular NO production by inhibiting the expression and transcription of iNOS protein. They also significantly inhibited the expression and transcription of inflammatory factors TNF-α and IL-6. Additionally, Compounds A and C suppressed the activation of the NF-κB signaling pathway and inhibited the phosphorylation level of p38, ultimately down-regulating inflammation. Compound B suppressed the activation of the NF-κB signaling pathway, while Compound D inhibited the phosphorylation level of p38 and down-regulated the activation of the p38 MAPK signaling pathway. In a word, our investigation supports the potential application of natural diterpenes as lead compounds for developing anti-inflammatory agents.

Antibacterial effect of Cu2O/TiO2 photocatalytic composite on Pseudomonas marginalis pv. marginalis.

Cu2O/TiO2 visible-light photocatalytic composite was successfully synthesized by supercritical solvothermal route. Cu2O/TiO2 presented excellent bacterial inactivation activity for Pseudomonas marginalis pv. marginalis, which was related to the concentration of bacteria and the antibacterial time. The highest sterilization ratio reached up to 100% when the bacteria was treated with 80 µg/mL of Cu2O/TiO2 photocatalytic composite for 80 min, which could be further proved by the damage of integrity and shrink of the cell membrane in transmission electron microscopy (TEM) image. When the bacterial concentration was 1 × 105 CFU/mL, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined as 16 and 32 µg/mL by agar dilution, respectively. Meanwhile, the production of reactive oxygen species (ROS), glutathione reductase (GR) and glutathione (GSH) of Pseudomonas marginalis pv. marginalis treated by Cu2O/TiO2 were determined by DCFH-DA, DTNB and kinetic method, respectively, to evaluate the anti-oxidation capacity of bacteria cell. The enzyme activity of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) in bacteria treated with Cu2O/TiO2 were measured to further confirm the overproduction of ROS. Cu2O/TiO2 was demonstrated as the excellent visible-light photocatalyst for efficiently killing Pseudomonas marginalis pv. marginalis with the low dosage. Finally, the Cu2O/TiO2 composite photocatalytic material was applied to cucumber seedlings based on field experimental, and its inhibitory effect in practical application was judged by measuring the morphology, enzyme activity and resistance index of cucumber plants. It is of great significance to the practical application as a suitable and powerful antibacterial agent for Pseudomonas marginalis pv. marginalis and other bacteria.

Ceratopteris chunii and Ceratopteris chingii (Pteridaceae), two new diploid species from China, based on morphological, cytological, and molecular data.

Understanding how natural hybridization and polyploidizations originate in plants requires identifying potential diploid ancestors. However, cryptic plant species are widespread, particularly in Ceratopteris (Pteridaceae). Identifying Ceratopteris cryptic species with different polyploidy levels is a challenge because Ceratopteris spp. exhibit high degrees of phenotypic plasticity. Here, two new cryptic species of Ceratopteris, Ceratopteris chunii and Ceratopteris chingii, are described and illustrated. Phylogenetic analyses reveal that each of the new species form a well-supported clade. C. chunii and C. chingii are similar to Ceratopteris gaudichaudii var. vulgaris and C. pteridoides, respectively, but distinct from their relatives in the stipe, basal pinna of the sterile leaf or subelliptic shape of the fertile leaf, as well as the spore surface. In addition, chromosome studies indicate that C. chunii and C. chingii are both diploid. These findings will help us further understand the origin of Ceratopteris polyploids in Asia.

Documenting the Sporangium Development of the Polypodiales Fern Pteris multifida.

Reconstructing the development of sporangia in seed-free vascular plants provides crucial information about key processes enabling the production of spores that are important in the life cycle of these plants. By applying fluorescence imaging in intact tissues using dyes and confocal microscopy, this study aimed to reconstruct the key steps during the development of sporangia. Special emphasis was taken on the cell wall structures of tapetum and spore mother cells that have been challenged by microscopical documentation in the past. After staining the cell wall and cytoplasm using calcofluor white and basic fuchsin, the sporangium development of Pteris multifida was observed using confocal microscopy. The clear cell lineages from the sporangial initial cell to stalk, epidermis, inner tapetum, outer tapetum, and sporogenous cells were revealed by confocal imaging. The sporangium development improved in this work will be useful for a general understanding of fern spore formation.

Synthesis and biological evaluation of cytotoxic activity of novel podophyllotoxin derivatives incorporating piperazinyl-cinnamic amide moieties.

Podophyllotoxin, as a natural lignan isolated from the dried rhizomes and roots of several plant species of Podophyllum family, exhibits potent activity of interfering polymerization of tubulin and causes cancer cell apoptosis. Structure-activity relationship research revealed that modification at 4-position was tolerable for its potency. In the present study, podophyllotoxin derivatives incorporating piperazinyl-cinnamic amide moieties at 4-position were designed and synthesized. Their structures were confirmed by 1H NMR, 13C NMR, and mass spectral data. ADMET analysis proposed that these compounds had a good distribution and high clearance profile with little toxicity. The cytotoxicity of these derivatives was evaluated against four human cancer cell lines (MCF-7, A549, HeLa and PC-3) by MTT assay. Among all the compounds, compound 6e exhibited the best anti-proliferative properties with an IC50 = 0.08 ± 0.01 µM against MCF-7 cancer cell line. Further cellular mechanism studies by cell colony formation, mitochondrial membrane potential assay, nuclear morphology analysis and western blot confirmed that compound 6e could inhibit cancer cell proliferation and induce mitochondria-associated apoptosis in MCF-7 cells. Meanwhile, immunofluorescence assay revealed that compound 6e could apparently disrupt tubulin network in MCF-7 cells, and molecular docking further supported that compound 6e was able to bind into the colchicine site of tubulin. The above results might lay a foundation for further investigation for drug discovery based on podophyllotoxin.

Adiantic acid, a new unsaturated fatty acid with a cyclopropane moiety from Adiantum flabellulatum L.

Phytochemical investigation of Adiantum flabellulatum L. led to the isolation of four natural compounds, including a novel unsaturated fatty acid with a cyclopropane moiety, i.e. (S,E)-7-(2-octylcyclopropylidene)heptanoic acid (1), together with three known compounds, isoadiantol B (2), stigmast-4-en-6ß-ol-3-one (3), ß-sitosterol (4). Compound 3 was isolated from the A. flabellulatum L. for the first time. The structure of 1 was elucidated following a comprehensive analysis of spectroscopic analyses including MS, 1 D and 2 D NMR, and by a mass spectrometry experiment of the dimethyl disulfide (DMDS) adduct, while the known compounds were identified by comparisons with those reported in the literature. Enzyme evaluation of 1 indicated this compound possesses anti- protein tyrosine phosphatase (PTP1B) activity with an IC50 value of 6.99 ± 0.41 µM in vitro.

Incorporation of amino moiety to alepterolic acid improve activity against cancer cell lines: Synthesis and biological evaluation.

Aleuritopteris argentea (S. G. Gmél.) Fée is a medicinal fern consisting of an ent-labdane diterpene, i.e. alepterolic aicd, as the major metabolite. We recently isolated grams of alepterolic acid from A. argentea enabling subsequent structural modification. By incorporation of amino moiety to alepterolic acid, fifteen amide derivatives were synthesized, characterized, and further biological evaluated regarding their activity against four cancer cells and normal human liver cells. The potency of synthesized amides dramatically improved as compared to alepterolic aicd itself. The best hit (compound 11) inhibits HeLa cells with an IC50 of 7.39 ± 0.80 µM, and is nearly nontoxic to normal cells. Compound 11 exhibits an inhibitory effect on the colony forming ability of the four cancer cells, especially of HeLa cells. Moreover, it induces apoptosis of HeLa cells by decreasing mitochondrial membrane potential and altering expression of apoptosis-associated proteins. Release of cytochrome c, activation of caspases-3, caspases-9 and alteration of Bax/Bcl-2 balance was detected in the biological assays. These results imply that compound 11 can inhibit the proliferation of cervical cancer cell line HeLa and induce apoptosis through the mitochondrial pathway. These findings encourage further rational structural modification of 15- carboxyl group of alepterolic acid.

Synthesis of Novel Analogs of Thieno[2,3-d] Pyrimidin-4(3H)-ones as Selective Inhibitors of Cancer Cell Growth.

New 2,3-disubstituted thieno[2,3-d]pyrimidin-4(3H)-ones were synthesized via a one-pot reaction from 2H-thieno[2,3-d] [1,3]oxazine-2,4(1H)-diones, aromatic aldehydes, and benzylamine or 4-hydroxylbezylamine. The obtained compounds were tested in vitro for cancer cell growth inhibition. Compound 19 can inhibit all four types of tested cancer cells, i.e., MCF-7, A549, PC-9, and PC-3 cells. Most of the compounds inhibited the proliferation of A549 and MCF-7 cells. Compound 15 exhibited the strongest anti-proliferative effect against A549 cell lines with IC50 values of 0.94 µM, and with no toxicity to normal human liver cells. Its potency was further proved by cell clone formation assay, Hoechst 33258 staining, and evaluation on the effects of apoptosis-related proteins.

Correction: Total flavonoid concentrations of bryophytes from Tianmu Mountain, Zhejiang Province (China): Phylogeny and ecological factors.

[This corrects the article DOI: 10.1371/journal.pone.0173003.].

Total flavonoid concentrations of bryophytes from Tianmu Mountain, Zhejiang Province (China): Phylogeny and ecological factors.

The flavonoids in bryophytes may have great significance in phylogeny and metabolism research. However, to date there has been little research on bryophyte metabolites, especially flavonoids. To redress this somewhat, we determined flavonoid concentrations of bryophytes from Tianmu Mountain through a colorimetric assay and considered the factors influencing the results. This is the first time that the flavonoid contents of bryophytes have been examined in detail. The results revealed a range of total flavonoid concentrations in 90 samples collected from Tianmu Mountain from 1.8 to 22.3 mg/g (w/w). The total flavonoid contents of liverworts were generally higher than those of mosses; acrocarpous mosses had generally higher values than that of pleurocarpous mosses. The total flavonoid contents of bryophytes growing at lower light levels were general higher than those growing in full-sun. The total flavonoid contents of epiphytic bryophytes were highest, while those of aquatic bryophytes were the lowest. Total flavonoid contents of species growing at low-latitudes were much higher than those at high-latitude individuals. In conclusion, total flavonoid contents of bryophytes have some connection with plant phylogeny; more flavonoids might be contained in relatively primitive bryophytes. Meanwhile, the effects of ecological factors on total flavonoid contents of bryophytes exist; light and habitat (especially tree habitat and river habitat) might be representative factor.

Seasonal dynamics of total flavonoid contents and antioxidant activity of Dryopteris erythrosora.

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.

Chemical composition and bioactivities of flavonoids-rich extract from Davallia cylindrica Ching.

The flavonoids profiles and bioactivities of flavonoids-rich extract from Davallia cylindrica Ching were investigated. The total flavonoids content in D. cylindrica was determined as about 164.41 mg/g. The main flavonoids in D. cylindrica were tentatively identified as quercetin-3-O-rutinoside, quercetin 7-O-glucoside, quercetin 7-O-glucoside, kaempferol 3-O-rutinoside, and quercitrin by UV and ESI-MS spectra. Flavonoids-rich extract (0.258 mg/ml) from D. cylindrica showed similar or higher free radical (O2(-), DPPH and ABTS) scavenging potential with that of rutin (0.25 mg/ml). The reducing power of flavonoids-rich extract (0.258 mg/ml) was slightly stronger than that of 0.25mg/ml rutin. The flavonoids extract from D. cylindrica exhibited cytotoxic effects on A549 cells. It exhibited a dose-dependent inhibition against acetylcholinesterase.

Flavonoids profiles, antioxidant, acetylcholinesterase inhibition activities of extract from Dryoathyrium boryanum (Willd.) Ching.

The profiles and bioactivities of flavonoids extracted from Dryoathyrium boryanum (Willd.) Ching were investigated. The total flavonoids content in extract from D. boryanum is about 145.8mg/g. By means of HPLC-DAD-ESI-MS, the main flavonoids in D. boryanum were tentatively identified as 3-hydroxyphloretin 6'-O-hexoside, quercetin-7-hexoside, apigenin7-O-glucoside, luteolin 7-O-glucoside, apigenin 7-O-galactoside, acacetin 7-O-(α-D-apio-furanosyl) (1→6)-ß-d-glucoside, 3-hydroxy phloretin 6-O-hexoside, luteolin-6-C-glucoside. 0.21mg/ml flavonoids extract from D. boryanum showed very strong superoxide anion radical scavenging potential, which is higher than that of rutin (0.25mg/ml). The extract (0.21mg/ml of flavonoids) from D. boryanum exhibited similar DPPH scavenging potential with that of rutin (0.25mg/ml). However, rutin (0.25mg/ml) showed a significantly higher reducing power and ABTS scavenging potential than that of 0.21mg/ml flavonoids extract from D. boryanum. It had no effect on acetylcholinesterase. D. boryanum can be considered as a medicinal plant and the flavonoids from D. boryanum are excellent antioxidants.

Ultrastructural and cytochemical studies on oogenesis of the fern Pteridium aquilinum.

Egg development in Pteridium aquilinum var. latiusculum was studied using ultrastructural and cytochemical methods to examine structural features influencing fertilization in leptosporangiate ferns. Ultrastructural observations indicate a separation cavity is first formed above the egg during oogenesis with a pore region persistently connecting the egg and the ventral canal cell. The egg envelope is formed by deposition of amorphous materials in the separation cavity on the outer surface of plasmalemma. The egg envelope was not formed across the pore region; instead, a fertilization pore was formed. During oogenesis, the egg nucleus produced extensive evaginations containing osmiophilic bodies. Cytochemical experiments revealed that the egg envelope displays strong periodic acid-Schiff reaction indicative of polysaccharides, with negligible Sudan black B staining for lipids, suggesting that the egg envelope is composed principally of polysaccharides, and not lipids. The present manuscript provides new insights into egg structure and development of Pteridium, including discovery and characterization of the fertilization pore and observations on the chemical nature of the egg envelope, thus contributing to the understanding of the cytological mechanism of the sexual reproduction of ferns.

Flavonoid Contents and Free Radical Scavenging Activity of Extracts from Leaves, Stems, Rachis and Roots of Dryopteris erythrosora.

The present study was designed to explore the flavonoid contents and the radical scavenging activities of 50% ethanol extracts of leaves, stems, rachis and roots of Dryopteris erythrosora. The total flavonoid contents in various parts were determined as: stems > roots > rachis > leaves. All extracts from different parts of D. erythrosora showed strong bioactivities. The DPPH free radical scavenging abilities of extracts were determined as: stems > root > rachis >> leaves. This trend is reciprocal proportion to the total flavonoid contents in leaves, stems, rachis and roots of D. erythrosora extracts. The superoxide anion scavenging abilities of ethanol extracts were determined as: stems > leaves > rachis > root. It illustrated that there are strong superoxide anion scavengers in D. erythrosora. It is worth to separate and identify these components in future.