Detection of chlorophylls in spores of seven ferns.

Fern spores were traditionally classified into chlorophyllous (green) and nonchlorophyllous (nongreen) types based on the color visible to the naked eye. Recently, a third type, "cryptochlorophyllous spores", is recognized, and these spores are nongreen under white light but contain chlorophylls. Epifluorescence microscopy was previously used to detect chlorophylls in cryptochlorophyllous spores. In addition to epifluorescence microscopy, current study performed some other approaches, including spore-squash epifluorescence, absorption spectra, laser-induced fluorescence emission spectra, thin layer chromatography (TLC), and ultra-high performance liquid chromatography with ultraviolet and mass spectrometric detection (UHPLC-UV-MS) in order to detect chlorophylls of spores of seven ferns (Sphaeropteris lepifera, Ceratopteris thalictroides, Leptochilus wrightii, Leptochilus pothifolius, Lepidomicrosorum buergerianum, Osmunda banksiifolia, and Platycerium grande). Destructive methods, such as TLC and UHPLC-UV-MS, successfully detected chlorophylls inside the spores when their signals of red fluorescence under epifluorescence microscope were masked by spore wall. Although UHPLC-UV-MS analysis was the most sensitive and reliable for determining the chlorophylls of spores, spore-squash epifluorescence is not only reliable but also cost- and time-effective one among our study methods. In addition, we first confirmed that Lepidomicrosorium buergerianum, Leptochilus pothifolius, Leptochilus wrightii, and Platycerium grande, produce cryptochlorophyllous spores.

New and Cytotoxic Components from Antrodia camphorata.

The solid-state cultured products of Antrodia camphorata as health foods has been blooming for the past few decades in Taiwan. In continuing our studies on the chemical constituents of the solid-state cultured products of this fungus, 6-methoxy-4-methyl-2,3-(methylenedioxy)phenol (1) and 4,4'-(ethane-1,2-diyl)bis(2,3,6-trimethoxyphenol)(2) together with 2,3,6-trimethoxy-4-methylphenol (3), 1(10→6)abeo-ergosta-5,7,9,22-tetraen-3α-ol (4), citreoanthrasteroid B (5) and dankasterones A (6) and B (7) were purified by a series of column chromatography. Their structures were elucidated by spectral data analysis. For bioactivity assay, compounds 4-7 showed significant cytotoxicity toward murine colorectal CT26 and human leukemia K562 cancer cell lines with IC50 values ranging from 6.7 to 15.3 µM and from 12.5 to 23.1 µM, respectively.

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography-electrospray ionization-tandem mass spectrometry.

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and (34)S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC-MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r(2)>0.99) over a range of concentrations (SAM 0.01-2.0µM, SAH 0.002-0.10µM, Met 0.05-4.0µM, GSH 0.17-20.0µM, GSSG 0.07-20.0µM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.

Simultaneous determination of sulphur metabolites in Arabidopsis thaliana via LC-ESI-MS/MS and ³4S-metabolic labelling.

INTRODUCTION: Sulphur-containing metabolites play an important role in metabolism and homeostasis. Determination of these metabolites is challenging owing to their low concentrations and the interference in mass spectrometry analysis. OBJECTIVE: To develop a sensitive and accurate method based on liquid chromatography, electrospray ionisation, tandem mass spectrometry (LC-ESI-MS/MS) and ³4S-metabolic labelling for quantification of methionine, reduced glutathione, oxidised glutathione in Arabidopsis thaliana. METHODOLOGY: A hydroponic set-up was used for the in vivo ³4S-metabolic labelling of A. thaliana. The ³4S-labelled metabolites biosynthesised in plant were extracted and used as internal standards. Tissue was extracted with perchloric acid (PCA) or PCA containing a known amount of the analytes for recovery analysis. Tissue extract mixed with extract of ³4S-labelled A. thaliana in an appropriate ratio was subjected to a LC system and electrospray ionisation-mass spectrometric (ESI-MS) analysis. Quantification of metabolites was measured by comparing the ³4S/³4S ratios obtained for samples with the calibration curves. RESULTS: Calibration curves showed linearity with regression coefficients in the range of 0.9994-0.9999. Analyte recoveries were approximately 100%. The coefficients of variation of intra-assay and inter-assay were less than 4.2% and 5%, respectively. The ranges for the limits of detection determined for Met, GSSG and GSH were 10 fmol, < 10 fmol and 1.12 fmol and the limits of quantification determined for Met, GSSG and GSH were 0.44 pmol, 0.16 pmol and 34 fmol, respectively. CONCLUSION: The validated method for determination of methionine, reduced glutathione and oxidised glutathione was effectively applied to measure metabolite dynamics of sulphur-containing metabolites at the whole-plant level.

Novel terpenoids from Calocedrus macrolepis var. formosana.

Calocetriol (1), diacetylcalocediol (2), and ferrugimenthenol (3) were isolated from the bark of Calocedrus macrolepis var. formosana. Among them, 1 and 2 are secoabietane-type diterpenoids, and 3, with a novel C(20)-C(10) skeleton, is classified as a meroterpenoid. The structures of 1-3 were elucidated by spectroscopic analyses, and their biological activities were also evaluated. Compound 3 exhibited significant cytotoxic activity against human oral epidermoid carcinoma KB cells with an IC(50) value of 9.0±0.1 µM.

Phenylpropanoids and lignoids from the whole plant of Vaccinium emarginatum and their cytotoxicity against prostate cancer cells.

One new naturally occurring quinone, 3',4'-dihydroxy-1,2,6-trimethoxy-[1,1'-biphenyl]-4(1H)-one (1), one new diarylpropane, emarginone A (2), and one new neolignan, emarginone B (3), along with eighteen known compounds have been isolated from the chemical investigation of the EtOAc-soluble fraction of the Vaccinium emarginatum whole plant methanolic extract. The new structures were elucidated by combined analysis of spectroscopic analytical methods and comparison with the literature data obtained from known analogues. In addition, the cytotoxicity of compounds 2, 4, and 14-20 against Du145 and PC-3 prostate cancer cell lines using MTT cell proliferation assay was evaluated. Compounds 2 and 19 showed most potent cytotoxicity against Du145 with IC50 values of 7.53 and 6.63 µg/mL, respectively. Furthermore, compounds 2, 17, and 19 also exhibited significant cytotoxicity against PC-3 with IC50 values ranging from 3.44-6.64 µg/mL.

18-nor-Podocarpanes and podocarpanes from the Bark of Taiwania cryptomerioides.

Seven nor- and podocarpane-type diterpenes were isolated from the bark of Taiwania cryptomerioides Hayata, including three 18-nor-podocarpanes: 18-nor-1beta,4alpha,14-trihydroxy-13-methoxy-8,11,13-podocarpatriene (1), 18-nor-1beta,4alpha,13,14-tetrahydroxy-8,11,13-podocarpatrien-7-one (2), 18-nor-1beta,4alpha,14-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (3), 1beta,14,19-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (4), 1beta,13,14,18-tetrahydroxy-8,11,13-podocarpatrien-7-one (5), 18-acetoxy-1beta,13,14-trihydroxy-8,11,13-podocarpatrien-7-one (6), and 1beta,14,18-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (7). Their structures were determined by application of 1D and 2D NMR spectroscopy and other techniques. Podocarpane-type diterpenes do not occur extensively in nature, and the presumed oxidative enzyme in this plant will be of interest to identify.

MALDI MS analysis, disk diffusion and optical density measurements for the antimicrobial effect of zinc oxide nanorods integrated in graphene oxide nanostructures.

Graphene oxide-zinc oxide hybrid nanostructures were synthesized and they demonstrated significant and promising antimicrobial activity on pathogenic bacteria. The combination of graphene oxide with zinc oxide nanorods showed an impressive antibacterial effect under intense scrutiny as compared with individual graphene oxide or zinc oxide nanomaterials. The characterization and investigation of GO-ZnO nanorod hybrid nanostructures were conducted using UV, FTIR, XRD, SEM, EDX and TEM measurements. The antimicrobial activity of the above hybrid material was evaluated by various methods including MALDI-MS analysis, a disk diffusion assay and optical density measurements.

New Furanone and Sesquiterpene from the Pericarp of Calocedrus formosana.

One new γ-lactone, namely calolactone (1), together with one new drimane-type sesquiterpene, namely caloterpene (2), were isolated from the pericarp of Calocedrus formosana Florin. Their structures were elucidated by spectroscopic and mass spectrometric analysis.

Copper exposure interferes with the regulation of the uptake, distribution and metabolism of sulfate in Chinese cabbage.

Exposure of Chinese cabbage (Brassica pekinensis) to enhanced Cu(2+) concentrations (1-10 microM) resulted in leaf chlorosis, a loss of photosynthetic capacity and lower biomass production at > or = 5 microM. The decrease in pigment content was likely not the consequence of degradation, but due to hindered chloroplast development upon Cu exposure. The Cu content of the root increased with the Cu(2+) concentration (up to 40-fold), though only a minor proportion (4%) was transferred to the shoot. The nitrate uptake by the root was substantially reduced at > or = 5 microM Cu(2+). The nitrogen content of the root was affected little at lower Cu(2+) levels, whereas that in the shoot was decreased at > or = 5 microM Cu(2+). Cu affected the uptake, distribution and metabolism of sulfate in Chinese cabbage. The total sulfur content of the shoot was increased at > or = 2 microM Cu(2+), which could be attributed mainly to an increase in sulfate content. Moreover, there was a strong increase in water-soluble non-protein thiol content in the root and, to a lesser extent, in the shoot at > or = 1 microM, which could only partially be ascribed to a Cu-induced enhancement of the phytochelatin content. The nitrate uptake by the root was substantially reduced at > or = 5 microM Cu(2+), coinciding with a decrease in biomass production. However, the activity of the sulfate transporters in the root was slightly enhanced at 2 and 5 microM Cu(2+), accompanied by enhanced expression of the Group 1 high affinity transporter Sultr1;2, and the Group 4 transporters Sultr4;1 and Sultr4;2. In the shoot, there was an induction of expression of Sultr4;2 at 5 and 10 microM Cu(2+). The expression of APS reductase was affected little in the root and shoot up to 10 microM Cu(2+). The upregulation of the sulfate transporters may be due not only to greater sulfur demand at higher Cu levels, but also the consequence of interference by Cu with the signal transduction pathway regulating the expression and activity of the sulfate transporters.

Allelopathy on bark of downed logs of Chamaecyparis Obtusa sieb. and Zucc. var. formosana (Hayata) Rehder.

Chamaecyparis obtusa Sieb. and Zucc. var. formosana (Hayata) Rehder is the dominant species in the temperate forest of Yuanyang Lake Nature Reserve (YYL), Taiwan. Although downed logs of C. obstusa var. formosana occupy only a small percentage of the forest floor area in YYL, they are important regeneration substrates. Seedlings of this species often grow without competition on the new downed logs, and a few broadleaf trees grow with them. We hypothesized that the bark of the newly fallen logs possesses allelopathic potential that provides a habitat especially suitable for seedling establishment. Eight different seeds including those from Lactuca sativa L. (lettuce), Bidens pilosa (an invasive weed), and six species in YYL were planted on the bark of the downed logs in an incubator for germination tests. Two dominant species in the forest of YYL, C. obtusa var. formosana and Rhododendron formosanum, were able to grow normally, but the others, Pieris taiwanensis, Barthea formosana, Chamaecyparis formosensis, Miscanthus transmorrisonensis, lettuce, and B. pilosa were growth inhibited. A bioactivity-guided isolation was designed to isolate allelochemicals from the bark. Salicylic acid, one of the inhibiting substances, was isolated and identified by gas chromatography/mass spectroscopy (GC/MS), proton nuclear magnetic resonance ((1)H NMR), and infrared (IR). Bioassay of salicylic acid confirmed a phytotoxic effect. The results suggest that the dominance of C. obtusa var. formosana seedlings on bark could be partly due to allelopathy.