Determination of indican, isatin, indirubin and indigotin in Isatis indigotica by liquid chromatography/electrospray ionization tandem mass spectrometry

The roots and leaves of Isatis indigotica, named 'Ban-Lan-Gen' and 'Da-Qing-Ye', respectively, are widely used for the treatment of influenza, viral pneumonia, mumps, pharyngitis, and hepatitis. The indoxyl derivatives detected in the roots and leaves of I. indigotica have been reported to be biologically active. In the present study, a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed to determine indican, isatin, indirubin and indigotin in the roots and leaves of I. indigotica. The method has been validated for linearity, precision and accuracy. Using multiple reaction monitoring (MRM), the limits of detection (LODs) were determined as 0.004 ng for indican, 0.01 ng for isatin, 0.01 ng for indrubin and 0.03 ng for indigotin, while the limits of quantitation (LOQs) were 0.015 ng for indican, 0.04 ng for isatin, 0.04 ng for indirubin and 0.1 ng for indigotin. Compared with previously reported methods, the current method is more rapid, selective and sensitive. This is the first report of the LC/MS/MS determination of indican, isatin, indirubin and indigotin.     

柱前衍生化反相高效液相色谱法测定板蓝根中的氨基酸

建立了板蓝根中指标性成分精氨酸、脯氨酸和谷氨酸的柱前衍生化反相高效液相色谱分析方法。以2,4-二硝基氟苯为衍生化试剂,将氨基酸在碱性条件下衍生化,然后直接进样进行高效液相色谱测定。采用的色谱条件为：色谱柱Sinochrom ODS-BP柱(250 mm×4.6 mm i.d.,5 μm),流动相醋酸钠缓冲溶液(pH 6.4)-乙腈(体积比为850∶150),检测波长360 nm,采用外标法定量。该文同时对谷氨酸、精氨酸和脯氨酸进行定性,并对精氨酸和脯氨酸进行定量。精氨酸和脯氨酸的进样量分别为0.627～5.016 μg和0.874～7.000 μg时,其峰面积与进样量呈良好的线性关系(r分别为0.9996和0.9995)。精氨酸和脯氨酸的平均回收率分别为98.5%(相对标准偏差（RSD）为2.5%（n=8))和98.4%(RSD为2.3%（n=8))。对板蓝根样品进行了测定,结果表明,板蓝根中精氨酸的含量最高,脯氨酸次之,谷氨酸含量很少。该法简便、准确、可靠,适用于板蓝根药材的质量控制。     

彩色不锈钢与氧化着色

通过化学或电化学手段与技术促使不锈钢表面发生氧化生成的钝化膜,不仅能够保护金属免受腐蚀,还能产生光干涉效应,使不锈钢不再是单一的银白色,而是呈现红橙黄绿蓝靛紫等不同的颜色,扩展了不锈钢的应用范围.     

Evidence for yellow light suppression of lettuce growth

Researchers studying plant growth under different lamp types often attribute differences in growth to a blue light response. Lettuce plants were grown in six blue light treatments comprising five blue light fractions (0, 2, 6% from high-pressure sodium [HPS] lamps and 6, 12, 26% from metal halide [MH] lamps). Lettuce chlorophyll concentration, dry mass, leaf area and specific leaf area under the HPS and MH 6% blue were significantly different, suggesting wavelengths other than blue and red affected plant growth. Results were reproducible in two replicate studies at each of two photosynthetic photon fluxes, 200 and 500 mumol m-2 s-1. We graphed the data against absolute blue light, phytochrome photoequilibrium, phototropic blue, UV, red:far red, blue:red, blue: far red and 'yellow' light fraction. Only the 'yellow' wavelength range (580-600 nm) explained the differences between the two lamp types.     

Micro-Raman spectroscopy of natural and synthetic indigo samples

In this work indigo samples from three different sources are studied by using Raman spectroscopy: the synthetic pigment and pigments from the woad (Isatis tinctoria) and the indigo plant (Indigofera tinctoria). 21 samples were obtained from 8 suppliers; for each sample 5 Raman spectra were recorded and used for further chemometrical analysis. Principal components analysis (PCA) was performed as data reduction method before applying hierarchical cluster analysis. Linear discriminant analysis (LDA) was implemented as a non-hierarchical supervised pattern recognition method to build a classification model. In order to avoid broad-shaped interferences from the fluorescence background, the influence of 1st and 2nd derivatives on the classification was studied by using cross-validation. Although chemically identical, it is shown that Raman spectroscopy in combination with suitable chemometric methods has the potential to discriminate between synthetic and natural indigo samples.     

Effect of light quality on the accumulation of photosynthetic pigments, proteins and mycosporine-like amino acids in the red alga Porphyra leucosticta (Bangiales, Rhodophyta)

The effect of different light qualities (white, blue, green, yellow and red light) on photosynthesis, measured as chlorophyll fluorescence, and the accumulation of photosynthetic pigments, proteins and the UV-absorbing mycosporine-like amino acids (MAAs) was studied in the red alga Porphyra leucosticta. Blue light promoted the highest accumulation of nitrogen metabolism derived compounds i.e., MAAs, phycoerythrin and proteins in previously N-starved algae after seven days culture in ammonium enriched medium. Similar results were observed in the culture under white light. In contrast, the lowest photosynthetic capacity i.e., lowest electron transport rate and lowest photosynthetic efficiency as well as the growth rate were found under blue light, while higher values were found in red and white lights. Blue light favored the accumulation of the MAAs porphyra-334, palythine and asterina-330 in P. leucosticta. However, white, green, yellow and red lights favored the accumulation of shinorine. The increase of porphyra-334, palythine and asterina-330 occurred in blue light simultaneous to a decrease in shinorine. The accumulation of MAAs and other nitrogenous compounds in P. leucosticta under blue light could not be attributed to photosynthesis and the action of a non-photosynthetic blue light photoreceptor is suggested. A non-photosynthetic photoreceptor could be also involved in the MAAs interconversion pathways in P. leucosticta.     

FIBER-OPTIC PLANT TISSUES: SPECTRAL DEPENDENCE IN DARK-GROWN AND GREEN TISSUES*

Abstract— The fiber-optic properties of etiolated plant tissues can be used to detect and characterize pigment absorption in vivo. Transmission spectra of light guided through several monocot and dicot etiolated tissues show a decreasing red/far red ratio with increasing tissue length. Absorption bands attributable both to vacuolar pigments such as anthocyanins and to chloroplast pigments lead to the conclusion that the guided light passes through both vacuole and cytoplasm. As etiolated tissue becomes green under white light treatment, the red/far red ratio also changes, the nature of the change depending upon the tissue involved. The blue/red ratio also changes both with increasing length of etiolated tissue and during the greening process, with the changes again dependent on the tissue involved. The spectral dependence of the light-guiding phenomenon in dark grown and green plants may have implications for physiological responses mediated by phytochrome.     

Phytochrome-regulated Starch Degradation in Germinating Turions of Spirodela polyrhiza

Abstract— Turions of the duckweed Spirodela polyrhiza contain about 70% starch on the dry weight basis. The rate of starch degradation in nondormant turions was highest in continuous red light (cR) followed by continuous blue light (cB), whereas continuous far red light (cFR) is almost ineffective. Continuous R could be substituted by repeated R pulses; this effect was not photoreversible by FR pulses applied after hourly R pulses. This suggested that R-dependent high irradiance response is the mode of phytochrome action in mediating starch degradation. Comparing the mode of phytochrome action with that of phytochrome mutants of other plants it can be assumed that phytochrome B is the photoreceptor involved. Application of the translation inhibitor cycloheximide immediately stopped the phytochrome-dependent starch degradation demonstrating the involvement of de novo synthesis of proteins.     

CONTROL BY PHYTOCHROME OF CHLOROPHYLL SYNTHESIS IN SEEDLINGS OF SORGHUM VULGARE

The strong effect of light pretreatments on the synthesis of chlorophyll-a and-b in the shoot of Sorghum vulgare (kept under saturating white light) can be attributed to phytochrome only. No specific blue light effect was found. The phytochrome system appears to function perfectly normally under these conditions. Escape from reversibility is not detectable up to approximately 40 min after the onset of an inductive red light pulse. Thereafter, escape is fast, being completed at approximately 2.5 h after the inductive light pulse.     

IS PHYTOCHROME INVOLVED IN THE LIGHT-MEDIATED CAROTENOGENESIS IN FUSARIUM AQUAEDUCTUUM AND NEUROSPORA CRASSA?*

Abstract— In the two fungal species ( Fusarium and Neurospora ) carotenogenesis has been described as a blue light effect. Generally, phytochrome was believed to be absent in fungi. Recently, work was reported suggesting that this might not strictly be the case. Therefore, we very thoroughly tested these two organisms for any red light effect on carotenogenesis. No action of phytochrome was found, either as a 'low irradiance response' or a 'high irradiance response'. Also, no sequential interaction between blue and red light and between UV and red light was observed. Therefore, we conclude that phytochrome is not involved in carotenogenesis in Fusarium and Neurospora.     

PHOTOBIOLOGIE DU MESOCOTYLE D'AVOINE–IV. SPECTRES D'ACTION ET EFFICACITE QUANTIQUE DE LA LUMIERE

Abstract— The wavelengths most active in influencing the growth of segments of oat mesocotyl are 435 nm (blue), 665 nm (red) and 730 nm (far red). Using a high-energy source (1.5 × 10¹⁸ quanta/cm²), an inhibition in all areas of the spectrum except the far red was obtained. The efficiency of the different radiations was studied: blue light is the most active, red and far red have about the same efficiency; green is a thousand times less active. The effect induced by blue light cannot be directly attributed to phytochrome, because the wavelength of maximum effect does not correspond with that of maximum absorption of the pigment (there is 55 nm difference); also, blue light is more active than the red radiation.     

Antiviral enantiomers of a bisindole alkaloid with a new carbon skeleton from the roots of Isatis indigotica

A pair of indole alkaloid enantiomers with a novel bisindolylacetamide skeleton, insatindibisindolamides A and B (1a and 1b), was isolated from an aqueous extract of Isatis indigotica roots. The enantiomers were separated by chiral HPLC. Their structures and absolute configurations were elucidated by extensive spectroscopic analysis, including 2D NMR, X-ray crystallography, and electronic CD (ECD) calculation. The proposed biosynthetic pathway and preliminary investigations of the biological activity of compounds 1a and 1b are also discussed.     

PHOTOINHIBITION OF GROWTH OF- A YELLOW AND A COLORLESS FORM OF CHLAMYDOMONAS REINHARDTII*

Abstract— Long-term growth on acetate in darkness of a permanently yellow and a white-in-darkness mutant of Chlamydomonus reinhardtii showed no requirement for light. Room light or moderate intensities of white light severely inhibited the white mutant, but allowed continuous growth of the yellow form. Blue light (460 nm) stops motility and growth, and induces clumping, in the white form at energy levels which only reduce the growth rate of the yellow mutant. Green light (550 nm) had no effect on either strain, while red light (630 nm), at equal energy levels, affected the white mutant only, however, not as severely as blue light. The mechanism of action of blue and red light is unknown. Evidence suggested the inhibition is not due to cell exudates.     

IDENTITY OF THE PHOTORECEPTORS FOR THE PERCEPTION OF IRRADIANCE IN PHOTOSYNTHETIC LIGHT ACCLIMATION IN TOMATO*

The photoreceptors involved in the photosynthetic acclimation of tomato (Lycopersicon esculentum Mill.) to increased irradiance were investigated. Plants were transferred from 100 p.mol m^?2 s^?1 cool white fluorescent light to higher irradiances of white light or white light supplemented with blue, red, green or yellow light. In these experiements light of all wavelengths tested was capable of causing acclimation as measured by the rate of light-saturated photosynthesis. It was concluded that the photosynthetic system rather than the blue-absorbing photoreceptor or phytochrome system acts as the photoreceptor for increased irradiance. No acclimation was observed in response to increased CO₂ levels, but increasing light integral at a constant irradiance was effective in bringing about acclimation. We conclude that acclimation is a response to increased photosynthetic light capture rather than increased photosynthetic carbon fixation, and involves a photon counting mechanism.     

THE INFLUENCE OF LIGHT UPON SEED GERMINATION. POSSIBLE INTERPRETATIONS OF DATA

Abstract— The response of seeds to irradiation by white light varies with the species: induction of germination, inhibition of germination or both according to the time-irradiation. The low energy reaction (LER) which acts via phytochrome can explain many cases of influence of light upon germination. It seems possible that a second reaction, the high energy reaction (HER), acts through another photoreceptor.     

Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.     

Electrochemical monitoring of indigo preparation using Maya’s ancient procedures

The preparation of indigo from Indigofera suffruticosa following the procedures attributed to ancient Mayas was electrochemically monitored using the voltammetry-of-microparticles approach. The mechanism formation of indigotin and indirubin from its precursors, indican and isatan, is discussed. Comparison of voltammetric profiles for differently prepared and commercial indigos and genuine Maya Blue samples suggests that the preparation procedure of indigo changed during the Late Classical Maya period. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microwave treatment of eight seconds protects cells of Isatis indigotica from enhanced UV-B radiation lesions

To determine the role of microwaves in the stress resistance of plants to enhanced ultraviolet-B (UV-B) radiation, Isatis indigotica Fort. seeds were subjected to microwave radiation for 8 s (wavelength 125 mm, power density 1.26 mW mm(-2), 2450 MHz). Afterwards they were cultivated in plastic pots in an artificial-glass greenhouse maintained at 25 degrees C, 70% relative humidity, and 400 micromol mol(-1) CO2, under visible-light conditions of 1500 micromol m(-2) s(-1) for 8 h day(-1). When the seedlings were 10 days old, they were subjected to 10.08 kJ m(-2) UV-B (PAR: 220 micromol m(-2) s(-1)) radiation for 8 days. Changes in a number of physiological and biochemical characteristics and in the thermal decomposition enthalpy of biomass were measured and used as indicators of the protective capacity of microwave radiation in this experiment. Our results revealed that microwave pretreatment of seeds enhanced UV-B stress resistance in the seedlings by decreasing the concentration of malondialdehyde (MDA) and increasing the concentration of ascorbic acid (AsA) and UV-B-absorbing compounds, increasing the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), and increasing the energy accumulation of photosynthesis. All these results suggest that microwave radiation enhances plant metabolism and results in increased UV-B stress resistance. This is the first investigation reporting the use of microwave pretreatment to protect the cells of Isatis indigotica from UV-B-induced lesions.     

Raman microscopy of Greek icons: identification of unusual pigments

Five Greek icons, made between the 15th and 18th centuries and now belonging to the Victoria and Albert Museum collections, were analysed by energy-dispersive X-ray fluorescence (EDXRF), optical microscopy and Raman microscopy in order to determine the stratigraphy of the artworks and the identity of the pigments used. Together with common pigments, such as red lake, vermilion, red lead, red iron oxide, orpiment, yellow ochre, lead white, chalk, gypsum, anhydrite, Prussian blue, indigo and a copper-containing green, a few unusual materials were identified, specifically pararealgar (a yellow arsenic sulphide, As4S4), its precursor the chi-phase, and lead tin yellow type II (PbSn(1-x)SixO3). Attention is drawn to the complementarity of the techniques used for the pigment identifications.