Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry monitoring of anthocyanins in extracts from Arabidopsis thaliana leaves

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.     

Anthocyanin Content and UVB Sensitivity in Brassica rap

Three genotypes of rapid-cycling Brassica rapa that differ in anthocyanin content were grown in the presence and absence of elevated levels of shortwave ultraviolet (UVB, 280–325 nm) radiation. After 41 days, UVB exposure reduced leaf length and plant height of all genotypes. Plants with low levels of anthocyanin experienced a reduction in flower number twice as great as in genotypes with normal or elevated levels of anthocyanins; however, the absence of differences in flower production by genotypes with normal and elevated levels of anthocyanins suggests that factors other than anthocyanin pigmentation contribute to UVB responses in this species.     

Anthocyanin Pigments: Beyond Aesthetics

Anthocyanins are polyphenol compounds that render various hues of pink, red, purple, and blue in flowers, vegetables, and fruits. Anthocyanins also play significant roles in plant propagation, ecophysiology, and plant defense mechanisms. Structurally, anthocyanins are anthocyanidins modified by sugars and acyl acids. Anthocyanin colors are susceptible to pH, light, temperatures, and metal ions. The stability of anthocyanins is controlled by various factors, including inter and intramolecular complexations. Chromatographic and spectrometric methods have been extensively used for the extraction, isolation, and identification of anthocyanins. Anthocyanins play a major role in the pharmaceutical; nutraceutical; and food coloring, flavoring, and preserving industries. Research in these areas has not satisfied the urge for natural and sustainable colors and supplemental products. The lability of anthocyanins under various formulated conditions is the primary reason for this delay. New gene editing technologies to modify anthocyanin structures in vivo and the structural modification of anthocyanin via semi-synthetic methods offer new opportunities in this area. This review focusses on the biogenetics of anthocyanins; their colors, structural modifications, and stability; their various applications in human health and welfare; and advances in the field.     

UVB Radiation Induced Increase in Quercetin: Kaempferol Ratio in Wild-Type and Transgenic Lines of Petunia

The use of genetically modified plants offers unique opportunities to study the role of specific flavonoids in plant UVB protection. Along with a parental wild-type Mitchell Petunia, two transgenic lines with altered flavonoids were also examined; Lc with enhanced levels of antho-cyanins due to the action of a maize flavonoid regulatory gene Leaf color, and AFLS that carries an antisense fla-vonol synthase construct and is known to have reduced flavonol levels in flowers. All three lines were grown in near ambient sunlight, sunlight lacking UVB (280–320 nm) radiation and sunlight with 25% added UVB. Ultra-violet-B radiation induced significant reductions in the rates of leaf expansion and seedling growth in all three lines. The presence of anthocyanins did not appear to afford Lc plants any special protection from UVB. Ul-traviolet-B treatment induced increases in total flavonol content in young plants of all three lines, and this effect decreased with increasing leaf age. Notably, increasing UVB levels led to an increase in the ratio of quercetin: kaempferol with all three cultivars. The AFLS transgenic, contrary to expectations based on its genetic construction, had normal levels of flavonols in the leaves and the highest Q:K ratio of the three cultivars. This transgenic was the least susceptible to UVB, which may indicate an enhanced protective role for quercetin. Because both quercetin and kaempferol have similar UVB screening properties, quercetin may exert this role by other means.     

Methods of analysis for anthocyanins in plants and biological fluids

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. They are responsible for most of the red, blue, and purple colors of fruits, vegetables, flowers, and other plant tissues or products. The analysis of anthocyanins is complex as a result of their ability to undergo structural transformations and complexation reactions. In addition, they are difficult to measure independently of other flavonoids, as they have similar structural and reactivity characteristics. Anthocyanins are generally extracted with weakly acidified alcohol-based solvents, followed by concentration (under vacuum), and purification of the pigments. Paper and/or thin-layer chromatography and UV-Vis spectroscopy have traditionally been used for the identification of anthocyanins. Capillary zone electrophoresis, a hybrid of chromatography and electrophoresis, is gaining popularity for the analysis of anthocyanins; however, liquid chromatography (LC) has become the standard method for identification and separation in most laboratories and may be used for both preparative and quantitative analysis. LC with mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are possibly the most powerful methods for the structural elucidation of anthocyanins available, to date. At present, the most satisfactory method for mixture analysis is the multistep method of separation, isolation, and quantification by LC with peak identification by MS and high-field NMR.     

Effects of Light Environment During Culture on UV-lnduced Cyclobutyl Pyrimidine Dimers and Their Photorepair in Rice (Oryza sativa L.)

Abstract— We examined the effects of a light environment during culture of rice plants ( Oryza sativa ) on the steady-state cyclobutyl pyrimidine dimer (CPD) level, CPD induction by challenge UVB exposure and the ability to photorepair CPD. The steady-state CPD level in plants grown under visible radiation with supplemental UVB radiation in a growth chamber was several times higher than in plants grown without supplemental UVB radiation, whereas in outdoor-grown plants, it was not enhanced by supplemental UVB radiation. The susceptibility to CPD induction by challenge UVB exposure was highest in dark-grown plants and decreased with increasing irradiance of visible radiation at low and high levels and outdoors. Chronic UVB radiation reduced the susceptibility to UV-induced CPD in plants grown both indoors and outdoors. There was a significant negative correlation between CPD levels induced by challenge UVB exposure and the content of UV-absorbing compounds. The UV-induced CPD could be reduced by subsequent blue radiation in all samples except in dark-grown seedlings. The higher the irradiance of visible radiation in the culture, the greater the ability to photorepair CPD. Chronic UVB radiation did not increase the ability to photorepair CPD.     

Photoprotection in tadpoles of the common frog, Rana temporaria

The common frog, Rana temporaria, spawning at altitudes up to 2600 m, is potentially threatened by UVB radiation. In laboratory experiments we found a specifically UVB absorbing substance (UVAS) in the skin of tadpoles with maximum absorption between 290 and 300 nm. The production of UVAS is induced by both visible light and ultraviolet radiation. The concentrations of UVAS found in tadpoles from the field are about as high as those found in tadpoles from laboratory experiments with UVB radiation under simulated mid-summer conditions. The existence of two powerful sun screen factors (UVAS and melanin pigmentation) may explain the high resistance of R. temporaria tadpoles to the intensive UVB radiation at high altitudes.     

Non-destructive analysis of pigments and other organic compounds in lichens using Fourier-transform Raman spectroscopy: a study of Antarctic epilithic lichens

Lichens in Antarctic habitats are subjected to environmental extremes, including UVB radiation, desiccation and low temperatures, as well as to rapid fluctuations in these. Lichens synthesise a variety of chemical compounds in response to their environmental conditions which contribute towards their colour, and which act as protectants against physiological stresses. The fluorescence generated by the lichens at 532 nm can be used in epifluorescence microscopy to identify their presence on substrata but this can severely affect the Raman spectra using visible excitation. The advantage of the near infrared excitation used in FT-Raman spectroscopy in minimising fluorescence emission facilitates the molecular characterisation of lichen encrustations without having to remove the thallus from its substrate or remove or otherwise damage any part of the thallus. Spectroscopic biomarkers are proposed which allow the lichens to be characterised by the identification of characteristic lichen substances; the use of these biomarkers for the preliminary taxonomic identification of Antarctic lichens is examined and some potential pitfalls are described.     

Anthocyanins: Promising Natural Products with Diverse Pharmacological Activities

Anthocyanins are natural products that give color to plants. As natural plant pigments, anthocyanins also have a series of health-promoting benefits. Many researchers have proved that anthocyanins have therapeutic effects on diseases, such as circulatory, nervous, endocrine, digestive, sensory, urinary and immune systems. Additionally, a large number of studies have reported that anthocyanins have an anticancer effect through a wide range of anti-inflammatory and antioxidant effects. The anti-disease impact and mechanism of anthocyanins are diverse, so they have high research value. This review summarizes the research progress of anthocyanins on the pharmacological agents of different diseases to provide references for subsequent research.     

Rat lens glycolysis after in vivo exposure to narrow band UV or blue light radiation

UV radiation and short wavelength visible light are known to damage various tissues in the eye. This paper investigates the effect on rat lens glycolysis after in vivo exposure with 90 kJ m⁻² narrow band UV radiation (UVB, 300 nm) and 90 kJ m⁻² blue light (435 nm) radiation. After exposure, all lenses were incubated in Medium 199. Samples of culture medium were withdrawn after 2, 4, 6 h and 5, 10, 20 h in two UVB studies and after 5, 10 and 20 h in a blue light study. Lactate is the major end product of lens glycolysis. Lactate was determined with a modified enzymatic-photometric method. Intralenticular lactate was determined in one UVB experiment. In the UVB experiments we found a lower lactate production in the exposed lenses 2–6 h after exposure. There was an accumulation of lactate inside UVB-exposed lenses after 6 h incubation compared with their contralateral lenses. No significant effect on lactate production was observed in the blue light experiment. Conclusions. UVB induced a reversible inhibition of glycolysis. UVB also induced an accumulation of lactate inside the lens. Blue light tended to increase glycolysis.     

Ultraviolet B irradiation modulates the immune system of fish (Rutilus rutilus, Cyprinidae). II: Blood

The effects of a single dose of ultraviolet B (UVB) radiation (0.4 J/cm2) on immunological functions by blood leukocytes and on hematological parameters was studied in roach (Rutilus rutilus), a teleostean fish. The respiratory burst of phorbol 12-myristate 13-acetate stimulated whole blood phagocytes increased significantly after UVB irradiation but spontaneous cytotoxicity of blood leukocytes toward 51chromium-labeled K562 target cells was not markedly altered. Differential cell counting revealed that UVB exposure significantly increased the proportion of granulocytes and significantly decreased the proportion of lymphocytes in the peripheral blood, whereas hematocrit and the total number of white and red blood cells were unchanged. Plasma cortisol concentration increased in UVB-exposed fish. Severe handling stress caused similar, although not as potent, effects on the measured parameters of fish blood as UVB irradiation. These observations suggest that in fish UVB brings about a stress response, which may account for the observed alterations in the immune parameters and leukocyte composition of blood. Exposure of fish to strong visible light induced no alterations in immunological or hematological parameters, making it unlikely that ultraviolet radiation mediates its effects through visual perception.     

The cascade mechanisms of nitric oxide as a second messenger of ultraviolet B in inhibiting mesocotyl elongations

In this report, a number of physiological aspects was examined during developmental growth of maize seedling's mesocotyl. It was found that ultraviolet B (UVB) radiation was able to significantly induce nitric oxide synthase (NOS) activities and speedup the release of apparent nitric oxide (NO) of mesocotyl and that exogenous NO donor's rhizospheric treatments may mimic the responses of the mesocotyl to UVB radiation, such as the inhibition of mesocotyl elongation, the decrease in exo- and endoglucanase activities and the increase in protein content of cell wall of mesocotyl. When the seedlings were treated with N-nitro-L-arginine, an inhibitor of NOS, the mesocotyl elongation was promoted, the exo- and endoglucanase activities were raised and the protein content was reduced. However, under UVB radiation, the effects of exogenous NO on several physiological aspects of mesocotyl were similar to those of exogenous reactive oxygen species (ROS) eliminator, N-acetyl-cysteine. All the physiological changes were associated with either the exogenous NO supply or the activities of NOS in plant. Accordingly, it is assumed that reduction in mesocotyl length caused by UVB radiation was possibly achieved through modification of the chemical properties of the cell wall polysaccharides, which was induced by NO and ROS synergically mediated changes in exo- and endo-beta-D-glucanases activities in cell walls, and NO was one of the main signaling molecule of UVB radiation in inhibiting mesocotyl elongations. So NO might function as both a second messenger and an antioxidant of UVB radiation during developmental growth of the mesocotyl.     

Light‐mediated DNA Repair Prevents UVB‐induced Cell Cycle Arrest in Embryos of the Crustacean Macrobrachium olfersi

High levels of ultraviolet‐B (UVB) radiation can negatively affect aquatic animals. Macrobrachium olfersi is a prawn that lives in clear freshwaters and during the breeding season, females carry eggs in an external brood pouch. Therefore, we hypothesize that eggs are also exposed to environmental UVB radiation. The aim of this study was to investigate whether UVB radiation induces DNA damage and compromises cell cycle in embryos of M. olfersi. In laboratory, UVB irradiance (310 mW. cm^?2) that embryos receive in the natural environment was simulated. After irradiation, embryos were kept under different light conditions in order to recognize the presence of cell repair. UVB radiation induces DNA damage, specifically thymine dimers. After 48 h of UVB exposure, a significant decrease in the level of these dimers was observed in embryos kept under visible light while it remained constant in the dark. Moreover, under visible light and darkness, a decrease in proliferation was observed after 48 h of irradiation. An increase in PCNA expression and decrease in p53 expression were observed after, respectively, 1 and 48 h of exposure. Our results showed that UVB radiation disturbs the cell cycle and induces DNA damage in M. olfersi embryos. However, under visible light these embryos showed successful DNA repair.     

Protective Effect of Tropical Highland Blackberry Juice (Rubus adenotrichos Schltdl.) Against UVB‐Mediated Damage in Human Epidermal Keratinocytes and in a Reconstituted Skin Equivalent Model

Solar ultraviolet (UV) radiation, particularly its UVB (280–320 nm) spectrum, is the primary environmental stimulus leading to skin carcinogenesis. Several botanical species with antioxidant properties have shown photochemopreventive effects against UVB damage. Costa Rica's tropical highland blackberry (Rubus adenotrichos) contains important levels of phenolic compounds, mainly ellagitannins and anthocyanins, with strong antioxidant properties. In this study, we examined the photochemopreventive effect of R. adenotrichos blackberry juice (BBJ) on UVB‐mediated responses in human epidermal keratinocytes and in a three‐dimensional (3D) reconstituted normal human skin equivalent (SE). Pretreatment (2 h) and posttreatment (24 h) of normal human epidermal keratinocytes (NHEKs) with BBJ reduced UVB (25 mJ cm^?2)‐mediated (1) cyclobutane pyrimidine dimers (CPDs) and (2) 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) formation. Furthermore, treatment of NHEKs with BBJ increased UVB‐mediated (1) poly(ADP‐ribose) polymerase cleavage and (2) activation of caspases 3, 8 and 9. Thus, BBJ seems to alleviate UVB‐induced effects by reducing DNA damage and increasing apoptosis of damaged cells. To establish the in vivo significance of these findings to human skin, immunohistochemistry studies were performed in a 3D SE model, where BBJ was also found to decrease CPDs formation. These data suggest that BBJ may be developed as an agent to ameliorate UV‐induced skin damage.     

Anthocyanins as antimicrobial agents of natural plant origin

Anthocyanins are particularly abundant in different fruits, especially in berries. The beneficial effects of these compounds for human health have been known from at least the 16th century. Despite the great number of papers devoted to the different biological effects exerted by anthocyanins only a limited number of studies is focused on the antimicrobial activity of these compounds. Anthocyanin content of berry fruits varies from 7.5 mg/100 mg fresh fruit in redcurrant (Ribes rubum) up to 460 mg/100 g fresh fruit in chokeberry (Aronia melanocarpa). After consumption, anthocyanins are intensively metabolized, mainly in the intestines and liver. Glucorination, methylation and sulfation are the most typical metabolic reactions. Antimicrobial activity of crude extracts of plant phenolic compounds against human pathogens has been intensively studied to characterize and develop new healthy food ingredients as well as medical and pharmaceutical products. However, there is very little information available about the antimicrobial activity of the pure anthocyanins. In the last part of this review we present the collection of papers describing the anthocyanin profiles of different fruits (mainly berries) and the antimicrobial properties of the identified compounds. Generally, anthocyanins are active against different microbes, however Gram-positive bacteria usually are more susceptible to the anthocyanin action than Gram-negative ones. Mechanisms underlying anthocyanin activity include both membrane and intracellular interactions of these compounds. Antimicrobial activity of berries and other anthocyanin-containing fruits is likely to be caused by multiple mechanisms and synergies because they contain various compounds including anthocyanins, weak organic acids, phenolic acids, and their mixtures of different chemical forms. Therefore, the antimicrobial effect of chemically complex compounds has to be critically analyzed.     

Requirement for Metalloproteinase‐dependent ERK and AKT Activation in UVB‐induced G1‐S Cell Cycle Progression of Human Keratinocytes

UVB (280–315 nm) in natural sunlight represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here we demonstrate that low doses of UVB induce keratinocyte proliferation and cell cycle progression of human HaCaT keratinocytes. Different from UVA, UVB irradiation induced extracellular signal‐regulated kinase (ERK) and AKT activation and their activation are both required for UVB‐induced cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVB exposure and is upstream of ERK/AKT/cyclin D1 pathway activation and cell cycle progression following UVB radiation. Furthermore, metalloproteinase (MP) inhibitor GM6001 blocked UVB‐induced ERK and AKT activation, cell cycle progression, and decreased the EGFR phosphorylation, demonstrating that MPs mediate the EGFR/ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB radiation. In addition, ERK or AKT activation is essential for EGFR activation because ERK or AKT inhibitor blocks EGFR activation following UVB radiation, indicating that EGFR/AKT/ERK pathways form a regulatory loop and converge into cell cycle progression following UVB radiation. Identification of these signaling pathways in UVB‐induced cell cycle progression of quiescent keratinocytes as a process mimicking tumor promotion in vivo will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer.     

UVB/UVA radiation activates a 48 kDa myelin basic protein kinase and potentiates wound signaling in tomato leaves

We investigated the effect of UV radiation on early signaling events in the response of young tomato plants (Lycopersicon esculentum) to wounding. Ultraviolet-C (< 280 nm) and UVB/UVA (280-390 nm) radiation both induced 48 kDa myelin basic protein kinase activity in leaves. The activation was associated with phosphorylation of tyrosine residues on the kinase, which is indicative of protein kinases of the mitogen-activated protein kinase family. Ultraviolet-C irradiation resulted in a strong proteinase inhibitor synthesis, as reported previously (Conconi et al., Nature 383, 826-829, 1996). Under the conditions used, UVB/UVA radiation did not induce proteinase inhibitor synthesis but resulted in a strong potentiation of systemic proteinase inhibitor synthesis in response to wounding. The UVB/UVA-irradiated plants that were subsequently wounded accumulated 2.5-4-fold higher levels of proteinase inhibitor I when compared to wounded non-irradiated plants. The potentiating effect was most prominent in the systemic unwounded leaf of a wounded plant. Levels of 12-oxo-phytodienoic acid and jasmonic acid that have been well documented to increase in response to wounding were not detected in response to UVB/UVA irradiation alone. The effect of UVB/UVA radiation in potentiating plant defense signaling should be further considered as a factor that may influence the ecological balance between plants and their predators.     

UVB Radiation Affects Growth,Reproduction and Tissue Structure of Daphnia magna Across Several Temperatures

We examined the effects of daily exposure to UVB on growth, reproduction and histological characteristics of Daphnia magna over two generations at 20, 22, 25 and 30°C. Animals were exposed to 16 h of UVA and photosynthetically active radiation daily. Treated animals received 6 h of UVB during the light phase. Parental (P) generation growth and reproduction was impaired by exposure to UVB at all temperatures, with the poorest production at 30°C. First brood size decreased with UVB exposure; it was lowest at 30°C. Although F1 length at birth increased with P generation age, F1 produced by UVB‐exposed mothers were smaller at all temperatures. The F1 generation was followed at 20 and 25°C; at both temperatures UVB exposure reduced F1 growth and reproduction. F1 growth and F2 production were lowest when both P and F1 generations were exposed to UVB. UVB exposure damaged ovarian and gut tissue at both 25 and 30°C; the consequences of this exposure were more severe at 30°C. The observed tissue damage may relate directly to the UVB‐induced impairment of growth and reproduction. This study provides new insights into the effects of UVB on an important component of the pelagic zooplankton.     

Effects of Pulsed Electric Field on Secondary Metabolism of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. Gamay Fréaux Suspension Culture and Exudates

Plant cell cultures provide a large potential for the production of secondary metabolites. Through the application of different physical and chemical cell stress factors, we investigated the production of the secondary metabolites in plant cell cultures. The effects of pulsed electric field (PEF) and ethephon on growth and secondary metabolism, particularly anthocyanins and phenolic acids synthesis, were investigated by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. Anthocyanins were measured by spectrophotometer and extracellular phenolic acids were determined by high-performance liquid chromatography. The compounds were identified by liquid chromatography–mass spectrometry and nuclear magnetic resonance. After the treatments with PEF and ethephon, the concentrations of anthocyanins and phenolic acids in cell culture were higher than in the control, without loss of biomass. The combination of PEF treatment and ethephon improved secondary metabolites formation. Production levels of extracellular phenolic acids, 3-O-glucosyl-resveratrol were increased by PEF and ethephon treatments. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane’s dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolites biosynthesis in plant cell cultures.     

Optimization of a Sustainable Protocol for the Extraction of Anthocyanins as Textile Dyes from Plant Materials

Anthocyanins are the largest group of polyphenolic pigments in the plant kingdom. These non-toxic, water-soluble compounds are responsible for the pink, red, purple, violet, and blue colors of fruits, vegetables, and flowers. Anthocyanins are widely used in the production of food, cosmetic and textile products, in the latter case to replace synthetic dyes with natural and sustainable alternatives. Here, we describe an environmentally benign method for the extraction of anthocyanins from red chicory and their characterization by HPLC-DAD and UPLC-MS. The protocol does not require hazardous solvents or chemicals and relies on a simple and scalable procedure that can be applied to red chicory waste streams for anthocyanin extraction. The extracted anthocyanins were characterized for stability over time and for their textile dyeing properties, achieving good values for washing fastness and, as expected, a pink-to-green color change that is reversible and can therefore be exploited in the fashion industry.