Anthraquinones from the Fungus Dermocybe sanguinea as Textile Dyes

This study is based on the multidiciplinary approach of using natural colorants as textile dyes. The author was interested in both the historical and traditional aspects of natural dyeing as well as the modern industrial applications of the pure natural compounds. In the study, the anthraquinone compounds were isolated as aglycones from the ectomycorrhizal fungus Dermocybe sanguinea. The endogenous beta-glucosidase of the fungus was used to catalyse the hydrolysis of the O-glycosyl linkage in emodin- and dermocybin-1-beta-D-glucopyranosides. The method, in which 10.45 kg of fresh fungi was starting material, yielded two fractions: 56.0 g of Fraction 1 (94% of the total amount of pigment,) consisting almost exclusively of the main pigments emodin and dermocybin, and 3.3 g of Fraction 2 (6%) consisting mainly of the anthraquinone carboxylic acids. The anthraquinone compounds in Fractions 1 and 2 were separated by one- and two-dimensional thin-layer-chromatography (TLC) using silica plates. 1D TLC showed that neither an acidic nor a basic solvent system alone separated completely all the anthraquinones isolated from D. sanguinea, in spite of the variation of the rations of the solvent components in the systems. Thus, a new 2D TLC technique was developed, applying n-pentanol-pyridine-methanol (6:4:3, v/v/v) and toluene-ethyl acetate-ethanol-formic acid (10:8:1:2, v/v/v/v) as eluents. Fifteen different anthraquinone derivatives were completely separated from one another. Emodin, physcion, endocrocin, dermolutein, dermorubin, 5-chlorodermorubin, emodin-1-beta-D-glucopyranoside, dermocybin-1-beta-D-glucopyranoside and dermocybin, and five new compounds, not earlier identified in D. sanguinea, 7-chloroemodin, 5,7-dichloroemodin, 5,7-dichloroendocrocin, 4-hydroxyaustrocorticone and austrocorticone, were separated and identified on the basis of their Rf-values, UV/Vis spectra and mass spectra. One substance remained unidentified, because of its very low concentration. The anthraquinones in Fractions 1 and 2 were preparatively separeted by liquid-liquid partition, with isopropylmethyl ketone and aqueous phosphate buffer as the solvent system. Advantage was taken of the principle of stepwise pH-gradient elution. The multiple liquid-liquid partition (MLLP) offered an excellent method for the preparative separation of compounds, which contain acidic groups such as the phenolic OH and COOH groups. Due to their strong aggregation properties, these compounds are, without derivatization, very difficult to separate on a preparative scale by chromatographic methods. By the MLLP method remarkable separations were achieved for the components in each mixture. Emodin and dermocybin were both obtained from Fraction 1 in a purity of at least 99%. Pure emodin and dermocybin were applied as mordant dyes to wool and polyamide and as disperse dyes to polyester and polyamide, using the high temperature (HT) technique. A mixture of dermorubin and 5-chlorodermorubin was applied as an acid dye to wool. In these experiments, synthetic dyes were used as references. Experiments were also performed using water extract of the air-dried fungi as dye liquor for wool and silk. The main colouring compounds in the crude water extract were emodin and dermocybin, which indicated that the O-glycosyl linkages in emodin- and dermocybin-1-beta-D-glucopyranosides were broken by the beta-glucosidase enzyme. Apparently, the hydrolysis occurred during the drying of the fungi and during the soaking of the dried fruit bodies overnight when preparing the dyebath. The colour of each dyed material was investigated in terms of the CIELAB L*, a* and b* values, and the colour fastness to light, washing and rubbing was tested according to the ISO standards. In the mordant dyeing experiments, emodin dyed wool and polyamide yellow and red, depending on the pH of the dyebath. Dermocybin gave purple and violet colours. The colour fastness of the mordant-dyed fabrics varied from good to moderate. The fastness properties of the natural anthraquinone carboxylic acids on wool were good, indicating the strength of the ionic bonds between the COO- groups of the dyes and the NH3+ groups of the fibres. In the disperse dyeing experiments, emodin dyed polyester bright yellow and dermocybin bright reddish-orange, and the fabrics showed excellent colour fastness. In contrast, emodin and dermocybin successfully dyed polyamide brownish-orange and wine-red, respectively, but with only moderate fastness. In industrial dyeing processes, natural anthraquinone aglycone mixtures dyed wool and silk well even at low concentrations of mordants, i.e. with 10% of the weight of the fibre (owf) of KAl(SO4)2 and 1 or 0.5% owf of other mordants. This study showed that purified natural anthraquinone compounds can produce bright hues with good colour-fastness properties in different textile materials. Natural anthraquinones have a significant potential for new dyeing techniques and will provide useful alternatives to synthetic dyes.

Spectral Tuning and Adaptation to Different Light Environments of Mysid Visual Pigments

In the present thesis, questions of spectral tuning, the relation of spectral and thermal properties of visual pigments, and evolutionary adaptation to different light environments were addressed using a group of small crustaceans of the genus Mysis as a model. The study was based on microspectrophotometric measurements of visual pigment absorbance spectra, electrophysiological measurements of spectral sensitivities of dark-adapted eyes, and sequencing of the opsin gene retrieved through PCR. The spectral properties were related to the spectral transmission of the respective light environments, as well as to the phylogentic histories of the species. The photoactivation energy (Ea) was estimated from temperature effects on spectral sensitivity in the long-wavelength range, and calculations were made for optimal quantum catch and optimal signal-to-noise ratio in the different light environments. The opsin amino acid sequences of spectrally characterized individuals were compared to find candidate residues for spectral tuning. The general purpose was to clarify to what extent and on what time scale adaptive evolution has driven the functional properties of (mysid) visual pigments towards optimal performance in different light environments. An ultimate goal was to find the molecular mechanisms underlying the spectral tuning and to understand the balance between evolutionary adaptation and molecular constraints. The totally consistent segregation of absorption maxima (λmax) into (shorter-wavelength) marine and (longer-wavelength) freshwater populations suggests that truly adaptive evolution is involved in tuning the visual pigment for optimal performance, driven by selection for high absolute visual sensitivity. On the other hand, the similarity in λmax and opsin sequence between several populations of freshwater M. relicta in spectrally different lakes highlights the limits to adaptation set by evolutionary history and time. A strong inverse correlation between Ea and λmax was found among all visual pigments studied in these respects, including those of M. relicta and 10 species of vertebrate pigments, and this was used to infer thermal noise. The conceptual signal-to-noise ratios thus calculated for pigments with different λmax in the Baltic Sea and Lake Pääjärvi light environments supported the notion that spectral adaptation works towards maximizing the signal-to-noise ratio rather than quantum catch as such. Judged by the shape of absorbance spectra, the visual pigments of all populations of M. relicta and M. salemaai used exclusively the A2 chromophore (3, 4-dehydroretinal). A comparison of amino acid substitutions between M. relicta and M. salemaai indicated that mysid shrimps have a small number of readily available tuning sites to shift between a shorter - and a longer -wavelength opsin. However, phylogenetic history seems to have prevented marine M. relicta from converting back to the (presumably) ancestral opsin form, and thus the more recent reinvention of marine spectral sensitivity has been accomplished by some other novel mechanism, yet to be found