Akiyoshi Fukushima

On the mechanism of the stable red colour expression of cellulose-bound carthamin

Abstract Four Carthamus pigments were mixed separately with cellulose powder or cellulose ion-exchangers in a neutral solution and their bonding capacities were compared. Only carthamin was adsorbed by cellulose powder at a specifically marked high level. Test pigments tended to bind with anion exchange celluloses rather than cationic ones, though some discrepancies were found in the tendency. Carthamin showed less selective affinity for ion-exchange cellulose. It was efficiently taken up by ECTEOLA-cellulose (3·02), PEI-cellulose (2·34), CM-cellulose (2·43), PAB-cellulose (2·82) and SE-cellulose (1·67%) per milligram of them. Precarthamin, safflor yellow A and safflor yellow B were preferentially bound to ECTEOLA-, PEI- and PAB-cellulose. The above-listed pigments showed little or no affinity for CM-Sephadex C-25. A conspicuous augmentation in the adsorption rate was also emphasized, particularly in carthamin, by increasing the amount of cellulose powder and ion-exchange celluloses. Carthamin was separable from the reddish cellulose-carthamin complex with aqueous methanol, ethanol, ethylacetate, acetone, N-vinylpyrrolidone and urea, while the pigment bound to ECTEOLA-cellulose was not. The ion-exchange cellulose-trapped carthamin could only be recovered by using diluted formic acid, acetic acid or ammonia. The experimental findings are discussed in relation to the Saito Effect proposed for the stable colour expression of cellulose-bound carthamin. The possibility of using bound carthamin as a red food colour is also disputed.

Decomposition of carthamin in aqueous solutions: influence of temperature, pH, light, buffer systems, external gas phases, metal ions, and certain chemicals

ZusammenfassungÄußere Faktoren stehen im Verdacht, die chemische Natur des Carthamin zu beeinflussen; es wurde daher der Versuch unter Modellbedingungen im wäßrigen Milieu unternommen. Das Pigment wurde leicht bei höheren Temperaturen in orangegelbe und gelbe Verbindungen zersetzt. Die Stabilität von Carthamin war pH-abhängig, wobei die rote Färbung die stabilere ist (pH 1,5–5,5). Gegenüber dem sichtbaren und dem UV-Licht nahm die Farbstärke innerhalb 1–40 min rund um die Hälfte ab. Der Verlust der roten Farbe von Carthamin ist augenscheinlich bedingt durch das Puffersystem, die äußere Atmosphäre und einige Chemikalien. Metallionen in Konzentration von 0,03–3,0 mmol erniedrigen die Farbstärke, wobei Eisenionen den größten Effekt haben, nämlich daß die Abbaurate das 47fache der Kontrolle beträgt. Die Reaktionen wurden spektrometrisch überwacht, um die Carthaminzusammensetzung zu kontrollieren.SummaryExternal factors suspecting to influence the chemical nature of carthamin in aqueous media were assessed under experimentally designed model systems. The pigment was more readily decomposed to orange-yellow or yellow compounds at higher temperature. The stability of carthamin was evidently pH-dependent, with increased degradation occurring above and below the pH range in which the red pigmentation is most stable (pH 1.5–5.5). On exposure to visible and ultraviolet light, the red colour in aqueous solutions decreased with half-lives of approximately 1 and 40 min, respectively. Loss of carthamin red coloration also occurred without perceptible dependence on the buffer systems, external gas phases or certain chemicals. Metal ions at a level of 0.03–3.0 mmol concentration caused an increase in the degradation rate compared to that of the control with no metal cations. Fe2+ had the greatest effect, reducing the rate of degradation approximately 47-fold compared to the control. The reaction systems were surveyed spectrophotometrically to check carthamin composition patterns.

An improved method for large-scale isolation of a water-soluble safflor pigment from dyer's saffron flowers

Abstract An improved method for large-scale isolation of a water-soluble safflor pigment was assessed. The pigment was separated from the alcoholic extract of the bright-yellow florets of dyers saffron by fixing it with BD-cellulose in an acidic solution and purified through successive column chromatographies. Thus, a chromatographically pure sample obtained was analyzed for confirming the identity with an authentic safflor yellow B, then it was mixed separately with BN-, BND-, QAE- and ECTEOLA-cellulose in an acidic buffer solution, and the trapping capacity by the anion-exchange celluloses was compared. The arrested pigment was released by diluted methanol, ethanol and acetone, and its solubility rate could be elevated further by mixing formic acid or acetic acid into the liberation media. Data were evaluated in reference to practical utilization of the synonymous colouring matters as colour additives for processed foods or soft drinks.

Effect of exogenous substances on carthamin red coloration

Abstract Effect of exogenous substances on carthamin colour modification was examined in solutions containing the pigment and alcohols, ethers, ketones, esters, carboxylic acids, fatty acids, amino acids, amides or amines. Alcohols, amino acids, amines, carboxylic acids and ethers effectively promoted reddening of the aqueous acetone comprising carthamin. Ketones and amides had little or no effect on the bathochromic colour shift. Esters and fatty acids, except for formic and acetic acids, exerted hypsochromic effects on the carthamin red. Based on the experimental findings, a possible mechanism of the carthamin colour modification was briefly debated.

A New Affinity-trapping Method for Isolation of Quinoidal Chalcone Pigments from Aqueous Extracts of Dyer's Saffron Flowers

Summary A new method was contributed to isolate water-soluble quinoidal chalcone pigments from aqueous extracts of dyers saffron florets. Carthamin, precarthamin, safflor yellow A and safflor yellow B were fixed with DEAE-, BD- and BND-cellulose and they were released from the cellulose-pigment complexes by using organic acids for checking their physicochemical characteristics, followed by estimating their recovery rate. Safflor yellow B was separated from a crude extract of dyers saffron florets by trapping it with BD-cellulose and purified through successive column chromatographies. The chromatographically purified sample was analyzed for identification with authentic safflor yellow B. Based on the experimental findings it was concluded that the pigment can be obtained by the affinitytrapping method without appreciable change of the chromatographic, spectrophotometric and tinctorial characteristics. The newly established method was practically applied to the quantitative determination of safflor yellow B content in dyers saffron flowers. The results proved that the amount of the pigment changed during flower ageing and that the quantitative expression was closely related with floret elongation.

Comparative Studies on the Activities of a Carthamin-Synthesizing Enzyme, Monophenol Monooxygenase and Peroxidase in Vegetative Tissues of Safflower

Summary Homogeneity of a carthamin-synthesizing enzyme sample obtained from matured florets or etiolated seedlings of safflower was confirmed by comparing electrophoretic migration pattern on polyacrylamide disc gels. Both enzyme samples were separately incubated in an incubation medium and the reaction product was analyzed for the enzyme homogeneity verification. Heterogeneity of the carthamin-synthesizing enzyme with native monophenol monooxygenase and peroxidase was checked on electrophoreograms prepared from protein extracts of the florets and the seedlings. Distribution and variation of the activities of a carthamin-synthesizing enzyme, monophenol monooxygenase and peroxidase were investigated with the extracts from florets under the different growth stage or of various parts of the tubular flower. Stability of a carthamin-synthesizing enzyme was tested in varied experiments using a preparation from safflower seedlings. Based on these observations, some of the implications were discussed mainly with respect to physiological role of the enzymes. in polyphenol metabolism in Carthamus plant organs under different growth conditions.

On a color change from yellow to red in the floral tissues of a dyer's saffron cultivar

Summary By using a spot scanning technique, the color change from yellow to red manifested in the floral tissues of Mogami-Benibana, a cultivar of dyers saffron ( Carthamus tinctorius L.), was investigated. Stamens and pistils were separated from fresh florets of the herbaceous flowering plant for microscopic observation of the zones of red color development. In pistils, color change is localized in the stigma and the peripheral region of the stylar canal. Observations of the fibrovascular bundle are especially indicative that the pollination process coincides with induction of the red shift. GA 3 was found to be a stimulator of tissue reddening, while IAA showed no effect. All red products isolated from the test samples were identical with authentic carthamine.

An Advanced Technique for Monitoring the Color Change from Yellow to Red in Floral Tissues of Dyer's Saffron

Summary Corollas, styles, anthers, and ovaries separated from fresh capitula of dyers saffron ( Carthamus tinctorius L.) were laid on microscope slides, and their reddening capacities were investigated by a technique of spot scanning. Styles reddened the most, followed by corollas, anthers, and finally ovaries with little or no capacity for tissue reddening. Red color accumulated more in larger styles than in smaller ones. Styles were divided longitudinally into three parts, and the appearance of red color in each part was scanned. The most activity was found in the middle part; reddening of the upper part ranked second, and reddening was least in the basal part. The observed differences in red-color-accumulating capacities are discussed briefly in relation to precursor translocation and enzyme activity distribution.

Influence of UV-light on carthamin accumulation and floret elongation in a cultivar of saffron thistle (Carthamus tinctorius L.)

Pre-matured florets of Benibana, a cultivar of saffron thistle (Carthamus tinctorius L.) was irradiated with UV-B (280–320 nm) or UV-C (254 nm) light for 48 h at 23±1 °C and the influence of UV-light on carthamin accumulation and floret elongation was investigated. UV-C light enhances carthamin accumulation most prominently, showing a specific value of 52.3 nmol carthamin·dm−3·h−1·25µm−2 (13.9 times of control), while it restricts floret elongation by a light-suppression manner (net elongation: 0.058 mm·h−1, one ninth of control). UV-B light is also promotive for the red colour appearance (25.0 nmol carthamin·dm−3·h−1·25 µm−2, 6.7 times of control) with suppressing floret elongation (net elongation: 0.17 mm·h−1, one third of control). Heterogeneous productivity of carthamin was seen in floret tissues after continuous treating under UV-C light. Carthamin accumulation, heterogeneous carthamin productivity and decrease of floret elongation restraint under UV-lights are discussed.

Scanning electron microscopic observation of stamen and pistil from a cultivar of Carthamus tinctorius : Influence of pollination process

Summary Florets of Mogami-Benibana, a cultivar of Carthamus tinctorius L., were collected from the flowering heads at the stage of pre-blooming or of post-blooming and separated into several groups. Stamen and pistil thus obtained were subjected to scanning electron microscopy and localized the damaged regions on the surface of the gamete generating tissues. Both stamen and pistil are wounded drastically after floret blooming: various gaps, scratches, tears, disturbed puckers, and broken and/or crushed hairs are scattered all over the outer surface, indicating that they are occurred during the pollination process — the resulting tissue damage triggers directly the reddening of florets. Hormonal control of the flower reddening appeared to involve the transpigmentation process: GA 3 is inducible the flower reddening through promoting activities of β-D-glucosidase and glucose oxidase. The experimental findings are discussed in relation to the induction of a bathochromatic color change in matured florets, where floral tissues lost their fresh and vivid appearance.