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Zoology Publications from Victoria University of Wellington—Nos. 42 to 46

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A. Epiphasic pigments

The upper brown hydrocarbon layer with thin-layer chromatography using five parts petroleum ether to one part methanol as solvent gave ten bands of pigments. Starting from the uppermost, these are referred to as follows: A yellow band (A) close to the front line; an orange band (A'); a yellow-pink band (A"); a light pink band (B); an intense violet-red band (B'); an orange band (C); a green band (D); page 3a pink band (E); a yellow band (E') and a greeny-yellow band (F).

Table 1 details the characteristics of these bands.

Identification of the Epiphasic Pigments

Comparison of the absorption peaks obtained for A (450 and 440 millimicrons) as mentioned above and those given by Fox (1953) suggests that band A (fig. 2) is a flavin or lyochrome. The wavelength of the absorption peak of band A' (fig. 7) suggests that this pigment is β-carotene. The nature of A" (fig. 3) is still not known. However, the peaks between 450 and 500 millimicrons and the colour of the pigment suggest that it might be a carotenoid compound. The values given by Fox (1953) for cyanidin were 510 and 269 millimicrons. The characteristics of B' (fig. 2) and its absorption peaks identify the pigment as cyanidin which is an anthocyan. Band B (fig. 1) only differs from B' by the last peak. Band B is probably also an anthocyan compound. Further work is needed, however, to fully identify this pigment. The yellow-orange colour of pigment C (fig. 4) and the absorption peaks noted earlier when dissolved in ethanol suggest that band C contains a group of two or three flavones, but this needs to be verified by further separation, purification and chemical analysis.

The absorption curve of D (fig. 6) in petroleum ether corresponds to that of phaeophytin, (660 millimicrons) as illustrated by Goodwin (1965). The green colour and the typical absorption curve show that this band is a chlorophyll (phaeophytin). By comparing the values obtained for E (fig. 4) and those of the known pigments of the two groups, anthocyans and flavonoid compounds, not all the absorption peaks correspond exactly. The value 267 millimicrons corresponds to one peak of pelargonidin. Apart from this peak pelargonidin also has peaks at 504.5; 450; 400.5 and 311 millimicrons. The range of maximum wavelength of the second peak of E is between 250-270 millimicrons. This corresponds to that of either flavones or flavonols. However the colour of the pigment, its solubility in water or cold methanol, its insolubility in fat solvents and its change of colour to green in the presence of ammonia shows that the pigment is more an anthocyan than a flavone. I have called this pigment "Isactinin" in the meantime. It could however be an incidental breakdown product of anthocyans accumulated as a result of the phytophagous nutrition of the animals.

Peaks of band E' (fig. 2) suggest that this pigment is probably a flavone compound. The peak for chrysin is 266 millimicrons, and that of quercitin 258 millimicrons. Pigment F (fig. 1) has peaks at 267 and 255 which are very near to those of the above-mentioned pigments. However, the peaks of F do not correspond exactly to any of the known pigments. This unidentified pigment which has most of the properties of flavone and is similar to quercitin and chrysin is tentatively called "olivacitin" because of its faint greenish-yellow colour.

In all previous investigations of animal pigments, most workers had to identify the pigments tentatively on the basis of absorption spectra together with any available chemical and physical characteristics. The two new pigments of bands E and F do not have all the characteristics of known pigments. However some of their properties indicate the group of compounds to which they may belong.

B. Hypophasic Pigments

The bottom layer of the extract when developed on a thin layer chromatogram showed three faint bands which decolorised very rapidly. (The solvent used was five parts petroleum ether to one part methanol). Nevertheless, one can distinguish three layers which did not move very far from the initial spot. This is to be expected page 4 Graphs of Absorption Spectra page 5 because the mixture of pigments obtained was extracted in the aqueous-alcoholic layer. The three bands in ascending order are:

  • Band G — greenish-yellow
  • Band H — also greenish-yellow
  • Band I — yellow

Identification of the Hypophasic Pigments

The individual pigments of the hypophasic layer have yet to be identified conclusively. However, some of their constituents and physical properties suggest the presence of xanthophylls. In a crude extract, xanthophylls are preferentially soluble in 90% methanol. In the presence of two immiscible solvents, petroleum ether and 90% methanol shaken in a separating funnel, xanthophylls are expected to become concentrated in the lower aqueous-alcoholic layer. The colours obtained from this lower layer also agree with those obtained by other workers for xanthophylls. If the lower layer pigments are xanthophyll, the coloration of Isactinia olivacea agrees with Goodwin's (1962) statement that in coelenterates the predominant carotenoid pigments are xanthophyll and acidic carotenoids.