The isolation, purification, and characterization of cis-zeta-carotene and the demonstration of its conversion to acyclic, monocyclic and dicyclic carotenes by a soluble enzyme system obtained from the plastids of tangerine tomato fruits

Cis-ζ-carotene was isolated, purified in several chromatographic systems, and then identified as an intermediate in the biosynthesis of poly-cis-carotenes. The structure of cis-ζ-carotene was tentatively established from its visible light-absorption spectrum, and by a comparison of the infrared spectrum with that of trans-ζ-carotene. Confirmation of the identity of this compound was obtained by high resolution mass spectroscopy. The presence of the cis-configuration was indicated by a bathochromic shift of 6–10 nm in the visible spectrum when the compound was subjected to iodinecatalyzed photoisomerization. The infrared spectrum also showed characteristic peaks for the cis-configuration. Proof of the conversion of cis-ζ-[¹⁴C]carotene to trans-ζ-carotene, proneurosporene, prolycopene, neurosporene, lycopene, β- and γ carotenes was obtained on incubation with soluble enzyme systems obtained from plastids of fruits of two different tangerine varieties of tomato. Proof for the formation of each of the carotenes was provided by column and thin-layer chromatography A close correspondence of radioactivity and optical density was observed for each carotene. Additional proof was obtained by gas-liquid chromatography of each hydrogenated carotene. A coincidence of mass and radioactivity was observed for each carotene.     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to more unsaturated acyclic, monocyclic, and dicyclic carotenes by a cell-free preparation of red tomato fruits

This paper reports the conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C|phytofluene and the conversion of the latter compound to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from the plastids of red tomato fruits. Each of these radioactive compounds was also converted to labeled neurosporene, lycopenc, α-carotene, and β-carotene by the same enzyme system. The incorporation of each substrate into more unsaturated carotenes was carried out under nitrogen at pH 7.5–8.2 (borate buffer), at 25 °C in the dark.Proof of the formation of the above carotenes from each of the three radioactive substrates was demonstrated by cochromatography with authentic nonradioactive carotenes on an alumina chromatographic column. A close correspondence between radioactivity and light absorbance for each carotene was observed. Confirmation of these conversions was achieved by cochromatography with authentic samples on thinlayer plates. Final proof for the formation of the acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed.Maximum conversion of cis- and trans-phytofluenes to more unsaturated carotenes by the red tomato fruit enzyme system appears to be dependent upon the presence of NADP⁺, FAD, and Tween 80. The formation of the carotenes is also increased in the presence of Mg²⁺ or Mn²⁺ ions.     

Extraction and utilization of carotenes and xanthophylls

Commercial methods are described for extracting carotenes and xanthophylls from alfalfa, carrots and palm oil. Utilization of these carotenoids as vitamin A precursors and coloring agents in margarines, dough products, Pharmaceuticals, stock and poultry feeds is discussed.     

Sex, light and carotenes: the development of Phycomyces.

Phycomyces blakesleeanus is known for the elaborate behaviour of its sporangiophores (fruiting bodies). Sporangiophore development is exquisitely sensitive to blue light, easy to describe quantitatively, pliable to genetic and biochemical research, and reminiscent in many details of other photoresponses in the same and in other organisms. The developmental and behavioural processes of Phycomyces share a number of genes. A combinatorial use of gene expression appears to be the basis for the complexities observed in this fungus.     

The biosynthesis of cyclic carotenes

1. The incorporation of (3RS)-[2-(14)C,(4R)-4-(3)H(1)]mevalonic acid into various cyclic carotenes in the fruit of the tomato mutant delta has been studied. The results confirm our previous view that the alpha-ionone ring of alpha-carotene does not arise by isomerization of a beta-ionone residue, and show that the same is also true for the alpha-ionone ring of delta- and in-carotene and alpha-zeacarotene. 2. The incorporation of (3RS)-[2-(14)C,2-(3)H(2)]mevalonic acid into alpha- and beta-carotene in carrot roots has been studied. The results show that the beta-ionone ring of beta-carotene does not arise by isomerization of the alpha-ionone residue of alpha-carotene. 3. These experiments show that alpha- and beta-ionone rings in cyclic carotenes are formed independently, probably by elimination of different protons from the same carbonium ion intermediates.     

Anticarcinogenesis activity of natural carotenes

Natural carotene sample obtained from palm oil was proved to suppress the promoting stage of two-stage carcinogenesis of mouse skin, and also inhibit the proliferation of human malignant tumor cells, such as neuroblastoma GOTO cells, stomach cancer HGC-27 cells, and pancreatic cancer PANC-I cells. Among the major constituents of palm carotene, alpha-carotene showed stronger anti-proliferative effect than beta-carotene. The present results indicate that further investigation for not only beta-carotene but also other kinds of natural carotenes, such as alpha-carotene, should be carried out.     

Spectrophotometric measurement of carotenes, xanthophylls, and chlorophylls in extracts from plant seeds

The method of spectrophotometric measurement of carotenoid and chlorophyll content in extracts from plant seeds was modified. The pigments were extracted with a mixture of petroleum ether (PE) and tetrahydrofuran (THF) (PE to THF ratio 4: 1). Equations for calculations of β-carotene, lutein, chlorophyll a and b content in PE: THF mixture were obtained using specific absorption coefficients of solutions of particular pigments. It was shown that subtraction of chlorophyll contribution from the absorption spectrum of seed extracts yields spectra, which could be used in some cases for calculation of carotene and xanthophyll contents.     

The stereospecific biosynthesis of phytoene and polyunsaturated carotenes

1. The incorporation of [2-(14)C,(5R)-5-(3)H(1)]mevalonic acid and [2-(14)C,5-(3)H(2)]-mevalonic acid into phytoene, phytofluene, zeta-carotene, neurosporene, alpha-, beta-, gamma- and delta-carotene and lycopene by slices of fruit from two tomato mutants (delta and tangerine) and into alpha- and beta-carotene by bean leaves has been studied. 2. In the formation of phytoene, all the pro-R-hydrogen atoms from C-5 of mevalonic acid are retained whereas two pro-S-hydrogen atoms are lost. 3. Possible mechanisms for the condensation of two molecules of all-trans-geranylgeranyl pyrophosphate are outlined. 4. In each dehydrogenation step from phytoene to the fully unsaturated carotenes, one pro-R-hydrogen atom from C-5 of mevalonic acid is lost, indicating that the sequential dehydrogenation is stereospecific and in the same sense at each step.     

天然胡萝卜素开发利用状况和设想

近年来,胡萝卜素的药理作用研究日益深入,大量研究表明胡萝卜素是维生素A的重要来源,具有抗氧化、抗癌、抗辐射、增强免疫力等生理和药用价值。不同植物种类之间和品种之间存在显著差异,通过育种途径来提高植物中胡萝卜素的含量是可行的。提出了开发利用高胡萝卜素含量植物——茶树的设想。     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to poly-cis-acyclic carotenes by a cell-free preparation of tangerine tomato fruit plastids

The conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C]phytofluene and the conversion of the latter to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from plastids of tangerine tomato fruits is reported. Each of these compounds is also converted to cis-ζ-carotene, proneurosporene, prolycopene, neurosporene, lycopene, and γ- and β-carotenes. [¹⁴C]Prolycopene was also incubated with the above enzyme system. No conversion of this compound to trans-lycopene or cyclic carotenes was observed. Proof for the formation of the above carotenes from each of the substrates mentioned above was obtained by cochromatography with authentic samples on an alumina column. A close correspondence between radioactivity and light absorbance of each carotene was observed. Further proof for the formation of acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed in each case.     

Co-oxidation of carotenes requires one soybean lipoxygenase isoenzyme.

The type II lipoxygenase (optimum pH 6.5) from soybeans was purified and separated into two fractions either by chromatography on DEAE-Sephadex or by isoelectric focusing. In the presence of linoleic acid and oxygen both fractions co-oxidise canthaxanthine or beta-carotene as effectively as a combination of these fractions. Oxygenation of linoleic acid and co-oxidation of canthaxanthine by type II lipoxygenase is stimulated by 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid but not by 13-hydroxy-cis-9,trans-11-octadecadienoic acid or 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid.     

Tocopherols and carotenes are differently distributed in subfractions of high-density lipoprotein

An apolipoprotein (apo) E-rich and an apo E-poor fraction of high-density lipoprotein (HDL) were isolated from four healthy men by heparin-Sepharose affinity chromatography. On a cholesterol basis, the apo E-poor HDL fraction contained a third more α- and γ-tocopherol and about a third less α- and β-carotene than the apo E-rich HDL fraction. Plasma concentrations of HDL cholesterol were highly correlated with the contribution of the apo E-rich HDL subfraction to total HDL α-tocopherol (r = − 0.990, P < 0.001).