Péter Molnár

Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes

Although a number of plant carotenoid cleavage dioxygenase (CCD) genes have been functionally characterized in different plant species, little is known about the biochemical role and enzymatic activities of members of the subclass 4 (CCD4). To gain insight into their biological function, CCD4 genes were isolated from apple (Malus×domestica, MdCCD4), chrysanthemum (Chrysanthemum×morifolium, CmCCD4a), rose (Rosa×damascena, RdCCD4), and osmanthus (Osmanthus fragrans, OfCCD4), and were expressed, together with AtCCD4, in Escherichia coli. In vivo assays showed that CmCCD4a and MdCCD4 cleaved β-carotene well to yield β-ionone, while OfCCD4, RdCCD4, and AtCCD4 were almost inactive towards this substrate. No cleavage products were found for any of the five CCD4 genes when they were co-expressed in E. coli strains that accumulated cis-ζ-carotene and lycopene. In vitro assays, however, demonstrated the breakdown of 8′-apo-β-caroten-8′-al by AtCCD4 and RdCCD4 to β-ionone, while this apocarotenal was almost not degraded by OfCCD4, CmCCD4a, and MdCCD4. Sequence analysis of genomic clones of CCD4 genes revealed that RdCCD4, like AtCCD4, contains no intron, while MdCCD, OfCCD4, and CmCCD4a contain introns. These results indicate that plants produce at least two different forms of CCD4 proteins. Although CCD4 enzymes cleave their substrates at the same position (9,10 and 9′,10′), they might have different biochemical functions as they accept different (apo)-carotenoid substrates, show various expression patterns, and are genomically differently organized.

Inhibition of multidrug resistance of cancer cells by natural diterpenes, triterpenes and carotenoids

The multidrug resistance (MDR) proteins are member of the ATP-binding cassette superfamily and are present in a majority of human tumors. Their activity is a crucial factor leading to therapeutic failure. It is likely that compounds which inhibit the function of the MDR-efflux proteins such as MDR1 will improve the cytotoxic action of anticancer chemotherapy. Therefore, a search for MDR reversing compounds was conducted among three classes of plant derived compounds such as diterpenes, triterpenes and carotenoids in a hope to find inhibitors without adverse effects in these natural compounds. The inhibition of efflux activity was determined by measuring the accumulation of substrate analogues such as rhodamine in tumor cells in the presence of potential inhibitors. Thus we determined the effect of structurally unrelated diterpenes, triterpenes and carotenoids on reversal of multidrug resistance in MDR-1 gene-transfected L1210 mouse lymphoma cells and MDR mediated multidrug resistance of human breast cancer cells MDA-MB-231 (HTB-26) and MCF-7. The majority of diterpenes, cycloartane triterpenes and carotenoids isolated from vegetables and medicinal plants were able to enhance rhodamine 123 accumulations of MDR-cells. Synergistic interaction was found between epirubicine and resistance modifier terpenoids in vitro. It is supposed that these MDR modulators bind into transmembrane domains and the action of ABC transporters is inhibited by induced conformational changes.

Reversal of Multidrug Resistance by Natural Substances from Plants

The multidrug resistance (MDR) proteins that belong to the ATP-binding cassette superfamily such as P-glycoprotein (P-gp) and MRP1, are present in a majority of human tumors and constitute an important cause of therapeutic failure. Selective inhibitors of the MDR-efflux proteins may improve the effectiveness of cancer chemotherapy. Their mechanism of action was believed to be a competition between resistance modifiers and drugs for the same binding site of P-gp. In our previous work we studied modulation of MDR in cancer cells expressing P-gp or MRP1 by selected carotenoids, flavonoids and extracts from medically important Chinese plants. Capsanthin and capsorubin, carotenoids isolated from paprika, were identified as potent P-gp inhibitors, while lycopene, lutein, antheraxanthin and violaxanthin induced moderate effects. Among flavonoids, effective modulators were rotenone, chrysin, phloretin and sakuranetin. Some chloroform extracts of Chinese herbs were also found to inhibit MDR efflux pumps. The effects of the modulators on P-gp activity were studied by measuring rhodamine 123 uptake in several cancer cells such as the human MDR1 gene-transfected mouse lymphoma cells (L1210) and human breast cancer cells MDA-MB-231 expressing the MRP1 pump (HTB26). Additionally, the ability to alter biophysical properties of lipid bilayers by selected carotenoids was studied by differential scanning calorimetry. The antiproliferative effects as well as the MDR reversal activity of the studied compounds, applied in combination with anticancer drugs, were also discussed.

Non-covalent associations of cyclomaltooligosaccharides (cyclodextrins) with carotenoids in water. A study on the α- and β-cyclodextrin/ψ, ψ-carotene(lycopene) systems by light scattering, ionspray ionization and tandem mass spectrometry

Water-soluble complexes of the dietary carotenoid ψ,ψ-carotene (lycopene 1) with cyclomaltohexaose (α-cyclodextrin, αCD) and cyclomaltoheptaose (β-cyclodextrin, βCD) have been prepared and characterized via multiangle light scattering (MALS), ionspray/electrospray ionization (IS/ESI) mass spectrometry (MS) and tandem MS. MALS experiments point out that large aggregates of particles, on the nanometer-size scale, are present in water, with meaningful differences in the shape of the αCD/1 aggregates with respect to βCD/1 analogues. The true 1:1 αCD/1 inclusion complex has been observed by IS/ESIMS and confirmed by tandem MS. The structure of CD/1 aggregations in water is proposed which are consistent with the combined MALS and MS experimental results.

Naturally occurring di-cis-violaxanthins from viola tricolor: Isolation and identification by 1H NMR spectroscopy of four di-cis-isomers

Abstract From blossoms of Viola tricolor , four new naturally occurring geometrical isomers of violaxanthin (5,6,5′,6′-diepoxy-5,6,5′,6′-tetrahydro-β,β-carotene-3,3′-diol) have been isolated in the crystalline state. By two-dimensional 1 H NMR methods, the new pigments are shown to be the 9,9′-, 9,13′-, 9,15- and 9,13-di- cis isomers. Thermal and iodine-catalysed photochemical stereomutations of the new natural pigments have also been studied.

Occurrence of 15-cis-violaxanthin in Viola tricolor

Abstract A new cis isomer in the violaxanthin series has been isolated from the blossoms of Viola tricolor and identified by MS, IR and UV as the central-mono cis form. It was converted to all- trans -violaxanthin by stereomutation. The CD correlation between 15- cis -violaxanthin and natural violaxanthin (5,6,5′,6′-diepoxy-5,6,5′,6′-tetrahydro- β,β-caroten-3,3′-diol) provided the basis for assignment of the absolute configurations 3 S , 5 R , 6 S , 3′ S , 5′ R , 6′ S . Trans—cis isomerization of all- trans -violaxanthin also resulted in 15- cis -violaxanthin. In addition a quantitative determination of the carotenoids was conducted.

Isolation and characterization of 3,5,6-trihydroxy-carotenoids from petals ofLilium tigrinum

SummaryReinvestigation by HPLC of the petals ofLilium tigrinum and the isolation of some minor compounds is reported. Using HPLC-controlled, preparative-column chromatography, 5,6-diakarpoxanthin (6), 6-epikarpoxanthin (2), 5,6-diacapsokarpoxanthin (8), and 9Z-antheraxanthin (9Z-13) were isolated and characterized. Based on spectroscopic data the absolute configurations of6 and8 were identical with those originating from paprika, thus the 5,6-diakarpoxanthin (6) and 5,6-diacapsokarpoxanthin (8) have the 3S, 5S, 6S configuration and 6-epikarpoxanthin (2) has the 3S, 5R, 6S configuration.

(Z/E)-photoisomerization of C40-carotenoids by iodine

The compositions of stereoisomeric mixtures of β-carotene (1), zeaxanthin (2), physaliene (3), natural violaxanthin (4), ‘semi-synthetic’ violaxanthin (5), capsorubin (6), capsanthin (7) and lutein-epoxide (8) have been investigated. The effect of end groups with different structures and stereochemistry on the cis-trans equilibrium, the rates of photo-induced cis–trans rearrangement of the zeaxanthin (2) set, and the ‘specific rate’(per unit light energy at a given wavelength) for several 13-cis carotenoids (2, 4, 6, 7, 9) have been determined.

Novel carotenoid 3,6-epoxides from red paprika, Capsicum annuum

Abstract The new carotenoid pigments (2),(3),(7),(9) and (10) have been isolated from red paprika; the 3,6-epoxy-5-hydroxy end group present in the pigments (2) and (9), hitherto has not been reported amongst natural carotenoids.

Carotenoids with anti‐Helicobacter pylori activity from Golden delicious apple

Previously it was reported that hypophasic carotenoids of Golden delicious apple peel showed potent anti‐H. pylori activity (MIC50 = 36 µg/mL), comparable to metronidazole (MIC50 = 45 µg/mL). To further investigate the involved active carotenoids of the apple peel extracts, seven carotenoids were isolated for the current study: (all‐E)‐luteoxanthin, (all‐E)‐neoxanthin, (9′Z)‐neoxanthin, (all‐E)‐antheraxanthin, (all‐E)‐violaxanthin, (9Z)‐violaxanthin and (all‐E)‐lutein. The MIC50 values of (all‐E)‐luteoxanthin, (all‐E)‐neoxanthin and (9′Z)‐neoxanthin were 7.9, 11 and 27 µg/mL, respectively. Other carotenoids and β,β‐carotene did not exhibit potent anti‐H. pylori activity (MIC50 > 100 µg/mL). An examination of structure and function suggested that active carotenoids contained a monofuranoid ring or an allenic bond in addition to an epoxy group and an additional two or three hydroxyl substituents on the side group. Copyright