Carotenylflavonoids , a novel group of potent , dual-functional antioxidants

We report here on the synthesis and antioxidant properties of novel covalently linked flavonoidcarotenoid hybrids, hereinafter referred to as carotenylflavonoids. By this strategy the essential properties of the protecting systems are improved. Compared with the parent carotenoids or flavonoids, e.g. ß-carotene and a hydroxyflavone, these molecular combinations exhibit improved photoprotective properties, as seen from their UV spectra, and they outperform the individual constituents in their antioxidant properties. The dual functionality is revealed from the time evolution of the peroxidation inhibition assay which clearly displays both phenolic and carotenoidpolyenic contributions to the antioxidant efficiency. Furthermore, these compounds have amphiphilic character, in contrast to symmetrical carotenes and hydroxylated flavones. The antioxidative potential of the carotenylflavonoids is high. They protect cumene against peroxidation more than β-carotene by a factor of about 2.3-2.5, and more than the individual components.


Introduction
Carotenoids exert many important functions, 1,2 and among them are the outstanding antioxidant effects in lipid phases by free radical scavenging or singlet oxygen quenching. 3,4,5With regard to antioxidant activity in biological systems carotenoids appear to be involved both in the protection against singlet oxygen and triplet oxygen (as radical chain-breaking antioxidants).Carote-have to be introduced and finally removed again.In these experiments the benzoyl group fulfilled all requirements.For the investigation of antioxidant capacities the method of recording the cumene hydroperoxide formation at 150 torr partial pressure of oxygen was used as has been described in detail 23 .This procedure allows a quantitative assessment of antioxidant capacities, described e.g. in protection factors or inhibition times, and, in addition, a correct time evolution of this behavior is obtained thereby affording parameters like induction periods, inhibition times as well as differential behavior of phenolic and polyenic function.
The antioxidant properties of a compound are evaluated by recording the protection against peroxidation of suitable substrates, e.g.methyl linoleate or cumene.In this work cumene is being preferred as described by Schmidt 23 .
The diagram containing the plots of cumene hydroperoxides evolution against time is shown in Figure 1.
ARKAT USA, Inc.A useful comparative discussion of the antioxidant capacities is done by the aid of inhibition times.The inhibition time is determined in the peroxide/time diagram (Fig. 1): the straight line of the second stronger rise intersects the time abscissa in the inhibition time (e.g. the final points of 3 define a straight line which intersects at 3.2 h).
It is obvious from Figure 1 that both reference compounds 1 and 4 are moderate carotenoid antioxidants which distinctly inhibit the amount of cumene hydroperoxide, as most carotenoids do, but with short inhibition times which are 1.5 and 1.8 h, respectively.The non-polyenic 4'hydroxy-6-methylflavone 7, however, lacking the carotenoid structure, displays a markedly increased antioxidant power with 2.5 h inhibition time and again reduced amount of hydroperoxides.This is due to the completely changed molecular structure: the hydroxyflavone acts primarily as a phenol and in minor contribution as an enone.This differential behavior of phenols (about > 2 h inhibition time) and carotenoids (about < 2 h inhibition time) is a useful mechanistic indicator at concentrations up to 2 x 10 -3 M, and differentiates clearly between e.g.astaxanthin and α-tocopherol 23 .
All reference compounds are, however, outperformed by the two carotenylflavonoids 3 and 6 which reach inhibition times of 3.2 and 3.0 h, respectively (see Fig. 2).The time evolution clearly displays both phenolic (the slow onset with very small gradient during the inhibition time) and carotenoid-polyenic (the gradually increasing gradient) contributions to the antioxidant efficiency.The total amount of peroxides is less compared with the other compounds.The differential behavior of phenolics and carotenoids is due to the fact that degradation of carotenoids yields fragments which still show antioxidant capacity whereas phenolics are superior when being present but after being consumed there is much less or no longer any protection (BHT and alphatocopherol show steep rises after being consumed!) 23.Thus it is justified to speak of dual antioxidant function in case of 3 and 6.
The difference in efficiency between 3 and 6 reflects a well-known fact: carotenoids get more and more unstable and fragile with increasing chain-length.Thus, compound 3 with shorter chain-length lives longer in the reaction mixture and exerts its superior function.In Figure 2 the qualities of these novel antioxidants are visually compared by plotting the inhibition time for each of the compounds.The fact that the combinations 3 and 6 of carotenoid and hydroxylated flavone are superior to the individual chromophores but do not equal the precise sum of the inhibition times of these building blocks may be explained by the conjugative connection: novel πsystems have been built up with their own specific characteristics.

Conclusions
The covalent combination of flavonoids with carotenoids creates potent, amphiphilic molecular entities which display their dual-functional antioxidative activity clearly in peroxide/time diagrams.Phenolic-flavonoid activity is responsible for the major part of the inhibition time whereas polyenic-carotenoid performance controls the gradually increasing peroxide accumulation which never reaches the peroxide amounts of the unprotected reference experiment and suppresses larger quantities of peroxides for hours.The intrusion of these amphiphilic antioxidants into lipid membranes will open new strategies for membrane protection.

1-(2-Hydroxy-5-methylphenyl)-3-phenylpropane-1,3-dione (18)
. 27 6.7 g (26 mmol) 2-acetyl-4-methylphenyl benzoate 17 are dissolved in 25 ml anhydrous pyridine in a 250 ml flask equipped with a drying tube.The mixture is heated to 50 °C.To the solution 2.5 g (45 mmol) of hot pulverized 85 % potassium hydroxide are added, and the mixture is stirred mechanically for 15 min, while a voluminous precipitate of the yellow potassium salt of the diketone forms.The mixture is cooled to room temperature and acidified with 100 ml of 10 % acetic acid.The diketone separated as a light-yellow amorphous precipitate which is collected on a filter and dried.Yield: 5.3 g, (21 mmol, 79 %) 18, C 16 H 14 O 3 , M = 254.3g/mol, mp.91 °C. 27,31 o a solution of 5.2 g (20 mmol) of 1-(2-hydroxy-5-methylphenyl)-3-phenylpropane-1,3-dione 18 in 30 ml of glacial acetic acid, contained in a 250 ml roundbottomed flask, 1.5 ml of concentrated sulphuric acid is added under shaking.The mixture is heated on a steam bath for 1 h with occasional shaking and is then poured under vigorous stirring onto 200 g of crushed ice.After the ice has melted, the crude flavone is collected on a filter, washed with water (about 400 ml) until free from acid, and finally dried at 50 °C.The flavone is obtained as white needles.Yield: 4.5 g, (19   20). 324.2 g (18 mmol) 6-methylflavone 19 are dissolved in 50 ml carbon tetrachloride and heated for 30 min.3.5 g (20 mmol) N-bromosuccinimide and 1.5 g azo-bisisobutyronitrile (AIBN) are added.The mixture is heated under reflux on a steam bath for 6 h.The suspension is slightly cooled and the succinimide is filtered off.The filtrate is cooled down and the 6-bromomethylflavone 20 crystallized as a light yellow amorphous solid.Yield: 3.8 g, (12 mmol, 67 %) 20, C 16 H 11 BrO 2 , M = 315.2g/mol, mp.128 °C.

1-(2-Hydroxyphenyl)-3-p-tolyl-propane-1,3-dione (25)
. 35 20 g (0.083 mol) 2-Acetyl-phenyl 4methylbenzoate 24 are dissolved in 75 ml anhydrous pyridine in a 250 ml flask equipped with a drying tube.The mixture is heated to 50 °C.To the solution 7.0 g (0.12 mol) of hot pulverized 85 % potassium hydroxide are added, and the mixture is stirred mechanically for 15 min, while a voluminous precipitate of the yellow-brown potassium salt of the diketone is formed.The mixture is cooled down to room temperature and acidification with 100 ml of 10 % acetic acid yields the diketone as yellow needles which are collected on a filter and dried.Yield: 16 g, (0.063 mol, 80 %)