- Email: [email protected]
Antioxidant activity of phenolic meroditerpenoids from marine algae
ELSEVIER
Journal of Photochemistry and Photobiology B: Biology 26 (1994) 159-164
Antioxidant
activity of phenolic meroditerpenoids marine algae
from
Mario Foti”p*, Mario Piattelli”, Vincenzo Amico”, Giuseppe Rubertobp* “Dipartimento di Scienze Chimiche dell’Universit~ degli Siudi di Carania, Vile A. Doria 6, I-95125 Carania, Italy bIstiruto de1 CNR per lo Studio delle Sosfanze Naturali di Interesse Alimentare e Chimico-Fannaceudco, Via de1 Santuario I-95028 Valverde (CT), Italy
110,
Received 18 February 1994; accepted 22 June 1994
Abstract The overall ‘0, quenching rate constants k, for three meroditerpenoids from Mediterranean marine algae of the genus were measured using 1,4_dimethylnaphthalene 1,4-endoperoxide as a thermal source of IO2 (‘A,). The most active of the compounds tested had a k, value which is comparable with that of cu-tocopherol. These metabolites also act as inhibitors of methyl linoleate peroxidation, while their O;- quenching activity is rather low, similar to cY-tocopherol. Cystoseiru
Keywords: Singlet oxygen; ‘Oz quenching rate constants; Oz.- quenching rate constants; Dye-sensitized photochemical oxidation; Peroxidation inhibition; Algal metabolites; Qwtoseira
1. Introduction
In recent years, a number of investigations on the effect of light, singlet oxygen and active free radicals (O;-, OH’ and lipid peroxyl radicals LOO’) on biological systems have appeared [l-7]. The results have shown that radical reactions are detrimental to health, being involved in the aging process as well as in pathogenesis of a number of human disease states, namely atherosclerosis, rheumatoid arthritis, muscular dystrophy, cataracts, some neurological disorders and possibly cancer [1,2,8-lo]. Cell membranes, DNA and proteins are the main target molecules of free radicals and singlet oxygen. Membranes are chiefly affected by peroxyl radicals that attack polyunsaturated phospholipids at the bisallylic methylenes thus compromising their integrity [2,11]. Radical reactions modify the purine and pyrimidine bases of DNA [12], whereas proteins are oxidized at the metal binding sites of amino acid residues leading, in the case of some amino acids, to loss of ammonia and the formation of carbonyl derivatives [12]. Lipid radical peroxidation, yielding evilsmelling substances as end products, is the main cause of the qualitative decay of foods rich in fatty acids [13]. -
*Corresponding
authors.
loll-1344/94/$07.00 0 1994 Elsevier Science S.A. All rights reserved SSDI 1011-1344(94)07038-P
Many natural or synthetic compounds act as antioxidants by trapping singlet oxygen or radicals, thereby terminating chains of reactions [11,14,15]. Phenols are particularly effective antioxidants for polyunsaturated fatty acids because they can easily transfer a hydrogen atom to lipid peroxyl radicals (reaction (1)) [16,17]. The aryloxyl radical, formed in reaction (l), is usually too sluggish to act as a chain carrier [l&18], but can easily couple with another radical to give non-radical products (reactions (2) and (3)). LOO‘+ ArOH Are’+ LOO’ ArO’ + ArO’ -
LOOH -t-AI-O
(I)
non-radical products
(2)
non-radical products
(3)
The mechanism of singlet oxygen quenching by phenols probably involves, in polar solvents, an electron transfer from phenol to singlet oxygen resulting in the formation of a radical ion pair [19]; the same mechanism has also been observed with aromatic amines [20] and thioanisoles [21]. This process is reversible and the back electron transfer leads to the starting phenol and triplet oxygen [l]. However, the radical ion pair can also react and, consequently, the overall quenching rate constant k, for a ‘0, scavenger is the sum of the physical quenching (k,) and the chemical reaction (k,) rate constants, k, = k, + k,.
160
M. Foti et al. I J. Photo&m.
Photobiol.
B: Biol. 26 (1994) 159-164
One of the most studied natural antioxidants is (Ytocopherol, the most active of the four tocopherols which make up vitamin E and the major lipid-soluble radical-trapping antioxidant present in human blood [ll]. cY-Tocopherol is an efficient ‘0, scavenger with a predominant physical component of the quenching rate constant [22], although many oxidation products are formed during its interaction with ‘0, [23,24]. The observation that many metabolites, previously isolated form Mediterranean seaweeds belonging to the genus Cystoseiru [25], have structures reminiscent of those of tocopherols prompted us to plan a screening of their scavenger properties against singlet oxygen, superoxide anion and peroxyl radicals, with the aim of identifying effective and safe natural antioxidants of potential value in the food industry. In the present paper, we describe the results obtained with three Cystoseira compounds l-3 [26-281.
bCH,
2. Materials and methods 2.1. General Metabolites l-3 were available from previous work. 1,4_Dimethylnaphthalene 1,4-endoperoxideqEP, 5) was prepared from 1,4_dimethylnaphthalene (DMN) according to the procedure described in Ref. 1291. A xanthine oxidase suspension is commercially available (Grade I, Sigma). All chemicals were of analytical grade and were used without further purification. UV measurements were carried out on a Beckman DU-65 spectrophotometer. High-resolution mass spectrometry (HRMS) was determined at 70 eV on a Kratos MS50s instrument. IR and nuclear magnetic resonance (NMR) spectra were recorded on Perkin-Elmer FTIR 1720 X and Bruker AC 250 instruments respectively. Preparative (PLC) and thin layer chromatography (TLC) were performed on precoated silica gel plates (Merck).
2.2. Determination of ‘0,
quenching rate constants
The procedure described followed. EP was employed by thermal decomposition, zofuran (DPBF) as standard reaction sequence underlies EP -
DMN +‘02
‘0,s
decay
DPBF + ‘02 z
by Saito et al. [20] was to generate singlet oxygen with 2,5-diphenyl-3,Cbencompound. The following this method
reaction
Q+‘O,*
Q+30,
Q + lo,*
reaction
where Q is any quencher. The quenching rate constants were obtained by the method of Young et al. [30]. Solutions of DPBF (lo-’ M), EP (1O-3 M) and varying amounts of scavenger (l-3, O-12 mM) in the solvent of choice were kept at 25 “C. The progress of the reaction was monitored by measuring the decrease in the absorbance maximum in the UV spectrum of DPBF (411 nm, in methanol). The slopes of the pseudofirst-order curves within the first 10 min of reaction in the absence (I$) and presence (Vo) of scavenger are related to the rate constant k, through the equation 1311 K/v,
= I + k,[Q]I(k,,[DPBF]
+ kd)
M. Foti et al, /J.
Photochem.
Photobiol.
Thus k, is calculated from the slope of the quenching plot, V,/Y, vs. [Q], using the values of ‘0, lifetimes, l/k,, reported in the literature [32] (10.25 ps in methanol, 83 ps ‘in acetonitrile, 30 ps in benzene and 769 ps in carbon tetrachloride) and the values of k, in the different solvents [33] (1.03 X lo9 M-l s-l in methanol, 1.36 x lo9 M-l s-’ in acetonitrile, 6.90 X 10’ M-l s-’ in benzene and 2.84~10~ M-’ s-’ in carbon tetrachloride). 2.3. Determination of O,‘- quenching rate constants Xanthine (5 x 10e5 M), cytochrome c (6.4~ 10T5 M) and various amounts of scavengers (O-200 mM) were dissolved in a buffer solution (NaH,PO, (low3 M), adjusted at pH 7.4 with 2 N NaOH) containing 15% propanol. Each solution (3 ml) was placed in a thermostatically controlled UV cell (25 “C) and 6 ~1 of xanthine oxidase solution (obtained by 1:3 dilution of the stock solution with sodium phosphate buffer) was added. The progress of the reaction was followed by monitoring the absorbance at 550 nm. The increase in absorbance per minute (AOD min-l) in the described experimental conditions was 0.020 min-’ in the absence of scavenger. Assuming that O;- is consumed only by cytochrome c and scavenger, the quenching constant of the scavenger, kinh, is calculated from the equation AOD,IAODo = I+ ki,,[Q]/kq,[cyt] where the subscripts 0 and Q refer to the absence and presence respectively of the scavenger, k,, is the rate constant of cytochrome c reduction by O,‘- (lo6 M-’ s-l) and [Q] and [cyt] are the concentrations of scavenger and cytochrome respectively. 2.4. Peroxidation of trans-pcarotene Methyl linoleate (4 X lo-* M), trans-p-carotene (6 x 10m6M), 2,2’-azobis(2-methylpropionitrile) (AIBN, 3 x 10e4 M) and various amounts of scavenger (O-O.25 mM) were dissolved in cyclohexane saturated, at room temperature, with methanol and 0,. Each solution, protected against light, was kept at 50 “C in a thermal bath and the rate of disappearance of p-carotene was followed by monitoring the decrease in absorbance at 450 nm at 15 min intervals.
B: Biol. 26 (1994) 159-164
161
run was carried out with 20 mg and an irradiation period of 3 h. PLC of the reaction mixture, using dichloromethane-ether (4:l) as eluant gave, with 7 mg of 1, 8 mg of 4 as a yellow oil (yield 61% based on the starting material converted). The oxidation product has the following physical properties: HRMS [M’] 440.2566 (calculated for C&H,,O, 440.2562); IR timax) (film) (cm-l) 3320, 1680, 1660, 1640, 1600; ‘H NMR (CDCl,) 6 6.57 (lH, m, H-5’), 6.51 (lH, m, H-3’), 5.23 (lH, t, J=7 Hz, H-2). 3.97 (lH, m, H-14), 3.19 (2H, d, 5=7 Hz, H-l), 3.03 (lH, S, H-6), 2.98 (2H, S, H-4), 2.97 (lH, m, H-13a), 2.06 (3H, d, J=2 Hz, 6’-Me), 1.95 (lH, m, H-13b), 1.15 (3H, s, H-17), 1.05 (3H, s, H-16), 0.94 (3H, s, H-19), 0.91 (3H, s, H-18), 1.65 (3H, s, H-20).
3. Results and discussion The total absolute second-order rate constants of singlet oxygen (‘A,) quenching for the algal metabolites l-3 were measured in different solvents. Figs. l-4 illustrate typical quenching plots in the different solvents, and Table 1 shows the overall quenching rate constants of ‘0,. It is readily apparent that the quenching process is strongly affected by the solvent. For instance, the value of k, for compound 1 ranges from 1.6X lo6 M-’ S -' in carbon tetrachloride to 2.0~ 10’ M-’ s-’ in methanol, with a 125-fold increase in the polar solvent. This remarkable solvent effect suggests the involvement of a charge transfer transition state in the quenching mechanism of singlet oxygen [1,19-211. Compound 3, on irradiation in the presence of 0, and a sensitizer (rose bengal or methylene blue), gives only traces of decomposition products and therefore we suggest that the value of k, represents essentially the physical quenching rate constant. In contrast, for the same experimental conditions, 2 and 1 suffer ex-
5-o I
1
2.5. Dye-sensitized photochemical oxidation of l-3 A methanol solution containing the algal metabolite (2x 10e3 M) and a ‘0, sensitizer (methylene blue or rose bengal, lop5 M) was irradiated with a 100 W tungsten lamp at room temperature with oxygen bubbling. At regular time intervals an aliquot was taken and examined by TLC. The results were essentially the same with either photosensitizer. In the case of 1, a
0
3
6
concentration
9
12
(mM)
Fig. 1. Variation in the ratio V,,IV, as a function of quencher concentration in CH,OH at 25 “C: Compound 1 (+), compound 2 (A) and compound 3 (0).
162
M. Foti et al. I J. Photochem.
Photobiol.
B: Biol. 26 (1994) 159-164
Table 1 ‘02 quenching rate constants (ko) of compounds 1-3 in different solvents at 25 “C?
10.7
Compound
Solvent
kQxlO-’ (M-’
? Y
7.5
4.3
1.0 0
3
6
concentration
9
12
(mM)
Fig. 2. Variation in the ratio V,lVo as a function of quencher concentration CH&N at 25 “C: compound 1 (+), compound 2 (A) and compound 3 (0).
2.9
2.3
1.6
1.0 0
3
6
concentration
9
12
(mM)
Fig. 3. Variation in the ratio V,/Vo as a function of quencher concentration in CbH6 at 25 “C: compound 1 (+), compound 2 (A) and compound 3 (0).
3.5
2.9 Y ;_
1 1 1 1
Methanol Acetonitrile Benzene Carbon tetrachloride
2 2 2 2
Methanol Acetonitrile Benzene Carbon tetrachloride
1.4kO.2 1.03*0.04 0.47 f 0.02 0.089 f 0.003
3 3 3 3
Methanol Acetonitrile Benzene Carbon tetrachloride
0.99 * 0.03 0.51 f 0.04 0.024 f 0.002 0.065 f 0.009
a-Tocopherol
Methanol
20*2 19.4 f 0.4 0.96 k 0.04 0.16 f 0.02
24kl
“The rate constants are the average of four measurements. Errors are given as f lo.
3.5
? PO
s-‘)
2.3
1.6
1.0 0
3
9
6
concentration
12
(mM)
Fig. 4. Variation in the ratio V,/Vo as a function of quencher concentration in CCL, at 25 “C: compound 1 (+), compound 2 (A) and compound 3 (0).
tensive oxidation; the former gives an intractable mixture, but the main oxidation product of the latter was isolated and characterized as 4 based on its spectral properties. HRMS indicated the elemental composition C,,H,,O, revealing the loss of a carbon atom (and four hydrogens) from the parent compound, while the presence in the molecule of a quinone moiety was evident from IR absorptions at 1660 and 1640 cm-l. The ‘H NMR spectrum showed, in comparison with that of 1, the following main differences: (a) disappearance of the signal for the methoxyl group; (b) replacement of the AB system due to the aromatic protons at 3’ and 5’ with two multiplets at 6.57 and 6.51 ppm; (c) modification of the position and multiplicity of the signals due to the protons of the methyl group on the ring at position 6’ and the methylene at position 1. The different behaviour of the three compounds in the dye-sensitized photochemical oxidation suggests that the chemical component (k,) of the observed rate constants is greater for compounds 1 and 2, which have a free phenolic OH, than compound 3. The ability of metabolites l-3 to quench lipid peroxyl radicals has also been investigated. Compounds 1 and 2 act as efficient scavengers, being able to inhibit the peroxidation of p-carotene mediated by methyl linoleate peroxyl radicals in the presence of 0, and a radical initiator. The oxidation percentages of p-carotene after 30 and 75 min of reaction, presented in Table 2, prove that the antioxidant activity of 1 and 2 is nearly half that of a-tocopherol, while 3 lacks any effect. The quenching activity of compounds 1 and 2 towards superoxide anion, generated by the system xanthinexanthine oxidase, is rather low (the value of the quenching rate constant is around 10 M-’ s-l), whereas 3 is
M Foti ei al. I .I. Photochem. Table 2 Percentages of oxidation of p-carotene presence and absence of scavengers”
times
75 min
Blank
4.5
19.1
Compound
c=1.6~10’+
M
1.9 0.2
5.8 1.0
Compound 2 c=3.OxlO+ c=2.5~10-~
M M
2.3 0.3
7.1 1.1
NA
3
c+Tocopherol c=2.9~10-~ c=l.S~lO-~
M M
‘Experiments with methanol.
were performed NA, not active.
163
in the
30 min
Compound
8: Biol. 26 (1994) 159-164
Acknowledgements at different
Sample
1 c=2.9x10-5 M
Photobiol.
1.2 0.0 at 50 “C in cyclohexane
NA
2.2 0.2 saturated
completely unreactive. Similarly, the value reported in the literature for the O,‘- quenching rate constant of cu-tocopherol is quite small (6 M-’ s-l) [l]. Finally, we wish to draw attention to the fact that, in the Cystoseiru species, the mean concentration of (Ytocopherol is around 10 mg kg-’ of alga (dry weight) [34], while that of the meroditerpenoids of the type considered in the present paper is much higher; for instance, in C. stricta, the content of 1 exceeds 500 mg kg-’ [26,35]. Therefore, the possibility that these compounds perform a physiological function somehow linked to their antioxidative properties must be considered.
4. Conclusions
The algal meroditerpenoids l-3 are very effective singlet oxygen scavengers, the most active (1) having a ‘0, quenching rate constant similar to that of (Ytocopherol. A large solvent effect has been observed in the values of the rate constants, in agreement with the quenching mechanism, reported in the literature, which initially involves an electron transfer from phenol to singlet oxygen [1,19-211. Unlike 3, which lacks free phenolic OH and is essentially a physical quencher, 1 and 2 suffer extensive degradation in the dye-sensitized photo-oxidation, indicating participation of a chemical process to the overall rate constants of singlet oxygen quenching. Moreover, 1 and 2 have a strong peroxyl radical quenching activity, the former being able to reduce the rate of peroxidation of p-carotene by 60%-70% at a concentration of 2.9~ lo-’ M. Similar to a-tocopherol, the algal metabolites have rather low, if any, O;- quenching activity.
This work was supported by the Consiglio Nazionale delle Ricerche (CNR, Roma) under the scheme “Progetto Strategic0 - Innovazione produttiva nelle piccole e medie imprese” and by a MURST 40% grant. The authors thank Dr. M. Recupero for skilful technical assistance.
References
111 A.
Breccia, M.A.J. Rodgers and G. Semeraro (eds.), Oqgen and Sulfur Radical in Chemistry and Medicine, Oxygen Radicals in Chemise nnd Biology (Chap. l), Edizioni Scientifiche Lo Scarabeo, Bologna, 1986, pp. 20-52, and references cited therein. 121 A.W. Girotti, Photodynamic peroxidation in biological systems, Photochem. Photobiol., 51 (1990) 497-509, and references cited therein. and P. Hochstein, Alterations in erythrocyte t31 C. Rice-Evans membrane fluidity by phenylhydrazine-induced peroxidation of lipids, B&hem. Biophys. Rex Commun., 100 (1981) 1537-1542. K.B. Heller and C.W.M. Haest, Progressive 141 B. Deuticke, oxidative damage in erythrocytes after pulse treatment with tbutylhydroperoxide, B&him. Biophys. Acta, 899 (1987) 113-124. killing of non151 G.J. Kantor and C. Ritter, Sunlight-induced dividing human cells in culture, Photochem. PhotobioZ., 37 (1983) 533-538. and G.C. Goyal, Photosensitized lysis of I61 L.I. Grossweiner liposomes by hematoporphyrin derivative, Photochem. Photobiol., 37 (1983) 529-532. in G. Moreno [71 B.E. Johnson, Skin and related photoreactions, et al. (eds.), Photosensitisation, NATO ASI Series Vol. H 15, Springer-Verlag, Berlin, 1988, pp. 253-264. S. Parthasarathy, T.E. Carew, J.C. Khoo and [81 D. Steinberg, J.L. Witztum, Modifications of low density lipoprotein that increase its atherogenicity, New Engl. J. Med., 320 (1989) 915-924. [91 B. Halliwell, Oxidants and human disease: some new concepts, FASEB. J., I (1987) 358-364. enzymes, and carcinogenesis, PO1 Y. Sun, Free radicals, antioxidant Free Radicals Biol. Med., 8 (1990) 583-599. L.R.C. Barclay, Model biomembranes: quantitative studies of Pll peroxidation, antioxidant action, partitioning, and oxidative stress (1992 Syntex Award Lecture), Can. J. Chem., 71 (1993) l-16, and references cited therein. P21 E.R. Stadtman, Protein oxidation and aging, Science, 257 (1992) 1220-1224, and references cited therein. and N.P. Das, Studies on the control of lipid iI31 L. Ramanathan oxidation in ground fish by some polyphenolic products, J. &ic. Food Chem., 40 (1992) 17-21, and references cited therein. of higher plants, Phytochemistty, 1141 R.A. Larson, The antioxidant 27 (1988) 969-978. 1151 M.S. Taga, E.E. Miller and D.E. Pratt, Chia seeds as a source of natural lipid antioxidants, J. Am. Oil Chem. Sot., 61 (1984) 928-931. G.W. Burton and K.U. Ingold, Autoxidation of biological PI molecules. 1. The antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in vitro, J. Am. Chem. Sot., 103 (1981) 6472-6477. L.J. Devall, B. Tait, B.K. Trivedi, P71 W.A. Pryor, J.A. Cornicehi, D.T. Witiak and M. Wu, A rapid screening test to determine the antioxidant potencies of natural and synthetic antioxidants, J. Org. Chem., 58 (1993) 3521-3532.
164
M. Foti et al. I J. Photochem.
Photobiol.
V.W. Bowry and R. Stocker, Tocopherol-mediated peroxidation. The prooxidant effect of .vitamin E on the radical-initiated oxidation of human low-density lipoprotein, J. Am. Chem. Sot., 115 (1993) 6029-6044. K. Mukai, K. Daifuku, K. Okabe, T. Tanigaki and K. Inoue, PI Struchire-activity relationship in the quenching reaction of singlet oxygen by tocopherol (Vitamin E) derivatives and related phenols. Finding of linear correlation between the rates of quenching of singlet oxygen and scavenging of peroxyl and phenoxyl radicals in solution,J. Org. Chem., 56 (1991) 41884192, and references cited therein. PO1 I. Saito, T. Matsuura and K. Inoue, Formation of superoxide ion via one-electron transfer from electron donors to singlet oxygen, J. Am. Chem. Sot., 105 (1983) 3200-3206, and references cited therein. of single WI K. Inoue, T. Matsuura and I. Saito, Importance electron-transfer in singlet oxygen reaction in aqueous solution. Oxidation of electron-rich thioanisoles, Tetrahedron, 41 (1985) 2177-2181. F.H. Doleiden, A.M. Trozzolo and A.A. ml S.R. Fahrenholtz, Iamola, On the quenching of singlet oxygen by cr-tocopherol, Photochem. PhotobioZ., 20 (1974) 505-509. photov31 M. d’Ischia, C. Costantini and G. Prota, Dye-sensitized oxidation of vitamin E revisited. New 7-oxaspiro[4,5]dec-l-ene3,6-dione products by oxygenation and ring contraction of (Ytocopherol, J. Am. Chem. Sot., 113 (1991) 8353-8356. G. Misuraca and v41 C. Costantini, M. d’Ischia, A. Napolitano, G. Prota, Photodynamic degradation of vitamin E induced by psoralens, Biochim. Biophys. Acta, 1116 (1992) 291. WI M. Piattelli, Chemistry and taxonomy of Sicilian Cystoseim species, New .I. Chem., 14 (1990) 777-782.
WI
B: Biol. 26 (1994) 159-164
WI
v71
PI P91 1301
1311
[321
I331
[341 [351
V. Amico, F. Cunsolo, M. Piattelli, G. Ruberto and F.R. Fronczek, Balearone: a metabolite of the brown alga Cystoseira baleatica, Tetrahedron, 40 (1984) 1721-1725. V. Amico, F. Cunsolo, M. Piattelli, G. Ruberto and L. Mayol, Strictaketal: a new tetraprenyltoluquinol with a heterotetracyclic diterpene moiety from the brown alga Cystoseira shicta, J. Nat. Prod., 50 (1987) 449-454. V. Amico, F. Cunsolo, M. Piattelli and G. Ruberto, Five novel tetraprenylhydroquinols from the brown alga Cystoseira a/geriensis, PhytochemisQ, 23 (1984) 2017-2020. H.H. Wasserman and D.L. Larsen, Formation of 1,4-endoperoxide from the dye-sensitized photo-oxygenation of alkylnaphthalenes,J. Chem. Sot., Chem. Commun., 5 (1972) 253-254. R.H. Young, K. Wehrly and R.L. Martin, Solvent effect in dye-sensitized photooxidation reactions, J. Am. Chem. Sot., 93 (1971) 5774-5779. C.S. Foote, T.-Y. Ching and G.G. Geller, Chemistry of singlet oxygen, XVIII. Rates of reaction and quenching of ru-tocopherol and singlet oxygen, Photochem. Photobiol., 20 (1974) 511-513. S.L. Studer, W.E. Brewer, M.L. Martinez and P.-T. Chou, Time-resolved study of the photooxygenation of 3-hydroxyflavone, J. Am. Chem. Sot., 111 (1989) 7643-7644. F. Wilkinson and J.G. Brummer, Rate constants for the decay and reactions of the lowest electronically excited singlet state of molecular oxygen in solution, J. Phys. Chem., Ref Data, 10 (1981) 809-999. M. Piattelli, G. Ruberto and V. Amico, unpublished results, 1992. V. Amico, P. Neri, M. Piattelli and G. Ruberto, Linear diterpenoids from Cystoseira balerica, Phytochemirtry, 26 (1987) 2637-2639.
Journal of Photochemistry and Photobiology B: Biology 26 (1994) 159-164
Antioxidant
activity of phenolic meroditerpenoids marine algae
from
Mario Foti”p*, Mario Piattelli”, Vincenzo Amico”, Giuseppe Rubertobp* “Dipartimento di Scienze Chimiche dell’Universit~ degli Siudi di Carania, Vile A. Doria 6, I-95125 Carania, Italy bIstiruto de1 CNR per lo Studio delle Sosfanze Naturali di Interesse Alimentare e Chimico-Fannaceudco, Via de1 Santuario I-95028 Valverde (CT), Italy
110,
Received 18 February 1994; accepted 22 June 1994
Abstract The overall ‘0, quenching rate constants k, for three meroditerpenoids from Mediterranean marine algae of the genus were measured using 1,4_dimethylnaphthalene 1,4-endoperoxide as a thermal source of IO2 (‘A,). The most active of the compounds tested had a k, value which is comparable with that of cu-tocopherol. These metabolites also act as inhibitors of methyl linoleate peroxidation, while their O;- quenching activity is rather low, similar to cY-tocopherol. Cystoseiru
Keywords: Singlet oxygen; ‘Oz quenching rate constants; Oz.- quenching rate constants; Dye-sensitized photochemical oxidation; Peroxidation inhibition; Algal metabolites; Qwtoseira
1. Introduction
In recent years, a number of investigations on the effect of light, singlet oxygen and active free radicals (O;-, OH’ and lipid peroxyl radicals LOO’) on biological systems have appeared [l-7]. The results have shown that radical reactions are detrimental to health, being involved in the aging process as well as in pathogenesis of a number of human disease states, namely atherosclerosis, rheumatoid arthritis, muscular dystrophy, cataracts, some neurological disorders and possibly cancer [1,2,8-lo]. Cell membranes, DNA and proteins are the main target molecules of free radicals and singlet oxygen. Membranes are chiefly affected by peroxyl radicals that attack polyunsaturated phospholipids at the bisallylic methylenes thus compromising their integrity [2,11]. Radical reactions modify the purine and pyrimidine bases of DNA [12], whereas proteins are oxidized at the metal binding sites of amino acid residues leading, in the case of some amino acids, to loss of ammonia and the formation of carbonyl derivatives [12]. Lipid radical peroxidation, yielding evilsmelling substances as end products, is the main cause of the qualitative decay of foods rich in fatty acids [13]. -
*Corresponding
authors.
loll-1344/94/$07.00 0 1994 Elsevier Science S.A. All rights reserved SSDI 1011-1344(94)07038-P
Many natural or synthetic compounds act as antioxidants by trapping singlet oxygen or radicals, thereby terminating chains of reactions [11,14,15]. Phenols are particularly effective antioxidants for polyunsaturated fatty acids because they can easily transfer a hydrogen atom to lipid peroxyl radicals (reaction (1)) [16,17]. The aryloxyl radical, formed in reaction (l), is usually too sluggish to act as a chain carrier [l&18], but can easily couple with another radical to give non-radical products (reactions (2) and (3)). LOO‘+ ArOH Are’+ LOO’ ArO’ + ArO’ -
LOOH -t-AI-O
(I)
non-radical products
(2)
non-radical products
(3)
The mechanism of singlet oxygen quenching by phenols probably involves, in polar solvents, an electron transfer from phenol to singlet oxygen resulting in the formation of a radical ion pair [19]; the same mechanism has also been observed with aromatic amines [20] and thioanisoles [21]. This process is reversible and the back electron transfer leads to the starting phenol and triplet oxygen [l]. However, the radical ion pair can also react and, consequently, the overall quenching rate constant k, for a ‘0, scavenger is the sum of the physical quenching (k,) and the chemical reaction (k,) rate constants, k, = k, + k,.
160
M. Foti et al. I J. Photo&m.
Photobiol.
B: Biol. 26 (1994) 159-164
One of the most studied natural antioxidants is (Ytocopherol, the most active of the four tocopherols which make up vitamin E and the major lipid-soluble radical-trapping antioxidant present in human blood [ll]. cY-Tocopherol is an efficient ‘0, scavenger with a predominant physical component of the quenching rate constant [22], although many oxidation products are formed during its interaction with ‘0, [23,24]. The observation that many metabolites, previously isolated form Mediterranean seaweeds belonging to the genus Cystoseiru [25], have structures reminiscent of those of tocopherols prompted us to plan a screening of their scavenger properties against singlet oxygen, superoxide anion and peroxyl radicals, with the aim of identifying effective and safe natural antioxidants of potential value in the food industry. In the present paper, we describe the results obtained with three Cystoseira compounds l-3 [26-281.
bCH,
2. Materials and methods 2.1. General Metabolites l-3 were available from previous work. 1,4_Dimethylnaphthalene 1,4-endoperoxideqEP, 5) was prepared from 1,4_dimethylnaphthalene (DMN) according to the procedure described in Ref. 1291. A xanthine oxidase suspension is commercially available (Grade I, Sigma). All chemicals were of analytical grade and were used without further purification. UV measurements were carried out on a Beckman DU-65 spectrophotometer. High-resolution mass spectrometry (HRMS) was determined at 70 eV on a Kratos MS50s instrument. IR and nuclear magnetic resonance (NMR) spectra were recorded on Perkin-Elmer FTIR 1720 X and Bruker AC 250 instruments respectively. Preparative (PLC) and thin layer chromatography (TLC) were performed on precoated silica gel plates (Merck).
2.2. Determination of ‘0,
quenching rate constants
The procedure described followed. EP was employed by thermal decomposition, zofuran (DPBF) as standard reaction sequence underlies EP -
DMN +‘02
‘0,s
decay
DPBF + ‘02 z
by Saito et al. [20] was to generate singlet oxygen with 2,5-diphenyl-3,Cbencompound. The following this method
reaction
Q+‘O,*
Q+30,
Q + lo,*
reaction
where Q is any quencher. The quenching rate constants were obtained by the method of Young et al. [30]. Solutions of DPBF (lo-’ M), EP (1O-3 M) and varying amounts of scavenger (l-3, O-12 mM) in the solvent of choice were kept at 25 “C. The progress of the reaction was monitored by measuring the decrease in the absorbance maximum in the UV spectrum of DPBF (411 nm, in methanol). The slopes of the pseudofirst-order curves within the first 10 min of reaction in the absence (I$) and presence (Vo) of scavenger are related to the rate constant k, through the equation 1311 K/v,
= I + k,[Q]I(k,,[DPBF]
+ kd)
M. Foti et al, /J.
Photochem.
Photobiol.
Thus k, is calculated from the slope of the quenching plot, V,/Y, vs. [Q], using the values of ‘0, lifetimes, l/k,, reported in the literature [32] (10.25 ps in methanol, 83 ps ‘in acetonitrile, 30 ps in benzene and 769 ps in carbon tetrachloride) and the values of k, in the different solvents [33] (1.03 X lo9 M-l s-l in methanol, 1.36 x lo9 M-l s-’ in acetonitrile, 6.90 X 10’ M-l s-’ in benzene and 2.84~10~ M-’ s-’ in carbon tetrachloride). 2.3. Determination of O,‘- quenching rate constants Xanthine (5 x 10e5 M), cytochrome c (6.4~ 10T5 M) and various amounts of scavengers (O-200 mM) were dissolved in a buffer solution (NaH,PO, (low3 M), adjusted at pH 7.4 with 2 N NaOH) containing 15% propanol. Each solution (3 ml) was placed in a thermostatically controlled UV cell (25 “C) and 6 ~1 of xanthine oxidase solution (obtained by 1:3 dilution of the stock solution with sodium phosphate buffer) was added. The progress of the reaction was followed by monitoring the absorbance at 550 nm. The increase in absorbance per minute (AOD min-l) in the described experimental conditions was 0.020 min-’ in the absence of scavenger. Assuming that O;- is consumed only by cytochrome c and scavenger, the quenching constant of the scavenger, kinh, is calculated from the equation AOD,IAODo = I+ ki,,[Q]/kq,[cyt] where the subscripts 0 and Q refer to the absence and presence respectively of the scavenger, k,, is the rate constant of cytochrome c reduction by O,‘- (lo6 M-’ s-l) and [Q] and [cyt] are the concentrations of scavenger and cytochrome respectively. 2.4. Peroxidation of trans-pcarotene Methyl linoleate (4 X lo-* M), trans-p-carotene (6 x 10m6M), 2,2’-azobis(2-methylpropionitrile) (AIBN, 3 x 10e4 M) and various amounts of scavenger (O-O.25 mM) were dissolved in cyclohexane saturated, at room temperature, with methanol and 0,. Each solution, protected against light, was kept at 50 “C in a thermal bath and the rate of disappearance of p-carotene was followed by monitoring the decrease in absorbance at 450 nm at 15 min intervals.
B: Biol. 26 (1994) 159-164
161
run was carried out with 20 mg and an irradiation period of 3 h. PLC of the reaction mixture, using dichloromethane-ether (4:l) as eluant gave, with 7 mg of 1, 8 mg of 4 as a yellow oil (yield 61% based on the starting material converted). The oxidation product has the following physical properties: HRMS [M’] 440.2566 (calculated for C&H,,O, 440.2562); IR timax) (film) (cm-l) 3320, 1680, 1660, 1640, 1600; ‘H NMR (CDCl,) 6 6.57 (lH, m, H-5’), 6.51 (lH, m, H-3’), 5.23 (lH, t, J=7 Hz, H-2). 3.97 (lH, m, H-14), 3.19 (2H, d, 5=7 Hz, H-l), 3.03 (lH, S, H-6), 2.98 (2H, S, H-4), 2.97 (lH, m, H-13a), 2.06 (3H, d, J=2 Hz, 6’-Me), 1.95 (lH, m, H-13b), 1.15 (3H, s, H-17), 1.05 (3H, s, H-16), 0.94 (3H, s, H-19), 0.91 (3H, s, H-18), 1.65 (3H, s, H-20).
3. Results and discussion The total absolute second-order rate constants of singlet oxygen (‘A,) quenching for the algal metabolites l-3 were measured in different solvents. Figs. l-4 illustrate typical quenching plots in the different solvents, and Table 1 shows the overall quenching rate constants of ‘0,. It is readily apparent that the quenching process is strongly affected by the solvent. For instance, the value of k, for compound 1 ranges from 1.6X lo6 M-’ S -' in carbon tetrachloride to 2.0~ 10’ M-’ s-’ in methanol, with a 125-fold increase in the polar solvent. This remarkable solvent effect suggests the involvement of a charge transfer transition state in the quenching mechanism of singlet oxygen [1,19-211. Compound 3, on irradiation in the presence of 0, and a sensitizer (rose bengal or methylene blue), gives only traces of decomposition products and therefore we suggest that the value of k, represents essentially the physical quenching rate constant. In contrast, for the same experimental conditions, 2 and 1 suffer ex-
5-o I
1
2.5. Dye-sensitized photochemical oxidation of l-3 A methanol solution containing the algal metabolite (2x 10e3 M) and a ‘0, sensitizer (methylene blue or rose bengal, lop5 M) was irradiated with a 100 W tungsten lamp at room temperature with oxygen bubbling. At regular time intervals an aliquot was taken and examined by TLC. The results were essentially the same with either photosensitizer. In the case of 1, a
0
3
6
concentration
9
12
(mM)
Fig. 1. Variation in the ratio V,,IV, as a function of quencher concentration in CH,OH at 25 “C: Compound 1 (+), compound 2 (A) and compound 3 (0).
162
M. Foti et al. I J. Photochem.
Photobiol.
B: Biol. 26 (1994) 159-164
Table 1 ‘02 quenching rate constants (ko) of compounds 1-3 in different solvents at 25 “C?
10.7
Compound
Solvent
kQxlO-’ (M-’
? Y
7.5
4.3
1.0 0
3
6
concentration
9
12
(mM)
Fig. 2. Variation in the ratio V,lVo as a function of quencher concentration CH&N at 25 “C: compound 1 (+), compound 2 (A) and compound 3 (0).
2.9
2.3
1.6
1.0 0
3
6
concentration
9
12
(mM)
Fig. 3. Variation in the ratio V,/Vo as a function of quencher concentration in CbH6 at 25 “C: compound 1 (+), compound 2 (A) and compound 3 (0).
3.5
2.9 Y ;_
1 1 1 1
Methanol Acetonitrile Benzene Carbon tetrachloride
2 2 2 2
Methanol Acetonitrile Benzene Carbon tetrachloride
1.4kO.2 1.03*0.04 0.47 f 0.02 0.089 f 0.003
3 3 3 3
Methanol Acetonitrile Benzene Carbon tetrachloride
0.99 * 0.03 0.51 f 0.04 0.024 f 0.002 0.065 f 0.009
a-Tocopherol
Methanol
20*2 19.4 f 0.4 0.96 k 0.04 0.16 f 0.02
24kl
“The rate constants are the average of four measurements. Errors are given as f lo.
3.5
? PO
s-‘)
2.3
1.6
1.0 0
3
9
6
concentration
12
(mM)
Fig. 4. Variation in the ratio V,/Vo as a function of quencher concentration in CCL, at 25 “C: compound 1 (+), compound 2 (A) and compound 3 (0).
tensive oxidation; the former gives an intractable mixture, but the main oxidation product of the latter was isolated and characterized as 4 based on its spectral properties. HRMS indicated the elemental composition C,,H,,O, revealing the loss of a carbon atom (and four hydrogens) from the parent compound, while the presence in the molecule of a quinone moiety was evident from IR absorptions at 1660 and 1640 cm-l. The ‘H NMR spectrum showed, in comparison with that of 1, the following main differences: (a) disappearance of the signal for the methoxyl group; (b) replacement of the AB system due to the aromatic protons at 3’ and 5’ with two multiplets at 6.57 and 6.51 ppm; (c) modification of the position and multiplicity of the signals due to the protons of the methyl group on the ring at position 6’ and the methylene at position 1. The different behaviour of the three compounds in the dye-sensitized photochemical oxidation suggests that the chemical component (k,) of the observed rate constants is greater for compounds 1 and 2, which have a free phenolic OH, than compound 3. The ability of metabolites l-3 to quench lipid peroxyl radicals has also been investigated. Compounds 1 and 2 act as efficient scavengers, being able to inhibit the peroxidation of p-carotene mediated by methyl linoleate peroxyl radicals in the presence of 0, and a radical initiator. The oxidation percentages of p-carotene after 30 and 75 min of reaction, presented in Table 2, prove that the antioxidant activity of 1 and 2 is nearly half that of a-tocopherol, while 3 lacks any effect. The quenching activity of compounds 1 and 2 towards superoxide anion, generated by the system xanthinexanthine oxidase, is rather low (the value of the quenching rate constant is around 10 M-’ s-l), whereas 3 is
M Foti ei al. I .I. Photochem. Table 2 Percentages of oxidation of p-carotene presence and absence of scavengers”
times
75 min
Blank
4.5
19.1
Compound
c=1.6~10’+
M
1.9 0.2
5.8 1.0
Compound 2 c=3.OxlO+ c=2.5~10-~
M M
2.3 0.3
7.1 1.1
NA
3
c+Tocopherol c=2.9~10-~ c=l.S~lO-~
M M
‘Experiments with methanol.
were performed NA, not active.
163
in the
30 min
Compound
8: Biol. 26 (1994) 159-164
Acknowledgements at different
Sample
1 c=2.9x10-5 M
Photobiol.
1.2 0.0 at 50 “C in cyclohexane
NA
2.2 0.2 saturated
completely unreactive. Similarly, the value reported in the literature for the O,‘- quenching rate constant of cu-tocopherol is quite small (6 M-’ s-l) [l]. Finally, we wish to draw attention to the fact that, in the Cystoseiru species, the mean concentration of (Ytocopherol is around 10 mg kg-’ of alga (dry weight) [34], while that of the meroditerpenoids of the type considered in the present paper is much higher; for instance, in C. stricta, the content of 1 exceeds 500 mg kg-’ [26,35]. Therefore, the possibility that these compounds perform a physiological function somehow linked to their antioxidative properties must be considered.
4. Conclusions
The algal meroditerpenoids l-3 are very effective singlet oxygen scavengers, the most active (1) having a ‘0, quenching rate constant similar to that of (Ytocopherol. A large solvent effect has been observed in the values of the rate constants, in agreement with the quenching mechanism, reported in the literature, which initially involves an electron transfer from phenol to singlet oxygen [1,19-211. Unlike 3, which lacks free phenolic OH and is essentially a physical quencher, 1 and 2 suffer extensive degradation in the dye-sensitized photo-oxidation, indicating participation of a chemical process to the overall rate constants of singlet oxygen quenching. Moreover, 1 and 2 have a strong peroxyl radical quenching activity, the former being able to reduce the rate of peroxidation of p-carotene by 60%-70% at a concentration of 2.9~ lo-’ M. Similar to a-tocopherol, the algal metabolites have rather low, if any, O;- quenching activity.
This work was supported by the Consiglio Nazionale delle Ricerche (CNR, Roma) under the scheme “Progetto Strategic0 - Innovazione produttiva nelle piccole e medie imprese” and by a MURST 40% grant. The authors thank Dr. M. Recupero for skilful technical assistance.
References
111 A.
Breccia, M.A.J. Rodgers and G. Semeraro (eds.), Oqgen and Sulfur Radical in Chemistry and Medicine, Oxygen Radicals in Chemise nnd Biology (Chap. l), Edizioni Scientifiche Lo Scarabeo, Bologna, 1986, pp. 20-52, and references cited therein. 121 A.W. Girotti, Photodynamic peroxidation in biological systems, Photochem. Photobiol., 51 (1990) 497-509, and references cited therein. and P. Hochstein, Alterations in erythrocyte t31 C. Rice-Evans membrane fluidity by phenylhydrazine-induced peroxidation of lipids, B&hem. Biophys. Rex Commun., 100 (1981) 1537-1542. K.B. Heller and C.W.M. Haest, Progressive 141 B. Deuticke, oxidative damage in erythrocytes after pulse treatment with tbutylhydroperoxide, B&him. Biophys. Acta, 899 (1987) 113-124. killing of non151 G.J. Kantor and C. Ritter, Sunlight-induced dividing human cells in culture, Photochem. PhotobioZ., 37 (1983) 533-538. and G.C. Goyal, Photosensitized lysis of I61 L.I. Grossweiner liposomes by hematoporphyrin derivative, Photochem. Photobiol., 37 (1983) 529-532. in G. Moreno [71 B.E. Johnson, Skin and related photoreactions, et al. (eds.), Photosensitisation, NATO ASI Series Vol. H 15, Springer-Verlag, Berlin, 1988, pp. 253-264. S. Parthasarathy, T.E. Carew, J.C. Khoo and [81 D. Steinberg, J.L. Witztum, Modifications of low density lipoprotein that increase its atherogenicity, New Engl. J. Med., 320 (1989) 915-924. [91 B. Halliwell, Oxidants and human disease: some new concepts, FASEB. J., I (1987) 358-364. enzymes, and carcinogenesis, PO1 Y. Sun, Free radicals, antioxidant Free Radicals Biol. Med., 8 (1990) 583-599. L.R.C. Barclay, Model biomembranes: quantitative studies of Pll peroxidation, antioxidant action, partitioning, and oxidative stress (1992 Syntex Award Lecture), Can. J. Chem., 71 (1993) l-16, and references cited therein. P21 E.R. Stadtman, Protein oxidation and aging, Science, 257 (1992) 1220-1224, and references cited therein. and N.P. Das, Studies on the control of lipid iI31 L. Ramanathan oxidation in ground fish by some polyphenolic products, J. &ic. Food Chem., 40 (1992) 17-21, and references cited therein. of higher plants, Phytochemistty, 1141 R.A. Larson, The antioxidant 27 (1988) 969-978. 1151 M.S. Taga, E.E. Miller and D.E. Pratt, Chia seeds as a source of natural lipid antioxidants, J. Am. Oil Chem. Sot., 61 (1984) 928-931. G.W. Burton and K.U. Ingold, Autoxidation of biological PI molecules. 1. The antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in vitro, J. Am. Chem. Sot., 103 (1981) 6472-6477. L.J. Devall, B. Tait, B.K. Trivedi, P71 W.A. Pryor, J.A. Cornicehi, D.T. Witiak and M. Wu, A rapid screening test to determine the antioxidant potencies of natural and synthetic antioxidants, J. Org. Chem., 58 (1993) 3521-3532.
164
M. Foti et al. I J. Photochem.
Photobiol.
V.W. Bowry and R. Stocker, Tocopherol-mediated peroxidation. The prooxidant effect of .vitamin E on the radical-initiated oxidation of human low-density lipoprotein, J. Am. Chem. Sot., 115 (1993) 6029-6044. K. Mukai, K. Daifuku, K. Okabe, T. Tanigaki and K. Inoue, PI Struchire-activity relationship in the quenching reaction of singlet oxygen by tocopherol (Vitamin E) derivatives and related phenols. Finding of linear correlation between the rates of quenching of singlet oxygen and scavenging of peroxyl and phenoxyl radicals in solution,J. Org. Chem., 56 (1991) 41884192, and references cited therein. PO1 I. Saito, T. Matsuura and K. Inoue, Formation of superoxide ion via one-electron transfer from electron donors to singlet oxygen, J. Am. Chem. Sot., 105 (1983) 3200-3206, and references cited therein. of single WI K. Inoue, T. Matsuura and I. Saito, Importance electron-transfer in singlet oxygen reaction in aqueous solution. Oxidation of electron-rich thioanisoles, Tetrahedron, 41 (1985) 2177-2181. F.H. Doleiden, A.M. Trozzolo and A.A. ml S.R. Fahrenholtz, Iamola, On the quenching of singlet oxygen by cr-tocopherol, Photochem. PhotobioZ., 20 (1974) 505-509. photov31 M. d’Ischia, C. Costantini and G. Prota, Dye-sensitized oxidation of vitamin E revisited. New 7-oxaspiro[4,5]dec-l-ene3,6-dione products by oxygenation and ring contraction of (Ytocopherol, J. Am. Chem. Sot., 113 (1991) 8353-8356. G. Misuraca and v41 C. Costantini, M. d’Ischia, A. Napolitano, G. Prota, Photodynamic degradation of vitamin E induced by psoralens, Biochim. Biophys. Acta, 1116 (1992) 291. WI M. Piattelli, Chemistry and taxonomy of Sicilian Cystoseim species, New .I. Chem., 14 (1990) 777-782.
WI
B: Biol. 26 (1994) 159-164
WI
v71
PI P91 1301
1311
[321
I331
[341 [351
V. Amico, F. Cunsolo, M. Piattelli, G. Ruberto and F.R. Fronczek, Balearone: a metabolite of the brown alga Cystoseira baleatica, Tetrahedron, 40 (1984) 1721-1725. V. Amico, F. Cunsolo, M. Piattelli, G. Ruberto and L. Mayol, Strictaketal: a new tetraprenyltoluquinol with a heterotetracyclic diterpene moiety from the brown alga Cystoseira shicta, J. Nat. Prod., 50 (1987) 449-454. V. Amico, F. Cunsolo, M. Piattelli and G. Ruberto, Five novel tetraprenylhydroquinols from the brown alga Cystoseira a/geriensis, PhytochemisQ, 23 (1984) 2017-2020. H.H. Wasserman and D.L. Larsen, Formation of 1,4-endoperoxide from the dye-sensitized photo-oxygenation of alkylnaphthalenes,J. Chem. Sot., Chem. Commun., 5 (1972) 253-254. R.H. Young, K. Wehrly and R.L. Martin, Solvent effect in dye-sensitized photooxidation reactions, J. Am. Chem. Sot., 93 (1971) 5774-5779. C.S. Foote, T.-Y. Ching and G.G. Geller, Chemistry of singlet oxygen, XVIII. Rates of reaction and quenching of ru-tocopherol and singlet oxygen, Photochem. Photobiol., 20 (1974) 511-513. S.L. Studer, W.E. Brewer, M.L. Martinez and P.-T. Chou, Time-resolved study of the photooxygenation of 3-hydroxyflavone, J. Am. Chem. Sot., 111 (1989) 7643-7644. F. Wilkinson and J.G. Brummer, Rate constants for the decay and reactions of the lowest electronically excited singlet state of molecular oxygen in solution, J. Phys. Chem., Ref Data, 10 (1981) 809-999. M. Piattelli, G. Ruberto and V. Amico, unpublished results, 1992. V. Amico, P. Neri, M. Piattelli and G. Ruberto, Linear diterpenoids from Cystoseira balerica, Phytochemirtry, 26 (1987) 2637-2639.