Vol. 52 No. 4/2005, 903–907

We recently reported that kinobeon A, produced from safflower cells, suppressed the free radical-induced damage of cell and microsomal membranes. In the present study, we investigated whether kinobeon A quenches singlet oxygen, another important active oxygen species. Kinobeon A inhibited the singlet oxygen-induced oxidation of squalene. The second-order rate constant between singlet oxygen and kinobeon A was 1.15 x 10(10) M(-1)s(-1) in methanol containing 10% dimethyl sulfoxide at 37 degrees C. Those of alpha-tocopherol and beta-carotene, which are known potent singlet oxygen quenchers, were 4.45 x 10(8) M(-1)s(-1) and 1.26 x 10(10) M(-1)s(-1), respectively. When kinobeon A was incubated with a thermolytic singlet oxygen generator, its concentration decreased. However, this change was extremely small compared to the amount of singlet oxygen formed and the inhibitory effect of kinobeon A on squalene oxidation by singlet oxygen. In conclusion, kinobeon A was a strong singlet oxygen quencher. It reacted chemically with singlet oxygen, but it was physical quenching that was mainly responsible for the elimination of singlet oxygen by kinobeon A. Kinobeon A is expected to have a preventive effect on singlet oxygen-related diseases of the skin or eyes.

Active oxygen species, such as free radicals and singlet oxygen ( 1 O 2 ), induce cellular injury via the accumulation of oxidative damage to DNA, lipids, and protein, and/or by the induction of uncontrolled signal transduction (Halliwell & Gu�eridge, 1999;Klotz et al., 2000).Therefore, oxidative stress is suggested to be a cause of various diseases (Halliwell & Gu�eridge, 1999).On the other hand, antioxidants, such as α-tocopherol, ascorbic acid and ubiquinol, protect biological systems from oxidative stress (Halliwell & Gu�eridge, 1999).We recently found a new antioxidant, kinobeon A (Kanehira et al., 2003).Kinobeon A is a unique red compound produced from safflower (Carthamus tinctorius L.) cells cultured under specific conditions (Wakayama et al., 1994) and has not been found in natural saf-flowers, other plants, animals or microorganisms.Safflower is a valuable plant used as an edible fat, as a Chinese medicine, in cosmetics, and in foodstuffs as a colorant.Kinobeon A inhibited the oxidation of rat liver microsomal membrane induced by the Fe 2+ -ADP/NADPH system, protected bovine kidney cell cultures from oxidative stress (hydrogen peroxide, tert-butyl hydroperoxide), and scavenged the superoxide anion produced in the hypoxanthine/xanthine oxidase system (Kanehira et al., 2003).However, the quenching of 1 O 2 by kinobeon A is still not well established.In vivo, 1 O 2 is produced by exposure to sunlight and from neutrophils and eosinophils.Neutrophils use 1 O 2 when they kill bacteria to protect biological systems (Nakano et al., 1998;Tatsuzawa et al., 1998;1999;2000 al., 2003).On the contrast, 1 O 2 is also considered to be a causative factor of various skin diseases and eye diseases (Halliwell & Gu�eridge, 1999).Therefore, the search for a novel effective 1 O 2 quencher is important. 1O 2 is consumed by an antioxidant via a chemical addition reaction and/or physical quenching, such as electron transfer.Kinobeon A has many double bonds, which may be able to react with 1 O 2 .
In the present study, we investigated the potential of kinobeon A as 1 O 2 quencher.
Effect of kinobeon A, α-tocopherol or β-carotene on 1 O 2 -induced squalene oxidation.Squalene was purified by HPLC using a CAPCELLPAK C18 column (20 × 250 mm, 5 µm, Shiseido Co. Ltd, Tokyo, Japan) and methanol as the mobile phase (flow rate: 10 ml/min), as reported previously (Nakano et al., 1998).Purified squalene was dissolved in chloroform and stored at -80°C prior to use.Kinobeon A was dissolved in dimethyl sulfoxide.The concentration of kinobeon A was calculated by using its molar absorption coefficient at 520 nm (1.95 × 10 5 M -1 cm -1 ; our data).α-Tocopherol and β-carotene were dissolved in methanol.The concentration of NEPO was calculated as previously reported (Nakano et al., 1998).A�er the solvent was removed from the chloroform solution of squalene under reduced pressure, methanol was added.The reaction was started by the addition of a NEPO solution (final concentration: 100 µM) to the mixture containing 1 mM squalene and 1-100 µM kinobeon A, 10-1000 µM α-tocopherol or 1-13 µM β-carotene in methanol/dimethyl sulfoxide (9 : 1, v/v; total volume: 1.0 ml) at 37°C.An aliquot of the reaction mixture (50 µl) was removed every 30 min for 90 min and injected into the HPLC using a CAPCELLPAK C18 (4.6 × 250 mm, 5 µm, Shiseido Co., Ltd, Tokyo, Japan) as an analytical column and methanol as the mobile phase (flow rate: 2 ml/min).Squalene hydroperoxide was detected in a hydroperoxide-specific assay using the chemiluminescence of isoluminol (Yamamoto et al., 1987;Nakano et al., 1998).
Reaction between kinobeon A and 1 O 2 or AMVN-derived radical.Kinobeon A (10 µM) was incubated with 1 mM NEPO in methanol containing 10% dimethyl sulfoxide at 37°C.AMVN (1 mM) was also used instead of NEPO to compare the 1 O 2induced oxidation with the radical-induced oxidation of kinobeon A. An aliquot of the reaction mixture (20 µl) was removed every 15 min for 75 min and injected into the HPLC system.A CAPCELL-PAK C18 (4.6 × 250 mm, 5 µm) and methanol/water (3 : 2, v/v) were used as a column and the mobile phase, respectively.The flow rate was 1.0 ml/min.Kinobeon A was detected at 520 nm.

Kinetic analysis of NEPO thermolysis in methanol containing 10% dimethyl sulfoxide at 37°C
NEPO, a thermolytic 1 O 2 generator, was used in the present study.The kinetics of the decomposition of NEPO in methanol containing 10% dimethyl sulfoxide at 37°C was examined first, as described previously (Nakano et al., 1998), to clarify the total amount of 1 O 2 formed by the thermal decomposition of NEPO in each experiment.The wavelength of the maximum absorbance (λ max ), molar absorption coefficient at λ max of 3-(4'-methyl-1'-naphthyl)-propionic acid (a molecule remained a�er 1 O 2 was produced from NEPO) and the first order rate constant of NEPO thermolysis were obtained as 287.4 ± 0.6 nm, 7800 ± 70 M -1 cm -1 , and (1.91 ± 0.09) × 10 -4 s -1 (mean ± S.D., n = 3), respectively.Since the thermolysis of NEPO is a first order reaction, the total amount of 1 O 2 formed can be calculated using eqn. 1.
where t (in seconds) stands for reaction time.

Suppression of 1 O 2 -induced squalene oxidation by kinobeon A
The effect of kinobeon A on the 1 O 2 -induced oxidation of squalene in organic solvent was investigated.Squalene was used since it is one of the most vulnerable lipids to 1 O 2 (Nakano et al., 1998).Kinobeon A inhibited the 1 O 2 -induced oxidation of squalene dose-dependently (Fig. 1A).α-Tocopherol and β-carotene, known as strong 1 O 2 quenchers (Di Mascio et al., 1989;Kaiser et al., 1990;Tatsuzawa et al., 2000), were used to verify the quenching abilities of kinobeon A. They also inhibited the 1 O 2 -induced oxidation of squalene in a dose-dependent manner (Fig. 1B and C).The half inhibitory concentrations (IC 50 s) of α-tocopherol and β-carotene to 1 O 2 -induced oxidation of squalene in the present experimental system were 316 µM and 13 µM, respectively (Table 1).That of kinobeon A was 15 µM (Table 1).Thus, the IC 50 of kinobeon A was similar to that of β-carotene and much smaller than that of α-tocopherol.Judging from these results, kinobeon A can act as a potent 1 O 2 quencher.

Kinetic analysis of reaction between each quencher and 1 O 2
Moreover, second-order rate constants between 1 O 2 and each quencher in methanol involving 10% dimethyl sulfoxide were roughly calculated using eqn. 2 (Young et al., 1971;Kohno et al., 1995).
where S 0 and S Q represent slopes of the formation of squalene hydroperoxide plo�ed as a function of time in the absence and presence of each quencher, respectively.k d denotes the first-order rate constant of 1 O 2 decay.In the present study, 1.8 × 10 5 s -1 (k d in methanol) (Young et al., 1971) was used as the k d for the calculation of second-order rate constant, since k d is slightly lower in dimethyl sulfoxide (3.3-5.2 × 10 4 s -1 ) than in methanol (0.9-2.0 × 10 5 s -1 ) (Bellus, 1978).
[Q] and k Q represent the initial concentration of each quencher and second-order rate constant of the reaction between 1 O 2 and the quencher involving physical quenching and a chemical reaction, respectively.k Q values in the present study were shown in Table 1.Reported k Q values of α-tocopherol and β-carotene were 2.5 × 10 8 M -1 s -1 in n-butanol at 35°C   Y. Kambayashi and others (Kohno et al., 1995) and 1.4 × 10 10 M -1 s -1 in ethanol/ chloroform/water (50 : 50 : 1, by vol.) (Di Mascio et al., 1989), respectively.These values were comparable to those obtained in the present study, although a different kind of organic solvent was used.These data also showed the high potential of kinobeon A as an 1 O 2 quencher.

Reaction of kinobeon A with 1 O 2
The change in the concentration of kinobeon A was followed using HPLC to elucidate the mechanism by which kinobeon A eliminates 1 O 2 (Fig. 2A).The concentration of kinobeon A (retention time: 5.3 min) decreased slightly during the incubation of 10 µM kinobeon A with 1 mM NEPO in methanol containing 10% dimethyl sulfoxide at 37°C.This decrease was slightly greater than that in the control system without NEPO.The new peak, however, did not appear on the chromatograms.The concentration of kinobeon A was also decreased by 1 mM AMVN, known as an oil-soluble radical initiator (Niki, 1990), in the same solvent at 37°C.In this case, an unknown peak (Fig. 2A; retention time: 9 min) was observed and its area slightly increased time-dependently (Fig. 2C).It might be an oxidation product of kinobeon A. The reaction between kinobeon A (10 µM) and 1 O 2 (5 mM NEPO) was also examined spectrophotometrically.Only a micromolar amount of kinobeon A was lost and new absorbance did not appear, although 2.49 mM 1 O 2 had formed for 60 min at 37°C (not shown).

DISCUSSION
In the present study, we showed the potential of kinobeon A as an 1 O 2 quencher.The total amount of 1 O 2 formed for 90 min was 64.3 µM (value obtained by calculation using eqn. 1) when 1 mM squalene was incubated with 100 µM NEPO in methanol containing 10% dimethyl sulfoxide at 37°C.On the other hand, 1.02 ± 0.28 µM (mean ±S.D., n = 9) of squalene hydroperoxide was formed in 90 min.Approximately 1.59% of the 1 O 2 formed contributed to the oxidation of squalene.Thus, the decay of 1 O 2 was significantly faster than the rate of oxidation product formation from squalene by 1 O 2 .It was reported that the rate constants of 1 O 2 decay were 0.9-2.0 × 10 5 s -1 and 3.3-5.2× 10 4 s -1 in methanol and dimethyl sulfoxide, respectively (Bellus, 1978).The k Q of squalene was reported to be 2.66 × 10 6 M -1 s -1 in n-butanol at 35°C (Kohno et al., 1995).Since 1 mM squalene was used in the present study, the k Q [Q] was 2.66 × 10 3 s -1 .These values were consistent with the present results, although different solvents were used in each system.The total amount of 1 O 2 formed from 1 mM NEPO in 75 min in this experimental system was 577 µM.The loss of kinobeon A within 75 min in this system was 0.99 µM.If 1 O 2 reacts with kinobeon A at a ratio of 1 : 1, 0.17% of the 1 O 2 formed was consumed by kinobeon A. This value was extremely small in comparison with the inhibitory efficiency of kinobeon A on the 1 O 2 -induced oxidation of squalene, even if most 1 O 2 was lost via decay (quenching by organic solvent).Therefore, most of the 1 O 2 consumed by kinobeon A would be physically quenched without a chemical reaction under the present conditions.
On the other hand, a 46.1% decrease in kinobeon A was observed in methanol containing 10% dimethyl sulfoxide during the incubation of 10 µM kinobeon A with 1 mM AMVN for 75 min at 37°C.The initial rate of peroxyl radical formation from AMVN was calculated as follows; R i = 2ek d [AMVN]; the initial rate of the thermal decomposition of AMVN was estimated to be approximately linear, since AMVN has a long half-life (2.8 days, calculated from reference (Niki et al., 1986)).R i , e and k d stand for the initial rate of peroxyl radical formation by AMVN thermolysis, the efficiency of free radical generation from AMVN and the rate constant of decomposition of AMVN, respectively.The ek d in benzene at 37°C is 2 × 10 -6 s -1 (Niki et al., 1986).Therefore, R i is calculated from the equation; 4 × 10 -6 × [AMVN] (Ms -1 ).Eighteen micromolar peroxyl radical is formed from 1 mM AMVN in benzene for 75 min at 37°C.This value would not be significantly different from that obtained in methanol containing 10% dimethyl sulfoxide.The total amount of peroxyl radicals formed in the AMVN system would be much smaller than that of 1 O 2 formed in the NEPO system, even if the kinetic chain length was about 10.On the basis of the above result, kinobeon A can trap (peroxyl) radicals efficiently.This is consistent with the finding that kinobeon A inhibited the oxidative damage to rat liver microsomal membrane induced by Fe 2+ -ADP/NADPH (Kanehira et al., 2003).These results show that kinobeon A chemically reacts efficiently with the peroxyl radical, but only slightly with 1 O 2 .
The present study shows that kinobeon A can react chemically with 1 O 2 , but mainly quenches

Figure 1 .
Figure 1.Inhibition of 1 O 2 -induced squalene oxidation by each quencher.(A) Kinobeon A, (B) α-tocopherol, and (C) β-carotene.Squalene (1 mM) was incubated with 100 µM NEPO and an 1 O 2 quencher in methanol containing 10% dimethyl sulfoxide for 90 min at 37°C.The ratio of squalene hydroperoxide formed in the presence of the quencher to that in its absence (control) is shown.Results are expressed as means ± S.D. (n = 3).On some points, error bars are not seen since they are very small.The structure of each quencher is also shown.

Figure 2 .
Figure 2. Reaction of kinobeon A with 1 O 2 or peroxyl radical.(A) Representative chromatogram from the kinobeon A analysis.Kinobeon A (10 µM) was incubated with AMVN (1 mM) in methanol containing 10% dimethyl sulfoxide at 37°C.(B) Change in kinobeon A concentration during the incubation with 1 mM NEPO or 1 mM AMVN in methanol containing 10% dimethyl sulfoxide at 37°C.A control experiment without NEPO or AMVN was also performed.Results are expressed as the ratio to the initial concentration of kinobeon A and as means ± S.D. (n = 3).Squares: control; circles: 1 mM NEPO; triangles: 1 mM AMVN.(C) Change in the unknown peak area during the reaction between kinobeon A and 1 mM AMVN.The initial concentration of kinobeon A was 10 µM.

Scheme 1 .
Scheme 1. Proposed pathway of scavenging of singlet oxygen by kinobeon A.