The introduction of pin-up oxygen on C60, such as in the oxidized fullerenes C60O and C60On, induced noticeable increase in the antioxidant activity as compared to pristine C60. The water-soluble inclusion complexes of fullerenes C60O and C60Onreacted with linoleic acid peroxyl radical 1.7 and 2.4 times faster, respectively.
KeywordsFullerene C60 Oxidized fullerene C60O Antioxidant γ-Cyclodextrin PVP
Fullerenes and its derivatives are well known as a new class of antioxidants and they have attracted considerable attention in biologic applications due to their high reactivity toward radicals , especially reactive oxygen species (ROS) such as superoxide , hydroxyl radical , peroxyl radicals , and nitric oxide . These harmful radicals attack lipids, proteins, DNA, and other biologic tissues and organs. It has been found that water-soluble fullerenes can be used as potential antioxidants and neuroprotective drugs against degenerative diseases related to oxidative stress [6–11]. Thus, water-soluble fullerenes, including host–guest inclusion complexes, are promising candidates for practical use as antioxidants. However, such a radical scavenging ability has not been well investigated systematically for functionalized fullerenes, and the development of more efficient and easily accessible fullerene antioxidant derivatives has become an urgent requirement.
Materials and Apparatuses
Fullerene C60and oxidized fullerene C60Onwere purchased from Frontier Carbon Corporation. Polyvinylpirrolidone (PVP K 30) was purchased from Wako Pure Chemical Industries, Ltd. Other reagents and organic solvents as well as pure water were all commercially available and used as received. UV-visible spectra were measured on a JASCO V-550 equipped with a thermal controller. LCMS analysis was performed on a SHIMADZU LCMS-2010EV. Ball mill grinding for the preparation ofγ-cyclodextrin inclusion complexes was carried out using a FRITSCH pulverisette 6. DFT calculation of molecular orbital energy levels were performed using Spartan ‘04 software at B3LYP/6-31G* level of theory.
Synthesis of PVP/C60and its Oxidized Derivatives
A toluene solution (10 mL) of fullerene C60(8 mg) was added to an ethanol solution (5 mL) of PVP (1 g) and stirred for 12 h at room temperature under air. After evaporation of the solvent, drying of the residue under vacuum at room temperature for 18 h gave PVP/C60quantitatively (1 g) as a brown solid.
Synthesis ofγ-CD/C60and C60O
Fullerene C60 (10 mg) and γ-cyclodextrin (70 mg) in an agate vessel (50 mL) together with a mixing ball made of zirconia (0.3 g × 30) were vigorously mixed by using ball mill at a rate of 650 rpm for 30 min. The milling was repeated by addition of ethanol (5 mL) for 30 min. After drying the ethanol, pure water (5 mL) was added and mixed again for 30 min. The mixture was centrifuged and the obtained solution was filtered through a membrane filter (0.45 and 0.1 μm) to give a clear purple solution. The concentration of solution and the yield were estimated to be 1.40 mM and 31.7%, respectively, by the use of the molar absorption coefficient ε = 5.06 × 104 M−1 cm−1 determined at λmax 329 nm for the cyclohexane solution according to the previously reported method b]. The concentration and the yield for C60O were 682 μM and 25.1%, respectively (ε = 3.25 × 104 M−1 cm−1 at λmax 322 nm in cyclohexane).
β-Caroten Bleaching Method
Chloroform solutions of 11 μL ofβ-carotene (1.0 mg/mL), 4.4 μL of linoleic acid (0.1 g/mL) and 22 μL of Tween 40 (0.2 g/mL) were mixed in a quartz cell equipped with a screw-on cap, and then the solvent was removed in vacuo. An aliquot of the emulsion was immediately diluted with 2.4 mL of phosphate buffer solution (0.018 M, pH 7.0), and 0.1 mL of antioxidant (7.5–75 nmol, equivalent to C60) in deionized water was added to the diluted mixture. The solution was mixed well and heated at 50°C under air in a quartz cell on a UV spectrometer in order to monitor the decrease in the absorbance ofβ-carotene at 460 nm.
Results and Discussion
In conclusion, we have found a meaningful key in developing new applicable antioxidants using fullerenes by means of a simple and conventional technique that can enhance their antioxidant activity by simply introducing pin-up oxygen on the fullerene cage.
1The C60On, instead of C60O2due to the difficulty in availability, was used to investigate the effect of the number of pin-up oxygen on C60as well as the scope for the practical use. The component ratio of C60Onwas determined by LCMS (mass spectra and peak area) as follows: C60, 22; C60O, 33; C60O2, 27; C60O3, 14; C60O4, 5%.
2Calculated HOMO levels of C60, C60O, and C60O2(e) are −5.99, −5.95, and −5.99 eV, respectively, and less related to the present observation.
The authors thank Dr. Y. Tajima (RIKEN, FLOX Corp.) for generous gift of C60O.