SpringerOpen Newsletter

Receive periodic news and updates relating to SpringerOpen.

Open Access Nano Express

Effects of Pin-up Oxygen on [60]Fullerene for Enhanced Antioxidant Activity

Kenji Matsubayashi, Tadashi Goto, Kyoko Togaya, Ken Kokubo* and Takumi Oshima

Author Affiliations

Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

For all author emails, please log on.

Nanoscale Research Letters 2008, 3:237-241  doi:10.1007/s11671-008-9142-4


The electronic version of this article is the complete one and can be found online at:


Received:16 May 2008
Accepted:12 June 2008
Published:4 July 2008

© 2008 to the authors

Abstract

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.

Keywords:
Fullerene C60; Oxidized fullerene C60O; Antioxidant; γ-Cyclodextrin; PVP

Introduction

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 [1], especially reactive oxygen species (ROS) such as superoxide [2], hydroxyl radical [3], peroxyl radicals [4], and nitric oxide [5]. 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.

In this article, we first report that the introduction of pin-up oxygen on C60, such as that in the oxidized fullerene (fullerene epoxide) C60On, induces significant increase in the antioxidant activity as compared to pristine C60. The relative radical scavenging rate constant krrs was kinetically determined using a β-carotene bleaching assay in the presence of water-soluble polyvinylpirrolidone (PVP)-entrapped [12] and γ-cyclodextrin (CD)-capped [13] C60 and C60On (n = 1 and 0–4) [14] inclusion complexes (Fig. 1).

thumbnailFigure 1. Plausible structure of water-soluble complexes of [60]fullerene monoepoxide C60O and structure of major isomers of C60O2 (cis-1ande)

Experimental

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 [13]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

The water-soluble fullerene inclusion complexes were synthesized by modified literature method [12]. The formation of γ-CD/C60O has been confirmed only by a mass spectrum [15]. Thus, we confirmed its formation (obtained as a brownish water solution including an excess of free γ-CD) and determined the concentration of solution using a UV-vis spectrometer by comparison of the peak absorbance around 360 nm in water to that of pristine C60O in cyclohexane (Fig. 2a). On the other hand, PVP/C60O and C60On have not been reported so far and this is the first report (Fig. 2b).

thumbnailFigure 2. UV-vis spectra of (a) γ-CD/C60O (blue line) and γ-CD/C60 (green line) and (b) PVP/C60O (blue line) and PVP/C60 (green line) in water (10 μM)

The β-carotene bleaching assay is one of the common methods used in the field of food chemistry for evaluating antioxidant activity. The method is based on the discoloration of the yellowish color of a β-carotene solution due to the breaking of π-conjugation by the addition of lipid peroxyl radical (LOO·) generated from the autoxidation of linoleic acid under air atmosphere [16-18]. The assay was performed according to an optimally modified procedure (Fig. 3) [19].

thumbnailFigure 3. β-Carotene bleaching assay with linoleic peroxyl radical; (a) decay curves of absorbance at 460 nm (Abs460) and (b) plots of ln (Abs0/Abst) versus time in the presence of antioxidants (10 μM), where Abs0 is initial Abs460 and Abst is Abs460 at time t. Vitamin E was used as a positive control

Figure 4 shows the dependency of the pseudo-first-order rate constants, kobs, for the discoloration of β-carotene on the antioxidant concentration of PVP and CD complexes of C60 and oxidized C60O. Here, the rate (Rf) of discoloration of β-carotene by the LOO· radical is given by Eq. 1 [18], where kc and kf denote the second-order rate constants for the radical scavenging of β-carotene and fullerene antioxidant, respectively.

(1)

It was found that the β-carotene bleaching was significantly suppressed by the increasing amount of antioxidants, although C60O was more effective than C60 in all tested ranges of concentration. It was also noted that the entrapped PVP and CD exerted no appreciable effect on the antioxidant activity of guest fullerenes. To the best of our knowledge, this is the first result of the higher antioxidant activity of C60O in comparison with pristine C60, despite the decreasing of π-conjugation. The concentration-dependent antioxidant activities %AOA [19] (= 100 × {kobs of control − kobs of fullerene}/kobs of control) of PVP/C60 and C60O were 50% and 68% in 10 μM for antioxidant, and 73% and 81% in 30 μM, respectively.

thumbnailFigure 4. Effects of antioxidant concentration on the observed pseudo-first-order rate constants kobs of β-carotene bleaching with linoleic acid peroxyl radical at 50°C. Values of kobs were obtained by monitoring the absorbance of β-carotene aqueous solution (8.2 μM) at 460 nm. The dotted horizontal line indicates the value of kobs in the absence of antioxidants as a control

Here, it is more convenient to define the absolute antioxidant activity of fullerenes toward the LOO· radical by considering the relative radical scavenging rate constants krrs (= kf/kc) of fullerenes versus β-carotene, as given in Eq. 2 [18], where R0 is the bleaching rate in the absence of antioxidants ([fullerene] = 0 in Eq. 1).

(2)

As shown in Fig. 5, the plots of the ratio R0/Rf versus the ratio of [fullerene]/β-carotene] gave a good regression line with intercept = 1 for each of the antioxidants, C60, C60O, and a commercially available mixture of fullerene oxide C60On.12 The dotted line indicates the value in the absence of antioxidants as a control (slope = 0). The slopes, krrs = 0.79 (for C60), 1.33 (for C60O), and 1.93 (for C60On), represent the efficiency of the antioxidants and thus C60O and C60On react with the LOO· radical approximately 1.7 and 2.4 times faster than C60. There is a clear tendency that the introduction of pin-up oxygen on C60 increases its antioxidant activity.

thumbnailFigure 5. Plots of the ratio of β-carotene bleaching rates in the presence (Rf) or absence (R0) of fullerene antioxidants Rf/R0 versus ratio of concentration [fullerene]/[β-carotene] for PVP-entrapped C60, C60O, and C60On. The slope of each linear regression line corresponds to the relative radical scavenging rate constant krrs relative to that of β-carotene. The dotted horizontal line indicates the value in the absence of antioxidants as a control

In order to clarify the reason for the significant effect of the pin-up oxygen on the antioxidant activity of C60, we calculated the energy level of LUMO and HOMO for the C60, C60O, and C60O2 as well as the energy level of SOMO for the LOO· and L· radical (Fig. 6). It was found that the pin-up oxygen lowers the LUMO level relative to those of pristine C60. According to the Klopman and Salem equation [20] as well as the frontier molecular orbital (FMO) theory, the energy (ΔE) gained in the orbital interactions is inversely proportional to the energy difference |LUMO–SOMO|. Thus, C60O can enjoy greater stabilization than C60 in capturing LOO·, or possibly linoleic acid radical L· first formed in autoxidation, thus enhancing the antioxidant activity.12

thumbnailFigure 6. Frontier molecular orbital interaction between LUMO of fullerenes C60, C60O, and C60O2(e) and SOMO of linoleic acid peroxyl radical (LOO·) or linoleic acid radical (L·) calculated by B3LYP/6-31G* level of theory

Conclusion

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.

Footnotes

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.

Acknowledgment

The authors thank Dr. Y. Tajima (RIKEN, FLOX Corp.) for generous gift of C60O.

References

  1. Krusic PJ, Wasserman E, Keizer PN, Morton JR, Preston KF:

    Science. 1991, 254:1183.

    COI number [1:CAS:528:DyaK38XhvFemurk%3D]

    Publisher Full Text OpenURL

  2. Monti D, Moretti L, Salvioli S, Straface E, Malorni W, Pellicciari R, et al.:

    Biochem. Biophys. Res. Commun.. 2000, 277:711.

    COI number [1:CAS:528:DC%2BD3cXnslCqu7w%3D]

    Publisher Full Text OpenURL

  3. Jin H, Chen WQ, Tang XW, Chiang LY, Yang CY, Schloss JV, et al.:

    J. Neurosci. Res.. 2000, 62:600.

    COI number [1:CAS:528:DC%2BD3cXotVWhsr4%3D]

    Publisher Full Text OpenURL

  4. Xiao L, Takada H, Gan XH, Miwa N:

    Bioorg. Med. Chem. Lett.. 2006, 16:1590.

    COI number [1:CAS:528:DC%2BD28Xht1ymsr0%3D]

    Publisher Full Text OpenURL

  5. Huang SS, Tsai SK, Chih CL, Chiang L-Y, Hsieh HM, Teng CM, et al.:

    Free Radic. Biol. Med.. 2001, 30:643.

    COI number [1:CAS:528:DC%2BD3MXisVWnsbc%3D]

    Publisher Full Text OpenURL

  6. Lai Y-L, Murugan P, Hwang KC:

    Life Sci.. 2003, 72:1271.

    COI number [1:CAS:528:DC%2BD38XpvVaiurk%3D]

    Publisher Full Text OpenURL

  7. Tajima Y, Osawa S, Arai H, Takeuchi K:

    Mol. Cryst. Liq. Cryst.. 2000, 340:559.

    COI number [1:CAS:528:DC%2BD3cXns1Sjt74%3D]

    Publisher Full Text OpenURL

  8. Giesa S, Gross JH, Gleiter R, Krätschmer W:

    Eur. Mass Spectrom.. 1998, 4:189. Publisher Full Text OpenURL

  9. Al-Saikhan MS, Howard LR, Miller JC:

    J. Food Sci.. 1995, 60:341.

    COI number [1:CAS:528:DyaK2MXltFyisr0%3D]

    Publisher Full Text OpenURL

  10. Sacchett G, Maietti S, Muzzoli M, Scaglianti M, Manfredini S, Radice M, et al.:

    Food Chem.. 2005, 91:621. Publisher Full Text OpenURL

  11. Tsuchihashi H, Kigoshi M, Iwasuki M, Niki E:

    Arch. Biochem. Biophys.. 1995, 323:137.

    COI number [1:CAS:528:DyaK2MXoslylu7Y%3D]

    Publisher Full Text OpenURL

  12. Takada H, Kokubo K, Matsubayashi K, Oshima T:

    Biosci. Biotechnol. Biochem.. 2006, 70:3088.

    COI number [1:CAS:528:DC%2BD2sXktVKisQ%3D%3D]

    Publisher Full Text OpenURL