Ultraviolet (UV) irradiation generates reactive oxygen species, which can have a series of biological effects on human skin cells, resulting in cosmetic skin damage such as pigmentation.
Fullerene, a carbon allotrope, is characterised as an antioxidant and is reported to react with various reactive chemical species such as free radicals. To prove the practical effectiveness of RadicalSponge (Polyvinylpyrrolidone (PVP)-wrapped fullerene), we performed two clinical tests on 18 female volunteers.1 A gel containing 1% RadicalSponge was prepared and applied on the face twice daily. After eight weeks of application, the melanin index significantly reduced compared to before the treatment.2 A tanning site was created on the volunteers by exposure to a 1.5 minimal erythema dose (MED) of UV irradiation on the left upper inner arm. The vehicle- and 1% RadicalSpongecontaining creams were applied on each tanning site twice a day, in the morning and at night. After eight weeks of application, the melanin index of the exposed area treated with 1% RadicalSponge cream significantly decreased compared with that of the control cream site. These data suggest that the product has a brightening effect because of its ability to scavenge UV-induced free radicals.
Ultraviolet (UV) irradiation is widely known to harm the human body. However, the real cause of the damage is not UV irradiation itself but free radicals such as reactive oxygen species (ROS), including active oxygen, which are generated in abundance on the surface of the skin by UV irradiation. Examples of ROS include super oxide anion radical (O2 –), hydroxide radical (OH), and lipoperoxide. Many studies suggest that these reactive molecules cause not only cosmetic problems such as pigmentation, wrinkles, and erythema, but also dermatological diseases such as malignant skin tumours, cutaneous vasculitis, and acne.1 At present, two approaches for maintaining healthy and bright skin are applied. One is the use of general sunscreen products containing UVprotecting agents such as titanium dioxide, zinc oxide, or avobenzone for blocking UV irradiation, which is the cause of radical action. The second is the application of pharmacologic reagents such as hydroquinone, arbutin, and vitamin C derivatives as symptomatic treatments for melanin that has already been generated in the skin and decreased the brightness of the skin. These two approaches are effective in maintaining the whiteness of the skin. However, ROS present in the skin that accelerate photoageing cannot be removed. We therefore proposed a complementary new approach by combining treatment with an antioxidant, fullerene, with the two conventional approaches (Fig. 1).
Fullerene, an antioxidant molecule
Fullerene, similar to diamond, is a carbon allotrope (a molecule consisting of a single element). It is also known by the generic chemical name C60, a polyhedron including 60 carbon atoms. The diameter of a C60 molecule is approximately 0.7 nm, and its structure resembles a truncated icosahedron (Fig. 2). Fullerene was accidentally discovered in 1985 by Dr Kroto at the University of Sussex (Great Britain), who was studying interstellar materials (trace substances in space), and by Dr Curl and Dr Smalley of Rice University (US) who were studying spectroscopy of clusters.2 Drs Kroto, Curl, and Smalley were awarded the Nobel Prize in Chemistry in 1996 for their contributions to the discovery of the fullerene (C60) and the prediction of its structure. In 1991, a paper published in Science3 shocked researchers in the field of life sciences. It stated that the fullerene (C60) absorbs, eliminates, and detoxifies free radicals. The Vitamin C60 BioResearch Corporation, established in 2003, was the first company to manufacture products that effectively utilise the biological properties of fullerenes. We focused our research on the attractive antioxidant property of fullerenes. In one of our studies, the antioxidant capacity of the fullerene was higher than that of vitamins C and E, which are commonly used as cosmetic ingredients (Fig. 3). We hypothesised that fullerenes are potential cosmetic ingredients. However, it was difficult to blend them with cosmetics owing to their insolubility in water. We therefore developed a technique for stable dissolution of fullerenes in water by wrapping them with a water-soluble polymer (product name: RadicalSponge) (Fig. 4). This technology facilitates the blending of fullerenes with various types of cosmetics, thus allowing fullerenes to be used in cosmetic products.
Safety of fullerenes
We previously evaluated the safety of biofullerene, the major ingredient of RadicalSponge (now referred to as ‘PVPwrapped fullerene’). We also found that biofullerene is safe for use as a cosmetic ingredient, from the results of all the tests, including single-dose oral toxicity, primary skin irritation, cumulative skin irritation, eye irritation, skin sensitisation, skin photosensitisation, contact phototoxicity, bacterial reverse mutation, chromosomal aberration, and human patch tests, which were conducted for application of biofullerene as an additive in a quasi-drug in Japan.4,5 PVP-wrapped fullerene can permeate into the epidermis via the corneum but not the dermis, as noted in the human skin biopsy specimens obtained 24 hours after PVP-wrapped fullerene was administered. This finding suggests that the toxicity of fullerenes to the body is negligible when PVP-wrapped fullerene is applied to the surface of the skin. According to our records, up to the end of 2010, a cosmetic ingredient containing fullerenes developed by us, has been used in more than 1000 cosmetic items, and all cosmetic product manufacturers are satisfied with our product. Therefore, we are confident that our products are safe.
Inhibitory effect
The ability of PVP-wrapped fullerene to inhibit UVA-induced melanin production was tested in human melanoma cells (HMV-II). The cells were treated with or without varying concentrations of PVPwrapped fullerene and UV irradiated five times at 0.4 J/cm2 (total, 2 J/cm2). PVPwrapped fullerene significantly reduced UVA-induced melanin production in a dose-dependent manner and suppressed the formation of dendrites, which transport melanin. Melanin is induced by active oxygen generated by UV rays. It is thought that fullerenes scavenge ROS, and thereby suppress melanin production (Fig. 5).
Clinical tests
To prove the clinical efficacy of PVPwrapped fullerene, a clinical trial was executed by the trust organisation (DRC Co. Ltd.). The study population comprised 18 healthy women of 34-49 years of age. Examination areas were the face and upper inner arm (Fig. 6). This study was conducted in accordance with the tenets of the Declaration of Helsinki, and was reviewed and approved by the institutional review board of Vitamin C60 Bioresearch Corporation. All the subjects provided written informed consent before they were enrolled in this study.
Results of clinical trial (face)
During the trial, the subjects applied 0.4 mL of 1% PVP-wrapped fullerene serum on their entire face twice a day. At 0, 4, and 8 weeks after the treatment was initiated, the brightening of the skin of the cheek was evaluated by a colorimeter (Konika Minolta, Japan), which can measure pigment colour and skin redness as the melanin, and hemoglobin indices, respectively. During the four weeks of application, the melanin index significantly reduced compared to the melanin index before the treatment, and decreased further for up to eight weeks of use. The hemoglobin index was also significantly reduced eight weeks after treatment. This suggests that PVP-wrapped fullerene suppressed redness of the skin caused by inflammation (Fig. 7). It is thought that the fullerene has an anti-inflammatory effect, because there is a report that the antioxidant substance is effective for the control of the inflammatory reaction.
Results of clinical trial (upper inner arm)
PVP-wrapped fullerene-containing cream (1%) was evaluated against solar-induced radiation (UVA+B)-induced tanning of the skin of the upper inner arm. The minimal erythema dose (MED) was defined as the lowest dose needed to cause erythema with a sharp border in the arm at 24 hours after irradiation. Before the trial, the MEDs for all the subjects were measured. All the subjects received 1.5 MED at the two sites of the upper inner arm. Vehicle- and 1% PVP-wrapped fullerenecontaining creams were applied on each tanning site twice a day, in the morning and at night. At 0, 1, 2, 4, and 8 weeks after irradiation, the brightening effect was evaluated by examining each tanned site with a colorimeter. The results of the examinations and a representative example are shown in Figure 8. After eight weeks of application, the melanin index of the area treated with 1% PVP-wrapped fullerene cream had significantly decreased compared with that of the control cream site. These data suggest that PVP-wrapped fullerene has a brightening effect because of its ability to scavenge UV-induced free radicals. No adverse events were noted during each examination period.
Conclusion
It is well known that fullerenes have a strong antioxidant effect. However, it is difficult to apply this substance in cosmetics because of its insolubility in water. Therefore, we developed a watersoluble, polymer wrapped complex (RadicalSponge) to increase water solubility and the stability of fullerenes in cosmetic formulations. The antioxidant activity, melanogenesis inhibition, and brightening efficacy of PVP-wrapped fullerene were evaluated by chemical, in vitro, and clinical tests, respectively. These data suggest that PVP-wrapped fullerene has a brightening effect because of its ability to scavenge UV-induced free radicals. Over 1000 cosmetic items worldwide have used PVP-wrapped fullerene as one of the ingredients, and these products have attested to the beneficial effect and safety of the fullerene (RadicalSponge). The number of fullerene cosmetics will probably increase in the future.
Acknowledgements
We would like to thank Prof. Dr. Nobuhiko Miwa (Faculty of Environmental Sciences, Prefectural University of Hiroshima), Prof. Dr. Takumi Oshima, and Dr. Ken Kokubo (Technology faculty, Osaka University) for providing professional consultation, data, and analysis. We would also like to thank Celless Co. Ltd., Osaka, Japan for preparation of RadicalSponge-containing serum for our clinical tests. PPCC
References
1 Bickers DR, Athar M. Oxidative stress in the pathogenesis of skin disease. J Invest Dermatol 2006; 126: 2565-75. 2 Kroto HW, Heath JR, O’Brien SC, Curl RF, Smalley RE. C60: Buckminsterfullerene. Nature 1985; 318: 162-3. 3 Krusic PJ, Wasserman E, Keizer PN, Morton JR, Preston KF. Radical reactions of C60. Science 1991; 254: 1183-5. 4 Mori T, Takada H, Ito S, Matsubayashi K, Miwa N, Sawaguchi T. Preclinical studies on safety of fullerene upon acute oral administration and evaluation for no mutagenesis. Toxicology 2006; 225: 48-54. 5 Aoshima H, Saitoh Y, Ito S, Yamana S, Miwa N. Safety evaluation of highly purified fullerenes (HPFs): Based on screening of eye and skin damages. J Toxicol Sci 2009; 34 (5): 555-62.
