Ascorbyl 2-phosphate tocopherol (APT) is a free radical scavenger synthesised by connecting vitamin E with vitamin C (ascorbic acid [AA]) through a phosphate linkage.
APT has been developed as an amphipathic antioxidant vitamin and is a water and oil soluble derivative of ascorbate and tocopherol, which is slightly soluble in oil but has a surface-active ability allowing the derivative to be used as an emulsifier and multi-layer liquid crystal nano capsule clathrate. Astaxanthin or other anti-oxidants like fullerene were encapsulated in APT, giving a multi-layer liquid crystal emulsification nano capsule containing anti-oxidant, called “APT-capsule”. Although the reaction rate constant of fullerene against superoxideanion radical seems to be lower than those of ascorbic acid (vitamin C) or tocopherol (vitamin E), the ability is known to last longer than other anti-oxidants. For finding out the useful application of the unique antioxidation ability of fullerene, our laboratory showed that fullerene can suppress UVB-induced ascorbyl radicals generated from skin tissue treated with AA.1 Since fullerene was expected to be useful for stabilising a vitamin C derivative capsule having multi-layer liquid crystal structure,2 fullerene was attempted to be encapsulated in the vitamin C derivative nano capsule, and the properties of this unique nano-capsule were evaluated. Figure 1 shows the multi-layer liquid crystal structure of self emulsifying ascorbyl 2- phosphate tocopherol (APT)/lipid nano capsules with fullerene (APT-capsule). Since the liquid crystal structure of APT was constructed from water and lipid multiple layers (e.g. water-lipid-water-lipid, etc.), lipid-soluble anti-oxidants such as fullerene and astaxanthin were encapsulated stably in the lipid layers and lipid core. APT has been developed as an amphipathic antioxidant vitamin and is a water and oil soluble derivative of ascorbate and tocopherol, which is slightly soluble in oil but has a surface-active ability allowing the derivative to be used as an emulsifier and multi-layer liquid crystal nano capsule clathrate. Furthermore, this capsule is hydrolysed from the capsule surface by an enzyme like a phosphatase in tissue, and ascorbate, tocopherol and astaxanthin are released slowly from this capsule. The efficacy of this vitamin CE derivative nano capsule containing fullerene on UVB and laser irradiations was investigated. Furthermore, the stability of astaxanthin encapsulated nano-capsule was examined. When the APT-capsule containing AA at a high concentration was irradiated by UVB, the amount of AA radicals was observed. However, when the APT-capsule containing antioxidant was irradiated, the generation of AA radicals was found to be reduced significantly, showing that the antioxidant controlled the generation of AA radicals by ultraviolet rays with the presence of amphipathic vitamin C. The APT-capsule was also found to inhibit the decomposition of astaxanthin, under a hyper oxidation condition of lipids. The experiments suggested that APTFcapsule inhibited the oxidisation of both water-soluble and lipid-soluble antioxidants,1 and its anti-oxidation property was due to a possible synergistic effect of amphipathic vitamins and antioxidant.2 Being encapsulated in an APT-capsule, unstable antioxidants such as astaxanthin or fullerene were preserved stably by the redox barrier of APT.
ESR analysis
The ESR peak intensities ratio (EPI) of the free radical on skin tissue samples after UVB irradiation are shown in Figure 2. ESR is an abbreviated designation of “electron spin resonance”. ESR is one convenient analysis method which can detect each kind of free radical species easily and directly from 3D skin tissue in a short period of time. The relative EPI of HO• (hydroxyl radical), H• (hydrogen radical), O2• (superoxide anion radical) (0 hours) and ascorbyl radical (24 hours) detected in the skin tissue after UVB irradiation. HO•, H• and O2• were significantly increased by UVB irradiation, compared to the control (non-UVB-irradiated specimens) at 0 hours (Fig. 2a). The application of AA (100 mmol/L), APT (100 mmol/L), APT (100 mmol/L) with fullerene (30 ?mol/L) significantly decreased H•, O2•, and HO•, compared to the negative control (P<0.001, Fig. 2a). However, AA• (ascorbyl radical) increased with the application of AA (100 mmol/L, P<0.01, against the positive control) at 24 hours after the irradiation (Fig. 2b). AA• inhibited with the application of 100 mmol/L APT and 30 ?mol/L fullerene in Fig. 2b (against the positive control) at 48 hours after the irradiation. Especially, the nano capsule of 100 mmol/L APT with 30 ?mol/L fullerene strongly inhibited AA• (Fig. 2b), compared to the positive control (P<0.01) at 48 hours.
Cell viability ratio
Figure 3 shows NIH-3T3 cell viability ratio after UVB irradiation (100 J/m2) for 48 hours. The cell viability ratio of the positive control with UVB irradiation (+UV) showed a significant decrease to 46%, compared to that of the negative control without UV irradiation (no UV) (P<0.001). Although 30 ?mol/L fullerene +UV group increased its cell viability ratio significantly (69%), 100 ?mol/L APT +UV group increased the ratio significantly (80%), and 100 mmol/L APT with 30 ?mol/L fullerene + UV group showed an increase significantly (91%), compared to the vehicle + UV control.
Apoptosis index
Figure 4 shows relative Yo-Pro-1 (apoptosis index) fluorescence intensity ratio (RYI) as an apoptosis index of NIH-3T3 cell after UVB irradiation for 48 hours. Apoptosis is a cell damage index by UVB. The vehicle control with UV irradiation (+UV) showed a significant increase in apoptosis index to 64% (mean RYI), compared to the negative control (P<0.01). Fullerene (30 ?mol/L) group showed a decrease to 178% (non-significant), compared to the vehicle control + UV. The mean RYI of the vehicle control + UV decreased significantly to 132% with the application of 100 ?mol/L APT. Nano-capsule containing 100 mmol/L APT and 30 ?mol/L fullerene significantly decreased the mean RYI to 119% (P<0.05).
Laser irradiation
Figure 5 shows the relative fluorescence intensity (RFI) of CM-H2DCFDA dye as an index of ROS in skin tissue after laser irradiation (3 J/cm2) for 48 hours. The vehicle control UV irradiation (+UV) showed a significant increase to 48% compared to the negative control (P<0.001). Although the 30 ?mol/L fullerene group shows a decrease to 183% (not significant) compared to the vehicle control + UV. The mean RFI of the vehicle control + UV decreased significantly to 149% with the application of 100 mmol/L APT (P<0.05). The nano-capsule of APT 100 mmol/L with fullerene 30 ?mol/L significantly decreased the mean RYI to 143% (P<0.01).
Lightness value (L-value of Lab colour analysis)
Figure 6 shows the L-value changes of 1% astaxanthin stored at 40°C for 80 days. L-value is an index to express whiteness of skin, when astaxanthin disintegrates, this numerical value rises. The L-value of 1% astaxanthin in APT-F emulsion (no nanocapsule) was found to increase significantly (L-value: 213), value in APT-F multi-layer liquid-crystal decreased significantly (L-value: 198, P<0.001), compared to the vehicle control (L-value: 239) at 80 days.
Conclusion
The results suggested that fullerene in APT nano-capsule (APTF-capsule) inhibited ascorbyl radicals in skin tissue that was irradiated by UVB and laser rays,1 and fullerene in the capsule more significantly inhibited apoptosis and cell death induced by UVB and laser irradiation than the solo application of individual components.2 Additionally, the APTF-capsule more significantly inhibited the colour change of encapsulated astaxanthin stored at 40°C for 80 days than the mono-layer emulsification capsule. This multi-layer APT nano-capsule containing fullerene is a promising basic tool for developing antiageing products including cosmetics and medicines, because this tool has an effective redox balance, which allows watersoluble and lipid-soluble redox molecules to synergistically work for expressing its antioxidant ability. In addition, the type and level of antioxidant in this capsule were able to be changed by user option.
References
1 Ito S, Itoga K, Yamato M, Akamatsu H, Okano T. The co-application effects of fullerene and ascorbic acid on UV-B irradiated mouse skin. Toxicology 2010; 267 (1-3): 27-38. 2 Ito S. Possibility of antioxidation vitamin derivative capsules, Nanotec, producing novel cosmetics in Japan. CMC Publishing 2009; (5): 148-54. 3 Takayama H, Hamner CE, Caccitolo JA, Hisamochi K, Pearson PJ, Schaff HV. A novel antioxidant, EPC-K1, stimulates endothelial nitric oxide production and scavenges hydroxyl radicals. Circ J 2003; 67 (12): 1046-52. 4 Ito S, Mori T, Kanazawa H, Sawaguchi T. Differential effects of the ascorbyl and tocopheryl derivative on the methamphetamine-induced toxic behavior and toxicity. Toxicology 2007; 240 (1-2): 96-110. 5 Fujimoto T, Ito S et al. Effect of fullerene as an antioxidant during laser therapy. J Am Acad Dermatol 2010; 62 (2): 149.
