Potential of an ascorbyl derivative probed

Under normal conditions, L-ascorbic acid (vitamin C) has a strong reducing effect and undergoes oxidisation to become discoloured when used in drugs.

Synthesis of ester derivatives with modification of carbon 2, the most reactive site of ascorbic acid, has been performed in Japan since the 1960s, and many esters of ascorbic acid are now used as food additives, stock feed additives, and cosmetic ingredients. Among the C2 ester derivatives of ascorbic acid, the most commonly used worldwide is L-ascorbic acid-2-phosphate ester (AP). APs were first developed in the 1970s as ingredients of medical cosmetics that prevent pigmentation, and their tyrosinase inhibitory effect prevents melanin production by the pigment cells of the skin.

In the 1980s, APs were found to prevent the death of prawns caused by a high seawater temperature, and now approximately 3,000 t is used worldwide as the ascorbic acid source in aquaculture feed, livestock feed, pet food, and cell culture media. Industrial production of Na L-ascorbyl-2-phosphate-6-palmitate (Showa Denko KK, Japan), an ascorbic acid-2-phosphate-6-palmitate Na (APP) with ester modification of a long-chain fatty acid of AP, started in 2005. APs are water soluble, but the lipid solubility of APP was increased by modification of palmitate, a long chain fatty acid, to carbon 6, markedly improving uptake by skin tissue and cells. The structural formulae of ascorbic acid, AP, and APP are shown in Figure 1.

Ascorbyl 2-Phosphate 6-Palmitate, Apprecier (Showa Denko KK) is a novel vitamin C derivative, trisodium salt of ascorbyl 2-phosphate 6-palmitate (APP), newly designed ascorbic acid amphiphilic derivative of ascorbyl 2-phosphate (AP), conjugated with a long acyl chain (C16: palmitoyl residue). AP has been proved very efficacious for skincare because of its capability of scavenging reactive oxygen species and promoting collagen synthesis. Thanks to its moderate hydrophobicity, APP penetrates effectively into dermis and is enzymatically converted to ascorbic acid quickly during permeation.

APP retains the stability of AP and in vivo ascorbic acid activity, along with improved tissue permeability and intracellular delivery of ascorbic acid due to reduced affinity for water. APP has a strong surfactant effect because of being water soluble, a required property, as well as being lipophilic. By employing these characteristics, self-emulsifying encapsulated materials can be created using APPs. Because APPs are strong antioxidants that are equivalent to ascorbic acid and can be used for ascorbic acid film coating for microcapsules to allow the production of microcapsules with a free radical barrier, it has become increasing clear that their characteristics have various applications. In this article, some of the interesting properties of APP are discussed.

APP effects

The main effect of APP was examined in detail by Kato, etc. (Showa Denko KK) and Miwa N. (Hiroshima Prefectural University, Japan).

They experimented in order to evaluate APP, and reported the following effects of APP.

Improved delivery of ascorbic acid.

Prevention of lipid-peroxidation caused by UVB irradiation.

Enhancement of collagen synthesis.

Inhibition of tyrosinase.

APP improved delivery of ascorbic acid
Changes occur in intracellular ascorbate (ascorbic acid) concentration in normal human epidermal keratinocytes (HaCaT) during the cultivation in the medium containing 0.1mM of trisodium ascorbyl 2- phosphate 6-palmitate (APP), ascorbyl 2- phosphate (AP), or ascorbyl 2-glucoside (AG). APP showed its superiority in enriching intracellular ascorbic acid, which suggested a quick conversion to ascorbic acid and an outstanding ascorbic acid delivery into the cell. (Miwa N., Hiroshima Prefectural University, Japan)

Prevention of intradermal lipid-peroxidation
Skin sections taken from hairless mice were cultivated in a medium containing 3% sodium ascorbyl 2-phosphate (APS) or 3% trisodium ascorbyl 2-phosphate 6-palmitate (APP) for two hours, and irradiated by UVB at 20 kJ/m2. The amount of intradermal TBARS (thiobarbituric acid reacting substances; a good indicator of lipid peroxidation) was measured 22 hours after the irradiation when the TBARS amount was expected to be the maximum. APP effectively suppressed the generation of intradermal TBARS, while the TBARS amount was increased by 1.6 times in non-treated skin.

Enhancement of collagen synthesis
The collagen content in the incised wound in guinea pigs was measured in the same experiment as shown previously. The wounded section of the skin was taken, defatted, homogenised, and treated with pepsin followed by concentrated hydrochloric acid to hydrolyse collagen. After the centrifugation the concentration of hydroxyproline in the supernatant was measured. The hydroxyproline content was significantly increased in APP-applied skin, which suggested that the collagen synthesis was accelerated by APP.

Inhibition of tyrosinase
Enzymatic inhibition of various skinbrightening agents against intracellular tyrosinase was examined using melanoma B16F10. The melanoma cells were cultivated for 96 hours in DMEM medium containing 10% FBS and 30 mM trisodium ascorbyl 2-phosphate 6-palmitate (APP), 2 mM kojic acid, or 1 mM ascorbyl 2-glucoside (AG), with a medium renewal at 48 hours. After the cultivation the cells were harvested, treated with cold methyl alcohol, and the tyrosinase activity was detected by a treatment with 0.1% dihydroxyphenylalanine (DOPA) for 90 minutes. After being washed the cells were further stained by nuclear fast red solution for a better microscopic observation.

Addition of 30 mM APP during the cultivation suppressed the intracellular tyrosinase activity.

Vitamin C derivatives

Examples of applications that employ the surfactant of APP are shown in the photos. Various surfactants are found in products that we use in daily life, such as cosmetics, foods, clothes, and paints.

Some surfactants have an irritant effect on humans and we would prefer to avoid using them. APP can be used more safely compared with common synthetic surfactants, because there are no synthetic residues or metabolites, and they are completely broken down into vitamin C and oils or fats in vivo.

They are also useful as vitamins, so they can be employed as radical scavengers or as nutrients. Our laboratory has devised creams, bath foams, toothpastes, shampoos, hair mousses, dishwashing liquids, cakes, ice creams, bread, mayonnaises, and bubble-making liquids that employ the surfactant activity of APP.

The enforcement examples of ascorbic acid, AP, and APP are shown in Figure 2. Testers who used these products have given favourable feedback, indicating that they were “soft” or “smooth”. The reasons for this are not clear, but it is thought that free radicals normally have a negative effect on the skin and on taste sensation.

Our environment is full of active oxygen species derived from tap water, sunlight, photocatalysts, drugs, agricultural chemicals, or glues used in buildings and furniture, so it appears that APP improve the touch and taste of products because of their powerful active-oxygen scavenging effect.

Potential applications that are not shown in the photographs include using ascorbic acid solubilising aid for drugs, paints, photographic paper, fabrics, gauzes and sutures. We have also been seeking themes in other fields to actively promote application studies. It should be noted, however, that APPs are still only approved as cosmetic ingredients, and their commercial use in foods, stock feed and drugs has not been approved due to lack of a legal registration system.

Oxidant effect of APP

After APPs are converted into ascorbic acid by phosphatase, thus developing a antioxidant effect, they also scavenge active oxygen species in vitro to a lesser extent. Ascorbic acid is also a strong antioxidant in vitro and it therefore reduces other substances, while oxidising itself to be converted into ascorbic acid radicals and pro-oxidants. This is the reason for the common advice that application of concentrated vitamin C lotion should be avoided before sunbathing in the summer, since vitamin C scavenges hydroxyl radicals produced in the skin by ultraviolet light, while ascorbic acid radicals accumulate.

Overproduction of ascorbic acid radicals due to the application of lotions means that scavenging by in vivo antioxidants (such as glutathione, a-lipoic acid, ubiquinone, etc) is inadequate. To investigate active oxygen scavenging by APP in vitro, hydroxyl radicals were generated by UVB irradiation of a mixture of water and DMPO, after which APP, ascorbic acid or water-soluble vitamin E was used to examine the scavenging of active oxygen species by the ESR-ST method. DMPO is a spin trap agent used for detection of radicals by the ESR-ST method, because it traps and stores the unpaired electrons of short-lived hydroxyl radicals.

DMPO with unpaired electrons becomes a paramagnetic substance, and the ESR device is magnetic field resonance apparatus that detects free radicals as unique wave patterns by creating a strong magnetic field around unpaired electrons to align their movement, while simultaneously amplifying the signals made by resonation of electrons using electromagnetic waves.

Number 1 in Figure 3 shows the wave pattern of hydroxyl radicals generated by UVB irradiation and detected by the ESR-ST method. Number 2 indicates the wave pattern obtained after vitamin C was an ascorbic acid 1% solution. It shows that hydroxyl radicals were scavenged, but ascorbic acid radicals were detected. Number 3 is the wave pattern when watersoluble vitamin E derivatives were added to ascorbic acid 1% solution instead of vitamin C, showing a scavenging effect of these hydroxyl radicals (although it was weaker than that of vitamin C). Number 4 is the wave pattern obtained when APP were added to ascorbic acid 1% solution, confirming the scavenging of hydroxyl radicals generated by UVB irradiation and the absence of ascorbic acid radicals. Ascorbic acid radicals were generated when a 1% ascorbic acid solution was exposed to UVB irradiation – APP prevented the generation of these radicals.

Therefore, APP are safe vitamin C derivatives for cutaneous use. (Fig. 3)

Anti-wrinkle effect of APPs

APPs are absorbed by the skin at high concentrations, preventing the oxidisation of lipids caused by UV and increasing collagen production. Therefore, APPs are expected to have an anti-wrinkle effect. In fact, Terashima et al at Tokyo Metropolitan Police Hospital, have clinically confirmed such as effect in a study using APP lotion with the formula shown in Table 1.

Twenty adult females aged between 31 and 62 (average: 50.3) years were asked to apply 1% APP lotion to the face twice daily (morning and night) after washing, and a cast of the outer canthus of the left eye was obtained from each subject before and after the five-month period. Then wrinkles in the casts were detected by two-dimensional image analysis using an imaging unit (Asahibiomed).

The monthly changes of wrinklereduction parameters are shown in Figure 4. Among the percent wrinkle area, maximum wrinkle width, maximum wrinkle depth, and number of wrinkles, 1% APP lotion significantly improved the percent area, maximum width, and a reduction in the number of wrinkles after five months. Therefore, lessening of wrinkles by the 1% APP lotion was confirmed. A typical analysis of casts from a subject with improvement of wrinkles is shown in Figure 5.

Thus, this clinical trial of APP showed that continuous application for five months significantly improved the percent wrinkle area, the maximum wrinkle width, and the number of wrinkles. These results confirmed the wrinkle-reducing effect of APPs in adult women.

Production of microcapsules using APPs

APPs are amphiphilic agents that form liquid crystals when combined with lipids as surfactants. The liquid crystal structure of APP is formed by multiple layers of water and lipid (i.e., water-lipid-water-lipid, see Fig. 6), so water-soluble materials can be mixed in the aqueous layers.

Microcapsules made in this fashion become self-emulsifying and addition of water leads to emulsification as dispersion. When the self-emulsifying process occurs, APPs in the form of microcapsules become dispersed in water ascorbic anionic dispersion, so electrophoresis is easily performed. Using this phenomenon, watersoluble materials with no electric charge, such as lipids, peptides, sugar chains, and antibodies, can be introduced as ions by iontophoresis. We have already succeeded in producing an APP-based self-emulsifying encapsulated lipid preparation (Nanomic), in which CoQ10, astaxanthin, VCIP, and fullerene are encapsulated by APP. With these APP microcapsules, the stability of both the APPs themselves and the encapsulated lipids is improved.

When encapsulated, APP/lipids, which contain various water-soluble and fat-soluble antioxidant molecules, the stability of both APP in the coating and also the molecules inside the microcapsules is greatly improved. This concurrent stabilisation of the APP coating and encapsulated antioxidant molecules is thought to depend on reproducing the redox balance between the various antioxidant molecules in cells. The stability of a 1% APP solution and of APP microcapsules is shown. Also, the stability of VCIP in emulsifying dispersions (VCIP encapsulated with APP) is shown. Carotinoids with strong singlet-oxygen scavenging ability, such as astaxanthin, are unstable when added to drugs, and undergo oxidative decomposition to become discoloured relatively easily even when antioxidants are also added, but their stability is improved by encapsulation with APPs.

Free radicals

We developed various encapsulated APP/lipid samples and assessed their freeradical scavenging ability by the ESR-ST method (spin trap agent: DMPO). We investigated the in vitro free-radical scavenging effect of APP microcapsules using hydroxyl radicals generated by the Fenton reaction, free radicals generated by reaction of xanthine with xanthine oxidase, and methyl radicals generated by the addition of methanol.

We also investigated the active oxygenscavenging effect of 10% nanomic C using hydroxyl radicals and methyl radicals generated simultaneously by a Fenton reaction system to which methanol had been added. It was confirmed that Nanomic C scavenged hydroxyl radicals and methyl radicals generated in this reaction system (Fig. 7).

An experiment using the ESR-ST method showed that Nanomic Q could scavenge super oxide generated by the xanthine-xanthine oxidase system. We plan to develop a Nanomic series that is able to scavenge many kinds of free radicals and mix these agents to meet the needs of cosmetic manufacturers and doctors. The applications and effects of members of the Nanomic series that are already available are shown in Table 2.

As described above, we showed that APP/lipid microcapsules could scavenge hydroxyl radicals, super oxide, and methyl radicals. Interestingly, a few ascorbic acid radicals were generated when ascorbic acid was added at an equimolar concentration, but ascorbic acid radicals were not generated when APP/lipid microcapsules were added, indicating the strong free-radical scavenging ability of the microcapsules.

APPs are ascorbic acid derivatives with surfactant properties that show efficient tissue uptake, and thus are new functional materials that possess strong activeoxygen scavenging activity and also promote collagen synthesis. In the field of cosmetic dermatology, the number of clinics that recommend APP lotions or creams as home-care products after laser treatment is increasing, and these clinics have received favourable feedback from their patients. APPs contain lipid groups, and therefore provide a stronger skin barrier compared with ordinary watersoluble ascorbic acid derivatives and also cause less drying of the skin than other vitamin C-containing lotions. Because APP are only bound to one lipid molecule, they are less sticky when applied to the skin compared with conventional fat-soluble vitamin C preparations.

Currently, many ascorbic acid derivatives are used in various fields. APPs are the latest ascorbic acid derivatives with the properties of vitamin C. We hope to establish an anti-ageing drug with an effective redox balance, in which watersoluble and fat-soluble redox molecules resonate with each other due to the amphiphilic properties of APP, and we plan to expand our research into various industrial fields such as cosmetics and drugs.