L-Ascorbic Acid 2-Glucoside

Vitamin C - Physiological Activities - Physiological Activities of AsA - Antiscorbutic activity (Enhancement of collagen synthesis) AsA acts as a cofactor for prolyl and lysyl hydroxylase, which are essential enzymes in collagen synthesis. - Antioxidant activity AsA removes free-radicals generated in the body and plays a critical role in the redox system. - Antiviral activity - Enhancement of immune system AsA enhances lymphocyte proliferation, neutrophil function and IFN production. - Inhibition of melanin synthesis L-ascorbic acid (AsA)

Reducing Action and Stractural Change of Ascorbic Acid

Development of Stable Vitamin C Derivatives AsA is unstable to heat, light and oxidation. AsA Active site (Antioxidant activity etc.) Vitamin C activity: Yes Vitamin C activity: No Vitamin C activity: No L-ascorbic acid 2-phosphate (AA-2P) L-ascorbic acid 2-sulfate (AA-2S) 2-O-octadecyl-L-ascorbic acid (CV-3611)

A Novel Stable Form of Vitamin C; L-Ascorbic Acid 2-Glucoside Liquefied starch AsA Cyclodextrin glucanotransferase (CGTase) n L-Ascorbic acid 2-maltooligoside (AA-2Gn) Glucoamylase L-Ascorbic acid 2-glucoside (AA-2G)

Major Properties of AA-2G a-glucosidase AA-2G AsA Properties ・Since C2-hydroxyl group of AsA is masked with glucose, AA-2G is nonreducible and very stable to oxidative conditions. ・AA-2G is readily hydrolyzed by a-glucosidase releasing AsA.

AA-2G Releases AsA by the Action of a -Glucosidase AA-2G Exhibits Physiological Activities of AsA in vivo and in vitro AA-2G AsA Hydrolysis Physiological Activities of AA-2G - Antiscorbutic activity in guinea pigs - Enhancement of collagen synthesis in cultured human skin fibroblasts - Enhancement of immune responses in cultured murine and human lymphocytes - Inhibition of melanin synthesis in melanoma cells a-glucosidase Cell membrane Intracellular AsA Physiological Activities

Contents 1. Properties of AA-2G 2. Bioavailabilities of AA-2G in the Cosmetics ・Promotion of Collagen Synthesis ・Whitening ・Antioxidant Activity 3. Skin Penetration of AA-2G 4. Conclusion

Properties of AA-2G

Stability of AA-2G in an Aqueous Solution pH 7.0, 50℃ pH 5.0, 37℃ Residual (%) AA-2G soln. (70 mM) Residual (%) AA-2G soln. (2%) AsA soln. (2%) AsA soln. (70 mM) Storage period (hr) Storage period (days) ■Method: 1) Aqueous solutions of AA-2G or AsA were adjusted to pH 5.0 or pH 7.0. These solutions were incubated at 37 or 50℃. 2) The amount of AA-2G or AsA in the aqueous solutions was continuously monitored.

Stability of AA-2G and AA-2P at Various pHs Residual (%) AA-2G soln. (0.2%) AA-2P soln. (0.2%) pH ■Method: 1) 0.2% solutions of AA-2G or AA-2P were adjusted to various pHs. These solutions were stored at 50℃ for 20 days. 2) The amount of AA-2G or AA-2P in each solution was measured by HPLC.

Stability of AA-2G in the Presence of Cu2+ AsA AA-2G ◆　　　CuSO4 0 mM ■　　　CuSO4 10 mM ◆　　　CuSO4 0 mM ■　　　CuSO4 10 mM ■Method: 1) 100 mM solution of AsA or AA-2G was incubated with or without 10 mM CuSO4 solution in 0.1 M acetate buffer (pH 6.5). 2) The amount of AsA or AA-2G in the reaction mixture was continuously monitored at 265 nm for AsA or 260 nm for AA-2G. [Yamamoto et al., Chem. Pharm. Bull., 38 (11), 3020 (1990)]

Heat Stability of AA-2G under Various pH Condition 1: Measured at OD420 nm, 2: Measured by HPLC, 3: glucose ■Method: 1) 2% solutions of AA-2G or AsA were adjusted to various pH. These solutions were heated at 125℃ for 60 min (autoclave). 2) After heating, color and compositions of each solution were measured.

Physicochemical Properties of AA-2G and AsA AA-2G AsA Molecular weight 338.27 176.13 Melting point 158.5 - 159.5℃ 190.0℃ Heat of fusion 38.5 kJ/mol 37.9 kJ/mol Specific rotation [α] (c = 5) +189.6°(at pH 2.0) + 22.1°(at pH 2.3) +246.3°(at pH 7.1) +119.9°(at pH 7.1) Water solubility (at 25℃) 125 g/100 g･H2O 33 g/100 g･H2O UV maximum absorbance 238 nm (at pH 2.0) 243 nm (at pH 2.0) 260 nm (at pH 7.0) 265 nm (at pH 7.0) pH (1.0%w/v) 2.3 2.6 Dissociation constant pK1 3.0 4.1 pK2 - 11.8 Reducing activity - ＋ 20 D

Bioavailabilities of AA-2G in the Cosmetics

Efficacy of AA-2G in Cosmetics 1. Promotion of Collagen Synthesis

Stimulatory Action of AA-2G on Collagen Synthesis Control AsA AA-2P AA-2G Collagen/total protein (%) 1 3 5 8 1 3 5 8 1 3 5 8 1 3 5 8 Cultivation (days) ■Method: 1) Human skin fibroblasts were incubated in the absence or presence of each collagen stimulator (0.25 mM) for 1 - 8 days. 2) The relative rate of collagen synthesis to total protein synthesis was determined. [Kumano and Yamamoto et al., Biol. Pharm. Bull., 21 (7), 662 (1998)]

Stimulatory Action of AA-2G on Collagen Synthesis ■ ▲ : AsA : AA-2P : AA-2G : AA-2G + castanospermine Intracellular AsA content (DPM/dish) Incubation days ■Method: 1) Human skin fibroblasts were cultured in the presence of 14C-AsA, 14C-AA-2P or 14C-AA-2G. 2) On the indicated day, the cells were broken up by sonication. The supernatants obtained by centrifugation were applied to thin layer chromatography (silica gel). 3)These were run using benzene, methanol, acetone and acetic acid solvent (9:9:1:2). The radioactivity in the fraction corresponding to AsA was measured. [Kumano and Yamamoto et al., Biol. Pharm. Bull., 21 (7), 662 (1998)]

AA-2G Releases AsA by the Action of a -Glucosidase AA-2G Exhibits Physiological Activities of AsA in vivo and in vitro AA-2G AsA Hydrolysis Physiological Activities of AA-2G - Antiscorbutic activity in guinea pigs - Enhancement of collagen synthesis in cultured human skin fibroblasts - Enhancement of immune responses in cultured murine and human lymphocytes - Inhibition of melanin synthesis in melanoma cells a-glucosidase Cell membrane Intracellular AsA Physiological Activities

Efficacy of AA-2G in Cosmetics 2. Whitening

Effects of AsA in the Process of Melanin Formation Inhibition Inhibition Tyrosinase Tyrosinase Reducing action L-Tyrosine L-Dopa Dopaquinone AsA Autoxidation Dopachrome 5,6-Dihydroxyindole Indole-5,6-quinone Polymerization Melanin (Black Color) Reduced Melanin (Colorless) Reducing action AsA

Inhibitory Effect of AA-2G on Melanin Formation 4.0 ■Method: 1) B16 melanoma cells were cultured with AA-2G or AsA for 12 hr. 2) The cells were treated with theophyline (0.5 mM) for 48 hr. 3) The cells were lysed with TCA, intracellular melanin content was measured. 3.5 Melanin content (mg/4 x 105 cells) AA-2G 3.0 AsA 2.5 2.0 0.1 0.5 2.5 Initiation of Culture Concentration (mM) 12 hr 48 hr AA-2G, AsA theophyline [Miyai and Yamamoto et al., Nishinihon J.Dermatol., 58 (3), 439 (1996)]

Inhibitory Effect of AA-2G on Melanin Formation Control AA-2G (2.5 mM) (× 200) ■Method: 1) B16 melanoma cells were treated with theophylline (0.5 mM) after 12 hr in the absence or presence of AA-2G (2.5 mM). 2) After 48 hr, the cells were reacted with DOPA for 1.5 hr and stained by Nuclear Fast Red. [Miyai and Yamamoto et al., Nishinihon J.Dermatol., 58 (3), 439 (1996)]

Inhibitory Effect of AA-2G on Melanin Formation ■Method: 1) B16 melanoma cells were cultured with AsA, AA-2P or AA-2G (2 mM) for hours indicated in the figure. 2) The cells were treated with theophyline (0.5 mM) for 24 hr. 3) The cells were lysed with TCA, intracellular melanin content was measured. AsA AA-2P Melanin content (%) AA-2G Initiation of Culture Time (hr) 3 - 36 hr 24 hr AsA, AA-2P, or AA-2G theophyline [Kumano and Yamamoto et al., J. Nutr. Sci. Vitaminol., 44, 345 (1998)]

Reducing Action of AA-2G on Existing Melanin (Lightening of Melanin Pigment) Day 1 Day 2 ■Method: 1) B16 melanoma cells were cultured until confluent (48 hr). 2) The cells were treated with AsA, AA-2P or AA-2G (2 mM) for 1-2 days. Theophyline (0.5 mM) was added to the culture during the last 24 hr. 3) The cells were lysed with TCA and intracellular melanin content was measured. Melanin content (%) Initiation of Culture 48 hr 1-2 days AsA, AA-2P, or AA-2G [Kumano and Yamamoto et al., J. Nutr. Sci. Vitaminol., 44, 345 (1998)]

Inhibitory Effect of AA-2G on Melanin Formation AA-2G Inhibits Melanin Formation Through the Liberation of AsA by a-Glucosidase AA-2G a-glucosidase a-glucosidase AsA AsA - Inhibition of tyrosinase activity Lightening of Melanin Pigment by Reducing Action - Reducing Action Inhibition Dopa- quinone Melanin Reduced L-tyrosine L-Dopa (black) Melanin tyrosinase Melanocyte

Efficacy of AA-2G in Cosmetics 3. Antioxidant Activity

Production of Reactive Oxygen by UV and Skin Damage Immunosuppression Damage of Keratinocyte Damage of Fibroblast Inhibition of Collagen Production Cross-linking between Collagen Reactive oxigen Keratinocyte Fibroblast Collagen Langerhans cell T cell UV Th B ICAM-1 LFA-1

Protective Effects of AA-2G from UV-Induced Cell Damage Suppressive Effect of AA-2G on UV-Induced Skin Damage Protective Effects of AA-2G from UV-Induced Cell Damage Human Skin Keratinocytes ■Method: 1) Human skin keratinocytes were cultured with AA-2G, AA-2P, or AsA (0.2 mM) for 24 hr. 2) The cells were irradiated with UVB at 17 mJ/cm2 , then cultured for 24 hr. 3) The number of viable cells was determined by using a hematocytometer. Number of viable cells (x 104) Initiation of Culture None AA-2G AA-2P AsA Non- irradiation UV irradiation 24 hr 24 hr AsA, AA-2P, or AA-2G UV-Irradiation [Kumano and Yamamoto et al., J. Nutr. Sci. Vitaminol., 44, 345 (1998)]

Suppressive Effect of AA-2G on UV-Induced Skin Damage Human Skin Fibroblasts ■Method: 1) Human skin fibroblasts were cultured until confluent. 2) The cells were irradiated with UVB at 292 mJ/cm2, then AA-2G, AA-2P, or AsA (0.2 mM) was added to the culture. 3) After 7 days, the number of viable cells was determined by DNA assay method. DNA (mg) Human skin fibroblasts None AsA AA-2P AA-2G Non- irradiation UV irradiation 7 days UV-Irradiation AsA, AA-2P, or AA-2G [Kumano and Yamamoto et al., J. Nutr. Sci. Vitaminol., 44, 345 (1998)]

Suppressive Effect of AA-2G on UV-Induced Skin Damage Suppresses Hydroxy Radicals Non- treatment None 100 0.1 1.0 AsA (mM) AA-2G (mM) H2O2 treatment Catalase (U/ml) Number of viable cells (x 104/well) Human Keratinocyte Cell Line ■Method: 1) Human keratinocyte cell line, SCC, were pretreated with AA-2G, AsA, or catalase for 7 hr. 2) Hydrogen peroxide (0.4 mM) was added to the culture. 3) After 2 hr, the number of viable cells was determined by neutral red assay method. [Miyai and Yamamoto et al., Biol. Pharm. Bull, 20 (6), 632 (1997)] UV irradiation OH radicals production Lipid peroxidation, Damage of cell membrane Cell death H2O2 treatment

Suppressive Effect of AA-2G on UV-Induced Skin Damage AA-2G Suppresses Lipid Peroxidation Induced by UV Irradiation (Hydroxy Radicals Production) Human Skin Keratinocytes ■Method: 1) AA-2G (3 mM), a-tocopherol (3 mM), or AA-2G (3 mM) + a-tocopherol (3 mM) was added to a suspension of human skin keratinocytes. 2) After incubation for 1 hr, the suspension was irradiated with UVB at 3 J/cm2. 3) The quantity of peroxidized lipids was determined by phenol indoleamine method. Peroxidized Lipid (Abs 586 nm) Non- irradiation None AA-2G VitE AA-2G + VE UV irradiation VitE: a-Tocopherol [Kumano and Yamamoto et al., J. Nutr. Sci. Vitaminol., 44, 345 (1998)]

Suppressive Effect of AA-2G on UV-Induced Skin Damage AsA Released from AA-2G Suppresses Lipid Peroxidation Through Scavenging Hydroxy Radicals Human Skin Fibroblasts Hydroxy radicals (%) ■Method: 1) Human skin fibroblasts were cultured in the presence or absence of AsA (0.2 mM) for 24 hr. 2) The cells were disrupted by sonication. 3) Hydroxy radicals were detected by Electron Spin Resonance (ESR) method. ESR conditions Temperature: room temp. Microwave powder: 40 mW Modulation amplitude: 0.75 G Time constant: 0.3 sec. Scan rate: 60 G/41.9 sec. Receiver gain: 1 x 106 [Kumano and Yamamoto et al., J. Nutr. Sci. Vitaminol., 44, 345 (1998)]

Efficacy of AA-2G in Cosmetics in vivo

Suppressive Effect of AA-2G on UV-Induced Skin Damage UV-Induced Erythema in Guinea Pig Skin Guinea Pigs ■Method: 1) Creams containing AsA derivatives (14.8 mmol/g) or placebo were applied to the shaved dorsal skin of guinea pigs. 2) After 4 hr, the skin site was irradiated with UVB at 0.35 J/cm2. 3) 16 hr after UV irradiation, erythema of the skin was evaluated by microscopy. Erythema index AA-2S: L-ascorbic acid 2-sulfate AA-PAL: L-ascorbic acid palmitate [Miyai and Yamamoto et al., Nishinihon J.Dermatol., 58 (3), 439 (1996)]

Suppressive Effect of AA-2G on UV-Induced Skin Damage UV-Induced Erythema and Pigmentation in Human Skin Human Subjects : Erythema : Pigmentation ■Method: 1) Creams containing 2% AA-2G or and placebo were applied at two places on the inside of the right arm of 15 healthy adults. 2) The creams were applied three times a day for 6 consecutive days. 3) During this treatment, the arms were irradiated with UVA and UVB once per day for a total of 3 days. Number of cases [Miyai and Yamamoto et al., Nishinihon J.Dermatol., 58 (3), 439 (1996)]

Suppressive Effect of AA-2G on UV-Induced Skin Damage Sunburn Cells Induced by UV Irradiation Human Subjects (7 healthy males) Sunburn cells (cells/specimen) Non- irradiation 2% AA-2G cream Placebo UV irradiation ■Method: 1) 2% AA-2G cream or placebo cream was applied to the inside of the upper part of one arm once a day for 20 days. 2) 5 hr after the last application of the cream, the arms were irradiated with UVB. 3) 24 hr after irradiation, skin samples were taken from the irradiated areas by shave biopsy, then fixed in 2% formalin. 4) The number of sunburn cells in the skin specimens was calculated under a microscope. [Kumano and Yamamoto et al., J. Nutr. Sci. Vitaminol., 44, 345 (1998)]

Skin Penetration of AA-2G

Percutaneous Absorption of AA-2G and Its Conversion to AsA in the Skin Human Subjects ■Method: 1) 2% AA-2G cream or 3% AA-2P cream was applied to the legs of five subjects, then covered with cling film for 14 hr. 2) Placebo cream was applied to one other subject under the same conditions. 3) Urine samples were taken every 2 hr for 26 hr. 4) AsA content in urine was measured by HPLC. 3% AA-2P cream AsA content in urine (mg/100 ml) 2% AA-2G cream Placebo cream (hr) Cream applied Cream removed [Kumano and Yamamoto et al., J. Nutr. Sci. Vitaminol., 44, 345 (1998)]

Intradermal AsA Distribution in Human Skin of AA-2G Cream AA-2G cream (after day 1) AA-2G cream (after day 3) Placebo cream (after day 3) AsA ■Method: 1) 2% AA-2G cream or 3% AA-2P cream was applied to the forearm of the male subject, then covered with cling film for 12 hr. 2) Placebo cream was applied to the subject under the same conditions. 3) The skin samples fixed with formalin were stained by the Reticulin silver impregnation method on the indicated day. AA-2P cream (after day 1) AA-2P cream (after day 3) [Kumano and Yamamoto et al., J. Nutr. Sci. Vitaminol., 44, 345 (1998)]

Conclusion

Potential Use of AA-2G 1. Maintenance of the Skin Resilience - Enhancement of collagen synthesis 2. Whitening Effect - Inhibition of tyrosinase activity - Antioxidant activity (Reducing action) 3. Prevention of Inflammation and Photoaging in the Skin - Antioxidant activity ( Scavenging action of hydroxy radicals)

Conclusion - AA-2G was originally developed as a melanin suppressing pharmaceutical agent (quasi-drugs) for whitening cosmetics in Japan. - AA-2G is also very effective in maintaining the skin resilience　and suppressing skin cell damage due to UV (one of the major causes of skin aging). - AA-2G has been shown to have very dramatic effects on fundamental biological processes. Its application in skincare products, other than as a whitening agent, such as sunscreen products for everyday use, cosmetics to prevent photoaging, and antiwrinkle cosmetics is supported by several studies.