Overview of AA2G

2-O--glucopyranosyl-L-ascorbic acid

May, 2014

TABLE OF CONTENTS
1. Introduction ................................................................................................ 1
2. Functions and applications ........................................................................ 1
2.1. AA2G reduces cell damage induced by H2O2 ..................................... 1
2.2. The effect of AA2G on SA--gal activity in NHDF cells ....................... 3
2.3. The effect of AA2G on SIRT1 expression in NHDF cells .................... 4
2.4. The effect of AA2G on p53 and p21 expression in NHDF cells ........... 5
2.5. Reduce UVB damage ......................................................................... 6
2.6. Effects on DNA synthesis and cell proliferatio..................................... 6
3. Stability studies.......................................................................................... 7

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1. Introduction

2-O--glucopyranosyl-L-ascorbic acid (AA2G) was produced by the action of the

cyclomaltodextrin glucanotransferase). AA2G is very stable in aqueous solutions and
exerts its biological activities after being hydrolyzed to ascorbic acid by mammalian
-glucosidase upon addition to cell cultures or gastrointestinal absorption. After receiving
approval as a quasi drug, AA2G has been used in cosmetics and as a food additive.
AA2G offers several advantages. First, in humans, AA2G is more readily absorbed. For
instance, AA2G is superior to ascorbyl ester derivatives in cosmetic applications because
it can be easily absorbed by the skin and easily degraded in vivo to release L-AA and
D-glucose, both of which are readily and safely absorbed. In addition, because of the
simple reaction steps, high regiospecificity, and low production cost, enzymatic production
of AA2G is more attractive than chemical synthesis of the other VC derivatives. Therefore,
AA2G is considered the best VC derivative and has recently received increasing attention.
AA2G exhibits almost all of the in vivo bioactivity of VC. When products containing AA2G
are used on the skin, the action of -glucosidase gradually releases VC, providing the
benefits of VC over a prolonged period of time. Moreover, AA2G can enhance antibody
production and collagen synthesis due to its structural integrity, so it plays an important
role in maintaining skin elasticity and repairing damaged skin. Most importantly, because
of the protection of the C-2 glycosyl, AA2G is extremely stable in response to prolonged
oxidation and heating in aqueous solutions and is non-reductive in its intact form.

2. Functions and applications

2.1. AA2G reduces cell damage induced by H2O2

We investigated the effects of AA2G on NHDF cell growth and against H2O2-induced cell
damage. Pretreatment of NHDF cells withAA2G for 72 h significantly promoted the cell
growth in a dosedependent manner (Fig. 1A, open squares). AA2G at 200 M increased
the NHDF cell growth by 140% when compared to untreated NHDF cells. Similar results
were obtained when the culture medium containing AA2G was replaced frequently (every
24 h) during the pretreatment culture (Fig. 1B, open squares). Pretreatment of NHDF cells
with ascorbic acid for 72 h by frequent medium exchange also promoted the cell growth.
In this culture system, significant promotion of NHDF cell growth was observed even at
low concentrations where AA2G did not promote the cell growth (Fig. 1B, open circles).
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However, its growth-promotion potential was not observed when ascorbic acid was added
only at the initiation of 72 h-pretreatment (Fig. 1A, open circles).

Fig.1
When NHDF cells were exposed to 250 MH2O2 for 24 h, cell survival decreased to 40%
of untreated NHDF cells, indicating that H2O2 induced oxidative cell damage. Pretreatment
of NHDF cells with AA2G significantly protected against cell damage induced by H2O2 in a
dose-dependent manner (Fig. 2A, B, open squares). Protective effects of AA2G against
H2O2-induced cell damage were similarly observed regardless of pretreatment conditions.
When NHDF cells were pretreated with 100 M AA2G for 72 h, only marginal cell damage
was observed after H2O2 exposure. Pretreatment with ascorbic acid for 72 h by frequent
medium exchange also significantly protected NHDF cells against cell damage induced by
H2O2 exposure even at low concentrations (Fig. 2B, open circles). However, as expected
from the proliferation data, the protective effect of ascorbic acid disappeared when
ascorbic acid was added at the initiation of 72 hpretreatment (Fig. 2A, open circles).
These results suggest that the protective effect of AA2G against H2O2-induced cell
damage is longer-lasting than that of ascorbic acid, reflecting stability of AA2G and
sustained release of ascorbic acid from AA2G.
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Fig. 2

2.2. The effect of AA2G on SA--gal activity in NHDF cells

We then examined activity of the cellular senescence biomarker SA--gal in
H2O2-exposed NHDF cells. Pretreatment with AA2G for 72 h slightly but significantly
decreased SA--gal activity in H2O2-unexposed NHDF cells (Fig. 3A, open squares).
When NHDF cells were exposed to 100 M H2O2 for 2 h, SA--gal activity significantly
increased to 150170% as compared with unexposed cells (Fig. 3A,B). A 72
h-pretreatment of NHDF cells with ascorbic acid or AA2G before H2O2 exposure
significantly inhibited the increase in SA--gal levels in a dose-dependent manner (Fig.
3A). AA2G at 100 M and 200 M reduced SA--gal activity comparable to that of control
culture without H2O2 exposure. Next, we examined whether AA2G or ascorbic acid
reduces SA--gal activity after H2O2 exposure. As shown in Fig. 3B, a 48 h-posttreatment
with AA2G dose-dependently inhibited the increase in SA--gal levels induced by H2O2
exposure. Posttreatment with ascorbic acid also reduced the increase in SA--gal levels.
However, no clear doseresponse effect was observed. These data suggest that both
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ascorbic acid and AA2G prevent oxidative stress-induced cellular senescence.However,

AA2G showed a greater potential for preventing cellular senescence than ascorbic acid.

Fig.3
2.3. The effect of AA2G on SIRT1 expression in NHDF cells
Since AA2G efficiently reduced H2O2-induced increase in SA--gal levels in NHDF cells,
we further analyzed its anti-aging effects.For this purpose, we investigated the expression
levels of SIRT1, which is the human homolog of yeast silent mating type information
regulator (Sir2) and a protein that modulates lifespan. Neither ascorbic acid nor AA2G
alone affected SIRT1 expression when they were added to cell cultures (Fig. 4). In
contrast, when NHDF cells wereexposed to H2O2, SIRT1 expression levels were
significantly decreased compared to unexposed cells. Pretreatment of NHDF cells with
200 M AA2G for 72 h significantly inhibited the H2O2-induced decrease in SIRT1
expression. However, pretreatment with 200 M ascorbic acid did not inhibit the
H2O2-induced decrease of the SIRT1 expression. These results suggest that AA2G
prevents oxidative stress-induced cellular senescence in terms of both SA--gal activity
and SIRT1 expression.
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Fig.4
2.4. The effect of AA2G on p53 and p21 expression in NHDF cells
To further clarify the anti-aging effect of AA2G, we examined the expression levels of p53
and p21 in H2O2-exposed NHDF cells (Fig. 5). Constitutive expression of p53 was
observed in AA2G untreated cells. Its expression level was elevated by H2O2 exposure,
and reached maximum levels at 4 h postexposure. Pretreatment with 200 M AA2G for 72
h resulted in a decrease in both constitutive and H2O2-induced expression of p53.
Exposure of NHDF cells to H2O2 also resulted in increased expression of p21 in AA2G
untreated cells compared with that in H2O2-unexposed cells. The expression reached
maximum levels at 4 h postexposure. These results suggest that cell cycle arrest at G1
phase is induced by H2O2-exposure. Pretreatment with 200 M AA2G inhibited the
expression of p21 in the H2O2-exposed cells.

Fig.5

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2.5. Reduce UVB damage

Environmental exposure to ultraviolet light B (UVB, wave lengths 290-320 nm) of the solar
spectrum causes major damage, including an inflammatory response, in skin. In the
present study, we estimated the ability of a stable derivative of ascorbic acid, ascorbic
acid 2-O-alpha-glucoside (AA2G), to reduce UVB damage, using the human keratinocyte
cell line, SCC, established from squamous cell carcinoma. By pre- (9 h) and
post-cultivation with AA2G, a significant preventive effect on the decrease in the absolute
number of surviving cells by exposure to UVB (typical dose, 20 mJ/cm2) was measurable
by a neutral red-uptake assay. The release of lactate dehydrogenase from the cell
membrane damaged by UVB was inhibited by AA2G. In agarose gel electrophoresis,
relatively high molecular weight DNA fragments were detected in irradiated cells after 6 h
post-irradiation, suggesting that the mechanism of cell death was necrosis. Quantitative
analysis of DNA content by flow cytometry indicated that AA2G suppressed both an
increase in debris with degraded nuclei and a decrease in cells in G1 and S phases, but
not in the G2/M phase, by UVB exposure. These data suggest that AA2G shows a
photoprotective effect against UVB-induced damage in human epithelial cells.

2.6. Effects on DNA synthesis and cell proliferatio

We examined the effects of L-ascorbic acid and its analogues on DNA synthesis and cell
proliferation. We also investigated the signal transduction pathways involved in the
induction of mitogenesis by L-ascorbic acid and its analogues using primary cultures of
adult rat hepatocytes. Following a 4-h serum-free cultivation, both L-ascorbic acid and its
stable analogue, L-ascorbic acid 2-glucoside, time- and dose-dependently stimulated
hepatocyte DNA synthesis and cell proliferation, with EC values of 6.4610 M and
3.3410 M, respectively. Dehydroascorbic acid (10 M-10 M) weakly stimulated
hepatocyte mitogenesis, whereas isoascorbic acid (10 M-10 M) had no effect.
Hepatocyte mitogenesis induced by L-ascorbic acid or L-ascorbic acid 2-glucoside was
dose-dependently abolished by treatment with monoclonal antibodies against insulin-like
growth factor (IGF)-I receptor, but not by treatment with monoclonal antibodies against
insulin receptor or IGF-II receptor. Western blot analysis showed that both L-ascorbic acid
and L-ascorbic acid 2-glucoside significantly stimulated IFG-I receptor tyrosine kinase
activity within 3 min, and mitogen-activated protein (MAP) kinase activity within 5 min.
These results demonstrate that both L-ascorbic acid and L-ascorbic acid 2-glucoside
induce DNA synthesis and cell proliferation in primary cultures of adult rat hepatocytes by
interacting with the IGF-I receptor site and by activating the receptor tyrosine kinase/MAP
kinase pathway.

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3. Stability studies

Ascorbic acid 2-glucoside (AA2G) has been widely used in cream and lotion types of
cosmetic products. Thus, the degradation of AA2G caused by the temperature change
and pH variation was very critical for determining the bio-functionality of cosmetics.
Response surface methodology (RSM) was introduced to study the influence of
temperature and pH on the stability of AA2G. The optimal condition of retaining AA2G with
the highest stability was determined to be 55.3 and pH 6.4.

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Reference
1. Mutsuko Taniguchi , Norie Arai, Keizo Kohno, Shimpei Ushio, Shigeharu Fukuda.
Anti-oxidative and anti-aging activities of 2-O--glucopyranosyl-L-ascorbic acid on
human dermal fibroblasts. J European Journal of Pharmacology 674 (2012) 126-131.
2. Ruizhi Han & Long Liu & Jianghua Li & Guocheng Du & Jian Chen. Functions,
applications and production of 2-O-D-glucopyranosyl-L-ascorbic acid. J Appl Microbiol
Biotechnol (2012) 95:313320.
3. Wen-Ying Huang, Pei-Chi Lee, Ling-Kuei Huang, Li-Ping Lu, Wayne C. Liao. Stability
studies of ascorbic acid 2-glucoside in cosmetic lotion using surface response
methodology. J Bioorganic & Medicinal Chemistry Letters 23 (2013) 15831587.

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