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Topical L‐Ascorbic Acid: Percutaneous Absorption Studies

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  • Kansas State University and North Carolina State University

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Background: Reactive oxygen species generated by ultraviolet light result in photocarcinogenic and photoaging changes in the skin. Antioxidants protect skin from these insults. Objective: This study defines formulation characteristics for delivering L-ascorbic acid into the skin to supplement the skin's natural antioxidant reservoir. Methods: L-ascorbic acid or its derivatives were applied to pig skin. Skin levels of L-ascorbic acid were measured to determine percutaneous delivery. Results: L-ascorbic acid must be formulated at pH levels less than 3.5 to enter the skin. Maximal concentration for optimal percutaneous absorption was 20%. Tissue levels were saturated after three daily applications; the half-life of tissue disappearance was about 4 days. Derivatives of ascorbic acid including magnesium ascorbyl phosphate, ascorbyl-6-palmitate, and dehydroascorbic acid did not increase skin levels of L-ascorbic acid. Conclusions: Delivery of topical L-ascorbic acid into the skin is critically dependent on formulation characteristics.
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© 2001 by the American Society for Dermatologic Surgery, Inc. Published by Blackwell Science, Inc.
ISSN: 1076-0512/01/$15.00/0 Dermatol Surg 2001;27:137–142
Topical L-Ascorbic Acid: Percutaneous Absorption Studies
Sheldon R. Pinnell, MD,* Huanshu Yang, MD,
Mostafa Omar, PhD,
Nancy Monteiro Riviere, PhD,
Holly V. DeBuys, MD,* Linda C. Walker,*
Yaohui Wang, MD,
§
and Mark Levine, MD
§
*
Duke University Medical Center, Durham, North Carolina,
PhytoCeuticals, Elmwood Park, New Jersey,
College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, and
§
National Institute of Diabetes & Digestive & Kidney Diseases, NIH, Bethesda, Maryland
background.
Reactive oxygen species generated by ultraviolet
light result in photocarcinogenic and photoaging changes in the
skin. Antioxidants protect skin from these insults.
objective.
This study defines formulation characteristics for
delivering L-ascorbic acid into the skin to supplement the skin’s
natural antioxidant reservoir.
methods.
L-ascorbic acid or its derivatives were applied to pig
skin. Skin levels of L-ascorbic acid were measured to determine
percutaneous delivery.
results.
L-ascorbic acid must be formulated at pH levels less
than 3.5 to enter the skin. Maximal concentration for optimal
percutaneous absorption was 20%. Tissue levels were saturated
after three daily applications; the half-life of tissue disappear-
ance was about 4 days. Derivatives of ascorbic acid including
magnesium ascorbyl phosphate, ascorbyl-6-palmitate, and dehy-
droascorbic acid did not increase skin levels of L-ascorbic acid.
conclusions.
Delivery of topical L-ascorbic acid into the skin
is critically dependent on formulation characteristics.
IN THE PRESENCE of our oxygen-rich atmosphere,
ultraviolet light generates reactive oxygen species in
skin. In addition to sunlight, other inflammatory in-
sults including smoking and pollution generate reac-
tive oxygen species. Reactive oxygen species, in turn,
cause oxidation of nucleic acids, proteins, and lipids.
Reactive oxygen species alter DNA,
1–7
as well as its
repair,
8
and trigger cytokine cascades that result in
photoaging
9,10
and photocarcinogenesis.
11
The body protects itself naturally from reactive ox-
ygen species by using antioxidants to neutralize them
before they cause damage to the skin and its compo-
nents. Vitamin C, or L-ascorbic acid, is the most
abundant antioxidant in skin.
12
Although most plants
and animals synthesize L-ascorbic acid to protect
themselves from free radical attack, a gene necessary
for its synthesis, L-gulono-
-lactone oxidase, has been
mutated in humans.
13
As a result, humans rely on di-
etary intake for their supply.
14
L-ascorbic acid is quite
water soluble and serves as the major aqueous phase
reductant in the body.
15
Since skin relies on antioxidants for protection
against reactive oxygen species, and since skin pre-
dominantly receives and must deal with the free radi-
cal assault resulting from UV light, increasing the anti-
oxidant defense of skin becomes an attractive strategy
for increased photoprotection.
16
If antioxidants could
be delivered in high concentration through the stratum
corneum barrier into the skin, then the antioxidant
protective reservoir could be increased and photopro-
tection might be enhanced. Indeed, our laboratory has
described a stable aqueous formulation of L-ascorbic
acid that gets into skin and provides photoprotection
against both UVB and UVA-psoralen phototoxicity by
a mechanism that is clearly not a sunscreen effect.
17
Moreover, we have demonstrated that topical L-ascor-
bic acid protected against UV immunosuppression and
tolerance to contact antigen in mice.
18
In order to maximize the protective effects of topi-
cal L-ascorbic acid in skin, we have undertaken this
study of formulation composition and kinetics so that
we can maximize the amount of L-ascorbic acid deliv-
ered into the skin.
Materials and Methods
L-ascorbic acid (pharmaceutical grade) was purchased from
Roche (Nutley, NJ). All concentrations of L-ascorbic acid
were made fresh and stabilized in 2% ZnSO
4
, 0.5% biofla-
vonoids, 1% hyaluronic acid, 0.1% citrate in glass-distilled
water. pH was adjusted with triethanolamine. Commercial
formulations of 13% magnesium ascorbyl phosphate (Vivi-
H. Yang, MD, N. Monteiro-Riviere, PhD, H.V. DeBuys, MD, L.C.
Walker, Y. Wang, MD, and M. Levine, MD have indicated no signifi-
cant interest with commercial supporters. S.R. Pinnell, MD is a consult-
ant for Skinceuticals (Dallas, TX). M. Omar, PhD is president of Phyto-
Ceuticals (Elmwood Park, NJ).
Address correspondence and reprint requests to: Sheldon R. Pinnell,
MD, Duke University Medical Center, Department of Medicine, Divi-
sion of Dermatology, P.O. Box 3135, Durham, NC 27707, or e-mail:
pinne002@mc.duke.edu.
138
pinnell et al.: topical l-ascorbic acid
Dermatol Surg 27:2:February 2001
fying Serum C, Dr. Mary Lupo Skin Care Products, New
Orleans, LA) and 10% ascorbyl-6-palmitate (C-Esta Serum,
Jan Marini Skin Research, San Jose, CA) were obtained
fresh from the manufacturer and opened just prior to test-
ing. Samples were tested as is; the content was not con-
firmed. Dehydroascorbic acid 1 M (17.4%) was prepared
by taking 1 ml of a 1 M ascorbate solution, adding bromine
55
l, vortexing vigorously for 30 seconds, and then imme-
diately bubbling with nitrogen for 10 minutes.
19
The institu-
tional review board of the College of Veterinary Medicine at
North Carolina State University approved the animal exper-
iments.
Experiments were conducted in white Yorkshire pigs.
The skin was shaved with an electric shaver 24 hours before
the experiment began to allow healing of any skin nicks.
Two hundred
l of formulation, the maximal volume of the
chamber, was placed under a Hill Top Chamber (Hill Top
Co., Cincinnati, OH) for 22–24 hours. The chamber is semi-
occlusive and protects the material from smearing. Although
the chamber may enhance percutaneous absorption, in sev-
eral cases in the experimental results, the baseline skin levels
of L-ascorbic acid were not increased. In some experiments
the chamber was replaced with fresh solution and changed
each day. In clinical practice, vitamin C solutions are usually
applied daily. At the end of the experiment, the formulation
was washed vigorously from the skin with water. Washed
skin was tape stripped 15 times to remove surface contami-
nation and stratum corneum. The tape strips were dis-
carded. Studies have shown that tape stripping removes the
stratum corneum layers (data not shown) and removes sur-
face radioactivity of topically applied substances bound to
the stratum corneum.
20
,
21
Full-thickness 6 mm punch biopsy
specimens of skin were taken and placed immediately into
liquid nitrogen. Tissue was shattered in liquid nitrogen and
weighed aliquots extracted and stabilized in 60% methanol,
1 mM EDTA in water, centrifuged, and kept at
70
C until
analyzed. Samples were analyzed in a blinded manner for
vitamin C by high-performance liquid chromatography
(HPLC) with coulometric electrochemical detection.
22
,
23
The
method is specific for L-ascorbic acid and has a sensitivity of
50 fmol. The results are expressed as mean
standard devi-
ation. Unless otherwise noted,
n
3. The
P
values were cal-
culated by two-tailed Student’s
t
-test with equal variance.
Results
pH
Fifteen percent formulations of L-ascorbic acid were
tested at pH levels between 2.0 and 5.0 (Figure 1). Tis-
sue levels of L-ascorbic acid were enhanced only at for-
mulation pH levels less than 3.5. The pKa for L-ascorbic
acid is 4.2. Apparently the molecule must be un-ionized
for percutaneous absorption to occur. Low pH is es-
sential for absorption and delivery is enhanced as the
pH is reduced to 2.0. The effect of the solutions on
skin pH is unknown.
Concentration
L-ascorbic acid concentrations were tested from 5 to
30.0% (Figure 2). pH was adjusted to 3.2. Tissue lev-
els of L-ascorbic acid increased and were maximal at
20%. For unknown reasons, concentration levels
higher than 20% resulted in decreased tissue levels.
Kinetics
Fifteen percent L-ascorbic acid at pH 3.2 was applied
daily for 1–5 days (Figure 3). After 3 days, tissue levels
were apparently saturated. Levels achieved were ap-
proximately 20 times normal tissue levels.
Washout
Fifteen percent L-ascorbic acid at pH 3.2 was applied
daily for 5 days to saturate skin levels (Figure 4). Skin
levels of L-ascorbic acid were then measured at daily
intervals, with no further topical application of L-ascor-
bic acid, to measure L-ascorbic acid remaining in the
tissues. Half-life of L-ascorbic acid in tissues was found
to be approximately 4 days.
Figure 1. Effect of pH on percutaneous absorption. 15% L-ascorbic
acid at different pHs were applied to pig skin for 24 hours. Skin
levels of L-ascorbic acid are expressed as mean SD (n 3). *Av-
erage (n 2).
Figure 2. Effect of concentration on percutaneous absorption.
Varying concentrations of L-ascorbic acid pH 3.2 were applied to
pig skin for 24 hours. Skin levels of L-ascorbic acid are expressed as
mean SD (n 3).
Dermatol Surg 27:2:February 2001
pinnell et al.: topical l-ascorbic acid
139
Ascorbic Acid Derivatives
Because L-ascorbic acid is an unstable molecule to
formulate for topical use, more stable derivatives of
L-ascorbic acid have been utilized in topical formula-
tions. Although esters of ascorbic acid are more stable
and readily converted to L-ascorbic acid after oral in-
gestion, it is not clear that derivatives, after topical ap-
plication, are absorbed into the skin or converted to
L-ascorbic acid after penetration. We have tested com-
mercially available high concentration formulations of
magnesium ascorbyl phosphate and ascorbyl-6-palmi-
tate to see if topical application resulted in elevated
skin levels of L-ascorbic acid (Figure 5). Neither ester
significantly increased L-ascorbic acid skin levels.
Dehydroascorbic Acid
Since dehydroascorbic acid can be enzymatically con-
verted to L-ascorbic acid in the body, we asked
whether topical dehydroascorbic acid could preferen-
tially raise skin L-ascorbic acid levels. Neither 20 mM
nor 1 M solutions of dehydroascorbic acid were effec-
tive. Skin levels of L-ascorbic acid were 7.51
3.34
pmol/mg for 20 mM dehydroascorbic acid and 8.70
2.13 pmol/mg for 1 M dehydroascorbic acid (
n
4)
and 9.24
3.55 for control skin.
Discussion
L-ascorbic acid is the most plentiful antioxidant in
body fluids
15
and in the skin.
12
It efficiently neutralizes
reactive oxygen species including superoxide anion,
24
hydroxyl radical,
25
singlet oxygen,
26
and peroxyni-
trite.
27
It is a particularly efficient antioxidant because
in one electron transfer reaction, its free radical inter-
mediate, ascorbic acid free radical, has low pro-oxi-
dant activity
28
and is enzymatically regenerated back
to L-ascorbic acid. Moreover, L-ascorbic acid’s effi-
ciency extends to lipophilic antioxidants as well; it re-
generates oxidized vitamin E molecules.
29,30
Maxi-
mum skin levels of L-ascorbic acid from ingestion are
regulated by active transport mechanisms that pre-
clude increasing levels by further ingestion.
31
In this
study we identify formulation characteristics that al-
low us to bypass these controls and increase the skin
reservoir by direct topical application.
This study reveals the critical importance of formu-
lation pH for percutaneous absorption of L-ascorbic
acid. Not until the pH was 3.5 or lower were cutane-
ous levels increased. Since the pKa of L-ascorbic acid
is 4.2, the molecule apparently must be un-ionized for
delivery across the stratum corneum barrier.
32
Percu-
taneous absorption of varying L-ascorbic acid concen-
trations formulated at acid pH increased steadily to a
maximum of 20%. Higher concentrations were less ef-
fective for unknown reasons. Daily application for 3
days of 15% L-ascorbic acid formulated at pH 3.2 re-
sulted in saturating skin concentrations of L-ascorbic
acid at more than 20 times control values. After satu-
rating the skin reservoir, the L-ascorbic acid was ap-
Figure 3. Time course of percutaneous absorption. 15% L-ascorbic
acid pH 3.2 was applied daily to pig skin for varying times. Skin
levels of L-ascorbic acid are expressed as mean SD (n 3).
Figure 4. Washout of skin L-ascorbic acid. Skin levels of L-ascorbic
acid were saturated by five daily applications to pig skin of 15%
L-ascorbic acid pH 3.2. After varying intervals skin levels of L-ascor-
bic acid were determined. Skin levels of L-ascorbic acid are ex-
pressed as mean SD (n 3).
Figure 5. Percutaneous absorption of ascorbic acid derivatives.
15% L-ascorbic acid pH 3.2 (VC-15), 10% ascorbyl-6-palmitate
(C-Esta), and 12% magnesium ascorbyl phosphate (Mag-C) were
applied to pig skin for 24 hours. Skin levels of L-ascorbic acid are
expressed as mean SD (n 10). P values are expressed versus
control. The P value of VC-15 versus each product is also 0.0005.
140
pinnell et al.: topical l-ascorbic acid
Dermatol Surg 27:2:February 2001
parently stabilized and remained in the tissue with a
half-life approaching 4 days. We have no data about
the relative distribution of ascorbic acid in the skin. A
persistent reservoir of antioxidant provides an impor-
tant and attractive photoprotection strategy when
contrasted to sunscreens which must be applied daily.
In the body both L-ascorbic acid and dehydroascor-
bic acid can be transported into cells, the latter con-
verted efficiently into L-ascorbic acid by glutathione
and enzymatic reduction.
33
L-ascorbic acid requires a
specific protein to be transported into cells. Hexose
transporters transport dehydroascorbic acid. Indeed
dehydroascorbic acid is preferentially accumulated in
comparison to L-ascorbic acid in HaCaT, a human
keratinocyte cell line.
34
Our experiments to deter-
mine whether dehydroascorbic acid was preferable to
L-ascorbic acid for topical use failed to reveal any in-
crease in skin levels of L-ascorbic acid with dehydro-
ascorbic acid.
Topical magnesium ascorbyl phosphate and ascor-
byl-6-palmitate in the tested formulations failed to in-
crease skin levels of L-ascorbic acid. Previous studies
have documented the marginal percutaneous absorp-
tion of magnesium ascorbyl phosphate;
35
as a charged
molecule, it would not be expected to traverse the stra-
tum corneum. Previous studies of ascorbyl-6-palmitate
failed to demonstrate protection against photoaging in
mouse skin;
36
in comparison, L-ascorbic acid was pro-
tective even though the formulation used was not op-
timal for percutaneous delivery. Although ascorbyl-
6-palmitate appears to readily enter skin,
36
its conversion
to L-ascorbic acid may be inefficient. Ascorbyl-6-pal-
mitate appears to remain on the extracellular surface
of cells and may not be readily converted to L-ascorbic
acid.
37
Indeed in human skin fibroblast culture 10
5
M
L-ascorbic acid, which is the physiologic concentration
in humans, stimulated cell growth, whereas similar lev-
els of ascorbyl-6-palmitate were toxic.
38
Topical antioxidants have been previously demon-
strated to be photoprotective for skin. Topical L-ascor-
bic acid has been shown to decrease UVB erythema in
pig
17
and human skin.
39
It also lessened UVA-psoralen
phototoxic injury in pig skin.
17
In hairless mice, topi-
cal L-ascorbic acid decreased photoaging changes in
human
40
and hairless mouse skin.
36
In addition, it pre-
vented UVB immunosuppression and tolerance to di-
nitrochlorobenzene (DNCB).
18
In persons with either
basal cell carcinoma or squamous cell carcinoma, se-
rum levels of L-ascorbic acid were below control lev-
els.
41
Topical
-tocopherol decreased photoinjury in
skin
36,42–47
and prevented UV immunosuppression.
45,48,49
Herbal antioxidants, including silymarin, a flavonoid
present in the thistle plant,
50
and grape seed polyphe-
nols
51
have been shown to prevent UV-induced squa-
mous cell skin cancer in mice.
Although all UV light can produce oxidative stress
in skin, UVA is more efficient. The peak UV spectrum
for generation of singlet oxygen from trans-urocanic
acid, a known photoreceptor in skin, is about 350
nm.52 The UV spectrum is similar to that previously
demonstrated to generate photoaging changes in mouse
skin.53 UVA from artificial light sources has been dem-
onstrated to generate photoaging changes in sun-pro-
tected skin.54,55 Similar changes have been demonstrated
using only long-wave UVA (340–400 nm).56 Studies in
skin cells,57,58 as well as human skin,10 implicate acti-
vation of matrix metalloproteinase by a mechanism in-
volving singlet oxygen, AP-1, and NF-B. In prelimi-
nary studies, antioxidants reverse activation of AP-1.58,59
Previous studies have demonstrated that photoaging
changes are even more pronounced in smokers than
sunbathers, and the combination of smoking and sun
exposure was most damaging of all.60 Presumably smok-
ing and UV exposure are both damaging to skin by
generating reactive oxygen species. In smokers, serum
ascorbic acid levels were reduced;61 they required an
elevated minimum daily dose of L-ascorbic acid to
keep body stores saturated.26
In addition to its antioxidant effects, L-ascorbic
acid is important for wound healing.62,63 It is essential
for collagen synthesis; in addition to its cofactor re-
quirements for lysyl hydroxylase and prolyl hydroxy-
lase,64 it stimulates transcription of collagen genes.65 It
has been used as a skin lightener; it inhibits tyrosi-
nase.66 Topical L-ascorbic acid has been reported to
be useful for healing of skin resurfaced by CO2 laser;
it reduced postlaser erythema.67 Topical L-ascorbic
acid together with 20% glycolic acid used for 3
months improved striae alba.68
Topical L-ascorbic acid provides a safe and effec-
tive supplement to normal tissue stores to enhance
photoprotection, improve wound healing, and in-
crease antioxidant defenses. Details of formulation are
essential if it is to be maximally effective. It must be
formulated at high concentration and at a pH lower
than 3.5 to be effective. After being delivered into the
skin, L-ascorbic acid is stabilized and remains in the
tissue for a period of days. Magnesium ascorbyl phos-
phate and ascorbyl-6-palmitate are not effective sub-
stitutes for L-ascorbic acid in topical formulations. Al-
though they are effective vitamin C derivatives for oral
use, they are apparently ineffective for increasing tis-
sue vitamin C levels when applied to the skin.
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... Ascorbic acid is one of the most potent antioxidants in the skin, with a structure predisposing it to oxidation and instability under either aerobic or anaerobic conditions [17,18]. To address this, stabilized forms of AA have been developed, including l-ascorbyl 2-phosphate or l-ascorbyl 6-palmitate [19,20]. However, the advantages of their chemical stability are sometimes at the expense of a much lower biological activity than their base form, for example the αtocopherol salt [11]. ...
... However, the advantages of their chemical stability are sometimes at the expense of a much lower biological activity than their base form, for example the αtocopherol salt [11]. Indeed, some are not cleaved by the skin to the active antioxidants, ascorbic acid or αtocopherol; thus, they have lower activity [19,21]. Ester or ether derivatives of AA, for example magnesium-L-ascorbyl-2-phosphate, 3-O-ethyl-l-ascorbic acid, have been used to stabilize AA and are reported to be enzymatically converted to AA within the skin [22][23][24][25][26]. ...
... Ester or ether derivatives of AA, for example magnesium-L-ascorbyl-2-phosphate, 3-O-ethyl-l-ascorbic acid, have been used to stabilize AA and are reported to be enzymatically converted to AA within the skin [22][23][24][25][26]. Although in vivo and in vitro studies demonstrated antioxidants effects of this type of prodrugs after enzymatic cleavage, this does not always lead to an increase levels of AA in the skin, for example magnesium ascorbyl phosphate, ascorbyl-6-palmitate and dehydroascorbic acid [19]. ...
Article
Objective: Deleterious effects of pollutants and ultraviolet radiation on the skin can be attenuated using formulations containing antioxidants. However, these have disadvantages, including chemical instability, photodegradation, poor bioavailability or biological activity. Here, two commercial formulations were evaluated: one optimized to stabilize and deliver ascorbic acid (AA) at 15% and the other containing a glucoside form of AA, namely ascorbic acid 2-glucoside (AA2G), at 1.8% and at a physiological pH. We compared the skin delivery, antioxidative effects and chemical stability of AA2G with AA in their respective formulations. Methods: Skin delivery was measured using fresh viable human skin explants, and oxidative stress was measured using a human reconstructed epidermal (RHE) model according to levels of malondialdehyde (MDA), superoxide dismutase (SOD) and catalase. Results: Ascorbic acid 2-glucoside was completely metabolized to AA by the skin before entering the receptor compartment. The skin contained parent and AA, indicating a reserve of AA2G was present for further metabolism. For AA2G and AA, maximum flux of AA-equivalents was at 12 h, with continued absorption over 24 h. The absolute amount in µg was higher in the skin after application of AA than after application of AA2G. This may suggest a greater antioxidative effect; however, according to all three measurements of oxidative stress, the protective effect of AA and AA2G was similar. Unlike AA, AA2G was chemically stable under storage conditions. Conclusion: A lower concentration of AA2G is as effective as the active metabolite, AA, in terms of antioxidant effects. AA2G was chemically stable and can be applied at a lower concentration than AA, thus avoiding the need for an acidic formulation with a pH below 3.5.
... Antioxidants can best be replenished or delivered to the skin by topical application giving far higher concentrations than attained by oral ingestion. For example, L-ascorbic acid (vitamin C) 15% gives 27-40 times the skin concentration resulting from high oral intake [66] , d-α-tocopherol 5% (the only one of 32 forms of vitamin E that is effective on skin), by a factor of 12 [67] , and selenium (L-selenomethionine 0.05%), by a factor of 8 [68] . These three antioxidants have been extensively studied and proven to prevent and even reverse photoaging of the skin in mice [67,68,69] , pigs [70] , and humans [71,72] , and to inhibit UV-induced skin cancer in mice [67,68,69,73] . ...
... However, stringent criteria are required for topical formulations to be stable, to be successfully absorbed, as to be active as antioxidants. Esterified forms of vitamins C [66] and E [67] are not absorbed transcutaneously, and the ester is not reduced to the -OH form required for antioxidant activity. Only the isomer d-α-tocopherol is effective; the other 31 isomers (eight "dl" isomer configurations and four α, β, γ, δ forms) are not [67] . ...
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The appearance of aging is determined primarily by extrinsic factors through exposure to environmental sunlight and airborne pollution. That solar ultraviolet B (λ = 290-320 nm) directly causes photoaging (with wrinkles, dryness, and mottled pigmentation) and skin cancer has been recognized for decades; the contribution by ultraviolet A (λ = 320-400 nm) was only more recently understood. New research further implicates visible light (λ = 400-700 nm) as well as the heat rays of infrared radiation (λ > 800 nm). Particularly in urban environments, airborne pollutants such as ozone (O3), polycyclic aromatic hydrocarbons, particulate matter (PM) in smog, and tobacco smoke contribute to photoaging and skin cancer. Furthermore, exposure simultaneously to both solar ultraviolet (UV) and these pollutants results in even greater synergistic damage. The volatile pollutants generate reactive oxygen species which oxidize surface lipids leading to deeper damaging inflammatory reactions. PM carries high concentrations of environmental organic compounds and trace metals. These pollutant-laden particles deliver toxins to the skin transcutaneously through hair follicles and through the blood after respiratory inhalation. The predominant natural mechanism of clearing these xenobiotic chemicals is through the ligand-activated transcription factor the arylhydrocarbon receptor (AHR) found on all skin cells. AHR activity regulates keratinocyte differentiation and proliferation, maintenance of epidermal barrier function, melanogenesis, and immunity. With chronic activation by UV exposure and pollutants, AHR signaling contributes to both extrinsic aging and carcinogenesis.
... 14 Vitamin C, L-ascorbic acid, is a hydrophilic antioxidant that acts as an ROS scavenger after UV exposure, and it reduces the formation of sunburn cells and erythema. 15,16 Efficacious percutaneous absorption of Lascorbic acid depends on its formulation and the delivery vehicle when applied topically to the skin. Among various derivatives and isoforms, lipophilic esterified L-ascorbic acid has exhibited the highest propensity for percutaneous absorption. ...
... 17 Adding α-tocopherol and ferulic acid stabilizes the formulation and enhances photoprotection. 15,16 Furthermore, L-ascorbic acid is involved in stimulating collagen biosynthesis. ...
... In addition to the aspects discussed so far, regarding oxidative stress in HT, vitamin C cannot be left aside, as it has high non-enzymatic antioxidant potential reported in literature. Thus, it is found in a reduced form, as ascorbic acid, or in an oxidized form, dehydroascorbic acid (DHA), and is not synthetized by humans; it is only obtained through diet [149,150]. Vitamin C has pleiotropic effects acting, besides as antioxidant substance, as an anti-inflammatory and immunesupporting component, as well as a cofactor for mono and di-oxygenase enzymes. All of the previous functions reduce cellular and organ harms [151]. ...
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A number of studies have shown that oxidative stress is related to the pathogenesis of several immunological diseases, such as Hashimoto’s thyroiditis (HT), although there is no plausible mechanism to explain it. Thus, we aimed at hypothesizing and providing some possible mechanisms linking oxidative stress to autoimmunity aspects and its implications for HT, as well as adjuvant therapeutic proposals to mitigate the deleterious effects. Our hypothesis is that deficient eating habits, autoimmune regulator gene predisposing gene, dysbiosis and molecular mimicry, unfolded proteins and stress in the endoplasmic reticulum, and thymus involution appear to be the main potential factors leading to HT oxidative stress. Likewise, we show that the use of minerals selenium and zinc, vitamins D and C, as well as probiotics, can be interesting adjuvant therapies for the control of oxidative damage and poor prognosis of HT. Further clinical trials are needed to understand the real beneficial and side effects of these supplements. Graphical abstract
... VitC itself demonstrates poor skin absorption when used in normal cosmetic formulations with skin compatible pH of 5-6. At pH of less than 4, vitC molecule is neutral because all hydroxyl groups on vitC molecule are not deprotonated, and the molecule can be more easily absorbed into the skin 20,22 . This, however, contradicts the recommended non-irritating pH range for skincare products. ...
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Delivering bioactive compounds into skin tissue has long been a challenge. Using ex vivo porcine and rat skins, here we demonstrate that a detachable dissolvable microneedle (DDMN) array, a special dissolvable microneedle that allows needle detachment from the base within 2 min post administration, can effectively embed a model compound into epidermis and dermis. Diffusion of the compound from the needle embedding sites to the nearby skin tissue is demonstrated at various post administration periods. The relationship between the time that a conventional dissolvable microneedle array is left on skin without needle detachment from the base and the degree of skin surface abrasion at each microneedle penetration spot is also demonstrated on skin of human volunteers. Co-loading glutathione with vitamin C (vitC) can stabilize vitC in the DDMN. DDMN loaded with vitC and glutathione can help erasing post-acne-hyperpigmentation spots.
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Background: Post acne scars following sebaceous injury and abnormal wound healing during the course of acne is a prevalent and challenging to treat condition. Aim of the work: To evaluate microneedling by dermapen with topical vitamin C versus microneedling with topical insulin in treating atrophic post-acne scars. Patients and methods: A split -face comparative study included 30 subjects with atrophic post-acne scars. Human insulin was topically applied to the left side of the face and on the right side, vitamin C serum was applied. Scars were assessed via the Acne Scar Assessment Scale (ASAS) and Scar quartile grading scale (SQGS). Results: After one month of 4 treatments, a statistically significant mean improvement in ASAS value was reported on both split sides of the face (2.13 and 1.83) compared to baseline (3.03 and 2.93) (p=0.005; p=0.001 respectively). When compared to baseline, the mean ASAS value improved significantly with a slight more improvement on the vitamin c treated side CONCLUSION: Topical insulin and vitamin c combined with microneedling, may both achieve comparable significant improvement for treating post acne scars. Insulin can be a promising novel anti-scarring therapy pending larger controlled studies to verify its efficacy. This article is protected by copyright. All rights reserved.
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Objective: This study aimed to develop and validate a rapid, simple, accurate, and precise analytical method for the quantification of L-AA in vitamin C serums. Moreover, the developed method was further applied to determine L-AA in eight different brands of vitamin C serums. A complementary study was also carried out to evaluate the stability of L-AA in the vitamin C serum samples after 15, 30, 45, and 60 days of storage at ambient temperature (15 ºC to 35 ºC). Methods: Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was applied. Results: Quantitative analyses were performed with a total chromatographic run time of 1.5 min by matrix-matched calibration, and the analytical curve was linear over the range of 1-1700 μg L-1 with a correlation coefficient of 0.9998. The limits of detection (LOD) and quantification (LOQ) were 0.3 and 1.0 μg L-1 , respectively. Intra- and inter-assay precisions, expressed in terms of relative standard deviation (RSD), ranged from 0.3% and 2.2%, respectively, and recoveries in two concentration levels (1 and 5 µg L-1 ) were 103.9% and 101.2%, respectively. The proposed analytical method was successfully applied to determine de L-AA content of eight commercial vitamin C serum samples. The stability of the target analyte in samples stored at ambient temperature (15 ºC to 35 ºC) was evaluated throughout 60 days with a 15-day interval between analyses. At 0 days, L-AA content in samples ranged from 1.05 - 169.91 mg L-1 , decreasing over time. Conclusion: The proposed method could be powerful in routine analyses to ensure the quality of L-AA vitamin C serums since it proved a simple, reliable, fast, precise, accurate, and sensitive analytical method.
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Delivering bioactive compounds into skin tissue has long been a challenge. Using ex vivo porcine and rat skins, here we demonstrate that a detachable dissolvable microneedle (DDMN) array, a special dissolvable microneedle that allows needle detachment from the base within 2 min post administration, can effectively embed a model compound into epidermis and dermis. Diffusion of the compound from the needle embedding sites to the nearby skin tissue is demonstrated at various post administration periods. The relationship between the time that a conventional dissolvable microneedle array is left on skin without needle detachment from the base and the degree of skin surface abrasion at each microneedle penetration spot is also demonstrated on skin of human volunteers. Co-loading glutathione with vitamin C (vitC) can stabilize vitC in the DDMN. DDMN loaded with vitC and glutathione can help erasing post-acne-hyperpigmentation spots.
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Vitamin C is a popular ingredient in over‐the‐counter cosmeceuticals due to its many biological functions in maintaining and improving skin health by treating UV damage, improving discoloration, and boosting collagen production. Several chemically modified derivatives of vitamin C have been developed in an attempt to increase the stability, percutaneous absorption, and overall activity of this ingredient in topical formulations. The goal of this review is to evaluate the differences between vitamin C derivatives that have been designed for cosmeceutical use and their efficacy.
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The pig has been a well accepted model for cutaneous absorption and toxicity studies because the integument is morphologically and functionally similar to human skin. We will review how the pig is utilized in this field, with particular reference to studies conducted in our laboratory. There has been considerable debate as to whether the topical effect of an antiinflammatory drug is due to direct penetration or is secondary to systemic absorption and recirculation. We addressed this question by studying the mechanism of topical penetration of piroxicam, a nonsteroidal antiinflammatory drug, in the pig. The pivotal role of the cutaneous vasculature on absorption will be further discussed and contrasted to in vitro systems. Pigs have been accepted as a model for studying iontophoretic drug delivery in humans. Transdermal delivery permits administration of therapeutic compounds that would not normally penetrate the skin. The pig is a principal animal model used in this field. The pathway a drug follows during delivery has been controversial and is of toxicological significance. We have defined the precise anatomical pathway which mercuric chloride traverses the stratum corneum during iontophoresis. In addition, new research methodologies have been applied in cutaneous toxicology, partly because of the driving force to replace in vivo animal models, with more humane in vitro approaches. The isolated perfused porcine skin flap (IPPSF), developed in our laboratory, is a perfused skin model which allows for in vitro cutaneous pharmacology and toxicology studies to be conducted in a viable system that has a morphological and functional microcirculation. A porcine skin model developed for use in phototoxicology will be characterized. Finally, studies involving use of this preparation to assess the toxicity of chemical vesicants will be discussed. These examples are illustrative of the dominant role of the pig as an animal model for human skin biology.
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L-ascorbic acid (ascorbate or vitamin C) is a required nutrient for humans. Absorption, transport, and disposition of ingested ascorbate involve the following: (1) bioavailability and absorption in the gastrointestinal tract; (2) presence in the circulation; (3) tissue distribution; (4) excretion; and, (5) metabolism. Fundamental to each of the above are ascorbate chemistry and mechanisms of transport of ascorbate across membranes. Ascorbate can be reversibly oxidized to dehydroascorbic acid, which can be irreversible degraded. Both reduced and oxidized forms cross cell membranes. Differences in transport kinetics, tissue specificity, and Na+ and energy dependence strongly support the existence of separate transport mechanisms. An important consideration in the analysis of ascorbate transport iss that of substrate availability. Reduced ascorbate is by far the most predominant form found in plasma and tissues. Dehydroascorbic acid is rapidly reduced intracellularly to ascorbate by both enzymatic and chemical mechanisms. Despite constitutively low levels of dehydroascorbic acid, conditions that promote oxidation of ascorbate can profound alter both the nature and availability of substrate. Elucidation of mechanisms that modulate the delivery of ascorbate to tissues and its utilization under different metabolic conditions will be invaluable for making recommendations for ascorbate ingestion. Published by Elsevier science Inc. 1998.
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In cancer chemoprevention studies, the identification of better anti- tumor-promoting agents is highly desired because they may have a wider applicability against the development of clinical cancers. Both epidemio- logical and animal studies have suggested that microchemicals present in the diet and several herbs and plants with diversified pharmacological properties are useful agents for the prevention of a wide variety of human cancers. Silymarin, a flavonoid isolated from milk thistle, is used clinically in Europe and Asia as an antihepatotoxic agent, largely due to its strong antioxidant activity. Because most antioxidants afford protection against tumor promotion, in this study, we assessed the protective effect of sily- marin on tumor promotion in the SENCAR mouse skin tumorigenesis model. Application of silymarin prior to each 12-O-tetradecanoylphorbol 13-acetate (TPA) application resulted in a highly significant protection against tumor promotion in 7,12-dimethylbenz(a)anthracene-initiated mouse skin. The protective effect of silymarin was evident in terms of reduction in tumor incidence (25, 40, and 75% protection, P < 0.001, X2 test), tumor multiplicity (76, 84, and 97% protection, P < 0.001, Wilcoxon rank sum test), and tumor volume (76, 94, and 96% protection, P < 0.001, Student's t test) at the doses of 3, 6, and 12 mg per application, respec- tively. To dissect out the stage specificity of silymarin against tumor promotion, we next assessed its effect against both stage I and stage II of tumor promotion. Application of silymarin prior to that of TPA in stage I or mezerein in stage II tumor promotion in dimethylbenz(a)anthracene- initiated SENCAR mouse skin resulted in an exceptionally high protective effect during stage I tumor promotion, showing 74% protection against tumor incidence (P < 0.001, X2 test), 92% protection against tumor multiplicity (P < 0.001, Wilcoxon rank sum test), and 96% protection against tumor volume (P < 0.001, Student's t test). With regard to stage II tumor promotion, silymarin showed 26, 63, and 54% protection in tumor incidence, multiplicity, and volume, respectively. Similar effect of silymarin to that in anti-stage I studies, were also observed when applied during both stage I and stage II protocols. In other studies, silymarin significantly inhibited: (a) TPA-induced skin edema, epidermal hyperpla- sia, and proliferating cell nuclear antigen-positive cells; ( b) DNA synthe- sis; and (c) epidermal lipid peroxidation, the early markers of TPA-caused changes that are associated with tumor promotion. Taken together, these results suggest that silymarin possesses exceptionally high protective ef- fects against tumor promotion, primarily targeted against stage I tumors, and that the mechanism of such effects may involve inhibition of promot- er-induced edema, hyperplasia, proliferation index, and oxidant state.
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The interaction between α-tocopherol and ascorbate in protecting membrane lipids from peroxidation was studied in unilamellar liposomes in which α-tocopherol was incorporated into the liposomal membrane, and ascorbate was trapped within the vesicles. Extravesi-cular ferricyanide was reduced by ascorbate-derived electrons, and this was enhanced by the presence of α-tocopherol in the lipid bilayer. When a water-soluble free radical initiator was added to the outside of liposomes, intravesicular ascorbate prevented oxidation of α-tocopherol, and this effect was associated with complete protection against peroxidation of membrane lipids. These results suggest that ascorbate-dependent recycling of α-tocopherol can protect biological membranes from peroxidation by oxidants originating across the membrane bilayer from ascorbate.
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We measured enzymic and non-enzymic antioxidants in human epidermis and dermis from six healthy volunteers undergoing surgical procedures. Epidermis was separated from dermis by currettage and antioxidants were measured by high-performance liquid chromatography (HPLC) or standard spectrophotometric methods. The concentration of every antioxidant (referenced to skin wet weight) was higher in the epidermis than in the dermis. Among the enzymic antioxidants, the activities of superoxide dismutase, glutathione peroxidase, and glutathione reductase were higher in the epidermis compared to the dermis by 126, 61 and 215%, respectively. Catalase activity in particular was much higher (720%) in the epidermis. Glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, which provide reduced nicotinamide adenine dinucleotide phosphate (NADPH), also showed higher activity in the epidermis than the dermis by 111% and 313%, respectively. Among the lipophilic antioxidants, the concentration of α-tocopherol was higher in the epidermis than the dermis by 90%. The concentration of ubiquinol 10 was especially higher in the epidermis, by 900%. Among the hydrophilic antioxidants, concentrations of ascorbic acid and uric acid were also higher in the epidermis than in the dermis by 425 and 488%, respectively. Reduced glutathione and total glutathione were higher in the epidermis than in the dermis by 513 and 471%. Thus the antioxidant capacity of the human epidermis is far greater than that of dermis. As the epidermis composes the outermost 10% of the skin and acts as the initial barrier to oxidant assault, it is perhaps not surprising that it has higher levels of antioxidants.