Role of Anthocyanins in Skin Aging and UV Induced Skin Damage

Abstract

The visible changes associated with chronological aging and chronic sun exposure, especially to the face, head, and neck areas, are particularly concerning for a significantpercentage of the general population. This fact, along with the powerful influence of advertisement and the popular press, has led to an increasing demand for natural and efficient cosmetic ingredients that claim to reduce manifestations of skin aging. More importantly, while skin cancers account for up to 40% of the newly diagnosed cancers in the United States, there are no natural preventive methods to avoid cutaneous malignancies associated with chronic sunlight exposure for individuals with pigmentary traits associated with high cancer risk. Consequently, new effective antiaging and chemopreventive agents are in high demand. Although many of the skin-protective claims attributed to botanical products still lack sufficient scientific evidence, the use of natural bioactives with potential antiaging and/or skin-protective properties continues to receive attention from consumers. During the last decade, a substantial body of knowledge has been produced in this area. Polyphenols and most recently, anthocyanins ave been reported as potentially effective agents to prevent signs of skin aging and protect the skin from external injuries caused by ultraviolet (UV) radiation role of UV radiation, reactive oxygen species (ROS), inflammation, and extracellular matrix (ECM) remodeling in skin pathophysiology has allowed researchers to propose the specific molecular targets for anthocyanins and/or anthocyanin-rich extracts. Although some of the current research describes promising skin-protective effects for anthocyanins, most of the proposed dermatological applications still await clinical validation. This chapter reviews the current scientific literature on the potential of anthocyanins in preserving skin health and preventing skin aging.

Full text

307
Role of Anthocyanins
inSkin Aging and UV-
Induced Skin Damage
Leonel E. Rojo, Diana E. Roopchand, Brittany Graf,
Diana M. Cheng, David Ribnicky, Bertold
Fridlender, and Ilya Raskin
11.1 INTRODUCTION
The visible changes associated with chronological aging and chronic sun exposure,
especially to the face, head, and neck areas, are particularly concerning for a sig-
nicant percentage of the general population. This fact, along with the powerful
inuence of advertisement and the popular press, has led to an increasing demand for
natural and efcient cosmetic ingredients that claim to reduce manifestations of skin
aging (Baumann etal., 2009). More importantly, while skin cancers account for up
to 40% of the newly diagnosed cancers in the United States (Afaq etal., 2005a), there
are no natural preventive methods to avoid cutaneous malignancies associated with
chronic sunlight exposure for individuals with pigmentary traits associated with
high cancer risk (Zanetti et al., 1996). Consequently, new effective antiaging and
chemopreventive agents are in high demand. Although many of the skin-protective
claims attributed to botanical products still lack sufcient scientic evidence, the
use of natural bioactives with potential antiaging and/or skin-protective properties
continues to receive attention from consumers. During the last decade, a substantial
body of knowledge has been produced in this area (Chiu and Kimball, 2003, Afaq
etal., 2002, 2005a, Afaq, 2011).
Polyphenols (Afaq etal., 2005a, Afaq and Katiyar, 2011, Kao etal., 2007, Kim
etal., 1998) and most recently, anthocyanins (Afaq et al., 2009, 2011, Lila, 2004,
11
CONTENTS
11.1 Introduction ..................................................................................................307
11.2 Skin Aging ....................................................................................................308
11.3 Anthocyanins and Skin Protection ............................................................... 309
11.4 Current and Future Work .............................................................................. 312
11.5 Conclusions ................................................................................................... 315
Acknowledgments .................................................................................................. 316
References .............................................................................................................. 316
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308 Anthocyanins in Health and Disease
Schmidt et al., 2008, Schreckinger et al., 2010, Tsoyi et al., 2008d) have been
reported as potentially effective agents to prevent signs of skin aging and protect the
skin from external injuries caused by ultraviolet (UV) radiation (Afaq etal., 2011,
2010, Schreckinger et al., 2010, Tsoyi etal., 2008c). A better understanding of the
role of UV radiation, reactive oxygen species (ROS), inammation, and extracel-
lular matrix (ECM) remodeling in skin pathophysiology has allowed researchers
to propose the specic molecular targets for anthocyanins and/or anthocyanin-rich
extracts. Although some of the current research describes promising skin-protective
effects for anthocyanins, most of the proposed dermatological applications still await
clinical validation. This chapter reviews the current scientic literature on the poten-
tial of anthocyanins in preserving skin health and preventing skin aging.
11.2 SKIN AGING
Skin aging affects the dermis, epidermis, and hypodermis of the skin (Gomez and
Berman, 1985, Giangreco etal., 2008). It not only makes the skin look different
but also makes it more vulnerable to external injuries (Giangreco etal., 2008). The
epidermis, the most external layer of the skin, is mainly composed of keratinocytes
and is directly exposed to environmental aggressions (Figure 11.1). The dermis, rich
in connective tissues (structural proteins), such as collagen and elastin, is under the
epidermis and gives the young skin its characteristic strength, extensibility, and elas-
ticity (Figure 11.1). Skin aging is an intrinsic biological process, which inevitably
starts once a person reaches puberty (Farage et al., 2009) and is manifested by the
appearance of skin wrinkles, dryness, thinning of the skin, loss of subcutaneous fat,
and uneven pigmentation (Giacomoni, 2008). Each individual’s genetic background
• Oxidative
damage
Ultraviolet
radiation
• Overexpression
of MMP's
• Activation of NF-
κB/AP-1
• Inflammation
• Oxidative
damage
• Overexpression
of MMP's
• Activation of NF-
κB/AP-1
• Inflammation
Merkel cells
Fibroblasts
Keratinocytes
Melanocyte
Collagen fibers
Elastin fibers
Sensory neuron
Langerhans cell
Epidermis
Dermis
FIGURE 11.1 (See color insert.) Schematic representation of the skin architecture and
mechanisms of skin damage.
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309Role of Anthocyanins inSkin Aging and UV- Induced Skin Damage
dictates when and how quickly the so-called “intrinsic aging process” unfolds
(Koehler etal., 2008). A number of factors can accelerate the intrinsic skin-aging
process, which includes weakened deoxyribonucleic acid (DNA)-repairing mech-
anisms, alterations of the mitochondrial function, slower repair of the ECM, and
alterations in cell cycle regulation. The most important extrinsic (accelerating) factor
of skin aging is exposure to UV radiation, although diet and smoking can also play a
key role in enhancing the appearance of signs of skin aging (Fazio etal., 1989, Fisher
etal., 2002, Sakuraoka etal., 1996). It is well known that the acute exposure to high
doses of UV radiation triggers the various inammatory pathways and oxidative
damage in the epidermis, dermis, and adnexal organs of the skin, especially UVB
radiation (290–320 nm) (Afaq etal., 2005a, Fisher etal., 2002, Ting et al., 2003).
The UVA radiation (320–400 nm) is less powerful than UVB, but can penetrate
deeper into the skin. In addition, the chronic exposure to high levels of UV radia-
tion can lead to accelerated skin aging (photoaging), hyperkeratosis or atrophy, and
precancerous lesions, such as squamous cell carcinomas (Afaq etal., 2005a, Farage
etal., 2009). One of the key molecular alterations associated with UV-induced skin
damage is the overexpression of metalloproteinases (MMPs), a family of zinc-depen-
dent endopeptidases capable of degrading proteins of the ECM, primarily collagen
and elastin. MMPs play an important physiological role in skin regeneration and
cell migration (adhesion/dispersion). However, repeated exposure to UV radiation
induces the overexpression of specic MMPs (e.g., MMP-9 and MMP-2) leading to
the degradation of skin collagen and elastin, incomplete repair of the ECM, loss of
skin elasticity and resilience, and the appearance of skin wrinkles. UV radiation also
triggers the increase in redox-sensitive transcription factors, including nuclear fac-
tor kappa-B, (NF-ĸB) and activator protein-1 (AP-1). Consequently, researchers are
actively looking for natural compounds or mixtures capable of blocking UV radia-
tion, suppressing UV-mediated oxidative damage, inhibiting UV-induced overexpres-
sion of MMPs, modulating NF-ĸB/AP-1 pathways, and decreasing skin inammation
(Figure 11.1).
11.3 ANTHOCYANINS AND SKIN PROTECTION
The known antioxidant power of anthocyanins has led researchers to study their
potential in preventing noncommunicable chronic diseases (Cao et al., 2000,
Chirinos et al., 2006, Grace et al., 2009, Prior and Wu, 2006, Rojo et al., 2012,
Schreckinger et al., 2010). However, the potential of anthocyanins in preventing
oxidative skin damage such as UV-induced erythema, skin cancer, and photoag-
ing have received less attention and only a relatively limited number of reports has
addressed this question (Afaq etal., 2005b, 2007, 2010, 2011). As the overexposure
to UVB radiation is among the most relevant risk factors for oxidative damage to
the skin, researchers have used various chemical and biological models to explore
the potential of anthocyanins in preventing UVB-induced skin damage. A recent in
vitro chemical study showed that a cosmetic formulation containing anthocyanins
from TNG73 purple sweet potato, at a concentration of 0.61 mg/100 g of cream,
could absorb up to 46% of the incident UV radiation (Chan etal., 2010). Although
this study was not performed using cellular or animal models of skin damage and
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310 Anthocyanins in Health and Disease
has not been clinically conrmed, the results suggest that the topical application
of anthocyanins from TNG73 purple sweet potato at very low doses may prevent
UV-induced skin damage by decreasing the amount of UVB radiation reaching
the epidermis. This mechanism of skin protection is not unexpected, considering
that anthocyanins also attenuate UV damage in plants (Woodall and Stewart, 1998,
Harvaux and Kloppstech, 2001). Anthocyanins absorb strongly in the visible and UV
spectrums, with maximum absorbances in the ranges of 500–550 and 280–320 nm
(Harborne, 1958). The UV absorption capacity of anthocyanins varies depending on
their specic aglycones, sugar conjugation, and acylation patterns. Consequently, in
some colored plant species, other C6–C3–C6 avonoids, but not anthocyanins, are
responsible for the UV protection (Woodall and Stewart, 1998). More importantly,
for anthocyanin-rich UV-blocking formulations, it has been reported that acylated
anthocyanins containing coumaric acid, caffeic acid, and ferulic acid display the
enhanced adsorption of UVB radiation (Harborne, 1958). Chan et al. (2010) also
concluded that acidic ethanol-extracted anthocyanins have better radical scavenging
ability, higher total phenolic content, and stronger reducing ability than acidic water-
extracted anthocyanins from TNG73 purple sweet potato.
A variety of cellular and animal models have been used to elucidate the phar-
macological mechanism by which anthocyanins prevent UV-induced damage to the
skin (Table 11.1). A recent study using the reconstituted human skin (EpiD5erm(TM)
FT-200) showed that pomegranate-derived extracts and juices rich in anthocyanins
prevented UVB-induced damage to the dermal structures (Afaq etal., 2009). In this
study, the pomegranate-derived products were applied to reconstituted human skin
1 h prior to a 12-h UVB (60 mJ/cm2) irradiation period. The pomegranate-derived
products signicantly inhibited protein oxidation, elevation of cyclobutane pyrimi-
dine dimers (CPD), and 8-dihydro-2’-deoxyguanosine (8-OHdG), suggesting the
protective effects against the oxidative damage to proteins and DNA. According to
the authors, anthocyanin-rich products from pomegranate also protected the ECM
of the skin by ameliorating the UVB-induced overexpression of various MMPs,
such as collagenase (MMP-1), gelatinase (MMP-2, MMP-9), stromelysin (MMP-3),
marilysin (MMP-7), and elastase (MMP-12). Similarly, another study showed that an
extract from the blueberry (Vaccinium uliginosum L.), rich in cyanidin-3-glucoside,
petunidin-3-glucoside, malvidin-3-glucoside, and delphinidin-3-glucoside, prevented
UVB-induced overexpression of MMPs and upregulated the UVB-induced suppres-
sion of collagen synthesis in human broblasts (Bae etal., 2009). These results sug-
gest that anthocyanins from the blueberry may offer protection against photoaging.
Another report by Cimino et al. (2006) showed that the anthocyanin cyanidin-3-
O-glucoside (C3G) inhibited UV-induced translocation of the transcription factors
NF-ĸB and AP-1 and other inammatory responses in keratinocytes. According to
these data, C3G could provide multifaceted protection against skin damage since
NF-ĸB and AP-1 are the key modulators of several cellular survival programs of
skin cells, including the synthesis of inammatory mediators, and effectors of both
innate and adaptive immunity. C3G was also found to prevent the UV-induced over-
expression of IL-8, caspase-3 activation, and DNA fragmentation in human kerati-
nocytes (Cimino etal., 2006). This evidence points to a potential protective role of
C3G-rich extracts, not only against UVB accumulative skin damage, but also against
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311Role of Anthocyanins inSkin Aging and UV- Induced Skin Damage
TABLE 11.1
Skin-Protective Effects Reported for Anthocyanins
Anthocyanin(s)
Tested Reported Mode of Action
Type of
Study
Associated Skin
Disease Reference
Anthocyanins
(+reduced
glutathione)
Reduction of erythema after
radiation therapy in patients
with breast cancer
Clinical/
human
Radiation of
dermatitis,
discomfort
associated with
breast irradiation
Enomoto
etal.
(2005)
Cyanidin-3-O-β-
glucopyranoside
Protection against UVA-
induced oxidative stress in
human keratinocytes
In vitro Photoaging,
hyperkeratosis,
skin atrophy,
precancerous
lesions, and skin
cancer
Tarozzi
etal.
(2005)
C3G Reduction of UVB-induced
translocation of NF- B and
AP-1, overexpression of the
cytokines IL-8, apoptosis, and
DNA fragmentation in
cultured human keratinocytes.
In vitro Photoaging,
UV-induced
erythema
Cimino
etal.
(2006)
Anthocyanins (+
proanthocyanidin)
from Jacquez
grapes
Reduction of IL-1α and PGE2,
malondialdehyde/4-
hydroxynonenal, protein
carbonyl groups, and oxidized
glutathione, in human
reconstructed dermis
In vitro Photoaging,
UV-induced
erythema
Tomaino
etal.
(2006)
Delphinidin Protection of human HaCaT
keratinocytes and mouse skin
against UVB-mediated
oxidative stress and apoptosis
In vitro
and
invivo
Photoaging and
skin cancer
Afaq et al.
(2007)
Anthocyanin-rich
extract from red
orange
Reduction of UVB-induced
translocation of NF- B and
AP-1, anti-inammation in
cultured human keratinocytes.
In vitro Photoaging,
UV-induced
erythema
Cimino
etal.
(2007)
Black soybean seed
anthocyanins
Prevention of UVB-induced
apoptotic cell death,
inammation, COX-2, and
PGE2. Decreased production
of NF- B and inhibition of
phosphatidylinositol 3-kinase/
Akt pathway
In vitro
and
invivo
Photoaging,
hyperkeratosis,
skin atrophy,
precancerous
lesions, and skin
cancer
Tsoyi etal.
(2008)
Blueberry
anthocyanins
Amelioration of UVB-induced
damage to human dermal
broblasts
In vitro Photoaging,
precancerous
lesions, and skin
cancer
Bae etal.
(2009)
(continued)
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312 Anthocyanins in Health and Disease
psoriasis, characterized by hyperactive NF-ĸB in keratinocytes. A similar effect was
documented using anthocyanins from bilberry and human keratinocytes as a model
of dermal UV-induced damage (Svobodova etal., 2008). This latter study showed
that anthocyanins from bilberry reduce UVA-stimulated ROS formation and lipid
peroxidation.
Analogous skin-protective mechanisms were documented in two separate publi-
cations from the same research group (Tsoyi etal., 2008a,b). According to these stud-
ies, anthocyanins from black soybean coats may offer protection against UV-induced
damage not only to cultured keratinocytes, but also in vivo to hairless mice skin.
At least two different modes of action were identied in these reports: (i) a reduc-
tion of UVB-induced elevation of cyclooxygenase-2 (COX-2) and prostaglandin E2
(PGE(2)) through a NF-ĸB-dependent pathways (Tsoyi et al., 2008b) and (ii) the
prevention of apoptotic cell death by inhibiting caspase-3 activation and reduction
of proapoptotic Bax protein levels (Tsoyi etal., 2008a). Delphinidin, an ubiquitous
anthocyanin, commonly found in edible berries (Escribano-Bailon et al., 2006,
Rojo etal., 2012, Schreckinger etal., 2010) has also shown the protective effect to
human HaCaT keratinocytes and mouse skin against UVB-mediated oxidative stress
and apoptosis (Afaq etal., 2009). Similarly, another anthocyanin, cyanidin-3-O-β-
glucopyranoside, was found to prevent UVA-induced damage to human keratino-
cytes (Tarozzi etal., 2005).
11.4 CURRENT AND FUTURE WORK
It is well known that oxidative damage, inammation, apoptotic cell death, and over-
expression of MMPs play a key role in skin aging and certain forms of UV-induced
skin damage. The accumulated scientic evidence suggests that anthocyanins may
offer the protection against UV-induced precancerous lesions and possibly delay the
appearance of signs of skin aging (Table 11.1). The protective effect of anthocyanins
and mode of action have been partially described in several in vitro and in vivo
TABLE 11.1 (continued)
Skin-Protective Effects Reported for Anthocyanins
Anthocyanin(s)
Tested Reported Mode of Action
Type of
Study
Associated Skin
Disease Reference
Bilberry
anthocyanins
Reduction of UVA-stimulated
oxidative damage to
keratinocytes
In vitro Photoaging,
hyperkeratosis,
skin atrophy,
precancerous
lesions, and skin
cancer
Bae et al.
(2009)
Anthocyanins from
TNG73 purple
potato
Absorption of 46% incident
UV radiation (0.61 mg/100 g
of cream)
In vitro Sun burns,
photoaging, and
UV-induced
erythema
Chan et al.
(2010)
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313Role of Anthocyanins inSkin Aging and UV- Induced Skin Damage
models of skin damage. However, the current preclinical evidence is seemingly
insufcient to conclude that anthocyanins are solely responsible for the skin-protec-
tive properties observed in vitro and in vivo (Table 11.1) because various polyphenols
that are different from anthocyanins may be present in the test materials used for
these studies.
Additional work is needed to address another important “innovation gap”; the
development of chemically stable and clinically effective anthocyanin-rich formula-
tions for dermatological applications. Only one clinical study was available at the
time this chapter was written. It reported that a multicomponent formulation con-
taining anthocyanins and glutathione signicantly reduced skin erythema after radi-
ation therapy in patients with breast cancer (Enomoto et al., 2005). Unfortunately,
the report provided very scarce information regarding the specic group of antho-
cyanins and doses used for topical applications.
Our research group has recently reported that anthocyanins, along with other
polyphenols, can be efciently separated from highly polar carbohydrates, bound,
concentrated, and stabilized into protein-rich, food matrixes, such as defatted soy-
bean our (DSF) and soy protein isolate (SPI), while preserving their pharmaco-
logical effects (Roopchand etal., 2012). The stability of anthocyanins captured in
this type of protein-rich matrices was veried up to 50 weeks (Figure 11.2) at 37°C.
This form of stabilized anthocyanins opens challenging avenues to develop stable
0
20
40
60
80
100
120
140
SPI Blueberry-SPI Maqui berry-SPI Cranberry-SPI
Relative MMP-9 activity (%)
*
*
*
FIGURE 11.2 Effect of anthocyanin-rich protein matrix on MMP-9 activity in vitro. The
polyphenols including anthocyanins from different sources were concentrated and stabilized
in SPI. The concentrations of anthocyanins bound to SPI are shown in Table 11.2. The SPI-
enriched matrices were suspended in water mixed with recombinant human MMP-9 (1 µg/
mL in PBS) and dye-quenched (DQ) gelatin (1 mg/mL in PBS), a uorescent quenched sub-
strate of MMP-9. Anthocyanin-enriched matrices (1.5 mg/mL) were incubated with 0.4 µg/
mL MMP-9, and 50 µg/mL DQ gelatin at 37°C for 30 min, centrifuged to precipitate solids,
and the supernatant was transferred to a 96-well plate to measure MMP-9 activity. The data
are reported as the percentage of the inhibition of MMP-9 activity relative to control (SPI).
The values correspond to the mean of the three replicates ± SD (*), P < 0.05, and t-test.
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314 Anthocyanins in Health and Disease
dermatological and cosmetic anthocyanin-rich formulations with the application
in cosmetics and food products. We also evaluated whether the SPI with electro-
statically bound and concentrated anthocyanins and other polyphenols from maqui
berry, blueberry, and cranberry retains the antioxidant capacity and human MMP-9
inhibitory activity of its components. According to our results, these anthocyanin-
rich matrices not only displayed a powerful antioxidant capacity (Table 11.2), but
also inhibited collagen degradation by human MMP-9 (Figure 11.3), a collagenase
known to participate in UV-induced skin damage. The molecular mechanisms by
TABLE 11.2
ORAC Antioxidant Capacity of Different Anthocyanin- Rich Soy Protein
Matrices from Fruits
Anthocyanins
(mg/g)
Phenolics
(mg/g)
ORACa (Trolox
Equivalents
(µmol/g)
Representative Kinetic Curve of
AAPHb-induced Fluorescence
Decay
Maqui
berry–SPI
32 80 1090 ± 130 30
20
10
Fluorescence
010 20 30 40
Min
50 60 70
Blueberry–
SPI
29 251 1380 ± 360
30
Fluorescence
20
10
010 20 30 40
Min
50 60 70
Cranberry–
SPI
16 290 1550 ± 200 30
20
10
Fluorescence
010 20 30 40
Min
50 60 70
SPI — — 230 ± 110
30
20
10
Fluorescence
010 20 30 40 50
Min
60 70
Note: SPI, Soy protein isolate.
a ORAC, Oxygen radical absorbance capacity.
b AAPH, 2,2′-Azobis (2-amidinopropane) hydrochloride.
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315Role of Anthocyanins inSkin Aging and UV- Induced Skin Damage
which polyphenols bound to SPI inhibit MMP-9 activity remain to be elucidated; our
hypothesis is that the specic and nonspecic mechanisms may explain this inhibi-
tion and are worthy of further investigations.
11.5 CONCLUSIONS
The previous publications and the presented data suggest that anthocyanins from
plants can prevent skin aging and UV-induced skin damage, particularly in formula-
tions that enhance their stability to temperature, pH, and light (Gironés-Vilaplana
etal., 2012, Roopchand etal., 2012, Schreckinger et al., 2010). For example, acyl-
ated anthocyanins have shown increased stability to pH offering a natural and safer
alternative to synthetic dyes for food and cosmetics (Giusti and Wrolstadb, 2003).
However, the skin-protective properties of acylated anthocyanins from plants are
scarcely studied (Schreckinger etal., 2010). Other authors have addressed this prob-
lem by stabilizing anthocyanins from Aristotelia chilensis in beverages using lemon
juice (Gironés-Vilaplana et al., 2012). Stabilizing anthocyanins by electrostatically
binding them to protein matrices may provide another strategy for protecting their
structural integrity, function, and color (Roopchand etal., 2012). We hope that in the
150 Anthocyanins
(a)
(b)
Total polyphenols
125
100
75
mg/Lmg/L
50
25
0
400
300
200
100
0
0 2 4 8 12 16
Weeks
22 24 28 38 50
FIGURE 11.3 Stability of blueber ry anthocyanins and polyphenols bound to DSF. The con-
centration of (a) monomeric anthocyanins and (b) total polyphenols are eluted from blue-
berry polyphenol-enriched DSF after the indicated number of weeks postincubation at 37°C.
Polyphenolic compounds were eluted from DSF with 75% methanol, 20% water, 5% acetic
acid solution, and the quantications were done as described elsewhere. (From Roopchand, D.
et al. 2012. Food Chemistry, 131, 1193–1200.)
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316 Anthocyanins in Health and Disease
future, the intense and beautiful colors of free or stabilized anthocyanins may offer
a possibility for developing naturally colored cosmetics with skin-protecting and
antiaging properties.
ACKNOWLEDGMENTS
This work was supported in part by the NIH training grant T32 AT004094 (support-
ing DER) and by P50AT002776-01 and 2P50AT002776-06 grants from the National
Center for Complementary and Alternative Medicine (NCCAM) and the Ofce of
Dietary Supplements (ODS) that funds the Botanical Research Center of Pennington
Biomedical Research Center and the Department of Plant Biology and Pathology at
Rutgers University.
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