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Topical application of spent coffee ground

Cite this: DOI: 10.1039/c6pp00045b
extracts protects skin from ultraviolet
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B-induced photoaging in hairless mice†

Hyeon-Son Choi,a Eu Ddeum Park,b Yooheon Park,c Sung Hee Han,c Ki Bae Hongc
and Hyung Joo Suh*d

Received 15th February 2016,
Accepted 5th May 2016
DOI: 10.1039/c6pp00045b
www.rsc.org/pps
The aim of this study was to evaluate the protective effect of spent coffee ground (SCG) on ultraviolet
(UV) B-induced photoaging in hairless mice. The oil fraction (OSCG) and ethanol extract (ESCG) of SCG
were prepared from SCG. OSCG contained a much higher level of caffeine (547.32 ± 1.68 μg mg−1) when
compared to the sum of its chlorogenic acid derivatives (∼119 μg mg−1), and pyrazines were the major
aromatic compounds in OSCG. OSCG effectively inhibited the UVB-induced increase in intracellular reac-
tive oxygen species in HaCaT cells. Topical application of OSCG or ESCG significantly reduced the UVB-
induced wrinkle formation in mice dorsal skin. The combined application of OSCG and ESCG (OEH) led
to a decrease in the wrinkle area by over 35% when compared with the UVB-treated control (UVBC). Epi-
dermal thickness was also reduced by 40%. This result was connected to the significant reduction in
transdermal water loss (27%) and erythema formation (48%) that result from UVB irradiation. Polarization-
sensitive optical coherence tomography (PS-OCT) and antibody-based histological analyses showed that
OSCG and ESCG effectively suppressed the UVB-induced decrease in collagen content. The level of type
1 collagen (COL1) in the OEH group was enhanced by around 40% compared with the UVB control group
(UVBC). This was attributed to the down-regulation of matrix metalloproteinases (MMP2, 9, and 13), which
are known to be responsible for collagen destruction. Our results indicate that topical treatment with
OSCG/ESCG protects mouse skin from UVB-induced photoaging by down-regulating MMPs; therefore,
suggesting the potential of SCG extracts as a topical anti-photoaging agent.

Introduction sitions can be changed depending on how the coffee is pre-

Instant coffee has been one of the most widely consumed beve-
rages for decades.1 Its consumption is continuously growing
with the development of the coffee industry and the increase
of information on its health benefits. Its unique aroma and
distinct flavor, which are derived from a range of volatile com-
pounds, make coffee a popular beverage.2 Coffee has been
known to have various biological activities, including both
harmful and beneficial effects. These effects are a result of the
presence of bioactive compounds, whose levels and compo-
a gated. Many studies focusing on the reutilization of SCG have
Department of Food Science and Technology, Seoul Women’s University, Seoul
01797, Republic of Korea
b
Kwang Dong Pharmaceutical Co., Ltd, Gyeonggi-do 17745, Republic of Korea
c
Institute for Biomaterials, Korea University, Seoul 07249, Korea
d
Department of Public Health Science, Graduate School, Korea University, Seoul
07249, Republic of Korea. E-mail: suh1960@korea.ac.kr; Fax: +82 2 940 2859;
Tel: +82 2 940 2764
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6pp00045b
pared.1 Caffeine, one of the major compounds in coffee, has
been known to have a stimulating effect with cardioactive pro-
perties. Other compounds such as chlorogenic acids are also
examples of bioactive compounds found in coffee.1 Instant
soluble coffee is prepared via processes such as roasting and
thermal water extraction from coffee beans. This process pro-
duces large quantities of insoluble residues called spent coffee
grounds (SCG). This coffee by-product has increasingly accu-
mulated with the growth of coffee consumption. SCG is pro-
duced in quantities that are double those of soluble coffee
every year,1 having the potential for environmental pollution.
Therefore, suitable ways to dispose of SCG need to be investi-
been carried out. Recent studies reported the potential use of
SCG as a source of biodiesel production and composting
materials.3,4 Furthermore, the biological activities of SCG such
as antioxidant and anticancer effects have also been studied.2
The skin is the outer covering that protects the body against
various harmful environmental stimuli such as pathogens,
chemical toxins, and mechanical stress.5 It is made up of several

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Paper
layers including the epidermis and dermis. The maintenance of
healthy skin results from optimal levels and ratios of the major
components comprising skin tissue. Collagen, a kind of protein
most commonly found in the skin, plays an important role in
the scaffolding of skin tissue; it provides structural support and
elasticity. In addition, it helps to form an intricate network of
fibrous tissue in which new cells can grow and regenerate.
Ultraviolet (UV) is a non-ionizing radiation with short wave-
lengths (100–400 nm).6
Due to its energy and reactivity, it has been used in medi-
cine, industry, and research, in the form of an artificial source
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of light like lamps. In addition, appropriate UV exposure via
sunlight is partially beneficial to the health of the body due to
its role in the synthesis of vitamin D.7 However, current
environmental concerns call for addressing the hazard of UV
exposure. Recently, the depletion of the ozone layer increases
the level of exposed solar UV, and excessive exposure to UV
acts as an environmental risk by having serious consequences
for the living organism.6 In particular, chronic UV exposure via
sunlight is harmful to the health of the skin and leads to pre-
mature photoaging or photodamage, accompanied by wrinkle
formation, increased thickness, and loss of elasticity.8 The
occurrence of UV-mediated photoaging is known to result
from the excessive production of reactive oxygen species
(ROS),9 which can cause oxidative stress or damage to the skin.
ROS have been known to promote the production of collagen-
ase, which destroys collagen.8 In addition to the undesirable
cosmetic effects, UV-mediated photodamage causes serious
problems such as sun burn and cancer.10 Therefore, many
commercial products have been used to block the exposure of
the skin to UV light. In addition, many studies have been per-
formed using natural sources to inhibit UV-mediated skin
photoaging in vitro and in vivo.11–14 However, the study of SCG
for use in UV-induced photoaging in mice skin is limited. In a
previous study, we investigated the effect of an orally adminis-
tered ethanol extract of SCG (ESCG) on UV-induced photo-
aging.15 In the current study, the effect of a topically applied
SCG-derived oil (OSCG) and ESCG on UVB-induced photoaging
was evaluated, and the preventive effect of SCG on UVB-
induced photoaging was examined.
Results
Flavonoids, caffeine, and aromatic compounds in OSCG
The levels of coffee compounds in OSCG were analyzed via
HPLC analysis. There was a big variation in the amounts of
Table 1 Caffeine and active flavonoids in ESCG and spent coffee ground (SCG)-derived oil (OSCG) after the extracted time
Contents 5-CQA Caffeine
Retention time (min) 19.825 28.399
OSCG (mg mL−1) 25.15 ± 2.76 547.32 ± 1.68
Values are means ± standard error of triple determinations. Different letters in the same column indicate significant differences (p < 0.05)
among samples as determined via the Duncan’s multiple range test. 5-CQA, 5-O-caffeoylquinic acid (neo-chlorogenic acid); 3-CQA, 3-O-
caffeoylquinic acid (chlorogenic acid); 4-CQA, 4-O-caffeoylquinic acid (crypto-chlorogenic acid); 3,5-diCQA, 3,5-dicaffeoylquinic acids.
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caffeine and its chlorogenic acid derivatives, which are known
to be major components of coffee. The caffeine level in OSCG
was 547.32 μg mg−1, which is over 4-fold more than the sum of
the four chlorogenic acid derivatives. The most abundant
among the tested chlorogenic acid isomers was 4-caffeoyl-
quinic acid (3-CQA, 47.94 ± 0.12 μg mg−1, Table 1). Among the
aromatic compounds of OSCG, pyrazines were found to be the
major aromatic compound group (Table 2). The total number
of aromatic components was 42, in which the pyrazine group
accounted for the highest proportion, i.e. 15 components
(Table 2). Other aromatic compounds such as furans, phenols,
and pyrroles were the next most abundant components.
Effect of OSCG on cell viability, ROS generation, MMP2, and
MMP9
The HaCaT cells did not show any decrease in cell viability
when treated with 500 μg per mL of OSCG, while cell viability
was decreased by around 20% with UVB exposure. However,
OSCG did not cause further reduction in the viability of UVB-
treated cells (Fig. 1A). This effect of OSCG on the cell viability
and UVB irradiation was verified by the trypan blue assay
(Fig. 1S†). This suggests that OSCG itself did not affect the via-
bility of cultured HaCaT cells in the presence or absence of
UVB irradiation within the tested ranges. UVB irradiation
caused a great increase (over 35%) in intracellular ROS pro-
duction when compared to the control (no UVB treatment),
which was significantly suppressed by OSCG (Fig. 1B). OSCG
(500 μg mL−1) inhibited ROS generation by 30% when com-
pared to the UVB control (Fig. 1B). In addition, OSCG signifi-
cantly inhibited mRNA expressions of the MMP2 and MMP9,
which are known to be collagen-degrading enzymes (Fig. 1C
and D).
Effect of OSCG/ESCG on body weight, organ weight, and food
intake
During the 12-week period in which OSCG and ESCG were
applied to the dorsal skin of hairless mice, body weight and
food intake were measured every week. All the groups did not
show any significant difference in body weight, but food intake
was slightly increased in the OSCG-treated group (Table 1S†).
No significant changes were observed in liver weight, but
kidney and heart weight slightly increased in the OSCG- and/
or ESCG-treated groups. For the spleen weight, the UVBC and
OSCG groups showed a slight increase in weight after 12 weeks
(Table 2S†).
3-CQA 4-CQA 3,5-diCQA
29.129 30.017 50.782
30.98 ± 0.29 47.94 ± 0.12 15.17 ± 0.38
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Table 2 Area percentage and number of aromatic components in increased two-fold when compared to the NC group (no UVB
spent coffee ground (SCG)-derived oil (OSCG) irradiation, Fig. 2). The application of OSCG and ESCG signifi-
cantly decreased the wrinkle formation induced by UVB, and
Number of
Groups Area (%) components the combined treatment of OSCG (0.5%) and ESCG (0.5%)
decreased wrinkle formation by 33% compared with the UVBC
Pyrazines 34.98 15 group (Fig. 2C). These results show that topical applications of
Pyrroles 11.26 5
Furans 21.80 8 ESCG and/or OSCG effectively inhibited UVB-induced wrinkle
Phenols 14.41 4 formation in mice dorsal skin. The topical effects of ESCG and
Aldehydes 3.53 3 OSCG on the mice tissues were evaluated via hematoxylin and
Acids 9.74 5
Alcohols 0.36 1 eosin (H&E) staining (Fig. 3). The UVB-treated group presented
Esters 1.38 1 hyperplasia with a relatively higher epidermal skin thickness
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Total sum 100.00
Effect of OSCG/ESCG application on dorsal skin wrinkles and
epidermal thickness in mice
The effect of OSCG/ESCG on UVB-induced wrinkle formation
was evaluated in mice dorsal skin. The UVB treatment induced
thick wrinkles in the dorsal skin, and its wrinkle area
42 (3-fold) than the control group, indicating UVB-induced
photoaging on the skin surface. OSCG and ESCG topical appli-
cations alleviated UVB-induced hyperplasia in epidermal skin
(Fig. 3A and B). A high dose of the OSCG/ESCG combination
(OEH) decreased the epidermal thickness by approximately
45% when compared to the UVBC group (Fig. 3B). These
results indicate that the application of OSCG and/or ESCG pro-
tects the skin against UVB-induced epidermal hyperplasia and
wrinkle formation.

Fig. 1 Effect of spent coffee ground (SCG)-derived oil (OSCG)/ethanol extract of SCG (ESCG) on cell viability, reactive oxygen species (ROS) gene-
ration, matrix metalloproteinase (MMP)2, and MMP9 in HaCaT cells. (A) Cells were treated with OSCG for 1 h and exposed to ultraviolet B (UVB).
After 24 h incubation, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was executed to determine cell viability at an absor-
bance of 570 nm. Cell viability is expressed as a percentage (%) of the non-treated control without UVB irradiation. (B) HaCaT cells were seeded in a
24-well plate at a density of 2 × 105 cells per well. OSCG-treated cells were exposed to UVB irradiation, and the intracellular ROS levels were exam-
ined by detecting the oxidative conversion of cell permeable 2’,7’-dichlorofluorescin diacetate (DCFA-DA) to fluorescent dichlorofluorescein (DCF).
(C, D) Total RNA was isolated from the cells using the Trizol® reagent. cDNA was synthesized and analyzed by a real-time polymerase chain reaction
(PCR) using a Power Taqman PCR Master Mix kit to determine expression levels of (C) MMP2 and (D) MMP9 mRNA. All results are expressed as % of
the control (without UVB irradiation). Values are presented as the mean ± standard error (SE) (n = 3). Different letters indicate significant differences
( p < 0.05) among samples using the Duncan’s multiple range test. NC, normal control (no UVB treatment); UVBC, UVB control (only UVB treatment).

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Fig. 2 Effect of spent coffee ground (SCG)-derived oil (OSCG)/ethanol extract of SCG (ESCG) on wrinkle formation in the dorsal skin of hairless
mice. Hairless mice were administered topical OSCG and ESCG on a daily basis for 12 weeks, and were irradiated with UVB light three times each
week. The dorsal skin of hairless mice was (A) photographed, and (B) replicas of the back skin were made using a Replica full kit. (C) The wrinkle area
was determined using the Image J software. NC, normal control; UVBC, UVB control; BC, base control; O, application of OSCG; EL, low dosage
application of ESCG; EH, high dosage application of ESCG; OEL, low dosage application of OSCG (0.5%) and ESCG (0.1%); OEH, high dosage appli-
cation of OSCG (0.5%) and ESCG (0.5%). All results are expressed as % of the control without UVB irradiation. Values correspond to the mean ± stan-
dard error (n = 5), and different letters indicate significant differences ( p < 0.05) among samples as determined via the Duncan’s multiple range test.

Effect of OSCG/ESCG topical application on the water-holding
capacity and erythema levels
To assess the effect of OSCG/ESCG on UVB-induced water loss,
the water-holding capacity of skin was examined. Single treat-
ment with ESCG or OSCG resulted in a slight increase in the
water-holding capacity. High doses of the combined groups
(OEH) effectively increased the water-holding capacity, which
was decreased by UVB; the water-holding capacity was
enhanced to a level that was almost the same as the normal
control (Fig. 4A). These results show that topical treatment
with OSCG and/or ESCG could protect skin from UVB-induced
water loss. Additionally, erythema formation, a skin problem
induced by UVB irradiation, was examined. A UVB-induced
increase in erythema formation was effectively inhibited by the
application of OSCG or ESCG. The OEH-treated group showed
a decrease of approximately 50% when compared to the UVBC
group (Fig. 4B). These results show that ESCG or OSCG can
suppress morbid symptoms of the skin such as erythema
formation.
Effect of OSCG/ESCG on MMP expression in hairless
mouse skin
We examined the effect of ESCG/OSCG topical treatments on
the mRNA expression levels of MMP2, 9, and 13, which are
genes related to collagen degradation. The mRNA levels of
all the genes were highly upregulated after UVB treatment

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Fig. 3 Effect of spent coffee ground (SCG)-derived oil (OSCG)/ethanol extract of SCG (ESCG) on epidermal thickness. (A) Excised skin fixed with
10% formalin was sectioned using a microtome to perform hematoxylin and eosin staining. (B) The epidermal thickness was measured using the Zen
2012 Blue Edition software. NC, normal control; UVBC, UVB control; BC, base control; O, application of OSCG; EL, low dosage application of ESCG;
EH, high dosage application of ESCG; OEL, low dosage application of OSCG (0.5%) and ESCG (0.1%); OEH, high dosage application of OSCG (0.5%)
and ESCG (0.5%). Values are displayed as the mean ± SE (n = 5), and different letters indicate significant differences ( p < 0.05) among samples as
determined via the Duncan’s multiple range test.

Fig. 4 Effect of spent coffee ground (SCG)-derived oil (OSCG)/ethanol extract of SCG (ESCG) on transepidermal water loss and erythema level. (A)
Transepidermal water loss in the dorsal skin was measured using the Tewameter TM 300. (B) The erythema level was examined using a Mexameter
MX 18. The values were indicated as the index value (erythema). NC, normal control; UVBC, UVB control; BC, base control; O, application of OSCG;
EL, low dosage application of ESCG; EH, high dosage application of ESCG; OEL, low dosage application of OSCG (0.5%) and ESCG (0.1%); OEH, high
dosage application of OSCG (0.5%) and ESCG (0.5%). The values are represented as the mean ± standard error (n = 5) and different letters indicate
significant differences ( p < 0.05) among samples as determined via the Duncan’s multiple range test.

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Fig. 5 Effect of spent coffee ground (SCG)-derived oil (OSCG)/ethanol extract of SCG (ESCG) on matrix metalloproteinase (MMP)2, MMP9, and
MMP13 expressions in hairless mouse. Total RNA was extracted from the skin samples using the Trizol® reagent. cDNA was synthesized, and (A)
MMP2, (B) MMP9, and (C) MMP13 expression levels were analyzed by real-time polymerase chain reaction (PCR) using a Power Taqman PCR Master
Mix kit. NC, normal control; UVBC, UVB control; BC, base control; O, application of OSCG; EL, low dosage application of ESCG; EH, high dosage
application of ESCG; OEL, low dosage application of OSCG (0.5%) and ESCG (0.1%); OEH, high dosage application of OSCG (0.5%) and ESCG (0.5%).
The values are represented as the mean ± standard error (n = 5) and different letters indicate significant differences ( p < 0.05) among samples as
determined via the Duncan’s multiple range test.

(2–3 fold) compared to the levels in the NC group (Fig. 5).
This UVB-induced upregulation of MMP2, 9, and 13 was sig-
nificantly decreased by the topical application of OSCG or
ESCG (Fig. 5). The highest suppression of these MMPs was
observed in the combined-treatment groups (OEL or OEH).
The MMP2 mRNA level in the group treated with OEH
decreased over 60% when compared to the UVBC group
(Fig. 5A). The mRNA levels of MMP9 and MMP13 were
suppressed with a high dose of OSCG and ESCG in combi-
nation (OEH) by 33 and 45%, respectively (Fig. 5B and C).
These results indicate that OSCG and/or ESCG effectively sup-
press the expression of MMPs and, thereby, inhibit UVB-
induced photoaging and one of its consequences, wrinkle
formation.

Fig. 6 Immunohistochemistry analysis and two-dimensional PS-OCT image for the effect of spent coffee ground (SCG)-derived oil (OSCG)/ethanol
extract of SCG (ESCG) on matrix metalloproteinase (MMP)2 and type I collagen (COL1) levels. Excised dorsal skins were subjected to immunohisto-
chemistry analysis using antibodies and a stereomicroscope at 63× magnification, and photographed. (A and B) MMP2 and (C and D) COL 1 levels
were reflected by brown spots and were quantified using the Image J software. (E) Optical image was obtained by polarization-sensitive optical
coherence tomography (PS-OCT). NC, normal control; UVBC, UVB control; BC, base control; O, application of OSCG; EL, low dosage application of
ESCG; EH, high dosage application of ESCG; OEL, low dosage application of OSCG (0.5%) and ESCG (0.1%); OEH, high dosage application of OSCG
(0.5%) and ESCG (0.5%). The values are represented as the mean ± standard error (n = 5) and different letters indicate significant differences ( p <
0.05) among samples as determined via the Duncan’s multiple range test.

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Effect of OSCG/ESCG on MMP2 and COL 1 levels in the
skin tissues
The protein levels of MMP2 and collagen 1 (COL1) in skin
tissue were directly examined via immunohistochemistry (IHC)
as an index for collagen synthesis. UVB exposure (UVBC)
induced clear brown spots, indicating high levels of MMP2
protein in the tissue (Fig. 6A and B). OSCG and/or ESCG
topical treatment significantly diminished the dispersion of
brown spots. This demonstrates the OSCG/ESCG-induced sup-
pression of MMP2 expression in the skin tissue. OEH treat-
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ment inhibited MMP2 expression by around 50% compared to
the UVBC group (Fig. 6B). On the other hand, the collagen
level (COL1) was significantly reduced by UVB treatment
(Fig. 6C and D). The application of OSCG and/or ESCG sup-
pressed the UVB-induced reduction in the collagen levels
(Fig. 6C and D). These results correlated with data from the
PS-OCT analysis (Fig. 6E) in which OSCG/ESCG suppressed the
UVB-induced loss of collagen thickness in the skin. These
results show that OSCG and/or ESCG protect skin from the
UVB-mediated reduction of collagen levels by suppressing
MMP expression.
Discussion
In this study, we examined the topical effect of SCG on UVB-
induced photoaging using HaCaT cells and hairless mice. In
our previous study, we showed the protective effect of orally-
administered ESCG prepared from SCG.15 However, evaluation
of OSCG, the oil fraction of SCG, has not previously been per-
formed, and the topical effects of ESCG also needed to be
examined. Therefore, the current study focused on topical
application as another means to evaluate the effect of OSCG
and ESCG on skin photoaging. Our data showed that topical
treatment with OSCG/ESCG suppressed UVB-induced photo-
aging; however, some differences in the components of OSCG
when compared to ESCG were observed. Our analysis showed
that the OSCG fraction also contains caffeine and chlorogenic
acids (Table 1). The caffeine level was much higher than that
of chlorogenic acids, as in the case of ESCG. However, the
amount of caffeine and chlorogenic acids derived from OSCG
was approximately 10-fold higher than the amount in ESCG.
The caffeine levels in OSCG and ESCG were 547.32 are
42.58 mg mL−1, respectively, while the total amount of chloro-
genic acids in the two samples were 119.25 and 50.75 mg
mL−1, respectively (Table 1).15 These results indicate that the
oil fraction of SCG could be a major source of phenolic com-
pounds, including caffeine and chlorogenic acids. Although it
had relatively higher levels of caffeine and chlorogenic acids,
OSCG showed lower ROS-scavenging activity (Fig. 1B) than that
shown for ESCG in previous work,15 considering the concen-
trations used in the assay. This discrepancy is considered to be
due to the solubility of OSCG, which may not be completely
dissolved in cell culture, making its active components
difficult for the cells to access. Therefore, the inhibitory effect
of OSCG on MMP2 and 9 mRNA expression might have been
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underestimated in the cultured cells (Fig. 1C and D). Our ana-
lysis of the aromatic compounds in OSCG showed that pyra-
zines and furans are the major volatile constituents (Table 2).
The most abundant volatile compounds from green coffee are
known be alcohol and esters, rather than pyrazines and
furans.16,17 These changes in the proportions of the volatile
compounds are due to processes such as roasting and
brewing, which can remove alcohols, esters, etc. Examination
of the fatty acid composition of OSCG showed that linoleic
acid and palmitic acid are the major fatty acids present (>75%,
Table 3S†). These results agree with data from many other
studies.18,19
Our in vivo test showed that OSCG and ESCG significantly
inhibited UV-induced wrinkle formation when applied
topically (Fig. 2). Combined treatment with OSCG and ESCG
showed a clear protective effect against UV-induced wrinkle
formation when compared to treatment with OSCG or ESCG
alone (Fig. 2). This was also more effective than previous
work.15 These results were corroborated by other evaluations of
UV-induced photoaging, which included measurement of the
epidermal thickness (Fig. 3). Wrinkle formation is associated
with the water-holding ability and collagen content of the
skin.20 As shown in Fig. 5, OSCG and ESCG attenuated the
decrease in these capacities that occurred as a result of UVB
irradiation. These phenotypic effects of topical applications of
OSCG and ESCG were recapitulated by the suppression of
MMP2, MMP9, and MMP13, which are responsible for the
degradation and levels of collagen (Fig. 5). The regulatory
effect of OSCG/ESCG on the levels of MMP2 and collagen was
directly verified in the skin (Fig. 6).
Caffeine may be the major active compound responsible for
the protective effect against UVB-induced photoaging, as it is
the most abundant phenolic compound in OSCG and ESCG.
Many studies have shown that caffeine protects the skin from
UV-induced damage. Huang et al. and Lu et al. reported the
inhibitory effects on UV-induced carcinogenesis.21,22 However,
the other compounds present, including chlorogenic acids and
aromatic substances, should not be excluded. A recent study
reported the protective effect of chlorogenic acids against UV-
induced erythema formation in guinea pig dorsal skin.23 Aro-
matic compounds including pyrazines could also contribute to
the suppression of UV-induced photoaging of the skin as ROS
scavengers, based on a recent study.7 Several studies reported
that coffee-derived diterpenes, cafestol and kahweol, protected
fibroblasts and hepatocytes from chemical-induced oxidative
stress.24,25 Cafestol and kahweol are other candidates that may
contribute to the suppression of UV-induced photoaging.
Other than coffee-derived compounds, many phytochemicals
have been studied for their anti-photoaging effects.11,12,14
Ellagic acid was shown to have inhibitory effects on UVB-
induced wrinkle formation and epidermal thickness in mice.11
Curcumin, carnosic acid, and baicalein have been reported to
have protective effects on UV-induced photoaging via the sup-
pression of MMP expression.11–14 UV-induced ROS production
has been implicated via oxidative damage in photoaging and
photocarcinogenesis.26,27 In particular, ROS generation has
Photochem. Photobiol. Sci.

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been known to activate a transcription factor called activator
protein-1 (AP-1), which inhibits the gene expression of pro-
collagen but promotes the gene expression of MMPs that degrade
extracellular matrix proteins.9,28 Therefore, OSCG and ESCG
are considered to exert protective effects against UV-induced
photoaging via the inhibition of MMPs such as MMP2, 9, and
13, and through the suppression of ROS production.
As the coffee industry grows due to the continual increase
in coffee consumption, an increasingly large quantity of SCG
will be generated. The quantity of this coffee by-product pro-
duced from soluble coffee production reaches 6 million tons
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per year.29 Since SCG may pollute the environment if it is not
properly treated or reused, there is a lot of concern on how to
recycle it. Therefore, studies investigating the characteristics of
SCG are of importance in order to determine its potential use.
Some alternatives, including media for microorganisms, agri-
cultural nutrients, and material to produce ethanol fuel have
been proposed.30–32 Even though this potential is underuti-
lized at an industrial level, these endeavors can be expanded to
various fields. In this sense, this study showed another poten-
tial use of the SCG as a functional material.
Conclusions
Our study showed that the topical application of SCG-derived
OSCG and ESCG effectively protected the skin against UVB-
induced photoaging in mice. The SCG-mediated protective
effect is considered to be due to the down-regulation of MMPs
and the suppression of ROS production, which are induced by
UVB irradiation. Therefore, we suggest that SCG, a coffee by-
product, could be used as a functional material in the food
sector as well as in cosmetics for skin health.
Experimental
Ethical statement
The animal protocol for this study was approved by the
Korea University Animal Care Committee and the Ethics
Committee of the Ministry of Education, Republic of Korea
(KUIACUC-2013-111) and conducted in accordance with Act
No. 27/2011 on the protection of animals against cruelty. Due
to the fact that neither human primary cells nor biopsies have
been used in this research, no written informed consent for
experiments on human subjects is required for this work.
Materials
SCG were obtained as a brewed ground from a coffee house
chain (Starbucks, Korea) and dried at 25 °C for 24 h. High per-
formance liquid chromatography (HPLC) grade methanol,
acetonitrile, and water were purchased from Burdick & Jackson
(Muskegon, MI, USA). Antibodies against mouse matrix metallo-
proteinase 2 (MMP2) and type-1 collagen (COL1) were
obtained from R&D Systems (Minneapolis, MN, USA). Horse-
radish peroxidase-conjugated goat anti-mouse immuno-
Photochem. Photobiol. Sci.
View Article Online
Photochemical & Photobiological Sciences
globulin G (IgG) was also purchased from R&D Systems.
Immortalized human keratinocyte HaCaT cells were obtained
from AmorePacific Corporation (Kyeonggi-do, Korea). An ROS
reagent, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), was pur-
chased from Sigma-Aldrich (St Louis, MO, USA). Trizol®
reagent was purchased from Invitrogen. Cell culture reagents
such as Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum (FBS), and antibiotics (100 U per mL penicillin
and 100 μg per mL streptomycin) were purchased from Gibco
BRL (Grand Island, NY, USA). Sequence-specific PCR primer
sets and the TaqMan MGB probe (FAM™ dye-labeled) were
purchased from Applied Biosystems; MMP2 (GenBank ID:
NM_008610.2), MMP9 (GenBank ID: NM_013599.2), MMP13
(GenBank ID: NM_008607.2) for animal; MMP2 (GenBank ID:
NM_004530.4) and MMP9 (GenBank ID: NM_004994.2) for
cell; glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
GenBank ID: NM_008084.2). A cell viability reagent, 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), was pur-
chased from Life Technologies (Waltham, MA USA). All other
reagents were purchased from Sigma-Aldrich.
Sample preparation
SCG were slightly roasted at 120 °C for 30 min. OSCG was
extracted via an expeller (National ENG Co., Ltd, Koyang,
Korea) from SCG at 60 MPa and 100 °C. OSCG was sealed and
stored at 4 °C until use. The residue resulting from the OSCG
extraction was extracted using 70% ethanol for 4 h to prepare
ESCG. ESCG extracts were centrifuged at 1000g for 15 min. The
supernatant was then concentrated by vacuum evaporation,
and freeze-dried (Fig. 2S†). OSCG and ESCG were added to a
base to prepare cosmetic formulations at concentrations of 0.1
or 0.5% (Table 3).
Cell culture
Immortalized human keratinocyte HaCaT cells were main-
tained at 37 °C in 95% humidity and 5% CO2 in DMEM con-
taining 10% FBS, 2 mM glutamine, and antibiotics. Subculture
was performed before cells reached their confluence via the
addition of trypsin and EDTA. The cells were incubated with
the respective treatments of OSCG during the experiments.
Cell viability assay
HaCaT cells were seeded on 24-well plates with 2 × 105 cells
per well. OSCG was added to the cells for 1 h, which was fol-
lowed by exposure of the cells to UVB at 30 mJ cm−2 by using a
UV lamp. After UVB-irradiated cells were incubated for 24 h at
37 °C, all wells were aspirated, refilled with MTT (1 mg mL−1)
solution, and incubated for 4 h at 37 °C. Aliquots of 100 g per
L sodium dodecyl sulfate (SDS) in 0.01 M HCl were added to
the wells overnight to dissolve the formazan crystals. The
absorbance of the samples at 570 nm was determined using a
microplate reader (Molecular Devices, LLC, Sunnyvale, CA,
USA). For the trypan blue assay, cells were trypsinized and col-
lected after 24 h incubation. Trypan blue dye (0.5%, Sigma-
Aldrich) was added for 2 min to stain the cells. Stained viable
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Photochemical & Photobiological Sciences Paper

Table 3 Composition of cosmetic formulations

Composition (%) BC O EL EH OEL OEH

OSCG 0 0.5 0 0 0.5 0.5

ESCG 0 0 0.1 0.5 0.1 0.5
Deionized water 69.774 69.274 69.674 69.274 69.174 68.774
Butylene glycol 6 6 6 6 6 6
Glycerin 3 3 3 3 3 3
Xanthan gum 0.05 0.05 0.05 0.05 0.05 0.05
Phenoxyethanol 0.2 0.2 0.2 0.2 0.2 0.2
Carbomer 0.06 0.06 0.06 0.06 0.06 0.06
Cetearyl alcohol 0.8 0.8 0.8 0.8 0.8 0.8
Glyceryl stearate 0.5 0.5 0.5 0.5 0.5 0.5
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Stearic acid 0.1 0.1 0.1 0.1 0.1 0.1

Polysorbate 60 0.6 0.6 0.6 0.6 0.6 0.6
PEG-100 stearate 0.6 0.6 0.6 0.6 0.6 0.6
Cetylethylhexanoate 6 6 6 6 6 6
Hydrogenated polydecene 7 7 7 7 7 7
Caprylic/capric triglyceride 2 2 2 2 2 2
Cyclohexasiloxane 3 3 3 3 3 3
Methylparaben 0.2 0.2 0.2 0.2 0.2 0.2
Propylparaben 0.1 0.1 0.1 0.1 0.1 0.1
Triethanol amine 0.016 0.016 0.016 0.016 0.016 0.016
Total 100 100 100 100 100 100

OSCG, spent coffee ground (SCG)-derived oil; ESCG, ethanol extract of SCG.

cells were observed and counted under a microscope (Olympus
CKX31, Seoul, Korea).
Analysis of intracellular ROS
HaCaT keratinocytes were seeded onto a 24-well plate (2 × 105
cells per well). The cells were pre-incubated with OSCG 30 min
and then exposed to UVB irradiation at 30 mJ cm−2 using a UV
lamp. The change in intracellular ROS levels was determined
via the DCFH-DA (5 μg mL−1) assay. The oxidative conversion
of cell-permeable DCFH-DA to fluorescent dichlorofluorescein
(DCF) was measured at an excitation wavelength of 485 nm
and an emission wavelength of 535 nm using a VICTOR3™
spectrofluorometer (PerkinElmer, MA, USA).
RNA extraction and real-time polymerase chain reaction (PCR)
Total RNA was extracted from the skin samples using the
Trizol® reagent (Invitrogen, Carlsbad, CA, USA). Complemen-
tary DNA (cDNA) was synthesized from 1 μg of total RNA using
the RevertAid™ First Strand cDNA Synthesis Kit (Thermo
Scientific Fisher, Waltham, MA, USA). RQ1 RNase-free DNase I
(Promega, Madison, WI, USA) was added to digest the DNA.
Real-time PCR was performed using cDNA and a Power
Taqman PCR Master Mix Kit (Applied Biosystems, Foster City,
CA, USA). The data were analyzed using the comparative CT
(2−ΔΔCT) method.33
Animal maintenance and experimental design
SKH-1 hairless mice (male, 6 weeks old) were purchased from
the Central Laboratory Animal Inc. (Seoul, Korea). The mice
were housed individually in plastic cages, with a grated stain-
less steel cover under the following conditions: 24 ± 1 °C, 60%
atmospheric humidity, and a 12 h light/12 h dark cycle. The
colony room was maintained as a pathogen-free facility. The
animals were provided with ad libitum access to water and
food for 1 week prior to their partition into the following
groups (5 mice per group): normal control (NC) group, UVB
control (UVBC) group, sample-treated groups (O, EL, EH, OEL,
and OEH). Topical base formulation (Table 3) was made by
modification from the previous study.34 The NC group is the
normal mice applied with base formulation without UVB
irradiation and sample. The UVBC group was exposed to gradi-
ent-UVB irradiation, and applied with base formulation
without sample. In sample-treated groups, the base formu-
lation containing OSCG (0.5%) or ESCG (0.1% or 0.5%), or
combinations of OSCG and ESCG was applied with UVB
irradiation. The food intake and body weight of mice were
recorded once a week during the experimental periods, and
the organ weight was measured after sacrifice.
UVB irradiation
The dorsal skin of the mice was subjected to UVB
(290–320 nm) irradiation using a UV lamp (FLB20SBL; Sankyo
Denki, Kanagawa, Japan). The UVB light used in the experi-
ment covered a wavelength range of 270 to 400 nm, containing
an output peak at 313 nm. Samples (OSCG and ESCG) were
applied to the experimental groups once every day, 24 h, 6 h
before, and 24 h after UVB exposure. Hairless mice were sub-
jected to UVB radiation three times a week for 12 weeks. The
UVB dose was set at 36 mJ cm−2 at the beginning of the study,
and it was gradually increased to 54, 72, 90, 108, 126, 144, 162,
180, on a weekly basis, and 198 mJ cm−2 at the tenth to twelfth
week. The dorsal wrinkles of mice were observed about
6 weeks after the first UVB irradiation. HaCaT cells were treated
with samples for 30 min and then exposed to UVB at a dose of
30 mJ cm−2 by using the UV lamp. Dosimetric amounts of the
UVB radiation were monitored using a Lutron UV-340 light
meter (Conrad Electronic AG, Hirschau, Germany).

This journal is © The Royal Society of Chemistry and Owner Societies 2016 Photochem. Photobiol. Sci.

Paper
Polarization-sensitive optical coherence tomography (PS-OCT)
The polarization-sensitive optical coherence interferometer is
based on the original free-space PS-OCT system.35 Images of
the skin epidermis were analyzed using Origin Pro 8.0 software
(Origin Lab Corporation, Northampton, MA, USA).
Macroscopic skin analysis
Silicone-based molds of the skin surfaces were prepared to
obtain replicas from the back skin of hairless mice using a
Replica full kit (Visioline VL650, Courage and Khazaka Elec-
Published on 10 May 2016. Downloaded by University of Sussex on 26/05/2016 03:08:00.
tronic GmbH, Koln, Germany). A flexible light guide 1500 (Carl
Zeiss, Jena, Germany) illuminated the replica at an angle of
20° to obtain the maximal formation of shadows behind the
wrinkles. The shadows were observed under a stereoscopic
microscope at 7× magnification (Axio Zoom v.16, Carl Zeiss)
and photographed using an Axio-Cam digital camera (Axio-
Cam HRc, Carl Zeiss). A binary image was obtained by extract-
ing the shadow regions at a constant gray level using the
Image J software (version 1.46r for Windows; NIH, Washington
DC, USA). The ratio of wrinkled area (RWA, %) and the ratio of
the sum of the shadow area to the measured area were deter-
mined from the image obtained from each mouse.
Histological analysis
The dorsal skin (4 × 4 cm) was rapidly excised from the sacri-
ficed animals. It was fixed in 10% formalin, dehydrated in 70,
95, and 100% ethanol, cleared in xylene, and embedded in
paraffin. Serial 4 mm areas were obtained via microtome sec-
tioning, and the sections were used for histological analyses.
All light microscopic and immunohistochemical analyses were
executed using a stereomicroscope at 63× magnification (Axio
Zoom v.16; Carl Zeiss) and photographed using an Axio-Cam
digital camera (Axio-Cam HRc; Carl Zeiss). Specific immuno-
histological staining was performed to detect the brown spots
of MMP2 and COL1, and analyzed using the Image J software.
The epidermal thickness was calculated using the Zen 2012
Blue Edition software (ZEISS, München, Germany).
Measurement of transepidermal water loss and erythema level
Transepidermal water loss (TEWL) was measured using the
Tewameter TM 300 (Courage and Khazaka Electronic GmbH)
according to the manufacturer’s protocol under the following
conditions: 40–60% humidity at 20–25 °C. Erythema was
measured at 660 nm (the spectral absorption peak of hemo-
globin) and 568 nm to avoid the influence of other colors on
the readings. Its levels were quantified by using a Mexameter
MX 18 (Courage and Khazaka Electronic GmbH), indicated as
the index value (erythema).
Determination of chemical composition in spent coffee
grounds
The fatty acid methyl esters (FAMEs) were prepared by saponi-
fication and methylation. A portion of 0.2 g of OSCG was
added to an Erlenmeyer flask followed by the subsequent
addition of 4 mL of 0.5 N NaOH/methanol. This was heated to
Photochem. Photobiol. Sci.
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Photochemical & Photobiological Sciences
reflux for 10 min. After adding 5 mL of 14% boron trifluoride/
methanol, the mixture was incubated for 2 min, followed by
the addition of 5 mL of hexane for 1 min. After the reaction,
the Erlenmeyer flask was cooled and the samples were trans-
ferred into a test tube. Saturated sodium chloride solution was
added, and a layer of hexane was obtained and analyzed by a
Younglin M600D gas chromatograph (Younglin Co. Ltd,
Anyang, Korea) with a HP-INNOWax column (30 m × 0.25 mm
× 0.25 µm film thickness) and a flame ionization detector.
Temperatures of the oven were 50 °C at 3 min (initial tempera-
ture), 250 °C at 5 min (final temperature) and the temperature
gradient was 10 °C min−1. The temperatures of the injector,
and the detector were 220 and 275 °C, respectively. The helium
flow rate was 1 mL min−1, and the split ratio was 1 : 100. The
flavor composition of OSCG was analyzed using the simul-
taneous distillation extraction (SDE) method based on the
Likens and Nickerson device. Thirty grams of coffee oil and
150 mL of distilled water were added to a distillation flask,
and 100 mL of dichloromethane was added thereafter. The
extract was extracted during a 2-hour period while maintaining
the apparatus at 40 °C in a water bath and 130 °C in an oil
bath, consecutively. After dehydration of the extract by sodium
sulfate (anhydrous), it was maintained at 40 °C and concen-
trated using a Kuderna-Danish concentrator. The final volume
of the concentrate was 1.0 mL, which was then analyzed using
a gas chromatograph (GC)/mass selective detector (MSD). An
Agilent 6890 N gas chromatograph (GC) coupled to an Agilent
5975 inert mass selective detector (MSD) (Agilent Technology,
Palo Alto, USA) with a J&W INNOWAX column (60 m ×
0.25 mm × 0.25 µm film thickness) was used for the analysis
of aromatic components. The oven temperature program was
initiated at 40 °C, held for 2 min, then increased to 230 °C at a
rate of 5 °C min−1, and maintained for a further 20 min. The
temperatures of the injector and the detector were 230 °C and
280 °C, respectively, with a helium flow rate of 1.0 mL min−1
and a 10 : 1 split ratio.
Statistical analysis
Data were analyzed using the Statistical Package for Social
Science (SPSS) for Windows (version 12.0, SPSS Inc., Chicago,
USA). All data were reported as the mean ± standard deviation
(SD) or standard error (SE). Values of p < 0.05 were considered
statistically significant by one-way analysis of variance (ANOVA)
and the Duncan’s multiple range test.
Conflict of interest
The authors declare that there are no conflicts of interest.
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