The Journal of Nutrition
Nutrition and Disease
Olive Leaf Extract and Its Main ComponentOleuropein Prevent Chronic Ultraviolet BRadiation-Induced Skin Damage andCarcinogenesis in Hairless Mice1–3
Yoshiyuki Kimura4* and Maho Sumiyoshi5
4Division of Biochemical Pharmacology, Department Basic Medical Research, and 5Division of Functional Histology, Department of
Functional Biomedicine, Ehime University Graduate School of Medicine, Toon City Ehime 791-0295, Japan
Abstract
Chronic exposure to solar UV radiation damages skin, increasing its thickness and reducing its elasticity, and causes skin
cancer. Our aim in this study was to examine the effects of an olive leaf extract and its component oleuropein on skin
damage and the incidence of skin tumors caused by long-term UVB irradiation in hairless mice. Male hairless mice (5 wk
old) were divided into 6 groups, including a non-UVB group, a vehicle-treated UVB group (control), 2 olive leaf extract-
treated UVB groups, and 2 oleuropein-treated UVB groups. Five groups were UVB irradiated (36–180 mJ/cm2) 3 times
each week for 30 wk and skin thickness and elasticity after UVB irradiation were measured every week. Olive leaf extract
(300 and 1000 mg/kg) and oleuropein (10 and 25 mg/kg) were administered orally twice daily every day for 30 wk. The
extract and oleuropein significantly inhibited increases in skin thickness and reductions in skin elasticity, and skin
carcinogenesis and tumor growth. Furthermore, they prevented increases in the expression of matrix metalloproteinase
(MMP)-2, MMP-9, and MMP-13 as well as in levels of vascular endothelial growth factor (VEGF) and cyclooxygenase-2
(COX-2) in the skin. Based on histological evaluation, they prevented increases in the expression of Ki-67 and CD31-
positive cells induced by the irradiation. These results suggest that the preventative effects of the olive leaf extract and
oleuropein on chronic UVB-induced skin damage and carcinogenesis and tumor growth may be due to inhibition of the
expression of VEGF, MMP-2, MMP-9, and MMP-13 through a reduction in COX-2 levels. J. Nutr. 139: 2079–2086, 2009.
Introduction
Chronic exposure to solar UVradiation has serious effects on thestructure and function of skin. The number of cases of non-melanoma skin cancers is estimated at.700,000 and is expectedto rise as more UV radiation reaches the earth because ofdepletion of the ozone layer (1–3). A case study in Australiashowed a significant inverse relationship between the risk of skincancer and a high intake of fish, vegetables in general, crucif-erous vegetables, and b-carotene- and vitamin C-containingfoods (4). Thus, there has been great interest in the use of dietarysupplements in the form of complementary and alternativemedicines derived from naturally occurring botanicals for theprevention of UV irradiation-induced photodamage including
skin cancer. The Mediterranean diet, rich in fruits, vegetables,and fish, has been associated with a lower incidence of diseasesand an overall improvement in health (5–8). These findings wereattributed to the high consumption of olive oil and olive leaf(Olea europaea L. Oleaceae). The polyphenolic compounds inolive oil are hydroxytyrosol and tyrosol, with oleuropein presentin minor quantities and mainly found in the olive itself (9). Oliveoil, oleuropein, and its derivatives have a variety of biochemicalroles, including antiinflammatory effects (10–14), antithrombicactions (15), prevention of LDL oxidation (16,17) and plateletaggregation (18), antihyperglycemic activity (19), and anti-ischemic and hypolipidemic effects (20). Furthermore, it hasbeen reported that oleuropein and/or olive oil inhibited tumorgrowth (21–24) and that the topical application of olive oilprevented UVB-induced carcinogenesis (25,26). However, theeffects of an orally administered olive leaf extract and its maincomponent oleuropein on long-term UVB-induced photoaging(for example, increases in skin thickness and reductions in skinelasticity) and carcinogenesis have not been fully studied in vivo.This study examined the effects of the oral administration of anolive leaf extract and oleuropein on skin thickness, elasticity, and
1 Supported in part by Grants-in-Aid for Scientific Research (C) (no. 1990694 to
M. Sumiyoshi, and no. 20590700 to Y. Kimura) from the Ministry of Education,
Culture, Sports, Science and Technology, and the Nihon Funmatsu Pharmacy Co.2 Author disclosures: Y. Kimura and M. Sumiyoshi, no conflicts of interest.3 Supplemental Figures 1–3 are available with the online posting of this article at
jn.nutrition.org.
* To whom correspondence should be addressed. E-mail: [email protected].
ac.jp.
0022-3166/08 $8.00 ã 2009 American Society for Nutrition.
Manuscript received January 22, 2009. Initial review completed March 9, 2009. Revision accepted August 19, 2009. 2079First published online September 23, 2009; doi:10.3945/jn.109.104992.
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the incidence of tumors caused by long-term, low-dose UVBirradiation in hairless mice.
Materials and Methods
Materials. The olive leaf extract (lot. no. 040526AG; Olea europaea L.Oleaceae) was supplied by Nihon Funmatsu Pharmacy. The amount of
oleuropein in the extract was measured using reverse-phase HPLC (a
JASCO LCSS-905, JASCO) under the following conditions: monitoringwavelength, 280 nm; flow rate, 1.0 mL/min; linear gradient profile,
solvent (A) 500 mg/L trifluoroacetic acid and (B) acetonitrile, 900–700
mL/L solvent A and 100–700 mL/L solvent B for 35 min; and column,
COSMOSIL 5C18 (150 3 4.0 mm i.d.) (Nacalai Tesque) (Supplemental
Fig. 1A). Oleuropein was obtained from EXTRASYNTHESE (Supple-
mental Fig. 1B). The amount of oleuropein in the extract used in this
study was calculated to be ~150 g/kg from a standard curve of an
authentic sample. Avoucher sample has been deposited at the Division ofBiochemical Pharmacology, Department of Basic Medical Research,
Ehime University Graduate SchoolMedicine. The extract and oleuropein
were suspended and dissolved in distilled water. Rat monoclonal anti-mouse Ki-67 antibody, rabbit polyclonal anti-rat biotin-labeled Ig
antibody, and peroxidase-labeled streptavidin were purchased from
DakoCytomation. Rabbit polyclonal anti-mouse CD31 and rabbit
polyclonal anti-human cyclooxygenase (COX)6-2 antibodies wereobtained from Spring Bioscience and Cell Signaling Technology, respec-
tively. Mouse monoclonal anti-rat matrix metalloproteinase (MMP)-13
(clone; LIPCO IID1) and anti-b-actin antibodies were purchased from
Lab Vision and Sigma-Aldrich Japan, respectively. The mouse vascularendothelial growth factor (VEGF) ELISA kit and tissue protein extrac-
tion reagent were acquired from R&D Systems and Pierce, respectively.
Other chemicals were of reagent grade and purchased from Wako PureChemical.
Mice. Male albino hairless HOS: HR-1 mice (4 wk old) were purchased
from Hoshino Laboratory Animals, housed for 1 wk in a temperature-controlled room at 25 6 18C and 60% relative humidity, and given free
access to standard nonpurified diet (7.7 g water, 54.4 g crude
carbohydrate, 24.6 g crude protein, 5.3 g crude lipid, 3.5 g crude fiber,
3.5 g mineral mixture, and 1 g vitamin mixture per 100 g diet, andenergy, 1505 kJ/100 g diet; Oriental Yeast) and water during the
experiments. The mice were treated according to the Ethical Guidelines
of the Animal Center, Ehime University Graduate School of Medicine,
and the experimental protocol was approved by the Animal StudiesCommittee of Ehime University.
Chronic UVB-induced skin damage and carcinogenesis. To exam-ine the effects of the olive leaf extract and its main component,
oleuropein, on skin damage induced by irradiation, a UVB lamp (15 W
type, UV maximum wavelength 312 nm; UV intensity, 100 mW per cm2;
Ieda Boueki) was used. Male hairless mice (5 wk old) were divided into 6groups: a non-UVB group, a vehicle-treated UVB group (control), 2 olive
leaf extract-treated UVB groups, and 2 oleuropein-treated UVB groups.
The extract (300 and 1000 mg/kg body weight) and oleuropein (10 and
25 mg/kg body weight) were administered orally twice using a gastrictube at 0800 and 2000 daily for 30 wk. Normal (non-UVB-irradiated
mice) and control (UVB-irradiated mice) were given distilled water alone
on the same schedule. Each group was defined as follows: non-UVBirradiation was normal, vehicle-treated UVB irradiated group was
control, olive leaf extract (300 and 1000 mg/kg body weight)-treated
UVB irradiated groups were OE-300 and OE-1000, and oleuropein-
treated (10 and 25 mg/kg body weight) groups were OL-10 and OL-25.The period of UV irradiation was varied to control the amount of UVB
energy applied to the dorsal region of the animal. The value of the
minimal erythema per mouse was ~36 mJ/cm2. The dose of UVB was
initially set at 36 mJ/cm2, then subsequently increased to 54 mJ/cm2 at
wk 1–4, 72 mJ/cm2 at wk 4–7, 108 mJ/cm2 at wk 7–11, 120 mJ/cm2 atwk 11–12, 132 mJ/cm2 at wk 12–13, 144 mJ/cm2 at wk 13–15, 156 mJ/
cm2 at wk 15–16, 168 mJ/cm2 at wk 16–17, and finally 180 mJ/cm2 at
wk 17–30. The frequency of irradiation was set at 3 times/wk before the
administration of vehicle (control), the indicated amounts of the oliveextract, or oleuropein. Skin thickness was assessed by measuring skin-
fold thickness by the described methods (27–29). Briefly, dorsal skin of
the hairless mice was lifted up by pinching gently under anesthetization
with pentobarbital and skin-fold thickness was measured using a QuickMini caliper (Mitutoyo). Skin elasticity was also assessed by measuring
skin stretch. Briefly, dorsal skin was lifted up and then the skin stretch
was measured using a Digimatic caliper (Mitustoyo). Skin thickness andelasticity after UVB irradiation were measured every week. During the
experiment, the UVB-irradiated dorsal skin of the mice was examined for
papillomas or tumors on a weekly basis. Growths .1 mm in diameter
that persisted for at least 2 wk were defined as tumors and recorded.Tumor data for individual mice were recorded until yield and size
stabilized, at which point the dimensions of all the tumors in each mouse
were recorded. Tumor volume was calculated as length 3 width2/2.
Skin VEGF, MMP-2, -9, and -13, and COX-2. At wk 30, the mice were
killed with an overdose of pentobarbital and all skin tissue was quickly
removed. The tissue was washed in PBS (pH 7.0) and cut into small
pieces. To measure the VEGF content of the skin, the small pieces (100mg) of tissue were homogenized with PBS (2 mL), the homogenate was
centrifuged at 2000 3 g for 10 min at 48C, and the amount of VEGF in
the supernatant was measured using a VEGF-ELISA kit. To evaluateMMP-2 and MMP-9 expression in the UVB-treated skin, small pieces
(100 mg) of skin were homogenized with tissue protein extraction
reagent (2 mL). After centrifugation as above, the MMP-2 (active and
inactive form) and MMP-9 (active and inactive form) in the supernatantwere separated by electrophoresis on a 75-g/L SDS-polyacrylamide gel
containing 1 g/L gelatin under nonreducing conditions. The gel waswashed
with 50 mmol/L Tris-HCl buffer (pH 7.5) containing 100 mmol/L NaCl
and 25 g/L Triton X-100 for 1.5 h and then incubated in 50 mmol/LTris-HCl buffer (pH 7.5) containing 10 mmol/L CaCl2 and 10 mmol/L
ZnCl2 at 378C for 20 h. The gel was stained with 2.5 g/L Coomassie
Brilliant Blue 250 (Sigma-Aldrich) and the stained gelatin-degraded zone
was quantified using NIH Image J 1.36. In addition, to measure theexpression of MMP-13 and COX-2 proteins in the UVB-irradiated skin,
small pieces (100 mg) of skin were homogenized with 50 mmol/L
Tris-HCl buffer (pH 7.5) containing 150 mmol/L NaCl, 1 mmol/LEDTA, 5 g/L sodium deoxycholate, 10 g/L Triton X-100, 2.5 mmol/L
sodium pyrophosphate, 1 mmol/L b-glycerophosphate, 1 mmol/L
Na3VO4, 1 g/L leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride.
After centrifugation, the supernatant (100mg of protein) was boiled for 5min, electrophoresed on a 75-g /L SDS-polyacrylamide gel, and subjected
to a Western blot analysis with anti-MMP-13 mouse monoclonal, anti-
COX-2 rabbit polyclonal, and anti-b-actin monoclonal antibodies.
Percent inhibition was calculated as 100 3 (control mean 2 treatedmean)/(control mean – normal mean)].
Diameter of blood vessels. The removed dorsal skin was washed in
PBS and the subcutaneous blood vessels were photographed using astereoscopic microscope. The diameters of the vessels were measured
using a Digimatic caliper and a Coordinating Area and Curvimeter
MACHINE (X-PLAN 360 dII, Ushitaka), respectively. Percent inhibitionwas calculated as explained above.
Thickness of the epidermis and dermis. The dorsal skin samples (~5
cm2) removed at wk 30 were fixed in 10% buffered formalin for at least24 h, progressively dehydrated in solutions containing an increasing
percentage of ethanol (700, 800, 950, and 1000 mL/L), cleared in
Histoclear (AS-one), embedded in paraffin under vacuum, sectioned
5 mm thick, deparaffinized, and stained with hematoxylin-eosin (HE)or Azan. Cross sections were selected from 3 plates per sample and
4 different microscopic fields (3200 magnification) per plate were
photographed. The thickness of the epidermis and dermis was measured
6 Abbreviations used: COX, cyclooxygenase; ECM, extracellular matrix; HE,
hematoxylin-eosin; MMP, matrix metalloproteinase; OE-300, olive leaf extract
(300 mg/kg body weight); OE-1000, olive leaf extract (1000 mg/kg body weight);
OL-10, oleuropein (10 mg/kg body weight); OL-25, oleuropein (25 mg/kg body
weight); VEGF, vascular endothelial growth factor.
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in the samples stained with HE and Azan using a Digimatic caliper.Percent inhibition was calculated as explained above.
Expression of Ki-67, CD31, and COX-2. To examine of the expression
of Ki-67 (a marker of cellular proliferation) (30), CD31 (a marker ofangiogenesis) (31), and COX-2, the paraffin-embedded skin sections
were deparaffinized and analyzed by immunohistochemical means using
anti-Ki-67 rat monoclonal, anti-CD31 rabbit polyclonal, and anti-COX-2 rabbit polyclonal antibodies, respectively. Four different microscopic
fields (3400 magnification) per plate were photographed, and Ki-67–
and COX-2–positive cells and CD31-positive areas were counted. Each
inhibition was calculated as 1003 (control mean 2 treated mean)/(control mean – normal mean).
Statistical analysis. All values are means 6 SEM. Data were analyzed
by 1-way ANOVA or repeated-measures ANOVA. When the F-test was
significant, means were compared using Fisher’s protected least signif-icant difference test with Stat View (SAS institute). Differences were
considered significant at P , 0.05.
Results
Skin thickness and elasticity, and diameter of blood
vessels following UVB irradiation. Skin thickness increased1.1-fold from wk 0 (baseline) to wk 30 (the end of the study) innormal mice. In the UVB-irradiated control mice, the skinthickness increased 4.2-fold from baseline to the end of thestudy. The skin thickness increased 2.4- and 2.1-fold from wk 0to wk 30 in the OE-300 and OE-1000 groups, respectively.Furthermore, in the OL-10 andOL-25 groups, the skin thicknessincreased 2.7- and 2.5-fold from wk 0 to 30 (Fig. 1A). Skinelasticity decreased by 31% from wk 0 to 30 in control mice butdid not change in the nonirradiated normal, OE-300, OE-1000,OL-10, and OL-25 groups (Fig. 1B). Skin thickness in UVB-irradiated control mice was greater than that in non-UVB–irradiated normal mice fromwk 5 to 30 of irradiation (P, 0.01;Fig. 1A). Skin thickness in the OE-300 and OE-1000 groups waslower than that in the control group from wk 7 to 30 (P, 0.01).UVB-induced increases in skin thickness in the OE-300 and OE-1000 groups were inhibited 62 and 54% at wk 7, and 55 and67% at wk 30, respectively, compared with controls (Fig. 1A).Skin thickness in the OL-25 group was also lower than that inthe control group from wk 7 to 30 (P , 0.01); the increase wasinhibited 46% at wk 7 and 56% at wk 30. Skin thickness in theOL-10 group was lower than that of control group from wk 11to 30 (P , 0.01); the increase was inhibited 44% at wk 11 and44% at wk 30. The inhibitory action on UVB-induced increasesin skin thickness did not differ between the extract- andoleuropein-treated UVB-irradiated mice (Fig. 1A). Skin elasticityin UVB-irradiated (control) mice was lower than that in normalmice from wk 12 to 30 (P, 0.01). Skin elasticity in the OE-300and OE-1000 groups was greater than that in the control groupfrom wk 12 to 30 (P , 0.01). Skin elasticity of the OE-300 andOE-1000 groups was 122 and 110% of the controls at wk 12and 153 and 151% of the controls at wk 30, respectively. Skinelasticity of the OL-10 and OL-25 groups was 112 and 124% ofthe control at wk 12 and 145 and 166% of the control at wk 30,respectively. UVB-induced changes in skin elasticity did notdiffer between the extract- and oleuropein-treated UVB-irradi-ated mice (Fig. 1B). The blood vessel diameter in UVB-irradiatedcontrol mice was significantly greater than that in normal miceand the blood vessel diameter in the OE-300, OE-1000, OL-10,and OL-25 groups was significantly lower than in the controlgroup (Supplemental Fig. 2A; Table 1).
UVB irradiation-induced carcinogenesis. Skin carcinogene-sis induced by chronic UVB irradiation was detected at wk 17.
TABLE 1 Effects of olive leaf extract and oleuropein on blood vessel diameter in UVB-irradiated mice1
Normal(no irradiation)
Control(UVB irradiation) UVB + OE-300 UVB + OE-1000 UVB + OL-10 UVB + OL-25
Diameter, mm 106.7 6 16.0c 302.9 6 14.8a 176.7 6 21.3bc 161.9 6 14.8bc 202.1 6 34.0b 139.4 6 8.9c
Inhibition,2 % 64 72 51 83
1 Values are means 6 SEM, n = 7. Means in a row with superscripts without a common letter differ, P , 0.05.2 Inhibition was calculated as 100 3 (control mean – treated mean)/(control mean 2 normal mean).
FIGURE 1 Effects of the olive leaf extract and its main component
oleuropein on skin thickness (A) and skin elasticity (B) in chronically
UVB-irradiated mice. Values are means 6 SEM, n = 7. Beginning at
wk 12, skin thickness (A) was greater in the control group and less in
the normal group than in all other groups (P , 0.05) and skin elasticity
(B) was less in the control group than in all other groups, which
differed slightly from one another and in some instances, significantly
(P , 0.05).
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The number of tumors per mouse in the OE-300 and OE-1000groups was significantly lower than that in the UVB-irradiatedcontrol group from wk 20 to 30; they were 71 and 77% lower atthe end of the study, respectively (Fig. 2A). The number oftumors per mouse in the OL-10 group was significantly lowerthan in the control group at wk 23, 26, and 30. The number oftumors per mouse in the OL-25 group was significantly lowerthan that in control group from wk 23 to 30; they were 43 and88% lower at the end of the study, respectively (Fig. 2A). Tumorincidence (percent mice with tumors) was prevented by the oraladministration of OE-300, OE-1000, OL-10, and OL-25 (Fig.2B). The total volume of tumors per mouse in the OE-300, OE-1000, and OL-25 groups was significantly lower than that incontrol group from wk 25 to 30; they were 70, 86, and 96%lower at wk 30, respectively (Fig. 2C). The total tumor volumein OL-10 groups was significantly lower than that in the control
group at wk 27 and 28,but not at wk 29 and 30 (Fig. 2C).Oleuropein at 10 mg/kg twice daily did not affect tumor size.
Expression of MMP-2, -9, and -13, VEGF, and COX-2. Theexpression of pro-MMP-2 (inactive form), MMP-2 (activeform), pro-MMP-9, MMP-9 (active from), and MMP-13 inUVB-irradiated control mice was greater than that of non-UVB–irradiated normal mice (P, 0.01); the expressions of pro-MMP-2, MMP-2, pro-MMP-9, and MMP-13 in the control groupwere 2.1-fold, 3.6-fold, 4.5-fold, and 2.7-fold those of thenormal group, respectively. MMP-9 expression was not detect-able in normal mice. The increases in expression of MMP-2 andpro-MMP-9 in the controls were significantly inhibited by OE-300, OE-1000, and OL-25 (Fig. 3A; Table 2). OL-25 alsoinhibited the increase in MMP-9 expression (Fig. 3A; Table 2).OE-300, OE-1000, and OL-25 inhibited the increase in theexpression of MMP-13 that occurred in the control mice (Fig.3B; Table 2). VEGF and COX-2 protein concentrations in theskin of the control group were 6.3- and 2.2- fold those of thenormal group (P , 0.05) and OE-300, OE-1000, and OL-25inhibited these increases (Fig. 3C; Table 2).
Thickness of the epidermis and extracellular matrix of the
dermis. Histological evaluation of HE- and Azan-stained sam-ples of dorsal skin revealed the development of tumors followingchronic UVB irradiation for 30 wk (Supplemental Fig. 3A,B).Furthermore, the thickness of the epidermis (HE-stained samples)and extracellular matrix (ECM) of the dermis (Azan-stainedsamples) in control mice were 14.6- and 2.1-fold those in normal
FIGURE 3 Representative picture of zymography for MMP-2 (active
and inactive from) and MMP-9 (active and inactive form) (A) and
Western blot analysis with anti-MMP-13 (B) and anti-COX-2 (C)
antibodies in the skin of a chronically UVB-irradiated control mouse.
FIGURE 2 Effects of olive leaf extract and oleuropein on tumor
incidence (A,B) and tumor volume (C) in chronically UVB-irradiated
mice. Values are means 6 SEM, n = 7. Means at a time without a
common letter differ, P , 0.05.
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mice (Supplemental Fig. 3A,B; Table 3). OE-300, OE-1000, andOL-25 significantly inhibited the thickening of the epidermiscaused by the exposure (Supplemental Fig. 3A; Table 3). Theincrease in the ECM of the dermis was inhibited by OE-300, OE-1000, OL-10, and OL-25 (Supplemental Fig. 3B; Table 3).
Expression of Ki-67, COX-2, and CD 31. The nuclear proteinKi-67 is an established marker of cellular proliferation (30),whereas CD31 is a marker of angiogenesis (31).We found that Ki-67–positive cells were localized to the stratum basale (basal layer)between the epidermis and dermis and the number of Ki-67–positive cells in control mice was 6.0-fold that of normal mice(Fig. 4A; Table 4). The oral administration of OE-300, OE-1000,and OL-25 significantly reduced the increase in Ki-67–positivecells that occurred in vehicle-treated UVB-irradiated control mice(Fig. 4A; Table 4). COX-2–positive cells were localized to theepidermis. On the other hand, CD31-positive area was localizedto the dermis. The COX-2–positive cell number and CD31-positive area in control mice were 21.0- and 7.6-fold those innormal mice (Fig. 4B,C; Table 4). The increases in COX-2–positive cells and the CD31-positive area were significantlyreduced by the administration of OE-300, OE-1000, and OL-25.
Discussion
Symptoms of cutaneous damage, including wrinkling andpigmentation, develop earlier in sun-exposed skin than in
unexposed skin, a phenomenon referred to as photoaging.More importantly, UV radiation is one of the most abundantcarcinogens in our environment and the development of non-melanoma skin cancers, the most common malignancy world-wide, represents a major consequence of excessive exposureresulting from depletion of the ozone layer and climate change.Therefore, protecting the skin against UV radiation is vital. In aseries of studies of the effects of natural products on UV-inducedskin damage, we found that ginseng saponins isolated from redginseng (32) and a nonsugar fraction of brown sugar (33)prevented UVB-induced photoaging. In the present study, weselected an olive leaf extract and its main component,oleuropein, and examined their effects on chronic UVB-inducedskin damage and carcinogenesis using hairless mice. The extractand oleuropein inhibited increases in skin thickness and reduc-tions in elasticity induced by long-term exposure to UVB.Furthermore, they reduced the incidence and growth of tumorsin exposed skin.MMP are a family of zinc-dependent proteolyticendopeptidases with ECM remodeling and degrading properties(34). They stimulate the growth, migration, invasion, angiogen-esis, and metastasis of cancer cells (35). Inomata et al. (36)reported that MMP-1 and MMP-3 were less active in irradiatedskin than in nonirradiated control skin, whereas type IVcollagen-degrading activity (MMP-1 and MMP-9 activity) wasstronger in wrinkle-bearing skin. Furthermore, they reportedthat MMP-2 and MMP-9 showed similar increases as detectedby gelatin zymography in chronically UVB-exposed skin (36).
TABLE 2 Effects of olive leaf extract and oleuropein on MMP, VEGF, and COX-2 expression in the skinof UVB-irradiated mice1
Normal(no irradiation)
Control(UVB irradiation) UVB + OE-300 UVB + OE-1000 UVB + OL-10 UVB + OL-25
Pro-MMP-2, % of control 47 6 6c 100 6 12ab 77 6 9bc 89 6 10abc 130 6 30a 83 6 10bc
Inhibition, % 43 21 257 32
MMP-2, % of control 28 6 8c 100 6 17a 53 6 8bc 60 6 11b 61 6 9b 36 6 4bc
Inhibition, % 65 56 54 89
Pro-MMP-9% of control 22 6 3c 100 6 9a 63 6 12b 56 6 9b 117 6 17a 59 6 8b
Inhibition, % 47 56 224 53
MMP-9% of control 0 6 0c 100 6 4ab 67 6 24c 42 6 12c 220 6 46a 21 6 9c
Inhibition, % 33 58 2120 79
MMP-13,2 % of normal 100 6 11c 272 6 52ab 126 6 25c 219 6 39bc 372 6 70a 93 6 4c
Inhibition, % 85 31 258 100
VEGF,3 pg/mg protein 37.2 6 5.2b 234.0 6 39.2a 64.5 6 13.1b 67.1 6 14.4b 208.4 6 49.3a 62.5 6 7.0b
Inhibition, % 86 85 13 87
COX-2,2 % of normal 100 6 11b 224 6 39a 116 6 11b 125 6 8b 223 6 50a 96 6 7b
Inhibition, % 87 80 1 100
1 Values are means 6 SEM, n = 7. Means in a row with superscripts without a common letter differ, P , 0.05.2 The expression of MMP-13 and COX-2 protein was expressed as percent (MMP-13:b-actin or COX-2:b-actin ratio) of normal.3 VEGF content was measured using a VEGF-ELISA kit and so expressed as pg/mg protein.
TABLE 3 Effects of olive leaf extract and oleuropein on the thickness of the epidermis and ECM of thedermis in UVB-irradiated hairless mice1
Normal(no irradiation)
Control(UVB irradiation) UVB + OE-300 UVB + OE-1000 UVB + OL-10 UVB + OL-25
Epidermis, mm 20.4 6 2.6b 297.4 6 73.1a 69.9 6 8.8b 59.6 6 14.6b 317.1 6 75.4a 76.4 6 16.8b
Inhibition, % 82 86 27 80
Dermis, mm 258.0 6 8.2b 549.2 6 72.6a 343.5 6 29.7b 368.1 6 33.2b 334.5 6 42.3b 287.6 6 26.0b
Inhibition, % 71 62 74 90
1 Values are means 6 SEM, n = 7. Means in a row with superscripts without a common letter differ, P , 0.05.
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FIGURE 4 Light micrographs of cells
stained with anti-Ki-67 rat monoclonal anti-
body to show cellular proliferation (A), anti-
human COX-2 rabbit monoclonal antibody
(B), and anti-mouse CD31 rabbit polyclonal
antibody to show angiogenesis (C) in normal
mice, chronically UVB-irradiated control mice,
olive leaf extract-treated UVB-irradiated mice,
and oleuropein-treated UVB-irradiated mice.
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VEGF is an angiogenic growth factor that induces the migrationand proliferation of endothelial cells and increases vascularpermeability (37–39). Moreover, the MMP-9 proteolytic systemmay also modulate active VEGF (40–42). It has been reportedthat inflammatory cells, including neutrophils, macrophages,and mast cells, especially those expressing MMP-9, can beaccomplices to neoplastic cells during squamous carcinogenesis(40,43–45). COX-2 expression is critical for chronic UV-inducedmurine skin carcinogenesis (46–48). One of the most frequentevents in carcinogenesis is the uncontrolled activation of the Rassignaling pathway and Lee et al. (49) reported that H-Rasupregulated MMP-9 and COX-2 expression through the acti-vation of extracellular signal-regulated kinase and the IkBkinase-IkBa-nuclear factor-kB signaling pathway, which maycontribute to the malignant progression of WB-F344 rat liverepithelial cells. Thus, UVB-induced skin carcinogenesis andtumor growth might be closely associated with the systems ofMMP, VEGF, and COX-2 expression (50). In this study, an oliveleaf extract and its major component, oleuropein, reduced theincidence and growth of tumors in chronically UVB-irradiatedmice. We found that olive leaf extract and oleuropein preventedchronic UVB-induced skin damage and carcinogenesis. Thesefindings suggest that the preventive actions of the extract andoleuropein on chronic UVB-induced skin damage (an increase inskin thickness and a reduction in elasticity) and tumor incidenceand tumor growth may be due to inhibition of the expression ofVEGF and/or MMP-2, -9, and -13 through a reduction in COX-2 levels. Oleuropein at a dose of 10 mg/kg inhibited the increasein skin thickness; however, it did not inhibit active MMP-9expression. The reason is unknown; therefore, further studies areneeded to examine the dose-response effect of oleuropein on skindamage induced by acute and single-dose UVB irradiation.Because the amounts of oleuropein in the olive leaf extract are~15%, 45 mg of oleuropein is contained in 300 mg of oliveextract. Therefore, the photoprotective effects of olive leafextract may be due to the photoprotective effects of oleuropein.It has been reported that oleuropein represents up to 14% (wt:wt) of the dry weight in unripe olive fruits and that Italiancommercial olive oil waste waters were the richest in totalpolyphenolic compounds with concentrations between 0.15 and0.4% (wt:v) (9,51,52). The amount of oleuropein in olive leaf isgreater than that in olive fruit and olive oil. On the other hand, ithas been reported that the topical application of olive oilprevented UVB-induced carcinogenesis (25,26). Therefore, an-other compound(s) in olive oil may also contribute toantiphotocarcinogenesis. Further studies are needed to comparethe effects of olive oil, olive leaf extract, and olive fruit extract onUVB-induced skin damage and carcinogenesis. This is the firststudy, to our knowledge, to show that the oral administration of
an olive leaf extract and its component, oleuropein, preventsUVB-induced skin photodamage, including photoaging andcarcinogenesis.
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TABLE 4 Effects of olive leaf extract and oleuropein on numbers of Ki-67- and COX-2–positive cells, andthe CD 31-positive areas (angiogenesis) in UVB-irradiated hairless mice1
Normal(no irradiation)
Control(UVB irradiation) UVB + OE-300 UVB + OE-1000 UVB + OL-10 UVB + OL-25
Ki-67–positive cells, n/field 37 6 7b 221 6 38a 84 6 12b 75 6 10b 198 6 51a 79 6 10b
Inhibition, % 74 79 13 77
COX-2–positive cells, n/field 7 6 3b 147 6 32a 28 6 12b 13 6 7b 47 6 10b 16 6 16b
Inhibition, % 85 96 71 94
CD31-positive areas, mm2/field 151.1 6 23.0c 1142.1 6 196.9a 380.0 6 124.9bc 247.9 6 60.0bc 486.5 6 73.5b 214.4 6 76.2bc
Inhibition, % 77 90 66 94
1 Values are means 6 SEM, n = 7. Means in a row with superscripts without a common letter differ, P , 0.05.
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