olive leaf extract and its main component oleuropein prevent

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.

2080 Kimura and Sumiyoshi

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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).

Oleuropein prevents ultraviolet B-induced carcinogenesis 2081

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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



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


COX-2,2 % of normal 100 6 11b 224 6 39a 116 6 11b 125 6 8b 223 6 50a 96 6 7b


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



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


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


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.

Literature Cited

1. Boring CC, Squire TS, Tony T. Cancer statistics, 1992. CA Cancer JClin. 1993;43:7–26.

2. Miller DL, Weinstock MA. Nonmelanoma skin cancer in the UnitedStates. Incidence. J Am Acad Dermatol. 1994;30:774–8.

3. O’Shaughnessy JA, Kelloff GJ, Gordon GB, Dannenberg AJ, Hong WK,Fabian CJ, Sigman CC, Bertagnolli MM, Stratton SP, et al. Treatmentand prevention of intraepithelial neoplasia: an important target foraccelerated new agent development. Recommendations of the AmericanAssociation for Cancer Research task force on the treatment andprevention of intraepithelial neoplasia. Clin Cancer Res. 2002;8:314–46.

4. Kune GA, Bannerman S, Field B, Watson LF, Cleland H, Meresnstein D,Vitetta L. Diet, alcohol, smoking, serum b-carotene, and vitamin A inmale nonmelanocytic skin cancer patients and controls. Nutr Cancer.1992;18:237–44.

5. Keys A. Wine garlic, and CHD in seven countries. Lancet. 1980;1:145–6.

6. Ferro-Luzzi A, Sette S. The Mediterranean diet: an attempt to define itspresent and past composition. Eur J Clin Nutr. 1989;43 Suppl 2:13–29.

7. Trichopoulou A, Costacou T, Bamia C, Trichopoulos D. Adherence to aMediterranean diet and survival in a Greek population. N Engl J Med.2003;348:2599–608.

8. Renaud S, De Lorgeril M, Delayer J, Guiodollet J, Jacquard F, MamelleN, Martin JL, Monjaud I, Salen P, et al. Cretan Mediterranean diet forprevention of coronary heart disease. Am J Clin Nutr. 1995;61 Suppl 6:S1360–7.

9. Amiot M, Fleuriet A, Macheix JJ. Importance and evolution of phenoliccompounds in olive during growth and maturation. J Agric Food Chem.1986;34:823–86.

10. Manna C, Della Ragione F, Cucciolla V, Borriello A, D’Angelo S,Galletti P, Zappia V. Biological effects of hydroxytyrosol, a polyphenolfrom olive oil endowed with antioxidant activity. Adv Exp Med Biol.1999;472:115–30.

11. Tuck KL, IIayball PJ. Major phenolic compounds in olive oil:metabolism and health effects. J Nutr Biochem. 2002;13:636–44.

12. Visioli F, Galli C. Biological properties of olive oil phytochemicals. CritRev Food Sci Nutr. 2002;42:209–21.

13. Bitler CM, Viale TM, Damaj R, Crea R. Hydrolyzed olive vegetationwater in mice has anti-inflammatory activity. J Nutr. 2005;135:1475–9.

14. Miles EA, Zoubouli P, Calder PC. Differential anti-inflammatory effectsof phenolic compounds from extra virgin olive oil identified in humanwhole blood cultures. Nutrition. 2005;21:389–94.

15. Carluccio MA, Siculclla L, Ancora MA, Scoditti E, Storelli C, Visioli F,Distante A, DeCaterina R. Olive oil and red wine antioxidantpolyphenols inhibit endothelial activation. Arterioscler Thromb VascBiol. 2003;23:622–9.

16. Wiseman SA, Mathot JN, De Fouw NJ, Tijburg JB. Dietary non-tocopherol antioxidants present in extra virgin olive oil increase the

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



Ki-67–positive cells, n/field 37 6 7b 221 6 38a 84 6 12b 75 6 10b 198 6 51a 79 6 10b


COX-2–positive cells, n/field 7 6 3b 147 6 32a 28 6 12b 13 6 7b 47 6 10b 16 6 16b


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


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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resistance of low density lipoproteins to oxidation in rabbits. Athero-sclerosis. 1996;120:15–23.

17. Visioli F, Galli C. Oleuropein protects low density lipoprotein fromoxidation. Life Sci. 1994;55:1965–71.

18. Petroni A, Blasevich M, Salami M, Papini N, Montedoro GF, Galli C.Inhibition of platelet aggregation and eicosanoid production by pheno-lic components of olive oil. Thromb Res. 1995;78:151–60.

19. Sato H, Genet C, Strehle A, Thomas C, Kobstein A, Wahner A,Mioskowski C, Auwerx J, Saladin R. Anti-hyperglycemic activity of aTGR5 agonist isolated from Olea europaea. Biochem Biophys ResCommun. 2007;362:793–8.

20. Andreadou I, Iliodromitis EK, Mikros E, Constantinou M, Agalias A,Magiatis P, Skaltsounis AL, Kamber E, Tsantili-Kakoulidou A, et al. Theolive constituent oleuropein exhibits anti-ischemic, antioxidative, andhypolipidemic effects in anesthetized rabbits. J Nutr. 2006;136:2213–9.

21. Hashim YZH, Gill CIR, McGlynn H, Rowland IR. Components of oliveoil and chemoprevention of colorectal cancer. Nutr Rev. 2005;63:374–86.

22. Hamdi HK, Castellon R. Oleuropein, a non-toxic olive iridoid, is ananti-tumor agent and cytoskeleton disruptor. Biochem Biophys ResCommun. 2005;334:769–78.

23. Menendez JA, Vazquez-Martin A, Colomer R, Brunet J, Carrasco-Pancorbo A, Garcia-Villaba R, Fernandez-Guierrez A, Segura-CarreteroA. Olive oil’s bitter principle reverses acquired autoresistance totrastuzumab (herceptin TM) in HER2-overexpressing breast cancercells. BMC Cancer. 2007;7:80.

24. Hashim YZH, Rowland IR, McGlynn H, Servili M, Selvaggini R,Taticchi A, Esposto S, Montedoro GF, Kaisalo L, et al. Inhibitory effectsof olive oil phenolics on invasion in human colon adenocarcinoma cellsin vitro. Int J Cancer. 2008;122:495–500.

25. Ichihashi M, Ahmed NU, Budiyanto A, Wu A, Bito T, Ueda M, OsawaT. Preventive effect of antioxidant on ultraviolet-induced skin cancer inmice. J Dermatol Sci. 2000;23 Suppl 1:S45–50.

26. Budiyanto A, Ahmed NU, Wu A, Bito T, Nikaido O, Osawa T, Ueda M,Ichihashi M. Protective effect of topically applied olive oil againstphotocarcinogenesis following UVB exposure of mice. Carcinogenesis.2000;21:2085–90.

27. Ibbotson SI, Morna MN, Nash JF, Kochvar IE. The effects of radicalscompared with UVB as initiating species for the induction of chroniccutaneous photodamage. J Invest Dermatol. 1999;112:933–8.

28. Park C-H, Lee MJ, Kim J-P, Yoo ID, Chung JH. Prevention of UVradiation-induced premature skin aging in hairless mice by the novelcompound melanocin A. Photochem Photobiol. 2006;82:574–8.

29. Kim YG, Sumiyoshi M, Sakanaka M, Kimura Y. Effects of ginsengsaponins isolated from red ginseng on ultraviolet B-induced skin agingin hairless mice. Eur J Pharmacol. 2009;602:148–56.

30. Urruticoechea A, Smith IE, Dowsett M. Proliferation marker Ki-67 onearly breast cancer. J Clin Oncol. 2005;23:7212–20.

31. Claffey KP, Abrams K, Shih SC, Brown LF, Mullen A, Keough M.Fibroblast growth factor 2 activation of stromal cell vascular endothe-lial growth factor expression and angiogenesis. Lab Invest. 2001;81:61–75.

32. Kim YG, Sumiyoshi M, Kawahira K, Sakanaka M, Kimura Y. Effects ofred ginseng extract on ultraviolet B-irradiated skin change in C57Blmice. Phytother Res. 2008;22:1423–7.

33. Sumiyoshi M, Hayashi T, Kimura Y. Effects of the nonsugar fraction ofbrown sugar on chronic ultraviolet B irradiation-induced photoaging inmelanin-possessing hairless mice. Nat Med (Tokyo). 2009;63:130–6.

34. Kleiner DE, Steler-Stevenson WG. Matrix metalloproteinases andmetastasis. Cancer Chemother Pharmacol. 1999;43:S42–51.

35. Egeblad M, Werb Z. New functions for the matrix metalloproteinases incancer progression. Nat Rev Cancer. 2002;2:161–74.

36. Inomata S, Matsunaga Y, Amano S, Takada K, Kobayashi K, TsunenagaM, Nishiyama T, Kohno Y, Fukuda M. Possible involvement ofgelatinases in basement membrane damage and wrinkle formation inchronically ultraviolet B-exposed hairless mouse. J Invest Dermatol.2003;120:128–34.

37. Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL,Delfino JJ, Siegel NR, Leimgruber RM, Feder J. Tumor vascularpermeability factors stimulates endothelial cell growth and angiogen-esis. J Clin Invest. 1989;84:1470–8.

38. Keck PJ, Hauser S, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT.Vascular permeability factor, an endothelial cell mitogen related toPDGF. Science. 1989;246:1309–12.

39. Leung DW, Cachianes G, Kuang W-J, Goeddek DV, Ferra N. Vascularendothelial growth factor is a secreted angiogenic mitogen. Science.1989;246:1306–9.

40. Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K,Tanzawa K, Thorpe P, Itohara S, et al. Matrix metalloproteinase-9triggers the angiogenic switch during carcinogenesis. Nat Cell Biol.2000;2:737–44.

41. Kaliski A, Maggiorella L, Cengel KA, Mathe D, Rouffiac V, Opolon P,Lassau N, Bourhis J, Deutsch E. Angiogenesis and tumor growthinhibition by a matrix metalloproteinase inhibitor targeting radiation-induced invasion. Mol Cancer Ther. 2005;4:1717–28.

42. Heissig B, Rafii S, Akiyama H, Ohki Y, Sato Y, Rafael T, Zhu Z, HicklinDJ, Okumura K, et al. Low-dose irradiation promotes tissue revascu-larization through VEGF release from mast cells and MMP-9-mediatedprogenitor cell mobilization. J Exp Med. 2005;202:739–50.

43. Coussens LM, Raymond WW, Bergers G, Laig-Webster M, BehrendtsenO, Werb Z, Caughey GH, Hanahan D. Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis. GenesDev. 1999;13:1382–97.

44. Coussens LM, Tinkle C, Hanahan D, Werb Z. MMP-9 supplied by bonemarrow-derived cells contributes to skin carcinogenesis. Cell. 2000;103:481–90.

45. van Kempen LC, Rhee J-S, Dehne K, Lee J, Edwards DR, Coussens LM.Epithelial carcinogenesis: dynamic interplay between neoplastic cellsand their microenvironment. Differentiation. 2002;70:610–23.

46. Fischer SM, Pavone A, Mikulec C, Langenbach R, Rundhaug JE.Cyclooxygenase-2 expression is critical for chronic UV-induced murineskin carcinogenesis. Mol Carcinog. 2007;46:363–71.

47. Chun K-S, Langenbach R. A proposed COX-2 and PGE2 receptorinteraction in UV-exposed mouse skin. Mol Carcinog. 2007;46:699–704.

48. Rundhaug JE, Fischer SM. Cyclooxygenase-2 plays a critical role in UV-induced skin carcinogenesis. Photochem Photobiol. 2008;84:322–9.

49. Lee KW, Kim M-S, Kang NJ, Kim D-H, Surh Y-J, Lee HJ, Moon A.H-Ras selectively up-regulates MMP-9 and COX-2 through activationof ERK1/2 and NF-kB: an implication for invasive phenotype in ratliver epithelial cells. Int J Cancer. 2006;119:1767–75.

50. Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G. Inflam-mation and cancer: how hot is the link? Biochem Pharmacol. 2006;72:1605–21.

51. Mulinacci N, Romani A, Galardi C, Pinelli P, Giaccherini C, Vincieri FF.Polyphenolic content in olive oil waste waters and related olive samples.J Agric Food Chem. 2001;49:3509–14.

52. Visioli F, Poli A, Galli C. Antioxidant and other biological activities ofphenols from olives and olive oil. Med Res Rev. 2002;22:65–75.


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olive leaf extract and its main component oleuropein prevent

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