[CANCER RESEARCH54, 701-708, February 1, 19941

ABSTRACT

A methanol extract of the leaves of the plant Rosmarinus officinalis L(rosemary) was evaluated for its effects on tumor initiation and promotionin mouse skin. Application of rosemary to mouse skin inhibited the covalent binding of benzo(a)pyrene [B(a)P] to epidermal DNA and inhibitedtumor initiation by B(a)P and 7,12-dimethylbenz[aJanthracene (DMBA).Topical application of 20 nmol B(a)P to the backs of mice once weekly for10 weeks, followed 1 week later by promotion with 15 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly for 21 weeks, resulted in

the formation of7.1 tumors per mouse. In a parallel group ofanimals thatwere treated topically with 1.2 or 3.6 mg of rosemary 5 mm prior to eachapplication of B(a)P, the number of tumors per mouse was decreased by54 or 64%, respectively. Application of rosemary to mouse skin also inhibited TPA-induced ornithine decarboxylase acfivity@TPA-mduced inflammation, arachidonic acid-induced inflammation, TPA-induced hyperplasia, and TPA-induced tumor promotion. Mice initiated with 200 nmolDMBA and promoted with S nmol TPA twice weekly for 19 weeks devel

o_ an average of 17.2 skin tumors per mouse. @fteatmentof the DMBAinitiated mice with 0.4, 1.2, or 3.6 mg of rosemary together with 5 nmol

TPAtwiceweeklyfor 19weeksinhibited the number ofTPA-mducedskintumors per mouse by 40, 68, or 99%, respectively. Topical application of

carnosol or ursolic acid belated from rosemary inhibited TPA-induced ear

inflammation, ornithine decarboxylase activity, and tumor promotion.Topical application of 1, 3, or 10 @amolcarnosol together with 5 nmol TPA

twice weekly for 20 weeks to the backs of mice previously initiated withDMBA inhibited the number ofsldn tumors per mouse by 38, 63, or 78%,

respectively. Topical application of 0.1, 0.3, 1, or 2 @amolursolic acidtogether with 5 nmol TPA twice weekly for 20 weeks to DMBA-initiatedmice inhibited the number of tumors per mouse by 45-61%.

INTRODUCTION

The leaves of the plant Rosmarinus officinalis L. are commonlyused as a spice, flavoring agent, and naturally occurring antioxidant(1, 2). In the 1950s, it was reported that an extract of rosemary leaves

contained high antioxidant activity (1, 3, 4) and shortly thereafterextracts from rosemary leaves were used commercially for their antioxidant activity (5, 6). Extracts of rosemary leaves have been used toprevent lipid autoxidation and to inhibit the oxidation of both animalfats and vegetable oils (6). The antioxidant activity of an extract fromrosemary leaves is comparable to that of butylated hydroxytolueneand butylated hydroxyanisole (7). Although carnosol, carnosic acid,rosmaridiphenol, rosmanol, isorosmanol, epirosmanol, and rosmari

quinone arc antioxidant compounds in rosemary leaves (8), about 90%of the antioxidant activity of rosemary can be attributed to carnosoland camosic acid (9).

Received 7/13/93; accepted 11/23/93.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

â€S̃upported in part by Grant CA49756 from the NIH.

2 William M. and Myrle W. Garbe Professor of Cancer and Leukemia Research.

3 To whom requests for reprints should be addressed.

Many compounds that possess antioxidant activity inhibit tumorpromotion in mouse skin (10—14).Butylated hydroxyanisole (10, iS),butylated hydroxytoluene (15), sodium selenite (16), a-tocopherol(17), glutathione (17), quercetin (18), ascorbyl palmitate (12), curcumm (13), and a green tea polyphenol fraction (14) are examples ofsubstances that possess antioxidant or reactive oxygen scavengingactivity and inhibit tumor promotion and/or biochemical events asso

ciated with tumor promotion in mouse skin (10—14,i8). In the presentstudy, we evaluated the effect of an extract of rosemary leaves (rosemary) on the initiation of skin tumors by B(a)P'@and DMBA as wellas the promotion of skin tumors by TPA. We also evaluated the effect

of carnosol and ursolic acid, isolated from the leaves of the rosemaryplant, on TPA-induced tumor promotion in mouse skin. We previouslypublished preliminary reports of these studies (i9, 20).

MATERIALS AND METhODS

Materials. TPA was purchased from Chemsyn Science Laboratories (Lenexa, KS). L-['4CjOrnithine (58 Ci/mmol) and [3H]B(a)P (62.8 Ci/mmol) werepurchased from Amersham Corp. (Arlington Heights, IL). DMBA was purchased from Calbiochem-Boehring (San Diego, CA). Aquasol was purchasedfrom NEN Research Products (Boston, MA). Acetone and dimethyl sulfoxidewere purchased from Burdick & Jackson Laboratories (Muskegon, MI). Arachidonic acid was purchased from Nu-Chek-Prep, Inc. (Elysian, MN). Thedried rosemary leaves were purchased from General Spice, Inc. (South Plain

field, NJ).Preparation of Methanol Extract of Ground Dried Leaves of Rosmari

ni's officinalis L A methanol extract of the ground dried leaves of the plantRosmarinus officinalis L. (rosemary extract;rosemary)was preparedaccordingto the previously reportedmethod of Wu et aL (7). Three kg of rosemaryleaves, which had been ground to a fine powder, were extracted with 18 litersmethanol at 60°Cfor 2 h. The extraction was performed in a custom-made30-liter steam jacketed, stainless steel extractor equipped with a Lightnin mixer

(Lightnin Group, Rochester, NY). After extraction, the mixture was filtered,

and the residue was extracted again with 12 liters fresh methanol. The combined filtrate was bleached with 600 g activated carbon and then filtered to

yield a light-brown filtrate. The methanol solution was concentrated to a final

volume of 2 liters with a vacuum rotary evaporator and then filtered to removeprecipitate. Three liters of water were added to the filtrate. The precipitate that

was formed after the addition of water was filtered and air dried to yield a

rosemary extract (rosemary) that was used for our studies. About 200—300gdry powdered rosemary was obtained from 3 kg of ground rosemary leaves.

HPLC Analysis of Rosemary ExtracL HPLC analysis was performedwith a Varian 5000 Liquid Chromatograph with an ACS model 750/14 mass

detector from Pens Industries (State College, PA). In this detector, the flow ofthe column moves through a nebulizer and then passes through a tube held ata temperature at which the solvent vaporizes and the solute forms small

droplets. The light scattered by the droplets is measured by a photomultiplier(21). The detector was operated at a temperatureof 50°Cand 16 psi airpressure. The column was a Whatman PartiSphere C,8 column (12.5 cm X0.46 cm internal diameter). The following ternary solvent system was utilized

4 The abbreviations used are: B(a)P, benzo(a)pyrene; DMBA, 7,12-dimethylbenz[aJ

anthracene; TPA, 12-O-tetradecanoylphorbol-13-acetate; BItT, butylated hydroxytoluene;BHA,butylatedhydroxyanisole.

701

Inhibition of Skin ilimorigenesis by Rosemary and Its Constituents Carnosol andUrsolic Acid'

Mou-Than Huang, Chi-Tang Ho, Zhi Yuan Wang, Thomas Ferraro, You-Rong Lou, Kathe Stauber, Wei Ma,Constantino Georgiadis, Jeffrey D. Laskin, and Allan H. Conney2―@Laboratory for Cancer Research, Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NewJersey 08855-0789 [M-T H., Z. V W, T F, Y-R. L., K S., W M., A. H. C.]; Department of Food Science, Cook College, Rutgers, The State University of New Jersey, NewBrunswick, New Jersey 08903 [C-T H.]; and Department of Environmental and Community Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey08854 [C. G., J. D. L.]

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ROSEMARY AND CANCER PREVENTION

for column elution: Solvent A, 1% acetic acid in water; solvent B, acetonitrile;and solvent C, 1% acetic acid in methanol. The mobile phase was programmedlinearly from 30% A170% C at the start of elution to 5% A/90% B over a 30mm interval, followed by 5% A/5% B/90% C for 5 mm and then 100% B foranother 15 mm. The flow rate was 0.7 ml/min. The retention times of carnosol,miltirone, carnosic acid, and ursolic acid were 7.9, 9.2, 15.7, and 30.8 mm,

respectively.HPLC analysis of several preparations of the rosemary extract indicated that

they contained 16.5—19.2%ursolic acid, 3.8—4.6%carnosol, 0.1-0.5% carnosicacid, and trace-0. 1% miltirone, respectively.

Preparation of Carnosol. The ground dried leaves of Rosmarinus officinaiLs L. (300 g) were extracted three times for 2—3h with 750 ml hexane at

25°Cin a 2-liter stainless steel vessel fitted with a mechanical stirrer. Thesolvent was evaporated under vacuum with a rotary evaporator to yield 11—13g of extract. The powdered rosemary extract was dissolved in 200 ml methanoland allowed to stand at room temperature for 1 week to allow the conversion

of carnosic acid to carnosol (22). After removing the methanol under vacuumwith a rotary evaporator, 0.5 g of the dry residue was dissolved in 2 ml

hexane:ether (3: 1) and was injected into a preparative column (550 mm X 25mm internal diameter) packed with activated silicic acid (Mallinckrodt, St.Louis, MO). The mobile phase, delivered by a piston pump, was hexane:ether(3: 1), and the flow rate was 2.5 mI/mm. The eluent was monitored by an UV

detector at 254 nm The carnosol fraction, which eluted between 12.5—14.5mm, was collected, and the solvent was evaporated. After removal of solvent,the carnosol fraction was recrystallized from methanol to yield carnosol of98% purity as determined by HPLC (described above). The identity of camosolwas confirmed by particle beam liquid chromatography/mass spectrometry in

the electron ionization mode on a Vestec Model 201 LC/MS (Vestec,Houston,TX) equipped with a universal interface. The mass spectrum of camosolshowed a molecular ion at m/z 330 (1 1%) and major fragmentation ions at m/z286 (100%), 284 (32%), 271 (33%), 215 (62%) and 269 (23%).

Preparation of Miltirone. Miltirone was synthesized by the Diels-Alderreaction between 3-isopropyl-O-benzoquinone and 6,6-dimethyl-1-vinylcyclohexane according to the method of Knapp and Sharma (23). The reactionmixture was subjected to silica gel column chromatography using ethyl ether

:pentane (3:97) as eluent to yield a red solid. After recrystallization fromhexane, miltirone (99% purity as determined by HPLC) was obtained. The

mass spectrum of miltirone by particle beam liquid chromatography/massspectrometry showed a molecular ion at m/z 282 (10%) and major fragmentation ions at 254 (55%), 239 (100%), 224 (19%), and 165 (12%).

Preparation of Ursolic Acid. Ten g of dried methanol extract of rosemary

leaves, prepared as described above, were dissolved in 200 ml boiling ethanol.Upon cooling, ursolic acid crystallized from the solvent. It was recrystallized

twice from ethanol to obtain ursolic acid of98% purity as determined by HPLC

(described above). The ursolic acid was identified by particle beam liquidchromatography/mass spectrometry to have a molecular ion at mJz 456 (27%)

and major fragmentation ions at 438 (30%), 423 (26%), 410 (30%), 395 (35%),

302 (90%), 248 (100%), 203 (98%), 189 (42%), and 133 (79%).

Animals. Female CD-i mice were purchased from Charles River Laboratories (Kingston, NY) and kept in our animal facility for 1—2weeks before use.Mice were fed a Purina Lab Chow 5001 diet ad libitum (Ralston-Purina Co.,St. Louis, MO) and kept on a 12-h light-dark cycle. Mice were provideddrinking water ad libitutn. The dorsal region of each mouse was shaved withelectric clippers at least 2 days before treatment with TPA, B(a)P, or DMBA.Only mice that did not show signs of hair regrowth were used.

Determination of @H]B(a)P-DNAAdducts in Mouse Epidermis. Thecovalent binding of [3H]B(a)P to epidermal DNA was performed as described

earlier (24).

Measurement of Mouse Ear Edema. TPA (0.5 nmol) either alone ortogether with test compound in 20 p3 acetone was applied to the right ear of25-day-old female CD-i mice (6—10mice per group). Control mice received20 pJ acetone. Five h later, the mice were killed by cervical dislocation, and

6-mm diameter ear punch biopsies were taken and weighed. The increase inweight of the ear punches was directly proportional to the degree of inflammation. In studies on arachidonic acid-induced edema of mouse ears, micewere treated on the right ear with 20 pA acetone or 0.3 mg arachidonic acid in20 p.1 acetone. The mice were sacrificed 1 h later by cervical dislocation, and

ear punches were taken and weighed (13).

Measurement of Epidermal Ornitbine Decarboxylase Activity. Thepreparation of epidermal homogenates and the determination of omithine

decarboxylase activity were performed as described previously (13). Mice (8—9weeks old) were treated with 200 p.1acetone, TPA in acetone, or rosemary andTPA in acetone. Five h after treatment, the mice were sacrificed by cervical

dislocation, and the dorsal area of the skin was removed. In order to separatethe epidermis from the dermis, the skins were plunged into a 58°Cwater bathfor 15 5, and then the skins were immediately submerged in an ice water bath

as described by Slaga et a!. (25). The epidermis was removed from the dermisby gentle scraping and placed in 1 ml 50 mr@ipotassium phosphate, pH 7.7

buffer containing 2 m@ dithiothreitol and 0.1 m@.iEDTA. The epidermis washomogenized on ice for 10 s using a Polytron homogenizer with the motor

speed setting at 4. Epidermal homogenates were centrifuged at 11,000 X g for30 mm at 4°C, and the supernatant fractions were removed and stored over

night at —20°Cprior to the determination ofornithine decarboxylase activity asdescribed earlier (13). Protein was determined by the protein-dye binding

procedure described by Bradford (26) using a Bio-Rad protein assay kit (Richmond, CA).

Morphological Examination ofTPA-treated Mouse Skin. Assay of TPAinduced epidermal hyperplasia was done by a slight modification of the pro

cedure of Smart et a!. (27). The dorsal skin of female CD-i mice (7—8weeksold, 3—4mice per group) was treated with 200 @lacetone, TPA (1 nmol), orTPA (1 nmol) and 3.6 mg rosemary in 200 @lacetone twice a day for 4 days.The mice were killed by cervical dislocation 18 h after the last dose. The dorsalskin was excised and fixed in 10% neutral buffered formalin. The skin sampleswere embedded in paraffin and processed for histology with hematoxylin andeosin staining. Inflammatory cell (leukocyte) infiltration that was absent (-),slight (+), moderate (+ +), or severe (+ + +) was characterized by diffuse

infiltration of mononuclear inflammatory cells into the dermis when comparedwith the acetone controls. Intercellular edema (accumulation of fluid between

the epidermal cells) was scored as present or absent. The number of nucleated

cell layers in the epidermis was determined at five locations per slide andaveraged. Thickness of the noncomifled cell layers of the epidermis was alsomeasured in a similar manner. The mean ± SE for each group of mice was

calculated. Morphometric analysis was performed using an automatic photo

microscope (X40).Skin Thmorigenesis Studies. For studies on the effects of rosemary or its

constituents on tumor initiation and promotion, the dorsal regions of femaleCD-i mice (7 weeks old) were shaved with an electric clipper. Two days later,groups of 30 mice were treated topically with 200 nmol DMBA in 200 @lacetone; control mice received 200 p.1 acetone alone. After 1 week, the mice

were treated topically with 200 pJ acetone, 5 nmol TPA in 200 pA acetone, or5 nmol TPA and rosemary or test compound in 200 pi acetone twice weekly

for 20 weeks. Tumors at least 1 mm in diameter were counted and recordedonce every 2 weeks, and the results were expressed as the average number of

tumors per mouse and percentage of tumor-bearing mice.For studies on the effect of rosemary on tumor initiation by B(a)P or

DMBA, mice were treated topically with 200 @lacetone or with rosemary in200 @lacetone 5 mm prior to each application of B(a)P (20 nmol) or DMBA(2 nmol) once a week for 10 weeks. One week later, the mice received 15 nmol

TPA in 200 @lacetone twice weekly for 20 weeks. In a second study, micewere treated topically with 200 @lacetone or 3.6 mg of rosemary in 200 piacetone at 120, 60, and 5 mm before the topical application of 200 nmol B(a)Pin 200@ acetone. One week later, the mice received 15 nmol TPA in 200 @l

acetone twice weekly for 20 weeks.Statistical Analyses. Student's t test was used for all statistical analyses.

RESULTS

Studies with Rosemary. Each preparation of dry, powdered rosemary extract (rosemary) was examined by HPLC and tested for itsability to inhibit TPA-induced increases in epidermal ornithine decarboxylase activity and TPA-induced mouse ear edema. The HPLCanalysis of several preparations of rosemary indicated that they contamed 16.5—19.2%ursolic acid, 3.8—4.6%carnosol, 0.1—0.5%carnosic acid, and a trace-0.i% miltirone. Examination of the biological

activity of several batches of rosemary obtained from rosemary leaves702

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Table 1 Inhibitory effect of rosemary on f3HJB(a)P-DNA adductformationinmouseepidermisIn

experiment 1, female CD-i mice were treated topically with 200 pi acetone aloneor rosemary in 200 pi acetone 5 mm before the topical application of [3H]B(a)P (20nmol,100

pCi) in 200 @.dacetone. In experiment 2, the mice were treated topically with 200piacetoneor 1.2 or 3.6 mg of rosemary in 200 @lacetone at 120, 60, and 5 mm before the

topical application of [3H]B(a)P (200 nmol, 100 pCi) in 200 gsl acetone. The micewerekilled15 h later. Skin was removed and radioactivity in epidermal DNA wasdetermined.The

data are expressed as mean ±SE (3—4mice pergroup).[3H]B(a)P

metabolites boundPercentageTreatment(jmollmg DNA)inhibitionExperiment

1Acetone0.74 ±0.13Rosemary

(1.2 mg) 0.52 ±0.0730Rosemary(3.6 mg) 0.34 ±0.13― 54

2032

Table 2 Inhibitory effect of topical application of rosemary on B(a)P-induced tumorinitiationIn

experiment 1, female CD-i mice were treated topically with 200 pAacetone or 1.2 or 3.6 mg of rosemary in 200 p1 acetone 5 mm prior to application of 20 nmol B(a)P in 200@lacetone once weekly for 10weeks. One week later, the mice were treated with 15 nmol TPA in 200 pAacetone twice weekly for 21 weeks. In experiment 2, mice were treated topically

with 200 @ilacetone or 3.6 mg rosemary in 200 @lacetone at 120, 60, and 5 min before the topical application of 200 nmol B(a)P in 200 pA acetone. One week later, the mice weretreated with 15 nmol TPA twice weekly for 21 weeks. Data are expressed as the mean ±SE from 30 mice pergroup.9

Weeks 13 Weeks 21WeeksTumors

Percentage mice Tumors Percentage mice Tumors Percentage miceTreatment per mouse with tumors per mouse with tumors per mouse withtumorsExperiment

1Acetone + acetone o― o o―@ 0.03 ±0.03―3Acetone

+ B(a)P 0.53 ±0.02 30 3.37 ±0.54 87 7.10 ±1.03 100Rosemary (i.2 mg) + B(a)P 0.45 ±0.26 17 1.96 ±0.07― 50 3.24 ±0.52― 90Rosemary (3.6 mg) + B(a)P 0.20 ±0.07 20 1.24 ±0.30― 48 2.57 ±0.48@' 73

00―0@a0473.35±0.80706.89 ±0.13100130.64±020b372.87 ±0.46―77

ROSEMARY AND CANCER PREVENTION

and 7.10 tumors per mouse after 2i weeks of promotion. In parallel

groups of mice, topical application of 1.2 or 3.6 mg rosemary S mmbefore each application of B(a)P decreased the number of tumors permouse by 15 or 62% after 9 weeks of promotion, 42 or 63% after 13weeks of promotion, and by 54 or 64% after 2i weeks of promotion,

respectively (Table 2, experiment 1). In an additional study, mice were

treated topically with acetone at 120, 60, and S mm before the topicalapplication of 200 nmol B(a)P. After i week, the mice were promotedwith 15 nmol TPA twice weekly for 21 weeks. These mice developedan average 0.77, 3.35, and 6.89 skin tumors per mouse after 9, i3, and21 weeks of promotion with TPA, respectively (Table 2, experiment2). A parallel group of mice that were treated topically with 3.6 mgrosemary in acetone at i20, 60, and S mm before the application ofB(a)P developed an average of 0.13, 0.64, and 2.87 skin tumors permouse after 9, 13, and 21 weeks of TPA promotion, respectively(Table 2, experiment 2). The results of this study indicated that rosemary decreased by 58—83%the number of skin tumors per mouse.

In another experiment, topical application of DMBA (2 nmol) to agroup of 30 mice once weekly for 10 weeks, followed by treatmentwith TPA (15 nmol) twice weekly for 7, ii, and 15 weeks producedan average of 3.1, 19.6, or 25 skin tumors per mouse, respectively

(Table 3). Topical application of i.2 or 3.6 mg of rosemary extract 5mm prior to each application of DMBA decreased the number ofDMBA-induced skin tumors per mouse by 48 or 84% after 7 weeks ofpromotion, by 27 or 37% after ii weeks of promotion, and by 28 or48% after iS weeks of promotion, respectively (Table 3).

Inhibitory Effect of Rosemary on TPA- and Arachidonic Acidinduced Inflammation of Mouse Skin. The antiinflammatory activity of rosemary was evaluated by determining its effect on TPA- andarachidonic acid-induced edema. Several batches of rosemary weretested for their abilities to inhibit TPA-induced ear edema, and the

various batches of rosemary were found to have a similar inhibitoryeffect on TPA-induced inflammation (Table 4, experiments 1—4).In atypical experiment, topical application of 0.5 nmol TPA in 20 @lacetone to the ear of a mouse increased the average weight of an earpunch (6-mm diameter) from 6.4 mg to 13.5 mg at S h after the dose(Table 4, experiment 1). Topical application of 0.04, 0.12, or 0.36 mgrosemary together with 0.5 nmol TPA to the ears of mice inhibited theTPA-induced edema of mouse ears by i7, 75, or 92%, respectively(Table 4, experiment i). During the course of these studies, we observed that rosemary also inhibited TPA-induced ear reddening. In

additional studies, topical application of 0.3 mg of arachidonic acid in20 ,.d acetone to the ears of mice rapidly induced edema formation,and maximum swelling was observed by 1 h. The results of our studies

Experiment 2Acetone 11.26 ±0.46Rosemary (3.6 mg) 8.98 ±061bRosemary (10.8 mg) 7.65 ±O.14'

a Statistically different from control acetone group (P < 0.10).

b Statistically different from control acetone group (P < 0.05).

C Statistically different from control acetone group (P < 0.02).

purchased on several occasions over a 5-year period revealed that theyall had about the same inhibitory effects on TPA-induced increases inornithine decarboxylase activity and TPA-induced ear inflammation.

Inhibitory Effect of Rosemary on the Covalent Binding of PH]-B(a)P to EpidermalDNA. In earlierstudies,it was foundthattheformation of [3H]B(a)P adducts in mouse epidermis was proportional

to the dose of [3H]B(a)P and reached a maximium level at i2—15hafter topical application of [3H]B(a)P (24). Topical application of 20nmol [3H]B(a)P resulted in the covalent binding of 0.74 pmol [3H]-B(a)P metabolites per mg of epidermal DNA at 15 h after application,and topical application of 1.2 or 3.6 mg rosemary S mm before theapplication of [3H]B(a)P inhibited covalent binding to DNA by 30 and54%, respectively (Fable 1, experiment 1). In an additional study,topical application of 200 nmol [3H]B(a)P to acetone-treated controlmice resulted in the covalent binding of 11.26 pmol [3H]B(a)P metabolites per mg of epidermal DNA at 15 h (Table 1, experiment 2).Topical application of 1.2 or 3.6 mg of rosemary at 120, 60, and S mmbefore the application of 200 nmol [3H]B(a)P resulted in the covalentbinding of 8.98 or 7.65 pmol [3HIB(a)P metabolites per mg of epidermal DNA, respectively (Table 1, experiment 2).

Inhibitory Effect of Rosemary on the Thmor-initiating Activityof B(a)P and DMBA. Topicalapplicationof B(a)P(20 nmol)onceweekly for 10 weeks followed by 15 nmol TPA twice weekly produced an average of 0.53 skin tumors per mouse after 9 weeks of TPApromotion, 3.37 skin tumors per mouse after 13 weeks of promotion,

Experiment 2Acetone + acetoneAcetone + B(a)P 0.77 ±0.28Rosemary (3.6 mg) + B(a)P 0.13 ±0.06

a Statistically different from the acetone + B(a)P group (P < 0.001).

b Statistically different from the acetone + B(a)P group (P < 0.005).

703

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Table 3 Inhibitory effect of topical application of rosemary on DMBA-inducedtumorinitiationFemaleCD-I mice (30 per group) were treated topically with 200 p.1 acetone or 1.2 or 3.6 mg of rosemary in 200 gil acetone 5 mm prior to application of 2 nmol of DMBAin200

pi acetone once weekly for 10 weeks. One week later, the mice were treated with 15 nmol TPA in 200 pi acetone twice weekly for 15 weeks. Data are expressed as themean±SE from 30 mice per group.7

Weeks 11 Weeks 15WeeksTumors

Percentage mice Tumors Percentage mice Tumors PercentagemiceTreatmentper mouse with tumors per mouse with tumors per mouse withtumorso―

0 o― 0 oao5319.6±1.7 100 25.0±1.61004114.2 ±18b 87 18.1 ±1.8―972412.4 ±1.6― 96 13.1 ±1.4― 86

TreatmentNo.of

miceWeightper punch

(mg)PercentageinhibitionExperiment

IAcetone46.4±0.2―TPA1213.5±0.4TPA

+ rosemary (0.04 mg)612.3 ±0.817TPA+ rosemary (0.12 mg)68.2 ±0.8―75TPA+ rosemary (0.36 mg)67.0 ±0.3―92Experiment

2Acetone65.80±0.22―TPA612.32±1.03TPA

+ rosemary (0.04 mg)69.83 ±1.4238TPA+ rosemary (0.18 mg)67.18 ±0.22―79Experiment

3Acetone67.12±0.32―TPA616.04±0.51TPA

+ rosemary (0.15 mg)68.51 ±0.53―84TPA+ rosemary (0.45 mg)67.71 ±0.62―93Experiment

4Acetone67.12±0.32―TPA616.04±0.51TPA

+ rosemary(0.15mg)67.95 ±0.62―91TPA+ rosemary (0.45 mg)66.91 ±O.IOa100a

StatisticallydifferentfromTPAalone group (P < 0.05).

Table 5 Inhthitory effect of rosemary on arachidonic acid-inducededemaofmouseearsThe

right ear of each female CD-i mouse was treated with 20 p1 acetone orrosemaryextractin 20 @dacetone 30 mm before 20 p.1 acetone alone or arachidonic acid (0.3mg)in

20 p.1 acetone. One h later, the mice were sacrificed and 6-mm diameter earpuncheswereweighed. Data are expressed as the mean ±SE from 10 mice pergroup.Weight

per punchPercentageTreatment(mg)inhibitionAcetone

6.52 ±0.17aArachidonicacid 10.98 ±0.56Arachidonicacid + rosemary (0.02 mg) 10.25 ±0.6316Arachidonicacid + rosemary (0.09 mg) 9.46 ±0.44―28Arachidonicacid + rosemary (0.45 mg) 8.58 ±Ø37a54a

Statisticallydifferentfrom arachidonicacid group(P < 0.05).

ROSEMARY AND CANCER PREVENTION

Acetone + acetoneAcetone + DMBA 3.1 ±0.7Rosemary (1.2 mg) + DMBA 1.6 ±0.5Rosemary (3.6 mg) + DMBA 0.5 ±0.2―

a Statistically different from the acetone + DMBA group (P < 0.01).

b Statistically different from the acetone + DMBA group (P < 0.05).

8-fold increase in the number of epidermal cell layers and in epidermal thickness. Inflammatory cell infiltration into the dermis and intercellular edema in the epidermis were also observed. Topical application of 3.6 mg rosemary together with i nmol TPA twice daily for4 days inhibited all of these TPA-induced effects.

Inhibitory Effect of Rosemary on TPA-induced Thmor Promotion in Mouse Epidermis. Mice initiated with 200 nmol DMBA andpromoted with 5 nmol TPA twice a week for 19 weeks had 17 tumorsper mouse, and 83% of the mice had tumors. In other groups ofsimilarly initiated mice, topical application of 0.4, 1.2, or 3.6 mgrosemary together with 5 nmol TPA twice a week for 19 weeks

inhibited the number of skin tumors per mouse by 40, 68, and 99%,respectively, and the percentage of mice with tumors was inhibited by57, 37, and 92%, respectively (Fig. 1). Additional groups of mice wereinitiated with DMBA and then treated with acetone alone or with 3.6mg of rosemary twice a week for 19 weeks. None of these animalsdeveloped tumors, indicating that rosemary was not a tumor promoter.In addition, mice were treated topically with 3.6 mg of rosemary andthen promoted with 5 nmol of TPA twice a week for 19 weeks. Noneof these animals developed tumors, indicating that rosemary was nota tumor initiator on mouse skin.

Studies with Carnosol and Ursolic Acid. Because of the stronginhibitory effects of rosemary on TPA-induced tumor promotion, weevaluated the effects of ursolic acid and camosol (major constituentsof rosemary) on TPA-induced inflammation, omithine decarboxylaseactivity, and tumor promotion. We also evaluated the effect of miltirone on TPA induction of omithine decarboxylase activity. The struclures of ursolic acid, camosol, and miltirone are shown in Fig. 2.Camosol has strong antioxidant activity but ursolic acid and miltironehave little or no antioxidant activity (7).

Inhibitory Effect of Carnosol and Ursolic Acid on TPA-inducedInflammation of Mouse Skin. The possible inhibitory effects of

camosol and ursolic acid on TPA-induced mouse ear edema wereexamined, and the results are shown in Table 7. Both camosol andursolic acid had strong antiinflammatory activity. Ursolic acid wassomewhat more active than camosol (Table 7).

Table 4 Inhibitory effect of topical application of rosemary on TPA-induced edemaof mouse ears

Both ears of CD-l female mice (23—25days old) were treated topically with 20 piacetone, TPA (0.5 nmol) in 20 @.dacetone, or TPA (0.5 nmol) and rosemary in 20 @.dacetone. Five hrs later, the mice were sacrificed and 6 mm diameter ear punches wereweighed. Data are expressed as the mean ±S.E. from 4—12mice per group.

indicated that topical application of 0.02, 0.09, or 0.24 mg rosemaryat 30 mm before 0.3 mg arachidonic acid inhibited arachidonic acidinduced ear edema by 16, 28, or 54%, respectively (Table 5).

Inhibitory Effect of Rosemary on TPA-induced Epidermal Ornithine Decarboxylase Activity. The effects oftopical application ofeach new batch of rosemary on TPA-induced increases in ornithinedecarboxylase activity in mouse epidermis was studied, and the results

are shown in Table 6. The various batches of rosemary had a similarinhibitory effect on TPA-induced increases in epidermal omithinedecarboxylase activity (Table 6, experiments 1—5).In these studies, it

was found that topical application of rosemary inhibited TPA-inducedincreases in epidermal omithine decarboxylase activity in a dosedependent manner. In a typical study, application of 0.4, 1.2, or 3.6 mgof rosemary together with S nmol TPA in 200 @lacetone to the backsof mice inhibited the TPA-induced increases in epidermal ornithinedecarboxylase activity by 67, 88, or 98%, respectively (Table 6, cx

periment 1).Inhibitory Effect of Rosemary on TPA-induced Hyperplasia in

Mouse Epidermis. The effects of topical application of rosemary onTPA-induced changes in cutaneous morphology were examined in 2

separate experiments. Application of 1 nmol TPA in 200 @lacetonetwice a day for 4 days to the dorsal surface of mice resulted in a 4- to

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Table 6 Inhibitory effect of topical application of rosemary on TPA-inducedornithinedecarboxylase activity in mouse epidermis

Female CD-i mice were treated topically with 200 @.dacetone, TPA (5 nmol) in 200pi acetone,or TPA (5 nmol) androsemaryin 200 p.1acetone.The micewerekilled 5 hlater, and epidermal ornithine decarboxylase activity was determined. In each experiment,the rosemary extract (rosemary) was obtained from a new batch of rosemary leaves. Dataare expressed as the mean ±SE from 3 mice per group.

Omithine decarboxylase activity Percentage(pmol CO@/h/mg protein) inhibition

Topical application of 1 or 2 @molursolic acid together with 5 nmolTPA twice weekly for 20 weeks inhibited TPA-induced formation ofskin tumors (tumors per mouse) by 45 or 61%, respectively (Fig. 4).

Lower doses of ursolic acid (0.1 or 0.3 p@mol)were shown to have asimilar inhibitory effect as the higher doses (1 or 2 p@mol).Topicalapplication of 0.i, 0.3, i, or 2 @molursolic acid together with S nmolTPA twice weekly for 8, 12, or 18 weeks inhibited TPA-inducedformation of skin tumors (number of tumors per mouse) by 52—86%,49—63%,or 44—61%,respectively (Table 9). There was no statisticallysignificant difference in number of tumors per mouse between any ofthe groups treated with ursolic acid (0.i—2p.mol). Doses of ursolicacid lower than 0.i @imolhad a smaller inhibitory effect than carnosol.

DISCUSSION

The results of the present study indicate that topical application ofrosemary inhibits B(a)P- and DMBA-induced initiation of tumors in

mouse skin as well as TPA-induced tumor promotion in DMBA

initiated mice (Tables 2 and 3; Fig. 1). Examination of the mechanisms of the inhibitory effect of rosemary on B(a)P-induced tumor

initiation indicates that topical application of rosemary inhibits covalent binding of B(a)P to epidermal DNA (Table 1). Singletary and

Nelshoppen (28) reported an inhibitory effect on mammary glandtumorigenesis of feeding an extract of rosemary leaves to rats treatedorally with DMBA. In that study, oral administration of the rosemaryextract inhibited the covalent binding of DMBA to total DNA and todeoxyguanosine in the mammary gland, but there was little or noeffect on the covalent binding of DMBA to deoxyadenosine (28).

Additional studies are needed to leam whether rosemary inhibits68 DMBA- and B(a)P-induced tumorigenesis by affecting the activity of

89 cytochrome P450 enzymes and/or phase II enzymes or whether rose

-@——— mary functions by additional mechanisms.

In the present study, we found that application of rosemary tomouse skin had a strong inhibitory effect on TPA-induced increases inornithine decarboxylase activity, inflammation, hyperplasia, and tumor promotion (Tables 4 and 6; Fig. 1). Analysis of rosemary extractsindicated that they contained 3—5%carnosol and 16—20%ursolic acid.We evaluated the effects of these substances on tumor promotion inmouse skin and found that camosol and ursolic acid are strong inhibitors of TPA-induced inflammation, ornithine decarboxylase activity, and tumor promotion in mouse skin (Tables 7 and 8; Figs. 3 and4). It is of interest that although camosol (a diphenolic diterpene; Fig.2) possesses high antioxidant activity, ursolic acid (a steroid-liketriterpene compound; Fig. 2) has little or no antioxidant activity (7).Camosol and camosic acid are thought to account for over 90% of theantioxidant properties of rosemary extract, and these compounds arepotent inhibitors of lipid peroxidation in microsomal and liposomal

678898

6687

100

ROSEMARY AND CANCER PREVENTION

628594

7192

Treatment

Experiment 1AcetoneTPATPA + rosemary (0.4 mg)TPA + rosemary (1.2 mg)TPA + rosemary (3.6 mg)

Experiment 2AcetoneTPATPA + rosemary (0.4 mg)TPA + rosemary (1.2 mg)TPA + rosemary (3.6 mg)

Experiment 3AcetoneTPATPA + rosemary (0.14 mg)TPA + rosemary (0.72 mg)TPA + rosemary (3.60 mg)

Experiment 4AcetoneTPATPA + rosemary (1.2 mg)TPA + rosemary (3.6 mg)

Experiment 5AcetoneTPATPA + rosemary (0.4 mg)TPA + rosemary (1.2 mg)TPA + rosemary (3.6 mg)

82 ±73―6592 ±9952172 ±198―

789 ±44―104 ±113―

220 ±69―2455±689

985 ±292―501 ±163―

89 ±3―

280 ±54―7816 ±3133156 ±863°1412 ±291°719 ±196―

110 ±iioa3906 ±14001195±391―402 ±113―

97 ±f@a6592 ±9952172 ±198°

786 ±44―144 ±140°

a Statistically different from TPA group (P < 0.05).

Inhibitory Effect of Carnosol and UrsolicAcid on TPA-inducedOrnithine Decarboxylase Activity in Mouse Epidermis. Topicalapplication of carnosol or ursolic acid had an inhibitory effect onTPA-induced omithine decarboxylase activity, but miltirone had little

or no effect (Table 8).Inhibitory Effect of Carnosol and Ursolic Acid on TPA-induced

Tumor Promotion in Mouse Epidermis. Mice that were initiatedwith 200 nmol DMBA and promoted with 5 nmol TPA twice weeklyfor 20 weeks had 20.2 skin tumors per mouse, and 93% of the micehad tumors. Topical application of 1, 3, or 10 p@molcamosol togetherwith 5 nmol TPA twice weekly for 20 weeks to mice previouslyinitiated with 200 nmol DMBA inhibited the number of skin tumorsper mouse by 37, 64, and 77%, respectively (Fig. 3).

21

4)

@ 14Fig. 1. Inhibitory effect of topical application of

rosemary on the tumor-promoting activity of TPAin mouse skin. Female CD-i mice were treatedtopically with 200 nmol DMBA in 200 @.dacetonefollowed 1 week later by topical application of 200@dacetone, 5 nmol TPA in 200 @.dacetone, or 5

nmol TPA and rosemary in 200 p@lacetone twiceweekly for 19 weeks. Points, mean ±SE from 30mice per group. , statistically different from TPAcontrol group (P < 0.05).

Weeks of TPA application

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Table 8 Inhibitory effect of topical application of carnoso4 miltirone, and ursolicacidonTPA-induced ornithine decarboxylase activity in mouseepidermisFemale

CD-i mice (8 weeks old) were treated topically with 200 @tlacetone,TPA(5nmol) in 200 p@lacetone, or TPA (5 nmol) and various amount of carnosol orursolicacid

in 200 ,.d acetone. Five h later, the mice were killed, and the epidermalornithinedecarboxylaseactivity was determined. Data are expressed as the mean ±SE from 3miceper

group.Ornithinedecarboxylase

activityPercentageTreatment(pmol CO@jh/mg protein) inhibition

per group.No.

ofWeight perpunchPercentageTreatmentmice(mg)inhibitionExperiment

1Acetone77.0±0.3―TPA816.0±0.4TPA

+ carnosol (0.1 pinol)8i2.2 ±0.9°42TPA+ carnosol (0.3 @mol)89.2 ±i.oa76TPA+ carnosol (1.0 p.mol)67.7 ±0.3°92TPA

+ ursolic acid (0.03 p.mol)812.5 ±i.oa39TPA+ ursolic acid (0.05 @.tmoI)811.8 ±1.1°46TPA+ ursolic acid (0.10 @smol)69.9 ±0.7―67TPA+ ursolicacid(0.20p@mol)68.0 ±0.4°89Experiment

2Acetone87.7±O.4@TPA813.1±1.0TPA

+ camosol (0.03 @.tmol)812.8 ±0.66TPA+ carnosol (0.10 @mol)89.2 ±0.6°72TPA+ carnosol (0.30 g.unol)89.5 ±1.0°67TPA+ camosol (1.00 @tmol)87.5 ±0.9°iOOTPA

+ ursolic acid (0.03 @moI)811.5 ±0.829TPA+ ursolic acid (0.15 @tmol)89.0 ±0.6°76TPA+ ursolic acid (0.30 @tmol)87.3 ±O.5a100Experiment

3Acetone86.8±0.2°TPA8i4.2±0.4TPA

+ ursolic acid (0.05 g.tmol)89.2 ±0.9a67TPA+ ursolic acid (0.25 p@mol)87.4 ±0.2°9ia

StatisticallydifferentfromTPAgroup (P < 0.05).

ROSEMARY AND CANCER PREVENTION

ExperimentAcetoneTPATPA + rosemary (0.72 mg)TPA + rosemary (3.60 mg)TPA + carnosol (0.72 mg; 2.2 @.tmol)TPA + carnosol (3.60 mg; 10.1 @smol)TPA + miltirone (0.72 mg; 2.6 p@mol)TPA + miltirone(3.60mg; 12.8 @tmol)

21 ±15―4796 ±355

104 ±i81°84 ±37―

i370 ±40i°435 ±96°59i2 ±1i633933 ±1141

116 ±ii6°8721 ±7105685±1863322 ±735°

97 ± @fJa

6592 ±9953923 ±396°2042 ±494@

ii ±ii―4200 ±15193079 ±2i02715 ±858i467 ±4613426 ±509i371 ±452

321 ±i3°4546 ±17752110 ±786i65O ±3374303 ±1i594217 ±5642576 ±68

i98 ±4@5696 ±2572215 ±69°2741 ±i42―3470 ±98°4040 ±695―3125 ±569°

7899729i

017

3563

4170

2735651868

546457

43

6152392945

Fig. 2. Structures of camosol, miltirone, and ursolic acid.

Table 7 Inhibitory effect of topical application of carnosol and ursolic acid onTPA-induced edema of mouse ears

The right ear of each female CD-i mouse (23—25days old) was treated topically with20 @.dacetone, TPA (0.5 nmol) in 20 p.1acetone, or 0.5 nmol TPA and various amount ofcamosol or ursolic acid in 20 gil acetone. Five h later, the mice were sacrificed and 6-mmdiameter ear punches were weighed. Data are expressed as the mean ±SE from 6—8mice

Experiment 2AcetoneTPATPA + carnosol (i.0 @mol)TPA + carnosol (2.0 prnol)

Experiment 3AcetoneTPATPA + carnosol (i.1 pinol)TPA + carnosol (iO.1 @smol)

Experiment 7Acetone 187 ±5°TPA 6784±78TPA + carnosol(3.0 @mol) 4188±65 38TPA + carnosol (10 @mol) 2176 ±50― 68TPA + ursolicacid(0.2 @tmol) 6759±440 0TPA + ursolicacid(0.6 @smol) 6132±10 10TPA + ursolic acid (2.0 p.mol) 3815 ±128° 45

a Statistically different from TPA group (P < 0.05).

Experiment 4AcetoneTPATPA + ursolic acid (0.03 pmol)TPA + ursolicacid(0.10 @tmol)TPA + ursolicacid(0.30 @smol)TPA + ursolic acid (1.00 @mol)TPA + ursolic acid (3.00 @mol)

Experiment 5AcetoneTPATPA + carnosol(3 @mol)TPA + carnosol (10 @smol)TPA + ursolic acid (0.2 @mol)TPA + ursolic acid (0.6 @tmol)TPA + ursolic acid (2.0 @imol)

Experiment 6AcetoneTPATPA + carnosol (3 @mol)TPA + carnosol (iO g@mol)TPA + ursolicacid(0.2 @mol)TPA + ursolicacid(0.6 @mol)TPA + ursolic acid (2.0 @mol)

706

inhibitors of arachidonic acid metabolism (30). Although we have notcompared the biological activities of camosol and curcumin in thesame experiment, the results of our studies suggest that curcumin maybe somewhat more active than carnosol as an inhibitor of TPA-induced tumor promotion (13).

Certain glucocorticoids strongly inhibit TPA-induced skin inflammation and tumor initiation as well as tumor promotion (31). Thesteroid-like structure of ursolic acid, its lack of antioxidant activity,and our data indicating that ursolic acid is a potent inhibitor of TPAinduced inflammation and tumor promotion in mouse skin (TablesMiltironeCarnosol

Ursolic acid

systems (9). Carnosol is also a good scavenger of peroxyl radicals and

superoxide anion radical as well as hydroxy radicals (9), and carnosol

has been demonstrated to inhibit mammalian 5-lipoxygenase activity(29). These observations, together with our studies indicating an inhibitory effect of camosol on arachidonic acid-induced inflammation,suggest that camosol may resemble other nonsteroidal phenolic antiinflammatory agents such as curcumin and quercetin that are potent

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Table 9 Inhibitory effect of topical application of ursolic acid on TPA-induced tumor promotion in CD-i mice previously initiated withDMBAFemaleCD-i mice (30 per group) were treated topically with 200 nmol DMBA in 200 @slacetone followed 1 week later by topical application of 200 @lacetone, 5 nmol TPAin200

@.lacetone, or 5 nmol TPA and ursolic acid in 200 @lacetone twice weekly for 18 weeks. Data are expressed as the mean ±SE from 30 mice per group. Values in theparenthesesarepercentage inhibition.8

Weeks I 2 Weeks 18WeeksTumors

Percentage mice Tumors Percentage mice Tumors PercentagemiceTreatmentper mouse with tumors per mouse with tumors per mouse withtumorsAcetone

o― 0 o― 0 00TPA2.8 ±0.7 66 15.3 ±2.4 90 20. 1 ±I .687Ursolic

acid (0.1 p.mol) + TPA 0.4 ±0.2a (86) 23 7.2 ±1.5°(53) 87 9.8 ±1.8°(51) 87Ursolic acid (0.3 @anol)+ TPA 1.3 ±0.5 (52) 30 7.5 ±1.4°(51) 77 11.3 ±l.9@'(44) 93Ursolic acid (1.0 @smol)+ TPA 1.0 ±O.4―(63) 33 7.9 ±19b (49) 70 11.0 ±2.3―(45) 77Ursolic acid (2.0 @imoI)+ TPA 0.9 ±o.4b(69) 23 5.7 ±1.6―(63) 53 7.8 ±1.9°(61)60a

Statistically different from the TPA group (P < 0.01).

b Statistically different from the TPA group (P < 0.05).

ROSEMARY AND CANCER PREVENTION

U

Fig. 3. Inhibitory effect of carnosol on TPAinduced tumor promotion in mouse skin. FemaleCD-i mice were treated topically with 200 nmolDMBA in 200 p.1acetone followed 1 week later bytopical application of5 nmolTPAin 200 p.1acetoneor 5 nmol TPA with camosol in 200 p.1acetonetwice weekly for 20 weeks. Points, mean ±SEfrom 30 mice per group. @,statistically differentfrom TPA control group (P < 0.05).

Weeks of ‘WAapplication

7—9;Fig. 4) suggest that ursolic acid may exert effects similar to thoseof steroidal, antiinflammatory compounds. Ursolic acid was shown toinhibit TPA-induced Epstein-Barr virus activation in Raji cells (32,33), to inhibit 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate-induced edema of mouse ears (34) and to inhibit TPA-induced tumorpromotion in mouse skin (35). The possibility that camosol and ursolic acid may inhibit tumor promotion by different mechanismssuggests that we explore possible synergistic effects of carnosol andursolic acid. Our observations that rosemary extract has a strongerinhibitory effect on TPA-induced tumor promotion than carnosol orursolic acid alone suggests that combinations of these compounds orcombinations of these compounds together with other constituents inrosemary are responsible for the inhibitory effect of rosemary ontumor promotion.

Leaves of the plant Rosmarinus officinalis L. are commonly used asa spice, flavoring agent, and naturally occurring antioxidant. Phenolicantioxidants from rosemary leaves are nonvolatile and quite stable at

high temperatures, whereas the commercial synthetic antioxidants

Fig. 4. Inhibitory effect of ursolic acid on TPAinduced tumor promotion in mouse skin. FemaleCD-i mice were treatedtopically with 200 nmolDMBA in 200 p.1acetone followed 1 week later bytopical application of 200 @.lacetone, 5 nmol TPAin 200 pJ acetone, or 5 nmol TPA and ursolic acidin 200 @LIacetone twice weekly for 20 weeks.Points, mean ±SE from 30 mice per group. @,statistically different from TPA control group (P <0.05).

BHT and BHA are unstable at high temperatures. Rosemary extractshave been used widely as an antioxidant during the preparation offrench fries and potato chips (36), and rosemary antioxidants havealso been used in poultry, lamb, veal, shellfish, sausages, salads, soup,breading, and other foods (36). Since the synthetic antioxidants BHAand BHT, at high dose levels, have been reported to have toxicity inanimals studies (37, 38), the naturally occurring antioxidants in rosemary have been used frequently in certain foods instead of BHT andBHA.

Antioxidants in the leaves of Rosmarinus officinalis L. includecamosol and carnosic acid (9), rosmanol, isorosmanol, epirosmanol,and rosmariquinone (8). Camosol, camosic acid, rosmanol, and rosmaridiphenol have the same structural backbone, whereas rosmarinicacid has a different structure (8). It is likely that a combination ofseveral components is responsible for the inhibitory effect of rosemaryon carcinogenesis.

Recent studies in our laboratory found that dietary rosemary inhibited B(a)P-induced forestomach and lung tumorigenesis, azoxymeth

20

Weeks of TPA application

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ROSEMARY AND CANCER PREVENTION

18. Kato, R., Nakadate, I., Yamamoto, S., and Sugimura, I. Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion and omithine decarboxylase activity by quercetin: possible involvement of lipoxygenase inhibition. Carcinogenesis(Land.), 4: 1301—1305,1983.

19. Huang, M-T., Ho, C-I., Cheng, S-i., Laskin, J. D., Stauber, K., Georgiadis, C., andConney, A. H. Inhibitory effect of an extract of leaves of Rosmarinus officinalis L. ontumor promotion by 12-O-tetradecanoylphorbol-i3-acetate on mouse skin. Proc. Am.Assoc. Cancer Res., 30: 180, 1989.

20. Huang, M-T., Ho, C-T., Ferraro, I., Wang, Z. Y., Stauber, K., Georgiadis, C., Laskin,J. D., and Conney, A. H. Inhibitory effect of an extract of leaves of Rosmarinusofficinalis L. and its constitutents, camosol and ursolic acid, on tumorigenesis in CD-imouse epidermis. Proc. Am. Assoc. Cancer Res., 33: 165, 1992.

21. Grossberger, I., and Rothschild, E. Determination of the triglyceride composition ofvegetable liquid oils by high performance liquid chromatography. LC-GC, 7:439—441,1989.

22. Schwarz, K., and Temes, W. Antioxidative constituents of Rosmarinus officinalis andSalvia officinalis. II. Isolation of carnosic acid and formation of other phenolicditerpenes. Z. Lebensm. Unters. Forsch., i95: 99—103,1992.

23. Knapp, S., and Sharma, S. Synthesis of the naturally occurring antioxidant rosmariquinone. J. Org. Chem., 50: 4996-4998, 1985.

24. Smart, R. C., Huang, M-T., Chang, R. L, Sayer, J. M., Jerina, D. M., Wood, A. W.,and Conney, A. H. Effect of ellagic acid and 3-O-decylellagic acid on the formationof benzo[ajpyrene-derived DNA adducts in vivo and on the tumorigenicity of 3-methylcholanthrene in mice. Carcinogenesis (Land.), 7: i669—i675, 1986.

25. Slaga,I. i., Das, S. B., Rice, J. M., and Thompson,S. Fractionationof mouseepidermal chromatin components. J. Invest. Dermatol., 63: 343—349,i974.

26. Bradford, M. M. A rapid and sensitive method for the quantitation of microgramsquantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.,72: 248—254,1986.

27. Smart, R. C., Huang, M-T., Monteiro-Riviere, N. A., Wong, C-Q., Mills, K., J., andConney, A. H. Comparison of the effect of sn-1,2-didecanoylglycerol and 12-0-tetradecanoylphorbol-13-acetate on cutaneous morphology, inflammation and tumorpromotion in CD-l mice. Carcinogenesis (Land.), 9: 2221—2226,1988.

28. Singletary, K. W., and Neishoppen, J. M. Inhibition of 7,12-dimethylbenz[a@anthracene (DMBA)-induced mammary tumorigenesis and of in viva formation of mammary DMBA-DNA adducts by rosemary extract. Cancer LeU, 60: 169-175, 199i.

29. Laughton, M. J., Evans, P. J., Moroney, M. A., Hoult, J. R. S., and Halliwell, B.Inhibition of mammalian 5-lipoxygenase and cyclooxygenase by flavonoids andphenolic dietary additives. Biochem. Pharmacol., 42: 1673—1681,1991.

30. Huang, M-T., Lysz, I., Ferraro, I., Abidi, I. D., Laskin, J. D., and Conney, A. H.Inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activitiesin mouse epidermis. Cancer Res., 51: 813—819,1991.

31. Slaga, I. J., and Seribner, J. D. Inhibition of tumor initiation and promotion byanti-inflammatory agents. J. Natl. Cancer Inst., 51: i723—i725,i973.

32. Okamoto, H., Yoshida, D., and MiZusaki, S. Inhibition of 12-0-tetradecanoylphorbol13-acetate-induced in Epstein-Barr virus early antigen in Raji cells. Cancer LeU., 19:47—53,1983.

33. Ohigashi, H., Takamura, H., Koshimizu, K., Tokuda, H., and Ito, Y. Search forpossible antitumor promoters by inhibition of 12-0-tetradecanoylphorbol-i3-acetateinduced Epstein-Barr virus activation; ursolic acid and oleanolic acid from an antiinflammatory Chinese medicinal plant, Glechoma hederaceae L Cancer LeE., 30:143—151,i986.

34. Hirota, H., Mori, I., Yoshida, M., and Iriye, R. Suppression of tumor promoterinduced inflammation of mouse ear by ursolic acid and 4,4-dimethylcholestane derivatives. Agric. Biol. Chem., 54: 1073—iO75,1990.

35. Tokuda, H., Ohigashi, H., Koshimizu, K., and Ito, Y. Inhibitory effects of ursolic andoleanolic acid on skin tumor promotion by i2-0-tetradecanoylphorbol-13-acetate.Cancer Lett., 33: 279—285,1986.

36. Fisher, C. Phenolic compounds in spices. In: C-T. Ho, C. Y. Lee, and M-T. Huang(eds.), Phenolic Compounds in Food and Their Effects on Health I: Analysis, Occurence, and Chemistry, Chap. 9, pp. i18—i29. Washington, DC: American ChemicalSociety, 1992.

37. Ito, N., Fukushima, S., Hagiwara, A., Shibata, M., and Ogiso, I. Carcinogenicity ofbutylated hydroxyanisole in F344 rats. J. NatI. Cancer Inst., 70: 343—352,1983.

38. Witschi, H., and Morse, C. C. Enhancement of lung tumor formation in mice bydietary butylated hydroxytoluene: dose-time relationships and cell kinetics. J. Nail.Cancer Inst., 71: 856—866,1983.5 Unpublished results.

708

ane-induced colon tumorigenesis, and DMBA-induced mammarygland tumorigenesis in mice.5 Further studies are needed to determinethe constituents of rosemary that are responsible for its inhibitoryeffect on tumorigenesis. Since rosemary is widely used in food preparations, it is important to more fully evaluate the pharmacological and

toxicological effects of this complex mixture.

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