Inhibition of Skin Ilimorigenesis by Rosemary and Its Constituents Carnosol and Ursolic Acid'

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[CANCERRESEARCH54, 701-708, February 1, 19941 Inhibition of Skin ilimorigenesis by Rosemary and Its Constituents Carnosol and Ursolic 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. Conney2â€•@

Laboratory for Cancer Research, Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 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, New Brunswick, New Jersey 08903 [C-T H.]; and Department of Environmental and Community Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 [C. G., J. D. L.]

ABSTRACT Many compounds that possess antioxidant activity inhibit tumor promotion in mouse skin (10â€”14).Butylated hydroxyanisole (10, iS), A methanol extract of the leaves of the plant Rosmarinus officinalis L butylated hydroxytoluene (15), sodium selenite (16), a-tocopherol (rosemary) was evaluated for its effects on tumor initiation and promotion (17), glutathione (17), quercetin (18), ascorbyl palmitate (12), curcu in mouse skin. Application of rosemary to mouse skin inhibited the cova lent binding of benzo(a)pyrene [B(a)P] to epidermal DNA and inhibited mm (13), and a green tea polyphenol fraction (14) are examples of tumor initiation by B(a)P and 7,12-dimethylbenz[aJanthracene (DMBA). substances that possess antioxidant or reactive oxygen scavenging Topical application of 20 nmol B(a)P to the backs of mice once weekly for activity and inhibit tumor promotion and/or biochemical events asso 10 weeks, followed 1 week later by promotion with 15 nmol 12-O-tetra ciated with tumor promotion in mouse skin (10â€”14,i8). In the present decanoylphorbol-13-acetate (TPA) twice weekly for 21 weeks, resulted in study, we evaluated the effect of an extract of rosemary leaves (rose the formation of7.1 tumors per mouse. In a parallel group ofanimals that mary) on the initiation of skin tumors by B(a)P'@and DMBA as well were treated topically with 1.2 or 3.6 mg of rosemary 5 mm prior to each as the promotion of skin tumors by TPA. We also evaluated the effect application of B(a)P, the number of tumors per mouse was decreased by of carnosol and ursolic acid, isolated from the leaves of the rosemary 54 or 64%, respectively. Application of rosemary to mouse skin also in plant, on TPA-induced tumor promotion in mouse skin. We previously hibited TPA-induced ornithine decarboxylase acfivity@TPA-mduced in published preliminary reports of these studies (i9, 20). flammation, arachidonic acid-induced inflammation, TPA-induced hyper plasia, and TPA-induced tumor promotion. Mice initiated with 200 nmol DMBA and promoted with S nmol TPA twice weekly for 19 weeks devel MATERIALS AND METhODS o_ an average of 17.2 skin tumors per mouse. @fteatmentofthe DMBA initiated mice with 0.4, 1.2, or 3.6 mg of rosemary together with 5 nmol Materials. TPA was purchased from Chemsyn Science Laboratories (Len TPAtwiceweeklyfor 19weeksinhibited the number ofTPA-mducedskin exa, KS). L-['4CjOrnithine (58 Ci/mmol) and [3H]B(a)P (62.8 Ci/mmol) were tumors per mouse by 40, 68, or 99%, respectively. Topical application of purchased from Amersham Corp. (Arlington Heights, IL). DMBA was pur carnosol or ursolic acid belated from rosemary inhibited TPA-induced ear chased from Calbiochem-Boehring (San Diego, CA). Aquasol was purchased inflammation, ornithine decarboxylase activity, and tumor promotion. from NEN Research Products (Boston, MA). Acetone and dimethyl sulfoxide Topical application of 1, 3, or 10 @amolcarnosol together with 5 nmol TPA were purchased from Burdick & Jackson Laboratories (Muskegon, MI). Ara twice weekly for 20 weeks to the backs of mice previously initiated with chidonic acid was purchased from Nu-Chek-Prep, Inc. (Elysian, MN). The DMBA inhibited the number ofsldn tumors per mouse by 38, 63, or 78%, dried rosemary leaves were purchased from General Spice, Inc. (South Plain respectively. Topical application of 0.1, 0.3, 1, or 2 @amolursolicacid field, NJ). together with 5 nmol TPA twice weekly for 20 weeks to DMBA-initiated Preparation of Methanol Extract of Ground Dried Leaves of Rosmari mice inhibited the number of tumors per mouse by 45-61%. ni's officinalis L A methanol extract of the ground dried leaves of the plant Rosmarinus officinalis L. (rosemary extract;rosemary)was preparedaccording to the previously reportedmethod of Wu et aL (7). Three kg of rosemary INTRODUCTION leaves, which had been ground to a fine powder, were extracted with 18 liters methanol at 60Â°Cfor 2 h. The extraction was performed in a custom-made The leaves of the plant Rosmarinus officinalis L. are commonly 30-liter steam jacketed, stainless steel extractor equipped with a Lightnin mixer used as a spice, flavoring agent, and naturally occurring antioxidant (Lightnin Group, Rochester, NY). After extraction, the mixture was filtered, (1, 2). In the 1950s, it was reported that an extract of rosemary leaves and the residue was extracted again with 12 liters fresh methanol. The com contained high antioxidant activity (1, 3, 4) and shortly thereafter bined filtrate was bleached with 600 g activated carbon and then filtered to extracts from rosemary leaves were used commercially for their an yield a light-brown filtrate. The methanol solution was concentrated to a final tioxidant activity (5, 6). Extracts of rosemary leaves have been used to volume of 2 liters with a vacuum rotary evaporator and then filtered to remove prevent lipid autoxidation and to inhibit the oxidation of both animal precipitate. Three liters of water were added to the filtrate. The precipitate that fats and vegetable oils (6). The antioxidant activity of an extract from was formed after the addition of water was filtered and air dried to yield a rosemary leaves is comparable to that of butylated hydroxytoluene rosemary extract (rosemary) that was used for our studies. About 200â€”300g dry powdered rosemary was obtained from 3 kg of ground rosemary leaves. and butylated hydroxyanisole (7). Although carnosol, carnosic acid, HPLC Analysis of Rosemary ExtracL HPLC analysis was performed rosmaridiphenol, rosmanol, isorosmanol, epirosmanol, and rosmari with a Varian 5000 Liquid Chromatograph with an ACS model 750/14 mass quinone arc antioxidant compounds in rosemary leaves (8), about 90% detector from Pens Industries (State College, PA). In this detector, the flow of of the antioxidant activity of rosemary can be attributed to carnosol the column moves through a nebulizer and then passes through a tube held at and camosic acid (9). a 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 50Â°Cand 16 psi air Received 7/13/93; accepted 11/23/93. pressure. The column was a Whatman PartiSphere C,8 column (12.5 cm X The costs of publication of this article were defrayed in part by the payment of page 0.46 cm internal diameter). The following ternary solvent system was utilized charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

â€˜Supported in part by Grant CA49756 from the NIH. 4 The abbreviations used are: B(a)P, benzo(a)pyrene; DMBA, 7,12-dimethylbenz[aJ 2 William M. and Myrle W. Garbe Professor of Cancer and Leukemia Research. anthracene; TPA, 12-O-tetradecanoylphorbol-13-acetate; BItT, butylated hydroxytoluene; 3 To whom requests for reprints should be addressed. BHA,butylatedhydroxyanisole. 701

for column elution: Solvent A, 1% acetic acid in water; solvent B, acetonitrile; Measurement of Epidermal Ornitbine Decarboxylase Activity. The and solvent C, 1% acetic acid in methanol. The mobile phase was programmed preparation of epidermal homogenates and the determination of omithine linearly from 30% A170% C at the start of elution to 5% A/90% B over a 30 decarboxylase activity were performed as described previously (13). Mice (8â€”9 mm interval, followed by 5% A/5% B/90% C for 5 mm and then 100% B for weeks old) were treated with 200 p.1acetone, TPA in acetone, or rosemary and another 15 mm. The flow rate was 0.7 ml/min. The retention times of carnosol, TPA in acetone. Five h after treatment, the mice were sacrificed by cervical miltirone, carnosic acid, and ursolic acid were 7.9, 9.2, 15.7, and 30.8 mm, dislocation, and the dorsal area of the skin was removed. In order to separate respectively. the epidermis from the dermis, the skins were plunged into a 58Â°Cwater bath HPLC analysis of several preparations of the rosemary extract indicated that for 15 5, and then the skins were immediately submerged in an ice water bath they contained 16.5â€”19.2%ursolic acid, 3.8â€”4.6%carnosol, 0.1-0.5% carnosic as described by Slaga et a!. (25). The epidermis was removed from the dermis acid, and trace-0. 1% miltirone, respectively. by gentle scraping and placed in 1 ml 50 mr@ipotassium phosphate, pH 7.7 Preparation of Carnosol. The ground dried leaves of Rosmarinus offici buffer containing 2 m@ dithiothreitol and 0.1 [email protected]. The epidermis was naiLs L. (300 g) were extracted three times for 2â€”3hwith 750 ml hexane at homogenized on ice for 10 s using a Polytron homogenizer with the motor 25Â°Cin a 2-liter stainless steel vessel fitted with a mechanical stirrer. The speed setting at 4. Epidermal homogenates were centrifuged at 11,000 X g for solvent was evaporated under vacuum with a rotary evaporator to yield 11â€”13 30 mm at 4Â°C, and the supernatant fractions were removed and stored over g of extract. The powdered rosemary extract was dissolved in 200 ml methanol night at â€”20Â°Cpriorto the determination ofornithine decarboxylase activity as and allowed to stand at room temperature for 1 week to allow the conversion described earlier (13). Protein was determined by the protein-dye binding of carnosic acid to carnosol (22). After removing the methanol under vacuum procedure described by Bradford (26) using a Bio-Rad protein assay kit (Rich with a rotary evaporator, 0.5 g of the dry residue was dissolved in 2 ml mond, CA). hexane:ether (3: 1) and was injected into a preparative column (550 mm X 25 Morphological Examination ofTPA-treated Mouse Skin. Assay of TPA mm internal diameter) packed with activated silicic acid (Mallinckrodt, St. induced epidermal hyperplasia was done by a slight modification of the pro Louis, MO). The mobile phase, delivered by a piston pump, was hexane:ether cedure of Smart et a!. (27). The dorsal skin of female CD-i mice (7â€”8weeks (3: 1), and the flow rate was 2.5 mI/mm. The eluent was monitored by an UV old, 3â€”4miceper group) was treated with 200 @lacetone,TPA (1 nmol), or detector at 254 nm The carnosol fraction, which eluted between 12.5â€”14.5 TPA (1 nmol) and 3.6 mg rosemary in 200 @lacetonetwice a day for 4 days. mm, was collected, and the solvent was evaporated. After removal of solvent, The mice were killed by cervical dislocation 18 h after the last dose. The dorsal the carnosol fraction was recrystallized from methanol to yield carnosol of skin was excised and fixed in 10% neutral buffered formalin. The skin samples 98% purity as determined by HPLC (described above). The identity of camosol were embedded in paraffin and processed for histology with hematoxylin and was confirmed by particle beam liquid chromatography/mass spectrometry in eosin staining. Inflammatory cell (leukocyte) infiltration that was absent (-), the electron ionization mode on a Vestec Model 201 LC/MS (Vestec,Houston, slight (+), moderate (+ +), or severe (+ + +) was characterized by diffuse TX) equipped with a universal interface. The mass spectrum of camosol infiltration of mononuclear inflammatory cells into the dermis when compared showed a molecular ion at m/z 330 (1 1%) and major fragmentation ions at m/z with the acetone controls. Intercellular edema (accumulation of fluid between 286 (100%), 284 (32%), 271 (33%), 215 (62%) and 269 (23%). the epidermal cells) was scored as present or absent. The number of nucleated Preparation of Miltirone. Miltirone was synthesized by the Diels-Alder cell layers in the epidermis was determined at five locations per slide and reaction between 3-isopropyl-O-benzoquinone and 6,6-dimethyl-1-vinylcyclo averaged. Thickness of the noncomifled cell layers of the epidermis was also hexane according to the method of Knapp and Sharma (23). The reaction measured in a similar manner. The mean Â± SE for each group of mice was mixture was subjected to silica gel column chromatography using ethyl ether calculated. Morphometric analysis was performed using an automatic photo :pentane (3:97) as eluent to yield a red solid. After recrystallization from microscope (X40). hexane, miltirone (99% purity as determined by HPLC) was obtained. The Skin Thmorigenesis Studies. For studies on the effects of rosemary or its mass spectrum of miltirone by particle beam liquid chromatography/mass constituents on tumor initiation and promotion, the dorsal regions of female spectrometry showed a molecular ion at m/z 282 (10%) and major fragmen CD-i mice (7 weeks old) were shaved with an electric clipper. Two days later, tation ions at 254 (55%), 239 (100%), 224 (19%), and 165 (12%). groups of 30 mice were treated topically with 200 nmol DMBA in 200 @l Preparation of Ursolic Acid. Ten g of dried methanol extract of rosemary acetone; control mice received 200 p.1 acetone alone. After 1 week, the mice leaves, prepared as described above, were dissolved in 200 ml boiling ethanol. were treated topically with 200 pJ acetone, 5 nmol TPA in 200 pA acetone, or Upon cooling, ursolic acid crystallized from the solvent. It was recrystallized 5 nmol TPA and rosemary or test compound in 200 pi acetone twice weekly twice from ethanol to obtain ursolic acid of98% purity as determined by HPLC for 20 weeks. Tumors at least 1 mm in diameter were counted and recorded (described above). The ursolic acid was identified by particle beam liquid once every 2 weeks, and the results were expressed as the average number of chromatography/mass spectrometry to have a molecular ion at mJz 456 (27%) tumors per mouse and percentage of tumor-bearing mice. and major fragmentation ions at 438 (30%), 423 (26%), 410 (30%), 395 (35%), For studies on the effect of rosemary on tumor initiation by B(a)P or 302 (90%), 248 (100%), 203 (98%), 189 (42%), and 133 (79%). DMBA, mice were treated topically with 200 @lacetone or with rosemary in Animals. Female CD-i mice were purchased from Charles River Labora 200 @lacetone 5 mm prior to each application of B(a)P (20 nmol) or DMBA tories (Kingston, NY) and kept in our animal facility for 1â€”2weeks before use. (2 nmol) once a week for 10 weeks. One week later, the mice received 15 nmol Mice were fed a Purina Lab Chow 5001 diet ad libitum (Ralston-Purina Co., TPA in 200 @lacetonetwice weekly for 20 weeks. In a second study, mice St. Louis, MO) and kept on a 12-h light-dark cycle. Mice were provided were treated topically with 200 @lacetone or 3.6 mg of rosemary in 200 pi drinking water ad libitutn. The dorsal region of each mouse was shaved with acetone at 120, 60, and 5 mm before the topical application of 200 nmol B(a)P @ electric clippers at least 2 days before treatment with TPA, B(a)P, or DMBA. in 200 acetone. One week later, the mice received 15 nmol TPA in 200 @l Only mice that did not show signs of hair regrowth were used. acetone twice weekly for 20 weeks. Determination of @H]B(a)P-DNAAdductsin Mouse Epidermis. The Statistical Analyses. Student's t test was used for all statistical analyses. covalent binding of [3H]B(a)P to epidermal DNA was performed as described earlier (24). RESULTS Measurement of Mouse Ear Edema. TPA (0.5 nmol) either alone or together with test compound in 20 p3 acetone was applied to the right ear of Studies with Rosemary. Each preparation of dry, powdered rose 25-day-old female CD-i mice (6â€”10mice per group). Control mice received mary extract (rosemary) was examined by HPLC and tested for its 20 pJ acetone. Five h later, the mice were killed by cervical dislocation, and ability to inhibit TPA-induced increases in epidermal ornithine decar 6-mm diameter ear punch biopsies were taken and weighed. The increase in weight of the ear punches was directly proportional to the degree of inflam boxylase activity and TPA-induced mouse ear edema. The HPLC mation. In studies on arachidonic acid-induced edema of mouse ears, mice analysis of several preparations of rosemary indicated that they con were treated on the right ear with 20 pA acetone or 0.3 mg arachidonic acid in tamed 16.5â€”19.2%ursolic acid, 3.8â€”4.6%carnosol, 0.1â€”0.5%carno 20 p.1 acetone. The mice were sacrificed 1 h later by cervical dislocation, and sic acid, and a trace-0.i% miltirone. Examination of the biological ear punches were taken and weighed (13). activity of several batches of rosemary obtained from rosemary leaves 702

Table 1 Inhibitory effect of rosemaryformationin on f3HJB(a)P-DNA adduct and 7.10 tumors per mouse after 2i weeks of promotion. In parallel epidermisIn mouse groups of mice, topical application of 1.2 or 3.6 mg rosemary S mm experiment 1, female CD-i mice were treated topically with 200 pi acetone alone ornmol,100 rosemary in 200 pi acetone 5 mm before the topical application of [3H]B(a)P (20 before each application of B(a)P decreased the number of tumors per piacetonepCi) in 200 @.dacetone. In experiment 2, the mice were treated topically with 200 mouse by 15 or 62% after 9 weeks of promotion, 42 or 63% after 13 or 1.2 or 3.6 mg of rosemary in 200 @lacetone at 120, 60, and 5 mm before the weeks of promotion, and by 54 or 64% after 2i weeks of promotion, topicalwerekilled application of [3H]B(a)P (200 nmol, 100 pCi) in 200 gsl acetone. The mice determined.The15 h later. Skin was removed and radioactivity in epidermal DNA was respectively (Table 2, experiment 1). In an additional study, mice were data are expressed as mean Â±SEgroup).[3H]B(a)P (3â€”4miceper treated topically with acetone at 120, 60, and S mm before the topical PercentageTreatment metabolites bound application of 200 nmol B(a)P. After i week, the mice were promoted inhibitionExperiment (jmollmg DNA) with 15 nmol TPA twice weekly for 21 weeks. These mice developed 1Acetone an average 0.77, 3.35, and 6.89 skin tumors per mouse after 9, i3, and 0.13Rosemary 0.74 Â± 30Rosemary(1.2 mg) 0.52 Â±0.07 21 weeks of promotion with TPA, respectively (Table 2, experiment (3.6 mg) 0.34 Â±0.13â€• 54 2). A parallel group of mice that were treated topically with 3.6 mg rosemary in acetone at i20, 60, and S mm before the application of Experiment 2 Acetone 11.26 Â±0.46 B(a)P developed an average of 0.13, 0.64, and 2.87 skin tumors per Rosemary (3.6 mg) 8.98 Â±061b 20 mouse after 9, 13, and 21 weeks of TPA promotion, respectively Rosemary (10.8 mg) 7.65 Â±O.14' 32 (Table 2, experiment 2). The results of this study indicated that rose a Statistically different from control acetone group (P < 0.10). b Statistically different from control acetone group (P < 0.05). mary decreased by 58â€”83%thenumber of skin tumors per mouse. C Statistically different from control acetone group (P < 0.02). In another experiment, topical application of DMBA (2 nmol) to a group of 30 mice once weekly for 10 weeks, followed by treatment purchased on several occasions over a 5-year period revealed that they with TPA (15 nmol) twice weekly for 7, ii, and 15 weeks produced all had about the same inhibitory effects on TPA-induced increases in an average of 3.1, 19.6, or 25 skin tumors per mouse, respectively ornithine decarboxylase activity and TPA-induced ear inflammation. (Table 3). Topical application of i.2 or 3.6 mg of rosemary extract 5 Inhibitory Effect of Rosemary on the Covalent Binding of PH]- mm prior to each application of DMBA decreased the number of B(a)P to EpidermalDNA. In earlierstudies,it was foundthatthe DMBA-induced skin tumors per mouse by 48 or 84% after 7 weeks of formation of [3H]B(a)P adducts in mouse epidermis was proportional promotion, by 27 or 37% after ii weeks of promotion, and by 28 or to the dose of [3H]B(a)P and reached a maximium level at i2â€”15h 48% after iS weeks of promotion, respectively (Table 3). after topical application of [3H]B(a)P (24). Topical application of 20 Inhibitory Effect of Rosemary on TPA- and Arachidonic Acid nmol [3H]B(a)P resulted in the covalent binding of 0.74 pmol [3H]- induced Inflammation of Mouse Skin. The antiinflammatory activ B(a)P metabolites per mg of epidermal DNA at 15 h after application, ity of rosemary was evaluated by determining its effect on TPA- and and topical application of 1.2 or 3.6 mg rosemary S mm before the arachidonic acid-induced edema. Several batches of rosemary were application of [3H]B(a)P inhibited covalent binding to DNA by 30 and tested for their abilities to inhibit TPA-induced ear edema, and the 54%, respectively (Fable 1, experiment 1). In an additional study, various batches of rosemary were found to have a similar inhibitory topical application of 200 nmol [3H]B(a)P to acetone-treated control effect on TPA-induced inflammation (Table 4, experiments 1â€”4).Ina mice resulted in the covalent binding of 11.26 pmol [3H]B(a)P me typical experiment, topical application of 0.5 nmol TPA in 20 @l tabolites per mg of epidermal DNA at 15 h (Table 1, experiment 2). acetone to the ear of a mouse increased the average weight of an ear Topical application of 1.2 or 3.6 mg of rosemary at 120, 60, and S mm punch (6-mm diameter) from 6.4 mg to 13.5 mg at S h after the dose before the application of 200 nmol [3H]B(a)P resulted in the covalent (Table 4, experiment 1). Topical application of 0.04, 0.12, or 0.36 mg binding of 8.98 or 7.65 pmol [3HIB(a)P metabolites per mg of epi rosemary together with 0.5 nmol TPA to the ears of mice inhibited the dermal DNA, respectively (Table 1, experiment 2). TPA-induced edema of mouse ears by i7, 75, or 92%, respectively Inhibitory Effect of Rosemary on the Thmor-initiating Activity (Table 4, experiment i). During the course of these studies, we ob of B(a)P and DMBA. TopicalapplicationofB(a)P(20 nmol)once served that rosemary also inhibited TPA-induced ear reddening. In weekly for 10 weeks followed by 15 nmol TPA twice weekly pro additional studies, topical application of 0.3 mg of arachidonic acid in duced an average of 0.53 skin tumors per mouse after 9 weeks of TPA 20 ,.d acetone to the ears of mice rapidly induced edema formation, promotion, 3.37 skin tumors per mouse after 13 weeks of promotion, and maximum swelling was observed by 1 h. The results of our studies

initiationIn Table 2 Inhibitory effect of topical application of rosemary on B(a)P-induced tumor 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 @lacetoneonce 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 were treated with 15 nmol TPA twice weekly for 21 weeks. Data aregroup.9 expressed as the mean Â±SE from 30 mice per WeeksTumors Weeks 13 Weeks 21

Percentage mice Tumors Percentage mice Tumors Percentage mice tumorsExperimentTreatment per mouse with tumors per mouse with tumors per mouse with 1 @ 3AcetoneAcetone + acetone oâ€• o oâ€• 0.03 Â±0.03â€• + B(a)P 0.53 Â±0.02 30 3.37 Â±0.54 87 7.10 Â±1.03 100 Rosemary (i.2 mg) + B(a)P 0.45 Â±0.26 17 1.96 Â±0.07â€• 50 3.24 Â±0.52â€• 90 Rosemary (3.6 mg) + B(a)P 0.20 Â±0.07 20 1.24 Â±0.30â€• 48 2.57 Â±0.48@' 73

Experiment 2 Acetone + acetone 00â€•[email protected] Acetone + B(a)P 0.77 Â±0.28 0.13100130.64 Â±0.80706.89 Â± Rosemary (3.6 mg) + B(a)P 0.13 Â±0.06 Â±020b372.87 Â±0.46â€•77 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

initiationFemale Table 3 Inhibitory effect of topical application of rosemary on DMBA-inducedtumor in200 CD-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 DMBA meanÂ±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 the SE from 30 mice per group.7 WeeksTumors Weeks 11 Weeks 15 miceTreatment Percentage mice Tumors Percentage mice Tumors Percentage pertumorsoâ€• mouse with tumors per mouse with tumors per mouse with Acetone + acetone o530 oâ€• 0 oa Acetone + DMBA 3.1 Â±0.7 10041 19.6Â±1.7 100 25.0Â±1.6 Rosemary (1.2 mg) + DMBA 1.6 Â±0.5 9724 14.2 Â±18b 87 18.1 Â±1.8â€• Rosemary (3.6 mg) + DMBA 0.5 Â±0.2â€• 12.4 Â±1.6â€• 96 13.1 Â±1.4â€• 86 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 epider Table 4 Inhibitory effect of topical application of rosemary on TPA-induced edema of mouse ears mal thickness. Inflammatory cell infiltration into the dermis and in Both ears of CD-l female mice (23â€”25days old) were treated topically with 20 pi tercellular edema in the epidermis were also observed. Topical appli acetone, TPA (0.5 nmol) in 20 @.dacetone, or TPA (0.5 nmol) and rosemary in 20 @.d cation of 3.6 mg rosemary together with i nmol TPA twice daily for acetone. Five hrs later, the mice were sacrificed and 6 mm diameter ear punches were weighed. Data are expressed as the mean Â±S.E. from 4â€”12mice per group. 4 days inhibited all of these TPA-induced effects. Inhibitory Effect of Rosemary on TPA-induced Thmor Promo of per punch inhibitionExperimentTreatmentNo. miceWeight (mg)Percentage tion in Mouse Epidermis. Mice initiated with 200 nmol DMBA and IAcetone46.4 promoted with 5 nmol TPA twice a week for 19 weeks had 17 tumors 0.2â€•TPA1213.5 Â± per mouse, and 83% of the mice had tumors. In other groups of 0.4TPA Â± similarly initiated mice, topical application of 0.4, 1.2, or 3.6 mg 0.817TPA+ rosemary (0.04 mg)612.3 Â± 0.8â€•75TPA+ rosemary (0.12 mg)68.2 Â± rosemary together with 5 nmol TPA twice a week for 19 weeks 0.3â€•92Experiment+ rosemary (0.36 mg)67.0 Â± inhibited the number of skin tumors per mouse by 40, 68, and 99%, respectively, and the percentage of mice with tumors was inhibited by 2Acetone65.80 0.22â€•TPA612.32 Â± 57, 37, and 92%, respectively (Fig. 1). Additional groups of mice were 1.03TPA Â± initiated with DMBA and then treated with acetone alone or with 3.6 1.4238TPA+ rosemary (0.04 mg)69.83 Â± 0.22â€•79Experiment+ rosemary (0.18 mg)67.18 Â± mg of rosemary twice a week for 19 weeks. None of these animals developed tumors, indicating that rosemary was not a tumor promoter. 3Acetone67.12 In addition, mice were treated topically with 3.6 mg of rosemary and 0.32â€•TPA616.04 Â± 0.51TPA Â± then promoted with 5 nmol of TPA twice a week for 19 weeks. None 0.53â€•84TPA+ rosemary (0.15 mg)68.51 Â± of these animals developed tumors, indicating that rosemary was not 0.62â€•93Experiment+ rosemary (0.45 mg)67.71 Â± a tumor initiator on mouse skin. 4Acetone67.12 Studies with Carnosol and Ursolic Acid. Because of the strong 0.32â€•TPA616.04Â±0.51TPA Â± inhibitory effects of rosemary on TPA-induced tumor promotion, we

0.62â€•91TPA+ rosemary(0.15mg)67.95 Â± evaluated the effects of ursolic acid and camosol (major constituents O.IOa100a+ rosemary (0.45 mg)66.91 Â± of rosemary) on TPA-induced inflammation, omithine decarboxylase StatisticallydifferentfromTPAalone group (P < 0.05). activity, and tumor promotion. We also evaluated the effect of milt irone on TPA induction of omithine decarboxylase activity. The struc lures of ursolic acid, camosol, and miltirone are shown in Fig. 2. indicated that topical application of 0.02, 0.09, or 0.24 mg rosemary Camosol has strong antioxidant activity but ursolic acid and miltirone at 30 mm before 0.3 mg arachidonic acid inhibited arachidonic acid have little or no antioxidant activity (7). induced ear edema by 16, 28, or 54%, respectively (Table 5). Inhibitory Effect of Carnosol and Ursolic Acid on TPA-induced Inhibitory Effect of Rosemary on TPA-induced Epidermal Or Inflammation of Mouse Skin. The possible inhibitory effects of nithine Decarboxylase Activity. The effects oftopical application of camosol and ursolic acid on TPA-induced mouse ear edema were each new batch of rosemary on TPA-induced increases in ornithine examined, and the results are shown in Table 7. Both camosol and decarboxylase activity in mouse epidermis was studied, and the results ursolic acid had strong antiinflammatory activity. Ursolic acid was are shown in Table 6. The various batches of rosemary had a similar somewhat more active than camosol (Table 7). inhibitory effect on TPA-induced increases in epidermal omithine decarboxylase activity (Table 6, experiments 1â€”5).In these studies, it was found that topical application of rosemary inhibited TPA-induced Table 5 Inhthitory effect of rosemaryedemaof on arachidonic acid-induced increases in epidermal omithine decarboxylase activity in a dose earsThe mouse dependent manner. In a typical study, application of 0.4, 1.2, or 3.6 mg rosemaryextractright ear of each female CD-i mouse was treated with 20 p1 acetone or mg)in in 20 @dacetone 30 mm before 20 p.1acetone alone or arachidonic acid (0.3 of rosemary together with S nmol TPA in 200 @lacetoneto the backs puncheswere20 p.1 acetone. One h later, the mice were sacrificed and 6-mm diameter ear of mice inhibited the TPA-induced increases in epidermal ornithine weighed. Data are expressed as the mean Â±SEgroup.Weight from 10 mice per decarboxylase activity by 67, 88, or 98%, respectively (Table 6, cx PercentageTreatment per punch periment 1). inhibitionAcetone (mg) Inhibitory Effect of Rosemary on TPA-induced Hyperplasia in 0.17aArachidonic 6.52 Â± Mouse Epidermis. The effects of topical application of rosemary on 0.56Arachidonicacid 10.98 Â± 16Arachidonicacid + rosemary (0.02 mg) 10.25 Â±0.63 TPA-induced changes in cutaneous morphology were examined in 2 28Arachidonicacid + rosemary (0.09 mg) 9.46 Â±0.44â€• separate experiments. Application of 1 nmol TPA in 200 @lacetone 54a acid + rosemary (0.45 mg) 8.58 Â±Ã˜37a twice a day for 4 days to the dorsal surface of mice resulted in a 4- to Statisticallydifferentfrom arachidonicacid group(P < 0.05). 704

Table 6 Inhibitory effect of topical application of rosemary on TPA-inducedornithine Topical application of 1 or 2 @molursolic acid together with 5 nmol decarboxylase activity in mouse epidermis TPA twice weekly for 20 weeks inhibited TPA-induced formation of Female CD-i mice were treated topically with 200 @.dacetone,TPA (5 nmol) in 200 pi acetone,orTPA (5 nmol) androsemaryin 200 p.1acetone.Themicewerekilled 5 h skin tumors (tumors per mouse) by 45 or 61%, respectively (Fig. 4). later, and epidermal ornithine decarboxylase activity was determined. In each experiment, Lower doses of ursolic acid (0.1 or 0.3 p@mol)were shown to have a the rosemary extract (rosemary) was obtained from a new batch of rosemary leaves. Data similar inhibitory effect as the higher doses (1 or 2 p@mol).Topical are expressed as the mean Â±SE from 3 mice per group. application of 0.i, 0.3, i, or 2 @molursolic acid together with S nmol Omithine decarboxylase activity Percentage TPA twice weekly for 8, 12, or 18 weeks inhibited TPA-induced Treatment (pmol CO@/h/mg protein) inhibition formation of skin tumors (number of tumors per mouse) by 52â€”86%, Experiment 1 Acetone 82 Â±73â€• 49â€”63%,or44â€”61%,respectively(Table 9). There was no statistically TPA 6592 Â±995 significant difference in number of tumors per mouse between any of TPA + rosemary (0.4 mg) 2172 Â±198â€• 67 the groups treated with ursolic acid (0.iâ€”2p.mol). Doses of ursolic TPA + rosemary (1.2 mg) 789 Â±44â€• 88 TPA + rosemary (3.6 mg) 104 Â±113â€• 98 acid lower than 0.i @imolhad a smaller inhibitory effect than carnosol.

Experiment 2 Acetone 220 Â±69â€• DISCUSSION TPA 2455Â±689 TPA + rosemary (0.4 mg) 985 Â±292â€• 66 TPA + rosemary (1.2 mg) 501 Â±163â€• 87 The results of the present study indicate that topical application of TPA + rosemary (3.6 mg) 89 Â±3â€• 100 rosemary inhibits B(a)P- and DMBA-induced initiation of tumors in mouse skin as well as TPA-induced tumor promotion in DMBA Experiment 3 Acetone 280 Â±54â€• initiated mice (Tables 2 and 3; Fig. 1). Examination of the mecha TPA 7816 Â±313 nisms of the inhibitory effect of rosemary on B(a)P-induced tumor TPA + rosemary (0.14 mg) 3156 Â±863Â° 62 TPA + rosemary (0.72 mg) 1412 Â±291Â° 85 initiation indicates that topical application of rosemary inhibits cova TPA + rosemary (3.60 mg) 719 Â±196â€• 94 lent binding of B(a)P to epidermal DNA (Table 1). Singletary and Nelshoppen (28) reported an inhibitory effect on mammary gland Experiment 4 Acetone 110 Â±iioa tumorigenesis of feeding an extract of rosemary leaves to rats treated TPA 3906 Â±1400 orally with DMBA. In that study, oral administration of the rosemary TPA + rosemary (1.2 mg) 1195 Â±391â€• 71 extract inhibited the covalent binding of DMBA to total DNA and to TPA + rosemary (3.6 mg) 402 Â±113â€• 92 deoxyguanosine in the mammary gland, but there was little or no Experiment 5 effect on the covalent binding of DMBA to deoxyadenosine (28). Acetone 97 Â±f@a Additional studies are needed to leam whether rosemary inhibits TPA 6592 Â±995 TPA + rosemary (0.4 mg) 2172 Â±198Â° 68 DMBA- and B(a)P-induced tumorigenesis by affecting the activity of TPA + rosemary (1.2 mg) 786 Â±44â€• 89 cytochrome P450 enzymes and/or phase II enzymes or whether rose TPA + rosemary (3.6 mg) 144 Â±140Â° -@â€”â€”â€” mary functions by additional mechanisms. a Statistically different from TPA group (P < 0.05). In the present study, we found that application of rosemary to mouse skin had a strong inhibitory effect on TPA-induced increases in Inhibitory Effect of Carnosol and UrsolicAcid on TPA-induced ornithine decarboxylase activity, inflammation, hyperplasia, and tu Ornithine Decarboxylase Activity in Mouse Epidermis. Topical mor promotion (Tables 4 and 6; Fig. 1). Analysis of rosemary extracts application of carnosol or ursolic acid had an inhibitory effect on indicated that they contained 3â€”5%carnosol and 16â€”20%ursolic acid. TPA-induced omithine decarboxylase activity, but miltirone had little We evaluated the effects of these substances on tumor promotion in or no effect (Table 8). mouse skin and found that camosol and ursolic acid are strong in Inhibitory Effect of Carnosol and Ursolic Acid on TPA-induced hibitors of TPA-induced inflammation, ornithine decarboxylase activ Tumor Promotion in Mouse Epidermis. Mice that were initiated ity, and tumor promotion in mouse skin (Tables 7 and 8; Figs. 3 and with 200 nmol DMBA and promoted with 5 nmol TPA twice weekly 4). It is of interest that although camosol (a diphenolic diterpene; Fig. for 20 weeks had 20.2 skin tumors per mouse, and 93% of the mice 2) possesses high antioxidant activity, ursolic acid (a steroid-like had tumors. Topical application of 1, 3, or 10 p@molcamosol together triterpene compound; Fig. 2) has little or no antioxidant activity (7). with 5 nmol TPA twice weekly for 20 weeks to mice previously Camosol and camosic acid are thought to account for over 90% of the initiated with 200 nmol DMBA inhibited the number of skin tumors antioxidant properties of rosemary extract, and these compounds are per mouse by 37, 64, and 77%, respectively (Fig. 3). potent inhibitors of lipid peroxidation in microsomal and liposomal

4) Fig. 1. Inhibitory effect of topical application of @ rosemary on the tumor-promoting activity of TPA 14 in mouse skin. Female CD-i mice were treated topically with 200 nmol DMBA in 200 @.dacetone followed 1 week later by topical application of 200 @dacetone, 5 nmol TPA in 200 @.dacetone, or 5 nmol TPA and rosemary in 200 p@lacetonetwice weekly for 19 weeks. Points, mean Â±SE from 30 mice per group. , statistically different from TPA control group (P < 0.05).

Weeks of TPA application 705

inhibitors of arachidonic acid metabolism (30). Although we have not compared the biological activities of camosol and curcumin in the same experiment, the results of our studies suggest that curcumin may be somewhat more active than carnosol as an inhibitor of TPA-in duced tumor promotion (13). Certain glucocorticoids strongly inhibit TPA-induced skin inflam mation and tumor initiation as well as tumor promotion (31). The steroid-like structure of ursolic acid, its lack of antioxidant activity, and our data indicating that ursolic acid is a potent inhibitor of TPA Carnosol Miltirone induced inflammation and tumor promotion in mouse skin (Tables

Table 8 Inhibitoryacidon effect of topical application of carnoso4 miltirone, and ursolic epidermisFemaleTPA-induced ornithine decarboxylase activity in mouse TPA(5 CD-i mice (8 weeks old) were treated topically with 200 @tlacetone, ursolicacidnmol) in 200 p@lacetone,or TPA (5 nmol) and various amount of carnosol or ornithinedecarboxylasein 200 ,.d acetone. Five h later, the mice were killed, and the epidermal miceper activity was determined. Data are expressed as the mean Â±SE from 3 group.Ornithinedecarboxylase

PercentageTreatment activity (pmol CO@jh/mg protein) inhibition Experiment Acetone 21 Â±15â€• Ursolic acid TPA 4796 Â±355 TPA + rosemary (0.72 mg) 104 Â±i81Â° 78 Fig. 2. Structures of camosol, miltirone, and ursolic acid. TPA + rosemary (3.60 mg) 84 Â±37â€• 99 TPA + carnosol (0.72 mg; 2.2 @.tmol) i370 Â±40iÂ° 72 Table 7 Inhibitory effect of topical application of carnosol and ursolic acid on TPA + carnosol (3.60 mg; 10.1 @smol) 435 Â±96Â° 9i 59i2 Â±1i63 TPA-induced edema of mouse ears TPA + miltirone (0.72 mg; 2.6 p@mol) 0 TPA + miltirone(3.60mg; 12.8 @tmol) 3933 Â±1141 17 The right ear of each female CD-i mouse (23â€”25days old) was treated topically with 20 @.dacetone, TPA (0.5 nmol) in 20 p.1acetone, or 0.5 nmol TPA and various amount of Experiment 2 camosol or ursolic acid in 20 gil acetone. Five h later, the mice were sacrificed and 6-mm Acetone 116 Â±ii6Â° diameter ear punches were weighed. Data are expressed as the mean Â±SE from 6â€”8mice TPA 8721 Â±710 per group.No. TPA + carnosol (i.0 @mol) 5685Â±186 35 punchPercentageTreatmentmice(mg)inhibitionExperimentofWeight per TPA + carnosol (2.0 prnol) 3322 Â±735Â° 63

Experiment 3 1Acetone77.0 Acetone 97 Â± @fJa 0.3â€•TPA816.0 Â± TPA 6592 Â±995 0.4TPA Â± TPA + carnosol (i.1 pinol) 3923 Â±396Â° 41 0.9Â°42TPA+ carnosol (0.1 pinol)8i2.2 Â± TPA + carnosol (iO.1 @smol) 2042 Â±494@ 70 i.oa76TPA+ carnosol (0.3 @mol)89.2 Â± 0.3Â°92TPA+ carnosol (1.0 p.mol)67.7 Â± Experiment 4 Acetone ii Â±iiâ€• i.oa39TPA+ ursolic acid (0.03 p.mol)812.5 Â± TPA 4200 Â±1519 1.1Â°46TPA+ ursolic acid (0.05 @.tmoI)811.8 Â± TPA + ursolic acid (0.03 pmol) 3079 Â±2i0 27 0.7â€•67TPA+ ursolic acid (0.10 @smol)69.9 Â± TPA + ursolicacid(0.10 @tmol) 2715 Â±858 35 0.4Â°89Experiment+ ursolicacid(0.20p@mol)68.0 Â± TPA + ursolicacid(0.30 @smol) i467 Â±461 65 TPA + ursolic acid (1.00 @mol) 3426 Â±509 18 2Acetone87.7 TPA + ursolic acid (3.00 @mol) i371 Â±452 68 [email protected] Â± 1.0TPA Â± Experiment 5 0.66TPA+ camosol (0.03 @.tmol)812.8 Â± Acetone 321 Â±i3Â° 0.6Â°72TPA+ carnosol (0.10 @mol)89.2 Â± TPA 4546 Â±1775 1.0Â°67TPA+ carnosol (0.30 g.unol)89.5 Â± TPA + carnosol(3 @mol) 2110 Â±786 54 0.9Â°iOOTPA+ camosol (1.00 @tmol)87.5 Â± TPA + carnosol (10 @smol) i65O Â±337 64 TPA + ursolic acid (0.2 @mol) 4303 Â±1i59 5 0.829TPA+ ursolic acid (0.03 @moI)811.5 Â± TPA + ursolic acid (0.6 @tmol) 4217 Â±564 7 0.6Â°76TPA+ ursolic acid (0.15 @tmol)89.0 Â± TPA + ursolic acid (2.0 @imol) 2576 Â±68 43 O.5a100Experiment+ ursolic acid (0.30 @tmol)87.3 Â± Experiment 6 3Acetone86.8 Acetone i98 Â±4@ 0.2Â°TPA8i4.2 Â± TPA 5696 Â±257 0.4TPA Â± TPA + carnosol (3 @mol) 2215 Â±69Â° 61 0.9a67TPA+ ursolic acid (0.05 g.tmol)89.2 Â± TPA + carnosol (iO g@mol) 2741 Â±i42â€• 52 0.2Â°9ia + ursolic acid (0.25 p@mol)87.4 Â± TPA + ursolicacid(0.2 @mol) 3470 Â±98Â° 39 StatisticallydifferentfromTPAgroup(P < 0.05). TPA + ursolicacid(0.6 @mol) 4040 Â±695â€• 29 TPA + ursolic acid (2.0 @mol) 3125 Â±569Â° 45

systems (9). Carnosol is also a good scavenger of peroxyl radicals and Experiment 7 superoxide anion radical as well as hydroxy radicals (9), and carnosol Acetone 187 Â±5Â° TPA 6784Â±78 has been demonstrated to inhibit mammalian 5-lipoxygenase activity TPA+ carnosol(3.0 @mol) 4188Â±65 38 (29). These observations, together with our studies indicating an in TPA + carnosol (10 @mol) 2176 Â±50â€• 68 TPA+ ursolicacid(0.2 @tmol) 6759Â±440 0 hibitory effect of camosol on arachidonic acid-induced inflammation, TPA + ursolicacid(0.6 @smol) 6132Â±10 10 suggest that camosol may resemble other nonsteroidal phenolic anti TPA + ursolic acid (2.0 p.mol) 3815 Â±128Â° 45 inflammatory agents such as curcumin and quercetin that are potent a Statistically different from TPA group (P < 0.05). 706

U Fig. 3. Inhibitory effect of carnosol on TPA induced tumor promotion in mouse skin. Female CD-i mice were treated topically with 200 nmol DMBA in 200 p.1acetone followed 1 week later by topical application of5 nmolTPAin 200 p.1acetone or 5 nmol TPA with camosol in 200 p.1acetone twice weekly for 20 weeks. Points, mean Â±SE from 30 mice per group. @,statistically different from TPA control group (P < 0.05).

Weeks of â€˜WAapplication

7â€”9;Fig.4) suggest that ursolic acid may exert effects similar to those BHT and BHA are unstable at high temperatures. Rosemary extracts of steroidal, antiinflammatory compounds. Ursolic acid was shown to have been used widely as an antioxidant during the preparation of inhibit TPA-induced Epstein-Barr virus activation in Raji cells (32, french fries and potato chips (36), and rosemary antioxidants have 33), to inhibit 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate-in also been used in poultry, lamb, veal, shellfish, sausages, salads, soup, duced edema of mouse ears (34) and to inhibit TPA-induced tumor breading, and other foods (36). Since the synthetic antioxidants BHA promotion in mouse skin (35). The possibility that camosol and ur and BHT, at high dose levels, have been reported to have toxicity in solic acid may inhibit tumor promotion by different mechanisms animals studies (37, 38), the naturally occurring antioxidants in rose suggests that we explore possible synergistic effects of carnosol and mary have been used frequently in certain foods instead of BHT and ursolic acid. Our observations that rosemary extract has a stronger BHA. inhibitory effect on TPA-induced tumor promotion than carnosol or Antioxidants in the leaves of Rosmarinus officinalis L. include ursolic acid alone suggests that combinations of these compounds or camosol and carnosic acid (9), rosmanol, isorosmanol, epirosmanol, combinations of these compounds together with other constituents in and rosmariquinone (8). Camosol, camosic acid, rosmanol, and ros rosemary are responsible for the inhibitory effect of rosemary on maridiphenol have the same structural backbone, whereas rosmarinic tumor promotion. acid has a different structure (8). It is likely that a combination of Leaves of the plant Rosmarinus officinalis L. are commonly used as several components is responsible for the inhibitory effect of rosemary a spice, flavoring agent, and naturally occurring antioxidant. Phenolic on carcinogenesis. antioxidants from rosemary leaves are nonvolatile and quite stable at Recent studies in our laboratory found that dietary rosemary inhib high temperatures, whereas the commercial synthetic antioxidants ited B(a)P-induced forestomach and lung tumorigenesis, azoxymeth

20 Fig. 4. Inhibitory effect of ursolic acid on TPA induced tumor promotion in mouse skin. Female CD-i mice were treatedtopically with 200 nmol DMBA in 200 p.1acetone followed 1 week later by topical application of 200 @.lacetone,5 nmol TPA in 200 pJ acetone, or 5 nmol TPA and ursolic acid in 200 @LIacetonetwice weekly for 20 weeks. Points, mean Â±SE from 30 mice per group. @, statistically different from TPA control group (P < 0.05).

Weeks of TPA application

DMBAFemale Table 9 Inhibitory effect of topical application of ursolic acid on TPA-induced tumor promotion in CD-i mice previously initiated with in200 CD-i mice (30 per group) were treated topically with 200 nmol DMBA in 200 @slacetonefollowed 1 week later by topical application of 200 @lacetone,5 nmol TPA [email protected],or 5 nmol TPA and ursolic acid in 200 @lacetonetwice weekly for 18 weeks. Data are expressed as the mean Â±SE from 30 mice per group. Values in the percentage inhibition.8 WeeksTumors Weeks I 2 Weeks 18

miceTreatment Percentage mice Tumors Percentage mice Tumors Percentage tumorsAcetone per mouse with tumors per mouse with tumors per mouse with 0TPA oâ€• 0 oâ€• 0 0 87Ursolic 2.8 Â±0.7 66 15.3 Â±2.4 90 20. 1 Â±I .6 acid (0.1 p.mol) + TPA 0.4 Â±0.2a (86) 23 7.2 Â±1.5Â°(53) 87 9.8 Â±1.8Â°(51) 87 Ursolic acid (0.3 @anol)+ TPA 1.3 Â±0.5 (52) 30 7.5 Â±1.4Â°(51) 77 11.3 Â±l.9@'(44) 93 Ursolic acid (1.0 @smol)+TPA 1.0 Â±O.4â€•(63) 33 7.9 Â±19b (49) 70 11.0 Â±2.3â€•(45) 77 60aUrsolic acid (2.0 @imoI)+ TPA 0.9 Â±o.4b(69) 23 5.7 Â±1.6â€•(63) 53 7.8 Â±1.9Â°(61) Statistically different from the TPA group (P < 0.01). b Statistically different from the TPA group (P < 0.05). 707

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1994 American Association for Cancer Research. ROSEMARY AND CANCER PREVENTION ane-induced colon tumorigenesis, and DMBA-induced mammary 18. Kato, R., Nakadate, I., Yamamoto, S., and Sugimura, I. Inhibition of 12-O-tetradec gland tumorigenesis in mice.5 Further studies are needed to determine anoylphorbol-13-acetate-induced tumor promotion and omithine decarboxylase ac tivity by quercetin: possible involvement of lipoxygenase inhibition. Carcinogenesis the constituents of rosemary that are responsible for its inhibitory (Land.), 4: 1301â€”1305,1983. effect on tumorigenesis. Since rosemary is widely used in food prepa 19. Huang, M-T., Ho, C-I., Cheng, S-i., Laskin, J. D., Stauber, K., Georgiadis, C., and Conney, A. H. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1994 American Association for Cancer Research. Inhibition of Skin Tumorigenesis by Rosemary and Its Constituents Carnosol and Ursolic Acid

Mou-Tuan Huang, Chi-Tang Ho, Zhi Yuan Wang, et al.

Cancer Res 1994;54:701-708.

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