Screening of Mammalian DNA Polymerase Inhibitors from Rosemary Leaves and Analysis of the Anti-inflammatory and Antiallergic Effects of the Isolated Compounds

Abstract

In this study, on screening mammalian DNA polymerase (pol) inhibitors from the extracts of 20 edible plants, we found that leaves of rosemary (Rosmarinus officinalis L.) showed the strongest pol inhibition; furthermore, we isolated four components, carnosic acid, carnosol, ursolic acid, and rosmarinic acid, from an extract of the leaves. Carnosic acid inhibited the activities of all 11 mammalian pols tested. In descending order of their inhibitory effect on pol λ, which is a DNA repair/recombination pol, the four compounds ranked as follows: carnosic acid > ursolic acid > carnosol > rosmarinic acid. The inhibition of pol λ by these compounds was significantly correlated with both in vivo anti-inflammatory and antiallergic effects, including on 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory mouse ear edema, and immunoglobulin E-induced passive cutaneous anaphylactic reaction in mice. These results indicate that rosemary and its constituent, carnosic acid, are potential therapeutic food candidates for inflammatory and allergic diseases.

Introduction

DNA-dependent DNA polymerase (pol) (E.C. 2.7.7.7) catalyzes the addition of deoxyribonucleotides to the 3’-hydroxyl terminus of primed double-stranded DNA (dsDNA) molecules (Kornberg and Baker, 1992). The human genome encodes at least 15 DNA pols that function in cellular DNA synthesis (Bebenek and Kunkel, 2004; Hubscher et al., 2002). Eukaryotic cells contain three replicative pols (α, δ, and ε), one mitochondrial pol (γ), and at least 11 non-replicative pols (β, ζ, η, θ, ι, κ, λ, μ, ν, terminal deoxynucleotidyl transferase (TdT), and REV1) (Lange et al., 2011; Loeb and Monnat, 2008). Pols have a highly conserved structure, with their overall catalytic subunits showing little variation among species; conserved enzyme structures that are preserved over time usually perform important cellular functions that confer evolutionary advantages. Based on sequence homology, eukaryotic pols can be divided into four main families: A, B, X, and Y (Loeb and Monnat, 2008). Family A includes mitochondrial pol γ as well as pols θ and ν; family B includes the three replicative pols, α, δ, and ε, and also pol ζ; family X includes pols β, λ, and μ, as well as TdT; and family Y includes pols η, ι, and κ, in addition to REV1 (Lange et al., 2011). We have studied selective inhibitors of eukaryotic pols derived from natural products, including food materials and components, over the past 15 years and have discovered more than 100 inhibitors of mammalian pols (Mizushina, 2009; Mizushina, 2011).

During our pol inhibitor studies, we found that pol λ-selective inhibitors such as curcumin derivatives (Mizushina et al., 2002) can suppress inflammation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (Mizushina et al., 2003). Although tumor promoters, including TPA, are classified as compounds that promote tumor formation (Hecker, 1978), they can also cause inflammation and are commonly used as artificial inducers of inflammation to screen for anti-inflammatory agents (Fujiki and Sugimura, 1987). The tumor promoter TPA is frequently used to search for new types of anti-inflammatory compounds. TPA not only causes inflammation, but also influences mammalian cell proliferation (Nakamura et al., 1995), suggesting that the molecular basis of inflammation stems from pol reactions related to cell proliferation. However, this relationship needs to be investigated more closely.

Recently, we found that inhibition of pol λ activity by 20 essential oils showed high correlation with in vivo antiallergic effects, such as an immunoglobulin E (IgE)-induced passive cutaneous anaphylactic (PCA) reaction in mice, based on mast cell degranulation (Mitoshi et al., 2012). It has been speculated that these results and phenomena indicate anti-inflammatory/antiallergic activities that may have been caused by pol λ inhibition.

In this study, we first screened for mammalian pol inhibitors from 20 edible plants, extracted the plants using 30% ethanol, and found that an extract from leaves of rosemary (Rosmarinus officinalis L.) was the strongest inhibitor among the plant extracts tested. We then isolated mammalian pol inhibitors from rosemary leaves and investigated whether these compounds had anti-inflammatory and antiallergic properties. The relationship between the inhibition of members of the four families of mammalian pols and the biological activities of the isolated compounds is discussed.

Materials and Methods

Materials    We obtained dried samples from 20 edible plants, including whole Asiatic dayflower (Commelina communis L.), husks of cacao (Theobroma cacao L.) beans, whole Chinese plantain (Plantago asiatica L.), Chinese quince (Chaenomeles sinensis Koehne) fruit, skin of Chinese soapberry (Sapindus mukurossi Gaertn.) fruit, corms of cobra lily (Arisaema spp.), young buds of fat hen (Chenopodium album var. centrorubrum Makino), gambir (Uncaria gambir Roxburgh) foliage, leaves and buds of globe artichoke (Cynara scolymus L.), aerial parts of green purslane (Portulaca oleracea L.), hiba arborvitae (Thujopsis dolabrata Sieb. et Zucc.) foliage, Indian lotus (Nelumbo nucifera Gaertn.) fruit, Japanese horse chestnut (Aesculus turbinata Blume) fruit, leaves of kidachi aloe (Aloe arborescens Mill.), leaves of Kuma bamboo grass (Sasa veitchii Rehd.), leaves of perilla (Perilla frutescens Britton var. acuta Kudo, P. frutescens Britton var. crispa Decaisne), seeds of proso millet (Panicum miliaceum L.), leaves of rosemary (Rosmarinus officinalis L.), silk tree (Albizia julibrissin Durazz.) bark, and leaves of summer savory (Satureja hortensis L.), from Nagaoka Perfumery Co. Ltd. (Osaka, Japan). A chemically synthesized DNA template, poly(dA), was purchased from Sigma-Aldrich Inc. (St Louis, MO, USA), and a customized oligo(dT)₁₈ DNA primer was produced by Sigma-Aldrich Japan K.K. (Hokkaido, Japan). Radioactive [³H]-labeled 2’-deoxythymidine-5’-triphosphate (dTTP; 43 Ci/mmol) was obtained from Moravek Biochemicals Inc. (Brea, CA, USA). All other reagents were purchased from Nacalai Tesque Inc. (Kyoto, Japan) and were of analytical grade.

Cells    Two cell lines, murine macrophage RAW264.7 and rat basophilic leukemia RBL-2H3, were obtained from the American Type Culture Collection (Manassas, VA, USA). RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 4.5 g/L glucose, 10% FBS, 5 mM l-glutamine, 50 units/mL penicillin, and 50 units/mL streptomycin. RBL-2H3 cells were cultured in Eagle's minimum essential medium supplemented with 4.5 g/L glucose, 10% FBS, 5 mM l-glutamine, 50 units/mL penicillin, and 50 units/mL streptomycin. The cells were cultured at 37°C in a humidified atmosphere of 5% CO₂/95% air.

Animals    Female 6-week-old ICR mice (body weight, 25 – 27 g) were obtained from Japan SLC, Inc. (Hamamatsu, Japan), and maintained on a standard diet (MF; Oriental Yeast Co. Ltd., Osaka, Japan), with water provided ad libitum. Mice that had been bred in-house with free access to food and water were used for all experiments. All of the mice were maintained under a 12-h light/dark cycle and housed at room temperature (25°C). This study was approved by the Institutional Animal Care and Use Committee of Kobe Gakuin University and was performed according to the guidelines outlined in the Care and Use of Laboratory Animals of Kobe Gakuin University. The animals were anesthetized with pentobarbital before undergoing cervical dislocation.

Instrumental analyses    ¹H- and ¹³C-nuclear magnetic resonance (NMR) data were collected using a Bruker DRX400 system (Bruker Biospin GmbH, Rheinstetten, Germany). Chemical shift δ values were reported in parts per million (ppm) relative to tetramethylsilane as an internal standard. Mass spectra were obtained on an API QSTAR Pulsar i spectrometer (Applied Biosystems, Foster City, CA, USA).

Measurement of inhibitory activities of pols and other DNA metabolic enzymes    The 11 mammalian pols α, β, γ, δ, ε, η, ι, κ, λ, μ, and TdT, plant (cauliflower) pol α, and three prokaryotic pols, E. coli pol I, Taq pol, and T4 pol, were prepared as described previously (Myobatake et al., 2012). The reaction mixtures for these pols have been described previously (Myobatake et al., 2012; Mizushina et al., 1996; Mizushina et al., 1997). For pol reactions, poly(dA)/oligo(dT)₁₈ (A/T, 2:1) and dTTP were used as the DNA template-primer substrate and nucleotide (dNTP; 2’-deoxynucleotide-5’-triphosphate) substrate, respectively. For the TdT reactions, oligo(dT)₁₈ (3’-OH) and dTTP were used as the DNA primer substrate and nucleotide substrate, respectively. Test compounds were dissolved in distilled dimethyl sulfoxide (DMSO) to various concentrations and sonicated for 30 s. Subsequently, 4-µL aliquots were mixed with 16 µL of each enzyme (0.05 units) in 50 mM Tris-HCl (at pH 7.5) containing 1 mM dithiothreitol, 50% glycerol (v/v), and 0.1 mM ethylenediaminetetraacetic acid, and were held at 0°C for 10 min. Subsequently, 8 µL inhibitor-enzyme mixtures were added to 16-µL aliquots of standard enzyme reaction mixture, and incubated at 37°C for 60 min, except for Taq pol, which was incubated at 74°C for 60 min. Activity without the inhibitor was considered 0%, and the inhibitory activity was determined for each inhibitor concentration. One unit of pol activity was defined as the amount of each enzyme that catalyzed incorporation of 1 nmol dTTP into synthetic DNA template primers in 60 min at 37°C under standard reaction conditions (Mizushina et al., 1996; Mizushina et al., 1997).

The inhibitory activities of other DNA metabolic enzymes, including calf primase of pol α, T7 RNA polymerase, mouse IMP dehydrogenase (type II), T4 polynucleotide kinase, and bovine deoxyribonuclease I, were also measured as described previously (Kuriyama et al., 2013; Mizushina et al., 1996; Mizushina et al., 1997).

The 50% inhibitory concentration (IC₅₀ value) of the enzyme inhibitor was determined by constructing a dose-response curve and examining the effect of different concentrations of inhibitor on reversing enzyme activity [functional antagonist assay using GraphPad Prism version 5.0 software (GraphPad Software, San Diego, CA, USA)].

Measurement of tumor necrosis factor-α (TNF-α) expression activity    RAW264.7 cells were plated in 12-well plates at a density of 5 × 10⁴ cells/well and incubated for 24 h. The cells were then pretreated with the test compounds for 30 min, at a final concentration of 1 or 5 µM in 0.5% DMSO, before the addition of 100 ng/mL of lipopolysaccharide (LPS), a major component of the outer membranes of gram-negative bacteria. After LPS stimulation for 24 h, the cell culture medium was collected to measure the concentration of secreted TNF-α using a commercially available enzyme-linked immunosorbent assay (ELISA) system (Bay Bioscience Co. Ltd., Kobe, Japan) in accordance with the manufacturer's protocol.

Measurement of anti-inflammatory activity    A mouse inflammatory test was performed according to Gschwendt's method (Gschwendt et al., 1984). In brief, an acetone solution containing a test compound at a concentration of 250 or 500 µg/20 µL or a vehicle control of 20 µL of acetone was applied to the inner part of the mouse ear and, 30 min later, an acetone solution of TPA, which is a chemical edema inducer, was applied at a concentration of 0.5 µg/20 µL to the same part of the ear. Acetone, followed by TPA application, served as the control. After 7 h, a 6-mm diameter disk was obtained from the ear and weighed. Anti-inflammatory activity was determined as the percentage difference in ear disk weight compared to that for the controls, as follows: percentage activity = ([TPA only] − [test compound plus TPA])/([TPA only] − [vehicle]) × 100.

Measurement of anti-β -hexosaminidase release activity    It has been reported that the release of β-hexosaminidase correlates well with the release of histamine, a major component of mast cell granules. Mast cell degranulation was determined using a β-hexosaminidase release assay as described by Razin et al. (1983). Briefly, RBL-2H3 cells at 8 × 10⁴ cells/well in 24-well plates were washed with Tyrode's buffer (137 mM NaCl, 5.6 mM glucose, 11.9 mM NaHCO₃, 2.7 mM KCl, and 0.32 mM NaH₂PO₄) containing 1 mM CaCl₂ and 0.5 mM MgCl₂. Each test compound was added individually to a final concentration of 10 µM in 0.5% DMSO. The cells were then stimulated with 5 µM A23187 and incubated for 30 min. The cell supernatant and total cell lysate were dissolved in 2% Triton X-100, collected, and mixed with the substrate solution (2 mM p-nitrophenyl-N-acetyl-β-d-glucosaminide in 0.1 M sodium citrate buffer, pH 4.5). The mixture was incubated for 90 min at 37°C, and the reaction terminated by adding stopping buffer composed of 0.2 M glycine buffer at pH 11.0. The absorbance at 405 nm was measured using a Vmax-K microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The test compound-mediated anti-β-hexosaminidase release activity was expressed as percentage inhibition, calculated using the following formula: % activity = ([β-hexosaminidase release without test compound-β-hexosaminidase release with test compound]/ β-hexosaminidase release without test compound) × 100.

Measurement of anti-anaphylactic activity    The PCA reaction was measured as described by Sato et al. (2012). Mice were sensitized by an intradermal injection of 0.1 µg of antidinitrophenyl (DNP) IgE in the ear and were intravenously challenged with 0.2 mL (1 mg/mL) of DNP-labeled human serum albumin containing 2% Evans blue dye 4 h later. Either a test compound (100 mg/kg) or saline was administered orally 2 h before the antigen challenge, with saline used in the control group. The mice were subsequently sacrificed, and the ears removed and weighed 30 min after the challenge. The ears were dissolved in 200 µL of 1 N KOH and were incubated overnight at 37°C. To measure the amount of Evans blue dye present in the exudates, the dissolved tissue solution was added to 400 µL of a mixture of acetone and 0.6 N phosphoric acid (5:13 by volume) and the optical density at 620 nm measured. The amount of dye in the exudates was calculated from an Evans blue standard curve, and the results expressed as a percentage for the mean exudate dye from the treated mice compared with controls.

Results

Screening of mammalian pol inhibitors extracted from 20 edible plants    First, 20 dried edible plants, extracted using 30% ethanol under reflux for 2 h, were prepared. Ethanol at 30% is not flammable or explosive, and can extract both hydrophobic and hydrophilic compounds; therefore, this solvent is considered to be a safe and effective extractant for industrial use. The inhibitory activities of extracts of 20 species of plants toward mammalian pols was investigated, using pol α as a representative DNA replicative pol and pol λ as a representative DNA repair/recombination-related pol (Lange et al., 2011; Loeb and Monnat, 2008). The inhibitory activity of 10 µg/mL of each edible plant extract toward calf pol α and human pol λ is shown in Fig. 1. Among the 20 plant extracts tested, rosemary leaves (Rosmarinus officinalis L.) showed the strongest inhibition of both pols α and λ, with inhibitory activities of 93.7 and 86.4%, respectively. Therefore, further experimentation focused on rosemary leaves.

Fig. 1.

Effects of 30% ethanol extracts of 20 edible plants on the inhibitory activity of mammalian pols α and λ. Each extract (10 µg/mL) was incubated with calf pol α or human pol λ (0.05 units each). Inhibition of pol activity by the vehicle control was taken as 0%. Data are shown as the mean ± SD (n = 3).

Isolation of mammalian pol inhibitors from the rosemary leaf extract    For further tests, 500 g of rosemary leaves was extracted with 3 L of 30% ethanol under reflux for 2 h. As shown in Fig. 2, the evaporated extract was partitioned between chloroform (1 L) and distilled water (1 L), adjusted to pH 7, and the organic layer evaporated. The fraction was subjected to silica gel column chromatography and eluted with chloroform:dichloromethane:meth anol (v:v:v, 5:5:1.5). The active fractions were defined such that the 10 µg/mL of extract in the fraction had over 50% inhibitory activity against both mammalian pols α and λ, and four active fractions were obtained. Active fraction 1 and fraction 2 were independently purified by two rounds of silica gel column chromatography, and white powdery compounds 1 (46 mg) and 2 (25 mg) were obtained from them (Fig. 2). The active fractions 3 and 4 were independently purified by silica gel column chromatography, and white powdery compounds 3 (160 mg) and 4 (12 mg) were obtained (Fig. 2).

Fig. 2.

Method for purification of compounds 1 – 4, mammalian pol inhibitors, from rosemary leaf extract.

Determination of the structure of compounds 1 – 4 purified from rosemary leaves    By high-resolution mass spectrometry, the molecular formulae of compounds 1 to 4 were determined to be C₂₀H₂₈O₄, C₂₀H₂₆O₄, C₃₀H₄₈O₃, and C₁₈H₁₆O₈, respectively. From the ¹H and ¹³C NMR spectral data, compounds 1 – 4 were identified as the polyphenols carnosic acid, carnosol, ursolic acid, and rosmarinic acid, respectively (Fig. 3). These spectroscopic data of compounds 1 – 4 were consistent with previous reports such as Cantrell et al., 2005, Brieskorn et al., 1964, Abe et al., 1996, and Simpol et al., 1994. Therefore, we focused on these four identified compounds for further study.

Fig. 3.

Structures of the purified compounds 1–4 from rosemary leaves. (A) Carnosic acid (compound 1), (B) carnosol (compound 2), (C) ursolic acid (compound 3), and (D) rosmarinic acid (compound 4).

Effect of the isolated compounds 1 – 4 on the activities of mammalian pols Initially, 10 µM solutions of compounds 1 – 4 purified from rosemary leaves were investigated to determine whether they inhibited the activities of mammalian pol species. The purification grade of these compounds was greater than 98%, as determined by NMR analysis (data not shown). We used the mammalian pols, pols α, γ, κ, and λ as representatives of pol families B, A, Y, and X, respectively (Lange et al., 2011; Loeb and Monnat, 2008). Since pol α consists of four subunits, the native complex of pol α subunits was purified from calf thymus tissue. Recombinants of human pol γ catalytic subunit were purified from Baculovirus and monomers of human pols κ and λ were purified from Escherichia coli. We found that pol α activity was inhibited more than 50% by carnosic acid (1), pols γ and κ were inhibited more than 50% by carnosic acid (1) and carnosol (2), and pol λ was inhibited more than 50% by carnosic acid (1) and ursolic acid (3) (Fig. 4). Rosmarinic acid (4) inhibited the activity of these pols more weakly, with less than 20% inhibition of activity. In terms of the inhibitory effect on pols α, γ, and κ, the ranking in descending order was as follows: carnosic acid (1) > carnosol (2) > ursolic acid (3) > rosmarinic acid (4). On the other hand, the ranking of the inhibitory effect on pol λ was as follows: carnosic acid (1) > ursolic acid (3) > carnosol (2) > rosmarinic acid (4). These results indicated that the mammalian pol inhibitory effect of carnosic acid (1) showed a different pattern from that of compounds 2 – 4. When activated DNA (bovine deoxyribonuclease I-treated DNA) was used as the DNA template/primer substrate instead of synthesized DNA (poly (dA)/oligo (dT)₁₈ (A/T = 2/1)), and dNTP was used as the nucleotide substrate instead of dTTP, the inhibitory effects of these compounds did not change (data not shown).

Fig. 4.

Inhibitory effects of compounds 1 – 4 from rosemary leaves on the activity of mammalian pols. Each compound (10 µM) was incubated with calf pol α (B-family pol), human pol γ (A-family pol), human pol κ (Y-family pol), or human pol λ (X-family pol) (0.05 units each). Inhibition of pol activity by the vehicle control was taken as 0%. Data are shown as the mean ± SD (n = 3).

In particular, carnosic acid (1) showed the strongest inhibition of the four compounds tested on mammalian pols. Therefore, further studies focused on carnosic acid (1).

Effect of carnosic acid (1) on the activities of various pols and other DNA metabolic enzymes    We isolated 10 mammalian pol species, including pols α, β, γ, δ, ε, η, ι, κ, λ, μ, and purchased TdT. Pols ζ, θ, ν, and REV1 were not available at the time of the study. Table 1 shows the inhibitory effect (IC₅₀ value) of carnosic acid (1) on the 11 mammalian pols obtained. This compound inhibited the activity of all of the mammalian pols, with IC₅₀ values between 4.6 and 8.2 µM. Specifically, 50% inhibition of the A, B, X, and Y families of pols was observed at carnosic acid (1) doses of 6.8, 7.2 – 7.8, 4.6 – 7.1, and 7.5 – 8.2 µM, respectively. Therefore, this compound had an inhibitory effect on the mammalian pols at a similar concentration for all four pol families. For comparison, the IC₅₀ value for petasiphenol, which is a curcumin-derived polyphenol and a known mammalian pol λ specific inhibitor from the Japanese vegetable Fuki (Petasites japonicus), was 7.8 µM (Mizushina et al., 2002). Thus, the pol λ inhibitory activity of carnosic acid (1) was 1.7-fold more potent than that of petasiphenol, highlighting a need for further study of this compound.

Table 1. IC₅₀ values of carnosic acid (1) on the activities of mammalian pols, plant and prokaryotic pols, and various DNA metabolic enzymes.

Enzyme	IC50 value (µM)
— Mammalian pols —
(A family of pols)
Human pol γ	6.8 ± 0.40
(B family of pols)
Calf pol α	7.2 ± 0.42
Human pol δ	7.8 ± 0.47
Human pol ε	7.4 ± 0.44
(X family of pols)
Rat pol β	4.9 ± 0.29
Human pol γ	4.6 ± 0.26
Human pol μ	5.2 ± 0.31
Calf TdT	7.1 ± 0.42
(Y family of pols)
Human pol η	8.2 ± 0.50
Mouse pol ι	7.7 ± 0.46
Human pol κ	7.5 ± 0.44
— Plant pol —
Cauliflower pol α	>100
— Prokaryotic pols —
E. coli pol I	>100
Taq pol	>100
T4 pol	>100
— Other DNA metabolic enzymes —
Calf primase of pol α	>100
T7 RNA polymerase	>100
Mouse IMP dehydrogenase (type II)	>100
T4 polynucleotide kinase	>100
Bovine deoxyribonuclease I	>100

The compound was incubated with each enzyme (0 . 05 units). Data, mean ± SD (n = 3).

In contrast to its inhibition of mammalian pols, carnosic acid (1) had no effect on plant pols, such as cauliflower pol α, or prokaryotic pols, such as E. coli pol I, Taq pol, or T4 pol (Table 1). The three-dimensional structures of eukaryotic pols are likely to differ greatly from those of prokaryotic pols. In addition, this compound did not inhibit the activity of other DNA metabolic enzymes, including calf primase pol α, T7 RNA polymerase, mouse IMP dehydrogenase (type II), T4 polynucleotide kinase, or bovine deoxyribonuclease I (Table 1). From these results, carnosic acid (1) can be classified as a selective inhibitor of mammalian pols.

We also performed specific assays to determine whether the inhibition of pol induced by carnosic acid (1) resulted from its ability to bind to DNA or to the enzyme. The interaction of carnosic acid (1) with dsDNA was investigated through examination of the thermal transition of dsDNA. The melting temperature (T_m) of the dsDNA was measured with an excess of carnosic acid (1; 100 µM) using a spectrophotometer equipped with a thermoelectric cell holder. No thermal transition representing T_m was observed within the concentration range of the compound used in the assay, whereas a typical intercalating compound used as a positive control (ethidium bromide, 15 µM) produced a clear thermal transition (data not shown). To determine whether the inhibitory effect of carnosic acid (1) resulted from nonspecific adhesion to mammalian pols or from selective binding to specific sites, we investigated whether excess nucleic acid (poly(rC)) or protein (bovine serum albumin (BSA)) could block the inhibitory effect. Poly (rC) and BSA had little or no influence on the pol inhibitory effect of carnosic acid (1) (data not shown), suggesting that it selectively binds to the pol molecule. These observations indicated that carnosic acid (1) does not act as a DNA intercalating agent or as a template/primer substrate, but can directly bind to the pol and inhibit its activity.

Inhibitory effect of compounds 1 – 4 on LPS-induced TNF- α production by cultured cells In a previous study, we found a relationship between pol λ inhibitors and their ability to suppress the protein expression of TNF-α, a pleiotropic inflammatory cytokine, by LPS-stimulated cells (Mizushina et al., 2013). The binding of the pro-inflammatory cytokine TNF-α to the TNF-α receptor can activate the nuclear factor-κB (NF-κB) signaling pathway, thereby initiating an inflammatory response, as evident in various inflammatory diseases (Aggarwal, 2003). Therefore, we investigated whether compounds 1 – 4 could inhibit LPS-induced TNF-α production in a cell culture system using RAW264.7 mouse macrophages.

In cultured RAW264.7 cells, none of the compounds tested showed cytotoxicity at 10 µM (data not shown). RAW264.7 cells produced 560 pg/mL of TNF-α after LPS treatment. The concentration of TNF-α produced in the vehicle control was defined as 0% inhibition. As shown in Fig. 5A, 5 µM of carnosic acid (1) and ursolic acid (3) showed approximately 80% and 50% suppression of TNF-α production, respectively, and the suppressive effect of these compounds was ranked as follows: carnosic acid (1) > ursolic acid (3) > carnosol (2) > rosmarinic acid (4). The suppression of LPS-stimulated TNF-α production by these compounds showed the same tendency as the inhibition of mammalian pol λ activity (Fig. 4).

Fig. 5.

Effects of compounds 1 – 4 from rosemary leaves on anti-inflammatory activities. (A) In vitro inhibition of LPS-induced TNF-α production by the mouse macrophage cell line RAW264.7. RAW264.7 cells were pretreated with either 1 or 5 µM of each compound or with a vehicle control (base TNF-α concentration, 45 pg/mL) for 30 min and then treated with 100 ng/mL LPS for 24 h (LPS-induced TNF-α concentration, 560 pg/mL). The TNF-α concentration in the cell culture medium was measured by ELISA. Inhibition of TNF-α production by the vehicle control was taken as 0%. Data are shown as the mean ± SD (n = 4). (B) In vivo anti-inflammatory activity on TPA-induced edema in the mouse ear. Each compound (250 or 500 µg) was applied individually to one ear of a mouse and, after 30 min, TPA (0.5 µg) was applied to both ears. Edema was evaluated after 7 h. The inhibitory effect is expressed as a decrease in the percentage of edema. The degree of inflammation in the vehicle control was taken as 0% inhibition. Data are shown as the mean ± SD (n = 6).

Effect of compounds 1 – 4 on TPA-induced inflammation in vivo    Next, we examined the anti-inflammatory effect of compounds 1 – 4 in vivo using the mouse ear inflammatory test. Edema was induced in the mouse ear by application of TPA (0.5 µg), resulting in a 241% increase in the weight of the ear disk 7 h after application. The anti-inflammatory effect of the vehicle control was defined as 0%. As shown in Fig. 5B, carnosic acid (1) had the strongest anti-inflammatory effect on TPA-induced inflammation among the compounds tested and showed 45.0 and 64.4% inhibition of swelling at doses of 250 and 500 µg/ear, respectively. The anti-inflammatory effect was ranked in the following order: carnosic acid (1) > ursolic acid (3) > carnosol (2) > rosmarinic acid (4). The in vivo anti-inflammatory effect of these compounds was of the same order of magnitude as the inhibition of TNF-α production in LPS-activated cultured cells (Fig. 5A). The anti-inflammatory effect of these compounds was also of the same order of magnitude as their in vitro inhibitory effect on mammalian pol λ (Fig. 4). In comparison, 500 µg/ear of petasiphenol (a pol λ specific inhibitor) and glycyrrhetinic acid (an anti-inflammatory agent) had an inhibitory effect of just 42 and 40%, respectively, in this assay (Mizushina et al., 2003). Thus, carnosic acid (1) was approximately 1.6-fold more potent than these compounds and may have potential as an anti-inflammatory agent.

Effects of compounds 1 – 4 on β-hexosaminidase release in cultured cells    β-Hexosaminidase release has frequently been used as an indicator for evaluating the extent of degranulation by mast cells. The release of histamine and other chemical mediators from mast cells is an important process in initiating an immediate anaphylactic reaction. Thus, the effect of compounds 1 – 4 on the release of the chemical mediator β-hexosaminidase was investigated in rat basophilic leukemia RBL-2H3 cells treated with the calcium ionophore A23187. The results confirmed that these compounds did not influence RBL-2H3 cell growth and did not inhibit β-hexosaminidase enzyme activity when they were used at a concentration of 10 µM (data not shown). The degree of degranulation was calculated from the β -hexosaminidase activity in the supernatant and cell lysate. Carnosic acid (1) at 10 µM exhibited >70% inhibition of mast cell degranulation, as measured by β -hexosaminidase activity, while rosmarinic acid (4) did not influence release activity (Fig. 6A). The strength of the inhibitory effect was ranked as follows: carnosic acid (1) > ursolic acid (3) > carnosol (2) > rosmarinic acid (4). As A23187 induces calcium ion flux and protein kinase C activities, the latter being essential for mast cell degranulation (Gordon, Burd, and Galli, 1990), these compounds with inhibitory effects on β -hexosaminidase release activity may have some effect on protein kinase C activities. Further studies are necessary to explain the inhibitory mechanism of the compounds on mast cell degranulation.

Fig. 6.

Effects of compounds 1 – 4 from rosemary leaves on antiallergic activities. (A) In vitro inhibition of β -hexosaminidase release activity in RBL-2H3 cells treated with A23187. Each compound was added at 10 µM. Inhibition of β -hexosaminidase release by the vehicle control was taken as 0%. Data are shown as the mean ± SD (n = 4). (B) In vivo inhibition of allergic activity measured by mouse PCA reaction. Each compound (100 mg/kg) was orally administered to the mice, and the IgE-dependent PCA reaction was investigated. Inhibition of anaphylactic activity by the vehicle control was taken as 0%. Data are shown as the mean± SD (n = 6).

Effects of compounds 1 – 4 on antiallergic activity in vivo    The in vivo IgE-mediated PCA reaction is used to study the mechanism of the immediate hypersensitivity reaction. As shown in Fig. 6B, compounds 1 to 4 inhibited the PCA reaction in mice by 67.1%, 18.8%, 44.0%, and 1.5%, respectively, when used at 100 mg/kg. Pol λ inhibitory activity by these compounds is positively correlated with antiallergic activities, such as inhibition of β-hexosaminidase activity, and inhibition of the PCA reaction (Fig. 4 and Fig. 6). Tranilast, a commonly used antiallergic drug that targets mast cell degranulation and inhibits the PCA reaction, caused 23.9% inhibition in this PCA reaction assay at 100 mg/kg (data not shown); the inhibitory effect of carnosic acid (1) was 2.8fold stronger, suggesting strong potential for use as an antiallergic compound.

Discussion

In this study, we found that extracts from rosemary leaves exhibited the strongest inhibitory activity on mammalian pols α and λ among the 20 edible plants tested (Fig. 1). Four polyphenol components isolated from rosemary (Fig. 3) were found to be mammalian pol inhibitors with both anti-inflammatory and antiallergic activity (Figs. 4 – 6).

Rosmarinus officinalis    L. (family Lamiaceae), commonly called rosemary, is a woody perennial herb with fragment evergreen needle-like leaves that are often used in cooking. It is native to the Mediterranean region and is now widespread in European countries. It has been found to act both as a stimulant and as a mild analgesic and has been used in folk medicine to treat headaches, epilepsy, poor circulation, and many ailments for which stimulants are prescribed. Its extracts have been incorporated into drugs and cosmetics and are used for flavors and fragrance in foods (Aruoma et al., 1996). The leaves of rosemary possess a variety of bioactivities, including anti-inflammatory activity (Altinier et al., 2007).

Carnosic acid (Fig. 3A), isolated from rosemary extract, is a proelectrophilic diterpene that shows multiple actions similar to those of other diterpenoid compounds such as carnosol (Fig. 3B). This compound has been reported to possess anti-inflammatory activity, such as the suppression of the nuclear translocation of NFκB and its upstream signaling and factors involved upstream in its signaling pathway including Syk/Src, phosphoinositide 3-kinase, Akt, inhibitor of κBα (IκBα) kinase, and IκBα for NF-κB activation (Oh et al., 2012). How this compound can have multipotential pharmacological properties is not well understood. The results of the present study show that mammalian pols, especially pol λ, may be novel molecular targets of carnosic acid, and that the pol λ inhibitory activity exhibited by this compound might be related to these biological activities.

Inflammation is an initial host immune reaction, mediated by inflammatory cytokines such as TNF-α (Ronis et al., 2008). Macrophages play an important role in host defense against noxious substances (Pierce, 1990). Macrophage activation by LPS, a major component of the outer membranes of gram-negative bacteria, leads to increased production of pro-inflammatory cytokines, thereby mediating the major cytotoxic and proapoptotic mechanisms that participate in the innate response in many mammals (Boscá et al., 2005). However, cytokine overproduction by activated macrophages has been implicated in the pathophysiology of several inflammatory diseases, including rheumatoid arthritis, atherosclerosis, chronic hepatitis, pulmonary fibrosis, and inflammatory brain diseases (Boscá et al., 2005). Therefore, LPS-stimulated macrophages serve as a useful model to study inflammation and the potential mechanism of action of anti-inflammatory compounds.

The prevalence of allergic diseases such as allergic rhinitis, atopic dermatitis, asthma, and food allergies has increased in many countries (Wüthrich, 1989). Immunologically active mast cells and basophils express the high-affinity receptor for IgE on their surfaces, and play critical roles in various biological processes associated with allergic diseases (Stevens and Austen, 1989). The interaction of multivalent antigens with surface-bound IgE triggers the secretion of the mediators stored in cytoplasmic granules and causes the de novo synthesis of cytokines (Plaut et al., 1989), which in turn activates the migration of neutrophils and macrophages, causing tissue inflammation (Gordon et al., 1990).

Eukaryotic cells are reported to contain 15 pol species that belong to four families: family A (pols γ, θ, and ν), family B (pols α, δ, ε, and ζ), family X (pols β, λ, µ, and TdT), and family Y (pols η, ι, and κ, and REV1) (Lange et al., 2011; Loeb and Monnat, 2008). As shown in Figs. 4 – 6, analysis of the pol inhibitors isolated from rosemary showed a strong correlation between pol λ (pol X family) inhibitory activity and a decrease in inflammatory and allergic activity. However, the correlation between the inhibition of pols α, γ, and κ (pol B, A, and Y families, respectively) and anti-inflammatory/antiallergic properties was less strong. In the X family of pols, pol λ and pol β appear to carry out similar functions (Garcia-Diaz et al., 2002). Pol β is involved in the short-patch base excision repair (BER) pathway, and plays an essential role in neural development (Hubscher et al., 2010). Pol λ, the other enzyme of interest, is capable of synthesizing DNA de novo, and in a template-dependent manner. Furthermore, it shows TdT as well as 5’-deoxyribose-5-phosphate lyase activity (Ramadan et al., 2004). Pol λ is implicated in V(D)J recombination (Nick McElhinny et al., 2005), translesion synthesis (Maga et al., 2007), and BER (Markkanen et al., 2012). Moreover, studies with eukaryotic cells and reactive oxygen species indicate that pol λ functions as a backup for pol β in BER (Braithwaite et al., 2010) and protects cells from oxidative damage (Zucca et al., 2013). There is also evidence that pol λ is required for cell cycle progression and that it is functionally connected to the S-phase DNA damage response machinery in cancer cells (Zucca et al., 2013).

In addition to causing inflammation, TPA influences cell proliferation and has tumor-promoting activity (Nakamura et al., 1995). Therefore, anti-inflammatory agents are expected to suppress DNA replication/repair/recombination in nuclei in relation to the action of TPA. Because pol λ is a repair/recombinationrelated pol (Garcia-Diaz et al., 2002), our finding that it is a molecular target of rosemary components, such as carnosic acid, is in agreement with this expected mechanism for its anti-inflammatory/antiallergic action. However, the detailed mechanisms by which carnosic acid inhibits mammalian pol λ, and their relationship to its anti-inflammatory and antiallergic effects, is unclear; we are pursuing further studies to elucidate these mechanisms.

Conclusions

To our knowledge, this is the first report that carnosic acid, isolated from rosemary (Rosmarinus officinalis L.), can potently inhibit the activities of mammalian pols. Carnosic acid may be an effective nutrient because of its anti-inflammatory and/or antiallergic effects, through the inhibition of mammalian pols, especially pol λ, leading to improvement in health. The carnosicacid-enriched fraction of rosemary leaves, or purified carnosic acid, may be considered suitable for incorporation into functional foods with anti-inflammatory/antiallergic properties or for use in cosmetics.

Acknowledgements    We are grateful for the following donations: calf pol α by Dr. M. Takemura of Tokyo University of Science (Tokyo, Japan); rat pol β and human pols δ and ε by Dr. K. Sakaguchi of Tokyo University of Science (Chiba, Japan); human pol γ by Dr. M. Suzuki of Nagoya University School of Medicine (Nagoya, Japan); mouse pol η and human pol ι by Dr. F. Hanaoka of Gakushuin University (Tokyo, Japan) and Dr. C. Masutani of Nagoya University (Nagoya, Japan); human pol κ by Dr. H. Ohmori of Kyoto University (Kyoto, Japan); and human pols λ and µ by Dr. O. Koiwai of Tokyo University of Science (Chiba, Japan).

This study was supported in part by the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities, 2012 – 2016. Y.M. acknowledges the Hyogo Science and Technology Association (Japan) and a Grant-in-Aid for Scientific Research (C) (no. 24580205) from MEXT. I.K. acknowledges a Grant-in-Aid for Young Scientists (B) (no. 23710262) from MEXT.

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