Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-γ

Biochimica et Biophysica Acta 1568 (2001) 74^82

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Inhibitory e¡ects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-Q Rie Ishii a , Masakazu Horie a , Koichi Saito a , Munehisa Arisawa b , Susumu Kitanaka c; * b

a Saitama Institute of Public Health, 639-1 Kamiokubo Saitama, Saitama 338-0824, Japan Laboratory of Herbal Garden, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani Toyama, Toyama 930-0194, Japan c College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan

Received 11 June 2001; received in revised form 27 August 2001; accepted 27 August 2001

Abstract An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-Q (IFN-Q). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC50 of 10.7 WM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-Q for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[14 C]arginine to L-[14 C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Euphorbiaceae ; Phloroglucinol derivative; Macrophage; Nitric oxide; Mallotus japonicus

1. Introduction Macrophages play an important role in non-speci¢c host defense mechanisms [1]. Among a variety of mediators released by activated macrophages [2], nitric oxide (NO) has been identi¢ed as a potent molecule that may exert regulatory or cytotoxic e¡ects on tumor cells and microbes [3^5]. The enzyme responsible for the synthesis of nitric oxide in macrophages is an inducible nitric oxide synthase (iNOS), which does not exist ordinarily but is strongly induced upon exposure to bacterial endotoxin and in£ammatory cytokines [6,7]. The excessive production of NO during in£ammation may also lead to severe damage to host cells and tissues [8]. Glucocorticoids, representative anti-in£ammatory agents, strongly inhibit NO

* Corresponding author. Fax: +81-47-465-5440. E-mail address : [email protected] (Susumu Kitanaka).

production [9]. This, therefore, suggests that inhibition of chronic NO production in macrophages could be a target for potential anti-in£ammatory drugs. Mallotus japonicus Mueller Arg. (Euphorbiaceae) is a dioecious and deciduous tree that is distributed throughout tropical and temperate Asia. Parts of the tree have been used for a long time in folk medicine; the bark is a medication for ulcers and for cancer and the leaves are used as treatment for pimples. An extract of the tree's pericarps is reported to exhibit anti-tumor activity [10,11], anti-tumor-promoting activity [12], and anti-viral activity [13,14]. Several rottlerin-like phloroglucinol derivatives have been isolated from the pericarps and identi¢ed as active compounds [10,13^16]. However, the physiological and pathological functions of this plant and its components have not been fully clari¢ed. In an extensive survey of plant extracts, we found that an 80% aqueous acetone extract of the pericarps (MJE) inhibited NO production by activated macrophages that had been treated

0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 2 0 3 - 3

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2. Materials and methods

NG -(1-iminoethyl)-L-ornithine hydrochloride (L-NIO) from Wako Pure Chemical Industries (Osaka, Japan). 125 I-Labeled protein A and L-[U-14 C]arginine were purchased from Amersham Pharmacia Biotech UK (Amersham, UK). All other chemicals and reagents were of the purest commercial grade available.

2.1. Materials

2.2. Cell culture

The compounds 2,6-dihydroxy-3-methyl-4-methoxyacetophenone (1), mallotophenone (2), mallotojaponin (3), butyrylmallotolerin (4), mallotochromene (5), mallotochromanol (7), isobutyrylmallotochromanol (8), and isomallotochromanol (10) were isolated from the pericarps of M. japonicus as described previously [10,13^16]. In brief, dried pericarps of M. japonicus were extracted with 80% aqueous acetone, suspended in water, and extracted with diethyl ether, ethyl acetate, and n-butanol, successively. Compounds 1^5, 7, 8, and 10 were obtained by separation of the diethyl ether fraction by column chromatography on silica gel. Mallotochroman (6) and isomallotochromene (9) were derived from 3 by cyclization of its side chain [13]. Structural formulae of these compounds are shown in Fig. 1. Ham's F12 medium, L-NADPH, £avin adenine dinucleotide (FAD), and £avin mononucleotide (FMN) were obtained from Sigma (St. Louis, MO, USA), and fetal bovine serum from Gibco BRL (Grand Island, NY, USA), IFN-Q from Genzyme (Cambridge, MA, USA), LPS (Escherichia coli, O55: B5) from Difco (Detroit, MI, USA), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (carboxy-PTIO) from Dojindo Laboratories (Kumamoto, Japan), tetrahydrobiopterin (BH4 ) from Alexis (San Diego, CA, USA), and L-arginine and

RAW 264.7 cells, a mouse macrophage-like cell line transformed with the Abelson leukemia virus, were obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were maintained by culturing in Ham's F12 medium supplemented 10% heat-inactivated fetal bovine serum in a CO2 incubator (5% CO2 ^95% humidi¢ed air) at 37³C. The cells were seeded onto plastic petri dishes (Falcon, No. 1001 ; Becton Dickinson) and passaged twice a week.

with lipopolysaccharide (LPS) and interferon-Q (IFN-Q). The present paper describes our results and the mechanisms by which the extract and its constituents act.

2.3. NO production assay and induction iNOS in macrophages The cells were plated at 1.2U106 cells/ml onto a 35 mm petri dish (Sumitomo Bakelite, No. 1135R ; Tokyo, Japan), and then incubated at 37³C for 2 h. MJE or a test compound and both 100 ng/ml LPS and 10 U/ml IFN-Q were added to the culture medium, and the cells were incubated at 37³C for 8 h. After incubation, the cells were chilled on ice, scraped from the dish using a cell scraper, collected in a microfuge tube and then centrifuged at 2800Ug for 1 min. The supernatant was placed in a new microfuge tube for analysis of nitrite (NO3 2 ) oxidized NO. The cells were washed twice with phosphate-bu¡ered sa-

Fig. 1. Chemical structures of the phloroglucinol derivatives examined in the present study.

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line without divalent cations (PBS) by repeated centrifugation at 2800Ug. The ¢nal cell pellet was extracted with 35 Wl of a lysis bu¡er, comprised of 1% (v/v) Triton X-100, 0.1 mM EDTA and 1% aprotinin (Sigma) in 20 mM HEPES^NaOH bu¡er (pH 7.5), at 4³C for 30 min. The cell lysate was centrifuged at 5500Ug at 4³C for 1 min. A 30 Wl volume of the resultant supernatant was placed in a new microfuge tube. The ¢nal cell extract was used for Western blot analysis. 2.4. iNOS activity in intact macrophages preactivated with LPS and IFN-Q The inhibitory e¡ects of MJE and the compounds on iNOS activity in intact cells were examined by a previously reported method [17]. In brief, macrophages were treated with 100 ng/ml LPS and 10 U/ml IFN-Q at 37³C for 16 h, the cells were collected, washed three times with PBS, and suspended at 4U105 cells/ml in modi¢ed Hanks' balance solution. A 500 Wl volume of cells was reseeded onto a 24well plate (Sumitomo Bakelite, No. 8024R). After incubation for 10 min, MJE or one of the compounds was added, followed by incubation at 37³C for 5 min, and then by the addition of 100 WM carboxy-PTIO and 1 mM L-arginine to start the reaction. The cells were incubated for 0^60 min, and the amount of NO3 2 in the culture supernatant was determined. 2.5. iNOS activity in cell extracts in vitro Analysis of iNOS activity in the cell extracts in vitro was performed as described [18] with a slight modi¢cation. Brie£y, RAW 264.7 cells (1.2U107 cells) were incubated in 10 ml culture medium containing 100 ng/ml LPS and 10 U/ml IFN-Q. After incubation for 16 h, the cells were scraped from the dish using a cell scraper, collected in a microfuge tube and then centrifuged at 2800Ug for 1 min. After two washes, the cells were extracted using 300 Wl of lysis bu¡er as described above. The cell lysate was centrifuged at 5500Ug for 5 min. The supernatant fraction was used as a crude preparation of NO synthase. Enzyme inhibition was analyzed in the 50 Wl reaction mixture, comprised of 1 mM L-NADPH, 4 WM BH4 , 1 WM FAD, 1 WM FMN, 4 WM L-[U-14 C]arginine in 25 mM Tris^HCl bu¡er (pH 7.4). The mixture was preincubated at 37³C for 3 min, and then MJE or the compound was added to the mixture together with a 50 Wg protein cell lysate as the enzyme source. This was followed by incubation for 0^15 min. The reaction was terminated by the addition of 50 mM HEPES^EDTA bu¡er (pH 5.5), and then a suspension of ion exchange resin (AG 50W-X8 resin; Bio-Rad, Hercules, CA, USA) was added. The mixture was transferred to the spin cup set in a new microfuge tube, and centrifuged at 5500Ug for 1 min. Radioactivity in the ¢ltrate was quantitated on a liquid scintillation analyzer (LSC-3500 ; Aloka, Tokyo, Japan). Background was determined by the

level of radioactivity in a reaction mixture from which the cell lysate was omitted. This amount was subtracted from the level detected in the experimental samples. Disintegration per minute was converted to citrulline production and expressed in pmol per mg protein cell extract per min. 2.6. Western blot analysis of iNOS protein Western blot analysis was performed according to a previously reported method [19] with a slight modi¢cation. A 60 Wg protein cell extract obtained as described in Section 2.3 was boiled in an SDS sample bu¡er, loaded onto a 5^20% gradient slab gel (Atto, Tokyo, Japan), and electrophoresed at 20 mA/gel. The proteins in the gel were electrotransferred onto a polyvinylidene £uoride membrane (Immobilon-P ; Millipore, Bedford, MA, USA). The proteins on the membrane were probed with a monoclonal anti-macrophage iNOS antibody (Transduction Laboratories, Lexington, KY, USA), then with rabbit anti-mouse IgG (Zymed Laboratories, San Francisco, CA, USA), and ¢nally with 125 I-labeled protein A. After air-drying, the membrane was placed on an X-ray ¢lm, and the bands on the membrane were photographed. The radioactivity of the bands corresponding to iNOS was quantitated on an auto well Q-counter (ARC-300 ; Aloka). 2.7. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of iNOS mRNA The cells were cultured at 1.2U106 cells/ml at 37³C for 2 h, and then MJE or one of the compounds was added to the culture medium together with 100 ng/ml LPS and 10 U/ml IFN-Q, and the cells were incubated for 6 h. Total RNA was isolated from the cell pellet using an RNA isolation kit (Qiagen, Hilden, Germany). The ¢nal amount of RNA was determined by absorbance at 260 nm. Fifty nanograms of the RNA samples were reverse-transcribed into cDNA by RT-PCR using oligo(dT)12ÿ18 primer. The PCR samples contained 25 Wl of the reaction mixture, comprised of 50 mM KCl, 5 mM MgCl2 , 0.16 mM dNTP, 5.0 units of Taq DNA polymerase (Takara Shuzo, Shiga, Japan), and 5 pmol of sense and antisense primers in 10 mM Tris^HCl (pH 8.3). The sense primer for iNOS was 5P-ACCTACTTCCTGGACATTACGACCC-3P, and the antisense primer was 5P-AAGGGAGCAATGCCCGTACCAGGCC-3P. The sense primer for L-actin was 5PGTGGGCCGCTCTAGGCACCAA-3P, and the antisense primer was 5P-CTCTTTGATGTCACGCACGATTTC-3P [20]. The PCR reaction was performed under the following conditions: 25 cycles of denaturation at 94³C for 1 min, annealing at 57³C for 1 min and extension at 72³C for 1.5 min, using a thermal cycler (GeneAmp PCR Systems 9700; PE Applied Biosystems, USA). The PCR products were run on a 2% agarose gel and visualized by ethidium

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bromide staining. The bands in the gel were photographed and their £uorescence intensity was analyzed using a densitometer (CS-930 ; Shimadzu Scienti¢c Instrument and Equipment, Kyoto, Japan). 2.8. Nitrite assay A 100 Wl aliquot of the culture supernatant was placed in duplicate in a well of a 96-well £at bottom plate. A standard solution of NaNO2 was also placed in other wells on the same plate. To quantitate NO3 2 , 50 Wl of Griess reagent, 1% sulfanilamide in 5% H3 PO4 , and 0.1% N-1naphthylethylenediamine dihydrochloride were added to each well [21]. After 10 min, the reaction products were colorimetrically quantitated at 550 nm after subtraction of the background absorbance at 630 nm, using an MTP-120 microplate reader (Corona Electric, Japan). 2.9. Protein assay Protein concentrations were determined according to Bradford, using bovine serum albumin as the standard [22]. 2.10. Statistical analysis Results were analyzed for statistical signi¢cance by Dunnett's test for multiple comparison. 3. Results 3.1. Inhibitory e¡ects on NO production by activated macrophages When the cells were incubated with 100 ng/ml LPS and

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10 U/ml IFN-Q for 8 h, NO production increased sharply. The addition of MJE caused concentration-dependent inhibition of NO production by macrophages during incubation at 37³C for 8 h (IC50 : 28.9 Wg/ml) (Fig. 2A). The diethyl ether, ethyl acetate, and n-butanol fractions showed 65%, 58%, and 32% inhibition, respectively, at 30 Wg/ml. However, water fraction did not inhibit NO production. Compounds 2, 3, 4, 5, 7, 8, and 10 showed inhibitory activities (Fig. 2). Of these compounds, 10 (IC50 : 10.7 WM) showed the strongest inhibitory activity. To study the structure activity relationships, we synthesized 6 and 9 from 3 by cyclization of its side chain. Compound 9 also exhibited strong inhibition of NO production (IC50 : 14.3 WM). However, 1, a constituent unit of these phloroglucinol derivatives, and 6 did not inhibit production. Time course experiments showed that the release of NO3 2 was ¢rst observed 6 h after incubation with 100 ng/ ml LPS and 10 U/ml IFN-Q. The amount of NO3 2 subsequently increased essentially linearly with time, up to 16 h, whereas the control cells without LPS and IFN-Q did not release NO3 2 (Fig. 3A). MJE (30 Wg/ml), 3 (30 WM), 7 (30 WM), 9 (30 WM), and 10 (30 WM) exhibited continuous inhibition of NO production for up to 16 h (Fig. 3). MJE (30 Wg/ml) and all the compounds (30 WM) examined in the present study did not a¡ect cellular viability, determined by monitoring the mitochondrial reduction of tetrazolium to formazan and morphological observation (data not shown). Hence, inhibition of NO production is unlikely to be due to toxicity of MJE or the compounds. All of the experiments were performed under conditions that did not a¡ect cell viability. 3.2. Inhibitory e¡ects on iNOS activity We wished to determine whether the inhibitory e¡ects of MJE and the phloroglucinol derivatives on NO produc-

Fig. 2. Inhibitory e¡ects of MJE and the phloroglucinol derivatives on NO production. Cells were treated with various concentrations of MJE, compounds 1, 2, and 3 (A), compounds 4, 5, 6, and 9 (B), and compounds 7, 8, and 10 (C) in the presence of 100 ng/ml LPS+10 U/ml IFN-Q at 37³C for 8 h. The symbols correspond to: (A) MJE (a), 1 (7), 2 (O), and 3 (R), (B) 4 (E), 5 (F), 6 (7), and 9 (b), and (C) 7 (8), 8 (b), and 10 (O). The results are the means þ S.E. for two independent experiments comprising duplicate assays. Statistical signi¢cance : *P 6 0.01 vs. corresponding controls (cells treated with 100 ng/ml LPS+10 U/ml IFN-Q).

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Fig. 3. Time course of inhibition of NO production by MJE and the phloroglucinol derivatives. Cells were treated with MJE or the phloroglucinol derivatives in the presence of 100 ng/ml LPS+10 U/ml IFN-Q at 37³C for up to 16 h. The results are the means þ S.E. for two independent experiments comprising duplicate assays. The symbols correspond to: (A) control cells (treated with LPS+IFN-Q) (7), 30 Wg/ml MJE (a), 30 WM 3 (R), and +NONE (absence of LPS or IFN) (b), (B) control cells (7), 30 WM 7 (8), 30 WM 9 (b), and 30 WM 10 (O). Statistical signi¢cance: *P 6 0.01 vs. corresponding controls (cells treated with 100 ng/ml LPS+10 U/ml IFN-Q).

tion were direct e¡ects on the intrinsic enzyme activity of iNOS, or whether these e¡ects were mediated by some other mechanism. In an intact cell assay, carboxy-PTIO converts c NO to c NO2 , and then to NO3 2 in a fully stoichiometric manner [23]. As shown in Fig. 4, LPS and IFN5 Q treated cells produced 129.3 pmol NO3 2 /min/2.0U10 3 cells under these conditions, and NO2 increased linearly at 37³C for up to 60 min. This NO3 2 production was completely abolished by 1 mM L-NIO, a representative inhibitor of iNOS [24] (Fig. 4A). MJE and the tested compounds exhibited continuous inhibition of iNOS activity for up to 60 min. However, 5 did not show inhibitory activity at either 30 min or 60 min (Fig. 4). In addition, 1 and 6 showed no e¡ects on NO production by activated

macrophages and did not inhibit iNOS activity (data not shown). MJE and the active compounds were inhibitory in a concentration-dependent manner (Fig. 5). At 30 and 100 Wg/ml, MJE reduced this enzyme activity to 84 and 72%, respectively. Signi¢cant inhibition was also seen after treatment with 100 WM 10 (60.0%), 8 (51.4%), 4 (49.8%), 7 (48.6%), 3 (44.8%), 9 (41.0%), and 2 (29.7%). Furthermore, inhibition of iNOS enzyme activity in the cell extracts was determined by the conversion of L-[U14 C]arginine to L-[U-14 C]citrulline. L-[U-14 C]Citrulline production increased linearly with time under these conditions (data not shown). MJE, 10, 8, and 4 showed 13.8%, 48.8%, 34.7%, and 31.5% inhibition, respectively, at 100 Wg/ml or 100 WM (Table 1).

Fig. 4. Time courses of the inhibitory e¡ects of MJE and the phloroglucinol derivatives on iNOS activity in preactivated macrophages. Cells were pretreated with 100 ng/ml LPS+10 U/ml IFN-Q at 37³C for 16 h and suspended in HBSS (+). Subsequently, cells were incubated with MJE (100 Wg/ml) or the active compounds (100 WM 2, 3^5, 7^10), and after incubation for 5 min, 100 WM carboxy-PTIO and 1 mM L-arginine were added to the cell culture, followed by incubation for 0^60 min. The symbols correspond to: (A) control (+100 WM carboxy-PTIO and 1 mM L-arginine) (9997999), +L-NIO (- - -7- - -), MJE (a), and 2 (O), (B) control (7), 3 (R), 4 (E), 5 (F), and 9 (b), and (C) control (7), 7 (8), 8 (b), and 10 (O). The results are presented as the means þ S.E. for two di¡erent experiments involving duplicate assays. Statistical signi¢cance: *P 6 0.01 vs. corresponding controls (cells treated with 100 ng/ml LPS+10 U/ml IFN-Q).

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Fig. 5. Concentration dependence of the inhibitory e¡ects of MJE and the phloroglucinol derivatives on the iNOS activity in preactivated macrophages. Assays were performed as described in the legend to Fig. 4. The cells were incubated for 60 min after the addition of both 100 WM carboxy-PTIO and 1 mM L-arginine to the cell culture. The symbols correspond to: (A) MJE (a), 2 (O), and 3 (R), (B) 4 (E), 5 (F), and 9 (b), and (C) 7 (8), 8 (b), and 10 (O). The results are presented as the means þ S.E. for two di¡erent experiments involving duplicate assays. Statistical signi¢cance: *P 6 0.05, **P 6 0.01 vs. corresponding controls (cells treated with 100 ng/ml LPS+10 U/ml IFN-Q).

3.3. Inhibitory e¡ects on iNOS protein induction We next investigated whether MJE and the active compounds a¡ected induction of iNOS protein. MJE showed signi¢cant inhibition of iNOS protein levels ^ when 1, 3, 10, and 30 Wg/ml of MJE were added, 14, 31, 61, and 90%

Table 1 E¡ects of MJE and the phloroglucinol derivatives on the conversion of [14 C]arginine to [14 C]citrulline by the cell extract

Control MJE

4

8

10

L-NIO

Reagent conc. (Wg/ml or WM)

L-Citrulline

production (pmol/mg protein cell extract/min)

100 30 10 100 30 10 100 30 10 100 30 10 1000

186.1 þ 10.2 160.5 þ 14.3* 190.3 þ 5.6 187.6 þ 10.4 127.4 þ 8.4** 164.2 þ 7.6* 182.8 þ 6.2 121.5 þ 6.8** 166.7 þ 11.4* 187.1 þ 6.4 95.2 þ 5.8** 162.4 þ 5.4* 181.4 þ 5.2 2.1 þ 0.2*

RAW 264.7 cells (1.2U107 cells) were incubated for 16 h in 10 ml of medium containing 100 ng/ml LPS and 10 U/ml IFN-Q. After two washes, the cells were extracted using lysis bu¡er, and centrifuged at 5500Ug for 5 min at 4³C. A reaction mixture containing [U14 C]arginine was preincubated at 37³C for 3 min, and then MJE, one of the compounds, or L-NIO was added to the mixture together with a 50 Wg protein cell extract, followed by incubation for 15 min. Conversion of [U-14 C]arginine to [U-14 C]citrulline was determined as described in Section 2.5. The results are presented as the means þ S.E. for two di¡erent experiments involving duplicate assays. Statistical signi¢cance: *P 6 0.05, **P 6 0.01, vs. corresponding controls.

inhibition was obtained, respectively (Fig. 6A). Compounds 10 (IC50 : 1.6 WM), 9 (IC50 : 2.7 WM), 4 (IC50 : 4.3 WM), 8 (IC50 : 6.5 WM), 3 (IC50 : 14.5 WM), 7 (IC50 : 16.8 WM), 5 (IC50 : 23.8 WM), and 2 (inhibition at 30 WM: 45.7%) markedly reduced the amount of iNOS protein (Fig. 6). However, 1 and 6 had little or no e¡ect on iNOS production (data not shown). 3.4. Inhibitory e¡ects on iNOS mRNA expression We examined by RT-PCR analysis whether or not MJE, 3, 9 and 10, which showed signi¢cant e¡ects on iNOS protein induction, also inhibited iNOS mRNA expression in RAW 264.7 cells treated with LPS and IFN-Q. The quantity of iNOS mRNA and L-actin mRNA, a housekeeping gene, was estimated from the £uorescence intensity of the bands corresponding to iNOS mRNA (456 bp) and L-actin mRNA (540 bp) in agarose gels. We determined the degree of inhibition using the iNOS/L-actin ratio as an adjustment for minor di¡erences in RT e¤ciency among the samples. Treatment with LPS and IFN-Q for 6 h increased the level of iNOS mRNA expression (Fig. 7). The induced mRNA level decreased with increasing concentrations of MJE and the active compounds. At 3, 10, and 30 Wg/ml, MJE reduced iNOS mRNA expression to 75, 68, and 47%, respectively. At 30 WM, compounds 9 and 10 almost totally blocked the mRNA expression, and low level inhibition of iNOS mRNA was seen at concentrations as low as 3 WM. This inhibition was speci¢c because reduction in mRNA expression of L-actin was not a¡ected by treatment with MJE or the active compounds. RT-PCR analysis indicated that MJE and the active compounds suppressed iNOS protein induction by reducing its mRNA level in RAW 264.7 cells.

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Fig. 6. Inhibition of iNOS induction by MJE and the phloroglucinol derivatives. Cells were treated with various concentrations of MJE or the active compounds in the presence of 100 ng/ml LPS+10 U/ml IFN-Q for 8 h. Cell lysates were then prepared and assayed as described in the text. The amounts of the iNOS protein were calculated from the radioactivity of the bands corresponding to iNOS protein. We used an iNOS standard in the immunoblot analysis in order to normalize the iNOS amounts between di¡erent gels. The results are expressed relative to the amount of iNOS in 60 Wg of the control cells (100%: treated with LPS+IFN-Q, but MJE or other compounds not added). The symbols correspond to: (A) MJE (a), 2 (O), and 3 (R), (B) 4 (E), 5 (F), and 9 (b), and (C) 7 (8), 8 (b), and 10 (O). In the lower panel the bands corresponding to the 130 kDa iNOS protein are indicated. The numbers correspond to: 1, control (absence of LPS or IFN-Q); 2, control (presence of LPS+IFN-Q); 3, MJE 1 Wg/ml; 4, MJE 3 Wg/ml; 5, MJE 10 Wg/ml; 6, MJE 30 Wg/ml; 7, 3 1 WM; 8, 3 3 WM; 9, 3 10 WM; 10, 3 30 WM; 11, 5 1 WM; 12, 5 3 WM; 13, 5 10 WM; 14, 5 30 WM; 15, 7 1 WM; 16, 7 3 WM; 17, 7 10 WM; 18, 7 30 WM; 19, 10 1 WM; 20, 10 3 WM; 21, 10 10 WM; 22, 10 30 WM.

Fig. 7. E¡ects of MJE and the phloroglucinol derivatives on iNOS mRNA expression. Cells were treated with various concentrations of MJE or the derivatives in the presence of 100 ng/ml LPS+10 U/ml IFN-Q for 6 h, and the cells were subsequently prepared and assayed as described in the text. The symbols correspond to: (A) MJE (a), and 3 (R), (B) 9 (b), and 10 (O). The lower panel shows the PCR products corresponding to the iNOS (456 bp) and L-actin (540 bp) bands. The amounts of iNOS mRNA in A and B are shown as % values relative to L-actin, individually. The results are for a typical example of repeated experiments.

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4. Discussion We found that MJE inhibited NO production over the course of macrophage activation, and that the major inhibitory activity was due to the phloroglucinol derivatives. The degree of inhibitory e¡ects of these phloroglucinol derivatives on NO production was, in order, 10 s 9 s 4 s 8W3W7 s 2 s 5, at a concentration of 30 WM. Compound 1 showed little or no inhibition, whereas 2 showed 26.1% inhibition at 30 WM. These results revealed that dimer formation was necessary for manifestation of the inhibitory activity. Compound 3 exhibited somewhat stronger inhibition than 2, suggesting that the presence of an isoprenyl side chain at the C-3 position led to stronger inhibition than in its absence. Moreover, inhibition by 3 was stronger than by 5. This result indicated that cyclization of the side chain with a hydroxy group at the ortho-position to an acetyl group might decrease the inhibitory activity. On the other hand, 9, which was cycled from a hydroxy group at the para-position to an acetyl group, showed signi¢cantly greater inhibition than 3. Based on these results, the presence of a side chain and the position of its cyclization were presumed to be the most important factors for the inhibitory activity. We found that the presence of a free hydroxy group at the chroman ring increased inhibition of NO production (7 s 5, 10 s 9). Of the test compounds, 10, 8, 4, and 7, which have a free hydroxy group on the side chain or the chroman ring, exhibited signi¢cant inhibition of the iNOS activity. The presence of a free hydroxy group was clearly essential for the direct e¡ects on iNOS activity. The inhibitory e¡ects on iNOS protein induction in LPS and IFN-Q activated macrophages were, in order, 10 s 9 s 4 s 8 s 3 s 7 s 5 s 2. This trend was similar to that of the inhibitory e¡ects on NO production during incubation for 8 h. Therefore, these inhibitory e¡ects of phloroglucinol derivatives on NO production by macrophages are attributable mainly to the inhibition of iNOS induction rather than direct e¡ects on iNOS activity. At the gene level, the promoter of the mouse gene encoding iNOS contains consensus sequences that bind several transcription factors including the nuclear factor UB [25] and the IFN regulatory factor [26]. The RT-PCR analysis in the present study indicated that LPS and IFN-Q treatment increased the level of iNOS mRNA expression, and that MJE and the phloroglucinol derivatives inhibited this increase. Inhibition of iNOS induction by MJE and the phloroglucinol derivatives, therefore, may be mediated through the suppression of these transcription activating factors, thereby inhibiting iNOS transcription. An alternative scenario involves protein kinase C (PKC). PKC inhibitors have been reported to inhibit iNOS induction in macrophages [27^29]. The phloroglucinol derivatives examined in the present study were similar to rottlerin in their chemical structures. Rottlerin is an isolate of Kamala, a crude drug consisting of glandular hairs from

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the pericarps of Mallotus philippinensis [30]. As the purported PKC N speci¢c inhibitor [31], rottlerin signi¢cantly attenuated NO production in a pancreatic islet L-cell line [32]. Likewise, PKC was reportedly involved in the stabilization of the iNOS mRNA [33]. We previously reported that the phloroglucinol derivatives in the present study inhibited tumor promotion by 12-O-tetradecanoylphorbol 13-acetate, a PKC activator, and that 9 most strongly inhibited among these compounds [12]. In the light of these reports, we speculate that MJE and the derivatives inhibit iNOS mRNA levels and protein expression by down-regulating PKC activity. Inhibitors of NO production by macrophages act mainly through two mechanisms: one is the inhibition of iNOS expression, and the other is the inhibition of enzyme activity. Most iNOS inhibitors act through either of these mechanisms. The inhibition of both the induction of iNOS and the activity of iNOS seems to be a characteristic of MJE and the phloroglucinol derivatives. The present ¢ndings encourage further studies to clarify the signaling pathway for the inhibition of iNOS protein induction by MJE and the phloroglucinol derivatives. Identi¢cation of other potential anti-in£ammatory constituents of the pericarps of M. japonicus is also of interest.

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