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Bioactive compounds from liverworts: Inhibition of lipopolysaccharide-induced inducible NOS mRNA in RAW 264.7 cells by Herbertenoids and Cuparenoids.


The inhibition of lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) by herbertenoids and cuparenoids isolated from liverworts in RAW 264.7 macrophages was evaluated. Among compounds tested, herbertenediol, cuparenediol, 1,2-diacetoxyherbertene and 2-hydroxy-4-methoxycuparene exhibited significant activity. For 2-hydroxy-4-methoxycuparene, chosen as representative compound, the strong inhibitory activity was related to the inhibition on LPS-induced iNOS mRNA. The structure-activity relationship will be discussed.

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Liverworts; Herbertenoids; Cuparenoids; RAW 264.7 cell; iNOS


Herbertenoids are the sesquiterpenes having 1 (Fig. 1) as skeleton and distributed in a few liverwort genera such as Herbertus (Herbertaceae), Mastigophora (Lepicoleaceae), and Plagiochila (Plagiochilaceae), while cuparenoids (e.g., 2) are the ubiquitous sesquiterpenoids found in many liverworts, Bazzania, Lepidozia, Plagiochila, Radula, Reboulia, Marchantia, Rucciocarpos, and red algae belonging to the genus Laurencia (Ceramiales, Rhodomelaceae) such as Laurencia microcladia (Asakawa, 1995; Kladi et al., 2005). Their role in the producing organisms is not yet clearly demonstrated but due to their wide biological activities (Asakawa, 1995; Kladi et al., 2005; Harinantenaina et al., 2004a), it is obvious that they function as chemical defenses against predators. In the course of our systematic search of biologically active metabolites from liverworts, we previously reported the antibacterial properties of some herbertenoids isolated from Mastigophora diclados collected in Madagascar (Harinantenaina et al., 2004a). In the present communication, we wish to report the inhibitions of NO production of lipopolysaccharide (LPS)-stimulated RAW 264.7 cells by seven herbertenoids (3-7, 10, 11) and two cuparenoids (8, 9) isolated from liverworts, as well as the inhibition of LPS-induced inducible nitric oxide synthase (iNOS) by the representative compounds (9).

NOS catalyzes the conversion of L-arginine and oxygen stimulators into L-citrulline and nitric oxide (NO) (Marletta, 1993). Overproduction of NO is involved in inflammatory response-induced tissue injury and the formation of carcinogenic N-nitrosamines (Miwa et al., 1987; Mulligan et al., 1991). Since a large amount of NO generated by induced iNOS upon stimulation of endotoxins or cytokins is implicated in pathological responses at the quantities of nM (Nevin et al., 2002), inhibition of iNOS is very important to control inflammatory diseases. Using LPS-stimulated RAW264.7 macrophage cells is one of the approaches to evaluate inhibitory activity of chemical compounds in iNOS.


Materials and methods

Isolation of compounds 3-11

Compounds 3-5, 10 and 11 were isolated from Mastigophora diclados collected in Madagascar (Harinantenaina et al., 2004a) Compound 6 was isolated from Herbertus sakuraii (Hashimoto et al., unpublished results). Compounds 7 were obtained by acetylation of compound 5. Compounds 8 and 9 were isolated from Lejeunea aquatica and Bazzania decrescens, respectively (Toyota et al., 1997; Harinantenaina et al., 2004b). Their structures were determined by a combination of 2D NMR spectrometry and chemical analysis.

Cell culture

RAW 264.7 cells, a mouse macrophage cell line, were grown in RPMI 1640 supplemented with 10% fetal bovine serum, kanamycin (50 [micro]g/mL), and ampicillin (60 [micro]g/mL) at 37 [degrees]C in the atmosphere of 5% C[O.sub.2] and 95% air.

Inhibition of NO production by 3-11

One hundred microliter of RAW-264.7 cells (8 x [10.sup.5] cells/mL) were pipetted in each well and cultured in 96-well culture plate. After 24 h incubation, cells were pretreated by adding 50 [micro]L of medium containing different concentrations of each compound previously diluted in DMSO (the final DMSO concentration was less than 0.1%, and at this concentration, DMSO did not show any NO-induction/or NO-inhibition with or without stimulation with LPS, Harinantenaina et al., 2005; Rockett et al., 1998) to each well before 1 h incubation. Fifty microliters of vehicle or LPS purified from Pantoea agglomerans (4 [micro]g/mL) was added. The cells were further incubated at 37 [degrees]C for 24 h. The supernatant (35 [micro]L) of each well was taken, then mixed with Griess reagents [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphthylethylenediamine-HC1] and after 10min incubation at room temperature, the absorbance at 550 nm was measured by using BIO-RAD model 550 Microplate reader. NO concentration was determined by measuring the amount of nitrite in the cell culture supernatant using Griess reagents and [IC.sub.50] values were calculated.

Reverse transcription-polymerase chain reaction (RT-PCR)

iNOS mRNA was amplified by RT-PCR analysis of its synthesized specific primers. 500 [micro]L RAW cells ([10.sup.6] cells/mL) were added to each well and cultured in 24-well culture plate. After 24 h incubation, 250 [micro]L of each sample, previously dissolved in DMSO and diluted in the medium in three dilutions (20, 10, and 0.1 [micro]M), was added to each well. Then 1 hour later, 250 [micro]L LPS (4 [micro]g/mL) or vehicle was added to each well. The cells were further incubated for 8h. Total RNA was isolated from RAW cells using TRIzol and the concentration of purified total RNA was determined by absorbance at 260 nm (data not shown). The RNA samples were reverse-transcribed into cDNA by following method: total RNA (2.5 [micro]g), oligo (dT) primer (0.5 [micro]L) and the final volume was brought up to 7.5 [micro]L with RNase-free water was heated at 70[degrees]C for 10min, 5 x RT buffer (3 [micro]L), 10mM dNTPS (1.5 [micro]L), ReverTra Ace (MMLV Reverse Transcriptase RNaseH-) (75u/0.75 [micro]L), RNase inhibitor (0.4 [micro]L) and water (1.9 [micro]L) were then added to the mixture, and incubated at 42 [degrees]C for 60 min, then 99 [degrees]C for 10 min.

PCR amplification was performed in a reaction volume of 10 [micro]L containing 3 [micro]L of the appropriate cDNA, 1 [micro]L of iNOS or [beta]-actin primer (5 pmol/[micro]L), 5 [micro]L of master mix and 1 [micro]L of water. The sense primer for iNOS was 5'-GTAGAAAGTCCAGCCGCAC-3' and the anti-sense primer was 5'-GTAGCTGCCGCTCTCATCCAG-3'. The sense primer for [beta]-actin was 5'-CCAACCGTGAAAAGATGACC-3' and the antisense was 5'-CAGGAGGAGCAATGATCTTG-3'. The primer sequence were determined for iNOS cDNA (BC062378) and [beta]-actin cDNA (AK078935).

For iNOS and [beta]-actin, the PCR procedures were carried out using a DNA Engine OpticonTM System Continuous for Fluorescence Detector (MJ Researcher Incorporated, USA). The PCR was carried out with 20 cycles for [beta]-actin and 25 cycles for iNOS to obtain the results within the exponential range. The PCR amplification was performed after 10 min incubation at 95 [degrees]C under the following condition: 20-25 cycles of denaturation at 95 [degrees]C for 10s, annealing at 60 [degrees]C for 10s and extension at 72 [degrees]C for 20s, using a thermal cycler. Final amount of RT-PCR products for iNOS mRNA (203 bp) and [beta]-actin (660 bp) were calculated and the ratios of iNOS/[beta]-actin were described.


RT-PCR products for iNOS mRNA and [beta]-actin were analyzed by electrophoresis on 2.5% agarose gel. Electrophoresis was run for 15 min using a Mupid apparatus and the gel was stained by ethidium bromide, read under UV light. The active compound affected iNOS mRNA expression, but did not affect constitutive gene expression, such as [beta]-actin. The electrophoresis for iNOS and [beta]-actin were illustrated in Fig. 2.


Statistical analysis

The results represented three independent experiments and are expressed as mean [+ or -] S.E.M. The data were analyzed by t-test using SPSS software (12.0 version). The differences were considered statistically significant at p-value less than 0.05.


Effects of compounds 3-11 on LPS-induced production of NO

Pretreatment of the RAW 264.7 cells with compounds 3-11 at 30, 20, 10, 5, 2.5, 1.75 [micro]M was carried out before LPS addition (see Material and methods). The nitrite concentrations in the conditioned medium after 24 h incubation were measured. Compounds 3-11 showed inhibition of LPS-induced production of nitrite and their [IC.sub.50] values were reported in Table 1.

Effect of the representative compound (9) on LPS-induced iNOS mRNA

Expression incubation of compound 9 in three different concentrations (20, 10, 0.1) and LPS (1 [micro]g/mL) for 8h markedly induced iNOS expression in RAW cells. Analysis of the RT-PCR of the iNOS mRNA level (Fig. 2) displayed that the suppression of iNOS gene expression by compound 9 was dose dependent. At the concentration 20 [micro]M, compound 9 completely inhibited the iNOS mRNA expression.


The highest inhibition of the LPS-induced NO production was attributed to 2-hydroxy-4-methoxycuparene (9, [IC.sub.50] = 4.1 [micro]M) and the lowest to [alpha]-herbertenol (3, [IC.sub.50] = 76 [micro]M). The only difference in the structures of compounds 3 and 4 is the ortho or para position of the hydroxyl group regarding to the aromatic methyl. From the ortho to a para position, the activity increased six times (Table 1). It is interesting to note that compound 9, which has strong NO inhibition, has a hydroxyl and a methoxyl at ortho position to the methyl group at C-12. Furthermore, the NO inhibitions of herbertenediol (5) and cuparenediol (8), which have two phenolic hydroxyls at C-l and C-2 positions, are comparable (8.03 and 9.2 [micro]M, respectively). Slight difference could be observed between the inhibition of 5 and its acetylated product (7, 7.01 [micro]M). Unexpectedly, the two dimeric compounds (10 and 11) did not show higher inhibitions comparing to the monomer (5). These findings suggest that the replacement of each methyl groups of the two monomers to a methylene for compound 10 decreased the activity. This fact was also observed in the NO inhibition of compound 11 (less than that observed in 10), in which two methylenes were present in the molecule. For compound 6, oxidation of the methyl group at C-12 to an aldehyde decreased the activity about four times. Moreover, acetylation of compound 5 slightly increased the inhibition [[IC.sub.50] from 8.03 [micro]M (5) to 7.01 [micro]M (7)]. Taken together, these results showed the evidence that not only the phenolic hydroxyl in the molecule, but also its location with regard to the aromatic methyl is the structure requirement of herbertenoids and cuparenoids for inhibition of NO production. Noteworthy, the NO inhibitory activities against LPS-induced macrophage cells of compounds 4, 5, 7, 8, 9-11 are more potent than the standard compound (L-[N.sub.6]-l-iminoethyl) lysine ([IC.sub.50] = 18.6 [micro]M).

In order to elucidate the mechanisms of active herbertenoids and/or cuparenoids for inhibition of the NO production in LPS-induced macrophage RAW 264.7, the mRNA expression affected by treatment of 9 (chosen as representative compound, for its strong inhibition) was examined in relation to iNOS mRNA expression along LPS-stimulated RAW 264.7 cells. As detected by electrophoresis on agarose gel (Fig. 2), the inhibition of iNOS mRNA expression of compound 9 was dose dependent. Complete inhibition was observed at 20 [micro]M. From these results, we could conclude that the inhibition of compound 9 on LPS-stimulated NO production in RAW cells was related with the suppression of the iNOS mRNA.

Our results showed that: (1) strong NO inhibitions of herbertenoids and cuparenoids were observed if the hydroxyl groups are located at meta and/or para to C-12 methyl, (2) apart from the presence of phenolic hydroxyl group, the aromatic methyl seems to be important for the inhibitions. Moreover, (3) oxidation of the aromatic methyl to an aldehyde decreased the inhibition of NO production. Further investigations should be done to prove if the stable conformation of the dimers could affect the activity of the compounds. (4) The inhibition of NO production of herbertenoids and cuparenoids could be elucidated as the results of the iNOS mRNA suppression.


The authors are grateful to the Japan Society for Promotion of Science (JSPS) for granting a postdoctoral fellowship to L. Harinantenaina (No. P05164). This work was supported by a "Open Research Center" Project of the Ministry of Education, Culture, Sports, Science and Technology.


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Harinantenaina, L., Asakawa, Y., 2004a. Chemical constituents of Malagasy liverworts, Part II: mastigophoric acid methyl ester of biogenetic interest from Mastigophora diclados (Lepicoleaceae Subf. Mastigophoroideae). Chem. Pharm. Bull. 52, 1382-1384.

Harinantenaina, L., Kurata, R., Asakawa, Y., 2004b. Chemical constituents of Malagasy liverworts, Part III: sesquiterpenoids from Bazzania decrescens and Bazzania madagassa. Chem. Pharm. Bull. 53, 515-518.

Hashimoto, T., Asakawa, Y., Unpublished results.

Harinantenaina, L., Quang, D.N., Nishizawa, T., Hashimoto, T., Kohchi, C., Soma, G-I., Asakawa, Y., 2005. Bis(bibenzyls) from liverworts inhibit lipopolysaccharide induced inducible NOS in RAW 264.7 cells: a study of structure--activity relationships and molecular mechanism. J. Nat. Prod. 68 (12), 1779-1781.

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Miwa, M., Stuehr, D.J., Marletta, M.A., Wishnok, J.S., Tannenbaum, S.R., 1987. Nitrosation of amines by stimulated macrophages. Carcinogenesis 8, 955-958.

Mulligan, M.S., Hevel, J.M., Marlletta, M.A., Ward, P.A., 1991. Tissue injury caused by deposition of immune complexes is L-arginine dependent. Proc. Natl. Acad. Sci. USA 88, 6338-6342.

Nevin, B.J., Broadley, K.J., 2002. Nitric oxide in respiratory diseases. J. Pharmacol. Ther. 95, 259-293.

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Liva Harinantenaina (a), Dang Ngoc Quang (a,d), Takashi Nishizawa (b), Toshihiro Hashimoto (a), Chie Kohchi (b,c), Gen-Ichiro Somab (b,c), Yoshinori Asakawa (a,*)

(a) Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan

(b) Institute for Health and Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan

(c) Institute of Drug Delivery Systems, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

(d) Faculty of Chemistry, Hanoi University of Education, 136 Xuan Thuy Road, Cau Giay, Hanoi, Viet Nam

*Corresponding author. Tel.: +81 88 622 9611; fax: +8188 655 3051.

E-mail address: (Y. Asakawa).
Table 1. Effect of compounds 3-11 on NO production by LPS-induced RAW
264.7 macrophages

Compounds [IC.sub.50] ([micro]M)

3: [alpha]-Herbertenol 76
4: [beta]-Herbertenol 12.23
5: Herbertenediol 8.03
6: Herbertenal 34
7: 1,2-Diacetoxy-herbertene 7.01
8: Cuparendiol 9.2
9: 2-Hydroxy-4-methoxycuparene 4.1
10: Mastigophorene C 10.18
11: Mastigophorene D 15.16
(L-[N.sub.6]-1-Iminoethyl)lysine 18.6
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Title Annotation:nitric oxide synthase, mouse leukaemic monocyte macrophage cell line
Author:Harinantenaina, Liva; Quang, Dang Ngoc; Nishizawa, Takashi; Hashimoto, Toshihiro; Kohchi, Chie; Soma
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Aug 1, 2007
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