Anti-Inflammatory Activity of Phenylpropanoids and Phytoquinoids from Illicium Species in RBL-2H3 Cells

Home , Anisatin

Anti-Inflammatory Activity of mediate phase and the latter is released in the late phase of type I allergic reactions [9], [10]. Phenylpropanoids and Phytoquinoids from Illicium Species in RBL-2H3 Cells This study was conducted to investigate the anti-inflammatory activity of the compounds isolated from Illicium species on mast Takuya Matsui1,2, Chihiro Ito1, Masataka Itoigawa3, Tadashi Okada2, cells. We analyzed the effect of six phenylpropanoids (1±6) and Hiroshi Furukawa1 six phytoquinoids (7±12), isolated from three Illicium species (Fig.1), on histamine release and TNF-a production and/or secretion from A23187-stimulated rat basophilic leukemia RBL-2H3 Abstract cells.

Two phenylpropanoids, 1-allyl-3,5-dimethoxy-4-(3-methyl-but- Among the compounds tested, phenylpropanoids 4 and 6 and 2-enyloxy)benzene (4) and 4-allyl-2,6-dimethoxy-3-(3-methyl- phytoquinoids 7 and 8 at a concentration of 50 mM markedly in-

2-butenyl)phenol (6), and two phytoquinoids, 4R-(±)-illicinone- hibited histamine release from RBL-2H3 cells (96.9 2.9%, 84.3 Letter A(7) and 2S,4R-(±)-illicinone-B (8), isolated from plants of the 4.1%, 99.4 1.0% and 98.3 1.9% inhibition, respectively), Illicium species significantly inhibited histamine release from whereas the inhibition rate of the standard reference epigalloca- rat basophilic leukemia (RBL-2H3) cells stimulated with thechin gallate (EGCG) was 28.6 6.0% (Fig. 2A). These com- A23187. Furthermore, these compounds caused a decline in pounds showed a dose-dependent inhibitory effect in the con- TNF-a levels in culture supernatants of RBL-2H3 cells following centration range of 1±50 mM on histamine release without ap- treatment with A23187. The results indicate that these com- parent cytotoxicity at the highest concentration (50 mM) pounds might be useful as anti-inflammatory agents against (Fig. 2B). The IC50 values of phenylpropanoids 4 and 6 were calcu- mast cell-mediated inflammatory diseases. lated as 20.2 0.4 mM and 23.1 3.3 mM, respectively. The IC50 values of phytoquinoids 7 and 8 (16.2 2.2 mM and 15.7 Abbreviations 0.6 mM, respectively) were only slightly lower than those of phe- MTT assay: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- nylpropanoids 4 and 6. These results show that phenylpropa- lium bromide noids 4 and 6 and phytoquinoids 7 and 8 have an inhibitory ef- TNF-a: tumor necrosis factor-a fect on histamine release from mast cells.

Supporting information available online at Based on the results obtained from the histamine release experi- http://www.thieme-connect.de/ejournals/toc/plantamedica ments, phenylpropanoids 4 and 6 and phytoquinoids 7 and 8 at 50 mM were assayed for a possible effect on TNF-a production and/or secretion from RBL-2H3 cells stimulated with A23187. Japanese star anise (Illicium anisatum L., Japanese name, Sikimi) The concentrations of TNF-a in the cultured supernatant from (Illiciaceae) has been reported to be neurologically and gastroin- RBL-2H3 cells pretreated with phytoquinoids 7 and 8 were re- testinally toxic [1], [2]. In our previous studies, several phenyl- duced by 87.4% and 83.1% compared to A23187-stimulated RBL- propanoids and phytoquinoids isolated from Illicium species [3], 2H3 cells (Fig. 3). Phenylpropanoids 4 and 6 also significantly re- [4], [5] were shown to have cancer chemopreventive activity [6]. duced TNF-a levels by 80.8 % and 30.0%, respectively (Fig. 3). In other reports, phenylpropanoids from Curcuma zedoaria (Zin- These findings suggest that phenylpropanoids 4 and 6 and phy- giberaceae) and Stella dichotoma L. var. lancolata (Caryophylla- toquinoids 7 and 8 have the potential to attenuate late-phase al- ceae) were reported to inhibit TNF-a release from mast cells [7], lergic reactions in mast cells. The inhibitory effect of phenylpro- [8], suggesting an anti-inflammatory effect. However, the effect panoids 4 and 6 on TNF-a production and/or secretion supports of other compounds isolated from Illicium plants on mast cells earlier reports [7], [8], [11], [12], [13]. Furthermore, the present is not yet fully understood. Mast cells play a central role in type report is the first to show that phytoquinoids suppress the pro- I allergic inflammatory reactions and in chronic inflammation duction and/or secretion of TNF-a from mast cells. associated with fibrosis. Histamine and TNF-a are the major che- mical mediators in mast cells. The former is released in the im- One structural feature common to the active phenylpropanoids 4 and 6 is the presence of a prenyl moiety on the aromatic ring. Furthermore, the O-prenylated 4 showed a greater inhibition of TNF-a activity than the C-prenylated 6. The presence of the O- Affiliation: 1 Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya, or C-prenyl moiety in the molecule seems to influence the inhi- Aichi, Japan ´ 2 Department of Physiology, Aichi Medical University, Nagakute- bitory activity towards TNF-a production and/or secretion from 3 cho, Aichi-gun, Aichi, Japan ´ Faculty of Human Wellness, Tokai Gakuen Uni- RBL-2H3 cells. Phenylpropanoids 1, 2 and 3 have no prenyl group, versity, Tempaku, Nagoya, Aichi, Japan and the inhibitory effect of compounds 1 and 2 on histamine re- Correspondence: Dr. Takuya Matsui ´ Department of Medicinal Chemistry, Fa- lease was lower than that of compound 3 (Fig. 2A). Based on this culty of Pharmacy ´ Maijo University ´ Yagotoyama 150 ´ Tempaku-ku ´ Nagoya 468±8503 ´ Japan ´ Phone: +81-52-832-1781 ´ Fax: +81-52-834-8090 ´ E-mail: observation, the methylenedioxy ring system in these aromatic [email protected] molecules does not appear to be necessary for inhibitory activity.

Received December 23, 2005 ´ Revised April 5, 2007 ´ Accepted April 24, 2007 On the other hand, among the phytoquinoids (7±12) which are Bibliography: Planta Med Georg Thieme Verlag KG Stuttgart ´ New York ´ constructed from 2,5-cyclohexadien-1-one or its dihydro skele- DOI 10.1055/s-2007-981528 ´ Published online 2007 ´ ISSN 0032-0943 ton, only phytoquinoids 7 and 8 inhibited histamine release and Fig. 1 Structures of phenylpropanoids (1±6) and phytoquinoids (7±12) isolated from Illicium species. Letter

Fig. 2 Inhibitory effects of phenylpropanoids and phytoquinoids on histamine release from RBL-2H3 cells. A Com- parison of six phenylpropanoids (1±6) and six phytoquinoids (7±12) at a concentration of 50 mM for the inhibition of histamine release from RBL-2H3 cells in response to A23187 (2 mM). RBL-2H3 cells were pretreated with the test compounds for 30 min at 37 8C and were stimulated with A23187 for 30 min at 37 8C. The white bar shows the standard reference epigallocathechin gallate (EGCG). Bars represent the mean + SD (n = 5). Significantly different from EGCG, **, p < 0.01. B Dose-response analysis of phenylpropanoids 4 and 6 and phytoquinoids 7 and 8 at concentrations of 1, 5, 10, 25 and 50 mM. Results are shown as mean + SD (n = 3). * and **, p < 0.05 and p < 0.01, respectively for percent histamine release from RBL-2H3 cells treated with A23187 (2 mM) in the presence versus absence of the test compounds (not shown).

Letter ¼ Planta Med Fig. 3 Inhibition of TNF-a production and/or secretion from RBL-2H3 cells by phenylpropanoids 4 and 6 and phytoquinoids 7 and 8 at a concentration of 50 mM. Bars represent the mean + SD (n = 3). * and **, p < 0.05 and p < 0.01, respectively for TNF-a levels in the culture supernatant from RBL-2H3 cells treated with A23187 (2 mM) in the presence of the test compounds (black bar) versus that in the absence of the test compounds (white bar). Letter TNF-a production and/or secretion. The inhibitory activities of Cell culture: The RBL-2H3 cells obtained from the National Insti- compounds 7 and 8 on histamine release and the observed de- tute of Health Sciences (Japan Collection of Research Biosources; crease of the TNF-a concentration were similar, suggesting that cell number JCRB0023) were plated on a 96-well plate at a den- the enone and dienone moieties in these molecules may not sity of 1105 cells/well and cultured in Isocove's modified Dul- have a biological effect. The presence of an oxirane ring on the becco's medium (IMDM; Sigma-Aldrich Co.; St. Louis, MO, USA) prenyl group, as shown in phytoquinoid 9, conferred a weaker in- supplemented with 10% heat-inactivated FCS (Gibco BRL; Grand hibition of histamine release (Fig. 2A). Further, the lack of activ- Island, NY, USA), penicillin (Meiji Seika Kaisha, Japan) at 100 U/ ity of phytoquinoids 10, 11 and 12 demonstrates the importance mL, streptomycin (Meiji Seika Kaisha) at 100 mg/mL and 2 mM of the methylenedioxy moiety as well as the prenyl group in this glutamine (Nissui Pharmaceutical Co., Tokyo, Japan) in 5% CO2. structural system, as shown for the active compounds 7 and 8. Histamine release assay: The supernatants of stimulated RBL- As mentioned above, the present results suggest that the pres- 2H3 cells were collected and centrifuged at 400 g for 5 min at ence of a prenyl moiety (C5-terpenoid), not only on the aromatic 4 8C. Residual histamine present in the cells was released by dis- but also on the cyclic-enone molecules, seems to be necessary for rupting the cells with 1% perchloric acid, followed by centrifuga- the anti-inflammatory activity. tion at 9000g for 5 min at 4 8C. The histamine content was determined by HPLC, coupled with post-column derivatization We previously showed that the presence of a prenyl moiety at- fluorometry. A 5 mL sample of the supernatant was injected onto tached to the aromatic rings of naturally occurring xanthones, an ODS HPLC column (CAPCELL PAK C18 UG120 S5; Shiseido, Ja- coumarins, flavonoids and phenylpropanoids plays an important pan), with a mobile phase of 10 mM sodium heptanesulfonate role in anti-tumor promoting activity [6], [14], [15], [16], [17]. containing 20 mM phosphate buffer (pH 3.2) and methanol at a Terencio et al. [18] also reported that prenylhydroquinones are ratio of 75:25 (v/v). The fluorescence intensity was measured at structural analogues of marine products that inhibit TNF-a pro- 455 nm (excitation at 340 nm) and the quantity of histamine re- duction and the release of nitrite and PGE2, suggesting that the lease is expressed as the peak area. Inhibition is expressed as a prenyl moiety has anti-inflammatory activity. Iwata et al. [19] percentage. also reported that prenylated orcinol derivatives inhibit histamine release from rat peritoneal mast cells. Studies examining TNF-a release assay: The TNF-a release assay was modified as de- the mechanism and detailed structure-activity relationships of scribed previously [7]. RBL-2H3 cells (1 105 cells/well) were in- the anti-inflammatory activity of these compounds are now in cubated with the test compounds at 50 mM and A23187 (2 mM) at progress. 37 8C for 6 hours, respectively. TNF-a levels in the culture supernatant taken from non-treated and treated RBL-2H3 cells were In conclusion, 1-allyl-3,5-dimethoxy-4-(3-methyl-but-2-enyloxy)- determined using a rat TNF-a ELISA kit (Quantikine; R&D Sys- benzene (4), 4-allyl-2,6-dimethoxy-3-(3-methyl-2-butenyl)phe- tems; Minneapolis, MN, USA) according to the manufacturer's nol (6), 4R-(±)-illicinone-A (7) and 2S,4R-(±)-illicinone-B (8) protocol. isolated from Illicium species had a protective effect against A23187-induced histamine release and TNF-a production and/ Statistical analysis: Data are expressed as the mean standard or secretion from RBL-2H3 cells. These findings indicate that deviation (SD). Statistical analysis was performed using the these compounds might be valuable anti-inflammatory agents. Welch test for statistical significance.

Materials and Methods Supporting information

Plant materials: Phenylpropanoids and phytoquinoids were Bioassay protocols are available as Supporting Information. isolated from Illicium tashiroi Maxim, I. anisatum L. and I. arborescens Hayata, respectively (Fig.1) [3], [4], [5]. The purity and structure of the test compounds were confirmed using melting point data as well as IR, UV, mass and 1H-NMR spectra.

Letter ¼ Planta Med References

1 Kudo Y, Oka JI, Yamada K. Anisatin, a potent GABA antagonist, isolated from Illicium anisatum. Neurosci Lett 1981; 25: 83±8. 2 Ize-Ludlow D, Ragone S, Bruck IS, Bernstein JN, Duchowny M, Pena BM. Neurotoxicities in infants seen with the consumption of star anise tea. Pediatrics 2004; 114: 653 ±6. 3 Yakushijin K, Sekikawa J, Suzuki R, Morishita T, Furukawa H, Murata H. Novel phytoquinoids from Illicium tashiroi Maxim. Chem Pharm Bull 1980; 28: 1951±4. 4 Yakushijin K, Tohshima T, Kitagawa E, Suzuki R, Sekikawa J, Morishita T et al. Studies on the constituents of the plants of Illicium species. III. Structure elucidation of novel phytoquinoids, illicinones and illifu- nones from Illicium tashiroi Maxim and I. arborescens Hayata. Chem Pharm Bull 1984; 32: 11±22. 5 Yakushijin K, Tohshima T, Suzuki R, Murata H, Lu ST, Furukawa H.

Letter Studies on the constituents of the plants of Illicium species. II. Struc- tures of phenolic components. Chem Pharm Bull 1983; 31: 2879±83. 6 Itoigawa M, Ito C, Tokuda H, Enjo F, Nishino H, Furukawa H. Cancer chemopreventive activity of phenylpropanoids and phytoquinoids from Illicium plants. Cancer Lett 2004; 214: 165±9. 7 Matsuda H, Tewtrakul S, Morikawa T, Nakamura A, Yoshikawa M. Anti- allergic principles from Thai zedoary: structural requirements of cur- cuminoids for inhibition of degranulation and effect on the release of TNF-a and IL-4 in RBL-2H3 cells. Bioorg Med Chem 2004; 12: 5891±8. 8 Morikawa T, Sun B, Matsuda H, Wu LJ, Harima S, Yoshikawa M. Bioac- tive constituents from Chinese natural medicines. XIV.New glycosides of b-carboline-type alkaloid, neolignan, and phenylpropanoid from Stellaria dichotoma L. var. lanceolata and their antiallergic activities. Chem Pharm Bull (Tokyo) 2004; 52: 1194±9. 9 Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev 1997; 77: 1033±79. 10 Schwartz LB. Mast Cells and basophils. In: Inflammatory Mechanisms in Allergic Diseases, Zweiman B, Schwartz LB, editors. New York: Mar- cel Dekker; 2002: 3±42. 11 Bremner P, Tang S, Birkmayer H, Fiebich BL, Munoz E, Marquez N et al. Phenylpropanoid NF-kB inhibitors from Bupleurum fruticosum. Planta Med 2004; 70: 914±8. 12 Cho MK, Jang YP, Kim YC, Kim SG. Arctigenin, a phenylpropanoid di- benzylbutyrolactone lignan, inhibits MAP kinases and AP-1 activation via potent MKK inhibition: the role in TNF- a inhibition. Int Immuno- pharmacol 2004; 4: 1419±29. 13 Díaz AM, Abad MJ, Fernµndez L, Silvµn AM, De Santos J, Bermejo P. Phenylpropanoid glycosides from Scrophularia scorodonia: in vitro anti-inflammatory activity. Life Sci 2004; 74: 2515±26. 14 Ito C, Itoigawa M, Furukawa H, Ichiishi E, Mukainaka T, Okuda M et al. Anti-tumor-promoting effects of phenylpropanoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis. Cancer Lett 1999; 142: 49±54. 15 Ito C, Itoigawa M, Furukawa H, Rao KS, Enjo F, Bu P et al. Xanthones as inhibitors of Epstein-Barr virus activation. Cancer Lett 1998; 132: 113 ± 7. 16 Ito C, Itoigawa M, Furukawa H, Tokuda H, Okuda Y, Mukainaka T et al. Anti-tumor-promoting effects of 8-substituted 7-methoxycoumarins on Epstein-Barr virus activation assay. Cancer Lett 1999; 138: 87±92. 17 Ito C, Itoigawa M, Tan HT, Tokuda H, Yang Mou X, Mukainaka T et al. Anti-tumor-promoting effects of isoflavonoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis. Cancer Lett 2000; 152: 187±92. 18 Terencio MC, Ferrµndiz ML, Posadas I, Roig E, de Rosa S, De Giulio A et al. Suppression of leukotriene B4 and tumour necrosis factor a release in acute inflammatory responses by novel prenylated hydroquinone derivatives. Naunyn Schmiedebergs Arch Pharmacol 1998; 357: 565 ± 72. 19 Iwata N, Wang N, Yao X, Kitanaka S. Structures and histamine release inhibitory effects of prenylated orcinol derivatives from Rhododendron dauricum. J Nat Prod 2004; 67: 1106±9.

Letter ¼ Planta Med 1

Supporting Information to:

Anti-Inflammatory Activity of Phenylpropanoids and Phytoquinoids from Illicium Species in RBL-2H3 Cells

Takuya Matsui1,2

Chihiro Ito1

Masataka Itoigawa3

Tadashi Okada2

Hiroshi Furukawa1

Affiliation: 1 Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya, Aichi, Japan

2 Department of Physiology, Aichi Medical University, Nagakute-cho, Aichi-gun, Aichi, Japan

3 Faculty of Human Wellness, Tokai Gakuen University, Tempaku, Nagoya, Aichi, Japan

Correspondence: Dr. Takuya Matsui

Department of Medicinal Chemistry, Faculty of Pharmacy

Maijo University

Yagotoyama 150

Tempaku-ku

Nagoya 468-8503

Japan

Tel: +81-52-832-1781

Fax: +81-52-834-8090

E-mail : [email protected]

Plants, Materials and Methods

Plants

Leaves of Illicium tashiroi Maxim. (Japanese name, Yaeyama-shikimi), I. anisatum L.

(Japanese name, Shikimi) and I. arborescens Hayata (Japanese name, Akabana-shikimi)

(Illicaceae) were collected on the Iriomote island (Japan), Mie Prefecture (Japan), and

Taiwan, respectively. Voucher specimens (I. tashiroi Maxim; YAKU021, I. anisatum L;

YAKU022, and I. arborescens Hayata; YAKU026) are preserved in the Faculty of Pharmacy,

Meijo University, Japan. The plant materials of I. tashiroi Maxim and I. anisatum L. were

identified by the late Dr. Hiroyuki Murata. The plant material of I. arborescens Hayata were

identified by Dr. Chang-Sheng Kuoh. The samples were dissolved in dimethyl sulfoxide

(DMSO) and were added to the culture medium to give a final DMSO concentration of 0.1%

v/v. This concentration of DMSO had no significant effect on the growth of the cell line tested

(data not shown).

Materials

A23187, a Ca-ionophore, was purchased from Calbiochem (La Jolla, CA, USA). All other

chemicals were of analytical grade and were obtained from Sigma-Aldrich, Co. (St. Louis,

MO, USA).

Cell stimulation

Cell stimulation was performed with reference to the method described by Yamashita et al.

[1]. Briefly, RBL-2H3 cells were cultured in IMDM containing defined concentrations of the

test compounds for 30 min at 37 °C. The cells treated with the test compounds were

stimulated with 2 µM A23187 for 30 min at 37 °C. Cell viability was evaluated by the MTT

assay [2].

References

1 Yamashita K, Suzuki Y, Matsui T, Yoshimaru T, Yamaki M, Suzuki-Karasaki M et al.

Epigallocatechin gallate inhibits histamine release from rat basophilic leukemia (RBL-2H3)

cells: role of tyrosine phosphorylation pathway. Biochem Biophys Res Commun 2000; 274:

603-8.

2 Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and

rapid dye method for measuring cell growth/cell kill. J Immunol Methods 1989; 119: 203-10.