J. Korean Soc. Appl. Biol. Chem. 52(5), 437-442 (2009) Article

Anti-inflammatory Effect and HPLC Analysis of Extract from Edible setidens

Sung-Hyun Lee†, Mee Jung Jung†, Seong-Il Heo, and Myeon-Hyeon Wang* School of Biotechnology, Kangwon National University, Chuncheon 200-701, Republic of Korea Received December 8, 2008; Accepted August 31, 2009

The anti-inflammatory effect of (C. setidens) was evaluated for its potential to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells. The ethanol (EtOH) extract of C. setidens exhibited strong anti-inflammatory activities in the NO production by LPS-stimulated RAW 264.7 cells. The individual fractions tested were, in order of most-to-least potent in anti-inflammatory activity: n-butanol (n-BuOH)>ethanol (EtOH)>water

(H2O)>ethyl acetate (EtOAc)>dichloromethane (CH2Cl2). The n-BuOH soluble fraction, which exhibited the strongest anti-inflammatory activity, was further purified by repeated MCI gel, silicagel, and RP-18 gel column chromatography. Syringin, isolated from C. setidens roots for the first time, were found to inhibit NO production in LPS-induced RAW 264.7 cells. High performance liquid chromatography (HPLC) was used for the analysis of the syringin in the EtOH extract of Cirsumn species. Key words: anti-imflammatory, Cirsium setidens, MTT assay, nitric oxide, RAW 264.7 cells, syringin

The Cirsium (), all members of the Compositae including C. steidens has attracted a lot of attention as family, have been used in traditional folk medicine as a functional health-maintenance and disease-prevention diuretic, antiphlogistic, hemostatic, and detoxifying agents food. However, the potential anti-inflammatory effects of [Lee, 1966; Kim, 1984]. A great deal of research has been C. setidens roots have not been fully investigated. conducted regarding the phytene-1,2-diol, b-sitosterol, Inflammation is a beneficial host response to foreign petolinarigenin, epilupeol acetate, apigenin, linaroside, challenge or tissue injury that leads to the recovery of siparunoside, notisopreonoids, triterpene hydorperosices, tissue structure and function [Zhou et al., 2007]. acyclic diterpenes, sesquiterpene, monoglalactosydiacy Inflammation is involved in a complex web of intercellular glycerol and sterol glycoside harbored by , and a cytokine signals. Anti-inflammatory compounds have variety of activities were exhibited by these species [Lee investigated the potential inhibitory effects of natural et al., 1994; Perez et al., 2001; Chung et al., 2002; Lee et products in vitro using, lipopolysaccharide (LPS)-stimulated al., 2002; Lee and Lee, 2005; Nazaruk and Jakoniuk, macrophage. 2005]. In the present study, the anti-inflammatory activity of Cirsium setidens Nakai (Compositae), a perennial herb, the ethanol (EtOH) extract of C. setidens, along with its has been used to treat edema, bleeding hemoptysis, and organic solvent soluble fractions such as dichloromethane cancer [Lee et al.,2002]. C. setidens is used as a food and (CH2Cl2), ethyl acetate (EtOAc), n-buthanol (n-BuOH), a traditional fermented vegetable food, and Bibimbap and water (H2O) were evaluated according to nitric oxide (NO) production in LPS-induced RAW 264.7 cells. The †The first two authors are equally contributed to this work. isolation and identification of syringin from the active n- BuOH fraction of C. setidens roots was investigated. *Corresponding author Moreover, we conducted a quantitative analysis of two Phone: +82-33-250-6486; Fax: +82-33-241-6480 Cirsium species, namely C. setidens and C. japonicum, E-mail: [email protected] by analyzing syringin with HPLC-PDA.

Abbreviations: n-BuOH, n-butanol; CH2Cl2, dichloromethane; EtOAc, ethyl acetate; EtOH, ethanol; H2O, water; HPLC, high- Materials and Methods performance liquid chromatography; LPS, lipopolysaccharide; NO, nitric oxide extraction and purification. Roots of Cirsium doi:10.3839/jksabc.2009.076 setidens Nakai were collected in September, 2006 in 438 Sung-Hyun Lee et al.

South Korea. The air-dried and chopped powder of gel (12 nm S-75 µm, YMC Co. LTD., Kyoto, Japan).

C. setidens Nakai (2.2 kg) was extracted with EtOH (18 L TLC was performed on precoated Kiesel gel 60 F254 plate ×3) at 80oC for 3 h. The total filtrate was concentrated and (0.25 µm, Merck, Darmstadt, Germany). dried in vacuo at 40oC to render the EtOH extract (225.7 Cell culture and cell viability assay. RAW 264.7 g). The extract was then suspended in distilled water and murine macrophages were obtained from Korean Cell sequentially partitioned CH2Cl2 (18.5 g), EtOAc (6.1 g), Line Bank (KCLB, Seoul, Korean). These cells were n-BuOH (41.7 g), and H2O (152.5 g). Each extract was cultured in RPMI 1640 containing 10% FBS, penicillin tested for its anti-inflammatory activity in lipopolysaccharide (100 U/mL), and streptomycin (100 µg/mL) in a 95% air, o (LPS)-induced RAW 264.7 cells, and the n-BuOH 5% CO2 humidified atmosphere at 37 C. Cell viability fraction exhibited strong activity. Therefore, n-BuOH was determined by 3-[4,5-dimethylthia-zol-2-yl]-2,5- (41.7 g) fraction loaded on a MCI gel column diphenyl tetrazolium bromide (MTT, Sigma, St. Louis, chromatography and eluted with methanol (MeOH)/H2O MO) assay. RAW 264.7 macrophages were plated at a (0:100, 40:60, 60:40, 100:0%) in a gradient mode to density of 2×105 cells in a 96-well cell culture plate with yield 4 fractions (CM1-4). Fraction CM2 (3.26 g) was 180 µL of culture medium, and incubated for 24 h. The chromatographed on a Sephadex LH-20 column cells incubated with C. setidens EtOH extract and its chromatography, using a solvent of MeOH to yield 7 fractions (1 mg/mL), and syringin (0.5, 1, 5 and 10 µM) fractions (CM2S1-7). Fraction CM2S2 (2.17 g) was for 20 h, and then 20 mL of MTT solution (2 mg/mL) chromatographyed on a silica gel column with was added to each well and incubated 2 h further. The

CH2Cl2:MeOH (10:1→1:1, 0:1, gradient condition) to supernatant was carefully removed using a needle and yield 6 fractions (CM2S21-6). The fraction CM2S25 200 µL of DMSO was added to each well to dissolve (0.66 g) was rechromatographed through the repeated crystals. The absorbance at 550 nm was measured with a silica gel and RP-18 gel column chromatographies. microplate reader (Bioteck Instrucments, Winooski, VT). Finally, a lignan compound 1 (syringin, 113 mg) was Nitrite (NO) assay. RAW264.7 macrophages were isolated. plated at a density of 2×105 cells in a 96-well cell culture Chemical structure of compound 1. Pale yellow- plate with 180 µL of culture medium, and incubated for o 20 white amorphous powder; mp 185.8-186.0 C; []α D −16.9 24 h. The cells were pre-treated with EtOH extract, its (c 0.23, MeOH); LR-FABMS (positive ion mode) m/z soluble solvent fraction, and syringin (0.5, 1, 5 and 10 + 1 395 [M+Na] ; H-NMR (400 MHz, DMSO-d6) δ: 6.75 µM) and stimulated with LPS (2 µg/mL) for 20 h. The (2H, s, H-3,5), 6.54 (1H, brd, J=15.9 Hz, H-7), 6.32 (1H, nitrite concentration in the medium was measured according dt, J=5.6, 15.9 Hz, H-8), 4.85 (1H, s, Glc-1), 4.23 (2H, to the Griess reaction, and the calculated concentration dd, J=1.3, 5.6 Hz, H-9), 3.85 (6H, s, OCH3), 3.77 (1H, was taken as an indicator of NO production. Briefly, 100 dd, J=2.3, 12.0 Hz, Glc-6a), 3.67 (1H, dd, J=5.0, 12.0 µL of each supernatant was mixed with 100 µL of Griess Hz, Glc-6b), 3.48 (1H, ddd, J=2.7, 7.1 Hz, Glc-2), 3.42 reagent (1% sulfanilamide in 5% phosphoric acid and (1H, d, J=2.3 Hz, Glc-4), 3.41 (1H, d, J=2.3 Hz, Glc-5), 0.1% naphthylethylenediamine dihydrochloride in distilled 13 3.20 (1H, m, Glc-3); C-NMR (100 MHz, DMSO-d6) δ: water), and an absorbance of the mixture at 550 nm was 153.3 (C-2, 6), 134.8 (C-1), 134.3 (C-4), 130.2 (C-7), determined with a microplate reader (Bioteck Instrucments, 129.1 (C-8), 104.4 (C-3,5), 104.3 (Glc-1), 77.3 (Glc-3), Winooski, VT). 76.8 (Glc-5), 74.7 (Glc-2), 70.2 (Glc-4), 62.6 (C-9), 61.4 HPLC analysis. The EtOH extacts of C. setidens roots

(Glc-6), 56.0 (OCH3). and Cirsium japonicum roots were analyzed by high General experimental procedures. Polarimeter was performance liquid chromatography (HPLC) using a measured on Perkin-Elmer 341 and FAB-MS was Waters system consisting of an M600E solvent delivery measured on Autospec. M363 series mass spectrometer. pump and M-996 photodiode array detector (PDA). The 1H and 13C-NMR spectra were measured on Bruker DPX system was controlled by Millenium-32 software. The 400 (400 MHz for 1H, 100 MHz for 13C, Karisruhe, column was Apollo C18 (4.6×250 mm; 5 mm particle Germany) spectrometer (Bruker). Chemical shift was size, Alltech Associates Inc, Deerield, IL). Elution was based on the decision of solvent peak (δH 2.50 and δC 39.5 performed in gradient mode with CH3CN/H2O (6:94- for DMSO-d6). DEPT, HMQC and HMBC spectra were 17:83, 0-30 min) at a flow rate of 1.0 mL/min. The C. measured on Bruker DPX 400. To perform column setidens (3 g) and C. japonicum (3 g) roots were extracted chromatography used Si gel [BW-820MH (S), Fuji silysia with 100 mL of EtOH by reflux and evaporated in vacuo. chemical Ltd., Kasugai, Japan], Sephadex LH-20 (25- The residue was dissolved in 50 mL of MeOH and 100 µm, GE Healthcare Bio-Sciences, Uppsala, Sweden), filtered with a 0.45 mm filter. The resulting solution was MCI gel (CHP20P, Supelco, Bellefonte, PA) and RP-18 used HPLC analysis. Isolation of syringin from the Cirsium setidens 439

Fig. 2. Structure of the compound 1 isolated from roots of C. setidens.

were investigated. As summarized in Fig. 1B, the order of

the nitrite-scavenging activity of EtOH, CH2Cl2, EtOAc,

n-BuOH and H2O, from highest to lowest active, was n-

BuOH>EtOH>H2O>EtOAc>CH2Cl2, indicating that the n-BuOH soluble fraction of the EtOH extract from C. setidens is a significant effect on inflammation. The EtOAc

and H2O fractions showed strong scavenging activities, slightly weaker than that of the n-BuOH fraction.

However, the CH2Cl2 soluble fraction showed only weak activity. Isolation and identification of compound from C. setiden. The combination of MCI gel, silica gel, Fig. 1. Effects of EtOH extract of C. setidens root and Sephadex LH-20, and RP-18 column chromgaography its fractions on the cell viability of RAW 264.7 conducted with n-BuOH soluble portions of EtOH macrophages. (A) RAW 264.7 cells were treated with 1 extracts of C. setiden, resulted in the isolation of mg/mL of EtOH extract of C. setidens and its fractions in compound 1 (Fig. 2). the presence or absence of LPS (2 µg/mL) for 20 h. Cell By analysis of the 1H-NMR spectrum for 1, an aromatic viability (2.0×105 cells/mL) was measured after 20 µL of MTT solution (2 mg/mL) was added to each well and proton at δ 6.75 and methylene protons at δ 6.54 and 6.32 incubated 2 h. (B) The RAW 264.7 cells were incubated were observed. The configuration of the glycosidic with EtOH extract of C. setidens root and its fractionations linkage was determined to be β on the basis of the J1,2 (1 mg/mL) in the presence or absence of LPS (2 µg/mL) value of the anometic proton of 7.5 Hz (δ 4.85). The 13C- for the indicated time. NO production was determined by NMR spectrum of 1 showed signals corresponding to an measuring the formation of nitrite. anomeric carbon at δ 104.3, methoxy carbons at δ 56.0, and a typical glucose moiety. The FAB-MS of 1 showed Statistical analysis. Each experiment was performed in an [M+Na]+ ion at m/z 395. On the basis of these results, triplicate. Results are expressed as the mean±standard the structure of 1 was determined the syringin from C. deviation (S.D.). setidens. This compound has been isolated from several , such as Codonopsis tangshen Olive, Acanthopanax Results trifoliatus, and Styrax japonica [Mizutani et al., 1988; Kiem et al., 2003; Min et al., 2004; Kim et al., 2007]. Cytotoxic effects and anti-inflammatory activities of Cytotoxic effects and anti-inflammatory activities of C. setidens. The cytotoxic effects of the C. setidens isolated compound from C. setidens. As summarized in EtOH extract and its soluble fractions on RAW 264.7 cell Fig. 3A, we investigated the effects of the isolated viability in the absence of LPS (2 µg/mL) were determined compound, syringin, on RAW 264.7 cell viability in the using an MTT assay (Fig. 1A). Pretreatment of absence of LPS, using an MTT assay. Pretreatment of unstimulated cells with the EtOH extract and its fractions unstimulated cells with the testing compound, to 50 µM, n-BuOH and H2O fractions did not significantly did not significantly compromise the cell viability. The compromise cell viability. The anti-inflammatory effects inhibitory effect of syringin on NO production was of the EtOH extract and its solvent partitioned fractions assessed by measuring the nitrite in LPS-induced RAW

CH2Cl2, EtOAc, n-BuOH and H2O on LPS-induced cells 264.7 cells (Fig. 3B). The syringin decreased in the NO 440 Sung-Hyun Lee et al.

Fig. 4. HPLC chromatogram of syringin.

Fig. 3. Effects of syringin (0.5, 1, 5 and 10 µM) on the cell viability of RAW 264.7 macrophages. (A) RAW 264.7 cells were treated with syringin in the presence or absence of LPS (2 µg/mL) for 20 h. Cell viability (2.0× 105 cells/mL) was measured after 20 µL of MTT solution (2 mg/mL) was added to each well and incubated 2 h. (B) The RAW264.7 cells were incubated with syringin (0.5, 1, 5 and 10 µM) in the presence or absence of LPS (2 µg/ mL) for the indicated time. NO production was determined by measuring the formation of nitrite.

production in a dose-dependent manner (0.5, 1, 5, 10 µM Fig. 5. (A) HPLC chromatogram of the roots of C. concentrations of syringin), showing nitrite values of setidens. (B) HPLC chromatogram of the roots of C. japonicum. Fig. 1. 39.73±2.32, 37.33±0.46, 32.67±2.41, and 25.47±1.15 µM, respectively. HPLC determination of sytingin from C. setidens Discussion and C. japonicum. The standard curve for syringin is Y= 7224.6X+37.93 (R2=0.999). Syringin was retained for The Genus Cirsium comprises a group of medicinal 20.53 min (Fig. 4). Figure 5A and 5B demonstrate the plants. The and stems of many species are edible, satisfactory resolution for syringin isolated from C. and are used in teas, soups, and salads, among others setidens and C. japonicum extracts, respectively. In the [Park et al., 2006]. Two hundred fifty species of the HPLC profile of the sample solution, the retention time of Genus Cirsium have thus far been identified in the the expected peaks of syringin was the same as that of worldwide [Bureš et al., 2004; Park et al., 2006]. Some standard compounds. These results are supported by the species have been shown to exhibit significant antioxidant, spike test. Table 1 shows the syringin content in the roots antidepressant, antimicrobial and pharmacological activities of the Cirsium species. The syringin contents were [Loizzo et al., 2004; Nazaruk and Jakoniuk, 2005; Park et measured in the roots of C. setidens (0.305 µg/mg) and C. al., 2006; Jeong et al., 2008]. Moreover, the roots of C. japonicum (0.160 µg/mg), resspectively. setidens have been shown to exhibit significant Isolation of syringin from the Cirsium setidens 441

Table 1. Syringin Content of The Roots of Cirsium LPS-induced RAW 264.7 cells. The results demonstrate Species by HPLC that EtOH extract and its isolated syringin compound are Sample Peak area Content (µg/mg) anti-inflammatory substances, and can serve as lead C. Setidens 132116 0.305 extracts and compounds in the development of anti- C. japonicum 69545 0.160 inflammatory foods and drugs. Acknowledgments. This research was financially cytotoxicity and hepatoprotective effects, even though the supported by the Ministry of Commerce, Industry and anti-inflammatory effect in LPS-induced RAW 264.7 Energy (MOCIE) and Korea Industrial Technology cells has not been studied [Lee et al., 2002; Lee et al., Foundation (KOTEF) through the Human Resource 2008]. From the anti-inflammatrory effects of C. setidens, Training Project for Regional Innovation. EtOH extract of C. setidens and its n-BuOH fraction could significantly inhibit NO production by LPS- References stimulated RAW 264.7 cells. The n-BuOH fraction, Bureš P, Wang YF, Horová L, and Suda J (2004) Genome moreover, did not significantly compromise cell viability. side variation in central european species of Cirsium However, both CH2Cl2 and EtOAc fractions exhibited (Compositae) and the irnatural hybrids. Ann Bot 94, 353- weak anti-inflammatory activities in the NO production 363. by LPS-stimulated RAW 264.7 cells. Therefore, the Choi JW, Shin KM, Park HJ, Jung HJ, Kim HJ, Lee YS, active n-BuOH soluble fraction was subjected to further Rew JH, and Lee KT (2004) Anti-inflammatory and chemical analaysis, and after successive column antinociceptive effects of sinapyl alcohol and its gluco- chromatography, one known compound was isolated. side syringin. Planta Med 70, 1027-1032. This compound was readily identified by comparison of Chung AK, Kwon HC, Choi SZ, Min YD, Lee SO, Lee reported spectroscopic data as syringin. WB, Yang MC, Lee KH, Nam JH, Kwak JJ, and Lee Syringin has been isolated and separated from KR (2002) Norisoprenoids from Cirsium rhinoceros. Kor Acanthopanax species using HPLC [Kim et al., 2006]. J Pharmacogn 33, 81-84. Chung HK (1996) TNF a-secretion inhibitor containing However, neither isolation nor HPLC analysis of syringin syringin or its aglcone. Korea, Patent, WO9, 604, 921, from Circium species was reported. In this paper, we Feb22. quantitatively analyzed syringin in two Cirsium species, Hiroshi S and Junzo S (1984) Remedy for psychogenic namely C. setidens and C. japonicum, by HPLC-PDA. asynodia. Japan, Patent, 982, 143, 850, Feb23. The syringin content in C. setidens was found to be Jeong DM, Jung HA, and Choi JS (2008) Comparative anti- significantly higher than that in C. japonicum. Syringin oxidant activity and HPLC profiles of some selected has various pharmacological effects and little toxicity. Korean thistles. Arch Pharm Res 31, 28-33. Various pharmaceutical effects of syringin have been Kiem PV, Minh CV, Dat NT, Cai XF, Lee JJ, and Kim YH reported [Yan et al., 2004]. According to two Japanese (2003) Two new phenylpropanoid glycosides from the patents, syringin is effective for the treatment of psychogenic stem bark of Acanthopanax trifoliatus. Arch Pharm Res behavior disorder, and has a hypnosis-inducing action 26, 1014-1017. Kim HM, Kim JS, Lee SH, Lee SH, Lee SJ, Lee GP, Kang [Hiroshi and Junzo, 1984; Setsuko et al., 2006]. SS, and Cho SH (2006) Quantitative analysis of lignans Pharmaceutical compositions containing syringin have in the fruits of Acanthopanax species by HPLC. Food been found to have hepato-protective activities [Lee et Sci Biotechnol 15, 778-780. al., 2008]. Syringin and its aglycone has a TNF-α- Kim JG (1984) Korean folk medicine. pp. 37, Namsan- secretion-inhibiting effect, an anti-hypersensitivity effect dang, Seoul. as well as anti-inflammatory effects on auto-immune Kim MR, Moon HT, Lee DG, and Woo ER (2007) A new diseases. In addition, histochemical stainging observed lignin glycoside from the stem bark of Styrax japonica severe infiltration of inflammatory cells into ciliary body, S. et Z. Arch Pharm Res 30, 425-430. retina, iris and vitreous of an eye of a normal rat, but Lee HB, Kwak JH, Zee OP, and Yoo SJ (1994) Flavonoids littleinfiltration of inflammatory cells into an eye of a from Cirsium rhinoceros. Arch Pharm Res 17, 273-277. Lee JH and Lee KR (2005) Phytochemical constituents of syringin-treated rat. Similar phenomena were showed in Cirsium nipponicum (MAX.) Makino. Kor J Pharma- the rest of rats, which indicated that syringin suppressed cogn 36, 145-150. effectively the inflammation of rat eyes. [Chung, 1996]. Lee SH, Heo SI, Lee MJ, and Wang MH (2008) Antioxi- EtOH extract of C. setidens and its n-BuOH fraction dant and hepatoprotective activities of Cirsium setidens shows potent inhibition of NO in LPS-induced RAW NAKAI against CCl4-induced liver damage. Am J Chi- 264.7 cells. Syringin [Choi et al., 2004] inhibits NO in nese Med 36, 107-114. 442 Sung-Hyun Lee et al.

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