Anti-Inflammatory Activity of Sugiol, A Diterpene Isolated from ...

Anti-Inflammatory Activity of Sugiol, A DiterpeneIsolated from Calocedrus formosana BarkKuo-Ping Chao 1Kuo-Feng Hua 2Hsien-Yeh Hsu 2Yu-Chang Su 3Shang-Tzen Chang 4Original Paper300AbstractSugiol is a diterpene which was isolated and purified from alcoholextracts of the bark of Calocedrus formosana Florin (Cupressaceae).Although sugiol has low inhibitory activity against theDPPH radical, it could effectively reduce intracellular reactiveoxygen species (ROS) production in lipopolysaccharide (LPS)-stimulated macrophages. The present study investigated the potentialanti-inflammatory activity of sugiol, and the relationshipbetween signal transduction and inflammatory cytokines invitro. A dose of 30 mM of sugiol was effectively inhibitory forproIL-1b, IL-1b and TNF-a production, suggesting that sugiol isbioactive against inflammation. Moreover, sugiol reveals a capacityfor suppressing the activation of mitogen-activated proteinkinases (MAPKs), including extracellular signal-regulated kinase(ERK), c-Jun NH 2 -terminal kinase (JNK), and p38 mitogenactivatedprotein kinase (p38) activated by LPS-stimulation inJ774A.1 murine macrophages. A low dosage of 10 mM of sugiolcompletely inhibited ERK1/2 phosphorylation, while 30 mM effectivelyinhibited JNK1/2 and p38 phosphorylation in LPS-stimulatedmacrophages. In addition, sugiol significantly inhibitedLPS-induced ROS production. Our studies suggest that sugiol'sefficacy in inhibiting the inflammatory cytokines of IL-1b andTNF-a could be attributed to a reduction of the ROSthat leads toa decrease in the phosphorylation of MAPKs.Key wordsCalocedrus formosana ´ Cupressaceae ´ sugiol ´ anti-inflammatoryactivity ´ TNF-a ´ IL-1b ´ MAPKsAbbreviationsLPS: lipopolysaccharideROS: reactive oxygen speciesMAPK: mitogen-activated protein kinaseERK: extracellular signal-regulated kinaseJNK: C-Jun NH 2 -terminal protein kinasep38: p38 mitogen-activated protein kinaseIntroductionCalocedrus formosana Florin (Cupressaceae) is an endemic treethat grows at elevations of 800 ±1500 meters in Taiwan's centralmountains. Sugiol is abundant in the bark of C. formosana, but inthe forestry industry the bark has always been discarded. Manystudies have investigated the bioactivity of sugiol, including astrong antitumor activity on EBV-EA induction [1], and weak antimicrobialand aldose reductase (AR) inhibitory activities [2],[3]. Many natural products exhibiting a good anti-inflammationAffiliation1 Department of Biological Science and Technology, Chung Hwa College of Medical Technology, Tainan, Taiwan2 Faculty of Medical Technology, Institute of Biotechnology in Medicine, National Yang-Ming University, Taipei,Taiwan3 Division of Wood Cellulose, Taiwan Forestry Research Institute, Taipei, Taiwan4 School of Forestry and Resource Conservation, National Taiwan University, Taipei, TaiwanCorrespondenceDr. Shang-Tzen Chang ´ School of Forestry and Resource Conservation ´ National Taiwan University ´ No. 1,Section 4 Roosevelt Road ´ Taipei 106 ´ Taiwan ´ Republic of China ´ Phone: +886-2-236-30231-3196 ´Fax: +886-2-236-54520 ´ E-mail: peter@ntu.edu.twReceived June 22, 2004 ´ Accepted November 15, 2004BibliographyPlanta Med 2005; 71: 300±305 ´ Georg Thieme Verlag KG Stuttgart ´ New YorkDOI 10.1055/s-2005-864094ISSN 0032-0943

capacity have been isolated from plants, but there have beenfew discussions about their molecular mechanism. Resultsfrom our preliminary study demonstrated that the n-hexanefractions of alcoholic extracts from C. formosana, fromwhichlarge amounts of sugiol were isolated, exhibited a significantanti-inflammatory capacity (unpublished results). In this study,we have isolated sugiol (Fig. 1) and investigated its biologicalfunctions, including antioxidant and anti-inflammatory activitiesin macrophages.Innate immunity is triggered by pathogen-associated molecularpatterns shared by groups of microbial pathogens, which can berecognized by pattern recognition receptors in host cells [4]. Thelipopolysaccharide (LPS), or endotoxin, represents a well-knownpathogen-associated molecular pattern, localized on Gram-negativebacteria cell walls [5]. LPSactivates macrophages by bindingto toll-like receptor 4 and stimulates the production of inflammatorycytokines, including TNF-a and IL-1b proteins. Whilemediation of inflammation against pathogen infection by TNF-aand IL-1b proteins could be beneficial to the host, overexpressionof these cytokines may cause serious disease, including septicshock. Hence, suppression of TNF-a and IL-1b protein productioncould aid in the treatment of septic shock.It is well known that the mitogen-activated protein kinases(MAPKs) play an important role in regulating cell growth andsurvival, including mitogenic and stress responses. Our recentstudy revealed that LPSstimulated reactive oxygen species(ROS) production, MAPKs activation, and proIL-1b/IL-1b proteinexpression in murine macrophages [6]. This prompted us to investigatewhether sugiol affects LPS-induced signal transductionin the regulation of cytokine production. Herein we present evidencethat sugiol inhibited LPS-induced TNF-a and IL-1b expressionas well as ROSand MAPKs activation in macrophages. This isthe first report to demonstrate that sugiol has an anti-inflammatoryactivity in macrophages.chemicals and solvents used in this study were of the highestquality available.Extraction and purificationThe bark particles of C. formosana (10 kg d. w.) were treated withethanol (95 % v/v, 10 days repeated 3 times) at room temperature,and then the extracts were concentrated to give the alcoholic extracts(AE; ca. 410 g). 205 g of AE were successively extractedwith n-C 6 H 14 ,CH 2 Cl 2 , EtOAc, BuOH and H 2 O, which then yieldedabout 64.8 g of n-C 6 H 14 -soluble fraction. The n-hexane fractionwas applied on the top of a 160 g silica gel column, then elutedwith n-hexane/EtOAc (95/5, 90/10, 85/15, 80/20, 75/25, 70/30,60/40, 40/60, 20/80, 0/100) followed by elution with EtOAc/ethanol(100/0, 90/10, 80/20, 70/30, 60/40, 50/50, 40/60, 20/80, 0/100) to give 22 subfractions. Each eluted volume of subfractionwas 1000 mL, except for the H1 subfraction, which was 4000mL. Each collected subfraction was rotary evaporated at 50 8C todryness, resulting in a white precipitate (678 mg) from subfractionH5. Recrystallization of this precipitate with ethanol gavesugiol which was then purified by semi-preparative HPLC (HitachiL-7100 pump, a UV detector model Jasco UV 975, l = 254 nm)on a Zorbax SIL column (25 cm in length, 4.6 mm i.d., 5.0 mm).The pure sugiol (> 98 %) was obtained at t R = 5.14 min. The separationconditions were as follows: flow rate, 2 mL/min; mobilephase, EtOAc/EtOH = 1/1. The structure of sugiol was confirmedby comparison of its physical and spectral data (optical rotation,EI-MS, 1 H-NMR) with previously reported values [7]. The opticalrotation was determined using a Horiva STA-300 polarimeter.[a] D 25 : + 28.38 (c 1.0, CHCl 3 ). The NMR spectra were recorded on aBruker Avance-300 MHz FT-NMR. 1 H-NMR (CDCl 3 ): d = 0.99 (3H,s, H-18), 1.00 (3H, s, H-19), 1.19 (3H, s, H-20), 1.20 (3H, d, J =7.0Hz, H-16), 1.24 (3H, d, J = 7.0 Hz, H-17), 1.83 (dd, J = 4.5, 13.0 Hz,H-5), 2.18 (dd, J = 13.0, 18.0 Hz, H-6b), 2.62 (1H, dd, J = 4.5, 1.8Hz, H-6a), 3.13 (1H, sept, J = 7.0 Hz, H-15), 6.68 (1H, s, H-11),7.89 (1H, s, H-14). The mass spectra (MS) were obtained on a FinniganMAT-95Smass spectrometer. EI-MS: m/z calcd. forC 20 H 28 O 2 :300; found: 300.Original Paper301Materials and MethodsMaterialsThe samples of C. formosana were collected in September 2003from the Lien Hua-Chin Research Center located in Nantou Countyin central Taiwan. The species was identified by Mr. Yen-RayHsui of the Taiwan Forestry Research Institute, and voucher specimens(reg. #WCCL-04-B006) were deposited at the laboratoryof wood chemistry (School of Forestry and Resource Conservation,National Taiwan University). Murine macrophage J774A.1cells were obtained from ATCC (Rockville, MD); LPS(fromEscherichia coli 0111:B4), SB203580, curcumin (> 95 %), monoclonalanti-MAP kinase, activated (diphosphorylated ERK1/2) antibody,monoclonal anti-JNK kinase, activated (diphosphorylatedJNK) antibody, and monoclonal anti-p38 MAP kinase, activated(diphosphorylated p38) antibody were obtained from Sigma Co.Mouse TNF-a and IL-1b enzyme-linked immunosorbent assay(ELISA) kits were purchased from R & D Systems, Inc. (Minneapolis,MN). Anti-IL-1b polyclonal antibody, anti-ERK1 polyclonalantibody, anti-JNK1 polyclonal antibody, anti-p38 polyclonal antibody,anti-rabbit IgG-HRP, and anti-mouse IgG-HRP were obtainedfrom Santa Cruz Biotechnology (Santa Cruz, CA). The otherCell cultureThe J774A.1 cells were propagated in RPMI 1640 medium supplementedwith 10% heat-inactivated fetal bovine serum and 2 mML-glutamine (Life Technologies, Inc., MD) and cultured in a 37 8C,5% CO 2 incubator. Sugiol was dissolved in dimethyl sulfoxide(DMSO) and the final DMSO concentration was 0.1 % in all culturescontaining this agent; the same amount of vehicle was addedto the control cultures.MTT assay for cell viabilityThe cytotoxicity of sugiol was assessed using the microculturetetrazolium (MTT) assay [8]. After culturing on 96 well platesfor 24 h, the cells were washed twice and incubated with 100 mLof 1 mg/mL of MTT for 2 h at 378C. The medium was discardedand 100 mL lysis buffer were then added. After 30 min incubation,the absorbance was measured at 570 nm using a microplatereader.Measurement of cytokine productionTo investigate the inhibitory effect of sugiol on TNF-a and proIL-1b/IL-1b protein production from LPS-stimulated J774A.1 cells,the cells were pretreated with sugiol (5 to 30 mM) for 30 min atChao K-P et al. Anti-Inflammatory Activity of ¼ Planta Med 2005; 71: 300 ±305