Food Chemistry 136 (2013) 1047–1054

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Food Chemistry

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Suppression of LPS-induced inflammatory activities by Rosmarinus officinalis L.

Mi-Hee Yu a,1, Jun-Hyeok Choi a,1, In-Gyeong Chae a, Hyo-Gwon Im a,b, Seun-Ah Yang c, Kunal More d,In-Seon Lee a, Jinho Lee d,⇑a Department of Food Science and Technology, Keimyung University, Daegu 704-701, Republic of Koreab Daegu Thechno Park Bio Industry Center, Daegu 704-801, Republic of Koreac The Center for Traditional Microorganism Resources, Keimyung University, Daegu 704-701, Republic of Koread Department of Chemistry, Keimyung University, Daegu 704-701, Republic of Korea

a r t i c l e i n f o

Article history:Received 27 April 2012Received in revised form 18 July 2012Accepted 29 August 2012Available online 12 September 2012

Keywords:Rosmarinus officinalis L.RosemaryAnti-inflammationInducible nitric oxide synthase (iNOS)Cyclooxygenase-2 (COX-2)Macrophage RAW 264.7 cells40-Methoxytectochrysin

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.08.085

⇑ Corresponding author. Address: Department of Csity, 1000 Sindang-Dong, Dalseo-Gu, Daegu 704-701, R580 5183; fax: +82 53 580 5056.

E-mail address: jinho@kmu.ac.kr (J. Lee).1 These authors contributed equally to this work.

a b s t r a c t

Rosemary (Rosmarinus officinalis L.) has been used in folk medicine to treat headaches, epilepsy, poor cir-culation, and many other ailments. It was found that rosemary could act as a stimulant and mild analge-sic and could reduce inflammation. However, the mechanisms underlying the anti-inflammatory effectsof rosemary need more study to be established. Therefore, in this study, the effects of rosemary on theactivation of nuclear factor kappa beta (NF-kB) and mitogen-activated protein kinases (MAPKs), theexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the productionof nitric oxide (NO), prostaglandin E2 (PGE2), and cytokine in lipopolysaccharide (LPS)-stimulated RAW264.7 cells were investigated. A methanol extract of rosemary and its hexane fraction reduced NO gen-eration with an IC50 of 2.75 and 2.83 lg/ml, respectively. Also, the methanol extract and the hexane frac-tion inhibited LPS-induced MAPKs and NF-kB activation associated with the inhibition of iNOS or COX-2expression. LPS-induced production of PGE2 and tumour necrosis factor-alpha (TNF-a) were blocked byrosemary. Rosemary extract and its hexane fraction are important for the prevention of phosphorylationof MAPKs, thereby blocking NF-kB activation, which in turn leads to decreased expression of iNOS andCOX-2, thus preventing inflammation.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Inflammation is a complex process regulated by a cascade ofcytokines, growth factors, nitric oxide (NO) and prostaglandinsproduced by activated macrophages. Activated macrophages playan important role in inflammatory diseases via production of cyto-kines, tumour necrosis factor-alpha (TNF-a) and other inflamma-tory mediators such as NO and prostaglandin E2 (PGE2). Excessproduction of inflammatory mediators is involved in many dis-eases including rheumatoid arthritis, atherosclerosis, asthma andpulmonary fibrosis. Thus, agents that down-regulate these pro-inflammatory mediators would be beneficial in the treatment ofinflammation (Alderton, Cooper, & Knowles, 2001; Kawano et al.,2006; Pearson et al., 2001; Tracey & Cerami, 1993; Uto, Fujii, &Hou, 2005; Wang & Mazza, 2002; Xie & Nathan, 1994).

Rosmarinus officinalis L., commonly called Rosemary, is a woodyperennial herb spice that belongs to the Family Labiatae (Inatani,Nakatani, & Fuwa, 1983) with fragrant evergreen needle-like

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hemistry, Keimyung Univer-epublic of Korea. Tel.: +82 53

leaves, which are often used in cooking. It is native to the Mediter-ranean region and is now widely spread in European countries. Ithas been known to act as a stimulant and mild analgesic, andhas been used in folk medicine to treat headaches, epilepsy, poorcirculation, and many ailments (Uto et al., 2005).

Leaves of rosemary have shown a variety of bioactivities such asantioxidation (Erkan, Ayranci, & Ayranci, 2008; Richheimer,Bernart, King, Kent, & Beiley, 1996; Wada et al., 2004), antitumour(Singletary, MacDonald, & Wallig, 1996), anti-HIV (Aruoma et al.,1996), and anti-inflammation (Kuo et al., 2011). The relevant mainconstituents are polyphenolics including carnosic acid, carnosol,rosemarinic acid and ursolic acid, etc. Carnosol and carnosic acid,major phenolic diterpenoid components of rosemary, have beenstated to inhibit nitric oxide (NO) production in LPS-activated mac-rophages. The inhibitory effects of carnosic acid on LPS-induced NOand TNF-a production are caused by the suppression of iNOS andCOX-2 expression (Kuo et al., 2011). In addition, it blocks LPS-induced nuclear translocation of NF-jB p65 by the suppressionof IjBa phosphorylation and degradation. Carnosol decreasesLPS-induced iNOS mRNA and down-regulates the inhibitor NF-jB(IjBa) kinase (IKK) activity on the mouse macrophage RAW264.7 cell line. Also, it blocks the LPS-caused activation of p38and p44/42 MAP kinase(Lo, Liang, Lin-Shiau, Ho, & Lin, 2002).However, antioxidative and anti-inflammatory activity of either

1048 M.-H. Yu et al. / Food Chemistry 136 (2013) 1047–1054

carnosic acid or carnosol alone is weaker than rosemary extract(Benincá, Dalmarco, Pizzolatti, & Fröde, 2011; Cuvelier, Richard, &Berset, 1996). The discrepancy was assumed as synergistic interac-tions of carnosic acid, carnosol and other phenolic compounds inrosemary extract (Cuvelier et al., 1996; Kuo et al., 2011). We triedto find a new component which contributes to the antioxidativeand anti-inflammatory effect of rosemary extract. Extraction, frac-tionation, activity monitoring, purification and structure identifica-tion were performed. The effects of rosemary on the activation ofnuclear factor kappa beta (NF-jB) and MAPKs, the expression ofiNOS and COX-2, and the production of NO, PGE2, and cytokine inLPS-challenged RAW 264.7 murine macrophages were used foractivity evaluation.

2. Materials and methods

2.1. Chemicals and reagents

All chemicals were obtained from Sigma and Aldrich Chemical(St. Louis, MO, USA) unless otherwise indicated. Carnosol was pro-vided by Cayman Chemical Company (Ann Arbor, MI, USA). Cellculture reagents were purchased from Gibco BRL (Rockville, Md.,USA) and fetal bovine serum (FBS) was from Hyclone (Logan, Utah,USA). Anti-NF-kB p65, iNOS, COX-2, MAPKs, monoclonal or poly-clonal antibodies were purchased from Cell Signaling TechnologyInc. (Danvers, MA, USA). RAW 264.7 murine macrophage cells wereobtained from the Korea Cell Line Bank (Seoul, Korea).

2.2. Preparation of methanol extracts and their fractions of rosemary

The leaves of rosemary were purchased from Yangyeongsi herbmedicine market, Dae-gu, South Korea. Air-dried herb was ex-tracted with ten-fold volume of 80% methanol in water at roomtemperature for 24 h, and the extraction was repeated three times.The extracts were collected and concentrated under reduced pres-sure at 55 �C. The concentrate was suspended to water and was ex-tracted with equal volumes of n-hexane, chloroform, ethyl acetateand n-butanol, successively. After the solvent was removed underreduced pressure, each fraction was lyophilized to give powder(Fig. 1).

2.3. Isolation of active components from n-hexane fractions

The n-hexane fraction was further fractionated using silica gelcolumn chromatography. Three types of solvent mixtures wereused as eluents for purification of active components. Eluents were

100 g of chopped samples

Extract for 24 hr ×3 with 1 L of 80% MeOH

Evaporate under reduced pressure at 55℃

Freeze Drying

Powdered MeOH extract

Filter with Wattman No. 1 & 3 filter paper

Fig. 1. Fractionation procedure of methanol extracts fr

n-hexnane:ethyl acetate(3:1), n-hexane:dichloromethane:diethylether(3:1:1), and n-hexane:ether(2:1). Two biological assays, DPPHradical and NO inhibition, were used to evaluate the isolated frac-tions for the purification of a single component.

2.4. Identification of active components

The isolated active components were identified by comparisonof HPLC, 1H NMR, 13C NMR, and MS data with authentic com-pounds. The liquid chromatography (HPLC) profile was obtainedusing Waters HPLC 2690. 1H NMR and 13C NMR spectra were re-corded on a Bruker AVANEC 400 (400 MHz) spectrometer andchemical shifts (d) are reported in ppm using tetramethylsilaneas an internal standard. Silica gel column chromatography wasperformed using Merck silica gel 60 (230–400 mesh). Mass spec-trum was obtained using the Waters Micromass Quattro microAPI Mass spectrometer coupled with Waters ACQUITY UPLCsystem.

2.5. Scavenging of a-a-diphenyl-b-picrylhydrazyl (DPPH) radical

The free radical scavenging activity of the samples was mea-sured by 2,2-diphenyl-1-picrylhydrazyl (DPPH). In its radical form,DPPH� absorbs at 517 nm, but upon reduction by antioxidant or aradical species, its absorption decreases. Briefly, a 0.15 mM solu-tion of DPPH� in ethanol was prepared, and 200 ll of this solutionwas added to 800 ll of sample solution in ethanol at differentconcentrations. After 30 min, the absorbance was measured at517 nm. Lower absorbance of the reaction mixture indicates higherfree radical scavenging activity. The free radical scavenging activityof each solution was then calculated as percent inhibition accord-ing to the following equation:

DPPH � scavenging effect ð%Þ ¼ ððAControl � ASample=AControlÞ � 100Þ

2.6. Cell culture

RAW 264.7 murine macrophage cells were cultured in DMEMsupplemented with 10% FBS containing 100 U/ml of penicillinand 100 lg/ml of streptomycin at 37 �C in a 5% CO2 humidifiedincubator.

Aqueous Phase

CHCl3 fraction Aqueous Phase

EtOAc fraction Aqueous Phase

Water fraction

Powdered MeOH extract in D.W.

Hexane fraction

Partition with Hexane

Partition with CHCl3

Partition with EtOAc

BuOH fraction

Partition with BuOH

om Rosmarinus officinalis L. by solvent separation.

M.-H. Yu et al. / Food Chemistry 136 (2013) 1047–1054 1049

2.7. Cell viability

Cell viability was assessed using a modified MTT assay. Briefly,cells (1 � 105 cells/well) were seeded in a 96-well plate and treatedwith Rosemary. Following treatment, 10 ll of a MTT solution(5 mg/ml in phosphate buffered saline) was added to each welland further incubated for 4 h at 37 �C. Subsequently, 100 ll of di-methyl sulfoxide (DMSO) was added to each well to solubilizeany deposited formazon. The optical density (OD) of each wellwas measured at 550 nm with a microplate reader (Molecular De-vices, Spectra max 340PC, USA).

2.8. Quantification of NO production

NO concentration in the cultured medium was determined viathe Griess reaction. Specifically, 100 ll of supernatant from eachwell was mixed with 100 ll of Griess reagent (1% sulfanilamide,0.1% naphthylethylendiamine in 2.5% phosphoric acid) in a sepa-rate 96-well plate. After an incubation of 10 min at room temper-ature, the optical density was determined at 540 nm with amicroplate reader.

2.9. Gelatin zymography assay

Conditioned medium was electrophoresed in a polyacrylamidegel containing 0.1% (w/v) gelatin. The gel was then washed at roomtemperature for 30 min with 2.5% Triton X-100 and subsequentlyincubated at 37 �C for 24 h in a buffer containing 10 mM CaCl2,0.01% NaM3 and 50 mM tris–HCl (pH 7.5). The gel was stained with0.2% Coomassie brilliant blue and photographed on a light box.Proteolysis was detected as a white zone in a dark blue field.

2.10. Prostaglandin E2 (PGE2) analysis

Prostaglandin E2 (PGE2) levels were measured using an immu-noenzymatic method (Cayman Chemicals, San Diego, CA, USA)according to the manufacturer’s specifications. Briefly, RAW264.7 macrophages (1 � 105 cells/well) were incubated with LPS(100 ng/ml) in the absence or presence of extracts and fractionsof R. officinalis L. (2.5, 5, 10 lg/ml) for 24 h. The PGE2 level in thesupernatants (50 ll) was estimated using a specific enzyme immu-noassay kit.

2.11. Tumour necrosis factor-alpha (TNF-a) analysis

The levels of TNF-a in the supernatants and in the mediumwere assessed with commercially available ELISA kits (invitrogencorporation, Carlsbad, CA, USA), according to the manufacturer’sinstructions. The ELISA assay was performed in duplicate.

2.12. Western blot analysis for iNOS, COX-2, NF-kB, and MAPKs

RAW 264.7 cells were plated at a density of 3 � 106 cells/ml in a6-well cell culture plate with 2 ml of culture medium and incu-bated for 24 h. The cells were pre-treated with rosemary for 1 hand stimulated with LPS (100 ng/ml) for specified time periods.Cells were harvested by scraping the cells from cultured dishesusing a cell scraper and were collected. Cellular lysates were pre-pared in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 2 mMEDTA, 150 mM NaCl, 0.5% deoxycholate, 0.1% sodium dodecylsul-fate (SDS), 1 mM NaF, 1 mM Na3VO4, 1 mM phenyl methyl sulfonylfluoride (PMSF), 1 mM dithiothreitol (DTT), 1 lg/ml leupeptin,1 lg/ml aprotinin, and 1% NP-40. The cells were disrupted and ex-tracted at 4 �C for 30 min. After centrifugation at 13000 rpm for15 min, the supernatant was obtained as the cell lysate. Proteinconcentrations were measured using a Bio-Rad protein kit. Ali-

quots of cellular proteins (10 lg/lane) were electrophoresed on10% SDS–polyacrylamide gel electrophoresis (PAGE) and trans-ferred to an Immobilon-P-membrane (Millipore, USA). The mem-brane was allowed to react with a specific antibody anddetection of specific proteins was carried out by enhanced chemi-luminescence following the manufacturer’s instructions. Loadingdifferences were normalized using a polyclonal anti-b-actinantibody.

2.13. HPLC analysis of rosemary extracts, carnosic acid, and carnosol

For HPLC analysis of methanol extract and hexane extract andquantification of carnosic acid (CA) and carnosol (CAR), a C18 re-versed-phase Xbridge analytical column (Waters Xbridge C18,5 lm � 150 mm � 4.6 mm) was used. Two mobile phases wereused in the current study: solvent A, water; and solvent B, 0.1%TFA in acetonitrile. The gradient for HPLC analysis was linearlychanged as follows (total 40 min): 30% B at 0 min, 40% B at3 min, 50% B at 5 min, 53% B at 7 min, 58% B at 10 min, 62% B at14 min, 68% B at 19 min, 75% B at 24 min, 100% B at 30 min. Theflow rate was set to 1.0 ml/min at constant temperature (25 �C).The detector was set at 254 nm. CA and CAR standards were madefor the calibration curves. Individual CA and CAR in the extractswere tentatively identified by comparison of their retention times.

2.14. Synthesis of 40-methoxytectochrysin

40-Methoxytectochrysin was synthesized according toZembower et al. (Zembower & Zhang, 1998) procedure with somemodifications.

2.14.1. 4,6-Dimethoxy-2-(40-methoxybenzoyloxy)acetophenone (1)4-Methoxybenzoyl chloride (414 ll, 3.05 mmol) was added to a

solution of 20-hydroxy-40,60-dimethoxyacetophenone (500 mg,2.54 mmol) in pyridine (3 ml), and the solution was heated to100 �C for 10 min. The solution was cooled to room temperatureand then diluted with ethyl acetate (30 ml). The solution waswashed with 1 N HCl (10 ml), then the aqueous phase was re-extracted with EtOAc (20 ml), and the combined organic layerswere washed with saturated NaCl (10 ml) and then dried overmagnesium sulfate, filtered, and concentrated. The product waspurified by flash chromatograpy to obtain 838 mg in 99% yield.

1H NMR (CDCl3, 400 MHz) d 2.7 (s, 3 H), 3.82 (s, 3 H), 3.86 (s, 3H), 3.88 (s, 3 H), 6.36 (d, 1 H, J = 2.4 Hz), 6.39 (d, 1 H, J = 2.4 Hz),6.96 (d, 2 H, J = 8.8 Hz), 8.09 (d, 2 H, J = 8.8 Hz).

2.14.2. 1-(40-Methoxyphenyl)-3-(20 0-hydroxy-30 0,60 0-dimethoxyphenyl)-1,3-propanedione (2)

To a solution of compound (1) (100 mg, 3.02 mmol) in dry THF(3 ml), lithium hexamethyldisilylazide (1.0 M solution in THF)(596 ll, 6.05 mmol) was added dropwise at �40 �C. After stirringfor 1 h, the temperature of the reaction mixture was slowly in-creased to 0 �C for 0.5 h. The reaction mixture was further stirredat RT for 5 h. The reaction was quenched with 5% citric acid solu-tion (10 ml) and the product was extracted with ethyl acetate(30 ml). The aqueous phase was re-extracted with ethyl acetate(20 ml). The organic layers were combined and washed with brine(10 ml). The organic layer was dried over magnesium sulfate andconcentrated under vacuum to give the titled compound quantita-tively. It was used for the next step without further purification.

2.14.3. 5,7,40-Trimethoxyflavone (3)To a solution of compound (2) (109 mg, 0.329 mmol) in 1 ml of

glacial acetic acid was added 2 ml of 20% H2SO4 in acetic acid at100 �C. The reaction mixture was stirred at 100 �C for 10 min.The reaction was quenched with saturated Na2CO3 solution

1050 M.-H. Yu et al. / Food Chemistry 136 (2013) 1047–1054

(5 ml). The product was extracted with ethyl acetate (20 ml) twice.The combined organic layers were washed with brine (10 ml),dried over magnesium sulfate, filtered, and evaporated to afford90 mg of the titled compound in 87% yield.

1H NMR (CDCl3, 400 MHz) d 3.86 (s, 3 H), 3.89 (s, 3 H), 3.92 (s, 3H), 6.33 (d, 1 H, J = 2.4 Hz), 6.52 (d, 1 H, J = 2.4 Hz), 6.59 (s, 1 H),6.97 (d, 2 H, J = 8.8 Hz), 7.78 (d, 2H, J = 8.8 Hz).

2.14.4. 7,40-Dimethoxy-5-hydroxyflavone (40-methoxytectochrysin)A 1 M solution of BBr3 (431 ll, 0.432 mmol) was added to a

solution of compound (3) (90.0 mg, 0.288 mmol) in anhydrousCH2Cl2 (5 ml) dropwise over 15 min at room temperature with stir-ring. The reaction mixture was further stirred at RT for 5 h. Thereaction was quenched by adding ethanol (10 ml). The solventwas removed in vacuo. The yellow residue was purified by flashchromatography to obtain 37 mg of the titled compound in 43%yield.

1H NMR (CDCl3, 400 MHz) d 3.87 (s, 3 H), 3.89 (s, 3 H), 6.36 (d, 1H, J = 2.4 Hz), 6.47 (d, 1 H, J = 2.4 Hz), 6.57 (s, 1 H), 7.00 (d, 2 H,J = 9.2 Hz), 7.83 (d, 2 H, J = 9.2 Hz), 12.81 (s, 1 H); EI-MS m/z 331(M+1).

2.15. Statistical analysis

All experiments were done at least in triplicate. Data were ex-pressed as the mean ± S.D. and differences between means fortwo groups were determined by unpaired Student’s t-test. Theminimum significance level was set at a P value of <0.05 for allanalyses.

3. Results

3.1. Effects of rosemary extract on cell viability and LPS-induced NOproductions

To investigate the effect of rosemary extracts on cell viability,methanol extract of rosemary was dissolved in water and re-extracted with several solvents sequentially. RAW 264.7 cell wastreated with methanol extracts and other solvent extracts in thepresence of LPS (100 ng/ml) for 24 h. The cytotoxicity of rosemaryextracts was measured by MTT assay. The methanol extracts andother solvent extracts showed higher than 80% cell viability exceptbutanol and water fractions at a concentration of 10 lg/ml(Table 1). Carnosic acid and carnosol, the active ingredients ofrosemary, showed higher than 90% cell viability up to 10 lM.

The inhibitory effect of the methanol extracts and other solventextracts on LPS-induced NO production was measured using Griessreagent. As shown in Table 1, methanol extract and hexane fractioninhibited NO production up to 88.31% and 94.61% at 10 lg/ml,respectively. However, chloroform, ethyl acetate, n-butanol andwater fraction were not as effective as methanol extract and hex-ane fraction.

3.2. Effects of methanol extract (Rosemary M) and hexane extract(Rosemary H) on LPS-induced iNOS, COX-2 expression and PGE2, TNF-a production

Since LPS-induced RAW 264.7 cells have been used to modelmacrophage-mediated inflammatory events in vitro, the effects ofRosemary M and Rosemary H on inflammatory cytokine and pro-tein levels were examined. As shown in Fig. 2A, Rosemary M signif-icantly decreased the levels of iNOS protein in LPS-stimulated cellsin a concentration-dependent manner, but did not affect COX-2protein levels. Rosemary H, on the other hand, did not affect iNOS

protein expression, but reduced the level of COX-2 protein slightlywhen the concentration was over 5 lg/ml.

The inhibitory effects of Rosemary M and Rosemary H on LPS-induced PGE2 and TNF-a productions in RAW 264.7 cells are pre-sented in Fig. 2B and C. The LPS-stimulated production of PGE2

was considerably reduced by treatment with Rosemary M at a con-centration of 10 lg/ml. However, Rosemary H suppressed the levelmore strongly in a concentration-dependent manner (Fig. 2B).

LPS stimulation caused a substantial production of TNF-a. Thepre-treatment with Rosemary M did not affect the TNF-a produc-tion. However, Rosemary H markedly inhibited the LPS-inducedTNF-a production at a concentration of 10 lg/ml (Fig. 2C).

3.3. Effects of Rosemary M and Rosemary H on LPS-induced NF-jB andMAPK activations

NF-jB is a central transcription factor that regulates the expres-sion of a large number of inflammatory-related genes. Many ofthese genes have additional cis-regulatory elements controlled byMAPKs-activated transcription factors. Therefore, the effect ofRosemary M and Rosemary H on the LPS-stimulated nucleartranslocation of the p65 subunits were examined by Western blotanalysis (Fig. 3A). Both Rosemary M and Rosemary H inhibitedLPS-induced nuclear translocations of p65.

All three members of the MAPKs were phosphorylated at30 min after LPS treatment (Fig. 3B). Rosemary M inhibited thephosphorylation of ERK1/2 and JNK at a concentration of 10 lg/ml. On the other hand, the phosphorylation of ERK1/2 and p38MAPK were blocked by Rosemary H in a dose-dependent manner.These results suggest that the regulations of LPS-induced iNOS andCOX-2 expression by Rosemary M and Rosemary H are related tothe inhibition of ERK1/2, p38 MAPK and JNK signaling pathways.

3.4. Effects of CA and CAR on LPS-induced NO and PGE2 production

The effects of CA and CAR on LPS-induced production of NO andPGE2 were studied. As shown in Fig. 4A, the LPS-stimulated accu-mulation of NO in the culture medium was suppressed by pretreat-ment with CA (P < 0.05 at 5 lM, P < 0.01 at 10 lM) and CAR(P < 0.001 at 1–10 lM) in a concentration-dependent manner.The LPS-stimulated production of PGE2 was reduced by about70% after treatment with a 10 lM concentration of CA, but it wasnot affected by CAR up to 10 lM concentration (Fig. 4B).

3.5. Quantification of CA and CAR in Rosemary M and Rosemary H

The amounts of CA and CAR in Rosemary M and Rosemary Hwere quantified by HPLC based on calibration curves (data notshown). The amount of CA was higher than that of CAR in bothRosemary M and Rosemary H (Table 2). The amount of CA in Rose-mary H (184 lg/mg) was almost 2 times higher than in RosemaryM (94 lg/mg), whereas the amounts of CAR in Rosemary M (3.7%)and Rosemary H (2.5%) were similar.

3.6. Fractionation and identification of active components in RosemaryH

Hexane fractions were further fractionated by flash columnchromatography using a hexane:ethyl acetate (3:1) mixture as aneluent (Fig. 5). Among 9 fractions, fraction 6, 7 and 8 showed bothan antioxidative effect and an inhibition of NO production (datanot shown). Active components in fraction 8 were not stable en-ough to be purified and identified. Active compounds in fraction6 were purified and identified by comparing HPLC, MS, 1H NMRand 13C NMR spectra with authentic compounds. One was

Table 1Effects of methanol extract from Rosmarinus officinalis L. and its fractions on cell viability and LPS-induced NO production in RAW 264.7 cells.

Rosmarinus officinalis L. Cell viability (%) NO inhibition rate (%)a

5 lg/ml 10 lg/ml 5 lg/ml 10 lg/ml

MeOH extract 93.57 ± 1.38 86.16 ± 1.64 55.09 ± 15.18 88.31 ± 1.98Hexane fraction 92.33 ± 2.36 91.69 ± 6.65 74.12 ± 16.03 94.61 ± 2.56CHCl3 fraction 79.42 ± 0.49 84.36 ± 4.34 16.64 ± 15.37 33.51 ± 1.31EtOAc fraction 84.80 ± 2.39 84.88 ± 2.55 17.58 ± 3.12 27.38 ± 2.99BuOH fraction 79.89 ± 1.52 79.20 ± 0.85 1.05 ± 1.35 10.39 ± 2.17Water fraction 78.81 ± 0.44 79.81 ± 1.32 2.54 ± 4.82 12.59 ± 3.53Carnosic acidb 98.93 ± 9.84 90.51 ± 9.23 92.50 ± 2.57 99.55 ± 0.11Carnosolb 98.62 ± 1.74 91.60 ± 9.86 92.46 ± 0.14 93.79 ± 0.12

Data represent the mean ± SEM of three independent experiments (n = 3).a The culture supernatants were isolated and analysed for nitrite levels.b The concentration unit was lM.

Fig. 2. Effects of methanol extract (Rosemary M) and hexane fraction (Rosemary H) from rosemary on LPS-induced iNOS, COX-2 expressions and PGE2, TNF-a production inRAW 264.7 cells. Cells were pretreated with various concentrations of methanol extract (Rosemary M) or hexane fraction (Rosemary H) from rosemary for 1 h and induced byLPS (100 ng/ml) for an additional 23 h. Total cell lysates (20 lg) were examined for iNOS and COX-2 protein expressions by Western blotting. b-Actin was used as an internalcontrol (A). The conditioned supernatants were prepared and cytokine level was evaluated by enzyme-linked immunosorbent assay for PGE2 (B), TNF-a (C). Results werepresented as means ± S.D. in triplicate. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 vs LPS alone.

M.-H. Yu et al. / Food Chemistry 136 (2013) 1047–1054 1051

identified as carnosol and the other was 4’-methoxytechtochrysin(Supplementary data).

3.7. Antioxidative activity of 40-methoxytechtochrysin

The antioxidative activity of 40-methoxytechtochrysin wasstudied by DPPH radical scavenging ability and compared with that

of carnosol (data not shown). 40-Methoxytechtochrysin showed anantioxidative effect similar to carnosol (97.5% vs. 95.0%. respec-tively). A combination of 4’-methoxytechtochrysin and carnosolin different compositions showed a slight synergistic effect. 10%,20%, 40%, and 80% 40-methoxytechtochrysin mixtures showedbetter antioxidative effects compared to carnosol or 40-methyoxytechtochryin alone (Table 3.).

Fig. 3. Effects of methanol extract (Rosemary M) and hexane fraction (Rosemary H) from rosemary on LPS-induced NF-kB and MAPK activations in RAW 264.7 cells. Cellswere pretreated with various concentrations of Rosemary M or Rosemary H for 1 h and induced by LPS (100 ng/ml) for an additional 30 min. Nuclear extracts were preparedand examined for NF-kB expression by Western blotting. b-Actin was used as an internal control (A). The phosphorylation levels of ERK, JNK and p38 MAPK were measured byWestern blotting (B).

Fig. 4. Effects of carnosic acid (CA) and carnosol (CAR) (A) on LPS-induced nitric oxide and PGE2 production in RAW 264.7 cells. Cells were pretreated with variousconcentrations of CA and CAR for 1 h and induced by LPS (100 ng/ml) for an additional 23 h. The conditioned supernatants were prepared and NO (A) and PGE2 (B) productionwere evaluated. Results were presented as means ± S.D. in triplicate. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 vs LPS alone.

1052 M.-H. Yu et al. / Food Chemistry 136 (2013) 1047–1054

4. Discussion

It has been known that iNOS and COX-2, responsible for synthe-sizing NO and PGE2, respectively, are important enzymes thatmediate inflammatory processes. Improper up-regulation of iNOS

and COX-2 has been related to certain types of inflammatory disor-ders and human diseases. iNOS is up-regulated mainly by NF-kBactivation in macrophages with LPS-stimulation (Xie, Kashiwabara,& Nathan, 1994). Since NF-kB plays the most critical role in theinduction of iNOS by LPS, the suppression of NF-kB activation

Table 2CA and CAR contents in RM and RH.

RT (min) Contents (lg/mg)

Carnosic acid 16.052 –RM 16.060 93.85RH 15.979 184.00

Carnosol 14.142 –RM 14.236 37.30RH 14.149 25.05

Hexane fraction (Rosemary H)

F1 F’ F4 F5 F6 F7 F8 F9

F2 F3

F61 F62

F81 F82

(a)

(b) (c) (d)

Fig. 5. Scheme for fractionation of Rosemary Hexane fraction. Eluting solventmixtures: (a) hexane:ethyl acetate (3:1), (b) hexane:ethyl acetate (7:1), (c)hexane:dichloromethane:diethyl ether (3:1:1), (d) hexane:diethyl ether (2:1).

Table 3Synergistic effects of 40-methoxytechtochrysin and carnosol on LPS-induced NOproduction in RAW 264.7 cells.

Carnosol–40-methoxytechtochrysin (%) NO inhibition rate (%)A

5 lg/ml

0–100 94.99 ± 0.22d

10–90 86.58 ± 1.69g

20–80 101.25 ± 15.37b

30–70 98.55 ± 0.18c

40–60 90.22 ± 0.90f

50–50 93.84 ± 1.31de

60–40 104.26 ± 0.76a

70–30 93.25 ± 0.65e

80–20 100.75 ± 0.43b

90–10 105.21 ± 0.66a

100–0 97.50 ± 0.47c

Data represent the mean ± SEM of three independent experiments (n = 3), statisticalsignificance was calculated (letters that differ indicate, p < 0.05).

A The culture supernatants were isolated and analysed for nitrite levels.

M.-H. Yu et al. / Food Chemistry 136 (2013) 1047–1054 1053

possibly accounts for the inhibitory effect of Rosemary M and H onNO production in RAW 264.7 cells. Even though both rosemary Mand H inhibited the LPS-induced activation of NF-kB in RAW 264.7cells, the inhibitory activity of Rosemary H was much weaker evenat 10 lM concentration. This inhibitory activity difference corre-lates with the result that only Rosemary M reduces the inductionof iNOS significantly.

According to previous research, CAR inhibited the LPS-inducedexpression of iNOS at much lower concentrations than CA (below2.5 lM for CAR and above 10 lM for CA) (Kuo et al., 2011; Loet al., 2002). Rosemary H contains twice the amount of CA andone third less CAR compared to Rosemary M. The concentrationranges of the active components were too low for Rosemary H

(0.8 lM of CAR and 5.6 lM of CA at 10 lg/ml) to show inhibitoryactivity.

The LPS-induced expression of COX-2 was reduced by RosemaryH but not Rosemary M. This result can be accounted for by two rea-sons. One of the reasons is the low concentration of CA in Rose-mary M. According to Kuo et al. (2011), CA reduces COX-2expression between 5 to 10 lM concentrations, and could be ob-tained only from Rosemary H. Second, it might have resulted fromthe inability of Rosemary M to suppress all three MAPK signallingarms, which is prerequisite for complete blockage of COX-2 induc-tion (Hou, Yanagita, Uto, Masuzaki, & Fujii, 2005). Blocking bothp38 MAPK and MEK caused stronger inhibition of COX-2 expres-sion than blocking JNK and MEK. Rosemary M inhibited LPS-induced phosphorylation of ERK and JNK but not p38 MAPK, whileRosemary H inhibited LPS-induced phosphorylation of ERK andp38 MAPK, but not JNK. In this context, the dose-dependent inhibi-tion of PGE2 production by Rosemary H could be explained partlyby the inhibition of COX-2 activity.

However, there is a possibility that the inhibition of expressionof iNOS and COX-2 could be affected by other components such asrosmanol, which was reported to have an effect on iNOS and COX-2expression at low concentrations (Lai et al., 2009).

LPS-induced TNF-a production was inhibited by Rosemary Hbut not Rosemary M. The p38 MAPK regulates LPS-induced produc-tion of proinflammatory cytokines like TNF-a, IL-1b, IL-6 in macro-phage (Beyaert et al., 1996; Foey et al., 1998). This suggests thatRosemary H may inhibit the LPS-induced TNF-a productionthrough the inhibition of the activation of p38 MAPK.

Plant-derived phytochemicals are an important and promisinggroup of potential anti-inflammatory agents because of their lowtoxicity and apparent benefit in acute and chronic diseases. Rose-mary leaf extracts and essential oil showed antioxidant, antimicro-bial and anti-inflammatory effects (Aruoma et al., 1996; Kuo et al.,2011; Moreno, Scheyer, Romano, & Vojnov, 2006). Carnosic acidand carnosol are the most abundant diterpenes in rosemary leaves(del Baño et al., 2003; Moreno et al., 2006). The relative amounts ofcarnosic acid and carnosol were 9.4%, 3.7% in Rosemary M and18.4%, 2.5% in Rosemary H, respectively.

Lo et al. (2002) showed that carnosol reduced the generation ofNO in a concentration-dependent manner. Carnosol reduced NOgeneration with an IC50 of 9.4 lM (about 3.1 lg/ml) as an approx-imation value coming from one of three separate experiments. Car-nosic acid and rosmarinic acid showed less inhibitory effect overthe same concentration range. These data were similar to ourstudy, although our results showed more potency (Fig. 4). Rose-mary M and Rosemary H showed the inhibition of NO generationat lower concentration. Rosemary M showed 55% inhibition at5 lg/ml which contained 1.4 lM of CA and 0.26 lM of CAR.Rosemary H showed 74% inhibition at 5 lg/ml which contained2.8 lM of CA and 0.38 lM of CAR. As previously mentioned, eventhough Rosemary H showed very weak inhibition of iNOS expres-sion, its NO inhibition rate was higher than Rosemary M. Cuvelieret al. (1996) had pointed out that the observed anti-oxidativeeffects of rosemary extracts from various origins did not correlatewell with the amount of known active compounds. These resultssuggest that there are other components that contribute to theanti-oxidative effects of extracts. The fractionation of Rosemary Hbased on DPPH radical scavenging activity and inhibitory activityof NO production resulted in three active compounds. Onecompound was not stable enough to be purified for identifica-tion. The other two compounds were found as carnosol and 40-methoxytectochrysin. The structure of 40-methoxytectochrysinwas confirmed by comparing spectroscopic data with authenticcompound which was synthesized. The antioxidative activity of40-methoxytechtocrysin was reported to be very weak in AOMexperiment (Inatani et al., 1983). However, its antioxidative

1054 M.-H. Yu et al. / Food Chemistry 136 (2013) 1047–1054

activity was found to be the same as that of carnosol in DPPH test.Also, it showed some synergistic antioxidative effect with carnosolwhen they are mixed in a certain composition. These results sug-gest that one of the underestimated components that contributesto the high antioxidative effect of rosemary extracts was 40-methoxytechtocrysin.

These findings showed that the anti-inflammatory effects ofrosemary extract results from the synergistic effect of many com-pounds in the herb. This work has added experimental support thatplants can be used against inflammatory disorders. Also, it wasfound that rosemary could be a cheap primary material to obtainimportant compounds with anti-inflammatory properties.

Acknowledgements

This work was supported by the Korean Science and Engineer-ing Foundation (KOSEF) grant funded by the Korean government(MEST) (No. 353-2009-1-F00018), and Technology DevelopmentProgram for Agriculture and Forestry, Ministry for Food, Agricul-ture, Forestry, and Fisheries (MIFAFF).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2012.08.085.

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