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Journal of Ethnopharmacology 123 (2009) 149–154

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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nti-inflammatory activity of Chrysanthemum indicum extract in acute andhronic cutaneous inflammation

o Yeon Lee, Goya Choi, Taesook Yoon, Myeong Sook Cheon,yung Kil Choo, Ho Kyoung Kim ∗

enter of Herbal Resources Research, Korea Institute of Oriental Medicine, 483 Exporo, Yuseong-gu, Daejeon 305-811, Republic of Korea

r t i c l e i n f o

rticle history:eceived 23 July 2008eceived in revised form 2 January 2009ccepted 2 February 2009vailable online 14 February 2009

eywords:hrysanthemum indicumkin inflammation

a b s t r a c t

Aim of the study: Although Chrysanthemum indicum Linné (Compositae) has long been used in traditionalKorean, Chinese, Japanese medicine to treat various immune-related diseases the underlying mecha-nism(s) by which these effects are induced remains to be defined in vivo model system. We investigatedthe effects of 70% ethanolic extract from Chrysanthemum indicum Linné (CIE) on skin inflammation inmice.Materials and methods: Production of pro-inflammatory cytokines (TNF-� and IL-1�), activation ofmyeloperoxidase, and histological assessment were examined in acute and chronic skin inflammationusing 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear edema.

nti-inflammatory activity2-O-tetradecanoyl-phorbol-13-acetateTPA)ermatitis

Results: CIE inhibited topical edema in the mouse ear, following administration at 200 mg/kg (i.p.), lead-ing to substantial reductions in skin thickness and tissue weight, inflammatory cytokine production,neutrophil-mediated myeloperoxidase activity, and various histopathological indicators. Furthermore,CIE was effective at reducing inflammatory damage induced by chronic TPA exposure.Conclusions: These results demonstrate that CIE is an effective anti-inflammatory agent in murine phorbolester-induced dermatitis, and suggest that the extract may have therapeutic potential in a variety ofimmune-related cutaneous diseases.

. Introduction

As the primary interface between the body and the externalnvironment, the skin provides the first line of defense against trau-atic injury and invasion by microbial pathogens. In addition to its

roperties as a physical barrier, the skin has many active defenseechanisms (Kupper and Fuhlbrigge, 2004) and regulation of theseechanisms is crucial, as inappropriate or misdirected immune

ctivity is implicated in the pathogenesis of a large variety of inflam-atory skin disorders. While some of these conditions are easily

emedied, treatments for chronic inflammatory diseases such assoriasis and atopic dermatitis are not 100% successful (Chi et al.,003).

High levels of inflammatory cytokines and reactive oxygenpecies are proposed to contribute to the pathophysiological

echanisms associated with various inflammatory skin disorders

Trouba et al., 2002). It is widely recognized that cutaneous inflam-ation is produced and maintained by the interaction of various

nflammatory cell populations that migrate to the inflammation

∗ Corresponding author. Tel.: +82 42 868 9502; fax: +82 42 863 9434.E-mail address: hkkim@kiom.re.kr (H.K. Kim).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.02.009

© 2009 Elsevier Ireland Ltd. All rights reserved.

site in response to the release of soluble pro-inflammatory medi-ators such as cytokines, prostaglandins, and leukotriene (Brigantiand Picardo, 2003; Lee et al., 2003). Therefore, pro-inflammatorycytokines, such as tumor necrosis factor (TNF)-�, play importantroles in the pathogenesis of skin disorders, and the modulation oftheir production may be an effective therapy for the treatment ofskin diseases.

Current therapies focus on treating symptoms of skin disorderswith a combination of moisturizers, antihistamines, antibiotics,and corticosteroids, with the aim of repairing barrier function, andreducing itch, secondary infections, and inflammation. However,steroids can disrupt a number of cytokine networks involved in lym-phocyte function, resulting in immunosuppression, and long-termtopical use can decrease collagen synthesis, leading to skin atro-phy (Oikarinen et al., 1998). Because of these risks, new therapeuticapproaches are being intensively investigated.

Chrysanthemum indicum Linné (Compositae) is a traditionalherb used to treat various immune-related disorders, hyperten-

sion symptoms and several infectious diseases in Korean andChinese medicine (Cheng et al., 2005; Shunying et al., 2005).Previous studies have reported that Chrysanthemum indicum pos-sesses antibacterial, antiviral, antioxidant, and immunomodulatoryproperties (Ren et al., 1999; Yan et al., 1999; Wang et al., 2000).

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he phytochemical profile of this plant shows the presence ofavonoids, terpenoids and phenolic compounds (Yoshikawa et al.,000). The anti-inflammatory effects of total flavonoids of this plantnd its partial mechanism have reported (Zhang et al., 2005). Sev-ral chemical compounds isolated from Chrysanthemum indicumowers were also found to exhibit inhibitory activity against bothitric oxide (NO) in lipopolysaccharide-activated macrophages, andat lens aldose reductase (Yoshikawa et al., 2000; Matsuda et al.,002). Also, preliminary studies on anti-inflammatory and anti-ociceptive effects of aqueous extract of Chrysanthllum indicumave reported (Amos et al., 2002). As several of these resultsnd the ethnomedicinal uses of this plant indicate possible anti-nflammatory properties, this stimulated our interest to study theffect of this plant on experimentally induced inflammatory condi-ions in mice.

Here, we describe the results of experiments that test thebility of a Chrysanthemum indicum extract (CIE) in reducingurine cutaneous inflammation induced by exposure to the well-

haracterized protein kinase C activator and tumor promoter,2-O-tetradecanoyl-phorbol-13-acetate (TPA). In mouse models,opical application of TPA has been used to screen for anti-nflammatory steroids and nonsteroidal agents (De Young et al.,989; Stanley et al., 1991). Our studies demonstrate that CIE pos-esses potent anti-inflammatory activity in both acute and chronicontact dermatitis models and acts by blocking pro-inflammatoryytokine production and neutrophil-mediated myeloperoxidaseMPO) activity.

. Materials and methods

.1. Preparation of extract from Chrysanthemum indicum

The flowers of Chrysanthemum indicum as a dried herbere obtained from an oriental drug store (Omniherb Co.) in

eoungcheon, South Korea and authenticated based on its micro-copic and macroscopic characteristics by “The Classificationnd Identification Committee of the Korea Institute of Orientaledicine”. The committee was composed of nine experts in the

elds of plant taxonomy, botany, pharmacognosy and herbology.voucher specimen (no. KIOM0077026) was deposited at the

erbarium of the Department of Herbal Resources Research inorea Institute of Oriental Medicine (Daejeon, Korea). The driedowers of Chrysanthemum indicum were extracted twice with 70%thanol (with 2 h reflux), and the extract was then concentratednder reduced pressure. The decoction was filtered, lyophilized,nd stored at 4 ◦C. The yield of dried extract from starting crudeaterials was about 12.35%.

.2. Animals

Specific pathogen-free 5-week-old male C57BL/6J mice wereurchased from Dae Han Biolink Co. (Korea). On arrival, mice wereandomized and transferred to cages containing sawdust beddingfive mice per cage), were given laboratory chow (Orient Co., Korea)nd water ad libitum, and were used for experimentation whenheir weight was between 18 and 20 g. They were acclimatized inn animal facility at KIOM at least 7 days prior to experimentationnder conditions of 20–22 ◦C, 40–60% relative humidity and a 12 h

ight/12 h dark cycle.

.3. Chemicals

TPA and indomethacin were purchased from Sigma Chemical Co.St. Louis, MO, USA). All other chemicals and reagents were of theighest commercial grade available.

acology 123 (2009) 149–154

2.4. Ear edema measurement

Edema was expressed as the increase in ear thickness and earweight due to inflammatory challenge. Ear thickness was measuredwith a micrometer (Mitutoyo Series 293) before and after inductionof an inflammatory response. The micrometer was applied near thetip of the ear just distal to the cartilaginous ridges, and the thicknesswas recorded in �m. To minimize variation due to technique, a sin-gle investigator performed all measurements throughout any oneexperiment. To evaluate ear weight, animals were anesthetized, and6 mm2 diameter ear punch biopsies were collected using a 5/16 in.leather punch, and the biopsies were individually weighed on aMettler–Toledo (AB-204-S) balance.

2.5. Mouse model of acute inflammation

The mouse model of acute inflammation used in these stud-ies was a slight modification of a previously described procedure(Stanley et al., 1991). Edema was induced on the right ear by topi-cal application of 2.5 �g/ear TPA in 20 �l acetone. To examine theeffect of CIE on ear edema following intraperitoneal (i.p.) challenge,groups of mice were injected with CIE, vehicle (saline, negative con-trol), or indomethacin (5 mg/kg, positive control) at 30 min prior toTPA challenge. Ear thicknesses and weights were measured prior toand 6 h after topical application of TPA (De Young et al., 1989).

2.6. Mouse model of chronic inflammation

The effect of CIE on chronic skin inflammation was evaluatedusing a previously described procedure that was slightly modi-fied (Burke, 2001). Briefly, 20 �l of a solution of TPA (2.5 �g/ear × 6times) or acetone (vehicle) was topically applied to the inner andouter surfaces of both ears of each mouse with a micropipette onalternate days. CIE (200 mg/kg), vehicle (saline, negative control), orindomethacin (5 mg/kg, positive control) was given i.p. once a dayfor 10 days each morning immediately after TPA application. Onday 10, the mice were sacrificed at 6 h after treatment, and 6 mm2

diameter ear punch biopsies were collected and weighed.

2.7. Cytokine level measurements

Serum levels of the cytokine proteins interleukin (IL)-1� andTNF-� were determined 6 h after TPA application using a standardsandwich enzyme-linked immunosorbent assay (ELISA) kit (R&DSystems, Minneapolis, MN, USA). Briefly, a 96-well plate coatedwith biotinylated anti-mouse cytokine monoclonal antibody wasused. One hundred microliters of serial dilutions of the standard ormouse sera samples were added to each well and incubated for 2 hat 37 ◦C. After five washes, 100 �l horseradish peroxidase solutionwas added to each well, and the plates were allowed to develop at37 ◦C for 2 h. After five washes, the plate was maintained at 37 ◦Cfor 30 min to react with 100 �l of a substrate solution. The reactionwas stopped by the addition of 100 �l blocking solution, and theabsorbance at 450 nm was read using a microplate reader (Molec-ular Devices, Sunnyvale, CA, USA). The results were expressed asarbitrary units of relative value.

2.8. Determination of myeloperoxidase activity

Plasma MPO activity was assessed 24 h after repeated topi-cal application of TPA using the mouse MPO ELISA kit (Hycult

Biotechnology, The Netherlands). Briefly, a 96-well plate coatedwith biotinylated anti-mouse MPO monoclonal antibody was used.One hundred microliters of serial dilutions of the standard or mouseplasma samples were added to each well and incubated for 2 h at37 ◦C. After three washes, 100 �l horseradish peroxidase solution

pharmacology 123 (2009) 149–154 151

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Fig. 1. Effect of CIE on TPA-induced ear thickness and weight changes in an acuteinflammation model. Mice were treated with normal saline (TPA + S), CIE (TPA + CIE),or indomethacin (TPA + Indo) for 1 h prior to topical application of acetone (vehicle)

D.Y. Lee et al. / Journal of Ethno

as added to each well, and the plates were allowed to developt 37 ◦C for 1 h. The plate was again washed three times, thenept at 37 ◦C for 30 min to react with 100 �l of substrate solution.he reaction was stopped by the addition of 100 �l blocking solu-ion, and the absorbance at 450 nm was read with a microplateeader. The results were expressed as arbitrary units of relativealue.

.9. Histology

For histological assessment of cutaneous inflammation, biop-ies from control and treated ears of mice in each treatment groupere collected and fixed in 4% paraformaldehyde (0.1 M phos-hate buffer, pH 7.4). The ear samples were dehydrated with 15,0, 25, and 30% serial sucrose solutions. A series of 10 �m earross-sections were prepared by a freezing microtome. The sectionsere stained with hematoxylin and eosin (H&E) for the evalua-

ion of leukocyte accumulation and edema. A representative areaas selected for qualitative light microscopic analysis of the cell-ediated inflammatory response. To minimize bias, the sample

nalysis was blinded.

.10. Acute toxicity

Different doses of CIE prepared in saline were given orally toroups of 10 mice. For 30 days subsequent to treatment, the ani-als were observed daily, and dead animals were subjected to

ostmortem examination for determination of the cause of death.

.11. Statistical analysis

All data were expressed as mean ± S.E.M., and statistical signifi-ance was determined via Student’s t-test with p < 0.05 consideredo be significant.

. Results

.1. Examining the effect of CIE on TPA-induced cutaneousnflammation

We assessed the anti-inflammatory activity of CIE in a murineodel of TPA-induced acute irritant contact dermatitis. Increased

kin thickening is often the first hallmark of skin irritation andocal inflammation. This parameter is one indicator of num-er of processes that occur during skin inflammation, including

ncreased vascular permeability, edema and swelling within theermis, and proliferation of epidermal keratinocytes. Exposure toPA on the ear of the mouse resulted in marked increases inoth skin thickness (Fig. 1A) and tissue weight (Fig. 1B). Topi-al application of acetone (vehicle) or CIE alone did not alter thekin thickness significantly, however, CIE significantly inhibitedhe phorbol ester-induced increases in both skin thickness andeight, indicating the therapeutic effect of this extract (Fig. 1A and).

Next, we investigated H&E-stained ear sections from TPA-reated animals. TPA application resulted in a marked increase inar thickness, with clear evidence of edema, epidermal hyperpla-

ia, and substantial inflammatory cell infiltration in the dermisith accompanying connective tissue disruption (Fig. 2A and B). CIE

reatment reduced ear thickness and associated pathological indi-ators to an extent comparable to the positive control indomethacinFig. 2C and D). These results directly illustrate the effects of CIEithin the target tissue, providing further evidence that CIE ame-

iorates TPA-induced contact dermatitis.

or TPA in acetone. Ear thickness (A) and ear weights (B) were measured 6 h afterTPA treatment. Each point represents the mean ± S.E.M. of the difference betweenear thickness/weight before and after challenge. N = 10 mice per group; *p < 0.01compared to vehicle, and **p < 0.01 compared to TPA alone.

3.2. Examining the effect of CIE on pro-inflammatory cytokineproduction

To assess the efficacy of CIE at the molecular level, we investi-gated the pro-inflammatory cytokine-inhibitory effect of CIE. Asshown in Fig. 3, topical application of TPA caused a dramaticincrease in the production of IL-1� and TNF-� 6 h after challenge.In contrast, treatment with TPA plus CIE or indomethacin reducedboth IL-1� and TNF-� cytokine levels significantly. Thus, CIE may actby reducing the levels of activated cellular infiltrates and secretionof cytokines, thereby reducing cutaneous inflammation.

3.3. Examining the effect of CIE on prolonged inflammationinduced by repeated TPA application

As a second in vivo measure of the anti-inflammatory activityof CIE, the extract was administered in a mouse model of chronicskin inflammation induced by repeated exposure to phorbol ester.Skin inflammation is persistent in this model (Stanley et al., 1991;Burke, 2001), which makes it useful for assessing whether CIEresolves existing inflammatory lesions. Exposure to TPA resulted

in marked increases in both skin thickness (Fig. 4A) and tissueweight (Fig. 4B). Interestingly, CIE significantly inhibited thesephorbol ester-induced increases, indicating a therapeutic effect ofthis extract in the chronic model (Fig. 4A and B). Consistent with theedema parameters, CIE reduced the level of MPO activity, an indi-

152 D.Y. Lee et al. / Journal of Ethnopharmacology 123 (2009) 149–154

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ig. 2. Representative micrographs of H&E–stained mouse ear cross-sections in thelus either normal saline (B), CIE (C), or indomethacin (D). Note the edema, polymo

n inflammatory cells and edema in ears following CIE treatment. Representative se

ator of polymorphonuclear leukocyte influx, by 76% in the chronicnflammation model (Fig. 5). These findings support the ability ofIE to resolve an existing, persistent inflammatory lesion inducedy multiple topical TPA applications, with an efficacy comparableo that of indomethacin.

.4. Acute toxicity

No animals died during the acute toxicity test, nor were anydverse effects detected in animals treated with different doses ofIE. This indicates that CIE was nearly nontoxic in mice up to an oralose of 2.0 g/kg body weight.

. Discussion

One previous study reports that acute and chronic humantopic dermatitis lesions contain activated T cells and degran-lated mast cells (Leung and Soter, 2001). Given this complex

nflammatory pathology, it has been difficult to establish relativelyhort-term animal models for pre-clinical drug testing. Althoughot an allergen-driven model, the phorbol ester-induced mousear inflammation model produces edema and leads to recruit-

ent of polymorphonuclear leukocytes under acute conditions,ith macrophages and T cells predominating by day 7 of the chronicodel (Stanley et al., 1991; Alford et al., 1992). Since this modelimics several aspects of human atopic dermatitis, it is a reliable

n vivo model system, providing opportunities to evaluate preven-

TPA model. Ears were harvested 6 h post-treatment with acetone vehicle (A), TPAuclear cell influx and epidermal hyperplasia in TPA-treated ears and the reductionfrom five animals in each group are shown (400× magnification).

tive treatment in acute disease and treatment of established lesionsin chronic disease.

This study provides evidence that CIE acts as an anti-inflammatory agent in mouse models of TPA-induced skininflammation. In both acute and chronic irritant contact dermati-tis mouse models, CIE markedly reduced cutaneous inflammation.This was supported by observed reductions in skin thicknessand weight, amelioration of several histopathological indicators,decreased release of pro-inflammatory cytokines, and diminishedneutrophil activation. Together, these finding suggest that CIEmay be an important therapeutic strategy for the treatment ofinflammatory skin diseases. These results are supported by thefindings of Wang et al. (2000), who showed that CIE effectivelyenhanced the phagocytic activity of macrophages in mice. Inaddition, Cheng et al. (2005) provided evidence for the anti-inflammatory properties of a CIE and its fractions in mouse auricleedema. Despite these reports describing the anti-inflammatoryactivities of Chrysanthemum indicum, little is known about itsdetailed mechanisms in vivo model system. Moreover, none ofstudies about the anti-inflammatory effect in chronic model havebeen published. Therefore, this study represents the first report onthe anti-inflammatory activity of Chrysanthemum indicum and itsaction mechanisms using acute and chronic skin inflammation in

mouse model.

Our present study clearly demonstrates that TPA exposureresulted in increased secretion of IL-1� and TNF-�, suggesting thatboth cytokines mediate inflammatory signaling and play a pivotalrole in TPA-induced acute irritant contact dermatitis, which is con-

D.Y. Lee et al. / Journal of Ethnopharmacology 123 (2009) 149–154 153

Fig. 3. Effect of CIE on TPA-induced IL-1� and TNF-� production. Mice were treatedwith normal saline (TPA + S), CIE (TPA + CIE), or indomethacin (TPA + Indo), for 1 hprior to topical application of acetone (vehicle) or TPA in acetone. Serum was taken6 h after TPA treatment and examined for the production of the pro-inflammatorycot

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Fig. 4. Effect of CIE on TPA-induced ear thickness and weight changes in the chronicinflammation model. Mice were treated with repeated topical applications of TPA.

fied in ethanol extract of Chrysanthemum indicum (Gao et al., 2008).It is well known that several compounds, such as acacetin, api-genin, etc., showed anti-inflammatory properties (Pan et al., 2006;Nicholas et al., 2007).

Fig. 5. Effect of CIE on TPA-induced MPO activity. Mice were treated with repeated

ytokines IL-1� (A) and TNF-� (B) using ELISA. The data shown are the mean ± S.E.M.f the percent change relative to vehicle. N = 10 mice per group; *p < 0.01 comparedo vehicle, and **p < 0.05 compared to TPA alone.

istent with previously published results (Ueda et al., 2004; Otuki etl., 2005). We also show that CIE negatively interfered with theseytokines. Moreover, we demonstrate that CIE inhibits MPO acti-ation in a mouse model of chronic skin inflammation. MPO isn enzyme found in the azurophilic granules of neutrophils andther cells of myeloid origin, and is commonly used as an indexf granulocyte infiltration. MPO inhibition is indicative of anti-nflammatory activity in the chronic inflammation model (Ajuebort al., 2000).

Interestingly, our histological analysis clearly confirmed thatIE inhibited the influx of polymorphonuclear leukocytes to theouse ear skin following TPA application. We propose that thearked inhibition of ear edema, MPO activity, and the activation

nd migration of leukocytes in response to topical TPA treatmentay be related to the inhibition of pro-inflammatory cytokine

elease by CIE. This concept correlates well with previous findingshat cells in injured skin, such as dermal dendritic cells, epider-

al Langerhans cells, melanocytes, fibroblasts, and leukocytes arenown to be sources and targets of cytokines (Grone, 2002). Takenogether, these results support the notion that CIE possesses anti-nflammatory properties. Indeed, neutrophil accumulation plays aritical role in cutaneous inflammatory diseases such as dermatitis,

nd is related to the pathological mechanism of disease (Bradley etl., 1982).

We could assume that the anti-inflammatory activity of CIEs related to a synergistic action of all constituents contained in

Ear thickness (A) and ear weights (B) were measured on day 10. Each point repre-sents the mean ± S.E.M. of the difference between ear thickness/weight before andafter challenge. N = 10 mice per group; *p < 0.01 compared to vehicle, and **p < 0.01compared to TPA alone.

ethanol extract. It is reported that seven compounds includingacacetin, apigenin, and their derivatives were isolated and identi-

topical applications of TPA and neutrophil activation levels were determined by anMPO activity assay of mouse plasma on day 10. TPA exposure resulted in a markedincrease in plasma MPO levels as compared with vehicle alone. The data shown arethe mean ± S.E.M. of the percent change relative to vehicle. N = 10 mice per group;*p < 0.01 compared to vehicle, and **p < 0.01 compared to TPA alone.

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In summary, we report that CIE has anti-inflammatory activitiesn both acute and chronic irritant contact dermatitis in vivo. To ournowledge, this is the first published report describing the activityf CIE in these models. Our results indicate that the inhibitory effectf CIE may be due, at least in part, to the inhibition of IL-1� andNF-� and to the subsequent blockade of leukocyte accumulation.his suggests that CIE may be a good candidate for the treatment ofnflammatory skin diseases or may be useful towards the develop-

ent of new anti-inflammatory cutaneous therapies.

cknowledgements

This work was supported by the ‘Construction of the Basis forractical Application of Herbal Resources’ funded by the Ministryf Education, Science and Technology (MEST) of Korea to the Koreanstitute of Oriental Medicine (KIOM). We thank ‘The Classifica-ion and Identification Committee’ of the Korea Institute of Oriental

edicine for the critical authentication of plant materials and forelpful discussions.

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