Journal of Ethnopharmacology 121 (2009) 28–34

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Journal of Ethnopharmacology

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Anti-inflammatory effects of Asparagus cochinchinensis extract in acute andchronic cutaneous inflammation

Do Yeon Lee1, Byung Kil Choo1, Taesook Yoon, Myeong Sook Cheon,Hye Won Lee, A. Yeong Lee, Ho Kyoung Kim ∗

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

a r t i c l e i n f o

Article history:Received 12 February 2008Received in revised form 20 June 2008Accepted 10 July 2008Available online 18 July 2008

Keywords:Asparagus cochinchinensisSkin inflammationAnti-inflammatory activity12-O-tetradecanoyl-phorbol-13-acetate(TPA)Dermatitis

a b s t r a c t

Aims of study: Although Asparagus cochinchinensis Merrill (Liliaceae) has long been used in traditionalKorean and Chinese medicine to treat inflammatory diseases, the underlying mechanism(s) by whichthese effects are induced remains to be defined. We investigated the effects of 70% ethanolic extract fromAsparagus cochinchinensis Merrill (ACE) on skin inflammation in mice.Materials and methods: Production of pro-inflammatory cytokines (tumor necrosis factor (TNF)-�, inter-leukin (IL)-1�), activation of myeloperoxidase, and histological assessment were examined in acuteand chronic skin inflammation using 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse earedema. We also performed acetic acid-induced vascular permeability test.Results: ACE 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 (MPO) activity, and various histopathological indicators. Further-more, ACE was effective at reducing inflammatory damage induced by chronic TPA exposure and evoked

a significant inhibition of vascular permeability induced by acetic acid in mice.Conclusion: These results demonstrate that ACE is an effective anti-inflammatory agent in murine phorbol

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. Introduction

As the primary interface between the body and the externalnvironment, the skin provides the first line of defence againstroad injury and invasion by microbial pathogens and trauma. Inddition to its properties as a physical barrier, the skin has manyctive defence mechanisms (Kupper and Fuhlbrigge, 2004). How-ver, the regulation of these mechanisms is crucial, as inappropriater misdirected immune activity is implicated in the pathogene-

is of a large variety of inflammatory skin disorders. While somef these are easily remedied, no completely successful treatmentsxist for chronic inflammatory diseases, such as psoriasis and atopicermatitis (Chi et al., 2003).

Abbreviations: ACE, Asparagus cochinchinensis extract; TPA, 12-O-tetradecanoyl-horbol-13-acetate; TNF, tumor necrosis factor; MPO, myeloperoxidase activity; IL,

nterleukin; ELISA, enzyme-linked immunosorbent assay; H&E, haematoxylin andosin.∗ Corresponding author. Tel.: +82 42 868 9502; fax: +82 42 863 9434.

E-mail address: hkkim@kiom.re.kr (H.K. Kim).1 These authors contributed equally to this work.

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378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2008.07.006

uggest that the compound may have therapeutic potential in a variety ofeases.

© 2008 Elsevier Ireland Ltd. All rights reserved.

High levels of inflammatory cytokines and reactive oxygenpecies are proposed contributors to the pathophysiological mech-nisms associated with various inflammatory skin disordersTrouba et al., 2002). It is widely recognized that cutaneous inflam-

ation is produced and maintained by the interaction of variousnflammatory cell populations that migrate to the inflammationite in response to the release of soluble pro-inflammatory medi-tors such as cytokines, prostaglandins, and leukotrienes (Brigantind Picardo, 2003; Lee et al., 2003). Therefore, pro-inflammatoryytokines, such as tumor necrosis factor (TNF)-�, play importantoles in the pathogenesis of skin disorders, and the modulation ofheir production may be an effective therapy for the treatment ofkin diseases.

Current therapies focus on treating symptoms of skin disordersith a combination of moisturizers, antihistamines, antibiotics, and

orticosteroids, with the aims of repairing barrier function, andeducing itch, secondary infections, and inflammation. However,

teroids can disrupt a number of cytokine networks involved inymphocyte function, resulting in immunosuppression; and theirong-term topical use can decrease collagen synthesis, leading tokin atrophy (Oikarinen et al., 1998). Because of these risks, newherapeutic approaches are being intensively investigated.

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The Asparagus cochinchinensis Merrill (Liliaceae) root, a tra-itional herbal medicine with sedative/tranquilizing side effectsHuang, 1993a), is used to treat various immune-related disordersuch as hepatitis, dermatitis, asthma, and brain disease. The activeompound of this plant, �-sitosterol, has been observed to have aositive effect on mouse S-180 leukemia and lung cancer (Huang,993b). The crude aqueous extract of Asparagus cochinchinensisnhibits ethanol-induced cytotoxicity in human hepatoma cellsKoo et al., 2000) and decreases TNF-� secretion from substance-- and lipopolysaccharide-stimulated primary cultures of mousestrocytes (Kim et al., 1998). As several of these results indicateossible anti-inflammatory properties for the root, we investigatedhis activity.

Here, we address whether Asparagus cochinchinensis extractACE) reduces murine cutaneous inflammation induced by expo-ure to the well-characterized protein kinase C activator andumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA).opical application of TPA has been used to screen for anti-nflammatory steroids and nonsteroidal agents. Our studiesemonstrate that ACE possesses potent anti-inflammatory activity

n both acute and chronic contact dermatitis models via blockade ofro-inflammatory cytokine production and neutrophil-mediatedyeloperoxidase (MPO) activity.

. Materials and methods

.1. Preparation of extract from Asparagus cochinchinensis

Asparagus cochinchinensis was collected from the Koreanrovince Songjeong-ri in June 2007. Botanical identification wasonfirmed by morphological characteristics and by analysis of ITSequences, which showed a 100% identification with the Asparagusochinchinensis ITS region (accession no. AB195579) in the GenBankatabase (http://www.ncbi.nlm.nih.gov/BLAST/). A voucher speci-en (no. KIOM200701000240) was deposited at the herbarium of

he Department of Herbal Resources Research (Korea Institute ofriental Medicine, Daejeon, Korea). The dried and pulverized rootsf Asparagus cochinchinensis were extracted three times 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 15.43%.

.2. Animals

Specific pathogen-free 5-week-old male C57BL/6J mice wereurchased from Dae Han Biolink Co. (Korea). On arrival, random-

zed mice were 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 (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 from Sigma Chemical Co. (St Louis,O, USA). All other chemicals and reagents were of the highest

ommercial grade available.

.4. Ear edema measurement

Edema was expressed as the increase in ear thickness and eareight due to inflammatory challenge. Ear thickness was mea-

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macology 121 (2009) 28–34 29

ured with a micrometer (Mitutoyo Series 293) before and after thenduction of inflammatory response. The micrometer was appliedear the tip of the ear just distal to the cartilaginous ridges andhe thickness was recorded in micrometer. To minimize variationue to technique, a single investigator performed all measurementshroughout any one experiment. To evaluate ear weight, animalsere anesthetized, 6-mm2 diameter ear punch biopsies collectedsing a 5/16 in. leather punch, and the biopsies were individuallyeighed on a Mettler-Toledo (AB-204-S) balance.

.5. Mouse model of acute inflammation.

The mouse model of acute inflammation employed here was alight modification of a previously described procedure (Stanley etl., 1991). Edema was induced on the right ear by topical applica-ion of 2.5 �g/ear TPA (in 20 �l acetone). To examine the effect ofCE on ear edema following intraperitoneal (i.p.) challenge, groupsf mice were injected with ACE, vehicle (saline, negative control),r indomethacin (5 mg/kg, positive control) at 30 min prior to TPAhallenge. Ear thicknesses and weights were measured 6 h afteropical application of TPA (De Young et al., 1989).

.6. Mouse model of chronic inflammation

The effect of ACE on chronic skin inflammation was evaluatedy a slight modification of a previous described procedure (Burke,001). Briefly, 20 �l of a solution of TPA (2.5 �g/ear × 6 times) orcetone (vehicle) were topically applied to the inner and outer earurfaces of both ears of each mouse with a micropipette on alter-ate days. ACE (200 mg/kg), vehicle (saline, negative control), or

ndomethacin (5 mg/kg, positive control) was given once a day (i.p.)or 10 days each morning immediately after TPA application. Onay 10, the mice were sacrificed at 6 h after treatment, and 6-mm2

iameter ear punch biopsies were collected and weighed.

.7. ELISA assay procedure

Serum levels of the cytokine proteins interleukin (IL)-1� andNF-� were determined 6 h after TPA application using a standardandwich enzyme-linked immunosorbent assay (ELISA) kit accord-ng to the manufacturer’s instructions (R&D Systems, Minneapolis,

N, USA). Briefly, a 96-well plate coated with biotinylated anti-ouse monoclonal antibody was used. One hundred microliters

f serial dilutions of the standard or sera samples were added toach well and incubated for 2 h at 37 ◦C. After five washes withash solution, 100 �l avidin–horseradish peroxidase solution were

dded to each well, and the plates were allowed to develop at 37 ◦Cor 2 h. After five washes, the plate was maintained at 37 ◦C for0 min to react with 100 �l of a substrate solution. The reaction wastopped by the addition of 100 �l blocking solution, and then thebsorbance at 450 nm was read with a microplate reader (molecu-ar device). The results were expressed as arbitrary units of relativealue.

.8. Determination of myeloperoxidase activity

Plasma MPO activity was assessed 24 h after repeated topicalpplication of TPA using the mouse MPO ELISA kit according tohe manufacturer’s instructions (Hycult Biotechnology, The Nether-ands). Briefly, a 96-well plate coated with biotinylated anti-mouse

onoclonal antibody was used. One hundred microliters of serialilutions of the standard or plasma samples were added to eachell and incubated for 2 h at 37 ◦C. After three washes with wash

olution, 100 �l avidin–horseradish peroxidase solution was addedo each well, and the plates were allowed to develop at 37 ◦C for

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Fig. 1. Effect of ACE on TPA-induced ear thickness and weight changes in anacute inflammation model. Mice were treated with normal saline (TPA + S), ACE(TPA + ACE), or indomethacin (TPA + Indo) for 1 h prior to topical application of ace-tone (vehicle) or TPA in acetone. Ear thickness (A) and ear weights (B) were measuredab*b

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h. The plate was again washed three times, then kept at 37 ◦Cor 30 min to react with 100 �l of substrate solution. The reactionas stopped by the addition of 100 �l blocking solution, and the

bsorbance at 450 nm was read with a microplate reader (molecu-ar device). The results were expressed as arbitrary units of relativealue.

.9. Acetic acid-induced vascular permeability

Acetic acid-induced vascular permeability in ICR mice was per-ormed as described previously (Whittle, 1964). Briefly, 1 h afterral administration of ACE (200 mg/kg), indomethacin (5 mg/kg),r an equivalent volume of vehicle (3%, v/v Tween 80), 0.2 ml ofvans blue dye (0.25% in normal saline) was administered intra-enously through the tail vein. Thirty minutes later, the animalseceived 1 ml/100 g of acetic acid (0.6%, v/v) i.p. Treated animalsere sacrificed 30 min after acetic acid injection and the peritoneal

avity washed with normal saline (3 ml) into heparinized tubes andentrifuged. The dye content of the supernatant was measured at10 nm using a microplate reader (molecular device). The resultsere expressed as arbitrary units of relative value.

.10. 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% para-formaldehyde (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 was prepared by a freezing microtome. The sectionsere stained with haematoxylin and eosin (H&E) for the evaluation

f leukocyte accumulation and edema. A representative area waselected for qualitative light microscopic analysis of the inflamma-ory cellular response. To minimize bias, the investigator did notnow which group was being analyzed.

.11. Acute toxicity

Different doses of ACE prepared in saline were given orally toroups of 10 mice each. For 30 days subsequent to treatment, thenimals were observed daily, and dead animals were subjected toostmortem examination for determination of the cause of death.

.12. Statistical analysis

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

. Results

.1. Effect of ACE on TPA-induced cutaneous inflammation

We assessed the anti-inflammatory activity of ACE in a TPAodel of acute irritant contact dermatitis. Increased skin thick-

ning is often the first hallmark of skin irritation and localnflammation. This parameter is one indicator of number of pro-esses that occur during skin inflammation, including increasedascular permeability, edema and swelling within the dermis, androliferation of epidermal keratinocytes. Ear edema was measured

n the dorsal skin prior to and at 6 h following treatments. Exposureo TPA resulted in marked increases in both skin thickness (Fig. 1A)nd tissue weight (Fig. 1B). Topical application of acetone (vehicle)r ACE alone did not alter the skin thickness significantly. However,CE significantly inhibited the phorbol ester-induced increases in

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t 6 h after TPA treatment. Each point represents the mean ± S.E.M. of the differenceetween ear thickness/weight before and after challenge. N = 10 mice per group;P < 0.01 compared to vehicle, and **P < 0.01 compared to TPA alone as determinedy the Student’s t-test.

oth skin thickness and weight, indicating the therapeutic effect ofhis extract (Fig. 1A and B).

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 hyperplasia,nd substantial inflammatory cell infiltration in the dermis withccompanying connective tissue disruption (Fig. 2A and B). ACEreatment reduced ear thickness and associated pathological indi-ators to an extent comparable to the positive control indomethacinFig. 2C and D). These results provide further evidence that ACEmeliorates TPA-induced contact dermatitis, directly illustrating itsffects within the target tissue.

.2. Effect of ACE on pro-inflammatory cytokine production

To assess the efficacy of ACE at the molecular level, we inves-igated the pro-inflammatory cytokine-inhibitory effect of ACE.s shown in Fig. 3, topical application of TPA caused a dramatic

ncrease in the production of IL-1� and TNF-� by 6 h after challenge.n contrast, treatment with TPA plus ACE or indomethacin reducedoth IL-1� and TNF-� cytokine levels significantly. Thus, ACE mayeduce the levels of activated cellular infiltrates and secretion ofytokines, thereby reducing cutaneous inflammation.

D.Y. Lee et al. / Journal of Ethnopharmacology 121 (2009) 28–34 31

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

n inflammatory cells and edema in ears with ACE treatment. Sections shown are re

.3. Effect of ACE on prolonged inflammation induced by repeatedPA application

As a second in vivo measure of the anti-inflammatory activityf ACE, the extract was administrated in a mouse model of chronickin inflammation induced by repeated exposure to phorbol ester.kin inflammation in this model is persistent, which makes thisodel useful for assessing whether ACE resolves existing inflam-atory lesions (Burke, 2001; Stanley et al., 1991). Exposure to TPA

esulted in marked increases in both skin thickness (Fig. 4A) and tis-ue weight (Fig. 4B). In contrast, ACE significantly inhibited thesehorbol ester-induced increases, indicating a therapeutic effect ofhis extract in the chronic model (Fig. 4A and B). Consistent with thedema parameters, ACE reduced the level of MPO activity, an indi-

ator of polymorphonuclear leukocyte influx, by 39% in the chronicnflammation model (Fig. 5). These findings support the ability ofCE to resolve an existing, persistent inflammatory lesion inducedy multiple topical TPA applications, with an efficacy comparableo that of indomethacin.

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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 reduction

ntative of observations from five animals in each group (400× magnification).

.4. Effect of ACE on acetic acid-induced vascular permeability

As a third in vivo measure of the anti-inflammatory activityf ACE, the extract was administrated in a mouse model of vas-ular permeability induced by acetic acid. It is well known thathe increase of vascular permeability induced by acetic acid corre-ponds to the early exudative stage of inflammation, one of the mostmportant processes in the inflammatory pathological mechanismWhittle, 1964; Winter et al., 1962). ACE treatment signifi-antly inhibited acetic acid-induced vascular permeability in miceFig. 6).

.5. Acute toxicity

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

32 D.Y. Lee et al. / Journal of Ethnopharmacology 121 (2009) 28–34

Fig. 3. Effect of ACE on TPA-induced IL-1� and TNF-� production. Mice were treatedwith normal saline (TPA + S), ACE (TPA + ACE), 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-inflammatorycytokines IL-1� (A) and TNF-� (B) using ELISA. The data shown are the mean ± S.E.M.ocS

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Fig. 4. Effect of ACE on TPA-induced ear thickness and weight changes in the chronicinflammation model. Mice were treated with repeated topical applications of TPA.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 as determined by the Student’s t-test.

Fig. 5. Effect of ACE on TPA-induced MPO activity. Mice were treated with repeatedtopical applications of TPA. Neutrophil activation levels were determined by an MPO

f the percent change relative to acetone-vehicle. N = 10 mice per group; *P < 0.01ompared to vehicle, and **P < 0.05 compared to TPA alone as determined by thetudent’s t-test.

. Discussion

This study provides evidence that ACE acts as an anti-nflammatory agent in mouse models of skin inflammation. In bothcute and chronic irritant contact dermatitis mouse models, ACEarkedly reduced cutaneous inflammation. This was supported

y observed reductions in skin thickness and weight, ameliora-ion of several histopathological indicators, decreased release ofro-inflammatory cytokines, and diminished neutrophil activation.esults from our study demonstrate that ACE inhibits phorbol ester-

nduced increases in ear edema as well as acetic acid-inducedascular permeability. Together, these finding suggest that ACE maye an important therapeutic strategy for the treatment of inflam-atory skin diseases.One previous study reports that acute and chronic human

topic 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 mouse ear

nflammation model produces edema and leads to recruitment ofolymorphonuclear leukocytes acutely, with macrophages and Tells predominating by day 7 of the chronic model (Alford et al.,992; Stanley et al., 1991). Since this model mimics several aspectsf human atopic dermatitis, it is a reliable in vivo model system,

roviding opportunities to evaluate preventive treatment in acuteisease and treatment of established lesions in chronic disease.

Our present study clearly demonstrates that TPA exposureesulted in increased secretion of IL-1� and TNF-�, suggesting that

activity assay of mouse plasma on day 10. TPA exposure resulted in a marked increasein plasma MPO levels as compared with acetone vehicle alone. The data shownare the mean ± S.E.M. of the percent change relative to acetone-vehicle. N = 10 miceper group; *P < 0.01 compared to vehicle, and **P < 0.01 compared to TPA alone asdetermined by the Student’s t-test.

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

Fig. 6. Effect of ACE on acetic acid-induced vascular permeability in mice. Micerag

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Okoli, C.O., Akah, P.A., Nwafor, S.V., Anisiobi, A.I., Ibegbunam, I.N., Erojikwe, O., 2007.

eceived oral administrations of ACE or indomethacin before i.p. injection of aceticcid. The data shown are mean ± S.E.M. of the percent inhibition. N = 10 mice perroup; *P < 0.01 compared to TPA alone as determined by the Student’s t-test.

oth cytokines mediate inflammatory signaling and play a piv-tal role in TPA-induced acute irritant contact dermatitis, a resulthat is consistent with results from others (Otuki et al., 2005;eda et al., 2004). We also showed that ACE negatively inter-

ered with these cytokines, which are known to play importantoles in the inflammatory process. These results were supportedy the findings of Koo et al., who showed that ACE significantlynhibits the secretion of TNF-� in dose-dependent manner (Koo etl., 2000). We demonstrated that ACE inhibits MPO activation inmouse model of chronic skin inflammation. MPO is an enzyme

ound in the azurophilic granules of neutophils and other cellsf myeloid origin, and is commonly used as an index of granulo-yte infiltration. MPO inhibition is indicative of anti-inflammatoryctivity in the chronic inflammation model (Ajuebor et al.,000).

Interestingly, our histological analysis of the ear clearly con-rmed that ACE inhibited the influx of polymorphonuclear

eukocytes to the mouse ear skin following TPA application. Weropose that the marked inhibition by ACE against ear edema, MPOctivity, activation and migration of leukocytes in response to top-cal TPA may be related to inhibition of pro-inflammatory cytokineelease. This concept correlates well with previous findings thatells in the injured skin, such as dermal dendritic cells, epider-al Langerhans cells, melanocytes, fibroblasts, and leukocytes, are

nown to be sources and targets of cytokines (Grone, 2002). Takenogether, these results support the notion that ACE 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 have provided what we believe to be the first reportf the anti-inflammatory effects of ACE in in vivo skin inflamma-ion, and our results suggest that ACE is a potential therapy for thereatment of skin inflammation.

Acetic acid-induced vascular permeability causes an immediateustained reaction that is prolonged over 24 h and leads to exu-ation of fluid rich in plasma proteins (Whittle, 1964; Winter etl., 1962). It is therefore used as a well-characterized mouse modelf acute inflammation, and inhibition of vascular permeability is

onsidered a major feature for the suppression of the exudativehase of acute inflammation (Okoli et al., 2007). Here, we demon-trate that ACE had significant anti-inflammatory effects in thisodel, indicating that ACE may affect membrane-stabilization to

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macology 121 (2009) 28–34 33

educe vascular permeability and/or inhibits various inflammatoryediators.In summary, this study reports for the first time to our knowl-

dge that ACE has anti-inflammatory activities in both acute andhronic irritant contact dermatitis. The results presented in thistudy also suggest that the inhibitory effect of ACE may be due, ateast in part, to the inhibition of IL-1� and TNF-� and to the sub-equent blockade of leukocyte accumulation. Our results suggesthat ACE may be a good candidate for the treatment of inflamma-ory skin diseases or may be useful towards the development ofew anti-inflammatory cutaneous therapies.

cknowledgements

This study, which is part of a larger project, was achieved withhe support of the Conservation Technology Research and Develop-

ent Project hosted by the National Research Institute of Culturaleritage of the Korean Cultural Heritage Administration. The studyas also partially supported by grants from the Construction of theasis of the Practical Application of Herbal Resources funded byOST. We express our gratitude to these funding agencies.

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