Journal of Ethnopharmacology 121 (2009) 2834

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Anti-inammatory effects of Asparagus cochinchinensis extract in acute and chronic cutaneous inammation
Do Yeon Lee 1 , Byung Kil Choo 1 , 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

a b s t r a c t
Aims of study: Although Asparagus cochinchinensis Merrill (Liliaceae) has long been used in traditional Korean and Chinese medicine to treat inammatory diseases, the underlying mechanism(s) by which these effects are induced remains to be dened. We investigated the effects of 70% ethanolic extract from Asparagus cochinchinensis Merrill (ACE) on skin inammation in mice. Materials and methods: Production of pro-inammatory cytokines (tumor necrosis factor (TNF)- , interleukin (IL)-1 ), activation of myeloperoxidase, and histological assessment were examined in acute and chronic skin inammation using 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear edema. 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.), leading to substantial reductions in skin thickness and tissue weight, inammatory cytokine production, neutrophil-mediated myeloperoxidase (MPO) activity, and various histopathological indicators. Furthermore, ACE was effective at reducing inammatory damage induced by chronic TPA exposure and evoked a signicant inhibition of vascular permeability induced by acetic acid in mice. Conclusion: These results demonstrate that ACE is an effective anti-inammatory agent in murine phorbol ester-induced dermatitis, and suggest that the compound may have therapeutic potential in a variety of immune-related cutaneous diseases. 2008 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 12 February 2008 Received in revised form 20 June 2008 Accepted 10 July 2008 Available online 18 July 2008 Keywords: Asparagus cochinchinensis Skin inammation Anti-inammatory activity 12-O-tetradecanoyl-phorbol-13-acetate (TPA) Dermatitis

1. Introduction As the primary interface between the body and the external environment, the skin provides the rst line of defence against broad injury and invasion by microbial pathogens and trauma. In addition to its properties as a physical barrier, the skin has many active defence mechanisms (Kupper and Fuhlbrigge, 2004). However, the regulation of these mechanisms is crucial, as inappropriate or misdirected immune activity is implicated in the pathogenesis of a large variety of inammatory skin disorders. While some of these are easily remedied, no completely successful treatments exist for chronic inammatory diseases, such as psoriasis and atopic dermatitis (Chi et al., 2003).

Abbreviations: ACE, Asparagus cochinchinensis extract; TPA, 12-O-tetradecanoylphorbol-13-acetate; TNF, tumor necrosis factor; MPO, myeloperoxidase activity; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; H&E, haematoxylin and eosin. 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. 0378-8741/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.07.006

High levels of inammatory cytokines and reactive oxygen species are proposed contributors to the pathophysiological mechanisms associated with various inammatory skin disorders (Trouba et al., 2002). It is widely recognized that cutaneous inammation is produced and maintained by the interaction of various inammatory cell populations that migrate to the inammation site in response to the release of soluble pro-inammatory mediators such as cytokines, prostaglandins, and leukotrienes (Briganti and Picardo, 2003; Lee et al., 2003). Therefore, pro-inammatory cytokines, such as tumor necrosis factor (TNF)- , play important roles in the pathogenesis of skin disorders, and the modulation of their production may be an effective therapy for the treatment of skin diseases. Current therapies focus on treating symptoms of skin disorders with a combination of moisturizers, antihistamines, antibiotics, and corticosteroids, with the aims of repairing barrier function, and reducing itch, secondary infections, and inammation. However, steroids can disrupt a number of cytokine networks involved in lymphocyte function, resulting in immunosuppression; and their long-term topical use can decrease collagen synthesis, leading to skin atrophy (Oikarinen et al., 1998). Because of these risks, new therapeutic approaches are being intensively investigated.

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The Asparagus cochinchinensis Merrill (Liliaceae) root, a traditional herbal medicine with sedative/tranquilizing side effects (Huang, 1993a), is used to treat various immune-related disorders such as hepatitis, dermatitis, asthma, and brain disease. The active compound of this plant, -sitosterol, has been observed to have a positive effect on mouse S-180 leukemia and lung cancer (Huang, 1993b). The crude aqueous extract of Asparagus cochinchinensis inhibits ethanol-induced cytotoxicity in human hepatoma cells (Koo et al., 2000) and decreases TNF- secretion from substanceP- and lipopolysaccharide-stimulated primary cultures of mouse astrocytes (Kim et al., 1998). As several of these results indicate possible anti-inammatory properties for the root, we investigated this activity. Here, we address whether Asparagus cochinchinensis extract (ACE) reduces murine cutaneous inammation induced by exposure to the well-characterized protein kinase C activator and tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Topical application of TPA has been used to screen for antiinammatory steroids and nonsteroidal agents. Our studies demonstrate that ACE possesses potent anti-inammatory activity in both acute and chronic contact dermatitis models via blockade of pro-inammatory cytokine production and neutrophil-mediated myeloperoxidase (MPO) activity. 2. Materials and methods 2.1. Preparation of extract from Asparagus cochinchinensis Asparagus cochinchinensis was collected from the Korean province Songjeong-ri in June 2007. Botanical identication was conrmed by morphological characteristics and by analysis of ITS sequences, which showed a 100% identication with the Asparagus cochinchinensis ITS region (accession no. AB195579) in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). A voucher specimen (no. KIOM200701000240) was deposited at the herbarium of the Department of Herbal Resources Research (Korea Institute of Oriental Medicine, Daejeon, Korea). The dried and pulverized roots of Asparagus cochinchinensis were extracted three times with 70% ethanol (with 2 h reux), and the extract was then concentrated under reduced pressure. The decoction was ltered, lyophilized, and stored at 4 C. The yield of dried extract from starting crude materials was about 15.43%. 2.2. Animals Specic pathogen-free 5-week-old male C57BL/6J mice were purchased from Dae Han Biolink Co. (Korea). On arrival, randomized mice were transferred to cages containing sawdust bedding (ve mice per cage), were given laboratory chow (Orient Co., Korea) and water ad libitum, and were used for experimentation when their weight was between 18 and 20 g. They were acclimatized in an animal facility (KIOM) at least 7 days prior to experimentation under conditions of 2022 C, 4060% relative humidity and a 12-h light/12-h dark cycle. 2.3. Chemicals TPA and indomethacin were from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals and reagents were of the highest commercial grade available. 2.4. Ear edema measurement Edema was expressed as the increase in ear thickness and ear weight due to inammatory challenge. Ear thickness was mea-

sured with a micrometer (Mitutoyo Series 293) before and after the induction of inammatory response. The micrometer was applied near the tip of the ear just distal to the cartilaginous ridges and the thickness was recorded in micrometer. To minimize variation due to technique, a single investigator performed all measurements throughout any one experiment. To evaluate ear weight, animals were anesthetized, 6-mm2 diameter ear punch biopsies collected using a 5/16 in. leather punch, and the biopsies were individually weighed on a Mettler-Toledo (AB-204-S) balance. 2.5. Mouse model of acute inammation. The mouse model of acute inammation employed here was a slight modication of a previously described procedure (Stanley et al., 1991). Edema was induced on the right ear by topical application of 2.5 g/ear TPA (in 20 l acetone). To examine the effect of ACE on ear edema following intraperitoneal (i.p.) challenge, groups of mice were injected with ACE, vehicle (saline, negative control), or indomethacin (5 mg/kg, positive control) at 30 min prior to TPA challenge. Ear thicknesses and weights were measured 6 h after topical application of TPA (De Young et al., 1989). 2.6. Mouse model of chronic inammation The effect of ACE on chronic skin inammation was evaluated by a slight modication of a previous described procedure (Burke, 2001). Briey, 20 l of a solution of TPA (2.5 g/ear 6 times) or acetone (vehicle) were topically applied to the inner and outer ear surfaces of both ears of each mouse with a micropipette on alternate days. ACE (200 mg/kg), vehicle (saline, negative control), or indomethacin (5 mg/kg, positive control) was given once a day (i.p.) for 10 days each morning immediately after TPA application. On day 10, the mice were sacriced at 6 h after treatment, and 6-mm2 diameter ear punch biopsies were collected and weighed. 2.7. ELISA assay procedure Serum levels of the cytokine proteins interleukin (IL)-1 and TNF- were determined 6 h after TPA application using a standard sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturers instructions (R&D Systems, Minneapolis, MN, USA). Briey, a 96-well plate coated with biotinylated antimouse monoclonal antibody was used. One hundred microliters of serial dilutions of the standard or sera samples were added to each well and incubated for 2 h at 37 C. After ve washes with wash solution, 100 l avidinhorseradish peroxidase solution were added to each well, and the plates were allowed to develop at 37 C for 2 h. After ve washes, the plate was maintained at 37 C for 30 min to react with 100 l of a substrate solution. The reaction was stopped by the addition of 100 l blocking solution, and then the absorbance at 450 nm was read with a microplate reader (molecular device). The results were expressed as arbitrary units of relative value. 2.8. Determination of myeloperoxidase activity Plasma MPO activity was assessed 24 h after repeated topical application of TPA using the mouse MPO ELISA kit according to the manufacturers instructions (Hycult Biotechnology, The Netherlands). Briey, a 96-well plate coated with biotinylated anti-mouse monoclonal antibody was used. One hundred microliters of serial dilutions of the standard or plasma samples were added to each well and incubated for 2 h at 37 C. After three washes with wash solution, 100 l avidinhorseradish peroxidase solution was added to each well, and the plates were allowed to develop at 37 C for

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1 h. The plate was again washed three times, then kept at 37 C for 30 min to react with 100 l of substrate solution. The reaction was stopped by the addition of 100 l blocking solution, and the absorbance at 450 nm was read with a microplate reader (molecular device). The results were expressed as arbitrary units of relative value. 2.9. Acetic acid-induced vascular permeability Acetic acid-induced vascular permeability in ICR mice was performed as described previously (Whittle, 1964). Briey, 1 h after oral administration of ACE (200 mg/kg), indomethacin (5 mg/kg), or an equivalent volume of vehicle (3%, v/v Tween 80), 0.2 ml of Evans blue dye (0.25% in normal saline) was administered intravenously through the tail vein. Thirty minutes later, the animals received 1 ml/100 g of acetic acid (0.6%, v/v) i.p. Treated animals were sacriced 30 min after acetic acid injection and the peritoneal cavity washed with normal saline (3 ml) into heparinized tubes and centrifuged. The dye content of the supernatant was measured at 610 nm using a microplate reader (molecular device). The results were expressed as arbitrary units of relative value. 2.10. Histology For histological assessment of cutaneous inammation, biopsies from control and treated ears of mice in each treatment group were collected and xed in 4% para-formaldehyde (0.1 M phosphate buffer, pH 7.4). The ear samples were dehydrated with 15%, 20%, 25%, and 30% serial sucrose solutions. A series of 10- m ear cross-sections was prepared by a freezing microtome. The sections were stained with haematoxylin and eosin (H&E) for the evaluation of leukocyte accumulation and edema. A representative area was selected for qualitative light microscopic analysis of the inammatory cellular response. To minimize bias, the investigator did not know which group was being analyzed. 2.11. Acute toxicity Different doses of ACE prepared in saline were given orally to groups of 10 mice each. For 30 days subsequent to treatment, the animals were observed daily, and dead animals were subjected to postmortem examination for determination of the cause of death. 2.12. Statistical analysis All data were expressed as mean S.D., and statistical signicance was determined via Students t-test with p < 0.05 considered to be signicant. 3. Results 3.1. Effect of ACE on TPA-induced cutaneous inammation We assessed the anti-inammatory activity of ACE in a TPA model of acute irritant contact dermatitis. Increased skin thickening is often the rst hallmark of skin irritation and local inammation. This parameter is one indicator of number of processes that occur during skin inammation, including increased vascular permeability, edema and swelling within the dermis, and proliferation of epidermal keratinocytes. Ear edema was measured in the dorsal skin prior to and at 6 h following treatments. Exposure to TPA resulted in marked increases in both skin thickness (Fig. 1A) and tissue weight (Fig. 1B). Topical application of acetone (vehicle) or ACE alone did not alter the skin thickness signicantly. However, ACE signicantly inhibited the phorbol ester-induced increases in

Fig. 1. Effect of ACE on TPA-induced ear thickness and weight changes in an acute inammation model. Mice were treated with normal saline (TPA + S), ACE (TPA + ACE), or indomethacin (TPA + Indo) for 1 h prior to topical application of acetone (vehicle) or TPA in acetone. Ear thickness (A) and ear weights (B) were measured at 6 h after TPA treatment. Each point represents the mean S.E.M. of the difference between 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 determined by the Students t-test.

both skin thickness and weight, indicating the therapeutic effect of this extract (Fig. 1A and B). Next, we investigated H&E-stained ear sections from TPAtreated animals. TPA application resulted in a marked increase in ear thickness, with clear evidence of edema, epidermal hyperplasia, and substantial inammatory cell inltration in the dermis with accompanying connective tissue disruption (Fig. 2A and B). ACE treatment reduced ear thickness and associated pathological indicators to an extent comparable to the positive control indomethacin (Fig. 2C and D). These results provide further evidence that ACE ameliorates TPA-induced contact dermatitis, directly illustrating its effects within the target tissue. 3.2. Effect of ACE on pro-inammatory cytokine production To assess the efcacy of ACE at the molecular level, we investigated the pro-inammatory cytokine-inhibitory effect of ACE. As shown in Fig. 3, topical application of TPA caused a dramatic increase in the production of IL-1 and TNF- by 6 h after challenge. In contrast, treatment with TPA plus ACE or indomethacin reduced both IL-1 and TNF- cytokine levels signicantly. Thus, ACE may reduce the levels of activated cellular inltrates and secretion of cytokines, thereby reducing cutaneous inammation.

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Fig. 2. Representative micrographs of H&E-stained mouse ear cross-sections in the acute TPA model. Ears were harvested 6 h post-treatment with acetone vehicle (A), TPA plus either normal saline (B), ACE (C), or indomethacin (D). Note the edema, polymorphonuclear cell inux and epidermal hyperplasia in TPA-treated ears and the reduction in inammatory cells and edema in ears with ACE treatment. Sections shown are representative of observations from ve animals in each group (400 magnication).

3.3. Effect of ACE on prolonged inammation induced by repeated TPA application As a second in vivo measure of the anti-inammatory activity of ACE, the extract was administrated in a mouse model of chronic skin inammation induced by repeated exposure to phorbol ester. Skin inammation in this model is persistent, which makes this model useful for assessing whether ACE resolves existing inammatory lesions (Burke, 2001; Stanley et al., 1991). Exposure to TPA resulted in marked increases in both skin thickness (Fig. 4A) and tissue weight (Fig. 4B). In contrast, ACE signicantly inhibited these phorbol ester-induced increases, indicating a therapeutic effect of this extract in the chronic model (Fig. 4A and B). Consistent with the edema parameters, ACE reduced the level of MPO activity, an indicator of polymorphonuclear leukocyte inux, by 39% in the chronic inammation model (Fig. 5). These ndings support the ability of ACE to resolve an existing, persistent inammatory lesion induced by multiple topical TPA applications, with an efcacy comparable to that of indomethacin.

3.4. Effect of ACE on acetic acid-induced vascular permeability As a third in vivo measure of the anti-inammatory activity of ACE, the extract was administrated in a mouse model of vascular permeability induced by acetic acid. It is well known that the increase of vascular permeability induced by acetic acid corresponds to the early exudative stage of inammation, one of the most important processes in the inammatory pathological mechanism (Whittle, 1964; Winter et al., 1962). ACE treatment signicantly inhibited acetic acid-induced vascular permeability in mice (Fig. 6).

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

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Fig. 3. Effect of ACE on TPA-induced IL-1 and TNF- production. Mice were treated with normal saline (TPA + S), ACE (TPA + ACE), or indomethacin (TPA + Indo), for 1 h prior to topical application of acetone (vehicle) or TPA in acetone. Serum was taken 6 h after TPA treatment and examined for the production of the pro-inammatory cytokines IL-1 (A) and TNF- (B) using ELISA. The data shown are the mean S.E.M. of the percent change relative to acetone-vehicle. N = 10 mice per group; *P < 0.01 compared to vehicle, and **P < 0.05 compared to TPA alone as determined by the Students t-test. Fig. 4. Effect of ACE on TPA-induced ear thickness and weight changes in the chronic inammation model. Mice were treated with repeated topical applications of TPA. Ear thickness (A) and ear weights (B) were measured on day 10. Each point represents the mean S.E.M. of the difference between 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 determined by the Students t-test.

4. Discussion This study provides evidence that ACE acts as an antiinammatory agent in mouse models of skin inammation. In both acute and chronic irritant contact dermatitis mouse models, ACE markedly reduced cutaneous inammation. This was supported by observed reductions in skin thickness and weight, amelioration of several histopathological indicators, decreased release of pro-inammatory cytokines, and diminished neutrophil activation. Results from our study demonstrate that ACE inhibits phorbol esterinduced increases in ear edema as well as acetic acid-induced vascular permeability. Together, these nding suggest that ACE may be an important therapeutic strategy for the treatment of inammatory skin diseases. One previous study reports that acute and chronic human atopic dermatitis lesions contain activated T cells and degranulated mast cells (Leung and Soter, 2001). Given this complex inammatory pathology, it has been difcult to establish relatively short-term animal models for pre-clinical drug testing. Although not an allergen-driven model, the phorbol ester-induced mouse ear inammation model produces edema and leads to recruitment of polymorphonuclear leukocytes acutely, with macrophages and T cells predominating by day 7 of the chronic model (Alford et al., 1992; Stanley et al., 1991). Since this model mimics several aspects of human atopic dermatitis, it is a reliable in vivo model system, providing opportunities to evaluate preventive treatment in acute disease and treatment of established lesions in chronic disease. Our present study clearly demonstrates that TPA exposure resulted in increased secretion of IL-1 and TNF- , suggesting that

Fig. 5. Effect of ACE on TPA-induced MPO activity. Mice were treated with repeated topical applications of TPA. Neutrophil activation levels were determined by an MPO activity assay of mouse plasma on day 10. TPA exposure resulted in a marked increase in plasma MPO levels as compared with acetone vehicle alone. The data shown are the mean S.E.M. of the percent change relative to acetone-vehicle. N = 10 mice per group; *P < 0.01 compared to vehicle, and **P < 0.01 compared to TPA alone as determined by the Students t-test.

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reduce vascular permeability and/or inhibits various inammatory mediators. In summary, this study reports for the rst time to our knowledge that ACE has anti-inammatory activities in both acute and chronic irritant contact dermatitis. The results presented in this study also suggest that the inhibitory effect of ACE may be due, at least in part, to the inhibition of IL-1 and TNF- and to the subsequent blockade of leukocyte accumulation. Our results suggest that ACE may be a good candidate for the treatment of inammatory skin diseases or may be useful towards the development of new anti-inammatory cutaneous therapies.

Acknowledgements This study, which is part of a larger project, was achieved with the support of the Conservation Technology Research and Development Project hosted by the National Research Institute of Cultural Heritage of the Korean Cultural Heritage Administration. The study was also partially supported by grants from the Construction of the Basis of the Practical Application of Herbal Resources funded by MOST. We express our gratitude to these funding agencies.

Fig. 6. Effect of ACE on acetic acid-induced vascular permeability in mice. Mice received oral administrations of ACE or indomethacin before i.p. injection of acetic acid. The data shown are mean S.E.M. of the percent inhibition. N = 10 mice per group; *P < 0.01 compared to TPA alone as determined by the Students t-test.

both cytokines mediate inammatory signaling and play a pivotal role in TPA-induced acute irritant contact dermatitis, a result that is consistent with results from others (Otuki et al., 2005; Ueda et al., 2004). We also showed that ACE negatively interfered with these cytokines, which are known to play important roles in the inammatory process. These results were supported by the ndings of Koo et al., who showed that ACE signicantly inhibits the secretion of TNF- in dose-dependent manner (Koo et al., 2000). We demonstrated that ACE inhibits MPO activation in a mouse model of chronic skin inammation. MPO is an enzyme found in the azurophilic granules of neutophils and other cells of myeloid origin, and is commonly used as an index of granulocyte inltration. MPO inhibition is indicative of anti-inammatory activity in the chronic inammation model (Ajuebor et al., 2000). Interestingly, our histological analysis of the ear clearly conrmed that ACE inhibited the inux of polymorphonuclear leukocytes to the mouse ear skin following TPA application. We propose that the marked inhibition by ACE against ear edema, MPO activity, activation and migration of leukocytes in response to topical TPA may be related to inhibition of pro-inammatory cytokine release. This concept correlates well with previous ndings that cells in the injured skin, such as dermal dendritic cells, epidermal Langerhans cells, melanocytes, broblasts, and leukocytes, are known to be sources and targets of cytokines (Grone, 2002). Taken together, these results support the notion that ACE possesses antiinammatory properties. Indeed, neutrophil accumulation plays a critical role in cutaneous inammatory diseases such as dermatitis, and is related to the pathological mechanism of disease (Bradley et al., 1982). We have provided what we believe to be the rst report of the anti-inammatory effects of ACE in in vivo skin inammation, and our results suggest that ACE is a potential therapy for the treatment of skin inammation. Acetic acid-induced vascular permeability causes an immediate sustained reaction that is prolonged over 24 h and leads to exudation of uid rich in plasma proteins (Whittle, 1964; Winter et al., 1962). It is therefore used as a well-characterized mouse model of acute inammation, and inhibition of vascular permeability is considered a major feature for the suppression of the exudative phase of acute inammation (Okoli et al., 2007). Here, we demonstrate that ACE had signicant anti-inammatory effects in this model, indicating that ACE may affect membrane-stabilization to

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