traditional herbal medicine and allergic...

Traditional Herbal Medicine and Allergic Asthma

Guest Editors: Bi-Fong Lin, Bor-Luen Chiang, Yan Ma, Jin-Yuarn Lin, and Miaw-Ling Chen

Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine


Guest Editors: Bi-Fong Lin, Bor-Luen Chiang, Yan Ma,Jin-Yuarn Lin, and Miaw-Ling Chen

Copyright 2015 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in Evidence-Based Complementary and Alternative Medicine. All articles are open access articlesdistributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the original work is properly cited.

Editorial Board

Mona Abdel-Tawab, GermanyJon Adams, AustraliaGabriel A. Agbor, CameroonUlysses P. Albuquerque, BrazilSamir Lutf Aleryani, USAAther Ali, USAM. Ali-Shtayeh, PalestineGianni Allais, ItalyTerje Alraek, NorwayShrikant Anant, USAIsabel Andjar, SpainLetizia Angiolella, ItalyVirginia A. Aparicio, SpainMakoto Arai, JapanManuel Arroyo-Morales, SpainHyunsu Bae, Republic of KoreaGiacinto Bagetta, ItalyOnesmo B. Balemba, USAWinfried Banzer, GermanyPanos Barlas, UKVernon A. Barnes, USASamra Bashir, PakistanPurusotam Basnet, NorwayJairo Kennup Bastos, BrazilSujit Basu, USAArpita Basu, USAGeorge D. Baxter, New ZealandAndre-Michael Beer, GermanyAlvin J. Beitz, USALouise Bennett, AustraliaMaria Camilla Bergonzi, ItalyAnna R. Bilia, ItalyYong C. Boo, Republic of KoreaMonica Borgatti, ItalyFrancesca Borrelli, ItalyGeoffrey Bove, USAGloria Brusotti, ItalyArndt Bussing, GermanyRainer W. Bussmann, USAAndrew J. Butler, USAGioacchino Calapai, ItalyGiuseppe Caminiti, ItalyRaffaele Capasso, ItalyFrancesco Cardini, ItalyOpher Caspi, Israel

Subrata Chakrabarti, CanadaPierre Champy, FranceShun-Wan Chan, Hong KongIl-Moo Chang, Republic of KoreaChun T. Che, USAKevin Chen, USAEvan P. Cherniack, USASalvatore Chirumbolo, ItalyW. Chi-shing Cho, Hong KongJae Youl Cho, KoreaKathrine B. Christensen, DenmarkShuang-En Chuang, TaiwanY. Clement, Trinidad And TobagoPaolo Coghi, ItalyMarisa Colone, ItalyLisa A. Conboy, USAKieran Cooley, CanadaEdwin L. Cooper, USAOlivia Corcoran, UKMuriel Cuendet, SwitzerlandRoberto K. N. Cuman, BrazilVincenzo De Feo, ItalyRoco De la Puerta, SpainLaura De Martino, ItalyNunziatina De Tommasi, ItalyMartin Descarreaux, USAAlexandra Deters, GermanyFarzad Deyhim, USAManuela Di Franco, ItalyClaudia Di Giacomo, ItalyAntonella Di Sotto, ItalyM. Dijoux-Franca, FranceLuciana Dini, ItalyTieraona L. Dog, USACaigan Du, CanadaJeng-Ren Duann, TaiwanNativ Dudai, IsraelThomas Efferth, GermanyAbir El-Alfy, USATobias Esch, USAGiuseppe Esposito, ItalyKeturah R. Faurot, USAYibin Feng, Hong KongNianping Feng, ChinaPatricia D. Fernandes, Brazil

Josue Fernandez-Carnero, SpainAntonella Fioravanti, ItalyFabio Firenzuoli, ItalyPeter Fisher, UKFilippo Fratini, ItalyBrett Froeliger, USAMaria pia Fuggetta, ItalyJoel J. Gagnier, CanadaSiew Hua Gan, MalaysiaMary K. Garcia, USASusana Garcia de Arriba, GermanyDolores G. Gimenez, SpainGabino Garrido, ChileIpek Goktepe, QatarMichael Goldstein, USAYuewen Gong, CanadaSettimio Grimaldi, ItalyGloria Gronowicz, USAMaruti Ram Gudavalli, USAAlessandra Guerrini, ItalyNarcis Gusi, SpainSvein Haavik, NorwaySolomon Habtemariam, UKAbid Hamid, IndiaMichael G. Hammes, GermanyKuzhuvelil B. Harikumar, IndiaCory S. Harris, CanadaJan Hartvigsen, DenmarkThierry Hennebelle, FranceLise Hestbaek, DenmarkEleanor Holroyd, AustraliaMarkus Horneber, GermanyChing-Liang Hsieh, TaiwanBenny T. K. Huat, SingaporeRoman Huber, GermanyHelmut Hugel, AustraliaCiara Hughes, UKAttila Hunyadi, HungarySumiko Hyuga, JapanH. Stephen Injeyan, CanadaChie Ishikawa, JapanAngelo A. Izzo, ItalyChris J. Branford-White, UKSuresh Jadhav, IndiaG. K. Jayaprakasha, USA

Gao jianli, ChinaStefanie Joos, GermanyZeev L Kain, USAOsamu Kanauchi, JapanWenyi Kang, ChinaShao-Hsuan Kao, TaiwanJuntra Karbwang, USAKenji Kawakita, JapanDeborah A. Kennedy, CanadaYoun C. Kim, Republic of KoreaC.-H. Kim, Republic of KoreaYoshiyuki Kimura, JapanToshiaki Kogure, JapanJian Kong, USATetsuya Konishi, JapanKarin Kraft, GermanyOmer Kucuk, USAVictor Kuete, CameroonYiu W. Kwan, Hong KongKuang C. Lai, TaiwanIlaria Lampronti, ItalyLixing Lao, Hong KongChristian Lehmann, CanadaMarco Leonti, ItalyLawrence Leung, CanadaShahar Lev-ari, IsraelMin Li, ChinaXiu-Min Li, USAChun G. Li, AustraliaBi-Fong Lin, TaiwanHo Lin, TaiwanChristopher G. Lis, USAGerhard Litscher, AustriaI-Min Liu, TaiwanYijun Liu, USAVctor Lopez, SpainThomas Lundeberg, SwedenFilippo Maggi, ItalyValentina Maggini, ItalyGail B. Mahady, USAJamal Mahajna, IsraelJuraj Majtan, SlovakiaFrancesca Mancianti, ItalyCarmen Mannucci, ItalyFulvio Marzatico, ItalyMarta Marzotto, ItalyJames H. McAuley, AustraliaKristine McGrath, Australia

James S. McLay, UKLewis Mehl-Madrona, USAPeter Meiser, GermanyKarin Meissner, GermanyAlbert S Mellick, AustraliaA. Guy Mensah-Nyagan, FranceAndreas Michalsen, GermanyOliver Micke, GermanyRoberto Miniero, ItalyGiovanni Mirabella, ItalyDavid Mischoulon, USAFrancesca Mondello, ItalyAlbert Moraska, USAGiuseppe Morgia, ItalyMark Moss, UKYoshiharu Motoo, JapanKamal D. Moudgil, USAYoshiki Mukudai, JapanFrauke Musial, GermanyMinKyun Na, Republic of KoreaHajime Nakae, JapanSrinivas Nammi, AustraliaKrishnadas Nandakumar, IndiaVitaly Napadow, USAMichele Navarra, ItalyIsabella Neri, ItalyPratibha V. Nerurkar, USAKaren Nieber, GermanyMenachem Oberbaum, IsraelMartin Offenbaecher, GermanyJunetsu Ogasawara, JapanKi-Wan Oh, Republic of KoreaYoshiji Ohta, JapanOlumayokun A. Olajide, UKThomas Ostermann, GermanyStacey A. Page, CanadaSiyaram Pandey, CanadaBhushan Patwardhan, IndiaBerit S. Paulsen, NorwayPhilip Peplow, New ZealandFlorian Pfab, GermanySonia Piacente, ItalyAndrea Pieroni, ItalyRichard Pietras, USAAndrew Pipingas, AustraliaJose M. Prieto, UKHaifa Qiao, USAWaris Qidwai, Pakistan

Xianqin Qu, AustraliaCassandra L. Quave, USAE. Ferreira Queiroz, SwitzerlandRoja Rahimi, IranKhalid Rahman, UKCheppail Ramachandran, USAElia Ranzato, ItalyKe Ren, USAMan H. Rhee, Republic of KoreaLuigi Ricciardiello, ItalyDaniela Rigano, ItalyJose L. Ros, SpainPaolo Roberti di Sarsina, ItalyMariangela Rondanelli, ItalyOmar Said, IsraelAvni Sali, AustraliaMohd Z. Salleh, MalaysiaA. Sandner-Kiesling, AustriaManel Santafe, SpainTadaaki Satou, JapanClaudia Scherr, SwitzerlandG. Schmeda-Hirschmann, ChileAndrew Scholey, AustraliaRoland Schoop, SwitzerlandSven Schroder, GermanyHerbert Schwabl, SwitzerlandVeronique Seidel, UKSenthamil R. Selvan, USAFelice Senatore, ItalyHongcai Shang, ChinaKaren J. Sherman, USARonald Sherman, USAKuniyoshi Shimizu, JapanKan Shimpo, JapanYukihiro Shoyama, JapanMorry Silberstein, AustraliaK. N. S. Sirajudeen, MalaysiaGraeme Smith, UKChang-Gue Son, KoreaRachid Soulimani, FranceDidier Stien, FranceCon Stough, AustraliaAnnarita Stringaro, ItalyShan-Yu Su, TaiwanBarbara Swanson, USAGiuseppe Tagarelli, ItalyOrazio Taglialatela-Scafati, ItalyTakashi Takeda, Japan

Ghee T. Tan, USAHirofumi Tanaka, USALay Kek Teh, MalaysiaNorman Temple, CanadaMayankThakur, GermanyMenaka C. Thounaojam, USAEvelin Tiralongo, AustraliaStephanie Tjen-A-Looi, USAMicha Tomczyk, PolandLoren Toussaint, USAYew-Min Tzeng, TaiwanDawn M. Upchurch, USAKonrad Urech, SwitzerlandTakuhiro Uto, JapanSandy van Vuuren, South Africa

Alfredo Vannacci, ItalySubramanyam Vemulpad, AustraliaCarlo Ventura, ItalyGiuseppe Venturella, ItalyPradeep Visen, CanadaAristo Vojdani, USADawnWallerstedt, USAShu-Ming Wang, USAYong Wang, USAChong-Zhi Wang, USAJ. L. Wardle, AustraliaKenji Watanabe, JapanJ. Wattanathorn, ThailandMichael Weber, GermanySilvia Wein, Germany

Janelle Wheat, AustraliaJenny M. Wilkinson, AustraliaDarren Williams, Republic of KoreaChristopher Worsnop, AustraliaHaruki Yamada, JapanNobuo Yamaguchi, JapanJunqing Yang, ChinaLing Yang, ChinaEun J. Yang, Republic of KoreaKen Yasukawa, JapanAlbert S. Yeung, USAArmando Zarrelli, ItalyChris Zaslawski, AustraliaRuixin Zhang, USA

Contents

Traditional Herbal Medicine and Allergic Asthma, Bi-Fong Lin, Bor-Luen Chiang, Yan Ma, Jin-Yuarn Lin,and Miaw-Ling ChenVolume 2015, Article ID 510989, 2 pages

Ethanol Extract of Perilla frutescens Suppresses Allergen-SpecificTh2 Responses and Alleviates AirwayInflammation and Hyperreactivity in Ovalbumin-Sensitized Murine Model of Asthma,Miaw-Ling Chen, Chi-Heng Wu, Li-Shiuan Hung, and Bi-Fong LinVolume 2015, Article ID 324265, 8 pages

Characteristics Associated with Utilization of Asthma-Related Traditional Chinese Medicine Servicesamong Asthma Children in Taiwan: A Nationwide Cohort Study, Shiou-Ian Lin, Tung-Hu Tsai,Yiing-Jenq Chou, and Nicole HuangVolume 2015, Article ID 108961, 8 pages

Triterpenoids and Polysaccharide Fractions of Ganoderma tsugae Exert Different Effects on AntiallergicActivities, Miaw-Ling Chen, Chia-Chien Hsieh, Bor-Luen Chiang, and Bi-Fong LinVolume 2015, Article ID 754836, 10 pages

Farnesol, a Sesquiterpene Alcohol in Herbal Plants, Exerts Anti-Inflammatory and Antiallergic Effectson Ovalbumin-Sensitized and -Challenged Asthmatic Mice, Chi-Mei Ku and Jin-Yuarn LinVolume 2015, Article ID 387357, 12 pages

Two Years versus One Year of TianjiuTherapy in Sanfu Days for Chronic Asthma: A Clinical EfficacyObservation Trial, Li Bing Zhu, Wei Zhang, Vivian Wong, Ziea Eric, Kwai Ching Lo, Wai Chung Chan,To Yau, and Lei LiVolume 2014, Article ID 807598, 9 pages

EditorialTraditional Herbal Medicine and Allergic Asthma

Bi-Fong Lin,1 Bor-Luen Chiang,2 Yan Ma,3 Jin-Yuarn Lin,4 and Miaw-Ling Chen5

1Department of Biochemical Science and Technology, National Taiwan University, Taipei 10617, Taiwan2Graduate Institute of Immunology, National Taiwan University, Taipei 10048, Taiwan3Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology & Immunology,Vienna General Hospital, Medical University of Vienna, Vienna, Austria4Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan5Department of Nutrition and Health Science, Chang Jung Christian University, Tainan City 71101, Taiwan

Correspondence should be addressed to Bi-Fong Lin; [email protected]

Received 12 March 2015; Accepted 12 March 2015

Copyright 2015 Bi-Fong Lin et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The frequency of allergic diseases such as asthma and allergicrhinitis has increased rapidly during the past decade; how-ever, the exact mechanisms have still not been established.Both air pollution and change of diet habit have been thoughtto play an important role in increasing prevalence of atopicdiseases. Atopic diseases were mediated predominantly bytype 2 T helper- (Th2-) mediated activity including allergen-specific IgE antibody and eosinophils. Allergic asthma is achronic disease with the characteristics of immune responsemediated by type 2 T helper- (Th2-) related cytokines andIgE antibody. Allergic airway inflammation has been char-acterized by the infiltration of Th2 cells and eosinophils,subsequently followed by the bronchial constriction andmucus secretion.

Conventional treatments for allergic asthma includesteroids, leukotriene antagonists, bronchodilators, and mostrecent anti-IgE antibody. All these drugs are still withcertain shortcomings such as side effects, effectiveness, andcost. It has become more and more important to developnovel therapeutic approaches for the treatment of allergicasthma. Complementary medical approaches such Chineseherb medication and acupuncture have been suggested toplay a role in the immune regulation of diseases [1]. Morestudies have focused on exploring the possibility of thesecomplementary therapeutic approaches for the treatmentof immunological diseases [2]. All these complimentarytherapeutic approaches have been regarded as having less sideeffects and being used as the adjuvant therapy for the diseases

[3]. Furthermore, many researchers also aim to identify theactive components of herb medicine for the purification anddevelopment of drugs.

In this special issue, we have five articles including thesurvey of traditional Chinese medicine application for thetreatment of allergic asthma and also the study on thetraditional Tianjiu therapy for the treatment of asthmaticpatients. Furthermore, three articles on studying the activecomponents of herb medicine have been included in theissue. S.-I. Lin et al. analyzed the use of Chinese traditionalmedicine for the treatment of allergic asthma in Taiwan.They collected 20,800 newly diagnosed asthmatic childrenand found out 20% of them actually used traditional Chinesemedicine as the treatment of their asthma. In addition, theyalso analyzed the most frequent used herbal medicine forallergic asthma in the article.

A variety of components purified from medicinal herbshave been found to exert the immune modulatory effect onmany diseases [4]. Among the components, polyphenols,triterpenoids, and polysaccharides are found to be the mosteffective in the anti-inflammatory or immune modulationof the diseases. M.-L. Chen et al. have identified bothtriterpenoids and polysaccharide portion for the treatmentof allergic diseases. The results showed that triterpenoidsportion of Ganoderma tsugae exerted anti-inflammatoryactivity and polysaccharide portion had the immune stim-ulatory effect instead. C.-M. Ku and J.-Y. Lin also identifiedan active component of farnesol, a sesquiterpene alcohol,

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015, Article ID 510989, 2 pageshttp://dx.doi.org/10.1155/2015/510989

http://dx.doi.org/10.1155/2015/510989

2 Evidence-Based Complementary and Alternative Medicine

exerting anti-inflammatory activity and alleviating airwayinflammation in murine model of asthma. M.-L. Chen etal. also identified ethanol extract of Perilla frutescens whichalso exerted anti-inflammatory activity and also alleviatedallergic airway inflammation. Finally, L. Zhu et al. studied theeffect of Tianjiu therapy in Sanfu Days for the treatment ofasthmatic children.The results suggested that Tianjiu therapycould decrease the dose of bronchodilator during the asthmaattack. However, the symptoms of allergic asthma did notshow significant improvement after treatment. More studiesare needed for the application of Tianjiu therapy for thetreatment of allergic asthma in the future.

The prevalence of clinical immunological diseases suchas autoimmune diseases and allergic diseases is increasingin recent years. More and more efforts have been dedicatedto develop novel therapeutic approaches for the treatment ofthese immune-mediated diseases. Complimentary medicinesuch as herb drugs or acupuncture has been suggested to beuseful for the treatment of a variety of clinical immunologicaldiseases [5]. Many of these herb medicinal componentshave been found to exert anti-inflammatory activity andmodulation of immune response. Although more studies areneeded to identify the novel compounds and the treatment,it might still be the future target for the novel therapeuticdevelopment. Particularly, these herb medicinal drugs arenoted with fewer side effects, which might also be useful forpossible adjuvant therapy.

Bi-Fong LinBor-Luen Chiang

Yan MaJin-Yuarn Lin

Miaw-Ling Chen

References

[1] M. George, M. Topaz, C. Rand et al., Inhaled corticosteroidbeliefs, complementary and alternative medicine, and uncon-trolled asthma in urban minority adults, Journal of Allergy andClinical Immunology, vol. 134, pp. 12521259, 2014.

[2] T. P. Huang, P. H. Liu, A. S. Lien, S. L. Yang, H. H. Chang, andH. R. Yen, Characteristics of traditional Chinese medicine usein children with asthma: a nationwide population-based study,Allergy, vol. 68, no. 12, pp. 16101613, 2013.

[3] W. S. Silvers and H. K. Bailey, Integrative approach to allergyand asthma using complementary and alternative medicine,Annals of Allergy, Asthma & Immunology, vol. 112, pp. 280285,2014.

[4] C. J. Chang, Y. H. Yang, Y. C. Liang et al., A novel phycobilipro-tein alleviates allergic airway inflammation by modulatingimmune response,American Journal of Respiratory and CriticalCare Medicine, vol. 183, pp. 1525, 2011.

[5] W. Morgan, H. Center, C. Arms-Chavez, and S. G. LoBello,Complementary and alternative medicine use and asthma:relation to asthma severity and comorbid chronic disease,TheJournal of Asthma, vol. 51, pp. 333338, 2014.

Research ArticleEthanol Extract of Perilla frutescens SuppressesAllergen-Specific Th2 Responses and Alleviates AirwayInflammation and Hyperreactivity in Ovalbumin-SensitizedMurine Model of Asthma

Miaw-Ling Chen,1 Chi-Heng Wu,2 Li-Shiuan Hung,2 and Bi-Fong Lin2

1Department of Nutrition and Health Sciences, College of Health Sciences, Chang Jung Christian University, No. 1, Changda Road,Guiren District, Tainan City 71101, Taiwan2Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Sec. 4, No. 1 Roosevelt Road,Taipei 10617, Taiwan

Correspondence should be addressed to Miaw-Ling Chen; [email protected] and Bi-Fong Lin; [email protected]

Received 15 August 2014; Accepted 10 November 2014

Academic Editor: Bor-Luen Chiang

Copyright 2015 Miaw-Ling Chen et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

This studywas to investigate the effects of different fractions of Perilla frutescens (Pf) leaves extracted bywater or ethanol on asthma.BALB/c mice sensitized intraperitoneally and challenged with ovalbumin (OVA) were divided into six groups. Each group of micewas tube-feedingwith 0 (control), 80 g (PfWL), or 320 g (PfWH)water extracts or 80 g (PfEL) or 320g (PfEH) ethanol extractsof perilla leaves daily for 3 weeks. A negative control group (PBS) was neither sensitized nor treated with Pf. The effects of perillaleave extracts on allergic immune response were evaluated. The results showed that OVA-specific IL-5 and IL-13 secretions fromOVA-stimulated splenocytes were significantly suppressed in the ethanol extract groups PfEL and PfEH. Serum level of anti-OVAIgE tended to be lower in the PfEH group.The inflammatory mediators, such as eotaxin and histamine, and total cells, particularlyeosinophils in bronchoalveolar lavage fluid (BALF), were also decreased in the PfEL and the PfEH groups.Therefore, the PfEL andthe PfEH groups had significantly lowermethacholine-induced hyperresponsiveness (AHR). In conclusion, ethanol extracts, ratherthan water extract, of perilla leaves could significantly suppress Th2 responses and airway inflammation in allergic murine modelof asthma.

1. Introduction

Allergic asthma is a chronic disease that clinically augmentsbronchial hyperresponsiveness and inflammation. The asth-matic inflammation is clearly associated with the high level oftype 2 T cell (Th2) cytokines that induced immunoglobulin(Ig) E production and eosinophilic infiltration [1]. The Th2cytokines IL-4, IL-5, and IL-13 are the major cytokines fordevelopment of atopic diseases, such as asthma, rhinitis,and dermatitis [2]. IL-4 promotes the immunoglobulin classswitch from IgM to IgE [3]. IL-5 induces eosinophilia activityand infiltration, which is the critical response in the allergicasthma [4]. IL-13 acts to induce airway hyperresponsiveness(AHR) that contributes to atopic disease [5].The suppression

of Th2 responses is a feasible attempt to attenuate thesymptoms of allergic asthma. Therefore, biologic targetedtherapies have been developed to target the specificmolecularpathways to treat asthma, especially those targets at IL-4,IL-5, IL-13, and IgE [6]. However, due to complex clinicalsymptoms and multiple mechanisms involved, the outcomeof these trials has not been satisfied. Epidemiological studiesindicate that patients often turn to complementary and alter-native therapies, including dietary supplements [7]. Studiesalso showed that dietary oil, adlay, and medicinal herbs, suchas Ganoderma and Andrographis, decreased Th2 cytokinesproductions and thus alleviated allergic responses in amurinemodel of asthma [812], suggesting the potential applicationof traditional herbal medicine for immunomodulation.


http://dx.doi.org/10.1155/2015/324265


Perilla frutescens (Pf) leaf is a kind of aromatic vegetables,which is also used as the traditional medicine in Asia. Perillahas been demonstrated to exert antiobesity, anti-dyslipi-demia, antioxidant, and anti-infammation [1319]. Recently,some researches focused on its antiallergic effects [2023].The studies suggested water extract of Pf inhibited histaminerelease from mast cells [24]. Subcutaneous injection withperilla seed water/ethanol extract, as a traditional orientaltherapeutic herbal acupuncture, seemed to reduce IgE, IL-4,IL-5, and IL-13 in BALF inOVA-induced asthma inmice [18].

However, they are either intraperitoneal injection [24],type II allergy evaluated by ear-passive cutaneous anaphylaxisor edema [20, 21], or lack of the important outcome oftreatment in asthma, airway hyperresponsiveness (AHR)result [22]. There are few studies investigating the effects oforal supplementation of perilla extracts on overall allergicresponses of asthmatic murine model. In addition, the crudepolysaccharide isolated from Pf by hot water extraction wasshown to increase nitric oxide andTNF and IL-6 productionboth in vitro and in vivo [25]. Therefore, whether water orethanol extracts of Perilla frutescens exert antiasthma effectsvia oral supplementation was investigated in this study usingOVA-sensitized and challenged BALB/c mice.

2. Materials and Methods

2.1. Preparation of Extracts of Perilla frutescens. The crudeextracts of Perilla frutescens (L.) Britt leaves were extractedas follows. Dried Perilla frutescens leaves were extracted with5-fold (w/v) hot water for 60min. The water extracts (PfW)were filtered through filter paper and concentrated using arotary evaporator and then freeze-dried. The yield of PfWwas 21.70% (w/w, dry basis). The ethanol extracts of driedPerilla frutescens leaves (PfE) were extracted with 15-fold(w/v) 95% ethanol for 24 hr. The PfE were filtered by filterpaper and then evaporated in a rotary evaporator to removethe solvent. The yield of PfE was 9.22% (w/w, dry basis).

2.2. Animal Study, Sensitization, andAirwayChallenge. FemaleBALB/c mice were purchased from the Animal Center atthe National Taiwan University College of Medicine, Taipei,Taiwan, and maintained at the Department of BiochemicalScience and Technology at the National Taiwan University.Animal care and handling conformed to accepted guideline[26].

The allergic asthma model, as well as the diet, was manip-ulated as described [11]. As shown in Figure 1, 8-week-oldfemale BALB/c mice were sensitized intraperitoneally (i.p.)with 10 g ovalbumin (OVA; Sigma, St. Louis, Mo) in alumadjuvant (Imject Alum; Pierce, Rockford, IL). After threetimes OVA-sensitization, sensitized mice were grouped ran-domly into six groups for tube-feeding with 0 g (Control),80 g (PfWL), or 320 g (PfWH) water extract or 80g(PfEL) or 320 g (PfEH) ethanol extract of perilla leaves dailyfor 3 weeks. During these 3 weeks, the OVA-sensitized micewere challenged twice with 5% aerosolizedOVA in PBS bufferby inhalation. In addition, mice without Pf supplement, thatreceived alum without OVA, and that inhaled aerosolizedPBS are used as the negative control group (PBS). After 3

Day 634228140

PfW or PfE supplementation from day 42 to day 62

6249 56

AHR Sacrifice

OVA sensitizationi.p.

Airway OVA challengeinhalation

Figure 1: Schematic diagram of the experimental protocol. Eight-week-old female BALB/c mice were sensitized intraperitoneally(i.p.) by OVA for three times (10, 30, and 30g/mouse) on days0, 14, and 28. OVA-sensitized mice were challenged with 5%aerosolized OVA three times on days 49, 56, and 62. After 3 weeksof supplementation, mice were sacrificed and the BALF, serum, andsplenocytes of mice were collected for further analysis.

weeks of Pf supplement, mice that received a third challengewere euthanized byCO

2inhalation and the BALF, serum, and

splenocytes of mice were collected for further analysis.

2.3. Determination of AirwayHyperresponsiveness (AHR). Air-way function was measured by the whole-body plethysmog-raphy as described previously [11]. Twenty-four hours afterthe secondary challenge, AHR was measured when micewere stimulated with methacholine (Sigma) using whole-body plethysmography (Buxco,Wilmington, NC). Mice wereplaced in the main chamber of a whole-body plethysmogra-phy and challenged with aerosolized methacholine, increas-ing concentration from 12.5 to 50mg/mL in PBS buffer.The AHR is expressed as the enhanced pause (Penh) valuesmeasured during each 3min period.

2.4. Determination of Anti-OVA Antibodies. Serum anti-OVA IgE antibody titers were measured by ELISA methodas previously described [27]. Briefly, 96-well plates werecoated with 10 g OVA/mL NaHCO

3buffer. After overnight

incubation at 4C and being blocked with 1% bovine serumalbumin (BSA)/PBS buffer for 2 hours at room temperature,the serum samples and positive control sera were appro-priately diluted with blocking buffer and added to the 96-well plate. After 2 hours, the biotin-conjugated anti-mouseIgE (PharMingen, San Diego, CA) was added for 2 hoursof incubation.Then, streptavidin-conjugated peroxidases andthe enzyme substrate, 2,2-azino-bis-3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), were added and incubated for 20minat room temperature.The antibody levels of the samples werecompared with the positive control sera. The positive controlwas a pool of serum collected from OVA-sensitized micewith strong response (optical density > 1). The results of theantibody titer were expressed in ELISA units (EU), EU =( sample blank)/(positive blank).

2.5. Collection of BALF and Splenocytes. The BALF wascollected with 5 instillations of 0.5mL saline. Approximately2.5mL of fluid was recovered with each sample and the

Evidence-Based Complementary and Alternative Medicine 3

volume did not differ significantly among groups. The fluidwas collected and kept at 80C for eotaxin and histamineanalysis. The cell pellet was resuspended in 250 L salinecontaining 10% BSA. The total cells were counted with ahemocytometer using the trypan blue dye exclusion method.BALF cells with a concentration of 2 105 were cytocen-trifuged and then stained with Lius stain for eosinophilcounts.

The splenocytes were prepared by aseptically removingspleens from sensitized and challenged BALB/c mice [27].Splenocytes were counted with a hemocytometer using thetrypan blue dye exclusion method. A concentration of 5 106 cells/mL was cultured in 48-well plates in RPMI-1640medium supplemented with TCMmedium in the absence orpresence of antigen, 50 and 100g/mL OVA, for 48 hours.The supernatants in cell cultures were collected and stored at80C for cytokines analysis.

2.6. Cytokines Assay. The cytokine levels in splenocytesculture supernatants were measured by sandwich ELISAmethods. Briefly, the anti-cytokine antibodywas coated in the96-well plates (Nunc, Roskilde, Denmark). After overnightincubation at 4C and being blocked with 1% BSA/PBS bufferfor 30min, the samples and standards were added to the 96-well plates for 2 hours of incubation. The biotin-conjugatedantibodies were added and incubated. After washing, thestreptavidin-conjugated peroxidase was added for 1 hour.Thesubstrate ABTS was added to each well for 20min.The plateswere read in a microplate autoreader (microplate autoreader;Bio-Tek Instrument, Inc. Winooski, VT) at 405 nm.

2.7. Eotaxin and Histamine Assay. The eotaxin concentra-tion in BALF was determined by mouse eotaxin sandwichELISA kit (R&D Systems, Minneapolis, MN). The eotaxinconcentration was assayed according to the manufacturersinstructions. After the color reagent was added, the platewas incubated at room temperature for 20min for colordevelopment. The absorbance was measured at 630 nm inthe microplate autoreader. The eotaxin level in BALF wasdetermined using the standard curve.

The histamine level was determined byHistamine-ELISAkit (IBL Hamburg, Germany).Themanipulation was accord-ing to manufacturers instructions for use. In brief, theculture supernatants and plasma standards were acylatedwith acylation reagent first. Then, aliquots of 50L acylatedsample and acylated standards were loaded into the 96-microplate wells, respectively. Aliquots of 50L enzymeconjugate and 50 L antiserum were pipetted into each wellto react for 3 hours. The plate was washed four times and200L tetramethylbenzidine (TMB) substrate solution wasadded for 30min. Then the reaction was stopped by adding100 L of stop solution into each well. The optical densitywas detected by the microplate autoreader at 450 nm. Thehistamine concentration is determined using the standardcurve.

2.8. Statistical Analysis. Data were expressed as mean SD. The significance of difference between the Pf and thecontrol groups was analyzed statistically by Students -test

of the SAS program system (Strategic Application Software;SAS windows version 8.2, SAS Institute Inc., Cary, NC)throughout the study.

3. Results

3.1. Ethanol Extracts of Perilla frutescens Suppressed Th2Responses of OVA-SensitizedMice. After feeding of two dosesof water extract of Pf (PfWL and PfWH) or ethanol extractof Pf (PfEL and PfEH) for 3 weeks, splenocytes were isolatedfrom OVA-sensitized mice and stimulated with OVA toexamine the effects of PfW and PfE on allergen-specific Th2responses. The Th1 cytokines IL-2 and IFN were not sig-nificantly affected (data not shown). However, Th2 cytokinesIL-5 and IL-13 productions of OVA-stimulated splenocytes inthe PfEL and the PfEH groups were significantly lower thanthose of the control group, as shown in Figure 2. The OVA-stimulated IL-4 productions were low and not affected byeither water or ethanol extracts. In addition, PHA-stimulatedIL-13 productions by splenocytes were also suppressed byPfE (data not shown). These data suggested that ethanolextracts of Pf could inhibit allergen-specific stimulated Th2cells activity.

The IgE antibody produced by Th2 cell-activated Bcells tended to be lower in serum of OVA-sensitized micesupplemented with PfEH (Figure 3). The serum levels OVA-specific IgG

1and IgG

2a were not significantly affected (datanot shown). This data suggested that ethanol extracts ofPerilla frutescensmight have potential to suppress serum IgElevels in OVA-sensitized mice.

3.2. Ethanol Extracts of Perilla frutescens Decrease CellsInfiltration in AirwayAllergic Inflammation of OVA-SensitizedMice. The BALF was collected after OVA-inhalation chal-lenge prior to sacrifice and the cell number in BALF wasdetermined and shown in Figure 4. Few total cells andeosinophils were in the PBS negative control group. Theinfiltrated cells number increased after OVA-challenge asshown in the control group. The total cell number in BALFsignificantly decreased in asthmatic mice supplemented withPfEH (Figure 4(a)). Particularly, the number of eosinophils,the major cells contributing to airway inflammation ofallergic asthma,was decreased by Pf supplement, significantlyin both the PfEL and the PfEH groups (Figure 4(b)). Thesedata suggested that Perilla frutescens inhibited the infiltrationof inflammatory cells to BALF in asthmatic mice, especiallythe ethanol extracts.

The proinflammatory mediators such as histamine andeotaxin in BALF of OVA-sensitized mice were also sup-pressed by Pf, as shown in Figure 5.The histamine levels weresignificantly lower in both the PfEL and the PfEH groups.The eosinophil chemotactic protein eotaxin in BALF wasalso reduced in the PfEH group. Th2 cytokines in BALFof OVA-sensitized mice were also measured but were notfound significantly lower by Pf in this experiment (datanot shown). These results indicated that ethanol extracts ofPerilla frutescens alleviated inflammatory cell infiltration andthus reduced the allergic inflammation in airway of OVA-sensitized and challenged mice.


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Figure 2: The Th2 cytokines produced by OVA-stimulated splenocytes from OVA-sensitized/challenged BALB/c mice supplemented withdifferent extracts of Perilla frutescens.OVA-sensitizedmice were fedwith water (PfWL or PfWH) or ethanol (PfEL or PfEH) extracts ofPerillafrutescens for 3 weeks, respectively. Splenocytes were isolated fromOVA-sensitized/challengedmice and stimulated with OVA (100g/mL forIL-4, 50 g/mL for IL-5 and IL-13) for 48 hours.The productions ofTh2 cytokines in supernatant were determined by ELISA. Values representmean SD, = 78 for each OVA-sensitized group, and = 6 for the PBS group as negative control. Statistical analysis was performed withStudents -test, < 0.05, < 0.01 compared with the control group.

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Figure 3: Serum levels of OVA-specific IgE in OVA-sensitizedBALB/cmice supplementedwith different extracts of Perilla frutescens.OVA-sensitized mice were fed with water or ethanol extracts ofPerilla frutescens for 3 weeks, respectively. Sera of mice werecollected and the OVA-IgE levels were determined by ELISA. Valuesrepresent mean SD, = 78 for each OVA-sensitized group, and = 5 for the PBS group as negative control. Statistical analysiswas performed with Students -test, < 0.01, #0.05 < < 0.1compared with the control group.

3.3. Ethanol Extracts of Perilla frutescens Decrease AirwayHyperresponsiveness (AHR) of OVA-Sensitized Mice. OVA-sensitized mice supplemented with two doses of water orethanol extracts, respectively, were challenged with 50mg/mLaerosolized OVA to further induce AHR. The results shownin Figure 6 demonstrated that the control group had signif-icantly higher AHR, measured as Penh value, than the PBSnegative control group after methacholine challenge. Ethanolextract of Pf significantly reduced AHR as significantly lowerPenh values were detected in both the PfEL and the PfEHgroups.Water extract of Pf tended to have lowAHR ( < 0.1)at high dose (PfWHgroup) though the low dose PfWL groupdid not reach statistical significance. This data indicates thatPerilla frutescens extracts possess inhibitory effects of airwayhyperresponsiveness, possibly due to lower cell infiltrationand inflammation in bronchiole and lung of allergic asthma.

4. Discussion

Allergic asthma is a chronic inflammatory disease in airway,which was induced by Th2-prone responses. The presentallergic asthma murine model demonstrated that Perillafrutescens extracts, especially the ethanol extracts, decreaseTh2 cytokines production, serum IgE level, cells infiltration,allergic mediator secretions, and AHR.

Early study examined the effects of different extracts ofPf on TNF levels in inflammatory mice and found thatwater extracts had stronger inhibition than n-hexane orethyl acetate extracts [28]. Water extract of Pf inhibited thehistamine released from rat peritoneal mast cells in vitro[29]. Glycoprotein derived from the hot water extract of Pfdemonstrated the effective component of Perilla frutescens


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Figure 4: Total cells and eosinophils number in BALF of OVA-sensitized/challenged BALB/c mice supplemented with different extractsof Perilla frutescens. OVA-sensitized mice were fed with water or ethanol extracts of Perilla frutescens for 3 weeks, respectively, and BALFwas collected after aerosolized OVA (50mg/mL) inhalation challenge. Total cells (a) and eosinophils number (b) were determined. Valuesrepresent mean SD, = 78 for each OVA-sensitized group, and = 5 for the PBS group as negative control. Statistical analysis wasperformed with Students -test, < 0.05, < 0.01 compared with the control group.

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Figure 5: Histamine and eotaxin contents in BALF of OVA-sensitized/challenged BALB/c mice supplemented with the different extractsof Perilla frutescens. OVA-sensitized mice were fed with water or ethanol extracts of Perilla frutescens for 3 weeks, respectively, and BALFwas collected after aerosolized OVA (50mg/mL) inhalation challenge. The concentrations of histamine (a) and eotaxin (b) in BALF weredetermined by ELISA. Values represent mean SD, = 78 for each OVA-sensitized group, and = 5 for the PBS group as negative control.Statistical analysis was performed with Students -test, < 0.05 compared with the control group.


Methacholine (mg/mL)0 10 20 30 40 50 60

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Figure 6: The AHR of OVA-sensitized/challenged BALB/c micesupplemented with different extracts of Perilla frutescens. OVA-sensitized mice were fed with water or ethanol extracts of Perillafrutescens for 3 weeks, respectively, and AHR was determined afteraerosolized OVA (50mg/mL) inhalation challenge. Values representmean SEM, = 78 for each OVA-sensitized group, and = 5 forthe PBS group as negative control. Statistical analysis was performedwith Students -test, #0.05 < < 0.1, < 0.05, < 0.01compared with the control group.

Britton to inhibit mast cells degranulation [30].These studiessuggested the potential antiallergic effect of Pf. Further,our study showed that the ethanol extract of Pf had moreeffective inhibition than water extract on the histaminerelease in BALF of OVA-challenged mice. We suggested thatthis inhibition might be related to the suppression of Th2activities.

It has been known that IL-4 plays a critical role in IgEproduction. IL-4 receptor was significantly associated withasthma risk [31, 32]. Allergen-stimulated IL-4 secretions bysplenocytes from sensitized mice were low in this studyand not suppressed by Pf, which might explain the reasonwhy sera IgE levels were not significantly lowered by Pf.The tendency of lower serum IgE in the PfEH group mightbe partially due to lower Th2 activities such as allergen-stimulated IL-5 and IL-13 productions. IL-13 polymorphismswere consistently associated with asthma and serum IgE inasthma populations [33]. Our study showed that the ethanolextracts of Pf significantly decreased IL-5 and IL-13 produc-tions from splenocytes of OVA-sensitized mice. Blocking thebinding of IgE to its receptors is the most effective therapystrategy for allergic disease [34]. The association betweenIL-13 and IgE productions in asthma [35, 36] suggests that

ethanol extracts of Pf suppressed IL-13 secretion and thustended to reduce IgE production [37].

IL-13 induced the eotaxin release by airway epithelial cellsin vitro [38]. Our data also indicated that ethanol extractsof Pf decreased eotaxin production in BALF, consistent withits lower allergen-induced IL-13 secretion. The inflammatorycells, such as eosinophils, recruited into airway are the majorclinical manifestations in allergic asthma. IL-5 is best charac-terized for eosinophilia that dominates airway inflammationon allergic asthma [39]. When asthmatic patients were givenanti-IL-5 (mepolizumab), bronchial mucosal eosinophilsdecreased [40]. Furthermore, eosinophils not only act asterminal effector cells but also act to actively amplify allergicresponses by promoting Th2 cell immunity [41], indicat-ing the close relationship between eosinophils and airwayhyperresponsiveness [42]. Th2-cell-derived cytokine IL-5,together with eotaxin, plays the critical roles in the inductionof airway hyperreactivity and the development of chronicairway wall remodeling [43]. Present report indicated thathigh dose of ethanol extracts of Pf inhibited eosinophils andeotaxin levels in BALF and also decreased AHR. These datasuggested that ethanol extracts of Pf exert attenuate airwayhyperresponsiveness and inflammation in allergic asthmathrough inhibition of eosinophils and proallergic mediators.

However, the percentage of eosinophil in total cell inBALF was low in this study. It may be due to the OVA-chal-lenge protocol. Continuous daily inhalation or intranasallychallenge with allergen may drive more eosinophils in BALF.Our previous studies showed 10% eosinophils in BALF ofmice when challenged with OVA inhalation every three days[11], but only 3% eosinophils were counted with challengeevery seven days. The majority of cell populations in thisstudy were neutrophils/basophils, which were 54% in thecontrol group and 3545% in the Pf groups.

Study showed that water extracts of perilla leaves improveatopic dermatitis [20] and identified the active constituentto be luteolin [44]. Rosmarinic acid extracts in Pf stronglyinhibited hexosaminidase release from RBL-2H3 mast cells[45] and were shown to decrease the neutrophils andeosinophils recruitment in nasal lavage fluid of seasonal aller-gic rhinoconjunctivitis patients [46]. Recent report revealedthat 80% ethanol extracts of purple perilla leaves contains8.47% (w/w) of rosmarinic acid which is the major phenolicacid in perilla and commonly found in aromatic plants [47].Our data suggested that the water extracts of Pf attenuatedAHR in OVA-sensitized mice, but they are less effectivethan ethanol extract. It may be due to the same doses ofdifferent extracts used in our study. Recent report indicatedthat ethanol extracts of Pf significantly decreased TNFproduction in BALF from LPS-induced airway inflammationand suggested that phenylpropanoids may contribute to theinhibitory activity of ethanol extracts of Pf on the lunginflammatory response [48]. As Perilla frutescent leaves arearomatic vegetables and can be consumed raw, cooked, orpickled [49], their application is feasible and expectable.In our study, the most effective dose for decreasing Th2responses and AHR is 80320mg PfE daily for mouse.According to the dose translation from animal to human[50], it corresponds to 1.56 g (520 pieces) of fresh Perilla


frutescens leaves daily for a 60 kg adult and 0.73 g (2.510pieces) for 20 kg child, respectively.

In conclusion, ethanol extracts of Perilla frutescent leavescould downregulate Th2 activities to secrete less IL-5 and IL-13 and thus lower serum IgE level when counting allergenchallenge. The cell infiltration, particularly eosinophils, andproinflammatory mediators such as histamine and eotaxinin BALF were significantly suppressed. As a result, AHRis alleviated by the extracts of Perilla frutescent leaves,suggesting that Perilla frutescent leaves are a potential herbalmedicine for immunomodulation.

Conflict of Interests

The authors have declared that no conflict of interests exists.

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Research ArticleCharacteristics Associated with Utilization of Asthma-RelatedTraditional Chinese Medicine Services among Asthma Childrenin Taiwan: A Nationwide Cohort Study

Shiou-Ian Lin,1 Tung-Hu Tsai,1,2,3 Yiing-Jenq Chou,4 and Nicole Huang1,3,5

1 Institute of Traditional Medicine, National Yang-Ming University, No. 155, Section 2, Li-Nong Street, Taipei 112, Taiwan2Graduate Institute of Acupuncture Science, China Medical University, No. 91 Hsueh-Shih Road, Taichung 404, Taiwan3Department of Education and Research, Taipei City Hospital, No. 145 Zhengzhou Road, Datong District, Taipei 103, Taiwan4 Institute of Public Health, School of Medicine, National Yang-Ming University, No. 155, Section 2, Li-Nong Street, Taipei 112, Taiwan5 Institute of Hospital and Health Care Administration, School of Medicine, National Yang-Ming University, The Medical Building II,No. 155, Section 2, Li-Nong Street, Taipei 112, Taiwan

Correspondence should be addressed to Nicole Huang; [email protected]

Received 3 June 2014; Accepted 10 November 2014

Academic Editor: Bor-Luen Chiang

Copyright 2015 Shiou-Ian Lin et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction. Previous studies have demonstrated the advantages of TCMuse among asthmatic children. However, there is a paucityof epidemiologic reports on features of TCMusers among asthmatic children.This cohort study aimed to investigate childs, parents,and providers characteristics associated with the use of asthma-related TCM services among newly diagnosed asthmatic children.Materials and Methods. A nationally representative cohort of one million National Health Insurance beneficiaries was used. Thenewly diagnosed asthma children who received asthma medication from western medicine providers from 2005 to 2010 wereselected as our sample for analysis. Generalized estimating equation was applied to identify the childs, parents, and providerscharacteristics associated with the use of asthma-related TCM among the newly diagnosed asthmatic children. Results. Of 20,080children who were enrolled and followed up for one year, 4,034 children used TCM for asthma-related treatment. Children withprior experience of TCM, pre-school and school aged children, boys, those with more severe asthma or poorer health, with higherincome parents were more likely to use asthma-related TCM. Herbal medicine was the most common modality among asthmaticchildren.Conclusions.Therewere only 20%newly diagnosed asthmatic children usingTCM.Thefindingsmay shed light on possibleintegration of TCM with western medicine services.

1. Introduction

Asthma is one most prevalent chronic condition among chil-dren. Global prevalence ranges from 1% to 18% across coun-tries [1]. In addition to the large financial burdens, asthmaalso leads to serious health consequences and compromiseslife quality of children [1]. Poorly controlled childhoodasthma may increase use of steroids, risk of emergency roomvisits or hospitalizations, persistently decrease lung function,or even lead to COPD later in adulthood [1]. Accordingto the guideline published by the Global Initiative forAsthma (GINA), currentwesternmedications of asthma suchas steroid, beta-2 adrenergic agonist, leukotriene modifier,

theophylline, and anti-IgE therapies are themainstream treat-ments of asthma. Recent empirical evidences also show goodcomparative effectiveness of integrating traditional Chineseherbal medicines to asthma management as complementarytherapies, particularly among children [26]. TraditionalChinese herbal medicines such as ASHMI [2], mMMDT [3],Ding-Chuan [4], and STA-1 [5, 6] are proven safe and having apositive effect on symptoms and/or lung function in children.

Recently, the rising popularity of complementary andalternative medicine (CAM) has facilitated the researchand development of scientifically sound CAM therapies.Numerous studies have investigated the utilization patternsof CAM among children [711] and found that CAM


http://dx.doi.org/10.1155/2015/108961


utilization is associated with children and parental charac-teristics, including age, gender, ethnicity, health conditions,household income, and parental use of CAM. Various CAMmodalities children received included traditional Chinesemedicine (TCM), herbal treatments, chiropractors,messages,and mind-body therapies [9]. However, not all CAM havebeen scientifically proven effective. Therefore, it is essentialto investigate whether and to what extent some scientificallyproven CAM therapies are used by children in addition totheir mainstream medical therapies.

Of the existing literature, several studies focus specificallyon CAM utilization of asthmatic children. The prevalence ofCAM visits among asthmatic children ranged from 13% inCanada [12] to 89% in USA [13]. CAM treatments of asthmaencompass many therapies, such as mind-body techniques,nutritional and herbal supplements, TCM, exercise, mes-sages, and homeopathy [14]. CAM use in asthmatic childrenhas been associated with childrens age, asthma severity,parents education level, parental income level, and insurancecoverage [12, 13, 1518].

Nevertheless, not only did characteristics of children andparents matter, but also the characteristics of their westernmedicine providers may influence their use of CAM [1921].Western medicine (WM) physicians may play a significantrole in influencing parents decision in seeking CAM ther-apies. WM providers knowledge of, attitude towards, andpractice of CAM may vary widely by their demographics,professional characteristics, and characteristics of the med-ical care system. Environmental factors such as availabilityof WM and CAM providers in the areas may create either afriendlier or a more hostile atmosphere for integration [22].Hence, the role of providers in use of CAM among asthmaticchildren shall not be overlooked.

Furthermore, the existing findings on asthmatic childrenare mostly conducted in health care systems where CAMservices are not covered by insurance, or in hospital settings.None of these studies are conducted in a health care systemwhere a major domain of CAM, TCM, is covered by a socialhealth insurance. It may be interesting to investigate theutilization pattern of asthma-related TCM among asthmaticchildren in the National Health Insurance (NHI) programin Taiwan, where TCM is comprehensively covered by theNHI program. This study aimed to investigate the use ofTCM among the newly diagnosed asthmatic children andits associated characteristics of children, parents, and WMproviders.

2. Materials and Methods

2.1. Data Source. The data source is the Taiwan NationalHealth Insurance Research Database (NHIRD).TheNationalHealth Insurance program is a single-payer mandatory uni-versal insurance system. In 2011, the enrollment rate was99% of the total population [23]. It is well known for itscomprehensive benefit coverage. It not only covers ambu-latory and inpatient services of western medicine, but alsoreimburses TCM ambulatory care services. The NHI TCMcoverage includes Chinese herbal medicine, acupuncture,

traumatology, and massage. The dataset consists of individ-uals enrolment and claims information, including patientsidentification number, gender, birthdate, date of service, diag-nosis, medication, treatment procedure, and expenditures.The dataset also records providers information, includinggender, age, specialty of physicians, and ownership andaccreditation level of physicians practice location. Individualand provider identifiers were encrypted before releasing tothe researchers.

2.2. Study Design and Sample. This study was a retrospectivecohort study. Of all the NHI enrollees in 2005 throughoutTaiwan from the NHIRD, a representative cohort of 1 millionrandomly sampled enrollees was used. The newly diagnosedasthmatic children who were aged 018 years and receivedhis/her primary diagnosis of asthma and asthma medica-tion from western medicine providers from 2005 to 2010were included for analyses. Asthma medications in westernmedicine were defined according to the GINA guideline.Thechildrenwho had received any diagnosis of asthmawithin thefive years before the index year were excluded. InternationalClassification of Diseases, Ninth Revision, Clinical Modifica-tion codes 493.xx were used to identify asthma children. Atotal of 20,080 children were newly diagnosed with asthma in20052010. Each child had been followed for one year sincehis/her first diagnosis of asthma for any asthma-related TCMvisit.

2.3. Variables. Asthma-related TCM visits were defined asvisits of primary TCMdiagnostic code of respiratory diseases(ICD-code: 460466, 470478, 480488, 490496, 500508,510519). Asthma is a chronic respiratory disease and maycoexist with or have other related respiratory conditions,such as respiratory tract infection [24], allergic rhinitis [25],chronic rhinitis [26], chronic bronchitis, and emphysema[27]. According to the fundamentals of TCM theories, liter-atures, and clinical practices, TCM normally treat diseasesfrom a more holistic perspective. TCM users were definedas those who had any asthma-related TCM visits duringthe 1-year follow-up period. TCM nonusers refer to thosewithout any asthma-related TCM visits during the 1-yearfollow-up period. Sensitivity analyseswere also conducted forvarious definitions of asthma-related TCM visits. The resultsremained robust. Furthermore, specific types of asthma-related TCM modalities children received and the top 10commonly prescribed Chinese herbal medicinal formulasduring the follow-up period among the asthmatic childrenwere also analyzed.

Characteristics of children, parents, and WM providerswere compared between TCM users and nonusers. Childsage, gender, health status (any hospitalization prior to theindex year), previous TCM experience, level of medicalresources of residential location, asthmatic WM medica-tion used, frequency of WM visits, and incident year ofasthmawere constructed. Asthmamedications used includedcontroller medications, oral and systemic corticosteroids.Controller medications include LABAs, xanthines, ICS,leukotriene modifiers, immunomodulators, and cromolyn


sodium. Controller medications were used for controllingpersistent asthma. Oral or systemic corticosteroids wereused for quickly reliving asthma exacerbations. Level ofmedical resources was defined as densities of westernmedicaldoctors and TCM doctors. Density of TCM andWMdoctorswere defined as number of TCM/WM doctors per 10,000population and classified into four categories: (1) high densityof TCM doctors and high density of WM doctors, (2) highdensity of TCM and low density of WM doctors, (3) lowdensity of TCM and high density ofWMdoctors, and (4) lowdensity of TCM and low density of WM.

In addition, socioeconomic status of parents was definedusing the insurance wage and category of the insured.For people with well-defined monthly wages, we catego-rized them into 3 groups: 40000NTD, 2000039999NTD,


Table 1: Continued.

Child and parentalcharacteristics

TCM user(%)

TCMnonuser (%)

Chi-square value

Parents insurablewage/category


Table 2: Children, parent, and provider characteristics associated with TCM uses.

Childs characteristics Crude OR (95% CI) Adjusted OR (95% CI)Age (ref.: infant and toddler)

Preschool 2.11 (1.85, 2.42) 1.72 (1.49, 1.99)

School 2.03 (1.77, 2.32) 1.50 (1.28, 1.75)

Adolescent 1.45 (1.23, 1.72) 0.91 (0.75, 1.11)Sex (ref.: girls)

Boys 1.09 (1.01, 1.17) 1.12 (1.04, 1.20)

Previous use of TCM (ref.: no)Yes 6.18 (5.73, 6.66) 6.33 (5.83, 6.87)

Previous hospitalization (ref.: no)Yes 1.17 (1.07, 1.29) 1.16 (1.05, 1.29)

Controller medication used (ref.: no)Yes 1.23 (1.14, 1.32) 1.07 (0.98, 1.16)

Systematic or oral corticosteroid used (ref.: no)Yes 1.10 (1.02, 1.17) 1.08 (1.00, 1.18)

Number of outpatient visits in one year (ref.:


Table 3: Top 10 Chinese herbal medicines used.

Top 10 Herbal formulas Frequency (%)1 Shin-Yi-Qing-Fei-Tang 21.42 Xiao-Qing-Long-Tang 16.33 Cang-Er-Zi-San 14.54 Shin-Yi-San 13.95 Ma-Xing-Gan-Shi-Tang 12.46 Xing-Su-San 8.77 Ge-Gen-Tang 7.98 Yin-Qiao-San 7.49 Ding-Chuan-Tang 5.910 Zhi-Sou-San 5.5

CAM were also the strongest predictor of future CAM visitsin the Young-HUNT studies [34]. Asthmatic patients accus-tomed to TCM therapies or believing in TCM treatmentsmaylead to further integrative use of WM and TCM. Hence, itis important to increase awareness of or positive attitudestowards scientifically proved TCM care and build trust ofTCM among asthmatic children and parents. More effectivepromotion or educational strategies or program about TCMmay help to reduce nonfinancial barriers to TCM services.

Furthermore, in spite of low financial barriers to TCMcare under the NHI program, children with higher incomeparents were significantly more likely to receive TCM carein addition to their WM asthmatic treatments. This may alsobe due to other barriers. A possible barrier is that parentsworkload may be so hard that they did not have spare time tobring their children to visit other doctors. Further studies ofother nonfinancial barriers (time or travel costs) are neededto facilitate better or more efficient integration of TCM andWM asthmatic treatments.

All of the provider factors studied are not significantlyassociated with TCM use among asthmatic children. Mostimportantly, the persistently low utilization of TCM acrossWMproviders of different characteristics may reflect that theissue of integration of TCM andWM is generally overlookedin routine management of pediatric asthma. Furthermore,limited availability of clinical trials may be one plausiblebarrier to better integration of TCM and WM. Whether ornot to useTCMto treat or control asthmamayheavily dependon therapeutic effects and safety of TCM. Therefore, moreclinical trials should be encouraged in this regard.

This study has several strengths. First, using the NationalHealth Insurance data of a large national representativesample in Taiwan can help us avoid some of the knownshortcomings of survey data. The National Health Insurancecovered both TCM and WM universally. Using the medicalrecords from National Health Insurance data may amelioratethe selection bias from the survey data. Second, in contrast toother studies on this topic, the large sample size and relativelycomprehensive data allow us to go beyond prior research byinvestigating not only children and parental characteristics,but also WM provider characteristics in their influences onthe utilization of TCM care among the newly diagnosedasthma children. Third, taking advantages of the detailed

information on TCM modalities and herbal medications inthe NHI databases, we were able to identify the commonTCM medications and therapies prescribed to the newlydiagnosed asthmatic children in Taiwan. The findings mayserve as important references in clinical management ofpediatric asthma and for future effectiveness researches inTCM.

Several limitations should also be noted. First, the NHIdatabase only allows us to estimate the use of TCM servicescovered by the NHI program.TheNHI program covers TCMservices provided by the NHI contracted TCM providers,including extracted TCMpowder preparations, acupuncture,moxibustion, and traumatology manipulative therapy. How-ever, other TCM services or products including crude herbalmedicine, herb tea bags, pills, capsules, and other CAMmodalities are not covered by the NHI program and theseinsurance noncovered services are self-paid by patients. Theprevalence of people who had used any self-paid TCM orCAM service in one year was 6.05% [35]. Those self-paidTCM services or CAM services are not included for analysesin this study. Not being able to include self-paid TCM orCAM services in this studymay lead to an underestimation ofthe CAM use among asthmatic children in Taiwan. However,as the CAM ranges widely, not many modalities have beenproven effective scientifically. As the TCM services coveredby the NHI program are well accepted CAM modalities andpatients costs in using these services are very low, studyingthe TCM utilization patterns under the NHI program mayhelp to go beyond the previous investigations of generalCAM uses. Second, a possible misclassification bias may beof concern. Because a claim may have up to 3 diagnoses,defining asthma-related TCM visits based on one set ofrespiratory diagnoses may be questionable. Therefore, sen-sitivity analyses were conducted on various definitions ofTCM visits containing only diagnoses of respiratory diseases,any diagnoses of respiratory diseases, diagnoses of asthma,and diagnoses of asthma and bronchitis. All major statisticalresults from sensitivity analyses remained significant. Third,due to data limitations, using a single measure (i.e., theNHI programs payroll and occupation-based categories)to analyze the association between a parents SES and thedecision on TCM care did not allow us to fully explore thisassociation. In addition, possible confounding biases such asknowledge level may also exist.

5. Conclusion

Adopting TCM care for asthma management among thenewly diagnosed children in Taiwan was low. The low preva-lence of TCM use is commonly seen across children treatedby different characteristics of WM providers. This may sug-gest there is still room to promote collaboration betweenWMand TCM providers. Instead of targeting specific physiciangroups, putting more efforts generally in integration betweenTCM and WM in the management of pediatric asthma isneeded. More efforts in improving understanding of TCMsuch as the educational programs of TCM andmore researchefforts in showing evidence-based effectiveness of TCM


modalities may help to build more confidence or positiveattitudes towards TCM among physicians and parents and tofacilitate the integration TCM into the mainstream medicinein pediatric asthma management.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

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Research ArticleTriterpenoids and PolysaccharideFractions of Ganoderma tsugae Exert DifferentEffects on Antiallergic Activities

Miaw-Ling Chen,1 Chia-Chien Hsieh,2 Bor-Luen Chiang,3 and Bi-Fong Lin4

1Department of Nutrition and Health Sciences, College of Health Sciences, Chang Jung Christian University, No. 1 Changda Road,Gueiren District, Tainan 71101, Taiwan2Department of Human Development and Family Studies, College of Education, National Taiwan Normal University,No. 162 Hepting East Road, Section 1, Taipei 10610, Taiwan3Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, No. 7 Chung San South Road,Taipei 10002, Taiwan4Department of Biochemical Science and Technology, College of Life Science, National Taiwan University,No. 1 Roosevelt Road, Section 4, Taipei 10617, Taiwan

Correspondence should be addressed to Bi-Fong Lin; [email protected]

Received 14 August 2014; Accepted 18 October 2014

Academic Editor: Jin-Yuarn Lin

Copyright 2015 Miaw-Ling Chen et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

This study was to investigate antiallergic effects of triterpenoids (Gt-TRE) and polysaccharide (Gt-PS) extracts from Ganodermatsugae, using mast cell line RBL-2H3, T cell line EL4, primary T cells, and transfected RAW264.7 macrophage cells. The resultsshowed that histamine secreted from activated RBL-2H3 mast cells was significantly suppressed by Gt-TRE but not Gt-PS.Interleukin- (IL-) 4 secreted from activated EL4 cells was significantly suppressed by Gt-TRE but not Gt-PS. Further primaryCD4+ T cells cultures also confirmed that Gt-TRE (5 50 g/mL) significantly suppressedTh2 cytokines IL-4 and IL-5 secretionsbut had no effect on Th1 cytokines IL-2 and interferon (IFN)-. Gt-PS did not affect IL-4 and IL-5 secretions until higher doses(400, 500 g/mL) and significantly suppressed IFN secretions but enhanced IL-2 at these high doses. The reporter gene assayindicated that Gt-TRE inhibited but Gt-PS enhanced the transcriptional activity of NF-B in activated transfected RAW264.7 cellsand transfected EL4 cells. IL-4 secreted by this transfected EL-4 cells was also significantly decreased by Gt-TRE but not by Gt-PS,suggesting that these two fractions may exert different effects on NF-B related cytokines expression. These data suggested thattriterpenoids fraction of Ganoderma tsugaemight be the main constituents to alleviate allergic asthma.

1. Introduction

The worldwide increase in prevalence of allergic diseasessuch as asthma [1, 2] poses a significant health problem anda demand for drug/diet therapy. Allergic asthma is charac-terized by histamine secretion by mast cell, bronchial hyper-responsiveness, and airway inflammation by accumulation ofeosinophils, lymphocytes and mast cells, and higher serumIgE levels [3]. The allergic immune responses in asthmaarise from an imbalance of helper T (Th) cells. Th1 cellsand their cytokines IL-2 and IFN enhance Th1 genera-tion and inhibit Th2 function, whereas Th2 cells and their

cytokines inhibit Th1 generation and rise allergic responses.IL-4 secreted by Th2 cells stimulates B cells class switchto produce allergic immunoglobulin (Ig) E and promoteneutrophil- and eosinophil-infiltrated inflammation. Thesecells were also activated by anotherTh2 cytokine IL-5.There-fore, downregulating Th2 cell differentiation by cytokineadministration is frequently used as a therapy for allergicdiseases [4]. Several studies indicated dietary factors, suchas frying oil, adlay, and andrographolide from Andrographispaniculata suppressed Th2 immune responses in the Th2-skewed ovalbumin- (OVA-) sensitized BALB/c mice [57], and polysaccharide from fruiting bodies of Ganoderma


http://dx.doi.org/10.1155/2015/754836


lucidum has been shown tomediate cytokines production [8].Therefore, the immunomodulatory effects of G. tsugae roseour interest.

The polypore genus Ganoderma had been widely usedfor Chinese medicine in Asian countries for a long time.It has been known for many biological activities of Gan-oderma, such as antitumor, immunoregulation, hepatopro-tection, anticholesterol synthesis and anti-inflammation [914]. Although the major active ingredients are polysaccha-rides, triterpenoids and proteins, polysaccharides are moststudied [15]. The polysaccharides with immunity enhance-ment effects have been isolated from the water extract ofG. lucidum mycelia and fruiting bodies [16]. It has beendemonstrated that polysaccharide extracts of G. lucidumexert immunomodulating activities by inducing cytokineexpression via TLR4 signaling pathways, activation of den-dritic cells, and innate immunity by NF-B pathways [1719].The different ingredients may exert diverse bioactive func-tions [20]. Although recent review articles also summarizedthe health benefits of Ganoderma, especially triterpenoids[21], the immunomodulatory effects of triterpenoids stillneed to be clarified.

G. lucidum is the most studied species of the Gano-derma. However, another species of medicinal mushrooms,G. tsugae, is most widely cultivated in Taiwan and used asfunctional foods. Our previous study showed that G. tsugaesupplementation significantly enhanced Th1/Th2 balance intheTh2-skewedOVA-sensitized and challengedBALB/cmiceas an allergic inflammationmodel [22]. Further study showedthat G. tsugae supplementation significantly alleviated his-tamine, prostaglandin (PGE) 2 and eotaxin, a protein that canactivate eosinophils and airway hyperresponsiveness, levelsin bronchoalveolar lavage fluid (BALF) in OVA-sensitizedallergic BALB/c mice [23]. However, the eosinophils in BALFand Th2 cytokines IL-4 and IL-5 and were not significantlysuppressed by G. tsugae [22, 23]. Since early in vitro studysuggested that triterpenoids suppressed histamine releasefrom mast cells [24], our study further demonstrated thattriterpenoids fraction from G. tsugae significantly inhibitedeosinophils, IL-4, IL-5, PGE2, eotaxin levels, and thus airwayhyperactivity [25].

There are studies that indicated that polysaccharide com-ponent promotes Th1 immune responses [18, 26]. There-fore, whether histamine or IL-4 production was affected bypolysaccharide or triterpenoids ofG. tsugaewere investigatedin this study using a mast cell line and a murine T cellline. To further investigate whether cytokine productionswere affected by polysaccharide or triterpenoids via T cellpolarization, the primary CD4+ T cells isolated from theDO11.10 transgenic mice w

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