Epigallocatechin Gallate

Transcription

1 Epgallocatechn Gallate The Major Causatve Agent of Green Tea-Induced Asthma* Toshhro Shra, MD ; Atsuhko Sato, MD, FCCP; and Yukhko Hara, PhD We descrbe three patents who worked n green tea factores and developed asthmatc and nasal symptoms after exposure to green tea dust. To clarfy what component(s) of green tea leaves mght be responsble for causng asthma, we prepared catechns, the major components of green tea leaves. Epgallocatechn gallate (EGCg; MW: 48 daltons), a major catechn, was purfed by hgh-performance lqud chromatography. Subjects ncluded three patents wth green tea-nduced asthma, fve asthmatcs wth no prevous exposure to tea dust,and fve healthy controls.it was found that all three patents exhbted an mmedate skn and bronchal response to EGCg. Prausntz-Kstner test wth EGCg was also postve. However, none of the asthmatc and healthy controls showed a postve reacton. These results ndcate that EGCg s a causatve agent of green tea-nduced asthma and suggest that an rge-medated response s, at least n part, responsble for causng ths type of occupatonal asthma. (Chest 1994; 106:1801-0) DSCG=dsodum cromoglycate; EGCg=epgallocak -hn gallate; IAR=mmedate asthmatc reacton; LAR=late asthmatc reacton; P-K=Prausntz-Kstner Key words: catechns; causatve agent and IgE-m edated; epgallocatechn gallate; green teal-nduced asthma; Prausntz-Kstner test In 1970, Uragoda frst reported a case of occupatonal asthma assocated wth tea fluff and desgnated tea maker's asthma.' Subsequently, several authors have descrbed smlar cases assocated wth tea dust or tea fluff. 2 ' 4 However, to our knowledge, no report has yet dentfed whch tea component(s) cause the bronchal response. In ths study, we descrbe three patents wth green tea-nduced asthma and present data ndcatng that epgallocatechn gallate (EGCg), the major catechn component n green tea leaves, s a causatve agent. Subject s MATERIAL AND METHODS Subjects conssted of thr ee groups: nona topc nonasthmatc volunteers (n=), asthmatcs wth no prevous exposure to gre en tea dust (asthmatc controls) (n= ), and patents wth green teanduced asthma (n= 3). The clncal characterstcs for patents wth green tea-nduced asthma are summarzed n Table 1. All patents had worked at dfferent green tea factores n Shzuoka Prefecture. Ther asthmatc symptoms (dyspnea, cough, and wheezng) and nasal symptoms (rhnorrhea and obstructon) began wthn 1 h after startng work and subsded on returnng home. Furthermore, all three patents exhbted bronchal hyp erresponsveness as determned by an acetylcholne or methacholne nhalaton test.. 6 Only one patent (patent 3) developed an asthmatc attack aft er drnkng green tea. *From the Second Dvson, Department of Internal Medcne, Hamamatsu Unv ersty School of Med cne, Hamamatsu, Japan (Drs. Shra and Sato), and the Food Research Laboratores, Mtsu Norn Co, Fujeda, Japan (Dr. Hara). Manuscrpt receved May 7, 1993; revson accepted May 19, Reprnt requests: Dr. Shra, Second Department of Int ernal Medcne, Hamamatsu Unversty School of Medcne, 3600 Handa-cho, Hamamatsu , Japan Preparaton of Green Tea Compon ents Powdered Green Tea Extract: A powdered green tea extract was prepared by bolng 300 g of green tea leaves n water for mn and then allowng the extract to ar dry. Crude Catechns and Noncatechn Components: The proc e dure for purfyng cat echns s llustrated n Fgure 1. Brefly, the powdered green tea extract was dssolved n boled water, followed by addng an equal porton of chloroform. After the aq ueous layer had separated out, three equal portons of ethyl acetate were added. Crude catechns of 91 %purty were obtaned from the ethyl acetate layer by evaporaton and lyophlzaton. Noncatechn components that consst of unknown substances other than catechns were also prepared from the aqueous layer by evapora ton. Catechn Compon ents (EGCg, EGC, ECg, and EC): The crude catechns were dssolved n water, fltered (0.4 JLm, MI I pore, Yonezawa, Japan), and placed on a reverse-phase dstrbuton column (Waters Col, model LC00A, cartrdge column of C18 ) usng a mxture of acetone, tetrah ydrofuran, and wat er (12:10:78, percent by volum e). The proc edure produced four peaks (Fg 1). Each peak fracton was evaporated by a stream of ntrogen gas. Through the use of nfrared and ultravolet absorpton and elementary analyss, resultant wht e resdues were found to consst of (- ) epgallocatechn gallate (EGCg), (- )-epgallocatechn (EGC), (-)-epcatechn gallate (ECg), or (- )-epcatechn (EC). Other Tea Extracts Extracts of black tea and oolong tea were prepared by strrng 1 g of each tea n 20 ml of 0.9% saln e soluton for 30 mn, followed by fltraton (0.4 JLm). Skn Test Each tea component was dssolved n salne soluton at concentratons of 1 ng/ml to 1 mg/ml and fltered (0.4 JLm). Intradermal skn tests were performed wth 0.02 ml of eac h solu- CHEST/106 /6 /DECEMBER,

2 Table I-Clncal Characterstcs of Patents Wth Green Tea-Induced Asthma Patent No./Sex/ Age, yr Postve Skn Reacton* I/M /2 + 2/ F/60 + 3/F/47 Duraton of Exposure to Tea Dust Pror to Onset, yr *Skn reactons were defned by utlzng 44 nhalant allergens. IProvocaton concentraton of acetylcholne causng a 20% fall n FEV,. t PC20 of methacholne. ton. All skn test reactons were evaluated at 1 mn and at 6 h postnject on. A postve reacton was defned as a flare great er than 20 mm and/ or a wheal grea ter than 9 mm n dameter." Salne soluton or 0.01 % hstam ne base soluton was employed as a negatv e or postve control, respect vely. Inhalaton Challenge Subjects were beng treated wth only ntermttent!j2-agonst nhalat on before the study, and they were asked to not take that medcaton for at least 12 h pror to the nhalaton challenge. Each tea component preparaton was nh aled as a mst usng a nebulzer (output: 0.16 ml/mn) (Nsshyo, Tokyo, Jap an ) for 2 mn. A 20% fall n FEV, compared wth the baseln e value was evaluated as postve. To establsh the relablt y of the baseln e values, changes n FE V, aft er salne soluton nhalaton were evaluated hourl y for 12 h. F EV, varablty after salne soluton nhalaton was less than % n all subjects. Inhalaton challenges wth eac h preparat on were subseq uently perf ormed. Th e concentraton nhal ed was ncreased n tenfold ncrements, from 10 ng/ml to 1 mg/ml. To detect a lat e asthamatc reacton (LAR), sprome try values were taken hourl y for 12 h. POWDERED GREEN TEA EXTRACT : 100g n lo00ml of boled water AQUEOUS LAYER Addton of equal porton of chloroform Addton of three equal portons of ethyl acetate ETHYL ACETATE LAYER Concentraton by evaporaton Lyophl zaton CRUDE CATECHINS : 28.9 g (P urty : 91%) EGC 3.g Hgh performancelqud chromtography EGC CHLOROFORM LAYER AQUEOUS LAYER NON-CATECHIN COMPONENT S -to= r--- =:: ECg o 20 ~ o 60 mn Concentraton and Lophlzaton EC 2. 0g EGCg 11.7g. Concentrat on by evaporaton FIGURE 1. Separaton procedure to obtan catechns from powdered gree n tea extract. Th e fnal step conssted of hgh-performance lqu d chromatography. ECg 3.0g Duraton of Symptoms, yr Total IgE, IU/mL PC20,t mg/ ml Open Oral Challenge 0.78t Subjects were gven an oral admnstra ton of 1 ml of powde red gree n tea extract dssolved n water at a concentra ton of 10 mg /ml, and sprome try values were tak en over a 12-h perod. Prausnt z-kstner Test Wth approprate consent, serum was obtaned fro m patents wth green tea-nduced asthma, and 0.1 ml of the serum was transferred ntradermally nto the back of a faml y member who prevously had shown a negat ve skn reacton to powd ered green tea extract or EGCg. Twenty-four hours later, each skn ste was challenged by a 0.02 ml ntradermal njecton of powdered gree n tea extract or EGCg at a conce ntraton of 1 mg/ml. As a contro l, other skn stes were senstzed wth serum nac tvated at 6 C for 2 h. Skn Test R ESULTS All patents wth green tea-nduced asthma showed an mmedate postve reacton to powdered gr een tea extract, crude catechns, EGCg, and noncatechn components, and two of three paten ts had a postve reacton to EGC, ECg, and EC (Tables 2 and 3). However, none of the normal and asthmatc controls reacted to these preparatons (Table 2). No lat e ntradermal reactons were de tected n any group. Inhalaton Challenge Tme courses of seral FEV1 measurements for the three pat ents wth green tea -nduced asthma were examned (Fg 2). In patents 1 and 2, an mmedate asthmatc reacton (IAR) was nduced by nhalaton of powd ered green tea extract, crude catechns, and EGCg at a concentraton of 1 mg jml. When paten t 3 was challenged wth EGCg at a concentraton of 0.01 mg jml, such a severe attack occu rred that subsequent sprometrc analyss cou ld not be completed. No LARs were observ ed n any patent. Inhalaton challeng es wth EGC, ECg, EC, and noncatechn components were performed n patents 1 and 2 and faled to produce a bronchal response. None of the preparatons caused postve reactons n healthy and asthmatc controls (Table 4). Oral Challenge Patent 3 demonstrated a postve reacton to an 1802 MajorCausatve Agent of Green Tea-Induced Asthma (Shra, Sata, Hara)

3 Table 2-Comparson of Skn Test Reactvty Intrade rmal Injectate* Powdered Green Tea Noncatechn Oolong Black Subjects Extract Crude Catechns EGCg EGC ECg EC Components Tea Tea Green tea-nduced 3/3 3/ 3 3/3 2/ 3 2/ 3 2/ 3 3/3 3/3 3/ 3 asthmat cs (n=3 ) Asthmat cs wth no 0/ 0/ 0/ 0/ 0/ 0/ 0/ 0/ 0/ prevous exposure to tea dust (n =) Normal controls 0/ 0/ 0/ 0/ 0/ 0/ 0/ 0/ 0/ (n=) *Each powder preparaton was dssolved n 0.9%salne soluton at a concentraton of I mg/ml. Each value ndcates the number of subjects who showed an mmedate skn reacton. oral challenge wth powdered gre en tea extract. A maxmum fall n FEVI of 37% occurred 30 mn after the exposur e wth no late reacton (Fg 2). Patents 1 and 2 exhbted no bronchal reacton to oral challenge. Effects of Prem edcatons on Bronchal Responses to Powdered Green Tea Extract The effects of bronchodlators and dsodum cromoglycate (DSCG) on the bronchal response to powd ered green tea extract were studed n a sngleblnd way n patent 1. The paten t receved 300 mg of amnophyllne orally 12 and 4 h pror to the nhalaton. Th e blood level of amnophyllne at the tm e of nhalaton was 8.7 JLg/mL. Salbutamol sulfate (1. mg) was nhaled 1 h before the challenge. Forty mllgrams of DSCG was nhaled mmedately before the challenge. It was found that treatment wth each medcaton completely nhbted lars (Fg 3). Prausntz-Kustner Test The Prausntz-Kstner (P-K) test revealed a postve reacton to powdered green tea extract and EGCg n each famly member, whle an njecton of ether salne soluton or heat-treated serum caused no reacton. Other Tea Extracts All three patents wth green tea-nduced asthma had an solated mmedate skn reacton to the extracts from black tea and oolong tea. We also per form ed nhalaton challeng es wth these tea extracts n patent 1, and he showed an solated IAR (F g 4). DISCUSSION We have descrbed three patents wth green teanduced asthma and demonstrated that EGCg s a causatve agent. Tea s classfed accordng to the method of manufacturng: unf ermented tea (gree n tea), semfermented tea (oolong tea), and fermented tea (black tea). All three types are derved from the same tea plant, Cam ella snens s, and contan EGCg: 8% (green tea), 4% (oolong tea), and 1% (black tea) by weght. 8 Thus, t s possble that EGCg s also a causatve agent n black tea-assocated asthmatc cases that have been prevously reported.l-v" Ths would.. ~ -. c 2 2 C,) C,) 0 J 0 C,) ~ - ~-2 0 ~-2 1 J :> w s w "-- "--40 Tme (hr) T me (br } Tme (hr) Inhalaton challenge Oral challenge Pat ent 1 Pat ent 2 Patent 3 F IGURE 2. Th e nhalat on and oral challenge tm e courses. In pa ten ts I and 2, an mmedate asthma tc reacton occurred after nh alng powdered gree n tea extract (crcles), crude catechns (squares), and EGCg (trangles) at a concentraton of 1 mg/ml. Patent 3 dem onstrated a postve reacton to powdered green tea extrac t oral cha llenge at a concentraton of 10 mg /ml. CHEST / 106/ 6/ DECEMBER,

4 Table 3-Threshold Concentraton (p.g/ml) That Produced an Immedate Skn Reacton Intradermal Injectate Patent Powder ed Green Tea Crude No. Extract Catechns EGCg EGC ECg EC I , Neg* Neg Neg , *Neg=negatvc. be supported by the postve mmedate skn reactons to black tea and oolong tea extracts demonstrated n our three patents and also the exhbted bronchal response to challenges wth these extracts n one of the patents. These fndngs suggest that EGCg s a causatve agent n several knds of tea-assocated asthma. However, there are some dscrepances between our cases and those prevously reported by Roberts and Thornpson'' and Carter and Malo. 4 They reported that skn prck tests to black tea dust extracts were negatve, and thatsome of the r patents showed late and prolonged mmedate asthmatc responses. These dscrepances may result from dfferent subjects studed, preparatons tested, or methods used for challenges. It s known that the ntradermal testng method that we used provdes ncreased senstvty compared wth prck testng. Furthermore, they performed nhalaton challenges by tppng tea dust between two contaners, whch may have resulted n nhalaton of more tea products than our nhalaton challenge. Catechns are the source of the astrngent taste of green tea and are the major soluble component of green tea, occupyng 1% by weght of dred Japanese green tea leaves. Catechns consst of several dfferent polyphenols, and EGCg accounts for more than half of the total catechn. Recent studes have shown that tea catechns have hypocholesterolemtc.f antoxdatve.l? and antmutagenc'! effects. Occupatonal asthma due to small molecular weght molecules remans complcated. Both mmunologc and nonmmunologc mechansms have been proposed. For example, occupatonal asthma nduced by trmelltc anhydrde s consdered to be medated by IgE, IgG, and nonspecfc rrtant stmulatlon.p In western red cedar nduced asthma, ar- Table 4-Comparson of Inhalaton Challenge Reactvty* Inhaled Preparatons Powdered Green Crude Subjects Tea Extract Catechns EGCg Green tea-ndu ced 2/2 2/2 3/3 asthmatcs (n=3) Asthmatcs wth 0/ ND 0/ no prevous exposure to tea dust (n=) Normal controls 0/ ND 0/ (n=) *Each value ndcates the rato of subjects showng an IAR. ND=not done.... Gl c co s: o -20 ~ No therepy OSCG.---4 Amnophyllne Selbutamol sulfate -40 (mn) Tme after nhalaton FIGURE 3. The effect of premedcatons on thebronchal response to powdered green tea extract n patent 1. Amnophyllne (300 mg, oral), salbutamol sulphate (1. mg, nhaled), and dsodum cromoglycate (DSCG) (40 mg, nhaled) demonstrated an mmedate nhbtory effect on the asthmatc reacton. (hr) ' I o 2, 3 4 Tme (h r ) FIGURE 4. The results of nhalaton challenge to the extracts from black tea (damonds) and oolong tea (trangles) n patent 1. The nhalaton of both extracts provoked an mmedate, but not late asthmatc reacton Major CausatveAgentof GreenTea-Induced Asthma (Shra, Sato, Hara)

5 HO "'0 ~ O H O(;{ ~ 0 0 H FIGURE. The chemcal structure of (- )-epgallocatechn gallate (EGCg). way hyperreactvty may be related to a stmulcausng reflex, ~ - a d r e blockade, n e r g drect c actvaton of the complement pathway, and a specfc IgE-medated response. IS Our patents exhbted an mmedate skn and bronchal response to EGCg, and a postve reacton to EGCg was shown n a P-K test. These fndngs ndcate that an IgE-medated reacton s, at least n part, responsble for causng bronchal and dermal responses n green tea-nduced asthma. Snce EGCg s a small molecular weght substance (48 daltons) (Fg ), one possblty s that t may act as a hapten by combnng wth a host proten(s). Although coworkers of our patents n the same factores had no respratory symptoms, t s probable that there are a certan number of workers sufferng from the symptoms due to exposure to green tea dust n ths area, where substantal amounts of green tea are produced and manufactured. The mechansms ~ O H 1: and the prevalence of green tea-nduced asthma should be nvestgated further because medcatons ncludng admnstraton of bronchodlators and DSCG enable patents to return to ther workplaces when they cannot afford to change the job. REFERENCES 1 Uragoda CG. Tea maker's asthma. Br J Ind Med 1970; 27: Ebhara I. Study on the nhalat ve allergy of clae of leaves: nhalatve allergy of the clae of tea leaves. J Sc Labour 197; 3 Roberts JA, Thompson NG Tea-dust nduced asthma. Eur Respr J 1988; 1: Carter A, Malo JL. Occupatonal asthma due to tea dust. Thorax 1990; 4: Muranaka M, Nakajma K, Suzuk S. Bronchal responsveness to acetylcholne n patents wth bronchal asthma after longterm treatm ent wth gold salt. J Allergy Cln Immunol 1981; 67: Takshma T, Hda W, Sasak H, Suzuk S, Sasak T. Drectwrtng recorder of the dose-response curves of the arway to methacholne. Chest 1981; 80: Voorhorst R. Perfecton of skn testng technque. Allergy 1980; 3: Ikegaya K, Nakagawa. Healthy and useful componentsof green tea. In: Ohsh S, ed. All about Japan ese tea (Shn Chagyo Zensho). 8th ed. Shzuoka, Shzuoka Tea Assocaton, 1988; Muramatsu K, Fuku yo M, Hara Y. Effect of green tea catechns on plasma cholesterol level n cholesterol-fed rats. J Nutr Sc Vtamnol 1986; 32: Matsuzak T, Hara Y. Antoxdatve actvt y of tea leaf catechns. J Agr Chern Soc Jpn 198; 9: Kada T, Kaneko K, Matsuzak S, Matsuzak T, Hara Y. Detecton and chemcal dentfcaton of natural boantmutagens: a case of the green tea factor. Mutat Res 198; 10: Zess CR, Patterson R, Pruzansky JJ, Mller MM, Rosenberg M, Levtz D. Trm elltc anhydrde-nduced arway syndromes: clncal and mmunologc studes. J Allergy Cln Imm unol 1977; 60: Chan-Yeung M. Immunologc and nonmunologc mechansms n asthma due to western red cedar (Thuja plcata). J Allergy Cln Immunol 1982; 70:32-8 CHEST DECEMBER,