: 59 2 2000 1 - = A bs tra c t= Occupational as thma caus ed by s ev eral kinds of herb materials Soo- Keol Lee, M.D., Jung- Hee Seo, Hyeon- Kyeong Cho, M.D., Sun- Sin Kim, M.D., Dong- Ho Nahm, M.D. and Hae- Sim Park, M.D. Department of A llergy and Clinical Immunology A jou University, School of M edicine, Suwon, Korea There have been a few cases of occupational allergy caused by herb materials. In this study, we report a case of occupational asthma and rhinitis sensitized by six kinds of herb materials- Ostericum (Kangwhal), Angelica (Danggui), Cnidium (Chunkung), Pinellia (Banha), Zingerber (Kunkang) and Discoreae (Sanyak) in a pharmacist working at a pharmacy The patient had shown negative responses to 80 common inhalant and food allergens, but strong positive responses to six herb material extracts, Kangwhal, Danggui, Chunkung, Banha, Kunkang and Sanyak, were noted on skin- prick test. Bronchoprovocation test showed a dual asthmatic response to Danggui extract. Serum specific IgE antibodies to Chunkung, Banha and Sanyak were detected by enzyme- linked immunosorbent assay (ELISA) with no specific IgE binding to Kangwhal, Danggui and Kunkang extracts. In order to further characterize the allergic components of these three extracts, sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS- PAGE) and electroblotting studies were performed. One IgE binding components (60 kda) were detected within Chunkung extract, two (10, 25 kda) in Banha and four (33, 34, 65, 98 kda) in Sanyak. It is suggested that Chunkung, Banha and Sanyak may induce IgE- mediated bronchoconstriction in an exposed worker. Further studies are needed to investigate the pathogenic mechanisms induced by Kangwhal, Danggui and Kunkang.(Korean J Med 59:213-219, 2000) Key Words : Herbs; Asthma; Occupational diseases; IgE,,,,, 300 1).,, IgE 2). : 1999 7 29 : 1999 8 13 :, 5, - (442-721) E-mail : hspark@madang.ajou.ac.kr ( ) HMP- 98- M- 1-0001 - 213 -
Korean Journal of Medicine : Vol. 59, No. 2, 2000 (Ostericum), (Angelica), (Cnidium) (Umbiliferae),,. (Pinellia) (Araceae),, (Zingerber) (Zingerberaceae),, (Discoreae) (Discoreaceae), 3)., 1984 4), 1994 5), 1994 6).,. :,, 35 : : ( ) : 15, 5 (89 ),. 8 (92 ),.,.,,,,,. :. :. :. : 200/mm3, IRMA (DPC, LA, CA, USA) IgE 1332 IU/mL.. 80, Table 1. The results of skin- prick test of six herb materials and amylase Allergens Kangwhal Danggui Chunkung Kunkang Sanyak Banha Amylase Histamine Control (T able 1). methacholine PD20 0.66 CIU. 1: 5 w/v 24, 646 (Devilbiss, Somerset, PA, USA) 1.0 cc 10, 1: 100,000 v/v 10 1 (FEV1: forced expiratory volume in one second) (MMFR: maximal mid- expiratory flow rate) 1 10 3, 30 1, 6 spirometer(multispiro Irvine, PA, USA). Skin- prick test Wheal/ Erythema(mm) 30 17/72 48 19 10/50 42 19 9/57 44 5 3/23 22 18 8/61 35 42 21/72 60 3 3/4 4 6 3/4 4 1: 100,000 v/v, 1: 10,000 v/v Figure 1. T he results of bronchoprovocation tests with Danggui extract and placebo. 0-214 -
Soo- Keol Lee, et al : Occupational asthma caused by several kinds of herb materials, 1:1000 v/v FEV1 45.6%, 10. 6 (Figure 1).,. :.. 1. modified Coca (1:5 w/v) 24 4, 4 40 3,500 rpm., dialysis membrane(spectrum Medical Indust, Houston, T exas, USA) 4 24. Nalgen syringe filter(rochester, New York, NY, USA), glycerine(sigma, St. Louis, MO, USA). Bio- Rad protein assay(bio- Rad Lab., Richmond CA, USA) Bradford 7), 2.2 mg/ml, 1.2 mg/ml, 5.2 mg/ml, 6.4 mg/ml, 2.0 mg/ml, 1.0 mg/ml. 2. IgE IgE. 96- well assay plate(corning, New York, NY, USA) Carbonate buffer(ph 9.6) well 100 L 4 24 PBST (phosphate buffered saline and 0.1% T ween 20) 3, 3% BSA- PBST (bovine serum albumin- tween phosphate buffered saline) well 350 L 1. PBST 3 6 7 3% BSA- PBST 1:5 v/v, well 50 L 3. PBST 3 Biotin- labelled goat anti- human IgE (Vector Lab., Burlingame, CA, USA) 1:500 v/v well 50 L 1 30 3. 1:1000 v/v streptavidine- peroxidase(sigma, St. Louis, MO, USA) well 50 L 30 5. (T MB: 3 3' 5 5' - tetraethylbenzidine one tablet, phosphate citrate buffer 10mL, 30% H2O2 2 L) well 75 L 10 H2SO4 plate reader 450 nm. 7 2 cut- off,. IgE, IgE, IgE (Table 2). Table 2. Specific Ig E bindings to six herb material ex tracts in sera of the patient and controls Absorbance value Allergen Kangw hal Dangui Chunkung Banha Kunkang Sanyak Patient 0.174 0.147 1.173 1.105 0.133 1.183 Control 0.102 0.102 0.109 0.060 0.100 0.108 T he absorbance values of control sera were derived from mean value of seven healthy controls. 3. (ELISA inhibition test) 1:5 v/v, 1, 10, 100, 1000 g/ml 4 24 ELISA plate 1 well 100 L IgE, - 215 -
: 59 2 480 2000 Chunkung Figure 2. Chunkung- ELISA inhibition test with additions of Chunkung, Danggui, house dust mite (HDM), Banha and Sanyak extracts. Figure 3. Banha- ELISA inhibition test with additions of Banha, Sanyak, house dust mite (HDM), Kanghwal and Chunkung extracts. PBS (%). ( - sample )/, 100. Figure 2 100 g/ml 50%.,,,. Figure 3, Figure 4. 10 g/ml 50%,,,,,. 100 g/ml 50%,.,,. 4. SDS-PAGE immunoblotting, 20 g/ml, 15 g/ml 8 g/ml sample buffer(0.5m tris- HCl, ph 6.8, 1.0 ml, glycerol 1.6 ml, 10% w/v SDS 1.6 ml, mercaptoethanol 0.4 ml, 0.5%(w/v), bromophenol blue 0.4 ml, 3.0 ml) 3. 10 l standard marker(4-250 kda) (Novex, San Diego, CA, USA) mini- protein Figure 4. Sanyak- ELISA inhibition test with additions of Sanyak, Banha, house dust mite (HDM), Kanghwal and Danggui extracts. electrophoresis cell(bio- Rad Lab., Hercules, CA, USA) tris- glycine gradient gel(12% acrylamide) 125V, 30 ma 1 20. gel colloidal blue(norvex, San Diego, CA, USA). 12, 24. 12. Figure 5, 12% SDS- PAGE - 216 -
5 : 1 Figure 5. 12% SDS-PAGE finding of three herb materials. T he standards used were myosin (250 kda), bovine serum albumin (98 kda), glutamic dehydrogenase (64 kda), alcohol dehydrogenase (50 kda), carbonic anhydrase (36 kda), myoglobin (30 kda), lysozome (16 kda). M : marker protein, Ba : Banha, Ch : Chunkung, and S : Sanyak. Figure 6. IgE- binding components of three herb materials analyzed by 12% SDS- PAGE and immunoblot analysis. The standards used were myosin (250 kda), bovine serum albumin (98 kda), glutamic dehydrogenase (64 kda), alcohol dehydrogenase (50 kda), carbonic anhydrase (36 kda), myoglobin (30 kda), lysozome (16 kda). M : marker protein, Ch : Chunkung, Ba : Banha, S : Sanyak, B : buffer, and 1-5 control sera.. Immunoblottiong gel colloidal blue, nitrocellulose membrane(bio- Rad) transfer buffer (T ris 3.0 gm, Glycine 14.5 gm, methanol 100cc, 1000 cc) 10 Electroblotting Bio- Rad Mini trans- blot electrophoretic transfer cell 30V, 200mA 2 blotting nitrocellulose membrane(bio- Rad) 5% BSA- T BST 2, 5% BSA- T BST 1:5 v/v 12. Biotinlabelled goat anti- human IgE (Vector Lab., Burlingame, CA, USA) 1:500 1 30. streptavidine alkaline phosphatase(sigma, St. Louis, MO, USA) 1:1,000 v/v, 30 66 L NBT(nitrobluetetrazolium) 33 L BCIP (5- bromo- 4- chloro- 3- indolyl phosphate, Sigma, St. Louis, MO, USA) substrate (100 mm NaCl, 100 mm tris ph 9.5, 5mM MgCl2). membrane. IgE 60 kda, 10, 25, kda. 33, 34, 65, 98 kda (Figure 6). - 217 -
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