KOREAN J. FOOD SCI. TECHNOL. Vol. 41, No. 6, pp. 717~721 (2009) š w» z ÁyÁy»ÁÁ 1 * w tƒœw, 1 w m y l The Korean Society of Food Science and Technology Anti-allergic Effect of Zizania latifolia Turcz Extracts Eun-Jung Lee, Eun-Yeong Whang, Key Whang, In-Seon Lee, and Seun-Ah Yang 1 * Deptartment of Food and Technology, Keimyung University 1 The Center for Traditional Microorganism Resources, Keimyung University Abstract The anti-allergic effect of the methanol extract of Zizania latifolia Turcz was investigated. Methanol extract of Z. latifolia Trucz did not display any cytotoxicity activities in the IgE-sensitized RBL-2H3 cells. The extract was found to inhibit compound 48/80-induced degranulation and antigen-induced β-hexosaminidase release as well as PMA plus A23187-induced TNF-α production in RBL-2H3 mast cells in a dose-dependent manner. Release of β-hexosaminidase, a marker for the release of histamine in mast cells, was inhibited with an IC 50 value of 73.8 µg/ml. These results suggest that Z. latifolia extracts may be useful for the prevention of type I allergic reaction. Key words: Zizania latifolia Turcz, β-hexosaminidase, degranulation, TNF-α, RBL-2H3 cells» y x y, y y w w w wš, ù» y y ƒ w ã ƒwš.»» w x, wx x j 4ƒ x(i~iv) ù m» w w w w w w w w z ü ù x 1x» w,», mv v»» y 1x» (type I allergic reaction) w(1-3). s x» w v s (4). s t xw š šey IgE FceRI IgE p ww, s ü e ƒ wš k ù kù xq, ƒwz š interleukin- 4(IL-4), IL-6, IL-13, TNF-α ƒ me w, x jš, x n ƒj,» j (5-8). s yy w IgE mw w w compound 48/80, protein kinase *Corresponding author: Seun-Ah Yang, The Center for Traditional Microorganism Resources, Keimyung University, Daegu 704-701, Korea Tel: 82-53-580-6449 Fax: 82-53-580-6449 E-mail: seunahy@kmu.ac.kr Received August 3, 2009; revised August 21, 2009; accepted September 8, 2009 C(PKC) activator phorbol 12-myristate 13-acetate(PMA), calcium ionophore(a23187) w» š (9-11). Rat RBL-2H3 s t IgE sww, IgE z w w k w(12), w» z RBL-2H3 sƒ s š. w w w k w e (13-15).» k w,» ƒwš x w w z yw š. x w»,,,, ù (16-20). š(zizania latifolia) ù, ƒ w w t, t š. š, šx, t,,,, y ƒ ù zƒ, j z jš šš. w, ù, ywt ƒ t ù z š w(21,22). ù w š z w ù, z t ƒš. s y g t w s, DNA fragmentation s š y w w šƒ, š zšƒ ƒƒ w wvz ƒ ƒ š(23,24). š w w» w» w, RBL- 717
718 w twz 41 «6y (2009) 2H3 s w s yy w wz mw. x š(zizania latifolia) w k w. 100 g 30 k ƒw 24 e wš, 3z w. (Whatman No. 3, Maidstone, England) w rotary vacuum evaporator(ut-1000, EYELA, Tokyo, Japan) w z w 15.4 g, 20 o C w w. s w RBL-2H3(KCLB No. 22256) s Rat y» s Korea Cell Line Bank(Seoul, Korea) l, 10% fetal bovine serum 1% streptomycin/ penicillin w MEM w 37 o C, 5% CO 2 w w. s d IgE s w š MTT assay dw. RBL-2H3 s(3 10 4 cells/well) 450 ng/ml DNP-specific IgE(Sigma, St. Louis, MO, USA) w 37 o C, 5% CO 2 w w z (10-100 µg/ml) š s w. 12 z ƒ well 5 mg/ml MTT 10 µl ƒwš 4 37 o C incubator w. wš 100 µl DMSO well formazan wg ELISA reader(spectra MAX 340 pc, Molecular Device, Sunnyvale, CA, USA) w 550 nm Ÿ dw. s Ÿ Ÿ w ù kü. RBL-2H3 s k RBL-2H3 s 24-well plate(2 10 5 cells/well) 4 w z, 200 µl incubation buffer(119 mm NaCl, 5 mm KCl, 0.4 mm MgCl 2, 25 mm PIPES, 40 mm NaOH, 5.6 mm glucose, 1 mm CaCl 2, 0.1% bovine serum albumin, ph 7.2) š 10 w. 0.1% DMSO ù 25 µl ƒ ƒ 37 o C 10 g. z, compound 48/80 (Sigma) 25 µl ƒw 20, 40 g. z, s xk x (DM IRE2, Leica, Wetzlar, Germany) 600 w w. 200 w k yw, 3z x x yw. β-hexosaminidase d RBL-2H3 s(2 10 5 cells/well) 450 ng/ml IgE wì CO 2 incubator 12 w s g. k s siraganian buffer(119 mm NaCl, 5 mm KCl, 0.4 mm MgCl 2, 25 mm PIPES, 40 mm NaOH, ph 7.2) w z, ƒ well 160 µl siraganian buffer(119 mm NaCl, 5 mm KCl, 0.4 mm MgCl 2, 25 mm PIPES, 40 mm NaOH, 5.6 mm glucose, 1 mm CaCl 2, 0.1% bovine serum albumin, ph 7.2) š 20 w. ƒ 5µM wortmannin(wako Pure Chemical Industries, Tokyo, Japan) 10 jš, 10 µg/ml DNP-BSA(Alpha Diagnostic International, San Antonio, TX, USA) ƒw z 10 w s yy g ice bath g. d 25 µl 96-well plate»š, substrate buffer(p-nitrophenyl-n-acetyl-β-d-glucosaminide 1 mm in citrate buffer 0.1 M, ph4.5) 25 µl z 37 o C 1 g. z, ƒ well stop solution (0.1 M Na 2 CO 3 /NaHCO 3, ph 10) 200 µl ƒw jš, ELISA reader w 405 nm Ÿ dw. TNF-α d RBL-2H3 s 24-well plate(2 10 5 cells/well) w z 12 w. MEM w š, A23187(1 µm) PMA(50 ng/ml) w 4 w. óù z d w 70 o C w ƒ TNF-α ELISA kit(invitrogen Corporation, CA, USA) w dw. ELISA reader w 450 nm Ÿ dw TNF-α w. m w š x w m Student s t-test w. m z p 0.05 (p<0.05) m š qw. š š s IgE RBL-2H3 s w š w» w MTT assay ww, Fig. 1 ùkü. s w x s ùkü. š x 0-100 µg/ml 12 w s ùkü. s k w š z Compound 48/80 formaldehyde w cross-linked phenethylamine mixed polymer s w s e s ü g s ü e ƒg s k w(25,26). Compound 48/80 w s xk x ùx s wš, ñ t, compound 48/80 yy k ù s xk s wš j»ƒ, s ü ù s (Fig. 2). Compound 48/80 w š w, k ù s xk yƒ, compound 48/80 w ü s ƒƒ ³ew st w. w, 50 100 µg/ml š w compound 48/80 w s k xw w.
š w» z 719 Fig. 1. Cytotoxic effects of Zizania latifolia extract on RBL-2H3 cells. RBL-2H3 cells (3 10 4 cells) were cultured with 450 ng/ml of DNP-specific IgE overnight. After treatment with varying doses of Z. latifolia extract, cells were cultured for 12h. The cytotoxicity of Z. latifolia extract on RBL-2H3 cells was determined by MTT assay. The value represents mean±sd of three different experiments. β-hexosaminidase w š z sƒ wwš k tryptase w» w w w w w yw w (27). β-hexosaminidase sƒ w yy k wì s k t š (28,29). RBL-2H3 s β-hexosaminidase y dw, š w β-hexosaminidase z w. 10, 20, 50, 100 µg/ml š s β- hexosaminidase y w ƒƒ 97, 77, 58, 37% ùkü, w yw (Fig. 3), IC 50 73.8 µg/ml ùkû. w, 2.14 µg/ml wortmannin 29% ùkü β-hexosaminidase w g yw. Fig. 3. Inhibitory effects of Zizania latifolia extract on β- hexosaminidase release from RBL-2H3 cells induced by IgE with DNP-BSA. RBL-2H3 cells (2 10 5 cells) were sentitized with 450 ng/ml of DNP-specific IgE overnight and pretreated with varying doses of Z. latifolia extract for 10 min. Cells were stimulated with 10 µg/ml of DNP-BSA for 10 min. The degranulation was determined by measurement of the activity of β-hexosaminidase in culture media. The value represents mean±sd of three different experiments. Wortmannin (WM, 2.14 µg/ml) was used as a positive control. *Values are significantly different from control (*p<0.05, **p<0.01). š TNF-α z TNF-α s s w,» w w w (30). š y y sl me TNF-α g ELISA w. RBL- 2H3 s TNF-α w 5.06 pg/ml û, A23187 PMA w 87.14 pg/ml x ƒ yw. 10, 50, Fig. 2. Light microphotographs using inverted microscopy (magnification; 600) of RBL-2H3 cells without (1, 2, 3) or with (4, 5, 6) treatment of 50 µg/ml compound 48/80 for 40 min. The Z. latifolia extract were pretreated for 10 min (2, 5: 100 µg/ml, 3, 6: 50 µg/ml) prior to the stimulation with compound 48/80. Arrow shows degranulated cells.
720 w twz 41 «6y (2009) Fig. 4. Effects of Zizania latifolia extract on PMA plus A23187- induced cytokine production in RBL-2H3 cells. The RBL-2H3 cells (2 10 5 cells) were incubated overnight in 24-well plate. The cells were pretreated with various concentrations of Z. latifolia extract and then stimulated with PMA (50 ng/ml) plus A23187 (1 µm) for 4 h. The level of cytokine in the supernatant was measured by using ELISA. The value represents the mean±sd of three independent experiments. Luteolin (1.43 µg/ml) was used positive control. *Values are significantly different from control (*p<0.05, **p<0.01). 100 µg/ml š w ƒƒ 79.76, 65.38, 53.4 pg/ml d, A23187 PMA w w 91.5, 75, 61.3% g ùkù TNF-α g yw. w, 1.43 µg/ml luteolin TNFα 32.5 pg/ml ùkù w 37.3% yw (Fig. 4). s šey IgE ww w xw s adenylate cyclaseƒ yy camp ƒj, camp y protein kinase yyjš, yy protein kinase e n y g qjš s w g k w (31). w, s MAPK ERK yj NF-κB yyg TNF-α, IL-6, IL-8 me x ƒk(32). x w» zƒ š polyphenol curcumin(33), flavonoid natsudaidain(34), šp xanthone mangostin (35), ù sesquiterpene lactone(18) saponin (19). ù x š w w» z ùkü d w». š k z w» w» w šl w» y ƒ wš, w s ü y» e w w w vƒ ƒ. š k w» y w» w, s RBL-2H3 s w, š w z IgE-w, A23187 PMA w s yy g β-hexosaminidase, TNF-α dw. š yy s β-hexosaminidase, TNF-α w yw. w, s k w ùkù xkw y w w w» w š w s compound 48/80 w, š w w k x w. š w w s k w mw» j q, š w»» ƒ w. t» w m y l w. 1. Patterson R, Dykewicz MS, Grammer LC, Greenberger PA, Lawrence ID, Walker CL, Wong S, Zeiss CR. Classification of immediate-type, life-threatening allergic or pseudoallergic reactions. Chest 98: 257-259 (1990) 2. Coombs, RRA, Gell, PGH. Classification of Allergic Reactions Responsible for Clinical Hypersensitivity and Disease. Clinical Aspects of Immunology, 3 rd ed. Blackwell Scientific Publications, London, UK. pp. 761-779 (1975) 3. Siraganian RP, Hook WA, Levine BB. Specific in vitro histamine release from basophils by bivalent haprens: The evidence for activation by simple bridging of membrane bound antibody. Immunochemistry 12: 149-157 (1975) 4. Ahn KM. Role of mast cells in allergic inflammation and innate immunity. Korean J. Pediatr. 47: 1137-1141 (2004) 5. Schroeder JT, Kagey-Sobotka A, Lichtenstein LM. The role of the basophil in allergic inflammation. Allergy 50: 463-472 (1995) 6. Beaven MA, Rogers J, Moore JP, Hesketh TR, Smith GA, Metcalfe JC. The mechanism of the calcium signal and correlation with histamine release in 2H3 cells. J. Biol. Chem. 259: 7129-7136 (1984) 7. Shakoory B, Fitzgerald SM, Lee SA, Chi DS, Krishnaswamy G. The role of human mast cell-derived cytokines in eosinophil biology. J. Interf. Cytok. Res. 245: 271-281 (2004) 8. Salamon P, Shoham NG, Gavrieli R, Wolach B, Mekori YA. Human mast cells release interleukin-8 and induce neutrophil chemotaxis on contact with activated T cells. Allergy 60: 1316-1319 (2005) 9. Metcalfe DD, Kaliner M, Donlon MA. The mast cell. Crit. Rev. Immunol. 3: 23-74 (1981) 10. Chand N, Pillar J, Diamantis W, Perhach JL, Sophia RD. Inhibition of calcium ionophore (A23187) stimulated histamine release from rat peritoneal mast cells by azelastine: Implications for its mode of action. Eur. J. Pharmacol. 96: 227-233 (1983) 11. Takei M, Umeyama A, Shoji N, Arihara S, Endo K. Mechanism of inhibition of IgE-dependent histamine release from rat mast x
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