KOREAN J. FOOD SCI. TECHNOL. Vol. 41, No. 5, pp. 560~565 (2009) The Korean Society of Food Science and Technology w ³ z z k 1 Á Á Á z Á ùá xá k Á Á½ * w w w, 1 w t In vivo Toxicity and Immunoadjuvant Activity of Korean Mistletoe (Viscum album coloratum) Extract Fermented with Lactobacillus Taek Joon Yoon 1, Woong Suk Yang, Sung Min Park, Hoe Yune Jung, An Na Lee, Jin Hyuk Jung, Tae Bong Kang, Yung Choon Yoo, and Jong Bae Kim* Institute for Biomedical Research, Han-Dong University 1 Deptartment of Food & Nutrition, Yuhan University Abstract In this study, Korean mistletoe extract (KM-110) was fermented with two strains of Lactobacillus (FKM-110) and then toxicity, lectin content, and immune activities were investigated. The lectin content of FKM-110 was about 53-71% lower than that of KM-110. When mice were subcutaneously administered with KM-110 and FKM-110, the LD 50 obtained for KM-110 treatment was 50-100 mg/kg as compared to 150-200 mg/ml for FKM-110. Each preparation stimulated macrophages directly and enhanced productivity of inflammatory cytokines such as TNF-α and IL-1β. FKM- 110 treatment resulted in lower cytokine production compared to KM-110. When mice were immunized with Keyhol limpet hemocyanin (KLH) antigen along with KM-110 or FKM-110 administration, higher antibody titers to KLH were observed in the KM-110 or FKM-110 groups compared to mice immunized with KLH alone, thereby showing no difference between KM-110 and FKM-110. Therefore, fermentation of Korean mistletoe extract with these Lactobacillus strains decreased toxicity in vivo while the enhancement of immune activity by KM-110 and FKM-110 was similar. These data suggest that KM-110 fermentation tended to decrease lectin content and in vivo toxicity. In addition, other components in the fermented mistletoe extract appear to stimulate immuno-adjuvant activity instead of lectin. Key words: mistletoe extract, fermentation, lectin, toxocity, immuno-adjuvant (Mistletoe, Viscum album) ƒ ù w w», ù sw w l w w (1-3). w Viscum w Viscum alum loranthacea, w w Viscum alum coloratum w š (4). p, w 1920 l z w e /»k» t xk q š (2,5). wr, ù w (Viscum album Coloratum) ù»,», w», m, šx,, em w w š(3), w y t y w ƒ w š (3,6-9). *Corresponding author: Jong Bae Kim, Institute for Biomedical Research, Han-Dong University, Pohang, Gyeongbuk 795-940 Korea Tel: 82-54-260-1350 Fax: 82-54-261-6705 E-mail: jbkim@handong.edu Received June 20, 2009; revised August 11, 2009; accepted August 12, 2009 560 w s y ƒ ƒ w p(lectin) w (3,4,6,7,10,11). p p w p-i, -II -III wš, p ƒ B-chain cytotoxic A chain hetero dimmer xk wš (10,11). p w s A-chain w 60S ribosomal subunit y w w ƒ (12), p w s apoptosis w (7,13). w p s p- w w s l tumour necrosis factor-α(tnfα), interleukin-1β(il-1β) interferon-γ(ifn-γ) ƒ cytokine w p y j y (11). p cytokine ùkü» p w» w w ù, x ¾ xk ƒ š (11,14). w w s w w p w t y, w» w wù ³ zw w (2,11,14). y w ù Iscador Lactobacillus plantarum z k
mw p w t y k (11,14). Iscador š w, ³ z w p w z j w 10 w ù, ƒ ƒ ƒ w š w y y w š š (2,11,14,15). Iscador, ³ z w s z ƒ ³ z w dimer xk p A B chain z ùkü w p peptides w k s ƒ x w š (15). w, ³ z z z ƒ w z ³ w z ƒ w wš (5,11,14). w Iscador w y w, macrophage natural killer cell(nk-cell) ww y y,, p s y,, w s z w š (11,14,15). Iscador w x, s w s z y y w s w y j z ƒ šw (11,16,17). ü y w w, y w ƒ y w w š (3,6-9,13), w w z s p p w (3,8) y w p w šw (9,18). w w w y (8,9,18) ³ z in vivo z z z w, z» ƒ mwš w. z x w ù w 12-1 w š, w (KM-110)» š (21) w w. KM-110 z w s w 2 ³ w w. w, Lactobacillus casei KCTC 2180, Lactobacillus plantarum KCTC 3103 1%ƒ 20 mg/ml KM-110 z, ³ 37 o C 30 o C ƒƒ 1, 2 3 w. z, ƒ ³ z w š, r ƒƒ FKM-LC FKM-LP ew. PBS w 50 mg/ml stock solution z 20 o C w x w. x z 6-8 BALB/c ICR ( ) w x (Seoul, Korea) w w x w. ƒ 5 x r (Samyang Co Ltd, Incheon, Korea) œ w š, 22 o C, 50%, 12 k p w w. w z y 561 s s w RPMI-1640 Eagle s minimal essential medium(emem), fetal bovine serum(fbs), vitamin solution, non-essential amino acid, L-glutamic acid, thiogly-collate Gibco(Carlsbad, CA, USA) w. s clon26-m3.1 lung carcinoma 7.5% FBS, vitamin solution, sodium pyruvate, non-essential amino acid, L-glutamine w EMEM, lymphocytes 7.5% FBSƒ w RPMI-1640 ƒƒ w 5% CO 2, 95% 37 o C»(Thermo Fisher Scientific Inc. Waltham, MA, USA) w. w w keyhol Lympet hemocyanin (KLH) Sigma-Aldrich (St. Louis, MO, USA) w. Lecin w p(kml-c) w» t twosite sandwich ELISA w (19). w, KML-C w j w 8E12-3E9(10 µg/ml) 50 ml ELISA plate (Rochester, NY, USA) ƒ well coating z, washing BSA w blocking w z, t 50 ng-5 µg/ml KML-C ƒw g. KML-C w detetion w t horseradish peroxidase(hrp: Sigma, St. Louis, MO, USA)ƒ w 8B11-2C5-HRP w ƒw g. z ƒ well wš HRP w» 3,3',5,5' tetramethylbenzidine(tmb; Sigma, St. Louis, MO, USA) ƒw z, 450 nm Ÿ d w. In vivo group 7 ICR (, 7 ) KM- 110, FKM-LP FKM-LC ƒƒ 200, 150, 100 50 mg/kg vw w š, z 5 w x y d w. Macrophage l Cytokine BALB/c 1% thioglycollate 1mL wš 3 z k z, RPMI-1640 w ü s(peritoneal exudative cells, PEC) w. z w PEC 24 well culture plate 1.5Ü10 6 /ml w w. z ƒ ƒ(10 µg/ml)wš 24 w. ƒ cytokine w lipopolysaccharide(lps: Sigma, St. Louis, MO, USA) w. z, macrophage z wš, TNF-α IL-1β ƒ cytokine ELISA kit(pharmingen, CA, USA) w e d w. w w ƒ d w w w KLH w. 6 BALB/ c 10 µg KLH y KLH 100 µg KM-110 ƒ ³ ƒƒ z FKM-110 y ww 2 2z vw w. z 1 l 7 ¾ 2 l x w š, x z w ƒ d ¾ 20 o C w. x ü KLH w w ƒ» š indirect ELISA (20) d w š, w ƒ x w OD 2 ƒ w.
562 w t wz 41 «5y (2009) Fig. 1. Consentration of lectin in fermented KM-110 determined by two-site Sandwich ELISA. (A) FKM-LC; fermented with Lactobacillus casei KCTC 2180, (B) FMK-LP; fermented with Lactobacillus plantanum KCTC 3103. *p<0.01 compared with the KM-110 group by student s two-tailed t-test. m w x m Microsoft Excel v (Redmond, WA, USA) Student s two-tailed t-test w. š ³ zw w lectin y 2 ³ w w 1-3 z k z, lectin w y w (Fig. 1). 10 mg/ml KM- 110 600 ng/ml lectin w š, KM- 110 Lactobacillus casei KCTC 2180 24, 48, 72 z FKM-LC lectin w ƒƒ 428, 398, 383 ng/ml z lectin w w w ùkû ù, m. wr, KM-110 Lactobacillus plantarum KCTC 3103 ³ w 24-72 z k FKM-LP, z 320 ng/ml lectin w. FKM-LC lectin w KM-110 w 71.3-63.8% š, FKM-LP 53.3%. ³ z w lectin w š ³ z z lectin w yƒ š, lectin w w z (5,17,21). z k, ³ k y z w ƒ w ƒw. lectin ƒ w» y y j ƒ w š š» (5,10,17,18). ù wr, lectin w w (11). x wš q w lectin w t y t ƒ š. w d x wš z Iscador p w w š(11,14) š, x z t Isorel Helixor w p w w w w p w w û ƒ ùkû ( t ). z ƒ z w x p p w w wš. x 1 p w p ƒ š ƒ ³ w p w in vivo z KM-110 w x ww. In vivo ³ z k in vivo x w. x w 3 z w. 7 kg KM-110, FKM-LC FKM-LP ƒƒ 200, 150, 100 50 mg vw wš 5 w (Table 1). KM-110 200 150 mg/kg w, n 2 x 7 ƒ w, 100 mg/kg n 1 1, 2 3, n 3 2 ƒ w x 1 ƒ w. 50 mg/kg n 2 1 w ù 6 w. KM-110 n w 50% ƒ w lethal dose(ld 50 ) 50 mg/ kg 100 mg/kg. wr, FKM-LC FKM- LP 200 mg/kg n ƒƒ 1 3 ƒ w š, 150 mg/kg ƒƒ 1 2 ƒ w. z k LD 50 150 mg/kg 200 mg/kg ƒ. w w, 6 z l w w (piloeection) x. w x w TNF-α cytokine w (22). w LD 50 ƒ¾ n w x ù, n 3 z y
w z y 563 Table 1. Motality in mice administered subcutaneously with KM-110 or FKM-110s Days after treatment Dose (mg/kg) 1 2 3 4 5 Final mortality KM-110 4/7 7/7 0 0 0 7 200 KM-LC 2/7 5/7 6/7 6/7 6/7 6 KM-LP 1/7 3/7 4/7 4/7 4/7 4 KM-110 3/7 7/7 0 0 0 7 150 KM-LC 0/7 2/7 2/7 2/7 2/7 2 KM-LP 0/7 1/7 1/7 1/7 1/7 1 KM-110 1/7 4/7 6/7 6/7 6/7 6 100 KM-LC 0/7 0/7 0/7 0/7 0/7 0 KM-LP 0/7 0/7 0/7 0/7 0/7 0 KM-110 0/7 2/7 2/7 2/7 2/7 2 50 KM-LC 0/7 0/7 0/7 0/7 0/7 0 KM-LP 0/7 0/7 0/7 0/7 0/7 0 Fig. 2. Comparison on the induction of cytokines from peritoneal macrophage stimulated with mistletoe and fermented mistletoe extracts. Each mistletoe extracts was co-cultured with macrophage for 24 h. The content of cytokines in cultured supernatant was assayed by ELISA. (A) FKM-LC; fermented with Lactobacillus casei KCTC 2180, (B) FMK-LP; fermented with Lactobacillus plantanum KCTC 3103. KM-110 25 mg/kg w, FKMs 100 mg/kg w. š cytokine w ƒ w p wš š(10,11,18), z p w (Fig. 1). ³ z š p ³ z w A B chain ù, peptides ë ƒ x w (5,15). in vivo z z ƒ û» t ü, z p w A- B-chain w, cytokine w p x w ƒ. Macrophage l cytokines w y macrophage y y j (3,8,14,16). y y macrophage w ƒ cytokine w sƒ w w w w w s y j w w (8,11,23). ww» y y» v ù, cytokine ƒ w cachexia, autoimmune disorder e septic shock(qx j) j» w (24). sƒ w cytokine t TNF-α IL-1β, p TNF-α w ƒ w cytokine (24,25). x Table 1 w z in vivo z w w» w macrophage l cytokine TNF-α IL-1β e ƒ z mw (Fig. 2). x Lactobacillus casei KCTC 2180 ³ z FKM-LP Lactobacillus plan-
564 w t wz 41 «5y (2009) Fig. 3. Comparison on the production of KLH-specific antibody between mistletoe and fermented mistletoe extracts. Five Balb/c mice per group were immunized s.c. twice at the interval of 2 weeks with KLH with or with each mistletoe extracts. (A) FKM-LC; fermented with Lactobacillus casei KCTC 2180, (B) FMK-LP; fermented with Lactobacillus plantanum KCTC 3103. The fermentation time by each Lactobacillus was adjusted to 24, 48, and 72 hr. tarum KCTC 3103 ³ z FKM-LP KM-110 w û cytokine š, wù w z w p» (11,14,16). z cytokine Table 1 z in vivo z w w» ƒ (11,16,24). FKMs-110 s z w y wù w w z w (5,20). x y z w e z mw» w w KLH ƒ z KLH w w mw (Fig. 3)., w w KM-110 100 µg n» x KM-110 z w w (20)., w KLH y ƒ yww z 7 ¾ l x z, wx KLH w w ƒ ELISA d w. Fig. 3 w ƒ w KLH w z, KLH w w ƒ d w z z KM-110 w z. x w z ³ z w. ³ z Iscador w w, z z z ƒ w z ³ w z ƒ ƒ (5,15). ³ z w p ƒ wš y ùkü p z y p w y w w» w ƒ w w. Fig. 3 p z ƒ y š š (6,10,14,18) wš, z w p w z z KM-110 w y ùkü, z w» w xk(native form) p x p w, y ƒ p w w w (16,26-28). ƒ w y ƒ p w, ƒ ƒ w y p ww w» (26-28). y ùkü p polysaccharides, oligosaccharides, peptides alkaloids š (4,26-28). w polysaccharides y oligosaccharides w š(26,28) š ù, p w y w. ³ z y» t w» w w w w z ³ z y mw p p w t y w ƒ v w ƒ. ³ w g l w j z ƒ w y š š(29-31) m t š w t,» w ³» t ƒ w y». Lactobacillus casei KCTC 2180 Lact. plantarum KCTC 3103 z w FKM-110 p w y, z mw. ƒ ³ w KM-110 1-3 z k, p z w 53-71% w ùký w. w
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