KOREAN J. FOOD SCI. TECHNOL. Vol. 40, No. 5, pp. 574~579 (2008) The Korean Society of Food Science and Technology v s z v s ƒ BALB/c mice y e w ½ Á«x Á½ yá y 1 Á 1 Á x 2 Á½ * w w t z sƒ» l, 1 w v s, 2 w w t w Immunomodulatory Effects of Propolis and Fermented-propolis in BALB/c Mice Yoon Hee Kim, Hyuck-Se Kwon, Dae Hwan Kim, IL-Hwan Park 1, Sang-Jae Park 1, Hyun-Kyung Shin 2, and Jin-Kyung Kim* Center for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University 1 Korea Agricultural Propolis Inc. 2 Department of Food Science and Nutrition, Hallym University Abstract Propolis is the generic term for the resinous substance collected by honey bees from a variety of plant sources. In this study, we have assessed the immunomodulatory properties of propolis (P) and fermented-propolis (FP) in BALB/ c mice. Mice were subjected to gavage once a day (for 14 days) with 50, 100, 200 mg/kg body weight P, FP, or vehicle. Lymphocytes were isolated from the spleen and mesenteric lymph nodes (MLN) and the immune cell proportions, proliferative activities, and cytokine production were evaluated. The P- and FP-administration induced similar, but differential, alterations in the percentage of immune cell populations and their biological functions, including cytokine production and NK cell cytotoxicity. The proportion of CD4 + and CD8 + T cells in the spleen was increased slightly in the P- and FP-administered mice as compared to the vehicle-treated mice. In MLN, the percentage of CD4 + T cells was increased significantly in the 200 mg/kg P-treated mice. The mice which were treated with P and FP evidenced significantly increased interferon-γ and interleukin-4 production in concanavalin A-stimulated splenocytes, whereas the production of theses cytokines was not shown to be induced by P-treatment. In addition, NK cell activity was also increased dramatically by the administration of P and FP. Collectively, these findings showed that P and FP are widespectrum immunomodulators, which may modulate both innate and adaptive immune responses. Key words: propolis, immunomodulatory effect, cytokines, NK cell activity 20» w w w s³ ƒwš, y y,,, sww ƒ z p ƒ y ƒwš.»y yw ƒ mv v y j ƒwš (1). w y w, y y w ƒ jš, w ƒ w š (2). *Corresponding author: Jin-Kyung Kim, Center for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University, Chuncheon, Gangwon-do 200-702, Korea Tel: 82-33-248-3106 Fax: 82-33-248-3103 E-mail: kimjin@hallym.ac.kr Received July 5, 2008; revised August 15, 2008; accepted August 20, 2008 s, natural killer(nk) s T v y ƒ k s l y w interleukin(il)-2 interferon(ifn)-γ me (cytokine) g, y ƒ j (3-5). v s ã ww ³ ù l yw» w k yw g,, š beeswax, resin, flavonoids, organic acids, caffeic acid w w wš. v s» 300 l ù.» 1» Plink Historia naturalis v s ƒ m,, e w š šwš (6,7). š l y v s w w w (8-10), w³(11), w l (12), w (13), w (14,15) w y(16 y x. v s z w w, v s z w z k w w š ƒ. 574
v s z v s ƒ BALB/c mice y e w 575 v s z v s x n w y e w ³ wš w. x x w v s z v s w v s ( ) l œ w. v s (P) 100 g 500 ml ƒw 60 o C 3,, w, 4 o C û e g. e w z œ w v s 18 g. z v s (FP) 100 g 500 ml ƒw 60 o C 3, w z 100 ml w. 45 g ƒw z œ w v s 50 g 10%(w/v) s 500 ml ƒw.» Saccharomyces cerevisiae w 37 o C 2 z g v ƒwš 60 o C 2, w z œ, w. z v s 100 g 13 g. x w v s z v s 100%. x x n x 5 f BALB/c g l( )(Pyoungtaek, Korea) l œ w w x 7, y k z, w. 20.9-22.6 o C, 50-55%,» 12 (08:00-20:00) w š, g. 7 y» e 7 x w, ƒ x 8 w. 95% ww v s z v s 1 kg, ƒƒ 50, 100 š 200 mg/kg body weight(b.w.) 3 w w, 14 n w. x n w v s z v s ethanol 5% w š, 5% ethanol n w. (spleen) v (mesenteric lymph node) l v x k w v ³ wš, ƒ v RPMI 1640 medium(hyclone, Logan, UT, USA) ü 40 µm stainless steel mesh(bd Falcon, Franklin Lakes, NJ, USA) w s w. s RPMI 1640 medium ƒw 4 o C, 1,200 rpm 5 e z, RBC lysis buffer(ebioscience, San Diego, CA, USA) x w v. v 10% fetal bovine serum(fbs), 100 units/ml penicillin, 100 µg/ml streptomycin w complete RPMI 1640 medium ƒw k z, Guava Viacount Kit(Guava Technologies, Hayward, CA, USA) w s d w. v d v CellTiter 96 AQ ueous ONE Solution Assay Kit(Promega, Madison, WI, USA) w d w (17). v w v 1 10 5 cells/200 µl/well 96 well plate w v concanavalin A(ConA, 5 µg/ml) ƒw š, v anti-cd3ε(1 µg/ml) anti-cd28(1 µg/ml) j w ƒ w 48 w. ƒ well cell titer 20 µl ƒw 2 ƒ z, SpectraMaxM2 Microplate reader(molecular Devices, Sunnyvale, CA, USA) 490 nm OD d w v d w. v d v fluorescence-activated cell sorting(facs) w d w (18). v 1 10 4 cells/200 µl/well 96 well round bottom plate w fluorescein isothiosyanate(fitc)-conjugated hamster antimouse CD3, phycoerythrin(pe)-conjugated rat anti-mouse B220, PE-conjugated rat anti-mouse CD4, PE-conjugated rat anti-mouse CD8, FITC-conjugated rat anti-mouse pan NK j w ƒ w. ƒ j w ƒ 30 z, flow cytometer(guava Technologies) w s d w. v me d v me Park, z d w (17). v 24 well plate 8 10 5 cells/500 µl/well w. v ConA(5 µg/ml), v anti-cd3ε(1 mg/ml) anti-cd28(1 mg/ml) j w ƒƒ 48 g s d. s d IL-2, IL-4, IL-5, IFN-γ ELISA Kit(eBIOSCIENCE) w d w. Natural killer(nk) s y d NK s y Combe, w d w (19). v NK s pan NK selection kit(stem Cell Technologies, Vancouver, CA, USA) w w. w NK s RPMI 1640 medium xk g s (effector cell) w. t s YAC-1 s w. NK s YAC-1 s 20:1 96 well round bottom plate w, 37 C 4 o w, NK s y Guava EasyCyte TM CellToxicity Kit(Guava Technologies) w d w. m w mean±sem ùkü, GraphPad Prism 4.0 software(graphpad Software Inc., San Diego, CA, USA) w one-way ANOVA w š, ƒ m Boneferroni multiple comparison post test w. p<0.05 m q w. š v s v s z v s n ƒ e w mw» w, v v mw (Tables 1, 2). v v s z v s n (vehicle-n ) w CD4 + CD8 + T v
576 w t wz 40 «5y (2008) Table 1. Proportion of lymphocytes isolated from spleen in BALB/c mice treated with propolis or fermented-propolis for 14 days Group Lymphocyte subsets CD3 + CD3 + CD4 + CD3 + CD8 + B220 + pannk + Vehicle 41.92±4.14 24.91±3.53 15.77±0.65 48.13±1.00 8.75±0.75 P-50 48.65±1.61 30.34±1.35 16.88±0.65 45.99±0.82 8.71±0.41 P-100 51.49±1.01 31.07±1.26 *18.49±0.39* 44.99±0.70 8.73±0.40 P-200 49.85±0.35 30.50±0.24 18.01±0.10 *44.03±1.00* **6.67±0.38** FP-50 *55.15±0.97* *33.85±0.60* **19.90±0.60** 42.26±0.82 **6.33±2.45** FP-100 50.55±1.45 31.33±0.72 17.74±0.61 45.41±0.64 7.43±0.40 FP-200 50.07±1.90 31.38±1.48 17.58±0.51 44.66±0.72 *7.29±0.78* P: Propolis treated group FP: Fermented-propolis treated group (*) p<0.05, (**) p<0.01 vs. vehicle treated group Table 2. Proportion of lymphocytes isolated from mesenteric lymph node in BALB/c mice treated with propolis or fermented-propolis for 14 days. Group Lymphocyte subsets CD3 + CD3 + CD4 + CD3 + CD8 + B220 + Vehicle 45.02±2.53 25.37±0.89 15.62±1.97 17.93±2.42 P-50 36.47±4.96 27.95±3.10 10.94±1.90 22.00±1.22 P-100 49.03±3.14 29.11±2.54 12.09±1.43 27.63±0.25 P-200 **70.24±1.91** **47.60±4.04** *20.51±2.64* 15.52±1.23 FP-50 48.13±4.32 34.99±4.37 09.77±1.82 21.83±3.05 FP-100 58.74±3.25 **46.71±1.75** 12.52±4.71 26.46±1.85 FP-200 53.43±5.63 29.83±3.65 *20.32±3.71* 20.53±1.90 P: Propolis treated group FP: Fermented-propolis treated group (*) p<0.05, (**) p<0.01 vs. vehicle treated group ƒw w, B220 + B v NK s v s z v s n w w w. v w š (200 mg/kg) v s z v s (50, 100, 200 mg/kg) n w CD4 + T v ƒw w š, B220 B s ƒ n + w (Table 2)., v s n, z v s n ƒ» s y ( ). Takagi (20) v s n ƒ x CD4 + CD8 + T v ƒ k š šw š, Andreia (18) Trypanosoma cruzi w v s k (50 mg/kg) 14 n w v CD4 + CD8 + T v ƒw š šw. v s n ƒ s y y g w me wš, me w T v ƒw. v, v s z v s n v w v d w, w v s z v s n w v ƒ w (Fig. 1). Sá-Nunes š(21) v s k (2.5-10 mg/kg) 3 n w v w w w. v s y caffeic acid phenethyl ester(cape) 20 mg/kg BALB/c 14 n w v w ùkû (17)., v s n w v w ƒ w v s n n» w š, w w z ƒ v w. v s n ƒ v e w w x š ƒ, ƒ š. v me CD4 + T s w me Th-1 Th-2 T v. Th-1 T v sü (intracellular infection) w s IL-2 IFN-γ wš, Th-2 T v (extracellular infection) w w IL-4 IL-5 w, ü w (4,21). v s z v s n w v me sƒw» w, ƒ v 48 w z, me d w (Fig. 2, 3). v s n w v me w ƒ w (Fig. 2). w, z v s
v s z v s ƒ BALB/c mice y e w 577 Fig. 1. Effects of propolis and fermented-propolis on proliferation of lymphocytes isolated from spleen (A) and mesenteric lymph nodes (B). Values are means±sem from triplicate wells. (**) p<0.01 and (***) p<0.001 vs. vehicle-treated group. Fig. 2. Effects of propolis and fermented-propolis on cytokine releases by splenocytes. Values are means±sem from triplicate wells. (A) IL-2, (B) IFN-γ, (C) IL-4, (D) IL-5. (*) p<0.05, (**) p<0.01 and (***) p<0.001 vs. vehicle-treated group. n IL-2, IFN-γ IL-4 ƒ w ƒwš. IL-5 w v s z v s z w (Fig. 2D). w v Th-1 Th-2 me z v s n ƒ v s n w w wš. v me w, IL-2 IFN-γ Th-1 me v s z v s n (50, 100, 200 mg/kg) w w ƒw w (Fig. 3A, 3B). IL-4 IL- 5 Th-2 me š (200 mg/kg) v s n z v s n w ƒ (Fig. 3C, 3D). w v s z v s n ƒ v v me w ùkü. IL-2 T v, B v NK s v y ¾ w me, IFN-γ CD8 + T v ù NK s w s ù w w. IL-4 B v w w wš, naive CD4 + T v l Th-2 T v y w me. IL-5 (innate immunity) w y y w me. x v s z v s n ƒ v v Th-1/Th-2 me w ü z ùkü ƒ w š.
578 w t wz 40 «5y (2008) Fig. 3. Effects of propolis and fermented-propolis on cytokine releases by lymphocytes isolated from mesenteric lymph nodes. Values are means±sem from triplicate wells. (A) IL-2, (B) IFN-γ, (C) IL-4, (D) IL-5. (*) p<0.05, (**) p<0.01, (***) p<0.001 vs. vehicle-treated group. Fig. 4. Effects of propolis and fermented-propolis on cytotoxicity of NK cells isolated from spleen. Values are means±sem from triplicate wells. (***) p<0.001 vs. vehicle-treated group. NK s y NK s s ü w s s ww ù, w ù s k s s y y k me sww w s w w wš s (22). v s z v s n w NK s y w w d w» w ƒ l NK s w s YAC-1 s œ w NK s YAC-1 s w d w (Fig. 4). v s n NK s y w 100 mg/kg n 200 mg/kg n ƒw. z v s n NK s y w w ùkû. x v s z v s n ƒ NK s y ƒ jš, z v s NK s y ƒ k w z ƒ ùkû. v s sww t y z w ƒ y w š. v s, y CAPEƒ w (23,24), w (8,9,25) w y (26) x. t sww z ƒ w y w š. v s v s zw y w w w, ƒ y e w, wš w w. w z v s v s w s me NK s y w ùkû. w z v s y ùkü y swwš wš ù, y w w ƒ w w. v s z v s n w y e w mw» w ww. v s z v s kg 50, 100, 200 mg/kg BALB/c 14
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