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1 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 , Korea Tel: Fax: 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

2 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 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 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 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 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

3 576 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± ± ± ± ±0.75 P ± ± ± ± ±0.41 P ± ±1.26 *18.49±0.39* 44.99± ±0.40 P ± ± ±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 ± ± ± ± ±0.40 FP ± ± ± ±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± ± ± ±2.42 P ± ± ± ±1.22 P ± ± ± ±0.25 P-200 **70.24±1.91** **47.60±4.04** *20.51±2.64* 15.52±1.23 FP ± ± ± ±3.05 FP ±3.25 **46.71±1.75** 12.52± ±1.85 FP ± ±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 ( 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).

4 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.

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.

5 578 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

6 v s z v s ƒ BALB/c mice y e w 579 n w z, w x n» w v v, v y me d w. w v l NK s w YAC-1 s ww NK s y d w. v s z v s n w v v T v (CD3 + ) ƒw w, CD4 + CD8 + T v ƒ» w. v l w v v s z v s n w ƒ w. Th-1/Th-2 me e w w v z v s v s w IFN-γ, IL-2, IL-4 j y w. v Th-2 me š (200 mg/kg) v s z v s n. v s z v s n w YAC-1 s ww NK s y ƒw., v s z v s n ü» k ƒ ƒ x l(w t z sƒ» l) w v s. x 1. Salem ML. Immunomodulatory and therapeutic properties of the Nigella sativa L. Seed. Int. Immunopharmacol. 5: (2005) 2. Ryu HS, Kim J, Kim HS. Enhancing effect of Sorghum bicolor L. Moench (Sorghum, su-su) extracts on mouse spleen and macrophage cell activation. J. Korean Food Nutr. 19: (2006) 3. Agarwal BB, Traquna PR, Eessalu TE. Modulation of receptor and cytotoxic response of tumor necrosis factor-l by various lectins. J. Biol. Chem. 261: (1986) 4. Belardelli F, Ferrantimi M. Cytokines as a link between innate and adaptive antitumor immunity. Trends Immunol. 23: (2002) 5. Diefenbach A, Rauler DH. The innate immune response to tumors and its role in the induction of T-cell immunity. Immunol. Rev. 188: 9-21 (2002) 6. Kumazawa SH, Yoneda M, Shibata I, Kanaeda J, Hamasaka T, Nakayama TS. Direct evidence for the plant origin of Brazilian propolis by the observation of honeybee behavior and phytochemical analysis. Chem. Pharm. Bull. 51: (2003) 7. Bankova V. Recent trends and important developments in propolis research. Evid. Based Complement. Alternat. Med. 2: (2005) 8. Bazo AP, Rodrigues MAM, Sforcin JM, de Camargo JLV, Ribeiro LR, Salvadori DMF. Protective action of propolis on the rat colon carcinogenesis. Teratogen. Carcin. Mut. 22: (2002) 9. El-khawaga O-AY, Salem TA, Elshal MF. Protective role of Egyptian propolis against tumor in mice. Clin. Chim. Acta 338: (2003) 10. Orsolic N, Kosalec I, Ivan B. Synergistic antitumor effect of polyphenolic components of water soluble derivative of propolis against ehrlich ascites tumour. Biol. Pharm. Bull. 28: (2005) 11. Ivanovska N, Neychev H, Stefanova Z, Bankova V, Popov S. Influence of cinnamic acid on lymphocyte proliferation, cytokine release, and Klebsiella infection in mice. Apidologie 26: (1995) 12. Sforcin, JM, Fernandes JA, Lopes CAM, Bankova V, Funari SR. Seasonal effect on Brazilian propolis antibacterial activity. J. Ethnopharmacol. 73: (2000) 13. Vynograd N, Vynograd I, Sosnowski Z. A comparative multi-centre study of the efficacy of propolis, acyclovir and placebo in the treatment of genital herpes (HSV). Phytomedicine 7: 1-6 (2000) 14. Khayyal MT, El-Ghazaly MA, El-Khatib AS. Mechanisms involved in the anti-inflammatory effect of propolis extract. Drug Exp. Clin. Res. 19: (1993) 15. Ledon N, Casaco A, Gonzalez R, Merino N, Gonzalez A, Tolon Z. Antipsoriatic, anti-inflammatory, and analgesic effects of an extract of red propolis. Acta Pharmacol. Sin. 18: (1997) 16. Jeong IY. Antioxidant activity and radioprotection of two flavonoids from propolis. J. Korean Soc. Food Sci. Nutr. 34: (2005) 17. Park JH, Lee JK, Kim HS, Chung ST, Eom JH, Kim KA, Chung SJ, Paik SY, Oh HY. Immunomodulatory effect of caffeic acid phenethyl ester in BALB/c mice. Int. Immunopharmacol. 4: (2004) 18. Andreia PD, Bianca PO, Fàtima HMG, Solange L, De Castro. Treatment of Trypanosoma cruzi-infected mice with propolis promotes changes in the immune response. J. Ethnopharmacol. 103: (2006) 19. Combe CL, Curiel TJ, Moretto MM, Khan IA. NK cells help to induce CD8 + T cell immunity against Toxoplasma gondii in the absence of CD4 + T cells. Infect. Immun. 73: (2005) 20. Takagi Y, Choi IS, Yamashita T, Nakamura T, Suzuki I, Hasegawa T, Oshima M, Gu YH. Immune activation and radioprotection by propolis. Am. J. Chin. Med. 33: (2005) 21. Sá-Nunes A, Faccioli LH, Sforcin JM. Propolis: Lymphocyte proliferation and IFN-gamma production. J. Ethnopharmacol. 87: (2003) 22. Wu D, Pae M, Ren Z, Guo Z, Smith D, Meydani SN. Dietary supplementation with white button mushroom enhances natural killer cell activity in C57BL/6 mice. J. Nutr. 137: (2007) 23. Song JJ, Gu Cho J, Hwang SJ, Gun Cho C, Park SW, Chae SW. Inhibitory effect of caffeic acid phenethyl ester (CAPE) on LPSinduced inflammation of human middle ear epithelial cells. Acta Otolaryngol. 8: 1-5 (2008) 24. Jung WK, Lee DY, Choi YH, Yea SS, Choi I, Park SG, Seo SK, Lee SW, Lee CM, Kim SK, Jeon YJ, Choi IW. Caffeic acid phenethyl ester attenuates allergic airway inflammation and hyperresponsiveness in murine model of ovalbumin-induced asthma. Life Sci. 82: (2008) 25. Ribeiro U Jr, Safatle-Ribeiro AV. Caffeic acid phenethyl ester (CAPE) may be a promising adjuvant treatment in gastric cancer. J. Clin. Gastroenterol. 41: (2007) 26. Hsu LY, Lin CF, Hsu WC, Hsu WL, Chang TC. Evaluation of polyphenolic acid esters as potential antioxidants. Biol. Pharm. Bull. 28: (2005)

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14.531~539(08-037).fm G Journal of the Korea Concrete Institute Vol. 20, No. 4, pp. 531~539, August, 2008 š x y w m š gj p { sƒ z 1) * 1) w w Evaluation of Flexural Strength for Normal and High Strength Concrete with Hooked