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1 KOREAN J. FOOD SCI. TECHNOL. Vol. 42, No. 2, pp. 233~239 (2010) The Korean Society of Food Science and Technology œ ù w y w xá«á Á Á «2 Á 2 Á½ Á Á x 1, * w œw, 1 w œw, 2 Comparison of Anticancer Activities of Berberis koreana Extracts Obtained by Different Extraction Processes Ji Hye Ha, Min Cheol Kwon, Yong Chang Seo, Woon Yong Choi, Eul Kwon Chung 2, Ae Ran Chung 2, Jin Chul Kim, Ju Hee Ahn, and Hyeon Yong Lee 1, * Department of Biotechnology and Bioengineering, Kangwon National University 1 Research Institute of Bioscience and Biotechnology, Kangwon National University 2 Myung Jin Farm Coperation Abstract This study was conducted to examine the anticancer activities of Berberis koreana extracts according to different extraction processes. The highest extraction yield obtained was 8.26% following extraction by ultrasonification at 60 khz and 60 o C followed by high pressure at 500 MPa. Generally, the extracts from the ultrasonification process showed relatively low cytotoxicities against the human normal cell line, HEK293 showing as low as 15%. This extract inhibited the growth of the digestive related organs cell lines, human stomach adenocarcimoma cell and human epithelial adenocarcinoma cell by up to 80% when administered at 1.0 mg/ml, and showed of selectivity. It was also found that this extract induced the production of nitric oxide levels as high µm from macrophages. For the in vivo experiment using ICR mice, the total serum IgG levels of mice treated with B. koreana extracts from ultrasonification extraction were increased by up to 57 ng/ml. The survival time of this group was longer than that of the other group after the injection of Sarcoma-180 and the increment of their body weights was also greatly suppressed. In addition, the extract showed the highest tumor inhibition activities, leading to a reduction of 78.47%. These results indicate that the highest activities of B. koreana associated with this extraction process can be significantly improved. Key words: Bereris koreana, high-pressure process, Sarcoma-180, antitumor activity, Nitric oxide, NK cell l, m, w, w w, w³ y» y (1) ù (Berberis koreana Palibin) ù ù ù ü p» s r w w y w³ y š (2), ù v NK s y z (3) s y (4) š. w ù ethyl alcohol z w w y, z 2-(3,4-dihydroxybenzyl)-ethyl alcohol x (5). w ù w w» y y w ƒ, w y w. ù š œ *Corresponding author: Hyeon Yong Lee, College of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon , Korea Tel: Fax: hyeonl@kangwon.ac.kr Received December 8, 2009; revised January 14, 2010; accepted January 15, 2010 ù w w y w wš» œ ƒ w w» w y w ù ƒ w e w»» w (6). w q œ q z w y z š š ƒ (7-9) w œ mw ù» k. x ¾» t ù z w ƒ w w w ƒ w š w. ƒ w s w w w wš w y w. s w» w w y w wù w z mw e ƒ ƒ w š w (10). x e,, w mw š ù e w ü w e w e (immunotheraphy) w š. w e w w 233

2 234 w t wz 42 «2y (2010)» z g ü» w s wš w w w ü x w w» e z g w e š š (11), w mw e z w w w š (12). w ù w y» t v w. ù y y y w š œ q ww ù œ wì in vitro, in vivo x mw ù w y sƒw ü ù» t ƒƒe» wš w. x x w ù» sw w p Berberis koreana 2007, w Ÿ x l w. š ù v w 100 g œ s w z š e(ilshin autoclave, Daejeon, Korea) w 5,000 bar 25 o C 5 š w w y þƒ»ƒ v j 10 w 60 o C 12 2z w. q y þƒ»ƒ v j 10 š 60 o C 12 2z w z 60 khz q œ 1 ƒ w. q w š» š œ z y þƒ ww 60 khz q œ 1 ww z w. 100 g ù v 100 o C 12 2z y þƒ w. ƒƒ z»(rotary vacuum evaporator N-N series, Eyela, Tokyo, Japan) w w z w x w. s Roswell Park Memorial Institute Medium(RPMI 1640) Alpha Minimum Essential Medium(α- MEM) Fetal bovine serum(fbs) Horse serum x Gibco C.(NY, USA) l w, Hepes buffer, Gentamycin sulfate Trypsin-EDTA Sigma (St. Louis, MO, USA) w w. s w SRB (sulphorhodamine B) Sigma l w x w. s x s w d w» w s HEK293(human embryonic kidney cell, ATCC, Manassas, VA, USA) w, s w y sƒw» w s AGS(human stomach adenocarcinoma, ATCC, USA) s Caco-2(human epithelial adenocarcinoma, ATCC) w.» s 37 o C, 5% CO 2 w 10% heating inactivated FBS s ww RPMI1640 w 4-5 z x w. Nitric oxide(no) macrophage(j774.1, ATCC) w d w. In vivo x x p (Seongnam, Korea) l w 4 ICR(female, g) o C, 55±10% ƒ w» w z x w. 12 w, ³ šx w. In vivo x s Sarcoma-180(CCL 8, ATCC) ü 6-7 w w. s in vitro w y d SRB assay(13) w ù s w s s w y sƒw. SRB assay s w s ù d w s HEK293 y» s AGS Caco cells/ml 4 96 well plate ƒ well 100 µl ƒw 24 (37 o C, 5% CO 2 )w z, 0.2, 0.4, 0.6, 0.8, 1.0 mg/ml w 100 µl ƒw 48 w. z wš ƒ 10%(w/v) TCA(Trichloroacetic acid) 100 µl ƒw 4C 1 ew z o 4-5z w TCA wš plate w ƒ well 1%(v/v) acetic acid 0.4%(w/v) SRB 100 µl ƒwš 30 g. w SRB 1% acetic acid 4-5z, k z 10 mm Tris buffer 100 µl ƒw ü z 540 nm microplate reader(thermo max, Molecular Devices, Sunnyvale, CA, USA) w Ÿ d w. Selectivity SRB assay w ƒƒ s w s ƒ s w w y d w z ƒ s w s y w. s Nitric Oxide d J774.1 macrophage(mouse) w, y y callus ƒ mw y y s nitric oxide microplate assay mw w (14). 10% FBS sww RPMI1640 w macrophage cells/ml w 24 well plate w x w,» s w d x ùkù šw, x s y s w w x w. ƒ 0.5 mg/ml 24 well plate ƒw humidified 5% CO 2 incubator 37 o C, 48 w. z 50 µl w v Griess (1% sulfanilamide/0.1% N-(1-naphthyl)-ethylenediamine digydochloride/2.5% H 3 PO 4 ) ƒw 10 k z microplate reader (Molecular Devices) w 540 nm Ÿ d w. Nitrite t sodium nitrite w 32 µm l 0.25 µm¾ RPMI1640 w w t š w

3 w. Nitric oxide(no) Lipopolysaccharide(LPS) w. w w (IgG) sƒ w (antibody) w ww w, x (immunoglobulin, Ig) f, w B v s t w B s w ƒw. ICR mouse w w 15 3 mouse l x w z, l x w Mouse IgG quantitative ELISA kit (Bethyl laboratories, Inc., Montgomery, TX, USA) w d w. Coating antibody coating buffer 1:100 w ƒ well 100 µl wš, 1 w z ƒ well antibody solution wš wash solution 3z w. Blocking w ƒ well blocking solution 200 µl wš, 30 z wash solution 3z w. Blocking z sample 50 w ƒ 100 µl w z 1 w z wash buffer 5z w. z HRP conjugate 1:100,000 conjugate diluent w w z ƒ 100 µl wš, 1 w z wash buffer w 5z w. TMB» yww z ƒ well 100 µl w z 5-30 w z j» w 2N H 2 SO µl w. ELISA reader w 450 nm Ÿ d w (15). Sarcoma-180 šx w in vivo w y d Sarcoma-180 s 10% heating inactivated FBS w w RPMI 1640» w. 0.25% trypsin-edta flask l w z 2 10 cells/mouse w µl mouse 1 ml syringe w vw w.» mouse ü 6-7 mw sarcoma-180 s w x mouse šx x mouse n 200 µl vw w. s z 21 1 n 0.5 ml, 30, 100 mg/kg w ƒƒ n w. x j š group (negative control group), z group(positive control group), k z n w group(treated sample group) w x ww mouse d w š (survival rate) d w, šx mouse s 30 z k mw e z šx d w w» d w (16). œ ù w y 235 Table 1. Yields of Berberis koreana extraction by different extraction processes Sample Solvent Temp. ( o C) Extraction process Pressure (bar) Ultrasonification (khz) Yield (%, w/w) WE 1) ±0.26 HPE 2) 60 5, ±0.34 Water USE 3) ±0.36 HUE 4) 60 5, ±0.41 Each value were compared with control at p<0.05 by the student s t- test. 1) B. koreana bark extracts by normal extraction. 2) B. koreana bark extracts under high pressure condition. 3) B. koreana bark extracts by ultrasonification extraction. 4) B. koreana bark extracts by ultrasoniciation extraction after high pressure extraction., q š ƒƒ 6.79% 7.25% w ƒw y w. š š œ mw ww 1.7 ƒw š(17) ù w š m w q w y mw œ mw y w wš» w. q œ w ù ƒ» w y w q k»x mw ƒw. š œ q œ ww w ù 8.26% ƒ ùkü. w 1.59 ƒw ù w š œ mw s q š ü ¾ w kƒ q ƒ w ü ¾ w z ùkü. s d w y Fig. 1 s HEK293 w s m x m SPSS package program(ver. 12.0, SPSS Inc, Chicago, IL, USA) paired t-test w Data s³±t (Mean±standard error) ùkü. š œ ù v ƒ œ Table 1 ùkþ. 5.21% ƒ û Fig. 1. Cytotoxicity of B. koreana extracts from different extraction processes on human embryonic kidney cell, HEK 293. WE: B. koreana bark extracts by normal extraction. HPE: B. koreana bark extracts by high pressure extraction. USE: B. koreana bark extracts by ultrasonification extraction. HUE: B. koreana bark extracts by high pressure and ultrasoniciation extraction. Each value were compared with control at p<0.05 by student t-test. Mean values ±standard deviation from triplicate separated experiments are shown.

4 236 w t wz 42 «2y (2010) Fig. 2. Inhibition effect (bar chart) of B. koreana extracts from different extraction processes on the growth of human stomach adenocarcinoma cell, AGS and its selectivity (line chart). See the Fig. 1 for abbreviation. Each value was compared with the control at p<0.05 by student s t-test. Mean values±standard deviation from triplicate separated experiments are shown. w ù s d w ƒ œ ƒw š 1.0 mg/ml ù 26.52% ƒ. q 21.54%, š 23.54% ƒ œ mw s z y w. ù ƒ ƒ w» œ ù z w» y s ƒ k». p ù š, q w š 18.02% ƒ û ùkü,, q š 0.6 mg/ml ƒw ql w, š, q w p w ƒ w ql, wz» š ƒ ƒ w w z y ù ký. s s AGS Caco-2 w y» s w in vitro w y sƒw ƒ ƒ Fig. 2 Fig. 3 ùkü. s w ù s w y d, ù š 1.0 mg/ml 67.3% ƒ û y ùkü, š, q ƒƒ 71.82% 68.32% w y y w. w w selectivity ùkü w q, š 3.0 selectivity ùkü yw w y y w. w š, q w š 1.0 mg/ml ƒ 74.86% w y, 3.5 selectivity ùkþ. p š 4.15 selectivity ùkü w 38.91% ƒ w k s z y w. s ù w y d ƒ š 59.03% s w y ùkü w w ƒ y ùkü, ù š, q 0.4 mg/ml 4.14 Fig. 3. Inhibition effect (bar chart) of B. koreana extracts from different extraction processes on the growth of human epithelia adenocarcinoma cell, Caco-2 and its selectivity (line chart). See the Fig. 1 for abbreviation. Each value was compared with the control at p<0.05 by student s t-test. Mean values±standard deviation from triplicate separated experiments are shown. selectivity ùkü ƒ yw s w k z ùkü. y, y w w y š y» s w w y š w y w s selectivityƒ w w w(18,19) ù» ƒ y» s w k z ƒ š ƒ y w. s w s d š q ww œ w s w ù q y z ü š s w j ƒw» ƒ. s Nitric Oxide d ƒ œ w ù w s NO y w Fig. 4 ùkü. NO interferon-γ, TNF-γ TNF-α ƒ cytokine LPS(E. coli derived lipopolysaccharide) ³ ü w inducible NO synthase(inos) w. w NOƒ w w» s w (20).» w NO ƒ s apoptosis wš(21) antigen-presenting cell (APC) w T cell w p w y y macrophage TNF-α s x j š (22). ƒ œ ù LPS n w s l NO y w J774.1 s LPS w p w z NO d w LPS w w 9.1%, š 17.4%, q 12.5% š š, q w 24% ƒw. w ù ƒ mw LPS ³ ü wì NO k y z ùkü. m w ù w z ùkü

5 œ ù w y 237 Fig. 4. Stimulation of nitric oxide production by adding 0.5 mg/ ml of B. koreana extracts from different extraction processes. See the Fig. 1 for abbreviation. Each value were compared with the control at p<0.05 by student s t-test. Mean values±standard deviation from triplicate separated experiments are shown. p š, q w mw ù v LPS wì n w NO d µm ƒ e š, q w œ mw w y» w y w. w w (IgG) sƒ w (antibody, Ab) w ww w, x (immunoglobulin, Ig) f. w B v st w, t (surface immunoglobulin) š, B v w (antigen receptor) w. B v s t w w w w, p w w w B v j v s (plasma cell) y st ƒ š w s w. B v w x ù w ww w ww ù w.» in vitro x w y ùkù ù š, q w w in vivo x w. ù ƒƒ 30 mg/kg/day, 100 mg/ kg/day w n w, 15 3 x w IgG w d w Table 2 ùkü. n w x ú w ƒw, š, q w 100 mg/kg/day x ng/ml ƒ Fig. 5. Survival rate of ICR mice inocultared by Sarcoma-180 in feed of B. koreana extracts from different extraction processes. WE30: B. koreana bark extracts by normal extraction at 30 mg/kg/ day concentration, WE100: B. koreana bark extracts by normal extraction at 100 mg/kg/day concentration, HUE30: B. koreana bark extracts by high pressure and ultrasoniciation extraction at 30 mg/kg/ day concentration, HUE100: B. koreana bark extracts by high pressure and ultrasoniciation extraction at 100 mg/kg/day concentration. Each value were compared with the control at p<0.05 by student s t- test. Mean values±standard deviation from triplicate separated experiments are shown. w w. n n x w 20% ƒw e in vivo x mw ù y z ƒ y w. Sarcoma-180 šx w in vivo w y d Fig. 5 Sarcoma-180 n w z ƒƒ n w 45 ¾» d w w ƒ w, ù 30 mg/kg/day x s³ 27.5, 100 mg/kg/day x s³ 30 w w ƒƒ 23.64%, 30% ƒ. w š, q w 100 mg/kg/days n x ü ƒ, ƒ w 40% ùkü ù š, q w z j y w % z, m w z» mw(23) ù z Table 2. Effect Berberis koreana extracts from different processes on total serum IgG level in ICR mice Time (days) Sample* (ng/ml) Con 21.71±0.65 b 19.32±0.21 a 16.26±0.49 b 16.95±0.51 b 15.54±0.26 a WE30 1) 26.77±0.80 b 25.17±0.43 c 29.83±0.33 a 31.25±0.34 a 33.22±0.37 a WE100 2) 35.10±1.05 b 39.34±0.43 a 38.12±0.42 a 42.94±0.47 a 39.09±0.51 a HUE30 3) 30.57±0.34 a 28.21±0.48 c 33.44±0.57 c 37.03±0.63 c 46.44±0.43 a HUE100 4) 36.85±0.63 c 42.94±0.47 a 54.20±0.60 a 55.13±0.94 c 57.25±0.97 c Value are expressed as mean±sdm of dada obtained from three independent experiments. a p<0.001, b p<0.005, c p<0.01 Vs. control. 1) B. koreana bark extracts by normal extraction at 30 mg/kg/day. 2) B. koreana bark extracts by normal extraction at 100 mg/kg/day. 3) B. koreana bark extracts by high pressure and ultrasoniciation extraction at 30 mg/kg/day. 4) B. koreana bark extracts by high pressure and ultrasoniciation extraction at 100 mg/kg/day.

6 238 w t wz 42 «2y (2010) z ùkü š w. w šx ù n w w w x û, ù š, q w ù w w y ùkü. ù 30 mg/kg/day x 34.54% ƒ û y ù kü w š, q w 100 mg/kg/day x 74.87% w y ùkþ. š q œ ww w œ w ù v y Sarcoma-180 w šx w w y z sƒw.» œ mw ù w 1.7 ƒw, s w 80% w ùkù z y. p, ù š, q w y» s AGS Caco-2 w 70% s wy ùkü, š 4 selectivity ù kü k s z ùkü.» w š w z w y ùkü, p s AGS w 50 mg/ml š 76% s w y ùkü (24). w w ù y» s w y š w. w w y d š, q w œ ù q, š œ mw w nitric oxide IgG ƒ j y w. š q œ y» w, p š œ ù y berberine ƒ k (25,26) š, q w œ ù y z w e. In vivo x mw œ ù w y sƒ mw» œ mw ù 40% 75% šx y ùkü z w y y w, wz w» w» y y. ww p w w. x 1. Yoo SJ, Lee KB, Kwak JH. Studies on the seasonal variation of berberine contents in Berberis koreana. Korean J. Pharmacogn. 17: (1986) 2. Jung HK, Kim YJ, Park BK, Park SC, Jeong YS, Hong JH. Antioxidative and antimicrobial activities of medicinal plant extracts for screening phytobiotic material. J. Korean Soc. Food Sci. Nutr. 36: (2007) 3. Jin L, Han JG, Ha JH, Jeong HS, Kwon MC, Ahn JH, Kim JC, Choi GP, Chung EK, Lee HY. Effect of immune activity on Berberis koreana Palibin by ultra high pressure low temperature process. Korean J. Medicinal Crop Sci. 16: (2008) 4. Syed Abdul Q, Kwon MC, Han JG, Ha JH, Chung HS, Ahn JH, Lee HY. Effect of different extraction protocols on anticancer and antioxidant activities of Berberis koreana bark extracts. J. Biosci. Bioeng. 107: (2009) 5. Kim YK, Kwak BM. Analysis of cytotoxic constituent of Berberis koreana Palibin. Mokchae Konghak 26: (1998) 6. Jin L, Ha JH, Jeong MH, Chung EK, Chung AR, Kim JC, Ahn JH, Lee HY. Enhancement of the antioxidant and anticancer activities of Berberis koreana bark using a low temperature and high-pressure extraction process. Korean J. Food Sci. Technol. 41: (2009) 7. Toma M, Vimatoru M, Panjwnyk L, Mason TJ. Investigation of effects of ultrasound on vegetal tissues during solvent extraction. Ultrason. Sonochem. 8: (2001) 8. Vinatoru M. An overview of the ultrasonically assisted extraction of bioactive principles from herbs. Ultrason. Sonochem. 8: (2001) 9. Chung K. Kim WI, Hong IK, Park KA. Ultrasonic energy effects on squalene extraction from amaranth seed. Appl. Chem. 4: (2000) 10. Choi IP. Natural killer cell therapy. Korean J. Hepatol. 14: (2008) 11. Jeong JY, Kim OH, Lee YS. Effects of the ex-vivo immunotherapy on the mammary gland tumorigenesis induced by 7,12-dimethylbenz(a)antheracene (DMBA) in rats. J. Toxicol. Pub. Health 14: (1998) 12. Kim EM, Jeong HJ, Park EH, Cheong SJ, Lee CM, Jang KY, Kim DW, Lim ST, Sohn MH. Evaluation of the radioimmunotherapy using I-131 labeled vascular endotherlial growth factor receptor 2 antibody in melanoma xenograft murine model. Nucl. Med. Mol. I. 42: (2008) 13. Houghton P, Fang R, Techatanawat I, Steventon G, Hylands PJ, Lee CC. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods 42: (2007) 14. Sung JH, Choi DH, Kim DH, Chun BG, Choi SG. White ginseng saponin upregulated the production of TNF-α, IL-1β, and NO in primary cultures of mixed glial cells. J. Ginseng Res. 28: (2004) 15. Kim CH, Kwon MC, Han JG, Ha JH, Jeong HS, Choi GP, Park UY, Nam JH, Hwong B, Lee HY. Immune activities of Rhodiola sachalinensis A. Bor extracts isolated with various extraction process. Korean J. Medicinal Crop Sci. 16: (2008) 16. Kim JH, Kim CH, Kim HS, Kwon MC, Song YK, Seong NS, Lee SE, Yi JS, Kwon OW, Lee HY. Effect of aqueous extracts from Rubus coreanus Miquel and Angelica gigas Nakai on antitumor and anti-stress activities in mice. Korean J. Med. Sci. 14: (2006) 17. Jeong MH, Kim SS, Ha JH, Jin L, Lee HJ, Kang HY, Park SJ, Lee HY. Enhancement of anticancer activity of Acer mono by high pressure extraction process. J. Korean Soc. Food Sci. Nutr. 38: (2009) 18. Jeong HS, Han JG, Ha JH, Jin L, Kim SS, Jeong MH, Choi GP, Park UY, Lee HY. Enhancement of anticancer activities of Ephedra sinica, Angelica gigas by ultra high pressure extraction. Korean J. Medicinal Crop Sci. 17: (2009) 19. Kim CH, Kwon MC, Syed Abdul Q, Hwang B, Nam JH, Lee HY. Toxicity reduction and improvement of anticancer activities from Rhodiola sacchalinensis A. Bor by ultra high pressure extraction process. Korean J. Medicinal Crop Sci. 15: (2007) 20. Choi BG, Kim SW. Studies on the regulation of nitric oxide synthesis in murine mononuclear phagocytes. Korean J. Environ. Toxicol. 15: (2000) 21. Kim KH, Roh SG, Park HR, Choi WC. Inhibition of apoptosis by nitric oxide in MCF-7 cells. J. Life Sci. 19: (2009) 22. Kim YS, Park KM, Song KS, Nam KY, Park JD. Anticancer activites of red ginseng acidic prolysaccharide by activation of

7 œ ù w y 239 macrophage and natural killer cells. Yakhak Hoeji 46: (2002) 23. Kim HM, Oh GT, Han SB, Hong DH, Hwang BY, Kim YH, Lee JJ. Comparative studies of adriamycin and 28-deacetyl sendanin on in vitro growth inhibition of human cell lines. Arch. Pharm. Res. 17: (1994) 24. Song JH, Lee HS, Hwang JK, Chung TY, Hong SR, Park KM. Physiological activities of Phellinus ribis extracts. Korean J. Food Sci. Technol. 35: (2003) 25. Kwon MC, Han JG, Ha JH, Oh SH, Jin L, Jeong HS, Choi GP, Hwang B, Lee HY. Immno-regulatory effects on Centella asiatica L. Urban extraction solvent associated with ultrasonification process. Korean J. Medicinal Crop Sci. 16: (2008) 26. Jin L, Ha JH, Jeong MH, Chung EK, Chung AR, Kim JC, Ahn JH, Lee HY. Enhancement of the antioxidant and anticancer activities of Berberis koreana bark by using a low temperature and high-pressure extraction process. Korean J. Food Sci. Technol. 41: (2009)

12.077~081(A12_이종국).fm

12.077~081(A12_이종국).fm J. of Advanced Engineering and Technology Vol. 1, No. 1 (2008) pp. 77-81 y w» e wx Á w œw Fabrication of Ceramic Batch Composition for Porcelain by Using Recycled Waste Ceramic Powder Hyun Guen Han, and