Antimicrobial and Antitumor Activities of Seed Extracts of Camellia sinensis L.

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(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 37, No. 1, pp. 108~112 (2005). ©The Korean Society of Food Science and Technology. N¾Z «¶ ºÂb~ RW 5 «·RW Jö^ÁRÒî1Áã^2ÁB?R1,* *¢& ®&", 1&v ÿ>¶ö>8, 2zJa%8Fö. Antimicrobial and Antitumor Activities of Seed Extracts of Camellia sinensis L. Won-Ho Yoon, Jae-Hoon Choi1, Keyong-Ho Lee2, and Chang-Han Kim1,* Department of Food Science and Technology, Seoil College 1 Animal Resources Research Center, Konkuk University 2 Kolon Central Research Park Antimicrobial and antitumor activities of Camellia sinensis L seed extracts were investigated. Seed extracts showed antifungal activities against Candida albicans IFO 1594 and Cryptococcus neoformans. Inhibition zone of 20 mm was shown by 70% ethanol extract against C. albicans IFO 1594 at 100 mg/mL. Antifungal activity of seed extract was not decreased by heating at 80 and 100oC for 30 min or at 121oC for 15 min, indicating heatstability of seed component. Growth-inhibitory effects were observed in 70 and 10% of tumor cell line SK-OV3 and normal cell line NIH/3T3 at 50 µg/mL, respectively. Key words: antimicrobial activity, antitumor activity, Camellia sinensis L., seed. B. . V r^ö & J; . N¾Z «¶ö & Ú º «¶~ ¢>Wª, « F~ z' ßW" æOÖ Wö & ©", ^ z¢ Oæ(9), 7«~ j> jÖj j 17«~ jÖ~ Ò& '·ö öò jî¢ ~£Ï bê ÏF > ®rj " ® (10). V¢B º N¾Z ¢ «¶f "b ªÒ~ 70% öêR" b j Ï~ ºÂbj B~, ºÂb~ W" « ·Wö &~ rj~ .. N¾Z" bf *^ê'b 40³ 600« &, j&, ÂÁN&æOö ª~ ÖÒ¾¢öBº 5³ 6« ¶¢, Ã¶¾ |r;j N´j NÚ b ÂÎ Ïf .* ¢&¦V F ¶Î îzN V^rò (1). N ~ "ºWªbº rÎ, ¾, jÖ, jæ" 5 ZV î ®b zWª f &æ ÒW" £ Ò·Ïj ¾æÚº ©b > ® . Nº J¾Æ ; ö V~º ÒWbîB B & > ®b, Ï ~.OÎ"(2), Öz ·Ï(3), W(4)f zW(5,6) ~ · VË ®º ©b > ® . N~ "º zWªf Ò¾¦(polyphenol)~ zb~ ¢« EGCG(epigallocatechin-3-gallate) rJ^ ® . º~ Nöº 150200 mg~ EGCG& >Ú ®b N¢ ±j~º Ò² f ~öê ºO î >& ®bæ z× Î"& ® . f ~ ´ö & òÒö & º saponin Wªö ~ êÎ"(7), "Ã Î"f Öz Î "(8)& ®º ©b >î . ¾ «¶º ~ öV>. Òò 5 O» Òò þö ÒÏ òº 2003j 10úö B ²Ò(* Î ËW)öB 35j N¾Z(Camellia sinensis L.)~ . f ´j ª· Aj ÒÏ~& . º «¶f "b ªÒ~ ¾² ªê ê ÿÖV . Î òº ïË&~ B þj ~& . Ï ê ºÂb~ B «¶, "b~ ªö 10 gö 10Vï~ 70% öêR" Ã~ >¢ &~ NöB 6*O 3² ºÂ~ Whatman No. 2 "æ¢ ÒÏ~ "~& . ''~ "j ~ ö ªÒ ê çòj rotary evaporator 6{³»~ Ï ¢ j*® BÎ ê ÿÖ~ ' ºÂb~ >Nj êÖ~& . N´~ ãÖê *f ?f O»b ºÂ~ B ~& .. *Corresponding author: Chang-Han Kim, Animal Resources Research Center, Konkuk University, 1, Hwayang-Dong, KwangjinGu, Seoul 143-701, Korea Tel: 82-2-450-3679 Fax: 82-2-455-1044 E-mail: [email protected] 108.

(2) N¾Z «¶ ºÂb~ W 5 «·W. 109. Table 1. List of strains used for antimicrobial activity test Strain Gram positive bacteria Bacillus subtilis IAM 1069 Staphylococcus aureus IFO 12732 Micrococcus luteus ATCC 11880 Enterococcus hirae ATCC 10541 Gram negative bacteria Escherichia coli ATCC 10536 Salmonella typhimurium SL 1102 Pseudomonas fluorescens IAM 12001 Pseudomonas aeruginosa ATCC 15442 Vibrio parahaemolyticus ATCC 17802 Yeast Candida albicans IFO 1594 Cryptococcus neoformans Saccharomyces cerevisiae IFO 1009 Fungus Penicillium nalgiovens Trichophyton mentagrophytes Aspergillus niger ATCC 9642 Alternaria alternata Rhizoctonia solani. ÒÏ" 5 Væ þö ÒÏB "º ² ·W 4«, ² rW 5«, ÎÎ 3«, G 5«j ÒÏ~& "~ «~f Væ 5 V ·f Table 1ö ¾æÚî . Væº "~ ßWö ² B~ 121oCöB 15ª* &{~ ÒÏ~& . Î þ f 3>b >¯~& . «·^

(3) þö ÒÏ «·^"º SK-MES-1(human lung carcinoma), SW-156(human kidney carcinoma), Farrow(human melanoma carcinoma), HEP-2(human larynx carcinoma), SKOV-3(human ovary carcinoma) 5 HEC-1B(human endometerial adenocarcinoma)¢ ÒÏ~&b, GibcoÒ(Life Technologies, INC., Rockville, ML, USA)~ Væ¢ Ï~ V·~& . SK-MES-1, SW-156, Farrow, SK-OV-3 5 HEC-1B ^"º RPMI 1640, HEP-2 ^"º MEM Væö 10% fetal bovine serum(FBS, heat inactivated)j F Væ¢ ÒÏ~ 37oCö B 5% CO2 V·VöB V·~& . RW /; ' ºÂb~ ³ê¢ 50 mg/mLf 100 mg/mL C ê 0.45 µm membrane filter B~ ò ÒÏ~& . æ ö & in vitro W G;f paper disc method(11)¢ Ï~& . ¯, B Væ¢ petri dishö 15 mLO ª"~ w Ê, brothöB ^f 37oCöB 24*, ÎÎ, Gº 27oCöB 48* *V· '« þj Z'b Î& ~ ¾ b 7[Ï Væ¢ 5 mLO V[Ï Væ *ö ª" ~ 27~ ï6Væ¢ ò î . ' ºÂbj B paper discö 50 µLO >Î ê, Væö RJ¹ Â r, ^f 37oC, ÎÎº 30oC, Gº 27oC~ incubatoröB 24-48* V·~ disc "æ~ inhibition zone(mm)j G;~& .. Cultivation condition Nutrient agar/broth, 30oC Nutrient agar/broth, 37oC Nutrient agar/broth, 37oC Nutrient agar/broth, 37oC Nutrient agar/broth, 37oC Nutrient agar/broth, 37oC Nutrient agar/broth, 37oC Nutrient agar/broth, 37oC Nutrient agar/broth (3% NaCl), 37oC YM agar/broth, 30oC YM agar/broth, 30oC YM agar/broth, 30oC Potato dextrose agar/broth, 27oC Potato dextrose agar/broth, 27oC Potato dextrose agar/broth, 27oC Potato dextrose agar/broth, 27oC Potato dextrose agar/broth, 27oC. RWbî~ n;W /; W jv' ÚÂ «¶~ bºÂb~ n;Wj Ò~V *~ 50 mg/mL~ ³ê Ã~>ö B ê 80oC, 100oCöB '' 30ª ÿn >»çöB ¾Ò òf 121oCöB 15ª* autoclave ò¢ ï'Î ê 8 mm paper discö 50 µLO > 5 Î ê ¸f Wj ¾æÞ Candida albicans, Cryptococcus neoformans¢ ¦;b ÒÏ ~ paper disc method¢ Ï~ inhibition zone(mm)j G; ~& (12). «·RW /; N¾Z «¶f "b ºÂbö & «·Wj rjV *~ MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay¢ Ï~& (14). ¯ '' «·^" 2Ü105 cells/mL¢ 100 µLO 96 well plateö 7«~ 37oC, CO2 incubatoröB 24* ÿn V· ê, '. ³ê~ ò¢ 100 µL 7«~ 48* ÿn ÂV . V· ¾V 4 * *ö MTT solution(0.5 mg/mL) £ 50 µLO '' Î& ê 37oCöB 4* º& V·~ formazan ;Wj FêV , ©j ÏÊV *~ dimethyl sulfoxide(DMSO)¢ ' well 100 µLO Î& ê plate shakeröB 20ª* v>~&. . Multi well scanning spectrophotometer(microplate autoreader, Bio-Tek instrument, USA) 570 nmöB 7ê¢ G; ~& . «·Wf ^WË &N ï&~& . &N 50% ç ¢ r¢ «·W ®º ©b 6;~& .. Ö 5 V Ï ê ºÂb >N Ï ö V «¶f "b~ ºÂb~ >Nf Table 2f ? . ºÂb~ >Nj jv Ö" «¶~ ãÖº 70% öêR º.

(4) ®"²æ B 37 ² B 1 ^ (2005). 110. mm~ inhibition zonej ¾æÚî . «¶ ºÂbf ²·W Micrococcus luteus, Enterococcus hirae, ²rW Salmonella typhimurium, G Alternaria alternata, Rhizoctonia solaniöB ÛBÎ"& ¾æÒb ß® ÎÎ Candida albicans, Cryptococcus neoformansö &Bê Î"& ¾æÒ . ºÂÏ. ö V Nº ¾æ¾æ p~ . "b ºÂbf Enterococcus hirae öº þö ÒÏ Ú¶ "öBê W ¾æ¾æ p~ . ËW Ê¿Þ" çöB ´ ºÂb" «¶ ºÂb~ ãÖ ~ö V ÛBÎ"~ N& ¾æÒ . ¯, ´ ºÂb~ ãÖº " ^~ö & Wj & , «¶ ºÂböBº ÎÎf G~ ¢¦ ³ö &B W ¾æÒ . ~ö V W Nº ´ " «¶~ Wbî B ©¢ ÒòB .. Table 2. Extraction yields from the seed and skin of Camellia sinenesis L. by solvents Sample. Solvent. Extraction yield (%, w/w). Seed. 70% Ethanol Water. 15.9 30.6. Skin. 70% Ethanol Water. 12.3 21.8. Âb 15.9%, b ºÂb 30.6%~ >Nj & . "b~ ã Öº 70% öêR ºÂb 12.3%, b ºÂb 21.8%~ >N j & . «¶f "b~ ãÖ b ºÂb 70% öêR ºÂ b >N ¸² ¾æÒ .. RWbî~ n;W N¾Z «¶f "b 7 ÎÎö & jv' ¸f W j «¶ b ºÂb~ n;Wj Ò~V *~ 80oC, 100oCöB 30ª, 121oCöB 15ª* ¾Ò ê Candida albicans, Cryptococcus neoformansö & inhibition zonej G; Ö"¢ Table 4ö ¾æÚî . &æö~ V& 121oCöB 15 ª* ¾Ò ãÖ, 11-12 mm º*, W~ ºf 78-85%~ ª¢ ¾æÚÚ Wj Fæ~º ãËj ¾æ Úî . & Nê& Ã&ö V¢ &æö~ V& ² 6 ²~º ãËj ¾æÚîæò W º ö jv' n; ©b 6>î .. RW «¶f "b~ W Ö"º ´" jv~ Table 3" ? ¾æÚî . Ï ö V Wf 70% öêR ºÂb b ºÂb GÛBÎ"& Ö>~² ¾æÒ ³êö V Wê 50 mg/mLöB 100 mg/mL ¸jöö V¢ GÛ BÎ"º ¸jr . ´ ºÂbf ^ö &B Staphylococcus aureus, Micrococcus luteus, Enterococcus hirae, Salmonella typhimurium, Pseudomonas fluorescens, G Penicillium nalgiovensöB Wj & 7ö 70% öêR ºÂb ~ 100 mg/mL ³êöB Staphylococcus aureusö &~ 22. Table 3. Antimicrobial activity of various solvent extracts from the leaf, seed and skin of of Camellia sinenesis L. Inhibition zone (mm)1) Strains. Leaf 2). Gram positive bacteria Bacillus subtilis Staphylococcus aureus Micrococcus luteus Enterococcus hirae Gram negative bacteria Escherichia coli Salmonella typhimurium Pseudomonas fluorescens Pseudomonas aeruginosa Vibrio parahaemolyticus Yeast Candida albicans Cryptococcus neoformans Saccharomyces cerevisiae Fungus Penicillium nalgiovens Trichophyton mentagrophytes Aspergillus niger Alternaria alternata Rhizoctonia solani. Seed. Skin. W1. W2. E1. E2. W1. W2. E1. E2. W1. W2. E1. E2. -3) 15 14. 17 9 15. 20 9 15. 22 11 17. -. 10 9. -. 10 11. 10. 12. 9. 9. 14 9 -. 14 10 -. 9 -. 10 -. -. 10 -. -. 10 -. -. -. -. -. -. -. -. -. 12 12 -. 20 19 -. 13 12 -. 21 19 -. -. -. -. -. -. -. 11 -. 12 -. 9 -. 10 10. 10 9. 12 11. -. -. -. -. 1) Diameter, 2)Concentration of test sample by various solvent; W1: water extract (2.5 mg/disc), W2: water extract (5.0 mg/disc), E1: 70% ethanol extract (2.5 mg/disc) E2: 70% ethanol extract (5.0 mg/disc). 3) No inhibitory zone was formed..

(5) N¾Z «¶ ºÂb~ W 5 «·W. 111. Table 4. Thermal stability of the water extract from the seed of Camellia sinenesis L. Strain Candida albicans Cryptococcus neoformans. Inhibition zone (mm) o. No heat. 80 C/30 min. 100oC/30 min. 121oC/15 min. 141) 13. 13 13. 13 12. 12 11. Concentration of test sample: 50 mg/mL. 1) Diameter.. Fig. 1. Growth inhibition of 70% ethanol extract (500 µg/mL) from the seed of Camellia sinensis L. on various tumor cell lines in MTT assay.. Fig. 2. Growth inhibition of the water extract (500 µg/mL) from the seed of Camellia sinensis L. on various tumor cell lines in MTT assay.. «·RW "b 5 «¶~ ºÂö V «·^ ÃÛBÎ"º Fig. 1" Fig. 2öB º :f ? . "b~ ãÖº «·^ Ã&N ¾Ò³êöB 50% ~& . 7öBê ¶§ z^ HEC-1B ^öB &Ë Ôf ÃÛBNj ¾æÚî. . 70% öêR ºÂböBº «·^"î £*~ Nº ®b¾ £ 30-40% ;ê~ ÃÛBN ¾æÒ, b ºÂb öBº ÿ¢ ³êöB £ 10% ;ê~ ÛBÎ"& ¾æ¾ " b~ ãÖº 70% öêR ºÂ ö «·^ Ãbî º Â>º ©b . «¶~ ºÂÏ ö V «·^ Ã &º "b ºÂb ÿ¢ ³êöB Ö> Î"& ¾æÒ . «¶ ºÂ b "bf îR&æ 70% öêR 5 b" ?f W Ï. öB~ ºÂ «·^ö & ÃÛBÎ"º «¶& " b ê ÿ¢ ³ê &j Ö> ÛBÎ"¢ ¾æÚî . ¯ «¶ ºÂbf ¶§z ^ HEC-1B «·^"ò B~. Fig. 3. Growth inhibition of the seed extract (100 µg/mL) of Camellia sinensis L. on various tumor cell lines in MTT assay.. Fig. 4. Growth inhibition of the water extract from the seed of Camellia sinensis L. on tumor cell line SK-OV-3 and normal cell line NIH/3T3 in MTT assay.. º Î «·^"öB £ 90% ç~ ÃÛBÎ"& ¾æ Ò . «¶~ ãÖº b ºÂ öê W ç® Fæ> îb, Ï & W¢> ÃÛB Wbî ¾ ºÂ>º ©b ÒòB . «¶¢ b ºÂ~ 100 µg/mL ³ê «·^ö & ÃÛBÎ"¢ G;~& (Fig. 3). ³êöB " r b º Âb~ «·Wf b¦z^ FarrowöB 67.1%, êvz^ HEP-2öB 92.4%f Â²z^ SK-OV-3öB 97.1% ¾æÒ 70% öêR ºÂ bîf b ºÂb «· W ² ÎÚr . «¶ b ºÂb~ ' ³êö & «·^ ÃÛBÎ" G ;~ ' «·^"º ÛBÎ"& ±f Â²z ^ SKOV-3 ~&, ëW G;f ;ç^" NIH/3T3 ^¢ Ï~ ¦ï~& (Fig. 4). Ö"º 10-50 µg/mL³êöBê ;ç^" NIH/3T3 ^öBº ÃÛBÎ"& ¾æ¾æ p~ . ¾ ' «·^ SK-OV-3^º 50 µg/mL ö Bê £ 70%~ ÃÛBÎ"& ¾æÒ . V¢B N¾Z «¶.

(6) ®"²æ B 37 ² B 1 ^ (2005). 112. ~ b ºÂbf ;ç^ «·^ö &~ F ëWj ¾æÚº bî 6B . æ.ræ N¾Z «¶ ¦V~ z 5 W B ¢^f ìº ©b 6B . N¾Z «¶º æO ³¦~ Òòï ôf ©b rJ^ ® . Na (14)~ ö ~~ , ^ 5 k ê Òò~ ï ¸² ¾æ Ò, æOïf B jÝ ©b >î . «¶¦V rJê Òòf Thea senensis L.öB~ theasapogenol A, E, dihydropiverogenin A 5 camelliagenin C& ® (15). ¶ f 5 zWj ¾æÞ N¾Z~ b º Âb¦V Òò Ú ®rj {~& (Ö" B). V ¢B ; > º ìb¾ N¾Z «¶~ b ºÂb~ 5 zW Wªf Òò~ ©b º; > ®Æb, ö & «¶~ b ºÂb~ º&' ;B 5 ÿ;" þ «· Wö & WV*ö & « jº~Ò¢ 'B .. 6Ò~ º 2004jê B¢& Fj æöö ~ Úê © ö 6Òãî .. º. £. º öV>º N¾Z ¢ Ï~ W" «·Wj jv~ Ï&ËWö &~ öï~¶ ~& . W" «·Wj rjV *~ 70% öêR" b ºÂb~ Wj G;~& . Wf 7ë" «~ ÷ö, ÎÎ, G 17"ö &~ G;~&, «· Wf 6«~~ Ú z^", 1«~~ ;ç^"ö &~ Ò~& . N¾Z ´, «¶f "b~ Wj jv Ö "º «¶~ 70% öêR" b ºÂböB ÎÎö &~ W ¸² ¾æÒ . ß®, Candida albicans, Cryptococcus neoformansö &~ Wj & . «¶ b ºÂbj 80, 100oCöB 30ª, 121oCöB 15ª* ¾Ò ê Wj ¦ ï Ö", &Nêö V Nº ² ¾æ¾æ p~ . «· Wj ¦ï~V *~ MTT assay¢ Û Wj G; ~& . "b ºÂböB *& W ìîæò «¶ ºÂbö Bº Wj ¾æÚî . WÏ 70% öêR" b º Âb~ «·^ ÃÛBÎ"& ¸² ¾æÒb 7öB b ºÂb 70% öêR ºÂb ¸² «· W ¾æ Ò . N¾Z «¶¦V ºÂ Wbî~ «·^f ;ç ^ö & ^ëWj MTT assay jv Ö" 50 µg/mL ~ ³êöB Â²z ^ SK-OV-3º 70%~ ÛBNj ¾æ Þ > ;ç^öBº 10% òb ¾æ¾ ;ç^. «·^ö &~ F ëWj ¾æÚº ©b .. ^. ò. 1. Yang JK, Gang BK, Kim JM, Park YG, Choi MS. Physico-chemical properties and composition of fatty acids from seed oil of Camellia sinensis L. J. Korean Tea Soc. 6: 83-91 (2000) 2. Matsumoto N, Ishigaki OF, Iwashima H, Hara Y. Reduction of blood glucose levels by tea catechin. Biosci. Biotechnol. Biochem. 57: 517-525 (1993) 3. Yeo SG, Ahn CW, Lee YW, Lee TK, Park YH, Kim SB. Antioxidative effects of tea extracts from green tea, oolong tea, black tea. J. Korean Soc. Food Nutr. 24: 299-304 (1995) 4. Yeo SG, Ahn CW, Lee YW, Park YB, Park YH, Kim SB. Antimicrobial effects of tea extracts from green tea, oolong tea, black tea. J. Korean Soc. Food Nutr. 24: 293-298 (1995) 5. Choe WK, Park JH, Kim SH, Lee DY, Lee YC. Antitumor effects of green tea catechin on different cancer cells. Korean J. Nutr. 32: 838-843 (1999) 6. Yoshinobu A, Seiichi O, Tononori O, Tetsuya E, Takeshi O, Yoshiyuki N. Effect of (-)-epigallocatechin gallate on leukemic blast cells from patients with acute myeloblastic leukemia. Life Sci. 60: 135-142 (1996) 7. Minoru T, Sumito Y, Kanoko Y, Hajime O, Tadaxhi YG, Katsunori K, Jan AB. Theasaponin E1 destroys the salt tolerance of yeasts. J. Biosci. Bioeng. 90: 637-642 (2000) 8. Sur P, Chaudhuri T, Vedasiromoni JR, Gomes A, Ganguly DK. Antiinflammatory and antiooxidant property of saponins of tea (Camellia sinensis (L.) O. Kuntze) root extract. Phytother. Res. 15: 174-176 (2001) 9. Wu KY, Weng YX, Fei XQ, Yang WQ, Sun XZ. Comparison of antisenile effects of seed oil of Camellia grijsii and certain other oil from woody crops on 2BS cell culture. Forest Res. 11: 355360 (1998) 10. Liu XG, Xia HY. Analysis of amino acids in fruit and processing by-products of Camellia oleifera. Chem. Ind. Forest Prod. 17: 5155 (1997) 11. Bauer AW, Kibby MM, Sherria JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45: 493-496 (1966) 12. Baek JW, Chung SH, Moon GS. Antimicrobial activities of ethanol extracts from Korean Bamboo culms and leaves. Korean J. Food Sci. Technol. 34: 1073-1078 (2002) 13. Ruvinstein LV, Shoemaker RH, Paul KD, Simon RM, Tosini S, Scudiero DA, Monks A, Boyd MR. Comparison of in vitro anticancer drug screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human cell lines. J. Matl. Cancer Inst. 82: 1113-1118 (1990) 14. Rah HH, Baik SO, Han SB. Bock JY. Chemical compositions of the seed of Korean green tea plant (Camellia sinecis L.) J. Korean Agric. Chem. Soc. 35: 272-275 (1992) 15. Yosioka I, Nishimura T, Matsuda A, Kitagawa I. Saponin and sapogenol. II. Seeds sapogenols of Thea sinensis L (2). Theasapogenol A. Chem. Pharmcol. Bull. 18: 1621-1632 (1970) (2004j 12ú 14¢ %>; 2005j 1ú 24¢ j).

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