Acetylcholinesterase Inhibitory Activity and Protective Effect against Cytotoxicity of Perilla Seed Methanol Extract

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(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 36, No. 6, pp. 1026~1031 (2004). ©The Korean Society of Food Science and Technology. zêR ºÂb~ acetylcholinesterase ÛBW 5 ^

(2) ëW ^Î Rö\Áê'ÁnæJÁB9¦Á;«z1Á~* ]®\ö ®8Ë\¦, 1^*L ®'·. Acetylcholinesterase Inhibitory Activity and Protective Effect against Cytotoxicity of Perilla Seed Methanol Extract Won-Hee Choi, Min-Young Um, Ji-Yun Ahn, Sung-Ran Kim, Myung-Hwa Kang1, and Tae-Youl Ha* Food Function Research Division, Korea Food Research Institute 1 Department of Food Science and Nutrition, Hoseo University Acetylcholinesterase inhibitory activity and protective effect against cytotoxicity of PC 12 cell induced by betaamyloid protein and glutamate were examined in perilla seed methanol extract and its solvent fractions. Methanol extract of perilla seed showed dose-dependent acetylcholinesterase inhibitory activity, with n-butanol fraction showing strongest activity. Perilla seed methanol extract also decreased glutamate- and β-amyloid protein (Aβ)induced cytotoxicities of PC12 cells dose-dependently. Formation of TBARS induced by FeSO4-H2O2 in rat brain was significantly reduced by perilla seed methanol extract, with strongest protective activity formation of TBARS shown in n-butanol fraction. Results suggest perilla seed methanol extract may attenuate actylcholinesterase activity and cytotoxicity induced by glutamate and β-amyloid protein through suppression of oxidative stress. Key words: perilla seed, acetylcholinesterase, cytotoxicity, glutamate, β-amyloid protein. B. . generationj Fê (10,11). öò jî¢ ò ³~ Aβ~ »' f acetylcholine~ Wj &ÚbB(12) òã^~ ÖW ã*>î~ Ösj .¾~ º &ò~ VÛ 5 æVË Ë¢ FB > ® >Ú ® (13,14). *Òr æ BBB ~ ~òBB acetylcholinej ª~º acetylcholinesterase¢ &~º acetylcholinesterase inhibitor¢ ôf & ÷7>Ú zb¾ "öº Aβj targetb ~ Aβ~ W" »'j ÛB~º z> BBj * K B® ê ¯> ® . Þ, ÖÒ¾¢ F~ Fæ¶ö º ';Nê r ¾Ò Ê {O»b F¢ OF~(15,16), F OF j>'b >>>º ¦Ö> OF;öº z^ ~ Ã ÛB Î", .{ &~ 5 .*Ã BF, vòBj / êÊº ~ Úö FÎ Wª ï F>Ú ® î æ ;öBº F~ & ®z æOÖ~ ÖN¢ ïj "º ; Öz Î"& ® > ® (17). 6 f ~ Ï ª³>¦V Æz¾j j~ &æ ~ ÖzWª" Êr, monoterpene~ ~ ß>Wª ª ÒNb ÚÚöB &æ ÒWj ¾æÞ. > ® (18,19). V¢B þöBº ~ ÖzÎ"ö "Ï~, ºÂ>ö &~ ò³~ Ö W ã*>î acetylchloine~ >&j .~º AChE W ÛBÎ"¢ ¦Æ~ L-glutamate 5 Aβb FêB PC12 ~ ^Ò ÛBÎ"¢ ¦Æ~¶ ~& .. ¯W òî~~ ~¾ ~ º &âÚ VÛK~ ËöB ¦V *>' æVË~ Ë¢ ¾æÚº W î~b J¾Æ~ _z Ò²öB ~ ;>«j »Êº º 7~ ~¾ (1). ~ ~ ööº · &J B V> ®b¾ W ~ º " ò.&W ~ f rF~ ^, ß® rF~^ ÷f >(senile plaques)" òã RF«(neurofibrillary tangles)j "º ßûb (2). Senile plaque~ "º WWªf precursor proteinb¦V Fê>º β-amyloid protein (Aβ)bB fibrillar 5 β-sheet w÷> º ãË ® (3,4). rF~^ ÷öº ôf ö&J. > ®b 7 FK &JB Aβ~ "ê » 'j > ®º(5) Aβ~ "ê »'f apoptosis pathway ¢ WzÊ(6,7) free radicalj Fê~ Öz' ÊÞ.Ê ö ~ ã^¢ æ³'b 2ZÊ(8-9) æVË 5 VÛËKj 6ê'b ç ~² b æ³' neurode-. *Corresponding author: Tae-Youl Ha, Food Function Research Division, Korea Food Research Institute, San 46-1, Baekhyon-dong, Bundang-gu, Songnam-si, Kyunggi-do 463-420, Korea Tel: 82-31-780-9054 Fax: 82-31-780-9225 E-mail: [email protected] 1026.

(3) ºÂ>~ acetylcholinesterase ÛBW. 1027. Òò 5 O». þÒò þöB ÒÏ (2002jÖ)º ãçÎê ÖÓb ¦V «~&, β-amyloid protein(Aβ25-35), L-glutamate, MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] 5 acetylcholinesterase f Sigma(St. Louis, MO, USA)¦V «~& . ò~ ºÂ 5 ª³b B. º ;F, ^;~ , r ¾Ò r OF»b îæ~ ò ÒÏ~& . îæB º ªê~ 10V(w/ v)~ 80% zêRj Î&~ NöB 150 rpmb 16* shaking~ ºÂ Ê "æ(Toyo No. 2) "~& 80% zêR Ïj ò7ï~ 5V¢ &~ 3* shaking . r "~& . f ÎjB 40oC ~~ 6{³»V¢ Ò Ï~ ³» Ê ÿÖ~ zêR ºÂ> ÒÏ~& . 6 zêR ºÂj silica gel(Merk)j Ïê open column ö loading~ W ' Ï BN'b ª³~ 6 { ³»~ hexane ³ª, chloroform ³ª, ethyl acetate ³ª, n-butanol ³ª, methanol ³ªj '' B~& . ºÂ>" ' ª³>f dimethylsulfoxide(DMSO) ¢; ³ê& >² ê ªCö ÒÏ~& . Acetylchoinesterase ÛBW /; Acetylcholinesterase(AChE)~ Wf Ellman ~ O»(20)j wÏ~& . ¯, 3 mLÏ cuvettö 37oC N 0.1 M phosphate buffer(pH 8.0) 3.0 mL &~, VîÏ 0.075 M acetylcholin iodide 20 µL, 0.01 M dithiobisnitrobenzonic acid (DTNB)/phosphate buffer(pH 7.0) 100 µL¢ '' &~ ¾ b r ' ;³ê~ ò¢ Î&~ acetylcholinesterase(Sigma, USA)¢ Î&~ Î²>wj ·~&b Î²Wêº 412 nmöB Î²>w.V¦V 5ª*~ 7ê æz¢ G;~ ÖÂ~&. . ò~ AChE ÛBWêº ò¢ &~æ pf ç~ Î ²Wêö & ò Î&ê~ Î²Wê¦V ~Ö~& . ^

(4) V· þöB ÒÏ ^"º rat pheochromocytoma PC12 B ATCCöB ª·Aj ÒÏ~& . ª·Af ^º 10%~ horse serum(Gibco, NY, USA)" 5%~ FBS(Gibco, NY, USA), 100 unit/mL penicillin, 100 unit/mL streptomycin Î&B RPMI 1640(Gibco, NY, USA)Væ¢ ÒÏ~ 5% CO2& / >º zÛê ç~ 37oC~ V·VöB 2-3¢ î medium change¢ ~ 1"¢î ê&V·j Û~ þ ÒÏ ræ V·~& . ^

(5) ëW ^Ë /; V· ^ j 96 well plateö 2.5Ü104 cell/well >&b 7 «~ 24* ê '' 20 mM~ L-glutamatef 25 µM~ Aβ25-35 ¢ Î&~ ^ëWj FBV ' þò¢ ³êê Î&~ 48* V·j V . V· Â ^~ Nf MTT assayö ~~ G;~& (21). ¯, ' wellö 0.1 mg~ MTT¢ &" 37oCöB 4* z V·~ MTT¢ ~ öB WB formazanj DMSO¢ Ï~ ÏÎ ê microplate reader(Molecular devices, Sunnyvale, CA, USA)¢. Fig. 1. Acetylcholinesterase inhibitory activity of perilla seeds methanol extracts. Values with different superscripts are significantly different (p < 0.05).. Ï~ 540 nmöB~ 7ê¢ G;~& . Glutamate 6º Aβ25-35 F¾ ^ëWö & ' ò~ ^ëW ^Ëf & ~ Nö & WªN(%) ¾æÚî . Öz æîW ÛBË /; Rat(Sprague-Dawley, ëW, 6"_)~ whole brainj 'Â~ îz~ ÒÏ~& Öz' ÊÞ.ÊöbBº FeSO4-H2O2 ¢ ÒÏ~& . ¯, òç îö FeSO4-H2O2f ' ò¢ ³êê Î& êö 37oCöB 20ª incubation ê, 20% acetic acid 1.5 mL" 8.1% SDS 0.2 mL Ò 0.8% TBA 1.5 mLj Î& ê 100oCöB 20ª* &~& . ï' ê 3,000 rpmöB 15ª* öªÒ Ê ç[j 535 nmöB 7ê¢ G;~ Öz' ÊÞ.Ê ÛBWj ~Ö~& .. Ö 5 8 Acetylchoinesterase(AChE) W ÛBÎ. zêR ºÂ> 5 ' Ï ê ª³> AChE Wö ~º 'Ëj ªC Ö", ºÂ>~ Î&³ê& ¸j> AChE ÛBW F~'b Ã&~&b Ïï ~' >w j ¾æÚî (Fig. 1). AChEº acetylcholinej ª~º Î² B ò7 AChE~ ³ê& Ã&> acetylcholine ï 6 ²~ amyloid z>~ ãëW Ã&~ ~ & FBF &ËW ¸ >Ú ® (13,14). Tacrine, donepezil, rivastigmine ~ AChE inhibitor¢ æVË ¶çB ~¶ö ² RbB ò7~ acetylcholine ïj Ã&V º rF~^÷ ~¶ ö²B æVË" ÛVË~ BF ®î º ôf (22-24)& ® . 6öB " r. zêR ºÂ>ê in vitroöB AChE Wj ÛB~º · Ï ®º ©b ¾æÒb º zêR ºÂ> æVË BFö Î"'¢ &ËWj Ò~ ® . zêR ºÂ>~ ' Ï ª³>ö & AChE &Wj AChE inhibitor(22) tacrine" jv Ö"(Fig. 2), 100 nM~ tacrinef 46%~ AChE ÛBÎ"¢ ¾æÚî *>'b zêR ºÂ >öB ª³>öB ¸f ÛB Wj ¾æÚî . ª³> 7öBº n-butanol[öB 59.8% &Ë ¸f ÛBNj &. rb ethyl acetate, chloroform~ Bb ¾æÒb¾ methanol³ªf £ 10%B &Ë Ôf AChE ÛBÎ"¢ ¾æÚî. . öº phenolic compound, Êr, monoterpene~ ~.

(6) 1028. ®"²æ B 36 ² B 6 ^ (2004). Fig. 2. Acetylcholinesterase inhibitory activity of various solvent fractions of perilla seed methanol extract. Values with different superscripts are significantly different (p < 0.05).. Fig. 4. Effect of perilla seed methanol extract on L-glutamate induced cytotoxicity in PC12 cells. *p < 0.05, **p < 0.01, ***p < 0.001, compares with control group. Table 1. Effects of various solvent fractions of perilla seed methanol extract on L-glutamate and β-amyloid protein fragment 25-35 induced cytotoxicity of PC12 cells Treatment Fractions (100 µg/mL) Control Chloroform Ethyl acetate n-Butanol Methanol. 20 mM Glutamate 1). 52.5Û2.5b1) 62.8Û6.4a2) 66.7Û4.9a 67.9Û5.8a 62.9Û5.8a. 1). 25 µM βA25-35 1)1). 64.2Û1.5NS) 62.5Û0.9 65.7Û1.6 67.7Û2.4 63.9Û3.1. 1). Values are mean standard errors. Values with different superscripts within same column are significantly different (p < 0.05). NS) Not significant. 2). Fig. 3. Cytotoxicity of L-glutamate in PC12 cells. *p < 0.05, **p < 0.01, ***p < 0.001, compares with control group.. ß>Wª F>Ú ® >Ú ®b¾(18,19) AChE Û BÎ"f~ &Wö &Bº Ëê ¸f Wj &~ nbutanol ³ªj 7b AChE ÛBWj ¾æÚº >î~ { , ö & & jº~ ÒòB . L-glutamateö Fê* ^

(7) Ò ÛBÎ Glutamateº ªW ã* >îB ò³~ " »'f ò^~ Ò" æWj .¾~ æVËj ¶çÊº © b >Ú ® (25). þöBº PC12 cellöB glutamate FêB ^ëWö ~º zêR ºÂ> 5 ª³> ~ 'Ëj ¦Æ~& . ÖF, glutamate¢ Î&~æ pf ãÖ ~ ^Nj 100% ~j r, glutamate¢ 10, 20 5 30 mM >&b Î&®j r~ Nf '' 70.3%, 52.5% Ò 17.6% 6²~ glutamate~ ³ê& Ã&> PC12 ~ N 6²~& (Fig. 3). zêR ºÂ> glutamate ö ~~ FêB PC12~ Òj ÛB~ºæ~ ¦¢ ¦Æ~ V *~ PC12 ^ö £ 50% *ê~ ^Nj ¾æÚ º 20 mM~ glutamatef ºÂ> ò¢ ÿö Î&~ 48 * V· ê ^Nj G;~ Ö"¢ ¾æÚî (Fig. 4). 50 µg/mL~ zêR ºÂ>f 53.1%~ Nj ¾æ ÚÚ glutamateö FêB ^Ò ÛBÎ"& &V>æ p~ b¾ 100, 250 5 500 µg/mL ³ê~ ºÂ>f ³ê& Ã& > N '' 61.4, 62.8, 69.3%¢ ¾æÚÚ Ã&~º ã Ëj ¾æÚî . 6 ' Ï ª³>j 100 µg/mL ³ê Î &~ glutamateö ~~ Fê>º ^Ò ÛBÎ"¢ . Ò Ö"(Table 1), glutamateò ¾Ò &ö j~ ' ª ³> Î&öº F~'b ¸f Nj ¾æÚî(p < 0.05), ª³>*öº Ûê' F~Nº ìîb¾ ethyl acetate 5 nbutanol ³ªöB £* ¸f ãËj ¾æÚî . Probucol, flupirtine, retigabine, vitamin E ~ ÖzBº glutamateö ~ FêB PC12 ^~ ^ëWj jzÒ > ® º & ô >Ú ®b(25-27) ÖzB f glutamate ö ~ æî"Öz>, free radical~ W b oxidative stress¦V ^¢ ^~º ·Ïj rJ^ ®. (28,29). V¢B þöBê zêR ºÂ>ö F> Ú ®º ÖzWª *f ?f ·Ï > ®j ©b Òò B . β-amyloid proteinö ~~ Fê* ^

(8) Ò ÛBÎ Aβº rF~^ î~öB ¾æ¾º brain senile plague ; W~ "B WWª òã^öB oxidative stress¢ F B~ ã^~ ¶ j .¾~ ¾j&Bº 6ê' æVË~ ç j .¾~º ©b rJ^ ® . þöBº PC12 cellö ®ÚB Aβ25-35ö FêB ^Òö &~ ºÂ> ÚÆ 'Ëj ~º æ¢ Ò~& . Aβ25-35¢ Î &~æ pf ãÖ~ ^Nj 100% ~j r, 25 µM ~ Aβ25-35j Î&~&j röº 64.1%~ Nj ¾æîb 100 µM~ ³êöBº 50.6%~ Nj ¾æÚÚ Aβ25-35~ ³ ê& Ã&> PC12~ N F~'b 6²~& (Fig. 5). 25 µM~ Aβ25-35" zêR ºÂ>j Î&~ 48*.

(9) ºÂ>~ acetylcholinesterase ÛBW. Fig. 5. Cytotoxicity of β-amyloid protein (Aβ25-35) in PC12 cells. *p < 0.05, **p < 0.01, ***p < 0.001, compares with control group.. 1029. Fig. 7. Oxidative stress inhibitory activity of perilla seeds methanol extracts. Values with different superscripts are significantly different (p < 0.05).. Fig. 6. Effect of perilla seed methanol extract on β-amyloid protein fragment 25-35 induced cytotoxicity in PC12 cells. *p < 0.05, **p < 0.01, ***p < 0.001, compares with control group.. V· ê ^Nj Ò Ö"(Fig. 6) ºÂ>~ Î&³ ê& Ã&> ^N Ã&~ zêR ºÂ> f Aβ25-35ö ~~ FêB ^Òj ÛB~º ©b ¾æ Ò . ¯, Aβ(25-35)ö ~~ 6²B ^ Nf ºÂ>j 125 µg/mL 6º 250 µg/mL ³ê ¾Òb N ' ' 71.3, 79.1% Ã&~& (p < 0.05, p < 0.01). Aβö ~ ã^~ Òö & V*f 1) Ca2+f K~ ^Ú çW~ 2Z(30-31) 2) Öz' stress(8-9) 3) proapoptotic bax~ upregulation 5 neuroprotective Bcl~ downregulation(32-33) Bn > ®b resveratrol(34), green tea catechin(35), ferulic acid(36)f ?f Â ÖzB f Aβö ~ ^Òj Û B rJ^ ® . Þ, zêR ºÂ>~ ' Ï ª ³> 100 µg/mLj Aβ25-35 25 µM" ÿö Î&~ ^ Nj Ò Ö"(Table 1) ' ª³> f Aβ25-35ö ~~ F êB ^Òö &~ Â] ÛBÎ"& ¾æ¾æ p~ . º zêR ºÂ>~ Î"& ÚÊ ¢ Wªö ~ © jî¢ Wª~ çßÎ"ö ~ &ËWê VB > ìb ö & Ëê~ ê ®º & ºB . òç~ Öz' stress ÛBW Î. zêR ºÂ>~ glutamate 6º Aβ25-35ö ~~ Fê B ^Òj ÛB~º Î"& Öz Î"ö ~ ©æ~ ¦¢ ¦Æ~V *~ rat brain homogenateö FeSO4-H2O2 Öz' ÊÞ.Ê¢ FBB zêR ºÂ> 5 ª³> TBARS(thiobarbituric acid reactive substances)~ Wö ÚÆ 'Ëj ~ºæ¢ Ò~& . Ö" 0.5, 1.0, 5 1.5 mg~. Fig. 8. Oxidative stress inhibitory activity of various solvent fraction of perilla seed methanol extract. Values with different superscripts are significantly different (p < 0.05).. zêR ºÂ>j Î&®j r~ TBARS W ÛBWf '' 49.0, 66.6 Ò 71.0% ºÂ>~ Î&³ê& Ã& ö V¢B ÛBW Ã&~& (Fig. 7). 6 ª³>ö & TBARS W ÛBWj Ò Ö" chloroform[" methanol [öBº '' 18.7%, 51.9%~ ÛBNj &b ß® ethyl acetate ª³ª" n-butanol ª³>öBº '' 68.3%f 81.6% ~ Ö ¸f ÛBWj ¾æÚî (Fig. 8). " Nagatsu(18) f f zêR ºÂ>~ öj^rÞ ª³b¦ V 5-(3,4-dihidroxyphenyl-methyl) oxazolidine-2,4-dion" 3-(3,4dihidroxyphenyl) lactamide 2«~ îÚ Öz Wªj ªÒ~ & (37) öêR ºÂ>~ öj^rÞ ª ³öB Ö> ÖzKj G;~& . þöBê z êR ºÂ>, ethyl acetate 5 n-butanol ³ªöB Öz' ÊÞ .Ê ÛBW {>Ú, ~ Ö"f ¢~j r > ®î . Tada (38)ê ¦V 2&æ~ îÚ Öz Wª(vinyl caffeate, trans-p-menth-8-en-7yl cafefeate) j ªÒ ~& . Þ, Yan (36)~ öBº PC 12 cellj Aβö ÂVj r reactive oxygen species(ROS) ;W Ã&~º © b ¾æÒb, Choi (35)f rat~ hippocampal neuronö Aβj ¾ÒbB æî "Öz> Ã& ~& . 6 Ú " r, zêR ºÂ>f PC12 cell.

(10) ®"²æ B 36 ² B 6 ^ (2004). 1030. öB glutamate 6º Aβö ~~ FêB Öz' ÊÞ.Ê¢ ÛB~º Öz ·Ïö ~~ ^Òj ÛB~º ©b Òò>î .. º. £. zêR ºÂ> acetylcholinesterase(AChE)~ W 5 PC 12 cell~ ^Òö ~º 'Ëj ¦Æ~& . AChEö & ÛBWf zêR ºÂ>~ ³ê& ¸j> F~ 'b ¸~b ºÂ>~ ª³> 7öBº n-butanol[ &Ë ¸f ÛBNj ¾æÚî . L-glutamate 6º β-amyloid protein (Aβ)b FêB PC 12 cellö & ^Ò ÛBÎ"ê ºÂ >~ ³ê& ¸j> F~'b Ã&~& . 6 rat brain ö FeSO4-H2O2 Öz' ÊÞ.Ê¢ FBB ºÂ>~ TBARS W ÛBWj Ò Ö" zêR ºÂ>f ³ê& ¸ jî> Öz' ÊÞ.Êö & ÛBW Ã&~&b ª ³> 7öBº n-butanol[öB &Ë ¸f ÛBWj ¾æÚî. . ç~ Ö" Ú " r zêR ºÂ>f AChE Wj ÛB~ glutamate 6º Aβö ~~ FêB PC12 cell ~ ^Òj ÛB~ Î"º zêR ºÂ>~ ÖzKö V >ê ®j ©b Òò>î .. ^. ò. 1. Choi KG. The long-term care and dementia policy in welfare states. Soc. Welfare Policy 17: 55-75 (2003) 2. Selkoe DJ. Translating cell biology into therapeutic advances in Alzheimer's disease. Nature 399: A23-A31 (1999) 3. Tanzi RE, Gaston S, Bush A, Romano D, Pettingell W, Peppercorn J, Paradis M, Gurubhagavatula S, Jenkins B, Wasco W. 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