Improvement of Learning Behavior of Mice by an Antiacetylcholinesterase and Neuroprotective Agent NX42, a Laminariales-Alga Extrac

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(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 36, No. 6, pp. 974~978 (2004). ©The Korean Society of Food Science and Technology. Acetylcholinesterase ÛB 5 ã^

(2) ^ Wj <º îÏ ºÂb NX42~ îÖÊ ÛËK Ëç Î /^*Á Steven M. Stein1 5L z\², 1òZL ~. Improvement of Learning Behavior of Mice by an Antiacetylcholinesterase and Neuroprotective Agent NX42, a Laminariales-Alga Extract Bong Ho Lee* and Steven M. Stein1 Laboratory of Aging and Degenerative Diseases, Hanbat National University 1 Department of Medicine, University of Washington Brown-alga-derived natural agent NX42, mainly composed of algal polysaccharides and phlorotannins, showed mild but dose-dependent inhibition of acetylcholinesterase with IC50 = 600-700 µg/mL. Phlorotannin-rich fraction of NX42 showed substantial increase of the activity by more than one order of magnitude (IC50 = 54 µg/mL) and significant protection of SK-N-SH cells from oxidative stress by H2O2. Learning trials of mice for 5 consecutive days revealed electric-shock treatment during learning period significantly retarded learning process, whereas NX42-treated mice showed significant resistance against learning deficiency possibly mainly due to anticholinesterase and neuroprotective activities of phlorotannin. Key words: learning behavior, anticholinesterase, neuroprotective, alga extract. B. . º ©ö & ÖJ ~, Ö "Ï~¢ ~º Ãç. (10). ¾ Ãç f, ªç'bº ;çº*ö ³ öò jî¢ «ö æË ®º ©ê jò ò¢, Ïê ÒÏF > ®º Wªf ¾Zª ìº n*W" ãBWj <ºº © Ö;' (11). V¢B, ãÃæËf ?f ã Ööº &æ targetöò ;~² ·Ï~º ~£® º Ï &Ë n* Wªb W>Ú ËV Ï &Ë ©b B, æVËj BF B"º ÿö ã^~ 2Z¢ æ Ò > ®º Wª z× :²ç ~ > ® . ÖÒ¾¢f ¢j j ÿjj æöB &¦V 6Ò Ï>ÚJº ~ " Ú ;®b '7 j A ® . 7öBê ß®, OöB “ ”(12)¢ ® Òº .~ (Laminaria japonica, Undaria pinnatifida, Eisenia bicyclis, Ecklonia kurome )f ª~ç Îv î Ï(13)ö ³~º « B, ö FB '· Wª ö & ; Ãê Î" ö & "' Ö"& »'Nö V¢(14-17) z× FÏWö & & ¸jæ ® . " Ú Laminaria japonicaö FB z(fucoidan)¢º ëß. ~ Wª~ ã^ ^Wö &~ > ® (1617). Þ, î Ïö ³~º .~ 7öB, Eisenia bicyclis ¾ Ecklonia kurome f &¦ª rZÖ~ öò ÒÏ>Ú z b¾ " Ú, ~ö FB 2êò(phlorotannin)(18)¢º ëß Wª~ · ÒWö & &. ã*bî~ ¢« j^Ö Û 5 VÛK" ? f æVËö 7º j º ©f ¾ rJ^ ®b (1-6), acetylcholinesterase ÛBB ê~ ~£® ãÃ ~. ~¶~ æVËj BF~º Ïê 6> ® . "öº ~ ç~ öS~ vòö ·*¶[R'(PET) »j 'Ï ~ 6> ®º ÖöÊV¢B ÛBB ê~ ~£® Aricept¢ Ï ãÖ, B vòöB~ j^Ö ³ê¢ Ã&Ò öò jî¢, ö V¢ æVË BFB º © «ÃB : ® (7). ¾, 6> ®º ÖöÊV¢B Û BB ~ ëW" ¦·Ï( Êâæ, Æ, *VÃ, * ëW ) b ~, MCI(mild cognitive impairment), ARCD(agerelated cognitive decline) f ?f ãÃæË Û A® º > ôf Ò² ö 'Ï~º ê¢ &æ ® (8-9). ã ÃæË~ ãÖ, &¦ª "ê ;' ÊÞ.Ê¾ z ~ ¾æ¾, ö ®ÚB ®Þ" ~ ¯> *Corresponding author: Bong Ho Lee, Laboratory of Aging and Degenerative Diseases, Hanbat National University, San 16-1, Dukmyung-dong, Yuseong-gu, Daejeon 305-719, Korea Tel: 82-42-821-1542 Fax: 82-42-821-1593 E-mail: [email protected] 974.

(3) Acetylcholinesterase ÛB 5 ã^ ^ Wj <º îÏ ºÂb NX42~ îÖÊ ÛËK Ëç Î". Úæ ® . Wª~ α2-n2¢®" ÛB W(19) 5 ÖzW(20)ö & & B : ® . 6, zb~ Â ²"BB~ ÆÒ' ÒWö & ¢N~ ê ¢ "¶ ö ~B BB : ® (21-22). F¯ j :ûb " r, .~ö FB Wª j '.® Ïb òVËj ^~ æËKj çßÒ > ®º ÆÒK ©b 6>Ú, öBº &z B . ºÂbB B Ö> ®º “NX42” W ªö & j^ÖöÊV¢B ÛBÎ", ò^^Î" 5 æVË ï&¢ Û~ &ËWj ¦Ã~¶ ~& .. Òò 5 O» ò PNI(pacific nutritional inc., Vancouver, WA, USA)Ò¦V &zB . ºÂb(NX42)j B AjB ÒÏ~& . 2 êò ª³j áV *B NX42(10 g)¢ .Öö(100 g)ö b ~ ~~ öB 1* ÿn ºÂ ê "~& . ºÂ j 3² > ê, "j Îv z~, Ã~> 2 L¢ ÒÏ ~ ^¿~, ê ~ 0.83 g~ .ïªö(Fraction 1)j áî . Þ, .Öö ºÂ>æ pf ¦ªj ê~ Fraction 2(8.55 g)¢ áîb, .Öö [~ ^¿ö ÒÏB > Ï [j ê ³» ê ÿÖ ~ Fraction 3(0.61 g)j áî . Fraction 2f 3j z~ NX42öB Fraction 1j B ¾^æ ª³b ~& (Fraction 4, 8.61 g). NX42f Fraction 1~ WªWf Table 1" ? . £ 5 ^

(4). þö ÒÏB ZV"~ 5 ¢>Ï 5 Î², Vî f Î v SigmaÒ¦V «~& : Acetylcholinesterase(AChE) [EC 3.1.1.7], Sigma C-5021; ATCh (acetylthiocholine chloride), B3128; DTNB [5,5'-dithio-bis(2-nitrobenzoic acid)], D-8130; Fucoidan, F-5631. ^ V·ö ÒÏ Væ~º Gibco BRL (USA) B®j ÒÏ~& . SK-N-SH ^º American Type Culture Collectionb¦V «~& . Acetylcholinesterase ÛB W /; Î² Wf 25oCöB, Ellman »(23)j æ;Î UV 7 ê ªC»b G;~& . DTNB Ï (900 µL of 5.55 mM DTNB in 50 mM potassium phosphate buffer, pH 7.4)j '' ~ cuvetteö I, ATCh Ï(25 µL of a buffer of ATCh of varying concentration)j Î&~& . Î²Ï(75 µL of AChE, diluted in 50 mM, pH 7.4, potassium phosphate buffer to give 0.005 unit)j Î&b Î²>w B>î, 412 nmö Table 1. Nutritional components of NX42 and its ethyl acetate extract Component Carbohydrate (Fucoidan) Lipid Protein Ash Phlorotannin Moisture. Content (%) NX42. Fraction 1. 34.5 (5.8) 1.1 6.9 34.0 20.1 3.4. 3.1 9.5 1.7 2.1 82.3 1.3. 975. B 60.* 7ê æzNj .V >w³ê ~, Î²W~ æ â~ . ' òö & Î²W ÛBNf r" ? f ö ~~ êÖ>î . Inhibition(%) = 100 − (ST/CT)Ü100 CT 5 ST º '', ò& ìj r~ .V>w³ê 5 ò Ò ~öB~ >w³ê¢ ~ . ã^

(5) ^W ï& 60 mm petri dish ö SK-N-SH(1Ü105 cells) ^¢ ¦OÎ ê, 0.5% DMEM Væ v~~& . 24* ê 7 n~ H2O2 ³êº 30 µM, ' sample~ ³êº 30 µg/mL >ê I Ú"î . 20* êö haemacytometer ^ >¢ ^î . ^ ^ Î" >~º reduced(%), Îj®º ^>V& = {1− (£¾Ò ê/£¾Ò *)}Ü100b ÖÂ~& .. þÿb 5 ÒG > ICR îÖÊ(5"_, 18-20 g)¢ bÒÞÊ()¦ V «~& . b" Òòö & 7"j ¶F² ~&b, 23Û1oC, Ûê 55Û5%, 12* «z"V¢ Fæ~B 10 îÒO 3B~ (&, ÊÞ.Ê, ÊÞ.Ê + NX42)b ¾*Ú ÒG~& . òÏf '' NX42 1%¢ Ã~>ö * ç~ ò î . ÊÞ.Ê+NX42öº òÏj, & 5 ÊÞ.Êöº Ã~>¢ 4"* 1¢ 1² ¢ ÒÏ~ ãR ~&b, ò Rïf 100 mg/kg >ê ~&. . Û V* 5"öº ÊÞ.Ê" ÊÞ.Ê+NX42ö ~ 15²~ *VÏÏ( ² 0.6 mA, 5 sec)j 7¢* ³b &~&b, &f *VÏÏj &~æ p~ . Morris >% þ òR · 5"ö Morris >7 þj ^òö ~ ~ ~& (24). ö;~ >(100 cm diameter, 35 cm height)ö 23oC~ bj 15 cm p , ªF¢ Î&~ ® R«~² ~& . B~ 2Ñ"(12 cm diameter, 15 cm height) j >¢ 4ª ª~ > 0.5 cm j¾ö *~V . >~ :ãöº &æ «~~ B Â] ; ~ ê;j *~B '' ª ¢ â~ , ''~ îÖ Êº 5¢* ¢ 3²~ 2Ñ"j dj&º Ûj ~& .. Û, îÖÊº ¢; ·6öB >~ : OËj Ë ~ ¹rb, NÎê 2Ñ"j dj&º ²º>º * V >î . îÖÊ& 2Ñ"j dº W~, 10.* & ®ê v, r ê *ræ Ïª Zj *~ cage æ~ V . Û· 6¢öº 2Ñ"j B~, C 90.*~ >'* ÿn 2Ñ" ¹®~ ªj >'~ º *~ jNj G;~& . Ûê Î Vº mean + SEMb ¾æÚîb, ÛêªCf Student's t-test¢ ÒÏ~& .. Ö 5 8 NX42~ acetylcholinesterase ÛBÎ NX42~ acetylcholinesterase ÛBÎ"¢ G; Ö", 600-700 µg/mL ~ ³êöB 50% ÛBNj ¾æÚî (Fig. 1). ~£®.

(6) 976. ®"²æ B 36 ² B 6 ^ (2004). Fig. 1. Inhibition of Acetylcholinesterase by NX42 (A) and ethyl acetate extract of NX42 (B).. ö j £ Î"æò Ïï ~Wj & . " 2 êòb WB Fraction 1(Table 1)f *&~² ¸f ÛB Î"(IC50 = ~50 µg/mL)¢ ¾æÚî . Þ, NX42öB 2 êò Wª BB fraction 4f z~ ãÖ, 2 mg/mL~ ³êræê ÛBÎ"& ¾æ¾æ pj IC50 G; ®&Ë ~ & . V¢B, NX42~ acetylcholinesterase ÛBÎ"& *'b 2êò Wªö V~º ©b {>î . 2êò ºÂbf 10«~ dibenzo-1,4-dioxin FêÚ W>Ú ®º ©b rJ^ ®Ú(18-22), B B~ zb j ªÒ~ '' ~ zb ÛBÎ"ö V~º ;ê 5 ÛB kineticsö & º&' ¢ ºê 7 . H2O2ö ~ Fê* ÖzÊÞ.Ê¦V SK-N-SH ã^

(7) ^ Î Human neuroblastoma SK-N-SH ^º " ò^^Î " , ò^ ·Ï£b screening j ~V * in vitro Î ^B 6Ò ÒÏ> ® (25-28). 30 µM~ H2O2¢ ¾ Ò ãÖ, ^~ f 35% >&b ÎÚrb¾, NX42 (30 µg/mL)¢ ¾Ò ãÖ, 77% >&b ²>î. (Fig. 2). NX42¦V ºÂB 2êò ª³(Fraction 1)~ ã Ööê 82% ~ ¸f j ¾æÚî . ¾ NX42öB 2êòj B ª³(Fraction 4) 5 z~ ãÖöº H2O2ò ¾Ò ãÖö j~ £* ¸f j &b¾ F ~' >&f jîî (p > 0.05). V¢B H2O2ö ~ FBB ÖzÊÞ.Ê¦V~ NX42~ SK-N-SH ^ ^Î"º 2 êò Wªö V~º ©b {>î . NX42öº 20% ;ê~ 2êò Wª F>Ú ®b, 2êò Wªf Ö> Öz Wj &æ ®º ©b B : ®Ú(20), SK-N-SH ^ö & ^ Î"º H2O2 ö ~ B>º WÖ² ¢:¢j B~º Î"ö V ~ º ©b 'B . Þ, z Wª P/L f/ö ~ B>º W.~ Úb^ Oj ÛBb &Ö² W î.ö V ò^~ "ÃW 2Z¢ Î"'b ïj&. º Ö"& BB : ®b¾(16-17), .&Úb^¾ W .& Ò~æ pº in vitro Öz 2Z öBº Î"¢ ¾æÚæ pº ©b ¾æÒ . ¾ in vivo öBº . ºÂbö FB z Wª Öz ·Ïj <º 2 êò Wª" þ 7ÿ'b ò^¢ ^~º V. Fig. 2. Cell viability of H2O2-treated SK-N-SH cell. Cell count after 20 hr incubation with (a) no H2O2 treatment, (b) 30 µM H2O2 only, (c)~(f) 30 µM H2O2 + 30 µg/mL of NX42, Fraction 1, Fraction 4, or Fucoidan, respectively. ***p < 0.001 compared with (b).. > ®j ©b 'B . >% þö ~ îÖÊ ÛËK ï& . ºÂb~ acetylcholinesterase ÛBÎ"f ã^^ W ÊÞ.Ê öB~ ÛËKö ÚÊ Î"¢ ~º æ rjV *~ >7 þ >¯>î . ÛV* 5 ¢* ¢ 15²O~ *VÏÏj Af ÊÞ.Êf 2Ñ"ö ê~º ²º>º *ö ®ÚB *VÏÏj Aæ pf & ö j *& Ã&¢ & (2-5¢, p < 0.01)(Fig. 3). º >B *VÏÏ ÊÞ.Ê¢ FB~ *& ÛË K & ºb ·Ï®rj ~ . ¾, NX42¢ 4" * S ê *V ÏÏj Af ÊÞ.Ê + NX42f ÛËK &ºö & *& &Wj , 1¢f 2¢ öº F~Wf ìæò ÊÞ.Êö j~ ²* BFB ãËj &b, 3, 4, 5¢öº F~W ®º BFj &. (p < 0.05 6º 0.01). ÛV* 6¢ >º Æf 2Ñ"j B ~, 2Ñ" ®î~ ªj >'~º *~ jNj G;~ & . ÊÞ.Ê~ ãÖ, &ö j~ F~W ®² f * jN(p < 0.01)j >, ÊÞ.Ê+NX42f & " FÒ >&j & . ÊÞ.Ê" jv~&j r, ÊÞ .Ê+NX42f F~W ®² Z *j î (Fig. 4)(p < 0.01). Cholinergic system~ Wz& "~K, Û 5 ËVVÛKj ËçÎ º ©f ¾ rJ^ ® (1-6). 6, REM(rapid eye movement) > ö j^Ö~ · ¸ º Ò f, REM > çß>º j^Ö ëÊ~ VË' FWö V ~ VÛ~ ;Wö Ö 7º j ~º ©b C > ® (3,29). Þ, Sanford (30)ö ~~ >>º *V ÏÏf îÖÊö² ¢ FB~ REM >j 6²Ê, F*' ßW 5 ÏÏ~ ãþ;êö V¢ ;êö N& ¾ º ©b : ® . V¢B öB ¾æÂ :f ? *VÏÏj Af îÖÊ (ÊÞ.Ê) º ÛË K~ 6²º REM >~ 6²f þ ¾æ¾º j^Ö~ ³ê &~ö ~ VÛ ;W ";~ O ©b C B . ¾ NX42¢ S îÖÊ (ÊÞ.Ê+NX42)f ÛËK 6²ö & F~' &j &b, º NX42ö FB 2êò Wª ö ~ acetylcholinesterase ~ ÛB 5 j^Ö ³ê çß Î"& V~º ©b CB . NX42¢ W~º Wª 7, 2êò Wªò acetyl-.

(8) Acetylcholinesterase ÛB 5 ã^ ^ Wj <º îÏ ºÂb NX42~ îÖÊ ÛËK Ëç Î". 977. ÛB W 5 ã^Wö ~ ©b CB .. ^. Fig. 3. Water maze test on fear-stressed mice. Time spent by to find the hidden platform was measured as escape latency during learning trial. Empty square (control group), empty triangle (stress group), filled triangle (stress + NX42 group). *p < 0.05, **p < 0.01.. Fig. 4. Time spent in the platform quadrant. After removing the platform, percent of time spent in the platform quadrant was measured. **p < 0.01.. cholinesterase ÛBW 5 SK-N-SH ^Wj ¾æÚî . ¾, z Wª ò.&ö ¢~º 'Ë ¢¦ > ®bæ(16-17), in vivo öBº *7' V·j Û~ ò VË~ ^·Ïö V &ËWê ' > ® . ö V¢ B>~² ªÒB 2êò 5 zj ÒÏ îÖ Ê æËK ï& ~ ê³ & ºê> ® .. º. £. ~ 5 2êò j "Wªb ~º .ºÂb NX42& æËK Ëçö ~º 'Ëj ï&~V * in vitro 5 ÿb þj >¯~& . Ö" NX42º acetylcholinesterase ö &~ Nz~æò Ïï~' ÛBÎ"(IC50 = 600-700 µg/ mL)¢ ¾æÚî . NX42¦V ºÂB 2êò ª³f * &® ¸f Ïï ~' ÛB Î"(IC50 = 54 µg/mL)¢ ¾æÚî. . >, 2êò BB ª³" zf ÛBÎ"& ìî . NX42 5 2êò ª³f "Öz>²ö ~ FB B ÖzÊÞ.Ê ~öB~ SK-N-SH ^~ 2Z¢ F~ W ®² ÛB >, 2êò BB ª³" zf ^Î"¢ ¾æÚæ p~ . ÊÞ.Ê ~ö ®º îÖÊ ~ ÛËKö ~º Î"¢ ï& Ö", NX42¢ S î ÖÊ~ ãÖöº S~æ pf ãÖö j~ F~W ®² B FB ÛËKj ¾æÚîb, º in vitro þ Ö"¢ : ûb " r, NX42ö FB 2êò~ acetylcholinesterase. ò. 1. Wenk GL. The nucleus basalis magnocellularis cholinergic system: one hundred years of progress. Neurobiol. Learn. Mem. 67: 85-95 (1997) 2. Power AE, Vazdarjanova A, McGaugh JL. Muscarinic cholinergic influences in memory consolidation. Neurobiol. Learn. Mem. 80: 178-193 (2003) 3. Rasmusson DD. The role of acetylcholine in cortical synaptic plasticity. Behav. Brain Res. 115: 205-218 (2000) 4. Fagen ZM, Mansvelder HD, Keath JR, McGehee DS. Short- and long-term modulation of synaptic inputs to brain reward areas by nicotine. Ann. N.Y. Acad. Sci. 1003: 185-195 (2003) 5. Disterhoft JF, Matthew OM. Modulation of cholinergic transmission enhances excitability of hippocampal pyramidal neurons and ameliorates learning impairments in aging animals. Neurobiol. Learn. Mem. 80: 223-233 (2003) 6. Yasuji K, Takatoshi H, Dai W, Ira P, Shigetada N. Impairment of reward-related learning by cholinergic cell ablation in the striatum. Proc. Natl. Acad. Sci. USA. 100: 7965-7970 (2003) 7. Tsukada H, Nishiyama S, Fukumoto D, Ohba H, Sato K, Kakiuchi T. Effects of acute acetylcholinesterase inhibition on the cerebral cholinergic neuronal system and cognitive function: functional imaging of the conscious monkey brain using animal PET in combination with microdialysis. Synapse 52: 1-10 (2004) 8. Delagarza VW. New drugs for Alzheimer’s disease. Am. Fam. Phys. 58: 1175-1182 (1998) 9. Roman GC, Rogers SJ. Donepezil: a clinical review of current and emerging indications. Expert Opin. Pharmacother. 5: 161-180 (2004) 10. Dubois B, Albert ML. MCI or prodromal Alzheimer’s disease? Lancet Neurol. 3: 246-248 (2004) 11. Tracy HM. Disorders of memory: mild cognitive impairment. Neuro Invest. 102: 4-18 (2003) 12. Chang IM. Cheonyeonyakmul. Vol. I. Dongyangeugwahakdaejeon (Treatise on Asian Herbal Medicines). Haksulphyeonsookwan, Seoul, Korea (2003) 13. Kang JW. Illustrated Encyclopedia of Fauna and Flora of Korea. Vol. 8. Sam Hwa Publishing Co., Seoul, Korea (1968) 14. Taira K, Tanaka H, Arakawa M, Nagahama N, Uza M, Shirakawa S. Sleep health and lifestyle of elderly people in Ogimi, a village of longevity. Psych. Clinic. Neurosci. 56: 243-244 (2002) 15. Morita K, Nakano T. Seaweed accelerates the excretion of dioxin stored in rats. J. Agric. Food Chem. 50: 910-917 (2002) 16. Uhm CS, Kim KB, Lim JH, Pee DH, Kim YH, Kim H, Eun BL, Tockgo YC. Effective treatment with fucoidan for perinatal hypoxic-ischemic encephalopathy in rats. Neurosci. Lett. 353: 2124 (2003) 17. Ruehl ML, Orozco JA, Stoker MB, McDonagh PF, Coull BM, Ritter LS. Protective effects of inhibiting both blood and vascular selectins after stroke and reperfusion. Neurol. Res. 24: 226-232 (2002) 18. Glombitza KW, Gerstberger G. Phlorotannins with dibenzodioxin structural elements from the brown alga Eisenia arborea. Phytochemistry 24: 543-551 (1985) 19. Fukuyama Y, Kodama M, Miura I, Kinzyo Z, Kido M, Mori H, Nakayama Y, Takahashi M. Structure of an anti-plasmin inhibitor, eckol, isolated from the brown alga Ecklonia kurome OKAMURA and inhibitory activities of its derivatives on plasmin inhibitors. Chem. Pharm. Bull. 37: 349-353 (1989) 20. Nakamura T, Nagayama K, Uchida K, Tanaka R. Antioxidant activity of phlorotannins isolated from the brown alga Eisenia bicyclis. Fish. Sci. 62: 923-926 (1996) 21. Shibata T, Fujimoto K, Nagayama K, Yamaguchi K, Nakamura T. Inhibitory activity of brown algal phlorotannins against hyaluronidase. Int. J. Food Sci. Technol. 37: 703-709 (2002) 22. Shibata T, Nagayama K, Tanaka R, Yamaguchi K, Nakamura T. Inhibitory effects of brown algal phlorotannins on secretory phospholipase A2s, lipoxygenases, and cycloxygenases. J. Appl. Phy-.

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