Page 44 - journal-8-23-Full

ÒıÙ «“√ “√°“√·æ∑¬ ¬·º√ “√ · °“√≈– ∑¬åå∑“߇≈◊Õ° ªï∑’Ë ¯©∫—∫∑’Ë Ú-Û æƒ…¿“§¡-∏—𫓧¡ ÚııÛ
°“√ ∑ å π‰∑¬
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√ “√°“√ ∑¬å·ºπ‰∑¬·≈–°“√ ∑¬å∑“å·ºπ‰∑¬·≈–°“√·æ∑¬å∑“߇≈◊Õ°
«“√ “√°“√·æ∑¬å·ºπ‰∑¬·≈–°“√·æ∑¬
à«πª√–°Õ∫ ¢Õß‚ª√μ’π¥â«¬ BCA protein assay kit ·≈–«—¥§à“ radio-

Ù.Ú °“√∑¥ Õ∫ Cell viability activity
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‡≈’Ȭ߇´≈≈å Caco-2 „π 96-well plate ∑’Ë¡’§«“¡Àπ“ ˆ. °“√∑¥ Õ∫ƒ∑∏‘Ϭ—∫¬—È߇Õπ‰´¡å pancreatic lipase
·πàπ Ò, ‡´≈≈å/À≈ÿ¡ ‚¥¬‡æ“–‡≈’Ȭ߇´≈≈å„π CO incu- ‡μ√’¬¡ “√≈–≈“¬¢Õß “√ °—¥ ¡ÿπ‰æ√∑’˧«“¡‡¢â¡¢âπ
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bator ‡ªìπ‡«≈“ Ò «—π ®“°π—Èππ”‡´≈≈å Caco-2 ¡“‡≈’Ȭ߄π μà“ß Ê ·≈â«ªî‡ªμ¡“ ª√‘¡“μ√ Úı ¡§≈. ‡μ‘¡ buffer
Õ“À“√‡≈’Ȭ߇´≈≈å„π ¿“«–∑’Ë¡’·≈–‰¡à¡’ “√ °—¥ ¡ÿπ‰æ√º ¡Õ¬Ÿà (Tris-HCl, NaCl, CaCl ) ∑’Ë¡’§à“ pH ¯. ·≈â«®÷ß‡μ‘¡‡Õπ‰´¡å
2
‡ªìπ‡«≈“ Ò «—π ‚¥¬„π™—Ë«‚¡ß∑’Ë ÚÚ ‡μ‘¡ “√≈–≈“¬ .ı% MTT pancreatic lipase Úı ¡§≈. „π ¢—ÈπμÕπ ÿ¥∑⓬ ‡μ‘¡

ª√‘¡“μ√ Ò ¡§≈. ≈߉ª ‡¡◊ËÕ§√∫ ÚÙ ™—Ë«‚¡ß ¥Ÿ¥Õ“À“√ 4-methylumbelliferyl oleate ´÷Ë߇ªìπ “√μ—Èßμâπ¢Õ߇Õπ‰´¡å
‡≈’Ȭ߇´≈≈åÕÕ° ·≈â«‡μ‘¡ “√≈–≈“¬ DMSO-EtOH (Ò:Ò v/v) pancreatic lipase ª√‘¡“μ√ Ò ¡§≈. ·≈â«π”‰ª∫à¡∑’ËÕÿ≥À¿Ÿ¡‘

ª√‘¡“μ√ Ú ¡§≈./À≈ÿ¡ ∫ࡉ«â∑’ËÕÿ≥À¿Ÿ¡‘ Û˜˚´ ‡ªìπ‡«≈“ Úı˚´ ‡ªìπ‡«≈“ Û π“∑’ ®“°π—Èπ𔉪«—¥°“√∑”ß“π¢Õ߇Õπ‰´¡å
Ò π“∑’ ·≈â«π”‰ª«—¥§à“°“√¥Ÿ¥°≈◊π· ß∑’˧«“¡¬“«§≈◊Ëπ 595 pancreatic lipase ‚¥¬«‘∏’ fluorometry ∑’Ë excitation Ûˆ
π“‚π‡¡μ√ π“‚π‡¡μ√ ·≈– emission ıÛı π“‚π‡¡μ√ √“¬ß“πº≈¥â«¬

ı. °“√¬—∫¬—Èß°“√¥Ÿ¥´÷¡§Õ‡≈ ‡μÕ√Õ≈¢Õß “√ °—¥ §à“ IC „πÀπ૬ ¡§°./¡≈. °“√∑¥ Õ∫π’È„™â orilstat ‡ªìπ
50
˜
¡ÿπ‰æ√ positive control
«—¥°“√¥Ÿ¥´÷¡§Õ‡≈ ‡μÕ√Õ≈‚¥¬ª√–‡¡‘π√–¥—∫¢ÕߧՇ≈ ˜. °“√∑¥ Õ∫ƒ∑∏‘Ϭ—∫¬—È߇Õπ‰´¡å HMG CoA reduc-
‡μÕ√Õ≈∑’Ëμ‘¥©≈“°¥â«¬ “√°—¡¡—πμ√—ß ’∑’˺à“π‡¢â“ Ÿà‡´≈≈å Caco-2 tase
„™â ezetimibe ‡ªìπ positive control ∑¥ Õ∫¥â«¬«‘∏’ in vitro enzymatic assay ‚¥¬„™â HMG

ı.Ò °“√‡μ√’¬¡ cholesterol micelles CoA reductase assay kit «—¥°“√∑”ß“π¢Õ߇Õπ‰´¡å
‚¥¬‡μ√’¬¡ Ú mM taurocholate, ı μM phospha- 3-hydroxy-3-methyl glutaryl CoA reductase (HMG CoA
3
tidylcholine, Ò μM cholesterol ·≈– Ò (Ci/ml [1,2- H] reductase) ‚¥¬‡Õπ‰´¡å™π‘¥π’È®–‡√àß°“√‡ª≈’ˬπ “√μ—Èßμâπ §◊Õ
cholesterol ¡“≈–≈“¬„π§≈Õ‚√øÕ√å¡ ·≈â«π”¡“º ¡°—π„π HMG Co A ‰ª‡ªìπº≈‘μ¿—≥±å´÷Ë߉¥â·°à °√¥‡¡«“‚≈π‘° Co-
À≈Õ¥·°â« ®“°π—Èπ 𔉪√–‡À¬¿“¬„μâ·°Á ‰π‚μ√‡®π ·≈– enzyme A ·≈– NADP ¥—ßπ—Èπ „π°“√∑¥ Õ∫º≈¢Õß “√ °—¥
‡°Á∫‰«â∑’ËÕÿ≥À¿Ÿ¡‘ -Ú˚´ ®π°«à“®–π”¡“„™â„π°“√∑¥ Õ∫ ‡¡◊ËÕ ¡ÿπ‰æ√μàÕ°“√∑”ß“π¢Õ߇Õπ‰´¡å HMG CoA reductase

®–π”¡“„™â„Àâ ≈–≈“¬„π DMEM/F12 ·≈– sonicate ·≈â«π” ®–ª√–‡¡‘π°“√≈¥≈ߢÕß NADPH ‚¥¬«—¥°“√¥Ÿ¥°≈◊π· ß∑’Ë
‰ª°√Õߺà“π .Ú μm filter membrane §«“¡¬“«§≈◊Ëπ ÛÙ π“‚π‡¡μ√ √“¬ß“πº≈‡ªìπ %°“√¬—∫¬—Èß

ı.Ú °“√π”‡¢â“§Õ‡≈ ‡μÕ√Õ≈ (cholesterol up- °“√∑¥ Õ∫π’È„™â pravastatin ‡ªìπ positive control
take) „π‡´≈≈å Caco-2 ¯. °“√·¬°À“ “√∫√‘ ÿ∑∏‘Ï
‡≈’Ȭ߇´≈≈å Caco-2 „π 24-well plate ‚¥¬‡ª≈’ˬπ ¯.Ò °“√·¬° “√ °—¥ GC-2

Õ“À“√‡≈’Ȭ߇´≈≈å∑ÿ° Ú-Û «—π ‡¡◊ËÕ‡´≈≈å¡’Õ“¬ÿ Ú-Û —ª¥“Àå ‡´≈≈å “√ °—¥ GC-2 πÈ”Àπ—° Û.˜˘ °√—¡ π”¡“·¬°¥â«¬
Caco-2 ®–‡°‘¥°“√ differentiation ‡ªìπ monolayer ´÷Ëß «‘∏’ column chromatography ‚¥¬„™â silica gel 60 πÈ”Àπ—°

‡≈’¬π·∫∫‡´≈≈å∑’˺π—ß≈”‰ â‡≈Á° ‡¡◊ËÕ®–«—¥°“√¥Ÿ¥´÷¡À√◊Õ°“√ ÚÚ °√—¡ ‡ªìπμ—«¥Ÿ¥´—∫·≈–™–¥â«¬ à«πº ¡√–À«à“߇Œ°‡´π
π”‡¢â“§Õ‡≈ ‡μÕ√Õ≈„À⇪≈’ˬπÕ“À“√‡≈’Ȭ߇´≈≈凪ìπ™π‘¥ serum ·≈–Õ–´’‚μπ„π≈—°…≥– gradient elution ‚¥¬‡√‘Ë¡®“°
3
free-medium ∑’Ë¡’ [1,2- H] cholesterol micelles ·≈– “√ -Ò% Õ–´’‚μπ μ√«® Õ∫º≈°“√·¬°‚¥¬¥Ÿ®“° TLC
°—¥ ¡ÿπ‰æ√‡ªìπ à«πª√–°Õ∫ 𔉪∫à¡„π CO incubator chromatogram ·≈–√«¡ fraction ∑’Ë„Àâ TLC pattern
2
‡ªìπ‡«≈“ Û ™—Ë«‚¡ß ®“°π—Èπ ¥Ÿ¥Õ“À“√‡≈’Ȭ߇´≈≈åÕÕ° ∑”°“√ ‡À¡◊Õπ°—π‡¢â“¥â«¬°—𠉥â∑—ÈßÀ¡¥ ˘ fraction (A1-A9)
≈â“߇´≈≈å¥â«¬Õ“À“√‡≈’Ȭ߇´≈≈凬Áπ ·≈â«∑”„À⇴≈≈å·μ°¥â«¬ .Ò π” fraction A4 ¡“°√Õߺà“π sephadex LH20 ‚¥¬

M NaOH ·≈– Ò °./≈. SDS ®“°π—Èπ𔉪«—¥§«“¡‡¢â¡¢âπ ™–¥â«¬ à«πº ¡¢Õ߉¥§≈Õ‚√¡’‡∑π·≈–‡¡∑“πÕ≈ (Õ—μ√“ à«π