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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
                                                                                                              ¯
                    ‡≈’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
                                                      2
             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≈’Ë¬π      ¯.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
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