TWI749435B - 促進膽固醇代謝相關基因表現之化合物之用途 - Google Patents
促進膽固醇代謝相關基因表現之化合物之用途 Download PDFInfo
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- TWI749435B TWI749435B TW108148424A TW108148424A TWI749435B TW I749435 B TWI749435 B TW I749435B TW 108148424 A TW108148424 A TW 108148424A TW 108148424 A TW108148424 A TW 108148424A TW I749435 B TWI749435 B TW I749435B
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- cholesterol
- cholesterol metabolism
- expression
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Abstract
本發明提供一種拉里西諾醇4'-O-β-D-吡喃葡萄糖苷或腺嘌呤用於製備促進膽固醇代謝相關基因表現之醫藥組合物之用途。該膽固醇代謝相關基因包括SCARB1、APOA1、及LDLR。藉由促進該些基因表現,拉里西諾醇4'-O-β-D-吡喃葡萄糖苷及腺嘌呤能降低個體因膽固醇代謝不良而罹患心血管疾病的風險。
Description
本發明係關於一種化合物用於製備血膽固醇含量調節劑之用途,特別係關於一種糖苷或核苷類化合物用於製備促進膽固醇代謝相關基因表現之醫藥組合物之用途。
人體中的膽固醇代謝涉及多個器官,其中最重要器官是肝臟。依據先前研究,細胞的膽固醇含量由一種複雜的平衡所調節,其中涉及膽固醇的內源性合成,細胞對膽固醇的攝取,以及細胞內膽固醇自細胞被排出至血液。
膽固醇於血液中運輸時是以膽固醇脂的形式與其他脂質和蛋白質一起包裝在囊泡中。腸道將經由飲食攝入的膽固醇組裝成乳糜微粒,這些微粒通過血液運輸最終被肝臟吸收,肝臟也是人體內膽固醇合成的主要場所。肝臟將飲食中及新合成的膽固醇包裝到低密度脂蛋白(low-density lipoprotein,LDL)中,這些脂蛋白被分泌到血液中以運輸到其他組織。該些組織的細胞通過LDL受體介導的內吞作用吸收LDL,從而獲得並利用來自LDL的膽固醇。另一方面,高密度脂蛋白(high-density lipoprotein,HDL)透過從周邊組織中獲取多餘膽固醇並將其輸送至肝臟來促進血管健康。在肝臟中,膽固醇被轉化為膽汁酸並被排出體外。
膽固醇動態平衡對人類的健康至關重要。當膽固醇的代謝有缺陷,可成導致在血液中循環的膽固醇過多。例如,LDL受體的缺陷會減少細胞對LDL的攝取,從而使得LDL在血液中累積,導致膽固醇沉積在動脈壁,最終造成動脈粥樣硬化和冠狀動脈疾病。由於血中膽固醇水平與冠狀動脈疾病間有著很強的負相關,血中膽固醇水平是臨床上評估心血管疾病風險的重要指標。
有鑑於此,為降低個體因膽固醇代謝不良而罹患心血管疾病的風險,開發一種可促進膽固醇代謝的組合物,實有其必要。
緣此,本發明之一目的在提供一種組合物用於製備促進膽固醇代謝相關基因表現之醫藥品之用途,其中該組合物包含一藥學上可接受的載體,及一選自由拉里西諾醇4'-O-β-D-吡喃葡萄糖苷、腺嘌呤、及其組合所組成群組之化合物。
在本發明之一實施例中,該組合物調節之膽固醇代謝相關基因編碼一低密度脂蛋白受體(LDLR),且該化合物之濃度至少為50μg/mL。
本發明之另一目的在提供一種拉里西諾醇4'-O-β-D-吡喃葡萄糖苷(lariciresinol 4'-O-β-D-glucopyranoside)用於製備促進膽固醇代謝相關基因表現之醫藥組合物之用途。
在本發明之一實施例中,該由拉里西諾醇4'-O-β-D-吡喃葡萄糖苷調節之膽固醇代謝相關基因編碼一清除者受體B類1型(scavenger receptor class B member 1,SRB1)或一低密度脂蛋白受體(low-density lipoprotein(LDL)receptor,LDLR)。
在本發明之一實施例中,該醫藥組合物含有濃度至少為50μg/mL之拉里西諾醇4'-O-β-D-吡喃葡萄糖苷。
本發明之另一目的在提供一種腺嘌呤(adenine)用於製備促進膽固醇代謝相關基因表現之醫藥組合物之用途。
在本發明之一實施例中,該由腺嘌呤調節之膽固醇代謝相關基因編碼一低密度脂蛋白受體(LDLR)。
在本發明之一實施例中,該醫藥組合物含有濃度至少為50μg/mL之腺嘌呤。
本文揭露施予有效量之拉里西諾醇4'-O-β-D-吡喃葡萄糖苷或腺嘌呤能顯著促進細胞內膽固醇代謝相關基因表現。因此,該二化合物可用於製備促進膽固醇代謝相關基因表現之組合物,其能降低個體因膽固醇代謝不良而罹患心血管疾病的風險,進而改善膽固醇代謝異常者或高膽固醇飲食者的心血
管健康。該組合物可為粉末、顆粒、溶液、膠體或膏體,且可製成醫藥品、食品、飲品、或營養補充劑,藉由口服或其他方式給予一個體。
以下將配合圖式進一步說明本發明的實施方式,下述所列舉的實施例係用以闡明本發明之特點及應用,而非用以限定本發明之範圍,任何熟習此技藝者,在不脫離本發明之精神和範圍內,當可做些許更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。
圖1顯示HepG2細胞以本發明一實施例之苦蕎麥種皮萃取物或其二次萃取物處理24小時後,相對於控制組細胞(不予處理)的SCARB1、APOA1、及LDLR基因的相對表現量;FT表示苦蕎麥種皮萃取物,FTE表示乙酸乙酯層萃取物,FTB表示正丁醇層萃取物,FTW表示水層萃取物。
圖2顯示化合物FT-01之質譜圖。
圖3顯示化合物FT-01之氫核磁共振(1H-NMR)光譜。
圖4顯示化合物FT-02之質譜圖。
圖5A顯示化合物FT-02之1H-NMR光譜。
圖5B顯示化合物FT-02之碳核磁共振(13C-NMR)光譜。
圖5C顯示化合物FT-02之氫-氫關聯性(correlation spectroscopy,COSY)光譜。
圖5D顯示化合物FT-02之碳-氫異核單量子相關(heteronuclear single quantum correlation,HSQC)光譜。
圖5E顯示化合物FT-02之碳-氫異核多鍵相關(heteronuclear multiple bond correlation,HMBC)光譜。
圖6顯示化合物FT-03之質譜圖。
圖7顯示化合物FT-03之1H-NMR光譜。
圖8顯示HepG2細胞以化合物FT-01、FT-02、或FT-03處理24小時後,相對於控制組細胞(不予處理)的SCARB1、APOA1、及LDLR基因的相對表現量。
本發明提供一種拉里西諾醇4'-O-β-D-吡喃葡萄糖苷或腺嘌呤用於製備促進膽固醇代謝相關基因表現之醫藥組合物之用途。本發明亦提供一種包含一藥學上可接受的載體,以及任一前述化合物或其組合之組合物用於製備促進膽固醇代謝相關基因表現之醫藥品之用途。以下實施例顯示對肝細胞施予任一前述化合物或包含該些化合物之組合物,可以促進包括SCARB1、APOA1、及LDLR等基因的表現。
除非另有說明,本文中所使用之「一」、「該」及類似用語應理解為包含單數及複數形式。
本文中所使用數值為近似值,所有實驗數據皆表示在20%的範圍內,較佳為在10%的範圍內,最佳為在5%的範圍內。
本文所述的苦蕎麥(bitter buckwheat)是屬於蓼科(Polygonaceae)蕎麥屬(Fagopyrum)的食用植物,學名為Fagopyrum tataricum,又名韃靼蕎麥(Tartary buckwheat)或綠色蕎麥。相比同屬的普通蕎麥(Fagopyrum esculentum),苦蕎麥的味道更苦,含有更多蘆丁(rutin),但缺少水楊醛(salicylaldehyde)和萘(naphthalene)。苦蕎麥與普通蕎麥通常被視為穀物,但與屬於禾本科(Poaceae)的常見穀物不同,因此它們與小麥無關。
本文所述的醫藥組合物或醫藥品可利用熟習此技藝者所詳知的技術而製備成一適合於非經腸道地(parenterally)或口服地(orally)投藥的劑型(dosage form),其包括但不限於:注射品(injection)[例如,無菌的水性溶液(sterile aqueous solution)或分散液(dispersion)]、粉末(sterile powder)、錠劑(tablet)、片劑(troche)、口含錠(lozenge)、丸劑(pill)、膠囊(capsule)、分散性粉末(dispersible powder)、細顆粒(granule)、溶液、懸浮液(suspension)、乳劑(emulsion)、糖漿(syrup)、酏劑(elixir)、濃漿(slurry)以及類似之物。
本文所述的醫藥組合物可由非經腸道途徑(parenteral routes)來投藥,其包括但不限於:腹膜內注射(intraperitoneal injection)、皮下注射(subcutaneous injection)、肌肉內注射(intramuscular injection)、及靜脈內注射(intravenous injection)。
本文所述的醫藥組合物可包含一廣泛使用於藥物製造技術之藥學上可接受的載體(pharmaceutically acceptable carrier)。該藥學上可接受的載體可
包含一或多種選自於由下列所構成之群組中的試劑:溶劑(solvent)、乳化劑(emulsifier)、懸浮劑(suspending agent)、分解劑(decomposer)、黏結劑(binding agent)、賦形劑(excipient)、安定劑(stabilizing agent)、螯合劑(chelating agent)、稀釋劑(diluent)、膠凝劑(gelling agent)、防腐劑(preservative)、潤滑劑(lubricant)、吸收延遲劑(absorption delaying agent)、脂質體(liposome)以及類似之物。該些試劑的選用與數量落在熟習此項技術者的專業素養與例行技術範疇內。
前述藥學上可接受的載體包含一選自於由下列所構成之群組中的溶劑:水、生理鹽水(normal saline)、磷酸緩衝鹽溶液(phosphate buffered saline,PBS)、含糖溶液、含醇水溶液(aqueous solution containing alcohol)、及它們的組合。
自Thermo Fisher Scientific購買DMEM培養基(Gibco Dulbecco's modified Eagle’s medium)、胎牛血清(Gibco fetal bovine serum,FBS)、青黴素/鏈黴素(Gibco penicillin/streptomycin)、及磷酸緩衝鹽溶液(Gibco PBS)。
溶劑係購自台灣默克公司,包括乙酸乙酯(ethyl acetate)、甲醇(methanol)、正丁醇(ethanol)、乙腈(acetonitrile)、丙酮(acetone)、二氯甲烷(dichloromethane)、氯仿-d 1 (氘化程度99.5%)、甲醇-d 6 (氘化程度99.5%)、重水(deuterium oxide,氘化程度>99.8%)、及二甲基亞碸-d 6 (dimethyl sulfoxide-d 6 ,氘化程度>99.9%)。
化合物分離係利用管柱層析法(column chromatography)及薄層層析法(thin layer chromatography,TLC)。中壓液相層析(medium pressure liquid chromatography,MPLC)系統係為CombiFlash ® Rf+(Teledyne ISCO);管柱填充材料係選用自Sephadex LH-20(Amersham Biosciences)、Diaion HP-20(Mitsubishi Chemical)、Merck Kieselgel 60(40-63μm,Art.9385)、及Merck LiChroprep® RP-18(40-63μm,Art.0250)。高效液相層析(high performance liquid chromatography,HPLC)系統裝配Hitachi L-2310系列幫浦、Hitachi L-2420紫外光-可見光偵測器(偵測波長為200nm至380nm)、及D-2000 Elite資料處理軟體;管柱係選用自
分析級Discovery® HS C18(250×4.6mm,5μm;SUPELCO)與Mightysil RP-18 GP 250(250×4.6mm,5μm;Kanto Chemical),以及半製備級Discovery® HS C18(250×10.0mm,5μm;SUPELCO)與製備級Discovery® HS C18(250×21.0mm,5μm;SUPELCO)。薄層層析片係塗覆矽膠60 F254(0.25mm;Merck)或RP-18 F254S(0.25mm;Merck)之鋁片,其配合紫外燈UVP UVGL-25(波長為254nm及365nm)進行分析。
化合物的化學結構係以質譜法(mass spectrometry,MS)及核磁共振光譜法(nuclear magnetic resonance spectrometry,NMR)確定。具體而言,使用電噴灑離子化串聯質譜(ESI-MS/MS)及二維離子阱串聯傅立葉轉換質譜(Bruker amaZon SL system及Thermo Scientific Orbitrap Elite system);並使用400MHz Varian 400 FT-NMR取得一維與二維NMR光譜,以四甲基矽烷(tetramethylsilane,TMS)作為內部標準品(δ=0)。NMR光譜以δ表示化學位移(chemical shift),單位為ppm;偶合常數(J)以Hz為單位;s表示單峰(singlet),d表示二重峰(doublet),d表示三重峰(triplet),q表示四重峰(quartet),p表示五重峰,m表示多重峰(multiplet),brs表示寬峰。
以下實施例所用細胞係購自美國典型培養物保存中心(American Type Culture Collection,ATCC)之人類肝癌細胞株HepG2(ATCC HB-8065)。HepG2細胞在37℃、5%二氧化碳的條件下培養於添加10% FBS及1%青黴素/鏈黴素的DMEM培養基,以下稱細胞培養基。
基於定量聚合酶鏈鎖反應(quantitative polymerase chain reaction,簡稱qPCR)測定HepG2細胞中膽固醇代謝相關基因的表現量,其步驟簡述如下。依據廠商使用說明,利用RNA萃取套組(RNA Extraction Kit;Geneaid)自細胞分離出核醣核酸(RNA),於37℃下以反轉錄酶SuperScript® III Reverse Transcriptase(Invitrogen)將2000ng RNA反轉錄為cDNA。其後,利用qPCR套組(KAPA CYBR FAST qPCR Kit(2X);KAPA Biosystems)以及目標基因與作為內部對照之甘油醛3-磷酸脫氫酶(Glyceraldehyde 3-phosphate dehydrogenase)基因GAPDH之引子對(表1)在PCR反應儀(Step One Plus Real-Time PCR system;
Applied Biosystems)對前述cDNA進行qPCR,並分析PCR產物的解鏈曲線(melting curve)。
最終,使用2-△△CT方法測定目標基因的相對表現量。該方法以GAPDH基因的循環閾值(CT)作為內部對照之參考基因的循環閾值,按照以下公式計算相對倍數變化:△CT=實驗組或控制組的目標基因的CT-內部對照的CT
△△CT=實驗組的△CT-控制組的△CT
倍數變化=2-△△Ct平均值
數據表示為平均值±標準差(SD)。統計分析係使用Excel軟體中的STDEV函數計算各基因相對表現量的標準差,並以單尾學生t檢定(TTEST)判定數據間差異在統計上的顯著性。
為取得具降膽固醇活性之化合物,首先製備一苦蕎麥種皮萃取物。簡言之,先洗淨及乾燥苦蕎麥全穀的外殼,即苦蕎麥種皮,及使用粉碎機將其粉碎。其次,以水為溶劑對苦蕎麥種皮粉碎物進行萃取。該溶劑與該苦蕎麥種皮粉碎物混合之重量比範圍為15:1至10:1。萃取溫度為介於70℃至90℃。本實施例中萃取時間為1至2小時。
為移除殘餘固體物,經上述萃取步驟所得之苦蕎麥種皮萃取物冷卻至室溫後,可進一步以400目(mesh)濾網過濾而獲得一上清液。該上清液可進一步在40℃至60℃進行減壓濃縮而獲得一濃縮產物(FT)。
其後,自該苦蕎麥種皮萃取物中進一步分離出富含降膽固醇活性成分之二次萃取物,其步驟簡述如下。首先,藉由乙酸乙酯與水等比例(體積比1:1)液相分配的方式萃取10L苦蕎麥種皮萃取物三次。將所得乙酸乙酯層萃取液合併,再經減壓濃縮乾燥可獲得一乙酸乙酯層萃取物(FTE)約1.9g。餘下水層萃取液以正丁醇與水等比例(體積比1:1)液相分配方式萃取三次。所得正丁醇層萃取液及水層萃取液分別合併,再經減壓濃縮乾燥可得到一正丁醇層萃取物(FTB)約11.5g及一水層萃取物(FTW)約75.0g。
為評估降膽固醇作用,對人類肝癌細胞株HepG2施以含20μg/mL前述苦蕎麥種皮萃取物、乙酸乙酯層萃取物、正丁醇層萃取物、或水層萃取物之2mL細胞培養基,或者僅用細胞培養基處理細胞(控制組)。各組細胞於37℃培養24小時後用於基因表現量分析,目標基因包括編碼清除者受體B類1型(SRB1)之SCARB1基因、編碼載脂蛋白A1(apolipoprotein A1,ApoA1)之APOA1基因、及編碼低密度脂蛋白受體(LDLR)之LDLR基因。
以上述三基因作為膽固醇代謝指標基因的原因在於,SRB1是肝細胞攝取高密度脂蛋白(HDL)所需的受體,其存在有助於來自周邊組織的膽固醇透過HDL進入肝細胞被代謝;ApoA1是HDL的主要構成蛋白之一,其生成有利HDL形成,因此促進膽固醇被HDL運送至肝細胞以被進一步代謝;LDLR是低密度脂蛋白(LDL)的受體,其存在有助於肝細胞及其他細胞攝取血液中的LDL以減少血中膽固醇累積。
圖1顯示前述各組HepG2細胞中各目標基因的相對表現量;圖中* **及***分別表示相比控制組為p<0.05、p<0.01及p<0.001。依據圖1,相比控制組,施予苦蕎麥種皮萃取物(FT)只能提升HepG2細胞的LDLR基因的表現;施予乙酸乙酯層萃取物顯著減少SCARB1、APOA1、及LDLR基因之表現;但正丁醇層萃取物(FTB)之處理增加SCARB1、APOA1、及LDLR基因之表現分別約18%、23%、及60%,且水層萃取物(FTW)之處理增加SCARB1、APOA1、及LDLR基因之表現分別約119%、51%、及56%。
因此,依據生物活性導引分離方法(bioassay guided fractionation)進一步自該正丁醇層萃取物及水層萃取物中分離出降膽固醇活性成分。使用填充Sephadex LH-20之管柱及甲醇沖提液,對正丁醇層萃取物(約11.5g)進行管柱層析,分離得到6個劃分層(分別標記為BF1至BF6)。其後,以二氯甲烷與甲醇依體積比7:1之混合液為沖提液,對BF2劃分層進行矽膠管柱層析,所得流出液再經薄層層析片分離,可得到10個劃分層(分別標記為BF2-1至BF2-10)。BF2-5劃分層以水與甲醇之混合液在中壓液相層析系統進行極性漸減沖提,所得流出液再經薄層層析片分離,可得到6個劃分層(分別標記為BF2-5-1至BF2-5-6)。BF2-5-6劃分層進一步以甲醇與水依體積比3:7之混合液作為移動相進行高效液相層析(使用C18管柱),可分離得化合物FT-02約8.0mg。
此外,使用填充Diaion HP-20之管柱及水與甲醇漸減極性梯度混合之沖提液,對水層萃取物(約75.0g)進行管柱層析,分離得到6個劃分層(分別標記為WF1至WF6)。其後,WF2劃分層以甲醇再結晶法得到化合物FT-01約200mg。以二氯甲烷與甲醇依體積比7:1之混合液為沖提液,對WF2劃分層於再結晶後的母液進行矽膠管柱層析,可分離得化合物FT-03約5.0mg。
化合物FT-01、FT-02、及FT-03的化學結構係以質譜法及核磁共振光譜法(NMR)確定。圖2及圖3分別顯示FT-01的質譜圖及核磁共振光譜;圖4及圖5A-5E分別顯示FT-02的質譜圖及核磁共振光譜;圖6及圖7分別顯示FT-03的質譜圖及核磁共振光譜。依據圖2-3判定化合物FT-01是蘆丁(rutin);依據圖4及5A-5E判定化合物FT-02是拉里西諾醇4'-O-β-D-吡喃葡萄糖苷(lariciresinol 4'-O-β-D-glucopyranoside);依據圖6-7判定化合物FT-03是腺嘌呤(adenine)。下表2顯示各該化合物的結構。
為驗證化合物FT-01、FT-02、及FT-03調節膽固醇代謝相關基因表現之效果,對人類肝癌細胞株HepG2施以含50μg/mL該三種化合物任一者之2mL細胞培養基,或者僅用細胞培養基處理細胞(控制組)。各組細胞於37℃培養24小時後用於基因表現量分析,目標基因包括SCARB1、APOA1、及LDLR基因。
圖8顯示前述各組HepG2細胞中各目標基因的相對表現量;圖中* **及***分別表示相比控制組為p<0.05、p<0.01及p<0.001。依據圖8,相比控制組,施予FT-01(即蘆丁)無法提升SCARB1、APOA1、及LDLR基因的表現。相對地,施予FT-02(即拉里西諾醇4'-O-β-D-吡喃葡萄糖苷)顯著提升SCARB1及LDLR基因之表現分別約34%及127%。此外,FT-03(即腺嘌呤)之處理亦顯著增加LDLR基因的表現約135%。此結果驗證FT-02及FT-03促進膽固醇代謝相關基因表現之效果。
綜上所述,本發明揭露施予有效量之拉里西諾醇4'-O-β-D-吡喃葡萄糖苷及腺嘌呤能顯著促進細胞內膽固醇代謝相關基因表現。因此,該二化合物可用於製備促進膽固醇代謝相關基因表現之組合物,其能降低個體因膽固醇代謝不良而罹患心血管疾病的風險,進而改善膽固醇代謝異常者或高膽固醇飲食者的心血管健康。該組合物可為粉末、顆粒、溶液、膠體或膏體,且可製成醫藥品、食品、飲品、或營養補充劑,藉由口服或其他方式給予一個體。
<110> 大江生醫股份有限公司
<120> 促進膽固醇代謝相關基因表現之化合物之用途
<130> 108B0461-I1
<160> 8
<170> PatentIn version 3.5
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Claims (6)
- 一種組合物用於製備促進膽固醇代謝相關基因表現之醫藥品之用途,其中該組合物包含一藥學上可接受的載體,及一選自由拉里西諾醇4'-O-β-D-吡喃葡萄糖苷、腺嘌呤、及其組合所組成群組之化合物,其中該膽固醇代謝相關基因編碼為一低密度脂蛋白受體(LDLR)。
- 如申請專利範圍第1項所述之用途,其中該化合物之濃度至少為50μg/mL。
- 一種拉里西諾醇4'-O-β-D-吡喃葡萄糖苷用於製備促進膽固醇代謝相關基因表現之醫藥組合物之用途,其中該膽固醇代謝相關基因編碼為一清除者受體B類1型(SRB1)或一低密度脂蛋白受體(LDLR)其中至少一。
- 如申請專利範圍第3項所述之用途,其中該拉里西諾醇4'-O-β-D-吡喃葡萄糖苷之濃度為至少50μg/mL。
- 一種腺嘌呤用於製備促進膽固醇代謝相關基因表現之醫藥組合物之用途,其中該膽固醇代謝相關基因編碼為一低密度脂蛋白受體(LDLR)。
- 如申請專利範圍第5項所述之用途,其中該腺嘌呤之濃度至少為50μg/mL。
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