TWI733458B - 用於預防、改善或治療肥胖症及/或糖尿病代謝症候群之包含餘甘子萃取物和大麥萃取物(ib複合物)作為有效成分之複合物之組成物 - Google Patents
用於預防、改善或治療肥胖症及/或糖尿病代謝症候群之包含餘甘子萃取物和大麥萃取物(ib複合物)作為有效成分之複合物之組成物 Download PDFInfo
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- TWI733458B TWI733458B TW109116980A TW109116980A TWI733458B TW I733458 B TWI733458 B TW I733458B TW 109116980 A TW109116980 A TW 109116980A TW 109116980 A TW109116980 A TW 109116980A TW I733458 B TWI733458 B TW I733458B
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- indian gooseberry
- barley malt
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Abstract
本發明係關於一種用於預防、改善或治療代謝症候群之組成物,包含作為一有效成分的一印度醋栗(amla)萃取物及一大麥芽萃取物之一複合物。特別是,根據本發明的該印度醋栗萃取物及該大麥芽萃取物的該複合物,而非每一種單一物質,抑制胰脂肪酶的酵素活性,從而抑制攝取的脂肪的消化及吸收,並促進包括IRS、PI3K在內的GLUT4訊號。印度醋栗萃取物及大麥芽萃取物的該複合物可減輕體重、降低血糖、血液中胰島素及血液中糖化血紅蛋白,從而顯示出在改善肥胖症或糖尿病方面的一協同作用,並因此可用作預防或治療諸如肥胖症及糖尿病等代謝症候群的一醫藥組成物或一保健功能食品。
Description
本發明是關於一種用於預防、改善或治療代謝症候群之包含作為一有效成分的天然萃取物之組成物。
肥胖症是指脂肪在體內過度累積的疾病,並且可能是由多種原因引起的,諸如遺傳因素、缺乏運動、壓力、荷爾蒙失調及西方化飲食等。最近,由於能量攝取及消耗的不平衡,肥胖症的發病率每年都在增加,且根據2017年國民健康與營養調查(National Health and Nutrition Survey),韓國19歲以上的肥胖症率是34.1%,並且相較於2001年的29.2%增加了4.9%。肥胖症本身可能是一種健康問題,但因為與代謝症候群(諸如高血脂症、高血壓、2型糖尿病及高血糖等)密切相關,目前被認為是嚴重的健康問題。
最近,以各種方式提供了用於治療或改善肥胖症的方法,其中一種方法是肥胖症治療劑。代表性的肥胖症治療劑包括諸如苯丁胺(Phentermine)、苯甲曲秦(Phendimetrazine)及馬吲哚(Mazindol)等的食慾抑制劑,以及諸如奧利司他(Orlistat)的脂肪吸收抑制劑,但是食慾抑制劑透過刺激中樞神經系統而具有諸如心悸、胸痛、暈眩、焦慮及無意識等的副作用的問題,脂肪吸收抑制劑具有諸如抑制脂溶性維生素吸收及消化失調等的胃腸道副作用。
除了使用這些肥胖症治療劑的藥物療法以外,還進行了限制能量攝取的飲食療法、增加能量消耗的運動療法、心理療法、行為療法及外科手術療法等,但是作為治療肥胖症的較佳方法,已經提出透過運動促進能量消耗並結合副作用較少的治療肥胖症的藥物是最安全且最有效的方法。
因此,迫切需要開發用於抑制或治療肥胖症或糖尿病的物質,該物質對於人體是安全的且顯示出優異的功效。
於整個說明書中參考及引用了許多文獻。所引用的文獻的揭示內容透過引用其整體併入本文中,並且更清楚地描述了本發明所屬技術領域的程度以及本發明的內容。
[專利文獻]KR 10-1830048 B
本發明人試圖開發出顯示出對肥胖症或糖尿病的治療及抑制功效並且沒有副作用的天然物質,結果證實了在各種天然原料中的印度醋栗(Indian gooseberry)萃取物及大麥芽(barley sprout)萃取物具有對於諸如肥胖症或糖尿病的代謝症候群具有優異的治療功效,並且發現透過最大化每種原料的功效的混合比對代謝症候群的治療具有協同作用,從而完成了本發明。
因此,本發明的目的是提供一種用於預防、改善或治療代謝症候群之組成物,包括作為有效成分的印度醋栗萃取物及大麥芽萃取物之複合物。
本發明的另一個目的是提供一種用於製備預防、改善或治療代謝症候群之組成物之方法,包括作為有效成分的印度醋栗萃取物及大麥芽萃取物之複合物。
透過以下對本發明、申請專利範圍及附圖的詳細描述,本發明的其他目的及優點將更加明顯。
本發明的一態樣是提供一種用於預防、改善或治療代謝症候群之組成物,包含作為有效成分的印度醋栗(餘甘子,Emblica officinalis)萃取物及大麥芽(大麥,Hordeum vulgare)萃取物之複合物。
印度醋栗(餘甘子,Phyllanthus emblica L.)也稱為餘甘子(Emblica officinalis Gaertn)或Amla,取決於每個地區或語言的不同,其名稱也不同。例如,它也被稱為Amalaki、Emblic Leafflower、Yougan(油甘)、Anmaroku(奄摩勒)、Emblic.myrobalan、馬六甲(Malaka)、馬六甲樹(Malacca tree)、Alonla、Amila、Amilaki、Amila chatra、Nellikai、Nelli、Tasha、Kayruk、Kemurak及Mak Kham pom等。印度醋栗是一種野生分佈的植物,並在廣泛的地區自然生長,諸
如包括尼泊爾在內的南亞及包括馬來西亞及中部及南部在內的東南亞等。它在台灣及喜馬拉雅山的海拔1500毫米以上的山坡上大量種植。
印度醋栗是一種高度為3~8m的落葉喬木,葉子長約10mm且寬約2~3mm,在4月至5月間開著帶有檸檬香氣的黃綠色小花。果實為扁平球形,平均尺寸為18~25mm,成熟時為淺黃色、富光澤及帶有六根黃綠色的細繩。新鮮果實的味道酸甜、苦澀、餘味甜。乾燥後的果實變為黑色,即使乾燥也不會削弱營養成分,因此可以分配乾燥的果實。Amla也被稱為amalaka,在尼泊爾被認為是神聖的樹。
眾所皆知,印度醋栗(Amla)含有維生素C、礦物質、胺基酸、單寧及蘆丁(rutin)等。印度醋栗的維生素C含量是柳橙的20倍且對熱非常穩定,因此,即使長期間暴露在高溫下,維生素也幾乎不會被破壞。維生素C的耐熱性被認為是源自一起含有的丹寧成分,並且這些成分顯示出抗氧化劑、抗腫瘤及抗發炎性活性等。此外,印度醋栗(Amla)中包含諸如鞣花酸、沒食子酸及槲皮素等各種生理成分。
大麥芽(Hordeum vulgare L.)是指透過以大麥的年輕順序播種大麥種子而生長的幼葉狀態。於一實施例中,大麥芽在播種後約8-15天可為約10-20cm的嫩葉。大麥是大約10,000年前在歐亞大陸最早種植的穀物之一,被譽為人類耕種的最古老的農作物之一。大麥芽是眾所周知的營養豐富的食物來源,其具有高含量的多種維生素(包含維生素A、維生素B及維生素C)以及礦物質(諸如鈣、鎂及鉀等),並且含有大量的膳食纖維,因此產業上用作保健功能食品及醫療用品的可能性正在增加。在美國及日本等地,將大麥幼葉冷凍乾燥並製成粉狀,從而被開發出來並作為保健食品出售,而在韓國,它被商業化並以粉
狀、丸劑、綠汁、茶及化妝品等的形式作為一般食品及保健功能食品出售。在藥理活性方面,大麥芽透過各種生物活性物質表現出諸如抗氧化活性、排便順暢、改善膽固醇量、改善血糖量、改善高血脂症功能及肥胖症的抑制作用等的各種功能。這些生物活性作用被認為源自包括皂草苷(saponarin)及盧他那林(lutonarin)等的類黃酮(flavonoids)。
本發明的組成物包含作為有效成分的印度醋栗及大麥芽萃取物,並且本文所用的術語「萃取物」的含義包含透過擠壓原料或將原料以萃取溶劑處理而取得的萃取結果,或配製的加工產品(例如,粉狀)。
當透過將萃取溶劑處理原料而取得用於本發明的組成物中的萃取物時,可以使用各種萃取溶劑,例如可以使用極性溶劑或非極性溶劑。可以使用(i)水、(ii)醇(較佳為甲醇、乙醇、丙醇、丁醇、正丙醇、異丙醇、正丁醇、1-戊醇、2-丁氧基乙醇或乙二醇)、(iii)乙酸、(iv)DMFO(二甲基甲醯胺)及(v)DMSO(二甲基亞碸)等作為極性溶劑,可以使用丙酮、乙腈、乙酸乙酯、乙酸甲酯、氟烷、戊烷、己烷、2,2,4-三甲基戊烷、癸烷、環己烷、環戊烷、二異丁烯、1-戊烯、1-氯丁烷、1-氯戊烷、鄰二甲苯、二異丙醚、2-氯丙烷、甲苯、1-氯丙烷、氯苯、苯、二乙醚、二乙基硫醚(diethyl sulfide)、氯仿、二氯甲烷、1,2-二氯乙烷、苯胺、二乙胺、醚、四氯化碳及四氫呋喃(THF)等作為非極性溶劑。
較佳地,用於本發明的萃取物可以透過擠壓原料或使用選自由水、具有1-4個碳原子的低級醇及其混合物所組成之群組的任何一種作為萃取溶劑來萃取,但不限於此。
此外,本文使用的術語「萃取物」具有如上所述在本領域中通常用作粗萃取物的含義,並且在廣義上包括將萃取物另外分餾的分餾物。換句話說,它不僅包括透過擠壓或使用上述萃取溶劑而取得的萃取物,而且還包括透過對其進行另外純化過程而取得的萃取物。例如,透過使萃取物通過具有一特定分子量截留值的超濾膜而取得的分餾物,以及透過另外進行的各種純化方法所取得的分餾物,諸如透過各種色譜法(根據尺寸、電荷、疏水性或親和力用於分離)等分離。
此外,本發明的萃取物可以是一種隨後進行另外處理的萃取物,例如其中一種透過過濾或進行濃縮或乾燥處理而除去溶劑的萃取物,或者是經過全部過濾、濃縮及乾燥的一種萃取物。例如,過濾可以使用濾紙或使用減壓過濾器,而濃縮可以使用減壓濃縮器,並且乾燥可以進行噴霧乾燥或冷凍乾燥等,以取得粉狀萃取物。
根據本發明的一實施例,如上製備的印度醋栗萃取物可以包含1至25mg/g的鞣花酸(ellagic acid),並且取決於分析方法可以在酸性水解狀態下包含1至5mg/g的鞣花酸,並可以在不進行酸性水解的狀態下包含5至25mg/g的游離鞣花酸。此外,如上製備的大麥芽萃取物可以包含6至11mg/g的皂草苷。
於本發明的組成物中,可以包含作為混合物或複合物的前述印度醋栗萃取物及大麥芽萃取物。混合物或複合物與每種單一物質相比可能具有協同作用。更具體地,印度醋栗萃取物及大麥芽萃取物的重量比可以為4:1至1:1,例如4:1、2:1或1:1的重量比,但不限於此。
根據一實施例,本發明的組成物可以透過以下一種方法製備包括:以包含擠壓及乾燥製程從印度醋栗中取得印度醋栗萃取物;以包括擠壓及
乾燥製程從大麥芽中取得大麥芽萃取物;以及透過以4:1至1:1的重量比混合印度醋栗萃取物及大麥芽萃取物以製備複合物。
本發明的印度醋栗萃取物及大麥芽萃取物的這種混合物或複合物透過抑制胰脂酶的酵素活性,抑制脂肪的消化及吸收、抑制脂肪的合成及促進脂肪分解而保留了抑制脂肪累積及減肥的功效;或透過調節血糖吸收並促進葡萄糖代謝而保留了降低血糖、胰島素及糖化血紅蛋白的功效,從而顯示出預防、改善或治療諸如肥胖症或糖尿病的代謝症候群的功效。
於本文中,術語「胰脂肪酶(pancreatic lipase)」是將胰腺分泌的三酸甘油酯(TG)水解為甘油單酸酯(MG)及脂肪酸的脂肪酶,也稱為胰腺三酸甘油酯脂肪酶(pancreatic triacylglycerol lipase),是促進細胞內三酸甘油酯吸收的酶。因此,胰脂肪酵素活性抑制能力可以用作評估體內脂肪吸收的指標。
於本文中,術語「cAMP(環腺苷單磷酸)(pancreatic triacylglycerol lipase)」透過活化腺苷酸環化酶或抑制細胞間cAMP磷酸二酯酶作用而具有增加的細胞間濃度,因為β-腎上腺素拮抗劑、前列腺素E2(PGE2)及組織胺與細胞表面受體結合。cAMP透過活化為脂肪酶的激素敏感性脂肪酶(HSL)參與促進三酸甘油酯的分解及促進發熱(heat generation)。因此,cAMP量可以用作評估脂肪分解的指標。
於本文中,術語「甘油釋放(Glycerol release)」表示釋放的甘油的量,並且當累積在脂肪細胞中的脂肪小球中存在三酸甘油酯(TG)時,分成甘油及脂肪酸。因此,游離甘油的含量可以用作評估脂肪小球中三酸甘油酯(TG)的分解的指標。
於本文中,術語「IRS(胰島素受體基質)」是一種分布在諸如肌肉、肝臟及脂肪組織等的全身組織中的胰島素受體基質,並且參與胰島素訊號傳遞途徑以進行調節糖及脂質代謝的功能。
於本文中,術語「PI3K(磷脂肌醇-3-激酶)(Phosphoinositide 3-Kinase)」是參與胰島素訊號傳遞途徑的磷酸化,並與胰島素受體基質(IRS(胰島素受體基質))相互作用,以調節血糖吸收及抑制脂肪合成。
於本文中,術語「GLUT4(葡萄糖運輸蛋白4)」是主要存在於脂肪細胞及肌肉細胞中的葡萄糖受體,並且透過依賴於胰島素將葡萄糖從細胞轉移到細胞膜中而成為抑制由葡萄糖代謝引起的脂肪合成的指標。當發生由脂肪累積引起的胰島素抵抗時,GLUT4的轉運就會發生不順,而GLUT4表達的增加透過將葡萄糖轉移到細胞內部並將其用作能量來調節體內葡萄糖代謝。
再者,本發明提供一種用於預防、治療諸如肥胖症或糖尿病的代謝症候群的醫藥組成物,透過包含含有有效成分的組成物,並加入藥學上可接受的載體、賦形劑或稀釋劑等配製成藥物單位劑型。
於本文中,藥學上可接受的載體通常用於配製,並包括乳糖、葡萄糖(dextrose)、蔗糖、山梨糖醇、甘露醇、澱粉、阿拉伯膠、磷酸鈣、藻酸鹽、明膠、矽酸鈣、微晶纖維素、聚乙烯吡咯烷酮、纖維素、水、糖漿、甲基纖維素、羥基苯甲酸甲酯、羥基苯甲酸丙酯、羥基苯甲酸丙酯、滑石粉、硬脂酸鎂及礦物油等,但不限於此。
此外,當配製包含有效成分的組成物時,可以使用常用的稀釋劑或賦形劑(諸如填充劑、增量劑、黏合劑、濕潤劑、崩散劑及表面活性劑等)來製備。
另外,藥物劑型可以透過配製成口服製劑(諸如粉末、顆粒、片劑、膠囊、懸浮液、乳劑、糖漿劑及氣霧劑等)、外用製劑、栓劑及無菌注射溶液的形式來使用。
可以透過將至少一種賦形劑與萃取物或粉末混合來製備用於口服給藥的固體製劑,例如可以透過混合碳酸鈣、蔗糖或乳糖及明膠等來製備澱粉。此外,除了簡單的賦形劑以外,還可以使用諸如硬脂酸鎂及滑石粉的潤滑劑。
在腸胃外給藥的製劑中,可包含無菌水溶液、非水溶劑、懸浮液、乳劑、冷凍乾燥製劑及栓劑。
可以使用諸如丙二醇、聚乙二醇及橄欖油的植物油以及可與油酸乙酯一起注射的酯等的植物油作為非水溶劑及懸浮液。
可以使用維比索爾(witepsol)、聚乙烯二醇(macrogol)、聚山梨醇酯61(tween 61)、可可脂、月桂酸酯及甘油明膠等作為栓劑的基礎化合物。此外,可以使用諸如硬脂酸鎂及滑石粉的潤滑劑。
本發明的醫藥組成物可以取決於所需方法為口服或非腸道給藥(例如,靜脈內、皮下、腹膜內或局部施用),且其劑量取決於患者的狀況及體重、疾病程度、年齡、性別、藥物形式、給藥途徑及期限而變化,但本領域技術人員可以適當地選擇之。
此外,本發明提供了一種用於預防或改善諸如肥胖症或糖尿病的代謝症候群的保健功能食品組成物,其透過包含含有有效成分的組成物並添加食物補充添加劑而配製成保健功能食品。
於本文中,能夠添加萃取物的食物,包括例如各種食物、飲料、橡皮糖、茶、維生素複合物及保健功能食品等。本發明的保健功能食品組成物除含有萃取物外,對其他成分沒有特別限制,可以含有作為附加成分的諸如普通食品、飲料等的各種調味劑或天然碳水化合物等。
於天然碳水化合物中,可以存在諸如單醣(例如葡萄糖、果糖)、雙糖(例如麥芽糖、蔗糖)、多醣(例如糊精及環糊精等)以及糖醇(諸如木糖醇、山梨糖醇、赤藻糖醇(erythritol)等)的常見醣類。
除此之外,可以有利地使用天然調味劑(索馬甜(thaumatin)、甜葉菊萃取物(例如萊鮑迪甙A(rebaudioside A)及甘草酸苷(glycyrrhizin)等)及合成調味劑(糖精及阿斯巴甜等)作為調味劑。
除此以外,本發明的萃取物、粉末或複合物可含有各種營養素、維生素、礦物質(電解質)、調味劑(諸如合成調味劑及天然調味劑)、著色劑及增強劑、果膠酸及其鹽、有機酸、保護性膠體增稠劑、pH調節劑、穩定劑、防腐劑、甘油、醇及用於碳酸飲料的碳化劑等。
而且,本發明的萃取物、粉末或複合物可含有用於製備天然果汁及蔬菜飲料的果肉。該成分可以單獨使用或結合使用。
由本發明的印度醋栗萃取物及大麥芽萃取物所組成的複合物顯示出抑制脂肪的消化及吸收的功效,更具體地,它顯示出抑制消化及吸收的功效,並且促進包括IRS及PI3K的GLUT4訊號傳導,從而抑制葡萄糖代謝中的脂肪合成,並透過調節cAMP的表達促進脂肪分解,從而增加釋放到細胞外的甘油的量,以抑制脂肪的累積,並調節血糖的吸收及促進葡萄糖的代謝,從而減輕體
重、降低血糖、胰島素及糖化血紅蛋白,從而保持了預防、改善或治療諸如肥胖症或糖尿病的代謝症候群的優異功效。
此外,本發明的印度醋栗萃取物及大麥芽萃取物是由無毒、無副作用及對人體無害的天然萃取物組成的複合物,可用於有效地預防、改善或治療代謝症候群。
圖1顯示對包括印度醋栗萃取物及大麥芽萃取物的11種天然物質的胰脂肪酶抑制活性的圖。
圖2顯示將印度醋栗萃取物及大麥芽萃取物的複合物作為單一物質進行比較時對胰脂肪酶抑制活性的協同作用的圖。
圖3顯示取決於印度醋栗萃取物及大麥芽萃取物的混合比例的胰脂肪酶抑制活性的圖。
圖4顯示取決於印度醋栗萃取物及大麥芽萃取物的混合比例的細胞內cAMP量的圖。
圖5顯示取決於印度醋栗萃取物及大麥芽萃取物的混合比例的細胞內甘油釋放的圖。
圖6顯示將印度醋栗萃取物及大麥芽萃取物的複合物作為單一物質進行比較時對細胞內P-IRS1/IRD1的蛋白質表達率的協同作用的圖。
圖7顯示將印度醋栗萃取物及大麥芽萃取物的複合物作為單一物質進行比較時對細胞內P-PI3K/PI3K的蛋白質表達率的協同作用的圖。
圖8顯示將印度醋栗萃取物及大麥芽萃取物的複合物作為單一物質進行比較時對細胞內GLUT4的蛋白質表達率的協同作用的圖。
圖9顯示將印度醋栗萃取物及大麥芽萃取物的複合物作為單一物質進行比較時對細胞內脂肪累積的抑制的協同作用的圖。
圖10顯示將印度醋栗萃取物及大麥芽萃取物的複合物作為單一物質進行比較時得自實驗動物的血糖(葡萄糖)的圖。
圖11顯示將印度醋栗萃取物及大麥芽萃取物的複合物作為單一物質進行比較時得自實驗動物的血液中的胰島素的圖。
圖12顯示將印度醋栗萃取物及大麥芽萃取物的複合物作為單一物質進行比較時得自實驗動物的血液中的糖化血紅蛋白(血紅素Alc;HbAlc)的圖。
於下文中,將透過實施例更詳細地描述本發明。這些實施例旨在更具體地說明本發明,並且對於本領域技術人員顯而易見的是,本發明的範圍不受這些實施例的限制。
1.測試物質的製備方法
1.1 印度醋栗(Amla)萃取物的製備(比較例1)
將印度醋栗(Amla,尼泊爾產)的果實洗淨並擠壓後,取得印度醋栗果實的萃取物。然後,在過濾及濃縮然後乾燥之後,製備用於本發明的印度醋栗萃取物。
1.2 大麥芽萃取物的製備(比較例2)
收成大麥芽(韓國產)並擠壓後,除去諸如纖維素的不溶性纖維,將其過濾然後乾燥。將乾燥的粉末均質化以製備用於本發明的大麥芽萃取物。
1.3 複合物的製備(實施例1至實施例5)
將在比較例1至比較例2中製備的印度醋栗(Amla)萃取物及大麥芽萃取物以如下表1的重量比結合以製備複合物。
1.4 候選材料的製備方法
透過國內外文獻審查,研究了抗肥胖症或抗糖尿病活性物質,並選擇了考慮到國內原材料的可獲得性及作為食品成分的攝入的可獲得性而取得的天然材料作為候選材料並用於研究。候選材料A、B、C及D分別代表荷葉、桔梗花、柘樹(Cudrania tricuspidata)及肉桂,透過加入熱水萃取並進行噴霧乾燥,然後用於研究。E及F代表桑葉及蓮(Nelumbo nucifera),並透過加入熱水萃取,然後濃縮並進行噴霧乾燥而用於研究。G、H及I代表甜菜根、石榴及酸櫻桃,將它們濃縮並在噴霧過程中進行噴霧,然後用於研究。
1.5標記成分含量確認
為了進行印度醋栗(Amla)萃取物及大麥芽萃取物的標記成分含量分析及原料標準化,對於印度醋栗(Amla)萃取物(比較例1-1、比較例1-2、比較例1-3)及大麥芽萃取物(比較例2-1、比較例2-2、比較例2-3)的每種原料生產了三種原料。
印度醋栗(Amla)萃取物中的標記成分設置為「鞣花酸」,大麥芽萃取物中的標記成分設置為皂草苷,然後進行分析。取決於分析方法,將印度醋栗(Amla)萃取物的標記成分的含量排列在表2及表3中,將大麥芽萃取物的標記成分的含量排列在表4中。
更具體地,如下分析每種萃取物的標記成分的含量。
用於定量印度醋栗(Amla)萃取物中的鞣花酸的分析方法如下。
在酸性水解後,使用高效色譜法測定於印度醋栗(Amla)萃取物中鞣花酸的含量。使用Capcellpak C18 UG120(4.6mm X 50mm,5μm)作為管柱,並使用0.85%磷酸鹽在蒸餾水中的6:4混合溶液及甲醇(A)以及甲醇(B)作為移動相透過梯度方法分離,並用紫外光檢測器在370nm波長下檢測鞣花酸。結果,根據實驗方法1.1製備的印度醋栗(Amla)萃取物中的鞣花酸的含量被確認在1-5mg/g的範圍內。
在用於定量印度醋栗(Amla)萃取物中的游離鞣花酸的分析方法中,在未進行酸性水解的條件下的分析方法如下。
在甲醇超音波萃取後,使用高效色譜法測定印度醋栗(Amla)萃取物中游離鞣花酸的含量。使用Capcellpak C18 UG120(4.6mm X 50mm,5μm)作為管柱,並使用0.85%磷酸鹽在蒸餾水中的6:4混合溶液及甲醇(A)及甲醇(B)作為移動相透過梯度方法分離,並用紫外光檢測器在370nm波長下檢測鞣花酸。結果,根據實驗方法1.1製備的印度醋栗(Amla)萃取物中的游離鞣花酸含量被確認在5-25mg/g的範圍內。
用於定量大麥芽萃取物的皂草苷的分析方法如下。
使用高效色譜法測定在大麥芽萃取物中皂草苷的含量。使用Capcellpak C18 UG120(4.6mm X 50mm,5μm)作為管柱,並使用0.1%甲酸(A)及甲醇(B)作為移動相透過梯度方法分離,並用紫外光檢測器在340nm波長下檢測皂草苷。結果,根據實驗方法1.2製備的大麥芽萃取物中皂草苷的含量被確認在6-11mg/g的範圍內。
2.療效評估
2.1 胰脂肪酶抑制活性的測定
胰脂肪酶是一種將三酸甘油酯水解為單甘油酯(MG)及脂肪酸的三酸甘油酯(TG)的酶,可促進脂肪的消化,並有助於腸上皮細胞吸收分解產物。於是,由於三酸甘油酯的分解抑制脂肪被腸細胞及消化道的吸收,因此,當胰脂肪酶的活性被抑制時,胰脂肪酵素活性的抑制能力是預測抗肥胖症活性的非常有用的試驗方法。於本實施例中,如下測量胰脂肪酶的抑制活性。首先,添加169μL的三羥甲基氨基甲烷(Tris)緩衝液(100mM Tris-HCl,5mM CaCl2,pH 7.0)及20μL樣品並混合到6μL酶溶液中,其中豬胰脂肪酶以0.5g/200mL的濃度溶解在酶緩衝液(10mM MOPS,1mM EDTA,pH6.8),然後在37℃下培養15分鐘。然後,在加入5μL基質溶液(在二甲基甲醯胺中的10mM對硝基苯基丁酸酯)並在37℃下培養30分鐘後,使用紫外光可見分光光度計在405nm處測定吸收度。胰脂肪酶抑制活性(%)如下式1所示,X表示添加了樣品的吸收度,Y表示沒有添加樣品的吸收度。測定值顯示為三個重複實驗的平均值。
式1胰脂肪酶抑制活性(%)=(1-(X-Y)/Y)x 100
2.2 複合物對胰脂肪酶抑制活性的協同作用的確認
為了研究本發明的印度醋栗萃取物及大麥芽萃取物的複合物與作為單一物質處理的情況相比對胰脂肪酶的抑制作用的協同作用,在相同濃度
下分析單一物質及複合物的活性,並利用Colby方程式確認複合物的協同作用。Colby方程式如下式2所示,當測定值超過Colby方程式得出的E值時,可以識別出協同作用。E表示複合物(A+B)的預測作用,A表示單一物質A的作用(%),B表示單一物質B的作用(B)。
式2 E=A+B-(AB/100)
2.3 細胞培養及分化誘導
與作為單一物質處理的情況相比,確認了本發明的印度醋栗萃取物及大麥芽萃取物的複合物在脂肪細胞分化過程中影響脂肪合成及脂肪分解的功效。於本實施例中,從美國典型培養物保藏中心(ATCC;美國馬里蘭州羅克維爾)分配3T3-L1細胞並進行實驗。使用含有10%新生小牛血清(NCS)的高葡萄糖Dulbeco改良的Eagle's培養基(DMEM)、1%青黴素-鏈黴素、1%L-谷氨醯胺、1%丙酮酸鈉、1%羥乙基哌嗪乙硫磺酸(hepes)及1%NEAA混合物,且每2天更換一次培養液,在37℃及5%CO2的培養箱中培養3T3-L1前脂肪細胞(賽默飛世爾科技公司(Thermo Fisher Scientific Inc.),美國賓夕法尼亞州匹茲堡)。當細胞以單層附著在培養瓶底部上80%或更多時,使用PBS溶液洗滌細胞表面,並加入0.25%胰蛋白酶-EDTA,將其在培養箱中放置3分鐘,並分離細胞。然後,透過使用離心機(GYROZEN 416G)以1600rpm離心5分鐘來收集細胞,並且為了分化細胞,使用DMEM培養液(包括10%胎牛血清(FBS)及1%青黴素-鏈黴素、1%L-谷氨醯胺、1%丙酮酸鈉、1%羥乙基哌嗪乙硫磺酸(hepes)1%NEAA混合物及健他黴素(gentamycin))將1x105細胞/孔的細胞平均地等分在6孔盤(TPP)中。當100%匯合時,開始在含有10%胎牛血清(FBS)及1%青黴素-
鏈黴素、1%L-谷氨醯胺、1%丙酮酸鈉、1%羥乙基哌嗪乙硫磺酸(hepes)1%NEAA混合物及健他黴素(gentamycin)的DMEM培養液中透過混合成脂肪形成的雞尾酒試劑(MDI溶液)的3-異丁基-1-甲基黃嘌呤(IBMX,0.5mM)、胰島素(10μg/mL)及地塞米松(DEX,1μM)誘導分化。分化期總共持續9天,在分化的前3天,更換相同的培養液,在分化的中間3天,將培養液每天更換為僅含胰島素(10μg/mL)的10%FBS的DMEM,而在分化的後3天,將其每天更換為含有10%FBS的DMEM培養液。在脂肪生成機制中,每天在開始分化的同時對樣品進行處理,而在脂肪分解機制中,從分化結束前3天開始對樣品進行3天的處理。
2.4 複合物對細胞間cAMP量的協同作用的確認
cAMP透過活性作為脂肪酶的激素敏感性脂肪酶(HSL)來促進三酸甘油酯的分解及促進發熱。於本實施例中,使用cAMP ELISA試劑盒(Cell biolabs公司,美國加利福尼亞州聖地亞哥)以測定細胞間cAMP量,並如下進行測定。首先,將1x106細胞添加在裂解緩衝液中並均質化,並以13000rpm離心5分鐘,將上清液作為樣品。在每孔中將樣品及標準試劑等分50μL,將過氧化物酶cAMP追蹤劑共軛試劑等分25μL,然後將兔抗cAMP多株抗體試劑等分50μL,並置放上盤蓋在室溫下放置2小時。在2小時內,用洗滌緩衝液等分250μL進行5次洗滌操作,然後在每孔中等分100μL在室溫下回溫的基質溶液,然後放置在室溫下20分鐘。在20分鐘內,在每孔中等分100μL終止溶液以終止反應,並在450nm波長處測定吸收度。
2.5 複合物對細胞間甘油釋放的協同作用的確認
脂肪分解是脂肪細胞中的三酸甘油酯水解為游離酸及甘油的過程,為了確認對脂肪細胞中的脂肪分解的功效,測定了脂肪分解過程中甘油的增加。於本實施例中,為了測定在脂肪細胞分化過程中甘油釋放的量的功效,使用游離甘油試劑採用磷酸甘油氧化酶-TRINDER酶反應法透過諸如McGowan等的該方法測定培養液中的甘油含量,其測定如下。首先,對每種樣品進行濃縮處理,分別收集培養液並在分化後期(第9天)使用,將培養液(1mL)及游離甘油試劑(800μL)混合並在37℃的加熱板上放置10分鐘反應,然後使用ELISA讀取器(分子儀器(Molecular Devices),美國)在540nm波長處測定光學密度。透過使用游離甘油作為標準試劑製備的標準曲線來測定甘油含量,並且透過布拉德福法(Bradford method)使用BSA作為標準試劑來測定蛋白質含量。
2.6 複合物對細胞間IRS、PI3K及GLUT4的協同作用的確認
IRS(胰島素受體基質)是分布在全身組織中的胰島素受體基質,參與胰島素訊號傳遞途徑並調節葡萄糖及脂質代謝,而PI3K(磷脂肌醇-3-激酶)是參與胰島素訊號傳遞途徑的酶,並透過與IRS相互作用來調節血糖吸收及抑制脂肪合成的功能。GLUT4(葡萄糖運輸蛋白4)是一種主要存在於脂肪細胞及肌肉細胞中的葡萄糖受體,並透過胰島素的作用促進葡萄糖從細胞轉移到細胞膜中。當內臟脂肪累積引起胰島素抵抗時,GLUT4從細胞質到細胞膜的轉運就會發生不順。於本實施例中,為了測定IRS、PI3K及GLUT4的蛋白質表達,使用布拉德福試劑(Bradford assay)對細胞溶解產物的蛋白質定量進行西方墨點法(western blotting)分析。將蛋白質(40μg)裝入10%的Mini-PROTEAN®TGXTM預鑄膠片(Bio-Rad)中,然後使用Trans-Blot®TurboTM轉移系統(Bio-Rad)進行轉移。將膜在阻斷緩衝液(含有1%Tween®20的三羥甲基氨基甲烷(Tris)緩
衝鹽水中的5%脫脂牛奶)中阻斷1小時,清洗後將其與IRS、PI3K及GLUT4一抗反應。洗滌HRP聚合的二抗(細胞傳訊(Cell Signaling),1:3000)並使之反應1小時後,將其洗滌並使用EzWest Lumi plus(ATTO,日本東京)顯色,並使用Ez-Capture II(ATTO)及CS Analyzer 3.0軟體(ATTO)分析。
2.7 複合物對細胞間脂肪累積的協同作用的確認
為了測定本發明的印度醋栗萃取物及大麥芽萃取物的複合物從前脂肪細胞分化為脂肪細胞的過程中對脂肪細胞分化的功效,進行了油紅O染色。換句話說,根據細胞培養及分化誘導協定,每天更換含有10%FBS的DMEM培養液,該溶液包含脂肪形成的雞尾酒試劑(adipogenic cocktail)及萃取物,在分化後期(第9天)分別進行油紅O染色。吸取培養液,並用PBS溶液洗滌兩次,然後完全地吸出PBS溶液,加入10%福馬林(formalin)並在室溫下固定5分鐘,然後吸出10%福馬林,再將新的10%福馬林加入,並在室溫下固定2小時或更長時間。然後,吸出福馬林,並加入60%異丙醇且立即吸出,然後將培養瓶完全乾燥,並加入油紅O溶液以使脂肪小球染色60分鐘。染色後,用蒸餾水洗滌4次,並用顯微鏡及照相機觀察脂肪小球的細胞間累積。為使脂肪小球累積的含量合格,在充分乾燥的情況下添加100%異丙醇,洗去油紅O染料,然後使用ELISA讀取器(分子儀器(Molecular Devices),美國)在520nm波長處測定光學密度。然後,將100%異丙醇用作空白。
2.8 複合物對肥胖症誘導的小鼠的體重變化的功效的確認
確認本發明的印度醋栗萃取物及大麥芽萃取物的複合物從高脂肪飲食對肥胖症誘導的小鼠的體重變化的功效。從Saeron Bio公司(韓國義王市)供應約20g的4週雄性C57BL/6J小鼠作為實驗動物。它們在明及暗為12小時(明
/暗循環)、溫度為23±2℃、相對溼度為50±5%的條件下經過1週的適應期後用於實驗。在適應期間,允許自由食用AIN-93G飲食及飲用水,並透過隨機方法測定體重以將每組分離8隻小鼠。在適應期間結束時,將樣品攝入量作為自由飲食攝入量進行15週,並每周測定飲食攝入量及體重,實驗結束後,體重增加除以同期的飲食攝入量(總食物消耗量)以計算出食物利用率(FER)。實驗組的類別及實驗飲食的組成如表5所示,飲食效率的方程式如下式3所示。
式3 FER=體重增加(g)/總食物消耗量(g)x100
2.9 複合物對肥胖症誘導的小鼠器官及脂肪組織重量變化的功效的確認
確認本發明的印度醋栗萃取物及大麥芽萃取物的複合物從高脂肪飲食中對肥胖症誘導的小鼠的器官及脂肪組織的重量變化的功效。對組織切除,在集血、切除器官(肝臟、腎臟、脾臟)及白色脂肪組織(皮下脂肪、內
臟脂肪(附睾脂肪及腹膜內脂肪))之後,用生理食鹽水溶液洗滌並用濾紙除去水份,然後測定重量。
2.10 複合物對肥胖症誘導的小鼠血液中葡萄糖、胰島素及HbAlc變化的功效的確認
確認本發明的印度醋栗萃取物及大麥芽萃取物的複合物從高脂肪飲食對肥胖症誘導的小鼠血液中葡萄糖、胰島素及HbAlc的變化的功效。為了進行血液分析,在實驗結束時,在禁食12小時後用異氟烷麻醉實驗動物,透過肝靜脈收集血液,並透過全血及離心(14,000rpm,20分鐘,4℃)分離的血清分析血液,且使用ELISA試劑盒(Biovision)進行測定。
3.實驗結果
3-1.11種天然物質對胰脂肪酶的抑制活性
測定了實驗方法中建議的製備方法製備的11種天然物質對胰脂肪酶的抑制活性。如圖1及表6所示,在相同濃度下,比較例1的樣品的脂肪酶抑制活性最高為83.77±0.27%,比較例2的樣品的脂肪酶抑制活性其次為42.81±2.09%。此結果確認了在11種天然物質對胰脂肪酶的抑制活性中,印度醋栗萃取物及大麥芽萃取物是最優異的,且印度醋栗萃取物及大麥芽萃取物透過充當胰脂肪酶抑制劑來降低體內的脂肪吸收,並保留了抗肥胖症活性。
3-2.印度醋栗及大麥芽的複合物對胰脂肪酶抑制活性的協同作用
測定了實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物的單一物質及複合物對胰脂肪酶的抑制活性。比較例1表示印度醋栗萃取物的胰脂肪酶抑制活性,比較例2表示大麥芽萃取物的胰脂肪酶抑制活性,實施例1-1代表相同濃度的複合物的胰脂肪酶抑制活性,而利用Colby方程式確認複合物的協同作用而測定的結果由實施例1-2代表。如表2及表7所示,印度醋栗萃取物的胰脂肪酶抑制活性為94.42±0.66%,大麥芽萃取物的功效為40.83±3.14%,相同濃度的複合物的胰脂肪酶抑制活性為112.64±3.20%,而複合物的胰脂肪酶抑制活性優於單一物質。使用Colby方程式推導的複合物的協同作用的預期值為96.70%,而實施例1-2顯示出130.06±2.21%的優異功效,約為預期值的134%或更高。這確認了印度醋栗萃取物及大麥芽萃取物的複合物在抑制胰脂肪酶的酵素活性方面具有比每種單一物質顯著的協同作用,且抑制了攝入脂肪的消化及吸收並保留了抗肥胖症活性。
3-3.以不同重量比結合印度醋栗及大麥芽的複合物對胰脂肪酶抑制活性的確認
為了選擇透過實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物的複合物的最佳比例,測定了胰脂肪酶抑制活性。根據實驗結果3-2,透過確認了印度醋栗及大麥芽在複合物中比每種單一物質均具有協同作用,考慮了製備過程及經濟可行性,然後選擇了顯示出優異功效的印度醋栗及大麥芽的複合物的最佳比例。如圖3及表8所示,在相同濃度下,根據印度醋栗及大麥芽的混合比例為4:1、2:1及1:1時,胰脂肪酶的抑制活性分別為103.45±2.14、108.37±1.92及105.62±0.40,這是相似的,並比在1:2及1:4測定到的83.30±1.63及60.98±0.33優異。結果確認了印度醋栗萃取物及大麥芽萃取物的複合物具有比每種單一物質顯著的協同作用,可以透過以4:1~1:1的印度醋栗:大麥芽的比例(比任何其他混合比例都高)作為優異的胰脂肪酶抑制劑來抑制脂肪的消化及吸收。
3-4.以不同重量比結合印度醋栗及大麥芽的複合物對細胞間cAMP量的功效的確認
以不同重量比結合由實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物來測定細胞間cAMP量。如圖4所示,在相同濃度下,印度醋栗及大麥芽的單一物質分別顯示出1479.3±16.8及1300.0±81.9的活性,而根據印度醋栗及大麥芽的混合比例為4:1及2:1時,cAMP量分別為2472.3±163.8及2508.3±106.3,與單一物質相比具有顯著的協同作用。隨後,以1:1、1:2及1:4測定的優異活性依次為2132.7±149.4、1763.7±101.1及1469.7±24.5。結果確認了印度醋栗萃取物及大麥芽萃取物的複合物具有比每種單一物質顯著的協同作用,可以透過cAMP表達調節促進脂肪分解。
3-5.以不同重量比結合印度醋栗及大麥芽的複合物對細胞間甘油釋放的功效的確認
以不同重量比結合由實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物來測定細胞間甘油釋放。如圖5所示,在相同濃度下,印度醋栗及大麥芽的單一物質分別顯示出0.55±0.03及0.47±0.03的活性,而根據印度醋栗及大麥芽的混合比例為4:1及2:1時,甘油釋放分別為1.01±0.17及1.01±0.01,與單一物質相比具有顯著的協同作用。隨後,以1:1、1:4及1:2
測定的優異活性依次為0.77±0.06、0.69±0.06及0.60±0.09。結果確認了印度醋栗萃取物及大麥芽萃取物的複合物具有比每種單一物質分解累積於脂肪細胞中存在於脂肪小球中的三酸甘油酯(即中性脂肪)顯著的協同作用,從而增加甘油釋放。
3-6.印度醋栗及大麥芽複合物對細胞間IRS1的協同作用
結合由實驗結果中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物來確認細胞間P-IRS1/IRS1的表達率。如圖6所示,在相同濃度下,印度醋栗及大麥芽的單一物質分別顯示出0.42及0.32的表達率,以及實施例3的印度醋栗及大麥芽的複合物的表達率為0.86,與單一物質相比顯示出顯著的協同作用。此外,50、100μg/mL的治療組分別顯示出0.51及0.63的表達率,而印度醋栗及大麥芽的複合物以濃度依賴的方式增加了細胞間P-IRS1/IRS1的表達率,結果確認了印度醋栗萃取物及大麥芽萃取物的複合物可透過增加P-IRS1/IRS1的表達率來調節血糖吸收及從葡萄糖代謝中抑制脂肪合成。
3-7.印度醋栗及大麥芽複合物對細胞間PI3K的協同作用
結合由實驗結果中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物來確認細胞間P-PI3K/PI3K的表達率。如圖7所示,在相同濃度下,印度醋栗及大麥芽的單一物質分別顯示出1.93及1.26的表達率,以及實施例3的印度醋栗及大麥芽的複合物的表達率為4.31,與單一物質相比顯示出顯著的協同作用。此外,50、100μg/mL的治療組分別顯示出1.05及1.47的表達率,而印度醋栗及大麥芽的複合物以濃度依賴的方式增加了細胞間P-PI3K/PI3K的表達率,結果確認了印度醋栗萃取物及大麥芽萃取物的複合物可調節血糖吸收及從葡萄糖代謝中抑制脂肪合成。
3-8.印度醋栗及大麥芽複合物對細胞間GLUT4的協同作用
結合由實驗結果中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物來確認細胞間GLUT4的表達率。如圖8所示,在相同濃度下,印度醋栗及大麥芽的單一物質分別顯示出0.18及0.11的表達率,以及實施例3的印度醋栗及大麥芽的複合物的表達率為0.38,與單一物質相比顯示出顯著的協同作用。此外,50、100μg/mL的治療組分別顯示出0.14及0.33的表達率,而印度醋栗及大麥芽的複合物以濃度依賴的方式增加了細胞間GLUT4的表達率,結果確認了印度醋栗萃取物及大麥芽萃取物的複合物可透過促進GLUT4傳訊來調節血糖吸收及從葡萄糖代謝中抑制脂肪合成。
3-9.印度醋栗及大麥芽複合物對細胞間脂肪累積的抑制的協同作用
結合由實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物進行油紅O染色,以測定對脂肪細胞分化的功效。如圖9所示,在相同濃度下,印度醋栗及大麥芽的單一物質分別顯示出0.60±0.02及1.10±0.05的細胞間三酸甘油酯含量,以及實施例3的印度醋栗及大麥芽的複合物為0.29±0.03,與單一物質相比顯示出顯著的協同作用。此外,於50、100μg/mL的治療組中分別為0.88±0.02及0.57±0.03,並確認了印度醋栗及大麥芽的複合物以濃度依賴的方式抑制累積在脂肪細胞中的脂肪。
3-10.印度醋栗及大麥芽複合物對肥胖症誘導的小鼠的體重變化的功效的確認
為了確認高脂肪飲食對肥胖症誘導的小鼠的體重變化的功效,將實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物混合使用,
測定了實驗動物的體重。如表9所示,透過高脂肪飲食誘導肥胖症的控制組體重增加了30.92±3.53,以及實施例3的印度醋栗及大麥芽的複合物顯示出以濃度依賴的方式減輕體重,根據100、200及400mg/kg的給藥組分別為28.13±3.93、25.78±3.35及24.70±1.85。另外,透過比較飲食效率,確認了與控制組相比,在印度醋栗及大麥芽複合物中以濃度依賴的方式降低,從而確認了印度醋栗萃取物及大麥芽萃取物的複合物在減輕體重方面顯示出優異的功效。
3-11.印度醋栗及大麥芽複合物對肥胖症誘導的小鼠的器官及脂肪組織重量變化的功效的確認
為了確認高脂肪飲食對肥胖症誘導的小鼠的器官及脂肪組織重量變化的功效,該高脂肪飲食是透過實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物的複合物,測定了實驗動物的器官(肝臟、腎臟、脾臟)及白色脂肪組織(皮下脂肪、內臟脂肪(附睾脂肪及腹膜內脂肪))。如表10所示,高脂肪飲食誘導肥胖症的控制組的白色脂肪組織的總量及皮下脂肪組織及內臟脂肪組織的重量分別為7.06±0.72、3.57±0.62及3.49±0.32,以及與
控制組相比,在實施例3的印度醋栗及大麥芽的複合物中,根據100、200及400mg/kg的給藥組,白色脂肪組織的總量分別減輕為5.52±0.86、5.37±0.31及3.95±0.41,皮下脂肪組織的重量減輕為2.62±0.43、2.31±0.18及1.54±0.31,而內臟脂肪組織的重量減輕為2.90±0.67、3.06±0.14及2.41±0.15,從而確認了印度醋栗及大麥芽的複合物以濃度依賴的方式減輕了脂肪組織的重量,並確認了印度醋栗萃取物及大麥芽萃取物的複合物在抑制體內脂肪累積方面顯示出優異的功效。
3-12.印度醋栗及大麥芽複合物對肥胖症誘導的小鼠的血糖變化的功效的確認
為了確認高脂肪飲食對肥胖症誘導的小鼠的血糖變化的功效,該高脂肪飲食是透過實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物的複合物,測定了實驗動物的血糖。如圖10所示,印度醋栗及大麥芽的
單一物質以400mg/kg的相同濃度分別顯示出4.74±0.64及6.99±0.79的血糖值,而在實施例3的印度醋栗及大麥芽複合物中血糖為4.63±0.51,與單一物質相比顯示出明顯的降低功效。此外,在100、200及400mg/kg的治療組中,確認了印度醋栗及大麥芽的複合物以濃度依賴的方式降低血糖,從而確認了印度醋栗萃取物及大麥芽萃取物的複合物在降低血糖方面顯示出優異的功效。
3-13.印度醋栗及大麥芽複合物對肥胖症誘導的小鼠的血液中胰島素改變的功效的確認
為了確認高脂肪飲食對肥胖症誘導的小鼠的胰島素改變,該高脂肪飲食是透過實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物的複合物,測定了實驗動物血液中的胰島素。如圖11所示,印度醋栗及大麥芽的單一物質以400mg/kg的相同濃度分別顯示出0.67±0.01及0.76±0.02的血液中胰島素數值,而在實施例3的印度醋栗及大麥芽複合物中血液中的胰島素為0.56±0.01,與單一物質相比顯示出明顯的降低功效。此外,在100、200及400mg/kg的治療組中,確認了印度醋栗及大麥芽的複合物以濃度依賴的方式降低胰島素,從而確認了印度醋栗萃取物及大麥芽萃取物的複合物在降低血液中胰島素方面顯示出優異的功效。
3-14.印度醋栗及大麥芽複合物對肥胖症誘導的小鼠的血液中糖化血紅蛋白(HbAlc)的功效的確認
為了確認高脂肪飲食對肥胖症誘導的小鼠的血液中糖化血紅蛋白的改變,該高脂肪飲食是透過實驗方法中建議的製備方法製備的印度醋栗萃取物及大麥芽萃取物的複合物,測定了實驗動物血液中的糖化血紅蛋白。如圖12所示,印度醋栗及大麥芽的單一物質以400mg/kg的相同濃度分別顯示出7.50±
0.11及9.67±0.57的血液中糖化血紅蛋白,而在實施例3的印度醋栗及大麥芽複合物中血液中的胰島素為7.15±0.22,與單一物質相比顯示出明顯的降低功效。此外,在100、200及400mg/kg的治療組中,確認了印度醋栗及大麥芽的複合物以濃度依賴的方式降低HbAlc,從而確認了印度醋栗萃取物及大麥芽萃取物的複合物在降低血液中HbAlc方面顯示出優異的功效。
Claims (13)
- 一種用於預防、改善或治療代謝症候群之組成物,包含一印度醋栗(餘甘子,Emblica officinalis)萃取物及一大麥芽(大麥,Hordeum vulgare)萃取物之一複合物,其中該印度醋栗萃取物及該大麥芽萃取物是有效成分,該印度醋栗萃取物或該大麥芽萃取物是經由擠壓或使用選自由水、1-4碳原子的低級醇及其混合物所組成之群組的一萃取溶劑所萃取而得,且該印度醋栗萃取物與該大麥芽萃取物的重量比(印度醋栗萃取物:大麥芽萃取物)為4:1至1:1。
- 如請求項1所述的組成物,其中該印度醋栗萃取物與該大麥芽萃取物的重量比(印度醋栗萃取物:大麥芽萃取物)為4:1、2:1或1:1。
- 如請求項1所述的組成物,其中該印度醋栗萃取物及該大麥芽萃取物的該複合物與每一種單一萃取物相比具有一協同作用。
- 如請求項1所述的組成物,其中該印度醋栗萃取物及該大麥芽萃取物的該複合物藉由抑制脂肪的消化、吸收及合成、藉由促進脂肪分解或藉由抑制胰脂肪酶的酵素活性而保留了抑制脂肪累積及減肥的功效;或藉由調節血糖吸收並促進葡萄糖代謝而保留了降低血糖、胰島素及糖化血紅蛋白的功效。
- 如請求項1所述的組成物,其中該代謝症候群為肥胖症(obesity)或糖尿病(diabetes)。
- 如請求項1至5中任一項所述的組成物,其中該印度醋栗萃取物在酸性水解狀態下包含1至5mg/g的鞣花酸(ellagic acid)。
- 如請求項1至5中任一項所述的組成物,其中該印度醋栗萃取物在非酸性水解狀態下包含5至25mg/g的鞣花酸(ellagic acid)。
- 如請求項1至5中任一項所述的組成物,其中該大麥芽萃取物包含6至11mg/g的皂草苷(saponarin)。
- 如請求項1至5中任一項所述的組成物,其中該印度醋栗萃取物或該大麥芽萃取物是經由擠壓而得。
- 如請求項1至5中任一項所述的組成物,其中該印度醋栗萃取物或該大麥芽萃取物為一乾燥粉末的形式。
- 一種包括根據請求項1至5中任一項所述的組成物之醫藥組成物。
- 一種包括根據請求項1至5中任一項所述的組成物之食品組成物。
- 一種用於製備預防、改善或治療代謝症候群之組成物之方法,包括:以包括擠壓及乾燥製程從印度醋栗中取得一印度醋栗萃取物;以包括擠壓及乾燥製程從大麥芽中取得一大麥芽萃取物;以及以4:1至1:1的重量比混合該印度醋栗萃取物及該大麥芽萃取物以得到其複合物。
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