TW201906539A - 抗氧化膳食增補劑及其相關方法 - Google Patents
抗氧化膳食增補劑及其相關方法 Download PDFInfo
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- TW201906539A TW201906539A TW107138812A TW107138812A TW201906539A TW 201906539 A TW201906539 A TW 201906539A TW 107138812 A TW107138812 A TW 107138812A TW 107138812 A TW107138812 A TW 107138812A TW 201906539 A TW201906539 A TW 201906539A
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Abstract
一種包含薑黃、檞皮素及迷迭香或聖羅勒、山葵、及青花菜籽萃取物之均衡及預定比例混合物的增補劑，其刺激抗氧化反應元件(ARE)、醌還原酶、及/或誘發相關基因表現，例如，血紅素氧化酶-1(HMOX-1)表現。該成份的混合物，能夠被形成一種適合施加至一主體的膳食增補劑，或形成於該增補劑內。該膳食增補劑能夠加以調配，以致於薑黃、檞皮素及迷迭香係以預定比例1：3：5出現，或者聖羅勒、山葵、及青花菜籽萃取物以預定比例1：1：0.2出現。額外的成份能夠被包含在該增補劑中。該增補劑能夠加乘地影響被施加增補劑之主體之細胞內的天然抗氧化反應途徑。也提供相關的使用方法。
Description
本發明關於膳食增補劑及施加方法，尤其是包括特定比例之薑黃(turmeric)、檞皮素(quercetin)及迷迭香(rosemary)、或聖羅勒(holy basil)、山葵(wasabi)及青花菜籽(broccoli seed)萃取物混合物而能夠刺激抗氧化反應元件(Antioxidant Response Element，ARE)或醌還原酶(Quinone Reductase，QR)、及/或誘發相關基因表現，例如，血紅素氧化酶-1(heme oxygenase-1，HMOX-1)表現的膳食增補劑。
自由基在多種病態及疾病中扮演著重要的角色，例如，發炎、過敏反應、關節退化、關節炎、骨質疏鬆、心血管疾病、癌症及相似之類。具體地說，在一個例子中，一般相信自由基對於脂質產生破壞，經常造成低密度脂蛋白過氧化作用及啟動動脈硬化斑塊，這能夠導致心血管疾病及動脈粥狀硬化。在另一個例子中，自由對於蛋白質分子產生破壞，能夠造成組織結構改變及免疫失調，這能夠導致關節炎及連結組織損壞(改變皮膚外觀及功能)。因此，許多注意注 意力被集中在膳食增補劑及食物內添加抗氧化劑，以便清除在生化系統內的自由基。
許多組成物已被認證具有抗氧化能力。例如，來自薑黃的薑黃素(curcumin)，一種香料，已經顯示出體內及體外的抗氧化性及抗發炎性。某些研究集中在曝露牛主動脈內皮細胞於薑黃素內，指示出薑黃素在這類內皮細胞內係一種HMOX-1的強力誘發劑，這增加了血紅素加血酶的活性。Motterlinin R等人，薑黃素，一種抗氧化劑及抗發炎劑，誘發血紅素加血酶-1及保護內皮細胞對抗氧化壓力。Free Radic Biol Med.，4月15日；28(8)：1303-12(2000)。然而，這些研究並未掌控薑黃素的全部能力。
其他非相關性嘗試已加以進行，以便結合薑黃素及其他成份，例如檞皮素及迷迭香，以改善特定發炎相關疾病。例如Darland的美國專利第6,210,701號(該專利於此併入本文以供參考)，以約2：1：2的比例，結合了薑黃素、檞皮素及迷迭香，以便改善這類疾病。這個參考案也提供：在這個比例之下觀察到，以這個比例結合的成份的氧自由基吸收能力(ORAC)，相對於個體成份對於ORAC的能力，係有較佳的結果。然而，這個參考案並未思及ORAC結果與其他功能性(如ARE或HMOX-1表現)的關係，或對其有相似結果，甚至未意識到薑黃素對於這些表現的效果。事實上，美國農業部最近已經移除其大規模的ORAC資料庫，這反映出一個假設：ORAC活性對於樣品的實際健康 利益並無反應。因此，在這個領域內仍保留探討及改良的空間。
一種增補劑及相關方法，提供一種配方，其包含均衡而預定比例之薑黃、檞皮素及迷迭香的組合物或混合物，或包含預定比例的聖羅勒、山葵及青花菜籽萃取物之組合物或混合物。該增補劑刺激抗氧化反應元件(ARE)、醌還原酶(QR)及/或誘發相關基因表現，例如，血紅素氧化酶-1(HMOX-1)表現。這種成份的配方或混合物，能夠加乘地影響被施加該增補劑之主體細胞內的天然抗氧化反應途徑。
在一實施例中，該配方係以易於施加至主體之錠劑、膠囊內膳食增補劑作為形式或其他形式。該增補劑能夠包含薑黃、檞皮素及迷迭香的混合物，可選擇地連同其他成份。這種膳食增補劑能夠加以調配，以致於薑黃、檞皮素及迷迭香係以預定比例1：3：5出現於該增補劑內。
在另一實施例中，包含薑黃、檞皮素及迷迭香之該膳食增補劑，係能夠被施加至細胞或主體內。該施加之結果係：該混合物能夠刺激細胞或主體內的ARE。ARE係一種DNA序列，其係Nrf2轉錄因子的結合位址。對抗氧化壓力之細胞防衛的主要機制，係Nrf2 ARE發信途徑的活化作用。這種途徑，尤其是Nrf2，控制了基因表現，其蛋白質產物，透 過反應及藉由增強細胞抗氧化能力，係涉及反應性氧化物種之損害及還原。因此，該增補劑能夠降低氧化壓力及/或對抗反應性氧化物種。
在另一實施例中，該薑黃、檞皮素及迷迭香混合物的施加，能夠誘發基因表現，例如HMOX-1表現。接著，具有這類表現的細胞能被被誘發，進而提供一種保護酶的抗氧化電池，例如，HMOX-1。
在另一實施例中，提供一種增補人類膳食的方法，該方法包括：一種增補劑，其包含薑黃、檞皮素及迷迭香混合物，可選擇性地連同其他成份；及施加該增補劑至人類，以便增補人類的膳食。可選擇地，該人類可能缺乏抗氧化劑，需要抗氧化增補劑。又可選擇地，該人類可能正在經歷氧化壓力或曝露於反應性氧化物種之下。
本發明提供一種配方，其具有加乘比例的薑黃、檞皮素及迷迭香混合物，可選擇地連同其他成份，以便刺激ARE及/或誘發HMOX-1表現，接著，能夠降低氧化壓力及/或對抗反應性氧化物種。又，該增補劑能夠改良抗氧化劑及營養狀態，減低自由基損害，及能夠用來清除其所附帶的疾病及病態。
在一實施例中，該薑黃、檞皮素及迷迭香混合物係以足夠的比例出現，以便降低人體8-異前列腺素尿濃度(urinary level of 8-isoprostane)及過氧化氫酶血清濃度(blood serum level of catalase)。
在一實施例中，該薑黃、檞皮素及迷迭香混合物係以足夠的比例出現，以便增加人體內麩胺基硫過氧化酶(glutathione peroxidase)及過氧化物歧化酶(Superoxide dismutase)之一者的產生。
在一實施例中，該配方係錠劑、膠囊作為形式的膳食增補劑或其他易於施加至主體形式。該增補劑能夠為聖羅勒、山葵及青花菜籽萃取物的混合物，可選擇地連同其他成份。該配方係加以製備，以致於該聖羅勒、山葵及青花菜籽萃取物混合物在增補劑內係預定比例1：1：0.2。
在另一實施例中，一種可供包含在膳食增補劑內的混合物，可以包含聖羅勒、山葵及青花菜籽萃取物，其中當該增補劑被施加至一主體時，該聖羅勒、山葵及青花菜籽萃取物在該增補劑內的含量及比例，係足夠至少刺激醌還原酶活性及誘發HMOX-1表現之一者。
在一實施例中，該聖羅勒、山葵及青花菜籽萃取物的混合物係足夠的比例，以便降低人體8-異前列腺素尿濃度。
在一實施例中，該聖羅勒、山葵及青花菜籽萃取物的混合物係足夠的比例，以便降低人體過氧化氫酶血清濃度。
在另一實施例中，提供一種增補人類膳食的方 法，該方法包括：該方法包括：提供一種膳食增補劑，其包含聖羅勒、山葵及青花菜籽萃取物，其中該聖羅勒、山葵及青花菜籽萃取物在該增補劑中係以1：1：0.2之比例加以出現；及口服該膳食增補劑至人體，以致於刺激醌還原酶活性及誘發人類細包HMOX-1表現之一者。
本發明其他目的、優點及特色，在參照現有實施例及圖式之描述下，將會更加完全地被瞭解及讚同。
在本發明實施例詳細解說之前，要瞭解的是，本發明並不局限於以下描述所敍述或圖式所圖解之詳細操作、或詳細結構及元件排列。本發明可由許多其他實施例加以實施及執行，或以未在本文中明確揭示之可替換方式加以施行。又，應該瞭解的是，在本文中所使用之片語及專業用語，係為了描述之目的而提供，而不應被視為一種限制。”包含”及”包括”及其變型之使用，表示涵蓋該詞之後所列出的品項及其等價者，以及額外的品項及其等價者。又，數字編號可用來描述許多實施例的描述。除非另有明確聲明，數字編號之使用不應被解釋本發明被限制於任何具體元件排列順序或數目。使用數字編號也不得解釋為：任何可能被結合至編號步驟或元件之額外的步驟及元件，係被排除於本發明範圍。
第一圖係一圖表，顯示在不同濃度下的ARE評估中，薑黃、檞皮素及迷迭香混合物的加乘效果相對於所預期 的混合物添加效果。
第二圖係一圖表，顯示在ARE評估中，薑黃、檞皮素及迷迭香混合物的加乘效果相對於所預期的混合物添加效果以及薑黃、檞皮素及迷迭香個體成份的效果。
第三圖係一圖表，顯示在各種比例下ARE評估中，薑黃、檞皮素及迷迭香混合物的加乘效果。
第四圖係一圖表，顯示在各種濃度下ARE評估中，薑黃、檞皮素及迷迭香混合物的加乘效果，以及個別成份的效果。
第五圖係一圖表，顯示薑黃、檞皮素及迷迭香混合物的EC50值。
第六圖係一圖表，顯示在1：3：5比例下薑黃、檞皮素及迷迭香混合物，針對HMOX-1基因表現的加乘效果，以及個體成份的效果。
第七圖係一圖表，顯示具有50μg不同比例之聖羅勒、山葵及青花菜籽混合物的效果。
第八圖係一圖表，顯示1：1：0.2比例之聖羅勒、山葵及青花菜籽混合物的醌還原酶(QR)劑量依賴性活化作用。
第九圖係一圖表，顯示聖羅勒、山葵及青花菜籽混合物針對於HMOX-1基因表現的效果。
第十圖係用於臨床研究的時間線圖。
本發明提供一種包含有薑黃、檞皮素及迷迭香組合物或混合物的配方。該薑黃、檞皮素及迷迭香能夠以1：3：5之比例，出現在該混合物中，及通常該配方中。可替換地，該配方可以包含1：1：0.2比例的聖羅勒、山葵、青花菜籽萃取物。該配方本身能夠合併到膳食增補劑內，適合以多種傳輸載體(詳如下述)施加至一主體，例如，人類或其他哺乳動物。該混合物基本形式也能夠合併到一傳輸載體，例如溶液或濃縮液，適合用於活體試驗或應用。
該薑黃、檞皮素及迷迭香能夠從多種來源取得。例如，薑黃能夠從薑黃(Curcuma longa)加以取得、萃取或其他方式獲得，其為一種薑科(ginger family,Zingiberaceae)的有根莖草本多年生植物。在某些情況下，其他薑科植物可取代薑黃。薑黃能夠以多種含量出現於該配方及/或混合物中。例如，薑黃能夠以下述底限的配方或膳食增補劑的重量百分比加以出現：1%、2%、3%、4%或5%，及相對應的上限係如下述：12%、15%、17%或20%。如另一個例子，該薑黃能夠以毫克重量範圍出現於該配方或膳食增補劑內，其底限如下：10mg、20mg、30mg、40mg或50mg，及相對應的上限如下：130mg、140mg、150mg、160mg或170mg。
檞皮素能夠從多種水果、蔬菜及草加以取得、萃取或以其他方式取得，例如：柑橘類(citrus fruits)、蘋果(例 如Malus domestica)、洋葱(例如但不限於：Allium cepa)、洋芫荽(parsley)、鼠尾草(sage)、茶(tea)、藍莓(blueberries)、黑莓(blackberries)、山桑(bilberries)、巴西芸香(Fava d’anta)(例如，Dimorphandra mollis)及槐樹(Sophora japonica)。檞皮素能夠以多種含量出現於該配方及/或混合物中。例如，檞皮素能夠以下述底限的配方或膳食增補劑的重量百分比加以出現：3%、4%或5%，及相對應的上限係如下述：33%、35%或40%。如另一個例子，該檞皮素能夠以毫克重量範圍出現於該配方或膳食增補劑內，其底限如下：20mg、30mg、40mg或50mg，及相對應的上限如下：440mg、450mg、460mg或470mg。
迷迭香能夠從迷迭香(Rosmarinus officinalis)加以取得、萃取或以其他方式獲得，其係一種薄荷科(mint family,Lamiaceae)草本多年生植物。其他薄荷科植物在某些情況下係可取代迷迭香。迷迭香能夠以多種含量出現於該配方及/或混合物中。例如，迷迭香能夠以下述底限的配方或膳食增補劑的重量百分比加以出現：4%、5%、6%或7%，及相對應的上限係如下述：45%、50%、55%或60%。如另一個例子，該檞皮素能夠以毫克重量範圍出現於該配方或膳食增補劑內，其底限如下：45mg、50mg、55mg、40mg或60mg，及相對應的上限如下：740mg、750mg、760mg或770mg。
聖羅勒、山葵及青花菜籽萃取物也能夠從多種來 源加以取得。聖羅勒能夠從薄荷科的神羅勒(Ocimum sanctum)植物(又稱為Tulsi或tulas)加以取得、萃取或以其他形式加以獲得。其他的羅勒，例如(但不限於)：甜羅勒(sweet basil,Ocimum basilicum)在某些情況下係得以取代聖羅勒。聖羅勒可由許多來源取得，其包含印第安那州Noblesville市的Verdure Sciences公司。
山葵材料可以從山葵加以取得，其為一種純粹的根莖乾燥粉末。山葵係十字花科(Brassicaceae family)一個成員，該科包含甘藍類(cabbages)、辣根(horseradish)、芥末(mustard)。其他來自十字花科的植物材料，在某些情況得以取代山葵(Wasabia japonica)。山葵粉末可以從許多來源加以取得，其包含加州Vista市的B&D Nutritional Ingredients公司。
青花菜(Brassica oleracea italica)萃取物，或青花菜籽萃取物，係取自於十字花科的植物。該青花菜籽萃取物係加以標準化成為13%的蘿蔔硫苷(glucoraphanin，其為一種硫化葡萄糖苷(glucosinolate)；及磺胺胡蘿蔔素前質(precursor of sulforaphane)，其為一種異硫氰酸鹽(isothiocyanate)。青花菜係一種物種，其包含甘藍、青花菜、花椰菜(cauliflower)、羽衣甘藍(kale)、芽球甘藍(Brussel sprouts)、皺葉甘藍(savoy)、芥藍(Chinese kale)。在某些情況下，其他來自十字花科的植物材料，也可以取代青花菜籽。青花菜籽萃取物可以從加州Vista市的B&D Nutritional Ingredients公司加以取 得。
該混合物能夠以多種方式混合在該配方中。例如，在該配方包含下述其他成份的場合中，該混合物能夠直接與這些其他成份加以混合，通常是以其任何可能的物理形式來均勻地混合於整個配方中。在某些時候，該混合物當然能夠先預混，及添加到其他成份內，以產生該配方。若想要的話，該混合物成份能夠在其他成份內的微膠囊、塗層、或其他結構中加以黏聚。
如前文所述，該配方能夠被包含在膳食增補劑內。該增補劑能夠為錠劑、粉末、膠體或液體形式(錠劑，基於本發明的緣故及在本申請案揭露內容全文中，係指任何形式的固態口服劑量，其包含但不限於：錠劑、小囊、膠囊、粉末等等)。該增補劑能夠被調配成粉末，以利與可食用的液體加以混合，如牛乳、果汁、水，或可食用的膠體或糖漿加以混合，而可供混合成為其他膳食液或食物。該增補劑也能夠與其他食物或液體一同調配，以便提供預測量的增補食品，例如，單次進食的棒狀體。取決於其應用，加味劑、賦形劑、黏結劑、蛋白質、綜合碳水化合物、防腐劑、螫合劑及相似之類者，也能夠添加。
當實施成為膳食增補劑時，該配方能夠以一或更多錠劑來施加，每次兩次。當然若想要的話，該膳食增補劑能夠以其他形式及所想要的劑量來施加。
該膳食增補劑能夠使用任何其他成份的藥學可接受形式加以調配，例如，濃縮物、植物性化合物、維生素、礦物質及其他營養素，包含鹽類及其衍生物。例如，適合使用於該配方及增補劑內的維生素包含維生素A、維生素B1、維生素B2、維生素B6、維生素B12、菸鹼酸/菸鹼醯胺、泛酸、葉酸、生物素、膽鹼、維生素C、維生素D及維生素E，可選擇地衍生自植物及其他天然來源。除了上列的維生素之外，可使用於該配方及增補劑內的礦物質能夠包含硼、鈣、鉻、銅、碘、鎂、錳、鉬、鉀、硒、釩、鋅。其他的維生素及礦物質也能夠加以使用。
可選擇地，該配方，尤其是本文所述的混合物，能夠施加至：已被識別為正在或易於缺乏抗氧化劑及相關營養素的、易於或正在經歷氧化壓力的、已遭受到反應性氧化物種的、及/或易於或證實為具有疾病及相關病態的人類、主體或生物系統。又可選擇地，主體能夠進行環境試驗，以便評估該主體是否從該所需配方的施加取得利益，或該配方是否能夠簡單地作為規則增補劑飲食來施加。
本實施例的配方係說明(但不欲加以限制)如下：
包含有薑黃、檞皮素及迷迭香混合物(TQR混合物，在本文中也稱為混合物A)的配方，進行試驗以便評估該混合物的ARE。具體地說，該三種個別的薑黃、檞皮素及迷 迭香成份係自身或組合成混合物來進行試驗。在試驗中，使用習知的用於測定ARE測定的檢定法。一般而言，該檢定包含：穩定地以一載體來傳輸的人類肝細胞株(HepG2)，購於維吉尼亞州Manassas市的ATCC公司。該載體含有四個位在最小啟動子(minimal promoter)上游的重複的ARE DNA序列(5’-GTGACTCAGCA-3’(SEQ ID NO：1))，其使用螢烏賊螢光酵素基因(firefly luciferase gene)作為報告基因(reporter)。然後，這些細胞以個別成份的薑黃、檞皮素及迷迭香、以及各種濃度的TQR混合物加以處理48小時(在本例子中及下述其他例子中)。人體肝細胞加以溶解，添加螢光酵素基材(購自加州Hayward市的Biotium公司)，對於全部的樣品取得相關的發光值。結果顯示成為a%反應vs.控制組。該控制物質係10μM的磺胺胡蘿蔔素。
一般而言，該結果指出：組合的TQR混合物(混合物A)處理，明顯地誘發較高的ARE活性(相較於個別成份的所預期累加活性)，甚至在不同濃度下亦然。例如，如第一圖所示，所預期的累加反應係藉由每一個成份單獨試驗時的%反應來總合計算。若無加乘效應發生，實際混合物的%反應，據估計應該大約相等於累加%反應。然而，在兩種試驗濃度的混合物處理中，亦即，15μg、50μg，吾人觀察到：比預期者高出3~5倍的反應。結果，吾人結論為：三種成份，薑黃、檞皮素及迷迭香，加乘地運作來活化TQR混合物內的ARE。
如第二圖所示，薑黃、檞皮素及迷迭香三種成份 之每一者、以及TQR混合物組合，係分別地在ARE檢定中加以試驗。在考慮到重量百分率之下，50μg/ml濃度下的實際TQR混合物反應明顯地較高，大約高出50μg/ml濃度下所預期累加效應%(簡單地由薑黃、檞皮素及迷迭香%反應的累加計算)反應之2.5倍。這再次證實TQR混合物對於ARE有加乘效應。
包含薑黃、檞皮素及迷迭香混合物的配方(即TQR混合物、混合物A)，係加以試驗，以便驗證及發現該成份的加乘比例。具體地說，多種薑黃：檞皮素：迷迭香比例係在例一的ARE檢定中加以試驗，以便測定最大加乘組合。全部的混合物都在50μg/ml濃度下進行試驗。如第三圖所示，吾人發現：被試驗之TQR混合物的最大加乘比例(most synergistic ratio)，包含1：3：5的薑黃：檞皮素：迷迭香比例。這是非預期性的，因為先前顯示的是：在比例中有較高濃度薑黃，應該改良全部的加乘效果，但基於某些原因，較低相對比例的薑黃增強了該加乘效果，可能因為薑黃對於以高於10~20μg/ml濃度來培養中細胞有毒性。
包含薑黃、檞皮素及迷迭香混合物(亦即TQR混合物，混合物A)的配方，進一步地加以試驗，以評估及驗證該成份在不同濃度下的加乘1：3：5比例。具體地說，如第四圖所示，對於各種濃度(μg/ml)的每一個別的薑黃、檞皮素及迷 迭香成份，在ARE檢定中進行試驗，並與不同TQR濃度下1：3：5比例之相對應濃度(μg/ml)的TQR混合物進行比較。依照該試驗之結果，吾人觀察到：當TQR混合物在薑黃、檞皮素及迷迭香1：3：5比例下濃度為50μg/ml時，該TQR混合物在ARE檢定中的%反應，至少高出薑黃、檞皮素及迷迭香個別任一者在所述濃度下之相對應%反應的1、2、3、4、5、6、7或8倍。例如，檞皮素展示出約50%的反應，但TQR混合物展示出約375%的反應。
也被觀察到的是，當TQR混合物在薑黃、檞皮素及迷迭香1：3：5比例下濃度為60μg/ml時，該TQR混合物在ARE檢定中的%反應，至少高出薑黃、檞皮素及迷迭香個別任一者在所述濃度下之相對應%反應的1、2、3、4、5、6或7倍。例如，檞皮素展示出約75%的反應，但TQR混合物展示出約525%的反應。
又被觀察到的是，當TQR混合物在薑黃、檞皮素及迷迭香1：3：5比例下濃度為70μg/ml時，該TQR混合物在ARE檢定中的%反應，至少高出薑黃、檞皮素及迷迭香個別任一者在所述濃度下之相對應%反應的1、2、3、4、5、6、7或7.5倍。例如，檞皮素展示出約80%的反應，但TQR混合物展示出約600%的反應。
進一步地觀察到，當TQR混合物在薑黃、檞皮素及迷迭香1：3：5比例下濃度為80μg/ml時，該TQR混合物在 ARE檢定中的%反應，至少高出薑黃、檞皮素及迷迭香個別任一者在所述濃度下之相對應%反應的1、2、3、4、5、6或7倍。例如，檞皮素展示出約90%的反應，但TQR混合物展示出約525%的反應。
進一步的試驗顯示：TQR混合物以劑量依賴方式來活化ARE。這說明於第五圖的EC50值的圖表。
為了進一步地試驗上述例子所展示出的加乘效果，在第二檢定中，對於薑黃、檞皮素及迷迭香的個別的、以及1：3：5比例之TQR混合物(混合物A)的血紅素氧化酶-1基因表現，進行評估。一般而言，在第二檢定中，以薑黃(1.3μg/ml)、檞皮素(3.9μg/ml)及迷迭香(6.5μg/ml)分別地、及以TQR混合物(15μg/ml)，對於人類乳癌MCF-7進行處理一整夜。在第二天，從被處理的細胞中隔離出mRNA，並執行2步驟的RT-qPCR。在第六圖示出的結果證明：各種成份的每一者誘發出的這些細胞HMOX-1表現，增加了三倍。如果全部的成份被添加在一起，累加的效果可能被預期為約11.9倍。但是，TQR混合物的實際反應係增加了50.9倍。基於此，吾人估算：TQR混合物的反應，高出薑黃、檞皮素及迷迭香的HMOX-1表現反應累加效果之至少1、2、3、4或5倍。
試驗包含有聖羅勒、山葵及青花菜籽混合物(混 合物B)的配方，以便評估其降低醌還原酶(QR)酶(相位II)的能力。QR係一種酶，其活性藉由上調ARE的相同訊號來上調。具體地說，試驗這種材料本身、以及組合成為各種聖羅勒：山葵：青花菜籽萃取物比例，2：1：0.1，2：1：0.2，1：1：0.1，3：3：0.6，1：1：0.6，1：1：0.5及1：2：0.5。在該試驗中，使用用於測定QR活性的習用檢定法。一般而言，該檢定包含購於維吉尼亞州Manassas市的ATCC公司的老鼠肝細胞株Hepa1c1c7。然後，在本例中，該細胞以個別的聖羅勒、山葵及青花菜籽材料加以處理，及以各種比例的該混合物加以處理48小時。老鼠肝細胞係加以溶解，及放置在含有葡糖6磷酸鹽(Glucose 6 phosphate)、黃素腺嘌呤雙核苷酸(Flavin adenine dinucleotide，”FAD”)、菸鹼醯腺嘌呤雙核苷酸(Nicotinamide adenine dinucleotide phosphate，”NADP”)、葡糖6磷酸鹽去氫酶(Glucose 6 phosphate dehydrogenase)、MTT及維生素K3(menadione)的QR基材上，在室溫下培育3~4分鐘，以利發展出紫色(QR活性的指標)。QR活性係正比於610nM下的吸收值。混合物比例的結果在第七圖中係以%反應vs.控制組來表示。控制物質係1M的磺胺胡蘿蔔素。
聖羅勒：山葵：青花菜籽2：1：0.1比例係加以選取用於臨床試驗，詳如下述。進行這個比例下醌還酶活化作用的獨立研究，及其結果示於第八圖。
混合物B(聖羅勒、山葵及青花菜籽混合物)對於 抗氧化基因表現的效果係示於第九圖。如第九圖所示，濃度5μg聖羅勒、5μg山葵及5μg青花菜籽萃取物係加以組合。執行RT-qPCR。依照例四相關敘述的相同方法，每一樣品分別降低約1.5倍的HMOX，但是，其反應係約全部三者出現的2倍。
分別以兩種劑量(300mg及600mg)製備四種不同植物基底的混合物(混合物A、B、C及D)。征召六十個(60)健康人士來參與本研究。參加人士隨意分成四組，所以15個參與者分別派發四種混合物之一者。被選擇來服用混合物A及B之參與者的結果，將於下文進一步地討論。被選擇來服用混合物C及D之參與者的結果係無關於本發明，因為混合物C及D的含有異於混合物A及B的混合材料。
假使老化被考量為氧化壓力模型，則相對較老的次族群被選取為參與者，其年齡介於45~65歲。各種種族背景並未加以考慮。然而，位在加州Buena Park市Southbay Pharma研究中心的參與者候選人族群，包括大約40%白人、20%西班牙語裔、30%亞洲/大洋洲裔、及10%其他人。將女性排除，避免對於結果的荷爾蒙效應。參與者係加以挑選，排除慢性病患或重大健康疾病。進一步地，參與者候選人實行被推蔫的食物檢查表(RFC)及62-條食物習慣問答(FFQ)，以便決定它們在目前膳食指南中的個別的食物消耗水準。選擇顯示出低水果及蔬菜攝取的參與者，進一步地篩選。
某些醫藥及食物可能改變氧化壓力生物標記的水準，這起因它們活化或抑制Nrf2/ARE途徑的能力。為了避免來自於普通醫藥及食物的潛在干擾，例如錯誤陽性結果，參與者在血液及尿液取樣前至少10小時，不允許進食以下的品項： 1.他丁類(statins，例如，辛伐他丁、氟伐他丁)，因為它們可能活化或抑制Nrf2/ARE途徑，這將會影響ARE受體；2.NSAIDS，包含阿斯匹靈；3.一氧化氮(eNOS)活化劑，包含他丁；4.司來吉蘭(Selegiline)，因為其可能活化ARE；5.PDE5抑制劑，例如威而鋼®，因為其可能活化ARE(經證實上調HMOX-1及增加血管NO)；6.血管收縮素II受體阻斷劑替米沙坦(Telmisartan)(用於治療血壓)；及7.辣椒素(辛辣食物)、咖啡、茶及花草茶，因為它們可能活化ARE途徑。
收集基本健康及醫藥資訊之後，參與者在禁食一夜(至少10小時)之後，收集血液及尿液來測定氧化壓力生物標記及基因表現的基線水準。在起始樣品被收集之後，參與者就被分發到群A或B之一者。總結每一群的時間研究之圖表係示於第十圖。A群接受低劑量的混合物A十四日，然後高劑量 的混合物B十四日，接著十四日為高劑量混合物A加上維生素、礦物質及色素混合物。群B接收低劑量混合物B14天。在14天低劑量相位之後，群B接收14天的高劑量混合物B供應。在14天期間完成之後而在進行高劑量或添加增補劑之前(假設有任一者跟隨)，大約間隔14天來收集經禁食的血液及尿液樣品。關於高及低劑量混合物A及B的進一步細目，係如下述表一。
R/Q/T=迷迭香/檞皮素/薑黃
HB/W/BS=聖羅勒/山葵/青花菜籽
從收集的血液及/或尿液樣品，進行六個生物標記及/或基因表現水準的分析，以便測定每一種植物基料混合物針對刺激ARE及/或誘發相關基因表現的效率。被分析的生物標記/基因表現的指標為：鯨蠟二醛(melodialdehye)、異前列腺素、過氧化物歧化酶、麩胺基硫過氧化物、血紅素氧化酶-1及過氧化氫酶。
由色度法及光譜法所測量的鯨蠟二醛(“MDA”)係加以分析。MDA可能是反應性氧物種(reactive oxygen species，”ROS”)所產生之聚不飽和脂質的降解產物，因此其為氧化壓力狀態的生物標記。MDA分析之後進行下述方法：製備數種溶液，包含：TMOP=20mM PBS緩衝劑內10mM(確實的TMOP濃度係由以下測定：先測量在滅絕係數(extinction coefficient)156,000M‧cm-1之下與TBARS反應之後的532nm吸收物質，典型地不超過重量的6%)。使用TMOP，以PBS稀釋成為0.0，0.2，0.4及0.6μg的標準溶液。
BHT=溶解0.5克BHT於100mL MeOH(0.0227M)。
TBA=溶解0.5克TBA於~80mL超純水。若殘餘存留，則劇烈搖晃及加熱至~50℃。若加熱則冷卻至室溫，以超純水稀釋至100.0mL(0.5%)。
TCA，溶解70g TCA於超純水中，以超純水稀釋至總容積100mL(70%)。部份的70%TCA稀釋液以超純水稀釋 至10%。
MDA分析的製備也包含：對於待測樣品標記為一式三份的清潔的耐煮沸微管(clean boil-proof microtubes)，每一份針對於一個標準。將VWR阻斷乾燥器打開，溫度設定60℃。在本程序中，標記為耐煮沸微管、15.0μL的BHT溶液，被添加到試管底部。按照順序來添加270μL的TMOP標準或血漿樣品、15.0μL的10% TCA、300.0μL的TBA溶液。在全部的標準品及樣品被處理之後，試管同時被安放在VWR阻斷加熱器，在60℃下培育90分鐘。冷卻後，添加300.0μL的70% TBA、然後900.0mL的氯仿及劇烈搖晃樣品，然後，在13,000g下離心3分鐘。將上方的水層傳輸到半微光析槽(semi-micro cuvet)，以430~630nm掃瞄來測量UV~Vis光譜。
將UV~Vis光譜轉換成為3rd衍生光譜(3rd。針對標準品及樣讀取在542nm下的d3A/dx3值。從標準品來建構標準曲線，及TBARS(MDA)濃度係利用線性回歸公式來測定。
異前列腺素係藉由自由基催化機制來產生，其係花生油酸過氧化作用的似前列腺素產物。在考量其自由基獨立成型之下，異前列腺素-8異PGF2α係一種生物標記，其可能反應全體氧化壓力狀態。
異前列腺素-8異PGF2α(Isoprostane 8-iso PGF2α)係藉由尿液異前列腺素ELISA套組(Urinary Isoprostane ELISA Kit)來分析，其購自密西根州牛津市的 Oxford Biomedical研究中心，產品編號為EA85。在使用ELISA套組之前，尿液樣品針對肌酸酐(creatinine)加以正規化，允許合理的樣品對樣品之比較。肌酸酐尿液濃度係由以下的程序來測量：
製備以下三種溶液：
苦味酸(picric acid)=在真空下，乾燥含水份-濕氣的苦味酸2小時。將9.156克苦味酸溶解在1.0L的超純水中(如果有殘餘物則加以過濾，0.04M)。儲放在棕色瓶子內，或以鋁箔紙包覆瓶子，安放在黑暗環境中。
NaOH=溶解30克NaOH於1.0L超純水(0.75N)。
肌酸酐=溶解0.2645克肌酸酐‧HCl於10.0mL 1N HCl及90.0mL超純水(2.0mg/mL肌酸酐)。以99.0mL超純水來稀釋1.0mL的2.0mg/mL肌酸酐(0.020mg/mL肌酸酐)。儲放在冰箱中。稀釋成0.0mg/mL、0.005mg/mL、0.010mg/mL、0.015mg/mL、0.020mg/mL肌酸酐標準物。將該標準物分開成為小容積，及分別放入小瓶儲放在冰箱中。
進一步的製備包含：標記為一式三份的清潔的半微光析槽(每一樣品一式兩份)。每一試管添加1485.0μL超純水，每一試管內放入15.0μL樣品，加蓋及在尿液樣品1/100X稀釋液下混合該溶液。如果樣品的520nm吸收係超出計量範圍，則調整稀釋因子，重複該檢定。本程序包含：添加1.5mL超純水(三份，試劑空白)至半微光析槽、或1.5mL 1/100X尿 液樣品、或1.5mL標準溶液。然後，0.5mL 0.75N的NaOH被添加到每一光析槽中，接著，添加0.5mL 0.04M的苦味酸，然後將樣品混合均勻並在室溫下培育30分鐘。測量每一標準物及樣品在520nm下的UV-Vis吸收。藉由讀取針對標準品及樣品在520nm之吸收、減去試劑空白之吸收，來計算結果。從標準品來建構標準曲線，樣品內肌酸酐濃度係藉由線性回歸公式來計算。然後，應用稀釋因子(100或經調整值)。
過氧化物分歧酶(“SODs”)係抗氧化酶，其對於曝露在氧之下的細胞係相當重要。SOD將損壞的過氧化物加以轉換，成為較無害的氧氣及過氧化氫。SOD係由加州聖地牙哥市之eBioscience公司購得的人類Cu/ZnSOD白金ELISA BMS222/BMS222TEN來測量。
麩胺基硫過氧化物(“GSH-Px”)係一種酶，其保護器官免於過氧化物的氧化破壞。更具體地說，其將脂質氫過氧化物、自由氫過氧化物加以還原，成為較無害的化合物，例如，乙醇或水。這種酶係由奧勒岡州波特蘭市的Oxis研究中心的Bioxytech® GPx-340TM色度檢定(產品編號21017)，來加以測量。
血紅素氧化酶(“HMOX-1”)係一種必要酶，其催化血紅素，避免其造成氧化壓力。這種酶將血紅素分開，形成較無害的膽綠素。HMOX-1係藉由使用賓州Plymouth Meeting市Enzo Life Sciences國際公司之HO-1(人類)、EIA套 組(產品編號ADI EKS-800)來測定。
最近，過氧化氫酶係一種對於曝露在氧之下的細胞相當重的酶，因為其保護細胞，避免由ROS所造成的氧化損害。過氧化氫酶催化反應性氫過氫化物，成為無害的水及氧氣。依照可購自於麻州劍橋市之Abcam公司之Catalase Human ELISA套組，來分析過氧化氫酶。
含有5：3：1迷迭香、檞皮素及薑黃之混合物A，以兩種有序的劑量濃度(300mg/日、600mg/日)，被提供給予群A。在收集基線血液及尿液樣品之後，15個參與者給予低劑量樣品增補劑，每一者含有150mg混合物A。群A參與者給予指示：服用該增補劑每日兩次，及推蔫他們與早餐一起食用每日劑量的第一部份，及與晚餐一起食用每日劑量的第二部份，以便服用總量為300mg/日的混合物A14日。在第15天，在整夜禁食後(至少10小時)，收集血液及尿液樣品。然後，給予相同的參與者14天的高劑量增補劑，每一者含有300mg混合物A，及推蔫他們從第15日起，食用這種增補劑，每日兩次，與早餐一同食用每日劑量的第一部份，及與晚餐一起食用每日劑量的第二部份，以便服用總量為600mg/日的混合物A 14日。
在第29天，在整夜禁食後(至少10小時)，再次收集血液及尿液樣品。再次給予群A參與者14天的高劑量增補 劑，預先提醒他們每日食用2次。此外，群A參與者給予含有下表成份之維生素、礦物質及色素混合物。群A參與者持續食用該高劑量增補劑，連同維生素、礦物質及色素混合物增補劑，14天。大約在第43天時，在整夜禁食後(至少10小時)，再次收集群A參與者的血液及尿液樣品。
*最近美國植物營養報告：鑑定該缺陷指出：平均10個美國人 中有8個(76%)係有”植物營養缺陷”。該”缺陷”代表，在典型植物營養攝取基礎下的植物營養攝取缺點，其符合被認為是審慎數量之水果及蔬菜的膳食。混合物”有色”部份的合理性，在於藉由提供5種顏色範疇(綠、紅、白、紫/藍、黃/橘)的水果及蔬菜，來減輕這種缺陷。
含有5：5：1比例之聖羅勒、山葵及青花菜籽的混合物B，以兩種有次序的劑量水準(300mg/日、600mg/日)，被提供給予群B。在收集基線血液及尿液樣品之後，15個參與者給予低劑量樣品增補劑，每一者含有150mg混合物B。群B參與者給予指示：服用該增補劑每日兩次，及推蔫他們與早餐一起食用每日劑量的第一部份，及與晚餐一起食用每日劑量的第二部份，以便服用總量為300mg/日的混合物B 14日。在第15天，在整夜禁食後(至少10小時)，收集血液及尿液樣品。然後，給予相同的參與者14天的高劑量增補劑，每一者含有300mg混合物B，及推蔫他們從第15日起，食用這種增補劑，每日兩次，與早餐一同食用每日劑量的第一部份，及與晚餐一起食用每日劑量的第二部份，以便服用總量為600mg/日的混合物B 14日。在第29日，在整夜禁食(至少10小時)之後，再次收集血液及尿液樣品。
從參與者所收集之樣品，在生物標記/基因表現 水準方面所偵測到的差異，係由基線測量來計算。從基線到服用14天低劑量增補劑所得樣品之間的改變，係指定為”L-BL”。低劑量相位後所得樣品、及服用高劑量增補劑14天後所得樣品之間的差異，係指定為”H-L”。就群A而言，藉由測定樣品在高劑量服用14天期間之後、及連同維生素、礦物質及色素混合混之高劑量增補劑服用14天期間之後的差異，來測量另一差別。這種差別指定為”C-H”。
計算差別，及使用配對t-試驗來比較基線及低劑量(L-BL)、低劑量及高劑量(H-L)、組合配方及高劑量(C-H)(針對群A)之間的生物標記差異。又，氧化壓力及生物標記之間的相關性係加以評估，及估計單變量統計(p-值、加熱圖)。使用統計分析軟體JMP(SAS機構)來分析資料。具最有利級寬及效果之處理，係以灰色亮點標示。
因為MDA係藉由色度及光譜方法加以測量，故相關性不良，並不進一步考慮。
分析尿液樣品從群A參與者取得的差別L-BL指示出8-異前腺素濃度有統計上顯著下降，這是有利的效果。從低劑量(300mg/日)增加劑量至高劑量(600mg/日)，顯示出8-異前列腺素增加(H-L=1.287)，這指示出：系統內氧化壓力增加。有趣的是，高劑量混合物A及維生素、礦物質及色素混合物的組合，矯正了單獨攝食高劑量混合物A時所偵測之已增加的氧化壓力。這些有利的結果(C-H=-0.830，p=0.077)也有統計上的顯著性。
混合物增補劑在低劑量下顯示，在尿液8-異前列腺素中的8-異前列腺素下降(L-BL=-0.892ng/mL，p=0.077)，這是有利的效果，相對地，高劑量下並無尿液8-異前列腺素效果(H-L=0.008，p=0.160)。
利用這些樣品，混合物A及B顯示出針對於SOD的正反應。就混合物A而言，低劑量下(L-BL=0.007)，指示出酶產物係有利地增加。混合物A在高劑量下(H-L=-0.026)，SOD 下降，因此，並未反應於有利方向。高劑量混合物A及維生素、礦物質及色素混合物之組合，增加SOD產物，因此，矯正單獨高劑量下的不利驅勢(C-H=0.041，p=0.104)。低劑量下的混合物B增補劑，也未顯示出有利的SOD產物增加(L-BL=0.001，p=0.472)。
高劑量混合物A及維生素、礦物質及色素混合物之組合，在GSH-Px產物方面，表現最有利。C-H差別係0.216，連帶p值0.223。就低及高劑量混合物A，GSH-Px產物方面有輕微下降。
高劑量混合物A及維生素、礦物質及色素混合物之組合，顯示在誘發HMOX-1產物(C-H=0.093,p=0.0762)方面係有利的驅勢。低劑量混合物A顯示，HMOX-1產物輕微下降，當以高劑量來增補時，HMOX-1產物並無改變。低劑量混合物B的增補劑，也顯示這種酶有利地增加(L-BL=0.187,p=0.100)。
以混合物A來增補的主體，測量其過氧化氫酶活性。低劑量混合物A(L-BL)顯示，過氧化氫酶有更加優勢的下降，相較於高劑量(H-L)或組合劑量(C-H)，推定有較強的效應。
本說明書所引證的全部的專利、專利申請及參考文獻，均併入本文以供參考。如果有衝突產生，將以本說明書的描述(包含定義)來解釋。
上述係本發明目前實施例之描述。許多變動及改 變係能夠被完成而不離開本發明精神及較寬大範圍，其係被界定如後附申請專利範圍，而依照包含均等論之專利法原理來加以解釋。本揭示內容係基於說明目的而提供，不應被解釋成為全部本發明實施例之排他性描述，或限制申請專利範圍於以這些實施例相關內容來說明或描述之具體元件。例如(及未限制)，任何所述發明之個體元件係可由提供實質相似功能或提供足夠操作之可替換元件來取代。這包含(例如)目前已知之可替換元件、例如一般精於本項技藝人士已熟知者，及可能在未來加以開發之可替換元件，例如，一般於本項技藝人士在研發時，識別出的可替換者。進一步地，所揭示的實施例包含多數個特徵及/或元件，它們可能個別地或互助合作來提供優點加成。除在申請專利範圍內另有陳述之範圍以外，本發明並未限制只包含這些全部特徵、或提供全部所述優點之實施例。請求權利之元件的任何單數的參考符號，例如，使用冠詞”一”(a,an)或”該”(the,said)，並不解釋為限制該元件為單數。任何代號，如以”X、Y、Z至少一者”來請求時，意指包含個別X、Y或Z之任一者，及X、Y及Z之任意組合，例如，X、Y、Z；X、Y；X、Z；及Y、Z。
<110> 美商‧通路實業集團國際公司(ACCESS BUSINESS GROUP INTERNATIONAL LLC)
<120> 抗氧化膳食增補劑及其相關方法
<140> TW102142795
<141> 2013-11-25
<150> US 61/729,919
<151> 2012-11-26
<160> 1
<170> PatentIn Version 3.5
<210> 1
<211> 11
<212> DNA
<213> 人工序列
<220>
<223> 人工序列之說明：合成寡核苷酸
<400> 1
Claims (15)
- 一種膳食增補劑，其包括：聖羅勒，山葵；及青花菜籽萃取物，其中該聖羅勒、山葵及青花菜籽萃取物在該增補劑內的比例分別為1：1：0.2，且其中當該聖羅勒、山葵及青花菜籽萃取物被施加至一主體時，協同作用至少刺激醌還原酶產生與誘發血紅素氧化酶-1(HMOX-1)表現其中之一者。
- 如申請專利範圍第1項的膳食增補劑，其中該聖羅勒、山葵及青花菜籽萃取物在該增補劑內的含量，分別為136mg、136mg與28mg。
- 如申請專利範圍第1項的膳食增補劑，其中該聖羅勒、山葵及青花菜籽萃取物在該增補劑內的含量，分別為273mg、273mg與54mg。
- 如申請專利範圍第1項的膳食增補劑，其中該聖羅勒係至少以取得與萃取其中之一者獲自神羅勒 (Ocimum sanctum)。
- 如申請專利範圍第1項的膳食增補劑，其中該山葵係取自山葵( Wasabia japonica)。
- 如申請專利範圍第1項的膳食增補劑，其中青花菜籽萃取物係萃取自青花菜( Brassica oleracea italica)。
- 如申請專利範圍第1項的膳食增補劑，進一步包含維生素與礦物質至少其中之一者。
- 如申請專利範圍第1項的膳食增補劑，進一步包含加味劑、賦形劑、黏結劑、蛋白質、綜合碳水化合物、防腐劑、及螫合劑至少其中之一者。
- 如申請專利範圍第1項的膳食增補劑，其中該增補劑為一膠囊、一錠劑、粉末或液態形式中至少其一者。
- 一種可包含在膳食增補劑之內的混合物，該混合物由聖羅勒、山葵及青花菜籽萃取物組成，其中該聖羅勒、山葵及青花菜籽萃取物在該增補劑內的含量及比例，在該增補劑被施加至一主體時，係足夠來刺激醌還原酶活性及誘發HMOX-1表現之至少一者。
- 如申請專利範圍第10項的混合物，其中該聖羅勒、山葵及青花菜籽萃取物在該增補劑內的比例分別為1：1：0.2。
- 如申請專利範圍第10項的混合物，其中該聖羅勒、山葵及青花菜籽萃取物在該增補劑內的比例，係足夠來降低人體內8-異前列腺素的尿液濃度。
- 如申請專利範圍第10項的混合物，其中該聖羅勒、山葵及青花菜籽萃取物在該增補劑內的比例，係足夠來增加人體內過氧化物歧化酶。
- 一種含有聖羅勒、山葵及青花菜籽萃取物比例分別為1：1：0.2之混合物的用途，其係用於製備用來增補人類膳 食以刺激有需求之人類細胞中醌還原酶產生與/或誘發血紅素氧化酶-1(HMOX-1)表現的膳食增補劑。
- 如申請專利範圍第14項的用途，其中該增補劑係為一膠囊、錠劑及液態形式之至少一者。
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- 2013-11-25 US US14/088,765 patent/US9517249B2/en active Active
- 2013-11-25 TW TW102142795A patent/TWI660680B/zh active
- 2013-11-25 CN CN201380061555.0A patent/CN104797144B/zh active Active
- 2013-11-25 TW TW107138812A patent/TWI671014B/zh active
- 2013-11-25 JP JP2015544164A patent/JP6825809B2/ja active Active
- 2013-11-25 KR KR1020157013277A patent/KR102173501B1/ko active IP Right Grant
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2015
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| JP2016501522A (ja) | 2016-01-21 |
| US10201583B2 (en) | 2019-02-12 |
| JP6825809B2 (ja) | 2021-02-10 |
| TW201440668A (zh) | 2014-11-01 |
| JP2019068825A (ja) | 2019-05-09 |
| JP2021000099A (ja) | 2021-01-07 |
| US20190083566A1 (en) | 2019-03-21 |
| KR20200128438A (ko) | 2020-11-12 |
| WO2014082027A3 (en) | 2014-08-28 |
| CN104797144A (zh) | 2015-07-22 |
| TWI671014B (zh) | 2019-09-11 |
| US9517249B2 (en) | 2016-12-13 |
| WO2014082027A2 (en) | 2014-05-30 |
| HK1208309A1 (zh) | 2016-03-04 |
| KR102173501B1 (ko) | 2020-11-04 |
| KR20150089009A (ko) | 2015-08-04 |
| JP6930957B2 (ja) | 2021-09-01 |
| CN104797144B (zh) | 2017-10-20 |
| US20140147526A1 (en) | 2014-05-29 |
| US20170106041A1 (en) | 2017-04-20 |
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