TWI698243B - 樟芝菌絲體活性物質用於改善慢性阻塞性肺病的用途 - Google Patents
樟芝菌絲體活性物質用於改善慢性阻塞性肺病的用途 Download PDFInfo
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Abstract
本發明提供一種樟芝菌絲體活性物質用於製備改善慢性阻塞性肺病之組合物的用途。所述樟芝菌絲體活性物質係以下列步驟製備而成:(a)取一樟芝菌絲體(Antrodia cinnamomea
)於平板培養基上,於15-30℃之溫度下培養4-10天;(b)將步驟(a)培養後之樟芝菌絲體接種至一燒瓶內,於15-30℃、pH 2-6之環境培養3-14天;(c)將步驟(b)培養後之樟芝菌絲體接種於一發酵槽內,於15-30℃、pH 2-6之環境下攪拌培養3-21天,形成含有該樟芝菌絲體活性物質之一樟芝菌絲體發酵液。
Description
本發明關於一種樟芝菌絲體活性物質的製備方法及其用途。具體來說,關於製備一種用於改善慢性阻塞性肺病的樟芝菌絲體活性物質,以及其在食品或醫藥品中的用途。
慢性阻塞性肺疾病(Chronic Obstructive Pulmonary Disease,簡稱COPD) 目前居全球死亡原因的第4位,是一種常見、多發、高致殘率和高致死率的慢性呼吸系統疾病。全世界慢性阻塞性肺病的患者有超過三億人。
吸菸是導致COPD的主要原因,然近年因我國機車、汽車的排廢氣以及工業上發電與燃油燃煤的使用增加,導致Particulate Matter (PM) 2.5 的空氣汙染上升,更加重此一疾病的發生。長期暴露在污染的環境下,會使肺部產生發炎反應,導致氣流通道變窄而產生肺組織功能障礙。
COPD的治療方式目前包含戒菸、肺康復治療、吸入性支氣管擴張藥、使用皮質類固醇、長期供氧或進行肺移植等。2015年最新通過的新藥係合併過去兩種使用於COPD的肌肉鬆弛劑,截至目前仍未有突破性的新藥發展。因此,研究有效治療慢性阻塞性肺病的方法仍是重要的發展方向。
樟芝(Antrodia cinnamomea
),又稱為樟菇、樟菰、樟內菇、牛樟菇、牛樟芝、紅樟、紅樟芝,只生長於台灣山區海拔450-1500公尺間的牛樟樹幹腐朽之心材內壁或枯死倒伏之牛樟木材陰暗潮濕之表面,屬於台灣國寶級的珍貴藥用真菌。目前仍缺乏樟芝對改善慢性阻塞性肺病之研究。
本發明之提供一種樟芝菌絲體活性物質及其製備方法,其可用於製備具改善慢性阻塞性肺病之組合物。相較於一般西藥及治療方法,本發明揭露的液態發酵樟芝菌絲體活性物質的製備方法更為安全、簡便,製成之樟芝菌絲體活性物質更天然、安全,並可有效改善COPD。
根據本發明之一實施例,提供一種樟芝菌絲體活性物質用於製備改善慢性阻塞性肺病之組合物的用途,其中樟芝菌絲體活性物質係以下列步驟製備而成:
(a)取樟芝菌絲體(Antrodia cinnamomea
)於平板培養基上,於15-30℃之溫度下培養4-10天;
(b)將步驟(a)培養後之樟芝菌絲體接種至燒瓶內,於15-30℃、pH 2-6之環境培養3-14天;
(c)將步驟(b)培養後之樟芝菌絲體接種於發酵槽內,於15-30℃、pH 2-6之環境下攪拌培養3-21天,形成含有樟芝菌絲體活性物質之樟芝菌絲體發酵液。
一實施例中,製備樟芝菌絲體活性物質的步驟更包括步驟(d):將樟芝菌絲體發酵液冷凍乾燥後磨粉,形成含有樟芝菌絲體活性物質之樟芝菌絲體凍乾粉。
一實施例中,製備樟芝菌絲體活性物質的步驟更包括步驟(e):將樟芝菌絲體凍乾粉以至少一溶劑萃取,形成含有樟芝菌絲體活性物質之樟芝菌絲體萃取液。
一實施例中,製備樟芝菌絲體活性物質的步驟更包括步驟(f):將樟芝菌絲體萃取液乾燥,以獲得樟芝菌絲體活性物質。
一實施例中,步驟(e)中的溶劑係選自水或醇類。
一實施例中,當步驟(e)之溶劑為水時,樟芝菌絲體活性物質的有效濃度介於 0.25-1 mg/ml。
一實施例中,當步驟(e)之該溶劑為醇類時,樟芝菌絲體活性物質的有效濃度介於25-100 μg/ml。
一實施例中,上述步驟(b)之燒瓶培養為震盪培養,且轉速為110-130 rpm。
一實施例中,步驟(c)中的發酵槽進一步通入一氣體,此氣體包括空氣、氧氣、二氧化碳、氦氣或其組合,發酵槽的槽壓為0.5-1.0 kg/cm2
且通氣速率為0.01-1.5VVM。
一實施例中,所述組合物為醫藥組合物,此醫藥組合物進一步包含藥學上可接受之載劑、賦形劑、稀釋劑或輔劑。
一實施例中,所述組合物為食品添加劑。
為使本發明之上述及其它方面更為清楚易懂,下文特舉實施例,並配合所附圖式詳細說明。
實施例一 樟芝菌絲體培養
本實施例採用之樟芝菌絲體(Antrodia cinnamomea
)係購自財團法人食品工業發展研究所(FIRDI)生物資源保存及研究中心(BCRC,前稱CCRC),編號為CCRC-35396。然而,可用於本發明的樟芝品種並不限定於此單獨一種,其更可包括BCRC編號35396、35398、35716、36401、36711、36795、37848、37849、37850、37889、37890、37891、37893、37894、37941或及其他生物材料寄存資料庫的樟芝菌種,本發明並不對其限制。
(1)平板培養:將樟芝菌絲體接種於平板上,於15-30℃下培養4-10天(本實施例中,係於25℃下培養7天)。平板培養基的成份可包含馬鈴薯糊精培養基(Potato Dextrose Agar, PDA)、碳源及氮源,並無特別限制。
(2)燒瓶培養:刮取(1)平板上之菌絲體接種於燒瓶中,並以15-30℃、pH 2-6及轉速110-130 rpm的條件震盪培養3-14天(本實施例中,於25℃、pH 5、轉速120 rpm之下震盪培養10天)。此震盪培養係以下表1所示之培養基進行培養。
上述培養基配方中,黃豆粉及肉桂粉為綜合性碳氮源,其可選自穀類(如:麥粉)或豆類(如:黃豆粉、綠豆粉、大豆粉、肉桂粉等);醣類可為葡萄糖、果糖、麥芽糖、蔗糖等;無機鹽類可為硫酸鎂、磷酸氫二鉀、磷酸二氫鉀、硫酸鐵等。特別說明的是,表1培養基配方僅為其中一種範例,使用時成份可依需求調整,或搭配市售培養基使用,並無特別限制。
(3)發酵槽培養:將(2)中燒瓶培養的菌絲體接種於發酵槽內,以15-30 ℃、槽壓0.5-1.0 kg/cm2
、pH 2-6及攪拌速度50-150 rpm條件下,以0.1-1.5 VVM的通氣速率培養3-21天,形成一樟芝菌絲體發酵液(本實施例中,係在25℃、槽壓0.5 kg/cm2
、pH 4,攪拌速度80 rpm及1.0 VVM(空氣)的條件下,培養14天),即得樟芝菌絲體發酵液。發酵槽培養使用之培養基可與步驟(2)燒瓶培養使用之培養基相同。此樟芝菌絲體發酵液內即含本發明之樟芝菌絲體活性物質。樟芝菌絲體發酵液可進一步藉由冷凍乾燥步驟製備為樟芝菌絲體發酵液凍乾粉;
(4)萃取物製備:將樟芝菌絲體發酵液凍乾粉分為兩份,分別加入20倍重量之蒸餾水及乙醇進行萃取。以水為溶劑的萃取物以溫度100℃加熱三十分鐘,待冷卻後離心取上清液,經冷凍乾燥法進行乾燥得樟芝發酵菌絲體水萃物。以乙醇為溶劑的萃取物,以超音波震盪1小時,離心後取上清液,將上清液經減壓濃縮得樟芝發酵菌絲體醇萃物;
經由萃取步驟,可取得含較高濃度之樟芝菌絲體活性物質的水萃物及醇萃物。樟芝菌絲體活性物質的態樣包含樟芝菌絲體發酵液(菌絲體與澄清液)、發酵液凍乾粉、水萃物、醇萃物、水萃物及醇萃物之混合物或其他劑型。以下實施例二中,係以水萃物及醇萃物作為樟芝菌絲體活性物質態樣。實施例二 樟 芝菌絲體水萃物及醇萃物改善 COPD 之分析
目前已有實驗利用香菸煙霧萃取物(Cigarette smoke extract,CSE)及人類肺癌上皮細胞A549成功建立COPD模式。可參照Miaomiao Chen et al. (2015). Azithromycin attenuates cigarette smoke extract-induced oxidative stress injury in human alveolar epithelial cells.MOLECULAR MEDICINE REPORTS,
11: 3414-3422. 此篇論文,其以人類肺癌上皮細胞A549對CSE進行細胞存活率分析(MTT assay),用以評估抗生素Azithromycin對COPD的改善結果。CSE對肺泡上皮的損傷是慢性阻塞性肺病機制中的重要過程。 因此,若能抑制CSE對肺泡上皮的傷害即能達到改善慢性阻塞性肺病的效果。本實驗中亦以人類肺癌上皮細胞A549對CSE進行細胞存活率分析(MTT assay),評估樟芝菌絲體活性物質對COPD的改善結果。
(1)收集人類肺癌上皮A549細胞,調整細胞懸浮液濃度,以50,000-100,000/mL 密度植種於96孔盤內;
(2)將(1)之96孔盤置於37℃、5% CO2
培養箱培養16-24小時後,以實施例一所獲得,不同劑量的樟芝菌絲體水萃物(0、0.25、0.5、1 mg/ml)及醇萃物(0、25、50、100 μg/ml)處理。樟芝菌絲體萃取物溶解於二甲基亞石楓(Dimethyl sulfoxide, DMSO)中,檢測時DMSO之濃度不超過0.1%以避免其毒性影響細胞生長;對照組處理0.1% DMSO,每個濃度進行三重複,結果置於37℃、5% CO2
培養箱培養24小時;
(3)上述(2)之結果先以400×g 離心10 min,移除上清液,再加入香菸煙霧萃取物CSE 12.5%誘導A549的細胞毒性,對照組不處理。接著置於37℃、5% CO2
培養箱培養6小時;
(4)將(3)之上清液移除,每孔加入100 μl MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 溶液,於37℃反應 2小時後,終止培養,小心移除孔內MTT溶液;
(5)於(4)中每孔加入100 μl DMSO,震盪2 min,於570 nm 測定吸光值。此吸光度代表細胞還原MTT的能力(甲䐶的形成量),即粒線體的活性(活細胞數目)。於570 nm的吸光度越高,表示細胞存活率越高。
統計分析:實驗結果數據以平均值(Mean)±標準差(SD)表示。試驗數據以Student's t-test 統計分析其組間差異。若p < 0.05則視為具有統計上的顯著差異。
水萃物之實驗結果如第1圖及下表2所示。香菸煙霧萃取物CSE會誘導A549細胞毒性,造成細胞存活率下降。然而,加入樟芝發酵菌絲體水萃物可顯著性抑制細胞損傷,提昇存活率,且加入的濃度越高,存活率越高。採用本發明實施例一中的樟芝菌絲體水萃物具有改善COPD之作用,有效濃度介於0.5-1 mg/ml。
醇萃物之實驗結果如第2圖及下表3所示。結果同樣表明,加入樟芝發酵菌本發明實施例一中的樟芝菌絲體醇萃物具有改善COPD之作用,有效濃度介於50-100 μg/ml。
本發明之樟芝活性物質經上述實驗,已證實具有改善COPD之作用。其可與藥學上可接受之載劑、賦形劑、稀釋劑或輔劑結合,製備醫藥組合物,亦可作為食品添加劑之用。
雖然本發明已用實施例揭露如上,然其並非用以限制本發明。本領域之通常知識者,於參酌以上教示後,當能對上述實施例的內容進行適當修改,而仍然能達到本案所主張之功效。因此,本發明的保護範圍應以其後所附之申請專利範圍修正本為準。
第1圖繪示樟芝菌絲體水萃物改善香菸煙霧萃取物誘導肺泡上皮A549細胞傷害之能力的結果,其中AcH(L)、AcH(M)及AcH(H)係分別指樟芝菌絲體水萃物濃度為0.25 mg/ml、0.5 mg/ml及1.0 mg/ml。
第2圖繪示樟芝菌絲體醇萃物改善香菸煙霧萃取物誘導肺泡上皮A549細胞傷害之能力的結果,其中AcMe(L)、AcMe(M)及AcMe(H)係分別指樟芝菌絲體醇萃物濃度為25 μg/ml、50 μg/ml及100 μg/ml。
Claims (6)
- 一種樟芝菌絲體活性物質用於製備改善慢性阻塞性肺病之組合物的用途,其中該樟芝菌絲體活性物質係透過抑制肺泡上皮細胞死亡,以改善慢性阻塞性肺病,該樟芝菌絲體活性物質之製備方法包括下列步驟:(a)取一樟芝菌絲體(Antrodia cinnamomea)於平板培養基上,於15-30℃之溫度下培養4-10天;(b)將步驟(a)培養後之樟芝菌絲體接種至一燒瓶內,於15-30℃、pH 2-6之環境培養3-14天;(c)將步驟(b)培養後之樟芝菌絲體接種於一發酵槽內,於15-30℃、pH 2-6之環境下攪拌培養3-21天,形成含有該樟芝菌絲體活性物質之一樟芝菌絲體發酵液;(d)將該樟芝菌絲體發酵液冷凍乾燥後磨粉,形成含有該樟芝菌絲體活性物質之一樟芝菌絲體凍乾粉;(e)將該樟芝菌絲體凍乾粉以水或醇類萃取,形成含有該樟芝菌絲體活性物質之至少一樟芝菌絲體萃取液;以及(f)將該樟芝菌絲體萃取液乾燥,以獲得該樟芝菌絲體活性物質;其中,當步驟(e)之該溶劑為水時,該樟芝菌絲體活性物質的有效濃度介於0.5-1mg/ml;當步驟(e)之該溶劑為醇類時,該樟芝菌絲體活性物質的有效濃度介於50-100μg/ml。
- 如申請專利範圍第1項所述之用途,其中步驟(b)之燒瓶培養為震盪培養,且轉速為110-130rpm。
- 如申請專利範圍第1項所述之用途,其中步驟(c)中該發酵槽進一步通入一氣體,該氣體包括空氣、氧氣、二氧化碳、氦氣或其組合,該發酵槽的槽壓為0.5-1.0kg/cm2且通氣速率為0.01-1.5VVM。
- 如申請專利範圍第1項所述之用途,其中該組合物為醫藥組合物,該 醫藥組合物進一步包含藥學上可接受之載劑、賦形劑、稀釋劑或輔劑。
- 如申請專利範圍第1項所述之用途,其中該組合物為食品添加劑。
- 如申請專利範圍第1項所述之用途,其中該樟芝菌絲體活性物質係藉由抑制香煙煙霧所引發之肺泡上皮細胞死亡,以改善慢性肺阻塞肺病。
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