TW202003004A - Use of Antrodia cinnamomea mycelia active substance for ameliorating chronic obstructive pulmonary disease - Google Patents

Use of Antrodia cinnamomea mycelia active substance for ameliorating chronic obstructive pulmonary disease Download PDF

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TW202003004A
TW202003004A TW107117897A TW107117897A TW202003004A TW 202003004 A TW202003004 A TW 202003004A TW 107117897 A TW107117897 A TW 107117897A TW 107117897 A TW107117897 A TW 107117897A TW 202003004 A TW202003004 A TW 202003004A
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陳勁初
李宜蓁
林定威
張曉苓
陳彥博
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葡萄王生技股份有限公司
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Abstract

A use ofAntrodia cinnamomea mycelia active substance is provided. TheAntrodia cinnamomea mycelia active substance is for the treatment of chronic obstructive pulmonary disease, and is prepared by the following steps: (a)culturing aAntrodia cinnamomea mycelium in a plate media at 15 to 30℃ for 4 to 10 days; (b)inoculating the mycelium of step(a) to a flask and culturing it at 15 to 30℃ with a pH of 2 to 6 for 3 to 14 days; (c)inoculating the mycelium of step(b) to a fermenter tank and culturing it at 15 to 30℃ with a pH of 2 to 6 for 3 to 21 days, so as to obtain aAntrodia cinnamomea mycelium fermentation liquid containing saidAntrodia cinnamomea mycelium active substance.

Description

樟芝菌絲體活性物質用於改善慢性阻塞性肺病的用途Use of Antrodia camphorata mycelium active substance for improving chronic obstructive pulmonary disease

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。The invention relates to a preparation method and application of active substances of Antrodia camphorata mycelium. Specifically, it relates to the preparation of an active substance of Antrodia camphorata mycelia for improving chronic obstructive pulmonary disease, and its use in food or medicine.

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。Chronic Obstructive Pulmonary Disease (COPD) is currently the fourth leading cause of death in the world. It is a common, frequent, high disability rate and high mortality rate of chronic respiratory diseases. There are more than 300 million people with chronic obstructive pulmonary disease worldwide.

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。Smoking is the main cause of COPD. However, in recent years, due to the increased emissions of locomotives and automobiles in China and the increased use of power generation and fuel coal in industry, the air pollution of Particulate Matter (PM) 2.5 has increased, and this disease is more important. . Long-term exposure to a polluted environment can cause an inflammatory reaction in the lungs, resulting in narrowing of airflow channels and resulting in lung tissue dysfunction.

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。COPD treatment methods currently include smoking cessation, lung rehabilitation, inhaled bronchodilators, corticosteroid use, long-term oxygen supply, or lung transplantation. The newly approved new drug series in 2015 merged the two muscle relaxants used in COPD in the past, and so far there has been no breakthrough new drug development. Therefore, research on effective treatment of chronic obstructive pulmonary disease is still an important development direction.

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。 Antrodia cinnamomea ( Antrodia cinnamomea ), also known as camphor, camphor, camphor mushroom, cinnamomum , cinnamomea , red camphor, red camphor, only grows in the decayed trunk of Antrodia camphorata at an altitude of 450-1500 meters in the mountainous area of Taiwan The inner wall of the heartwood or the dark and damp surface of the withered and fallen camphor wood belongs to the precious medicinal fungi of Taiwan national treasure level. At present, there is still a lack of research on Zhangzhi to improve chronic obstructive pulmonary disease.

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。The invention provides an active substance of Antrodia camphorata mycelium and a preparation method thereof, which can be used for preparing a composition with improved chronic obstructive pulmonary disease. Compared with the general western medicine and the treatment method, the preparation method of the liquid fermented Antrodia camphorata mycelium active substance disclosed by the present invention is safer and simpler, and the prepared Antrodia camphorata mycelium active substance is more natural and safe, and can be effectively improved 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:According to an embodiment of the present invention, there is provided a use of an active substance of Antrodia camphorata mycelium for preparing a composition for improving chronic obstructive pulmonary disease, wherein the active substance of Antrodia camphorata mycelium is prepared by the following steps:

(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;(a) Take Antrodia cinnamomea on a plate medium and incubate at a temperature of 15-30℃ for 4-10 days;

(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;(b) Inoculate the Antrodia camphorata mycelium cultured in step (a) into the flask, and cultivate it for 3-14 days in an environment of 15-30°C and pH 2-6;

(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。(c) Inoculate the mycelium of Antrodia camphorata after cultivation in step (b) in a fermentation tank, and stir and cultivate for 3-21 days at an environment of 15-30°C and pH 2-6 to form an activity containing Antrodia camphorata mycelia Substance of Antrodia camphorata mycelium fermentation broth.

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。In one embodiment, the step of preparing the active substance of Antrodia camphorata mycelia further includes step (d): freeze-drying the fermentation broth of Antrodia camphorata mycelia and grinding it to form antrodia camphorata mycelium containing the active substance of Antrodia camphorata mycelia Body lyophilized powder.

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。In one embodiment, the step of preparing the active substance of Antrodia camphorata mycelium further includes step (e): extracting the freeze-dried powder of Antrodia camphorata mycelium with at least one solvent to form Antrodia camphorata containing the active substance of Antrodia camphorata mycelium Silk extract.

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。In one embodiment, the step of preparing the active substance of Antrodia camphorata mycelia further includes step (f): drying the extract of the mycelium of Antrodia camphorata to obtain the active substance of the mycelium of Antrodia camphorata.

一實施例中,步驟(e)中的溶劑係選自水或醇類。In one embodiment, the solvent in step (e) is selected from water or alcohols.

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。In one embodiment, when the solvent in step (e) is water, the effective concentration of the active substance of Antrodia camphorata mycelium is between 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。In one embodiment, when the solvent in step (e) is an alcohol, the effective concentration of the active substance of Antrodia camphorata mycelium is between 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。In one embodiment, the flask culture in the above step (b) is shaking culture, and the rotation speed is 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。In one embodiment, the fermentation tank in step (c) is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium, or a combination thereof, the fermentation tank has a tank pressure of 0.5-1.0 kg/cm 2 and aeration rate It is 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。In one embodiment, the composition is a pharmaceutical composition, and the pharmaceutical composition further includes a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.

一實施例中,所述組合物為食品添加劑。In one embodiment, the composition is a food additive.

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。In order to make the above and other aspects of the present invention clearer and more understandable, the embodiments are specifically described below in conjunction with the accompanying drawings.

實施例一 樟芝菌絲體培養Example 1   Antrodia camphorata mycelium culture

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。The Antrodia cinnamomea used in this example was purchased from the Biological Resource Preservation and Research Center (BCRC, formerly CCRC) of the Food Industry Development Institute (FIRDI) of the consortium, and the number is CCRC-35396. However, the Antrodia camphorata varieties that can be used in the present invention are not limited to this single one, but can also include BCRC numbers 35396, 35398, 35716, 36401, 36711, 36795, 37848, 37849, 37850, 37889, 37890, 37891, 37893, The species of Antrodia camphorata strains of 37894, 37941 or other biological material storage databases are not limited by the present invention.

(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。(1) Plate culture: Antrodia camphorata mycelium is inoculated on the plate and cultivated at 15-30°C for 4-10 days (in this example, it is cultivated at 25°C for 7 days). The components of the plate medium may include potato dextrin medium (Potato Dextrose Agar, PDA), carbon source and nitrogen source, and are not particularly limited.

(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。(2) Flask culture: scrape (1) inoculate the mycelium on the plate into the flask, and culture with shaking at 15-30°C, pH 2-6 and rotation speed 110-130 rpm for 3-14 days (this implementation In the example, it was cultured with shaking at 25°C, pH 5, and rotation speed of 120 rpm for 10 days). This shaking culture was carried out with the medium shown in Table 1 below.

表1 培養基配方

Figure 107117897-A0304-0001
Table 1 Medium formula
Figure 107117897-A0304-0001

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。In the above medium formula, soybean powder and cinnamon powder are comprehensive carbon and nitrogen sources, which can be selected from cereals (such as wheat flour) or beans (such as soybean powder, mung bean powder, soybean powder, cinnamon powder, etc.); sugars can be It is glucose, fructose, maltose, sucrose, etc.; inorganic salts can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferric sulfate, etc. In particular, the formula of the medium in Table 1 is just one example, and the ingredients can be adjusted according to needs or used with commercially available medium without special restrictions.

(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;(3) Fermentation tank culture: inoculate the mycelium cultured in the flask in (2) into the fermentation tank at 15-30 ℃, tank pressure 0.5-1.0 kg/cm 2 , pH 2-6 and stirring speed 50-150 Under the condition of rpm, incubate at aeration rate of 0.1-1.5 VVM for 3-21 days to form a fermentation liquid of Antrodia camphorata mycelium (in this example, it was stirred at 25°C, tank pressure 0.5 kg/cm 2 , pH 4 and stirred Under the conditions of speed 80 rpm and 1.0 VVM (air), culture for 14 days), the fermentation liquid of Antrodia camphorata mycelium is obtained. The culture medium used for fermentation tank culture may be the same as the culture medium used for flask culture in step (2). The Antrodia camphorata mycelium fermentation liquid contains the active substance of Antrodia camphorata mycelium according to the present invention. The fermentation liquid of Antrodia camphorata mycelium can be further prepared as a freeze-dried powder of the fermentation liquid of Antrodia camphorata mycelium by a freeze-drying step;

(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;(4) Preparation of extract: The lyophilized powder of the fermentation solution of Antrodia camphorata mycelium is divided into two parts, and 20 times the weight of distilled water and ethanol are added to extract. The extract using water as a solvent is heated at a temperature of 100°C for thirty minutes. After cooling, the supernatant is taken by centrifugation and dried by freeze-drying to obtain an aqueous extract of fermented mycelia of Antrodia camphorata. The ethanol-extracted extract was shaken with ultrasound for 1 hour. After centrifugation, the supernatant was taken, and the supernatant was concentrated under reduced pressure to obtain an alcohol extract of Antrodia camphorata fermentation mycelium;

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 芝菌絲體水萃物及醇萃物改善 COPD 之分析 Through the extraction step, water extracts and alcohol extracts containing higher concentrations of active substances of Antrodia camphorata mycelium can be obtained. The active substances of Antrodia camphorata mycelia include the fermentation liquid of mycelium of Antrodia camphorata (mycelium and clarified liquid), lyophilized powder of fermentation broth, water extract, alcohol extract, water extract and mixture of alcohol extract Or other dosage forms. In the second embodiment below, the water extract and the alcohol extract are used as the active substance of Antrodia camphorata mycelium. Example 2 Analysis of water extract and alcohol extract of Antrodia camphorata mycelia to improve COPD

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。There have been experiments using Cigarette smoke extract (CSE) and human lung cancer epithelial cells A549 to successfully establish a COPD model. Refer to 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. This paper uses human lung cancer epithelial cells A549 to CSE MTT assay was performed to evaluate the improvement of COPD by antibiotic Azithromycin. CSE damage to the alveolar epithelium is an important process in the mechanism of chronic obstructive pulmonary disease. Therefore, if it can suppress the damage of CSE to alveolar epithelium, it can achieve the effect of improving chronic obstructive pulmonary disease. In this experiment, the human lung cancer epithelial cell A549 was also used to perform cell survival analysis (MTT assay) on CSE to evaluate the improvement of COPD by the active substances of Antrodia camphorata mycelium.

(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;(1) Collect human lung cancer epithelial A549 cells, adjust the concentration of the cell suspension, and plant them in 96-well dishes at a density of 50,000-100,000/mL;

(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;(2) Place the 96-well plate of (1) in a 37°C, 5% CO 2 incubator for 16-24 hours, and obtain the water extract of Antrodia camphorata mycelium with different doses obtained in Example 1 (0, 0.25, 0.5, 1 mg/ml) and alcohol extracts (0, 25, 50, 100 μg/ml). Antrodia camphorata mycelium extract is dissolved in Dimethyl sulfoxide (DMSO), the concentration of DMSO does not exceed 0.1% when tested to avoid its toxicity affecting cell growth; the control group is treated with 0.1% DMSO, each concentration Three replicates were performed and the results were placed in a 37°C, 5% CO 2 incubator for 24 hours;

(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;(3) The result of (2) above was first centrifuged at 400 × g for 10 min, the supernatant was removed, and then the cigarette smoke extract CSE 12.5% was added to induce the cytotoxicity of A549, the control group was not treated. Then put it in 37℃, 5% CO 2 incubator for 6 hours;

(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;(4) Remove the supernatant from (3), add 100 μl MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution to each well, and react at 37℃ for 2 hours After that, stop the culture and carefully remove the MTT solution in the well;

(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。(5) Add 100 μl DMSO to each well in (4), shake for 2 min, and measure the absorbance at 570 nm. This absorbance represents the ability of cells to reduce MTT (formation of formazan), that is, the activity of mitochondria (number of viable cells). The higher the absorbance at 570 nm, the higher the cell survival rate.

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。Statistical analysis: The experimental result data is expressed as mean (Mean) ± standard deviation (SD). The test data was analyzed by Student's t-test statistics to analyze the differences between groups. If p <0.05, it is regarded as having statistically significant difference.

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。The experimental results of the water extract are shown in Figure 1 and Table 2 below. Cigarette smoke extract CSE can induce A549 cytotoxicity, resulting in decreased cell survival rate. However, the addition of water extracts of fermented mycelia of Antrodia camphorata can significantly inhibit cell damage and improve survival rate, and the higher the concentration added, the higher the survival rate. Using the water extract of Antrodia camphorata mycelium in Example 1 of the present invention has the effect of improving COPD, and the effective concentration is between 0.5-1 mg/ml.

表2 樟芝菌絲體水萃物測試結果

Figure 107117897-A0304-0002
(n = 3) #表示與對照組(control)統計上具顯著差異(p<0.05) *表示與負對照組(CSE)統計上具顯著差異(p<0.05)Table 2 Test results of water extract of Antrodia camphorata mycelium
Figure 107117897-A0304-0002
(n = 3) # indicates statistically significant difference from the control group (control) (p<0.05) * indicates statistically significant difference from the negative control group (CSE) (p<0.05)

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。The experimental results of alcohol extracts are shown in Figure 2 and Table 3 below. The results also show that the addition of the Antrodia camphorata mycelium alcohol extract in the first embodiment of the present invention can improve the COPD, and the effective concentration is between 50-100 μg/ml.

表3 樟芝菌絲體醇萃物測試結果

Figure 107117897-A0304-0003
(n = 3) #表示與對照組(control)統計上具顯著差異(p<0.05) *表示與負對照組(CSE)統計上具顯著差異(p<0.05)Table 3 Test results of alcohol extract of Antrodia camphorata mycelium
Figure 107117897-A0304-0003
(n = 3) # indicates statistically significant difference from the control group (control) (p<0.05) * indicates statistically significant difference from the negative control group (CSE) (p<0.05)

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。The active substance of Antrodia camphorata according to the present invention has been confirmed to have the effect of improving COPD through the above experiments. It can be combined with pharmaceutically acceptable carriers, excipients, diluents or adjuvants to prepare pharmaceutical compositions and can also be used as food additives.

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。Although the present invention has been disclosed as above using embodiments, it is not intended to limit the present invention. Those of ordinary knowledge in the art, after referring to the above teachings, should be able to make appropriate modifications to the content of the above-mentioned embodiments and still achieve the effect claimed in this case. Therefore, the scope of protection of the present invention shall be subject to the appended amendment to the scope of the patent application.

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。Figure 1 shows the results of the water extract of Antrodia camphorata mycelium improving the ability of cigarette smoke extract to induce the injury of alveolar epithelial A549 cells, where AcH(L), AcH(M) and AcH(H) refer to Antrodia camphorata respectively The concentration of silk extracts was 0.25 mg/ml, 0.5 mg/ml and 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。Figure 2 shows the results of the mycelium alcohol extract of Antrodia camphorata improving the ability of cigarette smoke extract to induce damage to alveolar epithelial A549 cells, where AcMe(L), AcMe(M) and AcMe(H) refer to Antrodia camphorata respectively Serum alcohol extract concentration was 25 μg/ml, 50 μg/ml and 100 μg/ml.

Claims (11)

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。An active substance of Antrodia camphorata mycelium is used for preparing a composition for improving chronic obstructive pulmonary disease, wherein the active substance of Antrodia camphorata mycelium is prepared by the following steps: (a) Take an Antrodia camphorata mycelium ( Antrodia cinnamomea ) cultured on a plate medium at a temperature of 15-30℃ for 4-10 days; (b) Inoculate the mycelium of Antrodia camphorata after cultivation in step (a) into a flask at 15-30℃, Cultivation in pH 2-6 environment for 3-14 days; (c) Inoculate the Antrodia camphorata mycelium cultured in step (b) in a fermentation tank, and stir at 15-30℃, pH 2-6 Within 3-21 days, a fermentation liquid of Antrodia camphorata mycelia containing one of the active substances of the Antrodia camphorata mycelia was formed. 如申請專利範圍第1項所述之用途,其中製備該樟芝菌絲體活性物質的步驟更包括步驟(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。The use as described in item 1 of the scope of the patent application, wherein the step of preparing the active substance of the mycelium of Antrodia camphorata further includes step (d): freeze-drying the fermentation liquid of the mycelium of Antrodia camphorata to form a powder containing the camphor Antrodia camphorata mycelium freeze-dried powder, one of the active substances of Zhi mycelium. 如申請專利範圍第2項所述之用途,其中製備該樟芝菌絲體活性物質的步驟更包括步驟(e):將該樟芝菌絲體凍乾粉以至少一溶劑萃取,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。The use as described in item 2 of the patent application scope, wherein the step of preparing the active substance of the mycelium of Antrodia camphorata further includes step (e): extracting the freeze-dried powder of the mycelium of Antrodia camphorata with at least one solvent to form At least one extract of Antrodia camphorata mycelium active substance. 如申請專利範圍第3項所述之用途,其中製備該樟芝菌絲體活性物質的步驟更包括步驟(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。The use as described in Item 3 of the scope of the patent application, wherein the step of preparing the active substance of the Antrodia camphorata mycelium further includes step (f): drying the extract of the Antrodia camphorata mycelium to obtain the mycelium of Antrodia camphorata Active substance. 如申請專利範圍第3或4項所述之用途,其中在步驟(e)中,該溶劑係選自水或醇類。The use as described in item 3 or 4 of the patent application scope, wherein in step (e), the solvent is selected from water or alcohols. 如申請專利範圍第5項所述之用途,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 0.5-1 mg/ml。The use as described in item 5 of the patent application scope, wherein when the solvent in step (e) is water, the effective concentration of the active substance of the mycelia of Antrodia camphorata is between 0.5-1 mg/ml. 如申請專利範圍第5項所述之用途,其中當步驟(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 50-100 μg/ml。The use as described in item 5 of the patent application scope, wherein when the solvent in step (e) is an alcohol, the effective concentration of the active substance of the mycelia of Antrodia camphorata is between 50-100 μg/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110-130 rpm。The use as described in item 1 of the patent application scope, wherein the flask culture in step (b) is shaking culture and the rotation speed is 110-130 rpm. 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0.5-1.0 kg/cm2 且通氣速率為0.01-1.5 VVM。The use as described in item 1 of the patent application scope, wherein in step (c) the fermentation tank is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium or a combination thereof, and the fermentation tank has a tank pressure of 0.5 -1.0 kg/cm 2 and the ventilation rate is 0.01-1.5 VVM. 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。The use as described in item 1 of the patent application scope, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent or adjuvant. 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。The use as described in item 1 of the patent application scope, wherein the composition is a food additive.
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