TWI723368B - Method for manufacturing and the use of paecilomyces hepiali mycelia active substance for preventing and/or ameliorating acute lung injury - Google Patents
Method for manufacturing and the use of paecilomyces hepiali mycelia active substance for preventing and/or ameliorating acute lung injury Download PDFInfo
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本發明關於一種蝙蝠蛾擬青黴菌絲體活性物質、其製備方法及用途。具體來說，關於一種用於改善急性肺損傷的蝙蝠蛾擬青黴菌絲體活性物質，其製備方法，以及其在食品或醫藥品中的用途。The present invention relates to an active substance from Paecilomyces hepialum mycelium, a preparation method and application thereof. Specifically, it relates to an active substance of Paecilomyces hepipiens mycelium for improving acute lung injury, its preparation method, and its use in food or medicine.
衛生褔利部公布2017年十大死因中，其中第一大死因惡性腫瘤中的氣管、支氣管和肺癌、第三大死因肺炎，以及第七大死因慢性下呼吸道疾病，共四項十大死因與肺部發炎有著密不可分的相關性。The Ministry of Health and Welfare announced the top ten causes of death in 2017. Among them, malignant tumors are trachea, bronchus, and lung cancer, the third leading cause of death, pneumonia, and the seventh leading cause of death from chronic lower respiratory diseases. There are a total of four top ten causes of death. Inflammation of the lungs is inextricably related.
急性肺損傷(acute lung injury; ALI)與其更嚴重的急性呼吸窘迫症(acute respiratory distress syndrome; ARDS)為臨床常見的急性肺發炎的代表性疾病，皆引發呼吸衰節而導致死亡，並與許多衍生的呼吸道疾病相關。Acute lung injury (ALI) and its more serious acute respiratory distress syndrome (ARDS) are representative diseases of clinically common acute lung inflammation. Both cause respiratory failure and lead to death. Related to derived respiratory diseases.
美國公告的十大死因中，也有三項與肺部發炎相關的疾病，包括肺與支氣管癌、慢性呼吸道疾病、流感與肺炎。更進一步統計分析發現，在美國平均每年ALI與ARDS的發生率分別為79與59人/每100,000人，其死亡率約為 43%，也就是美國每年約有75,000人因肺部發炎相關疾病死亡。由於空氣污染日趨嚴重環境與平均餘命逐年提高的情況下，推測在美國未來25年內，ALI/ARDS的發生率將成長2倍以上。Among the ten major causes of death announced in the United States, there are also three diseases related to lung inflammation, including lung and bronchial cancer, chronic respiratory diseases, influenza and pneumonia. Further statistical analysis found that the average annual incidence of ALI and ARDS in the United States is 79 and 59 people per 100,000 people, respectively, and the mortality rate is about 43%. That is, about 75,000 people die from lung inflammation-related diseases in the United States each year. . As air pollution is getting worse, the environment and the average remaining life are increasing year by year, it is speculated that the incidence of ALI/ARDS will more than double in the next 25 years in the United States.
造成ALI的危險因子分二大類，分別是直接型因子與間接型因子。直接型因子是指危險因子源自於肺臟，包括細菌或病毒引起感染性肺炎、大量吸入胃酸或異物、肺部挫傷等；間接型因子是指危險因子非源自於肺臟，包括敗血症、長期酒精與藥物濫用、輸入人工血漿等。其中，細菌感染為ALI主要的危險因子，革蘭氏陰性菌為其中一大類，其外套膜的主成分為內毒素，又名脂多醣(lipopolysaccride; LPS)。The risk factors that cause ALI are divided into two categories, namely direct factors and indirect factors. Direct factor refers to the risk factor derived from the lungs, including bacteria or viruses that cause infectious pneumonia, a large amount of gastric acid or foreign body inhalation, lung contusion, etc.; indirect factor refers to the risk factor that does not originate from the lungs, including sepsis, long-term alcohol And drug abuse, artificial plasma input, etc. Among them, bacterial infection is the main risk factor for ALI, and gram-negative bacteria are one of a large category. The main component of its mantle is endotoxin, also known as lipopolysaccride (LPS).
ALI因機制複雜，致死率高，目前臨床上尚無確切有效的控制藥物。但常用的治療方法有機械通氣、β2腎上腺素受體刺激劑、抗凝、溶栓、表面活性劑及手術等。因此，研究有效治療ALI的方法仍是重要的發展方向。Due to its complex mechanism and high fatality rate, there is no clinically effective control drug for ALI. However, the commonly used treatment methods include mechanical ventilation, β2 adrenergic receptor stimulators, anticoagulation, thrombolysis, surfactants, and surgery. Therefore, research on effective treatment of ALI is still an important development direction.
蝙蝠蛾擬青黴(Paecilomyces hepiali)為冬蟲夏草中分離出之伴生菌株，屬於叢梗孢科(Moniliaceae)擬黴菌屬(Paecilomyces)，在中國已被列為可用於保健食品的真菌菌種名單中。已有文獻證實蝙蝠蛾擬青黴菌發酵液具有的功效性，包含抗發炎、抗氧化、免療調節、抗憂鬱、抗糖尿病與止痛的作用。然而，目前尚未有蝙蝠蛾擬青黴對改善ALI之研究。Paecilomyces hepiali is a companion strain isolated from Cordyceps sinensis, belonging to the genus Paecilomyces of the Moniliaceae family (Moniliaceae), and has been listed as a fungus species that can be used in health food in China. The literature has confirmed the efficacy of the fermentation broth of Paecilomyces hepipiens, including anti-inflammatory, anti-oxidant, immunotherapy, anti-depression, anti-diabetic and analgesic effects. However, there is no research on the improvement of ALI by Paecilomyces hepipiens.
本發明之提供一種蝙蝠蛾擬青黴菌絲體活性物質及其製備方法，其可用於製備具改善急性肺損傷之組合物。相較於一般西藥及治療方法，本發明揭露的液態發酵蝙蝠蛾擬青黴菌絲體活性物質的製備方法更為安全、簡便，製成之蝙蝠蛾擬青黴菌絲體活性物質更天然、安全，並可有效預防及/或改善急性肺損傷(ALI)。The present invention provides an active substance from Paecilomyces hepialis mycelium and a preparation method thereof, which can be used to prepare a composition for improving acute lung injury. Compared with general western medicines and treatment methods, the preparation method of the liquid-fermented Paecilomyces hepialis mycelium active substance disclosed in the present invention is safer and simpler, and the prepared Paecilomyces hepialis mycelium active substance is more natural and safer. And can effectively prevent and/or improve acute lung injury (ALI).
根據本發明之一實施例，提供用於製備預防及/或改善急性肺損傷之蝙蝠蛾擬青黴菌絲體活性物質的製備方法，其係包括下列步驟：According to an embodiment of the present invention, there is provided a preparation method for preparing an active substance of Paecilomyces hepialis mycelium for preventing and/or ameliorating acute lung injury, which comprises the following steps:
(a)取蝙蝠蛾擬青黴(Paecilomyces hepialid) 菌絲體於平板培養基上，於15至30℃之溫度下培養1至2周；(a) Take Paecilomyces hepialid mycelium on a plate medium and culture it at a temperature of 15 to 30°C for 1 to 2 weeks;
(b)將步驟(a)培養後之蝙蝠蛾擬青黴菌絲體接種至燒瓶內，於15至30℃、pH 2至8之環境培養3至14天；(b) Inoculate the mycelium of Paecilomyces hepipipes after culturing in step (a) into a flask, and cultivate for 3 to 14 days in an environment of 15 to 30°C and
(c)將步驟(b)培養後之蝙蝠蛾擬青黴菌絲體接種於發酵槽內，於15至30℃、pH 2至8之環境下攪拌培養3至21天，形成含有蝙蝠蛾擬青黴菌絲體活性物質之蝙蝠蛾擬青黴菌絲體發酵液。(c) Inoculate the mycelium of Paecilomyces hepipialis cultured in step (b) in a fermentation tank, and culture it with stirring at 15-30°C and pH 2-8 for 3-21 days to form Paecilomyces hepipialis The active substance of the mycelium is the fermentation broth of Paecilomyces hepialis mycelium.
一實施例中，製備蝙蝠蛾擬青黴菌絲體活性物質的方法更包括步驟(d)：將蝙蝠蛾擬青黴菌絲體發酵液冷凍乾燥後磨粉，形成含有蝙蝠蛾擬青黴菌絲體活性物質之蝙蝠蛾擬青黴菌絲體凍乾粉。In one embodiment, the method for preparing the active substance of Paecilomyces hepipialis mycelium further includes the step (d): freeze-drying the fermentation broth of Paecilomyces hepipialis mycelium and then grinding to form the active substance containing Paecilomyces hepialis mycelium. The material is the freeze-dried powder of Paecilomyces hepialis mycelium.
一實施例中，製備蝙蝠蛾擬青黴菌絲體活性物質的方法更包括步驟(e)：將蝙蝠蛾擬青黴菌絲體凍乾粉以至少一溶劑萃取，形成含有蝙蝠蛾擬青黴菌絲體活性物質之蝙蝠蛾擬青黴菌絲體萃取液。In one embodiment, the method for preparing the active substance of Paecilomyces hepipialis mycelium further includes step (e): extracting the lyophilized powder of Paecilomyces hepipialis mycelium with at least one solvent to form a mycelium containing Paecilomyces hepialis The active substance is Paecilomyces hepialum mycelium extract.
一實施例中，製備蝙蝠蛾擬青黴菌絲體活性物質的步驟更包括步驟(f)：將蝙蝠蛾擬青黴菌絲體萃取液乾燥，以獲得蝙蝠蛾擬青黴菌絲體活性物質。In one embodiment, the step of preparing the active substance of Paecilomyces hepialis mycelium further includes step (f): drying the extract of Paecilomyces hepialis mycelium to obtain the active substance of Paecilomyces hepialis mycelium.
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，上述步驟(b)之燒瓶培養為震盪培養，且轉速為110至130 rpm。In one embodiment, the flask culture in the above step (b) is shaking culture, and the rotation speed is 110 to 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至1.0 kg/cm2 且通氣速率為0.01至1.5VVM。In one embodiment, the fermentation tank in step (c) is further vented with a gas, the gas includes air, oxygen, carbon dioxide, helium, or a combination thereof, the tank pressure of the fermentation tank is 0.5 to 1.0 kg/cm 2 and the aeration rate It is 0.01 to 1.5VVM.
根據本發明，另提供一種蝙蝠蛾擬青黴菌絲體活性物質，其係以上述製備方法製造而成。According to the present invention, there is also provided an active substance of Paecilomyces hepipiens mycelium, which is manufactured by the above-mentioned preparation method.
根據本發明，再提供一種用於改善急性肺損傷之組合物，其係包含具有效濃度的上述蝙蝠蛾擬青黴菌絲體活性物質，以及藥學上可接受之載劑、賦形劑、稀釋劑或輔劑。According to the present invention, there is further provided a composition for improving acute lung injury, which contains an effective concentration of the active substance of the above-mentioned Paecilomyces hepialis mycelium, and pharmaceutically acceptable carriers, excipients, and diluents Or adjuvant.
根據本發明，再提供一種上述蝙蝠蛾擬青黴菌絲體活性物質的用途，其係用於製備改善急性肺損傷之組合物。According to the present invention, there is further provided a use of the above-mentioned active substance from Paecilomyces hepipiens mycelium, which is used to prepare a composition for improving acute lung injury.
一實施例中，所述組合物為醫藥組合物或食品添加劑。In one embodiment, the composition is a pharmaceutical composition or 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 easier to understand, the following specific embodiments are described in detail in conjunction with the accompanying drawings.
實施例一 蝙蝠蛾擬青黴菌絲體培養Example 1: Culture of Paecilomyces hepipipes mycelium
本實施例使用之蝙蝠蛾擬青黴菌絲體，係由採集而得之台灣野生蝙蝠蛾擬青黴子實體，經分離而得其菌絲，並繼代保存於平板培養基上。此菌株經台灣食品工業發展研究所做鑑定證實其基因序列為蝙蝠蛾擬青黴(Paecilomyces hepiali )，並已公開寄存於財團法人食品工業發展研究所之生物資源研究中心(BCRC)，寄存編號為BCRC 930203。但本發明所述之蝙蝠蛾擬青黴活性物質不限於由此菌株所得，亦可使用其他來源的蝙蝠蛾擬青黴，並不限制。The mycelium of Paecilomyces hepipialis used in this embodiment is collected from the fruiting bodies of Paecilomyces hepipialis from Taiwan, and the hyphae are obtained after separation and stored on a plate medium for subsequent generations. This strain has been identified by Taiwan Food Industry Development Research and confirmed that its gene sequence is Paecilomyces hepiali (Paecilomyces hepiali), and has been publicly deposited in the Bioresources Research Center (BCRC) of the Food Industry Development Research Institute, with the deposit number BCRC 930203. However, the active substance of Paecilomyces hepipiens according to the present invention is not limited to those obtained from this strain, and Paecilomyces hepipialis from other sources can also be used without limitation.
(1)平板培養：將蝙蝠蛾擬青黴菌絲體接種於平板上，於15至30℃下培養1至2周(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: Inoculate the mycelium of Paecilomyces hepipiens on a plate, and culture at 15 to 30°C for 1 to 2 weeks (in this example, culture at 25°C for 7 days). The components of the plate medium may include 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至30℃、pH 2至8及轉速110至130 rpm的條件震盪培養3至14天(本實施例中，於25℃、pH 6、轉速120 rpm之下震盪培養7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) The mycelium on the plate is inoculated into the flask, and shake culture for 3 to 14 days under the conditions of 15 to 30°C,
表1 培養基配方
上述培養基配方中，綜合性碳氮源可選自穀類(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, the comprehensive carbon and nitrogen source 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 glucose, fructose, maltose, sucrose Etc.; inorganic salts can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, iron sulfate and the like. In particular, the medium formula in Table 1 is only one example, and the ingredients can be adjusted according to requirements during use, or used with commercially available medium, and there is no special restriction.
(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至30 ℃、槽壓0.5至1.0 kg/cm2
、pH 2至8及攪拌速度50至150 rpm條件下，以0.1至1.5 VVM的通氣速率培養3至21天，形成一蝙蝠蛾擬青黴菌絲體發酵液(本實施例中，係在25℃、槽壓0.5 kg/cm2
、pH 5，攪拌速度80 rpm及1.0 VVM(空氣)的條件下，培養14天)，即得蝙蝠蛾擬青黴菌絲體發酵液。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(本實施例之作法)，亦可另外配置適當培養基。此蝙蝠蛾擬青黴菌絲體發酵液內即含本發明之蝙蝠蛾擬青黴菌絲體活性物質。蝙蝠蛾擬青黴菌絲體發酵液可進一步藉由乾燥製備為蝙蝠蛾擬青黴菌絲體發酵液凍乾粉，此處乾燥包含但不限於冷凍乾燥、噴霧乾燥、熱風乾燥、滾筒乾燥或其他適用本發明之乾燥方法。於本實施例中，100 L的蝙蝠蛾擬青黴菌絲體發酵液使用冷凍乾燥方法，可製得約5kg凍乾粉。(3) Fermentation tank culture: Inoculate the mycelium cultured in the flask in (2) into the fermentation tank at 15 to 30 ℃, tank pressure 0.5 to 1.0 kg/cm 2 ,
(4)萃取物製備：將蝙蝠蛾擬青黴菌絲體發酵液凍乾粉分為兩份，分別加入10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121℃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蝙蝠蛾擬青黴發酵菌絲體水萃物。以乙醇為溶劑的萃取物，以超音波震盪3小時，離心後取上清液，將上清液經減壓濃縮得蝙蝠蛾擬青黴發酵菌絲體醇萃物。(4) Extract preparation: Divide the freeze-dried powder from the fermentation broth of Paecilomyces hepialis mycelium into two parts, and add 10 times the weight of distilled water and ethanol for extraction. The water-solvent extract was heated at 121°C for 30 minutes, and after cooling, the supernatant was centrifuged to obtain the supernatant, which was dried by freeze-drying to obtain an aqueous extract of the fermentation mycelium of Paecilomyces hepialis. The extract with ethanol as solvent was oscillated by ultrasonic for 3 hours, centrifuged and the supernatant was taken, and the supernatant was concentrated under reduced pressure to obtain an alcohol extract of the fermentation mycelium of Paecilomyces hepialis.
經由萃取步驟，可取得含較高濃度之蝙蝠蛾擬青黴菌絲體活性物質的水萃物及醇萃物。蝙蝠蛾擬青黴菌絲體活性物質的態樣包含蝙蝠蛾擬青黴菌絲體發酵液(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、水萃物或醇萃物凍乾粉或其他劑型。以下實施例二中，係以蝙蝠蛾擬青黴菌絲體凍乾粉作為活性物質態樣。實施例二 蝙蝠蛾擬青黴菌絲體凍乾粉改善急性肺損傷 (ALI) 之分析 Through the extraction step, water extracts and alcohol extracts containing higher concentrations of active substances from Paecilomyces hepialis mycelium can be obtained. The active substance of Paecilomyces hepialis mycelium includes the fermentation broth (mycelium and clarified liquid) of the mycelium of Paecilomyces hepipipes, freeze-dried powder of the fermentation broth, water extract, alcohol extract, water extract and alcohol Extract mixture, water extract or alcohol extract freeze-dried powder or other dosage forms. In the following example 2, the lyophilized powder of Paecilomyces hepipiens mycelium is used as the active substance. Example 2 Analysis of the improvement of acute lung injury (ALI) by freeze-dried powder of Paecilomyces hepialis mycelium
細菌感染為ALI主要的危險因子，革蘭氏陰性菌為其中一大類，其外套膜的主成分為內毒素，又名脂多醣 (lipopolysaccride; LPS)。目前已有實驗利用內毒素誘發的ALI疾病動物模式。可參照Yunhe Fu et al. (2017), Protective effect of TM6 on LPS-induced acute lung injury in mice,SCIENTIFIC REPORTS , 7:572. 根據此篇論文中利用LPS建立老鼠的急性肺部發炎模式，在鼻腔內intranasally (i.n.)引入LPS 50 μg / 20 μL造成急性肺損傷，後續進行肺部病理、肺泡沖洗液總細胞數、蛋白質濃度及各種細胞激素之變化分析，用以評估合成肽(cell-permeable TIR domain-derived decoy peptide)對ALI的改善結果。在許多致病源中 LPS 是廣泛被接受造成肺部急性發炎最佳的誘發物，也是最接近臨床上急性發炎的模式。因此，若能抑制LPS造成的肺部發炎、免疫調解作用及機制即能達到改善ALI的效果。本實驗中亦以LPS建立小鼠的急性肺部發炎模式，進行肺部病理、肺泡沖洗液總細胞數、蛋白質濃度及各種細胞激素之變化分析，評估蝙蝠蛾擬青黴菌絲體活性物質對ALI的改善結果。Bacterial infection is the main risk factor for ALI. Gram-negative bacteria are one of the major categories. The main component of the mantle is endotoxin, also known as lipopolysaccride (LPS). There have been experiments using animal models of ALI disease induced by endotoxin. You can refer to Yunhe Fu et al. (2017), Protective effect of TM6 on LPS-induced acute lung injury in mice, SCIENTIFIC REPORTS , 7:572. According to this paper, LPS is used to establish the acute lung inflammation model in mice, in the nasal cavity LPS 50 μg / 20 μL was introduced intranasally (in) to cause acute lung injury. Follow-up analysis of lung pathology, total cell number of alveolar washing fluid, protein concentration and various cytokines were used to evaluate the synthetic peptide (cell-permeable TIR). domain-derived decoy peptide) to improve the results of ALI. Among many pathogenic sources, LPS is widely accepted as the best inducer of acute lung inflammation, and it is also the closest clinical model of acute inflammation. Therefore, if the lung inflammation and immune mediation effects and mechanisms caused by LPS can be inhibited, the effect of improving ALI can be achieved. In this experiment, LPS was also used to establish the acute lung inflammation model in mice, and the changes in lung pathology, total cell number of alveolar washing fluid, protein concentration and various cytokines were analyzed to evaluate the effect of active substances from Paecilomyces hepipiens mycelium on ALI. Improved results.
(1) 運用BALB/c 小鼠進行動物實驗，分為腹腔注射及口服投予樣品組兩大組。每大組再分為控制組、LPS組、Ph(蝙蝠蛾擬青黴活性物質)組、Dexamethasone (***, DEX， 溶於生理食鹽水中，濃度為5 μg/g，腹腔注射50 μL)組，共計4組，每組使用6隻小鼠。(1) BALB/c mice were used for animal experiments, and they were divided into two groups: intraperitoneal injection and oral administration of the sample group. Each group is further divided into control group, LPS group, Ph (Paecilomyces hepialid active substance) group, Dexamethasone (dexamethasone, DEX, dissolved in saline, concentration of 5 μg/g, intraperitoneal injection of 50 μL) group , A total of 4 groups, each group uses 6 mice.
(2) 利用腹腔注射投予蝙蝠蛾擬青黴菌絲體凍乾粉後(蝙蝠蛾擬青黴菌絲體凍乾粉溶於水:酒精=1:1溶液，濃度為0.5 mg/g，腹腔注射50 μL)，30分鐘後由鼻腔投予內毒素(LPS)。24小時後，犠牲動物取後檢體進行後述分析。(2) After administering the freeze-dried powder of Paecilomyces hepipiens mycelium by intraperitoneal injection (the lyophilized powder of Paecilomyces hepialis mycelium is dissolved in water: alcohol=1:1 solution, the concentration is 0.5 mg/g, intraperitoneal injection 50 μL), 30 minutes later, endotoxin (LPS) was administered through the nasal cavity. Twenty-four hours later, the animals were taken and the specimens were analyzed as described later.
(3) 利用口服投予蝙蝠蛾擬青黴菌絲體凍乾粉每天一次，共計三次(蝙蝠蛾擬青黴菌絲體凍乾粉溶於水:酒精=1:1溶液，濃度為0.5 mg/g，餵食體積為200 μL)，第3天口服投予蝙蝠蛾擬青黴30分鐘後由鼻腔投予內毒素 (LPS)。24小時後，犠牲動物取後檢體進行後述分析。(3) The lyophilized powder of Paecilomyces hepipialis mycelium was administered orally once a day, three times in total (The lyophilized powder of Paecilomyces hepialis mycelium was dissolved in water: alcohol=1:1 solution, and the concentration was 0.5 mg/g , The feeding volume is 200 μL), and 30 minutes after oral administration of Paecilomyces hepipiens on the 3rd day, endotoxin (LPS) was administered through the nose. Twenty-four hours later, the animals were taken and the specimens were analyzed as described later.
(4) 肺組織病理形態觀察：取一半未進行肺泡灌洗小鼠的右肺立即用福馬林液固定，製成石蠟切片，常規HE染色，在光學顯微鏡下觀察其病理組織學變化。(4) Observation of lung tissue pathology: half of the right lungs of mice without alveolar lavage were immediately fixed with formalin, made into paraffin sections, stained with conventional HE, and histopathological changes were observed under an optical microscope.
(5) 氣管肺泡灌洗術 (Bronchoalveolar lavage; BAL)： 另一半小鼠用1 ml PBS經氣管插管灌洗三次，以取得支氣管肺泡腔中的內含物沖出取得沖洗液(BAL fluid; BALF)。離心後，細胞的部分進行血球分類與計數；上清液的部分，利用布拉德福蛋白質定量法(Bradford protein assay)進行蛋白質濃度分析。(5) Bronchoalveolar lavage (BAL): The other half of the mice were lavaged three times with 1 ml PBS through tracheal intubation to obtain the contents in the bronchoalveolar cavity to flush out the flushing fluid (BAL fluid; BALF). After centrifugation, the part of the cells was sorted and counted; the part of the supernatant was analyzed for protein concentration by Bradford protein assay.
(6) 白血球計數與種類分析：利用流式細胞儀分析：為靈敏度高且人為誤差小的分析方法，將血液與BALF檢體利用白血球的表面具有專一性抗原的特性，進行白血球分型，包括有(1)分析BALF中細胞總數；(2)白血球 (CD45)總數；(3)吞噬細胞(CD45+/CD11b+)總數；(4)嗜中性球(CD45+/Ly6G+)總數；(5)巨噬細胞[(CD45+/CD11b+)-(CD45+/Ly6G+)]總數。(6) White blood cell count and type analysis: Analysis by flow cytometry: For the analysis method with high sensitivity and small human error, the blood and BALF specimens are used for white blood cell surface with the characteristics of specific antigen to perform white blood cell typing, including Yes (1) analyze the total number of cells in BALF; (2) the total number of white blood cells (CD45); (3) the total number of phagocytes (CD45+/CD11b+); (4) the total number of neutrophils (CD45+/Ly6G+); (5) macrophages The total number of cells [(CD45+/CD11b+)-(CD45+/Ly6G+)].
統計方法：實驗數據皆以平均值及標準差(Mean ± standard deviation, SD) 表示，統計以 one-way ANOVA 進行分析，並以 LSD事後檢定比較各組差異，分析結果若p小於0.05，視為具有統計上的顯著意義。Statistical method: All experimental data are expressed as mean and standard deviation (Mean ± standard deviation, SD). Statistics are analyzed by one-way ANOVA, and differences between groups are compared by LSD post-test. If the analysis result is less than 0.05, it is regarded as It is statistically significant.
第1圖為蝙蝠蛾擬青黴菌絲體活性物質(經由腹腔注射)之急性肺損傷模式測試結果。其中蝙蝠蛾擬青黴菌絲體凍乾粉(Ph mycelia, 0.5 mg/g)與Dexathemethasone (DEX, 5 μg/g)組係經由腹腔注射投予至BALB/c小鼠 30 分鐘後，再利用內毒素(LPS)經由鼻腔吸入誘發急性肺發炎。結果如第1圖所示， 對照組(Control)組未見明顯改變， 肺泡結構多數保持完整。LPS only組(負對照組)肺組織病變明顯，肺泡腔結構破壞、融合，而Ph mycelia組及Dex組小鼠肺部病情情況明顯減輕，接近於對照組。上述結果顯示蝙蝠蛾擬青黴菌絲體活性物質可有效改善肺部發炎病理變化。Figure 1 shows the results of the acute lung injury model test of the active substance of Paecilomyces hepipipes mycelium (via intraperitoneal injection). Among them, the lyophilized powder of Paecilomyces hepialis (Ph mycelia, 0.5 mg/g) and Dexathemethasone (DEX, 5 μg/g) were injected into BALB/c mice by intraperitoneal injection for 30 minutes, and then reused. Inhalation of toxins (LPS) through the nasal cavity induces acute lung inflammation. The results are shown in Figure 1. There was no significant change in the control group, and most of the alveolar structure remained intact. LPS only group (negative control group) had obvious lung tissue lesions, and the structure of alveolar cavity was destroyed and fused, while the lung disease of the Ph mycelia group and Dex group was significantly reduced, which was close to the control group. The above results show that the active substance of Paecilomyces hepialum mycelium can effectively improve the pathological changes of lung inflammation.
此外，於腹腔注射蝙蝠蛾擬青黴菌絲體凍乾粉後分析內毒素誘發肺部發炎所導致的蛋白質滲出反應，發現蝙蝠蛾擬青黴菌絲體凍乾粉可有效降低蛋白質滲出反應，參照第2圖及下表2。In addition, after intraperitoneal injection of the lyophilized powder of Paecilomyces hepipialis mycelium, the protein exudation reaction caused by endotoxin-induced lung inflammation was analyzed, and it was found that the lyophilized powder of Paecilomyces hepipialis mycelium can effectively reduce the protein exudation reaction. Figure 2 and Table 2 below.
表2 腹腔注射蝙蝠蛾擬青黴菌絲體活性物質蛋白質滲出測試結果
於腹腔注射蝙蝠蛾擬青黴菌絲體凍乾粉後分析內毒素誘發肺部發炎所導致的(A)白血球(CD45)、(B) 吞噬細胞 (CD45+/CD11b+)、及(C) 嗜中性球 (CD45+/Ly6G+)浸潤反應，發現蝙蝠蛾擬青黴菌絲體凍乾粉可有效改善白血球、吞噬細胞及嗜中性白血球 (PMN) 浸潤如第3圖(A), (B), (C)及下表3-5。After intraperitoneal injection of the lyophilized powder of Paecilomyces hepipiens mycelium, the endotoxin-induced lung inflammation was analyzed for (A) white blood cells (CD45), (B) phagocytes (CD45+/CD11b+), and (C) neutrophils (CD45+/Ly6G+) infiltration reaction, it is found that the freeze-dried powder of Paecilomyces hepipiens mycelium can effectively improve the infiltration of white blood cells, phagocytes and neutrophils (PMN) as shown in Figure 3 (A), (B), (C ) And the following table 3-5.
表3 腹腔注射蝙蝠蛾擬青黴菌絲體活性物質之白血球浸潤測試結果
表4 腹腔注射蝙蝠蛾擬青黴菌絲體活性物質之吞噬細胞浸潤測試結果
表5 腹腔注射蝙蝠蛾擬青黴菌絲體活性物質之嗜中性球浸潤測試結果
相似的，蝙蝠蛾擬青黴菌絲體凍乾粉(Ph mycelia, 0.5 mg/g)可經由口服投予至BALB/c小鼠3天後，再利用內毒素(LPS)經由鼻腔吸入誘發急性肺發炎。其測試結果如第4圖所示，對照組(Control)組未見明顯改變，肺泡結構多數保持完整。LPS only組(負對照組)肺組織病變明顯，肺泡腔結構破壞、融合，而Ph mycelia組及Dex組小鼠肺部病情情況明顯減輕。顯示蝙蝠蛾擬青黴菌絲體凍乾粉以口服方式施用，亦可有效改善肺部發炎病理變化。Similarly, the lyophilized powder of Paecilomyces hepialis mycelium (Ph mycelia, 0.5 mg/g) can be administered orally to BALB/c mice for 3 days, and then endotoxin (LPS) is used to induce acute lung injury via nasal inhalation. fever. The test results are shown in Figure 4. There was no significant change in the control group, and most of the alveolar structure remained intact. LPS only group (negative control group) had obvious lung tissue pathological changes, and the structure of alveolar cavity was destroyed and fused, while the lung disease of mice in Ph mycelia group and Dex group was significantly reduced. It shows that the lyophilized powder of Paecilomyces hepipiens mycelium can be used orally to improve the pathological changes of lung inflammation.
於口服投予蝙蝠蛾擬青黴菌絲體凍乾粉後分析內毒素誘發肺部發炎所導致的蛋白質滲出反應，發現蝙蝠蛾擬青黴菌絲體凍乾粉可有效降低蛋白質滲出反應，請參照第5圖及下表6。After oral administration of the lyophilized powder of Paecilomyces hepipialis mycelium, the protein exudation reaction caused by endotoxin-induced lung inflammation was analyzed. It was found that the lyophilized powder of Paecilomyces hepipialis mycelium can effectively reduce the protein exudation reaction. Please refer to the article Figure 5 and Table 6 below.
表6 口服投予蝙蝠蛾擬青黴菌絲體活性物質蛋白質滲出測試結果
於口服投予蝙蝠蛾擬青黴菌絲體凍乾粉後分析內毒素誘發肺部發炎所導致的(A)白血球(CD45)、(B) 吞噬細胞 (CD45+/CD11b+)、及(C) 嗜中性球 (CD45+/Ly6G+)浸潤反應，發現蝙蝠蛾擬青黴菌絲體凍乾粉可有效改善白血球、吞噬細胞及嗜中性白血球 (PMN) 浸潤如第6圖(A), (B), (C)及下表7-9。After oral administration of the lyophilized powder of Paecilomyces hepipiens mycelium, analysis of endotoxin-induced lung inflammation caused (A) white blood cells (CD45), (B) phagocytes (CD45+/CD11b+), and (C) mesophilia Infiltration reaction of sex bulbs (CD45+/Ly6G+), it was found that the lyophilized powder of Paecilomyces hepipiens mycelium can effectively improve the infiltration of white blood cells, phagocytes and neutrophils (PMN) as shown in Figure 6 (A), (B), ( C) and Table 7-9 below.
表7 口服投予蝙蝠蛾擬青黴菌絲體活性物質白血球浸潤測試結果
表8 口服投予蝙蝠蛾擬青黴菌絲體活性物質吞噬細胞浸潤測試結果
表9 口服投予蝙蝠蛾擬青黴菌絲體活性物質嗜中性球浸潤測試結果
本發明之蝙蝠蛾擬青黴菌絲體活性物質經上述實驗，已證實具有改善急性肺損傷(ALI)之作用，當可預期可達到預防急性肺損傷之作用。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。實施例三：組合物的製備 The active substance of Paecilomyces hepialum mycelium of the present invention has been proved to have the effect of improving acute lung injury (ALI) through the above experiments, and it can be expected to achieve the effect of preventing acute lung injury. It can be combined with pharmaceutically acceptable carriers, excipients, diluents or adjuvants to prepare pharmaceutical compositions, and can also be used as food additives. Example 3: Preparation of the composition
本發明提供一組合物，其包含蝙蝠蛾擬青黴菌絲體活性物質，以製備為醫藥組合物，亦可作為保健營養食品。The present invention provides a composite comprising active substances from Paecilomyces hepipiens mycelium, which is prepared as a pharmaceutical composition and can also be used as a health and nutritious food.
該組合物進一步包含添加劑。在一較佳的實施態樣中，該添加劑可為賦型劑、防腐劑、稀釋劑、填充劑、吸收促進劑、甜味劑、潤滑劑、黏稠劑、或其組合。該賦型劑可選自檸檬酸鈉、碳酸鈣、磷酸鈣、蔗糖或其組合。該防腐劑可延長醫藥組合物的儲藏期限，例如苯甲醇、對羥基苯甲酸(parabens)、二氧化矽或其組合。該稀釋劑可選自水、乙醇、丙二醇、甘油或其組合。該填充劑可選自乳糖、牛乳糖、高分子量舉乙二醇或其組合。該吸收促進劑可選自二甲基亞碸(DMSO)、月桂氮卓酮、丙二醇、甘油、聚乙二醇或其組合。該甜味劑可選自安塞甜(Acesulfame K)、阿斯巴甜(aspartame)、糖精(saccharin)、三氯蔗糖／蔗糖素(sucralose)、紐甜(neotame)或其組合。該潤滑劑可選自硬脂酸鎂或***膠。該黏稠劑可為玉米澱粉。除上述所列舉的添加劑以外，在不影響組合物的醫藥效果前提下，可依需求選用適合的其他添加劑。The composition further contains additives. In a preferred embodiment, the additives can be excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, lubricants, thickeners, or combinations thereof. The excipient can be selected from sodium citrate, calcium carbonate, calcium phosphate, sucrose or a combination thereof. The preservative can extend the shelf life of the pharmaceutical composition, such as benzyl alcohol, parabens, silicon dioxide or a combination thereof. The diluent can be selected from water, ethanol, propylene glycol, glycerin or a combination thereof. The filler can be selected from lactose, nougat, high molecular weight ethylene glycol, or a combination thereof. The absorption enhancer can be selected from dimethyl sulfide (DMSO), azone, propylene glycol, glycerin, polyethylene glycol, or a combination thereof. The sweetener can be selected from Acesulfame K, aspartame, saccharin, sucralose, neotame or a combination thereof. The lubricant can be selected from magnesium stearate or gum arabic. The thickener may be corn starch. In addition to the additives listed above, other suitable additives can be selected according to requirements without affecting the medicinal effect of the composition.
該組合物於醫藥領域中可開發為不同商品。在一較佳實施態樣中，該組合物為一藥品、飼料、飲料、營養補充品、乳製品、食品或保健食品。The composition can be developed into different commodities in the field of medicine. In a preferred embodiment, the composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.
該組合物可根據受施予者之需要，而採用不同形態。在一較佳實施態樣中，該組合物的形態為粉劑、錠劑、造粒、栓劑、微膠囊、安瓶(ampoule/ampule)、液劑噴劑或塞劑。The composition can adopt different forms according to the needs of the recipient. In a preferred embodiment, the composition is in the form of powder, lozenge, granulation, suppository, microcapsule, ampoule/ampule, liquid spray or suppository.
本發明的組合物可使用於動物或是人類。在不影響效果的前提下，該組合物可製為任何藥物型態，並根據藥物型態以適用的途徑施予該動物或人類。The composition of the present invention can be used in animals or humans. Under the premise of not affecting the effect, the composition can be prepared into any pharmaceutical form, and administered to the animal or human by an applicable route according to the pharmaceutical form.
組合物製備Composition preparation
本發明的蝙蝠蛾擬青黴菌絲體活性物質若應用於食品用途，則以下組合物1的態樣作為例示性實例。If the active substance of Paecilomyces hepialum mycelium of the present invention is applied to food applications, the following aspect of
組合物1：取蝙蝠蛾擬青黴菌絲體活性物質凍乾粉(20 wt%)與作為潤滑劑的硬脂酸鎂(8wt%)、作為防腐劑的二氧化矽(7 wt%)充分混合，並溶於純水(65 wt%)中，存放於4℃備用。前述wt%係指各成分佔組合物總重之比例。Composition 1: Take the lyophilized powder (20 wt%) of the active substance of Paecilomyces hepipiens mycelium, magnesium stearate (8wt%) as a lubricant, and silicon dioxide (7 wt%) as a preservative and mix thoroughly , And dissolve in pure water (65 wt%), store at 4℃ for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.
本發明的蝙蝠蛾擬青黴菌絲體活性物質若以液體劑型應用於醫藥用途，則以下組合物2的態樣作為例示性實例。 組合物2：取蟬花菌絲體活性物質凍乾粉(20 wt%)與作為甜味劑的蔗糖素(8wt%)、作為潤滑劑的***膠(7 wt%)、作為賦型劑的蔗糖(10 wt%)充分混合，並溶於純水(55 wt%)中，存放於4℃備用。前述wt%係指各成分佔組合物總重之比例。If the active substance of Paecilomyces hepialum mycelium of the present invention is applied in a liquid dosage form for medical purposes, the following
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為準。Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Those of ordinary knowledge in the field, after considering the above teachings, can make appropriate modifications to the content of the above embodiments, and still achieve the effects claimed in this case. Therefore, the scope of protection of the present invention should be subject to the scope of the patent application appended thereafter.
第1圖繪示蝙蝠蛾擬青黴菌絲體活性物質(經由腹腔注射)之急性肺發炎肺損傷模式測試結果。Figure 1 shows the results of the acute lung inflammation lung injury model test results of the active substance of Paecilomyces hepialis mycelium (via intraperitoneal injection).
第2圖繪示蝙蝠蛾擬青黴菌絲體活性物質(經由腹腔注射)之蛋白質滲出測試結果。Figure 2 shows the results of the protein exudation test of the active substance from Paecilomyces hepialis mycelium (via intraperitoneal injection).
第3圖繪示蝙蝠蛾擬青黴菌絲體活性物質(經由腹腔注射)的(A)白血球浸潤、(B)吞噬細胞及(C)嗜中性白血球(PMN)測試結果。Figure 3 shows the test results of (A) leukocyte infiltration, (B) phagocytic cells and (C) neutrophil (PMN) of the active substance of Paecilomyces hepipiens mycelium (via intraperitoneal injection).
第4圖繪示蝙蝠蛾擬青黴菌絲體活性物質(經由口服投予)之急性肺發炎肺損傷模式測試結果。Figure 4 shows the results of the acute lung inflammation lung injury model test results of the active substance from Paecilomyces hepipiens mycelium (administered orally).
第5圖繪示蝙蝠蛾擬青黴菌絲體活性物質(經由口服投予)之蛋白質滲出測試結果。Figure 5 shows the results of the protein exudation test of the active substance from Paecilomyces hepialis mycelium (administered orally).
第6圖繪示蝙蝠蛾擬青黴菌絲體活性物質(經由口服投予)的(A)白血球浸潤、(B)吞噬細胞及(C)嗜中性白血球(PMN)測試結果。Figure 6 shows the test results of (A) leukocyte infiltration, (B) phagocytic cells and (C) neutrophil (PMN) of the active substance of Paecilomyces hepialis mycelium (administered orally).
TW中華民國 財團法人食品工業發展研究所生物資源保存及研究中心 2019/03/06 BCRC930203TW The Bioresource Conservation and Research Center of the Food Industry Development Research Institute of the Republic of China 2019/03/06 BCRC930203
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Non-Patent Citations (1)
| Title |
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| Thakur, Asmitananda et al. "Pro-apoptotic effects of Paecilomyces hepiali, a Cordyceps sinensis extract on human lung adenocarcinoma A549 cells in vitro." Journal of cancer research and therapeutics vol. 7,4 (2011): 421-426. * |
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