CN110522771A - Antrodia camphorata mycelium active material is used to improve the purposes of chronic obstructive pulmonary disease - Google Patents

Antrodia camphorata mycelium active material is used to improve the purposes of chronic obstructive pulmonary disease Download PDF

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CN110522771A
CN110522771A CN201810761894.4A CN201810761894A CN110522771A CN 110522771 A CN110522771 A CN 110522771A CN 201810761894 A CN201810761894 A CN 201810761894A CN 110522771 A CN110522771 A CN 110522771A
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antrodia camphorata
camphorata mycelium
active material
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culture
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陈劲初
李宜蓁
林定威
张晓苓
陈彦博
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Grape King Bio Ltd
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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06Fungi, e.g. yeasts
    • A61K36/07Basidiomycota, e.g. Cryptococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10Preparation or pretreatment of starting material
    • A61K2236/19Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment

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Abstract

The present invention provides a kind of Antrodia camphorata mycelium active material and is used to prepare the purposes for improving the composition of chronic obstructive pulmonary disease.The Antrodia camphorata mycelium active material is prepared with the following steps: (a) being taken an Antrodia camphorata mycelium (Antrodia cinnamomea) on plating medium, is cultivated 4-10 days at a temperature of 15-30 DEG C;(b) Antrodia camphorata mycelium after step (a) culture is seeded in a flask, in 15-30 DEG C, environment culture 3-14 days of pH 2-6;(c) Antrodia camphorata mycelium after step (b) culture is inoculated in a fermentation tank, stir culture 3-21 days in the environment of 15-30 DEG C, pH2-6 forms the Antrodia camphorata mycelium fermented liquid containing the Antrodia camphorata mycelium active material.

Description

Antrodia camphorata mycelium active material is used to improve the purposes of chronic obstructive pulmonary disease
Technical field
A kind of preparation method and its usage of the present invention about Antrodia camphorata mycelium active material.Specifically, about preparation A kind of Antrodia camphorata mycelium active material and its purposes in food or pharmaceuticals for improving chronic obstructive pulmonary disease.
Background technique
Chronic Obstructive Pulmonary Disease (Chronic Obstructive Pulmonary Disease, abbreviation COPD) is current The 4th of the global cause of death is occupied, is a kind of common, multiple, high disability rate and high lethality rate chronic respiratory disease.Entirely The patient of world's chronic obstructive pulmonary disease has more than 300,000,000 people.
Smoking is the main reason for leading to COPD, so in recent years because of locomotive, the waste air of automobile and industrial power generation and combustion Oily coal-fired use increases, and causes the air pollution of Particulate Matter (PM) 2.5 to rise, more aggravates this disease Occur.It is chronically exposed in the environment of pollution, lung can be made to generate inflammatory response, airflow channel is caused to narrow and generate lung tissue Dysfunction.
The therapeutic modality of COPD includes smoking cessation at present, lung rehabilitation, imbedibility bronchodilators, solid using cortex class Alcohol, long-term oxygen supply or progress lung transplantation etc..2015 it is newest by new drug be to merge two kinds of muscle for being used in COPD in the past Relaxant does not have breakthrough new drug development yet so far.Therefore, research effectively treats the method for chronic obstructive pulmonary disease still It is important developing direction.
Antrodia (Antrodia cinnamomea), also known as camphor tree mushroom, camphor tree wild rice, mushroom in camphor tree, cinnamomum kanahirai hay mushroom, Antrodia camphorata, red camphor tree, Red antrodia, the rotten heartwood inner wall of the cinnamomum kanehirai dry rot being only grown between height above sea level 450-1500 meters of TaiWan, China mountain area or it is withered fall The dark moist surface of the Cinnamomum kanahirai hay timber of volt, belongs to the precious medicinal fungi of TaiWan, China national treasure grade.Still lack antrodia pair at present Improve the research of chronic obstructive pulmonary disease.
Summary of the invention
The present invention provides a kind of Antrodia camphorata mycelium active material and preparation method thereof, and can be used for preparing tool improves chronic resistance The composition of plug property tuberculosis.Compared to general Western medicine and treatment method, the invention discloses liquid state fermentation Antrodia camphorata mycelium activity The preparation method of substance is safer, easy, and manufactured Antrodia camphorata mycelium active material is more natural, safe, and can be effectively improved COPD。
An embodiment according to the present invention provides a kind of Antrodia camphorata mycelium active material and is used to prepare improvement chronic obstructive The purposes of the composition of tuberculosis, wherein Antrodia camphorata mycelium active material is prepared with the following steps:
(a) Antrodia camphorata mycelium (Antrodia cinnamomea) is taken on plating medium, at a temperature of 15-30 DEG C Culture 4-10 days;
(b) Antrodia camphorata mycelium after step (a) culture is seeded in flask, in 15-30 DEG C, the environment culture of pH 2-6 3-14 days;
(c) Antrodia camphorata mycelium after step (b) culture is inoculated in fermentation tank, in the environment of 15-30 DEG C, pH2-6 Stir culture 3-21 days, form the Antrodia camphorata mycelium fermented liquid containing Antrodia camphorata mycelium active material.
In one embodiment, the step of preparing Antrodia camphorata mycelium active material further includes step (d): will be Antrodia camphorata mycelium fermented It is milled after liquid freeze-drying, forms the Antrodia camphorata mycelium freeze-dried powder containing Antrodia camphorata mycelium active material.
In one embodiment, the step of preparing Antrodia camphorata mycelium active material further includes step (e): Antrodia camphorata mycelium is lyophilized Powder forms the Antrodia camphorata mycelium extract liquor containing Antrodia camphorata mycelium active material with an at least solvent extraction.
In one embodiment, the step of preparing Antrodia camphorata mycelium active material further includes step (f): Antrodia camphorata mycelium is extracted Liquid is dry, to obtain Antrodia camphorata mycelium active material.
In one embodiment, the solvent in step (e) is selected from water or alcohols.
In one embodiment, when the solvent of step (e) is water, the effective concentration of Antrodia camphorata mycelium active material between 0.25-1mg/ml。
In one embodiment, when the solvent of step (e) is alcohols, the effective concentration of Antrodia camphorata mycelium active material between 25-100μg/ml。
In one embodiment, the flask culture of above-mentioned steps (b) is shake culture, and revolving speed is 110-130rpm.
In one embodiment, the fermentation tank in step (c) is further passed through a gas, this gas includes air, oxygen, dioxy Change carbon, helium or combinations thereof, the slot pressure of fermentation tank is 0.5-1.0kg/cm2And Ventilation Rate is 0.01-1.5VVM.
In one embodiment, the composition is medical composition, this medical composition further includes pharmaceutically acceptable Carrier, excipient, diluent or adjuvant.
In one embodiment, the composition is food additives.
Detailed description of the invention
Antrodia camphorata mycelium water extraction object, which is shown, in Fig. 1 improves cigarette smoke extract induction alveolar epithelium A549 cell damage Ability as a result, wherein AcH (L), AcH (M) and AcH (H) respectively refer to Antrodia camphorata mycelium water extraction object concentration be 0.25mg/ml, 0.5mg/ml and 1.0mg/ml.
Antrodia camphorata mycelium alcohol extraction object, which is shown, in Fig. 2 improves cigarette smoke extract induction alveolar epithelium A549 cell damage Ability as a result, wherein AcMe (L), AcMe (M) and AcMe (H) respectively refer to Antrodia camphorata mycelium alcohol extraction object concentration be 25 μ g/ml, 50 μ g/ml and 100 μ g/ml.
Specific embodiment
To keep above and other aspect of the invention apparent understandable, special embodiment below, and cooperate institute's accompanying drawings It is described in detail.
One Antrodia camphorata mycelium culture of embodiment
The Antrodia camphorata mycelium (Antrodia cinnamomea) that the present embodiment uses is purchased from juridical person's development of food industry Research institute's (FIRDI) living resources save and research center (BCRC, preceding title CCRC), number CCRC-35396.However, available In antrodia kind of the invention, that it's not limited to that is individually a kind of, may also include BCRC number 35396,35398,35716, 36401,36711,36795,37848,37849,37850,37889,37890,37891,37893,37894,37941 or and its The antrodia strain in allogene material registered data library, the present invention do not limit it simultaneously.
(1) plate culture: Antrodia camphorata mycelium is inoculated on plate, and 4-10 days (the present embodiment are cultivated at 15-30 DEG C In, it is to be cultivated 7 days at 25 DEG C).The ingredient of plating medium may include detrine culture medium (PotatoDextrose Agar, PDA), carbon source and nitrogen source, there is no particular restriction.
(2) flask culture: scraping (1) plate on mycelium be inoculated in flask, and with 15-30 DEG C, pH 2-6 and turn Fast 110-130rpm condition shake culture 3-14 days (in the present embodiment, the shake culture under 25 DEG C, pH5, revolving speed 120rpm 10 days).This shake culture is that culture medium shown in following table 1 is cultivated.
1 culture medium prescription of table
In above-mentioned culture medium prescription, soybean powder and ground cinnamon are comprehensive carbon nitrogen source, can be selected from cereal (such as: flour) or Beans (such as: soybean powder, mung bean flour, soy meal, ground cinnamon);Carbohydrate can be glucose, fructose, maltose, sucrose etc.;It is inorganic Salt can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferric sulfate etc..It illustrates, 1 culture medium prescription of table is only One of example, ingredient can adjust on demand when use, or the commercially available culture medium of collocation uses, and there is no particular restriction.
(3) fermentation tank culture: the mycelium of flask culture in (2) is inoculated in fermentation tank, with 15-30 DEG C, slot pressure 0.5-1.0kg/cm2, under the conditions of pH 2-6 and mixing speed 50-150rpm, with the Ventilation Rate culture 3-21 of 0.1-1.5VVM It, forming an Antrodia camphorata mycelium fermented liquid (is in 25 DEG C, slot pressure 0.5kg/cm in the present embodiment2, pH4, mixing speed 80rpm And under conditions of 1.0VVM (air), cultivate 14 days) to get Antrodia camphorata mycelium fermented liquid.The culture medium that fermentation tank culture uses It can be identical as the culture medium that step (2) flask culture uses.I.e. containing antrodia camphorata mycelium of the invention in this Antrodia camphorata mycelium fermented liquid Body active material.Antrodia camphorata mycelium fermented liquid further can be prepared as Antrodia camphorata mycelium fermented liquid freeze-drying by being freeze-dried step Powder;
(4) prepared by extract: Antrodia camphorata mycelium fermented liquid freeze-dried powder being divided into two parts, is separately added into the distillation of 20 times of weight Water and ethyl alcohol are extracted.The extract taken water as a solvent is heated 30 minutes with 100 DEG C of temperature, centrifuging and taking supernatant after cooling Antrodia fermentation mycelium water extraction object is dried to obtain in liquid, freeze-dried method.Using ethyl alcohol as the extract of solvent, shaken with ultrasonic It swings 1 hour, supernatant is taken after centrifugation, antrodia fermentation mycelium alcohol extraction object is concentrated under reduced pressure to obtain in supernatant;
Via extraction step, the water extraction object and alcohol extraction object of the Antrodia camphorata mycelium active material containing higher concentration can be obtained.Camphor tree The aspect of camphorata mycelium active material includes Antrodia camphorata mycelium fermented liquid (mycelium and clarified solution), fermentation liquid freeze-dried powder, water extraction Object, alcohol extraction object, water extraction object and alcohol extract the mixture or other dosage forms of object.It is to extract object and alcohol extraction object with water in following embodiment two As Antrodia camphorata mycelium active material aspect.
Two Antrodia camphorata mycelium water of embodiment, which extracts object and alcohol extraction object, improves the analysis of COPD
Have experiment at present and utilizes cigarette smoke extract (Cigarette smoke extract, CSE) and Human Lung Cancer Epithelial cell A549 is successfully established COPD mode.It can refer to Miaomiao Chenet al. (2015) .Azithromycin attenuates cigarette smoke extract-induced oxidative stress injury in human This paper of alveolar epithelial cells.MOLECULAR MEDICINE REPORTS, 11:3414-3422., Cell survival rate analysis (MTT assay) is carried out to CSE with Human Lung Cancer epithelial cell A549, to assess antibiotic Improvement result of the Azithromycin to COPD.CSE is important in chronic obstructive pulmonary disease mechanism to the damage of alveolar epithelium Process.Therefore, if can inhibit CSE that can achieve the effect that improve chronic obstructive pulmonary disease to the injury of alveolar epithelium.This experiment In cell survival rate analysis (MTT assay) is also carried out to CSE with Human Lung Cancer epithelial cell A549, assessment Antrodia camphorata mycelium is living Improvement result of the property substance to COPD.
(1) Human Lung Cancer epithelium A549 cell is collected, cell suspending liquid concentration is adjusted, it is close with 50,000-100,000/mL Degree is planted in 96 porose discs;
(2) 96 porose discs of (1) are placed in 37 DEG C, 5%CO2After incubator culture 16-24 hours, obtained with embodiment one , at Antrodia camphorata mycelium water extraction object (0,0.25,0.5,1mg/ml) and the alcohol extraction object (0,25,50,100 μ g/ml) of various dose Reason.Antrodia camphorata mycelium extract is dissolved in dimethyl Asia Shi Feng (Dimethyl sulfoxide, DMSO), DMSO when detection Concentration is no more than 0.1% to avoid the growth of its toxic effect cell;Control group handles 0.1%DMSO, and each concentration carries out triple It is multiple, as a result it is placed in 37 DEG C, 5%CO2Incubator culture 24 hours;
(3) result of above-mentioned (2) is first centrifuged 10min with 400 × g, removes supernatant, adds cigarette smoke extract CSE12.5% induces the cytotoxicity of A549, and control group is not handled.Then 37 DEG C, 5%CO are placed in2Incubator culture 6 hours;
(4) supernatant of (3) is removed, 100 μ l MTT (3- (4,5-Dimethylthiazol-2-yl)-are added in every hole 2,5-diphenyltetrazolium bromide) solution, after 37 DEG C are reacted 2 hours, termination culture is carefully removed in hole MTT solution;
(5) 100 μ l DMSO are added in every hole in (4), shake 2min, measure light absorption value in 570nm.This inhales brightness and represents Ability (the first of cell reduction MTTForming amount), i.e. the activity (number of viable cells) of grain wire body.It is got in the suction brightness of 570nm Height indicates that cell survival rate is higher.
Statistical analysis: experimental result data is indicated with average value (Mean) ± standard deviation (SD).Test data with Student's t-test statisticallys analyze its group difference.It is considered as if p < 0.05 with statistically significant difference.
Water extracts the experimental result of object as shown in Fig. 1 and the following table 2.Cigarette smoke extract CSE can induce A549 cytotoxicity, Cell survival rate is caused to decline.However, be added antrodia fermentation mycelium water extraction object can conspicuousness inhibit cellular damage, promoted survival Rate, and the concentration being added is higher, survival rate is higher.Being had using the Antrodia camphorata mycelium water extraction object in the embodiment of the present invention one is improved The effect of COPD, effective concentration is between 0.5-1mg/ml.
2 Antrodia camphorata mycelium water of table extracts object test result
(n=3)
# indicates statistically to have significant difference (p < 0.05) with control group (control)
* it indicates statistically to have significant difference (p < 0.05) with negative control group (CSE)
Alcohol extracts the experimental result of object as shown in Fig. 2 and the following table 3.As a result it again shows that, it is real that the antrodia zymophyte present invention is added The Antrodia camphorata mycelium alcohol extraction object applied in example one has the function of improvement COPD, and effective concentration is between 50-100 μ g/ml.
3 Antrodia camphorata mycelium alcohol of table extracts object test result
Group Survival rate (%)
Control group (Control) 100±2.93
CSE 84.51±0.70#
CSE+ Antrodia camphorata mycelium alcohol extracts 25 μ g/ml of object 84.63±1.91
CSE+ Antrodia camphorata mycelium alcohol extracts 50 μ g/ml of object 86.90±2.20
CSE+ Antrodia camphorata mycelium alcohol extracts 100 μ g/ml of object 89.92±2.89*
Antrodia camphorata mycelium alcohol extracts 100 μ g/ml of object 103.66±2.47
(n=3)
# indicates statistically to have significant difference (p < 0.05) with control group (control)
* it indicates statistically to have significant difference (p < 0.05) with negative control group (CSE)
Antrodia active material of the invention has proven to have the function of improving COPD through above-mentioned experiment.It can with pharmaceutically Acceptable carrier, excipient, diluent or adjuvant combine, and prepare medical composition, can also be used as food additives and be used.
Although the present invention has used embodiment disclosed above, so it is not intended to limiting the invention.This field general technology people Member when can be appropriately modified to the content of above-described embodiment, and still can reach the present invention and lead after considering teachings above in light of actual conditions The effect of opening.Therefore, protection scope of the present invention should be subject to claims.

Claims (11)

1. a kind of Antrodia camphorata mycelium active material is used to prepare the purposes for improving the composition of chronic obstructive pulmonary disease, wherein should Antrodia camphorata mycelium active material is prepared with the following steps:
(a) it takes an Antrodia camphorata mycelium on plating medium, is cultivated 4-10 days at a temperature of 15-30 DEG C;
(b) Antrodia camphorata mycelium after step (a) culture is seeded in a flask, in 15-30 DEG C, the environment culture 3- of pH 2-6 14 days;
(c) Antrodia camphorata mycelium after step (b) culture is inoculated in a fermentation tank, is stirred in the environment of 15-30 DEG C, pH2-6 Culture 3-21 days is mixed, the Antrodia camphorata mycelium fermented liquid for containing the Antrodia camphorata mycelium active material is formed.
2. purposes as described in claim 1, wherein the step of preparing the Antrodia camphorata mycelium active material further includes step (d): will be milled after the Antrodia camphorata mycelium fermented liquid freeze-drying, form the antrodia for containing the Antrodia camphorata mycelium active material Mycelium freeze-dried powder.
3. purposes as claimed in claim 2, wherein the step of preparing the Antrodia camphorata mycelium active material further includes step (e): by the Antrodia camphorata mycelium freeze-dried powder with an at least solvent extraction, being formed containing the Antrodia camphorata mycelium active material extremely A few Antrodia camphorata mycelium extract liquor.
4. purposes as claimed in claim 3, wherein the step of preparing the Antrodia camphorata mycelium active material further includes step (f): the Antrodia camphorata mycelium extract liquor is dry, to obtain the Antrodia camphorata mycelium active material.
5. purposes as described in claim 3 or 4, wherein in step (e), the solvent is selected from water or alcohols.
6. purposes as claimed in claim 5, wherein when the solvent of step (e) is water, the Antrodia camphorata mycelium activity The effective concentration of substance is between 0.5-1mg/ml.
7. purposes as claimed in claim 5, wherein when the solvent of step (e) is alcohols, the Antrodia camphorata mycelium is living The effective concentration of property substance is between 50-100 μ g/ml.
8. purposes as described in claim 1, wherein the flask culture of step (b) is shake culture, and revolving speed is 110- 130rpm。
9. purposes as described in claim 1, wherein fermentation tank described in step (c) is further passed through a gas, the gas packet Air, oxygen, carbon dioxide, helium or combinations thereof are included, the slot pressure of the fermentation tank is 0.5-1.0kg/cm2And Ventilation Rate is 0.01-1.5VVM。
10. purposes as described in claim 1, wherein the composition is medical composition, and the medical composition is further Include pharmaceutically acceptable carrier, excipient, diluent or adjuvant.
11. purposes as described in claim 1, wherein the composition is food additives.
CN201810761894.4A 2018-05-25 2018-07-12 Antrodia camphorata mycelium active material is used to improve the purposes of chronic obstructive pulmonary disease Pending CN110522771A (en)

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