CN110337991A - A kind of preparation and application of the inhibiting tumour cells agent based on Antrodia camphorata extract - Google Patents
A kind of preparation and application of the inhibiting tumour cells agent based on Antrodia camphorata extract Download PDFInfo
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- CN110337991A CN110337991A CN201910530075.3A CN201910530075A CN110337991A CN 110337991 A CN110337991 A CN 110337991A CN 201910530075 A CN201910530075 A CN 201910530075A CN 110337991 A CN110337991 A CN 110337991A
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- antrodia camphorata
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- tumour cells
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention discloses the preparations and application of a kind of inhibiting tumour cells agent based on Antrodia camphorata extract, including Antrodia camphorata, the Antrodia camphorata can prepare antrodia mycelia in the health medicine for preparing inhibiting tumour cells agent and extract Antrodia camphorata total triterpene, the antrodia mycelia is cultivated in the medium by Antrodia camphorata and is obtained, and the Antrodia camphorata total triterpene is concentrated and is centrifuged by antrodia mycelia and obtained.The preparation and application of the inhibiting tumour cells agent based on Antrodia camphorata extract, with good antitumous effect, and Antrodia camphorata fructification, mycelium and its liquid medium can be made with anti-hepatitis, protect liver, adjust immune, anti-aging, anti-inflammatory effect health food and drug, have very big Development volue and application prospect.
Description
Technical field
The present invention relates to field of biotechnology, the system of specially a kind of inhibiting tumour cells agent based on Antrodia camphorata extract
Standby and application.
Background technique
Malignant tumour is a kind of disease for seriously endangering human health and life.Root according to the statistics of the World Health Organization, 2012
Year, global ten thousand people about more than 1200 suffered from cancer, wherein more than 850 ten thousand people die of the disease.At the same time, newfound patient is being just
With annual 1800000 speed increase.China's third time human mortality's reason sample survey results are shown: malignant tumour has become
The first place of China's city dweller's cause of death accounts for the 25% of all dead sums.In recent years, related epidemiology of tumor, morbidity
The basis such as mechanism, clinical diagnosis and treatment and clinicing aspect etc. all achieve greater advance, filter out a variety of anti-tumor drugs.So
And oneself has at present the generally existing therapeutic efficiency of anti-tumor drug is low, poor selectivity, toxicity are big and oncocyte drug resistance etc. is prominent
It goes wrong.Therefore, toxicity is low, curative effect is high anticancer active constituent is found from natural animal and plant just into country's surgery in recent years
One of the hot spot of worker research；
Antrodia camphorata (Antrodia camphorata) also known as cinnamomum kanahirai hay mushroom, camphor tree mushroom or antrodia are ability quilts the 1990s
The novel species that biochemical boundary delivers is the distinctive fungi of TaiWan, China.Antrodia camphorata contain there are many physiologically active ingredient, as polysaccharide body,
Triterpene compound, Sudismase, adenosine, protein (contain immune protein), vitamin, microelement, nucleic acid, agglutinin,
Amino acid, steroid, cellulose, blood pressure stabilization substance etc., but most important physiologically active ingredient is polysaccharide (callose)
And triterpene compound, fructification, mycelium and the liquid fermentation production of Antrodia camphorata wild and cultivated are living rich in physiology
Property substance, almost non-toxic side effect, can be processed into antitumor, anti-hepatitis, protect liver, improve immunity, anti-aging and
The health food and drug of antiinflammation have very big Development volue and application prospect, mesh in Antrodia camphorata fructification extract
Preceding about discovery has 11 kinds of triterpenes, and mycelium then has 5 kinds of triterpenes, the triterpenes Zhankuic of sub-entities separation
AcidA, B, C and find successively 2 kinds with noval chemical compound Zhankuic acid D that lumistane (Ergostane) is skeleton,
Zhankuic acid E and equally from fructification separate using lumistane as the triterpene compound of skeleton: Antcin A,
The anticancer effect that Antcin B, Antcin C, Antcin E and Antcin F have nothing in common with each other.Therefore we have proposed one kind to be based on
The preparation and application of the inhibiting tumour cells agent of Antrodia camphorata extract.
Summary of the invention
The purpose of the present invention is to provide the preparation and application of a kind of inhibiting tumour cells agent based on Antrodia camphorata extract,
To solve the problems mentioned in the above background technology.
To achieve the above object, the invention provides the following technical scheme: a kind of tumour cell based on Antrodia camphorata extract
The preparation and application of inhibitor, including Antrodia camphorata, the Antrodia camphorata can in the health medicine for preparing inhibiting tumour cells agent
Prepare antrodia mycelia and extract Antrodia camphorata total triterpene, the antrodia mycelia cultivated in the medium by Antrodia camphorata and
, the Antrodia camphorata total triterpene is concentrated and is centrifuged by antrodia mycelia and obtained.
Preferably, the breeding method of the antrodia mycelia is as follows:
1), bio-safety station is irradiated and alcohol disinfecting 20 minutes by ultraviolet light, with blade by fresh Cinnamomum kanahirai hay
Sesame mushroom body wraps Antrodia camphorata mushroom body by removing on linden, and with clean gauze or polybag, is put into bio-safety station；
2), the Antrodia camphorata mushroom body surface face cleaning gauze wiping for speckling with 75% ethyl alcohol, is cut mushroom body surface skin with sharp pocket knife
It goes, the inside pulp is divided into 0.5 centimeter of fritter, is inoculated in culture dish, is closed the lid, setting cultivation temperature is 24.5
DEG C cultivated into 27.5 DEG C of environment, after Antrodia camphorata culture medium is covered by mycelia, adjustment cultivation temperature be 18.5 DEG C extremely
23.5 DEG C are continued culture until growing Antrodia camphorata fructification, and Antrodia camphorata fructification is collected after growing Antrodia camphorata fructification and is obtained
Antrodia camphorata without wood cultivation；
3) it, takes the Antrodia camphorata without wood cultivation to carry out sterile chopping fermentation to cultivate or using rice solid medium to ox
Antrodia carries out solid cultivation.
Preferably, the extraction step of the Antrodia camphorata total triterpene is as follows:
1), sample pretreatment: antrodia mycelia 10 is weighed to 20g, drying to constant weight, pulverizes and sieves, and is added 10 to 20
The dissolution of times water, is made testing sample solution；
2) it, is dissolved in solvent in testing sample solution obtained, and active carbon is added, filtering with microporous membrane takes filtrate standby
With；
3) 3 times of 95% alcohol refluxs of amount, are added in sample filtrate obtained to extract 3 times, each 1h；
4), by sample crude extract obtained concentration, centrifugation, standing 10-20min, precipitating is eluted 2-4 times using water, mistake
Filter to obtain precipitating；
5), precipitating obtained is added in 50ml measuring bottle, solvent constant volume is added, saturated sodium bicarbonate solution 5-10ml is added
Extraction 3-4 time, centrifuging and taking precipitate, dry Antrodia camphorata total triterpene substance extract.
Preferably, the condition that the fermentation is cultivated is that glucose 20g/L, wheat bran leachate 60g/L is added towards triangular flask,
VB10.14g/L, KH2PO41g/L, MgSO40.05g/L and the Antrodia camphorata 30g cultivated without wood, and mixture is in triangular flask
Natural pH, liquid amount 200mL/500mL cultivate 12 in 28 DEG C of constant temperature incubation environment by the stirring of revolving speed 110r/min
It.
Preferably, the condition of the rice solid medium is the Antrodia camphorata 30g and rice: water=4:7 without wood cultivation
Culture medium in do not add any other nutriment, cultivated 55 days under illumination condition.
Preferably, the solvent is to be uniformly mixed and obtain according to 1.15:10 in ursolic acid standard items and ethyl acetate.
It compared with prior art, the beneficial effects of the present invention are: should the inhibiting tumour cells agent based on Antrodia camphorata extract
Preparation and application, there is good antitumous effect, and Antrodia camphorata fructification, mycelium and its liquid medium can be made
At with anti-hepatitis, protect liver, adjust immune, anti-aging, anti-inflammatory effect health food and drug, there is very big Development volue
And application prospect.
Detailed description of the invention
Fig. 1 is that Antrodia camphorata extracting solution of the present invention can induce death of neoplastic cells figure；
Fig. 2 is cell inhibitory rate lab diagram of the present invention；
Fig. 3 is Antrodia camphorata extract of the present invention to tumour cell IC50 result curve figure；
Fig. 4 is that tumor cell migration of the present invention tests negative control figure；
Fig. 5 is that Antrodia camphorata extract of the present invention inhibits tumor cell migration figure；
Fig. 6 is flow cytomery result schematic diagram of the present invention；
Fig. 7 is that Antrodia camphorata extract of the present invention remarkably promotes apoptosis of tumor cells schematic diagram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Fig. 1-7 is please referred to, the present invention provides a kind of technical solution: a kind of inhibiting tumour cells based on Antrodia camphorata extract
The preparation and application of agent, including Antrodia camphorata, the Antrodia camphorata can be prepared in the health medicine for preparing inhibiting tumour cells agent
Antrodia mycelia and extraction Antrodia camphorata total triterpene, the antrodia mycelia are cultivated in the medium by Antrodia camphorata and are obtained, institute
Antrodia camphorata total triterpene is stated to be concentrated and be centrifuged by antrodia mycelia and obtained.
The breeding method of the antrodia mycelia is as follows:
1), bio-safety station is irradiated and alcohol disinfecting 20 minutes by ultraviolet light, with blade by fresh Cinnamomum kanahirai hay
Sesame mushroom body wraps Antrodia camphorata mushroom body by removing on linden, and with clean gauze or polybag, is put into bio-safety station；
2), the Antrodia camphorata mushroom body surface face cleaning gauze wiping for speckling with 75% ethyl alcohol, is cut mushroom body surface skin with sharp pocket knife
It goes, the inside pulp is divided into 0.5 centimeter of fritter, is inoculated in culture dish, is closed the lid, setting cultivation temperature is 24.5
DEG C cultivated into 27.5 DEG C of environment, after Antrodia camphorata culture medium is covered by mycelia, adjustment cultivation temperature be 18.5 DEG C extremely
23.5 DEG C are continued culture until growing Antrodia camphorata fructification, and Antrodia camphorata fructification is collected after growing Antrodia camphorata fructification and is obtained
Antrodia camphorata without wood cultivation；
3) it, takes the Antrodia camphorata without wood cultivation to carry out sterile chopping fermentation to cultivate or using rice solid medium to ox
Antrodia carries out solid cultivation, and the condition that the fermentation is cultivated is that glucose 20g/L, wheat bran leachate is added towards triangular flask
60g/L, VB10.14g/L, KH2PO41g/L, MgSO40.05g/L and the Antrodia camphorata 30g cultivated without wood, and mixed in triangular flask
Conjunction object is nature pH, liquid amount 200mL/500mL, by the stirring of revolving speed 110r/min, in 28 DEG C of constant temperature incubation environment
Culture 12 days, the condition of the rice solid medium are Antrodia camphorata 30g and rice: water=4:7 culture without wood cultivation
Any other nutriment is not added in base, is cultivated 55 days under illumination condition.
The extraction step of the Antrodia camphorata total triterpene is as follows:
1), sample pretreatment: antrodia mycelia 10 is weighed to 20g, drying to constant weight, pulverizes and sieves, and is added 10 to 20
The dissolution of times water, is made testing sample solution；
2) it, is dissolved in solvent in testing sample solution obtained, and active carbon is added, filtering with microporous membrane takes filtrate standby
With the solvent is to be uniformly mixed and obtain according to 1.15:10 in ursolic acid standard items and ethyl acetate；
3) 3 times of 95% alcohol refluxs of amount, are added in sample filtrate obtained to extract 3 times, each 1h；
4), by sample crude extract obtained concentration, centrifugation, standing 10-20min, precipitating is eluted 2-4 times using water, mistake
Filter to obtain precipitating；
5), precipitating obtained is added in 50ml measuring bottle, solvent constant volume is added, saturated sodium bicarbonate solution 5-10ml is added
Extraction 3-4 time, centrifuging and taking precipitate, dry Antrodia camphorata total triterpene substance extract.
Experiment:
It cultivates and obtains 3 plants of excellent Antrodia camphorata strains, number is NZZN-1, NZZN-2, NZZN-3 respectively.NZZN-1 strain
Containing active material highest, rapidly, thallus is of moderate size for growth, is suitable for the research and development of limitation high-end health products.NZZN-2 is excellent
Strain has mycelia long, and the big feature of Antrodia camphorata thallus after maturation, triterpenes content is moderate, the need suitable for mass production
It asks.There is NZZN-3 excellent species growth to be rich in triterpene isoreactivity substance rapidly, is easy to laboratory and expands numerous, is easy to implement a large amount of
The advantages of culture.
Triterpene substance detection method in Antrodia camphorata body are as follows: by the conventional method that total triterpene contents measure, with triterpenes
Conjunction object ursolic acid is reference substance, is color developing agent with glacial acetic acid-vanillic aldehyde and perchloric acid, with spectrophotometry under 548nm wavelength
The content for measuring total triterpene, weighs Antrodia camphorata powder 10g, is dissolved in the solvent of certain volume, ultrasonic extraction certain time and one
Determine number (arrangement of Extraction solvent, solvent usage, extraction time and extraction time according to orthogonal arrage), filtering, filtrate recycling is molten
Agent, with chloroform dissolution and constant volume is in 50mL measuring bottle, and precision draws chloroform solution 10mL, sets in separatory funnel, with saturation NaHCO3
Solution extracts 4 times, each 10mL, and combining extraction liquid is set in 50mL measuring bottle, is diluted to scale with saturation NaHCO3, sample is made
Solution.5% vanillic aldehyde of 0.40mL-glacial acetic acid solution is added in the solution for drawing certain volume after being evaporated in 100 DEG C of water-baths
With 1.00mL perchloric acid, is moved into ice-water bath after 60 DEG C of heating water bath 15min, add 5.00mL glacial acetic acid, shake up postposition
In room temperature.The absorbance for measuring sample solution after 15min under 548nm wavelength with ultraviolet-uisible spectrophotometer, with standard song
Collimation method calculates content.
Triterpene substance is abundant in Antrodia camphorata body, has 70-200 kind to differ, and carries out Antrodia camphorata with the extraction process of optimization
Total triterpene extracts, the results show that when incubation time was less than 28 days, in mycelium triterpene content with the extension of incubation time and
It gradually increases, and 28 days whens mycelial triterpene content highest, triterpenes content highest in NZZN-1, up to (53.124 ±
2.342) triterpenes content takes second place in mg/g, NZZN-3, is triterpenes object in (50.167 ± 2.614) mg/g, NZZN-2
Matter content is (49.167 ± 2.155) mg/g, and compared with traditional handicraft, Antrodia camphorata triterpene substance total amount improves more.
This research has detected Antrodia camphorata extract to Leukemia K562 cell, hepatocellular carcinoma H22, cervical cancer cell
Hela, stomach cancer cell BGC823, Colon Carcinoma, lung cell A549, esophageal cancer cell ECA-109, human tongue carcinoma are thin
The In-vitro Inhibitory Effect of born of the same parents Tca8113.Wherein, the most obvious to the inhibiting effect of hepatocellular carcinoma H22, as a result as follows:
1) Antrodia camphorata extracting solution inducing death of neoplastic cells
In HepG2 source of people liver cancer cells, cell is handled with three kinds of antrodia mycelia extracting solutions respectively, it is whole with 8 μ g/ml
Concentration culture is for 24 hours.Inspection is observed and is taken pictures under the microscope, as a result as shown in Figure 1.
The result shows that the extracting solution of NZZN-1, NZZN-2 and NZZN-3, which show very strong induction tumour cell, becomes contracting circle
Ability can induce tumor mortality.
2) Antrodia camphorata extracting solution inhibits tumor cell proliferation
The HepG2 cell suspension of 100 μ L is uniformly added into 96 orifice plates, be placed in incubator cultivate 24 hours (37 DEG C,
5%CO2).Then the FIPs albumen of final concentration of 20 μ g/ml is added into the every hole of culture plate, PBS is then added in cellular control unit.
Culture plate is incubated in the incubator 24 hours.Then 20 μ L cell Proliferation detection reagent (CellTiter are added in every hole
AQ ueous One Solution, G3582, Promega).Continue after being incubated for 4 hours, with the suction at microplate reader measurement 450nm
Luminosity.Versus cell vigor is expressed as (OD450 (processing group)/OD450 (control group)) * 100%, as a result as shown in Fig. 2.Knot
Fruit shows that three kinds of Antrodia camphorata extracts have apparent inhibiting effect to the proliferation of tumour cell.503nhibiting concentration graphpad
The calculating of 5.0 software of prism is got, as a result as shown in Figure 3.IC50 the result shows that, three kinds of Antrodia camphorata extracts are to tumour cell
Toxicity is big, i.e., inhibitory effect is preferable.
3) Antrodia camphorata extracting solution inhibits tumor cell migration
Cell scarification is to measure one of the method for Tumor Cell Migration characteristic.On the cell monolayer of culture, scratch is caused
Then wound is added the experiment conditions cultures such as drug, observes its influence to tumor cell migration.First with marker in six holes
Board bottom portion equably draws at least three horizontal lines, is divided into 0.5-1cm.About 5x10 is added in hole5A HepG2 cell, inoculation
Principle is to be incubated overnight rear cell confluency rate to reach 100%.Later with the white tip pipette tips firmly equably scratch in culture dish,
PBS is washed twice, then the cell under removal stroke is added serum free medium culture 24 hours.Microscope sight is just being set with Nikon
It examines cell moving distance and takes pictures.As a result as shown in Figure 4, Figure 5.
It can be arrived with clear view, in negative control, tumour cell is just migrated to mid line region after 24 hours, and passes through three kinds
The migration of the tumour cell of Antrodia camphorata extract-treated is obviously suppressed, and wherein NZZN-1 inhibits migration effect the most obvious,
NZZN-3 takes second place.
4) Antrodia camphorata extracting solution promotes apoptosis of tumor cells
Adherent, 300g is digested with the pancreatin without EDTA, 4 DEG C of centrifugation 5min collect cell.Cell is washed with the PBS of pre-cooling
2 times, it is both needed to 300g, 4 DEG C of centrifugation 5min every time.Collect 1~5 × 105 cells.Then 100 microlitres of 1 × Binding are added
Cell is resuspended in Buffer.5 microlitres of Annexin V-EGFP and 5 microlitres of PI dyeing liquors are added later, are mixed gently.It is kept away in room temperature
Light reaction 10min.Then 400 microlitres of 1 × Binding Buffer are added, mix, sample uses fluidic cell in 1 hour
Analyzer BD Calibur is detected.The fluorescence of FITC and PI is soft with CellQuest respectively in FL1 and FL2 Air conduct measurement
Part is analyzed, and draws double-colored scatter plot (two-color dot plot), FITC is abscissa, and PI is ordinate.Each sample
Acquire 10000events.
Fig. 6 is result schematic diagram of the untreated cell after flow cytometer detection, and the corresponding meaning that represents of four quadrants has marked
Fig. 7 shows three kinds of Antrodia camphorata extracts to the function and effect of apoptosis of tumor cells in the figure.The results show that in final concentration 4,
8ug/ml, after processing cell 24 hours, PC, NZZN-1, NZZN-2, NZZN-3 can promote HepG2 Apoptosis, NZZN-1
Effect is especially significant, and apoptosis rate is up to 60%；3 kinds of extracting solutions, concentration for the treatment of is higher, induces cell apoptosis effect and is more obvious.
Antrodia camphorata health care product can be fabricated to oral solution or Sobering-up buccal tablet；
1) manufacture craft of compound Antrodia camphorata oral solution is as follows:
Using Antrodia camphorata fructification or mycelium as antrodia raw material, hot water extraction successively is carried out to Antrodia camphorata raw material and ethyl alcohol soaks
It mentions, obtains Antrodia camphorata water extract and Antrodia camphorata ethanol extract.Antrodia raw material and water is added, extracts 30-60 at 80-95 DEG C
Minute, filtering takes filtrate；Repeat above step at least twice；Merge resulting filtrate, obtains antrodia aqueous extract.Take by
The antrodia raw material taken with hot water extraction is added ethyl alcohol, flows back at 80-90 DEG C, each 1-1.5 hour, filtering；At least
It is repeated once previous step；Merge resulting filtrate, ethyl alcohol is volatilized clean, vacuum microwave drying crushes, obtains powdered camphor tree
Sesame ethanol extract；During hot water extraction, the ratio of antrodia raw material and water is 1:10；During alcohol steep, antrodia raw material with
The mass ratio of ethyl alcohol is 1:4-8.The process for being dried to powder is that the antrodia aqueous extract is concentrated in vacuo into cream at 60-80 DEG C
Then shape uses micro-wave vacuum at 100-120 DEG C, crush, or by the antrodia aqueous extract vacuum at 60-80 DEG C
It is condensed into concentrate, is spray-dried at 100-120 DEG C.There is sugar-type oral solution to contain Aspartame 0.001%-0.01%；Sweet tea
Synanthrin 0.001%-0.01%；Honey 0.001%-0.01%；Xanthan gum 0.01%-0.5%；Gellan gum 0.01%-0.5%；
Pectin 0.01%-0.5%；Fructose syrup 2%-8%.
2) manufacture craft of compound Antrodia camphorata Sobering-up buccal tablet is as follows:
Proportionally, Antrodia camphorata extract 600-800 parts, 100-200 parts of L-cysteine, 250-400 parts of filler, profit
Antrodia camphorata water extract and Antrodia camphorata alcohol extracting in 50-100 parts of humectant, 1-20 parts of magnesium stearate, Antrodia camphorata extract and oral solution
Take the mixture of object consistent.Filler is at least one of mannitol, microcrystalline cellulose, lactose, starch and Icing Sugar.By Antrodia camphorata
After mixing, wetting agent softwood is added in extract, L-cysteine and filler, and the granulation of 18 meshes makes pellet in 50-
It is 30-60 minutes dry at a temperature of 60 DEG C, magnesium stearate is added into dried particle and is uniformly mixed, in 3- after 18 mesh sieves
It is tabletted under 10 kilograms of pressure, obtain Antrodia camphorata lozenge.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. a kind of preparation and application of the inhibiting tumour cells agent based on Antrodia camphorata extract, including Antrodia camphorata, it is characterised in that:
The Antrodia camphorata can prepare antrodia mycelia in the health medicine for preparing inhibiting tumour cells agent and extraction Antrodia camphorata is total
Triterpene, the antrodia mycelia are cultivated in the medium by Antrodia camphorata and are obtained, and the Antrodia camphorata total triterpene is by Antrodia camphorata mycelia
Body is concentrated and is centrifuged and obtains.
2. the preparation and application of a kind of inhibiting tumour cells agent based on Antrodia camphorata extract according to claim 1,
Be characterized in that: the breeding method of the antrodia mycelia is as follows:
1), bio-safety station is irradiated and alcohol disinfecting 20 minutes by ultraviolet light, with blade by fresh Antrodia camphorata mushroom
Body wraps Antrodia camphorata mushroom body by removing on linden, and with clean gauze or polybag, is put into bio-safety station；
2), the Antrodia camphorata mushroom body surface face cleaning gauze wiping for speckling with 75% ethyl alcohol, is cut mushroom body surface skin with sharp pocket knife, will
The inside pulp is divided into 0.5 centimeter of fritter, is inoculated in culture dish, closes the lid, set cultivation temperature be 24.5 DEG C extremely
It is cultivated in 27.5 DEG C of environment, after Antrodia camphorata culture medium is covered by mycelia, adjustment cultivation temperature is 18.5 DEG C to 23.5
DEG C continue culture until growing Antrodia camphorata fructification, Antrodia camphorata fructification is collected after growing Antrodia camphorata fructification and is not necessarily to
The Antrodia camphorata of wood cultivation；
3) it, takes the Antrodia camphorata without wood cultivation to carry out sterile chopping fermentation to cultivate or using rice solid medium to Antrodia camphorata
Carry out solid cultivation.
3. the preparation and application of a kind of inhibiting tumour cells agent based on Antrodia camphorata extract according to claim 1,
Be characterized in that: the extraction step of the Antrodia camphorata total triterpene is as follows:
1), sample pretreatment: antrodia mycelia 10 is weighed to 20g, drying to constant weight, pulverizes and sieves, and 10 to 20 times of water are added
Testing sample solution is made in dissolution；
2) it, is dissolved in solvent in testing sample solution obtained, and active carbon is added, filtering with microporous membrane takes filtrate spare；
3) 3 times of 95% alcohol refluxs of amount, are added in sample filtrate obtained to extract 3 times, each 1h；
4), by sample crude extract obtained concentration, centrifugation, standing 10-20min, precipitating is eluted 2-4 times using water, is filtered
Precipitating；
5), precipitating obtained is added in 50ml measuring bottle, solvent constant volume is added, saturated sodium bicarbonate solution 5-10ml extraction is added
3-4 times, centrifuging and taking precipitating, dry Antrodia camphorata total triterpene substance extract.
4. the preparation and application of a kind of inhibiting tumour cells agent based on Antrodia camphorata extract according to claim 2,
Be characterized in that: the condition that the fermentation is cultivated is that glucose 20g/L, wheat bran leachate 60g/L is added towards triangular flask,
VB10.14g/L, KH2PO41g/L, MgSO40.05g/L and the Antrodia camphorata 30g cultivated without wood, and mixture is in triangular flask
Natural pH, liquid amount 200mL/500mL cultivate 12 in 28 DEG C of constant temperature incubation environment by the stirring of revolving speed 110r/min
It.
5. the preparation and application of a kind of inhibiting tumour cells agent based on Antrodia camphorata extract according to claim 2,
Be characterized in that: the condition of the rice solid medium is the Antrodia camphorata 30g and rice: water=4:7 culture without wood cultivation
Any other nutriment is not added in base, is cultivated 55 days under illumination condition.
6. the preparation and application of a kind of inhibiting tumour cells agent based on Antrodia camphorata extract according to claim 3,
Be characterized in that: the solvent is to be uniformly mixed and obtain according to 1.15:10 in ursolic acid standard items and ethyl acetate.
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