CN106309511A - Effective part of agaricus gennadii for inhibiting CDC25 enzyme as well as preparation method and application of effective part - Google Patents
Effective part of agaricus gennadii for inhibiting CDC25 enzyme as well as preparation method and application of effective part Download PDFInfo
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- CN106309511A CN106309511A CN201610761207.XA CN201610761207A CN106309511A CN 106309511 A CN106309511 A CN 106309511A CN 201610761207 A CN201610761207 A CN 201610761207A CN 106309511 A CN106309511 A CN 106309511A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation 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
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation 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
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
Abstract
The invention discloses an effective part of agaricus gennadii for inhibiting CDC25 enzyme as well as a preparation method and application of the effective part, and belongs to the technical field of plant extracts. The effective part is a mixture of one or two or more of an agaricus gennadii petroleum ether part, an agaricus gennadii ethyl acetate part, an agaricus gennadii n-butyl alcohol part and an agaricus gennadii water extraction part according to any mass ratio. The preparation method comprises the following steps: smashing the agaricus gennadii, adding an ethanol solution with the volume percentage concentration of 65 to 95 percent for reflux extraction, and concentrating and drying an obtained extracting solution to obtain an extract; adding distilled water into the extract for dispersion to obtain an extract dispersion solution, extracting the extract dispersion solution through petroleum ether, ethyl acetate and n-butyl alcohol which serve as solvents in sequence, and volatilizing the solvents to obtain the agaricus gennadii petroleum ether part, the agaricus gennadii ethyl acetate part and the agaricus gennadii n-butyl alcohol part; concentrating and drying the extracted extract dispersion solution to obtain the agaricus gennadii water extraction part. The application of the effective part of the agaricus gennadii for inhibiting the CDC25 enzyme in preparation of a medicine for inhibiting the CDC25 enzyme can be used for preparing an anti-tumor medicine.
Description
Technical field
The present invention relates to circle spore mushroom suppression CDC25 enzyme effective site and preparation method and application, belong to plant extract technology
Field.
Background technology
Circle spore mushroom Agaricus gennadii belongs to Agaricales Agaricus edibilis Agaricus (Agaricus), another name " circle spore torr handle
Mushroom " [see: Yang Hui, Li Na. the investigation [J] of circle spore mushroom. the Nature, 1996(6): 48.].Circle spore mushroom is umbrella shape, has
Strong mushroom fragrance.Milky during lamella children, after fade to pink to pitchy, single heavy 58-350g, maximum up to 2-
5kg.It is grown on summer and autumn under shrubbery sand ground, lakeside phragmites communis clump in the soil of 20-70cm, underground energy parachute-opening, Dan Sheng, scattered or clump
Raw, be grown in more western Xinjiang and Localities In Southwest [see: Wang Junyan. Xinjiang edible and medical fungi resource and DEVELOPMENT PROSPECT [J]. micro-
Biology is circulated a notice of, 1997(3): 176-177.].Since last century the nineties, it has been found that and belonged to middle Agaricus blazei Murrill together
Active component and its various active.But the chemical composition of the round spore mushroom Agaricus gennadii in belonging to together and phase
Close activity to be still in blank.
Malignant tumor is one of major disease of serious harm human health, it has also become the first cause that the mankind are lethal.Dislike
The preventing and treating of property cancerous cell has become the important topic that medical circle is paid close attention to.But, Incidence is hidden, and the cause of disease is complicated, sick
Reason change is various, and its treatment still is limited to multiformity and the repeatability of Cancerous disease.Although the carcinogen of different cancers because of
May be not quite similar, but its structure observation all shows similar cell cycling disorder and the growth of rapid uncontrollable cell
And transfer.Therefore, to participate in cell cycle regulating some family proteins research promote in treatment of cancer become important because of
Element.Wherein, cell division cycle kinases (Cdk/cyclins), as the major regulatory albumen of eukaryotic cell cycle, is responsible for promotion
The cell division of respective stage.CDC25 Phospoprotein enzyme (CDC25 phosphatases) swashs as cell division cycle protein
The activated protein enzyme of enzyme family (Cdk/cyclins), has played highly important regulation effect in the division cycle of eukaryotic cell.
At present clinical research shows, the protease of this family all shows excessive expression in kinds cancer tissue, and often with
Poor cancer early prediction is relevant, and these all make CDC25 Phospoprotein enzyme become the cancer treatment drugs with great potential
Target.In recent years, many research and development that enzymology is related inhibitors and cancer drug about CDC25 Phospoprotein enzyme family
Provide considerable information.
Applicant finds under study for action: the cost of material of antitumor drug is high at present, especially for preparation suppression CDC25
The raw material of Chinese medicine of enzyme medicine is the deficientest.In prior art, circle spore mushroom only has edibility because of its delicious flavour.Not yet
See about circle spore mushroom suppression CDC25 enzyme active in terms of research and application.
Summary of the invention
The technical problem to be solved in the present invention is: overcome the deficiencies in the prior art, it is provided that circle spore mushroom suppression CDC25 enzyme has
Imitating position and preparation method and application, this circle spore mushroom suppression CDC25 enzyme effective site can effectively suppress CDC25A enzyme and CDC25B enzyme
Activity, be used for preparing suppression CDC25 enzyme medicine, there is preferable antitumor action.
The technical solution adopted for the present invention to solve the technical problems is: this circle spore mushroom suppression CDC25 enzyme effective site,
It is characterized in that: selected from circle spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract, circle spore mushroom n-butanol portion, circle
The mixture of one or more any mass ratio in spore mushroom aqueous phase extraction position.
This circle spore mushroom suppression CDC25 enzyme effective site, it is characterised in that: selected from circle spore mushroom ethyl acetate extract and circle
One in spore mushroom petroleum ether part, circle spore mushroom n-butanol portion, circle spore mushroom aqueous phase extraction position is by any mass ratio
Mixture.
The preparation method of this circle spore mushroom suppression CDC25 enzyme effective site, it is characterised in that comprise the steps: circle
Spore mushroom is pulverized, and adds concentration expressed in percentage by volume 65% ~ 95% ethanol solution reflux, extract, is concentrated by gained extracting solution, is dried, obtains leaching
Cream；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction successively
Extract dispersion liquid, just must justify spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract, circle spore mushroom after flinging to solvent respectively
Butanol position；Extract dispersion liquid after extraction is concentrated, is dried, obtains round spore mushroom aqueous phase extraction position.
Described reflux, extract, is ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: will
Round spore mushroom after pulverizing adds concentration expressed in percentage by volume 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filters, filtering residue
Add concentration expressed in percentage by volume 65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.
The application of this circle spore mushroom suppression CDC25 enzyme effective site, it is characterised in that: at preparation suppression CDC25 enzyme medicine
The application of aspect.
Described preparation suppression CDC25 enzyme medicine be by circle spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract,
The mixing of one or more any mass ratio in circle spore mushroom n-butanol portion, circle spore mushroom aqueous phase extraction position
Thing, the oral formulations being mixed with pharmaceutic adjuvant or ejection preparation.
Described ejection preparation is lipidosome injection, nanoparticle injection or micro-balloon injection.
Described oral formulations is powder, tablet, granule, capsule or solution.
Applicant is described as follows for the present invention: in prior art, and circle spore mushroom only possesses edible valency because of its delicious flavour
Value, has no that it has the relevant report of antitumor efficacy.Applicant is found by numerous studies: the extracting section thing in circle spore mushroom
Also there is antibacterial, antineoplastic action, but its effect is inconspicuous, it is impossible to determine its concrete effective site.Therefore why pass through
The solvent of sample, use how preparation method, the effective site that suppression CDC25 enzymatic activity effect is best could be obtained, be have to
The problem solved.Applicant is found by research: use preparation method of the present invention, the round spore mushroom petroleum ether part that filters out,
Circle spore mushroom ethyl acetate extract, circle spore mushroom n-butanol portion and circle spore mushroom aqueous phase extraction position, its suppression CDC25 enzyme effect
Fruit is preferably.Analyzing further through applicant, circle spore mushroom ethyl acetate extract is imitated for the suppression of CDC25A enzyme and CDC25B enzyme
Fruit is optimum.
In preparation method, round spore mushroom used is selected from Herb or cap part.Extractum is crude extract, uses different molten
Agent extraction extract dispersion liquid, refer to be added by petroleum ether successively extract dispersion liquid extract and separate obtain petroleum ether extraction liquid, general
Ethyl acetate adds extract dispersion liquid extract and separate and obtains acetic acid ethyl acetate extract, n-butyl alcohol adds extract dispersion liquid extraction point
From obtaining butanol extraction liquid；Take petroleum ether extraction liquid, acetic acid ethyl acetate extract and butanol extraction liquid respectively volatilize removal molten
After agent, obtain round spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract, circle spore mushroom n-butanol portion；To remain after extraction
Remaining extract dispersion liquid (solvent is water), volatilization is removed solvent and is i.e. obtained round spore mushroom aqueous phase extraction position.Aqueous phase extraction position is also
Can be referred to as aqueous phase extract, aqueous phase extraction position is soluble in water；And relative, petroleum ether part, ethyl acetate extract and just
Butanol position correspondence is soluble in petroleum ether, ethyl acetate and n-butyl alcohol.Wherein, circle spore mushroom petroleum ether part contains sterols,
Circle spore mushroom ethyl acetate extract contains terpenoid；Circle spore mushroom n-butanol portion contains saponin and phenolic compound；Circle
Polysaccharide and organic acid compound are contained in spore mushroom aqueous phase extraction position.Fling to solvent i.e. volatilize removal solvent.Flinging to solvent can
To use normal heating to make solvent (petroleum ether, ethyl acetate and n-butyl alcohol) volatilize.Preferably, flinging to solvent uses decompression dense
Contracting, the method efficiency is high, and is avoided that heat-sensitive ingredients loses the property of medicine because of high temperature.
Preferably dosage form is oral formulations or ejection preparation.Oral formulations and ejection preparation are optimal route of administration, herein
Described oral formulations is through intestines and stomach drug-delivery preparation, it is preferred that oral formulations is slow-release controlled-release type oral formulations.Can also be
Other of parenteral routes, such as respiratory tract administration preparation (spray, aerosol, powder spray)；Percutaneous drug delivery preparation (externally used solution
Agent, lotion, liniment, ointment, plaster, paste, patch)；Film drug-delivery preparation (eye drop, nasal drop, ophthalmic ointment, rinsing the mouth
Agent, sublingual tablet)；Cavity/canal drug administration preparation (suppository).Ejection preparation can be conventional injection preparation；Preferably, the note of the present invention
Penetrating preparation is lipidosome injection, nanoparticle injection or micro-balloon injection, and above injection has been respectively adopted liposome, nanometer
Grain, microsphere as pharmaceutical carrier, can extend medicine carrying microgranule circulation time in vivo, extend medicine carrying microgranule stopping at absorption site
Stay the time, control the medicine burst effect at the release initial stage, strengthen drug effect.Preferably, as carrier in nanoparticle injection
Nanoparticle is nanorize colloidal particle (i.e. nano-micelle).
Compared with prior art, the round spore mushroom of the present invention suppresses CDC25 enzyme effective site and preparation method and application to be had
Provide the benefit that:
1, this circle spore mushroom suppression CDC25 enzyme effective site can effectively suppress CDC25A enzyme and the activity of CDC25B enzyme, is used for making
Standby suppression CDC25 enzyme medicine, has preferable antitumor action.Applicant determines through numerous studies: only have in prior art
The round spore mushroom of edibility, also has antineoplastic effect.And applicant is enterprising at CDC25A enzyme and CDC25B enzyme first
Row medicine efficacy screening, is just determining round spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract, circle spore mushroom by numerous studies
The mixture of one or more any mass ratio in butanol position, circle spore mushroom aqueous phase extraction position is respectively provided with preferably
Suppression CDC25A enzyme and CDC25B enzymatic activity, its CDC25A enzyme survival rate 22.6% ~ 40.9%, CDC25B enzyme survival rate 35.9% ~
48.6%, it is possible to be used for developing into antitumor Tibetan medicine new drug.Owing to each effective site of different solvents extraction acquisition is to CDC25A enzyme
Rejection ability of living with CDC25B enzyme is different, and applicant is by further determining: pressing down of circle spore mushroom ethyl acetate extract
CDC25 enzyme effect processed is optimum, its CDC25A enzyme survival rate 22.6%, CDC25B enzyme survival rate 35.9%, and circle spore mushroom acetic acid
The tumour inhibiting rate performance optimum at ethyl ester position.In sum, applicant finds new effect of circle spore mushroom first, and passes through
Determining the kind of its enzyme suppressed, CDC25A enzyme and CDC25B enzyme are respectively provided with relatively by design preparation method finally obtaining
The effective site of good inhibition, above procedure has paid substantial amounts of creative work.
2, the preparation method of this circle spore mushroom suppression CDC25 enzyme effective site is extracted conveniently, and extraction efficiency is high.Preparation method
In, applicant designs addition mass percentage concentration 65% ~ 95% ethanol solution and carries out ultrasonic assistant reflux, extract, improves extraction
Efficiency；In extraction, use petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction extract dispersion liquid successively, use above solvent
The ethyl acetate extract of extraction order gained, circle spore mushroom n-butanol portion, circle spore mushroom aqueous phase extraction position have preferably
The effect of suppression CDC25 enzyme.
3, the present invention has expanded suppression CDC25A enzyme and the raw material channel of CDC25B enzyme, expands the purposes of round spore mushroom,
Make circle spore mushroom develop into suppression CDC25A enzyme and the new raw material of CDC25B enzyme medicine, be remarkably improved the additional of round spore mushroom
Value.
Detailed description of the invention
Embodiment 1 ~ 3 is round spore mushroom suppression CDC25 enzyme effective site and the detailed description of the invention of preparation method of the present invention, its
Middle embodiment 1 is most preferred embodiment.
Embodiment 1
Preparation method, comprises the steps: to pulverize circle spore mushroom, uses concentration expressed in percentage by volume 75% ~ 85% ethanol solution ultrasonic
Ripple extracts 1 hour, then filters to obtain ultrasonic extract and filtering residue, adds concentration of volume percent 75% ~ 85% second in filtering residue
Alcoholic solution reflux, extract, 3 times, each 0.5 ~ 1 hour, filters to obtain reflux extracting liquid, merges ultrasonic extract and reflux, extract,
Liquid, concentrate drying obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate the most successively
With n-butyl alcohol as solvent extraction extract dispersion liquid, obtain petroleum ether extraction liquid, acetic acid ethyl acetate extract and n-butyl alcohol extraction respectively
Take liquid, after flinging to solvent respectively, obtain circle spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract, circle spore mushroom n-butyl alcohol
Position；After being extracted by extract dispersion liquid, remaining liq concentrates, is dried, and obtains round spore mushroom aqueous phase extraction position.
Embodiment 2
Preparation method, comprises the steps: to pulverize circle spore mushroom, uses concentration expressed in percentage by volume 85% ~ 95% ethanol solution ultrasonic
Ripple extracts 3 hours, then filters to obtain ultrasonic extract and filtering residue, adds concentration of volume percent 85% ~ 95% second in filtering residue
Alcoholic solution reflux, extract, 3 times, each 1 ~ 1.5 hour, filters to obtain reflux extracting liquid, merges ultrasonic extract and reflux, extract,
Liquid, concentrate drying obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate the most successively
With n-butyl alcohol as solvent extraction extract dispersion liquid, obtain petroleum ether extraction liquid, acetic acid ethyl acetate extract and n-butyl alcohol extraction respectively
Take liquid, after flinging to solvent respectively, obtain circle spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract, circle spore mushroom n-butyl alcohol
Position；After being extracted by extract dispersion liquid, remaining liq concentrates, is dried, and obtains round spore mushroom aqueous phase extraction position.
Embodiment 3
Preparation method, comprises the steps: to pulverize circle spore mushroom, uses concentration expressed in percentage by volume 65% ~ 75% ethanol solution ultrasonic
Ripple extracts 0.5 hour, then filters to obtain ultrasonic extract and filtering residue, adds concentration of volume percent 65 ~ 75% second in filtering residue
Alcoholic solution reflux, extract, 2 times, each 1.5 ~ 2 hours, filters to obtain reflux extracting liquid, merges ultrasonic extract and reflux, extract,
Liquid, concentrate drying obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate the most successively
With n-butyl alcohol as solvent extraction extract dispersion liquid, obtain petroleum ether extraction liquid, acetic acid ethyl acetate extract and n-butyl alcohol extraction respectively
Take liquid, after flinging to solvent respectively, obtain circle spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract, circle spore mushroom n-butyl alcohol
Position；After being extracted by extract dispersion liquid, remaining liq concentrates, is dried, and obtains round spore mushroom aqueous phase extraction position.
Performance test
Just respectively take the dried round spore mushroom petroleum ether part of 0.4mg, circle spore mushroom ethyl acetate extract, circle spore mushroom
Butanol position, circle spore mushroom aqueous phase extraction position, be each dissolved in formation DMSO in the dimethyl sulfoxide (DMSO) of 100 L molten
Liquid, ultrasonic 5 minutes.Take the DMSO solution of 1 L sample dissolution the most respectively, be separately added into buffer 99 L, respectively obtain for examining
Survey circle spore mushroom suppression CDC25 enzyme round spore mushroom petroleum ether part buffer solution, circle spore mushroom ethyl acetate extract delay
Dissolved liquid, the buffer solution of circle spore mushroom n-butanol portion and the buffer solution at circle spore mushroom aqueous phase extraction position.Buffer
Formula is: take respectively 0.2423gTris-HCl, 0.5138gNaCl, 0.0330gBSA, 0.0154gDTT, 0.0167g EDTA in
In 50mL beaker, add distilled water and dissolve and mix, add 0.5mol/L NaOH aqueous solution by pH regulator to 8.4, then fixed
Hold to 100mL volumetric flask, in 4 DEG C of preservations, as stock buffer (buffer).
One, suppression CDC25A enzyme and the experiment of CDC25B enzyme.
Major experimental material is:
CDC25A enzyme, CDC25B enzyme, Le Bo bio tech ltd, Beijing product；
Tris-HCl, Shanghai Jie Mei gene Pharmaceutical Technology Co., Ltd；
EDTA, Curie bio tech ltd, Shanghai；
DTT, Shanghai Sheng Gong biological engineering limited company；
BSA, Jiang Lai bio tech ltd, Shanghai；
DMSO, Shanghai Yi Ka Bioisystech Co., Ltd；
Na Cl(analytical pure), Tianjin red rock chemical reagent factory；
NaOH(analytical pure), Hedong District, Tianjin red rock chemical reagent work.
Major experimental instrument is:
FLUOstar Omega full-automatic multi-functional microplate reader, Bai Qi bio tech ltd, Guangzhou, Guangdong Province；
MP511 type pH meter electricity meter, company limited is believed in Shanghai three；
BRAND8 road liquid-transfering gun, Zhejiang Hangzhou converges your Instrument Ltd.；
500 L liquid-transfering guns, 50 L liquid-transfering guns, Shanghai Rong Tai biochemical instrument company limited；
Electronic balance XPE105, prunus mume (sieb.) sieb.et zucc. Teller-torr benefit Instrument Ltd..
The buffer solution of the round spore mushroom petroleum ether part obtained with embodiment 1, circle spore mushroom ethyl acetate extract slow
The buffer solution at dissolved liquid, the buffer solution of circle spore mushroom n-butanol portion and circle spore mushroom aqueous phase extraction position is tester.
It represents experiment for circle spore mushroom difference effective site suppression CDC25A enzyme and the activity of CDC25B enzyme.Its method is to take respectively
2.7 L 330 μm ol/L FDP, 1.8 L 100 μm ol/L testers, 2 L 0.1 μm ol/L CDC25A/CDC25B, 11.5 L
FDP buffer forms the experimental group of 18 L systems；Then 2.7 L 330 μm ol/L FDP, 2 L 0.1 μm ol/L are taken
CDC25A/CDC25B, 13.3 L FDP buffer form the matched group 1 of 18 L systems；Take again 2.7 L 330 μm ol/L FDP,
1.8 L 100 μm ol/L testers, 13.5 L FDP buffer form the matched group 2 of 18 L systems；Needs are added CDC25A/
The system of CDC25B enzyme hatches 60 min under the conditions of 37 DEG C, the most enzyme-added, reacts 60 min under similarity condition, and question response is complete
Entirely, detection product fluorescence intensity at 520nm under 485 nm exciting light effects.Experimental group, matched group 1 and matched group
2 are carried out under the same conditions, and record experimental result also calculates enzyme survival efficiency.Enzyme survival efficiency (%)=experimental group measured value/right
According to organizing 1 measured value × 100%, wherein enzyme survival efficiency is the lowest, illustrates that tester is to histone acetylation enzyme CDC25A/CDC25B
Inhibition the strongest.
The active testing result of table 1 circle spore mushroom suppression CDC25 enzyme effective site
。
* P ＜ 0.01 in table 1, by table 1 it can be seen that justify spore mushroom petroleum ether part, circle spore mushroom ethyl acetate portion
Position, circle spore mushroom n-butanol portion and circle spore mushroom aqueous phase extraction position exist for CDC25A enzyme and CDC25B enzyme and preferably press down
Effect processed；The effect wherein justifying spore mushroom ethyl acetate extract suppression CDC25A enzyme and CDC25B enzyme is optimum.
Two, anti-tumor activity test.
Mtt assay is utilized to measure the circle position, the spore mushroom each position cytotoxic activity to human tumor cells.Example 1 respectively
The gained circle each position of spore mushroom is appropriate, as test-compound, after adding DMSO dissolving, utilizes mtt assay to measure respectively people's colon
Cancer HCT-8, Human hepatocarcinoma Bel-7402 cell, people's gastric cancer BGC-803 cell, human pulmonary epithelial cells and human ovarian cancer A2780
The suppression ratio of cell growth.
Experimental technique: by the cell of exponential phase, after 0.25% pancreas enzyme-EDTA digestion, be configured to certain density list
Cell suspension, according to the difference of vitro growth rates, is inoculated in 96 orifice plates by 800 ~ 2000/hole, and every hole adds cell suspension
100 µL.After 24h, adding containing variable concentrations test-compound and the fresh culture of coordinative solvent comparison, every hole adds 100 L
(DMSO final concentration < 0.1%), every kind of test-compound sets 5 ~ 7 dosage groups, often organizes and at least sets 3 parallel holes, in 37 DEG C of continuation
After cultivating 72h, abandoning supernatant, every hole adds the freshly prepared serum-free medium containing 0.5mg/mL MTT of 100 L, continues
Cultivating 4h, after abandoning supernatant, every hole adds 200 L DMSO and dissolves MTT first hairpin precipitation, microoscillator vibration mixing, uses enzyme
Mark instrument measures optical density value (OD) under the conditions of reference wavelength 450nm, detection wavelength 570nm, the tumor processed with solvent control
Cell is matched group, calculates the test-compound suppression ratio to tumor cell by below equation, and by middle effect Equation for Calculating IC50:
Suppression ratio=(matched group mean OD value-administration group mean OD value) ÷ matched group mean OD value × 100%.By in preparation method
Extractum and the round spore mushroom petroleum ether part of gained, circle spore mushroom ethyl acetate extract, circle spore mushroom n-butanol portion, circle
Spore mushroom aqueous phase extraction position carries out tumor cell in vitro cytotoxic activity screening, the results are shown in Table 2.
Table 2 tumor cell in vitro cytotoxic activity the selection result
。
Note: 1), HCT-8 be human colon cancer cell, Bel-7402 is human liver cancer cell, and BGC-823 is gastric carcinoma cells,
A549 is human lung carcinoma cell, and A2780 is Proliferation of Human Ovarian Cell.2), > 50 show do not have anti-cell cytotoxic activity.Can be seen by table 2
Go out: circle spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract, circle spore mushroom n-butanol portion, circle spore mushroom aqueous phase extraction
Take position and really have preferable antineoplastic effect.
Three, external S180Tumor-bearing mice anti-tumor experiment.
1, the S at each position of embodiment 1180Tumor-bearing mice anti-tumor experiment method is as follows:
A) 5 ~ 6 week old kunming mice, selects Mus to be grouped at random, often group 10, often group male and female half and half (sub-cage rearing)；Group names
For: blank group, cyclophosphamide group and administration group；Administration group includes: circle spore mushroom petroleum ether part basic, normal, high dosage group,
Circle spore mushroom ethyl acetate extract basic, normal, high dosage group, circle spore mushroom n-butanol portion basic, normal, high dosage group, circle spore mushroom
Aqueous phase extraction position basic, normal, high dosage group；Every mice is carried out skin degerming；
B) in right fore subcutaneous vaccination S180Tumor liquid 0.2ml(S180Oncocyte number is 2.0 × 106~2.2×106In the range of)；
C) inoculation was administered after 24 hours；Blank group gastric infusion 10mL/kg injection normal saline；Cyclophosphamide group abdominal cavity
Injection cyclophosphamide 20mg/kg；Administration group uses the prepared round spore mushroom petroleum ether part of gastric infusion embodiment 1, circle spore mushroom
Mushroom ethyl acetate extract, circle spore mushroom n-butanol portion and circle spore mushroom aqueous phase extraction position；After first administration, the next day claim mice
Body weight first；Often group is administered once daily, and each dosage refers to following table, successive administration 14 days；
D) after last is administered 1 hour, putting to death mice, claim mice last body weight, stripping tumor mass is weighed, and calculates tumour inhibiting rate.Tumour inhibiting rate=
(blank group average tumor weight-administration group average tumor weight) ÷ blank group average tumor weight × 100%, calculates knot by tumour inhibiting rate
Really typing table 3.
The each position of table 3 embodiment 1 is to mice S180The impact of solid tumor
2, the S at each position of embodiment 2180Tumor-bearing mice method is with embodiment 1, data inputting table 4.
The each position of table 4 embodiment 2 is to mice S180The impact of solid tumor
3, the S at each position of embodiment 3180The method of tumor-bearing mice anti-tumor experiment is with embodiment 1, data inputting table 5.
The each position of table 5 embodiment 3 is to mice S180The impact of solid tumor
Add up through applicant: administration group is compared with blank group, and Mouse Weight change there was no significant difference (P > 0.05).Table 3 ~
In 5: S180Refer to mouse ascites oncocyte, S in table 3 ~ 5180Tumour inhibiting rate numerical value the biggest expression antitumous effect is the best.By table 3 ~ 5
It can be seen that first, embodiment 1 ~ 3 all has preferable antitumous effect, in embodiment 1 ~ 3 during same area same dose, respectively
Antitumous effect between group is without significant difference.Secondly, compared with blank group, middle and high dose of spore mushroom ethyl acetate extract of circle
Amount group, circle spore mushroom n-butyl alcohol middle and high dosage group, circle spore mushroom aqueous phase extraction position middle and high dosage group, Treated with Chemotherapy with Cyclophosphamide
Group, all can significantly inhibit S180The growth of tumor.
Embodiment 4
Circle spore mushroom suppression HDAC1 enzyme effective site is prepared as tablet, uses following steps: will circle spore mushroom ethyl acetate portion
Position 300mg and starch 100mg mixing, the gelatinized corn starch 40mg adding mass percent concentration 10% makes soft material, and sieve to obtain wet granular,
It is dried to obtain dry granule, granulate, adds magnesium stearate 4 mg mixing, tabletting, to obtain final product.
Embodiment 5
The preparation method of electuary: by weight ratio circle spore mushroom aqueous phase is extracted 5 parts of position, sucrose 1 part, the mixing of 3 parts of dextrin,
Add appropriate mass percent 95% ethanol solution 2 ~ 5 parts, stirring while adding, prepare soft material, soft material is dried, crosses 16 mesh sieves, point
Fill and get final product.
Embodiment 6
Pellet capsule is composed of the following raw materials by weight: circle spore mushroom n-butanol portion 30 parts, 5 parts of lecithin, Bile Salts 5
Part, microcrystalline Cellulose 30 parts；
The preparation method of pellet capsule: by proportioning by circle spore mushroom n-butanol portion, lecithin, Bile Salts and microcrystalline cellulose
Element is mixed, and pours ethanol water into and stirs evenly acquisition soft material, is poured into by soft material in extruder and extrudes, through round as a ball acquisition granule, extrusion
Rotating speed 250r/min, round as a ball rotating speed 800r/min, round as a ball time 20min, be dried, cross 24 ~ 30 mesh sieves acquisition micropills, filled by micropill
Load in capsule shells, to obtain final product.
Embodiment 7
The preparation method of lipidosome injection: 1) under nitrogen protection, by 50g cholesterol succinate, 250g distearyl phosphorus
Acyl ethanolamine, 40g soybean lecithin, 50g poloxamer-188 and 10g circle spore mushroom n-butanol portion are dissolved in 1500ml
Volume ratio is in the ethanol of 1:1 and the organic solvent of n-butyl alcohol, and stirring makes it dissolve acquisition suspension；By suspension by decompression
Organic solvent is flung in concentration, it is thus achieved that immobilized artificial membrane；
2) under nitrogen protection, adding 8000ml pH in immobilized artificial membrane is the phosphate buffered solution of 6.8, and stirring makes immobilized artificial membrane
Eluting abundant swelling hydration, through 0.22 μm filtering with microporous membrane, obtain the liposome of round spore mushroom n-butanol portion；
3) aseptically, in the liposome of circle spore mushroom n-butanol portion, add 100g trehalose, stir, super
Sonicated 0.5 ~ 1 hour, injects and uses water constant volume, through 0.22 μm filtering with microporous membrane, fill, obtains round spore mushroom n-butyl alcohol
The lipidosome injection at position.
Embodiment 8
The preparation method of nanoparticle injection: take 25g circle spore mushroom ethyl acetate extract and 100g poloxamer188 3000ml
Anhydrous alcohol solution, adds the ethanol solution of 30ml 1mol/L zinc chloride, and stirring mixing, ultrasonic Treatment makes it in 0.5 ~ 1 hour
Dissolve, it is thus achieved that mixed liquor；Solvent is removed in the volatilization of mixed liquor concentrating under reduced pressure, puts into-20 DEG C of refrigerator freezings 2 hours；Take out filling
Penetrate and use water constant volume, ultrasonic Treatment 0.5 ~ 1 hour, through 0.22 μm filtering with microporous membrane, fill, obtain round spore mushroom ethyl acetate
The nanoparticle injection at position.
The above, be only presently preferred embodiments of the present invention, is not the restriction that the present invention makees other form, appoints
What those skilled in the art changed possibly also with the technology contents of the disclosure above or be modified as equivalent variations etc.
Effect embodiment.But every without departing from technical solution of the present invention content, the technical spirit of the foundation present invention is to above example institute
Any simple modification, equivalent variations and the remodeling made, still falls within the protection domain of technical solution of the present invention.
Claims (8)
1. justify spore mushroom suppression CDC25 enzyme effective site, it is characterised in that: selected from circle spore mushroom petroleum ether part, circle spore mushroom
Ethyl acetate extract, circle spore mushroom n-butanol portion, circle spore mushroom aqueous phase extract any of one or more in position
The mixture of mass ratio.
2. suppress CDC25 enzyme effective site according to the round spore mushroom described in claim 1, it is characterised in that: selected from circle spore mushroom
Ethyl acetate extract extracts in position with circle spore mushroom petroleum ether part, circle spore mushroom n-butanol portion, circle spore mushroom aqueous phase
A kind of mixture by any mass ratio.
3. the preparation method of the round spore mushroom suppression CDC25 enzyme effective site described in claim 1, it is characterised in that under including
State step: pulverized by circle spore mushroom, add concentration expressed in percentage by volume 65% ~ 95% ethanol solution reflux, extract, by dense for gained extracting solution
Contract, be dried, obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, successively with petroleum ether, ethyl acetate and n-butyl alcohol
As solvent extraction extract dispersion liquid, spore mushroom petroleum ether part, circle spore mushroom ethyl acetate portion after flinging to solvent respectively, must be justified
Position, circle spore mushroom n-butanol portion；Extract dispersion liquid after extraction is concentrated, is dried, obtains round spore mushroom aqueous phase extraction position.
The preparation method of round spore mushroom the most according to claim 2 suppression CDC25 enzyme effective site, it is characterised in that: institute
The reflux, extract, stated is ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: the circle after pulverizing
Spore mushroom adds concentration expressed in percentage by volume 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filters, and filtering residue adds volume hundred
Divide concentration 65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.
5. the application of the round spore mushroom suppression CDC25 enzyme effective site described in claim 1, it is characterised in that: press down in preparation
Application in terms of CDC25 enzyme medicine processed.
The application of round spore mushroom the most according to claim 4 suppression CDC25 enzyme effective site, it is characterised in that: described system
Standby suppression CDC25 enzyme medicine is by circle spore mushroom petroleum ether part, circle spore mushroom ethyl acetate extract, circle spore mushroom n-butyl alcohol
The mixture of one or more any mass ratio in position, circle spore mushroom aqueous phase extraction position, mixes with pharmaceutic adjuvant
Close the oral formulations or ejection preparation made.
The application of round spore mushroom the most according to claim 5 suppression CDC25 enzyme effective site, it is characterised in that: described
Ejection preparation is lipidosome injection, nanoparticle injection or micro-balloon injection.
The application of round spore mushroom the most according to claim 5 suppression CDC25 enzyme effective site, it is characterised in that: described
Oral formulations is powder, tablet, granule, capsule or solution.
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| CN110938105A (en) * | 2019-11-18 | 2020-03-31 | 青海民族大学 | Extraction and separation method of active ingredients of agaricus tabularis |
| CN110959851A (en) * | 2019-11-18 | 2020-04-07 | 青海晨菲制药有限公司 | Application of agaricus bisporus gallic acid in functional food |
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