CN106176843A - Willow herb suppresses HDAC1 enzyme effective site and preparation method and application - Google Patents
Willow herb suppresses HDAC1 enzyme effective site and preparation method and application Download PDFInfo
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Abstract
Willow herb suppresses HDAC1 enzyme effective site and preparation method and application, belongs to plant extract technical field.The mixture of one or more any mass ratio in willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extraction position.Willow herb herb is pulverized, adds concentration of volume percent 65% ~ 95% ethanol water reflux, extract, gained extracting solution is concentrated, is dried, obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction extract dispersion liquid successively, after flinging to solvent, obtain willow herb ethyl acetate extract, willow herb n-butanol portion；Extract dispersion liquid after extraction is concentrated, is dried, obtains willow herb aqueous phase extraction position.The willow herb suppression HDAC1 enzyme effective site of present invention application in terms of preparation suppression HDAC1 enzyme medicine, it is possible to be used for preparing antitumor drug.
Description
Technical field
The present invention relates to willow herb suppression HDAC1 enzyme effective site and preparation method and application, belong to plant extract technical field.
Background technology
Willow herb (Epilobium angustifolium L.) be Oenotheraceae (Onagraceae) willow herb belong to perennial slightly
Strong herbaceous plant, is Tibetan medicine's tradition medicinal plants.It is mainly born in hillside border, sylvan life and the river valley of height above sea level 2800-4250 rice
Wet meadow.It is distributed in Southwestern China, northwest, North China to northeast.North temperate zone blazons, and North America, Europe to Japan, Asia Minor are extremely
The ground such as Himalaya also have.Willow herb herb bitter in the mouth, nontoxic, there are detumescencing diuresis, stimulating milk secretion, the function of intestine moistening.Cure mainly hypogalactia, gas
Impractical swollen.Lattice erg Sang Zhaxi records it in " practical Tibetan medicine name storehouse " and has clearing heat secreting bile, antidiarrheal, insecticidal function.
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.Histon deacetylase (HDAC) (HDAC) is an albuminoid enzyme, and structural modification and gene expression regulation to chromosome play
Important effect.Generally, the acetylation of histone beneficially DNA dissociates with histone octamer, nucleosomal structure
Lax, so that various transcription factor and collaborative transcription factor can be specific binding with DNA binding site, turning of activated gene
Record.In cancerous cell, the overexpression of HDAC causes the enhancing of deacetylation effect, by recovering histone positive charge, thus
Increase the gravitation between DNA and histone, make the nucleosome relaxed become very tight, be unfavorable for the expression of specific gene, bag
Include some tumor suppressor genes.Antibiotic FR 901228 (histone deacetylase inhibitors,
HDACi) then can be by improving chromatin specific region acetylation of histone, thus regulating cell apoptosis and differentiation associated protein
Express and stability, inducing cell apoptosis and differentiation, become the antitumor drug that a class is new.HDACi is not only to multiple blood system
System tumor and solid tumor have good therapeutical effect, and have the of a relatively high selectivity of tumor cell and the advantage of low toxicity.
Applicant finds under study for action: the raw material of antitumor drug is less at present, especially for preparation suppression HDAC1 enzyme
The raw material of Chinese medicine of medicine is the deficientest, finds that the raw material of new drug is problem in the urgent need to address.Willow herb plant contain Polyphenols,
The multiple chemical compositions such as flavone, triterpenic acid, tannin compound, in this area willow herb primary efficacy be clearing heat secreting bile, detumescencing diuresis,
Stimulating milk secretion, intestine moistening, there is presently no the correlational study in terms of suppression HDAC1 enzyme.
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 the willow herb suppression effective portion of HDAC1 enzyme
Position and preparation method and application, this willow herb suppression HDAC1 enzyme effective site can effectively suppress the activity of HDAC1 enzyme, be used for preparing suppression
HDAC1 enzyme medicine, has preferable antitumor action.
The technical solution adopted for the present invention to solve the technical problems is: this willow herb suppression HDAC1 enzyme effective site, it is special
Levy and be: selected from willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extraction position in one or more
The mixture of any mass ratio.
This willow herb suppression HDAC1 enzyme effective site, it is characterised in that: selected from willow herb n-butanol portion, or willow herb n-butyl alcohol
The mixture of any mass ratio in position and willow herb aqueous phase extraction position.
The preparation method of this willow herb suppression HDAC1 enzyme effective site, it is characterised in that comprise the steps: willow herb complete
Grass meal is broken, adds concentration of volume percent 65% ~ 95% ethanol water 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 molten the most successively
Agent extraction extract dispersion liquid, obtains willow herb ethyl acetate extract, willow herb n-butanol portion after flinging to solvent respectively；By the leaching after extraction
Cream dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
Described reflux, extract, is ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: will
Willow herb herb after pulverizing adds concentration of volume percent 65% ~ 95% ethanol water ultrasonic extraction 0.5 ~ 3 hour, filters,
Filtering residue adds concentration of volume percent 65% ~ 95% ethanol water reflux, extract, 1 ~ 3 time, and united extraction liquid to obtain final product.
The application of this willow herb suppression HDAC1 enzyme effective site, it is characterised in that: in terms of preparation suppression HDAC1 enzyme medicine
Application.
Described preparation suppression HDAC1 enzyme medicine is by willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase
The mixture of one or more any mass ratio in extraction position, 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.
The present invention is described as follows:
In prior art, willow herb normal usage is treatment cold, fever and skin infection, has no that it has the relevant report of antitumor efficacy.
Applicant is found by numerous studies: the extracting section thing in willow herb also has antibacterial, antineoplastic action, but its effect is not
Substantially, it is impossible to determine its concrete effective site.Therefore by what kind of solvent, use how preparation method, could obtain and press down
The effective site that HDAC1 enzymatic activity effect processed is best, is the problem that have to solve.Applicant is found by research: use this
Invention preparation method, the willow herb ethyl acetate extract filtered out, willow herb n-butanol portion and willow herb aqueous phase extraction position, the most right
Preferable effect is had in suppression HDAC1 enzyme.Wherein, willow herb n-butanol portion suppression HDAC1 enzymatic activity effect is optimum.
In preparation method, it is possible to use only reflux, extract,.Preferably employ ultrasonic assistant reflux, extract, there are higher receipts
Rate and extraction efficiency.The concrete operations of ultrasonic assistant reflux, extract, are: after ultrasonic extraction, filter and obtain ultrasonic extract
And filtering residue, in filtering residue, add concentration of volume percent 65% ~ 95% ethanol water reflux, extract, merge ultrasonic extract and return
Stream extracting solution.Further instruction, the concrete operations of reflux, extract, are: to the medicine being heated at reflux in device after being directly added into pulverizing
Filtering residue after material or addition ultrasonic extraction, adds ethanol water, heating extraction, lets cool and filter to obtain an extracting solution and filter
Slag；In filtering residue, add ethanol water again, heat, carry out second time reflux, extract, let cool and filter to obtain secondary raffinate and filter
Slag (is reflux, extract, 2 times) above；Merge repeatedly extracting solution, obtain reflux extracting liquid.Preferably, each reflux, extract, 0.5 ~ 2
Hour；During reflux, extract, added ethanol water did not had medical material surface 1cm ~ 2cm every time.The temperature of reflux, extract, is alcohol reflux
The ordinary temperature extracted, need to be higher than the boiling point of ethanol, preferably 80 DEG C ~ 100 DEG C.
In preparation method, extractum is crude extract, uses different solvent extraction extract dispersion liquids, refers to successively by stone
Oil ether adds extract dispersion liquid extract and separate and obtains petroleum ether extraction liquid, ethyl acetate adds extract dispersion liquid extract and separate obtains
Obtain acetic acid ethyl acetate extract, n-butyl alcohol is added extract dispersion liquid extract and separate acquisition butanol extraction liquid；Take ethyl acetate extraction
Taking liquid and butanol extraction liquid volatilizees after removing solvent respectively, gained is i.e. selected from willow herb ethyl acetate extract and willow herb n-butyl alcohol portion
Position；Remaining extract dispersion liquid (solvent is water) after extracting, volatilization is removed solvent and is i.e. obtained willow herb aqueous phase extraction position.Aqueous phase extracts
Taking position and may also be referred to as aqueous phase extract, aqueous phase extraction position is soluble in water；And relative, petroleum ether, ethyl acetate and just
Butanol is organic facies, and petroleum ether part, ethyl acetate extract be corresponding with n-butanol portion is soluble in petroleum ether, ethyl acetate and just
Butanol.Wherein, willow herb ethyl acetate extract contains alkaloid, flavone compound；Willow herb n-butanol portion contains saponin and phenol
Compounds；Polysaccharide and organic acid compound are contained in willow herb aqueous phase extraction position.Fling to solvent i.e. volatilize removal solvent.Fling to
Solvent can use normal heating to make solvent (petroleum ether, ethyl acetate and n-butyl alcohol) volatilize.Preferably, fling to solvent employing subtract
Pressure concentrates, and 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.Enter according to drug delivery system
Row classification, it is preferred that the ejection preparation of the present invention is lipidosome injection, nanoparticle injection or micro-balloon injection；Other,
The injection of the present invention can also be microcapsule injection, polymer micelle injection, microemulsion or Submicroemulsion injection, submicron note
Penetrating agent or gel injection, the injection of above drug delivery system can extend pharmaceutical carrier circulation time in vivo, prolongation
Medicine carrying microgranule absorption site the time of staying, control medicine release the initial stage burst effect.Pharmaceutic adjuvant includes that medicine carries
Body, and solvent, solubilizing agent, cosolvent, emulsifying agent, suspending agent, clarifier, deflocculant, correctives, coloring agent, preservative,
Chemosterilant, adsorbent, filter aid, antioxidant, pH adjusting agent, isoosmotic adjusting agent, diluent, binding agent, wetting agent, disintegrate
One in agent, lubricant, fluidizer, antitack agent, slow releasing agent, controlled release agent, coating material, filmogen, capsule material or
Multiple.
Compared with prior art, what the willow herb suppression HDAC1 enzyme effective site of the present invention and preparation method and application were had has
Benefit effect is:
1, this willow herb suppression HDAC1 enzyme effective site can effectively suppress the activity of HDAC1 enzyme, is used for preparing suppression HDAC1 enzyme medicine
Thing, has preferable antitumor action.In this area, willow herb has promoting diuresis to eliminate damp pathogen, relieving distension of regulating the flow of vital energy, the merit of promoting blood flow to regulate menstruation.Application
People finds that antitumor drug exists the problem of scarcity of raw material under study for action, and finds that willow herb also has antineoplastic merit by research
Effect, by design preparation method, carries out medicine efficacy screening, by determining the kind of the enzyme that willow herb is suppressed and optimal effectively portion
Position, applicant, during solution problem of pinpointing the problems above, has paid substantial amounts of creative work.Applicant exists first
Carry out medicine efficacy screening on HDAC1 enzyme, determine willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb water by numerous studies
The mixture of one or more any mass ratio in extraction position is respectively provided with the effect preferably suppressing HDAC1 enzyme mutually
Really, HDAC1 enzyme survival rate 18.94% ~ 36.02%, it is possible to be used for developing into antitumor Tibetan medicine new drug.Owing to different solvents extraction obtains
The each effective site obtained is different to HDAC1 inhibition of enzyme activity ability, and applicant is by further determining: willow herb n-butyl alcohol
The suppression HDAC1 enzyme effect at position is optimum, its HDAC1 enzyme survival rate 18.94%.In sum, applicant finds first
New effect of willow herb, and by determining the kind of its enzyme suppressed, design preparation method also finally obtains HDAC1 enzyme
Being respectively provided with the effective site of preferable inhibition, above procedure has paid substantial amounts of creative work.
2, the preparation method of this willow herb suppression HDAC1 enzyme effective site is extracted conveniently, and extraction efficiency is high.In preparation method,
Applicant designs addition concentration of volume percent 65% ~ 95% ethanol water and carries out ultrasonic assistant reflux, extract, improves and carries
Take efficiency；In extraction, use petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction extract dispersion liquid successively, use above molten
The agent extraction order willow herb ethyl acetate extract of gained, willow herb n-butanol portion, willow herb aqueous phase extraction position have and preferably press down
The effect of HDAC1 enzyme processed.
3, the present invention has expanded the raw material channel of suppression HDAC1 enzyme, expands the purposes of willow herb, makes willow herb develop into and press down
The new raw material of HDAC1 enzyme medicine processed, is remarkably improved the added value of willow herb.
Detailed description of the invention
Embodiment 1 ~ 3 is willow herb suppression HDAC1 enzyme effective site and the detailed description of the invention of preparation method, the Qi Zhongshi of the present invention
Execute example 1 for most preferred embodiment.
Embodiment 1
Preparation method, comprises the steps: to pulverize willow herb herb, uses concentration of volume percent 75% ~ 85% ethanol water
Ultrasonic extraction 1 hour, then filters to obtain ultrasonic extract and filtering residue, add in filtering residue concentration of volume percent 75% ~
85% ethanol solution reflux, extract, 3 times, each 0.5 ~ 1 hour, filters to obtain reflux extracting liquid, merges ultrasonic extract and backflow
Extracting solution, concentrate drying obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, acetic acid the most successively
Ethyl ester and n-butyl alcohol, as solvent extraction extract dispersion liquid, obtain willow herb petroleum ether part, willow herb ethyl acetate after flinging to solvent
Position, willow herb n-butanol portion；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
Embodiment 2
Preparation method, comprises the steps: to pulverize willow herb herb, uses concentration of volume percent 85% ~ 95% ethanol water
Ultrasonic extraction 3 hours, then filters to obtain ultrasonic extract and filtering residue, add in filtering residue concentration of volume percent 85% ~
95% ethanol solution reflux, extract, 3 times, each 1 ~ 1.5 hour, filters to obtain reflux extracting liquid, merges ultrasonic extract and backflow
Extracting solution, concentrate drying obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, acetic acid the most successively
Ethyl ester and n-butyl alcohol, as solvent extraction extract dispersion liquid, obtain willow herb petroleum ether part, willow herb ethyl acetate after flinging to solvent
Position, willow herb n-butanol portion；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
Embodiment 3
Preparation method, comprises the steps: to pulverize willow herb herb, uses concentration of volume percent 65% ~ 75% ethanol water
Ultrasonic extraction 0.5 hour, then filters to obtain ultrasonic extract and filtering residue, add in filtering residue concentration of volume percent 65 ~
75% ethanol solution reflux, extract, 2 times, each 1.5 ~ 2 hours, filters to obtain reflux extracting liquid, merges ultrasonic extract and backflow
Extracting solution, concentrate drying obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, acetic acid the most successively
Ethyl ester and n-butyl alcohol, as solvent extraction extract dispersion liquid, obtain willow herb petroleum ether part, willow herb ethyl acetate after flinging to solvent
Position, willow herb n-butanol portion；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
Performance test
Respectively take 0.4mg dried willow herb petroleum ether part, willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb
Aqueous phase extraction position, is each dissolved in the dimethyl sulfoxide (DMSO) of 100 L and forms DMSO solution, ultrasonic 5 minutes.Distinguish again
Take the DMSO solution of 1 L sample dissolution, be separately added into buffer 99 L, respectively obtain for detecting willow herb suppression HDAC1 enzyme
The buffer solution of willow herb petroleum ether part, the buffer solution of willow herb ethyl acetate extract, the buffer solution of willow herb n-butanol portion
Buffer solution with willow herb aqueous phase extraction position.The formula of buffer is: the Tris-HCl of 50mM/L, 0.137 mM/L
The MgCl of KCl, 1mM/L of NaCl, 2.7mM/L2, 0.01% polysorbas20, pH of buffer 8.0.
One, anti-HDAC1 enzyme experiment.
Major experimental material is:
HDAC1 enzyme, Cisbio Bioassays Products；
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；
NaCl(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 City three；
BRAND8 road liquid-transfering gun, converge your Instrument Ltd. in Hangzhou, Zhejiang province；
500 L liquid-transfering guns, 50 L liquid-transfering guns, Shanghai City Rong Tai biochemical instrument company limited；
Electronic balance XPE105, prunus mume (sieb.) sieb.et zucc. Teller-torr benefit Instrument Ltd..
Suppress HDAC1 enzyme effective site as test medicine with the willow herb that embodiment 1 obtains.It represents experiment for willow herb oil
Ether position, willow herb ethyl acetate extract, willow herb n-butanol portion and the activity of willow herb aqueous phase extraction position suppression HDAC1 enzyme.Its
Method is, the most separately take the willow herb petroleum ether part (label D) of 4 L, the buffer solution (label E) of willow herb ethyl acetate extract,
The buffer solution (label F) of willow herb n-butanol portion and willow herb aqueous phase extraction position buffer solution (label G), carry out respectively as
Lower operation: the 4 L buffer solution (D, E, F, G) taken are placed in 96 orifice plates, then are separately added into 2 L HDAC1 enzymes, room temperature bar
React 5min under part, add 4 L H3(1-21) K9 substrate, 37 DEG C, incubator is cultivated 60 minutes.Add 5 L SA-XL665
With 5 L H3K9me0 antibody, 665nm measured value, obtain experimental group absorbance.
The buffer solution of 4 L is placed in 96 orifice plates, then is separately added into 2 L HDAC1 enzymes, under room temperature condition, react 5min,
Add 4 L H3(1-21) K9 substrate, 37 DEG C, incubator is cultivated 60 minutes.Add 5 L SA-XL665 and 5 L H3K9me0
Antibody, 665nm measured value, obtain matched group absorbance.Calculating HDAC1 enzyme survival rate: survival rate (%)=experimental group absorbance/matched group
Absorbance × 100%.
The active testing result of table 1 willow herb suppression HDAC1 enzyme effective site suppression HDAC1 enzyme
。
* P ＜ 0.01 in table 1, by table 1 it can be seen that willow herb ethyl acetate extract, willow herb n-butanol portion and willow herb
All there is preferable inhibition at aqueous phase extraction position for HDAC1 enzyme.
Two, anti-tumor activity test.
Mtt assay is utilized to measure the willow herb suppression HDAC1 enzyme effective site cytotoxic activity to human tumor cells.Example
The 1 each position of gained willow herb is appropriate, as test-compound, after adding DMSO dissolving, utilizes mtt assay to measure respectively human colon carcinoma
HCT-8, Human hepatocarcinoma Bel-7402 cell, people's gastric cancer BGC-803 cell, human pulmonary epithelial cells and human ovarian cancer A2780 are thin
The suppression ratio of intracellular 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
100µL.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 training
Supporting 4h, after abandoning supernatant, every hole adds 200 L DMSO and dissolves MTT first hairpin precipitation, microoscillator vibration mixing, uses microplate reader
Optical density value (OD) is measured, the tumor cell processed with solvent control under the conditions of reference wavelength 450nm, detection wavelength 570nm
For matched group, calculate the test-compound suppression ratio to tumor cell by below equation, and by middle effect Equation for Calculating IC50: suppression
Rate=(matched group mean OD value-administration group mean OD value) ÷ matched group mean OD value × 100%.By the extractum in preparation method,
And the willow herb petroleum ether part of gained, willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extract position and carry out
Tumor cell in vitro cytotoxic activity screens, and 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: willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extract position preferable antineoplastic effect really.
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: willow herb petroleum ether part basic, normal, high dosage group, willow herb
Ethyl acetate extract basic, normal, high dosage group, willow herb n-butanol portion basic, normal, high dosage group, willow herb aqueous phase extraction position low,
Middle and high dosage group, willow herb n-butanol portion and willow herb aqueous phase extraction position mass ratio 1:1 mixture basic, normal, high dosage group, willow
Blue aqueous phase extraction position and willow herb ethyl acetate extract mass ratio 1:1 mixture basic, normal, high dosage group, willow herb n-butanol portion
With willow herb ethyl acetate extract mass ratio 1:1 mixture 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 willow herb petroleum ether part of gastric infusion embodiment 1, willow herb acetic acid second
Esteratic site, willow herb n-butanol portion and willow herb aqueous phase extraction position, willow herb n-butanol portion and willow herb aqueous phase extraction position quality
Than 1:1 mixture, willow herb aqueous phase extraction position and willow herb ethyl acetate extract mass ratio 1:1 mixture, willow herb n-butanol portion
With willow herb ethyl acetate extract mass ratio 1:1 mixture；After first administration, the next day claim mice body weight first；Often group is administered 1 every day
Secondary, 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).
In table 3 ~ 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, and embodiment 1 ~ 3 all has preferable antitumous effect, in embodiment 1 ~ 3 during same area same dose,
Antitumous effect between each group is without significant difference.Secondly, compared with blank group, the middle and high dosage of willow herb ethyl acetate extract
Group, willow herb n-butyl alcohol high dose group, willow herb n-butanol portion high dose group, willow herb aqueous phase extraction position middle and high dosage group, willow herb
N-butanol portion and willow herb aqueous phase extraction position mass ratio 1:1 mixture middle and high dosage group, willow herb aqueous phase extraction position and willow herb
Ethyl acetate extract mass ratio 1:1 mixture middle and high dosage group, willow herb n-butanol portion and willow herb ethyl acetate extract mass ratio
1:1 mixture middle and high dosage group, Treated with Chemotherapy with Cyclophosphamide group, all can significantly inhibit S180The growth of tumor.
Embodiment 4
Willow herb effective site is prepared as tablet, uses following steps: willow herb ethyl acetate extract 300mg and starch 100mg mixes
Even, the gelatinized corn starch 40mg adding mass percent concentration 10% makes soft material, and sieve to obtain wet granular, 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 willow herb aqueous phase is extracted 5 parts of position, sucrose 1 part, the mixing of 3 parts of dextrin, adds
Appropriate mass percent 95% ethanol water 2 ~ 5 parts, stirring while adding, prepare soft material, soft material is dried, crosses 16 mesh sieves, subpackage
Obtain.
Embodiment 6
Pellet capsule is composed of the following raw materials by weight: willow herb n-butanol portion 30 parts, 5 parts of lecithin, Bile Salts 5 parts,
Microcrystalline Cellulose 30 parts；
The preparation method of pellet capsule: willow herb n-butanol portion, lecithin, Bile Salts and microcrystalline Cellulose are mixed by proportioning
All, pour ethanol water into and stir evenly acquisition soft material, soft material is poured in extruder and extrude, through round as a ball acquisition granule, extrude 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, be filled into by micropill
In capsule shells, to obtain final product.
Embodiment 7
The preparation method of lipidosome injection:
1) under nitrogen protection, by 50g cholesterol succinate, 250g DSPE, 40g Semen sojae atricolor ovum phosphorus
Fat, 50g poloxamer-188 and 10g willow herb n-butanol portion are dissolved in ethanol that 1500ml volume ratio is 1:1 and n-butyl alcohol
In organic solvent, stirring makes it dissolve acquisition suspension；Suspension is flung to organic solvent by concentrating under reduced pressure, it is thus achieved that phospholipid
Film；
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 willow herb n-butanol portion；
3) aseptically, in the liposome of willow herb n-butanol portion, add 100g trehalose, stir, ultrasound wave
Process 0.5 ~ 1 hour, inject and use water constant volume, through 0.22 μm filtering with microporous membrane, fill, obtain the fat of willow herb n-butanol portion
Liposome injection.
Embodiment 8
The preparation method of nanoparticle injection: take 25g willow herb ethyl acetate extract and 100g poloxamer188 3000ml without
Water-ethanol dissolves, and adds the ethanol water 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 willow herb ethyl acetate extract
Nanoparticle injection.
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. willow herb suppression HDAC1 enzyme effective site, it is characterised in that: selected from willow herb ethyl acetate extract, willow herb n-butanol portion,
The mixture of one or more any mass ratio in willow herb aqueous phase extraction position.
2. suppress HDAC1 enzyme effective site according to the willow herb described in claim 1, it is characterised in that: selected from willow herb n-butyl alcohol portion
Position, or the mixture of any mass ratio of willow herb n-butanol portion and willow herb aqueous phase extraction position.
3. the preparation method of the willow herb suppression HDAC1 enzyme effective site described in claim 1, it is characterised in that include following step
Rapid: willow herb herb is pulverized, add concentration of volume percent 65% ~ 95% ethanol water reflux, extract, by dense for gained extracting solution
Contract, be dried, obtain extractum；Extractum is added distilled water dispersion, obtain extract dispersion liquid, use the most successively petroleum ether, ethyl acetate and
N-butyl alcohol, as solvent extraction extract dispersion liquid, obtains willow herb ethyl acetate extract, willow herb n-butanol portion after flinging to solvent respectively；
Extract dispersion liquid after extraction is concentrated, is dried, obtains willow herb aqueous phase extraction position.
4. the preparation method of HDAC1 enzyme effective site is suppressed according to the willow herb described in claim 3, it is characterised in that: described
Reflux, extract, is ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: the willow herb after pulverizing is complete
Grass adds concentration of volume percent 65% ~ 95% ethanol water ultrasonic extraction 0.5 ~ 3 hour, filters, and filtering residue adds volume hundred
Proportion by subtraction concentration 65% ~ 95% ethanol water reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.
5. the application of the willow herb suppression HDAC1 enzyme effective site described in claim 1, it is characterised in that: in preparation suppression
Application in terms of HDAC1 enzyme medicine.
The application of willow herb the most according to claim 5 suppression HDAC1 enzyme effective site, it is characterised in that: described preparation presses down
HDAC1 enzyme medicine processed be by willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extraction position in one or
The mixture of two or more any mass ratioes, the oral formulations being mixed with pharmaceutic adjuvant or ejection preparation.
The application of willow herb the most according to claim 6 suppression HDAC1 enzyme effective site, it is characterised in that: described injection
Preparation is lipidosome injection, nanoparticle injection or micro-balloon injection.
The application of willow herb the most according to claim 6 suppression HDAC1 enzyme effective site, it is characterised in that: described is oral
Preparation is powder, tablet, granule, capsule or solution.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115737549A (en) * | 2022-11-24 | 2023-03-07 | 北京鑫开元医药科技有限公司 | Injection with HDAC (Histone deacetylase) inhibitory activity and preparation method and application thereof |
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| CN115737549A (en) * | 2022-11-24 | 2023-03-07 | 北京鑫开元医药科技有限公司 | Injection with HDAC (Histone deacetylase) inhibitory activity and preparation method and application thereof |
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