CN106176845A - Willow herb suppresses CDC25 enzyme effective site and preparation method and application - Google Patents

Willow herb suppresses CDC25 enzyme effective site and preparation method and application Download PDF

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CN106176845A
CN106176845A CN201610780012.XA CN201610780012A CN106176845A CN 106176845 A CN106176845 A CN 106176845A CN 201610780012 A CN201610780012 A CN 201610780012A CN 106176845 A CN106176845 A CN 106176845A
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willow herb
extract
herb
willow
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林鹏程
吴疆
曾擎屹
刘晓翠
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Qinghai Nationalities University
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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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • 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
    • 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
    • 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
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

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Abstract

Willow herb suppresses CDC25 enzyme effective site and preparation method and application, belongs to plant extract technical field.One or more in willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extraction position are by the mixture of any mass ratio.After being pulverized by willow herb herb, using the extracting solution that concentration of volume percent 65% ~ 95% ethanol solution reflux, extract, obtains, united extraction liquid, concentrate drying 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 the most successively, after flinging to solvent, obtain willow herb ethyl acetate extract, willow herb n-butanol portion；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.The willow herb suppression CDC25 enzyme effective site of present invention application in terms of preparation suppression CDC25 enzyme medicine, it is possible to be used for preparing antitumor drug.

Description

Willow herb suppresses CDC25 enzyme effective site and preparation method and application
Technical field
Willow herb suppresses CDC25 enzyme effective site and preparation method and application, belongs 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 meters 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.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 raw material of antitumor drug is less at present, especially for preparation suppression CDC25 The raw material of Chinese medicine of Phospoprotein enzyme (being called for short CDC25 enzyme) medicine is the deficientest.Willow herb plant contains Polyphenols, flavone, triterpene The multiple chemical compositions such as acid, tannin compound, in this area, willow herb primary efficacy is clearing heat secreting bile, detumescencing diuresis, stimulating milk secretion, profit Intestinal, but there is presently no the correlational study of anti-tumor aspect.
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 CDC25 enzyme Position and preparation method and application, this willow herb suppression CDC25 enzyme effective site can effectively suppress CDC25A enzyme and the activity of CDC25B enzyme, use In preparation suppression CDC25 enzyme medicine, there is preferable antitumor action.
The technical solution adopted for the present invention to solve the technical problems is: this willow herb suppression CDC25 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 Mixture by any mass ratio.
Preferably, this willow herb suppression CDC25 enzyme effective site, it is characterised in that: extract position, or willow selected from willow herb aqueous phase Blue aqueous phase extraction position and willow herb ethyl acetate extract press the mixture of any mass ratio.
The preparation method of this willow herb suppression CDC25 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 solution and extracts, and is concentrated by gained extracting solution, is dried, obtains extractum；Will Extractum adds water-dispersible, obtains extract dispersion liquid, uses petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction extractum the most successively Dispersion liquid, obtains willow herb ethyl acetate extract, willow herb n-butanol portion after flinging to solvent respectively；After extract dispersion liquid is extracted Remaining liq concentrates, is dried, and obtains willow herb aqueous phase extraction position.
Preferably, described it is extracted as ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: Willow herb herb after pulverizing adds concentration of volume percent 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filters, Filtering residue adds concentration of volume percent 65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, and united extraction liquid to obtain final product.
The application of this willow herb suppression CDC25 enzyme effective site, it is characterised in that: in terms of preparation suppression CDC25 enzyme medicine Application.
Described suppression CDC25 enzyme medicine is willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extraction position In one or more by the mixture of any mass ratio, the oral formulations or the note system that are mixed with pharmaceutic adjuvant penetrate agent 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 willow herb normal usage is clearing heat secreting bile, antidiarrheal, kills Effect of worm, has no that it has the relevant report of antitumor efficacy.Applicant is found by numerous studies: the extracting section in willow herb Thing also has antibacterial, antineoplastic action, but the effective site purity in its extract is relatively low.What kind of by solvent extracted Taking, the effective site that could obtain suppression CDC25 enzymatic activity effect best is the problem that have to solve.Applicant is by research Find: use preparation method of the present invention, the aqueous phase extraction in the ethyl acetate extract filtered out, n-butanol portion and surplus solution Take position, CDC25 enzyme is had preferable inhibition.
In preparation method, it is possible to use only reflux, extract,.Preferably employing ultrasonic assistant reflux, extract, this extracting method has There are higher yield and extraction efficiency.The concrete operations of ultrasonic assistant reflux, extract, are: after ultrasonic extraction, filter acquisition super Sound wave extracting solution and filtering residue, add mass percent concentration 65% ~ 95% ethanol water reflux, extract, in filtering residue, merges ultrasound wave Extracting solution and reflux extracting liquid.Further instruction, the concrete operations of reflux, extract, are: be directly added into being heated at reflux in device Medical material after pulverizing or the filtering residue after addition ultrasonic extraction, add ethanol water, heating extraction, let cool and filter once to carry Take liquid and filtering residue；Again in filtering residue add ethanol water, heat, carry out second time reflux, extract, let cool filter secondary carries Take liquid and filtering residue (being reflux, extract, 2 times above)；Merge repeatedly extracting solution, obtain reflux extracting liquid.Preferably, backflow carries every time Take 0.5 ~ 2 hour；During reflux, extract, added ethanol solution did not had medical material surface 1cm ~ 2cm every time.The temperature of reflux, extract, is second The ordinary temperature that alcohol reflux extracts, 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.Wherein, willow herb ethyl acetate extract contains biology Alkali, flavone compound；Willow herb n-butanol portion contains saponin and phenolic compound；Willow herb aqueous phase extraction position contain polysaccharide and Organic acid compound.Fling to solvent i.e. volatilize removal solvent.Fling to solvent can use normal heating make solvent (petroleum ether, Ethyl acetate and n-butyl alcohol) volatilization.Preferably, flinging to solvent and use concentrating under reduced pressure, the method efficiency is high, and is avoided that temperature-sensitive Property composition loses the property of medicine because of high temperature.
Preferably dosage form is oral formulations or note system penetrates agent or ejection preparation.Oral formulations and ejection preparation are optimal administration Approach, oral formulations described herein 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 is (eye drop, nasal drop, ophthalmically acceptable Ointment, gargarism, sublingual tablet)；Cavity/canal drug administration preparation (suppository).Ejection preparation can be conventional injection preparation.According to medicine Transmission system is classified, it is preferred that the ejection preparation of the present invention is lipidosome injection, nanoparticle injection or microsphere injections Agent；Other, the injection of the present invention can also be microcapsule injection, polymer micelle injection, microemulsion or submicron emulsion injection Agent, submicron injection or gel injection, the injection of above drug delivery system can extend pharmaceutical carrier following in vivo The ring time, extend medicine carrying microgranule absorption site the time of staying, control medicine release the initial stage burst effect.Pharmaceutic adjuvant Including pharmaceutical carrier, 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, disintegrating agent, lubricant, fluidizer, antitack agent, slow releasing agent, controlled release agent, coating material, filmogen, capsule material In one or more.
Compared with prior art, what the willow herb suppression CDC25 enzyme effective site of the present invention and preparation method and application were had has Benefit effect is:
1, this willow herb suppression CDC25 enzyme effective site can effectively suppress CDC25A enzyme and the activity of CDC25B enzyme, presses down for preparation CDC25 enzyme medicine processed, 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, tune of invigorating blood circulation The merit of warp.Applicant finds that antitumor drug exists the problem of scarcity of raw material under study for action, and finds that willow herb also has by research There is antineoplastic effect, by design preparation method, carry out medicine efficacy screening, by determining the kind of the enzyme that willow herb is suppressed With optimal effective site, on CDC25A enzyme and CDC25B enzyme, carry out medicine efficacy screening first, determine willow herb second by numerous studies One or more mixing by any mass ratio in acetoacetic ester position, willow herb n-butanol portion, willow herb aqueous phase extraction position Compound is respectively provided with preferably suppression CDC25A enzyme and CDC25B enzymatic activity, suitably develops into antitumor Tibetan medicine new drug.Further, Applicant is by determining: willow herb aqueous phase extraction position suppression CDC25 enzyme effect is optimum, equal to CDC25A enzyme and CDC25B enzyme There are preferable inhibition, CDC25A enzyme survival rate 38.36%, CDC25B enzyme survival rate 37.3%；Further, willow herb aqueous phase extraction portion Position, and willow herb aqueous phase extraction position and willow herb ethyl acetate extract are by the mixture of any mass ratio, can effectively suppress tumor Growth.
2, the preparation method of this willow herb suppression CDC25 enzyme effective site is extracted convenient, is the side that determines after deliberation of applicant Method.In preparation method of the present invention, the extraction order of solvent is that applicant determines after deliberation, and order can not overturn replacement, use with The upper solvent extraction order willow herb ethyl acetate extract of gained, willow herb n-butanol portion and willow herb aqueous phase extraction position have preferably The effect of suppression CDC25 enzyme.
3, the application in terms of willow herb suppression CDC25 enzyme effective site preparation suppression CDC25 medicine, has expanded suppression CDC25A The raw material channel of enzyme and CDC25B enzyme, expands the purposes of willow herb, makes willow herb develop into suppression CDC25A enzyme and CDC25B enzyme The new raw material of medicine, is remarkably improved the added value of willow herb.
Detailed description of the invention
Embodiment 1 ~ 3 is willow herb suppression CDC25 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 solution to surpass Sound wave extracts 1 hour, then filters to obtain ultrasonic extract and filtering residue, adds concentration of volume percent 75% ~ 85% in filtering residue Ethanol 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, take acetic acid ethyl acetate extract and butanol extraction liquid flings to solvent respectively, To willow herb ethyl acetate extract, willow herb n-butanol portion；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb water Extract position mutually.
Embodiment 2
Preparation method, comprises the steps: to pulverize willow herb herb, uses concentration of volume percent 85% ~ 95% ethanol solution to surpass Sound wave extracts 3 hours, then filters to obtain ultrasonic extract and filtering residue, adds concentration of volume percent 85% ~ 95% in filtering residue Ethanol 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, take acetic acid ethyl acetate extract and butanol extraction liquid flings to solvent respectively, To willow herb ethyl acetate extract, willow herb n-butanol portion；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow herb water Extract position mutually.
Embodiment 3
Preparation method, comprises the steps: to pulverize willow herb herb, uses concentration of volume percent 65% ~ 75% ethanol solution to surpass Sound wave extracts 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, 3 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, take acetic acid ethyl acetate extract and butanol extraction liquid are flung to molten respectively Agent, obtains willow herb ethyl acetate extract, willow herb n-butanol portion；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains willow Blue aqueous phase extraction position.
Obtain willow herb petroleum ether part after embodiment 1 ~ 3 gained petroleum ether extraction liquid is flung to solvent respectively, give over to follow-up Performance test experiment uses.
Performance test
Respectively take 0.4mg dried willow herb petroleum ether part, willow herb ethyl acetate extract, willow herb aqueous phase extraction position, respectively Formation DMSO solution in the dimethyl sulfoxide (DMSO) being dissolved in 100 L, ultrasonic 5 minutes.Take 1 L sample dissolution the most respectively DMSO solution, each addition buffer 99 L, respectively obtain delaying of the willow herb petroleum ether part for detecting willow herb suppression HDAC1 enzyme Dissolved liquid, the buffer solution of willow herb petroleum ether part, the buffer solution of willow herb ethyl acetate extract and willow herb aqueous phase extraction position Buffer solution.The formula of buffer is: take respectively 0.2423gTris-HCl, 0.5138gNaCl, 0.0330gBSA, 0.0154gDTT, 0.0167g EDTA, in 50mL beaker, adds distilled water and dissolves and mix, and adds 0.5mol/L NaOH water Solution, by pH regulator to 8.4, is then settled in 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..
With each position of willow herb that embodiment 1 obtains as tester, detection willow herb each position suppression CDC25A enzyme and CDC25B The activity of enzyme.
Its method is: take 2.7 L 330 μm ol/L FDP, 1.8 L 100 μm ol/L testers, 2 L 0.1 μm ol/ respectively L CDC25A/CDC25B, 11.5 L FDP buffer form the experimental group of 18 L systems；Then 2.7 L 330 μm ol/L are taken FDP, 2 L 0.1 μm ol/L 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 comparison of 18 L systems Group 2；By needing the system adding CDC25A/CDC25B enzyme to hatch 60min under the conditions of 37 DEG C, the most enzyme-added, under similarity condition Reaction 60min, question response is complete, detection product fluorescence intensity at 520nm under 485nm exciting light effect.Experiment Group, matched group 1 and matched group 2 are carried out under the same conditions, and without tester in matched group 1, record experimental result also calculates Enzyme survival efficiency.Enzyme survival efficiency (%)=experimental group measured value/matched group 1 measured value × 100%, wherein enzyme survival efficiency is more Low, illustrate that tester is the strongest to the inhibition of histone acetylation enzyme CDC25A/CDC25B.
Table 1 willow herb puies forward the active testing result of suppression CDC25 enzyme effective site
。
* P ＜ 0.01 in table 1, by table 1 it can be seen that willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb water There is inhibition for CDC25A enzyme CDC25B enzyme in extraction position mutually, and inhibition be substantially better than willow herb petroleum ether part and Matched group.
Two, anti-tumor activity test.
Mtt assay is utilized to measure the willow herb suppression CDC25 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 A-2780 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, and A549 is Human lung carcinoma cell, A2780 is Proliferation of Human Ovarian Cell.2), > 50 show do not have anti-cell cytotoxic activity.Willow is can be seen that by table 2 Blue ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extraction position have 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 ethyl acetate portion Position and willow herb aqueous phase extraction position 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 ethyl acetate portion Position and willow herb aqueous phase extraction position mass ratio 1:1 mixture；After first administration, the next day claim mice body weight first；Often group is given every day Medicine 1 time, each dosage refers to table 3, 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) 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) 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 aqueous phase extraction position high dose group, willow herb n-butanol portion and willow herb aqueous phase extraction portion Position mass ratio 1:1 mixture middle and high dosage group, willow herb aqueous phase extraction position mixes with willow herb ethyl acetate extract mass ratio 1:1 Thing middle and high dosage group, willow herb ethyl acetate extract and willow herb aqueous phase extraction position mass ratio 1:1 mixture middle and high dosage group ring Phosphamide chemotherapy 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 concentration of volume percent 10% makes soft material, and sieve to obtain wet granular, is dried to obtain dry granule, granulate, adds The mixing of magnesium stearate 4mg, 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 volume percent concentration 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: 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 is anhydrous Ethanol dissolves, and adds the ethanol solution of 30mL 1mol/L zinc chloride, and stirring mixing, ultrasonic Treatment makes it molten in 0.5 ~ 1 hour Solve, 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；Taking-up injects 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 CDC25 enzyme effective site, it is characterised in that: selected from willow herb ethyl acetate extract, willow herb n-butanol portion, One or more in willow herb aqueous phase extraction position are by the mixture of any mass ratio.
2. suppress CDC25 enzyme effective site according to the willow herb described in claim 1, it is characterised in that: extract selected from willow herb aqueous phase Position, or willow herb aqueous phase extraction position and willow herb ethyl acetate extract are by the mixture of any mass ratio.
3. the preparation method of the willow herb suppression CDC25 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 solution and extract, gained extracting solution is concentrated, does Dry, obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol the most successively As solvent extraction extract dispersion liquid, after flinging to solvent respectively, obtain willow herb ethyl acetate extract, willow herb n-butanol portion；Will leaching Remaining liq after the extraction of cream dispersion liquid concentrates, is dried, and obtains willow herb aqueous phase extraction position.
4. the preparation method of CDC25 enzyme effective site is suppressed according to the willow herb described in claim 3, it is characterised in that: described It is extracted as ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: the willow herb herb after pulverizing adds Entering concentration of volume percent 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filter, it is dense that filtering residue adds percent by volume Degree 65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.
5. the application of the willow herb suppression CDC25 enzyme effective site described in claim 1, it is characterised in that: in preparation suppression Application in terms of CDC25 enzyme medicine.
The application of willow herb the most according to claim 5 suppression CDC25 enzyme effective site, it is characterised in that: described suppression CDC25 enzyme medicine be willow herb ethyl acetate extract, willow herb n-butanol portion, willow herb aqueous phase extraction position in one or both with Upper by the mixture of any mass ratio, the oral formulations being mixed with pharmaceutic adjuvant or note system penetrate agent or ejection preparation.
The application of willow herb the most according to claim 6 suppression CDC25 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 CDC25 enzyme effective site, it is characterised in that: described is oral Preparation is powder, tablet, granule, capsule or solution.
CN201610780012.XA 2016-08-30 2016-08-30 Willow herb suppresses CDC25 enzyme effective site and preparation method and application Pending CN106176845A (en)

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US20070036834A1 (en) * 2001-08-29 2007-02-15 Pauletti Giovanni M Method for augmentation of intraepithelial and systemic exposure of therapeutic agents having substrate activity for cytochrome P450 enzymes and membrane efflux systems following vaginal and oral cavity administration
JP4531637B2 (en) * 2005-06-15 2010-08-25 株式会社 ヒロインターナショナル Anti-menopausal agent comprising a plant-derived extract
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Application publication date: 20161207