CN106176984A - Radix Corydalis conspersae effective site and its preparation method and application - Google Patents
Radix Corydalis conspersae effective site and its preparation method and application Download PDFInfo
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Abstract
Radix Corydalis conspersae effective site and its preparation method and application, belongs to plant extract technical field.The mixture of one or more any mass ratio in Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion, Radix Corydalis conspersae aqueous phase extraction position.Radix Corydalis conspersae herb is pulverized, adds concentration of volume percent 65% ~ 95% ethanol solution 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 Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion；Extract dispersion liquid after extraction is concentrated, is dried, obtains Radix Corydalis conspersae aqueous phase extraction position.The Radix Corydalis conspersae effective site of present invention application in terms of preparation suppression CDC25 enzyme and suppression HDAC1 enzyme medicine, it is possible to be used for preparing antitumor drug.
Description
Technical field
Radix Corydalis conspersae effective site and its preparation method and application, belongs to plant extract technical field.
Background technology
Radix Corydalis conspersae (Corydalis conspersa Maxim.) is that Papaveraceae Genus Corydalis [sees: Chinese Plants
Will. volume 32. Beijing: Science Press, 1999:266], it is Tibetan medicine's tradition medicinal plants.It is distributed in China and produces Gan Suxi
South, the middle and south, Qinghai, Cycas szechuanmsis and west area and Eastern Tibet and middle part, be born in height above sea level (3800 ~ 5700) rice many
Stone river bank and high mountain gravel ground." China's Tibetan medicine " records its herb for pestilence epidemic disease, fire burn, the heat symptom-complex of red bar disease.Cure mainly
Hot spreading venereal diseases, gastritis, gastric ulcer, cold, fever, external treatment skin infection.
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.But, although different cancers is carcinogenic
Reason may be not quite similar, but its structure observation all shows similar cell cycling disorder and rapid uncontrollable cell
Growth and transfer.Therefore, the research to some family proteins participating in cell cycle regulating becomes to attach most importance in promoting treatment of cancer
Want factor.Wherein, cell division cycle kinases (Cdk/cyclins), as the major regulatory albumen of eukaryotic cell cycle, is responsible for
Promote the cell division of respective stage.CDC25 Phospoprotein enzyme (CDC25 phosphatases) is as cell division cycle egg
The activated protein enzyme of white kinase families (Cdk/cyclins), has played highly important regulation in the division cycle of eukaryotic cell
Effect.Clinical research at present shows, the protease of this family all shows the expression of excess in kinds cancer tissue, and past
Past and poor cancer early prediction is relevant, and these all make CDC25 Phospoprotein enzyme become the treatment of cancer with great potential
Drug targets.In recent years, many enzymologies about CDC25 Phospoprotein enzyme family are related inhibitors and cancer drug
Research and development provide considerable information.
Applicant finds under study for action: the raw material of antitumor drug is less at present, especially for preparation suppression CDC25 enzyme
The raw material of Chinese medicine of medicine is the deficientest.Radix Corydalis conspersae plant contains the multiple chemistry such as alkaloids, polysaccharide, chromocor compound and becomes
Point, prior art is mainly used in treating hot spreading venereal diseases, gastritis, gastric ulcer, cold, fever, external treatment skin infection.At present, not yet
See the research in terms of Radix Corydalis conspersae suppression CDC25 enzyme and HDAC1 enzyme and the report of 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 Radix Corydalis conspersae effective site and
Preparation method and application, this Radix Corydalis conspersae effective site can effectively suppress CDC25 enzyme medicine and the activity of HDAC1 enzyme, be used for making
Standby suppression CDC25 enzyme medicine and suppression HDAC1 enzyme medicine, have preferable antitumor action.
The technical solution adopted for the present invention to solve the technical problems is: this Radix Corydalis conspersae effective site, it is characterised in that:
One or two in Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion, Radix Corydalis conspersae aqueous phase extraction position
The mixture of any mass ratio more than kind.
This Radix Corydalis conspersae effective site, it is characterised in that: selected from Radix Corydalis conspersae ethyl acetate extract.
The preparation method of this Radix Corydalis conspersae effective site, it is characterised in that comprise the steps: Radix Corydalis conspersae herb powder
Broken, add concentration of volume percent 65% ~ 95% ethanol solution reflux, extract, gained extracting solution is concentrated, is dried, obtain extractum；Will
Extractum adds distilled water dispersion, obtains extract dispersion liquid, divides as solvent extraction extractum with petroleum ether, ethyl acetate and n-butyl alcohol successively
Dissipate liquid, after flinging to solvent, obtain Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion；By the extract dispersion liquid after extraction
Concentrate, be dried, obtain Radix Corydalis conspersae aqueous phase extraction position.
Preferably, described reflux, extract, is ultrasonic assistant reflux, extract, the concrete behaviour of ultrasonic assistant reflux, extract,
As: it is little that the Radix Corydalis conspersae herb after pulverizing adds concentration of volume percent 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3
Time, filtering, 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 Radix Corydalis conspersae effective site, it is characterised in that: at preparation suppression CDC25 enzyme medicine and suppression HDAC1
Application in terms of enzyme medicine.
Described preparation suppression CDC25 enzyme medicine and suppression HDAC1 enzyme medicine are by Radix Corydalis conspersae ethyl acetate extract, speckle
Brightly yellowish violet n-butanol portion, Radix Corydalis conspersae aqueous phase extract the mixture of one or more any mass ratio in 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.
Applicant is described as follows for the present invention: in prior art, and Radix Corydalis conspersae normal usage is treatment cold, fever
And skin infection, have no that it has the relevant report of antitumor efficacy.Applicant is found by numerous studies: the part in Radix Corydalis conspersae carries
Take thing and also there is antibacterial, antineoplastic action, but its effect is inconspicuous, it is impossible to determine its concrete effective site.Therefore lead to
Cross what kind of solvent, use how preparation method, the effective site that suppression CDC25 enzymatic activity effect is best could be obtained, be must
The problem that need solve.Applicant is found by research: use preparation method of the present invention, the Radix Corydalis conspersae ethyl acetate filtered out
Position, Radix Corydalis conspersae n-butanol portion and Radix Corydalis conspersae aqueous phase extraction position, not only preferable for suppression CDC25 enzyme effect, with
Time for suppression HDAC1 enzyme also have preferable effect.
In preparation method, ultrasonic extraction is to carry out under room temperature.Filter and reflux, extract, refers to: after ultrasonic extraction, mistake
Filter obtains ultrasonic extract and filtering residue, adds mass percent concentration 65% ~ 95% ethanol water reflux, extract, in filtering residue, closes
And ultrasonic extraction and reflux, extract, extracting solution.The concrete operations of reflux, extract, are: after being heated at reflux in device addition pulverizing
Medical material or ultrasonic extraction after filtering residue, add ethanol water, heating extraction, let cool and filter to obtain an extracting solution and filter
Slag；In filtering residue, add ethanol solution, heat, carry out second time reflux, extract, let cool filter secondary raffinate and filtering residue (with
Above it is reflux, extract, 2 times)；Merge repeatedly extracting solution, obtain reflux, extract, extracting solution.Preferably, each reflux, extract, 0.5 ~ 2 is little
Time；During reflux, extract, added ethanol solution did not had medical material surface 1cm ~ 2cm every time.The temperature of reflux, extract, is alcohol reflux
Ordinary temperature, 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
Take liquid and butanol extraction liquid volatilizees after removing solvent respectively, it is thus achieved that Radix Corydalis conspersae ethyl acetate extract and Radix Corydalis conspersae n-butyl alcohol
Position；Remaining extract dispersion liquid (solvent is water) after extracting, volatilization is removed solvent and is i.e. obtained Radix Corydalis conspersae aqueous phase extraction position.
Aqueous phase extraction position may also be referred to as aqueous phase extract, and aqueous phase extraction position is soluble in water；And relative, petroleum ether, acetic acid second
Ester and n-butyl alcohol are organic facies, and petroleum ether part, ethyl acetate extract are corresponding with n-butanol portion is soluble in petroleum ether, acetic acid second
Ester and n-butyl alcohol.Wherein, Radix Corydalis conspersae ethyl acetate extract contains alkaloid, flavone compound；Radix Corydalis conspersae n-butyl alcohol portion
Position is containing saponin and phenolic compound；Polysaccharide and organic acid compound are contained in Radix Corydalis conspersae aqueous phase extraction position.Fling to solvent
Solvent is removed in i.e. volatilization.Flinging to solvent can use normal heating to make solvent (petroleum ether, ethyl acetate and n-butyl alcohol) volatilize.Excellent
Choosing, to fling to solvent and use concentrating under reduced pressure, the method efficiency is high, and is avoided that heat-sensitive ingredients loses the property of medicine because of high temperature.
Preferably, suppression CDC25 enzyme medicine prepared by Radix Corydalis conspersae and suppression HDAC1 enzyme medicine, for oral formulations or note
Penetrate preparation.Oral formulations and ejection preparation are optimal route of administration, and oral formulations described herein is through intestines and stomach drug-delivery preparation, excellent
Choosing, oral formulations is spacetabs type or control release type oral formulations, comprises slow releasing agent or controlled release agent in pharmaceutic adjuvant.Can also be non-
Other of enteral administration, 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, micro-emulsion injecta, Submicroemulsion injection, Asia
Microgranule injection or gel injection, when the injection of above drug delivery system can extend pharmaceutical carrier circulation in vivo
Between, extend medicine carrying microgranule absorption site the time of staying, control medicine release the initial stage burst effect.Pharmaceutic adjuvant includes
Pharmaceutical carrier and solvent, solubilizing agent, cosolvent, emulsifying agent, suspending agent, clarifier, deflocculant, correctives, coloring agent, anti-
Rotten agent, chemosterilant, adsorbent, filter aid, antioxidant, pH adjusting agent, isoosmotic adjusting agent, diluent, binding agent, moistening
In agent, disintegrating agent, lubricant, fluidizer, antitack agent, slow releasing agent, controlled release agent, coating material, filmogen, capsule material
One or more.
Compared with prior art, what Radix Corydalis conspersae effective site of the present invention and its preparation method and application was had is useful
Effect is:
1, this Radix Corydalis conspersae effective site can effectively suppress CDC25 enzyme and the activity of HDAC1 enzyme, is used for preparing suppression CDC25 enzyme
Medicine and suppression HDAC1 enzyme medicine, have preferable antitumor action.Applicant determines through numerous studies: merit in prior art
Effect is treatment treatment cold, fever and the Radix Corydalis conspersae of skin infection, also has antineoplastic effect.And applicant exists first
Carry out medicine efficacy screening on CDC25A enzyme, CDC25B enzyme and HDAC1 enzyme, determine Radix Corydalis conspersae ethyl acetate portion by numerous studies
Position, Radix Corydalis conspersae n-butanol portion, Radix Corydalis conspersae aqueous phase extract the mixed of one or more any mass ratio in position
Compound is respectively provided with preferably suppression CDC25A enzyme and CDC25B enzymatic activity, its CDC25A enzyme survival rate 8.7 ~ 20.7%, CDC25B enzyme
Survival rate 19.6 ~ 31.8%, and there is a preferable inhibition at three above position equally for HDAC1 enzyme, and HDAC1 enzyme is survived
Rate 21.80 ~ 29.35 %, it is possible to be used for developing into antitumor Tibetan medicine new drug.The each effective site obtained due to different solvents extraction
Rejection ability alive to CDC25A enzyme and CDC25B enzyme is different, and applicant is by further determining: Radix Corydalis conspersae acetic acid second
The suppression CDC25 enzyme effect of esteratic site is optimum, and its CDC25A enzyme survival rate 8.7%, CDC25B enzyme survival rate 19.6%, to HDAC1
Enzyme can reach preferable inhibition, HDAC1 enzyme survival rate 22.88% equally.In sum, applicant finds first
New effect of Radix Corydalis conspersae, and by determining the kind of its enzyme suppressed, design preparation method and finally obtain right
CDC25 enzyme and HDAC1 enzyme are respectively provided with the effective site of preferable inhibition, and above procedure is to have paid substantial amounts of creative work
's.
2, the preparation method of this Radix Corydalis conspersae effective site is extracted conveniently, and extraction efficiency is high.In preparation method, applicant sets
Meter adds concentration of volume percent 65% ~ 95% ethanol solution and carries out ultrasonic assistant reflux, extract, improves extraction efficiency；Extraction
In, use petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction extract dispersion liquid successively, use above solvent extraction sequentially institute
Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion and Radix Corydalis conspersae aqueous phase extraction position have and preferably press down
CDC25 enzyme processed and the effect of HDAC1 enzyme.
3, the present invention has expanded suppression CDC25 enzyme medicine and the raw material channel of suppression HDAC1 enzyme medicine, expands speckle brightly yellowish
The purposes of violet, makes Radix Corydalis conspersae develop into suppression CDC25 enzyme medicine and the new raw material of suppression HDAC1 enzyme medicine, can significantly carry
The added value of high Radix Corydalis conspersae.
Detailed description of the invention
Embodiment 1 ~ 3 is the detailed description of the invention of the Radix Corydalis conspersae effective site of the present invention and preparation method thereof, Qi Zhongshi
Execute example 1 for most preferred embodiment.
Embodiment 1
Preparation method, comprises the steps: to pulverize Radix Corydalis conspersae herb, uses concentration of volume percent 75% ~ 85% ethanol molten
Liquid ultrasonic extraction 1 hour, then filters, filtering residue reflux, extract, 3 times, and each 0.5 hour, united extraction liquid, concentrate drying obtained
Extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol conduct the most successively
Solvent extraction extract dispersion liquid, takes acetic acid ethyl acetate extract and butanol extraction liquid solvent flashing respectively, obtains Radix Corydalis conspersae second
Acetoacetic ester position, Radix Corydalis conspersae n-butanol portion；After being extracted by extract dispersion liquid, remaining liq concentrates, is dried, and obtains Radix Corydalis conspersae
Aqueous phase extraction position.
Embodiment 2
Preparation method, comprises the steps: to pulverize Radix Corydalis conspersae herb, uses concentration of volume percent 85% ~ 95% ethanol molten
Liquid ultrasonic extraction 3 hours, then filters, filtering residue reflux, extract, 3 times, and each 1 hour, united extraction liquid, concentrate drying must soak
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, takes acetic acid ethyl acetate extract and butanol extraction liquid solvent flashing respectively, obtains Radix Corydalis conspersae acetic acid
Ethyl ester position, Radix Corydalis conspersae n-butanol portion；After being extracted by extract dispersion liquid, remaining liq concentrates, is dried, and obtains Radix Corydalis conspersae water
Extract position mutually.
Embodiment 3
Preparation method, comprises the steps: to pulverize Radix Corydalis conspersae herb, uses concentration of volume percent 65% ~ 75% ethanol molten
Liquid ultrasonic extraction 0.5 hour, then filters, filtering residue reflux, extract, 2 times, and each 2 hours, united extraction liquid, concentrate drying obtained
Extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol conduct the most successively
Solvent extraction extract dispersion liquid, takes acetic acid ethyl acetate extract and butanol extraction liquid solvent flashing respectively, obtains Radix Corydalis conspersae second
Acetoacetic ester position, Radix Corydalis conspersae n-butanol portion；After being extracted by extract dispersion liquid, remaining liq concentrates, is dried, and obtains Radix Corydalis conspersae
Aqueous phase extraction position.
Embodiment 1 ~ 3 extraction gained petroleum ether extraction liquid is flung to solvent respectively, obtains embodiment 1 ~ 3 Radix Corydalis conspersae petroleum ether
Position, gives over to follow-up performance test and uses.
Performance test
Respectively take 0.4mg dried Radix Corydalis conspersae petroleum ether part, Radix Corydalis conspersae ethyl acetate extract, the positive fourth of Radix Corydalis conspersae
Alcohol position, Radix Corydalis conspersae aqueous phase extraction position, be each dissolved in the dimethyl sulfoxide (DMSO) of 100 L and form DMSO solution,
Ultrasonic 5 minutes.Take the DMSO solution of 1 L sample dissolution the most respectively, be separately added into buffer 99 L, respectively obtain Radix Corydalis conspersae stone
Oil the buffer solution at ether position, the buffer solution of Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion buffering molten
Liquid and the buffer solution at Radix Corydalis conspersae aqueous phase extraction position.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
Even, add 0.5mol/L NaOH aqueous solution by pH regulator to 8.4, be 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；
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 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 Radix Corydalis conspersae that embodiment 1 obtains as tester, detection Radix Corydalis conspersae each position suppression CDC25A enzyme
Activity with CDC25B 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 60 min under the conditions of 37 DEG C, the most enzyme-added, under similarity condition
Reacting 60 min, question response is complete, detection product fluorescence intensity at 520 nm under 485 nm exciting light effects.Right
According to group be added without tester at similarity condition under carry out: enzyme survival efficiency (%)=experimental group measured value/matched group 1 measured value
×100%；Wherein enzyme survival efficiency is the lowest, and the test sample inhibition to histone acetylation enzyme CDC25A/CDC25B is described
The strongest.
Table 1 Radix Corydalis conspersae effective site suppression CDC25A enzyme and the active testing result of CDC25B enzyme
。
* P ＜ 0.01 in table 1, by table 1 it can be seen that Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butyl alcohol portion
Preferable inhibition, wherein Radix Corydalis conspersae second is there is in position and Radix Corydalis conspersae aqueous phase extraction position for CDC25A enzyme CDC25B enzyme
The inhibition at acetoacetic ester position is optimum.
Two, anti-HDAC1 enzyme experiment.
Major experimental material and key instrument are with experiment one.The main distinction: HDAC1 enzyme, Cisbio Bioassays
Products.
The Radix Corydalis conspersae effective site obtained with embodiment 1 is as test medicine.It represents experiment for Radix Corydalis conspersae acetic acid second
Esteratic site, Radix Corydalis conspersae ethyl acetate extract and the activity of Radix Corydalis conspersae aqueous phase extraction position suppression HDAC1 enzyme.Its method is,
The most separately take the Radix Corydalis conspersae of 4 L containing the buffer solution (label E) of ethyl acetate extract, the buffering of Radix Corydalis conspersae n-butanol portion
Solution (label F) and the buffer solution (label G) at Radix Corydalis conspersae aqueous phase extraction position, proceed as follows: 4 will taken respectively
L buffer solution (E, F, G) is placed in 96 orifice plates, then is separately added into 2 L HDAC1 enzymes, reacts 5min, add 4 under room temperature condition
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, obtains 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 2 Radix Corydalis conspersae effective site suppression HDAC1 enzyme
。
* P ＜ 0.01 in table 1, by table 2 it can be seen that Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae ethyl acetate
All there is preferable inhibition at position and Radix Corydalis conspersae aqueous phase extraction position for HDAC1 enzyme.
Three, anti-tumor activity test.
Mtt assay is utilized to measure the Radix Corydalis conspersae effective site cytotoxic activity to human tumor cells.Example 1 institute respectively
Obtaining Radix Corydalis conspersae extractum, petroleum ether part, ethyl acetate extract, n-butanol portion and aqueous phase extraction position appropriate, DMSO dissolves
After, utilize mtt assay to measure respectively human colon carcinoma HCT-8, Human hepatocarcinoma Bel-7402 cell, people's gastric cancer BGC-803 cell, people's lung
Adenocarcinoma A549 cell and the suppression ratio of human ovarian cancer A-2780 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
100µL.After 24 h, adding containing variable concentrations 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, continues cultivation 72 in 37 DEG C
After h, abandoning supernatant, every hole adds the freshly prepared serum-free medium containing 0.5mg/mL MTT of 100 L, continues to cultivate 4 h,
After abandoning supernatant, every hole adds 200 L DMSO and dissolves MTT first hairpin precipitation, and microoscillator vibration mixing, by microplate reader in ginseng
Examine wavelength 450 nm, under the conditions of detection wavelength 570 nm, measure optical density value (OD), with the tumor cell that solvent control processes be
Matched group, with the below equation computerized compound suppression ratio to tumor cell, and by middle effect Equation for Calculating IC50: suppression ratio=(right
According to group mean OD value-administration group mean OD value) ÷ matched group mean OD value × 100%.By the extractum in preparation method, Yi Jisuo
Radix Corydalis conspersae petroleum ether part, Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion, Radix Corydalis conspersae aqueous phase extraction
Take position and carry out tumor cell in vitro cytotoxic activity screening, the results are shown in Table 3.
Table 3 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: Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion, Radix Corydalis conspersae aqueous phase extract position to be had the most anti-really
The effect of tumor.
Four, 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；Group names is: blank
Group, cyclophosphamide group, administration group；Administration group includes: Radix Corydalis conspersae petroleum ether part basic, normal, high dosage group, Radix Corydalis conspersae acetic acid
Ethyl ester position basic, normal, high dosage group, Radix Corydalis conspersae n-butanol portion basic, normal, high dosage group, Radix Corydalis conspersae 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) it is administered after step b) is inoculated 24 hours；Blank group gastric infusion 10mL/kg injection normal saline；Cyclophosphamide
Group intraperitoneal injection of cyclophosphamide 20mg/kg；The administration group prepared Radix Corydalis conspersae petroleum ether part of employing gastric infusion embodiment 1,
Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion and Radix Corydalis conspersae 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 table 4, 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 4.
The each position of table 4 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 5.
The each position of table 5 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 6.
The each position of table 6 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 4 ~ 6: S180Referring to mouse ascites oncocyte, in table 4 ~ 6, tumour inhibiting rate numerical value the biggest expression antitumous effect is the best.By table 4 ~ 6
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, Radix Corydalis conspersae ethyl acetate extract is basic, normal, high
Dosage group, Radix Corydalis conspersae n-butyl alcohol middle and high dosage group, Radix Corydalis conspersae 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
Radix Corydalis conspersae effective site is prepared as tablet, uses following steps: by Radix Corydalis conspersae ethyl acetate extract 300mg and shallow lake
Powder 100mg mixes, and 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
Grain, granulate, add the mixing of magnesium stearate 4mg, tabletting, to obtain final product.
Embodiment 5
The preparation method of electuary: by weight ratio Radix Corydalis conspersae 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: Radix Corydalis conspersae 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 Radix Corydalis conspersae 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 Radix Corydalis conspersae 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 Radix Corydalis conspersae n-butanol portion；
3) aseptically, in the liposome of Radix Corydalis conspersae 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 Radix Corydalis conspersae n-butyl alcohol
The lipidosome injection at position.
Embodiment 8
The preparation method of nanoparticle injection: take 25g Radix Corydalis conspersae 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；Mixed liquor concentrating under reduced pressure is flung to solvent, 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 Radix Corydalis conspersae 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. Radix Corydalis conspersae effective site, it is characterised in that: selected from Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butyl alcohol portion
The mixture of one or more any mass ratio in position, Radix Corydalis conspersae aqueous phase extraction position.
2. according to the Radix Corydalis conspersae effective site described in claim 1, it is characterised in that: selected from Radix Corydalis conspersae ethyl acetate portion
Position.
3. the preparation method of the Radix Corydalis conspersae effective site described in claim 1, it is characterised in that comprise the steps: speckle
Brightly yellowish violet herb is pulverized, and adds concentration of volume percent 65% ~ 95% ethanol solution reflux, extract, is concentrated by gained extracting solution, does
Dry, obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol as molten successively
Agent extraction extract dispersion liquid, obtains Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion after flinging to solvent；After extracting
Extract dispersion liquid concentrate, be dried, obtain Radix Corydalis conspersae aqueous phase extraction position.
4. according to the preparation method of the Radix Corydalis conspersae effective site described in claim 3, it is characterised in that: described backflow carries
It is taken as ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: the Radix Corydalis conspersae herb after pulverizing
Adding concentration of volume percent 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filter, filtering residue adds percent by volume
Concentration 65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.
5. the application of the Radix Corydalis conspersae effective site described in claim 1, it is characterised in that: suppress CDC25 enzyme in preparation and press down
Application in terms of HDAC1 enzyme medicine processed.
The application of Radix Corydalis conspersae effective site the most according to claim 5, it is characterised in that: described preparation suppression CDC25
Enzyme medicine and suppression HDAC1 enzyme medicine are by Radix Corydalis conspersae ethyl acetate extract, Radix Corydalis conspersae n-butanol portion, Radix Corydalis conspersae
The mixture of one or more any mass ratio in aqueous phase extraction position, the oral system being mixed with pharmaceutic adjuvant
Agent or ejection preparation.
The application of Radix Corydalis conspersae effective site the most according to claim 6, it is characterised in that: described ejection preparation is fat
Liposome injection, nanoparticle injection or micro-balloon injection.
The application of Radix Corydalis conspersae effective site the most according to claim 6, it is characterised in that: described oral formulations is scattered
Agent, tablet, granule, capsule or solution.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108478634A (en) * | 2018-03-30 | 2018-09-04 | 青海民族大学 | A kind of method of general flavone in extraction Radix Corydalis conspersae |
| CN115944670A (en) * | 2022-12-20 | 2023-04-11 | 青海师范大学 | Method for extracting total alkaloids of corydalis impatiens |
| CN115993405A (en) * | 2022-07-14 | 2023-04-21 | 青海民族大学 | Method for measuring content of coptisine and berberine hydrochloride at different parts of corydaline maculosa medicinal material |
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| CN105560390A (en) * | 2015-12-03 | 2016-05-11 | 北京中医药大学 | Corydalis edulis extract, pharmaceutical composition containing same and uses thereof |
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| CN105560390A (en) * | 2015-12-03 | 2016-05-11 | 北京中医药大学 | Corydalis edulis extract, pharmaceutical composition containing same and uses thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108478634A (en) * | 2018-03-30 | 2018-09-04 | 青海民族大学 | A kind of method of general flavone in extraction Radix Corydalis conspersae |
| CN108478634B (en) * | 2018-03-30 | 2021-02-09 | 青海民族大学 | Method for extracting total flavonoids from corydalis impatiens |
| CN115993405A (en) * | 2022-07-14 | 2023-04-21 | 青海民族大学 | Method for measuring content of coptisine and berberine hydrochloride at different parts of corydaline maculosa medicinal material |
| CN115993405B (en) * | 2022-07-14 | 2023-09-05 | 青海民族大学 | Method for measuring content of coptisine and berberine hydrochloride at different parts of corydaline maculosa medicinal material |
| CN115944670A (en) * | 2022-12-20 | 2023-04-11 | 青海师范大学 | Method for extracting total alkaloids of corydalis impatiens |
| CN115944670B (en) * | 2022-12-20 | 2024-05-28 | 青海师范大学 | Extraction method of corydalis amabilis total alkaloids |
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Application publication date: 20161207 |