CN106236870A - Natural pond, northwest Herba Potentillae Chinensis suppresses CDC25 enzyme effective site and preparation method and application - Google Patents
Natural pond, northwest Herba Potentillae Chinensis suppresses CDC25 enzyme effective site and preparation method and application Download PDFInfo
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- CN106236870A CN106236870A CN201610760951.8A CN201610760951A CN106236870A CN 106236870 A CN106236870 A CN 106236870A CN 201610760951 A CN201610760951 A CN 201610760951A CN 106236870 A CN106236870 A CN 106236870A
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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)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
Abstract
Natural pond, northwest Herba Potentillae Chinensis suppresses CDC25 enzyme effective site and preparation method and application, belongs to plant extract technical field.Selected from natural pond, northwest Herba Potentillae Chinensis n-butanol portion.Natural pond, northwest Herba Potentillae Chinensis 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, gained butanol extraction liquid is flung to solvent, obtains natural pond, northwest Herba Potentillae Chinensis n-butanol portion.The application in terms of preparation suppression CDC25 enzyme medicine of natural pond, the northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site, has preferable antitumous effect.
Description
Technical field
The present invention relates to natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site and preparation method and application, belong to plant extract
Technical field.
Background technology
Natural pond, northwest Herba Potentillae Chinensis (Comarum salesovianum (Stepn.) Asch. et Gr.) is Rosaceae
(Rosaceae) natural pond Potentilla (Comarum palustre L.) perennial semishrub plant [sees: China of the Chinese Academy of Sciences
Flora editorial board. Chinese Higher plant [M]. Beijing: Science Press, 2003(6) .], it is tradition medicinal plants.Its
Mainly it is born in the wet meadow in hillside border, sylvan life and river valley of height above sea level 2800-4250 rice.It is distributed in Southwestern China, northwest, North China extremely
Northeast.North temperate zone blazons, and North America, Europe also have to Himalaya and other places [see: GBIF to Japan, Asia Minor
(Global Biodiversity Information Facility). Comarum salesovianum (Stephan)
Asch. & Graebn, 2015. www.gbif.org/ species/3020536.].It is bright that academy of science of Mongolia finds that it has
Aobvious anti-inflammatory activity [sees: Odontuya G, Banzragchgarav O, Murata T, et al.
Antibacterially active phenolic lipid derivatives from Comarum salesovianum
(Steph.) Aschers. et Gr. [J]. Phytochemistry Letters, 2015,13:360-364.]；One
Japan Patent shows that it has staphylococcus inhibition, can prevent acne and allergic dermatitis [see: Y. Nobuo,
S. Hiroyuki. JPO, 2011,JP 2011001329-A .]。
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.
Natural pond, northwest Herba Potentillae Chinensis plant contains the multiple chemical compositions such as Polyphenols, flavone, triterpenic acid, tannin compound, mainly
There is antibacterial anti-inflammatory effect.At present, there is not yet natural pond, northwest Herba Potentillae Chinensis and effective site anti-CDC25A and CDC25B enzyme effect is ground
Study carefully the report with application 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 natural pond, northwest Herba Potentillae Chinensis suppression CDC25
Enzyme effective site and preparation method and application, this natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site can effectively suppress CDC25A enzyme and
The activity of CDC25B enzyme, is used for preparing suppression CDC25 enzyme medicine, has preferable antitumous effect.
The technical solution adopted for the present invention to solve the technical problems is: this natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme is effective
Position, it is characterised in that: selected from natural pond, northwest Herba Potentillae Chinensis n-butanol portion.
The preparation method of this natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site, it is characterised in that comprise the steps:
Natural pond, northwest Herba Potentillae Chinensis is pulverized, adds 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 as molten successively
Agent extraction extract dispersion liquid, flings to solvent by gained butanol extraction liquid, obtains natural pond, northwest Herba Potentillae Chinensis n-butanol portion.
Preferably, described it is extracted as ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are:
Natural pond, northwest Herba Potentillae Chinensis after pulverizing adds concentration of volume percent 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, mistake
Filter, filtering residue adds concentration expressed in percentage by volume 65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, and united extraction liquid to obtain final product.
The application of this natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site, it is characterised in that: at preparation suppression CDC25 enzyme
Application in terms of medicine.
Described preparation suppression CDC25 enzyme medicine, is to mix system with pharmaceutic adjuvant by natural pond, northwest Herba Potentillae Chinensis n-butanol portion
The oral formulations become 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 natural pond, northwest Herba Potentillae Chinensis normal usage is antiphlogistic antibacterial,
Have no that it has the relevant report of antitumor efficacy.Applicant is found by numerous studies: the extracting section in the Herba Potentillae Chinensis of natural pond, northwest
Thing also has antibacterial, antineoplastic action, but its effect is inconspicuous, it is impossible to determine its concrete effective site.Therefore pass through
What kind of solvent, use how preparation method, the effective site that suppression CDC25 enzymatic activity effect is best could be obtained, be necessary
Problem to be solved.Applicant is found by research: use preparation method of the present invention, the Herba Potentillae Chinensis n-butyl alcohol portion, natural pond, northwest of gained
All there is preferable inhibition position to CDC25A enzyme and CDC25B enzyme.
In preparation method, described ultrasonic extraction is to carry out under room temperature.Filter and reflux, extract, refers to for 1 ~ 3 time: be ultrasonic
After ripple extracts, filter and obtain ultrasonic extract and filtering residue, in filtering residue, add mass percent concentration 65% ~ 95% ethanol solution return
Stream extraction 1 ~ 3 time, merges ultrasonic extraction and reflux, extract, extracting solution.Reflux, extract, refers to: to being heated at reflux in device addition
Medical material after pulverizing and ethanol solution, heating, reflux, extract, let cool and filter to obtain an extracting solution and medicinal residues；Add in medicinal residues
Ethanol solution, heats, carries out second time reflux, extract, let cool and filter to obtain secondary raffinate and medicinal residues；Merge repeatedly extracting solution, i.e.
Obtain reflux, extract, extracting solution.Preferably, each reflux, extract, 0.5 ~ 2 hour；During reflux, extract, added ethanol solution did not had every time
Medical material surface 1cm ~ 2cm.The temperature of reflux, extract, is the ordinary temperature of alcohol reflux, need to be higher than the boiling point of ethanol, preferably
It it is 80 DEG C ~ 100 DEG C.
In preparation method, using and pulverized by natural pond, northwest Herba Potentillae Chinensis herb, extractum is natural pond, northwest Herba Potentillae Chinensis crude extract, uses
Different solvent extraction extract dispersion liquids, refers to that petroleum ether adds extract dispersion liquid extract and separate successively and obtains petroleum ether extraction
Take liquid, ethyl acetate is added extract dispersion liquid extract and separate obtain acetic acid ethyl acetate extract, n-butyl alcohol is added extractum dispersion
Liquid extract and separate obtains butanol extraction liquid；Take butanol extraction liquid and fling to solvent, obtain n-butanol portion.Test in the present invention
In, applicant is for the ease of carrying out follow-up performance test, by residue leaching after petroleum ether extraction liquid, acetic acid ethyl acetate extract and extraction
Cream dispersion liquid, flings to solvent respectively and obtains natural pond, northwest Herba Potentillae Chinensis petroleum ether part and natural pond, northwest Herba Potentillae Chinensis ethyl acetate extract, northwest
Herba Potentillae Chinensis aqueous phase extraction position, natural pond, in order to each position is carried out follow-up performance test.Fling to solvent and can use normal heating
Solvent (petroleum ether, ethyl acetate and n-butyl alcohol) is made to volatilize.Preferably, flinging to solvent and use concentrating under reduced pressure, the method efficiency is high,
And it is avoided that heat-sensitive ingredients loses the property of medicine because of high temperature.Wherein, the composition of natural pond, northwest Herba Potentillae Chinensis n-butanol portion comprises saponin
And phenolic compound.
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, 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 suspending agent, clarifier, deflocculant, correctives,
Toner, preservative, chemosterilant, adsorbent, filter aid, antioxidant, pH adjusting agent, isoosmotic adjusting agent, diluent, bonding
Agent, wetting agent, disintegrating agent, lubricant, fluidizer, antitack agent, slow releasing agent, controlled release agent, coating material, filmogen, capsule
One or more in material.
Compared with prior art, natural pond, the northwest Herba Potentillae Chinensis of the present invention suppresses CDC25 enzyme effective site and preparation method and application institute
Have the beneficial effect that
1, this natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site can effectively suppress CDC25A enzyme and the activity of CDC25B enzyme, uses
In preparation suppression CDC25 enzyme medicine, there is preferable antitumor action.Applicant determines through numerous studies: in prior art only
There is natural pond, the northwest Herba Potentillae Chinensis of antiphlogistic antibacterial effect, also there is antineoplastic effect.And applicant first at CDC25A enzyme and
Carry out medicine efficacy screening on CDC25B enzyme, determined by numerous studies: natural pond, northwest Herba Potentillae Chinensis n-butanol portion has preferably suppression
CDC25A enzyme and CDC25B enzymatic activity, its CDC25A enzyme survival rate 24.6%, CDC25B enzyme survival rate 29.4%, it is possible to be used for developing
Become antitumor Tibetan medicine new drug.In sum, applicant finds new effect of natural pond, northwest Herba Potentillae Chinensis first, and by research
Determining the kind of its enzyme suppressed, design preparation method also finally obtains CDC25A enzyme and CDC25B enzyme is respectively provided with and is preferably pressed down
The effective site of effect processed, above procedure has paid substantial amounts of creative work.
2, the preparation method of this natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site is extracted conveniently, and extraction efficiency is high.Preparation
In method, applicant designs addition concentration of volume percent 65% ~ 95% ethanol solution and carries out ultrasonic assistant reflux, extract, improves
Extraction efficiency；In extraction, successively with petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction extract dispersion liquid, use with
Natural pond, the northwest Herba Potentillae Chinensis n-butanol portion of upper solvent extraction order gained has the effect preferably suppressing CDC25 enzyme.
3, the present invention has expanded suppression CDC25A enzyme and the raw material channel of CDC25B enzyme, expands the use of natural pond, northwest Herba Potentillae Chinensis
On the way, make natural pond, northwest Herba Potentillae Chinensis develop into suppression CDC25A enzyme and the new raw material of CDC25B enzyme medicine, be remarkably improved natural pond, northwest
The added value of Herba Potentillae Chinensis.
Detailed description of the invention
Embodiment 1 ~ 3 is natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site and the specific embodiment party of preparation method of the present invention
Formula, wherein embodiment 1 is most preferred embodiment.
Embodiment 1
Preparation method, comprises the steps: to pulverize natural pond, northwest Herba Potentillae Chinensis, uses concentration of volume percent 75 ~ 85% ethanol solution
Ultrasonic extraction 1 hour, filters to obtain ultrasonic extract and filtering residue, and it is molten that filtering residue adds concentration of volume percent 75% ~ 85% ethanol
Liquid reflux, extract, 3 times, each 0.5 hour, obtains reflux extracting liquid, merges sound wave extracting solution and reflux extracting liquid, concentrate drying obtain
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, obtains petroleum ether extraction liquid, acetic acid ethyl acetate extract and butanol extraction liquid respectively；By gained just
Butanol, before immunoassay liquid flings to solvent, obtains natural pond, northwest Herba Potentillae Chinensis n-butanol portion.
Embodiment 2
Preparation method, comprises the steps: to pulverize natural pond, northwest Herba Potentillae Chinensis, uses concentration of volume percent 85 ~ 95% ethanol solution
Ultrasonic extraction 3 hours, filters to obtain ultrasonic extract and filtering residue, and it is molten that filtering residue adds concentration of volume percent 85% ~ 95% ethanol
Liquid reflux, extract, 3 times, each 1 hour, obtains reflux extracting liquid, merges sound wave extracting solution and reflux extracting 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, obtains petroleum ether extraction liquid, acetic acid ethyl acetate extract and butanol extraction liquid respectively；By positive for gained fourth
Alcohol extract flings to solvent, obtains natural pond, northwest Herba Potentillae Chinensis n-butanol portion.
Embodiment 3
Preparation method, comprises the steps: to pulverize natural pond, northwest Herba Potentillae Chinensis, uses concentration of volume percent 65 ~ 75% ethanol solution
Ultrasonic extraction 0.5 hour, filters to obtain ultrasonic extract and filtering residue, and filtering residue adds concentration of volume percent 65% ~ 75% ethanol
Solution reflux, extract, 2 times, each 2 hours, obtains reflux extracting liquid, merges sound wave extracting solution and reflux extracting liquid, concentrate drying obtain
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, obtains petroleum ether extraction liquid, acetic acid ethyl acetate extract and butanol extraction liquid respectively；By gained just
Butanol, before immunoassay liquid flings to solvent, obtains natural pond, northwest Herba Potentillae Chinensis n-butanol portion.
Divide remaining extractum after gained petroleum ether extraction liquid, acetic acid ethyl acetate extract, n-butanol extraction in embodiment 1 ~ 3
Scattered liquid flings to solvent respectively, obtains natural pond, northwest Herba Potentillae Chinensis petroleum ether part, natural pond, northwest Herba Potentillae Chinensis ethyl acetate extract, Zhao Wei mound, northwest
Dish n-butanol portion, natural pond, northwest Herba Potentillae Chinensis aqueous phase extract position, give over to performance test part and use.
Performance test
With embodiment 1 example, respectively take natural pond, 0.4mg dried northwest Herba Potentillae Chinensis petroleum ether part, natural pond, northwest Herba Potentillae Chinensis acetic acid
Ethyl ester position, natural pond, northwest Herba Potentillae Chinensis n-butanol portion, natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position, be each dissolved in the two of 100 L
Formation DMSO solution in methyl sulfoxide (DMSO), ultrasonic 5 minutes.Take the DMSO solution of 1 L sample dissolution the most respectively, be separately added into
Buffer 99 L, respectively obtains the effective portion of Herba Potentillae Chinensis petroleum ether, natural pond, northwest for detecting natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme
The buffer solution of position, the buffer solution of natural pond, northwest Herba Potentillae Chinensis ethyl acetate effective site, the effective portion of Herba Potentillae Chinensis n-butyl alcohol, natural pond, northwest
The buffer solution of position and the buffer solution of natural pond, northwest Herba Potentillae Chinensis water solublity effective site.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 dissolves and mixes, and adds 0.5mol/L NaOH aqueous solution by pH regulator to 8.4, is then settled to 100mL capacity
In Ping, 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 the buffer solution of natural pond, northwest Herba Potentillae Chinensis n-butyl alcohol effective site that embodiment 1 obtains as tester.It represents real
Test as natural pond, northwest Herba Potentillae Chinensis difference effective site suppression CDC25A enzyme and the activity of CDC25B enzyme.Its method is, takes 2.7 L respectively
330 μm ol/L FDP, 1.8 L 100 μm ol/L testers, 2 L 0.1 μm ol/L CDC25A/CDC25B, 11.5 L FDP
Buffer forms the experimental group of 18 L systems；Then 2.7 L 330 μm ol/L FDP, 2 L 0.1 μm ol/L CDC25A/ are taken
CDC25B, 13.3 L FDP buffer form the matched group 1 of 18 L systems；Take 2.7 L 330 μm ol/L FDP, 1.8 L again
100 μm ol/L testers, 13.5 L FDP buffer form the matched group 2 of 18 L systems；Needs are added CDC25A/CDC25B
The system of enzyme hatches 60 min under the conditions of 37 DEG C, the most enzyme-added, reacts 60 min under similarity condition, and question response is complete, inspection
Survey product fluorescence intensity at 520 nm under 485 nm exciting light effects.Experimental group, matched group 1 and matched group 2 exist
Carrying out under the same terms, record experimental result also calculates enzyme survival efficiency.Enzyme survival efficiency (%)=experimental group measured value/comparison
Organizing 1 measured value × 100%, wherein enzyme survival efficiency is the lowest, illustrates that tester is to histone acetylation enzyme CDC25A/CDC25B's
Inhibition is the strongest.
The active testing result of natural pond, table 1 northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site
。
* P ＜ 0.01 in table 1, by table 1 it can be seen that natural pond, northwest Herba Potentillae Chinensis n-butanol portion for CDC25A enzyme and
There is preferable inhibition in CDC25B enzyme.
Two, anti-tumor activity test.
Mtt assay is utilized to measure the position, Herba Potentillae Chinensis each position, natural pond, the northwest cytotoxic activity to human tumor cells.Treating excess syndrome respectively
Execute each position of Herba Potentillae Chinensis, natural pond, example 1 gained northwest in right amount as tested material, after DMSO dissolves, utilize mtt assay to measure respectively and people is tied
Intestinal cancer HCT-8, people's hepatocarcinoma Bel7402 cell, people's gastric cancer BGC-803 cell, human pulmonary epithelial cells and human ovarian cancer A-
The suppression ratio of 2780 cell growths.
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 tested material and the fresh culture of coordinative solvent comparison, every hole adds 100 L(DMSO
Final concentration < 0.1%), every kind of tested material sets 5 ~ 7 dosage groups, often organizes and at least sets 3 parallel holes, continues to cultivate 72 h in 37 DEG C
After, abandon 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
Natural pond, northwest Herba Potentillae Chinensis petroleum ether part, natural pond, northwest Herba Potentillae Chinensis ethyl acetate extract, natural pond, northwest Herba Potentillae Chinensis n-butanol portion, west
Herba Potentillae Chinensis aqueous phase extraction position, natural pond, north carries out tumor cell in vitro cytotoxic activity screening, the results are shown in Table 2.
Table 2 tumor cell in vitro cytotoxic activity the selection result
。
Note: 1), HCT-8 be human colon cancer cell, Bel-7402 is human liver cancer cell, and BGC-823 is gastric carcinoma cells,
A549 is human lung carcinoma cell, and A2780 is Proliferation of Human Ovarian Cell.2), > 50 show do not have anti-cell cytotoxic activity.Can be seen by table 2
Go out: natural pond, northwest Herba Potentillae Chinensis n-butanol portion has preferable antineoplastic effect really, and the most remarkably productive.
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: basic, normal, high dose of natural pond, northwest Herba Potentillae Chinensis n-butanol portion
Amount group, natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position basic, normal, high dosage group, natural pond, northwest Herba Potentillae Chinensis n-butanol portion and natural pond, northwest committee
Mound dish aqueous phase extraction position mass ratio 1:1 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 natural pond, the northwest Herba Potentillae Chinensis n-butanol portion that gastric infusion embodiment 1 prepares；First
After secondary administration, the next day claim mice body weight first；Often group is administered once daily, and each dosage refers to following table, successive administration 14
My god；
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
。
After first administration, the next day record Mouse Weight change；Through applicant add up: administration group compared with blank group,
Mouse Weight change there was no significant difference (P > 0.05).In table 3 ~ 5: S180Refer to mouse ascites oncocyte, tumour inhibiting rate number in table 3
It is worth the biggest expression antitumous effect the best., embodiment 1 ~ 3 all have preferable antitumous effect by table 3 ~ 5 it can be seen that first,
The antitumous effect at each position of embodiment 1 ~ 3 is without significant difference.Secondly, compared with blank group, natural pond, the northwest positive fourth of Herba Potentillae Chinensis
Alcohol basic, normal, high dosage group, Treated with Chemotherapy with Cyclophosphamide group, all can significantly inhibit S180The growth of tumor.
Embodiment 4
Natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site is prepared as tablet, uses following steps: by natural pond, northwest Herba Potentillae Chinensis just
Butanol position 300mg and starch 100mg mixing, the gelatinized corn starch 40mg adding mass percent concentration 10% makes soft material, sieves wet
Granule, is dried to obtain dry granule, granulate, adds magnesium stearate 4 mg mixing, tabletting, to obtain final product.
Embodiment 5
Pellet capsule is composed of the following raw materials by weight: natural pond, northwest Herba Potentillae Chinensis n-butanol portion 30 parts, 5 parts of lecithin, Calculus Bovis gallbladder
Acid 5 parts of sodium, microcrystalline Cellulose 30 parts；
The preparation method of pellet capsule: by proportioning by natural pond, northwest Herba Potentillae Chinensis n-butanol portion, lecithin, Bile Salts and crystallite
Cellulose 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, by micro-
Ball is filled in capsule shells, to obtain final product.
Embodiment 6
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 natural pond, 10g northwest Herba Potentillae Chinensis 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；Suspension is led to
Cross concentrating under reduced pressure and fling to organic solvent, 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 natural pond, northwest Herba Potentillae Chinensis n-butanol portion；
3) aseptically, northwestwards in the liposome of natural pond Herba Potentillae Chinensis n-butanol portion, adding 100g trehalose, stirring is all
Even, ultrasonic Treatment 0.5 ~ 1 hour, inject and use water constant volume, through 0.22 μm filtering with microporous membrane, fill, obtain natural pond, northwest committee
The lipidosome injection of mound dish n-butanol portion.
Embodiment 7
The preparation method of nanoparticle injection: take natural pond, 25g northwest Herba Potentillae Chinensis n-butanol portion and 100g poloxamer188 is used
3000ml anhydrous alcohol solution, adds the ethanol solution of 30ml 1mol/L zinc chloride, stirring mixing, ultrasonic Treatment 0.5 ~ 1
Hour it is made to 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；
Taking-up injects uses water constant volume, ultrasonic Treatment 0.5 ~ 1 hour, through 0.22 μm filtering with microporous membrane, fill, obtains natural pond, northwest committee
The nanoparticle injection of mound dish n-butanol portion.
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 (7)
1. natural pond, northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site, it is characterised in that: selected from natural pond, northwest Herba Potentillae Chinensis n-butanol portion.
2. the preparation method of natural pond, the northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site described in claim 1, it is characterised in that bag
Include following step: pulverized by natural pond, northwest Herba Potentillae Chinensis, add concentration of volume percent 65% ~ 95% ethanol solution and extract, gained is carried
Take liquid to concentrate, be dried, obtain extractum；Extractum is added distilled water dispersion, obtain extract dispersion liquid, successively with petroleum ether, ethyl acetate and
Gained butanol extraction liquid, as solvent extraction extract dispersion liquid, is flung to solvent by n-butyl alcohol, obtains natural pond, the northwest positive fourth of Herba Potentillae Chinensis
Alcohol position.
3., according to the preparation method of natural pond, the northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site described in claim 2, its feature exists
In: described is extracted as ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: the west after pulverizing
Natural pond, north Herba Potentillae Chinensis adds concentration of volume percent 65% ~ 95% ethanol solution ultrasonic extraction 0.5 ~ 3 hour, filters, and filtering residue adds
Concentration of volume percent 65% ~ 95% ethanol solution reflux, extract, 1 ~ 3 time, united extraction liquid, to obtain final product.
4. the application of natural pond, the northwest Herba Potentillae Chinensis suppression CDC25 enzyme effective site described in claim 1, it is characterised in that: in system
Application in terms of standby suppression CDC25 enzyme medicine.
The application of natural pond, northwest the most according to claim 4 Herba Potentillae Chinensis suppression CDC25 enzyme effective site, it is characterised in that: institute
State preparation suppression CDC25 enzyme medicine, be the oral system being mixed by natural pond, northwest Herba Potentillae Chinensis n-butanol portion and pharmaceutic adjuvant
Agent or ejection preparation.
The application of natural pond, northwest the most according to claim 5 Herba Potentillae Chinensis suppression CDC25 enzyme effective site, it is characterised in that: institute
The ejection preparation stated is lipidosome injection, nanoparticle injection or micro-balloon injection.
The application of natural pond, northwest the most according to claim 5 Herba Potentillae Chinensis suppression CDC25 enzyme effective site, it is characterised in that: institute
The oral formulations stated is powder, tablet, granule, capsule or solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610760951.8A CN106236870A (en) | 2016-08-30 | 2016-08-30 | Natural pond, northwest Herba Potentillae Chinensis suppresses CDC25 enzyme effective site and preparation method and application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610760951.8A CN106236870A (en) | 2016-08-30 | 2016-08-30 | Natural pond, northwest Herba Potentillae Chinensis suppresses CDC25 enzyme effective site and preparation method and application |
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| CN (1) | CN106236870A (en) |
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2016
- 2016-08-30 CN CN201610760951.8A patent/CN106236870A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| G. ODONTUYA ETAL: "Antibacterially active phenolic lipi d derivatives from Comarum salesovianum (Steph.) Aschers. et Gr.", 《PHYTOCHEMISTRY LETTERS》 * |
| 高世宝等: "沼委陵菜属植物化学成分和药理活性研究进展 ", 《安徽农业科学》 * |
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