CN106177019A - Natural pond, northwest Herba Potentillae Chinensis suppresses HDAC1 enzyme effective site and preparation method and application - Google Patents

Natural pond, northwest Herba Potentillae Chinensis suppresses HDAC1 enzyme effective site and preparation method and application Download PDF

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CN106177019A
CN106177019A CN201610761172.XA CN201610761172A CN106177019A CN 106177019 A CN106177019 A CN 106177019A CN 201610761172 A CN201610761172 A CN 201610761172A CN 106177019 A CN106177019 A CN 106177019A
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northwest
natural pond
herba potentillae
potentillae chinensis
extract
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林鹏程
吴疆
高世宝
丁玉
包婷雯
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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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • 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
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • 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
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

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Abstract

Natural pond, northwest Herba Potentillae Chinensis suppresses HDAC1 enzyme effective site and preparation method and application, belongs to plant extract technical field.One or both in natural pond, northwest Herba Potentillae Chinensis n-butanol portion, natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position are by the mixture of any mass ratio.Natural pond, northwest Herba Potentillae Chinensis is pulverized, adds concentration expressed in percentage by volume 65% ~ 95% ethanol solution reflux, extract, gained extracting solution is concentrated, is dried, obtain extractum；Extractum adding distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and n-butyl alcohol as solvent extraction extract dispersion liquid successively, volatilization obtains natural pond, northwest Herba Potentillae Chinensis n-butanol portion after removing solvent；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains Herba Potentillae Chinensis aqueous phase extraction position, natural pond, northwest.Natural pond, the northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site of present invention application in terms of preparation suppression HDAC1 enzyme medicine, it is possible to be used for preparing antitumor drug.

Description

Natural pond, northwest Herba Potentillae Chinensis suppresses HDAC1 enzyme effective site and preparation method and application
Technical field
The present invention relates to natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 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.It is main The wet meadow in hillside border, sylvan life and river valley of height above sea level 2800-4250 rice to be born in.It is distributed in Southwestern China, northwest, North China to east North.North temperate zone blazons, and North America, Europe also have [see: GBIF (Global to Japan, Asia Minor to the ground such as Himalaya Biodiversity Information Facility). Comarum salesovianum (Stephan) Asch. Graebn, 2015. www.gbif.org/ species/3020536.].Academy of science of Mongolia finds that it has obvious antiinflammatory and lives Property [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 Fructus Vitis viniferae ball Bacterium 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.Histon deacetylase (HDAC) (HDAC) is an albuminoid enzyme, and structural modification and gene expression regulation to chromosome play Important effect.HDAC1 enzyme is a variant in histone deacetylase.Generally, the acetylation of histone is favourable In dissociating of DNA and histone octamer, nucleosomal structure relaxes, so that various transcription factor and collaborative transcription factor can be with DNA binding site is specific binding, transcribing of activated gene.In cancerous cell, the overexpression of HDAC causes deacetylation to be made Enhancing, by recovering histone positive charge, thus increase the gravitation between DNA and histone, make the nucleosome relaxed become Obtain the tightst, be unfavorable for the expression of specific gene, including some tumor suppressor genes.Antibiotic FR 901228 (histone deacetylase inhibitors, HDACi) then can by improving chromatin specific region acetylation of histone, Thus regulating cell apoptosis and the differentiation expression of associated protein and stability, inducing cell apoptosis and differentiation, become a class new Antitumor drug.HDACi not only has good therapeutical effect to multiple hematological system tumor and solid tumor, and has tumor The of a relatively high selectivity of cell and the advantage of low toxicity.
Applicant finds under study for action: the cost of material of antitumor drug is higher at present, especially for preparation suppression The raw material of Chinese medicine of HDAC1 enzyme medicine is the deficientest.In prior art, effect of natural pond, northwest Herba Potentillae Chinensis only antiphlogistic antibacterial.There is not yet To about natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme active in terms of research and application.
Summary of the invention
The technical problem to be solved in the present invention is: overcome the deficiencies in the prior art, it is provided that natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 Enzyme effective site and preparation method and application, this natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site can effectively suppress the work of HDAC1 enzyme Property, it is used for preparing suppression HDAC1 enzyme medicine, there is preferable antitumor action.
The technical solution adopted for the present invention to solve the technical problems is: this natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme is effective Position, it is characterised in that: selected from natural pond, northwest Herba Potentillae Chinensis n-butanol portion, natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position in one or Two kinds by the mixture of any mass ratio.
The application of this natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site, it is characterised in that: selected from natural pond, northwest Herba Potentillae Chinensis just Butanol position and the mixture of natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position mass ratio 1:1.
The preparation method of this natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 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 the most successively As solvent extraction extract dispersion liquid, take butanol extraction liquid volatilization and remove solvent, obtain natural pond, northwest Herba Potentillae Chinensis n-butanol portion；Will After extraction, remaining extract dispersion liquid concentrates, is dried, and obtains Herba Potentillae Chinensis aqueous phase extraction position, natural pond, northwest.
Described is extracted as ultrasonic assistant reflux, extract,；The concrete operations of ultrasonic assistant reflux, extract, are: will pulverize After natural pond, northwest Herba Potentillae Chinensis add concentration of volume percent 65% ~ 95% ethanol solution ultrasonic extraction 1 ~ 3 hour, filter, filtering residue Add concentration of volume percent 65% ~ 95% ethanol solution reflux, extract, 2 ~ 3 times, united extraction liquid, to obtain final product.
The application of this natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site, it is characterised in that: at preparation suppression HDAC1 enzyme Application in terms of medicine.
Described preparation suppression HDAC1 enzyme medicine is to pass through: by natural pond, northwest Herba Potentillae Chinensis n-butanol portion, natural pond, northwest Herba Potentillae Chinensis water One or both in extraction position are by the mixture of any mass ratio, the oral formulations being mixed with pharmaceutic adjuvant or note mutually System penetrates agent.
Described ejection preparation is lipidosome injection, nanoparticle injection or micro-balloon injection.
Described oral formulations is powder, tablet, granule, capsule or solution.
The present invention is described as follows:
In prior art, natural pond, northwest Herba Potentillae Chinensis normal usage is antiphlogistic antibacterial, has no that it has the relevant report of antitumor efficacy.Shen Ask someone to be found by numerous studies: the extracting section thing in the Herba Potentillae Chinensis of natural pond, northwest also has an antineoplastic action, but its effect Inconspicuous, it is impossible to determine its concrete effective site.Therefore by what kind of solvent, use how preparation method, could obtain The effective site that suppression HDAC1 enzymatic activity effect is best, is the problem that have to solve.Applicant is found by research: use Preparation method of the present invention, natural pond, the northwest Herba Potentillae Chinensis n-butanol portion filtered out and natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position, the most right Preferable effect is had in suppression HDAC1 enzyme.
In preparation method, add mass percentage concentration 65% ~ 95% ethanol solution extracting can be extraction, reflux, extract, or super Sound wave auxiliary reflux extracts.Preferably employing ultrasonic assistant reflux, extract, the method has higher yield and extraction efficiency.Institute The ultrasonic extraction stated is to carry out under room temperature.After ultrasonic extraction, filter and obtain ultrasonic extract and filtering residue, add in filtering residue Mass percent concentration 65% ~ 95% ethanol solution reflux, extract, 2 ~ 3 times, merges ultrasonic extraction and reflux, extract, extracting solution.Return Stream extraction refers to for 2 ~ 3 times: add the medical material after pulverizing and ethanol solution to being heated at reflux in device, and heating, reflux, extract, let cool Filter to obtain an extracting solution and filtering residue；In filtering residue, add ethanol solution, heat, carry out second time reflux, extract, let cool filtration Obtain secondary raffinate and filtering residue；Merge repeatedly extracting solution, obtain reflux, extract, extracting solution.Preferably, each reflux, extract, 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 that alcohol reflux carries The ordinary temperature taken, need to be higher than the boiling point of ethanol, preferably 80 DEG C ~ 100 DEG C.
In preparation method, natural pond, northwest used Herba Potentillae Chinensis selects herb to pulverize.Extractum is crude extract, uses different solvents Extraction extract dispersion liquid, refers to that petroleum ether adds extract dispersion liquid extract and separate successively and obtains petroleum ether extraction liquid, by second Acetoacetic ester adds extract dispersion liquid extract and separate and obtains acetic acid ethyl acetate extract, n-butyl alcohol adds extract dispersion liquid extract and separate Obtain butanol extraction liquid；If take natural pond, northwest Herba Potentillae Chinensis petroleum ether extraction liquid, natural pond, northwest Herba Potentillae Chinensis acetic acid ethyl acetate extract and Natural pond, northwest Herba Potentillae Chinensis butanol extraction liquid volatilizees after removal solvent respectively, it will correspondence obtains Herba Potentillae Chinensis petroleum ether portion, natural pond, northwest Position, natural pond, northwest Herba Potentillae Chinensis ethyl acetate extract and natural pond, northwest Herba Potentillae Chinensis n-butanol portion；Remaining extract dispersion liquid after extracting (solvent is water), volatilization is removed solvent and is i.e. obtained Herba Potentillae Chinensis aqueous phase extraction position, natural pond, northwest.Aqueous phase extraction position may also be referred to as water Phase extract, aqueous phase extraction position is soluble in water；And relative, petroleum ether, ethyl acetate and n-butyl alcohol are organic facies, petroleum ether Position, ethyl acetate extract and n-butanol portion are corresponding is soluble in petroleum ether, ethyl acetate and n-butyl alcohol.Wherein, natural pond, northwest committee Mound dish n-butanol portion contains saponin and phenolic compound；Polysaccharide and organic acid are contained in Herba Potentillae Chinensis aqueous phase extraction position, natural pond, northwest Compound.Volatilization is removed solvent and normal heating can be used to make solvent (petroleum ether, ethyl acetate and n-butyl alcohol) volatilize.Preferably , volatilization is removed solvent and is used concentrating under reduced pressure, and the method efficiency is high, and is avoided that heat-sensitive ingredients loses the property of medicine because of high temperature.
Preferably dosage form is oral formulations or note system penetrates agent.Oral formulations and ejection preparation are optimal route of administration, herein Described oral formulations is through intestines and stomach drug-delivery preparation, it is preferred that oral formulations is slow-release controlled-release type oral formulations.Can also be Other of parenteral routes, such as respiratory tract administration preparation (spray, aerosol, powder spray)；Percutaneous drug delivery preparation (externally used solution Agent, lotion, liniment, ointment, plaster, paste, patch)；Film drug-delivery preparation (eye drop, nasal drop, ophthalmic ointment, rinsing the mouth Agent, sublingual tablet)；Cavity/canal drug administration preparation (suppository).Ejection preparation can be conventional injection preparation.Enter according to drug delivery system Row classification, it is preferred that the ejection preparation of the present invention is lipidosome injection, nanoparticle injection or micro-balloon injection；Other, The injection of the present invention can also be microcapsule injection, polymer micelle injection, microemulsion or Submicroemulsion injection, submicron note Penetrating agent or gel injection, the injection of above drug delivery system can extend pharmaceutical carrier circulation time in vivo, prolongation Medicine carrying microgranule absorption site the time of staying, control medicine release the initial stage burst effect.Pharmaceutic adjuvant includes that medicine carries Body, also includes solvent, solubilizing agent, cosolvent, emulsifying agent, suspending agent, clarifier, deflocculant, correctives, coloring agent, anticorrosion Agent, chemosterilant, adsorbent, filter aid, antioxidant, pH adjusting agent, isoosmotic adjusting agent, diluent, binding agent, wetting agent, In disintegrating agent, lubricant, fluidizer, antitack agent, slow releasing agent, controlled release agent, coating material, filmogen, capsule material one Plant or multiple.
Compared with prior art, natural pond, the northwest Herba Potentillae Chinensis of the present invention suppresses HDAC1 enzyme effective site and preparation method and application institute Have the beneficial effect that
1, this natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site can effectively suppress the activity of HDAC1 enzyme, is used for preparing suppression HDAC1 enzyme medicine, has preferable antitumor action.In this area, natural pond, northwest Herba Potentillae Chinensis has effect of antiphlogistic antibacterial.Application People finds that antitumor drug exists the problem of scarcity of raw material under study for action, and finds that natural pond, northwest Herba Potentillae Chinensis also has anti-by research Effect of tumor, by design preparation method, carries out medicine efficacy screening, by determining enzyme that natural pond, northwest Herba Potentillae Chinensis suppressed Kind and optimal effective site, applicant, during solution problem of pinpointing the problems above, has paid substantial amounts of creative labor Dynamic.Applicant carries out medicine efficacy screening first on HDAC1 enzyme, by numerous studies determine 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, natural pond, northwest Herba Potentillae Chinensis aqueous phase extract in position The mixture of one or more any mass ratio is respectively provided with the effect preferably suppressing HDAC1 enzyme, HDAC1 enzyme survival rate 10.76 ~ 31.19 %, it is possible to be used for developing into antitumor Tibetan medicine new drug.The each effective site pair obtained due to different solvents extraction HDAC1 inhibition of enzyme activity ability is different, and applicant is by further determining: natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position Suppression HDAC1 enzyme effect is optimum, and HDAC1 enzyme survival rate is 21.02 ~ 35.02%.In sum, applicant finds first New effect of natural pond, northwest Herba Potentillae Chinensis, and by determining the kind of its enzyme suppressed, design preparation method also finally obtains HDAC1 enzyme is respectively provided with the effective site of preferable inhibition, and above procedure has paid substantial amounts of creative work.
2, the preparation method of this natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site is extracted conveniently, and extraction efficiency is high.Preparation In method, applicant designs addition concentration expressed in percentage by volume 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, more than employing Natural pond, northwest Herba Potentillae Chinensis n-butanol portion and natural pond, the northwest Herba Potentillae Chinensis aqueous phase extraction position of solvent extraction order gained have preferably The effect of suppression HDAC1 enzyme.
3, the present invention has expanded the raw material channel of suppression HDAC1 enzyme medicine, expands the purposes of natural pond, northwest Herba Potentillae Chinensis, makes west Natural pond, north Herba Potentillae Chinensis develops into the new raw material of suppression HDAC1 enzyme medicine, is remarkably improved the added value of natural pond, northwest Herba Potentillae Chinensis.
Detailed description of the invention
Embodiment 1 ~ 3 is natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 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 molten Liquid ultrasonic extraction 1 hour, filters to obtain ultrasonic extract and filtering residue, and filtering residue adds concentration of volume percent 75% ~ 85% ethanol Solution reflux, extract, 3 times, each 0.5 ~ 1 hour, obtains reflux extracting liquid, merges sound wave extracting solution and reflux extracting liquid, concentration are done Dry 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；Take butanol extraction liquid volatilization and remove solvent, obtain natural pond, northwest Herba Potentillae Chinensis n-butanol portion； After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains Herba Potentillae Chinensis aqueous phase extraction position, natural pond, northwest.
Embodiment 2
Preparation method, comprises the steps: to pulverize natural pond, northwest Herba Potentillae Chinensis, uses concentration of volume percent 85% ~ 95% ethanol molten Liquid ultrasonic extraction 3 hours, filters to obtain ultrasonic extract and filtering residue, and filtering residue adds concentration of volume percent 85% ~ 95% ethanol Solution reflux, extract, 2 times, each 1.5 ~ 2 hours, obtains reflux extracting liquid, merges sound wave extracting solution and reflux extracting liquid, concentration are done Dry 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, take after solvent is removed in butanol extraction liquid volatilization and obtain Herba Potentillae Chinensis n-butyl alcohol portion, natural pond, northwest Position；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains Herba Potentillae Chinensis aqueous phase extraction position, natural pond, northwest.
Embodiment 3
Preparation method, comprises the steps: to pulverize natural pond, northwest Herba Potentillae Chinensis, uses concentration of volume percent 65% ~ 75% ethanol molten Liquid ultrasonic extraction 0.5 hour, filters to obtain ultrasonic extract and filtering residue, and filtering residue adds concentration of volume percent 65% ~ 75% second Alcoholic solution reflux, extract, 2 times, each 1 ~ 1.5 hour, obtains reflux extracting liquid, merges sound wave extracting solution and reflux extracting liquid, concentration It is dried to obtain extractum；Extractum is added distilled water dispersion, obtains extract dispersion liquid, use petroleum ether, ethyl acetate and positive fourth the most successively Alcohol, as solvent extraction extract dispersion liquid, takes after solvent is removed in butanol extraction liquid volatilization and obtains Herba Potentillae Chinensis n-butyl alcohol portion, natural pond, northwest Position；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains Herba Potentillae Chinensis aqueous phase extraction position, natural pond, northwest.
For in embodiment 1 ~ 3, gained petroleum ether extraction liquid after extraction is flung to solvent and obtains natural pond, northwest Herba Potentillae Chinensis petroleum ether Position, flings to solvent by gained acetic acid ethyl acetate extract after extraction and obtains natural pond, northwest Herba Potentillae Chinensis ethyl acetate extract, give over to follow-up property Can test use.
Performance test
Respectively take natural pond, 0.4mg dried northwest Herba Potentillae Chinensis petroleum ether part, natural pond, northwest Herba Potentillae Chinensis ethyl acetate extract, west Natural pond, north Herba Potentillae Chinensis n-butanol portion, natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position, be each dissolved in the dimethyl sulfoxide of 100 L (DMSO) formation DMSO solution in, ultrasonic 5 minutes.Take the DMSO solution of 1 L sample dissolution the most respectively, be separately added into buffer 99 L, respectively obtain natural pond, northwest Herba Potentillae Chinensis petroleum ether part for detecting natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme buffer solution, The buffer solution of natural pond, northwest Herba Potentillae Chinensis ethyl acetate extract, the buffer solution of natural pond, northwest Herba Potentillae Chinensis n-butanol portion and natural pond, northwest committee The buffer solution at dish aqueous phase extraction position, mound.The formula of buffer is: the Tris-HCl of 50mM/L, the NaCl of 0.137 mM/L, The MgCl of KCl, 1mM/L of 2.7mM/L2, 0.01% polysorbas20, pH of buffer 8.4.
One, anti-HDAC1 enzyme experiment.
Major experimental material is:
HDAC1 enzyme, Cisbio Bioassays Products；
Tris-HCl, Shanghai Jie Mei gene Pharmaceutical Technology Co., Ltd；
EDTA, Curie bio tech ltd, Shanghai；
DTT, Shanghai Sheng Gong biological engineering limited company；
BSA, Jiang Lai bio tech ltd, Shanghai；
DMSO, Shanghai Yi Ka Bioisystech Co., Ltd；
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 City three；
BRAND8 road liquid-transfering gun, converge your Instrument Ltd. in Hangzhou, Zhejiang province；
500 L liquid-transfering guns, 50 L liquid-transfering guns, Shanghai City Rong Tai biochemical instrument company limited；
Electronic balance XPE105, prunus mume (sieb.) sieb.et zucc. Teller-torr benefit Instrument Ltd..
Suppress HDAC1 enzyme effective site as test medicine with natural pond, the northwest Herba Potentillae Chinensis that embodiment 1 obtains.It represents experiment The activity experiment of suppression HDAC1 enzyme.Its method is, the most separately takes the buffer solution of natural pond, the northwest Herba Potentillae Chinensis n-butanol portion of 4 L (label B) and natural pond, northwest Herba Potentillae Chinensis aqueous phase extract the buffer solution (label C) at position, proceed as follows respectively: 4 will taken L buffer solution (B, C) is placed in 96 orifice plates, then is separately added into 2 L HDAC1 enzymes, reacts 5min, add 4 L under room temperature condition H3(1-21) K9 substrate, cultivates 60 minutes in incubator by 37 DEG C.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 natural pond, table 1 northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site suppression HDAC1 enzyme
。
* P ＜ 0.01 in table 1, by table 1 it can be seen that natural pond, northwest Herba Potentillae Chinensis n-butanol portion and natural pond, northwest Herba Potentillae Chinensis water All there is preferable inhibition at extraction position for HDAC1 enzyme mutually.
Two, anti-tumor activity test.
Mtt assay is utilized to measure thin to human tumor cells of natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site effective site Born of the same parents' cytotoxic activity.Take each position of Herba Potentillae Chinensis, natural pond, Example 1 gained northwest respectively appropriate, as test-compound, after DMSO dissolves, Mtt assay is utilized to measure respectively human colon carcinoma HCT-8, Human hepatocarcinoma Bel-7402 cell, people's gastric cancer BGC-803 cell, people's lung gland Cancer A549 cell and the suppression ratio of human ovarian cancer A2780 cell growth.
Experimental technique: by the cell of exponential phase, after 0.25% pancreas enzyme-EDTA digestion, be configured to certain density list Cell suspension, according to the difference of vitro growth rates, is inoculated in 96 orifice plates by 800 ~ 2000/hole, and every hole adds cell suspension 100 µL.After 24 h, 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 72 h, abandoning supernatant, every hole adds the freshly prepared serum-free medium containing 0.5mg/mL MTT of 100 L, continues Cultivating 4 h, after abandoning supernatant, every hole adds 200 L DMSO and dissolves MTT first hairpin precipitation, microoscillator vibration mixing, uses enzyme Mark instrument measures optical density value (OD) under the conditions of reference wavelength 450nm, detection wavelength 570nm, the tumor processed with solvent control Cell is matched group, with the below equation computerized compound suppression ratio to tumor cell, 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 natural pond, the northwest Herba Potentillae Chinensis petroleum ether part of gained, natural pond, northwest Herba Potentillae Chinensis ethyl acetate extract, natural pond, northwest Herba Potentillae Chinensis n-butyl alcohol Position, natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position carry 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, natural pond, northwest Herba Potentillae Chinensis aqueous phase extract position preferable antineoplastic effect really.
Three, external S180Tumor-bearing mice anti-tumor experiment.
1, the S at each position of embodiment 1180Tumor-bearing mice anti-tumor experiment method is as follows:
A) 5 ~ 6 week old kunming mice, selects Mus to be grouped at random, often group 10, often group male and female half and half (sub-cage rearing)；Group names For: blank group, cyclophosphamide group and administration group；Administration group includes: 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 prepared natural pond, the northwest Herba Potentillae Chinensis petroleum ether part of gastric infusion embodiment 1, west Natural pond, north Herba Potentillae Chinensis ethyl acetate extract, natural pond, northwest Herba Potentillae Chinensis n-butanol portion and natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position；First After administration, the next day claim mice body weight first；Often group is administered once daily, and 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, the S at each position of embodiment 2180Tumor-bearing mice method is with embodiment 1, data inputting table 4.
The each position of table 4 embodiment 2 is to mice S180The impact of solid tumor
。
3, the S at each position of embodiment 3180The method of tumor-bearing mice anti-tumor experiment is with embodiment 1, data inputting table 5.
The each position of table 5 embodiment 3 is to mice S180The impact of solid tumor
。
Add up through applicant: administration group is compared with blank group, and Mouse Weight change there was no significant difference (P > 0.05). In table 3 ~ 5: S180Referring to mouse ascites oncocyte, in table 3, tumour inhibiting rate numerical value the biggest expression antitumous effect is the best.Can by table 3 ~ 5 To find out: first, embodiment 1 ~ 3 all has preferable antitumous effect, and the antitumous effect at each position of embodiment 1 ~ 3 is without the poorest Not.Secondly, compared with blank group, natural pond, northwest Herba Potentillae Chinensis n-butyl alcohol middle and high dosage group, Herba Potentillae Chinensis aqueous phase extraction portion, natural pond, northwest Position high dose group, natural pond, northwest Herba Potentillae Chinensis n-butanol portion and the extraction position mass ratio 1:1 mixing of natural pond, northwest Herba Potentillae Chinensis aqueous phase are middle and 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 water Extraction position 300mg and starch 100mg mixing mutually, the gelatinized corn starch 40mg adding mass percent concentration 10% makes soft material, sieves Wet granular, is dried to obtain dry granule, granulate, adds magnesium stearate 4 mg mixing, tabletting, to obtain final product.
Embodiment 5
The preparation method of electuary: by weight ratio natural pond, northwest Herba Potentillae Chinensis aqueous phase is extracted 5 parts of position, sucrose 1 part, 3 parts of dextrin Mixing, adds appropriate mass percent 95% ethanol solution 2 ~ 5 parts, stirring while adding, prepares soft material, is dried by soft material, crosses 16 mesh Sieve, subpackage and get final product.
Embodiment 6
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 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 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 8
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 (8)

1. natural pond, northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site, it is characterised in that: selected from natural pond, northwest Herba Potentillae Chinensis n-butanol portion, One or both in Herba Potentillae Chinensis aqueous phase extraction position, natural pond, northwest are by the mixture of any mass ratio.
The application of natural pond, northwest the most according to claim 1 Herba Potentillae Chinensis suppression HDAC1 enzyme effective site, it is characterised in that: choosing The mixture of position mass ratio 1:1 is extracted from natural pond, northwest Herba Potentillae Chinensis n-butanol portion and natural pond, northwest Herba Potentillae Chinensis aqueous phase.
3. the preparation method of natural pond, the northwest Herba Potentillae Chinensis suppression HDAC1 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, 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 butanol extraction liquid volatilization removal solvent and are just obtaining natural pond, northwest Herba Potentillae Chinensis Butanol position；After extracting, remaining extract dispersion liquid concentrates, is dried, and obtains Herba Potentillae Chinensis aqueous phase extraction position, natural pond, northwest.
The preparation method of natural pond, northwest the most according to claim 3 Herba Potentillae Chinensis suppression HDAC1 enzyme effective site, 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, 2 ~ 3 times, united extraction liquid, to obtain final product.
5. the application of natural pond, the northwest Herba Potentillae Chinensis suppression HDAC1 enzyme effective site described in claim 1, it is characterised in that: in preparation Application in terms of suppression HDAC1 enzyme medicine.
The application of natural pond, northwest the most according to claim 5 Herba Potentillae Chinensis suppression HDAC1 enzyme effective site, it is characterised in that: institute Stating preparation suppression HDAC1 enzyme medicine is to pass through: by natural pond, northwest Herba Potentillae Chinensis n-butanol portion, natural pond, northwest Herba Potentillae Chinensis aqueous phase extraction position In one or both by the mixture of any mass ratio, the oral formulations or the note system that are mixed with pharmaceutic adjuvant penetrate agent.
The application of natural pond, northwest the most according to claim 6 Herba Potentillae Chinensis suppression HDAC1 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 6 Herba Potentillae Chinensis suppression HDAC1 enzyme effective site, it is characterised in that: institute The oral formulations stated is powder, tablet, granule, capsule or solution.
CN201610761172.XA 2016-08-30 2016-08-30 Natural pond, northwest Herba Potentillae Chinensis suppresses HDAC1 enzyme effective site and preparation method and application Pending CN106177019A (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
G. ODONTUYA ET AL.: "Antibacterially active phenolic lipid derivatives from Coma rum 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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