CN104587140B - Black false hellebore estrogen receptor antagon sample new role - Google Patents
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Abstract
The present invention relates to a kind of new applications of Chinese medicine black false hellebore extract, in particular to the estrogen receptor antagon sample effect of black false hellebore extract.
Description
Technical field：
The present invention relates to a kind of new application of Chinese medicine black false hellebore extract, in particular to the estrogen receptor of black false hellebore extract is short of money
The effect of anti-agent sample.
Background technique：
Controversies in hormone replacement in the elderly (estrogen replacement therapy, ERT) is treatment postmenopausal syndrome
Preferred therapeutic scheme, but prolonged application leads to endometrial hyperplasia, breast cancer etc..Selective estrogen receptor modulators
(Selective estrogen receptor modulators, SERM) can alleviate perimenopausal symptom, pre- preventing bone rarefaction
With the urinary incontinence, stablize vagina environment, but does not increase the incidence of malignant tumour.SERM mainly passes through estrogen receptor
(estrogen receptor, ER) hypotype, the mechanism such as regulator, target promoter realize its tissue selectivity altogether, are playing bone
While bone, cardiovascular protective effect, reduce the side effect to mammary gland and reproductive system.Wherein ICI 182,780 is a kind of
Novel competitiveness, " pure " ER antagonist, ER signal can be blocked to pass after not having part ER agonism and ER combination
Guiding path, lower reconciliation degradation tumour ER, does not cause endometrial proliferation and hot flush occurrence rates is low rapidly.In recent years, many experiments
Studies have shown that phytoestrogen (phytoestrogen, PE) is non-steroid, plant origin with estrogen active natural
Compound has estrogen and antiestrogenic dual regulation.As the deep and Chinese medicine of phytoestrogen research is in cream
It is more and more applied in adenocarcinoma patients, finds and be similar to the Chinese medicine that ICI 182,780 has estrogen receptor antagon effect
Or active constituent, it has a vast market foreground.
Black false hellebore (Veratrum nigrum L.) is the root and rhizome of dicotyledon medicine liliaceous plant RADIX ET RHIZOMA VERATRI NIGRI
(Falsehellebore Root and Rhizome).Toil is trembled with fear, toxic.It is emetic with eliminating the phlegm, the effect of desinsection.Black false hellebore
Platymiscium is relatively abundant in china natural resources, black false hellebore be used as already in medical drugs, and treatment cancer is also used in folk remedy
Disease.Veratrum mainly contains steroid alkaloid, Pretty class compound etc..Pharmacological research confirms that its steroid alkaloid has the enhancing heart
Myotility, reduce blood pressure, antithrombotic the effects of；Pretty class compound has antitumor, inhibition coagulating platelets effect.But it has no
Report it with antiestrogenic sample effect.This research is tested by inside and outside investigates the effect of black false hellebore estrogen receptor antagon sample,
Scientific basis is provided to develop new tumour endocrine therapeutic agents, serves clinic.
Summary of the invention：
It is an object of the present invention to provide the new applications of black false hellebore, are especially to provide the estrogen receptor antagon sample of black false hellebore
Effect.For this purpose, the present invention provides application of the black false hellebore in the drug that preparation has the effect of estrogen receptor antagon sample.
Application of the present invention with the effect of estrogen receptor antagon sample is that black false hellebore is able to suppress not into mouse mouse
The increase of uterus weight and uterovaginal growth and development.
Application of the present invention has the effect of estrogen receptor antagon sample, is that (1) black false hellebore has inhibition prematurity mouse
Uterine coefficient growth effect, (2) black false hellebore have inhibit each raised effect of dosage group Uterine coefficient of ginseng.
Application of the present invention has the effect of estrogen receptor antagon sample, is that (1) black false hellebore has reduction prematurity mouse
Estradiol (estrogen, E in serum2) content, it increases follicle-stimulating hormone (follicle-stimulating hormone, FSH)
With the effect of lutropin (luteinizing hormone, LH) content, (2) black false hellebore antagonism has estrogen-like action people
Join E in the raising serum of each dosage group2Content, and reduce the effect of FSH and LH content.
Application of the present invention has the effect of estrogen receptor antagon sample, is that (1) black false hellebore has inhibition prematurity mouse
Uterus and the normal growth and development of vagina effect, there is (2) black false hellebore antagonism each dosage group of estrogen-like action ginseng to promote
The growth and development of uterus and vagina tissue acts on.
Application of the present invention has the effect of estrogen receptor antagon sample, is that (1) black false hellebore can reduce prematurity mouse
Uterus and vagina tissue ER α, ER β protein level expression.(2) black false hellebore being capable of each dosage group promotion uterus of antagonism ginseng and yin
The effect that estrogen receptor ERs is expressed in road tissue.
Application of the present invention has the effect of estrogen receptor antagon sample, is black false hellebore, to the uterus system of ovariectomized mouse
Number does not generate obvious inhibiting effect；As black false hellebore compatibility positive drug Estradiol Valerate (Estradiol valerate, EV), performance
Obviously inhibit the rush uterus weight effect of positive drug EV out；When black false hellebore compatibility has the Chinese medicine ginseng of estrogen action, performance
Obviously inhibit the rush uterus weight effect of ginseng administration group out.
Application of the present invention has the effect of estrogen receptor antagon sample, is the performance as black false hellebore compatibility positive drug EV
Obviously inhibit E in the elevating blood of positive drug EV out2The effect of content；When black false hellebore compatibility has the Chinese medicine ginseng of estrogen action
When, it shows obviously to inhibit E in the elevating blood of ginseng administration group2The effect of content.
Application of the present invention has the effect of estrogen receptor antagon sample, is the performance as black false hellebore compatibility positive drug EV
Positive drug EV is obviously inhibited to promote the effect of uterus vagina growth and development out；When black false hellebore compatibility has the Chinese medicine people of estrogen action
When ginseng, the effect for obviously inhibiting ginseng administration group to promote the growth and development of uterus vagina is shown；
Application of the present invention has the effect of estrogen receptor antagon sample, is the performance as black false hellebore compatibility positive drug EV
Obviously inhibit the effect of positive drug EV enhancing estrogen receptor ERs expression out；When black false hellebore compatibility has the Chinese medicine of estrogen action
When ginseng, the effect for obviously inhibiting the enhancing estrogen receptor ERs expression of ginseng administration group is shown.
Application of the present invention has the effect of estrogen receptor antagon sample, is water extract, alcohol extract, ethyl acetate extraction
Object, veratridine alkali and jervine have the function of inhibiting MCF-7 cell Proliferation.
Black false hellebore of the present invention, the raw medicinal material including black false hellebore and the drug products with raw medicinal material preparation, such as Chenopodiaceae
Reed extract, and using the extract as the pharmaceutical preparation of active pharmaceutical ingredient.Wherein black false hellebore extract includes water extract, alcohol
Extract, preferably 95% ethanol extract, ethyl acetate extract, veratridine alkali, Jervine.
Wherein the preparation method of water extract can be：
By black false hellebore pulverizing medicinal materials at coarse powder, it is added the distilled water of 6 times of amounts, refluxing extraction 2-3 times, 1 hour every time.According to reality
Border administration concentration is concentrated under reduced pressure or is diluted to respective concentration, is used for inside and outside experimental study.
Wherein the preparation method of alcohol extract can be：
By black false hellebore pulverizing medicinal materials at coarse powder, it is added the 60-80% ethyl alcohol of 6 times of amounts, refluxing extraction 2-3 times, 1 hour every time.
According to practical administration concentration, it is concentrated under reduced pressure or is diluted to respective concentration, is used for inside and outside experimental study.
Wherein the preparation method of other extracts can be：
By black false hellebore pulverizing medicinal materials at coarse powder, the ammonia spirit that 2 times of amount volume fractions are 28% is added, infiltrates 1 hour；Again plus
Enter chloroform-methanol (volume ratio 4:1) 20 times of mixed volume amounts are placed in 70 DEG C of water-baths and reflux 2 hours are added, and filter, and are added
Mixed solvent rinses filter residue, collects filtrate, is concentrated under reduced pressure or is diluted to respective concentration, studies for experiment in vitro.
The present invention further provides short of money in preparation estrogen receptor by the pharmaceutical composition of active constituent of black false hellebore extract
Application in the drug of anti-agent sample effect.
Pharmaceutical composition of the invention, wherein shared weight percent can be 0.01- to black false hellebore extract in the composition
99.99%, remaining is pharmaceutically acceptable carrier.Pharmaceutical composition of the invention, exists in a unit, the unit
Dosage form refers to the unit of preparation, such as every of tablet, every capsule of capsule, and every bottle of oral solution, every bag of granule etc..
Pharmaceutical composition of the invention can be any pharmaceutical dosage form, these dosage forms include：Tablet, sugar coated tablet,
Film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral solution, mouth containing agent, granule, electuary, ball
Agent, powder, paste, sublimed preparation, suspension, pulvis, solution, injection, suppository, ointment, emplastrum, creme, spray, drop
Agent, patch.Preparation of the invention, preferably peroral dosage form, such as：Capsule, tablet, oral solution, granule, pill, powder,
Sublimed preparation, paste etc..
Pharmaceutical composition of the invention, the preparation of oral administration can contain common excipient, such as adhesive, filling
Agent, diluent, tablet agent, lubricant, disintegrating agent, colorant, flavoring agent and wetting agent when necessary can be coated tablet.
Applicable filler includes cellulose, mannitol, lactose and other similar fillers.Suitable disintegrating agent packet
Include starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant includes, such as firmly
Fatty acid magnesium.Suitable pharmaceutically acceptable wetting agent includes lauryl sodium sulfate.
Can be by mixing, filling, commonly method prepares solid oral composition for tabletting etc..Carrying out mixing repeatedly can make to live
Property substance be distributed in entirely using in those of a large amount of fillers composition.
The form of oral liquid for example can be aqueous or oily suspensions, solution, emulsion, syrup or elixir,
Or it can be a kind of dry products that can be compounded with water or other suitable carriers before use.This liquid preparation can contain
Conventional additive, such as suspending agent, such as sorbierite, syrup, methylcellulose, gelatin, hydroxyethyl cellulose, carboxymethyl
Cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifier, such as lecithin, anhydro sorbitol monooleate or I
Primary glue；Non-aqueous carrier (they may include edible oil), for example, apricot kernel oil, fractionated coconut oil, such as glycerol ester oiliness
Ester, propylene glycol or ethyl alcohol；Preservative, such as para hydroxybenzene methyl esters or propylparaben or sorbic acid, and if need
It wants, contains conventional flavouring agent or colorant.
For injection, the fluid unit dosage form of preparation contains active material and sterile carrier of the invention.According to carrier
And concentration, this compound can be suspended or be dissolved.The preparation of solution is usually by the way that active material is dissolved in a kind of load
In body, disinfection is filtered before being loaded into a kind of suitable bottle or ampoule, is then sealed.For example a kind of local anaesthesia of auxiliary material
Agent, preservative and buffer are also soluble in this carrier.It, can be after being packed into bottle by this in order to improve its stability
Kind composition frost, and under vacuum remove water.
Suitable pharmaceutically acceptable load is optionally added pharmaceutical composition of the invention when being prepared into medicament
Body, the pharmaceutically acceptable carrier are selected from：Mannitol, sorbierite, sodium pyrosulfite, sodium hydrogensulfite, sodium thiosulfate, salt
Sour cysteine, thioacetic acid, methionine, injection Vitamin B_6 DTA disodium, Ethylenediaminetetraacetic Acid Calcium Salt, carbonate, the acetic acid of monovalence alkali metal
Salt, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, wheat
Bud sugar, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and its
Derivative, alginates, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surface-active
Agent, polyethylene glycol, cyclodextrin, beta-cyclodextrin, phospholipid material, kaolin, talcum powder, calcium stearate, magnesium stearate etc..
Composition of the invention determines usage and dosage according to the patient's condition when in use, can often taken three times per day, each 1-
20 doses, such as：1-20 bags or grain or piece.
Application of the invention is obtained by following tests research, and related experiment is as follows：
One, experimental material
(1) instrument and equipment
BMJ-1 biological tissue embedding machine (Tianli Aeronautics Electromechanical Co., Tianjin)；QPJ-C cycle type slicer (Tianjin
Tian Li Aviation ElctroMechanical Co., Ltd)；KPJ-1A bakes piece machine (Tianli Aeronautics Electromechanical Co., Tianjin)；ZPJ-1A opens up piece machine (day
Jin Tianli Aviation ElctroMechanical Co., Ltd)；GHP-9080 water isolation type constant incubator (the permanent Science and Technology Ltd. in Shanghai one)；It is just setting aobvious
Micro mirror (OLYPUS product BX50)；Vortex mixer (Beijing Jin Bei moral Trade Co., Ltd. KA-1000)；Electric heating constant temperature air blast is dry
Dry case (Hengfeng Medical Instruments Co., Ltd., Huangsih City SFG-02.400)；Automatic dual pure water distiller (the sub- flourish biochemical instruments in Shanghai
Device factory SZ-93)；Full-automatic microplate reader (Thermo company MK3 type)；Refrigerated centrifuge (Sigma company 3K15).Biohazard Safety Equipment
(Shanghai Lishen Scientific Equipment Co., Ltd. HFsafe-TE1200)；CO2 incubator (Shanghai Lishen Scientific Equipment Co., Ltd.
HF151UV)。
(2) drug and reagent
Black false hellebore is purchased from Tongling Feng Yuan, Anhui prepared slices of Chinese crude drugs company, is extracted with water, and is diluted after merging mother liquor twice with distilled water
To 2.25g/L；
Ginseng is purchased from Changchun Chinese medicine Co., Ltd, is ground into coarse powder, and refluxing extraction twice, 1 hour every time, merges twice
Filtrate, 50 DEG C are concentrated under reduced pressure into 0.6g/mL；
Black false hellebore 0.000001~1mg/mL of water extract, 0.000001~1mg/mL of Water extracts from Ginseng, ginseng black false hellebore, which is closed, decocts
0.000001~1mg/mL of water extract, is prepared by China Academy of Traditional Chinese Medicine, Institute of Basic Theory；
ICI182,780 are purchased from Shanghai Han Xiang Biotechnology Co., Ltd, with DMSO hydrotropy, then are diluted to distilled water
0.25mg/mL；
Estradiol and (30%) acrylamide are purchased from Sigma company；
Estradiol Valerate (Progynova) is purchased from Beyer Co., Ltd；
Bush dye liquor and eosin stain are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；
Two-step method immunohistochemical kit, DAB colour reagent box and 0.01M PBS, which are purchased from Wuhan doctor's moral bioengineering, to be had
Limit company；
PIPA lysate, 5 × sample-loading buffer, the dedicated skimmed milk power of closing and ECL luminous agent are purchased from Beijing Puli's Lay base
Because of Technology Co., Ltd.；
Rabbit-anti mouse ER α polyclonal antibody is purchased from Beijing flourishing age Zhong Fang Bioisystech Co., Ltd；
Rabbit-anti mouse ER β polyclonal antibody is purchased from Beijing flourishing age Zhong Fang Bioisystech Co., Ltd；
Horseradish enzyme peroxidase labelling goat anti-rabbit igg and albumen Marker (14-100kDa) are purchased from China fir gold in Beijing
Bridge Technology Co., Ltd. product；
Pvdf membrane is purchased from Millipore company；
Bovine serum albumin(BSA) is purchased from Roche product；
DMEM (high sugar), Gibco company is purchased from without phenol red DMEM, superfine fetal calf serum FBS and 0.25% pancreatin；
Active carbon filtering fetal calf serum CDT-FBS (SH30068.03) is purchased from Hyclone company；
Thiazolyl blue MTT and dimethyl sulfoxide DMSO is purchased from Beijing Ke Hai Rong Jing Biotechnology Co., Ltd；
Dimethylbenzene is purchased from Beijing Chemical Plant；
Methylene blue is purchased from three factory of Shanghai reagent；
Paraformaldehyde is purchased from Beijing Yili Fine Chemicals Co., Ltd..
(3) cell
MCF-7 Human Breast Cancer Cells are purchased from Beijing consonance cell centre.
Two, experiment content：
(1), black false hellebore has the effect of estrogen receptor antagon sample
1. experimental method (prematurity mouse model)
1.1 experimental animal
Kunming mice 50, female 21 days, weight 9-12g after birth, is purchased from Military Medical Science Institute, SPF grades, permits
Card number is：SCXK (army) 2012-0004.
1.2 experimental groups and administration
Health, female mice 30 of birth 21 days are taken, adaptive feeding 2 days, blank control is randomly divided into according to weight
Group (Con), ICI group, black false hellebore group (VT), every group 10.ICI 5mgkg-1 is injected intraperitoneally in ICI group, and Chenopodiaceae is given in black false hellebore group stomach-filling
Reed aqueous extract 0.045gkg-1, blank control control group give isometric distilled water.Successive administration 5 days, once a day.
Put to death fasting 12h before drawing materials
1.3 materials
Mouse after the last administration for 24 hours, pluck eyeball and take blood, and 3000rpm is centrifuged 15min by weighing, separates serum, -80 DEG C of storages
It is spare.Mouse takes off neck and puts to death, and cuts open the belly win uterus and vagina rapidly, Uterine coefficient is calculated after weighing, and divide and take left uterine angle,
Uterine neck proximal end vagina is partially placed into 4% paraformaldehyde solution fixed PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM to be made and immunohistochemistry inspection
It surveys, right uterine angle, uterine neck distal end portio vaginalis point is immediately placed on -80 DEG C of refrigerations and detects to Western-bolt.
1.4 statistical analysis
Data are analyzed and processed using SPSS17.0 software, each group of data is with mean ± standard deviationIt indicates,
With P<0.05 has the standard of significant difference as statistics.
2. experimental result
Influence of 2.1 black false hellebores to prematurity Mouse Uterus coefficient, the results are shown in Table 1；
Influence (n=10) of 1 black false hellebore of table to prematurity Mouse Uterus coefficient
Note：Compared with normal group, * indicates P<0.05, * * indicates P<0.01, * * * P<0.001.
The result shows that 0.045g/kg black false hellebore homospecificity receptor antagonist ICI182,780 is similar, is able to suppress not into mouse
The Uterine coefficient of mouse tentatively infers it with costimulatory receptor antagonist-like effect.
(2), the estrogen action of black false hellebore antagonism estradiol and ginseng extract
1. experimental method
1.1 prematurity mouse models
1.1.1 experimental animal
Kunming mice 120, female 21 days, weight 9-12g after birth, is purchased from Military Medical Science Institute, SPF grades, permits
Card number is：SCXK (army) 2012-0004.
1.2.2 experimental group and administration
Health, female mice 120 of birth 21 days are taken, adaptive feeding 2 days, blank control is randomly divided into according to weight
Group, ICI group, black false hellebore group, ginseng 12g/kg group, ginseng 12g/kg+ICI 5mg/kg group, ginseng 12g/kg+ black false hellebore 0.045g/kg
Group, ginseng 18g/kg group, ginseng 18g/kg+ICI5mg/kg, ginseng 18g/kg+ black false hellebore 0.045g/kg group, ginseng 24g/kg group,
Ginseng 24g/kg+ICI5mg/kg group, ginseng 24g/kg+ black false hellebore 0.045g/kg group.Every group 10.Each dosage group of ginseng fills respectively
Stomach gives ginseng aqueous extract 12,18,24gkg-1, and ICI 5mgkg-1 is injected intraperitoneally in ICI group, and black false hellebore is given in black false hellebore group stomach-filling
Aqueous extract 0.045gkg-1, ginseng compatibility ICI group and black false hellebore group are given respectively while giving above-mentioned ginseng dosage
ICI the and 0.045gkg-1 black false hellebore of 5mgkg-1, blank control control group give isometric distilled water.Successive administration 5
It, once a day.Put to death fasting 12h before drawing materials.
1.2 ovariectomized mouse models
1.2.1 experimental animal
Kunming mice 140, unpregnancy female, weight 22-25g, be purchased from Military Medical Science Institute, SPF grades, credit number
For：SCXK (army) 2012-0004.
1.2.2 experimental group
According to weight be randomly divided into sham-operation group (Sham), OVX group, EV group, EV0.154mg/kg+ICI5mg/kg group,
EV0.154mg/kg+ black false hellebore 0.045g/kg group, ginseng 18g/kg group, ginseng 18g/kg+ICI5mg/kg group, ginseng 18g/kg+
Black false hellebore 0.045g/kg group, ginseng 24g/kg group, ginseng 24g/kg+ICI5mg/kg group, ginseng 24g/kg+VT0.045g/kg group.
Ginseng aqueous extract 18,24gkg-1 is given in stomach-filling to each dosage group of ginseng respectively, and ICI 5mgkg-1, Chenopodiaceae is injected intraperitoneally in ICI group
Reed group stomach-filling gives black false hellebore aqueous extract 0.045gkg-1, ginseng compatibility ICI group and black false hellebore group and is giving above-mentioned ginseng dosage
While give ICI the and 0.045gkg-1 black false hellebore of 5mgkg-1 respectively, blank control control group gives isometric distillation
Water.Successive administration 4 weeks, once a day.Put to death fasting 12h before drawing materials.
1.2.3 modeling and administration
Mouse web portion hair is shaved, 2% chloral hydrate anesthesia is injected intraperitoneally according to 0.2mL/10g；With iodine disinfection mouse abdomen
Median abdominal incision is removed in portion, cuts off bilateral ovaries, tubal ligation and Concomitant Vessels, rinses closure wound.Sham-operation group is not cut
Except ovary, the ovary peripheral adipose tissue of same size is only cut off.Postoperative intramuscular injection penicillin for three days on end, 50,000 U/ are only；Art
Restorative raising afterwards, continuous 5d make vaginal smear, reject the individual for occurring emotionally reacting.Satisfactory animal, by mouse with
Machine is divided into 14 groups, every group 10.
Physiological saline is given in blank control group stomach-filling, and 0.154g/kg is given in positive group stomach-filling, and the intraperitoneal injection of ICI group is given
0.045g/kg is given in 5mg/kg, black false hellebore group stomach-filling, ginseng low, middle and high dose groups respectively stomach-filling give 12g/kg, 18g/kg,
24g/kg, successive administration 7 days.
1.3 materials
Mouse after the last administration for 24 hours, pluck eyeball and take blood, and 3000rpm is centrifuged 15min by weighing, separates serum, -80 DEG C of storages
It is spare.Mouse takes off neck and puts to death, and cuts open the belly win uterus and vagina rapidly, Uterine coefficient is calculated after weighing, and divide and take left uterine angle,
Uterine neck proximal end vagina is partially placed into 4% paraformaldehyde solution fixed PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM to be made and immunohistochemistry inspection
It surveys, right uterine angle, uterine neck distal end portio vaginalis point is immediately placed on -80 DEG C of refrigerations and detects to Western-bolt.
1.4 pathological study
Uterus and vagina tissue are after the fixed 48h of 4% formaldehyde, conventional to be dehydrated, specimens paraffin embedding slices, row hematoxylin-eosin
Dyeing.Histopathological examination is carried out under an optical microscope.
1.5 hematological examination
Mice serum E2, LH, FSH level detects by the prosperous equation Biotechnology Co., Ltd in Beijing.
1.6 immunohistochemistry
Histotomy is prepared by " 1.4 " item method, it is conventional to dewax to water, 3% dioxygen water seal endogenous peroxydase,
PBS washing, 0.01mol/L sodium citrate buffer solution (pH6.0) repair antigen, exposure antigenic determinant.PBS washing, cow's serum
Room temperature closes 30min, and rabbit-anti mouse ER α/ER β polyclonal antibody (1: 30 dilution) is added dropwise, with two-step method immunohistochemical kit,
DAB colour reagent box carries out ER α/ER β immunohistochemical staining.Microscopically observation color developing effect, until being steamed when brown color
Distilled water stops colour developing, and dehydration, dimethylbenzene is transparent, resin mounting.Immunohistochemical detection is carried out under an optical microscope.Using
IPP (Image Pro Plus 6.0, USA) image analysis system carries out semi-quantitative analysis, calculates IOD value.
1.7 Western blot
Uterus and the vagina soluble egg of RIPA reagent (50~100mg is organized, and adds 500~1000 μ L of lysate) extracting
It is white, SDS- polyacrylamide gel is prepared, it is wet after electrophoresis to go on pvdf membrane.5% (TBST dilution) skimmed milk power shaken at room temperature
TBST washes film, primary antibody ER α (1 after closing 2h：100) or ER β (1: 1000) or 4 DEG C of GAPDH (1: 1000) overnight.TBST washes film,
Horseradish enzyme marks goat anti-rabbit igg (1: 5000) to be incubated at room temperature 2h, ECL reaction 1min is added after washing film, using gel imaging system
Overall view examines Blot results.
1.8 statistical analysis
Data are analyzed and processed using SPSS17.0 software, each group of data is with mean ± standard deviationIt indicates,
With P<0.05 has the standard of significant difference as statistics.
2. experimental result
2.1 prematurity mouse models
2.1.1 black false hellebore antagonism ginseng the results are shown in Table 2 to the increasing action of prematurity Mouse Uterus coefficient；
Increasing action (n=10) of the 2 black false hellebore antagonism ginseng of table to prematurity Mouse Uterus coefficient
Note：Compared with Normal group, * indicates P<0.05, * * indicates P<0.01, * * * P<0.001；Compatibility ICI be applied alone
Each dosage group of ginseng compares, and ### indicates P<0.001；For compatibility black false hellebore compared with each dosage group of ginseng is applied alone, △ indicates P<0.05,
△ △ indicates P<0.01, △ △ △ indicates P<0.001.
The result shows that：(1) black false hellebore has the function of inhibiting the growth of the Uterine coefficient of prematurity mouse, and function and effect are similar
In estrogen receptor antagon ICI182,780；(2) black false hellebore, which has, inhibits each raised effect of dosage group Uterine coefficient of ginseng, with
Increasing for ginseng dosage, inhibiting effect gradually weakens, function and effect be similar to estrogen receptor antagon ICI182,780；
Illustrate that black false hellebore harms the estrogen-like action of ginseng, it may be possible to play a role by antagonising oestrogen receptors.
2.1.2 black false hellebore antagonism ginseng increases the influence of estrogen level in prematurity mice serum, the results are shown in Table 3；
3 black false hellebore antagonism ginseng of table increases the influence (n=10) of estrogen level in prematurity mice serum
Note：Compared with Normal group, * indicates P<0.05, * * indicates P<0.01, * * * P<0.001；Compatibility ICI be applied alone
Each dosage group of ginseng compares, and ### indicates P<0.001；For compatibility black false hellebore compared with each dosage group of ginseng is applied alone, △ indicates P<0.05,
△ △ indicates P<0.01, △ △ △ indicates P<0.001.
The result shows that：(1) compared with blank control group, black false hellebore has E in reduction prematurity mice serum2Content increases
The effect of LH and FSH content, function and effect and trend are similar to classical estrogen receptor antagon ICI；(2) black false hellebore antagonism
E2 content in raising serum with each dosage group of estrogen-like action ginseng, and the effect of LH and FSH content is reduced, it is in dosage
Correlation, it is consistent with the trend of each dosage effect of ICI antagonism ginseng.
2.1.3 black false hellebore antagonism ginseng promotes prematurity Mouse Uterus vagina growth and development effect
Pass through pathological study, the results showed that, there is black false hellebore antagonism ginseng to promote prematurity Mouse Uterus and vagina
Growth and development effect acts on and is similar to estrogen receptor antagon ICI182, and 780, the results are shown in attached figure 1, and 2.
2.1.4 black false hellebore antagonism ginseng promotes the table of estrogen receptor ERs albumen in prematurity Mouse Uterus and vagina tissue
It reaches, the results are shown in Table 4；
4 black false hellebore antagonism ginseng of table promotes estrogen receptor ERs protein expression in prematurity Mouse Uterus and vagina tissue
It influences (n=10)
Note：Compared with Normal group, * indicates P<0.05, * * indicates P<0.01, * * * P<0.001；Compatibility ICI be applied alone
Each dosage group of ginseng compares, and ### indicates P<0.001；For compatibility black false hellebore compared with each dosage group of ginseng is applied alone, △ indicates P<0.05,
△ △ indicates P<0.01, △ △ △ indicates P<0.001.
The result shows that：(1) black false hellebore can reduce the uterus of prematurity mouse and the table of vagina tissue ER α, ER β protein level
Reach, function and effect be similar to estrogen receptor antagon ICI182,780.(2) black false hellebore being capable of each dosage group promotion of antagonism ginseng
The effect that estrogen receptor ERs is expressed in uterus and vagina tissue, function and effect are similar to estrogen receptor antagon
ICI182,780, and the influence to ER beta receptor is better than ER α.
2.2 ovariectomized mouse models
2.2.1 black false hellebore antagonism ginseng the results are shown in Table 5 to the increased effect of ovariectomized mouse Uterine coefficient；
5 black false hellebore antagonism ginseng of table effect (n=10) increased to the ripe Mouse Uterus coefficient of removal ovary
Note：Compared with sham-operation group, * indicates P<0.05, * * indicates P<0.01, * * * P<0.001；Compared with OVX group, # table
Show P<0.05, ## indicates P<0.01, ### indicates P<0.001；For compatibility ICI compared with each dosage group of ginseng is applied alone, △ indicates P<
0.05, △ △ indicates P<0.01, △ △ △ indicates P<0.001；Compatibility black false hellebore is compared with being applied alone each dosage group of ginseng, ▲ expression P<
0.05, ▲ ▲ indicate P<0.01, ▲ ▲ ▲ indicate P<0.001.
The experimental results showed that not generating obvious inhibiting effect to the Uterine coefficient of ovariectomized mouse when black false hellebore is used alone；
As black false hellebore compatibility positive drug EV, the rush uterus weight effect for obviously inhibiting positive drug EV is shown；When black false hellebore compatibility is with female
When the Chinese medicine ginseng of hormonal action, the rush uterus weight effect for obviously inhibiting ginseng administration group is shown；It is said by above data
Bright black false hellebore has the effect of antiestrogenic sample.
2.2.2 black false hellebore antagonism ginseng inhibits the effect in ovariectomized mouse uterus and vagina tissue atrophy
Pass through pathological study, the results showed that, there is black false hellebore antagonism ginseng to inhibit ovariectomized mouse uterus and vagina
The effect of Telatrophy acts on and is similar to estrogen receptor antagon ICI182, and 780.
2.2.3 black false hellebore antagonism ginseng increases estrogen level in ovariectomized mouse serum, the results are shown in Table 6；
6 black false hellebore antagonism ginseng of table increases the influence (n=10) of estrogen level in ovariectomized mouse serum
Note：Compared with sham-operation group, * indicates P<0.05, * * indicates P<0.01, * * * P<0.001；Compared with OVX group, # table
Show P<0.05, ## indicates P<0.01, ### indicates P<0.001；For compatibility ICI compared with each dosage group of ginseng is applied alone, △ indicates P<
0.05, △ △ indicates P<0.01, △ △ △ indicates P<0.001；Compatibility black false hellebore is compared with being applied alone each dosage group of ginseng, ▲ expression P<
0.05, ▲ ▲ indicate P<0.01, ▲ ▲ ▲ indicate P<0.001.
The experimental results showed that positive drug EV group and ginseng administration group, which have, promotes E in blood2Content, simultaneously reduce FSH and
The effect of LH content shows obviously to inhibit E in the elevating blood of positive drug EV as black false hellebore compatibility positive drug EV2Content
Effect；When black false hellebore compatibility has the Chinese medicine ginseng of estrogen action, the elevating blood for obviously inhibiting ginseng administration group is shown
Middle E2The effect of content；Illustrate that black false hellebore has the effect of antiestrogenic sample by above data.
2.1.4 black false hellebore antagonism ginseng increases estrogen receptor ERs protein expression in ovariectomized mouse uterus and vagina tissue,
It the results are shown in Table 7；
7 black false hellebore antagonism ginseng of table increases estrogen receptor ERs protein expression (n in ovariectomized mouse uterus and vagina tissue
=10)
Note：Compared with sham-operation group, * indicates P<0.05, * * indicates P<0.01, * * * P<0.001；Compared with OVX group, # table
Show P<0.05, ## indicates P<0.01, ### indicates P<0.001；For compatibility ICI compared with each dosage group of ginseng is applied alone, △ indicates P<
0.05, △ △ indicates P<0.01, △ △ △ indicates P<0.001；Compatibility black false hellebore is compared with being applied alone each dosage group of ginseng, ▲ expression P<
0.05, ▲ ▲ indicate P<0.01, ▲ ▲ ▲ indicate P<0.001.
The experimental results showed that positive drug EV group and ginseng administration group have promote in target organ uterus and vagina estrogen by
The effect of body ERs expression as black false hellebore compatibility positive drug EV shows that positive drug EV is obviously inhibited to enhance estrogen receptor ERs
The effect of expression；When black false hellebore compatibility has the Chinese medicine ginseng of estrogen action, show that ginseng administration group is obviously inhibited to enhance
The effect of estrogen receptor ERs expression；Illustrate that black false hellebore has the effect of antiestrogenic sample by above data, and is swashed based on female
The receptor-mediated antiestrogenic sample effect of element.
(3), the experiment in vitro research of the estrogen action of black false hellebore antagonism estradiol and ginseng extract
1. experimental method
1.1 experimental groups and administration
Medicine ordinance：Black false hellebore 0.000001~1mg/mL of water extract, 0.000001~1mg/mL of Water extracts from Ginseng, ginseng
Black false hellebore, which is closed, decocts 0.000001~1mg/mL of water extract, is prepared by China Academy of Traditional Chinese Medicine, Institute of Basic Theory；Each water mentions
Object, estradiol and estrogen receptor antagon ICI182,780 are prepared with without phenol red DMEM.The administration concentration of estradiol is 10- 8M, estrogen receptor antagon ICI182,780 administration concentration is 0.1 μM.
5mg/mL MTT：5mg MTT is weighed, is dissolved in 1ml 0.01M PBS liquid, is removed with 0.22 μm of filtering with microporous membrane
Bacterium, it is ready-to-use.
The culture of 1.2 cells
MCF-7 human breast cancer cell routine culture is placed in 37 DEG C, 5%CO in DMEM high glucose medium2, saturated humidity
Culture in incubator.
1.3MTT screening
3d is changed to cultivate under the conditions of without phenol red DMEM (containing 5%CDT-FBS) before experiment starts, intracellular female sharp to exhaust
Element.Logarithmic growth phase cell is selected to be added without phenol red DMEM after pancreatin digestion, be inoculated in 96 orifice plates with the concentration in 3000/hole
It is interior, 200 holes μ l/.Culture after cell is adherent, is separately added into black false hellebore water extract, Water extracts from Ginseng, ginseng black false hellebore and closes pan-fried water for 24 hours
0.0001mg/ml~0.1mg/ml concentration of extract, cultivates 48h respectively, and every hole is added 20 μ l 5mg/mL MTT, continues to be incubated for
Culture solution is carefully sucked after 4h, 150 μ l of DMSO is added in every hole.Each hole light absorption is measured at 490nm with enzyme-linked immunosorbent assay instrument
Value calculates mean absorbance values and proliferation rate.Every group sets 3 multiple holes, is repeated 3 times.
2. experimental result
2.1 black false hellebore water extract the influence to MCF-7 cell proliferation, the results are shown in Table 8；
Pass through external MTT experiment, the results showed that：Black false hellebore Aqueous extracts significantly inhibit in the range of 0.00001-0.1mg/mL
MCF-7 cell Proliferation prompts black false hellebore water extract to have the effect of antiestrogenic sample.
Influence of 8 black false hellebore of table to MCF-7 cell proliferation rate
Note：Compared with blank control group, * indicates P<0.05, * * indicates P<0.01.
2.2 black false hellebore antagonism ginsengs act on the rush increment of MCF-7 cell, the results are shown in Table 9；
By external MTT experiment, influence of the black false hellebore compatibility ginseng to MCF-7 cell Proliferation is investigated, the experimental results showed that, Chenopodiaceae
The rush MCF-7 cel l proliferation of reed inhibition ginseng shows that antagonism becomes apparent after single pan-fried merging compared with closing and decocting.
9 black false hellebore antagonism ginseng of table is to MCF-7 cell proliferation
Note：Compared with blank control group, * indicates P<0.05, * * indicates P<0.01.
Influence of 2.3 veratrum alkaloids to MCF-7 cell proliferation, the results are shown in Table 10 and table 11；
According to the experimental result of black false hellebore water extract, the main active veratrum alkaloid pair in black false hellebore is further investigated
The influence of MCF-7 cell Proliferation, the experimental results showed that, veratridine alkali and jervine have inhibition in certain concentration range
The effect of MCF-7 cell Proliferation prompts veratridine alkali and jervine to have the effect of antiestrogenic sample.
The influence of 10 veratridine alkali of table and jervine to MCF-7 cell Proliferation
Detailed description of the invention：
Effect of Fig. 1 black false hellebore antagonism ginseng to prematurity Mouse Uterus growth and development
Effect of Fig. 2 black false hellebore antagonism ginseng to prematurity mouse vagina growth and development
Influence of Fig. 3 black false hellebore antagonism ginseng to the uterus ER α, ER β protein expression of prematurity mouse
Influence of Fig. 4 black false hellebore antagonism ginseng to the vagina ER α, ER β protein expression of prematurity mouse
Wherein a：Blank control group, b：ICI group, c are black false hellebore group, and d is ginseng 12g/kg group, and e is ginseng 12g/kg+ICI
5mg/kg group, f are ginseng 12g/kg+ black false hellebore 0.045g/kg group, and g is ginseng 18g/kg group, and h is ginseng 18g/kg+ICI5mg/
Kg, i are ginseng 18g/kg+ black false hellebore 0.045g/kg group, and j is ginseng 24g/kg group, and k is ginseng 24g/kg+ICI5mg/kg group, l
For ginseng 24g/kg+ black false hellebore 0.045g/kg group.
The effect of Fig. 5 black false hellebore antagonism ginseng recovery ovariectomized mouse metratrophia
The effect of Fig. 6 black false hellebore antagonism ginseng recovery ovariectomized mouse vaginal atrophy
a:Sham-operation group (Sham) b:OVX group c:EV group d:EV0.154mg/kg+ICI5mg/kg group e:EV0.154mg/kg
+ black false hellebore 0.045g/kg group f:Ginseng 18g/kg group g:Ginseng 18g/kg+ICI5mg/kg group h:Ginseng 18g/kg+ black false hellebore
0.045g/kg group i:Ginseng 24g/kg group j:Ginseng 24g/kg+ICI5mg/kg group k:Ginseng 24g/kg+VT0.045g/kg group
Specific embodiment：
By the following examples, the present invention is further illustrated, but not as limitation of the present invention
Embodiment 1
By black false hellebore pulverizing medicinal materials at coarse powder, it is added the distilled water of 6 times of amounts, refluxing extraction 2-3 times, 1 hour every time.According to reality
Border administration concentration is concentrated under reduced pressure or is diluted to respective concentration, is used for inside and outside experimental study.
Embodiment 2
By black false hellebore pulverizing medicinal materials at coarse powder, it is added the 60-80% ethyl alcohol of 6 times of amounts, refluxing extraction 2-3 times, 1 hour every time.
According to practical administration concentration, it is concentrated under reduced pressure or is diluted to respective concentration, is used for inside and outside experimental study.
Embodiment 3
By black false hellebore pulverizing medicinal materials at coarse powder, the ammonia spirit that 2 times of amount volume fractions are 28% is added, infiltrates 1 hour；Again plus
Enter chloroform-methanol (volume ratio 4:1) 20 times of mixed volume amounts are placed in 70 DEG C of water-baths and reflux 2 hours are added, and filter, and are added
Mixed solvent rinses filter residue, collects filtrate, is concentrated under reduced pressure or is diluted to respective concentration, studies for experiment in vitro.
Embodiment 4, tablet
Black false hellebore water extract 100g, microcrystalline cellulose 50g, superfine silica gel powder 3g, magnesium stearate 1.5g
Preparation method is as follows：
Former, auxiliary material is taken to sieve with 100 mesh sieve respectively；Notoginsenoside R, microcrystalline cellulose are taken, is mixed, is made with 60% appropriate amount of ethanol
For adhesive softwood, the pelleting of 20 meshes is crossed, 60 DEG C of dryings are taken out, and 30 mesh sieves are crossed, and superfine silica gel powder and stearic acid is added
Magnesium, mix, tabletting, be made 1000 to get.
Embodiment 5, capsule
Capsule takes black false hellebore alcohol extract 100mg, and appropriate amount of starch, the auxiliary materials such as magnesium stearate, granulation, whole grain, loading 1 is added
Capsule to get.
Embodiment 6, oral solution
Oral solution takes black false hellebore water extract 100mg, and suitable amount of sucrose is added, and preservative adds water to 1000ml, is distributed into 10ml
One to get oral solution.
Embodiment 7, granule
Granule takes black false hellebore alcohol extract 100mg, is added appropriate dextrin, Steviosin, dry granulation, whole grain, packing to get.
Embodiment 8, injection
The black false hellebore extract 150g of injection, embodiment 3 is dissolved in water, another sodium chloride, ethyl-para-hydroxybenzoate heating
Water dissolution, mixes, and adjusts pH value 5-7.Water for injection is diluted to 1000ml, is filtered with hollow-fibre membrane, filling, sterilizing to get.
Claims (4)
1. using black false hellebore extract as sole active agent in the drug that preparation has the effect of estrogen receptor antagon sample
Using, wherein described have the effect of estrogen receptor antagon sample, be black false hellebore extract have inhibit prematurity Mouse Uterus with
The effect of vagina normal growth and development；Wherein black false hellebore extract is water extract, alcohol extract, veratridine alkali, jervine；Wherein water mentions
The preparation method of object is：By black false hellebore pulverizing medicinal materials at coarse powder, the distilled water of 6 times of amounts is added, refluxing extraction 2-3 times, 1 is small every time
When；The preparation method of alcohol extract is：By black false hellebore pulverizing medicinal materials at coarse powder, the 60-80% ethyl alcohol of 6 times of amounts, refluxing extraction 2-3 is added
It is secondary, 1 hour every time.
2. application according to claim 1, wherein black false hellebore extract has antagonism ginseng to prematurity Mouse Uterus coefficient
Increasing action and black false hellebore extract have antagonism ginseng promote the growth and development effect of prematurity Mouse Uterus vagina.
3. the application using black false hellebore extract as sole active agent in the drug of preparation treatment breast cancer, wherein black false hellebore is extracted
Object is water extract, alcohol extract, veratridine alkali, jervine；Wherein the preparation method of water extraction is：By black false hellebore pulverizing medicinal materials at thick
Powder, be added 6 times amount distilled water, refluxing extraction 2-3 times, 1 hour every time；Alcohol extract preparation method is：By black false hellebore pulverizing medicinal materials
At coarse powder, it is added the 60-80% ethyl alcohol of 6 times of amounts, refluxing extraction 2-3 times, 1 hour every time.
4. application according to claim 3, wherein there is black false hellebore extract antagonism ginseng to promote MCF-7 cel l proliferation.
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