CN106580932B - Bakuchiol is preparing the purposes in the drug for preventing and/or treating benign prostatic hyperplasis - Google Patents
Bakuchiol is preparing the purposes in the drug for preventing and/or treating benign prostatic hyperplasis Download PDFInfo
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Abstract
The embodiment of the invention discloses Bakuchiols to prepare the purposes in the drug for preventing and/or treating benign prostatic hyperplasis.Since Bakuchiol passes through the expression of ER β in promotion epithelial cell, inhibit the expression of aromatizing enzyme in interstitial cell, it can inhibit BPH process, so as to prevent and/or treat benign prostatic hyperplasis, be used to prepare prevention and/or treat the drug of benign prostatic hyperplasis.Further, drug extract provided by the present invention can equally prevent and/or treat benign prostatic hyperplasis.
Description
Technical field
The present invention relates to pharmaceutical technology field, in particular to Bakuchiol prepare for prevent and/or treat it is benign before
Purposes in the drug of column gland hyperplasia.
Background technique
Benign prostate hyperplasia (Benign Prostatic Hyperplasia, BPH) belongs to outside middle-aging male uropoiesis
The common disease type of section.Epidemic data shows that the incidence of BPH increases with the growth at age, in recent years, with people
The trend further increased is presented in the quickening of the aged speed of mouth, the quantity and ratio of BPH patients.Usually initially occur at 40 years old with
Disease incidence may be up to 83% when disease incidence is greater than 50%, 80 years old when afterwards, by 60 years old, and become influences ageing male quality of life extensively
With the most common disease in the urological system of health.As prostate volume constantly increases, to oppress prostatic urethra, draw
Bladder outlet obstruction is played, patient is caused the symptoms such as different degrees of dysuria occur, serious person may occur in which bilateral upper urinary product
Water simultaneously leads to renal insufficiency, easily leads to blood urine, acute urinary retention, vesical calculus, renal failure if treatment is not in time
With the severe complications such as prostate cancer, the quality of life of elderly men is seriously affected.
BPH is one complicated, the histological change process including body of gland and interstitial tissue hyperplasia.In normal prostatic
In, epithelium mainly expresses the β hypotype (abbreviation ER β) of AR (androgen receptor) and ER (estrogen receptor), and interstitial mainly expresses ER
Alpha hypotype (abbreviation ER α) and aromatizing enzyme；The visible a small amount of aromatizing enzyme of epithelium and ER α when hyperplasia, interstitial also can be detected AR and
ER β prompts the development of AR, ER α/β and aromatizing enzyme and hyperplasia of prostate closely related.Androgen is (such as: testosterone
Testosterone, protona DHT) or estrogen (17 beta estradiols, E2) specifically in conjunction with AR or ER, and activate
AR or ER and enter nucleus, in conjunction with DNA sequence dna ARE or ERE on target gene, in a variety of auxiliary adjustment factors (cofactor)
Under the action of RNA polymerase, activation target gene transcription plays classical " genome effect "；In addition, the AR or ER of activation are also
Core can not be entered and the cascade reaction of downstream kinase is quickly activated to play " Non-genomic responses ".By adjusting turning for target gene
Record, the hero/intracellular a variety of accesses of estrogen participation, and the behaviors such as regulating cell proliferation, differentiation.
Prostate is Androgen-dependent Organs, and AR is the inhibition of cell Proliferation in the normal prostatic epithelium of studies have shown that
The factor, and AR then promotes epitheliosis in the epithelial cell of hyperplasia or prostate gland cancer cell.Simultaneously AR or Epithelial and stromal
Promotive factor has N-cadherin (nervous system type calcium mucin), the Snail expression for inhibiting epithelial cell special, promotes interstitial
The effect of Vimentin (vimentin) expression of cell-specific.In terms of growth factor regulation, IL-6, EGF, IGF-1, TGF β
Etc. the transcriptional activity that can obviously raise AR；And the promoter of some growth factors (such as IGF-1) also contains multiple androgens and answers
Answer element (ARE) sequence.
In recent years, effect of the estrogen in hyperplasia of prostate more and more attention has been paid to and pay attention to.Seemingly with androgens, female
Hormone is played a role by specifically binding estrogen receptor ER.The studies have shown that of the BPH rat models of estrogen induction is increased
It dedifferentes mark molecule SMemb expression in raw benign prostatic tissue to increase, basal cell is in Vimentin positive staining；Prompt
ER takes part in plasticity cell de-differentiation and EMT (Epithelial and stromal conversion).In addition to direct effect, ER also participates in having mediated epithelium-
The interaction of interstitial, the proliferation of indirect adjustments and controls cell and the expression of related gene.For example, estradiol can activate ER in epithelium
α promotes synthesis and the paracrine action of its transforming growth factor TGF β, and then promotes the differentiation of smooth muscle cell in interstitial.
In addition, intracorporal androgen can be converted into the active form estradiol of estrogen by aromatizing enzyme.Fragrance
The increase for changing raising or the expression of enzymatic activity can be such that estrogen concentrations increase, and then enhance estrogen and imitate in the effect of target organ
Fruit.In prostate, aromatizing enzyme is mainly expressed in interstitial cell, and Aromatase Expression obviously increases when BPH occurs.
Since BPH is a kind of high-incidence disease of elderly men, patient does not have the more of surgical indication, and drug becomes BPH
The first choice for the treatment of.
Summary of the invention
The present inventor passes through extensively the study found that Bakuchiol inhibits interstitial by the expression of ER β in promotion epithelial cell
The expression of aromatizing enzyme in cell can inhibit BPH process, and complete the present invention based on this.
The first aspect of the present invention provides Bakuchiol in preparation for preventing and/or treating benign prostatic hyperplasis
Purposes in drug.Wherein, the chemical structural formula of Bakuchiol is as follows:
In a kind of specific embodiment of the first aspect of the present invention, the Bakuchiol passes through up-regulation epithelial cell ER
β expression and/or the proliferation for lowering interstitial cell Aromatase Expression inhibition prostatic cell.
In another specific embodiment of the first aspect of the present invention, wherein the drug gives object in need
Daily dosage 0.01-1000mg/kg weight, preferably 0.1-100mg/kg weight, more preferably 1- are calculated as with Bakuchiol
100mg/kg weight.
In another specific embodiment of the first aspect of the present invention, wherein the Bakuchiol is with Bakuchiol
What the form of monomer provided, or provided in the form of the plant extracts comprising Bakuchiol.
Preferably, the Bakuchiol is provided in the form of the Fructus Psoraleae extract comprising Bakuchiol.
Herein, described " psoralea corylifolia " refers to that Effects of Bu Gu rouge, Effects of Bu Gu rouge are legumes psoraleae
The fruit of Psoralea colrylifolia L., warm-natured first recorded in Lei Gong's Treatise on Preparation and Broiling of Materia Medica, acrid flavour, hardship are returned kidney, the spleen channel, are had
Tonify the kidney and support yang, receive gas antidiarrheal the effect of.It is chiefly used in treating waist and knee crymodynia, spermatorrhoea, the enuresis, frequent micturition, Diarrhoea, cold of insufficiency type cough etc.
Disease.Bakuchiol (Bakuchiol) is one of main liposoluble constituent of psoralea corylifolia, and content generally exists in psoralea corylifolia
11.2-51.4mg/g.It is had been reported that in addition, the extracting mode of Fructus Psoraleae extract in the prior art more, herein without superfluous
It states, those skilled in the art can obtain Fructus Psoraleae extract by existing any way.
The second aspect of the present invention provides a kind of for preventing and/or treating the pharmaceutical composition of benign prostatic hyperplasis
Object, it includes Bakuchiol and pharmaceutically acceptable carrier or excipient.
In a kind of specific embodiment of the second aspect of the present invention, the total weight based on described pharmaceutical composition, institute
The content for stating Bakuchiol is 1-99%, preferably 20-80%, more preferably 40-60%.
In another specific embodiment of the second aspect of the present invention, the pharmaceutically acceptable carrier or figuration
Agent is selected from solvent, diluent, dispersing agent, suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, bonding
Agent, lubricant, stabilizer, hydrating agents, emulsification accelerator, buffer, absorbent, colorant, flavouring agent, sweetener, ion are handed over
Change agent, release agent, smears, corrigent and antioxidant.
In another specific embodiment of the second aspect of the present invention, described pharmaceutical composition is formulated as powder, piece
Any one dosage form in agent, pulvis, capsule, pill, pill, injection, emulsion, suspension or tincture.It is above-mentioned various
The drug of dosage form can be prepared according to the conventional method of pharmaceutical field.
Herein, term " treatment " has its general sense, and particularly refers to herein to having suffered from the present invention
The mammalian subject (being preferably people) of the benign prostatic hyperplasis disease is handled using drug of the invention, to right
The effects of disease generates treatment, cures, alleviates, mitigating.Similarly, as used herein, term " prevention " has one
As meaning, and particularly refer to herein to benign prostatic hyperplasis disease of the present invention may be suffered from or to the present invention
There is the benign prostatic hyperplasis disease mammalian subject of risk to be handled using drug of the invention, to
The effects of disease generation is prevented, prevents, prevents, separates.
Herein, it is " pharmaceutically acceptable " indicate when with usual dosage using when do not have tangible toxic effect,
So as to ratifying by government or with its comparable international organization or be approved for animal, more specifically to people, or
It is registered in pharmacopeia.
" pharmaceutically acceptable carrier or excipient " available in pharmaceutical composition of the present invention can be pharmaceutical preparation neck
The selection of any conventional carrier in domain, specific support will depend on the administration mode or disease type that are used to treat particular patient
And state.The preparation method of said synthetic processes for specific administration mode is completely in the knowledge of drug field technical staff
In range.
As used herein, term " pharmaceutical composition " has its general sense.In addition, " pharmaceutical composition " of the invention
Can also exist or provide in the form of health care product, functional food, food, food additives etc..It is special that pharmaceutical field can be used
It is the routine techniques in formulation art, obtains medicine group of the invention by extraction separation and purification means common in pharmaceutical production
The effective component for closing the raw material of object, optionally mixes with one or more of pharmaceutically acceptable carriers or excipient, then shape
At required dosage form, to prepare pharmaceutical composition of the invention.Pharmaceutical composition according to the present invention, to can be adapted for mouth
The pharmaceutical preparation of clothes administration, parenteral or local administration, topical administration, is particularly suitable for being administered orally.It is applied for oral
Dosage form may include for example tablet, pill, hard or soft capsule agent, solution, suspension, emulsion, syrup, powder, pulvis,
Granula subtilis, granule, pilule, elixir etc., however it is not limited to this.Other than active constituent, these preparations also may include diluent
(such as lactose, dextrose, sucrose, mannitol, D-sorbite, cellulose and glycine), lubricant (such as silica,
Talcum, stearic acid or its magnesium salts, calcium salt and polyethylene glycol).Tablet also may include adhesive, such as aluminium-magnesium silicate, gelatinized corn starch, bright
Glue, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine.It when necessary, also may include medicinal addition
Agent, such as disintegrating agent (such as starch, agar, alginic acid or its sodium salt), absorbent, colorant, flavouring agent, sweetener.Tablet
It can be prepared according to common mixing, the method for being granulated or being coated.
As it can be seen that Bakuchiol inhibits the table of aromatizing enzyme in interstitial cell by the expression of ER β in promotion epithelial cell
It reaches, BPH process can be inhibited, so as to prevent and/or treat benign prostatic hyperplasis, be used to prepare prevention and/or treatment
The drug of benign prostatic hyperplasis.Further, drug extract provided by the present invention equally can prevent and/or treat
Benign prostatic hyperplasis.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
It is influence of the Bakuchiol to BPH-1 and WPMY-1 cell Proliferation expressed by Fig. 1.Wherein, A is Bakuchiol suppression
The result of BPH-1 cell Proliferation processed；B is the result that Bakuchiol inhibits WPMY-1 cell Proliferation；C is that Bakuchiol inhibits BPH-
Result of the 1 cell PCNA in the expression of albumen and gene level；D is that Bakuchiol inhibits WPMY-1 cell PCNA in albumen and base
Because of the result of horizontal expression.
It is influence of the Bakuchiol to AR and ER protein expression expressed by Fig. 2.Wherein, A is that Bakuchiol is thin to BPH-1
The influence that AR is expressed in albumen and gene level in born of the same parents；B is for Bakuchiol to AR in WPMY-1 cell in albumen and gene level
The influence of expression；C is the influence that Bakuchiol expresses ER β in BPH-1 cell in albumen and gene level；D is Bakuchiol
The influence that ER α in WPMY-1 cell is expressed in albumen and gene level；E is Bakuchiol to LNCaP cell AR nuclear translocation
Influence (left side) and quantitative result (right side).
It is influence of the Bakuchiol to ER β expression and transcriptional activity in BPH-1 cell expressed by Fig. 3.Wherein, A is to mend
Bone fat phenol promotes ER β mRNA level in-site expression in BPH-1 cell with concentrationdependent manner；B is Bakuchiol in a manner of Time Dependent
Promote the expression of ER β mRNA level in-site in BPH-1 cell；C is the expression that Bakuchiol promotes ER β protein level in BPH-1 cell；
D is that siER β inhibits ER β expression in BPH-1 cell；E is that siER α inhibits ER alpha expression in BPH-1 cell；F is Bakuchiol pair
The influence of ERE transcriptional activity in BPH-1 cell；G is when inhibiting the expression of ER α or Er β, and Bakuchiol is to ERE in BPH-1 cell
The influence of transcriptional activity；Wherein, compared with the control group, * P < 0.05, * * P < 0.01；Compared with E2, #P < 0.05.
It is Bakuchiol expressed by Fig. 4 to the expression of aromatizing enzyme in WPMY-1 cell and the influence of transcriptional activity.Its
In, A is influence of the Bakuchiol to aromatizing enzyme protein expression；B is Bakuchiol with concentrationdependent manner inhibition WPMY-
The influence of the expression of aromatizing enzyme gene level in 1 cell；C is that Bakuchiol is inhibited in a manner of Time Dependent in WPMY-1 cell
Aromatizing enzyme is in protein expression；D be various concentration Bakuchiol to aromatizing enzyme in WPMY-1 cell in gene level
The inhibiting effect of expression.
It is the influence for the BPH rat prostate that Bakuchiol induces estrogen and androgen expressed by Fig. 5.Wherein, A is each group
Rat prostate aspect graph；B is influence of the Bakuchiol to BPH rat prostate index (PI)；C is that Bakuchiol is big to BPH
The influence diagram of mouse prostata tissue pathomorphism；D is the knot of PCNA, AR, aromatizing enzyme, ER β protein expression in prostata tissue
Fruit；E is the influence of expression and distribution of the α-SMA in prostata tissue；F is expression and distribution of the PCNA in prostata tissue
Influence.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
The influence that embodiment 1, mtt assay detection Bakuchiol are proliferated prostate epithelial cell and prostatic stroma cell
With 5 × 103The density in/hole is respectively by prostate epithelial cell (BPH-1) and prostatic stroma cell (WPMY-1)
It is laid in 96 orifice plates, it is 1nmol/L, 10nmol/L, 100nmol/L, 1 μ that 100 μ L concentration are separately added into after 37 DEG C of overnight incubations
The Bakuchiol (bakuchiol, ba) of mol/L, 10 μm of ol/L concentration, setting plus the control group of Bakuchiol, continue to cultivate
72h.Every hole adds 10 μ L MTT solution (5mg/ml is dissolved with PBS, pH=7.4), is incubated for 4 hours, inhales and abandons culture solution, every hole adds
100 μ l DMSO vibrate 10 minutes, melt crystal sufficiently.Each hole absorbance value is measured at 570nm wavelength with microplate reader,
Using blank group as standard, each acute drug group cell proliferation rate is calculated, and determine the best use dosage.Experimental result A as shown in figure 1
With in Fig. 1 shown in B, Bakuchiol can inhibit BPH-1 cell (figure in 1nmol/L-10 μm of ol/L concentration to some extent
A in 1), the proliferation (B in Fig. 1) of WPMY-1 cell.And inhibitory effect becomes apparent from low concentration.
Embodiment 2, PCR and western blot method detect the influence that Bakuchiol expresses PCNA respectively
With 2 × 106The density in/hole is respectively by prostate epithelial cell (BPH-1) and prostatic stroma cell (WPMY-1)
Be laid in 6 orifice plates, after 37 DEG C of overnight incubations be added 1.5mL concentration be 1nmol/L, 10nmol/L, 100nmol/L, 1 μm of ol/L,
The Bakuchiol of 10 μm of ol/L, setting is not plus the control group of Bakuchiol, culture extracted cell total rna after 24 hours, then instead
Transcription, PCR detect expression quantity of the PCNA with respect to GAPDH (primer sequence is referring to table 1).Culture 48 hours, is split with RIPA Buffer
It solves liquid and extracts total protein of cell, the albumen of different molecular weight is separated by electrophoresis, goes to pvdf membrane, primary antibody (antibody information is then added
Referring to table 2), 4 DEG C of overnight incubations wash film with TBST.Secondary antibody is incubated for 1 hour, and immune response and chemiluminescence, development, fixing are
It is stringent to be operated referring to kit by experimental procedure, detect influence of the Bakuchiol to PCNA protein expression.
The results show that Bakuchiol inhibits PCNA in BPH-1 cell to mend in the expression (C in Fig. 1) of albumen and gene level
Expression (in Fig. 1 D) of the PCNA in albumen and gene level in bone fat phenol inhibition WPMY-1 cell.
1 primer sequence of table
2 antibody information of table
The expression and activity of embodiment 3, Bakuchiol to ER and AR in prostate epithelial cell and prostatic stroma cell
Adjustment effect
(1) PCR and western blot method detects the influence that Bakuchiol expresses ER and AR respectively
The steroid hormone receptor that BPH-1 cell is mainly expressed has AR and ER β, and WPMY-1 cell mainly expresses AR and ER
α.Inventor detects influence of the Bakuchiol to AR and ER expression in both cells respectively.
With 2 × 106The density in/hole is respectively by prostate epithelial cell (BPH-1) and prostatic stroma cell (WPMY-1)
Be laid in 6 orifice plates, after 37 DEG C of overnight incubations be added 1.5mL concentration be 1nmol/L, 10nmol/L, 100nmol/L, 1 μm of ol/L,
The Bakuchiol of 10 μm of ol/L concentration, setting is not plus the blank group of Bakuchiol, culture extracted cell total rna after 24 hours,
Then reverse transcription, PCR detect in BPH-1 cell that AR and ER β is in the expression quantity of mRNA level in-site, AR and ER α exists in WPMY-1 cell
The expression quantity of mRNA level in-site (primer sequence is referring to table 1).Culture 48 hours extracts the total egg of cell with RIPA Buffer lysate
It is white, the albumen of different molecular weight is separated by electrophoresis, goes to pvdf membrane, primary antibody (antibody information is referring to table 2) then is added, 4 DEG C of incubations
Overnight, film is washed with TBST.Secondary antibody is incubated for 1 hour, and immune response and chemiluminescence, development, fixing are stringent referring to kit, is pressed
Experimental procedure operation detects expression quantity of the AR and ER β in the opposite GAPDH of protein level, WPMY-1 cell in BPH-1 cell respectively
Middle AR and ER α is in expression quantity of the protein level with respect to GAPDH.
The results show that expression no obvious effect (in Fig. 2 A) of the Bakuchiol to AR in BPH-1 cell, thin to WPMY-1
The expression of AR protein level has slight facilitation (B in Fig. 2) in born of the same parents, but does not obviously raise to the expression of mRNA level in-site.
In terms of ER, Bakuchiol can raise the expression (C in Fig. 2) of ER β on mRNA and protein level in BPH-1 cell, but to WPMY
The effect of ER α is unobvious (D in Fig. 2) in cell.
(2) influence of the detection Bakuchiol to LNCaP cell AR nuclear translocation
10%FBS RPMI 1640 cultivates LNCaP cell (a kind of prostate cancer cell line), is handled with containing 10% active carbon
The RPMI 1640 of serum is planted in LNCaP cell inoculation to 96 hole black ELISA Plates after plate cell adherent 24 hours.Setting is not
The control group of dosing, the positive controls of DHT and Bakuchiol experimental group, dosing cell kind plate 24 hours it is adherent after change liquid, point
The Bakuchiol that 100 μ L concentration are 10 μm of ol/L is not added, 100 μ L concentration are the DHT of 1 μm of ol/L.Pei Ji is discarded, is added more than 4%
200 hole μ L/ of polyformaldehyde is fixed.It inhales and abandons paraformaldehyde, wash cell with PBS.It is thin with the PBS permeabilization of the X-100 containing 0.5%Triton
Born of the same parents, 200 hole μ L/ 1%BSA are closed 60 minutes.Primary antibody is diluted with antibody diluent, 4 DEG C of refrigerators is placed in and is incubated overnight.It inhales and abandons one
It is anti-, cell is washed three times with PBST.With antibody diluent dilute secondary antibody and Hoechst 33342 (secondary antibody dilution ratio 1:1000,
33342 dilution ratio 1:10000 of Hoechst), room temperature is protected from light 2 hours.It inhales and abandons secondary antibody and dyestuff, HCS detects AR and Hoechst
33342 fluorescence intensities (antibody information is referring to table 2).As a result see E in Fig. 2.
E, which can be seen that Bakuchiol, from Fig. 2 can promote LNCaP cell AR to be indexed into nucleus from cytoplasm, and double clear
Testosterone is consistent.Embody the androgenic activity of Bakuchiol.
(3) concentration and time-dependent effect of the Bakuchiol to ER β expression and Active Regulation in BPH-1 cell
Based in Fig. 2 C Bakuchiol promote ER β expression as a result, inventor further studies its concentration and Time Dependent
Effect.With 2 × 106Prostate epithelial cell (BPH-1) is laid in 6 orifice plates by the density in/hole respectively, is divided after 37 DEG C of overnight incubations
Not Jia Ru 1.5mL concentration be 1nmol/L, 10nmol/L, 100nmol/L, 1 μm of ol/L, 10 μm of ol/L Bakuchiol, setting is not
Add the control group of Bakuchiol, culture extracted cell total rna after 24 hours, and then reverse transcription, PCR detects ER β in BPH-1 cell
In the expression quantity of mRNA level in-site (primer sequence is referring to table 1).
With 2 × 106Prostate epithelial cell (BPH-1) is laid in 6 orifice plates by the density in/hole respectively, 37 DEG C of overnight incubations
Afterwards be added 1.5mL concentration be 10 μm of ol/L Bakuchiol, setting plus Bakuchiol blank group, respectively culture 0 hour,
Extract cell total rna after 6 hours, 12 hours, 24 hours, then reverse transcription, PCR detect BPH-1 cell in ER β in mRNA level in-site
Expression quantity (primer sequence is referring to table 1).
With 2 × 106Prostate epithelial cell (BPH-1) is laid in 6 orifice plates by the density in/hole respectively, 37 DEG C of overnight incubations
It is separately added into the Bakuchiol that 1.5mL concentration is 10nmol/L, 100nmol/L, 1 μm of ol/L afterwards, setting is not plus Bakuchiol
Control group is cultivated 48 hours, extracts total protein of cell with RIPA Buffer lysate, the albumen of different molecular weight is separated by electrophoresis,
Pvdf membrane is gone to, primary antibody (antibody information is referring to table 2) then is added, 4 DEG C of overnight incubations wash film with TBST.It is small that secondary antibody is incubated for 1
When, immune response and chemiluminescence, development, fixing are operated by experimental procedure strictly referring to kit, detect BPH-1 cell
Middle ER β is in expression quantity of the protein level with respect to GAPDH.
The result shows that Bakuchiol inhibits ER β mRNA level in-site in BPH-1 cell in a manner of concentration dependant and Time Dependent
It expresses (B in A and Fig. 3 in Fig. 3), on protein level, 10nM to 1 μM of Bakuchiol can be obviously promoted expression (Fig. 3 of ER β
Middle C).
(4) detection of promoter activity
Whether inventor further analyzes Bakuchiol regulation active to estrogen response element in BPH-1 (ERE)
It is specifically beta mediated by ER.
(i) with the RPMI culture solution containing 1%FBS by BPH-1 cell, 90% density is inoculated with 24 orifice plates, after cell is adherent, presses
Liquid culture is changed for 24 hours after transfecting siER β or siER α, 6h respectively according to Lipofectamine2000 (invitrogen) specification, PCR
Detecting ER β or ER α in BPH-1, in the expression of mRNA level in-site, it is opposite that western blot method detects ER β or ER α in BPH-1
The expression of GAPDH.
The results show that siER β, siER alpha specific inhibit ER β of background level in BPH-1 cell, ER alpha expression (D in Fig. 3
With E in Fig. 3).
(ii) with the RPMI culture solution containing 1%FBS by BPH-1 cell, 90% density is inoculated with 24 orifice plates, after cell is adherent,
ERE-luceferase reporter plasmid (0.15 μ is transfected according to Lipofectamine2000 (invitrogen) specification
The hole g/), ER α/β (hole 0.075g/), be separately added into 500 μ L after siER α/β (hole 10pM/) and Renilla (0.075 hole μ g/) 6h
The estradiol that concentration is 10nmol/L, 100nmol/L, the Bakuchiol of 1 μm of ol/L and concentration are 10nmol/L, 100nmol/L
(E2) it is handled.Lysate is added in incubation afterwards for 24 hours, detects Firefly luciferase and Renillar respectively
Luciferase fluorescence values numerical value calculates the relative fluorescence under different condition.Whole measured values are at least repeated 3 times, statistics
Every hole data carries out data analysis.
The results show that individually E2 or 1 μm of ol/L Bakuchiol of addition can promote the activity (F in Fig. 3) of ERE.Inhibit ER α
Expression, E2 or 1 μm of ol/L Bakuchiol still have a facilitation to ERE transcriptional activity, while when the two is added, psoralea corylifolia
Phenol and E2 compete ER β, obviously lower (G in Fig. 3) when the transcriptional activity of ERE is handled compared to independent E2.In contrast, inhibit ER β
Expression, E2 can promote ERE transcriptional activity, but 1 μm of ol/L Bakuchiol loses the facilitation to ERE transcriptional activity,
When the two is added simultaneously, Bakuchiol does not compete ER α with E2, the indifference opposite sex change when transcriptional activity of ERE is handled compared to independent E2
Change (G in Fig. 3).
The influence of embodiment 4, Bakuchiol to Aromatase Expression in WPMY-1 cell
With 2 × 106WPMY-1 cell is laid in 6 orifice plates by the density in/hole, and 1.5mL concentration is added after 37 DEG C of overnight incubations
For the Bakuchiol of 10nmol/L, 100nmol/L, 1 μm of ol/L, 10 μm of ol/L, setting plus the control group of Bakuchiol, culture
48 hour, total protein of cell is extracted with RIPA Buffer lysate, the albumen of different molecular weight is separated by electrophoresis, goes to pvdf membrane,
Then primary antibody (antibody information is referring to table 2) is added, 4 DEG C of overnight incubations wash film with TBST.Secondary antibody be incubated for 1 hour, immune response and
Chemiluminescence, development, fixing are stringent to be operated referring to kit by experimental procedure, is detected aromatizing enzyme in WPMY-1 cell and is existed
Expression quantity of the protein level with respect to GAPDH.The results show that Bakuchiol has the work for inhibiting Aromatase Expression in WPMY-1 cell
With (A in Fig. 4), therefore further regulation and mechanism of the research Bakuchiol to Aromatase Expression.
With 2 × 106WPMY-1 cell is laid in 6 orifice plates by the density in/hole, is separately added into 1.5mL after 37 DEG C of overnight incubations
Concentration is the Bakuchiol of 10nmol/L, 100nmol/L, 1 μm of ol/L, 10 μm of ol/L, setting plus the control group of Bakuchiol,
Culture extracted cell total rna after 24 hours, then reverse transcription, and aromatizing enzyme is in mRNA level in-site in PCR detection WPMY-1 cell
Expression quantity (primer sequence is referring to table 1).Culture 48 hours extracts total protein of cell, electrophoretic separation with RIPA Buffer lysate
The albumen of different molecular weight goes to pvdf membrane, and primary antibody (antibody information is referring to table 2) then is added, and 4 DEG C of overnight incubations use TBST
Wash film.Secondary antibody is incubated for 1 hour, and immune response and chemiluminescence, development, fixing are stringent referring to kit, is grasped by experimental procedure
Make, aromatizing enzyme is in expression quantity of the protein level with respect to GAPDH in detection WPMY-1 cell.The results show that Bakuchiol is with dense
Spend the expression (D in B, Fig. 4 in Fig. 4) that dependence form inhibits aromatizing enzyme albumen and gene level.
With 2 × 106WPMY-1 cell is laid in 6 orifice plates by the density in/hole, and 1.5mL concentration is added after 37 DEG C of overnight incubations
For the Bakuchiol of 10 μm of ol/L, setting does not add the control group of Bakuchiol, respectively after culture 0 hour, 24 hours, 48 hours
With RIPA Buffer lysate extract total protein of cell, the albumen of different molecular weight is separated by electrophoresis, goes to pvdf membrane, then plus
Enter primary antibody (antibody information is referring to table 2), 4 DEG C of overnight incubations wash film with TBST.Secondary antibody is incubated for 1 hour, immune response and chemistry hair
Light, development, fixing are stringent to be operated referring to kit by experimental procedure, and aromatizing enzyme is in albumen water in detection WPMY-1 cell
The expression quantity of flat opposite GAPDH.The results show that Bakuchiol inhibits the expression of aromatizing enzyme protein level in the form of Time Dependent
(C in Fig. 4).
Embodiment 5, Bakuchiol inhibit the BPH rat process of estrogen and androgen induction
(1) foundation of BPH rat model
8 SPF grades of week old Wistar male rats, weight start after a week in 270 ± 20g, experimental animal adaptive feeding
Castration operation.6 experimental animals are randomly selected as Normal group, sham-operation are carried out to it, as sham-operation group
(shame).It randomly selects 12 rats and does castration operation, extract bilateral testes and its epididymis completely, and inject penicillin 1.2
Ten thousand units/only.
(2) the hyperplasia of prostate index of experiment with computing animal
12 are randomly divided into 2 groups, respectively model group, Bakuchiol group through the postoperative rat of castration, open within postoperative 21 days
Begin to be administered, 0.1mL/ days corn oil is injected intraperitoneally in sham-operation group, and 0.1mL estradiol/testosterone (E is injected intraperitoneally in sham-operation group2/ T,
1:100, E2 concentration are 100 μ g/ml) modeling, Bakuchiol group intraperitoneal injection 0.1ml E2(1:100, E2 concentration are 100 μ g/ to/T
) and Bakuchiol 5mg/kg ml.Successive administration 28 days, completion in 24 hours was drawn materials after the last administration.Take out prostata tissue, each group
Rat prostate aspect graph is referring to A in Fig. 5；It is dynamic for calculating each group experiment with the weight in wet base of electronic balance accurate weighing prostate
The prostate index of object, according to formula: weight in wet base/rat body weight × 100% of prostate calculates human prostate prostate index
(PI).It is compared as shown in A and B in Fig. 5 with sham-operation group, model group prostate volume increases, and PI index obviously increases；Administration
Prostate volume and PI are decreased obviously after Bakuchiol, close to sham-operation group；The jelly of two sides prostata tissue is cut in liquid nitrogen
In, western blot and PCR experiment for the later period；Prostate notopodium is put in 4% paraformaldehyde and is fixed, to paraffin
Embedding, for doing pathology piece and immunohistochemistry.
(3) influence of the Bakuchiol to BPH rat prostate tissue pathomorphism
The prostata tissue wax stone of paraffin embedding is switched to 4 μm/piece with slicer.Dimethylbenzene makes paraffin section de-waxing, warp
To washing after ethyl alcohol at different levels, haematoxylin dyeing 5min, a large amount of tap water are rinsed.Acidic alcohol breaks up 30 seconds.Tap water impregnates
15min.Set Yihong liquid dyeing 2min.Dehydrated, transparent, neutral resin sealing.Microscopic examination result is referring to C in Fig. 5 under microscope.Control
The expansion of group (sham-operation group) acinus is unobvious, and high column is presented in acinar epithelial cells more, and interstitial components are less, smooth muscle cell
Have no hyperplasia.Compared with the control group, interstitial components increased significantly model group, and acinus volume increases, and glandular epithelium is disorganized,
Connective tissue proliferation is more obvious, occurs stratified epithelium in acinus, and prompting a large amount of hyperplasia of galandular epithelium and connective tissue is BPH
One of main feature.Compared with model group, interstitial components are reduced Bakuchiol administration group, and glandular epithelium is disorganized
The phenomenon that obtain different degrees of alleviation, local acinus arrangement is more intensive, close to Normal group.
(4) influence of the Bakuchiol to PCNA, AR, aromatizing enzyme, ER β protein expression in BPH rat prostate tissue
It is removed from liquid nitrogen BPH rat prostate tissue sample in the present embodiment step (2), with 150 μ LRIPA of addition
Buffer lysate, extracts tissue total protein after ultrasonication, BCA method measures protein concentration, different molecular weight is separated by electrophoresis
Albumen goes to pvdf membrane, and primary antibody (antibody information is referring to table 2) then is added, and 4 DEG C of overnight incubations wash film with TBST.Secondary antibody is incubated for
1 hour, immune response and chemiluminescence, development, fixing were operated by experimental procedure strictly referring to kit, detect BPH rat
PCNA, AR, aromatizing enzyme, ER β are in expression quantity of the protein level with respect to GAPDH in prostata tissue.The results show that and model group
Compare, Bakuchiol is able to suppress the protein expression of PCNA, AR, aromatizing enzyme, and can promote the protein expression of ER β
(D in Fig. 5).
(5) in prostata tissue α-SMA and PCNA expression and distribution
α-SMA is the labelled protein of smooth muscle cell, and PCNA is the labelled protein of cell Proliferation.Further pass through immune group
The expression and distribution situation of both weave chemistry method detections.Electricity of the prostate section cut after paraffin embedding at 60 DEG C
It does and bakes 1 hour in hot insulating box, extremely washed after successively immersing dimethylbenzene dewaxing, ethyl alcohol at different levels, in 3% hydrogenperoxide steam generator
10min puts citric acid antigen and repairs 100 DEG C of reaction 10min in liquid, make its natural cooling 15 minutes, heats 100 DEG C again
10min closes 30min with 10% cow's serum.The primary antibody diluted (being shown in Table 3) is added dropwise to the surface of tissue, 4 DEG C were incubated for
Night.The secondary antibody (being shown in Table 4) diluted, 37 DEG C of incubation 30min are added dropwise.Dilution three is resisted and is added drop-wise to tissue (table 5), 37 DEG C incubate
Educate 20min.DAB colour developing, haematoxylin are redyed.By dehydration of alcohols at different levels, dimethylbenzene is transparent, neutral gum mounting.The results show that
The layer of smooth muscle cells thickness of the model group α-SMA positive staining of BPH obviously increases compared with sham-operation group, Bakuchiol group
Layer of smooth muscle cells thickness more significantly reduces (E in Fig. 5) compared with model group；The PCNA of model group is equal in epithelium and interstitial
It can be seen that expression, positive rate obviously raises compared with the control group；After Bakuchiol is administered, the positive rate of PCNA in interstitial and epithelium
It is substantially reduced (F in Fig. 5).
3 IHC primary antibody dilution ratio of table
4 IHC secondary antibody dilution ratio of table
The anti-dilution ratio of 5 IHC of table tri-
Prepared by the medicine for preventing and/or treating benign prostatic hyperplasis to Bakuchiol provided by the present invention above
Purposes in object is described in detail.Specific embodiment used herein carries out the principle of the present invention and embodiment
It illustrates, method and its central idea of the invention that the above embodiments are only used to help understand.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification also fall into the protection of the claims in the present invention.
Claims (7)
1. Bakuchiol is preparing the purposes in the drug for preventing and/or treating benign prostatic hyperplasis.
2. purposes as described in claim 1, which is characterized in that the Bakuchiol by up-regulation epithelial cell ER β expression and/
Or lower the proliferation that interstitial cell Aromatase Expression inhibits prostatic cell.
3. purposes as described in claim 1, which is characterized in that the drug gives the daily dosage of object in need to mend
Bone fat phenol is calculated as 0.01-1000mg/kg weight.
4. purposes as claimed in claim 3, which is characterized in that the drug gives the daily dosage of object in need to mend
Bone fat phenol is calculated as 0.1-100mg/kg weight.
5. purposes as claimed in claim 4, which is characterized in that the drug gives the daily dosage of object in need to mend
Bone fat phenol is calculated as 1-100mg/kg weight.
6. purposes as described in claim 1, which is characterized in that the Bakuchiol is provided in the form of Bakuchiol monomer
, or provided in the form of the plant extracts comprising Bakuchiol.
7. purposes as described in claim 1, which is characterized in that the Bakuchiol is mentioned with the psoralea corylifolia comprising Bakuchiol
The form of object is taken to provide.
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| CN1321483A (en) * | 2000-04-29 | 2001-11-14 | 湖北医科大学附属第一医院 | Chinese medicine-psoralen preparation for preventing and curing hyperplasia of prostate |
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| CN1321483A (en) * | 2000-04-29 | 2001-11-14 | 湖北医科大学附属第一医院 | Chinese medicine-psoralen preparation for preventing and curing hyperplasia of prostate |
Non-Patent Citations (3)
| Title |
|---|
| Phytoestrogens from Psoralea corylifolia reveal estrogen receptor-subtype selectivity;D. Xin等;《Phytomedicine》;20101231;第17卷;第126-131页，尤其是第128页左栏第6-7段，第127页左栏第6段，第128页左栏第3段 |
| 中药抑制良性前列腺增生作用机制研究进展;陶蕊等;《中国科技论文在线精品论文》;20141231;第7卷(第23期);第2307-2316页，尤其是第2308页第4-5段，第2309页第1段 |
| 补骨脂酚拮抗AR转录活性抑制雄激素诱导的前列腺癌细胞LNCaP的增殖;苗琳等;《天津中医药》;20130531;第30卷(第5期);第291-293页，尤其是第293页左栏第2段至右栏第1段 |
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