CN110327431A - Application and drug of the Germinatus Phragmitis extract in the drug of preparation treatment inflammation - Google Patents
Application and drug of the Germinatus Phragmitis extract in the drug of preparation treatment inflammation Download PDFInfo
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Abstract
The invention discloses a kind of application of Germinatus Phragmitis extract in the drug of preparation treatment inflammation, particularly, the application in the drug of preparation treatment vascular smooth muscle cells inflammation.The Germinatus Phragmitis extract can be also used for the drug that disease relevant to the inflammation is treated in preparation, and the disease includes hypertension, atherosclerosis, coronary heart disease, myocardial infarction, cerebral apoplexy etc..The present invention also provides a kind of for treating the drug of inflammation, and the drug includes Germinatus Phragmitis extract and pharmaceutically acceptable carrier or auxiliary material.Present invention effective component extracting from asparagus can be realized above-mentioned application, have highly important medicine and economics meaning.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, espespecially application and medicine of the Germinatus Phragmitis extract in the drug of preparation treatment inflammation
Object.
Background technique
Inflammation is body for stimulating a kind of defense reaction including injury, tissue damage and infectious agent, is
A kind of immune response of adaptability comprising chronic inflammation and acute inflammation.The oxidative stress and inflammation of body are mutually promoted, phase
Mutually inhibit, the two has inseparable inner link.The activation of inflammatory signals intermediate is to modify ROS/GSH by adjusting
For mercaptan (R-SH) group come what is realized, the generation of excessive active oxygen (ROS) destroys body redox equilibrium, and is recorded by consideration convey
Factor κ B (NF- κ B) is exaggerated inflammatory reaction, thus induction of pro-inflammatory cell factor such as TNF-α, IL-1, IL-6, cyclooxygenase 2
(COX2) and the transcription of nitric oxide synthase type (iNOS) etc., and then inflammation is aggravated.
NF- κ B, which is a kind of, to be a transcription factor egg with the albumen in conjunction with specific sequence in immunoglobulin kappa light chain
White family, including 5 subunits: Rel (cRel), p65 (RelA, NF- κ B3), RelB and p50 (NF- κ B1), p52 (NF- κ
B2).NF- κ B fine-tunes the expression of different genes by way of different dimers, the most common NF- κ B dimerization
Body is the heterodimer of p65 and p50 composition.NF- κ B signal access is constituted by the cell signal of core of NF- kB protein.It grinds
Study carefully the adjustment process for showing that NF- κ B participates in inflammation, therefore amplification approach-NF- κ B signal channel by inhibiting inflammation, thus
Inhibit anticusp inflammation factor transcribes the therapeutic effect, it can be achieved that inflammation.NF- κ B related inhibitors are developed into human treatment
The important directions of inflammation and tumour.The mankind have found kind of the relevant inhibitor of NF- κ B more than 170 so far.NF- κ B signal channel
The effect for amplifying the reaction is played in the inflammatory reaction caused by a variety of diseases, such as: hypertension, atherosclerosis, coronary disease
The cardiovascular diseases such as disease, myocardial infarction, cerebral apoplexy.Inflammatory reaction relevant to hypertension is illustrated by taking hypertension as an example below
The effect in middle NF- κ B signal channel:
In recent years with living-pattern preservation, the disease incidence of cardiovascular disease especially hypertension is risen year by year.High blood
It is also the common cause for causing other cardiovascular disease incidence rates and the death rate to increase that the disease incidence of pressure, which rises,.Diuretics, blood vessel
Anatransferase inhibitors (ACEI), calcium ion antagonist (CCB), AT1R antagonist (ARB) etc. are that clinical application is most
Five big hypotensors, it is rapid-action, act on it is clearly strong, take and carry it is convenient.But its Amplitude of Hypotensive is larger, and decompression is not
Steadily, blood pressure is easy rebound after drug withdrawal, cannot maintain very well, is easy to cause cranial vascular disease.
The reason of essential hypertension is mainly characterized by peripheral vascular resistance increase, and peripheral vascular resistance increases includes two
A aspect.First be hypertensive patient inner skin cell function it is impaired because endothelial cell is vital antiotasis tune
Device is saved, endothelial cell is impaired to make the reduction of vasodilation degree, and blood pressure is caused to increase；On the other hand, it is in hypertension sufferer body
Proinflammatory and pro-thrombotic state, the state can allow the tension of blood vessel to become larger, blood pressure caused to increase.It follows that reducing blood
The local inflammation degree of tubing is the important decompression mode of pathology, physiology.Renin-angiotensin system (RAS) is people
Body adjusts the important endocrine system of human blood-pressure, and Angiotensin II (Ang II) is the strongest contracting blood of RAS known activity
Pipe biologically active peptide, -1 receptor of Angiotensin II (AT1R) are the main effects devices of RAS, can mediate the physiology of Ang II
Effect, thus oxidative stress, inflammation, cell Proliferation and the migration etc. of induction of vascular smooth muscle cell (VSMCs).Ang II is current
Have become one of the main stimulus for inducing various cardiovascular disease pathologic basis.It can be seen that inhibiting Ang II induction
VSMCs oxidative stress, inflammation and excess cell proliferation and migration can be used as treatment cardiovascular disease, especially a hypertension
Important target spot.
Therefore, finding can inhibit the drug in NF- κ B signal channel, and for inhibiting inflammation, and treatment is related to inflammatory reaction
Disease have a very important significance.
Summary of the invention
The present inventor is in long-term R&D and production, it has unexpectedly been found that Germinatus Phragmitis extract can inhibit NF- κ B signal
Channel to reduce the transcription of pro-inflammatory cytokine such as COX2 and iNOS etc., and then mitigates inflammation, plays antiinflammation.Cause
This can be used for preparing the drug for the treatment of inflammation, especially can be reduced the expression of AT1R, can be used for preparing the medicine for the treatment of VSMCs inflammation
Object.
Therefore, the first aspect of the invention provides application of the Germinatus Phragmitis extract in the drug of preparation treatment inflammation.
Particularly, application of the Germinatus Phragmitis extract in the drug of preparation treatment VSMCs inflammation is provided.
The Germinatus Phragmitis extract is to extract to obtain to asparagus using water-miscible organic solvent by solvent extraction method.
It should be understood readily by those skilled in this art, extracting water-miscible organic solvent used in asparagus is mentioning for this field routine
Take solvent, such as water-ethanol mixed liquor, petroleum ether, ether, acetone, ethyl alcohol, methanol that water and ethyl alcohol are mixed with arbitrary proportion.
It should be understood readily by those skilled in this art, the solvent extraction method includes infusion method, percolation, decocting method, adds
Heat reflow method, ultrasonic oscillation extraction method, microwave loss mechanisms etc..
Part preferred embodiment according to the present invention, the Germinatus Phragmitis extract are to extract asparagus institute by water-ethanol mixed liquor
The asparagus ethanol extract obtained.
According to the present invention, the Germinatus Phragmitis extract generates inhibiting effect to AT1R protein expression and NF- κ B access.
According to the present invention, the Germinatus Phragmitis extract inhibits the reduction of I κ B α, inhibits the phosphorylation of p65, and reduces proinflammatory thin
The generation of intracellular cytokine iNOS and COX2.To which the Germinatus Phragmitis extract can be used for inhibiting NF- κ B signal channel.
According to the present invention, the preparation method of the asparagus ethanol extract includes the following steps:
(1), suitable water-ethanol solution is added after 0~6 DEG C of freeze grinding and extracts for asparagus, then controls 40
~50 DEG C of 2~4h of extracting at constant temperature；
(2), step (1) repeats 2~4 times, merges alcohol extract, is concentrated, after ethyl alcohol recycling, vacuum freeze drying is extracted
Liquid obtains powder, i.e. asparagus ethanol extract.
Preferably, in the step (2), alcohol extract is concentrated at 45 DEG C using Rotary Evaporators.
It should be understood readily by those skilled in this art, the terminal of the ethyl alcohol recycling is the taste that almost can't smell alcohol, or
Person's Rotary Evaporators drip slower.
Preferably, in the step (2), it is lower than -50 DEG C in temperature, vacuum refrigeration is dry under conditions of vacuum degree is less than 15Pa
It is dry.
Preferably, water-ethanol mixed liquor is added after 4 DEG C of freeze grindings and extracts for asparagus, what the step (1) used
In water-ethanol mixed liquor, the volume fraction of ethyl alcohol is 75%, and Extracting temperature is 45 DEG C, is repeated 3 times.
Under the conditions of said extracted, more active materials can be obtained, to have more preferable effect.
According to the present invention, the mass volume ratio of the asparagus and water-ethanol mixed liquor is 1:(50~100) (g/mL)
According to the present invention, raw material used in the Germinatus Phragmitis extract is green asparagus.
According to the present invention, the second aspect of the invention provides the Germinatus Phragmitis extract in preparation treatment and the inflammation
Application in the drug of relevant disease.The disease illustratively, but can be hypertension, Atherosclerosis without limitation
Change, coronary heart disease, myocardial infarction and cerebral apoplexy.
The third aspect of the invention is to provide a kind of for treating the drug of diseases associated with inflammation, and the drug includes asparagus
Extract and pharmaceutically acceptable carrier or auxiliary material.
In order to make drug discharge active component rapidly, continuously and in a very long time, drug of the invention can be with
According to those disclosed conventional method manufactures in the art.
The administration route of drug of the invention is oral, nasal inhalation, parenteral administration.
The preparation of drug can be powder, particle, tablet, emulsion, syrup, aerosol, soft capsule, hard capsule, sterilizing note
Penetrate agent and sterilized powder etc..
More preferred, the preparation of the drug is oral preparation.Preferably tablet or capsule.
It should be understood readily by those skilled in this art, the drug can individually be taken, it can also mix and take with other drugs；Institute
State drug can for effective component only containing the single preparations of ephedrine of Germinatus Phragmitis extract, or effective component be Germinatus Phragmitis extract and
The compound preparation of other drugs, for example compound preparation is made together with certain drugs for hypertension.
Herein, term " pharmaceutically acceptable " refers to that the compound is physiologically acceptable when compound is to human administration
, it is not to be included in comprising the allergic reactions such as gastrointestinal disturbance, dizziness or these similar anaphylactoid systemic anaphylaxis
Interior.
In the present invention, " pharmaceutically acceptable carrier " includes but is not limited to: adhesive (such as microcrystalline cellulose, alginic acid
Salt, gelatin and polyvinylpyrrolidone), filler (such as starch, sucrose, glucose and anhydrous lactic acid), disintegrating agent (as be crosslinked
PVP, crosslinked carboxymethyl fecula sodium, croscarmellose sodium and low-substituted hydroxypropyl cellulose), lubricant (stearic acid
Magnesium, aluminum stearate, talcum, polyethylene glycol, sodium benzoate), wetting agent (such as glycerol), surfactant (such as hexadecanol) and
Sorbefacient, corrigent, sweetener, diluent, coating agent etc..
According to the present invention, in the drug of effective dose, the concentration of the Germinatus Phragmitis extract is 50~200 μ g/mL, excellent
It is selected as 100 μ g/mL.
Compared with prior art, the positive effect of the present invention is that:
(1), Germinatus Phragmitis extract of the invention can inhibit NF- κ B signal channel, reduce proinflammatory cytokine COX2 and iNOS
Transcription, and then mitigate inflammation, play antiinflammation, can be used for preparing treatment inflammation drug.
(2), Germinatus Phragmitis extract inhibits NF- κ B access and proinflammatory cytokine iNOS, COX2 by the expression of reduction AT1R
Expression, play antiinflammation, can inhibit VSMCs inflammatory reaction caused by Ang II, can be used for preparing treatment VSMCs inflammation
Drug.
(3), Germinatus Phragmitis extract of the invention can be used for preparing treatment disease relevant to inflammation (such as hypertension, artery
Atherosis, coronary heart disease, myocardial infarction and cerebral apoplexy) drug, especially treat hypertension drug.
Detailed description of the invention
Fig. 1 is the influence that EEA causes AT1R to express Ang II.
Fig. 2 is the influence that EEA causes I κ B alpha expression to Ang II.
Fig. 3 is the influence that EEA causes p-p65 to express Ang II.
Fig. 4 is the influence that EEA causes COX2 to express Ang II.
Fig. 5 is the influence that EEA causes iNOS to express AngII.
In figure: in abscissa, control indicates that control group, EEA indicate EEA processing group, and AngII indicates Ang II processing group,
EEA+AngII indicates EEA and AngII processing group.
In ordinate, respectively with the protein expression of AT1R, I κ B α, p-p65, COX2 and iNOS in control group VSMCs cell
For 1 unit, the protein expression of processing group EEA, AngII and EEA+AngII are using the expression multiple relative to processing group come table
Show.
Specific embodiment
In following embodiment:
The preparation of embodiment 1, asparagus ethanol extract
The preparation method of asparagus ethanol extract includes the following steps:
(1), then 1000mL the water-ethanol mixed liquor (body of ethyl alcohol is added through 4 DEG C of freeze grindings at homogenate in 10g asparagus
Fraction is 75%), to control in 45 DEG C of extracting at constant temperature 3h；
(2), step (1) is repeated 3 times, and merges alcohol extract, and concentrated extracting solution at 45 DEG C of Rotary Evaporators is recycled to ethyl alcohol
Afterwards, it is lower than -50 DEG C in temperature, vacuum degree is less than vacuum freeze drying extracting solution under conditions of 15Pa, obtains powder, i.e. asparagus second
Alcohol extracting thing.
The preparation of embodiment 2, asparagus ethanol extract
The preparation method of asparagus ethanol extract includes the following steps:
(1), then 1000m the water-ethanol mixed liquor (body of ethyl alcohol is added through 0 DEG C of freeze grinding at homogenate in 10g asparagus
Fraction is 50%), to control in 45 DEG C of extracting at constant temperature 2h；
(2), step (1) is repeated 4 times, and merges alcohol extract, and concentrated extracting solution at 45 DEG C of Rotary Evaporators is recycled to ethyl alcohol
Afterwards, it is lower than -50 DEG C in temperature, vacuum degree is less than vacuum freeze drying extracting solution under conditions of 15Pa, obtains powder, i.e. asparagus second
Alcohol extracting thing.
The preparation of embodiment 3, asparagus ethanol extract
The preparation method of asparagus ethanol extract includes the following steps:
(1), then 500mL the water-ethanol mixed liquor (body of ethyl alcohol is added through 6 DEG C of freeze grindings at homogenate in 10g asparagus
Fraction is 90%), to control in 50 DEG C of extracting at constant temperature 2h；
(2), step (1) is repeated 2 times, and merges alcohol extract, and concentrated extracting solution at 45 DEG C of Rotary Evaporators is recycled to ethyl alcohol
Afterwards, it is lower than -50 DEG C in temperature, vacuum degree is less than vacuum freeze drying extracting solution under conditions of 15Pa, obtains powder, i.e. asparagus second
Alcohol extracting thing.
Embodiment 4, cell culture
Rat aorta VSMC cell line A7r5 is purchased from ATCC (cat#ATCC CRL-1444, Virginia Ma Nasa
This).
Rat aorta VSMC cell line A7r5 cell addition 10%FBS and 1% antibiotic (Pen .- Strep and
Gentamicin) DMEM culture medium in grow, until the convergence degree of cell reaches 80%, A7r5 cell can be used for subsequent reality at this time
Test analysis.
It is all test the A7r5 cell that uses for the 4th generation and the 11st instead of between cell.
Implement 5: western blot test Western Blot
Processing group (one), EEA and AngII processing group (EEA+Ang II)
(1), A7r5 fused cell is placed in tranquillization culture medium (+1% antibiotic of DMEM+1% fetal calf serum), then plus
Enter appropriate EEA (the final concentration of 100.0 μ g/mL for making EEA in system) processing 1h, adding appropriate Ang II (makes Ang in system
Final concentration of 1.0 μm of ol/L of II) processing 23h, then the Laemmle buffer by A7r5 cell in 50 μ L heat (contains
The Triton-X-100 of the DTT (reducing agent) and 0.2% (v/v) of 50mmol/L) in cracking, then scrape cell with cell scraper,
Cell pyrolysis liquid is collected to get Western-blot sample.
(2), cell cracking is run in 9% (v/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Liquid then in transferring film to nitrocellulose membrane, carries out immunoblotting reaction with COX2, iNOS, I κ B α, AT1R antibody respectively, makes
Use α-tubulin as internal reference.
Phospho-p65 (p-p65) antibody is normalized to total p65.Final concentration of 0.4 μ of α-tubulin antibody in system
G/mL, the final concentration of 1.0 μ g/mL of other antibody.Use goat antirabbit IRDye 680RD and donkey anti-mouse 800CW as two
It is anti-.Protein band is detected using Licor Odyssey biometric imager, and is carried out using Image Studio Lite 5.2
Density measurement.
Processing group (two), control group (control)
Basic step is identical as processing group (one), and difference is, A7r5 cell does not use at EEA and AngII in step (1)
Reason, obtained Western-blot sample loading on every clotting glue according to the method for step (2), and carry out subsequent detection point
Analysis.
Processing group (three), EEA processing group (EEA)
Basic step is identical as processing group (one), and difference is, handles after EEA processing without using Ang II in step (1).
Processing group (four), AngII processing group (Ang II)
Basic step is identical as processing group (one), and difference is, handles in step (1) without using EEA, only uses Ang II couple
A7r5 cell is handled.
Testing result:
(1), influence of the EEA to AT1R expression in the VSMCs of Ang II induction processing.
Ang II can cause increment, oxidative stress and the inflammation of VSMCs in conjunction with receptor AT1R.Experiment has detected EEA pairs
The influence of AT1R expression, as a result as shown in Figure 1.
As seen from Figure 1, Ang II processing makes the expression of AT1R in VSMCs significantly raised (p < 0.01), and EEA is handled
It significantly reduces it and expresses (p < 0.01), and the expression of AT1R is allowed to be reduced to foundation level.This shows that EEA can lower AT1R
Expression, EEA may have antiproliferative, anti-oxidant and anti-inflammatory activity in VSMCs.
(2), EEA inhibits inflammation caused by Ang II by NF- κ B signal access
COX2 and iNOS is proinflammatory cytokines molecule relevant to NF- κ B signal access.Experiment has further inquired into EEA pairs
The influence that NF- κ B signal access mainly forms, as a result as shown in Figures 2 and 3.
As shown in Figure 2, the experimental results showed that EEA can alleviate the reduction of IkBa content caused by Ang II, while such as Fig. 3 institute
Show, the phosphorylation level of p65 can be reduced.Because the reduction of IkBa content and the phosphorylation of p65 are the passes of NF- κ B signal access
Key step, therefore this is the result shows that the conduction of NF- κ B signal access is weakened by EEA.
(3), EEA has downward effect to VSMCs the proinflammatory cytokine iNOS and COX2 of Ang II induction processing
By detecting the expression of intracellular proinflammatory cytokines molecule COX2 and iNOS, EEA is had studied to inflammation caused by AngII
Influence, as a result as shown in Figure 4 and Figure 5.
The expression of COX2 and iNOS is all obviously increased after Ang II processing it can be seen from Fig. 4 and Fig. 5, and EEA processing can be with
Inhibit Ang II to act on the up-regulation of above two pro-inflammatory molecular, illustrates that EEA has certain anti-inflammatory activity.
Therapeutic effect of the embodiment 6:EEA to hypertensive patient
84 volunteers (42 males, women 42,35-67 years old age).Body mass index (BMI) model of these volunteers
It encloses for 20-30kg/m2.Wherein 51 volunteers are diagnosed with metabolic syndrome or according to the diagnosis mark of fat research association
Standard is with the syndrome before metabolism.Volunteer does not have stomach medical history in the recent period, is not pregnant, without major disease, operation or serious
Diet diversity, and currently without using any drug or nutritional supplement.
84 volunteers are randomly divided into three groups (every group of subject quantity n=28), first group of subject takes daily
With 1.6g embodiment 1 prepare EEA, second group of subject take daily 1.6g embodiment 2 preparation EEA, third group by
Examination person takes the EEA of the preparation of 1.6g embodiment 3 daily.
It takes 10 weeks, measured systolic pressure and diastolic pressure index at 0,5,10 week respectively.The systolic pressure of statistical measurement and relax
Pressure data are opened, as shown in table 1.
1 subject of table measured systolic pressure and diastolic blood pressure data at 0,5,10 week
Table 1 statistics indicate that, using asparagus ethanol extract of the invention, the blood pressure of hypertensive patient can be significantly reduced,
Has the function of good treatment hypertension, therefore asparagus ethanol extract of the invention can be used for preparing the medicine for the treatment of hypertension
Object.
Claims (10)
1. application of the Germinatus Phragmitis extract in the drug of preparation treatment inflammation.
2. application according to claim 1, which is characterized in that the inflammation is VSMCs inflammation.
3. application according to claim 1, which is characterized in that the Germinatus Phragmitis extract is mentioned by water-miscible organic solvent
It takes obtained by asparagus.
4. application according to claim 3, which is characterized in that the water-miscible organic solvent is water-ethanol mixed liquor, is mentioned
Obtain asparagus ethanol extract.
5. application according to claim 4, which is characterized in that the preparation method of the asparagus ethanol extract includes as follows
Step:
(1), then suitable water-ethanol mixed liquor is added through 0~6 DEG C of freeze grinding at homogenate in asparagus, controls at 40~50 DEG C
2~4h of extracting at constant temperature；
(2), it repeats step (1) 2~4 time, merges alcohol extract, concentration, after ethyl alcohol recycling, vacuum freeze drying extracting solution is obtained
To powder, i.e. asparagus ethanol extract.
6. application according to claim 5, which is characterized in that in the water-ethanol mixed liquor of the step (1), second
The volume fraction of alcohol is 75%, and Extracting temperature is 45 DEG C, repeats to extract 3 times.
7. application according to claim 5, which is characterized in that in the step (1), the asparagus and water-ethanol mixing
The mass volume ratio of liquid is 1:(50~100) (g/mL).
8. application according to claim 5, which is characterized in that in the step (2), be lower than -50 DEG C in temperature, vacuum degree
Less than vacuum freeze drying under conditions of 15 Pa.
9. application of the Germinatus Phragmitis extract in the drug that disease relevant to inflammation is treated in preparation, which is characterized in that the disease
Including hypertension, atherosclerosis, coronary heart disease, myocardial infarction and cerebral apoplexy.
10. a kind of for treating the drug of inflammation, which is characterized in that the drug includes Germinatus Phragmitis extract, and pharmaceutically may be used
The carrier or auxiliary material of receiving.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111789803A (en) * | 2020-08-28 | 2020-10-20 | 江南大学 | Method for improving tyrosinase inhibition effect of asparagus polyphenol through hydrothermal treatment |
| CN114381421A (en) * | 2022-02-09 | 2022-04-22 | 河北医科大学 | Lysozyme-induced mouse vascular smooth muscle cell inflammation model and establishment method and application thereof |
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| CN114381421A (en) * | 2022-02-09 | 2022-04-22 | 河北医科大学 | Lysozyme-induced mouse vascular smooth muscle cell inflammation model and establishment method and application thereof |
| CN114381421B (en) * | 2022-02-09 | 2023-08-15 | 河北医科大学 | Lysozyme-induced mouse vascular smooth muscle cell inflammation model and establishment method and application thereof |
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