CN107898774A - Isoliquiritigenin liposome is preparing the urgency for the treatment of adriamycin induction（Slowly）Application in property myocardium toxicity medicine - Google Patents
Isoliquiritigenin liposome is preparing the urgency for the treatment of adriamycin induction（Slowly）Application in property myocardium toxicity medicine Download PDFInfo
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Abstract
The invention discloses isoliquiritigenin liposome to prepare the urgency for the treatment of adriamycin induction（Slowly）Application in property myocardium toxicity medicine.After suitable cholesterol, lecithin and isoliquiritigenin are placed in mixing by the present invention, chloroform methanol mixed liquor is added, uniform lipid dry film is prepared using rotating thin film evaporation.Adriamycin is injected after pre-processing C57BL/6 mouse by isoliquiritigenin liposome, myocardial enzymes are horizontal to be reduced；Mouse weight reduces trend and slows down, and verification isoliquiritigenin can weaken myocardium toxicity caused by adriamycin, to human normal cell's safety, low toxicity.
Description
Technical field
The invention belongs to field of Chinese herbal, and in particular to a kind of isoliquiritigenin liposome is weakening the cardiac muscle of adriamycin induction
New opplication in toxicity.
Background technology
Tumour and heart disease are two Etiologicals for now resulting in human death, and when both appear in one at the same time
When with patient, the existence and prognosis of patient will be seriously affected.Unfortunately, with the raising of cancer morbidity and the death rate,
Also it is being stepped up with the relevant cardiac toxic for the treatment of of cancer.Cardiotoxity seriously affects the life quality of tumor patient,
Importantly, the development of toxicity may cause the adjustment of antineoplaston scheme or even stop, the existence of patient is influenced.It is anti-swollen
The cardiac toxic of tumor medicine is divided into 2 two types of type 1 and type according to the effect difference produced to cardiac muscle cell：type 1
Type (such as anthracycline causes) myocardium toxicity refers to can be with the necrosis of inducing cardiomyocytes or apoptosis, and this type is irreversible；
2 types of type (such as Herceptin causes) myocardium toxicity refers to that dysfunction by causing cardiac muscle cell rather than induction are thin
Born of the same parents are dead, so this type is reversible.Cardiac toxic performance is complicated various caused by antitumor drug, and common performance includes
Arrhythmia cordis, myocardial ischemia, coronary artery disease, hypertension and myocardial dysfunction.Common causes the anti-swollen of cardiac toxic
Tumor medicine mainly has two major classes：One kind is chemotherapeutics, including anthracycline (Doxorubicin, epirubicin, daunorubicin etc.), alkane
Agent (endoxan, ifosfamide etc.), anti-cell micro-pipe agent (taxol), anti-metabolism (fluorouracil) etc., it is another kind of
It is targeted drug, such as Herceptin and Lapatinib.
Adriamycin belongs to anthracene nucleus medicament, is widely used in the treatment of sarcoma, lymthoma, leukaemia and breast cancer, is mesh
Before cause the common chemotherapeutics of cardiac toxic.Cardiac toxic caused by anthracene nucleus medicament, clinical manifestation are generally divided into 3 classes：①
Acute and subacute cardiac toxic, it is more rare, during administration or of short duration electrocardiogram can occur in a few hours after administration and change
Become, disappear after drug withdrawal, show as arrhythmia cordis (supraventricular tachycardia, room property ectopic cardiac rhythm), myocardial infarction, heart failure more
And pericarditis-myocarditis syndrome, ECG change mainly include change, QRS wave low-voltage and the QT interval prolongations of ST-T
Deng.2. chronic cardiac toxicity, occurs generally after end of chemotherapy in some months to 1 year, clinical relatively conventional, a left side is mainly shown as
Ventricular dysfunction, the death rate are higher.3. Delayed onset cardiac toxic, occurs, is mainly shown as several years more after end of chemotherapy
Invisible ventricular dysfunction, congestive heart failure and arrhythmia cordis etc..Chronic and Delayed onset cardiac toxic mainly and Ah
The dosage of mycin accumulation is related.To the mechanism of adriamycin cardiotoxicity of medicine, it is now recognized that main include embedded nuclear dna suppression
Protein synthesis, produce active oxygen radical, suppress type Ⅱ topoisomerase and influence DNA and repair and then cause that cardiac muscle cell's is dead
Die, nearest research finds that 2 β of topoisomerase (Top2 β) is the main molecules target spot of cardiotoxicity induced by anthracyclines effect.
Isoliquiritigenin belongs to licoflavone class medicine, main pharmacological action have antiulcer, anti-inflammatory, antibacterial, anti parasitic,
Antitumor, anti-oxidant and estrogen-like action etc..HAX-1 (HCLS1associated X-1) is one newly discovered
(1997 find) anti-apoptotic proteins, because containing the anti-apoptotic domain similar to mitochondria anti-apoptotic proteins Bcl-2, therefore by
Think to participate in the regulation and control of Apoptosis.One in vitro study prompting most earlier than 2006：Mistakes of the HAX-1 in cardiac muscle cell
Degree expression can obviously reduce the cardiac muscle cell apoptosis that anoxia _ reoxygenation is induced, this protective effect can by HAX-1 with
Caspase 9 be combined with each other and suppresses its activity to confirm.HAX-1 has been reported in the work that anti-apoptotic is played in multiple tissues
With, but whether it participates in adjusting cardiac muscle cell apoptosis process and studies seldom.
Therefore, for tumor patient, when carrying out antineoplaston with adriamycin, it is necessary to use myocardial protective agent.
Good myocardial protective agent should be in the case where not influencing adriamycin antineoplastic action and cardiac muscle cell can be protected without prejudice.
The content of the invention
In order to can induce cardiac muscle cell apoptosis, necrosis, the heart when solving and carrying out antineoplaston with adriamycin in the prior art
Myofibrosis increases, and myocardial contractive power reduces, and the problem of ultimately result in Ventricular Remodeling and congestive heart failure, the present invention carries
For application of the isoliquiritigenin liposome in urgency (slow) the property myocardium toxicity medicine for preparing treatment adriamycin induction, isoliquiritigenin fat
Plastid property is stablized, and into after in vivo, degradation cycle is longer, lasting medicine, has preferable biocompatibility, and have been reported
Isoliquiritigenin can suppress the growth of tumour.Therefore, isoliquiritigenin liposome is high to reducing Cardiotoxicity caused by adriamycin
Effect low toxicity.
In order to realize the above object technical scheme is as follows：
The present invention provides isoliquiritigenin liposome in urgency (slow) the property myocardium toxicity medicine for preparing treatment adriamycin induction
Application.
Preferably, the preparation method of the isoliquiritigenin liposome described in above application is as follows：
Weigh cholesterol, lecithin and purity to be placed in container for 98% isoliquiritigenin, add chloroform-methanol mixed liquor,
Chloroform and methanol volume ratio=2 in the chloroform-methanol mixed liquor:1, revolved using rotating thin film evaporation in 50-60 DEG C of condition
It is 0.1-0.2mm lipid dry films that transformation of ownership film 20-50min, which obtains uniform thickness, dries up residual solvent with nitrogen, adds PH=
7 phosphate buffered saline solution, makes lipid film fully be swollen aquation, and ultrasound 5min to obtain the final product, surpasses under conditions of ultrasonic power 400W
In sound according to ultrasound 5 seconds, stop 5 seconds frequency carry out；The cholesterol and lecithin mass ratio are cholesterol：Lecithin=1:5；
The isoliquiritigenin and lecithin molar ratio are isoliquiritigenin：Lecithin=1:10.
The present invention proves that isoliquiritigenin liposome weakens the effect of myocardial damage by following experiment：
1. injecting adriamycin after isoliquiritigenin liposome pretreatment C57BL/6 mouse, myocardial enzymes are horizontal to be reduced, anti-apoptotic
Albumen HAX-1 levels are gradually increased with the concentration increase of the isoliquiritigenin of pretreatment；2. the cardiac muscle cell of experimental group 1,2
After H9C2 adds 50 μM of isoliquiritigenin processing 24h, it is separately added into the culture medium of 1 μM of adriamycin and cultivates 24h, 2 are tested after 24h
Group adds a certain amount of HAX-1CRISPR gene knockouts plasmid processing 24h, the myocardium enzyme of the cardiac muscle cell after adriamycin processing
Level is higher than isoliquiritigenin pretreated group, and after the processing of HAX-1CRISPR gene knockouts plasmid is added, myocardium enzyme level is higher than
Isoliquiritigenin pretreated group, it is above-mentioned test result indicates that, isoliquiritigenin liposome can be thin by adjusting HAX-1 expression inhibitings cardiac muscle
The damage of born of the same parents is so as to have the function that to weaken Adriamycin cardiotoxicity；3. after isoliquiritigenin liposome pretreatment C57BL/6 mouse
Adriamycin is injected, mouse weight reduces trend and slows down.
The present invention has the following advantages：
1) present invention prepares that isoliquiritigenin liposome technical maturity is reliable, and raw material sources are extensive and superior in quality.
2) the existing medicine for reducing heart failure can cause bone marrow suppression, Liver and kidney function exception etc., while the medicine will not drop
The anticancer drug effect of low anthracene nucleus medicament.Isoliquiritigenin liposome property is stablized, and has preferable biocompatibility, and have been reported
Isoliquiritigenin can suppress the growth of tumour.Therefore, isoliquiritigenin liposome is high to reducing Cardiotoxicity caused by adriamycin
Effect low toxicity.
3) isoliquiritigenin liposome play reduce myocardium toxicity while, can also by righting reinforcing, it is clearing heat and detoxicating,
The effects such as resolving sputum promoting blood circulation improve the immune function of body.
4) after isoliquiritigenin liposome property stabilization enters in vivo, degradation cycle is longer, lasting medicine.
5) using isoliquiritigenin liposome as medicine, the Immunogenicity that connection aglucon can be avoided to bring, while significantly
Degree reduces pharmacy cost.
6) present invention uses conventional administration formulation, technology maturation, and simple production process is easy.
7) present invention uses conventional administration mode, convenient drug administration, no pain.
Brief description of the drawings
Fig. 1 isoliquiritigenin liposomes, which slow down mouse weight, reduces trend；
It is horizontal that Fig. 2 isoliquiritigenin liposomes reduce myocardial enzymes；
Influence of Fig. 3 isoliquiritigenin liposomes to HAX-1 protein expressions, wherein A measure HAX-1 for Western blot
Protein expression, B measure protein density for gel imaging analysis software；
The influence that Fig. 4 isoliquiritigenin liposomes express HAX-1 protein mRNAs；
Fig. 5 isoliquiritigenin liposomes adjust myocardial enzymes level by HAX-1 albumen；
Embodiment
By combination attached drawing described further below it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
Isoliquiritigenin used in following embodiments, purity 98%, Chengdu Man Site bio tech ltd, lot number：
120928。
【Embodiment 1】Isoliquiritigenin liposome prepares and characterization detection
(1) prepared by isoliquiritigenin liposome
1. preparation method：
Weigh cholesterol, lecithin and isoliquiritigenin to be placed in container, add chloroform-methanol mixed liquor, the chloroform-first
Chloroform and methanol volume ratio=2 in alcohol mixed liquor:1, the rotating thin film evaporation rotation film 20- under the conditions of 50-60 DEG C
50min, is made uniform lipid dry film；The lipid thickness of dry film is 0.1-0.2mm；Residual solvent is dried up with nitrogen, then is added
Enter the phosphate buffered saline solution of PH=7, lipid film is fully swollen aquation, the ultrasound 5min under conditions of ultrasonic power 400W,
To obtain the final product；It is described ultrasound according to ultrasound 5 seconds, stop 5 seconds frequency carry out；The cholesterol and lecithin mass ratio are cholesterol：
Lecithin=1:5；The isoliquiritigenin and lecithin molar ratio are isoliquiritigenin：Lecithin=1:10.
The particle diameter of the above-mentioned isoliquiritigenin liposome being prepared is 224.8 ± 4.5nm, its envelop rate ＞ 55%.
2. Formulation and optimization
Reference literature and single factor experiment prerun analysis, determine the factor and level of orthogonal experiment.Using entrapment efficiency as
Evaluation index, prescription screening and optimization are carried out using orthogonal experiment.
(2) isoliquiritigenin liposome characterizes
1. morphological observation and particle size measure
Using ordinary optical microscope and transmission electron microscope phosphotungstic acid negative staining observation liposome.Using instruments for measuring particle diameter by use of laser
Measure average grain diameter.
2. liposome encapsulation measures
1) uv scan
Medicine, drug containing liposome and blank liposome are used into methanol-distilled water (1 respectively:1, V/V) mixed liquor dissolved dilution
Afterwards, scanned in 200~500nm, medicine and drug containing liposome have absorption maximum at 370nm, and blank liposome is in the wavelength model
Enclose no absworption peak.
2) prepared by standard curve
Precision is weighed in 60 DEG C of dry isoliquiritigenin 1mg to constant weight put 10ml volumetric flasks, adds methanol to dissolve on a small quantity simultaneously
Scale is diluted to as reference substance storing solution (100 μ gmL-1), accurately pipette standard solution 0.125,0.25,0.5,0.75,
1.0ml is in 10mL volumetric flasks, with methanol-distilled water (1:1, V/V) mixed liquor is diluted to scale.It is horizontal stroke with concentration C (μ g/ml)
Coordinate, absorbance A are ordinate, draw standard curve.
3) entrapment efficiency determination
Dextran gel column chromatography.Column chromatography condition：Sephadex G-50 columns (2.6cm × 30cm), redistillation washing
De-, flow velocity 0.5mlmin-1, carries out at room temperature.The total dose of liposome (W is total) is first measured, then uses Gel-filtration
Separation drug containing liposome and free drug simultaneously measure the amount (W trips) of free drug, according to the Chinese Pharmacopoeia liposome bag of 2000 editions
Envelope rate calculation formula calculates, i.e.,：/ W is always × 100% by envelop rate ﹦ (total-W trips of W).W is total：The amount of total medicine, W trips：Fat is not wrapped into
The amount of the free drug of plastid.Specifically it is summarized as follows：Precision measure liposome 5ml, be diluted to 25ml with double distilled water, after by its
50mL volumetric flasks methanol dilution is put to scale, is shaken up to be measured.Isoliquiritigenin liposome 5ml upper props, redistillation washing are taken again
It is de-, monitored with 370nm and collect free isoliquiritigenin eluting peak, be diluted to 25ml with double distilled water, then put 50ml capacity
Bottle methanol dilution is to scale and measures the amount of free drug, computational envelope rate.
4) rate of recovery is investigated
A series of isoliquiritigenin standard solution of various concentrations is configured, pipettes 5ml upper props respectively, according to above-mentioned layer 3)
Analysis condition carries out column chromatography, collects free drug component, and dilution, constant volume, surveys absorption value at 370nm, calculates the column rate of recovery.Match somebody with somebody
A series of isoliquiritigenin standard solution of various concentrations is put, is mixed respectively with blank liposome, standard mixed liquor is obtained, pipettes respectively
5ml upper props, the same terms column chromatography, measures the component absorption value of free drug, calculates column sample recovery rate.
5) study on the stability
3 batches of samples are put respectively under the natural conditions of room temperature and refrigerator in DEG C place 1,2,3 and investigate its stability June,
Specific inspection target is as follows：1. appearance：Liposome solutions belong to the thermodynamic unstable system of high degree of dispersion, grain in storage process
Son has the tendency such as self-assemble and fusion, causes liposomal particle size distribution to change, and long-term place can assemble, flocculate and be layered
Precipitation.According to particle observation under sample appearance and microscope as a result, evaluating it in experiment.Liposome appearance is not stratified, without heavy
Form sediment, be denoted as (-)；It is not stratified, there is a little flocculation resilient after shaking, be denoted as (±) layering, there is irreversible precipitation to be denoted as (+) degree；
2. pH value：For liposome during storage, phospholipid oxidation hydrolysis, which produces free aliphatic acid etc., declines pH value, it is therefore necessary to
The pH value of liposome is monitored, to reflect the stabilization envelop rate of liposome；3. envelop rate is the finger for evaluating lipid weight
One of mark, liposome is influenced, easy to leak during storage by pharmaceutical properties and membrane stability etc., therefore the bag of liposome
Envelope rate can reflect its stability.
(3) result
1. Formulation and optimization
Reference literature and single factor experiment prerun analysis result, determine cholesterol：Lecithin (mass ratio), isoliquiritigenin:
Lipid (molar ratio) and phosphate buffer pH value determine the horizontal extent of each factor as 3 investigation factors, such as table 1, with
Entrapment efficiency is evaluation index, and designing 9 prescriptions according to orthogonal design table prepares liposome.The influence factor size of envelop rate
For A>B>C, the optimal collocation of each factor is A2B1C3, i.e. cholesterol：Lecithin (mass ratio) is 1:5, isoliquiritigenin：Lipid
(molar ratio) is 1:10, PBS pH value is 7.2.Therefore, the prescription that preliminary screening goes out is：
(1) cholesterol 25mg；
(2) phosphatidase 1 00mg；
(3) isoliquiritigenin 10mg；
(4) pH value is 7.4 phosphate buffer 20ml, such as table 2.
Table 1, experimental factor and level
Table 2, orthogonal design and interpretation of result
2. isoliquiritigenin liposome characterizes
1) morphological observation and particle size measure
By transmission electron microscope photo as it can be seen that after the suitably dilution of isoliquiritigenin liposome suspension, with the phosphorus that mass fraction is 1%
Wolframic acid negative staining, observes form under transmission electron microscope, and as a result visible lipid sees rounding in vitro, is mostly spherical and spherical particle, greatly
It is small more uniform.Particle size distribution range is narrow and meets normal distribution law, and particle diameter is 224.8 ± 4.5nm.
2) prepared by uv scan, standard curve, the rate of recovery is investigated
Medicine and drug containing liposome have absorption maximum at 370nm, and blank liposome is in the wave-length coverage without absworption peak.With
Concentration C (μ g/ml) is abscissa, and absorbance A is ordinate, draws standard curve, obtains λmaxAbsorbance at=372nm wavelength
A and drug concentration C (μ gmL-1) regression equation be：A=0.0456C+0.0066 (r=0.9991), linear range are：
1.0~5.0 μ gmL-1.The column rate of recovery, its average value are 100.35%, RSD 1.24%.Column sample recovery rate, it is average
It is worth for 99.66%, RSD 0.86%, such as table 3.
Table 3, isoliquiritigenin liposome determination of recovery rates result (n=5)
3) entrapment efficiency determination
By Sephadex G-50 post separations on the isoliquiritigenin liposome sample of preparation, free Licochalcone is collected
A eluting peaks, and measure the amount of medicine, it is 55.73% (n=4) to obtain the liposome envelop rate that is averaged by the calculation formula of envelop rate.
4) study on the stability
Liposome places 1,2,3 respectively at 4 DEG C and room temperature under the conditions of inflated with nitrogen and is showed no within 6 months layering cohesion etc. now
As being still homogenous suspension, and pH value and envelop rate prompt it to have good stability, such as table 4 without significant changes.
Table 4, isoliquiritigenin liposome study on the stability result (n=3)
Note：Liposome appearance is not stratified, and no precipitation, is denoted as (-)；It is not stratified, there is a little flocculation resilient after shaking, be denoted as
(±) is layered, and has irreversible precipitation to be denoted as (+) degree
(4) conclusion
It is above-mentioned test result indicates that, the preparation method of isoliquiritigenin liposome is simple and practicable, and success rate is higher, and property is steady
It is fixed.
【Embodiment 2】Isoliquiritigenin liposome weakens the Effect study of Adriamycin cardiotoxicity
(1) animal packet and processing
6-8 weeks C57BL/6 male mice every group 10, is divided into Normal group, model control group, isoliquiritigenin processing
Group.(1) normal saline is injected intraperitoneally in continuous 7 days in Normal group daily；(2) the continuous 6 days daily abdominal cavities of model control group
Inject normal saline, the 7th day intraperitoneal injection DOX (adriamycin) 20mg/kg；(3) isoliquiritigenin liposome low dosage is tested
10mg/kg isoliquiritigenin liposomes are injected intraperitoneally in group daily, continuous 6 days, DOX (adriamycin) 20mg/kg are injected intraperitoneally within the 7th day；
(4) isoliquiritigenin liposome low dosage experimental group is injected intraperitoneally 20mg/kg isoliquiritigenin liposomes daily, continuous 6 days, the 7th day
DOX (adriamycin) 20mg/kg is injected intraperitoneally；Put to death mouse within 13rd day, take blood, heart, liver, kidney to be detected.
(2) experiment detection
1. detection mouse weight daily, observes mouse state and records；
2. myocardium enzyme CK, CK-MB, LDH, AST Concentration Testing
Collect mouse blood, according to commercial reagents box operating instruction detection serum myocardial zymogram concentration (kit by
Bioengineering Research Institute's offer is built up in Nanjing)；
3.Western blot measure anti-apoptotic proteins HAX-1 expression
Heart is ground in liquid nitrogen rapidly after heart is taken out, the heart is carried out in appropriate lysate and protease inhibitors
Dirty homogenate cracks 20min, 12 000r/min on ice, and 4 DEG C of centrifugation 15min, take supernatant, using BCA protein content detection reagents
Box measures protein content, and the protein content of all samples is diluted to isoconcentration.20 μ g samples are taken to be mixed with 5 × sample-loading buffer
It is even, 5min is boiled, SDS-PAGE electrophoresis is carried out per 20 μ g of hole loading.Electrophoresis finishes, and removes gel, the powered transferring film of albumen one is extremely
Pvdf membrane (80V about 2h).Pvdf membrane is placed in confining liquid, room temperature closing 1h.Corresponding primary antibody, 4 DEG C of overnight incubations are added, TBST is washed
Film 3 times, then adds secondary antibody again, and room temperature shaker is incubated 2h, and TBST washes film 3 times again.ECL chemiluminescences develop the color.Using gel into
As analysis software (Bio-Rad companies, the U.S.) measure protein density.
4.qPCR measure HAX-1mRNA expression
Reference literature, HAX-1mRNA expressions are measured using qPCR.Mouse heart is collected by Trizol RNA extraction examinations
Agent box extracts total serum IgE, using ultraviolet light spectrophotometric determination 260nm and 280nm OD value, estimates total rna concentration and pure
Degree.HAX-1 primers are synthesized by Shanghai Shen works biotech firm.HAX-1 gene primer sequences：Sense primer：5′-
GATATCTATCCGACAGGGTAGCGTA-3 ', anti-sense primer：5′-GGTACCTGGGATGACAGACAGAGC-3′；Use M-MLV
Reverse transcriptase is by total serum IgE (1.2 μ g) reverse transcription into cDNA.2 μ l cDNA templates are added to the SYBR Green containing primer
To be expanded in PCR premixed liquids, final volume is 25 μ l.Use CFX Connect fluorescence quantitative PCR instruments (U.S. BIO-RAD
Company) quantitative PCR is carried out, detect the Ct values of each group target gene.Melt curve analysis are analyzed to identify the specificity of amplified production, 2% fine jade
Sepharose electrophoresis confirms the length of amplified production.MRNA relative quantifications are carried out using 2- Δ Δ Ct methods, are interior using β-actin
Join gene.
(3) result
1. changes of weight
Isoliquiritigenin liposome pre-processes the Adriamycin cardiotoxicity model mice changes of weight curve of 5 days, as Fig. 1 can
See, increase over time, the mouse of isoliquiritigenin liposome pretreatment and the model group of unused isoliquiritigenin liposome processing
Mouse it is slow compared to weight loss trend, and mouse weight and isoliquiritigenin liposome are into dose-effect relationship.
2. myocardium enzymic change
As shown in Figure 2, increase over time, the myocardium toxicity model group serum center creatase CK of adriamycin processing,
CK-MB, LDH, AST concentration are apparently higher than Normal group, and with the processed myocardium toxicity mouse core of isoliquiritigenin liposome
The myocardium toxicity model group of creatase concentration ratio adriamycin processing is remarkably decreased.
3.HAX-1 albumen changes
As shown in Figure 3, adriamycin treatment group mouse anti-apoptotic proteins HAX-1 is horizontal is significantly lower than normal group, and with pre-
The concentration increase of the isoliquiritigenin of processing, HAX-1 levels gradually increase.
The horizontal changes of 4.HAX-1mRNA
As shown in Figure 4, adriamycin treatment group mouse HAX-1mRNA is horizontal is significantly lower than normal group, and with pretreatment
The concentration increase of isoliquiritigenin, HAX-1mRNA levels gradually increase.
(4) conclusion
It is above-mentioned test result indicates that, isoliquiritigenin liposome can by suppress the apoptosis of cardiac muscle cell so as to reach weaken Ah
The effect of mycin myocardium toxicity.
In summary result, the conclusion tested, it is believed that isoliquiritigenin liposome is to weaken Adriamycin cardiotoxicity
Active drug.
【Embodiment 3】Isoliquiritigenin is by adjusting HAX-1 expression inhibiting myocardial cell injuries
(1) cell culture and packet
H9C2 cells (contain the chain of 10% calf serum, the penicillin of 100U/ml and 100U/ml with DMEM in high glucose culture medium
Mycin), the secondary culture in 37 DEG C, the cell incubator of saturated humidity and 5%CO2.H9C2 in exponential phase is thin
Born of the same parents (1 × l04) be inoculated in 96 orifice plates, after cultivating 24h, model control group continues to train in the culture medium added with 1 μM of adriamycin
18h is supported, is added after the isoliquiritigenin processing 24h of 50 μM of the addition of experimental group 1,2 in the culture medium of 1 μM of adriamycin and cultivates 24h, 24h
Experimental group 2 subsequently adds a certain amount of HAX-1CRISPR gene knockouts plasmid processing 24h afterwards, Normal group culture containing
In the culture medium of 10% calf serum.After culture, supernatant cell is collected.
(2) experiment detection
Myocardium enzyme CK, CK-MB, LDH, AST Concentration Testing：Collect supernatant, examined according to the operating instruction of commercial reagents box
Survey cell supernatant myocardial zymogram concentration (kit builds up Bioengineering Research Institute by Nanjing and provides)；
(3) result
As shown in Figure 5, the myocardium enzyme level of the cardiac muscle cell after adriamycin processing is higher than isoliquiritigenin pretreated group, and
After adding the processing of HAX-1CRISPR gene knockouts plasmid, myocardium enzyme level is higher than isoliquiritigenin pretreated group.
(4) conclusion
It is above-mentioned test result indicates that, isoliquiritigenin liposome can be by adjusting the damage of HAX-1 expression inhibiting cardiac muscle cells
So as to have the function that to weaken Adriamycin cardiotoxicity.
In summary result, the conclusion tested, it is believed that isoliquiritigenin liposome is to weaken Adriamycin cardiotoxicity
Active drug.
Claims (2)
1. isoliquiritigenin liposome is preparing the urgency for the treatment of adriamycin induction（Slowly）Application in property myocardium toxicity medicine.
2. application according to claim 1, it is characterised in that the preparation method of the isoliquiritigenin liposome is：Claim
Take cholesterol, lecithin and purity to be placed in for 98% isoliquiritigenin in container, add chloroform-methanol mixed liquor, the chloroform-first
Chloroform and methanol volume ratio=2 in alcohol mixed liquor:1, using rotating thin film evaporation in 50-60 DEG C of condition rotation film 20-50
It is 0.1-0.2mm lipid dry films that min, which obtains uniform thickness, dries up residual solvent with nitrogen, adds the phosphoric acid buffer of PH=7
Salting liquid, makes lipid film fully be swollen aquation, under conditions of 400 W of ultrasonic power 5 min of ultrasound to obtain the final product, according to super in ultrasound
Sound 5 seconds, the frequency stopped 5 seconds carry out；The cholesterol and lecithin mass ratio are cholesterol：Lecithin=1:5；It is described different sweet
Careless element and lecithin molar ratio are isoliquiritigenin：Lecithin=1:10.
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| CN201711377750.0A CN107898774A (en) | 2017-12-19 | 2017-12-19 | Isoliquiritigenin liposome is preparing the urgency for the treatment of adriamycin induction（Slowly）Application in property myocardium toxicity medicine |
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| CN201711377750.0A CN107898774A (en) | 2017-12-19 | 2017-12-19 | Isoliquiritigenin liposome is preparing the urgency for the treatment of adriamycin induction（Slowly）Application in property myocardium toxicity medicine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113876696A (en) * | 2021-11-16 | 2022-01-04 | 中国人民解放军海军军医大学第一附属医院 | Clarithromycin hydrogel capable of being locally injected, preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104887650A (en) * | 2014-03-03 | 2015-09-09 | 陈建萍 | New use of isoliquiritigenin and derivative |
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| CN104887650A (en) * | 2014-03-03 | 2015-09-09 | 陈建萍 | New use of isoliquiritigenin and derivative |
Non-Patent Citations (2)
| Title |
|---|
| 何华等: "甘草黄酮对阿霉素细胞毒性的影响及其构效关系研究", 《中草药》 * |
| 张晶等: "异甘草素脂质体的制备及稳定性考察", 《中国医院药学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113876696A (en) * | 2021-11-16 | 2022-01-04 | 中国人民解放军海军军医大学第一附属医院 | Clarithromycin hydrogel capable of being locally injected, preparation method and application thereof |
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