CN103816218A - Application of clinopodium chinense total flavones in preparing medicines for protection effect of adriamycin-induced cardiotoxicity - Google Patents
Application of clinopodium chinense total flavones in preparing medicines for protection effect of adriamycin-induced cardiotoxicity Download PDFInfo
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- CN103816218A CN103816218A CN201410026172.6A CN201410026172A CN103816218A CN 103816218 A CN103816218 A CN 103816218A CN 201410026172 A CN201410026172 A CN 201410026172A CN 103816218 A CN103816218 A CN 103816218A
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Abstract
The invention discloses application of clinopodium chinense total flavones in preparing medicines for the protection effect of adriamycin-induced cardiotoxicity. The clinopodium chinense total flavones can be medicine compositions of clinopodium chinense total flavones, including various preparations of oral or parenteral administration forms. When being used in oral administration, the compositions can be tablets, capsules, soft capsules, oral liquid, syrup, granules, dropping pills, oral disintegrating tablets, sustained-release tablets, sustained-release capsules, controlled-release tablets and controlled-release capsules; when being used in parenteral administration, the compositions can be in the forms of water injection, lyophilized powder injection, sterile powder injection and infusion. Tablets and water injection are preferable dosage forms of the medicine compositions provided by the invention.
Description
Technical field
The invention belongs to medical technical field, more specifically, the present invention relates to a kind of medicinal active ingredient in the application of preparing in medicine, relate in particular at Herba Clinopodii Polycephali total flavones and prepare the application in medicine.
Background technology
Amycin is anthracycline antibiotics, finds and be applied to the treatment of kinds cancer from the sixties in 20th century, remains now one of the most effective cancer therapy drug.Be applicable to the kinds of tumors such as malignant lymphoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, bone and soft tissue sarcoma.But due to serious cardiac toxicity, its clinical practice is under some influence.In recent years, more to the research of doxorubicin cardiotoxicity, doxorubicin is divided into acute toxicity, subacute toxicity and chronic toxicity.Acute poisoning betides single use or after the course for the treatment of, common sympton has hypotension, arrhythmia, cardiac dysfunction etc., myocardial infarction can occur once in a while, often with liver, renal dysfunction.Therefore assessing doxorubicin cardiotoxicity degree and find can efficient solution just become current problem demanding prompt solution except the material of its cardiac toxicity, but still lacks effectively prevention and Therapeutic Method at present.
The Labiatae Clinopodium plant whole world has more than 20 to plant, and is mainly distributed in the province such as Zhejiang, Jiangsu in China.This platymiscium mostly is medication among the people, and wherein Herba Clinopodii Polycephali and the Clinopodium Polycephalum Herba Clinopodii (Herba Clinopodii Polycephali) are recorded as Herba Clinopodii medicine by " Chinese Pharmacopoeia " version in 2010, have effect of astringing to arrest bleeding.Modern pharmacology research shows, it has multiple pharmacological effect, as antiinflammatory, and antioxidation, antibacterial activity.Recently, it has protective effect to cardiovascular system also to have report.In Herba Clinopodii Polycephali, flavonoid content is abundant.By systematic study Herba Clinopodii Polycephali total flavones (Total flavones from Clinopodium chinense (Benth.) O.Ktze; TFCC) myocardial cell injury to amycin induction and protective effect and the molecular mechanism of cardiac toxicity; Herba Clinopodii Polycephali total flavones and adriamycin medication significantly improve the cardiac toxicity of amycin, and do not affect its antitumor action.
Summary of the invention
The invention discloses the application of Herba Clinopodii Polycephali total flavones in the medicine for the preparation of myocardial cell protection effect;
More specifically, the invention discloses the application of Herba Clinopodii Polycephali total flavones in the protective effect medicine for the preparation of amycin induction cardiac toxicity;
Above-mentioned application of the present invention, described Herba Clinopodii Polycephali total flavones is pharmaceutical composition, makes pharmaceutical composition by Herba Clinopodii Polycephali total flavones and one or more pharmaceutically acceptable carriers or excipient.
The medicine of the protective effect of amycin induction cardiac toxicity of the present invention is the pharmaceutical composition of Herba Clinopodii Polycephali total flavones, and the amount of the contained active component Herba Clinopodii Polycephali of described pharmaceutical composition minimum unit total flavones is 2.5-100mg.
Herba Clinopodii Polycephali total flavones pharmaceutical composition of the present invention is any acceptable dosage form clinically, comprises the various dosage forms of oral and parenteral form.When oral, can be tablet, capsule, soft capsule, oral liquid, syrup, granule, drop pill, oral cavity disintegration tablet, slow releasing tablet, slow releasing capsule, controlled release tablet, controlled release capsule; During for parenteral approach, can be liquid drugs injection, freeze-dried powder, aseptic powder injection, transfusion.Pharmaceutical composition preferred tablet of the present invention and liquid drugs injection dosage form.
Aforementioned pharmaceutical compositions, the optional adaptive pharmaceutical excipient for oral formulations of described pharmaceutically acceptable carrier or excipient, comprises filler, binding agent, lubricant, disintegrating agent, cosolvent, surfactant, absorption carrier etc.
Aforementioned pharmaceutical compositions, the optional adaptive pharmaceutical excipient for injection of described pharmaceutically acceptable carrier or excipient, comprises solvent, antioxidant, cosolvent, adsorbent, osmotic pressure regulator, PH regulator.
Pharmaceutical composition minimum unit refers to a slice, a capsule, one bag of granule or an injection etc.
Any method that dosage form of the present invention can be used pharmaceutical preparation technology to learn the known usual use of those skilled in the art produces and this is not particularly limited.
For example, tablet of the present invention can, by using suitable method granulation well known in the art, dry and sieve main medicament and excipient, binding agent etc., add lubricant etc. then to mix and form tablet in gained mixture.Pelletize can be undertaken by any suitable method well known in the art, for example wet granulation, non-slurry pelletizing or heating pelletize.Suitable limiting examples comprises that use high-speed stirred comminutor, fluidized granulation drying machine, Squeezinggranulator or cylinder compactor carry out these prilling process.In addition, for example method dry and screening can be carried out according to the needs that carry out pelletize.The mixture of main medicament, excipient, binding agent, lubricant etc. also can be formed directly in tablet.
If need film coating, can use any film coating device known in the art, and as film coating substrate, suitable example comprises sugar-coat base, hydrophilic film coating base, enteric film coating base and sustained release film coat base.
Accompanying drawing explanation
The protective effect of the rat heart damage of Fig. 1 Herba Clinopodii Polycephali total flavones to amycin induction; Rat is divided into normal group, TFCC (80mg/kg) group, DOX group and DOX+TFCC (20,40,80mg/kg) group.Rat administration finishes to be handled as follows respectively afterwards.The measurement heart is heavy, body weight, calculates heart body ratio; Get blood and measure the content of serum cardiac three enzymes; The dirty HE of the carrying out staining analysis of coring, the difference between more each group.(A) TFCC can improve heart weight and the weight loss that amycin causes.(B) TFCC reduces the cardiac muscle three enzymes risings that amycin causes.(C) TFCC alleviates the myocardium pathology damage that amycin causes.
The myocardial cell injury protective effect of Fig. 2 Herba Clinopodii Polycephali total flavones to amycin induction; (A) the reduction myocardial cell survival rate that amycin (0 μ M to3 μ M) can concentration dependent.(B) TFCC (0 μ g/ml to50 μ g/ml) is on not impact of myocardial cell survival.(C) cardiomyocyte viability of protection amycin (the 1 μ M) induction that TFCC can concentration dependent reduces.(D) TFCC can reduce the release of the cell conditioned medium LDH of amycin (1 μ M) induction.
Fig. 3 Herba Clinopodii Polycephali total flavones and the amycin coupling result to antioxidation related enzyme activity in cardiac muscular tissue and myocardial cell; Collect normal group, rat heart muscle tissue and the H9C2 myocardial cell of model group and Herba Clinopodii Polycephali total flavones and amycin coupling group, measure wherein CAT, MDA, the activity of SOD and GSH-Px.Amycin group MDA level raises, CAT, SOD and GSH-Px activity decreased; Herba Clinopodii Polycephali total flavones can weaken this change.
Fig. 4 Herba Clinopodii Polycephali total flavones alleviates the generation of the myocardial cell activity oxygen-derived free radicals of amycin induction.By immunofluorescence dyeing and flow cytometry analysis, respectively organize the generation of myocardial cell ROS.Amycin group ROS produces significantly to be increased, and TFCC can reduce ROE level.
Fig. 5 Herba Clinopodii Polycephali total flavones suppresses cardiac muscular tissue and the apoptosis of cardiac muscle of amycin induction; (A) Hoechst33342 fluorescence staining detects apoptosis and necrosis.(B) fluorimetry detects Caspase-3 activity.(C) TFCC alleviates cardiac muscular tissue's apoptosis of amycin induction.(D) TFCC alleviates the apoptosis of cardiac muscle of amycin induction.
Fig. 6 Herba Clinopodii Polycephali total flavones and amycin coupling do not affect amycin antitumor action.
The specific embodiment
Below in conjunction with the specific embodiment, the present invention is described in further details, does not form further restriction of the present invention.
The rat heart muscle disease model that in embodiment 1.1 bodies, amycin is induced is set up
The SD rat (Beijing dimension tonneau China, Beijing, China) in Mus age in 6-8 week.Animal adapt to 1 week and during freely obtain food and water.80 rats are divided into 6 groups immediately: Normal group (control); Herba Clinopodii Polycephali total flavones list administration group (TFCC, 80mg/kg); Amycin model group (DOX, 3mg/kg); Amycin and Herba Clinopodii Polycephali total flavones administering drug combinations group (20,40,80mg/kg).The oral pure water that gives of normal group and model group, other each group gives the TFCC of same volume respective concentration.After 15 days, model group and administering drug combinations group lumbar injection amycin are injected every other day, and totally 3 times, the normal saline of other group injection same volumes.Last administration, after 14 days, is processed.
Embodiment 1.2 cardiac weights and weight ratio and myocardium three enzymes detect
Weigh in, abdominal aortic blood under chloral hydrate anesthesia.Blood sample in latter one hour of collection under 3000g centrifugal 10 minutes, LDH in blood plasma subsequently, AST, the activity of CK is according to manufacturers instruction, measure by the commercial detection test kit of buying from Jiancheng Bio-engineering Institute (Nanjing, China).
Result is as shown in Fig. 1-A, and compared with normal group, amycin group cardiac weight and body weight obviously decline, and administering drug combinations group can significantly rise.As Fig. 1-B, the cardiac muscle three enzyme content in amycin group blood significantly rise, and administering drug combinations group can significantly reduce.
Embodiment 1.3 morphological examinations
Heart tissue is fixed with 4% paraformaldehyde.Hematoxylin and eosin dyeing for the tissue slice of 5mm.Under optical microscope, carry out morphological examination.
Result as shown in Fig. 1-C, normal group and Herba Clinopodii Polycephali but administration group: organizational structure normal.Model group: fracture is dissolved in large area cardiac muscle fiber, and volume is hemorrhage.Amycin+Herba Clinopodii Polycephali total flavones group: fracture is dissolved in a small amount of cardiac muscle fiber.
Embodiment 2: the protective effect of Herba Clinopodii Polycephali total flavones to external myocardial cell
The H9c2 cell injury model of embodiment 2.1 amycin inductions
The subclonal cell line of rat myocardial cell H9C2(period of embryo BD1X rat heart tissue) (Chinese Academy of Sciences's Shanghai cell bank) in Eagle ' the s culture medium of Dalbeck improvement of adding hyclone, 5%CO at 37 ℃
2condition under cultivate.Cell goes down to posterity and the set of successive transfer culture to 90% before experiment frequently.Cell is with 1*10
5density be inoculated in 96 orifice plates cultivate 36h.After Herba Clinopodii Polycephali total flavones (0,0.25,0.5,1,2,3 μ mol) pretreatment 4h, every hole adds 5mg/ml MTT (the every hole of 0.1mg/) and hatches 4h..Abandon supernatant, every hole adds 150 μ l DMSO dissolves, and 570nm microplate reader (Spectrafluor, TECAN, Sunrise, Austria) detects absorbance.
Result as shown in Fig. 2-A, the dependent reduction cell survival rate of doxorubicin concentration, wherein amycin 1 μ mol effect 24h, cell survival rate is 50.97%, is defined as Model Condition.
Embodiment 2.2 impacts of Herba Clinopodii Polycephali total flavones on H9c2 myocardial cell
H9c2 myocardial cell is with 1*10
5density be inoculated in 96 orifice plates cultivate 36h.After Herba Clinopodii Polycephali total flavones (0,6.25,12.5,25,50 μ g/ml) pretreatment 4h, every hole adds 5mg/ml MTT (the every hole of 0.1mg/) and hatches 4h..Abandon supernatant, every hole adds 150 μ l DMSO dissolves, and 570nm microplate reader (Spectrafluor, TECAN, Sunrise, Austria) detects absorbance.
Result as shown in Fig. 2-B, Herba Clinopodii Polycephali total flavones (0,6.25,12.5,25,50 μ g/ml) to myocardial cell without propagation and toxic action.
The protective effect of the H9c2 myocardial cell toxicity of embodiment 2.3 Herba Clinopodii Polycephali total flavones to amycin induction
The subclonal cell line of rat myocardial cell H9C2(period of embryo BD1X rat heart tissue) with 1*10
5density be inoculated in 96 orifice plates. after Herba Clinopodii Polycephali total flavones (0,6.25,12.5,25,50 μ g/ml) pretreatment 4h, amycin 1 μ M effect 24h, every hole adds 5mg/ml MTT (the every hole of 0.1mg/) and hatches 4h afterwards.Abandon supernatant, every hole adds 150 μ l DMSO dissolves, and 570nm microplate reader (Spectrafluor, TECAN, Sunrise, Austria) detects absorbance.
Result as shown in Fig. 2-C, the cardiomyocyte cell death of the protection amycin induction that Herba Clinopodii Polycephali total flavones can concentration dependent.Wherein Herba Clinopodii Polycephali total flavones dosage 25 μ g/ml reach maximum protection effect.
Embodiment 2.4LDH discharges mensuration
The subclonal cell line of rat myocardial cell H9C2(period of embryo BD1X rat heart tissue) with 1*10
5density be inoculated in 96 orifice plates.After Herba Clinopodii Polycephali total flavones (0,6.25,12.5,25,50 μ g/ml) pretreatment 4h, amycin 1 μ M effect 24h,, collect afterwards supernatant.LDH level is measured according to LDH test kit description.
Result is as shown in Fig. 2-D, and 1 μ M DOX can significantly improve LDH and discharge.And add after 25 μ g/ml TFCC pretreatment, LDH activity can significantly reduce.LDH is that dead mark appears in cell, and result proves that TFCC can protect the cardiomyocyte cell death of DOX induction.
Cardiac muscular tissue and normal saline are pressed 1:9 homogenate, and the centrifugal 10min of 4000d retains supernatant; After H9C2 myocardial cell drug treating completes, use Ultrasound Instrument fragmentation, centrifuging and taking supernatant.By specification detects GSH-PX in tissue and cell subsequently, MDA, and SOD, the activity of CAT, measures by the commercial detection test kit of buying from Jiancheng Bio-engineering Institute (Nanjing, China).
As shown in Figure 3, amycin can cause that cardiac muscular tissue and cell MDA level raise to result, SOD, and CAT, the level of GSH-PX reduces, and Herba Clinopodii Polycephali total flavones can alleviate MDA level, improves SOD, CAT, GSH-PX level.
Embodiment 4H9C2 myocardial cell ROS produces mensuration
ROS level detects according to total ROS detection kit operating instruction.After TFCC (25 μ g/ml) pretreatment 4h, amycin 1 μ M effect 24h, collecting cell washing once by 1 × washing liquid afterwards, the carboxy-H2DCFDA (final concentration 25 μ M) that uses afterwards 100 μ l in the dark 37 ℃ hatch 30min.Flow cytometer detects.Cell is put on slide, process the same, finally as for fluorescence microscopy Microscopic observation.
As shown in Figure 4, compared with normal group, the ROS that TFCC pretreatment can reduce DOX induction discharges result, and DOX processed group can improve ROS level.ROS generation can impel cell injury and Apoptosis process, and result proves that TFCC can protect the myocardial cell oxidative stress damage of DOX induction.
Embodiment 5 Herba Clinopodii Polycephali total flavones reduce the cardiomyocyte apoptosis of amycin induction
Embodiment 5-1Hoechst33342 dyeing
Utilize Hoechst33342 dyeing, and detect cell death level by fluorescence microscope.H9c2 cell was in slide 36 hours.After TFCCB (25 μ g/ml) pretreatment 4h, amycin 1 μ M effect 24h, cell is hatched 15 minutes in 5mg/ml Hoechst33342 lucifuge afterwards, and washes 2 times with PBS, obtains image with just putting fluorescence microscope.
Result is as shown in Fig. 5-A, and by just putting fluorescence microscope imaging, normal group cell is dyed to low blueness, and amycin group can be observed sapphirine fluorescence, and TFCC pretreated group can obviously be improved the metamorphosis that amycin causes.
Embodiment 5-2CASPASE-3 activity analysis
Caspase-3 is active in the caspase-3 staining kit (BioVision, CA, USA) of fluorescence-activation, measures by operating instruction.After TFCCB (25 μ g/ml) pretreatment 4h, amycin 1 μ M effect 24h, afterwards 300 μ L (1 × 10
6cells/mL) Cell sap add 1 μ L FITC-DEVD-FMK substrate in the dark 37 ℃ hatch 1h.Fluorescence is measured under excitation wavelength 485nm and emission wavelength 535nm by microplate reader.
Result is as shown in Fig. 5-B, and 1 μ M DOX can improve caspase-3 activity, and can suppress the caspase-3 activation that amycin is induced after adding TFCC.
Embodiment 5-3TUNEL dyeing
The fixing section of cardiac muscular tissue, myocardial cell is processed as 5-2, carries out to specifications TUNEL dyeing, is just putting under fluorescence microscope and is obtaining photo.
Result is as shown in Fig. 5-C, and DOX group cardiac muscular tissue and H9C2 apoptosis of cardiac muscle are obvious, and obviously reduce cardiomyocyte apoptosis after adding TFCC pretreatment.
Embodiment 6 Herba Clinopodii Polycephali total flavones and the impact of amycin coupling on amycin antitumor action
Adopt respectively oral cancer, hepatocarcinoma, breast cancer cell, by 1*10
5o'clock in 96 orifice plates, cultivate 36h, after TFCC (25 μ g/ml) pretreatment 4h, amycin 1 μ M effect 24h, every hole adds 5mg/ml MTT (the every hole of 0.1mg/) and hatches 4h afterwards.Abandon supernatant, every hole adds 150 μ l DMSO dissolves, and 570nm microplate reader (Spectrafluor, TECAN, Sunrise, Austria) detects absorbance.
Result is as Fig. 6, and than normal group, amycin group cells survival rate obviously declines, tool significance, and Herba Clinopodii Polycephali total flavones and amycin coupling group and amycin group do not have difference.Be that Herba Clinopodii Polycephali total flavones does not affect amycin antitumor action.
Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here without also giving exhaustive to all embodiments.And these belong to apparent variation that spirit of the present invention extended out or variation still among protection scope of the present invention.
Claims (7)
1. the application of Herba Clinopodii Polycephali total flavones in the medicine of preparing myocardial cell protection effect.
2. the application of Herba Clinopodii Polycephali total flavones in the protective effect medicine for the preparation of amycin induction cardiac toxicity.
3. the arbitrary described application of claim 1～2, wherein Herba Clinopodii Polycephali total flavones is the pharmaceutical composition of Herba Clinopodii Polycephali total flavones, is made up of Herba Clinopodii Polycephali total flavones and one or more pharmaceutically acceptable carriers or excipient.
4. application according to claim 3, the amount of the contained active component Herba Clinopodii Polycephali of wherein said pharmaceutical composition minimum unit total flavones is 2.5-100mg.
5. application according to claim 3, wherein said Herba Clinopodii Polycephali total flavones pharmaceutical composition is any acceptable dosage form clinically.
6. according to the application one of claim 1-4 Suo Shu, wherein said dosage form comprises the various dosage forms of oral and parenteral form; When oral, can be tablet, capsule, soft capsule, oral liquid, syrup, granule, drop pill, oral cavity disintegration tablet, slow releasing tablet, slow releasing capsule, controlled release tablet, controlled release capsule; During for parenteral approach, can be liquid drugs injection, freeze-dried powder, aseptic powder injection, transfusion.
7. require the described application of one of 1-4 according to power; the optional adaptive pharmaceutical excipient for oral formulations of wherein said pharmaceutically acceptable carrier or excipient, comprises filler, binding agent, lubricant, disintegrating agent, cosolvent, surfactant, absorption carrier etc.; Also the optional adaptive pharmaceutical excipient for injection, comprises solvent, antioxidant, cosolvent, adsorbent, osmotic pressure regulator, PH regulator.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107115372A (en) * | 2016-02-25 | 2017-09-01 | 任立群 | A kind of antineoplastic pharmaceutical compositions containing Folium Apocyni Veneti general flavone |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107115372A (en) * | 2016-02-25 | 2017-09-01 | 任立群 | A kind of antineoplastic pharmaceutical compositions containing Folium Apocyni Veneti general flavone |
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