CN110051671B - Application of purslane amide E in preparation of medicine for treating ischemic heart disease - Google Patents
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
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Abstract
The invention discloses an application of purslane amide E or a prodrug thereof in preparing a medicament for treating ischemic heart disease.
Description
Technical Field
The invention relates to application of purslane amide E, in particular to application of purslane amide E or a prodrug thereof in preparing a medicament for treating ischemic heart disease.
Background
Artemisia capillaris Meconopsis horridula hook.f. et Thoms. Purslane amide E (Oleracein E, OE) is tetrahydroisoquinoline alkaloid extracted from Artemisia Spinosa, and has antioxidant, neuroprotective, antibacterial and antiviral effects.
CN101234121B discloses the application of oleracein alkaloids in the preparation of antioxidants and neuron protective agents. The oleracein alkaloid is preferably monomer of oleracein A, oleracein B, or oleracein E.
CN103948596A discloses an application of purslane amide E in preparing anti-senile dementia drugs, and relates to five applications of purslane amide E in prolonging life of senile dementia, enhancing metabolic capability of senile dementia, relieving oxidative stress of senile dementia, protecting nerve cells of senile dementia and improving memory capability of senile dementia.
So far, no report on the application of the purslane amide E in the preparation of the medicine for treating the ischemic heart disease exists.
Disclosure of Invention
In view of the above, the present invention aims to provide a novel medical use of purslane amide E or a prodrug thereof. The invention adopts the following technical scheme to achieve the purpose.
Use of oleracein E, or a prodrug thereof, for the manufacture of a medicament for the treatment of ischemic heart disease, the oleracein E having a structure according to formula (I):
according to the use of the present invention, preferably, the medicament forms a pharmaceutical preparation for the treatment of ischemic heart disease; the pharmaceutical preparation comprises the purslane amide E or the prodrug thereof and also comprises pharmaceutically acceptable auxiliary materials.
According to the use of the present invention, preferably, the pharmaceutical formulation has the oleracein E or a prodrug thereof as the only active ingredient.
According to the use of the invention, preferably, the medicament forms a pharmaceutical preparation for increasing left ventricular ejection fraction and short axis shortening rate, reducing CK-MB in serum, and increasing SOD and GSH-Px in serum.
According to the use of the present invention, preferably, the medicament forms a pharmaceutical preparation for reducing the degree of inflammation and the degree of fibrosis of the myocardial tissue.
According to the use of the present invention, preferably, the medicament forms a pharmaceutical preparation for inhibiting apoptosis of cardiac muscle cells.
According to the use of the present invention, preferably, the medicament forms a pharmaceutical preparation for reducing the intracellular calcium ion concentration.
According to the use of the present invention, preferably, the medicament forms a pharmaceutical preparation for the treatment of ischemic heart disease; the content of the purslane amide E or the prodrug thereof in the unit pharmaceutical preparation is 65-260 mg.
According to the application, the content of the purslane amide E or the prodrug thereof in the unit pharmaceutical preparation is 65-150 mg.
According to the application, the content of the purslane amide E or the prodrug thereof in the unit pharmaceutical preparation is 70-130 mg.
The purslane amide E or the prodrug thereof can be used for preparing medicines with the effect of treating ischemic heart diseases. The purslane amide E or the prodrug thereof can improve left ventricular ejection fraction and short axis shortening rate, reduce CK-MB in serum and increase SOD and GSH-Px in serum. The purslane amide E or the prodrug thereof can reduce the inflammation degree and the fibrosis degree of myocardial tissues. The purslane amide E or the prodrug thereof can inhibit myocardial apoptosis. The purslane amide E or prodrug thereof can reduce the calcium ion concentration in cells.
Drawings
FIG. 1 is a graph showing the change of CK-MB in serum of each group of mice in example 1.
FIG. 2 is a graph showing the change in SOD in serum of each group of mice in example 1.
FIG. 3 is a graph showing the change in serum GSH-Px in each group of mice in example 1.
FIG. 4A is a graph showing the results of HE staining of a myocardial tissue section of a mouse in the sham-operated group in example 1.
FIG. 4B is a graph showing HE staining results of a myocardial tissue section of a mouse model group in example 1.
FIG. 4C is a graph showing HE staining results of myocardial tissue sections of mice in the gold multi-ginkgo leaf extract group of example 1.
FIG. 4D is a graph of the results of HE staining of myocardial tissue sections from OE high dose groups of mice in example 1.
FIG. 4E is a graph of HE staining of myocardial tissue sections from mice in dose groups in OE in example 1.
FIG. 4F is a graph of the results of HE staining of myocardial tissue sections from OE low dose groups of mice in example 1.
FIG. 5A isExample 6 intervention of blank control group H2O2Graph of fluorescence intensity (40 ×) of calcium ion of H9c2 cells after stimulation.
FIG. 5B is H for model set intervention in example 62O2Graph of fluorescence intensity (40 ×) of calcium ion of H9c2 cells after stimulation.
FIG. 5C is H of OE high dose group intervention in example 62O2Graph of fluorescence intensity (40 ×) of calcium ion of H9c2 cells after stimulation.
FIG. 5D is H of dose group intervention in OE in example 62O2Graph of fluorescence intensity (40 ×) of calcium ion of H9c2 cells after stimulation.
FIG. 5E is H of intervention in the OE Low dose group of example 62O2Graph of fluorescence intensity (40 ×) of calcium ion of H9c2 cells after stimulation.
FIG. 5F is H of intervention of the positive drug quercetin group in example 62O2Graph of fluorescence intensity (40 ×) of calcium ion of H9c2 cells after stimulation.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.
The structure of oleracein E of the invention is shown below:
in the invention, the molecular formula of the purslane amide E is C12H13NO3And the molecular weight is 219.237. The purslane amide E is a known compound, and can be extracted and separated from flowers, fruits, leaves, stems and roots of plants, and can be prepared according to a method disclosed in the literature "chemical composition of Tibetan medicine safflower Artemisia selengensis (Wuhaifeng et al, natural product research and development, 2011), or according to a method disclosed in the literature" synthesis of antioxidant purslane amide E and derivatives thereof "(Liu Bo Shu treatise on Master position of Shandong university, 2010).
In the present invention, a prodrug of oleracein E refers to a compound that is inactive or less active in vitro and that exerts its pharmacological effects by releasing oleracein E in vivo through enzymatic or non-enzymatic conversion.
In the invention, the purslane amide E or the prodrug thereof has the effect of treating the ischemic heart disease and can be used for preparing the medicine with the effect of treating the ischemic heart disease. According to some embodiments of the invention, the purslane amide E or prodrug thereof can increase left ventricular ejection fraction and short axis shortening rate, reduce CK-MB in serum, and increase SOD and GSH-Px in serum. According to further embodiments of the present invention, purslane amide E or prodrugs thereof of the present invention can reduce the degree of inflammation and the degree of fibrosis of myocardial tissue. According to still further embodiments of the present invention, purslane amide E or prodrugs thereof of the present invention may inhibit myocardial apoptosis. According to still further embodiments of the present invention, purslane amide E or prodrugs thereof of the present invention may reduce the calcium ion concentration within the cell.
In the present invention, the drug for treating ischemic heart disease may be oleracein E and/or a prodrug thereof as the only active ingredient; other active ingredients having an effect of treating ischemic heart disease may be contained, or active ingredients not having an effect of treating ischemic heart disease per se but capable of assisting in the action of purslane amide E and/or a prodrug thereof to treat ischemic heart disease may be contained.
In the invention, the medicine for treating ischemic heart disease can be a raw material medicine or a pharmaceutical preparation.
In the invention, the medicament forms a medicinal preparation with the effect of treating the ischemic heart disease. In a unit pharmaceutical preparation, the content of the purslane amide E or the prodrug thereof is 65-260 mg, preferably 65-150 mg, and more preferably 70-130 mg. A unit pharmaceutical preparation refers to a preparation and application unit, such as a tablet, a bag of granules, a capsule, a bottle of oral liquid, etc. When the content of the oleracein E or a prodrug thereof in a unit pharmaceutical preparation is within the above range, administration is facilitated, and the effect of treating ischemic heart disease is also exerted.
In the invention, the dosage form of the pharmaceutical preparation is not limited, and can be tablets, granules, capsules, pills, oral liquid, injection and the like. The pharmaceutical formulation may further comprise pharmaceutically acceptable excipients. The kind of the pharmaceutically acceptable auxiliary material is not limited. The adjuvants can be filler, correctant, lubricant, etc. Fillers are also known as diluents, such as wheat starch, tapioca starch, corn starch, potato starch, dextrin, lactose, and the like. Examples of flavoring agents include, but are not limited to, sucralose, isomaltulose, aspartame, acesulfame k and the like. Examples of lubricants include, but are not limited to, magnesium stearate, talc, aerosil, magnesium lauryl sulfate, and the like.
The experimental reagents, instruments, biological materials and detection indexes used in the following examples are as follows:
DMEM basal medium, Fetal Bovine Serum (FBS), 0.25% trypsin-EDTA and dimethyl sulfoxide are all conventional commercial products. Quercetin was purchased from Sigma, USA.
Glutathione peroxidase (Glutathione peroxidase, GSH-Px). Superoxide Dismutase (SOD).
CCK8 Cell proliferation and toxicity test Kit (Cell Counting Kit-8) was purchased from the institute of Homond chemistry (Dojindo) of Japan. The flow-type apoptosis detection kit was purchased from solibao corporation. The active oxygen detection kit was purchased from BD corporation, usa.
Rat H9c2 cardiomyocytes: culturing with DMEM complete medium (containing 90% DMEM basal medium and 10% fetal bovine serum FBS); the cell culture conditions were: 37 ℃ and 5% CO2And culturing at saturated humidity.
ICR mice: 25-28 g, 7-8 weeks old, purchased from Beijing Sibeifu biology, Inc.; feeding in SPF grade environment.
OD (optical density) is the optical density, also called the transmittance, and represents the optical density absorbed by the test object, and OD ═ lg (1/trans), where trans is the transmission of the test object.
Example 1 evaluation of the drug efficacy of Portulamide E for the treatment of ischemic Heart diseases
1. Modeling, grouping and administering drugs
1.1 Molding
100 healthy developing male ICR mice were taken and randomly divided into M groups of 84 mice and N groups of 16 mice.
The M groups of mice are anesthetized by intraperitoneal injection of 50mg/kg pentobarbital sodium, the M groups of mice are fixed, the hair of the precordial mouse is removed, an animal respirator (the respiratory frequency is 100 times/min, the respiratory rate is 1:1, and the tidal volume is 2mL/kg) is connected, the chest is opened layer by layer between the 3 rd and 4 th costal cavities on the left side of the mice, the heart is exposed, the blood vessel of the left coronary artery of the mice is observed at the position 1-2 mm below the left auricle, and the sterile suture needle with the specification of 7-0 is used for suturing the chest layer by layer after the blood vessel of the left coronary artery is ligated, and the sterile suture needle with the specification of 5-0 is used for suturing the chest.
The method comprises the following steps of carrying out intraperitoneal injection on N groups of mice for anesthesia, fixing the N groups of mice, removing the hair of the precordial mice, connecting an animal respirator (the respiratory frequency is 100 times/min, the respiratory suction ratio is 1:1, and the tidal volume is 2mL/kg), opening the chest layer by layer between the 3 rd and 4 th costal cavity on the left side of the mouse and exposing the heart, observing the left coronary artery blood vessel of the mouse at the position 1-2 mm below the left auricle, threading a sterile suture needle with a specification of 7-0 on the left coronary artery blood vessel without ligation, and suturing the closed chest layer by a sterile suture needle with a specification of 5-0.
The mice of the above groups were fed with free water after surgery and were fed conventionally.
1.2 grouping and administration
After 24h of modeling, 12 mice in the N groups are selected as a pseudo-operation group; m groups of 60 mice were selected and randomly and evenly divided into 5 groups, which were: a model group, a gold multi-ginkgo leaf extract group, an OE low dose group (a oleracein E low dose group), an OE medium dose group (a oleracein E medium dose group), and an OE high dose group (a oleracein E high dose group); the above groups were administered by continuous gavage for 7 days.
The above groups were administered as follows:
the sham operation group: 0.5% CMC-Na;
model group: 0.5% CMC-Na;
gold multiple ginkgo leaf extract group: the dosage of the gold multi-ginkgo leaf extract is 120 mg/kg;
OE low dose group (oleracein E low dose group): the dose of the oleracein E is 10 mg/kg;
dose group in OE (dose group in oleracein E): the dose of the purslane amide E is 20 mg/kg;
OE high dose group (oleracein E high dose group): the dose of oleracein E was 40 mg/kg.
The oleracein E and the gold multi-ginkgo leaf extract are dissolved in 0.5% of CMC-Na, and the gavage volume is converted by 0.1mL/10g of mouse body weight.
2. Test items and results
2.1 cardiac ultrasound examination
7 days after administration, each group of mice was anesthetized with isoflurane, fixed in the recumbent position, and the left ventricular end diastolic internal diameter (LVEDd) and the left ventricular end systolic internal diameter (LVEDs) of the hearts of each group of mice were examined by M-mode ultrasound, and the left ventricular Ejection Fraction (EF) and the short axis shortening rate (FS) were calculated. The short axis shortening rate (FS) was calculated from the left ventricular end diastolic internal diameter (LVEDd) and the left ventricular end systolic internal diameter (LVEDs) of ICR mouse hearts. The short axis shortening (FS) is calculated as: FS% ([ (LVEDd-LVEDs)/LVEDd ] × 100%. The results of the ultrasonic testing of the hearts of the mice in each group are shown in table 1.
TABLE 1 ultrasonic testing results of the heart of each group of mice
Note that: comparing to model group, P <0.05, P <0.01, P <0.001
2.2 blood Biochemical index detection
After 7 days of intragastric administration, a full-automatic biochemical analyzer detects creatine kinase isoenzyme (CK-MB), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum. The biochemical detection results of the blood of each group of mice are shown in figures 1-3.
The CK-MB in the serum of the model group mouse is obviously increased, which indicates that the myocardial cells of the model group mouse are seriously damaged; compared with the model group, the CK-MB in the serum of mice in the OE low dose group, the OE medium dose group and the OE high dose group is remarkably reduced (P < 0.001).
Compared with the model group, the serum SOD and GSH-Px of mice in the OE low-dose group, the OE medium-dose group and the OE high-dose group are both obviously improved, particularly the OE high-dose group is improved most obviously, and the OE high-dose group has equivalent effect with the gold multi-ginkgo leaf extract group.
2.3 detection of cardiac pathologies
2.3.1 tissue Paraffin embedding
(1) Material drawing and fixing: soaking the heart of a mouse in 4% paraformaldehyde for over 72 hours;
(2) washing with water: taking out the heart from 4% paraformaldehyde, transversely cutting along the ligature, dividing the heart into two parts, washing with running water, and soaking with double distilled water;
(3) dewatering, transparency and wax dipping: sequentially putting the tissue treated in the step (2) into 75% alcohol for 4 h-85% alcohol for 2 h-90% alcohol for 2 h-95% alcohol for 1 h-absolute ethyl alcohol I for 30 min-absolute ethyl alcohol II for 30 min-xylene I for 5-10 min-xylene II for 5-10 min-wax I for 1 h-wax II for 1 h-wax III, and heating to 60 ℃ for 1 h; wherein, I, II and III only represent the treatment for the second time, for example, 1h of wax means the first treatment for 1h of wax, 1h of wax II means the second treatment for 1h of wax, and 1h of heating wax III to 60 ℃ means the third treatment for 1h of wax to 60 ℃.
(4) Embedding: embedding the tissues soaked with the wax in an embedding machine, and cooling at room temperature;
(5) slicing, pasting and copying: the trimmed wax block was sliced on a paraffin slicer to a thickness of 4 μm. The sections were floated on a spreading machine at 40 ℃ warm water to flatten the tissue, attached to a slide, and placed in a 60 ℃ oven for baking. Taking out after water baking and wax baking and roasting for standby at normal temperature.
2.3.2HE staining
(1) Dewaxing to water: sequentially placing the slices in xylene I20 min-xylene II 20 min-absolute ethanol I10 min-absolute ethanol II 10 min-95% ethanol 5 min-90% ethanol 5 min-80% ethanol 5 min-70% ethanol 5 min; wherein, I and II only represent the treatment for the second time, for example, the absolute ethyl alcohol I10 min means the first treatment for 10min, and the absolute ethyl alcohol II 10min means the second treatment for 10 min.
(2) Dyeing: hematoxylin staining solution (staining cell nucleus) for 8min, washing with tap water, 1% hydrochloric acid alcohol differentiation for several seconds, washing with tap water, returning blue with 0.6% ammonia water, and washing with running water. Dyeing in eosin dye liquor (cytoplasm dyeing) for 1-3 min;
(3) dewatering and sealing: and (3) putting the slices into 95% alcohol I for 5min to 95% alcohol II for 5min to absolute ethyl alcohol I for 5min to absolute ethyl alcohol II for 5min to xylene I for 5min to xylene II for 5min to dehydrate and transparent, taking the slices out of the xylene, slightly drying the slices, sealing the slices with neutral gum, and observing the slices by a microscope. Wherein, I and II only represent the treatment for the second time, for example, the absolute ethyl alcohol I5 min means the first treatment for 5min, and the absolute ethyl alcohol II 5min means the second treatment for 5 min. The results of HE staining of myocardial tissue sections of each group of mice are shown in FIGS. 4A to 4F.
3. Conclusion of the experiment
The heart ultrasonic detection results of all groups of mice show that compared with a model group, the left ventricular Ejection Fraction (EF) and the short axis shortening rate (FS) of the gold multi-ginkgo leaf extract group are both obviously improved, and have extremely obvious difference (P is less than 0.001); the left ventricular Ejection Fraction (EF) and the short axis shortening rate (FS) of the OE high dose group are both significantly improved, with very significant differences (P < 0.001); the left ventricular Ejection Fraction (EF) and the minor axis shortening rate (FS) of the dose group in OE are both remarkably improved, and have remarkable difference (P is less than 0.05); the left ventricular Ejection Fraction (EF) of the OE low dose group was significantly increased with significant differences (P < 0.05).
The biochemical detection result of the blood of each group of mice shows that compared with the model group, the CK-MB of the gold multi-ginkgo leaf extract group is obviously increased, and has extremely obvious difference (P is less than 0.001); CK-MB in the serum of mice in the OE low-dose group and the OE high-dose group is remarkably reduced, and has extremely remarkable difference (P is less than 0.01); CK-MB in the serum of mice of the dose group in OE is remarkably reduced, and has a remarkable difference (P < 0.05).
The blood biochemical detection results of all groups of mice show that compared with a model group, the SOD of the gold multi-ginkgo leaf extract group is obviously increased, and the gold multi-ginkgo leaf extract group has extremely obvious difference (P is less than 0.01); SOD in serum of mice in the OE medium dose group and the OE high dose group are obviously increased, and have obvious difference (P is less than 0.05).
The biochemical detection result of the blood of each group of mice shows that compared with the model group, the GSH-Px of the gold multi-ginkgo leaf extract group is obviously increased, and has extremely obvious difference (P is less than 0.001); the serum of OE high-dose group mice is obviously increased in GSH-Px, and has extremely obvious difference (P is less than 0.001); the serum GSH-Px of mice in the dose group in OE is obviously increased, and has obvious difference (P < 0.05).
HE staining results of mouse myocardial tissue sections of all groups show that compared with a model group, the inflammation degree and the fibrosis degree of the myocardial tissue of the gold multi-ginkgo leaf extract group are obviously reduced; the degree of inflammation and fibrosis of myocardial tissues in the OE high-dose group are obviously reduced; the degree of inflammation and the degree of fibrosis of the myocardial tissue of the dose group in the OE are obviously reduced; the degree of inflammation and fibrosis of the myocardial tissue in the OE low dose group was significantly reduced.
The experiments show that OE (purslane amide E) can remarkably improve left ventricular Ejection Fraction (EF) and short axis shortening rate (FS), remarkably reduce creatine kinase isoenzyme (CK-MB), remarkably increase two antioxidant enzymes of SOD and GSH-Px in serum, and remarkably reduce inflammation degree and fibrosis degree of myocardial tissues, which indicates that purslane amide E has the effect of treating ischemic heart disease and can be used for preparing the medicine for treating the ischemic heart disease.
Example 2 CCK8 cell proliferation assay
1. Experimental methods and results
The effect of different concentrations of OE (oleracein E) on H9c2 cardiomyocyte proliferation activity was determined using CCK8 reagent (Cell Counting Kit, Cell Counting Kit 8).
Taking H9c2 cells in logarithmic phase, preparing cell suspension, adjusting cell concentration to 5 × 105Perml, 100. mu.l per well in 96-well plates. Culturing the cells for 24h to make the cells completely adhere to the walls, diluting OE (purslane amide E) mother solution into 10 muM, 20 muM, 40 muM, 80 muM, 160 muM and 320 muM with complete culture medium, and setting 6 auxiliary holes for each concentration; setting a blank control group; after 24h of administration, 100. mu.l of serum-free medium containing 10% CCK8 was added to each well, and OD was measured at 450nm after 2h of incubation in an incubator, andthe inhibition rate of each group was calculated by a formula. The effect of OE on H9c2 cell proliferation is shown in table 1.
TABLE 1 Effect of OE on H9c2 cell proliferation
| Grouping | Inhibition ratio (%) |
| Blank control group | 100.00±5.22 |
| OE(10μM) | 69.84±4.624 |
| OE(20μM) | 98.50±4.198 |
| OE(40μM) | 110.8±3.05 |
| OE(80μM) | 106.6±1.798 |
| OE(160μM) | 97.69±5.474 |
| OE(320μM) | 101.8±2.626 |
2. Conclusion of the experiment
Table 1 shows that OE does not inhibit proliferation of H9c2 cells below 160. mu.M, but with increasing OE concentration, OE significantly inhibited proliferation of H9c2 cells when OE concentration reached 320. mu.M. This indicates that OE at 160. mu.M or less had no inhibitory effect on H9c2 cells, and OE administration was relatively safe.
Example 3 CCK8 test for determining oxidative stress in cells
1. Experimental methods and results
H pairs of OE (Portulamide E) at various concentrations were determined using CCK8 reagent (Cell Counting Kit, Cell Counting Kit8)2O2Whether the damaged H9c2 cardiomyocytes had a protective effect.
Taking H9c2 cells in logarithmic phase, preparing cell suspension, and adjusting cell concentration to 1 × 105Perml, 100. mu.l per well in 96-well plates. Culturing the cells for 24h to ensure that the cells are completely attached to the wall, diluting OE (purslane amide E) mother solution into 5 mu M, 10 mu M and 20 mu M with complete culture medium, and setting 6 auxiliary holes for each concentration; setting a blank control group, and additionally setting a model group and a positive medicine quercetin group (the concentration of quercetin is 10 mu M); after 24H of administration, the culture medium was discarded and 100. mu.l of 50. mu.M H was added to each well2O2Stimulating for 2h, adding 100 μ l of serum-free medium containing 10% CCK8 into each well, measuring OD value at 450nm after incubating for 2h in an incubator, and calculating the inhibition rate of each group according to the formula. Each pair is H2O2The effect of induced H9c2 cell proliferation is seen in table 2.
Table 2 groups for H2O2Effect of induced proliferation of H9c2 cells
| Grouping | Protective Rate (%) |
| Blank control group | 100.0±1.988 |
| Model set | 47.15±1.508 |
| Positive drug quercetin group (10. mu.M) | 72.53±2.284 |
| OE(20μM) | 78.23±2.908 |
| OE(10μM) | 74.04±1.833 |
| OE(5μM) | 62.98±4.048 |
2. Conclusion of the experiment
Table 2 shows that OE was for H at 20. mu.M, 10. mu.M, 5. mu.M compared to the model group2O2The damage caused by the traditional Chinese medicine preparation has a good improvement effect and is dose-dependent. OE at 20 μ M for H2O2Amelioration of the resulting lesions and the positive drug quercetin group (10 μ M) for H2O2The improvement effect of the damage is equivalent.
Example 4 flow cytometry assay for apoptosis
1. Experimental methods and results
Taking H9c2 cells in logarithmic phase, preparing cell suspension, and adjusting cell concentration to 1 × 105Perml, add to 6 well plates, 2ml per well. Culturing the cells for 24h to ensure that the cells are completely attached to the wall, diluting OE (purslane amide E) mother solution into 5 mu M, 10 mu M and 20 mu M with complete culture medium, and setting 3 auxiliary holes for each concentration; setting a blank control group, and additionally setting a model group and a positive medicine quercetin group (the concentration of quercetin is 10 mu M); after 6H of administration, the culture medium was discarded, and 2ml of 50. mu.M H was added to each well2O2Stimulating for 1h, and operating according to the method of an Annexin V-FITC/PI detection kit. Briefly, the0.25% Trypsin (EDTA-free) digested single cell suspension experiment was performed to collect 1X 10 total cells6And (4) respectively. The cells were washed twice with PBS, centrifuged at 350rpm for 5min, and collected. Adding Annexin V binding buffer suspension cells, adding 5 mu l of Annexin V-FITC and 5 mu l of PI, uniformly mixing, keeping out of the sun, reacting at room temperature for 15min, and analyzing by a flow cytometer to obtain the apoptosis rate. Each pair is H2O2The effect of induced apoptosis of H9c2 cells is seen in table 3.
Table 3 groups H2O2Effect of induced apoptosis of H9c2 cells
| Grouping | Apoptosis Rate (%) |
| Blank control group | 14.63±3.099 |
| Model set | 44.20±5.767 |
| Positive drug quercetin group (10. mu.M) | 27.97±7.353 |
| OE(20μM) | 21.72±2.705 |
| OE(10μM) | 28.75±4.587 |
| OE(5μM) | 29.65±5.949 |
2. Conclusion of the experiment
Table 1 shows that OE inhibited the cardiomyocyte apoptosis compared to the model group, and that the rate of apoptosis was inversely proportional to the dose, i.e. the rate of apoptosis of H9c2 cardiomyocytes decreased gradually with increasing concentration of the administered drug.
Example 5 flow cytometry assay for cellular reactive oxygen species
1. Experimental methods and results
Taking H9c2 cells in logarithmic phase, preparing cell suspension, and adjusting cell concentration to 1 × 105Perml, add 6 plates, 2ml per well. Culturing the cells for 24h to ensure that the cells are completely attached to the wall, diluting OE (purslane amide E) mother solution into 5 mu M, 10 mu M and 20 mu M with complete culture medium, and setting 3 auxiliary holes for each concentration; setting a blank control group, and additionally setting a model group and a positive medicine quercetin group (the concentration of quercetin is 10 mu M); after 6H of administration, the culture medium was discarded, and 2ml of 50. mu.M H was added to each well2O2Stimulating for 1 h. All cells in the well plate were collected, centrifuged, the supernatant was decanted, and washed twice with 1mL of pre-cooled PBS. DCFH-DA was diluted with serum-free medium at a ratio of 1:1000 to a final concentration of 10. mu.M and a cell concentration of one million to two million/ml, and cultured in a 37 ℃ incubator for 20 min. And (4) reversing and mixing uniformly every 3-5 min to ensure that the probe is fully contacted with the cells. Cells were washed three times with serum-free cell culture medium to remove DCFH-DA well without entering the cells. Detecting by a flow cytometer to obtain the active oxygen detection result. Each pair is H2O2See table 4 for the effect of induced reactive oxygen species in H9c2 cells.
Table 4 groups H2O2Effect of induced reactive oxygen species in H9c2 cells
| Grouping | Percentage of ROS content (%) |
| Blank control group | 36.63 |
| Model set | 100.0 |
| Positive drug quercetin group (10. mu.M) | 18.69 |
| OE(20μM) | 20.55 |
| OE(10μM) | 28.26 |
| OE(5μM) | 54.12 |
2. Conclusion of the experiment
Table 4 shows that OE significantly reduced H at 20. mu.M, 10. mu.M, 5. mu.M, compared to the model group2O2Induced Reactive Oxygen Species (ROS) content in H9c2 cells. OE reduction of H at 20. mu.M2O2Effect of induced Reactive Oxygen Species (ROS) content in H9c2 cells with reduction of H by the Positive drug quercetin group (10 μ M)2O2The effect of induced Reactive Oxygen Species (ROS) content in H9c2 cells was comparable.
Example 6 laser confocal calcium ion assay
1. Experimental methods and results
Taking H9c2 cells in logarithmic phase, preparing cell suspension, and adjusting cell concentration to 1 × 105Perml, add 6 plates, 2ml per well. Culturing the cells for 24h to ensure that the cells are completely attached to the wall, diluting OE (purslane amide E) mother solution into 5 mu M, 10 mu M and 20 mu M with complete culture medium, and setting 3 auxiliary holes for each concentration; a blank control group is set up and,additionally, a model group and a positive medicine quercetin group (the concentration of quercetin is 10 μ M) are set; after 6H of administration, the culture medium was discarded, and 2ml of 50. mu.M H was added to each well2O2Stimulating for 1 h; after incubation for 2h in a constant temperature incubator at 37 ℃, taking out the six-hole plate, removing the cell culture solution, and adding 500 mu L PBS to each hole to wash the cells for three times; diluting Fluo-4AM mother liquor to 0.5-5 mu M of working solution by using PBS; adding 1mL of diluted Fluo-4AM working solution into each hole; incubating at 37 ℃ for 30 min; and (4) observing under a laser confocal microscope. H for each group intervention2O2The results of the fluorescence intensity (40 ×) of calcium ions of H9c2 cells after stimulation are shown in FIGS. 5A to 5F.
2. Conclusion of the experiment
Compared to the model set, OE can reduce calcium ion concentration in cells, and as the dose of OE increases, the efficacy of OE in reducing calcium ion concentration increases. The effect of OE at 20. mu.M on reducing intracellular calcium ion concentration was comparable to that of the group of positive drugs quercetin (10. mu.M).
The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.
Claims (7)
1. Use of oleracein E for the manufacture of a medicament for the treatment of ischemic heart disease, said oleracein E having the structure according to formula (I):
the medicament forms a pharmaceutical preparation for treating ischemic heart disease; the pharmaceutical preparation comprises the purslane amide E and also comprises pharmaceutically acceptable auxiliary materials; the pharmaceutical preparation takes the purslane amide E as the only active ingredient; the content of the purslane amide E in the unit pharmaceutical preparation is 65-260 mg.
2. The use according to claim 1, wherein the medicament forms a pharmaceutical preparation for increasing left ventricular ejection fraction and short axis shortening rate, decreasing CK-MB in serum, increasing SOD and GSH-Px in serum.
3. Use according to claim 1, wherein the medicament forms a pharmaceutical preparation for reducing the degree of inflammation and the degree of fibrosis of myocardial tissue.
4. Use according to claim 1, wherein the medicament forms a pharmaceutical preparation for inhibiting cardiomyocyte apoptosis.
5. Use according to claim 1, wherein the medicament forms a pharmaceutical preparation for reducing the intracellular calcium ion concentration.
6. The use of claim 1, wherein the amount of oleracein E in a unit pharmaceutical formulation is 65-150 mg.
7. The use of claim 1, wherein the amount of oleracein E in a unit pharmaceutical formulation is 70-130 mg.
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Non-Patent Citations (3)
| Title |
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| 藏药多刺绿绒蒿抗心肌缺血作用与化学成分研究;郭志琴;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20140915(第9期);第E056-1013页 * |
| 藏药多刺绿绒蒿的化学和药理学研究进展;赵凤等;《中国中药杂志》;20171031;第42卷(第19期);第3676-3683页 * |
| 附子化学成分研究;李小红等;《中国实验方剂学杂志》;20131031;第19卷(第19期);第86-89页 * |
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