Traditional Chinese medicine composition for treating cerebral apoplexy

Abstract

Active medicine materials of the traditional Chinese medicine composition comprise saffron, nutmeg, clove, sandalwood and apium graveolens according to the weight ratio of 100: n1-n2: n3-n4: n5-n6: n7-n8, n1, n2, n3, n4, n5, n6, n7 and n8 are all natural numbers, n1 is larger than or equal to 100 and smaller than or equal to 300, n3 is larger than or equal to 100 and smaller than or equal to 400, n5 is larger than or equal to 200 and smaller than or equal to 800, n7 is larger than or equal to 50 and smaller than or equal to 400, and n7 is larger than or equal to 400. The composition can prevent, treat, slow down or control stroke-heart syndrome, middle cerebral artery embolism and myocardial injury of subjects, and has popularization prospects.

Description

Technical Field

[0001] The invention belongs to the field of Chinese medicine preparations and relates to a Chinese medicine composition for treating cerebral stroke. Background Art

[0002] Stroke is a group of diseases that cause brain tissue damage due to sudden rupture of brain blood vessels or blood vessel blockage that prevents blood from flowing into the brain. In recent years, stroke has become one of the diseases with the highest mortality rate and the highest disability rate in my country, and my country has become the country with the largest number of strokes. At different stages after acute stroke, stroke damage to other organs will occur. Clinically, heart damage is more likely to occur in the early stages of stroke. Severe heart damage is the second leading cause of death in the early stages of stroke, with the characteristics of rapid onset and high mortality. Post-stroke cardiac complications are heart damage that occurs within 2-3 days of stroke, manifested as acute electrocardiogram changes, acute coronary syndrome and even heart failure. Clinical studies have found that there is a connection between heart disease and ischemic stroke - "stroke-heart syndrome", that is, acute ischemic stroke will have a direct adverse effect on the heart. It refers to cardiovascular complications that occur within 30 days after the occurrence of stroke, including acute myocardial injury, acute coronary syndrome, Takotsubo syndrome, heart failure, electrocardiogram changes, various arrhythmias and sudden cardiac death. Nearly one-third of stroke deaths are attributed to cardiac causes, and the risk of short-term mortality increases 2-3 times for stroke patients who suffer severe cardiac complications early in their lives.

[0003] The treatment of stroke-heart syndrome is currently limited to surgical treatment and drug treatment. In addition, clinical drugs for the treatment of stroke-heart syndrome can only provide symptomatic treatment. Therefore, it is urgent to find a targeted drug. Summary of the invention

[0004] Natural products have always played an important role in drug development. The present invention found that after giving experimental animals a group of natural product drug compositions (saffron, nutmeg, cloves, sandalwood and fragrant celery), it had a good intervention effect on stroke-heart syndrome caused by stroke. Through cell experiment verification, it was found that the administration serum of this group of drugs can improve the cell viability of myocardial cells and alleviate their apoptosis. The present invention reveals that the action pathway of this group of drugs in treating stroke-heart syndrome is the PPAR signaling pathway related to energy metabolism through a proteomics method based on stable isotope dimethylation labeling, and verifies that it can reduce myocardial damage, and is expected to become a first-line drug for treating stroke-heart syndrome, providing a scientific basis for the compatibility of traditional Chinese medicine compound prescriptions.

[0005] 1. The drug combination of saffron, nutmeg, cloves, sandalwood and celery can be used as a drug for the treatment of stroke-heart syndrome.

[0006] 2. The drug combination of saffron, nutmeg, cloves, sandalwood and celery acts on the PPAR signaling pathway, reduces fat accumulation in heart tissue and alleviates myocardial damage. This mechanism can become a therapeutic mechanism for treating stroke-heart syndrome.

[0007] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a traditional Chinese medicine composition, wherein the active medicinal materials of the traditional Chinese medicine composition include saffron, nutmeg, clove, sandalwood and celery.

[0008] In some embodiments, the weight ratio of saffron, nutmeg, cloves, sandalwood and celery in the active medicinal materials of the traditional Chinese medicine composition is 100:n1-n2:n3-n4:n5-n6:n7-n8, wherein n1, n2, n3, n4, n5, n6, n7 and n8 are all natural numbers, 100≤n1<n2≤300, 100≤n3<n4≤400:200≤n5<n6≤800:50≤n7<n8≤400.

[0009] In some embodiments, 120≤n1<n2≤150, 150≤n3<n4≤200: 300≤n5<n6≤500: 100≤n7<n8≤150.

[0010] In some embodiments, the saffron is selected from the stigma of Crocus sativus;

[0011] The nutmeg is selected from the seeds of nutmeg;

[0012] The cloves are selected from the buds of cloves;

[0013] The sandalwood is selected from the heartwood of the sandalwood tree trunk;

[0014] The celery is selected from the fruit of celery.

[0015] In some embodiments, the pharmaceutical composition further contains pharmaceutical excipients.

[0016] The second aspect of the present invention provides a method for preparing the Chinese medicine composition according to the first aspect of the present invention, the preparation method comprising the following steps:

[0017] Grind saffron, nutmeg, cloves, sandalwood and celery and mix them to obtain a mixture;

[0018] The mixture is mixed with water to prepare water-flooded pills;

[0019] The water-flooded pellets are dried to obtain dried water-flooded pellets.

[0020] The third aspect of the present invention provides use of the Chinese medicine composition described in the second aspect of the present invention in preparing a preparation for improving the health of a subject;

[0021] The improvement of the subject's health condition is selected from the following situations:

[0022] Prevent, treat, slow down or control stroke in subjects;

[0023] Prevent, treat, slow down or control stroke-heart syndrome in subjects;

[0024] Prevent, treat, slow down or control middle cerebral artery embolism in subjects;

[0025] Prevent or slow myocardial damage in subjects with middle cerebral artery embolism;

[0026] Reduce the levels of Caspase 3 protein, cleaved Caspase3 protein, Caspase 9 protein, cleaved Caspase 9 protein, BAX protein, Bcl-2 protein and Cytochrome C protein in the heart tissue of subjects with middle cerebral artery embolism.

[0027] In some embodiments, the subject is selected from humans and rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 AD are the electrocardiograms of rats in the normal group, model group, positive drug group and drug administration group (medium dose drug administration group). The model group had a large change in heart rate during the stroke process, and the positive drug group could stabilize the heart rate of reperfusion and short-term prognosis.

[0029] Figure 2 AD are the heart rate detection diagrams of each group at different periods of the MCAO experiment, and D is the medium-dose drug group. Compared with the normal group, the model group had obvious electrocardiogram abnormalities during the ischemia and reperfusion period, and the abnormal heart rate in the medium-dose drug group was significantly reduced compared with the model group and the positive drug group. * indicates that compared with the normal group, P < 0.05, ## indicates that compared with the plug after the insertion of the line, P < 0.01, and # indicates that compared with the plug after the insertion of the line, P < 0.05.

[0030] Figure 3 AD are the results of H&E staining of cardiac pathological sections in each group. Under a ×40 microscope, the myocardial fibers in the normal group were short cylindrical, with eosinophilic cytoplasm, myofibrils of varying thickness, and light and dark stripes. However, myocardial fiber rupture occurred in the model group, and no myocardial cell rupture occurred in the drug-treated group.

[0031] Figure 4A and B are the serum biochemical indexes of each group in the animal experiment. The levels of CK-MB and LDH in the model group increased, while the drug-treated group showed an obvious regression trend. This shows that the present invention has an alleviating effect on myocardial injury. * indicates that compared with the normal group, P < 0.05, # indicates that compared with the model group, P < 0.05

[0032] Figure 5 The weight changes of rats in the MCAO animal experiment before and after modeling are shown. The modeling process will cause the rats to lose weight, but there is no obvious difference between the drug administration group and the model group, indicating that the present invention has little effect on the weight of rats.

[0033] Figure 6 The picture is a photo of H9c2 cell apoptosis protein detected by Western Blot. Caspase3, cleavedCaspase 3, Caspase9, cleavedCaspase 9, BAX, Bcl-2, and Cytochrome C in the model group increased to varying degrees, while those in the drug-administered group decreased after treatment with the present invention.

[0034] Figure 7 The effect of conditioned medium on the survival rate of H9c2 cells measured by CCK-8 method is shown. The survival rate of H9c2 cells in the model group was significantly decreased compared with the control group, and the low, medium and high dose groups of composition 1 were significantly increased compared with the model group. *** indicates that P < 0.005 compared with the normal group, and ### indicates that P < 0.005 compared with the model group

[0035] Figure 8 The flow cytometry technique is used to detect the effect of conditioned medium on H9c2 cell apoptosis. Q2-1 is dead cells, Q2-2 is late apoptotic cells, Q2-3 is normal cells, and Q2-4 is early apoptotic cells. Compared with the normal group, the apoptosis of H9c2 cells in the model group increased significantly, while the apoptosis of cells in the drug group after low, medium, and high doses was reduced.

[0036] Fig. 9 Flow chart of chemical proteomics technology based on stable isotope dimethylation labeling.

[0037] Fig.10 In the figure, the upper figure shows the differential proteins in the model group compared with the normal group; the lower figure shows the differential proteins in the drug-treated group compared with the model group.

[0038] Fig.11 The changes of PPARγ protein in the PPAR signaling pathway are shown. PPARγ in the model group decreased to varying degrees, which was alleviated after drug administration.

[0039] Fig.12The heart tissue oil red stained sections of the normal group, model group and drug administration group. The heart of the rats in the model group had obvious fat accumulation compared with the normal group, and the gaps between the myocardial fibers increased. After administration of the present invention, the lipid accumulation phenomenon was significantly reduced with the increase of the drug administration concentration. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0041] Example 1: Preparation of Chinese medicine composition

[0042] 1. Source of medicinal materials

[0043] Saffron is selected from the dried stigma of Crocus sativus L., a plant of the Iridaceae family as specified in the "Chinese Pharmacopoeia 2020".

[0044] Nutmeg is selected from the dried seed kernel of Myristica fragrans Houtt. of the Myristicaceae family as specified in the "Chinese Pharmacopoeia 2020".

[0045] Cloves are selected from the dried buds of Eugenia caryophyllata Thunb., a plant of the Myrtaceae family, as specified in the Chinese Pharmacopoeia 2020. The buds are picked when they turn from green to red and dried in the sun. Sandalwood is selected from the dried heartwood of the trunk of Santalum album L., a plant of the Santalaceae family, as specified in the Chinese Pharmacopoeia 2020.

[0046] The celery is selected from the dried mature fruit part of the umbelliferae plant Cuminumcyminum L. specified in the "95 Tibetan Medicine Standards". The fruits are harvested when they are ripe in late summer and early autumn and dried in the sun.

[0047] (II) Preparation method of composition 1

[0048] The recipe is as follows:

[0049]

[0050] The preparation steps are as follows:

[0051] (1) Medicinal pieces

[0052] The origins of five medicinal materials, including saffron, nutmeg, cloves, sandalwood and fragrant celery, were identified and carefully selected and processed to ensure their quality and efficacy. Finally, impurities were removed and the medicinal materials were cleaned according to the formula ratio.

[0053] (2) Dispensing and mixing

[0054] Collect the five raw and auxiliary materials that need to be mixed in the medicine dispensing box, namely saffron, nutmeg, cloves, sandalwood, and fragrant celery, put them into the medicine dispensing box in turn, and mix them with a stainless steel shovel. Put the mixed raw and auxiliary materials into a clean medicine bag, seal it, and keep it for later use.

[0055] (3) Crushing and mixing

[0056] The mixed raw and auxiliary materials are put into a pulverizer (mixer) for pulverization, crushed into fine powder and passed through an 80-mesh sieve to obtain medicinal powder (screened material).

[0057] (4) Mixing

[0058] The obtained powder was put into a mixer and mixed for 3 minutes.

[0059] (5) Pill making

[0060] The required amount of master pellets is calculated according to the total amount of drug powder, and then pellets are made in each coating machine, purified water is used as a binder, and an appropriate amount of drug powder is added to make pellets. During the pelleting process, the size, appearance, and weight of the pellets are observed at any time.

[0061] (6) Drying

[0062] Spread the pills evenly on the drying tray and place them in the drying oven for drying. According to the properties of the material, the machine turns the pills once every 10-30 minutes to test the moisture content of the material. According to its actual moisture content, set the drying temperature and time. After the pills are turned over, the drying is finished.

[0063] (7) Polishing and drying

[0064] Mix and stir with appropriate amount of polish, honey, etc. Add the polish, take out the pills after they are smooth, and put them into a clean drying tray to dry.

[0065] Example 2: Drug efficacy determination

[0066] 1. Experimental Materials

[0067] 1. Animal husbandry

[0068] The experimental animals were Wistar male rats with an average body weight of 235 g, and the license number was SCXK(Gan)2023-004.

[0069] 2. Cell culture

[0070] Normal rat cardiomyocytes H9c2 (purchased from China Center for Type Culture Collection) were cultured in 1640 medium (purchased from Shanghai Yuanpei Biotechnology Co., Ltd.), supplemented with 10v / v% horse serum (HS, purchased from Thermo Fisher Scientific), 5v / v% fetal bovine serum (FBS, purchased from Thermo Fisher Scientific), and 1v / v% penicillin-streptomycin double antibody solution (PS, purchased from Thermo Fisher Scientific). Rat adrenal pheochromocytoma cells PC12 (purchased from China Center for Type Culture Collection) were cultured in DMEM medium (purchased from Shanghai Yuanpei Biotechnology Co., Ltd.), supplemented with 10v / v% fetal bovine serum (FBS, purchased from Thermo Fisher Scientific), and 1v / v% penicillin-streptomycin double antibody (PS, same as above, purchased from Thermo Fisher Scientific).

[0071] (II) Middle cerebral artery occlusion (MCAO) model

[0072] Rats were anesthetized by inhalation of isoflurane (R510-22-10, RWD) at a concentration of 2% for induction and maintenance of anesthesia at a flow rate of 2 L / min. The rats were fixed in the supine position. A midline incision was made in the neck. The muscles and fascia were separated along the inner edge of the sternocleidomastoid muscle. The left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were separated and ligated at the distal and proximal ends of the CCA and the ECA for later use. A slipknot was hung on the CCA for later use. The CCA was temporarily clamped with forceps, and then a small incision was made 4 mm away from the bifurcation of the CCA. The tether (2636-50A2, Cinontech) was inserted into the ICA. At this time, the tether was gently tied with the thin wire wrapped around the distal end of the CCA. The tether was gently pushed with ophthalmic forceps, and the distance was calculated from the bifurcation of the blood vessel. When the insertion depth was 18 mm, the thin wire at the distal end of the CCA was tied, the wound was sutured, and the rats were kept in a single cage for observation. Reperfusion was performed after 45 minutes of ischemia, and the ligature was pulled out 1 cm, and the excess ligature was cut off, and the model was successfully established.

[0073] (III) Electrocardiogram and heart rate measurement

[0074] The rats were divided into normal group, model group, positive drug group, drug administration group (low dose), drug administration group (middle dose) and drug administration group (high dose), with 6 rats in each group.

[0075] Normal group: Wistar male rats, no medication.

[0076] Model group: rat model obtained in step (ii) without drug administration.

[0077] Positive drug group: Compound Danshen dripping pills (27 mg / pill, the weight is the weight of the finished drug, purchased from Tasly Pharmaceutical Group Co., Ltd.) were ground into powder and the required mass was weighed, and 5w / v% carboxymethylcellulose sodium suspending agent (CMC-Na, purchased from Beijing Solebow Technology Co., Ltd.) was added to make a suspension, and the rat model obtained in step (ii) was given 1 hour in advance (no more medication after modeling). The rat gavage dose was 0.0213 g / rat.

[0078] Specifically, 5 w / v% sodium carboxymethylcellulose and normal saline were used as the solution to prepare a solution of compound danshen dripping pills with a concentration of 21.3 mg / mL, which was then used for testing.

[0079] Administration group: The above composition 1 was added with 5w / v% sodium carboxymethylcellulose to prepare a suspension of the required concentration, and administered to the rat model obtained in step (ii) 1 hour in advance. The oral dose for rats was 0.013g / rat for the low dose, 0.026g / rat for the medium dose, and 0.053g / rat for the high dose.

[0080] During the modeling process, the electrocardiograms were measured in four groups: before the insertion of the plug, after the insertion of the plug, before the removal of the plug, and after the removal of the plug. The measurement method is as follows:

[0081] During the modeling process, a bioinformation acquisition system (BL-420F bioinformation acquisition system purchased from Chengdu Taimeng, TM002) was used for electrocardiogram measurement. The rat was in a supine position, and the electrocardiogram lead wires were buried under the skin of the rat's limbs in the order of upper left-yellow, lower left-green, upper right-red, and lower right-yellow. The BL-420F bioinformation acquisition system was connected, and "ECG" in "Channel 1" in "Input Signal" was selected, and the "II Lead" switch was turned on for real-time ECG monitoring. The main monitoring indicators were: abnormal ECG waveform, heart rate, and amplitude. The ECG indicators were collected at four time points: before plugging the wire plug, after plugging the wire plug, before pulling the wire plug, and after pulling the wire plug.

[0082] Typical electrocardiograms of each group can be found in Figure 1 It can be seen that the model group has a large change in heart rate during the stroke process, the positive drug group can stabilize the heart rate of reperfusion and short-term prognosis, and the present invention also has a significant effect on stabilizing the heart rate. See the typical heart rate diagram of each group. Figure 2 It can be seen that compared with the normal group, the model group had obvious electrocardiogram abnormalities during the ischemia and reperfusion period, and the abnormal heart rate in the medium-dose drug group was significantly reduced compared with the model group and the positive drug group.

[0083] (IV) Biochemical and tissue index testing

[0084] 1. Collection of tissue and serum samples

[0085] For the four groups of rats in the aforementioned MCAO model (normal group, model group, positive drug group, medium-dose administration group), the first part was taken from each group, the chest cavity was opened after cervical dislocation, the heart was removed, and the residual blood was washed clean in physiological saline (purchased from Chenxin Pharmaceutical Co., Ltd.) and stored in a -80°C refrigerator. For each of the four groups of rats, the second part was taken, the surgical scissors were passed through the nasal cavity, the skull was cut upward along the nasal cavity, and the complete brain was taken out and stored in a 4°C refrigerator. For each of the four groups of rats, the third part was taken, the rats were anesthetized with isoflurane (purchased from Hebei Jinfuda Pharmaceutical Co., Ltd.) and the abdominal cavity was opened. After exposing the abdominal aorta, whole blood was extracted with a 10mL syringe, centrifuged (6000rpm, 4°C, 30min), and the upper serum was aspirated and stored in a 4°C refrigerator.

[0086] 2. H&E stained sections:

[0087] The heart tissue preserved in 4% paraformaldehyde (purchased from Biosharp) was dehydrated, embedded in paraffin, cut into 3 μm sections and stained with H&E using conventional hematoxylin and eosin staining solutions.

[0088] H&E stained sections of each group, see Figure 3 . It can be seen that under the ×40 microscope, the myocardial fibers in the normal group were short cylindrical, with eosinophilic cytoplasm, myofibrils of varying thickness, and light and dark stripes. However, myocardial fiber rupture occurred in the model group. No myocardial cell rupture occurred in the drug-treated group.

[0089] 3. Myocardial enzyme spectrum detection:

[0090] The whole blood was centrifuged first, and then the supernatant was aspirated and the creatine kinase isoenzyme (CK-MB) was measured using a creatine kinase isoenzyme detection kit (purchased from Nanjing Jiancheng Bioengineering Institute), and the lactate dehydrogenase (LDH) was measured using a lactate dehydrogenase detection kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0091] Results Figure 4 The levels of CK-MB and LDH in the model group increased, while those in the drug-treated group showed an obvious regression trend, which shows that the present invention has an alleviating effect on myocardial injury.

[0092] 4. Weight changes

[0093] The weight of the rats was recorded once before the experiment and once again 24 hours after MCAO. The weight change curve was drawn based on the recorded results (n=4). The results showed that the modeling process would cause the rats to lose weight, but there was no significant difference between the drug administration group and the model group, indicating that the present invention had little effect on the weight of the rats. Figure 5 .

[0094] The present invention established a middle cerebral artery occlusion (MCAO) model in experimental animals, and combined electrocardiogram waveform, heart rate, electrocardiogram amplitude detection, H&E staining of cardiac tissue pathological sections, and a series of serum biochemical indices such as creatine kinase isoenzymes and lactate dehydrogenase to systematically evaluate the efficacy of saffron, nutmeg, clove, sandalwood and celery combined for the treatment of stroke-heart syndrome at the animal level. The results showed that this group of drugs had the effect of stabilizing the heart rate of stroke model rats and reducing the occurrence of abnormal waveforms, reducing tendon rupture of myocardial fibers, restoring multiple biochemical indices to normal, having a significant therapeutic effect, and having little effect on the weight of rats. (See Figures 1 to 5 )

[0095] (V) Western Blot

[0096] Using antibodies as probes, antibodies can react with the antigenic epitopes of the target protein attached to the solid support, and then react with the second antibody of the enzyme. After substrate color development, a specific protein in the protein mixture extracted from the tissue can be identified and semi-quantitatively analyzed. The heart tissues of the MCAO rats in the above groups were removed and weighed, cut into tissue fragments, and 1 mL TritonTM X-100 (TEAB preparation, purchased from Sigma-Aldrich) was added to grind the tissues, centrifuged, and the supernatant was taken. Ultrasonic disruption was performed until the solution was clear, and the supernatant was taken after centrifugation again to obtain the desired protein sample. The protein was quantified to 5 mg / mL (solvent was PBS) using the BCA protein assay (Pierce TM BCA protein assay kit, purchased from Thermo Fisher Scientific), and 10 μL of the sample was added to each well in the SDS-PAGE gel (purchased from Kangwei Century) for electrophoresis. The protein blot was transferred to a PVDF membrane, blocked with 5% skim milk diluted with TBST (purchased from Solarbio), incubated with primary antibody after washing, incubated with secondary antibody after washing, and then developed with ECL colorimetric reagent. After washing, the membrane was exposed and photographed in an automatic imaging system (purchased from Tanon).

[0097] The following primary antibodies were used:

[0098] Rabbit polyclonal antibody to Bax, purchased from Affinity;

[0099] Rabbit polyclonal antibody to Bcl-2, purchased from Affinity;

[0100] Rabbit polyclonal antibody to Caspase 3, purchased from Affinity;

[0101] Rabbit polyclonal antibody to Cleaved-Caspase 3, purchased from Affinity;

[0102] Rabbit polyclonal antibody to Caspase 9, purchased from Affinity;

[0103] Rabbit polyclonal antibody to Cleaved-Caspase 9, purchased from Affinity;

[0104] Rabbit polyclonal antibody to Cytochrome C, purchased from Affinity;

[0105] Rabbit polyclonal antibody to beta Actin, used as an internal control, was purchased from Affinity.

[0106] The secondary antibody was Goat Anti-Rabbit IgG (H+L) HRP, purchased from Affinity.

[0107] The results are as follows Figure 6 As shown, Caspase3, cleaved Caspase 3, Caspase9, cleavedCaspase 9, BAX, Bcl-2, and Cytochrome C in the model group were increased to varying degrees, while those in the drug-administered group decreased after treatment with the present invention.

[0108] (VI) Determination of the effect of PC12 cells on H9c2 cells

[0109] 1. Cell Co-culture

[0110] PC12 cells are rat adrenal medullary pheochromocytoma differentiation cell lines, which can be differentiated by the action of nerve growth factor, and have the general characteristics of neuroendocrine cells. Not only is the morphology similar to the neuronal phenotype, but the function can also characterize the specific functions of nerve cells. Because of its passable characteristics, it is widely used in neurophysiology and neuropharmacology research. The present invention acts on cardiac complications after stroke, so a model of co-culture of PC12 cells and human normal myocardial cells H9c2 cells is selected at the cellular level to simulate the regulatory effect of the brain on the heart in vivo, reflecting the indirect therapeutic effect of the present invention on the heart.

[0111] (1) In order to culture H9c2 cells with the culture medium of PC12 and simulate the influence of nerve cells on cardiomyocytes, normal culture medium of PC12 (control group) was obtained, and the cell confluence was observed under a microscope. After the PC12 cells grew to 70% to 80% confluence, 1640 culture medium (10v / v% HS, 5v / v% FBS, 1v / v% PS) was replaced and placed in a 37°C constant temperature incubator for 24 hours. After 24 hours, the metabolites and culture medium containing PC12 cells (without cells) were collected.

[0112] (2) To simulate the hypoxic state of neural cells, obtain PC12 hypoxic culture medium (model group): observe the cell confluence under a microscope, replace 1640 culture medium (1% PS) when PC12 cells grow to 70%-80% confluence, and place in a hypoxic incubator (5% CO 2 , 1% O 2 After culturing for 24 h in 4% nitrogen and trace gases, the metabolites and culture medium containing PC12 cells (without cells) were collected.

[0113] (3) Obtaining PC12 drug-containing culture medium: After rats were continuously gavaged for one week (the dosage of the positive drug Compound Danshen Dropping Pills and the aforementioned composition 1 was 0.0213 g / rat / day, once a day, and the drug dosage was calculated as finished drug), drug-containing serum from the abdominal aorta of rats was taken from each group. After the serum protein was precipitated, the concentration of drug-containing serum protein from the abdominal aorta of rats treated with Compound Danshen Dropping Pills was diluted to 20 w / v% with 1640 culture medium (1% PS) as the PC12 drug-containing culture medium corresponding to Compound Danshen Dropping Pills. The concentration of drug-containing serum protein from the abdominal aorta of rats treated with composition 1 was diluted to 10 w / v%, 20 w / v% and 40 w / v% with 1640 culture medium (1% PS) as the culture medium for the low, medium and high dose groups. The cell confluence was observed under a microscope. After the PC12 cells grew to a confluence of 70%-80%, the culture medium for the low, medium and high dose groups was replaced and placed in a hypoxic incubator (5% CO 2 , 1% O 2 The cells were cultured in 1% 1640 medium (1% PS) for 24 hours, and the culture medium (without cells) was replaced with 1% 1640 medium for 24 hours. The cells were placed in a hypoxic incubator for another 24 hours. Metabolites and culture medium containing PC12 cells were collected after 24 hours.

[0114] 2. CCK-8 detection of cell viability

[0115] The original culture medium of H9c2 cells was discarded, 0.5 mL of trypsin (purchased from Gibco) was added, digested for 2 min, and then 2.5 mL of complete culture medium was added to terminate the digestion to obtain a cell suspension. 2 μL of trypan blue dye (purchased from Solarbio) and 18 μL of cell suspension were mixed and counted on a cell counter. According to the results, H9c2 cells were diluted to 1×10 5 / mL, add the diluted cell suspension (100 μL / well) into the 96-well plate, let it stand for 10 minutes, and then transfer the 96-well plate to a constant temperature incubator.

[0116] Modeling and drug administration were performed 12 hours after H9c2 cells were plated, and 100 μl / well of different culture media were added to each group. The specific situation is as follows: the experimental groups were divided into a control group (the aforementioned PC12 normal culture medium), a model group (the aforementioned PC12 hypoxic culture medium), a positive drug group (the aforementioned PC12 drug-containing culture medium corresponding to the compound Danshen dripping pills), a low-dose drug administration group (10% PC12 drug-containing culture medium), a medium-dose drug administration group (20% PC12 drug-containing culture medium), and a high-dose drug administration group (40% PC12 drug-containing culture medium). PC12 hypoxic culture medium was diluted to 75% with 1640 culture medium (that is, 1 / 3 of 1640 culture medium was added to PC12 hypoxic culture medium) as the model group, and the collected PC12 drug-containing culture medium was used as the drug administration group and added to a 96-well plate. CCK-8 (purchased from AbMol BioScience) and serum-free culture medium were prepared into a working solution at a ratio of 1:9. The original culture medium in the 96-well plate after modeling and administration for 24 hours was discarded, and CCK-8 was added at 100 μl / well. After reacting in a constant temperature incubator for 1 hour, the absorbance at 450 nm was measured by an enzyme reader.

[0117] Results Figure 7 The relative survival rates of H9c2 cells in the model group, the compound Danshen dripping pill group, the low-dose group (10% PC12 drug-containing culture medium), the medium-dose group (20% PC12 drug-containing culture medium), and the high-dose group (40% PC12 drug-containing culture medium) were calculated with the control group as 100%. It was found that the H9c2 cells in the model group decreased significantly compared with the control group, and the low, medium, and high-dose groups of the above-mentioned composition 1 increased significantly compared with the model group, indicating that the present invention has a protective effect on myocardial cells under the pathological model.

[0118] 3. Detection of cell apoptosis rate by flow cytometry

[0119] 1.5 × 10 5H9c2 cells (cell counter, purchased from Countstar Biotech), modeling and drug administration were performed 12 hours later. The specific situation is as follows: the experimental groups are control group C (the aforementioned PC12 normal culture medium), model group M (after dilution of the aforementioned PC12 hypoxic culture medium), positive drug group (Compound Danshen Dropping Pills) Dintonic (PC12 drug-containing culture medium corresponding to the aforementioned Compound Danshen Dropping Pills), low-dose drug administration group (10% PC12 drug-containing culture medium, TFPP) TFPP-L, medium-dose drug administration group (20% PC12 drug-containing culture medium) TFPP-M, high-dose drug administration group (40% PC12 drug-containing culture medium) TFPP-H. PC12 hypoxic culture medium was diluted to 75% with 1640 culture medium (1% PS) as the model group, and the collected PC12 drug-containing culture medium was used as the drug administration group and added to a 96-well plate.

[0120] The FITC-Annexin V / PI apoptosis kit (purchased from BD Biosciences, used to detect cell apoptosis rate) was used for determination: 24 hours after the above-mentioned drug intervention, the culture medium in the 6-well plate was discarded, and 1 mL of PBS (purchased from Shanghai Double Helix Biotechnology Co., Ltd.) was added to wash once, and 200 μL of trypsin was added to each well for digestion for 2 minutes, and then 1 mL of 1640 culture medium (containing 10% HS, 5% FBS, 1% PS) was added to terminate the digestion. H9c2 cells in each well plate were collected, centrifuged (3000rpm, 3min), and the supernatant was discarded. After adding PBS to resuspend the cells, centrifuged (3000rpm, 3min), the supernatant was discarded, and the cells were gently resuspended after repeated washing. According to Table 1, 5 μL of FITC-Annexin V and 5 μL of PI staining solution were added and gently mixed. Incubate at room temperature (20-25°C) for 10-15min. Resuspend the cells 2-3 times during the incubation process to improve the staining effect. After the incubation, 400 μL of 1× Annexin V binding solution was added to resuspend the cells, and the cells were immediately tested on a flow cytometer (purchased from BD Biosciences).

[0121] Table 1. Statistics of reagent addition

[0122]

[0123] Results Figure 8Flow cytometry was used to detect H9c2 cell apoptosis. 10%, 20%, and 40% PC12 drug-containing culture medium were used as low, medium, and high dose groups. It was found that the apoptosis of H9c2 cells in the model group was significantly increased compared with the normal group, while the apoptosis of cells in the drug-treated group after low, medium, and high doses was reduced. See Table 2 for details. Q2-1 is dead cells, Q2-2 is late apoptotic cells, Q2-3 is normal cells, and Q2-4 is early apoptotic cells.

[0124] Table 2. Statistics of apoptosis rate

[0125]

[0126] The present invention adopts the method of serum pharmacology, by co-culturing rat adrenal pheochromocytoma cells PC12 with neuroendocrine function and rat normal myocardial cells H9c2, combined with CCK-8, flow cytometry, and Western Blot to evaluate the efficacy at the cellular level. The results showed that this group of drugs can significantly increase the survival rate of H9c2 cells and reduce their apoptosis level. Therefore, it can be considered that this drug is safe for the rat heart and the whole body while showing obvious activity in treating stroke-heart syndrome. (See Figure 6-8 )

[0127] 7. Chemical proteomics

[0128] 1. Sample processing

[0129] 8M urea (purchased from Sigma) prepared with triethylammonium bicarbonate buffer (TEAB, purchased from Sigma-Aldrich) was used in the same way as step (V). The rat heart tissue protein in the model group and the normal group in the MCAO model was diluted to 10 mg / mL, 100 μL of protein diluent was added to 1 μL of dithiothreitol (purchased from Sigma-Aldrich), and the mixture was reacted in the dark for 30 minutes on a constant temperature mixer. 3 μL of iodoacetamide (purchased from Sigma) was added and the mixture was reacted in the dark for 30 minutes on a constant temperature mixer. After adding 0.5 μL of iodoacetamide, the mixture was reacted in the dark for 15 minutes on a constant temperature mixer. 15 μL of sample was added to TEAB, and rLys C (recombinant lysyl endopeptidase, purchased from Enzyme Source) was added and the mixture was reacted in the constant temperature mixer for 4 hours. After the reaction was completed, 10 μL of TEAB was added for dilution. Add 3.75 μL trypsin (purchased from Shanghai Yaxin) and 2 μL calcium chloride (TEAB diluted, purchased from Solarbio) and react on a constant temperature mixer for 16 h. After the reaction, take 50 μg of protein and add TEAB to 100 μL. Add 4 μL 4% CH 2 O, 4 μL 4% CD 2 O, 4 μL 4% 13 CD 2O (purchased from Sigma-Aldrich), formaldehyde and its isotope substitution substances, 4 μL NaBH 3 CN, 4 μL NaBD 3 CN (purchased from Sigma-Aldrich), sodium cyanoborohydride and its isotope substitution substances were reacted in a constant temperature mixer for 1 hour. After the reaction, 16 μL of 1% ammonia water (purchased from Sigma-Aldrich) was added to each sample and reacted on a constant temperature mixer for 15 minutes. After the reaction, 5% formic acid (purchased from Anpel laboratory technologies) was added to terminate the reaction. The light-labeled, medium-labeled and heavy-labeled samples of the same group were combined and dried using a vacuum rotary concentrator. The samples were stored at -80°C.

[0130] 2. LC-MS / MS analysis

[0131] LC-MS / MS analysis was performed on a Q-Exactive Orbitrap mass spectrometer (purchased from Thermo Fisher Scientific) coupled to an Ultimate 3000LC system according to the instrument manual. Briefly, the flow rate through the column was set to 0.3 μL / min, and the applied distal spray voltage was set to 2.8 kV. MS2 data collection was performed using a full scan (350-1800 MW) followed by a data-dependent MS2 scan of the 20 most abundant ions by enabling dynamic exclusion.

[0132] 3. MS data analysis

[0133] The obtained data were searched in pFind. The searched data were imported into Excel for processing, and proteins with Ratio values ​​≥ 1.5 were selected as up-regulated proteins, and proteins with Ratio values ​​≤ 0.67 were selected as down-regulated proteins for differential protein network analysis. The differential proteins were imported into DAVID (https: / / davidbioinformatics.nih.gov / ) for KEGG pathway analysis.

[0134] See the flowchart of chemical proteomics technology based on stable isotope dimethylation labeling for details. Fig. 9 .

[0135] Compared with the normal group, the differentially expressed proteins in the model group are shown in Fig.10 (Top), Differential proteins between the treatment group and the model group are shown in Fig.10 (Below) This shows that after drug administration, the metabolic pathway disorder caused by the model was intervened, that is, the pathway of drug intervention is closely related to the pathway caused by the disease model.

[0136] 4. Western Blot Detection

[0137] The method is the same as step (V), using the following primary antibodies:

[0138] Rabbit polyclonal antibody to PPAR gamma, purchased from Affinity;

[0139] Rabbit polyclonal antibody to beta Actin, used as an internal control, was purchased from Affinity.

[0140] The secondary antibody was Goat Anti-Rabbit IgG (H+L) HRP, purchased from Affinity.

[0141] The results are as follows Fig.11 As shown, the first three columns are the control group, the middle three columns are the model group, and the last three columns are the drug-administered group. It can be seen that the PPARγ in the model group in the PPAR signaling pathway is reduced to varying degrees, and is alleviated after the treatment of the present invention.

[0142] 5. Oil Red O Staining Sections

[0143] Prepare frozen sections of cardiac tissue for each group according to step (III), and incubate the sections in propylene glycol at room temperature for 5 minutes. Then incubate the sections in 60°C Oil Red O solution for 6-10 minutes. After the incubation, differentiate the sections with 85% propylene glycol for 1 minute, rinse the sections with distilled water twice, then stain the tissue sections with hematoxylin dye for 1-2 minutes, rinse the sections with distilled water twice, and seal the sections with synthetic resin after rinsing until the sections are transparent.

[0144] Oil red stained sections of heart tissues from the normal group, model group, and drug-treated group are shown in Fig.12 It can be seen that the hearts of the rats in the model group have obvious fat accumulation compared with the normal group, and the gaps between the myocardial fibers are enlarged. After the administration of the present invention, as the dosage concentration increases, the phenomenon of lipid accumulation is significantly reduced.

[0145] Peroxisome proliferator-activated receptors (PPAR) are nuclear receptors that act as ligands to activate transcription factors. PPARγ is most highly expressed in metabolic tissues (including white and brown adipose tissue) and is primarily involved in the transport of fat. In the early stages of cardiometabolic disorders, PPARγ is downregulated, resulting in reduced transport of fatty acids to mitochondria, thereby increasing lipid accumulation in cells. Administration of this drug restores energy metabolism to normal.

[0146] In order to explore the mechanism of action of the drug combination of saffron, nutmeg, clove, sandalwood and celery in treating stroke-heart syndrome, the present invention requires a "panoramic view" to see what specific changes in the protein network have occurred in the heart cells intervened by this group of drugs. To this end, the present invention plans to use a chemical proteomics method based on stable isotope dimethylation labeling to reveal the action pathway of this group of drugs in treating stroke-heart syndrome. Experiments have found that stroke-heart syndrome upregulates the PPAR ("peroxisome proliferator-activated receptor") signaling pathway related to energy metabolism, and the administration of this group of drugs downregulates the PPAR signaling pathway, which indicates that the drug exerts its efficacy through fatty acid metabolism mediated by the PPAR signaling pathway. Based on the above results, the present invention found through oil red stained pathological sections that stroke-heart syndrome causes lipid substances to accumulate in the heart, and the administration of a drug combination of saffron, nutmeg, clove, sandalwood and celery can improve this lipid accumulation phenomenon, which proves the correctness of the pathway. (See Figure 9-12 ).

[0147] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A Chinese medicine composition, wherein the active medicinal materials of the Chinese medicine composition include saffron, nutmeg, cloves, sandalwood and celery.

2. The Chinese medicine composition according to claim 1, characterized in that The active medicinal materials of the traditional Chinese medicine composition include saffron, nutmeg, cloves, sandalwood and celery in a weight ratio of 100: n1-n2: n3-n4: n5-n6: n7-n8, wherein n1, n2, n3, n4, n5, n6, n7 and n8 are all natural numbers, 100≤n1<n2≤300, 100≤n3<n4≤400: 200≤n5<n6≤800: 50≤n7<n8≤400.

3. The Chinese medicine composition according to claim 2, characterized in that: 120≤n1<n2≤150,150≤n3<n4≤200:300≤n5<n6≤500:100≤n7<n8≤150.

4. The Chinese medicine composition according to claim 1, characterized in that: The saffron is selected from the stigma of Crocus sativus; The nutmeg is selected from the seed kernel of nutmeg; The cloves are selected from the buds of cloves; The sandalwood is selected from the heartwood of the sandalwood tree trunk; The celery is selected from the fruit of celery.

5. The Chinese medicine composition according to claim 1, characterized in that: The pharmaceutical composition also contains pharmaceutical excipients.

6. A method for preparing the Chinese medicine composition according to any one of claims 1 to 5, comprising the following steps: Grind saffron, nutmeg, cloves, sandalwood and celery and mix them to obtain a mixture; The mixture is mixed with water to prepare water-flooded pills; The water-flooded pellets are dried to obtain dried water-flooded pellets.

7. Use of the Chinese medicine composition according to any one of claims 1 to 5 in preparing a preparation for improving the health status of a subject; The improvement of the subject's health condition is selected from the following situations: Prevent, treat, slow down or control stroke in subjects; Prevent, treat, slow down or control stroke-heart syndrome in subjects; Prevent, treat, slow down or control middle cerebral artery embolism in subjects; Prevent or slow myocardial damage in subjects with middle cerebral artery embolism; Reduce the levels of Caspase 3 protein, cleaved Caspase3 protein, Caspase 9 protein, cleaved Caspase 9 protein, BAX protein, Bcl-2 protein and Cytochrome C protein in the heart tissue of subjects with middle cerebral artery embolism.

8. The use according to claim 7, characterized in that The subjects are selected from humans and rats.

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