Application of traditional chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and their differentiation in cardiomyocytes

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Application of traditional Chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Traditional Chinese medication composition (versions). Method of treatment and prevention of cardiovascular disease, including introduction to patients who need it of efficient quantity of traditional Chinese medical composition, and mesenchymal stem cells, obtained from bone marrow.

EFFECT: composition is efficient for promotion of in vivo survival of obtained from bone marrow mesenchymal stem cells and their differentiation in cardiomyocytes.

15 cl, 8 dwg, 1 tbl, 1 ex

The scope of the invention

The present invention relates to a new use of traditional Chinese medicinal composition, in particular it relates to the use of traditional Chinese medicinal composition for obtaining a medicinal product for promotion survival in vivo bone-marrow-derived mesenchymal stem cells and their differentiation into cardiomyocytes. The invention also relates to a traditional Chinese medicinal composition, which has a stimulating effect on the survival of bone-marrow-derived mesenchymal stem cells in vivo and their differentiation into cardiomyocytes, and to a method for prevention or treatment of cardiovascular diseases traditional Chinese medicinal composition.

Background of the invention

Cardiovascular diseases cause the death of 12 million people, which is approximately 1/4 of all deaths worldwide per year, and they become one of the most dangerous diseases of people. In myocardial infarction or heart failure traditional therapy, including medications, different interventions, including surgery, may not cause regeneration of the missing cells of the myocardium, and due to the fact that the deficit of myocardial cells causes niobra emuu restructuring of the myocardium, this ultimately leads to heart failure and death. In recent years, regenerative medicine using stem cells has achieved great progress in the treatment of cardiovascular diseases, and the method is also referred to as cell cardiomyoplasty, i.e. it achieves the restoration of the damaged myocardium by transplantation of stem cells or cells of the myocardium or by mobilizing stem cells peripheral blood or myeloid stem cells to migrate to the damaged parts of the myocardium. Holding cell cardiomyoplasty by stem cell transplantation is a possible way to improve hemodynamic parameters and correction neurohumoral disorders caused by myocardial infarction. Various earlier experiments on animals show that stem cells have the ability to regenerate cells of the myocardium and can improve perfusion zones of infarction and cardiac function (Schuster M.D., Kocher A. A., Seki, T., Martens T.P., G. Xiang, Homma, S., et al. Myocardial neovascularization by bone marrow angioblasts results in cardiomyocyte regeneration. Am J Physiol Heart Circ Physiol 2004; 287: H525-532).

Although stem cells used for clinical studies of the recovery of the myocardium after exposure to ischemia/reperfusion and inflammatory factors that led to the death of the donor glue is OK in terms of regional myocardial ischemia, and therefore, the development of cell cardiomyoplasty stayed low survival rate of transplanted cells. Studies show that a large number of cells were killed after transplantation in the diseased heart, and there was considerable loss of cells within 24 h, but 15% of the transplanted cells survived after 12 weeks (Muller-Ehmsen j, Whittaker p, Kloner R.A., Dow J.S., T. Sakoda, Long, T.I., et al. Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium. J Mol Cell Cardiol 2002; 34: 107-11)[PMID: 11851351].

Acute myocardial infarction can cause severe regional myocardial ischemia, inflammatory response, oxidative stress and apoptosis, which can significantly reduce the survival rate of the transplanted cells. Thus, the regional protection of transplanted stem cells or exclusion of their death is important for clinical application. Currently, there are several ways to increase the survival of transplanted cells: (1) processing thermal shock can increase the stability of the transplanted cells to damage by ischemia/reperfusion in vivo, to increase the survival rate after transplantation in heart (Suzuki K., Smolenski R.T., J. Jayakumar, B. Murtuza, Brand N.J., Yacoub M.H. Heat shock treatment enhances graft cell survival in skeletal myoblast transplantation to the heart. Circulation 2000; 102: III216-221)[PMID: 11082390]); (2) bone-marrow-derived mesenchymal stem cells modified virus is m Akt, prevent remodeling and restore heart function after a heart attack (Mangi A.A., Noiseux n, Kong d, He h, Pezvani M., Ingwall, J. S., et al. Mesenchymal stem cells modifier with Akt prevent remodeling and restor performance of infracted hearts. Nat Med 2003; 9: 1195-1201)[PMID: 12910262]; (3) injecting the plasmid vector in regional itemizedoverlay myocardium, which leads to overexpression of oxygenase-1 Ferragamo, the decrease in the number of myocardial cells, infiltrated with mononuclear cells and suppressive regulation of expression of inflammatory factors (Tang Y.L., Tang Y., Zhang C., Qian K.P., L.P. Shen, Phillips I. Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated Ferroheme Oxygenase-1 vector. J Am Coll Cardiol 2005; 46: 1339-1350)[PMID: 1619885]. All of the above tools based on the level of donor cells, however, the key to the fate of transplanted cells in the heart is the microenvironment of the regional myocardium affected by a heart attack, so that the interference pursued in the microenvironment of the affected myocardial infarction, could be more effective in the promotion of the survival of the transplanted cells and exert a biological effect.

The present invention represents a further improvement of China patent No. 01131203.3 and patent applications No. 200410048292.2, which are fully included in the present description by reference. The invention relates to a new use of traditional Chinese medicinal composition to receive medication for promotion of the survival of bone-marrow-derived mesenchymal stem cells in vivo and differentiate into cardiomyocytes, which improves the quality of regional microenvironment using invasive therapy and effectively contribute to the survival and biological effects of the transplanted cells.

A brief description of the invention

The aim of the present invention is the use of traditional Chinese medicinal composition for obtaining a medicinal product for promotion survival of bone-marrow-derived mesenchymal stem cells in vivo and differentiation into cardiomyocytes. Traditional Chinese medicinal composition consists of the following crude drugs (in parts by weight):

Ginseng 3-10

Leech 3-11

Ground beetle 5-10

The frankincense tree (processed) 1-5

Root red peony 3-9

The core of dalbergia 1-5

Sandalwood 1-5

Scorpio 3-9

Wypasek cicadas 3-12

Centipede 1-3

Borneol 1-7

The seeds of the palm (baked with stirring) 3-10;

preferably the traditional Chinese medicinal composition consists of the following crude drugs (in parts by weight):

Ginseng 6

Leech 10

Ground beetle 7

The frankincense tree (processed) 2

Root red peony 5

The core of dalbergia 2

Sandalwood 2

Scorpio 7

Wypasek cicadas 7

Centipede 1

Borneol 5

The seeds of the palm(baked with stirring) 5;

or:

Ginseng 10

Leech 8

Ground beetle 7

The frankincense tree (processed) 2

Root red peony 5

The core of dalbergia 2

Sandalwood 2

Scorpio 9

Wypasek cicadas 7

Centipede 1

Borneol 5

The seeds of the palm (baked with stirring) 5;

or:

Ginseng 6

Leech 11

Ground beetle 7

The frankincense tree (processed) 2

Root red peony 5

The core of dalbergia 2

Sandalwood 2

Scorpion 3

Wypasek cicadas 7

Centipede 1

Borneol 5

The seeds of the palm (baked with stirring) 5;

or:

Ginseng 5,5

Leech of 10.375

Ground beetle 6,875

The frankincense tree (processed) 2,25

Root red peony 4,75

The core of dalbergia 2,375

Sandalwood 2,25

Scorpio 6,875

Wypasek cicadas 6,875

Centipede 1,375

Borneol 1,375

The seeds of the palm (baked with stirring) 4,625;

more preferably the active ingredients of the above traditional Chinese medicinal composition is composed of the following ingredients:

a. Scorpio, leech, centipede, ground beetles, wypasek cicadas and powder processed from the frankincense tree, which has an average particle size less than 100 microns;

b. powder borneol;

c. volatile oil extracted from the core of dalbergia and sandalwood;

d. Conde is zirovanii alcohol extract of ginseng, extracted by ethanol;

e. condensed water extract, which is obtained in the following way:

the extraction of the remnant core of dalbergia and sandalwood water after extraction component c of them, digestion root red peony and baked under stirring seeds of the palm in the water, extraction of the residue ginseng water after extraction component d from it, filtering all of the above extracts, their mixing, then concentration.

The invention, furthermore, reveals that a medicinal preparation containing the above Chinese medical composition as an active component, is a capsule, tablet, pill, oral liquid dosage form, a soft capsule or homeopathic ball, saturated liquid dosage form.

Another aim of the invention is to ensure the application of the above traditional Chinese medicinal composition for obtaining a medicinal product for the treatment of cardiovascular diseases mesenchymal stem cells derived from autologous bone marrow, while it is preferable that cardiovascular disease is a myocardial infarction, it is preferable acute myocardial infarction.

Another aim of the invention is the provision of Trad the traditional Chinese medicinal composition, which promotiom survival in vivo mesenchymal stem cells derived from autologous bone marrow, and their differentiation into cardiomyocytes, and traditional Chinese medicinal composition, which is used for the treatment of cardiovascular diseases in combination with mesenchymal stem cells derived from autologous bone marrow, and cardiovascular disease is a myocardial infarction, it is preferable acute myocardial infarction.

In the traditional Chinese medicinal composition according to the invention Latin names and the method of processing of raw materials as an active ingredient obtained from the Dictionary of Traditional Chinese Medicines (first edition, Shanghai Scientific and Technical Publishers, July 1977) and the Chinese Pharmacopoeia (edition 2005, Publishing house of the Chemical Industry).

Traditional Chinese medicinal composition according to the invention can be prepared in any conventional pharmaceutically acceptable dosage forms such as hard capsules, tablets, pills, orally taken liquid form, soft capsules, homeopathic balls, saturated liquid dosage form, etc. in accordance with a customary method, for example the technology of obtaining registered in CI is iska medicinal pharmaceutical preparations (Feng-Bi-tin, Shanghai Scientific Publishing house, December 1997, 1st edition).

The preparations according to the invention can optionally also contain included the usual pharmaceutically acceptable excipients, such as fillers, disintegrating agents, binding agents, glidant, antioxidants, flavouring agents, sweetening agents, suspendresume agents etc. Excipients include, for example, starch, sucrose, lactose, dextrin, pre gelatinizing starch, crosspollination etc. or other Chinese drugs, pharmaceutically acceptable excipients (excipients of different dosage forms, are registered in Chinese medicinal pharmaceutical preparations (Feng-Bi-tin, Shanghai Scientific Publishing house, December 1997, 1st edition).

Preferably, the method of obtaining preparative forms according to the invention is the following: cleaning 5 raw materials in the above proportions leeches, Scorpio, wyporska of crickets, ground beetles and millipedes; drying at a low temperature; sedimentation; extraction of volatile oils of sandalwood and core of dalbergia; defending the residue and the aqueous solution; extraction of ginseng by heat twice in the vessel under reflux with 70% ethanol, 3 hours the first time and 2 hours the second time, combining the extracts and complete the extraction of ethanol; the unification of the rest of the ginseng residue sandalwood and core of dalbergia in aqueous solution; adding thereto root red peony and palm seeds (roasted with stirring; adding a desired amount of water to the double boiling for 3 hours the first time and 2 hours the second time; combine broth, filtering, concentrating the filtrate to a paste with a relative density of 1.20-1.25 range (60ºC); then adding alcohol ginseng extract; mixing; drying at a low temperature, grinding into fine powder; joint grinding of the frankincense tree (processed) and 5 materials from leeches and etc. into fine powder; grinding borneol and joint grinding gradually with the above fine powders to thorough mixing; spray volatile oils and powders; mixing; filling of capsules for the manufacture of 1000 capsules.

Or, preferably, the preparations according to the invention was prepared as follows:

(a) the proportion by weight of raw materials is as follows: ginseng - 3-10 parts, leech - 3-11 parts, ground beetles - 5-10 parts frankincense tree (processed) - 1-5 parts, root red peony - 3-9 parts, the core of dalbergia - 1-5 parts sandalwood - 1-5 parts, Scorpion - 3-9 parts, wypasek cicadas - 3-12 parts, centipede - 1-3 parts, borneol - 1-7 parts, baked with stirring SEM is on the palm - 3-10 parts;

b) method of comminution for medical supplies:

selecting and washing five larval medicines from Scorpion, leeches, centipedes, ground beetles and wyporska cicadas, then merging them with the processed frankincense tree in accordance with the recipe, grinding mortar to obtain a powder of large particles, which can reach a size greater than 80 mesh; Surmeli grinding powder of large particles using technologies such as ultra-fine spray to obtain the medicinal powder particle size less than 100 microns; the introduction of medicinal materials in terms of comminution after cleaning, drying and sterilization.

C) methods of extraction, concentration and drying:

adding water to the core of dalbergia and sandalwood, the extraction of volatile oils from them with subsequent extraction with water, boiling the root, red peony and seeds of the palm, filtering the aqueous solution, the assertion; the extraction of ginseng water after extraction of its ethanol extract ethanol from the alcohol solution and concentrating it in the ethanol extract, filtering the aqueous solution of ginseng and combining it with other aqueous solutions, mixing and concentration in the aqueous extract;

d) method of delivery:

supply of ultra-fine powders in granulares is dried with a fluidized layer, then spraying the extract from stage c) for granulation; polishing granules, adding fine powder of borneol, atomization of volatile oils extracted from the core of dalbergia and sandalwood, filling in the capsule machine for filling capsules after thorough mixing.

Or, preferably, the preparations according to the invention was prepared as follows:

(a) the proportion by weight of raw materials is as follows: ginseng - 3-10 parts, leech - 3-11 parts, ground beetles - 5-10 parts frankincense tree (processed) - 1-5 parts, root red peony - 3-9 parts, the core of dalbergia - 1-5 parts sandalwood - 1-5 parts, Scorpion - 3-9 parts, wypasek cicadas - 3-12 parts, centipede - 1-3 parts, borneol - 1-7 parts, baked with stirring the seeds of the palm - 3-10 parts;

b) method of comminution for medical supplies:

selecting and washing five larval medicines from Scorpion, leeches, centipedes, ground beetles and wyporska cicadas, then merging them with the processed frankincense tree in accordance with the recipe, grinding mortar to obtain a powder of large particles, which can reach a size greater than 80 mesh; Surmeli grinding powder of large particle technology ultra-fine spray to obtain the medicinal powder size less than 100 microns; introduction l the drug materials under the conditions of comminution after cleaning, drying and sterilization.

C) methods of extraction, concentration and drying:

adding water to the core of dalbergia and sandalwood, the extraction of volatile oils from them with subsequent extraction with water, boiling the root, red peony and seeds of the palm, filtering the aqueous solution, the assertion; the extraction of ginseng water after extraction of its ethanol extract ethanol from the alcohol solution and concentrating it in the ethanol extract, filtering the aqueous solution of ginseng and combining it with other aqueous solutions, mixing and concentration in the aqueous extract, and then direct the spray drying to obtain dried spray powders;

d) method of delivery:

supply of ultra-fine powders and the resulting spray powder from step (c) granulating drying with fluidized layer, then spraying solvent to obtain granules; polishing granules, adding fine powder of borneol, atomization of volatile oils extracted from the core of dalbergia and sandalwood, filling in the capsule machine for filling capsules after thorough mixing.

The dosage of the composition according to the invention, calculated on the total mass of raw materials as the active component, is 0.8 to 3 g per reception, 2-4 times per day, preferably SOS is to place 1,11-2,22 g per reception, 3 times a day.

A large number of experimental data presented in the framework of the invention, have shown that the use of a group of medicines according to the invention may promote cell cardiomyoplasty undertaken derived from autologous bone marrow mesenchymal stem cells. Identifying profiles of gene expression after myocardial infarction microchip found that the use of low-dose group of medicines according to the invention can lead to positive changes in gene expression, including stimulating the regulation of anti-inflammatory, protivopolozhnyh and protivovirusnyh genes. Thus, it is believed that the use of a group of medicines according to the invention for therapeutic purposes can improve regional microenvironment after acute myocardial infarction in order to significantly improve the survival and differentiation of implanted mesenchymal cells derived from bone marrow. Therefore, the experimental data of the invention also show that the use of a group of medicines according to the invention for the treatment can effectively improve regional internal microenvironment after acute myocardial infarction and stimulate cell cardiomyoplasty undertaken derived from autologous bone marrow maseng the normal stem cells, through this having a positive impact on clinical use of transplantation of bone-marrow-derived mesenchymal stem cells.

Another aim of the invention is a method of treatment or prevention of cardiovascular disorders traditional Chinese medicinal composition, comprising an introduction to the needy in the patient an effective amount of a traditional Chinese medicinal composition. Cardiovascular disease preferably is a myocardial infarction, preferably represents an acute myocardial infarction. For injection can be applied to conventional approaches used in this field.

Description of figures

Fig. 1. Staining with hematoxylin-eosin (HE) and staining trichromes on Massone affected myocardial regions in four groups of experimental animals under light microscopy. The pictures show that all groups - the first group (control group), the second group (treated only a low dose of a medicinal product according to the invention) and in the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow), manifested severe fibrosis and inflammatory cell infiltration in the affected infarct areas on the merits was not the detected cardiomyocytes. However, in the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination with the medicinal product according to the invention) slight fibrosis and inflammatory cell infiltration. The increase in Fig. 1A is 400, and the increase in Fig. 1B is 40x.

Fig. 2. Survival potential of mesenchymal stem cells derived from bone marrow transplanted into the heart. In Fig. 2A indicated that the transplanted cells stained with the dihydrochloride of 4',6-diamino-2-phenylindole (DAPI), was observed in the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow), but in the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination medicinal product according to the invention) had a lot of transplanted cells, DAPI stained. Fig. 2B shows that there is a statistically significant difference between the survival potential of the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow) and in the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination medicinal product p. the invention). *P<0.0001), image magnification is 400x.

Fig. 3. Differentiation of transplanted into the body of mesenchymal stem cells derived from bone marrow, in cardiomyocyte and vascular structures. In Fig. 3A and 3B shows that some cells labeled with DAPI, Express α-SCA (α-sarcomeric actin) and cTnT (cardiac troponin T). Fig. 3C illustrates that some of DAPI-positive cells Express VSMA (vascular smooth muscle actin) and vascular endothelial specific factor VEGF, indicating the involvement of the formation of blood vessels. In Fig. 3D shows that the differentiation potential of mesenchymal stem cells derived from bone marrow, in the cardiomyocytes of the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow), with statistical comparison of the ratio of DAPI-positive cells differentiate into cardiomyocytes, with the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination medicinal product according to the invention) revealed a significant difference. *P<0.0001), increased Fig. 3A, Fig. 3B and Fig. 3C is 400x. Note: MSC refers to mesenchymal stem cells derived from bone marrow, VWF is the factor a background of Villebranda, SM-actin, not only the et vascular smooth muscle actin, and Overlay refers to the results of the overlay staining three fields of view.

Fig. 4. Expression of connexins in the implanted cells in vivo. Fig. 4A shows that the cells labeled with DAPI, Express connexin 43(Sh). Fig. 4B shows a significant difference in the expression of connexin 43, when comparing the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow) with the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination medicinal product according to the invention). *P<0.0001), increased Fig. 4A is 400x. Note: Overlay refers to the results of the overlay staining three fields of view.

Fig. 5. The density of the capillary network in the area of the infarct and surrounding the infarct zone 6 weeks after transplantation

6 weeks after transplantation, the density of the capillary network in the area of the infarct and surrounding the infarct zone of the second group (treated only a low dose of a medicinal product according to the present invention) and the density of the capillary network in the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow) have no significant differences from control group (*P>0,05, **P>0,05), but both values of density were lower than even what ertai group (treated by transplantation of mesenchymal stem cells, derived from bone marrow, in combination with the medicinal product according to the invention), (respectively,^#P<0,0001,^##P<0,0001).

Fig. 6. The area of the defect of myocardial perfusion detected single photon emission computed tomography (SPECT) after 1 week and 6 weeks after transplantation.

In Fig. 6A shows a typical graph of each group. In Fig. 6B shows the initial results of SPECT, where there is no significant difference between the area of the defect of myocardial perfusion in four groups (*P=0,984). 6 weeks after the transplantation area of the defect of myocardial perfusion in the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination medicinal product according to the invention) is significantly decreased by 22.1±9,3%; when compared with the control group, the second group (treated only a low dose of a medicinal product according to the present invention) and the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow) there is a stark difference (n=7, *P<0,0001).

Fig. 7. Anti-apoptotic action of a medicinal product according to the present invention

(A) Apoptotic cells with damaged DNA in the nucleus, which was determined using antibodies against the binding protein in the myocardium surrounding area information the KTA in pigs and tagging "nick"-the end indirectly limit deoxynucleotidyl-transferase (dUTP) deoxyuridine-triphosphate (TUNEL). At the end of the observation were few apoptotic nuclei in the second group (treated only a low dose of a medicinal product according to the present invention) and in the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination medicinal product according to the invention) (marked by arrow). Increase 20 times. (B) Statistical indicators of apoptosis (AI) of the fourth group. Compared with the control group AI in the second group (treated only a low dose of a medicinal product according to the present invention) sharply decreased (*P<0,0001). In addition, the AI in the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination medicinal product according to the invention) was obviously lower than in the second group (treated only a low dose of a medicinal product according to the present invention) (*P<0,0001). But compared with the control group AI of the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow) were not significantly different (**P=0,289).

Fig. 8. Detection of the level of oxidative stress in the myocardium surrounding the area infarc is a, at the end of the experiment

The activity of superoxide dismutase (SOD) in the second group (treated only a low dose of a medicinal product according to the present invention) and in the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination with the medicinal product according to the invention) increased significantly compared with the control group (*P<0,05,^#P<0,05), but there were obvious differences in the activity of superoxide-dismutase between the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow) and control group (**P=0,449). (B) the Content of malondialdehyde (MDA) in the second group (treated only a low dose of a medicinal product according to the present invention) and in the fourth group (treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination with the medicinal product according to the invention) was significantly compared with the control group (*P<0,05,^#P<0,05). No significant differences between the third group (treated only by transplantation of mesenchymal stem cells derived from bone marrow) and control group (P=of € 0.195).

Specific embodiments of

Example 1: Getting cartonnage of the preparation according to the invention

a) Preparation of raw materials:

ginseng 55g

leeches 103,75 g

ground beetle 68.75 kilopascals g

the frankincense tree (processed) 22,5 g

root red peony and 47.5 g

the core of dalbergia 23,75 g

sandalwood 22,5 g

Scorpio 68.75 kilopascals g

wypasek cicadas 68.75 kilopascals g

centipede of 13.75 g

borneol of 13.75

the seeds of the palm (baked with stirring) 46,25 g;

b) method of comminution for medicinal materials:

selecting and washing five larval medicines from Scorpion, leeches, centipedes, ground beetles and wyporska cicadas, then merging them with the processed frankincense tree in accordance with the recipe, grinding mortar to obtain a powder of large particles, which can reach a size greater than 80 mesh; Surmeli grinding powder of large particles using technologies such as ultra-fine spray to obtain the medicinal powder particle size of less than 30-40 microns; the introduction of medicinal materials in terms of comminution after cleaning, drying and sterilization.

C) methods of extraction, concentration and drying:

adding water to the core of dalbergia and sandalwood, the extraction of volatile oils from them with subsequent extraction with water, boiling the root, red peony and seeds of the palm for 3 hours each, combine broths, filtering, United on the pitch, defending his; double-extraction of ginseng appropriate amount of 70% ethanol for 3 hours each time, the Association of ethanol solutions, the complete removal of the ethanol, then the extraction residue ginseng water, the concentration of ethanol solution in ethanol extract with relative density of 0.9 to 1.1 (60°C)filtering the aqueous solution of ginseng and combining it with other aqueous solutions, mixing and concentration of solution in the aqueous extract with relative density of 0.9 to 1.1 (60°C), the assertion;

d) method of delivery:

supply of ultra-fine powders in a granulating drying with fluidized layer, then spraying the extract stage c) for granulation; polishing granules, adding fine powder of borneol, atomization of volatile oils extracted from the core of dalbergia and sandalwood, filling in the capsule machine for filling in 1000 capsules after thorough mixing.

The dosage of the drug according to the invention, calculated on the total mass of raw materials as the active component, is 2-4 capsules per reception, 3 times a day.

Experimental example. Catalytic role of a medicinal product according to the invention in the application of mesenchymal stem cells derived from bone marrow

Materials and methods

The animal is

Chinese minisini at the age of 10 months, weighing 30±5 kg were provided by the Center of Experimental Animals of the Chinese Agricultural University. All manipulations with animals were conducted humanely in accordance with the "guidelines for the handling and use of laboratory animals"published by the National Institutes of Health, USA. All of the experimental program were supported by the Committee for the Handling of Laboratory Animals of the Chinese Academy of Medical Sciences and approved by the Ethics Committee in relation to the Experimental Animal hospital of traditional Chinese medicine Fu-Wye Beijing United Medical College.

Isolation and cultivation of porcine mesenchymal stem cells derived from bone marrow

Pigs were anestesiologi intramuscular injection of ketamine and diazepam, respectively, at a dose of 25 mg/kg and 1 mg/kg In aseptic conditions trained skin in the region of the crest of the left Ilium, which covered surgical wipes, and a syringe containing a total of 12,500 units of heparin, and needles from the Ilium took 50 ml of bone marrow. All animals before returning to the premises for the content intramuscularly were injected with 0.3 mg of buprenorphine.

Methods of isolating and culturing mesenchymal article is elovich cells, bone-marrow-derived, represented the previously described methods with minor modifications. Briefly, extracted bone marrow was diluted with an equal volume of PBS (saline phosphate buffer)was added to a colloidal suspension of silicon dioxide (solution Percoll for separation of 1.077 g/ml, Sigma Company), mononuclear cells were separated by centrifugation at 800 g for 30 minutes at 4°C. After two washing cellular precipitate PBS, the cells were cultured at a density of 5×10⁵/cm²in normal medium (DMEM containing (modified by Dulbecco Wednesday Needle) (Gibco Company), 10% fetal calf serum (Gibco), 100 U/ml penicillin and streptomycin) in a humid incubator with 5% carbon dioxide at 37°C. After 3 days remove hematopoietic cells, fibroblasts and other non-adherent cells by replacement of the culture medium. Withheld peeled and stuck mesenchymal cells derived from bone marrow were cultured for further proliferation. The culture was replaced every 3 days during the experiment. After 10 days of culturing adherent cells formed a homogeneous cell clone. When the cells reached a confluence of 80%, by adhering to the cells was added a solution of 0.25% trypsin and 0.02% EDTA (ethylenediaminetetraacetic acid) (Sigma) for their resuspendable, when the effectiveness of passirani is 1:3 for further cultivation.

Obtaining models of myocardial infarction and transplanted cells and stimulating the action of a medicinal product according to the invention

28 Chinese mini-pigs were divided into 4 groups: the first group served as control (n=7), the second group received treatment only a low dose of a medicinal product according to the invention, n=7), the third group was treated only by transplantation of mesenchymal stem cells derived from bone marrow, n=7), the fourth group were treated by transplantation of mesenchymal stem cells derived from bone marrow, in combination with the medicinal product according to the invention, n=7).

After the merge cells reached 80%, they were separated from the culture flasks, resuspendable in DMEM (Gibco)containing 10% fetal calf serum, were marked by dihydrochloride 4',6-diamino-2-phenylindole (DAPI) (50 μg/ml, Sigma) for 30 minutes at 37°C. Cells were washed 6 times in PBS for washing unbound DAPI and then selected 3×10⁷cells of each animal and put them in warm DMEM for a few minutes before transplantation. The method of tagging was of great importance, because he had to ensure that the staining of all nuclei transplanted cells.

Pigs were anestesiologi intramuscular injection of ketamine and diazepam in a dose of 25 mg/kg and 1 mg/kg, produced kanyoro is in the trachea, connected to the ventilator for its holding, the anesthesia was supported by intravascular injection of ketamine and diazepam. Produced by thoracotomy sternotomy in the midline, were isolated left anterior descending branch (LAD) coronary artery to the first extending at an angle opposite branches and selected branch tied plastic ring ligature to provide education of the ischemic zone. Before ligation of the coronary artery was intravenously injected with 2 mg/kg lidocaine and intravenous administration would continue until the end of the operation when a maintenance dose of lidocaine 0.5 mg/min Occlusion of the left anterior descending branch (LAD) coronary artery was continued for 90 minutes to provide a model of myocardial infarction-reperfusion.

After 30 minutes of reperfusion, in the zone of the infarct and surrounding areas were injected with 500 μl of the suspension of mesenchymal stem cells derived from autologous bone marrow (3×10⁷cells). Animals in the control group was made DMEM injection of the same amount.

After the transplant incision of the chest sutured, and into the cavity mediastinum was placed catheter 18 gauge to restore a negative intrathoracic pressure and trenirovki residual blood and irrigating solution. After this stopped the introduction of drugs and intoniruyuschy the trachea cannula was removed, taking into account the practicality in relation to wound healing. The drain tube from the chest cavity was removed in the absence of gas leakage or residual blood. All animals were treated with antibiotics after surgery by intramuscular injections of cyclosporine at a dose of 1.0 g 2 times a day for 3 days, with intramuscular injection of animals with buprenorphine for pain relief 2 times a day 0.3 mg per dose for 3 days.

The animals received the drug for treatment according to the invention in the dosage based on the previous experiment, starting 3 days before and continuing until 4 days after transplantation of mesenchymal stem cells derived from bone marrow dose was 0.05 g/kg/D.

Magnetic resonance imaging (MRI)

After one and six weeks after transplantation parameters of cardiac function in experimental animals received respectively by using dynamic MRI and enhanced MRI. Magnetic resonance imaging was performed clinically used by the scanner 1,5T MRI [Siemens, Germany] with the radio frequency coil. Experimental animals were anestesiologi intramuscular injection of ketamine and diazepam dose was 25 mg/kg and 1 mg/kg In MRI was used wirelessly synchronized with ECG spin echo. Using dynamic MRI and corresponding reinforcing MRI scanned one layer every 4 mm, and was 6-sloev, since the level of butterfly valve. Identifiable view in the horizontal and sagittal plane to determine the correct planes along the short axis was determined by the image along the long axis every 60°. Image dynamic MRI has been fixed fast gradient echo (TrueFisp) in combination with the use of parallel technology-sensitive pulse sequence encoding (TSENSE). The parameters of the typical images were as follows: cycle time (TR)=41,7 MS, echo time (TE)=1,39 MS, bandwidth (BW)=965 Hz/PCB (pixel), flip angle (FA)=48°, image matrix=109×192, spatial resolution=3.2 mm×2.0 mm, slice thickness (SL)=6.0 mm, parallel factor=3. Echo-imaging device with the imaging technology TSENSE used to obtain images of perfusion first thread 3-4 short axis and four-chamber perfusion images (TR=6,0 MS, TE=1,2 MS, FA=30°, spatial resolution=2.8 mm×2.8 mm, SL=10.0 mm, parallel factor=2, 4-5 images on a single heartbeat, all levels represented pulses before the first saturation). The first scan was obtained image to about 60 cardiac cycles. Washing was performed with 0.1 mmol Gd-DTPA (Schering AG) with 20 ml of 0.9% NaCl (flow rate 4 ml/s). Intravenous were injected with 0.1 mmol/kg of Magnevist solution after intravenous perfusion, after 5 minutes of VNU is rivanna were injected with 0.2 mmol/kg of gadolinium complex with diethylenetriaminepentaacetic acid (Gd-DTPA), and then directly made pictures using enhanced MRI. For T1 weighting used inversion retrieving a sequence of light flashes (PSIR). PSIR used for the correction of T1. Typical imaging parameters were: TR=700 MS, TE=4,8 MS, BW=130 kHz, in-plane resolution=1,8×1.3 mm, image matrix=156×256, SL=8 mm Re-photographed all of the images obtained using dynamic MRI and enhanced MRI was performed 6 weeks after stem cell transplantation. Produces slices along the short axis in accordance with the original removed the original images, in accordance with the anatomical distribution by regions. In addition, to provide control without pathological changes investigated 5 falsely operated animals using the same structure of the experiment using MRI.

Single photon emission computed tomography (SPECT)

Single photon emission computed tomography myocardial conducted after one and six weeks after transplantation of the cells to identify the zone of the defect of myocardial perfusion. After 40-60 minutes after intravenous injection of 296 MBq (8 MCI) Tc-methoxyisobutylisonitrile, with γ the camera performed SPECT of myocardium with the use of low-energy γ camera with two heads (Varicum, GE) call the transformer high resolution with 20% energy Windows, dened on γ peak of 140 KEV. Received 32 image 40 with each frame received matrix 64×64, range projections from the right anterior oblique 45° ~ to the left posterior oblique 45°, for a total of 180°. SPECT reconstructed low-pass cavity filter Butterworth, cutoff frequency was 0.45, and type 5, by correcting the cardiac axis for reconstructing image data on the short axis, vertical long axis and horizontal long axis, in the triaxial plane. The area of perfusion defect was calculated using techniques video animation flash method.

Histological analysis

To determine the ability of transplanted mesenchymal stem cells derived from bone marrow to differentiate into cardiomyocytes and cells of blood vessels, frozen sections of cardiac tissue were analyzed by fluorescent immune analysis of serial sections with a thickness of 5 μm. Antibodies for detection included: antibodies to vascular endothelial cell-specific factor (VWF 1:50, DAKO), α-smooth muscle actin (SM-actin, 1:50, DAKO), α-musculoskeletal to actin (1:50, DAKO), cardiac troponin T (cTn-T, 1:50, Sigma), connexin 43 (1:50, Sigma). After rinsing the sections of PBS were incubated with labeled with rhodamine or isothiocyanates of fluorescein goat anti-mouse (GAM) or rabbit IgG. Finally took pictures of the laser skaniruesh the m confocal microscope.

To determine the survival and capacity to differentiate in vivo transplanted mesenchymal stem cells derived from bone marrow and myocardium of the left ventricle was cut into 8 slices from top to bottom, and randomly chose 5 frozen sections with a thickness of 5 μm of each piece. Under fluorescent microscope investigated 5 selected randomly horizontal planes of each of frozen sections for counting DAPI - and cTn-T-positive cells. cTn-T-positive cells were considered to be ready to differentiate into cardiomyocytes. Randomly chose 5 slices infarction zone for detecting the intensity of intracellular staining of connexin 43 and analyzed using image analysis.

Revealed the density of the capillary network in the area of the infarct and surrounding areas using the method of preparation of the fabric described in the publication Weidner n, Semple JP, Welch W.R., Folkman J. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma (N Engl J Med 1991; 324: 1-8) [PMID: 1701519]. Sections were stained with antibody to VWF (1:200, DAKO). Chose 5 and 8, respectively slices of areas of the heart and surrounding tissues of each experimental animal, the sections were analyzed by research staff who were not involved in the processing of cells for counting blood vessels with positive staining. For the calculation in each slice was chosen 5 is Olya with high optical gain, the results were represented as the number of capillaries in each field with high optical gain.

Tagging NIC-end, mediated deoxynucleotidyl-transferase (dUTP) deoxyuridine-triphosphate (TUNEL) to detect apoptosis

Applicants used the TUNEL analysis (Rochhe, Germany) for detection of apoptosis in tissue infarction. At the end of the experiment, the applicants had received all animals slices around the area affected heart tissue embedded in paraffin slices were deparaffinization trypsinization, incubated with labeled dUTP end deoxynucleotidyl transferase (TdT) and fluorescein at 37°C in a humid chamber for 60 minutes. Then incubated with specific alkaline phosphatase antibody conjugate associated with hydrofluorination within 30 minutes; 3,3-diaminobenzidin (DAB) was used for color staining for TUNEL analysis, containing the core segments destroyed DNA was stained in blue. To determine the ratio of apoptotic nuclei in sections of the tissue section was subjected to protivorechivuyu cardiospecific monoclonal antibody to desmin (1:100, DAKO), the tissue section was examined under a microscope at the magnification of 400 times, counted more than 100 cardiocytes at least eight fields with high optical gain; the index of apoptosis refers to the percentage of the number of apoptotic cells mi is the card of the total number of cardiocytes in the field of view.

The activity of antioxidant enzymes and lipid peroxides

To identify the level of oxidative stress in myocardial infarction applicants received tissue of the myocardium surrounding the infarction area, at the end of the experiment, revealed superoxide dismutase way using xanthine oxidase (Nanjing Jiancheng on Company); lipid peroxides were expressed as MDA level in the myocardium detected by the method using thiobarbituric acid (Nanjing Jiancheng on Company).

Statistical analysis

Continuous variables were expressed as average ± standard deviation criteria Chi-square (χ²used to analyze the frequency difference between the third and fourth group. After a test, homogeneity of variance and normality were conducted analysis of variance to determine differences between each group at each phase (baseline were tested one week after transplantation, six weeks after transplantation was the final identification); analyzed data from 6 weeks after transplantation, compared with the data one week after transplantation. Compared various parameters in two groups of minimum significant difference (LSD). Data were resolved by way of Bonferroni; the difference was considered highly significant when P<0,05. All data were analyzed with ispolzovaniem software SPSS13.0.

Results

Before the successful collection of all parameters were killed in one animal, respectively in the control group, second group and third group; data on dead animals were not included in statistical analysis.

Histological analysis

6 weeks after transplantation of cells staining HE showed that in the control group, the heart attack caused severe fibrosis with chronic infiltration of inflammatory cells, a rare survival of myocardial cells; in the second and third groups, the situation was similar. In contrast, in the fourth group was found insignificant fibrosis and infiltration of inflammatory cells, and a slight survival of myocardial cells in the heart (Fig. 1).

In the fourth group, there were obviously a greater number of positive cells labeled with DAPI, than in the third group (308,9±88,2, compared to 73.2±21,3, P<0,0001)(Fig. 2A-C).

6 weeks after transplantation analysis of immunofluorescence in the third and fourth groups showed that the positive cells labeled with DAPI, expressed specific for myocardium and specific microvascular proteins, including α-actin skeletal muscle, cardiac troponin T, the factor a background of Villebranda and actin in vascular smooth muscle, indicating that part of the implanted mesenchymal stem cells, recip is the R from the bone marrow, differentiated into cardiac muscle and microvessels (Fig. 3A-C). In particular, in the fourth group frequency differentiation of implanted mesenchymal stem cells derived from bone marrow was significantly higher than in the third group (45,8±5.1%as compared to 8.7±2,4%, P<0,0001)(Fig. 3D).

In addition, intercellular connection positive cells labeled with DAPI in the areas of heart attack, was investigated by detecting expression of connexin 43. The results showed that the expression of connexin 43 in the fourth group was significantly longer than its expression in the third group (16,1±1.4, in comparison with 4.7±1,8, P<0,0001)(Fig. 4A-C).

The density of the capillary network

The density of the capillary network in the areas of infarct and border zones was determined in accordance with the immunohistochemical staining of VWF antibodies; there was no significant difference in the density of the capillary network in the control group compared with the second group and the third group (1,8±0,5/HPF (on the field with high optical gain), compared with 2.0±0,6, compared with 1.8 ą 0.8, P>0,05). However, compared with the third group density of the capillary network in the area of infarction in the fourth group increased by 105% (3,7±1,0/HPF, P<0,0001). The density of the capillary network in the area bordering the zone of infarction, in the fourth group were 8.9±1,9/HPF, which was significantly higher than in the other three groups (4,9±1,3/HPF, 5,1±0,9, 5,2±1,4, < of 0.0001)(Fig. 5).

Magnetic resonance imaging and single photon emission computed tomography

For analysis from each group were selected 36 segments, positively the number of segments with movement disorders to calculate the frequency thickening of the wall. One week after transplantation, the number of segments with movement disorders of the 36 selected segments was in the control group, second group, third group and fourth group respectively 8,2±3,0; 8,3±3,1; 8,7±3,9 and 8.9±3,6, equal to, respectively, of 22.8%, and 23.1%, 24.2% and 24.7% of the total number; between groups was not statistically significant difference (P=0,983). All segments with disabilities used to measure the frequency of regional wall thickening. One week after transplantation among the 4 groups were not statistically significant differences between other parameters, including the frequency of regional wall thickening (P=0,915), the fraction of the expulsion of the left ventricle (LVEF, P=0,996), the size of the zone of infarction (P=0,991), konechnoparametricheskie volume of the left ventricle (EDV, P=0,852), konechnostyami volume of the left ventricle (ESV, P=0,990), the mass index of the left ventricle (LVmass index, P=0.791). 6 weeks after transplantation parameters of cardiac function in the fourth group, except for EDV and ESV, improved significantly (P<0,0001) compared with the control group. Indicators fu the functions of the left ventricle and changes in ventricular geometry are presented in table 1.

The initial level is one week after transplantation, the outcome refers to six weeks after transplantation; LVEF represents the fraction of the expulsion of the left ventricle; EDV is konechnoparametricheskie volume of the left ventricle; ESV is konechnostyami volume of the left ventricle; dyskinetic segments; wall thickness (%); size of the zone of infarction; mass index left ventricular (LV mass index);

*P>0,05 (comparison between the four groups one week after transplantation);^#P>0,05 (the control group in comparison with the second group at 6 weeks after transplantation);

**P>0,05 (konechnoparametricheskie volume and konechnostyami volume in the second group and the third group at 6 weeks after transplantation compared with those in the control group);

^##P<0,0001 (the second group and the third group compared with the control group at 6 weeks after transplantation).

In Fig. 6 shows a typical image obtained with SPECT for the detection zone of perfusion defect after one and six weeks after transplantation. The results of SPECT source of the first week after transplantation showed that there were significant differences among the four g the SCP (respectively 50,7±14.5 percent, compared with 52.7±15.5 per cent, compared to 51.8±16.5%, in comparison with 49,4±16,0%, P=0,984). 6 weeks after transplantation the results of SPECT outcome of the experiment showed that the average size of the area of perfusion defect in the control group, second group and third group was 47,8±11,1%, 50,7±12,5%, 47,3±13,2% (n=6, P=0,899), whereas the average size of perfusion defects in the fourth group was 22.1±9.3 percent, which significantly decreased compared with the first three groups (n=7, P<0,0001).

Cell apoptosis around the myocardium affected by heart attack

By staining the cells of the myocardium proteins that bind specific marker, followed by the end-labeled DNA, it was found that the composition according to the invention significantly reduced the number of apoptotic cells in the zone of infarction of the left ventricle, in the second group and the fourth group, in comparison with their number in the control group (apoptotic index of 6.1±1,4; 2,4±0,9, compared with 10.1±1,8, P<0,0001), and apoptotic index in the fourth group was statistically significantly lower than in the second group (P<0,0001). However, the apoptotic index in the third group did not show significant differences compared with the control group (P=0,289) (Fig. 7).

Assessment of the level of oxidative stress

At the end of the experiment the activity of SOD (superoxide dismutase) in the periphery zone of infarction in the second group of the fourth group were significantly higher than in the control group (98,7±9,8; 105,1±7,0 to of 83.4±8,8 U/mg protein, P<0,05); it showed that the group receiving treatment with the medicinal product according to the invention, could be enhanced acceptor activity "fighting" free radicals; however, between the third group and the control group was not statistically significant difference (87,4±10,2 U/mg protein, P=0,449). Compared with the corresponding levels of MDA in the area of myocardial infarction in the second group and the fourth group levels were significantly reduced (6,1±0,7, 6,0±0,6, compared to 9.0±0.8 nmol/mg protein, P<0,05), indicating that the drug according to the invention could significantly reduce lipid peroxidation and cell damage caused by them; there was a statistically significant difference between the control group and the third group (8,5±0.8 nmol/mg protein, P=of € 0.195) (Fig. 8).

Discussion

The results of the experiment showed that: (1) injection of mesenchymal stem cells derived from autologous bone marrow immediately after acute myocardial infarction/reperfusion did not affect the limited survival and ability to differentiation of the transplanted cells and did not contribute to significant improvement of cardiac function; (2) the application of a low dose of the drug according to the present invention in a short period of time also will not have a mn is a significant beneficial effect on cardiac function however, on the basis of a medicament according to the present invention, the injection of mesenchymal stem cells derived from autologous bone marrow immediately after acute myocardial infarction/reperfusion significantly increased the survival and the ability to differentiation of the transplanted cells, compared with the group that transplanted cells in vivo. In addition, the drug of the present invention in combination with stem cell transplantation may also reduce the size of the zone of infarction, to promote angiogenesis, to improve heart function and fix the restructuring of the ventricular tissue.

The most important data of this experiment is that on the basis of the short-term treatment low dose of the composition according to the invention the injection of mesenchymal stem cells derived from bone marrow directly into the myocardium after acute myocardial infarction and reperfusion in vivo significantly increased survival and ability to differentiation of implanted cells than in the group that only implanted mesenchymal stem cells derived from bone marrow, and at the same time also significantly improved cardiac function. This suggests that transplantation, survival and differentiation of stem cells appear the t pronounced dependence on the microcirculation of the myocardium after acute infarction.

This experiment showed that the drug and transplantation of mesenchymal stem cells derived from bone marrow, have significantly improved the survival and differentiation of cells compared to the group receiving only the mesenchymal stem cells derived from bone marrow. In addition, the use of low doses of the compositions of the present invention did not cause significant improvement in cardiac function. On the basis of the above results we can conclude that the improvement of regional microenvironment using medicinal product according to the invention after acute myocardial infarction improves survival and biological activity of cardiac cells. Despite the fact that a single application of a low dose of the drug according to the invention had no significant effects, it can significantly improve the survival and differentiation of implanted mesenchymal stem cells derived from bone marrow. The results of the experiments showed that short-term use of low doses of a medicinal product according to the invention may facilitate cardiomyoplasty implantation of mesenchymal stem cells from autologous bone marrow. Identifying profiles of gene expression after myocardial infarction using the technology of gene microarrays show is about, separate application of a low dose of the drug according to the invention can lead to positive changes in gene expression, including stimulating the regulation of anti-inflammatory, protivopolozhnyh and protivovirusnyh genes (data not shown). Based on the above studies, the authors consider that the use of a low dose of the drug according to the invention after acute myocardial infarction in a short period of time can improve regional microenvironment of the myocardium after acute infarction, in order to ensure sustainable survival and differentiation of implanted mesenchymal stem cells derived from bone marrow.

Summarizing the above, it can be argued that the authors discovered that the use of a low dose of the drug according to the invention in a short period of time can effectively improve regional microenvironment after acute myocardial infarction, to promote cell cardiomyoplasty by implantation of mesenchymal stem cells derived from autologous bone marrow, and this has implications for the clinical use of transplantation of stromal stem cells in the bone marrow.

1. The use of traditional Chinese medicinal composition for obtaining a medicinal product for which hromatirovanija survival of bone-marrow-derived mesenchymal stem cells in vivo and differentiate into cardiomyocytes, characterized in that the traditional Chinese medicinal composition obtained from the following crude drugs, parts by weight:

2. The use according to claim 1, where the traditional Chinese medicinal composition obtained from the following crude drugs, parts by weight:

3. The use according to claim 1, where the traditional Chinese medicinal composition obtained from the following crude drugs, parts by weight:

4. The use according to claim 1, where the traditional Chinese medicinal composition obtained from the following crude drugs, parts by weight:

5. The use according to claim 1, where the traditional Chinese medicinal composition obtained from the following crude drugs, parts by weight:

6. The use of traditional Chinese medicinal composition for receiving the Oia drug for promotion survival in vivo and differentiation of mesenchymal stem cells, derived from bone marrow, where the medicinal product contains traditional Chinese medicinal composition as defined in any one of claims 1 to 5, as an active ingredient and is a capsule, tablet, pill, oral liquid dosage form, a soft capsule or homeopathic ball, saturated liquid dosage form.

7. The use according to any one of claims 1 to 5, characterized in that the traditional Chinese medicinal composition is used for obtaining a medicinal product for the treatment of cardiovascular diseases mesenchymal stem cells derived from autologous bone marrow.

8. The use according to claim 7, where cardiovascular disease is a myocardial infarction.

9. The use according to claim 7, where cardiovascular disease is an acute myocardial infarction.

10. Traditional Chinese medicinal composition defined in any one of claims 1 to 5, which promotiom survival in vivo mesenchymal stem cells derived from bone marrow, and their differentiation into cardiomyocytes.

11. Traditional Chinese medicinal composition defined in any one of claims 1 to 5, in combination with mesenchymal stem cells derived from autologous bone marrow for the treatment of cardiovascular diseases.

12. Proc. of the traditional Chinese medicinal composition according to claim 11, where cardiovascular disease is a myocardial infarction, preferably acute myocardial infarction.

13. Method for the treatment or prevention of cardiovascular diseases, including introduction to the patients an effective amount of a traditional Chinese medicinal composition defined in any one of claims 1 to 6, and mesenchymal stem cells derived from bone marrow.

14. The method according to item 13, where cardiovascular disease is a myocardial infarction, preferably acute myocardial infarction.

15. The method according to item 13, where mesenchymal stem cells derived from bone marrow, are mesenchymal stem cells derived from autologous bone marrow.