阅读说明：本技术 一种具有预防和/或治疗冠状动脉微血管病变功效的药物组合物 (Pharmaceutical composition with effect of preventing and/or treating coronary artery microangiopathy ) 是由 郝盼盼 刘燕萍 于 2018-11-07 设计创作，主要内容包括：本发明提供一种具有预防和/或治疗冠状动脉微血管病变(CMVD)功效的药物组合物。该组合物包含单体化合物红景天苷和丹参乙酸镁,其可提高CMVD时冠状动脉左前降支血流储备(CFR),以及升高CMVD时左室血管密度、改善心肌微循环,用于预防和/或治疗CMVD。(The invention provides a pharmaceutical composition with the efficacy of preventing and/or treating coronary artery microangiopathy (CMVD). The composition comprises monomer compounds of salidroside and magnesium salvianolate, can improve coronary artery left anterior descending branch blood flow reserve (CFR) in CMVD, and increase left ventricular blood vessel density in CMVD, improve myocardial microcirculation, and can be used for preventing and/or treating CMVD.)

1. A pharmaceutical composition comprises monomer compounds of salidroside and magnesium salvianolate.

2. The pharmaceutical composition of claim 1, wherein the mass ratio of salidroside to magnesium salvianoacetate is 1: 99-99: 1, preferably 1 to 99: 1, more preferably 1-2: 1, most preferably 1.6: 1.

3. a nanoparticle comprising the pharmaceutical composition of claim 1 or 2.

4. The nanoparticle according to claim 3, wherein the nanoparticle is a chitosan nanoparticle, and the pharmaceutical composition according to claim 1 or 2 is encapsulated by chitosan;

preferably, the molecular weight of the chitosan is 300-2000 kDa, preferably 1800-2000kDa, and the degree of deacetylation is more than 92%;

preferably, in the pharmaceutical composition, the mass ratio of salidroside to magnesium salvianoacetate is 1-2: 1, preferably 1.6: 1;

preferably, the mass ratio of chitosan to pharmaceutical composition is 0.5-1.5: 1, preferably 1-2: 1, more preferably 1 to 1.5: 1;

preferably, the particle size of the nanoparticles is 100-600nm, preferably 200-450 nm.

5. A method of preparing nanoparticles as claimed in claim 3 or 4, comprising the steps of:

(1) dissolving chitosan in a glacial acetic acid solution, and adjusting the pH value with a saturated sodium hydroxide solution to obtain a solution I;

(2) adding salidroside and magnesium salvianolate into sodium tripolyphosphate aqueous solution for dissolving to obtain mixed solution II;

(3) while stirring by magnetic force, dropwise adding the solution II into the solution I, and spontaneously forming nanoparticles through electrostatic action of anions and cations; centrifuging, washing, and freeze drying.

6. The method according to claim 5, wherein the pH is adjusted to 5.0 to 6.0 in the step (1); preferably, the pH is adjusted to 5.5;

preferably, in the step (1), the mass-to-volume ratio of chitosan to glacial acetic acid is (12-18) mg: 8 mL;

preferably, in step (1), the concentration of glacial acetic acid is 10%.

7. The method according to claim 5, wherein in the step (2), the mass ratio of salidroside to magnesium salvianoacetate is 1-2: 1, preferably 1.6: 1;

preferably, in the step (2), the concentration of the sodium tripolyphosphate is 0.25 mg/mL;

preferably, in the step (2), the mass volume ratio of the salidroside and the magnesium salvianoacetate to the sodium tripolyphosphate is 13 mg: 4 mL;

preferably, the mass ratio of the chitosan to the sodium tripolyphosphate is 12-18: 1;

preferably, the concentration ratio (mg/mL) of chitosan to sodium tripolyphosphate is 6-9: 1.

8. the production method according to claim 5, wherein in the step (3), the dropping speed of the solution II is 1 drop/5 seconds;

preferably, in step (3), the centrifugation conditions are high speed centrifugation at 15000 rpm for 30 minutes at 4 ℃.

9. Use of a pharmaceutical composition according to claim 1 or 2 or a nanoparticle according to claim 3 or 4 for the preparation of a medicament for the prevention and/or treatment of coronary microvascular pathologies.

10. Use of the pharmaceutical composition of claim 1 or 2 or the nanoparticle of claim 3 or 4 for the manufacture of a medicament for improving myocardial microcirculation and/or increasing left ventricular vascular density and/or increasing left anterior descending blood flow reserve in a mammal.

Technical Field

The invention relates to the field of medicines, in particular to a pharmaceutical composition with the effect of preventing and/or treating coronary artery microangiopathy.

Background

Coronary microvascular disease (CMVD) refers to the clinical syndrome of labored angina or myocardial ischemia laboratory evidence caused by structural and/or functional abnormalities of the anterior and arterioles of the Coronary artery under the action of various pathogenic factors. Symptoms in CMVD patients are mainly manifested by chest pain attacks associated with exertion, and simple dependence on symptoms makes it difficult to distinguish CMVD patients from those with severe coronary stenosis, but the clinical features described below suggest that patients have a greater likelihood of CMVD. Firstly, women are common and account for about 56% -79% of CMVD patients, but most of the women with CMVD patients have the initial symptoms after menopause, and have no difference with the traditional women with coronary heart disease patients. Second, symptoms are mostly fatigue-induced, but not rare with a combination of resting chest pain, and fewer CMVD patients who simply present with resting chest pain. Impaired coronary dilation, increased sympathetic stimulation and sensitivity, and motor-mediated coronary constriction can lead to the development of coronary microvascular dysfunction, and reduced coronary flow reserve. Patients repeatedly attack angina pectoris for a long time to affect life quality. Patients, especially female patients, with significant reductions in coronary flow reserve or myocardial perfusion reserve have a higher incidence of adverse cardiovascular events.

Conventional anti-ischemic therapies are currently proposed for drug therapy using beta blockers, calcium antagonists and nitrates. Other drugs including angiotensin converting enzyme inhibitors, statins, ivabradine, ranolazine and estrogen may be selected if symptoms persist, however no specific drug treatment is available clinically.

Rhodiola rosea, also known as Rhodiola crenulata and Rhodiola crenulata, is a plant of Rhodiola of Crassulaceae (Crassulaceae), which is a perennial herb or subshrubular plant, and is named because the immersion liquid of roots, stems and flowers is red. The environment-friendly solar energy-saving solar water heater is mainly distributed in Himalaya mountainous areas of northern hemisphere, northwest Asia and North America, and has extremely strong environment adaptability and vitality in alpine, dry, anoxic and strong ultraviolet irradiation and large day-night temperature difference areas with the altitude of 1600-4000 meters. More than 90 types of rhodiola rosea are produced in the whole world, and more than 70 types of rhodiola rosea are abundant in wild resources in China, and are mainly produced in northwest of Yunnan province, Tibet and other places. Rhodiola root is used as a medicine by root and stem, has sweet, astringent and cold property, and is commonly used for clearing lung and relieving cough, stopping bleeding, treating traumatic injury, burn and scald, impotence, diabetes and the like in folk. The main components of radix Rhodiolae include Salidroside, tyrosol, rhodiola phenol, radix Rhodiolae, Salidroside, polysaccharide, flavone, amino acids, trace elements, trace volatile oil, etc. Modern research on rhodiola rosea begins in the 50 th of the 20 th century, and people find that rhodiola rosea has the similar effect as ginseng and acanthopanax and does not have the over-excitation effect of ginseng or the constipation effect of acanthopanax, so that the rhodiola rosea is more and more widely valued by people. The basic and clinical researches show that the traditional Chinese medicine rhodiola rosea has biological effects of resisting inflammation, resisting oxidative stress, resisting platelet aggregation, resisting atherosclerosis and the like, and is widely applied to clinical application. The salidroside, the most main active ingredient, has the effects of resisting oxidative stress, resisting aging, reducing blood lipid, reducing blood glucose, regulating immunity, and the like.

The Saviae Miltiorrhizae radix is dried root of Salvia milithiorrhiza Bge, which is a perennial herb of Labiatae, and has bitter taste and slightly cold nature, and has effects of promoting blood circulation for removing blood stasis, nourishing blood for tranquillization, cooling blood for resolving carbuncle, expelling pus and promoting granulation. The effective components of Saviae Miltiorrhizae radix are liposoluble diterpene compounds and water soluble phenolic acid compounds, including Saviae Miltiorrhizae radix polyphenol, tanshinol and tanshinone, wherein the liposoluble tanshinone can improve blood circulation, and has antibacterial and antiinflammatory effects, and the water soluble Saviae Miltiorrhizae radix polyphenol has antioxidant stress, antithrombotic and cytoprotective effects. The salvianolate contains 80% of Magnesium salvianolate (Magnesium tanshinoate B), and 20% of Magnesium tanshinolate homologues such as rosmarinic acid, lithospermic acid, and tanshinol. The magnesium salvianolate is the main component in the water-soluble extract of the traditional Chinese medicine salvia miltiorrhiza bunge, and has biological effects of resisting oxidation, resisting inflammation, protecting vascular endothelium, inhibiting proliferation and migration of vascular smooth muscle cells and the like.

Disclosure of Invention

The invention mainly aims to provide a salidroside and magnesium salvianolate composition for preventing and/or treating coronary artery microvascular lesion (CMVD), double-drug-loaded chitosan nanoparticles encapsulating the composition and a preparation method thereof. The chitosan nanoparticle of the pharmaceutical composition can improve the left anterior descending branch blood flow reserve (CFR) of coronary artery in CMVD, increase the left ventricular blood vessel density in CMVD and improve myocardial microcirculation, and can be used for preventing and/or treating CMVD.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, the present invention provides a pharmaceutical composition comprising the monomeric compound salidroside and magnesium salvianoate or both the monomeric compound salidroside and the monomeric compound magnesium salvianoate as the only active ingredients (or active ingredients); the composition has effects of preventing and/or treating CMVD; can increase CFR in CMVD, increase left ventricular blood vessel density in CMVD, and improve myocardial microcirculation.

Rhodiola rosea and salvia miltiorrhiza are two important traditional Chinese medicinal materials, the components of the two traditional Chinese medicinal materials are complex, for example, the main components of the rhodiola rosea comprise tyrosol, rhodiola rosea phenol, rhodiola rosea, salidroside, polysaccharide, flavone, amino acid, trace elements, trace volatile oil and other compounds, and the contents of the components are often related to the species and the production places of the rhodiola rosea; the components of Salvia miltiorrhiza are more complex, and the root mainly contains fat-soluble diterpenoid components and water-soluble phenolic acid components, and also contains other components such as flavonoids, triterpenes, sterols and the like. Wherein, most of the fat-soluble components of the salvia miltiorrhiza are conjugated quinone and ketone compounds, and mainly comprise: ectotanshinone I, Tanshinone IIA, IIB, IV, V, VI, Cryptotanshinone, Hydroxytanshinone, methyl salvianate, methine salvianolate, salvianolate A, salvianolate B, salvianolate C, salvianolate A, salvianolate B, salvianolate C, salvianolate I, salvianolate II, salvianolate III, 3 alpha-Hydroxytanshinone IIA, salvianolate C, salvianolate B, salvianolate C, salvianolate B, salvianolate C, salvianolate, Agrocybe aegerita (Saloelenone), salvianic acid spiroketal lactone (1socryptotanshinone), salvianol (salviol), and salvianic aldehyde (Tanshinalde-hyde). The water-soluble components of the salvia miltiorrhiza mainly comprise phenolic acid substances: salvianic acid A, also known as Salvianic acid D (+) -13, 4-dihydroxybenzalacetic acid, Salvianic acid B, Salvianic acid C, beta-sitosterol, ursolic acid Protocatechualdehyde (protocathecolone), caffeic acid, isoferulic acid, rosmarinic acid, methyl rosmarinate, Russian alcohol, tigrinine, carnosol, dihydroisotanshinone I, delta '-salvianone (delta' -hydrohemilone), delta '-tanshinone IIA (delta' -hydromicolone IIA), salvianolone (salvinone E), salvianolide (tanhintone), salviandiol A (tanhindiol A), salviandiol B, salviandiol C, and neotanshinone I (salvianolic acid I).

The prior method usually adopts the extract or medicinal powder of salvia miltiorrhiza or rhodiola rosea for treating diseases, but the extract and the medicinal powder of traditional Chinese medicines are all mixtures with complex components, measurable and known components are limited, most of the components are unknown components, and the treatment efficacy depends on the overall action of the mixture. The quality control mainly depends on index components or effective components, the composition change of the extract causes the change of the drug effect or the curative effect, the process always has uncontrollable factors and also always has potential safety hazards, and the further development and the application of the traditional Chinese medicinal materials in modern sustainable fields are not facilitated.

Compared with the prior art, the medicinal composition adopts the monomer compound with a determined structure as the component, and the inventor finds that the combination of salidroside and magnesium salvianoacetate has particularly good curative effect on preventing and/or treating CMVD.

Particularly, when salidroside and magnesium salvianolate are matched for use according to the following contents, the effect is achieved: the mass ratio of salidroside to magnesium salvianoacetate is 1: 99-99: 1, preferably 1 to 99: 1.

in particular, when the ratio is 1-2: 1, further 1.6:1, a better effect can be achieved.

In a second aspect of the invention, the invention provides a nanoparticle comprising two monomeric compounds, salidroside and magnesium salvianoacetate.

The nanoparticles are chitosan nanoparticles, and the chitosan is a pharmaceutical composition consisting of two monomer compounds of salidroside and magnesium salvianolate.

Preferably, the molecular weight of the chitosan is 300-2000 kDa, preferably 1800-2000kDa, and the deacetylation degree is more than 92%.

Preferably, in the pharmaceutical composition, the mass ratio of salidroside to magnesium salvianoacetate is 1-2: 1, preferably 1.6: 1.

in the research process, the invention discovers that when the mass ratio of salidroside to magnesium salvianoacetate is 1.6:1, better pharmacological research results and better bioavailability can be obtained when the salidroside and the magnesium salvianoacetate are prepared into chitosan nanoparticles.

Preferably, the mass ratio of chitosan to pharmaceutical composition is 0.5-1.5: 1, preferably 1-2: 1, more preferably 1 to 1.5: 1.

in the research results of the invention, when the mass ratio of the chitosan to the pharmaceutical composition is 1-1.5: 1, good encapsulation efficiency and drug loading are more easily obtained, and the balance between the drug loading and the encapsulation efficiency is better realized.

Preferably, the particle size of the nanoparticles is concentrated at 100-600nm, preferably 200-450 nm.

In a third aspect of the present invention, the present invention also provides a method for preparing the above nanoparticle, comprising the steps of:

(1) dissolving chitosan in a glacial acetic acid solution, and adjusting the pH value with a saturated sodium hydroxide solution to obtain a solution I;

(2) adding salidroside and magnesium salvianolate into sodium Tripolyphosphate (TPP) water solution for dissolving to obtain mixed solution II;

(3) while stirring by magnetic force, dropwise adding the solution II into the solution I, and spontaneously forming nanoparticles through electrostatic action of anions and cations; centrifuging, washing, and freeze drying.

Preferably, the pH value is adjusted to 5.0-6.0 in the step (1); preferably, the pH is adjusted to 5.5;

preferably, in the step (1), the mass-to-volume ratio of chitosan to glacial acetic acid is (12-18) mg: 8 mL;

preferably, in step (1), the concentration of glacial acetic acid is 10%.

Preferably, in the step (2), the mass ratio of salidroside to magnesium salvianoacetate is 1-2: 1, preferably 1.6: 1.

preferably, in step (2), the concentration of sodium tripolyphosphate is 0.25 mg/mL.

Preferably, in the step (2), the mass volume ratio of the salidroside and the magnesium salvianoacetate to the sodium tripolyphosphate is 13 mg: 4 mL.

Preferably, in the step (3), the dropping speed of the solution II is 1 drop/5 seconds.

Preferably, in step (3), the centrifugation conditions are high speed centrifugation at 15000 rpm for 30 minutes at 4 ℃.

Preferably, the mass ratio of the chitosan to the sodium tripolyphosphate is 12-18: 1.

preferably, the concentration ratio (mg/mL) of chitosan to sodium tripolyphosphate is 6-9: 1.

the chitosan is a macromolecular polymer with cations, the molecular weight of the chitosan is 300-2000 kDa, particularly 1500-2000kDa, the deacetylation degree is more than 92%, and the chitosan and sodium tripolyphosphate with long-chain anions spontaneously form nanoparticles through electrostatic interaction between anions and cations, and experiments show that when the concentration ratio of the chitosan to the sodium tripolyphosphate is 6-9: 1 or the mass ratio of 12-18: 1, the particle diameter is 200-450nm, the absolute value of Zeta potential is about 30mV, below which the solution is clear and cannot form nanoparticles, and above which large-particle aggregates are easily formed.

In a more preferred embodiment, the nanoparticles are prepared by the following method:

(1) dissolving 12-18 mg of chitosan with the molecular weight of 300-2000 kDa in 8mL of 10% glacial acetic acid solution, and adjusting the pH value to 5.0-6.0 by using saturated sodium hydroxide solution to obtain solution I;

(2) adding 8mg of salidroside and 5mg of magnesium salvianolate into 4mL of 0.25mg/mL sodium Tripolyphosphate (TPP) aqueous solution, and uniformly mixing and stirring (1200 revolutions per minute) to obtain solution II;

(3) dropping the mixed solution II into the solution I at the speed of 1 drop/5 seconds, and spontaneously forming nano particles through the electrostatic action of anions and cations to obtain a suspension; centrifuging the suspension at 15000 rpm at 4 deg.C for 30min, collecting precipitate, washing with triple distilled water for 3 times, each time for 5min, and lyophilizing.

The encapsulation rate of salidroside in the nano-particles prepared by the method can reach 87.6 percent, and the encapsulation rate of magnesium salvianolate can reach 79.1 percent; the nanoparticle is in a spheroid shape, and the particle size is concentrated between 100 nm and 600 nm. Pharmacological experiment results of the invention show that the nano-particles not only effectively improve CFR of a CMVD pig model, but also improve left ventricular vascular density, and show that the pharmaceutical composition improves myocardial microcirculation by promoting angiogenesis, thereby improving coronary blood flow reserve during CMVD and improving myocardial ischemia and anoxia.

In a fourth aspect of the present invention, the present invention also provides the use of the above pharmaceutical composition or the above nanoparticle for the preparation of a medicament for the prevention and/or treatment of CMVD.

And the application of the pharmaceutical composition or the nanoparticles in preparing a medicament for improving myocardial microcirculation and/or increasing left ventricular vascular density and/or increasing left anterior descending blood flow reserve of mammals.

Preferably, the medicament is an oral preparation, and the mammal is preferably a human, a pig and the like.

Compared with the prior art, the invention has the following beneficial effects:

the existing oral medication of salidroside or magnesium salvianolate is often marked by only taking salidroside or magnesium salvianolate as an effective component, and the actual use still is the mixed effect result of a medicine extract or medicinal material powder containing a plurality of components, so that the result often causes difficult quality control, unstable medicine effect and low bioavailability, and unknown components easily bring potential safety hazards. At present, no pharmaceutical composition taking salidroside and magnesium salvianoacetate monomeric compounds as main components exists, and the invention discovers that the salidroside and magnesium salvianoacetate pharmaceutical composition is applied to CMVD treatment/prevention by improving myocardial microcirculation for the first time.

According to the invention, the salidroside and magnesium salvianolate composition is prepared into chitosan nanoparticles, chitosan can be attached to the surface of a mucous membrane and can open the tight connection of epithelial cells, so that the chitosan can be used as an accelerant of a macromolecular drug to improve the bioavailability and achieve the purposes of slow release and long acting, and the chitosan and sodium tripolyphosphate spontaneously form nanoparticles through the electrostatic action of anions and cations, thereby realizing the entrapment of salidroside and magnesium salvianolate. Wherein, the encapsulation rate of salidroside can reach 87.6 percent, and the encapsulation rate of magnesium salvianolate can reach 79.1 percent; the nanoparticle is in a spheroid shape, and the particle size is concentrated between 100 nm and 600 nm. Pharmacological experiment results of the invention show that the nano-particles not only effectively improve CFR of a CMVD pig model, but also improve left ventricular vascular density, and show that the pharmaceutical composition improves myocardial microcirculation by promoting angiogenesis, thereby improving coronary blood flow reserve during CMVD and improving myocardial ischemia and anoxia.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows schematic view (. times.10) of chitosan nanoparticle transmission electron microscope of the present invention⁵)。

Fig. 2 is a particle size distribution diagram of chitosan nanoparticles of the present invention.

Fig. 3 is a schematic diagram of coronary artery left anterior descending branch flow reserve (CFR) measurement.

FIG. 4 is the effect of chitosan nanoparticles of the present invention on CMVD mini pig model CFR; wherein, P<Sham control group 0.01 vs;^#P<0.05vs. model set.

FIG. 5 shows the chitosan nanoparticle pair CMVD miniature pig model left chamber of the inventionEffect of vascular density (CD34 immunohistochemical staining was observed under 20-fold ocular and photographed); wherein, A: CD34 immunohistochemical labeling of vascular endothelium (20-fold ocular); b: and (5) quantitatively analyzing the blood vessel density. P<Sham control group at 0.05 vs;^##P<0.01vs. model set. .

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.