Sodium salvianolate A hydrate and its preparation method

Abstract

The present invention provides a sodium salt hydrate of salvianolic acid A and a preparation method thereof. Specifically, the sodium salt hydrate of salvianolic acid A provided by the present invention is a crystal formed by salvianolic acid A, salvianolic acid A anion, metal ions, and water molecules, which has higher stability, better water solubility, lower production cost, and higher purity.

Description

Technical Field

[0001] The present invention belongs to the field of chemistry. Specifically, the present invention relates to a sodium salvianolate A hydrate and a preparation method thereof. Background Art

[0002] With the continuous improvement of people's living standards, the proportion of animal foods in the diet has gradually increased. The saturated fatty acids and cholesterol in them form insoluble lipoproteins, which deposit on the arterial blood vessel wall and gradually form plaques. As the plaques grow, atherosclerosis occurs and the blood vessels are gradually blocked, leading to ischemia. In addition, due to various reasons such as cold and heat changes, exercise, and emotional excitement, the plaques may fall off and gradually flow with the blood to the capillaries, blocking the capillaries and thus blocking the blood flow, resulting in ischemia of the corresponding tissues and organs, thereby causing ischemic injury.

[0003] Anticoagulants, thrombolytics, antiplatelet drugs, mechanical thrombectomy, vascular stents, bypass surgery and other drug and surgical means can inhibit the formation of thrombus or remove thrombus. After reperfusion, sufficient oxygen in the blood will cause the production of "reactive oxygen species" such as free radicals in the reperfused part, resulting in "reperfusion injury", which is also harmful to the body and may even endanger life.

[0004] The above-mentioned ischemia / reperfusion injury can occur in the cardiovascular and cerebrovascular systems, liver, kidneys, nervous system, necrotic limb parts of diabetic patients, etc. in the human body, posing a great threat to human health.

[0005] Salvia miltiorrhiza Bunge is a traditional Chinese medicine derived from the dried roots and rhizomes of the plant Salvia miltiorrhiza in the Labiatae family. The water-soluble phenolic acid compounds in it are one of the main active ingredients, including salvianolic acid A, salvianolic acid B, lithospermic acid, rosmarinic acid, danshensu, etc. Research shows that salvianolic acid A is the most active compound in Salvia miltiorrhiza (Lu, Y.; Foo, L.Y., Polyphenolics of Salvia--a review. Phytochemistry 2002, 59, (2), 117 - 40; Liu, G.T.; Zhang, T.M.; Wang, B.E.; Wang, Y.W., Protective action of seven natural phenolic compounds against peroxidative damage to biomembranes. Biochemical Pharmacology 1992, 43, (2), 147 - 152; Jiang, B.; Li, D.; Deng, Y.; Teng, F.; Chen, J.; Xue, S.; Kong, X.; Luo, C.; Shen, X.; Jiang, H.; Xu, F.; Yang, W.; Yin, J.; Wang, Y.; Chen, H.; Wu, W.; Liu, X.; Guo, D.A., Salvianolic acid A, a novel matrix metalloproteinase-9 inhibitor.prevents cardiac remodeling in spontaneously hypertensive rats. PLoS One 2013, 8, (3), e59621), and its activities include anti-inflammatory, antioxidant, etc. In vitro and in vivo experiments show that it has good therapeutic effects on various diseases.

[0006] However, due to its strong antioxidant effect, the stability of salvianolic acid A is extremely poor, and it is very easy to degrade due to oxidation, hydrolysis and other effects during storage, making it difficult to meet the requirements for its development as a drug. In addition, because the water-soluble components of Salvia miltiorrhiza have similar structures and properties, and have strong water solubility, their purification is very difficult. The preparation process of high-purity salvianolic acid A samples with low cost and high efficiency has always been a difficult problem.

[0007] Chinese Patent Document CN200810223651.1 discloses a preparation process for 90% salvianolic acid A. However, a purity of 90% is difficult to support its development as a new chemical drug (since in the general dosage range, new chemical drugs require qualitative research on impurities with a content exceeding 0.1% and safety research on impurities exceeding 0.15%, so it may involve a large amount of quality, impurity and safety research, significantly increasing the drug safety risk and greatly increasing the R & D difficulty and cost), especially when developed as an injection, there are insurmountable difficulties.

[0008] Chinese Patent Document CN 201310487751.6 prepared a freeze-dried powder of salvianolic acid A with a purity that can exceed 97% and can be scaled up to industrial production scale. However, this product is an amorphous freeze-dried powder, and salvianolic acid A in this state has poor stability and needs to be stored at below -20 °C for long-term stability, which will significantly increase the difficulty and various costs in the actual production, storage, transportation and use processes.

[0009] Chinese Patent Document CN 201710055331.9 developed a preparation method for the crystallization of salvianolic acid A with a purity of over 99.5%, and the stability in the crystalline state is also significantly improved. However, the water solubility of this crystal is poor and it takes a long time to dissolve, which is difficult to meet the requirements for use as an injection preparation in clinical practice (it needs to dissolve quickly in an aqueous solution).

[0010] Due to the extremely low oral bioavailability of salvianolic acid A (the oral bioavailability in beagle dogs is only 1.47 - 1.84%) (Sun, J., et al. (2013). "Pharmacokinetic study of salvianolic acid A in beagle dog after oral administration by a liquid chromatography - mass spectrometry method: a study on bioavailability and dose proportionality." J. Ethnopharmacol. 148(2): 617 - 623.), in order to ensure effectiveness, the development of new drugs needs to be mainly based on non - oral preparations. Since cardiovascular and cerebrovascular diseases have an acute onset, intravenous injection preparations are the preferred dosage form.

[0011] Therefore, those skilled in the art are committed to developing a salvianolic acid A raw material drug and its production process with high purity, strong stability, good water solubility, easy preparation, and meeting drug development (especially for intravenous injection drugs) for the prevention and treatment of ischemic cardiovascular and cerebrovascular diseases and oxidative stress - induced damage diseases of body tissues and organs. Summary of the Invention

[0012] The object of the present invention is to provide a sodium salt hydrate of salvianolic acid A, a preparation method thereof, and uses thereof.

[0013] In the first aspect of the present invention, there is provided a sodium salt hydrate of salvianolic acid A represented by formula I,

[0014]

[0015] The molecular formula of the sodium salt hydrate of salvianolic acid A is C 26 H 22 O 10 ·C 26 H 21 O 10 ·Na·8(H₂O).

[0016] In another preferred example, the powder X-ray diffraction pattern of the sodium salt hydrate of salvianolic acid A has characteristic peaks at the following 2θ values: 12.00 ± 0.1, 18.86 ± 0.1, 19.02 ± 0.1, and 22.28 ± 0.1.

[0017] In another preferred example, the powder X-ray diffraction pattern of the sodium salt hydrate of salvianolic acid A further includes characteristic peaks at the following 2θ values: 16.66 ± 0.1, 17.78 ± 0.1, 21.88 ± 0.1, 24.22 ± 0.1, 25.50 ± 0.1, and 25.80 ± 0.1.

[0018] In another preferred example, the powder X-ray diffraction pattern of the sodium salt hydrate of salvianolic acid A has characteristic peaks at the following 2θ values:

[0019] 6.76±0.1, 6.98±0.1, 7.12±0.1, 8.50±0.1, 10.80±0.1, 11.38±0.1, 12.00±0.1, 12.28±0.1, 12.68±0.1, 13.70±0.1, 13.98±0.1, 14.30±0.1, 16.26±0.1, 16.66±0.1, 17.02±0.1, 17.48±0.1, 17.78±0.1, 18.12±0.1, 18.38±0.1, 18.86±0.1, 19.02±0.1, 19.42±0.1, 19.92±0.1, 20.18±0.1, 20.88±0.1, 21.52±0.1, 21.72±0.1, 21.88±0.1, 22.08±0.1, 22.28±0.1, 22.74±0.1, 23.10±0.1, 23.58±0.1, 23.92±0.1, 24.22±0.1, 24.76±0.1, 24.98±0.1, 25.20±0.1, 25.50±0.1, 25.80±0.1, 26.32±0.1, 27.04±0.1, 27.78±0.1, 27.96±0.1, 28.22±0.1, 29.16±0.1, 29.64±0.1, 30.36±0.1, 30.54±0.1, 31.34±0.1, 31.64±0.1, 31.86±0.1, 32.28±0.1, 32.54±0.1, 32.80±0.1, 33.56±0.1, 34.44±0.1, 35.34±0.1, 35.98±0.1, 36.24±0.1, 36.52±0.1, 37.96±0.1, 38.30±0.1, 38.60±0.1, 39.14±0.1, 41.10±0.1, 43.80±0.1, and 45.04±0.1..

[0020] In another preferred embodiment, the sodium salt hydrate of salvianolic acid A has an X-ray powder diffraction pattern (XRPD) substantially as Figure 4 shown.

[0021] In another preferred embodiment, the sodium salt hydrate of salvianolic acid A shown in Formula I is prepared by the preparation method described in the third aspect of the present invention.

[0022] In the second aspect of the present invention, there is provided a crystal of sodium salt hydrate of salvianolic acid A, which is determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are as follows: α = γ = 90.0°, β = 117.5±0.1°.

[0023] In another preferred embodiment, the unit cell parameters are as follows: α = γ = 90.00°, β = 117.480°. Preferably, the unit cell volume The number of asymmetric units Z = 4 within the unit cell.

[0024] In another preferred example, the absolute configuration of the sodium salt hydrate crystal of salvianolic acid A is:

[0025]

[0026] In another preferred example, the sodium salt hydrate crystal of salvianolic acid A is determined to be in the C2 space group by single crystal X-ray diffraction.

[0027] In the third aspect of the present invention, a preparation method of the sodium salt hydrate of salvianolic acid A is provided, and the method includes the steps:

[0028] (1) Prepare a salvianolic acid A solution having a first temperature;

[0029] (2) Add a sodium salt to the salvianolic acid A solution obtained in step (1), and stir to dissolve the sodium salt;

[0030] (3) Cool the solution in step (2) to a second temperature for crystallization to obtain the sodium salt hydrate of salvianolic acid A;

[0031] Wherein, the first temperature is 40 - 80°C; the second temperature is 0 - 20°C.

[0032] In another preferred example, the first temperature is 40 - 60°C; preferably about 50 - 60°C.

[0033] In another preferred example, the sodium salt added in step (2) is a strong base strong acid salt containing sodium (strong acid salt of sodium), such as sodium chloride, sodium sulfate, sodium bisulfate, sodium bromide, sodium dihydrogen phosphate, etc.

[0034] In another preferred example, in the salvianolic acid A solution obtained in step (1), the mass concentration of salvianolic acid A ≥ 5%; more preferably, ≥ 10%; most preferably, ≥ 20%.

[0035] In another preferred example, in the salvianolic acid A solution obtained in step (1), the mass concentration of salvianolic acid A is about 5% - 20%; preferably about 5% - 15%.

[0036] In another preferred example, the cooling rate in step (3) is 0.1°C / min - 3°C / min; preferably the cooling rate is 0.1°C / min - 2°C / min; more preferably the cooling rate is 0.1°C / min - 0.3°C / min.

[0037] In another preferred example, in step (3), the temperature is first decreased at a rate of 1-2 °C / min to about 20 °C, and after maintaining this temperature until crystallization turbidity appears, the temperature is further decreased at a rate of 0.1-1 °C / min to 0-4 °C and held at this temperature.

[0038] In another preferred example, the second temperature is 0-20 °C; preferably, the second temperature is 1-15 °C; more preferably, the second temperature is 4-10 °C.

[0039] In another preferred example, during the cooling and crystallization process in step (2), continuous stirring is carried out.

[0040] In another preferred example, after the temperature is decreased to the second temperature in step (2), crystallization is carried out for 5-48 h; preferably, crystallization is carried out for 8-36 h; more preferably, crystallization is carried out for 8-24 h.

[0041] In another preferred example, the method further includes the step of subjecting the obtained sodium salt hydrate of salvianolic acid A to vacuum drying treatment. Preferably, the temperature of the vacuum drying treatment is 10-30 °C.

[0042] In another preferred example, the method further includes the step of subjecting the obtained sodium salt hydrate of salvianolic acid A to recrystallization treatment; preferably, the recrystallization step includes:

[0043] (a) Dissolving the sodium salt hydrate of salvianolic acid A in an aqueous solvent having a first temperature to obtain a solution of sodium salt of salvianolic acid A;

[0044] (b) Cooling the solution in step (a) to the second temperature for crystallization to obtain a refined product of the sodium salt hydrate of salvianolic acid A;

[0045] wherein, the first temperature is 40-80 °C; the second temperature is 0-20 °C.

[0046] In another preferred example, the first temperature is 45-70 °C; preferably about 50 °C.

[0047] In another preferred example, in the solution obtained in step (a), the mass concentration of salvianolic acid A is about 5%-20%; preferably about 5%-15%.

[0048] In another preferred example, the cooling rate in step (b) is 0.1 °C / min - 1 °C / min; preferably the cooling rate is 0.1 °C / min - 0.5 °C / min; more preferably the cooling rate is 0.1 °C / min - 0.3 °C / min.

[0049] In another preferred example, the second temperature is 0-20 °C; preferably, the second temperature is 1-15 °C; more preferably, the second temperature is 4-10 °C.

[0050] In another preferred embodiment, during the cooling and crystallization process in step (b), continuous stirring is carried out.

[0051] In another preferred embodiment, after cooling to the second temperature in step (b), crystallization is carried out for 5 - 48 h; preferably, for 8 - 36 h; more preferably, for 12 - 24 h.

[0052] In another preferred embodiment, the method further includes a step of subjecting the obtained refined product of sodium salvianolate A hydrate to vacuum drying treatment. Preferably, the temperature of the vacuum drying treatment is 10 - 30°C.

[0053] In another preferred embodiment, in the method, the HPLC purity of the salvianolic acid A raw material is ≥70%; preferably, the HPLC purity of the salvianolic acid A raw material is ≥80%; alternatively, the HPLC purity of the salvianolic acid A raw material is between about 75% - 95%.

[0054] In another preferred embodiment, the mass fraction of water in the aqueous solvent is ≥70%; preferably ≥80%; more preferably ≥90%.

[0055] In another preferred embodiment, the aqueous solvent is selected from: water, ethanol aqueous solution, acetone aqueous solution, etc.

[0056] In another preferred embodiment, in step (2), the molar ratio of salvianolic acid A to the sodium salt is 1 - 3:3 - 1; preferably 1 - 3:1 - 2; most preferably 2:1.

[0057] In a fourth aspect of the present invention, there is provided the use of the sodium salvianolate A hydrate described in the first aspect of the present invention or the crystal of the sodium salvianolate A hydrate described in the second aspect of the present invention for preparing a pharmaceutical composition for treating or preventing diseases, wherein the diseases are selected from: cardiovascular and cerebrovascular diseases, oxidative stress injury, immune system diseases, hyperlipidemia, diabetic complications, etc.

[0058] The diseases are ischemic cardiovascular and cerebrovascular diseases, and related diseases caused by oxidative stress injury of organs or tissues (such as cardiovascular and cerebrovascular, liver, kidney, nervous system, etc.) (such as the related injuries of the above organs or tissues caused by ischemia / reperfusion).

[0059] In a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0060] The sodium salvianolate A hydrate described in the first aspect of the present invention or the crystal of the sodium salvianolate A hydrate described in the second aspect of the present invention; and a pharmaceutically acceptable carrier.

[0061] In another preferred embodiment, the pharmaceutical composition is composed of the sodium salvianolate A hydrate described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0062] In another preferred example, the pharmaceutical composition is an injection.

[0063] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. Description of the Drawings

[0064] Figure 1 are the X-ray single crystal diffraction crystal data of sodium salvianolate A hydrate;

[0065] Figure 2 is the stereoscopic structure ellipsoid diagram of the asymmetric unit of the single crystal of sodium salvianolate A hydrate;

[0066] Figure 3 is the projection diagram of the crystal cell stacking of the single crystal of sodium salvianolate A hydrate along the b direction;

[0067] Figure 4 is the powder diffraction pattern of the crystal of sodium salvianolate A hydrate;

[0068] Figure 5 is the list of 2θ values of the powder diffraction of sodium salvianolate A hydrate;

[0069] Figure 6 is the micrograph (10× eyepiece + 4× objective) of the single crystal of sodium salvianolate A hydrate prepared by the present invention;

[0070] Figure 7 is the micrograph (10× eyepiece + 4× objective) of the crystalline powder of the sodium salvianolate A complex prepared according to the method of patent document CN201910643821.X;

[0071] Figure 8 shows the protective effect of the crystal of sodium salvianolate A hydrate on the damage of HepG2 cells caused by hydrogen peroxide. Detailed Embodiments

[0072] After extensive and in-depth research, the inventor of the present invention unexpectedly prepared a complex composed of salvianolic acid A, salvianolic acid A anion, sodium ion, and water molecule. Through X-ray single crystal diffraction, X-ray powder diffraction, etc., the crystal form, crystal water and other characteristics of this type of complex crystal were determined, which proved that the sodium salt hydrate of salvianolic acid A prepared in the present invention is a new substance with a new crystal form and its purity exceeds 99%. The present invention also discloses a preparation method of this type of new crystal form. Using salvianolic acid A with a purity of about 80% as the starting material to prepare the sodium salt hydrate of salvianolic acid A of the present invention, and this sodium salt hydrate of salvianolic acid A can be applied to the treatment of ischemic cerebrovascular diseases and ischemic cardiovascular diseases. Moreover, this sodium salt hydrate of salvianolic acid A has the characteristics of low production cost, high product purity, good stability, and good water solubility.

[0073] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0075] Although any methods and materials similar or equivalent to those described in the present invention may be used in the practice or testing of the present invention, preferred methods and materials are exemplified herein.

[0076] Specifically, the present invention prepared a complex (pure substance) composed of salvianolic acid A, salvianolic acid A anion, sodium ion, and water molecule, obtained its single crystal, and studied the therapeutic effect of this complex on ischemic cerebrovascular diseases and ischemic cardiovascular diseases with common positive drugs on the market as controls.

[0077] Compared with the amorphous salvianolic acid A freeze-dried powder, this series of crystalline complexes have the characteristics of high purity, good stability, and low production cost. Compared with the reported salvianolic acid A crystals, this series of crystalline complexes are single crystals, have good water solubility, and low production cost, and are more suitable for developing them into intravenous injection preparations. Compared with the positive drugs butylphthalide sodium chloride injection, salvianolic acid polyphenols for injection, and salvianolic acid polyphenolate for injection, this series of crystalline complexes have the effects of significantly reducing the cerebral / myocardial infarction area, significantly reducing the mortality rate of model animals, and significantly improving the limb function recovery of model animals with cerebral ischemia / myocardial ischemia. This series of crystalline complexes can be applied to the treatment of ischemic heart disease and ischemic cerebrovascular diseases and used as the active pharmaceutical ingredient (API) of related drug injection preparations.

[0078] The properties of the sodium salt hydrate of salvianolic acid A of the present invention can be studied by the following various methods and instruments. For example, X-ray single crystal diffraction, X-ray powder diffraction, differential scanning calorimetry, and thermogravimetric analysis (TGA) can be used for analysis. These analysis methods can be conventional methods in the art.

[0079] For example, X-ray single crystal diffraction analysis can be performed using a Bruker D8 Venture single crystal X-ray diffractometer, and the test conditions are: CuKα radiation, scanning.

[0080] Through X-ray single crystal diffraction analysis, the single crystal characteristics and structural types of the sodium salt hydrate of salvianolic acid A of the present invention were obtained.

[0081] Specifically, the absolute configuration of the sodium salt hydrate crystal of salvianolic acid A was measured as:

[0082]

[0083] For the sodium salt hydrate crystal of salvianolic acid A, it was determined by X-ray single crystal diffraction to be monoclinic, space group C2, and the unit cell parameters are: α = γ = 90.0°, β = 117.5°.

[0084] The method of X-ray powder diffraction for determining the crystal form described in the present invention is known in the art. The specific detection conditions of the present invention can refer to the X-ray powder diffraction patterns of the crystals of the present invention. The results show that each crystal of the present invention has specific characteristic peaks in the X-ray powder diffraction (XRPD) pattern.

[0085] There is no rule to follow as to whether a certain compound can combine with a solvent to form a solvate or combine with several molecules of the solvent. When the molecular structures are the same but the crystal forms are different, it is possible to have different bioavailability, solubility, dissolution rate, chemical and physical stability, melting point, color, filterability, density, and fluidity. Some polymorphs are difficult to formulate due to their shape or hygroscopicity. X-ray powder diffraction pattern is a necessary method for crystal form identification, but not the only one. For hydrate crystal forms, due to the different ways in which water combines with the compound, it may result in similar X-ray powder diffraction patterns, but different other characterization data, such as DSC spectra. For the same drug, different crystal forms or different solvates will exhibit differences in stability, fluidity, hygroscopicity, solubility, and compressibility, all of which will have an important impact on the application of the drug, thus greatly affecting the bioavailability and usability of the drug.

[0086] For example, Patent Documents CN201910643821.X and CN201710067693.X describe a salvianolic acid A salt complex, which contains salvianolic acid A molecules, salvianolic acid A anions, and cations in its structure, but does not contain crystal water, and its water solubility is tested as "sparingly soluble". In order to compare the differences between the "salvianolic acid A salt complex" described in this patent document and the "sodium salt hydrate of salvianolic acid A" described in the present invention, referring to its method, a salvianolic acid A sodium salt complex was prepared. Figure 6 and Figure 7 Visible are the micrographs of the single crystal of the sodium salt hydrate of salvianolic acid A and the salvianolic acid A sodium salt complex of the present invention. As can be seen from the figure, the sodium salt hydrate of salvianolic acid A prepared in the present invention is a clearly visible "single crystal", while the salvianolic acid A sodium salt complex is a "crystalline powder" and no obvious "single crystal" is seen. The differences between the sodium salt hydrate of salvianolic acid A prepared in the present invention and the salvianolic acid A sodium salt complex of the comparative patent are as follows:

[0087] The former is a single crystal, a pure substance, with a stable chemical composition and good water solubility;

[0088] The latter is a crystalline powder, a mixture, and theoretically cannot guarantee the stability of its chemical composition (such as the ratio of salvianolic acid A molecules to sodium salvianolate), and has poor water solubility;

[0089] According to pharmaceutical registration regulations, the most basic characteristic of an injection raw material drug is that its composition is fixed and it is a pure substance. The single crystal of the sodium salt hydrate of salvianolic acid A prepared in the present invention has significant advantages when used as an injection raw material drug compared with the crystalline powder of the salvianolic acid A sodium salt complex of the comparative patent.

[0090] The present invention also provides a crystallization process for salvianolic acid A, by which a new crystal form of sodium salt hydrate of salvianolic acid A is obtained. Compared with the amorphous solid powder obtained by the drying process, this crystallization has the advantages of high purity, low production cost and better stability, and can meet the production requirements of new drug raw materials for injection. Compared with the salvianolic acid A crystals reported in the literature, the sodium salt hydrate crystal of salvianolic acid A obtained in the present invention has the advantages of high product purity, low production cost and good water solubility of the product, and is suitable for the development of salvianolic acid A injection preparations.

[0091] Active ingredient

[0092] As used herein, the term "active ingredient" or "active compound" refers to the sodium salt hydrate of salvianolic acid A of the present invention.

[0093] The present invention discloses a sodium salt hydrate crystal of salvianolic acid A and a preparation method thereof.

[0094] The salvia miltiorrhiza medicinal material is prepared into a water extract of salvia miltiorrhiza with reference to the preparation method of "Preparation of Water Extract of Salvia Miltiorrhiza" in the Chinese Pharmacopoeia (Part I) (2020 Edition). The latter is reacted to prepare a crude product of salvianolic acid A, and then after resin column chromatography, a sample of salvianolic acid A with a purity of ≥80% is obtained. Then, the sample is subjected to the processes of crystallization and recrystallization to prepare a high-purity sodium salt hydrate crystal of salvianolic acid A. This crystal is composed of salvianolic acid A, salvianolic acid A anion, cation, and water molecules. This series of complexes are all new pure substances and have a brand-new crystal form. Compared with the amorphous salvianolic acid A powder, it has the characteristics of good stability and solubility. Compared with the salvianolic acid A crystal, it has the characteristic of good water solubility. The sodium salt hydrate of salvianolic acid A prepared in the present invention is more suitable for the development of intravenous injection preparations, and its application in the treatment of ischemic stroke and ischemic cardiovascular diseases has significantly better effects than the positive drugs butylphthalide sodium chloride injection and salvianolate for injection.

[0095] Pharmaceutical composition and administration method

[0096] Since salvianolic acid A can be used to treat the following diseases: cardiovascular diseases (such as ischemic heart disease, stroke), hyperlipidemia, diabetes and its complications, etc. Therefore, the sodium salt hydrate of salvianolic acid A of the present invention can be used to treat or prevent the above diseases.

[0097] The pharmaceutical composition of the present invention contains the sodium salt hydrate of salvianolic acid A of the present invention within a safe and effective amount range and a pharmaceutically acceptable excipient or carrier. The "safe and effective amount" herein refers to: the amount of the sodium salt hydrate of salvianolic acid A is sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the crystal form of the present invention per dose, more preferably, it contains 10-200 mg of the crystal form of the present invention per dose. Preferably, the "per dose" is an injection dose or a capsule or a tablet.

[0098] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy or stability of the active ingredient, etc. Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as wetting agents (such as sodium dodecyl sulfate), disintegrants, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0099] There is no particular limitation on the administration mode of the sodium salt hydrate of salvianolic acid A or the pharmaceutical composition of the present invention. Representative administration modes include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. Preferably, administration is by injection, such as intravenous injection.

[0100] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or calcium phosphate, or is mixed with the following components: (a) fillers or volume-increasing agents, for example, starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxyethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizing agents, for example, paraffin wax; (f) absorption accelerators, for example, quaternary amine compounds; (g) wetting agents, for example, cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0101] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and the release of the active ingredient in such a composition can be delayed and released in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and wax-like substances. If necessary, the active ingredient can also be in the form of microcapsules with one or more of the above excipients.

[0102] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0103] In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents and fragrances.

[0104] In addition to the active ingredient, the suspension may contain suspending agents, for example, ethoxylated isooctadecanol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum monostearate and agar or mixtures of these substances.

[0105] The composition for parenteral injection may contain physiologically acceptable sterile aqueous or non - aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for re - dissolving into sterile injectable solutions or dispersions. Suitable aqueous and non - aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof. The sodium salt hydrate of salvianolic acid A of the present invention is particularly suitable for injectable administration due to its excellent solubility.

[0106] The dosage forms of the crystalline form of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary under sterile conditions.

[0107] The sodium salt hydrate of salvianolic acid A of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0108] When using the pharmaceutical composition, a safe and effective amount of the crystalline form of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is an effective dosage considered pharmaceutically. For a person weighing 60 kg, the daily dosage is usually 1 - 2000 mg, preferably 10 - 500 mg. Of course, the specific dosage should also consider factors such as the route of administration and the health status of the patient, which are all within the scope of the skills of a skilled physician.

[0109] The main advantages of the present invention are as follows:

[0110] (1) The sodium salt hydrate crystal of salvianolic acid A prepared by the present invention has stronger stability compared with the freeze - dried powder of salvianolic acid A, and is more resistant to high temperature, high humidity, strong light irradiation, etc.

[0111] (2) The sodium salt hydrate crystal of salvianolic acid A prepared by the present invention has better water solubility and a significantly faster dissolution rate, and is suitable for use as an injection.

[0112] (3) The method for preparing the crystal of salvianolic acid A provided by the present invention can use a raw material of salvianolic acid A with low purity (about 80% is sufficient) as the starting material for crystallization, thus greatly reducing the cost of preparing the crystal of salvianolic acid A.

[0113] (4) The method for preparing the crystal of salvianolic acid A provided by the present invention has high product purity, high yield, and is convenient for industrial production.

[0114] The present invention will be further described in detail below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels without special description.

[0115] Example 1 Preparation of the sodium salt hydrate crystal of salvianolic acid A

[0116] Weigh 25 kg of Salvia miltiorrhiza Bunge herbs, and prepare the water extract of Salvia miltiorrhiza Bunge according to the preparation method of "Water Extract of Salvia miltiorrhiza Bunge" on page 415 of "Pharmacopoeia of the People's Republic of China" (2020 Edition, Volume I). The product is further prepared into a product of salvianolic acid A (solution) with a purity ≥ 80% according to the method described in Chinese patent document CN201310487751.6, and calculate the amount of salvianolic acid A in the product (the actually measured HPLC purity ≥ 90%, about 220 g of salvianolic acid A).

[0117] Add a quantitative amount of sodium chloride solid (1 / 2 the amount of substance of salvianolic acid A) to the salvianolic acid A solution, dissolve it, and concentrate it to 20% (calculated based on salvianolic acid A) at 60 °C. Cool it to 20 °C at a cooling rate of about 1 °C / min, and continuously stir during this period. Continue to stir until the solution becomes turbid, and then further cool the whole system to 4 °C at a cooling rate of 0.5 °C / min, and continue to stir and crystallize for 8 h.

[0118] After crystallization, filter, and vacuum dry the obtained crystals at room temperature in a dryer with phosphorus pentoxide as the desiccant to obtain the crystalline powder of the sodium salt of salvianolic acid A (about 150 g). Calculated based on salvianolic acid A, its HPLC purity is 99.6%. Select crystalline particles with a particle size ≥ 0.5 mm for X-ray single crystal diffraction analysis and X-ray powder diffraction analysis.

[0119] Figure 1 is the X-ray single crystal diffraction crystal data of the sodium salt hydrate of salvianolic acid A;

[0120] Figure 2It is the stereoscopic structure ellipsoid diagram of the asymmetric unit of the single crystal of sodium salvianolate A hydrate;

[0121] Figure 3 It is the projection diagram of the crystal cell packing of the single crystal of sodium salvianolate A hydrate along the b direction;

[0122] Figure 4 It is the powder diffraction pattern of the crystal of sodium salvianolate A hydrate;

[0123] Figure 5 It is the list of 2θ values of the powder diffraction of sodium salvianolate A hydrate;

[0124] Figure 6 It is the micrograph (10× eyepiece + 4× objective) of the single crystal of sodium salvianolate A hydrate prepared in this example.

[0125] Figure 7 It is the micrograph (10× eyepiece + 4× objective) of the crystalline powder of the sodium salvianolate A complex prepared by the method of patent document CN201910643821.X.

[0126] Through X-ray single crystal diffraction, the absolute configuration of sodium salvianolate A hydrate can be determined as:

[0127]

[0128] The TGA test results show that the water content of the crystal water in the crystal is consistent with the single crystal diffraction test results.

[0129] Example 2 Comparison of the stability of the crystal of sodium salvianolate A hydrate with the crystal of salvianolic acid A and the freeze-dried powder of salvianolic acid A

[0130] Stability is one of the key characteristics for judging whether a compound has drug-forming properties. Only when a compound has a certain stability can it remain stable throughout the shelf life (usually not less than 2 years), without degrading into other impurities, thereby increasing the safety risk of the drug and reducing the efficacy of the drug.

[0131] Generally, for a compound, with the increase of temperature, light intensity, humidity, etc., its stability gradually decreases, or its physical and chemical properties change significantly, resulting in the unqualified quality of the drug itself. Although reducing the storage temperature, packaging conditions, etc. of the drug can relatively increase its stability, this process will significantly increase the cost of the drug, as well as the difficulties in the production, storage, transportation and use processes, and significantly reduce the commercial value of the drug.

[0132] The most accurate stability test needs to simulate the real storage environment of the drug during use (such as constant temperature, constant humidity and light protection at 25°C), but it is also possible to predict the stability of the product by "accelerating" the degradation process of the compound to be tested under more severe conditions. The commonly used methods include the "stress testing".

[0133] In this experiment, the sodium salt hydrate crystal of salvianolic acid A prepared in Example 1, the crystal of salvianolic acid A (the preparation method refers to Chinese Patent Document: 201710055331.9), and the freeze-dried powder of salvianolic acid A (the preparation method refers to Chinese Patent Document: 201310487751.6) were used to conduct a comparative study on the stability of the three.

[0134] This example refers to the test method described on page 457 of Part IV of the Chinese Pharmacopoeia 2020 Edition, which is briefly described as follows:

[0135] Place the test sample in a suitable open container (such as a weighing bottle or petri dish), spread it into a thin layer with a thickness ≤ 5 mm, and conduct the following tests:

[0136] High temperature test: The test sample is placed in an open clean container and sampled for HPLC detection after being placed at 60 °C for 10 days, and compared with the sample at 0 day.

[0137] High humidity test: The test sample is placed in a constant humidity closed container and sampled for HPLC detection after being placed at 25 °C under a relative humidity of 90 ± 5% for 10 days, and the weight of the sample before and after placement is recorded to investigate the moisture absorption of the sample. (Select a saturated solution of KNO3 to be placed in a closed container together to create a high humidity environment: relative humidity 92.5% at 25 °C), and compared with the sample at 0 day.

[0138] Strong light irradiation test: The test sample is placed in an open light box equipped with fluorescent lamps and sampled for HPLC detection after being irradiated at an illuminance of 4500 lx ± 500 lx for 10 days. Pay attention to the appearance change of the test sample and compare it with the sample at 0 day.

[0139] The test results of the influencing factors of the sodium salt hydrate crystal of salvianolic acid A, the crystal of salvianolic acid A, and the freeze-dried powder of salvianolic acid A are shown in Table 1.

[0140] Table 1. Statistical table of product quality before and after the influencing factor tests of high temperature, high humidity, and strong light irradiation

[0141]

[0142] From the results of this example, it can be seen that both the sodium salt hydrate crystal of salvianolic acid A and the crystal of salvianolic acid A have good stability. After high temperature and strong light irradiation tests, the products degrade very little, and in the high humidity test, the products are not easy to absorb moisture, still maintaining a good appearance state and high purity, which is suitable for developing as the raw material drug of a new drug. In contrast, the freeze-dried powder of salvianolic acid A is unstable under high temperature and strong light conditions, degrades severely, absorbs moisture severely under high humidity conditions, and has already shown an oily droplet shape, and the appearance is judged to be unqualified, so no further HPLC detection was carried out.

[0143] The results of this example are consistent with the common knowledge that the crystallization stability is generally stronger than that of the amorphous state, indicating that the druggability of the crystal is significantly better than that of the amorphous state.

[0144] Example 3 Comparison of the water solubility of sodium salvianolate A hydrate crystal, salvianolic acid A crystal and salvianolic acid A freeze-dried powder

[0145] The oral bioavailability of salvianolic acid A is very low. As a therapeutic drug for ischemic cardiovascular and cerebrovascular diseases, when used in the acute attack period of patients, the intravenous injection preparation is its preferred dosage form. Since the intravenous injection preparation can almost only use water as a solvent, the water solubility of the sample is very important for the druggability of the compound.

[0146] In this example, the sodium salvianolate A hydrate crystal prepared in Example 1, salvianolic acid A crystal (the preparation method refers to Chinese Patent Document: 201710055331.9), and salvianolic acid A freeze-dried powder (the preparation method refers to Chinese Patent Document: 201310487751.6) were used to compare and study the water solubility of the products and explore the feasibility of using them as raw materials for intravenous drip.

[0147] The method for solubility determination refers to the method described in the Fourth Part of the Chinese Pharmacopoeia (2020 Edition) (in order to develop it into an intravenous injection preparation, only its solubility in water was studied), as follows:

[0148] The above several samples were respectively crushed and passed through the No. 5 pharmacopoeia sieve. The samples were respectively weighed and different volumes of pure water were added. According to the experimental method and judgment criteria described in the pharmacopoeia (weigh the finely powdered test sample, in a certain volume of solvent at 25°C ± 2°C, shake vigorously for 30 seconds every 5 minutes; observe the dissolution situation within 30 minutes. If there are no visible solute particles, it is considered completely dissolved), the dissolution performance is divided into very soluble, soluble, dissolved, slightly soluble, sparingly soluble, and very sparingly soluble. The solubility of several samples is described as follows:

[0149] Salvianolic acid A freeze-dried powder:

[0150] "Very soluble" judgment: 0.12 g of the sample was insoluble in 0.1 mL of water for 30 min;

[0151] "Soluble" judgment: 0.12 g of the sample was dissolved in 1 mL of water in 5 min;

[0152] "Dissolved" judgment: 0.12 g of the sample was instantly dissolved (≤10 s) in 3 mL of water.

[0153] Conclusion: The water solubility of salvianolic acid freeze-dried powder is "soluble".

[0154] Salvianolic acid A crystal:

[0155] "Very easily soluble" determination: 0.12 g of crystals do not dissolve in 0.1 mL of water within 30 min;

[0156] "Easily soluble" determination: 0.12 g of crystals do not dissolve in 1 mL of water within 30 min;

[0157] "Soluble" determination: 0.12 g of crystals do not dissolve in 3 mL of water at 5 min, 10 min, 15 min, 20 min, 25 min, and dissolve at 30 min.

[0158] Conclusion: The water solubility of salvianolic acid A crystals is "soluble". Considering the clinical use, 0.12 g of the sample dissolves in 3 mL of water at 25 - 30 min, which is difficult to meet the clinical requirements.

[0159] Salvianolic acid A sodium hydrate crystals:

[0160] "Very easily soluble" determination: 0.12 g of crystals do not dissolve in 0.1 mL of water within 30 min;

[0161] "Easily soluble" determination: 0.12 g of crystals dissolve in 1 mL of water at 5 min;

[0162] "Soluble" determination: 0.12 g of crystals dissolve instantly (≤10 s) in 3 mL of water.

[0163] Conclusion and discussion: The water solubility of salvianolic acid A sodium hydrate is "easily soluble". Considering the clinical use, 0.12 g of the drug dissolves instantly in 3 mL of water, which can meet the clinical requirements.

[0164] The structure of this example shows that both salvianolic acid A lyophilized powder and salvianolic acid A sodium salt hydrate crystals have good water solubility and are suitable for development as intravenous injection preparations. The water solubility of salvianolic acid A crystals is poor and it is difficult to directly develop them into intravenous injection preparations. If they are to be developed into intravenous injection preparations, more improvements are needed in the pharmaceutical production industry and formulation technology.

[0165] Salvianolic acid A lyophilized powder and salvianolic acid A sodium salt hydrate crystals have good water solubility. The former may be because the lyophilized powder has a loose and porous structure, which is conducive to water entering the interior and accelerating the dissolution rate; while the latter may be because the hydrate itself contains water molecules around the salvianolic acid A molecules and there is sodium salt in it, making it easier to dissolve quickly in water.

[0166] Example 4 Protective effect of salvianolic acid A sodium hydrate on hepatocyte injury caused by oxidative stress

[0167] In this example, HepG2 hepatocytes were planned to be used to study the protective effect of salvianolic acid A sodium hydrate crystal on cell damage caused by H2O2 oxidative stress. H2O2 was used to create oxidative stress in the experiment, and the MTT method was used to determine the apoptosis of cells. The specific method can be found in the reference (Zhang Yeni, et al., Establishment of an oxidative stress model of HepG2 cells induced by hydrogen peroxide. Food Research and Development, 2018, 39(05): 160-164).

[0168] The effect of salvianolic acid A sodium hydrate crystal on HepG2 cells caused by hydrogen peroxide is shown in Figure 8 .

[0169] The results showed that after treatment with H2O2, the viability of HepG2 cells was significantly reduced (to about 20%) compared with the untreated CK group. After adding salvianolic acid A sodium hydrate to the system, the viability of the cells gradually increased, and with the increase of the added concentration, a certain drug-dose effect (2.5-100 mg / kg) was presented, and significant differences were shown. At a dose of 100 mg / kg, the protective effect of salvianolic acid A almost reached the maximum value (to about 85%) and did not increase with the increase of the dose.

[0170] Example 5 Protective effect of salvianolic acid A sodium hydrate crystal on myocardium in ischemic cardiovascular diseases

[0171] In this example, a best-selling drug on the market was selected as the positive drug to compare the effects of salvianolic acid A sodium hydrate crystal and the positive drug on the myocardial infarction area in an animal model of ischemic heart disease.

[0172] The salvianolic acid A sodium hydrate crystal in this example was prepared by the method in Example 1. The positive drug was salvianolate for injection (Green Valley Pharmaceutical), and its main component was salvianolic acid B magnesium salt-based (content 85%) water-soluble phenolic compounds of Salvia miltiorrhiza. It is one of the best-selling drugs for myocardial ischemia on the market, and there are a large number of literature reports on its activity.

[0173] In this experiment, SD rats were selected, and the model construction method was as follows:

[0174] Ischemia without reperfusion model:

[0175] The rats were fasted for 12 h. After weighing, they were anesthetized by intraperitoneal injection of 300 mg / kg of 15% chloral hydrate. After shaving and disinfecting the left chest wall, a transverse incision was made between the 3rd and 4th ribs on the left side of the sternum. The muscle layers were bluntly separated with hemostatic forceps and the pleura was punctured. Then, the sternum was clamped horizontally with two hemostatic forceps, and the sternum was cut between the two forceps. A retractor was inserted to spread the ribs, and the pericardium was incised to expose the heart. The great cardiac vein could be seen in the anterior interventricular groove. Taking this as a landmark, a 2×6 curved round needle with 6-0 medical polyamide monofilament thread was used to bypass the deep surface of the artery about 2-3 mm from the root under the left auricle, and the needle emerged from the groove beside the conus arteriosus pulmonalis, about 1 mm deep and about 2 mm wide. The two ends of the silk thread were passed out from a polyethylene tube with a diameter of 2 mm for standby. After stabilizing for 10 min, a cotton swab stick was inserted into the PE tube, the silk thread was tightened, and the PE tube was pushed forward with the cotton swab stick until the coronary artery blood flow was blocked, and the construction of the ischemia model was completed.

[0176] After myocardial ischemia, the rats were administered once via the caudal vein, and the rats were sacrificed 24 h later to detect the myocardial infarction area.

[0177] Ischemia-reperfusion model:

[0178] For the construction of the ischemia-reperfusion model, the previous steps were the same as those of the ischemia without reperfusion model. After myocardial ischemia, the cotton swab stick was pulled out from the PE tube, and reperfusion could be achieved. After reperfusion, the rats were administered once via the vein, and the rats were sacrificed 24 h later to detect the myocardial infarction area.

[0179] The results of the effects of sodium salvianolate A hydrate and the positive drug on the myocardial infarction area in the animal model of ischemic heart disease are shown in Table 2.

[0180] Table 2 Protective effects of sodium salvianolate A hydrate crystal and positive drug on the heart in the myocardial ischemia model

[0181]

[0182] *p < 0.05, **p < 0.01, ***p < 0.001 vs. the solvent control group

[0183] Salvianolate is derived from the water-soluble components of Salvia miltiorrhiza, which is a large variety of traditional Chinese medicine and one of the best-selling cardiovascular disease treatment drugs on the market. The content of magnesium salt of salvianolic acid B exceeds 85%. Both animal experiments and clinical human trials have shown its good therapeutic effects in the field of cardiovascular diseases, and the results of this example also support this view.

[0184] In this example, both sodium salvianolate A hydrate crystal and salvianolate had good protective effects on the myocardium of ischemic rats. The drug dose for the former to produce the maximum therapeutic effect was 10 mg / kg, and that for the latter was 60 mg / kg. Moreover, the maximum drug effect of the former was also better than that of the latter, indicating that it may have a lower drug dose and better therapeutic effects.

[0185] Example 6 Protective Effect of Salvianolic Acid A Sodium Salt Hydrate Crystal on the Brain in Ischemic Cerebrovascular Diseases

[0186] In this example, a best-selling drug on the market was selected as the positive drug to compare the effects of salvianolic acid A sodium salt hydrate crystal and the positive drug on the cerebral infarction area in animal models of stroke. The positive drug was butylphthalide sodium chloride injection, which is a drug recommended for the treatment of ischemic stroke in the "Chinese Guidelines for the Diagnosis and Treatment of Ischemic Stroke" (2018 edition).

[0187] In this experiment, SD rats were selected to construct an animal model of ischemic stroke. The method is as follows:

[0188] Ischemia without reperfusion model: Middle cerebral artery embolization - electrocoagulation method in rats

[0189] The SD rats were weighed and anesthetized by intraperitoneal injection of 15% chloral hydrate at 300 mg / kg. They were fixed on the rat operating table in the left lateral position, the left temporal top and face were shaved, and after disinfection with 75% ethanol, the skin was incised between the left eye and left ear. The temporal muscle and masseter muscle were bluntly separated to expose the pterygoid plate of the temporal bone. Under the operating microscope, a bone window of 2 mm × 2 mm was drilled with a cranial drill at 1 mm near the junction of the temporal bone and squamous temporal bone close to the oral side, and the skull was pried open with a crowbar. At this time, a relatively straight blood vessel with fewer branches could be seen through the dura mater, which was the middle cerebral artery. The blood flow was completely blocked by cauterization with bipolar electrocoagulation forceps between 1 mm within the olfactory tract and the inferior cerebral vein, and then hemostasis was performed. The temporal muscle and skin were sutured in turn, and then drugs were administered by tail vein injection. After the rats woke up, they were put back into the cage and continued to be fed for 24 h.

[0190] Ischemia-reperfusion model: Middle cerebral artery embolization - suture method in rats

[0191] After weighing, the rats were anesthetized by intraperitoneal injection of 15% chloral hydrate at 300 mg / kg, fixed supine on the rat operating table, the neck was shaved, and disinfected with 75% ethanol. The skin was longitudinally incised with surgical scissors, and one side of the common carotid artery was surgically exposed. The external carotid artery and pterygopalatine artery were separated and ligated along the common carotid artery. An arterial clip was placed at the proximal end of the common carotid artery to block the blood flow. A small incision was made at the distal end with ophthalmic surgical scissors, and a suture was inserted. The suture was slowly pushed in (about 20 mm) until the anterior cerebral artery and then withdrawn about 2 mm to reach the middle cerebral artery orifice, and the suture and blood vessel were ligated, and the skin was sutured. After cerebral ischemia, drugs were administered by tail vein injection once, and the rats were put back into the cage for feeding.

[0192] The results of the effects of salvianolic acid A sodium salt hydrate crystal and the positive drug on the cerebral infarction area in animal models of ischemic stroke are shown in Table 3.

[0193] Table 3 Effects of Salvianolic Acid A Sodium Salt Hydrate and Positive Drug on Cerebral Infarction Area in Animal Models of Ischemic Stroke

[0194]

[0195] *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle control group

[0196] Butylphthalide is a best-selling drug for the treatment of ischemic stroke in the market and is recommended by the Guidelines for the Diagnosis and Treatment of Ischemic Stroke in China (2018 Edition). Its efficacy has been recognized by the industry, and the results of this example also support its therapeutic effect.

[0197] The results of this example show that sodium salt of salvianolic acid A crystal and butylphthalide both have good effects in treating ischemic stroke in two models of ischemia-reperfusion and ischemia without reperfusion. Salvianolic acid A reaches the maximum therapeutic effect at 10 mg / kg, while butylphthalide reaches the maximum therapeutic effect at 3 mg / kg, and the maximum effect of the former is significantly better than that of the latter.

[0198] Example 7 Effects of sodium salt hydrate of salvianolic acid A on serum MDA, SOD, IL-1β and TNF-α in rats with cerebral ischemia-reperfusion

[0199] In this example, the rat serum in Example 6 was selected, and kits were used to detect the levels of cytokines related to inflammation, oxidation, etc. The kits are classified as follows:

[0200] Rat malondialdehyde (MDA) ELISA kit: Shanghai Xinyu Biotechnology Co., Ltd., specification: 96T; Rat superoxide dismutase (SOD) ELISA kit: Shanghai Xinyu Biotechnology Co., Ltd., specification: 96T; Rat interleukin 1β (IL-1β) ELISA kit: Shanghai Xinyu Biotechnology Co., Ltd., specification: 96T; Rat tumor necrosis factor α (TNF-α) ELISA kit: Shanghai Xinyu Biotechnology Co., Ltd., specification: 96T.

[0201] The detection results are shown in Table 4:

[0202] Table 4 Effects of sodium salt hydrate of salvianolic acid A and butylphthalide on the contents of MDA, SOD, IL-1β and TNF-α in rat serum

[0203]

[0204] *p < 0.05, **p < 0.01 vs vehicle control group

[0205] The results of this detection indicate that salvianolic acid A can increase SOD and reduce the activities of MDA, IL-1β, TNF-α, etc., which shows antioxidant and anti-inflammatory activities. Compared with the solvent control group, there are significant differences at high doses. At the maximum effective dose, the positive drug butylphthalide does not have significant anti-inflammatory and antioxidant activities, indicating that its therapeutic mechanism is not related to the above factors.

[0206] Ischemia-reperfusion injury is one of the common injuries in the body. During the reperfusion process, the addition of fresh oxygen makes the body prone to generate highly reactive free radicals, which attack key organelles such as cell membranes and mitochondria, causing cell necrosis, apoptosis, etc., and then leading to damage to the corresponding organs.

[0207] Ischemia-reperfusion injury occurs widely in various parts of the body. In addition to the heart, blood vessels, liver, kidneys, nervous system, lungs, and organ transplantation processes, it may also occur. The strong anti-inflammatory and antioxidant activities of sodium salt hydrate of salvianolic acid A indicate that it is very likely to be applied to the prevention and treatment of the above diseases.

[0208] All the documents mentioned in this invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Sodium salvianolate A hydrate represented by Formula I, Among them, The sodium salvianolate A hydrate is a crystal, and the crystal is determined to be monoclinic by X-ray single crystal diffraction; Moreover, the powder X-ray diffraction pattern of the sodium salvianolate A hydrate has characteristic peaks at the following 2θ values: 12.00±0.1, 18.86±0.1, 19.02±0.1, and 22.28±0.

1.

2. The sodium salt hydrate of salvianolic acid A according to claim 1, wherein, The powder X-ray diffraction pattern of the sodium salvianolate A hydrate further includes characteristic peaks at the following 2θ values: 16.66±0.1, 17.78±0.1, 21.88±0.1, 24.22±0.1, 25.50±0.1, and 25.80±0.

1.

3. The sodium salt hydrate of salvianolic acid A according to claim 1, characterized in that, The crystal was determined to be monoclinic by single-crystal X-ray diffraction, and the unit cell parameters are as follows: α = γ = 90.0°, β = 117.5 ± 0.1°.

4. The sodium salt hydrate of salvianolic acid A according to claim 1 or 3, characterized in that The crystal is determined to be in the C2 space group by X-ray single crystal diffraction.

5. A preparation method of sodium salvianolate A hydrate, characterized in that, The sodium salvianolate A hydrate is a crystal, and the crystal is determined to be monoclinic by X-ray single crystal diffraction. The method includes the steps: (1) Prepare a salvianolic acid A solution having a first temperature; (2) Add a sodium salt to the salvianolic acid A solution obtained in step (1), and stir to dissolve the sodium salt; (3) Cool the solution in step (2) to a second temperature for crystallization to obtain the sodium salvianolate A hydrate; wherein, the first temperature is 40 - 80°C; the second temperature is 0 - 20°C.

6. The method according to claim 5, wherein In the salvianolic acid A solution obtained in step (1), the mass concentration of salvianolic acid A is 5% - 20% 7. The method according to claim 5, characterized in that The sodium salt added in step (2) is sodium chloride.

8. Use of the sodium salvianolate A hydrate according to claim 1 or 3 for preparing a pharmaceutical composition for treating or preventing a disease, wherein the disease is a cardiovascular and cerebrovascular disease or oxidative stress injury of an organ or tissue.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: The sodium salvianolate A hydrate according to claim 1; and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is an injection.

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