WO2023143633A2 - Salvianolic acid a sodium salt hydrate and preparation method therefor - Google Patents

Abstract

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

Description

A kind of salvianolic acid A sodium salt hydrate and preparation method thereof technical field

The invention belongs to the field of chemistry. Specifically, the invention relates to a salvianolic acid A sodium salt hydrate and a preparation method thereof.

Background technique

With the continuous improvement of people's living standards, the proportion of animal foods in the diet is gradually increasing. The saturated fatty acids and cholesterol form insoluble lipoproteins, which are deposited on the walls of arteries and gradually form plaques, which gradually grow and form arteries. Atherosclerosis gradually blocks blood vessels causing ischemia. In addition, due to various reasons such as changes in cold and heat, exercise, and emotional agitation, plaques may fall off and gradually flow to capillaries with blood flow, and then block capillaries to block blood flow, resulting in ischemia of corresponding tissues and organs. resulting in ischemic injury.

Anticoagulant drugs, thrombolytic drugs, antiplatelet drugs, mechanical thrombectomy, vascular stents, bypass surgery and other drugs and surgical methods can inhibit the formation of thrombus or remove thrombus. Sufficient oxygen in the blood after recanalization will lead to partial reperfusion The generation of "active oxygen" such as free radicals and "reperfusion injury" are also harmful to the body and may even be life-threatening.

The above-mentioned ischemia/reperfusion injury can occur in the cardiovascular and cerebrovascular, liver, kidney, nervous system, necrotic limbs of diabetic patients, etc., and poses a great threat to human health.

Salvia miltiorrhiza is derived from the dry root and rhizome of Salvia miltiorrhiza, in which water-soluble phenolic acid compounds are one of the main active ingredients, including salvianolic acid A, salvianolic acid B, Shikonian acid, rosmarinic acid, Danshensu, etc. Studies have shown that salvianolic acid A is the most active compound in Danshen (Lu, Y.; Foo, L.Y., Polyphenols 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), its activities include anti-inflammation, anti-oxidation, etc., and in vivo and in vitro tests have shown that it has a good therapeutic effect on various diseases.

However, due to its strong antioxidant effect, salvianolic acid A has extremely poor stability and is easily degraded by oxidation, hydrolysis, etc. during storage, making it difficult to meet the needs of 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 low-cost, efficient and high-purity salvianolic acid A sample preparation process has always been a problem.

Chinese patent document CN200810223651.1 discloses a preparation process of 90% salvianolic acid A. However, the purity of 90% is difficult to support its development as a new chemical drug (because in the general dosage range, chemical drugs require qualitative research on impurities with a content exceeding 0.1%, and safety research on impurities exceeding 0.15%, so a large number of impurities may be involved. Quality, impurity and safety research, which significantly increases drug safety risks, greatly increases the difficulty and cost of research and development), Especially when developing as an injection, there are insurmountable difficulties.

Chinese patent document CN 201310487751.6 prepared salvianolic acid A freeze-dried powder, the purity can exceed 97%, and can be scaled up to industrial production scale. However, this product is an amorphous freeze-dried powder. In this state, the stability of salvianolic acid A is poor. It needs to be placed below -20°C for long-term storage to maintain long-term stability. In actual production, storage and transportation The difficulty and various costs will be significantly increased during use.

Chinese patent document CN 201710055331.9 has developed a method for preparing salvianolic acid A crystals, the purity of which can reach more than 99.5%, and the stability in the crystalline state is also significantly improved. However, the water solubility of the crystal is poor, and it takes a long time to dissolve, and it is difficult to meet the requirement of clinical use as an injection preparation (it needs to be quickly dissolved in an aqueous solution).

Due to the extremely low oral bioavailability of salvianolic acid A (the oral bioavailability of 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.Ethnopharmaco.l148(2):617-623.), in order to ensure effectiveness, its new drug development needs to be based on non-oral preparations host. Due to the acute onset of cardiovascular and cerebrovascular diseases, intravenous injection preparations are the preferred dosage form.

Therefore, those skilled in the art are committed to developing a salvianolic acid A raw material drug and its production process that are high in purity, strong in stability, good in water solubility, easy to prepare, and satisfy drug development (especially intravenous injection drugs), It is used for the prevention and treatment of ischemic cardiovascular and cerebrovascular diseases and oxidative stress damage diseases of body tissues and organs.

Contents of the invention

The object of the present invention is to provide a kind of salvianolic acid A sodium salt hydrate, its preparation method and application.

The first aspect of the present invention provides a salvianolic acid A sodium salt hydrate shown in formula I,

The molecular formula of the salvianolic acid A sodium salt hydrate is C ₂₆ H ₂₂ O ₁₀ ·C ₂₆ H ₂₁ O ₁₀ ·Na·8(H ₂ O).

In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A sodium salt 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.

In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A sodium salt hydrate also 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.

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

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. .

In another preferred example, the salvianolic acid A sodium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 4 .

In another preferred example, the salvianolic acid A sodium salt hydrate represented by formula I is prepared by the preparation method described in the third aspect of the present invention.

The second aspect of the present invention provides a salvianolic acid A sodium salt hydrate crystal, which is determined to be a monoclinic system by X-ray single crystal diffraction, and the unit cell parameters are: α=γ=90.0°, β=117.5±0.1°.

In another preferred example, the unit cell parameters are: α=γ=90.00°, β=117.480°. Preferably, the unit cell volume The number of asymmetric units in the unit cell is Z=4.

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

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

The third aspect of the present invention provides a kind of preparation method of salvianolic acid A sodium salt hydrate, said method comprises steps:

(1) preparing the salvianolic acid A solution with the first temperature;

(2) adding sodium salt in the salvianolic acid A solution that step (1) obtains, stirring makes described sodium salt dissolve;

(3) cooling the solution of step (2) to a second temperature for crystallization, thereby obtaining the salvianolic acid A sodium salt hydrate;

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

In another preferred embodiment, the first temperature is 40-60°C; preferably about 50-60°C.

In another preference, the sodium salt added in the step (2) is a sodium-containing strong base strong salt (sodium strong salt), such as sodium chloride, sodium sulfate, sodium bisulfate, sodium bromide, Sodium dihydrogen phosphate, etc.

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

In another preference, in the salvianolic acid A solution obtained in the step (1), the quality of salvianolic acid A is The molar concentration is about 5%-20%; preferably about 5%-15%.

In another preferred example, the cooling rate in the 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℃/min.

In another preferred example, in the step (3), the temperature is first lowered to about 20°C at a rate of 1-2°C/min, and the temperature is maintained until crystal turbidity appears, and then the temperature is further lowered at a rate of 0.1-1°C/min To 0-4 ℃ and keep warm.

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.

In another preferred example, during the cooling and crystallization process in the step (2), the stirring is continued.

In another preferred example, after the temperature is lowered to the second temperature in the step (2), crystallization takes 5-48 hours; preferably, crystallization takes 8-36 hours; more preferably, crystallization takes 8-24 hours.

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

In another preferred example, the method further includes the step of recrystallizing the obtained salvianolic acid A sodium salt hydrate; preferably, the recrystallization step includes:

(a) dissolving salvianolic acid A sodium salt hydrate in an aqueous solvent having a first temperature to obtain salvianolic acid A salt solution;

(b) cooling the solution of step (a) to a second temperature for crystallization, thereby obtaining the refined product of salvianolic acid A sodium salt hydrate;

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

In another preferred embodiment, the first temperature is 45-70°C; preferably about 50°C.

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

In another preferred example, the cooling rate in the 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℃/min.

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.

In another preferred example, during the cooling and crystallization process in the step (b), the stirring is continued.

In another preferred example, after the temperature is lowered to the second temperature in the step (b), crystallization takes 5-48 hours; preferably, crystallization takes 8-36 hours; more preferably, crystallization takes 12-24 hours.

In another preferred example, the method further includes the step of vacuum drying the obtained refined product of salvianolic acid A sodium salt hydrate. Preferably, the vacuum drying temperature is 10-30°C.

In another preferred example, 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%; or, the HPLC purity of the salvianolic acid A raw material is at Between about 75% -95%.

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

In another preferred example, the aqueous solvent is selected from water, ethanol aqueous solution, acetone aqueous solution and the like.

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

The fourth aspect of the present invention provides the use of salvianolic acid A sodium salt hydrate described in the first aspect of the present invention or the salvianolic acid A sodium salt hydrate crystal described in the second aspect of the present invention, for the preparation of treatment or A pharmaceutical composition for preventing diseases, the diseases are selected from the group consisting of cardiovascular and cerebrovascular diseases, oxidative stress damage, immune system diseases, hyperlipidemia, diabetes complications and the like.

The diseases are ischemic cardiovascular and cerebrovascular diseases, related diseases caused by oxidative stress damage of organs or tissues (such as cardiovascular and cerebrovascular, liver, kidney, nervous system, etc.) associated tissue damage).

The fifth aspect of the present invention provides a pharmaceutical composition, comprising:

The salvianolic acid A sodium salt hydrate described in the first aspect of the present invention or the salvianolic acid A sodium salt hydrate crystal described in the second aspect of the present invention; and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition consists of the salvianolic acid A sodium salt hydrate described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

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

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Fig. 1 is the X-ray single crystal diffraction crystal data of salvianolic acid A sodium salt hydrate;

Fig. 2 is the three-dimensional structure ellipsoid diagram of salvianolic acid A sodium salt hydrate single crystal asymmetric unit;

Fig. 3 is the unit cell stacking projection diagram of salvianolic acid A sodium salt hydrate single crystal along the b direction;

Fig. 4 is the powder diffractogram of salvianolic acid A sodium salt hydrate crystallization;

Fig. 5 is the list of powder diffraction 2θ values of salvianolic acid A sodium salt hydrate;

Fig. 6 is the single crystal photomicrograph of salvianolic acid A sodium salt hydrate prepared by the present invention (10 * eyepiece+4 * objective lens);

Fig. 7 is a photomicrograph of the crystalline powder of salvianolic acid A sodium salt complex prepared according to the method of patent document CN201910643821.X (10 × eyepiece + 4 × objective lens);

Figure 8 shows the protective effect of salvianolic acid A sodium salt hydrate crystals on HepG2 cell damage caused by hydrogen peroxide.

Detailed ways

After extensive and in-depth research, the inventor unexpectedly prepared a compound composed of salvianolic acid A, salvianolic acid A negative ions, sodium ions and water molecules. Through X-ray single crystal diffraction, X-ray powder diffraction, etc., the crystal form, crystal water and other characteristics of this compound crystal have been determined, which proves that the salvianolic acid A sodium salt hydrate prepared by the present invention is a new substance , has a new crystal form, and its purity exceeds 99%. The invention also discloses a preparation method of the new crystal form, using salvianolic acid A with a purity of about 80% as the starting material to prepare the salvianolic acid A sodium salt hydrate of the present invention, the salvianolic acid A sodium salt hydrate It can be applied to the treatment of ischemic cerebrovascular disease and ischemic cardiovascular disease. Moreover, the salvianolic acid A sodium salt hydrate has the characteristics of low production cost, high product purity, good stability and good water solubility.

Before describing the present invention, it is to be understood that the invention is not limited to the specific methods and experimental conditions described, Because such methods and conditions can be changed. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, the scope of the present invention being limited only by the appended claims.

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 to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may 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 and in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

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

Specifically, the present invention prepared a compound (pure substance) composed of salvianolic acid A, salvianolic acid A negative ion, sodium ion, and water molecule, obtained its single crystal, and compared it with common positive drugs on the market , studied the therapeutic effect of the complex on ischemic cerebrovascular disease and ischemic cardiovascular disease.

Compared with the freeze-dried powder of amorphous salvianolic acid A, this series of crystalline complexes has 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 for injection, and salvianolic acid salt for injection, this series of crystalline complexes can significantly reduce the size of brain/myocardial infarction, significantly reduce the mortality rate of model animals, and significantly Improve the effect of limb function recovery in cerebral ischemia/myocardial ischemia model animals. The series of crystalline complexes can be applied to the treatment of ischemic heart disease and ischemic cerebrovascular disease, and can be used as raw materials (API) of related drug injection preparations.

The properties of salvianolic acid A sodium salt hydrate of the present invention can be studied in various ways and instruments as follows, such as using X-ray single crystal diffraction, X-ray powder diffraction, differential scanning calorimetry, and thermogravimetric analysis (TGA) analyze. These analysis methods can be conventional methods in the art.

For example, X-ray single crystal diffraction analysis can use Bruker D8 Venture single crystal X-ray diffractometer, test conditions: CuKα radiation, scanning.

Through X-ray single crystal diffraction analysis, the single crystal characteristics and structure type of salvianolic acid A sodium salt hydrate of the present invention are obtained.

Specifically, the absolute configuration of the salvianolic acid A sodium salt hydrate crystal was determined to be:

The salvianolic acid A sodium salt hydrate crystal is determined to be a monoclinic crystal system by X-ray single crystal diffraction, C2 space group, and the unit cell parameters are: α=γ=90.0°, β=117.5°.

The method of X-ray powder diffraction for determining the crystal form of the present invention is known in the art. For the specific detection conditions of the present invention, reference may be made to the X-ray powder diffraction patterns of each crystal 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.

Whether a certain compound can combine with a solvent to become a solvate or combine with several molecules of solvent has no rules to follow. Different crystal forms with the same molecular structure may 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 shape or hygroscopicity. X-ray powder ray diffraction spectrum is a necessary way to identify the crystal form, but it is not the only way. Hydrate crystal forms may have similar X-ray powder ray diffraction patterns due to the different ways of combining water and compounds, but have different other characterization data, such as DSC spectra. The same drug, different crystal forms or different solvates will show differences in stability, fluidity, hygroscopicity, solubility, and compressibility, which will have an important impact on the application of the drug, thus greatly affecting the drug's performance. Bioavailability and ease of use.

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 negative ions, and cations in its structure, but does not contain crystal water. Test Its water solubility is "slightly soluble". In order to compare the difference between the "salvianolic acid A salt complex" described in this patent document and the "salvianolic acid A sodium salt hydrate" described in the present invention, referring to the method, the salvianolic acid A sodium salt complex was prepared, as shown in Fig. 6 and FIG. 7 can be seen the photomicrographs of the salvianolic acid A sodium salt hydrate single crystal and the salvianolic acid A sodium salt complex of the present invention. It can be seen from the figure that the salvianolic acid A sodium salt hydrate prepared by the present invention is a clearly visible "single crystal", while the salvianolic acid A sodium salt complex is a "crystalline powder" without obvious "single crystal" . The difference between the salvianolic acid A sodium salt hydrate prepared by the present invention and the salvianolic acid A sodium salt compound of the comparative patent is as follows:

The former is single crystal, pure substance, stable chemical composition, and good water solubility;

The latter is a crystalline powder or a mixture, and the stability of its chemical composition cannot be guaranteed theoretically (such as the ratio of salvianolic acid A molecule to salvianolic acid A sodium), and its water solubility is poor;

According to the pharmaceutical registration regulations, the most basic feature of injection raw materials is that the components are fixed and pure. Compared with the patented salvianolic acid A sodium salt complex crystalline powder, the salvianolic acid A sodium salt hydrate single crystal prepared by the present invention has significant advantages when used as an injection raw material.

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

active ingredient

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

The invention discloses salvianolic acid A sodium salt hydrate crystallization and a preparation method thereof.

Salvia miltiorrhiza is prepared by referring to the preparation method of "Preparation of salvia miltiorrhiza water extract" in "Chinese Pharmacopoeia" (Part 1) (2020 edition). The latter reacts to prepare the crude product of salvianolic acid A, and after resin column chromatography, a salvianolic acid A sample with a purity ≥ 80% is obtained. Then the sample is subjected to crystallization and recrystallization processes to prepare high-purity salvianolic acid A sodium salt hydrate crystals. The crystal is composed of salvianolic acid A, salvianolic acid A anions, cations and water molecules. The series of complexes are new pure substances, and have a new Compared with the amorphous salvianolic acid A powder, it has the characteristics of good stability and good solubility. Compared with the salvianolic acid A crystal, it has the characteristics of good water solubility. The salvianolic acid A sodium salt hydrate prepared by the present invention is more suitable for the development of intravenous injection, and it is applied to the treatment of ischemic stroke and ischemic cardiovascular disease, and the effect is significantly better than the positive drug butylphthalide sodium chloride Injection and salvianolate for injection.

Pharmaceutical compositions and methods of administration

Because 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 salvianolic acid A sodium salt hydrate of the present invention can be used for treating or preventing the above-mentioned diseases.

The pharmaceutical composition of the present invention comprises the salvianolic acid A sodium salt hydrate of the present invention within a safe and effective dose range and pharmaceutically acceptable excipients or carriers. Wherein, "safe and effective dose" refers to: the amount of salvianolic acid A sodium salt hydrate is enough to obviously improve the condition without serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the crystalline form/dose of the present invention, more preferably 10-200 mg of the crystalline form/dose of the present invention. Preferably, the "one dose" is an injection dose or a capsule or tablet.

"Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low enough toxicity. "Compatibility" here means that each component 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. Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as wetting agents (such as ten Dialkyl sodium sulfate), disintegrants, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The mode of administration of salvianolic acid A sodium salt hydrate or pharmaceutical composition of the present invention is not particularly limited, representative mode of administration includes (but not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular) or subcutaneous), and topical administration. Preferably, it is administered by injection, such as intravenous injection.

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 dicalcium phosphate, or with: (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents and the release of the active ingredient from such compositions may be in a certain part of the alimentary canal in a delayed manner. Examples of usable embedding components are polymeric substances and waxy substances. The active ingredient can also be in the form of microcapsules with one or more of the above-mentioned excipients, if necessary.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tincture. In addition to the active ingredient, 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, etc.

Besides such inert diluents, the compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredient, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof. The salvianolic acid A sodium salt hydrate of the present invention is particularly suitable for injection because of its excellent solubility.

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 under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required, if necessary.

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

When using a pharmaceutical composition, a safe and effective amount of the crystal form of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is the pharmaceutically effective dosage, for a human with a body weight of 60kg In other words, the daily dosage is usually 1-2000 mg, preferably 10-500 mg. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

The main advantages of the present invention are:

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

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

(3) The method for preparing salvianolic acid A crystallization provided by the present invention can adopt low-purity salvianolic acid A raw material (about 80%) to carry out crystallization as starting material, therefore greatly reduces salvianolic acid A The cost of crystallization preparation.

(4) The method for preparing salvianolic acid A crystals provided by the present invention has high product purity and high yield, which is convenient for industrial production.

Below in conjunction with specific embodiment, further elaborate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without detailed conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercially available channels unless otherwise specified.

The preparation of embodiment 1 salvianolic acid A sodium salt hydrate crystallization

Weigh 25 kg of Danshen medicinal material, and prepare Danshen water extract by referring to the preparation method of "Salvia miltiorrhiza water extract" on page 415 of "Chinese Pharmacopoeia" (2020 edition one), and further refer to the product described in Chinese patent document CN201310487751.6) According to the method, the salvianolic acid A product (solution) with a purity of ≥80% is prepared, and the amount of salvianolic acid A in the product is calculated (the actual HPLC purity is ≥90%, and the salvianolic acid A is about 220g).

Add quantitative sodium chloride solid (the amount of salvianolic acid A 1/2 substance) in the salvianolic acid A solution, after dissolving, concentrate to 20% (in salvianolic acid A) at 60 ℃. Cool down to 20°C at a cooling rate of about 1°C/min, with constant stirring. Continue to stir until the solution becomes turbid, then further lower the temperature of the whole system to 4°C at a cooling rate of 0.5°C/min, and continue to stir and crystallize for 8 hours.

Filtration after crystallization, the obtained crystals are vacuum-dried at room temperature in a desiccator using phosphorus pentoxide to obtain salvianolic acid A sodium salt crystalline powder (about 150g). In terms of salvianolic acid A, its HPLC purity is 99.6%. %. . Crystal particles with a particle size of ≥0.5 mm are selected for X-ray single crystal diffraction analysis and X-ray powder diffraction analysis.

Fig. 1 is the X-ray single crystal diffraction crystal data of salvianolic acid A sodium salt hydrate;

Fig. 2 is the three-dimensional structure ellipsoid diagram of salvianolic acid A sodium salt hydrate single crystal asymmetric unit;

Fig. 3 is the unit cell stacking projection diagram of salvianolic acid A sodium salt hydrate single crystal along the b direction;

Fig. 4 is the powder diffractogram of salvianolic acid A sodium salt hydrate crystallization;

Fig. 5 is the list of powder diffraction 2θ values of salvianolic acid A sodium salt hydrate;

Fig. 6 is a photomicrograph of the salvianolic acid A sodium salt hydrate single crystal prepared in this example (10× eyepiece + 4× objective lens).

Fig. 7 is a photomicrograph (10× eyepiece + 4× objective lens) of the crystalline powder of salvianolic acid A sodium salt complex prepared according to the method of patent document CN201910643821.X.

Through X-ray single crystal diffraction, it can be determined that the absolute configuration of salvianolic acid A sodium salt hydrate is:

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

Example 2 Comparison of the stability of salvianolic acid A sodium salt hydrate crystals, salvianolic acid A crystals, and salvianolic acid A freeze-dried powder

Stability is one of the key characteristics for judging whether a compound is druggable. Only when the compound has a certain stability can it remain stable throughout the shelf life (generally not less than 2 years) and will not degrade into other impurities that will increase the The safety risk of the drug reduces the efficacy of the drug.

Generally, with the increase of temperature, light intensity, humidity, etc., the stability of general compounds will gradually decrease, or the physical and chemical properties will change significantly, which will lead to the failure of the drug itself. Although reducing the storage temperature and packaging conditions of the drug can relatively increase its stability, this process will significantly increase the cost of the drug, as well as the difficulty of production, storage, transportation, and use, and significantly reduce the commercial value of the drug.

The most accurate stability test needs to simulate the real storage environment of the drug during use (such as constant temperature, constant humidity, and dark storage at 25°C), but it can also "accelerate" the degradation of the test compound through harsher conditions Process, to predict the stability of the product, which commonly used methods include "influence factor test".

In this test, salvianolic acid A sodium salt hydrate crystals prepared in Example 1, salvianolic acid A crystals (refer to Chinese patent document: 201710055331.9 for the preparation method) and salvianolic acid A freeze-dried powder (refer to the Chinese patent document for the preparation method) : 201310487751.6), and compared the stability of the three.

This embodiment refers to the test method described on page 457 of the fourth part of the "Chinese Pharmacopoeia" 2020 edition, which is briefly described as follows:

Place the test product in an appropriate open container (such as a weighing bottle or a petri dish), spread it into a thin layer with a thickness ≤ 5mm, and conduct the following test:

High temperature test: The test product is placed in a clean container with an opening, placed at 60°C for 10 days, then sampled for HPLC testing, and compared with the sample at 0 days.

High-humidity test: the test product is placed in a closed container with constant humidity, placed at 25°C for 10 days at a relative humidity of 90±5%, then sampled for HPLC testing, and the weight of the sample before and after placement is recorded to investigate the quality of the sample. Hygroscopic condition. (Choose KNO ₃ saturated solution to place together in an airtight container to create a high-humidity environment: 92.5% relative humidity at 25°C), and compare with the sample at 0 days.

Strong light irradiation test: the test sample is placed in a light box equipped with a fluorescent lamp, and after 10 days of illumination under the condition of illuminance 4500lx±500lx, samples are taken for HPLC detection, pay attention to the appearance changes of the test sample, and compare with the sample at 0 days.

Table 1 shows the test results of salvianolic acid A sodium salt hydrate crystals, salvianolic acid A crystals and salvianolic acid A freeze-dried powder.

Table 1. Product quality statistics before and after the test of high temperature, high humidity, and strong light exposure factors

From the results of this example, it can be seen that salvianolic acid A sodium salt hydrate crystals and salvianolic acid A crystals have good stability, and the product degradation is very little after high temperature and strong light tests, and the product is not easy to produce in high humidity tests. Absorbs moisture, still maintains a good appearance and high purity, and is suitable for the development of raw materials for new drugs. In contrast, the salvianolic acid A freeze-dried powder is unstable under high temperature and strong light conditions, and it degrades seriously. It absorbs moisture seriously under high humidity conditions, and it is in the shape of oil droplets. The appearance judgment has been unqualified, so it has not been tested. Enter One-step HPLC detection.

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.

Example 3 Water Solubility Comparison of Salvianolic Acid A Sodium Salt Hydrate Crystalline and Salvianolic Acid A Crystalline Salvianolic Acid Freeze-dried Powder

The oral bioavailability of salvianolic acid A is very low, and as a drug for the treatment of ischemic cardiovascular and cerebrovascular diseases, when it is used in patients with acute attacks, intravenous injection preparations are the preferred dosage form. Since intravenous formulations use almost exclusively water as a solvent, the aqueous solubility of the sample is important for the druggability of the compound.

This example uses salvianolic acid A sodium salt hydrate crystals prepared in Example 1, salvianolic acid A crystals (for the preparation method, refer to Chinese patent literature: 201710055331.9) and salvianolic acid A freeze-dried powder (for the preparation method, refer to Chinese patent literature : 201310487751.6), comparatively research the water solubility of the product, and explore its feasibility as a raw material for intravenous infusion.

The method of solubility determination refers to the method described in "Chinese Pharmacopoeia" (2020 Edition IV) (in order to develop it into an intravenous injection preparation, only its solubility in water is studied), as follows:

The above several samples were pulverized respectively and passed through No. 5 Pharmacopoeia sieve. Weigh the samples respectively, add different volumes of pure water, according to the experimental method and judgment standard described in the Pharmacopoeia (weigh the test sample that has been ground into a fine powder, put it in a solvent with a certain capacity at 25 ° C ± 2 ° C, every 5 minutes Shake vigorously for 30 seconds; observe the dissolution within 30 minutes. If there are no visible solute particles, it is considered completely dissolved. Very slightly soluble. Describe the solubility of several samples as follows:

Salvianolic acid A freeze-dried powder:

"Extremely soluble" judgment: 0.12g sample is insoluble in 0.1mL water for 30min;

"Easily soluble" judgment: 0.12g sample is dissolved in 1mL water for 5min;

"Dissolution" judgment: 0.12g sample is dissolved in 3mL water instantly (≤10s).

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

Salvianolic acid A crystallization:

"Extremely soluble" judgment: 0.12g crystals are insoluble in 0.1mL water for 30min;

"Easily soluble" judgment: 0.12g of crystals in 1mL of water, 30min insoluble;

"Dissolution" judgment: 0.12g of crystals in 3mL of water, insoluble in 5min, insoluble in 10min, insoluble in 15min, insoluble in 20min, insoluble in 25min, dissolved in 30min.

Conclusion: The water solubility of salvianolic acid A crystal is "dissolved". Combined with the clinical use situation, 0.12g sample is dissolved in 3mL water for 25-30min, which is difficult to meet the clinical needs.

Salvianolic acid A sodium salt hydrate crystallization:

"Extremely soluble" judgment: 0.12g crystals are insoluble in 0.1mL water for 30min;

"Easily soluble" judgment: 0.12g of crystals are dissolved in 1mL of water for 5 minutes;

"Dissolution" judgment: 0.12g of crystals dissolved in 3mL of water instantly (≤10s).

Conclusion and discussion: The water solubility of salvianolic acid A sodium salt hydrate is "easy soluble". Combined with the clinical use, 0.12g of the drug can be dissolved in 3mL of water for an instant, which can meet its clinical needs.

The structure of this example shows that both salvianolic acid A freeze-dried powder and salvianolic acid A sodium salt crystalline hydrate have good water solubility and are suitable for development as intravenous injection preparations. The water solubility of salvianolic acid A crystals is poor, so it is difficult to directly develop it as an intravenous injection preparation. If it is developed into an intravenous injection preparation, more improvements need to be made in the preparation production industry and the prescription process.

Salvianolic acid A freeze-dried powder and salvianolic acid A sodium salt hydrate crystal have better water solubility, the former may be because the freeze-dried powder has a loose and porous structure, which is conducive to water entering the interior to accelerate the dissolution rate; while the latter may be Because the hydrate itself contains water molecules located around the salvianolic acid A molecule, and there is a sodium salt in it, which makes it easier to dissolve in water quickly.

Example 4 Protective effect of salvianolic acid A sodium salt hydrate on liver cell damage caused by oxidative stress

This example plans to use HepG2 liver cells to study the protective effect of salvianolic acid A sodium salt hydrate crystals on cell damage caused by H ₂ O ₂ oxidative stress. The test uses H ₂ O ₂ to create oxidative stress, and the MTT method is used to measure cell apoptosis. For specific methods, refer to the literature (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).

The effect of salvianolic acid A sodium salt hydrate crystallization on HepG2 induced by hydrogen peroxide is shown in Figure 8.

The results showed that after H ₂ O ₂ treatment, the viability of HepG2 cells was significantly decreased (to about 20%) compared with the untreated CK group. After adding salvianolic acid A sodium salt hydrate to the system, the viability of the cells gradually increased, and with the increase of the added concentration, there was a certain drug-dose effect (2.5-100mg/kg), and there was a significant difference. At the dose of 100 mg/kg, the protective effect of salvianolic acid A almost reached the maximum (to about 85%), and no longer increased with the increase of the dose.

Example 5 Salvianolic Acid A Sodium Salt Hydrate Crystals Protect Myocardium in Ischemic Cardiovascular Diseases

In this example, a best-selling drug on the market was selected as the positive drug, and the effects of salvianolic acid A sodium salt hydrate crystals and the positive drug on the myocardial infarct size in ischemic heart disease model animals were compared.

The salvianolic acid A sodium salt hydrate crystals in this example were prepared by the method in Example 1. The positive drug is salvianolic acid salt for injection (Green Valley Pharmaceutical). One of the ischemic drugs, there are a large number of literatures reporting its activity.

SD rats were used in this experiment, and the model construction method is as follows:

Ischemia-nonperfusion model:

Rats were fasted for 12 hours, weighed and anesthetized with 15% chloral hydrate 300 mg/kg intraperitoneally. After the left chest wall was shaved and disinfected, the skin was cut transversely between 3-4 ribs on the left side of the sternum, the muscle layers were bluntly separated with hemostats and the pleura was punctured, and then two hemostats were used to clamp the sternum transversely. Cut the sternum between the forceps, insert the retractor to expand the ribs, cut the pericardium to expose the heart, the great cardiac vein can be seen in the anterior interventricular groove, as a sign, and use a 2×6 tape under the left atrial appendage about 2-3mm away from the root Thread round needle No. 6-0 medical cotton monofilament thread bypasses the deep surface of the artery, and exits the needle in the paraconical groove of the pulmonary artery, with a depth of about 1mm and a width of about 2mm. Pass out the two ends of the silk thread from a polyethylene pipe with a diameter of 2mm for later use. After stabilizing for 10 minutes, insert a cotton swab stick into the PE tube, tighten the silk thread and push the PE tube forward with the cotton swab stick until the coronary artery blood flow is blocked to complete the construction of the ischemia model.

After myocardial ischemia, the rats were administered via the tail vein once, and the rats were sacrificed 24 hours later to detect the myocardial infarct size.

Ischemia-reperfusion model:

The construction of the ischemia-reperfusion model is the same as the ischemia non-perfusion model in the early stage. After myocardial ischemia, the cotton swab stick Pull out from the PE tubing to achieve reperfusion. After reperfusion, the rats were administered intravenously once, and the rats were sacrificed 24 hours later to detect the myocardial infarct size.

Table 2 shows the effects of salvianolic acid A sodium salt hydrate and positive drugs on the size of myocardial infarction in ischemic heart disease model animals.

Table 2 The protective effect of salvianolic acid A sodium salt hydrate crystals and positive drugs on the heart in myocardial ischemia model

*p<0.05, **p<0.01, ***p<0.001vs vehicle control group

Salvianolic acid salt is derived from the water-soluble components of Salvia miltiorrhiza, which belongs to a large variety of traditional Chinese medicine and is one of the best-selling drugs for treating cardiovascular diseases in the market. The content of salvianolic acid B magnesium salt exceeds 85%. Both animal experiments and clinical human experiments have shown that it has a good therapeutic effect in the field of cardiovascular diseases, and the results of this example also support this view.

Salvianolic acid A sodium salt hydrate crystallization and salvianolic acid salt in this embodiment all produced better protective effect on the myocardium of ischemic rats, wherein the drug dose was 10 mg/kg when the former produced the maximum therapeutic effect, and the later The former is 60mg/kg, and the maximum drug effect of the former is better than that of the latter, indicating that it may have a lower dosage and better therapeutic effect.

Example 6 The protective effect of salvianolic acid A sodium salt hydrate crystals on the brain in ischemic cerebrovascular disease

In this example, a best-selling drug on the market was selected as the positive drug, and the effects of the salvianolic acid A sodium salt hydrate crystal and the positive drug on the cerebral infarct size of the stroke model animals were compared. Butylphthalide Sodium Chloride Injection was selected as the positive drug, which is the recommended drug for the treatment of ischemic stroke in the "Guidelines for the Diagnosis and Treatment of Ischemic Stroke in China" (2018 Edition).

In this experiment, SD rats were selected to construct the experimental animal model of ischemic stroke. The method is as follows:

Ischemia-nonperfusion model: rat middle artery embolization-electrocoagulation

SD rats were weighed, anesthetized by intraperitoneal injection of 15% chloral hydrate 300mg/kg, fixed on the left side On the operating table of rats, the top of the left temporalis and the face were shaved, after being disinfected with 75% ethanol, the skin was cut between the left eye and the left ear, the temporalis muscle and the masseter muscle were bluntly separated, and the temporal bone wing plate was exposed. Under the microscope, a bone window of 2 mm × 2 mm was ground with a skull drill at the joint of the temporal bone and temporal squamous bone 1 mm close to the mouth side, and the skull was pried open with a crowbar. At this time, a straighter blood vessel with fewer branches can be seen through the dura mater, which is the middle cerebral artery. Use bipolar electric coagulation forceps to cauterize the place between 1 mm inside the olfactory tract and the inferior cerebral vein, completely block the blood flow and stop the bleeding, suture the temporal muscle and skin in turn, and then inject the drug into the tail vein. After the rats regained consciousness, they were put back into the cages and continued to be fed for 24 hours.

Ischemia-reperfusion model: middle artery embolization-suture method in rats

After weighing, rats were anesthetized with 300 mg/kg intraperitoneal injection of 15% chloral hydrate, fixed on the rat operating table in supine position, shaved the neck, sterilized with 75% ethanol, cut the skin longitudinally with surgical scissors, and separated and exposed one side of the neck Common artery, separate and ligate the external carotid artery and pterygognathic artery along the common carotid artery, place an arterial clip at the proximal end of the common carotid artery to block the blood flow, cut a small opening with ophthalmic surgical scissors at the distal end, and insert the plug Slowly push the suture (about 20mm) into the anterior cerebral artery and then pull it back about 2mm to the middle cerebral artery ostium, ligate the suture and blood vessels, and suture the skin. After brain tissue ischemia, the rats were injected into the tail vein once, and then put back into the cage for feeding.

Table 3 shows the effects of salvianolic acid A sodium salt hydrate crystals and positive drugs on the cerebral infarct size in ischemic stroke model animals.

Table 3 The effect of salvianolic acid A sodium salt hydrate and positive drugs on the size of cerebral infarction in ischemic stroke model animals

*p<0.05, **p<0.01, ***p<0.001vs vehicle control group

Butylphthalide is a best-selling drug for the treatment of ischemic stroke in the market. It is recommended by the "Guidelines for the Diagnosis and Treatment of Ischemic Stroke in China" (2018 Edition). therapeutic effect.

The results of this example show that salvianolic acid A sodium salt crystallization and butylphthalide in ischemia-reperfusion and ischemia In the two models of perfusion, both have a good effect on the treatment of ischemic stroke. Salvianolic acid A reached the maximum therapeutic effect at 10 mg/kg, while butylphthalide reached the maximum therapeutic effect at 3 mg/kg, and the maximum effect of the former was significantly better than that of the latter.

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

In this example, the rat serum in Example 6 was selected, and a kit was used to detect the levels of cytokines related to inflammation and oxidation. Kits are classified as follows:

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. Company, specification: 96T.

The test results are shown in Table 4:

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

*p<0.05, **p<0.01vs vehicle control group

The results of this test show that salvianolic acid A can increase SOD, reduce MDA, IL-1β, TNF-α and other activities, which are manifested as anti-oxidation and anti-inflammatory activities. There was a significant difference at the high dose compared to the vehicle control group. At the maximum effective dose, the positive drug butylphthalide did not have significant anti-inflammatory and antioxidant activities, indicating that its therapeutic mechanism has nothing to do with the above factors.

Ischemia-reperfusion injury is one of the common injuries in the body. The addition of fresh oxygen during reperfusion makes the body prone to generate highly active free radicals, which attack key organelles such as cell membranes and mitochondria, causing cell necrosis, apoptosis, etc. This in turn leads to damage to the corresponding organs.

Ischemia-reperfusion injury occurs widely in various parts of the body, except for the cardiovascular and cerebrovascular, liver, kidney, nervous system, lung, and organ transplantation. Salvianolic acid A sodium salt hydrate has a strong anti-inflammatory effect , antioxidant activity, indicating that it is very likely to be applied to the prevention and treatment of the above diseases.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, Those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. Salvianolic acid A sodium salt hydrate shown in a kind of formula I,
   
2. Salvianolic acid A sodium salt hydrate as claimed in claim 1, is characterized in that, the powder X-ray diffraction spectrum of described salvianolic acid A sodium salt 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.
3. Salvianolic acid A sodium salt hydrate as claimed in claim 2, is characterized in that, the powder X-ray diffraction pattern of described salvianolic acid A sodium salt hydrate also comprises the characteristic peak at following 2θ value place: 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.
4. A salvianolic acid A sodium salt hydrate crystal is characterized in that, the salvianolic acid A sodium salt hydrate crystal is measured as a monoclinic system by X-ray single crystal diffraction, and the unit cell parameters are: α=γ=90.0°, β=117.5±0.1°.
5. The salvianolic acid A sodium salt hydrate crystal as claimed in claim 4, is characterized in that, the absolute configuration of the salvianolic acid A sodium salt hydrate crystal is:
   
6. The salvianolic acid A sodium salt hydrate crystal as claimed in claim 4, is characterized in that, the salvianolic acid A sodium salt hydrate crystal adopts X-ray single crystal diffraction to measure it as C2 space group.
7. A kind of preparation method of salvianolic acid A sodium salt hydrate, it is characterized in that, described method comprises steps:

   (1) preparing the salvianolic acid A solution with the first temperature;

   (2) adding sodium salt in the salvianolic acid A solution that step (1) obtains, stirring makes described sodium salt dissolve;

   (3) cooling the solution of step (2) to a second temperature for crystallization, thereby obtaining the salvianolic acid A sodium salt hydrate;

   Wherein, the first temperature is 40-80°C; the second temperature is 0-20°C.
8. The purposes of the salvianolic acid A sodium salt hydrate described in claim 1 or the salvianolic acid A sodium salt hydrate crystal described in claim 4 are used for preparing a pharmaceutical composition for treating or preventing diseases, and the diseases are selected from: Cardiovascular and cerebrovascular diseases, oxidative stress damage of organs or tissues, immune system diseases, hyperlipidemia, complications of diabetes, etc.
9. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises:

   The salvianolic acid A sodium salt hydrate described in claim 1 or the salvianolic acid A sodium salt hydrate crystal described in claim 3; and a pharmaceutically acceptable carrier.
10. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is an injection.