WO2022233310A1 - Salvianolic acid a salt hydrate, preparation method therefor, and use thereof - Google Patents

Salvianolic acid a salt hydrate, preparation method therefor, and use thereof Download PDF

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WO2022233310A1
WO2022233310A1 PCT/CN2022/091078 CN2022091078W WO2022233310A1 WO 2022233310 A1 WO2022233310 A1 WO 2022233310A1 CN 2022091078 W CN2022091078 W CN 2022091078W WO 2022233310 A1 WO2022233310 A1 WO 2022233310A1
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salvianolic acid
salt hydrate
crystal
salvianolic
sodium
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PCT/CN2022/091078
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French (fr)
Chinese (zh)
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叶天健
阚士东
张志杰
潘永存
黄旭阳
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台州永健医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C07C67/52—Separation; Purification; Stabilisation; Use of additives by change in the physical state, e.g. crystallisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C07C67/60—Separation; Purification; Stabilisation; Use of additives by treatment giving rise to chemical modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
    • C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the invention belongs to the field of chemistry, in particular, the invention relates to salvianolic acid A salt hydrate and its preparation method and application.
  • Cardiovascular and cerebrovascular diseases are the number one killer of human health. With the gradual improvement of people's living standards, the proportion of high-fat, high-sugar, and high-protein diets has gradually increased. Especially animal fat, a series of substances represented by "low-density lipoprotein" formed with cholesterol in the body gradually deposit on the inner wall of blood vessels, causing blood vessel blockage and reducing blood supply to key organs such as the heart and brain. Lead to insufficient oxygen supply, thereby affecting the normal physiological functions.
  • Salvia is a commonly used Chinese herbal medicine and one of the medicinal plants with the largest trade volume in the world. Salvia has a wide range of physiological activities, and its products are widely used in medicine, health care products, cosmetics, functional foods and other fields. According to the "Pharmacopoeia”, Danshen has the effects of "activating blood and removing blood stasis, clearing the meridian and relieving pain, clearing the heart and eliminating vexation, cooling blood and eliminating carbuncle", and is widely used in the treatment of cardiovascular and cerebrovascular diseases.
  • the main active components in Salvia are fat-soluble tanshinone compounds represented by tanshinone IIA, and water-soluble phenolic acid compounds represented by salvianolic acid B. Since traditional Chinese medicine is mainly used in the way of "decoction", its water-soluble components have received more attention. Studies have shown that salvianolic acid A is the most active compound in Salvia miltiorrhiza. It has anti-inflammatory, antioxidant, free radical scavenging and other physiological activities, and has potential therapeutic effects on cardiovascular and cerebrovascular diseases.
  • salvianolic acid A in Salvia miltiorrhiza is extremely low, only about 0.01-0.1%.
  • the study by Kan S. et al. (Kan S.etc.J Food Sci 79:C499-504) showed that the water-soluble components of Salvia miltiorrhiza salvianolic acid B and shikalic acid can be chemically transformed into salvianolic acid A, thereby increasing its content and reduce its cost.
  • CN201310487751.6 discloses a method for preparing salvianolic acid A amorphous freeze-dried powder with a purity of over 97% by means of column chromatography and freeze-drying.
  • CN200810223651.1 discloses a method for preparing two kinds of salvianolic acid A crystals, and the purity of the final product can reach more than 90%.
  • CN201710055331.9 discloses a preparation method of salvianolic acid A crystal, the purity of which is more than 99.5%, and the stability is significantly better than that of salvianolic acid A freeze-dried powder, but its water solubility is poor, and the dissolution process needs to be assisted by methods such as heating and ultrasound , there is a big disadvantage when it is developed as an injection.
  • its preparation requires more than 97% salvianolic acid A as a starting material, which leads to high production cost, and it is difficult to use it as a requirement for large-scale commercial production of medicines.
  • Salvianolic acid A amorphous powder is less stable and easily degraded even at room temperature.
  • the preparation of salvianolic acid A into crystals can significantly improve its stability.
  • oral bioavailability 1.47-1.84% oral bioavailability 1.47-1.84%
  • the intravenous preparation is the most suitable dosage form.
  • the intravenous preparation is a high-risk dosage form, it needs to be prepared into a high-purity sample to ensure its safety and quality controllability, and it needs to be convenient for clinical use.
  • the purpose of the present invention is to provide a salvianolic acid A salt hydrate, its preparation method and use.
  • a first aspect of the present invention provides a salvianolic acid A salt hydrate shown in formula I,
  • M is a cation; 0 ⁇ m ⁇ 20, 0 ⁇ n ⁇ 10, 0 ⁇ x ⁇ 20, 0 ⁇ y ⁇ 150.
  • n 2x.
  • n 3x.
  • the M is selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion, or ammonium ion.
  • M is a sodium ion, and 5 ⁇ m ⁇ 15, 1 ⁇ n ⁇ 10, 1 ⁇ x ⁇ 10, 20 ⁇ y ⁇ 100.
  • M is a sodium ion, and m is 11, n is 5, x is 5, and y is 56.
  • M is a potassium ion, and 1 ⁇ m ⁇ 5, 1 ⁇ n ⁇ 5, 1 ⁇ x ⁇ 5, 1 ⁇ y ⁇ 10.
  • M is a potassium ion, and m is 1, n is 1, x is 1, and y is 8.
  • M is a calcium ion, and 1 ⁇ m ⁇ 5, 1 ⁇ n ⁇ 5, 0.5 ⁇ x ⁇ 5, 5 ⁇ y ⁇ 15.
  • M is a calcium ion, and m is 1, n is 1, x is 0.5, and y is 9.
  • M is a magnesium ion, and 1 ⁇ m ⁇ 5, 1 ⁇ n ⁇ 5, 0.5 ⁇ x ⁇ 5, 5 ⁇ y ⁇ 15.
  • M is a magnesium ion, and m is 1, n is 1, x is 0.5, and y is 7.
  • M is an ammonium ion, and 1 ⁇ m ⁇ 5, 1 ⁇ n ⁇ 5, 1 ⁇ x ⁇ 5, 5 ⁇ y ⁇ 15.
  • M is an ammonium ion, and m is 2, n is 2, x is 2, and y is 11.
  • the salvianolic acid A salt hydrate is selected from the following group:
  • Salvianolic acid A sodium salt hydrate A sodium salt hydrate:
  • Salvianolic acid A calcium salt hydrate:
  • Salvianolic acid A ammonium salt hydrate
  • the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A sodium salt hydrate has a characteristic endothermic peak at 86-96°C.
  • the differential scanning calorimetry spectrum of the salvianolic acid A sodium salt hydrate also has a characteristic endothermic peak at 116-126°C.
  • the differential scanning calorimetry spectrum of the salvianolic acid A sodium salt hydrate is substantially as shown in FIG. 6 .
  • the powder X-ray diffraction pattern of the salvianolic acid A sodium salt hydrate has characteristic peaks at the following 2 ⁇ values: 16.56 ⁇ 0.1, 17.90 ⁇ 0.1, 19.04 ⁇ 0.1, 25.97 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A sodium salt hydrate further includes characteristic peaks at the following 2 ⁇ values: 12.00 ⁇ 0.1, 16.18 ⁇ 0.1, 17.07 ⁇ 0.1, 17.54 ⁇ 0.1 , 18.87 ⁇ 0.1, 19.89 ⁇ 0.1, 21.04 ⁇ 0.1, 21.57 ⁇ 0.1, 22.66 ⁇ 0.1, and 22.93 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A sodium salt hydrate has characteristic peaks at the following 2 ⁇ values:
  • the salvianolic acid A sodium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 4 .
  • the salvianolic acid A sodium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
  • the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A potassium salt hydrate has a characteristic endothermic peak at 95-105°C.
  • the differential scanning calorimetry spectrum of the salvianolic acid A potassium salt hydrate also has a characteristic endothermic peak at 142-153°C.
  • the differential scanning calorimetry spectrum of the salvianolic acid A potassium salt hydrate is basically as shown in FIG. 11 .
  • the powder X-ray diffraction pattern of the salvianolic acid A potassium salt hydrate has characteristic peaks at the following 2 ⁇ values: 18.89 ⁇ 0.1, 24.35 ⁇ 0.1, 24.61 ⁇ 0.1, and 26.13 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A potassium salt hydrate further includes characteristic peaks at the following 2 ⁇ values: 16.40 ⁇ 0.1, 21.59 ⁇ 0.1, 22.62 ⁇ 0.1, 32.84 ⁇ 0.1 , , and 36.35 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A potassium salt hydrate has characteristic peaks at the following 2 ⁇ values:
  • the salvianolic acid A potassium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 9 .
  • the salvianolic acid A potassium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
  • the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A calcium salt hydrate has a characteristic endothermic peak at 90-100°C.
  • the differential scanning calorimetry spectrum of the salvianolic acid A calcium salt hydrate is substantially as shown in FIG. 17 .
  • the powder X-ray diffraction pattern of the salvianolic acid A calcium salt hydrate has characteristic peaks at the following 2 ⁇ values: 18.92 ⁇ 0.1, 22.59 ⁇ 0.1, 24.35 ⁇ 0.1, and 27.49 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A calcium salt hydrate further includes characteristic peaks at the following 2 ⁇ values: 16.49 ⁇ 0.1, 21.47 ⁇ 0.1, 23.74 ⁇ 0.1, 25.70 ⁇ 0.1 , and 26.10 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A calcium salt hydrate has characteristic peaks at the following 2 ⁇ values:
  • the salvianolic acid A calcium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 16 .
  • the salvianolic acid A calcium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
  • the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A magnesium salt hydrate has a characteristic endothermic peak at 100-115°C.
  • the differential scanning calorimetry spectrum of the salvianolic acid A magnesium salt hydrate is basically as shown in Figure 23.
  • the powder X-ray diffraction pattern of the salvianolic acid A magnesium salt hydrate has characteristic peaks at the following 2 ⁇ values: 21.65 ⁇ 0.1, 24.18 ⁇ 0.1, 24.49 ⁇ 0.1, 25.32 ⁇ 0.1, 25.91 ⁇ 0.1, and 27.89 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A magnesium salt hydrate further includes characteristic peaks at the following 2 ⁇ values: 16.28 ⁇ 0.1, 17.88 ⁇ 0.1, 19.18 ⁇ 0.1, 22.50 ⁇ 0.1 , and 22.93 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A magnesium salt hydrate has characteristic peaks at the following 2 ⁇ values:
  • the salvianolic acid A magnesium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 21 .
  • the salvianolic acid A magnesium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
  • the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A ammonium salt hydrate has a characteristic endothermic peak at 91-101°C.
  • the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A ammonium salt hydrate has a characteristic endothermic peak at 132-142°C.
  • the differential scanning calorimetry spectrum of the salvianolic acid A ammonium salt hydrate is basically as shown in FIG. 30 .
  • the powder X-ray diffraction pattern of the salvianolic acid A ammonium salt hydrate has characteristic peaks at the following 2 ⁇ values: 14.21 ⁇ 0.1, 18.83 ⁇ 0.1, 21.33 ⁇ 0.1, 24.25 ⁇ 0.1, 24.53 ⁇ 0.1, and 25.97 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A ammonium salt hydrate further includes characteristic peaks at the following 2 ⁇ values: 13.34 ⁇ 0.1, 13.64 ⁇ 0.1, 16.30 ⁇ 0.1, 17.90 ⁇ 0.1 , 18.06 ⁇ 0.1, 19.08 ⁇ 0.1, and 21.83 ⁇ 0.1.
  • the powder X-ray diffraction pattern of the salvianolic acid A ammonium salt hydrate has characteristic peaks at the following 2 ⁇ values:
  • the salvianolic acid A ammonium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 28 .
  • the salvianolic acid A ammonium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
  • a second aspect of the present invention provides a salvianolic acid A salt hydrate crystal, and the salvianolic acid A salt hydrate crystal is selected from the following group:
  • the absolute configuration of the salvianolic acid A sodium salt hydrate crystal is:
  • the absolute configuration of the salvianolic acid A potassium salt hydrate crystal is:
  • the absolute configuration of the salvianolic acid A calcium salt hydrate crystal is:
  • the relative configuration of the salvianolic acid A magnesium salt hydrate crystal is:
  • the absolute configuration of the salvianolic acid A ammonium salt hydrate crystal is:
  • the salvianolic acid A sodium salt hydrate crystal is determined to be the C2 space group by X-ray single crystal diffraction.
  • the salvianolic acid A potassium salt hydrate crystal is determined to be the C2 space group by X-ray single crystal diffraction.
  • the salvianolic acid A calcium salt hydrate crystal is determined to be the C2 space group by X-ray single crystal diffraction.
  • the salvianolic acid A magnesium salt hydrate crystal is determined to be C2 space group by X-ray single crystal diffraction.
  • the salvianolic acid A ammonium salt hydrate crystal is determined to be P2 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 salt hydrate shown in formula I, the method comprises the steps:
  • step (2) adding basic compound to the salvianolic acid A solution obtained in step (1), so that part of salvianolic acid A and the basic compound carry out a salt-forming reaction;
  • step (3) cooling the solution after the salt-forming reaction in step (2) to a second temperature for crystallization, thereby obtaining the salvianolic acid A salt hydrate;
  • the first temperature is 40-80°C; the second temperature is 0-20°C.
  • the first temperature is 45-70°C; preferably about 50°C.
  • the mass concentration of salvianolic acid A is about 5%-20%; preferably, it is about 5%-15%.
  • step (2) about 5%-80% (w/w) of salvianolic acid A and a basic compound are subjected to a salt-forming reaction; preferably, about 10%-60% (w/w) salvianolic acid A and the basic compound are subjected to a salt-forming reaction; more preferably, about 15%-50% (w/w) of the salvianolic acid A and the basic compound are subjected to a salt-forming reaction.
  • the cooling rate in the step (2) 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.
  • the second temperature is 0-20°C; preferably, the second temperature is 1-15°C; more preferably, the second temperature is 4-10°C.
  • stirring is continued.
  • crystallization is performed for 5-48 hours; preferably, crystallization is performed for 8-36 hours; more preferably, crystallization is performed for 12-24 hours.
  • the method further includes the step of vacuum drying the obtained salvianolic acid A salt hydrate.
  • the vacuum drying treatment temperature is 10-30°C.
  • the method further includes a step of recrystallization of the obtained salvianolic acid A salt hydrate; preferably, the recrystallization step includes:
  • step (b) cooling the solution of step (a) to the second temperature to carry out crystallization, thereby obtaining the salvianolic acid A salt hydrate refined product;
  • the first temperature is 40-80°C; the second temperature is 0-20°C.
  • the first temperature is 45-70°C; preferably about 50°C.
  • the mass concentration of salvianolic acid A is about 5%-20%; preferably, it is about 5%-15%.
  • 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°C/min.
  • the second temperature is 0-20°C; preferably, the second temperature is 1-15°C; more preferably, the second temperature is 4-10°C.
  • stirring is continued.
  • crystallization is performed for 5-48 hours; preferably, crystallization is performed for 8-36 hours; more preferably, crystallization is performed for 12-24 hours.
  • the method further includes the step of vacuum drying the obtained salvianolic acid A salt hydrate refined product.
  • the vacuum drying treatment temperature is 10-30°C.
  • the basic compound is selected from: a sodium-containing basic compound, a potassium-containing basic compound, a calcium-containing basic compound, a magnesium-containing basic compound, and an ammonium-containing basic compound.
  • the sodium-containing basic compound is selected from: sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium malate, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium acetate.
  • the potassium-containing basic compound is selected from the group consisting of potassium hydroxide, potassium carbonate, potassium bicarbonate, potassium malate, potassium citrate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and Potassium acetate.
  • the calcium-containing basic compound is selected from calcium carbonate, calcium bicarbonate, calcium oxide, calcium hydroxide, calcium gluconate, and calcium acetate.
  • the magnesium-containing basic compound is selected from the group consisting of: magnesium hydroxide, magnesium oxide, magnesium carbonate, magnesium bicarbonate, basic magnesium carbonate, magnesium malate, magnesium citrate, and magnesium acetate.
  • the ammonium-containing basic compound is selected from the group consisting of ammonia gas, ammonia water, ammonium carbonate, and ammonium bicarbonate.
  • 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 Between about 75%-95%.
  • the mass fraction of water in the aqueous solvent is ⁇ 70%; preferably ⁇ 80%; more preferably ⁇ 90%.
  • the aqueous solvent is selected from the group consisting of water, aqueous ethanol, aqueous acetone and the like.
  • the basic compound is a weak acid salt, preferably a strong base weak acid salt.
  • the anion of the salt is selected from: carbonate ion, bicarbonate ion, silicate ion, metaaluminate ion, acetate ion, etc.; and/or,
  • the cation of the salt is selected from: sodium ion, potassium ion, magnesium ion, lithium ion, ammonium ion, calcium ion.
  • the molar ratio of salvianolic acid A and the basic compound is 1-3:3-1; preferably: 1-3:1-2; most preferably 2:1.
  • the fourth aspect of the present invention provides a use of the salvianolic acid A salt hydrate described in the first aspect of the present invention or the salvianolic acid A salt hydrate crystal described in the second aspect of the present invention, for the preparation of therapeutic or A pharmaceutical composition for preventing diseases selected from cardiovascular and cerebrovascular diseases, immune system diseases, hyperlipidemia, diabetic complications and the like.
  • the fifth aspect of the present invention provides a pharmaceutical composition, comprising:
  • the pharmaceutical composition is composed of the salvianolic acid A salt hydrate described in the first aspect of the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is an injection.
  • Figure 1 is the X-ray single crystal diffraction crystal data of salvianolic acid A sodium salt hydrate
  • FIG. 2A stereostructure ellipsoid diagram of asymmetric unit
  • FIG. 2B stereostructure ellipsoid diagram of a single host molecule
  • 3 is a unit cell stacking projection view along the b-axis of salvianolic acid A sodium salt hydrate.
  • Figure 4 is the powder diffraction pattern of salvianolic acid A sodium salt hydrate.
  • Figure 5 is a list of powder diffraction 2 ⁇ values of salvianolic acid A sodium salt hydrate.
  • Figure 6 is the DSC spectrum of salvianolic acid A sodium salt hydrate.
  • Figure 7 is the X-ray single crystal diffraction crystal data of salvianolic acid A potassium salt hydrate
  • 8A is a three-dimensional structure ellipsoid diagram obtained by X-ray single crystal diffraction of salvianolic acid A potassium salt hydrate obtained by the present invention
  • 8B is a unit cell stacking projection view along the b-axis direction of the salvianolic acid A potassium salt hydrate obtained by the present invention
  • Fig. 9 is the powder diffraction pattern of the salvianolic acid A potassium salt hydrate obtained by the present invention
  • Fig. 10 is the powder diffraction 2 ⁇ value list of the salvianolic acid A potassium salt hydrate obtained by the present invention
  • Fig. 11 is the DSC spectrum of the salvianolic acid A potassium salt hydrate obtained by the present invention
  • Figure 12 is the X-ray single crystal diffraction crystal data of salvianolic acid A calcium salt hydrate
  • FIG. 13A stereostructure ellipsoid diagram of asymmetric unit
  • FIG. 13B stereostructure ellipsoid diagram showing calcium coordination
  • Fig. 15 is the powder diffraction pattern of the salvianolic acid A calcium salt hydrate obtained by the present invention
  • Fig. 17 is the DSC spectrum of the salvianolic acid A calcium salt hydrate obtained by the present invention
  • Fig. 18 is the X-ray single crystal diffraction crystal data of the salvianolic acid A magnesium salt hydrate obtained by the present invention
  • Fig. 19 is the three-dimensional structure ellipsoid diagram obtained by the X-ray single crystal diffraction of the salvianolic acid A magnesium salt hydrate obtained by the present invention
  • Fig. 21 is the powder diffraction pattern of the salvianolic acid A magnesium salt hydrate obtained by the present invention
  • Figure 22 is a list of powder diffraction 2 ⁇ values of the salvianolic acid A magnesium salt hydrate obtained in the present invention
  • Fig. 23 is the DSC spectrum of the salvianolic acid A magnesium salt hydrate obtained by the present invention
  • Figure 24 is the X-ray single crystal diffraction crystal data of the salvianolic acid A ammonium salt hydrate obtained in the present invention
  • Figure 25 ball-and-stick diagram of the three-dimensional structure of the asymmetric unit
  • Figure 26 the three-dimensional structure ellipsoid diagram of a single host molecule
  • 27 is a unit cell stacking projection view along the b-axis direction of the salvianolic acid A ammonium salt hydrate obtained in the present invention
  • Fig. 28 is the powder diffraction pattern of the salvianolic acid A ammonium salt hydrate obtained by the present invention
  • Figure 29 is a list of powder diffraction 2 ⁇ values of the salvianolic acid A ammonium salt hydrate obtained in the present invention
  • Fig. 30 is the DSC spectrum of the salvianolic acid A ammonium salt hydrate obtained by the present invention
  • Figure 31 shows the crude salvianolic acid A product (A), the salvianolic acid A sample (B) after column chromatography, the salvianolic acid A sodium salt hydrate (C) after primary crystallization and the salvianolic acid A after recrystallization during the implementation of the invention HPLC chromatogram of sodium salt hydrate (D) (purity calculated as salvianolic acid A)
  • Fig. 32A is a photomicrograph (10 ⁇ eyepiece+4 ⁇ objective lens) of the single crystal of salvianolic acid A sodium salt hydrate obtained by the method of the present invention
  • Fig. 32B is the salvianolic acid A obtained by the method of patent document CN201910643821.X Micrograph of sodium salt complex crystalline powder (10x eyepiece + 4x objective).
  • salvianolic acid A a kind of salvianolic acid A, salvianolic acid A negative ion, metal ion (respectively sodium ion, potassium ion, calcium ion, magnesium ion, ammonium ion), water molecule composition the complex.
  • metal ion refractive index
  • DSC differential scanning calorimetry
  • TGA thermogravimetric analysis
  • the prepared salvianolic acid A salt hydrate is a new substance with a new crystal form and its purity exceeds 99%.
  • the present invention also discloses a preparation method of the new crystal form.
  • the salvianolic acid A salt hydrate of the present invention is prepared by using salvianolic acid A with a purity of about 80% as the starting material, and the salvianolic acid A salt hydrate can be applied for the treatment of ischemic cerebrovascular disease and ischemic cardiovascular disease. Moreover, the salvianolic acid A salt hydrate has the characteristics of low production cost, high product purity, good stability and good water solubility.
  • the present invention prepares a compound (which is composed of salvianolic acid A, salvianolic acid A negative ions, metal ions (respectively sodium ions, potassium ions, calcium ions, magnesium ions, and ammonium ions) and water molecules. pure substance), obtained its single crystal, and compared with the common positive drugs in the market, the therapeutic effect of the complex on ischemic cerebrovascular disease and ischemic cardiovascular disease was studied.
  • a compound which is composed of salvianolic acid A, salvianolic acid A negative ions, metal ions (respectively sodium ions, potassium ions, calcium ions, magnesium ions, and ammonium ions) and water molecules. pure substance), obtained its single crystal, and compared with the common positive drugs in the market, the therapeutic effect of the complex on ischemic cerebrovascular disease and ischemic cardiovascular disease was studied.
  • this series of crystalline compounds Compared with amorphous salvianolic acid A freeze-dried powder, this series of crystalline compounds has the characteristics of high purity, good stability and low production cost. Compared with the reported salvianolic acid A crystals, the series of crystal complexes are single crystals, and have the characteristics of good water solubility and low production cost, and are more suitable for development into intravenous injection preparations. Compared with the positive drug butylphthalide and sodium chloride injection, salvianolic acid for injection, and salvianolate for injection, this series of crystalline complexes can significantly reduce the area of brain/myocardial infarction, significantly reduce the mortality rate of model animals, and significantly reduce the size of cerebral/myocardial infarction.
  • the effect of improving limb function recovery in cerebral ischemia/myocardial ischemia model animals The series of crystalline compounds can be applied to the treatment of ischemic heart disease and ischemic cerebrovascular disease, and can be used as APIs for related drug injection preparations.
  • the properties of the salvianolic acid A salt hydrate of the present invention can be studied by the following methods and instruments, such as X-ray single crystal diffraction, X-ray powder diffraction, differential scanning calorimetry, and thermogravimetric analysis (TGA) for analysis. . These analytical methods can be conventional methods in the art.
  • a compound can bind a solvent to become a solvate or bind several molecules of solvent.
  • the crystal form is 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 because of their shape or hygroscopicity.
  • X-ray powder ray diffraction pattern is a necessary way to identify the crystal form, but it is not the only way. Hydrate crystal forms, due to the different ways in which water binds to the compound, may result in similar X-ray powder ray diffraction patterns, but different other characterization data, such as DSC patterns.
  • 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. Its water solubility is "slightly soluble".
  • the salvianolic acid A sodium salt complex was prepared, Figure 32A And FIG. 32B shows the photomicrograph of the salvianolic acid A sodium salt hydrate single crystal and the salvianolic acid A sodium salt complex of the present invention.
  • 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 complex of the comparative patent is as follows:
  • the former is single crystal, pure substance, stable in chemical composition and good in water solubility
  • the latter is a crystalline powder and mixture, which theoretically cannot guarantee the stability of its chemical composition (such as the ratio of salvianolic acid A molecule and salvianolic acid A sodium), and has poor water solubility;
  • the most basic feature of APIs for injections is that the composition is fixed and pure.
  • the salvianolic acid A sodium salt complex crystalline powder of the comparative patent the salvianolic acid A sodium salt hydrate single crystal prepared by the present invention has significant advantages when used as an injection raw material drug.
  • the salvianolic acid A potassium, calcium, magnesium, and ammonium salt hydrate crystals (single crystals) prepared by the present invention are similar to the salvianolic acid A salt complex crystalline powder described in the comparative patent. Advantage.
  • the invention also provides a crystallization process of salvianolic acid A, through which a new crystal form of salvianolic acid A salt hydrate is obtained.
  • the crystal 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 requirements of the new pharmaceutical raw materials for injection.
  • the salvianolic acid A salt hydrate crystals obtained by the present invention have the advantages of high product purity, low production cost, good product water solubility, etc., and are suitable for the development of salvianolic acid A injection preparations. .
  • active ingredient or “active compound” refers to the salvianolic acid A salt hydrate of the present invention.
  • the invention discloses a series of salvianolic acid A salt hydrate crystals and a preparation method thereof (such as salvianolic acid A sodium salt hydrate, salvianolic acid A potassium salt hydrate, salvianolic acid A calcium salt hydrate, salvianolic acid A calcium salt hydrate, salvianolic acid A sodium salt hydrate, salvianolic acid A potassium salt hydrate A magnesium salt hydrate, salvianolic acid A ammonium salt hydrate).
  • the medicinal materials of Salvia miltiorrhiza were prepared with reference to the preparation method of "Preparation of Salvia miltiorrhiza Aqueous Extract" in "Chinese Pharmacopoeia" (Part I) (2020 Edition).
  • the latter is reacted to prepare a crude product of salvianolic acid A, which is then subjected to resin column chromatography to obtain a salvianolic acid A sample with a purity of ⁇ 80%.
  • the sample is then subjected to the process of crystallization and recrystallization to prepare high-purity salvianolic acid A salt hydrate crystals.
  • the crystal is composed of salvianolic acid A, salvianolic acid A negative ions, cations, and water molecules.
  • the series of compounds are all new pure substances and have a new crystal form. Compared with the amorphous salvianolic acid A powder, they have 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 salt hydrate prepared by the 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 solution and salvianolate for injection.
  • compositions and methods of administration are provided.
  • salvianolic acid A can be used to treat the following diseases: cardiovascular disease (such as ischemic heart disease, stroke), hyperlipidemia, diabetes and its complications and so on. Therefore, the salvianolic acid A salt hydrate of the present invention can be used to treat or prevent the above-mentioned diseases.
  • the pharmaceutical composition of the present invention comprises the salvianolic acid A salt hydrate of the present invention and a pharmaceutically acceptable excipient or carrier within a safe and effective amount.
  • the "safe and effective amount” refers to: the amount of salvianolic acid A salt hydrate is sufficient to significantly improve the condition without causing serious side effects.
  • 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.
  • the "one dose” is one injection dose or one capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid filler or gelling substances which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity. "Compatibility” as used herein 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 moieties include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, 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.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as wetting agents (such as ten Sodium dialkyl sulfate), disintegrating agent, coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as talc
  • solid lubricants such as stearic acid
  • the mode of administration of the salvianolic acid A salt hydrate or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.
  • administration is by injection, such as intravenous injection.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • 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 compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostea
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using 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 compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. If desired, the active ingredient may also be in microencapsulated form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally employed 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, and the like.
  • inert diluents conventionally employed 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, dimethylform
  • compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or 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 excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the salvianolic acid A salt hydrate of the present invention is particularly suitable for injection administration due to 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 that may be required if necessary.
  • the salvianolic acid A salt hydrate of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the crystalline form of the present invention is suitable for mammals (such as humans) in need of treatment, wherein the dose is a pharmaceutically effective dose when administered, and for a 60kg body weight human.
  • the daily dosage is usually 1-2000 mg, preferably 10-500 mg.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the salvianolic acid A salt hydrate crystal prepared for the first time in the present invention has stronger stability and is more resistant to high temperature, high humidity, strong light irradiation, etc.;
  • the salvianolic acid A salt hydrate crystal prepared by the present invention has better water solubility, has a significantly faster dissolution rate, and is suitable for use as an injection.
  • the method for preparing salvianolic acid A crystals provided by the present invention can use low-purity salvianolic acid A raw materials (about 80%) as the starting material for crystallization, so that the salvianolic acid A is greatly reduced. Cost of crystal preparation.
  • the method for preparing salvianolic acid A crystals provided by the present invention has high product purity and high yield, and is convenient for industrial production.
  • Step 1 Preparation of crude salvianolic acid A
  • the salvianolic acid A sample with HPLC purity ⁇ 80% prepared by the method is used for the subsequent crystallization method to prepare the starting material of different salvianolic acid A salt hydrates.
  • salvianolic acid A solution with a purity of ⁇ 80% (containing about 16 g of salvianolic acid A) to a concentration of 15%, heat it to 50°C in a water bath, slowly add about 532 mg of sodium carbonate powder, and wait for the two to react completely (reaction about 15 minutes, wherein 31% of salvianolic acid A can be reacted to generate salvianolic acid A sodium salt), the solution is cooled to 20°C, the cooling rate is 0.1°C/min, during which constant stirring, and then further cooled to 5°C, Stir continuously for 24h to obtain salvianolic acid A sodium salt hydrate (precipitation).
  • the "salvianolic acid A sodium salt hydrate" obtained above is used for X-ray single crystal diffraction, X-ray powder diffraction, DSC, TGA analysis and determination of solubility and stability, as well as anti-cerebral ischemia, myocardial Ischemia and other pharmacodynamic tests are used.
  • Figure 1 is the X-ray single crystal diffraction crystal data of salvianolic acid A sodium salt hydrate
  • FIG. 2A stereostructure ellipsoid diagram of asymmetric unit
  • FIG. 2B stereostructure ellipsoid diagram of a single host molecule
  • 3 is a unit cell stacking projection view along the b-axis of salvianolic acid A sodium salt hydrate.
  • Figure 4 is the powder diffraction pattern of salvianolic acid A sodium salt hydrate.
  • Figure 5 is a list of powder diffraction 2 ⁇ values of salvianolic acid A sodium salt hydrate.
  • Figure 6 is the DSC spectrum of salvianolic acid A sodium salt hydrate.
  • the TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
  • salvianolic acid A solution with a purity of ⁇ 80% (containing 16 g of salvianolic acid A) to a concentration of 10%, heat it to 50° C. in a water bath, slowly add about 1117 mg of potassium carbonate powder, and wait for the two to react completely (the reaction is about 10 minutes, 50% of the salvianolic acid A can be reacted to generate salvianolic acid A potassium salt), the solution is cooled to 20°C, the cooling rate is 0.3°C/min, and the stirring is continued, then further cooled to 5°C, continuously Stir for 24h to obtain salvianolic acid A potassium salt hydrate (precipitation).
  • Figure 7 is the X-ray single crystal diffraction crystal data of salvianolic acid A potassium salt hydrate
  • Figure 8A is a three-dimensional structure ellipsoid obtained by X-ray single crystal diffraction of salvianolic acid A potassium salt hydrate
  • Fig. 8B is a unit cell stacking projection view along the b-axis of salvianolic acid A potassium salt hydrate
  • Fig. 9 is the powder diffraction pattern of salvianolic acid A potassium salt hydrate
  • Figure 10 is a list of powder diffraction 2 ⁇ values of salvianolic acid A potassium salt hydrate
  • Figure 11 is the DSC spectrum of salvianolic acid A potassium salt hydrate
  • the TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
  • Figure 12 is the X-ray single crystal diffraction crystal data of salvianolic acid A calcium salt hydrate
  • Fig. 13A stereostructure ellipsoid diagram of asymmetric unit
  • Fig. 13B stereostructure ellipsoid diagram showing calcium coordination
  • Fig. 14 is a unit cell stacking projection view along the b-axis of salvianolic acid A calcium salt hydrate
  • Figure 15 is the powder diffraction pattern of salvianolic acid A calcium salt hydrate
  • Figure 16 is a list of powder diffraction 2 ⁇ values of salvianolic acid A calcium salt hydrate
  • Figure 17 is the DSC spectrum of salvianolic acid A calcium salt hydrate.
  • the TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
  • salvianolic acid A solution with a purity of ⁇ 80% (containing 16 g of salvianolic acid A) to a concentration of 10%, take a water bath at 50 ° C, slowly add about 470 mg of magnesium hydroxide powder, and wait for the two to react completely (the reaction is about 10 minutes, wherein 50% of salvianolic acid A can be reacted to generate salvianolic acid A magnesium salt), the solution is cooled to 20 ° C, the cooling rate is 0.2 ° C/min, during which constant stirring, then further cooled to 5 ° C, stirring Crystallize for 24h to obtain salvianolic acid A magnesium salt hydrate (precipitation).
  • Figure 18 is the X-ray single crystal diffraction crystal data of salvianolic acid A magnesium salt hydrate
  • Figure 19 is a three-dimensional structure ellipsoid figure obtained by X-ray single crystal diffraction of salvianolic acid A magnesium salt hydrate
  • Fig. 20 is a unit cell stacking projection view along the b-axis of salvianolic acid A magnesium salt hydrate
  • Figure 21 is the powder diffraction pattern of salvianolic acid A magnesium salt hydrate
  • Figure 22 is a list of powder diffraction 2 ⁇ values of salvianolic acid A magnesium salt hydrate
  • Figure 23 is the DSC spectrum of salvianolic acid A magnesium salt hydrate
  • the TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
  • Figure 24 is the X-ray single crystal diffraction crystal data of salvianolic acid A ammonium salt hydrate
  • Figure 25 ball-and-stick diagram of the three-dimensional structure of the asymmetric unit
  • Figure 26 the three-dimensional structure ellipsoid diagram of a single host molecule
  • Fig. 27 is a unit cell stacking projection view along the b-axis of salvianolic acid A ammonium salt hydrate
  • Fig. 28 is the powder diffraction pattern of the salvianolic acid A ammonium salt hydrate obtained by the present invention
  • Figure 29 is a list of powder diffraction 2 ⁇ values of the salvianolic acid A ammonium salt hydrate obtained in the present invention
  • Fig. 30 is the DSC spectrum of the salvianolic acid A ammonium salt hydrate obtained by the present invention
  • the TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
  • Influencing factor test is an important method for drug stability investigation. It can have a preliminary understanding of drug stability in a relatively short period of time, and is of great significance to subsequent new drug development. All APIs and preparations must be tested for influencing factors.
  • the salvianolic acid A salt (sodium, potassium, calcium, magnesium, ammonium) hydrate used in this embodiment is prepared by the method in the corresponding embodiment of the present invention.
  • the salvianolic acid A crystal is prepared by the preparation method of the salvianolic acid A crystal described in the patent document CN201710055331.9 (Ye Tianjian et al., a crystal form of salvianolic acid A and its preparation method).
  • Salvianolic acid A freeze-dried powder is prepared by the preparation method of salvianolic acid A recorded in patent document CN201310487751.6 (Kan Shidong, a kind of preparation method of salvianolic acid A)
  • test sample Place the test sample in a suitable open container (such as a weighing bottle or a petri dish), spread it into a thin layer with a thickness of ⁇ 5mm, and carry out the following tests:
  • a suitable open container such as a weighing bottle or a petri dish
  • High temperature test The test sample was placed in a clean container with the opening of the test sample, placed at a temperature of 60 °C for 10 days, and then sampled for HPLC detection, and compared with the sample at 0 days.
  • High-humidity test The opening of the test sample is placed in a constant-humidity airtight container, and it is placed under the condition of 25°C under the condition of relative humidity of 90 ⁇ 5% for 10 days, and then sampled for HPLC detection, and the weight of the sample before and after being placed is recorded to investigate the quality of the sample. Moisture condition. (The KNO 3 saturated solution was chosen to be placed together in a closed container to create a high humidity environment: 92.5% relative humidity at 25°C), and compared 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 is irradiated for 10 days under the condition of illuminance 4500lx ⁇ 500lx. After 10 days, take samples for HPLC detection, pay attention to the appearance change of the test sample, and compare it with the sample at 0 days.
  • the salvianolic acid A salt hydrate crystal prepared by the present invention has significantly better stability than freeze-dried powder, and is more suitable for the use of raw materials for new drug development.
  • Example 8 Comparative study on water solubility of salvianolic acid A (sodium, potassium, calcium, magnesium, ammonium) salt hydrate, salvianolic acid A crystal, and salvianolic acid A freeze-dried powder
  • solubility determination refers to the method described in the "Chinese Pharmacopoeia” (2020 edition four parts) (in order to develop it into an intravenous preparation, only its solubility in water is studied), as follows:
  • Salvianolic acid A freeze-dried powder:
  • Judgment of "dissolving” 0.12 g of crystals in 3 mL of water, insoluble in 5 minutes, insoluble in 10 minutes, insoluble in 15 minutes, insoluble in 20 minutes, insoluble in 25 minutes, and dissolved in 30 minutes.
  • Salvianolic acid A sodium salt hydrate A sodium salt hydrate:
  • Salvianolic acid A calcium salt hydrate:
  • Judgment of "dissolving” 0.12 g of crystals in 3 mL of water, insoluble in 5 minutes, insoluble in 10 minutes, insoluble in 15 minutes, and dissolved in 20 minutes.
  • Salvianolic acid A ammonium salt hydrate
  • the clinical dosage of salvianolic acid A is about 50-100 mg/piece.
  • salvianolic acid A sodium salt hydrate and salvianolic acid A magnesium salt hydrate can fully meet the clinical needs.
  • Salvianolic acid A potassium salt hydrate, salvianolic acid A ammonium salt hydrate, and salvianolic acid A calcium salt hydrate can basically meet the clinical needs.
  • salvianolic acid A crystal is used directly, it is difficult to meet its clinical needs as an intravenous preparation.
  • Example 9 Effects of salvianolic acid A salt hydrate and positive drugs on cerebral infarct size in ischemic stroke model animals
  • the best-selling drug on the market was selected as the positive drug, and the effect of salvianolic acid A salt hydrate crystal and the positive drug on the cerebral infarct size of the stroke model animal was compared.
  • the positive drugs were butylphthalide and sodium chloride injection and salvianolic acid for injection.
  • the former is the drug for ischemic stroke recommended in the "Guidelines for Diagnosis and Treatment of Ischemic Stroke in China" (2018 Edition), and the latter is the injection preparation of water-soluble phenolic compounds of Salvia miltiorrhiza represented by salvianolic acid B. References It is also reported to have good activity.
  • Ischemia-nonperfusion model middle artery embolization-coagulation in rats
  • SD rats were weighed and anesthetized by intraperitoneal injection of 15% chloral hydrate 300 mg/kg.
  • the left side was fixed on the operating table of the rat, and the left temporal top and face were shaved. After disinfection with 75% ethanol, the left eye and Between the left ear, the skin was incised, the temporalis muscle and masseter muscle were bluntly separated, and the temporal bone wing plate was exposed. Under the operating microscope, a 2mm ⁇ 2mm bone was ground with a skull drill at the junction of the temporal bone and the temporal squamous bone 1mm near the oral side. window, use a spudger to pry open the skull.
  • Ischemia-reperfusion model middle artery embolization-suture method in rats
  • the rats were anesthetized by intraperitoneal injection of 15% chloral hydrate 300 mg/kg, supine and fixed on the rat operating table, the neck was shaved, sterilized with 75% ethanol, the skin was cut longitudinally with surgical scissors, and the side of the neck was exposed by surgical separation.
  • the common artery was separated and ligated along the common carotid artery, and the external carotid artery and pterygo-maxillary artery were ligated.
  • An arterial clip was placed at the proximal end of the common carotid artery to block the blood flow.
  • a small opening was cut with ophthalmic scissors at the distal end, and a plug was inserted.
  • mice were administered by tail vein injection once, and then they were put back into the cage.
  • Butylphthalide is recognized as an effective drug for the treatment of ischemic stroke. It is recommended by the "Guidelines for the Diagnosis and Treatment of Ischemic Stroke in China” (2018 edition), and its sales in 2020 will exceed 6 billion yuan. The results show that salvianolic acid A sodium salt hydrate and positive drug butylphthalide can significantly reduce cerebral infarction in model animals in ischemia-reperfusion and ischemia-nonperfusion models compared with the "vehicle control group" without drugs. area.
  • salvianolic acid A sodium salt hydrate reached the maximum therapeutic effect (therapeutic ceiling) at a dose of 10 mg/kg, while butylphthalide reached the maximum therapeutic effect (therapeutic ceiling) at a dose of 3 mg/kg. And whether in ischemia-reperfusion or ischemia-nonperfusion models, the maximum efficacy of salvianolic acid A sodium salt hydrate was significantly higher than that of butylphthalide.
  • salvianolic acid A sodium salt hydrate for the treatment of ischemic stroke is significantly better than the control group without medication, and also significantly better than the positive drug D. phthalide.
  • Example 10 Effects of salvianolic acid A sodium salt hydrate and positive drugs on myocardial infarction size in ischemic heart disease model animals
  • the best-selling drug on the market is selected as the positive drug, and the effect of salvianolic acid A salt hydrate crystal and the positive drug on the myocardial infarction area of the ischemic heart disease model animal is compared.
  • the positive drug selects Salvia miltiorrhiza polyphenolate for injection (Lvgu Pharmaceutical), whose main component is salvianolic acid B magnesium salt (content 85%) water-soluble phenolic compounds of Salvia miltiorrhiza, which is currently the best-selling cardiac muscle in the market.
  • salvianolic acid B magnesium salt content 85%
  • the rats were fasted for 12 hours, and then anesthetized by intraperitoneal injection of 15% chloral hydrate 300 mg/kg after weighing.
  • a transverse incision was made between the 3-4 ribs on the left side of the sternum.
  • the skin was bluntly separated with a hemostat and the pleura was punctured.
  • two hemostats were used to clamp the sternum horizontally. Cut the sternum between the forceps, insert a retractor to spread the ribs, cut the pericardium to expose the heart, the great cardiac vein can be seen in the anterior interventricular groove, and use this as a sign, and use a 2 ⁇ 6 belt about 2-3mm from the root under the left atrial appendage.
  • Thread round needle 6-0 medical cotton monofilament thread bypasses the deep surface of the artery and exits the needle in the groove next to the conus pulmonary artery, with a depth of about 1mm and a width of about 2mm.
  • the two ends of the silk thread were passed out from a polyethylene tube with a diameter of 2 mm for use. After stabilizing for 10 minutes, insert a cotton swab into the PE tube, tighten the silk thread and push the PE tube forward with the cotton swab until the coronary blood flow is blocked, and the ischemia model is constructed.
  • the rats were given one administration to the tail vein, and the rats were sacrificed 24 hours later, and the size of myocardial infarction was detected.
  • the construction of the ischemia-reperfusion model is the same as the ischemia-non-perfusion model in the early stage.
  • the cotton swab is pulled out from the PE tube to achieve reperfusion.
  • the rats were intravenously administered once, and the rats were sacrificed 24 hours later to detect the myocardial infarction area.
  • Salvia polyphenolate is a phenolic compound derived from Salvia miltiorrhiza, and it is an effective drug for the treatment of ischemic cardiovascular disease.
  • the results showed that salvianolic acid A sodium salt hydrate and salvianolic acid salt in the ischemia-reperfusion model and ischemia-nonperfusion model, compared with the "vehicle control group" without medication, can significantly reduce the experimental animals. myocardial infarction size.
  • the maximum effect dose of salvianolic acid A sodium salt hydrate is 10mg/kg, and the maximum effect dose of salvianolic acid salt is 40mg/kg.
  • the former's maximum healing effect (efficacy ceiling) is significantly better than the latter.
  • Example 11 Effects of salvianolic acid A sodium salt hydrate and positive drugs on thrombosis in animals
  • the best-selling drug on the market was selected as the positive drug, and the effects of salvianolic acid A salt hydrate crystals and the positive drug on thrombosis in animals were compared.
  • the positive drug was selected as Butylphthalide Sodium Chloride Injection (Shi Pharmaceutical Group).
  • the present embodiment selects SD rats as experimental animals, and the concrete test method is as follows:
  • Rats were randomly divided into 5 groups with 10 rats in each group. After weighing, the rats were anesthetized by intraperitoneal injection of 15% chloral hydrate 300 mg/kg. After anesthesia, the rats were administered by tail vein injection according to their body weight, and the administration volume was 1 ml/kg.
  • the rat was fixed on the mouse board in a supine position, and the tongue was pulled out to prevent suffocation caused by falling back of the tongue.
  • the neck was shaved, disinfected with 75% alcohol, the neck skin was cut longitudinally, the right common carotid artery and the left external jugular vein were separated, and one end of the prepared polyethylene tube was inserted into the left external jugular vein first, and the other end was inserted into the left external jugular vein.
  • the right common carotid artery is inserted, the arterial clip is opened, and blood flows from the right common carotid artery through the tube back to the left external jugular vein.
  • the silk thread was quickly taken out and weighed. The total weight minus the dry weight of the silk thread was the wet weight of the thrombus.
  • Example 12 Effects of salvianolic acid A sodium salt hydrate and positive drugs on inflammatory factors in the blood of ischemic stroke and ischemic heart disease model animals
  • the best-selling drug on the market was selected as the positive drug, and the effects of salvianolic acid A salt hydrate crystal and the positive drug on blood inflammatory factors in ischemic stroke and ischemic heart disease model animals were compared.
  • the positive drug for the ischemic heart disease model was selected as salvianolate for injection (Lvgu Pharmaceutical) and the positive drug for the ischemic stroke model was selected as butylphthalide and sodium chloride injection (Shi Pharmaceutical Group).
  • SD rats were selected as the test animals, and the specific test methods were referred to the construction methods of "ischemia-reperfusion model" in Examples 13 and 14, respectively.
  • drugs or control
  • 3 ml of blood was collected from the femoral artery, centrifuged at 3000 revolutions for 10 min, and the serum was collected.
  • both salvianolic acid A sodium salt hydrate and the positive drug salvianolic acid salt can reduce the concentration of inflammatory factors in the blood of myocardial ischemia model animals and increase the activity of SOD, among which the effect of salvianolic acid A sodium salt hydrate Better than salvia polyphenolate. This may be one of the reasons for their therapeutic effect.
  • salvianolic acid A solution with a purity of ⁇ 80% (containing 16 g of salvianolic acid A) to a concentration of 20%, take a water bath at 50°C, slowly add about 343 mg of sodium carbonate powder, and wait for the two to react completely (reaction for about 15 minutes). , 20% of the salvianolic acid A can be reacted to generate salvianolic acid A sodium salt), the solution is slowly cooled to 20 °C, the cooling rate is 0.2 °C/min, and the stirring is continued, and then further cooled to 5 °C, continuously Stir for 24h to obtain salvianolic acid A sodium salt hydrate (precipitation).
  • the amount of sodium carbonate powder added was changed to 1373 mg (reaction for about 20 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A sodium salt).
  • the final product "salvianolic acid A sodium salt hydrate crystal" was 4.256 g, and its HPLC purity was 99.5% in terms of salvianolic acid A.
  • the basic sodium compound used actually provides sodium ions, as long as it can be ensured that the basic sodium compound and salvianolic acid A can react. Therefore, the basic sodium compound adopted, in addition to the sodium carbonate used in the present invention, weak acids such as sodium hydroxide, sodium bicarbonate, sodium malate, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium acetate and sodium
  • weak acids such as sodium hydroxide, sodium bicarbonate, sodium malate, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium acetate and sodium
  • the formed strong base and weak acid salt can be used to prepare the salvianolic acid A sodium salt hydrate crystal described in the present invention.
  • salvianolic acid A solution with a purity of ⁇ 80% (containing 16 g of salvianolic acid A) to a concentration of 20%, take a water bath at 50°C, slowly add about 447 mg of potassium carbonate powder, and wait for the two to react completely (reaction for about 25 minutes). , 20% of salvianolic acid A can be reacted to generate salvianolic acid A potassium salt), the solution is slowly cooled to 20°C, the cooling rate is 0.2°C/min, during which stirring is continued, and then further cooled to 5°C, continuously Stir for 24h to obtain salvianolic acid A potassium salt hydrate (precipitation).
  • the amount of potassium carbonate powder added was changed to 1788 mg (reaction for about 20 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A potassium salt).
  • the final product "salvianolic acid A potassium salt hydrate crystal" was 3.221 g, and its HPLC purity was 99.5% based on salvianolic acid A.
  • the basic potassium compound used in addition to the potassium carbonate used in the present invention, is potassium hydroxide, potassium bicarbonate, potassium malate, potassium citrate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium acetate Strong bases and weak acid salts formed by isoweak acids and potassium can be used in the present invention to prepare the salvianolic acid A potassium salt hydrate crystals of the present invention.
  • salvianolic acid A solution with a purity of ⁇ 80% (containing 16 g of salvianolic acid A) to a concentration of 20%, take a water bath at 50 ° C, slowly add about 324 mg of calcium carbonate powder, and wait for the two to react completely (the reaction is about 15 minutes). , 20% of the salvianolic acid A can be reacted to generate salvianolic acid A calcium salt), the solution is slowly cooled to 20°C, the cooling rate is 0.2°C/min, during which stirring is continued, and then further cooled to 5°C, continuously Stir for 24h to obtain salvianolic acid A calcium salt hydrate (precipitation).
  • the amount of calcium carbonate powder added was changed to 1296 mg (reaction for about 20 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A calcium salt).
  • the final product "salvianolic acid A calcium salt hydrate crystal" was 5.501 g, and its HPLC purity was 99.5% in terms of salvianolic acid A.
  • the basic calcium compound used actually provides calcium ions, as long as it can be ensured that the basic calcium compound and salvianolic acid A can react. Therefore, the basic calcium compound adopted, in addition to the calcium carbonate used in the present invention, the salts formed by weak acids such as calcium bicarbonate, calcium oxide, calcium hydroxide, calcium gluconate, calcium acetate and calcium, all can be applied to the present invention , for the preparation of the salvianolic acid A calcium salt hydrate crystals described in the present invention
  • salvianolic acid A solution with a purity of ⁇ 80% (containing 16 g of salvianolic acid A) to a concentration of 20%, take a water bath at 50 ° C, slowly add about 188 mg of magnesium hydroxide powder, and wait for the two to react completely (the reaction is about 15 minutes, wherein 20% of salvianolic acid A can be reacted to generate salvianolic acid A magnesium salt), the solution is slowly cooled to 20 °C, the cooling rate is 0.2 °C/min, during which constant stirring, then further cooled to 5 °C, Stir continuously for 24h to obtain salvianolic acid A magnesium salt hydrate (precipitation).
  • the amount of magnesium hydroxide powder added was changed to 751 mg (reaction for about 20 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A magnesium salt).
  • the final product "salvianolic acid A magnesium salt hydrate crystal" is 0.522 g, and its HPLC purity is 99.5% based on salvianolic acid A.
  • the basic magnesium compound used actually provides magnesium ions, as long as the basic magnesium compound and salvianolic acid A can be guaranteed to react. Therefore, the basic magnesium compound adopted, in addition to the magnesium hydroxide used in the present invention, weak acids such as magnesium hydride, magnesium carbonate, magnesium bicarbonate, basic magnesium carbonate, magnesium malate, magnesium citrate, magnesium acetate and magnesium form
  • the salt of salvianolic acid A can be used in the present invention to prepare the salvianolic acid A magnesium salt hydrate crystal of the present invention.
  • the amount of ammonium carbonate powder added was changed to 1.244 mg (reaction for about 10 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A ammonium salt).
  • the final product "salvianolic acid A ammonium salt hydrate crystal" is 1.688 g, and its HPLC purity is 99.5% in terms of salvianolic acid A.
  • the basic ammonium (ammonia) compound used actually provides ammonium ions, as long as the basic ammonium (ammonia) compound can be guaranteed to react with salvianolic acid A. Therefore, the basic ammonia (ammonia) compound adopted, in addition to the ammonium carbonate used in the present invention, ammonia, ammonia water, ammonium bicarbonate, etc. can provide ammonium ions, and can react with salvianolic acid A compounds, all It can be used in the present invention to prepare the salvianolic acid A ammonium salt hydrate crystal of the present invention.

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Abstract

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

Description

丹酚酸A盐水合物、其制备方法及用途Salvianolic acid A salt hydrate, its preparation method and use 技术领域technical field
本发明属于化学领域，具体地说，本发明涉及丹酚酸A盐水合物与其制备方法及用途。The invention belongs to the field of chemistry, in particular, the invention relates to salvianolic acid A salt hydrate and its preparation method and application.
背景技术Background technique
心脑血管疾病是人类健康的头号杀手。随着人们生活水平的逐渐提高，高脂、高糖、高蛋白在饮食中所占的比例逐渐增加。尤其是动物性脂肪，其在体内与胆固醇形成的以“低密度脂蛋白”为代表的一系列物质，逐渐沉积于血管内壁，引起血管堵塞、降低心脏、大脑等关键脏器的供血量，导致其氧供应不足，从而影响正常生理功能的发挥。Cardiovascular and cerebrovascular diseases are the number one killer of human health. With the gradual improvement of people's living standards, the proportion of high-fat, high-sugar, and high-protein diets has gradually increased. Especially animal fat, a series of substances represented by "low-density lipoprotein" formed with cholesterol in the body gradually deposit on the inner wall of blood vessels, causing blood vessel blockage and reducing blood supply to key organs such as the heart and brain. Lead to insufficient oxygen supply, thereby affecting the normal physiological functions.
更严重的，因为血管狭窄、栓塞、栓塞脱落、栓塞堵塞毛细血管等导致的“缺血/再灌注”过程，会导致心肌细胞和大脑神经细胞的严重损伤，轻者会影响心脏和大脑的生理功能，引起各种心脑血管疾病，留下瘫痪、半身不遂、冠心病、心绞痛等并发症，重者可以直接导致死亡！More serious, because of the "ischemia/reperfusion" process caused by vascular stenosis, embolism, embolism shedding, and capillary blockage by embolism, it will lead to serious damage to myocardial cells and brain nerve cells, and in mild cases, it will affect the physiology of the heart and brain. function, causing various cardiovascular and cerebrovascular diseases, leaving paralysis, hemiplegia, coronary heart disease, angina pectoris and other complications, and severe cases can directly lead to death!
丹参是一种常用中药材，是全球贸易额最大的药用植物之一。丹参具有广泛的生理活性，其产品广泛的应用于药品、保健品、化妆品、功能性食品等领域。《药典》记载，丹参具有“活血祛瘀、通经止痛、清心除烦、凉血消痈”的功效，广泛的应用于心脑血管疾病的治疗。Salvia is a commonly used Chinese herbal medicine and one of the medicinal plants with the largest trade volume in the world. Salvia has a wide range of physiological activities, and its products are widely used in medicine, health care products, cosmetics, functional foods and other fields. According to the "Pharmacopoeia", Danshen has the effects of "activating blood and removing blood stasis, clearing the meridian and relieving pain, clearing the heart and eliminating vexation, cooling blood and eliminating carbuncle", and is widely used in the treatment of cardiovascular and cerebrovascular diseases.
丹参中的主要活性成分是以丹参酮ⅡA为代表的脂溶性丹参酮类化合物，和以丹酚酸B为代表的水溶性酚酸类化合物。由于中药的用药主要以“水煎煮”的方式进行，其水溶性成分受到了更加广泛的关注。研究表明，丹酚酸A是丹参中活性最高的化合物，其具有抗炎、抗氧化、清除自由基等多种生理活性，对心脑血管疾病具有潜在的治疗作用。目前以丹参为主要活性成分的中成药，已经广泛的应用于心脑血管疾病的治疗,如：丹红注射液、复方丹参滴丸、注射用丹参多酚酸、注射用丹参多酚酸盐等。The main active components in Salvia are fat-soluble tanshinone compounds represented by tanshinone IIA, and water-soluble phenolic acid compounds represented by salvianolic acid B. Since traditional Chinese medicine is mainly used in the way of "decoction", its water-soluble components have received more attention. Studies have shown that salvianolic acid A is the most active compound in Salvia miltiorrhiza. It has anti-inflammatory, antioxidant, free radical scavenging and other physiological activities, and has potential therapeutic effects on cardiovascular and cerebrovascular diseases. At present, Chinese patent medicines with Danshen as the main active ingredient have been widely used in the treatment of cardiovascular and cerebrovascular diseases, such as: Danhong injection, compound Danshen dripping pills, salvianolic acid for injection, salvianolic acid salt for injection, etc. .
丹酚酸A在丹参药材中含量极低，仅占0.01-0.1％左右。Kan S.等(Kan S.etc.J Food Sci 79:C499-504)的研究表明，丹参水溶性成分丹酚酸B和紫草酸，可以通过化学转化的方式生成丹酚酸A，从而增加其含量，降低其成本。CN201310487751.6公开了经过柱色谱层析、冷冻干燥的方式，制备纯度超过97％的丹酚酸A无定型冻干粉的方法。CN200810223651.1公开了制备丹酚酸A两种晶体的方法，最终产物纯度可达90％以上。CN201710055331.9公开了一种丹酚酸A结晶的制备方法，其纯度超过99.5％，稳定性显著好于丹酚酸A冻干粉，但其水溶性较差，溶解过程需要升温、超声等方式辅助，将其作为注射剂开发时存在较大劣势。并且，其制备需要以97％以上的丹酚酸A为起始物料，导致其生产成本较高，难以其作为药品大规模化商业生产的要求。The content of salvianolic acid A in Salvia miltiorrhiza is extremely low, only about 0.01-0.1%. The study by Kan S. et al. (Kan S.etc.J Food Sci 79:C499-504) showed that the water-soluble components of Salvia miltiorrhiza salvianolic acid B and shikalic acid can be chemically transformed into salvianolic acid A, thereby increasing its content and reduce its cost. CN201310487751.6 discloses a method for preparing salvianolic acid A amorphous freeze-dried powder with a purity of over 97% by means of column chromatography and freeze-drying. CN200810223651.1 discloses a method for preparing two kinds of salvianolic acid A crystals, and the purity of the final product can reach more than 90%. CN201710055331.9 discloses a preparation method of salvianolic acid A crystal, the purity of which is more than 99.5%, and the stability is significantly better than that of salvianolic acid A freeze-dried powder, but its water solubility is poor, and the dissolution process needs to be assisted by methods such as heating and ultrasound , there is a big disadvantage when it is developed as an injection. In addition, its preparation requires more than 97% salvianolic acid A as a starting material, which leads to high production cost, and it is difficult to use it as a requirement for large-scale commercial production of medicines.
丹酚酸A无定型粉末稳定性较差，即使在室温下也很容易降解。将丹酚酸A制备成结晶，可以显著提高其稳定性。但是，由于丹酚酸A口服生物利用度非常低 (口服生物利用度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 Ethnopharmacol148(2):617-623.)。考虑到心脑血管疾病发病时病人需要抢救且可能处于休克状态，静脉注射制剂是其最适宜的剂型。而由于静脉注射制剂是一种高风险剂型，需要将其制备成高纯度样品保证其安全和质量可控性，而且需要方便临床使用。Salvianolic acid A amorphous powder is less stable and easily degraded even at room temperature. The preparation of salvianolic acid A into crystals can significantly improve its stability. However, due to the very low oral bioavailability of salvianolic acid A (oral bioavailability 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.). Considering that the patient needs to be rescued and may be in a state of shock when cardiovascular and cerebrovascular diseases are onset, the intravenous preparation is the most suitable dosage form. However, since the intravenous preparation is a high-risk dosage form, it needs to be prepared into a high-purity sample to ensure its safety and quality controllability, and it needs to be convenient for clinical use.
因此，本领域的技术人员致力于开发一种产物纯度高、稳定性好、水溶解性好且易于工业化生产的丹酚酸A(或以丹酚酸A为主要成分的复合物)原料，以满足其作为注射制剂原料药的需求。Therefore, those skilled in the art are devoted to developing a raw material of salvianolic acid A (or a compound with salvianolic acid A as the main component) with high product purity, good stability, good water solubility and easy industrial production. To meet its needs as a raw material for injection preparations.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种丹酚酸A盐水合物、其制备方法及用途。The purpose of the present invention is to provide a salvianolic acid A salt hydrate, its preparation method and use.
本发明的第一方面，提供了一种式I所示的丹酚酸A盐水合物，A first aspect of the present invention provides a salvianolic acid A salt hydrate shown in formula I,
Figure PCTCN2022091078-appb-000001
Figure PCTCN2022091078-appb-000001
其中，M为阳离子；0＜m≤20，0＜n≤10，0＜x≤20，0＜y≤150。Wherein, M is a cation; 0<m≤20, 0<n≤10, 0<x≤20, 0<y≤150.
在另一优选例中，1≤m≤20，1≤n≤10，1≤x≤20，1≤y≤150。In another preferred example, 1≤m≤20, 1≤n≤10, 1≤x≤20, and 1≤y≤150.
在另一优选例中，当M为1价阳离子时，n＝x。In another preferred example, when M is a monovalent cation, n=x.
在另一优选例中，当M为2价阳离子时，n＝2x。In another preferred example, when M is a divalent cation, n=2x.
在另一优选例中，当M为3价阳离子时，n＝3x。In another preferred example, when M is a trivalent cation, n=3x.
在另一优选例中，所述M选自下组：钠离子、钾离子、镁离子、钙离子、或铵离子。In another preferred example, the M is selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion, or ammonium ion.
在另一优选例中，所述式I中，M为钠离子，并且5≤m≤15，1≤n≤10，1≤x≤10，20≤y≤100。优选地，M为钠离子，并且m为11，n为5，x为5，y为56。In another preferred example, in the formula I, M is a sodium ion, and 5≤m≤15, 1≤n≤10, 1≤x≤10, 20≤y≤100. Preferably, M is a sodium ion, and m is 11, n is 5, x is 5, and y is 56.
在另一优选例中，所述式I中，M为钾离子，并且1≤m≤5，1≤n≤5，1≤x≤5，1≤y≤10。优选地，M为钾离子，并且m为1，n为1，x为1，y为8。In another preferred example, in the formula I, M is a potassium ion, and 1≤m≤5, 1≤n≤5, 1≤x≤5, 1≤y≤10. Preferably, M is a potassium ion, and m is 1, n is 1, x is 1, and y is 8.
在另一优选例中，所述式I中，M为钙离子，并且1≤m≤5，1≤n≤5，0.5≤x≤5，5≤y≤15。优选地，M为钙离子，并且m为1，n为1，x为0.5，y为9。In another preferred example, in the formula I, M is a calcium ion, and 1≤m≤5, 1≤n≤5, 0.5≤x≤5, 5≤y≤15. Preferably, M is a calcium ion, and m is 1, n is 1, x is 0.5, and y is 9.
在另一优选例中，所述式I中，M为镁离子，并且1≤m≤5，1≤n≤5，0.5≤x≤5，5≤y≤15。优选地，M为镁离子，并且m为1，n为1，x为0.5，y为7。In another preferred example, in the formula I, M is a magnesium ion, and 1≤m≤5, 1≤n≤5, 0.5≤x≤5, 5≤y≤15. Preferably, M is a magnesium ion, and m is 1, n is 1, x is 0.5, and y is 7.
在另一优选例中，所述式I中，M为铵离子，并且1≤m≤5，1≤n≤5，1≤x≤5，5≤y≤15。优选地，M为铵离子，并且m为2，n为2，x为2，y为11。In another preferred example, in the formula I, M is an ammonium ion, and 1≤m≤5, 1≤n≤5, 1≤x≤5, 5≤y≤15. Preferably, M is an ammonium ion, and m is 2, n is 2, x is 2, and y is 11.
在另一优选例中，所述丹酚酸A盐水合物选自下组：In another preferred embodiment, the salvianolic acid A salt hydrate is selected from the following group:
丹酚酸A钠盐水合物：Salvianolic acid A sodium salt hydrate:
Figure PCTCN2022091078-appb-000002
Figure PCTCN2022091078-appb-000002
丹酚酸A钾盐水合物：Salvianolic Acid A Potassium Salt Hydrate:
Figure PCTCN2022091078-appb-000003
Figure PCTCN2022091078-appb-000003
丹酚酸A钙盐水合物：Salvianolic acid A calcium salt hydrate:
Figure PCTCN2022091078-appb-000004
Figure PCTCN2022091078-appb-000004
丹酚酸A镁盐水合物：Salvianolic Acid A Magnesium Salt Hydrate:
Figure PCTCN2022091078-appb-000005
Figure PCTCN2022091078-appb-000005
丹酚酸A铵盐水合物：Salvianolic acid A ammonium salt hydrate:
Figure PCTCN2022091078-appb-000006
Figure PCTCN2022091078-appb-000006
在另一优选例中，所述丹酚酸A钠盐水合物的差示扫描量热法图谱(DSC)在86-96℃有特征吸热峰。In another preferred embodiment, the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A sodium salt hydrate has a characteristic endothermic peak at 86-96°C.
在另一优选例中，所述丹酚酸A钠盐水合物的差示扫描量热法图谱还在116-126℃有特征吸热峰。In another preferred embodiment, the differential scanning calorimetry spectrum of the salvianolic acid A sodium salt hydrate also has a characteristic endothermic peak at 116-126°C.
在另一优选例中，所述丹酚酸A钠盐水合物的差示扫描量热法图谱基本如图6所示。In another preferred embodiment, the differential scanning calorimetry spectrum of the salvianolic acid A sodium salt hydrate is substantially as shown in FIG. 6 .
在另一优选例中，所述丹酚酸A钠盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：16.56±0.1、17.90±0.1、19.04±0.1、25.97±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: 16.56±0.1, 17.90±0.1, 19.04±0.1, 25.97±0.1.
在另一优选例中，所述丹酚酸A钠盐水合物的粉末X-射线衍射图谱还包括在以下2θ值处的特征峰：12.00±0.1、16.18±0.1、17.07±0.1、17.54±0.1、18.87±0.1、19.89±0.1、21.04±0.1、21.57±0.1、22.66±0.1、和22.93±0.1。In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A sodium salt hydrate further includes characteristic peaks at the following 2θ values: 12.00±0.1, 16.18±0.1, 17.07±0.1, 17.54±0.1 , 18.87±0.1, 19.89±0.1, 21.04±0.1, 21.57±0.1, 22.66±0.1, and 22.93±0.1.
在另一优选例中，所述丹酚酸A钠盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：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:
7.12±0.1、8.49±0.1、10.72±0.1、11.39±0.1、12.00±0.1、12.26±0.1、12.74±0.1、13.68±0.1、14.03±0.1、14.28±0.1、16.18±0.1、16.56±0.1、17.07±0.1、17.54±0.1、17.90±0.1、18.87±0.1、19.04±0.1、19.57±0.1、19.89±0.1、20.15±0.1、21.04±0.1、21.57±0.1、21.89±0.1、22.30±0.1、22.66±0.1、22.93±0.1、23.68±0.1、24.29±0.1、24.71±0.1、25.04±0.1、25.62±0.1、25.97±0.1、26.44±0.1、27.19±0.1、27.63±0.1、28.16±0.1、29.56±0.1、和36.29±0.1。7.12±0.1, 8.49±0.1, 10.72±0.1, 11.39±0.1, 12.00±0.1, 12.26±0.1, 12.74±0.1, 13.68±0.1, 14.03±0.1, 14.28±0.1, 16.18±0.1, 16.56±0.1, 17.07± 0.1, 17.54±0.1, 17.90±0.1, 18.87±0.1, 19.04±0.1, 19.57±0.1, 19.89±0.1, 20.15±0.1, 21.04±0.1, 21.57±0.1, 21.89±0.1, 22.30±0.1, 22.66±0.1, 22.93±0.1, 23.68±0.1, 24.29±0.1, 24.71±0.1, 25.04±0.1, 25.62±0.1, 25.97±0.1, 26.44±0.1, 27.19±0.1, 27.63±0.1, 28.16±0.1, 29.56±0.1, and 36.29 ±0.1.
在另一优选例中，所述丹酚酸A钠盐水合物物具有基本如图4所示的X-射线粉末衍射谱图(XRPD)。In another preferred embodiment, the salvianolic acid A sodium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 4 .
在另一优选例中，式I所示的丹酚酸A钠盐水合物通过本发明第二方面所述制备方法制备得到。In another preferred embodiment, the salvianolic acid A sodium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
在另一优选例中，所述丹酚酸A钾盐水合物的差示扫描量热法图谱(DSC)在95-105℃有特征吸热峰。In another preferred embodiment, the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A potassium salt hydrate has a characteristic endothermic peak at 95-105°C.
在另一优选例中，所述丹酚酸A钾盐水合物的差示扫描量热法图谱还在142-153℃有特征吸热峰。In another preferred example, the differential scanning calorimetry spectrum of the salvianolic acid A potassium salt hydrate also has a characteristic endothermic peak at 142-153°C.
在另一优选例中，所述丹酚酸A钾盐水合物的差示扫描量热法图谱基本如图11所示。In another preferred example, the differential scanning calorimetry spectrum of the salvianolic acid A potassium salt hydrate is basically as shown in FIG. 11 .
在另一优选例中，所述丹酚酸A钾盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：18.89±0.1、24.35±0.1、24.61±0.1、和26.13±0.1。In another preferred embodiment, the powder X-ray diffraction pattern of the salvianolic acid A potassium salt hydrate has characteristic peaks at the following 2θ values: 18.89±0.1, 24.35±0.1, 24.61±0.1, and 26.13±0.1.
在另一优选例中，所述丹酚酸A钾盐水合物的粉末X-射线衍射图谱还包括在以下2θ值处的特征峰：16.40±0.1、21.59±0.1、22.62±0.1、32.84±0.1、 和36.35±0.1。In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A potassium salt hydrate further includes characteristic peaks at the following 2θ values: 16.40±0.1, 21.59±0.1, 22.62±0.1, 32.84±0.1 , , and 36.35±0.1.
在另一优选例中，所述丹酚酸A钾盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A potassium salt hydrate has characteristic peaks at the following 2θ values:
7.11±0.1、12.06±0.1、12.90±0.1、13.48±0.1、13.87±0.1、14.37±0.1、16.40±0.1、16.61±0.1、17.86±0.1、18.23±0.1、18.89±0.1、19.24±0.1、21.59±0.1、22.12±0.1、22.62±0.1、23.78±0.1、24.35±0.1、24.61±0.1、25.26±0.1、25.55±0.1、26.13±0.1、28.58±0.1、29.62±0.1、30.02±0.1、30.45±0.1、31.35±0.1、32.45±0.1、32.84±0.1、33.17±0.1、33.98±0.1、35.22±0.1、36.35±0.1、43.06±0.1、和44.00±0.1。7.11±0.1, 12.06±0.1, 12.90±0.1, 13.48±0.1, 13.87±0.1, 14.37±0.1, 16.40±0.1, 16.61±0.1, 17.86±0.1, 18.23±0.1, 18.89±0.1, 19.24±0.1, 21.59± 0.1, 22.12±0.1, 22.62±0.1, 23.78±0.1, 24.35±0.1, 24.61±0.1, 25.26±0.1, 25.55±0.1, 26.13±0.1, 28.58±0.1, 29.62±0.1, 30.02±0.1, 30.45±0.1, 31.35±0.1, 32.45±0.1, 32.84±0.1, 33.17±0.1, 33.98±0.1, 35.22±0.1, 36.35±0.1, 43.06±0.1, and 44.00±0.1.
在另一优选例中，所述丹酚酸A钾盐水合物物具有基本如图9所示的X-射线粉末衍射谱图(XRPD)。In another preferred embodiment, the salvianolic acid A potassium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 9 .
在另一优选例中，式I所示的丹酚酸A钾盐水合物通过本发明第二方面所述制备方法制备得到。In another preferred example, the salvianolic acid A potassium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
在另一优选例中，所述丹酚酸A钙盐水合物的差示扫描量热法图谱(DSC)在90-100℃有特征吸热峰。In another preferred embodiment, the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A calcium salt hydrate has a characteristic endothermic peak at 90-100°C.
在另一优选例中，所述丹酚酸A钙盐水合物的差示扫描量热法图谱基本如图17所示。In another preferred example, the differential scanning calorimetry spectrum of the salvianolic acid A calcium salt hydrate is substantially as shown in FIG. 17 .
在另一优选例中，所述丹酚酸A钙盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：18.92±0.1、22.59±0.1、24.35±0.1、和27.49±0.1。In another preferred embodiment, the powder X-ray diffraction pattern of the salvianolic acid A calcium salt hydrate has characteristic peaks at the following 2θ values: 18.92±0.1, 22.59±0.1, 24.35±0.1, and 27.49±0.1.
在另一优选例中，所述丹酚酸A钙盐水合物的粉末X-射线衍射图谱还包括在以下2θ值处的特征峰：16.49±0.1、21.47±0.1、23.74±0.1、25.70±0.1、和26.10±0.1。In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A calcium salt hydrate further includes characteristic peaks at the following 2θ values: 16.49±0.1, 21.47±0.1, 23.74±0.1, 25.70±0.1 , and 26.10±0.1.
在另一优选例中，所述丹酚酸A钙盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A calcium salt hydrate has characteristic peaks at the following 2θ values:
6.79±0.1、7.14±0.1、8.58±0.1、10.77±0.1、11.78±0.1、12.30±0.1、12.85±0.1、13.66±0.1、14.13±0.1、14.29±0.1、16.14±0.1、16.49±0.1、17.22±0.1、17.84±0.1、18.02±0.1、18.92±0.1、19.85±0.1、20.19±0.1、21.10±0.1、21.47±0.1、21.89±0.1、22.59±0.1、22.87±0.1、23.74±0.1、24.35±0.1、24.71±0.1、25.02±0.1、25.70±0.1、26.10±0.1、26.42±0.1、27.08±0.1、27.49±0.1、28.32±0.1、29.48±0.1、30.41±0.1、30.97±0.1、31.26±0.1、32.03±0.1、32.52±0.1、33.07±0.1、33.69±0.1、34.02±0.1、34.55±0.1、36.11±0.1、38.28±0.1、40.00±0.1、40.45±0.1、41.17±0.1、41.83±0.1、43.77±0.1。6.79±0.1, 7.14±0.1, 8.58±0.1, 10.77±0.1, 11.78±0.1, 12.30±0.1, 12.85±0.1, 13.66±0.1, 14.13±0.1, 14.29±0.1, 16.14±0.1, 16.49±0.1, 17.22± 0.1, 17.84±0.1, 18.02±0.1, 18.92±0.1, 19.85±0.1, 20.19±0.1, 21.10±0.1, 21.47±0.1, 21.89±0.1, 22.59±0.1, 22.87±0.1, 23.74±0.1, 24.35±0.1, 24.71±0.1, 25.02±0.1, 25.70±0.1, 26.10±0.1, 26.42±0.1, 27.08±0.1, 27.49±0.1, 28.32±0.1, 29.48±0.1, 30.41±0.1, 30.97±0.1, 31.26±0.1, 32.03± 0.1, 32.52±0.1, 33.07±0.1, 33.69±0.1, 34.02±0.1, 34.55±0.1, 36.11±0.1, 38.28±0.1, 40.00±0.1, 40.45±0.1, 41.17±0.1, 41.83±0.1, 43.77±0.1.
在另一优选例中，所述丹酚酸A钙盐水合物物具有基本如图16所示的X-射线粉末衍射谱图(XRPD)。In another preferred example, the salvianolic acid A calcium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 16 .
在另一优选例中，式I所示的丹酚酸A钙盐水合物通过本发明第二方面所述制备方法制备得到。In another preferred embodiment, the salvianolic acid A calcium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
在另一优选例中，所述丹酚酸A镁盐水合物的差示扫描量热法图谱(DSC)在100-115℃有特征吸热峰。In another preferred embodiment, the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A magnesium salt hydrate has a characteristic endothermic peak at 100-115°C.
在另一优选例中，所述丹酚酸A镁盐水合物的差示扫描量热法图谱基本如 图23所示。In another preferred example, the differential scanning calorimetry spectrum of the salvianolic acid A magnesium salt hydrate is basically as shown in Figure 23.
在另一优选例中，所述丹酚酸A镁盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：21.65±0.1、24.18±0.1、24.49±0.1、25.32±0.1、25.91±0.1、和27.89±0.1。In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A magnesium salt hydrate has characteristic peaks at the following 2θ values: 21.65±0.1, 24.18±0.1, 24.49±0.1, 25.32±0.1, 25.91 ±0.1, and 27.89 ±0.1.
在另一优选例中，所述丹酚酸A镁盐水合物的粉末X-射线衍射图谱还包括在以下2θ值处的特征峰：16.28±0.1、17.88±0.1、19.18±0.1、22.50±0.1、和22.93±0.1。In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A magnesium salt hydrate further includes characteristic peaks at the following 2θ values: 16.28±0.1, 17.88±0.1, 19.18±0.1, 22.50±0.1 , and 22.93±0.1.
在另一优选例中，所述丹酚酸A镁盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A magnesium salt hydrate has characteristic peaks at the following 2θ values:
6.80±0.1、7.24±0.1、8.74±0.1、10.54±0.1、11.02±0.1、11.43±0.1、11.98±0.1、12.49±0.1、12.85±0.1、13.36±0.1、13.93±0.1、14.40±0.1、16.28±0.1、16.77±0.1、17.88±0.1、18.08±0.1、18.65±0.1、18.84±0.1、19.18±0.1、19.66±0.1、20.42±0.1、21.29±0.1、21.65±0.1、21.88±0.1、22.28±0.1、22.50±0.1、22.93±0.1、23.80±0.1、24.18±0.1、24.49±0.1、24.98±0.1、25.32±0.1、25.91±0.1、26.60±0.1、27.43±0.1、27.89±0.1、28.40±0.1、28.99±0.1、29.46±0.1、30.19±0.1、30.86±0.1、31.38±0.1、32.72±0.1、33.27±0.1、33.82±0.1、34.77±0.1、35.09±0.1、36.41±0.1、38.01±0.1、40.00±0.1、和44.03±0.1。6.80±0.1, 7.24±0.1, 8.74±0.1, 10.54±0.1, 11.02±0.1, 11.43±0.1, 11.98±0.1, 12.49±0.1, 12.85±0.1, 13.36±0.1, 13.93±0.1, 14.40±0.1, 16.28± 0.1, 16.77±0.1, 17.88±0.1, 18.08±0.1, 18.65±0.1, 18.84±0.1, 19.18±0.1, 19.66±0.1, 20.42±0.1, 21.29±0.1, 21.65±0.1, 21.88±0.1, 22.28±0.1, 22.50±0.1, 22.93±0.1, 23.80±0.1, 24.18±0.1, 24.49±0.1, 24.98±0.1, 25.32±0.1, 25.91±0.1, 26.60±0.1, 27.43±0.1, 27.89±0.1, 28.40±0.1, 28.99± 0.1, 29.46±0.1, 30.19±0.1, 30.86±0.1, 31.38±0.1, 32.72±0.1, 33.27±0.1, 33.82±0.1, 34.77±0.1, 35.09±0.1, 36.41±0.1, 38.01±0.1, 40.00±0.1, and 44.03 ± 0.1.
在另一优选例中，所述丹酚酸A镁盐水合物物具有基本如图21所示的X-射线粉末衍射谱图(XRPD)。In another preferred embodiment, the salvianolic acid A magnesium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 21 .
在另一优选例中，式I所示的丹酚酸A镁盐水合物通过本发明第二方面所述制备方法制备得到。In another preferred embodiment, the salvianolic acid A magnesium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
在另一优选例中，所述丹酚酸A铵盐水合物的差示扫描量热法图谱(DSC)在91-101℃有特征吸热峰。In another preferred example, the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A ammonium salt hydrate has a characteristic endothermic peak at 91-101°C.
在另一优选例中，所述丹酚酸A铵盐水合物的差示扫描量热法图谱(DSC)在132-142℃有特征吸热峰。In another preferred example, the differential scanning calorimetry (DSC) spectrum of the salvianolic acid A ammonium salt hydrate has a characteristic endothermic peak at 132-142°C.
在另一优选例中，所述丹酚酸A铵盐水合物的差示扫描量热法图谱基本如图30所示。In another preferred example, the differential scanning calorimetry spectrum of the salvianolic acid A ammonium salt hydrate is basically as shown in FIG. 30 .
在另一优选例中，所述丹酚酸A铵盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：14.21±0.1、18.83±0.1、21.33±0.1、24.25±0.1、24.53±0.1、和25.97±0.1。In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A ammonium salt hydrate has characteristic peaks at the following 2θ values: 14.21±0.1, 18.83±0.1, 21.33±0.1, 24.25±0.1, 24.53 ±0.1, and 25.97 ±0.1.
在另一优选例中，所述丹酚酸A铵盐水合物的粉末X-射线衍射图谱还包括在以下2θ值处的特征峰：13.34±0.1、13.64±0.1、16.30±0.1、17.90±0.1、18.06±0.1、19.08±0.1、和21.83±0.1。In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A ammonium salt hydrate further includes characteristic peaks at the following 2θ values: 13.34±0.1, 13.64±0.1, 16.30±0.1, 17.90±0.1 , 18.06±0.1, 19.08±0.1, and 21.83±0.1.
在另一优选例中，所述丹酚酸A铵盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：In another preferred example, the powder X-ray diffraction pattern of the salvianolic acid A ammonium salt hydrate has characteristic peaks at the following 2θ values:
6.67±0.1、6.95±0.1、7.10±0.1、10.62±0.1、11.66±0.1、11.96±0.1、12.18±0.1、12.79±0.1、13.34±0.1、13.64±0.1、14.21±0.1、16.30±0.1、17.15±0.1、17.90±0.1、18.06±0.1、18.83±0.1、19.08±0.1、19.61±0.1、20.15±0.1、21.33±0.1、21.83±0.1、22.44±0.1、22.95±0.1、23.43±0.1、23.64±0.1、24.25±0.1、24.53±0.1、25.06±0.1、25.50±0.1、25.97 ±0.1、26.37±0.1、26.86±0.1、27.41±0.1、28.14±0.1、29.34±0.1、30.71±0.1、31.20±0.1、32.66±0.1、33.63±0.1、36.11±0.1、38.17±0.1、和43.61±0.1。6.67±0.1, 6.95±0.1, 7.10±0.1, 10.62±0.1, 11.66±0.1, 11.96±0.1, 12.18±0.1, 12.79±0.1, 13.34±0.1, 13.64±0.1, 14.21±0.1, 16.30±0.1, 17.15± 0.1, 17.90±0.1, 18.06±0.1, 18.83±0.1, 19.08±0.1, 19.61±0.1, 20.15±0.1, 21.33±0.1, 21.83±0.1, 22.44±0.1, 22.95±0.1, 23.43±0.1, 23.64±0.1, 24.25±0.1, 24.53±0.1, 25.06±0.1, 25.50±0.1, 25.97±0.1, 26.37±0.1, 26.86±0.1, 27.41±0.1, 28.14±0.1, 29.34±0.1, 30.71±0.1, 31.20±0.1, 32.66± 0.1, 33.63±0.1, 36.11±0.1, 38.17±0.1, and 43.61±0.1.
在另一优选例中，所述丹酚酸A铵盐水合物物具有基本如图28所示的X-射线粉末衍射谱图(XRPD)。In another preferred embodiment, the salvianolic acid A ammonium salt hydrate has an X-ray powder diffraction pattern (XRPD) substantially as shown in FIG. 28 .
在另一优选例中，式I所示的丹酚酸A铵盐水合物通过本发明第二方面所述制备方法制备得到。In another preferred embodiment, the salvianolic acid A ammonium salt hydrate represented by formula I is prepared by the preparation method described in the second aspect of the present invention.
本发明的第二方面，提供了一种丹酚酸A盐水合物晶体，所述丹酚酸A盐水合物晶体选自下组：A second aspect of the present invention provides a salvianolic acid A salt hydrate crystal, and the salvianolic acid A salt hydrate crystal is selected from the following group:
丹酚酸A钠盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
Figure PCTCN2022091078-appb-000007
α＝γ＝90.0°,β＝91.6°； The salvianolic acid A sodium salt hydrate crystal is determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000007
α=γ=90.0°, β=91.6°;
丹酚酸A钾盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
Figure PCTCN2022091078-appb-000008
α＝γ＝90.0°,β＝115.16°； Salvianolic acid A potassium salt hydrate crystal, which was determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000008
α=γ=90.0°, β=115.16°;
丹酚酸A镁盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
Figure PCTCN2022091078-appb-000009
α＝γ＝90.0°,β＝116.1°； The salvianolic acid A magnesium salt hydrate crystal is determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000009
α=γ=90.0°, β=116.1°;
丹酚酸A钙盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
Figure PCTCN2022091078-appb-000010
α＝γ＝90.0°,β＝115.7°；和 The salvianolic acid A calcium salt hydrate crystal is determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000010
α=γ=90.0°, β=115.7°; and
丹酚酸A铵盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
Figure PCTCN2022091078-appb-000011
α＝γ＝90.0°，β＝96.9°。 Salvianolic acid A ammonium salt hydrate crystal, which was determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000011
α=γ=90.0°, β=96.9°.
在另一优选例中，所述丹酚酸A钠盐水合物晶体的绝对构型为：In another preferred embodiment, the absolute configuration of the salvianolic acid A sodium salt hydrate crystal is:
Figure PCTCN2022091078-appb-000012
Figure PCTCN2022091078-appb-000012
在另一优选例中，所述丹酚酸A钾盐水合物晶体的绝对构型为：In another preferred example, the absolute configuration of the salvianolic acid A potassium salt hydrate crystal is:
Figure PCTCN2022091078-appb-000013
Figure PCTCN2022091078-appb-000013
在另一优选例中，所述丹酚酸A钙盐水合物晶体的绝对构型为：In another preferred embodiment, the absolute configuration of the salvianolic acid A calcium salt hydrate crystal is:
Figure PCTCN2022091078-appb-000014
Figure PCTCN2022091078-appb-000014
在另一优选例中，所述丹酚酸A镁盐水合物晶体的相对构型为：In another preferred embodiment, the relative configuration of the salvianolic acid A magnesium salt hydrate crystal is:
Figure PCTCN2022091078-appb-000015
Figure PCTCN2022091078-appb-000015
在另一优选例中，所述丹酚酸A铵盐水合物晶体的绝对构型为：In another preferred embodiment, the absolute configuration of the salvianolic acid A ammonium salt hydrate crystal is:
Figure PCTCN2022091078-appb-000016
Figure PCTCN2022091078-appb-000016
在另一优选例中，所述丹酚酸A钠盐水合物晶体对其采用X-射线单晶衍射测定为C2空间群。In another preferred embodiment, the salvianolic acid A sodium salt hydrate crystal is determined to be the C2 space group by X-ray single crystal diffraction.
在另一优选例中，所述丹酚酸A钾盐水合物晶体对其采用X-射线单晶衍射测定为C2空间群。In another preferred embodiment, the salvianolic acid A potassium salt hydrate crystal is determined to be the C2 space group by X-ray single crystal diffraction.
在另一优选例中，所述丹酚酸A钙盐水合物晶体对其采用X-射线单晶衍射测定为C2空间群。In another preferred embodiment, the salvianolic acid A calcium salt hydrate crystal is determined to be the C2 space group by X-ray single crystal diffraction.
在另一优选例中，所述丹酚酸A镁盐水合物晶体对其采用X-射线单晶衍射测定为C2空间群。In another preferred embodiment, the salvianolic acid A magnesium salt hydrate crystal is determined to be C2 space group by X-ray single crystal diffraction.
在另一优选例中，所述丹酚酸A铵盐水合物晶体对其采用X-射线单晶衍射测定为P2空间群。In another preferred embodiment, the salvianolic acid A ammonium salt hydrate crystal is determined to be P2 space group by X-ray single crystal diffraction.
本发明的第三方面，提供了一种式I所示的丹酚酸A盐水合物的制备方法，所述方法包括步骤：The third aspect of the present invention provides a kind of preparation method of salvianolic acid A salt hydrate shown in formula I, the method comprises the steps:
(1)将丹酚酸A原料溶于具有第一温度的含水溶剂中，获得丹酚酸A溶液；(1) salvianolic acid A raw material is dissolved in the water-containing solvent with first temperature, obtains salvianolic acid A solution;
(2)向步骤(1)获得的丹酚酸A溶液中加入碱性化合物，使部分丹酚酸A和碱性化合物进行成盐反应；(2) adding basic compound to the salvianolic acid A solution obtained in step (1), so that part of salvianolic acid A and the basic compound carry out a salt-forming reaction;
(3)对步骤(2)成盐反应后的溶液降温至第二温度进行析晶，从而获得所述丹酚酸A盐水合物；(3) cooling the solution after the salt-forming reaction in step (2) to a second temperature for crystallization, thereby obtaining the salvianolic acid A salt hydrate;
其中，所述第一温度为40-80℃；所述第二温度为0-20℃。Wherein, the first temperature is 40-80°C; the second temperature is 0-20°C.
在另一优选例中，所述第一温度为45-70℃；优选地为约50℃。In another preferred embodiment, the first temperature is 45-70°C; preferably about 50°C.
在另一优选例中，所述步骤(1)中所得丹酚酸A溶液中，丹酚酸A的质量浓度为约5％-20％；优选地为约5％-15％。In another preferred example, in the salvianolic acid A solution obtained in the step (1), the mass concentration of salvianolic acid A is about 5%-20%; preferably, it is about 5%-15%.
在另一优选例中，所述步骤(2)中使约5％-80％(w/w)的丹酚酸A和碱性化合物进行成盐反应；优选地，使约10％-60％(w/w)的丹酚酸A和碱性化合物进行成盐反应；更优选地，使约15％-50％(w/w)的丹酚酸A和碱性化合物进行成盐反应。In another preferred example, in the step (2), about 5%-80% (w/w) of salvianolic acid A and a basic compound are subjected to a salt-forming reaction; preferably, about 10%-60% (w/w) salvianolic acid A and the basic compound are subjected to a salt-forming reaction; more preferably, about 15%-50% (w/w) of the salvianolic acid A and the basic compound are subjected to a salt-forming reaction.
在另一优选例中，所述步骤(2)中降温速率为0.1℃/min-1℃/min；优选地降温速率为0.1℃/min-0.5℃/min；更优选地降温速率为0.1℃/min-0.3℃/min。In another preferred embodiment, the cooling rate in the step (2) 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.
在另一优选例中，所述第二温度为0-20℃；优选地，所述第二温度为1-15℃；更优选地，所述第二温度为4-10℃。In another preferred embodiment, the second temperature is 0-20°C; preferably, the second temperature is 1-15°C; more preferably, the second temperature is 4-10°C.
在另一优选例中，所述步骤(2)中降温析晶过程中，不停搅拌。In another preferred embodiment, during the cooling and crystallization process in the step (2), stirring is continued.
在另一优选例中，所述步骤(2)中降温至第二温度后，析晶5-48h；优选地，析晶8-36h；更优选地，析晶12-24h。In another preferred example, after cooling to the second temperature in the step (2), crystallization is performed for 5-48 hours; preferably, crystallization is performed for 8-36 hours; more preferably, crystallization is performed for 12-24 hours.
在另一优选例中，所述方法还包括步骤，对获得的所述丹酚酸A盐水合物进行真空干燥处理。优选地，真空干燥处理温度为10-30℃。In another preferred embodiment, the method further includes the step of vacuum drying the obtained salvianolic acid A salt hydrate. Preferably, the vacuum drying treatment temperature is 10-30°C.
在另一优选例中，所述方法还包括步骤，对获得的所述丹酚酸A盐水合物进行重结晶处理；优选地，所述重结晶步骤包括：In another preferred example, the method further includes a step of recrystallization of the obtained salvianolic acid A salt hydrate; preferably, the recrystallization step includes:
(a)将丹酚酸A盐水合物溶于具有第一温度的含水溶剂中，获得丹酚酸A盐溶液；(a) dissolving salvianolic acid A salt hydrate in an aqueous solvent with a first temperature to obtain a salvianolic acid A salt solution;
(b)对步骤(a)的溶液降温至第二温度进行析晶，从而获得所述丹酚酸A盐水合物精制品；(b) cooling the solution of step (a) to the second temperature to carry out crystallization, thereby obtaining the salvianolic acid A salt hydrate refined product;
其中，所述第一温度为40-80℃；所述第二温度为0-20℃。Wherein, the first temperature is 40-80°C; the second temperature is 0-20°C.
在另一优选例中，所述第一温度为45-70℃；优选地为约50℃。In another preferred embodiment, the first temperature is 45-70°C; preferably about 50°C.
在另一优选例中，所述步骤(a)中所得溶液中，丹酚酸A的质量浓度为约5％-20％；优选地为约5％-15％。In another preferred example, in the solution obtained in the step (a), the mass concentration of salvianolic acid A is about 5%-20%; preferably, it is about 5%-15%.
在另一优选例中，所述步骤(b)中降温速率为0.1℃/min-1℃/min；优选地降温速率为0.1℃/min-0.5℃/min；更优选地降温速率为0.1℃/min-0.3℃/min。In another preferred embodiment, 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°C/min.
在另一优选例中，所述第二温度为0-20℃；优选地，所述第二温度为1-15℃；更优选地，所述第二温度为4-10℃。In another preferred embodiment, the second temperature is 0-20°C; preferably, the second temperature is 1-15°C; more preferably, the second temperature is 4-10°C.
在另一优选例中，所述步骤(b)中降温析晶过程中，不停搅拌。In another preferred embodiment, during the cooling and crystallization process in the step (b), stirring is continued.
在另一优选例中，所述步骤(b)中降温至第二温度后，析晶5-48h；优选地，析晶8-36h；更优选地，析晶12-24h。In another preferred example, after cooling to the second temperature in the step (b), crystallization is performed for 5-48 hours; preferably, crystallization is performed for 8-36 hours; more preferably, crystallization is performed for 12-24 hours.
在另一优选例中，所述方法还包括步骤，对获得的所述丹酚酸A盐水合物精制品进行真空干燥处理。优选地，真空干燥处理温度为10-30℃。In another preferred embodiment, the method further includes the step of vacuum drying the obtained salvianolic acid A salt hydrate refined product. Preferably, the vacuum drying treatment temperature is 10-30°C.
在另一优选例中，所述碱性化合物选自：含钠碱性化合物、含钾碱性化合物、含钙碱性化合物、含镁碱性化合物、和含铵碱性化合。In another preferred embodiment, the basic compound is selected from: a sodium-containing basic compound, a potassium-containing basic compound, a calcium-containing basic compound, a magnesium-containing basic compound, and an ammonium-containing basic compound.
在另一优选例中，所述含钠碱性化合物选自：氢氧化钠、碳酸钠、碳酸氢钠、苹果酸钠、磷酸钠、磷酸二氢钠、磷酸氢二钠、和乙酸钠。In another preferred example, the sodium-containing basic compound is selected from: sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium malate, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium acetate.
在另一优选例中，所述含钾碱性化合物选自：氢氧化钾、碳酸钾、碳酸氢 钾、苹果酸钾、柠檬酸钾、磷酸钾、磷酸氢二钾、磷酸二氢钾、和乙酸钾。In another preferred embodiment, the potassium-containing basic compound is selected from the group consisting of potassium hydroxide, potassium carbonate, potassium bicarbonate, potassium malate, potassium citrate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and Potassium acetate.
在另一优选例中，所述含钙碱性化合物选自：碳酸钙、碳酸氢钙、氧化钙、氢氧化钙、葡萄糖酸钙、和乙酸钙。In another preferred embodiment, the calcium-containing basic compound is selected from calcium carbonate, calcium bicarbonate, calcium oxide, calcium hydroxide, calcium gluconate, and calcium acetate.
在另一优选例中，所述含镁碱性化合物选自：氢氧化镁、氧化镁、碳酸镁、碳酸氢镁、碱式碳酸镁、苹果酸镁、柠檬酸镁、和乙酸镁。In another preferred embodiment, the magnesium-containing basic compound is selected from the group consisting of: magnesium hydroxide, magnesium oxide, magnesium carbonate, magnesium bicarbonate, basic magnesium carbonate, magnesium malate, magnesium citrate, and magnesium acetate.
在另一优选例中，所述含铵碱性化合物选自：氨气、氨水、碳酸铵、和碳酸氢铵。In another preferred embodiment, the ammonium-containing basic compound is selected from the group consisting of ammonia gas, ammonia water, ammonium carbonate, and ammonium bicarbonate.
在另一优选例中，所述方法中，丹酚酸A原料的HPLC纯度≥70％；优选地，丹酚酸A原料的HPLC纯度≥80％；或者，丹酚酸A原料的HPLC纯度在约75％-95％之间。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 Between about 75%-95%.
在另一优选例中，所述含水溶剂中水的质量分数≥70％；优选地≥80％；更优选地≥90％。In another preferred embodiment, the mass fraction of water in the aqueous solvent is ≥70%; preferably ≥80%; more preferably ≥90%.
在另一优选例中，所述含水溶剂选自：水、乙醇水溶液、丙酮水溶液等。In another preferred embodiment, the aqueous solvent is selected from the group consisting of water, aqueous ethanol, aqueous acetone and the like.
在另一优选例中，所述步骤(2)中，所述碱性化合物为弱酸盐，优选为强碱弱酸盐。In another preferred example, in the step (2), the basic compound is a weak acid salt, preferably a strong base weak acid salt.
在另一优选例中，所述步骤(2)中，所述盐的阴离子选自：碳酸根离子、碳酸氢根离子、硅酸根离子、偏铝酸根离子、醋酸根离子等；和/或，所述盐的阳离子选自：钠离子、钾离子、镁离子、锂离子、铵离子、钙离子。In another preferred example, in the step (2), the anion of the salt is selected from: carbonate ion, bicarbonate ion, silicate ion, metaaluminate ion, acetate ion, etc.; and/or, The cation of the salt is selected from: sodium ion, potassium ion, magnesium ion, lithium ion, ammonium ion, calcium ion.
在另一优选例中，所述步骤(2)中，丹酚酸A和碱性化合物的摩尔比为1－3：3-1；优选为：1－3：1-2；最优选地为2：1。In another preferred example, in the step (2), the molar ratio of salvianolic acid A and the basic compound is 1-3:3-1; preferably: 1-3:1-2; most preferably 2:1.
本发明的第四方面，提供了一种本发明第一方面所述丹酚酸A盐水合物或本发明第二方面所述的丹酚酸A盐水合物晶体的用途，用于制备治疗或预防疾病的药物组合物，所述疾病选自：心脑血管疾病、免疫系统疾病、高脂血症、糖尿病并发症等。The fourth aspect of the present invention provides a use of the salvianolic acid A salt hydrate described in the first aspect of the present invention or the salvianolic acid A salt hydrate crystal described in the second aspect of the present invention, for the preparation of therapeutic or A pharmaceutical composition for preventing diseases selected from cardiovascular and cerebrovascular diseases, immune system diseases, hyperlipidemia, diabetic complications and the like.
本发明的第五方面，提供了一种药物组合物，包含：The fifth aspect of the present invention provides a pharmaceutical composition, comprising:
本发明第一方面所述的丹酚酸A盐水合物或本发明第二方面所述的丹酚酸A盐水合物晶体；以及药学上可接受的载体。The salvianolic acid A salt hydrate described in the first aspect of the present invention or the salvianolic acid A salt hydrate crystal described in the second aspect of the present invention; and a pharmaceutically acceptable carrier.
在另一优选例中，所述药物组合物由本发明第一方面所述的丹酚酸A盐水合物和药学上可接受的载体构成。In another preferred embodiment, the pharmaceutical composition is composed of the salvianolic acid A salt hydrate described in the first aspect of the present invention and a pharmaceutically acceptable carrier.
在另一优选例中，所述药物组合物为注射剂。In another preferred embodiment, the pharmaceutical composition is an injection.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。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 (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.
附图说明Description of drawings
图1是丹酚酸A钠盐水合物的X射线单晶衍射晶体数据Figure 1 is the X-ray single crystal diffraction crystal data of salvianolic acid A sodium salt hydrate
图2A(不对称单位的立体结构椭球图)和图2B(单个主体分子的立体结构椭球图)是丹酚酸A钠盐水合物的X射线单晶衍射得到的立体结构椭球图。2A (stereostructure ellipsoid diagram of asymmetric unit) and FIG. 2B (stereostructure ellipsoid diagram of a single host molecule) are stereostructure ellipsoid diagrams obtained by X-ray single crystal diffraction of salvianolic acid A sodium salt hydrate.
图3是丹酚酸A钠盐水合物的延b轴方向的晶胞堆积投影图。3 is a unit cell stacking projection view along the b-axis of salvianolic acid A sodium salt hydrate.
图4是丹酚酸A钠盐水合物的粉末衍射图谱。Figure 4 is the powder diffraction pattern of salvianolic acid A sodium salt hydrate.
图5是丹酚酸A钠盐水合物的粉末衍射2θ值列表。Figure 5 is a list of powder diffraction 2Θ values of salvianolic acid A sodium salt hydrate.
图6是丹酚酸A钠盐水合物的DSC图谱。Figure 6 is the DSC spectrum of salvianolic acid A sodium salt hydrate.
图7是丹酚酸A钾盐水合物的X射线单晶衍射晶体数据Figure 7 is the X-ray single crystal diffraction crystal data of salvianolic acid A potassium salt hydrate
图8A是本发明得到的丹酚酸A钾盐水合物的X射线单晶衍射得到的立体结构椭球图8A is a three-dimensional structure ellipsoid diagram obtained by X-ray single crystal diffraction of salvianolic acid A potassium salt hydrate obtained by the present invention
图8B是本发明得到的丹酚酸A钾盐水合物的延b轴方向的晶胞堆积投影图8B is a unit cell stacking projection view along the b-axis direction of the salvianolic acid A potassium salt hydrate obtained by the present invention
图9是本发明得到的丹酚酸A钾盐水合物的粉末衍射图谱Fig. 9 is the powder diffraction pattern of the salvianolic acid A potassium salt hydrate obtained by the present invention
图10是本发明得到的丹酚酸A钾盐水合物的粉末衍射2θ值列表Fig. 10 is the powder diffraction 2θ value list of the salvianolic acid A potassium salt hydrate obtained by the present invention
图11是本发明得到的丹酚酸A钾盐水合物的DSC图谱Fig. 11 is the DSC spectrum of the salvianolic acid A potassium salt hydrate obtained by the present invention
图12是丹酚酸A钙盐水合物的X射线单晶衍射晶体数据Figure 12 is the X-ray single crystal diffraction crystal data of salvianolic acid A calcium salt hydrate
图13A(不对称单元的立体结构椭球图)和图13B(显示钙配位情况的立体结构椭球图)是本发明得到的丹酚酸A钙盐水合物的X射线单晶衍射得到的立体结构椭球图13A (stereostructure ellipsoid diagram of asymmetric unit) and FIG. 13B (stereostructure ellipsoid diagram showing calcium coordination) are obtained by X-ray single crystal diffraction of salvianolic acid A calcium salt hydrate obtained by the present invention Three-dimensional structure ellipsoid diagram
图14是本发明得到的丹酚酸A钙盐水合物的延b轴方向的晶胞堆积投影图14 is a unit cell stacking projection view along the b-axis direction of the salvianolic acid A calcium salt hydrate obtained in the present invention
图15是本发明得到的丹酚酸A钙盐水合物的粉末衍射图谱Fig. 15 is the powder diffraction pattern of the salvianolic acid A calcium salt hydrate obtained by the present invention
图16是本发明得到的丹酚酸A钙盐水合物的粉末衍射2θ值列表16 is a list of powder diffraction 2θ values of salvianolic acid A calcium salt hydrate obtained in the present invention
图17是本发明得到的丹酚酸A钙盐水合物的DSC图谱Fig. 17 is the DSC spectrum of the salvianolic acid A calcium salt hydrate obtained by the present invention
图18是本发明得到的丹酚酸A镁盐水合物的X射线单晶衍射晶体数据Fig. 18 is the X-ray single crystal diffraction crystal data of the salvianolic acid A magnesium salt hydrate obtained by the present invention
图19是本发明得到的丹酚酸A镁盐水合物的X射线单晶衍射得到的立体结构椭球图Fig. 19 is the three-dimensional structure ellipsoid diagram obtained by the X-ray single crystal diffraction of the salvianolic acid A magnesium salt hydrate obtained by the present invention
图20是本发明得到的丹酚酸A镁盐水合物的延b轴方向的晶胞堆积投影图20 is a unit cell stacking projection view along the b-axis direction of the salvianolic acid A magnesium salt hydrate obtained in the present invention
图21是本发明得到的丹酚酸A镁盐水合物的粉末衍射图谱Fig. 21 is the powder diffraction pattern of the salvianolic acid A magnesium salt hydrate obtained by the present invention
图22是本发明得到的丹酚酸A镁盐水合物的粉末衍射2θ值列表Figure 22 is a list of powder diffraction 2θ values of the salvianolic acid A magnesium salt hydrate obtained in the present invention
图23是本发明得到的丹酚酸A镁盐水合物的DSC图谱Fig. 23 is the DSC spectrum of the salvianolic acid A magnesium salt hydrate obtained by the present invention
图24是本发明得到的丹酚酸A铵盐水合物的X射线单晶衍射晶体数据Figure 24 is the X-ray single crystal diffraction crystal data of the salvianolic acid A ammonium salt hydrate obtained in the present invention
图25(不对称单位的立体结构球棍图)和图26(单个主体分子的立体结构椭球图)是本发明得到的丹酚酸A铵盐水合物的X射线单晶衍射得到的立体结构椭球图Figure 25 (ball-and-stick diagram of the three-dimensional structure of the asymmetric unit) and Figure 26 (the three-dimensional structure ellipsoid diagram of a single host molecule) are the three-dimensional structures obtained by X-ray single crystal diffraction of the salvianolic acid A ammonium salt hydrate obtained by the present invention Ellipsoid
图27是本发明得到的丹酚酸A铵盐水合物的延b轴方向的晶胞堆积投影图27 is a unit cell stacking projection view along the b-axis direction of the salvianolic acid A ammonium salt hydrate obtained in the present invention
图28是本发明得到的丹酚酸A铵盐水合物的粉末衍射图谱Fig. 28 is the powder diffraction pattern of the salvianolic acid A ammonium salt hydrate obtained by the present invention
图29是本发明得到的丹酚酸A铵盐水合物的粉末衍射2θ值列表Figure 29 is a list of powder diffraction 2θ values of the salvianolic acid A ammonium salt hydrate obtained in the present invention
图30是本发明得到的丹酚酸A铵盐水合物的DSC图谱Fig. 30 is the DSC spectrum of the salvianolic acid A ammonium salt hydrate obtained by the present invention
图31是发明实施过程中丹酚酸A粗品(A)、柱色谱后丹酚酸A样品(B)、一次结晶后丹酚酸A钠盐水合物(C)和重结晶后丹酚酸A钠盐水合物(D)的HPLC图谱(纯度以丹酚酸A计算)Figure 31 shows the crude salvianolic acid A product (A), the salvianolic acid A sample (B) after column chromatography, the salvianolic acid A sodium salt hydrate (C) after primary crystallization and the salvianolic acid A after recrystallization during the implementation of the invention HPLC chromatogram of sodium salt hydrate (D) (purity calculated as salvianolic acid A)
图32A是本发明方法制得的丹酚酸A钠盐水合物单晶的显微照片(10×目镜+4×物镜)；图32B是根据专利文献CN201910643821.X方法制得的丹酚 酸A钠盐复合物结晶性粉末的显微照片(10×目镜+4×物镜)。Fig. 32A is a photomicrograph (10× eyepiece+4× objective lens) of the single crystal of salvianolic acid A sodium salt hydrate obtained by the method of the present invention; Fig. 32B is the salvianolic acid A obtained by the method of patent document CN201910643821.X Micrograph of sodium salt complex crystalline powder (10x eyepiece + 4x objective).
具体实施方式Detailed ways
经过广泛而深入的研究，本发明人意外制得一种丹酚酸A、丹酚酸A负离子、金属离子(分别为钠离子、钾离子、钙离子、镁离子、铵离子)、水分子组成的复合物。经过X射线单晶衍射、X射线粉末衍射、差式扫描量热法(DSC)、热重分析法(TGA)等确定了该类复合物晶体的晶型、结晶水等特性，证明了本发明制备得到的丹酚酸A盐水合物，是一种新的物质，具有新的晶型，其纯度超过99％。本发明还公开了该类新晶型的制备方法，以纯度约80％的丹酚酸A为起始原料制备本发明的丹酚酸A盐水合物，该丹酚酸A盐水合物能够应用于缺血性脑血管疾病和缺血性心血管疾病的治疗。而且该丹酚酸A盐水合物具有生产成本低、产物纯度高、稳定性好、水溶性好的特点。After extensive and in-depth research, the inventors unexpectedly obtained a kind of salvianolic acid A, salvianolic acid A negative ion, metal ion (respectively sodium ion, potassium ion, calcium ion, magnesium ion, ammonium ion), water molecule composition the complex. Through X-ray single crystal diffraction, X-ray powder diffraction, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), etc., the crystal form, crystal water and other characteristics of the composite crystal have been determined, which proves the present invention The prepared salvianolic acid A salt hydrate is a new substance with a new crystal form and its purity exceeds 99%. The present invention also discloses a preparation method of the new crystal form. The salvianolic acid A salt hydrate of the present invention is prepared by using salvianolic acid A with a purity of about 80% as the starting material, and the salvianolic acid A salt hydrate can be applied for the treatment of ischemic cerebrovascular disease and ischemic cardiovascular disease. Moreover, the salvianolic acid A salt hydrate has the characteristics of low production cost, high product purity, good stability and good water solubility.
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。Before the present invention is described, it is to be understood that this invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. 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 invention will be limited only by the appended claims.
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。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 this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" 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 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。Although any methods and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, the preferred methods and materials are exemplified herein.
具体地，本发明制备了一种由丹酚酸A、丹酚酸A负离子、金属离子(分别为钠离子、钾离子、钙离子、镁离子、铵根离子)、水分子组成的复合物(纯净物)，得到了其单晶体，并以市场上的常见阳性药物为对照，研究了该复合物对于缺血性脑血管疾病和缺血性心血管疾病的治疗作用。Specifically, the present invention prepares a compound (which is composed of salvianolic acid A, salvianolic acid A negative ions, metal ions (respectively sodium ions, potassium ions, calcium ions, magnesium ions, and ammonium ions) and water molecules. pure substance), obtained its single crystal, and compared with the common positive drugs in the market, the therapeutic effect of the complex on ischemic cerebrovascular disease and ischemic cardiovascular disease was studied.
与无定型丹酚酸A冻干粉对比，该系列结晶复合物具有纯度高、稳定性好、生产成本低等特点。与已报道的丹酚酸A结晶对比，该系列结晶复合物表现为单晶、且具有水溶性好、生产成本低的特点，更适用于将其开发成静脉注射制剂。与阳性药物丁苯酞氯化钠注射液、注射用丹参多酚酸、注射用丹参多酚酸盐对比，该系列结晶复合物具有显著降低脑/心肌梗死面积、显著降低模型动物死亡率、显著改善脑缺血/心肌缺血模型动物肢体功能恢复的效果。该系列结晶复合物可应用于缺血性心脏病和缺血性脑血管疾病的治疗，作为相关药物注射制剂原料药(API)使用。Compared with amorphous salvianolic acid A freeze-dried powder, this series of crystalline compounds has the characteristics of high purity, good stability and low production cost. Compared with the reported salvianolic acid A crystals, the series of crystal complexes are single crystals, and have the characteristics of good water solubility and low production cost, and are more suitable for development into intravenous injection preparations. Compared with the positive drug butylphthalide and sodium chloride injection, salvianolic acid for injection, and salvianolate for injection, this series of crystalline complexes can significantly reduce the area of brain/myocardial infarction, significantly reduce the mortality rate of model animals, and significantly reduce the size of cerebral/myocardial infarction. The effect of improving limb function recovery in cerebral ischemia/myocardial ischemia model animals. The series of crystalline compounds can be applied to the treatment of ischemic heart disease and ischemic cerebrovascular disease, and can be used as APIs for related drug injection preparations.
可以采用如下多种方式和仪器对本发明的丹酚酸A盐水合物性质进行研究，例如采用X射线单晶衍射、X射线粉末衍射、示差扫描量热分析、热重分析法(TGA)进行分析。这些分析方法可以为本领域常规的方法。The properties of the salvianolic acid A salt hydrate of the present invention can be studied by the following methods and instruments, such as X-ray single crystal diffraction, X-ray powder diffraction, differential scanning calorimetry, and thermogravimetric analysis (TGA) for analysis. . These analytical methods can be conventional methods in the art.
例如X射线单晶衍射分析可以采用BrukerSMART APEX-II单晶X射线衍射仪，测试条件：CuKα辐射，石墨单色器，单导管直径Φ＝0.50mm，晶体与CCD探测器距离d＝60.3mm，管压40kV，管流30mA，扫描方式：φ/ω扫描。For example, X-ray single crystal diffraction analysis can use BrukerSMART APEX-II single crystal X-ray diffractometer, test conditions: CuKα radiation, graphite monochromator, single tube diameter Φ=0.50mm, crystal and CCD detector distance d=60.3mm, Tube pressure 40kV, tube flow 30mA, scanning mode: φ/ω scanning.
经X射线单晶衍射分析，获得了本发明的丹酚酸A盐水合物的单晶特征， 及结构类型。Through X-ray single crystal diffraction analysis, the single crystal characteristics and structure type of the salvianolic acid A salt hydrate of the present invention are obtained.
具体地，测得丹酚酸A钠盐水合物晶体的绝对构型为：Specifically, the absolute configuration of the salvianolic acid A sodium salt hydrate crystal was measured as:
Figure PCTCN2022091078-appb-000017
Figure PCTCN2022091078-appb-000017
测得丹酚酸A钾盐水合物晶体的绝对构型为：The absolute configuration of salvianolic acid A potassium salt hydrate crystals was measured as:
Figure PCTCN2022091078-appb-000018
Figure PCTCN2022091078-appb-000018
测得丹酚酸A钙盐水合物晶体的绝对构型为：The absolute configuration of the salvianolic acid A calcium salt hydrate crystal measured is:
Figure PCTCN2022091078-appb-000019
Figure PCTCN2022091078-appb-000019
测得丹酚酸A镁盐水合物晶体的相对构型为：The relative configuration of salvianolic acid A magnesium salt hydrate crystals was measured as:
Figure PCTCN2022091078-appb-000020
Figure PCTCN2022091078-appb-000020
测得丹酚酸A铵盐水合物晶体的绝对构型为：The absolute configuration of salvianolic acid A ammonium salt hydrate crystals was measured as:
Figure PCTCN2022091078-appb-000021
Figure PCTCN2022091078-appb-000021
测试的各丹酚酸A盐水合物晶体的晶胞参数如下：The unit cell parameters of each salvianolic acid A salt hydrate crystal tested are as follows:
丹酚酸A钠盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，C2 空间群，晶胞参数为：
Figure PCTCN2022091078-appb-000022
α＝γ＝90.0°,β＝91.6°； The salvianolic acid A sodium salt hydrate crystal was determined by X-ray single crystal diffraction to be monoclinic, C2 space group, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000022
α=γ=90.0°, β=91.6°;
丹酚酸A钾盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，C2空间群，晶胞参数为：
Figure PCTCN2022091078-appb-000023
α＝γ＝90.0°,β＝115.16°； Salvianolic acid A potassium salt hydrate crystal, which was determined by X-ray single crystal diffraction to be monoclinic, C2 space group, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000023
α=γ=90.0°, β=115.16°;
丹酚酸A镁盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，C2空间群，晶胞参数为：
Figure PCTCN2022091078-appb-000024
α＝γ＝90.0°,β＝116.1°； The salvianolic acid A magnesium salt hydrate crystal was determined by X-ray single crystal diffraction to be monoclinic, C2 space group, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000024
α=γ=90.0°, β=116.1°;
丹酚酸A钙盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，C2空间群，晶胞参数为：
Figure PCTCN2022091078-appb-000025
α＝γ＝90.0°,β＝115.7°；和 The salvianolic acid A calcium salt hydrate crystal was determined by X-ray single crystal diffraction to be monoclinic, C2 space group, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000025
α=γ=90.0°, β=115.7°; and
丹酚酸A铵盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，P2空间群，晶胞参数为：
Figure PCTCN2022091078-appb-000026
α＝γ＝90.0°,β＝96.9°。 The salvianolic acid A ammonium salt hydrate crystal was determined by X-ray single crystal diffraction to be monoclinic, P2 space group, and the unit cell parameters are:
Figure PCTCN2022091078-appb-000026
α=γ=90.0°, β=96.9°.
本发明所述测定晶型的X射线粉末衍射的方法在本领域中是已知的。本发明的具体检测条件可参考本发明各晶体的X射线粉末衍射图谱。结果表明，本发明的各晶体在X-射线粉末衍射(XRPD)图中具有特定的特征峰。Methods of determining the X-ray powder diffraction of a crystalline form according to the present invention are known in the art. For the specific detection conditions of the present invention, reference may be made to the X-ray powder diffraction pattern 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.
某一化合物能否结合溶剂成为溶剂合物或结合几分子溶剂，并没有规律可循。分子结构相同但晶型不同时，有可能具有不同的生物利用度、溶解度、溶解速率、化学物理稳定性、熔点、颜色、可滤性、密度和流动性。有些多晶型物由于形状或吸湿性而难于制成制剂。X射线粉末射线衍射谱图是晶型鉴定的必要方式，但不是唯一方式。水合物晶型，由于水与化合物结合的方式不同，可能导致其X射线粉末射线衍射谱图相似，但其他的表征数据不同，如DSC谱图。同一药物，不同的晶型或不同的溶剂合物，会表现出稳定性、流动性、吸湿性、溶解性、可压缩性的不同，都会对药物的应用有重要的影响，从而大大影响药物的生物利用度及使用便捷性。There is no rule to follow whether a compound can bind a solvent to become a solvate or bind several molecules of solvent. When the molecular structure is the same but the crystal form is 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 because of their shape or hygroscopicity. X-ray powder ray diffraction pattern is a necessary way to identify the crystal form, but it is not the only way. Hydrate crystal forms, due to the different ways in which water binds to the compound, may result in similar X-ray powder ray diffraction patterns, but different other characterization data, such as DSC patterns. 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, thereby greatly affecting the drug's performance. Bioavailability and ease of use.
例如，专利文献CN201910643821.X和CN201710067693.X中描述了一种丹酚酸A盐复合物，其结构中包含丹酚酸A分子、丹酚酸A负离子、和阳离子，但是不含结晶水，测试其水溶性为“略溶”。为了对比该专利文献所述“丹酚酸A盐复合物”与本发明所述“丹酚酸A盐水合物”的差别，参考其方法，制备了丹酚酸A钠盐复合物，图32A和图32B可见本发明丹酚酸A钠盐水合物单晶与丹酚酸A钠盐复合物的显微照片。由图可见，本发明所制备的丹酚酸A钠盐水合物为清晰可见的“单晶”，而丹酚酸A钠盐复合物为“结晶性粉末”，未见明显的“单晶”。本发明制备的丹酚酸A钠盐水合物与对比专利的丹酚酸A钠盐复合物的差异如下：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. 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 salt hydrate" described in the present invention, referring to its method, the salvianolic acid A sodium salt complex was prepared, Figure 32A And FIG. 32B shows the photomicrograph 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 complex of the comparative patent is as follows:
前者为单晶、纯净物、化学组成稳定，水溶解性好；The former is single crystal, pure substance, stable in chemical composition and good in water solubility;
后者为结晶性粉末、混合物、理论上并不能保证其化学组成的稳定性(如丹酚酸A分子与丹酚酸A钠的比例)，水溶解性较差；The latter is a crystalline powder and mixture, which theoretically cannot guarantee the stability of its chemical composition (such as the ratio of salvianolic acid A molecule and salvianolic acid A sodium), and has poor water solubility;
根据医药注册法规，注射剂原料药最基本的特征为组成成分固定，且为纯净物。本发明制备的丹酚酸A钠盐水合物单晶相比较对比专利的丹酚酸A钠盐 复合物结晶性粉末，作为注射剂原料药使用时，具有显著的优势。According to the pharmaceutical registration regulations, the most basic feature of APIs for injections is that the composition is fixed and pure. Compared with the salvianolic acid A sodium salt complex crystalline powder of the comparative patent, the salvianolic acid A sodium salt hydrate single crystal prepared by the present invention has significant advantages when used as an injection raw material drug.
同理，本发明所制备的丹酚酸A钾、钙、镁、铵盐水合物结晶(单晶)与对比专利所述的丹酚酸A盐复合物结晶性粉末相比，亦有类似的优势。Similarly, the salvianolic acid A potassium, calcium, magnesium, and ammonium salt hydrate crystals (single crystals) prepared by the present invention are similar to the salvianolic acid A salt complex crystalline powder described in the comparative patent. Advantage.
本发明还提供了一种丹酚酸A的结晶工艺，通过该结晶工艺，得到了一种丹酚酸A盐水合物的新晶型。同干燥工艺得到的无定型固体粉末相比，该结晶具有纯度高、生产成本低、更加稳定的优点，能够满足注射剂新药原料药的生产需求。与文献报道的丹酚酸A晶体相比，本发明得到的丹酚酸A盐水合物结晶，具有产物纯度高、生产成本低、产物水溶性好等优势，适用于丹酚酸A注射制剂的开发。The invention also provides a crystallization process of salvianolic acid A, through which a new crystal form of salvianolic acid A 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 requirements of the new pharmaceutical raw materials for injection. Compared with the salvianolic acid A crystals reported in the literature, the salvianolic acid A salt hydrate crystals obtained by the present invention have the advantages of high product purity, low production cost, good product water solubility, etc., and are suitable for the development of salvianolic acid A injection preparations. .
活性成分Active ingredient
如本文所用，术语“活性成分”或“活性化合物”指本发明的丹酚酸A盐水合物。As used herein, the term "active ingredient" or "active compound" refers to the salvianolic acid A salt hydrate of the present invention.
本发明公开了一系列丹酚酸A盐水合物结晶及其制备方法(如丹酚酸A钠盐水合物、丹酚酸A钾盐水合物、丹酚酸A钙盐水合物、丹酚酸A镁盐水合物、丹酚酸A铵盐水合物)。丹参药材参考《中国药典》(一部)(2020版)中“丹参水提物的制备”的制法制备丹参水提物。后者经过反应制备丹酚酸A粗品，再经过树脂柱层析后，得到纯度≥80％的丹酚酸A样品。再将该样品经过结晶、重结晶的过程，制备得到高纯度丹酚酸A盐水合物结晶。该结晶由丹酚酸A、丹酚酸A负离子、阳离子、和水分子组成。该一系列复合物皆为新的纯净物质，并具有全新的晶型，相比起无定型丹酚酸A粉末，其具有稳定性好、溶解性好的特点，相比较丹酚酸A结晶，其具有水溶性好的特点。本发明制备的丹酚酸A盐水合物更适用于静脉注射剂的开发，其应用于缺血性脑卒中和缺血性心血管疾病的治疗，效果显著好于阳性药物丁苯酞氯化钠注射液和注射用丹参多酚酸盐。The invention discloses a series of salvianolic acid A salt hydrate crystals and a preparation method thereof (such as salvianolic acid A sodium salt hydrate, salvianolic acid A potassium salt hydrate, salvianolic acid A calcium salt hydrate, salvianolic acid A calcium salt hydrate, salvianolic acid A sodium salt hydrate, salvianolic acid A potassium salt hydrate A magnesium salt hydrate, salvianolic acid A ammonium salt hydrate). The medicinal materials of Salvia miltiorrhiza were prepared with reference to the preparation method of "Preparation of Salvia miltiorrhiza Aqueous Extract" in "Chinese Pharmacopoeia" (Part I) (2020 Edition). The latter is reacted to prepare a crude product of salvianolic acid A, which is then subjected to resin column chromatography to obtain a salvianolic acid A sample with a purity of ≥80%. The sample is then subjected to the process of crystallization and recrystallization to prepare high-purity salvianolic acid A salt hydrate crystals. The crystal is composed of salvianolic acid A, salvianolic acid A negative ions, cations, and water molecules. The series of compounds are all new pure substances and have a new crystal form. Compared with the amorphous salvianolic acid A powder, they have 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 salt hydrate prepared by the 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 solution and salvianolate for injection.
药物组合物和施用方法Pharmaceutical compositions and methods of administration
由于丹酚酸A可用于治疗以下疾病：心血管疾病(如缺血性心脏病、脑卒中)、高脂血症、糖尿病及其并发症等等。因此，本发明的丹酚酸A盐水合物可以用于治疗或预防上述疾病。Because salvianolic acid A can be used to treat the following diseases: cardiovascular disease (such as ischemic heart disease, stroke), hyperlipidemia, diabetes and its complications and so on. Therefore, the salvianolic acid A salt hydrate of the present invention can be used to treat or prevent the above-mentioned diseases.
本发明的药物组合物包含安全有效量范围内的本发明的丹酚酸A盐水合物及药学上可以接受的赋形剂或载体。其中“安全有效量”指的是：丹酚酸A盐水合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明的晶型物/剂，更佳地，含有10-200mg本发明的晶型物/剂。较佳地，所述的“一剂”为一个注射剂量或一个胶囊或药片。The pharmaceutical composition of the present invention comprises the salvianolic acid A salt hydrate of the present invention and a pharmaceutically acceptable excipient or carrier within a safe and effective amount. The "safe and effective amount" refers to: the amount of salvianolic acid A salt hydrate is sufficient to significantly improve the condition without causing 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 one injection dose or one capsule or tablet.
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效或者稳定性等。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆 油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如润湿剂(如十二烷基硫酸钠)、崩解剂、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。"Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler or gelling substances which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity. "Compatibility" as used herein 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 moieties include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, 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.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as wetting agents (such as ten Sodium dialkyl sulfate), disintegrating agent, coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
本发明的丹酚酸A盐水合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。优选地，采用注射方式给药，如静脉注射。The mode of administration of the salvianolic acid A salt hydrate or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration 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.
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。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 compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性成分也可与上述赋形剂中的一种或多种形成微胶囊形式。Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using 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 compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. If desired, the active ingredient may also be in microencapsulated form with one or more of the above-mentioned excipients.
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally employed 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, and the like.
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。Besides these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。In addition to the active ingredient, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。本发明的丹酚酸A盐水合物由于其优异的溶解性，因此特别适用于注射给药。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 excipients include water, ethanol, polyols and suitable mixtures thereof. The salvianolic acid A salt hydrate of the present invention is particularly suitable for injection administration due to its excellent solubility.
用于局部给药的本发明的晶型物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。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 that may be required if necessary.
本发明的丹酚酸A盐水合物可以单独给药，或者与其他药学上可接受的化合物联合给药。The salvianolic acid A salt hydrate of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
使用药物组合物时，是将安全有效量的本发明的晶型物适用于需要治疗的 哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选10～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。When the pharmaceutical composition is used, a safe and effective amount of the crystalline form of the present invention is suitable for mammals (such as humans) in need of treatment, wherein the dose is a pharmaceutically effective dose when administered, and for a 60kg body weight human. In other words, the daily dosage is usually 1-2000 mg, preferably 10-500 mg. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
本发明的主要优点在于：The main advantages of the present invention are:
(1)本发明首次制备得到的丹酚酸A盐水合物结晶，与丹酚酸A冻干粉末相比，稳定性更强，更耐高温、高湿、强光照射等；(1) Compared with the salvianolic acid A freeze-dried powder, the salvianolic acid A salt hydrate crystal prepared for the first time in the present invention has stronger stability and is more resistant to high temperature, high humidity, strong light irradiation, etc.;
(2)本发明制备得到的丹酚酸A盐水合物结晶水溶性更好，且具有显著更快地溶解速率，适合作为注射剂使用。(2) The salvianolic acid A salt hydrate crystal prepared by the present invention has better water solubility, has a significantly faster dissolution rate, and is suitable for use as an injection.
(3)本发明提供的制备丹酚酸A结晶的方法，可以采用低纯度的丹酚酸A原料(约80％即可)为起始原料进行结晶，因此极大的降低了丹酚酸A结晶制备的成本。(3) The method for preparing salvianolic acid A crystals provided by the present invention can use low-purity salvianolic acid A raw materials (about 80%) as the starting material for crystallization, so that the salvianolic acid A is greatly reduced. Cost of crystal preparation.
(4)本发明提供的制备丹酚酸A结晶的方法，产物纯度高，收率大，方便工业生产。(4) The method for preparing salvianolic acid A crystals provided by the present invention has high product purity and high yield, and is convenient for industrial production.
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。Below in conjunction with specific embodiments, the present invention is described in further detail. 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 detailed conditions in the following examples are usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例1 制备纯度≥80％的丹酚酸A样品Example 1 Preparation of salvianolic acid A samples with a purity of ≥ 80%
步骤一：制备丹酚酸A粗品Step 1: Preparation of crude salvianolic acid A
称取丹参总酚酸提取物1000g(其中丹酚酸B含量约50g，迷迭香酸含量约5g)，溶解于2000mL水中，调节pH 5.0，加热回流(100℃)反应24h，HPLC检测，一般的，每1000g丹参提取物可制得含丹酚酸A约20g的粗品溶液。Weigh 1000 g of total phenolic acid extract of Salvia miltiorrhiza (including about 50 g of salvianolic acid B and about 5 g of rosmarinic acid), dissolved in 2000 mL of water, adjusted to pH 5.0, heated under reflux (100 ° C) and reacted for 24 h, detected by HPLC, generally Yes, every 1000g of Salvia miltiorrhiza extract can produce a crude solution containing about 20g of salvianolic acid A.
步骤二：柱色谱法纯化丹酚酸AStep 2: Purification of Salvianolic Acid A by Column Chromatography
选用大孔吸附树脂HZ816(华东理工大学华震生物科技科技有限公司)1000mL，将步骤一得到的丹酚酸A粗品溶液，调节pH 3.0，流经树脂后，再用6000mL水洗脱树脂柱；然后采用50％的乙醇溶液洗脱树脂柱，并分步收集流出液，500mL/瓶，HPLC检测各样品，收集HPLC纯度≥50％的样品溶液，合并，一般情况下可得含丹酚酸A约19.5g的丹酚酸A溶液。Select 1000mL of macroporous adsorption resin HZ816 (Huazhen Biotechnology Co., Ltd., East China University of Science and Technology), adjust the pH of the crude salvianolic acid A solution obtained in step 1 to 3.0, flow through the resin, and then elute the resin column with 6000mL of water; Then use 50% ethanol solution to elute the resin column, and collect the effluent step by step, 500mL/bottle, test each sample by HPLC, collect the sample solution with HPLC purity ≥ 50%, and combine them. Generally, salvianolic acid A can be obtained. About 19.5g of salvianolic acid A solution.
树脂再生后，再次重复该柱层析过程，收集HPLC纯度≥80％的样品溶液，合并，一般情况下可得含丹酚酸A约16g的丹酚酸A溶液。After the resin is regenerated, repeat the column chromatography process again, collect sample solutions with HPLC purity ≥80%, and combine them to obtain a salvianolic acid A solution containing about 16 g of salvianolic acid A in general.
采用本方法制备得到的HPLC纯度≥80％的丹酚酸A样品，用于后续结晶法制备不同丹酚酸A盐水合物的起始原料。The salvianolic acid A sample with HPLC purity ≥80% prepared by the method is used for the subsequent crystallization method to prepare the starting material of different salvianolic acid A salt hydrates.
实施例2 制备丹酚酸A钠盐水合物结晶Example 2 Preparation of salvianolic acid A sodium salt hydrate crystal
将纯度≥80％的丹酚酸A溶液浓缩(内含约16g丹酚酸A)至浓度15％，水浴加热至50℃，缓慢加入碳酸钠粉末约532mg，待两者反应完全后(反应约 15分钟，可使其中31％的丹酚酸A反应生成丹酚酸A钠盐)，将溶液降温至20℃，降温速率为0.1℃/min，其间不停搅拌，然后进一步降温至5℃，不断搅拌24h，得丹酚酸A钠盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing about 16 g of salvianolic acid A) to a concentration of 15%, heat it to 50°C in a water bath, slowly add about 532 mg of sodium carbonate powder, and wait for the two to react completely (reaction about 15 minutes, wherein 31% of salvianolic acid A can be reacted to generate salvianolic acid A sodium salt), the solution is cooled to 20°C, the cooling rate is 0.1°C/min, during which constant stirring, and then further cooled to 5°C, Stir continuously for 24h to obtain salvianolic acid A sodium salt hydrate (precipitation).
过滤沉淀，加入水于50℃溶解，配置成质量浓度为15％的溶液(以丹酚酸A计)，将溶液降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌析晶24h，析出丹酚酸A钠盐水合物(重结晶产物)。Filter the precipitation, add water to dissolve at 50 ° C, configure into a solution with a mass concentration of 15% (calculated by salvianolic acid A), cool the solution to 20 ° C, and the cooling rate is 0.2 ° C/min, during which stirring is continued, and then further The temperature was lowered to 5°C, and the mixture was stirred and crystallized for 24 hours, and the salvianolic acid A sodium salt hydrate (recrystallized product) was precipitated.
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A钠盐水合物结晶”11.563g，以丹酚酸A计，其HPLC纯度99.6％。Filter the precipitate and spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize, and dry at 25°C for 12h to obtain the product "Salvianolic Acid A Sodium Salt Hydrate Crystal" 11.563 g, in terms of salvianolic acid A, its HPLC purity is 99.6%.
将上述制得的“丹酚酸A钠盐水合物”，用于进行X射线单晶衍射、X射线粉末衍射、DSC、TGA分析及溶解性、稳定性等测定，以及抗脑缺血、心肌缺血等药效学试验使用。The "salvianolic acid A sodium salt hydrate" obtained above is used for X-ray single crystal diffraction, X-ray powder diffraction, DSC, TGA analysis and determination of solubility and stability, as well as anti-cerebral ischemia, myocardial Ischemia and other pharmacodynamic tests are used.
图1是丹酚酸A钠盐水合物的X射线单晶衍射晶体数据Figure 1 is the X-ray single crystal diffraction crystal data of salvianolic acid A sodium salt hydrate
图2A(不对称单位的立体结构椭球图)和图2B(单个主体分子的立体结构椭球图)是丹酚酸A钠盐水合物的X射线单晶衍射得到的立体结构椭球图。2A (stereostructure ellipsoid diagram of asymmetric unit) and FIG. 2B (stereostructure ellipsoid diagram of a single host molecule) are stereostructure ellipsoid diagrams obtained by X-ray single crystal diffraction of salvianolic acid A sodium salt hydrate.
图3是丹酚酸A钠盐水合物的延b轴方向的晶胞堆积投影图。3 is a unit cell stacking projection view along the b-axis of salvianolic acid A sodium salt hydrate.
图4是丹酚酸A钠盐水合物的粉末衍射图谱。Figure 4 is the powder diffraction pattern of salvianolic acid A sodium salt hydrate.
图5是丹酚酸A钠盐水合物的粉末衍射2θ值列表。Figure 5 is a list of powder diffraction 2Θ values of salvianolic acid A sodium salt hydrate.
图6是丹酚酸A钠盐水合物的DSC图谱。Figure 6 is the DSC spectrum of salvianolic acid A sodium salt hydrate.
经X射线单晶衍射，可以确定丹酚酸A钠盐水合物的绝对构型为：Through X-ray single crystal diffraction, the absolute configuration of salvianolic acid A sodium salt hydrate can be determined as:
Figure PCTCN2022091078-appb-000027
Figure PCTCN2022091078-appb-000027
TGA检测结果表明，晶体中结晶水含量与单晶衍射检测结果一致。The TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
实施例3 制备丹酚酸A钾盐水合物结晶Example 3 Preparation of salvianolic acid A potassium salt hydrate crystal
将纯度≥80％的丹酚酸A溶液浓缩(内含16g丹酚酸A)至浓度10％，水浴加热至50℃，缓慢加入碳酸钾粉末约1117mg，待两者反应完全后(反应约10分钟，可使其中50％的丹酚酸A反应生成丹酚酸A钾盐)，将溶液降温至20℃，降温速率为0.3℃/min，其间不停搅拌，然后进一步降温至5℃，不断搅拌24h，得丹酚酸A钾盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing 16 g of salvianolic acid A) to a concentration of 10%, heat it to 50° C. in a water bath, slowly add about 1117 mg of potassium carbonate powder, and wait for the two to react completely (the reaction is about 10 minutes, 50% of the salvianolic acid A can be reacted to generate salvianolic acid A potassium salt), the solution is cooled to 20°C, the cooling rate is 0.3°C/min, and the stirring is continued, then further cooled to 5°C, continuously Stir for 24h to obtain salvianolic acid A potassium salt hydrate (precipitation).
过滤沉淀，加入水于50℃溶解，配置成浓度10％的溶液(以丹酚酸A计)，将溶液降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌析晶36h，析出丹酚酸A钾盐水合物(重结晶产物)。Filter the precipitation, add water to dissolve at 50°C, configure into a solution with a concentration of 10% (calculated by salvianolic acid A), cool the solution to 20°C, and the cooling rate is 0.2°C/min. 5 ℃, stirring and crystallization for 36h, salvianolic acid A potassium salt hydrate (recrystallized product) was precipitated.
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A钾盐水合物结晶”11.212g，以丹酚酸A计，其HPLC纯度99.5％，保存待用。Filter the precipitate, spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize, and dry at 25°C for 12h to obtain the product "salvianolic acid A potassium salt hydrate crystal" 11.212 g, in terms of salvianolic acid A, its HPLC purity is 99.5%, and it is stored for use.
图7是丹酚酸A钾盐水合物的X射线单晶衍射晶体数据Figure 7 is the X-ray single crystal diffraction crystal data of salvianolic acid A potassium salt hydrate
图8A是丹酚酸A钾盐水合物的X射线单晶衍射得到的立体结构椭球图Figure 8A is a three-dimensional structure ellipsoid obtained by X-ray single crystal diffraction of salvianolic acid A potassium salt hydrate
图8B是丹酚酸A钾盐水合物的延b轴方向的晶胞堆积投影图Fig. 8B is a unit cell stacking projection view along the b-axis of salvianolic acid A potassium salt hydrate
图9是丹酚酸A钾盐水合物的粉末衍射图谱Fig. 9 is the powder diffraction pattern of salvianolic acid A potassium salt hydrate
图10是丹酚酸A钾盐水合物的粉末衍射2θ值列表Figure 10 is a list of powder diffraction 2θ values of salvianolic acid A potassium salt hydrate
图11是丹酚酸A钾盐水合物的DSC图谱Figure 11 is the DSC spectrum of salvianolic acid A potassium salt hydrate
经X射线单晶衍射，可以确定丹酚酸A钾盐水合物晶体的绝对构型为：Through X-ray single crystal diffraction, it can be determined that the absolute configuration of salvianolic acid A potassium salt hydrate crystal is:
Figure PCTCN2022091078-appb-000028
Figure PCTCN2022091078-appb-000028
TGA检测结果表明，晶体中结晶水含量与单晶衍射检测结果一致。The TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
实施例4 制备丹酚酸A钙盐水合物结晶Example 4 Preparation of salvianolic acid A calcium salt hydrate crystals
将纯度≥80％的丹酚酸A溶液浓缩(内含16g丹酚酸A)至浓度15％，水浴50℃，缓慢加入碳酸钙粉末约810mg，待两者反应完全后(反应约20分钟，可使其中50％的丹酚酸A反应生成丹酚酸A钙盐)，将溶液降温至20℃，降温速率为0.15℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌析晶24h，得丹酚酸A钙盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing 16 g of salvianolic acid A) to a concentration of 15%, take a water bath at 50 ° C, slowly add about 810 mg of calcium carbonate powder, and wait for the two to react completely (the reaction is about 20 minutes, 50% of the salvianolic acid A can be reacted to generate salvianolic acid A calcium salt), the solution is cooled to 20°C, the cooling rate is 0.15°C/min, during which stirring is continued, and then the temperature is further cooled to 5°C, stirring and crystallization 24h, salvianolic acid A calcium salt hydrate (precipitation) was obtained.
过滤沉淀，加入水于50℃溶解，配置成浓度18％的溶液(以丹酚酸A计)，将溶液降温至20℃，降温速率为0.5℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌析晶24h，得丹酚酸A钙盐水合物(重结晶产物)。Filter the precipitation, add water to dissolve at 50 ° C, configure into a solution with a concentration of 18% (calculated by salvianolic acid A), cool the solution to 20 ° C, and the cooling rate is 0.5 ° C/min, stirring continuously during the period, and then further cooling to 5 ℃, stirring and crystallization for 24h to obtain salvianolic acid A calcium salt hydrate (recrystallized product).
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A钙盐水合物结晶”14.003g，以丹酚酸A计，其HPLC纯度99.5％，保存待用。Filter the precipitate, spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize it, and dry it at 25°C for 12h to obtain the product "Salvianolic acid A calcium salt hydrate crystal" 14.003 g, in terms of salvianolic acid A, its HPLC purity is 99.5%, and it is stored for use.
图12是丹酚酸A钙盐水合物的X射线单晶衍射晶体数据Figure 12 is the X-ray single crystal diffraction crystal data of salvianolic acid A calcium salt hydrate
图13A(不对称单元的立体结构椭球图)和图13B(显示钙配位情况的立体结构椭球图)是丹酚酸A钙盐水合物的X射线单晶衍射得到的立体结构椭球图Fig. 13A (stereostructure ellipsoid diagram of asymmetric unit) and Fig. 13B (stereostructure ellipsoid diagram showing calcium coordination) are stereostructure ellipsoids obtained by X-ray single crystal diffraction of salvianolic acid A calcium salt hydrate picture
图14是丹酚酸A钙盐水合物的延b轴方向的晶胞堆积投影图Fig. 14 is a unit cell stacking projection view along the b-axis of salvianolic acid A calcium salt hydrate
图15是丹酚酸A钙盐水合物的粉末衍射图谱Figure 15 is the powder diffraction pattern of salvianolic acid A calcium salt hydrate
图16是丹酚酸A钙盐水合物的粉末衍射2θ值列表Figure 16 is a list of powder diffraction 2θ values of salvianolic acid A calcium salt hydrate
图17是丹酚酸A钙盐水合物的DSC图谱。Figure 17 is the DSC spectrum of salvianolic acid A calcium salt hydrate.
经X射线单晶衍射，可以确定丹酚酸A钙盐水合物晶体的绝对构型为：Through X-ray single crystal diffraction, it can be determined that the absolute configuration of the salvianolic acid A calcium salt hydrate crystal is:
Figure PCTCN2022091078-appb-000029
Figure PCTCN2022091078-appb-000029
TGA检测结果表明，晶体中结晶水含量与单晶衍射检测结果一致。The TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
实施例5 制备丹酚酸A镁盐水合物结晶Example 5 Preparation of salvianolic acid A magnesium salt hydrate crystal
将纯度≥80％的丹酚酸A溶液浓缩(内含16g丹酚酸A)，至浓度10％，水浴50℃，缓慢加入氢氧化镁粉末约470mg，待两者反应完全后(反应约10分钟，可使其中50％的丹酚酸A反应生成丹酚酸A镁盐)，将溶液降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌析晶24h，得丹酚酸A镁盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing 16 g of salvianolic acid A) to a concentration of 10%, take a water bath at 50 ° C, slowly add about 470 mg of magnesium hydroxide powder, and wait for the two to react completely (the reaction is about 10 minutes, wherein 50% of salvianolic acid A can be reacted to generate salvianolic acid A magnesium salt), the solution is cooled to 20 ° C, the cooling rate is 0.2 ° C/min, during which constant stirring, then further cooled to 5 ° C, stirring Crystallize for 24h to obtain salvianolic acid A magnesium salt hydrate (precipitation).
过滤沉淀，加入水于50℃溶解，配置成浓度10％的溶液(以丹酚酸A计)，将溶液降温至20℃，降温速率为0.5℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌析晶24h，得丹酚酸A镁盐水合物(重结晶产物)。Filter the precipitation, add water to dissolve at 50°C, configure into a solution with a concentration of 10% (calculated by salvianolic acid A), cool the solution to 20°C, and the cooling rate is 0.5°C/min. 5 ℃, stirring and crystallization for 24h to obtain salvianolic acid A magnesium salt hydrate (recrystallized product).
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A镁盐水合物结晶”2.256g，以丹酚酸A计，其HPLC纯度99.5％，保存待用。Filter the precipitate, spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize, and dry at 25°C for 12h to obtain the product "Salvianolic Acid A Magnesium Salt Hydrate Crystal" 2.256 g, in terms of salvianolic acid A, its HPLC purity is 99.5%, and it is stored for use.
图18是丹酚酸A镁盐水合物的X射线单晶衍射晶体数据Figure 18 is the X-ray single crystal diffraction crystal data of salvianolic acid A magnesium salt hydrate
图19是丹酚酸A镁盐水合物的X射线单晶衍射得到的立体结构椭球图Figure 19 is a three-dimensional structure ellipsoid figure obtained by X-ray single crystal diffraction of salvianolic acid A magnesium salt hydrate
图20是丹酚酸A镁盐水合物的延b轴方向的晶胞堆积投影图Fig. 20 is a unit cell stacking projection view along the b-axis of salvianolic acid A magnesium salt hydrate
图21是丹酚酸A镁盐水合物的粉末衍射图谱Figure 21 is the powder diffraction pattern of salvianolic acid A magnesium salt hydrate
图22是丹酚酸A镁盐水合物的粉末衍射2θ值列表Figure 22 is a list of powder diffraction 2θ values of salvianolic acid A magnesium salt hydrate
图23是丹酚酸A镁盐水合物的DSC图谱Figure 23 is the DSC spectrum of salvianolic acid A magnesium salt hydrate
经X射线单晶衍射，可以确定丹酚酸A镁盐水合物晶体的相对构型为：Through X-ray single crystal diffraction, the relative configuration of salvianolic acid A magnesium salt hydrate crystals can be determined as:
Figure PCTCN2022091078-appb-000030
Figure PCTCN2022091078-appb-000030
TGA检测结果表明，晶体中结晶水含量与单晶衍射检测结果一致。The TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
实施例6 制备丹酚酸A铵盐水合物结晶Example 6 Preparation of salvianolic acid A ammonium salt hydrate crystal
将纯度≥80％的丹酚酸A溶液浓缩(内含16g丹酚酸A)，至浓度15％，水浴50℃，缓慢加入碳酸铵粉末约777mg，待两者反应完全后(反应约15分钟，可使其中50％的丹酚酸A反应生成丹酚酸A铵盐)，将溶液降温至20℃，降温速率为0.4℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌析晶20h，得丹酚酸A铵盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing 16 g of salvianolic acid A) to a concentration of 15%, take a water bath at 50°C, slowly add about 777 mg of ammonium carbonate powder, and wait for the two to react completely (reaction for about 15 minutes). , 50% of the salvianolic acid A can be reacted to generate salvianolic acid A ammonium salt), the solution is cooled to 20 ° C, the cooling rate is 0.4 ° C/min, during which constant stirring, and then further cooled to 5 ° C, stirring to separate out Crystallization for 20h to obtain salvianolic acid A ammonium salt hydrate (precipitation).
过滤沉淀，加入水于60℃溶解，配置成浓度15％的溶液(以丹酚酸A计)，将溶液降温至20℃，降温速率为0.4℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌析晶24h，得丹酚酸A铵盐水合物(重结晶产物)。Filter the precipitation, add water to dissolve at 60°C, configure into a solution with a concentration of 15% (calculated by salvianolic acid A), cool the solution to 20°C, and the cooling rate is 0.4°C/min. 5 ℃, stirring and crystallization for 24h to obtain salvianolic acid A ammonium salt hydrate (recrystallized product).
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A铵盐水合物结晶”6.681g，以丹酚酸A计，其HPLC纯度99.6％，保存待用。Filter the precipitate, spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize it, and dry it at 25°C for 12h to obtain the product "salvianolic acid A ammonium salt hydrate crystal" 6.681 g, in terms of salvianolic acid A, its HPLC purity is 99.6%, and it is stored for use.
图24是丹酚酸A铵盐水合物的X射线单晶衍射晶体数据Figure 24 is the X-ray single crystal diffraction crystal data of salvianolic acid A ammonium salt hydrate
图25(不对称单位的立体结构球棍图)和图26(单个主体分子的立体结构椭球图)是丹酚酸A铵盐水合物的X射线单晶衍射得到的立体结构椭球图Figure 25 (ball-and-stick diagram of the three-dimensional structure of the asymmetric unit) and Figure 26 (the three-dimensional structure ellipsoid diagram of a single host molecule) are the three-dimensional structure ellipsoid diagrams obtained by X-ray single crystal diffraction of salvianolic acid A ammonium salt hydrate
图27是丹酚酸A铵盐水合物的延b轴方向的晶胞堆积投影图Fig. 27 is a unit cell stacking projection view along the b-axis of salvianolic acid A ammonium salt hydrate
图28是本发明得到的丹酚酸A铵盐水合物的粉末衍射图谱Fig. 28 is the powder diffraction pattern of the salvianolic acid A ammonium salt hydrate obtained by the present invention
图29是本发明得到的丹酚酸A铵盐水合物的粉末衍射2θ值列表Figure 29 is a list of powder diffraction 2θ values of the salvianolic acid A ammonium salt hydrate obtained in the present invention
图30是本发明得到的丹酚酸A铵盐水合物的DSC图谱Fig. 30 is the DSC spectrum of the salvianolic acid A ammonium salt hydrate obtained by the present invention
经X射线单晶衍射，可以确定丹酚酸A铵盐水合物晶体的绝对构型为：Through X-ray single crystal diffraction, the absolute configuration of salvianolic acid A ammonium salt hydrate crystal can be determined as:
Figure PCTCN2022091078-appb-000031
Figure PCTCN2022091078-appb-000031
TGA检测结果表明，晶体中结晶水含量与单晶衍射检测结果一致。The TGA test results showed that the crystal water content in the crystals was consistent with the single crystal diffraction test results.
实施例7Example 7
丹酚酸A盐(钠、钾、钙、镁、铵)水合物、丹酚酸A结晶及丹酚酸A冻干粉末的高温、高湿、强光照射影响因素试验。The influence factors of high temperature, high humidity and strong light irradiation on salvianolic acid A salt (sodium, potassium, calcium, magnesium, ammonium) hydrate, salvianolic acid A crystal and salvianolic acid A freeze-dried powder.
影响因素试验是药物稳定性考察的重要方法，可以在较短时间内对药物稳定性有初步的了解，对后续的新药开发具有重要的意义，所有的原料药与制剂都要进行影响因素试验。Influencing factor test is an important method for drug stability investigation. It can have a preliminary understanding of drug stability in a relatively short period of time, and is of great significance to subsequent new drug development. All APIs and preparations must be tested for influencing factors.
本实施例中采用的丹酚酸A盐(钠、钾、钙、镁、铵)水合物，采用本发明相应实施例中的方法制备。The salvianolic acid A salt (sodium, potassium, calcium, magnesium, ammonium) hydrate used in this embodiment is prepared by the method in the corresponding embodiment of the present invention.
丹酚酸A结晶，采用专利文献CN201710055331.9中记载的丹酚酸A结晶的制备方法制备(叶天健等，一种丹酚酸A的晶型及其制备方法)。The salvianolic acid A crystal is prepared by the preparation method of the salvianolic acid A crystal described in the patent document CN201710055331.9 (Ye Tianjian et al., a crystal form of salvianolic acid A and its preparation method).
丹酚酸A冻干粉末，采用专利文献CN201310487751.6中记载的丹酚酸A的制备方法制备(阚士东，一种丹酚酸A的制备方法)Salvianolic acid A freeze-dried powder is prepared by the preparation method of salvianolic acid A recorded in patent document CN201310487751.6 (Kan Shidong, a kind of preparation method of salvianolic acid A)
本实施例参考《中国药典》2020版四部第457页中所述的试验方法，简述如下：This example refers to the test method described in the fourth part of the "Chinese Pharmacopoeia" 2020 edition on page 457, which is briefly described as follows:
将供试品置于适当的开口容器中(如称量瓶或培养皿)，摊成厚度≤5mm的薄层，进行如下试验：Place the test sample in a suitable open container (such as a weighing bottle or a petri dish), spread it into a thin layer with a thickness of ≤5mm, and carry out the following tests:
高温试验：供试品开口置于洁净容器中，60℃温度下放置10天后取样HPLC检测，并与0天时的样品对比。High temperature test: The test sample was placed in a clean container with the opening of the test sample, placed at a temperature of 60 °C for 10 days, and then sampled for HPLC detection, and compared with the sample at 0 days.
高湿试验：供试品开口置于恒湿密闭容器中，在25℃条件下分别于相对湿度90±5％条件下放置10天后取样HPLC检测，并记录样品放置前后的重量，以考察样品的吸湿情况。(选择KNO 3饱和溶液共同放置于密闭容器中营造高湿环境：25℃时相对湿度92.5％)，并与0天时的样品对比。 High-humidity test: The opening of the test sample is placed in a constant-humidity airtight container, and it is placed under the condition of 25°C under the condition of relative humidity of 90±5% for 10 days, and then sampled for HPLC detection, and the weight of the sample before and after being placed is recorded to investigate the quality of the sample. Moisture condition. (The KNO 3 saturated solution was chosen to be placed together in a closed container to create a high humidity environment: 92.5% relative humidity at 25°C), and compared with the sample at 0 days.
强光照射试验：供试品开口放置于装有日光灯的光照箱内，于照度4500lx±500lx条件下照射10天后取样HPLC检测，留意供试品外观变化，并与0天时的样品对比。Strong light irradiation test: The test sample is placed in a light box equipped with a fluorescent lamp, and is irradiated for 10 days under the condition of illuminance 4500lx ± 500lx. After 10 days, take samples for HPLC detection, pay attention to the appearance change of the test sample, and compare it with the sample at 0 days.
丹酚酸A(盐钠、钾、钙、镁、铵)水合物、丹酚酸A结晶及丹酚酸A冻干粉末影响因素试验结果见表1。The test results of the influencing factors of salvianolic acid A (salt sodium, potassium, calcium, magnesium, ammonium) hydrate, salvianolic acid A crystal and salvianolic acid A freeze-dried powder are shown in Table 1.
表1 高温、高湿、强光照射影响因素试验前后的产品质量统计表Table 1 Product quality statistics before and after the test of high temperature, high humidity and strong light irradiation factors
Figure PCTCN2022091078-appb-000032
Figure PCTCN2022091078-appb-000032
结果表明，在高温、光照、高湿影响因素试验中，丹酚酸A几种盐水合物和丹酚酸A结晶的稳定性和形态特征远好于冻干粉。初始时两者的纯度基本一致(99.5％-99.6％对比99.4％)，经过高温试验后，丹酚酸A几种盐水合物和丹酚酸A结晶纯度降低皆≤0.5％，后者则降低至74.7％；光照试验后，丹酚酸A几种盐水合物和丹酚酸A结晶纯度降低皆≤0.2％，而后者降低了8.5％；高湿试验中，10天后丹酚酸A几种盐水合物和丹酚酸A结晶纯度降低皆≤0.1％，而后者由于吸湿严重，由初始的淡黄色粉末，变为黄色油滴状，明显已经不合格(因此，未进行HPLC分析)。The results showed that the stability and morphological characteristics of several salts of salvianolic acid A hydrate and salvianolic acid A crystals were much better than freeze-dried powders in the high temperature, light and high humidity influencing factors test. Initially, the purity of the two is basically the same (99.5%-99.6% vs. 99.4%). After the high temperature test, the hydrates of several salts of salvianolic acid A and the crystalline purity of salvianolic acid A were reduced by ≤0.5%, and the latter decreased. After the light test, the hydrates of several salts of salvianolic acid A and the crystal purity of salvianolic acid A decreased by ≤0.2%, while the latter decreased by 8.5%; in the high humidity test, several salvianolic acid A The reduction of crystal purity of salt hydrate and salvianolic acid A was ≤0.1%, and the latter changed from the initial light yellow powder to yellow oil drop due to serious moisture absorption, and was obviously unqualified (therefore, HPLC analysis was not carried out).
综上，本发明制备得到的丹酚酸A盐水合物结晶，其稳定性显著好于冻干粉，更适用于新药开发的原料药使用。In conclusion, the salvianolic acid A salt hydrate crystal prepared by the present invention has significantly better stability than freeze-dried powder, and is more suitable for the use of raw materials for new drug development.
实施例8 丹酚酸A(钠、钾、钙、镁、铵)盐水合物、丹酚酸A结晶、丹酚酸A冻干粉水溶解性对比研究Example 8 Comparative study on water solubility of salvianolic acid A (sodium, potassium, calcium, magnesium, ammonium) salt hydrate, salvianolic acid A crystal, and salvianolic acid A freeze-dried powder
丹酚酸A口服生物利用度非常低，注射剂是其商业开发的首选剂型。由于其主要适应症为心脑血管疾病，病人急性发病期一般采用静脉注射的方式用药，因此产品水溶性及溶解速度非常重要，将直接影响医生和患者在临床使用时的选择，进而影响到药品的销售及市场。The oral bioavailability of salvianolic acid A is very low, and injection is the preferred dosage form for its commercial development. Because its main indication is cardiovascular and cerebrovascular diseases, patients usually use intravenous injection in the acute onset period. Therefore, the water solubility and dissolution rate of the product are very important, which will directly affect the choice of doctors and patients in clinical use, which in turn affects the drug sales and marketing.
溶解度测定的方法参照《中国药典》(2020版四部)中所描述的方法(为了将其开发成静脉注射制剂，仅研究其在水中的溶解度)，如下：The method of solubility determination refers to the method described in the "Chinese Pharmacopoeia" (2020 edition four parts) (in order to develop it into an intravenous preparation, only its solubility in water is studied), as follows:
将上述几种样品，分别粉碎，过5号药典筛。分别称取样品，加入不同体 积的纯水，按照药典所描述的实验方法及判断标准(称取研成细粉的供试品，于25℃±2℃一定容量的溶剂中，每隔5分钟强力振摇30秒；观察30分钟内的溶解情况，如无目视可见的溶质颗粒时，即视为完全溶解)，溶解性能分为极易溶解、易溶、溶解、略溶、微溶、极微溶解。记述几种样品的溶解性，如下：The above samples were pulverized and passed through a 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 ground into fine powder, in a solvent of a certain capacity at 25 ℃ ± 2 ℃, every 5 minutes Shake vigorously for 30 seconds; observe the dissolution within 30 minutes, if there are no visible solute particles, it is regarded as completely dissolved), the dissolution performance is divided into extremely soluble, easily soluble, soluble, slightly soluble, slightly soluble, Very slightly soluble. Describe the solubility of several samples as follows:
丹酚酸A冻干粉：Salvianolic acid A freeze-dried powder:
“极易溶解”判断：0.12g样品于0.1mL水中，30min不溶；Judgment of "extremely soluble": 0.12g of sample is insoluble in 0.1mL of water for 30min;
“易溶”判断：0.12g样品于1mL水中，5min溶解；"Easy-soluble" judgment: 0.12g of sample is dissolved in 1mL of water in 5min;
“溶解”判断：0.12g样品于3mL水中，瞬间溶解(≤10s)。Judgment of "dissolution": 0.12g of sample dissolved in 3mL of water instantly (≤10s).
结论及讨论：丹酚酸冻干粉的水溶解性为“易溶”。0.12g、Conclusion and discussion: The water solubility of salvianolic acid freeze-dried powder is "easy to dissolve". 0.12g,
丹酚酸A结晶：Salvianolic acid A crystal:
“极易溶解”判断：0.12g结晶于0.1mL水中，30min不溶；Judgment of "extremely soluble": 0.12g of crystals are insoluble in 0.1mL of water for 30min;
“易溶”判断：0.12g结晶于1mL水中，30min不溶；"Easy to dissolve" judgment: 0.12g of crystals in 1mL of water, insoluble in 30min;
“溶解”判断：0.12g结晶于3mL水中，5min不溶，10min不溶，15min不溶，20min不溶，25min不溶，30min溶解。Judgment of "dissolving": 0.12 g of crystals in 3 mL of water, insoluble in 5 minutes, insoluble in 10 minutes, insoluble in 15 minutes, insoluble in 20 minutes, insoluble in 25 minutes, and dissolved in 30 minutes.
结论及讨论：丹酚酸A结晶的水溶解性为“溶解”。结合临床使用情况，0.12g样品于3mL水中25min溶解，难以满足临床需求。Conclusion and discussion: The water solubility of salvianolic acid A crystal is "dissolved". Combined with clinical use, 0.12g of sample was dissolved in 3mL of water for 25min, which was difficult to meet clinical needs.
丹酚酸A钠盐水合物：Salvianolic acid A sodium salt hydrate:
“极易溶解”判断：0.12g结晶于0.1mL水中，30min不溶；Judgment of "extremely soluble": 0.12g of crystals are insoluble in 0.1mL of water for 30min;
“易溶”判断：0.12g结晶于1mL水中，5min溶解；"Easy-soluble" judgment: 0.12g of crystals are dissolved in 1mL of water in 5min;
“溶解”判断：0.12g结晶于3mL水中，瞬间溶解(≤10s)。Judgment of "dissolution": 0.12g of crystals dissolved in 3mL of water instantly (≤10s).
结论及讨论：丹酚酸A钠盐水合物的水溶解性为“易溶”。结合临床使用情况，0.12g药品于3mL水溶瞬间溶解，能够满足其临床需求。Conclusion and discussion: The water solubility of salvianolic acid A sodium salt hydrate is "easy to dissolve". Combined with clinical use, 0.12g of the drug dissolves instantly in 3mL of water, which can meet its clinical needs.
丹酚酸A钾盐水合物：Salvianolic Acid A Potassium Salt Hydrate:
“极易溶解”判断：0.12g结晶于0.1mL水中，30min不溶；Judgment of "extremely soluble": 0.12g of crystals are insoluble in 0.1mL of water for 30min;
“易溶”判断：0.12g结晶于1mL水中，5min不溶，10min不溶，15min不溶，20min溶解；"Easy-soluble" judgment: 0.12g of crystals in 1mL of water, insoluble in 5min, insoluble in 10min, insoluble in 15min, and dissolved in 20min;
“溶解”判断：0.12g结晶于3mL水中，5min溶解。"Dissolved" judgment: 0.12 g of crystals were dissolved in 3 mL of water in 5 min.
结论及讨论：丹酚酸A钾盐水合物的水溶解性为“易溶”。结合临床使用情况，0.12g药品于3mL需5min才能溶解，基本能够满足其临床需求。Conclusion and discussion: The water solubility of salvianolic acid A potassium salt hydrate is "easy soluble". Combined with clinical use, it takes 5 minutes for 0.12g of drug to dissolve in 3mL, which can basically meet its clinical needs.
丹酚酸A钙盐水合物：Salvianolic acid A calcium salt hydrate:
“极易溶解”判断：0.12g结晶于0.1mL水中，30min不溶；Judgment of "extremely soluble": 0.12g of crystals are insoluble in 0.1mL of water for 30min;
“易溶”判断：0.12g结晶于1mL水中，30min不溶；"Easy to dissolve" judgment: 0.12g of crystals in 1mL of water, insoluble in 30min;
“溶解”判断：0.12g结晶于3mL水中，5min不溶，10min不溶，15min不溶，20min溶解。Judgment of "dissolving": 0.12 g of crystals in 3 mL of water, insoluble in 5 minutes, insoluble in 10 minutes, insoluble in 15 minutes, and dissolved in 20 minutes.
结论及讨论：丹酚酸A钙盐水合物的水溶解性为“溶解”。结合临床使用情况，0.12g样品于3mL水中20min溶解，基本能够满足临床需求。Conclusion and discussion: The water solubility of salvianolic acid A calcium salt hydrate is "dissolved". Combined with clinical use, 0.12g of sample was dissolved in 3mL of water for 20min, which basically met clinical needs.
丹酚酸A镁盐水合物：Salvianolic Acid A Magnesium Salt Hydrate:
“极易溶解”判断：0.12g结晶于0.1mL水中，30min不溶；Judgment of "extremely soluble": 0.12g of crystals are insoluble in 0.1mL of water for 30min;
“易溶”判断：0.12g结晶于1mL水中，5min溶解；"Easy-soluble" judgment: 0.12g of crystals are dissolved in 1mL of water in 5min;
“溶解”判断：0.12g结晶于3mL水中，瞬间溶解(≤10s)。Judgment of "dissolution": 0.12g of crystals dissolved in 3mL of water instantly (≤10s).
结论及讨论：丹酚酸A镁盐水合物的水溶解性为“易溶”。结合临床使用情况，0.12g药品于3mL水溶瞬间溶解，能够满足其临床需求。Conclusion and discussion: The water solubility of salvianolic acid A magnesium salt hydrate is "easy to dissolve". Combined with clinical use, 0.12g of the drug dissolves instantly in 3mL of water, which can meet its clinical needs.
丹酚酸A铵盐水合物：Salvianolic acid A ammonium salt hydrate:
“极易溶解”判断：0.12g结晶于0.1mL水中，30min不溶；Judgment of "extremely soluble": 0.12g of crystals are insoluble in 0.1mL of water for 30min;
“易溶”判断：0.12g结晶于1mL水中，5min不溶，10min不溶，15min溶解；"Easy to dissolve" judgment: 0.12g of crystals in 1mL of water, insoluble in 5min, insoluble in 10min, and dissolved in 15min;
“溶解”判断：0.12g结晶于3mL水中，5min溶解。"Dissolved" judgment: 0.12 g of crystals were dissolved in 3 mL of water in 5 min.
结论及讨论：丹酚酸A铵盐水合物的水溶解性为“易溶”。结合临床使用情况，0.12g药品于3mL需5min才能溶解，基本能够满足其临床需求。Conclusion and discussion: The water solubility of salvianolic acid A ammonium salt hydrate is "easy to dissolve". Combined with clinical use, it takes 5 minutes for 0.12g of drug to dissolve in 3mL, which can basically meet its clinical needs.
根据初步的药效学试验，丹酚酸A的临床应用制剂量约50-100mg/支。结合上述溶解性试验，单纯从溶解性方面考虑，丹酚酸A钠盐水合物与丹酚酸A镁盐水合物完全能够满足临床需求。丹酚酸A钾盐水合物、丹酚酸A铵盐水合物、丹酚酸A钙盐水合物基本能够满足临床需求。而丹酚酸A结晶直接使用时难以满足其作为静脉注射制剂的临床需求。According to the preliminary pharmacodynamics test, the clinical dosage of salvianolic acid A is about 50-100 mg/piece. Combined with the above solubility test, from the perspective of solubility alone, salvianolic acid A sodium salt hydrate and salvianolic acid A magnesium salt hydrate can fully meet the clinical needs. Salvianolic acid A potassium salt hydrate, salvianolic acid A ammonium salt hydrate, and salvianolic acid A calcium salt hydrate can basically meet the clinical needs. However, when salvianolic acid A crystal is used directly, it is difficult to meet its clinical needs as an intravenous preparation.
实施例9 丹酚酸A盐水合物与阳性药物在缺血性脑中风模型动物中对大脑梗死面积的影响Example 9 Effects of salvianolic acid A salt hydrate and positive drugs on cerebral infarct size in ischemic stroke model animals
本实施例中，选择市场上的畅销药物作为阳性药，对比丹酚酸A盐水合物结晶与阳性药物对脑中风模型动物大脑梗死面积的影响。阳性药物选择丁苯酞氯化钠注射液和注射用丹参多酚酸。其中前者为《中国缺血性脑卒中诊疗指南》(2018版)中推荐的缺血性脑卒中治疗药物，后者为以丹酚酸B为代表的丹参水溶性酚酸类化合物注射制剂，文献报道其亦具有较好的活性。In this example, the best-selling drug on the market was selected as the positive drug, and the effect of salvianolic acid A salt hydrate crystal and the positive drug on the cerebral infarct size of the stroke model animal was compared. The positive drugs were butylphthalide and sodium chloride injection and salvianolic acid for injection. The former is the drug for ischemic stroke recommended in the "Guidelines for Diagnosis and Treatment of Ischemic Stroke in China" (2018 Edition), and the latter is the injection preparation of water-soluble phenolic compounds of Salvia miltiorrhiza represented by salvianolic acid B. References It is also reported to have good activity.
本试验选用SD大鼠，构建缺血性脑卒中试验动物模型，方法如下：SD rats were used in this experiment to construct an experimental animal model of ischemic stroke. The methods are as follows:
缺血不灌注模型：大鼠中动脉栓塞-电凝法Ischemia-nonperfusion model: middle artery embolization-coagulation in rats
SD大鼠称重，用15％的水合氯醛300mg/kg腹腔注射麻醉，左侧侧卧固定在大鼠手术台，左侧颞顶部及面部剃毛，用75％乙醇消毒后在左眼和左耳之间，切开皮肤，钝性分离颞肌和咬肌，暴露颞骨翼板，于手术显微镜下，在颞骨和颞鳞骨结合靠近口侧1mm处用颅骨钻研磨出2mm×2mm的骨窗，用撬棒撬开颅骨。此时，透过硬脑膜即可见一条较直且分支较少的血管，即为大脑中动脉。用双极电凝镊在嗅束内1mm至大脑下静脉之间处烧灼，将血流彻底阻断后止血，依次缝合颞肌和皮肤，然后尾静脉注射给药。待大鼠苏醒后，放回笼中继续饲养24h。SD rats were weighed and anesthetized by intraperitoneal injection of 15% chloral hydrate 300 mg/kg. The left side was fixed on the operating table of the rat, and the left temporal top and face were shaved. After disinfection with 75% ethanol, the left eye and Between the left ear, the skin was incised, the temporalis muscle and masseter muscle were bluntly separated, and the temporal bone wing plate was exposed. Under the operating microscope, a 2mm×2mm bone was ground with a skull drill at the junction of the temporal bone and the temporal squamous bone 1mm near the oral side. window, use a spudger to pry open the skull. At this point, a straighter, less branched blood vessel, the middle cerebral artery, is visible through the dura mater. Use bipolar coagulation forceps to cauterize between 1 mm in the olfactory tract and the inferior cerebral vein, completely block the blood flow and stop the bleeding, suture the temporalis muscle and the skin in turn, and then inject the drug into the tail vein. After the rats were awake, they were put back into the cage to continue feeding for 24 hours.
缺血再灌注模型：大鼠中动脉栓塞-线栓法Ischemia-reperfusion model: middle artery embolization-suture method in rats
大鼠称重后用15％水合氯醛300mg/kg腹腔注射麻醉，仰卧固定在大鼠手术台上，颈部剃毛，75％乙醇消毒，手术剪纵向剪开皮肤，手术分离暴露一侧颈总动脉，沿颈总动脉上行分离并结扎颈外动脉和翼突颚动脉，在颈总动脉的近 心端放置动脉夹阻断血流，于远心端用眼科手术剪剪一个小口，插入栓线，缓缓推入栓线(约20mm)至前脑动脉再往回抽出约2mm即至大脑中动脉口，结扎栓线和血管，缝合皮肤。脑组织缺血后，尾静脉注射给药一次，放回笼中饲养。After weighing, the rats were anesthetized by intraperitoneal injection of 15% chloral hydrate 300 mg/kg, supine and fixed on the rat operating table, the neck was shaved, sterilized with 75% ethanol, the skin was cut longitudinally with surgical scissors, and the side of the neck was exposed by surgical separation. The common artery was separated and ligated along the common carotid artery, and the external carotid artery and pterygo-maxillary artery were ligated. An arterial clip was placed at the proximal end of the common carotid artery to block the blood flow. A small opening was cut with ophthalmic scissors at the distal end, and a plug was inserted. Line, slowly push the suture (about 20mm) to the anterior cerebral artery and then withdraw about 2mm back to the mouth of the middle cerebral artery, ligate the suture and blood vessels, and suture the skin. After cerebral ischemia, the mice were administered by tail vein injection once, and then they were put back into the cage.
丹酚酸A钠盐水合物及阳性药物在缺血性脑中风模型动物中对大脑梗死面积的影响结果见表2。The effects of salvianolic acid A sodium salt hydrate and positive drugs on cerebral infarct size in ischemic stroke model animals are shown in Table 2.
表2 本发明所得到的丹酚酸A钠盐水合物与阳性药物在缺血性脑中风模型动物中对脑梗死面积的影响Table 2 The effect of the salvianolic acid A sodium salt hydrate obtained by the present invention and the positive drug on the cerebral infarct size in ischemic stroke model animals
Figure PCTCN2022091078-appb-000033
Figure PCTCN2022091078-appb-000033
*p<0.05，**p<0.01，***p<0.001vs溶媒对照组*p<0.05, **p<0.01, ***p<0.001 vs vehicle control
丁苯酞是公认的治疗缺血性脑卒中的有效药物，被《中国缺血性脑卒中诊治指南》(2018版)所推荐，2020年的销售额超过60亿元人民币。结果显示，丹酚酸A钠盐水合物和阳性药物丁苯酞，在缺血再灌注和缺血不灌注模型中，相比较不用药的“溶媒对照组”都可以显著降低模型动物的脑梗死面积。Butylphthalide is recognized as an effective drug for the treatment of ischemic stroke. It is recommended by the "Guidelines for the Diagnosis and Treatment of Ischemic Stroke in China" (2018 edition), and its sales in 2020 will exceed 6 billion yuan. The results show that salvianolic acid A sodium salt hydrate and positive drug butylphthalide can significantly reduce cerebral infarction in model animals in ischemia-reperfusion and ischemia-nonperfusion models compared with the "vehicle control group" without drugs. area.
其中，丹酚酸A钠盐水合物在剂量为10mg/kg时，达到最大治疗效果(治疗天花板)，而丁苯酞在3mg/kg剂量时，达到最大治疗效果(治疗天花板)。且无论是在缺血再灌注还是缺血不灌注模型，丹酚酸A钠盐水合物的最大疗效，皆显著高于丁苯酞。Among them, salvianolic acid A sodium salt hydrate reached the maximum therapeutic effect (therapeutic ceiling) at a dose of 10 mg/kg, while butylphthalide reached the maximum therapeutic effect (therapeutic ceiling) at a dose of 3 mg/kg. And whether in ischemia-reperfusion or ischemia-nonperfusion models, the maximum efficacy of salvianolic acid A sodium salt hydrate was significantly higher than that of butylphthalide.
该结果表明，在低于3mg/kg剂量时，丁苯酞的效价强度高于丹酚酸A钠盐水合物，但其最大治疗效果显著低于后者。在实际应用中，最大疗效的意义显著大于效价强度。The results showed that the titer intensity of butylphthalide was higher than that of salvianolic acid A sodium salt hydrate at a dose lower than 3 mg/kg, but its maximum therapeutic effect was significantly lower than the latter. In practical applications, the significance of maximal efficacy is significantly greater than potency intensity.
综上，丹酚酸A钠盐水合物用于治疗缺血性脑卒中，无论是缺血再灌注模型还是缺血不灌注模型，都显著好于不用药对照组，也显著好于阳性药物丁苯 酞。In conclusion, salvianolic acid A sodium salt hydrate for the treatment of ischemic stroke, whether in ischemia-reperfusion model or ischemia-nonperfusion model, is significantly better than the control group without medication, and also significantly better than the positive drug D. phthalide.
实施例10 丹酚酸A钠盐水合物与阳性药物在缺血性心脏病模型动物中对心肌梗死面积的影响Example 10 Effects of salvianolic acid A sodium salt hydrate and positive drugs on myocardial infarction size in ischemic heart disease model animals
本实施例中，选择市场上的畅销药物作为阳性药，对比丹酚酸A盐水合物结晶与阳性药物对缺血性心脏病模型动物心肌梗死面积的影响。In this example, the best-selling drug on the market is selected as the positive drug, and the effect of salvianolic acid A salt hydrate crystal and the positive drug on the myocardial infarction area of the ischemic heart disease model animal is compared.
阳性药物选择注射用丹参多酚酸盐(绿谷制药)，其主要成分为丹酚酸B镁盐为主的(含量85％)丹参水溶性酚酸类化合物，是目前市场上最畅销的心肌缺血药物之一，有大量的文献报道其活性。The positive drug selects Salvia miltiorrhiza polyphenolate for injection (Lvgu Pharmaceutical), whose main component is salvianolic acid B magnesium salt (content 85%) water-soluble phenolic compounds of Salvia miltiorrhiza, which is currently the best-selling cardiac muscle in the market. One of the ischemic drugs, there are a large number of literatures reporting its activity.
本试验选用SD大鼠，其模型构建方法如下：SD rats were used in this experiment, and the model construction method was as follows:
缺血不灌注模型：Ischemia-nonperfusion model:
大鼠禁食12h，称重后用15％水合氯醛300mg/kg腹腔注射麻醉。左侧胸壁剃毛消毒后于胸骨左侧3-4根肋骨间横切口切开皮肤，用止血钳钝性分离各肌层并刺破胸膜，再用两把止血钳横行夹住胸骨，在两钳之间剪断胸骨，插入牵引器撑开肋骨，剪开心包膜暴露心脏，在前室间沟可见心大静脉，以此为标志，在左心耳下离根部约2-3mm用2×6带线圆针6-0号医用棉纶单丝线绕过动脉深面，在肺动脉圆锥旁沟出针，深约1mm，宽约2mm。把丝线两端从一直径2mm聚乙烯管中传出备用。稳定10min后，把一棉签棒插入PE管，拉紧丝线用棉签棒将PE管向前推，直至阻断冠状动脉血流，完成缺血模型构建。The rats were fasted for 12 hours, and then anesthetized by intraperitoneal injection of 15% chloral hydrate 300 mg/kg after weighing. After shaving and sterilizing the left chest wall, a transverse incision was made between the 3-4 ribs on the left side of the sternum. The skin was bluntly separated with a hemostat and the pleura was punctured. Then two hemostats were used to clamp the sternum horizontally. Cut the sternum between the forceps, insert a retractor to spread the ribs, cut the pericardium to expose the heart, the great cardiac vein can be seen in the anterior interventricular groove, and use this as a sign, and use a 2×6 belt about 2-3mm from the root under the left atrial appendage. Thread round needle 6-0 medical cotton monofilament thread bypasses the deep surface of the artery and exits the needle in the groove next to the conus pulmonary artery, with a depth of about 1mm and a width of about 2mm. The two ends of the silk thread were passed out from a polyethylene tube with a diameter of 2 mm for use. After stabilizing for 10 minutes, insert a cotton swab into the PE tube, tighten the silk thread and push the PE tube forward with the cotton swab until the coronary blood flow is blocked, and the ischemia model is constructed.
心肌缺血后，大鼠尾静脉给药一次，24h处死大鼠，检测其心肌梗死面积。After myocardial ischemia, the rats were given one administration to the tail vein, and the rats were sacrificed 24 hours later, and the size of myocardial infarction was detected.
缺血再灌注模型：Ischemia-reperfusion model:
缺血再灌注模型的构建，前期同缺血不灌注模型，心肌缺血后，将棉签棒从PE管中拔出，即可实现再灌注。再灌注后，大鼠静脉给药一次，24h后处死大鼠，检测其心肌梗死面积。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 is pulled out from the PE tube to achieve reperfusion. After reperfusion, the rats were intravenously administered once, and the rats were sacrificed 24 hours later to detect the myocardial infarction area.
丹酚酸A钠盐水合物及阳性药物在缺血性心脏病模型动物中对心肌梗死面积的影响结果见表3。The effects of salvianolic acid A sodium salt hydrate and positive drugs on myocardial infarction size in ischemic heart disease model animals are shown in Table 3.
表3 本发明所得到的丹酚酸A钠盐水合物与阳性药物在缺血性心脏病模型动物中对心肌梗死面积大小的影响Table 3 The effect of salvianolic acid A sodium salt hydrate and positive drug obtained by the present invention on myocardial infarction size in ischemic heart disease model animals
Figure PCTCN2022091078-appb-000034
Figure PCTCN2022091078-appb-000034
Figure PCTCN2022091078-appb-000035
Figure PCTCN2022091078-appb-000035
*p<0.05，**p<0.01，***p<0.001vs溶媒对照组*p<0.05, **p<0.01, ***p<0.001 vs vehicle control
丹参多酚酸盐是同样来源于丹参的酚酸类化合物，是一种有效的缺血性心血管疾病治疗药物。结果显示，丹酚酸A钠盐水合物、丹参多酚酸盐，在缺血再灌注模型和缺血不灌注模型中，与不用药的“溶媒对照组”相比，皆能显著降低试验动物的心肌梗死面积。Salvia polyphenolate is a phenolic compound derived from Salvia miltiorrhiza, and it is an effective drug for the treatment of ischemic cardiovascular disease. The results showed that salvianolic acid A sodium salt hydrate and salvianolic acid salt in the ischemia-reperfusion model and ischemia-nonperfusion model, compared with the "vehicle control group" without medication, can significantly reduce the experimental animals. myocardial infarction size.
其中丹酚酸A钠盐水合物的最大效应剂量皆为为10mg/kg，丹参多酚酸盐的最大效应剂量为40mg/kg。前者的最大治疗效果(疗效天花板)显著好于后者。Among them, the maximum effect dose of salvianolic acid A sodium salt hydrate is 10mg/kg, and the maximum effect dose of salvianolic acid salt is 40mg/kg. The former's maximum healing effect (efficacy ceiling) is significantly better than the latter.
实施例11 丹酚酸A钠盐水合物与阳性药物对动物体内血栓形成的影响Example 11 Effects of salvianolic acid A sodium salt hydrate and positive drugs on thrombosis in animals
本实施例中，选择市场上的畅销药物作为阳性药，对比丹酚酸A盐水合物结晶与阳性药物对动物体内血栓形成的影响。In this example, the best-selling drug on the market was selected as the positive drug, and the effects of salvianolic acid A salt hydrate crystals and the positive drug on thrombosis in animals were compared.
阳性药物选择丁苯酞氯化钠注射液(石药集团)。The positive drug was selected as Butylphthalide Sodium Chloride Injection (Shi Pharmaceutical Group).
本实施例选用SD大鼠作为试验动物，具体试验方法如下：The present embodiment selects SD rats as experimental animals, and the concrete test method is as follows:
实验前量取6cm长的4号手术丝线若干，将所有丝线称量后求出单根丝线的重量。准备长约10cm的硅胶管，管内放置一根长6cm的4号手术丝线，硅胶管两端连接长8cm的聚乙烯塑料管，结扎固定后向管内充满50u/ml肝素盐水，备用。Before the experiment, a number of No. 4 surgical silk threads with a length of 6 cm were measured, and the weight of a single silk thread was obtained after weighing all the threads. Prepare a silicone tube with a length of about 10 cm, place a No. 4 surgical wire with a length of 6 cm in the tube, and connect both ends of the silicone tube to a polyethylene plastic tube with a length of 8 cm. After ligation and fixation, the tube is filled with 50u/ml heparin saline for use.
大鼠随机分为5组，每组10只。大鼠称重后用15％水合氯醛300mg/kg腹腔注射麻醉，麻醉后根据体重尾静脉注射给药，给药体积为1ml/kg。Rats were randomly divided into 5 groups with 10 rats in each group. After weighing, the rats were anesthetized by intraperitoneal injection of 15% chloral hydrate 300 mg/kg. After anesthesia, the rats were administered by tail vein injection according to their body weight, and the administration volume was 1 ml/kg.
给药10min后开始手术，大鼠仰卧位固定于鼠板上，牵出舌头以防发生舌后坠引起窒息。颈部剃毛，用75％酒精消毒，纵向剪开颈部皮肤，分离右侧颈总动脉和左侧颈外静脉，将备制好的聚乙烯管一端先插入左侧颈外静脉，另一端插入右侧颈总动脉，打开动脉夹，血液从右侧颈总动脉流经管道返回左侧颈外静脉。开放15min后阻断血流，迅速取出丝线称重，总重量减去丝线干重量即是血栓湿重。After 10 minutes of administration, the operation was started. The rat was fixed on the mouse board in a supine position, and the tongue was pulled out to prevent suffocation caused by falling back of the tongue. The neck was shaved, disinfected with 75% alcohol, the neck skin was cut longitudinally, the right common carotid artery and the left external jugular vein were separated, and one end of the prepared polyethylene tube was inserted into the left external jugular vein first, and the other end was inserted into the left external jugular vein. The right common carotid artery is inserted, the arterial clip is opened, and blood flows from the right common carotid artery through the tube back to the left external jugular vein. After 15 minutes of opening, the blood flow was blocked, and the silk thread was quickly taken out and weighed. The total weight minus the dry weight of the silk thread was the wet weight of the thrombus.
丹酚酸A钠盐水合物与阳性药物对动物体内血栓形成的影响结果见表4。The effects of salvianolic acid A sodium salt hydrate and positive drugs on thrombosis in animals are shown in Table 4.
表4 本发明所得到的丹酚酸A钠盐水合物与阳性药物对动物体内血栓形成的影响Table 4 Influence of salvianolic acid A sodium salt hydrate obtained by the present invention and positive drugs on thrombosis in animals
Figure PCTCN2022091078-appb-000036
Figure PCTCN2022091078-appb-000036
*p<0.05，**p<0.01，***p<0.001vs溶媒对照组*p<0.05, **p<0.01, ***p<0.001 vs vehicle control
#p<0.05，##p<0.01，###p<0.001丹酚酸A钠盐水合物组vs丁苯酞组#p<0.05, ##p<0.01, ###p<0.001 Salvianolic acid A sodium salt hydrate group vs butylphthalide group
结果显示，相比较不用药的“溶媒对照组”，丹酚酸A钠盐水合物和阳性 药物丁苯酞，皆可以显著抑制血栓形成，具体表现在血栓湿重显著降低，且呈现一定的剂量效应关系。The results showed that, compared with the "vehicle control group" without medication, both salvianolic acid A sodium salt hydrate and the positive drug butylphthalide could significantly inhibit thrombus formation. effect relationship.
相比之下，丹酚酸A钠盐水合物抑制血栓形成的能力，好于阳性药物丁苯酞，这与其降低心肌梗死面积和脑梗死面积方面的效果是一致的。In contrast, the ability of salvianolic acid A sodium salt hydrate to inhibit thrombosis is better than that of the positive drug butylphthalide, which is consistent with its effect on reducing myocardial infarct size and cerebral infarction size.
实施例12 丹酚酸A钠盐水合物与阳性药物对缺血性脑中风和缺血性心脏病模型动物血液中炎症因子的影响Example 12 Effects of salvianolic acid A sodium salt hydrate and positive drugs on inflammatory factors in the blood of ischemic stroke and ischemic heart disease model animals
本实施例中，选择市场上的畅销药物作为阳性药，对比丹酚酸A盐水合物结晶与阳性药物对缺血性脑中风和缺血性心脏病模型动物血液炎症因子的影响。In this example, the best-selling drug on the market was selected as the positive drug, and the effects of salvianolic acid A salt hydrate crystal and the positive drug on blood inflammatory factors in ischemic stroke and ischemic heart disease model animals were compared.
缺血性心脏病模型阳性药物选择注射用丹参多酚酸盐(绿谷制药)和缺血性脑中风模型阳性药物选择丁苯酞氯化钠注射液(石药集团)。The positive drug for the ischemic heart disease model was selected as salvianolate for injection (Lvgu Pharmaceutical) and the positive drug for the ischemic stroke model was selected as butylphthalide and sodium chloride injection (Shi Pharmaceutical Group).
本实施例选用SD大鼠作为试验动物，具体试验方法分别参照实施例13、14中“缺血再灌注模型”的构建方法。造模完成并相应给予药物(或对照)60min后，经股动脉采血3ml，置离心机3000转速，离心10min留取血清，-80℃保存待测MDA、SOD、IL-1β和TNF-α。In this example, SD rats were selected as the test animals, and the specific test methods were referred to the construction methods of "ischemia-reperfusion model" in Examples 13 and 14, respectively. After the modeling was completed and drugs (or control) were administered for 60 min, 3 ml of blood was collected from the femoral artery, centrifuged at 3000 revolutions for 10 min, and the serum was collected.
丹酚酸A盐水合物结晶与阳性药物对动物血液炎症因子的影响结果见表5、表6。The effects of salvianolic acid A salt hydrate crystals and positive drugs on animal blood inflammatory factors are shown in Table 5 and Table 6.
表5 本发明所得到的丹酚酸A钠盐水合物与阳性药物对缺血性脑中风(缺血再灌注)模型动物血液中炎症因子的影响Table 5 Effects of salvianolic acid A sodium salt hydrate and positive drugs obtained by the present invention on inflammatory factors in the blood of ischemic cerebral apoplexy (ischemia-reperfusion) model animals
Figure PCTCN2022091078-appb-000037
Figure PCTCN2022091078-appb-000037
*p<0.05，**p<0.01vs溶媒对照组*p<0.05, **p<0.01 vs vehicle control
结果表明，丹酚酸A钠盐水合物，能够显著降低脑缺血大鼠血液中的炎症因子含量，增加SOD活性，效果好于不用药组和阳性药物丁苯酞组，这可能是其产生治疗作用的原因之一。The results show that salvianolic acid A sodium salt hydrate can significantly reduce the content of inflammatory factors in the blood of cerebral ischemia rats and increase the activity of SOD, and the effect is better than that of the non-medicated group and the positive drug butylphthalide group. One of the reasons for the therapeutic effect.
表6 本发明所得到的丹酚酸A钠盐水合物与阳性药物对缺血性心脏病(缺血再灌注)模型动物血液中炎症因子的影响Table 6 Effects of salvianolic acid A sodium salt hydrate and positive drugs obtained by the present invention on inflammatory factors in the blood of ischemic heart disease (ischemia-reperfusion) model animals
Figure PCTCN2022091078-appb-000038
Figure PCTCN2022091078-appb-000038
Figure PCTCN2022091078-appb-000039
Figure PCTCN2022091078-appb-000039
*p<0.05，**p<0.01，***p<0.001vs溶媒对照组*p<0.05, **p<0.01, ***p<0.001 vs vehicle control
结果表明，丹酚酸A钠盐水合物和阳性药物丹参多酚酸盐，都可以降低心肌缺血模型动物血液中的炎症因子浓度，增加SOD活性，其中丹酚酸A钠盐水合物的效果较丹参多酚酸盐更好。这可能是它们产生治疗效果的原因之一。The results showed that both salvianolic acid A sodium salt hydrate and the positive drug salvianolic acid salt can reduce the concentration of inflammatory factors in the blood of myocardial ischemia model animals and increase the activity of SOD, among which the effect of salvianolic acid A sodium salt hydrate Better than salvia polyphenolate. This may be one of the reasons for their therapeutic effect.
实施例13 制备丹酚酸A钠盐水合物结晶(探索碱性钠化合物加入量)Example 13 Preparation of salvianolic acid A sodium salt hydrate crystal (exploring the amount of basic sodium compound added)
将纯度≥80％的丹酚酸A溶液浓缩(内含16g丹酚酸A)，至浓度20％，水浴50℃，缓慢加入碳酸钠粉末约343mg，待两者反应完全后(反应约15分钟，可使其中20％的丹酚酸A反应生成丹酚酸A钠盐)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，不断搅拌24h，得丹酚酸A钠盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing 16 g of salvianolic acid A) to a concentration of 20%, take a water bath at 50°C, slowly add about 343 mg of sodium carbonate powder, and wait for the two to react completely (reaction for about 15 minutes). , 20% of the salvianolic acid A can be reacted to generate salvianolic acid A sodium salt), the solution is slowly cooled to 20 °C, the cooling rate is 0.2 °C/min, and the stirring is continued, and then further cooled to 5 °C, continuously Stir for 24h to obtain salvianolic acid A sodium salt hydrate (precipitation).
过滤沉淀，加入水50℃溶解，配置成浓度20％的溶液(以丹酚酸A计)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌并等待24h，得丹酚酸A钠盐水合物(重结晶产物)。Filter the precipitation, add water at 50°C to dissolve, configure into a solution with a concentration of 20% (calculated as salvianolic acid A), slowly cool the solution to 20°C, and the cooling rate is 0.2°C/min. 5 ℃, stirring and waiting for 24h to obtain salvianolic acid A sodium salt hydrate (recrystallized product).
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A钠盐水合物结晶”7.112g，以丹酚酸A计，其HPLC纯度99.5％。Filter the precipitate, spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize, and dry at 25°C for 12h to obtain the product "salvianolic acid A sodium salt hydrate crystal" 7.112 g, in terms of salvianolic acid A, its HPLC purity is 99.5%.
采用类似的方法，更改加入碳酸钠粉末的量1373mg(反应约20分钟，可使其中80％的丹酚酸A反应生成丹酚酸A钠盐)。最终可得产品“丹酚酸A钠盐水合物结晶”4.256g，以丹酚酸A计，其HPLC纯度为99.5％。Using a similar method, the amount of sodium carbonate powder added was changed to 1373 mg (reaction for about 20 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A sodium salt). The final product "salvianolic acid A sodium salt hydrate crystal" was 4.256 g, and its HPLC purity was 99.5% in terms of salvianolic acid A.
综合本实施例结果，加入碱性钠化合物的量，使其中20-80％丹酚酸A反应完毕，都可以制备纯度高达99.5％的丹酚酸A钠盐水合物结晶，差别在于其得率略有不同。Based on the results of this example, adding the amount of basic sodium compound so that 20-80% of salvianolic acid A has been reacted, salvianolic acid A sodium salt hydrate crystals with a purity of up to 99.5% can be prepared. The difference lies in the yield. Slightly different.
另外，可以看到，采用的碱性钠化合物，实际提供的是钠离子，只要能保证该碱性钠化合物与丹酚酸A能够起反应即可。因此，采用的碱性钠化合物，除了本发明中使用的碳酸钠外，氢氧化钠、碳酸氢钠、苹果酸钠、磷酸钠、磷酸二氢钠、磷酸氢二钠、乙酸钠等弱酸与钠形成的强碱弱酸盐，都能够用于制备本发明中所述的丹酚酸A钠盐水合物结晶。In addition, it can be seen that the basic sodium compound used actually provides sodium ions, as long as it can be ensured that the basic sodium compound and salvianolic acid A can react. Therefore, the basic sodium compound adopted, in addition to the sodium carbonate used in the present invention, weak acids such as sodium hydroxide, sodium bicarbonate, sodium malate, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium acetate and sodium The formed strong base and weak acid salt can be used to prepare the salvianolic acid A sodium salt hydrate crystal described in the present invention.
实施例14 制备丹酚酸A钾盐水合物结晶(探索碱性钾化合物加入量)Example 14 Preparation of salvianolic acid A potassium salt hydrate crystal (exploring the amount of basic potassium compound added)
将纯度≥80％的丹酚酸A溶液浓缩(内含16g丹酚酸A)，至浓度20％，水浴50℃，缓慢加入碳酸钾粉末约447mg，待两者反应完全后(反应约25分钟， 可使其中20％的丹酚酸A反应生成丹酚酸A钾盐)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，不断搅拌24h，得丹酚酸A钾盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing 16 g of salvianolic acid A) to a concentration of 20%, take a water bath at 50°C, slowly add about 447 mg of potassium carbonate powder, and wait for the two to react completely (reaction for about 25 minutes). , 20% of salvianolic acid A can be reacted to generate salvianolic acid A potassium salt), the solution is slowly cooled to 20°C, the cooling rate is 0.2°C/min, during which stirring is continued, and then further cooled to 5°C, continuously Stir for 24h to obtain salvianolic acid A potassium salt hydrate (precipitation).
过滤沉淀，加入水50℃溶解，配置成浓度20％的溶液(以丹酚酸A计)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌并等待24h，得丹酚酸A钾盐水合物(重结晶产物)。Filter the precipitation, add water at 50°C to dissolve, configure into a solution with a concentration of 20% (calculated as salvianolic acid A), slowly cool the solution to 20°C, and the cooling rate is 0.2°C/min. 5 ℃, stirring and waiting for 24h to obtain salvianolic acid A potassium salt hydrate (recrystallized product).
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A钾盐水合物结晶”5.508g，以丹酚酸A计，其HPLC纯度99.6％。Filter the precipitate, spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize, and dry at 25°C for 12h to obtain the product "salvianolic acid A potassium salt hydrate crystal" 5.508 g, in terms of salvianolic acid A, its HPLC purity is 99.6%.
采用类似的方法，更改加入碳酸钾粉末的量1788mg(反应约20分钟，可使其中80％的丹酚酸A反应生成丹酚酸A钾盐)。最终可得产品“丹酚酸A钾盐水合物结晶”3.221g，以丹酚酸A计，其HPLC纯度为99.5％。Using a similar method, the amount of potassium carbonate powder added was changed to 1788 mg (reaction for about 20 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A potassium salt). The final product "salvianolic acid A potassium salt hydrate crystal" was 3.221 g, and its HPLC purity was 99.5% based on salvianolic acid A.
综合本实施例结果，加入碱性钾化合物的量，使其中20-80％丹酚酸A反应完毕，都可以制备纯度高达99.5％的丹酚酸A钾盐水合物结晶，差别在于其得率稍有不同。Based on the results of this example, adding the amount of basic potassium compound so that 20-80% of the salvianolic acid A has been reacted, all can prepare salvianolic acid A potassium salt hydrate crystals with a purity of up to 99.5%, the difference lies in its yield slightly different.
另外，可以看到，采用的碱性钾化合物，实际提供的是钾离子，只要能保证该碱性钾化合物与丹酚酸A能够起反应即可。因此，采用的碱性钾化合物，除了本发明中使用的碳酸钾外，氢氧化钾、碳酸氢钾、苹果酸钾、柠檬酸钾、磷酸钾、磷酸氢二钾、磷酸二氢钾、乙酸钾等弱酸与钾形成的强碱弱酸盐，都能够应用于本发明中，用于制备本发明所述的丹酚酸A钾盐水合物结晶。In addition, it can be seen that the basic potassium compound used actually provides potassium ions, as long as it can be ensured that the basic potassium compound and salvianolic acid A can react. Therefore, the basic potassium compound used, in addition to the potassium carbonate used in the present invention, is potassium hydroxide, potassium bicarbonate, potassium malate, potassium citrate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium acetate Strong bases and weak acid salts formed by isoweak acids and potassium can be used in the present invention to prepare the salvianolic acid A potassium salt hydrate crystals of the present invention.
实施例15 制备丹酚酸A钙盐水合物结晶(探索碱性钙化合物加入量)Example 15 Preparation of salvianolic acid A calcium salt hydrate crystal (exploring the amount of basic calcium compound added)
将纯度≥80％的丹酚酸A溶液浓缩(内含16g丹酚酸A)，至浓度20％，水浴50℃，缓慢加入碳酸钙粉末约324mg，待两者反应完全后(反应约15分钟，可使其中20％的丹酚酸A反应生成丹酚酸A钙盐)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，不断搅拌24h，得丹酚酸A钙盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing 16 g of salvianolic acid A) to a concentration of 20%, take a water bath at 50 ° C, slowly add about 324 mg of calcium carbonate powder, and wait for the two to react completely (the reaction is about 15 minutes). , 20% of the salvianolic acid A can be reacted to generate salvianolic acid A calcium salt), the solution is slowly cooled to 20°C, the cooling rate is 0.2°C/min, during which stirring is continued, and then further cooled to 5°C, continuously Stir for 24h to obtain salvianolic acid A calcium salt hydrate (precipitation).
过滤沉淀，加入水50℃溶解，配置成浓度20％的溶液(以丹酚酸A计)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌并等待24h，得丹酚酸A钙盐水合物(重结晶产物)。Filter the precipitation, add water at 50°C to dissolve, configure into a solution with a concentration of 20% (calculated as salvianolic acid A), slowly cool the solution to 20°C, and the cooling rate is 0.2°C/min. 5 ℃, stirring and waiting for 24h to obtain salvianolic acid A calcium salt hydrate (recrystallized product).
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A钙盐水合物结晶”7.158g，以丹酚酸A计，其HPLC纯度99.7％。Filter the precipitate, spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize it, and dry it at 25°C for 12h to obtain the product "salvianolic acid A calcium salt hydrate crystal" 7.158 g, in terms of salvianolic acid A, its HPLC purity is 99.7%.
采用类似的方法，更改加入碳酸钙粉末的量1296mg(反应约20分钟，可使其中80％的丹酚酸A反应生成丹酚酸A钙盐)。最终可得产品“丹酚酸A钙盐水合物结晶”5.501g，以丹酚酸A计，其HPLC纯度为99.5％。Using a similar method, the amount of calcium carbonate powder added was changed to 1296 mg (reaction for about 20 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A calcium salt). The final product "salvianolic acid A calcium salt hydrate crystal" was 5.501 g, and its HPLC purity was 99.5% in terms of salvianolic acid A.
综合本实施例结果，加入碱性钙化合物的量，使其中20-80％丹酚酸A反应完毕，都可以制备纯度高达99.5％的丹酚酸A钙盐水合物结晶，差别在于其得 率有所不同。Based on the results of this example, adding the amount of basic calcium compound to make 20-80% of salvianolic acid A complete the reaction can prepare salvianolic acid A calcium salt hydrate crystals with a purity of up to 99.5%, the difference lies in its yield different.
另外，可以看到，采用的碱性钙化合物，实际提供的是钙离子，只要能保证该碱性钙化合物与丹酚酸A能够起反应即可。因此，采用的碱性钙化合物，除了本发明中使用的碳酸钙外，碳酸氢钙、氧化钙、氢氧化钙、葡萄糖酸钙、乙酸钙等弱酸与钙形成的盐，都可以应用于本发明中，用于制备本发明所描述的丹酚酸A钙盐水合物结晶In addition, it can be seen that the basic calcium compound used actually provides calcium ions, as long as it can be ensured that the basic calcium compound and salvianolic acid A can react. Therefore, the basic calcium compound adopted, in addition to the calcium carbonate used in the present invention, the salts formed by weak acids such as calcium bicarbonate, calcium oxide, calcium hydroxide, calcium gluconate, calcium acetate and calcium, all can be applied to the present invention , for the preparation of the salvianolic acid A calcium salt hydrate crystals described in the present invention
实施例16 制备丹酚酸A镁盐水合物结晶(探索碱性镁化合物加入量)Example 16 Preparation of salvianolic acid A magnesium salt hydrate crystal (exploring the amount of basic magnesium compound added)
将纯度≥80％的丹酚酸A溶液浓缩(内含16g丹酚酸A)，至浓度20％，水浴50℃，缓慢加入氢氧化镁粉末约188mg，待两者反应完全后(反应约15分钟，可使其中20％的丹酚酸A反应生成丹酚酸A镁盐)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，不断搅拌24h，得丹酚酸A镁盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing 16 g of salvianolic acid A) to a concentration of 20%, take a water bath at 50 ° C, slowly add about 188 mg of magnesium hydroxide powder, and wait for the two to react completely (the reaction is about 15 minutes, wherein 20% of salvianolic acid A can be reacted to generate salvianolic acid A magnesium salt), the solution is slowly cooled to 20 ℃, the cooling rate is 0.2 ℃/min, during which constant stirring, then further cooled to 5 ℃, Stir continuously for 24h to obtain salvianolic acid A magnesium salt hydrate (precipitation).
过滤沉淀，加入水50℃溶解，配置成浓度20％的溶液(以丹酚酸A计)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，搅拌并等待24h，得丹酚酸A镁盐水合物(重结晶产物)。Filter the precipitation, add water at 50°C to dissolve, configure into a solution with a concentration of 20% (calculated as salvianolic acid A), slowly cool the solution to 20°C, and the cooling rate is 0.2°C/min. 5 ℃, stirring and waiting for 24h to obtain salvianolic acid A magnesium salt hydrate (recrystallized product).
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A镁盐水合物结晶”1.105g，以丹酚酸A计，其HPLC纯度99.6％。Filter the precipitate, spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize, and dry at 25°C for 12h to obtain the product "Salvianolic Acid A Magnesium Salt Hydrate Crystal" 1.105 g, in terms of salvianolic acid A, its HPLC purity is 99.6%.
采用类似的方法，更改加入氢氧化镁粉末的量751mg(反应约20分钟，可使其中80％的丹酚酸A反应生成丹酚酸A镁盐)。最终可得产品“丹酚酸A镁盐水合物结晶”0.522g，以丹酚酸A计，其HPLC纯度为99.5％。Using a similar method, the amount of magnesium hydroxide powder added was changed to 751 mg (reaction for about 20 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A magnesium salt). The final product "salvianolic acid A magnesium salt hydrate crystal" is 0.522 g, and its HPLC purity is 99.5% based on salvianolic acid A.
综合本实施例结果，加入碱性镁化合物的量，使其中20-80％丹酚酸A反应完毕，都可以制备纯度高达99.5％的丹酚酸A镁盐水合物结晶，差别在于其得率有所不同。Based on the results of this example, adding the amount of basic magnesium compound to make 20-80% of the salvianolic acid A react, all can prepare salvianolic acid A magnesium salt hydrate crystals with a purity of up to 99.5%, the difference lies in the yield different.
另外，可以看到，采用的碱性镁化合物，实际提供的是镁离子，只要能保证该碱性镁化合物与丹酚酸A能够起反应即可。因此，采用的碱性镁化合物，除了本发明中使用的氢氧化镁外，化镁、碳酸镁、碳酸氢镁、碱式碳酸镁、苹果酸镁、柠檬酸镁、乙酸镁等弱酸与镁形成的盐，都可应用于本发明中，制备本发明所述的丹酚酸A镁盐水合物结晶。In addition, it can be seen that the basic magnesium compound used actually provides magnesium ions, as long as the basic magnesium compound and salvianolic acid A can be guaranteed to react. Therefore, the basic magnesium compound adopted, in addition to the magnesium hydroxide used in the present invention, weak acids such as magnesium hydride, magnesium carbonate, magnesium bicarbonate, basic magnesium carbonate, magnesium malate, magnesium citrate, magnesium acetate and magnesium form The salt of salvianolic acid A can be used in the present invention to prepare the salvianolic acid A magnesium salt hydrate crystal of the present invention.
实施例17 制备丹酚酸A铵盐水合物结晶(探索碱性铵(氨)化合物加入量)Example 17 Preparation of salvianolic acid A ammonium salt hydrate crystal (exploring the amount of basic ammonium (ammonia) compound added)
将纯度≥80％的丹酚酸A溶液浓缩(内含16g丹酚酸A)，至浓度20％，水浴50℃，缓慢加入碳酸铵粉末约311mg，待两者反应完全后(反应约20分钟，可使其中20％的丹酚酸A反应生成丹酚酸A铵盐)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步降温至5℃，不断搅拌24h，得丹酚酸A铵盐水合物(沉淀)。Concentrate the salvianolic acid A solution with a purity of ≥80% (containing 16 g of salvianolic acid A) to a concentration of 20%, take a water bath at 50 ° C, slowly add about 311 mg of ammonium carbonate powder, and wait for the two to react completely (the reaction is about 20 minutes. , 20% of the salvianolic acid A can be reacted to generate salvianolic acid A ammonium salt), the solution is slowly cooled to 20 °C, the cooling rate is 0.2 °C/min, and the stirring is continued, and then further cooled to 5 °C, continuously Stir for 24h to obtain salvianolic acid A ammonium salt hydrate (precipitation).
过滤沉淀，加入水50℃溶解，配置成浓度20％的溶液(以丹酚酸A计)，将溶液缓慢降温至20℃，降温速率为0.2℃/min，其间不停搅拌，然后进一步 降温至5℃，搅拌并等待24h，得丹酚酸A铵盐水合物(重结晶产物)。Filter the precipitation, add water at 50°C to dissolve, configure into a solution with a concentration of 20% (calculated as salvianolic acid A), slowly cool the solution to 20°C, and the cooling rate is 0.2°C/min. 5 ℃, stirring and waiting for 24h to obtain salvianolic acid A ammonium salt hydrate (recrystallized product).
过滤沉淀，并将其平铺至厚度≤5mm，放置于以五氧化二磷为干燥剂的干燥器内，抽真空，25℃干燥12h，得产品“丹酚酸A铵盐水合物结晶”2.229g，以丹酚酸A计，其HPLC纯度99.5％。Filter the precipitate, spread it to a thickness of ≤5mm, place it in a desiccator with phosphorus pentoxide as a desiccant, vacuumize, and dry at 25°C for 12h to obtain the product "salvianolic acid A ammonium salt hydrate crystal" 2.229 g, in terms of salvianolic acid A, its HPLC purity is 99.5%.
采用类似的方法，更改加入碳酸铵粉末的量1.244mg(反应约10分钟，可使其中80％的丹酚酸A反应生成丹酚酸A铵盐)。最终可得产品“丹酚酸A铵盐水合物结晶”1.688g，以丹酚酸A计，其HPLC纯度为99.5％。Using a similar method, the amount of ammonium carbonate powder added was changed to 1.244 mg (reaction for about 10 minutes, 80% of salvianolic acid A can be reacted to form salvianolic acid A ammonium salt). The final product "salvianolic acid A ammonium salt hydrate crystal" is 1.688 g, and its HPLC purity is 99.5% in terms of salvianolic acid A.
综合本实施例结果，加入碱性铵(氨)化合物的量，使其中20-80％丹酚酸A反应完毕，都可以制备纯度高达99.5％的丹酚酸A铵盐水合物结晶，差别在于其得率有所不同。Based on the results of this example, adding the amount of basic ammonium (ammonia) compound to make 20-80% of salvianolic acid A complete the reaction can prepare salvianolic acid A ammonium salt hydrate crystals with a purity of up to 99.5%. The difference is that Its yields vary.
另外，可以看到，采用的碱性铵(氨)化合物，实际提供的是铵根离子，只要能保证该碱性铵(氨)化合物与丹酚酸A能够起反应即可。因此，采用的碱性氨(氨)化合物，除了本发明中使用的碳酸铵外，氨气、氨水、碳酸氢铵等能够提供铵根离子，且能够与丹酚酸A起反应的化合物，都可以用于本发明，制备本发明所述的丹酚酸A铵盐水合物结晶。In addition, it can be seen that the basic ammonium (ammonia) compound used actually provides ammonium ions, as long as the basic ammonium (ammonia) compound can be guaranteed to react with salvianolic acid A. Therefore, the basic ammonia (ammonia) compound adopted, in addition to the ammonium carbonate used in the present invention, ammonia, ammonia water, ammonium bicarbonate, etc. can provide ammonium ions, and can react with salvianolic acid A compounds, all It can be used in the present invention to prepare the salvianolic acid A ammonium salt hydrate crystal of the present invention.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned herein are incorporated by reference in this application as if each document 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 (15)

  1. 一种式I所示的丹酚酸A盐水合物，A kind of salvianolic acid A salt hydrate shown in formula I,
    Figure PCTCN2022091078-appb-100001
    Figure PCTCN2022091078-appb-100001
    其中，M为阳离子；0＜m≤20，0＜n≤10，0＜x≤20，0＜y≤150。Wherein, M is a cation; 0<m≤20, 0<n≤10, 0<x≤20, 0<y≤150.
  2. 如权利要求1所述的丹酚酸A盐水合物，其特征在于，当M为1价阳离子时，n＝x；当M为2价阳离子时，n＝2x；当M为3价阳离子时，n＝3x。The salvianolic acid A salt hydrate according to claim 1, wherein when M is a monovalent cation, n=x; when M is a divalent cation, n=2x; when M is a trivalent cation , n=3x.
  3. 如权利要求1所述的丹酚酸A盐水合物，其特征在于，所述M选自下组：钠离子、钾离子、镁离子、钙离子、和铵离子。The salvianolic acid A salt hydrate of claim 1, wherein the M is selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion, and ammonium ion.
  4. 如权利要求1所述的丹酚酸A盐水合物，其特征在于，所述式I中，M为钠离子，并且5≤m≤15，1≤n≤10，1≤x≤10，20≤y≤100；优选地，M为钠离子，并且m为11，n为5，x为5，y为56；或者The salvianolic acid A salt hydrate according to claim 1, wherein, in the formula I, M is a sodium ion, and 5≤m≤15, 1≤n≤10, 1≤x≤10, 20 ≤y≤100; preferably, M is a sodium ion, and m is 11, n is 5, x is 5, and y is 56; or
    所述式I中，M为钾离子，并且1≤m≤5，1≤n≤5，1≤x≤5，1≤y≤10；优选地，M为钾离子，并且m为1，n为1，x为1，y为8；或者In the formula I, M is potassium ion, and 1≤m≤5, 1≤n≤5, 1≤x≤5, 1≤y≤10; preferably, M is potassium ion, and m is 1, n is 1, x is 1, and y is 8; or
    所述式I中，M为钙离子，并且1≤m≤5，1≤n≤5，0.5≤x≤5，5≤y≤15；优选地，M为钙离子，并且m为1，n为1，x为0.5，y为9；或者In the formula I, M is calcium ion, and 1≤m≤5, 1≤n≤5, 0.5≤x≤5, 5≤y≤15; preferably, M is calcium ion, and m is 1, n is 1, x is 0.5, and y is 9; or
    所述式I中，M为镁离子，并且1≤m≤5，1≤n≤5，0.5≤x≤5，5≤y≤15；优选地，M为镁离子，并且m为1，n为1，x为0.5，y为7；或者In the formula I, M is a magnesium ion, and 1≤m≤5, 1≤n≤5, 0.5≤x≤5, 5≤y≤15; preferably, M is a magnesium ion, and m is 1, n is 1, x is 0.5, and y is 7; or
    所述式I中，M为铵离子，并且1≤m≤5，1≤n≤5，1≤x≤5，5≤y≤15；优选地，M为铵离子，并且m为2，n为2，x为2，y为11。In the formula I, M is an ammonium ion, and 1≤m≤5, 1≤n≤5, 1≤x≤5, 5≤y≤15; preferably, M is an ammonium ion, and m is 2, n is 2, x is 2, and y is 11.
  5. 如权利要求1所述的丹酚酸A盐水合物，其特征在于，所述丹酚酸A盐水合物选自下组：The salvianolic acid A salt hydrate of claim 1, wherein the salvianolic acid A salt hydrate is selected from the group consisting of:
    丹酚酸A钠盐水合物：Salvianolic acid A sodium salt hydrate:
    Figure PCTCN2022091078-appb-100002
    Figure PCTCN2022091078-appb-100002
    丹酚酸A钾盐水合物：Salvianolic Acid A Potassium Salt Hydrate:
    Figure PCTCN2022091078-appb-100003
    Figure PCTCN2022091078-appb-100003
    丹酚酸A钙盐水合物：Salvianolic acid A calcium salt hydrate:
    Figure PCTCN2022091078-appb-100004
    Figure PCTCN2022091078-appb-100004
    丹酚酸A镁盐水合物：Salvianolic Acid A Magnesium Salt Hydrate:
    Figure PCTCN2022091078-appb-100005
    Figure PCTCN2022091078-appb-100005
    丹酚酸A铵盐水合物：Salvianolic acid A ammonium salt hydrate:
    Figure PCTCN2022091078-appb-100006
    Figure PCTCN2022091078-appb-100006
  6. 如权利要求3所述的丹酚酸A盐水合物，其特征在于，Salvianolic acid A salt hydrate as claimed in claim 3, is characterized in that,
    所述丹酚酸A盐水合物为丹酚酸A钠盐水合物，并且所述丹酚酸A钠盐水合物的差示扫描量热法图谱(DSC)在86-96℃有特征吸热峰；或者，The salvianolic acid A salt hydrate is salvianolic acid A sodium salt hydrate, and the differential scanning calorimetry spectrum (DSC) of the salvianolic acid A sodium salt hydrate has a characteristic endotherm at 86-96 ° C peak; or,
    所述丹酚酸A盐水合物为丹酚酸A钾盐水合物，并且所述丹酚酸A钾盐水合物的差示扫描量热法图谱(DSC)在95-105℃有特征吸热峰；或者，The salvianolic acid A salt hydrate is salvianolic acid A potassium salt hydrate, and the differential scanning calorimetry spectrum (DSC) of the salvianolic acid A potassium salt hydrate has a characteristic endotherm at 95-105 ° C peak; or,
    所述丹酚酸A盐水合物为丹酚酸A钙盐水合物，并且所述丹酚酸A钙盐水合物的差示扫描量热法图谱(DSC)在90-100℃有特征吸热峰；或者，The salvianolic acid A salt hydrate is salvianolic acid A calcium salt hydrate, and the differential scanning calorimetry spectrum (DSC) of the salvianolic acid A calcium salt hydrate has a characteristic endotherm at 90-100° C. peak; or,
    所述丹酚酸A盐水合物为丹酚酸A镁盐水合物，并且所述丹酚酸A镁盐水合物的差示扫描量热法图谱(DSC)在100-115℃有特征吸热峰；或者，The salvianolic acid A salt hydrate is salvianolic acid A magnesium salt hydrate, and the differential scanning calorimetry spectrum (DSC) of the salvianolic acid A magnesium salt hydrate has a characteristic endotherm at 100-115° C. peak; or,
    所述丹酚酸A盐水合物为丹酚酸A铵盐水合物，并且所述丹酚酸A铵盐水合物的差示扫描量热法图谱(DSC)在91-101℃有特征吸热峰。The salvianolic acid A salt hydrate is salvianolic acid A ammonium salt hydrate, and the differential scanning calorimetry spectrum (DSC) of the salvianolic acid A ammonium salt hydrate has a characteristic endotherm at 91-101 ° C peak.
  7. 如权利要求3所述的丹酚酸A盐水合物，其特征在于，Salvianolic acid A salt hydrate as claimed in claim 3, is characterized in that,
    所述丹酚酸A盐水合物为丹酚酸A钠盐水合物，并且所述丹酚酸A钠盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：16.56±0.1、17.90±0.1、19.04±0.1、25.97±0.1；或者，The salvianolic acid A salt hydrate is salvianolic acid A sodium salt hydrate, and the powder X-ray diffraction pattern of the salvianolic acid A sodium salt hydrate has characteristic peaks at the following 2θ values: 16.56±0.1, 17.90±0.1, 19.04±0.1, 25.97±0.1; or,
    所述丹酚酸A盐水合物为丹酚酸A钾盐水合物，并且所述丹酚酸A钾盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：18.89±0.1、24.35±0.1、24.61±0.1、和26.13±0.1；或者，The salvianolic acid A salt hydrate is salvianolic acid A potassium salt hydrate, and the powder X-ray diffraction pattern of the salvianolic acid A potassium salt hydrate has characteristic peaks at the following 2θ values: 18.89±0.1, 24.35±0.1, 24.61±0.1, and 26.13±0.1; or,
    所述丹酚酸A盐水合物为丹酚酸A钙盐水合物，并且所述丹酚酸A钙盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：18.92±0.1、22.59±0.1、24.35±0.1、和27.49±0.1；或者，The salvianolic acid A salt hydrate is salvianolic acid A calcium salt hydrate, and the powder X-ray diffraction pattern of the salvianolic acid A calcium salt hydrate has characteristic peaks at the following 2θ values: 18.92±0.1, 22.59±0.1, 24.35±0.1, and 27.49±0.1; or,
    所述丹酚酸A盐水合物为丹酚酸A镁盐水合物，并且所述丹酚酸A镁盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：21.65±0.1、24.18±0.1、24.49±0.1、25.32±0.1、25.91±0.1、和27.89±0.1；或者，The salvianolic acid A salt hydrate is salvianolic acid A magnesium salt hydrate, and the powder X-ray diffraction pattern of the salvianolic acid A magnesium salt hydrate has characteristic peaks at the following 2θ values: 21.65±0.1, 24.18±0.1, 24.49±0.1, 25.32±0.1, 25.91±0.1, and 27.89±0.1; or,
    所述丹酚酸A盐水合物为丹酚酸A铵盐水合物，并且所述丹酚酸A铵盐水合物的粉末X-射线衍射图谱在以下2θ值处具有特征峰：14.21±0.1、18.83±0.1、21.33±0.1、24.25±0.1、24.53±0.1、和25.97±0.1。The salvianolic acid A salt hydrate is salvianolic acid A ammonium salt hydrate, and the powder X-ray diffraction pattern of the salvianolic acid A ammonium salt hydrate has characteristic peaks at the following 2θ values: 14.21±0.1, 18.83±0.1, 21.33±0.1, 24.25±0.1, 24.53±0.1, and 25.97±0.1.
  8. 一种丹酚酸A盐水合物晶体，其特征在于，所述丹酚酸A盐水合物晶体选自下组：A salvianolic acid A salt hydrate crystal, characterized in that the salvianolic acid A salt hydrate crystal is selected from the following group:
    丹酚酸A钠盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
    Figure PCTCN2022091078-appb-100007
    α＝γ＝90.0°,β＝91.6°；     The salvianolic acid A sodium salt hydrate crystal is determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
    Figure PCTCN2022091078-appb-100007
    α=γ=90.0°, β=91.6°;
    丹酚酸A钾盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
    Figure PCTCN2022091078-appb-100008
    α＝γ＝90.0°,β＝115.16°；     Salvianolic acid A potassium salt hydrate crystal, which was determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
    Figure PCTCN2022091078-appb-100008
    α=γ=90.0°, β=115.16°;
    丹酚酸A镁盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
    Figure PCTCN2022091078-appb-100009
    α＝γ＝90.0°,β＝116.1°；     The salvianolic acid A magnesium salt hydrate crystal is determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
    Figure PCTCN2022091078-appb-100009
    α=γ=90.0°, β=116.1°;
    丹酚酸A钙盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
    Figure PCTCN2022091078-appb-100010
    α＝γ ＝90.0°,β＝115.7°；和     The salvianolic acid A calcium salt hydrate crystal is determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
    Figure PCTCN2022091078-appb-100010
    α=γ=90.0°, β=115.7°; and
    丹酚酸A铵盐水合物晶体，对其采用X-射线单晶衍射测定为单斜晶系，晶胞参数为：
    Figure PCTCN2022091078-appb-100011
    α＝γ＝90.0°,β＝96.9°。     Salvianolic acid A ammonium salt hydrate crystal, which was determined to be monoclinic by X-ray single crystal diffraction, and the unit cell parameters are:
    Figure PCTCN2022091078-appb-100011
    α=γ=90.0°, β=96.9°.
  9. 如权利要求8所述的丹酚酸A盐水合物晶体，其特征在于，所述丹酚酸A钠盐水合物晶体的绝对构型为：The salvianolic acid A salt hydrate crystal according to claim 8, wherein the absolute configuration of the salvianolic acid A sodium salt hydrate crystal is:
    Figure PCTCN2022091078-appb-100012
    Figure PCTCN2022091078-appb-100012
    所述丹酚酸A钾盐水合物晶体的绝对构型为：The absolute configuration of the salvianolic acid A potassium salt hydrate crystal is:
    Figure PCTCN2022091078-appb-100013
    Figure PCTCN2022091078-appb-100013
    所述丹酚酸A钙盐水合物晶体的绝对构型为：The absolute configuration of the salvianolic acid A calcium salt hydrate crystal is:
    Figure PCTCN2022091078-appb-100014
    Figure PCTCN2022091078-appb-100014
    所述丹酚酸A镁盐水合物晶体的相对构型为：The relative configuration of the salvianolic acid A magnesium salt hydrate crystal is:
    Figure PCTCN2022091078-appb-100015
    Figure PCTCN2022091078-appb-100015
    所述丹酚酸A铵盐水合物晶体的绝对构型为：The absolute configuration of the salvianolic acid A ammonium salt hydrate crystal is:
    Figure PCTCN2022091078-appb-100016
    Figure PCTCN2022091078-appb-100016
  10. 如权利要求8所述的丹酚酸A盐水合物晶体，其特征在于，The salvianolic acid A salt hydrate crystal according to claim 8, characterized in that,
    所述丹酚酸A钠盐水合物晶体对其采用X-射线单晶衍射测定为C2空间群；The salvianolic acid A sodium salt hydrate crystal is determined to be C2 space group by X-ray single crystal diffraction;
    所述丹酚酸A钾盐水合物晶体对其采用X-射线单晶衍射测定为C2空间群；Said salvianolic acid A potassium salt hydrate crystal is determined to be C2 space group by X-ray single crystal diffraction;
    所述丹酚酸A钙盐水合物晶体对其采用X-射线单晶衍射测定为C2空间群；The salvianolic acid A calcium salt hydrate crystal is determined to be C2 space group by X-ray single crystal diffraction;
    所述丹酚酸A镁盐水合物晶体对其采用X-射线单晶衍射测定为C2空间群；The salvianolic acid A magnesium salt hydrate crystal is determined to be C2 space group by X-ray single crystal diffraction;
    所述丹酚酸A铵盐水合物晶体对其采用X-射线单晶衍射测定为P2空间群。The salvianolic acid A ammonium salt hydrate crystal is determined to be P2 space group by X-ray single crystal diffraction.
  11. 一种丹酚酸A盐水合物的制备方法，其特征在于，所述方法包括步骤：A preparation method of salvianolic acid A salt hydrate, characterized in that the method comprises the steps:
    (1)将丹酚酸A原料溶于具有第一温度的含水溶剂中，获得丹酚酸A溶液；(1) salvianolic acid A raw material is dissolved in the water-containing solvent with first temperature, obtains salvianolic acid A solution;
    (2)向步骤(1)获得的丹酚酸A溶液中加入碱性化合物，使部分丹酚酸A和碱性化合物进行成盐反应；(2) adding basic compound to the salvianolic acid A solution obtained in step (1), so that part of salvianolic acid A and the basic compound carry out a salt-forming reaction;
    (3)对步骤(2)成盐反应后的溶液降温至第二温度进行析晶，从而获得所述丹酚酸A盐水合物；(3) cooling the solution after the salt-forming reaction in step (2) to a second temperature for crystallization, thereby obtaining the salvianolic acid A salt hydrate;
    其中，所述第一温度为40-80℃；所述第二温度为0-20℃。Wherein, the first temperature is 40-80°C; the second temperature is 0-20°C.
  12. 如权利要求11所述的方法，其特征在于，所述步骤(1)中所得丹酚酸A溶液中，丹酚酸A的质量浓度为约5％-20％；优选地为约5％-15％。The method according to claim 11, wherein, in the salvianolic acid A solution obtained in the step (1), the mass concentration of salvianolic acid A is about 5%-20%; preferably about 5%-20% 15%.
  13. 如权利要求11所述的方法，其特征在于，所述步骤(2)中降温速率为0.1℃/min-1℃/min；优选地降温速率为0.1℃/min-0.5℃/min；更优选地降温速率为0.1℃/min-0.3℃/min。The method according to claim 11, wherein the cooling rate in the step (2) 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℃/min-0.3℃/min.
  14. 一种权利要求1所述丹酚酸A盐水合物或权利要求8所述的丹酚酸A盐水合物晶体的用途，用于制备治疗或预防疾病的药物组合物，所述疾病选自：心脑血管疾病、免疫系统疾病、高脂血症、糖尿病并发症等。A use of the salvianolic acid A salt hydrate of claim 1 or the salvianolic acid A salt hydrate crystal of claim 8, for the preparation of a pharmaceutical composition for treating or preventing a disease, the disease is selected from: Cardiovascular and cerebrovascular diseases, immune system diseases, hyperlipidemia, diabetic complications, etc.
  15. 一种药物组合物，其特征在于，所述药物组合物包含：A pharmaceutical composition, characterized in that the pharmaceutical composition comprises:
    权利要求1所述的丹酚酸A盐水合物或权利要求8所述的丹酚酸A盐水合物晶体；以及药学上可接受的载体。The salvianolic acid A salt hydrate of claim 1 or the salvianolic acid A salt hydrate crystal of claim 8; and a pharmaceutically acceptable carrier.
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