A novel crystalline form of tanshinone hemihydrate, its preparation method, and its applications

Abstract

This invention discloses a novel crystalline form of tanshinone hemihydrate, its preparation method, and its applications, belonging to the fields of microbial fermentation and crystallization technology. The crystalline form of this invention, as determined by X-ray powder diffraction, infrared analysis, melting point determination, and nuclear magnetic resonance, is significantly different from the crystalline form of tanshinone in existing technologies. The novel crystalline form of tanshinone hemihydrate prepared by this invention exhibits solubility exceeding 580 mg / mL in water and organic solvents, along with excellent chemical stability and purity. It is easy to prepare on a large scale, simple to operate, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to a novel crystal form of tanshinone hemihydrate, its preparation method, and its application, belonging to the fields of microbial fermentation and crystallization technology. Background Technology

[0002] Tanshinone is mainly extracted from the traditional Chinese plant Salvia miltiorrhiza and is one of the main active ingredients of Salvia miltiorrhiza. Its scientific name is R-(+)-3-(3,4-dihydroxyphenyl)-2-hydroxypropionic acid and D-(+)-β-(3,4-dihydroxyphenyl)lactic acid. It is a dextrorotatory phenolic acid compound.

[0003] Currently, methods for synthesizing tanshinone are divided into chemical synthesis and biosynthesis. For example, patent CN116064348A discloses that by regulating the expression levels of the pathway enzymes of D-tanshinone, L-amino acid deaminase LAAD, phenylpyruvate reductase LaPPR, and glucose dehydrogenase GDH, the conversion rate of tanshinone prepared from dopamine as a substrate is improved. Patent CN110592038A discloses that Li Keyong prepared tanshinone using engineered bacteria that overexpress α-hydroxycarboxylic acid dehydrogenase and L-phenylalanine dehydrogenase. Patent CN103570547A discloses that β-(3,4-dihydroxyphenyl)pyruvate reacts with isopropanol in a solvent to generate tanshinone isopropyl ester under the conditions of second hydrochloric acid (12 mol / L) and reducing agent Zn-Hg.

[0004] Tanshinone, due to its unstable structure, is usually preserved as sodium tanshinone. Patent CN108744583B discloses the preparation of sodium tanshinone crystals from tanshinone tablets using ethanol. This process involves multiple steps, including ultrasound, addition of complex reagents, decoction reflux, overnight standing, filtration concentration, resin adsorption, and low-temperature membrane concentration, to obtain high-purity sodium tanshinone. This method is complex, costly, and time-consuming. Patent CN116143616A discloses a sodium tanshinone II crystal form, obtained by simultaneous or stepwise addition of ethanol / water, which has higher solubility than commercially available sodium tanshinone, reaching 218.76 mg / mL in a pH 6.8 phosphate buffer solution. Therefore, although the currently reported sodium tanshinone is more stable than tanshinone, sodium tanshinone salt crystals still suffer from low solubility or poor solubility in organic solvents.

[0005] Therefore, there is an urgent need to develop a tanshinone product and preparation method with improved stability and / or solubility. Summary of the Invention

[0006] To address the aforementioned problems, this invention first knocks out genes pheA, yeeP, efeU, yeeL, and ycgH, and overexpresses genes aroE and tyrA. fbrRecombinant strains were constructed using tyrB, HpaBC, and ldh, and crude tanshinone was prepared after fermentation and purification. Then, based on the crude tanshinone, a hemihydrate tanshinone compound with good solubility and stability was further prepared by using water and organic solvents.

[0007] The first objective of this invention is to provide a tanshinone hemihydrate crystal with the structural formula shown in Formula 1.

[0008]

[0009]

[0010] Among them, the X-ray powder diffraction of the tanshinone hemihydrate crystal is 2 θ Diffraction peaks are observed at ±0.02° at 6.398°, 10.919°, 12.567°, 16.597°, 18.825°, 22.574°, 24.232°, 26.594°, 27.275°, 28.684°, 29.401°, 31.372°, 32.642°, 33.267°, 35.042°, 36.225°, 36.711°, and 38.367°.

[0011] In one embodiment, tanshinone hemihydrate refers to a crystal in which every two tanshinone molecules are combined with one water molecule.

[0012] In one embodiment, in the X-ray powder diffraction pattern of the crystal, its 2 θ The ±0.01° angle value, interplanar spacing d, and relative intensity of diffraction peaks have the following characteristics:

[0013]

[0014] The unit cell parameters of the crystal are: α=β=90.00°, γ=120.00°.

[0015] In one embodiment, the tanshinone hemihydrate has a hexagonal crystal system, space group P65, and cell parameters: α=β=90.00°, γ=120.00°, unit cell volume The number of asymmetric units within the unit cell is Z = 6; its simplest chemical formula is C9H. 11 O 5.5 The molecular weight is 207.18; the crystal density is 1.519 mg / mm³. 3 ;

[0016] Where a, b, and c are the crystal axis lengths of the unit cell; α and β are the angles between the a-axis and b-axis and the c-axis, respectively; and γ is the angle between the a-axis and b-axis.

[0017] In one embodiment, the method for preparing the crystal includes:

[0018] The crude tanshinone was mixed with an organic solvent and dissolved; water was added, and the mixture was stirred, aged, filtered, and dried to obtain tanshinone hemihydrate crystals.

[0019] Optionally, the purity of tanshinone in the crude product is 75% to 95%.

[0020] Optionally, crude tanshinone can be extracted from tanshinone.

[0021] Optionally, crude tanshinone can be prepared by genetically engineered bacteria;

[0022] Alternatively, crude tanshinone can be prepared by chemical synthesis.

[0023] In one embodiment, the ratio of crude ginseng to organic solvent is 5g:7.5-15mL; the volume ratio of organic solvent to water is 7.5-15:0.1.

[0024] Optionally, the organic solvent includes one or more of ethyl acetate, isopropyl acetate, ethyl formate, and propyl formate.

[0025] In one embodiment, dissolution is achieved by heating at 40–60°C until complete dissolution;

[0026] Stir at 20-100 rpm for 1-3 hours;

[0027] Aging involves standing at 2–8℃ for 48–96 hours.

[0028] Drying includes hot air drying, vacuum drying, freeze drying, and microwave drying;

[0029] Alternatively, vacuum drying can be performed at 40–50°C for 6–10 hours.

[0030] A second objective of this invention is to provide a method for preparing tanshinone hemihydrate, the method comprising:

[0031] The crude tanshinone was mixed with an organic solvent and dissolved; water was added, and the mixture was stirred, aged, filtered, and dried to obtain the tanshinone hemihydrate.

[0032] Optionally, the purity of tanshinone in the crude product is 75% to 95%.

[0033] Optionally, the organic solvent includes one or more of ethyl acetate, isopropyl acetate, ethyl formate, and propyl formate;

[0034] Optionally, the ratio of crude tanshinone to organic solvent is 5g:7.5-15mL;

[0035] Optionally, the volume ratio of organic solvent to water is 7.5–15:0.1;

[0036] Optionally, the stirring is performed at 20–100 rpm for 1–3 hours;

[0037] Optionally, aging involves standing at 2–8°C for 48–96 hours;

[0038] Optionally, drying includes hot air drying, vacuum drying, freeze drying, and microwave drying;

[0039] Alternatively, vacuum drying can be performed at 40–50°C for 6–10 hours.

[0040] In one embodiment, optionally, crude tanshinone can be extracted from Salvia miltiorrhiza; optionally, crude tanshinone can be prepared by genetically engineered bacteria; optionally, crude tanshinone can be prepared by chemical synthesis.

[0041] In one embodiment, the method for preparing crude tanshinone using genetically engineered bacteria is as follows:

[0042] (1) Constructing genetically engineered bacteria:

[0043] Using *E. coli* as a host, the gene knockout of one or more of the following genes was performed: tyrR (transcriptional regulator), pykF (pyruvate kinase), pheA (branching acid isomerase-prephenylate dehydrogenase bifunctional enzyme), yeeP, efeU, yeeL, or ycgH; and glk (glucokinase) and aroG were overexpressed. fbr (DAHP synthase), aroE (shikimate dehydrogenase), tyrA fbr Genetically engineered bacteria were prepared by using one or more of the following: prephenylacetic acid dehydrogenase, tyrB (aromatic aminotransferase), HpaBC (4-hydroxyphenylacetic acid hydroxylase), and ldh (D-lactate dehydrogenase).

[0044] (2) Fermentation broth was obtained by fermentation of genetically engineered bacteria. The pH of the fermentation broth was adjusted to 2-3, the bacterial cells were removed by centrifugation, and the broth was concentrated. Tanshinone crude product was obtained by extraction and concentration with ethyl acetate.

[0045] In one embodiment, the promoter overexpressed in the genetically engineered bacteria includes: P j23119 P j23100 P j23102 P j23104 P j23101 ;

[0046] Optionally, with Pj23119 This is the promoter.

[0047] In one implementation, Escherichia coli MG1655, DH5α, JM109, and BL21(DE3) are used as hosts.

[0048] In one implementation, among the knocked-out genes, tyrR has a Gene ID of 945879; pykF has a Gene ID of 946179; pheA has a Gene ID of 947081; yeeP has a Gene ID of 946524; efeU has a Gene ID of 948956; yeeL has a Gene ID of 2847764; and ycgH has a Gene ID of 2847703.

[0049] In one implementation, the glk (glucokinase) in the overexpressed gene may be derived from Escherichiacoli str.K-12substr.MG1655 (Gene ID 946858), Xanthomonas arboricolapv.juglandis (Gene ID 67407047), Xanthomonas oryzae pv.Oryzicola (Gene ID 77337330), Marinobacter nauticus ATCC 49840 (Gene ID 31820944), or Cronobacter sakazakii (Gene ID 56729736), etc.

[0050] In one implementation, the overexpressed gene aroG fbr It may originate from Escherichia colistr.K-12substr.MG1655 (Gene ID 945605).

[0051] In one implementation, the aroE gene in the overexpressed gene may be derived from Escherichia colistr.K-12substr.MG1655 (Gene ID 947776).

[0052] In one implementation, the overexpressed gene contains tyrA. fbr It may originate from Escherichia colistr.K-12substr.MG1655 (Gene ID 947115).

[0053] In one implementation, the tyrB gene in the overexpressed gene may be derived from Escherichia colistr.K-12substr.MG1655 (Gene ID 948563).

[0054] In one implementation, the HpaBC in the overexpressed gene can be derived from Escherichia colistr.K-12substr.MG1655 (GeneBank: Z29081.2).

[0055] In one implementation, the ldh gene in the overexpressed gene may be derived from Limosilactobacillus reuteri strain JN516 (GenBank: MG653257.1).

[0056] In one embodiment, the fermentation in step (2) includes, in sequence, strain activation, seed culture, and fermenter culture.

[0057] In one embodiment, the fermentation tank culture is as follows: seed liquid is inoculated into the fermentation tank at an inoculation rate of 8-12%, the temperature is 35-38°C, the pH is 7.0±0.2, the dissolved oxygen is maintained at 18-22%, the glucose concentration in the tank is maintained at 0.4-0.6 g / L when glucose solution is added, and the fermentation time is 36-48 h.

[0058] In one embodiment, the crude tanshinone product prepared by genetically engineered bacteria achieves a purity of 90-95%.

[0059] A third objective of this invention is to provide a method for improving the solubility and / or stability of tanshinone, comprising the steps of:

[0060] The crude tanshinone was mixed with an organic solvent and dissolved; water was added, and the mixture was stirred, aged, filtered, and dried to obtain a hemihydrate tanshinone compound with improved solubility.

[0061] Optionally, the purity of tanshinone in the crude product is 75% to 95%.

[0062] Optionally, the ratio of crude tanshinone to organic solvent is 5g:7.5-15mL;

[0063] Optionally, the volume ratio of organic solvent to water is 7.5–15:0.1;

[0064] Optionally, the stirring is performed at 20–100 rpm for 1–3 hours;

[0065] Optionally, aging involves standing at 2–8°C for 48–96 hours;

[0066] Optionally, drying includes hot air drying, vacuum drying, freeze drying, and microwave drying;

[0067] Alternatively, vacuum drying can be performed at 40–50°C for 6–10 hours.

[0068] A fourth object of the present invention is to provide the application of any of the above-mentioned tanshinone hemihydrate crystals or the above-mentioned method for preparing tanshinone hemihydrate or the above-mentioned method for improving the solubility of tanshinone in the preparation of food, health products or pharmaceuticals.

[0069] A fifth object of the present invention is to provide a product containing any of the above-mentioned tanshinone hemihydrate compounds; the product is a food, medicine or health product.

[0070] In one embodiment, the food includes beverages and candies.

[0071] In one embodiment, the dosage form of the medicine includes, but is not limited to, liquid, solid, and semi-solid dosage forms;

[0072] Optionally, liquid preparations include, but are not limited to, solutions, injections, lotions, mixtures, and liniments;

[0073] Optionally, solid dosage forms include, but are not limited to, powders, granules, tablets, capsules, and films;

[0074] Optionally, semi-solid preparations include, but are not limited to, ointments, suppositories, and pastes.

[0075] The present invention also provides a direct drinking powder, comprising fruit powder, antioxidant, sweetener and the above-mentioned tanshinone hemihydrate compound.

[0076] In one embodiment, the fruit powder includes, but is not limited to, orange extract, cranberry extract, apple extract, banana extract, blueberry extract, and prickly pear extract.

[0077] In one embodiment, the antioxidants include, but are not limited to, L-ascorbic acid, tea polyphenols, catechins, astaxanthin, tocopherols, flavonoids, and butylated hydroxytoluene.

[0078] In one embodiment, the sweetener includes, but is not limited to, xylitol, sucrose, steviol glycosides, and fructose.

[0079] In one embodiment, the direct drinking powder contains, by weight percentage, 30-60% fruit powder, 2-4% L-ascorbic acid, 10-35% xylitol, and 0.1-1% tanshinone hemihydrate.

[0080] In one embodiment, food coloring and flavoring can also be added to the direct drinking powder.

[0081] In one embodiment, the formulation of a direct-drinking powder containing tanshinone hemihydrate simplifies the composition of the mixture and, in addition to enhancing its ability to relieve fatigue, improves cardiovascular function.

[0082] The present invention also provides a tablet comprising fruit powder, antioxidant, sweetener, filler and the above-mentioned tanshinone hemihydrate.

[0083] In one embodiment, the fruit powder includes, but is not limited to, orange extract, cranberry extract, apple extract, banana extract, blueberry extract, and prickly pear extract.

[0084] In one embodiment, the antioxidants include, but are not limited to, L-ascorbic acid, tea polyphenols, catechins, astaxanthin, tocopherols, flavonoids, and butylated hydroxytoluene.

[0085] In one embodiment, the sweetener includes, but is not limited to, xylitol, sucrose, steviol glycosides, and fructose.

[0086] In one embodiment, the filler includes, but is not limited to, starch and maltodextrin.

[0087] In one embodiment, food coloring and flavoring may also be added to the tablets.

[0088] In one embodiment, the tablets contain, by weight percentage, 30-60% fruit powder, 2-4% L-ascorbic acid, 5-15% xylitol, 10-20% starch, 3-5% maltodextrin, 0-0.1% food coloring, 0-0.1% food flavoring, and 0.1-1% tanshinone hemihydrate.

[0089] In one embodiment, the formulation of tablets containing tanshinone hemihydrate simplifies the composition of the composition and, in addition to improving the ability to relieve fatigue, also improves cardiovascular function.

[0090] The present invention also provides a gummy candy comprising concentrated fruit juice, an antioxidant, a cross-linking agent, a sweetener, and the above-mentioned tanshinone hemihydrate compound.

[0091] In one embodiment, the concentrated fruit juice includes, but is not limited to, orange juice, cranberry juice, apple juice, pear juice, blueberry juice, and prickly pear juice.

[0092] In one embodiment, the sweetener includes, but is not limited to, malt syrup, white sugar, xylitol, sucrose, steviol glycosides, and fructose.

[0093] In one embodiment, the crosslinking agent includes, but is not limited to, gelatin, carrageenan, pectin, and calcium lactate.

[0094] In one embodiment, the antioxidants include, but are not limited to, citric acid, L-ascorbic acid, tea polyphenols, catechins, astaxanthin, tocopherols, flavonoids, and butylated hydroxytoluene.

[0095] In one embodiment, food coloring and flavoring may also be added to the gummies.

[0096] In one embodiment, the gummies contain, by weight percentage: 20-35% fruit juice, 10-15% malt syrup, 10-16.8% white sugar, 10-15% gelatin, 5-10% carrageenan, 1-5% pectin, 0-1% citric acid, 0-0.5% calcium lactate, 0-1% L-ascorbic acid, 0-0.2% edible flavoring, and 0-1% tanshinone hemihydrate.

[0097] In one embodiment, the formulation of gummies containing tanshinone hemihydrate simplifies the composition of the composition and, in addition to enhancing the ability to relieve fatigue, improves cardiovascular function.

[0098] The present invention also provides a genetically engineered Escherichia coli strain that overexpresses the gene HpaBC (4-hydroxyphenylacetic acid hydroxylase).

[0099] In one embodiment, the genetically engineered bacteria also knock out one or more of the following genes: tyrR (transcriptional regulator), pykF (pyruvate kinase), pheA (branching acid isomerase-prephenyl acid dehydrogenase bifunctional enzyme), yeeP, efeU, yeeL, or ycgH.

[0100] In one embodiment, the genetically engineered bacteria overexpress the genes glk (glucokinase) and aroG. fbr (DAHP synthase), aroE (shikimate dehydrogenase), tyrA fbr One or more of the following: (prephenylate dehydrogenase), tyrB (aromatic aminotransferase), and ldh (D-lactate dehydrogenase).

[0101] In one embodiment, the genetically engineered bacteria, after knocking out one or more of the genes tyrR (transcription regulator), pykF (pyruvate kinase), pheA (branching acid isomerase-prephenylate dehydrogenase bifunctional enzyme), yeeP, efeU, yeeL, or ycgH, further overexpress glk (glucokinase) and aroG. fbr (DAHP synthase), aroE (shikimate dehydrogenase), tyrA fbr One or more of the following: (prephenylate dehydrogenase), tyrB (aromatic aminotransferase), and ldh (D-lactate dehydrogenase).

[0102] In one embodiment, the promoter overexpressed in the genetically engineered bacteria includes: P j23119 P j23100 P j23102 P j23104 P j23101 ;

[0103] Optionally, with P j23119 This is the promoter.

[0104] In one implementation, Escherichia coli MG1655, DH5α, JM109, and BL21(DE3) are used as hosts.

[0105] In one implementation, among the knocked-out genes, tyrR has a Gene ID of 945879; pykF has a Gene ID of 946179; pheA has a Gene ID of 947081; yeeP has a Gene ID of 946524; efeU has a Gene ID of 948956; yeeL has a Gene ID of 2847764; and ycgH has a Gene ID of 2847703.

[0106] In one implementation, the glk (glucokinase) in the overexpressed gene may be derived from Escherichiacoli str.K-12substr.MG1655 (Gene ID 946858), Xanthomonas arboricolapv.juglandis (Gene ID 67407047), Xanthomonas oryzae pv.Oryzicola (Gene ID 77337330), Marinobacter nauticus ATCC 49840 (Gene ID 31820944), or Cronobacter sakazakii (Gene ID 56729736), etc.

[0107] In one implementation, the overexpressed gene aroG fbr It may originate from Escherichia colistr.K-12substr.MG1655 (Gene ID 945605).

[0108] In one implementation, the aroE gene in the overexpressed gene may be derived from Escherichia colistr.K-12substr.MG1655 (Gene ID 947776).

[0109] In one implementation, the overexpressed gene contains tyrA. fbr It may originate from Escherichia colistr.K-12substr.MG1655 (Gene ID 947115).

[0110] In one implementation, the tyrB gene in the overexpressed gene may be derived from Escherichia colistr.K-12substr.MG1655 (Gene ID 948563).

[0111] In one implementation, the HpaBC in the overexpressed gene can be derived from Escherichia colistr. ATCC 11105 (ACCESSION Z29081; sequence positions 1112-3204).

[0112] In one implementation, the ldh gene in the overexpressed gene may be derived from Limosilactobacillusreuteri strain JN516 (GenBank: MG653257.1).

[0113] In one embodiment, fermentation includes, in sequence, strain activation, seed culture, and fermenter culture.

[0114] In one embodiment, the fermenter culture is as follows: seed liquid is inoculated into the fermenter at an inoculation rate of 8-12% v / v, the temperature is 35-38°C, the pH is 7.0±0.2, the dissolved oxygen is maintained at 18-22%, the glucose concentration in the tank is maintained at 0.4-0.6 g / L when glucose solution is added, and the fermentation time is 36-48 h.

[0115] The present invention also provides the use of the above-mentioned Escherichia coli genetically engineered bacteria in the preparation of tanshinone or any of the above-mentioned tanshinone hemihydrate crystals.

[0116] In one embodiment, the genetically engineered Escherichia coli overexpresses the gene HpaBC (4-hydroxyphenylacetic acid hydroxylase);

[0117] Optionally, the genetically engineered Escherichia coli may also knock out one or more of the following genes: tyrR (transcriptional regulator), pykF (pyruvate kinase), pheA (branching acid isomerase-prephenyl acid dehydrogenase bifunctional enzyme), yeeP, efeU, yeeL, or ycgH.

[0118] Optionally, the genetically engineered E. coli overexpresses the genes glk (glucokinase) and aroG. fbr (DAHP synthase), aroE (shikimate dehydrogenase), tyrA fbrOne or more of the following: (prephenylate dehydrogenase), tyrB (aromatic aminotransferase), and ldh (D-lactate dehydrogenase);

[0119] Optionally, the genetically engineered *E. coli* strain, in addition to knocking out one or more of the genes tyrR (transcription regulator), pykF (pyruvate kinase), pheA (branching acid isomerase-prephenylate dehydrogenase bifunctional enzyme), yeeP, efeU, yeeL, or ycgH, further overexpresses the genes glk (glucokinase) and aroG. fbr (DAHP synthase), aroE (shikimate dehydrogenase), tyrA fbr One or more of the following: (prephenylate dehydrogenase), tyrB (aromatic aminotransferase), and ldh (D-lactate dehydrogenase).

[0120] In one embodiment, the promoter overexpressed in the genetically engineered bacteria includes: P j23119 P j23100 P j23102 P j23104 P j23101 ;

[0121] Optionally, with P j23119 This is the promoter.

[0122] In one implementation, Escherichia coli MG1655, DH5α, JM109, and BL21(DE3) are used as hosts.

[0123] Beneficial effects of the present invention

[0124] Compared with existing crystal forms (sodium tanshinone), the tanshinone hemihydrate of this invention has high water solubility and is readily soluble in organic solvents such as alcohols, ethers, and acetonitrs. Its solubility in water reaches 700 mg / mL, in ethanol and methanol it reaches 760 mg / mL, in diethyl ether it reaches 580 mg / mL, and in acetonitrile it reaches 600 mg / mL. This is an effect not found in salt-forming crystal forms such as sodium tanshinone. Stability testing results for the tanshinone hemihydrate show that, under accelerated testing conditions, this crystal form exhibits good stability. Attached Figure Description

[0125] Figure 1 This is the X-ray powder diffraction pattern of the crystal form of the tanshinone hemihydrate prepared in this invention;

[0126] Figure 2 These are the unit cell parameters of the tanshinone hemihydrate crystal form prepared by this invention, obtained by powder diffraction analysis.

[0127] Figure 3This is a powder diffraction analysis of the molecular stereostructure ellipsoid of the tanshinone hemihydrate compound prepared in this invention.

[0128] Figure 4 This is the infrared spectrum (IR) of the crystalline form of the tanshinone hemihydrate compound prepared in this invention;

[0129] Figure 5 The 1H NMR spectrum of the tanshinone hemihydrate crystalline form prepared in this invention is shown.

[0130] Figure 6 The carbon nuclear magnetic spectrum (C1NMR) of the crystalline form of the tanshinone hemihydrate compound prepared in this invention is shown.

[0131] Figure 7 This is a high-performance liquid chromatography (HPLC) chromatogram of the tanshinone hemihydrate crystalline form prepared in this invention.

[0132] Figure 8 This invention utilizes the genetically engineered bacterium DSS-07 to ferment and prepare tanshinone. Detailed Implementation

[0133] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the amounts of solvent and related reagents used in the present invention are conventional amounts for the reaction, which can be determined by those skilled in the art based on existing technology; the reagents used in the present invention are conventional reagents that can be purchased from the market, and the starting materials and reactants used can all be prepared by existing technology or published existing literature or patents.

[0134] Experimental methods:

[0135] Gene editing method: The gene editing method used was based on the literature (Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang YJ, Chen T, Zhao X. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab Eng. 2015 Sep; 31:13-21.). The engineered plasmids involved in this method are pTargetF and pCas, where pTargetF carries an sgRNA expression plasmid, and pCas contains the Cas9 protein expression system and kanamycin resistance (working concentration: 50 mg / L).

[0136] Raw material source:

[0137] pTargetF was purchased from Newp Gene; (Cat.#V012144);

[0138] pCas was purchased from Newp Gene; (Cat.#V012287).

[0139] Detection method:

[0140] 1. Nuclear magnetic resonance (NMR) detection method

[0141] The samples were tested according to the 2020 edition of the Chinese Pharmacopoeia, including hydrogen and carbon spectra.

[0142] 2. Crystal form analysis methods

[0143] The samples were tested using XRD according to the 2020 edition of the Chinese Pharmacopoeia.

[0144] 3. Crystal stability testing methods

[0145] The method for stability testing can be found in Appendix XIXC of Part II of the 2020 edition of the Chinese Pharmacopoeia.

[0146] 4. Crystalline Infrared Method

[0147] The samples were tested using infrared spectroscopy according to the 2020 edition of the Chinese Pharmacopoeia.

[0148] 5. HPLC detection method

[0149] The column was packed with octadecylsilane-bonded silica gel (4.6 mm × 250 mm, 5 μm, or a column with equivalent performance); the mobile phase was acetonitrile (A)-0.025% phosphoric acid water (B), with gradient elution: 0-16 min: 10% A, 16-28 min: 10%–30% A, 28-38 min: 30% A, 38-38.1 min: 30%–10% A, 38.1-48 min: 10% A; the flow rate was 0.5 mL / min (first 16 min), 0.8 mL / min (16 min–48 min), the column temperature was 35 °C, the detection wavelength was 280 nm, and the injection volume was 10 μL.

[0150] 6. Solubility testing methods

[0151] The solubility of the samples was tested according to the 2020 edition of the Chinese Pharmacopoeia. The specific steps are as follows:

[0152] Weigh the finely powdered test sample or measure the liquid test sample, and place it in a solvent of a certain volume at 25℃±2℃. Shake vigorously for 30 seconds every 5 minutes. Observe the dissolution within 30 minutes. If there are no visible solute particles or droplets, it is considered to be completely dissolved.

[0153] The culture medium used in the examples:

[0154] Seed culture medium (g / L): glucose 20, yeast extract 3, peptone 2, (NH4)2SO4 1, K2HPO4·3H2O 2, MgSO4·7H2O 2, citric acid 1.5, glutamic acid 3, methionine 6, FeSO4·7H2O 0.01;

[0155] Fermentation medium (g / L): yeast extract 3.5, peptone 1, (NH4)2SO4 2, K2HPO4·3H2O 2, MgSO4·7H2O 2, glutamic acid 2, methionine 0.5, citric acid 3, MnSO4·7H2O 0.01, FeSO4·7H2O 0.03.

[0156] Example 1: Construction of genetically engineered Escherichia coli

[0157] Using Escherichia coli MG1655 as the starting strain, the gene knockout and integration process was carried out, specifically:

[0158] 1. Constructing the E. coli str.K-12substr.MG1655ΔtyrR::P23119-glk strain

[0159] Starting with Escherichia coli MG1655, the transcriptional regulator tyrR (Gene ID 945879) was knocked out, and the glucokinase glk (Gene ID 946858) was integrated into this gene at this location. The steps are as follows:

[0160] Using the *E. coli* MG1655 genome as a template, primers tyrR-up-F / tyrR-up-R and tyrR-down-F / tyrR-down-R (primers shown in Table 1) were used to amplify and recover the upstream and downstream homologous arms of tyrR; using the *E. coli* MG1655 genome as a template, primers glk-F / glk-R (primers shown in Table 1) were used to amplify and recover the promoter Pj23119-glk expression cassette; primers were used... The upstream and downstream homologous arms of tyrR and the Pj23119-glk expression cassette were integrated by overlapping PCR to obtain the tyrR-Pj23119-glk integration cassette. Using the commercial plasmid pTargetF as a template, the N20-tyrR plasmid targeting the knockout of the tyrR gene was constructed by reverse PCR using primers N20-tyrR-F / N20-tyrR-R (primers are shown in Table 1).

[0161] The tyrR-Pj23119-glk integration frame and N20-tyrR plasmid were introduced into E. coli MG1655 competent cells containing the pCas plasmid via electroporation. The N20-tyrR plasmid was lost, resulting in a strain with tyrR knocked out and glk overexpressed, named DSS-01(pCas). The pCas plasmid was not eliminated and was used for subsequent gene knockout and integration.

[0162] 2. Construct E.coli str.K-12substr.MG1655ΔtyrR-ΔpykF::P23119-glk-aroG fbr strain

[0163] Following the method in step 1, pykF-P is constructed. j23119 -aroG fbr The integration box (Gene ID 945605) and the N20-pykF plasmid targeting the knockout of the pykF (Gene ID 946179) gene (primers are shown in Table 1); pykF-P j23119 -aroG fbr The integration box and N20-pykF plasmid were introduced into DSS-01 (pCas) competent cells via electroporation. The N20-pykF plasmid was lost, resulting in pykF knockout and aroG overexpression. fbr The strain was named DSS-02(pCas). The pCas plasmid was not eliminated and was used for subsequent gene knockout and integration.

[0164] 3. Construct E.coli str.K-12substr.MG1655ΔtyrR-ΔpykF-ΔpheA::P23119-glk-aroG fbr -aroE strain

[0165] Following the method in step 1, pheA-P is constructed. j23119 The pheA-Pj23119-aroE integrative frame and the N20-pheA plasmid targeting the knockout of the pheA (Gene ID 947776) gene (primers are shown in Table 1) were introduced into DSS-02 (pCas) competent cells via electroporation. The N20-tyrR plasmid was lost, resulting in a strain that knocked out pheA and overexpressed aroE, named DSS-03 (pCas). The pCas plasmid was not eliminated and was used for subsequent gene knockout and integration.

[0166] 4. Construct E.coli str.K-12substr.MG1655

[0167] ΔtyrR-ΔpykF-ΔpheA-ΔyeeP::P23119-glk-aroG fbr -aroE-tyrA fbr strain

[0168] Following the method in step 1, yeeP-P is constructed. j23119 -tyrA fbr The integration box (Gene ID 947115) and the N20-yeeP plasmid targeting the knockout of the yeeP (Gene ID 946524) gene (primers are shown in Table 1); yeeP-Pj23119-tyrA fbr The integration box and N20-yeeP plasmid were introduced into DSS-03 (pCas) competent cells. The N20-yeeP plasmid was lost, resulting in yeeP knockout and tyrA overexpression. fbr The strain was named DSS-04(pCas). The pCas plasmid was not eliminated and was used for subsequent gene knockout and integration.

[0169] 5. Construct E.coli str.K-12substr.MG1655

[0170] ΔtyrR-ΔpykF-ΔpheA-ΔyeeP-ΔefeU::P23119-glk-aroG fbr -aroE-tyrA fbr -tyrB strain

[0171] Following the method in step 1, efeU-P is constructed. j23119 The efeU-Pj23119-tyrB integrative frame and the N20-efeU plasmid targeting the knockout of the efeU (Gene ID 948956) gene (primers are shown in Table 1) were introduced into DSS-04 (pCas) competent cells via electroporation. The N20-efeU plasmid was lost, resulting in a strain that knocked out efeU and overexpressed tyrB, named DSS-05 (pCas). The pCas plasmid was not eliminated and was used for subsequent gene knockout and integration.

[0172] 6. Construct E.coli str.K-12substr.MG1655

[0173] ΔtyrR-ΔpykF-ΔpheA-ΔyeeP-ΔefeU-ΔyeeL::P23119-glk-aroG fbr -aroE-tyrA fbr-tyrB-HpaBC strain

[0174] Following the method in step 1, using Escherichia coli ATCC 11105 as a template, yeeL-P was constructed. j23119 -HpaBC (ACCESSION Z29081; positions 1112-3204) integration frame and N20-yeeL plasmid targeting and knocking out the yeeL (Gene ID 2847764) gene (primers are shown in Table 1); yeeL-P j23119 The HpaBC integration frame and N20-yeeL plasmid were electroporated into DSS-05 (pCas) competent cells. The N20-efeU plasmid was lost, resulting in a strain that knocked out yeeL and overexpressed HpaBC, named DSS-06 (pCas). The pCas plasmid was not eliminated and was used for subsequent gene knockout and integration.

[0175] 7. Construct E.coli str.K-12substr.MG1655

[0176] ΔtyrR-ΔpykF-ΔpheA-ΔyeeP-ΔefeU-ΔyeeL-ΔycgH::P23119-glk-aroG fbr -aroE-tyrA fbr -tyrB-HpaBC-ldh strain

[0177] Following the method in step 1, using Limosilactobacillus reuteri strain JN516 as a template, ycgH-P was constructed. j23119 -ldh(GenBank:MG653257.1) integration box and N20-ycgH plasmid targeting and knocking out the ycgH (Gene ID 2847703) gene (primers are shown in Table 1); ycgH-P j23119 The N20-efeU plasmid was introduced into DSS-06 (pCas) competent cells via electroporation. The N20-efeU plasmid was lost, resulting in the DSS-07 (pCas) strain with ycgH knocked out and ldh overexpressed. The strain obtained by eliminating the pCas plasmid was named DSS-07.

[0178] Table 1 Primer Table

[0179]

[0180]

[0181]

[0182] Example 2: Production of Tanshinone using Recombinant Bacteria

[0183] The strains DSS-01(pCas), DSS-02(pCas), DSS-03(pCas), DSS-04(pCas), DSS-05(pCas), DSS-06(pCas), and DSS-07 prepared in Example 1 were used to produce tanshinone, and the steps are as follows:

[0184] (1) Activation of strain: After activating the strain by drawing lines on a plate in a glycerol tube for 12 hours, inoculate it into an Erlenmeyer flask (LB medium) and continue to expand the culture for 10 hours. The shaking speed is 200 rpm and the temperature is 37℃.

[0185] (2) Seed culture: The bacterial culture obtained by activating the above-mentioned strain was inoculated into the seed tank at a 5% inoculum rate for strain expansion culture. The culture temperature was 37℃, and the pH was maintained at 7.0±0.2 by automatic addition of ammonia water. The dissolved oxygen was maintained at 50% by automatic adjustment of the stirring speed. The culture was carried out until the OD reached the target value. 600 Reaching 15;

[0186] (3) Fermentation tank culture: Take the seed liquid and inoculate it into the fermentation tank at an inoculation amount of 10%. The culture temperature is 37℃. The pH is maintained at 7.0±0.2 by automatically adding ammonia water. The dissolved oxygen is maintained at 20% by automatically adjusting the stirring speed. The glucose concentration in the tank is maintained at about 0.5g / L by adding 80% (m / v, g / mL) glucose solution. The fermentation cycle is 48h.

[0187] The results are as follows Figure 8 As shown, after 48 hours of fermentation, the yield of tanshinone in the fermentation broth of strain DSS-07 reached 25.7 g / L.

[0188] In addition, after 48 hours of fermentation, the yields of tanshinone in the fermentation broths of DSS-01 (pCas), DSS-02 (pCas), DSS-03 (pCas), DSS-04 (pCas), DSS-05 (pCas), and DSS-06 (pCas) were 0, 0, 0, 0, and 3.4 g / L, respectively.

[0189] Example 3: Preparation of crude tanshinone

[0190] The fermentation broth prepared in Example 2 was subjected to post-processing, as follows:

[0191] The pH of the fermentation broth was adjusted to 2-3 (the range does not affect the results), and the cells were removed by centrifugation. The fermentation broth was concentrated by vacuum evaporation to 0.5 times its original weight, and extracted five times with an equal volume of ethyl acetate, achieving an extraction rate of 95% (1 - tanshinone content in the aqueous phase after extraction / tanshinone content in the original broth). The ethyl acetate extract was concentrated by vacuum evaporation to remove the ethyl acetate, yielding crude amorphous tanshinone (amorphous, i.e., paste, with a tanshinone purity of 83%).

[0192] Example 4: Preparation of Tanshinone Hemihydrate Crystal Form

[0193] Take 5g of the amorphous crude product prepared in Example 3, add 10mL of ethyl acetate, heat to 40℃ to completely dissolve, add 0.1mL of water while stirring, stir at 100rpm for 2 hours, and age at 2-8℃ (within which range does not affect the results) for 72 hours; filter with filter paper, and vacuum dry at 45℃ for 6 hours to obtain 3.2g of off-white crystals.

[0194] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.3%. Powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone, and its chemical structural formula is as follows:

[0195]

[0196] Example 5: Preparation of Tanshinone Hemihydrate Crystal Form

[0197] Based on Example 4, the amount of ethyl acetate was changed to 7.5 mL, and the remaining steps were the same as in Example 4, to prepare 3.6 g of off-white crystals.

[0198] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.5%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0199] Example 6: Preparation of Tanshinone Hemihydrate Crystal Form

[0200] Based on Example 4, the amount of ethyl acetate was changed to 15 mL, and the remaining steps were the same as in Example 4, to prepare 2.6 g of off-white crystals.

[0201] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.7%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0202] Example 7: Preparation of Tanshinone Hemihydrate Crystal Form

[0203] Based on Example 4, ethyl acetate was replaced with isopropyl acetate, and the remaining steps were the same as in Example 4, to prepare 2.6g of off-white crystals.

[0204] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.8%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0205] Example 8: Preparation of Tanshinone Hemihydrate Crystal Form

[0206] Based on Example 4, ethyl acetate was replaced with ethyl formate, and the remaining steps were the same as in Example 4, to prepare 2.6g of off-white crystals.

[0207] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.5%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0208] Example 9: Preparation of Tanshinone Hemihydrate Crystal Form

[0209] Based on Example 4, ethyl acetate was replaced with propyl formate, and the remaining steps were the same as in Example 4, to prepare 2.6g of off-white crystals.

[0210] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.6%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0211] Example 10: Changing the amount of water added

[0212] (1) Based on Example 4, the amount of water was changed to 0.5 mL, and the remaining steps were the same as in Example 4, and 3.9 g of off-white crystals were prepared.

[0213] The HPLC purity of tanshinone in the off-white crystals was determined to be 96.4%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0214] (2) Based on Example 4, the amount of water was changed to 0.02 mL, and the remaining steps were the same as in Example 4, and 1.4 g of off-white crystals were prepared.

[0215] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.5%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0216] The results showed that increasing the amount of water used reduced the purity of tanshinone crystals, while reducing the amount of water used affected the yield of tanshinone crystals.

[0217] Example 11: Changing the preparation method of crude tanshinone and the purity of tanshinone in the crude product.

[0218] Using the method disclosed in CN1670008A, crude tanshinone was extracted from Salvia miltiorrhiza, and the purity of tanshinone in the crude product was 80%.

[0219] The crude tanshinone was used to replace the amorphous crude product in Example 4, while the other steps and parameters remained the same as in Example 4. 2.1 g of off-white crystals were obtained.

[0220] The HPLC purity of tanshinone in the off-white crystals was determined to be 98.5%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0221] Example 12: Changing the preparation method of crude tanshinone and the purity of tanshinone in the crude product.

[0222] Tanshinone was prepared using genetically engineered bacteria, and crude tanshinone was obtained by resin adsorption purification as disclosed in CN1868994A. The purity of tanshinone in the crude product was 90%.

[0223] The crude tanshinone was used to replace the amorphous crude product in Example 4, while the other steps and parameters remained the same as in Example 4. 3.8 g of off-white crystals were obtained.

[0224] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.6%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0225] Example 13: Changing the preparation method of crude tanshinone and the purity of tanshinone in the crude product.

[0226] Tanshinone was prepared using the chemical synthesis method disclosed in CN102863328A, and crude tanshinone was obtained after preliminary purification. The purity of tanshinone in the crude product was 86%.

[0227] The crude tanshinone was used to replace the amorphous crude product in Example 4, while the other steps and parameters remained the same as in Example 4. 3.5 g of off-white crystals were obtained.

[0228] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.7%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0229] Example 14: Crude Tanshinone of Different Purities

[0230] (1) Based on Example 4, using different batches of amorphous crude tanshinone (with a purity of 75%), and the remaining steps were the same as in Example 4, 1.4 g of off-white crystals were prepared.

[0231] The HPLC purity of tanshinone in the off-white crystals was determined to be 98.3%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0232] (2) Based on Example 4, using different batches of amorphous crude tanshinone (with a purity of 95%), and with the remaining steps being the same as in Example 4, 4.5g of off-white crystals were prepared.

[0233] The HPLC purity of tanshinone in the off-white crystals was determined to be 99.8%; powder diffraction analysis showed that the off-white crystals were the hemihydrate form of tanshinone.

[0234] The results showed that the amorphous crude tanshinone product affected the purity and yield of tanshinone.

[0235] Comparative Example 1: No water was added during the crystallization process.

[0236] Based on Example 4, without adding 0.1 mL of water, the remaining steps are the same as in Example 4.

[0237] The results showed that crystallization could not occur without the addition of water.

[0238] Comparative Example 2: Crystallization using conventional methods

[0239] The sodium tanshinone II crystal form was prepared according to the method in Example 1 of patent CN116143616A.

[0240] Example 15: Performance Testing of Tanshinone Hemihydrate

[0241] The properties of the tanshinone hemihydrate compound prepared in Example 7 were tested, and the results are as follows:

[0242] 1. X-ray powder diffraction

[0243] The powder diffraction pattern of tanshinone hemihydrate is shown below. Figure 1 As shown, the results indicate that the XRD pattern exhibits diffraction peaks at 6.398°, 10.919°, 12.567°, 16.597°, 18.825°, 22.574°, 24.232°, 26.594°, 27.275°, 28.684°, 29.401°, 31.372°, 32.642°, 33.267°, 35.042°, 36.225°, 36.711°, and 38.367°, among which 2 θ The value error range is ±0.02°.

[0244] 2. Unit cell parameters

[0245] The unit cell parameters of tanshinone hemihydrate are as follows: Figure 2As shown, the results indicate that the crystal belongs to the hexagonal crystal system, with space group P65 and cell parameters as follows: α=β=90.00°, γ=120.00°, unit cell volume The number of asymmetric units within the unit cell is Z = 6. The final determined chemical formula is C9H. 11 O 5.5 The calculated molecular weight is 207.18. The calculated crystal density is 1.519 mg / mm³. 3 .

[0246] 3. Three-dimensional structure

[0247] The stereostructure of tanshinone hemihydrate is as follows: Figure 3 As shown, the results indicate that the compound contains half a water of crystallization and that the hydroxyl group at the α-position of carbon in the compound is in the R configuration.

[0248] 4. Infrared spectrum

[0249] The infrared spectrum of tanshinone hemihydrate is as follows: Figure 4 As shown, the results indicate that the length is 3470.94-3523.46 cm. -1 The peak is the hydroxyl stretching vibration peak, at 3198.62 cm⁻¹. -1 The peak corresponds to the stretching vibration of the carboxyl hydroxyl group, at 1739.85 cm⁻¹. -1 The peak represents the carbonyl stretching vibration, ranging from 1527.31 to 1606.04 cm⁻¹. -1 The peaks represent the stretching vibrations of the carbon-carbon double bond benzene ring, indicating the presence of hydroxyl, carbonyl, methyl, and benzene ring groups in the sample molecule. Therefore, the infrared spectral data of the standard sample are consistent with the molecular structure of tanshinone hemihydrate.

[0250] 5. NMR results

[0251] The 1H NMR spectrum of the tanshinone hemihydrate is as follows: Figure 5 As shown, the 1H NMR (600MHz, DMSO-d6) values ​​are: δ 12.37 (s, 1H), 8.69–8.60 (m, 2H), 7.00–6.94 (m, 2H), 6.66–6.45 (m, 3H), 5.18 (s, 1H), 4.10 (s, 1H), 2.76–2.71 (dd, J = 6.95Hz, 1H), 2.58 (s, 1H).

[0252] The carbon NMR spectrum results of the tanshinone hemihydrate are as follows: Figure 6 As shown, 13C NMR (600MHz, DMSO-d6) δ 175.12, 144.57, 143.47, 128.65, 119.92, 116.73, 115.04, 71.33, 38.62.

[0253] The tanshinone hemihydrates prepared in Examples 4-6 and 8-14 were tested. The results showed that the test results of Examples 4-6 and 8-14 were the same as those of Example 7, that is, the tanshinone hemihydrates prepared in Examples 4-14 were all the same substance.

[0254] 6. HPLC

[0255] HPLC analysis of tanshinone hemihydrate compounds, such as Figure 7 As shown, the results indicate that the purity can reach 99.8%.

[0256] 7. Stability

[0257] The stability of the new crystalline form of tanshinone hemihydrate was tested after 0 months, 3 months, and 6 months at 40℃±2℃ and 75%±5% relative humidity. The results are shown in Table 2. The results indicate that the new crystalline form of tanshinone hemihydrate has good stability.

[0258] Table 2 Stability test results

[0259]

[0260] 8. Solubility

[0261] The solubility of tanshinone hemihydrate prepared in Example 7 and sodium tanshinone II crystal form prepared in Comparative Example 2 was tested.

[0262] The results showed that the tanshinone hemihydrate prepared in Example 7 had a solubility of 700 mg / mL in water, 760 mg / mL in ethanol and methanol, 580 mg / mL in diethyl ether, and 600 mg / mL in acetonitrile; while the sodium tanshinone II crystal form prepared in Comparative Example 2 had a water solubility of 213 mg / mL and a solubility of only 15 mg / mL in ethanol and methanol.

[0263] Example 16: Preparation of direct drinking powder from tanshinone hemihydrate

[0264] The tanshinone hemihydrate crystal forms obtained in Examples 4-10 are used here. Taking Example 4 as an example, the direct drinking powder is prepared with the following formula:

[0265] The direct-drinking powder was prepared according to the following mass percentages: 15% orange powder, 20% cranberry powder, 15% apple powder, 15% banana powder, 3% L-ascorbic acid, 31.5% xylitol, and 0.5% tanshinone hemihydrate.

[0266] Example 17: Preparation of tablets from tanshinone hemihydrate

[0267] The tanshinone hemihydrate crystal forms obtained in Examples 4-10 are used here. Taking Example 4 as an example, tablets are prepared with the following formulation:

[0268] The tablets were prepared according to the following mass percentages: 10% orange powder, 39% cranberry powder, 5% apple powder, 5% banana powder, 3% L-ascorbic acid, 12.3% xylitol, 20% starch, 5% maltodextrin, 0.1% food coloring, 0.1% food flavoring, and 0.5% tanshinone hemihydrate.

[0269] Example 18: Preparation of filled gummy candies from tanshinone hemihydrate

[0270] The tanshinone hemihydrate crystal forms obtained in Examples 4-10 are used here. Taking Example 4 as an example, a sandwich candy is prepared with the following formula:

[0271] The following percentages by weight are used to prepare the filled soft candy: 35% orange juice (or cranberry juice, apple juice, or banana juice), 15% malt syrup, 16.8% white sugar, 15% gelatin, 10% carrageenan, 5% pectin, 1% citric acid, 0.5% calcium lactate, 1% L-ascorbic acid, 0.2% edible flavoring, and 0.5% tanshinone hemihydrate.

[0272] Example 19: Products prepared from tanshinone hemihydrate

[0273] A product containing tanshinone hemihydrate with the following chemical structural formula:

[0274]

[0275] Optionally, the product may be food, medicine, or health supplement.

[0276] Optionally, the drug uses tanshinone hemihydrate as the sole active ingredient, or tanshinone hemihydrate as one of the effective ingredients.

[0277] Optionally, the drug is a drug for treating cardiovascular and cerebrovascular diseases.

[0278] Optionally, the drug may have one or more functions such as anti-oxidation, anti-inflammation, anti-liver fibrosis, anti-atherosclerosis, and inhibition of thrombosis.

[0279] Optionally, the dosage form of the drug includes, but is not limited to, liquid, solid, and semi-solid dosage forms; further optionally, liquid dosage forms include, but are not limited to, solutions, injections, lotions, mixtures, and liniments; further optionally, solid dosage forms include, but are not limited to, powders, granules, tablets, capsules, and films; further optionally, semi-solid dosage forms include, but are not limited to, ointments, suppositories, and pastes.

[0280] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A tanshinone hemihydrate crystal, characterized in that, The structural formula is shown in Equation 1. ， Among them, X-ray powder diffraction of crystal 2 θ Diffraction peaks are observed at ±0.02° at 6.398°, 10.919°, 12.567°, 16.597°, 18.825°, 22.574°, 24.232°, 26.594°, 27.275°, 28.684°, 29.401°, 31.372°, 32.642°, 33.267°, 35.042°, 36.225°, 36.711°, and 38.367°.

2. The crystal according to claim 1, characterized in that, In the X-ray powder diffraction pattern of the crystal, its 2 θ The ±0.02° angle value, interplanar spacing d, and relative intensity of diffraction peaks have the following characteristics: ； The unit cell parameters of the crystal are: α=β=90.00°, γ=120.00°.

3. The crystal according to claim 1, characterized in that, Preparation methods include: The crude tanshinone was mixed with an organic solvent and dissolved; water was added, and the mixture was stirred, aged, filtered, and dried to obtain tanshinone hemihydrate crystals. Optionally, crude tanshinone can be extracted from tanshinone. Optionally, crude tanshinone can be prepared by genetically engineered bacteria; Alternatively, crude tanshinone can be prepared by chemical synthesis; Optionally, the ratio of crude tanshinone to organic solvent is 5g:7.5-15mL; the volume ratio of organic solvent to water is 7.5-15:0.

1. Optionally, the organic solvent includes one or more of ethyl acetate, isopropyl acetate, ethyl formate, and propyl formate.

4. The crystal according to claim 3, characterized in that, Dissolving involves heating at 40–60°C until completely dissolved. Stir at 20-100 rpm for 1-3 hours; Aging involves standing at 2–8℃ for 48–96 hours. Drying includes hot air drying, vacuum drying, freeze drying, and microwave drying; Alternatively, vacuum drying can be performed at 40–50°C for 6–10 hours.

5. A method for preparing a tanshinone hemihydrate compound, characterized in that, Including the following steps: The crude tanshinone was mixed with an organic solvent and dissolved. Add water, stir, age, filter, and dry to obtain tanshinone hemihydrate; Optionally, the organic solvent includes one or more of ethyl acetate, isopropyl acetate, ethyl formate, and propyl formate; Optionally, the ratio of crude tanshinone to organic solvent is 5g:7.5-15mL; Optionally, the volume ratio of organic solvent to water is 7.5–15:0.1; Optionally, the stirring is performed at 20–100 rpm for 1–3 hours; Optionally, aging involves standing at 2–8°C for 48–96 hours; Optionally, drying includes hot air drying, vacuum drying, freeze drying, and microwave drying; Alternatively, vacuum drying can be performed at 40–50°C for 6–10 hours.

6. A method for improving the solubility and / or stability of tanshinone, characterized in that, Including the following steps: The crude tanshinone was mixed with an organic solvent and dissolved; water was added, and the mixture was stirred, aged, filtered, and dried to obtain a hemihydrate tanshinone compound with improved solubility. Optionally, the ratio of crude tanshinone to organic solvent is 5g:7.5-15mL; Optionally, the volume ratio of organic solvent to water is 7.5–15:0.1; Optionally, the stirring is performed at 20–100 rpm for 1–3 hours; Optionally, aging involves standing at 2–8°C for 48–96 hours; Optionally, drying includes hot air drying, vacuum drying, freeze drying, and microwave drying; Alternatively, vacuum drying can be performed at 40–50°C for 6–10 hours.

7. The application of the tanshinone hemihydrate crystal according to any one of claims 1 to 4, or the method for preparing the tanshinone hemihydrate according to claim 5, or the method for improving the solubility of tanshinone according to claim 6, in the preparation of food, health products, or pharmaceuticals.

8. A product characterized in that, The product contains the tanshinone hemihydrate compound as described in any one of claims 1 to 4; the product is a food, medicine, or health product. Optionally, the food includes candy and beverages; the pharmaceutical includes liquid, solid, and semi-solid dosage forms. Optionally, liquid preparations include solutions, injections, lotions, mixtures, and liniments; Optionally, solid dosage forms include powders, granules, tablets, capsules, and films; Optionally, semi-solid preparations include ointments, suppositories, and pastes.

9. A genetically engineered Escherichia coli bacterium, characterized in that, The genetically engineered Escherichia coli strain overexpressed 4-hydroxyphenylacetic acid hydroxylase; Optionally, the genetically engineered Escherichia coli also has one or more genes knocked out from tyrR, pykF, pheA, yeeP, efeU, yeeL, and ycgH. Optionally, the engineered E. coli strain also overexpresses the genes glk and aroG. fbr ,aroE,tyrA fbr One or more of tyrB and ldh.

10. The use of the genetically engineered Escherichia coli of claim 9 in the preparation of tanshinone or the tanshinone hemihydrate crystals of any one of claims 1 to 4.

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