Crystal forms of ergothioneine

Abstract

The invention discloses a crystal form of ergothioneine and a preparation method thereof. According to the method provided by the invention, the anhydrous crystal form of ergothioneine can be efficiently and stably produced; the ergothioneine crystal form is high in purity, high in mass fraction, low in moisture content and high in crystallization yield, so that the ergothioneine crystal form has a wide application prospect in the fields of dietary supplements, foods or cosmetics.

Description

Crystalline forms of ergothioneine Technical Field

[0001] The invention belongs to the field of crystallization technology, and in particular relates to a crystal form of ergothioneine and a preparation method thereof. Background Art

[0002] Ergothioneine (EGT) is a rare, naturally occurring chiral amino acid derived from plants and capable of accumulating in animals. It is safe and non-toxic. It is a natural antioxidant with numerous physiological functions, including detoxification, maintaining DNA growth, protecting against radiation, fighting inflammation, resisting aging, protecting the nervous system, inhibiting developmental defects, and protecting the liver. Ergothioneine is present throughout the human body, with highest concentrations in the kidneys, liver, red blood cells, and semen, making it crucial for human health. Clinical studies are underway domestically and internationally on the effects of ergothioneine on cognition, mood, and sleep. Given EGT's physiological activity, it holds broad application prospects in dietary supplements, cosmetics, and pharmaceuticals.

[0003] The method of producing thioneine at present has chemical synthesis, biological extraction method and biological fermentation method.The thioneine prepared by existing method is mostly lamellar amorphous, and the crystallinity of crystal is not high, and because thioneine solubility in water is high, therefore crystallization yield is generally on the low side.The condition affecting thioneine crystallization has a lot, such as the purity of thioneine in the supersaturation of solution, solution, inorganic salt kind and addition thereof, anti-solvent kind and addition thereof, stirring mode and mixing degree, crystallization temperature etc., therefore yield and purity are high, crystallization time is short, energy consumption and material consumption are low, crystal size is evenly distributed The preparation of thioneine crystals and the selection of suitable crystal formation are the key contents that those skilled in the art need to study.

[0004] Summary of the Invention

[0005] According to the conditions required for the formation of ergothioneine crystals, the present invention controls the crystallization process to stably prepare an anhydrous ergothioneine crystal. The anhydrous ergothioneine crystal has the advantages of high yield (≥90%), high purity (>99.5%), short crystallization time, low energy consumption and material consumption, uniform crystal particle size distribution, etc., and the obtained anhydrous ergothioneine crystal has good fluidity and stability, and can be better applied in the fields of dietary supplements, food or cosmetics.

[0006] In one aspect, the present invention provides an anhydrous crystalline Form A of ergothioneine, having an X-ray powder diffraction pattern comprising peaks at diffraction angles (2θ) of 8.6°±0.1°, 19.0°±0.1°, 26.4°±0.1°, and 32.3°±0.1°.

[0007] In some embodiments, the anhydrous crystalline Form A further comprises an X-ray powder diffraction pattern of one or more peaks at diffraction angles (2θ) of 15.6°±0.1°, 20.5°±0.1°, and 24.9°±0.1°.

[0008] In some embodiments, the anhydrous crystalline Form A further comprises one or more peaks at diffraction angles (2θ) of 17.3°±0.1°, 22.3°±0.1°, and 34.7°±0.1°.

[0009] In some embodiments, the X-ray powder diffraction pattern of anhydrous crystalline Form A is shown in FIG1 .

[0010] On the other hand, the present invention provides a method for preparing the anhydrous crystalline form A as described above, comprising the following steps: dissolving thioneine in water to obtain an thioneine solution; adding one or more monovalent cation salts of formate, acetate, and citrate after concentration, stirring and allowing the temperature to reach equilibrium, and adding an organic solvent miscible with water or a mixed solvent of an organic solvent miscible with water and water to the solution, and continuing stirring; cooling to obtain thioneine slurry; and filtering and drying the thioneine slurry to obtain anhydrous crystalline form A.

[0011] In some embodiments, the thioneine for dissolving can be thioneine amorphous body. In some embodiments, concentrated makes the concentration of thioneine solution exceed 120g / L. Concentrated can be carried out using the mode of rotary evaporation concentration. In some embodiments, rotary evaporation concentration makes the concentration of thioneine solution exceed 200g / L, preferably 250g / L, more preferably after 290g / L, pH is adjusted to 6-8, preferably 6.5-7.5, more preferably 6.5-7.

[0012] In some embodiments, the monovalent cation salt of formate, acetate, or citrate is selected from sodium formate, potassium formate, ammonium formate, sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium citrate, and ammonium citrate; and the organic solvent is selected from one or more of lower alkyl alcohols or lower alkyl ketones. In some embodiments, the monovalent cation salt of formate, acetate, or citrate is selected from sodium formate, sodium acetate, potassium acetate, ammonium acetate, and sodium citrate; and the organic solvent is selected from one or more of ethanol, methanol, isopropanol, and acetone. In some embodiments, the organic solvent is selected from one or more of ethanol, methanol, and isopropanol, and the flow rate of the organic solvent is 5-15% BV / h, preferably 10% BV / h.

[0013] In some embodiments, the temperature is lowered at a low speed to 10-30° C., preferably 15-25° C. In some embodiments, the drying can be performed by vacuum drying.

[0014] On the other hand, the present invention provides a crystalline anhydrous Form B of ergothioneine, whose X-ray powder diffraction pattern includes peaks at diffraction angles (2θ) of 15.3°±0.1°, 22.8°±0.1°, 27.7°±0.1° and 33.5°±0.1°.

[0015] In some embodiments, the anhydrous crystalline Form B further comprises an X-ray powder diffraction pattern of one or more peaks at diffraction angles (2θ) of 11.2°±0.1°, 22.5°±0.1°, and 35.4°±0.1°.

[0016] In some embodiments, the anhydrous crystalline Form B further comprises an X-ray powder diffraction pattern of one or more peaks at diffraction angles (2θ) of 12.8°±0.1°, 28.4°±0.1°, 39.9°±0.1°.

[0017] In some embodiments, the X-ray powder diffraction pattern of anhydrous Form B is shown in FIG2 .

[0018] On the other hand, the present invention provides a method for preparing anhydrous crystalline Form B as described above, comprising the following steps: dissolving thioneine in water to obtain an thioneine solution; adding an organic solvent miscible with water or a mixed solvent of an organic solvent miscible with water and water to the solution under stirring, and cooling to obtain thioneine magma; and filtering and drying the thioneine magma to obtain anhydrous crystalline Form B.

[0019] In some embodiments, the ergothioneine used for dissolving can be an ergothioneine amorphous body. In some embodiments, dissolution is promoted by any one or more of the following combinations: heating, adjusting the pH to 6-7, or stirring; after dissolution, it is concentrated so that the concentration of the ergothioneine solution exceeds 120g / L. In some embodiments, heating can be carried out in a water bath at 30-80°C, preferably 35-70°C, more preferably 40-60°C. In some embodiments, the pH is adjusted to 6-8, preferably 6.5-7.5, more preferably 6.5-7. In some embodiments, pH adjustment can be carried out in a conventional manner, such as using sodium hydroxide or hydrochloric acid solution. In some embodiments, stirring can be carried out at 50-300rpm, preferably 100-200rpm, more preferably 150rpm to promote dissolution. In some embodiments, vacuum concentration is performed at 40-80°C, preferably 50-70°C, more preferably 60-70°C, so that the concentration of the ergothioneine solution exceeds 200 g / L, preferably 250 g / L, more preferably 290 g / L.

[0020] In some embodiments, the organic solvent is selected from one or more of lower alkyl alcohols or lower alkyl ketones. In some embodiments, the organic solvent is selected from one or more of ethanol, methanol, isopropanol, acetone, preferably selected from one or more of ethanol, methanol, isopropanol. In some embodiments, an organic solvent miscible with water or a mixed solvent of an organic solvent miscible with water and water can be added by streaming, such as adding with a peristaltic pump. In some embodiments, the rate of addition of an organic solvent miscible with water or a mixed solvent of an organic solvent miscible with water and water is less than 60% / h of the volume of thioneine solution, preferably less than 55% / h, more preferably less than 50% / h; the volume of an organic solvent miscible with water or a mixed solvent of an organic solvent miscible with water and water is 0.1-5 times, preferably 0.3-2 times, of the volume of thioneine solution.

[0021] In some embodiments, after adding a water-miscible organic solvent or a mixed solvent of a water-miscible organic solvent and water, the temperature is lowered to a temperature not higher than 20°C. In some embodiments, after adding a water-miscible organic solvent or a mixed solvent of a water-miscible organic solvent and water, the temperature is lowered to a temperature not higher than 20°C and maintained for more than 0.5 hours, or a temperature not higher than 15°C, preferably 12°C, and maintained for more than 0.5 hours, preferably 1 hour, more preferably 1.5 hours. In some embodiments, drying can be carried out by vacuum drying at 50-70°C, preferably 55-65°C.

[0022] On the other hand, the present invention provides a crystalline anhydrous Form C of ergothioneine, whose X-ray powder diffraction pattern includes peaks at diffraction angles (2θ) of 11.2°±0.1°, 18.1°±0.1°, 24.0°±0.1° and 26.9°±0.1°.

[0023] In some embodiments, the anhydrous crystalline Form C further comprises an X-ray powder diffraction pattern of one or more peaks at diffraction angles (2θ) of 12.8°±0.1°, 19.8°±0.1°, and 24.6°±0.1°.

[0024] In some embodiments, the anhydrous crystalline Form C further comprises an X-ray powder diffraction pattern of one or more peaks at diffraction angles (2θ) of 15.6°±0.1°, 19.0°±0.1°, 21.6°±0.1°.

[0025] In some embodiments, the X-ray powder diffraction pattern of anhydrous crystalline Form C is shown in FIG3 .

[0026] On the other hand, the present invention provides a method for preparing the anhydrous crystalline Form C as described above, comprising the following steps: dissolving ergothioneine in water, heating the mixture to 60-100° C., dropwise adding a water-miscible organic solvent or a mixed solvent of a water-miscible organic solvent and water and stirring; then rapidly cooling the mixture to 10-30° C., continuing stirring to obtain the anhydrous crystalline Form C.

[0027] In some embodiments, the organic solvent is selected from one or more lower alkyl alcohols or lower alkyl ketones, preferably one or more ethanol, methanol, isopropanol, and acetone. In some embodiments, the temperature is raised to 70-95°C, preferably 75-90°C; the organic solvent is selected from one or more ethanol, methanol, and isopropanol; and the temperature is rapidly lowered to 15-25°C.

[0028] In some embodiments, the anhydrous crystalline form is used to prepare food, beverages, supplements, nutritional products, and cosmetics.

[0029] In another aspect, the present invention provides a composition comprising an effective amount of the anhydrous crystalline form as described above, and a pharmaceutically acceptable carrier.

[0030] In some embodiments, the composition is used to prepare a food, a beverage, a supplement, a nutraceutical, or a cosmetic.

[0031] Compared with the prior art, the method provided by the present invention can efficiently and stably produce anhydrous thioneine crystalline form; and the thioneine crystalline form of the present invention has high purity, a mass fraction of more than 99.5%, a low moisture content (<0.3%), and a high crystallization yield (≥90%), and therefore can have a wide range of application prospects in the fields of dietary supplements, food or cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is an XRPD pattern of anhydrous crystalline Form A of the present invention.

[0033] FIG2 is an XRPD pattern of anhydrous crystalline Form B of the present invention.

[0034] FIG3 is an XRPD pattern of anhydrous crystalline Form C of the present invention.

[0035] FIG4 is a DSC graph of anhydrous crystalline Form A of the present invention.

[0036] FIG5 is a DSC graph of anhydrous crystalline Form C of the present invention. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention will now be described in detail with reference to the embodiments thereof. Although the present invention will be described in conjunction with the preferred embodiments, it should be understood that they are not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to cover substitutions, modifications and equivalents, which may be included within the spirit and scope of the present invention as defined in the claims. In addition, in the detailed description of the present invention, many specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention can be practiced without these specific details. In other cases, well-known methods, procedures, components and other features are not described in detail to avoid unnecessarily obscuring various aspects of the present invention.

[0038] As used herein, the term "or" is intended to include "and" and "or." In other words, the term "or" can also be replaced with "and / or."

[0039] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0040] As used herein, the term "comprises" or "includes" or variations thereof refers to instances where the term is used in its non-limiting sense, meaning that items following the term are included, but items not specifically mentioned are not excluded. It also includes the more restrictive verbs 'consisting essentially of' and 'consisting of.'

[0041] As used herein, the terms "about" and "approximately" provide flexibility in numerical values ​​by providing that a given value may be "slightly higher" or "less than" an endpoint. The flexibility of this term can be determined by the specific variables and is within the knowledge of those skilled in the art to determine based on experience and the relevant description herein.

[0042] As used herein, the term "administer" refers to the process of delivering the disclosed crystalline form or active ingredient to a subject. The crystalline form of the present invention can be administered in various suitable ways to exert the desired effect, including oral, intragastric, parenteral (referring to intravenous and intra-arterial and other suitable parenteral routes), topical administration, etc. The crystalline form of the present invention can be administered to a subject in an effective dose and / or at an effective frequency. In some embodiments, multiple doses of the crystalline form are administered over a period of time. The frequency of administration, duration, etc. of the crystalline form can vary according to any of a variety of factors, including the subject's response, desired effect, etc.

[0043] As used herein, the term "effective amount" refers to the amount required to achieve the effects taught herein. The amount to be administered can vary depending on factors such as individual sensitivity, individual age, sex, and weight, individual idiosyncrasies, and the like. In accordance with the present disclosure, a suitable single dose size is a dose that, when administered one or more times over an appropriate time period, achieves the effects described above.

[0044] As used herein, the term "pharmaceutically acceptable" refers to pharmaceutically, physiologically, dietary and / or nutritionally acceptable, and refers to those compositions or agents, materials or combinations of compositions and / or dosage forms thereof that are within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals, compatible with the other ingredients of the composition, without excessive toxicity, irritation, allergic response or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0045] In some embodiments, the crystalline forms of the present invention can be prepared as a composition together with a dietary or pharmaceutically acceptable carrier. In the present invention, the administration form of the composition involves a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the composition and is not harmful to the subject, i.e., suitable for consumption or nutritionally acceptable.

[0046] In some embodiments, the crystalline forms of the present invention may be administered with other supplements, such as vitamins, minerals, nootropics, and other supplements known in the art.

[0047] The following examples are illustrative of selected embodiments of the present invention and are not intended to limit the scope of the invention.

[0048] Experimental technique described in the following embodiment, if no special instructions, is conventional method;Described reagent and material, if no special instructions, all can be obtained from commercial sources.The rotating speed of stirring can be suitable rotating speed, such as 150rpm;Ethanol is anhydrous ethanol solution;The thioneine content of thioneine amorphous body can be 98%.

[0049] Example 1. Preparation of anhydrous crystal form A

[0050] 300g thioneine amorphous bodies are dissolved in 1.5L pure water, and by the mode of rotary evaporation concentration, the volume of solution is concentrated to 1L, so that wherein the concentration of thioneine is 300g / L;Then 1.8g sodium acetate is added in solution, stirring and dissolving, and regulate its pH=7.0, then dehydrated alcohol is added by peristaltic pump stream in solution, and flow rate is 10%BV / h, adds 0.5BV (about 0.5L) dehydrated alcohol altogether.Ethanol continues to stir 1 hour after adding, subsequently with the temperature falling rate of 5 ℃ / h, solution is cooled 20 ℃, then crystal is carried out suction filtration, oven dry, obtain 276g anhydrous thioneine crystalline finished product, yield is 92%, and content is 99.9%.

[0051] Example 2. Preparation of anhydrous crystal form B

[0052] 150g thioneine amorphous bodies are dissolved in 1L pure water, accelerated dissolution by stirring and water-bath (45-50 DEG C), then the pH value of regulating solution is 7.0, then by the mode (65 DEG C) of vacuum concentration, the concentration of thioneine is increased to 300g / L.Concentrated solution is transferred in 2L crystallizer, under continuous stirring, stream is added with peristaltic pump in crystallizer, and flow velocity is: 100mL / h, and total addition is 1000mL.After dehydrated alcohol adds and finishes, be cooled to 5 DEG C by the chuck of crystallizer, maintain 1 hour at this temperature, then stop stirring, the thioneine magma in crystallizer is emitted, crystal suction filtration, and put into vacuum drying oven at 60 DEG C and carry out drying, i.e. obtain anhydrous thioneine crystalline finished product 144g, yield is 96%, and content is 99.8%.

[0053] Example 3. Preparation of anhydrous crystal form B

[0054] 200g thioneine amorphous bodies are dissolved in 1L pure water, accelerated dissolution by stirring and water-bath (50-60 DEG C), then the pH value of regulating solution is 7.0, then by the mode (65 DEG C) of vacuum concentration, the concentration of thioneine is increased to 350g / L.Concentrated solution is transferred in 2L crystallizer, under continuous stirring, stream is added with peristaltic pump in crystallizer, flow velocity is: 200mL / h, and total addition is 1000mL.After methanol stream is added and terminates, 10 DEG C are cooled to by the chuck of crystallizer, maintained 1 hour at this temperature, then stop stirring, the thioneine magma in crystallizer is emitted, crystal suction filtration, and put into 60 DEG C of vacuum drying ovens and carry out drying, i.e., obtain anhydrous thioneine crystalline finished product 190g, yield is 95%, and content is 99.8%.

[0055] Example 4. Preparation of anhydrous crystal form B

[0056] 50g thioneine amorphous bodies are dissolved in 0.5L pure water, accelerated dissolution by stirring and water-bath (40~50 DEG C), then the pH value of regulating solution is 7.0, then by the mode (65 DEG C) of vacuum concentration, the concentration of thioneine is increased to 310g / L.Concentrated solution is transferred in 2L crystallizer, under continuous stirring, stream is added with peristaltic pump in crystallizer, and flow velocity is: 80mL / h, and total addition is 320mL.After isopropyl alcohol stream is added and finishes, 12 DEG C are cooled to by the chuck of crystallizer, maintained 1 hour at this temperature, then stop stirring, the thioneine magma in crystallizer is emitted, crystal suction filtration, and put into 60 DEG C of vacuum drying ovens and carry out drying, i.e., obtain anhydrous thioneine crystalline finished product 45.5g, yield is 91%, and content is 99.7%.

[0057] Example 5. Preparation of anhydrous crystal form C

[0058] 1g of thioneine was dissolved in 1mL of water and heated to 80-90°C to dissolve, cooled to 35°C, and 5mL of methanol was added dropwise; stirred for 30min. Then the temperature was rapidly cooled to 20°C and stirred for 30min. 0.95g of anhydrous thioneine crystals was finally obtained, with a yield of 95% and a content of 99.9%.

[0059] Comparative Example 1

[0060] 75g thioneine raw material dissolution is in 0.5L pure water, dissolved by stirring and water-bath (30-35 DEG C), and solution is transferred in the crystallizer of 2L, then after stirring 1 hour at 25 DEG C, stream is added with peristaltic pump in crystallizer, and flow velocity is: 100mL / h, and total addition is 1000mL.After absolute ethanol stream is added and finishes, be cooled to 10 DEG C by the chuck of crystallizer, maintain 1 hour at this temperature, then stop stirring, the thioneine magma in crystallizer is emitted, crystal suction filtration, and put into vacuum drying oven at 60 DEG C and dry, what is obtained is the thioneine solid 52.5g of amorphous body, and yield is 70%, and content is 98.5%.

[0061] The crystal forms prepared in the examples were subjected to X-ray diffraction (XRD), differential scanning calorimetry (DSC), elemental analysis, particle size analysis, quantitative nuclear magnetic resonance (NMR), Raman spectroscopy (Raman), infrared spectroscopy (IR), TGA, IPC-MS, DVS and other tests.

[0062] X-ray diffraction

[0063] The X-ray powder diffraction pattern was obtained using a SmartLab 3KW X-ray powder diffractometer under the following conditions: diffraction line: Cu_K-beta (40KV, 40mA), scanning rate: 20.00deg / min, scanning range: 5°~40°.

[0064] The XRPD pattern of the anhydrous crystalline Form A obtained in Example 1 is shown in FIG1 , and the XRPD data are shown in Table 1.

[0065] Table 1

[0066] The XRPD pattern of Form B obtained in Example 2 is shown in FIG2 , and the XRPD data are shown in Table 2.

[0067] Table 2

[0068] The XRPD results of the crystalline forms obtained in Examples 3 and 4 were basically consistent with those in Example 2.

[0069] The XRPD pattern of Form C obtained in Example 5 is shown in FIG3 , and the XRPD data are shown in Table 3.

[0070] Table 3

[0071] Differential Scanning Calorimetry

[0072] Differential scanning calorimetry was performed using a TA Q2000 module with a thermal analysis controller, and data were collected and analyzed using TA Instruments Thermal Solutions software. Approximately 1-5 mg of sample was accurately weighed into a special aluminum crucible with a lid, and sample analysis was performed from 40°C to approximately 300°C using a linear heating device at 10°C / min. During use, the DSC chamber was purged with dry nitrogen. The differential scanning calorimetry (DSC) curve of the anhydrous crystalline Form A of Example 1 is shown in Figure 4, which includes an endothermic peak at 269.61°C, and an error tolerance of ±3°C may exist. The differential scanning calorimetry (DSC) curve of the crystalline Form C of Example 5 is shown in Figure 5, which includes an endothermic peak at 273.30°C, and an error tolerance of ±3°C may exist.

[0073] Elemental analysis

[0074] Elemental analysis of Form C of Example 5 using an elemental analyzer revealed: C, 47.01%, H, 6.23%, N, 18.30%, and S, 13.59%. The elemental analysis results of the anhydrous Form A prepared in Example 1 and the Form B prepared in Examples 2-4 were substantially consistent with those of Example 5.

[0075] Quantitative NMR

[0076] The anhydrous form A of Example 1 was recorded in a spectrometer. 1 H NMR spectrum: 1 H NMRδ (ppm) 6.71, 6.67, 6.25, 4.70, 3.83, 3.79, 3.12.

[0077] The crystal form C of Example 5 was recorded in the spectrometer. 1 H NMR spectrum: 1 H NMR (400 MHz, deuterium oxide) δ (ppm) 6.71, 6.67, 6.25, 4.70, 3.83, 3.79, 3.14.

[0078] The NMR results of Form B obtained in Examples 2-4 were also basically consistent with those in Examples 1 and 5.

[0079] The other crystalline forms of the present invention were characterized in a similar manner.

[0080] Property analysis and comparison

[0081] Using thioneine crude product as raw material, prepare respectively these 3 kinds of crystal form finished products of A, B, C, and compare its character with amorphous body thioneine, result is as shown in Table 4:

[0082] Table 4 *: Prepared as a 10% aqueous solution and measured at 400nm

[0083] The preparation methods of ergothioneine generally include chemical synthesis, enzymatic catalysis, and fermentation. Fermentation is the current mainstream production method due to its low cost and ease of industrial scale-up. However, the fermentation broth is dark in color and high in impurities. After purification and concentration, the solution still has a certain color. The crude ergothioneine after initial crystallization is usually yellow.

[0084] As can be seen from the data in Table 4, 3 kinds of crystal formations prepared by the embodiment of the present invention are white transparent crystals, and the absorbance of its aqueous solution at 400nm is <0.01, and is odorless.And the amorphous body thioneine reference substance prepared with same raw materials, color is slightly yellow, and its absorbance is 0.105, much higher than several crystal formations provided by the present invention.In addition, amorphous body has obvious fishy smell because content and purity are not high. In addition, amorphous body hygroscopicity is strong, and moisture reaches 1.29%, is not easy for long-term preservation.And several crystal formations prepared by the embodiment of the present invention are all anhydrous crystals, and moisture content is all <0.2%, is not easy for hygroscopicity, is conducive to the stability of long-term preservation.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations to these embodiments without departing from the principles and purpose of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. An anhydrous crystalline form A of ergothioneine, characterized in that, The anhydrous crystalline Form A has an X-ray powder diffraction pattern including peaks at diffraction angles (2θ) of 8.6°±0.1°, 19.0°±0.1°, 26.4°±0.1°, and 32.3°±0.1°.

2. The anhydrous crystalline form A according to claim 1, characterized in that: The X-ray powder diffraction pattern of the anhydrous crystalline form A further includes one or more peaks at diffraction angles (2θ) of 15.6°±0.1°, 20.5°±0.1°, and 24.9°±0.1°.

3. The anhydrous crystalline form A according to claim 1 or 2, characterized in that: The X-ray powder diffraction pattern of the anhydrous crystalline form A further includes one or more peaks at diffraction angles (2θ) of 17.3°±0.1°, 22.3°±0.1°, and 34.7°±0.1°.

4. The anhydrous crystalline form A according to any one of claims 1 to 3, characterized in that: The X-ray powder diffraction pattern of the anhydrous crystalline form A is shown in FIG1 .

5. A method for preparing anhydrous crystalline form A as claimed in any one of claims 1 to 4, characterized in that: The following steps are involved: Dissolving thioneine in water to obtain an thioneine solution; adding one or more monovalent cation salts of formate, acetate and citrate after concentration, stirring and allowing the temperature to reach equilibrium, adding an organic solvent miscible with water or a mixed solvent of an organic solvent miscible with water and water to the solution, and continuing stirring; cooling to obtain thioneine slurry; filtering and drying the thioneine slurry to obtain the anhydrous crystalline form A.

6. The preparation method according to claim 5, characterized in that: Concentrating makes the concentration of the thioneine solution exceed 120g / L.

7. The preparation method according to claim 5 or 6, characterized in that: The monovalent cation salts of formate, acetate and citrate are selected from sodium formate, potassium formate, ammonium formate, sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium citrate and ammonium citrate; and the organic solvent is selected from one or more of lower alkyl alcohols or lower alkyl ketones.

8. The preparation method according to any one of claims 5 to 7, characterized in that: The cooling is performed at a low speed to a temperature of 10-30°C.

9. An anhydrous crystal form B of ergothioneine, characterized in that, The anhydrous crystalline Form B has an X-ray powder diffraction pattern including peaks at diffraction angles (2θ) of 15.3°±0.1°, 22.8°±0.1°, 27.7°±0.1°, and 33.5°±0.1°.

10. The anhydrous crystal form B according to claim 9, characterized in that The X-ray powder diffraction pattern of the anhydrous crystalline form B further includes one or more peaks at diffraction angles (2θ) of 11.2°±0.1°, 22.5°±0.1°, and 35.4°±0.1°.

11. The anhydrous crystalline form B according to claim 9 or 10, characterized in that: The X-ray powder diffraction pattern of the anhydrous crystalline form B further includes one or more peaks at diffraction angles (2θ) of 12.8°±0.1°, 28.4°±0.1°, and 39.9°±0.1°.

12. Anhydrous crystalline form B according to any one of claims 9 to 11, characterized in that The X-ray powder diffraction pattern of the anhydrous crystalline form B is shown in FIG2 .

13. A method for preparing anhydrous crystalline form B according to any one of claims 9 to 12, characterized in that: The following steps are involved: Dissolving thioneine in water to obtain a thioneine solution; adding an organic solvent miscible with water or a mixed solvent of an organic solvent miscible with water and water to the solution under stirring, cooling to obtain thioneine slurry; filtering and drying the thioneine slurry to obtain the anhydrous crystal type B.

14. The preparation method according to claim 13, characterized in that: The dissolution is promoted by any one or a combination of the following: heating, adjusting the pH to 6-7 or stirring; and concentrating after dissolution so that the concentration of the ergothioneine solution exceeds 120 g / L.

15. The preparation method according to claim 13 or 14, characterized in that: The organic solvent is selected from one or more of lower alkyl alcohols or lower alkyl ketones.

16. The preparation method according to any one of claims 13 to 15, characterized in that: After adding the water-miscible organic solvent or the mixed solvent of the water-miscible organic solvent and water, the temperature is lowered to a temperature not higher than 20°C.

17. An anhydrous crystalline form C of ergothioneine, characterized in that, The anhydrous crystalline Form C has an X-ray powder diffraction pattern including peaks at diffraction angles (2θ) of 11.2°±0.1°, 18.1°±0.1°, 24.0°±0.1°, and 26.9°±0.1°.

18. The anhydrous crystalline form C according to claim 17, characterized in that The X-ray powder diffraction pattern of the anhydrous crystalline Form C further includes one or more peaks at diffraction angles (2θ) of 12.8°±0.1°, 19.8°±0.1°, and 24.6°±0.1°.

19. Anhydrous crystalline form C according to claim 17 or 18, characterized in that The X-ray powder diffraction pattern of the anhydrous crystalline Form C further includes one or more peaks at diffraction angles (2θ) of 15.6°±0.1°, 19.0°±0.1°, and 21.6°±0.1°.

20. Anhydrous crystalline form C according to any one of claims 17 to 19, characterized in that The X-ray powder diffraction pattern of the crystalline form C is shown in FIG3 .

21. A method for preparing anhydrous crystalline Form C as claimed in any one of claims 17 to 20, characterized in that: The method comprises the following steps: dissolving ergothioneine in water, heating the temperature to 60-100° C., dripping an organic solvent miscible with water or a mixed solvent of an organic solvent miscible with water and water and stirring; then rapidly cooling the temperature to 10-30° C., continuing stirring, and obtaining the anhydrous crystalline form C.

22. The preparation method according to claim 21, characterized in that: The organic solvent is selected from one or more of lower alkyl alcohols or lower alkyl ketones.

23. The anhydrous crystalline form according to any one of claims 1 to 4, 9 to 12, and 17 to 20, characterized in that: The anhydrous crystal type is used for preparing food, beverages, supplements, nutritional products, and cosmetics.

24. A composition, characterized in that The composition comprises an effective amount of the anhydrous crystalline form according to any one of claims 1-4, 9-12, 17-20, and a pharmaceutically acceptable carrier.

25. The composition according to claim 24, characterized in that The composition is used for preparing food, beverage, supplement, nutrition product and cosmetic.

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