5-hydroxytryptophan crystal form as well as preparation method and application thereof

Abstract

The invention discloses a 5-hydroxytryptophan crystal form as well as a preparation method and application thereof, and belongs to the technical field of medical products. In a powder X-ray diffraction pattern of the 5-hydroxytryptophan crystal form, characteristic peaks exist at 2theta diffraction angles of 8.1 + / -0.2 degrees, 10.5 + / -0.2 degrees, 12.7 + / -0.2 degrees, 14.0 + / -0.2 degrees, 17.7 + / -0.2 degrees, 18.9 + / -0.2 degrees, 20.2 + / -0.2 degrees, 21.3 + / -0.2 degrees, 22.0 + / -0.2 degrees, 23.2 + / -0.2 degrees, 24.7 + / -0.2 degrees, 25.7 + / -0.2 degrees, 29.4 + / -0.2 degrees, 31.5 + / -0.2 degrees and 32.5 + / -0.2 degrees. The preparation process of the novel crystal form of 5-hydroxytryptophan is simple to operate, low in energy consumption, high in economical efficiency and high in reproducibility. The product has good chemical stability, the yield is 90% or above, and the purity reaches 99.5% or above. The compound has a good application prospect in preparation of products with better stability for resisting depression, losing weight, reducing blood pressure, preventing headache or relieving insomnia.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical products, and specifically relates to a 5-hydroxytryptophan crystal form and a preparation method and use thereof. Background Art

[0002] 5-HTP (5-hydroxytryptophan) is a natural amino acid with a chemical formula of C 11 H 12 N2O3, with a relative molecular mass of 220.225, is chemically named 5-hydroxy-3-indolyl-α-aminopropionic acid, with a CAS number of 56-69-9. It is a white to off-white fine powder at room temperature, and its structural formula is shown below. This drug has not yet been popularized in China and is in the new drug development stage.

[0003]

[0004] 5-Hydroxytryptophan (5-HTP) is an amino acid that exists in most natural protein foods but in small amounts. As a precursor of the neurotransmitter 5-hydroxytryptamine (5-HT), it will further generate 5-HT under the action of decarboxylase, which can then increase the content of 5-HT in the human body, which is of great clinical significance. Studies have found that 5-HTP has multiple functions, such as anti-depression, analgesia, improving sleep, regulating diet, and enhancing immunity. It is of great significance to develop a new 5-HTP crystal form with simple preparation process, low energy consumption, high economy, and high reproducibility.

[0005] Patent application CN117720454A discloses a 5-hydroxytryptophan dihydrate crystal, a preparation method and use thereof. Compared with commercially available 5-hydroxytryptophan, the 5-hydroxytryptophan dihydrate crystal has improved hygroscopic stability and water solubility, a simple product preparation process, a stable preparation process, high reproducibility, a yield of more than 81%, and a product purity of more than 99.2%. However, on the one hand, the purity of the 5-hydroxytryptophan dihydrate crystal decreased by 0.2%-0.4% during storage for 30 days at 30°C ± 5°C and RH 40 ± 5%, and the chemical stability needs to be further improved; on the other hand, the yield of the 5-hydroxytryptophan dihydrate crystal obtained by the preparation method disclosed in the patent application needs to be further improved. Summary of the invention

[0006] The object of the present invention is to provide a 5-hydroxytryptophan crystal form and a preparation method and use thereof.

[0007] The invention provides a 5-hydroxytryptophan crystal form, characterized in that, in its powder X-ray diffraction pattern, 2θ diffraction angles have diffraction peaks at 8.1±0.2°, 10.5±0.2°, 12.7±0.2°, 14.0±0.2°, 17.7±0.2°, 18.9±0.2°, 20.2±0.2°, 21.3±0.2°, 22.0±0.2°, 23.2±0.2°, 24.7±0.2°, 25.7±0.2°, 29.4±0.2°, 31.5±0.2° and 32.5±0.2°.

[0008] Furthermore, in its powder X-ray diffraction spectrum, the relative intensity value of the characteristic peak at 2θ diffraction angle is:

[0009]

[0010]

[0011] Furthermore, its powder X-ray diffraction pattern is as follows Figure 1 shown.

[0012] The present invention also provides a method for preparing the 5-hydroxytryptophan crystal form according to any one of claims 1 to 3, wherein the method is a cooling crystallization method; the cooling crystallization method comprises the following steps:

[0013] Step 1: heating the 5-hydroxytryptophan concentrate or the 5-hydroxytryptophan solution to form a clear solution;

[0014] Step 2: Cooling the clarified solution in step 1 and maintaining the temperature for a period of time;

[0015] Step 3: Maintain the temperature of step 2 and add alkali to adjust the pH to acidic;

[0016] Step 4: solid-liquid separation, taking the solid and drying it to obtain the 5-hydroxytryptophan crystal form.

[0017] Furthermore, in step 1, the concentration of the 5-hydroxytryptophan concentrate or the 5-hydroxytryptophan solution is 120-150 g / L, and the temperature of the concentrated solution after heating is 50-65° C.; in step 2, the cooling rate is 5-10° C. / h, the temperature after cooling is 40-55° C., and the constant temperature is maintained for 10-30 min; in step 3, the alkali is a sodium hydroxide solution, the concentration of the sodium hydroxide solution is preferably 10%-30%, and the pH is adjusted to 5.2-7.2; in step 4, the drying temperature is 40-50° C., and the drying time is 3-6 h.

[0018] Furthermore, in step 1, the concentration of the 5-hydroxytryptophan concentrate or the 5-hydroxytryptophan solution is 120, 135 or 150 g / L, and the temperature of the concentrated solution after heating is 50, 60 or 65°C; in step 2, the cooling rate is 5, 6, 7, 8, 9 or 10°C / h, the temperature after cooling is 45, 50 or 55°C, and the constant temperature is maintained for 10, 20 or 30 min; in step 3, the concentration of the sodium hydroxide solution is 10%, 20% or 30%, and the pH is adjusted to 5.2, 5.6, 6.1, 6.5 or 6.8; in step 4, the drying temperature is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50°C, and the drying time is 3, 4, 5 or 6 h.

[0019] Furthermore, in step 1, the 5-hydroxytryptophan concentrated solution is a concentrated solution obtained by acidifying, filtering, decolorizing with activated carbon, and concentrating the 5-hydroxytryptophan fermentation solution obtained by fermenting with recombinant Escherichia coli.

[0020] Furthermore, the acidification pH is 2.0-4.0, and the time is 10-40 min; preferably, the acidification pH is 3.0, and the time is 25 min.

[0021] Furthermore, the concentration temperature is 60-80°C, preferably 70°C.

[0022] Furthermore, the concentration multiple is 3.5 to 8 times.

[0023] The present invention also provides the use of the 5-hydroxytryptophan crystal form in preparing drugs for resisting depression or preventing headaches.

[0024] The present invention also provides a pharmaceutical composition, which is a preparation prepared by using the above-mentioned 5-hydroxytryptophan crystal form as an active ingredient and adding pharmaceutically acceptable auxiliary ingredients; the preparation is preferably a powder injection, tablet, granule or capsule.

[0025] The present invention also provides a use of the 5-hydroxytryptophan crystal form in preparing health food.

[0026] Furthermore, the health food includes products for losing weight, lowering blood pressure or relieving insomnia.

[0027] The present invention has achieved the following beneficial effects:

[0028] The 5-HTP crystal preparation process provided by the present invention is simple to operate, consumes less energy, is highly economical, and has high reproducibility. The product has good chemical stability, a yield of more than 90%, and a purity of more than 99.5%. The 5-HTP crystal can be used to prepare products with better stability for anti-depression, weight loss, lowering blood pressure, preventing headaches, or relieving insomnia, and has broad application prospects.

[0029] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0030] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the X-ray powder diffraction pattern of the new crystalline form of 5-HTP in Example 3 of the present invention.

[0032] Figure 2 This is the DSC-TG analysis chart of the new crystalline form of 5-HTP in Example 3 of the present invention.

[0033] Figure 3 This is the FT-IR image of the new crystalline form of 5-HTP in Example 3 of the present invention.

[0034] Figure 4 This is the Raman graph of the new crystalline form of 5-HTP in Example 3 of the present invention.

[0035] Figure 5 This is the solubility performance curve of the new 5-HTP crystal form in Example 3 of the present invention. DETAILED DESCRIPTION

[0036] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0037] The 5-HTP concentrate used in the specific embodiment of the present invention is a concentrate obtained by concentrating the 5-HTP fermentation broth produced according to the method described in the specific embodiment mode section of the patent application specification with application number 2025100437632.

[0038] The preparation steps of 5-HTP fermentation broth are as follows:

[0039] (1) Prepare LB solid medium. The ingredients of LB solid medium are: 10 g / L soy peptone, 5 g / L yeast extract powder, 10 g / L NaCl, and 18 g / L agarose.

[0040] (2) Activate the recombinant E. coli by streaking on LB solid medium.

[0041] (3) Pick 1-2 single colonies from the solid plate and place them in LB liquid culture medium. The composition of LB liquid culture medium is: soy peptone 12g / L, yeast extract powder 6g / L, NaCl 10g / L, ampicillin 0.1g / L. Culture in a shaking incubator at 30°C and 180rpm for 15h±2h.

[0042] (4) When OD 600 When the pH value is greater than 2.0, the microorganisms are inoculated into the seed tank at an inoculation amount of 1% for seed tank culture. The seed tank culture medium is: 5 g / L yeast extract powder, 10 g / L soy peptone, 10 g / L NaCl, and 0.1 g / L ampicillin. The temperature is 30°C, the pressure is 0.040 MPa, and the stirring speed is 250 rpm.

[0043] (5) When OD 600 When the pH value is greater than 2.0, the microorganism is inoculated into the fermentation tank at an inoculation amount of 10%. The fermentation medium is an optimized LB medium: 50 g / L yeast extract, 10 g / L soy peptone, 10 g / L NaCl, 13 g / L dipotassium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 20 g / L glucose, and 40 g / L corn steep liquor. 60% (W / V) glucose is continuously added for culture.

[0044] (6) When the OD in the fermenter 600 When the pH value is ≥45, 0.15 g / L isopropyl-β-D-thiogalactoside (IPTG) is added once and fermented for 45-50 h to obtain 5-hydroxytryptophan fermentation liquid.

[0045] The source of the recombinant Escherichia coli capable of producing 5-hydroxytryptophan is the patent application with publication number CN116287042A.

[0046] The concentration steps are as follows:

[0047] (1) Acidification of fermentation broth: After adding antioxidant to the 5-hydroxytryptophan fermentation broth, adjust the pH to 3.0 with sulfuric acid or acetic acid, and the acidification time is 25 min.

[0048] (2) Filtration: Filter the acidified fermentation broth using a ceramic membrane or a plate and frame filter, and collect the filtrate.

[0049] (3) Activated carbon decolorization: The filtrate in step (2) is decolorized with activated carbon and then the filtrate is collected.

[0050] (4) Concentration: The decolorized solution obtained in step (3) is concentrated by thin film evaporation, and the temperature during the concentration process is controlled at 70°C.

[0051] (5) Concentration: The concentrated solution was obtained by vacuum distillation with the temperature of the concentrated solution being controlled at 70°C.

[0052] Example 1: New 5-HTP crystal form and its preparation process

[0053] Heat 120g / L 5-HTP concentrate to 50°C to form a clear solution; cool the clear solution to 45°C at a rate of 5°C / h and maintain for 10 minutes; then slowly drip 10% sodium hydroxide solution into the 5-HTP concentrate while maintaining this temperature, and adjust the pH of the solution to 5.2; then filter, and dry the filter cake at 40°C for 3 hours to obtain a new crystalline form of 5-HTP. The molecular formula of the new crystalline form of 5-HTP is C 11 H 12 N2O3.

[0054] The X-ray powder diffraction pattern of the new 5-HTP crystal form has diffraction peaks at diffraction angles 2θ=8.1±0.2°, 10.5±0.2°, 12.7±0.2°, 14.0±0.2°, 17.7±0.2°, 18.9±0.2°, 20.2±0.2°, 21.3±0.2°, 22.0±0.2°, 23.2±0.2°, 24.7±0.2°, 25.7±0.2°, 29.4±0.2°, 31.5±0.2°, and 32.5±0.2°. DSC-TG results show that it starts to lose weight at 264°C. The product has a white powder appearance, a product purity of 99.59%, and a process yield of 93%.

[0055] Example 2: New 5-HTP crystal form and its preparation process

[0056] Heat 135g / L 5-HTP concentrate to 58°C to form a clear solution; cool the clear solution to 40°C at a rate of 10°C / h and maintain for 20 minutes; then slowly drip 20% sodium hydroxide solution into the 5-HTP concentrate while maintaining this temperature, and adjust the solution pH to 7.2; then filter, and dry the filter cake at 45°C for 4 hours to obtain a new crystalline form of 5-HTP. The molecular formula of the new crystalline form of 5-HTP is C 11 H 12 N2O3.

[0057] The X-ray powder diffraction pattern of the new 5-HTP crystal form has diffraction peaks at diffraction angles 2θ=8.1±0.2°, 10.5±0.2°, 12.7±0.2°, 14.0±0.2°, 17.7±0.2°, 18.9±0.2°, 20.2±0.2°, 21.3±0.2°, 22.0±0.2°, 23.2±0.2°, 24.7±0.2°, 25.7±0.2°, 29.4±0.2°, 31.5±0.2°, and 32.5±0.2°. DSC-TG results show that it starts to lose weight at 264°C. The product has a white powder appearance, a product purity of 99.68%, and a process yield of 95%.

[0058] Example 3: New 5-HTP crystal form and its preparation process

[0059] Heat 150g / L 5-HTP concentrate to 65°C to form a clear solution; cool the clear solution to 55°C at a rate of 8°C / h and maintain for 30 minutes; then slowly drip 30% sodium hydroxide solution into the 5-HTP concentrate while maintaining this temperature, and adjust the pH of the solution to 5.6; then filter, and dry the filter cake at 50°C for 6 hours to obtain a new crystalline form of 5-HTP. The molecular formula of the new crystalline form of 5-HTP is C 11 H 12 N2O3.

[0060] The X-ray powder diffraction pattern of the new 5-HTP crystal form has diffraction peaks at diffraction angles 2θ=8.1±0.2°, 10.5±0.2°, 12.7±0.2°, 14.0±0.2°, 17.7±0.2°, 18.9±0.2°, 20.2±0.2°, 21.3±0.2°, 22.0±0.2°, 23.2±0.2°, 24.7±0.2°, 25.7±0.2°, 29.4±0.2°, 31.5±0.2°, and 32.5±0.2°. DSC-TG results show that it starts to lose weight at 264°C. The product has a white powder appearance, a product purity of 99.95%, and a process yield of 92%.

[0061] The chemical properties of the new 5-HTP crystal form of the present invention are tested by experimental examples below.

[0062] Experimental Example 1: Chemical property detection of new 5-HTP crystal form

[0063] 1. The chemical stability of the new 5-HTP crystal prepared by the present invention was investigated. During storage for 30 days at 30°C ± 5°C and RH 40 ± 5%, the color, purity and morphology of the product did not change, indicating that the crystal had good chemical stability. The investigation results are shown in Table 1.

[0064] Table 1 Chemical stability study of 5-HTP crystals of the present invention

[0065]

[0066] 2. X-ray powder diffraction experiment of the new 5-HTP crystal obtained in the present invention. The X-ray powder diffraction pattern of the new 5-HTP crystal obtained in Example 3 is as follows: Figure 1 As shown, the X-ray powder diffraction patterns of the new 5-HTP crystal forms obtained in Examples 1 and 2 are consistent with those in Example 3 and are not provided again here.

[0067] 3. The new 5-HTP crystal obtained in the present invention is subjected to DSC-TG analysis. The specific method of DSC-TG analysis is a conventional method in the prior art. The DSC-TG analysis results of the new 5-HTP crystal obtained in Example 3 are as follows: Figure 2 As shown, its TG analysis chart shows that it starts to lose weight at 264°C, and the 5-HTP crystals obtained in Example 3 have excellent thermal stability. The results of Examples 1 and 2 are consistent with those of Example 3, and the pictures are not repeated here.

[0068] 4. Testing the FT-IR graph of the new 5-HTP crystal obtained by the present invention. The FT-IR graph of the new 5-HTP crystal obtained in Example 3 is as follows: Figure 3 The results of Examples 1 and 2 are consistent with those of Example 3, and the figures are not repeated here.

[0069] 5. Testing the Raman spectrum (Raman) of the new 5-HTP crystal obtained by the present invention. The Raman spectrum of the new 5-HTP crystal obtained in Example 3 is as follows: Figure 4 The results of Examples 1 and 2 are consistent with those of Example 3, and the figures are not repeated here.

[0070] 6. Testing the solubility of the new 5-HTP crystal obtained by the present invention, Figure 5 This is the solubility performance curve of the new 5-HTP crystal obtained in Example 3. The results of Examples 1 and 2 are consistent with those of Example 3, and the pictures are not provided repeatedly here.

Claims

1. A 5-hydroxytryptophan crystal form, characterized in that: In its powder X-ray diffraction pattern, 2θ diffraction angles have diffraction peaks at 8.1±0.2°, 10.5±0.2°, 12.7±0.2°, 14.0±0.2°, 17.7±0.2°, 18.9±0.2°, 20.2±0.2°, 21.3±0.2°, 22.0±0.2°, 23.2±0.2°, 24.7±0.2°, 25.7±0.2°, 29.4±0.2°, 31.5±0.2°, and 32.5±0.2°.

2. The 5-hydroxytryptophan crystal form according to claim 1, characterized in that In its powder X-ray diffraction spectrum, the relative intensity value of the characteristic peak at 2θ diffraction angle is:

3. The 5-hydroxytryptophan crystal form according to claim 2, characterized in that: Its powder X-ray diffraction pattern is shown in Figure 1.

4. A method for preparing the 5-hydroxytryptophan crystal form according to any one of claims 1 to 3, characterized in that: The method is a cooling crystallization method; the cooling crystallization method comprises the following steps: Step 1: heating the 5-hydroxytryptophan concentrate or the 5-hydroxytryptophan solution to form a clear solution; Step 2: Cooling the clarified solution in step 1 and maintaining the temperature for a period of time; Step 3: Maintain the temperature of step 2 and add alkali to adjust the pH to acidic; Step 4: solid-liquid separation, taking the solid and drying it to obtain the 5-hydroxytryptophan crystal form.

5. The method according to claim 4, characterized in that In step 1, the concentration of the 5-hydroxytryptophan concentrate or the 5-hydroxytryptophan solution is 120-150 g / L; the temperature of the concentrated solution after heating is 50-65° C.; In step 2, the cooling rate is 5-10°C / h; the temperature after cooling is 40-55°C; the constant temperature is maintained for 10-30 minutes; In step 3, the alkali is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is preferably 10% to 30%; the pH is adjusted to 5.2 to 7.2; In step 4, the drying temperature is 40-50° C. and the drying time is 3-6 hours.

6. The method according to claim 5, characterized in that In step 1, the concentration of the 5-hydroxytryptophan concentrate or the 5-hydroxytryptophan solution is 120, 135 or 150 g / L; the temperature of the concentrated solution after heating is 50, 60 or 65° C.; In step 2, the cooling rate is 5, 6, 7, 8, 9 or 10°C / h; the temperature after cooling is 45, 50 or 55°C; the constant temperature is maintained for 10, 20 or 30 minutes; In step 3, the concentration of the sodium hydroxide solution is 10%, 20% or 30%; the pH is adjusted to 5.2, 5.6, 6.1, 6.5 or 6.8; In step 4, the drying temperature is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50° C.; and the drying time is 3, 4, 5 or 6 h.

7. Use of the 5-hydroxytryptophan crystal form according to any one of claims 1 to 3 in the preparation of drugs for antidepression, headache prevention, weight loss, lowering blood pressure or relieving insomnia.

8. A pharmaceutical composition, characterized in that The invention discloses a preparation which is prepared by taking the 5-hydroxytryptophan crystal form described in any one of claims 1 to 3 as an active ingredient and adding pharmaceutically acceptable auxiliary ingredients; the preparation is preferably a powder injection, a tablet, a granule or a capsule.

9. Use of the 5-hydroxytryptophan crystal form according to any one of claims 1 to 3 in the preparation of health food for controlling body fat, maintaining healthy blood pressure levels or improving sleep.

10. A health food, characterized in that: The product is prepared by taking the 5-hydroxytryptophan crystal form described in any one of claims 1 to 3 as an active ingredient and adding auxiliary ingredients acceptable in the food field.

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