Stabilizer of 5-ALA liquid

Abstract

The invention relates to the field of stabilizers, in particular to a stabilizer for 5-ALA liquid. The stabilizer of the 5-ALA liquid comprises a preservative and an antioxidant, and is characterized by further comprising a pH buffering agent and pectin. The stability of the 5-ALA in a liquid environment is improved through methods of pH stabilization, oxidation resistance and medium adsorption. The degradation rate of the 5-ALA liquid preparation is lower than 5% after the 5-ALA liquid preparation is hermetically stored at room temperature for 100 days.

Description

Technical Field

[0001] The invention relates to the field of stabilizers, and in particular to a stabilizer for 5-ALA liquid. Background Art

[0002] 5-aminolevulinic acid (5-ALA), also known as 5-amino-4-ketovaleric acid, is a functional non-protein amino acid that exists naturally in organisms. It is a common precursor for the biosynthesis of all porphyrin compounds (heme, chlorophyll, vitamin B12 and cytochrome). It is widely present in bacteria, fungi, animals and plants, and has an important impact on cell energy metabolism. ALA has the advantages of being biodegradable, non-toxic and residue-free, and is widely used in medicine, pesticides, chemicals and other fields. In the agricultural field, 5-ALA has the effect of plant growth stimulant, which can promote the growth of crops and increase the yield and quality of agricultural products; in the feed field, it can improve anemia and improve the immunity of animals; in the medical field, ALA is used as a new generation of photodynamic drugs for cancer diagnosis and treatment; in addition, ALA is also used as an additive in cosmetics and health foods.

[0003] At present, in the production process of 5-ALA, the loss rate in extraction and purification is relatively high, while liquid preparations have obvious cost advantages. At the same time, many existing papers and other literature have reported that the stability of 5-ALA in liquid decreases with the increase of initial concentration, pH value and storage temperature, and light and dissolved oxygen will accelerate the decomposition of 5-aminolevulinic acid. It is known that the aqueous solution of 5-ALA is unstable. Existing studies have shown that 5-ALA is an α-aminoketone, which can automatically enolize under aerobic and slightly alkaline conditions to generate active oxygen free radicals. Under alkaline conditions, two molecules of 5-ALA can automatically form dihydropyrazine and 2,5-β-carboxyethylpyrazine. Therefore, dimerization condensation may be the main reason for the instability of high-concentration 5-ALA aqueous solutions.

[0004] In order to improve the stability and safety of 5-ALA during transportation and storage, Chinese patent application CN117799917A discloses a technology for preventing fat and corrosion during the transportation of 5-aminolevulinic acid, which uses microencapsulation technology to encapsulate 5-aminolevulinic acid powder in a biodegradable polymer to form tiny particles, and finally uses low-temperature vacuum drying technology to prepare it into a dry solid form; Chinese patent application CN117886641A discloses a plant nutrient solution, which includes 5-aminolevulinic acid, nutrients and other main active ingredients. In addition, the patent application discloses auxiliary agents such as spreaders, active solubilizers, stabilizers, preservatives, etc., which can not only make the calcium source, 5-aminolevulinic acid and nutrients fully dissolved and homogenized, but also ensure that the nutrient solution will not deteriorate or stratify during long-term storage, thereby increasing the stability of the nutrient solution.

[0005] However, the higher the concentration of 5-ALA, the worse the stability. Therefore, the study of high-concentration 5-ALA stabilizers is of great significance for storage, transportation and use. Therefore, the development of high-concentration 5-ALA stable liquid preparations is of great significance for the wide use of this product in the fields of medicine, pesticides, chemicals, etc. Summary of the invention

[0006] In order to solve the above technical problems, the present invention aims to provide a stabilizer for 5-ALA liquid to improve the stability of high-concentration 5-ALA in a liquid environment.

[0007] Another object of the present invention is to provide use of pectin for stabilizing 5-ALA liquid.

[0008] The stabilizer of 5-ALA liquid according to the present invention comprises a preservative and an antioxidant, and is characterized in that it also comprises a pH buffer and pectin.

[0009] In some embodiments of the present invention, the preservative comprises benzoic acid, sodium benzoate, sorbic acid, potassium sorbate or calcium propionate.

[0010] In some embodiments of the present invention, the pH buffer comprises sodium hydrogen phosphate-citric acid buffer, ethanol-ammonium acetate buffer, citric acid-sodium citrate buffer or glycine-hydrochloric acid buffer. In a specific embodiment, specifically, the pH buffer is citric acid-sodium citrate buffer.

[0011] In some embodiments of the present invention, the antioxidant includes vitamin C, vitamin E, glutathione, butylated hydroxyanisole (BHA), 2,6-di-tert-butylated cresol (BHT), sodium sulfite-cobalt chloride or sodium pyrosulfite. In a specific embodiment, specifically, the antioxidant is sodium sulfite-cobalt chloride or sodium pyrosulfite.

[0012] In some embodiments of the present invention, the concentration ratio of pectin to 5-ALA liquid is 1:1-3.

[0013] Beneficial effects of the present invention:

[0014] The stability of 5-ALA needs to be maintained in an acidic environment. In addition, the intensified intermolecular collision caused by high temperature is also one of the factors affecting its stability. For other active systems, stabilizers such as the most common EDTA and HEDTA are dissolved in an alkaline environment and are not suitable for 5-ALA. The inventors of the present invention unexpectedly found that pectin, which is used as a thickener and emulsifier in the field of food additives, has an unexpectedly excellent effect on the stability of 5-ALA.

[0015] The stability of 5-ALA in liquid environment is improved by stabilizing pH, anti-oxidation and medium adsorption. The degradation rate of 5-ALA liquid preparation is less than 5% when stored in a sealed container at room temperature for 100 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The stability of 5-ALA liquid formulations at different pH values ​​is shown. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0018] The invention provides a 5-aminolevulinic acid liquid stable preparation, which comprises at least four parts, namely, a preservative, a pH buffer, an antioxidant and pectin.

[0019] In some embodiments of the present invention, the preservatives used include: benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, calcium propionate, etc.

[0020] In some embodiments of the present invention, the pH buffers used include: disodium hydrogen phosphate-citric acid buffer (pH 3.5-4.2); ethanol-ammonium acetate buffer (pH 3.5-4.2); citric acid-sodium citrate buffer (pH 3.5-4.2); glycine-hydrochloric acid buffer (pH 3.5-4.2), etc.

[0021] In some embodiments of the present invention, the antioxidants used include: vitamin C, vitamin E, glutathione, butylated hydroxyanisole (BHA), 2,6-di-tert-butylated cresol (BHT), sodium sulfite-cobalt chloride, sodium metabisulfite, etc.

[0022] In some embodiments of the present invention, the water-soluble anionic polymer used includes: pectin.

[0023] The inventors of the present application unexpectedly discovered that when pectin is used as a water-soluble anionic polymer, the 5-ALA stabilizer of the present invention can better prevent the degradation of 5-ALA. As a polyuronic acid, the stability of pectin is closely related to the pH value of the environment. When the pH value is about 3.5, the carboxyl groups in the pectin molecules can be prevented from ionizing, and the pectin stability is the highest at this time. Without being limited by theory, we believe that the 5-ALA molecule contains -NH2 and the pectin molecule contains -COOH. Under an appropriate pH buffer system, such as pH 3.5, a complex reaction will occur between them, which can effectively protect the activity of 5-ALA and reduce its degradation.

[0024] 5-aminolevulinic acid concentration detection method:

[0025] 1. Detection reagents:

[0026] A. Preparation of color developer (stored at 4°C for no more than 12 hours): Take 9 mL of glacial acetic acid, add 1 mL of perchloric acid, mix well, and then add 0.2 g of p-dimethylaminobenzaldehyde to dissolve;

[0027] B. Sodium acetate solution: Take 50 mL of pure water, add 5.7 mL of glacial acetic acid, 8.2 g of sodium acetate, and dilute to 100 mL with pure water;

[0028] C. Sample diluent (used to dilute fermentation broth): pH 3.0 acetic acid solution.

[0029] 2. Testing steps:

[0030] (1) Dilute the fermentation broth with solution C to an appropriate concentration (about 1-10 mg / L);

[0031] (2) Take 250 μL of the diluted solution, add 125 μL of solution B, add 62.5 μL of acetylacetone, and cook in boiling water for 15 minutes;

[0032] (3) Cool the sample from step (2) to room temperature, add 400 μL of A, mix well, and react for 20 minutes;

[0033] (4) Detect the absorbance OD of the sample at a wavelength of 554 nm using a spectrophotometer 554 .

[0034] (5) 5-ALA standard curve: Weigh 0.1 g of 5-aminolevulinic acid hydrochloride standard, dissolve it in 100 mL of solution C, and dilute it to 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L standard solutions. Detect the absorbance OD of the standard at a wavelength of 554 nm according to the method of steps (2)-(3). 554 The absorbance is calculated and the corresponding linear calculation formula is generated.

[0035] (6) According to the sample OD 554 The concentration of 5-aminolevulinic acid hydrochloride was calculated using the corresponding formula and converted into 5-aminolevulinic acid concentration.

[0036] Example 1 Effect of pH on the Stability of 5-ALA Liquid Preparations

[0037] Take the 5-ALA fermentation supernatant (5-ALA concentration is 30 g / L), add sodium benzoate with a final concentration of 0.5%, adjust the pH value with hydrochloric acid, and set the pH gradient to: pH 3.0, pH 3.5, pH 3.7, pH 4.0, pH 4.2, pH 4.5, pH 5.0. The final concentration of 5-ALA is 27 g / L, and the effect of different pH values ​​on the liquid stability of 5-ALA at room temperature is tested.

[0038] The preparation was placed at room temperature for one week, and samples were taken every two days for testing. Figure 1 The stability of 5-ALA liquid preparations at different pH values ​​is shown, and the results show that pH 3.0-4.2 has the best effect on the stability of 5-ALA liquid. However, considering that in actual applications, the application scenario of pH 3.0 almost does not exist, and the cost of the stabilizer is increased, further lowering the pH value has limited effect on the stability of 5-ALA. Therefore, the pH value of the 5-ALA stabilizer of the present invention is 3.5-4.2.

[0039] Example 2 Effect of pH buffer system on the stability of 5-ALA liquid preparation

[0040] (1) Ethanol-ammonium acetate buffer system

[0041] Take 15.0mL of 5M acetic acid solution and 60mL of ethanol, add them to 900mL of 5-ALA fermentation supernatant (the supernatant was taken by centrifugation, the 5-ALA concentration was 30g / L, and sodium benzoate was added to a final concentration of 0.5%), adjust the pH to 3.5 with 10mol / L ammonium hydroxide solution, and make the volume to 1L. The final 5-ALA concentration was 27g / L.

[0042] (2) Glycine-HCl

[0043] Weigh 3.75 g of glycine and add it to 900 mL of 5-ALA fermentation supernatant (the supernatant is taken by centrifugation and sodium benzoate is added to a final concentration of 0.5%), adjust the pH to 3.5 with hydrochloric acid, and make the volume to 1 L.

[0044] (3) Citric acid-sodium citrate

[0045] Weigh 16.6 g of citric acid and 3.6 g of sodium citrate, add them to 900 mL of 5-ALA fermentation supernatant (centrifuge to take the supernatant, add sodium benzoate with a final concentration of 0.5%), adjust the pH to 3.5 with hydrochloric acid, and make the volume to 1 L.

[0046] After standing at room temperature for 7 days, the pH value and 5-ALA titer were tested.

[0047] Table 1. Effect of buffer system on 5-ALA stability

[0048]

[0049] As shown in Table 1, citric acid-sodium citrate buffer has the best effect on the liquid stability of 5-ALA.

[0050] Example 3 Effect of Antioxidants on the Stability of 5-ALA

[0051] Weigh 16.6 g of citric acid and 3.6 g of sodium citrate, add to 900 mL of 5-ALA fermentation supernatant (centrifuge to obtain the supernatant, 5-ALA concentration is 30 g / L, add sodium benzoate to a final concentration of 0.5%), adjust the pH to 3.5 with hydrochloric acid, make the volume to 1 L, the final 5-ALA concentration is 27 g / L, and set aside.

[0052] (1) Vitamin C

[0053] Take 200 mL of the above-mentioned 5-ALA liquid preparation, add 0.3 g of vitamin C, and adjust the pH to 3.5.

[0054] (2) Sodium sulfite-cobalt sulfate

[0055] Take 200 mL of the above-mentioned 5-ALA liquid preparation, add 0.6 g of sodium sulfite and 20 mg of cobalt sulfate, and adjust the pH to 3.5.

[0056] (3) BHA

[0057] Take 200 mL of the above-mentioned 5-ALA liquid preparation, add 40 mg BHA, and adjust the pH to 3.5.

[0058] (4) BHT

[0059] Take 200 mL of the above-mentioned 5-ALA liquid preparation, add 40 mg BHT, and adjust the pH to 3.5.

[0060] All preparations were placed at room temperature for 7 days and the concentration of 5-ALA was measured.

[0061] Table 2 Effect of antioxidants on the stability of 5-ALA

[0062]

[0063] As shown in Table 2, sodium sulfite-cobalt sulfate has the most significant effect on the stability of 5-ALA liquid preparation.

[0064] Implementation Case 4 Effect of Pectin on the Stability of 5-ALA

[0065] First, determine the optimal ratio of pectin and 5-ALA. Weigh 50g of pectin, add it to 500mL of deionized water, heat and stir until fully dissolved; take 167mL of 30g / L 5-ALA fermentation broth into 5 beakers, add 50mL, 25mL, 17mL, 12.5mL and 10mL of pectin solution respectively, adjust the pH value to 3.5, and adjust the volume to 217mL (5-ALA concentration 23g / L, pectin: 5-ALA 1:1, 1:2, 1:3, 1:4, 1:5 respectively), and test the 5-ALA concentration after leaving it at room temperature for 5 days.

[0066] Table 3. Effect of pectin on the stability of 5-ALA

[0067]

[0068]

[0069] The results showed that when the concentration ratio of pectin to 5-ALA was 1:1-3, the degradation rate of 5-ALA in 5 days was less than 20%, which could significantly reduce the degradation rate of 5-ALA.

[0070] Step 1: Weigh 16.6 g citric acid, 3.6 g sodium citrate, 3 g sodium sulfite, and 100 mg cobalt sulfate, add to 900 mL 5-ALA fermentation supernatant (centrifuge to obtain the supernatant, 5-ALA concentration is 30 g / L, add sodium benzoate to a final concentration of 0.5%), stir to dissolve, and adjust the pH to 3.5 as a 5-ALA standby solution.

[0071] Step 2: Weigh 10g of pectin, add it to 100mL of deionized water, heat and stir until fully dissolved; take 400mL of 5-ALA reserve solution, add 50mL of pectin solution, stir well, adjust the pH to 3.5, and make up to 500mL.

[0072] Step 3: Take the pectin solution in step 2 and add it to the solution in step 1 to make the volume 1 L. The final concentration of 5-ALA is 27 g / L, the final concentration of pectin is 10 g / L, and the ratio of pectin to 5-ALA is 1:2.7.

[0073] Step 4:

[0074] Take 200 mL of the 5-ALA liquid preparation prepared in Example 3, add 0.6 g of sodium sulfite and 20 mg of cobalt sulfate, and adjust the pH to 3.5 as a control.

[0075] After being placed at room temperature for 7 days, the 5-ALA titer of the experimental group was 27 g / L (no degradation), and that of the control group was 24 g / L.

[0076] The control group and the experimental group were sealed and stored at room temperature for 105 days, and samples were taken for testing. The results showed that the titer of the control group was 19.3 g / L (degradation rate 28.5%), and that of the experimental group was 21.9 g / L (degradation rate 18.9%).

[0077] In summary, after adding the pH buffer, after 7 days, the overall stability is better than that of each pH group in Example 1, among which the citric acid-sodium citrate buffer has the best effect on the liquid stability of 5-ALA.

[0078] On this basis, by adding antioxidants, the stability of each group was further improved after 7 days, among which sodium sulfite-cobalt sulfate had the most obvious effect on increasing the stability of 5-ALA liquid preparation.

[0079] On the basis of adding pH buffer and antioxidant, the stability of 5-ALA liquid preparation was significantly increased by adding pectin in a ratio of 1:1 to 3.

[0080] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A stabilizer for 5-aminolevulinic acid, comprising a preservative and an antioxidant, characterized in that: Also includes pH buffer, pectin.

2. The stabilizer of 5-aminolevulinic acid according to claim 1, characterized in that The preservative is one or more selected from benzoic acid, sodium benzoate, sorbic acid, potassium sorbate or calcium propionate.

3. The stabilizer of 5-aminolevulinic acid according to claim 1, characterized in that The antioxidant is one or more of vitamin C, vitamin E, glutathione, butylated hydroxyanisole, 2,6-di-tert-butylated cresol, sodium sulfite-cobalt chloride or sodium pyrosulfite.

4. The stabilizer of 5-aminolevulinic acid according to claim 3, characterized in that The antioxidant is sodium sulfite-cobalt chloride or sodium pyrosulfite.

5. The stabilizer of 5-aminolevulinic acid according to claim 1, characterized in that The pH buffer is disodium hydrogen phosphate-citric acid buffer, ethanol-ammonium acetate buffer, citric acid-sodium citrate buffer or glycine-hydrochloric acid buffer.

6. The stabilizer of 5-aminolevulinic acid according to claim 5, characterized in that The pH buffer is citric acid-sodium citrate buffer.

7. Application of pectin in stabilizing 5-aminolevulinic acid liquid.

8. The use according to claim 7, characterized in that: The concentration ratio of the pectin to the 5-aminolevulinic acid liquid is 1:1-3.

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