Preparation method of tylosin without histamine residue

Abstract

The invention belongs to the technical field of medical chemistry, and particularly relates to a preparation method of tylosin without histamine residues. The preparation method comprises the following steps: mixing tylosin fermentation liquor, a surfactant and a flocculant, performing ultrafiltration, and collecting filtrate; adding ethanol into the filtrate, enabling the filtrate to sequentially pass through a Ni-NTA His.Bind resin column and strongly acidic cation exchange resin, collecting effluent, adjusting the pH value, and drying, so as to obtain the product. According to the preparation method of the tylosin without histamine residue, the removal rate of histamine is obviously improved by utilizing the synergistic effect of lauroyl diethanolamine and betaine, so that the histamine content is less than or equal to 1.0 ppm, and the histamine residue is limited according to the enterprise standard; a continuous column chromatography mode is adopted, histamine contained in the tylosin fermentation liquor is effectively removed, effective components in the tylosin are reserved, and the method is easy and convenient to operate, low in cost and suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a method for preparing tylosin without histamine residue. Background Art

[0002] Tylosin, also known as Tainong, is a white or light yellow powder, slightly soluble in water, and the aqueous solution is alkaline; at 26°C, the solubility in aqueous solution is 5.0 mg / ml, and its solubility in water decreases with increasing temperature. Tylosin is a common livestock and poultry drug. There are three common forms on the market, including tylosin base, sulfate, and tartrate. According to the national veterinary drug standards, there are three contents, 5%, 10% and 50%. The 10% content is the most common in the market and can be used for disease treatment and to promote animal growth. At present, liquid deep fermentation is used both at home and abroad. After the fermentation liquid is treated with flocculants, solid-liquid separation is performed to obtain the fermentation filtrate. The fermentation filtrate is subjected to liquid-liquid extraction with butyl acetate to obtain an ester phase liquid. The ester phase liquid can be subjected to processes such as back extraction, concentration and spray drying to obtain tartaric acid or tylosin phosphate.

[0003] Histamine (HA), also known as tissue amine, has the molecular formula of C5H9N3. It is one of the autoactive substances and is mainly stored in mast cells and basophils in the human body. When the human body is stimulated by allergens, histamine is released, causing vasodilation, edema, lowered blood pressure, and various corresponding symptoms, which damages physical health. As an inflammatory mediator of immediate hypersensitivity reactions, when histamine in the blood reaches a certain level, anaphylactic shock or even death will occur.

[0004] There are few reports on methods for removing histamine. For example, Chinese invention patent application CN115508165A discloses an extraction method for removing histamine. The operation steps are as follows: S1. Sample preparation: a certain amount of fish meat, trichloroacetic acid solution, sodium hydroxide solution, n-pentanol, hydrochloric acid, saturated borax solution, distilled water, filter membrane, modified activated carbon; S2. Accurately weigh 10g of the mashed and mixed fish meat sample, put the fish meat into a 50ml small beaker, and use 20ml of 40-80% ethanol solution for extraction. The sample is treated at a temperature of 20-60°C and for 30-70 minutes, filtered, and 20 ml of trichloroacetic acid (10%) is added as an extractant. The extraction method for removing histamine uses an ethanol solution as an extractant, and according to the properties of histamine, histamine is easily soluble in an ethanol solution, so that the method for extracting histamine is simple and fast, greatly shortening the extraction time, reducing the cumbersome operation process, and meeting the qualitative and semi-quantitative determination of histamine by the colloidal gold method, but the histamine removal rate of this method is not high.

[0005] Since histamine has certain harm to the immune system, nervous system and cardiovascular system of animals, in view of this, there is an urgent need in the art to provide a method for preparing tylosin without histamine residue. Summary of the invention

[0006] The invention aims at solving the problems in the prior art and provides a method for preparing tylosin without histamine residue.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing tylosin without histamine residue comprises the following steps:

[0009] (1) firstly mixing tylosin fermentation broth, a surfactant and a flocculant, ultrafiltering and collecting the filtrate;

[0010] (2) Add ethanol to the filtrate, and then pass it through a Ni-NTA His·Bind resin column and a strongly acidic cation exchange resin in sequence, collect the effluent, adjust the pH, and dry it to obtain the product.

[0011] Preferably, the surfactant in step (1) comprises lauroyl diethanolamine and betaine.

[0012] Preferably, the mass ratio of lauroyl diethanolamine to betaine is 1-3:4-6.

[0013] More preferably, the mass ratio of lauroyl diethanolamine to betaine is 2-3:5-6.

[0014] Further preferably, the mass ratio of lauroyl diethanolamine to betaine is 1:2.

[0015] Preferably, the flocculant in step (1) is polyaluminium chloride or polyaluminium phosphochlorid.

[0016] More preferably, the flocculant is polyaluminium chloride.

[0017] Preferably, the volume mass ratio of the tylosin fermentation broth, the surfactant and the flocculant is 5-10 ml: 0.3-0.6 g: 0.02-0.04 g.

[0018] More preferably, the volume mass ratio of the tylosin fermentation broth, the surfactant and the flocculant is 8 ml:0.6 g:0.04 g.

[0019] Preferably, the mixing in step (1) requires stirring, the stirring speed is 300-500 rpm, and the stirring time is 60-90 min.

[0020] Preferably, the pore size of the ultrafiltration membrane in step (1) is 5-10 nm, the pressure of the ultrafiltration is 0.4-0.6 MPa, and the pH needs to be adjusted to 7.5-8.0 after the ultrafiltration is completed.

[0021] Preferably, the volume concentration of ethanol in step (2) is 60-80%, the volume ratio of the filtrate to ethanol is 1:2-4, and after the addition of ethanol is completed, stirring is required at 400-600 rpm for 1.5-2.5 hours, and then the ethanol is removed by concentration under reduced pressure, and the concentrate is collected.

[0022] More preferably, the volume concentration of the ethanol is 70%, and the volume ratio of the filtrate to the ethanol is 1:3.

[0023] Preferably, the strongly acidic cation exchange resin in step (2) is selected from one or more of the models SR-1, S-9 and 001×7FD.

[0024] Preferably, in step (2), the temperature of the Ni-NTA His·Bind resin column and the strongly acidic cation exchange resin are both 15-20° C., and the flow rate is both 1-3 BV / h.

[0025] Preferably, the pH adjustment process in step (2) comprises first adding 2-3% by volume phosphoric acid or tartaric acid to the effluent to adjust the pH to 3.0-4.0, allowing the effluent to stand, collecting the lower aqueous phase, and then adjusting the pH to 6.0-7.0.

[0026] Preferably, activated carbon decolorization and concentration are performed before drying in step (2), and the drying is vacuum drying or freeze drying.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention provides a method for preparing tylosin without histamine residue. The method selects a specific surfactant and mixes it with the tylosin fermentation broth, and utilizes the synergistic effect between lauroyl diethanolamine and betaine to significantly improve the histamine removal rate, so that the histamine content is ≤1.0ppm, which meets the enterprise standard limit for histamine residue.

[0029] (2) The present invention adopts a continuous column chromatography method to effectively remove the histamine contained in the tylosin fermentation broth, retaining the effective ingredients in tylosin, and is simple to operate, low in cost, and suitable for industrial production. DETAILED DESCRIPTION

[0030] It is worth noting that the raw materials used in the present invention are all common commercially available products. Among them, the tylosin fermentation broth is obtained from the fermentation broth obtained after the fermentation in the tylosin fermentation workshop of Ningxia Tairui Pharmaceutical Co., Ltd.; betaine, CAS No. 107-43-7, purchased from Aladdin; lauroyl diethanolamine, CAS No. 120-40-1, purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.; ethanol, purity 95%, purchased from Sinopharm Chemical Reagent Co., Ltd.; Ni-NTA His·Bind resin, purchased from Sigma; His Pur TM Cobalt resin was purchased from Thermo Fisher Scientific; SR-1, S-9 and 001×7FD strong acid cation exchange resins were all purchased from Zhejiang Zhengguang Industrial Co., Ltd.

[0031] Example 1

[0032] A method for preparing tylosin without histamine residue, comprising the following steps:

[0033] (1) First, tylosin fermentation broth (8 ml, potency 12386 U / ml), surfactant (0.6 g) and polyaluminium chloride (0.04 g) were stirred and mixed at 400 rpm for 80 min, wherein the surfactant was composed of lauroyl diethanolamine and betaine in a mass ratio of 1:2. After stirring, ultrafiltration was performed, the pore size of the ultrafiltration membrane was 10 nm, the pressure of the ultrafiltration was 0.6 MPa, the filtrate was collected, and the pH of the filtrate was adjusted to 7.8.

[0034] (2) Add 70% ethanol in a volume concentration of 2 times the volume of the filtrate to the filtrate, stir at 500 rpm for 2 hours, concentrate under reduced pressure to remove the ethanol, and collect the concentrate. At 20°C, pass the concentrate through a Ni-NTA His·Bind resin column and an S-9 strong acid cation exchange resin in sequence, both at a flow rate of 2.5 BV / h, and collect the effluent. Finally, add 3% tartaric acid to the effluent to adjust its pH to 3.5, let it stand, collect the lower aqueous phase, adjust the pH to 6.5, decolorize with activated carbon, concentrate under reduced pressure, and dry in vacuum to obtain the product.

[0035] Example 2

[0036] A method for preparing tylosin without histamine residue, comprising the following steps:

[0037] (1) First, tylosin fermentation broth (5 ml, potency 12386 U / ml), surfactant (0.3 g) and polyaluminium chloride (0.02 g) were stirred and mixed at 500 rpm for 60 min, wherein the surfactant was composed of lauroyl diethanolamine and betaine in a mass ratio of 1:6. After stirring, ultrafiltration was performed, the pore size of the ultrafiltration membrane was 5 nm, the pressure of the ultrafiltration was 0.6 MPa, the filtrate was collected, and the pH of the filtrate was adjusted to 7.5.

[0038] (2) Add 60% ethanol in a volume concentration of 3 times the volume of the filtrate to the filtrate, stir at 400 rpm for 2.5 hours, concentrate under reduced pressure to remove the ethanol, and collect the concentrate. At 15°C, pass the concentrate through a Ni-NTA His·Bind resin column and a SR-1 type strong acid cation exchange resin in sequence, both at a flow rate of 1.0 BV / h, and collect the effluent. Finally, add 3% tartaric acid to the effluent to adjust its pH to 3.0, let it stand, collect the lower aqueous phase, adjust the pH to 7.0, decolorize with activated carbon, concentrate under reduced pressure, and dry in vacuum to obtain the product.

[0039] Example 3

[0040] A method for preparing tylosin without histamine residue, comprising the following steps:

[0041] (1) First, tylosin fermentation broth (10 ml, total titer 12386 U / ml), surfactant (0.3 g) and polyaluminium chloride (0.02 g) were stirred and mixed at 500 rpm for 60 min, wherein the surfactant was composed of lauroyl diethanolamine and betaine in a mass ratio of 3:4. After stirring, ultrafiltration was performed, the pore size of the ultrafiltration membrane was 10 nm, the pressure of the ultrafiltration was 0.6 MPa, the filtrate was collected, and the pH of the filtrate was adjusted to 8.0.

[0042] (2) Add 4 times the volume of filtrate with 80% ethanol, stir at 600 rpm for 1.5 h, remove ethanol by vacuum concentration, and collect the concentrate. At 20°C, pass the concentrate through Ni-NTA His·Bind resin column and 001×7FD strong acid cation exchange resin in sequence, with a flow rate of 3.0 BV / h, and collect the effluent. Finally, add 3% tartaric acid to the effluent to adjust its pH to 4.0, let it stand, collect the lower aqueous phase, adjust the pH to 6.0, decolorize with activated carbon, concentrate under reduced pressure, and dry in vacuum to obtain the product.

[0043] Comparative Example 1

[0044] The same as Example 1, the only difference is that the surfactant is lauroyl diethanolamide.

[0045] A method for preparing tylosin without histamine residue, comprising the following steps:

[0046] (1) First, tylosin fermentation broth (8 ml, potency 12386 U / ml), surfactant (0.6 g) and polyaluminium chloride (0.04 g) were stirred and mixed at 400 rpm for 80 min, wherein the surfactant was lauroyl diethanolamide. After stirring, ultrafiltration was performed, the pore size of the ultrafiltration membrane was 10 nm, the pressure of the ultrafiltration was 0.6 MPa, the filtrate was collected, and the pH of the filtrate was adjusted to 7.8.

[0047] (2) Add 70% ethanol in a volume concentration of 2 times the volume of the filtrate to the filtrate, stir at 500 rpm for 2 hours, concentrate under reduced pressure to remove the ethanol, and collect the concentrate. At 20°C, pass the concentrate through a Ni-NTA His·Bind resin column and an S-9 strong acid cation exchange resin in sequence, both at a flow rate of 2.5 BV / h, and collect the effluent. Finally, add 3% tartaric acid to the effluent to adjust its pH to 3.5, let it stand, collect the lower aqueous phase, adjust the pH to 6.5, decolorize with activated carbon, concentrate under reduced pressure, and dry in vacuum to obtain the product.

[0048] Comparative Example 2

[0049] The same as Example 1, the only difference is that the surfactant is betaine.

[0050] A method for preparing tylosin without histamine residue, comprising the following steps:

[0051] (1) First, tylosin fermentation broth (8 ml, potency 12386 U / ml), surfactant (0.6 g) and polyaluminium chloride (0.04 g) were stirred and mixed at 400 rpm for 80 min, wherein the surfactant was betaine. After stirring, ultrafiltration was performed, the pore size of the ultrafiltration membrane was 10 nm, the pressure of the ultrafiltration was 0.6 MPa, the filtrate was collected, and the pH of the filtrate was adjusted to 7.8.

[0052] (2) Add 70% ethanol in a volume concentration of 2 times the volume of the filtrate to the filtrate, stir at 500 rpm for 2 hours, concentrate under reduced pressure to remove the ethanol, and collect the concentrate. At 20°C, pass the concentrate through a Ni-NTA His·Bind resin column and an S-9 strong acid cation exchange resin in sequence, both at a flow rate of 2.5 BV / h, and collect the effluent. Finally, add 3% tartaric acid to the effluent to adjust its pH to 3.5, let it stand, collect the lower aqueous phase, adjust the pH to 6.5, decolorize with activated carbon, concentrate under reduced pressure, and dry in vacuum to obtain the product.

[0053] Comparative Example 3

[0054] Same as Example 1, except that the Ni-NTA His·Bind resin column was replaced with His Pur TM Cobalt resin column.

[0055] A method for preparing tylosin without histamine residue, comprising the following steps:

[0056] (1) First, tylosin fermentation broth (8 ml, potency 12386 U / ml), surfactant (0.6 g) and polyaluminium chloride (0.04 g) were stirred and mixed at 400 rpm for 80 min, wherein the surfactant was composed of lauroyl diethanolamine and betaine in a mass ratio of 1:2. After stirring, ultrafiltration was performed, the pore size of the ultrafiltration membrane was 10 nm, the pressure of the ultrafiltration was 0.6 MPa, the filtrate was collected, and the pH of the filtrate was adjusted to 7.8.

[0057] (2) Add 70% ethanol in a volume concentration of 2 times the volume of the filtrate to the filtrate, stir at 500 rpm for 2 h, remove the ethanol by vacuum concentration, and collect the concentrate. TM The cobalt resin column and S-9 type strong acid cation exchange resin are both at a flow rate of 2.5 BV / h, and the effluent is collected. Finally, 3% volume concentration of tartaric acid is added to the effluent to adjust its pH to 3.5, and the effluent is allowed to stand, and the lower aqueous phase is collected, and the pH is adjusted to 6.5, and activated carbon is used for decolorization, reduced pressure concentration, and vacuum drying to obtain the product.

[0058] Comparative Example 4

[0059] The same as Example 1, the only difference is that the S-9 type strong acid cation exchange resin is replaced by the D001 type strong acid cation exchange resin.

[0060] A method for preparing tylosin without histamine residue, comprising the following steps:

[0061] (1) First, tylosin fermentation broth (8 ml, potency 12386 U / ml), surfactant (0.6 g) and polyaluminium chloride (0.04 g) were stirred and mixed at 400 rpm for 80 min, wherein the surfactant was composed of lauroyl diethanolamine and betaine in a mass ratio of 1:2. After stirring, ultrafiltration was performed, the pore size of the ultrafiltration membrane was 10 nm, the pressure of the ultrafiltration was 0.6 MPa, the filtrate was collected, and the pH of the filtrate was adjusted to 7.8.

[0062] (2) Add 70% ethanol in a volume concentration of 2 times the volume of the filtrate to the filtrate, stir at 500 rpm for 2 hours, remove the ethanol by decompression and concentrate, and collect the concentrate. At 20°C, pass the concentrate through a Ni-NTA His·Bind resin column and a D001 strong acid cation exchange resin column in sequence, with a flow rate of 2.5 BV / h, and collect the effluent. Finally, add 3% tartaric acid to the effluent to adjust its pH to 3.5, let it stand, collect the lower aqueous phase, adjust the pH to 6.5, decolorize with activated carbon, concentrate under reduced pressure, and dry in vacuum to obtain the product.

[0063] Comparative Example 5

[0064] The same as Example 1, except that the volume concentration of ethanol is 95%.

[0065] A method for preparing tylosin without histamine residue, comprising the following steps:

[0066] (1) First, tylosin fermentation broth (8 ml, potency 12386 U / ml), surfactant (0.6 g) and polyaluminium chloride (0.04 g) were stirred and mixed at 400 rpm for 80 min, wherein the surfactant was composed of lauroyl diethanolamine and betaine in a mass ratio of 1:2. After stirring, ultrafiltration was performed, the pore size of the ultrafiltration membrane was 10 nm, the pressure of the ultrafiltration was 0.6 MPa, the filtrate was collected, and the pH of the filtrate was adjusted to 7.8.

[0067] (2) Add 95% ethanol in a volume concentration of 2 times the volume of the filtrate to the filtrate, stir at 500 rpm for 2 hours, concentrate under reduced pressure to remove the ethanol, and collect the concentrate. At 20°C, pass the concentrate through a Ni-NTA His·Bind resin column and an S-9 strong acid cation exchange resin in sequence, both at a flow rate of 2.5 BV / h, and collect the effluent. Finally, add 3% tartaric acid to the effluent to adjust its pH to 3.5, let it stand, collect the lower aqueous phase, adjust the pH to 6.5, decolorize with activated carbon, concentrate under reduced pressure, and dry in vacuum to obtain the product.

[0068] Test Case

[0069] First, according to the record of tylosin in Volume 1 of the 2020 edition of the Chinese Veterinary Pharmacopoeia, the tylosin content (calculated as the sum of the contents of tylosin A, tylosin B, tylosin C and tylosin D) prepared in Examples 1-3 and Comparative Examples 1-5 was tested respectively.

[0070] Then, the histamine content of the tylosin prepared in Examples 1-3 and Comparative Examples 1-5 was measured respectively according to the HP1C method.

[0071] Test solution: accurately weigh 1.0 g of tylosin tartrate, add 10 ml of water, vortex mix, ultrasonicate for 10 min, centrifuge at 8000 r / min for 2 min, accurately measure 200 μl of supernatant, add 800 μl of acetonitrile, vortex mix, pass through 0.22 μm organic filter membrane, and set aside.

[0072] Histamine reference solution: Weigh approximately 33.4 mg of histamine dihydrochloride reference (purity ≥ 99.0%), accurately weigh, and place in a 200 ml brown volumetric flask, dilute to scale with 0.1 mol / l hydrochloric acid solution, shake well, and prepare a 100 mg / l stock solution, and then dilute to a concentration of 5.0 mg / l histamine reference solution.

[0073] The chromatographic conditions are as follows: the chromatographic column is a Waters Sunfire C18 column (250 mm×4.6 mm, 5 μm); the flow rate is 1.0 ml / min; the column temperature is 35° C.; the injection volume is 20 μl; the detection wavelength is 254 nm; a histamine standard is used as a reference; the mobile phase A is 100 ml of a 0.01 mol / l ammonium acetate solution containing 1% formic acid and added to 900 ml of acetonitrile; the mobile phase B is 900 ml of a 0.01 mol / l ammonium acetate solution containing 1% formic acid and added to 100 ml of acetonitrile, and the elution program is shown in Table 1.

[0074] Table 1 Elution program

[0075] Time / min Mobile phase A / % Mobile phase B / % 0 60 40 22 75 25 25 100 0 32 100 0 32.01 60 40 37 60 40

[0076] The test results of tylosin content and histamine content prepared in Examples 1-3 and Comparative Examples 1-5 are shown in Table 2.

[0077] Table 2 Content test data

[0078] serial number Tylosin content / % Histamine content / ppm Example 1 99.9 Not detected Example 2 99.8 Not detected Example 3 99.7 0.5 Comparative Example 1 92.9 12.3 Comparative Example 2 91.8 13.7 Comparative Example 3 91.4 13.8 Comparative Example 4 90.8 14.4 Comparative Example 5 89.5 17.6

[0079] As shown in Table 2, the tylosin content prepared in Examples 1-3 is as high as 99.7% or more, and the histamine content is less than 1.0 ppm, which meets the requirement of the enterprise standard for API histamine ≤ 6.9 ppm. However, the tylosin content prepared in Comparative Examples 1-5 is 89.5-92.9%, the API active ingredient loss is large, and the histamine content is 12.3-17.6 ppm, which does not meet the API (API) histamine control standard ≤ 6.9 ppm.

[0080] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing tylosin without histamine residue, characterized in that: The steps include: (1) firstly mixing tylosin fermentation broth, a surfactant and a flocculant, ultrafiltering and collecting the filtrate; (2) Add ethanol to the filtrate, and then pass it through a Ni-NTA His·Bind resin column and a strongly acidic cation exchange resin in sequence, collect the effluent, adjust the pH, and dry it to obtain the product.

2. The preparation method according to claim 1, characterized in that: The surfactant in step (1) includes lauroyl diethanolamine and betaine.

3. The preparation method according to claim 2, characterized in that: The mass ratio of lauroyl diethanolamine to betaine is 1-3:4-6.

4. The preparation method according to claim 1, characterized in that: The flocculant in step (1) is polyaluminium chloride or polyaluminium phosphochlorid.

5. The preparation method according to claim 1, characterized in that: The volume mass ratio of the tylosin fermentation broth, surfactant and flocculant is 5-10 ml: 0.3-0.5 g: 0.02-0.04 g; the mixing in step (1) requires stirring, the stirring speed is 300-500 rpm, and the stirring time is 60-90 min.

6. The preparation method according to claim 1, characterized in that: The pore size of the ultrafiltration membrane in the ultrafiltration in step (1) is 5-10 nm, the pressure of the ultrafiltration is 0.4-0.6 MPa, and the pH needs to be adjusted to 7.5-8.0 after the ultrafiltration is completed.

7. The preparation method according to claim 1, characterized in that: The volume concentration of ethanol in step (2) is 60-80%, and the volume ratio of the filtrate to ethanol is 1:2-4. After the addition of ethanol is completed, stirring is required at 400-600 rpm for 1.5-2.5 hours, and then concentrated under reduced pressure to remove ethanol, and the concentrate is collected.

8. The preparation method according to claim 7, characterized in that: The strongly acidic cation exchange resin in step (2) is selected from one or more of the models SR-1, S-9 and 001×7FD.

9. The preparation method according to claim 7, characterized in that: In step (2), the temperature of the Ni-NTA His·Bind resin column and the strongly acidic cation exchange resin is 15-20° C., and the flow rate is 1-3 BV / h.

10. The preparation method according to claim 1, characterized in that: The pH adjustment process in step (2) includes first adding phosphoric acid or tartaric acid with a volume concentration of 2-3% to the effluent to adjust the pH to 3.0-4.0, allowing it to stand, collecting the lower aqueous phase, and then adjusting the pH to 6.0-7.0; activated carbon decolorization and concentration are also performed before drying, and the drying is vacuum drying or freeze drying.