A high-purity ningnanmycin reference standard and its preparation method
Ningnanmycin is separated and purified by strong acid cationic resin and gel column, combined with methanol desalting treatment, simplifying the preparation process of Ningnanmycin, solving the problems of cumbersome steps and expensive equipment in the existing technology, and achieving stable production of high-purity Ningnanmycin standards, reducing quality control costs.
Patent Information
- Application Number
- CN202211704198.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-29
- Publication Date
- 2025-07-04
- Estimated Expiration
- 2042-12-29
AI Technical Summary
The prior art has cumbersome steps in the purification process of Ningnanmycin, expensive equipment, and it is difficult to mass produce stable standard products, resulting in inconsistent quality of Ningnanmycin, which increases the cost of quality control.
After adsorption and separation by strong acid cationic resin, the preparation process of ningnanmycin was simplified by using CM-25 gel and LH-20 gel, and the preparation process of ningnanmycin was simplified by discarding the high-performance liquid chromatography step to directly obtain high-purity ningnanmycin standard products.
It has achieved efficient and simple purification of Ningnanmycin, reduced production costs, provided a stable source of standard products, solved the problem of inconsistent quality of Ningnanmycin, and promoted the sustainable development of biopesticide enterprises.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of purification of agricultural antibiotics, and particularly relates to a high-purity ningnanmycin reference standard and a preparation method thereof. Background Art
[0002] Ningnanmycin is a patented technical product developed successfully by the Chengdu Institute of Biology, Chinese Academy of Sciences. This strain was isolated from the soil in Ningnan County, Sichuan Province and is a newly discovered cytosine nucleoside peptide-type new antibiotic. Therefore, its fermentation product was named ningnanmycin. Now, a series of ningnanmycin products such as 2% aqueous solution, 8% aqueous solution, 10% soluble powder, and bitter·ningnanmycin seed coating agent have been developed and registered for tobacco mosaic virus disease, tomato virus disease, pepper virus disease, rice damping-off, soybean root rot, rice stripe disease, apple blotch leaf spot, and cucumber powdery mildew. Currently, there are a total of 11 domestic registration certificates, including two registration certificates for 40% ningnanmycin technical material. In addition, ningnanmycin has also been widely promoted and applied in the prevention and control of Sclerotinia sclerotiorum of rape, Peronophythora litchi, and other crop virus diseases, stem rot, gummy stem blight, powdery mildew, etc.
[0003] Ningnanmycin is an antibiotic pesticide produced by microbial fermentation technology. Its characteristic is that its producing strain 16A-6 is a new variant of Streptomyces noursei, named Streptomyces noursei var. xichangensis. The antibiotic produced by this strain is named ningnanmycin, and its chemical name is 1-(4-sarcosyl-L-seryl-4-deoxy-β-D-glucuronamide) cytosine, with a molecular weight of 444 [mass spectrometry] and a molecular formula of C 16 H 25 N7O8. The structural formula is:
[0004]
[0005] According to the retrieval of the prior art, the common purification and preparation process of ningnanmycin is adsorption by 732 cation resin - chromatography on D110 macroporous resin - chromatography on neutral alumina column - preparative high-performance liquid chromatography to obtain a single ningnanmycin product. On this basis, after the crude extraction of ningnanmycin by 732 cation resin in the present invention, the original reported process is changed, and a more simple and easily available gel column is directly used to separate and purify ningnanmycin, and then further desalting treatment can obtain a high-purity ningnanmycin reference standard. This method is more convenient than the previously reported steps, and the used instrument and equipment are more easily available. At the same time, the preparative high-performance liquid chromatography step is abandoned, and it can be mass-produced, providing a stable source of reference standard for the detection of ningnanmycin products. Summary of the Invention
[0006] The present invention provides a high-purity ningnanmycin reference standard and a preparation method thereof. On the basis of roughly separating the ningnanmycin aqueous solution by using a strongly acidic cation exchange resin, the analytical solution is further separated and purified by using CM-25 gel, and desalting treatment is carried out by using methanol and LH-20 gel, and then concentration and freeze-drying are carried out to obtain a ningnanmycin reference standard with a purity ≥ 98%.
[0007] To achieve the invention purpose, the present invention adopts the following technical scheme: A preparation method of a high-purity ningnanmycin reference standard, wherein the ningnanmycin aqueous solution successively undergoes the steps of adsorption and separation by a strongly acidic cation resin, low-pressure concentration, desalting by methanol, separation by CM-25 gel, low-pressure concentration, desalting by methanol, desalting by LH-20 gel, concentration and freeze-drying to obtain a ningnanmycin reference standard with a purity ≥ 98%.
[0008] More specifically, the technical scheme adopted by the present invention is as follows:
[0009] A preparation method of a high-purity ningnanmycin reference standard, comprising the following technological steps:
[0010] Adsorption and separation by a strong-acid cation resin: After the ningnanmycin aqueous solution is left standing, the supernatant is taken and filtered, and the filtrate is reserved; the 732 cation resin is treated into the NH4 + type, the filtrate is passed through the resin, after adsorption saturation, the resin column is washed with pure water, and then eluted with ammonia water, and the analytical solution is collected;
[0011] Low-pressure concentration and desalting by methanol: 1) The analytical solution is concentrated, after concentration is completed, methanol is added to wash out ningnanmycin, and then concentration is continued, and the above steps 2 to 3 times are repeated, finally the methanol is evaporated to dryness, and the remaining solid matter is dissolved with pure water;
[0012] Separation by a CM-25 gel column: The CM-25 gel is fully swollen with deionized water and loaded into a glass chromatography column, the above concentrated solution is transferred into the gel column, gradient elution is carried out, first eluted with ultrapure water, then eluted with a sodium chloride solution, and finally eluted with a sodium chloride solution, and the fraction with a ningnanmycin purity ≥ 98% is collected;
[0013] Low-pressure concentration and desalting by methanol: 3) The collected fraction is concentrated, after concentration is completed, methanol is added to wash out ningnanmycin, and then concentration is continued, and the above steps 2 to 3 times are repeated, finally the methanol is evaporated to dryness, and the remaining solid matter is dissolved with pure water;
[0014] Desalting by LH-20 gel: 4) The concentrated solution is passed through the LH-20 gel, balanced with pure water, the mobile phase is pure water, the fraction is collected, the fraction is detected, and the fraction without salt is collected; the fraction with salt can be repeatedly desalted on the LH-20 again until no salt is detected, and the collected fraction without salt is concentrated under reduced pressure until it is evaporated to dryness;
[0015] Decompression concentration and freeze-drying: Concentrate the salt-free ningnanmycin fraction with a purity ≥ 98% prepared in step 5), and then freeze-dry the concentrated solution to obtain a ningnanmycin standard product with a purity ≥ 98%.
[0016] In the preferred implementation steps of the present invention, in step 1), the sample loading flow rate of the filtrate through the resin is 1 BV / h to 1.5 BV / h; the concentration of the ammonia water in the eluent is 2% to 3%; and the elution flow rate is 1.5 BV / h to 2.5 BV / h.
[0017] In the preferred implementation steps of the present invention, in step 2), the low-pressure concentration air pressure range is 0.1 to 0.3 atmospheres.
[0018] In the preferred implementation steps of the present invention, in step 3), the CM-25 gel is Sephadex CM-25 gel; the size of the glass chromatography column is 10 cm × 1000 mm; the sample loading volume is 20 ml to 50 ml; and the elution mobile phase is successively 2 L of pure water, 2 L of 0.2N to 0.3N NaCl aqueous solution, and 2 L of 0.4N sodium chloride solution.
[0019] In the preferred implementation steps of the present invention, in step 4), the low-pressure concentration air pressure range is 0.1 to 0.3 atmospheres.
[0020] In the preferred implementation steps of the present invention, in step 5), the size of the LH-20 gel is 60 mm * 1000 mm; and the sample loading volume each time is 20 to 50 ml.
[0021] The present invention also protects the ningnanmycin standard product prepared by the above preparation method, and the purity of the ningnanmycin standard product is ≥ 98%.
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0023] In the prior art, the purification and preparation process of ningnanmycin is to adsorb through 732 cation resin, chromatographically separate through D110 macroporous resin, chromatographically separate through neutral alumina column, and prepare through high-performance liquid chromatography to obtain a single ningnanmycin single product. On this basis, in the present invention, after the crude extraction of ningnanmycin by 732 cation resin, the original reported process is changed, and a more easily available gel column is directly used to separate and purify ningnanmycin, and then further desalting treatment can obtain a high-purity ningnanmycin standard product. Compared with the preparation method of the prior art, this method has more convenient steps, and the used instruments and equipment are more easily available. At the same time, the high-performance liquid chromatography preparation step is discarded, and it can be mass-produced, providing a stable standard product source for the detection of ningnanmycin products.
[0024] (2) The method of the present invention can be applied to the quality detection of ningnanmycin in current enterprise production, filling the blank of the current ningnanmycin reference standard, greatly reducing the quality control cost of ningnanmycin, effectively solving the phenomenon of uneven quality of ningnanmycin, and being conducive to the sustainable development of bio-pesticide enterprises. Description of the Drawings
[0025] The following is a further description in conjunction with the drawings.
[0026] Figure 1 It is the liquid chromatogram of ningnanmycin aqueous solution.
[0027] Figure 2 It is the liquid chromatogram of the ningnanmycin reference standard obtained in Example 1.
[0028] Figure 3 It is the mass spectrum of the ningnanmycin reference standard obtained in Example 1.
[0029] Figure 4 It is the 1H NMR spectrum of the ningnanmycin reference standard obtained in Example 1.
[0030] Figure 5 It is the 13C NMR spectrum of the ningnanmycin reference standard obtained in Example 1. Detailed Embodiments
[0031] The substantial features of the present invention are illustrated by the following examples. It should be understood that the examples are for illustration purposes only and do not limit the implementation of the invention. The scope and core content of the present invention are defined by the claims. Examples
[0032] Strong acid cation resin adsorption and separation: Let the ningnanmycin aqueous solution stand for 1 h, take the supernatant and filter it, and keep the filtrate for use; Treat the 732 cation resin into the NH4 + type, pass the filtrate through the resin at a flow rate of 1 BV / h. After adsorption saturation, wash the resin column with pure water, and then elute it with 2% ammonia water at an elution rate of 1.5 BV / h, and collect the eluate;
[0033] Vacuum concentration and methanol desalting: Concentrate the eluate obtained in 1). After concentration, add methanol to wash out ningnanmycin, and then continue to concentrate. Repeat the above steps 2-3 times. Finally, evaporate the methanol to dryness, and dissolve the remaining solid matter with pure water;
[0034] (3) CM-25 gel column separation: Sephadex CM-25 gel was fully swollen with deionized water and loaded into a glass chromatography column (10 cm × 1000 mm). The above concentrate was transferred to the gel column with a sample volume of 20 ml and gradient elution was performed. First, 2 L of ultrapure water was used for elution to remove most impurities such as pigments; then 2 L of 0.2 N sodium chloride solution was used for elution; finally, 0.4 N sodium chloride solution was used for elution, and the fractions with a purity of ≥98% of Ningnanmycin were collected;
[0035] (4) Concentration under reduced pressure and desalination with methanol: The fractions collected in 3) were concentrated. After the concentration was completed, methanol was added to wash out Ningnanmycin, and then the concentration was continued. The above steps were repeated 3 times. Finally, the methanol was evaporated to dryness and the remaining solids were dissolved in pure water;
[0036] (5) LH-20 gel desalting: The concentrate from 4) was passed through LH-20 gel (60mm*1000mm), balanced with pure water, about 20ml of sample was loaded each time, pure water was used as the mobile phase, and fractions were collected, each of which was about 100ml. The fractions were tested, and the fractions without salt were collected. The fractions with salt can be repeatedly desalted on LH-20 until the salt is undetectable. The collected fractions without salt were concentrated under reduced pressure until they were evaporated to dryness.
[0037] (6) Concentrating under reduced pressure and freeze-drying: Concentrating the salt-free Ningnanmycin fraction with a purity of ≥98% prepared in 5), and freeze-drying the concentrate to obtain a Ningnanmycin standard with a purity of 99.2%. Example
[0038] (1) Strong acid cationic resin adsorption separation: let the Ningnanmycin aqueous solution stand for 1 hour, filter the supernatant, and set aside the filtrate; treat the 732 cation into NH4 + Type, the filtrate is passed through the resin at a flow rate of 1.5BV / h. After adsorption saturation, the resin column is washed with pure water, and then analyzed with 2% ammonia water at a rate of 2BV / h, and the analyzed solution is collected;
[0039] (2) Concentration under reduced pressure and desalination with methanol: The analytical solution from 1) is concentrated. After the concentration is completed, methanol is added to wash out Ningnanmycin, and then the concentration is continued. The above steps are repeated twice. Finally, the methanol is evaporated to dryness and the remaining solids are dissolved in pure water;
[0040] (3) CM-25 gel column separation: Sephadex CM-25 gel was fully swollen with deionized water and loaded into a glass chromatography column (10 cm × 1000 mm). The above concentrate was transferred into the gel column with a sample volume of 30 ml and gradient elution was performed. First, 2 L of ultrapure water was used for elution to remove most impurities such as pigments; then 2 L of 0.2 N sodium chloride solution was used for elution; finally, 0.4 N sodium chloride solution was used for elution, and the fractions with a purity of ≥98% of Ningnanmycin were collected;
[0041] (4) Concentration under reduced pressure and desalination with methanol: The fractions collected in 3) were concentrated. After the concentration was completed, methanol was added to wash out Ningnanmycin, and then the concentration was continued. The above steps were repeated 3 times. Finally, the methanol was evaporated to dryness and the remaining solids were dissolved in pure water;
[0042] (5) LH-20 gel desalting: The concentrate from 4) was passed through LH-20 gel (60mm*1000mm), balanced with pure water, about 30ml of sample was loaded each time, pure water was used as the mobile phase, and fractions were collected, each of which was about 100ml. The fractions were tested, and the fractions without salt were collected. The fractions with salt can be repeatedly desalted on LH-20 until the salt is undetectable. The collected fractions without salt were concentrated under reduced pressure until they were evaporated to dryness.
[0043] (6) Concentrating under reduced pressure and freeze-drying: Concentrating the salt-free Ningnanmycin fraction with a purity of ≥98% prepared in 5), and freeze-drying the concentrate to obtain a Ningnanmycin standard with a purity of 98.6%. Example
[0044] (1) Strong acid cationic resin adsorption separation: let the Ningnanmycin aqueous solution stand for 1 hour, filter the supernatant, and set aside the filtrate; treat the 732 cation into NH4 + Type, the filtrate is passed through the resin at a flow rate of 1.5BV / h. After adsorption saturation, the resin column is washed with pure water, and then analyzed with 2% ammonia water at a rate of 2.5BV / h, and the analyzed solution is collected;
[0045] (2) Concentration under reduced pressure and desalination with methanol: The analytical solution from 1) is concentrated. After the concentration is completed, methanol is added to wash out Ningnanmycin, and then the concentration is continued. The above steps are repeated twice. Finally, the methanol is evaporated to dryness and the remaining solids are dissolved in pure water;
[0046] (3) CM-25 gel column separation: Sephadex CM-25 gel was fully swollen with deionized water and loaded into a glass chromatography column (10 cm × 1000 mm). The above concentrate was transferred into the gel column with a loading volume of 50 ml. Gradient elution was performed. First, 2 L of ultrapure water was used for elution to remove most impurities such as pigments; then 2 L of 0.2 N sodium chloride solution was used for elution; finally, 0.4 N sodium chloride solution was used for elution, and the fractions with a purity of ≥98% of Ningnanmycin were collected;
[0047] (4) Concentration under reduced pressure and desalination with methanol: The fractions collected in 3) were concentrated. After the concentration was completed, methanol was added to wash out Ningnanmycin, and then the concentration was continued. The above steps were repeated 3 times. Finally, the methanol was evaporated to dryness and the remaining solids were dissolved in pure water;
[0048] (5) LH-20 gel desalting: The concentrate from 4) was passed through LH-20 gel (60mm*1000mm), balanced with pure water, about 50mL of sample was loaded each time, pure water was used as the mobile phase, and fractions were collected, each of which was about 100mL. The fractions were tested, and the fractions without salt were collected. The fractions with salt can be repeatedly desalted on LH-20 until the salt is undetectable. The collected fractions without salt were concentrated under reduced pressure until they were evaporated to dryness.
[0049] (6) Concentrating under reduced pressure and freeze-drying: Concentrating the salt-free Ningnanmycin fraction with a purity of ≥98% prepared in 5), and freeze-drying the concentrate to obtain a Ningnanmycin standard with a purity of 98.1%.
[0050] Note: The amount of standard product used is relatively small, so the yield is not evaluated during the process.
[0051] The above contents are further descriptions of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. Without departing from the concept of the present invention, several substitutions or modifications made to these described embodiments should be deemed to belong to the protection scope of the present invention.
Claims
1. A preparation method of a high-purity ningnanmycin standard substance, characterized in that, The process steps include: 1) Adsorption and separation by strong acid cation resin: Let the ningnanmycin aqueous solution stand still, then take the supernatant for filtration, and keep the filtrate for use; Treat the 732 cation resin into NH4 + type, pass the filtrate through the resin, after adsorption saturation, wash the resin column with pure water, and then elute with ammonia water to collect the eluate; 2) Low-pressure concentration and methanol desalination: Concentrate the analytical solution from 1) and add methanol to wash out Ningnanmycin after concentration. Then continue to concentrate and repeat the above steps 2 to 3 times. Finally, evaporate the methanol to dryness and dissolve the remaining solids with pure water. 3) CM-25 gel column separation: CM-25 gel is fully swollen with deionized water and loaded into a glass chromatography column. The above concentrate is transferred into the gel column and gradient eluted, first with ultrapure water, then with sodium chloride solution, and finally with sodium chloride solution, and the fraction with Ningnanmycin purity ≥98% is collected; 4) Low-pressure concentration and methanol desalination: Concentrate the fractions collected in 3). After concentration, add methanol to wash out Ningnanmycin, and then continue to concentrate. Repeat the above steps 2 to 3 times. Finally, evaporate the methanol to dryness and dissolve the remaining solids with pure water; 5) LH-20 gel desalting: pass the concentrate from 4) through LH-20 gel, balance with pure water, use pure water as mobile phase, collect fractions, detect the fractions, and collect the fractions without salt; repeat the LH-20 desalting on the fractions with salt until the salt is undetectable, and concentrate the collected fractions without salt under reduced pressure until they are evaporated to dryness; 6) Concentrating under reduced pressure and freeze-drying: Concentrating the salt-free Ningnanmycin fraction with a purity of ≥98% prepared in 5), and freeze-drying the concentrate to obtain a Ningnanmycin standard with a purity of ≥98%.
2. The preparation method according to claim 1, wherein In step 1), the sample flow rate of the filtrate passing through the resin is 1 BV / h~1.5 BV / h; the concentration of the analytical liquid ammonia water is 2%~3%; the analytical flow rate is 1.5 BV / h~2.5 BV / h.
3. The preparation method according to claim 1, characterized in that, In step 2), the low-pressure concentrated gas pressure ranges from 0.1 to 0.3 atmospheres.
4. The preparation method according to claim 1, wherein In step 3), the CM-25 gel is Sephadex CM-25 gel; the size of the glass chromatography column is 10 cm×1000 mm; the loading volume is 20 ml~50 ml; the elution mobile phases are 2 L of pure water, 2 L of 0.2N~0.3N NaCl aqueous solution, and 2 L of 0.4N sodium chloride solution.
5. The preparation method according to claim 1, characterized in that, In step 4), the low-pressure concentrated gas pressure ranges from 0.1 to 0.3 atmospheres.
6. The preparation method according to claim 1, wherein In step 5), the size of the LH-20 gel is 60 mm*1000 mm; and the sample loading volume is 20-50 mL each time.
Citation Information
Patent Citations
High-purity zhongshengmycin F reference substance and preparation method thereof
CN112442096A