CN113831390B - High-purity melittin and preparation method thereof - Google Patents
High-purity melittin and preparation method thereof Download PDFInfo
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- CN113831390B CN113831390B CN202111116875.4A CN202111116875A CN113831390B CN 113831390 B CN113831390 B CN 113831390B CN 202111116875 A CN202111116875 A CN 202111116875A CN 113831390 B CN113831390 B CN 113831390B
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- 108010036176 Melitten Proteins 0.000 title claims abstract description 93
- VDXZNPDIRNWWCW-JFTDCZMZSA-N melittin Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(N)=O)CC1=CNC2=CC=CC=C12 VDXZNPDIRNWWCW-JFTDCZMZSA-N 0.000 title claims abstract description 93
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000004005 microsphere Substances 0.000 claims abstract description 13
- 238000005119 centrifugation Methods 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000004793 Polystyrene Substances 0.000 claims abstract description 11
- 229920002223 polystyrene Polymers 0.000 claims abstract description 11
- 239000008213 purified water Substances 0.000 claims abstract description 10
- 239000000945 filler Substances 0.000 claims abstract description 8
- 239000003659 bee venom Substances 0.000 claims description 44
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 36
- 239000000706 filtrate Substances 0.000 claims description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 20
- 239000003480 eluent Substances 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 10
- 230000001105 regulatory effect Effects 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 8
- 239000012043 crude product Substances 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 2
- 238000004007 reversed phase HPLC Methods 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 13
- 238000000746 purification Methods 0.000 abstract description 11
- 238000000926 separation method Methods 0.000 abstract description 10
- 239000003814 drug Substances 0.000 abstract description 5
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 229920005654 Sephadex Polymers 0.000 abstract description 3
- 239000012507 Sephadex™ Substances 0.000 abstract description 3
- 241000700605 Viruses Species 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- 229960001340 histamine Drugs 0.000 abstract description 2
- 150000002500 ions Chemical class 0.000 abstract description 2
- 238000001556 precipitation Methods 0.000 abstract description 2
- 238000002953 preparative HPLC Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 101000761020 Dinoponera quadriceps Poneritoxin Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000002349 favourable effect Effects 0.000 description 4
- 238000004237 preparative chromatography Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- GLRAHDCHUZLKKC-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid;hydrate Chemical compound O.CC#N.OC(=O)C(F)(F)F GLRAHDCHUZLKKC-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the technical field of medicines, and particularly discloses a high-purity melittin and a preparation method thereof. According to the preparation method of the high-purity melittin, the water-insoluble impurities are removed by centrifugation after the crude melittin is dissolved in purified water, and then the pH value of the centrifugate is adjusted to 3.0-4.0, so that part of the impurities in the crude melittin can be removed by precipitation under the pH value condition, and meanwhile, viruses can be inactivated under the pH value condition, and the pH value is adjusted to 4.5-5.5, thereby being beneficial to improving the stability of the melittin; and removing ions, histamine and other small molecular impurities by an ultrafiltration concentration method, and performing preparative high-performance liquid chromatography purification and separation by using DAC-100 chromatographic columns with polystyrene microspheres as fillers to finally obtain the high-purity melittin with the content of more than 99%, thereby improving the clinical medication safety of the refined melittin, effectively simplifying the preparation process of the melittin without repeated purification by using a sephadex column, and having extremely high popularization and application values.
Description
Technical Field
The invention relates to the technical field of melittin preparation, in particular to high-purity melittin and a preparation method thereof.
Background
Melittin (MLT) is the main active ingredient in bee venom, consisting of 26 amino acid residues, accounting for about 50% of the dry weight of bee venom. Melittin is used as a typical antibacterial peptide, has pharmacological activities of analgesia, anti-inflammatory, antivirus, anti-tumor and the like, and has higher scientific research value and wide medical application value.
Bee venom is a complex mixture, and enzyme substances with large molecular weight are easy to cause allergy, amine substances with small molecular weight are easy to cause pain, so that the substances are removed, the purity of the bee venom peptide is improved, and the bee venom has important significance for clinical popularization and application. However, the prior art has complicated steps and complex operation, and serious loss of melittin in the purification process, so that the preparation cost of melittin is high, and further development and application of melittin are hindered.
At present, the preparation method of the high-purity melittin comprises the following steps: dissolving melittin in water, centrifuging, adding ethanol into centrifugate to remove precipitate, extracting with n-butanol, removing precipitate with ethanol twice, purifying the obtained crude melittin water solution by multiple sephadex column chromatography, ultrafiltering, and freeze drying to obtain melittin with content of 45.34-87.83%, low melittin content, and complex preparation method with high production cost. Therefore, the research and development of the preparation method of the high-purity melittin, which is simple to operate and has higher yield and purity of the melittin, has very important significance for expanding the further development and application of the melittin in the field of medicine.
Disclosure of Invention
Aiming at the problems of complicated operation, high cost and low yield and purity of the prepared melittin existing methods for preparing melittin, the invention provides the high-purity melittin and the preparation method thereof, which mainly comprises the steps of removing impurities and ultrafiltering at a specific pH value after centrifuging a melittin aqueous solution, and separating and purifying by using a preparation chromatograph, thereby effectively improving the purity of the melittin.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
A preparation method of high-purity melittin comprises the following steps:
Step a, adding the bee venom crude product into purified water, uniformly dispersing, and centrifuging to obtain bee venom centrifugate;
step b, regulating the pH of the bee venom centrifugate to 3.0-4.0, standing for 1-2 h, and filtering to obtain a first filtrate; then, the pH value of the first filtrate is regulated to be 4.5-5.5, and the second filtrate is obtained by filtering;
Step c, carrying out ultrafiltration concentration on the second filtrate by using an ultrafiltration membrane with the molecular weight cutoff of 500 Da-1000 Da, and collecting a raffinate to obtain a bee venom ultrafiltrate;
step d, separating and purifying the bee venom ultrafiltrate by reverse phase high performance liquid chromatography, collecting target components, and combining eluents with the HPLC purity of more than 99 percent; wherein the preparation chromatographic column is DAC-100, the filler is polystyrene microsphere, the mobile phase A is trifluoroacetic acid aqueous solution, the mobile phase B is methanol solution containing trifluoroacetic acid or acetonitrile solution containing trifluoroacetic acid, and the gradient elution is carried out;
And e, carrying out ultrafiltration concentration on the melittin eluent by using an ultrafiltration membrane with the molecular weight cutoff of 500 Da-1000 Da, collecting the residual liquid, and freeze-drying to obtain a melittin product.
Compared with the prior art, the preparation method of the high-purity melittin provided by the invention has the advantages that the water-insoluble impurities are removed by centrifugation after the crude melittin is dissolved in purified water, then the pH value of the centrifugate is regulated to 3.0-4.0, so that partial impurities in the crude melittin can be removed by precipitation under the pH value condition, and meanwhile, viruses can be inactivated under the pH value condition, and the pH value is regulated to 4.5-5.5, thereby being beneficial to improving the stability of the melittin; removing ion and small molecular impurities such as histamine by ultrafiltration concentration, and performing preparative chromatography purification separation by using DAC-100 chromatographic column with polystyrene microsphere as filler to obtain high-purity melittin with content of more than 99%.
The invention can separate and remove the sensitization substances and ineffective impurities in the bee venom crude product only through simple processes such as centrifugation, pH adjustment, ultrafiltration, preparation chromatographic separation and the like, is favorable for realizing the purpose of rapidly producing refined bee venom products in large quantities, has the purity of more than 99 percent, improves the safety of clinical medication, does not need to be purified for many times through a sephadex column, effectively simplifies the preparation process of the bee venom peptide, reduces the cost and has extremely high popularization and application value.
The preparation chromatographic column is preferably a DAC-100 (100 mm 250 mm) preparation column manufactured by Jiangsu Hanbang technology Co., ltd; the filler is preferably polystyrene microsphere UniPS 20-300.
Preferably, in the step a, the mass ratio of the bee venom crude product to the purified water is 1: 8-10.
The preferable ratio of the crude bee venom to purified water can fully dissolve bee venom, remove water insoluble impurities as much as possible, and improve the purity of bee venom peptide.
Preferably, in the step a, the rotation speed of centrifugation is 4000 r/min-6000 r/min, and the centrifugation time is 15 min-30 min.
The preferred centrifugation speed and centrifugation time remove as much water insoluble impurities as possible.
Optionally, in the step b, 1.0mol/L hydrochloric acid solution is selected to adjust the pH of the bee venom centrifugate to 3.0-4.0.
The preferred acidic conditions can inactivate viruses while precipitating as much as possible of the impurities in the crude bee venom.
Optionally, in the step b, 1.0mol/L sodium hydroxide solution is selected to adjust the pH to 4.5-5.5.
The preferred pH value is favorable for improving the stability of the melittin in the purification process and ensuring the stable quality of the refined melittin product.
Optionally, in step b, after the first filtrate is adjusted to a pH of 4.5-5.5, a 0.45 μm filter membrane is used.
Preferably, in the step c, when the volume of the residual liquid to be filtered is 30% -40% of the volume of the second filtrate during ultrafiltration concentration, the concentration is stopped.
The preferred filtrate volume is beneficial to improving the purity and yield of melittin and simultaneously is beneficial to improving the efficiency of the subsequent high performance liquid chromatography separation and purification.
Preferably, in step c, the ultrafiltration pressure is between 0.4MPa and 0.8MPa.
The preferable operation pressure can ensure that impurities have higher transmittance and higher membrane flux, and can also avoid membrane pollution caused by overlarge pressure and improve ultrafiltration efficiency.
Preferably, in the step d, the polystyrene microsphere has a particle size of 20 μm and a pore size of
Preferably, in step d, the gradient elution is performed in the following sequence:
0min of mobile phase A65-60%, mobile phase B35-40%;
50% -45% of mobile phase A and 50% -55% of mobile phase B in 15 min.
Preferably, in step d, the flow rate is 280mL/min to 320mL/min.
Preferably, in the step d, the volume percentage of trifluoroacetic acid in the mobile phase A is 0.05% -0.2%.
Preferably, in the step d, the volume percentage of trifluoroacetic acid in the mobile phase B is 0.05% -0.2%.
In the high performance liquid chromatography separation and purification process, the selection and combination of various chromatographic conditions are important, and the separation degree, the peak time and the like of various components are directly influenced. The preferred chromatographic conditions of the invention are favorable for effectively separating the melittin from impurities, and have better separation effect in a shorter time.
Preferably, in the step e, when ultrafiltration concentration is performed, the concentration is stopped when the volume of the residual liquid to be filtered is the same as the volume of the bee venom ultrafiltration liquid obtained in the step c.
The preferred volume of the filtered liquid is favorable for subsequent freeze-drying of the melittin, shortens the freeze-drying time and obtains the melittin product with good physical form and smooth surface.
Preferably, in step e, the ultrafiltration pressure is between 0.4MPa and 0.8MPa.
The optimized operation pressure can improve the ultrafiltration efficiency and shorten the concentration time, thereby improving the production efficiency of melittin.
Optionally, in step c and step e, ultrafiltration is performed at room temperature.
The invention also provides a high-purity melittin, which is prepared by the preparation method of the high-purity melittin.
The invention effectively improves the purity of the melittin through a simple process, and ensures that the purity of the melittin reaches more than 99 percent, thereby improving the safety of clinical application of the melittin, being suitable for industrial mass production, having very important significance for expanding the development and application of the melittin in the medicine field and having wide market prospect.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In order to better illustrate the present invention, the following examples are provided for further illustration.
The polystyrene microspheres UniPS 20-300 and methacrylate microspheres UniPMM 20-500 used in the examples below were all purchased from Soy micro technology Co. The preparation chromatographic column is a DAC-100 (100 mm. Times.250 mm) preparation column manufactured by Jiangsu Hanbang technology Co., ltd.
The crude bee venom used in the following examples and comparative examples had a content of melittin of 46.7%.
Example 1
The embodiment provides a preparation method of high-purity melittin, which comprises the following steps:
step a, adding 25g of bee venom crude product into 250g of purified water, uniformly dispersing, centrifuging for 30min at the rotating speed of 4000r/min, and taking supernatant after the centrifugation is finished to obtain bee venom centrifugate;
step b, regulating the pH of the bee venom centrifugate to 3.5 by using 0.1mol/L hydrochloric acid solution, standing for 1.5h at room temperature, and filtering to obtain a first filtrate; then adjusting the pH value of the first filtrate to 5.5 by using 0.1mol/L sodium hydroxide solution, and filtering the first filtrate by using a 0.45 mu m filter membrane to obtain a second filtrate;
Step c, carrying out ultrafiltration concentration on the second filtrate by using an ultrafiltration membrane with the molecular weight cutoff of 1000Da, wherein the ultrafiltration pressure is 0.4MPa, stopping concentration when the volume of the filtered residual liquid is 30% of the volume of the second filtrate, and collecting the filtered residual liquid to obtain the bee venom ultrafiltrate;
Step d, separating and purifying the bee venom ultrafiltrate by a preparation chromatographic column, collecting target components, detecting the HPLC purity of the main peak eluent, and collecting and combining the eluents with the HPLC purity of more than 99% to obtain the bee venom peptide eluent;
wherein, the conditions for separation and purification by preparative chromatography are as follows:
chromatographic column: DAC-100, wherein the filler is polystyrene microsphere UniPS 20-300;
detection wavelength: 220nm;
Mobile phase a:0.05% aqueous trifluoroacetic acid;
Mobile phase B: acetonitrile solution containing 0.05% trifluoroacetic acid;
Flow rate: 300mL/min;
gradient elution, the elution sequence is:
0min mobile phase A65% mobile phase B35%;
15min mobile phase A50% and mobile phase B50%.
And e, carrying out ultrafiltration concentration on the obtained melittin eluent by using an ultrafiltration membrane with the molecular weight cut-off of 1000Da, wherein the ultrafiltration pressure is 0.4MPa, stopping ultrafiltration when the volume of the filtered residual liquid is the same as that of the melittin ultrafiltrate obtained in the step c, collecting the filtered residual liquid, and freeze-drying to obtain 7.9g of melittin freeze-dried powder, wherein the melittin content is 99.3%.
Example 2
The embodiment provides a preparation method of high-purity melittin, which comprises the following steps:
Step a, adding 25g of bee venom crude product into 200g of purified water, uniformly dispersing, centrifuging at a rotation speed of 5000r/min for 20min, and taking supernatant after centrifugation to obtain bee venom centrifugate;
Step b, regulating the pH of the bee venom centrifugate to 3.0 by using 0.1mol/L hydrochloric acid solution, standing for 1h at room temperature, and filtering to obtain a first filtrate; then adjusting the pH value of the first filtrate to 4.5 by using 0.1mol/L sodium hydroxide solution, and filtering by using a 0.45 mu m filter membrane to obtain a second filtrate;
step c, carrying out ultrafiltration concentration on the second filtrate by using an ultrafiltration membrane with the molecular weight cutoff of 1000Da, wherein the ultrafiltration pressure is 0.6MPa, stopping concentration when the volume of the filtered residual liquid is 35% of the volume of the second filtrate, and collecting the filtered residual liquid to obtain the bee venom ultrafiltrate;
Step d, separating and purifying the bee venom ultrafiltrate by a preparation chromatographic column, collecting target components, detecting the HPLC purity of the main peak eluent, and collecting and combining the eluents with the HPLC purity of more than 99% to obtain the bee venom peptide eluent;
wherein, the conditions for separation and purification by preparative chromatography are as follows:
chromatographic column: DAC-100, wherein the filler is polystyrene microsphere UniPS 20-300;
detection wavelength: 220nm;
Mobile phase a:0.2% aqueous trifluoroacetic acid;
Mobile phase B: acetonitrile solution containing 0.2% trifluoroacetic acid;
Flow rate: 280mL/min;
gradient elution, the elution sequence is:
mobile phase A60% and mobile phase B40% after 0 min;
15min mobile phase A45% and mobile phase B55%.
And e, carrying out ultrafiltration concentration on the obtained melittin eluent by using an ultrafiltration membrane with the molecular weight cut-off of 1000Da, wherein the ultrafiltration pressure is 0.6MPa, stopping ultrafiltration when the volume of the filtered residual liquid is the same as that of the melittin ultrafiltrate obtained in the step c, collecting the filtered residual liquid, and freeze-drying to obtain 8.1g of melittin freeze-dried powder, wherein the melittin content is 99.1%.
Example 3
The embodiment provides a preparation method of high-purity melittin, which comprises the following steps:
step a, adding 25g of bee venom crude product into 225g of purified water, uniformly dispersing, centrifuging at 6000r/min for 15min, and taking supernatant after centrifugation to obtain bee venom centrifugate;
Step b, regulating the pH of the bee venom centrifugate to 4.0 by using 0.1mol/L hydrochloric acid solution, standing for 2 hours at room temperature, and filtering to obtain a first filtrate; then adjusting the pH value of the first filtrate to 5.0 by using 0.1mol/L sodium hydroxide solution, and filtering the first filtrate by using a 0.45 mu m filter membrane to obtain a second filtrate;
Step c, carrying out ultrafiltration concentration on the second filtrate by using an ultrafiltration membrane with the molecular weight cutoff of 500Da, wherein the ultrafiltration pressure is 0.8MPa, stopping concentration when the volume of the filtered residual liquid is 30% of the volume of the second filtrate, and collecting the filtered residual liquid to obtain the bee venom ultrafiltrate;
Step d, separating and purifying the bee venom ultrafiltrate by a high-performance reversed phase liquid preparation chromatographic column, collecting target components, detecting the HPLC purity of the main peak eluent, and collecting and combining the eluents with the HPLC purity of more than 99% to obtain the bee venom peptide eluent;
wherein, the conditions for separation and purification by preparative chromatography are as follows:
chromatographic column: DAC-100, wherein the filler is polystyrene microsphere UniPS 20-300;
detection wavelength: 220nm;
mobile phase a:0.1% aqueous trifluoroacetic acid;
mobile phase B: acetonitrile solution containing 0.1% trifluoroacetic acid;
flow rate: 320mL/min;
gradient elution, the elution sequence is:
0min mobile phase A62%, mobile phase B38%;
15min mobile phase A47% mobile phase B53%.
And e, carrying out ultrafiltration concentration on the obtained melittin eluent by using an ultrafiltration membrane with the molecular weight cutoff of 500Da, wherein the ultrafiltration pressure is 0.8MPa, stopping ultrafiltration when the volume of the filtered residual liquid is the same as that of the melittin ultrafiltrate obtained in the step c, collecting the filtered residual liquid, and freeze-drying to obtain 8.3g of melittin freeze-dried powder, wherein the melittin content is 99.0%.
Comparative example 1
This comparative example provides a method for preparing high purity melittin, which comprises the same steps as in example 1, except that in step b, the melittin centrifugate is not adjusted in pH, the original solution of the melittin centrifugate is 4.95, and the melittin centrifugate is directly subjected to ultrafiltration in step c and subsequent steps d and e.
The prepared melittin is 5.8g, and the melittin content is 99.1%.
Comparative example 2
This comparative example provides a method for preparing high purity melittin, which is identical to example 1 in steps except that in step d the packing of the DAC-100 chromatographic column is replaced by methacrylate microsphere UniPMM 20-500, and the remaining conditions are identical.
The main peak eluent was checked for HPLC purity, with a maximum purity of 93.5%, and therefore, the eluent with main peak HPLC purity greater than 93% was collected and pooled.
The prepared melittin is 7.2g, and the melittin content is 93.2%.
Comparative example 3
The comparative example provides a preparation method of high-purity melittin, which has the same steps as in the example 1, except that the mobile phase in the conditions of preparation chromatography separation and purification in the step d is replaced by trifluoroacetic acid-acetonitrile-water, the volume percentage of the trifluoroacetic acid is 0.05%, the volume ratio of acetonitrile to water is 42:58, the isocratic elution is carried out for 15min, and the rest conditions are the same. The prepared melittin is 4.2g, and the melittin content is 99.0%.
In the above examples and comparative examples, the HPLC analysis conditions for analyzing the content of the produced melittin were as follows:
chromatographic column: agilent ZORBAX 300SB-C8 (4.6X250 mm,5 μm);
mobile phase a:0.1% aqueous trifluoroacetic acid;
mobile phase B: acetonitrile solution containing 0.1% trifluoroacetic acid;
gradient elution, elution sequence is as follows:
| Time (min) | Mobile phase A% | Mobile phase B% |
| 0.01 | 65.0 | 35.0 |
| 15.00 | 50.0 | 50.0 |
| 15.01 | 65.0 | 35.0 |
| 20.00 | 65.0 | 35.0 |
Detection wavelength: 220nm;
Flow rate: 1.0mL/min.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (7)
1. The preparation method of the high-purity melittin is characterized by comprising the following steps of:
Step a, adding the bee venom crude product into purified water, uniformly dispersing, and centrifuging to obtain bee venom centrifugate;
step b, regulating the pH of the bee venom centrifugate to 3.0-4.0, standing for 1-2 h, and filtering to obtain a first filtrate; then, the pH value of the first filtrate is regulated to be 4.5-5.5, and the second filtrate is obtained by filtering;
Step c, carrying out ultrafiltration concentration on the second filtrate by using an ultrafiltration membrane with the molecular weight cutoff of 500 Da-1000 Da, and collecting a raffinate to obtain a bee venom ultrafiltrate;
Step d, separating and purifying the bee venom ultrafiltrate by reverse phase high performance liquid chromatography, collecting target components, and combining eluents with the HPLC purity of more than 99 percent; wherein the preparation chromatographic column is DAC-100, the filler is polystyrene microsphere, the mobile phase A is trifluoroacetic acid aqueous solution, the mobile phase B is acetonitrile solution containing trifluoroacetic acid, and gradient elution is carried out; the particle size of the polystyrene microsphere is 20 mu m, and the aperture is 300A;
The volume percentage of trifluoroacetic acid in the mobile phase A is 0.05% -0.2%; the volume percentage of trifluoroacetic acid in the mobile phase B is 0.05-0.2%;
The gradient elution sequence is as follows:
0min, 65% -60% of mobile phase A and 35% -40% of mobile phase B;
15min, 50% -45% of mobile phase A and 50% -55% of mobile phase B;
And e, carrying out ultrafiltration concentration on the melittin eluent by using an ultrafiltration membrane with the molecular weight cutoff of 500 Da-1000 Da, collecting the residual liquid, and freeze-drying to obtain a melittin product.
2. The method for preparing high purity melittin according to claim 1, wherein in step a, the mass ratio of crude melittin to purified water is 1: 8-10.
3. The method for preparing high purity melittin according to claim 1, wherein in step a, the rotational speed of centrifugation is 4000r/min to 6000r/min and the centrifugation time is 15min to 30min.
4. The method for preparing high purity melittin according to claim 1, wherein in step c, concentration is stopped when the volume of the residual solution to be filtered is 30% -40% of the volume of the second filtrate; and/or
In the step c, the ultrafiltration pressure is 0.4MPa to 0.8MPa.
5. The method for preparing high purity melittin according to claim 1, wherein in step d, the flow rate is 280mL/min to 320mL/min.
6. The method for preparing high purity melittin according to claim 1, wherein in step e, concentration is stopped when the volume of the residual solution to be filtered is the same as the volume of the melittin ultrafiltration liquid; and/or
In the step e, the ultrafiltration pressure is 0.4MPa to 0.8MPa.
7. A high purity melittin produced by the method of producing a high purity melittin as claimed in any one of claims 1 to 6.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007114575A1 (en) * | 2006-03-31 | 2007-10-11 | Ki-Rok Kwon | Preparation of enzyme-free bee venom |
| KR20150049865A (en) * | 2013-10-31 | 2015-05-08 | 주식회사 휴메딕스 | Method for Isolating Melittin from Bee Venom |
| CN113166213A (en) * | 2018-12-05 | 2021-07-23 | 友生物工程株式会社 | Bee venom purification method including virus removal process and composition for preventing or treating inflammatory disease using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014196674A1 (en) * | 2013-06-07 | 2014-12-11 | 주식회사 청진바이오텍 | Preparation method for isolated, purified bee venom having allergic components isolated |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007114575A1 (en) * | 2006-03-31 | 2007-10-11 | Ki-Rok Kwon | Preparation of enzyme-free bee venom |
| KR20150049865A (en) * | 2013-10-31 | 2015-05-08 | 주식회사 휴메딕스 | Method for Isolating Melittin from Bee Venom |
| CN113166213A (en) * | 2018-12-05 | 2021-07-23 | 友生物工程株式会社 | Bee venom purification method including virus removal process and composition for preventing or treating inflammatory disease using the same |
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