Method for purifying melittin with high purity by using pH zone refining countercurrent chromatography

Abstract

The application relates to a high-purity melittin purification method using pH zone refining countercurrent chromatography. In order to overcome the defects of the prior art, a solvent system composed of 4-12% methyl tert-butyl ether, 42-50% n-butanol, 4-12% acetonitrile and 42-50% deionized water is used, a high-purity melittin solution is purified by using the pH zone refining countercurrent chromatography, and the finished high-purity melittin is obtained through concentration and drying. The application does not use solid fillers, does not cause loss to the sample, can almost recover all the melittin in the crude melittin, the yield is more than 90%, and the application is suitable for batch purification of high-purity melittin.

Description

Technical Field

[0001] This invention relates to a method for purifying high-purity melitrix peptides using pH-zone refining countercurrent chromatography. Background Technology

[0002] Honeybees (Apis mellifera L.) have long been considered beneficial insects, and their benefits extend beyond honey; bee venom is also an important natural resource. Bee venom, a natural venom from worker bees, is a complex mixture of various active polypeptides and enzymes, playing a crucial role in protecting the bee colony. Bee venom expelled by worker bees is a pale yellow, bitter, and irritating translucent liquid. The use of bee venom has a long history in both Eastern and Western traditional medicine. For example, in traditional Chinese medicine, bee therapy is a treatment for neurological diseases and inflammation. It involves injecting bee venom into the affected area or meridians using the stinger of a live bee to achieve therapeutic effects. This therapy has shown significant therapeutic effects on diseases such as rheumatoid arthritis. In recent years, the efficacy of bee therapy has been validated in modern medicine. The active polypeptides in bee venom, especially melittin, play a key role in its medical applications, showing potential therapeutic effects on many diseases, particularly rheumatoid arthritis, and demonstrating significant clinical value.

[0003] The biological activity of bee venom mainly comes from its abundant polypeptides and enzymes. Among these, melittin is the main component, accounting for 40%–60% of the dry weight of bee venom. Its molecular formula is C131H229N39O31, and it is the primary pain-inducing component of bee venom. Melittin consists of 26 amino acids, without disulfide bonds. The N-terminal 21 amino acids are hydrophobic, while the rest are hydrophilic. Its amino acid sequence is Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln.

[0004] Melipotassium venom peptides are polar polypeptides, readily soluble in polar solvents such as water and methanol. Due to the presence of several basic amino acids in their polypeptide chains, aqueous solutions of melipotassium venom peptides are alkaline. Melipotassium venom peptides possess a variety of biological activities and show therapeutic potential for many diseases. Numerous reports have confirmed their antibacterial, anti-inflammatory, antiviral, and antitumor biological activities.

[0005] The complex composition of natural bee venom in traditional medicine, leading to side effects and unstable efficacy, severely limits its application. Phospholipase A2 is a major allergen in bee venom, accounting for 10%–13% of its dry weight. Apamin, composed of 18 amino acids, although comprising only 1%–3% of the dry weight of crude bee venom, effectively blocks calcium and other neurotoxic substances. 2+ Activated K + Furthermore, bee venom contains small amine molecules such as histamine and catecholamines, which negatively impact its clinical application. Therefore, removing toxic substances from bee venom through separation and purification is crucial for its use in disease treatment.

[0006] Obtaining melittin from crude bee venom is currently the primary method for preparing melittin. This method typically utilizes chromatographic techniques to separate and purify the crude bee venom, thereby yielding the melittin. For example, gel column chromatography, ion exchange resin chromatography, or preparative chromatography can be used for the purification of crude bee venom. Besides isolating and preparing melittin from crude bee venom, various melittin peptides can also be expressed by introducing and expressing target genes into engineered bacteria using genetic engineering techniques. However, the antibacterial properties of melittin peptides significantly limit the application of bioengineering methods for their preparation. Typically, it is necessary to first synthesize the precursor or modified melittin peptide, followed by complex processing or purification to obtain high-purity, active melittin peptides.

[0007] Column chromatography is currently the most commonly used method for obtaining melitoxin from crude bee venom. For example, Chinese invention patent document CN113831390B, authorized on May 14, 2024, discloses a high-efficiency method... High-purity bee venom peptides and their preparation method. The method for preparing high-purity bee venom peptides provided by this invention involves dissolving crude bee venom in purified water, centrifuging to remove water-insoluble impurities, and then adjusting the pH of the centrifuged solution to [value missing]. A pH of 3.0–4.0 allows some impurities in crude bee venom to precipitate and be removed, while also inactivating viruses. Adjusting the pH to 4.5–5.5 further improves the stability of the bee venom. After ultrafiltration to remove ions and small molecule impurities such as histamine, preparative high-performance liquid chromatography (HPLC) is performed using a DAC-100 column packed with polystyrene microspheres for purification and separation. This yields high-purity bee venom peptides with a purity of over 99%, improving the safety of refined bee venom peptides for clinical use. Furthermore, it eliminates the need for multiple purification steps using dextran gel columns, effectively simplifying the preparation process and demonstrating high potential for widespread application.

[0008] However, column chromatography typically requires multiple chromatography columns to separate the melittin in sequence to obtain high-purity melittin. Before sample loading, a cumbersome coarse separation process involving multiple impurity removal and filtration steps is necessary. Furthermore, melittin undergoes irreversible adsorption with the solid column packing material, reducing the yield to only about 50%. In addition, preparative high-performance liquid chromatography (HPLC) equipment and column packing materials are expensive, increasing preparation costs.

[0009] On November 16, 2011, the online publication of the Master's Electronic Journal, Issue S2 of 2011, published a paper entitled "Separation and Preparation of Bee Venom Peptide by High-Speed ​​Countercurrent Chromatography". In Chapter 3 of the paper, solvent screening was carried out by measuring the K value (partition coefficient) of bee venom peptide in five solvent systems. The relevant results are presented in Tables 3-1 to 3-6. The K values ​​listed in the tables are all partition coefficients of bee venom peptide. The partition coefficients of other impurities in crude bee venom are not listed. Therefore, the separation factor cannot be calculated. Thus, the partition coefficient of bee venom peptide listed alone cannot indicate that the measured system can effectively separate bee venom peptide from other impurities.

[0010] Chapter 4 of this paper describes the separation using countercurrent chromatography with the selected solvent system. Other impurities in crude bee venom, such as melittin and phosphatase A2, all exhibit UV absorption at 254 nm. However, the countercurrent chromatography results shown in Figures 4-1 and 2 in this paper only show a single peak, and the positions of the impurity peaks are not displayed in the chromatograms. Furthermore, the fragment information with a mass-to-charge ratio of 2026.886 present in the mass spectrum shown in Figure 4-5 matches the molecular weight (2026.9 Da) of melittin, a typical important allergen in crude bee venom. Therefore, the countercurrent chromatograms shown in this paper only indicate that different solvent systems alter the elution time of this peak, but cannot support the conclusion that this method can effectively separate high-purity melittin from crude bee venom. Summary of the Invention

[0011] The technical problem to be solved by the present invention is how to overcome the above-mentioned defects of the prior art and provide a high-purity bee venom peptide purification method using pH zone purification countercurrent chromatography with high yield and low cost.

[0012] To solve the above-mentioned technical problems, the present invention provides a high-purity melioflavone peptide purification method using pH-zone countercurrent chromatography, comprising the following steps: (1) Solvent preparation: Measure 4-12% methyl tert-butyl ether, 42-50% n-butanol, 4-12% acetonitrile and 42-50% deionized water respectively. After thorough shaking and mixing and standing to separate the layers, place the upper and lower phases in different containers. Then add 160mM triethylamine to the upper phase as a retention agent and 160mM hydrochloric acid to the lower phase as an eluent. After mixing evenly, sonicate for 30 minutes to remove dissolved gases. All percentages are volume percentages. (2) Preparation of crude bee venom solution: The injection volume is 5-10% of the column volume. Use the lower phase containing eluent obtained in step (1) to dissolve the crude bee venom powder at a concentration of 25 mg / mL. Vortex for 3 min, sonicate for 3 min, and then filter through a 0.45 μm filter to obtain crude bee venom solution. (3) Pumping in the stationary phase: Pump 1-2 column volumes of the upper phase containing the retaining agent obtained in step (1) into the column of the high-speed countercurrent chromatograph at a flow rate of 2-4% of the column volume / min. At the same time, turn on the UV detector to start preheating. The pumped upper phase flows out from the detector outlet tube and enters the recovery container. (4) Injection and elution: Switch the injection six-way switching valve to the load position, push the crude bee venom solution obtained in step (2) into the injection loop through the syringe, then switch the injection six-way switching valve from the load position to the inject position, and at the same time start the countercurrent chromatograph and adjust the speed to 1000-1500 rpm. Then, pump the lower phase containing eluent obtained in step (1) into the high-speed countercurrent chromatograph at a flow rate of 2-4% of the column volume / min and run the detector to collect the 280 nm ultraviolet signal. (5) Detection and collection: The eluent of the countercurrent chromatograph was detected with a UV-Vis detector (280 nm). The high-purity meliotide component solution was collected according to the obtained chromatogram: when about 1-2 column volumes were eluted and the pH of the eluent decreased from 8 to 2, the high-purity meliotide was eluted and the high-purity meliotide solution was collected. ⑹. Cleaning: Pump 1-2 column volumes of 70% methanol solution into the high-speed countercurrent chromatograph at a flow rate of 2-4% of the column volume / min, then turn off the instrument; (7) Concentration and drying: The high-purity bee venom peptide solution obtained in step (5) is concentrated by rotary evaporation and then freeze-dried. The resulting solid is the finished high-purity bee venom peptide.

[0013] As an optimization, in step (1): the volume percentage of deionized water is 46%, the volume percentage of n-butanol is 46%, the volume percentage of acetonitrile is 4%, and the volume percentage of methyl tert-butyl ether is 4%.

[0014] As an optimization, in step (1), the volume percentage of deionized water is 42%, the volume percentage of n-butanol is 46%, the volume percentage of acetonitrile is 8%, and the volume percentage of methyl tert-butyl ether is 4%.

[0015] As an optimization, in step (1): the volume percentage of deionized water is 42%, the volume percentage of n-butanol is 46%, the volume percentage of acetonitrile is 4%, and the volume percentage of methyl tert-butyl ether is 8%.

[0016] As an optimization, in step (1): the volume percentage of deionized water is 42%, the volume percentage of n-butanol is 42%, the volume percentage of acetonitrile is 4%, and the volume percentage of methyl tert-butyl ether is 12%.

[0017] Practice has shown that using the solvent system with the above-mentioned ratio results in a higher purity of bee venom peptide.

[0018] The countercurrent chromatography used in this method does not use solid packing material, does not cause sample loss, and can recover almost all bee venom peptides from crude bee venom with a yield of over 90%, making it suitable for batch purification of bee venom peptides. Attached Figure Description

[0019] The following description, in conjunction with the accompanying drawings, further illustrates the method for purifying high-purity melioflavone peptides using pH-zone countercurrent chromatography: Figure 1 This is a chromatogram of bee venom peptides separated and purified from crude bee venom using the pH-zone purification elution mode of this invention. Detailed Implementation

[0020] Example 1: The present invention provides a method for purifying high-purity melioflavone peptides using pH-zone countercurrent chromatography, comprising the following steps: (1) Solvent preparation: Measure 4% methyl tert-butyl ether, 42% n-butanol, 4% acetonitrile and 50% deionized water respectively. After thorough shaking and mixing and standing to separate the layers, place the upper and lower phases in different containers. Then add 160mM triethylamine to the upper phase as a retention agent and 160mM hydrochloric acid to the lower phase as an eluent. After mixing evenly, sonicate for 30 minutes to remove dissolved gases. All percentages are volume percentages. (2) Preparation of crude bee venom solution: The injection volume is 5-10% of the column volume. Use the lower phase containing eluent obtained in step (1) to dissolve the crude bee venom powder at a concentration of 25 mg / mL. Vortex for 3 min, sonicate for 3 min, and then filter through a 0.45 μm filter to obtain crude bee venom solution. (3) Pumping in the stationary phase: Pump 1-2 column volumes of the upper phase containing the retaining agent obtained in step (1) into the column of the high-speed countercurrent chromatograph at a flow rate of 2-4% of the column volume / min. At the same time, turn on the UV detector to start preheating. The pumped upper phase flows out from the detector outlet tube and enters the recovery container. (4) Injection and elution: Switch the injection six-way switching valve to the load position, push the crude bee venom solution obtained in step (2) into the injection loop through the syringe, then switch the injection six-way switching valve from the load position to the inject position, and at the same time start the countercurrent chromatograph and adjust the speed to 1000-1500 rpm. Then, pump the lower phase containing eluent obtained in step (1) into the high-speed countercurrent chromatograph at a flow rate of 2-4% of the column volume / min and run the detector to collect the 280nm ultraviolet signal. (5) Detection and collection: The eluent of the countercurrent chromatograph was detected with a UV-Vis detector (280nm). The high-purity melitoxin component solution was collected according to the obtained chromatogram: when about 1-2 column volumes were eluted and the pH of the eluent decreased from 8 to 2, the high-purity melitoxin was eluted and the high-purity melitoxin solution was collected. ⑹. Cleaning: Pump 1-2 column volumes of 70% methanol solution into the high-speed countercurrent chromatograph at a flow rate of 2-4% of the column volume / min, then turn off the instrument; (7) Concentration and drying: The high-purity bee venom peptide solution obtained in step (5) is concentrated by rotary evaporation and then freeze-dried. The resulting solid is the finished product, high-purity bee venom peptide 633.13 mg, with a purity of 96.0% and a yield of 93.8%.

[0021] Example 2: The volume percentage of acetonitrile was changed to 8%, the volume percentage of deionized water was changed to 46%, the volume percentage of methyl tert-butyl ether remained at 4%, and the volume percentage of n-butanol remained at 42%. The operations described in steps (1) to (7) of Example 1 were repeated to obtain the high-purity bee venom peptide product, which was found to have a purity of 97.1%.

[0022] Example 3: The volume percentage of acetonitrile was changed to 12%, the volume percentage of deionized water was changed to 42%, the volume percentage of methyl tert-butyl ether was kept at 4%, and the volume percentage of n-butanol was kept at 42%. The operations described in steps (1) to (7) of Example 1 were repeated to obtain the high-purity bee venom peptide product, which was found to have a purity of 95.8%.

[0023] Example 4: The volume percentage of deionized water was changed to 46%, the volume percentage of n-butanol was changed to 46%, the volume percentage of acetonitrile remained at 4%, and the volume percentage of methyl tert-butyl ether remained at 4%. The operations described in steps (1) to (7) of Example 1 were repeated to obtain the high-purity bee venom peptide product, which was found to have a purity of 97.3%.

[0024] Example 5: The volume percentage of deionized water was changed to 42%, the volume percentage of n-butanol was changed to 46%, the volume percentage of acetonitrile was changed to 8%, and the volume percentage of methyl tert-butyl ether was kept at 4%. The operations described in steps (1) to (7) of Example 1 were repeated to obtain the high-purity bee venom peptide product, which was tested to have a purity of 97.3%.

[0025] Example 6: The volume percentage of deionized water was changed to 42%, the volume percentage of n-butanol was changed to 50%, the volume percentage of acetonitrile remained at 4%, and the volume percentage of methyl tert-butyl ether remained at 4%. The operations described in steps (1) to (7) of Example 1 were repeated to obtain the high-purity bee venom peptide product, which was found to have a purity of 95.5%.

[0026] Example 7: The volume percentage of deionized water was changed to 46%, the volume percentage of n-butanol was changed to 42%, the volume percentage of acetonitrile remained at 4%, and the volume percentage of methyl tert-butyl ether was changed to 8%. The operations described in steps (1) to (7) of Example 1 were repeated to obtain the high-purity bee venom peptide product, which was found to have a purity of 96.3%.

[0027] Example 8: The volume percentage of deionized water was changed to 42%, the volume percentage of n-butanol was kept at 42%, the volume percentage of acetonitrile was changed to 8%, and the volume percentage of methyl tert-butyl ether was changed to 8%. The operations described in steps (1) to (7) of Example 1 were repeated to obtain the finished product, high-purity bee venom peptide, which was tested to have a purity of 95.3%.

[0028] Example 9: The volume percentage of deionized water was changed to 42%, the volume percentage of n-butanol was changed to 46%, the volume percentage of acetonitrile remained at 4%, and the volume percentage of methyl tert-butyl ether was changed to 8%. The operations described in steps (1) to (7) of Example 1 were repeated to obtain the high-purity bee venom peptide product, which was found to have a purity of 98.2%.

[0029] Example 10: The volume percentage of deionized water was changed to 42%, the volume percentage of n-butanol was kept at 42%, the volume percentage of acetonitrile was kept at 4%, the volume percentage of methyl tert-butyl ether was changed to 12%, and the operations of steps (1) to (7) of Example 1 were repeated to obtain the finished product, high-purity bee venom peptide, whose purity was tested to be 97.5%.

[0030] from Figure 1 - The countercurrent chromatogram of the purified countercurrent chromatography separation of melittin from crude bee venom in the pH zone and the trend of pH value change with the elution process show that the high-purity melittin is eluted with a decrease in pH value. Figure 1 The small image on the right shows the HPLC-DAD detection results of the isolated high-purity meliothionein.

Claims

1. A method for purifying high-purity melitrix peptides using pH-zone countercurrent chromatography, comprising the following steps: (1) Solvent preparation: Measure 4-12% methyl tert-butyl ether, 42-50% n-butanol, 4-12% acetonitrile and 42-50% deionized water respectively. After thorough shaking and mixing and standing to separate the layers, place the upper and lower phases in different containers. Then add 160mM triethylamine to the upper phase as a retention agent and 160mM hydrochloric acid to the lower phase as an eluent. After mixing evenly, sonicate for 30 minutes to remove dissolved gases. All percentages are volume percentages. (2) Preparation of crude bee venom solution: The injection volume is 5-10% of the column volume. Use the lower phase containing eluent obtained in step (1) to dissolve the crude bee venom powder at a concentration of 25 mg / mL. Vortex for 3 min, sonicate for 3 min, and then filter through a 0.45 μm filter to obtain crude bee venom solution. (3) Pumping in the stationary phase: Pump 1-2 column volumes of the upper phase containing the retaining agent obtained in step (1) into the column of the high-speed countercurrent chromatograph at a flow rate of 2-4% of the column volume / min. At the same time, turn on the UV detector to start preheating. The pumped upper phase flows out from the detector outlet tube and enters the recovery container. (4) Injection and elution: Switch the injection six-way switching valve to the load position, push the crude bee venom solution obtained in step (2) into the injection loop through the syringe, then switch the injection six-way switching valve from the load position to the inject position, and at the same time start the countercurrent chromatograph and adjust the speed to 1000-1500 rpm. Then, pump the lower phase containing eluent obtained in step (1) into the high-speed countercurrent chromatograph at a flow rate of 2-4% of the column volume / min and run the detector to collect the 280 nm ultraviolet signal. (5) Detection and collection: The eluent of the countercurrent chromatograph was detected with a UV-Vis detector. The high-purity melitoxin component solution was collected according to the obtained chromatogram. When approximately 1-2 column volumes were eluted and the pH of the eluent decreased from 8 to 2, the high-purity melitoxin was eluted and the high-purity melitoxin solution was collected. ⑹. Cleaning: Pump 1-2 column volumes of 70% methanol solution into the high-speed countercurrent chromatograph at a flow rate of 2-4% of the column volume / min, then turn off the instrument; (7) Concentration and drying: The high-purity bee venom peptide solution obtained in step (5) is concentrated by rotary evaporation and then freeze-dried. The resulting solid is the finished high-purity bee venom peptide.

2. The method for purifying high-purity melilotinib using pH-zone countercurrent chromatography according to claim 1, characterized in that: In section (1): the volume percentage of deionized water is 46%, the volume percentage of n-butanol is 46%, the volume percentage of acetonitrile is 4%, and the volume percentage of methyl tert-butyl ether is 4%.

3. The method for purifying high-purity melioflavone peptides using pH-zone countercurrent chromatography according to claim 1, characterized in that: In step (1), the volume percentage of deionized water is 42%, the volume percentage of n-butanol is 46%, the volume percentage of acetonitrile is 8%, and the volume percentage of methyl tert-butyl ether is 4%.

4. The method for purifying high-purity melilotinib using pH-zone countercurrent chromatography according to claim 1, characterized in that: In section (1): the volume percentage of deionized water is 42%, the volume percentage of n-butanol is 46%, the volume percentage of acetonitrile is 4%, and the volume percentage of methyl tert-butyl ether is 8%.

5. The method for purifying high-purity melitriceps using pH-zone countercurrent chromatography according to claim 1, characterized in that: In section (1): the volume percentage of deionized water is 42%, the volume percentage of n-butanol is 42%, the volume percentage of acetonitrile is 4%, and the volume percentage of methyl tert-butyl ether is 12%.

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