CN110627891A - Method for hiding hemolytic toxic and side effects of melittin - Google Patents
Method for hiding hemolytic toxic and side effects of melittin Download PDFInfo
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Abstract
The invention discloses a method for concealing hemolytic toxic and side effects of bee venom hemolysate peptide, which takes natural bee venom dry powder or bee venom hemolysate crude peptide synthesized by adopting a biological polypeptide solid phase synthesis method as a raw material, and prepares the bee venom hemolysate peptide with the mass content of more than 98 percent through gel filtration chromatographic column separation and reversed phase high performance liquid chromatography purification, wherein a concealing agent is adopted for dissolution in the gel filtration chromatographic column separation process; then processing the melittin hemolytic peptide by one or more molecular sieves of an ultrafiltration membrane, a nanofiltration membrane and a semipermeable membrane to obtain the melittin without hemolytic activity; after the treatment by the method, the hemolytic toxic and side effects of the bee venom hemolyzing peptide can be concealed; it has hemolytic activity when treated with less than 1mmol/L organic acid and its salt buffer, as demonstrated by treatment with purified water (water for injection).
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a preparation method of high-purity and high-activity melittin, in particular to a method for concealing hemolytic toxic and side effects by using melittin.
Background
Bee venom is known as bee venom, which is the venom discharged from a worker bee when the worker bee stings the bee to an enemy. The bee venom therapy has a long history in the traditional Chinese medicine for treating diseases, and particularly has good curative effects on treating diseases such as rheumatism, rheumatoid arthritis, scapulohumeral periarthritis and the like. In recent years, with the continuous improvement of separation, purification and biosynthesis technologies, on the basis of researching the composition and structure of bee venom, the functions and action mechanisms of bee venom are discussed, so that the bee venom compound preparation lays a foundation for further clinical application and shows a better application prospect.
Peptide for bee venom lysis (Melittin) (or melittin) is one of the important candidate molecules with antibiotic against drug-resistant germs, has strong broad-spectrum antibacterial and anti-inflammatory activity, but has limited application due to hemolytic toxic and side effects and difficult natural acquisition. Therefore, we abolished their hemolytic activity while retaining their antibacterial activity. Bee venom hemolysis peptide can form an amphipathic alpha-helical structure, 4 of 5 basic amino acids are concentrated at the C-terminal end of a peptide chain (KRKR), and is a typical cationic antibacterial peptide. The melittin has strong inhibiting effect on gram-positive bacteria and gram-negative bacteria, and also has strong hemolytic activity, so that the clinical application of the melittin is limited.
The bee venom dry powder contains peptide, enzyme and non-peptide main active ingredients. The peptides mainly comprise melittin, melittin and mast cell degranulation polypeptide. Enzymes are mainly the main substances of the phospholipase A2 which produce allergic reactions after bee stings. Non-peptide substances include histamine, various biogenic amines, which are associated with pain following bee sting.
The bee venom hemolyzing peptide is the main component and active component of bee venom, and accounts for about 50% of dry weight of bee venom; has antiinflammatory, antibacterial, antiviral and antitumor effects. The melittin is white or white-like powder, has a molecular weight of 2840D, and consists of 26 amino acid residues, wherein the amino acid residue sequence of the primary structure is as follows:
NH2-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-COOH
since Habermann et al first isolated bee venom hemolyzing peptide consisting of 26 amino acids from bee venom in 1967, its amino acid sequence was analyzed, demonstrating no disulfide bond in its structure. The tertiary structure was observed in Anderson et al, 1980, and the results showed that the spatial structure of melittin can be divided into four regions: namely (1-10) an alpha helical structure at the N terminal, (11-13) a hinge structure in which C connects two helices at an angle of 120 degrees, (13-20) a C alpha helical structure, and (2 l-26) a positive charge region at the C terminal. These four monomers are linked by hydrophobic groups to form a stable tetrameric structure, 4 of the 5 basic amino acids are concentrated at the C-terminus of the peptide chain (krkrkr), which is typical of cationic antimicrobial peptides. The bee venom hemolytic peptide can form an amphipathic alpha-helical structure and has the characteristics of broad-spectrum antibacterial and antiviral functions, strong hemolytic activity and the like. However, when the melittin binds to the cell membrane and acts, it is arranged on the surface of the cell membrane in the form of a monomer, but still has a significant cytotoxic effect (hemolytic activity). Melittin is a broad-spectrum antibacterial peptide, and its antibacterial and antiviral effects are that melittin destroys bacterial cell membrane by combining with membrane to achieve the purpose of sterilization.
Biological broad-spectrum antibacterial peptides (antibiotics) widely exist in insects, plants, animals and human bodies, have nonspecific antibacterial, antifungal and antiviral effects, and can be called peptide antibiotics (peptiaceotics). The polypeptide has amino acid residues less than 40, can form alpha-helix structure, and contains more basic amino acids (with positive charges under physiological conditions) and more hydrophobic amino acids. The biological antibacterial peptide is combined on a bacterial membrane through electrostatic attraction of amphipathic positive charges and microbial cell membrane phospholipid molecule negative charges, a hydrophobic end is inserted into the cell membrane, and finally the biological antibacterial peptide is gathered together through intermolecular displacement in the membrane to form an ion channel, so that the bacteria lose membrane potential and die because normal osmotic pressure cannot be maintained. Due to the unique antibacterial mechanism, the bacteria are not easy to generate drug resistance; the biological antibacterial peptide has strong bactericidal capacity, wide antibacterial spectrum and less adverse reaction.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for concealing hemolytic toxic and side effects by bee venom hemolysate, which takes natural bee venom dry powder or bee venom hemolysate crude peptide synthesized by adopting a biological polypeptide solid phase synthesis method as a raw material, and prepares the bee venom hemolysate with the mass content of more than 98 percent by gel filtration chromatographic column separation and reversed phase high performance liquid chromatography purification, wherein a concealing agent is adopted for dissolution in the separation process of the gel filtration chromatographic column; then the melittin hemolytic peptide is treated by one or more molecular sieves of an ultrafiltration membrane, a nanofiltration membrane and a semipermeable membrane to obtain the melittin without hemolytic activity.
The natural bee venom dry powder contains 45-55% of bee venom fibrinolytic peptide.
The bee venom hemolytic peptide crude peptide synthesized by the biological solid phase synthesis method is prepared by adopting an amino acid solid phase synthesis method protected by FMOC (refer to a method in Lou Carpino 1972 (FMOC)), according to a literature method, a 2% crosslinking degree chloromethyl resin is used as a carrier, a C terminal is used as an amide polypeptide, bee venom hemolytic peptide consisting of 26 amino acid residues is sequentially synthesized according to an amino acid residue sequence of a primary structure, the reaction is carried out for 1 hour at 0 ℃ in anhydrous hydrofluoric acid containing 10% by mass of anisole, the bee venom hemolytic peptide is cut from the chloromethyl resin after the reaction is finished, and meanwhile, a protecting group of a side chain is removed, so that the bee venom hemolytic peptide crude peptide is prepared.
The masking agent is 1 mmol/L-1000 mmol/L organic acid solution or 1 mmol/L-1000 mmol/L organic acid-organic acid salt buffer solution; examples include, but are not limited to, acetic acid-ammonium acetate buffer solutions, acetic acid solutions.
The gel filtration chromatographic column separation specific operation is to balance the gel filtration chromatographic column by using a masking agent, dissolve natural bee venom dry powder or bee venom hemolyzed peptide crude peptide synthesized by adopting a biological solid phase synthesis method by using the masking agent, load the sample into the gel filtration chromatographic column, elute by using the masking agent as eluent, detect by using an ultraviolet detector at 280nm, collect a first peak, and freeze-dry the first peak.
The cut-off molecular weight of the ultrafiltration membrane is less than 10000 Da, and the cut-off molecular weight of the nanofiltration membrane is 100-1000 Da; the semi-permeable membrane has a cut-off of less than 10000 Da.
The melittin without hemolytic activity prepared by the method is a biological broad-spectrum antibacterial peptide; the biological antibacterial peptide is melittin (26P) with concealed hemolytic activity, and has broad-spectrum antibacterial, antifungal and antiviral activity, and no hemolytic activity.
The melittin without hemolytic activity can be prepared into injection drugs, such as melittin for injection and melittin injection; can also be made into external preparations such as spray, gel and liniment, and can be used for treating mosquito bite, relieving swelling and pain, and relieving itching; can also be used for treating infantile eczema.
The principle of bee venom hemolyzing peptide for concealing hemolytic toxic and side effects is as follows:
under the action of 1-1000 mmol/L organic acid and salt thereof, the bee venom hemolytic peptide recovers the 120-degree corner of the 11-13 peptide chain of the primary structure of the bee venom hemolytic peptide through the molecular sieve filtration (ultrafiltration membrane ultrafiltration process, nanofiltration membrane nanofiltration process and/or biological semipermeable membrane action process), has no electrostatic adsorption with erythrocyte membrane, loses the perforation effect, has no hemolysis, solves the toxic and side effect of the bee venom hemolytic peptide, has no influence on the perforation effect of prokaryotic cells, and keeps the sterilization effect unchanged (the perforation effect of the 13-26 peptides of the bee venom peptides).
The preparation method is simple in preparation process and easy to operate, the bee venom fibrinolytic peptide is dissolved by organic acid and salt buffer thereof, separated by a gel filtration chromatographic column, purified by reversed-phase high performance liquid chromatography, and subjected to molecular sieve treatment such as ultrafiltration, nanofiltration, semipermeable membrane and the like, so that the hemolytic toxic and side effects of the bee venom fibrinolytic peptide can be concealed; experiments have shown that organic acids and their salts, if treated with less than 1mmol/L of buffer, are again hemolytic and confirmed by treatment with purified water (water for injection).
Drawings
FIG. 1 shows the results of the purity of melittin by analytical high performance liquid chromatography;
FIG. 2 is a graph showing the results of measurement of the antibacterial activity of melittin having no hemolytic activity.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited to the above-described examples.
Example 1: the method for concealing hemolytic toxic and side effects by using bee venom hemolytic peptide and the hemolytic detection content are as follows:
1. taking 1.0g of bee venom dry powder containing 50% of bee venom hemolysate peptide, adding 10mL of acetic acid-ammonium acetate buffer solution (100 mmol/L) for dissolution, centrifuging for 10min at 2000 rpm, taking supernatant, performing primary separation by using a SephadexG-50 gel filtration chromatographic column (50 mm multiplied by 600 mm), balancing the chromatographic column by using 100mmol/L of acetic acid-ammonium acetate buffer solution, eluting by using 100mmol/L of acetic acid-ammonium acetate buffer solution under the condition that the flow rate is 1.0mL/min after sample loading, detecting by using an ultraviolet detector at 280nm, collecting a first peak, and freeze-drying; purifying by using a semi-preparative high performance liquid chromatograph, and collecting a peak with the maximum abundance;
wherein the semi-preparative high performance liquid chromatograph is purified by using a C18 reverse phase column (300 Å, 10 mu m, 19mm multiplied by 300mm), dissolving a product obtained by separating the gel filtration chromatographic column in 2mL of ultrapure water, centrifuging for 5min at 10000g, loading the product on the reverse phase column which is well balanced by BufferA (0.1% trifluoroacetic acid), performing gradient elution by BufferB (acetonitrile containing 0.1% trifluoroacetic acid), detecting at the flow rate of 10mL/min and 215nm, and collecting the peak with the maximum abundance;
2. and (3) sequentially carrying out ultrafiltration on the collected liquid by an ultrafiltration membrane with the molecular weight cutoff of 5000Da, nanofiltration by a nanofiltration membrane with the molecular weight cutoff of 200Da and dialysis by a dialysis membrane with the molecular weight cutoff of 3000Da to obtain the melittin (26P) without hemolytic activity.
The purity of melittin is 99.2% as determined by analytical high performance liquid chromatography (C18) HPLC; the results are shown in FIG. 1; the melittin prepared by amino acid analysis and determination by MALDI-TOF mass spectrometer is target product melittin hemolysate.
3. Hemolytic Activity test of melittin having No hemolytic Activity
The hemolytic activity was determined according to the activity assay of the national drug Standard WS1-XG-016 and 2001 bee venom quality Standard as follows:
adding a sodium chloride solution with the mass concentration of 0.9% into the bee venom hemolytic peptide prepared in the step 2 to prepare a bee venom hemolytic peptide solution with the concentration of 8 mu g/mL, wherein the bee venom hemolytic peptide solution is used as a test sample;
preparing anticoagulant rabbit blood: adding 20mL of newly collected rabbit blood into 2mL of 2% potassium oxalate solution, and slowly shaking to prepare anticoagulant rabbit blood; taking 8mL of anticoagulated rabbit blood, adding 10mL of 0.9% sodium chloride solution, and shaking up to obtain diluted rabbit blood;
taking 9 test tubes, wherein 10mL of the test sample is added into 3 test tubes respectively; adding 10mL of 0.9% sodium chloride solution into 3 pieces of the test pieces respectively to serve as negative controls; 10mL of water is added into 3 branches respectively to serve as positive control; placing each tube in 37 deg.C water bath, keeping the temperature for 20min, adding diluted rabbit blood 0.2mL, shaking, keeping the temperature in 37 deg.C water bath for 30min, taking out, placing in ice water bath for 5min, centrifuging at 2000 rpm for 5min, respectively sucking supernatant, and measuring absorbance A at 545nm wavelength; average value of negative controlA nc Not more than 0.03, mean value of positive controlA pc Should be 0.8 + -0.03, if not within this range, should be tested separately; results hemolytic activity was calculated as follows:
(A i -A nc )×100%
A
pc
-A
nc
in the formulaA i -the test article absorbency;
A nc -negative control absorbance;
A pc -positive control absorbance;
the results are shown in Table 1, and the hemolytic activity of the melittin prepared in step 2 is 0;
TABLE 1
。
Example 2: the method for concealing hemolytic toxic and side effects by using bee venom hemolytic peptide and the hemolytic detection are as follows:
1. adopting an FMOC (methyl methacrylate-vinyl acetate copolymer) protected amino acid solid-phase synthesis method, preparing a bee venom hemolytic peptide (26P) which is composed of 26 amino acid residues according to a literature method, sequentially synthesizing a polypeptide taking a 2% crosslinking degree chloromethyl resin as a carrier and an amino acid residue sequence with a C end as amide according to an amino acid residue sequence of a primary structure of the polypeptide, reacting in anhydrous hydrofluoric acid containing 10% by mass of anisole at 0 ℃ for 1h, cutting the bee venom hemolytic peptide from the resin, and removing a side chain protecting group to prepare the bee venom hemolytic peptide crude peptide; wherein the reagent cut from the resin by the melittin hemolysate is trifluoroacetic acid/phenol/water/thioanisole/1, 2-dimercaptoethanol (the volume ratio of trifluoroacetic acid, phenol, water, thioanisole and 1, 2-dimercaptoethanol is 8: 0.5: 0.25), the reaction is carried out for 5 hours at room temperature, after most of trifluoroacetic acid is removed by rotary evaporation, ether is dripped at 0 ℃ to obtain flocculent precipitate, and the melittin hemolysate crude peptide is obtained after centrifugation;
2. performing primary separation by using a SephadexG-25 gel filtration chromatographic column, balancing the column by using an acetic acid solution with the mass concentration of 30%, eluting by using acetic acid with the mass concentration of 30% at the flow rate of 1mL/min after sample loading, detecting by using an ultraviolet detector at 280nm, collecting a first peak, and performing freeze drying; purifying by using a semi-preparative high performance liquid chromatograph, and collecting a peak with the maximum abundance;
3. sequentially subjecting the collected liquid to ultrafiltration by an ultrafiltration membrane with the cut-off molecular weight of 5000Da, nanofiltration by a nanofiltration membrane with the cut-off molecular weight of 200Da and dialysis by a dialysis membrane with the cut-off molecular weight of 3000Da to obtain melittin (26P) without hemolytic activity;
the purity of melittin is 99.5% by HPLC of analytical high performance liquid chromatography (C18); determining the prepared melittin as target product melittin by amino acid analysis and determination with MALDI-TOF mass spectrometer;
4. hemolytic activity assay of melittin having no hemolytic activity, which was performed in the same manner as step 3 of example 1, by examining the hemolytic activity of melittin having no hemolytic activity prepared in step 3 of this example to be 0 (see Table 2);
TABLE 2
。
Example 3: the method for concealing hemolytic toxic and side effects by using bee venom hemolytic peptide and the hemolytic detection are as follows:
1. taking 1.0g of bee venom dry powder containing 52% bee venom hemolysate, adding 10mL of acetic acid-ammonium acetate buffer solution (200 mmol/L) for dissolution, centrifuging for 10min at 2000 rpm, taking supernatant, performing primary separation by using a SephadexG-50 gel filtration chromatographic column (50 mm multiplied by 600 mm), balancing the chromatographic column by using 200mmol/L of acetic acid-ammonium acetate buffer solution, eluting by using 200mmol/L of acetic acid-ammonium acetate buffer solution under the condition that the flow rate is 1.0mL/min after sample loading, detecting by using an ultraviolet detector at 280nm, collecting a first peak, and freeze-drying; purifying with semi-preparative high performance liquid chromatograph, collecting peak with maximum abundance, and drying the collected liquid;
2. step 1 the dried product is treated by a dialysis membrane
Adopting an import MD34-3.5(7) dialysis bag to cut off the molecular weight of 3500 (7000) Da; shearing a dialysis bag with the volume of 1-3m according to the feeding amount each time;
and (3) new dialysis bag treatment: boiling the dialysis bag with an aqueous solution containing 2% sodium bicarbonate and 1mol/L disodium ethylene diamine tetraacetate for 10min, cooling, and washing with water for injection for 3-5 times; boiling with 1mol/L disodium ethylene diamine tetraacetate solution for 10min, cooling, washing with water for injection for 3-5 times, soaking in water for injection at 4 deg.C, and storing; the treated dialysis bag should be worn with gloves.
Preparing a dialysate: weighing 7.7 g of ammonium acetate, adding 0.8L of water for injection to dissolve, and adding 10-20mL of acetic acid to adjust the pH value to 4.5-5.5; water for injection was added to 1L.
Dissolving bee venom powder: weighing the dried product obtained in the step 1, dissolving the dried product in the dialysate according to the proportion of 6 percent, and fully dissolving the dried product; taking out the treated dialysis bag, pricking one end with a rubber ring, filling 2/3 injectable water, inverting the dialysis bag for several times, removing water from the dialysis bag, and cleaning for 2-3 times; then putting the bee venom dissolved with the dried product into a dialysis bag (about 2/3), pricking the other end with a rubber band, and dialyzing in 5 times of the above dialysate; dialyzing and extracting for more than 30h, taking out the dialysis bag, wherein the solution in the container is bee venom hemolytic peptide dialysate I;
and (3) secondary dialysis: transferring the dialysis bag into injection water with 6 times of volume of the dialysis bag to continue dialysis for 30 h; taking out the dialysis bag, wherein the solution in the container is bee venom hemolysis peptide dialysate II; performing dialysis for 3-4 times; obtaining bee venom hemolytic peptide dialysate III and IV; namely melittin stock solution, ultraviolet content detection and hemolytic activity determination. The bee venom hemolytic peptide dialyzates I and II have no hemolytic activity through determination; the bee venom hemolytic peptide dialyzates III and IV have hemolytic activity, and the hemolytic activity of the solutions of 8 mu g/mL is more than 60 percent (see table 3); the result shows that the bee venom hemolysis peptide has hemolysis activity again after being treated by the water for injection;
TABLE 3
。
Example 4: detection of antibacterial Activity of melittin without hemolytic Activity
The method for detecting the antibacterial activity of the melittin without hemolytic activity prepared in the above embodiment comprises heating beef extract peptone agar medium in water bath for melting, cooling to about 50 deg.C, and aseptically sucking 60 μ L of bacterial liquid to be detected (OD)6000.3), adding the mixture into 20mL of beef extract peptone agar culture medium, quickly mixing uniformly, pouring the mixture into a sterile flat dish with the diameter of 9cm and the thickness of about 1.5mm, and horizontally placing the mixture at room temperature for solidification; punching round holes with diameter of 2.7mm on agar, adding 10 μ l of melittin without hemolytic activity into the holes, respectively, using sterile water as negative control, and using Amp as positive control; after the sample is added, the plate is placed in a refrigerator at 4 ℃, after the sample is fully diffused to the agar, the plate is placed upside down at 37 ℃ for overnight culture, and the result is observed the next day. The test bacteria in the test are staphylococcus aureus, and the test result is shown in figure 2; the result shows that the melittin without hemolytic activity has the antibacterial activity of inhibiting and killing staphylococcus aureus.
Determination of Minimum Inhibitory Concentration (MIC) of melittin without hemolytic activity
The test strains were cultured to logarithmic growth phaseThe bacterial liquid of (A) is diluted to 5X 106CFU/mL, adding into a 96-hole culture plate, adding 90 mul of bacterial liquid into each test hole, and then adding 10 mul/hole of the diluted melittin solution with different concentrations; the positive control is 100 mu L/hole bacterial liquid, and the negative control is corresponding 100 mu L culture medium; then slowly shaking and culturing at 37 ℃ for about 16h, and measuring OD by using a microplate reader630(ii) a The minimum concentration for inhibiting the growth of bacteria is MIC, and the result is shown in Table 4;
table 4: melittin MIC (minimum concentration to inhibit bacterial growth)
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention may be made by those skilled in the art without departing from the spirit of the present invention, and the technical solution of the present invention shall fall within the protection scope defined by the claims of the present invention.
Claims (6)
1. A method for concealing hemolytic toxic and side effects by using melittin hemolytic peptide is characterized by comprising the following steps: taking natural bee venom dry powder or bee venom hemolytic peptide crude peptide synthesized by adopting a biological polypeptide solid-phase synthesis method as a raw material, and preparing bee venom hemolytic peptide with the mass content of more than 98 percent by gel filtration chromatographic column separation and reversed-phase high performance liquid chromatography purification, wherein a masking agent is adopted for dissolution in the gel filtration chromatographic column separation process; then the melittin hemolytic peptide is treated by one or more molecular sieves of an ultrafiltration membrane, a nanofiltration membrane and a semipermeable membrane to obtain the melittin without hemolytic activity.
2. The method of claim 1 for sequestering hemolytic toxic side effects of melittin, comprising: the natural bee venom dry powder contains 45-55% of bee venom fibrinolytic peptide.
3. The method of claim 1 for sequestering hemolytic toxic side effects of melittin, comprising: the masking agent is 1 mmol/L-1000 mmol/L organic acid solution or 1 mmol/L-1000 mmol/L organic acid-organic acid salt buffer solution.
4. The method of sequestering hemolytic toxic side effects of melittin according to claim 3, wherein: the gel filtration chromatographic column separation specific operation is to balance the gel filtration chromatographic column by using a masking agent, dissolve natural bee venom dry powder or bee venom hemolyzing peptide crude peptide synthesized by adopting a biological solid phase synthesis method by using the masking agent, load the sample into the gel filtration chromatographic column, use the masking agent as eluent for elution, detect the peak at 280nm by using an ultraviolet detector, collect the first peak and freeze-dry the peak.
5. The method of sequestering hemolytic toxic side effects of melittin according to claim 4, wherein: the cut-off molecular weight of the ultrafiltration membrane is less than 10000 Da, and the cut-off molecular weight of the nanofiltration membrane is 100-1000 Da; the semipermeable membrane is a dialysis membrane with molecular weight cut-off less than 10000 Da.
6. The method of claim 1 for sequestering hemolytic toxic side effects of melittin, comprising: the bee venom hemolytic peptide crude peptide synthesized by the biological polypeptide solid phase synthesis method is prepared by adopting an amino acid solid phase synthesis method protected by FMOC, sequentially synthesizing bee venom hemolytic peptide consisting of 26 amino acid residues according to an amino acid residue sequence of a primary structure by taking chloromethyl resin with 2% of crosslinking degree as a carrier and taking polypeptide with a C end as amide, reacting for 1h at 0 ℃ in anhydrous hydrofluoric acid containing 10% by mass of anisole, cutting the bee venom hemolytic peptide from the chloromethyl resin after the reaction is finished, and removing a protecting group of a side chain.
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