Preparation method and application of low-pain and low-sensitivity bee venom active peptide

Abstract

The application discloses a preparation method and application of low-pain and low-sensitization bee venom active peptide. The method comprises the following steps: pretreating Apis cerana cerana bee venom, performing first-stage separation by using an ultrafiltration membrane with a molecular weight cut-off of 1-5 kDa, and collecting filtrate with a molecular weight lower than the cut-off; performing second-stage separation by using a gel filtration medium with a size exclusion limit of 500-1000 Da, removing small-molecule neurotransmitters and stimulating small molecules, collecting bee venom active peptide components with a range of 700-3000 Da, and obtaining the low-pain and low-sensitization bee venom active peptide through freeze drying. The method can simultaneously reduce the content of 5-hydroxytryptamine, histamine and other pain-related small molecules and phospholipase A1, hyaluronidase and other large-molecule potential sensitization proteins in the bee venom, and retain anti-inflammatory activity. The bee venom active peptide can be used for preparing anti-inflammatory preparations, low-stimulation bee venom active raw materials, external use repair preparations, tumor cell proliferation inhibitors or other bioactive peptide related products.

Description

Technical Field

[0001] This invention belongs to the field of bee venom polypeptide separation and purification technology, specifically relating to the preparation method of low-pain and low-allergenic bee venom active peptides derived from wasps, and their application in the fields of biomedicine and functional raw materials. Background Technology

[0002] Bee venom is a complex, naturally occurring active system secreted by the venom glands of bees, typically containing proteins, polypeptides, enzymes, small neurotransmitters, and other metabolites. Bee venom and its derived active components have potential applications in anti-inflammatory, immunomodulatory, antitumor, and antibacterial fields. However, direct application of crude bee venom often results in significant irritation, pain, and allergic reactions, limiting its industrial development as a bioactive raw material.

[0003] Hornet venom and honeybee venom differ significantly in species origin, main toxic components, and allergen composition. Hornet venom may contain neuroactive small molecules such as serotonin, histamine, tyramine, tryptamine, and dopamine derivatives, as well as phospholipase A1, hyaluronidase, and other macromolecular enzymes and proteins. Among these, small molecule neurotransmitters such as serotonin, histamine, tyramine, tryptamine, and dopamine derivatives, as well as amines, are considered to be the active components in bee venom closely related to local pain, stinging, nerve stimulation, and inflammatory responses; macromolecular proteins such as phospholipase A1, hyaluronidase, and antigen 5 protein are considered to be the main protein components in hornet venom closely related to the risk of sensitization.

[0004] In existing technologies, bee venom development mainly focuses on obtaining the effective parts of crude bee venom, monomeric bee venom peptides, or ordinary polypeptides, with less emphasis on systematically designing solutions to the technical challenge of "simultaneously removing pain-related small molecules and sensitization-related large molecular weight proteins while retaining active peptides." For wasp venom, if only crude venom or polypeptide components with a wide molecular weight range are retained, irritating components and sensitization-related proteins may still remain, making it difficult to meet the requirements for low irritation, low sensitization, and industrial application.

[0005] Therefore, there is an urgent need to establish a safe separation and preparation method for venom from the hornet, which can reduce the amount of pain-causing small molecules and potentially sensitizing large protein molecules while enriching and retaining bee venom peptide components with anti-inflammatory activity. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing low-pain and low-sensitivity bee venom active peptides and their applications. This method is based on the component risk characteristics of wasp venom and achieves "upper limit desensitization and lower limit depainization" through a two-stage molecular weight screening, thereby obtaining bee venom active peptide components with anti-inflammatory activity whose molecular weight is mainly distributed in the range of 700–3000 Da.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: a method for preparing a low-pain and low-allergenic bee venom active peptide, comprising the following steps: (1) Pretreatment of bee venom: Take the venom of the hornet, reconstitute it with water or buffer solution, centrifuge to remove impurities, and collect the supernatant; (2) Primary separation: The supernatant is separated by an ultrafiltration membrane with a molecular weight cutoff of 1–5 kDa (preferably 3 kDa), and the filtrate with a molecular weight cutoff is collected to reduce the amount of large molecular weight proteins such as phospholipase A1 and hyaluronidase and other components >3 kDa. (3) Secondary separation: The filtrate obtained in step (2) is separated by a gel filter medium with an exclusion limit of 500–1000 Da (preferably about 700 Da) to remove small molecule neurotransmitters such as 5-hydroxytryptamine and histamine, as well as stimulating small molecules, and the target peptide component of 700–3000 Da is collected. (4) Post-processing: freeze-dry the target peptide component to obtain low-pain and low-sensitivity bee venom active peptide.

[0008] Furthermore, the gel filtration medium is Sephadex G-10; the eluent can be water, ultrapure water, physiological saline or pharmaceutically acceptable buffer, preferably ultrapure water; the elution curve is detected by dual wavelengths of 280nm and 595nm, and the eluents are combined according to the target peak segment.

[0009] Furthermore, the pain-related small molecules such as 5-hydroxytryptamine and histamine in the low-pain and low-sensitivity bee venom active peptide are significantly reduced compared with crude bee venom, and the >3kDa protein band is significantly reduced or not obvious; the active peptide still has anti-inflammatory activity that inhibits LPS-induced NO release from RAW264.7 cells.

[0010] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention proposes a two-stage molecular weight screening process for venom of the basal wasp. First, large molecular proteins / potential sensitizing components are removed by 3kDa ultrafiltration, and then small molecular neurotransmitters / irritating components are removed by gel exclusion of approximately 700Da. The process logic is clear.

[0011] 2. This invention can obtain bee venom active peptide components with molecular weights mainly distributed in the range of 700–3000 Da, avoiding the problem of the coexistence of pain-inducing small molecules and sensitizing-related large molecular proteins in crude bee venom.

[0012] 3. The bee venom active peptides obtained in this invention reduce pain-related small molecules and sensitization-related large molecular proteins while retaining anti-inflammatory activity, and can be developed as low-irritant bee venom active raw materials.

[0013] 4. The process of this invention mainly uses ultrafiltration and gel filtration, does not rely on complex organic solvent systems, has mild preparation conditions, is easy to scale up, and is suitable for subsequent industrialization development by enterprises.

[0014] 5. The active peptides obtained in this invention can be used in anti-inflammatory preparations, topical repair preparations, tumor cell proliferation inhibitors, and functional bioactive peptide raw materials, and have good application prospects. Attached Figure Description

[0015] Figure 1 This is a flowchart illustrating the preparation process of the low-pain and low-allergenic bee venom active peptide of the present invention.

[0016] Figure 2 SDS-PAGE electrophoresis images of crude bee venom, <3kDa fraction and >3kDa fraction.

[0017] Figure 3 The elution curves at 280 nm and 595 nm are for fractions <3 kDa after gel filtration through Sephadex G-10 gel.

[0018] Figure 4 The image shows the HPLC detection results of pain-related small molecules such as 5-hydroxytryptamine and histamine in crude bee venom, small molecule components <700Da, and target peptide components with 700–3000Da.

[0019] Figure 5 The HPLC peptide fingerprint of the target peptide fraction with a value of 700–3000 Da shows that it has five main characteristic absorption peaks between 29 and 40 min.

[0020] Figure 6 The diagram shows the inhibitory effect of wasp venom on LPS-induced NO release in RAW264.7 cells.

[0021] Figure 7 The graph shows the inhibitory effect of the 700–3000 Da target peptide component on LPS-induced NO release in RAW264.7 cells.

[0022] Figure 8 This diagram illustrates the inhibitory effect of wasp venom on tumor cell proliferation.

[0023] Figure 9 The graph shows the inhibitory effect of the 700–3000 Da target peptide component on tumor cell proliferation.

[0024] Figure 10 This is a graph showing the safety evaluation of bee venom and the 700–3000 Da target peptide component on normal THLE-2 hepatocytes. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. All equivalent substitutions, parameter adjustments, or conventional improvements made based on the technical concept of the present invention should fall within the scope of protection of the present invention.

[0026] Example 1: Risk component analysis of crude bee venom from the hornet.

[0027] like Figure 1-4 As shown, this embodiment illustrates the presence of pain-related small molecules and potentially sensitizing large molecular weight proteins in the crude venom of the hornet, thus providing a technical basis for the safe preparation of the two-level molecular weight venom of the present invention.

[0028] 1. Preparation of crude bee venom samples: Crude venom was obtained by pulsed electrical stimulation method before the basal wasps were about to hibernate. The crude venom was resuspended in pre-cooled ultrapure water, centrifuged at 4℃ and 5000×g for 15 min, the supernatant was collected, and the crude bee venom was freeze-dried to obtain lyophilized powder, which was stored at -20℃.

[0029] 2. Detection of pain-related small molecules: The crude bee venom lyophilized powder was reconstituted, and 21 neurotransmitters, including 5-hydroxytryptamine, histamine, tyramine, tryptamine, and dopamine, were detected by LC-MS / MS.

[0030] 3. Sensitization-related macromolecule detection: The protein components in the crude venom of the wild hornet were systematically analyzed using SDS-PAGE combined with LC-MS / MS technology, with a focus on identifying potential allergens with molecular weights greater than 3kDa, including phospholipase A1 (PLA1) and hyaluronidase (HYAL).

[0031] 4. The test results showed that 10 neurotransmitters were detected in the crude venom of the *Vespa mandshurica*. Among them, serotonin, L-glutamate, and histamine were the three most abundant, with significantly higher relative abundance than the other seven components. All three are important active small molecules involved in nerve excitation, inflammation regulation, and pain signal transmission. The detection of sensitization-related macromolecules revealed the presence of allergenic proteins in the crude venom of *Vespa mandshurica*, including phospholipase A1 (33.3 kDa and 36.7 kDa), hyaluronidase A (43.7 kDa), antigen 5 protein (22.2 kDa), and venom dipeptidyl peptidase IV (88.4 kDa). These results indicate that the crude venom of *Vespa mandshurica* contains pain-related small molecules and potentially sensitizing macromolecular proteins, requiring safe separation and treatment.

[0032] Example 2: Preparation of low-pain and low-sensitivity bee venom active peptides.

[0033] This example illustrates the process of preparing a target peptide component of 700–3000 Da by continuous separation using 3 kDa ultrafiltration and approximately 700 Da gel size exclusion.

[0034] 1. Reconstitution of crude bee venom: Take the freeze-dried powder of crude bee venom from the base wasp, reconstitute it with pre-cooled ultrapure water to 20 mg / mL, mix thoroughly, centrifuge at 4℃ to remove insoluble matter, and collect the supernatant.

[0035] 2.3kDa Ultrafiltration Separation: The supernatant was added to an ultrafiltration centrifuge tube with a molecular weight cutoff of 3kDa, and centrifuged according to the manufacturer's instructions. The >3kDa cutoff and <3kDa filtrate were collected separately. Equal volumes of sample were analyzed by SDS-PAGE to verify the separation of the >3kDa and <3kDa protein components. (See Appendix) Figure 2 .

[0036] 3. G-10 Gel Filtration Separation: The <3kDa filtrate was further separated using Sephadex G-10 gel filtration chromatography. A 1.6cm × 30cm column was used, with pre-cooled ultrapure water as the eluent, a flow rate of 1mL / min, and collection time of 3 min per tube. The absorbance of each tube was measured at 280nm and 595nm, and dual-wavelength elution curves were plotted (see attached figure). Figure 3 .

[0037] 4. Target Peak Collection: Based on the elution curve, target peptide peaks with significant Coomassie Brilliant Blue G-250 characteristic absorption at a wavelength of 595 nm were screened and collected independently. These target peaks represent bee venom active peptides with a range of 700–3000 Da. After merging, the peaks were freeze-dried to obtain a low-pain and low-allergenic bee venom active peptide lyophilized powder.

[0038] 5. Results: SDS-PAGE results showed that no obvious >3kDa protein bands were observed in the <3kDa filtrate, indicating that the method can effectively remove large molecular weight allergenic proteins; the G-10 elution curve showed that the target peptide peak and the subsequent small molecule elution peaks could be distinguished, indicating that this method can obtain target peptide components of 700–3000 Da.

[0039] Example 3: Verification of the low analgesic and low sensitization characteristics of the target peptide component.

[0040] This example illustrates that the 700–3000 Da target peptide component obtained in Example 2 has the characteristic of reducing pain-related small molecules and sensitization-related large molecules compared to crude bee venom.

[0041] 1. Detection of pain-related small molecule residues: Crude bee venom, small molecule components smaller than 700 Da, and target peptide components of 700–3000 Da were collected separately. Using serotonin and histamine as representative small molecules, the contents of serotonin and histamine were determined by HPLC. Crude bee venom, small molecule components smaller than 700 Da, and target peptide components of 700–3000 Da were dissolved in ultrapure water to a concentration of approximately 1 mg / mL, filtered through an appropriate 0.22 μm filter membrane, and then injected for analysis. The HPLC system used was an SPD-15C or LC-15C, and the column was an UltimateLP-C18 column (4.6 × 250 mm, 5 μm, 300 Å). Mobile phase A was 0.1% TFA pure water, and mobile phase B was 0.1% TFA acetonitrile. The flow rate was 1 mL / min; the injection volume was 10 μL; the detection wavelength was 220 nm; and the elution program was 0–45 min, 3%–70% B, 45–51 min, 70% B. Each sample was tested in triplicate, and the mean and standard deviation were calculated. (See attached table). Figure 4 .

[0042] 2. Verification of Large Protein Residue: The SDS-PAGE results in Example 2 showed that no obvious >3kDa protein bands were observed in the <3kDa filtrate, proving that large protein / potential sensitizing components were effectively reduced after the first-stage ultrafiltration. Therefore, this example no longer requires SDS-PAGE of the target peptide fraction as a mandatory independent repeat experiment; when batch quality control or further evidence is needed, a repeat SDS-PAGE analysis can be performed on the final lyophilized 700–3000Da target peptide fraction to observe whether >3kDa protein bands are present.

[0043] 3. HPLC fingerprint analysis of the target peptide fraction: The 700–3000 Da target peptide fraction was dissolved in ultrapure water to a concentration of approximately 1 mg / mL. After appropriate centrifugation or filtration, the solution was injected for analysis. The HPLC system was an SPD-15C or LC-15C, and the column was an UltimateLP-C18 column (4.6 × 250 mm, 5 μm, 300 Å). Mobile phase A was 0.1% TFA pure water, and mobile phase B was 0.1% TFA acetonitrile. The flow rate was 1 mL / min; the injection volume was 10 μL; the detection wavelength was 220 nm; and the elution program was 0–45 min, 3%–70% B, 45–51 min, 70% B. The results showed that the 700–3000 Da target peptide fraction had five main characteristic absorption peaks between 29 and 40 min, which can be used as the HPLC fingerprint characteristics of the target peptide fraction to characterize its composition and batch stability. See [link to relevant documentation]. Figure 5 .

[0044] 4. Results: Compared with crude bee venom, the 5-hydroxytryptamine and histamine contents in the 700–3000 Da target peptide fraction were reduced by 96% and 57%, respectively. Combined with the SDS-PAGE results of Example 2, the >3 kDa protein bands in the target peptide fraction were significantly reduced or no obvious bands were observed. HPLC fingerprinting further showed that the target peptide fraction had five main characteristic absorption peaks between 29 and 40 min. These results indicate that the target peptide fraction obtained in this invention has low analgesic and low sensitization characteristics and possesses a peptide fraction fingerprint spectrum suitable for quality control.

[0045] Example 4: Evaluation of the anti-inflammatory activity and cell safety of the target peptide component.

[0046] This example illustrates that the 700–3000 Da target peptide component obtained in Example 2 retains anti-inflammatory activity while reducing the risk of pain and sensitization, and has good cellular safety at effective concentrations.

[0047] 1. Evaluation of anti-inflammatory activity of RAW264.7 cells: Logarithmically growing RAW264.7 cells were seeded in 96-well plates and cultured for 24 h. An in vitro inflammation model was established by adding 1 μg / mL LPS, and different concentrations of the 700–3000 Da target peptide fraction were added for treatment. 10 μg / mL dexamethasone was used as a positive control. The NO release level in the cell culture supernatant was measured after treatment, and cell viability at the same concentration was detected using CCK-8 assay. Simultaneously, crude wasp venom was examined to compare the changes in anti-inflammatory activity of the 700–3000 Da peptide fraction.

[0048] 2. Evaluation of antitumor expansion activity: Logarithmically growing tumor cells (HeLa and K562) were seeded into 96-well plates and cultured for 24 h. Then, different concentrations of crude bee venom and 700–3000 Da target peptide components were added and treated for 24 h. Cell viability was detected by CCK-8 assay and the half-maximal inhibitory concentration (IC50) was calculated.

[0049] 3. Safety evaluation of THLE-2 cells: THLE-2 normal human hepatocytes were seeded into 96-well plates and cultured for 24 h. Then, different concentrations of the 700–3000 Da target peptide component were added for 24 h. Cell viability was detected using the CCK-8 assay. Simultaneously, the toxicity of crude wasp venom to THLE-2 cells was investigated, and the safety-enhancing effect of the 700–3000 Da peptide component was compared.

[0050] 4. Results: The venom of the *Vespa mandshurica* showed no significant toxicity to RAW264.7 cells at a concentration of 30 μg / mL, and significantly inhibited LPS-induced NO release from RAW264.7 cells at a concentration of 10 μg / mL (IC50 value was 22 μg / mL). Figure 6The 700–3000 Da target peptide fraction showed no significant toxicity to RAW264.7 cells at 40 μg / mL, and significantly inhibited LPS-induced NO release from RAW264.7 cells at a concentration of 10 μg / mL, with an IC50 value of 23 μg / mL. It retained the main anti-inflammatory activity of the wasp venom. (See [link to article]). Figure 7 .

[0051] The half-maximal inhibitory concentrations (IC50) of *Vespa mandshurica* venom against THP-1 leukemia cells and HeLa cervical cancer cells were 34 μg / mL and 48 μg / mL, respectively. (See [link to relevant data]). Figure 8 The 700–3000 Da target peptide fraction had IC50 values ​​of 33 μg / mL and 56 μg / mL against cervical cancer HeLa cells and leukemia K562 cells, respectively, retaining the main antitumor activity of the venom of the wasp. (See below) Figure 9 The above results indicate that the bee venom active peptides obtained in this invention, in addition to their anti-inflammatory activity, also exhibit a certain inhibitory effect on tumor cell proliferation. This anti-tumor activity represents an extended biological activity of the bee venom active peptides beyond their anti-inflammatory effects.

[0052] Furthermore, in THLE-2 cells, the crude venom of the basal wasp exhibited concentration-dependent cytotoxicity, with a half-maximal inhibitory concentration (IC50) of 47 μg / mL. The target peptide fraction, exhibiting low analgesia and low sensitization, showed no significant toxicity at 80 μg / mL, demonstrating significantly improved safety. Figure 10 .

[0053] Example 5: Product form and formulation application examples.

[0054] This embodiment illustrates the application of the low-pain and low-sensitivity bee venom active peptide obtained in this invention. This embodiment is an exemplary description of the formulation application method and does not require animal experiments or actual commercial products as prerequisites; the specific dosage form, excipients, and dosage can be further optimized according to the anti-inflammatory, low-irritant active raw materials or tumor cell proliferation inhibition related applications.

[0055] The low-pain and low-allergenic bee venom active peptide lyophilized powder obtained in Example 2 can be dissolved at a predetermined concentration in a pharmaceutically, food-, or cosmetically acceptable carrier to prepare solutions, lyophilized powders, gels, creams, dressings, sprays, injectable prodrugs, or other topical / oral / investigative formulations.

[0056] In one application, the active peptide is used as an anti-inflammatory active ingredient in the preparation of compositions that inhibit the release of inflammatory mediators. In another application, the active peptide is used as a low-irritant bee venom active ingredient in topical repair, skin soothing, or functional bioactive peptide-related products. Furthermore, based on its inhibitory effect on tumor cell proliferation, the active peptide can also be used as a raw material or active component in related formulations for tumor cell proliferation inhibition research. Specific dosage forms, administration methods, excipient composition, and concentration ranges can be further optimized according to the intended use of the product.

[0057] The scope of protection of this invention is not limited to the above embodiments. For those skilled in the art, various changes, modifications, substitutions and variations can be made without departing from the principles and spirit of this invention. The scope of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a low-pain and low-allergenic bee venom active peptide, characterized in that: The steps include the following: (1) Take the venom of the hornet and redissolve it, then centrifuge it to remove impurities, and obtain the venom supernatant; (2) The bee venom supernatant was subjected to primary separation using an ultrafiltration membrane with a molecular weight cutoff of 1–5 kDa, and the filtrate with a molecular weight cutoff was collected. (3) The filtrate obtained in step (2) is subjected to secondary separation through a gel filter medium with an exclusion limit of 500–1000 Da to remove small molecule components and collect target peptide components with a molecular weight range of 700–3000 Da. (4) Freeze-dry the target peptide component to obtain a low-pain and low-sensitivity bee venom active peptide.

2. The method for preparing a low-pain and low-allergenic bee venom active peptide according to claim 1, characterized in that: The ultrafiltration membrane in step (2) has a molecular weight cutoff of 3 kDa, and the filtrate is a bee venom component with a molecular weight cutoff of <3 kDa.

3. The method for preparing a low-pain and low-allergenic bee venom active peptide according to claim 1, characterized in that: In step (3), the gel filter medium is Sephadex G-10, or a gel filter medium with the same or similar exclusion limits; water, ultrapure water, physiological saline or pharmaceutically acceptable buffer solution is used as the elution solution.

4. The method for preparing a low-pain and low-allergenic bee venom active peptide according to claim 1, characterized in that: During the secondary separation process, the absorbance of the eluent at 280 nm and / or 595 nm is simultaneously detected, and the target peak segments are merged according to the elution curve.

5. The method for preparing a low-pain and low-allergenic bee venom active peptide according to claim 1, characterized in that: The obtained low-pain and low-sensitivity bee venom active peptides showed that the 5-hydroxytryptamine content was reduced by no less than 90% compared to the crude bee venom of the common wasp, and the histamine content was reduced by no less than 50% compared to the crude bee venom of the common wasp.

6. The method for preparing a low-pain and low-allergenic bee venom active peptide according to claim 1, characterized in that: Among the obtained low-pain and low-sensitivity bee venom active peptides, the protein components with a molecular weight greater than 3kDa were below the detection limit, and phospholipase A1 and hyaluronidase were not detected.

7. A low-pain, low-allergenic bee venom active peptide composition obtained by the preparation method according to any one of claims 1-6, characterized in that: The composition mainly consists of bee venom peptides with a molecular weight of 700–3000 Da, which is defined by ultrafiltration and gel filtration separation processes; it has five characteristic absorption peaks in the HPLC chromatogram; and compared with crude bee venom, the content of pain-related small molecules is significantly reduced, and the content of sensitization-related large molecular proteins is below the detection limit.

8. The low-pain and low-allergenic bee venom active peptide composition according to claim 7, characterized in that: The composition exhibits anti-inflammatory activity by inhibiting LPS-induced nitric oxide release from RAW264.7 cells.

9. The use of the low-pain and low-sensitivity bee venom active peptide obtained by the preparation method according to any one of claims 1–6, or the composition according to any one of claims 7–8, in the preparation of anti-inflammatory agents, tumor cell proliferation inhibitors, low-irritant bee venom active raw materials, topical repair agents, functional bioactive peptide raw materials, and pharmaceutically acceptable formulations.

10. The application according to claim 9, characterized in that: The formulations may be in the form of lyophilized powder, solution, gel, cream, dressing, spray, oral formulation, or injectable prodrug.

Images