Wasp venom polypeptide extract as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a wasp venom polypeptide extract as well as a preparation method and application thereof. The method comprises the following steps: by taking wasps as a raw material and an ethanol water solution as an extraction solvent, soaking the wasps for a preset time, centrifuging, taking supernate, filtering to remove impurities, and freeze-drying filtrate to obtain wasp venom crude extract powder; ultrasonically dissolving with water, and filtering with an organic filter membrane to obtain a sample solution of the vespid venom crude extract; and then carrying out separation and purification on a reverse chromatographic column with phenylsilane bonded silica gel as a filler, collecting an eluent, and carrying out freeze drying to obtain the wasp venom polypeptide extract. The wasp venom polypeptide extract provided by the invention can inhibit perception, transmission and amplification of pain signals by regulating and controlling various ion channels such as TRPV1, Nav1.8 and Cav2.2, can quickly relieve pain, and can be used for treating chronic pain such as sciatica, rheumatic arthritis, musculoskeletal pain, neuropathic pain and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a polypeptide extract of wasp venom, a preparation method thereof and application thereof. BACKGROUND

[0002] Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, and is also a protective defense response of the body to harmful stimulation. Globally, 30% to 50% of the population suffers from chronic pain, such as diabetic neuropathic pain, sciatica, rheumatoid arthritis, musculoskeletal pain and cancer pain, which has become a global public health concern, seriously affecting people's life and health and quality of life. Pain management mainly relies on non-steroidal anti-inflammatory drugs, traditional opioid drugs, and auxiliary drugs such as antidepressants and anticonvulsants. However, the serious side effects and high cost of these therapies make them unable to meet the treatment needs of patients with pain, and there is an urgent need to open up new treatment methods.

[0003] At present, the research on innovative targets related to chronic pain mainly focuses on primary sensory neurons, and different ion channels have been explored in many aspects. Among them, the voltage-gated Cav2.2 (N-type) calcium channel and the Nav1.8 channel are clinically successful targets for pain relief. The Cav2.2 inhibitor Ziconotide and the Nav1.8 inhibitor Journavx have shown effective analgesic effects in clinical treatment, and do not cause addiction. As a result, a series of innovative targets for ion channels have attracted the layout of major pharmaceutical companies, and many inhibitors of Nav1.7 and P2X7 and other channels are in the clinical stage. Therefore, screening and identifying new ion channel inhibitors have important theoretical research and clinical application value for the development of analgesic drugs targeting different channels. SUMMARY

[0004] The present application aims at the deficiencies in the prior art, and provides a polypeptide extract of wasp venom, a preparation method thereof and application thereof. The polypeptide extract of wasp venom is taken as a research object, and a variety of methods such as reverse phase high performance liquid chromatography, mass spectrometry identification, whole cell membrane patch clamp recording and animal models are combined to analyze the ion channel targeting mechanism of the polypeptide extract of wasp venom and evaluate the analgesic activity, thereby further providing a theoretical guidance basis for developing analgesic products from wasp extracts.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The first aspect of the present application is to provide a preparation method of a polypeptide extract of wasp venom, comprising the following steps:

[0007] S1, using wasp as raw material, and an ethanol aqueous solution as an extraction solvent, the wasp is soaked for a preset time, then centrifuged, the supernatant is taken, impurities are removed by filtration, and the filtrate is freeze-dried to obtain a wasp venom crude extract powder; wherein the volume fraction of ethanol in the ethanol aqueous solution is 75%-80%;

[0008] S2, 5-10 times water is added by mass volume ratio, and the wasp venom crude extract powder is ultrasonically dissolved, then an organic filter membrane is passed through after being dissolved to obtain a sample solution of the wasp venom crude extract;

[0009] S3, the sample solution obtained in S2 is separated and purified on a reverse phase chromatography column with a filler of phenylsilane bonded silica gel, a binary mobile phase system is adopted, A is 0.1% trifluoroacetic acid, and B is acetonitrile, the separation and purification conditions are as follows: 0-20 min, A changes from 100% by volume to 85%, and B changes from 0 by volume to 15%, 20-45 min, A changes from 85% by volume to 60%, and B changes from 15% by volume to 40%, according to the ultraviolet absorption spectrum, the eluate with a retention time of 5.5-6.5 min, 6.6-7.5 min, 12.5-14 min and 18-19 min is collected, and freeze-drying is performed to obtain the wasp venom polypeptide extract.

[0010] Further, in step S1, the mass ratio of the wasp to the ethanol aqueous solution is 1:(1-1.2), and the soaking time is 24-36 h.

[0011] Further, the centrifugation process is 10000xg-12000xg, and the centrifugation time is 30 min.

[0012] Further, in step S2, the pore size of the organic filter membrane is 0.22-0.45 μm.

[0013] Further, in step S3, the diameter of the reverse phase separation chromatography column is 30 mm, the column length is 250 mm, and the particle size of the filler is 5 μm-10 μm.

[0014] Further, the detection wavelength in the separation and purification in step S3 is 215±2 nm, the injection amount is 0.8-1 g, and the flow rate is 30 mL / min.

[0015] The second aspect of the present application is to provide the application of the wasp venom polypeptide extract prepared by the preparation method in the preparation of an N-type voltage-gated calcium channel Cav2.2 inhibitor.

[0016] The third aspect of the present application is to provide the application of the wasp venom polypeptide extract prepared by the preparation method in the preparation of a Nav1.8 inhibitor.

[0017] The fourth aspect of the present application provides the use of the wasp venom polypeptide extract prepared by the preparation method in the preparation of a TRPV1 inhibitor.

[0018] The fifth aspect of the present application provides a pharmaceutical composition comprising the wasp venom polypeptide extract prepared by the preparation method and a pharmaceutical excipient.

[0019] The sixth aspect of the present application provides a wasp venom polypeptide spray comprising the wasp venom polypeptide extract or the wasp venom polypeptide extract prepared by the preparation method of the wasp venom polypeptide extract, a honeycomb extract, a Herba Veratrinae extract and menthol.

[0020] Compared with the prior art, the technical solution provided by the present application has the following beneficial effects:

[0021] (1) The wasp venom polypeptide extract prepared by the present application can regulate TRPV1, Nav1.8 and Cav2.2 ion channels, inhibit the perception, transmission and amplification of pain signals, and quickly relieve pain, and can be used for the treatment of chronic pain such as sciatica, rheumatoid arthritis, muscle and skeletal pain, and neuropathic pain.

[0022] (2) The preparation method of the wasp venom polypeptide extract is simple and easy to realize large-scale production, which brings a new treatment method for patients suffering from chronic pain. It also provides more theoretical guidance for the mechanism of the wasp venom polypeptide in relieving pain. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 HPLC chromatogram of wasp venom polypeptide extract;

[0024] Figure 2 Mass spectrum identification of wasp venom polypeptide extract;

[0025] Figure 3 Molecular weight distribution count of wasp venom polypeptide extract;

[0026] Figures 4a-4d Inhibition of Nav1.7, Nav1.8, Cav2.2 and TRPV1 channels by wasp venom polypeptide extract;

[0027] Figure 5 Inhibition of Nav1.7, Nav1.8, Cav2.2 and TRPV1 channels by wasp venom polypeptide extract;

[0028] Figure 6 Activity evaluation of wasp venom polypeptide extract in acetic acid writhing analgesia model;

[0029] Figure 7Evaluation of the I phase analgesic effect of the wasp venom polypeptide extract in a formalin pain model;

[0030] Figure 8 Evaluation of the II phase analgesic effect of the wasp venom polypeptide extract in a formalin pain model. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present application clearer, the specific embodiments of the present application will be further described in detail below with reference to the drawings. If the specific test methods, instruments, devices or conditions are not specified in the embodiments, they are all carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained from the market.

[0032] The present application provides a preparation method of a wasp venom polypeptide extract. The wasp can be from any region, and the specific steps are as follows:

[0033] Step S1, using wasps as raw materials and an ethanol aqueous solution as an extraction solvent, the mass ratio of wasps to the ethanol aqueous solution is 1:(1-1.2), the soaking time is 24-36 h, then centrifugation is carried out at 10000xg-12000xg for 30 min, the supernatant is taken, impurities are removed by filtration, and the filtrate is freeze-dried to obtain a wasp venom crude extract powder; wherein the volume fraction of ethanol in the ethanol aqueous solution is 75%-80%;

[0034] Step S2, 5-10 times of water is added according to the mass volume ratio, and the wasp venom crude extract powder is ultrasonically dissolved, then an organic filter membrane (0.22-0.45 μm) is passed after dissolution to obtain a sample solution of the wasp venom crude extract;

[0035] Step S3, the sample solution obtained in S2 is separated and purified on a reverse phase chromatography column with a phenylsilane bonded silica gel as the filler, the diameter of the reverse phase chromatography column is 30 mm, the column length is 250 mm, the particle size of the filler is 5 μm-10 μm, a binary mobile phase system with A being 0.1% trifluoroacetic acid and B being acetonitrile is used, and the separation and purification conditions are as follows: 0-20 min, A changes from 100% by volume to 85% by volume, and B changes from 0 by volume to 15% by volume, 20-45 min, A changes from 85% by volume to 60% by volume, and B changes from 15% by volume to 40% by volume, the eluate with a retention time of 5.5-6.5 min, 6.6-7.5 min, 12.5-14 min and 18-19 min is collected according to the ultraviolet absorption spectrum, and freeze-drying is carried out to obtain the wasp venom polypeptide extract.

[0036] The preparation method of the wasp venom polypeptide extract provided by the present application will be described in detail below with specific embodiments.

[0037] Example 1

[0038] Preparation of peptide extracts from wasp venom.

[0039] In this example, Vespa nigra was collected from the mountainous area of ​​southern Yunnan and Changjie Yi Township, Yongping County, Dali Prefecture, Yunnan.

[0040] A. Venom Collection: Take 100-200 live wasps and soak them in medical alcohol at a 1:1 mass ratio. After alcohol extraction for 24 hours, collect the supernatant and centrifuge at 12,000 × g for 30 minutes at 4°C. Remove impurities by filtering the supernatant through a 0.45 μm filter membrane and freeze-dry it in a vacuum freeze dryer to obtain a crude wasp venom extract powder.

[0041] B. Isolation, Purification, and Identification of Wasp Venom: Wasp venom extract was dissolved in ultrapure water to a concentration of 40 mg / mL. The extract was centrifuged at 8000 rpm for 20 min at 4°C and then filtered through a 0.22 μm filter. The prepared venom was isolated and purified on a Beijing Auno UV2000 chromatograph, with a sample load of 20 mL (800 mg) and a phenyl liquid chromatography column (5 μm, 30 mm × 250 mm). Eluent A was an aqueous solution (containing 0.1% TFA), and eluent B was an acetonitrile solution (containing 0.1% TFA). The elution gradient was as follows: 0-20 min, 0-15% eluent B, flow rate 30 mL / min, 20-45 min, 15-40% eluent B, flow rate 30 mL / min. The elution peak was collected at 215 nm, as shown in Table 1.

[0042] The collected elution peaks 4, 5, 7, and 9 were separated and purified on a Waters Alliance 2695 high performance liquid chromatograph with a sample volume of 50 μL. The chromatographic column was a reverse phase C18 column (Yuexu, 4.6 mm × 250 mm, 5 μm). Eluent A: aqueous solution (containing 0.1% TFA), eluent B: acetonitrile solution (containing 0.1% TFA). Elution gradient: 0-40 min, 0-25% eluent B, flow rate 1 mL / min, column temperature 35°C, peak collection at 215 nm. CCA matrix was dissolved in 50% acetonitrile to 10 mg / mL. 1.0 μL of CCA matrix solution was mixed with 0.5 μL of sample solution, and 0.5 μL of the mixture was applied to the sample. The mixture was dried at room temperature and analyzed by MALDI-TOF mass spectrometry.

[0043] C. Peaks 4, 5, 7, and 9 collected in step A were freeze-dried in an in-situ freeze dryer using the following method. The freeze-drying procedure is shown in Table 1 to obtain powdered samples for subsequent activity verification and product development.

[0044] Table 1. Freeze-drying procedures

[0045]

[0046] By Figure 1 and Figure 2 It can be seen that the wasp venom extract contains 10 elution peaks, and the polypeptide components in elution peaks 4, 5, 7 and 9 are identified by mass spectrometry. At the same time, the whole wasp extract contains rich polypeptides in the range of 500-1000 Da, as shown in Table 2.

[0047] Table 2. Inhibition activity of different elution peaks on ion pain channel

[0048]

[0049]

[0050] (Note: NA represents no activity)

[0051] Example 2

[0052] Identification of the inhibition activity of wasp venom polypeptide extract targeting ion channels.

[0053] 0.5 mg of wasp polypeptide extract (elution peaks 4, 5, 7 and 9) was weighed into 1 mL of deionized water as a mother liquor, which was diluted with each ion channel extracellular fluid for the experiment. The activity of Nav1.7, Nav1.8, Cav2.2 and TRPV1 channels was verified.

[0054] The recovered HEK293T cells can be used for transfection experiments after 3-4 passages of culture. When the cell density reaches 80-90%, discard the culture medium, rinse once with PBS, and add 2 mL of serum-free culture medium (Opti-MEM). Prepare solution A: 240 μL of serum-free medium + 10 μL of lipofectamine 2000 per well (total volume 250 μL), and let it stand at room temperature for 5 min. Prepare solution B: 250 μL of serum-free medium, add 4 ng of Nav1.7 (Nav1.8, Cav2.2 and TRPV1) channel plasmid, and let it stand at room temperature for 5 min. Mix A and B, and let it stand at room temperature for 20 min. Mix the Nav1.7-liposome and gently add it to the cells, and incubate in a 37°C, 5% CO2 incubator for 4-6 h. Then, the cells are cultured in DMEM medium containing 10% FBS for 12 h, and can be used for subsequent electrophysiological experiments.

[0055] Whole-cell patch-clamp recording was performed at room temperature (20-25 °C). The recording acquisition data amplifier was EPC10 USB Amplifier (HEKA, Elektronik, Lambrecht, Germany), and the data recording and control software was PatchMaster (HEKA, Elektronik, Lambrecht, Germany). Glass electrodes were pulled from microelectrode glass capillary (outer diameter 1.5 mm, inner diameter 1.05 mm, length 10 mm, Wuhan Micro-Explore Scientific Instrument Co., Ltd.) by two-step method using a PC-10 electrode puller (NARISHIGE, Tokyo, Japan). The electrode was adjusted to have a resistance of 2-3 MΩ in water. The cell was pressed to seal, and the resistance was generally > GΩ after complete sealing. The fast capacitance was compensated, and then the recording was adjusted to WHOLE-CELL mode. The three-way valve was opened to break the membrane, the slow capacitance was compensated, and the R-series resistance should be less than 10 MΩ. The cell was clamped at a specific voltage, the series resistance and leakage capacitance were compensated, and then the stimulation was given according to the set stimulation mode, and the channel current was recorded.

[0056] The cell was clamped at -100 mV, and the depolarization stimulation voltage was -10 mV with a duration of 20 ms. The results are shown in Figure 3 and Fig. 4, the venom polypeptide extract of the wasp has the strongest inhibitory activity on Cav2.2 channel, 0.5 mg / mL can inhibit 67.2% of the channel current, and the inhibition rate on Nav1.8 channel current is 45.63%; and has no obvious inhibitory effect on Nav1.7.

[0057] When determining the inhibitory activity of the venom polypeptide extract of the wasp on TRPV1, the cell was maintained at 0 mV, and was stimulated at -60 mV for 40 s, with an interval of 2 s between each stimulation. After a stable current was stimulated using 0.1 μM capsaicin (Cap), the candidate venom polypeptide extract of the wasp was added at the same concentration as capsaicin. The results are shown in Figure 4d showing that 0.5 mg / mL can inhibit 58.25% of the TRPV1 channel current.

[0058] Example 3

[0059] Pain-relieving activity evaluation of the venom polypeptide extract of the wasp in acetic acid writhing pain model.

[0060] Morphine was purchased from Shenyang First Pharmaceutical Factory (1 mL x 10 bottles), Liaowei Pharmaceutical Permit No. (1996) 002747.

[0061] Male Kunming mice 20g±2g; all drug groups were 6 per group. Mice were randomly divided into 3 groups, 6 in each group. Divided into saline group, positive control group and experimental group. The positive control drug is morphine (2mg / kg), the experimental drug is Polistes antennata venom polypeptide extract elution peak 5 (1mg / kg, 2mg / kg, 4mg / kg). Intraperitoneal administration, 15min after administration, mice were injected with 0.8% acetic acid, 0.2mL per injection. The mice were placed in open polyethylene cages (30cm×40cm×30cm), and the number of writhing was observed. The evaluation criteria for writhing reaction are as follows: abdominal depression, arching, hip lifting, hind leg stretching, and one arching and hind leg stretching as one complete writhing reaction. The number of writhing within 30min after injection of acetic acid was observed and recorded.

[0062] As shown in Figure 7 , the number of writhing in the control group was 28.17±3.71, and the number of writhing in the 1mg / kg, 2mg / kg and 4mg / kg Polistes antennata venom polypeptide extract groups was 18.67±7.58, 14.33±5.2 and 10.67±4.92 respectively. As a positive control, the number of writhing in the 2mg / kg morphine group was 11.4±9.86 (P<0.001). The results showed that in the acetic acid writhing pain model, Polistes antennata venom polypeptide extract could effectively reduce the number of writhing in mice compared with the control group, and the trend of reduction was more obvious with the increase of dose. At the highest dose of 4mg / kg, the analgesic effect of Polistes antennata venom polypeptide extract was particularly significant, with the number of writhing reduced to 10.67±4.92, close to the analgesic effect of the positive control drug 2mg / kg morphine (11.4±9.86, P<0.001).

[0063] Polistes antennata venom polypeptide extract obtained in Example 1 was prepared into a spray, and the analgesic effect was evaluated in the formalin analgesia model.

[0064] The composition of Polistes antennata venom polypeptide spray product: ethanol 40-50 parts, Polistes antennata venom polypeptide extract 1-20 parts (elution peaks 4, 5, 7, 9), alveolar extract 1-10 parts, Herba Lysimachiae extract 1-10 parts, menthol 1-10 parts.

[0065] The preferred formula in the example is composed of ethanol 50 parts, Polistes antennata venom polypeptide extract 15 parts (elution peaks 4, 5, 7, 9 mixed freeze-dried powder), alveolar extract 10 parts, Herba Lysimachiae extract 10 parts, menthol 5 parts.

[0066] Model construction: Male ICR mice, 17-20 g, were randomly divided into 3 groups, 6 mice in each group. They were divided into saline group, positive control group and experimental group. The positive control drug was morphine (2 mg / kg), intramuscular injection of 0.1 mL. The test drug was Polypeptide Extract of Vespa Venom Spray and Polypeptide Extract of Vespa Venom Elution Peak 5 (2 mg / kg, intramuscular injection of 0.1 mL). The drug was sprayed on the right leg of the mice by skin absorption administration. Thirty minutes after administration, 10 μL of 5% formalin solution was injected into the right toe of the mice, and the time of the mice to lick the foot was recorded every 5 min, and the licking time of the mice within 45 min after injection of formalin was observed and recorded.

[0067] In the formalin mouse pain experiment, the pain induced by formalin was divided into two phases, phase I (0-10 min) was the direct effect caused by stimulating nociceptors, and phase II (10-45 min) was the inflammatory response of prostaglandin. 10 μL of 5% formalin solution was injected into the right toe of the mice, and the licking time of the mice within 45 min was observed and recorded. The experiment was divided into control group, morphine group and experimental group. As shown in Figure 7 the licking time of the mice in the control group was 104.3±27.46 s, and the licking time of the mice in the Polypeptide Extract of Vespa Venom Spray group was 61.5±32.87 s; the corresponding licking time of the mice in the morphine (2 mg / kg) group was 66.17±14.96 s; these results showed that there was a significant difference in analgesic effect between the Polypeptide Extract of Vespa Venom Spray and the control group in the I phase of pain (p<0.05). As shown in Figure 8 the licking time of the mice in the control group was 132±74.87 s, and the licking time of the mice in the Polypeptide Extract of Vespa Venom Spray group was 26.5±13.17 s, and the corresponding licking time of the mice in the morphine (2 mg / kg) group was 55.5±23.28 s. These results showed that the analgesic effect of the Polypeptide Extract of Vespa Venom Spray group in the second phase of pain exhibited a more obvious analgesic effect, and the difference reached a high significant level (P<0.01). Compared with the control group, the duration of pain behavior of the Polypeptide Extract of Vespa Venom Spray was significantly shortened, and the intensity of pain response was also significantly reduced. The Polypeptide Extract of Vespa Venom Spray can significantly reduce the inflammatory pain behavior, and in addition to the anti-inflammatory mechanism to relieve pain, it can also regulate TRPV1, Nav1.8 and Cav2.2 ion channels to inhibit the perception, transmission and amplification of pain signals, and further quickly relieve pain.

[0068] Example 4

[0069] Safety test of Polypeptide Extract of Vespa Venom

[0070] White male guinea pigs 3, body weight 250g-300g, 24h before the test, the guinea pigs' back hair on both sides was cut, the left and right sides of the hair removal area were about 3cm x 3cm, the right side was the drug application area, and the left side was the blank control area. 0.5mL of the test substance was measured and directly dropped onto the skin of the drug application area of the animals, then covered with a layer of non-irritating plastic film, and fixed with non-irritating adhesive tape, the application time was 4h. After 4h of application, the drug application area was washed with warm water to remove the residue. Apply once a day for 14 consecutive days. Observe the local reaction of the skin 24h after each application, calculate the average score of each animal per day (irritation index) (average score of each animal per day = total score of erythema and edema of all test animals for 14d / number of test animals x 14), and determine the skin irritation intensity according to Table 2 of "Disinfection Technical Specification" (2002 edition).

[0071] The results are shown in Table 3, all test animals' skin in the drug application area had no erythema, edema and other abnormalities during the 14d observation period after exposure; the control area of the test animals had no abnormal phenomena during the observation period. The average score of irritation reaction of each animal per day (irritation index) was 0.

[0072] Table 3. Results of multiple intact skin irritation test

[0073]

[0074] In the case of no conflict, the above-mentioned embodiments and features in the embodiments can be combined with each other.

[0075] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a wasp venom polypeptide extract, characterized in that: The following steps are involved: S1. Using wasps as raw material and ethanol-water as extraction solvent, soaking the wasps for a predetermined time, centrifuging, collecting the supernatant, filtering to remove impurities, and freeze-drying the filtrate to obtain a crude wasp venom extract powder; wherein the volume fraction of ethanol in the ethanol-water solution is 75% to 80%; S2. adding 5 to 10 times the amount of water by mass to dissolve the crude venom extract powder by ultrasonication, and filtering the powder through an organic filter membrane after dissolution to obtain a sample solution of the crude venom extract; S3. The sample solution obtained in S2 is separated and purified on a reverse phase chromatography column filled with phenylsilane bonded silica gel, using a binary mobile phase system in which A is 0.1% trifluoroacetic acid and B is acetonitrile. The separation and purification conditions are as follows: from 0 to 20 min, the volume ratio of A changes from 100% to 85%, and the volume ratio of B changes from 0 to 15%; from 20 to 45 min, the volume ratio of A changes from 85% to 60%, and the volume ratio of B changes from 15% to 40%. The eluents with retention times of 5.5 to 6.5 min, 6.6 to 7.5 min, 12.5 to 14 min, and 18 to 19 min are collected according to the ultraviolet absorption spectrum, and the eluents are freeze-dried to obtain the wasp venom polypeptide extract.

2. The preparation method according to claim 1, wherein In step S1, the mass ratio of wasps to ethanol aqueous solution is 1: (1-1.2), and the soaking time is 24-36 hours; the centrifugation process is 10000×g-12000×g, and centrifugation is 30 minutes.

3. The preparation method according to claim 2, wherein In step S2, the pore size of the organic filter membrane is 0.22-0.45 μm.

4. The preparation method according to claim 1, wherein In step S3 , the diameter of the reverse phase separation chromatographic column is 30 mm, the column length is 250 mm, and the particle size of the filler is 5 μm to 10 μm.

5. The preparation method according to claim 1, wherein: The detection wavelength during separation and purification in step S3 is 215±2 nm, the injection volume is 0.8-1 g, and the flow rate is 30 mL / min.

6. Use of the wasp venom polypeptide extract prepared by the preparation method according to any one of claims 1 to 5 in the preparation of an N-type voltage-gated calcium channel Cav2.2 inhibitor.

7. Use of the wasp venom polypeptide extract prepared by the preparation method according to any one of claims 1 to 5 in the preparation of Nav1.8 inhibitors.

8. Use of the wasp venom polypeptide extract prepared by the preparation method according to any one of claims 1 to 5 in the preparation of TRPV1 inhibitors.

9. A pharmaceutical composition, characterized in that The invention comprises a wasp venom polypeptide extract prepared by the preparation method according to any one of claims 1 to 5, and pharmaceutical excipients.

10. A wasp venom polypeptide spray, characterized in that: The invention comprises a wasp venom polypeptide extract, a honeycomb extract, a radix scutellariae extract and menthol prepared by the preparation method according to any one of claims 1 to 5.

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