A kind of Trichosanthes kirilowii polypeptide with kidney-tonifying and yang-strengthening effects and its preparation method and application

Abstract

The present invention belongs to the field of biotechnology and specifically relates to a Trichosanthes kirilowii polypeptide having the efficacy of tonifying the kidney and strengthening yang, as well as its preparation method and application. The present invention prepares the Trichosanthes kirilowii polypeptide having the efficacy of tonifying the kidney and strengthening yang through processes such as pretreatment of Trichosanthes kirilowii raw materials, preparation of Trichosanthes kirilowii seed liquid, preparation of crude Trichosanthes kirilowii peptides, ion exchange resin chromatography, and high-performance liquid chromatography purification. The Trichosanthes kirilowii polypeptide provided by the present invention can regulate the expression of testosterone secretion-related genes and proteins in TM3 cells, enhance mitochondrial function, and promote the expression of mitochondrial biogenesis-related genes and proteins. It can significantly promote testosterone secretion in TM3 cells, has the efficacy of tonifying the kidney and strengthening yang, and expands the application range of Trichosanthes kirilowii.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Trichosanthes kirilowii polypeptide having kidney-tonifying and yang-strengthening effects and an application thereof. Background Art

[0002] Testosterone is an important male hormone, playing a key role in regulating sexual function, reproduction, muscle movement, and various physiological activities. Recent research suggests that testosterone deficiency may be associated with conditions such as obesity, cardiovascular disease, and even depression. Testosterone deficiency can occur in men of all ages. In adult men, this deficiency is generally attributed to a natural decline in endogenous testosterone levels due to aging and other modifiable factors. Currently, exogenous testosterone replacement therapy (TRT) still has certain side effects. Therefore, the search for substances that can promote endogenous testosterone synthesis is of great significance.

[0003] Commonly used traditional Chinese medicines for kidney tonification and yang enhancement include seahorse, epimedium, dodder seed, and Cistanche deserticola. However, these herbs all have side effects, and their long-term safety is uncertain. Trichosanthes kirilowii, a member of the genus Cucurbitaceae, is a traditional oilseed crop in China, widely cultivated and highly regarded. Trichosanthes kirilowii seeds are rich in protein (26.70%) and possess significant nutritional value. Besides being rich in arginine, leucine, and glutamic acid, their amino acid profile closely matches the essential amino acid content recommended by the World Health Organization. Currently, little research has been conducted on the effects and active ingredients of Trichosanthes kirilowii seeds, and further research is needed to explore their applications. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a Trichosanthes kirilowii polypeptide with kidney-tonifying and yang-strengthening effects, as well as a preparation method and application thereof. The preparation method provided by the present invention can obtain the Trichosanthes kirilowii polypeptide with kidney-tonifying and yang-strengthening effects from Trichosanthes kirilowii. The preparation process is simple and the polypeptide yield is high. The obtained Trichosanthes kirilowii polypeptide is safe, has a small molecule and can be better absorbed and utilized by the human body. It can significantly increase testosterone secretion in TM3 cells and can be developed as a substitute for traditional aphrodisiac drugs.

[0005] In a first aspect, the present invention provides a method for preparing a Trichosanthes polypeptide, comprising the following steps:

[0006] Step 1: Defatting and removing phenolic substances from the shelled and crushed Trichosanthes kirilowii seeds, and then drying and powdering to obtain Trichosanthes kirilowii seed powder;

[0007] Step 2: extracting the Trichosanthes kirilowii seed powder by heating water to obtain a protein-denatured Trichosanthes kirilowii seed liquid;

[0008] Step 3: performing a composite enzymatic hydrolysis on the Trichosanthes kirilowii seed liquid, inactivating the enzyme after the enzymatic hydrolysis, separating the solid and liquid, collecting the enzymatic hydrolyzate, and drying to obtain a crude Trichosanthes kirilowii peptide; the composite enzymatic hydrolysis adopts a neutral protease and an alkaline protease;

[0009] Step 4: dissolving the crude peptide of Trichosanthes kirilowii in water to prepare a crude peptide solution of Trichosanthes kirilowii, filtering, and subjecting the obtained filtrate to ion exchange resin chromatography, using double distilled water as eluent, collecting the eluate of the corresponding peak according to the absorbance curve, and then drying to obtain an ion exchange chromatography hydrolyzate; the ion exchange resin chromatography uses a cellulose anion exchange chromatography column;

[0010] Step 5: dissolving the ion exchange chromatography hydrolyzate in water, separating and purifying it by reverse-phase high-performance liquid chromatography, collecting protein polypeptides that can significantly promote testosterone secretion in TM3 cells, and obtaining Trichosanthes kirilowii polypeptides; the chromatographic column in the reverse-phase high-performance liquid chromatography is a C18 chromatographic column, the mobile phase A is 0.1%-0.2% trifluoroacetic acid-water solution, the mobile phase B is 0.1%-0.2% trifluoroacetic acid-acetonitrile solution, and the gradient elution is performed; the gradient elution procedure is as follows:

[0011] 0-3min, mobile phase: 95% mobile phase A + 5% mobile phase B;

[0012] 3-10 min, mobile phase: 80% mobile phase A + 20% mobile phase B;

[0013] 10-20 min, mobile phase is 50% mobile phase A + 50% mobile phase B;

[0014] 20-23 min, mobile phase is 20% mobile phase A + 80% mobile phase B.

[0015] Fructus Trichosanthis seeds contain abundant protein, and protein is more by-products in the enzymolyte mixed peptide after enzymolysis, and the screening difficulty of target polypeptide is large, and Fructus Trichosanthis is rich in grease and polyphenols as oil crops, and grease has a strong interference effect on the column of protein purification, and polyphenols can be combined with protein, hindering the acquisition of protein and reducing extraction rate. The preparation method of Fructus Trichosanthis polypeptide provided by the present invention is with Fructus Trichosanthis seeds as raw material, after defatting and removing phenol, by high temperature boiling, making protein denaturation, and then by enzymolysis, then through multi-stage separation and purification, in the separation and purification process at each level, by the control of the parameters such as the type, concentration, flow velocity of the eluent, finally prepare the Fructus Trichosanthis polypeptide with kidney-tonifying and yang-strengthening effect. The method can accurately determine the elution peak of target polypeptide by detecting absorbance curve, and by controlling the time of collecting eluent, the target polypeptide can be repeatedly and stably collected.

[0016] In step 1, drying and powdering the Trichosanthes kirilowii seeds is helpful for extracting the protein therein, and the dried Trichosanthes kirilowii seeds are crushed to a particle size of ≤0.5 mm.

[0017] The solid-liquid separation in step 3 can be performed by filtration or centrifugation, and the corresponding filtrate or supernatant is collected as the enzymatic hydrolyzate. The drying method in steps 3 and 4 can be direct drying, concentration followed by drying, or reduced pressure distillation followed by drying.

[0018] In step 4, double-distilled water is used as the eluent, and the yield of the enzymatic hydrolysate by ion exchange chromatography is the highest.

[0019] Significantly promoting the testosterone secretion of TM3 cells in step 5 means that the effect of the screened polypeptide on increasing the testosterone secretion of TM3 cells is statistically significantly different from the original testosterone secretion of TM3 cells.

[0020] Preferably, in step 1, the solvent for defatting the Trichosanthes seeds is n-hexane, and the weight-to-volume ratio of the Trichosanthes seeds to n-hexane is 1:3-6; the solvent used for removing phenolic substances is acetone, and the weight-to-volume ratio of the Trichosanthes seeds to acetone is 1:3-6.

[0021] Preferably, in the step 2, the mass ratio of the Trichosanthes kirilowii seed powder to water is 1:30-50, the temperature is 90-100° C., and the time is 3-10 h.

[0022] High temperature extraction at 90-100°C is beneficial to subsequent enzymatic hydrolysis.

[0023] Preferably, the neutral protease in step 3 is a metalloprotease derived from Bacillus subtilis.

[0024] Neutral protease derived from Bacillus subtilis is a metalloprotease. It is an endoprotease extracted from Bacillus subtilis after deep fermentation. It is pure natural, safe and non-toxic, has strong hydrolysis ability, and can decompose large molecular proteins into products such as polypeptides and amino acids.

[0025] Preferably, the alkaline protease is an endoprotease derived from Bacillus licheniformis.

[0026] Alkaline protease derived from Bacillus licheniformis is an enzyme preparation produced by Bacillus licheniformis through deep liquid fermentation. It can quickly decompose proteins and hydrolyze large molecular proteins into free amino acids and other products.

[0027] More preferably, the amount of alkaline protease added accounts for 20%-40% of the mass of the Trichosanthes kirilowii seed powder; the amount of neutral protease added accounts for 20%-40% of the mass of the Trichosanthes kirilowii seed powder; the pH of the composite enzymatic hydrolysis in step 3 is 7.5-8 or 9-9.5, the enzymatic hydrolysis temperature is 55-75°C, and the enzymatic hydrolysis time is 3-5h.

[0028] More preferably, the pH of the composite enzymatic hydrolysis in step 3 is 9-9.5, the enzymatic hydrolysis temperature is 65° C., and the enzymatic hydrolysis time is 5 h. Under this enzymatic hydrolysis process, the yield of the crude peptide from Trichosanthes kirilowii is the highest.

[0029] Preferably, the eluent flow rate in step 4 is 1-2 mL / min; and the ion exchange resin chromatography uses a DEAE-52 cellulose anion exchange chromatography column.

[0030] More preferably, the eluent flow rate in step 4 is 1 mL / min, and the eluent with a peak time of 135-250 min is collected.

[0031] At this flow rate, the peak time of the ion exchange chromatography hydrolyzate is 135-250 min.

[0032] Preferably, the chromatographic column in step 5 is Pursuit XRs C-18; the mobile phase A is 0.1% trifluoroacetic acid-water, the mobile phase B is 0.1% trifluoroacetic acid-acetonitrile, the mobile phase flow rate is 20 mL / min, and the eluate with a peak elution time of 10.0-12.3 min or the eluate with a peak elution time of 21.0-21.4 min is collected to obtain the Trichosanthes polypeptide.

[0033] Under these conditions, the Trichosanthes kirilowii polypeptides obtained from the eluate with a peak time of 10.0-12.3 min and the eluate with a peak time of 21.0-21.4 min both had a significant promoting effect on the secretion of testosterone in TM3 cells.

[0034] In a second aspect, the present invention provides the Trichosanthes kirilowii polypeptide prepared by the above method.

[0035] The Trichosanthes kirilowii polypeptide provided by the present invention is mainly composed of small molecule peptides, is safe for the human body, is well absorbed, can inhibit and promote testosterone secretion in TM3 cells, promote mitochondrial biogenesis, and has the effect of tonifying the kidney and strengthening yang. The mechanism by which the Trichosanthes kirilowii polypeptide promotes testosterone secretion in TM3 cells is related to regulating the expression of genes and proteins related to testosterone secretion and promoting the expression of genes and proteins related to mitochondrial biogenesis.

[0036] In a third aspect, the present invention provides applications of the Trichosanthes kirilowii polypeptide, including applications in the preparation of drugs for tonifying the kidney and strengthening yang; or

[0037] Use in drugs for promoting testosterone secretion in TM3 cells; or

[0038] Use in the preparation of a drug for regulating the expression of testosterone secretion-related genes and proteins in TM3 cells; or

[0039] Application in the preparation of drugs for regulating the expression of mitochondrial biogenesis-related genes and proteins in TM3 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 The absorbance curves of the ion exchange chromatography hydrolyzates obtained after elution with different eluents in the present invention;

[0042] Figure 2 is the absorbance curve of the Trichosanthes kirilowii polypeptide obtained after purification by reversed-phase high performance liquid chromatography;

[0043] Figure 3 This is the total ion current chromatogram of the Trichosanthes kirilowii polypeptide TSH1-3 obtained in Example 2 of the present invention;

[0044] Figure 4 The results of the water extraction process and enzymatic hydrolysis process of the crude peptide from Trichosanthes kirilowii in Example 1 of the present invention are as follows;

[0045] Figure 5 The effect of the Trichosanthes kirilowii polypeptide of the present invention on the survival rate of TM3 cells;

[0046] Figure 6 The effect of the ion exchange chromatography hydrolysate of the present invention on testosterone secretion in TM3 cells;

[0047] Figure 7 The effect of the Trichosanthes kirilowii polypeptide of the present invention on testosterone secretion in TM3 cells;

[0048] Figure 8 The effects of the Trichosanthes kirilowii polypeptides TSH1-3 and the synthetic peptide VTPVGSPR of the present invention on the secretion of androgen related to TM3 cells and the levels of ATP and mitochondrial potential membrane;

[0049] Figure 9 The effects of the Trichosanthes kirilowii polypeptides TSH1-3 and the synthetic peptide VTPVGSPR on the expression of genes related to testosterone synthesis in the present invention;

[0050] Figure 10 The effects of the Trichosanthes kirilowii polypeptides TSH1-3 and the synthetic peptide VTPVGSPR on the expression of testosterone synthesis-related proteins in the present invention;

[0051] Figure 11 The effects of the Trichosanthes kirilowii polypeptides TSH1-3 and the synthetic peptide VTPVGSPR on mitochondrial mtDNA replication in the present invention;

[0052] Figure 12 The effects of the Trichosanthes kirilowii polypeptides TSH1-3 and the synthetic peptide VTPVGSPR on the expression of proteins related to mitochondrial biogenesis;

[0053] Figure 13 This is the effect of the Trichosanthes kirilowii polypeptides TSH1-3 and the synthetic peptide segment VTPVGSPR in the present invention on the expression of genes related to mitochondrial biogenesis. DETAILED DESCRIPTION

[0054] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0055] (I) Reagents: n-Hexane (McLean H810749); acetone (Guangzhou Chemical Reagent Factory HB02); trifluoroacetic acid (TFA), (McLean T818778); acetonitrile (McLean A800362); ion exchange resin (DEAE-52, Solebol C8930); penicillin-streptomycin solution (100X), (Biyuntian), C0222; CCK8 detection reagent (Abbkine KTA1020); BCA protein quantification kit (Abiowell AWB0104); RIPA lysis buffer (strong) (Abiowell AWB0136); trypsin (Gibco 25200056); ECL Plus ultrasensitive luminescent solution (Abiowell AWB0005); alcalase 2.4L, Novozymes; Neutral protease Nuetrase 0.8L, Novozymes; Mouse testosterone (T) ELISA research kit, MM-0569M1, Jiangsu Enzyme Immunity Industry Co., Ltd.; Superoxide dismutase (SOD) kit G0101W, Suzhou Grace Biotechnology Co., Ltd.; Malondialdehyde (MDA) kit, G0109W, Suzhou Grace Biotechnology Co., Ltd.

[0056] (2) Equipment: The Pursuit XRs C-18 column for reversed-phase high performance liquid chromatography was purchased from Agilent Technologies, USA.

[0057] Example 1

[0058] This embodiment provides a Trichosanthes polypeptide.

[0059] The specific preparation method is as follows:

[0060] Step 1: Shell and crush the Trichosanthes kirilowii seeds. Add n-hexane at a material-liquid ratio of 1:5 (g:mL) to defat the seeds. Add acetone at a material-liquid ratio of 1:5 (g:mL) to remove phenolic substances. After air-drying, sieve through an 80-mesh sieve to obtain Trichosanthes kirilowii seed powder.

[0061] Step 2: Take 10 g of Trichosanthes kirilowii seed powder, mix it with purified water at a material-liquid ratio of 1:40 (g:mL), heat it in a 95°C water bath, and keep it warm for 5 hours to obtain Trichosanthes kirilowii seed liquid.

[0062] Step 3: Cool the Trichosanthes kirilowii seed liquid to 65°C, add 2mL alkaline protease and 2mL neutral protease, and enzymatically hydrolyze at pH 9 and 65°C for 5h. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95°C for 15min, then centrifuge, collect the solution, distill under reduced pressure, and freeze-dry to obtain the Trichosanthes kirilowii crude peptide.

[0063] Step 4: Dissolve the crude peptide from Trichosanthes kirilowii and dilute it to 20 mg / ml. After filtering with a 0.22 μm filter membrane, load the sample into a DEAE-52 cellulose anion exchange chromatography column through a pressure pump and elute with double distilled water at a flow rate of 1 mL / min. The absorbance curve of the eluate at 220 nm is as follows: Figure 1 As shown, the fractions with elution time between 135 and 250 min were collected, concentrated under reduced pressure and freeze-dried to obtain ion exchange chromatography hydrolysate TSH1.

[0064] Step 5: TSH1 was dissolved in double-distilled water to a concentration of 5 mg / mL. The product was filtered through a 0.22 μm filter membrane and separated and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using a Pursuit XRs C-18 column at a temperature of 35°C. The loading volume was 600 μL. Mobile phase A consisted of 0.1% trifluoroacetic acid in water and mobile phase B consisted of 0.1% trifluoroacetic acid in acetonitrile. The flow rate was 20 mL / min. The gradient elution sequence was detailed in the table below:

[0065] Table 1 Order of linear gradient elution of mobile phase

[0066] Time (min) A (0.1% TFA-water) B (0.1% TFA-acetonitrile) 0-3 95％ 5％ 3-10 80％ 20％ 10-20 50％ 50％ 20-23 20％ 80％ 23-25 95％ 5％

[0067] The absorbance curves of the eluate at 220 nm at different time periods are as follows: Figure 2 The eluate with an elution time of 21.0-21.4 min was collected, concentrated under reduced pressure, and freeze-dried to obtain the Trichosanthes kirilowii polypeptide TSH1-3.

[0068] The peptides of TSH1-3 components were identified by LC-MS / MS method and high-resolution mass spectrometry database, and their amino acid sequences were analyzed. Finally, 42 peptides with high credibility were obtained. The total ion currents are shown in the figure below. Figure 3 The amino acid sequences of the 42 peptides are shown in Table 2.

[0069] Table 2 Peptide composition of TSH 1-3

[0070]

[0071]

[0072] Comparative Examples 1-4

[0073] Comparative Examples 1-4 provide three ion exchange chromatography hydrolysates TSH2, TSH3, TSH4 and TSH5, respectively.

[0074] The crude peptide of Trichosanthes kirilowii prepared in step 3 of Example 1 was subjected to ion exchange resin chromatography according to step 4 of Example 1, except that sodium chloride with a concentration of 0.01 mol / L was used instead of double distilled water for elution, and the components with an elution time of 168-264 min were collected. After reduced pressure concentration and vacuum freeze-drying, the ion exchange chromatography hydrolyzate TSH2 was obtained.

[0075] The crude peptide of Trichosanthes kirilowii prepared in step 3 of Example 1 was subjected to ion exchange resin chromatography according to step 4 of Example 1, except that sodium chloride with a concentration of 0.05 mol / L was used instead of double distilled water for elution, and the components with an elution time of 116-292 min were collected. After reduced pressure concentration and vacuum freeze-drying, the ion exchange chromatography hydrolyzate TSH3 was obtained.

[0076] The crude peptide of Trichosanthes kirilowii prepared in step 3 of Example 1 was subjected to ion exchange resin chromatography according to step 4 of Example 1, except that sodium chloride with a concentration of 0.1 mol / L was used instead of double distilled water for elution, and the components with an elution time of 116-212 min were collected, and after concentration under reduced pressure and vacuum freeze-drying, the ion exchange chromatography hydrolyzate TSH4 was obtained; the components with an elution time of 308-368 min were collected, and after concentration under reduced pressure and vacuum freeze-drying, the ion exchange chromatography hydrolyzate TSH5 was obtained.

[0077] The absorbance curves of the ion exchange chromatography hydrolysates of Comparative Examples 1-4 are shown in Figure 1 .

[0078] Example 2

[0079] Example 2 provides a Trichosanthes kirilowii polypeptide TSH1-2.

[0080] The exchange chromatography hydrolysate TSH1 prepared in step 4 of Example 1 was separated and purified by reverse-phase high-performance liquid chromatography according to the conditions of step 5 of Example 1, except that the collection time of the TSH1-2 eluate was 10.0-12.3 min.

[0081] The absorbance curve of the Trichosanthes polypeptide of Example 2 is shown in Figure 2 .

[0082] Comparative Examples 5-7

[0083] Comparative Examples 5-7 provide a kind of Trichosanthes kirilowii polypeptides TSH1-1, TSH1-4 and TSH1-5 respectively.

[0084] The ion exchange chromatography hydrolyzate TSH1 prepared in step 4 of Example 1 was separated and purified according to the reverse-phase high-performance liquid chromatography conditions of step 5 of Example 1, except that the collection times of the eluates of TSH1-1, TSH1-4, and TSH1-5 were 6.7-7.0 min, 21.6-22.0 min, and 23.2-23.6 min, respectively.

[0085] The absorbance curves of the Trichosanthes polypeptides of Comparative Examples 5-7 are shown in Figure 2 .

[0086] Example 3

[0087] Example 3 provides a Trichosanthes polypeptide.

[0088] Step 1: Shell and crush the Trichosanthes kirilowii seeds. Add n-hexane at a material-liquid ratio of 1:6 (g:mL) to defat the seeds. Add acetone at a material-liquid ratio of 1:6 (g:mL) to remove phenolic substances. After air-drying, sieve through an 80-mesh sieve to obtain Trichosanthes kirilowii seed powder.

[0089] Step 2: Take 10 g of Trichosanthes kirilowii seed powder and add purified water in a material-liquid ratio of 1:5 (g:mL), heat in a 100°C water bath, and keep warm for 10 hours to obtain Trichosanthes kirilowii seed liquid.

[0090] Step 3: Cool the Trichosanthes kirilowii seed liquid to 50°C, add 4 mL of alkaline protease and 4 mL of neutral protease, and perform enzymatic hydrolysis at pH 7.5 and 55°C for 5 hours. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95°C for 15 minutes, then centrifuge, collect the solution, distill under reduced pressure, and freeze-dry to obtain the crude Trichosanthes kirilowii peptide.

[0091] The remaining steps were the same as in Example 1 to obtain the Trichosanthes polypeptide.

[0092] Example 4

[0093] Example 4 provides a Trichosanthes polypeptide.

[0094] Step 1: Shell the Trichosanthes kirilowii seeds and crush them. Add n-hexane at a material-liquid ratio of 1:3 (g:mL) to defat the seeds. Add acetone at a material-liquid ratio of 1:3 (g:mL) to remove phenolic substances. After air-drying, sieve through an 80-mesh sieve to obtain Trichosanthes kirilowii seed powder.

[0095] Step 2: Take 10 g of Trichosanthes kirilowii seed powder, mix it with purified water at a material-liquid ratio of 1:50 (g:mL), heat it in a 90°C water bath, and keep it warm for 3 hours to obtain Trichosanthes kirilowii seed liquid.

[0096] Step 3: Cool the Trichosanthes kirilowii seed liquid to 45°C, add 3mL alkaline protease and 3mL neutral protease, and enzymatically hydrolyze at pH 9.5 and 75°C for 3h. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95°C for 15min, then centrifuge, collect the solution, distill under reduced pressure, and freeze-dry to obtain the Trichosanthes kirilowii crude peptide.

[0097] The remaining steps were the same as in Example 1 to obtain the Trichosanthes polypeptide.

[0098] Example 5

[0099] This embodiment provides a crude peptide from Trichosanthes kirilowii.

[0100] Step 1: Shell and crush the Trichosanthes kirilowii seeds. Add n-hexane at a material-liquid ratio of 1:5 (g:mL) to defat the seeds. Add acetone at a material-liquid ratio of 1:5 (g:mL) to remove phenolic substances. After air-drying, sieve through an 80-mesh sieve to obtain Trichosanthes kirilowii seed powder.

[0101] Step 2: Take 10 g of Trichosanthes kirilowii seed powder, mix it with purified water at a material-liquid ratio of 1:40 (g:mL), heat it in a 95°C water bath, and keep it warm for 5 hours to obtain Trichosanthes kirilowii seed liquid.

[0102] Step 3: Cool the Trichosanthes kirilowii seed liquid to the enzymatic hydrolysis temperature, add 2 mL of alkaline protease and 2 mL of neutral protease, and enzymatically hydrolyze for 4 hours at a pH of 9 and a hydrolysis temperature of 75°C. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95°C for 15 minutes, then centrifuge, collect the solution, distill under reduced pressure, and freeze-dry to obtain the crude Trichosanthes kirilowii peptide.

[0103] Example 6

[0104] The difference from Example 5 is that the enzymatic hydrolysis temperature in step 3 is 65°C.

[0105] Example 7

[0106] The difference from Example 5 is that the enzymatic hydrolysis temperature in step 3 is 55°C.

[0107] Comparative Example 8

[0108] The difference from Example 5 is that the enzymatic hydrolysis temperature in step 3 is 45°C.

[0109] Calculate the yield of crude peptide from Trichosanthes kirilowii at different enzymatic hydrolysis temperatures. The yield calculation formula is:

[0110]

[0111] Protein content: After Dumas nitrogen determination, the protein content of defatted Trichosanthes kirilowii powder is 73.21%

[0112] Polypeptide content: The polypeptide content in the enzymatic hydrolysate was determined using the biuret method.

[0113] The results of Examples 5-7 and Comparative Example 8 are as follows Figure 4 As shown in Figure A, it can be seen that the enzymatic hydrolysis temperature has a great influence on the polypeptide yield. At an enzymatic hydrolysis temperature of 55-75°C, the polypeptide yield is higher, among which 65°C has the best effect.

[0114] Example 8

[0115] This embodiment provides a crude peptide from Trichosanthes kirilowii.

[0116] The Trichosanthes kirilowii seed solution prepared in step 2 of Example 5 was taken, cooled to 65°C, 2mL of alkaline protease and 2mL of neutral protease were added, and enzymatic hydrolysis was performed at pH 9 and a temperature of 65°C for 3h. After the enzymatic hydrolysis was completed, the enzyme was inactivated at 95°C for 15min, followed by centrifugation, the solution was collected, distilled under reduced pressure, and freeze-dried to obtain a crude Trichosanthes kirilowii peptide.

[0117] Example 9

[0118] The difference from Example 8 is that the enzymatic hydrolysis time in step 3 is 4 h.

[0119] Example 10

[0120] The difference from Example 8 is that the enzymatic hydrolysis time in step 3 is 5 h.

[0121] Comparative Example 9

[0122] The difference from Example 8 is that the enzymatic hydrolysis time in step 3 is 6 h.

[0123] Comparative Example 10

[0124] The difference from Example 8 is that the enzymatic hydrolysis time in step 3 is 7 h.

[0125] Comparative Example 11

[0126] The difference from Example 8 is that the enzymatic hydrolysis time in step 3 is 8 h.

[0127] The yield of crude peptide from Trichosanthes kirilowii was calculated for Examples 8-10 and Comparative Examples 9-11. Figure 4 As shown in B, it can be seen that the yield of crude peptide from Trichosanthes kirilowii is higher when the enzymatic hydrolysis time is 3-5h, among which the enzymatic hydrolysis time of 5h is the best.

[0128] Example 11

[0129] This embodiment provides a crude peptide from Trichosanthes kirilowii.

[0130] The Trichosanthes kirilowii seed solution prepared in step 2 of Example 5 was taken, cooled to 65°C, 2 mL of alkaline protease and 2 mL of neutral protease were added, and enzymatic hydrolysis was performed at pH 7.5 at a temperature of 65°C for 5 h. After the enzymatic hydrolysis was completed, the enzyme was inactivated at 95°C for 15 min, followed by centrifugation, the solution was collected, distilled under reduced pressure, and freeze-dried to obtain a crude Trichosanthes kirilowii peptide.

[0131] Example 12

[0132] The difference from Example 11 is that the enzymatic hydrolysis pH in step 3 is 8.

[0133] Example 13

[0134] The difference from Example 11 is that the enzymatic hydrolysis pH in step 3 is 9.

[0135] Example 14

[0136] The difference from Example 11 is that the enzymatic hydrolysis pH in step 3 is 9.5.

[0137] Comparative Example 12

[0138] The difference from Example 11 is that the enzymatic hydrolysis pH in step 3 is 8.5.

[0139] Comparative Example 13

[0140] The difference from Example 11 is that the enzymatic hydrolysis pH in step 3 is 10.

[0141] The yield of crude peptide from Trichosanthes kirilowii was calculated for Examples 11-14 and Comparative Examples 12-13. Figure 4 As shown in Figure C, it can be seen that the yield of Trichosanthes kirilowii crude peptide is higher when the enzymatic hydrolysis pH is 7.5-8 or pH 9-9.5, among which the effect is best when pH = 9.

[0142] Example 15

[0143] This embodiment provides a crude peptide from Trichosanthes kirilowii.

[0144] Step 1: Shell and crush the Trichosanthes kirilowii seeds. Add n-hexane at a material-liquid ratio of 1:5 (g:mL) to defat the seeds. Add acetone at a material-liquid ratio of 1:5 (g:mL) to remove phenolic substances. After air-drying, sieve through an 80-mesh sieve to obtain Trichosanthes kirilowii seed powder.

[0145] Step 2: Take 10 g of Trichosanthes kirilowii seed powder, mix it with purified water at a material-liquid ratio of 1:30 (g:mL), heat it in a 95°C water bath, and keep it warm for 5 hours to obtain Trichosanthes kirilowii seed liquid.

[0146] Step 3: The Trichosanthes kirilowii seed liquid prepared with different material-liquid ratios was cooled to 65°C, 2 mL of alkaline protease and 2 mL of neutral protease were added, and enzymatic hydrolysis was carried out at pH 9 and a temperature of 65°C for 5 hours. After the enzymatic hydrolysis was completed, the enzyme was inactivated at 95°C for 15 minutes, and then centrifuged, the solution was collected, distilled under reduced pressure, and freeze-dried to obtain the Trichosanthes kirilowii crude peptide.

[0147] Example 16

[0148] The difference from Example 15 is that the material-liquid ratio in step 2 is 1:40.

[0149] Example 17

[0150] The difference from Example 15 is that the material-liquid ratio in step 2 is 1:50.

[0151] Comparative Example 14

[0152] The difference from Example 15 is that the material-liquid ratio in step 2 is 1:10.

[0153] Comparative Example 15

[0154] The difference from Example 15 is that the material-liquid ratio in step 2 is 1:20.

[0155] The yield of crude peptide from Trichosanthes kirilowii was calculated for Examples 15-17 and Comparative Examples 14-15. Figure 4 As shown in D, it can be seen that the yield of Trichosanthes kirilowii crude peptide is higher when the material-liquid ratio is 1:30-50, among which 1:40 has the best effect.

[0156] Test Example 1 Evaluation of the efficacy of Trichosanthes polypeptide in promoting testosterone secretion

[0157] 1. Cell Culture

[0158] TM3 cells were cultured in DMEM medium enriched with 2.5% (v / v) fetal bovine serum (FBS) and 5% horse serum (HS), supplemented with 1% penicillin-streptomycin solution, and cultured at 37°C and 5% CO2 until they reached the logarithmic growth phase.

[0159] 2. Effects of ion exchange chromatography hydrolysates and Trichosanthes kirilowii polypeptides on TM3 cell survival

[0160] The survival rate of TM3 cells was determined using the CCK8 method. TM3 cell suspension in the logarithmic growth phase was plated in a 96-well plate, 100 μL per well, and the cell density in the well was approximately 10 5CFU / mL, in 5% CO2, 37 DEG C of incubators, cultivate 24h.After treating that cells adhere, carefully remove the supernatant in the hole, then add the DMEM culture fluid of 100uL, wherein sample group (TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, TSH1-5) is respectively containing 200μg / mL of TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4 and TSH1-5 (respectively made by embodiment 1, embodiment 2, comparative example 5-7), the DMEM culture fluid of control group and blank group does not contain above-mentioned polypeptide.Continue to put 96-well plate into incubator and cultivate 48h, then sample group and control group every hole add 10 μL CCK8 solution, in blank group, add equivalent deionized water, continue to incubate in incubator after 4 hours, be placed in microplate reader and measure the absorbance at 450nm, calculate cell viability. The formula for calculating cell viability is as follows:

[0161]

[0162] Among them, A0 is the absorbance value of the sample group, A1 is the absorbance value of the control group, and A2 is the absorbance value of the blank group.

[0163] Effects of TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, and TSH1-5 on the survival of TM3 cells Figure 5 As shown. Figure 5 It can be seen that each component has no significant effect on the cell survival rate of TM3 cells, indicating that they have no significant toxicity to TM3 cells.

[0164] 3. Effect of Trichosanthes polypeptide on testosterone secretion in TM3 cells

[0165] The testosterone secretion of TM3 cells was determined using an Elisa kit. TM3 cell suspension in the logarithmic growth phase was plated in a 96-well plate, 100 μL per well, and the cell density in the well was approximately 10 5CFU / mL, cultured in a 5% CO2, 37°C incubator for 24 h. After the cells adhered, the supernatant in the well was carefully removed, and then 100 uL of DMEM culture medium was added, wherein sample group 1 (TSH1, TSH2, TSH3, TSH4, TSH5) were DMEM culture medium containing 200 μg / mL of TSH1, TSH2, TSH3, TSH4 and TSH5 (prepared by Example 1 and Comparative Examples 1-4, respectively), sample group 2 (TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, TSH1-5) were DMEM culture medium containing 200 μg / mL of TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4 and TSH1-5 (prepared by Example 1, Example 2, Comparative Examples 5-7, respectively), and the DMEM culture medium of the control group (Control) did not contain the above polypeptide. After 48 hours of culture, the cell supernatant was collected and the testosterone content in the supernatant was measured using an Elisa kit. Figure 6-7 shown.

[0166] Figure 6 The effect of the ion exchange chromatography hydrolysate on testosterone secretion in TM3 cells is shown in FIG. Figure 6 It was found that all five components could enhance testosterone secretion in TM3 cells at a concentration of 200 μg / mL, among which TSH1 and TSH2 had comparable effects. Considering the yield, TSH1 component was selected for further screening.

[0167] Figure 7 The effect of the Trichosanthes kirilowii polypeptide on testosterone secretion in TM3 cells is described in the present invention; Figure 7 Results indicate that TSH1-2 and TSH1-3 significantly increase testosterone secretion in TM3 cells at a concentration of 200 μg / mL. However, TSH1-1, TSH1-4, and TSH1-5, all derived from TSH1, were less effective than TSH1 at 200 μg / mL. Within the same bar graph, different letters above any two columns indicate a significant difference (p < 0.05) between the mean values ​​of those two columns; otherwise, the difference is not significant. The TSH1-3 fractions exhibited the greatest effect in increasing testosterone secretion in TM3 cells, and therefore were selected for further investigation of their kidney-tonifying and yang-strengthening mechanisms.

[0168] 4. Effects of TSH1-3 and synthetic peptide VTPVGSPR on TM3 cell-related androgen secretion

[0169] Elisa kit was used to measure the secretion of androstenedione, testosterone, dihydrotestosterone and free testosterone by TM3 cells. 5The density of CFU / mL was evenly plated in a 96-well plate, 100 μL per well, and cultured in a 5% CO2, 37°C incubator for 24 hours. The culture medium was discarded, and then 100uL of DMEM culture medium was added. The sample groups (VTPVGSPR, 50, 100, 200) were DMEM culture medium containing 50 μg / mL, 100 μg / mL, 200 μg / mL of TSH1-3 and 200 μg / mL of synthetic peptide VTPVGSPR, respectively. The cell culture medium in the control group (Control) did not contain the above polypeptides. After continuing to culture in the incubator for 48 hours, the cell supernatant was collected and the content of androstenedione, testosterone, dihydrotestosterone and free testosterone in the supernatant was measured using an Elisa kit. The results are detailed in [ ]. Figure 8 .

[0170] Figure 8 The figure shows the effects of the Trichosanthes kirilowii polypeptides TSH1-3 and the synthetic peptide VTPVGSPR in the present invention on the secretion of androgen-related TM3 cells and the levels of ATP and mitochondrial potential membrane. As can be seen from the figure, both TSH1-3 and the synthetic peptide VTPVGSPR can promote the secretion of androstenedione, testosterone, dihydrotestosterone, and free testosterone, and the effect of TSH1-3 on their secretion is dose-dependent.

[0171] Testosterone is synthesized from androstenedione (ADS) under the action of 17β-hydroxysteroid dehydrogenase (17β-HSD). Dihydrotestosterone (DHT) is a potent metabolite of testosterone, which undergoes 5α reduction primarily in specific tissues such as the prostate, skin, and liver. This localized synthesis of DHT is crucial for the normal development of male characteristics during prenatal and puberty. According to the free hormone hypothesis (FHH), free testosterone can diffuse into cells and bind to androgen receptors. Testosterone bound to sex hormone-binding globulin (SHBG) cannot diffuse directly into tissues, so free testosterone levels better reflect its biological activity than total testosterone levels. Exploring the effects of Trichosanthes polypeptide on the secretion of the above substances will help us understand the mechanism and efficacy of Trichosanthes polypeptide in tonifying the kidney and strengthening yang.

[0172] 5. Effects of TSH1-3 and synthetic peptide VTPVGSPR on the expression of testosterone synthesis-related proteins and genes in TM3 cells

[0173] TM3 cells (5×10 5CFU) were incubated overnight in a twelve-well plate and then treated with different concentrations of TSH1-3 (50, 100, 200 μg / mL) and VTPVGSPR (200 μg / mL) at 37°C for 48 h. Total RNA was extracted from the cells using Trizol reagent, and a control group (Control) was set up, using the same volume of DMEM culture medium instead of polypeptide. The extracted RNA was reverse transcribed using an RNA PCR kit (CWBIO, China), and PCR reactions were performed. β-actin was used as a control gene, and 2 -△△Ct The relative mRNA expression level of the target gene was determined by the method. The relative expression of the target gene was calculated as the ratio of the experimental group to the control group (Control). The results are shown in Figure 2. Figure 9 shown.

[0174] TM3 cells (5×105 CFU / well) were incubated overnight in 12-well plates and then treated with various concentrations of TSH1-3 (50, 100, and 200 μg / mL) and VTPVGSPR (200 μg / mL) at 37°C for 48 hours. TM3 cells were then harvested, washed twice with PBS, and lysed with 200 μL of radioimmunoprecipitation assay (RIPA) buffer. The supernatant was centrifuged at 12,000 rpm for 15 minutes at 4°C to obtain the total protein extract. Protein concentration in the lysate was determined using a BCA protein assay kit. The total protein extract was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 12% SDS-PAGE gel and then transferred to a nitrocellulose (NC) membrane. The membrane was blocked with 5% nonfat dry milk in phosphate-buffered saline containing Tween 20 (PBST) for 1.5 hours at room temperature. The membrane was then incubated with the primary antibody overnight at 4°C. After washing three times with PBST, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1.5 h. Chemiluminescence signal detection was performed using enhanced chemiluminescence (ECL) detection reagent, and images were captured using a ChemiScope 6100 imaging system (Clinx, Shanghai, China). The bands were analyzed using image J software to obtain the relative expression levels of the relevant proteins. The results are shown in Figure 2. Figure 10 shown.

[0175] StAR, TSPO, CYP11A1 and 3β-HSD genes play an important role in the synthesis of testosterone. First, the steroidogenic acute regulatory protein (StAR) is activated, and then binds to the transport protein (TSPO) on the outer membrane of the mitochondria to accelerate the transport of cholesterol from the outer membrane to the inner membrane. Cholesterol, as the raw material for synthesizing testosterone, participates in the synthesis of testosterone in TM3 cells. Subsequently, cholesterol is converted into pregnenolone by cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1) on the inner membrane of the mitochondria. Pregnenolone is further transferred to the endoplasmic reticulum and converted into progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD). Progesterone is then catalyzed into androstenedione and ultimately converted into testosterone. By Figure 9 and Figure 10 The results showed that after intervention with TSH1-3 and the synthetic peptide VTPVGSPR, the expression levels of various proteins and related genes were significantly increased compared with the control group, and TSH1-3 promoted the expression of StAR, TSPO, CYP11A1 and 3β-HSD at the transcriptional and protein levels in a dose-dependent manner, indicating that TSH1-3 and the synthetic peptide VTPVGSPR can intervene in the testosterone synthesis pathway and promote the synthesis and secretion of testosterone.

[0176] 6. Effects of TSH1-3 and synthetic peptide VTPVGSPR on the expression of testosterone synthesis-related proteins and genes in TM3 cells

[0177] mtDNA was extracted from TM3 cells using the DNeasy Blood and Tissue Kit (Qiagen, Shanghai), and mtDNA copy number was quantified using specific TaqMan probes provided by Life Technologies. To assess mtDNA levels, probes targeting mitochondrial genes (ND1 and ND6) were used, and nuclear 18S was used as a normalization reference. The results are shown in Figure 11 .

[0178] TM3 cells (5×10 5Cells were incubated overnight in 12-well plates (370 μg / well) and then treated with various concentrations of TSH1-3 (50, 100, and 200 μg / mL) and VTPVGSPR (200 μg / mL) at 37°C for 48 hours. Cells were harvested and washed twice with PBS. TM3 cells were lysed with 200 μL of radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 12,000 rpm for 15 minutes at 4°C. The resulting supernatant was used as the total protein extract, and the protein concentration of the lysate was determined using a BCA protein assay kit. The total protein extract was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 12% SDS-PAGE gel and then transferred to a nitrocellulose (NC) membrane. The membrane was blocked with 5% nonfat dry milk in phosphate-buffered saline containing Tween 20 (PBST) for 1.5 hours at room temperature. Next, the membrane was incubated with the primary antibody overnight at 4°C. After washing three times with PBST, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1.5 hours at room temperature. Enhanced chemiluminescence (ECL) detection reagent was used for chemiluminescence signal detection, and images were captured using a ChemiScope 6100 imaging system (Clinx, Shanghai, China). The bands were analyzed using imageJ software to obtain the relative expression levels of the relevant proteins. The results are shown in Figure 12 .

[0179] TM3 cells (5×10 5 CFU / well) were incubated overnight in a 12-well plate and then treated with different concentrations of TSH1-3 (50, 100, 200 μg / mL) and VTPVGSPR (200 μg / mL) at 37°C for 48 h. Total RNA was extracted from the cells using 1 mL of Trizol reagent and reverse transcribed using an RNA PCR kit (CWBIO, China). Polymerase chain reaction was performed using specific primers. β-actin was used as a control gene, and 2 -△△Ct The relative mRNA expression level of the target gene was determined by the method, and the relative expression of the target gene was calculated as the ratio of the experimental group to the control group. Figure 13 .

[0180] Studies have shown that mitochondrial biogenesis is closely related to testosterone secretion, and inhibiting mitochondrial biogenesis will lead to a decrease in testosterone synthesis. Figure 11 As shown in Figure 2, the peptide components TSH1-3 from Trichosanthes kirilowii and the synthetic peptide VTPVGSPR can promote the expression of mitochondrial mtDNA. Figure 12 、 13As shown, after peptide treatment, the expression levels of genes and proteins related to mitochondrial biogenesis, PGC-1α, TFAM, TFB1M, TFB2M, NRF1, and NRF2, were significantly increased. Among them, PGC-1α can coordinate the expression of nuclear respiratory factors 1 and 2 (NRF1 and NRF2) and transcription factors A and B (TFAM, TFB1M, and TFB2M). TFAM, TFB1M, and TFB2M are proteins required for mitochondrial biogenesis. The experimental results show that Trichosanthes polypeptide promotes the secretion of the male hormone testosterone in TM3 cells, which may be achieved by regulating mitochondrial biogenesis.

[0181] In summary, the present invention uses mouse Leydig cells TM3 as a model to evaluate the efficacy of the Trichosanthes polypeptide provided by the present invention in promoting testosterone secretion. The results show that the Trichosanthes polypeptide provided by the present invention can significantly promote the secretion of testosterone in TM3 cells. Its mechanism of promoting testosterone secretion is related to regulating the expression of genes and proteins related to testosterone synthesis, and is also related to enhancing mitochondrial function and promoting the expression of genes and proteins related to mitochondrial biogenesis. Therefore, the Trichosanthes polypeptide provided by the present invention can promote testosterone secretion in TM3 cells, has a certain kidney-tonifying and aphrodisiac effect, and can be developed as a substitute for traditional aphrodisiac drugs.

[0182] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a Trichosanthes polypeptide, characterized in that: The preparation method specifically comprises the following steps: Step 1: Defatting and removing phenolic substances from the shelled and crushed Trichosanthes kirilowii seeds, and then drying and powdering to obtain Trichosanthes kirilowii seed powder; Step 2: extracting the Trichosanthes kirilowii seed powder by heating water to obtain a protein-denatured Trichosanthes kirilowii seed liquid; Step 3: performing a composite enzymatic hydrolysis on the Trichosanthes kirilowii seed liquid, inactivating the enzyme after the enzymatic hydrolysis, separating the solid and liquid, collecting the enzymatic hydrolyzate, performing reduced pressure distillation, and drying to obtain a Trichosanthes kirilowii crude peptide; the composite enzymatic hydrolysis adopts a neutral protease and an alkaline protease; the amount of the alkaline protease added accounts for 20%-40% of the mass of the Trichosanthes kirilowii seed powder; the amount of the neutral protease added accounts for 20%-40% of the mass of the Trichosanthes kirilowii seed powder; the pH of the composite enzymatic hydrolysis in step 3 is 7.5-8 or 9-9.5, the enzymatic hydrolysis temperature is 55-75° C., and the enzymatic hydrolysis time is 3-5 h; Step 4: dissolving the crude peptide of Trichosanthes kirilowii in water to prepare a crude peptide solution of Trichosanthes kirilowii, filtering, and subjecting the obtained filtrate to ion exchange resin chromatography, using double distilled water as eluent, collecting the eluate according to the peak time of the absorbance curve, and then concentrating and drying to obtain an ion exchange chromatography hydrolyzate; the ion exchange resin chromatography adopts a DEAE-52 cellulose anion exchange chromatography column; Step 5: dissolving the ion exchange chromatography hydrolyzate in water, separating and purifying it by reverse-phase high performance liquid chromatography, collecting the eluate with a peak time of 10.0-12.3 min or the eluate with a peak time of 21.0-21.4 min to obtain the Trichosanthes polypeptide; the chromatographic column in the reverse-phase high performance liquid chromatography is a Pursuit XRs C-18 chromatographic column, the mobile phase A is 0.1% trifluoroacetic acid-water solution, the mobile phase B is 0.1% trifluoroacetic acid-acetonitrile solution, and the gradient elution is performed; the gradient elution procedure is as follows: 0-3 min, mobile phase: 95% mobile phase A + 5% mobile phase B; 3-10 min, mobile phase: 80% mobile phase A + 20% mobile phase B; 10-20 min, mobile phase is 50% mobile phase A + 50% mobile phase B; 20-23 min, mobile phase: 20% mobile phase A + 80% mobile phase B.

2. The preparation method according to claim 1, characterized in that In the step 1, the solvent for defatting the Trichosanthes seeds is n-hexane, and the weight-to-volume ratio of the Trichosanthes seeds to the n-hexane is 1:3-6; the solvent used for removing phenolic substances is acetone, and the weight-to-volume ratio of the Trichosanthes seeds to the acetone is 1:3-6; and / or In the step 2, the water extraction is performed with heating, the mass ratio of the Trichosanthes kirilowii seed powder to water is 1:30-50, the temperature is 90-100° C., and the time is 3-10 h.

3. The preparation method according to claim 1, characterized in that The neutral protease in step 3 is a metalloprotease derived from Bacillus subtilis; and / or The alkaline protease is an endoprotease derived from Bacillus licheniformis.

4. The preparation method according to claim 1, characterized in that The pH of the composite enzymatic hydrolysis in step 3 is 9-9.5, the enzymatic hydrolysis temperature is 65° C., and the enzymatic hydrolysis time is 5 h.

5. The preparation method according to claim 1, characterized in that The eluent flow rate in step 4 is 1-2 mL / min.

6. The preparation method according to claim 5, characterized in that The eluent flow rate in step 4 is 1 mL / min, and the eluent with a peak time of 135-250 min is collected.

7. A Trichosanthes kirilowii polypeptide prepared by the preparation method according to any one of claims 1 to 6.

Images