Polypeptide of Trichosanthes cucumeroides having kidney-tonifying and yang-strengthening effects, method of preparation thereof, and use

Abstract

The present invention provides a trifoliate polypeptide having kidney-tonifying and stimulating effects, a method for preparing the same, and its use. [Solution] In the present invention, a Triticum vulgare polypeptide with kidney-tonifying and stimulating effects is obtained through processes such as pretreatment of Triticum vulgare raw materials, preparation of Triticum vulgare seed juice, preparation of Triticum vulgare crude peptides, and purification by ion exchange resin chromatography and high performance liquid chromatography. The Triticum vulgare polypeptide provided by the present invention can regulate the expression of testosterone secretion-related genes and proteins in TM3 cells, improve mitochondrial function, promote the expression of mitochondrial biogenesis-related genes and proteins, and significantly promote testosterone secretion in TM3 cells, thereby possessing the effect of kidney-tonifying and stimulating, and broadening the scope of use of Triticum vulgare.

Description

[Technical Field]

[0001] This invention belongs to the field of biotechnology and relates to Trichosanthes cucumeroides polypeptide, which has kidney-tonifying and yang-strengthening effects, and its use. [Background technology]

[0002] Testosterone is a vital hormone for men, playing a crucial role in regulating sexual function, reproduction, muscle movement, and various physiological activities. New research suggests that testosterone deficiency may be linked to conditions such as obesity, cardiovascular disease, and even depression. Testosterone deficiency can occur in men of any age. In adult men, this deficiency is often due to a natural decline in endogenous testosterone levels caused by aging and other modifiable factors. Currently, exogenous testosterone replacement therapy (TRT) still has certain side effects. Therefore, finding substances that can stimulate endogenous testosterone synthesis is extremely important.

[0003] Clinically, common Chinese herbal medicines used to tonify the kidneys and strengthen yang include seahorse, Epimedium, dodder, and Cistanche, but all have certain side effects, and it is difficult to guarantee their safety for the human body with long-term use. Trichosanthes cucumeroides, belonging to the Cucurbitaceae family, is a traditional Chinese oil crop that is widely cultivated and highly valued. Trichosanthes cucumeroides seeds are rich in protein (26.70%) and are very nutritious, containing abundant arginine, leucine, and glutamic acid, and their amino acid composition is close to the essential amino acid content recommended by the WHO. Currently, there is little research on the effects and active substances of Trichosanthes cucumeroides seeds, and further research and exploration of its applications are needed. [Overview of the Initiative]

[0004] To solve the above technical problems, the present invention provides a Trichosanthes cucumeroides polypeptide having kidney-tonifying and yang-enhancing effects, a method for preparing the same, and its use. The preparation method provided in the present invention makes it possible to obtain a Trichosanthes cucumeroides polypeptide having kidney-tonifying and yang-enhancing effects from Trichosanthes cucumeroides. The preparation process is simple, the polypeptide yield is high, the obtained Trichosanthes cucumeroides polypeptide is safe, its low molecular weight allows for good absorption and utilization in the body, it significantly increases testosterone secretion from TM3 cells, and it can be developed as a substitute for conventional yang-enhancing drugs.

[0005] In a first aspect, the present invention provides a method for preparing Trichosanthes cucumeroides polypeptide. The method comprises the following steps 1 to 5. Step 1: After removing the shells and crushing the seeds of the Trichosanthes cucumeroides, defatting and removing phenolic substances, the seeds are dried and powdered to obtain Trichosanthes cucumeroides seed powder. Step 2: The Trichosanthes cucumeroides seed powder is heated and extracted with water to obtain a Trichosanthes cucumeroides seed liquid after protein denaturation. Step 3: The Trichosanthes cucumeroides seed extract is enzymatically hydrolyzed with a complex enzyme. After the enzymatic hydrolysis is complete, the enzyme is inactivated, solid-liquid separation is performed, the hydrolyzed liquid is collected, dried, and crude Trichosanthes cucumeroides peptide is obtained. In the enzymatic hydrolysis using the complex enzyme, a neutral protease or alkaline protease is used. Step 4: Dissolve the crude peptide of Trichosanthes cucumeroides in water to prepare a crude peptide solution of Trichosanthes cucumeroides, filter it, and subject the resulting filtrate to ion exchange resin chromatography, using redistilled water as the eluent, collect the eluent corresponding to the peak according to the absorbance curve, and then dry it to obtain the ion exchange chromatography enzyme hydrolysate. In the ion exchange resin chromatography, a cellulose anion exchange chromatography column was used. Step 5: The ion-exchange chromatography enzyme hydrolysate is dissolved in water, separated and purified by reverse-phase high-performance liquid chromatography, and protein polypeptides capable of significantly promoting testosterone secretion from TM3 cells are collected to obtain Trichosanthes cucumeroides polypeptide. The column used in the reverse-phase high-performance liquid chromatography is a C18 column, mobile phase A is a 0.1%-0.2% trifluoroacetic acid aqueous solution, and mobile phase B is a 0.1%-0.2% trifluoroacetic acid-acetonitrile solution. Gradient elution is performed, and the gradient elution process is as follows: 0-3min, mobile phase: 95% mobile phase A + 5% mobile phase B; 3-10min, mobile phase: 80% mobile phase A + 20% mobile phase B; 10-20min, mobile phase: 50% mobile phase A + 50% mobile phase B; 20-23min, mobile phase: 20% mobile phase A + 80% mobile phase B That is the case.

[0006] The seeds of Trichosanthes cucumeroides are rich in protein. After the protein is enzymatically hydrolyzed, the mixed peptide of the hydrolyzed product contains many by-products, making it difficult to screen for the target polypeptide. In addition, Trichosanthes cucumeroides, as an oil crop, contains large amounts of oils and polyphenols. The oils exert a strong interference effect on the protein purification column, and the polyphenols bind to the protein, hindering protein acquisition and reducing the extraction yield. In the method for preparing Trichosanthes cucumeroides polypeptide provided in the present invention, Trichosanthes cucumeroides seeds are used as raw material, degreased to remove phenolic substances, the protein is denatured by high-temperature steaming, and then enzymatic hydrolysis and multi-stage separation and purification are performed. By controlling parameters such as the type, concentration, and flow rate of the eluent in each stage of the separation and purification process, Trichosanthes cucumeroides polypeptide with kidney-tonifying and yang-enhancing effects is finally prepared. In this method, the elution peak of the target polypeptide can be accurately determined by detecting the absorbance curve, and the target polypeptide can be repeatedly and stably collected by controlling the collection time of the eluent.

[0007] In Step 1, drying and pulverizing the seeds of the Japanese snake gourd helps in protein extraction. The dried Japanese snake gourd seeds should be ground to a particle size of ≤0.5 mm.

[0008] In step 3, solid-liquid separation can be performed by collecting the corresponding filtrate or supernatant (i.e., enzymatically hydrolyzed liquid) by filtration or centrifugation. The drying method in steps 3 and 4 may be direct drying, drying after concentration, or drying after vacuum distillation.

[0009] In step 4, using redistilled water as the eluent yields the highest yield of the enzymatically degraded product by ion exchange chromatography.

[0010] Significantly promoting testosterone secretion from TM3 cells in Step 5 means that the stimulating effect of the screened polypeptide on testosterone secretion from TM3 cells is statistically significant compared to the original testosterone secretion from TM3 cells.

[0011] Preferably, the degreasing solvent for the Trichosanthes cucumeroides seeds in step 1 is n-hexane, with a weight-to-volume ratio of 1:3-6 between the seeds and n-hexane, and the solvent used to remove phenolic substances is acetone, with a weight-to-volume ratio of 1:3-6 between the seeds and acetone.

[0012] Preferably, in the heated water extraction of step 2, the mass ratio of Trichosanthes cucumeroides seed powder to water is 1:30-50, the temperature is 90-100°C, and the time is 3-10 minutes.

[0013] High-temperature extraction at 90-100°C is beneficial for subsequent enzymatic decomposition.

[0014] Preferably, in step 3, the neutral protease is a metalloprotease derived from Bacillus subtilis.

[0015] Neutral protease derived from Bacillus subtilis is a metalloprotease, an endoprotease extracted after sufficient fermentation and culture of Bacillus subtilis. It is pure, natural, safe, non-toxic, has strong hydrolytic ability, and can break down high molecular weight proteins into products such as polypeptides and amino acids.

[0016] Preferably, the alkaline protease is an endoprotease derived from Bacillus richeniformis.

[0017] Alkaline proteases derived from Bacillus richeniformis are enzyme preparations obtained by thoroughly fermenting Bacillus richeniformis in liquid. They can rapidly decompose proteins and hydrolyze high molecular weight proteins into products such as free amino acids.

[0018] More preferably, the amount of alkaline protease added is 20%-40% of the mass of the Trichosanthes cucumeroides seed powder, and the amount of neutral protease added is 20%-40% of the mass of the Trichosanthes cucumeroides seed powder. In step 3, the pH of the complex enzyme decomposition is 7.5-8 or 9-9.5, the enzyme decomposition temperature is 55-75°C, and the enzyme decomposition time is 3-5 hours.

[0019] More preferably, the pH of the complex enzymatic degradation in step 3 is 9-9.5, the enzymatic degradation temperature is 65°C, and the enzymatic degradation time is 5h. This enzymatic degradation process yields the highest yield of Trichosanthes cucumeroides crude peptide.

[0020] Preferably, the flow rate of the eluent in step 4 is 1-2 mL / min, and a DEAE-52 cellulose anion exchange chromatography column is used in the ion exchange resin chromatography.

[0021] More preferably, the flow rate of the eluent in step 4 is 1 mL / min, and the eluent is collected with a retention time (time from sample injection until a peak appears) of 135-250 min.

[0022] At this flow rate, the retention time when the enzymatic hydrolysate is subjected to ion exchange chromatography is 135 - 250 min.

[0023] Preferably, the 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 flow rate of the mobile phase is 20 mL / min, and an eluent with a retention time of 10.0 - 12.3 min or an eluent with a retention time of 21.0 - 21.4 min is collected to obtain cucumis polypeptide.

[0024] Under such conditions, the cucumis polypeptide obtained from the eluent with a retention time of 10.0 - 12.3 min and the eluent with a retention time of 21.0 - 21.4 min both have a significant promoting effect on testosterone secretion from TM3 cells.

[0025] In a second aspect, the present invention provides a cucumis polypeptide prepared by the above method.

[0026] The cucumis polypeptide provided by the present invention is mainly composed of low molecular weight peptides, is safe for the human body, is easily absorbed, can promote testosterone secretion from TM3 cells and mitochondrial biosynthesis, and has the effect of tonifying the kidney and strengthening yang. The mechanism by which this cucumis polypeptide promotes testosterone secretion from TM3 cells is related to the regulation of the expression of genes and proteins related to testosterone secretion and the promotion of the expression of genes and proteins related to mitochondrial biosynthesis.

[0027] In a third aspect, the present invention provides the use of the above cucumis polypeptide. The use is Use in the preparation of a medicine for tonifying the kidney and strengthening yang, or Use in the preparation of a drug for promoting testosterone secretion from TM3 cells, or Use in the preparation of a drug for regulating the expression of genes and proteins related to testosterone secretion in TM3 cells, or Use in the preparation of drugs to regulate the expression of genes and proteins involved in mitochondrial biosynthesis in TM3 cells. Includes. [Brief explanation of the drawing]

[0028] To more clearly illustrate embodiments of the present invention or technical means of the prior art, the following drawings, which are necessary for describing the embodiments or the prior art, are briefly introduced. Clearly, the following drawings represent only a few embodiments of the present invention. Those skilled in the art can obtain other drawings based on these without any creative effort. [Figure 1] The absorbance curves of ion-exchange chromatography enzyme decomposition products obtained by elution using different eluents in this invention are shown. [Figure 2] The absorbance curve of the Trichosanthes cucumeroides polypeptide obtained by purification using reverse-phase high-performance liquid chromatography is shown. [Figure 3] The chromatogram of the total ion flow of the Trichosanthes cucumeroides polypeptide TSH1-3 obtained in Example 2 of the present invention is shown. [Figure 4] The results of screening the crude peptide of Trichosanthes cucumeroides in Example 1 of the present invention by a water extraction process and an enzymatic degradation process are shown. [Figure 5] This paper demonstrates the effect of the Trichosanthes cucumeroides polypeptide according to the present invention on TM3 cell viability. [Figure 6] This invention demonstrates the effect of ion-exchange chromatography enzyme degradation products on TM3 cell testosterone secretion. [Figure 7] This invention demonstrates the effect of the Trichosanthes cucumeroides polypeptide on TM3 cell testosterone secretion. [Figure 8] This paper demonstrates the effects of the Trichosanthes cucumeroides polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on TM3 cell-associated androgen secretion, ATP, and mitochondrial membrane potential levels. [Figure 9]This paper demonstrates the effects of the Trichosanthes cucumeroides polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on the expression of testosterone synthesis-related genes. [Figure 10] This paper demonstrates the effects of the Trichosanthes cucumeroides polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on the expression of testosterone synthesis-related proteins. [Figure 11] This paper demonstrates the effects of the Trichosanthes cucumeroides polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on mitochondrial mtDNA replication. [Figure 12] This paper demonstrates the effects of the Trichosanthes cucumeroides polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on the expression of mitochondrial biosynthesis-related proteins. [Figure 13] This paper demonstrates the effects of the Trichosanthes cucumeroides polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on the expression of mitochondrial biosynthesis-related genes. [Modes for carrying out the invention]

[0029] The technical means in embodiments of the present invention will be clearly and completely described below with reference to embodiments of the present invention. Clearly, the following embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments that a person skilled in the art could obtain without creative effort based on embodiments of the present invention are all within the scope of protection of the present invention.

[0030] (1) Reagents n-Hexane: Mclean H810749 Acetone: Guangzhou Chemical Reagent Plant HB02 Trifluoroacetic acid (TFA): Mclean T818778 Acetonitrile: Mclean A800362 Ion exchange resin: DEAE-52, Solarbio C8930 Penicillin-streptomycin solution (100X): Beyotime C0222 CCK8 detection reagent: Abbkine KTA1020 BCA Protein Quantification Kit: Abiowell AWB0104 RIPA solution (strong): Abiowell AWB0136 Trypsin: Gibco 25200056 ECL Plus Ultra-High Sensitivity Luminescent Solution: Abiowell AWB0005 Alkaline protease: 2.4L, Novozymes Neutral protease Nuetrase 0.8L, Novozymes Mouse Testosterone (T) ELISA Scientific Research Kit: MM-0569M1, Jiangsu Baimen Industrial Co., Ltd. Superoxide dismutase (SOD) kit: G0101W, Jiangsu Geruisi Biotechnology Co., Ltd. Malondialdehyde (MDA) Kit: G0109W, Jiangsu Geruisi Biotechnology Co., Ltd.

[0031] (2) Equipment The Pursuit XRs C-18 column for the reverse-phase high-performance liquid chromatography system was purchased from Agilent Technologies, Inc. in the United States.

[0032] Example 1 This embodiment provides a polypeptide of Trichosanthes cucumeroides. The specific preparation method is as follows: Step 1: The hulls were removed from the seeds of Trichosanthes cucumeroides and they were crushed. Degreasing was performed by adding n-hexane in a solid-liquid ratio of 1:5 (g:mL), then acetone in a solid-liquid ratio of 1:5 (g:mL) was added to remove phenolic substances, and after air drying, the mixture was sieved through an 80-mesh sieve to obtain Trichosanthes cucumeroides seed powder. Step 2: Take 10 g of Trichosanthes cucumeroides seed powder, add purified water in a solid-liquid ratio of 1:40 (g:mL), mix, heat in a 95°C water bath, and maintain the temperature for 5 hours to obtain Trichosanthes cucumeroides seed liquid. Step 3: The Trichosanthes cucumeroides seed extract was cooled to 65°C, 2 mL of alkaline protease and 2 mL of neutral protease were added, and enzymatic digestion was carried out at pH 9 and 65°C for 5 hours. After the enzymatic digestion was complete, the enzyme was inactivated at 95°C for 15 minutes, then the solution was collected by centrifugation, distilled under reduced pressure, and freeze-dried to obtain Trichosanthes cucumeroides crude peptide. Step 4: The crude peptide of Trichosanthes cucumeroides was dissolved, diluted to 20 mg / ml, filtered through a 0.22 μm filtration membrane, and then injected into a DEAE-52 cellulose anion exchange chromatography column using a pressure pump. Elution was performed with redistilled water (flow rate 1 mL / min). The absorbance curve of the eluent at 220 nm is shown in Figure 1. Components were collected during elution times of 135-250 min, concentrated under reduced pressure, and freeze-dried under vacuum to obtain the ion exchange chromatography enzyme hydrolysate TSH1. Step 5: TSH1 was dissolved in redistilled water to a concentration of 5 mg / mL, filtered through a 0.22 μm filtration membrane, and separated and purified by reversed-phase high-performance liquid chromatography. The column used was Pursuit XRs C-18, the column temperature was 35°C, the injection volume was 600 μL, mobile phase A was 0.1% trifluoroacetic acid-water, mobile phase B was 0.1% trifluoroacetic acid-acetonitrile, and the mobile phase flow rate was 20 mL / min. The gradient elution sequence is shown in the table below.

[0033] Table 1: Sequence of mobile phase linear gradient elution JPEG0007885987000001.jpg38170

[0034] Figure 2 shows the absorbance curves at 220 nm of the eluents at various time points. Eluents with elution times of 21.0–21.4 min were collected, concentrated under reduced pressure, and freeze-dried under vacuum to obtain Trichosanthes cucumeroides polypeptide TSH1-3.

[0035] Using LC-MS / MS and a high-resolution mass spectrometry database set, peptide fragments were identified for TSH1-3 components, and their amino acid sequences were analyzed. As a result, 42 peptide fragments with relatively high reliability were obtained. The total ion flow is shown in Figure 3. The amino acid sequences of the 42 peptide fragments are shown in Table 2.

[0036] Table 2: Components of TSH1-3 peptide fragments JPEG0007885987000002.jpg122170JPEG0007885987000003.jpg109170

[0037] Comparative Example 1-4 Comparative Examples 1-4 provide four types of ion-exchange chromatography enzyme degradation products: TSH2, TSH3, TSH4, and TSH5, respectively.

[0038] The crude peptide of Trichosanthes cucumeroides prepared in step 3 of Example 1 was taken and subjected to ion exchange resin chromatography according to step 4 of Example 1. The difference from step 4 of Example 1 was that instead of redistilled water, elution was performed with sodium chloride at a concentration of 0.01 mol / L, components were collected during the elution time of 168-264 min, and after vacuum concentration and vacuum freeze-drying, the ion exchange chromatography enzyme hydrolysate TSH2 was obtained.

[0039] The crude peptide of Trichosanthes cucumeroides prepared in step 3 of Example 1 was taken and subjected to ion exchange resin chromatography according to step 4 of Example 1. The difference from step 4 of Example 1 was that instead of redistilled water, elution was performed with sodium chloride at a concentration of 0.05 mol / L, components were collected during the elution time of 116-292 min, and after vacuum concentration and vacuum freeze-drying, the ion exchange chromatography enzyme hydrolysate TSH3 was obtained.

[0040] The crude peptide of Trichosanthes cucumeroides prepared in step 3 of Example 1 was taken and subjected to ion exchange resin chromatography according to step 4 of Example 1. The difference from step 4 of Example 1 was that instead of redistilled water, elution was performed with sodium chloride at a concentration of 0.1 mol / L, components were collected during elution times of 116-212 min, and after vacuum concentration and vacuum freeze-drying, the ion exchange chromatography enzyme hydrolysate TSH4 was obtained. Components were collected during elution times of 308-368 min, and after vacuum concentration and vacuum freeze-drying, the ion exchange chromatography enzyme hydrolysate TSH5 was obtained.

[0041] The absorbance curves of the ion-exchange chromatography-enzymatic decomposition products of Comparative Examples 1-4 are shown in Figure 1.

[0042] Example 2 Example 2 provides the Trichosanthes cucumeroides polypeptide TSH1-2.

[0043] The exchange chromatography enzyme hydrolysate TSH1 prepared in step 4 of Example 1 was taken and separated and purified under the reverse-phase high-performance liquid chromatography conditions of step 5 of Example 1. The difference from step 5 of Example 1 is that the collection time for the TSH1-2 eluent was 10.0-12.3 min.

[0044] The absorbance curve of the Trichosanthes cucumeroides polypeptide from Example 2 is shown in Figure 2. Comparative Example 5-7

[0045] Comparative Examples 5-7 provide Trichosanthes cucumeroides polypeptides TSH1-1, TSH1-4, and TSH1-5, respectively.

[0046] The ion-exchange chromatography enzyme hydrolysate TSH1 prepared in step 4 of Example 1 was taken and separated and purified under the reverse-phase high-performance liquid chromatography conditions of step 5 of Example 1. The difference from step 5 of Example 1 is that the collection times for the eluents 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.

[0047] The absorbance curves of the Trichosanthes cucumeroides polypeptides for Comparative Examples 5-7 are shown in Figure 2.

[0048] Example 3 Example 3 provides Trichosanthes cucumeroides polypeptide.

[0049] Step 1: The hulls were removed from the seeds of Trichosanthes cucumeroides and they were crushed. Degreasing was performed by adding n-hexane in a solid-liquid ratio of 1:6 (g:mL), then acetone in a solid-liquid ratio of 1:6 (g:mL) was added to remove phenolic substances, and after air drying, the mixture was sieved through an 80-mesh sieve to obtain Trichosanthes cucumeroides seed powder.

[0050] Step 2: Take 10 g of Trichosanthes cucumeroides seed powder, add purified water in a solid-liquid ratio of 1:5 (g:mL), mix, heat in a 100°C water bath, and maintain the temperature for 10 hours to obtain Trichosanthes cucumeroides seed liquid.

[0051] Step 3: The Trichosanthes cucumeroides seed extract was cooled to 50°C, 4 mL of alkaline protease and 4 mL of neutral protease were added, and the mixture was enzymatically digested at pH 7.5 and 55°C for 5 hours. After the enzymatic digestion was complete, the enzyme was inactivated at 95°C for 15 minutes, then the mixture was centrifuged, the solution was collected, and the mixture was freeze-dried by vacuum distillation to obtain the Trichosanthes cucumeroides crude peptide.

[0052] The other steps were the same as in Example 1, and a polypodium trichotomum polypeptide was obtained.

[0053] Example 4 Example 4 provides Trichosanthes cucumeroides polypeptide.

[0054] Step 1: The hulls were removed from the seeds of Trichosanthes cucumeroides and they were crushed. Degreasing was performed by adding n-hexane in a solid-liquid ratio of 1:3 (g:mL), then acetone in a solid-liquid ratio of 1:3 (g:mL) was added to remove phenolic substances, and after air drying, the mixture was sieved through an 80-mesh sieve to obtain Trichosanthes cucumeroides seed powder.

[0055] Step 2: Take 10 g of Trichosanthes cucumeroides seed powder, add purified water in a solid-liquid ratio of 1:50 (g:mL), mix, heat in a 90°C water bath, and maintain the temperature for 3 hours to obtain Trichosanthes cucumeroides seed liquid.

[0056] Step 3: The Trichosanthes cucumeroides seed extract was cooled to 45°C, 3 mL of alkaline protease and 3 mL of neutral protease were added, and enzymatic digestion was carried out at pH 9.5 and 75°C for 3 hours. After the enzymatic digestion was complete, the enzyme was inactivated at 95°C for 15 minutes, then the solution was collected by centrifugation, and the solution was collected by vacuum distillation and freeze-dried to obtain Trichosanthes cucumeroides crude peptide.

[0057] The other steps were the same as in Example 1, and a polypodium trichotomum polypeptide was obtained.

[0058] Example 5 This example provides a crude peptide from Trichosanthes cucumeroides.

[0059] Step 1: The hulls were removed from the seeds of Trichosanthes cucumeroides and they were crushed. Degreasing was performed by adding n-hexane in a solid-liquid ratio of 1:5 (g:mL), then acetone in a solid-liquid ratio of 1:5 (g:mL) was added to remove phenolic substances, and after air drying, the mixture was sieved through an 80-mesh sieve to obtain Trichosanthes cucumeroides seed powder.

[0060] Step 2: Take 10 g of Trichosanthes cucumeroides seed powder, add purified water in a solid-liquid ratio of 1:40 (g:mL), mix, heat in a 95°C water bath, and maintain the temperature for 5 hours to obtain Trichosanthes cucumeroides seed liquid.

[0061] Step 3: The seed extract of Trichosanthes cucumeroides was cooled to the enzymatic digestion temperature, 2 mL of alkaline protease and 2 mL of neutral protease were added, and enzymatic digestion was carried out at pH 9 for 4 hours. The enzymatic digestion temperature was 75°C. After the completion of enzymatic digestion, the enzyme was inactivated at 95°C for 15 minutes, then centrifuged, the solution was collected, and the solution was obtained by vacuum distillation and freeze-drying to obtain crude Trichosanthes cucumeroides peptide.

[0062] Example 6 The procedure is the same as in Example 5, except that the enzymatic decomposition temperature in Step 3 is 65°C.

[0063] Example 7 The procedure is the same as in Example 5, except that the enzymatic decomposition temperature in Step 3 is 55°C.

[0064] Comparative Example 8 The procedure is the same as in Example 5, except that the enzymatic decomposition temperature in Step 3 is 45°C.

[0065] Yield of Trichosanthes cucumeroides crude peptide at various enzymatic degradation temperatures Yield calculation formula:

number

[0066] The results for Examples 5-7 and Comparative Example 8 are shown in Figure 4A. As can be seen from Figure 4A, the enzymatic degradation temperature has a significant effect on polypeptide yield, with relatively high polypeptide yields at enzymatic degradation temperatures of 55-75°C, and the most effective temperature being 65°C.

[0067] Example 8 This example provides a crude peptide from Trichosanthes cucumeroides.

[0068] Prepared in Step 2 of Example 5 The seed extract of Trichosanthes cucumeroides was taken, cooled to 65°C, and 2 mL of alkaline protease and 2 mL of neutral protease were added. Enzymatic digestion was carried out at pH 9 and 65°C for 3 hours. After the enzymatic digestion was complete, the enzyme was inactivated at 95°C for 15 minutes, then the solution was collected by centrifugation, distilled under reduced pressure, and freeze-dried to obtain crude Trichosanthes cucumeroides peptide.

[0069] Example 9 Step 3 is the same as in Example 8, except that the enzymatic decomposition time is 4 hours.

[0070] Example 10 Step 3 is the same as in Example 8, except that the enzymatic decomposition time is 5 hours.

[0071] Comparative Example 9 The procedure is the same as in Example 8, except that the enzymatic decomposition time in Step 3 is 6 hours.

[0072] Comparative Example 10 The procedure is the same as in Example 8, except that the enzymatic decomposition time in Step 3 is 7 hours.

[0073] Comparative Example 11 Step 3 is the same as in Example 8, except that the enzymatic decomposition time is 8 hours.

[0074] The yields of the crude peptide from Trichosanthes cucumeroides in Examples 8-10 and Comparative Examples 9-11 were calculated. The results are shown in Figure 4B. As can be seen from Figure 4B, the yield of the crude peptide from Trichosanthes cucumeroides was relatively high at enzymatic degradation times of 3-5 hours, and the effect was highest at an enzymatic degradation time of 5 hours.

[0075] Example 11 This example provides a crude peptide from Trichosanthes cucumeroides.

[0076] The seed extract of Trichosanthes cucumeroides prepared in Step 2 of Example 5 was taken, cooled to 65°C, and 2 mL of alkaline protease and 2 mL of neutral protease were added. Enzymatic digestion was carried out at pH 7.5 and 65°C for 5 hours. After the enzymatic digestion was completed, the enzyme was inactivated at 95°C for 15 minutes, then the solution was collected by centrifugation, distilled under reduced pressure, and freeze-dried to obtain crude Trichosanthes cucumeroides peptide.

[0077] Example 12 Step 3 is the same as in Example 11, except that the enzymatic decomposition pH is 8.

[0078] Example 13 Step 3 is the same as in Example 11, except that the enzymatic decomposition pH is 9.

[0079] Example 14 The procedure is the same as in Example 11, except that the enzymatic decomposition pH in Step 3 is 9.5.

[0080] Comparative Example 12 The procedure is the same as in Example 11, except that the enzymatic decomposition pH in Step 3 is 8.5.

[0081] Comparative Example 13 The procedure is the same as in Example 11, except that the enzymatic decomposition pH in Step 3 is 10.

[0082] The yield of the crude peptide from Trichosanthes cucumeroides was calculated for Examples 11-14 and Comparative Examples 12-13. The results are shown in Figure 4C. As can be seen from Figure 4C, the yield of the crude peptide from Trichosanthes cucumeroides was relatively high when the enzymatic degradation pH was 7.5-8 or pH 9-9.5, and the effect was highest at pH=9.

[0083] Example 15 This example provides a crude peptide from Trichosanthes cucumeroides.

[0084] Step 1: The hulls were removed from the seeds of Trichosanthes cucumeroides and they were crushed. Degreasing was performed by adding n-hexane in a solid-liquid ratio of 1:5 (g:mL), then acetone in a solid-liquid ratio of 1:5 (g:mL) was added to remove phenolic substances, and after air drying, the mixture was sieved through an 80-mesh sieve to obtain Trichosanthes cucumeroides seed powder.

[0085] Step 2: Take 10 g of Trichosanthes cucumeroides seed powder, add purified water in a solid-liquid ratio of 1:30 (g:mL), mix, heat in a 95°C water bath, and maintain the temperature for 5 hours to obtain Trichosanthes cucumeroides seed liquid.

[0086] Step 3: The seed extracts of Trichosanthes cucumeroides prepared with different solid-liquid ratios were cooled to 65°C, 2 mL of alkaline protease and 2 mL of neutral protease were added, and enzymatic digestion was carried out at pH 9 and 65°C for 5 hours. After the enzymatic digestion was complete, the enzymes were inactivated at 95°C for 15 minutes, then the solution was collected by centrifugation, distilled under reduced pressure, and freeze-dried to obtain crude Trichosanthes cucumeroides peptide.

[0087] Example 16 Step 2 is the same as in Example 15, except that the solid-liquid ratio is 1:40.

[0088] Example 17 Step 2 is the same as in Example 15, except that the solid-liquid ratio is 1:50.

[0089] Comparative Example 14 Step 2 is the same as in Example 15, except that the solid-liquid ratio is 1:10.

[0090] Comparative Example 15 Step 2 is the same as in Example 15, except that the solid-liquid ratio is 1:20.

[0091] The yields of the crude peptide from Trichosanthes cucumeroides were calculated for Examples 15-17 and Comparative Examples 14-15. The results are shown in Figure 4D. As can be seen from Figure 4D, the yield of the crude peptide from Trichosanthes cucumeroides was relatively high when the solid-liquid ratio was 1:30-50, and the highest efficiency was observed at 1:40.

[0092] Test Example 1: Evaluation of the testosterone secretion-promoting effect of Trichosanthes cucumeroides polypeptide. 1.Cell culture TM3 cells were cultured in DMEM medium containing large amounts of 2.5% (v / v) fetal bovine serum (FBS) and 5% equine serum (HS), and then cultured in a 1% penicillin-streptomycin solution at 37°C and 5% CO2 until the logarithmic growth phase.

[0093] 2. Effects of ion-exchange chromatography enzyme hydrolysates and Trichosanthes cucumeroides polypeptide on TM3 cell viability The viability of TM3 cells was measured using the CCK8 method. A suspension of TM3 cells in the logarithmic growth phase was seeded at 100 μL / well into a 96-well plate, and the cell density in each well was approximately 10 5The cultures were CFU / mL and incubated for 24 hours in a 5% CO2, 37°C incubator. After the cells adhered to the wall, the supernatant in the well was carefully removed, and then 100 μL of DMEM culture medium was added. Here, the sample groups (TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, TSH1-5) were DMEM culture mediums containing 200 μg / mL of TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, and TSH1-5 (prepared in Example 1, Example 2, and Comparative Examples 5-7, respectively). The DMEM culture mediums of the control and blank groups did not contain the above polypeptides. Next, 96-well plates were placed in an incubator and incubated for 48 hours. Then, 10 μL of CCK8 solution was added to each well of the sample and control groups, and the same amount of deionized water was added to the blank group. After incubation for another 4 hours in the incubator, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The formula for calculating cell viability is as follows:

number

[0094] Figure 5 shows the effects of TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, and TSH1-5 on TM3 cell viability. As can be seen from Figure 5, each component does not have a significant effect on the cell viability of TM3 cells and does not exhibit significant toxicity to TM3 cells.

[0095] 3. Effects of Trichosanthes cucumeroides polypeptide on TM3 cell testosterone secretion Testosterone secretion from TM3 cells was measured using the Elisa kit. A suspension of TM3 cells in the logarithmic growth phase was seeded at 100 μL / well into a 96-well plate, with a cell density of approximately 10 in each well. 5The cells were cultured for 24 hours in an incubator at 5% CO2 and 37°C after the cells had adhered to the wall. After the cells had adhered to the wall, the supernatant in the well was carefully removed, and then 100 μL of DMEM culture medium was added. Here, sample group 1 (TSH1, TSH2, TSH3, TSH4, TSH5) was a DMEM culture medium containing 200 μg / mL of TSH1, TSH2, TSH3, TSH4, and TSH5 (prepared in Example 1 and Comparative Examples 1-4, respectively), sample group 2 (TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, TSH1-5) was a DMEM culture medium containing 200 μg / mL of TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, and TSH1-5 (prepared in Example 1, Example 2, and Comparative Examples 5-7, respectively), and the control group's DMEM culture medium did not contain the above polypeptides. After subsequent incubation for 48 hours, the cell supernatant was collected and the amount of testosterone in the supernatant was measured using the Elisa kit. The results are shown in Figure 6-7.

[0096] Figure 6 shows the effect of the ion-exchange chromatography enzyme hydrolysates according to the present invention on TM3 cell testosterone secretion. As can be seen from Figure 6, all five components can increase TM3 cell testosterone secretion at a concentration of 200 μg / mL. Here, the effects of TSH1 and TSH2 were equivalent, and TSH1 was selected for further screening along with the yield situation.

[0097] Figure 7 shows the effect of the Trichosanthes cucumeroides polypeptide according to the present invention on TM3 cell testosterone secretion. As can be seen from Figure 7, TSH1-2 and TSH1-3 significantly increased testosterone secretion from TM3 cells at a concentration of 200 μg / mL, whereas TSH1-1, TSH1-4, and TSH1-5, also derived from TSH1, had a lower promoting effect on TM3 cell testosterone secretion at a concentration of 200 μg / mL than TSH1. In the same bar graph, if the letters shown above any two columns are different, it indicates a significant difference between the mean values ​​of those two columns (p<0.05), otherwise it indicates no significant difference. Since the TSH1-3 component had the highest effect on increasing testosterone secretion from TM3 cells, we selected the TSH1-3 component and further studied its mechanism of kidney-tonifying and yang-strengthening effects.

[0098] 4. Effects of TSH1-3 and synthetic peptide fragment VTPVGSPR on TM3 cell-associated androgen secretion Androstenedione, testosterone, dihydrotestosterone, and free testosterone secreted from TM3 cells were measured using the Elisa kit. 10 TM3 cells in the logarithmic growth phase were used. 5 Cells were uniformly seeded in 96-well plates at a density of CFU / mL (100 μL / well) and cultured for 24 hours in a 5% CO2, 37°C incubator. After removing the culture medium, 100 μL of DMEM culture medium was added. The sample groups (VTPVGSPR, 50, 100, 200) were DMEM culture mediums containing 50 μg / mL, 100 μg / mL, and 200 μg / mL of TSH1-3 and 200 μg / mL of the synthetic peptide fragment VTPVGSPR, respectively. The control group's cell medium did not contain the above polypeptides. After subsequent 48 hours of incubation in the incubator, the cell supernatant was collected, and the content of androstenedione, testosterone, dihydrotestosterone, and free testosterone in the supernatant was measured using the Elisa kit. The results are shown in Figure 8.

[0099] Figure 8 shows the effects of the Trichosanthes cucumeroides polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on TM3 cell-associated androgen secretion, ATP, and mitochondrial membrane potential levels. As can be seen from Figure 8, both TSH1-3 and the synthetic peptide fragment VTPVGSPR can promote the secretion of androstenedione, testosterone, dihydrotestosterone, and free testosterone, and of these, TSH1-3 showed a dose-dependent effect on their secretion.

[0100] Testosterone is synthesized by androstenedione (ADS) under the action of 17β-hydroxysteroid dehydrogenase (17β-HSD). Dihydrotestosterone (DHT) is a potent metabolite of testosterone, primarily reduced to 5α in certain tissues such as the prostate, skin, and liver. This local synthesis process of DHT is crucial for the normal development of male characteristics during prenatal and adolescent stages. Based on the free hormone hypothesis (FHH), free testosterone can diffuse into cells and bind to androgen receptors, whereas testosterone bound to sex hormone-binding globulin (SHBG) cannot directly diffuse into tissues. Therefore, the level of free testosterone can better reflect biological activity compared to the total testosterone level. Investigating the effects of Trichosanthes cucumeroides polypeptide on the secretion of the above substances will help to understand the mechanism and effects of Trichosanthes cucumeroides polypeptide in tonifying the kidneys and strengthening yang.

[0101] 5. Effects of TSH1-3 and synthetic peptide fragment VTPVGSPR on the expression of TM3 cell testosterone synthesis-related proteins and genes. TM3 cells (5 x 10 5CFUs were incubated overnight in 12-well plates, then treated with various concentrations of TSH1-3 (50, 100, 200 μg / mL) and VTPVGSPR (200 μg / mL) at 37°C for 48 hours. Total RNA was then extracted from the cells using Trizol reagent to establish a control group, using the same volume of DMEM culture medium instead of polypeptides. The extracted RNA was reverse transcribed and PCR was performed using an RNA PCR kit (CWBIO, China), with β-actin used as the control gene. -△△Ct The relative mRNA expression level of the target gene was determined by a specific method. The relative expression level of the target gene was calculated as the ratio of the experimental group to the control group. The results are shown in Figure 9.

[0102] TM3 cells (5 x 10 5 After incubation of CFU / well overnight in a 12-well plate, TM3 cells were treated with various concentrations of TSH1-3 (50, 100, 200 μg / mL) and VTPVGSPR (200 μg / mL) at 37°C for 48 hours. TM3 cells were then collected, washed twice with PBS, and subsequently degraded in 200 μL of radioactive immunoprecipitation (RIPA) buffer. The cells were then centrifuged at 4°C and 12000 rpm for 15 minutes. The resulting supernatant was the total protein extract, and the protein concentration of the degraded solution was measured using a BCA protein assay kit. The total protein extract was separated by sodium lauryl sulfate polyacrylamide gel electrophoresis on a 12% SDS-PAGE gel and then transferred to a nitrocellulose (NC) membrane. The membrane was blocked for 1.5 hours at room temperature with 5% skim milk powder in phosphate-buffered saline (PBST) containing Tween 20. The membrane was then incubated overnight with primary antibody at 4°C. After washing three times with PBST, the membranes were incubated with two horseradish peroxidase (HRP)-labeled reagents for 1.5 hours at room temperature. Chemiluminescence signals were detected using enhanced chemiluminescence (ECL) detection reagents, images were acquired using a ChemiScope 6100 imaging system (Clinx, Shanghai, China), and the relative expression levels of the relevant proteins were obtained by analyzing the bands using imageJ software. The results are shown in Figure 10.

[0103] The StAR, TSPO, CYP11A1, and 3β-HSD genes play crucial roles in testosterone synthesis. First, the steroid-producing acute regulatory protein (StAR) is activated and then binds to the transporter protein (TSPO) on the outer mitochondrial membrane, facilitating the transport of cholesterol from the outer to the inner membrane. Cholesterol is involved in the synthesis of TM3 cells as a raw material for testosterone. Next, cholesterol is converted to pregnenolone on the inner mitochondrial membrane by the cell pigment P450 cholesterol side-chain cleavage enzyme (CYP11A1), and pregnenolone is further transferred to the endoplasmic reticulum and converted to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD). Finally, progesterone is catalyzed to androstenedione and ultimately converted to testosterone. As can be seen from the results in Figures 9 and 10, after intervention with TSH1-3 and the peptide fragment VTPVGSPR, the expression levels of each protein and the expression levels of related genes increased significantly compared to the control group. Furthermore, TSH1-3 dose-dependently promoted the transcriptional and protein-level expression of StAR, TSPO, CYP11A1, and 3β-HSD, indicating that TSH1-3 and the synthetic peptide fragment VTPVGSPR can be involved in the testosterone synthesis pathway and promote testosterone synthesis and secretion.

[0104] 6. Effects of TSH1-3 and synthetic peptide fragment VTPVGSPR on the expression of TM3 cell testosterone synthesis-related proteins and genes mtDNA was extracted from TM3 cells using the DNeasy blood and tissue kit (Qiagen, Shanghai), and the mtDNA copy number was quantified using a specific TaqMan probe from Life Technologies. To evaluate mtDNA levels, probes targeting mitochondrial genes (ND1 and ND6) were used, with nuclear 18S as the normalized reference. The results are shown in Figure 11.

[0105] TM3 cells (5 x 10 5(CFU / well) were incubated overnight in a 12-well plate and then treated with various concentrations of TSH1-3 (50, 100, 200 μg / mL) and VTPVGSPR (200 μg / mL) at 37 °C for 48 h. The cells were collected, washed twice with PBS, and then TM3 cells were lysed with 200 μL of radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 12,000 rpm for 15 min at 4 °C. The resulting supernatant was the total protein extract, and the protein concentration of the lysate was measured using a BCA protein assay kit. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separation was performed on a 12% SDS-PAGE gel for the total protein extract, and then transferred to a nitrocellulose (NC) membrane. The membrane was blocked with 5% non-fat dry milk in phosphate-buffered saline containing Tween 20 (PBST) at room temperature for 1.5 h. Next, the membrane was 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 signals were detected using an enhanced chemiluminescence (ECL) detection reagent, images were captured using a ChemiScope 6100 imaging system (Clinx, Shanghai, China), and bands were analyzed using image J software to analyze the relative expression levels of related proteins. The results are shown in Figure 12.

[0106] TM3 cells (5×10 5 (CFU / well) were incubated overnight on a 12-well plate and then treated with various 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, reverse transcribed using an RNA PCR kit (CWBIO, China), and then polymerase chain reaction was performed using specific primers. β-actin was used as a control gene, and the relative mRNA expression level of the target gene was determined by the 2 -△△Ct -method, and the relative expression level of the target gene was calculated as the ratio of the experimental group to the control group. The results are shown in Figure 13.

[0107] Studies have shown that mitochondrial biosynthesis is closely related to testosterone secretion, and inhibiting mitochondrial biosynthesis may lead to a decrease in testosterone synthesis. As shown in Figure 11, the Trichosanthes cucumeroides polypeptide component TSH1-3 and the synthetic peptide fragment VTPVGSPR can promote mitochondrial mtDNA expression. As shown in Figures 12 and 13, after treatment with the polypeptide, the expression levels of genes and proteins related to mitochondrial biosynthesis, PGC-1α, TFAM, TFB1M, TFB2M, NRF1, and NRF2, were significantly increased. Here, PGC-1α can modulate 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 necessary for mitochondrial biosynthesis. As can be seen from the experimental results, the promotion of androgen testosterone secretion by Trichosanthes cucumeroides polypeptide in TM3 cells may be achieved by modulating mitochondrial biosynthesis.

[0108] Based on the above, the present invention evaluated the testosterone secretion-promoting effect of the Trichosanthes cucumeroides polypeptide provided in the present invention on mouse testicular stromal cells TM3, using this as a model. As can be seen from the results, the Trichosanthes cucumeroides polypeptide provided in the present invention can significantly promote testosterone secretion in TM3 cells, and the mechanism of this testosterone secretion promotion is related to the regulation of the expression of testosterone synthesis-related genes and proteins, and is also related to the enhancement of mitochondrial function and the promotion of the expression of mitochondrial biosynthesis-related genes and proteins. Therefore, the Trichosanthes cucumeroides polypeptide provided in the present invention can promote testosterone secretion in TM3 cells, has a certain degree of kidney-tonifying and yang-strengthening effect, and can be developed as a substitute for conventional yang-strengthening drugs.

[0109] The above describes only preferred embodiments of the present invention and does not limit it. Any modifications, equivalent substitutions, or improvements made within the scope of the spirit and principles of the present invention should all be included within the scope of protection of the present invention.

Claims

1. A method for preparing Trichosanthes cucumeroides polypeptide comprising the following steps 1 to 5, Step 1: Remove the shells from the seeds of the Trichosanthes cucumeroides, crush them, defatt them to remove phenolic substances, then dry and powder them to obtain Trichosanthes cucumeroides seed powder. Step 2: The Trichosanthes cucumeroides seed powder is subjected to heated water extraction to obtain a Trichosanthes cucumeroides seed liquid after protein denaturation. Step 3: The Trichosanthes cucumeroides seed extract is enzymatically hydrolyzed with a complex enzyme. After the enzymatic hydrolysis is complete, the enzyme is inactivated, solid-liquid separation is performed, the hydrolyzed liquid is collected, and it is subjected to vacuum distillation and drying to obtain crude Trichosanthes cucumeroides peptide. In the enzymatic hydrolysis using the complex enzyme, a neutral protease and an alkaline protease are used. Step 4: Dissolve the crude peptide of Trichosanthes cucumeroides in water to prepare a crude peptide solution of Trichosanthes cucumeroides, filter it, and subject the resulting filtrate to ion exchange resin chromatography, using redistilled water as the eluent, collect the eluent according to the retention time of the absorbance curve, and then concentrate and dry it to obtain the ion exchange chromatography enzyme hydrolysate. In the ion exchange resin chromatography, a cellulose anion exchange chromatography column was used. Step 5: The ion-exchange chromatography enzyme hydrolysate is dissolved in water, separated and purified by reverse-phase high-performance liquid chromatography, and the eluent with a retention time of 10.0–12.3 min or eluent with a retention time of 21.0–21.4 min is collected to obtain the Trichosanthes cucumeroides polypeptide. The column used in the reverse-phase high-performance liquid chromatography is a C18 column, mobile phase A is a 0.1%–0.2% trifluoroacetic acid aqueous solution, and mobile phase B is a 0.1%–0.2% trifluoroacetic acid-acetonitrile solution. Gradient elution is performed, and the gradient elution process is as follows: 0-3min, mobile phase: 95% mobile phase A + 5% mobile phase B; 3-10min, mobile phase: 80% mobile phase A + 20% mobile phase B; 10-20min, mobile phase: 50% mobile phase A + 50% mobile phase B; 20-23min, mobile phase: 20% mobile phase A + 80% mobile phase B A preparation method characterized by the following:

2. In step 1, the degreasing solvent for the seeds of Trichosanthes cucumeroides is n-hexane, the weight-to-volume ratio of the seeds of Trichosanthes cucumeroides to n-hexane is 1:3-6, the solvent used to remove phenolic substances is acetone, the weight-to-volume ratio of the seeds of Trichosanthes cucumeroides to acetone is 1:3-6, and / or The preparation method according to claim 1, characterized in that, in step 2, when hot water extraction, the mass ratio of Trichosanthes cucumeroides seed powder to water is 1:30-50, the temperature is 90-100°C, and the time is 3-10 hours.

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

4. The preparation method according to claim 3, characterized in that the amount of alkaline protease added is 20%-40% of the mass of the Trichosanthes cucumeroides seed powder, the amount of neutral protease added is 20%-40% of the mass of the Trichosanthes cucumeroides seed powder, and in step 3, the pH of the complex enzyme decomposition is 7.5-8 or 9-9.5, the enzyme decomposition temperature is 55-75°C, and the enzyme decomposition time is 3-5 hours.

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

6. The preparation method according to claim 1, characterized in that the flow rate of the eluent in step 4 is 1-2 mL / min, and a DEAE-52 cellulose anion exchange chromatography column is used in the ion exchange resin chromatography.

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

8. The preparation method according to claim 1, characterized in that the 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, and the flow rate of the mobile phase is 20 mL / min.

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