Trichosanthes kirilowii polypeptide having nephrotonic effect, preparation method therefor and use thereof

Abstract

To provide a Trichosanthes kirilowii polypeptide having a nephrotonic effect, a preparation method and use thereof.SOLUTION: In the present invention, the Trichosanthes kirilowii polypeptide with nephrotonic effect is obtained by pre-treatment of Trichosanthes kirilowii raw materials, preparation of Trichosanthes kirilowii seed liquid, preparation of crude Trichosanthes kirilowii peptide, ion-exchange chromatography, HPLC purification and other processes. The Trichosanthes kirilowii polypeptide provided in the present invention can regulate the expression of testosterone secretion-related genes and proteins in TM3 cells, improve the mitochondrial function, promote the expression of mitochondrial biosynthesis-related genes and proteins, and significantly promote the testosterone secretion of TM3 cells, and has the effect of promoting kidney tone, thereby expanding the use range of Trichosanthes kirilowii.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology and relates to a trifoliate trifoliate polypeptide having kidney-tonifying and stimulating effects and its use. [Background technology]

[0002] Testosterone is an important hormone for men, playing a key role in regulating sexual function, reproduction, muscle movement, and various physiological activities. New 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 any age. In adult men, this deficiency is often attributed 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 promote endogenous testosterone synthesis is crucial.

[0003] Commonly used kidney-tonifying and yang-boosting herbal medicines include seahorse root, coccinellid, dodder, and Cistanche salicina, but all have certain side effects, making their safety difficult to guarantee for long-term use. Trifolium nigricans (Trifolium nigricans) is a widely cultivated and highly valued traditional Chinese oilseed crop belonging to the Cucurbitaceae family. Trifolium nigricans seeds are rich in protein (26.70%) and highly nutritious, containing abundant amounts of arginine, leucine, and glutamic acid. Their amino acid composition is close to the essential amino acid content recommended by the WHO. Currently, there is little research on the functions and active substances of Trifolium nigricans seeds, and further research and exploration of its applications is needed. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a Triticum vulgare polypeptide with kidney-tonifying and stimulating effects, as well as a preparation method and use thereof. The preparation method provided by the present invention enables Triticum vulgare polypeptide with kidney-tonifying and stimulating effects to be obtained from Triticum vulgare. The preparation process is simple, the polypeptide yield is high, and the obtained Triticum vulgare polypeptide is safe and has a small molecular weight, which allows for good absorption and utilization in the body. It significantly increases testosterone secretion from TM3 cells, making it suitable for development as an alternative to traditional stimulating drugs.

[0005] In a first aspect, the present invention provides a method for preparing a bitter gourd polypeptide, the method comprising the following steps 1 to 5: Step 1: The shelled and crushed seeds of Triticum vulgare are defatted, and phenolic substances are removed. Then, the seeds are dried and powdered to obtain Triticum vulgare seed powder. Step 2: extracting the bitter gourd seed powder with heated water to obtain a bitter gourd seed liquid after protein denaturation; Step 3: Enzymatically hydrolyzing the seed liquid of the bitter melon with a complex enzyme, and after completion of the enzymatic hydrolysis, inactivating the enzyme, performing solid-liquid separation, collecting and drying the enzymatic hydrolysis liquid, and obtaining a bitter melon crude peptide; and the hydrolysis by the complex enzyme uses a neutral protease or an alkaline protease. Step 4: The bitter melon crude peptide is dissolved in water to prepare a bitter melon crude peptide solution, which is then filtered. The resulting filtrate is subjected to ion exchange resin chromatography, using double distilled water as an eluent. The eluate corresponding to the peak in the absorbance curve is collected and then dried to obtain the ion exchange chromatography enzymatic digest. The ion exchange resin chromatography uses a cellulose anion exchange chromatography column. Step 5: The ion exchange chromatography enzymatic digest is dissolved in water, and separated and purified by reversed-phase high performance liquid chromatography to obtain the protein polypeptide that can significantly promote testosterone secretion from TM3 cells, to obtain the Triticum vulgare polypeptide, the column used in the reversed-phase high performance liquid chromatography is a C18 column, mobile phase A is a 0.1%-0.2% trifluoroacetic acid-water solution, and mobile phase B is a 0.1%-0.2% trifluoroacetic acid-acetonitrile solution, and 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 is.

[0006] The seeds of bitter melon are rich in protein. After enzymatic hydrolysis of proteins, the resulting peptide mixture contains many by-products, making screening for the target polypeptide difficult. Furthermore, as an oilseed crop, bitter melon contains large amounts of fats and polyphenols. Fat has a strong interfering effect on protein purification columns, and polyphenols bind to proteins, hindering protein extraction and reducing extraction yields. The method for preparing bitter melon polypeptides provided by the present invention uses bitter melon seeds as a raw material. After defatting to remove phenolic substances and steaming at high temperatures, the proteins are denatured. Subsequently, enzymatic hydrolysis and multi-stage separation and purification are carried out. By controlling parameters such as the type, concentration, and flow rate of the eluent during each stage of the separation and purification process, a bitter melon polypeptide with kidney-tonifying and stimulating effects is finally prepared. This method allows accurate determination of the elution peak of the target polypeptide by detecting the absorbance curve, and by controlling the collection time of the eluent, the target polypeptide can be repeatedly and stably collected.

[0007] In step 1, drying and powdering the seeds of Triticum vulgare helps in extracting the protein. Dried Triticum vulgare seeds can be ground to a particle size of ≤0.5 mm.

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

[0009] In step 4, the use of double-distilled water as the eluent results in the highest yield of enzymatic digests by ion exchange chromatography.

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

[0011] Preferably, the solvent for degreasing the seeds of Triticum vulgare in step 1 is n-hexane, and the weight / volume ratio of the seeds to n-hexane is 1:3-6; and the solvent for removing phenolic substances is acetone, and the weight / volume ratio of the seeds to acetone is 1:3-6.

[0012] Preferably, in the step 2, the mass ratio of the triticum vulgare 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 helps with subsequent enzymatic decomposition.

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

[0015] Bacillus subtilis-derived neutral protease is a metalloprotease, an endoprotease extracted after sufficient fermentation and cultivation of Bacillus subtilis. It is a pure, natural, safe, non-toxic enzyme with strong hydrolytic ability, capable of decomposing high molecular weight proteins into products such as polypeptides and amino acids.

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

[0017] Bacillus licheniformis alkaline protease is an enzyme preparation obtained by sufficient liquid fermentation of Bacillus licheniformis, and 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 bitter gourd seed powder, and the amount of neutral protease added is 20%-40% of the mass of the bitter gourd seed powder. In step 3, the pH of the complex enzyme hydrolysis is 7.5-8 or 9-9.5, the enzyme hydrolysis temperature is 55-75°C, and the enzyme hydrolysis time is 3-5 hours.

[0019] More preferably, the pH of the multi-enzyme hydrolysis in step 3 is 9-9.5, the enzymatic hydrolysis temperature is 65°C, and the enzymatic hydrolysis time is 5 hours. This enzymatic hydrolysis process can achieve the highest yield of bitter melon crude peptides.

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

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

[0022] At this flow rate, the retention time of the enzymatic digest when 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 the eluent with a retention time of 10.0-12.3 min or the eluent with a retention time of 21.0-21.4 min is collected to obtain the Triticum vulgare polypeptide.

[0024] Under these conditions, the trifoliate polypeptides obtained from the eluates with retention times of 10.0-12.3 min and 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 bitter gourd polypeptide prepared by the method described above.

[0026] The trifoliate gourd polypeptide provided by the present invention is mainly a low molecular weight peptide, which is safe and easily absorbed by the human body, and can promote testosterone secretion from TM3 cells and mitochondrial biosynthesis, and has the effect of tonicifying the kidneys and stimulating the yang. The mechanism by which the trifoliate gourd 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 a use of the bitter melon polypeptide, comprising: Use in preparing kidney-tonifying and yang-stimulating medicines; or Use in the preparation of a medicament for promoting testosterone secretion from TM3 cells; or Use in the preparation of a medicament for regulating the expression of genes and proteins related to testosterone secretion in TM3 cells; or Use in the preparation of a medicament for regulating the expression of genes and proteins related to mitochondrial biogenesis in TM3 cells. Includes: [Brief explanation of the drawings]

[0028] In order to more clearly describe the embodiments of the present invention or the technical solutions of the prior art, the following will briefly introduce the drawings necessary for describing the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. Those skilled in the art can obtain other drawings based on these drawings without any creative efforts. [Figure 1] 1 shows the absorbance curves of the enzymatic hydrolyzates obtained by ion exchange chromatography using different eluents in the present invention. [Figure 2] 1 shows the absorbance curve of the Triticum vulgare polypeptide obtained by purification using reversed-phase high-performance liquid chromatography. [Figure 3] 1 shows a chromatogram of total ion current of the bitter gourd polypeptide TSH1-3 obtained in Example 2 of the present invention. [Figure 4] 1 shows the results of screening crude peptides from Triticum vulgare in Example 1 of the present invention using a water extraction process and an enzymatic hydrolysis process. [Figure 5] 1 shows the effect of the Triticum vulgare polypeptide of the present invention on the viability of TM3 cells. [Figure 6] 1 shows the effect of the ion exchange chromatography enzymatic digest of the present invention on testosterone secretion from TM3 cells. [Figure 7] 1 shows the effect of the trifoliate gourd polypeptide of the present invention on testosterone secretion in TM3 cells. [Figure 8] 1 shows the effects of the Triticum vulgare 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]1 shows the effects of the Triticum vulgare polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on the expression of testosterone synthesis-related genes. [Figure 10] 1 shows the influence of the Triticum vulgare polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on the expression of testosterone synthesis-related proteins. [Figure 11] 1 shows the effects of the Triticum vulgare polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on mitochondrial mtDNA replication. [Figure 12] 1 shows the effects of the Triticum vulgare polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on the expression of mitochondrial biogenesis-related proteins. [Figure 13] 1 shows the effects of the Triticum vulgare polypeptide TSH1-3 and the synthetic peptide fragment VTPVGSPR according to the present invention on the expression of mitochondrial biogenesis-related genes. DETAILED DESCRIPTION OF THE INVENTION

[0029] The following embodiments of the present invention will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the following embodiments are only a part of the embodiments of the present invention, and are not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without any creative efforts are all included in the protection scope of the present invention.

[0030] (1) Reagents n-Hexane: Mclean H810749 Acetone: Guangzhou Chemical Reagent Factory 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 Quantitation Kit: Abiowell AWB0104 RIPA solution (strong): Abiowell AWB0136 Trypsin: Gibco 25200056 ECL Plus Ultra-Sensitive Luminescence Solution: Abiowell AWB0005 Alkaline protease: Alcalase 2.4L, Novozymes Neutrase Neutral Protease 0.8L, Novozymes Mouse Testosterone (T) ELISA Scientific Research Kit: MM-0569M1, Jiangsu Baimen Industrial Co., Ltd. Superoxide dismutase (SOD) kit: G0101W, Jiangsu Geruis Biotechnology Co., Ltd. Malondialdehyde (MDA) kit: G0109W, Jiangsu Geruis Biotechnology Co., Ltd.

[0031] (2) Equipment The reversed-phase high-performance liquid chromatograph column Pursuit XRs C-18 was purchased from Agilent Technologies, Inc., USA.

[0032] Example 1 This example provides a bitter gourd polypeptide. The specific preparation method is as follows. Step 1: The seeds of Triticum vulgare were dehusked and crushed. They were defatted with n-hexane at a solid-liquid ratio of 1:5 (g:mL), and phenolic substances were removed with acetone at a solid-liquid ratio of 1:5 (g:mL). The powder was air-dried and then sieved through an 80-mesh sieve to obtain Triticum vulgare seed powder. Step 2: 10 g of bitter melon seed powder was taken and mixed with purified water at a solid-liquid ratio of 1:40 (g:mL), heated in a 95°C water bath, and kept warm for 5 hours to obtain bitter melon seed liquid. Step 3: The solution from the seeds of Triticum vulgare was cooled to 65°C, and 2 mL of alkaline protease and 2 mL of neutral protease were added. The solution was hydrolyzed at pH 9 and 65°C for 5 hours. After hydrolysis, the enzymes were inactivated at 95°C for 15 minutes. The solution was then centrifuged, distilled under reduced pressure, and freeze-dried to obtain crude Triticum vulgare peptides. Step 4: The crude peptides from Triticum vulgare were dissolved and diluted to 20 mg / ml. After filtering through a 0.22 μm membrane, they were loaded onto a DEAE-52 cellulose anion-exchange chromatography column using a pressure pump and eluted with double-distilled water (flow rate 1 mL / min). The absorbance curve of the eluate at 220 nm is shown in Figure 1. The components between 135 and 250 min of elution were collected, concentrated under reduced pressure, and lyophilized to obtain the ion-exchange chromatography enzymatic digest TSH1. Step 5: TSH1 was dissolved in double-distilled water to a concentration of 5 mg / mL, filtered through a 0.22 μm filter, and purified by reversed-phase high-performance liquid chromatography (RPLC). The column was a 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, and 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: Mobile phase linear gradient elution sequence JPEG2026004192000002.jpg38170

[0034] The absorbance curves at 220 nm of the eluate at various times are shown in Figure 2. The eluate at 21.0-21.4 min of elution was collected, concentrated under reduced pressure, and then vacuum lyophilized to obtain the trifoliate polypeptide TSH1-3.

[0035] Using LC-MS / MS and a high-resolution mass spectrometry database set, we identified peptide fragments from the TSH1-3 components and analyzed their amino acid sequences. As a result, we obtained 42 peptide fragments with relatively high reliability. 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: Composition of peptide fragments of TSH1-3 JPEG2026004192000003.jpg122170JPEG2026004192000004.jpg109170

[0037] Comparative Examples 1-4 Comparative Examples 1-4 provide four ion exchange chromatographic enzymatic digests, TSH2, TSH3, TSH4 and TSH5, respectively.

[0038] The crude peptides from Triticum vulgare prepared in step 3 of Example 1 were subjected to ion exchange resin chromatography in accordance with step 4 of Example 1. The difference from step 4 of Example 1 was that elution was performed with 0.01 mol / L sodium chloride instead of double-distilled water, and the components between the elution times of 168 and 264 minutes were collected, concentrated under reduced pressure, and then vacuum lyophilized to obtain the ion exchange chromatographic enzymatic digest TSH2.

[0039] The crude peptides from Triticum vulgare prepared in step 3 of Example 1 were subjected to ion exchange resin chromatography in accordance with step 4 of Example 1. The difference from step 4 of Example 1 was that elution was performed with 0.05 mol / L sodium chloride instead of double-distilled water, and the components with an elution time of 116-292 min were collected, concentrated under reduced pressure, and vacuum lyophilized to obtain the ion exchange chromatographic enzymatic digest TSH3.

[0040] The crude peptides from Triticum vulgare prepared in step 3 of Example 1 were subjected to ion exchange resin chromatography in accordance with step 4 of Example 1. The difference from step 4 of Example 1 is that elution was performed with 0.1 mol / L sodium chloride instead of double-distilled water, and the components with an elution time of 116-212 min were collected and concentrated under reduced pressure and lyophilized to obtain the ion exchange chromatographic enzymatic digest TSH4, and the components with an elution time of 308-368 min were collected and concentrated under reduced pressure and lyophilized to obtain the ion exchange chromatographic enzymatic digest TSH5.

[0041] The absorbance curves of the enzymatic hydrolyzates obtained by ion exchange chromatography in Comparative Examples 1-4 are shown in FIG.

[0042] Example 2 Example 2 provides the bitter gourd polypeptides TSH1-2.

[0043] The exchange chromatography enzymatic digest TSH1 prepared in step 4 of Example 1 was separated and purified under the reversed-phase high-performance liquid chromatography conditions in step 5 of Example 1. The difference from step 5 of Example 1 was that the collection time for the TSH1-2 eluate was 10.0-12.3 min.

[0044] The absorbance curve of the bitter gourd polypeptide of Example 2 is shown in FIG. Comparative Examples 5-7

[0045] Comparative Examples 5-7 provide the bitter gourd polypeptides TSH1-1, TSH1-4 and TSH1-5, respectively.

[0046] The ion exchange chromatography enzymatic digest TSH1 prepared in Step 4 of Example 1 was separated and purified under the reversed-phase high-performance liquid chromatography conditions in Step 5 of Example 1. The difference from Step 5 of Example 1 is that the collection times for the TSH1-1, TSH1-4, and TSH1-5 eluates were 6.7-7.0 min, 21.6-22.0 min, and 23.2-23.6 min, respectively.

[0047] The absorbance curves of the bitter melon polypeptides of Comparative Examples 5-7 are shown in FIG.

[0048] Example 3 Example 3 provides a bitter gourd polypeptide.

[0049] Step 1: The seeds of Triticum vulgare were dehusked and crushed. They were defatted with n-hexane at a solid-liquid ratio of 1:6 (g:mL), and phenolic substances were removed with acetone at a solid-liquid ratio of 1:6 (g:mL). The resulting mixture was air-dried and then sieved through an 80-mesh sieve to obtain Triticum vulgare seed powder.

[0050] Step 2: 10 g of bitter melon seed powder was taken and mixed with purified water at a solid-liquid ratio of 1:5 (g:mL). The mixture was heated in a water bath at 100°C and kept at that temperature for 10 hours to obtain bitter melon seed liquid.

[0051] Step 3: The solution of the seeds of Triticum vulgare was cooled to 50°C, and 4 mL of alkaline protease and 4 mL of neutral protease were added. The solution was hydrolyzed at pH 7.5 and 55°C for 5 hours. After hydrolysis, the enzymes were inactivated at 95°C for 15 minutes. The solution was then centrifuged, distilled under reduced pressure, and freeze-dried to obtain crude Triticum vulgare peptides.

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

[0053] Example 4 Example 4 provides a bitter gourd polypeptide.

[0054] Step 1: The seeds of Triticum vulgare were dehusked and crushed. They were defatted with n-hexane at a solid-liquid ratio of 1:3 (g:mL), and phenolic substances were removed with acetone at a solid-liquid ratio of 1:3 (g:mL). The resulting mixture was air-dried and then sieved through an 80-mesh sieve to obtain Triticum vulgare seed powder.

[0055] Step 2: 10 g of bitter melon seed powder was taken and mixed with purified water in a solid-liquid ratio of 1:50 (g:mL), heated in a water bath at 90°C, and kept warm for 3 hours to obtain bitter melon seed liquid.

[0056] Step 3: The seed solution of Triticum vulgare was cooled to 45°C, and 3 mL of alkaline protease and 3 mL of neutral protease were added. The solution was hydrolyzed at pH 9.5 and 75°C for 3 hours. After hydrolysis, the enzymes were inactivated at 95°C for 15 minutes. The solution was then centrifuged, distilled under reduced pressure, and freeze-dried to obtain Triticum vulgare crude peptides.

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

[0058] Example 5 This example provides crude peptides from Triticum vulgare.

[0059] Step 1: The seeds of Triticum vulgare were dehusked and crushed. They were defatted with n-hexane at a solid-liquid ratio of 1:5 (g:mL), and phenolic substances were removed with acetone at a solid-liquid ratio of 1:5 (g:mL). The resulting mixture was air-dried and then sieved through an 80-mesh sieve to obtain Triticum vulgare seed powder.

[0060] Step 2: 10 g of bitter melon seed powder was taken and mixed with purified water in a solid-liquid ratio of 1:40 (g:mL), heated in a water bath at 95°C, and kept warm for 5 hours to obtain bitter melon seed liquid.

[0061] Step 3: The seed solution of Triticum vulgare was cooled to the enzymatic degradation temperature, and 2 mL of alkaline protease and 2 mL of neutral protease were added. The enzymatic degradation was carried out at pH 9 for 4 hours. The enzymatic degradation temperature was 75°C, and after the enzymatic degradation was completed, the enzymes were inactivated at 95°C for 15 minutes. Then, the solution was centrifuged, and the solution was collected, distilled under reduced pressure, and freeze-dried to obtain Triticum vulgare crude peptides.

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

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

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

[0065] Yields of crude peptides from Triticum vulgare at various enzymatic hydrolysis temperatures Yield calculation formula:

number

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

[0067] Example 8 This example provides crude peptides from Triticum vulgare.

[0068] Prepared in Step 2 of Example 5 The seed juice of Triticum vulgare was taken and 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 65°C for 3 hours. After enzymatic hydrolysis was completed, the enzymes were inactivated at 95°C for 15 minutes, and then centrifuged. The solution was collected, distilled under reduced pressure, and freeze-dried to obtain Triticum vulgare crude peptides.

[0069] Example 9 The procedure was the same as in Example 8, except that the enzymatic decomposition time in step 3 was 4 hours.

[0070] Example 10 The procedure was the same as in Example 8, except that the enzymatic decomposition time in step 3 was 5 hours.

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

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

[0073] Comparative Example 11 The procedure was the same as in Example 8, except that the enzymatic decomposition time in step 3 was 8 hours.

[0074] The yields of the bitter melon crude peptides from 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 yields of the bitter melon crude peptides were relatively high when the enzymatic hydrolysis time was 3-5 hours, and the effect was greatest when the enzymatic hydrolysis time was 5 hours.

[0075] Example 11 This example provides crude peptides from Triticum vulgare.

[0076] The seed juice of Triticum vulgare prepared in step 2 of Example 5 was taken and 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 7.5 and 65°C for 5 hours. After enzymatic hydrolysis was completed, the enzyme was inactivated at 95°C for 15 minutes. The solution was then centrifuged, collected, distilled under reduced pressure, and freeze-dried to obtain Triticum vulgare crude peptides.

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

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

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

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

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

[0082] The yields of the bitter melon crude peptides from Examples 11-14 and Comparative Examples 12-13 were calculated. The results are shown in Figure 4C. As can be seen from Figure 4C, when the enzymatic hydrolysis pH was 7.5-8 or 9-9.5, the yields of the bitter melon crude peptides were relatively high, with the effect being greatest at pH 9.

[0083] Example 15 This example provides crude peptides from Triticum vulgare.

[0084] Step 1: The seeds of Triticum vulgare were dehusked and crushed. They were defatted with n-hexane at a solid-liquid ratio of 1:5 (g:mL), and phenolic substances were removed with acetone at a solid-liquid ratio of 1:5 (g:mL). The resulting mixture was air-dried and then sieved through an 80-mesh sieve to obtain Triticum vulgare seed powder.

[0085] Step 2: 10 g of bitter melon seed powder was taken and mixed with purified water in a solid-liquid ratio of 1:30 (g:mL). The mixture was heated in a water bath at 95°C and kept at that temperature for 5 hours to obtain bitter melon seed liquid.

[0086] Step 3: The solution of the seeds prepared with different solid-liquid ratios was cooled to 65°C, and 2 mL of alkaline protease and 2 mL of neutral protease were added. Enzymatic hydrolysis was carried out at pH 9 and 65°C for 5 hours. After the enzymatic hydrolysis was completed, the enzymes were inactivated at 95°C for 15 minutes. The solution was then centrifuged, distilled under reduced pressure, and freeze-dried to obtain crude peptides of the seeds of the fruit.

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

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

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

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

[0091] The yields of the bitter melon crude peptides from Examples 15-17 and Comparative Examples 14-15 were calculated. The results are shown in Figure 4D. As can be seen from Figure 4D, when the solid-liquid ratio was 1:30-50, the yields of the bitter melon crude peptides were relatively high, and the effect was greatest at 1:40.

[0092] Test Example 1: Evaluation of the testosterone secretion promoting effect of Triticum vulgare polypeptide 1.Cell culture TM3 cells were cultured in DMEM medium containing a large amount of 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.

[0093] 2. Effects of ion exchange chromatography enzymatic digests and Triticum vulgare polypeptides on TM3 cell viability The viability of TM3 cells was measured using the CCK8 method. TM3 cell suspensions in the logarithmic growth phase were seeded into a 96-well plate at 100 μL / well, and the cell density in each well was approximately 10 5The cells were cultured 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 medium was added. Here, the sample group (TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, TSH1-5) was DMEM 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). The control and blank DMEM medium did not contain the above polypeptides. The 96-well plate was then placed in an incubator and incubated for 48 hours. After that, 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 further incubation in the incubator for 4 hours, the absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated. The formula for cell viability was as follows:

number

[0094] The effects of TSH1, TSH1-1, TSH1-2, TSH1-3, TSH1-4, and TSH1-5 on TM3 cell viability are shown in Figure 5. As can be seen from Figure 5, each component had no significant effect on TM3 cell viability, indicating that they were not significantly toxic to TM3 cells.

[0095] 3. Effect of Triticum aestivum polypeptide on testosterone secretion in TM3 cells Testosterone secretion from TM3 cells was measured using an Elisa kit. TM3 cell suspensions in the logarithmic growth phase were seeded into a 96-well plate at 100 μL / well, and the cell density in each well was approximately 10 5The cells were cultured for 24 hours in an incubator at 5% CO and 37° C. After the cells attached 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). The control DMEM culture medium did not contain any of the above polypeptides. After 48 hours of incubation, the cell supernatants were collected and the testosterone levels in the supernatants were measured using an ELISA kit. The results are shown in Figures 6-7.

[0096] Figure 6 shows the effect of the ion exchange chromatography enzymatic digests of the present invention on testosterone secretion in TM3 cells. As can be seen from Figure 6, all five components can increase testosterone secretion in TM3 cells at a concentration of 200 μg / mL. Since the effects of TSH1 and TSH2 were comparable, the TSH1 component was selected for further screening based on its yield.

[0097] Figure 7 shows the effect of the trifoliate trifoliate polypeptide of the present invention on testosterone secretion in TM3 cells. As can be seen from Figure 7, TSH1-2 and TSH1-3 significantly increased testosterone secretion in TM3 cells at a concentration of 200 μg / mL, whereas TSH1-1, TSH1-4, and TSH1-5, derived from the same TSH1, had a lower stimulatory effect on testosterone secretion in TM3 cells than TSH1 at a concentration of 200 μg / mL. In the same bar graph, different letters above any two columns indicate a significant difference between the mean values ​​of the two columns (p<0.05); otherwise, no significant difference is indicated. Because TSH1-3 had the greatest effect on increasing testosterone secretion in TM3 cells, it was selected to further investigate its kidney-tonifying and stimulating mechanism.

[0098] 4. Effects of TSH1-3 and synthetic peptide fragment VTPVGSPR on TM3 cell-associated androgen secretion Using ELISA kits, androstenedione, testosterone, dihydrotestosterone, and free testosterone secreted from TM3 cells were measured. TM3 cells in the logarithmic growth phase were cultured at 10 5 Cells were uniformly seeded into 96-well plates at a density of 100 μL / well and cultured in a 5% CO2, 37°C incubator for 24 h. The medium was then removed and 100 μL of DMEM medium was added. The sample groups (VTPVGSPR, 50, 100, and 200) contained DMEM medium 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 contained no polypeptides. After 48 h of culture in the incubator, the cell supernatants were collected and assayed for androstenedione, testosterone, dihydrotestosterone, and free testosterone using ELISA kits. The results are shown in Figure 8.

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

[0100] Testosterone is synthesized by androstenedione (ADS) under the action of 17β-hydroxysteroid dehydrogenase (17β-HSD). Dihydrotestosterone (DHT) is a potent metabolite of testosterone that is primarily reduced to 5α in certain tissues, such as the prostate, skin, and liver. This local DHT synthesis process is crucial for the normal development of male characteristics during prenatal and pubertal life. 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 diffuse directly into tissues. Therefore, free testosterone levels can better reflect biological activity compared with total testosterone levels. Investigating the effects of Triticum aestivum polypeptides on the secretion of these substances will help us understand the kidney-tonifying and stimulating mechanisms and effects of Triticum aestivum polypeptides.

[0101] 5. Effects of TSH1-3 and synthetic peptide fragment VTPVGSPR on the expression of testosterone synthesis-related proteins and genes in TM3 cells TM3 cells (5 x 10 5CFU) were incubated overnight on a 12-well plate and then treated with various concentrations of TSH1-3 (50, 100, 200 μg / mL) and VTPVGSP (200 μg / mL) at 37°C for 48 hours. Total RNA was extracted from the cells using Trizol reagent. A control group was set up using the same volume of DMEM culture medium instead of the polypeptide. Reverse transcription and PCR reactions were performed on the extracted RNA using an RNA PCR kit (CWBIO, China). β-actin was used as a control gene, and 2 -△△Ct The relative mRNA expression level of the target gene was determined using the NMR method. The relative expression level of the target gene was calculated as the ratio between the experimental group and the control group. The results are shown in Figure 9.

[0102] TM3 cells (5 x 10 5 After overnight incubation in 12-well plates, TM3 cells (CFU / well) were treated with various concentrations of TSH1-3 (50, 100, 200 μg / mL) and VTPVGSP (200 μg / mL) at 37°C for 48 h. The TM3 cells were then harvested and washed twice with PBS. Then, TM3 cells were lysed in 200 μL of radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 12,000 rpm for 15 min at 4°C. The resulting supernatant was used as the total protein extract, and the protein concentration of the lysate 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 with 5% nonfat dry milk in phosphate-buffered saline (PBST) containing Tween 20 at room temperature for 1.5 h. The membrane was then incubated with primary antibodies overnight at 4°C. After washing three times with PBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated antibody at room temperature for 1.5 h. The chemiluminescent signal was detected using enhanced chemiluminescence (ECL) detection reagents, and images were captured using a ChemiScope 6100 imaging system (Clinx, Shanghai, China). Bands were analyzed using Image J software to obtain the relative expression levels of related proteins. The results are shown in Figure 10.

[0103] StAR, TSPO, CYP11A1, and 3β-HSD genes play important roles in testosterone synthesis. First, steroidogenic acute regulatory protein (StAR) is activated, then binds to the mitochondrial outer membrane transporter protein (TSPO) and promotes cholesterol transport from the outer membrane to the inner membrane. Cholesterol, as a raw material for testosterone synthesis, is involved in the testosterone synthesis process in TM3 cells. Next, cholesterol is converted to pregnenolone on the inner mitochondrial membrane by the cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1). Pregnenolone is then transported to the endoplasmic reticulum and converted to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD). Progesterone is then catalyzed to androstenedione, which is 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 VTPVGSP, the expression levels of each protein and the expression amounts of related genes increased significantly compared to the control group, and TSH1-3 dose-dependently promoted the expression of StAR, TSPO, CYP11A1 and 3β-HSD at the transcription and protein levels, indicating that TSH1-3 and the synthetic peptide fragment VTPVGSP can be involved in the testosterone synthesis pathway and promote the synthesis and secretion of testosterone.

[0104] 6. Effects of TSH1-3 and synthetic peptide fragment VTPVGSPR on the expression of testosterone synthesis-related proteins and genes in TM3 cells 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 from Life Technologies. To assess mtDNA levels, probes for mitochondrial genes (ND1 and ND6) were used, with nuclear 18S as the normalization reference. The results are shown in Figure 11.

[0105] TM3 cells (5 x 10 5Cells (CFU / well) were incubated overnight in 12-well plates and then treated with various concentrations of TSH1-3 (50, 100, 200 μg / mL) and VTPVGSP (200 μg / mL) for 48 h at 37°C. Cells were harvested and washed twice with PBS. TM3 cells were then lysed in 200 μL of radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 12,000 rpm for 15 min at 4°C. The resulting supernatant was used as the total protein extract, and the protein concentration of the lysate 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 with 5% nonfat dry milk in phosphate-buffered saline (PBST) containing Tween 20 for 1.5 h at room temperature. The membrane was then incubated with primary antibodies overnight at 4°C. After washing three times with PBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1.5 h. Chemiluminescence signals were detected using enhanced chemiluminescence (ECL) detection reagents, and images were captured using a ChemiScope 6100 imaging system (Clinx, Shanghai, China). 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 x 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 VTPVGSP (200 μg / mL) at 37°C for 48 h. Total RNA was extracted from the cells using 1 mL Trizol reagent and reverse-transcribed using an RNA PCR kit (CWBIO, China), followed by polymerase chain reaction 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 amount of the target gene was calculated as the ratio between the experimental group and the control group. The results are shown in Figure 13.

[0107] Studies have shown that mitochondrial biogenesis is closely related to testosterone secretion, and inhibiting mitochondrial biogenesis can result in decreased testosterone synthesis. As shown in Figure 11, the trifoliate gourd 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 polypeptides, the expression levels of mitochondrial biogenesis-related genes and proteins PGC-1α, TFAM, TFB1M, TFB2M, NRF1, and NRF2 were significantly increased. Here, PGC-1α can regulate 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. These experimental results suggest that the promotion of trifoliate gourd polypeptides on the secretion of the androgen testosterone in TM3 cells may be achieved through regulating mitochondrial biogenesis.

[0108] Based on the above, the present invention evaluated the testosterone secretion-promoting effect of the trichosanthes trichosanthes polypeptide provided by the present invention using mouse testicular interstitial cells TM3 as a model. The results show that the trichosanthes trichosanthes polypeptide provided by the present invention can significantly promote testosterone secretion in TM3 cells, and the mechanism of its promotion is related to regulating the expression of testosterone synthesis-related genes and proteins, and also related to enhancing mitochondrial function and promoting the expression of mitochondrial biogenesis-related genes and proteins. Therefore, the trichosanthes trichosanthes polypeptide provided by the present invention can promote testosterone secretion in TM3 cells and has a certain kidney-tonifying and yang-stimulating effect, making it suitable for development as an alternative to traditional yang-stimulating drugs.

[0109] The above is only a preferred embodiment of the present invention, and does not limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a bitter gourd polypeptide, comprising the following steps 1 to 5: Step 1: The shelled and crushed seeds of Triticum vulgare are defatted, and phenolic substances are removed. Then, the seeds are dried and powdered to obtain Triticum vulgare seed powder. Step 2: Extracting the bitter gourd seed powder with heated water to obtain a bitter gourd seed liquid after protein denaturation; Step 3: Enzymatically hydrolyzing the seed liquid of the bitter melon with a complex enzyme, and after completion of the enzymatic hydrolysis, inactivating the enzyme, performing solid-liquid separation, collecting the enzymatic hydrolysis liquid, distilling it under reduced pressure, and drying it to obtain a bitter melon crude peptide. In the hydrolysis with the complex enzyme, a neutral protease or an alkaline protease is used. Step 4: The bitter melon crude peptide is dissolved in water to prepare a bitter melon crude peptide solution, which is then filtered. The resulting filtrate is subjected to ion exchange resin chromatography. Double distilled water is used as an eluent. The eluent is collected according to the retention time of the absorbance curve, and then concentrated and dried to obtain an ion exchange chromatography enzymatic digest. The ion exchange resin chromatography uses a cellulose anion exchange chromatography column. Step 5: The ion exchange chromatography enzymatic digest is dissolved in water, and separated and purified by reversed-phase high performance liquid chromatography to obtain the protein polypeptide that can significantly promote testosterone secretion from TM3 cells, to obtain the bitter melon polypeptide, the column in the reversed-phase high performance liquid chromatography is a C18 column, the mobile phase A is a 0.1%-0.2% trifluoroacetic acid-water solution, and the mobile phase B is a 0.1%-0.2% trifluoroacetic acid-acetonitrile solution, and 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:

2. In step 1, the solvent for degreasing the seeds of Triticum vulgare is n-hexane, and the weight / volume ratio of the seeds to n-hexane is 1:3-6; the solvent for removing phenolic substances is acetone, and the weight / volume ratio of the seeds to acetone is 1:3-6; and / or 2. The method of claim 1, wherein in step 2, during the hot water extraction, the mass ratio of Triticum vulgare seed powder to water is 1:30-50, the temperature is 90-100°C, and the time is 3-10 minutes.

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 licheniformis.

4. 4. The method of claim 3, wherein the amount of alkaline protease added is 20%-40% of the mass of the Winter Trillium seed powder, and the amount of neutral protease added is 20%-40% of the mass of the Winter Trillium seed powder. In step 3, the pH of the multi-enzyme hydrolysis is 7.5-8 or 9-9.5, the enzymatic hydrolysis temperature is 55-75°C, and the enzymatic hydrolysis time is 3-5 hours.

5. The method according to claim 4, wherein in step 3, the pH of the complex enzymatic hydrolysis is 9-9.5, the enzymatic hydrolysis temperature is 65°C, and the enzymatic hydrolysis time is 5 hours.

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

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 with a retention time of 135-250 min is collected.

8. 2. The method of claim 1, wherein 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 the eluent with a retention time of 10.0-12.3 min or the eluent with a retention time of 21.0-21.4 min is collected to obtain the Triticum vulgare polypeptide.

9. A bitter gourd polypeptide prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the bitter gourd polypeptide according to claim 8, For use in preparing kidney-tonifying and yang-stimulating medicines; or Use in the preparation of a medicament for promoting testosterone secretion from TM3 cells; or Use in the preparation of a medicament for regulating the expression of genes and proteins related to testosterone secretion in TM3 cells; or Use in the preparation of a medicament for regulating the expression of genes and proteins related to mitochondrial biogenesis in TM3 cells.

2. The use of claim 1, wherein the

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