Selenium-containing cicer arietinum peptides, methods of making and use thereof in the preparation of blood pressure lowering medicaments

Abstract

The application discloses selenium-containing chickpea peptides, a preparation method thereof and application of the selenium-containing chickpea peptides in preparation of blood pressure lowering drugs, and belongs to the technical field of bioactive peptides. Selenium-enriched Bacillus natto is used to ferment chickpeas to obtain selenium-containing chickpea protein hydrolysate, and then fermentation conditions are optimized and quantitative peptidomics technology is used to obtain selenium-containing chickpea peptides PQM and PQML. Experiments prove that the selenium-containing chickpea peptides have good in-vivo stability, bioavailability and blood pressure lowering activity, have an effect of inhibiting angiotensin converting enzyme (ACE) activity, and have potential for preventing or treating hypertension.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to selenium-containing chickpea peptides, their preparation methods, and their application in the preparation of antihypertensive drugs. Background Technology

[0002] Hypertension is one of the most common chronic non-communicable diseases worldwide, with its incidence showing a continuous upward trend. Its long-term presence can significantly increase the risk of cardiovascular and cerebrovascular diseases, kidney damage, and premature death. Angiotensin-converting enzyme (ACE), a zinc-dependent metalloproteinase, plays a central role in two key pathways regulating blood pressure: on the one hand, it catalyzes the conversion of angiotensin I (Ang I) to angiotensin II (Ang II) in the renin-angiotensin system (RAS), the latter being a potent vasoconstrictor that can significantly raise blood pressure; on the other hand, in the kallikrein-kinin system (KKS), ACE can also degrade the vasodilator bradykinin, thereby exacerbating the development of hypertension. Therefore, ACE has become a key target for antihypertensive therapy.

[0003] ACE inhibitory peptides are small bioactive peptides composed of 2 to 20 amino acid residues. They can effectively inhibit ACE activity and block the conversion of Ang I to Ang II, thereby achieving the effect of lowering blood pressure. In recent years, food-derived ACE inhibitory peptides have gradually become an important direction for drug substitution due to their natural source and high safety, and have broad market prospects. At present, a variety of ACE inhibitory peptides have been reported (such as milk peptides, fish collagen peptides, etc.), but these ACE inhibitory peptides still have the following limitations in practical applications: (1) Poor in vivo stability and easy degradation by digestive enzymes: Most natural blood pressure lowering peptides have small molecular weights and loose structures. After entering the gastrointestinal tract, they are easily hydrolyzed by pepsin, trypsin, etc., resulting in loss of activity and difficulty in maintaining sufficient angiotensin-converting enzyme (ACE) inhibitory effect in vivo; (2) Insufficient bioavailability and targeting: Conventional peptides have low absorption efficiency in the intestine after oral administration, and the plasma concentration is difficult to reach the effective level; and they lack structural modification or carrier systems, so they cannot be effectively located in the vascular endothelium or kidney, and the targeting of the action is limited.

[0004] Selenium is an essential trace element for the human body, possessing various physiological functions. Inorganic selenium is not easily absorbed directly by organisms and must undergo biological transformation to become a more readily usable organic selenium form. Studies have shown that Bacillus natto exhibits strong resistance to acids, alkalis, and high temperatures, and can efficiently convert inorganic selenium into a physiologically active organic selenium form. Chickpeas are the world's third most cultivated legume, rich in high-quality protein, with outstanding nutritional value, making them suitable as a fermentation substrate. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide selenium-containing chickpea peptides, their preparation method and their application in the preparation of antihypertensive drugs, so as to solve the technical problems of poor in vivo stability and low bioavailability.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] In a first aspect, the present invention discloses a selenium-containing chickpea peptide containing an amino acid sequence as shown in SEQ ID NO.1.

[0008] Preferably, the amino acid sequence of the selenium-containing chickpea peptide is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0009] In a second aspect, the present invention discloses a pharmaceutically acceptable salt of the above-mentioned selenium-containing chickpea peptide.

[0010] A third aspect of the present invention discloses an antihypertensive drug comprising the above-mentioned selenium-containing chickpea peptide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0011] Preferably, the above-mentioned selenium-containing chickpea peptide or a pharmaceutically acceptable salt of selenium-containing chickpea peptide is mixed with a pharmaceutically acceptable carrier to prepare a clinically acceptable injection, oral formulation, transdermal absorption formulation, or mucosal absorption formulation.

[0012] A fourth aspect of the present invention discloses an antihypertensive pharmaceutical composition comprising the above-mentioned selenium-containing chickpea peptide or a pharmaceutically acceptable salt thereof, and other antihypertensive drugs.

[0013] In a fifth aspect, the present invention discloses a method for preparing the above-mentioned selenium-containing chickpea peptide, which is prepared by Fmoc solid-phase synthesis.

[0014] A sixth aspect of the present invention discloses the use of the above-mentioned selenium-containing chickpea peptide, pharmaceutically acceptable salt of selenium-containing chickpea peptide, antihypertensive drug or antihypertensive drug composition in the preparation of an antihypertensive drug for prevention and / or treatment.

[0015] Preferably, the antihypertensive drug is a drug that promotes the release of NO from endothelial cells.

[0016] Preferably, the antihypertensive drug is a drug that inhibits the release of ET-1 from endothelial cells.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The selenium-enriched chickpea peptide provided by this invention is obtained by selenium enrichment fermentation using Bacillus natto as the fermentation strain and chickpea as the substrate. The selenium-enriched chickpea peptide has the following advantages: (1) Enhanced stability: Selenium enrichment modification forms disulfide bonds / selenium bridges in the peptide chain, which improves the peptide's tolerance to enzymatic hydrolysis in the gastrointestinal environment; (2) Improved bioavailability and targeting: The introduction of selenium not only improves the hydrophobic-hydrophobic interaction and hydrogen bond network of the peptide, but also promotes the stable circulation of the peptide in the blood; Combined with cell-level experiments, the selenium-enriched peptide is more easily taken up by endothelial cells such as HUVECs, enhancing the local antihypertensive targeting effect, and can be further coupled with delivery systems such as nanocapsules and liposomes to achieve targeted delivery of the peptide in the vascular endothelium; (3) Antihypertensive activity: Selenium itself has an antioxidant effect, and the fermented peptide retains some antioxidant residues, which helps to reduce endothelial oxidative stress induced by hypertension; While inhibiting ACE, the selenium-enriched peptide can comprehensively improve vascular function through multi-target synergistic effects such as increasing endothelial NO release and inhibiting ET-1 generation. ACE activity inhibition assays showed that the selenium-containing chickpea peptide possesses good ACE inhibitory activity. Thermodynamic properties of the interaction between the selenium-containing chickpea peptide and ACE were determined by isothermal titration calorimetry, revealing multiple binding sites on ACE. Spectroscopic studies of the interaction between the selenium-containing chickpea peptide and ACE showed that the peptide can bind to amino acid residues of ACE, disrupting the ACE hydrogen bond network. Cellular experiments demonstrated that the selenium-containing chickpea peptide has no toxic effect on HUVEC cells, promotes NO release from endothelial cells, and inhibits ET-1 release, thereby achieving a hypotensive effect superior to that of the positive control drug captopril. Therefore, the selenium-containing chickpea peptide provided by this invention has the potential to inhibit angiotensin-converting enzyme (ACE) activity and is suitable for the preparation of drugs for the prevention or treatment of hypertension. Attached Figure Description

[0019] Figure 1 The graph shows the inhibition of ACE activity by selenium-containing chickpea crude peptides under different conditions according to the present invention; where (a) inoculum size, (b) solid-liquid ratio, (c) fermentation time, and (d) fermentation temperature.

[0020] Figure 2 The chromatographic purity diagrams of the two synthetic peptides of the present invention are shown; where (a) is PQML and (b) is PQM.

[0021] Figure 3 These are the secondary mass spectra of the two synthetic peptides of the present invention; wherein, (a) PQML, (b) PQM;

[0022] Figure 4 IC50 values ​​of two synthetic peptides at different concentrations according to the present invention 50 Values; where (a) PQML, (b) PQM;

[0023] Figure 5 The heat flow curves for titrating ACE with PQM and PQML according to the present invention are shown; where (a) is PQM and (b) is PQML.

[0024] Figure 6 The CD spectra of ACE are shown in the presence and absence of the polypeptide of the present invention.

[0025] Figure 7 The following are fluorescence spectra of different concentrations of peptides and ACE according to the present invention; wherein, (a) fluorescence spectra of different concentrations of PQM and ACE, and (b) fluorescence spectra of different concentrations of PQML and ACE.

[0026] Figure 8 This is a diagram showing the combination mode of ACE and PQM in this invention; wherein, (a) is the two-dimensional combination mode of PQM and ACE, the green dashed line represents hydrogen bonding, and the red gear shape represents hydrophobic interaction; (b) the position of PQM in the three-dimensional structure of ACE (left), and the three-dimensional combination mode of PQM and ACE (right).

[0027] Figure 9 This is a diagram showing the bonding pattern of ACE and PQML in this invention; wherein, (a) is the two-dimensional bonding pattern of PQML and ACE, the green dashed line represents hydrogen bonding, the red gear shape represents hydrophobic interaction, (b) the position of PQML in the three-dimensional structure of ACE (left), and the three-dimensional bonding pattern of PQML and ACE (right).

[0028] Figure 10 Cell viability diagrams of HUVECs after treatment with different concentrations of selenium-containing ACE inhibitory peptides according to the present invention; wherein, (a) PQM, (b) PQML;

[0029] Figure 11 The figure shows the NO content of HUVEC after treatment with different concentrations of selenium-containing ACE inhibitory peptides according to the present invention; where (a) is PQM and (b) is PQML.

[0030] Figure 12 The graph shows the ET-1 content of HUVEC after treatment with different concentrations of selenium-containing ACE inhibitory peptides according to the present invention; where (a) is PQM and (b) is PQML. Detailed Implementation

[0031] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0032] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0033] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0034] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0035] In this article, "room temperature" is usually 20℃ to 33℃, for example, it can be 25℃ to 30℃, specifically 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃ or 33℃.

[0036] Throughout this document, the terms “peptide” or “polypeptide” have meanings well-known to those skilled in the art. Typically, a peptide or polypeptide is two or more amino acids linked by an amide bond, which is formed by the amino group of one amino acid and the carboxyl group of an adjacent amino acid. Polypeptides described herein may contain naturally occurring or non-naturally occurring amino acids. They can be modified into analogs, derivatives, functional mimics, pseudopeptides, and other compounds containing at least two amino acids. A polypeptide with a particular amino acid sequence may include modified amino acids and / or additional amino acids, unless the N- and / or C-terminus contain modifications that prevent the further addition of amino acids. Such modifications include, for example, N-terminal acetylation and / or C-terminal amidation.

[0037] In this document, the polypeptides may be modified, and modifications (generally without altering the primary structure) include: chemically derived forms of polypeptides, either in vivo or in vitro, including but not limited to acetylation, carboxylation, alkylation, acylation, and carbamylation. Modifications also include glycosylation, such as those polypeptides resulting from glycosylation modifications performed during or after polypeptide synthesis and processing. This modification can be accomplished by exposing the polypeptide to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include polypeptides modified to improve their resistance to proteolytic hydrolysis or optimize their solubility. The polypeptides of this invention may be recombinant polypeptides or synthetic polypeptides. The polypeptides of this invention may be chemically synthesized or recombinant. Accordingly, the polypeptides of this invention can be artificially synthesized using conventional methods or produced using recombinant methods. A preferred method is to use liquid-phase synthesis or solid-phase synthesis techniques. Another method is to produce the polypeptides of this invention using recombinant techniques. The polynucleotides of this invention can be used to express or produce recombinant polypeptides of this invention using conventional recombinant DNA techniques. Because the peptides of this invention are relatively short, it is possible to chain multiple peptides together, recombinantly express them to obtain the expression product, and then form the desired small peptides through methods such as enzymatic digestion. The peptides disclosed in this invention, including their salts, can also exist in their hydrated form or in the form of solvents containing them (e.g., ethanol, DMSO, etc.), and can be used for crystallization.

[0038] In this document, the term "amino acid" includes the standard 20 genetically encoded amino acids and their corresponding stereoisomers in "D" form (compared to the natural "L" form), ω-amino acids, other naturally occurring amino acids, unconventional amino acids (e.g., α, α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derived amino acids. When amino acids are explicitly listed as "alanine" or "Ala" or "A," the term refers to L-alanine and D-alanine, unless otherwise explicitly stated. Other unconventional amino acids may also be suitable components of the polypeptides of the present invention, provided that the desired functional properties are retained by the polypeptide. For the peptides shown, each encoded amino acid residue is represented by a single-letter name where appropriate, the single-letter name corresponding to the common name of a conventional amino acid. "Met" in the amino acid sequence stands for methionine.

[0039] In this article, the word “including” is used to mean “including but not limited to”.

[0040] In this article, the term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents. This term refers to pharmaceutical carriers that do not induce antibodies harmful to the individual receiving the composition and do not cause excessive toxicity after administration. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0041] In this article, "IC" is used. 50 "50% inhibition concentration" refers to the concentration of a drug or inhibitor required to inhibit a specified biological process by half.

[0042] In this article, “prevention” as used means preventing the onset of a disease and / or its accompanying symptoms or protecting a subject from acquiring a disease. “Treatment” as used includes delaying and halting the progression of a disease, or eliminating the disease, and does not require 100% suppression, eradication, or reversal.

[0043] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0044] This invention provides a selenium-enriched chickpea peptide, using Bacillus natto as the fermentation strain and chickpeas as the substrate, to design an ACE-inhibiting peptide produced through selenium-enriched fermentation. The yield of the selenium-enriched ACE-inhibiting peptide was increased by regulating fermentation conditions. Quantitative peptidomics was used to analyze the structural characteristics and ACE binding mechanism of the selenium-enriched peptide. Its physicochemical properties were characterized using isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), and fluorescence spectroscopy. Furthermore, its mechanism of action was explored through inhibition kinetics and molecular docking. This research provides a theoretical basis for the green production of functional antihypertensive peptides and promotes their industrial application in selenium-enriched functional foods and nutritional interventions.

[0045] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0046] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. In the following examples, chickpea flour was purchased from the Institute of Food Crops, Xinjiang Academy of Agricultural Sciences; angiotensin-converting enzyme (ACE) was purchased from Shanghai Maclean Biotechnology Co., Ltd.; FAPGG was purchased from Shanghai Maclean Biotechnology Co., Ltd.; HEPES buffer was purchased from Beijing Wanjia Shouhua Biotechnology Co., Ltd.; and all other raw materials used, unless otherwise stated, are conventional commercially available products with specifications in the art.

[0047] I. Extraction of crude polypeptides from selenium-containing chickpeas

[0048] 1. Extraction of chickpea protein (CPI)

[0049] 1) Accurately weigh 50g of chickpea flour, add 500mL of petroleum ether, stir at room temperature for 1h, let the resulting mixture precipitate for 1h, repeat defatting twice to obtain defatted chickpea flour, dry overnight, and store in a refrigerator at 4℃.

[0050] 2) Mix defatted chickpea flour and water at a liquid-to-solid ratio of 1:10 (g / mL). Adjust the pH to 9.0 with 0.5 mol / L NaOH, stir for 1 hour, centrifuge at 4,500 rpm for 20 minutes, collect the supernatant, suspend the resulting precipitate in half a volume of water, extract again, combine the two supernatants, adjust the pH to 4.5 with 0.5 mol / L HCl, centrifuge at 4,500 rpm for 20 minutes, collect the precipitate, wash twice with deionized water, resuspend in 200 mL of deionized water, adjust the pH to 7.0, and freeze-dry to obtain chickpea protein. Store the obtained chickpea protein at -20°C for later use.

[0051] 2. Preparation of selenium-enriched fermentation broth

[0052] 1) Weigh 1g of chickpea protein obtained in step 1 and dissolve it in 10mL of water. After sterilization, let it stand at room temperature to obtain CPI culture medium, and store it for later use.

[0053] 2) Take 10 mL of Bacillus natto bacterial suspension and inoculate it into LB medium. Incubate at 37°C for 24 h, activating it for three generations to ensure the Bacillus natto is in the logarithmic growth phase. Continue activation for 6 h after the third generation, then add 1.5 mL of LB medium at 1% of the inoculum amount. -5Selenium-enriched Bacillus natto suspension was obtained by culturing selenium-enriched Bacillus natto in sodium selenite (Na2SeO3) for 24 h.

[0054] 3) On a clean bench, add the selenium-enriched Bacillus natto bacterial suspension prepared in step 2) to the CPI culture medium obtained in step 1) at an inoculation rate of 3%, ferment for 36 h, centrifuge at 10,000 r / min for 15 min, filter the supernatant through a 0.45 μm aqueous filter membrane to obtain selenium-enriched Bacillus natto fermentation broth (Se-CPIF), and store it at 4℃ for later use.

[0055] 3. Extraction of crude polypeptides from selenium-containing chickpeas

[0056] CPIF and Se-CPIF supernatants were fractionated sequentially using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa, 3 kDa, and 1 kDa. The fermentation supernatant was first passed through a 10 kDa ultrafiltration membrane, then the filtrate through a 3 kDa ultrafiltration membrane, and finally through a 1 kDa ultrafiltration membrane. Centrifugation was performed at 4°C and 3,500 g for 10 min. After ultrafiltration and centrifugation, fractions with molecular weights >10 kDa, 3–10 kDa, 1–3 kDa, and <1 kDa were obtained. These fractions were collected and freeze-dried to obtain selenium-containing chickpea crude peptides for later use.

[0057] 4. Quantitative peptidomics method for identifying the structure of selenium-containing chickpea crude polypeptides

[0058] The structures of selenium-containing chickpea peptides synthesized by the Fmoc solid-phase method were screened and identified using membrane ultrafiltration combined with high-throughput quantitative peptidomics. The specific steps included:

[0059] 1) Desalting and purification of peptide samples. The selenium-containing crude chickpea peptides obtained in step 3 were desalted using a C18 solid-phase extraction column to remove interfering impurities and enrich the target peptides, providing a sample basis for subsequent mass spectrometry analysis.

[0060] 2) Mass spectrometry analysis. High-resolution liquid chromatography-tandem mass spectrometry (LC-MS / MS, Orbitrap platform) was used to separate peptide samples and acquire mass spectrometry data. DDA mode was used to obtain information on the parent ion and its fragments, and peptide sequence characteristics were obtained.

[0061] 3) Database Identification and Quantitative Analysis. Bioinformatics software (such as PEAKS Studio) was used to analyze the mass spectrometry data, and peptide sequences were identified by matching them to the Uniprot chickpea database. The identified peptide sequence information includes the peptides PQM and PQML shown in Table 1. The abundance of each peptide was calculated using non-standard quantitative methods to achieve relative quantitative analysis.

[0062] Table 1 Amino acid sequence list

[0063] name amino acid sequence Serial Number PQM PQSe-Met SEQ ID NO.1 PQML PQSe-MetL SEQ ID NO.2

[0064] 4) Functional Prediction and Screening. For the identified peptides, the activity of the peptide sequences obtained from the mass spectrometry identification of selenium-containing crude chickpea peptides was predicted using the PeptideRanker website (http: / / distilldeep.ucd.ie / PeptideRanker / ). After inputting the peptide amino acid sequence into the system, an activity score between 0 and 1 was obtained; the higher the score, the greater the potential to become a bioactive peptide. Furthermore, the peptides were compared with known bioactive peptide sequences in the "Bioactive peptides" module of the BIOPEP database (http: / / www.uwm.edu.pl / biochemia / index.php / pl / biopep) to predict their potential functional activities.

[0065] 5. Activity verification of selenium-containing chickpea crude peptides

[0066] Using FAPPGG as a mimic substrate for Ang I, the detection principle is as follows: ACE hydrolyzes FAPPGG to release N-[3-(furyl)acryloyl]-2-phenylalanine (N-[3-(2-furyl)acryloyl]-L-phenylalanyl (FAP) and glycylglycine (GG). As the peptides are released, the absorbance of FAPPGG at 340 nm decreases. The ACE inhibition rate can be obtained by measuring the absorbance at 340 nm. Using Design-Expert 8.0.6.1 software, and following the determination methods in Table 2 and the aforementioned extraction methods, the inhibitory effect of selenium-containing chickpea crude peptides on ACE activity was detected under different inoculum amounts (1%, 3%, 5%, 7%, and 9%), material-to-liquid ratios (10:1, 15:1, 20:1, 25:1, and 30:1), fermentation times (24, 30, 36, 42, and 48 h), and fermentation temperatures (27, 32, 37, 42, and 47 °C). The specific steps are as follows:

[0067] Table 2 shows the ACE inhibition rate determined by FAPGG using simulated substrates.

[0068] reagents sample / μL Blank control / μL ACE (0.1 U / mL) 10 10 FAPGG (1 mmol / L) 50 50 Selenium-containing chickpea crude polypeptide 40 0 HEPES buffer (80 mmol / L, pH 8.3) 0 40

[0069] The absorbance of the blank control and the sample at 340 nm was measured and recorded as A1 and A2, respectively. Then, the 96-well plate was placed in a shaking incubator at 37℃ for 30 min, and the absorbance was measured again and recorded as A3 and A4. Three parallel experiments were performed, and the average value of the results was taken. The ACE inhibition rate (E, %) was calculated according to formula (1).

[0070]

[0071] In the formula:

[0072] ΔA a The change in absorbance of the blank control over 30 minutes is expressed as ΔAa = A1 - A3; ΔA b ΔA represents the change in absorbance of the sample over 30 minutes. b =A2-A4.

[0073] The results of the determination of the inhibition of ACE activity by selenium-containing chickpea crude peptides under different conditions are as follows: Figure 1 As shown. By Figure 1 As shown in (a), the ACE inhibition rate reaches its maximum of 85% when the inoculum size is 3%. At lower inoculum sizes, the bacterial count is lower, resulting in limited protease secretion and a lower yield of selenium-containing chickpea crude polypeptides from protein hydrolysis, thus leading to a lower ACE inhibition rate. As the inoculum size gradually increases, the growth of Bacillus natto accelerates, and protease secretion increases, thereby promoting the production of selenium-containing chickpea crude polypeptides and increasing the ACE inhibition rate. However, when the inoculum size further increases, the limited nutrients in the culture medium restrict the growth of Bacillus natto and the secretion of protease, thereby reducing the production of selenium-containing chickpea crude polypeptides and causing a decrease in the ACE inhibition rate. Figure 1 As shown in (b), the ACE inhibition rate reaches its maximum of 89% when the liquid-to-solid ratio of distilled water to chickpea flour is 15:1 (mL / g). When the extraction liquid volume is too small, the water content in the culture medium is low, which is unfavorable for the growth and proliferation of selenium-enriched Bacillus natto, and the amount of protease produced is also low. Consequently, the content of selenium-enriched chickpea crude polypeptides produced by enzymatic hydrolysis is also low, resulting in a low ACE inhibition rate. As the extraction liquid volume gradually increases, it becomes suitable for the growth and proliferation of selenium-enriched Bacillus natto, and the ACE inhibition rate gradually increases. However, as the extraction liquid volume continues to increase, the nutrients in the culture medium are diluted, making it difficult for selenium-enriched Bacillus natto to meet its own nutritional needs for growth, and the ACE inhibition rate gradually decreases. Figure 1As shown in (c), the ACE inhibition rate reaches its maximum of 84% when the fermentation time is 36 hours. When the fermentation time is short, the protease is not fully released, resulting in a low content of selenium-enriched chickpea crude polypeptides released from the enzymatic hydrolysis. The content of selenium-enriched chickpea crude polypeptides gradually increases with the extension of fermentation time, leading to an increased ACE inhibition rate. When the fermentation time reaches a certain point, the selenium-enriched Bacillus natto enters a stable growth phase, and the small portion of selenium-enriched chickpea crude polypeptides produced during the early fermentation stage is decomposed by the protease, losing their ACE inhibitory activity, thus decreasing the ACE inhibition rate. Therefore, the optimal fermentation time is 36 hours. Figure 1 As shown in Figure (d), the ACE inhibition rate reaches its maximum value of 84% when the fermentation temperature is 37℃. When the fermentation temperature is too high or too low, the growth of selenium-enriched Bacillus natto is inhibited, the protease activity is weakened, the metabolic capacity is poor, the amount of selenium-enriched chickpea crude polypeptide produced by enzymatic hydrolysis is less, and the ACE inhibition rate is lower.

[0074] II. Solid-phase synthesis of selenium-containing chickpea peptides

[0075] The selected peptides (PQM, amino acid sequence shown in SEQ ID NO.1 of Table 1; PQML, amino acid sequence shown in SEQ ID NO.2 of Table 1) were synthesized in vitro using the Fmoc solid-phase method, following the C-terminus to N-terminus sequence. Dried crude peptides were prepared through amino acid ligation, peptide chain ligation, and peptide chain shearing. The chemically synthesized crude peptides were purified and analyzed using high-performance liquid chromatography coupled with high-resolution mass spectrometry. The peak region of the target peptide was determined by detecting the ion size of the target peptide based on its mass-to-charge ratio.

[0076] Test results as follows Figure 2 and Figure 3 As shown, the purity of the synthetic peptide PQML was 95.923%, and its relative molecular mass was 534.47 g / mol; the purity of the synthetic peptide PQM was 95.035%, and its relative molecular mass was 421.31 g / mol. These results indicate that the target compounds were obtained, and the synthesized peptides are suitable for subsequent activity assays, structural identification, or pharmacodynamic experiments.

[0077] The samples with a purity greater than 95% were freeze-dried to obtain pure selenium-containing chickpea peptides PQM and PQML.

[0078] III. Performance Evaluation of Selenium-Containing Chickpea Peptides

[0079] 1. Determination of the inhibitory effect of selenium-containing chickpea peptides on ACE activity

[0080] To further verify the inhibitory activity of the two synthesized pure selenium-containing chickpea peptides on ACE, the ACE inhibition rates of the two selenium-containing chickpea peptides (PQM and PQML) at different concentrations (0.1, 0.25, 0.5, 1, 2 and 3 mg / mL) were determined according to the above steps.

[0081] Test results as follows Figure 4 As shown, the calculated IC50 of selenium-containing chickpea peptide PQML is... 50 The concentration was 0.9327 mmol / L, and the IC50 of selenium-containing chickpea peptide PQM was... 50 The concentration was 0.9777 mmol / L. Based on the comprehensive experimental data analysis, both selenium-containing chickpea peptides synthesized by this method showed good ACE inhibitory activity and have potential application value.

[0082] 2. Determination of the thermodynamic properties of the interaction between selenium-containing chickpea peptides and ACE by isothermal titration calorimetry

[0083] Isothermal titration calorimetry (ITC) analysis, used in drug research, has been found to be an effective tool for analyzing the thermodynamic parameters (ΔG, ΔH, and ΔS) of peptide-ACE interactions and the dissociation constant K. d To illustrate the combination between them.

[0084] 300 μL of ACE (2 × 10⁻⁶) -4 Add 50 μL of 0.05 mmol / L peptide solution (PQM and PQML) to the sample cell, and aspirate 50 μL of PQM and PQML peptide solution into the syringe. Once the temperature stabilizes at 298 K, precisely add 50 μL of peptide solution in a stepwise loading mode, totaling 20 drops (2.5 μL each), at 120 s intervals, with a stirring speed of 350 rpm. The control group is HEPES buffer.

[0085] The isothermal titration calorimetry curves of the interaction between PQM and PQML and ACE are as follows: Figure 5 As shown, when the peptide sample is added dropwise to the calorimeter, PQM and PQML react with ACE, generating heat changes, which are presented by the heat flow curve. As PQM and PQML are continuously added, the binding of ACE with PQM and PQML tends to saturate, thus the peak value of the heat flow curve continuously decreases and eventually tends to stabilize. The values ​​of the heat flow curve are all negative, indicating that the reaction is exothermic. The thermodynamic parameters were analyzed using NanoAnalyze software, and the relevant data are shown in Table 3.

[0086] Table 3. Thermodynamic parameters of PQM, PQML and ACE binding

[0087]

[0088] According to the Gibbs free energy equation ΔG = ΔH – TΔS, the change in Gibbs free energy ΔG consists of an enthalpy change (ΔH) and an entropy change (-TΔS). ΔG < 0 and |-TΔS| < |ΔH| indicate that the reaction is a spontaneous reaction driven primarily by enthalpy change, and can proceed spontaneously under any conditions. ΔH < 0 indicates that the binding process of PQM, PQML, and ACE is exothermic. In biomolecular recognition, intermolecular interactions mainly encompass several key mechanical mechanisms, including electrostatic forces, hydrogen bonds, van der Waals forces, and hydrophobic interactions. Systematic analysis of thermodynamic parameters can reveal the binding patterns between small molecules and proteins, and further determine the contribution of these forces to the overall binding energy. ΔS < 0 and ΔH < 0 reflect the formation of van der Waals forces and hydrogen bonds in the binding of PQM, PQML, and ACE. Furthermore, the dissociation constant K... d The affinity of the peptide for ACE was characterized. Table 3 shows that the Ka of PQM-ACE... d The value is 2.248 × 10 -6 PQML-ACE's K d The value is 1.93 × 10 -6 K d The smaller the binding site, the more stable the binding. Compared to the PQM-ACE complex, the PQML-ACE complex is more stable and has a stronger ability to inhibit ACE activity. Furthermore, under 298K conditions, the number of binding sites (n) between the peptide and ACE is greater than 1, indicating the possibility of multiple binding sites on the ACE. PQM has 4.734 binding sites, while PQML has 6.217. A higher number of binding sites indicates that PQML is more likely to bind to ACE, which coincides with the K... d The analysis results correspond.

[0089] 3. Spectroscopic study on the interaction between selenium-containing chickpea peptides and ACE.

[0090] Circular dichroism (CD) chromatography, as an effective tool for characterizing conformational changes in biomolecules, can accurately resolve the dynamic evolution of protein secondary structure induced by ligand binding. To study the secondary structure of peptides, 200 μL of HEPES buffer was added for background testing. Selenium-enriched chickpea ACE synthetic peptides were prepared to a concentration of 1 mg / mL using HEPES buffer. 200 μL of the peptide sample was placed in a quartz cuvette at 25 °C, with a scan rate of 100 nm / min. Each sample was scanned three times, and its spectrum from 190 to 260 nm was recorded. The final CD spectrum was obtained by averaging the three consecutive scans. The results were smoothed, and the secondary structure content was calculated using the Chirascan software.

[0091] The secondary structure of proteins is a crucial factor influencing enzyme binding probability. The effect of peptides on the secondary structure of the original ACE was investigated using CD spectroscopy. The CD spectra of selenium-containing chickpea peptides after interaction with ACE were shown in the 190-260 nm range. Figure 6 As shown, the CD spectrum of the original ACE has a strong positive band at 195 nm, a zero crossover at 197 nm, and a strong negative band at 200 nm. These are characteristic absorption peaks of the α-helix structure. After adding selenium-containing chickpea peptide, the zero crossover point red-shifts, and the ellipticity value increases simultaneously, indicating the loss of the α-helix structure. This may be due to the flexible structure of ACE, which can contact a large number of peptides, resulting in the unfolding of the protein.

[0092] As shown in Table 4, ACE without selenium-containing chickpea peptides consists of 16.67% α-helices, 40.41% β-sheets, 16.99% β-turns, and 25.85% random coils. When the peptide binds to ACE, the content of β-turns and random coils does not change significantly. However, the binding of PQM and PQML to ACE leads to a decrease in the content of α-helices, β-sheets, and β-turns, and an increase in the content of random coils. This indicates that PQM and PQML bind to the amino acid residues of ACE, disrupting the hydrogen bond network of ACE, and the ACE structure may be unfolding, resulting in a stronger inhibitory effect on ACE activity.

[0093] Table 4. Secondary structure content of ACE in the presence and absence of polypeptides. A fluorescence spectroscopy-based detection platform was established to quantitatively characterize the binding efficacy of bioactive ligands to target proteins. Selenium-containing chickpea peptides PQM and PQML were prepared at gradient concentrations (0–450 μM, gradient difference 150 μM), and their spectral characteristics under intrinsic fluorescence were measured. Subsequently, they were thoroughly mixed with ACE (0.0718 μM), and 300 μL of the mixed sample was added to a quartz cuvette for measurement. The excitation source was calibrated to 280 nm, and the emission monochromator detection range was set to 300–500 nm. During the experiment, the aperture of both monochromators was maintained at 5 nm, and each sample underwent three independent scans.

[0094] The inherent fluorescence spectrum of ACE is produced at 280 nm, mainly attributed to three aromatic amino acid residues: tyrosine (Tyr), phenylalanine (Phe), and tryptophan (Trp), especially the Trp residue. Figure 7As shown, when different concentrations of two selenium-containing chickpea peptides were added at the same ACE concentration, the fluorescence intensity at 367 nm showed a dose-dependent decrease with increasing selenium-containing chickpea peptide concentration, and the fluorescence intensity was negatively correlated with the selenium-containing chickpea peptide concentration, indicating that the fluorescence intensity gradually decreased with increasing concentrations of the two selenium-containing chickpea peptides. This phenomenon is presumably related to the formation of complexes between ACE and selenium-containing chickpea peptides, which in turn causes the aromatic residues in ACE to be shielded, leading to fluorescence quenching. Therefore, with increasing concentrations of the two selenium-containing chickpea peptides, the formation of ACE-PQM and ACE-PQML peptide complexes gradually increases, further leading to a decrease in fluorescence intensity.

[0095] 4. Molecular docking analysis of selenium-containing chickpea peptides with ACE

[0096] The peptide structure was predicted using PEP-FOLD3 based on peptide sequence information (PQM, PQML), and named according to the peptide sequence. Subsequently, the peptide structure was optimized using the MOPAC program, and atomic charges were calculated based on the PM3 method for subsequent molecular docking studies. The ligand structure was processed using AutoDock Tools 1.5.6 to generate a pdbqt file for docking calculations.

[0097] The ligand-receptor interaction was simulated using AutoDock 4.2.6 software, with the center of the docking grid set as the protein active site to ensure coverage of the entire binding site. The grid size was set to 100×100×100, and 50 independent docking operations were performed. Other parameters used their default values. To improve the stability of the calculated complex, further conformational optimization was performed. The Amber14 force field was used to minimize the energy of the molecular system. The optimization process consisted of two stages: first, a 1000-step steepest descent method was performed to eliminate high-energy conformations; then, a 5000-step conjugate gradient method was performed to obtain a convergent low-energy conformation. The final result served as the model for subsequent analyses.

[0098] To explore the molecular mechanism of the interaction between PQM, PQML, and ACE, docking simulation studies were conducted using the flexible docking tool of AutoDock 4.2.6 software. Table 5 presents the molecular docking energy scores for peptides and ACE. According to the predictions of AutoDock software, the results show that the binding energies between ACE and the selenium-containing chickpea peptide PQM and PQML are -7.845 kcal / mol and -8.929 kcal / mol, respectively. Lower binding energies indicate more stable binding between the peptide and ACE, and the PQML-ACE complex is more stable. This result is consistent with the IC50-2000-2000 (IC50-2000) results. 50 Consistent with the ITC experimental results, this demonstrates the consistency of the binding strength of the two peptides to ACE across different experimental methods. Peptides with lower binding energies show greater binding strength in IC50. 50It exhibited stronger inhibitory activity and more stable binding in ITC experiments, which further verified the accuracy of the binding energy calculation and the mutual support between the results obtained from different experimental techniques.

[0099] Table 5. Molecular docking scoring results

[0100]

[0101] The results of ACE-PQM molecular docking are as follows Figure 8 As shown, PQM is deeply embedded in the narrow channel of the ACE active site. The selenium-containing chickpea peptide is stably bound within the ACE cavity composed of amino acids Trp279, Val380, Thr166, Asn374, Asp377, Ala354, Gln369, His513, Tyr523, Lys511, Phe457, Gln281, His353, Glu162, Cys352, Cys370, and Tyr520. Interaction analysis revealed that the peptide interacts with surrounding amino acids through hydrophobic interactions and hydrogen bonds, promoting its stable binding to ACE. Molecular docking studies showed that the purified peptide PQM binds to the ACE residue His353. Glu162 Cys352 Cys370 and Tyr520 Five amino acids formed hydrogen bonds, and PQM also formed hydrophobic interactions with 12 amino acids surrounding the ACE pocket: Asn374, Thr166, Asp377, Trp279, Val380, Ala354, Gln281, Lys511, Gln369, His513, Tyr523, and Phe457. These results indicate that PQM may interact with the active pocket of ACE, further enhancing the affinity of selenium-containing chickpea peptides for ACE and inhibiting ACE activity. In this study, the PQM binding sites Cys352, Cys370, Asn374, Thr166, Asp377, Trp279, Val380, Gln369, and Phe457 are not amino acid residues at the central active site of ACE; therefore, the interaction between PQM and ACE is non-competitive. The docking results between ACE and PQML are shown below. Figure 9As shown in the figure, PQML is deeply embedded in the narrow channel of the ACE active site. The selenium-containing chickpea peptide can stably bind to the cavity in ACE composed of amino acids Thr166, Glu376, Asp453, Trp279, Phe457, Tyr523, Phe527, Val380, Cys352, His353, Ala354, Cys370, Gln369, Glu162, Ala170, Asp377, Asn285, Tyr520, Lys511, Gln281, and Asn374. Further analysis of the interaction between the two shows that hydrophobic interactions and hydrogen bonds are formed between the peptide and the amino acids surrounding the pocket, promoting the stable binding of the peptide to ACE. Specifically, PQML binds to the Asn285 amino acids surrounding ACE. Tyr520 Lys511 Gln281 and Asn374 Six groups of hydrogen bonds were formed between amino acids. At the same time, PQML also formed hydrophobic interactions with 16 amino acids around the ACE pocket, namely Ala170, Asp377, Glu162, Gln369, Cys370, Ala354, Thr166, Glu376, His353, Cys352, Asp453, Trp279, Val380, Phe457, Phe527, and Tyr523, further enhancing the affinity between the peptide and ACE. In this study, the PQML binding sites Asn285, Asn374, Ala170, Asp377, Gln369, Cys370, Thr166, Glu376, Cys352, Asp453, Trp279, Val380, Phe457, and Phe527 are not amino acid residues of the ACE central active site. Therefore, the interaction between PQML and ACE is non-competitive. Meanwhile, PQM and PQML share multiple common binding sites with amino acid residues at the active site of ACE: Trp279, Val380, Thr166, Asn374, Asp377, Ala354, Gln369, Tyr523, Lys511, Phe457, Gln281, His353, Glu162, Cys352, Cys370, and Tyr520. This indicates that PQM and PQML may have similar binding modes to the active site of ACE, meaning that they may have similar molecular structures or functional groups in their spatial conformation, enabling them to interact with these specific amino acid residues. This suggests that they have similar molecular mechanisms of action in the ACE inhibition mechanism, possibly through hydrogen bonding, hydrophobic interactions, or other intermolecular forces, thereby stabilizing themselves at the active site of ACE. In addition, these common binding sites (such as Trp279, Val380, and His353) are key residues for ACE function, indicating that PQM and PQML may be potential drug molecules that can regulate ACE activity by binding to key ACE sites, thereby affecting physiological processes such as blood pressure regulation. PQM occupies 6 active site pockets, while PQML occupies 7, further demonstrating that PQML has a better inhibitory effect on ACE.

[0102] 5. Effects of selenium-containing chickpea peptides on the viability of human endothelial venous cells (HUVECs)

[0103] (1) Human endothelial vein cell culture

[0104] At 25cm 2HUVECs were cultured in culture flasks using DMEM medium (complete medium) containing 10% FBS and 1% antibiotics. The cells were incubated at 37°C in a 5% CO2 incubator, with the medium changed every 2–3 days. When cell confluence reached over 80%, the cells were digested with 0.25% trypsin solution. Microscopic observation revealed some cells were rounded. Complete medium was added to stop the digestion. The cells were gently pipetted and centrifuged at 1000 rpm for 3 minutes to collect the cells. The cells were then passaged or seeded into 96-well plates for further experiments.

[0105] (2) Cytotoxicity assay

[0106] Toxicity assays were performed according to the CCK-8 kit instructions. 100 μL of HUVECs (1 × 10⁻⁶) were added. 5 Cell suspensions (cells / mL) were seeded into 96-well plates and cultured adherently for 24 h. After the culture medium was discarded, 100 μL of selenium-containing chickpea peptide (PQM and PQML) solutions (0.1, 0.5, 1, 5, and 10 μM) were added to the sample groups. The blank control group was replaced with complete culture medium, and the positive control group was captopril. After culturing for another 4 h, 10 μL of CCK-8 solution was added to each well. The 96-well plates were incubated in an incubator for 4 h, and the OD value at 450 nm was detected by a microplate reader. Cell viability was calculated as shown in formula (2):

[0107]

[0108] A s Test wells (containing cells, culture medium, selenium-containing chickpea peptides, and CCK-8)

[0109] A c Control wells (containing cells, culture medium, and CCK-8)

[0110] A b Blank wells (containing culture medium and CCK-8)

[0111] Test results are as follows Figure 10 As shown, 10(a) shows the test results of the activity of selenium-containing chickpea peptide PQM on HUVEC cells. In the concentration range of 0.5 to 5 μM, the cell survival rate was above 95%, indicating that PQM has no toxic effect on HUVEC cells in this concentration range. Figure 10 (b) The results of the activity test of selenium-containing chickpea peptide PQML on HUVEC cells show that the cell viability was above 95% within the concentration range of 0.5–5 μM, indicating that PQML has no toxic effect on HUVEC cells within this concentration range. The positive control captopril showed no significant toxic effect on the cells. Therefore, subsequent activity evaluation experiments will be conducted on selenium-containing chickpea peptides PQM and PQML within the concentration range of 0.5–5 μM.

[0112] 6. Effect of selenium-containing chickpea peptides on NO content

[0113] The effect of selenium-containing chickpea peptides on NO content was detected using a nitric oxide (NO) assay kit (enzymatic method, catalog number A012-1-2). 100 μL of HUVECs (1×10⁵ cells / mL) suspension was seeded into 96-well plates and cultured for 24 h before the culture medium was discarded. 100 μL of ACE inhibitory peptide (PQM and PQML) solutions (0.1, 0.5, 1, 5, 10 μM) were added to the sample groups, double-distilled water was added to the blank groups, and captopril was used as the positive control. The plates were incubated for 24 h in a 5% CO₂ incubator at 37°C. 100 μL of the supernatant was collected, 200 μL of reagent one was added, and the mixture was stirred. Then, 100 μL of reagent two was added, and the mixture was vortexed thoroughly. After standing for 10 min, the plates were centrifuged at 1000 rpm for 15 min, and 160 μL of the supernatant was collected. The procedure is shown in Table 6 below.

[0114] Table 6 NO Content Determination Table

[0115] blank hole Standard Hole Measurement well Double distilled water 0.16 20 μmol / L 0.16 Supernatant 0.16 color developer 0.08 0.08 0.08

[0116] Add the solution to the 96-well plate according to the operation table, mix well, let stand for 15 min, and then measure the OD value at 550 nm using a microplate reader. Each experiment was repeated in triplicate. The NO content in the cells was calculated as shown in formula (3):

[0117]

[0118] Note: C 标准 The standard concentration is 20 μmol / L; N is the dilution factor in the pretreatment process for sample dilution, which is 4 for serum.

[0119] Test results as follows Figure 11 As shown, by Figure 11 (a) As shown, compared with the control group, the NO content in cells was significantly increased after treatment with captopril and different concentrations of selenium-containing chickpea peptide (P<0.05). When cells were treated with 0.2 μM, 1 μM, or 5 μM selenium-containing chickpea peptide, the release of NO from cells was significantly increased by 14.58%, 33.62%, and 3.66%, respectively. Notably, the NO content in the 1 μM PQM treatment group was significantly better than that in the captopril positive control, indicating that PQM has a very significant antihypertensive effect. These results indicate that PQM can promote the release of NO from endothelial cells, thereby achieving an antihypertensive effect. Figure 11(b) It was found that, compared with the control group, the NO content in cells was significantly increased after treatment with captopril and different concentrations of selenium-containing chickpea peptide (P<0.05). When cells were treated with 0.2 μM, 1 μM, or 5 μM selenium-containing chickpea peptide, the release of NO from cells was significantly increased by 39.77%, 59.31%, and 13.36%, respectively. Notably, the NO content in the 1 μM PQML treatment group was significantly better than that in the captopril positive control, indicating that PQML has a very significant hypotensive effect. These results indicate that PQML can promote the release of NO from endothelial cells, thereby achieving a hypotensive effect.

[0120] 7. Effect of selenium-containing chickpea peptides on ET-1 content

[0121] 100 μL of HUVECs (1×10⁻⁶) 5 Cell suspensions (cells / mL) were seeded into 96-well plates and cultured adherently for 24 h, after which the culture medium was discarded. Sample groups were treated with 100 μL of ACE inhibitory peptide (PQM and PQML) solutions (0.1, 0.5, 1, 5, 10 μM), blank groups were treated with culture medium, and the positive control group received captopril. Incubation was continued for 24 h in a 5% CO2 incubator at 37°C. Centrifuge the cell supernatant from each group at 4000 rpm for 20 min in a high-speed centrifuge. Then, transfer all reagents from the Human Endothelin 1 (ET-1) Quantitative Detection Kit (ELISA) to room temperature for 2 h to equilibrate. Take 5 mL of concentrated washing buffer and dilute it with distilled water at a ratio of 1:20 to 100 mL. Mix well and set aside. Remove the plate to be coated from the sealed bag. Set up one blank control well without adding any liquid. According to the calibrator concentration (0, 10, 20, 40, 80, and 160 pg / mL), set up two wells for each calibrator concentration. Add 15 μL of the corresponding calibrator to each well. Add 15 μL of the test supernatant directly to each of the remaining test wells. Add 100 μL of Asaay Diluent to all wells except the blank wells. Mix well, attach the sealing film, and incubate at 37℃ for 60 min. Discard the liquid in the wells, add 300 μL of washing buffer, let stand for 10 s, and then shake dry. Repeat three times and then pat dry. Next, add 100 μL of SA-HRP to each well (except for the blank control wells), mix well, cover with a sealing film, and incubate at 37°C for 60 min. Discard the liquid in the wells again, add 300 μL of washing buffer, let stand for 10 s, then shake dry. Repeat three times, then pat dry. Next, add 100 μL of TMB to each well, vortex to mix, and incubate at 37°C in the dark for 15 min. Add 100 μL of stop solution to each well, and measure the absorbance at 450 nm using a microplate reader. First, zero the plate using the blank control wells, then measure the absorbance of each well. Each experiment was performed in triplicate. Sample concentrations were determined after plotting a standard curve using ELISAcalc software.

[0122] Test results as follows Figure 12As shown, captopril and different concentrations of peptides exhibited significant inhibitory effects on ET-1 formation. Compared with the control group, the ET-1 secretion of cells decreased by 10.41%, 25.72%, and 15.58% in the PQM treatment groups at concentrations of 0.2 μM, 1 μM, and 5 μM, respectively; and the ET-1 secretion of cells decreased by 4.77%, 25.85%, and 15.61% in the PQML treatment groups at concentrations of 0.2 μM, 1 μM, and 5 μM, respectively. More importantly, the inhibitory effect of ET-1 secretion in the 1 μM PQM and PQML treatment groups was significantly better than that in the positive control group captopril, a result similar to that of NO analysis. Therefore, PQM and PQML have similar effects to captopril, possibly exerting a hypotensive effect by inhibiting the release of ET-1 from endothelial cells, but their activity is superior to captopril.

[0123] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A selenium-containing chickpea peptide, characterized in that, The amino acid sequence of selenium-containing chickpea peptide is PQSe-Met or PQSe-MetL.

2. The pharmaceutically acceptable salt of the selenium-containing chickpea peptide according to claim 1.

3. An antihypertensive drug, characterized in that, It comprises a pharmaceutically acceptable salt of the selenium-containing chickpea peptide of claim 1 or the selenium-containing chickpea peptide of claim 2, and a pharmaceutically acceptable carrier.

4. The antihypertensive drug according to claim 3, characterized in that, The selenium-containing chickpea peptide of claim 1 or the selenium-containing chickpea peptide of claim 2 is mixed with a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier to prepare a clinically acceptable injection, oral formulation, transdermal absorption formulation or mucosal absorption formulation.

5. A pharmaceutical composition for treating hypertension, characterized in that, It includes pharmaceutically acceptable salts of the selenium-containing chickpea peptide of claim 1 or the selenium-containing chickpea peptide of claim 2, as well as other antihypertensive drugs.

6. The method for preparing selenium-containing chickpea peptides according to claim 1, characterized in that, It was prepared by the Fmoc solid-phase synthesis method.

7. The use of the selenium-containing chickpea peptide of claim 1, the pharmaceutically acceptable salt of the selenium-containing chickpea peptide of claim 2, the antihypertensive drug of claim 3 or 4, or the antihypertensive drug composition of claim 5 in the preparation of a drug for the prevention and / or treatment of hypertension.

8. The application according to claim 7, characterized in that, The medication for the prevention and / or treatment of hypertension is a drug that promotes the release of NO from endothelial cells.

9. The application according to claim 7, characterized in that, The medication for the prevention and / or treatment of hypertension is a drug that inhibits the release of ET-1 from endothelial cells.

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