A torreya grandis active peptide and its application in the preparation of a product for reducing blood pressure

Abstract

The present invention discloses a torreya grandis active peptide, whose amino acid sequence is Val-Asn-Asp-Tyr-Leu-Asn-Trp. The present invention also discloses its application in the preparation of blood pressure-lowering products. The torreya grandis active peptide provided by the present invention has good ACE inhibitory ability, and the IC 50 is 0.19±0.01mg / mL, and it can be used as a functional active factor for developing health products and drugs with blood pressure-lowering effects. Through the preparation and research of the torreya grandis active peptide, the present invention can more effectively expand the utilization rate of torreya grandis resources and increase its added value, which has great economic and social significance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a torreya grandis active peptide and its application in the preparation of a product for lowering blood pressure. Background Art

[0002] Hypertension is one of the most common chronic diseases, often inducing other diseases such as coronary heart disease, myocardial infarction, stroke, atherosclerosis, etc., seriously threatening human health. [1, 2] Angiotensin-converting enzyme (ACE) is a zinc-containing dipeptide-carboxypeptidase that plays a key role in blood pressure regulation. ACE mainly acts on two systems, leading to an increase in blood pressure. In the renin-angiotensin system (RAS), ACE hydrolyzes the decapeptide angiotensin I (Ang I) without vasoconstrictive function to generate the octapeptide angiotensin II (Ang II). [3] Ang II is a potent vasoconstrictor, and it can act on two types of receptors of angiotensin II (AT1, AT2). [4] When Ang II binds to the AT1 receptor, it can promote the synthesis and release of aldosterone and the production of reactive oxygen species, resulting in an increase in blood pressure. [5] When it acts on the AT2 receptor, it plays a role in dilating blood vessels and lowering blood pressure. In the kallikrein-kinin system (KKS), ACE can inactivate bradykinin with antihypertensive activity. [6] As a result, blood pressure increases. Therefore, inhibiting or inactivating ACE is considered a way to relieve hypertension.

[0003] Torreya grandis Torreya grandis , a plant of the genus Torreya in the family Taxaceae, is a native species in China with a long planting history. [7] It is widely distributed in Zhejiang, Anhui, Jiangxi and other places in China. [8] Torreya grandis seeds are rich in oil (42.61%-54.39%), and the unsaturated fatty acid component is the most (accounting for 76.1 - 82.0% of the total fatty acids), which can be used for oil production. [9] After extracting oil from Torreya grandis seeds, the residual cake contains a large amount of protein, which is an excellent protein resource. However, there is little research on Torreya grandis seed cake protein at present, and the protein resource has not been fully utilized.

[0004] High-quality protein is an important raw material for bioactive peptides. Polypeptides are compounds formed by amino acids linked together by peptide bonds, usually consisting of 2-20 amino acids. Different amino acid compositions and arrangements endow amino acids with different biological activities.

[10] Researchers believe that foods or proteins rich in valine (Val), leucine (Leu), phenylalanine (Phe), proline (Pro), histidine (His), threonine (Thr), arginine (Arg), methionine (Met), lysine (Lys) and tyrosine (Tyr) are high-quality raw materials for preparing ACE inhibitory peptides. [11, 12] The total amount of these amino acids accounts for 50.34% of torreya grandis protein.

[13] Therefore, it is speculated that torreya grandis protein may be a good raw material for producing ACE inhibitory peptides.

[0005] The present invention aims to provide a torreya grandis active peptide and its application in the preparation of antihypertensive products, so as to provide technical support for expanding the utilization rate of torreya grandis resources, increasing its added value, and developing health care products and drugs with antihypertensive functions.

[0006] [1] Yu Zhenqiu, Chen Yun. Interpretation of the ISH 2020 International Hypertension Practice Guidelines [J]. Chinese Journal of Rural Medicine and Pharmacy, 2020, 27(23): 24-25.

[0007] [2] Wang Chunbo, Jiang Shiying. Current situation of hypertension prevention and control and health management strategies among adult residents in China [J]. Journal of Preventive Medicine of Chinese People's Liberation Army, 2020, 38(12): 35-36.

[0008] [3] Yan Ze. Research on the preparation technology of ACE inhibitory peptides from Ostrea tailienwhanensis [Z]. Dalian Ocean University, 2018.

[0009] [4] Pan Shunshun. Study on the preparation of angiotensin-converting enzyme inhibitory peptides from tea and their antihypertensive activities [D]. South China Agricultural University, 2018.

[0010] [5] Xu Z, Wu C, Sun-Waterhouse D, et al. Identification of post-digestion angiotensin-I converting enzyme (ACE) inhibitory peptides from soybean protein Isolate: Their production conditions and in silico molecular docking with ACE [J]. Food Chem, 2021, 345: 128855.

[0011] [6] Hongli Zhou. Research on angiotensin-converting enzyme inhibitory peptides from pumpkin seed protein [D]. Hunan Agricultural University, 2012.

[0012] [7] He Z, Zhu H, Li W, et al. Chemical components of cold pressed kernel oils from different Torreya grandis cultivars [J]. Food Chemistry, 2016, 209: 196 - 202.

[0013] [8] Shi L, Mao J, Zheng L, et al. Chemical characterization and free radical scavenging capacity of oils obtained from Torreya grandis Fort. ex. Lindl. and Torreya grandis Fort. var. Merrillii: A comparative study using chemometrics [J]. Industrial Crops and Products, 2018, 115: 250 - 260.

[0014] [9] Dong D, Wang H, Xu F, et al. Supercritical Carbon Dioxide Extraction, Fatty Acid Composition, Oxidative Stability, and Antioxidant Effect of Torreya grandis Seed Oil [J]. Journal of the American Oil Chemists' Society, 2014, 91(5): 817 - 825.

[0015]

[10] Dong Wei. Identification and activity exploration of ACE inhibitory peptides in distillers' grains [D]. Jiangnan University, 2020.

[0016]

[11] Xingfei L, Shunshun P, Wenji Z, et al. Properties of ACEinhibitory peptide prepared from protein in green tea residue and evaluationof its anti-hypertensive activity[J]. Process Biochemistry, 2020,92:277-287.

[0017]

[12] Rui X, Boye J I, Simpson B K, et al. Angiotensin I-convertingenzyme inhibitory properties of Phaseolus vulgaris bean hydrolysates: Effectsof different thermal and enzymatic digestion treatments[J]. Food researchinternational, 2012,49(2):739-746.

[0018]

[13] Luo X, Wu S, Xue J, et al. The bioactive peptide screening fromTorreya grandis meal protein hydrolysates[J]. Food bioscience, 2021,44:101419. Summary of the Invention

[0019] The object of the present invention is to provide a Torreya grandis active peptide and its application in the preparation of antihypertensive products to solve the deficiencies of the prior art.

[0020] The present invention adopts the following technical solutions:

[0021] In the first aspect of the present invention, a Torreya grandis active peptide is provided, and its amino acid sequence is Val-Asn-Asp-Tyr-Leu-Asn-Trp, abbreviated as VNDYLNW.

[0022] In the second aspect of the present invention, the above-mentioned Torreya grandis active peptide is provided for use in the preparation of antihypertensive products.

[0023] Furthermore, the product is a health product or a medicine.

[0024] Advantages of the present invention:

[0025] The torreya grandis active peptide provided by the present invention has good ACE inhibitory ability, and the IC 50 is 0.19 ± 0.01 mg / mL, and it can be used as a functional active factor for developing health products and drugs with blood pressure-lowering effects. Through the preparation and research of torreya grandis active peptide, the present invention can more effectively expand the utilization rate of torreya grandis resources, improve its added value, and has great economic and social significance. Description of the drawings

[0026] Figure 1 shows the ACE inhibitory activities of each ultrafiltration component (T0 represents the unseparated torreya grandis enzymolysate, and 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL represent the concentrations of each component).

[0027] Figure 2 is the chromatogram and ACE inhibitory activity of the T4 component separated by Sephadex G-25 column.

[0028] Figure 3 is the ACE inhibitory activities of 9 synthetic peptides.

[0029] Figure 4 is the HPLC diagram of VNDYLNW.

[0030] Figure 5 is the mass spectrum diagram of VNDYLNW.

[0031] Figure 6 is the molecular docking diagram of VNDYLNW.

[0032] Figure 7 is the inhibition mode diagram of VNDYLNW. Detailed implementation manners

[0033] The present invention will be further explained below in conjunction with the embodiments and the drawings. The following embodiments are only used to illustrate the present invention, but do not limit the implementation scope of the present invention.

[0034] Example 1 Preparation of torreya grandis active peptide

[0035] (1) Preparation of torreya grandis protein

[0036] First, the torreya grandis seeds are shelled and then cold-pressed and defatted by a hydraulic oil press under the conditions of room temperature, 50 Mpa, and 40 min to obtain torreya grandis seed cake. Then, the obtained torreya grandis seed cake is pulverized, and petroleum ether is used to stir and extract at a liquid-solid ratio of 1:5 (w / v, g / mL) at room temperature and 500 rpm for 2 h, and then allowed to settle at room temperature for 1 h. After extraction twice, the sediment is collected, dried at 50 °C until the water content is lower than 4 wt%, pulverized, and passed through a 60-mesh sieve to obtain torreya grandis powder, which is refrigerated at 4 °C for later use.

[0037] Mix torreya powder with deionized water at a ratio of 1:20 (w / v, g / mL), adjust the pH to 10 with 6 mol / L NaOH, and stir and extract at 50 °C and 500 rpm for 2 h. After the extraction is completed, cool the suspension to room temperature, centrifuge at 4000 rpm for 20 min at room temperature, and collect the supernatant. Adjust the pH of the supernatant to 4 with 6 mol / L HCl to precipitate proteins. After standing for 2 h, centrifuge at 4000 rpm for 20 min at room temperature, collect the precipitate, redissolve it with deionized water, adjust the pH of the solution to 7 with 6 mol / L NaOH, and freeze-dry at -80 °C for 48 h to obtain torreya protein.

[0038] (2)Preparation of torreya enzymatic hydrolysate

[0039] Prepare a 2 wt% aqueous solution of torreya protein with deionized water and let it stand for denaturation at 95 °C for 15 min. After cooling to room temperature, adjust the pH to 10 with 1 mol / L NaOH, then add alkaline protease at 5000 U / g (based on the mass of torreya protein in the solution) (the specification of alkaline protease is 200 U / mg, purchased from Solarbio), and continuously stir and enzymatically hydrolyze at 50 °C and 300 rpm for 4 h. After the enzymatic hydrolysis is completed, immediately let it stand for enzyme inactivation at 95 °C for 15 min, then centrifuge at 8500 rpm for 30 min at 4 °C, and collect the supernatant to obtain torreya enzymatic hydrolysate.

[0040] (3)Isolation and purification of torreya bioactive peptides

[0041] Ultrafilter the torreya enzymatic hydrolysate successively using ultrafiltration membranes with MWCO of 10 kDa, 3 kDa, and 1 kDa to obtain fractions T1: >10 kDa, T2: 3 - 10 kDa, T3: 1 - 3 kDa, T4: <1 kDa. Measure the ACE inhibitory activity of each fraction, as shown in Figure 1 . Purify the T4 fraction using a Sephadex G-15 column. The specific operation is as follows: Equilibrate the Sephadex G-15 column (1.6 cm × 70 cm) with ultrapure water at a flow rate of 0.6 mL / min. Filter the T4 fraction solution (10 mg / mL) using a 0.22 μm aqueous filter membrane, then load 3 mL of the T4 fraction solution onto the well-equilibrated Sephadex G-15 column. Use deionized water as the eluent, collect fractions using an automatic fraction collector at a flow rate of 0.6 mL / min (2 min / tube), and measure the absorbance at a wavelength of 280 nm. See the chromatogram and activity diagram in Figure 2 . The F2 fraction is the obtained torreya ACE inhibitory peptide.

[0042] Example 2 Screening and activity verification of torreya bioactive peptides

[0043] (1)LC-MS / MS Peptide Sequence Determination and Computer Simulation Screening

[0044] In order to clarify the structure-activity relationship of Torreya grandis ACE inhibitory peptides, the present invention selected the LC-MS / MS method to perform de novo sequencing on the F2 fraction, and a total of 359 peptides were obtained. Thirty-seven peptide segments with a peak area greater than 10 8 and a score greater than 95 were analyzed.

[0045] Using the BIOPEP website to speculate, 34 of these peptide segments have potential functional fragments for ACE; the ToxinPred website speculates that all 37 peptide segments are non-toxic; the Peptide Ranker website is used to predict the probability of the peptide segments having biological activity, and a total of 17 peptide segments with a score greater than 0.5 are obtained; the AHTpin website is used to analyze that 17 of these peptide segments are ACE inhibitors. In order to better select peptide segments containing potential ACE activity, the peptide segments are docked with the ACE protein through the Autodock Vina software to obtain the lowest binding energy of each peptide segment docking. Considering comprehensively, 11 peptide segments with a Peptide Ranker score > 0.5 and a free energy < -8.5 Kcal / mol are selected for synthesis, as shown in Table 1.

[0046]

[0047] (2)Polypeptide Synthesis

[0048] The selected peptide segments were synthesized by the solid-phase synthesis method, and the purity was > 95% (Nanjing Genscript Biotech Co., Ltd.).

[0049] (3)In Vitro Activity Verification

[0050] The method for determining the ACE inhibitory activity is as follows:

[0051] Mix 60 μL of hippuryl-histidyl-leucine (HHL) solution (2.5 mmol / L, prepared with borax-boric acid buffer at pH 8.3) with 20 μL of sample solution (diluted with borax-boric acid buffer at pH 8.3) or borax-boric acid buffer at pH 8.3, let it stand and incubate at 37 °C for 5 min, then add 40 μL of ACE solution (0.05 U / mL, prepared with borax-boric acid buffer at pH 8.3), mix well and let it stand and react at 37 °C for 1 h. After the reaction is completed, add 120 μL of HCl (1 mol / L) to terminate the reaction. After the reaction solution passes through a 0.45 μm aqueous filter membrane, use a Shimadzu LC-20A HPLC equipped with a C18 Inertsil ODS-SP (4.6 mm × 250 mm, 5 μm) chromatographic column to detect the content of hippuric acid generated. The mobile phase is acetonitrile / ultrapure water (volume ratio 1:3, each containing 0.1 v / v% trifluoroacetic acid), the flow rate is 1.0 mL / min, the detection wavelength is 228 nm, the column temperature is 30 °C, and the injection volume is 10 μL.

[0052] The calculation formula for ACE inhibitory activity is shown in Equation (1):

[0053]

[0054] In the formula, A0 is the peak area of hippuric acid in the blank sample (i.e., using buffer instead of sample), and A is the peak area of hippuric acid in the sample.

[0055] The preparation method of the above borax-boric acid buffer at pH 8.3 is as follows: Accurately weigh 12.37 g of boric acid powder, dissolve it with ultrapure water and make up the volume to 1000 mL. Accurately weigh 19.07 g of borax powder, dissolve it with ultrapure water and make up the volume to 1000 mL. Measure 175 mL of borax solution and 325 mL of boric acid solution respectively, mix them evenly, adjust the pH of the mixed solution to 8.3 with hydrochloric acid or sodium hydroxide, then add 17.532 g of sodium chloride to dissolve, and make up the volume to 1000 mL with ultrapure water.

[0056] Use the above ACE inhibitory activity determination method to determine the ACE inhibitory activity of the selected peptide segments. Among them, the peptide segments VVPF and FFDLK did not show ACE inhibitory activity. For the remaining 9 peptide segments, at a concentration of 0.2 mg / mL, the ACE inhibitory ability is shown in Figure 3 , among which, VNDYLDW has the best inhibitory effect, and its amino acid sequence is: Val-Asn-Asp-Tyr-Leu-Asn-Trp, and its IC 50 is 0.19 ± 0.01 mg / mL. Its HPLC chromatogram and mass spectrum are shown in Figure 4 、 Figure 5 respectively.

[0057] Example 3 Exploration of the inhibitory mechanism of Torreya grandis active peptide VNDYLNW

[0058] (1) Molecular docking

[0059] Download the crystal structure of ACE (1O8A) from the PDB database (http: / / www.rcsb.org), use it as the protein target, and perform molecular docking through the Autodock Vina program to identify the active site. The results show that the binding energy of VNDYLDW docked with ACE is -10 Kcal / mol; the peptide segment VNDYLDW binds to the amino acid residues ASN70, LYS368, SER355, HIS410, TYR394, and HIS383 of ACE, forming 6 hydrogen bonds ( Figure 6 ), and forms metal acceptor interactions with Zn 2+ , with the bond length ranging from 1.9 - 3.4 Å.

[0060] (2) Inhibition kinetics study

[0061] Determine the effect of different concentrations of VNDYLDW (0, 0.1, 0.4 mg / mL) on the reaction rate of ACE under different concentrations of HHL substrate (2, 3, 4, 5 mmol / L) with a certain concentration of ACE (0.05 U / mL). Use the double-reciprocal plotting method to analyze the type of inhibition ( Figure 7 ). The results show that as the peptide concentration increases, V max (V max maximum reaction rate) increases, and K m (K m Michaelis constant, which refers to the concentration of substrate HHL [S] when the enzymatic reaction reaches half of the maximum rate (V max )) decreases. The three curves neither intersect the x-axis nor the y-axis, showing mixed inhibition.

Claims

1. A torreya grandis active peptide, characterized in that, Its amino acid sequence is Val-Asn-Asp-Tyr-Leu-Asn-Trp.

2. Use of the torreya grandis active peptide according to claim 1 in the preparation of a blood pressure-lowering product, characterized in that, The product is a health food or a medicine.

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