Camellia oleifera seed cake ACE inhibitory peptide and application thereof

Abstract

The present invention discloses an ACE inhibitory peptide derived from camellia seed cake, the amino acid sequence of which is Val-Val-Val-Pro-Gln-Asn. The present invention also discloses its application in the preparation of antihypertensive drugs. In vitro experiments of the present invention show that the peptide segment has good ACE inhibitory activity, and its IC 50 The ACE inhibitory peptide obtained by the present invention has the characteristics of simple structure, safety, strong activity, etc., and can be used to prepare drugs with blood pressure lowering function.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an ACE inhibitory peptide from camellia seed cake and an application thereof. Background Art

[0002] Hypertension is a common chronic disease that can cause cardiovascular and cerebrovascular diseases, atherosclerosis, and kidney disease (Piovesana et al., 2018). With improvements in living standards, the incidence of hypertension has gradually increased. Angiotensin-converting enzyme (ACE) is a dipeptide carboxypeptidase that catalyzes the conversion of inactive angiotensin I (AngI) to angiotensin II (AngII), which can constrict blood vessels (Saleh et al., 2016), leading to elevated blood pressure. ACE inhibitors effectively block the conversion of Angiotensin I to Angiotensin II, thereby treating hypertension. Existing synthetic ACE inhibitors include benazepril, captopril, and enalapril (Lee and Hur, 2017). However, these synthetic ACE inhibitors can cause certain side effects, such as cough, rash, taste disturbances, and angioedema (Ahmed et al., 2020). Therefore, the search for natural ACE inhibitory peptides from food that offer health benefits and minimize side effects has become a research hotspot. Currently, many ACE inhibitory peptides have been isolated from cereals, legumes, nuts, vegetables, fruits, mushrooms and their by-products (Fan et al., 2022).

[0003] Camellia oleifera Abel is one of the world's four major woody oil crops (along with camellia, oil palm, olive, and coconut) (Liu et al., 2022), primarily distributed in China, India, Japan, and Southeast Asian countries (Luan et al., 2020). Camellia oil is rich in unsaturated fatty acids and various phenolic compounds, possessing excellent nutritional value (Zhou et al., 2021). Camellia seed meal is a byproduct of oil extraction. In China, approximately 2 million tons of camellia seed meal are produced annually, accounting for approximately 50% of camellia seed production (Zhang et al., 2019). Camellia seed meal contains 10–20% protein (Yao et al., 2019), making it a potential protein resource. However, camellia seed meal is currently used primarily for traditional purposes such as animal feed, detergents, and organic fertilizers (Yu et al., 2022), leaving it underutilized as a protein resource.

[0004] Plant-derived proteins are the source of a variety of bioactive peptides. Studies have found that peptides obtained after hydrolysis of camellia oleifera protein have antioxidant, anti-tumor, hypoglycemic, and hypotensive activities (Zheng et al., 2021). However, there are currently few studies on ACE-inhibitory peptides from camellia oleifera seed cake. Therefore, the development of ACE-inhibitory peptide products from camellia oleifera seed cake can increase the added value of camellia oleifera seed cake and achieve comprehensive utilization of resources.

[0005] Ahmed,M.,Verma,AK,andPatel,R.(2020).Collagen extraction and recentbiological activities of collagen peptides derived from sea-food waste:Areview.Sustainable Chemistry and Pharmacy 18,100315.

[0006] Fan, H., Liu, H., Zhang, Y., Zhang, S., Liu, T., and Wang, D. (2022). Review on plant-derived bioactive peptides: biological activities, mechanism of action and utilizations in food development. Journal of Future Foods 2, 143-159.

[0007] Lee,SY,and Hur,SJ(2017).Antihypertensive peptides from animal products,marine organisms,and plants.Food chemistry 228,506-517.

[0008] Liu,X.,Xie,M.,Hu,Y.,Li,S.,Nie,S.,Zhang,A.,Wu,H.,Li,C.,Xiao,Z.,andHu,C.(2022).Facile preparation of lignin nanoparticles from waste Camelliaoleiferashell:The solvent effect on the structural characteristic of ligninnanoparticles.Industrial Crops and Products 183,114943.

[0009] Luan,F.,Zeng,J.,Yang,Y.,He,X.,Wang,B.,Gao,Y.,and Zeng,N.(2020).Recentadvances in Camellia oleifera Abel:Areview of nutritional constituents,biofunctional properties,and potential industrial applications.Journal ofFunctionalFoods 75,104242.

[0010] Piovesana,S.,Capriotti,A.L.,Cavaliere,C.,La Barbera,G.,Montone,C.M.,Zenezini Chiozzi,R.,and Lagana,A.(2018).Recent trends and analyticalchallengesin plant bioactive peptide separation,identification andvalidation.Analytical andbioanalytical chemistry 410,3425-3444.

[0011] Saleh,A.S.,Zhang,Q.,and Shen,Q.(2016).Recent ResearchinAntihypertensive Activity of Food Protein-derived Hydrolyzates andPeptides.Critical reviews in food science and nutrition 56,760-787.

[0012] Yao,G.-l.,He,W.,Wu,Y.-g.,Chen,J.,Hu,X.-w.,and Yu,J.(2019).Purificationof Angiotensin-I-Converting Enzyme Inhibitory Peptides Derivedfrom Camelliaoleifera Abel Seed Meal Hydrolysate.Journal of Food Quality2019,1-9.

[0013] Yu,N.,Shao,S.,Huan,W.,Ye,Q.,Nie,X.,Lu,Y.,and Meng,X.(2022).Preparation of novel self-assembled albumin nanoparticles from Camellia seedcakewaste for lutein delivery.Food chemistry 389,133032.

[0014] Zhang,S.,Zheng,L.,Zheng,X.,Ai,B.,Yang,Y.,Pan,Y.,and Sheng,Z.(2019).Effect of steam explosion treatments on the functional properties andstructure ofcamellia(Camellia oleifera Abel.)seed cake protein.FoodHydrocolloids 93,189-197.

[0015] Zheng,Y.,Shi,P.,Li,Y.,Zhuang,Y.,Linzhang,Y.,Liu,L.,and Wang,W.(2021).Anovel ACE-inhibitory hexapeptide from camellia glutelin-2hydrolysates:Identification, characterization and stability profiles under different foodprocessing conditions.Lwt 147,111682.

[0016] Zhou, L., Luo, S., Li, J., Zhou, Y., Wang, 86,104678. Summary of the Invention

[0017] The purpose of the present invention is to provide a camellia seed cake ACE inhibitory peptide and application thereof, so as to solve the deficiencies of the prior art.

[0018] The present invention adopts the following technical solutions:

[0019] In a first aspect, the present invention provides a camellia seed cake ACE inhibitory peptide, the amino acid sequence of which is Val-Val-Val-Pro-Gln-Asn, abbreviated as VVVPQN.

[0020] The second aspect of the present invention provides the use of the above-mentioned camellia seed cake ACE inhibitory peptide in the preparation of antihypertensive drugs.

[0021] Beneficial effects of the present invention:

[0022] The present invention screened and obtained an ACE inhibitory peptide with the sequence of Val-Val-Val-Pro-Gln-Asn (VVVPQN) from Camellia oleifera protein. In vitro experiments showed that the peptide had good ACE inhibitory activity. 50The ACE inhibitory peptide obtained by the present invention has the characteristics of simple structure, safety, strong activity, etc., and can be used to prepare drugs with blood pressure lowering function. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The chromatogram shows the most active component (CPH3) separated by Sephadex G-25 gel.

[0024] Figure 2 Figure 2 is a graph showing the ACE inhibitory activity of VVVPQN.

[0025] Figure 3 HPLC chart of VVVPQN.

[0026] Figure 4 This is the primary mass spectrum of VVVPQN.

[0027] Figure 5 This is the secondary mass spectrum of VVVPQN.

[0028] Figure 6 This is the molecular docking diagram of VVVPQN.

[0029] Figure 7 It is the Lineweaver-Burk graph of VVVPQN versus ACE. DETAILED DESCRIPTION

[0030] The present invention will be further explained below in conjunction with the examples and drawings. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0031] Example 1 Preparation of ACE inhibitory peptide from camellia seed cake

[0032] (1) Extraction of Camellia oleifera protein

[0033] Camellia seeds were shelled and then cold-pressed and degreased using a hydraulic oil press at room temperature, 50 MPa, and 40 minutes to obtain camellia seed cake. The resulting camellia seed cake was then pulverized through a 60-mesh sieve to obtain camellia seed cake powder. The camellia seed cake powder was mixed with petroleum ether at a ratio of 1:5 (w / v, g / mL) and extracted with stirring at room temperature and 500 rpm for 2 hours. The mixture was then allowed to settle at room temperature for 1 hour to obtain a sediment. The mixing, stirring, extraction, and sedimentation steps were repeated three times, and the mixture was dried at 45°C to obtain defatted camellia seed cake powder. The defatted camellia seed cake powder was then mixed with 80% v / v ethanol at a ratio of 1:10 (w / v, g / mL) and extracted with stirring at 40°C and 500 rpm for 2 hours. The residue was then filtered at room temperature to obtain a residue. The mixing, stirring, extraction, and filtration steps were repeated twice, and the mixture was dried at 45°C until the moisture content was less than 4 wt%, resulting in defatted and desaponified camellia seed cake powder.

[0034] Camellia oleifera protein was prepared by alkali extraction and acid precipitation method: the defatted and desaponified camellia oleifera seed cake powder was passed through a 60-mesh sieve, mixed with deionized water at a ratio of 1:20 (w / v, g / mL), and extracted with stirring at 50°C, pH 10.0 (pH adjusted with 6 mol / L NaOH), and 500 r / min for 2 h. After cooling to room temperature, the mixture was centrifuged at room temperature and 4000 r / min for 15 min. The supernatant was collected, the pH was adjusted to 4.0 with 6 mol / HCl, and the mixture was allowed to stand for 1 h. The precipitate was collected and washed three times with 80 v / v% ethanol. After that, an appropriate amount of deionized water was added and the pH was adjusted to 7.0 with 6 mol / L NaOH to dissolve the mixture. The mixture was freeze-dried at -80°C for 48 h to obtain camellia oleifera protein.

[0035] (2) Preparation of Camellia oleifera protein hydrolysate

[0036] Camellia oleifera protein was added to deionized water to prepare a protein solution with a substrate concentration of 2 wt%, and the solution was placed in a 95°C water bath for denaturation for 15 min. After cooling to room temperature, the pH was adjusted to 7 with 1 mol / L NaOH and 1 mol / L HCl. 5000 U / g (based on the mass of camellia oleifera protein in the solution) of neutral protease (Beijing Solebaugh Technology Co., Ltd., enzyme activity 50000 U / g) was added. The solution was placed in a 45°C water bath at 300 r / min and stirred for enzymolysis for 4 h. The solution was placed in a 95°C water bath for enzyme inactivation for 10 min. After cooling to room temperature, the solution was centrifuged at 4°C and 8500 r / min for 30 min. The supernatant was collected to obtain camellia oleifera protein hydrolysate (CPH).

[0037] (3) Separation, purification and identification

[0038] The tea oil protein hydrolysate was filtered through a 0.45 μm aqueous microporous filtration membrane and then separated using ultrafiltration membranes with a molecular weight cutoff of 10 kDa and 3 kDa, respectively, to obtain three fractions: >10 kDa (CPH1), 3-10 kDa (CPH2), and <3 kDa (CPH3). The ACE inhibitory activity was determined separately, and the most active fraction (CPH3) was separated using Sephadex G-25 gel: the sample was prepared at 10 mg / mL, 3 mL was taken and passed through a 0.45 μm aqueous syringe filter membrane to remove impurities, and deionized water was used as the eluent at a flow rate of 0.8 mL / min. The absorbance was measured at a wavelength of 280 nm, and two fractions (F1 and F2) were collected. Figure 1 ), the ACE inhibitory activity was determined, among which the F2 component had better ACE inhibitory activity, and the amino acid sequence information of the F2 component was determined by LC-MS / MS technology.

[0039] (4) Peptide screening

[0040] Screen out peaks with an area greater than 10 8 For peptides with a score greater than 95, the peptide activity was predicted using the BIOPEP website, and the Autodock Vina program was used for molecular docking to screen out 7 peptides with potential ACE inhibitory activity.

[0041] Example 2 Peptide Synthesis and Activity Verification

[0042] The following solutions are prepared as follows:

[0043] Borate buffer: Dissolve 6.185 g of boric acid powder in ultrapure water and dilute to 500 mL. Dissolve 4.7675 g of borax powder in ultrapure water and dilute to 250 mL. Mix 325 mL of boric acid solution and 175 mL of borax solution. Adjust the pH of the mixture to 8.3 with hydrochloric acid or sodium hydroxide. Add 17.532 g of sodium chloride and dissolve, then dilute to 1000 mL.

[0044] HHL reagent: Dissolve 25 mg of hippocuronylhistidylleucine (HHL) powder in 11.64 mL of borate buffer to a concentration of 5 mmol / L (dilute when needed), aliquot into centrifuge tubes, and store at -20°C.

[0045] ACE reagent: Dissolve 0.1 U of ACE in 1 mL of borate buffer to make 0.1 U / mL (dilute when needed).

[0046] (1) Peptide synthesis: Seven peptides were synthesized by solid-phase synthesis with a purity higher than 95% (Nanjing GenScript Biotechnology Co., Ltd.).

[0047] (2) In vitro ACE activity assay: 60 μL HHL (2.5 mmol / L) and 20 μL sample (diluted with borate buffer) were mixed in a 1 mL centrifuge tube, and incubated at 37°C for 5 min. 40 μL ACE solution (0.05 U / mL) was added and mixed, and the mixture was allowed to react at 37°C for 1 h. Finally, 120 μL HCl (1 mol / L) was added to stop the reaction. The mixture was filtered through a 0.45 μm water-based needle filter membrane, and the hippuric acid content was determined by high performance liquid chromatography.

[0048] The HPLC conditions were as follows:

[0049] Mobile phase: acetonitrile / ultrapure water (volume ratio 1:3, each containing 0.1 v / v% trifluoroacetic acid)

[0050] Flow rate: 1 mL / min; detection wavelength: 228 nm; column temperature: 30°C; injection volume: 10 μL

[0051] The ACE inhibitory activity was calculated using the following formula:

[0052]

[0053] Where A 空白 is the hippuric acid peak area of ​​the blank sample (i.e., the sample is replaced by buffer), A 样品 is the hippuric acid peak area of ​​the sample.

[0054] The ACE inhibitory activity of 7 peptides was determined (Table 1). The results showed that at a concentration of 0.5 mg / mL, VVVPQN had the strongest ACE inhibitory activity, reaching 80.46%. Its IC 50 0.13 mg / mL ( Figure 2 The molecular weight of VVVPQN is 654.3Da, it is non-toxic and has good water solubility. Its HPLC chart is as follows Figure 3 As shown in the mass spectrum Figure 4 and Figure 5 shown.

[0055] Table 1 ACE inhibitory activity of different peptides at 0.5 mg / mL

[0056]

[0057] Example 3 Inhibitory Mechanism of VVVPQN

[0058] (1) Molecular docking: The ACE crystal structure (1O8A) was downloaded from the PDB database (http: / / www.rcsb.org / pdb / home / home.do), and VVVPQN was molecularly docked using the Autodock Vina program to analyze the molecular interaction mechanism between VVVPQN and ACE. The results showed that the binding energy of VVVPQN and ACE was -8.8 Kcal / mol; the peptide VVVPQN formed 8 hydrogen bonds with ACE residues HIS353, ASN374, GLU376, GLN281, THR282, GLU162, and ASN277 (including 2 hydrogen bonds with GLU376, Figure 6 ).

[0059] (2) Inhibition kinetics study: VVVPQN (0, 0.05, 0.2 mg / mL) and different concentrations of HHL (2, 3, 4, 5 mmol / L) were mixed with ACE (0.05 U / mL) according to the method of Example 2 to determine the content of hippuric acid. The inhibition kinetics were analyzed using a Lineweaver-Burk plot. The results showed that ( Figure 7 ), K m Unchanged (K m is the Michaelis constant, the enzyme-catalyzed reaction reaches its maximum speed (V max ) is the concentration of HHL substrate [S] at half the time, V max (V max The maximum reaction rate was decreased, so VVVPQN was considered to be a non-competitive inhibitor.

Claims

1. An application of an ACE inhibitory peptide from camellia seed cake in the preparation of a blood pressure-lowering drug, wherein: The amino acid sequence of the camellia seed cake ACE inhibitory peptide is Val-Val-Val-Pro-Gln-Asn.

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