Chlamydomonas reinhardtii antihypertensive peptide and preparation method and application of composition thereof
By preparing enzymatic peptide extracts and oligopeptides with strong ACE inhibitory activity from Chlamydomonas reinhibited, the problem of side effects of existing angiotensin-converting enzyme inhibitor drugs was solved, and a safe and efficient blood pressure regulation effect was achieved.
Patent Information
- Application Number
- CN202510339932.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-21
- Publication Date
- 2025-06-13
AI Technical Summary
Existing angiotensin-converting enzyme inhibitors have side effects in the treatment of hypertension, such as dry cough, edema, and hyperkalemia, and long-term use may bring health risks, leading to the need to develop safer and more effective angiotensin-converting enzyme inhibitors.
Enzymatic peptide extracts and ACE inhibitory oligopeptides with good angiotensin converting enzyme (ACE) inhibitory activity were prepared by using Chlamydomonas reinforcing as a starting material, such as IDYRY, through protease enzymatic lysis and separation and purification by multiple chromatography columns.
The prepared Chlamydomonas reincarnation peptide extract and ACE inhibitory oligopeptide showed significant ACE inhibitory activity and blood pressure lowering activity in vivo. The IC50 is 18.54 μmol/L, the in vivo pressure lowering amplitude can reach -29.67 mmHg, and it has good stability and safety.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for preparing a series of antihypertensive peptides using Chlamydomonas reinhardtii as a raw material and its applications in foods, health products, and pharmaceuticals. Background Art
[0002] According to the definition of the World Health Organization, in the case of not taking antihypertensive drugs, if the systolic blood pressure ≥ 140 mmHg and / or the diastolic blood pressure ≥ 90 mmHg are measured over three days, it is defined as hypertension. Further, according to the specific values of blood pressure, hypertension is divided into different grades: Grade 1 hypertension (mild): systolic blood pressure 140 - 159 mmHg and / or diastolic blood pressure 90 - 99 mmHg. Grade 2 hypertension (moderate): systolic blood pressure 160 - 179 mmHg and / or diastolic blood pressure 100 - 109 mmHg. Grade 3 hypertension (severe): systolic blood pressure ≥ 180 mmHg and / or diastolic blood pressure ≥ 110 mmHg.
[0003] The renin - angiotensin - aldosterone system is an important mechanism for regulating blood pressure. Renin produced by the kidneys will circulate throughout the body and convert angiotensinogen produced by the liver into angiotensin I, which is further converted into angiotensin II under the catalysis of angiotensin - converting enzyme. After angiotensin II acts on its receptor, the angiotensin II type 1 receptor, it elevates blood pressure by regulating the levels of aldosterone, nitric oxide, endothelin, and sodium - potassium balance. In this process, angiotensin - converting enzyme plays a central role. Therefore, angiotensin - converting enzyme inhibitors have always been the first - line drugs for treating hypertension. However, existing angiotensin - converting enzyme inhibitor drugs have side effects such as causing dry cough, edema, and hyperkalemia. Therefore, it is still very necessary to develop safer and more effective angiotensin - converting enzyme inhibitors, and it has long been a hot topic in related research.
[0004] Chlamydomonas reinhardtii is a single - celled green alga of the genus Chlamydomonas, retaining some characteristics of the common ancestor of eukaryotes, and thus has long been cultured by researchers as a model organism. Genetically engineered Chlamydomonas reinhardtii can be used to synthesize various high - value products such as polypeptides, recombinant proteins, lipids, and hydrogen. In addition, Chlamydomonas reinhardtii also contains a large amount of protein, with its protein content reaching more than 35%, and the amino acid composition is reasonable. It is an excellent protein source and raw material for bioactive peptides. However, there has been no report on the bioactive peptides of Chlamydomonas reinhardtii, especially ACE - inhibitory peptides. Therefore, ACE - inhibitory peptides derived from Chlamydomonas reinhardtii are still very valuable for research. Summary of the Invention
[0005] The present invention provides a Chlamydomonas reinhardtii antihypertensive peptide, an antihypertensive peptide composition, and a preparation method thereof; the present invention also provides the applications of the above - mentioned antihypertensive peptide and antihypertensive peptide composition in foods, health products, or pharmaceuticals.
[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] The present invention provides a Chlamydomonas reinhardtii enzymatically hydrolyzed peptide extract, characterized in that: the proportion of peptides with an average molecular weight of 0-1000 Da in the extract is not less than 70%, and it has good angiotensin-converting enzyme (ACE) inhibitory activity.
[0008] The present invention also provides a Chlamydomonas reinhardtii angiotensin-converting enzyme inhibitory oligopeptide, characterized in that: the oligopeptide is one or more of IDYRY, VVGFALVF, RNRNLYGS, VTTPSPR, PTGWYLM, PTRARYT, YISGRAF and VFAGVFR.
[0009] The present invention also provides a Chlamydomonas reinhardtii food, characterized in that: the Chlamydomonas reinhardtii food contains a Chlamydomonas reinhardtii enzymatically hydrolyzed peptide extract or any one or more Chlamydomonas reinhardtii ACE inhibitory peptide oligopeptides and their compositions
[0010] Furthermore, the present invention provides an application of a Chlamydomonas reinhardtii hypotensive peptide, characterized in that: the application of the Chlamydomonas reinhardtii enzymatically hydrolyzed peptide extract, or ACE inhibitory oligopeptide, or their composition in the preparation of an angiotensin-converting enzyme inhibitor, or a health product or drug related to the prevention and treatment of hypertension and cardiovascular diseases.
[0011] The health product or pharmaceutical preparation for an angiotensin-converting enzyme inhibitor, or the prevention and treatment of hypertension and cardiovascular diseases is characterized in that: it is made by using the extract, any one or more ACE inhibitory oligopeptides and their compositions supplemented with excipients permitted in foods or drugs.
[0012] Furthermore, the present invention provides a preparation method of a Chlamydomonas reinhardtii enzymatically hydrolyzed peptide extract, characterized in that: using Chlamydomonas reinhardtii as a raw material, breaking the cell wall by methods such as freeze-thaw, ultrasonic or high-pressure homogenization treatment, enzymatically hydrolyzing with a protease, and then obtaining the Chlamydomonas reinhardtii enzymatically hydrolyzed peptide extract through enzyme inactivation, centrifugation and freeze-drying. The protease is any one of neutral protease, flavor protease, bromelain, α-chymotrypsin, proteinase K, papain, alkaline protease and trypsin.
[0013] Preferably, the preparation method of the enzymatically hydrolyzed peptide extract of Chlamydomonas reinhardtii includes: the material-liquid ratio (Chlamydomonas reinhardtii powder: water, w / w) is 1:10 - 1:30, and the microalgae suspension is treated with an ultrasonic cell disruptor at a power of 200 - 800W. After circulating 3 - 5 times, protease is added at an enzyme-substrate ratio (protease / Chlamydomonas reinhardtii, w / w) of 1 - 10% for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: pH 7 - 10, temperature 40 - 65°C, and enzymatic hydrolysis time 1 - 12h; after enzymatic hydrolysis is completed, it is heated at 95°C for 15 min to inactivate the protease, centrifuged at 8000g for 10 min to obtain the supernatant, and freeze-dried to obtain the enzymatically hydrolyzed peptide extract of Chlamydomonas reinhardtii. The enzymatically hydrolyzed peptide extract is separated and purified using one or a combination of ultrafiltration, gel chromatography column, ion exchange chromatography column, and reverse phase chromatography column, and the refined enzymatically hydrolyzed peptide extract of Chlamydomonas reinhardtii is obtained by freeze-drying.
[0014] The antihypertensive peptide IDYRY (SEQ ID NO.1) is obtained from Chlamydomonas reinhardtii as a raw material through protease enzymatic hydrolysis and separation and purification by multiple chromatography columns. Its three-letter amino acid sequence is Ile-Asp-Tyr-Arg-Tyr, and the relative molecular mass measured by mass spectrometry is: 728.79 Da. Polypeptides with this sequence obtained through enzymatic hydrolysis separation and purification, microbial fermentation, or chemical synthesis can be applied to the preparation of antihypertensive drugs or drug compositions, health products, or ordinary foods.
[0015] Advantages of the present invention:
[0016] The preparation methods of various enzymatically hydrolyzed peptide extracts of Chlamydomonas reinhardtii provided by the present invention are simple, and have good in vitro ACE inhibitory activity and in vivo antihypertensive activity. The ACE inhibitory oligopeptide is a pentapeptide with a molecular weight of 728.79 Da, and its small molecular weight is easy to absorb. The said enzymatically hydrolyzed peptides and oligopeptides have good application prospects in the fields of foods, health products, and drugs with blood pressure regulation activity. A series of polypeptides are identified from its alkaline protease hydrolysate, among which the antihypertensive oligopeptide with the sequence IDYRY shows good ACE inhibitory activity and in vivo antihypertensive activity, with an IC50 of 18.54 μmol / L and an in vivo antihypertensive amplitude of up to -29.67 mmHg. The present invention provides a method for preparing Chlamydomonas reinhardtii antihypertensive products, and a series of antihypertensive polypeptides with novel sequences and good activities are identified. The present invention fully elucidates the application potential of Chlamydomonas reinhardtii as foods, health products, and drugs, provides a theoretical reference for the development of Chlamydomonas reinhardtii-related products, and has good application value. Description of the Drawings
[0017] Figure 1 . Preparation characterization and in vitro ACE inhibitory activity of different enzymatically hydrolyzed peptides of Chlamydomonas reinhardtii.
[0018] Figure 2. Total ion chromatogram of enzymatically hydrolyzed peptide extract from Chlamydomonas reinhardtii.
[0019] Figure 3 . Dose curve of the oligopeptide IDYRY from Chlamydomonas reinhardtii inhibiting ACE.
[0020] Figure 4 . Double reciprocal plot for determining the inhibition mode of IDYRY on ACE.
[0021] Figure 5 . pH, temperature, and in vitro simulated digestion stability of IDYRY inhibiting ACE.
[0022] Figure 6 . In vivo antihypertensive activity of enzymatically hydrolyzed peptides from Chlamydomonas reinhardtii and the oligopeptide IDYRY prepared by alkaline protease.
[0023] Figure 7 . Chromatogram for purity identification of antihypertensive peptides from Chlamydomonas reinhardtii.
[0024] Figure 8 . Mass spectrometry identification of antihypertensive peptides from Chlamydomonas reinhardtii. Detailed implementation mode
[0025] The present invention will be further explained and illustrated below in combination with examples and drawings. The purpose of the present invention is to use Chlamydomonas reinhardtii as a raw material, prepare an enzymatically hydrolyzed peptide extract from Chlamydomonas reinhardtii through protease hydrolysis, and isolate, purify, and screen to obtain an ACE inhibitory oligopeptide with a clear, novel, and good activity sequence. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various modifications to the present invention, and these equivalent forms also fall within the scope defined by the present invention.
[0026] Example 1 Preparation of enzymatically hydrolyzed peptides from Chlamydomonas reinhardtii
[0027] Weigh 10 g of Chlamydomonas reinhardtii dry powder into 200 mL of deionized water, adjust the pH of the system to 10, freeze at -20 °C, thaw at 23 °C, and repeat the freeze-thaw cycle 3 times. Then use an ultrasonic cell disruptor to ultrasonically disrupt for 30 min at 300 W (working for 15 s, with an interval of 15 s). Subsequently, adjust the pH to 7, and add cellulase, hemicellulase, pectinase, and lysozyme respectively according to the enzyme / substrate ratio (E / S, w / w) of 1%. After enzymatic hydrolysis at 50 °C for 2 h, centrifuge at 8000 g for 10 min to obtain the supernatant, and freeze-dry to obtain the Chlamydomonas reinhardtii protein extract.
[0028] Dissolve the Chlamydomonas reinhardtii protein extract in deionized water to obtain a solution with a protein concentration of 10 mg / mL. Add protease according to the ratio of E / S = 1% (w / w). The enzymatic hydrolysis conditions of the protease are shown in Table 1. After enzymatic hydrolysis for 4 h, heat at 95 °C for 15 min to inactivate the protease. Centrifuge at 8000 g for 10 min and take the supernatant, then freeze-dry to obtain the enzymatically hydrolyzed peptide of Chlamydomonas reinhardtii. The proteases and enzymatic hydrolysis conditions used are as follows:
[0029] Table 1. Temperature and pH conditions for preparing enzymatically hydrolyzed peptide of Chlamydomonas reinhardtii
[0030]
[0031] Physicochemical determinations were performed on several kinds of enzymatically hydrolyzed peptides of Chlamydomonas reinhardtii prepared. The results are shown in Figure 1 . As can be seen from A in Figure 1 , the enzymatically hydrolyzed peptides of Chlamydomonas reinhardtii prepared by protease K, alkaline protease, and dispase have a relatively high degree of hydrolysis (DH). Among them, protease K and alkaline protease hydrolyze the Chlamydomonas reinhardtii protein more thoroughly ( Figure 1 A in); there is no obvious difference in the peptide yields obtained by several different proteases ( Figure 1 B in); when measuring the molecular weight distribution of the enzymatically hydrolyzed peptides, it was found that the enzymatically hydrolyzed peptides with a molecular weight less than 1000 Da are dominant, accounting for about 70 - 90%. Among them, the enzymatically hydrolyzed peptide of Chlamydomonas reinhardtii prepared by alkaline protease has the highest proportion.
[0032] Example 2 Determination of in vitro ACE inhibitory activity of the enzymatically hydrolyzed peptide extract of Chlamydomonas reinhardtii by high performance liquid chromatography (HPLC)
[0033] Experimental method:
[0034] The principle of this method is as follows: Hip-His-Leu (HHL, Sigma-Aldrich) can be used as a substrate for angiotensin-converting enzyme (ACE) to be decomposed to produce hippuric acid. After adding different ACE inhibitors, the production amount of hippuric acid will decrease accordingly. By calculating the peak area of hippuric acid at 228 nm, the inhibitory activity of the inhibitor on ACE activity can be evaluated.
[0035] Experimental reagents:
[0036] ACE (0.1 U / mL), Hip-His-Leu (HHL), captopril, 0.1 M sodium borate solution (pH 8.3, containing 0.3 M sodium chloride)
[0037] Dissolve ACE in sodium borate buffer to a final concentration of 0.1 U / mL for determination. Dissolve the polypeptide sample in sodium borate buffer to prepare polypeptide solutions with different concentrations. Then, mix 20 μL of a certain concentration of polypeptide solution with 10 μL of ACE solution. Incubate the mixture at 37 °C for 5 min, and add 50 μL of 5 mM HHL (sodium borate buffer pH 8.3 containing 0.3 M sodium chloride) to the above mixture to start the reaction. Maintain the reaction at 37 °C for 60 min, and then add 150 μL of 1 M HCl to terminate the reaction. Filter the solution through a 0.22 μm filter membrane. Take 20 μL of the reaction solution and load it onto RP-HPLC, which is connected to an Eclipse XDB-C18 column (4.6 mm × 150 mm × 5 μm), and detect the concentration of hippuric acid (HA) by ultraviolet detection. Detect the absorbance of hippuric acid at 228 nm. All determinations are repeated three times. The ACE inhibition activity is calculated as follows:
[0038] ACE inhibition activity (%) = (A Control - A Inhibitor ) / A Control * 100%)
[0039] where A Inhibitor is the relative area of the hippuric acid (HA) peak obtained from the reaction of ACE and HHL with the inhibitor. A Control is the relative area of the hippuric acid (HA) peak obtained from the reaction of ACE and HHL without an inhibitor. IC 50 is defined as the polypeptide concentration that can inhibit half of the ACE activity.
[0040] Chromatographic conditions:
[0041] Detection wavelength: 228 nm; mobile phase: 78% ultrapure water (containing 0.05% TFA) + 22% acetonitrile (containing 0.05% TFA); flow rate: 0.8 mL / min.
[0042] As Figure 1 shown in D of, determined by the above method, the inhibition rates of the 8 Chlamydomonas reinhardtii enzymatically hydrolyzed peptide extracts prepared in Example 1 against ACE were the highest, approaching 80% at a concentration of 200 μg / mL, and all were above 50%. Among them, the ACE inhibition activities of the protease K, alkaline protease, trypsin, α-chymotrypsin, and neutral protease hydrolysates of Chlamydomonas reinhardtii were relatively high, and the ACE inhibition rates were 74.52%, 71.68%, 70.08%, 65.22%, and 60.89%, respectively.
[0043] Combining Example 1 and Example 2, the enzymatically hydrolyzed peptides prepared by Protease K and Alkaline protease have a high degree of hydrolysis, and the proportion of enzymatically hydrolyzed peptides with a molecular weight < 1000 Da is high; while the ACE inhibitory activities of the enzymatically hydrolyzed products prepared by Protease K, Alkaline protease, Trypsin, α-Chymotrypsin, and Neutral protease are relatively high, all exceeding 60%, and the proportion of enzymatically hydrolyzed peptides with a molecular weight < 1000 Da all exceeds 70%. Therefore, based on the comprehensive judgment of the degree of hydrolysis (DH), molecular weight distribution, and ACE inhibitory activity, it is preferred to prepare Chlamydomonas reinhardtii enzymatically hydrolyzed peptides with Trypsin, Alkaline protease, Protease K, α-Chymotrypsin, and Neutral protease, which have good application potential.
[0044] Example 3 Separation, purification and ACE inhibitory activity determination of Chlamydomonas reinhardtii peptides
[0045] Taking the Chlamydomonas reinhardtii enzymatically hydrolyzed peptide extract (CRPA) prepared by Alkaline protease in Example 1 as an example to illustrate the separation and purification process of the Chlamydomonas reinhardtii enzymatically hydrolyzed peptide extract. The Chlamydomonas reinhardtii enzymatically hydrolyzed peptide extract was first separated and purified by a gel chromatography column (Sephadex G15, 1.6×100 cm), with deionized water as the mobile phase and a flow rate of 1 mL / min. According to the elution curve, CRPA was divided into 4 components, namely G1, G2, G3, and G4. Among them, G4 had the best activity, and its in vitro ACE inhibition rate was 41.10% at 400 μg / mL.
[0046] Subsequently, the G4 fraction was separated and purified using a Welch Ultimate XB-C8 (4.6×250 mm×5 μm) chromatographic column, with methanol and ultrapure water as the mobile phase. The detection wavelengths were 214 nm and 280 nm, and the flow rate was 0.8 mL / min. Within the first 5 minutes, the methanol concentration was 15%, and then it gradually increased to 30% within the next 10 minutes, and then remained at 30% for the following 5 minutes. According to the elution curve, G4 was divided into 6 components, among which R2 and R3 had better activities, and the ACE inhibition rates were 40.72% and 53.23% at 400 μg / mL.
[0047] Example 4 Identification of the sequence of Chlamydomonas reinhardtii antihypertensive peptides by high-resolution mass spectrometry
[0048] To identify the peptide sequence, the fraction with good ACE inhibitory activity in Example 3 was dissolved in 50 mmol / L NH 4 HCO 3In the solution, dithiothreitol (DTT) was added to a final concentration of 10 mmol / L. It was reduced at 56 °C for 1 h and alkylated with 3-indoleacetic acid (IAA, 50 mmol / L) in the dark for 40 min. Then the sample was lyophilized and dissolved with 0.1% formic acid, and then subjected to LC-MS / MS analysis. 5 μL of the sample was loaded into a liquid chromatography system equipped with an Acclaim PepMap RPLC C18 column (150 μm × 15 cm, 3 μm, Dr. Maisch GmbH, Germany). The mobile phase was deionized water containing 0.1% formic acid (A) and 80% acetonitrile containing 0.1% formic acid (B). The system was eluted with a linear gradient at a flow rate of 600 nL / min: 4 - 8% B for 2 min; 8 - 28% B for 43 min; 20 - 40% B for 10 min; 40 - 95% B for 1 min, and 95 - 95% B for 10 min. The secondary mass spectrum was obtained by Q Exactive TM Hybrid Quadrupole-Orbitrap TM Mass Spectrometer (Thermo Fisher Scientific, USA) in the positive ion detection mode. The mass spectrometry precursor m / z was set to 300.0 - 1800.0 Da, and the secondary mass spectrum was searched in the Byonic database to determine the peptide sequence. The total ion chromatogram of the mass spectrum of fraction R2 is shown as Figure 2 shown, and IDYRY was detected at t R = 17.22 min in R2.
[0049] Example 5 Discovery of ACE-inhibitory oligopeptides from Chlamydomonas reinhardtii
[0050] After mass spectrometry identification, a total of 8790 polypeptide sequences were obtained from R2 and R3 in Example 4. A total of 754 peptides with a length of less than 10 amino acids were selected and subjected to molecular docking in autodock vina, and the top 10% of the peptides with the highest scores were selected for secondary docking. Eight oligopeptide sequences were selected by manually examining the interactions between the polypeptide and Glu162 and Gln281 in the active center of ACE and synthesized by solid-phase synthesis. Among them, IDYRY (ID-5) had the best activity. According to the method in Example 2, its ACE inhibitory activity was measured, and the inhibition rate of ACE was 80.04% at a concentration of 200 μg / mL (Table 2).
[0051] Table 2. Sequences, docking scores, and in vitro ACE inhibition rates of Chlamydomonas reinhardtii oligopeptides
[0052]
[0053]
[0054] Example 6 Determination of the IC 50 value of the antihypertensive peptide from Chlamydomonas reinhardtii
[0055] The ACE inhibitory rate of IDYRY was determined according to the method described in Example 2 at concentrations of 400, 100, 50, 10, 1, 0.1, 0.01, 0.001 μg / mL. Three parallels were set up in the experiment, and the average value was taken. The logarithm of the concentration was plotted against the inhibition rate ( Figure 3 ) to calculate the IC 50 value of the polypeptide. After parameter fitting, the IC 50 of the pentapeptide ID-5 from Chlamydomonas reinhardtii reached 18.54 μmol / L, indicating strong ACE inhibitory ability.
[0056] Example 7 Determination of the inhibition mode of the antihypertensive peptide from Chlamydomonas reinhardtii
[0057] Different reaction systems were set up to evaluate the inhibition mode of the antihypertensive peptide. The final concentration of HHL in the system was 4, 2, 1, 0.5, 0.25, 0.1 mM, and the final concentration of the antihypertensive peptide was 0.5 mg / mL and 0.1 mg / mL. The reaction and detection methods were as described in Example 2. The reciprocal of the production rate of hippuric acid was plotted against the reciprocal of the substrate concentration, and the double-reciprocal method was used to analyze the inhibition mode of the polypeptide on ACE. As Figure 4 shown, as the concentration of IDYRY changed, the double-reciprocal curve intersected with the negative half-axis of the x-axis at one point. Therefore, the inhibition mode of IDYRY on ACE was non-competitive inhibition.
[0058] Example 8 Stability evaluation of the antihypertensive peptide from Chlamydomonas reinhardtii
[0059] Thermal stability study: The antihypertensive peptide with a concentration of 80 μg / mL was placed at 0, 20, 40, 60, 80, 100 °C for 2 h, and then the in vitro ACE inhibitory activity of the antihypertensive peptide was measured according to the method described in Example 2.
[0060] pH stability study: The antihypertensive peptide with a concentration of 80 μg / mL was placed in environments with pH 2, 4, 6, 8, 10, 12 for 2 h, and then the in vitro ACE inhibitory activity of the antihypertensive peptide was measured according to the method described in Example 2.
[0061] Digestive enzyme stability study: After incubation in a simulated gastric environment (pH 1.3, 1% (m / v) pepsin dissolved in 1 mol / L HCl) and an intestinal environment (pH 6.8, 6.8 g K2PO4 and 1% (m / v) trypsin and α-chymotrypsin dissolved in 500 mL deionized water) for 2 hours, the in vitro ACE inhibitory activity of the antihypertensive peptide was measured according to the method described in Example 2.
[0062] As Figure 5As shown, extreme pH and temperature have no significant effect on the in vitro ACE inhibitory activity of IDYRY. During the simulated gastric digestion stage, the activity of the antihypertensive peptide decreases, but after simulated intestinal digestion, the activity rebounds to the initial level. This indicates that the identified antihypertensive peptide has good tolerance to pH and high temperature. Although digestive enzymes can affect the in vitro ACE inhibitory activity of the antihypertensive peptide, the antihypertensive peptide can still exhibit good in vitro ACE inhibitory activity after gastrointestinal digestion.
[0063] Example 9 Evaluation of the Antihypertensive Activity of Enzymatically Hydrolyzed Peptide Extracts from Chlamydomonas reinhardtii and IDYRY in Vivo
[0064] Using spontaneously hypertensive rats (SHR) as a hypertension model, the in vivo antihypertensive activities of the enzymatically hydrolyzed peptide extract from Chlamydomonas reinhardtii (CRPA) prepared with alkaline protease in Example 1 and the antihypertensive peptide IDYRY (ID-5) were evaluated. CRPA is an enzymatically hydrolyzed peptide from Chlamydomonas reinhardtii prepared with alkaline protease and has significant ACE inhibitory activity in vitro. Taking CRPA as a representative, the potential of enzymatically hydrolyzed peptides from Chlamydomonas reinhardtii for antihypertensive use was evaluated. At a dosage of 100 mg / kg, CRPA could significantly reduce the systolic blood pressure of SHR rats, with a blood pressure reduction amplitude of 29.67 mmHg, and its antihypertensive efficacy was comparable to that of captopril ( Figure 6 ). Similarly, the pentapeptide ID-5 from Chlamydomonas reinhardtii also showed significant antihypertensive effects. At 2 h after a dosage of 20 mg / kg, the systolic blood pressure of SHR rats decreased by 12.5 mmHg ( Figure 6 ), indicating that ID-5 has antihypertensive effects in vivo.
[0065] Example 10 Purity Identification of Antihypertensive Peptides from Chlamydomonas reinhardtii
[0066] Eight ACE inhibitory peptides were identified through Example 5, and eight polypeptides were synthesized using solid-phase synthesis technology. In this example, the purity of the above eight synthetic peptides was determined by high-performance liquid chromatography. Take 0.2 mg of the polypeptide sample, dissolve it in deionized water, and analyze the sample using a high-performance liquid chromatography system equipped with a NanoChrom Chromcore TM120 C18 (4.6 * 250 mm * 5 μM) chromatographic column. The sample injection volume was 40 μL, and the mobile phases were acetonitrile containing 0.1% trifluoroacetic acid and ultrapure water respectively. The flow rate was 1.0 mL / min, and the peak elution was detected at 214 nm. The chromatogram is shown in Figure 7 , and the results showed that the purity of the eight synthetic polypeptides was greater than 95%.
[0067] Example 11 Mass Spectrometry Identification of Antihypertensive Peptides from Chlamydomonas reinhardtii
[0068] The molecular weights of the 8 ACE inhibitory peptides synthesized in Example 5 were determined by mass spectrometry. For each polypeptide sample, 0.1 mg was taken and dissolved in 0.5 mL of ultrapure water. After passing the sample through a C18 reverse-phase column, the sample was analyzed with a Q-Exactive mass spectrometer. The sample injection volume was 1 μL, the carrier gas flow rate was 1.5 L / min, and the liquid-phase mobile phase was 50% H 2 O + 50% MeOH.
[0069] The results of mass spectrometry determination are shown in Figure 8 , and the quasi-molecular ion peaks of the 8 synthesized peptides corresponded to their molecular weights, without other impurity peaks, indicating that the molecular formula of the synthesized peptides was consistent with the theoretically calculated molecular formula, and the amino acid sequences of each synthesized peptide were accurate.
[0070] Chlamydomonas reinhardtii contains a large amount of protein, with its protein content reaching over 35%, and its amino acid composition being reasonable. It is an excellent protein source and raw material for bioactive peptides. In 2022, Chlamydomonas reinhardtii was approved as a new food raw material by the National Health Commission and is an important economic microalgae. The present invention provides a simple and green method for preparing enzymatically hydrolyzed peptides from Chlamydomonas reinhardtii, which can stably prepare a raw material of Chlamydomonas reinhardtii small-molecule peptides with a molecular weight < 1000 Da accounting for over 70%; meanwhile, the present invention also provides enzymatically hydrolyzed peptides from Chlamydomonas reinhardtii, identified single small-molecule peptides, and compositions, all of which exhibit good ACE inhibitory activity and in vivo blood pressure-lowering activity. The enzymatically hydrolyzed peptides from Chlamydomonas reinhardtii have a small molecular weight and are easily absorbed. According to the technical method provided by the present invention, it can be scaled up for pilot-scale or production, and has strong industrial practicability. The enzymatically hydrolyzed peptides from Chlamydomonas reinhardtii and their compositions can be used for the treatment and prevention of chronic diseases such as hypertension and heart failure, including patients at high risk of chronic heart failure, asymptomatic left ventricular dysfunction, hypertension, and cardiovascular events. Supplementing the enzymatically hydrolyzed peptides from Chlamydomonas reinhardtii and their compositions helps maintain a healthy blood pressure level, protect target organs such as the heart and kidneys, and can be applied to health products, drugs, or pharmaceutical preparations, etc. It has good market development prospects whether as food, health products, or drugs.
Claims
1. A Chlamydomonas reinhardtii enzymatic peptide, characterized in that: The peptides have an average molecular weight of 0-1000 Da, and the proportion of the peptides in the peptides is not less than 70%.
2. A Chlamydomonas reinhardtii oligopeptide capable of inhibiting angiotensin converting enzyme (ACE), characterized in that: The oligopeptide sequence is any one or more of IDYRY, VVGFALVF, RNRNLYGS, VTTPSPR, PTGWYLM, PTRARYT, YISGRAF and VFAGVFR.
3. A Chlamydomonas reinhardtii antihypertensive peptide or composition, characterized in that: The invention comprises the enzymatic peptide of claim 1 or any one or more oligopeptides of Chlamydomonas reinhardtii of claim 2, or a composition consisting of the enzymatic peptide of claim 1 and any one or more oligopeptides of claim 2.
4. A Chlamydomonas reinhardtii food, characterized in that: The Chlamydomonas reinhardtii food is added with the enzymatic peptides of claim 1, or any one or more oligopeptides of claim 2, or the antihypertensive peptides or the combination of claim 3.
5. An application of a Chlamydomonas reinhardtii antihypertensive peptide or composition, characterized in that: The use of the enzymatic peptide according to claim 1, the Chlamydomonas oligopeptide according to claim 2, or the composition according to claim 3 in the preparation of angiotensin converting enzyme inhibitors, or health products and pharmaceutical preparations related to the treatment or prevention of hypertension and cardiovascular diseases.
6. The use according to claim 5, characterized in that: The enzymatic peptides described in claims 1-3, or any one or more Chlamydomonas reinhardtii antihypertensive peptides, or the combination are added with any excipients that comply with the production permits for food or medicine and are used for health products or pharmaceutical preparations related to angiotensin converting enzyme inhibitors, or the treatment or prevention of hypertension and cardiovascular diseases.
7. A method for preparing the Chlamydomonas reinhardtii enzymatic peptide according to claim 1, characterized in that: Chlamydomonas reinhardtii is used as a raw material, the cell wall is broken by freeze-thaw, ultrasound or high-pressure homogenization, and the cell wall is hydrolyzed by protease. The cell wall is then inactivated, centrifuged and freeze-dried to obtain Chlamydomonas reinhardtii enzymatic peptides.
8. The extraction method according to claim 7, characterized in that: The method for extracting Chlamydomonas reinhardtii enzymatic peptides comprises: a solid-liquid ratio (Chlamydomonas reinhardtii powder: water, w / w) of 1:10-1:30, treating a microalgae suspension with an ultrasonic cell disruptor at a power of 200-800W, and after 3-5 cycles, adding a protease at an enzyme-substrate ratio (protease / Chlamydomonas reinhardtii, w / w) of 0.5-10% for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are: pH 7-10, temperature 40-65°C, and enzymatic hydrolysis time 1-12h; after the enzymatic hydrolysis is completed, heating at 95°C for 15min to inactivate the protease, centrifuging at 8000×g for 10min to obtain a supernatant, and freeze-drying to obtain a Chlamydomonas reinhardtii enzymatic peptide extract.
9. The extraction method according to claim 8, characterized in that: The supernatant after centrifugation can be further separated, purified and refined by ceramic membrane filtration, ultrafiltration membrane concentration, nanofiltration membrane concentration, Sephadex G-15 gel column, DEAE anion exchange column chromatography, C18 reverse phase column chromatography and the like.
10. The extraction method according to claim 7, characterized in that: The protease is preferably any one of trypsin, alkaline protease, proteinase K, chymotrypsin, and neutral protease.