Polypeptide, preparation method, xanthine oxidase inhibitor, and uric acid-lowering preparation

Abstract

The present invention relates to the field of active polypeptide technology, disclosing a polypeptide with XOD inhibitory activity. The polypeptide is selected from the short peptides KR5, FL7, RK8, or a combination thereof, and has amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively. The polypeptide exhibits excellent XOD inhibitory properties and can be developed as a XOD inhibitor and uric acid-lowering agent. Furthermore, the polypeptide provided by the present invention can be extracted from red tuna meat, enabling the high-value development of dark tuna meat.

Description

Technical Field

[0001] The present invention relates to the technical field of active polypeptides, and in particular to a polypeptide, a preparation method, a xanthine oxidase inhibitor, and a uric acid-lowering preparation. Background Art

[0002] Xanthine oxidase (XOD) catalyzes the conversion of hypoxanthine to xanthine, which is then oxidized to uric acid. Abnormally elevated XOD activity leads to excessive uric acid production, causing hyperuricemia. Hyperuricemia (HUA) can cause symptoms such as gout and kidney stones, and is closely linked to metabolic diseases such as cardiovascular disease, posing numerous risks. Xanthine oxidase inhibitors (XODs) target XOD to inhibit its activity, reducing the conversion of xanthine to uric acid and lowering uric acid production. Currently, the most commonly used XODs in clinical practice are allopurinol and febuxostat, but both have serious side effects. For example, allopurinol can cause fatal allopurinol hypersensitivity syndrome, while febuxostat can easily cause cardiovascular events. Therefore, there is a need to develop safer and more effective XOD inhibitors.

[0003] Obtaining polyphenolic compounds with xanthine oxidase inhibitory effects from natural plants has become an important research direction, such as flavonoids such as chrysin and apigenin, and flavonols such as galangin. However, these polyphenolic compounds often have problems such as weak stability, poor solubility, and low toxicity, which limit their application. Therefore, as the biological activity and safety of polypeptides are widely recognized, the development of polypeptides with xanthine oxidase inhibitory effects from animals or plants has gradually attracted the attention of researchers. For example, Hou Mengfan et al. (Preparation and process optimization of xanthine oxidase inhibitory peptides from oval pomfret [J], Food and Fermentation Industries, 2021, 47(23):185-192) used enzymatic hydrolysis to extract polypeptides with xanthine oxidase inhibitory effects from oval pomfret, and the XOD inhibition rate of the polypeptide was 50% to 60%. Summary of the Invention

[0004] The object of the present invention is to provide a polypeptide having xanthine oxidase inhibitory activity in vitro and a high inhibition rate.

[0005] The present invention provides the following technical solutions:

[0006] A polypeptide comprises one or more of the short peptides with amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0007] The polypeptide provided by the present invention is selected from three short peptides KR5, FL7, and RK8, or a combination of two or three thereof. The amino acid sequence of KR5 is SEQ ID NO. 1: Lys-Leu-Ala-His-Arg (KLAHR), the amino acid sequence of FL7 is SEQ ID NO. 2: Phe-Val-Gly-Gly-Leu-Arg-Leu (FVGGLRL), and the amino acid sequence of RK8 is SEQ ID NO. 3: Arg-Met-Pro-Met-Phe-Phe-Leu-Lys (RMPMFFLK).

[0008] After testing, KR5, FL7, and RK8 all have XOD inhibitory activity. At a low concentration of 1 mg / mL, the XOD inhibition rate of RK8 can reach 69.80%, and that of FL7 can reach 68.22%. When the concentration increases to 30 mg / mL, the inhibition rate of RK8 can reach 87.6%, and that of FL7 can reach 100%, indicating a significant XOD inhibition effect.

[0009] A method for preparing the above polypeptide comprises the following steps:

[0010] Tuna meat is boiled in water, the supernatant is separated, and products with a molecular weight <3 kD are cut from the supernatant, freeze-dried, and then purified. Unlike previously reported polypeptides with XOD inhibitory activity obtained through enzymatic hydrolysis, the polypeptides of the present invention can be obtained through aqueous extraction from tuna meat. Tuna red meat is preferred. Red tuna, the dark-colored meat during tuna processing, is often discarded due to its strong fishy odor and rough texture. Therefore, the technical solution of the present invention is of great significance for achieving high-value development of dark-colored meat and increasing the added value of products.

[0011] As a preferred embodiment of the method of the present invention, the mass ratio of tuna meat to water is 1:3-6.

[0012] As a preferred embodiment of the method of the present invention, the boiling temperature is 80-100° C. and the boiling time is 20-30 minutes.

[0013] Application of the above polypeptide in the preparation of xanthine oxidase inhibitors.

[0014] A xanthine oxidase inhibitor, comprising the above polypeptide.

[0015] As a preferred embodiment of the present invention, the inhibitor contains a short peptide with an amino acid sequence as shown in SEQ ID NO. 2, the inhibitor is in liquid form, and the concentration of the short peptide with an amino acid sequence as shown in SEQ ID NO. 2 in the inhibitor is ≥20 mg / mL.

[0016] Preferably, the inhibitor comprises a short peptide with an amino acid sequence as shown in SEQ ID NO. 2, is in liquid form, and the concentration of the short peptide with an amino acid sequence as shown in SEQ ID NO. 2 in the inhibitor is ≥ 30 mg / mL. When the concentration reaches 30 mg / mL, the XOD inhibition effect on FL7 can reach 100%.

[0017] A uric acid lowering preparation, comprising the above-mentioned xanthine oxidase activity inhibitor.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention provides a polypeptide with XOD inhibitory activity, selected from the short peptides KR5, FL7, RK8, or a combination thereof, with amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively. This polypeptide exhibits XOD inhibitory activity and can be developed as a XOD inhibitor and uric acid-lowering agent. Furthermore, the polypeptide provided by the present invention can be extracted from red tuna meat, enabling the high-value development of dark tuna meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the mass spectrum of the short peptide KR5.

[0021] Figure 2 This is the mass spectrum of the short peptide FL7.

[0022] Figure 3 This is the mass spectrum of the short peptide RK8. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention are further described below.

[0024] Unless otherwise specified, the raw materials used in the present invention can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are all conventional methods in the art.

[0025] The present invention provides a polypeptide having an XOD activity inhibitory effect, wherein the polypeptide is selected from one or more of the short peptides KR5, FL7 and RK8, wherein:

[0026] The amino acid sequence of KR5, SEQ ID NO. 1, is: Lys-Leu-Ala-His-Arg;

[0027] The amino acid sequence of FL7 is SEQ ID NO. 2: Phe-Val-Gly-Gly-Leu-Arg-Leu;

[0028] The amino acid sequence of RK8 is SEQ ID NO. 3: Arg-Met-Pro-Met-Phe-Phe-Leu-Lys.

[0029] The present invention also provides a xanthine oxidase inhibitor. In some embodiments, the xanthine oxidase inhibitor is a short peptide KR5 or FL7 or a short peptide RK8, or a combination of two or three thereof.

[0030] In some other embodiments of the xanthine oxidase inhibitor provided by the present invention, the xanthine oxidase inhibitor is a complex of short peptide KR5 or FL7 or short peptide RK8 or a combination of two or three thereof and other ingredients, such as conventional drug carriers, excipients, forming agents, etc.

[0031] In some embodiments of the xanthine oxidase inhibitor provided by the present invention, the xanthine oxidase inhibitor is in liquid form and contains the short peptide FL7; preferably, the concentration of the short peptide FL7 is ≥20 mg / mL, further, the concentration of the short peptide FL7 is ≥30 mg / mL, more preferably 30 mg / mL.

[0032] The present invention also provides a uric acid-lowering preparation. In some embodiments, the uric acid-lowering preparation is a short peptide KR5, FL7, or RK8, or a combination of two or three thereof. In some embodiments, the uric acid-lowering preparation is the aforementioned xanthine oxidase inhibitor, or a complex of the aforementioned xanthine oxidase inhibitor and other components, such as a carrier, excipient, or shaping agent.

[0033] The present invention also provides an embodiment of a method for preparing the polypeptide, comprising boiling tuna meat, such as tuna red meat, filtering and separating the supernatant, cutting off products with a molecular weight of <3KD, freeze-drying, and then purifying.

[0034] In some embodiments provided by the present invention, the mass ratio of tuna meat to water is 1:3-6.

[0035] In some embodiments provided by the present invention, the boiling temperature is 80-100° C., and the boiling time is 20-30 min.

[0036] The following is a further description of the acquisition of the polypeptide of the present invention and the XOD inhibition effect of the polypeptide through specific examples.

[0037] Example 1 Acquisition of polypeptides

[0038] (1) 400 g of bluefin tuna red meat was taken, added to 1600 mL of ddH2O and boiled for 20 min, and then centrifuged and filtered. The supernatant obtained by centrifugation was subjected to ultrafiltration, and the molecular segments of >10KD, 5-10KD, 5-3KD, and <3KD were intercepted, freeze-dried, and stored at -20°C for use. The yields of the molecular segments of >10KD, 5-10KD, 5-3KD, and <3KD were 1.42%, 0.24%, 0.17%, and 0.18%, respectively. At 1 mg / mL, the xanthine oxidase inhibition rates (%) of the molecular segments were 28.81±0.03, 35.18±0.12, 17.55±0.22, and 20.88±0.02, respectively.

[0039] (2) The peptide sequence analysis of the sample with a molecular weight less than 3KD was performed, and 458 peptides were identified and corresponded to tuna proteins. Among them, 4 short peptides that were not mapped to known species proteins were obtained, including KR5, FL7, and RK8, as shown in Table 1. The mass spectra of KR5, FL7, and RK8 are shown in Table 1. Figure 1 、 2 , as shown in 3.

[0040] Table 1 Four short peptides

[0041] short peptides Amino acid sequence ALC (%) quality local confidence (%) KR5 KLAHR 92 623.3867 100 99 94 81 89 FL7 FVGGLRL 92 760.4595 99 96 95 88 85 91 92 RK8 RMPMFFLK 91 1068.5613 90 85 83 95 98 96 90 90

[0042] The above short peptides are discovered for the first time and can be obtained in batches through conventional synthesis methods if the amino acid sequence is obtained.

[0043] Example 2 Comparison of XOD inhibition rates of polypeptides

[0044] The xanthine oxidase inhibition test of KR5, FL7 and RK8 is as follows:

[0045] The reaction system volume was 200 μL. 40 μL of sample and 80 μL of 8U XOD solution were added to a 96-well plate in sequence. The plates were incubated at 40°C for 30 minutes. Then, 80 μL of substrate xanthine solution was added and the plates were incubated in a constant temperature incubator for 15 minutes. The absorbance was then recorded at 292 nm. The experiment included a sample group (containing sample, enzyme, and substrate), a blank group (containing substrate and sample but not enzyme), a positive control group (containing allopurinol, enzyme, and substrate), and an enzyme group (containing enzyme and substrate but no sample). Each group contained three replicate wells, and the experiment was repeated three times. The final concentration of each sample was 100 μg / mL. The XOD inhibition rate was calculated as follows:

[0046] Inhibition rate (%) = [(A1-A0) / (B1-B0)] / ×100%;

[0047] A1: OD value of the well containing positive drug, XOD, XO, and buffer;

[0048] A0: blank control without XOD;

[0049] B1: OD value of the well containing XOD, XO, and buffer;

[0050] B0: Negative control wells containing XO and buffer.

[0051] The results are shown in Table 2.

[0052] Table 2 Effects of bluefin tuna water extract on XOD ( n=3)

[0053]

[0054] As can be seen from the table above, different short peptides have different XOD inhibitory activities. FR7 and RK8 showed good XOD inhibitory activity at the same concentration, while some peptides among the 458 peptides had no XOD inhibitory activity.

[0055] Example 3: XOD inhibitory activity test of FL7

[0056] The XOD inhibitory activities of FL7 at different concentrations are shown in Table 3.

[0057] Table 3 XOD inhibition rate at different FL7 concentrations (`x±s, n=3)

[0058] C mg / mL(mmol / L) 1(1.32) 5(6.58) 10(13.16) 20(26.32) 30(39.47) XOD inhibition rate (%) 68.22±0.12 64.22±0.32 75.13±0.02 78.33±0.20 100±0.29

[0059] As can be seen from the table above, within the test range, the XOD inhibition rate gradually increased with the increase of FL7 concentration, with a significant increase in the range of 20-30 mg / mL. When the FL7 concentration reached 30 mg / mL, the XOD inhibition rate reached 100%, and the half-inhibitory concentration of FL7 was calculated to be IC50 = 1.69 mg / mL.

[0060] Example 4 XOD inhibitory activity of RK8

[0061] The XOD inhibitory activities of RK 8 at different concentrations are shown in Table 4.

[0062] Table 4 XOD inhibition rate at different RK8 concentrations (`x±s, n=3)

[0063] C mg / mL(mmol / L) 1(1.87) 5(6.58) 10(13.16) 20(26.32) 30(56.07) XOD inhibition rate (%) 69.80±0.32 65.73±0.24 69.44±0.25 76.50±0.42 87.62±0.22

[0064] As can be seen from the table above, within the test range, as the RK8 concentration increases, the XOD inhibition rate gradually increases. When the RK8 concentration reaches 30 mg / mL, the XOD inhibition rate reaches 87.62%. The half-inhibitory concentration of RK8 is calculated to be IC50 = 1.82 mg / mL.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is one of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

2. A method for preparing the polypeptide according to claim 1, characterized in that: The polypeptide is synthesized according to the amino acid sequence of the polypeptide.

3. A xanthine oxidase inhibitor, characterized in that The inhibitor comprises the polypeptide according to claim 1.

4. The inhibitor according to claim 3, characterized in that The inhibitor contains a short peptide with an amino acid sequence as shown in SEQ ID NO.

2. The inhibitor is in liquid form, and the concentration of the short peptide with an amino acid sequence as shown in SEQ ID NO. 2 in the inhibitor is ≥20 mg / mL.

5. The inhibitor according to claim 3, characterized in that The inhibitor contains a short peptide with an amino acid sequence as shown in SEQ ID NO.

2. The inhibitor is in liquid form, and the concentration of the short peptide with an amino acid sequence as shown in SEQ ID NO. 2 in the inhibitor is ≥30 mg / mL.

6. A uric acid lowering preparation, characterized in that: The uric acid-lowering preparation contains the xanthine oxidase inhibitor according to any one of claims 3 to 5.

Images