CN117886888B - Sea cucumber peptide with uric acid reducing effect and application thereof - Google Patents
Sea cucumber peptide with uric acid reducing effect and application thereofInfo
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- CN117886888B CN117886888B CN202410281905.4A CN202410281905A CN117886888B CN 117886888 B CN117886888 B CN 117886888B CN 202410281905 A CN202410281905 A CN 202410281905A CN 117886888 B CN117886888 B CN 117886888B
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- sea cucumber
- uric acid
- peptide
- cucumber peptide
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Abstract
The application belongs to the technical field of active peptide development, and particularly relates to a sea cucumber peptide with uric acid reducing effect and application thereof. According to the application, sea cucumber is taken as a polypeptide source, and three sea cucumber peptides with uric acid reducing activity are obtained through enzymolysis, separation, purification and sequence identification: EKFPPPM, PPLVKPW, KDLGVLI.
Description
Technical Field
The invention belongs to the technical field of active peptide development, and particularly relates to a sea cucumber peptide with uric acid reducing effect and application thereof.
Background
Hyperuricemia is a chronic disease due to impaired purine metabolism or decreased uric acid excretion. At present, according to large epidemiological studies, hyperuricemia is becoming a serious public health problem, and is a risk factor for metabolic diseases such as gout, uric acid kidney stones, hypertension, diabetes mellitus and the like. In humans, elevated blood uric acid levels are associated with abnormal nucleic acid metabolism and decreased renal excretion in the body. Xanthine oxidase is a key enzyme regulating uric acid production. It is widely used in various organisms, and xanthine oxidase can reduce serum uric acid in vivo by catalyzing oxidation of hypoxanthine to xanthine and further oxidation to uric acid and inhibiting activity of xanthine oxidase. Therefore, maintenance of uric acid levels in human serum is extremely important for human health, and xanthine oxidase can be a key target for the treatment of hyperuricemia.
Sea cucumber can be used for continuing aging, eliminating fatigue, improving immunity, enhancing disease resistance, is rich in more than 50 natural precious active substances such as proteins, minerals, vitamins and the like, and 18 amino acids contained in sea cucumber can be used for enhancing the metabolic function of tissues, enhancing the activity of organism cells and improving the immunity of human bodies. Therefore, the sea cucumber peptide has great market prospect in treating hyperuricemia.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention aims to obtain the sea cucumber peptide with uric acid reducing function, which is used for developing medicines aiming at reducing uric acid.
The invention aims at realizing the following technical scheme:
in one aspect, the invention provides a sea cucumber peptide with uric acid reducing effect, wherein the amino acid sequence of the polypeptide is as follows: EKFPPPM, PPLVKPW, KDLGVLI.
On the other hand, the invention provides a preparation method of sea cucumber peptide, which comprises the following steps:
(1) Homogenizing sea cucumber flesh to obtain sea cucumber homogenate, and shearing and homogenizing the sea cucumber homogenate to obtain sea cucumber homogenate;
(2) Regulating the pH value of the sea cucumber homogenized liquid to 7.0, and freeze-drying the obtained sea cucumber homogenized liquid to obtain freeze-dried powder for later use;
(3) Preparing a solution from the sea cucumber freeze-dried powder, regulating the pH value to 8.0, adding alkaline protease, compound protease and papain at 55 ℃ for enzymolysis for 1h, and obtaining a sea cucumber protein enzymolysis solution;
(4) And regulating the pH value of the obtained sea cucumber protein enzymolysis liquid to 7.0, and freeze-drying the enzymolysis liquid to obtain sea cucumber peptide powder.
(5) Sea cucumber peptides are determined by separation, purification and sequence identification, and the sequences are EKFPPPM (SEQ ID NO. 1), PPLVKPW (SEQ ID NO. 2) and KDLGVLI (SEQ ID NO. 3) respectively.
Wherein the solution of the sea cucumber freeze-dried powder is 5%.
Wherein the total enzyme activities of alkaline protease, composite protease and papain are 3000U.
Wherein the addition ratio of the alkaline protease, the compound protease and the papain is 1:1:1.
In another aspect, the present invention provides a composition comprising 1 and/or 2 and/or 3 of the aforementioned 3 sea cucumber peptides.
On the other hand, the application provides application of sea cucumber peptide and a composition thereof in preparing an auxiliary uric acid reduction product.
Compared with the prior art, the invention has the beneficial effects that:
In order to obtain the sea cucumber xanthine oxidase inhibitory peptide, the invention adopts a compound enzymolysis mode, and polypeptide with xanthine oxidase inhibitory activity is obtained through a series of enzymolysis reactions, and the specific sequence is EKFPPPM, PPLVKPW, KDLGVLI, and the peptide sequences have xanthine oxidase inhibitory activity, so that the effect of reducing uric acid is achieved.
Drawings
FIG. 13 xanthine oxidase IC50 of sea cucumber peptides.
The composition of fig. 2 reduced uric acid and renal function impairment results in rats. A is uric acid lowering result; b is serum creatinine, C is urea nitrogen.
FIG. 3 effect of composition on rat serum and liver XOD activity and ADA activity. A is serum XOD activity; b is serum ADA activity; c is liver XOD activity; d is liver ADA activity.
FIG. 4 composition III is a graph of kidney pathology tissue of rats.
Detailed Description
The present invention will be described in detail by examples. It should be understood that the following examples are illustrative only of the present invention and are not intended to limit the present invention.
EXAMPLE 1 preparation of uric acid Activity-reducing sea cucumber peptide
(1) Homogenizing sea cucumber flesh to obtain sea cucumber homogenate, and shearing and homogenizing the sea cucumber homogenate to obtain sea cucumber homogenate;
(2) Regulating the pH value of the sea cucumber homogenized liquid to 7.0, and freeze-drying the obtained sea cucumber homogenized liquid to obtain freeze-dried powder for later use;
(3) Preparing a sea cucumber freeze-dried powder into a 5% solution, regulating the pH value to 8.0, adding alkaline protease, compound protease and papain at 55 ℃ for enzymolysis, wherein the total enzyme activity of the three proteases is 3000U, the adding ratio of the alkaline protease to the compound protease to the papain is 1:1:1, and the sea cucumber proteolytic liquid is obtained after enzymolysis of 1:1 h;
(4) Regulating the pH value of the obtained sea cucumber protein enzymolysis liquid to 7.0, and freeze-drying the enzymolysis liquid to obtain sea cucumber peptide powder;
(5) Three sea cucumber peptides are found through separation, purification and sequence identification, and the sequences are EKFPPPM (SEQ ID NO. 1), PPLVKPW (SEQ ID NO. 2) and KDLGVLI (SEQ ID NO. 3) respectively.
EXAMPLE 2 xanthine oxidase inhibitory Activity test for sea cucumber peptide Synthesis
The experiment adopts the method for measuring the inhibition rate of xanthine oxidase to characterize the uric acid reduction activity of a sample, and comprises the following specific steps: taking 250 mu L of a sample solution to be tested and 250 mu L of xanthine oxidase, adding the sample solution to be tested and 250 mu L of xanthine oxidase into a 2mL centrifuge tube, incubating the sample solution at 37 ℃ for 10 min, adding 750 mu L of xanthine solution to start the reaction, continuing incubating the sample solution at 37 ℃ for 15 min, and adding 400 mu L of 1M hydrochloric acid to stop the reaction after the reaction is finished. Positive and negative controls were made with 5. Mu.g/mL allopurinol and 50mM Tris-HCl buffer, respectively. The sample was passed through a 0.22 μm PES membrane. HPLC conditions were column: c18 column (250X 4.6 mm, 5 μm); mobile phase: 85% aqueous NH 4H2PO4 of 10mM and 15% methanol; flow rate: 0.8 mL/min; UV:290 nm. A50. Mu.M uric acid solution was used as a standard to determine the retention time of uric acid. Quantitative calculations were performed with corresponding peak areas based on retention time.
P（XOD）=(ccontrol-csample)/ccontrol×100%
The results of xanthine oxidase IC50 of the three synthetic peptides are shown in figure 1, and according to the results, the semi-inhibitory concentration of the cucumber xanthine oxidase inhibitory peptide EKFPPPM, PPLVKPW, KDLGVLI is 14.48+/-0.90, 19.81 +/-0.65, 24.52+/-0.27 and mM, the three synthetic peptides have good xanthine oxidase inhibitory concentration, the semi-inhibitory concentration represents the concentration of the substrate when the inhibitory rate is 50%, and the smaller the value is, the better the inhibitory effect is, and the relatively better the inhibitory activity of the short-chain polypeptide is. Sea cucumber inhibitory peptides can inhibit uric acid synthesis by inhibiting xanthine oxidase activity, thereby playing a role in uric acid lowering.
EXAMPLE 3 xanthine oxidase inhibitory Activity test of sea cucumber peptide powder
Because the cost of the synthetic peptide is relatively high, the sea cucumber peptide powder is directly adopted for subsequent experiments. The xanthine oxidase inhibition rates of the sea cucumber peptide powder and the compositions I to III containing the sea cucumber peptide powder were respectively determined. Wherein the composition I comprises the following raw materials in parts by weight: 10 parts of sea cucumber peptide, 10 parts of celery extract, 460 parts of skim milk powder, 70 parts of chrysanthemum extract, 10 parts of chitosan oligosaccharide, 2 parts of luteolin and 1 part of quercetin. The composition II comprises the following raw materials in parts by weight: 10 parts of sea cucumber peptide, 30 parts of celery extract, 460 parts of skim milk powder, 70 parts of chrysanthemum extract, 10 parts of chitosan oligosaccharide, 2 parts of luteolin and 1 part of quercetin. The composition III comprises the following raw materials in parts by weight: 10 parts of sea cucumber peptide, 50 parts of celery extract, 460 parts of skim milk powder, 70 parts of chrysanthemum extract, 10 parts of chitosan oligosaccharide, 2 parts of luteolin and 1 part of quercetin. The inhibition rate of xanthine oxidase of the sea cucumber peptide powder is 18.79+/-0.62%, and 10 parts by weight of the sea cucumber peptide powder (the concentration is 10 mg/mL) is far greater than the synthesized sea cucumber peptide sequence, which is probably due to the fact that the activity of the sea cucumber peptide is limited by the existence proportion of the sea cucumber peptide in the sea cucumber powder or the existence of other interfering substances, and the composition mode shows that the sea cucumber peptide has higher inhibition rate, namely the sea cucumber peptide has uric acid reducing potential.
Table 1 xanthine oxidase inhibition ratio of formulation raw materials
Example 4 animal experiments demonstrate uric acid lowering efficacy of composition III
32 SD male rats (body weight 200 g + -20 g) were selected, 23+ -3deg.C; 65.+ -. 5% humidity, light-dark cycle 12 h, keep ventilation good, rats were kept for 1 week to fit the laboratory environment before the experiment began. Rats were randomly divided into NC, HUA, allopurinol, and formula groups (8 rats per group). Then, NC rats are filled with gastric distilled water, other rats are filled with 1.5g/kg BW of potassium gastroxymate, the model is built 1 time a day for 7 continuous days, and when the concentration of uric acid in serum is more than 110 mu mol/L, the model building is successful.
HUA group, allopurinol group and formula group were administered 1 h after the administration of gastric lavage, and the rats were respectively given gastric distilled water, positive drug and uric acid reducing composition. Wherein, rats in NC group and HUA group only are given distilled water, allopurinol group is used for lavage positive drug allopurinol, the dosage is 20mg/kg BW, and the formula group is used for lavage composition III for lowering uric acid by 500mg/kg BW every day.
All rats were examined daily for body weight, diet and health. Retroorbital plexus blood samples were collected by intraperitoneal injection of 3% sodium pentobarbital (30 mg/kg BW) under anesthesia. Blood samples were centrifuged at 3000 Xg for 15min at 4℃to obtain serum for further physiological analysis. Serum samples were collected 3 times on days 7, 14 and 21 of administration, rats were sacrificed on day 28, blood, liver and kidney were collected and stored at-80 ℃ for detection. Kidney and liver metrics are based on fresh body weight: kidney or liver index (mg/g) =kidney or liver body weight (mg)/body weight (g).
And measuring uric acid level in the blood sample on the 7 th day, wherein the serum uric acid concentration is more than 110 mu mol/L, and the modeling is successful. Starting to perfuse the allopurinol and the formulation composition. Uric acid levels in serum were measured in 14 and 21, and after the rats were sacrificed, blood was collected to measure uric acid, creatinine and urea nitrogen in serum, as measured using commercial test kits. XOD and Adenosine Deaminase (ADA) activity in serum and liver was determined.
As shown in FIG. 2A, it can be seen that uric acid levels in hyperuricemia rats were all greater than 110. Mu. Mol/L after one week of gastric lavage, i.e., hyperuricemia modeling was considered successful. After three weeks of intervention, normal rat serum uric acid in NC group was 83.83 + -3.32 μmol/L, but serum uric acid in HUA group hyperuricemia rat reached 203+ -5.9 μmol/L, and rat uric acid content in formula group and allopurinol group was significantly reduced, wherein allopurinol group serum uric acid was reduced to 88.33+ -2.94 μmol/L, and gradually recovered to normal level compared with NC group. In the formula set, the serum uric acid is reduced to 113+/-4.09 mu mol/L, which indicates that the composition III has the potential of developing into an anti-hyperuricemia candidate drug.
In practice, serum creatinine and urea nitrogen concentrations are commonly used to characterize renal function status. As shown in fig. 2B and C, HUA group mice had significantly elevated serum creatinine and urea nitrogen levels compared to NC group. The urea nitrogen and creatinine levels of the formulation were significantly lower than those of the HUA group, and the results indicate that supplementing the compositions provided by the present study can alleviate hyperuricemia and associated kidney function impairment.
The effect of the composition on rat serum XOD activity and ADA activity is shown in fig. 3A, B. The serum XOD activity and ADA activity of the hyperuricemia rats in the HUA group are remarkably increased compared with those of the normal rats in the NC group, and after the allopurinol group is infused with the stomach-positive medicament, no remarkable difference exists between the serum XOD activity and ADA activity and the NC group, and the normal level is recovered. After the gastric lavage composition, the serum XOD activity and ADA activity of the formulation group were significantly reduced compared to the HUA group, which had therapeutic effects on serum XOD and ADA of hyperuricemia rats, although unlike allopurinol. These results indicate that in hyperuricemia rats, serum XOD activity and ADA activity are enhanced, leading to increased uric acid production, thereby inducing the occurrence of hyperuricemia. The composition can reduce the activity of serum XOD and ADA, thereby reducing the generation of uric acid by inhibiting the activity of uric acid synthesis key enzyme, and further relieving the symptoms of hyperuricemia rats.
The effect of the composition on liver XOD activity and ADA activity of rats, as shown in fig. 3C, D, the liver XOD activity of NC group normal rats was 15.15±0.45±0.45: 0.45U/g prot, the liver ADA activity of normal rats was 17.34±0.62: 0.62U/g prot, the liver XOD activity of HUA group hyperuricemia rats and normal rats was significantly increased to 29.79 ±0.21U/g prot, the liver ADA activity was significantly increased to 24.50±0.18: 0.18U/g prot, the liver XOD activity and ADA activity of hyperuricemia rats after the administration of allopurinol, the composition were significantly reduced, and the activity of uric acid production key enzymes in the liver was inhibited.
The histopathological image of the kidney is shown in fig. 4, the HUA group has some pathological changes, the glomerulus at the black arrow is atrophic, the tubular at the yellow arrow is dilated, the cell brush border is shed, and the like, but under the action of allopurinol, the kidney of the rat is improved, and compared with the NC group, the kidney glomerulus is plump and the cell compact gap is small. After the stomach is irrigated, compared with the hyperuricemia rats in the HUA group, the kidneys of the rats are also obviously improved. These results indicate that the formulated composition is capable of ameliorating the damage to renal function in hyperuricemic rats.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. A sea cucumber peptide with uric acid reducing effect, which is characterized in that the amino acid sequence of the sea cucumber peptide is as follows: EKFPPPM as shown in SEQ ID NO. 1.
2. A sea cucumber peptide with uric acid reducing effect, which is characterized in that the amino acid sequence of the sea cucumber peptide is as follows: PPLVKPW as shown in SEQ ID NO. 2.
3. A sea cucumber peptide with uric acid reducing effect, which is characterized in that the amino acid sequence of the sea cucumber peptide is as follows: KDLGVLI as shown in SEQ ID NO. 3.
4. A method for producing a sea cucumber peptide as claimed in any one of claims 1 to 3, characterized by the following method:
(1) Homogenizing sea cucumber flesh to obtain sea cucumber homogenate, and shearing and homogenizing the sea cucumber homogenate to obtain sea cucumber homogenate;
(2) Regulating the pH value of the sea cucumber homogenized liquid to 7.0, and freeze-drying the obtained sea cucumber homogenized liquid to obtain freeze-dried powder for later use;
(3) Preparing a 5% solution of the sea cucumber freeze-dried powder, regulating the pH value to 8.0, adding alkaline protease, compound protease and papain at 55 ℃ for enzymolysis for 1h, and obtaining a sea cucumber proteolytic liquid;
The adding ratio of the alkaline protease to the compound protease to the papain is 1:1:1;
(4) Regulating the pH value of the obtained sea cucumber protein enzymolysis liquid to 7.0, and freeze-drying the enzymolysis liquid to obtain sea cucumber peptide powder;
(5) The sea cucumber peptide is determined through separation, purification and sequence identification.
5. The method according to claim 4, wherein the total enzyme activity of alkaline protease, complex protease and papain is 3000U.
6. A composition comprising the sea cucumber peptide of claim 1 and/or claim 2 and/or claim 3.
7. Use of a sea cucumber peptide as claimed in any one of the claims 1-3 for the manufacture of a medicament for assisting in uric acid lowering.
8. Use of the composition according to claim 6 for the preparation of a medicament for assisting in lowering uric acid.
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| CN202410281905.4A CN117886888B (en) | 2024-03-13 | Sea cucumber peptide with uric acid reducing effect and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105077263A (en) * | 2015-09-25 | 2015-11-25 | 山东大学（威海） | Medicinal and edible sea cucumber polypeptide as well as preparation method and application thereof |
| CN117285596A (en) * | 2023-11-27 | 2023-12-26 | 中国科学院烟台海岸带研究所 | Hexapeptide RL6 with uric acid reducing function and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105077263A (en) * | 2015-09-25 | 2015-11-25 | 山东大学（威海） | Medicinal and edible sea cucumber polypeptide as well as preparation method and application thereof |
| CN117285596A (en) * | 2023-11-27 | 2023-12-26 | 中国科学院烟台海岸带研究所 | Hexapeptide RL6 with uric acid reducing function and application thereof |
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