An active polypeptide for alleviating kidney injury, its preparation method and application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHANGCHUN UNIV OF CHINESE MEDICINE
- Filing Date
- 2026-03-04
- Publication Date
- 2026-05-26
Smart Images
Figure CN121779501B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to an active polypeptide that alleviates kidney damage, its preparation method, and its application. Background Technology
[0002] The kidneys are the core organs for uric acid metabolism and excretion in the human body. Hyperuricemia has become a significant risk factor for acute kidney injury, chronic kidney disease, and even kidney failure. Urate crystal deposition, oxidative stress damage, and activated inflammatory responses can directly cause damage to renal tubular epithelial cells, renal interstitial fibrosis, and decreased glomerular filtration function, seriously threatening public health. Currently, most drugs used clinically to improve hyperuricemia-related kidney damage primarily work by inhibiting uric acid production or promoting uric acid excretion. However, these drugs generally suffer from problems such as increased metabolic burden on the liver and kidneys, gastrointestinal irritation, and insufficient safety profiles for long-term use, making them unsuitable for applications in food, health supplements, and long-term care settings.
[0003] Natural bioactive peptides have become a research hotspot in the fields of nephrology and metabolic regulation due to their advantages such as small molecular weight, easy absorption, good biocompatibility, and high safety. Marine biological resources are abundant and widely available, with unique protein compositions. Various physiologically active peptides can be prepared through enzymatic hydrolysis, making them high-quality raw materials for the development of functional foods, health products, and pharmaceuticals. While some peptides with uric acid-lowering or nephroprotective effects have been disclosed in existing technologies, marine-derived bioactive peptides that simultaneously possess high safety, well-defined structures, simple preparation processes, and suitability for industrial production remain relatively scarce. Furthermore, traditional enzymatic extraction processes generally suffer from low hydrolysis efficiency, low product purity, and unclear active components, hindering the development and application of related bioactive peptides in food, health products, and pharmaceuticals.
[0004] To address the aforementioned technical challenges, developing a marine-derived bioactive polypeptide that can effectively reduce kidney damage and has a simple and safe preparation process is of great significance and industrial value for meeting market demand for kidney care products and expanding the high-value application of marine biological resources. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an active polypeptide that alleviates kidney damage, along with its preparation method and applications. This active polypeptide is derived from the scallop and has significant effects in alleviating kidney damage. Furthermore, the preparation process is simple, safe, and suitable for industrial production and widespread application.
[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:
[0007] The present invention provides an active polypeptide that alleviates kidney damage, the amino acid sequence of which is shown in SEQ ID NO:1.
[0008] Furthermore, the amino acid sequence of the active polypeptide is Met-Ala-Leu-Asn-Asn-Ser-Gly-Glu-Thr-Tyr-Glu (MALNNSGETYE).
[0009] Furthermore, the molecular weight of the active polypeptide is 1.23 kDa.
[0010] The present invention also provides a pharmaceutical preparation containing the aforementioned active polypeptide.
[0011] Furthermore, the pharmaceutical preparation also contains pharmaceutically acceptable excipients.
[0012] Furthermore, the pharmaceutically acceptable excipient is at least one of solvents, wetting agents, emulsifiers, thickeners, excipients, suspending agents, disintegrants, fillers, lubricants, or diluents.
[0013] Furthermore, the pharmaceutical formulation can be formulated into any pharmacologically acceptable dosage form.
[0014] Furthermore, the dosage form of the pharmaceutical preparation includes tablets, liquids, capsules, powders, suppositories, granules, pills, sprays, or liniments.
[0015] Furthermore, the drug can be administered orally, intravenously, locally, intradermally, or subcutaneously.
[0016] The present invention also provides the use of the described active polypeptide or the described pharmaceutical preparation in the preparation of products related to alleviating kidney injury.
[0017] Furthermore, the product includes pharmaceuticals.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0019] The active polypeptide MALNNSGETYE (SEQ ID NO:1) provided by this invention is derived from the scallop and has high safety. This active polypeptide can significantly alleviate cisplatin-induced kidney damage and significantly reduce creatinine and urea nitrogen levels. The preparation method of this invention is simple and suitable for industrial production, solving the problems of low efficiency, low product purity, and unclear composition in traditional enzymatic hydrolysis processes. The obtained active polypeptide can be used in drugs for treating kidney damage, and has significant clinical application value and market prospects. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1 This is a schematic diagram of the molecular structure of the active polypeptide of the present invention;
[0022] Figure 2 This is a predicted three-dimensional structure diagram of the active polypeptide of the present invention (A represents the main chain backbone of the active polypeptide, and B represents the amino acid residues of the side chain further shown on the basis of the main chain backbone).
[0023] Figure 3 This is the result of the cell safety evaluation of the active polypeptide of the present invention. Detailed Implementation
[0024] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods.
[0025] The terms “comprising,” “including,” “containing,” “having,” “comprising,” or other variations thereof are intended to cover non-closed inclusion, and no distinction is made between these terms. The term “comprising” means that other steps and components may be added without affecting the final result. The term “comprising” also includes the terms “consisting of” and “substantially consisting of”. The compositions and methods / processes of the present invention may comprise, consist of, and substantially consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.
[0026] Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without a specified manufacturer are commercially available, conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. Those skilled in the art should understand that the invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.
[0027] The experimental data of this invention were analyzed using SPSS 20.0 software. All data were tested for normality and homogeneity of variance. The experimental data are expressed as x±s. One-way ANOVA was used to analyze the results. ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.
[0028] Example 1: Process for extracting active polypeptides from Yesso scallops
[0029] Ingredients: Ezo scallops ( Mizuhopecten yessoensis (Commonly known as Xia Yi Bei), purchased from Changchun Changbai Road Aquatic Products Market, No. 5 Changbai Road, Kuancheng District.
[0030] The specific steps for extracting the active polypeptide MALNNSGETYE (SEQ ID NO:1) from the scallop in this embodiment include:
[0031] (1) Raw material pretreatment: Take 200g of fresh scallop adductor muscle, rinse it three times with ultrapure water to remove surface mud and impurities, drain it and cut it into 1cm³ pieces; add pre-cooled 0.05mol / L Tris-HCl buffer (pH7.0) at a material-to-liquid ratio of 1:4 (g:mL), put it into a tissue homogenizer, homogenize it at 8000 r / min for 5min at 4℃ to obtain scallop meat homogenate; place the homogenate in a refrigerator at 4℃ for 2h, stirring once every 30min during the period, and then centrifuge at 8000 r / min at 4℃ for 20min, and take the supernatant as the crude scallop protein extract.
[0032] (2) Two-step enzymatic hydrolysis: First, add neutral protease (enzyme activity 1000U / mg) to the crude protein extract, with an enzyme amount of 1.5% according to the material-to-liquid ratio (g:mL); adjust the pH of the system to 7.0, place it in a constant temperature water bath shaker, and enzymatically hydrolyze at 45℃ and 150r / min for 4h; after the enzymatic hydrolysis is completed, heat the system to 95℃ and inactivate the enzyme in a water bath for 10min, cool it to room temperature, and centrifuge at 4℃ and 8000r / min for 15min, and take the supernatant. Then, add trypsin (enzyme activity 2500U / mg) to the supernatant, with an enzyme amount of 1.0% according to the material-to-liquid ratio (g:mL); adjust the pH of the system to 7.5, place it in a constant temperature water bath shaker, and enzymatically hydrolyze at 50℃ and 150r / min for 3h; after the enzymatic hydrolysis is completed, heat the system to 95℃ and inactivate the enzyme in a water bath for 10min, cool it to room temperature, and centrifuge at 4℃ and 8000r / min for 15min, and take the supernatant, which is the scallop enzymatic hydrolysate.
[0033] (3) Crude purification by gel chromatography: The Sephadex G-25 gel was swollen with ultrapure water for 24 h, degassed and loaded into the chromatography column. The chromatography column was equilibrated with 0.05 mol / L Tris-HCl buffer (pH 7.0) until the baseline was stable. 5 mL of scallop enzymatic hydrolysate was loaded onto the column and eluted with equilibration buffer at a flow rate of 0.8 mL / min. 5 mL was collected from each tube using an automatic collector, and the absorbance at 280 nm was measured. The eluents corresponding to the absorbance peak were combined and concentrated under reduced pressure at 4 °C to 1 / 5 of the original volume to obtain the crude peptide solution.
[0034] (4) HPLC purification: The crude peptide solution was purified by high performance liquid chromatography using an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm), at a column temperature of 30 °C, a flow rate of 1 mL / min, an injection volume of 20 μL, and a measurement wavelength of 220 nm. Mobile phase A was an aqueous solution of 0.1% TFA, and mobile phase B was an acetonitrile solution of 0.1% TFA. The gradient elution program was as follows: 0-10 min, 5%-15% B; 10-25 min, 15%-30% B; 25-35 min, 30%-40% B; 35-45 min, 40%-5% B. Based on the HPLC peak retention time (approximately 28 min), the eluent corresponding to the peak was collected and freeze-dried under vacuum to obtain high-purity active peptides with a purity ≥98%.
[0035] (5) Peptide sequence determination: The amino acid sequence was determined using the Edman degradation method. The coupling, cleavage and separation processes were completed using an automated protein sequencer. The amino acid sequence of the obtained active peptide was determined to be MALNNSGETYE (SEQ ID NO: 1, structural formula as shown in Figure 1). Figure 1 As shown, the molecular weight is 1.23 kDa, and the purity was determined by HPLC to be ≥98%. It should be stored at low temperature for future use.
[0036] (6) Structure prediction: The active peptide of SEQ ID NO:1 was predicted using the tertiary structure prediction tool PEP-FOLD (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD3 / ), and the results are as follows. Figure 2 As shown.
[0037] Example 2 Safety evaluation of active peptides
[0038] Fully adherent HeLa cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0101) were digested with 0.25% trypsin and cultured in high-glucose DMEM containing 5% FBS to achieve a cell density of 1×10⁻⁶ cells / cells. 5Cell suspension at 100 μL / mL was seeded into 96-well plates and cultured at 37°C with 5% CO2 saturated humidity for 24 h. The complete culture medium was then aspirated. The experimental groups were added to high-glucose DMEM medium diluted to concentrations of 0.2 mg / ml, 1 mg / ml, 4 mg / ml, and 10 mg / ml, respectively, to the polypeptide solution prepared in Example 1. The control group consisted of cells cultured in DMEM medium, and the blank group consisted of cell-free DMEM medium. The cells were cultured for another 24 h at 37°C with 5% CO2 saturated humidity. 10 μL of CCK-8 reagent (GLPbio) was added to each group, and the cells were incubated for 4 h. The absorbance (OD value) of each well was measured using an ELISA reader at 450 nm. Cell viability was calculated based on the mean absorbance of each group using the following formula:
[0039]
[0040] Experimental results are as follows Figure 3 As shown, the cell survival rates of the active peptides of the present invention at different concentrations and the control group were all above 100%, and the activity of the active peptides was improved compared with the blank group, indicating that the active peptides of the present invention have no cytotoxicity and good safety.
[0041] Example 3: Active peptides inhibit cisplatin-induced damage and apoptosis in human renal tubular epithelial cells.
[0042] Cell source: Human renal tubular epithelial cells (purchased from Wuhan Pronosei Life Science Co., Ltd., catalog number: CP-H193).
[0043] Culture medium: Complete culture medium for human renal tubular epithelial cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CM-H193).
[0044] Experimental Methods: Cultured human renal tubular epithelial cells were seeded at a density of approximately 10,000 cells / well in 96-well cell culture plates and incubated at 37°C with 5% CO2 for 1 day. Then, the cells were cultured for another 12 hours in complete human renal tubular epithelial cell culture medium. After changing the medium, cisplatin (25 μg / mL) was added to stimulate the cells, and different concentrations (0, 50, 100, 200, 500, and 1000 ng / mL) of bioactive peptides were added and incubated for 12 hours. Cell viability was assessed using the MTT assay after incubation.
[0045] Table 1. Protective effect of different concentrations of active peptides against cisplatin-induced human renal tubular epithelial cell injury.
[0046]
[0047] Compared with the normal group, *p < 0.05, **p < 0.01; compared with the control group, #p < 0.05, ##p < 0.01.
[0048] The detection results are shown in Table 1. The bioactive polypeptide of the present invention has a significant improvement effect on the damage of human renal tubular epithelial cells induced by cisplatin, and the effect is positively correlated with the polypeptide concentration.
[0049] Example 4 Inhibitory effect of bioactive polypeptide on cisplatin-induced kidney injury in mice
[0050] Source of mice: SPF-grade male ICR mice, 6 weeks old, body weight 25 - 30 g, purchased from Zhuhai Besttone Biotechnology Co., Ltd., with the use license number SYXK (Guangdong) 2025 - 0229. The feeding temperature was controlled at 20 - 25 °C, the humidity was 40% - 60%, the light and dark alternated for 12 h, and the experiment was started after 1 week of adaptive feeding.
[0051] Grouping and administration: Thirty male ICR mice were randomly divided into 5 groups: blank control group, model group, positive control group, low-dose and high-dose bioactive polypeptide groups. Among them:
[0052] The normal group and the model group were given intragastric administration of the corresponding volume of normal saline for 14 consecutive days;
[0053] Positive control group: Amifostine was intraperitoneally injected at a dose of 2 mg / kg for 14 consecutive days;
[0054] Low-dose bioactive polypeptide group: The bioactive polypeptide was intragastrically administered at a dose of 5 mg crude drug / kg for 14 consecutive days;
[0055] High-dose bioactive polypeptide group: The bioactive polypeptide was intragastrically administered at a dose of 20 mg crude drug / kg for 14 consecutive days.
[0056] On the 15th day, except for the normal group, the other groups were modeled by intraperitoneal injection of a cisplatin solution at a dose of 25 mg / kg; 72 hours later, the mice in each group were anesthetized with 1% pentobarbital, and then blood was collected by eye socket extraction into heparin sodium-treated blood collection tubes, centrifuged at 3000 rpm for 10 min, and the plasma was taken for standby; after weighing the bilateral kidney tissues, they were stored in 4% paraformaldehyde.
[0057] Index detection:
[0058] Calculate the kidney index of the 5 groups of mice (kidney index = bilateral kidney tissue weight / mouse body weight × 100%);
[0059] Take the plasma samples of the mice in each group and detect the creatinine and urea nitrogen contents in the serum of the mice in each group according to the instructions of the creatinine (CRE) and urea nitrogen (BUN) kits (purchased from Nanjing Jiancheng Bioengineering Institute, product serial numbers are C011 - 2 - 1 and C013 - 2 - 1).
[0060] Experimental results:
[0061] Table 2. Effects of bioactive peptides on kidney coefficient, creatinine, and blood urea nitrogen in mice (x±s, n=6)
[0062]
[0063] Compared with the normal group, #p<0.05, ##p<0.01, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001; compared with the positive control group, △p<0.05, △△p<0.01, △△△p<0.001.
[0064] The experimental results are shown in Table 2. Compared with the normal group, the kidney coefficient, creatinine and blood urea nitrogen in the model group were significantly increased (p<0.01 or p<0.001), indicating that the model was successfully constructed.
[0065] Compared with the model group, the positive control group, active peptide group, and all groups showed significantly lower renal coefficients, creatinine, and blood urea nitrogen levels (p < 0.01). Compared with the positive control group, there were no significant differences in creatinine levels among the active peptide group and the positive control group, except for the low-dose active peptide group (p > 0.05). These results indicate that cisplatin induction can significantly cause acute kidney injury. The low-dose, high-dose, and positive control groups of the active peptide of this invention can alleviate cisplatin-induced edema or congestion, as well as reduce the increase in creatinine and blood urea nitrogen levels. The peptide of this invention has a significant effect in alleviating kidney injury and can be used in the preparation of drugs for treating kidney injury.
[0066] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection claimed by this invention is defined by the appended claims and their equivalents.
Claims
1. An active polypeptide for alleviating kidney damage, characterized in that, The amino acid sequence of the active polypeptide is shown in SEQ ID NO:
1.
2. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation contains the active polypeptide as described in claim 1.
3. The pharmaceutical preparation according to claim 2, characterized in that, The pharmaceutical preparation also contains pharmaceutically acceptable excipients.
4. The pharmaceutical preparation according to claim 3, characterized in that, The pharmaceutically acceptable excipient is at least one of the following: wetting agent, emulsifier, thickener, excipient, suspending agent, disintegrant, filler, lubricant, or diluent.
5. The pharmaceutical preparation according to claim 2, characterized in that, The pharmaceutical preparation can be formulated into any pharmacologically acceptable dosage form.
6. The pharmaceutical preparation according to claim 5, characterized in that, The dosage forms of the pharmaceutical preparations include tablets, liquids, capsules, powders, suppositories, granules, pills, sprays, or liniments.
7. The use of an active polypeptide as described in claim 1 or a pharmaceutical preparation as described in any one of claims 2-6 in the preparation of a drug for relieving kidney injury.