A faucet fish bone collagen hypoglycemic oligopeptide

Abstract

The application relates to the field of biotechnology, and particularly relates to a blood sugar reducing oligopeptide Leu-Ala-Leu-Phe-Val-Pro-Arg (LALFVPR) of dragonhead fish bone collagen. The oligopeptide has significant blood sugar reducing activity, can significantly reduce the blood sugar of diabetic mice, and can repair the sugar metabolism function of the mice.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a hypoglycemic oligopeptide derived from lizardfish bone collagen. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia. Long-term hyperglycemia can lead to damage and dysfunction of organs and tissues such as the eyes, kidneys, cardiovascular system, liver, and nerves, making it a silent killer of human health. With the increasing number of diabetes patients, the situation is becoming increasingly uncontrollable. According to the 10th edition of the International Diabetes Federation (IDF) Diabetes Atlas, as of 2021, more than 537 million people worldwide had diabetes, distributed across all age groups. This number is projected to increase to 643 million by 2030, with one person dying from diabetes every five seconds globally. The global prevalence of diabetes continues to rise, and the exorbitant treatment costs have placed a huge economic burden on countries worldwide, posing a serious challenge to the health and well-being of people around the world. Oxidative stress caused by excessive ROS production under hyperglycemic stimulation is a significant factor in liver disease. Liver damage exacerbates the disorder of glucose and lipid metabolism, further increasing blood sugar levels, leading to abnormal glycogen accumulation and inducing hepatic microvascular complications, which are inextricably linked to the development of diabetes. Summary of the Invention

[0003] On the one hand, the present invention provides a hypoglycemic oligopeptide Leu-Ala-Leu-Phe-Val-Pro-Arg (LALFVPR) derived from lizardfish bone collagen. This oligopeptide has significant hypoglycemic activity and can significantly reduce blood glucose in diabetic mice and repair glucose metabolism function in mice.

[0004] On the other hand, the present invention provides a method for preparing a hypoglycemic oligopeptide Leu-Ala-Leu-Phe-Val-Pro-Arg (LALFVPR) derived from lizardfish bone collagen, which includes the following steps:

[0005] a) Enzymatic hydrolysis: Take the processed lizardfish bone collagen, add distilled water and mix well. Use papain to perform enzymatic hydrolysis at pH 8.0 and temperature 55℃. After the enzymatic hydrolysis is completed, boil in a water bath for 10 minutes to inactivate the protein. Centrifuge and take the supernatant to adjust the pH to neutral.

[0006] b) Filtration: The enzymatic hydrolysate was filtered through a 0.22 μm filter membrane, and then filtered through an ultrafiltration membrane with a molecular weight of 1 kDa. The filtrate was freeze-dried to obtain lyophilized powder.

[0007] c) Sephadex G-15 gel chromatography: Dissolve the lyophilized powder from step b) in distilled water and separate it on a Sephadex G-15 gel column; the eluent is distilled water, the eluent flow rate is 0.3 mL / min, the collection time for each tube is 3 min, and every 100 tubes constitute one cycle; place the collected eluent in a micro-volume cuvette and measure the UV absorbance at a wavelength of 280 nm; combine the collected liquids with the strongest absorbance values, freeze-dry them, and measure the DPPH· scavenging rate of each peak; select the component with the highest scavenging activity for subsequent separation.

[0008] d) Fully automated high-performance liquid chromatography (HPLC) preparation and separation: The most active component from step c) was divided into a 1 mg / mL solution, filtered through a 0.22 μm filter membrane, and the mobile phase was water and acetonitrile (containing 0.1% trifluoroacetic acid). The elution gradient of acetonitrile was 10%–95%, the injection volume was 5 mL, the flow rate was 3.0 mL / min, the elution time was 90 min, and the detection wavelengths were set to 215 and 280 nm. The elution peaks were collected, lyophilized, and the obtained oligopeptide sequence was determined by LC-MS / MS analysis to be Leu-Ala-Leu-Phe-Val-Pro-Arg (LALFVPR).

[0009] In some embodiments, the preparation of the tilapia bone collagen in step a) includes the following steps: cutting the fish bones into small pieces, removing non-collagenous substances, decalcifying, defatting, and extracting fish collagen.

[0010] In some embodiments, the preparation of pufferfish bone collagen in step a) includes the following steps: The processed fish bones are chopped and stirred with 0.1 mol / L NaOH solution at a material-to-liquid ratio of 1:20 (g / ml) for 48 hours, with the solution changed 4 times to remove non-collagenous substances; the bones are then washed with distilled water until neutral. Next, the fish bones are soaked in a 0.5 mol / L disodium ethylenediaminetetraacetate solution at a material-to-liquid ratio of 1:20 (g / ml) for 48 hours to remove calcium, with the solution changed 4 times. After treatment, the bones are washed several times. Then, the fish bones are stirred with 15% isopropanol solution at a material-to-liquid ratio of 1:20 (g / ml) until the fat in the fish bones is removed, and then the isopropanol is washed away. The entire experimental process is carried out at 4°C. The solution is then stirred with 1:30 ( Fish bone collagen was extracted by adding 0.5 mol / L acetic acid solution (containing 0.5% pepsin by weight) to the material solution at a ratio of g / ml and stirring at 4℃ for one day. After extraction, the solution was filtered through defatted cotton gauze. The extraction was repeated twice using the above method, and the filtrates were combined. NaCl solution was added to the filtrate for salting out, and the mixture was stirred continuously until no precipitate was washed out. The suspension was centrifuged at 4℃ and 12000 r / min. The precipitate at the bottom of the centrifuge tube was redissolved in 0.5 mol / L acetic acid solution. Finally, the mixture was placed in a dialysis bag and dialyzed until neutral. The mixture was then freeze-dried to obtain fish collagen.

[0011] In some implementations, the weight-to-volume ratio of pufferfish bone collagen to distilled water in step a) is 8:500 (g / ml).

[0012] In some implementations, the amount of papain added in step a) is 5500 U / g.

[0013] In some embodiments, the weight-to-volume ratio of lyophilized powder to distilled water in step c) is 30:1 (mg / ml).

[0014] This invention obtains a novel oligopeptide, Leu-Ala-Leu-Phe-Val-Pro-Arg (LALFVPR), by enzymatically hydrolyzing lizardfish bone collagen and then separating and identifying it using techniques such as gel chromatography and liquid chromatography-mass spectrometry. This oligopeptide has significant hypoglycemic activity and can significantly reduce blood glucose in diabetic mice and repair glucose metabolism in mice. Attached Figure Description

[0015] Figure 1 This is the mass spectrum of the oligopeptide LALFVPR in Example 1.

[0016] Figure 2 The graph shows the effect of the oligopeptide LALFVPR on blood glucose concentration in mice in Example 2.

[0017] Figure 3 The graph shows the effect of the oligopeptide LALFVPR on serum insulin concentration in diabetic mice in Example 3.

[0018] Figure 4 The graph shows the effect of oligopeptide LALFVPR on liver glycogen concentration in diabetic mice in Example 4.

[0019] Figure 5 The graph shows the effect of the oligopeptide LALFVPR on muscle glycogen concentration in diabetic mice in Example 5. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments; however, these do not constitute a limitation or restriction on the scope of the present invention.

[0021] There are no particular restrictions on the solvents used in this invention; commercially available conventional solvents can be used.

[0022] The preparation of collagen from tilapia bones according to this invention: The processed fish bones are cut into small pieces and stirred with 0.1 mol / L NaOH solution at a material-to-liquid ratio of 1:20 (g / ml) for 48 hours, with the solution changed 4 times to remove non-collagenous substances. The mixture is then washed with distilled water until neutral. Next, the fish bones are soaked in a 0.5 mol / L disodium ethylenediaminetetraacetate solution at a material-to-liquid ratio of 1:20 (g / ml) for 48 hours to remove calcium, with the solution changed 4 times. After treatment, the mixture is washed several times. Then, the fish bones are stirred with 15% isopropanol solution at a material-to-liquid ratio of 1:20 (g / ml) until the fat in the fish bones is removed. The isopropanol is then rinsed off. The entire experimental process is carried out at 4°C. The mixture is then stirred with 1:30 ( Fish bone collagen was extracted by adding 0.5 mol / L acetic acid solution (containing 0.5% pepsin by weight) to the material solution at a ratio of g / ml and stirring at 4℃ for one day. After extraction, the solution was filtered through defatted cotton gauze. The extraction was repeated twice using the above method, and the filtrates were combined. NaCl solution was added to the filtrate for salting out, and the mixture was stirred continuously until no precipitate was washed out. The suspension was centrifuged at 4℃ and 12000 r / min. The precipitate at the bottom of the centrifuge tube was redissolved in 0.5 mol / L acetic acid solution. Finally, the mixture was placed in a dialysis bag and dialyzed until neutral. The mixture was then freeze-dried to obtain fish collagen.

[0023] Example 1

[0024] a) Enzymatic hydrolysis: Take 0.8g of processed pufferfish bone collagen, add 50mL of distilled water and mix well. Perform enzymatic hydrolysis using papain at pH 8.0, temperature 55℃, and enzyme dosage of 5500U / g for 4 hours. After enzymatic hydrolysis, boil in a water bath for 10 minutes to inactivate the enzyme, centrifuge, and adjust the pH to neutral using the supernatant.

[0025] b) Filtration: The enzymatic hydrolysate was filtered through a 0.22 μm filter membrane, then filtered through an ultrafiltration membrane with a molecular weight of 1 kDa, and the filtrate was freeze-dried.

[0026] c) Sephadex G-15 gel chromatography: Dissolve 90 mg of the above lyophilized powder in 3 mL of water and separate using a Sephadex G-15 gel column. The eluent was distilled water at a flow rate of 0.3 mL / min, with a collection time of 3 min per tube, and 100 tubes constituting one cycle. Place the collected eluent in a microcuvette and measure the UV absorbance at 280 nm. Combine the tubes with the strongest absorbance values, freeze-dry, and measure the DPPH· scavenging rate of each peak. Select the component with the highest scavenging activity for subsequent separation.

[0027] d) Fully automated high-performance liquid chromatography (HPLC) preparation and separation: The above lyophilized powder was prepared into a 1 mg / mL solution, filtered through a 0.22 μm filter membrane, and the mobile phase was water and acetonitrile (containing 0.1% trifluoroacetic acid). The elution gradient of acetonitrile was 10%–95%, the injection volume was 5 mL, the flow rate was 3.0 mL / min, the elution time was 90 min, and the detection wavelengths were set to 215 and 280 nm. The elution peaks were collected and lyophilized.

[0028] e) LC-MS / MS Analysis: The solution was redissolved or diluted with 0.1% formic acid solution. After dissolution, the supernatant was collected by high-speed centrifugation and set aside. After desalting and lyophilizing C18, the solution was analyzed by mass spectrometry. Mass spectrometer model: Orbitrap Fusion Lumos (Thermofisher). Mass spectrometry method: positive ion detection mode, primary resolution 120000, AGC set to 3e6, scan range 300-1400 m / z. The 10 ions with the highest intensity from one MS spectrum were selected for MS / MS analysis, secondary resolution 15000, AGC set to 2e4, separation window 1.6 m / z. Liquid chromatography method: Eksigent C18 column (3 μm, 250 mm × 75 μm), phase A: water + 0.1% formic acid; phase B: acetonitrile + 0.1% formic acid, flow rate 300 nl / min, injection volume 4 μL, gradient elution for 78 min. The results were analyzed using PEAKS software for De-novo and database searches. During data processing, possible amino acid combinations were first calculated based on the molecular weight information of fragments in each MS / MS spectrum, and then compared to the protein database from which the samples originated. The samples used were from the Mytilus protein database in NCBI. The results consist of two parts: one part shows sequencing results where corresponding sequences could be found in the Mytilus protein database in NCBI; the other part shows peptides not found in the database, which were obtained through de novo analysis. The oligopeptide sequence was determined to be: Leu-Ala-Leu-Phe-Val-Pro-Arg, and its mass spectrum is shown below. Figure 1 As shown.

[0029] Example 2

[0030] Male C57BL / 6J mice weighing 18±2g were purchased and acclimatized for 7 days. The mice were randomly divided into three groups (n=8 per group): a normal control group, a model group, and an oligopeptide group. STZ was dissolved in pre-chilled citrate-sodium citrate buffer (0.1mol / L, pH=4.5), and the prepared solution was filtered through a 0.22μm filter and stored on ice. The model group and oligopeptide group were intraperitoneally injected with STZ at 55mg / kg once daily for 5 consecutive days, while the normal control group received an equal volume of buffer solution. Fasting blood glucose levels were continuously monitored from day 7 after injection to prevent relapse. Once blood glucose levels stabilized, the oligopeptide group was administered the solution. The normal control group and model group were gavaged with an equal volume of distilled water daily, while the oligopeptide group was gavaged with an oligopeptide solution at a dose of 80mg / kg. This gavage treatment continued for 4 weeks, and changes in fasting blood glucose were recorded after the start of administration. Results are shown below. Figure 2 As shown, from Figure 2 It can be seen that the blood glucose level of the normal group mice was significantly lower than that of the diabetic model mice, and the blood glucose level remained stable at a low level. After the oligopeptide was administered, the blood glucose level of the mice gradually decreased, indicating that the oligopeptide LALFVPR has a significant hypoglycemic effect.

[0031] Example 3

[0032] Blood was collected from the mouse tail vein using a disposable lancet, being handled gently to avoid stress. The first drop of blood was discarded. The collected blood was immediately centrifuged at 4°C and 8000 rpm for 5 minutes. The supernatant serum was aspirated, aliquoted, and stored in an ultra-low temperature freezer for later use. Serum insulin levels were determined according to the kit (Shanghai Fantai Biotechnology). Results are shown below. Figure 3 ,from Figure 3 It can be seen that the serum insulin level in the model group mice was significantly lower than that in the normal group; after oligopeptide administration, the insulin level in the model group was significantly higher than that in the model group.

[0033] Example 4

[0034] Liver samples were weighed from each group of mice, and the weight readings were recorded. Glycogen content was determined according to the instructions of the liver glycogen reagent kit (Shanghai Fantai Biotechnology). Results are shown in [Table missing]. Figure 4 .

[0035] Example 5

[0036] Gastrocnemius muscle samples were weighed from each group of mice, and the weight readings were recorded. Glycogen content was determined according to the instructions of the muscle glycogen kit (Shanghai Fantai Biotechnology). Results are shown below. Figure 5 .

[0037] Glycogen is the body's storage form of glucose, primarily found in skeletal muscle and the liver. It regulates blood glucose levels and maintains blood glucose balance through gluconeogenesis, glycogen synthesis, and glycogenolysis. Therefore, glycogen levels are closely related to the development of diabetes. Figure 4 and 5 As shown, compared with the normal group, the liver glycogen and muscle glycogen content in the model group was significantly reduced, indicating that the glycogen storage in the mice in the model group was low; compared with the model group, the content of the two types of glycogen in the oligopeptide group was significantly increased, indicating that the administration promoted glycogen synthesis in the mice.

[0038] Finally, it should be noted that the above-described embodiments are merely one specific example of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A blood glucose-lowering oligopeptide derived from the collagen of the bone of the dragonhead fish, characterized in that The sequence of the oligopeptide is Leu-Ala-Leu-Phe-Val-Pro-Arg, and the oligopeptide has significant hypoglycemic activity and can significantly reduce the blood sugar of diabetic mice and repair the sugar metabolism function of the mice.

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