A pearl polypeptide extract with antioxidant effect and a preparation method thereof

Abstract

The present application relates to the technical field of functional active substance extraction, and particularly relates to extraction of pearl polypeptide with antioxidant effect. The present application adopts ultrasonic-assisted acetic acid extraction and Sephadex G-25 gel filtration column separation and purification to prepare pearl polypeptide extract. The obtained pearl polypeptide extract contains peptide segments with amino acid sequences as shown in SEQ ID NO. 1-5. Analysis and determination show that the pearl polypeptide extract of the present application has significant antioxidant capacity and can effectively scavenge hydroxyl radicals and ABTS free radicals at low concentration. In addition, the pearl polypeptide extract prepared by the present application has high extraction rate and purity and can be widely used in the fields of food, biological medicine and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of functional active substance extraction technology, and in particular to the extraction of a pearl polypeptide with antioxidant properties. Background Technology

[0002] Human oxidation is an irreversible process. Free radicals are harmful compounds produced during the body's oxidation reactions. They are highly oxidizing and can damage the body's tissues and cells, leading to chronic diseases and aging effects. Free radical scavengers, also known as antioxidants, are substances that can eliminate the harmful substances produced during oxidation. The human body has a system for eliminating free radicals, but this system's ability weakens with age and changes in physical condition. Free radicals accumulate within cells, causing them to lose their function and eventually die during continuous oxidation, potentially leading to a series of diseases. However, supplementing with certain foods and special nutrients can help the body achieve antioxidant effects and even extend lifespan.

[0003] Pearls have been cherished since ancient times, used not only as precious ornaments but also in medicine and skincare, possessing properties such as clearing heat and detoxifying, calming the nerves, beautifying the skin, anti-oxidation, and delaying aging. Research shows that the main active components of pearls are small molecule peptides. Compared to expensive and less safe drugs, marine bioactive peptides derived from pearls have advantages such as high activity, specificity, and stability, making the preparation of antioxidant peptides from pearls a promising market opportunity. However, the main component of pearls is approximately 95% inorganic calcium carbonate, with organic components primarily consisting of proteins and polysaccharides, with protein content only reaching 5%. How to efficiently prepare antioxidant bioactive peptides from pearls has been a bottleneck hindering the industry's development.

[0004] Patent CN112390848A discloses a method for preparing a pearl powder polypeptide extract. This method uses room-temperature acetic acid solution for extraction, yielding a pearl powder polypeptide extract with a yield of 1.5%–1.8% and a purity of 40.05%–45.61%. At a high concentration of 300 mg / mL, the hydroxyl radical scavenging capacity is 75.25%–80.76%. However, the method disclosed in this patent has drawbacks such as low pearl powder polypeptide yield, low purity, unclear structural composition, and poor antioxidant activity of the extract. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a pearl polypeptide extract with antioxidant properties and its preparation method. The pearl polypeptide prepared by this method has high extraction rate and purity, clearly defined main components, and good antioxidant effect, and can be widely used in the fields of biomedicine and cosmetics.

[0006] First, the present invention provides a pearl polypeptide extract with antioxidant properties, wherein the pearl polypeptide extract includes peptides having the amino acid sequences shown in SEQ ID NO. 1~5.

[0007] The pearl polypeptide extract obtained in this invention has a molecular weight of less than 2 kDa. LC-MS / MS analysis identified five peptide segments with high content in the extracted pearl polypeptide extract, whose amino acid sequences are shown in SEQ ID NO. 1-5, and whose relative molecular masses are 1122.4829, 719.37147, 984.45526, 976.509031, and 953.0571, respectively, with relative percentage contents of 29.75%, 12.99%, 5.38%, 5.31%, and 4.12%, respectively. Analysis of the effect of pearl polypeptide on the scavenging ability of hydroxyl radicals and ABTS radicals revealed that pearl polypeptide has outstanding antioxidant capacity, effectively scavenging hydroxyl radicals and ABTS radicals. Its scavenging ability is close to that of commonly used antioxidant vitamin C. Specifically, at a low concentration of 5 mg / mL, it can scavenge 97.97% of hydroxyl radicals and 79.05% of ABTS radicals.

[0008] Secondly, the present invention also provides a method for preparing the above-mentioned pearl polypeptide extract, comprising the following steps:

[0009] (1) Extraction: Pearl powder and acetic acid are mixed and stirred, ultrasonically assisted extraction is performed, magnetic stirring is used, insoluble matter is removed by suction filtration, and acetic acid is removed by rotary evaporation to obtain pearl polypeptide concentrate.

[0010] (2) Dialysis: The pearl polypeptide concentrate was dialyzed and then freeze-dried to obtain crude pearl polypeptide;

[0011] (3) Purification: The crude pearl polypeptide was reconstituted in deionized water, filtered through a membrane to obtain a crude pearl polypeptide reconstituted solution, and then separated and purified by Sephadex G-25 gel filtration column, and freeze-dried to obtain pearl polypeptide extract.

[0012] Patent CN112390848A discloses a method for preparing a pearl powder polypeptide extract. The method uses room temperature acetic acid solution for extraction to obtain a pearl powder polypeptide extract with a yield of 1.5% to 1.8% and a purity of 40.05% to 45.61%. The pearl powder polypeptide extract has a hydroxyl radical scavenging capacity of 75.25% to 80.76% at a high concentration of 300 mg / mL.

[0013] This invention employs ultrasound-assisted acetic acid extraction of pearl polypeptides, followed by purification using a Sephadex G-25 gel filtration column to prepare a pearl polypeptide extract with high extraction rate and high purity. Specifically, the purity of the pearl polypeptide extract of this invention is more than twice that of existing technologies, and its main components have been identified by LC-MS / MS analysis. Furthermore, the pearl polypeptide extract prepared by the method of this invention has strong antioxidant capacity, exhibiting highly efficient antioxidant effects at low concentrations, and its scavenging ability is comparable to that of commonly used antioxidant vitamin C.

[0014] This invention employs an ultrasonic-assisted extraction, Sephadex G-25 gel filtration column separation and purification, and multiple freeze-drying techniques to improve the yield and purity of pearl polypeptide extracts, while better preserving the activity and structural integrity of the extracted pearl polypeptides.

[0015] Preferably, in the extraction step, the concentration of acetic acid is 1.5–2.5 mol / mL, and the ratio of pearl powder to acetic acid is 1:15–1:25.

[0016] As a preferred option, the ultrasonic-assisted extraction conditions in the extraction step are: ultrasonic power of 150-250W and ultrasonic time of 50-70min.

[0017] Preferably, the magnetic stirring time is 4 to 6 hours.

[0018] Preferably, the parameters for rotary evaporation in the extraction step are: rotary evaporation water bath temperature of 40-60℃ and rotary evaporation speed of 50-70 rpm.

[0019] Preferably, a dialysis bag with a molecular weight of 500 Da is used in the dialysis step.

[0020] As a further preferred option, the dialysis time is 12–24 hours, and the water change frequency is once every 2 hours.

[0021] Preferably, in the purification step, the concentration of the crude pearl polypeptide complex solution is 4–6 mg / mL, and the collection rate of the pearl polypeptide during the separation and purification process is 1.5–2.5 mL / min, with one tube collected every 3 min.

[0022] Furthermore, this invention proposes the application of the above-mentioned pearl polypeptide extract and the pearl polypeptide extract prepared using the preparation method of this invention in the fields of biomedicine and cosmetics.

[0023] Furthermore, this invention also proposes the application of the above-mentioned pearl polypeptide extract and the pearl polypeptide extract prepared using the preparation method of this invention in scavenging hydroxyl radicals and ABTS radicals.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) The pearl polypeptide extract of the present invention can effectively scavenge hydroxyl radicals and ABTS radicals at low concentrations, and has significant antioxidant effects;

[0026] (2) The present invention adopts the technical solution of ultrasonic-assisted extraction, Sephadex G-25 gel filtration column separation and purification and multiple freeze drying, which improves the yield and purity of pearl polypeptide extract, and better preserves the activity and structural integrity of the extracted pearl polypeptide, and the components of the pearl polypeptide extract are clear.

[0027] (3) The preparation process of the present invention is simple, requires no complex equipment or preparation steps, and the preparation conditions are mild and have universality;

[0028] (4) The pearl polypeptide extraction process of this invention adopts green extraction technology and does not use toxic organic solvents, resulting in high product safety. Attached Figure Description

[0029] To more clearly illustrate the embodiments of the present invention, the accompanying drawings will be briefly described below:

[0030] Figure 1 This is a flow chart of the preparation process of the pearl powder polypeptide extract of the present invention;

[0031] Figure 2 Basepeak plot for LC-MS / MS identification of pearl polypeptide extract in Experiment 1;

[0032] Figure 3 The graph shows the effect of different concentrations of pearl polypeptide extract on the hydroxyl radical scavenging rate in Experiment 2.

[0033] Figure 4 The graph shows the effect of different concentrations of pearl polypeptide extract on the ABTS free radical scavenging rate in Experiment 3. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0035] The purity detection method in the following examples is as follows: the purity of the collected liquid with an absorbance greater than 0.20 is determined by high performance liquid chromatography using a UV detector and a differential detector connected in series, wherein the mobile phase is pure water and the flow rate is 0.8 mL / min.

[0036] The extraction yield in the following examples is calculated as follows: (dry weight of extract / dry weight of pearl powder raw material) × 100%.

[0037] Example 1

[0038] This embodiment includes the following steps:

[0039] (1) Extraction: Prepare a 1.5 mol / mL dilute acetic acid solution using deionized water. Mix pearl powder with the dilute acetic acid solution at a ratio of 1:15 and stir rapidly. After the foam disappears, sonicate the pearl extract under the following conditions: 150 W power and 50 min sonication time, followed by magnetic stirring in the dark for 4 h. After removing insoluble matter by filtration, rotary evaporation is performed on the pearl extract under the following conditions: 40 °C water bath temperature and 50 rpm to remove the acetic acid solution and concentrate the extract to obtain concentrated pearl extract.

[0040] (2) Dialysis: The concentrated pearl extract after rotary evaporation was desalted by dialysis using a dialysis bag with a molecular weight of 500 Da. The water was changed every 2 hours. After dialysis for 20 hours, the crude pearl polypeptide was freeze-dried for preservation.

[0041] (3) Purification: 150 mg of crude pearl polypeptide powder was redissolved in 30 mL of deionized water, filtered through a membrane, and purified using a Sephadex G-25 gel filter column. The purified pearl polypeptide was collected at a rate of 1.5 mL / min and 3 min / tube. The absorbance of each tube of collected liquid was measured at 562 nm using a BCA kit and an elution curve was plotted. The extract was freeze-dried to obtain the pearl polypeptide extract.

[0042] Analysis and testing showed that the crude pearl polypeptide yield of the pearl polypeptide extract obtained in this example was 5.08%, the extraction rate was 3.157%, and the purity of the purified pearl polypeptide was 97.58%.

[0043] Example 2

[0044] The following differences exist between this embodiment and Embodiment 1, while the rest are the same and will not be described again here.

[0045] (1) Extraction: The acetic acid concentration is 2.0 mol / mL, and the pearl powder and dilute acetic acid solution are mixed at a material-to-liquid ratio of 1:20; the ultrasonic treatment conditions are: ultrasonic power of 200 W, ultrasonic time of 60 min; the light-proof magnetic stirring time is 5 h; the rotary evaporation conditions are: rotary evaporation water bath temperature of 50℃ and rotary evaporation speed of 60 rpm.

[0046] (2) Dialysis: The dialysis time is 22 hours.

[0047] (3) Purification: Collect purified pearl polypeptides at a rate of 2.0 mL / min and 3 min / tube.

[0048] Analysis and testing showed that the crude pearl polypeptide yield of the pearl polypeptide extract obtained in this example was 5.61%, the extraction rate was 3.203%, and the purity of the purified pearl polypeptide was 98.08%.

[0049] Example 3

[0050] The following differences exist between this embodiment and Embodiment 1, while the remaining steps are the same and will not be described again here.

[0051] (1) Extraction: The acetic acid concentration is 2.5 mol / mL, and the pearl powder and dilute acetic acid solution are mixed at a material-to-liquid ratio of 1:25; the ultrasonic treatment conditions are: ultrasonic power of 250W, ultrasonic time of 70min; the light-proof magnetic stirring time is 6h; the rotary evaporation conditions are: rotary evaporation water bath temperature of 60℃, rotary evaporation speed of 70rpm.

[0052] (2) Dialysis: The dialysis time is 24 hours.

[0053] (3) Purification: Collect purified pearl polypeptides at a rate of 2.5 mL / min and 3 min / tube.

[0054] Analysis and testing showed that the crude pearl polypeptide yield of the pearl polypeptide extract obtained in this example was 4.83%, the extraction rate was 3.084%, and the purity of the purified pearl polypeptide was 96.89%.

[0055] To identify the peptide composition, structure, and free radical scavenging ability of the pearl polypeptide of the present invention, the following experiments were conducted:

[0056] Experiment 1: Compositional Analysis of Pearl Peptide Extract

[0057] LC-MS / MS proteometry was used to identify the peptide composition and structure of pearl polypeptides. The mass spectrometry information was compared with PDB and NCBI databases, revealing five peptides with high content, as shown in Table 1.

[0058] Table 1. Composition analysis of pearl polypeptide extract

[0059]

[0060] Experiment 2: The scavenging ability of pearl polypeptides against hydroxyl radicals

[0061] To 1 mL of sample solution (the sample was freeze-dried pearl polypeptide extract solid powder dissolved in distilled water, with concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mg / mL), 1 mL of 6 mmol / L salicylic acid-anhydrous ethanol solution, 1 mL of 6 mmol / L ferrous sulfate solution, and 1 mL of 0.1% hydrogen peroxide solution were added sequentially. Distilled water was then added to bring the total volume to 5 mL. The solution was incubated in a water bath at 37°C for 30 min, cooled with cold water, and the absorbance was measured at 510 nm. Vitamin C was used as a control. The free radical scavenging rate formula is:

[0062] Clearance rate (%) = (A0 - (A0)) i -A j )) / A0×100%

[0063] Where A0 is the absorbance value of the blank control; A i A represents the absorbance of the sample. j The absorbance value is the value without the addition of the colorimetric reagent H2O2.

[0064] Based on experimental results Figure 3 As can be seen from the figure, the pearl polypeptide extract of the present invention has extremely strong hydroxyl radical scavenging ability. The hydroxyl radical scavenging rate gradually increases with increasing sample concentration. Specifically, at a sample concentration of 1 mg / mL, its hydroxyl radical scavenging rate exceeds that of the prior art pearl polymorphic extract at 300 mg / mL. Furthermore, at a sample concentration of 5 mg / mL, the hydroxyl radical scavenging rate reaches as high as 97.97%.

[0065] Experiment 3: The scavenging ability of pearl polypeptides against ABTS free radicals

[0066] Add 3.6 mL of ABTS solution to 0.4 mL of sample solution (sample was freeze-dried pearl polypeptide extract solid powder, dissolved in distilled water, concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg / mL). Mix well and react at room temperature in the dark for 5 min. Measure the absorbance at a wavelength of 734 nm. VC was used as a control. The free radical scavenging rate formula is:

[0067] Clearance rate (%) = ((A) b -A s ) / A b ) × 100%

[0068] Where A b A represents the absorbance of the mixture of ABTS solution and sample solvent solution; s The absorbance is the value of the mixture of the test solution and the ABTS solution.

[0069] Based on experimental results Figure 4 As can be seen from the figure, the pearl polypeptide extract of the present invention has a strong ABTS free radical scavenging ability. As the sample concentration increases, the ABTS free radical scavenging rate gradually increases. When the sample concentration is 5 mg / mL, the ABTS free radical scavenging rate reaches 79.05%.

[0070] The above description of the invention is merely a preferred embodiment of the invention and is not intended to limit the scope of the invention. Any modifications and improvements made to the technical solutions of the invention by those skilled in the art without departing from the spirit of the invention should fall within the protection scope of the invention.

Claims

1. A pearl polypeptide extract with antioxidant properties, characterized in that, The pearl polypeptide extract is composed of peptides with amino acid sequences shown in SEQ ID NO. 1~5.

2. A method for preparing the pearl polypeptide extract of claim 1, characterized in that, Includes the following steps: (1) Extraction: Pearl powder and acetic acid are mixed and stirred, ultrasonically assisted extraction is performed, magnetic stirring is used, insoluble matter is removed by suction filtration, and acetic acid is removed by rotary evaporation to obtain pearl polypeptide concentrate. (2) Dialysis: The pearl polypeptide concentrate was dialyzed and then freeze-dried to obtain crude pearl polypeptide; (3) Purification: The crude pearl polypeptide was reconstituted in deionized water, filtered through a membrane to obtain a crude pearl polypeptide solution, and then separated and purified by Sephadex G-25 gel filtration column. After freeze drying, the pearl polypeptide extract was obtained.

3. The method for preparing the pearl polypeptide extract according to claim 2, characterized in that, In the extraction step, the concentration of acetic acid is 1.5-2.5 mol / mL, and the ratio of pearl powder to acetic acid is 1:(15-25).

4. The method for preparing the pearl polypeptide extract according to claim 2, characterized in that, The ultrasonic-assisted extraction conditions in the extraction step are: ultrasonic power of 150-250W and ultrasonic time of 50-70min.

5. The method for preparing the pearl polypeptide extract according to claim 2, characterized in that, The magnetic stirring time in the extraction step is 4 to 6 hours.

6. The method for preparing the pearl polypeptide extract according to claim 2, characterized in that, The parameters for rotary evaporation in the extraction step are: water bath temperature of rotary evaporator is 40-60℃, and rotary evaporation speed is 50-70 rpm.

7. The method for preparing the pearl polypeptide extract according to claim 2, characterized in that, Dialysis was performed using dialysis bags with a molecular weight of 500 Da.

8. The method for preparing the pearl polypeptide extract according to claim 2, characterized in that, During the purification process, the collection rate of pearl polypeptides is 1.5–2.5 mL / min, with one tube collected every 3 minutes.

9. The application of the pearl polypeptide extract according to claim 1 or the pearl polypeptide extract prepared by any one of claims 2 to 8 in the preparation of biomedicine and cosmetics.

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