Antioxidative peptide extracted from prinsepia utilis royle as well as extraction method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to antioxidant peptide extracted from prinsepia utilis royle as well as an extraction method and application of the antioxidant peptide. The amino acid sequence of the antioxidant peptide is Lu-Met-Gly-Cys-Trp-Val-Val-Phe, and the amino acid sequence of the antioxidant peptide is Lu-Met-Gly-Cys-Trp-Val- The molecular mass is 954 Da. The antioxidant peptide is derived from prinsepia utilis royle fruits. The obtained (active) antioxidant peptide is an excellent antioxidant, has good fat solubility, can be dissolved in the prinsepia utilis royle oil, improves the free radical scavenging activity of the prinsepia utilis royle oil, improves the quality of the prinsepia utilis royle oil, prolongs the shelf life of the prinsepia utilis royle oil, and can be used in cosmetics, healthy foods and dietary supplements developed based on the prinsepia utilis royle oil.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an antioxidant peptide extracted from Prinsepia utilis, its extraction method and application. Background Art

[0002] Prinsepia utilis (scientific name: Prinsepia utilis), belonging to the genus Prinsepia of the Rosaceae family, is a small shrub plant mainly growing in the southwestern region of China and the Himalayas, especially common in the Qinghai-Tibet Plateau, Yunnan, Sichuan and other places. The fruit of Prinsepia utilis is small and dark blue or purple, usually ripe in autumn. It contains rich oils, unsaturated fatty acids, vitamin E, flavonoid compounds and other antioxidant components, so it has high development value in medicine, food and skin care.

[0003] Prinsepia utilis oil is a vegetable oil extracted from the fruit of Prinsepia utilis, which has attracted much attention due to its rich nutritional components and diverse functions. Prinsepia utilis oil is light yellow or light green, with a mild aroma and high permeability, and is widely used in the fields of food, health care and skin care. Prinsepia utilis oil has received extensive attention due to its rich unsaturated fatty acids and antioxidant components, but this oil is prone to oxidation during storage, resulting in a decline in quality and a shortening of the shelf life. At present, to extend the shelf life of this oil, chemical antioxidants are usually added, but this may bring safety problems.

[0004] Antioxidant polypeptides from natural sources are considered an effective way to replace chemical antioxidants. However, there is no relevant report on improving the stability of Prinsepia utilis oil. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides an antioxidant peptide extracted from Prinsepia utilis and its extraction method. The antioxidant peptide is extracted from Prinsepia utilis; through the extraction method in the present invention, a polypeptide with high antioxidant activity can be obtained. This polypeptide has good natural antioxidant properties and can improve the stability of Prinsepia utilis oil.

[0006] To achieve the purpose of the present invention, the specific technical solutions of the present invention are as follows:

[0007] One of the purposes of the present invention is to provide an antioxidant peptide extracted from Prinsepia utilis, and its amino acid sequence is Leu-Met-Gly-Cys-Trp-Val-Val-Phe.

[0008] Furthermore, the molecular mass of the antioxidant peptide is 954 Da; it has good lipid solubility.

[0009] Furthermore, the antioxidant peptide is derived from the fruit of Prinsepia utilis.

[0010] Another object of the present invention is to provide a method for extracting the above-mentioned antioxidant peptide, comprising the following steps:

[0011] Crush the dried Prinsepia utilis Royle fruits to obtain a powder of dried Prinsepia utilis Royle fruits; then add ethanol to the powder of dried Prinsepia utilis Royle fruits and perform ultrasonic treatment; after the ultrasonic treatment, centrifuge the mixture to separate the supernatant.

[0012] Pour the supernatant into a separating funnel, then add chloroform thereto, shake well and let it stand for layering; collect the lower organic phase; perform rotary evaporation on the collected organic phase at low temperature to remove the solvent and obtain a concentrated crude polypeptide extract.

[0013] Dialyze the concentrated crude polypeptide extract through a dialysis bag with a molecular weight cut-off of 3500 Da in PBS buffer, collect the dialysate, concentrate it by rotary evaporation to obtain a concentrated solution; freeze-dry the concentrated solution with a vacuum freeze dryer to obtain a dry powder of Prinsepia utilis Royle fruit polypeptide extract.

[0014] Dissolve the dry powder of Prinsepia utilis Royle fruit polypeptide extract with ethanol, load it onto a Superdex 30 Increase gel filtration column, elute with sterile water, measure the absorbance of the eluate at 215 nm after collecting the eluate, collect and freeze-dry a single peak to obtain a dry powder of Prinsepia utilis Royle fruit polypeptide complex.

[0015] Dissolve the dry powder of Prinsepia utilis Royle fruit polypeptide complex with ethanol, purify it by reverse high performance liquid chromatography, collect a single peak to obtain Prinsepia utilis Royle fruit polypeptide, namely the antioxidant peptide.

[0016] Preferably, the chromatographic conditions for the reverse high performance liquid chromatography purification are as follows: the chromatographic column is a PE C18 column, 150 mm × 4.6 mm; phase A: pure water, phase B: acetonitrile containing 0.1% trifluoroacetic acid; the injection volume is set to 30 μL; the column temperature is set to 25 ± 5 °C; the flow rate is selected as 1.0 mL / min; the detection wavelength is set to 215 nm.

[0017] Preferably, the elution conditions for the reverse high performance liquid chromatography purification are as follows: phase A: 0 - 8 min, 99% - 97%; 8 - 12 min, 97% - 96%; 12 - 16 min, 96% - 80%; 16 - 20 min, 80% - 99%.

[0018] The third object of the present application is to protect the application of the above-mentioned antioxidant peptide in the preparation of antioxidant products.

[0019] The fourth object of the present application is to protect the application of the above-mentioned antioxidant peptide in the preparation of antioxidants.

[0020] An antioxidant containing the above-mentioned antioxidant peptide.

[0021] The fifth object of the present application is to protect the application of the above-mentioned antioxidant peptide in extending the storage period of Prinsepia utilis Royle oil.

[0022] Furthermore, it can also be used in cosmetics, health foods and dietary supplements developed based on Prinsepia utilis Royle oil.

[0023] Compared with the existing technologies, the beneficial effects of the present invention are as follows:

[0024] (1) The obtained (active) antioxidant peptide is an excellent antioxidant, has good liposolubility, can be dissolved in Prinsepia utilis Royle oil, improves the free radical scavenging activity of Prinsepia utilis Royle oil, enhances the quality of Prinsepia utilis Royle oil, extends the shelf life of Prinsepia utilis Royle oil, and can be used in cosmetics, health foods and dietary supplements developed based on Prinsepia utilis Royle oil.

[0025] (2) The polypeptide extracted from Prinsepia utilis Royle has natural source consistency with Prinsepia utilis Royle oil itself, which helps to maintain the pure natural characteristics of the product, and is very popular in the fields of cosmetics and skin care products. The polypeptide of Prinsepia utilis Royle can produce a synergistic antioxidant effect with other components (such as polyphenols and vitamin E) in Prinsepia utilis Royle oil, further enhancing the antioxidant capacity and extending the shelf life of the oil.

[0026] (3) The antioxidant peptide is extracted by a natural method without adding exogenous chemical antioxidants, which helps to reduce the dependence on synthetic antioxidants and conforms to the trend of green environmental protection. Description of the Drawings

[0027] Figure 1 It is the molecular exclusion chromatography separation diagram of Prinsepia utilis Royle polypeptide;

[0028] Figure 2 It is the RP-HPLC chromatogram of Prinsepia utilis Royle polypeptide;

[0029] Figure 3 It is the amino acid sequence of Prinsepia utilis Royle polypeptide PUP-8;

[0030] Figure 4 It is the DPPH free radical scavenging activity of Prinsepia utilis Royle oil mixture;

[0031] Figure 5 It is the acid value change diagram of Prinsepia utilis Royle oil under natural storage conditions. Detailed Embodiments

[0032] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any manner.

[0033] Any feature disclosed in this specification (including claims and abstract), unless specifically stated, can be replaced by other equivalent or similar-purpose alternative features. That is, unless specifically stated, each feature is just an example among a series of equivalent or similar features.

[0034] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0035] In the following embodiments, the experimental methods, unless otherwise specified, are all conventional methods. The test materials used in the following embodiments, unless otherwise specified, are all obtained from commercial channels.

[0036] In this application, unless otherwise specified, % represents its mass percentage content, that is, wt%.

[0037] In the following embodiments, the antioxidant peptide (PUP-8) is the Prunus humilis polypeptide PUP-8.

[0038] Example 1:

[0039] Isolation, purification and identification of Prunus humilis polypeptide

[0040] Weigh 100 g of dried Prunus humilis fruits, place them in a traditional Chinese medicine grinder, pulverize them and pass through a 100-mesh sieve to obtain the powder of dried Prunus humilis fruits. Add 200 mL of 50% ethanol and perform ultrasonic treatment. The ultrasonic power is 40 kHz and the time is 40 min. After the ultrasonic treatment, centrifuge the mixture (12000 rpm, 10 min) and separate the supernatant.

[0041] Pour the supernatant into a separating funnel, add 100 mL of chloroform, shake well and let it stand for stratification. Collect the lower organic phase (containing liposoluble small peptides). Rotate and evaporate the collected organic phase at a low temperature (40 °C) to remove the solvent and obtain a concentrated polypeptide crude extract.

[0042] Prepare phosphate buffered saline (PBS buffer): 0.27 g of potassium dihydrogen phosphate (KH2PO4), 1.42 g of disodium hydrogen phosphate (Na2HPO4), 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), add about 800 mL of deionized water, stir well to dissolve, then add concentrated hydrochloric acid to adjust the pH to 6.8, and finally make up the volume to 1 L. Place the concentrated polypeptide crude extract in a dialysis bag with a molecular weight cut-off of 3500 Da and dialyze it in PBS buffer for 24 h. Collect the dialysate, concentrate it with a rotary evaporator, and freeze-dry the obtained concentrate with a vacuum freeze dryer to obtain the dry powder of Prunus humilis polypeptide extract.

[0043] Take 10 g of dry powder of the polypeptide extract from Prinsepia utilis Royle, dissolve it in 50% ethanol, prepare a solution with a concentration of 100 mg / mL, load it onto a Superdex 30 Increase gel filtration column, elute with sterile water, collect the eluate with an automatic collector, then measure the absorbance value of the eluate at 215 nm, collect and freeze-dry a single peak to obtain the Prinsepia utilis Royle polypeptide complex.

[0044] Take 100 mg of the dry powder of the Prinsepia utilis Royle polypeptide complex and dissolve it in 50% ethanol to prepare a solution with a concentration of 10 mg / mL. Purify it by reverse high performance liquid chromatography, collect a single peak, and obtain Prinsepia utilis Royle polypeptide (PUP-8). The chromatographic conditions are set as follows: The chromatographic column is a PE C18 column (150 mm × 4.6 mm), phase A: pure water, phase B: acetonitrile containing 0.1% trifluoroacetic acid; the injection volume is set at 30 μL; the column temperature is set at 25 ± 5 °C; the flow rate is selected at 1.0 mL / min; the detection wavelength is set at 215 nm. Elution conditions: Phase A: 0 - 8 min, 99% - 97%; 8 - 12 min, 97% - 96%; 12 - 16 min, 96% - 80%; 16 - 20 min, 80% - 99%.

[0045] Analyze the amino acid composition of PUP-8 by liquid chromatography - mass spectrometry (LC-MS-MS). Liquid phase A solution is an aqueous solution containing 0.1% formic acid, and liquid phase B solution is an acetonitrile solution containing 0.1% formic acid. The chromatographic column is equilibrated with liquid phase A. The sample is loaded by an automatic sampler, then gradient separated by the chromatographic column, with a flow rate of 0.3 mL / min and a column temperature of 70 °C. Mass spectrometry conditions: The sample is analyzed by a TripleTOF 5600+ mass spectrometer. Analysis duration: 18 min, detection mode: positive ion, primary mass spectrometry scanning range: 600 - 3500 m / z.

[0046] 100 g of dry Prinsepia utilis Royle is crushed and dissolved in 50% ethanol, and then extracted with chloroform to obtain 17.71 g of the crude polypeptide extract from Prinsepia utilis Royle. It is dissolved in PBS buffer, dialyzed with a 3500 Da dialysis bag, and the obtained dialysate is freeze-dried to obtain a total of 12.53 g of dry powder of the polypeptide extract. After 10 g of the polypeptide extract is subjected to size exclusion chromatography on a Superdex 30 Increase gel filtration column, 0.42 g of freeze-dried powder of the polypeptide complex is obtained. Finally, 100 mg of the freeze-dried powder of the polypeptide complex is purified by reverse high performance liquid chromatography (RP-HPLC) to obtain 9.36 mg of freeze-dried powder of PUP-8.

[0047] The separation results of size exclusion chromatography are as Figure 1 shown Figure 1This is the molecular exclusion chromatography separation diagram of Prinsepia utilis Royle polypeptide. According to the molecular weight, the polypeptide extract can obtain 3 elution peaks, which are labeled as A1-3 from large to small molecular weight. By separately collecting the elution peaks and measuring their free radical scavenging activities, the A2 fraction is selected as the Prinsepia utilis Royle polypeptide complex for further purification. After the fraction A2 is separated and purified by RP-HPLC ( Figure 2 RP-HPLC chromatogram of Prinsepia utilis Royle polypeptide), 4 fractions, namely B1-4, are obtained. After measuring the free radical scavenging activities, the B3 fraction with the highest free radical scavenging activity is selected as the antioxidant peptide, namely Prinsepia utilis Royle polypeptide, denoted as PUP-8. The amino acid sequence of the antioxidant peptide (PUP-8) is shown in SEQ ID NO:1, and the SEQ ID NO:1 sequence is: Leu-Met-Gly-Cys-Trp-Val-Val-Phe. The amino acid sequence is shown in Figure 3 .

[0048] Example 2

[0049] Synthesis of Prinsepia utilis Royle polypeptide PUP-8

[0050] In order to quickly obtain a large amount of PUP-8, a solid-phase synthesis method for PUP-8 is provided.

[0051] 1. Resin preparation. Resin activation: Weigh an appropriate amount of Wang resin (such as 0.5 mmol), wash it 3 times with dichloromethane (DCM), 5 minutes each time, to remove impurities and moisture. Amino deprotection: Add 20% piperidine / dimethylformamide (DMF) solution to the resin and shake for 15 minutes to remove the Fmoc protecting group on the resin. Then, wash it 5 times with DMF (1 minute each time) to ensure the removal of residual piperidine.

[0052] 2. Coupling of amino acids. Coupling of the first amino acid (Phe): Weigh Fmoc-Phe-OH (1.1 eq), DIEA (2 eq), HBTU (1 eq), and dissolve them in DMF respectively. After mixing, let it stand for 2 minutes to activate the amino acid. Add the activated amino acid solution to the resin and oscillate the reaction for 1 h. After the coupling is completed, wash the resin 3 times with DMF and DCM.

[0053] 3. Deprotection and washing. Remove the Fmoc group with 20% piperidine / DMF and shake for 15 minutes. Wash it 5 times with DMF and DCM.

[0054] 4. Repeat the coupling and deprotection steps. Sequentially couple other amino acids to the peptide chain one by one, and perform deprotection after each coupling. The sequence is: Val-Val-Trp-Cys-Gly-Met-Leu. Couple each step with an Fmoc-protected amino acid, add DIEA (basic catalyst) and HBTU for activation, then add to the resin and react for 1 h, and then wash the resin and remove the Fmoc protecting group.

[0055] 5. Removal of side-chain protecting groups and cleavage of the peptide chain. Prepare a TFA cleavage solution (TFA:H2O:TIPS = 95:2.5:2.5), immerse the resin in the TFA cleavage solution, and shake for 1 h. After cleavage, filter the solution, collect the filtrate, and add cold ether to precipitate the polypeptide. Perform freeze-drying, dissolve the precipitated polypeptide in an appropriate amount of water or perform freeze-drying again to obtain a solid polypeptide powder.

[0056] 6. Polypeptide purification. Purify the polypeptide using reverse-phase high-performance liquid chromatography (RP-HPLC), and monitor the polypeptide peak according to the detection wavelength (such as 220 nm). Collect the purified target fraction, concentrate and freeze-dry.

[0057] 7. Quality control. Use mass spectrometry (such as MALDI-TOF or ESI-MS) to detect the molecular weight of the polypeptide and confirm its accuracy. Test the purity again by HPLC to ensure that the target polypeptide meets the expected quality standards.

[0058] Example 3

[0059] Effect of Prinsepia utilis Royle polypeptide PUP-8 on free radical scavenging activity of Prinsepia utilis Royle oil

[0060] Weigh an appropriate amount of DPPH powder, dissolve it in absolute ethanol to prepare a DPPH solution with a concentration of 0.1 mM, store it in the dark and place it in a 4 °C refrigerator for later use. Add different masses of PUP-8 to Prinsepia utilis Royle oil, shake and dissolve it, and finally ensure that the addition amount of PUP-8 is 0.5%, 1%, 1.5% and 2%. Add 2.0 mL of DPPH solution to each test tube, and then add 2.0 mL of the sample solution (a mixture of Prinsepia utilis Royle oil and polypeptide). The control group adds 2.0 mL of absolute ethanol instead of the sample solution. All test tubes are placed in the dark at 25 °C for 30 min for reaction. Use a UV-visible spectrophotometer to measure the absorbance (A) of the reaction system at 517 nm. Measure the absorbance of the control group, the sample group and the blank group respectively. The DPPH free radical scavenging rate is calculated according to the following formula:

[0061]

[0062] where A 样品 is the absorbance of the sample group, A 空白 is the absorbance of the blank group, A 对照is the absorbance of the control group.

[0063] The results are as Figure 4 shown, Figure 4 is the DPPH free radical scavenging activity of the pricklyash fruit oil mixture. It can be Figure 4 seen that the pricklyash fruit oil itself has good free radical scavenging activity. For the pricklyash fruit oil with a concentration of 80 mg / mL, its DPPH free radical scavenging activity can reach 48.89%. After adding PUP-8, the DPPH free radical scavenging activity of the pricklyash fruit oil mixture is significantly enhanced. When the addition amount of PUP-8 reaches 1%, the DPPH free radical scavenging activity reaches 62.61%. Continuing to increase the concentration of PUP-8, the DPPH free radical scavenging activity of the pricklyash fruit oil mixture is not significantly enhanced. Therefore, in terms of the selection of the concentration for DPPH free radical scavenging, the optimal addition dose of PUP-8 is 1%.

[0064] Example 4

[0065] Effect of Pricklyash Fruit Polypeptide PUP-8 on the Quality of Pricklyash Fruit Oil

[0066] The cold resistance, heat resistance and centrifugal stability of the pricklyash fruit oil mixture were measured to evaluate the effect of PUP-8 on the quality of pricklyash fruit oil. 1% of PUP-8 was added to the pricklyash fruit oil, shaken and mixed evenly, and used as the pricklyash fruit oil mixture for standby. The pricklyash fruit oil was used as a reference substance to complete the measurement. Cold resistance: Take 5 g of the pricklyash fruit oil mixture sample in a dry PV tube, tighten the lid and place it in a refrigerator at -20 °C for 24 h. Take out the sample and restore it to room temperature, and observe whether there is layering. Each sample was measured 3 times repeatedly. Heat resistance: Take 5 g of the pricklyash fruit oil mixture sample in a dry PV tube, tighten the lid and place it in an incubator at 40 °C for 24 h. Take out the sample and restore it to room temperature, and observe whether there is layering. Each sample was measured 3 times repeatedly. Centrifugation experiment: Take 5 g of the pricklyash fruit oil mixture sample and centrifuge it at 4000 r / min for 30 min, and observe the layering situation. Each sample was measured 3 times repeatedly.

[0067] The results showed that the pricklyash fruit oil mixture after adding PUP-8 did not show layering after centrifugation at 4000 r / min for 30 min, indicating that PUP-8 derived from the pricklyash fruit has good liposolubility, can dissolve in the pricklyash fruit oil, and does not reduce the stability of the pricklyash fruit oil.

[0068] Example 5

[0069] Effect of Pricklyash Fruit Polypeptide PUP-8 on the Storage Quality of Pricklyash Fruit Oil

[0070] The storage quality of Prinsepia utilis Royle oil was evaluated by measuring its acid value and peroxide value. 1% of PUP-8 was added to the Prinsepia utilis Royle oil, shaken well and mixed, and used as the Prinsepia utilis Royle oil mixture for standby. The Prinsepia utilis Royle oil was used as a reference substance to complete the measurement. The samples were stored under natural conditions for 18 consecutive months, from March 2023 to September 2024. The changes in acid value and peroxide value during storage were tracked and detected every other month.

[0071] Determination of acid value: Ethanol-ether mixed solution (1:1, volume ratio): Equal amounts of ethanol and ether were mixed. 0.1M KOH ethanol solution: An appropriate amount of KOH was dissolved in absolute ethanol to prepare a 0.1M concentration, and its concentration was calibrated. Weigh 1 g of the Prinsepia utilis Royle oil mixture and put it into a conical flask. Add 50 mL of the ethanol-ether mixed solution and gently shake to completely dissolve the sample. Add 1 drop of phenolphthalein indicator, and the solution is colorless. Titrate with 0.1M KOH ethanol solution, shaking while titrating until the solution shows a persistent pink color for 30 s. Record the consumption volume of the KOH ethanol solution. The acid value is calculated according to the following formula:

[0072]

[0073] In the formula, V: the volume of the KOH ethanol solution (mL); C: the concentration of the KOH ethanol solution (mol / L); m: the mass of the sample (g).

[0074] Determination of peroxide value: Glacial acetic acid-chloroform solution (3:2, volume ratio): Mix glacial acetic acid and chloroform with a volume ratio of 3:2. Saturated KI solution: Prepare a saturated potassium iodide solution. 0.01M sodium thiosulfate solution: Dissolve an appropriate amount of sodium thiosulfate in distilled water to prepare a 0.01M concentration and calibrate it. Starch indicator: Prepare a fresh 0.5% starch solution. Weigh 1 g of the Prinsepia utilis Royle oil mixture sample and put it into a conical flask. Add 30 mL of the glacial acetic acid-chloroform solution, shake well, add 0.5 mL of the saturated KI solution, immediately cover the bottle cap and react in the dark for 1 min. Add 30 mL of distilled water and shake well. Titrate with 0.01M sodium thiosulfate solution until it turns light yellow, add 1 drop of starch indicator, continue titrating until the solution turns colorless after it turns blue. Record the consumption volume of the sodium thiosulfate solution. The peroxide value is calculated according to the following formula:

[0075]

[0076] In the formula, V: the consumption volume of sodium thiosulfate in the sample group (mL); V0: the consumption volume of sodium thiosulfate in the blank group (mL); C: the concentration of the sodium thiosulfate solution (mol / L); m: the mass of the sample (g)

[0077] The results showed that after storing the Prinsepia utilis Royle oil and the mixture of Prinsepia utilis Royle oil added with PUP-8 for 18 months under natural conditions, both the acid value and peroxide value increased. The increase in the acid value and peroxide value of the mixture of Prinsepia utilis Royle oil added with PUP-8 was weaker than that of Prinsepia utilis Royle oil, proving that the addition of PUP-8 could effectively improve the storage quality of Prinsepia utilis Royle oil.

[0078] Example 6

[0079] Effect of Prinsepia utilis Royle polypeptide PUP-8 and chemical antioxidants on the storage quality of Prinsepia utilis Royle oil

[0080] The storage quality of Prinsepia utilis Royle oil was evaluated by measuring the acid value and peroxide value in the oil. 1% of PUP-8, 1% of BHT, 1% of vitamin C, and 1% of vitamin E were respectively added to the commercially available Prinsepia utilis Royle oil, and after shaking and mixing evenly, it was used as the Prinsepia utilis Royle oil mixture for standby. The measurement was completed using Prinsepia utilis Royle oil as the control. The samples were stored under natural conditions for 16 consecutive months, from March 2023 to July 2024, and the changes in the acid value and peroxide value during the storage process were tracked and detected every 4 months. The calculation methods of the acid value and peroxide value were the same as those in Example 5, and the results are shown in Table 1.

[0081]

[0082]

[0083] As can be seen from Table 1, after adding PUP-8, the acid value of Prinsepia utilis Royle oil showed a slow upward trend. In July 2024, the acid value of Prinsepia utilis Royle oil was 1.48. Limited by solubility, BHT and Vc had little effect on inhibiting the increase in the acid value of Prinsepia utilis Royle oil. Ve showed a certain activity in inhibiting the increase in the acid value of Prinsepia utilis Royle oil, but the effect was weaker than that of PUP-8. For the effect on the peroxide value of Prinsepia utilis Royle oil, the above additives showed the same trend. Limited by solubility, BHT and Vc showed little inhibitory effect on the peroxide value of Prinsepia utilis Royle oil. The inhibitory effects of PUP-8 and Ve were comparable. Generally speaking, PUP-8 had the best effect of extending the shelf life of Prinsepia utilis Royle oil and ensuring its quality.

[0084] The above-described embodiments only represent the specific implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application.

[0085] This Background of the Invention section is provided to generally present the context of the present invention. Work of the presently named inventors, to the extent it is described in this Background of the Invention section, and aspects of the work that are not yet prior art as of the filing of this application, are neither expressly nor impliedly admitted to be prior art to the present invention.

Claims

1. An antioxidant peptide extracted from Prinsepia utilis Royle, characterized in that: Its amino acid sequence is Leu-Met-Gly-Cys-Trp-Val-Val-Phe.

2. The antioxidant peptide according to claim 1, characterized in that: The molecular mass of the antioxidant peptide is 954 Da.

3. The antioxidant peptide according to claim 1 or 2, characterized in that: The antioxidant peptide is derived from the fruits of Prinsepia utilis Royle.

4. The method for extracting the antioxidant peptide according to claim 1 or 2, characterized in that It includes the following steps: Crush the dried Prinsepia utilis Royle fruits to obtain dried Prinsepia utilis Royle fruit powder; then add ethanol to the dried Prinsepia utilis Royle fruit powder and perform ultrasonic treatment. After the ultrasonic treatment, centrifuge the mixture to separate the supernatant. Pour the supernatant into a separating funnel, then add chloroform to it, shake well and let it stand for stratification. Collect the lower organic phase; rotate and evaporate the collected organic phase at low temperature to remove the solvent and obtain a concentrated crude polypeptide extract. Dialyze the concentrated crude polypeptide extract through a dialysis bag with a molecular weight cut-off of 3500 Da in PBS buffer, collect the dialysate, rotate and evaporate to concentrate it to obtain a concentrated solution; freeze-dry the concentrated solution with a vacuum freeze dryer to obtain a dry powder of Prinsepia utilis Royle polypeptide extract. Dissolve the dry powder of Prinsepia utilis Royle polypeptide extract with ethanol, load it onto a Superdex 30 Increase gel filtration column, elute with sterile water, measure the absorbance value of the eluate at 215 nm after collecting the eluate, collect and freeze-dry a single peak to obtain a dry powder of Prinsepia utilis Royle polypeptide complex. Dissolve the dry powder of Prinsepia utilis Royle polypeptide complex with ethanol, purify it by reverse high performance liquid chromatography, collect a single peak to obtain Prinsepia utilis Royle polypeptide, namely the antioxidant peptide.

5. Use of the antioxidant peptide according to claim 1 or 2 in the preparation of antioxidant products.

6. Use of the antioxidant peptide according to claim 1 or 2 in the preparation of antioxidants.

7. An antioxidant, characterized in that, Containing the antioxidant peptide according to claim 1 or 2.

8. Use of the antioxidant peptide according to claim 1 or 2 in extending the storage period of Prinsepia utilis Royle oil.

9. The extraction method of the antioxidant peptide according to claim 5, wherein The chromatographic conditions for the reverse high performance liquid chromatography purification are as follows: The chromatographic column is a PE C18 column, 150 mm × 4.6 mm; Phase A: pure water, Phase B: acetonitrile containing 0.1% trifluoroacetic acid; the injection volume is set at 30 μL; the column temperature is set at 25 ± 5 °C; the flow rate is selected at 1.0 mL / min; the detection wavelength is set at 215 nm.

10. The extraction method of the antioxidant peptide according to claim 5, characterized in that, The elution conditions for the reverse high performance liquid chromatography purification are as follows: Phase A: 0 - 8 min, 99% - 97%; 8 - 12 min, 97% - 96%; 12 - 16 min, 96% - 80%; 16 - 20 min, 80% - 99%.

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