A method for separating and purifying a flaxseed cyclic peptide from flaxseed and antioxidant application thereof

Abstract

The application belongs to the field of natural product extraction, and particularly relates to a method for separating and purifying a flaxseed cyclic peptide from flaxseed and antioxidant application thereof. The application realizes selective enrichment of a hydrophobic cyclic peptide in an ionic liquid phase by using phosphate-quadric phosphonium cation synergistic hydrogen bond action of an ionic liquid, and saves traditional defatting and organic solvent extraction steps through three-stage purification of salting-out-dilution-precipitation.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction, specifically relating to a method for separating and purifying flaxseed cyclic peptides from flaxseed and their antioxidant applications. Background Technology

[0002] Flax, also known as linseed, is an annual herbaceous plant belonging to the genus *Linum* in the family Linaceae, and is one of the oldest crops in the world. Flaxseed, the seed of the flax plant, is rich in various functional components such as α-linolenic acid, lignans, flaxseed gum, and cyclic peptides. It possesses multiple benefits including antioxidant, anti-inflammatory, anti-cancer, antihypertensive, and cardiovascular disease prevention effects, making it a highly nutritious functional food. With the deepening of research in phytochemistry and molecular biology, a class of cyclic oligopeptides with unique structural characteristics in flaxseed—flaxseed cyclic peptides—has gradually become a research hotspot in the field of natural products. These compounds consist of 8-9 amino acid residues linked end-to-end by peptide bonds to form a cyclic structure. Their molecular weight is typically in the range of 1,000-1,200 Da, rich in hydrophobic amino acids, exhibiting significant hydrophobicity and stability. They can withstand gastrointestinal digestive enzymes and extreme pH environments, demonstrating excellent bioavailability and pharmacodynamic properties. Studies have shown that flaxseed cyclic peptides possess multiple biological activities, particularly excelling in anti-oxidative stress. The aromatic amino acid residues such as tyrosine and phenylalanine in its cyclic structure can effectively scavenge free radicals, block the lipid peroxidation chain reaction, and protect cell membrane integrity. Furthermore, flaxseed cyclic peptides exhibit significant antibacterial, anti-inflammatory, antihypertensive, and immunomodulatory activities, making them valuable in the development of functional foods, the preparation of natural preservatives, and the screening of antioxidant drugs. However, due to the low content of cyclic peptides in flaxseed and their coexistence with various components such as oils, proteins, and polysaccharides, efficient separation and purification has become a key bottleneck restricting their industrial application.

[0003] Currently, traditional extraction methods for flaxseed cyclic peptides mainly rely on a combination of organic solvent extraction and multi-step chromatographic separation. Early studies often used organic solvents such as methanol, ethanol, and ethyl acetate for repeated extraction, combined with liquid-liquid partitioning to remove oils and polar impurities, followed by stepwise purification using silica gel column chromatography, gel filtration chromatography, or reversed-phase high-performance liquid chromatography. However, these methods have significant drawbacks: large amounts of organic solvents are used, they are flammable and explosive, causing serious environmental pollution, and the extraction selectivity is poor, easily dissolving coexisting components such as oils, flavonoids, and lignans, leading to cumbersome and time-consuming subsequent separation and purification steps with low yields. Although supercritical carbon dioxide extraction technology can achieve solvent-free extraction, it requires high equipment investment, demanding operating parameters, and has limited solubility for moderately polar molecules such as cyclic peptides, making its extraction efficiency insufficient for industrial applications.

[0004] With the promotion of green chemistry, ionic liquids, as a novel green solvent system, have demonstrated unique advantages in the field of natural product extraction. Ionic liquids are room-temperature molten salts composed of organic cations and inorganic or organic anions, characterized by extremely low vapor pressure, high thermal stability, and strong structural designability. In particular, functional ionic liquids can achieve selective recognition and dissolution of target molecules through the rational combination of cations and anions. Aqueous two-phase systems, as a mild and efficient separation technique, utilize the partitioning differences between two immiscible aqueous phases to achieve selective enrichment of target substances. CN115558014A discloses a method for the separation and purification of flaxseed cyclic peptides based on an aqueous two-phase system; however, the purity of this method is low, with a maximum of only 80.4%. The emergence of ionic liquid-salt aqueous two-phase systems provides a new approach for bioseparation: by adding an appropriate amount of salt to an aqueous solution of an ionic liquid, phase separation can be induced, forming an ionic liquid enriched phase and a salt enriched phase. Due to the specific binding of ionic liquids to cyclic peptides, target molecules preferentially partition into the ionic liquid phase, separating from polar impurities such as sugars and inorganic salts, significantly simplifying the purification process. While ionic liquids and aqueous two-phase technology theoretically offer possibilities for the extraction of flaxseed cyclic peptides, existing techniques still face numerous challenges in practical applications. On one hand, conventional ionic liquids lack sufficient selectivity for dissolving flaxseed cyclic peptides, easily resulting in the accumulation of large amounts of oil and pigment impurities during extraction, affecting product color and purity. On the other hand, systematic research on the composition optimization of aqueous two-phase systems is lacking. The selection of salt type and concentration directly affects phase separation efficiency and the partition coefficient of cyclic peptides; improper parameter settings may lead to the loss of the target compound in the salt phase or severe emulsification.

[0005] Therefore, developing a method for separating and purifying flaxseed cyclic peptides that is highly selective, has a high extraction rate, high purity, and is environmentally friendly is of great significance for promoting the high-value utilization of flaxseed. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide a method for isolating and purifying flaxseed cyclic peptides from flaxseed. To achieve the above objective, this invention employs the following technical solution:

[0007] A method for isolating and purifying flaxseed cyclic peptides from flaxseed includes the following steps:

[0008] Step 1: After flaxseed is crushed, it is sieved and dried to obtain dry flaxseed powder;

[0009] Step 2: Thoroughly mix the quaternary phosphate ionic liquid and deionized water to obtain an aqueous solution of the quaternary phosphate ionic liquid;

[0010] Step 3: Mix dried flaxseed with an aqueous solution of quaternary phosphate ionic liquid and extract by ultrasonication; centrifuge the resulting mixture and collect the supernatant; add salt to the supernatant and let it stand to form a quaternary phosphate ionic liquid-salt aqueous two-phase system; separate the two phases using a separatory funnel and collect the quaternary phosphate ionic liquid enriched phase.

[0011] Step 4: Add deionized water to the quaternary phosphate ion liquid enriched phase described in Step 4, stir evenly, let stand, and allow flaxseed cyclic peptides to precipitate. Filter, and vacuum dry the collected flaxseed cyclic peptide precipitate to obtain flaxseed cyclic peptides.

[0012] The structural formula of the quaternary phosphorus salt ionic liquid is as follows: or .

[0013] In some implementations, the mass fraction of the quaternary phosphate ionic liquid in the aqueous solution of the quaternary phosphate ionic liquid in step 2 is 10-30%.

[0014] In some embodiments, the salt in step 3 is selected from one or more of sodium carbonate, potassium carbonate, potassium phosphate, sodium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium citrate, and sodium citrate.

[0015] In some embodiments, the mass-to-volume ratio of the dried flaxseed to the quaternary phosphate ion liquid aqueous solution in step 3 is 1 g:(5~15) mL; and the mass ratio of the dried flaxseed to salt is 1:(1~5).

[0016] In some implementations, the conditions for ultrasonic extraction in step 3 are: temperature of 25~50℃, power of 100~250W, and time of 10~60min; and the conditions for centrifugation are: speed of 4000~7000rpm and centrifugation time of 5~20min.

[0017] In some embodiments, the volume ratio of the deionized water in step 4 to the quaternary phosphate ionic liquid aqueous solution in step 3 is (10~20):1;

[0018] In some implementation schemes, The preparation method includes the following steps:

[0019] Diethyl 4-bromobutylphosphonate, triphenylphosphine, and toluene were added to a container, which was then placed in a microwave reactor and reacted at a temperature of 90–120 °C and a power of 300–500 W. After the reaction was completed, the mixture was filtered, the filter cake was washed with toluene, and dried to obtain the target product. The reaction formula is as follows: .

[0020] In some implementation schemes, The preparation method includes the following steps:

[0021] Diethyl 4-chlorobutylphosphonate, triphenylphosphine, and toluene were added to a container and then placed in a microwave reactor. The reaction was carried out at a temperature of 90-120℃ and a power of 300-500W. After the reaction was completed, the mixture was filtered, the filter cake was washed with toluene, and dried to obtain the target product.

[0022] This invention also protects the use of the above-mentioned flaxseed cyclic peptide as an antioxidant.

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

[0024] 1) This invention utilizes the synergistic hydrogen bonding effect of phosphate esters and quaternary phosphorus cations in ionic liquids to achieve selective enrichment of hydrophobic cyclic peptides in ionic liquid phases, and eliminates the need for traditional defatting and organic solvent extraction steps through a three-stage purification process of salting out, dilution and precipitation.

[0025] 2) The flaxseed cyclic peptide powder of this invention has an extraction rate of up to 93.2% and an HPLC purity of up to 97.5%. The extraction conditions are mild, with little environmental pollution, making it suitable for large-scale industrial production. Detailed Implementation

[0026] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0027] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The invention is further described below by way of specific embodiments. All chemical reagents used in the embodiments of this invention, unless otherwise specified, are obtained through conventional commercial means.

[0028] Extraction rate = (Mass of extracted flaxseed cyclic peptide powder / Mass of flaxseed cyclic peptides in flaxseed powder) x 100%;

[0029] The method for determining the content of flaxseed cyclic peptides in the raw material flaxseed powder and extract was as follows: A 1.0 mg / mL solution was prepared using flaxseed mixed cyclic peptides (14 types) (manufactured by Prairie Tide Chemicals, Canada) as the standard and acetonitrile as the solvent. The solution was filtered through a 0.45 μL filter membrane and injected into a 2 mL Waters vial for HPLC analysis. Chromatographic conditions: column temperature 30℃; UV detection wavelength 214 nm.

[0030] 1) The quaternary phosphorus salt ionic liquid used in this invention is (Hereinafter referred to as P-1), its preparation method includes the following steps: ,

[0031] Diethyl 4-bromobutylphosphonate (0.11 mol), triphenylphosphine (0.1 mol), and toluene (150 mL) were added to a two-necked round-bottom flask, which was then placed in a microwave reactor and stirred for 2 h at 100 °C and 450 W. After the reaction was completed, the mixture was filtered, and the filter cake was washed three times with toluene and dried under vacuum for 12 h to obtain the quaternary phosphate ionic liquid P-1, with a yield of 82.1%.

[0032] 1 H NMR (400 MHz, CDCl3)δ=7.78 (dd, 3H), 7.70-7.59 (m, 12H), 4.05 (p,4H), 3.18 (t, 2H), 1.85 (d, 4H), 1.76-1.67 (m, 2H), 1.16 (t, 6H).

[0033] 2) Based on a similar method, the raw materials Replace with It can be prepared (Hereinafter referred to as P-2); Example 1

[0034] A method for isolating and purifying flaxseed cyclic peptides from flaxseed, comprising the following steps:

[0035] Step 1: After the flaxseeds are crushed by a grinder and passed through a 70-mesh sieve, the sieved flaxseed powder is placed in an oven and dried at 50°C for 24 hours to obtain dried flaxseed powder.

[0036] Step 2: Add the quaternary phosphate ionic liquid P-1 to a beaker, slowly add deionized water to make the mass fraction of quaternary phosphate ionic liquid P-1 in the system 20%, and stir thoroughly to obtain an aqueous solution of quaternary phosphate ionic liquid P-1.

[0037] Step 3: Place 10g of dried flaxseed powder in a beaker, then add 100mL of quaternary phosphate ionic liquid P-1 aqueous solution, stir with a magnetic stirrer, and ultrasonically extract for 30 min at 30℃ and 200W power; centrifuge the resulting mixture at 6000rpm for 10 min and collect the supernatant; add 15.0g of potassium phosphate to the supernatant, let stand to form a quaternary phosphate ionic liquid-potassium phosphate aqueous two-phase system, then separate using a separatory funnel and collect the quaternary phosphate ionic liquid enriched phase;

[0038] Step 4: Add 1.0 L of deionized water to the quaternary phosphate ion liquid enrichment phase described in Step 4, stir evenly, and let stand for 10 h to precipitate flaxseed cyclic peptides. Filter, and vacuum dry the collected flaxseed cyclic peptide precipitate for 12 h to obtain flaxseed cyclic peptide powder with an extraction rate of 93.2% and an HPLC purity of 97.5%. Example 2

[0039] A method for isolating and purifying flaxseed cyclic peptides from flaxseed, comprising the following steps:

[0040] Step 1: After the flaxseeds are crushed by a grinder and passed through a 50-mesh sieve, the sieved flaxseed powder is placed in an oven and dried at 50°C for 24 hours to obtain dried flaxseed powder.

[0041] Step 2: Add the quaternary phosphate ionic liquid P-2 to a beaker, slowly add deionized water to make the mass fraction of quaternary phosphate ionic liquid P-2 in the system 15%, and stir thoroughly to obtain an aqueous solution of quaternary phosphate ionic liquid P-2.

[0042] Step 3: Place 10g of dried flaxseed powder in a beaker, then add 120mL of quaternary phosphate ionic liquid P-2 aqueous solution, stir with a magnetic stirrer, and ultrasonically extract for 30 min at 40℃ and 150W; centrifuge the resulting mixture at 6000rpm for 10 min and collect the supernatant; add 20.0g of potassium phosphate to the supernatant, let stand to form a quaternary phosphate ionic liquid-potassium phosphate aqueous two-phase system, then separate using a separatory funnel and collect the quaternary phosphate ionic liquid enriched phase;

[0043] Step 4: Add 1.0 L of deionized water to the quaternary phosphate ion liquid enrichment phase described in Step 4, stir evenly, and let stand for 10 h to precipitate flaxseed cyclic peptides. Filter, and vacuum dry the collected flaxseed cyclic peptide precipitate for 12 h to obtain flaxseed cyclic peptide powder with an extraction rate of 91.5% and an HPLC purity of 97.3%. Example 3

[0044] A method for isolating and purifying flaxseed cyclic peptides from flaxseed, comprising the following steps:

[0045] Step 1: After the flaxseeds are crushed by a grinder and passed through a 60-mesh sieve, the sieved flaxseed powder is placed in an oven and dried at 50°C for 24 hours to obtain dried flaxseed powder.

[0046] Step 2: Add the quaternary phosphate ionic liquid P-1 to a beaker, slowly add deionized water to make the mass fraction of quaternary phosphate ionic liquid P-1 in the system 20%, and stir thoroughly to obtain an aqueous solution of quaternary phosphate ionic liquid P-1.

[0047] Step 3: Place 10g of dried flaxseed powder in a beaker, then add 100mL of quaternary phosphate ionic liquid P-1 aqueous solution, stir with a magnetic stirrer, and ultrasonically extract for 30 min at 30℃ and 200W power; centrifuge the resulting mixture at 6000rpm for 10 min and collect the supernatant; add 20.0g of sodium carbonate to the supernatant, let stand to form a quaternary phosphate ionic liquid-sodium carbonate aqueous two-phase system, then separate using a separatory funnel and collect the quaternary phosphate ionic liquid enriched phase;

[0048] Step 4: Add 1.0 L of deionized water to the quaternary phosphate ion liquid enriched phase described in Step 4, stir evenly, and let stand for 10 h to precipitate flaxseed cyclic peptides. Filter, and vacuum dry the collected flaxseed cyclic peptide precipitate for 12 h to obtain flaxseed cyclic peptide powder with an extraction rate of 92.0% and an HPLC purity of 97.6%. Example 4

[0049] A method for isolating and purifying flaxseed cyclic peptides from flaxseed, comprising the following steps:

[0050] Step 1: After the flaxseeds are crushed by a grinder and passed through a 70-mesh sieve, the sieved flaxseed powder is placed in an oven and dried at 50°C for 24 hours to obtain dried flaxseed powder.

[0051] Step 2: Add the quaternary phosphate ionic liquid P-1 to a beaker, slowly add deionized water to make the mass fraction of quaternary phosphate ionic liquid P-1 in the system 20%, and stir thoroughly to obtain an aqueous solution of quaternary phosphate ionic liquid P-1.

[0052] Step 3: Place 10g of dried flaxseed powder in a beaker, then add 100mL of quaternary phosphate ionic liquid P-1 aqueous solution, stir with a magnetic stirrer, and ultrasonically extract for 30 min at 30℃ and 200W; centrifuge the resulting mixture at 6000rpm for 10 min and collect the supernatant; add 15.0g of potassium citrate to the supernatant, let stand to form a quaternary phosphate ionic liquid-potassium citrate aqueous two-phase system, then separate using a separatory funnel and collect the quaternary phosphate ionic liquid enriched phase;

[0053] Step 4: Add 1.0 L of deionized water to the quaternary phosphate ion liquid enriched phase described in Step 4, stir evenly, and let stand for 10 h to precipitate flaxseed cyclic peptides. Filter, and vacuum dry the collected flaxseed cyclic peptide precipitate for 12 h to obtain flaxseed cyclic peptide powder with an extraction rate of 90.1% and an HPLC purity of 97.2%.

[0054] Comparative Example 1

[0055] Based on Example 1, the quaternary phosphate ionic liquid P-1 was replaced with tributyl (octyl)phosphine bromide, and other operating steps and conditions were the same as in Example 1. The extraction rate of flaxseed cyclic peptides was 69.8%, and the HPLC purity was 92.1%.

[0056] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for isolating and purifying flaxseed cyclic peptides from flaxseed, comprising the following steps: Step 1: After flaxseed is ground, it is sieved and dried to obtain dry flaxseed powder; Step 2: Thoroughly mix the quaternary phosphate ionic liquid and deionized water to obtain an aqueous solution of the quaternary phosphate ionic liquid; Step 3: Mix dried flaxseed with an aqueous solution of quaternary phosphate ionic liquid and extract by ultrasonication; centrifuge the resulting mixture and collect the supernatant; add salt to the supernatant and let it stand to form a quaternary phosphate ionic liquid-salt aqueous two-phase system; separate the two phases using a separatory funnel and collect the quaternary phosphate ionic liquid enriched phase. Step 4: Add deionized water to the quaternary phosphate ion liquid enriched phase described in Step 4, stir evenly, let stand, and allow flaxseed cyclic peptides to precipitate. Filter, and vacuum dry the collected flaxseed cyclic peptide precipitate to obtain flaxseed cyclic peptides. The structural formula of the quaternary phosphorus salt ionic liquid is as follows: or .

2. The method according to claim 1, characterized in that, The mass fraction of the quaternary phosphate ionic liquid in the aqueous solution of the quaternary phosphate ionic liquid in step 2 is 10~30%.

3. The method according to claim 1, characterized in that, The salt mentioned in step 3 is selected from one or more of sodium carbonate, potassium carbonate, potassium phosphate, sodium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium citrate, and sodium citrate.

4. The method according to claim 1, characterized in that, The mass-to-volume ratio of the dried flaxseed to the quaternary phosphate ion liquid aqueous solution in step 3 is 1 g:(5~15) mL; the mass ratio of the dried flaxseed to salt is 1:(1~5).

5. The method according to claim 1, characterized in that, The conditions for ultrasonic extraction in step 3 are: temperature 25~50℃, power 100~250W, time 10~60min; the conditions for centrifugation are: speed 4000~7000rpm, centrifugation time 5~20min.

6. The method according to claim 1, characterized in that, The volume ratio of the deionized water in step 4 to the quaternary phosphate ionic liquid aqueous solution in step 3 is (10~20):

1.

7. The method according to claim 1, characterized in that, The preparation method includes the following steps: Diethyl 4-bromobutylphosphonate, triphenylphosphine, and toluene were added to a container, which was then placed in a microwave reactor and reacted at a temperature of 90–120 °C and a power of 300–500 W. After the reaction was completed, the mixture was filtered, the filter cake was washed with toluene, and dried to obtain the target product. The reaction formula is as follows: 。 8. The method according to claim 1, characterized in that, The preparation method includes the following steps: Diethyl 4-chlorobutylphosphonate, triphenylphosphine, and toluene were added to a container and then placed in a microwave reactor. The reaction was carried out at a temperature of 90-120℃ and a power of 300-500W. After the reaction was completed, the mixture was filtered, the filter cake was washed with toluene, and dried to obtain the target product.

9. A flaxseed cyclic peptide, characterized in that, It is prepared by the method described in any one of claims 1-8.

10. The use of the flaxseed cyclic peptide of claim 9 as an antioxidant.

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