Method for separating and purifying linseed cyclic peptide from linseed oil
Through the two-step regular-inverting phase purification method, combined with gradient elution technology, the problem of separation and purification of flax seed cyclic peptides was solved, and efficient separation and purification was achieved, which improved the utilization rate and product development potential of flax seed cyclic peptides.
Patent Information
- Application Number
- CN202510351806.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-24
- Publication Date
- 2025-07-22
AI Technical Summary
The prior art is difficult to efficiently isolate and purify flaxseed cyclic peptides, especially due to their numerous species and small differences in physical properties and polarity, which makes it difficult for conventional chromatography columns to separate the cyclic peptides of high purity, limiting the development and research of related products.
The normal-reverse phase two-step purification method was adopted. First, the crude flax seed cyclic peptide was extracted using silica gel column elution method, and then the liquid phase normal phase separation and reverse phase separation were prepared by rapid preparation of liquid phase, combined with gradient elution technology, the cyclic peptide containing methionine and methionine sulfoxide was collected and purified respectively. Finally, fractions with a purity of more than 60% were combined to obtain high-purity flax seed cyclic peptide.
The separation efficiency and purity of flaxseed cyclic peptides has been significantly improved, and the efficient separation and purification of a variety of cyclic peptides has been achieved, supporting the development of related products and basic research.
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Figure CN120349380A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of extraction and purification of natural products, and particularly relates to a method for separating and purifying linseed cyclopeptides from linseed oil. Background Art
[0002] Linseed cyclopeptides are a class of natural hydrophobic cyclic compounds composed of 8-9 hydrophobic α-amino acids. The amino acids are connected head to tail to form a ring, belonging to homocyclic peptides, and the molecular weight is approximately in the range of 960-1100 Da. Linseed cyclopeptides are a special concomitant present in linseeds and linseed rhizomes. So far, such compounds have only been found in linseeds and are a unique class of lipid concomitants in linseed oil. So far, more than 20 different linseed cyclopeptides have been isolated and identified from linseed oil. At present, the research on the activities of linseed cyclopeptides has confirmed that these compounds have a variety of activities, and the differences in the amino acid sequences of linseed cyclopeptides will have a profound impact on the corresponding activities. It is reported that cyclopeptide A, cyclopeptide B, and cyclopeptide E can exert an anti-inflammatory effect, and cyclopeptide A, cyclopeptide J, cyclopeptide C, and cyclopeptide E can inhibit the increase in the level of reactive oxygen species in RAW264.7 cells induced by hydrogen peroxide. Cyclopeptide A and cyclopeptide B can play an immunosuppressive role. Cyclopeptide F, cyclopeptide G, and cyclopeptide J (structural formulas are shown below) can inhibit the differentiation of osteoclasts to achieve the purpose of preventing osteoporosis. Thus, it can be seen that linseed cyclopeptides are high-value-added products in the future, and the research on their activities is of great significance for the development of related products.
[0003]
[0004] Chemical Structures and Oxidation Pathways of Common Linseed Cyclopeptides Among the currently discovered linseed cyclopeptides, except for cyclopeptide A and cyclopeptide S, most cyclopeptides contain 1-2 methionine residues. Methionine is sensitive to oxidation reactions. As oxidation proceeds, it will first be oxidized to methionine sulfoxide (MetO), and then continue to be oxidized to methionine sulfone (MetO2). The oxidation of methionine is related to the oxidation of linseed cyclopeptides. As oxidation proceeds, the physical properties such as the polarity and hydrophobicity of linseed cyclopeptides will change. Linseed cyclopeptides extracted from linseed oil rarely show a form of deep oxidation (i.e., MetO residues are oxidized to MetO2). According to the different oxidation purities of methionine, cyclopeptides can be classified into methionine-containing cyclopeptides and methionine sulfoxide-containing cyclopeptides, namely the Met site and the MetO site.
[0005] Due to the large number of linseed cyclic peptides and the small differences in physical properties and polarities among different types of cyclic peptides, it is difficult to separate and purify them by means of conventional single chromatographic column stationary phases, which limits the research on cyclic peptides with a single configuration. Currently, the main methods for separating and purifying linseed cyclic peptides are as follows: (1) Manual column chromatography method, which is complex in operation, requires the combination of multiple different stationary phases, involves a large number of solvent types, has low separation efficiency, and it is difficult to obtain a single type of cyclic peptide, and the purity of the separated product is relatively low; (2) Preparative high-performance liquid chromatography method, which has high requirements for equipment and reagents, takes a long time, has low yield, and the purity can reach more than 95%. However, the single injection volume is in the mg level, which severely limits the basic research and product development of high-purity cyclic peptide single substances.
[0006] Regarding the good biological activities of linseed cyclic peptides, a variety of linseed cyclic peptide compounds with relatively high purity are required in the development and research of related products. Considering the challenges faced by linseed cyclic peptide separation technology, it is necessary to develop a simple and efficient method for separating and purifying linseed cyclic peptides, which is of positive significance for improving the separation efficiency of linseed cyclic peptides, increasing the utilization rate of cyclic peptides, and promoting the progress of related fields. Summary of the Invention
[0007] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide a method for separating and purifying linseed cyclic peptides from linseed oil by using normal-phase - reverse-phase two-step purification method.
[0008] The technical solution adopted by the present invention is as follows: A method for separating and purifying linseed cyclic peptides from linseed oil, comprising the following steps: 1) Using silica gel column elution method for linseed oil raw materials to extract crude linseed cyclic peptides; 2) Performing rapid preparative liquid chromatography normal-phase separation on the mixed linseed cyclic peptides obtained in step 1), with the stationary phase being silica, the ultraviolet detector wavelength bands being 214 nm and 280 nm, adopting the method of gradient elution, collecting the eluent according to the column volume CV of the elution solution, collecting the Met part containing methionine cyclic peptides before 40CV, and collecting the MetO part containing methionine sulfoxide after 40CV to enrich cyclic peptides; 3) Performing reverse-phase separation on the two component cyclic peptides of the Met part and the MetO part obtained in step 2) respectively by using rapid preparative liquid chromatography, with the stationary phase being Phenyl-hexyl, adopting stepwise gradient elution, and the ultraviolet detector wavelength bands being 214 nm and 280 nm; 4) Measuring the purity of the fractions collected in step 3), and combining the fractions containing the same monomer and with a purity higher than 60% to obtain linseed cyclic peptides.
[0009] In some examples, the mobile phase A used in the gradient elution in step 2) is dichloromethane, and the mobile phase B is methanol.
[0010] In some examples, by volume percentage, during the gradient elution in step 2), the proportion of the mobile phase A is 100% - 70%.
[0011] In some examples, the flow rate of the gradient elution in step 2) is 10 - 30 mL / min.
[0012] In some examples, the mobile phase A for the stepwise gradient elution in step 3) is water, and the mobile phase B is acetonitrile.
[0013] In some examples, by volume percentage, the method of stepwise gradient elution in step 3) is: increase the mobile phase B by 5% within every 3 CV each time, and then hold for 3 CV, repeating the above operation.
[0014] In some examples, by volume percentage, during the stepwise gradient elution in step 3), the proportion of the mobile phase B is 15% - 80%.
[0015] In some examples, the flow rate of the stepwise gradient elution in step 3) is 10 - 40 mL / min.
[0016] In some examples, the chromatographic column selected for the normal-phase separation of liquid chromatography in step 2) is an amorphous silica gel normal-phase column.
[0017] In some examples, the size range of the amorphous silica gel normal-phase column is: column size 4 - 120 g, column specification 105.8 mm × 12.4 mm - 261.5 mm × 37.2 mm, particle size 40 - 63 μm.
[0018] In some examples, the chromatographic column selected for the reverse-phase separation of liquid chromatography in step 3) is a phenyl ethyl reverse-phase chromatographic column.
[0019] In some examples, the size range of the phenyl ethyl reverse-phase column is: column size 4 - 25 g, column specification 113.8 mm × 12.4 mm - 184 mm × 21.4 mm, particle size 20 - 45 μm.
[0020] In some examples, flaxseed oil with a moderate oxidation purity is selected as the raw material for the extraction of cyclic peptides.
[0021] In some examples, freshly squeezed flaxseed oil with a low oxidation degree is selected as the raw material for the extraction of cyclic peptides.
[0022] In some examples, flaxseed oil with a moderate oxidation purity is selected as the raw material for the extraction of cyclic peptides.
[0023] The above features can be combined arbitrarily without conflict.
[0024] The beneficial effects of the present invention are as follows: The present invention further optimizes the extraction process, enabling the entire extraction method to achieve effective coordination. The efficiency and purity of extracting linseed cyclopeptides have been greatly improved, resulting in remarkable progress. Description of the Drawings
[0025] Figure 1 It is the chromatogram of the rapid preparative liquid phase in the normal-phase separation process.
[0026] Figure 2 It is the HPLC chromatogram of the methionine-enriched fraction and the methionine sulfoxide-enriched fraction.
[0027] Figure 3 It is the chromatogram of the corresponding rapid preparative liquid phase during the reverse-phase separation of the methionine-enriched fraction.
[0028] Figure 4 It is the chromatogram of the corresponding rapid preparative liquid phase during the reverse-phase separation of the methionine sulfoxide-enriched fraction.
[0029] Figure 5 It is the HPLC chromatogram of some components with a relatively high content of partial single cyclopeptides obtained by reverse-phase separation. Detailed Description of the Invention
[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the following further elaborates on the present invention in detail in conjunction with the accompanying drawings and specific embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.
[0031] The present invention provides a method for separating and purifying linseed cyclopeptides from linseed oil. In some embodiments, it includes the following steps: Step.1 Mixed Cyclopeptide Extraction S1.1 Preparation of the silica gel column: Mix linseed oil and silica gel in a ratio of 5:1 to obtain the loading silica gel, and load it into a glass column with an inner diameter of 3 cm. The column is pre-filled with an equal amount of silica gel; S1.2 Gravity elution: Naturally remove the oil components in the silica gel column by gravity; S1.3 Elution of the silica gel column: The elution solvents for the silica gel column are successively 100% n-hexane, 20% ethyl acetate - n-hexane, 50% ethyl acetate - n-hexane, 100% ethyl acetate, and 10% methanol - dichloromethane; S1.4 Preparation of mixed cyclic peptides: Transfer the eluate rich in cyclic peptides to a round-bottom flask, and obtain the solid of mixed cyclic peptides after removing the solvent by concentration under reduced pressure and vacuum drying using a rotary evaporator.
[0032] Step.2 Rapid preparative liquid chromatography normal-phase separation S2.1 Sample pretreatment: Weigh the mixed cyclic peptides of flaxseed and place them in a round-bottom flask, add methanol solution, stir well, let stand, and ultrasonicate to fully dissolve the sample, obtaining a sample solution with a concentration of 50 - 200 mg / mL. S2.2 Chromatographic column pretreatment: Select a medium- and low-pressure silica gel normal-phase column, and equilibrate the chromatographic column with a dichloromethane-methanol solution with a volume concentration of 100% as the elution system solution. S2.3 Sample loading: Filter the sample obtained in step S2.2 and place it in a syringe, and use the syringe to load the sample onto the rapid separation liquid phase. S2.4 Elution: Elute the chromatographic column with a dichloromethane-methanol solution with a volume concentration of 100% - 90% as the elution system solution, set the flow rate to 10 - 30 mL / min, and collect the eluate according to the column volume of the elution solution. S2.5 Treatment of normal-phase separation eluate: Collect the separated eluate according to the peak elution time of the chromatogram (component 1 before 40 CV, and component 2, i.e., the MetO part, after 40 CV), transfer it to a round-bottom flask with methanol, and remove the solvent in the sample by concentration under reduced pressure and vacuum drying using a rotary evaporator, obtaining the solid of the mixed cyclic peptides after preliminary separation, the cyclic peptide Met part (component 1), and the cyclic peptide MetO part (component 2).
[0033] Step.3 Rapid preparative liquid chromatography reverse-phase separation S3.1 Sample pretreatment: Dissolve the two groups of solids obtained in S2.5 after preliminary separation with methanol respectively to prepare sample solutions with a concentration of 50 - 200 mg / mL. S3.2 Chromatographic column pretreatment: Select a medium- and low-pressure phenyl ethyl reverse-phase chromatographic column, and equilibrate the chromatographic column with an acetonitrile-formic acid aqueous solution with a volume concentration of 20% as the elution system solution. S3.3 Sample loading: Filter the sample solutions obtained in S3.5 respectively and place them in syringes, and use the syringes to load the samples onto the rapid separation liquid phase. S3.4 Elution: Elute the chromatographic column with an acetonitrile-formic acid aqueous solution with a volume concentration of 20% - 70% as the elution system solution, set the flow rate to 5 - 30 mL / min, and set the collection frequency to 5 - 25 mL / fraction. S3.5 Treatment of reverse-phase separation eluate: Number the obtained fractions, transfer the eluate to the corresponding round-bottom flask with methanol, and remove the solvent in the sample by concentration under reduced pressure and vacuum drying using a rotary evaporator, obtaining the solid of the cyclic peptides after two-step separation and purification.
[0034] Step 4 Purity determination and merging S4.1 Preparation for establishing the standard curve equation of linseed cyclopeptides: Using chromatographic grade methanol as the solvent, prepare gradient solutions of cyclopeptide standards corresponding to each cyclopeptide monomer. After filtering with a 0.45 μL filter membrane, inject them into 1.5 mL Waters injection vials for standby; S4.2 Establishing the standard curve equation of linseed cyclopeptides: Record the peak areas of cyclopeptide standards at different concentrations. Taking the peak area as the ordinate (Y) and the concentration of cyclopeptide standards as the abscissa (X), obtain the linear regression equation, which is the standard curve equation of linseed cyclopeptide monomers.
[0035] S4.3 Purity calculation: Calculate the purity of cyclopeptides in each fraction according to the standard curve equation.
[0036] S4.4 After HPLC purity determination, dissolve and merge the fractions with a unified monomer purity higher than 60% using methanol, and then further concentrate by vacuum distillation, weigh, and repeat S4.1 - 4.2 to determine the purity again.
[0037] In step S2.2, the size range of the amorphous silica normal phase column is: column size 4 - 120 g, column specification 105.8 mm × 12.4 mm - 261.5 mm × 37.2 mm, particle size 40 - 63 μm.
[0038] In step S2.4, the flow rate range can be 10 - 30 mL / min.
[0039] In step S2.4, when detecting during the normal phase separation of the high-speed liquid chromatography preparative instrument, mobile phase A is dichloromethane solution, mobile phase B is methanol solution, and a gradient elution program is adopted, with the proportion of organic phase B being 0% - 10%.
[0040] In step S3.2, the size range of the phenyl ethyl reverse phase column is: column size 4 - 25 g, column specification 113.8 mm × 12.4 mm - 184 mm × 21.4 mm, particle size 20 - 45 μm.
[0041] In step S3.4, the flow rate is 5 - 30 mL / min, and the collection frequency is 5 - 25 mL / fraction.
[0042] When performing high-speed liquid chromatography preparative detection and separation, two different detection wavelengths are selected: the detection wavelength for peptide bonds is 214 nm, and the detection wavelength for tryptophan residues is 280 nm.
[0043] To verify the effect of the method for separating and purifying flaxseed cyclopeptides, the present invention also uses HPLC to analyze the purity of flaxseed cyclopeptides, and calculates the purity of cyclopeptides by the external standard method. Among them, the chromatographic conditions of HPLC are as follows: water (mobile phase A), acetonitrile (mobile phase B); gradient elution program: the initial concentration of mobile phase B is 30%, which is increased to 92% at a rate of 1% per minute, and then maintained for 2 min, and the flow rate is 0.5 mL. The ultraviolet detection wavelength is 214 nm. The external standard method is used for the quantification of cyclopeptides: standard product gradient solutions of 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL are respectively prepared, filtered with a 0.45 μm filter membrane, and reserved. Example 1 S1. Screening of flaxseed oil Select flaxseed oil with moderate oxidation purity and containing both unoxidized cyclopeptides (containing methionine groups) and partially oxidized cyclopeptides (containing methionine sulfoxide groups) as the raw material to extract flaxseed cyclopeptides.
[0044] S2. Extraction of mixed cyclopeptides Mix 500 mL of flaxseed oil with 100 mL of silica gel, and load it into a glass column with an inner diameter of 3 cm. The column is pre-filled with 100 cm 3 silica gel, and the oil components in the silica gel column are removed naturally by gravity. The elution solvents of the silica gel column are 500 mL of 100% n-hexane, 300 mL of 20% ethyl acetate / n-hexane (V / V), 300 mL of 50% ethyl acetate / n-hexane (V / V), 300 mL of 100% ethyl acetate, and 300 mL of 10% methanol / dichloromethane (V / V) in sequence. Transfer the 100% ethyl acetate and 10% methanol / dichloromethane eluates rich in cyclopeptides to a round-bottom flask, and obtain a mixed cyclopeptide solid after removing the solvent by rotary evaporation under reduced pressure and vacuum drying.
[0045] S3. Normal-phase separation by rapid preparative liquid chromatography Weigh 1 g of the mixed flaxseed cyclopeptides and place them in a round-bottom flask, add 10 mL of dichloromethane solution, stir well, let it stand, and ultrasonicate to fully dissolve the sample to obtain a sample solution with a concentration of 100 mg / mL; Select a medium-pressure and low-pressure silica gel normal-phase column, and equilibrate the chromatographic column with dichloromethane as the elution system solution; after loading the sample, mobile phase A is dichloromethane, and mobile phase B is methanol. A gradient elution program is adopted, and the volume ratio of organic phase B is 0% - 15%. The flow rate is set at 25 mL / min, and the eluate is collected according to the column volume of the elution solution. The chromatogram of the rapid preparative liquid chromatography during the separation process is as Figure 1 shown; Collect the separated eluates according to 20 - 40 CV and 40 - 60 CV, transfer them to a round-bottom flask with methanol, and remove the solvent in the sample by concentration under reduced pressure and vacuum drying using a rotary evaporator, as Figure 2 shown, to obtain 430 mg of methionine-enriched cyclic peptide and 400 mg of methionine sulfoxide-enriched cyclic peptide.
[0046] S4. Rapid preparative liquid phase reverse phase separation Dissolve the methionine-enriched cyclic peptide and methionine sulfoxide-enriched cyclic peptide separately in a methanol solution to obtain a sample solution with a concentration of 200 mg / mL.
[0047] Select a medium and low pressure phenyl ethyl reverse phase chromatography column with a specification of 172.7 mm × 21.3 mm and a particle size of 40 - 63 μm. After equilibrating the chromatography column with an acetonitrile-formic acid aqueous solution (where the volume ratio of acetonitrile is 20%), load 2 mL of each of the two sample solutions respectively.
[0048] Use an acetonitrile-water system (the volume ratio of acetonitrile is 30% - 70%) as the elution system solution to elute the chromatography column, set the flow rate to 15 mL / min, start collecting after observing a chromatographic peak at 214 nm, and set the collection frequency to collect one fraction every 5 mL, obtaining 36 fractions each, for a total of 72 fractions. The chromatograms of the rapid preparative liquid phase during the separation process are as Figure 3 and Figure 4 shown.
[0049] Number the obtained fractions, transfer the eluate to the corresponding round-bottom flask with methanol, and remove the solvent in the sample by concentration under reduced pressure and vacuum drying using a rotary evaporator and record the weight.
[0050] S5. Result analysis As Figure 5 shown, after purity determination, the purity of cyclic peptide A, cyclic peptide E, and cyclic peptide O can reach over 90%, and in addition, fractions with an average purity of about 80% can be collected. By calculation, the recovery rates of these three cyclic peptides are: cyclic peptide A (81.5%) 261.1 mg, cyclic peptide E (95.3%) 176.7 mg, cyclic peptide O (37.9%) 21.6 mg.
[0051] Other fractions containing high-purity cyclic peptides are: cyclic peptide C (71.3%) 46.9 mg, cyclic peptide I* (74.8%) 32.7 mg, cyclic peptide F (65.3%) 26 mg, cyclic peptide P (63.7%) 31.6 mg, cyclic peptide L (62.0%) 16 mg.
[0052] The yields of the above-mentioned types of cyclic peptides are as follows: cyclic peptide C (66.8%), cyclic peptide I* (51.3%), cyclic peptide F (17.3), cyclic peptide P (21.5%), cyclic peptide L (7.2%).
[0053] Example 2 S1. Screening of linseed oil Same as Example 1.
[0054] S2. Extraction of mixed cyclic peptides Same as Example 1.
[0055] S3. Normal-phase separation by rapid preparative liquid chromatography Weigh 1 g of the mixed cyclic peptides from linseed and place them in a round-bottom flask. Add 10 mL of dichloromethane solution, stir well, let it stand, and sonicate to fully dissolve the sample, obtaining a sample solution with a concentration of 80 mg / mL; Select a medium-pressure and low-pressure silica gel normal-phase column, and equilibrate the chromatographic column with dichloromethane as the elution system solution; after loading the sample, mobile phase A is dichloromethane, mobile phase B is methanol, and a gradient elution program is adopted. The volume ratio of organic phase B is 0% - 12%, the flow rate is set at 15 mL / min, and the eluate is collected according to the column volume of the elution solution; Collect the eluate after separation according to 20 - 40 CV and 40 - 60 CV, transfer it to a round-bottom flask with methanol, and use a rotary evaporator to remove the solvent in the sample by vacuum concentration and vacuum drying to obtain 581.9 mg of methionine-enriched cyclic peptides and 183.5 mg of methionine sulfoxide-enriched cyclic peptides.
[0056] S4. Reverse-phase separation by rapid preparative liquid chromatography (1) Dissolve the methionine-enriched cyclic peptides and methionine sulfoxide-enriched cyclic peptides separately with methanol solution to obtain sample solutions with a concentration of 100 mg / mL.
[0057] (2) Select a medium-pressure and low-pressure phenyl ethyl reverse-phase chromatographic column with a specification of 184 mm × 21.4 mm and a particle size of 20 - 45 μm. After equilibrating the chromatographic column with an acetonitrile-formic acid aqueous solution (where the volume ratio of acetonitrile is 20%), load 2 mL of each of the two sample solutions respectively.
[0058] (3) Elute the chromatographic column with an acetonitrile-water system (the volume ratio of acetonitrile is 18% - 68%) as the elution system solution, set the flow rate at 15 mL / min, start collecting after observing a chromatographic peak at 214 nm, and set the collection frequency to collect one fraction every 25 mL, obtaining 13 methionine-enriched fractions and 10 methionine sulfoxide-enriched fractions respectively, totaling 23 fractions.
[0059] (4)Number the obtained fractions, transfer the eluate to the corresponding round-bottom flask with methanol, remove the solvent in the sample by rotary evaporation under reduced pressure and vacuum drying, and record the weight.
[0060] S5. Result analysis For the Met fraction, the cyclic peptides with higher purity in each fraction are: cyclic peptide C (82.5%) 22.2 mg, cyclic peptide A (93.3%) 92.2 mg, and cyclic peptide O (92.3%) 14.8 mg; For the MetO fraction, the cyclic peptides with higher purity in each fraction are: cyclic peptide C (73.3%) 12.7 mg, cyclic peptide E (98.5%) 67.7 mg, cyclic peptide I* (88.4%) 24.2 mg, and cyclic peptide A (81.3%) 36.5 mg.
[0061] The yields of the above types of cyclic peptides are: cyclic peptide C (47.6%), cyclic peptide E (73.1%), cyclic peptide I* (75.9%), cyclic peptide A (80.3%), cyclic peptide O (51.9%). Example 3 S1. Screening of linseed oil Select linseed oil with a lower oxidation purity, containing more unoxidized cyclic peptides (containing methionine groups) and a lower content of partially oxidized cyclic peptides (containing methionine sulfoxide groups) as the raw material to extract linseed cyclic peptides.
[0062] S2. Extraction of mixed cyclic peptides Same as Example 1.
[0063] S3. Normal-phase separation by rapid preparative liquid chromatography Same as Example 1.
[0064] S4. Reverse-phase separation by rapid preparative liquid chromatography (1)Dissolve the methionine-enriched cyclic peptide and methionine sulfoxide-enriched cyclic peptide in methanol solution respectively to obtain a sample solution with a concentration of 100 mg / mL.
[0065] (2)Select a medium- and low-pressure phenyl ethyl reverse-phase chromatographic column with a specification of 134.8 mm × 21.4 mm and a particle size of 40 - 63 μm. After equilibrating the chromatographic column with an acetonitrile-formic acid aqueous solution as the elution system solution (where the volume ratio of acetonitrile is 20%), load 2 mL of each of the two sample solutions respectively.
[0066] (3) Use an acetonitrile-water system (the volume ratio of acetonitrile is 20% - 70%) as the elution system solution to elute the chromatographic column. Set the flow rate to 15 mL / min. Start collecting after observing a chromatographic peak at 214 nm. Set the collection frequency to collect one fraction every 15 mL. After the Met fraction and the MetO fraction are separated by a phenylhexyl stationary phase, 16 fractions and 10 fractions are obtained respectively, for a total of 26 fractions.
[0067] (4) Number the obtained fractions, transfer the eluate to the corresponding round-bottom flask with methanol, and use a rotary evaporator to remove the solvent in the sample by reduced pressure concentration and vacuum drying, and record the weight.
[0068] S5. Result analysis For the Met fraction, the cyclic peptides with relatively high purity in each fraction are: cyclic peptide C (68.5%) 6.9 mg, cyclic peptide A (90.1%) 66.9 mg, cyclic peptide B (83.3%) 31.1 mg, cyclic peptide P (78.6%) 19.9 mg, and cyclic peptide O (94.3%) 24.3 mg.
[0069] For the MetO fraction, the cyclic peptides with relatively high purity in each fraction are: cyclic peptide F 6.3 mg, cyclic peptide C (69.3%) 15.5 mg, cyclic peptide E (93.5%) 47.6 mg, cyclic peptide I* (66.6%) 20.9 mg, and cyclic peptide A (90.3%) 40.7 mg.
[0070] The yields of the above types of cyclic peptides are: cyclic peptide F (20.4%), cyclic peptide C (30.6%), cyclic peptide E (51.4%), cyclic peptide I* (64.6%), cyclic peptide B (40.5%), cyclic peptide A (67.1%), cyclic peptide P (63.1%), cyclic peptide O (85.2%).
[0071] The above is a further detailed description of the present invention, and it should not be regarded as a limitation to the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention pertains, any simple deduction or replacement without departing from the concept of the present invention falls within the protection scope of the present invention.
Claims
1. A method for separating and purifying linseed cyclic peptides from linseed oil, characterized in that, It includes the following steps: 1) Use silica column elution method for flaxseed oil raw materials to extract crude flaxseed cyclopeptides; 2) Perform rapid preparative liquid chromatography normal-phase separation on the flaxseed mixed cyclopeptides obtained in step 1). The stationary phase is silica, the UV detector wavelength ranges are 214 nm and 280 nm. Adopt gradient elution method, collect the eluent according to the column volume CV of the elution solution. The Met part containing methionine cyclopeptide is collected before 40CV, and the MetO part containing methionine sulfoxide is collected after 40CV to enrich cyclopeptides; 3) Perform reverse-phase separation on the two-component cyclopeptides of the Met part and the MetO part obtained in step 2) respectively using rapid preparative liquid chromatography. The stationary phase is Phenyl-hexyl, adopt stepped gradient elution, and the UV detector wavelength ranges are 214 nm and 280 nm; 4) Determine the purity of the fractions collected in step 3), and combine the fractions containing the same monomer and with a purity higher than 60% to obtain flaxseed cyclopeptides.
2. The method according to claim 1, wherein In step 2), the mobile phase A for gradient elution is dichloromethane, and the mobile phase B is methanol.
3. The method according to claim 1 or 2, characterized in that, By volume percentage, in step 2) during gradient elution, the proportion of mobile phase A is 100% - 70%.
4. The method according to claim 1, wherein In step 2), the flow rate of gradient elution is 10 - 30 mL / min.
5. The method according to claim 1, characterized in that In step 3), the mobile phase A for stepped gradient elution is water, and the mobile phase B is acetonitrile.
6. The method according to claim 1 or 5, characterized in that, By volume percentage, the method of stepped gradient elution in step 3) is: increase the mobile phase B by 5% within 3 CV each time, and then hold for 3 CV, and cycle the above operations.
7. The method according to claim 6, characterized in that, By volume percentage, in step 3) during stepped gradient elution, the proportion of mobile phase B is 15% - 80%.
8. The method according to claim 1, wherein In step 3), the flow rate of stepped gradient elution is 10 - 40 mL / min.
9. The method according to claim 1, wherein In step 2), the chromatographic column selected for liquid chromatography normal-phase separation is an amorphous silica normal-phase column.
10. The method according to claim 1, wherein In step 3), the collection frequency is 5 - 25 mL / fraction.