A selenium-enriched pine pollen peptide with DPP-IV inhibitory activity and its application

Abstract

The present invention belongs to the technical field of food-derived active peptide development, and specifically relates to a selenium-enriched pine pollen peptide with DPP-IV inhibitory activity and its application. A plurality of pine pollen peptides with DPP-IV inhibitory activity were obtained by preparing, enzymolysis, extraction, separation and purification, and identification of selenium-enriched pine pollen protein, among which EMPKRIALP and MLRDYITREYIE had high inhibition rates. The DPP-IV selenium-enriched pine pollen inhibitory peptide in the present invention can be used for the development of health foods and medicines for the purpose of lowering blood sugar.

Description

Technical Field

[0001] The invention belongs to the technical field of development of food-derived active peptides, and particularly relates to a selenium-enriched pine pollen peptide with DPP-IV inhibitory activity and an application thereof. Background Art

[0002] Pine pollen is a commonly used Chinese medicine. It is the dried pollen of Pinus massoniana, Pinus tabulaeformis or several plants of the same genus. Pine pollen contains a large number of nutrients and bioactive substances, including more than 100 ingredients such as protein, amino acids, vitamins, minerals, nucleic acids and lecithin. Pine pollen is a pure natural material used for both medicine and food. It can delay aging, resist fatigue, lower blood lipids, and enhance human immunity. It has a high development and utilization value.

[0003] Selenium is an essential trace element for the human body. In nature, selenium exists in two states: inorganic selenium and organic selenium. Organic selenium generally exists in the form of selenomethionine, which is metabolized by the methionine metabolic pathway, participates in protein synthesis, and is easily stored and absorbed in tissues. After being absorbed by the human body, it can be quickly used by the human body, effectively improving the blood selenium status in the human body. The average content of selenium in an adult is 0.25 mg / kg, and the human body should not take less than 50 μg / day of selenium. The latest research results of American scientists believe that healthy people can prevent the occurrence of tumors to a certain extent by supplementing 200 μg / day of selenium or a little more. However, the maximum safe intake of selenium is 400 μg / day, and too much will cause poisoning. In addition, selenium also has the functions of regulating immunity, resisting viruses such as HIV, and protecting the brain. However, the selenium content in natural foods is generally low, and relying solely on food to take in selenium to maintain life and movement is far from meeting the needs of health. However, excessive intake of selenium can produce acute and chronic toxic effects on the body, and at the same time, there are many toxic and side effects such as the genetic toxicity of selenium (sodium selenite).

[0004] Dipeptidyl peptidase IV (DPP-IV) is an important target for regulating blood sugar. In the human body, DPP-IV has the function of degrading glucagon-like peptide-1 (GLP-1), which hinders GLP-1 from exerting its activity in lowering blood sugar, thus causing blood sugar to lose control. Therefore, the research and development of DPP-IV inhibitors has become an important way to treat type 2 diabetes. The blood sugar regulating effect of active peptides derived from grain protein has been proven to be closely related to the inhibitory activity of DPP-IV enzyme.

[0005] Food-borne DPP-IV inhibitory peptides mainly come from animals, plants and dairy products. The IC50 of the DPP-IV inhibitory peptide DTMYDT identified from sardine protein was 1.83 mg / mL; the IC50 of the DPP-IV inhibitory peptide LDKVFR from Atlantic salmon skin protein was 128.71 μmol / L. The peptides screened from dairy products all have high DPP-IV inhibitory activity. The IC50 of the DPP-IV inhibitory peptide ILDKVGINY identified from bovine α-lactalbumin was 1.0 mg / mL. In particular, the most active DPP-IV inhibitory peptide in casein is IPI, with an IC50 of 3.2-7.40 μmol / L. Compared with animal protein, plant protein has a wider source, is economical, has a short growth cycle, and is a renewable energy source. It is a high-quality source of food-borne DPP-IV inhibitory peptides.

[0006] Although some plant-derived inhibitory peptides have been reported to have DPP-IV inhibitory activity, such as the IC50 of the DPP-IV inhibitory peptide mixture screened from quinoa protein is 0.23 mg / mL-0.98 mg / mL, and the IC50 of the most active HPF purified is as low as 13.69 μg / mL; the IC50 of the DPP-IV inhibitory peptide mixture screened from oat protein is 0.68 mg / mL-0.99 mg / mL, and the IC50 of the most active LQAFEPLR purified is as low as 103.5 μmol / L. However, it is not clear whether pine pollen protein and its active peptides have lipid-lowering activity. This application uses pine pollen as the main source to screen and purify pine pollen peptides with DPP-IV inhibitory activity. Summary of the invention

[0007] In view of the deficiencies of the prior art and actual needs, the purpose of the present invention is to obtain pine pollen peptides with DPP-IV inhibitory activity for the development of drugs for the purpose of lowering blood sugar.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] The invention provides a pine pollen peptide with DPP-IV inhibitory activity. The amino acid sequence of the polypeptide is: EMPKRIALP, as shown in SEQ ID NO.4.

[0010] The present invention also provides a pine pollen peptide having DPP-IV inhibitory activity, wherein the amino acid sequence of the polypeptide is: MLRDYITREYIE, as shown in SEQ ID NO.6.

[0011] The pine pollen peptide is prepared by the following method:

[0012] (1) Preparation of pine pollen protein: Pine pollen and petroleum ether are mixed to obtain defatted pine pollen, which is then subjected to pH adjustment, dialysis, desalination, and drying to obtain pine pollen protein;

[0013] (2) Pine pollen protein hydrolysis: 4% compound enzyme was used to hydrolyze 2% pine pollen protein at pH 7;

[0014] (3) Extraction of pine pollen peptide complex: boil the solution after reaction in (2) to inactivate the protease, collect the supernatant by centrifugation, and obtain the crude extract of pine pollen peptide;

[0015] (4) Isolation and purification of pine pollen peptides: Ultrafiltration and liquid chromatography separation and purification, combined with Nano LC-MS / MS sequencing, were used to determine the composition of pine pollen peptides.

[0016] The specific preparation method is:

[0017] (1) Obtaining pine pollen: Take 20-50 grams of selenium-rich foliar fertilizer, add 40-150 kilograms of water, and spray it on the trunks, branches and crowns of 15-25 year old Masson pine trees before 8-10 am or 2-6 pm on a windless day. When the Masson pine is in bloom, selenium-rich pine pollen can be obtained after picking, drying, and removing impurities.

[0018] (2) Pine pollen protein extraction: Place pine pollen in an air flow wall breaking machine for low-temperature wall breaking treatment, sieve the product with a mesh size of 100-300 mesh, add purified water according to a ratio of 1: (2-10), add (0.01-0.5)% by weight of cellulase and (0.01-0.5)% by weight of α-amylase, heat to (20-37)℃±5℃, react for 2-10h for wall breaking treatment. Preferably, the low-temperature wall breaking temperature is 5-20℃, preferably, the wall breaking temperature is 10-15℃

[0019] Subsequently, the broken cell product was mixed with petroleum ether at a solid-liquid ratio of 1:1, and slowly stirred at room temperature for 0.5-2h (less than 600rpm). After vacuum filtration and drying, defatted pine pollen was obtained, and then added to water at (5-20)%, the pH was adjusted to 7-13, stirred for 1h, and centrifuged at 6000 rpm for 5 min to obtain a supernatant protein solution. The pH of the supernatant protein solution was adjusted to 4.5, and centrifuged at 6000 rpm for 5 min to collect the pine pollen protein precipitate. The precipitate was re-dissolved in water, the pH was adjusted to 7, and the salt ions were removed by dialysis for 24h, and then dried to obtain pine pollen protein.

[0020] (3) Pine pollen protein hydrolysis: Add pine pollen protein to ddH2O to a weight ratio of 1-5%, and place in a boiling water bath for 5 minutes to denature the protein to assist enzymatic hydrolysis; add 2-10% complex enzyme (the complex protease consists of neutral protease, papain and flavor protease, and the mass ratio of the three is 1: (1-2): 1) at a temperature of 50℃-65℃ and perform enzymatic hydrolysis for 1-4 hours.

[0021] (4) Pine pollen peptide complex extract: The pine pollen protein hydrolysate was fully stirred, boiled for 10 min to inactivate the enzyme, and centrifuged at 10,000 rpm for 10 min. The supernatant of the hydrolysate was collected as the crude pine pollen peptide extract. A portion of the crude pine pollen peptide extract was freeze-dried for activity detection.

[0022] (5) Isolation and purification of pine pollen peptides: The pine pollen peptide components were separated according to their size using an ultrafiltration membrane pore size. The crude pine pollen peptide extract was filtered through a 0.45 μM water membrane. After refrigerated centrifugation (8000 rpm, 4°C, 30 min), the pine pollen peptides were retained and separated into components >10 kDa, 3-10 kDa, and <3 kDa. The DPP-IV inhibitory activity was determined.

[0023] It also includes a sequencing step, using liquid chromatography for further purification, screening out DPP-IV inhibitory peptides with higher activity, and clarifying the amino acid sequence of its specific peptide segment. It includes using Nano LC-MS / MS to sequence the highly active pine pollen purified peptides, and using the BLAST® tool to clarify the pine pollen polypeptide sequence.

[0024] The present invention also provides the use of the two pine pollen peptides or their complexes or their synergistic use in different proportions in regulating the activity of DPP-IV protease.

[0025] The present invention also provides the use of the two pine pollen peptides or their complexes or their synergistic use in different proportions in the preparation of drugs for preventing or assisting in the treatment of diseases that benefit from DPP-IV inhibition.

[0026] The present invention also provides application of the pine pollen peptide in preparing medicines.

[0027] The activity detection method of the DPP-IV inhibitory peptide in the present invention is:

[0028] In a 96-well ELISA plate, 25 μL of peptide sample (dissolved in pH 8.0, 100 mmol / L Tris-HCl buffer) was mixed with 25 μL Gly-Pro-PNA (1.6 mmol / L), incubated at 37°C for 10 min, 50 μL DPP-Ⅳ (8 U / L) solution was added to start the reaction, the reaction was carried out at 37°C for 60 min, and then 100 μL sodium acetate buffer (1 mol / L, pH 4.0) was added to terminate the reaction, and the absorbance was measured at 405 nm. Tris-HCl buffer was used instead of DPP-Ⅳ solution as the blank group, and Tris-HCl buffer was used instead of peptide sample as the control group. The DPP-Ⅳ inhibition rate was calculated as follows:

[0029]

[0030] Among them, Ac, Ab, and As are the absorbance of the control group, blank group, and sample group, respectively. Diprotin A was used as a control.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) DPP-IV inhibitory peptides from selenium-enriched pine pollen were obtained by enzymatic hydrolysis and separation. The peptide sequences with the highest DPP-IV inhibitory activity were H4:EMPKRIALP and H6:MLRDYITREYIE, with IC50 values ​​of 1.7632 mM (1.859 mg / mL) and 1.567 mM (2.510 mg / mL), respectively.

[0033] (2) The above-mentioned DPP-IV inhibitory peptide can be used in the development of drugs for the purpose of lowering blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 DPP-IV inhibition rate of ultrafiltrated pine pollen peptide components of different molecular weights.

[0035] Figure 2 DPP-IV inhibition rate of pine pollen peptide components after chromatographic separation and purification. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not used to limit the present invention.

[0037] Selenium-rich foliar fertilizer: 1.5-2 parts of iron sulfate, 2.5-3 parts of boron fertilizer (borax), 10-20 parts of amino acid powder, 10-15 parts of biogas sludge, 1-2 parts of 6% sodium selenite solution, 5-8 parts of penetrant, 5-10 parts of isopentenyl adenine, 10-15 parts of sodium nitrophenolate, 3-5 parts of distilled water, and the rest is water.

[0038] Example 1: Preparation of pine pollen peptides with DPP-IV inhibitory activity

[0039] (1) How to obtain pine pollen: Take 35 grams (one bag) of selenium-rich foliar fertilizer, add 80-100 kilograms of water, and spray it on the trunks, branches and crowns of 15-25 year old Masson pine trees before 9 am or 4 pm on a windless day. When the Masson pine is in bloom, selenium-rich pine pollen can be obtained after picking, drying, and removing impurities.

[0040] (2) Pine pollen protein extraction: The above pine pollen was placed in an air flow wall breaking machine at 10-15°C for low-temperature wall breaking treatment, the sieve mesh number was 200 mesh, and the sieved product was added with purified water at a ratio of 1:5, and 0.1% by weight of cellulase and 0.1% by weight of α-amylase were added. The temperature was raised to 30°C±5°C and the wall breaking treatment was carried out for 6 hours.

[0041] Then, the broken cell product was mixed with petroleum ether at a solid-liquid ratio of 1:1, and slowly stirred at room temperature for 1 h (less than 600 rpm). After vacuum filtration and drying, defatted pine pollen was obtained, and then added to water at 10%, the pH was adjusted to 7-13, stirred for 1 h, and centrifuged at 6000 rpm for 5 min to obtain a supernatant protein solution. The pH of the supernatant protein solution was adjusted to 4.5, centrifuged at 6000 rpm for 5 min, and then the pine pollen protein precipitate was collected. The precipitate was re-dissolved with water, the pH was adjusted to 7, and the salt ions were removed by dialysis for 24 h, and then dried to obtain pine pollen protein.

[0042] (3) Pine pollen protein hydrolysis: Add pine pollen protein to ddH2O to a weight ratio of 2%, and boil in a boiling water bath for 5 min to denature the protein and assist enzymatic hydrolysis; add 4% complex enzyme (the complex protease consists of neutral protease, papain and flavor protease, with a mass ratio of 1:(1-2):1) at a temperature of 50℃-65℃, and perform enzymatic hydrolysis for 2 hours.

[0043] (4) Pine pollen peptide complex extract: The pine pollen protein hydrolysate was fully stirred, boiled for 10 min to inactivate the enzyme, and centrifuged at 10,000 rpm for 10 min. The supernatant of the hydrolysate was collected as the crude pine pollen peptide extract. A portion of the crude pine pollen peptide extract was freeze-dried for activity detection.

[0044] (5) Isolation and purification of pine pollen peptides: The pine pollen peptide components were separated according to their size using ultrafiltration membrane pore size. The crude pine pollen peptide extract was filtered through a 0.45 μM water membrane. After refrigerated centrifugation (8000 rpm, 4°C, 30 min), the pine pollen peptides were retained and separated into components >10 kDa, 3-10 kDa, and <3 kDa. The DPP-IV inhibitory activity was determined according to the above method.

[0045] Depend on Figure 1 The results showed that pine pollen protein had no DPP-IV inhibitory activity; compared with the crude pine pollen peptide extract without ultrafiltration separation treatment, the DPP-IV inhibition rate of the <3 kDa component increased significantly, from 27.34% to 42.52%; the DPP-IV inhibitory activity of the 3-10 kDa and >10 kDa components was significantly lower than that of the <3 kDa component, indicating that the pine pollen DPP-IV inhibitory peptides were aggregated in the components with a molecular weight of <3 kDa; ultrafiltration separation treatment effectively screened out pine pollen peptide components with smaller molecular weight and higher DPP-IV inhibitory activity.

[0046] Example 2 Further purification of pine pollen peptide

[0047] The component <3 kDa with the highest DPP-IV inhibition rate in the above-mentioned pine pollen peptide isolate was further purified by liquid chromatography to screen out DPP-IV inhibitory peptides with higher activity and to determine the amino acid sequence of its specific peptide segment.

[0048] Liquid chromatography separation conditions: mobile phase A is ultrapure water with 0.1% trifluoroacetic acid, mobile phase B is acetonitrile with 0.1% trifluoroacetic acid, injection volume is 50 μL, flow rate is 1 mL / min. Gradient elution was used: mobile phase 0 min, 5% B; 5 min, 10% B; 22 min, 20% B, 24 min, 60% B; 25 min, 5% B; 30 min, 5% B. The collected peptides were subjected to DPP-IV inhibition experiments, and the inhibitory activity of each component on DPP-IV was compared.

[0049] Nano LC-MS / MS was used to collect 15 pine pollen purified peptide separation peaks and sequenced them separately. Pine pollen purified peptides were chemically synthesized based on the sequencing results, and DPP-IV inhibitory activity was detected. 31.25ug of the synthesized pine pollen purified peptide was accurately weighed and dissolved in 25ul of the test system (1.25mg / ml), and the detection method was used as above. The inhibition rate is described in Table 1.

[0050] Table 1 DPP-IV inhibitory activity and sequencing results of 15 pine pollen purified peptides

[0051]

[0052] The DPP-IV inhibitory activity of the 6 pine pollen peptides with stronger DPP-IV inhibitory activity was detected by mixing the components <3 kDa before purification (1.25 mg / ml) and plotted. Figure 2 As shown, the inhibitory activity of the separated H2, H3, H4, and H6 against DPP-IV was significantly higher than that of the mixed components <3 kDa before purification, indicating that purification can screen out pine pollen peptide components with weaker DPP-IV inhibitory activity.

[0053] The sequencing results of the six pine pollen peptides with the strongest DPP-IV inhibitory activity are shown in Table 2. The highest DPP-IV inhibition rates are H4:EMPKRIALP and H6:MLRDYITREYIE. The H4 and H5 pine pollen purified peptides with similar molecular weights have quite different inhibition rates on DPP-IV. H4 has the highest inhibition rate and H5 has the lowest activity among the six pine pollen purified peptides. H5, on the other hand, has a DPP-IV inhibitory activity close to that of H1. This shows that molecular weight is not the only criterion for measuring the DPP-IV inhibitory activity of pine pollen peptides. The activity of functional peptides depends more on their complex spatial conformation and the binding site with the DPP-IV enzyme to determine their inhibitory activity.

[0054] Table 2 Pine pollen peptide sequences with the highest DPP-IV inhibitory activity

[0055]

[0056] Note: The amino acid abbreviations in the sequence are: Q-glutamine (Gln); M-methionine (Met); P-proline (Pro); E-glutamic acid (Glu); K-lysine (Lys); R-arginine (Arg); L-leucine (Leu); S-serine (Ser); I-isoleucine (Ile); H-histidine (His); V-valine (Val); G-glycine (Gly); D-aspartic acid (Asp); N-asparagine (Asn); T-threonine (Thr); A-alanine (Ala); F-phenylalanine (Phe); E-glutamic acid (Glu); Y-tyrosine (Tyr).

[0057] In summary, the present invention obtains functional short peptides EMPKRIALP and MLRDYITREYIE with high DPP-IV inhibitory activity by extracting pine pollen polypeptides and enzymatic separation. After the synthesis of the two peptide segments, the DPP-IV inhibitory activity was verified, and the IC50 of EMPKRIALP was 1.7632 mM (1.859 mg / mL), and the IC50 of MLRDYITREYIE was 1.567 mM (2.510 mg / mL).

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pine pollen peptide having DPP-IV inhibitory activity, characterized in that: The amino acid sequence of the pine pollen peptide is: EMPKRIALP, as shown in SEQ ID NO.

4.

2. A pine pollen peptide having DPP-IV inhibitory activity, characterized in that: The amino acid sequence of the pine pollen peptide is: MLRDYITREYIE, as shown in SEQ ID NO.

6.

3. A composition, characterized in that Comprising the pine pollen peptide according to claim 1 and / or claim 2.

4. The composition according to claim 3, characterized in that It also includes stabilizers, emulsifiers, and flavoring agents.

5. Use of the pine pollen peptide according to claim 1 or 2 in the preparation of a DPP-IV protease activity inhibitor product.

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