An α-glucosidase inhibitory peptide derived from ginkgo seeds and its screening method

Abstract

The present invention discloses an α-glucosidase inhibitory peptide derived from Ginkgo biloba seeds and a screening method thereof. The α-glucosidase inhibitory peptide is prepared from Ginkgo biloba seeds by enzymatic hydrolysis with Alcalase protease and screened by combining PeptideRanker, Pepsite 2 and molecular docking software. Its amino acid sequences are Leu-Ser-Met-Ser-Phe-Pro-Pro-Phe (LSMSFPPF), Val-Pro-Lys-Ile-Pro-Pro-Pro (VPKIPPP) and Met-Pro-Gly-Pro-Pro-Ser-Asp (MPGPPSD). Taking the inhibitory activity against α-glucosidase as an index, the IC 50 values of LSMSFPPF, MPGPSD and VPKIPPP are 0.42 mg / mL, 0.66 mg / mL and 1.08 mg / mL respectively. The present invention not only enriches the α-glucosidase inhibitory peptide library, but also provides a feasible strategy for efficiently screening bioactive polypeptides from food matrices.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide technology, specifically relating to an α-glucosidase inhibitory peptide derived from ginkgo and its rapid screening method. Background Technology

[0002] Type 2 diabetes is a metabolic disease characterized by hyperglycemia caused by insulin resistance, often accompanied by metabolic disorders of carbohydrates, proteins, and fats. Long-term hyperglycemia and metabolic disturbances can further induce systemic organ damage, including damage to the kidneys, cardiovascular system, eyes, nervous system, and skin infections. Therefore, lowering blood glucose levels within a scientifically sound framework plays a positive role in the prevention and treatment of diabetes. Inhibiting alpha-glucosidase activity is considered an effective method for treating type 2 diabetes. Clinically, most commonly used alpha-glucosidase inhibitors are chemically synthesized drugs. While these drugs are highly effective, long-term use can cause adverse reactions and side effects such as liver damage, myocardial infarction, edema, and anemia. Therefore, screening for safe, non-toxic natural active ingredients with alpha-glucosidase inhibitory activity has been a major focus of researchers.

[0003] Food-derived α-glucosidase inhibitory peptides have attracted widespread attention from researchers due to their natural, safe, and efficient characteristics. However, traditional peptide preparation processes involve a series of complex workflows, such as protein extraction and hydrolysis, separation of hydrolysates, and characterization and identification of active peptides. Furthermore, the yield of active peptides is generally low after separation and purification processes. This is detrimental to subsequent activity studies and industrial production.

[0004] The emergence of virtual screening technology has provided a new approach for the discovery of bioactive peptides. Virtual screening is a method for rapidly screening target bioactive molecules from a complex database by evaluating the interaction between ligands and receptors. This method has been widely used to screen and discover natural bioactive molecules. Ginkgo biloba, a traditional Chinese food, is rich in bioactivity and high in protein. However, there are no reports on using virtual screening technology to screen α-glucosidase inhibitory peptides from ginkgo biloba.

[0005] While existing technologies, such as Chinese patents with publication numbers CN102086465A and CN108486201A, report on ginkgo biloba hydrolysates, they focus on using enzymatic methods to obtain ginkgo polypeptides, without describing the activity of these polypeptides. Similarly, Chinese patent CN107418990A, although describing the inhibition of α-glucosidase activity by ginkgo polypeptides, does not provide the specific α-glucosidase inhibitory peptide sequence. Summary of the Invention

[0006] The purpose of this invention is to fill the gap in the specific α-glucosidase inhibitory peptide sequence in Ginkgo biloba and to develop a rapid screening method for Ginkgo biloba polypeptides with high efficiency in inhibiting α-glucosidase, providing a feasible strategy to solve the problems of complex processes and low yields in traditional polypeptide preparation.

[0007] To achieve the above objectives, the present invention employs the following method.

[0008] 1. Defatted Ginkgo Biloba Powder

[0009] The dried ginkgo biloba powder was added to n-hexane at a material-to-liquid ratio of 1:20 (g / mL), and the mixture was repeatedly extracted and defatted at 4℃. After drying at 45℃, defatted ginkgo biloba powder was obtained.

[0010] 2. Preparation of Ginkgo Biloba Protein Isolate

[0011] Add deionized water to defatted ginkgo powder at a material-to-liquid ratio of 1:50-100 (g / mL), mix thoroughly, adjust the pH of the system to 10.0 with 1 mol / L NaOH solution, and extract at 1000 rpm for 2-4 hours at room temperature. After extraction, centrifuge the extract at 8000g for 20 minutes to obtain the supernatant. Repeat the extraction twice. Adjust the pH of the supernatant to 4.62 with 1 mol / L HCl, let it stand at 4℃ for 2 hours, centrifuge at 8000g for 20 minutes, collect the precipitate, wash the precipitated protein repeatedly with deionized water 3 times, adjust the pH to 7.4, dialyze, and freeze-dry to obtain ginkgo protein isolate.

[0012] 3. Enzymatic hydrolysis of ginkgo protein isolate

[0013] A certain amount of ginkgo protein isolate was added to distilled water at a material-to-liquid ratio of 1:100-200 (g / mL). After stirring for 0.5-1 h, Alcalase protease was added to the system at an enzyme-to-protein ratio of 4000 U / g. The ginkgo protein isolate was enzymatically hydrolyzed at pH 8.0-9.0 and a temperature of 50℃ for 4-6 h. After the reaction was completed, the reaction system was heated in a water bath at 90℃ for 10 min to inactivate the protease. After cooling to room temperature, the hydrolysate was centrifuged at 8000g for 20 min, the supernatant was collected, and the ginkgo protein hydrolysate was obtained by freeze-drying.

[0014] 4. Ultrafiltration separation and structural analysis of ginkgo peptides

[0015] Ginkgo biloba protein hydrolysate was redissolved in distilled water at a ratio of 1:10-20 (g / mL). Five different molecular weight fractions with molecular weight cutoffs of <1000 Da, 1000-3000 Da, 3000-5000 Da, 5000-10000 Da, and >10000 Da were then retained using an ultrafiltration membrane. The fraction with molecular weight cutoff of <1000 Da was freeze-dried.

[0016] Peptide fractions with molecular weight <1000 Da were desalted, and their structural characteristics, including molecular weight, amino acid sequence, and composition, were determined by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). The separation conditions were as follows: a 3C18-CL-120 column with a pore size of 3-5 μm; mobile phase A was 2% acetonitrile and 0.1% trifluoroacetic acid aqueous solution; mobile phase B was 98% acetonitrile and 0.1% trifluoroacetic acid aqueous solution; the elution mode was gradient elution: 5% B at 0 min, 8% B at 1 min, 38% B at 51 min, 80% B at 52 min, 80% B at 55 min, 5% B at 56 min, and 5% B at 60 min; the relative molecular mass scan range for mass spectrometry was 100-1500; the scan mode was positive ion mode; and the Protein Pilot analysis tool was used to search the database to resolve the amino acid sequence.

[0017] 5. Screening of bioactive peptides

[0018] The above peptides were scored using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker), and potential active peptide sequences with scores greater than 0.5 were screened.

[0019] 6. Screening of α-glucosidase inhibitory peptides

[0020] Using Pepsite 2 (http: / / pepsite2.russelllab.org / ), the aforementioned potentially active peptides were docked with α-glucosidase (PDB ID: 3WY1) for evaluation. Peptide sequences with a P-value less than 0.05 were further screened, and molecular docking software was used to further verify the docking of these peptides. When a peptide interacts with α-glucosidase, it can be considered an α-glucosidase inhibitory peptide.

[0021] The amino acid sequences of the α-glucosidase inhibitory peptides obtained by screening were Leu-Ser-Met-Ser-Phe-Pro-Pro-Phe (LSMSFPPF), Val-Pro-Lys-Ile-Pro-Pro-Pro (VPKIPPP), and Met-Pro-Gly-Pro-Pro-Ser-Asp (MPGPPSD).

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention uses ginkgo as raw material. After defatting with n-hexane, the protein in ginkgo is extracted using an alkaline extraction and acid precipitation method. Then, it is enzymatically hydrolyzed with Alcalase protease. The hydrolysate is separated by ultrafiltration and further screened using ultra-high performance liquid chromatography-mass spectrometry, PeptideRanker, Pepsite 2, and molecular docking software to obtain α-glucosidase inhibitory peptides. Traditional methods for preparing peptides, from protein extraction, hydrolysis, peptide separation and purification to obtaining the specific peptide sequence, require at least three months or even longer. This process is cumbersome and time-consuming. This method uses PeptideRanker, Pepsite 2, and molecular docking software to screen for α-glucosidase inhibitory peptides, reducing the time required to obtain the peptide sequence to 30%, significantly saving time. Furthermore, the inhibitory effects of LSMSFPPF, VPKIPPP, and MPGPPSD on α-glucosidase (IC50) are shown in the figure. 50 The concentrations of 0.42 mg / mL, 1.08 mg / mL, and 0.66 mg / mL, respectively, are superior to those of the peptide (GEY(IC)) previously reported in the literature. 50 2.70 mg / mL), GYG (IC) 50 1.50 mg / mL) and PFP (IC 50 3.10 mg / mL))(Lee, HJ, Lee, HS, Choi, JW, Ra, KS, Kim, JM, & Suh, HJ (2011). Novel tripeptides with alpha-glucosidase inhibitory activity isolated from silk cocoon hydrolysate. Journal of Agricultural and Food Chemistry, 59(21), 11522-11525; Kang, MG, Yi, SH, Lee, JS (2013). Production and characterization of a new α-glucosidase inhibitorypeptide from Aspergillus oryzae N159-1, Mycobiology, 41:3, 149-154). This method not only realizes the high added value application of ginkgo, but also has high efficiency and low cost. Attached Figure Description

[0024] Figure 1 Pepsite 2 molecular docking score of peptide with α-glucosidase (PDB ID: 3WY1);

[0025] Figure 2 A two-dimensional schematic diagram of the interaction between VPKIPPP and the non-specific site of α-glucosidase (PDB ID: 3WY1);

[0026] Figure 3 A two-dimensional schematic diagram of the interaction between LSMSFPPF and the nonspecific site of α-glucosidase (PDB ID: 3WY1);

[0027] Figure 4 A two-dimensional schematic diagram of the interaction between MPGPPSD and the nonspecific site of α-glucosidase (PDB ID: 3WY1). Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0029] Example 1

[0030] 1. Defatted Ginkgo Biloba Powder

[0031] The dried ginkgo biloba powder was added to n-hexane at a material-to-liquid ratio of 1:20 (g / mL), and the mixture was repeatedly extracted and defatted at 4°C to obtain defatted ginkgo biloba powder.

[0032] 2. Preparation of Ginkgo Biloba Protein Isolate

[0033] Add deionized water to defatted ginkgo powder at a material-to-liquid ratio of 1:50-100 (g / mL), mix thoroughly, adjust the pH of the system to 10.0 with 1 mol / L NaOH solution, and extract at 1000 rpm for 2-4 hours at room temperature. After extraction, centrifuge the extract at 8000g for 20 minutes to obtain the supernatant. Repeat the extraction twice. Adjust the pH of the supernatant to 4.62 with 1 mol / L HCl, let it stand at 4℃ for 2 hours, centrifuge at 8000g for 20 minutes, collect the precipitate, wash the precipitated protein repeatedly with deionized water 3 times, adjust the pH to 7.4, dialyze, and freeze-dry to obtain ginkgo protein isolate.

[0034] 3. Enzymatic hydrolysis of ginkgo protein isolate

[0035] A certain amount of ginkgo protein isolate was added to distilled water at a material-to-liquid ratio of 1:100-200 (g / mL). After stirring for 0.5-1 h, Alcalase protease was added to the system at an enzyme-to-protein ratio of 4000 U / g. The ginkgo protein isolate was enzymatically hydrolyzed at pH 8.0-9.0 and a temperature of 50℃ for 4-6 h. After the reaction was completed, the reaction system was heated in a water bath at 90℃ for 10 min to inactivate the protease. After cooling to room temperature, the hydrolysate was centrifuged at 8000g for 20 min, the supernatant was collected, and the ginkgo protein hydrolysate was obtained by freeze-drying.

[0036] 4. Ultrafiltration separation and structural analysis of ginkgo peptides

[0037] Ginkgo biloba protein hydrolysate was redissolved in distilled water at a ratio of 1:10-20 (g / mL). Five different molecular weight fractions with molecular weight cutoffs of <1000 Da, 1000-3000 Da, 3000-5000 Da, 5000-10000 Da, and >10000 Da were then retained using an ultrafiltration membrane. The fraction with molecular weight cutoff of <1000 Da was freeze-dried.

[0038] After desalting, peptide fractions with molecular weight <1000 Da were analyzed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) to determine their structural characteristics, including molecular weight, amino acid sequence, and composition. The separation conditions were as follows: a 3C18-CL-120 column with a pore size of 3-5 μm; mobile phase A consisted of 2% acetonitrile and 0.1% trifluoroacetic acid aqueous solution; mobile phase B consisted of 98% acetonitrile and 0.1% trifluoroacetic acid aqueous solution; the elution mode was gradient elution: 5% B at 0 min, 8% B at 1 min, 38% B at 51 min, 80% B at 52 min, 80% B at 55 min, 5% B at 56 min, and 5% B at 60 min; the relative molecular mass scan range for mass spectrometry was 100-1500, and the scan mode was positive ion mode; the Protein Pilot analysis tool was used to search databases to resolve the amino acid sequence.

[0039] 5. Screening of bioactive peptides

[0040] The peptides were scored using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker), and potential active peptide sequences with scores higher than 0.5 were screened. The peptide sequences are shown in Table 1.

[0041] Table 1. PeptideRanker scores for peptides

[0042]

[0043]

[0044] 6. Screening of α-glucosidase inhibitory peptides

[0045] Using Pepsite 2 (http: / / pepsite2.russelllab.org / ), the above-mentioned potentially active peptides were docked with α-glucosidase (PDB ID: 3WY1) for evaluation. Peptide sequences with a P-value less than 0.05 were screened, and the results are shown in [Figure number missing]. Figure 1 Finally, the peptides obtained from the screening were further validated using molecular docking software. When a peptide interacts with α-glucosidase, it can be considered an α-glucosidase inhibitory peptide. The amino acid sequences of the aforementioned α-glucosidase inhibitory peptides are Leu-Ser-Met-Ser-Phe-Pro-Pro-Phe (LSMSFPPF), Val-Pro-Lys-Ile-Pro-Pro-Pro (VPKIPPP), and Met-Pro-Gly-Pro-Pro-Ser-Asp (MPGPPSD). Their interactions are shown in [the table below]. Figure 2-4As shown, the polypeptide VPKIPPP forms four hydrogen bonds with α-glucosidase, namely Pro 230, Asn 301, and Asp 333 on the A chain. VPKIPPP forms hydrophobic interactions with Val 334, Val 335, Met 302, Arg 340, Phe 297, Phe 397, Ala 232, Ala 343, and Pro 230 on the A chain of α-glucosidase. Furthermore, VPKIPPP also exhibits van der Waals forces with Glu 377, Thr 339, Leu 227, Leu 300, Pru 602, Gly 228, Arg 400, Tyr 389, Glu 396, Asn 233, Ala 229, and Glu 231 on the A chain of α-glucosidase. LSMSFPPF forms 12 hydrogen bonds with α-glucosidase, namely Ala 518, Phe 534, Phe 516, His 515, and Pro 512 on the A chain, and Ser 92 and Gly 94 on the B chain. LSMSFPPF also forms 11 hydrophobic interactions with α-glucosidase, namely Ala 533, Met 535, Ala 451, Ala 532, Val 513, Phe 516, and Pro 442 on the A chain. In addition, LSMSFPPF also exhibits van der Waals forces with Ala 454, Phe 455, Ala 514, Asp 440, Thr 517, Thr 519, Leu 525, Asn 447, and Arg 520 on the A chain of α-glucosidase, and Leu 93, His 91, and Lys 89 on the B chain. MPGPPSD forms 11 hydrogen bonds with α-glucosidase: Pro 442 and Asp440 on the A chain, and Arg457, Asn 4, and Gly 94 on the B chain. MPGPPSD also forms five hydrophobic interactions with the B chain of α-glucosidase: Lys 96, Trp 7, Phe 463, and Arg 457. Furthermore, MPGPPSD forms van der Waals forces with Asp 441 and Asn447 on the A chain of α-glucosidase, and Met 6, Asp 464, Lys 483, Leu 93, Leu 95, Val 47, Asn 46, Ala 43, Arg 456, His 459, Pro 460, and Gly 11 on the B chain.

[0046] 7. α-Glucosidase Inhibition Experiment

[0047] The α-glucosidase inhibitory activities of the three polypeptides synthesized via Foc solid-phase synthesis are shown in Table 2. The IC50 values ​​of LSMSFPPF, MPGPPSD, and VPKIPPP are also presented. 50The concentrations were 0.42 mg / mL, 0.66 mg / mL, and 1.08 mg / mL, respectively. These results are superior to those of the peptide (GEY(IC)) previously reported in the literature. 50 2.70 mg / mL), GYG (IC) 50 1.50 mg / mL) and PFP (IC 50 3.10mg / mL))(Lee, HJ, Lee, HS, Choi, JW, Ra, KS, Kim, JM, & Suh, HJ (2011). Novel tripeptides with alpha-glucosidase inhibitory activity isolated from silk cocoon hydrolysate. Journal of Agricultural and Food Chemistry, 59(21), 11522-11525; Kang, MG, Yi, SH, Lee, JS (2013). Production and characterization of a new α-glucosidase inhibitory peptide from Aspergillus oryzae N159-1, Mycobiology, 41: 3, 149-154).

[0048] Table 2. α-glucosidase inhibitory activities of the three peptides

[0049]

Claims

1. An α-glucosidase inhibitory peptide derived from ginkgo nuts, characterized in that: The amino acid sequence of the α-glucosidase inhibitory peptide is Leu-Ser-Met-Ser-Phe-Pro-Pro-Phe (LSMSFPPF), Val-Pro-Lys-Ile-Pro-Pro-Pro (VPKIPPP) or Met-Pro-Gly-Pro-Pro-Ser-Asp (MPGPPSD).

2. The method for screening α-glucosidase inhibitory peptides according to claim 1, characterized in that: (1) Defatted ginkgo powder The dried ginkgo biloba powder was added with n-hexane at a solid-liquid ratio of 1:20 (g / mL), and the powder was subjected to multiple extraction and defatting at 4°C, and then dried at 45°C to obtain defatted ginkgo biloba powder. (2) Preparation of Ginkgo Protein Isolate Deionized water was added to the defatted ginkgo powder at a material-liquid ratio of 1:50-100 (g / mL). After thorough mixing, the pH of the system was adjusted to 10.0 with 1 mol / L NaOH solution. The mixture was stirred at 1000 rpm at room temperature for 2-4 h. After the extraction was completed, the extract was centrifuged at 8000 g for 20 min to obtain a supernatant, and the extraction was repeated twice. The pH of the supernatant was adjusted to 4.62 with 1 mol / L HCl, and after standing at 4°C for 2 h, the supernatant was centrifuged at 8000 g for 20 min to collect the precipitate. The precipitated protein was repeatedly washed 3 times with deionized water, the pH was adjusted to 7.4, and the ginkgo protein isolate was obtained after dialysis and freeze-drying. (3) Enzymatic hydrolysis of ginkgo protein isolate A certain amount of ginkgo protein isolate was added to distilled water at a material-liquid ratio of 1:100-200 (g / mL), stirred for 0.5-1h, and then Alcalase protease was added to the system at an enzyme-substrate ratio of 4000U / g. The ginkgo protein isolate was enzymatically hydrolyzed at pH 8.0-9.0 and a temperature of 50°C. After the reaction was completed for 4-6h, the reaction system was heated in a water bath at 90°C for 10min to inactivate the protease. After cooling to room temperature, the hydrolyzate was centrifuged at 8000g for 20min, the supernatant was collected, and the ginkgo protein hydrolyzate was obtained after freeze-drying. (4) Ultrafiltration separation and structural analysis of ginkgo peptides The ginkgo protein hydrolysate was redissolved in distilled water at a ratio of 1:10-20 (g / mL), and then an ultrafiltration membrane was used to separate the five molecular weight fractions with molecular weight cut-offs of <1000Da, 1000-3000Da, 3000-5000Da, 5000-10000Da, and >10000Da. The fraction <1000Da was freeze-dried. After desalting, peptide components with a molecular weight of less than 1000 Da were analyzed by ultra-performance liquid chromatography coupled with mass spectrometry to determine their structural characteristics, including molecular weight, amino acid sequence, and composition. The separation conditions were as follows: a 3C18-CL-120 column with a pore size of 3-5 μm, mobile phase A consisting of 2% acetonitrile and 0.1% trifluoroacetic acid in water; mobile phase B consisting of 98% acetonitrile and 0.1% trifluoroacetic acid in water, with a gradient elution pattern of 5% B at 0 min, 8% B at 1 min, 38% B at 51 min, 80% B at 52 min, 80% B at 55 min, 5% B at 56 min, and 5% B at 60 min. The mass spectrometry scan range was 100-1500 in positive ion mode. The amino acid sequence was analyzed by database search using the Protein Pilot analysis tool. (5) Screening of active peptides The above peptides were scored using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker), and potential active peptide sequences with a score greater than 0.5 were screened; (6) Screening of α-glucosidase inhibitory peptides The above-mentioned potential active peptides were docked and scored with the α-glucosidase protein with PDB ID 3WY1 using Pepsite 2 (http: / / pepsite2.russelllab.org / ). After further screening to obtain peptide sequences with a P-value less than 0.05, the peptides obtained above were further docked and verified using molecular docking software. When the peptide interacted with α-glucosidase, it was considered to be an α-glucosidase inhibitory peptide.

3. Use of the α-glucosidase inhibitory peptide according to claim 1 in the preparation of hypoglycemic drugs.

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