Traditional Chinese medicine hippocampus polypeptide with strong antioxidant and alpha-glucosidase inhibitory activity

Abstract

The invention discloses two brand-new bioactive peptides with antioxidant and alpha-glucosidase inhibitory activity in traditional Chinese medicine hippocampus. The sequences of the novel bioactive peptides provided by the invention are respectively YLPPNW and SVWLGGSLL. According to the method, purity and structure identification is carried out on the new bioactive peptide obtained through separation by adopting liquid phase, primary mass spectrum and secondary mass spectrum, and a material basis is provided for further quality control and drug effect research of the traditional Chinese medicine hippocampus.

Description

Technical Field

[0001] The invention belongs to the field of extraction, separation and structure identification of traditional Chinese medicine, and specifically relates to the discovery of two peptides of the traditional Chinese medicine hippocampus with antioxidant and alpha-glucosidase inhibitory activities. Background Art

[0002] Seahorse is a marine bony fish, commonly known as water horse, dragon fish, horse head fish, etc. Taxonomically, it belongs to the class of fish, order of syngnathidae, family of syngnathidae, and genus of Hippocampus. The 2020 edition of the Chinese Pharmacopoeia includes 5 types of seahorses, namely, lined seahorse, three-spotted seahorse, seahorse, spiny seahorse, and small seahorse (sea maggot). As an extremely precious Chinese medicinal material, seahorse is known as "ginseng in the north and seahorse in the south". Its main effects are warming the kidney and strengthening yang, dispersing knots and removing blood stasis, etc. At present, seahorse has physiological effects such as anti-aging, antioxidant, anti-fatigue, anti-tumor, anti-inflammatory, neuroprotective, and antihypertensive. Unfortunately, there are few studies on the Chinese medicine seahorse bioactive peptides in lowering blood sugar. Summary of the invention

[0003] The purpose of the present invention is to discover two active polypeptides with antioxidant and α-glucosidase inhibition effects in the traditional Chinese medicine hippocampus.

[0004] 1. Two new bioactive peptides with antioxidant and α-glucosidase inhibitory effects from Chinese herbal medicine Hippocampus and a preparation method thereof, characterized in that two new bioactive peptides are prepared from Chinese herbal medicine Hippocampus through extraction, separation, prediction, synthesis, screening and other steps, and these two bioactive peptides have hypoglycemic activity, the sequence of the new bioactive peptide I is Tyr-Leu-Pro-Pro-Asn-Trp (YLPPNW), and its molecular formula is C 40 H 52 N8O9, whose structural formula is The sequence of the new bioactive peptide II is Ser-Val-Trp-Leu-Gly-Gly-Ser-Leu-Leu (SVWLGGSLL), and its molecular formula is C 44 H 70 N 10 O 12 , whose structural formula is

[0005] 2. Two new bioactive peptides with hypoglycemic activity from the traditional Chinese medicine hippocampus and a preparation method thereof, comprising the following steps:

[0006] (1) Preparation and extraction: The purchased dried seahorses were cleaned, the internal organs were removed, freeze-dried, crushed, and passed through a 60-mesh sieve to obtain seahorse bone powder. An appropriate amount of seahorse bone powder was weighed as raw material, and the seahorse bone powder was enzymatically hydrolyzed to obtain a crude extract of seahorse bioactive peptides by simulating the in vitro gastrointestinal digestion process.

[0007] (2) Separation and enrichment: After centrifuging the crude protein extract of hippocampus obtained by enzymatic hydrolysis, the supernatant is ultrafiltered through a 3 kDa ultrafiltration membrane to obtain bioactive peptides less than 3 kDa, which are then separated through polysaccharide gel G-25. The elution peak components are collected or combined and freeze-dried to obtain a crude extract of bioactive peptides.

[0008] (3) Tandem mass spectrometry and De novo sequencing: The crude bioactive peptide extracts in step 2 were subjected to sequence prediction using the De novo sequencing method of tandem mass spectrometry, and the predicted bioactive peptide sequences were subjected to bioinformatics screening;

[0009] (4) Solid phase synthesis: synthesizing the polypeptide screened in step 3;

[0010] (5) α-glucosidase inhibitory activity assay: The bioactive peptides synthesized in step 4 were screened for α-glucosidase inhibitory activity, and new peptides YLPPNW and SVWLGGSLL were screened out;

[0011] (6) Perform primary mass spectrometry, secondary mass spectrometry, and liquid chromatography on the new peptide in step 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the liquid phase diagram, primary mass spectrum diagram, and secondary mass spectrum diagram of the peptide YLPPNW.

[0013] Figure 2 These are the liquid phase diagram, primary mass spectrum diagram, and secondary mass spectrum diagram of the peptide SVWLGGSLL. DETAILED DESCRIPTION

[0014] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, the present invention is further explained below with reference to examples, but the scope of protection claimed by the present invention is not limited to the following implementation modes.

[0015] Embodiment 1:

[0016] Preparation and extraction: Take the purchased dried seahorse, clean it, remove the internal organs, freeze-dry it, crush it, and pass it through a 60-mesh sieve to obtain seahorse bone powder, weigh an appropriate amount of seahorse bone powder as raw material, and simulate the in vitro gastrointestinal digestion process to enzymatically hydrolyze the seahorse bone powder to obtain a crude extract of seahorse bioactive peptides, wherein the enzymatic hydrolysis conditions are as shown in Table 1.

[0017] Table 1.Detail conditions for pepsin and trypsin hydrolysis

[0018]

[0019] Embodiment 2:

[0020] Separation and enrichment: After centrifuging the crude protein extract of hippocampus obtained by enzymatic hydrolysis, the supernatant is ultrafiltered through a 3kDa ultrafiltration membrane to obtain bioactive peptides less than 3kDa, which are then separated through polysaccharide gel G-25, and the elution peak components are collected or combined, and freeze-dried to obtain a crude extract of bioactive peptides.

[0021] Embodiment 3:

[0022] Tandem mass spectrometry and De novo sequencing: First, the HT-F2 sample was dissolved in 50 mM ammonium bicarbonate solution, followed by reductive alkylation and desalting. Then, the sample was first eluted by a nanoflow ultra-high performance liquid chromatography system (Easy-nLC1200, Thermo Fisher Scientific, USA), and then by Q Exactive TM Hybrid Quadrupole-Orbitrap TM Mass spectrometer (Thermo Fisher Scientific, USA) was used for detection. Ultra-high performance liquid chromatography separation was performed on a RPLC C18 column (Acclaim PepMap, size 150 μm × 150 mm, particle size 3 μm) and a guard column (300 μm × 5 mm, particle size 5 μm), both from Thermo Company. Chromatographic conditions were: mobile phase A, 0.1% (v / v) formic acid in water; mobile phase B, 0.1% (v / v) formic acid in 80% acetonitrile; flow rate, 600 nL / min; column temperature, 25°C. The optimized elution gradient was: 4-8% B in 2 minutes, 8-28% B in 43 minutes, 28-40% B in 10 minutes, 40-95% B in 1 minute, and 95% B was kept for 10 minutes.

[0023] After chromatographic separation, peptides were identified by tandem mass spectrometry in the positive ion mode, with mass spectra recorded from 100 to 1500 m / z. The conditions for electrospray ionization (ESI) were as follows: spray voltage, 2.2 kV; capillary temperature, 320 °C; mass resolution, 70,000; AGC target, 3e6; maximum injection time, 100 ms. MS / MS was performed in HCD mode with the following conditions: normalized collision energy, 28.0; activation time, 6.000; resolution, 17,500; AGC target, 1e5; maximum injection time, 50 ms; Top N, 20; NCE / step NCE, 28.

[0024] The analysis and search of peptide identification from MS and MS / MS spectra were performed using PEAKS Studio (10.6) software (Beijing Biotech Biotechnology Co., Ltd.). The parameters were set as follows: protein modifications were carboxymethylation (C) (fixed), oxidation (M) (variable), and acetylation (N-terminal); enzyme specificity was set to none; maximum missed cleavage sites were set to 3; precursor ion mass tolerance was set to 20 ppm, and MS / MS tolerance was 0.02 Da. Only high-confidence identified peptides were selected for downstream protein identification analysis.

[0025] Embodiment 4:

[0026] Solid phase synthesis: 2-Chlorotrityl chloride resin (0.5 g) was transferred to a reaction vessel and swollen with 10 mL of dichloromethane (DCM) for 30 min to activate the resin. Subsequently, the first Fmoc-protected amino acid (3.75 mmol) and N,N-diisopropylethylamine (DIPEA, 7.5 mmol) were dissolved in 10 mL of DCM and added to the reaction vessel, and the mixture was stirred for 2 h. The resin was then washed with DCM to remove unreacted components. Methanol / DCM / DIPEA (volume ratio: 2 / 17 / 1) solution was added to block the resin, followed by washing with N,N-dimethylformamide (DMF). Fmoc deprotection was performed using 20% ​​piperidine / DMF solution for 30 min, followed by washing with DMF. The second Fmoc-protected amino acid (3.75 mmol), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 3.75 mmol) and DIPEA (7.5 mmol) were dissolved in 10 mL DCM, mixed well with the resin and reacted for 2 hours. The DMF washing, Fmoc deprotection and amino acid coupling steps were repeated until the sequence was complete. A solution of trifluoroacetic acid / triisopropylsilane / water (volume ratio: 95 / 2.5 / 2.5) was added to cleave the peptide from the resin, and ether was subsequently added to the resulting mixture. The precipitate was collected and purified by preparative liquid chromatography.

[0027] Embodiment 5:

[0028] α-glucosidase inhibitory activity assay: First, α-glucosidase (20 μL, 1.3 U / mL) was mixed with the peptide solution (20 μL), and the mixture was incubated at 37°C for 5 minutes, and then PNPG substrate (20 μL, 2.5 mmol / L) was added. After further incubation at 37°C for 15 minutes, Na2CO3 solution (80 μL, 0.2 mol / L) was immediately added to terminate the reaction, and finally the absorbance was measured at 405 nm by an ELISA reader. Acarbose was used as a positive control, and the inhibition rate (%) of each parallel synthesized peptide was calculated as follows:

[0029]

[0030] Among them, A a represents the absorbance of the sample group containing enzyme, A b represents the absorbance of the sample control group without enzyme, A c represents the absorbance of the control group without sample, A d represents the absorbance of the blank control group without sample and enzyme. Finally, the IC of a specific peptide was calculated using SPSS software. s0 value.

[0031] Results: The experimental results show that the IC inhibition rate of the bioactive peptide I and bioactive peptide II provided by the present invention on α-glucosidase 50 They are 0.29mM and 0.36mM respectively.

Claims

1. Two peptides with antioxidant and α-glucosidase inhibitory activities from the traditional Chinese medicine hippocampus, characterized by The sequence of the novel bioactive peptide I is Tyr-Leu-Pro-Pro-Asn-Trp (YLPPNW), and the molecular formula of the novel bioactive peptide I is C 40 H s2 N8O9, the structural formula of the new polypeptide I is The sequence of the novel bioactive peptide II is Ser-Val-Trp-Leu-Gly-Gly-Ser-Leu-Leu (SVWLGGSLL), and the molecular formula of the novel bioactive peptide II is C 44 H 70 N 10 O 12 , the structural formula of the new polypeptide II is 2. The active peptide of YLPPNW and SVWLGGSLL according to claim 1, characterized in that: (1) Two peptide sequences, YLPPNW and SVWLGGSLL, were screened for α-glucosidase inhibitory activity, IC 50 0.29mM and 0.36mM respectively; (2) The above two polypeptides have antioxidant activity. At a concentration of 3 mM, the antioxidant scavenging rate of YLPPNW against DPPH free radicals is 66.91%; at a concentration of 3 mM, the antioxidant scavenging rate of SVWLGGSLL against DPPH free radicals is 77.59%.

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