A long-stalked amygdalus enzymatic peptide for preventing chronic fatigue syndrome and its application

Abstract

The present invention discloses an enzymatic peptide of long-pedunculated almond for preventing chronic fatigue syndrome and its application. The present invention extracts long-pedunculated almond protein from long-pedunculated almond kernel, enzymatically hydrolyzes the long-pedunculated almond protein with alkaline protease to obtain long-pedunculated almond peptide, separates the peptide with a C18 chromatographic column, and identifies the composition of the enzymatic peptide with LC-MS / MS. According to the anti-fatigue activity in mice, based on LC-MS / MS identification and molecular docking results, a hydrolytic peptide with anti-fatigue activity is screened out, and its amino acid sequence from N-terminus to C-terminus is Glu-Val-Glu-Arg-Leu-Phe. The present invention has simple steps and provides a theoretical basis for the comprehensive utilization and development of long-pedunculated almond kernel.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptides, and in particular relates to a polypeptide derived from long-petaled almond kernel protein that can prevent chronic fatigue syndrome.

[0002] Background Field

[0003] Fatigue is a common symptom. Normal fatigue usually occurs after strenuous physical labor. It is often a protective signal that the body should rest and can be relieved by resting or changing lifestyle. Compared with normal fatigue, pathological fatigue does not improve with rest, and many patients with chronic diseases have this experience. It is more intense and lasts longer, which can cause serious damage to the individual's functional activities and quality of life. However, people ignore the impact of fatigue in their lives and often misunderstand it as sleepiness or even laziness. Therefore, they do not seek professional medical treatment, which further accelerates the negative impact of fatigue. In addition, frequent or difficult to recover fatigue may be a manifestation of underlying pathology and chronic diseases, such as chronic kidney disease, obstructive pulmonary disease, heart failure, multiple sclerosis, and cancer.

[0004] With people's increasing attention to health and the increasing importance of food safety, plant-derived anti-fatigue peptides play an extremely important role in preventing human sub-health with their unique advantages of high efficiency and safety. Compared with chemical synthetic drugs, plant-derived anti-fatigue peptides can not only effectively prevent the occurrence of fatigue in the body, but also have the natural advantage of low toxic side effects. Long-pedunculated almond is a perennial deciduous shrub of the subgenus Amygdalus in the genus Amygdalus of the Rosaceae family, also known as wild cherry. Long-pedunculated almond has strong cold and drought resistance, deep roots, and can adapt to various soil environments. The development of protein resources in long-pedunculated almond kernels is gradually developing. It contains more than 300 kinds of proteins, with a complete range of amino acids, and has multiple biological activities such as antioxidant, anti-fatigue and improving body immunity. It is a high-quality source of bioactive peptides. The research and development of long-pedunculated almond enzymatic peptide anti-fatigue peptides can further broaden its application range, help extend the long-pedunculated almond industry chain, and provide a research basis for the industrial development and utilization of long-pedunculated almonds in my country. Summary of the invention

[0005] The invention uses long-pedunculate almond kernels as raw materials, performs shelling pretreatment on the long-pedunculate almond kernels, defats the kernels with n-hexane, selects a water extraction method to prepare long-pedunculate almond protein, and uses alkaline protease to hydrolyze the long-pedunculate almond hydrolyzed peptides, evaluates the anti-fatigue activity of the long-pedunculate almond hydrolyzed peptides through a mouse anti-fatigue experiment, further separates and purifies the long-pedunculate almond hydrolyzed peptides, identifies the sequences of the long-pedunculate almond hydrolyzed peptides by using liquid chromatography-mass spectrometry (LC-MS / MS), and screens out the polypeptide fragments with the best anti-fatigue activity by using molecular docking technology.

[0006] The long-stalked tonsil enzymatic peptide for preventing chronic fatigue syndrome provided by the present invention has an amino acid sequence from N-terminus to C-terminus: Glu-Val-Glu-Arg-Leu-Phe, hereinafter referred to as EVERLF.

[0007] The enzymatic hydrolysis peptide of the present invention can be used to prepare medicine for preventing chronic fatigue syndrome. When the enzymatic hydrolysis peptide of the long-pedunculated Amygdalus is used, it can be obtained by artificial synthesis or separation and purification after enzymatic hydrolysis of long-pedunculated Amygdalus seed kernels.

[0008] The beneficial effects of the present invention are as follows:

[0009] The present invention uses long-pedunculated almond kernels as raw materials, uses alkaline protease to enzymolyze long-pedunculated almond protein, and obtains long-pedunculated almond enzymolysis peptides. The anti-fatigue activity of long-pedunculated almond enzymolysis peptides is studied by anti-fatigue experiments on mice, and the long-pedunculated almond enzymolysis peptides are separated and purified. The polypeptide sequence is determined by LC-MS / MS, and the molecular docking technology is used to screen out the peptide fragments with the best ability to prevent chronic fatigue syndrome. The preparation of long-pedunculated almond enzymolysis peptides by the present invention is not only simple in steps but also low in cost, and provides a basic basis for further broadening the application scope of long-pedunculated almond enzymolysis peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The effect of Amygdalus longifolia enzymatic peptide on the exhaustive swimming time of mice.

[0011] Figure 2 This is the purification chromatogram of the enzymatic peptides from Amygdalus longifolia.

[0012] Figure 3 It is the DPPH free radical scavenging activity of different components of Amygdalus longifolia enzymatic peptides. DETAILED DESCRIPTION

[0013] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0014] Example 1

[0015] 1. Preparation of Amygdaloids

[0016] After blanching, the long-pedunculated almond kernels were manually peeled, dried at 40°C, and crushed with a high-speed grinder to obtain long-pedunculated almond kernel powder. The long-pedunculated almond kernel powder was added to n-hexane at a solid-liquid ratio of 1:5 (g / mL), stirred in a fume hood for 40 minutes for defatting, and the n-hexane was poured out; the defatting was repeated 3 times, the n-hexane was filtered off, and the obtained long-pedunculated almond defatted kernel powder was dried at 25°C. The dried long-pedunculated almond defatted kernel powder and distilled water were mixed at a solid-liquid ratio of 1:16 (g / mL), stirred at room temperature for 90 minutes, and then centrifuged at 4000 rpm for 10 minutes. The supernatant was collected and filtered, and the long-pedunculated almond protein was obtained after freeze-drying, which was placed in a refrigerator at -20°C for later use.

[0017] 2. Preparation of Amygdalus longipes enzymatic peptides

[0018] According to the material-liquid ratio of 1g:25mL, the long-handled amygdalus protein powder was dissolved in distilled water, pretreated at 90℃ for 10 minutes, the temperature was adjusted to 57℃ and the pH was adjusted to 8.4 with 0.5M NaOH aqueous solution, enzymatic hydrolysis was performed with alkaline protease, the enzyme amount was 1192U / g, and the water bath was hydrolyzed for 4 hours. During the hydrolysis process, the solution pH was maintained at 8.4 with 0.5M NaOH aqueous solution. After the hydrolysis was completed, the enzyme was inactivated in a 100℃ water bath for 10 minutes, centrifuged at 8000 rpm for 10 minutes, the supernatant was taken, and freeze-dried into powder to obtain the long-handled amygdalus enzymatic peptide (APH), which was stored in a -20℃ refrigerator for later use.

[0019] 3. Determination of anti-fatigue activity of Amygdalus longipes enzymatic peptides

[0020] Thirty male Kunming mice were randomly divided into five groups according to their body weight, namely, control group (Con), American ginseng positive control group (AGS), Amygdalus longifolius enzymatic peptide high, medium and low dose groups (APH-H, APH-M, APH-L). The control group was given normal saline, and the positive control group was given American ginseng 0.6 mg / g; the Amygdalus longifolius enzymatic peptide high, medium and low dose groups were given 2 mg / g, 1 mg / g and 0.5 mg / g per day, respectively, with a dosing frequency of once a day for a total of 28 days. After the end of the dosing, the exhaustive swimming time was measured. Figure 1 As shown in the figure, compared with the mice in the Con group, the exhaustive swimming time of mice in the AGS, APH-M and APH-H groups was significantly increased (p<0.05), and it showed a dose-dependent relationship. The exhaustive swimming time of mice in the AGS, APH-L, APH-M and APH-H groups increased by 1.68, 1.23, 1.37 and 1.59 times, respectively, indicating that the long-stalked amygdaloid peptide significantly improved the exercise endurance of mice after administration. Long-stalked amygdaloid peptide can significantly increase the exhaustive swimming time of mice, indicating that long-stalked amygdaloid peptide has the effect of relieving fatigue in mice.

[0021] 4. Isolation, purification and identification of enzymatic peptides from Amygdalus longipes

[0022] The long-stalked Amygdalus enzymatic peptide was separated and purified using a C18 column. The mobile phase A was a 0.1% trifluoroacetic acid aqueous solution, and B was a 0.1% trifluoroacetic acid acetonitrile solution. The column was first equilibrated with 5% B phase, and then gradient elution was performed. The elution program was 5% to 90% B for 40 minutes. Samples were collected in sections during the elution process. After the long-stalked Amygdalus enzymatic peptide was purified by the C18 column, a total of five components (F1-F5) were collected. After freeze-drying, the DPPH scavenging activity of the five components was measured. The results are shown in Figure 2 , 3 As shown, the F4 component has good DPPH free radical scavenging activity. The peptide sequence of the F4 component was identified by Nano LC-MS / MS, and a total of 5 key anti-fatigue peptides FLDVLR, FLDLVR, EVERLF, EFLDRL, and EFLR were identified and screened. The amino acid sequences from N-terminus to C-terminus are shown in Table 1.

[0023] Table 1 Amino acid sequences of five key anti-fatigue peptides

[0024]

[0025]

[0026] 5. Molecular docking

[0027] The molecular docking method was used to predict the binding affinity between five key anti-fatigue peptides and anti-fatigue targets in order to screen the optimal anti-fatigue peptides and provide a reference for further experiments. The lower the Vina score, the higher the affinity between the ligand and the receptor. The threshold is generally selected as Affinity≤-5kcal / mol. If it is lower than this value, it is considered to have potential affinity. The conformation with the lowest affinity was used as the optimal docking conformation. The affinity and binding mode of the five key anti-fatigue peptides and the DLG4 target were analyzed by molecular virtual docking, and the cocrystal molecule Glutathione (PubChemCID: 124886) was selected as the positive control molecule to screen active peptides with better affinity than the positive control molecule. From the data in Table 2, it can be found that for the DLG4 target, the affinity scores of FLDVLR (Affinity = -5.1 kcal / mol), EVERLF (Affinity = -5.9 kcal / mol) and EFLR (Affinity = -5.2 kcal / mol) are all better than the positive control molecule Glutathione (Affinity = -4.6 kcal / mol), among which EVERLF has the lowest score and can form a more stable binding mode with the DLG4 target than the positive control molecule, and has the best anti-fatigue activity.

[0028] Table 2 Docking results of 5 key anti-fatigue peptides and DLG4 target

[0029]

Claims

1. A long-stalked amygdalus enzymatic peptide for preventing chronic fatigue syndrome, characterized in that: The amino acid sequence of the long-stalked amygdalus enzymatic peptide from the N-terminus to the C-terminus is Glu-Val-Glu-Arg-Leu-Phe.

2. Use of the long-petaled Amygdalus enzymatic peptide according to claim 1 in the preparation of a drug for chronic fatigue syndrome.

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