Torreya grandis peptide tgp-75 with hypoglycemic function and application thereof

Abstract

The application discloses a torreya grandis peptide TGPs-75 with a blood sugar reducing function, and the torreya grandis peptide TGPs-75 is prepared through the following steps: torreya grandis protein is subjected to enzymolysis by using alkaline protease; and after enzyme inactivation, the enzymolysis liquid is subjected to separation and purification through a macroporous resin column, the macroporous resin is DA201-C type, and 15v / v%, 35v / v%, 55v / v% and 75v / v% ethanol is used for elution in sequence; the 75v / v% ethanol eluate is collected and concentrated under reduced pressure to obtain 75v / v% ethanol elution components, namely the torreya grandis peptide TGPs-75. The torreya grandis peptide TGPs-75 can realize a good in-vivo blood sugar reducing effect, and has very important significance for developing health-care products and medicines with a blood sugar reducing function.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioactive peptides, and particularly relates to a torreya grandis peptide TGPs-75 with a blood glucose lowering function and application thereof. BACKGROUND

[0002] Diabetes is a chronic disease characterized by high blood sugar, which is becoming more and more common worldwide. At present, diabetes is mainly treated by artificially synthesized drugs, and long-term use will cause liver and gastrointestinal side effects, kidney function damage, etc., and the cost of synthetic drugs is high and expensive. Therefore, natural products with good blood glucose lowering function have attracted widespread attention.

[0003] Bioactive peptides are a class of peptide compounds that are beneficial to the life activities of the biological body or have physiological effects, and have the characteristics of safety, high efficiency, easy absorption and long-term use. Peptides with blood glucose lowering function are mostly used for developing drugs for preventing and treating diabetes or obesity.

[0004] Torreya grandis Merrillii belongs to the family Taxaceae and the genus Torreya, and is a coniferous tree widely distributed in Zhejiang, Anhui and Jiangxi provinces. Torreya grandis Merrillii seed oil is rich in unsaturated fatty acids and can be used for oil production. The residual cake after oil extraction contains a large amount of protein and is a good protein resource. High-quality protein is an important raw material for bioactive peptides. SUMMARY

[0005] The present application aims to provide a torreya grandis peptide TGPs-75 with a blood glucose lowering function and application thereof to solve the problems in the prior art.

[0006] The present application adopts the following technical solutions:

[0007] The present application provides a torreya grandis peptide TGPs-75 with a blood glucose lowering function in the first aspect, which is prepared by the following steps: enzymatic hydrolysis of torreya grandis protein with alkaline protease, and separation and purification of the enzymatic hydrolysate with a macroporous resin column after enzyme inactivation. The macroporous resin is DA201-C type, and 15v / v%, 35v / v%, 55v / v% and 75v / v% ethanol are used for elution in sequence. The 75v / v% ethanol eluate is collected and concentrated under reduced pressure to obtain the 75v / v% ethanol elution component, i.e. the torreya grandis peptide TGPs-75.

[0008] Further, the Torreya grandis peptide TGPs-75 is prepared by the following steps: Torreya grandis protein is weighed according to a substrate concentration of 3 w / v%, and Torreya grandis protein solution is prepared by adding distilled water; after being denatured in a 95°C water bath for 15 min and cooled to room temperature, the pH is adjusted to 10.0 by using 1.0 mol / L NaOH solution, and then alkaline protease is added, the alkaline protease is obtained by fermentation of Bacillus licheniformis, and the activity is 200 U / mg; the mass ratio of alkaline protease to Torreya grandis protein is 5.22:100; enzymolysis is carried out at 46°C and 300 r / min for 5.2 h; after the enzymolysis is completed, the enzyme is inactivated in a 95°C water bath for 10 min, and then cooled to room temperature; centrifugation is carried out at 4°C and 8000 r / min for 30 min; the supernatant is taken, and freeze-drying is carried out at-80°C for 48 h to obtain Torreya grandis enzymatic hydrolysate, which is stored at-20°C for standby use;

[0009] DA201-C macroporous resin column chromatography is selected, the size of the DA201-C macroporous resin column is 2.6 cm*60 cm, 100 mL of Torreya grandis enzymatic hydrolysate solution with a concentration of 10 mg / ml is added, the Torreya grandis enzymatic hydrolysate solution is prepared by using ultrapure water and is placed in a 4°C chromatography cabinet overnight; after the resin is adsorbed and balanced, the Torreya grandis enzymatic hydrolysate that is not successfully adsorbed is washed away by using ultrapure water, and then gradient elution is carried out by using 15 v / v%, 35 v / v%, 55 v / v% and 75 v / v% ethanol solutions in sequence, the flow rate is constant at 5 mL / min, the 75 v / v% ethanol eluate is collected, concentrated at 45°C under reduced pressure until there is no ethanol, and then freeze-dried at-80°C for 48 h to obtain the Torreya grandis peptide TGPs-75 in powder form.

[0010] Further, the Torreya grandis protein is prepared by the following steps: after the Torreya grandis seeds are shelled, cold pressing and degreasing are carried out by using a hydraulic oil press, the conditions are room temperature, 50 Mpa and 40 min, and Torreya grandis seed cake meal is obtained; then the obtained Torreya grandis seed cake meal is crushed through a 60-mesh sieve to obtain Torreya grandis seed cake meal powder; the Torreya grandis seed cake meal powder and petroleum ether are mixed at a solid-liquid ratio of 1:5, stirring is carried out at room temperature and 500 r / min for 2 h, and then suction filtration is carried out at room temperature; after two times of extraction, the filter residue is collected, and the Torreya grandis seed cake meal degreasing powder is obtained by baking at 45°C for 12 h, and is stored at 4°C for standby use;

[0011] The Torreya grandis protein is prepared by the following steps: defatted powder of the Torreya grandis seed cake obtained above is dissolved in distilled water according to a material-liquid ratio of 1:20, and the pH is adjusted to 10.0 by using 1.0 mol / L NaOH solution, and then the solution is stirred at 50°C and 500 r / min for 2 h, and then cooled to room temperature, and then centrifuged at 4000 r / min for 15 min, and then the supernatant is taken, and the pH is adjusted to 4.0 by using 1.0 mol / L HCl solution, and then the solution is statically placed for 2 h, and then centrifuged at 4000 r / min for 15 min, and then the precipitate is taken, and then washed with distilled water until neutral, and then frozen and dried at-80°C for 48 h to obtain the Torreya grandis protein, and then stored at-20°C for later use.

[0012] The second aspect of the present application provides application of the Torreya grandis peptide TGPs-75 with the blood glucose-lowering function in preparation of blood glucose-lowering health products and medicines.

[0013] The present application has the following advantages:

[0014] The present application provides a Torreya grandis peptide TGPs-75 with a blood glucose-lowering function, which is prepared by using Torreya grandis seed cake protein as raw material and by using alkaline protease for enzymolysis, and then by using DA201-C macroporous resin to sequentially elute 15 v / v%, 35 v / v%, 55 v / v% and 75 v / v% ethanol from the enzymolysis solution, and then by using the 75 v / v% ethanol elution component as the Torreya grandis peptide TGPs-75, which can achieve a good blood glucose-lowering effect in vivo, and is of great significance for development of health products and medicines with a blood glucose-lowering function.

[0015] The Torreya grandis peptide TGPs-75 has sufficient raw material source, simple preparation method and excellent blood glucose-lowering function. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is an elution curve of the Torreya grandis peptide on the DA201-C macroporous resin. DETAILED DESCRIPTION

[0017] The present application will be further explained by the following examples and drawings. The following examples are only used to illustrate the present application, but not used to limit the scope of the present application.

[0018] Example 1: Preparation of Torreya grandis enzymolysis product

[0019] Firstly, the Chinese torreya seed is shelled and then defatted by hydraulic press, under the conditions of room temperature, 50Mpa and 40min, to obtain Chinese torreya seed cake. Then the Chinese torreya seed cake is crushed to pass through a 60-mesh sieve to obtain Chinese torreya seed cake powder. The Chinese torreya seed cake powder and petroleum ether are mixed at a solid-liquid ratio of 1:5 (g / mL, w / v), stirred at room temperature and 500r / min for 2h, and then filtered under room temperature. The filtrate is collected after two times of extraction, and the residue is dried at 45℃ for 12h to obtain Chinese torreya seed cake defatted powder, which is stored at 4℃ for later use.

[0020] The Chinese torreya protein is extracted by alkali extraction and acid precipitation. The Chinese torreya seed cake defatted powder is dissolved in distilled water at a solid-liquid ratio of 1:20 (g / mL, w / v), and the pH is adjusted to 10.0 with 1.0mol / L NaOH solution. The solution is stirred at 50℃ and 500r / min for 2h, and then cooled to room temperature. The supernatant is obtained by centrifugation at 4000r / min for 15min. The pH is adjusted to 4.0 with 1.0mol / L HCl solution, and the solution is left to stand for 2h. The precipitate is obtained by centrifugation at 4000r / min for 15min, and then washed with distilled water until neutral. The Chinese torreya protein is obtained by freeze-drying at -80℃ for 48h, and stored at -20℃ for later use.

[0021] A certain amount of Chinese torreya protein is weighed according to a substrate concentration of 3% (g / mL, w / v) and dissolved in distilled water to prepare a Chinese torreya protein solution. The solution is denatured in a 95℃ water bath for 15min, and then cooled to room temperature. The pH is adjusted to 10.0 with 1.0mol / L NaOH solution, and then alkaline protease (purchased from Beijing Solabio Technology Co., Ltd., with product number B8360, obtained by fermentation of Bacillus licheniformis, and activity of 200U / mg) is added. The mass ratio of alkaline protease to Chinese torreya protein is 5.22:100. The solution is stirred at 46℃ and 300r / min for 5.2h. After enzyme inactivation in a 95℃ water bath for 10min, the solution is cooled to room temperature, centrifuged at 4℃ and 8000r / min for 30min, and the supernatant is obtained. The Chinese torreya enzymatic hydrolysate is obtained by freeze-drying at -80℃ for 48h, and stored at -20℃ for later use.

[0022] Example 2 Isolation and purification of Chinese torreya peptide

[0023] 1. Separation and purification by macroporous resin

[0024] A DA201-C macroporous resin column (2.6cm×60cm) is selected, and 100mL of Chinese torreya enzymatic hydrolysate solution (prepared with ultrapure water) with a concentration of 10mg / ml is added and placed in a 4℃ chromatography cabinet overnight. After the resin is adsorbed and balanced, the Chinese torreya enzymatic hydrolysate that is not successfully adsorbed is washed away with ultrapure water, and then gradient elution is performed with 15%, 35%, 55%, 75% and 95% (v / v) ethanol solutions in sequence, at a constant flow rate of 5mL / min.Figure 1 ), and the elution fractions were obtained by concentrating the elution solutions of different concentrations at 45°C under reduced pressure (rotary evaporator) to remove ethanol and then freeze-drying at -80°C for 48 h to obtain powders for determination of the inhibitory activities of α-glucosidase and α-amylase.

[0025] 2. Determination of α-glucosidase inhibition rate

[0026] 500 μL of samples of different concentrations (prepared with pH 6.8, 0.1 M phosphate buffer) were added with 500 μL of α-glucosidase solution (0.2 U / mL, prepared with pH 6.8, 0.1 M phosphate buffer), and then incubated at 37°C for 10 min, followed by adding 500 μL of pNPG solution (2.5 mmol / L, prepared with pH 6.8, 0.1 M phosphate buffer) and further incubating at 37°C for 40 min, and then immediately adding 2.0 mL of Na2CO3 (0.2 mol / L, prepared with ultrapure water) to terminate the reaction, and then measuring the absorbance at 405 nm, and the α-glucosidase inhibition rate was calculated according to the following formula:

[0027]

[0028] In the formula, Y represents the α-glucosidase inhibition rate, %, A0 represents the absorbance of the control group, A1 represents the absorbance of the sample group, and A2 represents the absorbance of the background group. The control group was not added with samples, but was replaced with the same volume of pH 6.8, 0.1 M phosphate buffer; and the background group was not added with α-glucosidase solution, but was replaced with the same volume of pH 6.8, 0.1 M phosphate buffer.

[0029] 3. Determination of α-amylase inhibition rate

[0030] Take different concentrations of samples 500 μL (with pH 6.8, 0.1M phosphate buffer), add 500 μL of α-amylase solution (0.2U / mL, prepared with pH 6.8, 0.1M phosphate buffer), incubated at 37°C water bath for 15 min, then add 2.0 mL 1wt% starch solution (prepared with pH 6.8, 0.1M phosphate buffer), further incubated at 37°C water bath for 5 min, then add 2 mL 3,5-dinitrosalicylic acid (DNS) reagent (DNS reagent preparation method as follows: A solution: 6.9 g of crystalline phenol is dissolved in 15.2 mL of 10wt% NaOH solution, distilled water is diluted to 69 mL, 6.9 g of sodium bisulfite is added to the solution; B solution: 255 g of potassium sodium tartrate is dissolved in 300 mL of 10wt% NaOH solution, then 880 mL of 1wt% 3,5-dinitrosalicylic acid solution is added; the two solutions are mixed to obtain the DNS reagent, which is stored in a brown bottle and used after 7-10 days, and is valid for one year in the brown bottle), to terminate the reaction. After the reaction is completed, the reaction mixture is placed in a boiling water bath for 15 min, cooled to room temperature, and diluted to 25 mL with distilled water. The absorbance is measured at 540 nm, and the α-amylase inhibition rate is calculated as follows:

[0031]

[0032] In the formula: y = α-amylase inhibition rate, %; A0 = control group absorbance; A1 = sample group absorbance; A2 = background group absorbance. The control group is not added with sample, and the same volume of pH 6.8, 0.1M phosphate buffer is used instead; the background group is not added with α-amylase solution, and the same volume of pH 6.8, 0.1M phosphate buffer is used instead.

[0033] Table 1 α-glucosidase inhibitory activity of different elution fractions

[0034]

[0035] Table 2 α-amylase inhibitory activity of different elution fractions

[0036]

[0037] The results show (Tables 1 and 2) that different concentrations of 75% ethanol elution fractions all have the highest α-glucosidase inhibitory activity and α-amylase inhibitory activity, which are recorded as Torreya grandis peptides TGPs-75 for subsequent experiments.

[0038] Example 3 In vivo evaluation of the blood glucose lowering efficacy of Torreya grandis peptides TGPs-75

[0039] 1 Test materials

[0040] 1.1 Sample preparation information

[0041] The Torreya grandis peptide TGPs-75 (mixture) prepared in Example 2 was prepared into a 2.00 mg / mL stock solution with ultrapure water, and used immediately after preparation.

[0042] Positive control: pioglitazone hydrochloride, white tablets, batch number 20220503, Jiangsu Deyuan Pharmaceutical Co., Ltd., stored in a cool and dark place. Prepare a 10.0 mg / mL stock solution with ultrapure water, and store at -20°C for future use.

[0043] 1.2 Experimental animals

[0044] Zebrafish were bred in fish water at 28°C (water quality: 200 mg of instant sea salt was added to each 1 L of reverse osmosis water, with an electrical conductivity of 450-550 μS / cm, a pH of 6.5-8.5, and a hardness of 50-100 mg / L CaCO3), and were provided by the fish breeding center of Hangzhou Huan Te Biological Technology Co., Ltd. The experimental animal use license number was SYXK(Zhe)2022-0004, and the feeding and management met the requirements of international AAALAC certification (certification number: 001458).

[0045] Wild-type AB strain zebrafish were bred by natural pair mating, and zebrafish at 5 days post-fertilization (5 dpf) were used for evaluation of the hypoglycemic efficacy of Torreya grandis peptide TGPs-75 in vivo.

[0046] 1.3 Instruments, consumables and reagents

[0047] Precision electronic balance (CP214, OHAUS, USA); blood glucose meter (ACCU-CHEK Performa, Roche Diagnostics (Shanghai) Co., Ltd., China).

[0048] Pure egg yolk powder (batch number 20200809, Zhejiang Aige Biological Technology Co., Ltd., China); D-(+)-glucose (batch number E2018008, Shanghai Aladdin Bio-Chem Technology Co., Ltd., China); anhydrous ethanol (batch number 20210901, National Pharmaceutical Group Chemical Reagent Co., Ltd., China); blood glucose meter test paper (batch number 478829, Roche Diagnostics (Shanghai) Co., Ltd., China).

[0049] 2 Detection method

[0050] Diabetic zebrafish model establishment: a diabetic zebrafish model was constructed by alternating administration of 0.15 wt% egg yolk powder for 8 h and 3 wt% glucose solution for 16 h during the day and night, and continuous feeding for 2 d, without feeding during the period. The combination of egg yolk powder and glucose feeding caused the zebrafish to exhibit high-sugar characteristics, and the significant increase in blood glucose level compared with the normal control group indicated that the zebrafish diabetic model was successfully constructed.

[0051] Randomly selected 180 tail 5dpf wild type AB strain zebrafish in beaker, 30 tails per beaker, respectively, set up blank control group: no treatment; Model control group: high fat and high sugar feed (same as above diabetic zebrafish model) treatment; Positive control group: 20.0 μg / mL hydrochloric acid and biguanide and high fat and high sugar feed (same as above diabetic zebrafish model) co-processing; Experimental group: different concentrations of torreya peptide TGPs-75 (7.81, 15.6, 31.2 μg / mL) and high fat and high sugar feed (same as above diabetic zebrafish model) co-processing. After 48h of experiment, 30 zebrafish per group were washed with ultrapure water for 3 times, then 3 tails per centrifuge tube, 10 centrifuge tubes per group (i.e. 10 times), 25uL anhydrous ethanol was added to 65℃ oven, dried, 3uL ultrapure water was added after drying, and the data was collected by blood glucose meter, the blood glucose value in zebrafish was analyzed, and the statistical analysis results of the index were used to evaluate the hypoglycemic effect of torreya peptide TGPs-75 in vivo.

[0052] 3 Test results

[0053] 3.1 Evaluation of the hypoglycemic effect of torreya peptide TGPs-75 in vivo

[0054] The blood glucose values of zebrafish treated with different concentrations of torreya peptide TGPs-75 and hydrochloric acid and biguanide are shown in Table 3. Compared with the blood glucose value of 1.12±0.03 mmol / L in the blank control group, the blood glucose value of the model control group was 2.17±0.11 mmol / L, indicating that the model was successfully established (P<0.001); compared with the model control group, the blood glucose values of zebrafish treated with different concentrations (7.81, 15.6, 31.2 μg / mL) of torreya peptide TGPs-75 and high sugar and high fat feed were significantly reduced (P<0.05), close to the blood glucose value of the blank control group (P>0.05), and lower than that of the positive control group of hydrochloric acid and biguanide (20 μg / mL) and high sugar and high fat feed (P<0.05). This indicates that torreya peptide TGPs-75 can improve the blood glucose level of diabetic zebrafish at a low dose level (7.81 μg / mL), and the hypoglycemic effect is better than that of hydrochloric acid and biguanide.

[0055] Table 3 Evaluation of the hypoglycemic effect of torreya peptide TGPs-75 in vivo (n=10)

[0056]

[0057] Note: compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0058] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A torreya grandis peptide TGPs-75 having a blood glucose-lowering function, characterized in that, The torreya grandis peptide TGPs-75 is prepared by the following steps: torreya grandis protein is extracted from torreya grandis seed cake by alkali extraction and acid precipitation, and then subjected to enzymatic hydrolysis by alkaline protease, and the enzymatic hydrolysate is purified by a macroporous resin column, wherein the macroporous resin is DA201-C, and the 75v / v% ethanol eluate is collected and concentrated under reduced pressure to obtain the torreya grandis peptide TGPs-75. 2.The Torreya grandis peptide TGPs-75 with blood glucose-lowering function of claim 1, characterized in that, The torreya grandis peptide TGPs-75 is prepared by the following steps: torreya grandis protein is extracted from torreya grandis seed cake by alkali extraction and acid precipitation, and then subjected to enzymatic hydrolysis by alkaline protease, and the enzymatic hydrolysate is purified by a macroporous resin column, wherein the macroporous resin is DA201-C, and the 75v / v% ethanol eluate is collected and concentrated under reduced pressure to obtain the torreya grandis peptide TGPs-75. The torreya grandis protein is prepared by the following steps: the torreya grandis seed is defatted by a hydraulic oil press at room temperature, 50Mpa and 40min to obtain torreya grandis seed cake; the obtained torreya grandis seed cake is crushed to pass through a 60-mesh sieve to obtain torreya grandis seed cake powder; the torreya grandis seed cake powder and petroleum ether are mixed at a solid-liquid ratio of 1:5, stirred at room temperature and 500r / min for 2h, and then filtered; the filtration residue is collected after two times of extraction, and dried at 45℃ for 12h to obtain torreya grandis seed cake defatted powder, which is stored at 4℃. 3.The torreya grandis peptide TGPs-75 with blood glucose-lowering function according to claim 2, characterized in that, The torreya grandis protein is prepared by the following steps: the torreya grandis seed is defatted by a hydraulic oil press at room temperature, 50Mpa and 40min to obtain torreya grandis seed cake; the obtained torreya grandis seed cake is crushed to pass through a 60-mesh sieve to obtain torreya grandis seed cake powder; the torreya grandis seed cake powder and petroleum ether are mixed at a solid-liquid ratio of 1:5, stirred at room temperature and 500r / min for 2h, and then filtered; the filtration residue is collected after two times of extraction, and dried at 45℃ for 12h to obtain torreya grandis seed cake defatted powder, which is stored at 4℃. The Torreya grandis protein is obtained by alkali extraction and acid precipitation: the Torreya grandis seed cake obtained above is defatted to obtain a powder, which is dissolved in distilled water to obtain a solution with a solid-liquid ratio of 1:20, and then the pH is adjusted to 10.0 by using a 1.0 mol / L NaOH solution, and then the solution is stirred at 50 ℃ and 500 r / min for 2 h, and then the solution is cooled to room temperature, and then centrifuged at 4000 r / min for 15 min, and then the supernatant is obtained, and then the pH is adjusted to 4.0 by using a 1.0 mol / L HCl solution, and then the solution is statically placed for 2 h, and then centrifuged at 4000 r / min for 15 min, and then the precipitate is obtained, and then the precipitate is washed to neutral by using distilled water, and then the precipitate is frozen at-80 ℃, and then dried for 48 h to obtain the Torreya grandis protein, and then the Torreya grandis protein is stored at-20 ℃ for later use.

4. Application of the Torreya grandis peptide TGPs-75 with a blood glucose lowering function according to any one of claims 1-3 in preparation of blood glucose lowering health products and medicines.

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