CN103626844B - Artificially synthesized wheat germ peptide and preparation method and application thereof - Google Patents

Abstract

The present invention relates to the field of biomedicine, and discloses an artificially synthesized wheat germ peptide with antioxidant activity, and discloses a preparation method and application of the wheat germ peptide as an antioxidant. The wheat germ peptide of the present invention is synthesized by Fmoc solid phase chemistry, contains 3 amino acid residues, has the sequence of Arg-Val-Phe, and has the following structural formula. Experiments show that the wheat germ peptide can protect oxidatively damaged nerve cells by maintaining cell membrane integrity, improving cell viability, and enhancing mitochondrial membrane enzyme activity, and has a significant protective effect on _{H2O2 -} induced oxidative stress and apoptosis of human nerve cells.

Description

A kind of artificially synthesized wheat germ peptide and its preparation method and application

technical field

The invention relates to the field of biomedicine, in particular to an artificially synthesized wheat germ peptide and its preparation method and application.

Background technique

According to statistics, the total number of elderly people over the age of 60 in the world has reached 600 million, and the elderly population in more than 60 countries has reached or exceeded 10% of the total population, and they have entered the ranks of aging societies. The rapid development of population aging and a series of problems caused by it have aroused the attention and attention of governments all over the world. As of now, my country's elderly population aged 60 and above has reached 185 million, accounting for 13.7% of the total population. It is estimated that by the end of the "Twelfth Five-Year Plan", the national elderly population will increase by more than 43 million, reaching 221 million. With the growth of age, many organs may experience dysfunction, and when this dysfunction develops to a certain extent, it will show symptoms, which is "geriatric disease". The number of patients with Alzheimer's disease (AD) and Parkinson's disease (PD), two major geriatric diseases in my country, has reached more than 15 million. Since there is no effective treatment drug at present, these diseases seriously reduce the quality of life of patients , It also brings huge pressure and burden to the whole society and family. According to recent epidemiological studies, if the elderly population supplements a certain dose of antioxidants for a long time, it can effectively reduce the incidence of cardiovascular and cerebrovascular diseases and cancer. Therefore, the role of reducing free radicals and anti-oxidation therapy in the treatment of PD and AD patients has been paid more and more attention by drug researchers and clinicians.

In recent years, research on antioxidant peptides as a non-enzymatic free radical scavenger is emerging. It has been reported that peptides with antioxidant activity enzymatically hydrolyzed or extracted from food protein sources include wheat germ peptide, soybean peptide, bovine whey protein peptide, corn peptide, wheat gluten peptide and so on.

Wheat germ is a by-product of wheat processing and is a living plant embryo. After proper proteolytic hydrolysis and purification, the wheat germ polypeptides produced are not only easy to digest and absorb, but also have the functions of scavenging free radicals and inhibiting lipid peroxidation, which can be used as potential antioxidants in the fields of food and medicine. develop. Current studies have confirmed that wheat germ peptide has a strong ability to scavenge free radicals in vitro. However, the extraction, separation and purification process of wheat germ peptide is too cumbersome and complicated, which cannot guarantee the stability of product quality.

Contents of the invention

In order to solve the problems in the prior art, the object of the present invention is to provide a synthetic wheat germ peptide and its preparation method, as well as its application as an antioxidant in the preparation of drugs for the prevention of diseases related to nerve cell oxidative stress.

In order to achieve the purpose of the present invention, the present invention firstly provides a kind of artificially synthesized wheat germ peptide with anti-oxidative stress activity, its amino acid sequence is Arg-Val-Phe, and its structural formula is as follows:

Further, it uses phenylalanine protected by N-fluorenyl methaneoxycarbonyl as raw material, through two condensation reactions, respectively connects valine and arginine protected by N-fluorenyl methaneoxycarbonyl in sequence to generate wheat germ peptide.

The present invention also provides a method for preparing the aforementioned wheat germ peptide, comprising the following steps:

(1) Resin swelling;

(2) Remove amino protection;

(3) Synthesis of dipeptides;

(4) Synthesis of tripeptides;

(5) Wheat germ peptide resin cutting;

(6) Precipitation of wheat germ peptide.

Wherein, the step (1) is to place 30 mmol of Fmoc-L-phenylalanine-King resin in a reactor, soak in 300 mL of dichloromethane, wait for the resin to swell for 30 minutes, and remove the dichloromethane by suction filtration.

Wherein, the step (2) is to soak the resin obtained in the step (1) by adding a mixed solution of hexahydropyridine and N,N-dimethylformamide with a volume ratio of 2:8, agitate it with nitrogen gas, and use N, N-dimethylformamide washes the resin.

Wherein, the step (3) is to mix 150 mL of a solution containing 18 mmol of Fmoc-L-valine with 100 mmol of N,N'-diisopropylcarbimide and 90 mmol of 1-hydroxybenzotriazole Mix 120mL of the solution in an ice bath and react for 0.5h; then mix it with the resin treated in step (2), add 16.5mmol of 4-dimethylaminopyridine, and react for 5h at room temperature; filter, and use N,N-di Methylformamide, dichloromethane, and methanol were washed three times, each time for 3 minutes; 450mL anhydrous pyridine and 90mL acetic anhydride were dissolved in 600mL N,N-dimethylformamide and poured into the resin, and reacted for 2 hours; the reaction was completed Afterwards, wash the resin three times with 1000 mL each of N,N-dimethylformamide, dichloromethane, and methanol in turn, 3 min each time, and dry in vacuum.

Wherein, the step (4) is to repeat steps (2)-(3), replace Fmoc-L-valine in step (3) with Fmoc-L-arginine, and connect Fmoc-L-arginine acid to complete the tripeptide synthesis, the resin was soaked in dichloromethane and ether and then drained.

Wherein, the step (5) is to place the obtained tripeptide-resin in a sand core reactor, add 750mL cutting reagent, react for 2h, filter, and concentrate the filtrate by rotary evaporation, add 600mL ice ether to the concentrated solution, and precipitate out, Centrifuge at 5000r/m for 10min, then discard the supernatant, and vacuum-dry the precipitate; wherein, the cutting reagent is a mixture of 2.5% water, 2.5% triisopropylsilane and 95% trifluoroacetic acid.

Wherein, the step (6) is to filter out the resin, add anhydrous ether to the filtrate, centrifuge with a centrifuge to obtain a solid, add anhydrous ether to wash, then centrifuge, repeat several times, desalt by HPLC, freeze-dry Obtain wheat germ peptide.

The present invention also provides the application of the artificially synthesized wheat germ peptide as an antioxidant in the preparation of drugs for preventing diseases related to nerve cell oxidative stress.

The beneficial effects that the present invention has are:

The present invention utilizes the Fmoc solid-phase chemical method to synthesize wheat germ peptide containing 3 amino acid residues, which solves the technical problem that the extraction, separation and purification process of natural wheat germ peptide is too cumbersome and complicated in the prior art and cannot guarantee stable product quality. And it was found through experiments that it can protect _nerve cells from oxidative damage by maintaining cell membrane integrity, improving cell viability, and enhancing _{mitochondrial} membrane enzyme activity. Oxidative stress and apoptosis have a significant protective effect, which provides new ideas and possibilities for the preparation of potential health foods or drugs for the treatment and prevention of neurodegenerative diseases mediated by oxidative stress.

Description of drawings

Fig. 1 shows the protective effect of the artificially synthesized wheat germ peptide preincubation of the present invention on the oxidative damage of human nerve cells induced by H ₂ O ₂ .

Fig. 2 is the effect of the artificially synthesized wheat germ peptide of the present invention on the LDH release rate of human nerve cells induced by H ₂ O ₂ .

Fig. 3 is the effect of preincubation of the artificially synthesized wheat germ peptide of the present invention on the morphology of human nerve cells induced by H ₂ O ₂ .

Fig. 4 is the liquid chromatography identification result of the artificially synthesized wheat germ peptide of the present invention.

Fig. 5 is the liquid phase mass spectrometry identification result of the artificially synthesized wheat germ peptide of the present invention.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

The synthesis of embodiment 1 wheat germ peptide

The specific synthesis steps of wheat germ peptide are as follows:

1. Resin swelling

Soak 30 mmol (0.5 mol/g) of Fmoc-L-phenylalanine-Wang resin (purchased from Shanghai Xinran Biotechnology Co., Ltd.) in dichloromethane, wait for the resin to expand, and filter off the dichloromethane. Wang resin was placed in a reactor, soaked in 300mL of dichloromethane, and the resin was swelled for 30 minutes, and the dichloromethane was removed by suction filtration.

2. Removal of amino protection

Add the mixed solution of hexahydropyridine and N,N-dimethylformamide with a volume ratio of 2:8 to soak the resin obtained in step (1), agitate with nitrogen gas, and wash the resin with N,N-dimethylformamide after the reaction . Take a small amount of resin and add 2-3 drops of each color test agent ABC (A solution: ninhydrin/absolute ethanol solution; B solution: pyridine; C solution: phenol/absolute ethanol solution) and heat at 100°C for 1-2min. If the color of the solution and resin is blue (some amino acids are purple), it means that the amino protection has been removed, and the next reaction can be carried out.

3. Synthesis of dipeptide (valine-phenylalanine)

A solution (150mL) dissolved in Fmoc-L-valine (18mmol) and dissolved in N,N′-diisopropylcarbimide (DIC, 100mmol) and 1-hydroxybenzotriazole (HOBt , 90mmol) solution 120mL were mixed under ice bath, and reacted for 0.5h. Then mix with the resin treated in step (2); add 4-dimethylaminopyridine (DMAP, 16.5mmol) and react at room temperature for 5h. Filter and wash with N,N-dimethylformamide, dichloromethane, and methanol three times, each time for 3 minutes. Dissolve 450mL of anhydrous pyridine and 90mL of acetic anhydride in 600mL of N,N-dimethylformamide and pour it into the resin for 2 hours of reaction; Wash the resin with 1000mL three times in turn, each time for 3 minutes, and dry it in vacuum. Take a small amount of resin for color inspection. The method is the same as that in step (2). If the color of the solution and resin is colorless, the reaction can be considered complete.

4. Synthesis of tripeptide (arginine-valine-phenylalanine)

Repeat steps (2)-(3), replace Fmoc-L-valine with Fmoc-L-arginine in step (3), connect Fmoc-L-arginine to complete the tripeptide synthesis, resin with two Soak in methyl chloride and ether and drain.

5. Wheat germ peptide resin cleavage

The obtained tripeptide-resin was placed in a sand core reactor, and 750 mL of cutting reagent (2.5% v/v water, 2.5% v/v triisopropylsilane, 95% v/v trifluoroacetic acid) was added and reacted for 2 h. It was then filtered and the filtrate was concentrated by rotary evaporation. Add 600mL of glacial ether to the concentrated solution to precipitate a precipitate. Centrifuge at 5000r/m for 10min, discard the supernatant, and dry the precipitate in vacuo.

6. Precipitation of wheat germ peptide

Filter off the resin, add anhydrous ether to the filtrate, centrifuge to obtain a solid, add anhydrous ether to wash, then centrifuge, repeat several times, desalt by HPLC, and freeze-dry to obtain wheat germ peptide.

7. Product Identification

The obtained wheat germ peptide was identified by liquid phase and liquid phase mass spectrometry, and the identification results are shown in Fig. 4 and Fig. 5 . It can be seen from the liquid chromatogram in Figure 4 that there is only one peak, and the resulting wheat germ peptide is a pure product; it can be seen from the liquid mass spectrogram in Figure 5 that the molecular weight obtained is 420.97, and this molecular weight is the molecular weight of [M+H]. Therefore, the molecular weight of wheat germ peptide is 419.97, which conforms to the molecular formula C ₂₀ H ₃₂ N ₆ O ₄ molecular weight MW420 of wheat germ peptide RVF. This proves that the product synthesized in Example 1 according to the synthesis method described in the present invention is wheat germ peptide, and the obtained wheat germ peptide is a pure product.

Example 2 Wheat Germ Peptide Antioxidative Stress Activation Detection

1. Detection of cell viability

SH-SY5Y cells were purchased from Shanghai Yansheng Biochemical Reagent Co., Ltd. SH-SY5Y cells were cultured in MEM medium containing 10% FBS at 37°C, 5% CO2 (v/v), and saturated humidity. When the cell density reaches 80% to 90%, discard the old medium, add an appropriate amount (limited to cover the bottom of the bottle) trypsin to digest for about 1 min, and observe under a microscope. It is found that the intercellular space increases and the shape becomes round. Immediately discard the digestion solution and add medium with serum to terminate the digestion. Gently and repeatedly blow the bottle wall cells with a pipette, do not use too much force or generate a lot of air bubbles. The cell suspension was divided into new bottles to continue culturing, the medium was changed every other day, and the cells were passaged every 2-3 days.

The cell viability was detected by MTT assay. Succinate dehydrogenase in the mitochondria of living cells can react with MTT to produce water-insoluble blue-purple crystalline formazan (Formazan) and deposit in cells, but dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve the crystalline formazan deposited in the cells. Within a certain range of cell numbers, the amount of MTT crystal formation is proportional to the number of cells. By measuring its absorbance value at 490-570nm wavelength, it can be Indirectly reflect the number and proliferation activity of living cells.

After the drug effect on the cells, add 40 μL of MTT solution (2 mg/mL), continue to incubate in the cell incubator for 4 hours, gently suck away the supernatant (be careful not to suck up the crystals), add 100 μL of dimethyl sulfoxide to each well, Shake for 5-10 minutes to fully dissolve the crystal violet, detect the optical density value at 490nm with a microplate reader, and calculate the cell survival rate according to the following formula:

2. Establishment of H ₂ O ₂ induced oxidative damage model of SH-SY5Y cells

After the cells grow to the logarithmic phase, trypsinize and collect the cells, adjust the cell density to 1×10 ⁵ cells/mL, inoculate 100 μL per well into a 96-well culture plate, and place at 37°C, 5% (v/v) After culturing overnight in the CO ₂ incubator, add H ₂ O ₂ diluted with MEM medium, the final concentrations were 50, 100, 150, 200, 250, 300, 400 μmol/L, and 6 parallel wells were set up in each group. After 12h and 24h respectively, the cell viability was detected by MTT.

3. Protective effect of wheat germ antioxidant peptides _on H2O2 _- induced oxidative damage of human neurons

Collect human neurons (SH-SY5Y) in the logarithmic phase, adjust the cell density to 1×10 ⁵ cells/mL, inoculate 100 μL per well into a 96-well cell culture plate, place at 37°C, 5% CO ₂ (v/ v) After cultivating overnight, group processing:

3.1. Control group: add fresh MEM medium;

3.2. Injury model group: MEM medium containing H ₂ O ₂ was added;

3.3. Wheat germ antioxidant peptide protection group: add MEM medium containing wheat germ antioxidant peptide and co _- incubate SH _- SY5Y cells, then add MEM medium containing H2O2;

After the culture, the cell morphology was observed with an inverted phase-contrast microscope (see Figure 3), and the cell viability was detected by the MTT method (see Figure 1).

3.4. Detection of LDH release rate

Take cells in the logarithmic growth phase, dilute the cell density to ¹ ×105 cells/mL, inoculate 100 μL per well into a 96-well cell culture plate, discard the old medium after overnight culture and wash with PBS, and divide each culture well into the following sections: Group: culture solution wells without cells, cell control wells without drug treatment, cell wells without drug treatment for subsequent lysis, and drug treatment wells, continue to culture according to the routine, until 1 hour before the scheduled detection time point, Add LDH release reagent to the sample maximum enzyme activity control well, mix well, and continue to incubate. After the culture, 120 μL of the supernatant was taken from each well, and measured according to the instructions of the LDH detection kit. The measurement results are shown in Figure 2, and the cytotoxicity or death rate was calculated:

4. Experimental results

It can be seen from Figure 1 that the cell proliferation activity of human nerve cells was significantly inhibited after being treated with 200 μmol/L H ₂ O ₂ for 24 hours. Compared with the control group, the cell survival rate dropped to 49.18±3.26% (p<0.01); when 150, After pre-incubation with 200 and 250 μmol/L wheat germ peptide for 4 hours, the cell viability increased to 59.67±3.10% (p<0.05), 66.15±3.98% (p<0.01) and 67.20±4.03% (p<0.01), respectively, And present a certain dose-dependent. It shows that wheat germ peptide can improve the proliferation activity of cells by enhancing the activity of mitochondrial oxidoreductase.

It can be seen from Figure 2 that compared with the control group, the LDH release rate in the H ₂ O ₂ model group increased by 56%, reaching 91.88±1.76% (p<0.01), suggesting that the integrity of the cell membrane was damaged, and a large amount of LDH leaked into the However, after pre-incubation with wheat germ peptide, the release rate of LDH decreased to 81.47±1.65% (p<0.05), 75.40±0.57% (p<0.01) and 71.88±1.98% (p<0.01), showing a certain dose dependence. It shows that wheat germ peptide effectively repairs the damaged cell membrane and inhibits the release of LDH.

It can be seen from Figure ₃ that the number of cells in the control group is large, the outline is clear, and there are long protrusions in the shape of spindle or polygon _; Short or even disappeared, while the cell morphology of the wheat germ peptide intervention group was significantly improved.

Through research on cell survival rate, LDH release rate, and cell morphological changes, it was found that wheat germ antioxidant peptide can protect nerve cells from oxidative damage by maintaining cell membrane integrity, improving cell viability, and enhancing mitochondrial membrane enzyme activity. This shows that the wheat germ antioxidant peptide has a significant protective effect on the oxidative stress and apoptosis of human neurons induced by H ₂ O ₂ , and the wheat germ antioxidant peptide can be used to treat and prevent neurodegenerative diseases mediated by oxidative stress Potential health food or medicine.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (3)

1. a preparation method with the wheat germ peptide of anti-oxidation stress activity for synthetic, is characterized in that, comprise the steps:

(1) resin swelling;

(2) amido protecting is removed;

(3) synthesis of dipeptides;

(4) synthesis of tripeptides;

(5) wheat germ peptide resin cutting;

(6) wheat germ peptide is separated out;

Wherein, described step (1), for the Fmoc-L-of 30mmol phenylalanine-king's resin is placed in reactor, is soaked with 300mL methylene dichloride, is treated that resin expands, swelling 30min, suction filtration removing methylene dichloride;

Described step (2) is for adding the hexahydropyridine and DMF mixing solutions soaking step (1) gained resin that volume ratio is 2:8, and use nitrogen to agitate, reaction terminates rear DMF washing resin;

Described step (3) is to be dissolved with the solution 150mL of 18mmol Fmoc-L-α-amino-isovaleric acid and to be dissolved with 100mmol N, and the solution 120mL of N'-diisopropylcarbodiimide and 90mmol 1-hydroxyl benzotriazole mixes under ice bath, reaction 0.5h; Then the mixed with resin processed with step (2), then add the DMAP of 16.5mmol, react 5h under room temperature; Filter, wash three times respectively with DMF, methylene dichloride, methyl alcohol, each 3min; 450mL anhydrous pyridine, 90mL diacetyl oxide are poured in resin after being dissolved in 600mL DMF, reaction 2h; Reaction terminates rear DMF, methylene dichloride, each 1000mL of methyl alcohol washing resin three times in turn, each 3min, vacuum-drying;

Described step (4) is repeating step (2)-(3), Fmoc-L-α-amino-isovaleric acid in step (3) is replaced with Fmoc-L-arginine, connect Fmoc-L-arginine and complete three peptide symthesis, drain after resin methylene dichloride and ether are soaked.

2. preparation method as claimed in claim 1, it is characterized in that, described step (5), for gained tripeptides-resin is placed in core reactor, adds 750mL cutting reagent, reaction 2h, filter, rotary evaporation concentrated filtrate, adds 600mL ice ether in concentrated solution, separate out precipitation, centrifugal 10min, then abandoning supernatant under 5000r/m, vacuum-drying precipitates; Wherein, described cutting reagent is volume fraction is 2.5% water, and 2.5% tri isopropyl silane and 95% trifluoroacetic acid mix.

3. preparation method as claimed in claim 1, it is characterized in that, described step (6) is elimination resin, anhydrous diethyl ether is added in filtrate, solid is obtained with after centrifuge, add anhydrous diethyl ether washing, more centrifugal, repeat can obtain wheat germ peptide through HPLC desalination, freeze-drying for several times.