WO2022068784A1

WO2022068784A1 - Selenium-rich cordyceps militaris derived active selenium peptide with neuron protection function, and preparation method therefor and use thereof

Info

Publication number: WO2022068784A1
Application number: PCT/CN2021/121134
Authority: WO
Inventors: 丁郁; 吴书建; 吴清平; 王涓; 朱振军; 陈梦霏; 廖茜妤; 张菊梅; 陈玲; 韦献虎; 古其会; 张友雄; 雷涛
Original assignee: 暨南大学; 广东省科学院微生物研究所(广东省微生物分析检测中心)
Priority date: 2021-05-08
Filing date: 2021-09-28
Publication date: 2022-04-07
Also published as: CN113248568B; CN113248568A

Abstract

Provided are a selenium-rich Cordyceps militaris derived active selenium peptide with a neuron protection function, and a preparation method therefor and the use thereof. The amino acid sequence of the selenium-rich Cordyceps militaris derived active selenium peptide is Val-Pro-Arg-Lys-Leu-(Se)Met or Arg-Tyr-Asn-Ala-(Se)Met-Asn-Asp-Tyr-Thr. The selenium-rich Cordyceps militaris derived active selenium peptide can be prepared through artificial chemical synthesis, and can also be obtained by means of separating and purifying the selenium-rich Cordyceps militaris derived protein enzymatic hydrolyzate. The selenium-rich Cordyceps militaris derived active selenium peptide of the present invention can effectively reduce excessive ROS accumulation and improve the activity of antioxidant enzymes (CAT and SOD) in the H ₂ O ₂ induced oxidative damage cells, and can be used for alleviating neurodegenerative diseases.

Description

Selenium-enriched Cordyceps militaris active selenopeptide with neuron protection function and preparation method and application thereof

Technical field:

The invention belongs to the field of active peptides, and in particular relates to a selenium-rich Cordyceps militaris active selenopeptide with neuron protection function and a preparation method and application thereof.

Background technique:

The body needs to maintain the body's antioxidant defense system through the action of reactive oxygen species (ROS), so as to maintain the body's constant redox state and ensure the body's internal environment is stable. When the body's redox balance is biased towards oxidation, it can cause disruption of redox signaling and control, as well as molecular damage, known as oxidative stress (OS), resulting in a variety of diseases. Among them, oxidative stress in the brain damages neuronal cells, which can trigger a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. These diseases obviously affect people's quality of life and bring huge economic burden to the society.

Studies have shown that there is a close relationship between neuronal damage and neurodegenerative diseases, and antioxidants that have protective effects on oxidatively damaged neurons can alleviate neurodegenerative diseases. Food-derived selenopeptides are natural antioxidants. Compared with similar peptides, selenopeptide has multiple biochemical functions of selenium and peptides, and has superior neuron protection activity and antioxidant activity. Various biological activities of selenopeptides (such as antioxidant, antihypertensive, anti-inflammatory and immunomodulatory effects) are involved in various biological processes in living organisms. Further research results show that selenopeptides can effectively interfere with the development of Alzheimer's disease and aging through antioxidant pathways. Selenopeptides can act as an oxidant to remove excessive accumulation of ROS in neuronal cells, and can also improve the anti-oxidative enzyme defense system of neuronal cells. Therefore, selenopeptides have great potential in preventing or treating age-related neurodegenerative diseases by protecting oxidatively damaged neuronal pathways.

At present, selenopeptides are mainly derived from the enzymatic hydrolysis of selenoproteins. Compared with inorganic selenium, organic selenium has lower toxicity, higher bioavailability and better antioxidant activity. Plants obtained by biofortification can convert inorganic selenium into organic selenium, mainly in the form of selenoproteins. The previous experimental results showed that Cordyceps militaris has a strong selenium accumulation ability, and the selenium content is as high as 117.7 mg/kg. Therefore, selenium-enriched Cordyceps militaris has a high selenoprotein content and is an excellent source of selenoprotein, which can be used for the preparation of food-derived selenopeptides.

The PC12 cell line is derived from a pheochromocytoma of the rat adrenal medulla and can be differentiated into cells with many properties similar to sympathetic neurons by in vitro nerve growth factor (NGF) treatment. research on neurodegenerative diseases. Many studies have reported that high concentrations of H ₂ O ₂ can cause oxidative stress damage to cells, usually as a study on the occurrence of neuronal oxidative damage in vitro. Therefore, to prepare bioactive selenopeptides from Se-enriched Cordyceps militaris, selenopeptides with neuronal protective effects can be screened through the PC12 cell model of H ₂ O ₂ -induced oxidative damage.

Invention content:

The first object of the present invention is to provide two active selenopeptides of selenium-rich Cordyceps militaris with neuron protection function, the amino acid sequences of which are Val-Pro-Arg-Lys-Leu-(Se)Met and Arg-Tyr-Asn- Ala-(Se)Met-Asn-Asp-Tyr-Thr.

The second object of the present invention is to provide the application of the above-mentioned active selenopeptide of Cordyceps militaris rich in selenium in the preparation of a medicine for protecting neuron cells.

The third object of the present invention is to provide a drug for protecting neuron cells, which contains the above-mentioned active selenopeptide of Cordyceps militaris as an active ingredient.

The neuron protection refers to the pre-protection effect on H ₂ O ₂ -induced oxidative damage cells, including that the selenopeptide has no obvious toxicity to PC12 cells, and improves the viability of PC12 cells after H ₂ O ₂ injury. Meanwhile, _{selenopeptides} can effectively reduce excessive ROS accumulation and enhance the activity of antioxidant enzymes in cells with H2O2 _- induced oxidative damage. Therefore, the drug for protecting neuron cells is a drug for protecting neuron cells from oxidative damage.

The fourth object of the present invention is to provide a method for preparing the above-mentioned active selenopeptide of selenium-enriched Cordyceps militaris, which is prepared and separated from selenium-enriched Cordyceps militaris.

The above-mentioned selenium-enriched Cordyceps militaris active selenopeptide can be artificially synthesized in solid phase by using the existing chemical technology. Taking Val-Pro-Arg-Lys-Leu-(Se)Met as an example, the Fmoc-SeMet-wang Resin resin is swollen and deprotected, and the next amino acid Fmoc-Leu-oh, amino acid co-condensing agent and organic base are weighed Add it into the reactor for condensation reaction, wash after reaction, repeat deprotection-weighing-deprotection washing-feeding-reaction-washing after reaction process until all amino acids are connected, and finally obtain polypeptide through detection, cleavage and purification.

In addition, the selenium-enriched Cordyceps militaris selenopeptide of the present invention can also be obtained from the selenium-enriched Cordyceps militaris protease hydrolyzed solution using separation methods such as ultrafiltration, Sephadex G-25 dextran gel chromatography, and reversed-phase high performance liquid chromatography. ,Specific steps are as follows:

(1) Add the selenium-rich Cordyceps militaris dry powder to a 5% acetic acid solution containing 0.1% β-mercaptoethanol, and after stirring, centrifugally filter the filtrate, add ammonium sulfate to 75% saturation, stand for precipitation, centrifuge, and collect the precipitation, After precipitation redissolving, dialysis, freeze-drying to obtain selenium-enriched Cordyceps militaris selenoprotein dry powder;

(2) take the selenoprotein dry powder, add ultrapure water to mix, use pepsin for enzymolysis, then use trypsin for enzymolysis, kill the enzyme, centrifuge, take the supernatant, and obtain the selenoprotein simulated gastrointestinal digestion enzymolysis solution;

(3) Dilute the enzymatic hydrolyzate with pure water and pass it through a 3kDa molecular weight ultrafiltration membrane, collect the <3kDa components and concentrate;

(4) Load the fraction <3kDa onto a Sephadex G-25 Sephadex column, elute with ultrapure water, collect the fractions, concentrate, and select the GF1 fraction with good neuronal cell protection effect;

(5) Load the GF1 component on a reversed-phase high-performance liquid chromatography column, use water-acetonitrile eluent for gradient elution, collect the components, concentrate, and select the most active selenopeptide component by UPLC-MS/MS Detected to obtain selenium-enriched Cordyceps militaris selenopeptide.

The fifth object of the present invention is to provide the application of selenium-enriched Cordyceps militaris in preparing the above-mentioned active selenopeptides of selenium-enriched Cordyceps militaris.

Compared with the prior art, the present invention has the following advantages and effects:

The active selenopeptide of selenium-enriched Cordyceps militaris has a clear structure and can be prepared by artificial chemical synthesis, or can be obtained by separating and purifying the proteolytic solution of selenium-enriched Cordyceps militaris. Meanwhile, the active selenopeptide of selenium-enriched Cordyceps militaris of the present invention has a pre-protective effect on H ₂ O ₂ -induced oxidative damage cells, and has great potential in relieving neurodegenerative diseases.

Description of drawings

Figure 1 shows the elution curve of Sephadex G-25 Sephadex gel chromatography of selenium-enriched Cordyceps militaris protease hydrolysate and the effect of different fractions on the viability of PC12 cells induced by H ₂ O ₂ oxidative damage.

Figure 2 shows the elution curve of the selenium-enriched Cordyceps militaris protease hydrolysate by reversed-phase liquid chromatography and the effect of different fractions on the viability of H ₂ O ₂ -induced oxidative damage PC12 cells.

Figure 3 is a secondary mass spectrum of active selenopeptides from Se-enriched Cordyceps militaris.

Figure 4 shows the effect of synthetic selenopeptides on the viability of PC12 cells induced by H ₂ O ₂ oxidative damage.

Figure 5 shows the effect of synthetic selenopeptides on ROS in H ₂ O ₂ -induced oxidative damage in PC12 cells.

Figure 6 shows the effects of synthetic selenopeptides on CAT and SOD in H ₂ O ₂ -induced oxidative damage in PC12 cells.

Detailed ways

In order to show the technical solutions, purposes and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

In the following examples, experiments are usually carried out in accordance with laboratory conditions, conventional conditions or conditions suggested by the manufacturer, if there are special experimental conditions, it will be noted otherwise.

Example 1. Selenopeptides Val-Pro-Arg-Lys-Leu-(Se)Met and Arg-Tyr-Asn-Ala-(Se)Met-Asn-Asp- Separation and purification of Tyr-Thr

(1) Preparation of selenium-enriched Cordyceps militaris protein dry powder: 5% acetic acid dissolution-75% ammonium sulfate precipitation method was used to extract selenoprotein from selenium-enriched Cordyceps militaris. The specific process is as follows. After the selenium-rich Cordyceps militaris is dried and powdered, 5% acetic acid solution (containing 0.1% β-mercaptoethanol) by volume is added at a solid-to-liquid ratio of 1:15 (g/mL) to dissolve → 4°C, stirring for 3h →4℃, centrifuge at 8500g for 10min →add ammonium sulfate to 75% saturation →stir for 0.5h →4℃, let stand for 15h →4℃, centrifuge at 8500g for 12min →pure water redissolve and precipitate →4℃, dialyze for 24h → freeze drying → Selenoprotein dry powder.

(2) Preparation of selenium-enriched Cordyceps militaris protease hydrolysate: prepare 10 mg/mL selenoprotein solution with selenoprotein dry powder → add 4% pepsin by mass, and enzymatically hydrolyze at pH 2.0 and temperature 37°C for 2 hours, every 0.5 hours Use 0.1M HCl and NaOH to adjust the pH once → adjust the pH value to 7.0, add 4% trypsin, enzymolysis at 37 °C for 2 hours, and use 0.1M HCl and NaOH to adjust the pH once every 0.5h → Boiling water bath to inactivate the enzyme 10min→cooled to room temperature→passed through 0.22μm filter membrane→packed, frozen at -80℃.

(3) Separation and purification of active selenopeptide: use a 3kDa molecular weight ultrafiltration membrane to collect the permeated <3kDa enzymatic hydrolysate, concentrate and store it; use Sephadex G-25 Sephadex column to separate and purify the <3kDa group 5 minutes, ultrapure water was eluted at a flow rate of 1 mL/min, and the detection wavelength was 280 nm. The elution curve is shown in Figure 1A. The fractions were collected for cytotoxicity and protective effect determination. The cells were seeded in a 96-well plate at a density of 1.5×10 ⁴ cells/well (100 μL medium per well) and incubated for 24 h, then 10 μg/well of selenopeptide (100 μL medium per well) was added for protection for 12 h, and then passed through CCK-8 The kit measures cell viability to determine the toxicity of the sample to cells. In the control group, no selenopeptide was added, and three replicates were performed for each treatment (n=3). At the same time, cells were seeded in a 96-well plate at a density of 1.5×10 ⁴ cells/well (100 μL medium per well) for 24 hours, and then 10 μg/well of selenopeptide (100 μL medium per well) was added for protection for 12 hours, and then 80 μM was added. H ₂ O ₂ was treated for 1 h to induce oxidative damage (100 μL per well), and finally the cell viability was determined by CCK-8 kit to determine the protective effect of the sample on H ₂ O ₂ -induced oxidative damage cells. The control group did not add selenopeptide and H ₂ O ₂ , and the injury group did not add selenopeptide but added H ₂ O ₂ . Each treatment was repeated three times (n=3). The GF1 fractions with the best protective effect on oxidatively damaged PC12 cells and no obvious cytotoxicity (Figure 1B and C) were collected, concentrated, and frozen at -80°C until use; The column was (C18column, 10×250 mm, 5 μm, Waters). Ultrapure water (mobile phase A)-acetonitrile (mobile phase B) for gradient elution: 0.01-1min, 5% acetonitrile; 1-35min, 5-40% acetonitrile; 35-36min, 40% acetonitrile; 36-40min, 40-5% acetonitrile; 40-45min, 5% acetonitrile, the flow rate is 0.7mL/min, the detection wavelength is 220nm, a total of 5 components are collected (Figure 2A), the collection has the best protection effect on oxidative damage PC12 cells and no obvious cells The toxic selenopeptide fraction GF1-2 (Fig. 2B and C) was concentrated to obtain a fraction containing the target active selenopeptide.

(4) The amino acid sequence of GF1-2 components was analyzed by UPLC-MS/MS technology, and the results of secondary mass spectrometry confirmed that they contained the target selenopeptide. Cordyceps militaris active selenopeptides, their sequences are Val-Pro-Arg-Lys-Leu-(Se)Met and Arg-Tyr-Asn-Ala-(Se)Met-Asn-Asp-Tyr-Thr.

Example 2. Artificial solid-phase synthesis of Val-Pro-Arg-Lys-Leu-(Se)Met or Arg-Tyr-Asn-Ala-(Se)Met-Asn-Asp-Tyr-Thr selenopeptide

Taking Val-Pro-Arg-Lys-Leu-(Se)Met as an example, the Fmoc-SeMet-wang Resin resin was swollen and deprotected, and the next amino acid Fmoc-Leu-oh was weighed into the reactor for condensation reaction. Washing after the reaction, repeat the process of deprotection-weighing-deprotection washing-feeding-reaction-washing after reaction until all amino acids are connected. The Val-Pro-Arg(pbf)-Lys(boc)-Leu-SeMet-wangresin resin peptide was obtained, and the peptide was removed from the resin by a cleavage solution and the side chain protecting group was removed to obtain a crude polypeptide. Finally, purified peptides (>95%) were obtained by reverse-phase high-performance preparative chromatography.

Arg-Tyr-Asn-Ala-(Se)Met-Asn-Asp-Tyr-Thr can be obtained by the same solid-phase synthesis method.

Example 3. Artificial solid-phase synthesis of Val-Pro-Arg-Lys-Leu-(Se)Met and Arg-Tyr-Asn-Ala-(Se)Met-Asn-Asp-Tyr-Thr induced oxidation by H ₂ O ₂ Protective effect of damaged PC12 cells

In this study, a PC12 cell model of H ₂ O ₂ -induced oxidative damage was used to evaluate selenopeptides (Val-Pro-Arg-Lys-Leu-(Se)Met and Arg-Tyr-Asn-Ala-(Se)Met-Asn-Asp-Tyr -Thr) protective effect on neurons. The cells were seeded in a 96-well plate at a density of 1.5×10 ⁴ cells/well (100 μL medium per well) for 24 h, then 10 μg/well of selenopeptide (100 μL medium per well) was added for protection for 12 h, and 80 μM H ₂ O ₂ treatment for 1 h induced oxidative damage (100 μL per well). The control group did not add selenopeptide and H ₂ O ₂ , and the injury group did not add selenopeptide but added H ₂ O ₂ , and each treatment was repeated 3 times (n=3). Cell viability was determined by CCK-8 kit. Cell viability was measured immediately after selenopeptide pre-protection, showing that selenopeptide had no obvious cytotoxicity (Fig. 4A), while cell viability was measured immediately after H ₂ O ₂ injury, showing that selenopeptide had obvious protective effect (Fig. 4B). Therefore, the selenopeptide of the present invention has obvious neuron protection effect.

Example 4. Effects of synthetic selenopeptides on ROS in H ₂ O ₂ -induced oxidative damage in PC12 cells

In this study, fluorescent probe method (determined by DCFH-DA) was used to evaluate the effect of selenopeptides on ROS in PC12 cells induced by H ₂ O ₂ oxidative damage. The cells were seeded in a 96-well plate at a density of 1.5×10 ⁴ cells/well (100 μL medium per well) for 24 h, and then 10 μg/well selenopeptide (100 μL medium per well) was added for protection for 12 h. 10 μM DCFH-DA solution (100 μL per well) was then incubated with PC12 cells for 20 min in a 37° C. incubator. After incubation, the cells were washed twice with DMEM, and then treated with 80 μM H ₂ O ₂ for 1 h to induce oxidative damage (100 μL per well). The control group did not add selenopeptide and H ₂ O ₂ , and the injury group did not add selenopeptide but added H ₂ O ₂ . The damaged cells were washed twice with PBS, and the cell fluorescence was photographed (Ex=488 nm, Em=525 nm) using an automatic microplate reader (Biotek Cytation 5, USA). H ₂ O ₂ induced a significant increase in ROS in oxidatively damaged PC12 cells, while selenopeptide pretreatment could effectively reduce excessive ROS in oxidatively damaged PC12 cells (Figure 5). Excessive accumulation of ROS can cause oxidative stress, leading to oxidative damage to cells. Therefore, selenopeptides can protect neurons by scavenging excessive ROS accumulation in oxidative damage.

Example 5. Effects of synthetic selenopeptides on CAT and SOD in PC12 cells induced by H ₂ O ₂ oxidative damage

In this study, the H ₂ O ₂ -induced oxidative damage PC12 cell model was used to evaluate the effects of selenopeptides on CAT and SOD in oxidatively damaged cells. The cells were seeded in a plate at a density of 2.5×10 ⁵ cells/dish (10 mL medium per plate) for 24 h, and then 1 μg/mL selenopeptide (10 mL medium per dish) was added for protection for 12 h. Oxidative damage was induced by treatment with 80 μM H ₂ O ₂ for 1 h (10 mL per well). The control group did not add selenopeptide and H ₂ O ₂ , and the injury group did not add selenopeptide but added H ₂ O ₂ . Cells were collected and lysed according to the kit instructions, and finally CAT and SOD were determined.

Endogenous enzymes and antioxidant enzymes (such as CAT and SOD) play an important role in maintaining the body's redox balance. In oxidatively stressed cells, SOD can convert O ^2- into less toxic H ₂ O ₂ , while CAT can further convert H ₂ O ₂ into H ₂ O and O ₂ . Therefore, changes in the viability of CAT and SOD were used to evaluate the protective effect of selenopeptides _on H2O2 _- induced oxidative damage in PC12 cells. The measurement results are shown in Figure 6. Compared with the control group, the CAT and SOD activities of PC12 cells were significantly reduced after H ₂ O ₂ injury. However, _{selenopeptide} _pretreatment could effectively ameliorate the decreased viability of CAT and SOD in cells after H2O2 injury. Therefore, selenopeptides can effectively enhance the activity of antioxidant enzymes CAT and SOD in cells to protect neurons.

The above-mentioned embodiments only represent several preferred embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

A selenium-rich Cordyceps militaris active selenopeptide with neuron protection function, characterized in that the amino acid sequence is Val-Pro-Arg-Lys-Leu-(Se)Met or Arg-Tyr-Asn-Ala-(Se)Met-Asn -Asp-Tyr-Thr.
Application of the selenium-rich Cordyceps militaris active selenopeptide according to claim 1 in the preparation of a drug for protecting neuron cells.
The application according to claim 1, characterized in that, the drug for protecting neuron cells is a drug for protecting neuron cells from oxidative damage.
A medicine for protecting neuronal cells, characterized in that it contains the active selenopeptide of selenium-enriched Cordyceps militaris according to claim 1 as an active ingredient.
The drug for protecting neuron cells according to claim 4, wherein the drug for protecting neuron cells is a drug for protecting neuron cells from oxidative damage.
The medicament for protecting neuronal cells according to claim 5, wherein the protecting neuronal cells from oxidative damage comprises removing excess ROS in cells induced by H 2 O 2 and increasing the resistance of oxidatively damaged cells to oxidase activity.
A method for preparing active selenopeptide of selenium-enriched Cordyceps militaris according to claim 1, characterized in that it is prepared and separated from selenium-enriched Cordyceps militaris.
preparation method according to claim 7, is characterized in that, concrete steps are as follows:

(1) Add the selenium-rich Cordyceps militaris dry powder to a 5% acetic acid solution containing 0.1% β-mercaptoethanol, and after stirring, centrifugally filter the filtrate, add ammonium sulfate to 75% saturation, stand for precipitation, centrifuge, and collect the precipitation, After precipitation redissolving, dialysis, freeze-drying to obtain selenium-enriched Cordyceps militaris selenoprotein dry powder;

(2) take the selenoprotein dry powder, add ultrapure water to mix, use pepsin for enzymolysis, then use trypsin for enzymolysis, kill the enzyme, centrifuge, take the supernatant, and obtain the selenoprotein simulated gastrointestinal digestion enzymolysis solution;

(3) Dilute the enzymatic hydrolysis solution with pure water and pass it through a 3 kDa molecular weight ultrafiltration membrane, collect the <3 kDa components and concentrate;

(4) Load the fraction < 3 kDa onto a Sephadex G-25 Sephadex column, elute with ultrapure water, collect the fraction, concentrate, and select the GF1 fraction with good neuronal cell protection effect;

(5) Load the GF1 component on a reversed-phase high-performance liquid chromatography column, use water-acetonitrile eluent for gradient elution, collect the components, concentrate, and select the most active selenopeptide component by UPLC-MS/MS Detected to obtain selenium-enriched Cordyceps militaris selenopeptide.
Application of selenium-enriched Cordyceps militaris in preparing the active selenopeptide of selenium-enriched Cordyceps militaris according to claim 1.