WO2021082294A1 - Preparation of and method for immunomodulatory peptide - Google Patents

Abstract

Provided is a method for isolating and purifying an immunomodulatory peptide by using Pseudostellaria heterophylla. The method uses Pseudostellaria heterophylla as a raw material to obtain a specific immunity polypeptide after using protein extraction, the two-step enzymolysis of pepsin and trypsin, isolation and purification, and freeze-drying. The polypeptide has a molecular weight of 522 Da, and the full amino acid sequence thereof is: Arg-Gly-Pro-Pro-Pro. The present invention eliminates the defects present in immunomodulatory drugs, and eliminates public worries about artificial immunomodulators; in addition, the present invention lays the theoretical foundation for the development of immunity peptides based on food sources as well as for the exploration of the wide applications thereof in food and medicine.

Description

Preparation and method of immunomodulatory peptide Technical field

The invention relates to a method for separating and purifying immunoregulatory peptides by using Radix Pseudostellariae, belonging to the field of biotechnology.

Background technique

Immunity is a specific physiological response that occurs when the body comes into contact with "antigenic foreign bodies" or "different molecules", and its main purpose is to maintain the body's homeostasis. The immune system, which is responsible for the body's immune function, is a network composed of cells, tissues and organs, used to eliminate potentially harmful substances, such as bacteria, viruses, fungi, and protozoa, and to prevent the growth of cancer cells. In the body's survival, the immune system acts as the first line of defense against pathogens and provides protection before the body's functions are damaged. However, the immune system is also affected by many factors, including stress, unhealthy lifestyles, pathogens and antigens, etc., which can destroy the body's immune system. As a result, drugs used to regulate the human immune response came into being, such as cyclosporine, tacrolimus, glucocorticoids, plant alcohol, aristolochic acid, graphene and levamisole have been successfully applied to human immune response The adjustment. However, the toxic side effects and high cost of these drugs limit their use in patients, and most immunomodulatory drugs are not suitable for chronic or preventive use. Therefore, the discovery of new immunologically active peptides from food proteins has become an effective means of treatment and prevention.

technical problem

Taizi ginseng is the root tuber of the caryophyllaceae plant Haier ginseng, also known as Tongshen, Four-leaf ginseng, Mi ginseng, etc. It has been recorded in many traditional Chinese medicine books since the Qing Dynasty "Materia Medica Congxin". It can invigorate qi and invigorate the spleen, promote body fluid and nourish the lungs. In TCM treatment, it is mostly used for spleen deficiency, weak appetite, weakness after illness, insufficient qi and yin, spontaneous sweating and thirst, dry cough in the lungs. The main planting areas of Pseudostellaria are Fujian, Jiangsu, Shandong, Anhui, etc. Among them, Zherong, Fujian has been planted since the end of Qing Dynasty, and is well-known at home and abroad, and has the reputation of "Hometown of Pseudostellaria". So far, there have been many studies on the chemical composition and medicinal effects of P. sylvestris, but there are no relevant reports on the preparation of polypeptides by proteolysis of P. sylvestris.

Therefore, the present invention prepares high-efficiency immune polypeptides with specific amino acid sequences from the proteolysis of Radix Pseudostellariae. The polypeptide can be applied to the food and health products industry.

Technical solutions

In order to solve the above-mentioned problems, the present invention provides a method for separating and purifying immunomodulatory peptides by using Radix Pseudostellariae. The polypeptide obtained by the method has a specific amino acid sequence.

In order to achieve the above objectives, the following technical solutions are adopted:

An immunomodulatory peptide of the present invention, the amino acid sequence is Arg-Gly-Pro-Pro-Pro (RGPPP), and the preparation includes the following steps:

(1) Extraction of P. sylvestris protein: Grind P. sylvestris into powder, and extract P. sylvestris protein with alkali extraction and acid precipitation;

(2) Preparation of Pseudostellaria chinensis proteolysate: use pepsin and trypsin to carry out two-step enzymatic hydrolysis of Pseudosciaena serrata protein to obtain the hydrolysate of Pseudostellaria chinensis;

(3) Separation and purification of the proteolysate of Pseudosciaena sylvestris: Sephadex G25 chromatography and RP-HPLC reversed-phase high performance liquid chromatography to separate and purify the proteolysis of Pseudosciaena sylvestris, collect the most immunologically active components and freeze-dry them, and The amino acid sequence of the components was identified by Nano LC-MS/MS.

Step (1) The extraction of P. sylvestris protein is as follows: ground P. sylvestris into powder, and add the corresponding volume of 0.2-0.3 mol/L sodium hydroxide solution (NaOH) according to the mass concentration of P. sylvestris at 4.0-5.0%. Extract in a water bath at 50-60 °C for 1.0-3.0 h. After extraction, the supernatant was collected by centrifugation, and the pH value was adjusted to 2.5-3.5 with 1 mol/L hydrochloric acid (HCl). After standing for 1 to 3 h, the precipitate was collected by centrifugation and freeze-dried to obtain P. chinensis protein.

In step (2), the preparation of the proteolysate of Radix Pseudostellariae is as follows: the protein concentration of Radix Pseudostellariae is 1.0~2.0 w/v %, the pH is 1.5~2.5, the temperature is 30~40 ℃, the enzymolysis time is 4.0~6.0 h, and 8.0 is added. ~10.0 w/w% pepsin for enzymatic hydrolysis. After the reaction is over, adjust the pH to 6.5~7.5, add 8.0~10.0 w/w% trypsin for enzymatic hydrolysis for 2.0~4.0 h. After the reaction, place it immediately Boil in a boiling water bath for 10-20 minutes to terminate the reaction. After the reaction, the solution is centrifuged to collect the supernatant and freeze-dried to obtain the proteolysis product of Radix ginseng.

The specific operation of the separation and purification of the proteolytic product of Pseudostellaria chinensis in step (3) is as follows: separate and purify the proteolytic solution of Pseudosciaena sylvestris in step (2). Sephadex G25 uses deionized water as the eluent. The sample volume is 5 mL, the flow rate is 0.3 mL/min, and the detection wavelength is 214 nm. The immunological activity of the eluent corresponding to each absorption peak is measured, and the components with the highest immunological activity are collected and separated by reversed-phase high performance liquid chromatography; The separation of high performance liquid chromatography takes the acetonitrile solution with a concentration gradient of 0~55 v/v% containing 0.05% trifluoroacetic acid as the eluent for linear elution. The column used is Gemini 5μ C18, and the sample volume is 100 μL, the flow rate is 1 mL/min, the detection wavelength is 214 nm, the immunological activity of the eluate corresponding to each absorption peak is measured, and the components with the highest immunological activity are collected and freeze-dried; the components are identified by Nano LC-MS/MS The amino acid sequence. Using Nano LC-MS/MS to identify the amino acid sequence of the components, the amino acid sequence of the immunomodulatory peptide according to claim 1 is: Arg-Gly-Pro-Pro-Pro.

The specific operation of the determination of immune activity in step (3) is as follows: the immune activity is evaluated by promoting the proliferation activity of mouse spleen lymphocytes in vitro. The mouse spleen lymphocyte suspension was added to a 96-well plate, and phosphate buffer was used as a blank control. The final concentrations of the samples were 10 μg/mL, 50 μg/mL, and 100 μg/mL, respectively. After adding the sample, place it in a 37 °C 5% CO ₂ incubator for different periods of time, add 20 μL of 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium to each well Bromide salt (5 mg/mL), continue to incubate for 4 h, centrifuge to discard the supernatant, add 200 μL of dimethyl sulfoxide, shake at low speed for 10 min, and detect the absorbance at 570 nm.

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Beneficial effect

The beneficial effects of the present invention are: the present invention is based on the disadvantages of the existing immunomodulatory drugs, such as side effects and high cost, and is dedicated to finding a natural immunomodulator. It starts from Pseudoginseng and focuses on the effects of pepsin and trypsin. The two-step enzymatic hydrolysis process is controlled to prepare active peptides with specific peptide chain lengths, so that the immune activity can be efficiently realized. The enzymolysis technology adopted by the invention is simple and efficient, can track the activity of enzymolysis products to achieve targeted enzymatic digestion, avoids cost waste, and the obtained polypeptide has immunological activity, which provides a theoretical basis for its application in the food and health product industries.

Description of the drawings

Figure 1 shows the G25 elution profile of the proteolysate of Radix Pseudostellariae (a) and the immunological activity of each component (b).

Figure 2 shows the elution profile of RP-HPLC (a) and the immunological activity of each component (b).

Figure 3 shows the total ion current of F18.

Figure 4 shows the peptide fingerprint of immunomodulatory peptides.

Figure 5 shows the proliferation-promoting activity of immunomodulatory peptides.

Embodiments of the present invention

Example 1

The amino acid sequence of the immunomodulatory peptide of the present invention is Arg-Gly-Pro-Pro-Pro.

The preparation method is as follows:

Step 1: Use a Chinese medicine pulverizer to pulverize Radix sylvestris into powder, and pass through a 60-mesh sieve; weigh out 5.0 g of Radix sylvestris powder and add 105 mL 0.27 mol/L sodium hydroxide solution, leached at 52.1 ℃ for 1 h, centrifuged at 10,000 rpm for 20 min, then took the supernatant, adjusted pH to 3.0 with 1 mol/L hydrochloric acid, allowed to stand for 1 h, and centrifuged at 5,000 rpm for 20 min , Take the precipitate, freeze-dry it to become the ginseng protein.

The second step: Weigh 10.0 g of Radix ginseng protein, add 100 mL of deionized water, adjust the pH to 2.0, add 0.8 g of pepsin, and place at 37 ℃ for enzymatic hydrolysis for 5.0 h, then adjust the pH to 7.0, add 0.8 g of pancreas Put the protease in 37 ℃ for 3.0 h, boil in a boiling water bath for 10 min to terminate the reaction, centrifuge at 10,000 rpm for 20 min, take the supernatant, freeze-dry it to obtain the proteolytic product of Radix Pseudostellariae.

Step 3: Weigh 100 mg of the proteolytic product of Pseudostellaria chinensis, add 5 mL of deionized water, stir until it is completely dissolved, centrifuge at 8,000 rpm for 20 min, take the supernatant, pass through a 0.22 μm filter membrane, and use Sephadex G25 Separation and purification: deionized water was used as the eluent, the flow rate was 0.3 mL/min, the detection wavelength was 214 nm, the immunological activity of the eluent corresponding to each absorption peak was measured, and the elution curve of each peak was determined (Figure 1a) ) And immunological activity determination (Figure 1b).

Step 4: Collect G3, which is the most immunologically active component, for reversed-phase high performance liquid chromatography separation: Weigh 200 mg of G3, add 5 mL of deionized water, fully dissolve it and pass through a 0.22 μm filter membrane, and load 100 μL . In the separation of reversed-phase high performance liquid chromatography, linear elution was performed with an acetonitrile solution containing a volume fraction of 0.05% trifluoroacetic acid with a concentration gradient of 0~55 v/v% as the eluent. The column used was Gemini 5μ C18, and the flow rate was 1 mL/min, the detection wavelength is 214 nm, the immunological activity of the eluate corresponding to each absorption peak is measured, and the elution curve (Figure 2a) and immunological activity (Figure 2b) of each peak obtained are measured.

Step 5: Collect F18, the component with the highest immunological activity, and use Nano LC-MS/MS to identify the amino acid sequence of the component after freeze-drying. Using Nano LC-MS/MS to identify the amino acid sequence of the components, the total ion current diagram is shown in Figure 3, and then using MS/MS to identify the 77.35 min ion peak, the full amino acid sequence is obtained as (shown in Figure 4): Arg-Gly-Pro-Pro-Pro (molecular weight is 522 Da) is the immunomodulatory peptide of the present invention.

The sixth step: the immune activity is evaluated by promoting the in vitro proliferation activity of mouse spleen lymphocytes. The mouse spleen lymphocyte suspension was added to a 96-well plate, and phosphate buffer was used as a blank control. The final concentrations of the samples were 10 μg/mL, 50 μg/mL, and 100 μg/mL, respectively. After adding the sample, place it in a 37 °C 5% CO ₂ incubator for different periods of time, add 20 μL of 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium to each well Bromide salt (5 mg/mL), continue to incubate for 4 h, centrifuge to discard the supernatant, add 200 μL of dimethyl sulfoxide, shake at low speed for 10 min, and detect the absorbance at 570 nm. The immunomodulatory peptide of the present invention reaches a maximum spleen cell stimulation index of 1.25 when the stimulation time is 48 hours and the concentration is 100 μg/mL (Figure 5).

The foregoing descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made in accordance with the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims (5)

1. An immunomodulatory peptide, characterized in that the amino acid sequence of the immunological polypeptide is Arg-Gly-Pro-Pro-Pro.
2. A method for preparing immunomodulatory peptides according to claim 1, characterized in that: using Pseudostellaria chinensis as a raw material, protein is extracted from it, and the protein is subjected to enzymatic hydrolysis, separation, purification, and freeze-drying to obtain immune peptides.
3. The method for preparing immunomodulatory peptides according to claim 2, characterized in that: the specific steps of protein extraction are: ground ginseng into powder, and add 0.2 to 0.3 according to the mass concentration of ginseng powder of 4.0-5.0% mol/L sodium hydroxide solution, and then leached at 50-60 °C for 1.0-3.0 h; after leaching, the supernatant was collected by centrifugation, and the pH of the supernatant was adjusted to 2.5-3.5 with 1 mol/L hydrochloric acid. After 1~3 hours of sedimentation, centrifugation and freeze-drying to obtain the ginseng protein.
4. The method for preparing immunomodulatory peptides according to claim 2, wherein the specific steps of the enzymatic hydrolysis are: the protein concentration of Pseudostellaria heterophylla is 1.0~2.0w/v%, the pH is 1.5~2.5, and the temperature is 30~40. ℃, enzymolysis time is 4.0~6.0 h, add 8.0~10.0 w/w% pepsin for enzymolysis, after the reaction is over, adjust the pH to 6.5~7.5, add 8.0~10.0 w/w% trypsin for enzymolysis After the reaction is completed for 2.0 to 4.0 h, immediately place it in a boiling water bath and boil for 10 to 20 minutes, terminate the reaction, and collect the supernatant by centrifugation to obtain the immunomodulatory peptide enzymatic hydrolysis product.
5. The method for preparing immunomodulatory peptides according to claim 2, characterized in that: the specific steps of separation and purification are: Sephadex G25 chromatography and RP-HPLC reversed-phase high performance liquid chromatography are used to separate the enzymatic hydrolysis products , And monitor the immunological activity of the separated components, and collect the components with the best immunological activity; the separation of Sephadex G25 uses deionized water as the eluent, the sample volume is 5 mL, and the flow rate is 0.3 mL/min. The detection wavelength is 214 nm, and the immunological activity of the eluent corresponding to each absorption peak is measured, and the components with the highest immunological activity are collected for reversed-phase high performance liquid chromatography; the reversed-phase high performance liquid chromatography is separated with a volume fraction of 0.05% The concentration gradient of trifluoroacetic acid is 0~55 v/v% acetonitrile solution was used as the eluent for linear elution. The column used was Gemini 5μ C18, the sample volume was 100 μL, the flow rate was 1 mL/min, the detection wavelength was 214 nm, and the corresponding absorption peaks were measured. The immune activity of the eluate is collected and the component with the highest immune activity is collected; after freeze-drying, the amino acid sequence of the component is identified by Nano LC-MS/MS.