WO2021082294A1 - Peptide immunomodulateur, préparation et procédé associés - Google Patents

Peptide immunomodulateur, préparation et procédé associés Download PDF

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Publication number
WO2021082294A1
WO2021082294A1 PCT/CN2020/073062 CN2020073062W WO2021082294A1 WO 2021082294 A1 WO2021082294 A1 WO 2021082294A1 CN 2020073062 W CN2020073062 W CN 2020073062W WO 2021082294 A1 WO2021082294 A1 WO 2021082294A1
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pro
protein
immunomodulatory
freeze
collected
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PCT/CN2020/073062
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English (en)
Chinese (zh)
Inventor
汪少芸
杨倩
蔡茜茜
陈旭
田永奇
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福州大学
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Publication of WO2021082294A1 publication Critical patent/WO2021082294A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the invention relates to a method for separating and purifying immunoregulatory peptides by using Radix Pseudostellariae, belonging to the field of biotechnology.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an immunomodulatory peptide of the present invention is Arg-Gly-Pro-Pro-Pro (RGPPP), and the preparation includes the following steps:
  • 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.
  • NaOH sodium hydroxide solution
  • 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 °C, 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.
  • step (3) 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 5 ⁇ C18, and the sample volume is 100 ⁇ L, 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.
  • step (3) 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 ⁇ g/mL, 50 ⁇ g/mL, and 100 ⁇ g/mL, respectively.
  • 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.
  • 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.
  • 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 °C 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 °C 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 °C 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 ⁇ m 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 ⁇ m filter membrane, and load 100 ⁇ L . 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 5 ⁇ 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.
  • 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 ⁇ g/mL, 50 ⁇ g/mL, and 100 ⁇ g/mL, respectively.
  • 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 ⁇ g/mL ( Figure 5).

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé d'isolement et de purification d'un peptide immunomodulateur à l'aide de Pseudostellaria heterophylla. Le procédé utilise Pseudostellaria heterophylla comme matière première pour obtenir un polypeptide d'immunité spécifique après utilisation d'une extraction de protéine, une enzymolyse à deux étapes de pepsine et de trypsine, un isolement, une purification et une lyophilisation. Le polypeptide a un poids moléculaire de 522 Da, et sa séquence d'acides aminés complète est : Arg-Gly-Pro-Pro-Pro. La présente invention élimine les défauts présents dans des médicaments immunomodulateurs, et permet de résoudre des problèmes se rapportant aux immunomodulateurs artificiels ; de plus, la présente invention pose un fondement théorique pour le développement de peptides d'immunité à base de sources alimentaires ainsi que pour l'exploration de larges applications de ceux-ci dans des aliments et des médicaments.
PCT/CN2020/073062 2019-10-31 2020-01-19 Peptide immunomodulateur, préparation et procédé associés WO2021082294A1 (fr)

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CN110655556B (zh) * 2019-10-31 2021-05-18 福州大学 一种免疫调节肽的制备及方法
CN111978374B (zh) * 2020-07-29 2022-03-04 中国科学院华南植物园 一种免疫调节活性六肽及其制备方法和应用
CN111825745B (zh) * 2020-07-29 2022-03-04 中国科学院华南植物园 一种免疫调节活性七肽及其制备方法和应用
CN114195860B (zh) * 2021-12-16 2023-07-25 吉林国瑞医药有限公司 一种轮叶党参来源的抗血管生成及抗肿瘤活性肽及其制备方法和应用
CN114213516B (zh) * 2021-12-16 2023-07-25 吉林国瑞医药有限公司 一种轮叶党参来源的催乳及抗乳腺肿瘤的活性肽及其制备方法和用途

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* Cited by examiner, † Cited by third party
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CN110655556A (zh) * 2019-10-31 2020-01-07 福州大学 一种免疫调节肽的制备及方法

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CN104610430B (zh) * 2015-02-26 2018-02-27 江苏安惠生物科技有限公司 利用灵芝蛋白制备的抗氧化多肽及其制备方法
CN108840928B (zh) * 2018-06-26 2020-09-01 福州大学 一种太子参胰蛋白酶抑制剂及其制备方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110655556A (zh) * 2019-10-31 2020-01-07 福州大学 一种免疫调节肽的制备及方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
YANG QIAN, CAI XIXI, HUANG MUCHEN, JIA LEE, WANG SHAOYUN: "Immunomodulatory effects of Pseudostellaria heterophylla peptide on spleen lymphocytes via a Ca 2+ /CaN/NFATc1/IFN-γ pathway", FOOD & FUNCTION, R S C PUBLICATIONS, GB, vol. 10, no. 6, 19 June 2019 (2019-06-19), GB, pages 3466 - 3476, XP055809259, ISSN: 2042-6496, DOI: 10.1039/C9FO00577C *
YANG QIAN; CAI XIXI; HUANG MUCHEN; WANG SHAOYUN: "A specific peptide with immunomodulatory activity from Pseudostellaria heterophylla and the action mechanism", JOURNAL OF FUNCTIONAL FOODS, ELSEVIER BV, NL, vol. 68, 15 March 2020 (2020-03-15), NL, XP086145334, ISSN: 1756-4646, DOI: 10.1016/j.jff.2020.103887 *
YANG QIAN; HUANG MUCHEN; CAI XIXI; JIA LEE; WANG SHAOYUN: "Investigation on activation in RAW264.7 macrophage cells and protection in cyclophosphamide-treated mice of Pseudostellaria heterophylla protein hydrolysate", FOOD AND CHEMICAL TOXICOLOGY, PERGAMON, GB, vol. 134, 10 September 2019 (2019-09-10), GB, XP085885961, ISSN: 0278-6915, DOI: 10.1016/j.fct.2019.110816 *

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