CN112843237A - Novel leukocyte extract mixed factor and preparation method thereof - Google Patents

Novel leukocyte extract mixed factor and preparation method thereof Download PDF

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CN112843237A
CN112843237A CN202110126011.4A CN202110126011A CN112843237A CN 112843237 A CN112843237 A CN 112843237A CN 202110126011 A CN202110126011 A CN 202110126011A CN 112843237 A CN112843237 A CN 112843237A
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朱晗
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Hongzhijun Biotechnology Shanghai Co Ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/19—Cytokines; Lymphokines; Interferons
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    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61P37/04—Immunostimulants

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Abstract

The invention discloses a novel leukocyte extract mixed factor, which comprises 15-27 parts of leukocyte extract, 25-33 parts of platelet lysate, 5-10 parts of interleukin, 22-25 parts of stem cell extract and 5-23 parts of leukin according to volume percentage; the invention also discloses a preparation method of the novel leukocyte extract mixed factor, which comprises the following steps: s1 preparing a leukocyte extract; s2 preparing a platelet lysate; s3 preparing mixed factors; s4, drying the finished product; according to the invention, the leukocyte extract is added with the leukocyte, so that the generation quantity of leukocytes and the maturation speed of the leukocytes are improved after the mixed factors enter an animal body on the basis of the healing of the leukocyte extract, and the inflammation healing and the regulation on the immunocompetence are accelerated; in the preparation process of the mixed factor, the cultured white blood cells are used for supplementing the nucleotide and the white blood cell factor, so that the powder yield of the mixed factor can be increased, the treatment effect can be improved, and the treatment effect can be exerted more stably.

Description

Novel leukocyte extract mixed factor and preparation method thereof
Technical Field
The invention relates to the field of cell extraction, and particularly relates to a novel leukocyte extract mixed factor and a preparation method of the novel leukocyte extract mixed factor.
Background
Leukocytes are classified into neutrophils, eosinophils, basophils, monocytes, and lymphocytes. The first three are called granulocytes because they contain chromotropic particles in the cytoplasm, and they are seen microscopically to have a large volume and a small number of nuclei in blood cells. Its main functions are to phagocytize bacteria and defend diseases. Leukocytes can phagocytose foreign bodies, plasma cells can generate antibodies, and the leukocyte plays an important role in healing body injuries, resisting invasion of pathogens and immunizing diseases.
Interleukins are a class of cytokines that are produced by and act on a wide variety of cells. At present, at least 38 interleukins are found, which are respectively named as IL-1-IL 38, have complex functions, form a network and are overlapped in a complex way; plays an important role in a series of processes such as maturation, activation, proliferation and immunoregulation of immune cells, and in addition, the immune cells also participate in various physiological and pathological reactions of organisms.
The existing human leucocyte extract (leucocyte factor) is obtained by taking human leucocyte as a raw material through methods of extraction, adsorption, pH gradient elution and the like, the product does not contain protein and polypeptide, and a single peak with the purity of 100 percent can be obtained through High Pressure Liquid Chromatography (HPLC). Laboratory researches show that the leucocyte extract has the function of enhancing immunity, the immunocompetence of the leucocyte extract is represented by the enhancement of the capacities of rosette formation, rosette restoration, H-TdR doped lymphocyte transformation, leucocyte migration inhibition and the like, and the curative effect of clinical trials is obvious.
At present, leukocyte extracts are mostly applied to anti-inflammatory drugs or skin treatment. Immunotherapy of leukocytes also relies on the intact leukocyte organism. The mixed application of the leukocyte extracts is still to be developed.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a novel leukocyte extract mixed factor and a preparation method of the novel leukocyte extract mixed factor.
In order to achieve the purpose, the invention adopts the following technical scheme: a novel leukocyte extract mixed factor comprises 15-27 parts of leukocyte extract, 25-33 parts of platelet lysate, 5-10 parts of interleukin, 22-25 parts of stem cell extract and 5-23 parts of leukapheresis according to volume percentage.
Preferably, the interleukin selected is from the interleukin-1 family, including IL-1 α, IL-1 β, IL-1 receptor antagonists, IL-18, IL-36Ra, IL-36 α, IL-37, IL-36 β, IL-36 γ, IL-38, and IL-33.
Preferably, the leucogen comprises bisbenzylisoquinoline alkaloid, rubinic acid derivative and synergistic plant; the mass mixing ratio of the bisbenzylisoquinoline alkaloid to the rubioic acid derivative is 1:4.5-1: 3.
Preferably, the synergists include the serenosides, the anisenes and the aminopeptidases, and the mass mixing ratio of the serenosides, the anisenes and the aminopeptidases is 2:2: 3.
Preferably, the mixed factor is suitable for human immunity regulating oral medicine, externally applied skin inflammation medicine and injection organ inflammation medicine.
A preparation method of a novel leukocyte extract mixed factor comprises the following steps: s1 preparing a leukocyte extract; taking HClO for leucocyte4Cracking the solution, preparing suspension, filtering and adsorbing, washing out adsorbate on an adsorption net by using clear water, and then carrying out pH elution and drying to obtain dry leukocyte powder; preparing stem cell extract by the same steps; s2 preparing a platelet lysate; freeze-drying and crushing plasma, and filtering to obtain a platelet lysate; s3 preparing mixed factors; mixing the leukocyte extract, platelet lysate, interleukin, stem cell extract and leukocyte increasing extract to obtain a mixture; s4, drying the finished product; adding the mixed solution of S3 into cell culture medium, culturing with leukocyte at 35-37 deg.C for 2-3 days, taking out the culture, mixing with supporting solution, filtering, drying, and grinding into powder to obtain the final product of leukocyte extract mixed factor.
Preferably, in the step of preparing the mixing factor at S3, the temperature of the liquid mixing environment is 10 to 13 ℃.
The invention has the beneficial effects that: according to the invention, the leukocyte extract is added with the leukocyte, so that the generation quantity of leukocytes and the maturation speed of the leukocytes are improved after the mixed factors enter an animal body on the basis of the healing of the leukocyte extract, and the inflammation healing and the regulation on the immunocompetence are accelerated; in the preparation process of the mixed factor, the mixed factor is mixed with the white blood cells for culture and then is crushed to prepare the finished mixed factor, and the cultured white blood cells are used for supplementing the nucleotide and the white blood cell factor, so that the powder yield of the mixed factor can be increased, the treatment effect can be improved, and the treatment effect can be exerted more stably.
Drawings
FIG. 1 is a table of experimental data for three examples of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example one
A novel leukocyte extract mixed factor comprises 27 parts of leukocyte extract, 33 parts of platelet lysate, 10 parts of interleukin, 25 parts of stem cell extract and 5 parts of leukin according to volume percentage.
In this example, the interleukin selected is from the interleukin-1 family, including IL-1 α, IL-1 β, IL-1 receptor antagonists, IL-18, IL-36Ra, IL-36 α, IL-37, IL-36 β, IL-36 γ, IL-38, and IL-33, and in this example, a mixture of IL-18, IL-36Ra, IL-36 α, and IL-37 is used.
In this embodiment, the leucin includes bisbenzylisoquinoline alkaloids, alizarin derivatives and synergists; the mass mixing ratio of the bisbenzylisoquinoline alkaloid to the rubioic acid derivative is 1:4.
In this embodiment, the collaboration substance includes chamajin, anisylene and aminopeptin, and the mass mixing ratio of the chamajin, the anisylene and the aminopeptin is 2:2: 3.
In this embodiment, the mixed factor of the present invention is suitable for use in oral administration, external application type skin inflammation drug, and injection type organ inflammation drug for regulating human immunity.
A preparation method of a novel leukocyte extract mixed factor comprises the following steps: s1 preparing a leukocyte extract; taking HClO for leucocyte4Cracking the solution, preparing suspension, filtering and adsorbing, washing out adsorbate on an adsorption net by using clear water, and then carrying out pH elution and drying to obtain dry leukocyte powder; preparing stem cell extract by the same steps; s2 preparing a platelet lysate; freeze-drying and crushing plasma, and filtering to obtain a platelet lysate; s3 preparing mixed factors; mixing the leukocyte extract, platelet lysate, interleukin, stem cell extract and leukocyte increasing extract to obtain a mixture; s4, drying the finished product; adding the mixed solution of S3 into cell culture medium, culturing with leukocyte at 36 deg.C for 3 days, taking out the culture, mixing with supporting solution, filtering, drying, and grinding into powder to obtain the final product of leukocyte extract mixed factor.
In the present example, in the step of preparing the mixing factor at S3, the temperature of the liquid mixing environment was 12 ℃.
Example two
A novel leukocyte extract mixed factor comprises 15 parts of leukocyte extract, 25 parts of platelet lysate, 5 parts of interleukin, 22 parts of stem cell extract and 23 parts of leukin according to volume percentage.
In this example, the interleukin selected is from the interleukin-1 family, which includes IL-1 α, IL-1 β, IL-1 receptor antagonists, IL-18, IL-36Ra, IL-36 α, IL-37, IL-36 β, IL-36 γ, IL-38, and IL-33, and specifically a mixture of IL-18, IL-36Ra, IL-36 α, and IL-37.
In this embodiment, the leucin includes bisbenzylisoquinoline alkaloids, alizarin derivatives and synergists; the mass mixing ratio of the bisbenzylisoquinoline alkaloid to the rubioic acid derivative is 1:4.
In this embodiment, the collaboration substance includes chamajin, anisylene and aminopeptin, and the mass mixing ratio of the chamajin, the anisylene and the aminopeptin is 2:2: 3.
In this embodiment, the mixed factor of the present invention is suitable for use in oral administration, external application type skin inflammation drug, and injection type organ inflammation drug for regulating human immunity.
A preparation method of a novel leukocyte extract mixed factor comprises the following steps: s1 preparing a leukocyte extract; taking HClO for leucocyte4Cracking the solution, preparing suspension, filtering and adsorbing, washing out adsorbate on an adsorption net by using clear water, and then carrying out pH elution and drying to obtain dry leukocyte powder; preparing stem cell extract by the same steps; s2 preparing a platelet lysate; freeze-drying and crushing plasma, and filtering to obtain a platelet lysate; s3 preparing mixed factors; mixing the leukocyte extract, platelet lysate, interleukin, stem cell extract and leukocyte increasing extract to obtain a mixture; s4, drying the finished product; adding the mixed solution of S3 into cell culture medium, culturing with leukocyte at 36 deg.C for 3 days, taking out the culture, mixing with supporting solution, filtering, drying, and grinding into powder to obtain the final product of leukocyte extract mixed factor.
In the present example, in the step of preparing the mixing factor at S3, the temperature of the liquid mixing environment was 12 ℃.
EXAMPLE III
A novel leukocyte extract mixed factor comprises 25 parts of leukocyte extract, 30 parts of platelet lysate, 7 parts of interleukin, 23 parts of stem cell extract and 15 parts of leukocyte increasing substance by volume percentage.
In this example, the selected interleukin is from the interleukin-1 family, including IL-1 α, IL-1 β, IL-1 receptor antagonist, IL-18, IL-36Ra, IL-36 α, IL-37, IL-36 β, IL-36 γ, IL-38, and IL-33; in particular to a mixture of IL-18, IL-36Ra, IL-36 alpha, IL-37 and IL-36 beta.
In this embodiment, the leucin includes bisbenzylisoquinoline alkaloids, alizarin derivatives and synergists; the mass mixing ratio of the bisbenzylisoquinoline alkaloid to the rubioic acid derivative is 1:4.
In this embodiment, the collaboration substance includes chamajin, anisylene and aminopeptin, and the mass mixing ratio of the chamajin, the anisylene and the aminopeptin is 2:2: 3.
In this embodiment, the mixed factor of the present invention is suitable for use in oral administration, external application type skin inflammation drug, and injection type organ inflammation drug for regulating human immunity.
A preparation method of a novel leukocyte extract mixed factor comprises the following steps: s1 preparing a leukocyte extract; taking HClO for leucocyte4Cracking the solution, preparing suspension, filtering and adsorbing, washing out adsorbate on an adsorption net by using clear water, and then carrying out pH elution and drying to obtain dry leukocyte powder; preparing stem cell extract by the same steps; s2 preparing a platelet lysate; freeze-drying and crushing plasma, and filtering to obtain a platelet lysate; s3 preparing mixed factors; mixing the leukocyte extract, platelet lysate, interleukin, stem cell extract and leukocyte increasing extract to obtain a mixture; s4, drying the finished product; adding the mixed solution of S3 into cell culture medium, culturing with leukocyte at 36 deg.C for 3 days, taking out the culture, mixing with supporting solution, filtering, drying, and grinding into powder to obtain the final product of leukocyte extract mixed factor.
In the present example, in the step of preparing the mixing factor at S3, the temperature of the liquid mixing environment was 12 ℃.
The powder finished products of the three mixed factors are obtained according to the three embodiments, and an inflammation healing experiment and an immunity test experiment are carried out on the mouse, wherein the immunity test experiment is used for testing the rehabilitation ability of the mouse under the same environment, and the rehabilitation ability of the mouse is determined by taking the rehabilitation time of the mouse as a standard, so that a graph 1 is obtained.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (7)

1. A novel leukocyte extract mixed factor is characterized by comprising 15-27 parts of leukocyte extract, 25-33 parts of platelet lysate, 5-10 parts of interleukin, 22-25 parts of stem cell extract and 5-23 parts of leukin according to volume percentage.
2. The novel leukocyte extract cocktail of claim 1 wherein the selected interleukins are from the interleukin-1 family comprising IL-1 α, IL-1 β, IL-1 receptor antagonists, IL-18, IL-36Ra, IL-36 α, IL-37, IL-36 β, IL-36 γ, IL-38, and IL-33.
3. The novel leukocyte extract cocktail factor of claim 1 wherein the leukapheresis product comprises a bisbenzylisoquinoline alkaloid, an alizarin derivative, and a synergist; the mass mixing ratio of the bisbenzylisoquinoline alkaloid to the rubioic acid derivative is 1:4.5-1: 3.
4. The leukocyte extract MIfactor as claimed in claim 3, wherein the synergists comprise serenosides, anisenes and aminopeptidases, and the mass mixing ratio of the serenosides, the anisenes and the aminopeptidases is 2:2: 3.
5. The leukocyte extract factor cocktail according to claim 1, wherein said leukocyte extract factor is suitable for use as an immunological competence-modulating oral drug, an external-application-type skin inflammation drug, or an injectable-type organ inflammation drug.
6. A preparation method of a novel leukocyte extract mixed factor is characterized by comprising the following steps:
s1 preparing a leukocyte extract; taking HClO for leucocyte4Cracking the solution, preparing suspension, filtering and adsorbing, washing out adsorbate on an adsorption net by using clear water, and then carrying out pH elution and drying to obtain dry leukocyte powder; preparing stem cell extract by the same steps;
s2 preparing a platelet lysate; freeze-drying and crushing plasma, and filtering to obtain a platelet lysate;
s3 preparing mixed factors; mixing the leukocyte extract, platelet lysate, interleukin, stem cell extract and leukocyte increasing extract to obtain a mixture;
s4, drying the finished product; adding the mixed solution of S3 into cell culture medium, culturing with leukocyte at 35-37 deg.C for 2-3 days, taking out the culture, mixing with supporting solution, filtering, drying, and grinding into powder to obtain the final product of leukocyte extract mixed factor.
7. The method of claim 6, wherein the temperature of the liquid mixing environment is 10-13 ℃ in the step of S3.
CN202110126011.4A 2021-01-29 2021-01-29 Novel leukocyte extract mixed factor and preparation method thereof Pending CN112843237A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1210718A (en) * 1997-09-11 1999-03-17 中国医学科学院血液学研究所 Method for preparing leukocytic extract as immunopotentiator
CN101439088A (en) * 2008-07-22 2009-05-27 罗丹晓 Medicament for promoting leucocyte hyperplasia
CN104622777A (en) * 2015-02-15 2015-05-20 广州赛莱拉干细胞科技股份有限公司 Leukocyte extract and preparation method and application thereof
CN111375048A (en) * 2018-12-27 2020-07-07 北京第一生物化学药业有限公司 Application of spleen aminopeptide in preparation of medicines for treating leukopenia
CN111467374A (en) * 2020-04-28 2020-07-31 杨喻丹 Novel leukocyte extract mixed factor and preparation method thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1210718A (en) * 1997-09-11 1999-03-17 中国医学科学院血液学研究所 Method for preparing leukocytic extract as immunopotentiator
CN101439088A (en) * 2008-07-22 2009-05-27 罗丹晓 Medicament for promoting leucocyte hyperplasia
CN104622777A (en) * 2015-02-15 2015-05-20 广州赛莱拉干细胞科技股份有限公司 Leukocyte extract and preparation method and application thereof
CN111375048A (en) * 2018-12-27 2020-07-07 北京第一生物化学药业有限公司 Application of spleen aminopeptide in preparation of medicines for treating leukopenia
CN111467374A (en) * 2020-04-28 2020-07-31 杨喻丹 Novel leukocyte extract mixed factor and preparation method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
胡亚美等: "常用升白细胞药物", 《儿科药物治疗学》 *

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