CN115536732A - Beta-glucan thymopentin conjugate, preparation method and application thereof in immunoregulation, antitumor drugs and health care products - Google Patents
Beta-glucan thymopentin conjugate, preparation method and application thereof in immunoregulation, antitumor drugs and health care products Download PDFInfo
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- CN115536732A CN115536732A CN202211382134.5A CN202211382134A CN115536732A CN 115536732 A CN115536732 A CN 115536732A CN 202211382134 A CN202211382134 A CN 202211382134A CN 115536732 A CN115536732 A CN 115536732A
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of chemical synthetic medicines, and particularly relates to a preparation method of a beta-glucan thymopentin conjugate and application of the beta-glucan thymopentin conjugate in preparation of immunoregulation medicines and health-care products. The conjugate of beta-glucan and thymopentin is obtained by covalently coupling a reducing end hemiacetal group of the beta-glucan and a free amino group of thymopentin. The method has the advantages of high reaction efficiency, definite product structure, specific reaction site and no by-product. The invention synthesizes the beta-glucan thymopentin conjugate for the first time, the glycopeptide has obvious immunoregulation and anti-tumor effects, has no cytotoxicity to RAW264.7 macrophage and can obviously promote the phagocytosis capacity of the macrophage, has strong recovery capacity to the bone marrow suppression state of mice with low immunity induced by cyclophosphamide, has obvious tumor growth inhibition effect on 4T1 and B16F10 tumor model mice, and can be used for preparing immunopotentiators, anti-tumor medicaments, health care products and formula foods with special medical application.
Description
Technical Field
The invention belongs to the technical field of chemical synthetic medicines, and particularly relates to a beta-glucan thymopentin conjugate, a preparation method and application thereof in immune system regulation, antitumor drugs and health care products.
Background
Beta-glucans are a class of polysaccharides composed of glucose residues linked together in different ways via beta-glycosidic bonds and widely occur in nature. Beta-1, 3-backbones of natural origin and having/beta-1, 6-branching degree glucans are mainly present in various fungi, bacteria and seaweeds, and beta-glucans of different structures have different biological activities. Beta-glucan is widely reported to play roles in immunoregulation, anti-inflammation, antivirus, antitumor and the like by activating macrophages, dendritic cells and the like.
Thymopentin (TP 5), an active fragment of thymopoietin II which is a polypeptide compound separated from thymic hormone, can promote the differentiation and development of thymus and peripheral T lymphocytes, has the same all physiological functions as thymopoietin II, has a bidirectional regulation effect on the immune function of an organism, and is an important clinically immune balance regulator. The thymopentin consists of five amino acids of arginine, lysine, aspartic acid, valine and tyrosine, and the amino acid sequence of the thymopentin is Arg-Lys-Asp-Val-Tyr.
The thymopentin is a peptide drug, and the indication is to treat patients with primary or secondary T cell deficiency. Since it is easily degraded by gastric acid and is rapidly degraded by proteases and aminopeptidases in human blood, the plasma half-life is only about 30 seconds. Thymopentin is easy to be filtered and cleared by glomeruli due to its small molecular weight and is discharged into urine. At present, thymopentin is clinically injected with large dosage, high frequency and long treatment period, so that patients cannot tolerate the thymopentin easily and the compliance is poor. Therefore, the development of derivatives capable of prolonging the half-life of the thymopentin drug or improving the plasma stability has important research significance.
Aiming at the problems of short half-life period of the thymopentin and the like, the long-acting thymopentin analogue is mainly developed at home and abroad to prolong the half-life period of the thymopentin analogue so as to increase the retention time in vivo, or the long-acting preparation effect is achieved through dosage form modification optimization and the like. In addition, studies have shown that plasma stability can also be improved by functionally derivatizing the structure of thymopentin.
The technical problems existing in the prior art are as follows: at present, no beta-glucan thymopentin conjugate is developed and applied to immune system regulation, anti-tumor drugs and health care products and formula food application for special medical application.
The significance of solving the problems and the defects is as follows: (1) The beta-glucan thymic pentapeptide conjugate is prepared for the first time, and the preparation method has the characteristics of high reaction efficiency, clear product structure characteristics, specific reaction site, no by-product and the like, and can be industrialized on a large scale. (2) On one hand, the thymopentin is modified by beta-glucan, so that the molecular weight of the thymopentin is increased, the in vivo retention time is prolonged, and the enzyme degradation clearance rate in plasma is reduced; on the other hand, the beta-glucan is subjected to thymopentin modification, so that the two immune regulation effects can be synergistically exerted, and the effect of' 1+1 >. The beta-glucan thymopentin conjugate obtained based on the preparation method has obvious immune regulation and anti-tumor effects, and the effect is more obvious than that of singly using beta-glucan, singly using thymopentin and a mixture of beta-glucan and thymopentin. The invention can be used for preparing immunopotentiators, antitumor drugs, health products and formula foods with special medical application.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a conjugate of beta-glucan and thymopentin, a preparation method and application thereof in immune system regulation, anti-tumor drugs and health care products.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a β -glucan and thymopentin conjugate, the β -glucan and thymopentin conjugate having the structure:
wherein n = 1-100, m = 1-30 in the structural formula of the glycopeptide conjugate.
The conjugate of beta-glucan and thymopentin is a glyco-peptide conjugate (BG-TP) obtained by covalently coupling a hemiacetal group at the reducing end of beta-glucan and the amino group of thymopentin serving as an immunoregulation medicament, and the specific preparation method of the conjugate of beta-glucan and thymopentin is as follows:
step 1: dissolving beta-glucan (BG, 1e.q.) and thymopentin (TP 5, 10e.q.) in a dimethyl sulfoxide/acetic acid mixed reaction solvent (83, 17, v/v), and reacting in a water bath at 60 ℃ for 1 hour under the protection of nitrogen;
step 2: sodium cyanoborohydride (NaBH) 3 CN, 25e.q.) is dissolved in water, the reaction solution is added, and the reaction is continued for 24 hours; sodium cyanoborohydride (NaBH) was added again 3 CN, 25e.q.) solid powder, and the reaction was continued for 24 hours;
and step 3: and after the reaction is finished, adding 16% sodium chloride (NaCl) solution, uniformly mixing, adding 4-5 times of volume of absolute ethyl alcohol for alcohol precipitation, centrifuging for 10 minutes, adding the sodium chloride solution again to the precipitate until the precipitate is just dissolved, continuing alcohol precipitation, repeating for 3 times, collecting the precipitate, dialyzing for 48 hours, concentrating under reduced pressure, and freeze-drying to obtain the glycopeptide conjugate (BG-TP) in which the beta-glucan and the thymopentin are covalently coupled.
Preferably, the molecular weight of the beta-glucan is 0.1 kDa-300 kDa, the monosaccharide composition is mainly glucose, the content of the monosaccharide is more than 95 percent, the main chain is formed by connecting the glucose through beta-1, 3 glycosidic bonds, and meanwhile, the main chain is provided with beta-1, 6 branches, and the branching degree is 1 percent-30 percent.
Preferably, the selected peptides are Thymopentin (TP 5) and other peptide drug analogs containing Thymopentin active fragment (RKDVY).
Preferably, the volume ratio of the dimethyl sulfoxide to the acetic acid in the mixed reaction solvent is 83 (v/v).
Preferably, the equivalent ratio of beta glucan to thymopentin is 1; the beta-glucan and sodium cyanoborohydride (NaBH) 3 CN) in an equivalent ratio of 1; the concentration of the sodium chloride solution used in the alcohol precipitation and dialysis process is 4-32%.
A scheme for the preparation of conjugates of β -glucan and thymopentin is as follows:
further, the conjugate of the beta-glucan and the thymopentin is applied to medicines, health-care products and formula foods with special medical application for regulating an immune system and resisting tumors. The medicine, health product and food for special medical use are in the forms of injection, powder injection, oral liquid, tablet, capsule, soft capsule and powder.
Further, the application of the conjugate of the beta-glucan and the thymopentin in recovering an immune system is specifically shown in the fact that the conjugate has a remarkable improvement effect on a bone marrow suppression state of a cyclophosphamide-induced immunocompromised mouse model, promotes the phagocytic capacity of abdominal macrophages, improves the total number of lymphocytes in the spleen and the CD4+/CD8+ T ratio to recover the immune system, and promotes the secretion capacity of cytokines such as TNF-alpha, IL-1 beta, IFN-gamma and the like and antibodies such as IgG, sIgA and the like in serum of a mouse; the application of the beta-glucan and thymopentin conjugate in immunoregulation specifically is the non-cytotoxicity effect on RAW264.7 macrophages, the phagocytosis capacity and the cytokine release capacity of the conjugate can be remarkably promoted, and the effect is more remarkable than that of the single use of the beta-glucan, the single use of the thymopentin and the mixture of the beta-glucan and the thymopentin.
Furthermore, the conjugate of beta-glucan and thymopentin has the anti-tumor application, specifically has the effect of inhibiting tumor growth on 4T1 and B16F10 tumor model mice, and can promote the secretion capacity of cytokines such as TNF-alpha, IL-1 beta, IFN-gamma and the like in mouse serum.
To verify the technical effect of the conjugate of β -glucan and thymopentin, the inventors performed the following experiments:
the technical effect is as follows: the beta-glucan and thymic pentapeptide conjugate has an immunoregulation effect, shows no cytotoxicity to RAW264.7 macrophage, and can remarkably promote the phagocytic capacity and the cytokine release capacity of the conjugate. Provides basis for developing the glycopeptidyl conjugate BG-TP into immunopotentiators, health products, formula foods with special medical application and the like. The experimental verification steps are as follows:
the method comprises the following steps: after a certain number of RAW264.7 macrophages were cultured and resuspended in PBS at 5X 10 5 The number of the holes is inoculated in a 96-hole plate, and glycopeptide conjugate with different concentrations is added for stimulation for 24 hours; cell proliferation activity is detected by a CCK8 method, phagocytosis capacity is detected by a neutral red phagocytosis method, and cell cytokine secretion conditions such as TNF-alpha, IL-1 beta, IFN-gamma and the like in cell supernatant are detected by an Elisa kit. The result shows that the glycopeptidyl conjugate BG-TP has no cytotoxicity in a selected concentration range, the cell proliferation activity is over 80 percent, and the glycopeptidyl conjugate BG-TP can remarkably promote RAW264.7 macrophages to secrete cytokines such as IL-1 beta and the like along with dose dependence, and has remarkable effect compared with the single use of beta-glucan (BG), the single use of Thymopentin (TP) and the mixture of the beta-glucan and thymopentin (BG + TP).
The technical effect is as follows: the conjugate of beta-glucan and thymopentin has the capability of recovering an immune system, shows that the conjugate has a remarkable improvement effect on the bone marrow suppression state of a mouse with a low immune model, promotes the phagocytosis capability of macrophages in abdominal cavities, promotes the secretion of cytokines and antibodies in the blood serum of the mouse and maintains the immune steady-state capability. Provides basis for developing immunopotentiators, health products, special medical application formula foods and the like. The experimental verification steps are as follows:
step three: after 7-week-old Bal/C mice were acclimatized and raised for 2 weeks, beta-glucan and thymopentin conjugate (BG-TP) were administered at a dose of 4mg/kg once every 3 days by tail vein administration for a total of 9 th administration.
Step four: cyclophosphamide is continuously administrated for 3 days at a dose of 80mg/kg, a mouse model with low immunity is established by intraperitoneal injection, blood is taken from the tail tip every day after 3 days, the number of leucocytes is monitored by blood routine analysis, and the bone marrow suppression state of the model mouse is evaluated. The result shows that after the induction of cyclophosphamide, the white blood cell count is obviously reduced, and the glycopeptide conjugate BG-TP can improve the white blood cell count and relieve the bone marrow inhibition induced by cyclophosphamide.
Step five: killing the mice, extracting abdominal macrophages from abdominal cavities of the mice, and evaluating the phagocytic capacity of the abdominal macrophages of the mice in different administration groups; taking fresh spleen tissues, and detecting the total number of lymphocytes and the ratio of CD4+/CD8+ T in the spleen by using flow cytometry. The results show that CD4 in spleen of model mouse is induced by cyclophosphamide + Reduced T cell count, CD8 + Increased T cell number, resulting in CD4 + /CD8 + The ratio is reduced, and the glycopeptide conjugate BG-TP can obviously improve and recover CD4 + /CD8 + The ratio is close to normal value, which shows that the medicine has the functions of recovering the immune system and maintaining the immune homeostasis
Step six: the secretion of cytokines such as TNF-alpha, IL-1 beta, IFN-gamma and the like and antibodies such as IgG, sIgA and the like in mouse serum is detected by an Elisa kit. The results show that after cyclophosphamide induction, the secretion of cytokines such as TNF-alpha and IFN-gamma in the serum of a model mouse and antibodies such as IgG and sIgA is obviously reduced, and the glycopeptide conjugate BG-TP can be obviously improved and restored to a normal level, which shows that the conjugate has the function of obviously restoring the immunoregulation function.
The technical effect is three: the conjugate (BG-TP) of beta-glucan and thymopentin has anti-tumor effect, can inhibit tumor growth of 4T1 and B16F10 tumor model mice, can promote secretion of cytokines such as TNF-alpha, IL-1 beta and IFN-gamma in mouse serum, and has immunity and anti-tumor effect. Provides basis for developing a glycopeptide conjugate BG-TP into an immunopotentiator, an antitumor drug, a formula food with special medical application and the like. The experimental verification steps are as follows:
step seven: after 7-week-old C57 mice were acclimatized and raised for 2 weeks, the conjugates of beta-glucan and thymopentin (BG-TP) were administered to the mice at a dose of 4mg/kg once every 3 days by tail vein administration.
Step eight: after the 7 th dose, cultured B16F10 and 4T1 cells were resuspended in PBS, and the cell count was 5-45X 10 5 mL, subcutaneously inoculated in the right axilla of C57 mice at a dose of 100 microliters per mouse. Monitoring the growth of tumor volume every day until the tumor volume reaches 1500mm 3 The experiment was stopped. The result shows that the conjugate of beta-glucan and thymopentin (BG-TP) has obvious inhibition effect on the tumor growth of 4T1 and B16F10 tumor model mice.
Step nine: the mice are sacrificed, tumor tissues and other related organs are taken, and cytokines such as TNF-alpha, IL-1 beta and the like and IgG antibody secretion conditions in the serum of the mice are detected by an Elisa kit. The result shows that the secretion of cytokines such as TNF-alpha, IL-1 beta and the like and IgG antibodies in the serum of a B16F10 tumor model mouse is obviously reduced, and the glycopeptide conjugate BG-TP can be obviously improved and restored to a normal level, which indicates that the glycopeptide conjugate BG-TP has the function of obviously restoring the immunoregulation function and playing a role in resisting tumors.
The invention has the beneficial technical effects that: (1) On one hand, the thymopentin is modified by beta-glucan, so that the in-vivo retention time is increased, and the enzyme degradation clearance rate in plasma is reduced; on the other hand, the beta-glucan is subjected to thymopentin modification, so that the two immune regulation effects can be synergistically exerted, and the effect of' 1+1 >. (2) The method has the characteristics of high reaction efficiency, definite product structural characteristics, specific reaction site, no by-product and the like, and is suitable for synthesizing the carbohydrate compounds with different monosaccharide types, different molecular weights and different structural characteristics and the peptide drug covalent conjugates with different functions and drug effects. (3) The beta-glucan thymopentin conjugate obtained based on the preparation method has obvious immune regulation and anti-tumor effects, and the effect is obviously superior to that of the beta-glucan alone, the thymopentin alone and a mixture of the beta-glucan and the thymopentin. (4) The beta-glucan thymopentin conjugate obtained by the invention can be used for preparing immunopotentiators, antitumor drugs, health products and formula foods with special medical application.
Drawings
FIG. 1 is a scheme showing the synthesis of beta-glucan thymopentin conjugate (BG-TP);
FIG. 2 is a nuclear magnetic hydrogen spectrum of beta-glucan thymopentin conjugate (BG-TP);
FIG. 3 is a beta-glucan thymopentin conjugate (BG-TP) IR spectrum;
FIG. 4 is a UV full wavelength scan of a beta-glucan thymopentin conjugate (BG-TP);
FIG. 5 is a high resolution mass spectrum of a beta-glucan thymopentin conjugate (BG-TP);
FIG. 6 is a graph of the immunomodulatory effect of beta-glucan thymopentin conjugate (BG-TP) on RAW264.7 cells;
FIG. 7 is a graph of the immune recovery of beta-glucan thymopentin conjugate (BG-TP) in immunocompromised mice;
FIG. 8 is a graph of the inhibition of beta-glucan thymopentin conjugate (BG-TP) on a 4T1 tumor model.
FIG. 9 is a graph of the inhibition of beta-glucan thymopentin conjugate (BG-TP) on B16F10 tumor models.
Detailed Description
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
Example 1
The preparation method of beta-glucan thymopentin conjugate (BG-TP) is as follows (as shown in figure 1):
the method comprises the following steps: beta-glucan BG (100mg, 0.02mM, 1e.q.) and thymopentin TP5 (136 mg, 0.2mM, 10e.q.) were dissolved in 4mL of a dimethylsulfoxide/acetic acid mixed reaction solvent (83, 17, v/v), and reacted in a water bath at 60 ℃ for 1 hour under nitrogen protection;
step two: sodium cyanoborohydride (31.4 mg,0.5mM, 25e.q.) was dissolved in water, and the above reaction solution was added to continue the reaction for 24 hours; sodium cyanoborohydride (31.4 mg,0.5mM, 25e.q.) was added again as a solid powder, and the reaction was continued for 24 hours.
And step three, after the reaction is finished, adding 2mL of 16% sodium chloride solution, uniformly mixing, adding 4-5 times of volume of absolute ethyl alcohol for alcohol precipitation, centrifuging for 10 minutes, adding the sodium chloride solution again to the precipitate until the precipitate is just dissolved, continuing alcohol precipitation, repeating for 3 times, collecting the precipitate, dialyzing for 48 hours, concentrating under reduced pressure, and freeze-drying to obtain the glycopeptide conjugate (BG-TP) in which the beta-glucan and the thymopentin are covalently coupled.
Example 2
The structural characterization procedure of the beta-glucan thymopentin conjugate (BG-TP) was as follows:
(1) Nuclear magnetic hydrogen spectrum characterization: taking 5mg of glycopeptide conjugate (BG-TP), adding 500 mu L of heavy water, freeze-drying, repeating 3 times of heavy water exchange, adding 500 mu L of heavy water and transferring into a nuclear magnetic resonance tube, and performing nuclear magnetic resonance spectrum analysis at 25 ℃ by using deuterated acetone as an internal standard. As shown in FIG. 2, the characteristic signals of thymopentin appeared at 6.75 and 7.05 and 0.75 ppm, indicating that the glycopeptidyl conjugate (BG-TP) is formed by covalently coupling beta-glucan and thymopentin.
(2) And (3) infrared spectrum characterization: taking a proper amount of glycopeptide conjugate (BG-TP) to perform infrared spectrum characterization by a KBr tabletting method. The results are shown in FIG. 3, where a signature for thymopentin is shown at 1500, indicating that the glycopeptide conjugate (BG-TP) is formed by covalently coupling beta-glucan to thymopentin.
(3) Ultraviolet full-wavelength scanning characterization: a proper amount of glycopeptide conjugate (BG-TP) is taken to pass through a 200-600nm ultraviolet full-wavelength scanning, the result is shown in figure 4, thymopentin characteristic signals appear at 225 nm and 280nm, and the glycopeptide conjugate (BG-TP) is formed by covalently coupling beta-glucan and thymopentin.
(4) High-resolution mass spectrometry characterization: an appropriate amount of glycopeptide conjugate (BG-TP) was taken for high resolution mass spectrometry characterization, and the results are shown in FIG. 5, wherein fragment ions such as 1579.24 and 1633.25 with z =3 appear in the range of 984.71-2281.46, and the fragment ions are ion fragments formed by covalently coupling beta-glucan and thymopentin with different polymerization degrees.
Example 3
The beta-glucan and thymic pentapeptide conjugate (BG-TP) has an immunoregulation effect, shows no cytotoxicity to RAW264.7 macrophage, and can remarkably promote the phagocytic capacity and the cytokine release capacity of the RAW264.7 macrophage. Provides basis for developing the glycopeptidyl conjugate BG-TP into immunopotentiators, health products, formula foods with special medical application and the like. The specific verification experiment is as follows:
after a certain number of RAW264.7 macrophages were cultured and resuspended in PBS at 5X 10 5 The number of/well is inoculated in a 96-well plate, and the beta-glucan thymopentin conjugate (BG-TP) with the final concentration of 2 mu M, 6 mu M, 18 mu M, 54 mu M and 162 mu M is added for stimulation for 24 hours; cell proliferation activity is detected by a CCK8 method, phagocytosis capacity is detected by a neutral red phagocytosis method, and cell cytokine secretion conditions such as TNF-alpha, IL-1 beta, IFN-gamma and the like in cell supernatant are detected by an Elisa kit. As shown in fig. 6, the glycopeptide conjugate BG-TP has no cytotoxicity in a selected concentration range, has a cell proliferation activity of 80% or more, and can significantly promote the secretion of cytokines such as IL-1 β from RAW264.7 macrophages in a dose-dependent manner, and has a significant effect as compared with the use of β -glucan (BG) alone, thymopentin (TP) alone, and a mixture of β -glucan and thymopentin (BG + TP).
Example 4
The beta-glucan and thymic pentapeptide conjugate (BG-TP) has the capability of restoring the immune system, shows that the conjugate has the obvious improvement effect on the bone marrow inhibition state of a low-immunity model mouse, promotes the phagocytosis capability of abdominal macrophages, promotes the secretion capability of cytokines and antibodies in mouse serum and maintains the immune steady-state capability. Provides basis for developing immunopotentiators, health products, special medical application formula foods and the like. . The specific verification experiment is as follows:
after 7-week-old Bal/C mice were bred for 2 weeks, glycopeptide conjugates (BG-TP) were administered by tail vein administration at doses of 4mg/kg and 16mg/kg, respectively, once every 3 days. Cyclophosphamide is continuously administrated for 3 days at a dose of 80mg/kg, a mouse model with low immunity is constructed by intraperitoneal injection, blood is taken from the tail tip every day after 3 days, the number of leucocytes is monitored by blood routine analysis, and the bone marrow suppression state of the model mouse is evaluated. Mice were sacrificed and peritoneal macrophages were withdrawn from the abdominal cavity of the mice and evaluated for different dosingPhagocytic capacity of macrophage in abdominal cavity of mice; taking fresh spleen tissues, and detecting the total number of lymphocytes and the ratio of CD4+/CD8+ T in the spleen by using flow cytometry. The secretion of cytokines such as TNF-alpha, IL-1 beta, IFN-gamma and the like and antibodies such as IgG, sIgA and the like in mouse serum is detected by an Elisa kit. The results are shown in fig. 7, and after cyclophosphamide induction, the white blood cell count of the peripheral blood of the model mouse is obviously reduced; CD4 in spleen + Reduced T cell number, CD8 + Increased T cell number, resulting in CD4 + /CD8 + The ratio is decreased; the secretion of cytokines such as TNF-alpha, IFN-gamma and the like and antibodies such as IgG, sIgA and the like in serum is obviously reduced. The glycopeptide conjugate BG-TP can improve the white blood cell count and relieve the bone marrow depression induced by cyclophosphamide; significantly improve and recover CD4 + /CD8 + The ratio tends to normal value, and the immune regulation function of the immune system is recovered, so that the immune homeostasis is maintained.
Example 5
The beta-glucan thymopentin conjugate (BG-TP) has anti-tumor effect, can inhibit tumor growth of tumor model mice, can promote secretion of cytokine in mouse serum, and has immunity and anti-tumor effects. Provides basis for developing a glycopeptide conjugate BG-TP into an immunopotentiator, an antitumor drug, a formula food with special medical application and the like. The specific verification experiment is as follows:
after 7-week-old C57 mice were bred for 2 weeks, the glycopeptide conjugate (BG-TP) was administered to the mice at a dose of 4mg/kg once every 3 days by tail vein administration. After the 7 th dose, cultured B16F10 and 4T1 cells were resuspended in PBS, and the cell count was 5-45X 10 5 mL, subcutaneously inoculated in the right axilla of C57 mice at a dose of 100 microliters per mouse. Monitoring the growth of tumor volume every day until the tumor volume reaches 1500mm 3 The experiment was stopped. The mice are sacrificed, tumor tissues and other related organs are taken, and the secretion conditions of cytokines such as TNF-alpha, IL-1 beta, IFN-gamma and the like in the serum of the mice are detected by an Elisa kit. As shown in FIGS. 8 and 9, the conjugate of beta-glucan thymopentin (BG-TP) showed significant inhibitory effect on tumor growth in 4T1 and B16F10 tumor model mice. For TNF-alpha and IL-1 in serum of tumor model miceThe secretion of cytokines such as beta and the like and IgG antibodies is obviously reduced, and the glycopeptidyl conjugate BG-TP can be obviously improved and recovered to a normal level, which shows that the glycopeptidyl conjugate BG-TP has the function of obviously recovering the immunoregulation function and playing a role in resisting tumors.
In conclusion, the beta-glucan thymopentin conjugate (BG-TP) is obtained by covalently coupling a beta-glucan reduction end hemiacetal group and an amino group of thymopentin. On one hand, the glycopeptidyl conjugate increases the molecular weight, increases the retention time in vivo and reduces the enzyme degradation clearance rate in blood plasma by carrying out beta-glucan modification on thymopentin; on the other hand, the beta-glucan is modified by thymopentin, so that the two immunoregulation functions can be synergistically exerted, and the effect of ' 1+1> ' 2 ' is achieved. The method has the characteristics of high reaction efficiency, definite structural characteristics of products, specific reaction sites, no by-products and the like, and is suitable for synthesizing saccharide compounds with different monosaccharide types, different molecular weights and different structural characteristics and peptide drug covalent conjugates with different functions and drug effects. The glycopeptide conjugate obtained based on the strategy has obvious immune regulation and anti-tumor effects, and the effect is more obvious than that of singly using beta-glucan, singly using thymopentin and a mixture of the beta-glucan and the thymopentin. The glycopeptide conjugate has no cytotoxicity to RAW264.7 macrophage and can remarkably promote the phagocytosis capacity of the macrophage; the recovery capability on the bone marrow suppression state of a cyclophosphamide-induced immunocompromised mouse model is strong; has the function of inhibiting the growth of tumors of 4T1 and B16F10 tumor model mice. The glycopeptidyl conjugate can be used for preparing immunopotentiators, antitumor drugs, health products and formula foods with special medical application.
The above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A β -glucan thymopentin conjugate, wherein the β -glucan and thymopentin conjugate has the structure:
wherein n = 1-100, m = 1-30 in the structural formula of the glycopeptide conjugate.
2. A method of preparing the beta-glucan thymopentin conjugate (BG-TP) of claim 1, wherein: the beta-glucan thymopentin conjugate is obtained by covalently coupling a beta-glucan reducing end hemiacetal group and an amino group of an immunoregulation medicament thymopentin.
3. The method of claim 2, wherein the β -glucan thymopentin conjugate is prepared by the steps of:
(1) Dissolving beta-glucan (BG) and thymopentin (TP 5) in a dimethyl sulfoxide/acetic acid mixed reaction solvent, and reacting in a water bath at 60 ℃ for 1 hour under the protection of nitrogen;
(2) Sodium cyanoborohydride (NaBH) 3 CN) is dissolved in water, the reaction solution is added, and the reaction is continued for 24 hours; sodium cyanoborohydride (NaBH) was added again 3 CN) solid powder, and continuing the reaction for 24 hours;
(3) After the reaction is finished, adding an equal volume of sodium chloride (NaCl) salt solution, uniformly mixing, adding 4-5 times of volume of absolute ethyl alcohol for alcohol precipitation, centrifuging for 10 minutes, adding the sodium chloride solution again to the precipitate until the precipitate is just dissolved, continuing alcohol precipitation, repeating for 3 times, collecting the precipitate, dialyzing for 48 hours, concentrating under reduced pressure, and freeze-drying to obtain the glycopeptide conjugate (BG-TP) of the covalent coupling of beta-glucan and thymopentin.
4. The method of claim 3, wherein the beta-glucan thymopentin conjugate is prepared by: the molecular weight of the beta-glucan is 0.1-300 kDa, the monosaccharide composition is mainly glucose, the content of the monosaccharide is more than 95 percent, the main chain is formed by connecting the glucose through beta-1, 3 glycosidic bonds, and meanwhile, the main chain is provided with beta-1, 6 branches, and the branching degree is 1-50 percent.
5. The method of claim 3, wherein the selected peptide is Thymopentin (TP 5) and other peptide analogs of Thymopentin active fragment (RKDVY).
6. The method of claim 3, wherein the volume ratio of dimethyl sulfoxide to acetic acid in the mixed reaction solvent is 83 (v/v).
7. The method of claim 3, wherein the equivalent ratio of β -glucan to thymopentin is 1; the beta glucan is reacted with sodium cyanoborohydride (NaBH) 3 CN) the equivalent ratio is 1; the concentration of the sodium chloride solution used in the alcohol precipitation and dialysis processes is 4-32%.
8. Use of the conjugate of beta-glucan thymopentin of any one of claims 1-7 for the modulation of the immune system and anti-tumor drugs, nutraceuticals and special medical formulas.
9. Use according to claim 8, characterized in that: the beta-glucan thymopentin conjugate is applied to medicines, health-care products and special medical formula foods for regulating an immune system, and particularly has the effects of remarkably improving the bone marrow suppression state of a cyclophosphamide-induced immunocompromised mouse model, promoting the phagocytosis of abdominal macrophages, improving the total number of lymphocytes in the spleen and the CD4+/CD8+ T ratio to restore the immune system, and promoting the secretion capacity of cytokines such as TNF-alpha, IL-1 beta, IFN-gamma and the like and antibodies such as IgG, sIgA and the like in the serum of a mouse; the application of the beta-glucan and thymopentin conjugate in immunoregulation specifically is the non-cytotoxicity effect on RAW264.7 macrophages, the phagocytosis capability and the cytokine release capability of the conjugate can be remarkably promoted, the effect is more remarkable than that of the single use of the beta-glucan, the single use of the thymopentin and the mixture of the beta-glucan and the thymopentin, and the medicine, the health care product and the special medical application formula food are in the forms of injection, powder injection, oral liquid, tablets, capsules, soft capsules and powder.
10. Use according to claim 8, characterized in that: the beta-glucan thymopentin conjugate is applied to antitumor drugs, health-care products and special medical formula foods, particularly has the effect of inhibiting tumor growth of 4T1 and B16F10 tumor model mice, and can promote the secretion capacity of cell factors such as TNF-alpha, IL-1 beta, IFN-gamma and the like in serum of the mice, and the drugs, the health-care products and the special medical formula foods are in the forms of injections, powder injections, oral liquids, tablets, capsules, soft capsules and powder.
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