Galactomanninotetraose with anti-tumor activity and application of galactomanninotetraose

Abstract

The invention relates to galactomannan tetrasaccharide with anti-tumor activity and application of the galactomannan tetrasaccharide, and belongs to the technical field of active oligosaccharide drugs. The galactomanninotetraose ACP-3 synthesized by the invention has a good anti-tumor effect, is effective and safe, and has a very good development value. The galactomanninotetraose ACP-3 can obviously inhibit tumor cell proliferation in vitro and has no obvious toxicity to normal cells, so that the antitumor effect of the galactomanninotetraose ACP-3 has selectivity. In-vivo anti-tumor experiments show that the immunologic function of a body can be improved and tumor growth can be inhibited by single administration of the galactomanninotetraose ACP-3, and the galactomanninotetraose ACP-3 and CTX or PTX are combined for use, so that the effects of enhancing effects and reducing toxicity are achieved. The invention is helpful for developing oligosaccharide drugs with anti-tumor effect, and has important significance for research and development of corresponding carbohydrate drugs.

Description

Technical Field

[0001] The invention relates to galactomannotetraose with anti-tumor activity and application thereof, belonging to the technical field of active oligosaccharide drugs. Background Art

[0002] The high incidence and mortality of cancer have placed a heavy burden on society. Currently, the main treatment methods include surgery, radiotherapy, chemotherapy, etc. However, some disadvantages of traditional radiotherapy and chemotherapy drugs cause patients to suffer from infection, decreased immune function, liver and kidney damage, etc. Therefore, it is urgent to develop new anti-tumor drugs with significant efficacy and few side effects.

[0003] Polysaccharides are a class of macromolecules closely related to life activities. They are widely present in various animals, plants and microorganisms, and play an important role in cell communication, adhesion and molecular recognition. At the same time, polysaccharides are a class of compounds with extremely rich diversity, in which the connections and arrangements of various monosaccharides are numerous and complex. Polysaccharides are like a huge information library, attracting the attention of many scientists. A large number of pharmacological experiments have shown that polysaccharides have a wide range of biological activities and can be used as antioxidants, antibacterial agents, immunomodulators, antitumor agents, lipid-lowering drugs, hypoglycemic drugs or liver-protecting drugs. The biological activity of polysaccharides depends on its spatial structure, molecular weight, type of monosaccharides in the molecule, type of glycosidic bonds, the source of polysaccharide separation (type of organism, type of strain) and the culture conditions of microorganisms.

[0004] Antrodia cinnamomea (AC) is a medicinal and edible fungus unique to Taiwan, China, with anti-tumor and anti-inflammatory effects. The main active ingredients of AC include triterpenoids, polysaccharides, vitamins, nucleic acids, lectins, superoxide dismutase (SOD), adenosine, succinic acid and maleic acid derivatives, proteins (including immune proteins), ergosterol and quality. Among them, polysaccharides are considered to be one of the main sources of AC's active functions.

[0005] Chinese patent document CN 114686542 A (application number 202111595425.8) discloses a low molecular weight Antrodia cinnamomea exopolysaccharide and its preparation and application. The Antrodia cinnamomea S29 fermentation broth is collected by centrifugation, ultrafiltration is retained, trichloroacetic acid is added to remove protein, and alcohol precipitation is performed to obtain a low molecular weight polysaccharide. The molecular weight of the Antrodia cinnamomea exopolysaccharide is 2.6×10 4Da, which includes galactose, glucose, mannose and fucose, and the molar ratio of each component is 1:0.21:0.18:0.11, with good anti-tumor function and good safety. However, AC polysaccharides are mainly produced in the fruiting body stage, and the content is not high. At the same time, its growth environment requirements are very strict. It only grows on the Antrodia cinnamomea tree unique to Taiwan, China, and the growth rate is very slow. Therefore, it is extremely difficult to obtain AC polysaccharides by natural extraction methods, and the cost is high. In addition, naturally extracted AC polysaccharides are usually mixtures with microscopic heterogeneity. The above factors seriously hinder the further study of the biological activity of Antrodia cinnamomea polysaccharides. Therefore, it is particularly urgent to prepare and obtain oligosaccharide fragments related to AC polysaccharides with clear structures by chemical synthesis.

[0006] Perera et al. isolated a cold-water-soluble galactomannan (ACP) from AC with a molecular weight of up to 70 kDa, and found that this polysaccharide can significantly enhance the phagocytic activity of mouse macrophages, thereby improving the bactericidal activity. They used modern technical means such as nuclear magnetic resonance technology and liquid chromatography-mass spectrometry to analyze the structure of ACP and found that the polysaccharide is composed of only two monosaccharides, mannose and galactose, with a molar ratio of 3:1. They also speculated that the repeating unit of the polysaccharide molecule is an octose structure, and the bonding method between each sugar group is an α-glycosidic bond. In previous studies, our research group attempted and completed the synthesis of this repeating octose unit derivative (CN110128491A, application number 201910405954.3), and synthesized a galactomannan octaose derivative with the following structural formula:

[0007]

[0008] Taking into account that oligosaccharide fragments with different glycosyl sequences may have different biological activities, and the smaller the molecular weight of the oligosaccharide fragment, the lower the cost of synthesis, the present invention designed and synthesized galactomannotetraose derivatives and explored their biological activities in cellular immunity and anti-tumor. Summary of the invention

[0009] In view of the shortcomings of the prior art, the present invention provides a galactomannotetraose with anti-tumor activity and its application. Oligosaccharide fragments with different structures have different biological activities, and the galactomannotetraose prepared by the present invention has good immune activity and anti-tumor activity.

[0010] To achieve the above object, the present invention adopts the following technical solution:

[0011] A galactomannotetraose, the general structural formula is as follows:

[0012]

[0013] In the general formula, n is an integer from 1 to 6.

[0014] In a preferred technical solution of the present invention, the structural formula of the galactomannotetraose is as follows:

[0015]

[0016] The preparation method of the above-mentioned galactomannotetraose comprises the following steps:

[0017] Using dry ether as solvent, the disaccharide donor ACP-14 and the disaccharide acceptor ACP-8 are subjected to glycosylation coupling reaction under the catalysis of TfOH to obtain the fully protected tetrasaccharide ACP-15; then, the fully protected tetrasaccharide ACP-15 is subjected to a deprotection reaction in a mixed solution of tert-butyl alcohol-dichloromethane-water, and ACP-15 is directly placed in a hydrogen atmosphere and catalytically hydrogenated under the action of palladium carbon to obtain the target molecule galactomannotetraose ACP-3;

[0018]

[0019] Preferably, according to the present invention, the disaccharide donor ACP-14 uses the disaccharide glucosidase donor ACP-13 as a starting material, firstly dissolving the disaccharide ACP-13 in a mixed solution of dichloromethane and acetonitrile in a volume ratio of 1:1 (containing 10 equivalents of water), and hydrolyzing the C-1 glucosidase group under the catalysis of iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf); then, dissolving the hydrolyzate in dry dichloromethane under the condition of an ice-water bath, adding trichloroacetonitrile and a catalytic amount of hindered base DBU to react, and obtaining the α-configuration trichloroacetimidate donor ACP-14;

[0020]

[0021] Preferably, according to the present invention, the disaccharide acceptor ACP-8 is based on the monosaccharide ACP-4 as a starting material. First, a trichloroacetimidate group is introduced into the C-1 hydroxyl group of ACP-4 under the action of trichloroacetonitrile and a catalytic amount of a sterically hindered base 1,8-diazabicycloundec-7-ene (DBU) to obtain an α-configuration trichloroacetimidate donor ACP-5; then, the donor ACP-5 and the monosaccharide acceptor ACP-6 are dissolved in dry dichloromethane and reacted with After the molecular sieve is used to remove water, a glycosylation coupling reaction is carried out under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) at 0°C to obtain the α-configuration disaccharide ACP-7; then, the disaccharide ACP-7 is dissolved in a mixed solution of methanol and dichloromethane, and a 1M methanol solution of sodium methoxide is added to remove the acetyl group to obtain the disaccharide receptor ACP-8;

[0022]

[0023] The application of the galactomannotetraose in the preparation of medicines or foods with immunomodulatory effects.

[0024] The application of the above galactomannotetraose in the preparation of vaccines.

[0025] The application of the above galactomannotetraose in the preparation of anti-tumor drugs.

[0026] Preferably according to the present invention, the tumor is a tumor type in which cell apoptosis depends on activation of the JNK signaling pathway.

[0027] Further preferably, the activation of the JNK signaling pathway depends on the expression of ROS in cells.

[0028] Preferably according to the present invention, the tumor is liver cancer, lung cancer or breast cancer.

[0029] The application of the above galactomannotetraose combined with chemotherapy drugs in the preparation of anti-tumor drugs.

[0030] Preferably according to the present invention, the tumor is liver cancer, lung cancer or breast cancer.

[0031] Preferably according to the present invention, the chemotherapy drug is cyclophosphamide (CTX) or paclitaxel (PTX).

[0032] Beneficial effects:

[0033] The galactomannotetraose ACP-3 synthesized by the present invention is a tetrasaccharide structure in Antrodia cinnamomea galactomannan, with a clear structure, high solubility, good absorption performance, good immune activity, effective and safe anti-tumor effect, and good development value. Galactomannotetraose ACP-3 can significantly inhibit tumor cell proliferation in vitro and has no obvious toxicity to normal cells, indicating that its anti-tumor effect is selective. In vivo anti-tumor experiments show that galactomannotetraose ACP-3 can be administered alone to improve the body's immune function, inhibit tumor growth, and has a synergistic and toxic effect when used in combination with CTX or PTX. The present invention helps to develop oligosaccharide drugs with anti-tumor effects, which is of great significance for the research and development of corresponding carbohydrate drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the synthesis process of the disaccharide receptor ACP-8-1.

[0035] Figure 2 This is the synthesis process of the disaccharide donor ACP-11.

[0036] Figure 3 This is the synthesis process of galactomannotetraose ACP-2.

[0037] Figure 4 ACP-2 1H NMR spectrum.

[0038] Figure 5 This is the synthesis process of the disaccharide donor ACP-14.

[0039] Figure 6 This is the synthesis process of galactomannotetraose ACP-3-1.

[0040] Figure 7 ACP-3-1 1 H NMR spectrum.

[0041] Figure 8 This is the synthesis process of the disaccharide receptor ACP-8-2.

[0042] Fig. 9 This is the synthesis process of galactomannotetraose ACP-3-2.

[0043] Fig.10 This is the synthesis process of the disaccharide receptor ACP-8-3.

[0044] Fig.11 This is the synthesis process of galactomannotetraose ACP-3-3.

[0045] Fig.12 Cell survival rate curve of macrophages incubated with different oligosaccharides.

[0046] Fig.13 It is a bar graph showing the phagocytic ability of macrophages incubated with different oligosaccharides.

[0047] Fig.14 The bar graphs show the cell survival rates of Hep G2 cells (A), A549 cells (B), L02 cells (C), and BEAS-2B cells (D) incubated with different concentrations of tetrasaccharide ACP-3-1.

[0048] Fig.15 The changes of tumor volume (A) and tumor weight (B) of mice in different drug administration groups. In the figure, compared with the model group, #: P < 0.05, ##: P < 0.01, ###: P < 0.001, n = 6.

[0049] Fig.16 The figure shows the changes in the body weight of mice in different drug administration groups. In the figure, compared with the CTX positive group, *: P < 0.05, ***: P < 0.001, n = 6.

[0050] Fig.17 HE staining images of tumor tissues of mice in different drug administration groups.

[0051] Fig.18The results of spleen index (A) and thymus index (B) of mice in different drug administration groups are shown in the figure. Compared with the normal group, ***: P < 0.001, compared with the model group, #: P < 0.05, ##: P < 0.01, ###: P < 0.001, n = 6.

[0052] Fig.19 The expression levels of AST, ALT, Cre and BUN in the serum of mice in different drug administration groups. In the figure, compared with the normal group, #: P < 0.05, ###: P < 0.001, n = 6.

[0053] Fig. 20 The changes of tumor volume (A) and tumor weight (B) of mice in different drug administration groups. In the figure, compared with the blank group, **: P < 0.01, ***: P < 0.001, n = 6.

[0054] Fig.21 The figure shows the changes in the body weight of mice in different drug administration groups. In the figure, compared with the blank group, ***: P<0.001, n=6.

[0055] Fig. 22 HE staining images of tumor tissues of mice in different drug groups.

[0056] Fig.23 The results of spleen index (A) and thymus index (B) of mice in different drug administration groups are shown in the figure. Compared with the blank group, *: P<0.05, ***: P<0.001, n=6.

[0057] Fig.24 The expression levels of ALT, AST, Cre and BUN in the serum of mice in different drug administration groups. In the figure, compared with the blank group, *: P<0.05, n=6.

[0058] Fig.25 The Western blot detection diagram of p-JNK protein in Hep G2 cells; A is the Western blot development diagram, and B is the quantitative bar graph of p-JNK protein expression. In the figure, compared with the control group, *: P < 0.05, **: P < 0.01.

[0059] Fig.26 The cell survival rate of Hep G2 cells under different drug administration conditions is shown in the bar graph. In the figure, compared with the control group, **: P < 0.01; compared with the ACP-3-1 group, ##: P < 0.01.

[0060] Fig. 27The results of flow cytometry detection of Hep G2 cell apoptosis under different drug administration conditions; A is the flow cytometry detection diagram, and B is the quantified cell apoptosis rate bar graph. In the figure, compared with the control group, ***: P < 0.001; compared with the ACP-3-1 group, ##: P < 0.01.

[0061] Fig.28 The results of flow cytometry detection of reactive oxygen species in Hep G2 cells under different drug administration conditions; A is the flow cytometry detection graph, and B is the bar graph of the quantified reactive oxygen species level. In the figure, compared with the control group, **: P<0.01, ***: P<0.001.

[0062] Fig.29 The Western blot detection diagram of p-JNK and Cleaved-caspase3 proteins in Hep G2 cells; A is the Western blot development diagram, and B is the quantitative bar graph of the expression of p-JNK and Cleaved-caspase3 proteins. In the figure, compared with the control group, *: P < 0.05, **: P < 0.01; compared with the ACP-3-1 group, #: P < 0.05. DETAILED DESCRIPTION

[0063] The technical scheme of the present invention is further described below in conjunction with the examples and drawings, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the examples are all common commercially available products unless otherwise specified.

[0064] Cells: Macrophage Raw 264.7, human liver cancer cell Hep G2, human lung cancer cell A549, human normal liver cell L02, human normal lung epithelial cell BEAS-2B, mouse liver cancer cell H22, mouse breast cancer cell 4T1 can all be obtained commercially.

[0065] Drugs: Cyclophosphamide (CTX) and paclitaxel (PTX) are broad-spectrum anti-tumor drugs and are commercially available.

[0066] In previous studies, the inventors completed the chemical synthesis of galactomannooctaose ACP-1 and applied for an invention patent on May 16, 2019, with the application number 201910405954.3. The synthesis process of galactomannooctaose ACP-1 was described in detail in Example 4 of the patent. Considering that oligosaccharide fragments with different glycosyl sequences may have different biological activities, galactomannotetraose derivatives were designed and synthesized in the following examples, and their biological activities were explored.

[0067] Example 1: Synthesis of galactomannotetraose ACP-2

[0068] For the synthesis of the target molecule galactomannotetraose ACP-2, a [2+2] assembly strategy was adopted, that is, the disaccharide acceptor ACP-8-1 and the disaccharide donor ACP-11 were assembled to produce it.

[0069] like Figure 1 As shown in the figure, the preparation of the disaccharide acceptor ACP-8-1 is based on the monosaccharide ACP-4 as the starting material. First, ACP-4 is treated with trichloroacetonitrile and a catalytic amount of sterically hindered base 1,8-diazabicycloundec-7-ene (DBU) to introduce a trichloroacetimidate group on the C-1 hydroxyl group to obtain the α-configuration trichloroacetimidate donor ACP-5 with a yield of 96%. Next, the activated ester donor ACP-5 and the monosaccharide acceptor ACP-6-1 are dissolved in dry dichloromethane and reacted with After the molecular sieve was dehydrated, the glycosylation coupling reaction was carried out under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) at 0°C to obtain the α-configuration disaccharide ACP-7-1 with a yield of 87%. In the above glycosylation reaction, the configuration control of the α-glycosidic bond is achieved by the neighboring group participation effect of the 2-acetyl group (Ac). Then, the disaccharide ACP-7-1 was dissolved in a mixed solution of methanol and dichloromethane, and a 1M methanol solution of sodium methoxide was added to remove the acetyl group to obtain the disaccharide receptor ACP-8-1 with a yield of 94%.

[0070] like Figure 2 As shown, the preparation of disaccharide donor ACP-11 is to use disaccharide ACP-9 (the preparation method is recorded in patent 201910405954.3) as the starting material, and convert it into a trichloroacetimidate donor with higher reaction activity. First, disaccharide ACP-9 is dissolved in a mixed solution of dichloromethane and acetonitrile with a volume ratio of 1:1 (containing 10 equivalents of water), and the C-1 position glucosidic group is hydrolyzed under the catalysis of iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf) to obtain disaccharide ACP-10; then, under the condition of an ice water bath, disaccharide ACP-10 is dissolved in dry dichloromethane, trichloroacetonitrile and a catalytic amount of hindered base DBU are added to react to obtain disaccharide donor ACP-11, and the total yield of the two steps is 79%.

[0071] like Figure 3 As shown, the disaccharide acceptor ACP-8-1 and the disaccharide donor ACP-11 were dissolved in ether solution and After the molecular sieve was used to remove water, the reaction system was cooled to -30°C, and a catalytic amount of trifluoromethanesulfonic acid (TfOH) was added for coupling reaction to obtain the fully protected tetrasaccharide ACP-12 with a yield of 85%. Similarly, the participation effect of the benzoyl adjacent group at the C-2 position of the disaccharide donor and the solvent effect of ether ensure the formation of the α-glycosidic bond. Next, the fully protected tetrasaccharide ACP-12 was subjected to the removal of the acetyl and benzoyl groups by sodium methoxide solution, and then catalytic hydrogenation was carried out by palladium carbon in a hydrogen atmosphere to remove all benzyl groups and reduce the azide group (N3) to the amino group (NH2), and finally the target molecule galactomannotetraose ACP-2 was obtained with a two-step reaction yield of 81%. The structure of ACP-2 was confirmed by nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrometry, among which the nuclear magnetic hydrogen spectrum is shown in FIG. Figure 4 shown.

[0072] Example 2: Synthesis of galactomannotetraose ACP-3-1

[0073] For the synthesis of the target molecule galactomannan ACP-3-1, the [2+2] assembly strategy was also adopted, that is, the disaccharide acceptor ACP-8-1 and the disaccharide imidate donor ACP-14 were glycosylated to construct this target molecule.

[0074] like Figure 5 As shown, the disaccharide glucosidase donor ACP-13 (a reported compound, the preparation method is recorded in Patent 201910405954.3) is used as the starting material, the glucosidase group at its C-1 position is first hydrolyzed to 1-OH, and then the trichloroacetimidate group is introduced to obtain the α-configuration trichloroacetimidate donor ACP-14. The specific experimental operation is the same as the preparation of the disaccharide donor ACP-11, and the total yield of the two-step reaction is 69%.

[0075] like Figure 6 As shown, using dry ether as solvent, the disaccharide donor ACP-14 and the disaccharide acceptor ACP-8-1 were subjected to glycosylation coupling reaction under the catalysis of TfOH to obtain the fully protected tetrasaccharide ACP-15-1 with a yield of 73%. The four glycosidic bonds in the tetrasaccharide ACP-15-1 are all α-configuration. Finally, the fully protected tetrasaccharide ACP-15-1 was subjected to a deprotection reaction in a mixed solution of tert-butanol-dichloromethane-water. Since all the molecules are benzyl-protected, ACP-15-1 is directly placed in a hydrogen atmosphere and catalytically hydrogenated under the action of palladium carbon to obtain the target molecule galactomannotetraose ACP-3-1. Similarly, the structure of ACP-3-1 was determined by nuclear magnetic hydrogen spectrum, carbon spectrum, and mass spectrum, among which the nuclear magnetic hydrogen spectrum is shown in Figure 7 shown.

[0076] Example 3: Synthesis of galactomannotetraose ACP-3-2

[0077] The synthesis of the target molecule galactomannan ACP-3-2 was similar to that of Example 2, and the [2+2] assembly strategy was also adopted, that is, the disaccharide acceptor ACP-8-2 and the disaccharide imidate donor ACP-14 were subjected to glycosylation reaction to construct the target molecule.

[0078] like Figure 8 As shown, the preparation of disaccharide acceptor ACP-8-2, first, activated ester donor ACP-5 and monosaccharide acceptor ACP-6-2 are dissolved in dry dichloromethane, After the molecular sieves were used to remove water, a glycosylation coupling reaction was carried out under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) at 0°C to obtain the α-configuration disaccharide ACP-7-2. Then, the disaccharide ACP-7-2 was dissolved in a mixed solution of methanol and dichloromethane, and a 1M methanol solution of sodium methoxide was added to remove the acetyl group to obtain the disaccharide receptor ACP-8-2.

[0079] like Fig. 9 As shown, using dry ether as solvent, the disaccharide donor ACP-14 and the disaccharide acceptor ACP-8-2 were subjected to glycosylation coupling reaction under the catalysis of TfOH to obtain the fully protected tetrasaccharide ACP-15-2. Finally, the fully protected tetrasaccharide ACP-15-2 was subjected to a deprotection reaction in a mixed solution of tert-butyl alcohol-dichloromethane-water, and ACP-15-2 was directly placed in a hydrogen atmosphere and catalytically hydrogenated under the action of palladium carbon to obtain the target molecule galactomannotetraose ACP-3-2.

[0080] Example 4: Synthesis of galactomannotetraose ACP-3-3

[0081] The synthesis of the target molecule galactomannan ACP-3-3 was similar to that of Example 2, and the [2+2] assembly strategy was also adopted, that is, the disaccharide acceptor ACP-8-3 and the disaccharide imidate donor ACP-14 were subjected to glycosylation reaction to construct the target molecule.

[0082] like Fig.10 As shown, the preparation of the disaccharide acceptor ACP-8-3, first, the activated ester donor ACP-5 and the monosaccharide acceptor ACP-6-3 were dissolved in dry dichloromethane, and then After the molecular sieves were used to remove water, a glycosylation coupling reaction was carried out under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) at 0°C to obtain the α-configuration disaccharide ACP-7-3. Then, the disaccharide ACP-7-3 was dissolved in a mixed solution of methanol and dichloromethane, and a 1M methanol solution of sodium methoxide was added to remove the acetyl group to obtain the disaccharide receptor ACP-8-3.

[0083] like Fig.11As shown, using dry ether as solvent, the disaccharide donor ACP-14 and the disaccharide acceptor ACP-8-3 were subjected to glycosylation coupling reaction under the catalysis of TfOH to obtain the fully protected tetrasaccharide ACP-15-3. Finally, the fully protected tetrasaccharide ACP-15-3 was subjected to a deprotection reaction in a mixed solution of tert-butyl alcohol-dichloromethane-water, and ACP-15-3 was directly placed in a hydrogen atmosphere and catalytically hydrogenated under the action of palladium carbon to obtain the target molecule galactomannotetraose ACP-3-3.

[0084] Example 5: Evaluation of the immunological activity of galactomannotetrasaccharides ACP-2 and ACP-3-1

[0085] The immune activity of macrophage Raw 264.7 was evaluated by measuring the effects of galactomannotetrasaccharides ACP-2 and ACP-3-1 on the proliferation activity and phagocytic ability.

[0086] Cell culture: Prepare a medium containing FBS and 100 μg L -1 DMEM complete medium containing penicillin and streptomycin (FBS: medium = 1:9), and macrophage Raw 264.7 cells were placed in the DMEM complete medium and cultured in an incubator at 37° C. and 5% CO 2 until the cells grew to the logarithmic phase.

[0087] (1) Proliferation activity

[0088] The Raw 264.7 cells were cultured at a concentration of 1 × 10 cells / mL in DMEM complete medium containing 10% FBS. 5 cells / mL, and inoculated into a 96-well plate (100 μL per well). The 96-well plate was placed in an incubator at 37°C and 5% CO2 saturated humidity for 24 h, the culture medium was discarded, and the cells were incubated in DMEM complete medium (containing 10% FBS) containing different concentrations of oligosaccharides for 24 h. After adding CCK-8 reagent and culturing for 1 h, the absorbance was measured at 450 nm by a microplate reader.

[0089] The CCK-8 method was used to determine the effects of oligosaccharides at different concentrations (10, 25, 50, 100, 200 μg / mL) on the proliferation activity of macrophage Raw264.7. Fig.12As shown in the figure, the three oligosaccharides showed similar proliferation trends on macrophages Raw 264.7. When the concentration of the three oligosaccharides was 25 μg / mL, the proliferation activity of macrophages Raw 264.7 was the strongest, and the difference was statistically significant compared with the blank group (P<0.05). However, with the increase of oligosaccharide concentration (>25 μg / mL), the proliferation activity of macrophages Raw 264.7 showed a decreasing trend. In short, the order of the three oligosaccharides on the proliferation activity of macrophages Raw 264.7 was: tetrasaccharide ACP-3-1>octasaccharide ACP-1>tetrasaccharide ACP-2, among which galactomannotetrasaccharide ACP-3-1 had the best effect on the proliferation activity of macrophages Raw 264.7.

[0090] (2) Phagocytic ability

[0091] The Raw 264.7 cells were cultured at a concentration of 1 × 10 cells / mL in DMEM complete medium containing 10% FBS. 5 Cells / mL, cells were inoculated into 96-well plates, 100 μL per well. The 96-well plates were placed in an incubator at 37°C and 5% CO2 saturated humidity for 24 hours, and then the culture medium was discarded. 25 μg / mL oligosaccharide solution and LPS solution (1 μg / mL, positive control) were prepared with DMEM complete medium (containing 10% FBS), and macrophages Raw 264.7 were cultured with them for 24 hours, then the culture medium was removed and washed twice with PBS. 100 μL of prepared 0.075% neutral red was added to each well. After 4 hours, the liquid was aspirated and washed twice with PBS. Finally, 200 μL of cell lysis solution (C2H5OH:CH3COOH=1:1, v / v) was used for treatment, and after overnight culture, the absorbance value was measured at 540nm using an enzyme reader.

[0092] The effect of oligosaccharides on the phagocytic ability of macrophages Raw 264.7 was determined by detecting the uptake of neutral red. The experimental results are as follows: Fig.13 As shown in the results, compared with the blank group, octasaccharide ACP-1 and tetrasaccharide ACP-3-1 significantly increased the phagocytic ability of macrophages Raw 264.7 (P<0.05), and the effect of tetrasaccharide ACP-3-1 was the best. In addition, tetrasaccharide ACP-3-1 was more effective than the positive control LPS in enhancing the phagocytic ability of macrophages. In contrast, compared with the blank group, tetrasaccharide ACP-2 had no significant effect on the phagocytic ability of macrophages Raw264.7 (P>0.05).

[0093] Example 6: Effect of galactomannotetraose ACP-3-1 on tumor cell proliferation

[0094] (1) Cell culture

[0095] Hep G2 cells, A549 cells, L02 cells, and BEAS-2B cells were placed in DMEM complete medium containing 10% FBS, 100 U / mL penicillin, and 100 mg / mL streptomycin, respectively, and cultured in an incubator at 37° C. and 5% CO 2 until the cells grew to the logarithmic phase.

[0096] (2) Cell proliferation assay

[0097] The cell concentration was adjusted in DMEM complete medium containing 10% FBS. Hep G2 cells, A549 cells, L02 cells, and BEAS-2B cells were cultured at 1×10 5 The cells were inoculated at a concentration of 1.5 cells / mL in a 96-well culture plate. After the cells adhered to the wall, the culture medium was discarded and different concentrations of tetrasaccharide ACP-3-1 solutions (0, 10, 25, 50, 100, 200 μM) were prepared with DMEM complete culture medium containing 10% FBS. The cells were cultured for 24 h, 48 h and 72 h, respectively, and the cell proliferation was detected by CCK-8 method.

[0098] (3) Experimental results

[0099] like Fig.14 As shown in the figure, the tetrasaccharide ACP-3-1 significantly inhibited the proliferation of human lung cancer cells A549 and human liver cancer cells Hep G2 in a time- and dose-dependent manner, especially after 200μM tetrasaccharide ACP-3-1 was treated with human lung cancer cells A549 and human liver cancer cells Hep G2 for 72h, the inhibition rate was close to 50%. In contrast, 10-400μM tetrasaccharide ACP-3-1 did not inhibit the proliferation of human normal lung epithelial cells BEAS-2B and human normal liver cells L02, indicating that the tetrasaccharide ACP-3-1 has low toxicity to normal cells. In short, galactomannotetrasaccharide ACP-3-1 inhibits the proliferation of lung cancer and liver cancer cells in a time- and dose-dependent manner, and has no obvious toxicity to normal cells.

[0100] Example 7: Effect of galactomannotetraose ACP-3-1 on tumor growth in H22 tumor-bearing mice

[0101] (1) Animal grouping and drug administration

[0102] Female BALB / c mice, 6-8 weeks old, 18-22 g, were selected and adaptively fed for one week. The mice were divided into a normal group, a model group, a low-dose ACP-3-1 group, a high-dose ACP-3-1 group, a CTX-positive group, a low-dose ACP-3-1 + CTX group, and a high-dose ACP-3-1 + CTX group, with 6 mice in each group. The H22 cell concentration was adjusted to 1×10 7 Except for the mice in the normal group, 0.2 mL (2×106 When the tumor volume is greater than 100 mm 3 , and started to administer the drug. The administration conditions are as follows: normal group (normal saline), model group (normal saline), ACP-3-1 low-dose group (1 mg / kg), ACP-3-1 high-dose group (5 mg / kg), CTX positive group (30 mg / kg), ACP-3-1 low-dose (1 mg / kg) + CTX (30 mg / kg) group and ACP-3-1 high-dose (5 mg / kg) + CTX (30 mg / kg) group, according to the weight of mice, intraperitoneal injection once a day. The body weight and tumor volume were recorded every other day. Tumor volume calculation formula: length × width × width / 2. After one week of administration, peripheral blood of mice was collected, and blood biochemical indicators were detected by kit. The mice were killed, and the tumors, spleen, and thymus were dissected and weighed.

[0103] (2) HE staining of tumor tissue

[0104] Sampling: At the end of the experiment, all mice were killed and tumor tissues were collected.

[0105] Fixation: The tumor tissue was fixed in 4% paraformaldehyde for one week, and then the target area was smoothed.

[0106] Dehydration: The tissue was dehydrated through a series of ethanol treatments: 70% ethanol, 30 min; 80% ethanol, 30 min; 85% ethanol, 30 min; 90% ethanol, 30 min; 95% ethanol, 30 min; 100% ethanol, 30 min, twice. Xylene was added for 1 hour to remove the alcohol.

[0107] Paraffin penetration: Place the tissue in melted paraffin for 1 hour to allow the paraffin to completely penetrate the tissue.

[0108] Embedding: Place the waxed tissue block into the mold with the side to be cut facing down, add the melted paraffin into the mold, and embed the tissue in paraffin.

[0109] Sectioning: Place the paraffin block containing the embedded tissue on ice to cool down, and use a microtome to cut paraffin sections with a thickness of 3-5 μm from the paraffin block.

[0110] Spread and scoop the slices: Place the slices in a 40°C water bath for spreading. After the tissue is flattened, scoop the slices onto a glass slide.

[0111] Bake the paraffin sections at 60°C for 2 h.

[0112] Dewaxing: Dewax the tissue sections routinely to water: immerse the paraffin sections in xylene, 15 minutes, twice; 100% ethanol, 5 minutes, twice; 95% ethanol, 5 minutes; 90% ethanol, 5 minutes; 80% ethanol, 5 minutes; double distilled water, 5 minutes.

[0113] Staining: Stain the sections with hematoxylin for 5 minutes, rinse with distilled water for 5 minutes, differentiate with hydrochloric acid ethanol for a few seconds, rinse with distilled water for 3 minutes, stain with eosin for 5 minutes, rinse with distilled water for 3 minutes.

[0114] Gradient dehydration: Dehydrate the sections with gradient ethanol: 95% ethanol, 2 min, 2 times; 100% ethanol, 2 min, 2 times. And clear with xylene, 5 min, 2 times.

[0115] Mounting: The slides were mounted with neutral gum and then the staining was observed under an optical microscope (Nikon Eclipse E100, Japan).

[0116] (3) Experimental results

[0117] Effects of tetrasaccharide ACP-3-1 on tumor growth Fig.15 As shown. The tumor volume and tumor weight of mice in the model group increased significantly, while each drug-treated group could delay tumor growth. Among them, the tumor volume and tumor weight of mice in the low-dose ACP-3-1 group (1 mg / kg) were similar to those in the CTX-positive group. In addition, the combination of low-dose ACP-3-1 (1 mg / kg) and CTX (30 mg / kg) can significantly delay tumor growth in mice, indicating that the combination of tetrasaccharide ACP-3-1 and CTX can enhance its anti-tumor effect.

[0118] The changes of body weight of mice in each group over time are shown in Fig.16 As shown in the figure, the weight of mice in the CTX positive group decreased significantly, indicating that CTX has serious side effects on mice. Compared with the model group, the low and high dose groups of ACP-3-1 (1 mg / kg and 5 mg / kg) had no significant effect on the weight of mice. The changes in the weight of mice in the low dose (1 mg / kg) + CTX (30 mg / kg) group of ACP-3-1 also show that the tetrasaccharide ACP-3-1 can significantly reverse the trend of weight loss in mice caused by CTX and alleviate the side effects of CTX.

[0119] HE staining results of tumor tissue ( Fig.17) showed that the tumor cells in the model group grew vigorously, were closely arranged, had darker nuclei, a high nuclear-cytoplasmic ratio, and strong basophilia, and no obvious cell death occurred. After administration, the growth of tumor cells was inhibited to varying degrees, and the number of tumor cells was significantly reduced, the volume became smaller, the nuclei were lighter, the nuclear-cytoplasmic ratio became lower, and the basophilia was weakened. Among them, the nuclei of the CTX combined with low-dose ACP-3-1 group were severely ruptured, the cells shrank, and the cells showed obvious cell death.

[0120] The results of immune organ index determination are as follows Fig.18 As shown. Compared with the normal group, the thymus index of mice in the model group was significantly decreased (P<0.001), indicating that the proliferation of tumor cells has a destructive effect on the thymus. Compared with the model group, the thymus index of mice in the CTX-positive group was significantly decreased (P<0.001), indicating that CTX will further reduce the body's immune function. Although low-dose and high-dose tetrasaccharide ACP-3-1 can increase the thymus index to a certain extent, there is no statistical significance compared with the model group (P>0.05). In addition, the thymus index of mice was slightly increased by the combination of low-dose and high-dose tetrasaccharide ACP-3-1 and CTX. Compared with the normal group, the spleen index of mice in the model group was significantly increased (P<0.001), indicating that tumor cell proliferation can cause the spleen of mice to swell. Compared with the model group, the spleen index of mice in the low-dose and high-dose tetrasaccharide ACP-3-1 groups was significantly decreased (P<0.05). The above immune organ index analysis results showed that the tetrasaccharide ACP-3-1 can enhance the immune function of tumor-bearing mice, CTX has a strong toxicity to immune organs (P<0.001), and the combination of low and high doses of tetrasaccharide ACP-3-1 and CTX can improve the immune function of tumor-bearing mice to a certain extent.

[0121] In order to study the side effects of drugs on the body, we used a kit to detect the levels of ALT, AST, Cre and BUN in mouse serum. Among them, AST (aspartate aminotransferase) is mainly distributed in the myocardium, followed by tissues such as the liver, skeletal muscle and kidney. When the corresponding cells are damaged, AST is released into the blood, so its serum concentration increases, which is one of the clinical indicators of liver damage; ALT (alanine aminotransferase) is mainly present in various cells, especially liver cells, and is an important indicator for diagnosing liver diseases; Cre (serum creatinine) is a product of muscle metabolism in the human body and is mainly excreted from the body by glomerular filtration. Measuring blood creatinine concentration can reflect the glomerular filtration function; BUN (blood urea nitrogen) is a nitrogen-containing compound in plasma other than protein. It is excreted from the body by glomerular filtration and is clinically used as an indicator for judging glomerular filtration function. The measurement results are as follows Fig.19As shown in the figure, compared with the normal group, the levels of ALT, AST, Cre and BUN in the model group remained basically unchanged, indicating that the modeling would not cause liver and kidney damage in mice, while after CTX administration, the levels of ALT, AST, Cre and BUN in serum increased, indicating that CTX would cause liver and kidney damage. There was no significant change in the levels of ALT, AST, Cre and BUN in the serum of mice in the ACP-3-1 low-dose group and the ACP-3-1 high-dose group, indicating that the tetrasaccharide ACP-3-1 did not cause liver and kidney damage in tumor-bearing mice and had no hepato-renal toxicity. In the ACP-3-1 and CTX combined administration group, there was no significant change in the levels of AST and Cre in serum, and the levels of ALT and BUN were further reduced compared with the model group, indicating that the tetrasaccharide ACP-3-1 can alleviate the degree of liver and kidney damage caused by CTX to a certain extent.

[0122] Example 8: Effect of galactomannotetraose ACP-3-1 on tumor growth in 4T1 tumor-bearing mice

[0123] (1) Animal grouping and drug administration

[0124] Female BALB / c mice, 6-8 weeks old, 18-22 g, were selected and fed for one week. The mice were divided into a blank group, an ACP-3-1 group, a PTX group, and an ACP-3-1+PTX group. The 4T1 cell concentration was adjusted to 1×10 7 Except for the blank group, 0.2 mL (2×10 6 When the tumor volume is greater than 100 mm 3 , and started dosing. The dosing conditions are as follows: blank group (normal saline), ACP-3-1 group (1 mg / kg), PTX group (10 mg / kg), ACP-3-1 (1 mg / kg) + PTX (10 mg / kg) group, dosing according to the weight of mice, once every other day by tail vein injection. Weigh the mice every two days and measure the tumor volume. After the 5th dose, the mice were killed by removing the eyeballs and bleeding, and the blood biochemical indicators were tested using a kit. The spleen and thymus were dissected and weighed. The tissue was fixed in 4% paraformaldehyde for pathological histological determination.

[0125] (2) HE staining of tumor tissue

[0126] The method is the same as Example 5.

[0127] (3) Experimental results

[0128] The tumor growth of mice in each group was as follows Fig. 20As shown, the tumors of mice in the blank group grew rapidly, and the tumor growth was slowed down in each drug-treated group. The ACP-3-1+PTX group had the best effect in delaying the tumor growth of mice, indicating that the combined use of tetrasaccharide ACP-3-1 and PTX can enhance the anti-tumor effect.

[0129] Changes of body weight of tumor-bearing mice in each group over time Fig.21 As shown. Compared with the blank group, the weight of mice in the PTX group decreased significantly. Compared with the blank group, there was no significant difference in the weight of mice in the ACP-3-1 group and the ACP-3-1+PTX group. The above results show that PTX can cause serious side effects, while ACP-3-1 has no obvious side effects and can alleviate the trend of weight loss in mice caused by PTX to a certain extent.

[0130] HE staining results of tumor tissue sections also showed ( Fig. 22 ), the tumor cells of mice in the ACP-3-1 group, PTX group, and ACP-3-1+PTX group were obviously necrotic.

[0131] The results of immune organ index determination are as follows Fig.23 As shown. Compared with the blank group, the thymus index of mice in the PTX group was significantly reduced, indicating that PTX can damage the thymus. ACP-3-1 had no significant effect on the thymus index, and ACP-3-1 could alleviate the damage of PTX to the thymus to a certain extent. In addition, ACP-3-1 did not show obvious toxicity to the spleen.

[0132] Blood biochemical index results such as Fig.24 As shown in the figure, PTX increased the levels of BUN and Cre in serum, indicating that PTX caused certain toxicity to the kidneys. However, when PTX was used in combination with ACP-3-1, there was no significant change in BUN and Cre in serum, indicating that ACP-3-1 could reduce the renal toxicity caused by PTX. However, ACP-3-1 had no significant effect on BUN and Cre levels, indicating that ACP-3-1 had no significant toxicity to the kidneys. And the ALT and AST results also showed that ACP-3-1 had no significant toxicity to the liver.

[0133] The galactomannotetraose ACP-3-2 and ACP-3-3 prepared by the invention show similar immune activity and anti-tumor activity to galactomannotetraose ACP-3-1.

[0134] Example 9: ACP-3-1 induces tumor cell apoptosis via ROS-mediated JNK signaling pathway

[0135] 1. MAPK-related signaling pathways (p38, JNK and ERK1 / 2) play an important role in inducing cell apoptosis. The present invention studies whether the JNK signaling pathway is related to cell apoptosis induced by tetrasaccharide ACP-3-1.

[0136] (1) Western blot was used to detect the expression of p-JNK protein in tumor cells

[0137] The cell concentration was adjusted in DMEM complete medium containing 10% FBS. Hep G2 cells in the logarithmic growth phase were cultured at a density of 1×10 5 The cells were inoculated in a 6-well plate at a density of 100 μM / mL. After the cells adhered to the wall, the culture medium was discarded and different concentrations of ACP-3-1 solution (0, 100, 200 μM) prepared with DMEM complete culture medium containing 10% FBS were added and cultured for 72 hours. After the culture, the cells were washed 3 times with PBS, and the cell lysis solution containing protease inhibitors and phosphatase inhibitors was added to the cell culture medium and placed on ice for 30 minutes. The cell lysate was centrifuged at 4°C and 13000 rpm for 15 minutes, and the supernatant was collected to determine the protein concentration and perform Western blot detection. The proteins in the supernatant were separated by SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% skim milk powder at room temperature for 1 hour, the PVDF membrane was washed 3 times with TBST buffer. The specific primary antibody was added to the PVDF membrane, incubated overnight at 4°C, washed 3 times with TBST buffer, and the secondary antibody was added and incubated at room temperature for 1 hour. The cells were washed three times with TBST, and developer was added dropwise for color development. The density of the protein blot bands was quantified using Image J software.

[0138] The results are as follows Fig.25 As shown, the results showed that tetrasaccharide ACP-3-1 promoted the expression of p-JNK protein in Hep G2 cells in a concentration-dependent manner, indicating that tetrasaccharide ACP-3-1 activated the JNK signaling pathway.

[0139] (2) Further verification of the JNK signaling pathway using the JNK inhibitor SP600125

[0140] The cell concentration was adjusted in DMEM complete medium containing 10% FBS. Hep G2 cells in the logarithmic growth phase were cultured at a density of 1×10 5The cells were inoculated at a density of 100 μM / mL in a 6-well plate. After the cells adhered to the wall, the culture medium was discarded and different solutions prepared with DMEM complete culture medium containing 10% FBS were added for 72 hours. The solutions with different components and their concentrations were SP600125 solution (20 μM), ACP-3-1 solution (200 μM), ACP-3-1+SP600125 solution (200 μM+20 μM, SP600125 was added to the cells for pretreatment for 2 hours, and then ACP-3-1 solution was added for 72 hours). The DMEM complete culture medium containing only 10% FBS was used as the blank control group. After the culture was completed, the cell proliferation was detected by CCK-8 method, and the apoptotic cells were detected by flow cytometry.

[0141] The flow cytometry method is as follows: suspend the cells with 1 mL of 1×Binding Buffer, centrifuge at 300×g for 10 min, and discard the supernatant. Resuspend the cells with 1×Binding Buffer to a cell density of 1×10 6 Add 100 μL of cells (1×10 5 ). Add 5 μL Annexin V-FITC to the tube. Mix gently at room temperature, away from light, and react for 10 min. Add 5 μL PI, incubate at room temperature, away from light, and incubate for 5 min. Add PBS to 500 μL and mix gently. Detect using flow cytometer (ACEA NovoCyte, Hanzhou) within 1 h.

[0142] The proliferation of Hep G2 cells Fig.26 As shown in the figure, SP600125 inhibited the JNK signaling pathway and reversed the trend of decreased cell proliferation activity caused by ACP-3-1. Further flow cytometric analysis results showed that ( Fig. 27 )SP600125 significantly reversed the cell apoptosis induced by ACP-3-1.

[0143] The above analysis results showed that the tetrasaccharide ACP-3-1 induced tumor cell apoptosis by activating the JNK signaling pathway in Hep G2 cells.

[0144] 2. The JNK signaling pathway is a downstream pathway of reactive oxygen species (ROS), so the present invention also studied whether the activation of the JNK signaling pathway in Hep G2 cells is related to the generation of ROS induced by ACP-3-1.

[0145] (1) ROS determination

[0146] Hep G2 cells were cultured at a density of 1 × 10 cells / mL in DMEM complete medium containing 10% FBS. 5The cells were seeded at a density of 100 cells / mL in a 6-well plate. After the cells adhered to the wall, the culture medium was discarded and different concentrations of ACP-3-1 solution (0, 100, 200 μM) prepared with DMEM complete medium containing 10% FBS were added and cultured for 72 hours. The cells were then resuspended in the working solution of ROS fluorescent indicator DCFH-DA and incubated at 37°C for 20 minutes. Finally, the cells were washed three times with serum-free medium, and the intracellular ROS expression level was detected by flow cytometry.

[0147] Test results such as Fig.28 As shown, the results showed that the tetrasaccharide ACP-3-1 increased the generation of ROS in Hep G2 cells in a concentration-dependent manner.

[0148] (2) Using the antioxidant NAC to further verify the role of ROS in the JNK signaling pathway

[0149] The cell concentration was adjusted in DMEM complete medium containing 10% FBS. Hep G2 cells in the logarithmic growth phase were cultured at a density of 1×10 5 The cells were inoculated at a density of 100 μg / mL in a 6-well plate. After the cells adhered to the wall, the culture medium was discarded and different solutions prepared with DMEM complete medium containing 10% FBS were added for 72 hours. The solutions and concentrations of different components were NAC solution (10 mM), ACP-3-1 solution (200 μM), ACP-3-1 + NAC solution (200 μM + 10 mM, NAC was added to the cells for pretreatment for 2 hours, and then ACP-3-1 solution was added for 72 hours). The DMEM complete medium containing only 10% FBS was used as the blank control group. After the culture was completed, the expression of p-JNK protein in the cells was detected by Western blot method, and the density of the protein blot band was quantified by Image J software.

[0150] The results are as follows Fig.29 As shown in the figure, the antioxidant NAC treated Hep G2 cells and significantly inhibited the expression of pathway protein p-JNK and pro-apoptotic protein Cleaved-caspase-3 in cells activated by tetrasaccharide ACP-3-1. Therefore, it is preliminarily speculated that tetrasaccharide ACP-3-1 promoted the expression of ROS in cells, thereby activating the JNK signaling pathway and inducing tumor cell apoptosis.

Claims

1. A galactomannotetraose, characterized in that: The general structure is as follows: In the general formula, n is an integer from 1 to 6.

2. The galactomannotetraose according to claim 1, characterized in that The structural formula is as follows:

3. The method for preparing galactomannotetraose according to claim 1, characterized in that: The steps include: Using dry ether as solvent, the disaccharide donor ACP-14 and the disaccharide acceptor ACP-8 are subjected to glycosylation coupling reaction under the catalysis of TfOH to obtain the fully protected tetrasaccharide ACP-15; then, the fully protected tetrasaccharide ACP-15 is subjected to a deprotection reaction in a mixed solution of tert-butyl alcohol-dichloromethane-water, and ACP-15 is directly placed in a hydrogen atmosphere and catalytically hydrogenated under the action of palladium carbon to obtain the target molecule galactomannotetraose ACP-3; Preferably, the disaccharide donor ACP-14 is prepared by using the disaccharide glucosidase donor ACP-13 as a starting material. The disaccharide ACP-13 is first dissolved in a mixed solution of dichloromethane and acetonitrile in a volume ratio of 1:1, containing 10 equivalents of water, and the C-1 glucosidase group is hydrolyzed under the catalysis of iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf); then, the hydrolyzate is dissolved in dry dichloromethane under ice-water bath conditions, and trichloroacetonitrile and a catalytic amount of hindered base DBU are added to react to obtain an α-configuration trichloroacetimidate donor ACP-14; Preferably, the disaccharide acceptor ACP-8 is based on the monosaccharide ACP-4 as a starting material. First, a trichloroacetimidate group is introduced into the C-1 hydroxyl group of ACP-4 under the action of trichloroacetonitrile and a catalytic amount of a sterically hindered base 1,8-diazabicycloundec-7-ene (DBU) to obtain an α-configuration trichloroacetimidate donor ACP-5. Next, the donor ACP-5 and the monosaccharide acceptor ACP-6 are dissolved in dry dichloromethane and reacted with After the molecular sieve is used to remove water, a glycosylation coupling reaction is carried out under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) at 0°C to obtain the α-configuration disaccharide ACP-7; then, the disaccharide ACP-7 is dissolved in a mixed solution of methanol and dichloromethane, and a 1M methanol solution of sodium methoxide is added to remove the acetyl group to obtain the disaccharide receptor ACP-8; 4. Use of the galactomannotetraose according to claim 1 or 2 in the preparation of medicines or foods having immunomodulatory effects.

5. Use of the galactomannotetraose according to claim 1 or 2 in the preparation of vaccines.

6. Use of the galactomannotetraose according to claim 1 or 2 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that The tumor is a tumor type in which cell apoptosis depends on activation of the JNK signaling pathway; Further preferably, the activation of the JNK signaling pathway depends on the expression of ROS in cells.

8. The use according to claim 6, characterized in that The tumor is liver cancer, lung cancer or breast cancer.

9. Use of the galactomannotetraose according to claim 1 or 2 in combination with a chemotherapy drug in the preparation of an anti-tumor drug.

10. The use according to claim 9, characterized in that The tumor is liver cancer, lung cancer or breast cancer; Preferably, the chemotherapy drug is cyclophosphamide or paclitaxel.

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