Galactomanno-tetraose with antitumor activity and use thereof

Abstract

The present application relates to a kind of galactomannan tetrasaccharide with antitumor activity and its application, belong to active oligosaccharide pharmaceutical technical field.The galactomannan tetrasaccharide ACP-3 synthesized in the present application has good antitumor effect effective and safe, with good development value.The galactomannan tetrasaccharide ACP-3 in the present application can significantly inhibit tumor cell proliferation in vitro, and normal cells have no obvious toxicity, indicating that its antitumor effect has selectivity.In vivo antitumor experiment shows that galactomannan tetrasaccharide ACP-3 alone can improve the immune function of organism, inhibit tumor growth, and has synergistic effect and attenuates toxicity effect when combined with CTX or PTX.The present application is helpful to develop oligosaccharide drug with antitumor effect, and has important significance for the development of corresponding saccharide drug.

Description

TECHNICAL FIELD

[0001] The present application relates to a galactomannotetraose with anti-tumor activity and its application, belonging to the technical field of active oligosaccharide drugs. BACKGROUND

[0002] The incidence and mortality of cancer are high, which has caused serious burden to the society. At present, the treatment methods mainly include surgical treatment, radiotherapy, chemotherapy and other means. However, some drawbacks of traditional chemotherapeutic drugs cause infection, immune function decline, liver and kidney function damage and other problems in patients. Therefore, it is urgent to develop new anti-tumor drugs with significant efficacy and small side effects.

[0003] Polysaccharides are a class of macromolecules closely related to life activities, which widely exist 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 connection and arrangement of various monosaccharides are complex. Polysaccharides are like a huge information bank, attracting the attention of numerous scientists. A large number of pharmacological experiments show that polysaccharides have a wide range of biological activities, and can be used as antioxidants, antibacterial agents, immunomodulators, antitumor agents, hypolipidemic drugs, hypoglycemic drugs or hepatoprotective active drugs, etc. The biological activity of polysaccharides depends on its spatial structure, molecular weight, type of monosaccharide in the molecule, type of glycosidic bond, and isolation source of polysaccharides (type of organism, type of strain) and culture conditions of microorganisms, etc.

[0004] Antrodia cinnamomea Antrodia cinnamomea AC is a special fungus for both medicine and food in Taiwan area, which has the effects of anti-tumor and anti-inflammatory. The main active components of AC include triterpenoids, polysaccharides, vitamins, nucleic acids, lectins, superoxide dismutase (SOD), adenosine, succinic acid and maleic acid derivatives, proteins (including immunoprotein), ergosterol and suberin. Among them, polysaccharides are considered as one of the main sources of the active functions of AC.

[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 low molecular weight polysaccharide is obtained by adding trichloroacetic acid to remove protein after centrifugal collection of Antrodia cinnamomea S-29 fermentation broth and ultrafiltration retention, and 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, has good antitumor function and good safety. However, AC polysaccharide is mainly produced in the fruiting body stage, and the content is not high. At the same time, its growth environment is very strict, and it only grows on the specific cow-tangerine trees in Taiwan area, and the growth speed is very slow. Therefore, it is extremely difficult to obtain AC polysaccharide by natural extraction method, and the cost is high. In addition, the naturally extracted AC polysaccharide is usually a mixture, and there is microscopic heterogeneity. The above factors seriously hinder the further study of the biological activity of cow-tangerine polysaccharide. Therefore, it is particularly urgent to prepare AC polysaccharide related oligosaccharide fragments with clear structure 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 could significantly enhance the phagocytic activity of mouse macrophages, thereby improving the bactericidal activity. They used nuclear magnetic resonance technology and liquid chromatography-mass spectrometry and other modern technical means to analyze the structure of ACP, and found that the polysaccharide was composed of only mannose and galactose, and the molar ratio of the two was 3:1. It is speculated that the repeating unit of the polysaccharide molecule is an octasaccharide structure, and the bond between each glycosyl is an α-glycosidic bond. In the previous study, the synthesis of this repeating octasaccharide derivative was attempted and completed (CN110128491A, application number 201910405954.3), and a galactomannan octasaccharide derivative was synthesized, and the structural formula is as follows:

[0007]

[0008] Considering 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 required for synthesis, the present application designs and synthesizes a galactomannan tetrasaccharide derivative, and explores its biological activity in cellular immunity and antitumor activity. SUMMARY

[0009] In view of the shortcomings of the prior art, the present application provides a galactomannan tetrasaccharide with antitumor activity and its application. Different structures of oligosaccharide fragments have different biological activities, and the galactomannan tetrasaccharide prepared by the present application has good immunological activity and antitumor activity.

[0010] To achieve the above object, the present application adopts the following technical scheme:

[0011] A galactomannan tetrasaccharide, the general structural formula of which 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 application, the structure of the galactomannotetraose is as follows:

[0015]

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

[0017] The glycosylation coupling reaction of the disaccharide donor ACP-14 and the disaccharide acceptor ACP-8 is carried out in dry diethyl ether as a solvent under the catalysis of TfOH, to obtain a fully protected tetrasaccharide ACP-15; then, the fully protected tetrasaccharide ACP-15 is subjected to a protection group removal reaction in a mixed solution of tert-butyl alcohol-dichloromethane-water, and the ACP-15 is directly placed in a hydrogen atmosphere and subjected to catalytic hydrogenation under the action of palladium carbon, to obtain the target molecule galactomannotetraose ACP-3;

[0018] .

[0019] According to the present application, preferably, the disaccharide donor ACP-14 is prepared by taking the disaccharide glucosinolate donor ACP-13 as a starting material, 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 carrying out C-1 glucosinolate group hydrolysis under the catalysis of iodosobenzene sulfonate (NIS) and silver trifluoromethanesulfonate (AgOTf); then, the hydrolysis product is dissolved in dry dichloromethane under the condition of an ice water bath, and trichloroacetonitrile and a catalytic amount of a hindered base DBU are added for reaction, to obtain an α configuration trichloroacetimidate ester donor ACP-14;

[0020] .

[0021] According to the present application, preferably, the disaccharide acceptor ACP-8 is prepared by taking a monosaccharide ACP-4 as a starting material, first, the ACP-4 is subjected to introduction of a trichloroacetimidate group on a C-1 hydroxyl group under the action of trichloroacetonitrile and a catalytic amount of a hindered base 1,8-diazabicycloundec-7-ene (DBU), to obtain an α configuration trichloroacetimidate ester donor ACP-5; next, the donor ACP-5 is dissolved in dry dichloromethane with the monosaccharide acceptor ACP-6, water is removed by using a 4 Å molecular sieve, and glycosylation coupling reaction is carried out under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) at 0°C, to obtain an α configuration disaccharide ACP-7; then, the disaccharide ACP-7 is dissolved in a mixed solution of methanol and dichloromethane, and 1M sodium methoxide in methanol is added to remove acetyl groups, to obtain the disaccharide acceptor ACP-8;

[0022] .

[0023] Use of the above-mentioned galactomannotetraose in the preparation of a medicine with immunomodulatory effect.

[0024] Use of the above-mentioned galactomannotetraose in the preparation of a vaccine.

[0025] Use of the above-mentioned galactomannotetraose in the preparation of an antitumor drug.

[0026] According to the application, preferably, the tumor is a tumor type dependent on the activation of the JNK signaling pathway of apoptosis.

[0027] Further preferably, the activation of the JNK signaling pathway is dependent on the expression of ROS in the cell.

[0028] According to the application, preferably, the tumor is liver cancer, lung cancer or breast cancer.

[0029] Use of the above-mentioned galactomannotetraose in combination with a chemotherapeutic drug in the preparation of an antitumor drug.

[0030] According to the application, preferably, the tumor is liver cancer, lung cancer or breast cancer.

[0031] According to the application, preferably, the chemotherapeutic drug is cyclophosphamide (CTX) or paclitaxel (PTX).

[0032] Beneficial effects:

[0033] The galactomannotetraose ACP-3 synthesized in the application is a tetrasaccharide structure in the galactomannan of the Tianmagillate fungus, has a clear structure, high solubility, good absorption performance, good immunological activity, effective and safe antitumor effect, and has good development value. The galactomannotetraose ACP-3 can significantly inhibit tumor cell proliferation in vitro, and has no obvious toxicity to normal cells, indicating that the antitumor effect has selectivity. In vivo antitumor experiments show that the galactomannotetraose ACP-3 alone can improve the immune function of the body, inhibit tumor growth, and has a synergistic effect when used in combination with CTX or PTX. The application is helpful for the development of oligosaccharide drugs with antitumor effect, and has important significance for the development of corresponding saccharide drugs. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Synthesis process of disaccharide acceptor ACP-8-1.

[0035] Figure 2 Synthesis process of disaccharide donor ACP-11.

[0036] Figure 3 Synthesis process of galactomannotetraose ACP-2.

[0037] Figure 4 Synthesis process of galactomannotetraose 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] Figure 9 This is the synthesis process of galactomannotetraose ACP-3-2.

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

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

[0045] Figure 12 Cell survival curves of macrophages incubated with different oligosaccharides.

[0046] Figure 13 The figure is a bar graph showing the phagocytic ability of macrophages incubated with different oligosaccharides.

[0047] Figure 14 The cell survival rate bar graphs 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] Figure 15 The changes in tumor volume (A) and tumor weight (B) of mice in different drug-treated groups. P <0.05,##: P <0.01,###: P <0.001, n=6.

[0049] Figure 16 The changes in body weight of mice in different drug-treated groups are shown in the figure. Compared with the CTX-positive group, *: P <0.05,***: P <0.001, n=6.

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

[0051] Figure 18 The spleen index (A) and thymus index (B) of mice in different administration groups were determined. 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] Figure 19 The expression levels of AST, ALT, Cre and BUN in the serum of mice in different administration groups were determined. In the figure, compared with the normal group, #: P <0.05, ###: P <0.001, n=6.

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

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

[0055] Figure 22 The HE staining pictures of tumor tissues of mice in different administration groups were determined.

[0056] Figure 23 The spleen index (A) and thymus index (B) of mice in different administration groups were determined. In the figure, compared with the blank group, *: P <0.05, ***: P <0.001, n=6.

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

[0058] Figure 25 The Western blot detection figure of p-JNK protein in Hep G2 cells was determined; wherein, A was the Western blot development figure, and B was the quantification columnar chart of p-JNK protein expression amount. In the figure, compared with the control group, *: P <0.05, **: P <0.01.

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

[0060] Figure 27 The results of flow cytometry detection of Hep G2 cell apoptosis under different drug administration conditions are shown in Figure 1. A is the flow cytometry detection graph, 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] Figure 28 The results of flow cytometry detection of reactive oxygen species in Hep G2 cells under different drug administration conditions are shown in Figure 1. 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] Figure 29 The Western blot detection images of p-JNK and Cleaved-caspase3 proteins in Hep G2 cells are shown in Figure 1. A is the Western blot image, and B is the quantitative bar graph of the expression levels of p-JNK and Cleaved-caspase3 proteins. In the figure, compared with the control group, *: P <0.05,**: P <0.01; compared with ACP-3-1 group, #: P <0.05. DETAILED DESCRIPTION

[0063] The technical solution of the present invention will be further described below in conjunction with the examples and drawings, but the scope of protection 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 cells Hep G2, human lung cancer cells A549, human normal liver cells L02, human normal lung epithelial cells BEAS-2B, mouse liver cancer cells H22, and mouse breast cancer cells 4T1 are all commercially available.

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

[0066] In the previous study, the inventors completed the chemical synthesis of galactomanno octasaccharide ACP-1 and applied for an invention patent on May 16, 2019, with the application number 201910405954.3. The synthesis process of galactomanno octasaccharide ACP-1 is detailed in Example 4 of the patent. Considering that oligosaccharide fragments with different glycan sequences may have different biological activities, galactomanno tetrasaccharide derivatives were designed and synthesized in the following examples, and their biological activities were explored.

[0067] Example 1: Synthesis of galactomanno tetrasaccharide ACP-2

[0068] For the synthesis of the target molecule galactomanno tetrasaccharide ACP-2, a [2+2] assembly strategy was adopted, i.e., the assembly of disaccharide acceptor ACP-8-1 and disaccharide donor ACP-11.

[0069] As shown in Figure 1 , the preparation of disaccharide acceptor ACP-8-1 was based on monosaccharide ACP-4 as the starting material. First, ACP-4 was subjected to the introduction of a trichloroacetimidate group at the C-1 hydroxyl group under the action of trichloroacetonitrile and a catalytic amount of the hindered base 1,8-diazabicycloundec-7-ene (DBU), resulting in the trichloroacetimidate ester donor ACP-5 in the alpha configuration with a yield of 96%. Next, the activated ester donor ACP-5 was dissolved in dry dichloromethane, water was removed with 4 Å molecular sieves, and then a glycosylation coupling reaction was carried out at 0°C under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf), resulting in the disaccharide ACP-7-1 in the alpha configuration with a yield of 87%. In the above glycosylation reaction, the configuration control of the alpha-glycosidic bond was achieved through the ortho effect of the 2-acetyl (Ac) group. Then, the disaccharide ACP-7-1 was dissolved in a mixture of methanol and dichloromethane, and 1 M sodium methoxide in methanol was added to remove the acetyl group, resulting in the disaccharide acceptor ACP-8-1 with a yield of 94%.

[0070] As shown in Figure 2 , the preparation of disaccharide donor ACP-11 was based on disaccharide ACP-9 (the preparation method is described in patent 201910405954.3) as the starting material, which was converted into a trichloroacetimidate ester donor with higher reactivity. First, disaccharide ACP-9 was dissolved in a 1:1 volume ratio mixture of dichloromethane and acetonitrile (containing 10 equivalents of water), and the C-1 thioglycoside group was hydrolyzed under the catalysis of iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf), resulting in disaccharide ACP-10. Then, disaccharide ACP-10 was dissolved in dry dichloromethane under ice water bath conditions, and trichloroacetonitrile and a catalytic amount of the hindered base DBU were added for reaction, resulting in disaccharide donor ACP-11 with a total yield of 79% for the two steps.

[0071] As Figure 3 shown, the disaccharide acceptor ACP-8-1 and the disaccharide donor ACP-11 were dissolved in diethyl ether solution, and after water removal by 4 Å molecular sieves, the reaction system was cooled to -30 °C, and a catalytic amount of trifluoromethanesulfonic acid (TfOH) was added to carry out the coupling reaction, and the fully protected tetrasaccharide ACP-12 was prepared with a yield of 85%. Similarly, the benzyloxy group at C-2 of the disaccharide donor participates in the effect and the solvent effect of diethyl ether ensures the formation of α-glycosidic bond. Next, the fully protected tetrasaccharide ACP-12 was removed from the acetyl and benzoyl group by sodium methoxide solution, and then in the hydrogen atmosphere, catalytic hydrogenation was carried out by palladium on carbon to remove all benzyl groups and reduce azide group (N3) to amino group (NH2), and finally the target molecule galactomannan tetrasaccharide ACP-2 was obtained, and the two-step reaction yield was 81%. The structure of ACP-2 was confirmed by nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum, and the nuclear magnetic hydrogen spectrum is shown in Figure 4 .

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

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

[0074] As Figure 5 shown, the disaccharide acceptor ACP-8-1 and the disaccharide imidate donor ACP-14 were used for glycosylation reaction to construct the target molecule.

[0075] As Figure 6 shown, the disaccharide acceptor ACP-8-1 and the disaccharide imidate donor ACP-14 were used for glycosylation reaction to construct the target molecule. Figure 7

[0076] ​Example 3: Synthesis of Galactomanno-tetrasaccharide ACP-3-2

[0077] For the synthesis of the target molecule Galactomanno-tetrasaccharide ACP-3-2, the same [2+2] assembly strategy was adopted as in Example 2, i.e. the glycosylation reaction of disaccharide acceptor ACP-8-2 and disaccharide imino ester donor ACP-14 to construct the target molecule.

[0078] As shown in the scheme below, the preparation of disaccharide acceptor ACP-8-2, first, activated ester donor ACP-5 was dissolved in dry dichloromethane with monosaccharide acceptor ACP-6-2, after water removal with 4A molecular sieves, glycosylation coupling reaction was carried out at 0°C under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf), to prepare the disaccharide ACP-7-2 in α configuration. Then, the disaccharide ACP-7-2 was dissolved in a mixed solution of methanol and dichloromethane, and 1 M sodium methoxide in methanol was added to remove the acetyl group to obtain the disaccharide acceptor ACP-8-2. Figure 8

[0079] As shown in the scheme below, the preparation of disaccharide acceptor ACP-8-3, first, activated ester donor ACP-5 was dissolved in dry dichloromethane with monosaccharide acceptor ACP-6-3, after water removal with 4A molecular sieves, glycosylation coupling reaction was carried out at 0°C under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf), to prepare the disaccharide ACP-7-3 in α configuration. Then, the disaccharide ACP-7-3 was dissolved in a mixed solution of methanol and dichloromethane, and 1 M sodium methoxide in methanol was added to remove the acetyl group to obtain the disaccharide acceptor ACP-8-3. Figure 9

[0080] Example 4: Synthesis of Galactomanno-tetrasaccharide ACP-3-3

[0081] For the synthesis of the target molecule Galactomanno-tetrasaccharide ACP-3-3, the same [2+2] assembly strategy was adopted as in Example 2, i.e. the glycosylation reaction of disaccharide acceptor ACP-8-3 and disaccharide imino ester donor ACP-14 to construct the target molecule.

[0082] As shown in the scheme below, the preparation of disaccharide acceptor ACP-8-3, first, activated ester donor ACP-5 was dissolved in dry dichloromethane with monosaccharide acceptor ACP-6-3, after water removal with 4A molecular sieves, glycosylation coupling reaction was carried out at 0°C under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf), to prepare the disaccharide ACP-7-3 in α configuration. Then, the disaccharide ACP-7-3 was dissolved in a mixed solution of methanol and dichloromethane, and 1 M sodium methoxide in methanol was added to remove the acetyl group to obtain the disaccharide acceptor ACP-8-3. Figure 10

[0083] As shown in the scheme below, the preparation of disaccharide acceptor ACP-8-3, first, activated ester donor ACP-5 was dissolved in dry dichloromethane with monosaccharide acceptor ACP-6-3, after water removal with 4A molecular sieves, glycosylation coupling reaction was carried out at 0°C under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf), to prepare the disaccharide ACP-7-3 in α configuration. Then, the disaccharide ACP-7-3 was dissolved in a mixed solution of methanol and dichloromethane, and 1 M sodium methoxide in methanol was added to remove the acetyl group to obtain the disaccharide acceptor ACP-8-3. Figure 11 ​​​As shown, the glycosylation coupling reaction of the disaccharide donor ACP-14 and the disaccharide acceptor ACP-8-3 was carried out in dry diethyl ether as the solvent 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 the deprotection reaction in a mixed solution of tert-butyl alcohol-dichloromethane-water, and then ACP-15-3 was directly placed in a hydrogen atmosphere and subjected to catalytic hydrogenation in the presence of palladium carbon, to obtain the target molecule galactomannotetraose ACP-3-3.

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

[0085] The immunological activity of galactomannotetraoses ACP-2 and ACP-3-1 was evaluated by determining the effect of the galactomannotetraoses on the proliferation activity and phagocytic capacity of macrophage Raw 264.7.

[0086] Cell culture: DMEM complete medium (FBS: medium = 1:9) containing FBS and 100 μg·L -1 of streptomycin was prepared, and macrophage Raw 264.7 was placed in the DMEM complete medium and cultured in an incubator at 37℃ and 5% CO2until the cells grew to the logarithmic phase.

[0087] (1) Proliferation activity

[0088] The concentration of Raw 264.7 cells was adjusted to 1×10 5 cells / mL in DMEM complete medium containing 10% FBS, and inoculated into a 96-well plate (100 μL per well). After the 96-well plate was cultured in an incubator at 37℃ and 5% CO2saturated humidity for 24 h, the culture medium was discarded, and then the cells were incubated in DMEM complete medium (containing 10% FBS) containing different concentrations of oligosaccharides for 24 h. After CCK-8 reagent was added and cultured for 1 h, the absorbance was determined at 450 nm by an enzyme marker.

[0089] The CCK-8 method was used to determine the effect of oligosaccharides at different concentrations (10, 25, 50, 100, and 200 μg / mL) on the proliferation activity of macrophage Raw 264.7. The results are shown in Table 1. Figure 12As shown in Figure 3, the three oligosaccharides showed similar proliferation trend on macrophage Raw 264.7. When the concentration of the three oligosaccharides was 25 μg / mL, the proliferation activity of macrophage 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 the concentration of oligosaccharides (> 25 μg / mL), the proliferation activity of macrophage Raw 264.7 showed a decreasing trend. In summary, the order of the effect of the three oligosaccharides on the proliferation activity of macrophage Raw 264.7 was: tetrasaccharide ACP-3-1 > octasaccharide ACP-1 > tetrasaccharide ACP-2, wherein the galactomanno tetrasaccharide ACP-3-1 was the best for the proliferation activity of macrophage Raw 264.7.

[0090] (2) Phagocytic ability

[0091] The concentration of Raw 264.7 cells was adjusted to 1 × 10 5 The 96-well plate was placed in a 37 ℃, 5% CO2saturated humidity incubator for 24 h, and then the culture medium was discarded. Oligosaccharide solution (25 μg / mL) and LPS solution (1 μg / mL, positive control) were prepared with DMEM complete medium (containing 10% FBS), and then the macrophage Raw 264.7 was cultured with the prepared solution for 24 h. After that, the culture medium was removed, and the cells were washed twice with PBS. 100 μL of prepared 0.075% neutral red was added to each well. After 4 h, 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 540 nm by using a microplate reader.

[0092] The effect of oligosaccharides on the phagocytic ability of macrophage Raw 264.7 was determined by detecting the uptake amount of neutral red, and the experimental results are shown in Figure 4. Figure 13 As shown in Figure 4, compared with the blank group, octasaccharide ACP-1 and tetrasaccharide ACP-3-1 significantly improved the phagocytic ability of macrophage 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 positive control LPS in enhancing the phagocytic ability of macrophage. In contrast, compared with the blank group, tetrasaccharide ACP-2 had no obvious effect on the phagocytic ability of macrophage Raw 264.7 (P > 0.05).

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

[0094] (1) Cell culture

[0095] Hep G2 cells, A549 cells, L02 cells, BEAS-2B cells were respectively placed in DMEM complete medium containing 10% FBS, 100 U / mL penicillin, 100 mg / mL streptomycin, and cultured in a 37°C, 5% CO2 incubator 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, and Hep G2 cells, A549 cells, L02 cells, BEAS-2B cells were respectively inoculated in 96-well culture plates at a concentration of 1×10 5 After the cells adhered, the culture medium was discarded, and different concentrations of tetrasaccharide ACP-3-1 solution (0, 10, 25, 50, 100, 200 µM) containing 10% FBS were prepared in DMEM complete medium, and the cells were cultured. After 24 h, 48 h and 72 h of culture, the cell proliferation was detected by CCK-8 method.

[0098] (3) Experimental results

[0099] As shown in Figure 14 , 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 200 µM tetrasaccharide ACP-3-1 treatment of human lung cancer cells A549 and human liver cancer cells Hep G2 for 72 h, the inhibition rate was close to 50%. In contrast, 10-400 µM 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 tetrasaccharide ACP-3-1 has low toxicity to normal cells. In summary, galactomannan tetrasaccharide 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 galactomannan tetrasaccharide 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 grams, were first adaptively fed for one week. The mice were divided into normal group, model group, ACP-3-1 low dose group, ACP-3-1 high dose group, CTX positive group, ACP-3-1 low dose + CTX group, ACP-3-1 high dose + CTX group, 6 mice in each group. Adjust the concentration of H22 cells to 1×10 7cells / mL, and 0.2 mL (2×10 6 When the tumor volume is greater than 100 mm 3 Dosing was initiated at 4:00 p.m. The following dosing conditions were observed: normal group (normal saline), model group (normal saline), low-dose ACP-3-1 group (1 mg / kg), high-dose ACP-3-1 group (5 mg / kg), CTX-positive group (30 mg / kg), low-dose ACP-3-1 (1 mg / kg) + CTX (30 mg / kg) group, and high-dose ACP-3-1 (5 mg / kg) + CTX (30 mg / kg) group. Dosing was based on mouse body weight, with intraperitoneal injection once daily. Body weight and tumor volume were recorded every other day. Tumor volume was calculated using the formula: length × width × width / 2. One week after dosing, peripheral blood was collected from the mice, and biochemical markers were measured using kits. The mice were sacrificed, 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: Fix the tumor tissue in 4% paraformaldehyde for one week, and then flatten the tissue at the target site.

[0106] Dehydration: Tissues were dehydrated using a series of ethanol treatments: 70% ethanol for 30 min, 80% ethanol for 30 min, 85% ethanol for 30 min, 90% ethanol for 30 min, 95% ethanol for 30 min, and 100% ethanol for 30 min, twice. Xylene was then added for 1 h to remove the alcohol.

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

[0108] Embedding: Place the paraffin-transmitted tissue block into the mold with the side to be cut facing down, add 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 then use a microtome to cut paraffin sections with a thickness of 3-5 µm from the paraffin block.

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

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

[0112] Dewaxing: The tissue sections were dewaxed to water routinely: paraffin sections were sequentially immersed in xylene, 15 min, 2 times; 100% ethanol, 5 min, 2 times; 95% ethanol, 5 min; 90% ethanol, 5 min; 80% ethanol, 5 min; double distilled water, 5 min.

[0113] Staining: The sections were stained with hematoxylin for 5 min, and washed with distilled water for 5 min. Differentiated with hydrochloric acid ethanol for several seconds, and washed with distilled water for 3 min. Stained with eosin for 5 min, and washed with distilled water for 3 min.

[0114] Gradient dehydration: The sections were dehydrated with gradient ethanol: 95% ethanol, 2 min, 2 times; 100% ethanol, 2 min, 2 times. And transparent with xylene, 5 min, 2 times.

[0115] Mounting: The sections were mounted with neutral balsam, and then observed the staining condition with an optical microscope (Nikon Eclipse E100, Japan).

[0116] (3) Experimental results

[0117] The effect of tetrasaccharide ACP-3-1 on tumor growth is shown in Figure 15 The tumor volume and tumor weight of the model group mice were significantly increased, while the tumor growth of each administration group was delayed. Among them, the tumor volume and tumor weight of the ACP-3-1 low dose group (1 mg / kg) mice were similar to those of the CTX positive group. In addition, the combination of ACP-3-1 low dose (1 mg / kg) and CTX (30 mg / kg) can significantly delay the tumor growth of 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 Figure 16 The body weight of mice in the CTX positive group was significantly decreased, indicating that CTX had serious side effects on mice. Compared with the model group, ACP-3-1 low and high dose groups (1 mg / kg and 5 mg / kg) had no obvious effect on the body weight of mice. The change of body weight of mice in the ACP-3-1 low dose (1 mg / kg) + CTX (30 mg / kg) group also showed that tetrasaccharide ACP-3-1 can significantly reverse the trend of body weight loss in mice caused by CTX, and alleviate the side effects of CTX.

[0119] The HE staining results of tumor tissues are shown in Figure 17) showed that tumor cells in the model group grew vigorously and were densely arranged, with darker nuclear staining, a high nuclear-cytoplasmic ratio, and strong basophilia. No obvious cell death occurred. After drug administration, tumor cell growth was inhibited to varying degrees, with a significant decrease in the number of tumor cells, smaller size, lighter nuclear staining, a lower nuclear-cytoplasmic ratio, and decreased basophilia. In the CTX combined with low-dose ACP-3-1 group, cell nuclei were severely ruptured, cells shrank, and cells showed obvious signs of cell death.

[0120] Immune organ index test results are as follows Figure 18 Compared with the normal group, the thymus index of the model group mice was significantly reduced ( 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 reduced ( 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 when low and high doses of tetrasaccharide ACP-3-1 were used in combination with 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 mouse spleen swelling. Compared with the model group, the spleen index of mice in the low-dose and high-dose tetrasaccharide ACP-3-1 groups decreased significantly ( P <0.05). The above immune organ index analysis results show that tetrasaccharide ACP-3-1 can enhance the immune function of tumor-bearing mice, and CTX has a strong toxicity to immune organs ( P <0.001), low and high doses of tetrasaccharide ACP-3-1 combined with 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 test 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 the liver, skeletal muscle and kidney tissues. When the corresponding cells are damaged, AST is released into the blood, so its serum concentration increases, which is one of the indicators of liver damage in clinical practice; ALT (alanine aminotransferase) is mainly present in various cells, especially liver cells, and is an important indicator for diagnosing liver diseases; Cre (blood 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 used clinically as an indicator for judging glomerular filtration function. The measurement results are as follows: Figure 19As shown in the figure, compared with the normal group, the levels of ALT, AST, Cre, and BUN in the model group remained essentially unchanged, indicating that the modeling did not cause liver and kidney damage in mice. However, after CTX administration, serum ALT, AST, Cre, and BUN levels increased, indicating that CTX can cause liver and kidney damage. There were no significant changes in serum ALT, AST, Cre, and BUN levels in the low-dose and high-dose ACP-3-1 groups, indicating that the tetrasaccharide ACP-3-1 did not cause liver and kidney damage in tumor-bearing mice and was non-hepatorenal. In the group receiving combined ACP-3-1 and CTX, serum AST and Cre levels remained unchanged, and ALT and BUN levels were further reduced compared to the model group, indicating that the tetrasaccharide ACP-3-1 can alleviate the degree of CTX-induced liver and kidney damage to some 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, weighing 18-22 g, were selected and acclimated 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 of 4T1 cell suspension (2 × 10 6 When the tumor volume is larger than 100 mm 3 Dosing began after 48 hours. Dosing was based on body weight in the following groups: blank group (normal saline), ACP-3-1 group (1 mg / kg), PTX group (10 mg / kg), and ACP-3-1 (1 mg / kg) + PTX (10 mg / kg) groups. Mice were dosed every other day via tail vein injection. Mice were weighed every two days, and tumor volume was measured. After the fifth dose, the mice were sacrificed by removing their eyeballs and bleeding. Blood biochemical markers were measured using kits. The spleen and thymus were dissected and weighed. Tissues were fixed in 4% paraformaldehyde for histopathological analysis.

[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 Figure 20As shown, the tumors of mice in the blank group grew rapidly, and the tumor growth was slowed down in all the drug-treated groups. 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] The changes of body weight of tumor-bearing mice in each group over time Figure 21 As shown in the figure, compared to the blank group, mice in the PTX group showed a significant decrease in body weight. There were no significant differences in body weight between the ACP-3-1 group and the ACP-3-1 + PTX group compared to the blank group. These results suggest that PTX can cause serious side effects, while ACP-3-1 has no significant side effects and can, to some extent, alleviate the PTX-induced weight loss in mice.

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

[0131] Immune organ index test results such as Figure 23 As shown in Figure 2 . Compared with the blank control group, the thymic index of mice in the PTX group was significantly decreased, indicating that PTX damages the thymus. ACP-3-1 had no significant effect on the thymic index and, to a certain extent, alleviated PTX-induced thymic damage. Furthermore, ACP-3-1 showed no significant toxicity to the spleen.

[0132] Blood biochemical index results such as Figure 24 As shown, PTX increased serum BUN and Cre levels, indicating that PTX causes some renal toxicity. However, combined use of PTX and ACP-3-1 did not significantly alter serum BUN and Cre levels, suggesting that ACP-3-1 can mitigate PTX-induced renal toxicity. ACP-3-1 had no significant effect on BUN and Cre levels, indicating that ACP-3-1 has no significant renal toxicity. Furthermore, ALT and AST results also indicate that ACP-3-1 has no significant hepatic toxicity.

[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: Galactomannotetrasaccharide ACP-3-1 induces tumor cell apoptosis through ROS-mediated JNK signaling pathway

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

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

[0137] Hep G2 cells in logarithmic growth phase were inoculated in 6-well plates at a density of 1 x 10 5 After the cells were attached, 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 h. After the culture ended, the cells were washed with PBS for 3 times, and cell lysate containing protease inhibitors and phosphatase inhibitors was added to the cell culture solution, and lysed on ice for 30 min. The cell lysate was centrifuged at 4°C, 13000 rpm for 15 min, 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 at room temperature for 1 h, the PVDF membrane was washed with TBST buffer for 3 times. Specific primary antibody was added to the PVDF membrane, and incubated at 4°C overnight, washed with TBST buffer for 3 times, and secondary antibody was added, and incubated at room temperature for 1 h. Washed with TBST for 3 times, add developing solution for color development, and use Image J software to quantify the density of Western blot band.

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

[0139] (2) JNK inhibitor SP600125 was used to further verify the JNK signaling pathway

[0140] Hep G2 cells in logarithmic growth phase were inoculated in 6-well plates at a density of 1 x 10 5Hep G2 cells were seeded in 6-well plates at a density of 1×10

[0141] The method of flow cytometry detection was as follows: 1 mL of 1x Binding Buffer was used to suspend the cells, and the cells were centrifuged at 300xg for 10 min, and the supernatant was discarded. The cells were resuspended with 1x Binding Buffer to make the density of the cells reach 1x10 6 5 The cells were added to each tube at a density of 1x10 5 5 μL of Annexin V-FITC was added to the tube. The reaction was carried out at room temperature and in the dark, and the cells were gently mixed and reacted for 10 min. 5 μL of PI was added, and the reaction was carried out at room temperature and in the dark for 5 min. PBS was added to 500 μL, and the cells were gently mixed. The cells were detected by flow cytometry (ACEA NovoCyte, Hanzhou) within 1 h.

[0142] As shown in Table 1, the proliferation of Hep G2 cells was inhibited by ACP-3-1. Figure 26 As shown in Table 1, the proliferation of Hep G2 cells was inhibited by ACP-3-1. Figure 27 As shown in Table 1, the proliferation of Hep G2 cells was inhibited by ACP-3-1.

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

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

[0145] (1) ROS determination

[0146] The cells were seeded in 6-well plates at a density of 1×10 5Cells were seeded at a density of 100 cells / mL in 6-well plates. After cell attachment, the culture medium was discarded and various concentrations of ACP-3-1 (0, 100, and 200 µM) prepared in complete DMEM medium supplemented with 10% FBS were added and cultured for 72 hours. The cells were then resuspended in a working solution of the 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 intracellular ROS expression levels were measured by flow cytometry.

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

[0148] (2) The antioxidant NAC was used 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 Cells were seeded at a density of 100 cells / mL in 6-well plates. After attachment, the culture medium was discarded and the cells were cultured for 72 hours with solutions containing different compositions prepared in DMEM complete medium supplemented with 10% FBS. The solutions and concentrations of the different compositions were NAC solution (10 mM), ACP-3-1 solution (200 µM), and ACP-3-1 + NAC solution (200 µM + 10 mM; NAC was added to the cells for a 2-hour pretreatment, followed by ACP-3-1 solution for 72 hours). DMEM complete medium supplemented with 10% FBS alone served as a blank control. After culture, p-JNK protein expression in the cells was detected by Western blotting, and the density of the protein blot bands was quantified using Image J software.

[0150] The results are as follows Figure 29 As shown in the results, treatment of Hep G2 cells with the antioxidant NAC significantly inhibited the expression of the pathway protein p-JNK and the pro-apoptotic protein Cleaved-caspase-3 in cells activated by the tetrasaccharide ACP-3-1. Therefore, it is preliminarily speculated that the tetrasaccharide ACP-3-1 promotes 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 structural formula is as follows: In the general formula, n is an integer from 1 to 6.

2. The galactomannotetraose according to claim 1, wherein 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 a solvent, the disaccharide donor ACP-14 and the disaccharide acceptor ACP-8 underwent a glycosylation coupling reaction catalyzed by TfOH to produce the fully protected tetrasaccharide ACP-15. The fully protected tetrasaccharide ACP-15 was then deprotected in a mixed solution of tert-butanol, dichloromethane, and water. ACP-15 was then directly placed in a hydrogen atmosphere and catalytically hydrogenated over palladium on carbon to produce the target molecule, galactomannotetrose ACP-3. 。 4. The preparation method according to claim 3, wherein The disaccharide donor ACP-14 is prepared 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 at 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). The hydrolyzate is then dissolved in dry dichloromethane in an ice-water bath, and trichloroacetonitrile and a catalytic amount of a hindered base DBU are added to react to obtain the α-configuration trichloroacetimidate donor ACP-14. 。 5. The preparation method according to claim 3, wherein The disaccharide acceptor ACP-8 is prepared using the monosaccharide ACP-4 as a starting material. First, a trichloroacetimidate group is introduced into the C-1 hydroxyl group of ACP-4 in the presence of trichloroacetonitrile and a catalytic amount of the hindered base 1,8-diazabicycloundec-7-ene (DBU) to obtain the α-configured trichloroacetimidate donor ACP-5. Next, the donor ACP-5 and the monosaccharide acceptor ACP-6 are dissolved in dry dichloromethane, dehydrated with 4Å molecular sieves, and then glycosylated at 0°C under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to obtain the α-configured disaccharide ACP-7. Then, the disaccharide ACP-7 is dissolved in a mixed solution of methanol and dichloromethane, and a 1M methanolic solution of sodium methoxide is added to remove the acetyl group to obtain the disaccharide acceptor ACP-8. 。 6. Use of the galactomannotetrose according to claim 1 or 2 in the preparation of a medicine having an immunomodulatory effect.

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

8. Use of the galactomannotetrose according to claim 1 or 2 in the preparation of an anti-tumor drug; the tumor is liver cancer, lung cancer or breast cancer.

9. The use according to claim 8, characterized in that The tumor is a tumor type in which cell apoptosis depends on activation of the JNK signaling pathway.

10. The use according to claim 9, characterized in that The activation of the JNK signaling pathway is dependent on the expression of ROS in cells.

11. Use of the galactomannotetraose according to claim 1 or 2 in combination with a chemotherapy drug in the preparation of an anti-tumor drug; the tumor is liver cancer, lung cancer or breast cancer.

12. The use according to claim 11, characterized in that The chemotherapy drug is cyclophosphamide or paclitaxel.

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