Preparation method of sugarcane leaf polysaccharide and application of sugarcane leaf polysaccharide in immune regulation and tumor resistance

Abstract

The invention discloses a preparation method of sugarcane leaf polysaccharide and application of the sugarcane leaf polysaccharide in immune regulation and tumor resistance, and belongs to the technical field of biological medicine. Comprising the following steps: (1) adding water into sugarcane leaves for extraction, and sequentially performing centrifugation, concentration, alcohol precipitation and dialysis on a water extract to obtain sugarcane leaf crude polysaccharide I; (2) carrying out enzymolysis and deproteinization on the crude polysaccharide by using papain, adsorbing overnight by using AB-8 type macroporous resin, dialyzing a solution, and freeze-drying to obtain a crude polysaccharide II; and (3) purifying and eluting the crude polysaccharide II through an ion exchange column, concentrating, dialyzing, and separating and purifying through a gel chromatography column to obtain the sugarcane leaf polysaccharide SLP-D2N2. The obtained polysaccharide has remarkable biological activity, can enhance the activity of macrophages, plays an important role in regulating the tumor microenvironment and enhancing the anti-tumor immune response, and particularly can be in synergistic interaction with anti-tumor drugs such as cis-platinum and the like to improve the treatment effect.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for preparing sugarcane leaf polysaccharide and its application in regulating immunity and anti-tumor. Background Art

[0002] Cancer is one of the leading causes of death worldwide, with lung cancer causing the most cancer-related deaths. Lung cancer is mainly divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is a heterogeneous disease with diverse molecular and pathological characteristics, accounting for about 85% of all lung cancer cases. Currently, cancer treatment drugs usually have significant side effects. Cisplatin, as a classic chemotherapy drug, is commonly used to treat a variety of cancers, but its side effects such as drug resistance and organ toxicity limit its application.

[0003] Polysaccharides are long-chain polymers composed of multiple monosaccharide molecules connected by glycosidic bonds. They are very important macromolecules in cells and have multiple biological functions such as anti-tumor, lipid-lowering, anti-viral, antioxidant and immunomodulatory. Studies have shown that polysaccharides can enhance the body's immune function by enhancing the phagocytic ability of macrophages, promoting T cell proliferation and increasing B cell activity. Cytotoxic polysaccharides can directly kill tumor cells, or indirectly inhibit or kill tumor cells by enhancing the body's immune function as immunomodulators.

[0004] Sugarcane (Saccharum officinarum L.) is a herbaceous plant of the genus Saccharum in the Poaceae family, which is widely planted in tropical areas in southern my country. Sugarcane leaves are by-products in the sugarcane industry chain and contain rich chemical components, including polysaccharides, amino acids, reducing sugars, glycosides, organic acids and flavonoids. Sugarcane leaf polysaccharides (SLP) are one of the main components of sugarcane leaves and have multiple biological activities such as antioxidant, anti-tumor and hypoglycemic effects. Studies in recent years have shown that sugarcane leaf polysaccharides have a protective effect in the myocardial ischemia-reperfusion injury model and can prevent cardiovascular diseases by inhibiting necrosis and oxidative stress. In addition, sugarcane leaf polysaccharides also show antibacterial properties against a variety of pathogenic bacteria. However, the extraction purity of sugarcane leaf polysaccharides in the current existing technology is low. Therefore, it is crucial to provide a new method for preparing sugarcane leaf polysaccharides. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing sugarcane leaf polysaccharide and its application in regulating immune anti-tumor to solve the problems existing in the above-mentioned prior art. The present invention provides a new option for the development of anti-tumor drugs and the research of new treatment strategies, and has good application prospects.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a method for preparing sugarcane leaf polysaccharide, comprising the following steps:

[0008] (1) Extracting sugarcane leaves with water, and sequentially subjecting the water extract to centrifugation, concentration, alcohol precipitation, and dialysis to obtain crude sugarcane leaf polysaccharide I;

[0009] (2) The crude polysaccharide is hydrolyzed and deproteinized by papain, and then adsorbed by AB-8 macroporous resin overnight. The solution is dialyzed and freeze-dried to obtain crude polysaccharide II;

[0010] (3) The crude polysaccharide II is purified, eluted, concentrated, and dialyzed by an ion exchange column, and then separated and purified by a gel chromatography column to obtain the sugarcane leaf polysaccharide.

[0011] The second technical solution of the present invention is the sugarcane leaf polysaccharide prepared by the preparation method.

[0012] The third technical solution of the present invention is the use of the sugarcane leaf polysaccharide in the preparation of a drug for promoting macrophage proliferation.

[0013] A fourth technical solution of the present invention is the use of the sugarcane leaf polysaccharide in the preparation of a drug for promoting the polarization of macrophages to the M1 phenotype.

[0014] A fifth technical solution of the present invention is a drug for promoting polarization of macrophages to M1 phenotype, comprising the sugarcane leaf polysaccharide.

[0015] The sixth technical solution of the present invention is the use of the sugarcane leaf polysaccharide in the preparation of anti-tumor drugs.

[0016] The seventh technical solution of the present invention is an anti-tumor drug, comprising the sugarcane leaf polysaccharide and cisplatin.

[0017] Based on the above technical solution, the present invention has the following technical effects:

[0018] The sugarcane leaf polysaccharide SLP-D2N2 provided by the present invention not only provides a new way to utilize agricultural by-products at a high value, but also demonstrates its broad application prospects in the field of biomedicine. SLP-D2N2 can be used as an ideal synergist for chemotherapy drugs such as cisplatin by promoting immune cell activity and regulating the tumor microenvironment, reducing the side effects of drugs and improving the therapeutic effect, providing new ideas for the development of new tumor immunotherapy programs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 These are the purification elution curves of sugarcane leaf polysaccharides, where A is the ion exchange purification elution curve and B is the gel chromatography purification elution curve.

[0021] Figure 2 Total ion current of methylation analysis of sugarcane leaf polysaccharides, showing the methylation status of sugar residues of SLP-D2N2.

[0022] Figure 3 This is the structural analysis of sugarcane leaf polysaccharide SLP-D2N2, where A is a one-dimensional 1H NMR spectrum, B is a 13C NMR spectrum, C is a two-dimensional COSY NMR spectrum, D is a two-dimensional HSQC NMR spectrum, E is a two-dimensional HMBC NMR spectrum, F is a two-dimensional NOESY NMR spectrum, and G is a schematic diagram of the inferred structure of SLP-D2N2.

[0023] Figure 4 The results show the effect of sugarcane leaf polysaccharide SLP-D2N2 on the activity of mouse Raw264.7 macrophages. The CCK-8 method was used to detect the enhancing effect of different concentrations of SLP-D2N2 on macrophage activity.

[0024] Figure 5 In order to investigate the effect of sugarcane leaf polysaccharide SLP-D2N2 on the phenotype of mouse Raw264.7 macrophages, flow cytometry was used to detect the regulatory effect of SLP-D2N2 on the M1 and M2 phenotypes of macrophages.

[0025] Figure 6 This is the synergistic index (CI) analysis diagram of sugarcane leaf polysaccharide SLP-D2N2 synergizing with cisplatin, where CI<1 indicates a synergistic effect between the two drugs, CI=1 indicates an additive effect, and CI>1 indicates an antagonistic effect.

[0026] Figure 7 The anti-tumor effect of sugarcane leaf polysaccharide SLP-D2N2 in the nude mouse A549-luc tumor model was shown, and it significantly reduced tumor volume when used in combination with cisplatin.

[0027] Figure 8 This is the tumor volume change curve of the nude mouse A549-luc tumor model after 30 days of administration of different doses of SLP-D2N2 and cisplatin combined treatment.

[0028] Fig. 9 This is the final tumor volume of the nude mouse A549-luc tumor model after 30 days of administration of different doses of SLP-D2N2 and cisplatin combined treatment.

[0029] Fig.10Comparison of tumor weights in nude mouse A549-luc tumor model after 30 days of administration of different doses of SLP-D2N2 and cisplatin combination therapy.

[0030] Fig.11 The results of flow cytometric analysis of macrophage phenotype in A549-luc tumor tissue of nude mice showed that the SLP-D2N2 combined with cisplatin treatment group significantly increased the proportion of M1 macrophages (CD86+) and reduced the proportion of M2 macrophages (CD206+). DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0037] The embodiment of the present invention provides a method for preparing sugarcane leaf polysaccharide, comprising the following steps:

[0038] (1) Extracting sugarcane leaves with water, and sequentially subjecting the water extract to centrifugation, concentration, alcohol precipitation, and dialysis to obtain crude sugarcane leaf polysaccharide I;

[0039] (2) The crude polysaccharide is hydrolyzed and deproteinized by papain, and then adsorbed by AB-8 macroporous resin overnight. The solution is dialyzed and freeze-dried to obtain crude polysaccharide II;

[0040] (3) The crude polysaccharide II is purified, eluted, concentrated, and dialyzed by an ion exchange column, and then separated and purified by a gel chromatography column to obtain the sugarcane leaf polysaccharide SLP-D2N2.

[0041] In some specific embodiments, the Mw cutoff value of the dialysis is 3000 Da.

[0042] The embodiment of the present invention also provides sugarcane leaf polysaccharide prepared by the preparation method.

[0043] The embodiment of the present invention also provides the use of the sugarcane leaf polysaccharide in preparing a drug for promoting macrophage proliferation.

[0044] The embodiment of the present invention also provides the use of the sugarcane leaf polysaccharide in the preparation of a drug for promoting macrophage polarization to the M1 phenotype.

[0045] The embodiment of the present invention also provides a drug for promoting macrophage polarization to M1 phenotype, comprising the sugarcane leaf polysaccharide.

[0046] The embodiment of the present invention also provides the use of the sugarcane leaf polysaccharide in the preparation of anti-tumor drugs.

[0047] The embodiment of the present invention also provides an anti-tumor drug, comprising the sugarcane leaf polysaccharide and cisplatin.

[0048] The purified sugarcane leaf polysaccharide SLP-D2N2 of the present invention has significant biological activity, including promoting macrophage proliferation, inhibiting M2 macrophage polarization and promoting M1 polarization. In in vitro and in vivo experiments, the combined use of SLP-D2N2 and cisplatin can significantly inhibit the proliferation of tumor cells, especially in the mouse lung cancer cell A549-luc tumor model, the synergistic effect of SLP-D2N2 and cisplatin can significantly reduce tumor volume and improve the anti-tumor treatment effect. The present invention provides a new choice for the development of anti-tumor drugs and the research of new treatment strategies, and has good application prospects.

[0049] By subjecting sugarcane leaves to water extraction, alcohol precipitation, ion exchange purification and gel chromatography, SLP-D2N2 with a purity of 96.1% was finally obtained. Its molecular structure was analyzed by methylation and nuclear magnetic resonance, and it was mainly composed of a main chain composed of β-D-xylose residues and a small amount of branched structures. This structural feature gives SLP-D2N2 a unique advantage in enhancing immune function.

[0050] The specific preparation of the sugarcane leaf polysaccharide (SLP-D2N2) includes:

[0051] Crude extraction of sugarcane leaf polysaccharides: Sugarcane leaves were mixed with pure water, refluxed and extracted, and centrifuged to obtain an extract. The residue was filtered and discarded. The extract was concentrated to obtain a crude polysaccharide extract. Anhydrous ethanol was added to precipitate the supernatant until the final concentration was 70%. The precipitate was collected and marked. The solution was further dialyzed with water for 48 hours (Mw cutoff value: 3000Da). The dialyzate was freeze-dried to obtain crude polysaccharides.

[0052] Crude polysaccharide impurity removal: After dissolving in water, add papain for overnight enzymolysis. Use Sevag reagent to remove protein, add petroleum ether to collect the water phase and adsorb it with AB-8 macroporous resin overnight. The solution is dialyzed in distilled water with a 3000Da dialysis bag for 48 hours and then freeze-dried.

[0053] Ion purification: Dissolve in pure water, centrifuge and take the supernatant to purify through an ion exchange column, combine the eluates corresponding to the same elution peak, and concentrate by rotary evaporation to 1 / 5 of the original volume. Desalt by dialyzing in a 3000Da dialysis bag for 48 hours.

[0054] Gel purification: The crude polysaccharide was dissolved in pure water, and the supernatant was centrifuged and separated and purified by gel chromatography column at a flow rate of 1 mL / min. Pure water was used to elute 1.5 times the column volume. Every 12 mL was collected in one tube, and all the eluates were collected, and the eluates corresponding to the same elution peak were combined and concentrated by rotary evaporation to 1 / 5 of the original volume. Freeze drying was performed to obtain SLP-D2N2 with a purity of 96.1%.

[0055] Bioactivity verification: In in vitro and in vivo experiments, SLP-D2N2 was shown to effectively enhance the activity of mouse macrophage Raw264.7 and synergize with cisplatin to inhibit cancer cell proliferation in the lung cancer cell model A549-luc. In particular, in the mouse A549-luc tumor model, SLP-D2N2 significantly inhibited tumor volume growth, indicating that the polysaccharide can be used to synergize with chemotherapy drugs to enhance the anti-tumor therapeutic effect.

[0056] Example 1

[0057] Preparation of Sugarcane Leaf Polysaccharide

[0058] Sugarcane leaves were mixed with pure water at a solid-liquid ratio of 1:30 g / mL, w / v, heated to 80°C for reflux extraction, centrifuged to obtain an extract, and the residue was filtered and discarded. The extract was concentrated to 1 / 5 of the original volume to obtain a crude polysaccharide extract. Four times the volume of anhydrous ethanol was added to precipitate the supernatant. The precipitate was collected and labeled. The solution was further dialyzed with water for 48 hours (Mw cutoff: 3000Da). The dialyzate was freeze-dried to obtain crude polysaccharide I.

[0059] After the crude polysaccharide I was dissolved in water, 1% papain was added for overnight enzymatic hydrolysis. Deproteinization was performed using Sevag reagent (chloroform: n-butanol = 4:1 (v / v)), and petroleum ether was added to collect the aqueous phase for overnight adsorption using AB-8 macroporous resin. The solution was dialyzed in distilled water for 48 hours using a 3000Da dialysis bag and then freeze-dried to obtain crude polysaccharide II. The crude polysaccharide II was dissolved in pure water, and the supernatant was taken by centrifugation and purified by an ion exchange column at a flow rate of 4 mL / min; pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions were used in turn for isocratic elution, and one tube was collected every 15 mL, and all the eluates were collected. The total sugar content of the eluate in each collection tube was determined by the sulfuric acid-phenol method, and the ion purification elution curve was drawn as shown in the figure. Figure 1 As shown in A. Combine the eluents corresponding to the same elution peak (a total of three elution peak components were obtained, peak 1: tubes 7-19, peak 2: tubes 28-40, peak 3: tubes 49-58), and concentrate the ion elution peak 2 by rotary evaporation to 1 / 5 of the original volume. Use a 3000Da dialysis bag to dialyze for 48 hours to remove salt, centrifuge and take the supernatant for separation and purification through a gel chromatography column at a flow rate of 1mL / min. Use pure water to elute 1.5 times the column volume, collect one tube for every 12mL, and collect all the eluents. The gel purification elution curve is shown in Figure 1 As shown in B. The eluates corresponding to the same elution peak (three elution peak components were obtained, peak 1: tubes 19-36, peak 2: tubes 37-43, peak 3: tubes 44-53) were combined, and the elution peak 2 was concentrated by rotary evaporation to 1 / 5 of the original volume. Freeze drying was performed to obtain crude polysaccharide D2N2 (SLP-D2N2) with a purity of 96.1%.

[0060] Example 2

[0061] Analysis of the bonding structure (methylation) of SLP-D2N2

[0062] After acid hydrolysis and derivatization of SLP-D2N2, GC-MS was performed using a 7890A-5977B gas spectrometer (Agilent Technologies Inc. CA, UAS) to analyze the glycosidic bond connection type of SLP-D2N2. The Agilent gas chromatography system was used, the chromatographic column was BPX70 (30m×0.25mm×0.25μm, SGE, Australia), the carrier gas was high-purity helium, the injection volume was 1μL, and the split ratio was 10:1; the initial temperature of the column oven was 140℃ for 2.0min, and the temperature was programmed to 230℃ at 3℃ / min and maintained for 3min. The mass spectrometry system used a quadrupole mass spectrometry detection system (Agient5977B; Agilent Technologies, USA) from Aiglent, USA, equipped with an electron impact ion source (EI) and a MassHunter workstation. The electron impact ion source (EI) was used, and the full scan (SCAN) mode was used for detection. The mass scanning range (m / z) was 50-350. The total ion current of SLP-D2N2 is shown in the figure below. Figure 2 As shown. According to the characteristic fragment analysis after polysaccharide methylation, the connection mode is confirmed to be t-Ara(f), 2-Ara(f), 3-Ara(f), t-Gal(p), 5-Ara(f), 4-Xyl(p), 3-Glc(p), 3-Gal(p), 4-Gal(p), 4-Glc(p), 3,4-Xyl(p), 3,6-Gal(p).

[0063] Example 3

[0064] Comprehensive structural analysis of SLP-D2N2

[0065] The sugarcane leaf polysaccharide was accurately weighed and fully dissolved in D2O to prepare a 40 mg / mL SLP-D2N2 solution for nuclear magnetic resonance spectroscopy detection. Figure 3 As shown in AF, multiple coupled signal peaks were identified in the anomeric signal region of δ4.3-5.4ppm, indicating that the polysaccharide contains multiple sugar residues. The presence of anomeric signals was determined by combining the cross peaks in the anomeric region in the 13C NMR and HSQC spectra. The connection order between the residues was inferred based on the 13C and 1H chemical shifts of each sugar residue and the HMBC spectrum. The spatial correlation between the residues was further inferred by combining the NOESY spectrum. By combining the one-dimensional and two-dimensional NMR information and the methylation results, it was concluded that the main chain of the polysaccharide is mainly composed of →4)-β-D-Xylp-(1→ and a small amount of →3,4)-α-D-Xylp-(1→ connected to each other, and the side chain is composed of α-L-Araf-(1→ connected to the O-3 position of the sugar residue →3,4)-α-D-Xylp-(1→. The structure of SLP-D2N2 is shown in the figure. Figure 3As shown in G.

[0066] Example 4

[0067] SLP-D2N2 promotes the proliferation of mouse Raw264.7 macrophages

[0068] Mouse Raw264.7 macrophages are adherent cells. Cells were revived in fresh DMEM complete culture medium (containing 89% DMEM culture medium, 10% fetal bovine serum FBS, 1% 1U / mL penicillin-streptomycin) and cultured in a 37°C, 5% CO2 incubator. Mouse Raw264.7 macrophages in the logarithmic growth phase were centrifuged and resuspended in DMEM complete culture medium. Cells were counted and diluted to 1×10 4 / mL of cell suspension was inoculated into a 96-well culture plate. After the cells adhered to the wall, DMEM culture medium containing different concentrations of SLP-D2N2 (25, 75, 200 μg / mL) was added to the drug-treated group, and a blank control group was set up. After culturing for 24 hours, the supernatant was discarded, and 100 μL of DMEM culture medium containing 10% CCK-8 was added and incubated for 2 hours. A multifunctional microplate reader was used to detect the absorbance value A of each well at a wavelength of 450nm, and the effect of SLP-D2N2 on the proliferation activity of mouse Raw264.7 macrophages was calculated. The results are shown in Figure 4 As shown, compared with the control group, different concentrations of SLP-D2N2 could enhance the cell viability of mouse Raw264.7 macrophages, indicating that SLP-D2N2 has a pro-proliferative effect on mouse Raw264.7 macrophages.

[0069] Example 5

[0070] SLP-D2N2 induces mouse Raw264.7 macrophages to polarize toward M1

[0071] Mouse Raw264.7 macrophages were cultured in DMEM complete culture medium (containing 89% DMEM culture medium, 10% fetal bovine serum FBS, and 1% 1U / mL penicillin-streptomycin) at a density of 5*10 5 / mL. The A549-luc cell culture supernatant was used to induce Raw264.7 cells to differentiate into M2 type, and drugs were given at the same time as modeling.

[0072] 100ng LPS and 20ng IFN-γ were set as the positive control group, and unstimulated cells were set as the negative control group. The cells to be tested were collected by centrifugation and washed with PBS. The corresponding FITC, PE-conjugated Anti-mouse F4 / 80, and CD86 were added according to the concentrations recommended in the instructions and placed in a 4°C refrigerator away from light for 30 minutes. Centrifuge and wash, fix and break the membrane at room temperature, add APC-conjugated Anti-mouse CD206 for staining for 1 hour, centrifuge and wash, resuspend with 400μL PBS, sieve into a flow tube, and inject into an analytical flow cytometer (BD LSRFortessa) for sample detection.

[0073] The results are as follows Figure 5 As shown, compared with the positive control group, SLP-D2N2 increased the level of CD86 (M1 marker) in a dose-dependent manner, downregulated the level of CD206 (M2 marker) in Raw264.7, and the CD86 / CD206 ratio increased significantly. This indicates that treatment with SLP-D2N2 can inhibit the polarization of M2 macrophages induced by A549-luc supernatant and promote the polarization of cells to the M1 phenotype.

[0074] Example 6

[0075] SLP-D2N2 synergizes with cisplatin (Cis) to inhibit the growth of A549-luc at the cellular level

[0076] To verify the inhibitory effect of SLP-D2N2 combined with Cis on A549-luc cells after co-culture of mouse Raw264.7 macrophages and lung cancer cells, A549-luc and Raw264.7 macrophages in the logarithmic growth phase were obtained, digested, and centrifuged. They were resuspended in F-12K and DMEM complete culture medium, counted, and diluted to 1×10 4 / mL. The two cells were mixed in a 1:1 ratio and inoculated into a 96-well black culture plate. 100 μL of cell suspension was added to each well and cultured for 24 hours until the cells adhered to the wall. 100 μL of culture medium containing different concentrations of SLP-D2N2 (0, 10, 50, 75, 100, 150, 200 μg / mL), Cis (0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μM) and SLP-D2N2 combined with Cis drugs was added to each well. After 24 hours of culture, 100 μL of F-12K culture medium containing 15 mg / mL D-luciferin potassium salt was added and cultured for 10 minutes. The luminescence intensity of each well was detected at 540-560 nm using a multifunctional microplate reader to calculate the inhibitory effect of SLP-D2N2 on A549-luc cells and the survival rate of tumor cells. CompuSyn 1.0 software was used to calculate the synergy index (CI) of drug combinations at different concentrations, and the inhibition rate-combination index curve (Fa-CI) was drawn.

[0077] The results are as follows Figure 6 As shown, the data of CI<1 indicate that there is a significant synergistic effect between SLP-D2N2 and Cis drugs. When the concentration of SLP-D2N2 is 75μg / mL and Cis is 25μM, 76% of A549-luc tumor cells in the system can be inhibited, and the anti-tumor effect is better than the effect of either alone. At this time, CI=0.56125. This example proves that SLP-D2N2 can cooperate with Cis to enhance its anti-A549-luc therapeutic effect.

[0078] Example 7

[0079] SLP-D2N2 synergizes with Cis to exert anti-tumor effects at the animal level

[0080] A549-luc cells were used to establish a nude mouse A549-luc lung cancer model to evaluate the in vivo anti-tumor effect of SLP-D2N2 in combination with Cis. Forty-eight 5-week-old BALB / c nude male mice were randomly divided into 8 groups, with 6 mice in each group, including blank group, model group, Cis group (6 mg / kg, 3 mg / kg), SLP-D2N2 group (90 mg / kg), SLP-D2N2 combined with Cis administration group (LH-L, 10 mg / kg SLP-D2N2 + 3 mg / kg Cis; LH-M, 30 mg / kg SLP-D2N2 + 3 mg / kg Cis, LH-H, 90 mg / kg SLP-D2N2 + 3 mg / kg Cis). A549-luc tumor cells in the logarithmic growth phase with a cell density of 80-90% were routinely digested to make a concentration of about 5×10 7A cell suspension of 10 cells / mL was subcutaneously injected into the left armpit of the mouse, with an inoculation volume of 0.1 mL per mouse. After the inoculation, the nude mice were kept in an SPF animal room, and drug intervention began 2 weeks after tumor formation. SLP-D2N2 was administered once a day, once every three days for the first 15 days of Cis, and once every 7 days for the next 15 days. The model group was given the same volume of normal saline. While administering the drug, the long and short diameters of the tumors of each group of nude mice were measured and recorded, and the changes in tumor volume (V) were calculated, V = long diameter x short diameter. 2 / 2, draw the tumor growth curve. After 30 days of administration, the mice were euthanized, and the tumors were removed, photographed and weighed. Tumor inhibition rate (%) = (average tumor weight of the control group - average tumor weight of the treatment group) / average tumor weight of the control group × 100%. 20 mg of tumor was taken from the model group, Cis group (3 mg / kg), SLP-D2N2 group (90 mg / kg), and SLP-D2N2 combined with Cis high-dose group (LH-H), respectively, and the tumors were washed and cut into pieces, and collagenase IV was added to digest for 1 hour and sieved. Red blood cell lysis solution was added to crack the red blood cells on ice for 3 minutes, and washed with PBS. The cells were blocked on ice for 10 min, and the corresponding FITC-, PE-conjugated Anti-mouse F4 / 80 and CD86 were added at the concentrations recommended in the instructions, and the cells were incubated in a 4°C refrigerator away from light for 1 h. The cells were centrifuged and washed, and the membranes were fixed and broken at room temperature. The cells were resuspended by centrifugation, and the APC-conjugated antibody CD206 was added for staining for 1 h. The cells were centrifuged and washed, and the cells were resuspended in 400 μL PBS, sieved into flow tubes, and injected into analytical flow cytometer (BD LSRFortessa) for sample detection.

[0081] The results are as follows Figure 7 As shown in Figure 8, compared with the model group, the tumor volume of the drug-treated group was significantly reduced, and the tumor volume of the SLP-D2N2 combined with Cis treatment group showed a concentration gradient decrease, and there was a statistical difference. Fig. 9 The tumor volume of the combined drug group was significantly lower than that of the Cis alone drug group, which further indicated that the combined drug administration had a concentration-dependent therapeutic effect on the tumor growth of mice. Fig.10 Compared with the model group, the tumor weights of the 6 mg / kg Cis group and the combined drug groups were significantly reduced, and the tumor weights of the LH-M and LH-H groups were significantly reduced in a concentration-dependent manner compared with the Cis (3 mg / kg) alone treatment group. Flow cytometry analysis of tumor tissues showed that SLP-D2N2 significantly downregulated the M2 marker CD206 in macrophages and upregulated the M1 marker CD86. The ratio of M2 macrophages to M1 macrophages (CD206 / CD86) in the SLP-D2N2 combined with Cis treatment group was significantly lower than that in the model group ( Fig.11). This example further demonstrates at the animal level that SLP-D2N2 can inhibit the polarization of M2 macrophages and induce macrophage polarization to M1 phenotype, which helps to increase the anti-tumor efficacy of the combined drug Cis.

[0082] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing sugarcane leaf polysaccharide, characterized in that: The following steps are involved: (1) Extracting sugarcane leaves with water, and sequentially subjecting the water extract to centrifugation, concentration, alcohol precipitation, and dialysis to obtain crude sugarcane leaf polysaccharide I; (2) The crude polysaccharide is hydrolyzed and deproteinized by papain, and then adsorbed by AB-8 macroporous resin overnight. The solution is dialyzed and freeze-dried to obtain crude polysaccharide II; (3) The crude polysaccharide II is purified, eluted, concentrated, and dialyzed by an ion exchange column, and then separated and purified by a gel chromatography column to obtain the sugarcane leaf polysaccharide.

2. The preparation method according to claim 1, characterized in that: The Mw cut-off value for the dialysis was 3000 Da.

3. Sugarcane leaf polysaccharide prepared by the preparation method according to any one of claims 1 or 2.

4. Use of the sugarcane leaf polysaccharide as claimed in claim 3 in the preparation of a medicament for promoting macrophage proliferation.

5. Use of the sugarcane leaf polysaccharide as claimed in claim 3 in the preparation of a drug for promoting polarization of macrophages to the M1 phenotype.

6. A drug that promotes macrophage polarization to M1 phenotype, characterized in that: Including the sugarcane leaf polysaccharide as claimed in claim 3.

7. Use of the sugarcane leaf polysaccharide as claimed in claim 3 in the preparation of anti-tumor drugs.

8. An anti-tumor drug, characterized in that: The invention comprises the sugarcane leaf polysaccharide as claimed in claim 3 and cisplatin.

Images