Preparation method of sugarcane leaf polysaccharide and its application in regulating immunity and anti-tumor

Abstract

The present invention discloses a preparation method of sugarcane leaf polysaccharide and its application in regulating immunity and anti-tumor, belonging to the field of biomedicine technology. The method comprises the following steps: (1) extracting sugarcane leaves with water, and subjecting the aqueous extract to centrifugation, concentration, alcohol precipitation, and dialysis to obtain crude sugarcane leaf polysaccharide I; (2) enzymatically hydrolyzing and deproteinizing the crude polysaccharide with papain, adsorbing it with AB-8 type macroporous resin overnight, and lyophilizing the solution after dialysis to obtain crude polysaccharide II; (3) purifying, eluting, concentrating, and dialysis the crude polysaccharide II with an ion exchange column, and then separating and purifying it with a gel chromatography column to obtain the sugarcane leaf polysaccharide SLP-D2N2. The obtained polysaccharide has significant biological activity, can enhance the activity of macrophages, and plays an important role in regulating the tumor microenvironment and enhancing the anti-tumor immune response. In particular, it can synergize with anti-tumor drugs such as cisplatin to improve the therapeutic effect.

Description

Technical Field

[0001] The present 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 primarily divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC, a heterogeneous disease with diverse molecular and pathological features, accounts for approximately 85% of all lung cancer cases. Current cancer treatments often have significant side effects. Cisplatin, a classic chemotherapy drug, is commonly used to treat various cancers, but its use is limited by side effects such as drug resistance and organ toxicity.

[0003] Polysaccharides are long-chain polymers composed of multiple monosaccharide molecules linked by glycosidic bonds. They are very important macromolecules in cells and possess numerous biological functions, including anti-tumor, lipid-lowering, antiviral, antioxidant, and immunomodulatory properties. Studies have shown that polysaccharides can enhance immune function by enhancing the phagocytic capacity of macrophages, promoting T cell proliferation, and increasing B cell activity. Cytotoxic polysaccharides can directly kill tumor cells or indirectly inhibit or kill them by acting as immunomodulators and enhancing immune function.

[0004] Sugarcane (Saccharum officinarum L.) is an herbaceous plant of the genus Saccharum in the Poaceae family, widely cultivated in tropical regions of southern my country. Sugarcane leaves are a byproduct of the sugarcane industry chain and contain a rich variety of chemical components, including polysaccharides, amino acids, reducing sugars, glycosides, organic acids, and flavonoids. Sugarcane leaf polysaccharides (SLPs) are one of the main components of sugarcane leaves and have multiple biological activities, including antioxidant, antitumor, and hypoglycemic activities. Recent studies have shown that sugarcane leaf polysaccharides have a protective effect in a myocardial ischemia-reperfusion injury model and can prevent cardiovascular disease by inhibiting necrosis and oxidative stress. In addition, sugarcane leaf polysaccharides also exhibit antibacterial properties against a variety of pathogens. However, the extraction purity of sugarcane leaf polysaccharides in current existing technologies is relatively low. Therefore, it is crucial to provide a new method for preparing sugarcane leaf polysaccharides. Summary of the Invention

[0005] The present invention aims to provide a method for preparing sugarcane leaf polysaccharide and its application in immune regulation and anti-tumor treatment, thereby overcoming the above-mentioned problems in the prior art. The present invention provides a new option for the development of anti-tumor drugs and the research of novel 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) Sugarcane leaves were extracted with water, and the aqueous extract was centrifuged, concentrated, precipitated with alcohol, and dialyzed to obtain crude sugarcane leaf polysaccharide I;

[0009] (2) The crude polysaccharide is enzymatically hydrolyzed and deproteinized by papain, and then adsorbed on 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] The fifth technical solution of the present invention is a drug for promoting macrophage polarization to the 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 this invention not only provides a new approach for the high-value utilization of agricultural byproducts but also demonstrates its broad application prospects in the biomedical field. By promoting immune cell activity and modulating the tumor microenvironment, SLP-D2N2 can serve as an ideal synergist for chemotherapy drugs such as cisplatin, reducing drug side effects and enhancing therapeutic efficacy, providing new insights into the development of novel tumor immunotherapy options. 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

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

[0021] Figure 2 The total ion current (TIC) of the methylation analysis of sugarcane leaf polysaccharides shows the methylation status of the sugar residues of SLP-D2N2.

[0022] Figure 3 This is the structural analysis of sugarcane leaf polysaccharide SLP-D2N2, where A is the one-dimensional 1H NMR spectrum, B is the 13C NMR spectrum, C is the two-dimensional COSY NMR spectrum, D is the two-dimensional HSQC NMR spectrum, E is the two-dimensional HMBC NMR spectrum, F is the 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 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 a synergistic index (CI) analysis diagram of the synergistic effect of sugarcane leaf polysaccharide SLP-D2N2 and 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 conjunction 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] Figure 9 The final tumor volume of nude mouse A549-luc tumor model after 30 days of administration of different doses of SLP-D2N2 and cisplatin combination therapy.

[0029] Figure 10Comparison of tumor weights in nude mouse A549-luc tumor model 30 days after administration of different doses of SLP-D2N2 combined with cisplatin.

[0030] Figure 11 The results of flow cytometric analysis of macrophage phenotype in nude mouse A549-luc tumor tissue 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

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

[0036] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

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

[0038] (1) Sugarcane leaves were extracted with water, and the aqueous extract was centrifuged, concentrated, precipitated with alcohol, and dialyzed to obtain crude sugarcane leaf polysaccharide I;

[0039] (2) The crude polysaccharide is enzymatically hydrolyzed and deproteinized by papain, and then adsorbed on 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 embodiments of the present invention also provide sugarcane leaf polysaccharide prepared by the preparation method.

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

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

[0045] An embodiment of the present invention further provides a drug for promoting macrophage polarization to the 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 further provides an anti-tumor drug comprising the sugarcane leaf polysaccharide and cisplatin.

[0048] The purified sugarcane leaf polysaccharide SLP-D2N2 described in this invention exhibits significant biological activity, including promoting macrophage proliferation, inhibiting M2 macrophage polarization, and promoting M1 macrophage polarization. In both in vitro and in vivo studies, SLP-D2N2, when used in combination with cisplatin, significantly inhibited tumor cell proliferation. In particular, in the mouse lung cancer cell A549-luc tumor model, the synergistic effect of SLP-D2N2 and cisplatin significantly reduced tumor volume and enhanced anti-tumor therapeutic efficacy. This invention offers new options for the development of anti-tumor drugs and novel therapeutic strategies, and holds great promise for future applications.

[0049] SLP-D2N2, with a purity of 96.1%, was obtained through water extraction, alcohol precipitation, ion exchange purification, and gel chromatography from sugarcane leaves. Its molecular structure, analyzed by methylation and nuclear magnetic resonance, revealed a primary backbone composed of β-D-xylose residues with a small number of branching structures. This structural characteristic gives SLP-D2N2 unique advantages 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 purified water and subjected to reflux extraction. The extract was centrifuged and the residue was filtered and discarded. The extract was concentrated to obtain a crude polysaccharide extract. The supernatant was precipitated by adding anhydrous ethanol to a final concentration of 70%. The precipitate was collected and labeled. The solution was further dialyzed against water for 48 hours (Mw cutoff: 3000 Da). The dialyzed solution was lyophilized to obtain the crude polysaccharide.

[0052] Crude polysaccharide removal: Dissolve in water and add papain for overnight enzymatic hydrolysis. Deproteinize with Sevag reagent, add petroleum ether, collect the aqueous phase, and adsorb it with AB-8 macroporous resin overnight. The solution is dialyzed in distilled water using 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 on 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, centrifuged, and the supernatant was purified by gel chromatography at a flow rate of 1 mL / min. 1.5 column volumes of pure water were used for elution, with 12 mL of eluate collected in one tube. All eluates were pooled, and eluates corresponding to the same elution peak were combined and concentrated by rotary evaporation to 1 / 5 of the original volume. Freeze-dried, SLP-D2N2 was obtained with a purity of 96.1%.

[0055] Bioactivity Verification: In in vitro and in vivo studies, SLP-D2N2 was shown to effectively enhance the viability of Raw264.7 mouse macrophages and synergize with cisplatin to inhibit cancer cell proliferation in the A549-luc lung cancer cell model. Specifically, in the A549-luc mouse tumor model, SLP-D2N2 significantly inhibited tumor volume growth, demonstrating that this polysaccharide can be used in synergistic synergy with chemotherapy drugs to enhance anti-tumor therapeutic effects.

[0056] Example 1

[0057] Preparation of Sugarcane Leaf Polysaccharide

[0058] Sugarcane leaves were mixed with purified water at a solid-liquid ratio of 1:30 g / mL (w / v). The mixture was heated to 80°C and refluxed for extraction. The extract was centrifuged and the residue was filtered and discarded. The extract was concentrated to 1 / 5 of its original volume to obtain a crude polysaccharide extract. The supernatant was precipitated by adding four volumes of anhydrous ethanol. The precipitate was collected and labeled. The solution was further dialyzed against water for 48 hours (Mw cutoff: 3000 Da). The dialyzed solution was lyophilized 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 carried out using Sevag reagent (chloroform: n-butanol = 4:1 (v / v)). Petroleum ether was added to collect the aqueous phase and adsorbed overnight using AB-8 macroporous resin. The solution was dialyzed in distilled water using a 3000Da dialysis bag for 48 hours 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 4mL / min. Pure water, 0.1M, 0.2M and 0.3M NaCl solutions were used in turn for gradient elution, and one tube was collected every 15mL, and all 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 below. Figure 1 As shown in A. Combine the eluates 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 desalt, centrifuge and take the supernatant to separate and purify on 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 eluates. The gel purification elution curve is shown as follows Figure 1 As shown in Figure B, the eluates corresponding to the same elution peak (three peak fractions were obtained: Peak 1: tubes 19-36, Peak 2: tubes 37-43, and Peak 3: tubes 44-53) were combined and Peak 2 was concentrated by rotary evaporation to 1 / 5 of its 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, SLP-D2N2 was analyzed by GC-MS using an Agilent 7890A-5977B gas chromatography-mass spectrometer (Agilent Technologies Inc., CA, UAS) to analyze the glycosidic linkage types of SLP-D2N2. An Agilent gas chromatograph system was used, with a BPX70 column (30 m × 0.25 mm × 0.25 μm, SGE, Australia) and high-purity helium as the carrier gas. The injection volume was 1 μL, and the split ratio was 10:1. The column oven temperature was initially set at 140°C for 2.0 min, then programmed at 3°C / min to 230°C for 3 min. The mass spectrometer was an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA) equipped with an electron impact ionization (EI) source and a MassHunter workstation. The electron impact ion source (EI) was used in full scan (SCAN) mode, with a mass scan range (m / z) of 50-350. The total ion current of SLP-D2N2 is shown in the figure below. Figure 2 The connection patterns confirmed by characteristic fragment analysis after polysaccharide methylation are 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), and 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 Figure 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→, 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), and 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 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 450 nm, 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 in the results, 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 M1 polarization of mouse Raw264.7 macrophages

[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. A549-luc cell culture supernatant was used to induce Raw264.7 cells to differentiate into M2 type, and drugs were administered simultaneously with model establishment.

[0072] 100ng LPS and 20ng IFN-γ were set up as the positive control group, and unstimulated cells were used 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 incubated in a 4°C refrigerator in the dark for 30 minutes. Centrifuge and wash, fix and permeabilize the membrane at room temperature, add APC-conjugated Anti-mouse CD206 for staining for 1 hour, centrifuge and wash, resuspend in 400μL PBS, filter 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 and downregulated the level of CD206 (M2 marker) in Raw264.7 cells, with a significant increase in the CD86 / CD206 ratio. This indicates that SLP-D2N2 treatment can inhibit the polarization of M2 macrophages induced by A549-luc supernatant and promote the polarization of cells toward 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. The cells were resuspended in F-12K and DMEM complete culture medium, counted, and diluted to 1×10 4The two cell lines were mixed at a 1:1 ratio and seeded into a 96-well black culture plate. 100 μL of the cell suspension was added to each well and cultured for 24 hours to allow the cells to adhere. 100 μL of culture medium containing varying concentrations of SLP-D2N2 (0, 10, 50, 75, 100, 150, and 200 μg / mL), Cis (0, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μM), and SLP-D2N2 combined with Cis 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 incubated for 10 minutes. The luminescence intensity of each well was measured at 540-560 nm using a multi-function microplate reader to measure the inhibitory effect of SLP-D2N2 on A549-luc cells and calculate tumor cell survival rate. CompuSyn 1.0 software was used to calculate the synergistic index (CI) of drug combinations at different concentrations, and the inhibition rate-combination index curve (Fa-CI) was plotted.

[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 75 μg / mL and Cis is 25 μM, 76% of A549-luc tumor cells in the system can be inhibited. The anti-tumor effect is better than the effect of using either alone. At this time, CI=0.56125. This example proves that SLP-D2N2 can cooperate with Cis to enhance its therapeutic effect against A549-luc.

[0078] Example 7

[0079] SLP-D2N2 synergizes with Cis to exert anti-tumor effects in animals

[0080] A549-luc lung cancer model was established in nude mice using A549-luc cells 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), and 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 and prepared into a concentration of approximately 5×10 7The cell suspension of 10 cells / mL was subcutaneously injected into the left armpit of the mouse, and the inoculation volume was 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. At the same time as the administration, the long and short diameters of the tumors of the nude mice in each group were measured and recorded, and the changes in tumor volume (V) were calculated, V = long diameter x short diameter. 2 / 2, and the tumor growth curve was drawn. 30 days after 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 collected from each of 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), washed, minced, digested with collagenase IV for 1 hour, and sieved. Red blood cell lysis buffer was added to lyse the red blood cells on ice for 3 minutes, and then washed with PBS. Block on ice for 10 minutes, add the corresponding FITC, PE-conjugated Anti-mouse F4 / 80, and CD86 at the concentrations recommended in the instructions, incubate in a 4°C refrigerator in the dark for 1 hour, centrifuge and wash, fix and permeabilize the membrane at room temperature, resuspend by centrifugation, add APC-conjugated antibody CD206 for staining for 1 hour, centrifuge and wash, resuspend in 400 μL PBS, sieve into a flow tube, and inject into an 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. Figure 9 The tumor volume of the combined drug group was significantly lower than that of the Cis alone drug group, which further demonstrated that the combined drug treatment had a concentration-dependent therapeutic effect on tumor growth in mice. Figure 10 Compared with the model group, the tumor weight of the 6 mg / kg Cis group and each combined drug group was significantly reduced, and the tumor weight of the LH-M and LH-H groups showed a concentration-dependent decrease compared with the Cis (3 mg / kg) alone treatment group, with significant differences. Flow cytometry analysis of tumor tissue 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 ( Figure 11This example further demonstrates at the animal level that SLP-D2N2 can inhibit the polarization of M2 macrophages and induce macrophage polarization toward the M1 phenotype, thereby increasing the anti-tumor efficacy of the combined drug Cis.

[0082] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. The use of sugarcane leaf polysaccharide in the preparation of a drug that promotes macrophage polarization to the M1 phenotype, characterized in that: The preparation method of sugarcane leaf polysaccharide comprises the following steps: (1) Sugarcane leaves were extracted with water, and the water extract was centrifuged, concentrated, precipitated with alcohol, and dialyzed to obtain crude sugarcane leaf polysaccharide I; (2) The crude polysaccharide I was enzymatically hydrolyzed and deproteinized by papain, and then adsorbed on AB-8 macroporous resin overnight. The solution was 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. The specific step (3) includes: dissolving the crude polysaccharide II in pure water, centrifuging and taking the supernatant to purify it through an ion exchange column at a flow rate of 4 mL / min; using pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions in turn for gradient elution, collecting one tube for every 15 mL, collecting all the eluates, determining the total sugar content of the eluates in each collection tube by sulfuric acid-phenol method, drawing an ion purification elution curve, combining the eluates corresponding to the same elution peak, and obtaining three elution peak components, peak 1: tubes 7-19, peak 2: tubes 28-40, peak 3: tubes 49-58, concentrating the ion elution peak 2 by rotary evaporation to 1 / 5 of the original volume, dialyzing for 48 hours using a 3000 Da dialysis bag for desalting, centrifuging and taking the supernatant to separate and purify it through a gel chromatography column at a flow rate of 1 mL / min, using pure water to elute 1.5 times the column volume, and every 12 mL was collected into a tube, all the eluates were collected, a gel purification elution curve was drawn, and the eluates corresponding to the same elution peak were combined to obtain three elution peak components, peak 1: tubes 19-36, peak 2: tubes 37-43, and peak 3: tubes 44-53. The elution peak 2 was concentrated by rotary evaporation to 1 / 5 of the original volume and freeze-dried to obtain the sugarcane leaf polysaccharide.

2. A drug that promotes macrophage polarization to the M1 phenotype, characterized in that: The invention relates to a sugarcane leaf polysaccharide, wherein the preparation method of the sugarcane leaf polysaccharide comprises the following steps: (1) Sugarcane leaves were extracted with water, and the water extract was centrifuged, concentrated, precipitated with alcohol, and dialyzed to obtain crude sugarcane leaf polysaccharide I; (2) The crude polysaccharide I was enzymatically hydrolyzed and deproteinized by papain, and then adsorbed on AB-8 macroporous resin overnight. The solution was 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. The specific step (3) includes: dissolving the crude polysaccharide II in pure water, centrifuging and taking the supernatant to purify it through an ion exchange column at a flow rate of 4 mL / min; using pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions in turn for gradient elution, collecting one tube for every 15 mL, collecting all the eluates, determining the total sugar content of the eluates in each collection tube by sulfuric acid-phenol method, drawing an ion purification elution curve, combining the eluates corresponding to the same elution peak, and obtaining three elution peak components, peak 1: tubes 7-19, peak 2: tubes 28-40, peak 3: tubes 49-58, concentrating the ion elution peak 2 by rotary evaporation to 1 / 5 of the original volume, dialyzing for 48 hours using a 3000 Da dialysis bag for desalting, centrifuging and taking the supernatant to separate and purify it through a gel chromatography column at a flow rate of 1 mL / min, using pure water to elute 1.5 times the column volume, and every 12 mL was collected into a tube, all the eluates were collected, a gel purification elution curve was drawn, and the eluates corresponding to the same elution peak were combined to obtain three elution peak components, peak 1: tubes 19-36, peak 2: tubes 37-43, and peak 3: tubes 44-53. The elution peak 2 was concentrated by rotary evaporation to 1 / 5 of the original volume and freeze-dried to obtain the sugarcane leaf polysaccharide.

3. The use of sugarcane leaf polysaccharide in the preparation of anti-lung cancer drugs, characterized in that: The preparation method of sugarcane leaf polysaccharide comprises the following steps: (1) Sugarcane leaves were extracted with water, and the water extract was centrifuged, concentrated, precipitated with alcohol, and dialyzed to obtain crude sugarcane leaf polysaccharide I; (2) The crude polysaccharide I was enzymatically hydrolyzed and deproteinized by papain, and then adsorbed on AB-8 macroporous resin overnight. The solution was 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. The specific step (3) includes: dissolving the crude polysaccharide II in pure water, centrifuging and taking the supernatant to purify it through an ion exchange column at a flow rate of 4 mL / min; using pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions in turn for gradient elution, collecting one tube for every 15 mL, collecting all the eluates, determining the total sugar content of the eluates in each collection tube by sulfuric acid-phenol method, drawing an ion purification elution curve, combining the eluates corresponding to the same elution peak, and obtaining three elution peak components, peak 1: tubes 7-19, peak 2: tubes 28-40, peak 3: tubes 49-58, concentrating the ion elution peak 2 by rotary evaporation to 1 / 5 of the original volume, dialyzing for 48 hours using a 3000 Da dialysis bag for desalting, centrifuging and taking the supernatant to separate and purify it through a gel chromatography column at a flow rate of 1 mL / min, using pure water to elute 1.5 times the column volume, and every 12 mL was collected into a tube, all the eluates were collected, a gel purification elution curve was drawn, and the eluates corresponding to the same elution peak were combined to obtain three elution peak components, peak 1: tubes 19-36, peak 2: tubes 37-43, and peak 3: tubes 44-53. The elution peak 2 was concentrated by rotary evaporation to 1 / 5 of the original volume and freeze-dried to obtain the sugarcane leaf polysaccharide.

4. An anti-tumor drug, characterized in that: The invention comprises sugarcane leaf polysaccharide and cisplatin; the tumor is lung cancer; and the preparation method of the sugarcane leaf polysaccharide comprises the following steps: (1) Sugarcane leaves were extracted with water, and the water extract was centrifuged, concentrated, precipitated with alcohol, and dialyzed to obtain crude sugarcane leaf polysaccharide I; (2) The crude polysaccharide I was enzymatically hydrolyzed and deproteinized by papain, and then adsorbed on AB-8 macroporous resin overnight. The solution was 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. The specific step (3) includes: dissolving the crude polysaccharide II in pure water, centrifuging and taking the supernatant to purify it through an ion exchange column at a flow rate of 4 mL / min; using pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions in turn for gradient elution, collecting one tube for every 15 mL, collecting all the eluates, determining the total sugar content of the eluates in each collection tube by sulfuric acid-phenol method, drawing an ion purification elution curve, combining the eluates corresponding to the same elution peak, and obtaining three elution peak components, peak 1: tubes 7-19, peak 2: tubes 28-40, peak 3: tubes 49-58, concentrating the ion elution peak 2 by rotary evaporation to 1 / 5 of the original volume, dialyzing for 48 hours using a 3000 Da dialysis bag for desalting, centrifuging and taking the supernatant to separate and purify it through a gel chromatography column at a flow rate of 1 mL / min, using pure water to elute 1.5 times the column volume, and every 12 mL was collected into a tube, all the eluates were collected, a gel purification elution curve was drawn, and the eluates corresponding to the same elution peak were combined to obtain three elution peak components, peak 1: tubes 19-36, peak 2: tubes 37-43, and peak 3: tubes 44-53. The elution peak 2 was concentrated by rotary evaporation to 1 / 5 of the original volume and freeze-dried to obtain the sugarcane leaf polysaccharide.

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