Application of functional polysaccharide in wolfberry in preparation of immunomodulator and antitumor drugs

Abstract

The application discloses application of a functional polysaccharide of Lycium barbarum in preparation of an immunoregulator and an antitumor drug. The specific process steps are adopted to prepare the Lycium barbarum polysaccharide with a specific molecular weight of 50-100 kD. The Lycium barbarum polysaccharide with the specific molecular weight can promote LBP repolarization of macrophages, promote up-regulation of M1 type macrophages and down-regulation of M2 type macrophages, and has the advantages of increasing NO secretion and reducing survival rate of tumor cells co-incubated with BMDM cells. The Lycium barbarum functional polysaccharide can be used as a regulator for promoting macrophage repolarization and an antitumor immune enhancer.

Description

Technical Field

[0001] This invention relates to a traditional Chinese medicine, specifically the application of Lycium barbarum functional polysaccharides in the preparation of immunomodulators and antitumor agents, belonging to the field of pharmaceutical technology. Background Technology

[0002] Macrophages are important immune cells in the body, with functions including phagocytosis, processing and presenting antigens, and activating immune responses. Due to their high plasticity, macrophages can polarize into two different phenotypes under the influence of different stimuli. One is the M1 type, or classically activated macrophages, which exhibit strong antigen-presenting capacity and promote inflammatory responses, thus inhibiting tumor metastasis and growth in tumor regulation. The other is the M2 type, or alternatively activated macrophages, which can suppress inflammation and play a role in matrix remodeling, tissue repair, and angiogenesis, exhibiting pro-tumor activity in tumor regulation. Therefore, macrophage phenotypes are closely related to immune regulation and tumor development.

[0003] Goji berries are the dried, ripe fruit of *Lycium barbarum* L., grown in Ningxia. They are neutral in nature and sweet in taste, entering the liver and kidney meridians, and have the effects of nourishing the kidneys and lungs, and tonifying the liver and improving eyesight. Goji berry polysaccharide (LBP) is considered the main active ingredient of goji berries, possessing various pharmacological effects, such as immunomodulation, anti-tumor, antioxidant, anti-aging, and lowering blood sugar and lipids. Among these, the immunomodulatory effect of LBP is mainly manifested in its ability to regulate the function of various immune cells.

[0004] There are no reports on the effects of different molecular weights of Lycium barbarum polysaccharides on macrophage phenotype and their antitumor effects. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to screen for the most effective Lycium barbarum polysaccharide fraction by studying the effects of different molecular weights of LBP on macrophage phenotype and its anti-tumor effects, so as to provide a theoretical basis for the rational clinical use of Lycium barbarum polysaccharides and tumor immunotherapy.

[0006] This invention isolates mouse bone marrow-derived macrophages (BMDM) and treats them with different molecular weight Lycium barbarum polysaccharides (LBP) (<3kD, 3-50kD, 50-100kD, >100kD). The remodeling effects of different treatment groups on BMDM cells are investigated using flow cytometry and qPCR. The remodeled BMDM cells are co-cultured with tumor cells at different ratios, and the antitumor effect of BMDM repolarization is examined using a luciferase assay. The results show that Lycium barbarum significantly promotes macrophage LBP repolarization, upregulates M1-type BMDM and downregulates M2-type BMDM, and increases NO secretion while reducing the survival rate of tumor cells co-incubated with BMDM cells. This demonstrates that Lycium barbarum polysaccharides can induce M2-type macrophages to repolarize to M1-type, enhancing the killing effect of macrophages on tumor cells. Therefore, the Lycium barbarum functional polysaccharides provided by this invention can serve as regulators of macrophage repolarization and antitumor immune enhancers.

[0007] As a preferred embodiment, the functional polysaccharide of wolfberry described in this invention is prepared by the following method: 10 kg of dried wolfberry is weighed, and 80% ethanol is added at a solid-liquid ratio of 10:1. The mixture is heated and refluxed twice, each time for 1 hour, to obtain an ethanol extract and an ethanol-extracted residue. 15 times the volume of deionized water is added to the ethanol-extracted residue, and the mixture is heated and refluxed twice, each time for 1 hour. After filtration and concentration, crude wolfberry polysaccharide is obtained. After dissolving in deionized water, the crude wolfberry polysaccharide solution is fed into an ultrafiltration device via a circulating pump and ultrafiltered through a 100 kD cellulose membrane. The retentate is then circulated and ultrafiltered, and the collected retentate is the wolfberry polysaccharide sample solution with a molecular weight >100 kD. The permeate from the ultrafiltration membrane is the wolfberry crude polysaccharide with a molecular weight <100 kD. Similarly, the crude wolfberry polysaccharide is separated sequentially using 50 kD and 3 kD ultrafiltration membrane modules to obtain wolfberry polysaccharide sample solutions with molecular weights of 50-100 kD, 3-50 kD, and <3 kD. The four molecular weight ranges of wolfberry polysaccharides were concentrated by evaporation and then freeze-dried for later use.

[0008] This invention can formulate wolfberry polysaccharides and pharmaceutically acceptable carriers into tablet, capsule, granule or pill dosage forms.

[0009] In this invention, when making tablets, a carrier lactose or corn starch is added to the wolfberry polysaccharide, and a lubricant magnesium stearate is added if necessary. The mixture is then mixed evenly and compressed into tablets.

[0010] When making capsules, wolfberry polysaccharides and carrier lactose or corn starch are mixed evenly, granulated, and then filled into capsules.

[0011] In this invention, when making granules, wolfberry polysaccharide and diluent lactose or corn starch are mixed evenly, granulated, dried, and made into granules.

[0012] Beneficial effects: The wolfberry polysaccharide provided by this invention has the following advantages compared with the prior art:

[0013] This invention screens the effects of different molecular weight Lycium barbarum polysaccharides (<3kD, 3-50kD, 50-100kD, >100kD) on macrophage repolarization and antitumor activity. The screening results show that Lycium barbarum polysaccharides with a specific molecular weight of 50-100kD can remodel M2 macrophages into M1 macrophages and increase NO release levels in BMDM cells. Lycium barbarum polysaccharides with a specific molecular weight of 50-100kD can be applied to the field of macrophage remodeling. This invention uses the optimal 50-100kD molecular weight Lycium barbarum polysaccharide as an immunomodulator for macrophage repolarization and an antitumor immune enhancer. Attached Figure Description

[0014] Figure 1 This is the gate process for flow cytometry.

[0015] Figure 2 This illustrates the remodeling effect of LBP on the immunophenotype of BMDM cells. Figure (A) shows flow cytometry analysis of M1 and M2 BMDM cells at F4 / 80. + CD11b + Representative images showing the proportion of cells in the population; (B) M1 and M2 type BMDM at F4 / 80 + CD11b + Statistical results of the proportion of cells in the population; (C) M1 and M2 type BMDM in F4 / 80 + CD11b + The ratio of the proportions in the cell population; statistical results of the MFI of (D)CD86 and CD206.

[0016] Figure 3 The effect of LBP on gene expression levels in BMDM cells. (A) Statistical results of qPCR detection of M1 markers (Il1β, Nos2, Tnfα) gene expression levels in BMDM cells; (B) Statistical results of qPCR detection of M2 markers (Arg1, Chil3, Il4) gene expression levels in BMDM cells.

[0017] Figure 4 To illustrate the effect of LBP on NO release levels in BMDM cells, the figure shows (A) the standard curve and counting formula for NO; and (B) the statistical results of NO concentration in the supernatant of BMDM cells.

[0018] Figure 5The figure shows the direct killing effect of BMDM repolarization on tumor cells. In the figure, (A) is the statistical result of fluorescence intensity of each group after direct co-culture of 4T1-Luc and BMDM cells by ELISA reader; (B) is the statistical result of direct inhibition rate of BMDM on 4T1-Luc cells; (C) is the statistical result of fluorescence intensity of each group after direct co-culture of B16-Luc and BMDM cells by ELISA reader; and (D) is the statistical result of direct inhibition rate of BMDM on B16-Luc cells. Detailed Implementation

[0019] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0020] Example 1: Preparation of Lycium barbarum functional polysaccharides

[0021] Weigh 10 kg of dried wolfberries and add 80% ethanol at a solid-liquid ratio of 10:1. Extract twice by reflux, 1 hour each time, to obtain an ethanol extract and a residue. Add 15 times the volume of deionized water to the residue and extract twice by reflux, 1 hour each time. Combine the extracts and add anhydrous ethanol to bring the ethanol volume fraction to 80%. Let stand overnight at room temperature, centrifuge at 4500 rpm for 5 minutes to collect the precipitate, wash with anhydrous ethanol, and freeze-dry to obtain crude wolfberry polysaccharide. Dissolve the crude wolfberry polysaccharide in deionized water and pump the solution into an ultrafiltration device. Ultrafiltration is performed through a 100 kDa cellulose membrane. The retentate is then circulated for ultrafiltration; the collected retentate is the wolfberry polysaccharide sample solution with a molecular weight >100 kDa. The permeate from the ultrafiltration membrane is the crude wolfberry polysaccharide with a molecular weight <100 kDa. Similarly, by sequentially separating crude Lycium barbarum polysaccharides using 50kD and 3kD ultrafiltration membrane modules, Lycium barbarum polysaccharide sample solutions of 50-100kD, 3-50kD, and <3kD can be obtained. The Lycium barbarum polysaccharides in the four molecular weight ranges are then concentrated by evaporation, freeze-dried, and ready for use.

[0022] Example 2 Activity Experiment

[0023] 1.1 Reagents are listed in Table 1.

[0024] Table 1 Reagents

[0025]

[0026]

[0027] 1.2 The instruments and equipment are shown in Table 2.

[0028] Table 2 Instruments and Equipment

[0029] Clean bench Thermo Technologies, Inc. Cell incubator ThermoFisherScientific, USA High-speed refrigerated centrifuge Beijing Dalongxingchuang Experimental Instrument Co., Ltd. PCR instruments ThermoFisherScientific, USA ELISA reader PerkinElmer, USA microscope German company Zeiss Ultra-micro UV spectrophotometer Denovix, Inc. (USA)

[0030] 1.3 Animals and Cells

[0031] Male C57BL / 6 mice, 5-6 weeks old, were purchased from Nanjing Qinglongshan Animal Farm. They were housed in separate cages, fed standard feed, with free access to food and water, normal lighting, and a temperature controlled at 20-25℃. All operations and research procedures were carried out in accordance with the "Regulations on the Management of Laboratory Animals".

[0032] 4T1-Luc and B16-Luc cells were obtained from the ATCC cell bank and were cultured in RPMI 1640 complete medium containing 10% FBS (containing 1% antibiotics) in a cell culture incubator (37℃, 5% CO2) and passaged every 1-2 days.

[0033] 1.4 Primers

[0034] The qPCR primers were synthesized by Qingke Biotechnology Co., Ltd., and the primer sequences are shown in Table 3.

[0035] Table 3 Primer sequences

[0036]

[0037] 2. Experimental Methods

[0038] 2.1 Extraction and induction of BMDM cells

[0039] C57BL / 6 mice (5-6 weeks old, male) were euthanized by cervical dislocation. After soaking in alcohol for 3-5 minutes, the abdominal cavity was opened, and the femur was separated from the surrounding muscle and fibrous tissue. The femur was disinfected by alcohol immersion, washed three times with PBS (pH 7.4), and the cartilage at both ends was removed. A suitable amount of RPMI 1640 medium was drawn into a syringe and used to flush out bone marrow cells from the femur. Washing was repeated until the bone turned white. The bone marrow was ground using a plunger to pass through a 70μm (200 mesh) cell sieve. The collected filtrate was centrifuged at 400g for 4 minutes, and the supernatant was discarded. Red blood cell lysis buffer was added, and the mixture was mixed by pipetting and incubated at room temperature for 4 minutes. The reaction was terminated by adding PBS, centrifuged at 400g for 4 minutes, and the supernatant was discarded. The isolated bone marrow cells were stored in RPMI 1640 medium (containing 10% FBS and 1% penicillin-dextrose antibody). Complete culture medium (containing GM-CSF at a final concentration of 20 ng / mL) was added to promote cell differentiation, and cells were seeded in culture dishes, marking day 0. The medium was then changed by half every two days. On day 6, the medium was changed again to complete culture medium (containing GM-CSF, IL-4, and IL-13, all at a final concentration of 20 ng / mL), and cells were cultured for another two days to obtain M2 BMDM cells.

[0040] 2.2 Flow cytometry detection of the remodeling effect of LBP on the immunophenotype of BMDM cells

[0041] Preparation method of Lycium barbarum polysaccharide solution: Weigh 125 mg each of Lycium barbarum polysaccharide powder with different molecular weights (<3kD, 3-50kD, 50-100kD, >100kD) prepared in Example 1, dissolve in 50 mL of RPMI 1640 medium to prepare a 2.5 mg / mL Lycium barbarum polysaccharide solution. Before administration, dilute with RPMI 1640 medium to the required concentration for the experiment, and filter sterilize using a 0.22 μm filter membrane.

[0042] Preparation method of LPS solution: Dissolve 1 mg of LPS powder in 1 mL of sterile water to prepare a 1 mg / mL LPS solution. Before administration, dilute with RPMI 1640 medium to the required concentration for the experiment, and filter sterilize using a 0.22 μm filter membrane.

[0043] The induced BMDM cells were seeded in 6-well plates and divided into four groups: M0 group, control group, LPS group, and four LBP groups with molecular weights (<3kD, 3–50kD, 50–100kD, >100kD). The M0 group did not receive IL-4 or IL-13 induction. Cells were treated with 0.5 μg / mL LPS or 250 μg / mL LBP for 2 days. Cell supernatant was collected for subsequent NO detection.

[0044] Add 1 mL of PBS (pH 7.4) to each well of the culture dish. Scrape BMDM cells and collect them into Eppendorf tubes. Centrifuge at 400g for 5 min to wash away the culture medium, discard the supernatant, and repeat once. Resuspend the cells in 100 μL of cell suspension buffer (CSB). Add 0.1 μg of CD86 antibody, 0.25 μg of CD11b antibody, and 0.1 μg of F4 / 80 antibody, and stain on ice in the dark for 20 min. Add 1 mL of PBS to each tube, centrifuge at 400g for 5 min, wash away excess antibody, and repeat once. Discard the supernatant, add 500 μL of fixative and immediately pipette to resuspend the cells, and incubate at room temperature in the dark for 20 min. Centrifuge at 400g for 5 min and discard the supernatant. Add 1 mL of 1× permeabilization buffer (diluted with DEPC water to obtain 10× permeabilization buffer), resuspend, centrifuge, and repeat once. Resuspend cells in 100 μL CSB, add 2 μL CD206 antibody to each tube, stain on ice in the dark for 30 min, resuspend in 2 mL 2× permeabilization buffer, centrifuge, and discard the supernatant. Resuspend in 300 μL CSB, filter through a 200-mesh sieve, and analyze using flow cytometry. The specific method is as follows: first, viable cells are selected using the FSC-A and SSC-A channels; then, single cells are selected using the FSC-A and FSC-H channels; next, BMDM cells are selected using the F4 / 80 and CD11b markers; finally, F4 / 80 is analyzed. +CD11b + The expression levels of two biomarkers, CD86 and CD206, in the cell population were determined, and the results were analyzed using Flowjo software.

[0045] 2.3 Effect of qPCR on gene expression levels in BMDM cells

[0046] (1) Total RNA extraction:

[0047] The induced BMDM cells were seeded in 6-well plates and treated with either 0.5 μg / mL LPS or 250 μg / mL LBP for 2 days. Cells were collected into Eppendorf tubes and centrifuged at 1000 rpm for 5 min, discarding the supernatant. 1 mL of Trizol reagent was added to each tube and vigorously pipetted for 15 s to mix thoroughly. The mixture was incubated at room temperature for 2-3 min, then rotated at 4°C for 1-2 h. 200 μL of chloroform was added, and the mixture was vortexed for approximately 15 s until no clear separation occurred. The cells were then centrifuged at 12000 g for 15 min. 500 μL of the supernatant was transferred to a nuclease-free Eppendorf tube, along with 500 μL of isopropanol. The mixture was vortexed to mix thoroughly and then placed on ice for 5-10 min. The cells were then centrifuged at 12000 g for 10 min. The supernatant was discarded, and 1 mL of 75% ethanol (pre-chilled at 4°C) prepared with DEPC water was added, gently swirled to mix, and centrifuged at 7500 g for 5 min. Discard the supernatant, evaporate the remaining ethanol in a fume hood, add 25 μL of DEPC water, gently tap to mix, and place on ice. After determining the concentration using an ultra-micro UV spectrophotometer, store at -80°C for later use.

[0048] (2) cDNA synthesis:

[0049] cDNA was synthesized according to the kit instructions; the synthesis system is shown in Table 4. The reverse transcription procedure was as follows: the system was incubated at 42℃ for 15 min, then inactivated at 95℃ for 3 s. The product was stored at -20℃ for later use.

[0050] Table 4 cDNA Synthesis System

[0051] RNA 5 RandomPrimer 0.5 <![CDATA[AnchoredOligo(dT) 18 First]]> 0.5 E-Mix 1 R-Mix 1 gDNARemover(2×) 10 RNase-freeWater 2

[0052] (3) PCR amplification:

[0053] The PCR amplification system is shown in Table 5. The reaction system was thoroughly mixed, added to a 96-well PCR plate, centrifuged, and measured using a PCR machine. PCR reaction conditions are shown in Table 6. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. After measuring the CT value of the samples, 2... -ΔΔCT The relative expression levels of Il1β, Nos2, Tnfα, Arg1, Chil3, and Il4 in the sample were calculated using the method.

[0054] Table 5 PCR amplification system

[0055] cDNA 1 SYBRGreen (2×) 25 Forwardprimer 1 Reverseprimer 1 RNase-freeWater 22

[0056] Table 6 PCR reaction conditions

[0057]

[0058]

[0059] 2.4 Effect of LBP on NO release levels in BMDM cells as determined by Griess method

[0060] Take the BMDM cell supernatant collected in section 2.2, bring it to room temperature, and dilute the standards to 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 μM with RPMI 1640 medium containing 10% FBS. Add 50 μL of standards and samples to each well of a 96-well plate. Add 50 μL of room temperature Griess Reagant I and Griess Reagant II to each well. Measure the OD value at 540 nm using a microplate reader. Calculate the actual NO concentration value based on the fitted standard curve.

[0061] 2.5 Direct co-culture method to investigate the antitumor effect of BMDM repolarization

[0062] BMDM cells were co-cultured directly with 4T1-Luc or B16-Luc tumor cells in four different ratios in 96-well plates, with 1000 tumor cells / well and 0 BMDM cells / well, at a ratio of 1×10⁶ cells / well. 4 pcs / hole, 2×10 4 pcs / hole and 4×10 4 Cells / well. Each cell group was set up with a control group, an LPS group, and four LBP groups with different molecular weights (<3kD, 3–50kD, 50–100kD, >100kD). Cells were treated with 0.5 μg / mL LPS or 250 μg / mL LBP for 2 days. The culture medium was discarded, and the cells were rinsed with PBS. The PBS was then largely removed, and an appropriate amount of cell lysis buffer was added to cover the culture dish to ensure complete cell lysis. Luciferase reagent was added to each 96-well plate (50 μL / well), with one blank well and three auxiliary wells for each sample. Lysed cells were added to the luciferase solution at 20 μL / well. The plate was immediately incubated using a fluorescence microplate reader to detect the number of 4T1-Luc and B16-Luc cells based on the fluorescence intensity of the tumor cells.

[0063] 2.6 Transwell assay to investigate the antitumor effect of BMDM repolarization

[0064] BMDM cells were co-incubated with 4T1-Luc or B16-Luc tumor cells in four different ratios in 24-well Transwell dishes. BMDM cells were seeded in the upper chamber, and tumor cells were seeded in the lower chamber. The tumor cell count was 4000 cells / well, and the BMDM cell count was 0 cells / well, at a ratio of 4 × 10⁻⁶. 4 8 holes / hole, 8×10 4 1 / hole and 1.6×10 5 Cells / well. Each cell group was set up as a control, LPS group, and four LBP molecular weight groups (<3kD, 3–50kD, 50–100kD, >100kD). Treatment with 0.5 μg / mL LPS or 250 μg / mL LBP for 2 days. Subsequent procedures were the same as in section 2.5.

[0065] 2.7 Statistical Analysis

[0066] Data analysis and plotting were performed using GraphPad Prism 9.4.0 software. All results are expressed as mean ± standard deviation (SD). One-way ANOVA was used for data between groups. A p-value < 0.05 was considered statistically significant. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0067] 3. Experimental Results

[0068] 3.1 The effect of LBP on the remodeling of BMDM cell immunophenotype

[0069] This invention investigates the effects of LBP of different molecular weights on the remodeling effect of M2 type BMDM cells. The expression levels of M1 macrophage marker CD86 and M2 macrophage marker CD206 in different treatment groups were analyzed by flow cytometry.

[0070] The gating process of flow cytometry is described in [link to gating procedure]. Figure 1 Taking the 50-100kD LBP group as an example, first circle the live cells, then circle the single cells, and finally circle the F4 / 80 single cells. + CD11b + The cell population, namely macrophages, accounted for (95.22±1.34)%, indicating that BMDM induction was successful. This cell population was then analyzed.

[0071] The results of the flow cytometry experiment are shown below Figure 2 , Figure 2 In (A), the horizontal and vertical axes represent the fluorescence intensity of the M1-type marker CD86 and the M2-type marker CD206 molecules, respectively. Figure 2 (B) The vertical axis represents the M1 and M2 macrophages at F4 / 80. + CD11b+ The proportion of cells in the population Figure 2 (C) The vertical axis represents the ratio of M1 and M2 macrophages at F4 / 80. + CD11b + The ratio of the proportion of cells in the population. Figure 2 (D) The ordinate represents the mean fluorescence intensity (MFI) of CD86 and CD206, respectively. Group M0 consisted of macrophages without IL-4 and IL-13 induction. Compared to the control group, the CD206 MFI was increased, indicating that macrophages were induced by IL-4 and IL-13 to polarize towards the M2 type. Figure 2 D).

[0072] Compared with the control group, M1 macrophages (CD86) in the LPS group and LBP groups with different molecular weights showed higher activity levels. + CD206 - The proportion of M2 macrophages (CD86 MFI) and CD86 MFI both increased significantly. - CD206 + The proportion of CD206 MFI decreased significantly, except for the LBP group with a molecular weight less than 3kD. Figure 4 (B, D). This indicates that after treatment with LPS or LBP, the expression of the M1 surface marker CD86 in BMDM increased, while the expression of the M2 surface marker CD206 decreased. Compared with the control group, where the proportions of M1 and M2 cells were (8.57±0.37)% and (46.50±0.72)%, respectively, the LPS group showed the most significant repolarization effect, with the proportions of M1 and M2 macrophages being (34.73±1.50)% and (9.59±0.84)%, respectively. Among the LBP groups with different molecular weights, the 50–100 kDa LBP group showed the most significant repolarization effect, with the proportion of M1 macrophages reaching (23.70±0.70)% and the proportion of M2 cells decreasing to (20.97±0.76)%. Figure 2 B, D).

[0073] Statistical analysis of the M1 / M2 values ​​revealed that the M1 / M2 values ​​of both the LPS group and the LBP group were significantly higher than those of the control group. Figure 2 C) indicates that LPS and LBP generally promote the polarization of macrophages towards the M1 type.

[0074] 3.2 Effects of LBP on gene expression levels in BMDM cells

[0075] This invention investigates the effects of LBP of different molecular weights on gene expression levels in M2 type BMDM cells. The gene expression levels of M1 macrophage markers (Il1β, Nos2, Tnfα) and M2 macrophage markers (Arg1, Chil3, Il4) in different groups were analyzed by qPCR.

[0076] qPCR test results are shown below Figure 3 The vertical axis represents the relative expression level of mRNA. Compared with the control group, the relative mRNA expression levels of Il1β, Nos2, and Tnfα increased in most LPS and LBP groups. Figure 3 A), while the relative expression levels of Arg1, Chil3, and Il4 mRNA decreased ( Figure 3 B), and the effect of LBP with a molecular weight greater than 50 was more significant than that of other molecular weights, indicating that LPS and LBP promoted the gene expression of M1 macrophage markers and reduced the gene expression level of M2 markers.

[0077] In addition, the LBP group with a molecular weight of less than 50 kDa showed almost no upregulation of gene expression levels of M1 type markers. Figure 3 A), while the gene expression levels of the M2 biomarkers Arg1 and Chil3 in the LBP group with a molecular weight less than 3kD were not different from those in the control group, the LBP with a molecular weight greater than 100kD upregulated the expression level of the M2 biomarker Il4. Figure 3 B). Therefore, overall, LBPs with a molecular weight of 50–100 kDa are more effective than those with other molecular weights in remodeling macrophage phenotypes.

[0078] 3.3 Effect of LBP on NO release levels in BMDM cells

[0079] This invention investigates the effect of LBP of different molecular weights on NO release levels from M2 BMDM cells. NO concentrations in the supernatant of BMDM cells from different treatment groups were measured using a NO detection kit.

[0080] The NO test results are shown in the table below. Figure 4 , Figure 4 (A) The horizontal and vertical axes represent the NO concentration of the standard and the OD value at 540 nm, respectively. Figure 4 (B) The vertical axis represents the NO concentration in the samples. Compared with the control group, the supernatant of BMDM cells in the LPS group and the 50–100 kD LBP group contained higher concentrations of NO. The LPS group showed the most significant effect, with a NO concentration of (2.72 ± 0.12) μM. The LBP group with a molecular weight of 50–100 kD showed a slightly better effect than other molecular weight groups, with a NO concentration of (1.61 ± 0.19) μM, all within the linear range of the standard curve.

[0081] 3.4 Direct killing effect of BMDM repolarization on tumor cells

[0082] This invention investigates the direct tumor-killing effect of LBP of different molecular weights on remodeled M2 BMDM cells. BMDM cells and tumor cells were co-cultured and drugged. After adding luciferase, the fluorescence intensity of tumor cells in different treatment groups was detected using an ELISA reader, thereby analyzing the survival rate of tumor cells.

[0083] The results of the fluorescence microplate reader test are shown below. Figure 5 , Figure 5 (A, C) The vertical axis represents the relative fluorescence intensity of tumor cells. Figure 5 (B, D) The ordinate represents the tumor cell inhibition rate. For 4T1-Luc cells, in the absence of BMDM, there was no statistically significant difference between the LPS group and the control group. The fluorescence intensity of LBP (with a molecular weight greater than 3kD) was significantly reduced, indicating that LPS alone had no significant effect on 4T1-Luc cells, while LBP itself had a certain inhibitory effect on 4T1-Luc cell killing. Figure 5 A). When BMDM was directly co-incubated with 4T1-Luc cells, the fluorescence intensity in both the LPS and LBP groups was significantly reduced compared to the control group. Figure 5 A) indicates that LPS and LBP of different molecular weights can enhance the direct killing effect of BMDM on 4T1-Luc cells, and this effect is more significant than that of LPS or LBP alone.

[0084] The highest inhibitory rates of LBPs of different molecular weights (<3kD, 3–50kD, 50–100kD, >100kD) on 4T1-Luc cells were (57.05±1.48)%, (78.84±3.01)%, (98.68±0.11)%, and (94.16±0.18)%, respectively. This indicates that LBPs with a molecular weight of 50–100kD were more effective than those with other molecular weights. Figure 5 B). Furthermore, comparing the three cell ratios used in the experiment revealed that when BMDM:4T1-Luc = 10:1, the overall tumor cell inhibition rate was higher. Figure 5 B) suggests that the co-culture ratio of BMDM and tumor cells may affect the survival rate of tumor cells.

[0085] For B16-Luc cells alone, the fluorescence intensity of LBP groups with a molecular weight less than 100kD was not different from that of the control group, while the fluorescence intensity of LPS groups and LBP groups with a molecular weight >100kD was increased. Figure 5 C). When BMDM was co-cultured with B16-Luc cells, the fluorescence intensity of the LPS and LBP groups decreased to varying degrees. Figure 5C) It can be seen that LPS and LBP can enhance the killing effect of BMDM on B16-Luc cells.

[0086] Comparison of LBPs with different molecular weights revealed that the 50–100 kDa LBP group exhibited a more significant inhibitory effect on B16-Luc cells, with the highest inhibition rate of (46.80 ± 6.02)%. When BMDM:B16-Luc = 20:1, the overall tumor cell inhibition rate was higher than the other two cell ratio groups. Figure 5 D).

[0087] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of Lycium barbarum functional polysaccharides in the preparation of anti-melanoma drugs; The aforementioned wolfberry functional polysaccharide was prepared using the following method: 10 kg of dried wolfberries were weighed, and 80% ethanol was added at a solid-liquid ratio of 10:

1. The mixture was heated and refluxed twice, each time for 1 hour, to obtain an ethanol extract and an ethanol-extracted residue. 15 times the volume of deionized water was added to the ethanol-extracted residue, and the mixture was heated and refluxed twice, each time for 1 hour. The aqueous extracts were combined, and anhydrous ethanol was added to the aqueous extract to achieve an ethanol volume fraction of 80%. The mixture was allowed to stand overnight at room temperature, centrifuged at 4500 rpm for 5 minutes to collect the precipitate, washed with anhydrous ethanol, and freeze-dried to obtain crude wolfberry polysaccharide. The crude wolfberry polysaccharide was dissolved in deionized water, and the solution was fed into an ultrafiltration device via a circulating pump. The solution was then filtered through a 100 kDa fiber filter. Ultrafiltration using a vitamin membrane: The retentate is circulated and ultrafiltered, and the collected retentate is the Lycium barbarum polysaccharide sample solution with a molecular weight >100kD; the permeate from the ultrafiltration membrane is the crude Lycium barbarum polysaccharide with a molecular weight <100kD; similarly, the crude Lycium barbarum polysaccharide is separated by ultrafiltration membrane modules of 50kD and 3kD in sequence to obtain Lycium barbarum polysaccharide sample solutions of 50-100kD, 3-50kD and <3kD; the 50-100kD Lycium barbarum polysaccharide sample solution is evaporated and concentrated to obtain Lycium barbarum polysaccharides in the molecular weight range of 50-100kD, which are then freeze-dried for later use.

2. The application according to claim 1, comprising a drug prepared from wolfberry functional polysaccharide and a pharmaceutically acceptable carrier into tablets, capsules, granules, pills, mixtures, oral liquids or ointments.

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