Poplar phellinus polysaccharide SVP-50-1, and preparation method and application thereof

Abstract

The application discloses poplar phellinus polysaccharide SVP-50-1 and a preparation method and application thereof, and belongs to the field of biological medicines.The poplar phellinus polysaccharide SVP-50-1 is obtained by water extraction and 50% ethanol solution precipitation of poplar phellinus fruiting bodies.The poplar phellinus polysaccharide SVP-50-1 can regulate a tumor metabolic process, inhibit the activity of a tumor cell mitochondrial respiratory chain and an oxidative phosphorylation process, thereby inhibiting the energy source of tumor cells, and achieving an anti-tumor effect.The poplar phellinus polysaccharide SVP-50-1 can play a synergistic effect when combined with radiotherapy and chemotherapy drugs.The combined treatment strategy can significantly improve the curative effect of an existing tumor treatment, while reducing adverse reactions, and provides a new optimized scheme for clinical tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a poplar mulberry polysaccharide SVP-50-1, its preparation method, and its application. Background Technology

[0002] Breast cancer is the most common and deadliest malignant tumor among women, and its treatment remains a significant challenge. Metabolic reprogramming is a core characteristic of tumors, with oxidative phosphorylation showing significantly enhanced activity in breast cancer and other cancers, making it a potential intervention target. Studies have shown that highly malignant breast cancer cells, especially tumor stem cells, are highly dependent on OXPHOS for energy, and the expression of respiratory chain complexes is elevated in cancer tissue, suggesting that inhibiting OXPHOS may have therapeutic value. However, currently, there are no effective therapeutic methods to target the OXPHOS process, resulting in a gap in anti-tumor strategies targeting this pathway.

[0003] While the potential of OXPHOS inhibition in anti-tumor therapy has been recognized, developing drug components with a clear mechanism of action, the ability to specifically regulate this process, and stable biological activity remains challenging. For example, known metabolic interventions generally suffer from low selectivity, complex preparation processes, or heterogeneous active ingredients, hindering clinical translation. Therefore, there is an urgent need to find structurally defined, controllable in preparation, and effective inhibitory substances capable of inhibiting OXPHOS to fill the gaps in current breast cancer metabolic therapy.

[0004] As a medicinal fungus, *Phellinus linteus* (poplar fungus) is known to possess bioactive polysaccharide components, but current research has not clarified the role of specific polysaccharide components in regulating tumor OXPHOS and energy metabolism. Furthermore, existing polysaccharide components often face challenges in anti-tumor applications, such as unclear structures, poor reproducibility in preparation, and limited single-drug efficacy, thus restricting their clinical application. Therefore, developing a *Phellinus linteus* polysaccharide component with a clear structure, stable preparation method, and the ability to target OXPHOS and be suitable for combination therapy is of great significance for expanding the therapeutic avenues for breast cancer. Summary of the Invention

[0005] The purpose of this invention is to provide a poplar linteus polysaccharide SVP-50-1, its preparation method, and its applications, to solve the problems existing in the prior art. This invention provides a poplar linteus polysaccharide SVP-50-1 with a well-defined structure, which exerts its anti-tumor effect by inhibiting the mitochondrial respiratory chain and oxidative phosphorylation process in tumor cells. This polysaccharide can effectively inhibit tumor proliferation and invasion, and when used in combination with radiotherapy and chemotherapy, it can synergistically enhance efficacy and reduce toxicity, providing a new strategy for tumor treatment.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a poplar mulberry fungus polysaccharide SVP-50-1, the structure of which consists of 128 repeating units: The main chain repeating unit is →6)-β-D-Glc p -(1→、→2,4)-β-D-Man p -(1→、→2)-α-L-Ara p -(1→、→6)-β-D-Glc p -(1→、→6,3)-β-D-Glc p -(1→and→6,3)-β-D-Glc p -(1→ is composed of six sugar residues connected sequentially by (1→4), (1→2), (1→6), (1→3), and (1→3) glycosidic bonds; Branched β-D-Man p -(1→2)-α-L-Ara p -(1→、α-L-Fuc p -(1→ and β-D-Gal p -(1→ is sequentially linked to →2,4)-β-D-Man via (1→2), (1→6), and (1→6) glycosidic bonds respectively. p -(1→、→6,3)-β-D-Glc p -(1→、→6,3)-β-D-Glc p -(1→on sugar residue; The sugar chain structure is as follows: .

[0007] This invention also provides a method for preparing the aforementioned poplar mulberry polysaccharide SVP-50-1, comprising the following steps: After crushing the fruiting bodies of *Pinus linteus*, distilled water was added for boiling and extraction. The filtrate was concentrated and precipitated with ethanol solution. The precipitate was then decolorized, impurities removed, and separated by a DEAE cellulose column to obtain the *Pinus linteus* polysaccharide SVP-50-1. The ethanol solution has a volume concentration of 50% in the precipitation system.

[0008] Optionally, the mass-to-volume ratio of the fruiting body powder to the distilled water is 1 g: 10 mL.

[0009] The present invention also provides the application of the aforementioned poplar mulberry polysaccharide SVP-50-1 in the preparation of anti-breast cancer drugs.

[0010] Optionally, the drug is an inhibitor of oxidative phosphorylation energy metabolism in tumor cells.

[0011] The present invention also provides the application of the aforementioned poplar mulberry polysaccharide SVP-50-1 in combination with antitumor chemotherapy or radiotherapy drugs in the preparation of anti-breast cancer drugs.

[0012] Optionally, the chemotherapy drugs include docetaxel, doxorubicin, and cyclophosphamide.

[0013] The present invention also provides an anti-tumor tumor cell oxidative phosphorylation energy metabolism inhibitor, the active ingredient of which includes the aforementioned poplar mulberry polysaccharide SVP-50-1.

[0014] The present invention also provides an anti-breast cancer drug, the active ingredients of which include the aforementioned poplar mulberry polysaccharide SVP-50-1, docetaxel, doxorubicin and cyclophosphamide.

[0015] The present invention discloses the following technical effects: The poplar mulberry polysaccharide SVP-50-1 provided by this invention exhibits significant antitumor activity, effectively inhibiting the proliferation and invasion of tumor cells, thereby blocking the malignant progression of tumors. Its well-defined structure and controllable preparation method ensure the stability and bioactivity of the polysaccharide components, providing a reliable foundation for the development of antitumor drugs.

[0016] The poplar polysaccharide SVP-50-1 provided by this invention can regulate tumor metabolism, inhibit the activity of the mitochondrial respiratory chain and its oxidative phosphorylation process in tumor cells, thereby inhibiting the energy source of tumor cells and achieving an anti-tumor effect.

[0017] The poplar polysaccharide SVP-50-1 provided by this invention, when used in combination with radiotherapy and chemotherapy drugs, can exert a synergistic effect, not only improving the sensitivity of tumors to treatment but also reducing the toxic side effects of radiotherapy and chemotherapy, achieving a "reduced toxicity and increased efficacy" effect. This combined treatment strategy can significantly improve the efficacy of existing tumor therapies while reducing adverse reactions, providing a new and optimized solution for clinical tumor treatment. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 For the purification and molecular weight determination of SVP-50-1, (A) DEAE cellulose-52 elution curve; (B) HPGPC chromatogram of SVP-50-1 (ELSD detector). Figure 2The total ion gas chromatogram of SVP-50-1 analyzed by gas chromatography-mass spectrometry; Figure 3 One-dimensional nuclear magnetic resonance spectroscopy analysis of SVP-50-1; where, (A) 1 H NMR spectrum; (B) 13 C10 NMR spectrum; Figure 4 Hydrogen-hydrogen correlation spectrum of SVP-50-1 ( 1 H- 1 H COSY spectrum); Figure 5 The carbon-hydrogen heteronuclear single quantum correlation spectrum (HSQC spectrum) of SVP-50-1. Figure 6 The carbon-hydrogen heteronuclear multibond correlation spectrum (HMBC spectrum) of SVP-50-1. Figure 7 The predicted chemical structure of SVP-50-1; Figure 8 To investigate the effect of the SVP-50-1 combined with TAC regimen on the proliferation of MCF-7 cells, (A, C) the effect of the SVP-50-1 combined with TAC regimen on the proliferation of MCF-7 cells was detected by EdU assay; (B) the effect of the SVP-50-1 combined with TAC regimen on the viability of MCF-7 cells was detected by CCK-8 assay. Figure 9 The effects of SVP-50-1 combined with TAC regimen on the invasion and migration abilities of MCF-7 cells were investigated. (A, B) Effect of SVP-50-1 combined with TAC regimen on the migration of MCF-7 cells; (A, C) Effect of SVP-50-1 combined with TAC regimen on the invasion of MCF-7 cells. Figure 10 To illustrate the effect of SVP-50-1 combined with the TAC regimen on tumors in 4T1 tumor-bearing mice, (A) tumor images; (B) statistical graph of tumor volume changes; (C) statistical graph of tumor weight; (D) H&E staining images of tumors. Figure 11 Hematoxylin-eosin (H&E) staining of the heart, liver, spleen, lungs, and kidneys; Figure 12 The transcriptomic analysis results of MCF-7 cells are as follows: (A) Volcano plot of differentially expressed genes between the SVP-50-1H combined with TAC group and the control group; (B) Enrichment analysis results of significantly differentially expressed genes between the SVP-50-1H combined with TAC group and the control group. Figure 13To evaluate the activity of SVP-50-1 combined with TAC on tumor oxidative phosphorylation-related proteins and mitochondrial respiratory chain complex I-V in mice, (A) related protein bands; (BF) quantification of related protein expression levels; (GK) mitochondrial respiratory chain complex I-V activity. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] The Sanghuang used in this embodiment of the invention is poplar Sanghuang (… Sanghuangporus vaninii), the fruiting bodies of Phellinus igniarius were purchased from Yuchen Changbai Mountain Wild Ganoderma Technology Co., Ltd. (Baishan, China). The human breast cancer cell line (MCF-7) and mouse breast cancer cell line (4T1) were purchased from Wuhan Punosai Life Science and Technology Co., Ltd. (Wuhan, China). Sprague Dawley (SD) rats (weighing 180 - 220 g) and Kunming (KM) mice (weighing 18 - 22 g) were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd., with the license number SYXK (Ji) 2018 - 0023. Other reagents and materials were obtained through regular procurement without special instructions.

[0026] Example 1 Screening of the Anticancer Active Parts of Phellinus igniarius 1. Preparation of Each Extracted and Isolated Part of Phellinus igniarius After crushing 5 kg of the fruiting bodies of Phellinus igniarius, they were ultrasonically extracted 3 times with 10 - fold volume of 70% methanol solution (50 L) for 1 hour each time. The extraction solutions were combined and concentrated to dryness to obtain the methanol extract (denoted as SV - ME); another 5 kg of the fruiting bodies of Phellinus igniarius were crushed and decocted 3 times with 10 - fold volume of hot water (50 L) for 2 hours each time. The decoction solutions were combined and concentrated to 1 / 100 volume (partially concentrated to dryness to obtain the crude polysaccharide of Phellinus igniarius, denoted as SVP).

[0027] Alcohol precipitation: Ethanol was added step - by - step to the concentrated decoction solution to make the volume concentration of ethanol reach 30%, 50%, 70% and 90% in sequence, and polysaccharides were precipitated fractionally.

[0028] Depigmentation: The polysaccharides precipitated fractionally above were completely dissolved in water respectively, loaded onto D101 macroporous resin columns, and eluted with 8 - fold column volume of distilled water to remove pigments. The elution solutions were collected and concentrated under reduced pressure.

[0029] Impurity removal: The Sevag method was used to remove proteins, and then the supernatant was collected and concentrated. Dialysis was carried out with a dialysis bag with a molecular weight cut - off of 3500 Da to remove small - molecule impurities. After freeze - drying, the 30%, 50%, 70% and 90% alcohol - precipitated fractions of Phellinus igniarius polysaccharides (denoted as SVP - 30, SVP - 50, SVP - 70 and SVP - 90) were obtained respectively.

[0030] 2. Study on the Antibreast Cancer Activity of Each Part of Phellinus igniarius Clean-grade SD rats were randomly divided into 7 groups (n=6 per group): control group (Con), SV-ME group, SVP group, SVP-30 group, SVP-50 group, SVP-70 group, and SVP-90 group. They were housed in an SPF-grade environment (temperature 24±2℃, relative humidity 55±5%, 12-hour light / dark cycle, free access to food and water). The experimental protocol was approved by the Experimental Animal Ethics Committee of Jilin Agricultural University. After one week of acclimatization, each group was administered an equal volume of physiological saline (Con) and 200 mg / kg of SV-Me (SV-ME group), SVP (SVP group), SVP-30 (SVP-30 group), SVP-50 (SVP-50 group), SVP-70 (SVP-70 group), and SVP-90 (SVP-90 group) by gavage for 7 consecutive days, 2 mL daily. After fasting for 12 hours following the last administration, the patient was anesthetized with sodium pentobarbital, and blood was collected via the abdominal aorta. The serum was separated by centrifugation at 4000 r / min for 15 minutes, inactivated by water bath at 56 ℃ for half an hour, and then aliquoted for subsequent cell experiments.

[0031] After MCF-7 cells were revived and passaged, they were cultured at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of [missing value] / mL in 96-well plates. After cell adhesion, they were divided into the following groups and treated accordingly: control group; TAC group (TAC: docetaxel 30 μg / mL, doxorubicin 20 μg / mL, and cyclophosphamide 100 μg / mL); SV groups (containing 10% serum from rats containing SV-ME / SVP / SVP-30 / SVP-50 / SVP-70 / SVP-90); and combination groups [containing 5% serum from each SV group containing the drug + TAC (docetaxel 15 μg / mL, doxorubicin 10 μg / mL, and cyclophosphamide 50 μg / mL)]. Drug concentrations were determined in preliminary experiments. After treatment of each group, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C and 5% CO2 for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and cell viability was calculated for each group.

[0032] The results of MCF-7 cell viability assay are shown in Table 1.

[0033] Table 1. Effects of serum samples containing SV drugs, alone and in combination with TAC, on the viability of MCF-7 cells. Note: Data are expressed as mean ± standard deviation (n=5 per group). Compared with the model group, #P<0.05, ##P<0.01; compared with the TAC group, $$P<0.01.

[0034] As shown in Table 1, compared with the control group, only SVP-50 and SVP-70 significantly inhibited the cell viability of MCF-7 cells in various parts of *Phyllanthus linteus*, with SVP-50 showing the best effect. Furthermore, the combined use of SVP-50 and TAC exhibited a significant synergistic effect, with SVP-50 showing a significantly better inhibitory effect on MCF-7 cell viability than TAC alone. Therefore, the 50% alcohol-precipitated fraction of crude polysaccharide from *Phyllanthus linteus* was selected for further research.

[0035] Example 2: Preparation and structural characterization of SVP-50-1, a polysaccharide from the poplar tree *Phellinus linteus*. 1. Preparation of SVP-50-1, a polysaccharide from the poplar tree *Phellinus linteus* The SVP-50 prepared in Example 1 was further separated as follows: SVP-50 was dissolved in distilled water at a concentration of 25 mg / mL and loaded onto a DEAE cellulose-52 column (30 cm × 2.5 cm) for separation. Elution was performed sequentially using water and different concentrations of NaCl solution (0.1 M, 0.3 M, 0.5 M, 0.7 M, 1 M) as mobile phases at a flow rate of 1 mL / min, with each fraction collected for 10 minutes. The polysaccharide content of each fraction (tube) was determined using the phenol-sulfuric acid method, and the polysaccharide concentration was determined by detecting the absorbance at 490 nm. The fraction with the highest polysaccharide content was collected and dialyzed through a dialysis bag with a molecular weight cutoff of 3500 Da for 24 hours (with distilled water replaced every 8 hours). After lyophilization, HPGPC (high performance gel permeation chromatography) purity analysis was performed using a Tsk Gel 4000 PWXL column (7.8 mm × 300 mm) and a Waters 2424 evaporative light scattering detector (ELSD). The chromatographic conditions were: detector gain 100, pressure 30 MPa, drift tube temperature 90℃, carrier gas compressed air (pressure 5 bar), column temperature 40℃, mobile phase H2O, and flow rate 1 mL / min. The results showed a chromatographic peak with a single retention time, indicating a homogeneous polysaccharide, named SVP-50-1.

[0036] 2. Structural characterization of SVP-50-1, a polysaccharide from the poplar tree *Phellinus linteus*. (1) Molecular weight determination SVP-50 was separated using a DEAE cellulose-52 ion exchange chromatography column to obtain three components ( Figure 1 (A): SVP-50-1, SVP-50-2, and SVP-50-3. Among them, SVP-50-1 obtained by water elution has a symmetrical peak shape and high purity, indicating that it has the richest polysaccharide content. HPGPC chromatographic evaporative light scattering detection (ELSD) analysis showed that only a single symmetrical peak existed in the SVP-50-1 chromatogram. Figure 1(B), proving that it is a homogeneous polysaccharide. The linear equation fitted based on the dextran standard is [log(Mw) = -0.8817 X + 8.68, R] 2 = 0.9992] The average molecular weight of SVP-50-1 is calculated to be 198.35 kDa.

[0037] (2) Methylation and gas chromatography-mass spectrometry analysis Methylation analysis was performed on SVP-50-1 to obtain the total ion chromatogram of the sample. Figure 2 The secondary mass spectrometry fragment peaks of each sugar residue were observed. Based on literature and ion fragment information from the Center for Complex Carbohydrate Research (CCRC) database at the University of Georgia, the glycosidic bond types were inferred. Ultimately, seven glycosidic bond fragments were identified in SVP-50-1 (Table 2): 2-Ara... p (2-Linked arabinopyranosyl); T-Man p (Terminal mannopyranosyl); T-Fuc p (Terminal phrysopyranosyl); 2,4-Man p (2,4-linked mannopyranosyl); 6-Glc p (6-linked glucopyranosyl); 3,6-Glc p (3,6-linked glucopyranosyl); T-Gal p (Terminal galactopyranose).

[0038] Table 2. Methylation analysis data and corresponding glycosidic bond linkage modes of SVP-50-1 (3) Nuclear magnetic resonance spectroscopy analysis Based on the methylation analysis results of sugar residue fragments, combined with 1 H NMR, 13 C NMR and 1 H- 1 H COSY spectrum ( Figure 3 A and B, Figure 4 A total of 7 sugar residue fragments (AG) were identified.

[0039] Anterior proton region analysis: Seven characteristic peaks were detected in the 1H NMR spectrum of the anoproton region, with chemical shifts of δ 5.36, 5.12, 4.57, 5.26, 5.15, 4.97, and 4.99 ppm.

[0040] 1 H- 1 H COSY spectroscopy further analyzes the H signaling chain of each residue: Residue A: δ 5.36, 3.60, 3.69, 3.51, 3.90, 3.78 / 4.00 ppm; Residue B: δ 5.12, 4.25, 3.92, 3.86, 3.50, 3.79 / 3.96 ppm; Residue C: δ 4.57, 3.56, 3.56, 3.67, 3.35 / 3.94 ppm; Residue D: δ 5.26, 3.70, 3.96, 3.75, 4.16, 3.79 / 3.93 ppm; Residue E: δ 5.15, 3.98, 3.85, 3.71, 3.94, 3.78 / 3.90 ppm; Residue F: δ 4.97, 3.77, 3.87, 3.77, 4.05, 1.84 ppm; Residue G: δ 4.99, 3.79, 3.92, 3.98, 3.92, 3.70 / 3.69 ppm; 1 H- 13 C HSQC spectral analysis ( Figure 5 ): Residue A: 5.36 / 101.44, 3.60 / 72.54, 3.69 / 73.61, 3.51 / 70.51, 3.90 / 72.18, 3.78 / 68.25, 4.00 / 68.25, inferred to be →6)-Glcp-(1→; Residue B: 5.12 / 101.87, 4.25 / 77.30, 3.92 / 73.61, 3.86 / 76.81, 3.50 / 77.29, 3.79 / 60.55, 3.96 / 60.55, inferred to be →2,4)-Manp-(1→; Residue C: 4.57 / 105.10, 3.56 / 77.29, 3.56 / 73.61, 3.67 / 70.30, 3.35 / 63.25, 3.94 / 63.25, inferred to be →2)-Arap-(1→; Residue D: 5.26 / 100.61, 3.70 / 71.07, 3.96 / 77.29, 3.75 / 70.52, 4.16 / 72.18, 3.79 / 68.5, 33.93 / 68.5, inferred to be →3,6)-Glcp-(1→; Residue E: 5.15 / 100.86, 3.98 / 72.18, 3.85 / 71.07, 3.71 / 69.72, 3.94 / 72.55, 3.78 / 60.56, 3.90 / 60.56, inferred to be Manp-(1→ (terminus); Residue F: 4.97 / 100.00, 3.77 / 69.30, 3.87 / 70.52, 3.77 / 72.18, 4.05 / 63.23, 1.84 / 16.73, inferred to be Fucp-(1→(terminus); Residue G: 4.99 / 99.22, 3.79 / 69.72, 3.92 / 70.52, 3.98 / 70.31, 3.92 / 71.07, 3.70 / 60.56, 3.69 / 60.56, inferred to be Galp-(1→ (terminus); 1 H- 13 C HMBC spectral verification connection order ( Figure 6 ): The δ 5.36 / 76.81 and δ 5.12 / 77.29 signals indicate that H1 of residue A is associated with C4 of residue B, and H1 of residue B is associated with C2 of residue C, i.e., A→B→C sequential connection; The δ 4.57 / 68.53, δ 5.36 / 77.29, and δ 5.26 / 77.29 signals indicate that H1 of residue C is associated with C6 of residue A, H1 of residue A is associated with C3 of residue D, and H1 of residue D is associated with C3 of another residue D. That is, the sequence is C→A→D→D. Therefore, it can be inferred that the main chain structure of SVP-50-1 is: →A→B→C→A→D→D→; The δ 5.15 / 77.29 and δ 4.57 / 77.30 signals indicate that H1 of residue E is related to C2 of residue C, and H1 of residue C is related to C2 of residue B, that is, E→C→B forms a branch; The δ 4.97 / 68.53 and δ 4.99 / 68.53 signals indicate that H1 of residues F and G are associated with C6 of residue D, respectively, i.e., F→D and G→D form a branch; Based on the combined methylation and NMR data, the structure of SVP-50-1 is as follows: Figure 7 As shown, the sequence is →AB(EC)-CAD(F)-D(G)→. Based on the molecular weight calculated using data provided by HPGPC, the SVP-50-1 structure contains 128 of the aforementioned repeating sequences.

[0041] Table 3 Major sugar residues of SVP-50-1 1 H and 13 C10 NMR chemical shift data Example 3: Effects of Populus euphratica polysaccharide SVP-50-1 combined with TAC on MCF-7 cells I. Methods 1. Preparation of drug-containing serum in mice Five clean-grade SD rats were selected and housed in an SPF-grade environment (temperature 24±2 ℃, relative humidity 55±5%, 12-hour light / dark cycle, free access to food and water). The experimental protocol was approved by the Experimental Animal Ethics Committee of Jilin Agricultural University. After one week of acclimatization, SVP-50-1 (200 mg / kg, 2 mL daily) was administered by gavage for 7 consecutive days. After the last administration, the rats were fasted for 12 hours, anesthetized with sodium pentobarbital, and blood was collected via the abdominal aorta. Serum was separated by centrifugation at 4000 r / min for 15 minutes and then aliquoted for use.

[0042] 2. Cell viability assay (CCK-8 assay) MCF-7 cells were loaded at 5 × 10 4 SVP-50-1 was seeded at a density of 15 μg / mL in 96-well plates and incubated for 24 hours. After adhesion, the cells were divided into 6 groups: (1) control group; (2) TAC group (TAC: docetaxel 30 μg / mL, doxorubicin 20 μg / mL and cyclophosphamide 100 μg / mL); (3) SVP-50-1 low-dose group (SVP-50-1L, containing 5% rat serum containing the drug); (4) SVP-50-1 high-dose group (SVP-50-1H, containing 10% rat serum containing the drug); (5) SVP-50-1 low-dose combination group [SVP-50-1L (2.5% serum containing the drug) + TAC (docetaxel 15 μg / mL, doxorubicin 10 μg / mL and cyclophosphamide 50 μg / mL)]; (6) SVP-50-1 high-dose combination group [SVP-50-1H (5% serum containing the drug) + TAC (docetaxel 15 μg / mL, doxorubicin 10 μg / mL and cyclophosphamide 50 μg / mL)]; [μg / mL, doxorubicin 10 μg / mL and cyclophosphamide 50 μg / mL]. Drug concentrations were determined through preliminary experiments. After each group was treated, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37 ℃ with 5% CO2 for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. All cell experiments were conducted using these 6 concentration groups.

[0043] 3. 5-Ethynyl-2'-deoxyuridine (EdU) cell proliferation experiment MCF-7 cells (1×10) 5(EdU cells were seeded in 6-well plates and cultured for 24 hours. Different concentrations of the drug were then added and treated for another 24 hours. An equal volume of pre-prepared EdU working solution (20 μM) was added and incubated for 2 hours. The culture medium was discarded, and 1 mL of 4% paraformaldehyde was added to each well for fixation at room temperature for 20 minutes. After washing with PBS (containing 3% BSA), the cells were permeabilized with PBS containing 0.3% Triton X-100 for 20 minutes. After washing, 0.5 mL of Click reaction buffer (containing CuSO4, Azide 488, and Click additive) was added, and the cells were reacted at room temperature in the dark for 30 minutes. The reaction solution was discarded, and Hoechst 33342 staining solution was added and incubated in the dark for 10 minutes. After washing, the proliferation of MCF-7 cells was observed under a fluorescence microscope (EdU-positive green fluorescent markers for proliferating cells, and blue fluorescent markers for cell nuclei).

[0044] 4. Cell migration assay (Transwell assay) MCF-7 cells were seeded into 6-well plates and cultured to an appropriate density. After digestion, the cells were resuspended in serum-free medium (containing the corresponding drug; an equal volume of blank rat serum was added to the TAC group and the model group to balance the effect of the drug-containing serum group) and the concentration was adjusted to be uniform (1×10⁵ cells / well). 100 μL of cell suspension was added to the upper chamber of a Transwell plate, and 600 μL of medium containing 30% fetal bovine serum (FBS) was added to the lower chamber. The plates were incubated at 37°C for 24 hours. After incubation, the chambers were removed, the medium in the wells was discarded, and the cells were gently washed twice with PBS. The cells were fixed with methanol for 30 minutes and allowed to air dry. The plates were stained with 0.1% crystal violet for 20 minutes, and the unmigrated cells in the upper chamber were carefully wiped away with cotton swabs. The plates were then washed three times with PBS. Five fields of view were randomly selected under a microscope to count the number of migrating cells.

[0045] 5. Cell invasion assay (Matrigel method) The upper chamber of the Transwell was pre-coated with Matrigel (50 μL / chamber) and allowed to stand at room temperature for 3 hours to allow the gel to polymerize. MCF-7 cells were pretreated according to the migration assay method, and 1×10⁶ cells were collected. 5 Cell / well suspension was added to the upper chamber pre-coated with Matrigel, and 600 μL of culture medium containing 30% FBS was added to the lower chamber. The cells were incubated at 37°C for 12 hours. The culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 10 minutes and stained with 0.1% crystal violet for 20 minutes. The number of cells that penetrated the Matrigel and entered the lower chamber was observed and counted under a microscope.

[0046] All results were analyzed and expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, and the Student's t-test was used for pairwise comparisons. A p-value < 0.05 was considered statistically significant.

[0047] II. Results 1. Effects of SVP-50-1 combined with TAC on the proliferation of MCF-7 cells MCF-7 cell viability was assessed using the CCK-8 assay. Results showed that, compared to the control group, all treatment groups (containing different concentrations of SVP-50-1 rat serum combined with TAC) significantly inhibited MCF-7 cell proliferation. Compared to the TAC group, the SVP-50-1 combined with TAC group showed a significantly enhanced inhibitory effect on MCF-7 cell viability. Among them, the high-dose group of SVP-50-1 combined with TAC showed the most significant effect. Figure 8 (B). Further EdU assay was used to verify the effect of combined drug administration on cell proliferation. The results were consistent with the CCK-8 assay: the MCF-7 cell proliferation capacity of the SVP-50-1 combined with TAC group was significantly lower than that of the TAC group, indicating a synergistic inhibitory effect (B). Figure 8 (A, C). Conclusion: The combination of SVP-50-1 and TAC can significantly enhance anti-tumor activity, and may inhibit the proliferation of breast cancer cells through synergistic effect.

[0048] 2. Effects of SVP-50-1 combined with TAC on MCF-7 cell migration and invasion Tumor cells migrate and invade during progression. Therefore, this invention investigates the effects of SVP-50-1 combined with TAC on the migration and invasion of MCF-7 cells using a Transwell chamber assay. Figure 9 The results showed that, compared with the model group, all treatment groups significantly inhibited the migration and invasion of MCF-7 cells. Compared with the TAC group, the SVP-50-1 combined with TAC group showed significantly enhanced inhibitory effects on cell migration and invasion, with the high-dose SVP-50-1 combined with TAC group showing the most significant effect.

[0049] Example 4: Effects of Populus euphratica polysaccharide SVP-50-1 combined with TAC regimen on tumor growth in 4T1 tumor-bearing mice I. Methods Male Kunming (KM) mice were housed in an SPF-grade environment (temperature 24±2 ℃, relative humidity 55±5%, 12-hour light / dark cycle, with free access to food and water). The experimental protocol was approved by the Experimental Animal Ethics Committee of Jilin Agricultural University.

[0050] 1. Establishment and treatment of 4T1 tumor-bearing mouse model Mice were randomly divided into 7 groups (n=8 per group): (1) control group; (2) model group; (3) TAC group (TAC: 30 mg / kg, docetaxel: doxorubicin: cyclophosphamide mass ratio of 3:2:10); (4) SVP-50-1 low-dose group (SVP-50-1L: 200 mg / kg); (5) SVP-50-1 high-dose group (SVP-50-1H: 400 mg / kg); (6) TAC combined with SVP-50-1 low-dose group [TAC (30 mg / kg, docetaxel: doxorubicin: cyclophosphamide mass ratio of 3:2:10) + SVP-50-1L (200 mg / kg)]; (7) TAC combined with SVP-50-1 high-dose group [TAC (30 mg / kg, docetaxel: doxorubicin: cyclophosphamide mass ratio of 3:2:10) + SVP-50-1L (200 mg / kg)]; [400 mg / kg docetaxel:doxorubicin:cyclophosphamide in a mass ratio of 3:2:10 + SVP-50-1H (400 mg / kg)]. Except for the control group, mice in all other groups were injected with 4T1 cells under the right axilla to establish a tumor-bearing model. Tumor volume was monitored daily until the tumor grew to approximately 100 mm. 3 Drug administration began at the following times: SVP-50-1 was administered by gavage once daily; TAC was administered intraperitoneally every four days. The control and model groups received an equal volume of normal saline. Drug dosages were determined based on literature and preliminary experiments. During treatment, the tumor's long diameter (L) and short diameter (W) were measured daily, calculated using the formula V = 1 / 2 × L × W. 2 Tumor volume was calculated, and mice were continuously monitored until day 20 when they were sacrificed.

[0051] 2. Tumor inhibition rate and immune organ index measurement After the last administration, the mice were weighed using an electronic balance. Mice were anesthetized with sodium pentobarbital via intraperitoneal injection. Blood was collected through the eye and the serum was separated by centrifugation at 3500 r / min for 10 minutes at 4°C. The mice were then euthanized by cervical dislocation. The thymus, spleen, and tumor tissue were dissected and harvested (the remaining tissues were frozen for later use). The tissues were washed with sterile saline, blotted dry with filter paper, and weighed. The tumor inhibition rate was calculated using the following formula: Tumor inhibition rate (%) = (average tumor weight in the model group - average tumor weight in the treatment group) / average tumor weight in the model group (average tumor weight in the model group - average tumor weight in the treatment group) / average tumor weight in the model group × 100%.

[0052] 3. Hematoxylin-eosin (H&E) staining of tumors and various tissues Tumor tissues, as well as tissues from the heart, liver, spleen, lungs, and kidneys, were collected and fixed in 4% paraformaldehyde for 24 hours. After dehydration with graded ethanol, clearing with xylene, and embedding in paraffin, the sections were prepared to a thickness of 4 μm. Sections were stained with hematoxylin for 5 minutes, differentiated with hydrochloric acid and ethanol, counterstained with eosin for 2 minutes, and mounted with neutral resin. Histopathological changes (such as tumor necrosis areas, inflammatory cell infiltration, and organ damage) were observed under an optical microscope and photographed.

[0053] 4. Transcriptomics analysis Total RNA was extracted from tumor tissue using the Trizol method, and RNA concentration and purity were detected using a Nanodrop 2000 (Thermo Scientific). Hieff NGS was used. TM RNA sequencing libraries were prepared using the MaxUp Dual-Mode mRNA Library Construction Kit (Illumina® compatible) and sequenced on an Illumina HiSeq2000 sequencer. Sequencing data were aligned to the mouse reference genome (GRCm38) using Hisat2 (v2.1.0), with splice site information based on the Ensembl 92 database. Gene expression levels were expressed as FPKM (kilobase fragments per million aligned exons) and converted to log2 values ​​for subsequent analysis. Differentially expressed genes (DEGs) were screened using the edgeR software package, and multiple validation was corrected using the false discovery rate (FDR) (threshold FDR < 5%). Hierarchical clustering analysis was performed using R, and gene ontology (GO) functional enrichment analysis was conducted using topGO (v2.24.0), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using clusterProfiler (v3.0.5). GO entries and KEGG pathways with P < 0.05 were considered significantly enriched.

[0054] 5. Western blot analysis of proteins Mouse tumor tissue was collected, washed 2-3 times with pre-cooled PBS to remove blood, homogenized under ice bath conditions, and lysed for 30 minutes on ice with pre-cooled tissue protein lysis buffer. The supernatant was collected after centrifugation at 12000 r / min for 5 minutes at 4 ℃. Protein concentration was determined by BCA method. 30 μg of protein sample was denatured at 95 ℃ for 10 minutes, separated by SDS-PAGE electrophoresis, and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight at 4 ℃ with the following primary antibodies: ATP8 (Proteintech, 1:2000), COX2 (Invitrogen, 1:2000), CYTB (abcam, 1:1000), SDHC (Invitrogen, 1:1000), NDUFA12 (Thermo Fisher, 1:2000), and internal control β-actin (Proteintech, 1:20000). After washing three times with TBST, HRP-labeled goat anti-rabbit secondary antibody (abcam, ab205718, 1:10000) was added and incubated at room temperature for 2 hours. After ECL development, the protein bands were imaged using a scanner, and the gray values ​​of the protein bands were analyzed using Image Pro Plus 6.0.

[0055] 6. Detection of mitochondrial respiratory chain complex activity in tumor tissue After removing excess tumor tissue, the activity of mitochondrial respiratory chain complexes I-V in the tumor tissue was measured according to the kit instructions.

[0056] II. Results 1. Effects of SVP-50-1 combined with TAC on tumors in 4T1 tumor-bearing mice In vitro experiments confirmed that SVP-50-1 could enhance the inhibitory effect of TAC on MCF-7 cell proliferation. Therefore, 4T1 tumor-bearing mice were further selected to study the in vivo antitumor activity of SVP-50-1. Model establishment and animal condition: Before inoculation with 4T1 cells, the body weight of mice in each group was similar with no differences between groups. All mice had shiny fur, good mental state, and normal excretion. During treatment after model establishment, mice in the TAC group showed symptoms such as diarrhea, bloody stools, dark urine, disheveled fur, and lethargy, while mice in other groups were in good condition. Tumor inhibition effect ( Figure 10 (AC): The tumors in the model group grew rapidly, and all treatment groups inhibited tumor growth. Tumor inhibition rate: 48.14% in the TAC group, 39.55% in the low-dose SVP-50-1 group, and 49.34% in the high-dose SVP-50-1 group. Combined administration effect: The tumor inhibition rate of the SVP-50-1 combined with high-dose TAC group reached 75.44%, significantly higher than that of the TAC group; the tumor inhibition rate of the SVP-50-1 combined with low-dose TAC group was 64.2%. Pathological changes in tumor tissue were observed by H&E staining. Figure 10 In the model group (D), tumor cells were morphologically intact, with dense and neatly arranged nuclei and almost no necrotic cells, exhibiting typical tumor cell growth characteristics. In the TAC or SVP-50-1 monotherapy group, tumor cells showed varying degrees of damage and apoptosis, with a decrease in the number of normal cells; nuclear fragmentation and lymphocyte infiltration were observed; lipid droplets appeared in the tissue (reflecting abnormal tumor cell metabolism or damage). In the SVP-50-1 combined with TAC group, the number of blue-purple cell nuclei was significantly reduced, indicating that nuclear components (such as DNA) were gradually degraded by proteases and nucleases; the areas of apoptosis and necrosis were expanded, and the degree of tumor structural destruction was more significant than in the monotherapy group. Conclusion: Combined therapy significantly enhances the anti-tumor effect by synergistically inducing tumor cell nuclear lysis, exacerbating apoptosis, and structural destruction, showing superior pathological improvement compared to monotherapy. The combination of SVP-50-1 and TAC can significantly enhance anti-tumor activity through synergistic effects, providing a potential strategy for breast cancer treatment.

[0057] 2. H&E staining of heart, liver, spleen, lung, and kidney tissues H&E staining was performed on the heart, liver, spleen, lung, and kidney tissues of 4T1 tumor-bearing mice, and the results are as follows: Figure 11 As shown.

[0058] Depend on Figure 11It is known that SVP-50-1 monotherapy has no significant toxicity to the heart, liver, spleen, lungs, and kidneys. When used in combination with TAC, it can: reduce the cardiotoxicity of TAC (interstitial widening, capillary congestion); improve liver lobule structure and reduce lymphocyte infiltration; repair the white pulp-red pulp boundary in the spleen; alleviate pulmonary interstitial thickening and hemorrhage; protect glomerular morphology; and reverse TAC-induced kidney damage.

[0059] Note: SVP-50-1 enhances the safety of TAC treatment through multi-organ protection.

[0060] 3. Differentially expressed gene screening Differentially expressed genes (DEGs) were analyzed using DESeq (1.12.4). The screening criteria were set as follows: significance threshold: corrected P-value (Q-value) ≤ 0.05; fold change threshold: |log2FoldChange| ≥ 1 (i.e., gene expression up-regulation or down-regulation ≥ 2-fold).

[0061] Results: Compared with the model group, the high-dose group of SVP-50-1 combined with TAC screened out a total of 3039 DEGs: 771 genes were upregulated and 410 genes were downregulated. Figure 12 A).

[0062] 4. GO and KEGG enrichment analysis of DEGs Functional enrichment analysis was performed using Cluster Profiler (3.0.5), and GO enrichment analysis was performed using topGO (2.24.0). Significance threshold: corrected P-value (Q-value) < 0.05.

[0063] (1) GO enrichment analysis: Gene Ontology (GO) is an internationally standardized classification system for gene function, covering the following three dimensions: Biological Processes (BP): biological activities in which genes participate; Cellular Components (CC): cellular structures in which gene products are located; Molecular Functions (MF): molecular activities of gene products.

[0064] DEGs enrichment results of the SVP-50-1+TAC group vs. the model group ( Figure 12 B): BP: Exogenous peptide antigens are processed and presented through MHC class Ib molecules; antibody-dependent cytotoxicity regulation; positive regulation of antibody-dependent cytotoxicity; natural killer cell tolerance induction; TRAIL production; CC: MHC protein complex; MHC class I peptide loading complex; mitochondrial inner membrane protein complex; mitochondrial respiratory chain; respiratory chain complex; MF: Electron transfer activity; NADH dehydrogenase activity; primary transmembrane transporter activity; oxidoreductase activity, acting on NAD(P)H; cytochrome c oxidase activity; oxidoreductase activity acting on donor heme groups; (2) KEGG enrichment analysis: The KEGG database integrates genomic, chemical, and systemic functional information for pathway analysis.

[0065] KEGG enrichment results of the SVP-50-1+TAC group vs. the model group ( Figure 12 B): The most significant pathway is oxidative phosphorylation, which is significantly enriched at a higher level than other pathways.

[0066] Key differentially expressed proteins: Five significantly differentially expressed genes were screened in this pathway: ATP8; COX2; CYTB; SDHC; NDUFA12.

[0067] (3) Mechanism analysis: SVP-50-1 combined with TAC can inhibit the expression of five genes in tumor cells: ATP8, COX2, CYTB, SDHC and NDUFA12, thereby inhibiting the activity of the mitochondrial respiratory chain complex in tumor cells and its ability to produce ATP through oxidative phosphorylation, thus achieving the effect of inhibiting tumor cell growth.

[0068] 5. Mechanism Verification The activities of the above five proteins and respiratory chain complexes in tumor tissue were detected. The results are as follows: Figure 13 As shown, Western blot analysis revealed that, compared to the model group, the expression levels of ATP8, COX2, CYTB, SDHC, and NDUFA12 proteins were significantly reduced in the SVP-50-1 combined with high-dose TAC group. The activity assay of mitochondrial respiratory chain complexes I-V showed that, compared to the model group, the activity of mitochondrial respiratory chain complexes I-V was significantly inhibited in the SVP-50-1 combined with high-dose TAC group. In other words, SVP-50-1+TAC inhibited the expression of five proteins (ATP8, COX2, CYTB, SDHC, and NDUFA12) and the activity of mitochondrial respiratory chain complexes I-V in tumor tissue, consistent with transcriptomics results.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A polysaccharide SVP-50-1 from poplar mulberry fungus, characterized in that, The structure of the poplar mulberry polysaccharide SVP-50-1 consists of 128 repeating units: The main chain repeating unit is →6)-β-D-Glc p -(1→、→2,4)-β-D-Man p -(1→、→2)-α-L-Ara p -(1→、→6)-β-D-Glc p -(1→、→6,3)-β-D-Glc p -(1→and→6,3)-β-D-Glc p -(1→ is composed of six sugar residues connected sequentially by (1→4), (1→2), (1→6), (1→3), and (1→3) glycosidic bonds; Branched β-D-Man p -(1→2)-α-L-Ara p -(1→、α-L-Fuc p -(1→ and β-D-Gal p -(1→ is sequentially linked to →2,4)-β-D-Man via (1→2), (1→6), and (1→6) glycosidic bonds respectively. p -(1→、→6,3)-β-D-Glc p -(1→、→6,3)-β-D-Glc p -(1→on sugar residue; The sugar chain structure is as follows: 。 2. A method for preparing the poplar mulberry polysaccharide SVP-50-1 according to claim 1, characterized in that, Includes the following steps: After pulverizing the fruiting bodies of *Phyllanthus linteus*, distilled water was added for extraction. The filtrate was concentrated and precipitated with ethanol solution. The precipitate was then completely dissolved in water and loaded into a D101 macroporous resin column. The column was eluted with 8 column volumes of distilled water to remove pigments. The eluent was collected and concentrated under reduced pressure. Proteins were removed using the Sevag method. The supernatant was collected and concentrated. Dialysis was performed using a dialysis bag with a molecular weight cutoff of 3500 Da to remove small molecule impurities, yielding *Phyllanthus linteus* polysaccharide SVP-50. This polysaccharide was loaded into a DEAE cellulose-52 ion exchange chromatography column for separation. Water was used as the mobile phase for elution. The fraction with the highest polysaccharide content was collected and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain *Phyllanthus linteus* polysaccharide SVP-50-1. The ethanol solution has a volume concentration of 50% in the precipitation system.

3. The preparation method according to claim 2, characterized in that, The mass-to-volume ratio of the fruiting body powder to the distilled water is 1g:10mL.

4. The use of the poplar mulberry polysaccharide SVP-50-1 according to claim 1 in the preparation of an anti-breast cancer drug.

5. The application according to claim 4, characterized in that, The drug is an inhibitor of oxidative phosphorylation energy metabolism in breast cancer cells.

6. The use of the poplar mulberry polysaccharide SVP-50-1 according to claim 1 in combination with antitumor chemotherapy or radiotherapy drugs in the preparation of anti-breast cancer drugs.

7. The application according to claim 6, characterized in that, The chemotherapy drugs include docetaxel, doxorubicin, and cyclophosphamide.

8. A tumor cell oxidative phosphorylation energy metabolism inhibitor with anti-tumor properties, characterized in that, The active ingredient includes the poplar mulberry polysaccharide SVP-50-1 as described in claim 1.

9. A drug for treating breast cancer, characterized in that, The active ingredients include the poplar polysaccharide SVP-50-1, docetaxel, doxorubicin and cyclophosphamide as described in claim 1.

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