Fructus liquidambaris acidic polysaccharide LFP-2 as well as preparation method and application thereof

Abstract

The invention discloses a liquidambar formosana hance acidic polysaccharide LFP-2 as well as a preparation method and application thereof, and belongs to the field of biological medicines. According to the method, a method for extracting the liquidambar formosana hance polysaccharide by boiling water and precipitating the liquidambar formosana hance polysaccharide by ethanol is adopted, a DEAE cellulose column purification technology is combined, and the principle that macromolecular polysaccharide is insoluble in an organic solvent is utilized, so that the separation of the liquidambar formosana hance acidic polysaccharide is successfully realized, and the liquidambar formosana hance acidic polysaccharide LFP-2 with anti-osteosarcoma activity is obtained; the monosaccharide of the liquidambar formosana hance acidic polysaccharide LFP-2 is prepared from the following components in mole percent: 6.84% of rhamnose, 5.33% of arabinose, 3.76% of galactose, 7.02% of glucose and 77.06% of galacturonic acid. Experiments prove that the traditional Chinese medicine composition has a remarkable anti-osteosarcoma effect, and a new treatment method is provided for clinical treatment of osteosarcoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a L. lupulina acidic polysaccharide LFP-2 and a preparation method and application thereof. Background Art

[0002] Osteosarcoma is a malignant bone tumor that originates in bone tissue and is prevalent in adolescents and young adults. It is the most common malignant bone tumor in children, accounting for approximately 5% of all pediatric tumors. Osteosarcoma originates from mesenchymal cells and directly or indirectly forms tumorous bone-like tissue and bone tissue through the cartilage stage. It typically develops in the epiphyses of long bones, most commonly in the distal femur, proximal tibia, and proximal humerus. Osteosarcoma is highly malignant and can easily metastasize to the lungs through the bloodstream. Clinical treatment primarily involves surgical resection combined with neoadjuvant chemotherapy, with a 5-year survival rate of 60%-70%.

[0003] Current clinical treatments have significant limitations: surgical resection may lead to limb dysfunction, and chemotherapy drugs have significant toxic side effects and are prone to drug resistance, resulting in poor therapeutic effects for patients with advanced or metastatic disease. Traditional treatments are unable to meet clinical needs, and the development of new treatment strategies is urgently needed. In recent years, plant polysaccharides have shown potential application value in the anti-tumor field. They exert anti-tumor effects through multiple pathways, such as regulating the immune system, inhibiting tumor angiogenesis, and inducing tumor cell apoptosis, providing new ideas for the treatment of osteosarcoma.

[0004] Lulutong (Liquidambarformosana) is the dried, mature infructescence of Liquidambarformosana Hance, a plant in the Hamamelidaceae family. Modern pharmacological research has confirmed its anti-tumor, antibacterial, and anti-angiogenic activities. Given the unique advantages of plant polysaccharides in tumor treatment, the development of Lulutong polysaccharides with inhibitory effects on osteosarcoma is of great significance. Summary of the Invention

[0005] The present invention aims to provide a Pulsatilla acidic polysaccharide (LFP-2), its preparation method, and its application to address the aforementioned problems of the prior art. The present invention isolates and obtains a Pulsatilla acidic polysaccharide (LFP-2) with anti-osteosarcoma activity. Experimental studies have demonstrated its significant anti-osteosarcoma efficacy, providing a new therapeutic approach for the clinical treatment of osteosarcoma.

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

[0007] The invention provides a passiflora acidic polysaccharide LFP-2. The monosaccharide composition of the passiflora acidic polysaccharide LFP-2, calculated by mole percentage, comprises 6.84% of rhamnose, 5.33% of arabinose, 3.76% of galactose, 7.02% of glucose and 77.06% of galacturonic acid.

[0008] Optionally, the LFP-2 acidic polysaccharide comprises two fragments, wherein fragment 1 is the main structure, which is formed by →4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-6-Ome-(1→ connected to each other, and fragment 2 is formed by →4)-β-D-Galp-(1→ and →4,6)-β-D-Glcp-(1→ connected to each other, and α-L-Araf-(1→ or α-D-GalpA-(1→) is connected to the →4,6)-β-D-Glcp-(1→O-6 position;

[0009] The sugar chain structure of fragment 1 is shown below:

[0010]

[0011] The sugar chain structure of fragment 2 is shown below:

[0012] Wherein, R is α-L-Araf-(1→ or α-D-GalpA-(1→.

[0013] The present invention also provides a method for preparing the L. lulutong acidic polysaccharide LFP-2, comprising the following steps:

[0014] The dried P. lupulus was ultrafinely ground and soaked in ethanol. Distilled water was added according to the solid-liquid ratio of P. lupulus powder: distilled water = 1 g: 40 mL for boiling and extraction. The extracted filtrate was filtered and concentrated. After ultrahigh-speed centrifugation, the supernatant was taken. The supernatant was precipitated with ethanol solution and centrifuged again. The precipitate was collected and separated and purified by DEAE cellulose column to obtain the P. lupulus acidic polysaccharide LFP-2.

[0015] Optionally, during the precipitation with the ethanol solution, the volume concentration of ethanol in the system is 80%.

[0016] Optionally, the ultracentrifugation condition is 10000 rpm, 10 min.

[0017] Optionally, the condition for the re-centrifugation is 7000 rpm, 15 min.

[0018] Optionally, the eluent used in the separation and purification process is a 0.4 M NaCl solution.

[0019] The present invention also provides the use of the Lulutong acidic polysaccharide LFP-2 in preparing a medicine for treating osteosarcoma.

[0020] The present invention also provides a medicine for treating osteosarcoma, wherein the active ingredient includes the Lulutong acidic polysaccharide LFP-2.

[0021] Optionally, the dosage form of the drug includes tablets, granules and solutions.

[0022] The present invention discloses the following technical effects:

[0023] The present invention adopts the method of boiling water extraction and ethanol precipitation of P. lupulus polysaccharide, combined with DEAE cellulose column purification technology, and utilizes the principle that macromolecular polysaccharides are insoluble in organic solvents to successfully achieve the separation of P. lupulus acidic polysaccharide and obtain a P. lupulus acidic polysaccharide LFP-2 with anti-osteosarcoma activity.

[0024] Through cell experiments, the present invention confirmed that the acidic polysaccharide LFP-2 from Lulutong has an inhibitory effect on osteosarcoma cells, and the effect is better than that of cervical cancer HeLa cells, glioma SHG-44 cells and prostate cancer 145 cells; further analysis found that LFP-2 inhibits the growth of osteosarcoma by regulating pathways related to osteosarcoma cell apoptosis and cell colony formation ability, providing a new treatment method for the clinical treatment of osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] Figure 1 This is the molecular weight and uniformity detection diagram of LFP-2;

[0027] Figure 2 This is the monosaccharide composition detection diagram of LFP-2;

[0028] Figure 3 This is the FT-IR infrared detection image of LFP-2;

[0029] Figure 4 is the total ion current of LFP-2 sample;

[0030] Figure 5 : The one-dimensional nuclear magnetic resonance detection image of LFP-2, wherein A is the one-dimensional hydrogen spectrum of LFP-2, and B is the one-dimensional carbon spectrum of LFP-2;

[0031] Figure 6 This figure shows the effect of LFP-2 on the morphology and proliferation of four types of tumor cells observed under a microscope;

[0032] Figure 7 This is the result diagram of the effect of LFP-2 on the colony-forming ability of osteosarcoma U2OS cells;

[0033] Figure 8 This is the DAPI staining result of U2OS cells after LFP-2 treatment;

[0034] Figure 9 This is the result of AO / EB fluorescence staining after U2OS cells were treated with LFP-2. DETAILED DESCRIPTION

[0035] 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.

[0036] 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. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] 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.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Reagents and materials involved in the embodiments of the present invention:

[0041] Rhizoma L. Rhizoma was purchased from Beijing Tong Ren Tang as a dried medicinal piece.

[0042] Osteosarcoma cell line U2OS (CL-0236, Prosai Biotechnology Co., Ltd.), McCoy's 5A medium (PM150710, Prosai Biotechnology Co., Ltd.), and fetal bovine serum (164210, Prosai Biotechnology Co., Ltd.) were used. Reagents used in the experiment included AO / EB fluorescent staining solution (CA1142, Beijing Solebio Technology Co., Ltd., China), DAPI fluorescent staining solution (KGA1808-100, Jiangsu KeyGen Biotech Co., Ltd., China), and Annexin V-FITC / PI double staining apoptosis detection kit (KGA1102-100, Jiangsu KeyGen Biotech Co., Ltd., China).

[0043] Example 1 Extraction, separation and molecular weight detection of Psoralea corylifolia acidic polysaccharide LFP-2

[0044] 1. Extraction and separation of LFP-2 acidic polysaccharide from Psoralea corylifolia

[0045] Dried P. lupulus was ultrafinely ground and soaked in ethanol for 3 days. Then, distilled water was added at a ratio of 1 g of P. lupulus powder to 40 mL of distilled water. The mixture was heated to boiling point and extracted four times, each for 2 hours. The combined extracts were filtered twice through filter cloth and concentrated to 1 / 5 of their original volume. Ultracentrifugation was then performed (10,000 rpm, 10 minutes), and the supernatant was retained. The supernatant was added to four volumes of 95% ethanol to a final concentration of 80%. The mixture was stirred and allowed to stand overnight at 4°C to allow precipitation. The next day, the precipitate was collected by centrifugation (7,000 rpm, 15 minutes), dissolved in a small amount of water, and purified by separation on a DEAE cellulose column. The eluent was 0.4 M NaCl solution. The resulting liquid was freeze-dried and stored as P. lupulus acidic polysaccharide LFP-2.

[0046] 2. Structural identification of L. lulutong acidic polysaccharide LFP-2

[0047] (1) Molecular weight detection and monosaccharide composition of LFP-2

[0048] The molecular weight and monosaccharide composition of LFP-2 were determined by methylation technology. The molecular weight of LFP-2 was 22.926 kDa ( Figure 1 After the monosaccharide composition test, it was found that LFP-2 includes rhamnose, arabinose, galactose, glucose and galacturonic acid, which account for 6.84%, 5.33%, 3.76%, 7.02% and 77.06% of the molar percentage of LFP-2 respectively ( Figure 2 ).

[0049] (2) Functional group detection of LFP-2

[0050] Infrared spectroscopy was used to detect the main functional groups in LFP-2 to understand its structural characteristics.

[0051] LFP-2 infrared results analysis shows that ( Figure 3 ), at 3000-3700cm -1 There is an obvious strong absorption peak in the molecule, which is composed of intermolecular and internal hydrogen bonds, making it appear at 3306cm -1 There is a broad peak at 3306cm -1 and 2927cm -1 They represent the hydroxyl group (-OH) and CH on the sugar chain of LFP-2. -1 The C=O stretching vibration is strong, so 1643 cm -1 The peak at 1360 cm indicates that there may be acid-linked glucans in LFP-2. -1 The absorption peak at 1149cm is the CH angle vibration absorption of the methyl group connected to the carboxyl group. -1 The absorption peak at 1076 cm may be the vibration of peptide bonds CC and CO. -1 and 1015cm -1 The absorption peak is the angular vibration absorption peak of the hydroxyl group of pyranose ring, indicating that the monosaccharide exists in the form of pyranose. These are the characteristic peaks of polysaccharides. In addition, at 847cm -1 and 927cm -1 The absorption peak at indicates that the glycosidic bond in the LFP-2 sugar chain is in α-configuration.

[0052] (3) LFP-2 methylation detection

[0053] The total ion current of methylated LFP-2 is shown in the figure Figure 4 As shown, the polysaccharide linkages of LFP-2 were analyzed, and the results are shown in Table 1.

[0054] Table 1 Results of bonding structure analysis of LFP-2 polysaccharide samples

[0055]

[0056]

[0057] (4) NMR detection of LFP-2

[0058] The main sugar chain structure of LFP-2 was identified by nuclear magnetic resonance (NMR) technology to determine its specific sugar chain conformation and connection mode.

[0059] One-dimensional NMR results showed that LFP-2 polysaccharide 1The signals in H NMR are concentrated in 3-6 ppm. Usually, the anomeric hydrogen signals of β-glycosidic bond configuration are mainly distributed in δ4.3-4.8 ppm, and the anomeric hydrogen signals of α-glycosidic bond configuration are mainly distributed in δ4.8-5.8 ppm. 13 The chemical shift signal distribution in C NMR is relatively broad. The spectral lines have little overlap and can provide information about sugar residues. The anomeric carbon signal of polysaccharides in 13C NMR is concentrated at 95-110 ppm. The above results are used to determine the attachment position of the sugar chain and the specific group ( Figure 5 ).

[0060] Two-dimensional nuclear magnetic resonance imaging reveals the sugar residues in LFP-2 polysaccharide 1 H and 13 The chemical shifts of C are shown in Table 2.

[0061] Table 2 Chemical shifts of hydrogen and carbon isotopes of sugar residues

[0062]

[0063]

[0064] Note: nd: abbreviation of “not detected”, indicating that it was not identified.

[0065] Based on the analysis of one-dimensional and two-dimensional nuclear magnetic resonance information and methylation results, it was inferred that the LFP-2 polysaccharide may contain two fragments, of which fragment 1 is the main structure, formed by the interconnection of →4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-6-Ome-(1→), and fragment 2 is formed by the interconnection of →4)-β-D-Galp-(1→ and →4,6)-β-D-Glcp-(1→, and α-L-Araf-(1→ or α-D-GalpA-(1→) is connected to the →4,6)-β-D-Glcp-(1→O-6 position. Therefore, the possible structure of the polysaccharide chain is speculated as follows:

[0066] Snippet 1:

[0067]

[0068] Snippet 2:

[0069] R:α-L-Araf ORα-D-GalpA-(1→.

[0070] Example 2 Antitumor activity of L. lulutong acidic polysaccharide LFP-2

[0071] 1. Inhibitory activity of LFP-2 from Lulutong acidic polysaccharide against four types of tumor cells

[0072] Cell Lines and Cell Culture: Cervical cancer HeLa cells, glioma SHG-44 cells, prostate cancer DU145 cells, and osteosarcoma U2OS cells were cultured in a cell culture medium supplemented with 100 μg / mL streptomycin and 100 μg / mL penicillin G, along with 10% fetal bovine serum, at 37°C in a constant temperature and humidity environment with a CO concentration of 5%. When the cell density reached 90%, a 1:3 subculture was performed and culture continued.

[0073] Cell proliferation assay: cervical cancer HeLa cells, glioma SHG-44 cells, prostate cancer DU145 cells, and osteosarcoma U2OS cells were cultured at 2×10 3 The cells were seeded into 96-well plates at a density of 10 cells / well. After 24 hours of cell culture, the original culture medium was replaced with a drug culture medium containing different concentrations of LFP-2 (0, 200, 400 and 800 μg / mL). The experimental settings included negative and positive controls: the cells in the negative control group were cultured normally without the addition of drugs; the positive control group added doxorubicin hydrochloride (DOX) instead of LFP-2 drugs, and the concentration of DOX was 5 μM. After inoculation, all tumor cells were co-cultured with 5 μM DOX and different concentrations of LFP-2 (0, 200, 400, 800 μg / mL). Five replicates were set up for each treatment group, and the experiment needed to be repeated more than 3 times and tested in parallel. After 24 hours and 48 hours of co-incubation, cell viability was assessed using the CCK-8 cell proliferation detection kit. The CCK-8 reagent was added to the culture medium at a ratio of 1:10, and after incubation for 30 minutes, the absorbance value A at 450 nm was read on a microplate reader. The calculation formula is as follows:

[0074] Proliferation inhibition rate (%) = ([A 阴性对照 -A 实验组 ] / [A 阴性对照 -A 空白对照 ])×100%.

[0075] Microscopic observation results Figure 6 As shown, tumor cells co-cultured with LFP-2 had abnormal morphology, appeared shrunken and fragmented, and the cell density was significantly reduced.

[0076] The CCK-8 assay results, as shown in Table 3, show that LFP-2 exhibits a time- and concentration-dependent inhibitory effect on tumor cell proliferation. With increasing LFP-2 concentration, the increase in the proliferation inhibition rate of cervical cancer HeLa cells, glioma SHG-44 cells, and prostate cancer DU145 cells was less than that of osteosarcoma U2OS cells, with the maximum inhibition rate for U2OS cells being 89.65±0.77%. Therefore, LFP-2 has the strongest inhibitory effect on U2OS osteosarcoma cells, facilitating further investigation of the antitumor activity and mechanism of LFP-2 against osteosarcoma.

[0077] Table 3 Statistics of inhibition rate of tumor cell proliferation by LFP-2 from Psoralea corylifolia acidic polysaccharide (%)

[0078]

[0079] Note: The results are expressed as mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0080] 2. Inhibitory activity and anti-tumor mechanism of LFP-2 from Lulutong acidic polysaccharide on osteosarcoma U2OS cells

[0081] (1) LFP-2, a polysaccharide from Lulutong, inhibits the colony formation of osteosarcoma U2OS cells

[0082] Crystal violet staining experiment: Wash the cells three times with PBS buffer, cover the cells at the bottom of the cell culture plate with crystal violet stain, stain for 15 minutes, and then rinse the cells with PBS buffer. After washing, observe the cell colonies under a microscope and count the cells after confirming the formation of multiple single colonies. Perform 3 to 5 parallel wells for each concentration and take the average value for statistical analysis.

[0083] After 21 days of continuous drug administration and culture, crystal violet staining showed that the number of cell colonies in the control group was significantly higher than that in the LFP-2 administration group ( Figure 7 When the concentration of LFP-2 was 400 μg / mL and 800 μg / mL, there was almost no cell colony in the culture dish. Specific statistical information is shown in Table 4.

[0084] Table 4 Effect of L. lulutong acidic polysaccharide LFP-2 on the number of osteosarcoma cell U2OS colonies

[0085]

[0086] Note: The results are expressed as mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0087] (2) LFP-2, a polysaccharide from Lulutong, induces apoptosis of osteosarcoma cells U2OS

[0088] To verify the effect of LFP-2 on apoptosis of osteosarcoma U2OS cells, the present invention used 0 and 800 μg / mL of LFP-2 to perform DAPI fluorescence staining, acridine orange / ethidium bromide fluorescence staining and cell apoptosis experiments on U2OS cells, respectively.

[0089] DAPI fluorescence staining experiment: U2OS cells were 1×10 4 Cells were seeded at a density of 10 cells / well in a culture dish containing McCoy's 5A medium and cultured for 24 minutes. Subsequently, the original culture medium was replaced with drug culture medium and co-incubated with LFP-2 for 24 hours and 48 hours. Afterwards, the cells were washed with PBS, fixed with methanol, and washed again. 80 μL of DAPI was added to each well, stained in the dark for 5 minutes, and then washed once with PBS. Finally, photos were taken using a Nikon inverted fluorescence microscope (magnification of 100× and 200×), and 5 photos were retained for each drug concentration.

[0090] Acridine orange / ethidium bromide fluorescence staining experiment: U2OS cells were plated at 1×10 4 Cells were seeded at a density of 10 cells / well in a culture dish containing McCoy's 5A medium and cultured for 24 hours. Subsequently, the original medium was replaced with drug medium and co-incubated with LFP-2 for 24 and 48 hours. A 1:1 acridine orange (AO) / ethidium bromide (EB) staining solution was prepared and 20 μL of the staining solution was added to each well. The mixture was mixed thoroughly and the reaction was allowed to proceed in the dark for 5 minutes. Images were taken using a Nikon inverted fluorescence microscope (100× and 200× magnification), and five images were retained for each drug concentration.

[0091] Apoptosis assay: U2OS cells were cultured at 2×10 5 Cells were seeded at a density of 10 cells / well in a culture dish containing McCoy's 5A medium and cultured for 24 hours. Subsequently, the medium was replaced with LFP-2-containing drug medium and incubated with LFP-2 for 48 hours. Afterwards, the cells were washed twice with PBS, and the reagents were added according to the instructions and mixed thoroughly. The cells were allowed to react at room temperature in the dark for 5–15 minutes. The experiment was repeated three or more times.

[0092] DAPI fluorescence staining showed that after drug administration, the cell nuclear morphology began to change, chromatin aggregation, cell nuclear condensation, and the appearance of blue bright apoptotic bodies. The number of apoptotic bodies in the experimental group was significantly greater than that in the control group, and increased with the increase of drug concentration (see Figure 8 ).

[0093] AO / EB fluorescent staining can reflect the conditions of cell apoptosis at different stages. Four different cell morphologies can be seen under a fluorescence microscope. The nuclear chromatin of living cells appears green and has a typical shape. The nuclear chromatin of early apoptotic cells appears green, solidified or beaded. The nuclear chromatin of non-apoptotic dead cells is orange and has a normal morphology. The chromatin of apoptotic cells is orange and solid or beaded. In the treatment group, AO / EB staining showed that the number of apoptotic cells increased with increasing concentration, while the number of living cells decreased (see Figure 9 ).

[0094] Inducing apoptosis is an important pathway for inhibiting tumor cell proliferation. The results of apoptosis analysis are shown in Table 5. When U2OS cells were treated with 0 and 800 μg / mL of LFP-2, the number of viable cells decreased significantly, the apoptosis rate showed a significant change (P < 0.05), and the total apoptosis rate increased significantly, reaching a maximum of 36.51 ± 5.65%. Fluorescence staining and flow cytometry showed that LFP-2 significantly induced apoptosis in U2OS cells.

[0095] Table 5 Results of apoptosis induction of osteosarcoma U2OS cells by LFP-2 acidic polysaccharide

[0096]

[0097] Note: The results are expressed as mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0098] In summary, the present invention experimentally verified that L. lulutong acidic polysaccharide has significant anti-tumor activity against U2OS cells, inhibiting osteosarcoma growth and affecting cell colonies by regulating the cell apoptosis pathway. LFP-2 provides a new therapeutic method for the clinical treatment of osteosarcoma.

[0099] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A L. lulutong acidic polysaccharide LFP-2, characterized in that: The monosaccharide composition of the L. lulutong acidic polysaccharide LFP-2, in terms of molar percentage, includes 6.84% rhamnose, 5.33% arabinose, 3.76% galactose, 7.02% glucose and 77.06% galacturonic acid.

2. The LFP-2 acidic polysaccharide according to claim 1, characterized in that The Lulutong acidic polysaccharide LFP-2 comprises two fragments, wherein fragment 1 is the main structure, which is formed by the mutual connection of →4)-α-D-GalpA-(1→ and →4)-α-D-GalpA-6-Ome-(1→), and fragment 2 is formed by the mutual connection of →4)-β-D-Galp-(1→ and →4,6)-β-D-Glcp-(1→, and α-L-Araf-(1→ or α-D-GalpA-(1→) is connected at the O-6 position of →4,6)-β-D-Glcp-(1→; The sugar chain structure of fragment 1 is shown below: The sugar chain structure of fragment 2 is shown below: Wherein, R is α-L-Araf-(1→ or α-D-GalpA-(1→.

3. A method for preparing the L. lulutong acidic polysaccharide LFP-2 according to claim 1 or 2, characterized in that: The following steps are involved: The dried P. lupulus was ultrafinely ground and soaked in ethanol. Distilled water was added according to the solid-liquid ratio of P. lupulus powder: distilled water = 1 g: 40 mL for boiling and extraction. The extracted filtrate was filtered and concentrated. After ultrahigh-speed centrifugation, the supernatant was taken. The supernatant was precipitated with ethanol solution and centrifuged again. The precipitate was collected and separated and purified by DEAE cellulose column to obtain the P. lupulus acidic polysaccharide LFP-2.

4. The preparation method according to claim 3, characterized in that During the precipitation with the ethanol solution, the volume concentration of ethanol in the system is 80%.

5. The preparation method according to claim 3, characterized in that The ultracentrifugation conditions are 10000 rpm, 10 min.

6. The preparation method according to claim 3, characterized in that The conditions for the re-centrifugation are 7000 rpm and 15 min.

7. The preparation method according to claim 3, characterized in that The eluent used in the separation and purification process is 0.4 M NaCl solution.

8. Use of the Lulutong acidic polysaccharide LFP-2 according to claim 1 or 2 in the preparation of a medicament for treating osteosarcoma.

9. A drug for treating osteosarcoma, characterized in that: The active ingredient includes the Lulutong acidic polysaccharide LFP-2 according to claim 1 or 2.

10. The drug according to claim 9, characterized in that The dosage forms of the drug include tablets, granules and solutions.

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